key: cord-298064-itxuyetu authors: tang, rong; erdelez, sanda; may, emma; zhang, yishan title: the state of practice of covid‐19 tracking systems: an inventory study date: 2020-10-22 journal: proc assoc inf sci technol doi: 10.1002/pra2.397 sha: doc_id: 298064 cord_uid: itxuyetu in this poster, we report the preliminary results of an inventory of 149 publicly accessible active covid‐19 tracking systems. key findings include the frequency distribution of the systems' web domain names, the countries where the systems were created, the languages they support, the visual display format, the map platforms, and the data sources. these findings help to advance the knowledge of the data characteristics and design of pandemic surveillance/tracking systems. during the current covid-19 pandemic, numerous covid-19 tracking systems and dashboards (e.g., "covid-19 map" by johns hopkins university, "covid-view" by the cdc, "coronavirus [covid-19]" by who) have been developed, and used readily by public health professionals, researchers, and the general public. according to the world health organization (who), the objectives of covid-19 surveillance systems include: "(a) monitor trends in the disease where human-to-human transmission occurs; (b) rapidly detect new cases in countries where the virus is not circulating; (c) provide epidemiological information to conduct risk assessments at the national, regional and global levels; (d) provide epidemiological information to guide preparedness and response measures" (who, 2020). shah (2020) also indicated that "covid-19 has shown the importance of dashboards and visualization for everyone. various countries and even states/regions have created their own covid-19 dashboards to educate people on the pandemic." however, to date, there has been no in-depth examination of the functions and features of these tools, and no sufficient empirical evidence about the accessibility and usability of these tools for the general public, whose daily lives have been threatened by the virus. the main goal of this study is to investigate the primary functions, features, and data display characteristics of covid-19 tracking systems. the research questions for the study are as follows: the covid-19 tracking system inventory was developed from april 6 to may 21, 2020 by our research team. there is a total of 149 publicly accessible systems documented in the inventory. among them, 112 (75.17%) were found through using google as the search engine. twenty (13.42%) were found using youtube, 10 (6.71%) were found on news websites. other search tools used to source tracking systems include baidu (n = 2, 1.34%), bing (n = 1, .67%), weibo (n = 1, .67%), articles (n = 1, .67%), newsletters (n = 1, .67%), and more. search terms used include "covid 19," "coronavirus," "covid 19 map," "新冠疫情" "疫情地图" or other variations. all but three systems were retrieved using english, and three using chinese search terms. we also used search terms that included individual states in the us, for example, "covid 19 and california," "covid 19 and washington dc." 3 | results the most frequently occurred url domain extension was .gov (n = 52, 35%), followed by .com (n = 42, 28.2%), .org (n = 13, 8.7%), then by .edu (n = 4, 2.7%), .live (n = 3, 2.0%). other generic domain extensions that had only one occurrence include .us, info, io, app and tech. the .cn has six instances (n = 6, 4%). the remaining 24 sites included one instance from eu and 23 individual countries' url extensions. there were 37 originating countries. ninety-seven (64.67%) systems were from the us, eight systems were from china (5.33%), three were from the uk (2%), two each were from australia, germany, hong kong, india, japan, korea, and switzerland. our sample was biased towards tools in the u.s., as we performed u.s. state related searches. table 1 includes all the countries where tracking systems were created. twenty-four languages were used in the 149 systems, with several systems having multilingual versions. the most common language was english (n = 127, 85.23%), followed at a distant second by chinese (n = 13, 8.72%), spanish (n = 8, 5.37%), korean (n = 5, 3.36%), german (n = 3, 2.01%), japanese (n = 3, 2.01%), russian (n = 3, 2.01%), vietnamese (n = 3, 2.01%), and haitian creole (n = 2, 1.34%). the remaining 14 other languages were used by one system. data visualization features and display format were documented in terms of the presence or absence of maps, tables, various chart formats including line charts, column charts, bar graphs, area charts, and pie charts. more than 80% (n = 120) of the systems contained a map, mostly interactive maps, and over 76% (n = 114) of the systems had tables. the types of charts in the order of frequency, were line charts (n = 86, 57.72%), column charts (n = 84, 56.38%), bar graph (n = 45, 30.20%), area charts (n = 34, 22.82%), and pie charts (n = 28, 18.79%). figure 1 shows the proportion of the systems' visual display capabilities. twenty-six kinds of platforms were used for tracking systems' map displays. the most popular platform was arcgis by esri (n = 30, 20.13%), followed by here (n = 22, 14.77%), and openstreetmap (n = 22, 14.77%). figure 2 displays all the platforms used by the 149 tracking systems. a total of 71 data sources were used by the 149 systems. among them, 19 were repeated sources, 52 were single sources. the most frequent data source was local state governments in the u.s. (n = 63, 42.28%). the second most frequent data source was johns hopkins center for systems science and engineering (csse) (n = 35, 23.49%). government-provided data sources were utilized for 31 systems (20.81%). beyond that, sources such as who (n = 21, 14.09%), the cdc (n = 14, 9.40%), the national health commission of china (n = 10, 6.71%), and european centre for disease prevention and control (ecdc) (n = 9, 6.04%) were used by multiple tracking systems. examples for data sources that were used for one system only included lancet, стопкоронавирус, helsingin sanomat, and bbc. table 2 lists data sources that were used by more than one system. according to french and monahan (2020) , it is necessary to establish a broader understanding of covid-19 surveillance systems as "[t]his understanding could productively contemplate 'any tracking and monitoring, whether systematic or not, of health-related information,' as well as any tracking and monitoring that could be enrolled into the work of determining the health status of people and populations" (pp. 4-5) . with the current status of the covid-19 pandemic, it is essential that covid-19 tracking systems provide accurate, reliable, and timely data. with reports (e.g., madrigal & meyer, 2020) on how various tracking systems including the cdc and state governments in the u.s. mixing data about tests, and thereby producing an inaccurate picture of the outbreak status, our inventory study presents an overall understanding the state of practice of such systems. note that our inventory at this stage is heavily biased towards the u.s. dashboards, systems originating from countries with more than 10,000 coronavirus cases will be searched and included. our next steps will involve the heuristic evaluation and usability testing of selected systems. it is hoped that our findings will advance the knowledge of the data characteristics and design of pandemic surveillance/tracking systems. dis-ease surveillance: how might surveillance studies address covid-19? data visualization and dashboards in the era of covid-19 global surveillance for human infection with coronavirus disease (covid-19) key: cord-017621-pyn1enz2 authors: zaras, nikolaos v. title: case study – greece date: 2012-08-31 journal: biopreparedness and public health doi: 10.1007/978-94-007-5273-3_11 sha: doc_id: 17621 cord_uid: pyn1enz2 biological terrorism and the need for biological defence is a relatively new concept for greece. although defence against weaponized pathogens was part of cbrn training in the military, it was the 9/11 massacre followed by the anthrax letters horror that triggered a more active involvement of the greek public health sector. in that historical moment a third bullet was added to the already existing disease outbreak classification – naturally, accidental and now deliberate. these incidents and the subsequent 2004 olympic games in athens drove the greek government to focus on biodefence and revise existing civil emergency planning by inclusion of new emerging threats. naturally occurring or accidental outbreaks of a disease usually take place in both urban and country environments. big cities are usually the targets of bioterrorism due to the high density of population resulting in both physical and psychological casualties. if the disease does not start from one's own country then early warning might be possible, leading to preventive measures all the way from the borders into the community. the h1n1 virus pandemic is an example of this globalization of medical information that is useful to both countries and citizens. one important parameter of the epidemiology of infectious diseases is the movement of the populace either for professional, recreational or immigration reasons. in the past, moving from one location to another, even within the same country borders, took considerable time. in modern times, usually less than 24 h is needed to cross the world. we witnessed the contribution of faster travel to the spread of disease recently during the 2010 fl u pandemic. apart from the legal movement of a population, mass illegal immigration also poses a signi fi cant problem in certain parts of the world -e.g. in greece -in relation to the spread of a disease or reemergence of old diseases like malaria or tuberculosis. the geographical location of greece and its porous borders due to the signi fi cant coastline make it an attractive destination for those seeking a better living environment or as a way to enter other eu countries as a fi nal destination. greece receives a considerable number of tourists annually that exceeds its own population. greeks also travel globally for the reasons mentioned above. this constant movement of a populace makes epidemiologic surveillance and disease prevention extremely dif fi cult. the reality of disease transmission as a result of immigration and travel is re fl ected below in the results from the hellenic centre for disease control and prevention (cdcp) [ 2 ] and various relevant ngos addressing the health status of immigrants and transmission of old and new infectious diseases. the percentage of declared cases of speci fi c diseases attributed to greek citizens and immigrants is shown in table 11 .1 . timely information is crucial when it comes to a natural or accidental outbreak of a disease. this information might be bene fi cial to laboratory or institution workers or the population that needs to be protected. of course, in most cases, basic hygiene measures (personal or collective, at home or in a wider infrastructure such as schools) can prevent these diseases. defence against a deliberate outbreak of a disease requires intelligence. this type of medical intelligence is attributed to national intelligence service both civilian and military. usually international collaboration is mandatory when weaponized pathogens are the problem. risk identi fi cation and assessment contribute to national defence as well. it is a continuous process dealing with both the deliberate and non-deliberate forms of disease outbreaks. internal (sanitary institutions, police reports, etc.) and external (neighboring countries, world health organization, eu public health surveillance systems, etc.) hints can assist experts to perform a risk assessment leading to an alert of the public health system. current geopolitical instability and turmoil in our own region combined with the existing direct and indirect, overt and covert threats against western societies make bioterrorism attacks a potential risk. production of biological weapons is both easy and cost effective. of course we must discriminate between the production and weaponization of pathogens that is not as easy and needs specialized equipment. pathogen production does not require large factories and existing facilities in commercial infrastructure (food industry, drug industry) can be used for this purpose. on a smaller scale, pathogens can be cultivated in small laboratories or mobile caravans similar to those used to produce illegal drugs. identi fi cation of such illegal laboratories is very dif fi cult. viral pathogens are more dif fi cult to produce as compared to bacteria and also need some extra precautions and equipment. large quantities of biological weapons can still be produced in a short period of time (days or weeks) in small laboratories. according to kathleen c. bailey, former assistant director, of fi ce for disarmament and armaments control, who visited many biotechnology and pharmacology companies, a complete biolab requires no more than a room of 4.5 m × 4.5 m and a budget of usd 15,000 for supplies [ 1 ] . in such a room, trillions of bacteria can be quickly produced with low risk and with minimum personal protection equipment such as a gas mask and a plastic suit over clothing. dif fi culties relevant to the production of biological weapons include: dif fi culties in the protection of workers at all levels of production, transportation, • and fi nal dispersal of biological weapons; low level of training and expertise can lead to accidents and exposure to • pathogens; vaccination of those involved is not always protective/effective; • controlling the quality and quantity of produced material is dif fi cult; • dispersion is not without problems since dispersal device explosives, uv expo-• sure, or weather conditions such as rain or drying may have negative effects on pathogens or spores; storage of pathogens poses additional problems; speci fi c conditions are required • to maintain the ef fi cacy, and it is dif fi cult to maintain them in a form ready for dispersion over long periods of time. key stakeholders in public health preparedness and response systems are: epidemiologic monitoring is the systematic and continuous collection, analysis and interpretation of sanitary/medical information relevant to public health. the objectives of epidemiologic monitoring are: follow-up of tendencies (estimate the impact of a disease or health problem the system of mandatory reporting of diseases represents the basis of epidemiologic monitoring in most countries; usually it is supplemented by more specialized systems, networks or studies with speci fi c objectives. the objectives of this system are: speci fi c (for the system of mandatory reporting of diseases) -detection of spo-• radic cases; detection of epidemic cases generic (for every system of epidemiologic monitoring) -estimation of reper the reporting process can start from the clinical or laboratory doctor or the hospital's infectious diseases nurse but has to be sent immediately (by fax) to the regional health directorates and cdcp. the reporting form includes the following data: after reporting, evaluation of the validity/completeness of the reported elements will follow along with a thorough investigation of the case that will lead to a systematic/ rapid analysis and interpretation/export of the conclusions. brie fi ng of public health/ sanitary/medical/nursing services will follow a complete evaluation of the system. 1836: elementary mandatory reporting of diseases (newspaper of government, no. 83, 31/12/1836 ). 1911 -1915 : legislation on systematic mandatory reporting of diseases (cholera 1911, smallpox 1911, plague 1915) . 1950: "measures taken against infectious diseases justifying their reporting as mandatory", art. 1: mandatory reporting of diseases (rd 7/9-11-1950) . 1998: essential improvement of mandatory reporting system (national centre of epidemiologic surveillance and intervention). 2003: "organization and modernization of public health services and other provisions", art. 8, law 3172/6-8-2003: epidemiologic monitoring of pestiferous diseases is practiced and coordinated by cdcp. 2003: "regulations applied for regional systems of health and providence", art. 44, law 3204/23-12-2003: cdcp -each private or public medical institution or individual doctor, operating legally, is obliged to inform cdcp of each case of pestiferous disease that comes to his/her attention. hellenic personal data protection authority: 1997: "protection of individuals from the manipulation/exposure of data of per-• sonal character", art. 7, law 2472/1997: exceptionally, it is allowed: if it concerns subjects of health; -if it is executed by a health professional in duty of secrecy; -if it is essential for medical prevention. this system was set in operation in 1999 and revised in september 2004. it deals with common diseases with minor indications (usually). its scope is to support the health system through data gathering and processing, to make a clear estimate of diachronic trends and detect a possible epidemic elevation in an area or region. a large number of selected primary care doctors participate in this system/ programme. these doctors are distributed all over the country in the following networks: private doctors network (86 physicians); • regional health care centres/clinics (98 physicians); • social security institute health units network (44 physicians). • the diseases included in the system of illness observers at the fi rst degree health care centres are: whooping cough, measles, mumps, rubella, varicella, in fl uenza of infective etiology, respiratory infection with fever (>37.5 °c). a weekly report is done of the number of cases and patients. the report is done according to the clinical fi ndings and de fi nitions. all public sector services, in the case of a suspected or con fi rmed biological incident -deliberate or not -that needs to be treated, alert the civil protection operations' centre of gscp. gscp then activates the crisis management team (cmt) which consists of representatives from police, fire service, first aid national center (fanc) [ 7 ] , national defence general staff, centre for disease control and prevention (cdcp) and the gscp itself. gscp's representative coordinates the functions of the cmt through telephone or video conference. after the thorough evaluation of the severity of the incident and the classi fi cation with different color codes if necessary, cmt will conduct a meeting at the gscp building for better coordination of the operation. when an initial estimation has been made, medical directorates in various/all regions of the country are informed and guidelines are issued. medical directorates are obliged to report immediately to the gscp about any laboratory result following citizens' examinations and inform the public according to the guidelines of gscp. different missions are given to police and fire service depending on the incident's nature. if needed, national defence general staff contributes resources through its military hospitals, laboratories, mobile laboratories, medical personnel, services (mass vaccination) and equipment (direct supply of masks with fi lters against biological agents, personal protective suits, decontaminants, antidotes, drugs, mobile toilets, and decontamination facilities) or other supportive units (e.g. to clear or secure an area, for quick transportation or relocation of people, etc.). in case of a cbrn agent release, hellenic national defence general staff activates its special joint cbrn company which has the capability to be airborne and deploy anywhere in greece within 4 h (maximum), to conduct a cbrn search, survey, identi fi cation, sampling, decontamination, and provide specialized fi rst aid. for bioterrorism agents, this company has the capability to operate portable biological detectors that can identify pathogens of special interest, such as those causing anthrax or plague, within 30 min (up to 28 biological samples can be processed simultaneously). the platoon was established after the 2004 olympic games by merging the two specialized units (one fi eld unit operating in both hot and warm zones and one hospital-based unit deployed at the army general hospital of athens) that were created and deployed during the games in support of fi rst responders. the optimal function of the system include: systematic weekly reporting • sharing small amounts of information with constant fl ow from the laboratories syndromic surveillance (special systems) this system is activated in special conditions or when there is a speci fi c objective. it applies to the reporting of predetermined clinical conditions ("syndromes") and not diagnosed diseases (e.g. "respiratory infection with fever" instead of syndromic surveillance applies to the system of illness observers, early detection of epidemic elevations or individual incidents with public health importance hepatitis a (syndrome compatible with acute hepatitis) • septic/unexplained shock monitoring of hospital infections) and studies with speci fi c objectives also exist. these are clinical-laboratory networks for special pathogens, such as hospital bio-pathology laboratories, reference laboratories, specialized laboratories, and special clinical units • contagious spongiform encephalopathy (centres for spongiform encephalopathy • reporting) poliomyelitis (centres for poliovirus reporting) • meningitis (centres for meningitis reporting) weapons of mass destruction: costs versus bene fi ts supplement to the general plan for civil protection "xenokrates" with the specialized plan for the management of human casualties" 5. ministry of citizen protection key: cord-027721-hpzs6fvf authors: mcheick, hamid; sayegh, john; ajami, hicham title: context-aware healthcare adaptation model for copd diseases date: 2020-05-31 journal: the impact of digital technologies on public health in developed and developing countries doi: 10.1007/978-3-030-51517-1_27 sha: doc_id: 27721 cord_uid: hpzs6fvf nowadays, ubiquitous computing and mobile applications are controlling all our life’s aspects, from social media and entertainment to the very basic needs like commerce, learning, government, and health. these systems have the ability to self-adapt to meet changes in their execution environment and the user’s context. in the healthcare domain, information systems have proven their efficiency, not only by organizing and managing patients’ data and information but also by helping doctors and medical experts in diagnosing disease and taking precluding procedure to avoid serious conditions. in chronic diseases, telemonitoring systems provide a way to monitor the patient’s state and biomarkers within their usual life’s routine. in this article, we are combining the healthcare telemonitoring systems with the context awareness and self-adaptation paradigm to provide a self-adaptive framework architecture for copd patients. chronic obstructive pulmonary diseases (copd) have attracted research interest as a major public health problem, because according to the world health organization (who) [1] , copd is currently considered the fourth, and will soon become the third, most frequent cause of death worldwide. it is also a disabling disease and therefore associated with high costs for treating and managing patients. as the disease progresses, patients become more susceptible to respiratory exacerbations which cause frequent hospital admissions and, thus, have a huge impact on patients' quality of life and healthcare costs [2, 3] . monitoring patient's health conditions from home and transmitting these data to a healthcare center could be a great solution that facilitates the management of the growing number of patients with copd and reduce the burden on health services. this approach is called home tele-monitoring, which can be used for a timely assessment of acute exacerbation or as a mechanism to generate alarms to the patients and/or healthcare professionals when clinical changes that may constitute a risk to the patient occur [4] . there are many systematic reviews and studies on the topic of telemonitoring in respiratory patients, specifically in patients with copd [5] [6] [7] [8] . all these studies are focusing on proving the effectiveness of applying home telemonitoring on copd patients, by studying the services that could be provided and their impacts on the patient's quality-of-life. however, none of these studies has provided a comprehensive proposal for a telemonitoring system that helps to control the burden of copd. we aim to design a telemonitoring healthcare application that helps copd patients with self-management and improve their quality of life, therefore reducing pressures on healthcare resources. we must develop a system that uses this data to provide an effective intervention that prevents exacerbation through the early recognition of symptoms and prompt treatment which may reduce the risk of hospitalization and control the burden of copd. based on this healthcare requirement, we realized the need of combining context awareness and self-adaptation with health telemonitoring, which will give our system the ability to be aware of the patient's data and context, then to adapt the required changes and act accordingly. the remainder of this paper is structured as follows. section 2 introduces the concept of context-awareness and reviews the most common forms of self-adaptation frameworks. section 3 highlights the characteristics of self-adaptive systems. section 4 presents our self-adaptation healthcare system for copd. section 5 then validates the proposed approach. finally, sect. 6 presents our conclusions. 2 context awareness and self-adaptation systems the notion of context has appeared implicitly for the first time in the ubiquitous computing area in 1993 by weiser "all the information that should be taken into consideration for an adjustment" [9] . lieberman et al. [10] proposed another interpretation of the context that exists within the field of computing: "context can be considered to be everything that affects the computation". this definition focuses on the application instead of the user, but nowadays with the widespread of mobile applications that focus on the user's lifestyle, health, and activities the factors that affect the user and these that affect the computation process become almost the same. in software systems, context awareness notion is mostly coupled with selfadaptation capability otherwise, there is no point in collecting contextual data. selfadaptation is a set of simultaneous and successive processes as an organized reaction to changes in the resources or environment of the system [11] . self-adaptive systems dynamically modify their behaviors to respond effectively to changes in their operational environment [12] . in the next section, an overview of many self-adaptation frameworks is provided. rainbow framework provides general mechanisms for developing reusable selfadaptive systems at a variety of different levels [13] . model-driven approach is an automated self-adaptive model that supports adding and removing technical resources at run-time [14] . meta-self is a service-oriented framework that provides a solid platform for the development of sas [15] . this framework allows designers to identify system properties, architectural patterns, and different adaptation mechanisms. fusion is a reusable feature architecture that incorporates a learning-based adaptive cycle. the adaptation cycle consists of three main steps-detect, plan, and effect [14] . moses is a service-oriented framework that focuses on quality of service (qos) requirements at runtime. moses provides a reusable implementation strategy of the adaptation logic following the mape cycle (monitoring, analysis, planning, and execution) [14] . the contract-based adaptive software application framework (casa) [16] is specialized in handling resources instability. the framework assumes that a system should not make any assumptions about the resources that will be available and should be prepared for any resource availability scenario cases. service-oriented architectures (ssoa) is a software framework that specifies any kind of adaptation by decomposing of functionalities [17] . each of these functionalities shall be specialized to fit a particular purpose. caredroid is an adaptation framework for android context-aware applications [18] . this framework caredroid monitors the contexts at run-time, and active methods only when it intercepts calls to sensitive methods. 3 system requirements and self-adaptation characteristics and taxonomy the first step to designing a self-adaptive system is to well identify the system requirements; we will depend on w5h-pattern [19] , which presents six questions that would help us in eliciting adaptation requirements (table 1) . [20] , the change needs to be done in the application layer on both sides: user interface and physician interface when when do we need to make these changes? whenever an urgent update happens in the user contextual data like vital signs, environmental risk factors, and planned activities or periodical changes like the evaluation of treatment and decision support suggestions what what do we need to change? we need to update some system attributes that present the system state and these attributes in its turn could trigger new functions or activate new components why why these changes are required? in healthcare monitoring applications especially these related to chronic diseases, taking precluding actions is crucial in treatment plans. also being able to notify the patient and the medical experts about any threatening situation or abnormal signs make these kinds of applications more efficient who is any human intervention is required in the adaptation process? from the patient side, all his biomedical data and surrounding environments data will be collected from sensors. however, because physical activities do affect the copd patient's state, he needs to detect his planned physical activity (running, swimming) how how to determine what changes and actions are needed to be done in the adaptation process? ajami and mcheick [20] provided a rule-based reasoning engine, depending on these generated rules all the required actions and changes can be deduced christian et al. [21] presented a taxonomy of the different properties of self-adaptive software. we will analyze this work and do a projection on our system requirements and use the results to build our system. handte et al. [22] provided two perspectives of temporal aspects: (i) reactive is when we have to adapt whenever a change in the context does happen. (ii) proactive is when the monitored data is used to forecast system behavior or environmental state [21] . in our case, the adaptation will be reactive depending on the changes that happen in the user contextual data. reason: the adaptation could be triggered for three reasons: (i) change of the context, (ii) change in the technical resources, and (iii) change in the users. in our case, the adaptation is triggered due to contextual changes, which provide a potential solution for the multiscale nature of copd. level: in our system, the change needs to be done on the application layer, where we need to update the acceptable range for the different datasets or we need to activate new components or call new functions. technique: mckinley [23] provided two techniques for adaptive software: parameter adaptation and compositional adaptation. parameter adaptation achieves a modified system behavior by adjusting system parameters. whereas compositional adaptation enables the exchange of algorithms or system components dynamically at runtime. we will use the first approach because it is suitable for a rule-based system. adaptation control: two approaches for implementing the adaptation logic can be found in the literature. the internal approach, which twists the adaptation logic with the system resources. the external approach splits the system into adaptation logic and managed resources, the ibm autonomic computing initiative provided mape model [24] , which is an external, feedback control approach. another aspect of the adaptation logic is the degree of decentralization. we will follow a decentralized approach by implementing independent units that control different aspects of adaptation. 4 self-adaptation healthcare system for copd ajami and mcheick [20] have proposed an ontology-based approach to keep track of the physical status of patients, suggest recommendations and deliver interventions promptly, by developing a decision support system based on an ontological formal description that uses swrl rules. the main goal of this paper is to provide an adaptation architecture design for the application layer, which will address the connection between three different entities: 1 -the end-user application: which is supposed to provide a certain service for both patient and physician. 2 -the data sources (sensors and patient's records): that provides a continuous stream of contextual data and historical data about the patient. 3 -the rules base: which presents the knowledge base in our system (fig. 1) the adaptation engine consists of a central adaptation unit and multiple sub adaptation units. each subunit is responsible for monitoring and managing changes for a specific category tuple (data, rules, services). the system variables will be saved in a shared memory called the global state, which is a composition of sub-states. each sub-state is considered as a container for saving category-specific data and it will be updated and managed by the adaptation subunit that is responsible for monitoring the same category. we will divide all our sets of data, rules, and services into three categories ( table 2) : now each subunit will be responsible for a specific category of the tuple. therefore, we will have three subunits: 1 -the biometrics unit 2 -the environmental unit 3 -the activities unit (fig. 2 ). -the biometrics unit will be responsible for monitoring all the patient's biomarkers upcoming from the biometrics sensors, and it will read all the stored data in the other two sub-states (the environmental state & the activities state), then depending on the vital related rules in the rules engine, it will update the biometrics state with the safe ranges for all the vital biomarkers and the current measurements for them. the same workflow will be applied in both the environmental unit and the activities unit. -the central adaptation unit: it is considered the main core of our system; it will be responsible for monitoring the global state, which will get rid of the burden of dealing with the continuous streaming of the patient's biometrics and the environmental data. by collecting all the contextual data in the global state, each category in its sub-state, we will have access to all the current biometrics and external factors value with the safe range for each one of them as well the current physical activity and the planned list of activities. depending on the previous data, the central unit will be able to detect any potential risk or abnormal situations by comparing the current value of each factor in the substates with its normal range, which had been adapted by every sub adaptation unit. when an abnormal situation is detected, the central unit will detect what action should be taken to prevent an exacerbation in the patient's health state. in order to test and validate our proposed system, we implemented a simulation app using some data obtained from medical records to simulate the streamed data and a set of copd rules extracted in [25] to create some testing scenarios. the main focus of the validation process was on the efficiency of the system to provide continuous monitoring of the patient status, and the ability to apply and adapt the required changes to prevent any dangerous exacerbation. the testing scenarios we had performed, proofed the ability of our system to handle the complexity of monitoring the enormous amount of contextual data, and keep track of the latest updates in the global state. also, following an aspect-oriented approach facilitates the implementation of the adaptation logic, by separating the categories of data that each adaptation unit needs to be responsible for observing. after testing some rules that lead to call a sequential set of actions and multiple updates in the state units, the system was able to adapt the safe ranges for the different environmental and biometrical factors and detect suitable action in an abnormal situation. nevertheless, our system still needs to be tested when it is connected to the whole rules engine when all copd rules are inserted into the engine, which will be done in future work. in this paper, we have presented an architecture for a context-aware self-adaptive system that is used to develop a copd healthcare telemonitoring system. the system is backed out by a medical rules engine in the copd domain that is used as the knowledge base to determine the safe ranges for patient's biomarkers and external factors, then detect the precluding actions needed to be taken to prevent severe exacerbations in patient's health state. our main contribution in this work is providing a context-aware self-adaptive system architecture that is dealing with the huge variety and complexity of contextual data and different sets of services by implementing a decentralized adaptation unit, which makes the monitoring and adaptation task easier and less complex by applying the separation of concerns principle. the top 10 causes of death integrated care prevents hospitalisations for exacerbations in copd patients global strategy for the diagnosis management and prevention of copd, global initiative for chronic obstructive lung disease (gold the use of remote monitoring technologies in managing chronic obstructive pulmonary disease insufficient evidence of benefit: a systematic review of home telemonitoring for copd home telehealth for chronic obstructive pulmonary disease: a systematic review and meta-analysis home telemonitoring forrespiratory conditions: a systematic review systematicreview of telemedicine services for patients affected by chronic obstructive pulmonary disease (copd) telehealthcare for chronic obstructive pulmonary disease: cochrane review and meta-analysis out of context: computer systems that adapt to, and learn from, context. ibm syst a survey on engineering approaches for self-adaptive systems an architecture-based approach to self-adaptive software rainbow: cost-effective software architecture-based self-adaptation comparison of approaches for developing self-adaptive systems metaself -an architecture and a development method for dependable self-* systems casa a contract-based adaptive software architecture framework towards a generic context-aware framework for self-adaptation of service-oriented architectures caredroid: adaptation framework for android context-aware applications getting to requirements: the w5h challenge ontology-based model to support ubiquitous healthcare systems for copd patients a survey on engineering approaches for self-adaptive systems 3pc: system support for adaptive peer-to-peer pervasive computing composing adaptive software ibm: an architectural blueprint for autonomic computing a pervasive healthcare system for copd patients ), which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the creative commons license and indicate if changes were made. the images or other third party material in this chapter are included in the chapter's creative commons license, unless indicated otherwise in a credit line to the material. if material is not included in the chapter's creative commons license and your intended use is not permitted by statutory regulation or exceeds the permitted use key: cord-300467-zht8olyh authors: nkengasong, john n.; nsubuga, peter; nwanyanwu, okey; gershy-damet, guy-michel; roscigno, giorgio; bulterys, marc; schoub, barry; decock, kevin m.; birx, deborah title: laboratory systems and services are critical in global health: time to end the neglect? date: 2010-09-01 journal: am j clin pathol doi: 10.1309/ajcpmpsinq9brmu6 sha: doc_id: 300467 cord_uid: zht8olyh the $63 billion comprehensive global health initiative (ghi) emphasizes health systems strengthening (hss) to tackle challenges, including child and maternal health, hiv/aids, family planning, and neglected tropical diseases. ghi and other initiatives are critical to fighting emerging and reemerging diseases in resource-poor countries. hss is also an increasing focus of the $49 billion program of the us president’s emergency plan for aids relief and the global fund to fight aids, tuberculosis and malaria. laboratory systems and services are often neglected in resource-poor settings, but the funding offers an opportunity to end the neglect. to sustainably strengthen national laboratory systems in resource-poor countries, the following approaches are needed: (1) developing integrative national laboratory strategic plans and policies and building systems to address multiple diseases; (2) establishing public-private partnerships; (3) ensuring effective leadership, commitment, and coordination by host governments of efforts of donors and partners; (4) establishing and/or strengthening centers of excellence and field epidemiology and laboratory training programs to meet shortand medium-term training and retention goals; and (5) establishing affordable, scalable, and effective laboratory accreditation schemes to ensure quality of laboratory tests and bridge the gap between clinicians and laboratory experts on the use of test results. and malaria. laboratory systems and services are often neglected in resource-poor settings, but the funding offers an opportunity to end the neglect. to sustainably strengthen national laboratory systems in resource-poor countries, the following approaches are needed: (1) developing integrative national laboratory strategic plans and policies and building systems to address multiple diseases; (2) establishing publicprivate partnerships; (3) ensuring effective leadership, commitment, and coordination by host governments of efforts of donors and partners; (4) establishing and/or strengthening centers of excellence and field epidemiology and laboratory training programs to meet short-and medium-term training and retention goals; and (5) establishing affordable, scalable, and effective laboratory accreditation schemes to ensure quality of laboratory tests and bridge the gap between clinicians and laboratory experts on the use of test results. in may 2009, president barack obama announced a $63 billion comprehensive global health initiative (ghi) to focus attention on broad global health challenges, including child and maternal health, hiv/aids, family planning, and neglected tropical diseases, with cost-effective intervention. the ghi adopts an integrated approach to fighting diseases, improving health, and strengthening health systems. during the past 10 years, the field of global infectious diseases has witnessed tremendous challenges and opportunities, including the continuous struggle to control and prevent the hiv/aids pandemic and to tackle emerging or reemerging infectious diseases, including multidrug-resistant and extensively drugresistant tuberculosis (tb), 1 novel emerging infections such as severe acute respiratory syndrome, 2 the avian influenza virus h5n1, 3 and the current pandemic of novel influenza a (h1n1) virus. 4 these infections have been well contained in developed countries, thanks to well-established laboratorysupported surveillance systems. however, laboratory services and systems are weak in resource-poor countries (rpcs). the new ghi, together with the surge in funding and commitment for programs to fight hiv/aids, tb, malaria, and flu in rpcs (an estimated $10 billion per annum is devoted to tackling the hiv/aids pandemic 5,6 ), offers a monumental opportunity to strengthen laboratory systems in rpcs that could be used to combat multiple diseases. health system strengthening is a key area of focus for some of the major programs, including the ghi. for example, in 2009, the us president's emergency plan for aids relief (pepfar) dedicated $1.0 to $1.4 billion to supporting health systems, of which 6% is earmarked for strengthening laboratory systems. 7 the global fund to fight aids, tuberculosis and malaria has also allocated a similar proportion to strengthening laboratory services. 7 in addition, the world bank will be allocating $63.6 million to strengthen laboratory systems in a regional program in east africa. 8 together these increased resources offer an unprecedented prospect to strengthen, in a holistic and integrated way, multiple challenges facing the appalling national laboratory systems (nlss) in rpcs. strengthened nlss will be critical to delivering reliable laboratory services to meet the millennium development goals (mdgs) for health; fight multiple existing, emerging, and reemerging diseases; and achieve president obama's ghi objectives. despite substantial evidence that nlss (comprising public health, government, private, and mission hospitals) are a key component of the overall health system; are needed to achieve the mdgs for health; are required for meeting universal access for treatment of hiv/aids, tb, and malaria; and are critical for achieving the objectives of the world health organization's (who) international health regulation (ihr), they remain one of the most neglected components of the health system in rpcs. 9,10 major challenges facing nlss in rpcs include a shortage of skilled and trained personnel, an inadequate infrastructure (eg, consistent supply of electricity and water, physical infrastructure), a lack of equipment, inadequate supply-chain management for consumables and reagents, poor equipment maintenance, lack of clear policies, and insufficient leadership. 9,10 consequently, clinicians lack confidence in laboratory results 11 and often rely on clinical diagnosis and empirical treatment instead of laboratory-confirmed diagnosis. for example, few clinicians order sputum-smear microscopy for a tb diagnosis because the turnaround time for the results may be longer than the average hospital stay, and patients are often discharged or die before the results are received. in addition, clinical diagnosis without quality laboratory testing often results in significant misdiagnosis and overdiagnosis, leading to inadequate or inappropriate treatment, drug resistance, and increased mortality. 12, 13 reyburn and colleagues 14 found that among 4,670 patients admitted and treated for malaria in a tanzania hospital, fewer than 50% actually had malaria, as confirmed by blood smear. similarly, a study in ghana found that 40% of patients with a clinical diagnosis of malaria actually had bacterial sepsis, 15, 16 and the correct diagnosis of malaria leads to better treatment. 17 in january 2008, the maputo declaration for a global commitment to strengthen laboratory services and systems in rpcs was issued and endorsed. 18, 19 with the surge in funding, we believe that the following actions should be undertaken to strengthen nlss in a sustainable manner in rpcs. as illustrated in ❚figure 1❚, countries and partners need to develop national laboratory policies and strategic plans 19 as part of the overall health sector development investment. these plans should be comprehensive and not disease-specific and should integrate multiple diseases with the ultimate goal of establishing a functional tiered laboratory network in the country as proposed in ❚figure 2❚. laboratory plans should also be considered a key component of implementing the who ihr, which is a legally binding agreement of all member states of who to help the international community ❚figure 1❚ core cross-cutting elements of laboratory health systems that should be strengthened to support multiple diseases of public health importance. horizontal arrows indicate cross-cutting core elements that affect laboratories and need to be addressed in a national laboratory strategic plan, regardless of the disease. these core elements must be addressed at various levels of a tiered laboratory network. the vertical arrows are examples of major public health diseases that have seen a surge in funding that can drive the laboratory systems to be strengthened in resource-poor countries. prevent and respond to acute public health risks with a global impact, such as influenza, multidrug-resistant and extensively drug-resistant tb, and other diseases. 20 as shown in figure 1 , a comprehensive national laboratory strategic plan should focus on strengthening core cross-cutting elements of laboratory health systems, including the following: (1) a framework for training, retaining, and career development of laboratory workers; (2) infrastructure development; (3) supply-chain management of laboratory supplies and maintenance of laboratory equipment; (4) specimen referral systems in an integrated, tiered nls network; (5) standards for quality management systems and accrediting laboratories and facilities; (6) laboratory information system; (7) biosafety and waste management; and (8) a governance structure that will clearly address regulatory issues and define reporting structures, authority, and the relationship between private diagnostic and public health laboratories, to ensure a functional nls network as illustrated in figure 2 . these regulatory frameworks are critical for guiding the implementation of laboratory quality management systems, accreditation of laboratories, defining certification process for technicians, and monitoring and evaluation. ❚figure 3❚ is a proposed framework for strengthening laboratory systems in developing countries. if efforts are focused on strengthening core cross-cutting aspects of laboratory systems that are critical to ensure quality diagnostic services (microscopy, flow cytometry, molecular diagnosis, serology, clinical chemistry, and culture), sustainable nlss will emerge in rpcs that will benefit the fight against multiple emerging, reemerging, and existing diseases. in fact, because some hiv/ aids laboratory investments had strengthened laboratory core systems in some rpcs, they have been instrumental in the past in supporting the investigation of other disease outbreaks. 21 moreover, with support from the world bank's aids program and pepfar, rwanda developed a functional integrated national reference laboratory that is currently used to combat multiple diseases, including tb, malaria, and epidemic-prone diseases (eg, cholera), and priority diseases. finally, laboratory plans should aim at strengthening infrastructure in health facilities outside the public sector, especially private and mission hospitals that are critical to achieving universal access to care and treatment in rpcs. in fact, a sizable portion of persons in africa receive health care in the private and rural settings in which, in some countries, 40% of all health care and more than 70% of care in rural areas is provided in mission hospitals. 22 once developed, laboratory strategic plans can be implemented through a variety of ways. first, public-private partnerships (ppps) have been shown to be effective in strengthening ❚figure 2❚ potential structure of a national laboratory network system in resource-poor countries. in this structure, clear lines of functions, authority, and responsibility are outlined from the central public health laboratory (cphl) to the primary health center (phc). for example, the cphl sets policy, conducts planning, and establishes appropriate quality management systems. in larger countries, regional public health laboratories may need to take on the responsibilities of the cphl so as to reach the phc. health systems in rpcs. 23 therefore, ppps that focus on implementing established laboratory plans will be vital. in this respect, pepfar has entered into an $18 million ppp agreement with becton dickinson, a private medical diagnostic company, to strengthen laboratory capacity in 8 african countries in the areas of training, development of referral systems for transporting samples, and quality management schemes. 24 similarly, the abbott fund in tanzania is developing laboratory infrastructure in the country, including a stateof-the-art reference laboratory in dar es salaam, and upgrading all regional hospital laboratories in the country. 25 26 will be critical to developing laboratory services and systems in the private sector. increased funding to combat hiv/aids, tb, malaria, flu, and global infectious diseases means that multiple international partners and donors will be present in a given rpc. the unintended consequences of uncoordinated efforts can be fragmented laboratory health systems. to avoid this shortcoming, the maputo declaration calls for increased collaboration and coordination of donors and implementing partners' activities, based on their comparative advantages, within the context of countries' national strategic laboratory plans. therefore, host governments will need to show leadership and commitment by ensuring that laboratory plans are an integral part of the overall health systems strategy and investment for the country and that a functional department of laboratory services is established within the ministry of health, similar to other departments such as pharmacy and disease control. there is an acute shortage of a well-trained national laboratory workforce to meet the expanding hiv/aids, tb, malaria, and flu programs. the who estimates that to meet the mdgs, approximately 1 million new health workers (including staff working in laboratories) will need to be trained and retained by the year 2010. 27 in this regard, the pepfar reauthorization legislation that aims to train 140,000 health care workers is a highly appropriate initiative. 28 training and retention efforts for laboratory experts should focus on job redesign and use holistic approaches that address projected skill shortages, ie, based on an assessment and definition of critical gaps at the country or regional level. any effective training strategy must proactively involve stakeholders (eg, national governments, the private sector, development partners, and multilateral bodies). to partly address the short-term laboratory needs and support current ❚figure 3❚ proposed framework for strengthening laboratory systems globally showing the core elements of a laboratory system that need to be strengthened, with the surge in hiv/aids and tuberculosis funding, to provide broad-based, integrated, quality laboratory services and ensure sustainability of global laboratory investments. this approach will be needed to combat multiple global disease program needs, especially for infections that do not have specific funding. for example, for serologic diagnosis of any disease to be effective in a resource-poor country, there is a need to adequately address issues related to quality management, training and retention, equipment procurement and maintenance, supply chain management, laboratory information systems, biosafety, and policy. program goals, centers of excellence are needed to provide specialized staff needs. with funding from pepfar, the national institute of communicable diseases in johannesburg, south africa, recently established the african center for integrated laboratory training (acilt). the acilt was created after in-depth consultation on laboratory training needs of several african countries, in collaboration with the who regional office for africa. the courses used a competency-based approach for the trainees to obtain knowledge and skills at the national institute of communicable diseases; attendees then returned to their countries and developed competencies while working under supervision and received follow-up visits by the core training staff of acilt. this training approach is similar to the short-course component of the us centers for disease control and prevention-supported field epidemiology training programs in several rpcs. in these programs, field epidemiologists and public health laboratory staff are jointly trained in long courses (ie, 2-year public health leadership training) and short courses (ie, 1-to 2-week public health implementer training) to acquire skills and develop competencies while providing a public health service. 29 these were modeled on the us centers for disease control and prevention epidemic intelligence service. laboratory components of the field epidemiology training programs will need to be standardized and strengthened to address needs for technicians, managers, and policy makers. although implementing international laboratory standards, such as iso 15189, has been shown to be possible in developing countries, implementation is not easily scalable in the laboratories of most developing countries. thus, the who regional office for africa stepwise laboratory accreditation program and the thai accreditation scheme, both described in this issue of the journal, offer practical and affordable approaches to quality management systems that can be implemented and scaled up in developing countries. there is need to establish continuous laboratory medical education (cmle) for clinicians so as to bridge the gap between laboratory testing and uptake for patient management. regional societies for laboratory medicine or clinical pathology could serve as the appropriate vehicle to meet these critical gaps, including implementing cmle and certification of training for medical technologists. the new obama ghi and the increased funding for hiv/ aids, tb, malaria, and flu programs provide a monumental opportunity to build integrated and sustainable nlss in rpcs, which will help advance the goals of the mdgs for health, support universal access to treatment, meet the objectives of the maputo declaration, and advance the who ihr. the efforts of rpc governments and partners should be focused on developing an integrated and functional tiered nls through a comprehensive national laboratory strategic plan and policy, establishment of ppps, leadership and commitment of host countries that is needed to effectively coordinate donors and implement the efforts of partners, establishment of centers of excellence to meet urgent human capacity needs, and implementation of practical and affordable laboratory accreditation schemes and cmle for physicians. global efforts should center on strengthening critical cross-cutting core elements of laboratory systems (figure 3 ) that are vital to support multiple global health needs and will ensure sustainability. multidrug-resistant and extensively drug-resistant tuberculosis: a review identification of the novel coronavirus in patients with severe acute respiratory syndrome characterization of an avian influenza a (h5n1) virus isolated from a child with a fatal respiratory illness severe respiratory disease concurrent with circulation of h1n1 influenza the great funding surge hiv/aids: money matters aids: lessons learnt and myths dispelled cross-country laboratory network to raise east africa's defenses against disease are laboratory services coming of age in sub-saharan africa laboratory medicine in africa: a barrier to effective health care laboratory use in ghana: physician perception and practice achilles heel" of global efforts to combat infectious diseases diagnostic insufficiency in africa overdiagnosis of malaria in patients with severe febrile illness in tanzania: a prospective study high mortality of infant bacteraemia clinically indistinguishable from severe malaria overlap in the clinical features of pneumonia and malaria in african children improved diagnostic testing and malaria treatment practices in zambia world health organization. the maputo declaration on strengthening of laboratory systems critical role of developing national laboratory strategic plans as a guide to strengthen laboratory health systems in resource-poor settings outbreak news: avian influenza responding to the global human resources crisis public-private partnerships to build human capacity in low income countries: findings from the pfizer program the united states president's emergency plan for aids relief: bd and pepfar collaborate to strengthen laboratory systems in fight against hiv/aids and tb corporate volunteerism as a pilot approach to strengthening laboratory infrastructures in mbeya region abstract mopeg4165 international health partnership: a welcome initiative health in africa fund holds first close on $57m united states. president bush signs hr 5501, the tom lantos and henry j. hyde united states global leadership against hiv/ aids, tuberculosis and malaria reauthorization act of training programmes for field epidemiology key: cord-023104-dpftawj3 authors: boin, arjen title: the transboundary crisis: why we are unprepared and the road ahead date: 2018-07-22 journal: nan doi: 10.1111/1468-5973.12241 sha: doc_id: 23104 cord_uid: dpftawj3 modern societies rely on complex technological systems that are deeply intertwined with other complex systems that stretch across geographical, judicial and administrative borders. when threats emanate from this transboundary space, national governments are often surprised and discover that existing crisis management arrangements do not suffice. this article describes the political and administrative challenges that accompany transboundary crises. it argues that arrangements and processes that work reasonably well for “bounded” crises are unlikely to work in the case of transboundary crises. it formulates an agenda for political debate and academic research. the bottom line is that we need to rethink traditional crisis management arrangements in order to prepare for these increasingly common type of threats. much has been done in recent decades to prepare modern society for all sorts of crises and disasters. one might say that many societies have never been as prepared for crises and disasters as they are today. that is a good thing. but here is the bad news: modern societies are woefully underprepared for dealing with a type of threat that is on the rise. this is what i refer to as the transboundary crisis. the transboundary crisis effortlessly exceeds geographical, policy, cultural, public-private and legal boundaries that normally enable public managers to classify, contain and manage a crisis. it escalates rapidly and mutates constantly, creating confusion about causes and possible consequences. it ends up on many administrative tables, but it is not obvious which of those tables is or should be "in the lead." these features make a fast and adequate response difficult, to say the least. the transboundary crisis comes in many guises: • in 2002, a mysterious virus spread from china, via hong kong, to 37 countries causing hundreds of deaths. the sars virus caused a major health crisis in toronto. in all these examples, national governments were confronted with a crisis that had origins in faraway domains. however well prepared they may have been for traditional crises, they soon discovered that their response repertoire was insufficient in the face of the transboundary challenges. when the state has no answer to a crisis, consequences follow. in times of crisis, the public expects representatives of the state to take charge. it is, after all, a core task of the state to protect its citizens against the consequences of threat and calamity. if the state fails in this core task, the legitimacy of public institutions and the especially when the legitimacy of public institutions is already under question. the transboundary crisis is the ultimate nightmare for crisis managers. it marks the moment they discover their traditional crisis arrangements do not suffice in the light of the political-administrative challenges that this crisis brings. it sheds light on a structural governance deficit, which presents politicians with a pressing design, let alone management, challenge. this study offers a roadmap for a discussion about possible solutions. this roadmap hinges on a strategic choice between two options that emerge from our discussion of theory: move backward by decoupling from modern systems or move forward by strengthening transboundary crisis management capacities. transboundary crises may come in different guises, but they share common characteristics that make them difficult to manage: the transboundary crisis reaches across multiple countries and/or multiple policy areas. there is no defined geographical location (a "ground zero") or policy sector around which to organize. that creates diversity in perspective: what in one domain is experienced as a problem of scarcity may become a matter of public safety in another; what in one country is considered a local matter is chefsache in another country. the transboundary crisis brings a critical challenge to any administrative system that is based on boundaries and demarcation. by crossing borders, the transboundary crisis challenges borders. in a democratic state that is based on the principle of political accountability and makes use of the bureaucratic organization form, most organizations are organized around demarcated areas of expertise and authority. the bureaucratic organization is based on boundaries among task fields, responsibilities, divisions, departments and policy sectors. two mechanisms have been traditionally used to address blurring of borders: coordination (negotiating boundaries) and centralization (transcending boundaries). these mechanisms can be problematic in the best of times; they are especially problematic in a transboundary crisis. coordination mechanisms may work fine for complex problems and the traditional crisis. the mechanisms, however, do not work in the world of the transboundary crisis for two reasons. first, in a transboundary crisis, it is not clear who the critical actors are or should be, and what their authority in the matter is. second, it is hard to establish or negotiate ownership in a short time frame (time is always scarce in a crisis). we may thus say that the transboundary crisis robs bureaucracy of its most effective tool. centralizing emergency powers in the hands of a leader or a central body is the traditional catch-all solution. in the roman empire, unlimited powers were placed in the hands of a dictator. in modern democracy, crisis centralization is still a valued mechanism. but it comes with constraints: it is not easy to centralize power, and it does not happen often. moreover, the "high command" is not always defined clearly enough, and the mechanisms that should regulate such a concentration of power are cumbersome. a quick look at the legal terms that condition the authorization of exceptional violence (the deployment of special police units) makes clear that a tension exists between legal considerations and the required speed of action. this tension can often only be circumvented in an environment in which political actors know and trust each other. in a transboundary crisis, the nation state may not be the only actor. but how to centralize power in an international context? governments are reluctant to shift decision-making authority to international institutions. think, for example, of eu agencies, which in principle could play a decisive role during a transboundary crisis. but these organizations were never endowed with decision-making powers. we should, then, not be surprised that an eu agency such boin | 95 as frontex accomplished so little during the immigration crisis that peaked in 2015. in sum, transboundary crises pose a wide and deep challenge to the standing governance arrangements of democratic states. that is problematic. the state is left rudderless in a time when citizens look to their elected leaders and trusted institutions to navigate them through the storm. a transboundary crisis can thus rapidly become a crisis of legitimacy. a vicious cycle threatens. the effectiveness of the crisis response relies to a large extent on legitimacy. but the legitimacy of public institutions is already under attack. if institutions do not function effectively during a crisis, they lose even more legitimacy. the transboundary crisis makes vulnerable institutions even more vulnerable. what can be done to protect our country, our prosperity and our well-being in a world of new, unprecedented crises that effortlessly bypass existing lines of defence? to answer this question, we must understand the underlying drivers of the transboundary crisis. two books, both classics, provide a great starting point: barry turner's (1978) man-made disasters and charles perrow's (1984) normal accidents. both authors focus on the relentless modernization of socio-technical systems. we build increasingly complex systems that we connect to other complex systems with one goal: to enhance the efficiency of critical processes (such as food supply, transport, production chains, internet and energy), that is, to increase the speed of service delivery at ever-lower costs. the thesis that emerges from both books can be summarized as follows: these "highways of efficiency" become the "highways of failure" that allow, if not actually enable, routine disruptions to travel very quickly from one system to another. perrow's argument is simple and convincing. if a system becomes evermore complex, it also becomes evermore difficult to understand. when something goes wrong, it is not immediately clear for system operators what is happening. if a complex system is tightly connected with other systems, we know that even a small disturbance can affect the functioning of these other systems. by the time the problem is recognized in one system, others may have been infected. complexity and tight coupling create fertile ground for small incidents to jump from one domain to another and thus escalate into larger-scale crises. new threats exploit these "highways of failure." the rise of revolutionary technologies-artificial intelligence, dna editing, drones, 3d-printing, self-propelled cars, the "internet of things"-brings happiness and economic prosperity, but they also speed up failure and create unknown shortcuts for unprecedented threats. from this theoretical perspective, two options emerge. a country can move backward by decoupling from the modern complexity ecology or move forward by a strategy of protecting complexity. perrow opted for the way back: simplify and unbundle critical systems. if complexity and tight coupling are the problem, perrow's solution-decoupling-is the obvious solution: return to simpler systems that are isolated from other systems. by decoupling from modernity, many crises simply cannot happen. the choice for decoupling does not come free, however. withdrawal entails a decoupling from the benefits that complex systems generate. it is a costly affair, as economists are fond to explain. but this rational argumentation collides with the growing unease about the negative effects of modern systems. in times of uncertainty, the call for entrenchment behind hard borders may not be evidence based, but it is intuitively convincing and politically attractive. we should therefore expect that "decoupling" remains an option as long as modern systems generate uncontrollable hazards. an alternative (or complimentary) strategy is prepared in the face of complexity. if we accept that disturbances can and will emerge, perhaps we are better off investing in early detection and timely intervention. aaron wildavsky (1988) argued that enhanced resilience through a trial-and-error strategy, relying on the human capacity for innovation and learning, is a much more fruitful strategy than a short-sighted focus on prevention through entrenchment. resilience became a dominant feature of many policy efforts in the domain of safety and security. governments that are unable or unwilling to protect their citizens against systemic risks appeal to the ability of citizens (also organizations and communities) to "absorb shocks" and "bounce back" after a disruptive event. the underlying idea is that citizens know what is good for them. in this line of thinking, it is not necessary that government arranges full protection for all citizens from all those complex systems surrounding them. begins with understanding the many ways in which incidents can develop into cross-border threats. how are systems connected? how can a system be infected by a disruption in another system? how can one system be protected against another without undermining the fruits of the existing connection? we need to understand the vulnerabilities of complex and tightly coupled systems. vulnerability thinking is still in its infancy. intelligence services seek to recognize potential threats in a timely manner. hedge funds bet on "big data" to foresee political upheavals. the eu has more than 100 detection and early warning mechanisms in place (but nobody knows if they are effective). research funded by the eu (horizon 2020) focuses on understanding escalation mechanisms in critical infrastructures. complexity researchers study "tipping points." other researchers focus on the ability of people in control rooms to detect early aberrations in critical processes. the need for detection mechanisms is clearly and widely acknowledged, but we await major breakthroughs. the transboundary crisis will continue to surprise us for the time being. the transboundary crisis is difficult to comprehend. the causes are hidden in system complexity and pile up when the dominoes start falling. to understand how a threat unfolds, where exactly and how quickly, it is necessary to bring together as much relevant information as possible, that is, to authorize, analyse and share it with the right parties-quickly and effectively. where critical information about the broken levees in new orleans disappeared. it took the federal authorities more than 24 hours to understand that the city was under water. the solution does not lie in the development of new technologies. what is needed is an approach that helps information managers to quickly collect information from a variety of organizational domains. they must learn to locate sources of critical information; they must also learn to make sense of that information, which is likely to be difficult as the information emanates from very different sources. it is a truism that critical decisions must often be made quickly on the basis of very little information when in a crisis. from a legitimacy perspective (see below), it is important that those critical decisions are made by the appropriate officials or institutions. the transboundary crisis makes this quite tricky, as it challenges the underlying logic of bureaucracy (where responsibility is tied to a person, position or institution). when a crisis involves multiple actors, each with their own responsibilities, interests and working methods, it must be clear who is authorized to decide what. this rarely has been determined beforehand, as a transboundary crisis tends to involve a unique constellation of actors. as we have seen, the typical solution to this type of problem involves a combination of centralization and coordination. centralization is hard enough for the more routine crises; it is even less likely to work in the international arena. national governments do not lightly cede authority to international organizations (certainly not during a crisis where so much is at stake). the international arena has produced some innovative solutions: think of nato's article five boin | 97 (centralization in case of defined threats) and the european central bank (centralization by stealth). the most important problem is that crisis centralization tends to create serious legitimacy issues. if the ownership on critical issues is allocated to an official or institution, and thus not allocated somewhere else or taken away from someone, it is crucial that the "crisis owner" can rely on political and public support. if this is not the case, the legitimacy of public institutions may come under pressure. without a solid legitimacy basis, effective crisis management becomes difficult. centralization that has not been subjected to processes of democratic deliberation and control is risky in this regard. the european central bank became increasingly powerful during the financial crisis and is hardly subject to democratic control. the impotence of the european parliament is telling. the european central bank has made decisions that large groups of citizens perceive as unjust. the lack of democratic control opens the door for politicization of the crisis response. coordination, on the other hand, may work. but that requires a degree of "instant trust" that is often lacking between actors that have never worked together before. promising initiatives can be found at the interface of public and private. for instance, the netherlands has the ict response board in which private parties work with government to prepare for cyber disasters. the louisiana business emergency operations center is another innovative example. the question is whether such practices can be scaled to the international level. theoretical progress is being made. by formulating transboundary crisis management as a collective action problem, we can apply theoretical insights from this body of research. this type of research may provide a view of the conditions under which crisis cooperation is possible, and the strategies that may be helpful. one such strategy, for example, is based on that idea of "instant trust"-unilaterally taking the first step, without being sure of reciprocity (majchrzak, jarvenpaa, & hollingshead, 2007) . but, overall, we can state that mechanisms for the delineation of authority are lacking for transboundary crises. in the absence of serious political discussion, real progress will likely only happen after a disastrous encounter with a transboundary crisis. the transboundary crisis is, of course, not a new phenomenon. crises have always traversed boundaries. think of the plague sweeping across europe, the 1918 flu epidemic, food shortages in the roman empire or the two world wars. the optimist may claim that the western world has managed the really big threats in the postwar era. the pessimist may argue that the connections among international systems have spectacularly increased, problems have become more complex, and the number of actors has increased dramatically. even if the pessimist is only half right, we may justifiably ask whether we are sufficiently prepared. the good news is that crisis management has professionalized in the past few decades. since the beginning of this century (after the millennium computer bug and 9/11), western countries have heavily invested in crisis management capacity, both in the public and private sectors. municipalities, schools, hospitals, businesses-crisis management is now firmly on the radar just about everywhere. more, even though public bureaucracies are not designed to deal with exceptional situations, "work arounds" have been created to deal with routine crises. i have argued, however, that these existing crisis management structures are no match for the transboundary crisis. when the system under threat becomes spread too far, and workarounds do not we leave it to the national crisis institutions. we can instruct these institutions to pay more attention to the transboundary dimensions of crises. it will not be easy to introduce a new way of working in institutions that are built around traditional practices that work for traditional incidents and crises. these organizations do not have to unlearn standing practices; they must learn to deal with the transboundary crisis. we must, in other words, make those institutions "ambidextrous." this will require a lot of work, because national crisis institutions have little affinity with transnational and cross-border crisis management. perhaps it is better to invest in organizations that are already active in cross-border domains. nato and the european union have in recent years developed crisis management capabilities which, in principle, are intended for cross-border crises. the eu, in particular, has developed in embryonic form abilities needed for a transboundary crisis. but these capabilities and assets are scattered across the many agencies and commission parts of the eu. and the crisis performance of the eu is not always considered effective or legitimate. in particular, the response to the financial crisis and the immigration crisis has become sources of controversy. the eu will need time to become an effective and legitimate actor in the crisis domain. 1 3. build transboundary crisis management institutions. think in terms of a new organization, with new people, a respected leadership, real powers and a realistic budget. an institute that invents and tests new forms of crisis management practices. new processes and forms of organization that can effectively address the transboundary crisis. the advantage is that a brand new institution can devise novel methods or ways of thinking from scratch. that is, at the same time, the downside: it will take a long time and the outcome is by no means guaranteed. we conclude by setting out a research challenge. it is important to study the different guises in which the transboundary crisis comes, which allow for classification. it may well turn out that some types of transboundary crises are more amenable to certain interventions (such as centralization and coordination, decoupling or resilience) than others. it may well be that certain types are more politically or analytically challenging. the research challenge is to find out how characteristics relate to preparatory and management efforts. we may discover that certain types are intractable, whereas others may lend themselves to early detection and intervention. by studying cases of actual crises and near misses, we should be able to enhance our understanding of the transboundary crisis. acknowledgment i wish to thank emery roe for his extensive and constructive comments, which greatly improved this article. this study has benefitted from funding provided by the european union's horizon 2020 research and innovation programme under grant agreement no 649484 (transcrisis). the politics of crisis management managing hurricane katrina: lessons from a megadisaster coordinating expertise among emergent groups responding to disaster man-made disasters searching for safety how to cite this article: boin a. the transboundary crisis: why we are unprepared and the road ahead key: cord-302879-ao0n3x9b authors: vanhee, t.; lachiri, f.; van den steen, e.; bottenberg, p.; vanden abbeele, a. title: child behaviour during dental care under nitrous oxide sedation: a cohort study using two different gas distribution systems date: 2020-10-16 journal: eur arch paediatr dent doi: 10.1007/s40368-020-00569-z sha: doc_id: 302879 cord_uid: ao0n3x9b purpose: conscious sedation by inhalation of a mixture of nitrous oxide and oxygen (cs) is a technique used in dental care for anxious, handicapped or uncooperative patients. the very special objective of this cohort study is to compare the behaviour of young patients during dental care under cs in two hospitals using different gas distribution systems. methods: young patients were divided into four categories: young child (yc), phobic anxiety (pa), mental disorder (md), occasional indication (oi). differences in behaviour scale at various time points according to the sedation system used were established and compared using mann–whitney tests. results: this study showed that there is no difference in behaviour during dental care in yc after sedation. in pa, a significant difference in behaviour is only observed during local anaesthesia (p = 0.024). conclusion: no significant differences detected in children’s behaviour under conscious sedation using different gas administration systems. the delicate stage of local anaesthesia in pa patients can be facilitated with repeated sessions of dental care under conscious sedation. electronic supplementary material: the online version of this article (10.1007/s40368-020-00569-z) contains supplementary material, which is available to authorised users. conscious sedation by inhalation of a mixture of nitrous oxide and oxygen (cs) is an effective means for the management of uncooperative children requiring dental care in paediatric dentistry services in hospitals (bryan 2002) . the majority of cs dental care is successful in anxious children (> 90% of cases) (foley 2005; hennequin et al. 2012 ). this medical tool increases the cooperation of young patients by reducing anxiety during dental care (burnweit et al. 2004; galeotti et al. 2016 ). inhaled after a period of induction, the nitrous oxide mixed with pure oxygen, allows to decrease the painful perception during dental care (hammond and full 1984) . two gas distribution systems are used for cs in paediatric dental care: the mixing system with a bottle of n 2 o and a bottle of o 2 and the system using a fixed equimolar mixture of n 2 o and o 2 (emono) readily available in a single tank from the supplier. the mixing system is widely used for dental care around the world except in some countries like france while the emono system, widely used in general medical practice is more widespread in the french paediatric dental practice. both systems have advantages: the mixer system allows to manage the concentration of n 2 o (capped at 50%) and maintains pure o 2 available to speed up recovery. drawbacks are a higher level of sophistication and costs as it depends on an electronic system for mixing. furthermore, pure n 2 o is present at the operatory. in the emono system, the mixing is already done, the dentist has no risk of n 2 o overdose except in case of gas demixing, it can be operated independent of a power supply and at lower cost. whatever the system used, this does not affect the oxygen saturation of haemoglobin (primosch e al. 1999 ). the effect of nitrous oxide administration can be measured by the success or failure rate of the treatment or using behavioural observation with instruments like of venham scale (collado et al. 2006) . indeed, one of the first interests of cs in the context of paediatric dental care is to improve the cooperation of patients to be able to take care of them serenely and safely. behaviour is therefore an essential element in the estimation of the success of care under cs. despite extensive literature on dental care under cs in paediatric dentistry, no study has compared the behaviour of young patients during cs dental care based on the gas distribution system used. often we just find studies on one of the two systems but it has never been possible to compare the two on the same city. indeed, a system is more often developed in one region than in another. for example, in france, the premixed system with a naso-oral mask is the most used, while in the netherlands, it is the system with the two separate gases and a nasal mask. it is therefore difficult for the same observer to compare the behaviour of patients over the same period and in the same region. our situation in brussels, through the diversity of our linguistic communities, meant that, in the french-speaking university hospitals, the practitioners were trained in france and learned to use the emono system while in the dutch-speaking university hospitals, the practitioners were trained in the netherlands and have expertise in using the two-cylinder system. this study is therefore exceptional. the very special objective of this study is to compare the behaviour of children in care for dental care under cs according to the gas distribution system used. the observations were made in two cohorts of patients from paediatric dentistry department of two hospitals in the brussels (belgium) region, saint-pierre university hospital cesar de paepe site (cdp) and tandheelkundige kliniek (vub). inclusion criteria: every patient aged 2 to 18 admitted for dental consultation under conscious sedation between 10/11/2017 and 23/02/2018. the exclusion criteria correspond to the usual precautions required for dental care under conscious sedation. this study was reviewed and ethical permission was granted by the local hospital ethics committee of the saint-pierre university hospital in brussels (approval number o.m. 007) under the reference b076201734522. used at cdp, a fixed gas administration system (fix) is used (antafil ® , sol spa, monza, italy) consisting of a cylinder containing mixture of 50% o 2 and 50% n 2 o at a pressure of 185 bar at 15 °c. the flow rate is adjusted using a mechanical valve system to an average of 6 l/min. the gas is administered to the patient by a latex-free naso-oral mask (quadralite™, intersurgical, france) with a one-used antibacterial filter (clear-guard™, intersurgical, france) connected to the administration circuit (mapleson d deluxe bain breathing system, intersurgical, france) (picture 1 in esm). after at least 3 min of induction, the mask is placed on the nose, allowing access to the oral cavity for dental care. the second sedation system, used at vub, is parker's matrx mdm ® system (mix). this system is used with two gce medical bottles, one containing 100% o 2 at a pressure of 200 bar at 15 °c and the other containing 100% n 2 o at a pressure of 100 bar at 15 °c. each cylinder of gas is connected to the flowmeter. the gas is administered via a corrugated pipe from the flowmeter to the nasal mask (picture 2 in esm). this system is accompanied by a gauge, located on the flow meter, to adjust the percentage of oxygen and nitrous oxide that is administered to the patient. the flow rate is adjusted according to the respiratory rate of the patient. 6 l/min is recommended by the eapd guidelines. on the fix system, the flow can be adapted and we follow the inflation of the reservoir bag so that it is neither too tight, if not enough. it must be just flexible enough and allow visualisation of the respiratory rate by inflating and slightly deflating the reservoir bag. for the mix system, the maximum flow rate is set by the company but it is adapted at the start of the consultation before the titration by observing the inflation of the reservoir bag. patients were divided into four categories: young child (yc), phobic anxiety (pa), mental deficiency (md), occasional indication (oi) (collado et al. 2006) . yc are patients less than 5 years old, without mental development problems; pa are patients from the age of 5, without mental development problems, showing signs of anxiety or phobia during previous conventional dental care; md are patients from the age of 5, with a mental disability or a cognitive or behavioural disorder; oi are patients who could be treated conventionally but in which the dentist judged that they might benefit from cs for an intervention that could be more invasive such as an extraction after trauma or requested by the orthodontist. for this study, behaviour was assessed using the modified venham behavioural scale (picture 3 in esm) (hennequin et al. 2012 ), a hetero-assessment scale that measures the behaviour of young patients during conscious sedation dental care based on well-defined criteria (moura et al. 2016) . it is a descriptive scale of behaviour that can be used to record the behaviour of patients throughout the session according to a score ranging from 0 to 5 (veerkamp et al. 1993) . a training session was conducted using the visualisation of ten videos of young children with different behaviours during dental care. the behaviour score was noted at five time points: at the first contact with the patient, in the treatment room or in the waiting room (t0); when applying the mask on the face/ nose (t1); at the end of the induction, at least 3 min after the application of the mask (t2); when performing local anaesthesia (t3) and eventually during the intervention (t4). all observations were performed by a single, trained observer who did not participate in the clinical procedures. a form completed during the sessions included the patient's information: age, sex, medical history, session number, patient category, intervention performed and behavioural score. a treatment session was considered a success if sedation and care could be achieved and a failure when sedation or treatment could not be performed. the statistical analysis makes it possible to determine the differences in behavioural venham scores between the two populations during dental care after sedation at t2 and t3. the null hypothesis is that there is no difference between the means of the behavioural scores of the two groups at t2 and t3. behavioural scores are qualitative variables and we used the mann-whitney non-parametric u test for 2 independent samples for the statistical analyse or this variable. concerning the session number variable, the distribution does not follow a normal distribution, the medians of the two groups are compared with the mann-whitney nonparametric u test for 2 independent samples. the statistic analysis is performed using ibm spss v. 25.0 (spss inc., armonk, ny.) one observer attended 100 sessions of dental care under cs with 91 different patients. 44 patients at vub with the mix system (mix) and 47 patients at cdp with the fix system (fix), were treated by 6 operators, 3 at each site. in case a patient received several cs sessions, the last session was selected for the study. the total population observed had more female (55%) than male (45%) patients and the average age observed is 6.77 years. pa patients represent 62% of the population, followed by yc 27%. there were 8 md patients and only 2 oi cases. the success rate of acts performed was 95%, there was no significant difference between the two sites (p = 0.701, chi 2 test). among the cohort, five failures were noted, two in the md group, one in the pa group and two in yc group. these children were not cooperative and had to be referred to general anaesthesia. the median number of sessions was one for the mix and two for the fix cohort. the number of sessions was higher for the yc and pa group undergoing the fix protocol, 2 (p = 0.026) for yc and 3 (p < 0.001) for pa, mann-whitney test. the different independent variables are grouped in table 1 . in this table, we find the description of the cohorts in terms of age, gender, indication as well as the success of sedation and dental procedures performed during sedation. the distribution of venham scores in the yc group, the results are very comparable: at t2 and t3, there is no significant difference in behaviour between the two groups (p = 0.740 at t2 and p = 0.936 at t3) (fig. 1a) . for the pa group, the situation is different: more than half of fix pa had a score of 0 (21 patients) compared to mix pa (11 patients). then, there is a significant difference in behaviour between the two groups at t2 (p = 0.026). for the other scores, the number of pa presenting the scores 1, 2, 3 and 4 is more or less similar for the two groups. indeed, at t3, the mann-whitney non-parametric u test indicates that there is no significant difference in behaviour between the two groups (p = 0.223) (fig. 1b) . this study could demonstrate that no significant differences could be detected in children's behaviour under conscious sedation using different gas administration systems. slight behavioural differences could be observed due to possible operator or cohort effects. the strong points are: comparison between two gas administration systems with an identical measuring instrument used by the same observer. points of possible criticisms are: possible underpowered by the number of subjects, confounding effects such as cohort and operator. this confounding effects are reduced by video training for using the hetero-rating behaviour scale. all patients treated with conscious sedation have an initial consultation at the hospital to present them with the equipment, including the sedation mask. however, for many patients, and especially new patients, sedation is a new experience. faced with this novelty, some patients show opposing reactions that hamper their cooperation despite a preparatory consultation. these patients will then require steps to restore confidence and, if this is not enough and the dental situation deteriorates with pain and infections, it will be necessary to proceed to a step of dental care under general anaesthesia. the analysis of the number of sessions for the two categories of patients showed a significant difference for yc and highly significant for pa. repetition of sedation care allows uncooperative patients to better cope with long-term dental care (collado et al. 2006) . in a subsequent study, patients could be grouped according to the number of sessions already performed. then, pa patients would be observed during the first session under cs and during a second or third session. local anaesthesia is a special step in dental care because it causes an unpleasant sensation when the needle penetrates the mucosa, during the injection even if it is carried out with all the required precautions, including in particular an infiltration slow and controlled, and also at the time of the sensation of numbness of the tissues surrounding the injection area. this sensation can worry the patient and it is therefore essential to reassure him by effective verbal communication throughout the procedure. indeed, local anaesthesia is the most feared moment in dental care, even if the cause of anxious or phobic behaviour of the young child at the dentist is multifactorial locker et al. 2001) . the fear of the dentist is often associated with a negative experience, recalling the pain, during previous care. (risløv staugaard et al. 2017) . after observing children treated with both the two sedation systems, no differences in child behaviour attributable to a difference in gas administration method could be observed. the systems can be judged to be equivalent in clinical efficacy. the practitioner or hospital can then make a choice based on advantages and disadvantages found for each one of them. using the fixed gas administration system (fix), the advantage of the naso-oral mask is to involve the young child. by applying the tell-show-do technique for confidence-building, the patient participates with the practitioner in placing the mask on the mouth and nose. the mask being on the nose and the mouth: bilateral communication is possible between the practitioner and the young patient. the child can speak in the naso-oral mask while with the nasal mask only, if the child speaks, all the gas used for sedation escapes and the gas can no longer have its effect. the practitioner can communicate with the child and thus distract him from his environment (armfield and heaton 2013). the flow rate and the concentration being constant, the practitioner can concentrate easily on the care, taking into account the fact that the inhalation can be stopped at any time if necessary. using the gas mixing device (mix), the advantage of the nasal mask fixed and adapted to the patient is to reduce the gas leak. this gas leak can cause an adverse effect in healthcare professionals, frequently encountered during inhalation of n 2 o, including headache (field et al. 1993) . the gauge on the flow meter allows to adapt the flow rate according to the child's breathing throughout the treatment. a button also makes it possible to adapt the n 2 o concentration according to the observed heart rate. the practitioner can thus give the young child a concentration of less than 50% n 2 o. this system has a bottle of o 2 , recovery is by inhalation of pure o 2 for 5 min. using the fixed gas composition recovery is in ambient air or when needed a separate oxygen tank is available. in the literature, both systems have been analysed for air pollution. whether it is the premixed system or the two-cylinder system, air pollution remains below the recommended standards at european level (hennequin and onody 2004; gilchrist et al. 2007) . however, when the child cries, this atmospheric concentration of n 2 o increases. preparing the patient in terms of behaviour is therefore a prerequisite for management under conscious sedation. hennequin and onody (2004) specified in their article that demand valves can help to further reduce this environmental concentration of n2o. while in most parts of the world dentists use a two-bottle and nasal mask system, in france recent publications show that the use of the premixed system is still heavily used (prud'homme, dajean-trutaud, et al. 2019a, b) . each technique has its advantages and disadvantages, but being able to compare two teams, each with expertise in one of these methods, makes our study an exceptional work. this study was carried out before the covid-19 pandemic and therefore did not require advanced reflection about aerosol generating procedures. since march 2020, following the pandemic, great questions have been asked about the contamination of masks and circuits. for the premixed system (fix), the mask is for individual use, or even one-time use, and its cost is less than the nasal mask. in addition, it has a filter system as found in the operating room. for the two-bottle system, there is a choice between disposable or sterilizable masks. as it does not have an antibacterial filter, it is necessary to sterilise the entire circuit. the investment is greater because it is necessary to provide one circuit per patient and to have an adequate sterilisation service, such as central hospital sterilisation. the aapd advises the systematic use of the pulse oximeter for conscious sedation but this advice comes during 2018 and our data collection carried out in 2017-2018 (aapd 2018). then, we based ourselves on the eapd guidelines which specify that the use of pulse oximetry, although interesting, is not essential because it would cause additional stress to the child (hallonsten et al. 2005) . during our study, we wanted to observe the behaviour of children in a usual framework of care outside the study. while pulse oximetry is recorded for each patient at the vub site, this is not the case at cdp where the eapd guidelines were followed. we find this same notion in the belgian national recommendations in all cases, monitoring is performed by a dental assistant (mix) or a paediatric nurse (fix), both trained in the use of the sedation system and in monitoring the patient's respiratory functions (coté and wilson 2019) . in a subsequent study, the use of the heart rate function of the pulse oximeter is of great interest for a further assessment of patient stress (appelhans and luecken 2006) . despite the differences between sites and practitioners in terms of training in conscious sedation (obtained in dutch in the netherlands for vub practitioners and in french in france for cdp practitioners), very few significant differences were found in the statistical analysis. further study could be done with the same practitioner using both sedation systems on the same population. this approach would be interesting to compare only the two systems but we would lose the diversity factor present in this study. considering the limitations of the present study the following conclusions can be made: • no significant differences detected in children's behaviour under conscious sedation using different gas administration systems. • the delicate stage of local anaesthesia facilitated with repeated sessions of dental care under cs. guideline on use of nitrous oxide for pediatric dental patients heart rate variability as an index of regulated emotional responding management of fear and anxiety in the dental clinic: a review avis 9299-protoxyde d'azote. spf santé publique the success of inhalation sedation for comprehensive dental care within the community dental service nitrous oxide analgesia for minor pediatric surgical procedures: an effective alternative to conscious sedation? modification of behavior with 50% nitrous oxide/oxygen conscious sedation over repeated visits for dental treatment a 3-year prospective study guidelines for monitoring and management of pediatric patients before, during, and after sedation for diagnostic and therapeutic procedures effect of nitrous oxide on cerebral blood flow in normal humans a prospective study of the use of nitrous oxide inhalation sedation for dental treatment in anxious children inhalation conscious sedation with nitrous oxide and oxygen as alternative to general anesthesia in precooperative, fearful, and disabled pediatric dental patients: a large survey on 688 working sessions exposure to nitrous oxide in a paediatric dental unit eapd guidelines on sedation in paediatric dentistry nitrous oxide analgesia and children's perception of pain pollution level during inhalation sedation with a 50%n2o/50%o2 premix: comparison of two administration devices. ann francaises d'anesthesie et de reanim a clinical trial of efficacy and safety of inhalation sedation with a 50% nitrous oxide/oxygen premix (kalinox tm ) in general practice psychological disorder, conditioning experiences, and the onset of dental anxiety in early adulthood how do observational scales correlate the ratings of children's behavior during pediatric procedural sedation? determinants of early-vs late-onset dental fear in a longitudinal-epidemiological study effect of nitrous oxide-oxygen inhalation with scavenging on behavioral and physiological parameters during routine pediatric dental treatment assessment of an equimolar mixture of oxygen and nitrous oxide: effects in pediatric dentistry the meopaedent trial protocol-an observational study of the equimolar mixture of oxygen and nitrous oxide (emono) effects in paediatric dentistry the role of negative and positive memories in fear of dental treatment dental treatment of fearful children using nitrous oxide part 3: anxiety during sequential visits publisher's note springer nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations all authors declare having no financial interest or conflict of interest. key: cord-027118-2xm8nkmi authors: sevastianov, leonid a.; lovetskiy, konstantin p.; kulyabov, dmitry s. title: an effective stable numerical method for integrating highly oscillating functions with a linear phase date: 2020-06-15 journal: computational science iccs 2020 doi: 10.1007/978-3-030-50417-5_3 sha: doc_id: 27118 cord_uid: 2xm8nkmi a practical and simple stable method for calculating fourier integrals is proposed, effective both at low and at high frequencies. an approach based on the fruitful idea of levin, which allows the use of the collocation method to approximate the slowly oscillating part of the antiderivative of the desired integral, allows reducing the calculation of the integral of a rapidly oscillating function (with a linear phase) to solving a system of linear algebraic equations with a triangular or hermitian matrix. the choice of gauss-lobatto grid nodes as collocation points let to increasing the efficiency of the numerical algorithm for solving the problem. to avoid possible numerical instability of the algorithm, we proceed to the solution of a normal system of linear algebraic equations. the initial formulation of the method of numerical integration of highly oscillating functions by levin and his followers suggests a possible ambiguity in finding the antiderivative: any solution to the differential equation without boundary (initial) conditions can be used to calculate the desired value of the integral. levin's approach [1] to the integration of highly oscillating functions consists in the transition to the calculation of the antiderivative function from the integrand using the collocation procedure in physical space. in this case, the elements of the degenerate [2] differentiation matrix of the collocation method [3] are a function of the coordinates of the grid points, the matrix elements are calculated using very simple formulas. in books [3, 4] various options for the implementation of this method are considered, many applied problems are solved. the method proposed by levin both in the one-dimensional and in the multidimensional case was published by him in articles [1, 9] , and then he was thoroughly studied in [10] . the method is presented in great detail in the famous monograph [4] , which describes the evolution of numerical methods for integrating rapidly oscillating functions over the past fifteen years. there are a large number of works using various approaches in order to propose fast and stable methods for solving systems of linear algebraic equations (slae) that arise when implementing the collocation method. however, many of them [5] [6] [7] encounter difficulties in solving the corresponding systems of linear equations. in particular, the use in specific implementations of the levin collocation method in the physical space of degenerate chebyshev differentiation matrices, which also have eigenvalues differing by orders of magnitude, makes it impossible to construct a stable numerical algorithm for solving the resulting slaes. the approach to solving the differential equation of the levin method, described in [5, 6, 8] , is based on the approximation of the solution, as well as the integrand phase and amplitude functions in the form of expansion into finite series in chebyshev polynomials. moreover, to improve the properties of the algorithms, and hence the matrices of the corresponding slaes, three-term recurrence relations are used that connect the values of chebyshev polynomials of close orders. however, these improvements are not enough to ensure stable calculation of integrals with large matrix dimensions. in our work, we consider a method of constructing a primitive, based on the spectral representation of the desired function. we propose increasing the efficiency of the algorithm by reducing the corresponding system of linear equations to a form that is always successfully solved using the lu-decomposition method with partial selection of the leading element. consider the integral that often occurs in fourier analysis -in applications related to signal processing, digital images, cryptography and many other areas of science and technology. in accordance with the levin method, the calculation of this integral reduces to solving an ordinary differential equation as argued in [1] , the system (2) has a particular solution which is not rapidly oscillatory, and we shall look for an approximation to this particular solution by collocation with 'nice' functions, e.g. polynomials. if the unknown function p(x) is a solution of eq. (2), then the result of integration can be obtained according to the formula below we will consider the special case of integration of a highly oscillating function with a linear phase, reduced to the standard form this can be justified, in particular, by the fact that in many well-known publications [7, 11, 12] stable transformations are discussed in detail, which make it possible to proceed from a general integral with a nonlinear phase to an integral in standard form (on the interval [−1, 1]) with a linear phase. in the paper by levin [1] , to automatically exclude the rapidly oscillating component ce àixx of the general solution p x ð þ ¼ p 0 x ð þ þ ce àixg x ð þ , it is proposed to search for a numerical solution (2) based on the collocation method, using its expansion in a basis of slowly oscillating functions, rather than using difference schemes (or methods of the runge-kutta type). in this case, the following statement is true [2] : statement. the solution of eq. (2) obtained using the levin collocation method is a slowly oscillating function o x à1 ð þ for x ) 1. let us consider in more detail the problem of finding the antiderivative integrand, or rather, the approximating polynomial p x ð þ, satisfying condition (2) in a given number of points on the interval [−1, 1]. consider the spectral method of finding an approximating function in the form of expansion in a finite series in the basis of chebyshev polynomials of the first kind t k x ð þ f g 1 k¼0 , defined in the hilbert space of functions on the interval [−1, 1]. the application of the collocation method to solve the problem p 0 x ð þ þ ixp x ð þ ¼ f x ð þ leads to the need to fulfill the following equalities for the desired coefficients at the collocation points x 0 ; x 1 ; . . .; x n f g . the last statement is equivalent to the fact that the coefficients c k ; k ¼ 0; . . .; n should be a solution to the system of linear algebraic equations of the collocation method: we represent the values of the derivative of the desired function (polynomial) at the collocation points in the form of the product dp ¼ p 0 of the matrix d by the vector of values of p. recall that the chebyshev differentiation matrix d has the standard representation in the physical space [3] (7) we reduce it to a system of linear algebraic equations here e is an identity matrix, f is a vector of values of the amplitude function on the grid. denote by b the differentiation matrix in the frequency (spectral) space [13] , whose coefficients are explicitly expressed as where 0 i; j n and r i ¼ & denote by t the chebyshev matrix of mapping a point (vector) from the space of coefficients to the space of values of the function [14] . given that p ¼ tc is the vector of values of the desired function (also in physical space), the components of the derivative vector can be written as dp ¼ tbc [14] . as a result, we obtain the system of linear algebraic equations equivalent to system (9), which is valid for an arbitrary grid on the interval [−1, 1]. we write eq. (11) in detail where to reduce the formulas we used the notation t kj ¼ t k x j à á ; k; j ¼ 0; . . .; n. the product of a non-degenerate matrix t by a non-degenerate triangular matrix b þ ixe is a non-degenerate matrix. therefore, the system of linear algebraic eqs. (12) has a unique solution. statement 1. the solution of this system of linear algebraic equations with respect to the coefficients c ¼ c 0 ; c 1 ; . . .; c n ð þallows us to approximate the antiderivative function in the form of a series (5) and calculate the approximate value of the integral by formula (4). system (12) is valid for an arbitrary grid on the interval [−1, 1]. however, consideration of the collocation problem on a gauss-lobatto grid allows significant simplification of this system of linear algebraic equations. first, we multiply the first and last equations from (12) by 1= ffiffi ffi 2 p to obtain an equivalent "modified" system with a new matrixt (instead of t), which is good because it has the property of discrete "orthogonality" and, therefore, is non-degenerate. therefore, multiplying it on the left by its transposed one gives the diagonal matrix: we use this property and multiply the reduced (modified) system (12) on the left by the transposed matrixt t , thereby reducing it to the upper triangular form. indeed, the matrix of the resulting system is calculated as the product of the diagonal matrix by the triangular matrix, which, in turn, is the sum of the chebyshev differentiation matrix in the spectral space and the diagonal matrix. since the matrixt t is non-degenerate, the new system of linear algebraic equations is equivalent to system (12) and has a unique solution. taking into account the specific values of the chebyshev polynomials on the gauss-lobatto grid [15] , simplifies the system, bringing it to the form ð13þ . . .; n and symbol p 00 denotes a sum in which the first and last terms are additionally multiplied by 1/2. by the kronecker-capelli theorem, the system of linear algebraic eqs. (13) with a square matrix and a non-zero determinant is not only solvable for any vector of the right-hand side, but also has a unique solution. (13) has a stable solution. statement 3. to solve system (13) , no more than ( $ n 2 =4) operations of addition/subtraction and multiplication/division with a floating point are required. algorithms for solving systems of linear equations such as the gauss method or the lu-decomposition work well when the matrix of the system has the property of diagonal dominance. otherwise, standard solution methods lead to the accumulation of rounding errors. a stable solution to the system may be provided by the lu-decomposition method with a partial choice of a leading element. for the triangular matrix (13) , there is no forward pass (in which the leading element is selected) of the lu-decomposition, and the back pass of the method is always implemented without the selection of leading element. a solution to system (13) can still be unstable in the case when ix j j 2n. passing to the solution of the normal system, that is, to the problem of minimizing the residual ac àf 2 , multiplying the system of eqs. (13) on the left by the hermitian conjugate matrix we transform the matrix of the system (13) to the hermitian form. although the system of linear equations became more filled, since instead of upper triangle three-diagonal matrix a system of linear equations with all matrix elements filled appeared, its computational properties are cardinally improved. the resulting matrix of a system of linear algebraic equations is hermitian, its eigenvalues are real, and the eigenvectors form an orthonormal system. the method of lu-decomposition with a partial choice of the leading element, due to the properties of the resulting matrix, provides [17] the stability of the numerical algorithm for finding the only solution to the system. let us describe the sequence of operations of the presented algorithm for calculating the integral of a rapidly oscillating function of the form (1) with a linear phase. input data preprocessing. 1. if the integral is given on the interval a; b ½ , we pass to the standard domain of integration à1; 1 ½ by changing the variables x ¼ bàa fill by columns the chebyshev transformation matrix t from (12) using only one pass of the recursive method for calculating the values of chebyshev polynomials of the first kind of the n-th order. 3. calculate the vector of the right-hand side of system (13). 4. fill in the elements of the sparse matrix (13) , which depend only on the dimension n and the phase value x. 5. if x j j[ 2n, then go to step 6. otherwise go to step 7. 6. the matrix of system (13) is a matrix with a diagonal dominance and can be stably solved. the solution values at the boundary points are used to determine the desired antiderivative values. go to step 8. 7. multiply relation (13) on the left by the conjugate matrix to obtain a hermitian matrix (14) with diagonal dominance. in this case, to determine the values of the antiderivative at the boundary points the normal solution is stably determined using the lu-decomposition with a partial choice of the leading element. 8. we calculate the values of the antiderivative at the ends of the interval using the formulas p 1 ð þ ¼ p n j¼0 c j ; and p à1 ð þ ¼ p n j¼0;jàeven c j à p n j¼0;jàodd c j . the desired value of the integral is obtained using the formula i f ; x ð þ ¼ p 1 ð þe ix à p à1 ð þe àix . 6 numerical examples example 1. we give an example of calculating the integral when, for a good polynomial approximation of a slowly oscillating factor of the integrand, it is necessary to use polynomials of high degrees. this integral is given by olver ( [16] , p. 6) as an example of the fact that the gmres method allows one to calculate the integral much more accurately than the levin collocation method. however, in his article, solving the resulting system of linear algebraic equations requires o n 3 ð þ operations, as in the levin collocation method using the gaussian elimination algorithm (table 1) . a comparison of our results at 40 interpolation points with the results of [16] shows a significant gain in accuracy: the deviation from the exact solution is of the order of 10 à17 compared with the deviation of the order of 10 −7 in olver's article. the proposed algorithm to achieve an accuracy of 10 −13 in the calculation of the integral uses no more than 30 points (n 30þ for x = 1,…, 100. example 2. as a second example, we consider the integral from [16] , where the results of calculating the integrals depending on the number of approximation points are illustrated on fig. 1 [16] . à á and the integral can be written as: let us consider the calculation of this integral for various values of the parameter x using an algorithm that takes into account the linearity of the phase function ( table 2 ). the proposed algorithm to achieve an accuracy of 10 à16 when calculating the integral uses no more than 90 points (n 90) with x = 0.1, …, 100. a significant gain in the number of addition/subtraction and multiplication/division operations is achieved when the frequency value is greater than the number n, which ensures the diagonal dominance of the system of linear algebraic equations in the matrix (13) (fig. 2) . it is useful to compare the algorithm we developed for finding the integrals of rapidly oscillating functions with the results of [5] , which presents various and carefully selected numerical examples for various classes of amplitude functions. example 3. consider the calculation of the integral with an exponential function as the amplitude ð þ e ixx dx; a ¼ 16; 64; x ¼ 20; 1000: the exact value of the integral can be calculated by the formula i a; x ð þ ¼ 2ãe àa sinh a þ ix ð þ a þ ix ð þ [5] . the plot of the deviation of the integral calculated by us from the exact one depending on the number of collocation points (absolute error) is shown in fig. 3 . comparison with the results of [5] shows that the accuracy of calculating the integrals practically coincides with that of [5] . our advantage is the much simpler form of the matrix of a system of linear equations. in the best case, when |x| > n matrix of the system is a triangular matrix with a dominant main diagonal. if n > |x|, then the transition to the search for a normal solution to a system with a positively defined hermitian matrix allows us to create a numerically stable solution scheme. example 4. in this example [5] , the rapidly oscillating function e i2pax is considered as the amplitude one. it is clear that in this case, to achieve the same accuracy in calculating the integral as in the previous example, a larger number of collocation points will be required (fig. 4) . e i2pax e ixx dx; a ¼ 5; 10; x ¼ 20; 1000: ð19þ example 5. the example demonstrates the calculation of the integral in the case when the amplitude function is the generating function of the chebyshev polynomials of the first kind. 1 à a 2 1 à 2ax þ a 2 e ixx dx; a ¼ 0:8; 0:9; x ¼ 20; 1000: the behaviour of the amplitude function should lead to an almost linear dependence of the approximation accuracy on the number of points for various values of the parameters a and x. numerical experiments carried out confirm this assertion (fig. 5 ). moreover, the accuracy of calculating the integrals is not inferior to the accuracy of the methods of [5] . the example is rather complicated for interpolation by chebyshev polynomials. to achieve acceptable accuracy (10 à18 ), the deviation of the calculated value of the integral from the exact one requires about 300 approximation points both for small values of x ¼ 20 and for large x ¼ 1000 (fig. 6 ). example 7. we give an example of integration when the amplitude function has second-order singularities at both ends of the integration interval the value of this integral can be calculated in an analytical form: f x ð þ ¼ 3pj 2 x ð þ=x 2 . we present the numerical values of the integral for various values of the frequency: i 20 ð þ ¼ à0:00377795409950960, i 1000 ð þ¼à2:33519886790130 â 10 à7 (fig. 7) . similar to the previous example, to achieve good accuracy in calculating the integral, it is necessary to consider a large number of collocation points. however, for this type of amplitude functions, the method presented in the article works reliably both in the case of low and high frequencies. the given examples demonstrate that the dependence of the solution on the number of approximation points is similar to the dependence demonstrated in olver [17] and hasegawa [5] . the advantage of our approach is the simplicity of the algorithm and the high speed of solving the resulting very simple system of linear algebraic equations. if it is necessary to repeatedly integrate various amplitude functions at a constant frequency, multiple gains are possible due to the use of the same lu-decomposition backtracking procedure. a simple, effective, and stable method for calculating the integrals of highly oscillating functions with a linear phase is proposed. it is based on levin's brilliant idea, which allows the use of the collocation method to approximate the antiderivative of the desired integral. using the expansion in slowly oscillating polynomials provides a slowly changing solution of the differential equation. the transition from a solution in physical space to a solution in spectral space makes it possible to effectively use the discrete orthogonality property of the chebyshev mapping matrix on a gauss-lobatto grid. with this transformation, the uniqueness of the solution of the studied system is preserved, and its structure from a computational point of view becomes easier. there are a large number of works using various approaches aimed to offer fast and effective methods for solving slaes that arise when implementing the collocation method. however, many methods [5, 6] encounter instability when solving the corresponding systems of linear equations. when using chebyshev differentiation matrices in physical space, instability is explained primarily by the degeneracy of these matrices and the huge spread of eigenvalues of the matrix of the collocation method system. the approach to solving the differential equation based on the representation of the solution, as well as the phase and amplitude functions, in the form of expansion in finite series by chebyshev polynomials and the use of three-term recurrence relations [5, 6, 8] also does not provide a stable calculation for n > |x|. to overcome instability, various methods of regularizing the systems under study are proposed. in our work, we propose a new method for improving computational properties by preconditioning of the system in the spectral representation and by searching for its pseudo-normal solution. the proposed method has been reduced to solving a slae with a hermitian matrix. a number of numerical examples demonstrates the advantages of the proposed effective stable numerical method for integrating rapidly oscillating functions with a linear phase. procedures for computing one-and two-dimensional integrals of functions with rapid irregular oscillations filon and levin methods in: computing highly oscillatory integrals chebyshev polynomials computing highly oscillatory integrals a user-friendly method for computing indefinite integrals of oscillatory functions stability and error estimates for filon-clenshaw-curtis rules for highly oscillatory integrals an improved levin quadrature method for highly oscillatory integrals a well-conditioned levin method for calculation of highly oscillatory integrals and its application fast integration of rapidly oscillatory functions moment-free numerical integration of highly oscillatory functions a comparison of some methods for the evaluation of highly oscillatory integrals an alternative method for irregular oscillatory integrals over a finite range a practical guide to pseudospectral methods regularized computation of oscillatory integrals with stationary points integration of highly oscillatory functions fast, numerically stable computation of oscillatory integrals with stationary points gmres shifted for oscillatory integrals acknowledgement. the publication has been prepared with the support of the "rudn university program 5-100". key: cord-259958-46e7xb7b authors: marfori, cherie q.; klebanoff, jordan s.; wu, catherine z.; barnes, whitney a.; carter-brooks, charelle m.; amdur, richard l. title: reliability and validity of two surgical prioritization systems for reinstating non-emergent benign gynecologic surgery during the covid-19 pandemic date: 2020-07-30 journal: j minim invasive gynecol doi: 10.1016/j.jmig.2020.07.024 sha: doc_id: 259958 cord_uid: 46e7xb7b study objective: scientifically evaluate the validity and reproducibility of two novel surgical triaging systems, as well as offer modifications to the ments criteria for improved application in gynecologic surgeries design: retrospective cohort study setting: academic university hospital patients: 97 patients with delayed benign gynecologic procedures due to the covid-19 pandemic intervention(s): surgical prioritization was assessed using two novel scoring systems, the gyn-ments and mesas systems for all 93 patients included measurements and main results: the inter-rater reliability and validity of two novel surgical prioritization systems (gyn-ments and mesas) were assessed. gyn-ments scores were calculated by three raters and analyzed as continuous variables, with a lower score indicating more urgency/priority. the mesas score was calculated by two raters and analyzed as a 3-level ordinal variable with a higher score indicating more urgency/priority. all five raters were blinded to reduce bias. gyn-ments inter-rater reliability was tested using spearman r and paired t-tests were used to detect systematic differences between raters. weighted kappa indicated mesas reliability. concurrent validity with mesas and surgeon self-prioritization (ssp) was examined with spearman r and logistic regression. spearman r's for all gyn-ments rater pairs were above 0.80 (0.84 for 1 vs. 2, 0.82 for 1 vs. 3, 0.82 for 2 vs. 3, all p<.0001) indicating strong agreement. the weighted kappa for the 2 mesas raters was 0.57 (95% ci 0.40-0.73) indicating moderate agreement. when used together, both scores were significantly independently associated with ssp, with strong discrimination (auc 0.89). conclusions: inter-rater reliability is acceptable for both scoring systems, and concurrent validity of each is moderate for predicting ssp, but discrimination improves to a high level when they are used together. the respiratory illness caused by the novel coronavirus sars-cov-2 has upended global healthcare delivery. in march 2020, the american college of surgeons (acs), the us surgeon general, and the centers for medicare & medicaid services (cms) all made recommendations to cancel elective surgery throughout the us in order to preserve resources to treat those critically ill and to reduce transmission between doctors and patients. [1] [2] [3] however, the term elective does not mean unnecessary, and postponement of these surgeries may lead to significant morbidity or even mortality. surgeons are faced with the potentially overwhelming and morally exhausting task of prioritizing postponed patients within their departments and hospital systems. in response, the american college of surgeons (acs) developed a tiered ranking system for prioritization of elective surgeries based on the risk of delay and patient co-morbidities, the elective surgery acuity scale (esas). 4 while this system provides a framework, many hospitals and surgeons find implementation difficult given its vagueness. recognizing these limitations, the society for gynecologic surgeons (sgs) issued a joint statement which applied the acs tier to numerous benign gynecologic procedures to serve as a guide of acuity. 5 additionally, a third and novel scoring system by prachand et al serves to individually rank nonemergent surgeries coined medically-necessary, time-sensitive (ments) procedures. 6 this scoring system attempts to objectively prioritize surgeries by grading 21 factors within the broad categories of procedure variables, disease state, and comorbidities. all of these scoring systems are based on expert opinion and consensus, and none have been externally validated nor tested for reliability. at our institution, the division of benign gynecologic surgery incurred a large number of case cancellations with the orders to halt elective surgery. we recognized a need for a reproduceable system beyond a three-tiered scale to help triage and prioritize affected patients. we took great interest in the ments tool as a potential solution. while our institution elected not to adapt the ments criteria system-wide, our division sought to critically evaluate it as a solution to ethically and efficiently prioritize patients beyond the acs criteria. our objective was to scientifically evaluate the validity and reproducibility of both of these triaging systems, as well as offer modifications to the ments criteria for improved application in gynecologic surgeries. after obtaining irb exemption (irb#ncr202525), we performed a single-center retrospective cohort study evaluating the inter-rater reliability and validity of two novel prioritization systems, the sgs adaptation of the esas (mesas) and a modified version of the ments tool (gyn-ments). a total of 97 benign gynecologic procedures were affected between march 16, 2020 and april 30, 2020 in our tertiary academic institution in washington, dc, including cases from eight general obstetrician gynecologists, two minimally-invasive gynecologic surgeons, a urogynecologist, and a gynecologic oncologist. we excluded three patients who had not completed their preoperative evaluations and thus the severity of their disease was unclear. one additional patient was excluded given she suffered from a sub-arachnoid hemorrhage and was no longer eligible for her planned procedure. beginning march 16, as required by our hospital system, all patients were categorized by their respective surgeons into one of three categories based on their level of morbidity if delayed. level 1 indicated no morbidity with delay, level 2 indicated some morbidity with delay, and level 3 indicated significant morbidity and/or mortality with delay. only those deemed level 3 were initially allowed to proceed as scheduled. level 1 and 2 would be postponed until further notice, likely to extend 2-3 months. for the purpose of the validity analysis, those patients who were allowed to proceed with surgery will be referred to as "urgent ssp". the esas scale divides elective surgeries into three tiers, low acuity (tier 1) defined as "non life-threatening illness", intermediate acuity (tier 2) defined as "nonlife-threatening but with potential for future morbidity and mortality", and high acuity (tier 3). additionally, each tier is further divided into subtype a (healthy patients) or subtype b (unhealthy patients) to better discriminate patient risk and hospital resource use. the acs recommended proceeding with tier 3 surgery and postponing tier 1 and 2 surgery (or performing at an ambulatory surgery-center). 4 the joint statement published by sgs on april 28 applied the acs tiered system to numerous benign gynecologic procedures ( figure 1 ). 5 this framework categorizes specific surgical procedures taking into consideration their indications and severity of disease symptoms. two authors (wab and cmcb) assigned all patients into one of the three mesas tiers by reviewing the patient's electronic medical record. these authors were queried about their awareness of the alternative ments scoring system and, after finding them unfamiliar, were instructed to intentionally proceed in this manner to reduce potential bias towards another scoring system. the original ments scoring criteria attempts to objectively prioritize surgeries by grading 21 factors within the broad categories of procedure variables, disease state, and comorbidities. 6 the cumulative score ranges between 21 and 105 and serves as a rank in priority, with lower numbers equating to greater priority (table 1) . higher scores equate to poorer perioperative outcomes, higher hospital resource utilization, increased risk of covid-19 transmission, and an increased ability to safely defer surgery. when attempting to apply the ments model to our gynecologic patients, we felt adaptations could be made to improve clarity, objectivity, and validity. specific adaptations made to the original ments score included ( 3. instead of calculating "non-operative treatment effectiveness percentage" and "exposure risk", we transformed these two variables into five distinct categories pertinent to the gyn surgical patient including: whether and how many alternative therapies have been tried, the presence/severity of pain, the presence/severity of anemia, the impact on desired immediate fertility, and the impact on adjacent genitourinary and gastrointestinal systems. 6. removing the variable "influenza-like illness symptoms." in our opinion, any patient demonstrating these symptoms should continue to be delayed until resolution. 7. limiting the options for "exposure to known covid positive patient in the last 14 days" to improve reproducibility. at our institution, we implemented universal covid testing for all patients undergoing emergent and scheduled surgery. three authors (cqm, jsk, czw) adapted and applied the modified gyn-ments scoring system to all patients. all five authors were blinded to each other's scores to reduce bias. authors could not be blinded from the surgeons' self-prioritization (urgent ssp) given these surgeries were performed as scheduled and could be elicited from chart review. variables. gyn-ments scores were calculated by three reviewers and analyzed as continuous variables with possible scores ranging from 21-105. the lower the score, the more prioritized the surgery would be. the mesas score was calculated by two different reviewers and analyzed as a three-level ordinal variable (1/2/3) with a higher score indicating more urgency/prioritization. finally, the ssp score was made into a binary variable with those considered highest priority (urgent ssp) separated from those considered lower priority. reliability. the three gyn-ments raters' scores were examined using spearman r to determine the level of monotonic association, and paired t-tests to determine whether there were systematic differences between raters. a relevant systematic difference was indicated by a mean difference > 0.1 along with a significant difference on the paired t-test. if all spearman r's were above 0.80, this indicated strong inter-rater reliability. in order to determine which gyn-ments items had the worst reliability, agreement between raters' gyn-ments item scores were examined using percent exact agreement, rather than kappa, because raters used different numbers of categories on several items. concurrent validity. we took the mean of the gyn-ments scores and examined the spearman r of this score with the mean of the mesas scores, and with urgent ssp as measured by actual scheduling (a binary variable, yes/no). concurrent validity for the gyn-ments score was indicated by strong spearman r with both the mesas score and with urgent ssp. we also examined whether urgent ssp could be predicted independently using both the gyn-ments score and the mesas rating in a multivariable logistic regression model. if both were significant independent predictors, we then used the log-linear equation produced by the regression model to calculate each patient's probability of being classified as urgent ssp and examined the association of probability quartile with urgent ssp status using chi-square. we examined the distribution of mesas rating with the gyn-ments scores achieving the highest 67%, 75%, 80%, 85%, 90% and 95% of urgency levels using chi-square. sas (version 9.4, cary, nc) was used for data analysis, with p<.05 considered significant. the mean ± sd gyn-ments score for raters 1, 2, and 3 were 58.0 ± 4.8, 59.2 ± 4.3, and 58.0 ± 5.0, respectively. gyn-ments scores for raters 1 and 3 did not differ p=.002), after adjusting for the mesas score. when the probabilities derived from this model were coded into quartiles, the incidence of being urgent ssp was 75% in the highest priority quartile, 14% and 12% in the middle two quartiles, and 0% in the lowest quartile (p<.0001). the equation for calculating probabilities is in appendix i. the 67th, 75th, 80th, 85th, 90th, and 95th percentiles of gyn-ments score are shown in table 4 . in order to make it easier to visualize these relationships, we coded the gyn-ments into three levels: low, medium and high priority, based on quartiles: the lowest quartile of scores is highest priority, middle two quartiles are medium priority, and the highest quartile of scores is the lowest priority. we then compared these three levels of gyn-ments urgency and the three levels of sgs urgency, with the urgent ssp (table 5 ). comparing gyn-ments to urgent ssp, we found that from the highest priority quartile on gyn-ments to the lowest, 52%, 23%, and 4% of patients were urgent ssp (p=.0009). for patients with mesas levels of high, moderate, and low priority, the percentages found within the urgent ssp category were 88%, 36% and 10%, respectively (p<.0001). when looking only at the 12 of 24 patients who were deemed the "highest priority" by the ssp scheme and their surgeries performed without delay, the mesas system was able to capture 92% of these patients in its most urgent quartile, while the gyn-ments system captured only 67% in its most urgent quartile. despite finding overall high inter-rater reproducibility in the gyn-ments scoring system (spearman r 0.82-0.84), it does not appear this scoring system strongly discriminates the most urgent cases as determined by either the mesas system (spearman r 0.31) or when surgeons proceed using their instinct alone, the ssp (spearman r 0.46). the inter-rater reproducibility of the mesas tiered system was moderate (weighted kappa 0.57), and it appears to perform slightly better in discerning how surgeons instinctively prioritize (spearman r 0.53). in fact, the mesas system identified 92% of the most urgent ssp patients while the gyn-ments found only 67%. however, when used together, the two scoring systems had high discrimination in capturing clinicians' instinctive beliefs about urgency, and each contributed independently, suggesting that a) they capture distinct issues related to urgency, and b) their combined use may provide the optimal system for objectively rating surgical urgency. despite seeing merit in the original scoring system, we felt additional steps could be taken to make the ments model more objective, yielding higher inter-observer reliability. our goal was to create a modified gynecologic ments that would still score in comparable ranges to the original in the event our institution later decided to prioritize by this route. most modifications were made within the disease factors category. we felt the category should be given more weight of importance (with more scored items) and tailored to the disease burden gynecologic patients uniquely incur. thus, we expanded the number of scoring categories and created objective criteria for quantifying pain, anemia, fertility impact, and impact on adjacent organ systems such as the genitourinary and gastrointestinal systems. while we agree that exposure to known covid-19 should be considered, we recommend that, if available, all patients undergoing scheduled surgery be tested for covid-19 within 48 hours of their planned surgery and delayed if found positive. if testing is unavailable, we recommend screening all elective surgery patients for influenza-like symptoms, and surgery postponed if present. given the reports of significantly worsened morbidity and mortality when surgery is unwittingly performed in pre-symptomatic covid-19 patients, all attempts to identify these patients should be made. 9, 10 additionally, universal covid-19 testing protects health care workers from unnecessary exposure, as well as creates a binary ppe triaging system to "standard precautions" or "transmission-based precautions" to protect scare resources. despite our attempts to make the scoring system as objective as possible, many categories are open to wider interpretation than it may first seem. we recommend, prior to implementing the scoring system, an initial dialogue to "lay the ground rules" occurs to improve reviewer reproducibility and thus reliability across the cohort. for example: 1. lung disease. attempt to define what constitutes 'minimal' disease. our system did not capture smoking history other than a binary yes/no. quantifying risk of disease in a pack/year history calculation may prove beneficial as smoking impacts risks of both respiratory disease and wound healing with direct impact on outcomes. it is also becoming increasingly known that lung disease, such as chronic obstructive pulmonary disease (copd) and asthma, can worsen patient outcomes in the setting of covid, independent of their usual perioperative risks. 11 2. surgical team size. we agreed team size would be calculated by the minimum number of surgeons needed to perform a procedure safely and efficiently. in an academic teaching institution, the size of surgical teams can easily be twice the actual number needed. thus, all hysteroscopy (that did not require intraoperative sonography) was scored a one, and almost all laparoscopy a two. when constant uterine manipulation was needed, a score of three was given to laparoscopy. 3. or time. at our institution, we are asked to provide estimates of "wheels-in" to "wheels-out" rather than "incision-to-closure" time when posting cases. as long as consistency is applied across graders, this variable has the potential to be reliable. given surgeon notoriety around being poor predictors of needed surgical time, it is not surprising that this variable performed among the worst in inter-rater agreement. 1. pain. despite our attempt to objectively define pain using a vas system and incorporating ability to control this pain based on frequency of office or er visits, our inter-rater agreement remained fair (agreement = 57%) ( table 3) . this was due, in large part, to provider differences in documentation of pain, its impact on quality of life, and the ability to control pain with medical management. 2. alternative therapy. despite our attempt to objectively quantitate numbers of alternative therapies tried and incorporating the frequency of office visits to maintain therapy, our inter-rater agreement was poor (agreement = 34%) despite our extensive list of what constitutes alternative therapy in the table 2 footnotes. we feel this was due to difficulty in abstracting this information from charts. 3. prediction of peri-operative transfusion. we agree the presence of anemia can be inconsequential when it comes to procedures with a risk of low blood loss. the counter is true as well; high blood loss procedures can be tolerated when the patient has no baseline anemia. the most important question, particularly during a time of blood shortage, is whether and to what degree the patient will need perioperative blood products. finding an objective way to quantify this risk was difficult, a problem compounded by surgeons' inability to reliably predict estimates of ebl (agreement = 73%). 4. cardiovascular disease. while simplifying the determination of heart disease to represent the number of medications it takes to control it, care should be taken to avoid this simple assumption. patients with untreated hypertension may carry significantly more heart disease than patients well-controlled on three medications due simply to their access to health care and compliance with recommended therapy. 5. emphasis on hospital resource utilization with a bias towards the young, healthy patient. perhaps the biggest limitation to the gyn-ments scoring system is its favor towards quick procedures on young, healthy patients. despite our attempts to increase the weight of disease burden by adding more graded variables, young and healthy patients getting quick, elective procedures (such as tubal ligation or polypectomy) were consistently prioritized in this grading system. while not an invalid conclusion when a hospital system is severely limited in its capacity to do anything but the most quick and simple of procedures on healthy patients, it would be difficult to justify, for example, elective sterilization over treatment of debilitating pain from endometriosis, especially if patients have access to alternative contraception. in our institution, despite receiving some of the lowest gyn-ments scores, patients requesting sterilization are not being prioritized at this time. 6. we also must acknowledge that many of our changes to the original ments scoring system could be specific to the study institution which thus impacts the generalizability of the gyn-ments system. despite the extensive list of surgical examples given by major gynecologic surgical societies in the mesas table, we found inconsistencies with application between reviewers. it is clear there is still room for interpretation of disease severity, and thus acuity, making assignment of tiers prone to significant variation. additionally, at the end of this exercise, a high-volume institution could still have large numbers of patients within each cohort that still must be prioritized further, and this system provides no guidance as to how to perform this. finally, the mesas system appears to contain some inconsistencies within their surgical examples. for instance, endometriosis with poorly controlled pain and desire for fertility is categorized as a level 2 while myomectomy for an asymptomatic patient that is experiencing infertility is categorized as a level 3. a hysteroscopic polypectomy in the infertile patient is categorized more urgently (level 3) than a hysteroscopic evaluation or polypectomy in patients over 50 with inability to sample in the office (level 2) that have higher risks of malignant potential. while these surgical assignments have been agreed upon by major societal stakeholders, these discrepancies deserve attention. to a certain extent, both scoring systems depend on accurate and elaborate chart documentation. ideally, surgeons would grade their own patients to improve accuracy and overcome this obstacle. difficulty was encountered when reviewers graded each other's patients in categories such as efficacy of alternative therapies, immediacy of fertility desire, and severity of pain. this clearly impacts the interrater reliability and can lead to the false assumption that poor inter-rater reliability means a scoring system is invalid. finally, further evaluation in a setting with clinicians who did not develop the scoring system is warranted. to our knowledge, this is the first study to assess reliability and validity of previously published surgical scoring systems. additionally, we are the first to report application of these scoring systems in gynecologic patients and have made recommendations for implementation in this arena. more robust prospective data are needed to either confirm or refute our retrospective findings. further study should also evaluate the efficacy of utilizing both the gyn-ments and mesas system together to triage non-emergent procedures. we also feel strongly that a system to provide more emphasis on the disease variables of the gyn-ments scoring system would yield an even more valid triaging system. cqm, jsk, czw, and rla were responsible for study design and creation of the modified scoring system. cqm, jsk, czw, wab, and cmcb were responsible for chart review and data abstraction. rla was responsible for data analysis and interpretation. cqm, jsk, czw, wab, and cmcb were responsible for data interpretation. cqm, jsk, czw, wab, cmcb, and rla were responsible for manuscript drafting and editing. jsk was responsible for publication management. data sharing: all data collected for the study including the study protocol, a data dictionary defining each field, and de-identified patient data will be made available to qualified researchers using a data sharing platform upon reasonable request with publication. recommendations for management of elective surgical procedures american college of surgeons surgeon general. hospitals & healthcare systems, please consider stopping elective procedures until we can #flattenthecurve centers for medicare & medicaid services. cms releases recommendations on adult elective surgeries, non-essential medical, surgical, and dental procedures during covid-19 response american college of surgeons. covid-19: guidance for triage of non-emergent surgical procedures american college of surgeons joint statement on re-introduction of hospital and office-based procedures in the covid-19 climate il: society of gynecologic surgeons medically necessary, time-sensitive procedures: scoring system to ethically and efficiently manage resource scarcity and provider risk during the covid-19 joint statement on minimally invasive gynecologic surgery during the covid-19 pandemic society of american gastrointestinal and endoscopic surgeons. sages and eaes recommendations regarding surgical response to covid-19 crisis clinical characteristics and outcomes of patients undergoing surgeries during the incubation period of covid-19 infection. eclinicalmedicine author's reply: hazardous postoperative outcomes of unexpected covid-19 infected patients: a call for global consideration of sampling all asymptomatic patients before surgical treatment epidemiological, comorbidity factors with severity and prognosis of covid-19: a systematic review and meta-analysis key: cord-029290-vf5qebso authors: devonport, alex; khaled, mahmoud; arcak, murat; zamani, majid title: pirk: scalable interval reachability analysis for high-dimensional nonlinear systems date: 2020-06-13 journal: computer aided verification doi: 10.1007/978-3-030-53288-8_27 sha: doc_id: 29290 cord_uid: vf5qebso reachability analysis is a critical tool for the formal verification of dynamical systems and the synthesis of controllers for them. due to their computational complexity, many reachability analysis methods are restricted to systems with relatively small dimensions. one significant reason for such limitation is that those approaches, and their implementations, are not designed to leverage parallelism. they use algorithms that are designed to run serially within one compute unit and they can not utilize widely-available high-performance computing (hpc) platforms such as many-core cpus, gpus and cloud-computing services. this paper presents pirk, a tool to efficiently compute reachable sets for general nonlinear systems of extremely high dimensions. pirk can utilize hpc platforms for computing reachable sets for general high-dimensional non-linear systems. pirk has been tested on several systems, with state dimensions up to 4 billion. the scalability of pirk’s parallel implementations is found to be highly favorable. applications of safety-critical cyber-physical systems (cps) are growing due to emerging iot technologies and the increasing availability of efficient computing devices. these include smart buildings, traffic networks, autonomous vehicles, truck platooning, and drone swarms, which require reliable bug-free software that perform in real-time and fulfill design requirements. traditional simulation/testing-based strategies may only find a small percentage of the software defects and the repairs become much costly as the system complexity grows. hence, in-development verification strategies are favorable since they reveal the faults in earlier stages, and guarantee that the designs satisfy the specifications as they evolve through the development cycle. formal methods offer an attractive alternative to testing-and simulation-based approaches, as they can verify whether the specifications for a cps are satisfied for all possible behaviors from a set of the initial states of the system. reachable sets characterize the states a system can reach in a given time range, starting from a certain initial set and subjected to certain inputs. they play an important role in several formal methods-based approaches to the verification and controller synthesis. an example of this is abstraction-based synthesis [1] [2] [3] [4] , in which reachable sets are used to construct a finite-state "abstraction" which is then used for formal synthesis. computing an exact reachable set is generally not possible. most practical methods resort to computing over-approximations or under-approximations of the reachable set, depending on the desired guarantee. computing these approximations to a high degree of accuracy is still a computationally intensive task, particularly for high-dimensional systems. many software tools have been created to address the various challenges of approximating reachable sets. each of these tools uses different methods and leverages different system assumptions to achieve different goals related to computing reachable sets. for example, cora [5] and spaceex [6] tools are designed to compute reachable sets of high accuracy for very general classes of nonlinear systems, including hybrid ones. some reachability analysis methods rely on specific features of dynamical systems, such as linearity of the dynamics or sparsity in the interconnection structure [7] [8] [9] . this allows computing the reachable sets in shorter time or for relatively high-dimensional systems. however, it limits the approach to smaller classes of applications, less practical specifications, or requires the use of less accurate (e.g., linearized) models. other methods attack the computational complexity problem by computing reachable set approximations from a limited class of set representations. an example of limiting the set of allowed overapproximations are interval reachability methods, in which reachable sets are approximated by cartesian products of intervals. interval reachability methods allow for computing the reachable sets of very general non-linear and high-dimensional systems in a short amount of time. they also pose mild constraints on the systems under consideration, usually only requiring some kind of boundedness constraint instead of a specific form for the system dynamics. many reachability tools that are designed to scale well with state dimension focus on interval reachability methods: these include flow * [10] , capd [11] , c2e2 [12] , vnode-lp [13] , dynibex [14] , and tira [15] . another avenue by which reachable set computation time can be reduced, which we believe has not been sufficiently explored, is the use of parallel computing. although most reachability methods are presented as serial algorithms, many of them have some inherent parallelism that can be exploited. one example of a tool that exploits parallelism is xspeed [16] , which implements a parallelized version of a support function-based reachability method. however, this parallel method is limited to linear systems, and in some cases only linear systems with invertible dynamics. further, the parallelization is not suitable for massively parallel hardware: only some of the work (sampling of the support functions) is offloaded to the parallel device, so only a relatively small number of parallel processing elements may be employed. in this paper, we investigate the parallelism for three interval reachability analysis methods and introduce pirk, the parallel interval reachability kernel. pirk uses simulation-based reachability methods [17] [18] [19] , which compute rigorous approximations to reachable sets by integrating one or more systems of odes. pirk is developed in c++ and opencl as an open-source 1 kernel for pfaces [20] , a recently introduced acceleration ecosystem. this allows pirk to be run on a wide range of computing platforms, including cpus clusters, gpus, and hardware accelerators from any vendor, as well as cloud-based services like aws. the user looking to use a reachability analysis tool for formal verification may choose from an abundance of options, as our brief review has shown. what pirk offers in this choice is a tool that allows for massively parallel reachability analysis of high-dimensional systems with an application programming interface (api) to easily interface with other tools. to the best of our knowledge, pirk is the first and the only tool that can compute reachable sets of general non-linear systems with dimensions beyond the billion. as we show later in sect. 5, pirk computes the reachable set for a traffic network example with 4 billion dimension in only 44.7 min using a 96-core cpu in amazon aws cloud. consider a nonlinear system with dynamicsẋ = f (t, x, p) with state x ∈ r n , a set of initial states x 0 , a time interval [t 0 , t 1 ], and a set of time-varying inputs p defined over [t 0 , t 1 ]. let φ(t; t 0 , x 0 , p) denote the state of the system, at time t, of the trajectory beginning at time t 0 at initial state x 0 under input p. we assume the systems are continuous-time. the finite-time forward reachable set is defined as for the problem of interval reachability analysis, there are a few more constraints on the problem structure. an interval set is a set of the form [a, a] = {a : a ≤ a ≤ a}, where ≤ denotes the usual partial order on real vectors, that is the partial order with respect to the positive orthant cone. the vectors a and a are the lower and upper bounds respectively of the interval set. an interval set can alternatively be described by its center a * = 1 2 (a + a) and half-width [a] = 1 2 (a−a). in interval reachability analysis, the initial set must be an interval, and inputs values restricted to an interval set, i.e. p(t) ∈ [p, p], and the reachable set approximation must also be an interval (fig. 1) . furthermore, certain methods for computing interval reachable sets require further restrictions on the system dynamics, such as the state and input jacobian matrices being bounded or sign-stable. pirk computes interval reachable sets using three different methods, allowing for different levels of tightness and speed, and which allow for different amounts of additional problem data to be used. the contraction/growth bound method [4, 21, 22] computes the reachable set using component-wise contraction properties of the system. this method may be applied to input-affine systems of the formẋ = f (t, x) + p. the growth and contraction properties of each component of the system are first characterized by a contraction matrix c. the contraction matrix is a component-wise generalization of the matrix measure of the jacobian j x = ∂f /∂x [19, 23] , satisfying the method constructs a reachable set over-approximation by separately establishing its center and half-width. the center is found by simulating the trajectory of the center of the initial set, that is as φ(t 1 ; t 0 , x * , p * ). the half width is found by integrating the growth dynamicṡ [24] computes the reachable set by separating the increasing and decreasing portions of the system dynamics in an auxiliary system called the embedding system whose state dimension is twice that of the original system [25] . the embedding system is constructed using a decomposition function d(t, x, p,x,p), which encodes the increasing and decreasing parts of the system dynamics and satisfies d(t, x, p, x, p) = f (t, x, p). the evaluation of a single trajectory of the embedding system can be used to find a reachable set over-approximation for the original system. the monte carlo method computes a probabilistic approximation to the reachable set by evaluating the trajectories of a finite number m of pairs sample points (x (i) 0 , p (i) ) in the initial set and input set, and selecting the smallest interval that contains the final points of the trajectories. unlike the other two methods, the monte carlo method is restricted to constant-valued inputs, i.e. inputs of the form the interval reachable set is then approximated by the elementwise minimum and maximum of the x (i) 1 . this approximation satisfies a probabilistic guarantee of correctness, provided that enough sample states are chosen [26] . let [r, r] be the approximated reachable set, , δ ∈ (0, 1), and m ≥ ( 2n ) log 2n δ . then, with probability 1−δ, the approximation [r, r] satisfies p (r t0,t1 \[r, r]) ≤ , where p (a) denotes the probability that a sampled initial state will yield a final state in the set a, and \ denotes set difference. the probability that a sampled initial state will be sent to a state outside the estimate (the "accuracy" of the estimate) is quantified by . improved accuracy (lower ) increases the sample size as o(1/ ). the probability that running the algorithm will fail to give an estimate satisfying the inequality (the "confidence") is quantified by δ. improved confidence (lower δ) increases the sample size by o(log(1/δ)). the bulk of the computational work in each method is spent in ode integration. hence, the most effective approach by which to parallelize the three methods is to design a parallel ode integration method. there are several available methods for parallelizing the task of ode integration. several popular methods for parallel ode integration are parallel extensions of runge-kutta integration methods, which are the most popular serial methods for ode integration. pirk takes advantage of the task-level parallelism in the runge-kutta equations by evaluating each state dimension in parallel. this parallelization scheme is called parallelization across space [27] . pirk specifically uses a space-parallel version of the fourth-order runge-kutta method, or space-parallel rk4 for brevity. in space-parallel rk4, each parallel thread is assigned a different state variable to evaluate the intermediate update equations. after each intermediate step, the threads must synchronize to construct the updated state in global memory. space-parallel rk4 can use as many parallel computation elements as there are state variables: since pirk's goal is to compute reachable sets for extremely high-dimensional systems, this is sufficient in most cases. the space-parallel scheme is not hardware-specific, and may be used with any parallel computing platform. pirk is similarly hardware-agnostic: the pfaces ecosystem, for which pirk is a kernel, provides a common interface to run on a variety of heterogeneous parallel computing platforms. the only difference between platforms that affects pirk is the number of available parallel processing elements (pes). the parallelized implementations of the three reachability methods described in sect. 2.1 use space-parallel rk4 to perform almost all computations other than setting up initial conditions. we can therefore find the time and memory complexity of each method by analyzing the complexity of space-parallel rk4 and counting the number of times each method uses it. for a system with n dimensions, space-parallel rk4 scales linearly as the number of pes (denoted by p ) increases. in a computer with a single pe (i.e., p = 1), the algorithm reduces to the original serial algorithm. then, suppose that a parallel computer has p ≤ n pes of the same type. we assume a computational model under which instruction overhead and latency from thread synchronization are negligible, memory space has equal access time from all processing elements, and the number of parallel jobs can be evenly distributed among the p processing elements. 2 under this parallel random-access machine model [28] , the time complexity of space-parallel rk4 is reduced by a factor of p : each pe is responsible for computing n/p components of the state vector. therefore, for fixed initial and final times t 0 and t 1 , the time complexity of the algorithm is o( n p ). the parallel version of the contraction/growth bound method uses spaceparallel rk4 twice. first, it is used to compute the solution of the system's ode f for the center of the initial set x 0 . then, it is used to compute the growth/contraction of the initial set x 0 by solving the ode g of the growth dynamics. since this method uses a fixed number of calls of space-parallel rk4, its time complexity is also o( n p ) for a given t 0 and t 1 . the parallelized implementation of the mixed-monotonicity method uses space-parallel rk4 only once, in order to integrate the 2n-dimensional embedding system. this means that the mixed-monotonicity method also has a time complexity of o( n p ) for fixed t 0 and t 1 . however, the mixed-monotonicity method requires twice as much memory as the growth bound method, since it runs spaceparallel rk4 on a system of dimension 2n. the parallelized implementation of the monte carlo method uses spaceparallel rk4 m times, once for each of the m sampled initial states. the implementation uses two levels of parallelization. the first level is a set of parallel threads over the samples used for simulations. then, within each thread, another parallel set of threads are launched by space-parallel rk4. this is realized as one parallel job of m × n threads. consequently, the monte carlo method has a complexity of o( mn p ). since only the elementwise minima and maxima of the sampled states need to be stored, this method only requires as much memory as the growth bound method. a pseudocode of each parallel algorithm and a detailed discussion of their time and space complexities are provided in an extended version of this paper [29] . the extended version also contains additional details for the case studies that will be presented in the next section. in each of the case studies to follow, we report the time it takes pirk to compute reachable sets for systems of varying dimension using all three of its methods on a variety of parallel computing platforms. we perform some of the same tests using the serial tool tira, to measure the speedup gained by pirk's ability to use massively parallel hardware. we set a time limit of 1 h for all of the targeted case studies, and report the maximum dimensions that could be reached under this limit. the monte carlo method is given probabilistic parameters = δ = 0.05 in each case study where it is used. we use four aws machines for the computations with pirk: m4.10xlarge which has a cpu with 40 cores, c5.24xlarge which has a cpu with 96 cores, g3.4xlarge which has a gpu with 2048 cores, and p3.2xlarge which has a gpu with 5120 cores. for the computations with tira, we used a machine with a 3.6 ghz intel i7 cpu. we consider the road traffic analysis problem reported in [30] , a proposed benchmark for formal controller synthesis. we are interested in the density of cars along a single one-way lane. the lane is divided into n segments, and the density of cars in each segment is a state variable. the continuous-time dynamics are derived from a spatially discretized version of the cell transmission model [31] . this is a nonlinear system with sparse coupling between state variables. the results of the speed test are shown in the first row of figure 2 . the machines m4.10xlarge and c5.24xlarge reach up to 2 billion and 4 billion dimensions, respectively, using the growth/contraction method, in 47.3 min and 44.7 min, respectively. due to memory limitations of the gpus, the machines g3.4xlarge and p3.2xlarge both reach up to 400 million in 106 s and 11 s, respectively. the relative improvement of pirk's computation time over tira's is significantly larger for the growth bound method than for the other two. this difference stems from how each tool computes the half-width of the reachable set from the radius dynamics. tira solves the radius dynamics by computing the full matrix exponential using matlab's expm, whereas pirk directly integrates the dynamics using parallel runge-kutta. this caveat applies to sect. 5.2 as well. the second test system is a swarm of k identical quadrotors with nonlinear dynamics. the system dynamics of each quadrotor model are derived in a similar way to the model used in the arch-comp 18 competition [32] , with the added simplification of a small angle approximation in the angular dynamics and the neglect of coriolis force terms. a derivation of both models is available in [33] . similar to the n-link traffic model, this system is convenient for scaling: system consisting of one quadrotor can be expressed with 12 states, so the state dimension of the swarm system is n = 12k. while this reachability problem could be decomposed into k separate reachability problems which can be solved separately, we solve the entire 12k-dimensional problem as a whole to demonstrate pirk's ability to make use of sparse interconnection. the results of the speed test are shown in fig. 2 (second row) . the machines m4.10xlarge and c5.24xlarge reach up to 1.8 billion dimensions and 3.6 billion dimensions, respectively, (using the growth/contraction method) in 48 min and 32 min, respectively. the machines g3.4xlarge and p3.2xlarge both reach up to 120 million dimensions in 10.6 min and 46 s, respectively. the third test system is a modification of the quadrotor swarm system which adds interactions between the quadrotors. in addition to the quadrotor dynamics described in sect. 5.2, this model augments each quadrotor with an artificial potential field to guide it to the origin while avoiding collisions. this controller applies nonlinear force terms to the quadrotor dynamics that seek to minimize an artificial potential u that depends on the position of all of the quadrotors. due to the interaction of the state variables in the force terms arising from the potential field, this system has a dense jacobian. in particular, at least 25% of the jacobian elements will be nonzero for any number of quadrotors. table 1 shows the times of running pirk using this system on the four machines m4.10xlarge, c5.24xlarge, g3.4xlarge and p3.2xlarge in amazon aws. due to the high density of this example, we focus on the memory-light growth bound and the monte-carlo methods. pirk computed the reach sets of systems up to 120,000 state variables (i.e., 10,000 quadrotors). up to 1,200 states, all machines solve the problems in less than one second. some of the machines lack the required memory to solve the problems requiring large memory (e.g., 27.7 gb of memory is required to compute the reach set of the system with 120,000 state variables using the growth bound method). the fourth test system is a model for the diffusion of heat in a 3-dimensional cube. the model is based on a benchmark used in [7] to test a method for numerical verification of affine systems. a model of the formẋ = f (t, x, p) which approximates the heat transfer through the cube according to the heat equation can be obtained by discretizing the cube into an × × grid, yielding a system with 3 states. the temperature at each grid point is taken as a state variable. each spatial derivative is replaced with a finite-difference approximation. since the heat equation is a linear pde, the discretized system is linear. we take a fixed state dimension of n = 10 9 by fixing = 1000. integration takes place over [t 0 , t 1 ] = [0, 20] with time step size h = 0.02. using the growth bound method, pirk solves the problem on m4.10xlarge in 472 min, and in 350.2 min on c5.24xlarge. this is faster than the time reported in [7] (30 h) using the same machine. the remaining case studies focus on models of practical importance with low state dimension. although pirk is designed to perform well on high-dimensional systems, it is also effective at quickly computing reachable sets for low dimensional systems, for applications that require many reachable sets. the first such case study is single-track vehicle model with seven states, presented in [34] . we fix an input that performs a maneuver to overtake an obstacle in the middle lane of a 3-lane highway. to verify that the maneuver was safely completed, we compute reachable sets over a range of points and ensuring that the reachable set does not intersect any obstacles. we consider a step-size of 0.005 s in a time window between 0 and 6.5 s. we compute one reachable set at each time step, resulting in a "reachable tube" comprising 1300 reachable sets. pirk computed the reachable tube in 0.25 s using the growth bound method on an i7 cpu (fig. 3) . in order to compare pirk's performance to existing tools, we tested pirk's growth bound implementation on three systems from the arch-comp'18 category report for systems with nonlinear dynamics [32] . this report contains benchmark data from several popular reachability analysis tools (c2e2, cora, flow * , isabelle, spaceex, and symreach) on nonlinear reachability problems with state dimensions between 2 and 12. table 2 compares the computation times for pirk on the three systems to those reported by other tools in [32] . all times are in seconds. pirk ran on an i9 cpu, while the others ran on i7 and i5: see [32] for more hardware details. pirk solves each of the benchmark problems faster than the other tools. both of the i7 and i9 processors used have 6 to 8 cores: the advantage of pirk is its ability to utilize all available cores. using a simple parallelization of interval reachability analysis techniques, pirk is able to compute reachable sets for nonlinear systems faster and at higher dimensions than many existing tools. this performance increase comes from pirk's ability to use massively parallel hardware such as gpus and cpu clusters, as well as the use of parallelizable simulation-based methods. future work will focus on improving the memory-usage of the mixed monotonicity and monte-carlo based methods, including an investigation of adaptive sampling strategies, and on using pirk as a helper tool to synthesize controllers for high-dimensional systems. open access this chapter is licensed under the terms of the creative commons attribution 4.0 international license (http://creativecommons.org/licenses/by/4.0/), which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the creative commons license and indicate if changes were made. the images or other third party material in this chapter are included in the chapter's creative commons license, unless indicated otherwise in a credit line to the material. if material is not included in the chapter's creative commons license and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. symbolic models for nonlinear control systems with-out stability assumptions formal methods for discrete-time dynamical systems verification and control of hybrid systems: a symbolic approach feedback refinement relations for the synthesis of symbolic controllers an introduction to cora spaceex: scalable verification of hybrid systems numerical verification of affine systems with up to a billion dimensions ellipsoidal toolbox (et) sapo: reachability computation and parameter synthesis of polynomial dynamical systems flow * : an analyzer for non-linear hybrid systems computer assisted proofs in dynamics group, a c++ package for rigorous numerics c2e2: a verification tool for stateflow models implementing a rigorous ode solver through literate programming validated explicit and implicit runge-kutta methods tira: toolbox for interval reachability analysis xspeed: accelerating reachability analysis on multi-core processors computing bounded reach sets from sampled simulation traces trajectory based verification using local finite-time invariance reachability analysis of nonlinear systems using matrix measures pfaces: an acceleration ecosystem for symbolic control a lohner-type algorithm for control systems and ordinary differential inclusions locally optimal reach set over-approximation for non-linear systems simulation-based reachability analysis for nonlinear systems using componentwise contraction properties formal methods for control of traffic flow: automated control synthesis from finite-state transition models monotone flows and order intervals data-driven reachable set computation using adaptive gaussian process classification and monte carlo methods parallel numerical methods for ordinary differential equations: a survey an introduction to parallel algorithms pirk: scalable interval reachability analysis for high-dimensional nonlinear systems (2020) a benchmark problem in transportation networks a compartmental model for traffic networks and its dynamical behavior arch-comp18 category report: continuous and hybrid systems with nonlinear dynamics quadrotor dynamics and control rev 0.1 commonroad: vehicle models key: cord-257623-j8dqvbqw authors: norris, ken; terry, andrew; hansford, james p.; turvey, samuel t. title: biodiversity conservation and the earth system: mind the gap date: 2020-07-07 journal: trends ecol evol doi: 10.1016/j.tree.2020.06.010 sha: doc_id: 257623 cord_uid: j8dqvbqw one of the most striking human impacts on global biodiversity is the ongoing depletion of large vertebrates from terrestrial and aquatic ecosystems. recent work suggests this loss of megafauna can affect processes at biome or earth system scales with potentially serious impacts on ecosystem structure and function, ecosystem services, and biogeochemical cycles. we argue that our contemporary approach to biodiversity conservation focuses on spatial scales that are too small to adequately address these impacts. we advocate a new global approach to address this conservation gap, which must enable megafaunal populations to recover to functionally relevant densities. we conclude that re-establishing biome and earth system functions needs to become an urgent global priority for conservation science and policy. the loss of large vertebrates (megafauna) from the world's ecosystems has been occurring throughout human history. emerging evidence suggests these losses can have dramatic impacts on ecological functions, including the near collapse of biogeochemical cycles at biome and earth system scales. although the biodiversity conservation community increasingly recognises the need to restore ecological functions at landscape scales, it currently operates at scales that are too small to adequately address the functional consequences of megafaunal losses at biome and earth system scales. re-establishing biome and earth system functions needs to become a global priority for the biodiversity conservation community, and we will need a new global initiative to deliver this. known to provide essential ecosystem functions through regulation of terrestrial vegetation structure and dynamics at both landscape and biome scales. although individuals and small isolated populations can perpetuate localised interactions, functionality is driven by large populations and diverse assemblages, highlighting the vulnerability of megafauna-driven ecosystem regulation to population declines well before actual extinctions [18, 19] . the role of large herbivores (including terrestrial and arboreal mammals, large birds, and giant tortoises) in promoting plant regeneration and regulating vegetation composition through dispersal of plant propagules, especially over long distances, is crucial in most terrestrial systems [20] . however, there has been substantial human-caused trophic downgrading of frugivore communities worldwide [21] , and although coevolved plant species may persist beyond megafaunal extinction by exploiting alternative dispersal mechanisms, cascading effects of large glossary biogeochemical cycles: pathways through which chemical substances, for example, nitrogen or phosphorus, move through the biotic and abiotic components of ecological systems. biome: a distinct community of microorganisms, plants, and animals occupying an extensive geographical area, such as a tropical forest or desert. convention on biological diversity: a multilateral treaty for the conservation and sustainable use of biological diversity delivered through national strategies (www.cbd.int). convention on migratory species: an international agreement that aims to conserve migratory species within their migratory ranges (www.cms.int). earth system: physical, chemical, and biological processes interacting at a planetary scale and involving the atmosphere, land, oceans, and polar regions. ecological function: a process or set of processes that can change an ecological system over time, for example, seed dispersal, herbivory, or predation. ecosystem services: benefits that humans receive from ecosystems, for example, food, clean water, disease control, and cultural experiences. ecosystem structure and function: structure refers to the way an ecosystem is organised and includes its species composition, trophic structure or functional composition, and distribution of mass and energy between its components; function refers to the flow of mass and energy through an ecosystem. half-earth project: a call to protect half the global area of land and sea to reverse species extinctions and ensure long-term planetary health (www.half-earthproject.org). international convention for the regulation of whaling: an international agreement for the conservation of whale stocks and their sustainable exploitation. landscape: an area of land, its landforms, and their integration with human-made and natural elements, and typically covering a geographical area much smaller than a biome. megafauna: large-bodied animals. specific mass thresholds used vary between studies, but the term often refers to animals n44.5 kg. figure 1 . the functional roles played by large vertebrates (megafauna) across spatial scales. important ecological processes driven by megafauna include the long-distance dispersal of seeds; browsing, grazing and physical disturbance of plant communities by herbivores; and predation by large carnivores. large-scale movements by megafauna transport nutrients such as nitrogen and phosphorus across land and in the ocean when they urinate and defaecate, and when they die and decompose. these processes interact to drive biogeochemical cycles at biome (e.g., the amazon) and earth system scales. the loss of megafauna has had a significant impact on these processes, resulting in substantial reductions in nutrient flows at biome and earth system scales. vertebrate loss include plant community reorganisation, reduction of megafauna-dependent plant abundance, distribution, and population structure, and local extinctions [10, 12, 22, 23] . recent evidence also shows megafauna influence the dispersal of microbes [24] . more direct regulation of habitat structure, ecosystem state, and associated species diversity and richness by herbivores is widely recognised, including suppression of plant growth and regeneration through grazing and browsing, and further physical modification of vegetation and geomorphology by trampling and other damage. megaherbivore presence is often associated with increased landscape openness and heterogeneity, for example from closed-canopy forest to forest-grassland mosaic parkland landscapes [25, 26] , but can have numerous system-specific regulatory effects, such as a state shift between open-water wetlands and sphagnum bogs in the galápagos islands driven by presence or absence of the giant tortoise (chelonoidis nigra) [27] . megaherbivores modify water tables and soil methane emissions and affect evapotranspiration and land surface albedo [28] . megaherbivore extinction can also be associated with changed fire regimes, with the potential for increased fire frequency due to accumulation of uncropped plant material, and associated state shifts to more fire-resistant dominant vegetation communities [29, 30] . large carnivores also play an important role in regulating habitat structure through behaviourally mediated indirect interactions, by causing changes in prey distribution and associated mesoherbivore-vegetation interactions across landscapes (so-called landscapes of fear) [31] [32] [33] , although the dynamics of such carnivore-induced trophic cascades are further modified by local presence of megaherbivores [34] . megafaunal interactions such as propagule dispersal and nutrient transfer through faeces and urine play a further important role in regulating biogeochemical cycling. this is well recognised at landscape and biome scales. loss of seed and fruit dispersers in tropical forests has a negative impact on ecosystem carbon storage through reduction of tree biomass. for example, extinction of forest elephants (loxodonta cyclotis) would result in a 7% decrease in above-ground biomass in central african rainforests, reducing efficiency of carbon sequestration [11] . past megafaunal extinctions are predicted to have already reduced carbon storage capacity in globally important ecoregions such as the amazon [12] . megafaunal regulation of soil biogeochemical processes is particularly important in nutrient-poor cold or dry environments, and megafaunal disappearance in north eurasia is posited to have locked nutrients into slowly decomposing plant matter within permafrost soils and decreased system productivity [35] . megafauna play comparable functional roles in marine systems [36] . there is increasing recognition of the vital additional role played by megafauna in horizontal movement of carbon and nutrients both across landscapes and biomes and across system boundaries, thus scaling up the megafaunal keystone paradigm to wider continental and global contexts. megafauna are now known to make a disproportionate contribution to lateral nutrient transfer, with large herbivores and carnivores both acting as important carbon and nutrient vectors by excreting organic matter derived from one system into another [37, 38] . megafaunamediated translocation, either via feeding migrations or local-scale movements across system boundaries (between terrestrial, freshwater, and/or marine systems), can profoundly shape the ecology, productivity, and structure of recipient systems by increasing diffusion rates along concentration gradients and against hydrological flow directions [39] . this global megafaunadriven nutrient pump counters sedimentation, with large cetaceans recovering nutrients from the deep sea and acting as vertical and horizontal vectors [40] , and a further chain of system-boundary transfers by other large vertebrates progressively moving nutrients upstream and into continental interiors [41] . megafauna therefore regulate key earth-system processes megaherbivore: large-bodied herbivorous animals n1000 kg in mass, for example, hippopotami and elephants. mesoherbivore: medium-sized herbivorous animals in the mass range 50-500 kg, for example, red deer. montreal protocol: an international treaty designed to protect the ozone layer by phasing out substances responsible for ozone depletion. seascape: the equivalent of a landscape in the ocean. transboundary initiative: conservation initiatives that cross national borders, that is, that include two or more countries. and global interconnectivity, and megafaunal extinctions have caused major perturbations to biogeochemical cycles at biome and earth system scales [15, 41] . over the past 30 years, biodiversity conservation has been focused primarily on area-based protection and restoration of threatened species and populations. these activities have been supported by global indicators such as the international union for conservation of nature red list and the living planet index, which have enabled conservationists to prioritise the most threatened species, identify threatening processes, and monitor responses to conservation interventions [4, 5, 42, 43] . this approach has tended to focus on threatened populations with geographically restricted ranges (e.g., relict populations that were formally part of much larger, connected ranges; island endemics). however, it has become increasingly clear that a focus on population-level or single-species conservation (and typically for populations already in serious decline) will not be sufficient to protect or restore key ecological functions at larger spatial scales (e.g., landscapes and biomes) [8, 44] , and that species' ecological roles need to be a more prominent part of the conservation agenda [45, 46] . furthermore, recognition of the links between biodiversity, ecosystem services and human well-being means that conservation is increasingly part of a transformative global agenda that is considering possible futures for nature and people [47] . against this wider background, we now understand that megafauna can play a critical role in the restoration of landscapes with potentially wide-ranging benefits to biodiversity because of the key functions they perform [48] [49] [50] . this realisation is fundamental to the concept of rewilding, which aims to restore self-sustaining ecosystems that require minimal management interventions in the longer term. within this framework, reintroductions of regionally extinct species and novel introductions of surrogate or analogue species are often used to replace lost ecological functions associated with historical removal of megafauna [51] . for example, large-bodied carnivores have been reintroduced at various sites in europe and north america [e.g., grey wolves (canis lupus) in yellowstone national park] to restore top-down regulation of ecosystems through trophic cascades. a wide range of large-bodied herbivores have been used as grazers, browsers, and agents of disturbance across different european rewilding projects to replace the roles of regionally extirpated species such as european horse (equus ferus ferus), bison (bison bonasus), and aurochs (bos primigenius), and extant species of giant tortoise (e.g., aldabrechelys gigantea) have been used to restore herbivory and seed dispersal functions to vegetation communities on tropical islands such as the galápagos and mauritius that have lost their endemic tortoise species. even nonintentional replacement can restore at least some functionality of extinct taxa [52] , and the introduction of non-native megafauna can have functional consequences [e.g., pablo escobar's hippos (hippopotamus amphibius) in colombia] [53] . these species-level and landscape-level approaches represent contrasting conservation scales ( figure 2 ). when viewed in this way, it becomes apparent that conservation has progressively expanded its scale of operation over the past 30 years from small (species and populations) to larger (e.g., landscapes) spatial scales. however, this perspective also reveals a worrying gap between the scales over which conservation currently operates, and the scales over which ecological functions are changing in response to the ongoing loss of megafauna. the loss of ecological functions at biome and earth system scales is simply not adequately represented in contemporary approaches to conservation. for example, yellowstone, into which grey wolves were reintroduced, covers an area of roughly 9000 km 2 ; in contrast, amazonia covers an area of 7 000 000 km 2 , and the area covered by the biogeochemical cycles supporting it represents a significantly larger area again. consequently, our contemporary approach to biodiversity trends in ecology & evolution conservation focuses on spatial scales that are too small to adequately address changes in ecological function at biome or earth system scales. given the magnitude of changes in biogeochemical cycles at these scales since the late quaternary, the conservation community therefore urgently needs to address the gap between the scales over which conservation currently operates, and the scales over which ecological functions are changing. the depletion of megafauna has occurred over millennia, and the loss of associated ecological functions occurs across a range of spatial scales, including biome and earth system scales ( figure 1) . furthermore, although the scales over which contemporary conservation operates have arguably expanded over recent decades, these responses remain too localised to address the scale of the problem (figure 2 ). we need urgent action at biome and earth system scales: in other words, a genuinely integrated, global response. environmental science and policy have had a significant impact at global scales. for example, the montreal protocol, an international treaty that came into force in 1987, was established to protect the ozone layer by phasing out the production and use of numerous substances responsible for ozone depletion. over 190 countries participate in the treaty, and it has resulted in the phaseout of 99% of nearly 100 ozone-depleting chemicals. without this treaty, the ozone layer is predicted to have collapsed by the mid-21st century [54] , with hugely serious implications for human health. although important work to further mitigate the impact of ozone-depleting figure 2 . conservation scales. contemporary biodiversity conservation is focused on populations and species, and on landscapes and seascapes. associated actions typically cover areas of a few thousand square kilometres at most. at biome or earth system scales, limited attention is being given to re-establishing the key functional roles and relationships provided by megafauna. this conservation gap is particularly serious given recent evidence showing substantial reductions in nutrient flows at these spatial scales. chemicals is still necessary, recent research shows that the ozone layer is recovering [55] . some megafaunal conservation efforts have also operated at global scales. the international whaling commission (iwc) was set up under the international convention for the regulation of whaling in 1946. although some of its activities remain contentious, a moratorium on hunting was introduced in the 1980s, and whale sanctuaries were established in the indian ocean (1979) and southern ocean (1994), covering an area of over 50 million km 2 . a further sanctuary in the south atlantic is currently under discussion. several whale populations are showing signs of recent recovery [56] [57] [58] , and although many remain below their historical baselines, the iwc represents one of the few initiatives aimed at the conservation of megafauna at appropriately large spatial scales. despite examples of global initiatives that have delivered demonstrable environmental benefits, there is little overall evidence that biodiversity conservation is currently operating at the scales required to address functional consequences at biome and earth system scales. the majority of global conservation conventions, such as the cbd and convention on migratory species (cms), are implemented at national scales through a shared responsibility approach, which almost inevitably means that progress is piecemeal, and coordination and integration across systems and scales is poor. a number of transboundary initiatives have developed globally in recent years [59, 60] , which include the conservation of megafaunal species. these initiatives often reflect the long-held view that large-scale interventions are needed to restore ecologically functional communities [46] . furthermore, there have been recent calls by conservationists for a half-earth project to conserve half of the earth's biodiversity. it seems unlikely, however, that these projects will adequately address the depletion of megafauna and restore the biome or earth system functions they drive without including measures specifically designed to do so. indeed, where the conservation community has had some recent successes in conserving megafaunal populations, this can result in increased conflict between wildlife and local people unless adverse impacts can be appropriately managed [61, 62] . in addition, human infrastructure such as fences, roads, and other urbanisation of landscapes often significantly restricts animal movement [63, 64] , constraining the scales over which key ecological functions can operate and hence limiting restoration potential even if megafaunal populations are locally able to recover. these major constraints mean that addressing the loss of megafauna and its functional consequences requires a new global initiative. at its heart, we need large, transboundary functional units capable of delivering key ecological functions at earth system scales, and within which megafauna and their associated functional pathways can be maintained or restored. it is likely that surviving large intact terrestrial biomes, such as the amazonian, central african and russian forests, the sahel and the eurasian steppe, should represent key components of such an initiative, as would large protected marine areas such as the existing marine mammal sanctuaries designated by the iwc. coupling between terrestrial and marine regions is also important to incorporate into any global initiative that aims to effectively address megafaunadriven functionality, given that the biogeochemical cycles we wish to restore are themselves driven by both aquatic and terrestrial processes [15] . restoring a functional earth system in this way will not be achieved by simply protecting megafaunal species, but their populations must be enabled to recover to functionally relevant densities and have ecological impacts at functionally relevant scales. this represents a fundamental shift in the scale at which global conservation operates. such an endeavour will require unprecedented international agreement and cooperation, and an expansion and reframing of the current global conservation paradigm. identifying, protecting and restoring transboundary functional units will require an interdisciplinary approach to science and policy that has thus far been largely an academic exercise rather than a practical reality. implementation will be challenging, not least because it will require individual countries and their inhabitants to act as custodians of earth system functions from which we will all benefit. we recognise that previous global initiatives targeted at the atmosphere (montreal protocol) and oceans (iwc) are less complex in terms of national sovereignty than a global initiative that includes terrestrial and aquatic systems plus linkages between them. success will inevitably depend, therefore, on benefit sharing, equality, and social justice, which in turn will require us to reform the dominant political and economic ideologies that have shaped global society for over 50 years. while daunting, we already recognise the need to address these issues if we are to create a shared future for nature and people [47, 65] . we are simply arguing that restoring a functional earth system needs to be a key global priority for biodiversity conservation within this wider debate. the loss of megafauna due to human activities has been taking place for millennia, but it is only recently that we have begun to understand the implications of this loss for the structure and function of ecological systems at biome and earth system scales. although the biodiversity conservation community increasingly recognises the need to restore and conserve whole systems, its priorities and interventions remain focused on scales that are too small to address biome or earth system functions. we argue that a new global initiative is required to address the past and ongoing loss of megafauna and its functional implications. we acknowledge the significant challenges involved with designing and delivering such an initiative (see outstanding questions). the consequences of a failure to act are, however, beyond serious. the collapse of the ozone layer would have had health implications for millions of people globally. we face impacts of similar scale and magnitude due to the depletion of megafauna. there are also key dependencies with other global environmental initiatives. for example, the paris climate agreement requires the earth system to play its part in the global carbon cycle. the restoration of megafauna and their functional roles will need to be a key part of any naturebased climate solutions. as we write this paper, the world is managing a global coronavirus pandemic; a poignant reminder that nature shows little respect for human constructs like national borders. as a global biodiversity conservation community this is a lesson we need to learn, and quickly. the conservation and restoration of megafauna needs to be an urgent, global conservation priority, not only for their inherent biodiversity value, but to maintain a healthy planet that supports both nature and people. what are the functional roles of megafauna in historic and contemporary systems at biome and earth system scales? how has the depletion of megafauna impacted the function of ecological systems at biome and earth system scales, and what are the implications of these changes for the long-term structural and functional integrity of these systems? what are the human impacts of the depletion of megafauna and degradation in their functional roles at biome and earth system scales? where will the restoration of megafauna have the greatest impact on earth system processes? how can we restore megafauna populations and their key ecological functions at biome and earth systems scales, and what are the social and ecological barriers to restoration? how do we need to reform our social, economic and political systems to incentivise nations to cooperate to restore and protect functional ecological systems at biome and earth system scales, and to ensure costs and benefits are shared equitably? patterns, causes, and consequences of anthropocene defaunation defaunation in the anthropocene biodiversity losses and conservation responses in the anthropocene the impact of conservation on the status of the world's vertebrates reductions in global biodiversity loss predicted from conservation spending collapse of the world's largest herbivores status and ecological effects of the world's largest carnivores beyond species loss: the extinction of ecological interactions in a changing world projected losses of global mammal and bird ecological strategies dispersal limitation induces long-term biomass collapse in overhunted amazonian forests carbon stocks in central african forests enhanced by elephant disturbance megafauna extinction, tree species range reduction, and carbon storage in amazonian forests extinction in the anthropocene the accelerating influence of humans on mammalian macroecological patterns over the late quaternary global nutrient transport in a world of giants megafauna and ecosystem function from the pleistocene to the anthropocene the megabiota are disproportionately important for biosphere functioning flying foxes cease to function as seed dispersers long before they become rare loss of seed dispersal before the loss of seed dispersers pleistocene megafaunal extinctions and the functional loss of long-distance seed-dispersal services extinction-driven changes in frugivore communities on oceanic islands seed dispersal anachronisms: rethinking the fruits extinct megafauna ate implications of lemuriform extinctions for the malagasy flora megafauna decline have reduced pathogen dispersal which may have increased emergent infectious diseases the impact of the megafauna extinctions on savanna woody cover in south america combining paleo-data and modern exclosure experiments to assess the impact of megafauna extinctions on woody vegetation the ecological consequences of megafaunal loss: giant tortoises and wetland biodiversity biophysical feedbacks between the pleistocene megafauna extinction and climate: the first humaninduced global warming? ecological consequences of late quaternary extinctions of megafauna can trophic rewilding reduce the impact of fire in a more flammable world? phil wolves and the ecology of fear: can predation risk structure ecosystems? the landscape of fear: the missing link to understand top-down and bottom-up controls of prey abundance? energy landscapes and the landscape of fear megaherbivores modify trophic cascades triggered by fear of predation in an african savanna ecosystem mammoth steppe: a high-productivity phenomenon megafaunal impacts on structure and function of ocean ecosystems lateral diffusion of nutrients by mammalian herbivores in terrestrial ecosystems megafauna moves nutrients uphill the hippopotamus conveyor belt: vectors of carbon and nutrients from terrestrial grasslands to aquatic systems in sub-saharan africa whales as marine ecosystem engineers the legacy of the pleistocene megafauna extinctions on nutrient availability in amazonia global biodiversity: indicators of recent declines a mid-term analysis of progress toward international biodiversity targets biodiversity and resilience of ecosystem functions ecological function analysis: incorporating species roles into conservation ecological effectiveness: conservation goals for interactive species pervasive human-driven decline of life on earth points to the need for transformative change merging paleobiology with conservation biology to guide the future of terrestrial ecosystems restoration, reintroduction, and rewilding in a changing world introduced herbivores restore late pleistocene ecological functions science for a wilder anthropocene: synthesis and future directions for trophic rewilding research unintentional rewilding: lessons for trophic rewilding from other forms of species introductions ecosystem effects of the world's largest invasive animal what would have happened to the ozone layer if chlorofluorocarbons (cfcs) had not been regulated? challenges for the recovery of the ozone layer status of the world's baleen whales ecosystem modelling to quantify the impact of historical whaling on southern hemisphere baleen whales assessing the recovery of an antarctic predator from historical exploitation global opportunities and challenges for transboundary conservation yellowstone to yukon: transborder conservation across a vast international landscape factors influencing community participation in wildlife conservation human-elephant conflict: a review of current management strategies and future directions why did the elephant cross the road? the complex response of wild elephants to a major road in peninsular malaysia predicting landscape connectivity for the asian elephant in its largest remaining subpopulation protecting half of the planet could directly affect over one billion people usa. we would like to thank three anonymous reviewers and andrea stephens for very helpful comments on a previous version of our paper. key: cord-002474-2l31d7ew authors: lv, yang; hu, guangyao; wang, chunyang; yuan, wenjie; wei, shanshan; gao, jiaoqi; wang, boyuan; song, fangchao title: actual measurement, hygrothermal response experiment and growth prediction analysis of microbial contamination of central air conditioning system in dalian, china date: 2017-04-03 journal: sci rep doi: 10.1038/srep44190 sha: doc_id: 2474 cord_uid: 2l31d7ew the microbial contamination of central air conditioning system is one of the important factors that affect the indoor air quality. actual measurement and analysis were carried out on microbial contamination in central air conditioning system at a venue in dalian, china. illumina miseq method was used and three fungal samples of two units were analysed by high throughput sequencing. results showed that the predominant fungus in air conditioning unit a and b were candida spp. and cladosporium spp., and two fungus were further used in the hygrothermal response experiment. based on the data of cladosporium in hygrothermal response experiment, this paper used the logistic equation and the gompertz equation to fit the growth predictive model of cladosporium genera in different temperature and relative humidity conditions, and the square root model was fitted based on the two environmental factors. in addition, the models were carried on the analysis to verify the accuracy and feasibility of the established model equation. with the large-scale use of central air conditioning system and the improvement of people's living standard, more and more attention has been paid to the increasingly serious problem of indoor air pollution. studies showed that air handing unit is an important source of microorganisms for indoor biological pollution, and some microorganisms tend to stay in the dust of air conditioning units with the appropriate temperature and humidity environment. the microorganisms grow and then enter the indoor space through the air, resulting in the destruction of indoor air quality [1] [2] [3] [4] [5] [6] [7] [8] . national institute for occupational safety and health (niosh) conducted a study of 529 buildings, and the research results showed that among the sources of indoor air pollution, central air-conditioning system pollution accounted for 53% 9 . summary of measurement by professor fanger showed that air-conditioning systems accounted for 42% in the indoor pollution sources 10 . based on the supervision and inspection of air conditioning and ventilation system in the public places of china's 30 provinces, municipalities and autonomous regions by china academy of building research and the chinese center for disease control and prevention, it was found that more than 90% of the central air conditioning systems could not meet china's national sanitary standard 11 . thus, air conditioning system should eliminate negative impact caused by its own, on this basis, it may relate to positive effect of the ventilation. in recent years, h1n1, sars and other popular virus spread [12] [13] , and some researches showed that the hygienic, reasonable air conditioning systems were important to reduce damage [14] [15] . therefore, microbial pollution in central air conditioning system has become a critical topic in the field of indoor air pollution. studies showed that the filter, cooling coil, humidifier, and cooling tower in central air-conditioning system were easy to microbial breeding [16] [17] [18] [19] [20] [21] . in this study, a venue in dalian was selected as the research object. as the working condition of the air conditioning system was down, the environment parameters were measured, and microorganisms existing on the wind pipe, filtering net, surface cooler, and condensate water, on the floor and in the air were collected. besides, according to the tested microbial density and the identified genome sequence of collected microorganisms, the hygrothermal response experiment of dominant fungal was detected, and the fitting analysis was carried out based on the prediction model, followed by a series of statistical analysis. the aim of the present study was to clarify characteristics of the microorganisms in air conditioning systems, and the study would be helpful for policymakers and hvac engineers to develop the appropriate strategies and to conduct the bacteria and fungi characteristic assessments in hvac system. preliminary survey. the object of study is a venue in dalian, which covers a total area of 36400 m 2 and building area is 17320 m 2 . the aboveground part includes a swimming pool, ball training venues, gymnasium, the lounge room and the clinic. the underground part consists of a table tennis hall, air conditioning equipment rooms and reservoir area. the whole building is centralized-controlled by the central air conditioning room, which includes two air handling units. two measured units were all air system, which only had a coarse efficiency filter, and the unit is also provided with a heater, cooler and fan etc. both units are the primary return air system and the filters are removable types. the running time of the air conditioning system is from may to october, and the daily operation period is 08:00-21:00. all components are cleaned every two years. when the measurement was carried on, the unit a and b were both cleaned a year ago. both units were closed during the sample collection. measurement method. the actual measurement is divided into two parts: the environment parameter measurement and air unit sampling. first, the temperature, humidity, and co 2 concentration were automatically recorded by the temperature, humidity and co 2 monitor (mch-383sd, japan). second, the disinfected planktonic microorganism sampler (hkm series dw-20, china) was installed where fresh air and return air mixed. once installing the sampler, we loaded medium in the sampler and set the parameter of air flow in sampler as 2000 l loaded medium. after the sample collection, the petri dishes must be sealed for preservation. finally, according to the hygienic specification of central air conditioning ventilation system in public buildings of china 22 , we sampled the dust by using sterile non-woven (100 mm*100 mm) on components of unit a and b, respectively, and each sampling point covered a 15 cm*15 cm area at the sampling area. the non-woven fabrics were put into sterile water and stirred to make sure that organic substance on the non-woven was fully dissolved in sterile water. then, the samples of sterile water containing organic substances were prepared in 10 times and 100 times diluted concentration, respectively. there are 10 sampling points in unit a and 5 points in b, and two measuring point positions of the units are shown in fig. 1 . the microorganisms collected in the air were directly cultured, and the samples in the dust were 10 times and 100 times diluted and 100 μ l of the sample was inoculated into the two kinds of solid culture media. beef extract peptone medium was used for cultivating bacteria and potato dextrose agar was used for cultivating fungus 22, 23 . each dilution was done in 3 parallel samples to reduce the error, and final results showed the average value. the blank samples and test samples were set up for each of the cultures. if there is no colony detected on the blank sample, the test results are valid. both field test and laboratory measurements were performed in accordance with the hygienic specification of central air conditioning ventilation system in public buildings requirements 22 . genome sequencing. only a small part of microorganisms are cultivable. therefore, the traditional cultivation method can not harvest all the species in ecological samples 24 . fungal genome sequencing is an emerging method to identify the microbial genome, which could directly indicate related species information from environment samples 25 . fungal amplicon sequencing analysis was used in this study, because the existing research showed that fungal spores have stronger vitality than other microorganisms in the air, and fungi dominated the microorganism in air conditioning systems. therefore, this method was mainly used to identify fungi in this study 3,17-28 . environment parameters in air handling units. temperature, humidity and co 2 concentration of unit a and b are shown in table 1 . unit a is located in the ground floor (b1), and the unit b is located on the ground floor. compared to the unit b, the humidity of unit a is higher, and the temperature is lower. microbial colony analysis. the distribution density of bacteria and fungi in the unit a is obtained through statistics, as shown in fig. 2 . the concentration of airborne fungus was 44 cfu/m 3 , and the concentration of airborne bacteria was 16 cfu/m 3 . the unit a showed the obvious microbial contamination status, though all components and airborne microorganism meet the hygienic specification of central air conditioning ventilation system in public buildings of china 22 . the microbial distribution in filter net is central < edge < bottom and bacteria accounted for a larger proportion; the microbial distribution in surface cooler is center > against the wall > edge, and fungi accounted for a large. the fungal contamination in the air is more serious than the bacteria. the distribution density of bacteria and fungi in the unit b were obtained through statistics, as shown in fig. 3 . the concentration of airborne fungus was 22 cfu/m 3 , and the concentration of airborne bacteria was 80 cfu/m 3 . parts of the measuring point in the unit b were polluted seriously. the bacterial colonies in the corner and the ground of the surface cooler were beyond the hygienic index (≤ 100 cfu/cm 2 ) in the hygienic specification of central air conditioning ventilation system in public buildings of china regulates 22 . limited by unit placement, there were less measuring points in unit b, and we chose the same measuring points in both units for comparison (centre of surface cooler, surface cooler against wall, corner of surface, and ground of surface cooler). the comparison between unit a and b indicates that the bacterial density in unit a was less than that in the same sampling point in unit b, but the fungal density in unit a was more than that in the same sampling point in unit b. if the cleaning and disinfection is not enough before the air conditioning system running, it may make the fungus to enter the indoor environment, which results in make the pollution of indoor air. compared with cooling coil, the fungus contamination is worse in the floor dust and the air suspension. during the actual measurement, it is found that the unit internal is unprecedentedly narrow and low intensity of illumination in a closed state. according to the description by technicians, it is easy to trample damage to the underground pipes, which leads to the disinfection and cleaning work rarely in the unit. fungal genome sequencing analysis. in this study, we analysed the samples from the sampling points a1, b1, and b2 by amplicon sequencing information analysis, respectively named a1a, b1a, and b2a. all collected samples in the air conditioner were transferred to the eppendorf tubes and processed with the extraction step. samples were resusponded in tens buffer with sds and proteinase k as described by vinod 29 . after incubation at 55 °c, phenol/chloroform/isoamyl alcohol was added to remove proteins, and the nucleic acid was precipitated with isopropanol and sodium acetate (0.3 m). total dna was dissolved in 1 × te after washing with 75% ethanol. and then the quality and quantity tests were conducted by agarose gel electrophoresis, . for pcr product, the jagged ends of dna fragment would be converted into blunt ends by using t4 dna polymerase, klenow fragment and t4 polynucleotide kinase. then add an ' a' base to each 3' end to make it easier to add adapters. after all that, fragments too short would be removed by ampure beads. for genomics dna, we use fusion primer with dual index and adapters for pcr, fragments too short would be removed by ampure beads too. in both cases, only the qualified library can be used for sequencing. the quality and quantity of libraries were assessed using the 2130 bioanaylzer (agilent technologies) and the steponeplus real-time pcr system (applied biosystems). the raw data generated by miseq and hiseq 2000 sequencers was processed to eliminate the adapter pollution and low quality to obtain clean reads. the qualified clean data was used for the further bioinformatics analysis. firstly, paired-end reads with overlap were merged to tags by software flash (v1.2.11) 30 , and then tags were clustered to otu at 97% sequence similarity using usearch (v7.0.1090) 31 . secondly, taxonomic ranks were assigned to otu representative sequence using ribosomal database project (rdp) na, e bayesian classifier v.2.2 32 . at last, alpha diversity, beta diversity and the different species screening were analyzed based on otu and taxonomic ranks by mothur (v1.31.2) 33 . in order to fully understand the community structure of fungal sample and analyse fungus microbial diversity, while excluding errors that human operation brings, genome sequencing method in fields of molecular biology was employed in this study to obtain micro biological information. illumina company developed miseq method with higher flux and simple operation and lower cost for genome sequencing. besides, the synthesis of side edge sequencing method with higher reliability is more suitable for laboratory community structure. the high-throughput sequencing was found to be useful to characterize compositions and diversities of moulds. the gene sequence of the test samples from genome sequencing was dealed with, such as stitching and matching, and the sample had a total of 59309 high quality fungal sequences, with an average length of 219 bp. the optimized sequence and the average length of the sample are shown in table 2 . otu and abundance analysis. stitching and optimising the tags, in order to be the otu (operational taxonomic units) for species classification, gathering in the 97% similarity, and the statistics of each sample in the abundance of information in each otu were done 31, [34] [35] . rank otu curve is a form of species diversity in the sample, which can explain two aspects of sample diversity, that is, the richness and evenness of species in the sample. the richness of species in the samples represented by the horizontal length of the curve is wide, so that the sample species is more abundant. the uniformity of species in the samples from the curve reflects the longitudinal axis of the shape. that the curve is flat means that the sample has a higher composition of the species evenness. from fig. 4 , the species composition of b2a is the most abundant, and the uniformity is the highest. sample diversity analysis of observed species. alpha diversity is the analysis of species diversity in a single sample 33 , including the species observed index, shannon index, etc. the greater the two indices are, the more abundant species is in the sample. the species observed index reflects the richness of the community in the sample, which also refers to the number of species in the community, without taking into account the abundance of each species in the community. shannon index reflects the diversity of the community, and the species richness and evenness of species in the sample community. in the case of the same species richness, the greater the evenness of the species is in the community, the greater the diversity of the community is. observed species exponential dilution curve. random sample in the processed sequence, draw the sequence number for the abscissa and the corresponding species number can be detected as the ordinate, so as to form a curve production dilution curve, shown in fig. 5(a) . with the increase of the sample quantity, the number of species increase and gradually become stabilized. when the curve reaches the plateau, it can be considered that the depth of the sequencing has been basically covered all the species in the sample. at the same time, the observed species index can reflect the richness of community in the sample, that is, the number of species in the community. it can be seen that the distribution of fungal species richness is b2a > b1a > a1a. shannon exponential dilution curve. shannon index is affected not only by species richness in the sample community, but also by the evenness of species. in the case of the same species richness, the greater the evenness of the species is in the community, the more abundant diversity of the community is. it can be seen in the fig. 5 (b) that the fungal species diversity of the unit b is significantly more complex than the unit a, and the similarity of species diversity in two sampling points of unit b was very high. composition of microbial samples. figure 6 illustrates the species composition proportion of the three sampling points, and the proportion was redrew by removing the strains which were not detected in the sample. the results are shown in table 3 . the species with the largest proportion is the dominant fungi. according to the fungal genome sequencing analysis results, fungal components in different units at the same sampling were different, and that in the same unit at different sampling points were roughly similar. they were caused by the different environmental conditions. on the center of air cooling coil in unit a, candida accounted for 80%; on the center and against the wall of the air cooling coil in unit b, cladosporium accounted for 50%, accompanied by alternaria, emericella and other fungus. cladosporium is usually rich in outdoor air, but they will also grow on the indoor surfaces when the humidity is high. existing research shows that the cladosporium spore is an extremely important allergen in the airborne transmission, which could cause asthma attacks or similar respiratory disease in patients with allergic reactions 36 . some species of candida is a kind of conditional pathogenic fungi in the human body. growth prediction analysis of models. traditional microbial detection generally have the characteristics of hysteresis, which cannot play the role of prediction, but the use of mathematical models to predict the growth of microorganisms can timely and effectively predict the growth of microorganisms. therefore, it is very important to study the growth prediction model of the fungi in the air conditioning system. according to environmental conditions mentioned before, we established growth kinetics prediction model of cladosporium spp. to predict the rapid fungal growth in the experimental conditions, which can provide a theoretical basis for air microbial contamination prediction system and help evaluate the health risk inside buildings. the models were fitted by origin software (version 8) and matlab r2014a, and the fitting conditions of logistic model and gompertz model were compared under different temperature and humidity conditions. the fitting effect between these two models and the fitting results of the two models were compared, and the corresponding model parameters were obtained. in addition, the square root model was fitted based on the two environmental factors. experimental study on the hygrothermal response of fungus. laboratory studies have revealed that fungal growth and reproduction are affected by water, temperature, nutrients, micro-elements, ph, light, carbon dioxide, and oxygen tension 37 .the most relevant determinants of fungal proliferation in the building context are water/moisture and temperature, and to a certain extent those factors affect other environmental factors such as substrate ph, osmolarity, nutrient, material properties etc 37, 38 .in order to lay the foundation for the fitting model, and to study the growth characteristics of fungi in different temperature and relative humidity, we set an experimental study on the hygrothermal response of fungus. from the results of fungal genome sequencing and literature research 39-41 , we selected cladosporium spp. and penicillium spp. as the research objects which are both common in air conditioning systems.this paper mainly studied the status of microbial contamination in air handling units so that the air temperature of each part of the air handling unit should be considered. the temperature gradient of 20 °c − 25 °c − 30 °c and relative humidity gradient of 45%− 60%− 75%− 80% were selected as experimental hygrothermal conditions. the results of hygrothermal experiments are shown in figs 7, 8, 9. it can be known that growth rate of cladosporium spp. is faster than that of penicillium spp., in any experimental conditions, which is the essential characteristics of a strain, is hygrothermal response control method cannot change. these data indicated that low rh environments can reduce or even inhibit fungal growth. this observation agrees with findings by w. tang and pasanen 42,43 . growth prediction analysis based on logistic model. logistic model is a typical model of biological ecology, which has been widely used in the field of biological ecology 44 . according to the actual research, the following formula equation (1) was obtained after the appropriate change of the logistic equation. n was the colony growth diameter, cm; t was the microbial growth culture time, h; a 1 , a 2 , x 0 , p as the model parameters. it can be seen from the table 4 , the fitting curve of logistic model is similar to the experimental results. at 20 °c and 30 °c temperature conditions, the model's fitting effect is excellent, and r 2 is greater than 0.99; at 25 °c temperature conditions, the model fitting effect is not as good as other temperature conditions. predicting the growth of microorganisms. the pmp program developed by the ministry of agriculture to establish the model of pathogenic bacteria is the basic model for the study of gompertz equation. gompertz model has been widely used in the field of biology. gompertz model expression was as equation (2): c n was the colony growth diameter, cm; t was the microbial growth culture time, h; a, a, k, x c as the model parameters. it can be seen from the table 5 that the fitting curve of gompertz model is better fitted to the measured parameters. at the same temperature, with the increase of relative humidity, gompertz model fitting effect is better; the model is well fitted at the temperature of 25 °c and the fitting effect is better than 20 °c and 30 °c temperature conditions. the fitting of logistic model to the growth of the fungus is better than that of the gompertz model. the two models are tested by the deviation factor b f and the accuracy factor a f in the mathematical model test. staphylococcus xylosus were studied by mcmeekin 45 . they found that when t min is fixed, for each ϕ , the relationship between growth rate and temperature can be described by using the square root model. the combined effects of these two variables can be expressed by the modified equation (3): in the formula, u is the growth rate of fungus, cm/h; b 2 is the coefficient; t is the culture temperature, °c; t min is the most important parameter of square root equation, and it refers to the lowest temperature when the growth rate is zero, °c; ϕ is relative humidity of the cultivation, %. by using the logistic primary model, the predictive value of the growth rate of the cladosporium colony growth rate (instantaneous velocity) was obtained, as table 6 shows. through the model fitting, the parameters of the square root model could be obtained, as table 7 shows, and the model fitting of predicting growth of cladosporium was shown as fig. 10 . the model equation of b f value was between 0.90-1.05, indicating that the model used to predict the range of the experimental environment in cladosporium colony growth condition. at the same time, the a f value of the model was 1.05169 that is closed to 1, which shows that the model has high accuracy. table 7 . model fitting and model parameters of double factor square root. this study selected two central air conditioning systems at a venue in dalian as the objects. actual measurement and a series of studies were carried out on microbial pollution characteristic, and the results are shown as below: (1) the bacterial colony forming units of the two measuring points in unit b were 192 cfu/cm 2 and 828 cfu/cm 2 , respectively, which exceeded the hygienic specification of central air conditioning ventilation system in public buildings of china (≤ 100cfu/cm 2 ), and the rest of the test points met the relevant standards of china. the distribution of bacteria was more than fungi, and the concentration was higher. with the total characteristics of different distribution density, the area of dust associated microorganisms and the air pollution were more serious. (2) alternaria spp., candida spp., cercospora spp. and cladosporium spp. existed in both units. the candida spp. accounted for 80% in unit a, and the cladosporium spp. occupied 50% in unit b. the composition of fungi in b was more complicated. two dominant fungi are both deleterious to health, so the timely maintenance and cleaning are required. it is suggested that the operating space should be reserved in the air conditioning room, so as to avoid incomplete cleaning and disinfection. (3) within the experimental temperature and relative humidity, with the increase of relative humidity or temperature, the colony growth of the same strain showed an increasing trend. for the prediction model of the fungus growth, the study found that the overall fitting effect of logistic model is better, and r 2 values were greater than 0.97. logistic model for the cladosporium spp. growth was better than gompertz model. at the same time, considering the influence of temperature and relative humidity, the square root model can well predict the growth of cladosporium spp. it provides a theoretical basis for the growth of fungi in the air conditioning system under the hygrothermal environment conditions. why, when and how do hvac-systems pollute the indoor environment and what to do about it? the european airless project the control technology of microbial contamination in air conditioning system overview of biological pollution prevention methods in air conditioning system. heating ventilating and air conditioning relationships between air conditioning, airborne microorganisms and health. bulletin de l'academie nationale de medecine a review of the biological pollution characteristics, standards and prevention and control technologies of central air-conditioning system risks of unsatisfactory airborne bacteria level in air-conditioned offices of subtropical climates development of a method for bacteria and virus recovery from heating, ventilation, and air conditioning (hvac) filters microbial air quality at szczawnica sanatorium the national institute for occupational safety and health indoor environmental evaluation experience air pollution sources in offices and assembly halls quantified by the olf unit control and improvement of building indoor biological pollution a major outbreak of severe acute respiratory syndrome in hong kong epidemiological investigation of an outbreak of pandemic influenza a (h1n1) 2009 in a boarding school: serological analysis of 1570 cases a study on the effective removal method of microbial contaminants in building according to bioviolence agents performance analysis of a direct expansion air dehumidification system combined with membrane-based total heat recovery hvac systems as emission sources affecting indoor air quality: a critical review testing and analysis for microbes and particles in central air conditioning systems of public buildings. heating ventilating and air conditioning endotoxins and bacteria in the humidifier water of air conditioning systems for office rooms investigation and review of microbial pollution in air conditioning systems of public buildings. heating ventilating and air conditioning study on the microbial secondary pollution and control in air conditioning system research on the dust deposition in ventilation and air conditioning pipe. heating ventilating and air conditioning hygienic specification of central air conditioning ventilation system in public buildings indoor air part17: detection and enumeration of moulds-culture-based method macro genomics approach in environmental microbial ecology and genetic search application study of the microbiome in waiting rooms of a japanese hospital fungal colonization of automobile air conditioning systems changes in airborne fungi from the outdoors to indoor air; large hvac systems in nonproblem buildings in two different climates research on the microbial diversity analysis in the sediment and macro genomic library at the south pole, xiamen university isolation of vibrio harveyi bacteriophage with a potential for biocontrol of luminous vibriosis in hatchery environments fast length adjustment of short reads to improve genome assemblies highly accurate otu sequences from microbial amplicon reads ribosomal database project: data and tools for high throughput rrna analysis introducing mothur: open-source, platform-independent, community-supported software for describing and comparing microbial communities the silva ribosomal rna gene database project: improved data processing and web-based tools naive bayesian classifier for rapid assignment of rrna sequences into the new bacterial taxonomy study on the relationship between dominant fungi in the air and the allergic asthma in children field guide for the determination of biological contaminants in environmental samples separate effects of moisture content and water activity on the hyphal extension of penicillium rubens on porous media investigation and review of microbial pollution in air conditioning systems of public buildings. heating ventilating and air conditioning microorganisms and particles in ahu systems: measurement and analysis the indoor fungus cladosporium halotolerans survives humidity dynamics markedly better than aspergillus niger and penicillium rubens despite less growth at lowered steady-state water activity effects of temperature, humidity and air flow on fungal growth rate on loaded ventilation filters fungal growth and survival in building materials under fluctuating moisture and temperature conditions mathematical modeling of growth of salmonella in raw ground beef under isothermal conditions from 10 to 45 °c model for combined effect of temperature and salt concentration/water activity on the growth rate of staphylococcus xylosus the study is supported by the national nature science foundation of china (51308088), beijing key lab of heating, gas supply, ventilating and air conditioning engineering (nr2013k05), the fundamental research funds for the central universities (dut14qy24) and the urban and rural housing construction science and technology plan project (2014-k6-012). key: cord-027120-w6agcu63 authors: lago, andré sousa; dias, joão pedro; ferreira, hugo sereno title: conversational interface for managing non-trivial internet-of-things systems date: 2020-05-25 journal: computational science iccs 2020 doi: 10.1007/978-3-030-50426-7_29 sha: doc_id: 27120 cord_uid: w6agcu63 internet-of-things has reshaped the way people interact with their surroundings. in a smart home, controlling the lights is as simple as speaking to a conversational assistant since everything is now internet-connected. but despite their pervasiveness, most of the existent iot systems provide limited out-of-the-box customization capabilities. several solutions try to attain this issue leveraging end-user programming features that allow users to define rules to their systems, at the cost of discarding the easiness of voice interaction. however, as the number of devices increases, along with the number of household members, the complexity of managing such systems becomes a problem, including finding out why something has happened. in this work we present jarvis, a conversational interface to manage iot systems that attempts to address these issues by allowing users to specify time-based rules, use contextual awareness for more natural interactions, provide event management and support causality queries. a proof-of-concept was used to carry out a quasi-experiment with non-technical participants that provides evidence that such approach is intuitive enough to be used by common end-users. the internet of things (iot) is usually defined as the networked connection of everyday objects with actuating and sensing capabilities, often equipped with a collective sense of intelligence [21] . the integration of such objects creates a vast array of distributed systems that can interact with both the environment and the human beings around them, in a lot of different ways [21] . this flexibility of iot systems has enabled their use across many different product areas and markets, including smart homes, smart cities, healthcare, transportation, retail, wearables, agriculture and industry [17] . still, one of the most visible application of iot are customized smart spaces, such as smart homes as the current technology make it possible for consumers to create a customized iot experience based on off-the-shelf products [16] . the initial popularity of devices such as single-board computers and low-cost micro-controllers, followed by widespread cloud-based solutions controlled by mobile phones, it is now commonplace to remotely interact with a myriad of devices to perform automated tasks such as turning the lights on and opening the garage door just before one arrives home [16, 22] . but as the number of devices and interactions grows, so does the management needs of the system as a whole, as it becomes essential to understand and modify the way they (co)operate. in the literature this capability commonly known as end-user programming [6] , and once we discard trained system integrators and developers, two common approaches emerge: visual programming tools and conversational assistants [22] . visual programming solutions are usually deployed as centralized orchestrators, with access to the devices and components that comprise such systems. these platforms range from simplified if-then rules (e.g. ifttt 1 ) to exhaustive graphical interfaces (e.g. node-red 2 ) through which one can visualize, configure and customize the devices and systems' behaviour [7, 19, 20] . most visual approaches attempt to offer integration with third-party components (e.g., google calendar), so that their services can be used as part of the system's behavioural rules. these solutions, however, possess some disadvantages for non-technical endusers. consider a node-red system orchestrating an user's smart home with multiple devices. even in situations where there are only a couple of dozen rules defined, it can be challenging to understand why a specific event took place due to the overwhelming data flow that results from these. furthermore, just one dozen rules can already lead to a system not possible to visualize in a single screen [11] . the more rules one adds, the harder it becomes to conceptually grasp what the system can do. part of the reason is because they are built to be imperative, not informative; current solutions mostly lack in meta-facilities that enable the user or the system to query itself [5] . another common, and sometimes complementary, alternative to visual programming, is the many conversational assistants in the market, such as google assistant, alexa, siri and cortana, that are capable of answering natural language questions and which recently gained the ability to interact with iot devices (see [18] and [15] for a comparison of these tools). amongst the most common features they provide is allowing direct interaction with sensing and actuating devices, which enables the end-user to talk to their light bulbs, thermostats, sound systems, and even third-party services. the problem with these solutions is that they are mostly comprised of simple commands and queries directly to the smart devices (e.g. is the baby monitor on? ", "what is the temperature in the living room? ", or "turn on the coffee machine". these limitations mean that although these assistants do provide a comfortable interaction with devices, a huge gap is easily observable regarding their capabilities on managing a system as a whole and allowing the definition of rules for how these smart spaces oper-ate. even simple rules like "close the windows everyday at 8pm" or "turn on the porch light whenever it rains" are currently not possible, unless one manually defines every single one of them as a capability via a non-conversational mechanism. furthermore, most assistant are deliberately locked to specific vendor devices, thus limiting the overall experience and integration. one can conclude that although current smart assistants can be beneficial and comfortable to use, they do not yet have the complexity and completeness that other systems like node-red. meanwhile, visual programming environments are still far too technical for the common end user. in this paper, we propose a system that tackles the problem of managing iot systems in a conversational approach, towards shortening the existing feature gap between assistants and visual programming environments. parts of this work are from [13] master's thesis. the rest of this document is structured as follows: sect. 2 provides a summary of related works which identify open research challenges; in sect. 3 we propose our approach to support complex queries in conversational assistants, which implementation details are further presented in sect. 4; we proceed in sect. 5 to evaluate our approach using simulated scenarios and experimental studies. finally, sect. 6 drafts some closing remarks. there exists some work in this area that recognize the problem of controlling and managing iot infrastructures by an end-user via a several approaches. kodali et al. [12] present an home automation system to "increase the comfort and quality of life", by developing an android app that is able to control and monitor home appliances using mqtt, node-red, ifttt, mongoose os and google assistant. their limitations lie in that the flows must first have been first created in node-red, and the conversational interface is used just to trigger them, ignoring all the management activities. austerjost et al. [3] recognized the usefulness of voice assistants in home automation and developed a system that targets laboratories. possible applications reported in their paper include stepwise reading of standard operating procedures and recipes, recitation of chemical substance or reaction parameters to a control, and readout of laboratory devices and sensors. as with the other works presented, their voice user interface only allows controlling devices and reading out specific device data. he et al. [9] , concludes that, even with conversational assistants, most of iot systems have usability issues when faced with complex situations. as example, the complexity of managing devices schedules rises with the number of devices and the common conflicting preferences of household members. nonetheless, as concluded by ammari et al. [2] , controlling iot devices is one of the most common uses of such assistants. agadakos et al. [1] focus on the challenge of understanding the causes and effects of an action to infer a potential sequence. their work is based on a mapping the iot system' devices and potential interactions, measuring expected behaviours with traffic analysis and side-channel information (e.g. power) and detecting causality by matching the mapping with the collected operational data. this approach would potentially allow the end user to ask why is something happening, at the cost of modified hardware and a convoluted side-channel analysis. they did not attempted to port their findings into a conversational approach. braines et al. [4] present an approach based on controlled natural language (cnl) -natural language using only a restricted set of grammar rules and vocabulary -to control a smart home. their solution supports (1) direct question/answer exchanges, (2) questions that require a rationale as response such as "why is the room cold?" and (3) explicit requests to change a particular state. the most novel part of their solution is in trying to answer questions that require a rational response, however they depend on a pre-defined smart home model that maps all the possible causes to effects. from the above analysis, the authors were not able to found any solution that would simultaneously provide: (1) a non-trivial management of an iot system, (2) be comfortable and easy to use by a non-technical audience, and (3) allow the user to better understand how the system is functioning. by non-trivial we mean that it should be possible to define new rules and modify them via a conversational approach, achieving a de facto integration of multiple devices; not just directly interacting with its basic capabilities. the comfort would be for the user not to have to move or touch a device to get his tasks done (i.e. using voice), or edit a node-red visual flow. as to understanding their system's functioning, we mean the ability to grasp how and why something is happening in their smart space. this last point, combined with the other two, would ideally allow someone to simply ask why something happens. we propose the development of a conversational bot dedicated to the management of iot systems that is capable of defining and managing complex system rules. our prototype is called jarvis, and is available as a reproducible package [14] . jarvis's abilities reach across different levels of operational complexity, ranging from direct one-time actions (e.g. turn on the light) to repeating conditional actions (e.g. when it is raining, close the windows). jarvis also lets the user easily understand and modify the rules and cooperation of multiple devices in the system, through queries like why did the toaster turn on? in these cases, we incorporated jarvis with conversational awareness to allow for chained commands; the following dialogue exemplifies this particular feature: user: "why did the toaster turn on? " jarvis: "you told me to turn it on at 8 am." user: "okay, change it to 7:50 am." jarvis: "sure, toaster timer was changed." ... the reader would note that the second user's query would not make sense on its own. we believe that such features improve the user's experience since it avoids repeating information that has already been mentioned in the conversation, and presents a more natural (conversational) interaction. to ease the integration with nowadays systems and provide us an experimental reproducible environment, we integrated the interface with existing platforms such as google assistant 3 and slack 4 , amongst others. we made sure to provide the ability for jarvis to interact both via voice and text. to develop the conversational interface, we decided to opt for dialogflow 5 as this platform provides built-in integration with multiple popular frontends and there exists extensive documentation for this purpose [10] . in this case, we used (1) the slack team-communication tool, and (2) google assistant, so that both text and voice interfaces were covered. in the case of google assistant, the user may use any supported device paired with their account to communicate with jarvis, following a known query prefix such as "hey google, talk to jarvis". regardless of which type of interface is used, the result is converted to strings representing the exact user query and subsequently sent to dialogflow's backend (thus overcoming potential challenges due to speech recognition), which are then analyzed using natural language processing (nlp) techniques. advancement of the existent nlp techniques made available by dialogflow falls out-of-the-scope of this work. upon receiving a request, dialogflow can either produce an automatic response or send the parsed request to a fulfillment backend. this component is thus responsible for parsing the incoming strings into a machine understandable format (json). there are a few key concepts that are leveraged in our implementation: entity. things that exist in a specific iot ecosystem can be represented by different literal strings; for example, an entity identified by toggleable-device may be represented by "living room light" or "kitchen light". additionally, entities may be represented by other entities. dialogflow use of the @ symbol (i.e. @device) for entities, and provides some system's defaults; intent. an intent represents certain type of user interaction. for instance, an intent named turn on/off device may be represented by turn the @device on and turn the @device off. for a request such as "turn the kitchen light on", dialogflow understands that @device corresponds to kitchen light and provides that data to the fulfillment backend; context. contexts allow intents to depend on previous requests, enabling the creation of context-aware interactions. these are what supports queries such as "cancel that" or "change it to 8am". multiple intents, entities and contexts were defined in jarvis and the main ones are illustrated in fig. 2 . here we provide in detail one of its intents: usage creates an action that is performed upon a certain event, such as an activity of another device or a change of a device's status. definition @action:action when @event:event example turn the bedroom light on when the living room light turns off with the above definitions, this component takes requests and builds the corresponding objects containing all actionable information to be sent to the jarvis backend for further processing. for each of the defined intents, this component has an equivalent class responsible for (a) parsing the request, (b) validating its request parameters (e.g. device name or desired action), and (c) generating an appropriate response. an overview is provided in fig. 3 . should the request contain errors, an explanatory response is returned. when all the parameters are considered valid, but the intended device is unclear (e.g. user wants to turn on the light, but there is more than one light), the generated response specifically asks the user for further clarification in order to gain context. to tackle cancellation intents, we model all actions using the command design pattern, thus providing both a straightforward execute and undo mechanism, as well as an history of performed actions. for most intents, such as direct actions or "why did something happen?" queries, the effects are immediate. however, period actions, events and causality queries require a different design approach so that they can perform actions on the backend without the need for a request to trigger them. a period action is an intent be carried and then undone after a certain period (e.g. "turn on the light from 4 pm to 5 pm"). in these scenarios, we generate a state machine to differentiate between all the different action status, such as (a) nothing has executed yet (before 4pm), (b) only the first action was executed (after 4 but before 5pm), and (c) both have been executed (after 5pm). we use a combination of schedulers and threads to guarantee proper action, and abstract all these details inside the command pattern. the same strategy applies for rules such as "turn on the light every day at 5 pm", with the appropriate state machine and scheduler modifications. this mechanism is (obviously) different for events that are the result of intentions such as "turn on the kitchen light when the presence sensor is activated". here, we leverage a publish-subscribe approach by orchestrating multiple unique and identifiable message queues for the devices' actions and state transitions. upon startup of the backend, we create class listeners that subscribe the corresponding event queues of available devices, and callback the jarvis backend when appropriate. this orchestration management is dynamic, and depends on the specific rules that are defined. in the aforementioned intent, we would add an observer to look for messages on the presence sensor's event queue with the value on. causality queries. this relate to the user asking why something happened (e.g. "why did the light turn on? "). to implement them, we augment each command to determine whether it can cause a specific condition to be true. this per se solves some scenarios where the answer can be found by looking at the history of executed commands (e.g. "because you asked me to turn it on at 3:56pm"). however, there might exist multiple rules may have cause the condition to be true, in which case it is not enough to blame the latest logged command. instead, there are two possible approaches: (a) return the earliest possible cause, or (b) use a heuristic to infer the most relevant one. another non-trivial scenario is where the explanation is due to a chain of interconnected rules. here, it seems that one can (a) reply with the complete chain of events, (b) reply with the latest possible cause, or (c) engage in a conversation through which the user can explore the full chain of events as they deem adequate (e.g. "tell me more about things that are triggered by rain"). in this work, we opted to use the earliest possible cause for the first scenario, and the latest for the second; more complex alternatives can be found in [1, 4] . in order to understand how jarvis compares to other systems, we established a baseline based on (1) a visual programming language, and (2) a conversational interface. node-red was picked amongst the available visual programming solution, as it is one of the most popular visual programming solutions [8] . it follows a flow-based programming paradigm, providing its users with a web-based application through which they can manage rules via connections between nodes that represent devices, events and actions [20] . google assistant was selected for the conversational interface due to its naturality 6 . there are plenty of ways users can interact with it: (a) the standalone google apps, (b) built-in integration with android and chrome os, or (c) with standalone hardware such as the google home. we compare to this baseline according to two criteria: (1) the number of different features, and (2) their user experience in terms of easiness of usage and intuitiveness. for the first, we created a list of simulated scenarios to assess the ability to manage iot systems. we then performed a (quasi-)controlled experiment with users to assess the second criteria. table 1 summarizes the comparison of our prototype to the chosen baseline. it is important to clarify that one-time action w/ unclear device refers to actions like "turn on the light" with which jarvis asks the user to clarify which device he means based through responses such as "do you mean the bedroom or living room light?". a cancel last command refers to the ability of undoing the last action or rule creation by specifically saying that. finally, rules defined for device refers to the user performing queries that require introspection, such as "what rules are defined for the bedroom light?" it is easy to observe that our prototype provides several features that are not present in either the google assistant or node-red. both of these products do a lot more than these features, but especially with the assistant, the advantage is clear since the only kind of iot feature it supports is the one-time action. our second conclusion is that it is possible to bring some of the features currently available in visual programming environments to a conversational interface; the converse (how to bring conversational features to node-red), eludes the authors. we performed a (quasi-)controlled experiment with 17 participants, to gain insight into how end users responded to a conversational approach. our sample includes mostly participants without formal technological skills (14) , with ages ranging from 18 to 51. we made sure that (a) all were familiar with basic technologies, though, such as basic usage of smartphones and the internet, and (b) that even non-native english participants had adequate speaking and understanding skills. methodology. each participant was given 5 tasks (1 control task and 4 study tasks) to be performed with the help of jarvis, using google assistant as the system interface. as an example, this was one of the sets of tasks given to participants within a scenario with a living room light, bedroom light and a living room motion sensor : the only instructions given were that they should talk to the phone in a way that feels the most natural to them to complete the task at hand. besides the tasks, participants were also given the list of iot devices available in the simulated smart house that they would be attempting to manage through. as a way of increasing the diversity and reducing the bias of the study, we created two different sets of tasks that participants were assigned to randomly. each set also had different devices, with different smart house topologies. the participants were assigned to one of the test sets randomly. variable identification. for each of the tasks, we collected (1) if the participant was able to complete it, (2) the time taken to complete, and (3) the number of unsuccessful queries. this count was made separately for (a) queries that were not understood by the assistant's speech recognition capabilities (e.g. microphone malfunction, background noise), (b) queries where the user missed the intention or made a syntactic/semantic error (e.g. "turn up the lighting"), and (c) valid queries that an human could interpret, but that jarvis was unable to. subjective perception. after completing the tasks, we introduced a nonconversational alternative (node-red), explaining how all tasks could have been performed using that tool. we inquired the participants whether they perceived any advantages of jarvis over such a tool, and whether they would prefer jarvis over non-conversational tools. finally the participants were asked if they had any suggestions to improve jarvis and the way it handles system management. results. table 2 compiles the results observed during the study, each row representing a task given to the participant. each column means: -task: number of the task (0-4) and the task set number in parenthesis (1/2); -done: percentage of participants that completed the task successfully; -time: time in seconds that participants took to complete the task; -iq (ast): number of occurrences that queries were incorrect due to the google assistant not properly recognizing the user's speech; -iq (user): number of occurrences that queries were incorrect due to the user not speaking a valid query; -iq (jvs): number of occurrences that queries were incorrect due to jarvis not recognizing a valid query; -iq: total invalid queries, i.e. sum of iq (ast), iq (user) and iq (jvs). discussion. the complexity of the queries increases from task 0 to task 3 since the queries require more words or interactions. this is reflected by the corresponding increase in time in both task sets. the numbers related to incorrect queries show some occurrences at the (voice) assistant level, which means the speech recognition failed to correctly translate what the participants said. although this does not have implications on the evaluation of jarvis, it does indicate that this sort of systems might be harder to use due if they are not truly multilingual. directly comparing the time needed to complete a task to what would be needed to perform it in a visual programming language is meaningless; either the task is not defined, and that would require orders of magnitude longer than what we observe here, or the task is defined and the times will be obviously similar. similarly, we also observe a few instances of incorrect queries due to grammar mistakes or semantically meaningless, cf. iq (user), and therefore did not match the sample queries defined in dialogflow. nevertheless, there where grammatically incorrect user queries such as "turn on lights" but which still carries enough information to understand what the user's intent is. we consider more serious the number of valid sentences that were considered incorrect queries by jarvis, cf. iq (jvs). these could have been caused by either a mispronunciation of a device's name, or a sentence structure that is unrecognizable by the dialogflow configuration. this possibly represents the most serious threat to our proposal, to which we will later dedicate some thoughts on how to mitigate it. nonetheless, the success rate of all tasks is very high (always greater than 88%), which provides evidence that the system might be intuitive enough to be used without previous instruction or formation. these points were reflected by the participants' subjective perception, were they claimed jarvis to be easy to use, intuitive, and comfortable; ultimately, these would be the deciding factors for end-users to prefer jarvis over a non-conversational interface. an additional observation pertaining jarvis' answers, particularly those regarding causality queries, were state by some users, where they claimed that if the provided response was too long, it would become harder to understand it due to the sheer increase of conveyed information. a possible solution for this problem would be to use a hybrid interface that provides both visual and audio interactions, but there could be other approaches such as an interactive dialogue that shortens the sentences. threats to validity. empirical methods seem to be one of the most appropriate techniques for assessing our approach (as it involves the analysis of humancomputer interaction), but it is not without liabilities that might limit the extent to which we can assess our goals. we identify the following threats: (1) natural language capabilities, where queries like "enable the lights" might not be very common or semantically correct, but it still carries enough information so that a human would understand its intention. the same happen with device identification, such as when the user says turn on the bedroom lights, and the query fails due to the usage of the plural form. during our study, we observed many different valid queries that did not worked due to them not being covered by the dialogflow configuration; (2) coverage error, which refers to the mismatch between the target population and the frame population. in this scenario, our target population was (non-technical) end-users, while the frame population was all users that volunteered to participate; and (3) sampling errors are also possible, given that our sample is a small subset of the target population. repeating the experience would necessarily cover a different sample population, and likely attain different results. we mitigate these threats by providing a reproducible package [14] so other researchers can perform their own validation. in this paper we presented a conversational interface prototype able to carry several different management tasks currently not supported by voice assistants, with capabilities that include: (1) delayed, periodic and repeating actions, enabling users to perform queries such as "turn on the light in 5 min" and "turn on the light every day at 8 am"; (2) the usage of contextual awareness for more natural conversations, allowing interactions that last for multiple sentences and provide a more intuitive conversation, e.g. "what rules do i have defined for the living room light?"; (3) event management, that allows orchestration of multiples devices that might not necessarily know that each other exists, e.g. "turn on the light when the motion sensor is activated"; and (4) causality queries, to better understand how the current system operates, e.g. "why did the light turn on?" we conducted (quasi-)controlled experiments with participants that were asked to perform certain tasks with our system. the overall high success rate shows that the system is intuitive enough to be used by people without significant technological knowledge. it also shows that most challenges lie in the natural language capabilities of the system, as it is hard to predict them any user queries that have the same intrinsic meaning. we thus conclude that incorporating recent nlp advances (that were beyond the scope of this paper) would have an high impact in terms of making it more flexible to the many different ways (correct or incorrect) that users articulate the same intentions. nonetheless, by doing a feature comparison, we can observe that jarvis was able to implement many features that current conversational assistants are lacking, while simultaneously being more user-friendly than the available alternatives to iot management (such as visual programming approaches). as future work, we believe that our approach could be improved by sometimes engaging in a longer (but fragmented) conversation with the user, particularly when providing causality explanations. this would allow the user to understand more information at his own pace, but also because it would enable them to make changes to the rules as the conversation unfolds. butterfly effect: causality from chaos in the iot music, search, and iot: how people (really) use voice assistants introducing a virtual assistant to the lab: a voice user interface for the intuitive control of laboratory instruments conversational homes: a uniform natural language approach for collaboration among humans and devices a reactive and model-based approach for developing internet-of-things systems meta-design: a manifesto for end-user development end-user development fog at the edge: experiences building an edge computing platform when smart devices are stupid: negative experiences using home smart devices hands-on chatbots and conversational ui development: build chatbots and voice user interfaces with chatfuel, dialogflow, microsoft bot framework, twilio, and alexa skills visual dataflow modelling -some thoughts on complexity low cost smart home automation system using smart phone exploring complex event management in smart-spaces through a conversation-based approach andrelago13/jarvis: initial release alexa vs. siri vs. cortana vs. google assistant: a comparison of speech-based natural user interfaces an iot-based user-centric ecosystem for heterogeneous smart home environments from the internet of things to the internet of people conversational interface challenges. developing conversational interfaces for ios from iot mashups to model-based iot a survey on visual programming languages in internet of things internet of things strategies for the integration of smart technologies into buildings and construction assemblies key: cord-314779-f5nvspcg authors: roth, steffen; valentinov, vladislav title: east of nature. accounting for the environments of social sciences date: 2020-06-07 journal: ecol econ doi: 10.1016/j.ecolecon.2020.106734 sha: doc_id: 314779 cord_uid: f5nvspcg at the core of ecological economics is the image of the economy as an open system embedded in the natural environment whose carrying capacity is limited. the present paper revisits this image by drawing upon the constructivist implications of luhmann's social systems theory. to luhmann, the modern society consists of a multitude of social systems each bringing forth and observing their own environments. if the luhmannian vision is accepted, then ecological economics can be said to privilege the observational perspective of natural sciences. the unfortunate consequence of this privileging is the underestimation of a broad range of multidimensional sustainability risks which are foregrounded by the numerous alternative observational perspectives which are just as legitimate. it is argued that, rather than relativizing the sustainability concerns of the modern ecological economics, the luhmannian perspective generalizes and radicalizes them. in doing so, the latter perspective opens new possibilities not only for navigating these risks but also for envisioning new resources and solutions. the understanding of the present-day sustainability challenges, as well as the practice of sustainability accounting, rest on specific assumptions about the relationship of the economy to its outer environment. for many ecological economists, these assumptions are centred around the idea that the economy is an open system embedded in the environment which is usefully classified into societal and natural. karl william kapp (1985, p. 152) , an early contributor to the ecological economics literature, seminally attributed the phenomenon of social costs not only to the corporate decision-making under capitalism, but also to "the open-system character of the economy". this open system character of the economy has many implications, such as materiality and the relevance of multiple time-space scales, each calling for a multidisciplinary approach (luzzati, 2009; luzzati, 2010a luzzati, , 2010b . most importantly for the present purposes, the open systems character that makes the economy susceptible to the limits of the environmental carrying capacity. the vision of the economy as an open system embedded in a finite environment seems to also underlie, e.g., elkington (1997) triple bottom line concept. as elkington explained, "sustainable development involves the simultaneous pursuit of economic prosperity, environmental quality, and social equity. companies aiming for sustainability need to perform not against a single, financial bottom line but against the triple bottom line" (ibid, p. 397) . the logic of the environmental embeddedness of the economy seems straightforward and impeccable. if the environmental carrying capacity is limited, and if systems populating this environment overstrain this carrying capacity, it is logical that they are in trouble (luzzati et al., 2014, p. 100 ). this trouble, or sustainability risks, can be given an alternative formulation suggested by niklas luhmann's theory of social systems. while ecological economics was admittedly not a central concern to luhmann, he devoted a 1989 book to the ongoing ecological crisis, which he took to be a specific illustration of the more general theme of the precariousness of the relations of social systems, of all types, to their outer environments. to luhmann, system-environment relations are precarious because of the fact that the main function of social systems is to reduce, externalize, or blind out the complexity of their environment. while the complexity-reducing function is highly valuable for boundedly rational individuals, it exposes social systems themselves to sustainability risks. thus, luhmann (1989, p. 62) came to the conclusion that "the key to the ecological problems, as far as the economy is concerned, resides in the language of prices… the economy cannot react to disturbances that are not expressed in this language". this argument strikes a chord with much of the ecological economics literature (valentinov, 2014a (valentinov, , 2014b , despite this literature's systems-theoretic focus on open systems (e.g., kapp, 1985; luzzati, 2009 luzzati, , 2010a rather than on the operational closure which is a mainstay of luhmann's thought. yet, what may turn out to be problematic for ecological economics is luhmann's social systems theory. he believed social systems to construct their own environments in the course of their functioning. this means that, apart from this functioning, and apart from the ways systems observe reality, the nature of the environments in which the systems are embedded cannot be specified. against this backdrop, the key research question of the present paper pertains to the clarification of the nature of sustainability risks from the point of view of his social systems theory. luhmann designates the modern society as polycontextural, or, as roth et al. (2019) explain, "constructed by alternative and contingent observational perspectives that may be mutually conflicting and discrepant". from this perspective, a polycontextural society does not have any single, fixed, and definitely known environment. instead, there is a multitude of social systems each bringing forth their own environments. contrast this implication with those of elkington's (1997) triple bottom line concept. elkington rightly assumed that the practice of the traditional bottom line rested on problematic assumptions about the nature of the relations of the economy with its outer environment, both societal and natural. elkington has questioned these assumptions and replaced them by his own, thus substituting one type of certainty for another. the essential luhmannian insight, however, is that in a polycontextural society, such certainty does not exist at all. instead, there is a multitude of social systems each bringing forth their own environments. consequently, if the focus on any type of the environment happens to be dominant or privileged, the role of the other possible and equally legitimate types of environments, and the sustainability risks associated therewith, most likely will be underestimated. insofar as ecological economists can be said to privilege a certain observational perspective on the natural and societal environment, their professional knowledge, profound as it is, may yet turn out to be dangerously one-sided. the positive way of formulating the above contention is that the polycontextural regime of the modern society provides a space for a radical amplification and multiplication of sustainability concerns as the primary area of interest of many ecological economists. to make that case, the following sections reconsider the concept of nature underlying the ecological economics scholarship, contrast the notions of the environments embraced by the natural and social sciences, and on that basis, reconceptualize the triple bottom line approach in the emerging multienvironmental context. this reconceptualization opens new possibilities and envisions new resources for navigating sustainability risks. the paper concludes with a discussion of how these new possibilities and resources can be harnessed by strategic management tools. as a transdisciplinary field of study, "ecological economics addresses the relationships between ecosystems and economic systems in the broadest sense." (costanza, 1989, p . 1) while the latter focus has early been complemented by or extended to a social systems perspective, the primary goal of ecological economics (ee) has always remained the "sustainable wellbeing of both humans and the rest of nature" (costanza, 2020, p. 1) ; and in the pursuit of this worthy goal, the transdiscipline has consequently developed a high level of facility in incorporating and combining insights from economics, further social sciences, and the natural sciences. yet, the implication that economics is a social science may also be contested for an economics that accentuates the word ecological and aims to "recast economics as a life science" (røpke, 2004, p. 300; van passel, 2007, p. 31; costanza, 2020, p. 1) . there is hence a considerable tension between this organic view of economics and the increasingly popular demands for an advancement of its social sciences stream (spash, 2011 (spash, , 2012 , as it seems hard to impossible to reconciliate those who "remain wary (…) that the basis of human social relations can be reduced to, and essentialized within, the natural world" (hird, 2010, p. 738) with those convinced that "(d) etermining what is necessary for safeguarding environmental functions for future generations is a matter for the natural sciences." (hueting and reijnders, 2004, p. 255) . in fact, the contradiction could hardly be more blatant: on the one hand, proponents of the natural sciences camp suggest (ruth, 2006) and are criticised (van hecken et al., 2015) for building economic laws, social institutions, and ethical standards on natural scientific insights. on the other hand, the social sciences camp is accused of culturally turning its back on precisely these insights (hird, 2010) while holding that both money and nature, and hence the cornerstones of entire field, are social constructions (fourcade, 2011) . the fact of social construction may be derogatively registered by those who hold religious or metaphysical beliefs in the sanctity of nature as something primordial and antecedent to human activity (cf. blancke et al., 2015) . yet, the understandings of naturalness can be often themselves unmasked as social constructions framed by the prevailing institutional structure (cf. valentinov et al., 2019a valentinov et al., , 2019b . these and further severe contradictions notwithstanding, however, both streams of ecological economics agree on the general adequacy and utility of the attempted "integration and synthesis of economics and ecologynot the disciplines as they existed at the time, which were seen as too narrow in their conceptions of the issuesbut of the study of economic and social systems embedded in and interdependent with their ecological life support systems" (costanza, 2020, p. 2) . hence, the "recognition that the economy is embedded within society, which is embedded within the rest of nature" (ibid.) is widespread; and even if some reservation might apply to the implication that economy and society belong to the realm of nature, the convention to define sustainability along an economic, social, and ecological or environmental dimension (basiago, 1995; european commission, 2001 ) is well-established and has gained particular prominence under the "triple bottom line" label (elkington, 1994 (elkington, , 1998 mauerhofer, 2008; ahi et al., 2018; vatn, 2020 ) (see fig. 1 ). 1 as impactful, integrative, and intuitive this idea may be, it remains grounded in the assumption that the environment of society in general and the economy in particular is adequately referred to as nature. in the subsequent sections of this article, we shall contest this basic assumption, reclaim a social scientific approach to the concepts of environment and ecology, reconceptualises nature as the environmental view of the social subsystem called natural sciences, and extend this regional nature-perspective to a broader concept of environment. starting from its roots in the 1960s (e.g., boulding, 1966; daly, 1968 ) and likely extending well into the anticipated future of the discipline (costanza, 2020) , ecological economics has always been open to systems thinking. terms and ideas such as economic system, social system, system of life, ecosystem, or nature as system consequently belong to the basic conceptual equipment of the transdiscipline. yet, the dominant systems-theoretical paradigm of the transdiscipline seems to be anchored in the open systems theory going back to the groundbreaking work of the biologist ludwig von bertalanffy (cf. von bertalanffy, 1968). interestingly, the discipline of biology has brought forth an alternative and in a sense opposite systems-theoretical paradigm, the theory of operationally closed, "autopoietic" systems. in this context, autopoiesis means that "everything that is used as a unit by the system is produced as a unit by the system itself" (luhmann, 1990, p. 1 more differentiated models distinguish between larger numbers of nested systems, e.g., from the outside in, physical environment, society, institutionalpolitical system, and economy (luzzati, 2010b, p. 53) . 3). the latter paradigm, while anticipated by von bertalanffy himself, mainly originates from the work of natural scientists maturana and varela, 1992 who were searching for an encompassing biological definition of life. maturana and varela (ibid) came to understand the essence of life in terms of the interrelated phenomena of autopoiesis and operational closure, both of which have been later appropriated by luhmann. as autopoietic systems, living organisms "produce not only their structures, but also the elements of which they consist, within the network of these very elements" (luhmann, 2012, p. 76f) . as a result, autopoietic systems can be said to operate "only in the context of [their] own operations" (luhmann, 2018, p. 33) . if life is understood in terms of its autopoietic organization, it would follow that "(t)he structure of living systems and their actual (material) components are complementary yet distinct aspects of any biological explanation: they complement each other reciprocally but cannot be reduced to one another" (varela and maturana, 1972, p. 382) . we may read these lines as a reminder not to confuse our object of research with its footprint. life is not what it takes to live. as organisms, we are neither what we eat nor the air we breathe, and we are not our excrements and other ecological footprint either. and it is precisely this condition that explains why we do, and indeed can, crave for nutrition, water, oxygen, and all other environmental factors that we critically depend on. just as varela and maturana insisted on the dividing line between matter and life, niklas luhmann (1995 luhmann ( , 2013 drew and defended the distinction between the behaviour of autopoietic biological systems and the communicative autopoiesis of social systems, the latter of which he said to be reliant, yet not reducible to the level of individual behaviour. it is ironic that luhmann established this borderline between biological and social systems by drawing on a concept that was originally invented by biologists, and that this move exposed him to criticism from both natural and social scientists alike (see cadenas and arnold, 2015; king, 2001; mingers, 2002) , including humberto maturana: "i have had relatively long discussions with maturana on this point. he always told me that1 if one speaks of the autopoiesis of communication/ one has to show it. that is to say, one has to show that the concept really works in the domain of communication so that it is possible to state that an individual communicative act can come about only in the network of communication. it cannot be conceived as a one-time event. and it also cannot be conceived as produced externally, in a communication-free context, as it were-say, as a chemical artifact that then has a communicative effect. on the contrary, it must always be produced by and through communication. i believe that this claim does not create much difficulty. it is relatively easy to see-especially if one considers the linguistic tradition of saussure, for instance and all that came of it-that communication occurs via its own differences and has nothing to do with chemical or physical phenomena." (luhmann, 2013, p. 79) . the same intrinsic logic that draws the dividing line between biological and social systems as different forms of systems then also applies to the differentiation between different forms of social systems. thus, as much as biological and social systems constitute environments for each other, and as much as there is hence no overlap but only mutual influences or "irritation" between these two forms of systems, so too is there no overlap between different subsystems of the comprehensive social system we commonly refer to as society. if we now insist "that the economy is embedded within society" (costanza, 2020, p. 2) , then this claim is perfectly compatible with luhmannian social systems theory, in which the economy is a subsystem of society and thus a social system itself. a social systems-theoretical perspective also allows for the observation that the economic system is now confronted with two different forms of environment, namely one intra-societal and one extra-societal environment. there are, however, two major differences. first, a social systems-theoretical variant of the triple-bottom-line model depicted in fig. 1 , there would be no need for a border around the extra-social environment unless we intend to observe it as a(n eco)system and thus distinguish it from yet another environment (see fig. 2 ). the second big difference between an ecological economics and social systems-theoretical perspective is that the latter does not content itself with an ultimately cancelled negative definition of society as the complement of its economic subsystem. if we challenge the idea that society is properly defined as "economy and the rest of society", then we shift our focus from the economy to its intra-societal environment. in looking at this social ecology of the economy, however, we quickly realise that the economy is certainly not the only subsystem of society as politics, science, religion, art, or law clearly are social subsystems, too. as there is no system without environment, this discovery implies that all other systems are not only located in the intra-societal environment of the economy, but also locate the economy as much as all other in their respective intra-societal environments. in other words, the social ecology of the economy is made of numerous social systems, each of which has a different environment and thus a different view of the overall ecology of social systems. as each of these systems has a different view of the social ecology, they necessarily have a different view of the extra-societal environment, too. true, natural science defines this environment as nature; yet, from a religious point of view, this environment may be populated by spirits or gods, and most commonly referred to as one of these god's creation. for yet other subsystems, this environment might appear as an indefinite learning space or an equally gigantic though potentially shrinking stock of non-social resources. a truly social science that accounts for this multienvironmental condition would therefore need to refrain from over-identifying the extra-social environment with the environmental concepts and terminologies of the natural sciences. this is true because, first, it is hard to impossible to prove scientifically that one of the above-mentioned subsystems is essentially more important than the other(s). second, even if a scientific bias to science seems logical or at least comprehensible, it would still remain unclear why social sciences should favour an environmental concept of the natural sciences over a socialscientific one. in the subsequent section of this article, we shall therefore outline a social-scientific concept of environment, in the context of with the natural-scientific concept of nature is positioned as one environmental concept among others. regardless of whether we define ecological economics as a natural or a social science, it remains true that the economy is neither a natural nor a social science. as economy and science are two distinct though certainly interacting subsystems of society, the current situation, in which a transdisciplinary field attempts at almost coercing one social subsystem to adopt the environmental perspective of one branch of another subsystem, seems far from being natural. in looking at fig. 3 , the reductionist nature of this enterprise is even more evident: fig. 3 depicts a challenge and extension of the classical "3-d sustainability" (mauerhofer, 2008 ) models such as the triple-bottom-line and cognate frameworks. the basic feature of these models is that they locate the economy (the right-hand triangle) within society (the social environment of the economy triangle). society is then again embedded within an extra-social environment, which is commonly referred to as nature, ecosystem, or ecological environment. in order to observe ecosystems or nature, however, we need science in general and natural sciences in particular (luzzati et al., 2014, p. 100) . science, however, does not appear in its own models, which is as ironic as consequential as these models are used to argue that a sustainable economy must confuse its environment with the environment of the natural sciences. in fig. 3 , this condition is depicted as a prohibition to use an economic radar and obligation to use a natural scientific radar for environmental screening. as indicated earlier, in its more radical forms, this concurrent prohibition and obligation explicitly includes or is extended to the social environment, e.g., when ruth (2006, p. 339) suggests to establishing "the economic, legal, institutional and ethical basis" of sustainable human behaviour "on fundamental insights from the natural sciences". in revisiting fig. 3 , however, it becomes obvious that even this already enhanced version of the triple-bottom-line remains incomplete as economy and science are certainly not the only subsystems of society. moreover, it is just as sensible that these subsystems constantly influence each other. for instance, "(t)he political system depends upon informational and conceptual inputs and contributions from the other societal systems in the governance process-for example, the social and the natural sciences" (luks and siebenhüner, 2007, p. 418) , and the same is true vice versa. last not least, it is critical to realise that function systems constitute parts of the social environment for each other and that they are only one type of social systems that constitute the overall social environment, with other types being, for example, families or organizations. in figs. 3 and 4, the label "societal environment" is therefore written across the border between the "inner circle" from a social-scientific perspective, we therefore observe an entire ecology of interacting social subsystems and their corresponding environments. in the luhmannian terminology, what we observe is polycontexturality: a multitude of interdependent, yet mutually incommensurable and operationally closed systems bringing forth their unique definitions of both the societal and extra-societal environment. as important as the actually quite provincial natural environmental perspective might seem today against the backdrop of the urgent threats to its environment, so too is the perceived urgency no scientific argument to ignore, devalue, or even prohibit the environmental perspective of other subsystems of society in general and other subsystems of science in particular. however, from a social-scientific perspective, there is no need to ignore, devalue, or even prohibit the environmental perspective of the natural sciences either. rather, what is needed is a multifunctional perspective similar to the one depicted in fig. 4 . fig. 4 presents a social-scientific perspective on society and its internal and external environments. this perspective does not ignore, devalue, or even prohibit; to the contrary, it encourages the natural sciences to make universal claims about the nature of both the societal and the extra-societal environment of the economy or any other social system. yet, fig. 4 is also a reminder that universality must not be confused with exclusivity (luhmann, 1995, p. xlviii) . this means that each type of environment can harbour unique sustainability risks, many of which would remain unnoticed even by the modern sustainability concepts such as the triple bottom line concept. the relationship between the luhmannian systems theory and ecological economics is marked by a deep ambivalence, which, is not unique to the present paper. take the case of kenneth boulding, who was not only the author of the seminal "spaceship earth" metaphor but also an engaged systems theorist. whereas the spaceship metaphor evidently rested on boulding's firm belief in the certainty of the exhaustible and destructible nature of the environment of the economy, his systems-theoretical views were more open-ended. in his systemstheoretical treatise ecodynamics (boulding, 1978) , he titles one of the sections "the myth of the environment". in the section, he explains that "there is no such thing as an 'environment', if by this we mean a surrounding system that is independent of what goes on inside it. (…) it makes sense to divide the totality of the universe into parts that have some degree of independent dynamic pattern, but none of these parts is really independent of others: all interact. (…) everything is the environment of everything else" (ibid, p. 31). the luhmannian systems-theoretical perspective affirms the view that "there is no such thing as the environment", but for somewhat different reasons. if we agree that there is 1) no system without environment, 2) no ecology without a plurality of systems, and thus 3) no ecosystem without a plurality of environments, then we cannot assume the environment as a singular entity to be given with ontological certainty. it is rather the case that different social subsystems have different views of their environment, and that nature-the environment of the natural sciences-is nothing more or less than the environment of a subsystem of the scientific subsystem of society. does that mean that the luhmannian perspective relativizes the fundamental sustainability concerns of ecological economics? a sympathetic look at fig. 4 would suggest that these concerns are generalized and radicalized rather than relativized. while sustainability risks addressed by ecological economists are serious, they are observationally contingent. employing other observational perspectives may lead to the discovery of new dimensions of risks which are no less serious. on a more positive note, these risks may likewise engender entrepreneurial opportunities. just as the triple bottom line concept has provided inspiration for the development of sustainable business models (cf. joyce and paquin, 2016) , the polycontextural environment indicated in fig. 4 invites the use of strategic management tools for the purposes of the multienvironmental screening and accounting of the diverse logics and observational perspectives . these tools would open radically new possibilities for navigating multidimensional sustainability risks, particularly for those types of social systems (such as corporations) that are known to systematically blind out specific segments of environmental complexity or stakeholder interests valentinov et al., 2019a valentinov et al., , 2019b . take classical environmental scanning tools such as pest or its derivates pestle, steeple, steepled (see roth et al., 2017, p. 200f ), many of which give additional weight to ethics and the natural environment. while the individual letters p (politics), e (economy), s (society) and t (technology) have been continually supplemented or reshuffled, it is clear that critical aspects of the societal environment have escaped the attention of these and many other environmental scanning tools. health is one such factor, as the system does not seem to deserve a dedicated letter or specific attention and is typically lumped together with often both "political" and "social" issues. yet, the coronavirus crisis has shown in the most dramatic ways that such neglect may come at a cost. the same events have furthermore demonstrated the inescapable incommensurability of the individual spheres (kapp, 1977, p. 538; luzzati, 2009, p. 315) or subsystems of society (luhmann, 2002, p. 52; roth, 2019, p. 508) as emerging dilemmas or trade-offs between health on the one side and money or liberty on the other side clearly indicate that we are confronted here with some of "those questions that are in principle undecidable" (von foerster, 1992, p. 14) . in discussing the example of managerialized public health care, roth et al. (2019, p.8) argue that the "proliferation of this type of health care can be observed from multiple and equally legitimate points of view associated with the function systems, each of which potentially generates a distinct moral evaluation of this trend. business-like public health care may be politically endorsed (i.e. good) and economically efficient (i.e. good) while being inconsistent with medical professionalism and unchristian. it may at the same time present a setback for risk sport participants and probably even a scandal in the mass media system, while making no difference from an artistic or legal point of view. this diverse constellation of moral judgments shows the rise of business-like public health care to be simultaneously good, bad, and morally irrelevant. this example makes clear that functional differentiation precludes the automatic identification of economic, political, legal or scientific operations as inherently good or bad." in showing how quickly conventional judgments on the relative importance of the individual function systems may change, the 2020 coronavirus crisis, therefore, make a strong case that a better (mutual) understanding of how differently different function systems conceive of both their societal and extra-societal environment is essential. the basis of this understanding would be a strategic management tool that accounts for the, in principle, equal importance of all functions systems that could be acronymized "rhesamples" (roth et al., 2017, p. 201 ), a derivation from the initial letters of each of the 10 function systems: religion, health, economy, science, art, mass media, politics, law, education, and sport. as another implication for further research, a multiplication of sustainability concerns achievable by the proposed multienvironmental scanning would be capable of yielding information on the possible environmental shocks that might be suffered by specific social systems. that way, the introduction of multienvironmental scanning holds the potential to improve the resilience of the systems in question, a property which is often understood as the systemic capacity "to absorb change and disturbance and still maintain the same relationships" (holling, 1973, p. 14) . in the last decades, the concept of resilience rose to prominence in the context of the literature on the socio-ecological systems (ibid) and was applied to numerous other contexts. it was acknowledged that ensuring the resilience of the socio-ecological systems requires adaptive governance which must be conceptualized as multilevel and polycentric (van assche et al., 2019) . it is evident that the proposed multienvironmental scanning radically expands the observational capacity required for anticipating the possible shocks. while this may be a valuable contribution to the literature on the socio-ecological systems, the understanding of these systems may itself be transformed by the multiplication of observational perspectives suggested by a consistent application of the luhmannian insights. furthermore, the luhmannian systems theory may itself benefit from appreciating the way in which the polycontextural diversity of the modern society converges on the twofold understanding of the boundary between the social and the natural. as van assche et al. (2019) , p. 313) explain, "the ecological is the environment of the social in a double sense: internally and externally". the same understanding of the boundary is evidently applicable to the distinction between human organisms and what luhmann took to be psychic systems. there is room to argue that much of this complexity can be illuminated by the proposed multienvironmental scanning, especially given that the coevolution of organisms and psychic systems may itself be a reflection of the much more encompassing process of co-evolution of systems and environments constituting the regime of polycontexturality. finally, the diverse observational perspectives generate further insights going beyond risks and entrepreneurial opportunities. paradoxically as it sounds, these insights bring to the fore new solutions and new resources that have been invisible from the traditional or dominant perspectives. if contemporary societies are responsible for what we commonly refer to as ecological problems, then the solutions to these problems might require not ever-bigger natural-scientific efforts (shah, 2020) , but rather a shift of perspective to the environments of social sciences and a corresponding multienvironmental scanning that dislodges the problems while foregrounding the above opportunities that have not been clear before. the authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper. a quantitative approach for assessing sustainability performance of corporations methods of defining 'sustainability fatal attraction: the intuitive appeal of gmo opposition the economics of the coming spaceship earth ecodynamics: a new theory of societal evolution the autopoiesis of social systems and its criticisms what is ecological economics? ecological economics in 2049: getting beyond the argument culture to the world we all want on economics as a life science towards the sustainable corporation: win-win-win business strategies for sustainable development cannibals with forks: the triple bottom line of the 21st century business partnerships from cannibals with forks: the triple bottom line of 21st-century business a sustainable europe for a better world: a european union strategy for sustainable development. commission's proposal to the gothenburg european council cents and sensibility: economic valuation and the nature of "nature coevolution, symbiosis and sociology resilience and stability of ecological systems broad sustainability contra sustainability: the proper construction of sustainability indicators the triple layered business model canvas: a tool to design more sustainable business models environment and technology: new frontiers for the social and natural sciences the humanization of the social sciences the construction and demolition of the luhmann heresy ecological communication theories of distinction: redescribing the descriptions of modernity introduction to systems theory transdisciplinarity for social learning? the contribution of the german socio-ecological research initiative to sustainability governance 14. human needs, sustainable development, and public policy: learning from kw kapp economic development, environment and society: rediscovering karl william kapp economic development, environment and society: karl william kapp measuring the sustainability performances of the italian regions the tree of knowledge: the biological roots of human understanding 3-d sustainability: an approach for priority setting in situation of conflicting interests towards a sustainable development can social systems be autopoietic? assessing luhmann's social theory three pillars of sustainability: in search of conceptual origins the early history of modern ecological economics heal the world. a solution-focused systems therapy approach to environmental problems multiplying the division of labour: functional differentiation of the next key variables in management research csr beyond economy and society: a post-capitalist approach dissecting the empirical-normative divide in business ethics a quest for the economics of sustainability and the sustainability of economics social ecological economics: understanding the past to see the future new foundations for ecological economics the complexity-sustainability trade-off in niklas luhmann's social systems theory kapp's theory of social costs: a luhmannian interpretation the anti-gmo advocacy: an institutionalist and systems-theoretic assessment stakeholder theory: a luhmannian perspective the social, the ecological, and the adaptive. von bertalanffy's general systems theory and the adaptive governance of social-ecological systems towards a power-sensitive and socially-informed analysis of payments for ecosystem services (pes): addressing the gaps in the current debate assessing sustainability performance of farms: an efficiency approach mechanism and biological explanation institutions for sustainability-towards an expanded research program for ecological economics ethics and second-order cybernetics from nonprofit diversity to organizational multifunctionality: a systems-theoretical proposal he holds a habilitation in economic and environmental sociology awarded by the italian ministry of education, university, and research; a phd in sociology from the university of geneva; and a phd in management from the chemnitz university of technology. he is the field editor for social systems theory of systems research and behavioral science. the journals his research has been published in include germany, and senior researcher at the leibniz institute of agricultural development in transition economies in halle, germany. he has served on the editorial boards of several journals, such as the journal of economic issues, voluntas, and kybernetes. his research has been published in journals such as nonprofit and voluntary sector quarterly the authors are grateful to the editor and the anonymous reviewers for their close reading of our manuscript and their very helpful comments. key: cord-294877-bbs8a8jz authors: liu, chuanpeng; fan, dongjie; shi, yi; zhou, qiming title: a glimpse of enzymology within the idea of systems date: 2012-09-27 journal: sci china life sci doi: 10.1007/s11427-012-4371-2 sha: doc_id: 294877 cord_uid: bbs8a8jz nan next year will mark the 100th anniversary of the michaelis-menten equation [1] , one of the most important and well known models in enzymology. remarkable progress in enzymology over the past hundred years has not only provided deep insight into biological processes, but also has dramatically changed our lives. however, because of limitations in available techniques, enzymologists primarily investigate the in vitro properties and catalytic mechanisms of individual enzymes. thus, they often fail to address a fundamental scientific question: how are biological processes, most of which are chemical reactions catalyzed by enzymes, coordinated in space and time to produce a living organism [2]? fortunately, applications of new techniques and, in particular, wide acceptance of the idea of systems have enabled enzymologists to begin studying enzymes in the context of dynamic, complicated biological systems in recent years. many excellent reviews and research articles on enzymology have been published in the past few years. these articles highlight the latest developments in enzymology, and should prove helpful for understanding how to best integrate the idea of systems into enzymology research. in living organisms, protein post-translational modification (ptm) pathways are large and interconnected networks. via reversible or irreversible covalent modifications, allo-steric regulations of ptm enzymes and cross-talk of ptm pathways, ptm networks enable organisms to respond to stimuli faster than is possible through gene transcription regulation. therefore, ptm enzymes, as key nodes of biological networks, are a recent focus of attention. phosphorylation is often argued to be the most abundant ptm, and also is the most extensively studied because of the essential contribution of phosphorylation-dependent interaction networks to cellular regulation [3] . in the review by han et al. [4] , a novel receptor-like kinase subfamily in angiosperm, crrlk1-l subfamily, was described in detail. after discussing the structure, enzymological properties and biological function of crrlk1-l, the authors argued that crrlk1-l could initiate signal transduction largely independent of the known signaling pathways. however, a better understanding of its allosteric regulation and substrate specificity are still necessary for defining the pathways that it may modulate. unlike crrlk1-l, calcineurin is an extensively studied serine-threonine protein phosphatase because it plays a pivotal role in a wide series of crucial physiological processes. tu et al. [5] reviewed the recent literature on a new family of calcineurin regulators (rcans), and discussed the molecular mechanism for rcan-mediated regulation of calcineurin. after exemplifying the roles of rcans in a wide variety of diseases including alzheimer's disease, the authors suggested that rcans are potential targets for drug development. similar to rcans, glycogen synthase kinase-3 (gsk-3) is also involved in alzheimer's disease, but the underlying mechanism remains unclear. recently, a research article by ding et al. [6] provided a clue to this question. in their article, one member of sr protein family, 9g8, was identified as a new substrate protein of gsk-3. hence, the authors suggested that gsk-3 could inhibit 9g8-mediated alternative splicing of tau exon 10 by phosphorylating 9g8, consequently inhibiting the aggregation of tau exon 10. ubiquitination is another common ptm. by attachment of ubiquitin to the protein as a monomer or as polyubiquitin chains with different ubiquitin-ubiquitin linkages, the function and fate of the modified substrate can be drastically changed. ubiquitination is a reversible process regulated by a subtle balance of ubiquitin-modifying enzymes and deubiquitinating enzymes. two reviews and one research article were recently published, both the review by xie et al. [7] and the research article by sun et al. [8] focused on the deubiquitinating enzymes. xie et al. provided an overview of the recent advances in structural and functional studies of ubiquitin c-terminal hydrolase l1 (uch-l1), with a special emphasis on the protective effects of uch-l1 on neurons. sun and coworkers focused on nsp3, a newly-identified deubiquitinase from human coronavirus nl63. nsp3 has two papain-like protease (plp) core domains, and has both protease and deubiquitinase activities. by in vitro and in vivo methods, the authors demonstrated that the deubiquitinase site was located in the second plp domain and shared two catalytic residues with the protease site. furthermore, the authors found the deubiquitinase activity of nsp3 could induce the deubiquitination of rig-i and eris and then regulate antiviral innate immune responses by inhibition of both rig-i and tlr3-dependent induction of interferon. although ubiquitination in eukaryotic systems has been studied for decades, the prokaryotic analog of ubiquitin, ubiquitin-like protein (pup) was not identified until 2008. wang et al. [9] reviewed the latest advances in research of the pup-proteasome system and comprehensively discussed its catalytic and regulatory mechanism. the authors suggested that the pup-proteasome system could be regarded as a potential therapeutic target because of its extensive involvement in many important physiological pathways of pathogenic bacteria. acetylation and methylation of histones play fundamental roles in gene expression following environmental stimuli. while acetylation of histones converts chromatin from a closed to an open conformation that facilitates transcription, histone methylation can keep chromatin in either an activated or a repressed state, depending on the precise methylation site and the degree of modification. interestingly, the intense cross-talk between histone modifications drives distinct downstream functions. two recent reviews summarize the latest advances of research into two histone modifying enzymes. in the review by guo [10] , the structure and function of a new histone trimethylase, prdm9 (pr domain containing 9) are described. prdm9 catalyzes the h3k4 trimethylation, and possesses the activity of a transcription factor and presumably plays an important role in the initiation of recombination. this review strengthens our understanding of species evolution and genetic recombination. peng et al. [11] introduced the latest advances of research into sirtuin proteins. sirtuins are a highly conserved family of nad + -dependent deacetylase enzymes and are involved in many important biological processes, including cellular stress resistance, genomic stability, tumorigenesis and energy metabolism. in this review, the authors emphasized the structure and catalytic mechanism of sirtuins and discussed the relationship between sirtuins and aging. oxidation of side groups is a very important ptm for cellular proteins. although cys and met are the residues prone to oxidative modification, other residues also can be oxidized under more stringent conditions. for example, the ε-amino group of lysine may be oxidized to an aldehyde by lysyl oxidases (loxs). loxs are a family of multi-functional enzymes, and play an essential role in cell proliferation, cell chemotactic responses and tumor genesis. in their review, zhang et al. [12] summarized the latest knowledge of loxs, including their potential as targets for diagnosis and therapy of several human diseases such as connective tissue disease, exfoliation syndrome, disorders of copper metabolism, pelvic organ prolapse, and bone disorders. proteolysis is another one of the most common ptms. proteolysis needs to be very precisely controlled on multiple levels because of its irreversible nature. therefore, enzymes involved in proteolysis are regarded as key nodes in the living system, and have received increasing attention over the past decade. in eukaryotic cells, proteasome-dependent degradation is the general mode of selective protein degradation. while the ubiquitin-dependent proteasomal degradation pathway has been well studied, little attention has been directed toward the possible involvement of the proteasome in ubiquitin-independent proteolysis. however, increasing evidence shows that the ubiquitin-independent proteasomal proteolysis pathway is involved in the elimination of some short-lived regulated proteins, misfolded proteins, aged proteins and oxidized proteins as well as providing "quality control" of newly synthesized proteins. thus, it plays important roles in pathological processes such as cancer and neurodegenerative diseases. recent progress in understanding ubiquitin-independent proteasomal degradation and the underlying molecular mechanisms were discussed by chen et al. [13] . besides the proteasome, proteases also catalyze protein proteolysis. in a review by wu et al. [14] , a newlyidentified protease, tmprss6, was described. tmprss6 is a transmembrane serine protease which can regulate the expression of hepcidin, thereby affecting iron metabolism. in this review, the authors discussed the relationship between the structure and function of tmprss6, and the roles of tmprss6 in iron metabolism-related diseases. because proteases are involved in many diseases [15] , protease inhibitors have been of great interest for the development of novel therapies. in a recent research article, tian and coworkers [16] investigated the protease inhibitory activity and antineoplastic effect of buckwheat trypsin inhibitor (bti). the authors not only identified the active site of bti, but also obtained two bti mutants which showed much better stability to heat and ph than wild-type bti. their results will contribute to clarifying the physiological mechanism of bti and support the development of novel anticancer drugs. many biologists are now using "omics" tools to investigate organisms and construct various interaction networks. however, because biological systems are essentially built of highly coordinated sets of interaction networks across different scales, integration of the networks from the various 'omics' tools is a core paradigm for deep comprehension and ultimately reconstruction of these living systems. while ptm enzymes act as key nodes in protein-protein interaction networks as discussed above, the enzymes whose substrates are something other than proteins, can bridge protein networks and other networks together. therefore, the enzymes acting on non-protein substrates are of increasing interest in the systems biology era. glutamate synthase, lactate dehydrogenase (ldh) and dihydrofolate reductase (dhfr) are three important metabolism enzymes, and play key roles in metabolomics and proteomics data integration. although these enzymes had been extensively studied, the results presented in three recently published articles indicate that their biological functions are still to be clarified. in an article by lu et al. [17] , the authors investigated the roles of glutamate synthase in carbon and nitrogen metabolism of rice, and concluded that glutamate synthase was indispensable for efficient nitrogen assimilation in rice. meanwhile, new physiological functions of dhfr and ldh were discussed. while sun and coworkers [18] found dhfr was essential for the development of pharyngeal arches, li and coworkers [19] suggested that the reduction or loss of ldh-c4 activity in sperm might cause male infertility. these results indicate that our knowledge of "old" enzymes is not complete. based on the accumulated experiment data on metabolism enzymes, a general relationship between genomics, proteomics and metabolomics of microbes was discussed recently [20, 21] . phase ii drug/xenobiotic metabolizing enzymes, together with phase i enzymes, play a central role in the metabolism and detoxification of numerous exogenous and endogenous compounds. therefore, the characterization of xenobiotic metabolizing enzymes not only contributes to the understanding of the context of biological networks within organisms, but also is indispensable to elucidate the underlying mechanisms of the interplay between organisms and the environment. of particular importance is to establish the correlation between the genetic polymorphisms of xenobiotic metabolizing enzymes and interindividual variations in drug metabolism [22] . hao et al. [23] investigated the po-tential effect of all the known nonsynonymous single nucleotide polymorphisms (nssnps) of human phase ii metabolizing enzyme on protein function, and identified many residues that are likely to be functionally critical, but have not yet been studied experimentally. their results contribute to integrating polymorphisms into a physiologically based pharmacokinetic (pbpk) modeling framework. since dna is the carrier of genetic information, the maintenance of dna stability is essential in living cells. in two recent research articles, the inhibitory mechanisms of helicase and integrase were discussed. luo and coworkers [24] extensively investigated the effect of lomefloxacin on bloom syndrome helicase (blm). blm is an important member of the recq family of dna helicases and plays important roles in dna repair, recombination, transcription and telomere maintenance. the authors found that lomefloxacin could promote the binding of blm to dna, but would unexpectedly inhibit the helicase and atpase activities of blm. subsequently, the authors proposed a possible mechanism based on their kinetics and thermodynamics results which should prove useful for the design of inhibitors against dna helicase and improve our understanding of the mechanism of action of quinolone drugs. while luo et al. used in vitro methods to study blm, hu and coworkers [25] investigated the inhibitory mechanism of compound l708, 906 towards hiv-1 integrase by using in silico methods. they found the binding of l708, 906 to hiv-1 integrase could alter the mobility of the protein, and thereby result in the decrease of integrase activity. their results should be helpful for anti-hiv drug design. however, in silico results ultimately have to be supported by "real" experimental data. high-throughput screening assays with adequate sensitivity, reproducibility, and accuracy have been indispensable for the discovery of drugs targeting hiv-1 integrase. in a recent research article, he and coworkers [26] developed a novel method for measuring the disintegration activity of hiv-1 integrase. this method is rapid, sensitive and, most importantly, readily adapted to a high-throughput format, and hence is likely to be applicable to antiviral drug screening. as many chemical and physical factors can cause dna changes, multiple mechanisms have evolved to deal with dna lesions in living cells. translesion synthesis is a special pathway that enables the replication fork to bypass blocking lesions. as a key member of the translesion dna synthesis pathway, dna synthesis polymerase kappa (polκ) has received much attention. in a recent review, lu et al. [27] comprehensively described the structure, catalytic mechanism and biological function of polκ, which contributes to our understanding of translesion dna synthesis. homologous recombination repair is another powerful dna repair system. although homologous recombination has been studied for decades, the underlying mechanism is not yet clear. since the resolution of holliday-junction recombination intermediates is the critical step for homolo-gous recombination, unraveling the catalytic mechanism of resolvase will contribute to our understanding of homologous recombination. the resolvase from bacteriophage, t7 endonuclease i, is one of the most studied resolvases, and possesses resolvase, nicking enzyme and endonuclease activities. t7 endonuclease i is a homodimer comprising two identical active domains, each of which consists of parts of both subunits in the dimer. however, the function of each active domain is unclear. in a recent research article, fan and coworkers [28] prepared and characterized a t7 endonuclease i mutant with only a single active domain. their results showed that the t7 endonuclease i mutant with a single active domain could recognize the holliday-junction structure but failed to introduce two nicked sites simultaneously. taken together, this information is helpful for elucidating the catalytic mechanism of resolvase. while many biologists focus on biological networks in cells, tissues and organisms, environmental enzymologists are striving to explore the dynamic nature of ecosystems at the molecular level. the application of meta-omics and enzymology technologies has rapidly increased our knowledge of the global biogeochemical cycles of inorganic and organic compounds, organism-environment and organismorganism interactions. the convergence between enzymology and the ecology of the nitrogen cycle was discussed in recent reviews [29, 30] . zhang et al. [31] investigated the phosphorus uptake of brassica napus under low phosphorus stress using enzymological methods, and proposed an acid phosphatase-centric regulatory mechanism of phosphorus uptake. their results increase our understanding of the mechanisms of the phosphorus cycle. with increasing use of transgenic crops, more scientists are concerned about the environmental impact of transgenic organisms. in a recent research article, guo and coworkers [32] examined the influence of transgenic bt cotton on beet armyworm, which is not an insect which targets currently commercialized transgenic bt cotton varieties in china. their results show that feeding beet armyworm with bt cotton can cause significant changes of many important enzyme activities in the worm making it necessary to evaluate the environmental impact of bt cotton. while molecular level research on biological systems has evolved from investigating individual enzymes or pathways to studies on biological networks and even ecological networks, a new challenge for biologists is to add a dynamic property to the constructed networks. one strategy is to create an animation to display the dynamic change of biological networks using a series of snapshots of the biological networks captured by high-throughput methods at different points in time. however, because of technical limitations, it is impossible to capture the snapshots at a rate faster than the biochemical reactions, indicating that the "dynamic change of networks" provided by this strategy is actually discontinuous in time, and the biochemical events occurring between any two adjacent snapshots are missed. a better strategy is to incorporate biochemical reaction parameters into biological networks, by which biologists can construct the dynamic biological networks with more "living" properties. therefore, enzymological studies are crucial not only for the construction of biological networks as discussed above, but also for the elucidation of the dynamical properties of biological systems. although enzyme catalysis has been extensively studied, most of this knowledge comes from in vitro experiments under conditions in which substrate and product concentrations, mass transfer and diffusion are radically different from the living system. in addition, while canonical kinetic methods provide only ensemble-averaged molecular information, the conformational heterogeneity of an enzyme cannot be ignored in living cells because of the low abundance of enzymes, macromolecular crowding and confinement. therefore, characterization of enzymes in the context of a biological system is the prerequisite for understanding its dynamic properties. fortunately, thanks to the close collaboration of scientists and engineers from various disciplines, many novel techniques have recently been developed and applied to the investigation of the in vivo behavior of enzymes. for example, mass spectrometry based enzymatic assay techniques have been applied to inhibitor screening in vivo, identification of endogenous substrates, and in vivo enzymatic activities of orphan proteins [33] . light and fluorescence microscopy techniques are being used in measuring enzyme activity in single cells in a real-time manner [34, 35] . in particular, the development of single molecule detection techniques has made it possible to characterize enzyme catalysis at the single molecule level [3639] . mathematical modeling and model-based data analysis are indispensable for understanding the behavior of biological systems [40, 41] . xu and coworkers [42] proposed a generic rate equation for the dynamical modeling of metabolic kinetics, in which kinetic parameters are clear and precise enough to correlate to experimental data for purposes of constructing a database. the equation is applicable to an arbitrary number of substrates and products for computation of dynamic modeling, which makes the construction of omics scale networks using kinetic models much more feasible. because enzymes are the central components of biological networks, any change related to an enzyme, even a slight fluctuation in enzyme content or activity, will lead to local or global alteration of biological networks. for example, cd38 is an adp-ribosyl cyclase responsible for synthesizing cadpr and naadp. through these two secondary messengers, cd38 can regulate insulin secretion, susceptibility to bacterial infection, and even social behavior of mice (see the review by lee [43] ). therefore, enzymes can be used as markers to characterize the status of biological systems and trace the dynamic changes of biological networks. identification of highly specific and sensitive enzyme markers from biological networks for various purpos-es has been a major task for enzymologists. schizophrenia is a common and severe neuropsychiatric disorder. although many scientists believe that increased oxidative stress is relevant to the pathophysiology of schizophrenia, it is still debated whether oxidoreductases could be used as biomarkers for diagnosis and treatment of this disease [44] . in a recent research article, zhang and coworkers [45] performed a meta-analysis of data from early studies related to oxidative stress in schizophrenia, and found that superoxide dismutase (sod) activity significantly decreased in the disorganized type of schizophrenia patients, indicating that sod may be a potential biomarker of schizophrenia. their results provide an exciting opportunity for diagnosis and treatment of schizophrenia. thanks to intensive research efforts from an increasing number of laboratories, detailed information regarding the physics, chemistry and kinetics of enzymes is accumulating rapidly. however, theoretical developments in this area have lagged behind experimental data. significant evidence shows that in some cases, the observed behaviors of enzymes are incompatible with the classical theories of enzymatic catalysis. for example, when biologists began to study the dynamic properties of biological networks, the possible role of protein motion in enzyme catalysis was still controversial [46, 47] . therefore, elucidating the mechanisms of enzymatic catalysis remains one of the most intriguing biochemical phenomena. zhang and coworkers [48] investigated the effect of substrate-induced conformation changes on enzyme catalysis, and found that the activity and conformation of an enzyme is changed when the enzyme induced with one substrate was reacted with other substrates in subsequent reactions. based on their results, the authors ingeniously brought the two classical catalysis models, "induced fit" and "lock and key" together, and proposed a new model called "induced fit-lock and key" for enzyme action, potentially deepening our understanding of enzyme catalysis. one of the major goals of enzymology is to develop efficient biocatalytic systems for industrial, medical or environmental applications. along with the rapidly growing understanding of enzyme action in the context of biological systems, our capacity to design biocatalytic systems is growing at an unprecedented pace. while the early studies of biocatalysis were restricted to "one-step" biotransformations or optimization of an organism's own metabolic pathways, we are now able to engineer entire foreign biosynthetic pathways into host organisms to make non-native natural or even unnatural products [49, 50] . however, biocatalysis still has many challenges that need to be addressed, most of which are around enzyme discovery, design, and optimization. for example, how can we find or create enzymes that catalyze the desired chemical reaction of interest, even those not existing in nature? how can we create multiple-enzyme biocatalytic systems required for multistep biosynthesis of complex natural and unnatural products? how can we effectively integrate non-native pathways into the metabolism of a host? how can we improve the stress tolerance of biocatalytic systems to industrial reaction conditions? the following papers will be helpful for biologists to answer these questions. the identification and isolation of novel enzymes is an extremely active field in enzymology. there are two general experimental strategies for enzyme discovery, which are based on function (activity) or sequence, respectively. the function-based strategy is a straightforward way to obtain enzymes which have desired functions. for example, using molecular probes that bind to enzymes of interest in an activity-dependent manner, enzymologists not only can quantify the dynamics of an enzyme in vivo, but also can directly isolate and identify the enzyme with the desired function from cell or tissue samples [51] . this technique, termed activity-based protein profiling (abpp), is being widely applied to the discovery of novel enzymes and drugs [52, 53] . compared with the function-based strategy, the sequencebased strategy is readily performed using bioinformatics and pcr approaches, and is widely used in many laboratories. using the sequence-based strategy, sun and liu [54] successfully isolated a new carboxylesterase gene from geobacillus stearothermophilus. because of the fact that the enzyme exhibited a high thermal stability and broad ph tolerance, the authors suggested that this carboxylesterase had potential applications in the synthesis of stereospecific compounds and degradation of environmental contaminants. although these two experimental strategies have been very successful in enzyme discovery, biological experimentation alone is clearly inadequate to meet the challenge presented by the exponentially increasing number of unknown sequences. to address this challenge, a large-scale collaborative project, the enzyme function initiative (efi), was established in 2010. the efi is developing a computation-based multidisciplinary strategy for functional assignment of unknown enzymes in a high throughput manner [55] . while more and more novel enzymes are identified from diverse organisms, especially those in extreme environments, a new challenge for applied enzymologists is how to assign each enzyme to the job that suits it best. for example, early studies showed that the (r)-oxynitrilase from prunus salicina was one of the poorest oxynitrilases for the synthesis of (r)-cyanohydrins from hydrogen cyanide with aldehydes, and thereby not suitable for industrial applications. however, in a recent research article, liu and zong [56] reported that plum (r)-oxynitrilase could efficiently catalyze enantioselective transcyanation of acetyltrimethylsilane with acetone cyanohydrin, and both acetyltrimethylsilane conversion and enantiomeric excess of the product were above 99%. this finding implies that identifying new catalytic functions of known enzymes is an efficient way to increase the potential applications of enzymes in biotechnology. although enzymes are usually regarded as specific catalysts of biochemical reactions, the promiscuity of some enzymes is a common phenomenon, and the structural, mechanistic, and evolutionary implications of this were discussed in a recent review [57] . considering the fact that industrial reaction conditions are different from physiological conditions, the secondary capabilities of some enzymes may make them more suitable for industrial applications in certain cases. the interest in utilizing enzymes in industrial processes is constantly growing during the past several decades. however, natural enzymes are usually not optimally suited for industrial applications because they have evolved to work in the mild cellular environment rather than under harsh process conditions. fortunately, many strategies have been developed to overcome this problem, the most popular ones of which are directed evolution, rational design, chemical modification and immobilization. using these approaches, many biocatalysts with excellent properties were developed and used in various applications. for example, wu and coworkers [58] developed a novel capillary trypsin reactor by organic-inorganic hybrid monolith based immobilization. this trypsin reactor not only has high enzymatic activity, but also shows reliable mechanical stability and permeability. this reactor is promising for the development of an automated online digestion device for use in proteomics. the recent advances, new challenges and future trends of directed evolution, rational design, chemical modification and immobilization are comprehensively discussed in a number of reviews [59] [60] [61] [62] [63] . one of the major technological hurdles in enzymology research is how to control the expression of the enzyme of interest at the desired level. although many biologists hope to maximize the expression yield, some enzymologists who engage in metabolic engineering and synthetic biology are facing a greater challenge: how to optimize the absolute and relative levels of the enzyme components of the designed biosynthetic pathway, and how to balance the activities of endogenous and exogenous enzymes. for example, to make a taxol precursor in escherichia coli, the expression level of more than 30 endogenous and exogenous enzymes were optimized [64] . therefore, the development of novel expression strategies has an important impact on enzymological research. two recent research articles introduced two new enzyme expression strategies. a functional fragment of coagulation factor viii (bdd-fviii) has potential for use in the therapy of hemophilia a. however, because of its large size, it is difficult to deliver the bdd-fviii gene into cells by a single adeno-associated virus (aav) vector, slowing the development of gene therapy for hemophilia a. zhu and coworkers [65] presented a protein trans-splicing based dual-vector delivery system which is suitable for the expression of larger proteins in mammalian cells. using this delivery system, bdd-fviii was successfully expressed in hek-293 and cos-7 cells. these results not only pave the way for the development of gene therapy for hemophilia a, but also offer a new strategy for the expression of large for-eign proteins. wang et al. [66] inserted h. polymorphaderived ribosomal dna (rdna)-targeting element into a ppic9k-derived expression vector containing the pgap promoter, and developed a high-efficiency h. polymorpha expression system, by which the yield of recombinant t4 lysozyme was up to 0.5 g l 1 . besides describing a highefficiency expression system for production of heterologous protein, their results also provide a strategy for the construction of a high-efficiency expression system. beyond the studies of "one-step" biotransformations, the exploration and development of multiple-enzyme biocatalytic systems is of great interest and is an important challenge to scientists. in past years, researchers have successfully engineered and constructed many important multiple-enzyme biocatalytic systems in vivo and in vitro [67, 68] , some of which are even composed of tens of enzyme components. for example, cellulosome is a multi-enzyme complex from anaerobic microorganisms which consists of a large number of enzymatic components arranged around non-enzymatic scaffolding proteins. because of its potential biotechnological applications in the conversion of intractable cellulosic biomass into sugars, cellulosome is of wide interest in enzymology and biotechnology. in a recent review, wang et al. [69] summarized the latest advances and future challenges in the reconstitution of cellulosome. besides describing the structure and assembly mode of cellulosome, the authors introduced a general strategy for reengineering cellulosome which also could be applied in the design and development of other multiple-enzyme complexes. at the end of the 20th century, many people predicted that enzymology as a mature field of research over one century would inevitably decline in importance. however, because of the development of new techniques and theories, and the need for sustainable industrial development, the scope of enzymology has unexpectedly been expanding rapidly in the past decade. for example, under the idea of systems, enzymology is robustly being integrated with all areas of biology and other related disciplines, as discussed in the articles mentioned above and other reviews elsewhere. with the rapid evolution of enzymology from a traditional science which focused on simple enzymological characterization to the cross-discipline, high-content modern form, more and more scientists believe that enzymology not only has a booming present, but also will have a bright future. protein phosphorylation from the perspective of systems biology receptor-like kinase crrlk1-l subfamily: novel motifs in extracellular domain and biological functions in plants a new family of regulators of calcineurin (rcans) gsk-3 modulates 9g8-mediated alternative splicing of tau exon 10 the protective effects of ubiquitin c-terminal hydrolase l1 on neurons deubiquitinase activity and regulation of antiviral innate immune responses by papain-like proteases of human coronavirus nl63 progress in the study of prokaryotic ubiquitin-like protein (pup)-proteasome system the function of prdm9 on mammalian recombination hotspots advances in relationship between deacetylase (sirtuin) and aging lysyl oxidases related to human diseases the research advances in ubiquitinindependent degradation of proteins transmembrane serine proteases 6: a newly discovered hepcidin regulator proteolytic networks in cancer analysis of inhibitory activity and antineoplastic effect of wild type rbti and its mutants suppression of glutamate synthase genes significantly affects carbon and nitrogen metabolism in rice the effects of dihydrofolate reductase gene on the development of pharyngeal arches a mutation of testis-specific lactate dehydrogenase gene found in male patients with unexplained infertility systems biology of microbial metabolism regulation and control of metabolic fluxes in microbes genetic polymorphism in metabolism and host defense enzymes: implications for human health risk assessment phenotype prediction of nonsynonymous single nucleotide polymorphisms in human phase ii drug/xenobiotic metabolizing enzymes: perspectives on molecular evolution study on the mechanism of effects of lomefloxacin on biological properties of bloom syndrome helicase study on the binding mode and mobility of hiv-1 integrase with l708, 906 inhibitor development of a high-throughput assay for the hiv-1 integrase disintegration reaction advances of study on translesion dna synthesis polymerase kappa in mammalian cells preparation and characterization of t7 endonuclease i with single active domain enzymology and ecology of the nitrogen cycle enzymology under global change: organic nitrogen turnover in alpine and sub-arctic soils analysis of the contribution of acid phosphatase to p efficiency in brassica napus under low phosphorus conditions temporal allocation of metabolic tolerance to transgenic bt cotton in beet armyworm functional proteomics: application of mass spectrometry to the study of enzymology in complex mixtures imaging enzymes at work: metabolic mapping by enzyme histochemistry measuring enzyme activity in single cells single-molecule approaches to characterizing kinetics of biomolecular interactions singlebiomolecule kinetics: the art of studying a single enzyme probing single enzyme kinetics in real-time single molecule enzymology: watching the reaction classic and contemporary approaches to modeling biochemical reactions network-based functional modeling of genomics, transcriptomics and metabolism in bacteria generic enzymatic rate equation cyclic adp-ribose and naadp: fraternal twin messengers for calcium signaling oxidative stress in schizophrenia: an integrated approach a meta-analysis of oxidative stress markers in schizophrenia a 21st century revisionist's view at a turning point in enzymology protein dynamics and enzyme catalysis: insights from simulations induced fit-lock and key" model in enzymic reactions emerging biomedical applications of synthetic biology systems metabolic engineering of microorganisms for natural and non-natural chemicals activity-based probes: discovering new biology and new drug targets activity-based protein profiling for biochemical pathway discovery in cancer a potent and selective inhibitor of kiaa1363/aadacl1 that impairs prostate cancer pathogenesis heterologous expression and characterization of the carboxylesterase from geobacillus stearothermophilus the enzyme function initiative r)-oxynitrilase from prunus salicina catalysed synthesis of (r)-ketone-cyanohydrin by enantioselective transcyanation enzyme promiscuity: a mechanistic and evolutionary perspective a novel organic-inorganic hybrid monolith for trypsin immobilization advances in enzyme immobilisation strategy and success for the directed evolution of enzymes chemical modification in the creation of novel biocatalysts enzyme engineering for enantioselectivity: from trial-and-error to rational design? computational protein design: engineering molecular diversity, nonnatural enzymes, nonbiological cofactor complexes, and membrane proteins isoprenoid pathway optimization for taxol precursor overproduction in escherichia coli protein trans-splicing based dual-vector delivery of the coagulation factor viii gene expression, characterization, and antimicrobial ability of t4 lysozyme from methylotrophic yeast hansenula polymorpha a16 expanding nature's small molecule diversity via in vitro biosynthetic pathway engineering combinatorial biosynthesis of polyketides--a perspective research advances on the assembly mode of cellulosomal macromolecular complexes open access this article is distributed under the terms of the creative commons attribution license which permits any use, distribution, and reproduction in any medium, provided the original author(s) and source are credited key: cord-011871-hlykwius authors: himmler, sebastian; van exel, job; perry-duxbury, meg; brouwer, werner title: willingness to pay for an early warning system for infectious diseases date: 2020-03-16 journal: eur j health econ doi: 10.1007/s10198-020-01171-2 sha: doc_id: 11871 cord_uid: hlykwius early warning systems for infectious diseases and foodborne outbreaks are designed with the aim of increasing the health safety of citizens. as a first step to determine whether investing in such a system offers value for money, this study used contingent valuation to estimate people’s willingness to pay for such an early warning system in six european countries. the contingent valuation experiment was conducted through online questionnaires administered in february to march 2018 to cross-sectional, representative samples in the uk, denmark, germany, hungary, italy, and the netherlands, yielding a total sample size of 3140. mean willingness to pay for an early warning system was €21.80 (median €10.00) per household per month. pooled regression results indicate that willingness to pay increased with household income and risk aversion, while they decreased with age. overall, our results indicate that approximately 80–90% of people would be willing to pay for an increase in health safety in the form of an early warning system for infectious diseases and food-borne outbreaks. however, our results have to be interpreted in light of the usual drawbacks of willingness to pay experiments. electronic supplementary material: the online version of this article (10.1007/s10198-020-01171-2) contains supplementary material, which is available to authorized users. increasing the health safety of citizens is an important policy goal in countries across the world. recent infectious outbreaks of, for example, ebola, sars, bird flu, and salmonella, emphasise that improving safety cannot always be realised by countries separately [1] . recently, for example, the european union has initiated an interdisciplinary research network that investigates the potential for an international, integrated early warning system for identifying, containing and mitigating large infectious outbreaks more rapidly (http://www.compa re-europ e.eu/). establishing and maintaining such a system would likely entail considerable costs. to determine whether this would be money well spent, it is essential to consider all its potential benefits. the relevant benefits could include a reduction in disease burden, increased feeling of safety, or the mitigation of economic consequences of infectious diseases and food-borne outbreaks, which can be considerable for countries, organisations and individuals. for instance, the economic impact of the ebola crisis in 2014-2015 on sierra leone, guinea and liberia was estimated at $2.8 billion [2] . however, in general, reliable evidence and estimates of these potential benefits of an early warning system, separately or overall, are scarce and difficult to obtain, especially in the case of multinational initiatives. in light of this and the fact that the full potential benefits would include, besides aspects such as health gains, also elements like the improved feeling of health safety, it is not possible to quantify the overall benefits of such an international early warning system based on existing data. therefore, in this study, we aim to provide an indication of the perceived overall value of such a system in terms of improving citizen's feelings of health safety. for that purpose, we first develop a contingent valuation willingnessto-pay approach, which provides such a valuation, given beliefs and sentiments in the population regarding all different aspects of a warning system. second, we apply this approach in six selected countries across europe (i.e., denmark, germany, hungary, italy, the netherlands, and the uk) to derive a range of estimates and assess the potential implications of our results on an international level. this paper summarises our efforts to accomplish these goals and its remainder is divided into four sections. first, we briefly summarise the findings from a previous literature review surrounding the methods that have been applied in similar contexts, namely valuing health safety, to motivate the chosen approach further. after that, we consecutively report on the design and administration of our experiment, the data analysis, present the results of our study, and conclude the paper with a discussion of the limitations and implications of our findings. the introduction of an international integrated warning system to increase health safety would not be necessary if communicable or infectious diseases were not a significant factor in the global burden of disease. the burden of communicable diseases in europe project found an average disease burden in germany alone of 33,116 disability adjusted life years (dalys) per year for influenza and 19,115 dalys per year for salmonella [3] . on a european level, influenza was estimated to be responsible for 81.8 dalys lost per 100,000 population between 2009 and 2013, corresponding to 412,673 dalys using the eu population size from 2011 [4] . considering these substantial effects of infectious diseases, some of the potential benefits of an international integrated warning system become clearer. of course, the real benefits also depend on the translation from warnings to effective interventions that prevent or mitigate the consequences of outbreaks. besides possible health gains resulting from this, there are also less tangible benefits from having a warning system, which include an increase in health safety and feeling more secure. the valuation of these benefits may be less straightforward than calculating potential dalys averted. the valuation of interventions affecting safety is relevant both within and outside the health care setting. for example, environmental and transportation research is concerned with interventions, which aim to improve the safety of recipients. perry-duxbury et al. conducted a literature review in which they examined the methodologies of empirical research valuing safety from all relevant fields, including environment, transportation and health [5] . of the 33 papers reviewed, 22 were found to use the contingent valuation method to value the effects of safety-affecting interventions. the four papers in the field of health that empirically valued interventions increasing health safety, all used a form of stated preference methodology. these papers aimed to estimate the value of reducing mortality risks [6] , preventing child maltreatment deaths [7] , reducing the risk of sexually transmitted diseases [8] and vaccinations in pandemic outbreaks [9] . the first three papers used willingness to pay (wtp) contingent valuation method, while the last paper used a discrete choice experiment to elicit valuations. the literature review identified income to be a significant predictor of wtp in all included contingent valuation studies [10] . a higher level of education was associated with a higher wtp in six of the nine papers that included information on education. age and gender both also had strong correlations with wtp. however, these correlations were positive in some of the studies and negative in others. the literature review also reported results regarding relationships of wtp with risk (perception). for example, individuals that had been directly or indirectly exposed to the outcome of interest reported a higher wtp, as did those who had a higher level of perceived risk, were more knowledgeable or more concerned about the issue, or were more concerned than others about the outcome under study. finally, study design elements were shown to affect wtp estimates. for example, presenting scenarios with higher baseline risk was associated with a higher wtp. in addition, different studies found that presenting higher intervention costs or more information about the intervention in the scenario description also affected the estimated wtp. however, the direction of the effect differed between studies. the information provided by the literature review guided some of the methodological choices of our study, which are described next. to estimate the wtp for an international integrated early warning system for infectious diseases and food-borne outbreaks, we conducted contingent valuation experiments utilising general population samples from six european countries: denmark, germany, hungary, italy, the netherlands, and the uk. sampling and administration of the wtp questionnaire were conducted by a professional sampling agency, from february to march 2018, using an online survey format. the sampling agency recruited participants from existing online panels. the survey was administered to citizens aged between 18 and 65. individuals aged 65 and above were not included for two reasons: first, recruiting elderly respondents from online panels can be challenging in some of the included countries. second, we wanted to limit our population to the (income) taxpayers, as we used a tax increase as payment vehicle in the experiment. the samples were aimed to be representative for national populations regarding age, gender and level of education, with a sample size of around 500 individuals per country. participants were able to complete the questionnaire on a computer or mobile device. they did not receive a personal financial reward for engaging in the experiment but could choose a charity, which would receive a small donation after completing the survey. participants had to consent to their information being used for research purposes and were free to drop out of the experiment at any time. the reasoning behind the country selection was to cover a variety of cultural perspectives relevant to the valuation of safety and public intervention. the latter was assessed by applying the three most relevant dimensions of hofstede's cultural dimensions theory in this context: individualism vs collectivism, masculinity, and uncertainty avoidance [11] . the included countries furthermore constitute a mix of different levels of social and economic development in europe. the questionnaire, which was initially developed in english, was translated into danish, german, hungarian, italian, and dutch by professional translators and checked for comprehensibility and consistency by native speakers. in designing the experiment and payment scales, gbp and eur values were assumed to be equivalent, while monetary values and payment scales were converted from gbp into dkk and huf using the mean exchange rate from february 2018. in the case of hungary, this was additionally adjusted for purchasing power [12] . payment scales were rounded to natural integer values in all survey versions to prevent peculiar payment options. the payment scale of the uk survey and the equivalent monetary values for danish crowns and hungarian forint can be found in online appendix d. before the launch of the main survey, the questionnaire was tested in both a group of experts in infectious diseases and food-borne outbreaks associated with the compare research network (n = 22) and a representative sample of the public in the uk (n = 134) in january 2018. the length of the survey was slightly reduced following the pilot tests. after this stage of piloting, the questionnaire was fielded in a representative sample of uk citizens (n = 533). to test the payment scale used in the experiment, we administered two additional surveys (n = 500 each): one with smaller payment options, and the other asking for yearly contributions instead of monthly contributions. the validity of the results of the three survey versions was assessed based on whether wtp was influenced by income and based on a comparison to a reference point (home contents insurance 1 ), which was included in the surveys. the initial payment scale performed best and was therefore used in all surveyed countries. the general design of the wtp experiment followed the structure of an existing survey, which was purposely designed to elicit the wtp for a quality-adjusted life year (qaly) [13] . after a brief introduction to the topic at hand and the purpose and design of the questionnaire (see online appendix a), respondents had to state their age and gender before describing their current health using a generic health instrument (eq-5d-5l). the following part of the questionnaire started with a "warm-up" wtp exercise, where participants had to state their wtp for a pair of shoes. this elicitation task was included to familiarise respondents with the procedure and to test whether the chosen approach resulted in reasonable estimates for a common market good. next, respondents started with the central wtp task: valuing the early warning system. a two-stage procedure consisting of a two-step payment scale approach and an open-ended question was applied to elicit individuals' wtp. the motivation for this approach has been outlined elsewhere [13] [14] [15] . in summary, it intends to provide precise and direct maximum wtp valuations, using a stepwise procedure that helps respondents to form and articulate their preferences. the scenario outlined to respondents was that establishing and maintaining an international integrated warning system, which could contain and mitigate infectious disease and food-borne outbreaks, naming ebola, sars, bird flu and salmonella as examples, is not without costs. participants then were asked to imagine that the funding of such an international warning system would take place through national taxation in the participating countries. all eligible people in their country (aged 18 and above) would have to contribute via monthly instalments starting immediately. the payment was framed as a recurrent tax since most respondents in european countries are likely familiar with similar forms of payments. this scenario did not include information on the magnitude of the potential health benefits. the reasoning behind that was to emphasise the perceived feelings of health safety due to such a system in the elicitation rather than a particular hypothetical gain in health, also because the potential benefits are uncertain at this stage. this could provide a broad valuation based on the beliefs and attitudes of the respondents themselves. information on the types of local systems already in place and how these would be integrated into this international system was also omitted. not only would this be cognitively burdening, the chosen approach also conformed more closely to the general definition of the compare project and hence warning system at this stage. while this leaves respondents with imperfect information, this was intentional, as our goal was to value a warning system which features are not yet fully clear, in terms of the incremental feelings of health safety that comes with it. in the first step of the initial stage of the willingnessto-pay experiment, respondents were asked to indicate the amounts they would definitely be willing to pay per month for having this international, integrated warning system, using a payment scale ordered from low to high gbp or eur values (0, 1, 2, 3, 4, 5, 10, 15, 20, 25, 30, 40, 50, 60, 70, 80, 100, 120, 150, 200, more). the payment scale for the uk contained the same values in gbp, the version for denmark contained the same values converted to dkk (and rounded), and the version of hungary was adjusted for purchasing power and converted to huf (and rounded). the hungarian and the danish scale are included in appendix d. individuals who chose the "more" option on the payment scale subsequently had to indicate a value higher than 200 in an open-ended question. individuals who chose 0 as their maximum wtp had to select one of the following options to specify the reason for this answer, with the following predefined options: (i) not worth more than 0, (ii) unable to pay more than 0, (iii) government task, or (iv) the option to formulate another reason in an open text field. the former two options were considered to indicate a true wtp of zero, while "government task" was designated as a protest zero. entries in the open text field were evaluated and labelled as either true zero or protest zero. individuals who chose a value between 1 and 200 were subsequently asked to mark the amounts they would definitely not be willing to pay per month on the same payment scale, excluding the wtp values they had selected in the preceding step. jointly, these two steps generated a wtp interval between the highest amount that a respondent definitely was willing to pay and the lowest amount he or she was definitely not willing to pay. in the second stage of the wtp procedure, respondents had to indicate an exact amount within this interval that was closest to the maximum that they would be willing to pay per month. respondents could specify decimals in this second stage, not limiting the wtp to integer values. the elicited wtp amounts in the second step were taken as the best approximation of people's wtp for the (health safety benefits from an) international integrated early warning system for infectious diseases. throughout the two steps, participants were reminded to keep their ability to pay in mind (their net monthly household income) before indicating any interval or specific value to prevent ex-ante mitigation [16] . the design and the exact wording of the wtp questions can be found in appendix c. the questionnaire continued with two additional wtp valuation scenarios involving different degrees of risk reduction and disease severity, which will not be discussed in this paper. subsequently, respondents had to provide further sociodemographic information. estimates for household income were obtained in a two-step process. respondents first selected an income range before indicating an exact amount. missing exact income amounts were imputed based on the sample means of the income interval selected in the first step, if applicable. respondents were furthermore asked about whether they or their family had ever been exposed to an emerging infectious disease or outbreak (yes/no), and about their general awareness related to emerging infectious diseases and foodborne outbreaks, which was queried using 12 statements and a 7-point likert scale. the statements comprised of a collection of aspects found to be relevant in this context based on the findings from the literature review (see online appendix b). finally, respondents completed a brief version of the health-risk attitude scale (hras) [17] , which consists of six statements about resolving risky health decisions that need to be ranked on a 7-point likert scale ranging from "totally disagree" to "totally agree". the survey ended with a module asking respondents whether they had home contents insurance, the size of the corresponding yearly premiums and how they would value the described early warning system in comparison to their contents insurance (lower, roughly the same or higher). these results of this final module were intended to serve two purposes: first, they were used to test different types of payment scales before the rollout of the main survey. second, comparing the contents insurance premiums people actually pay and the stated relative value of early warning system and contents insurance serves as a validity check of the stated wtp values. in addition to the survey data, we collected country aggregate estimates on the relevant dimensions of hofstede's cultural dimension theory (masculinity, individualism, and uncertainty avoidance) and the level of trust in public institutions [18] . before analysing the data, we converted all monetary values from danish, uk, and hungarian respondents to euro values using the average exchange rates during the month of sampling (7.45 dkk/€, 1.14 £/€, 312 huf/€). in the next step, cross-country data validity and comparability were assessed by exploratory, descriptive analysis. we first inspected the proportions of and reasons for zero wtp answers, distinguishing between true and protest zeros. we excluded protest zeros and wtp outliers from the remainder of the analysis. the latter was defined as wtp values larger than 5% of monthly household income. descriptive statistics were calculated based on the remaining wtp valuations. linear regression analysis was conducted on the wtp valuations from all six countries to examine which factors influenced the wtp answers and whether the observed effects were in line with theoretical considerations as well as previous empirical findings of wtp determinants (see section "background"). the regression analysis thus functions as a validity check for our experimental design and wtp results. we also explored the suitability of tobit or two-part-models for the regression analysis, however using root mean squared error and mean absolute error as performance criteria revealed that standard linear regression provided the best model fit. calculations were conducted using the pooled total sample, as well as the separate country-level samples. descriptive analysis and regression analyses were performed using stata 15 the total number of completed surveys from the six chosen european countries was 3140. unfortunately, information on the response rate or the share of respondents starting, but not finishing the survey could not be obtained from the sampling agency. on average, it took respondents 18.9 min (sd 11.2) to complete the questionnaire. the six samples were well balanced regarding age, gender and education in their respective countries for the aimed subset of individuals aged between 18 and 65. descriptive statistics of the respondents per country are shown in table 1 . the average gross monthly household income ranged from €1214 in hungary to €6417 in denmark. employment status and educational attainment varied between countries, as to be expected. the sub-samples also differed considerably in the rate of past exposure to infectious diseases and food-borne outbreaks (10% in the uk vs 62% in hungary). overall, 14.8% of respondents stated a wtp of zero, with a share of 7.3% in italy at the lower end and 23.2% in hungary at the upper end. of those with a wtp of zero, most respondents chose the pre-specified option "government task" (57.3%) and only to a lesser extent the options "not worth it" (17.2%) and "unable to pay" (15.3%) to justify a wtp of zero, with considerable differences between countries. of the 47 qualitative responses in the category "other", 40 were classified to be similar to "government task" or as protest answers. the remaining seven qualitative responses were more related to whether the system would be worth installing. these, therefore, were included in the "not worth it" category, which, together with "unable to pay" category, represent true zeros. the entirety of "government task" and further protest answers (n = 306) was treated as protest zeros and, therefore, not included in the following wtp estimates and regression analysis. table 2 presents the share of zero values per country as well as the indicated reasons for the zero valuations. the share of protest zeros among zeros varied between 53.5% in the uk and 78.6% in hungary. individuals who provided protest answers had a significantly lower income (p = 0.010), higher age (p < 0.001), lower level of education (p = 0.046) and only little awareness of outbreaks (p < 0.001) in comparison to respondents with non-protest answers. turning to the actual wtp estimates, the elicited values for the lower interval of the first stage of the wtp exercise ("definitely be willing to pay") had a mean of €14.68 (sd 23.65). the corresponding mean for the upper interval ("definitely not willing to pay") was €42.63 (sd 67.15). the second stage produced a mean stated wtp for an international integrated early warning system for infectious diseases and food-borne outbreaks of €25.17 (median €10.07) per month per household. the standard deviation of €42.87 exemplifies a considerable heterogeneity in wtp within and across countries. several outliers with values up to €1000 per month influence the mean wtp. the proportion of respondents with a wtp above €100 in the analysed sample was 5.0% and ranged from 0.7% in hungary to 8.8% in italy. some of these outliers might represent the real wtp of respondents, while others may be deliberate or incidental overstatements. applying the above-described criterion, 4.8% of responses qualified as outliers (n = 121) and were excluded from the remainder of the analysis. doing so reduced the mean monthly wtp from €25.17 to €21.80 in the remaining sample of 2713 observations. table 3 presents the corresponding values and further summary statistics, while. figure 1 presents the distributions of all wtp values on country level. for readability, values over €100 (4% of the total sample) are trimmed off. the mean monthly wtp varied from €8.89 in hungary and €28.33 in denmark. results from the included reference point, home contents insurance, revealed that for 51.1% of insurance holders (68.9% had this type of insurance) the perceived value of the warning system was more or less equal to the value of the contents insurance. in the subgroup that provided information on their monthly premiums, the mean difference between wtp and stated insurance premium was €5.28 (50.7% within a €10 range). a higher perceived value of the warning system (24.7%) coincided with a wtp, which was larger than the insurance premium in 56.6% of cases. a lower perceived value (24.3%) fell in line with a relatively lower wtp in 56.5% of cases. table 4 column one lists the results of regressing the wtp values on multiple individual characteristics using the pooled data from all six countries, excluding protest answers and outliers. to account for the correlation of errors within countries, we used cluster-robust standard errors on country level in the regression models. the number of observations dropped from 2713 to 2583, as some respondents did not provide any information on their household income. as the wtp data were skewed, we also analysed the data using log-transformed wtp values. however, here we present the results using the raw wtp values as the general results, and implications of both approaches were highly similar. moreover, the linear specification avoided having to drop zero wtp values and provides a more straightforward interpretation. the log wtp results can be made available upon request. income had a highly significant and positive non-linear effect on the wtp, while age significantly reduced the wtp. education did not affect wtp. the highest levels of awareness of outbreaks and health risk aversion (hras) seemed to influence wtp, although the coefficient of the former was not significant. past exposure, marital status, or not being employed, did not significantly affect wtp. the remaining columns of table 4 present the results on country level. factors affecting wtp differed considerably between countries with some coefficients even switching signs. household income significantly increases wtp in all six countries, whereas age was significantly negatively associated with wtp in three of the countries. consistently positive (but not always significant) coefficients were found for the highest quartiles of outbreak awareness and hras, i.e. being relatively most aware of the associated risks and being relatively most health risk-averse in general. better health was associated with lower wtp throughout all countries. alongside the differences in coefficients, the explanatory power of our model changed substantially between countries. the r 2 varied between 0.117 for the german model and 0.247 for the italian model. differences in model fit as measured by aic/bic and rmse were even more substantial. when including variables in a stepwise procedure, the conclusions for the pooled regression were reasonably stable across model specifications (see online appendix e). adding country dummy variables to the pooled model slightly diminished the effect of income. the respective coefficients of hungary, italy and the netherlands were significant compared to the uk as reference category. this result indicates that even after controlling for socioeconomic characteristics, including income, wtp significantly differed between countries. hofstede's cultural dimensions masculinity, individualism, and uncertainty avoidance, as well as trust in public institution further explained these differences (see online appendix e). to estimate the value of an international integrated early warning system for infectious diseases and food-borne outbreaks aimed at increasing health safety, we developed a two-stage contingent valuation experiment. a survey containing the experiment was administered to balanced samples from denmark, germany, hungary, italy, the netherlands, and the uk. the share of respondents indicating a wtp of zero varied between 7.3% in italy and 23.2% in hungary, of which most were protest zeros. excluding protest answers and outliers (with a wtp exceeding 5% of income), the elicited overall mean monthly wtp per household was €21.80 (median = €10.00). this value ranged from €8.89 (median = €3.85) in hungary to €28.33 (median = €13.42) in denmark. the corresponding standard deviations were standard errors in parentheses; *p < 0.10, **p < 0.05, ***p < 0.01; outliers defined as wtp over 5% of monthly income a sum score rescaled from 0 to 100 substantial, expressing either diverse or ill-formed preferences. differences between countries can partly be explained by the variation in purchasing power, hofstede's cultural dimensions and trust in public institutions. the results, in general, indicate that the majority of respondents see a certain value in the early warning system. regression analyses showed that throughout countries and models, income, as expected, was the most important determinant of the wtp values elicited in our experiment. before discussing the implications of our findings, we must acknowledge several limitations inherent to our analysis and the contingent valuation approach. individual wtp estimates are susceptible to the design and framing of a wtp exercise (see for example [19] ). for instance, by instructing respondents to consider other similar contributions to inform their wtp (see online appendix c), we may have introduced a possible anchor point for some individuals, biasing our results [20] . furthermore, by listing very serious (but low probability) threats like ebola, sars and bird flu in the description of what the system aims to contain and mitigate, respondents may have overestimated the potential health gains of the system. however, as the aim of our analysis was to capture gains in their feelings of safety in the valuation, this is of less concern. a possibly more problematic concern of this type of contingent valuation studies is the respondent's sensitivity to the chosen payment scale [19, 21] . it has also been reported that valuations are relatively insensitive to framing the payment as a monthly or yearly instalment [22] . to reduce the effects of such potential biases in our study, we tested two additional versions of our survey, varying payment scale and frequency of payment, and chose the survey version, which provided the most internally consistent results. the two-stage approach, asking respondents for a value they would definitely pay and a value they would definitely not pay before the actual valuation, also aims to reduce midpoint bias and scale sensitivity. including a "more" option in the payment scale was intended to decrease endpoint bias. a further limitation of wtp studies, in general, is the hypothetical nature of the experiment itself. whether respondents would indeed pay the elicited amounts in real life is questionable. research has shown that hypothetical wtp questions typically lead to an overestimation of actual wtp [23, 24] . a limitation specific to our analysis is that the actual unit of valuation, an international integrated early warning system for infectious diseases and food-borne outbreaks, is also a hypothetical construct, as it is not in existence yet. the survey included a concise description of its general purpose (online appendix a), but we did not provide any more detailed information on the actual functioning and effectiveness of such a system. we also do not know about respondents' expectations concerning potential future (health safety) benefits through such a system. besides these tangible benefits, individuals might also have incorporated potential improvements in the feeling of safety due to the system in their wtp valuation, as well as other benefits. respondents may have unrealistic expectations regarding the potential (health) benefits of the early warning system, leading to distorted wtp valuations. however, as mentioned earlier, individuals make similar decisions without complete knowledge of real risks or benefits when deciding on specific types of insurance coverage. in both cases, they include perceived risks and benefits in their decision-making. one further noteworthy limitation of our study is the exclusion of individuals aged 65 and above. one could argue that the wtp would be higher in the excluded group as they are in general more vulnerable to infectious diseases. we do not find strong evidence for this hypothesis, considering that the coefficients of age-squared are not significant in general and small in size. future studies could investigate this age group further. despite these limitations, there are several aspects, which generate some confidence in the validity of the chosen design and our findings. for instance, the included warm-up exercise eliciting the wtp for the market good shoes provided plausible results, with means ranging between €61.09 in hungary and €138.54 in denmark. these results suggest that the respondents understood the question format and the wtp elicitation exercises and answering formats. results from the survey module about contents insurance furthermore indicate, that the stated wtp, i.e. the perceived value of the system, somewhat corresponded to an actual wtp. this can be inferred from comparing elicited wtp and premiums paid for home contents insurance (as reported by respondents) in relation to respondents' indication of their relative value. for example, respondents who indicated the values of the warning system and home contents insurance to be similar, the mean difference in premiums and estimated wtp was €5.28 with half of the differences lying within an (admittedly arbitrary) €10 range. the results from our regression analysis, moreover, demonstrated that, in general, wtp behaved as expected. wtp increased with income and to some extent with the awareness of outbreaks and risk aversion. the positive effect of the level of trust in public institutions and the significance of the included cultural dimensions were further reassuring findings. considering that most of the mentioned limitations are inherent to willingness-to-pay approaches, one could wonder whether other methodologies, not based on stated preferences, would have been the more appropriate methodological choice. such methods could entail using valuations of statistical life years or monetising the potential health gain using qaly threshold values [25] . however, there are two main reasons, concerning feasibility (mainly due to the limited current knowledge about the warning system) and scope of the analysis, why this is not the case. first, the statistical life year approach requires the availability of certain types of (international) data, which, at this stage of the compare project are not available, yet, if they can be provided at all, or are difficult to obtain in general. using qaly thresholds, on the other hand, requires the availability of threshold values in all countries of interest, while explicit threshold values are only available for the uk and the netherlands. noteworthy in this context is also that some of the estimates of the value of statistical life years and qalys are based on willingness-to-pay studies, which had similar drawbacks as our study. second, and more importantly, applying either of these methodologies would shift the focus exclusively on valuing direct health gains of the warning system. we opted for the current methodology and operationalisation as we intended to also capture the society's valuation of the perceived feeling of safety that comes with the envisaged system. the applied methodology is admittedly not perfect, with results also reflecting beliefs and imperfect information of respondents, which, next to methodological limitations, warrants caution in their interpretation. notwithstanding this, our study provides results, which have implications for policymakers and stakeholders in the context of interventions increasing health safety of the population in european countries. for instance, in a more general sense, our results indicate that most european citizens seem to value an early warning system when using additional taxation as a payment mechanism in an experimental setting. aggregating our wtp estimates to a national or international level can inform discussions about appropriate funding of the warning system, given current knowledge and perceptions of the effectiveness of such a system. while we stress the explorative nature of our study, based on the median wtp estimates from table 3 , the relevant number of households (excluding the share of protesters), and assuming 50% of those households would be eligible to pay the additional tax, an aggregate wtp of €6.5bn for all six included countries per year would be estimated (see online appendix f). considering that health care spending on prevention is rather modest in the included countries (compared to spending on curative care), this may be considered a high amount. while the mentioned limitations related to estimates of individual wtp apply to the aggregate as well, it can help to give such a number a bit more context. a study from the netherlands in 2007 estimated a yearly comprehensive national spending on preventive measures aimed at infectious diseases of €261.46 per capita (inflation-adjusted €333.16 in 2018) [26] . this national spending includes vaccinations but in particular, infrastructure aimed at protection from infectious diseases like waste disposal and clean water technologies. an early warning system could be seen as an add-on to this existing infrastructure. on a per capita level (17.3 m citizens), the aggregated wtp in the netherlands would be €23.71, which corresponds to 7.1% of the previously calculated comprehensive national spending on infectious disease prevention. assuming that the calculated aggregate wtp corresponds to actual yearly costs and that the early warning system would reduce the burden of disease of influenza of 81.8 dalys per 100,000 in the six included european countries by 20%, e.g. through rapid sequencing of new types of influenza and timely vaccinations, the costs per daly averted would amount to €164,190. this ratio does not yet include dalys averted in other infectious diseases or food-borne outbreaks, nor does it account for the economic burden of such outbreaks, which can be considerable [2] , or more intangible benefits like the increased feeling of safety. in terms of the methodology used in this study and the specification of the contingent valuation approach, future research should investigate the use of more precise assumptions about the actual benefits of such a warning system and how this impacts the wtp valuations. as soon as information on the effectiveness of a compare like warning system is available, it could also be of interest to explore the value of the direct health gains, e.g. using a statistical life year approach or different national estimates of the value of health gains. overall, our analysis provided first estimates of the perceived value of this type of early warning system in european countries. while the used approach is clearly not without limitations, the results of our analysis can be relevant to policymakers when discussing investments in health safety on a european level in general, and an early warning system for infectious diseases in particular. however, future research will have to provide further information on what this system would look like, the costs associated with installing and maintaining such a system, and how effective it would be at actually increasing health safety, i.e. reducing the risks of pandemics and outbreaks as well as mitigating their impact, among european citizens. only then, it is possible to assess whether the investment in such a system is money well spent and health and welfare improving. international engagement is critical to fighting epidemics world bank group: 2014-2015 west africa ebola crisis: impact update. world bank fisc rep the disease burden of hepatitis b, influenza, measles and salmonellosis in germany: first results of the burden of communicable diseases in europe study impact of infectious diseases on population health using incidence-based disabilityadjusted life years (dalys): results from the burden of communicable diseases in europe study, european union and european economic area countries how to value safety in economic evaluations in health care? a review of applications in different sectors willingness to pay to reduce mortality risks: evidence from a three-country contingent valuation study a comparison of willingness to pay to prevent child maltreatment deaths in ecuador and the united states the economic impact of project mars (motivating adolescents to reduce sexual risk) acceptance of vaccinations in pandemic outbreaks: a discrete choice experiment the value of safety: a literature review. forthcoming national cultures in four dimensions: a researchbased theory of cultural differences among nations willingness to pay for a quality-adjusted life-year: the individual perspective willingness to pay for improved respiratory and cardiovascular health: a multiple-format, stated-preference approach validity of openended and payment scale approaches to eliciting willingness to pay eliciting willingness to pay without bias using follow-up certainty statements: comparisons between probably/definitely and a 10-point certainty scale the development of the health-risk attitude scale measuring trust in european public institutions how you ask is what you get: framing effects in willingness-to-pay for a qaly anchoring effects on consumers' willingness-to-pay and willingness-to-accept evidence of range bias in contingent valuation payment scales framing the willingness-to-pay question: impact on response pattern and mean willingness to pay what experimental protocol influence disparities between actual and hypothetical stated values? eliciting willingness to pay without bias: evidence from a field experiment measuring the value of a statistical life: problems and prospects towards a comprehensive estimate of national spending on prevention we would like to thank johan von ophem and other discussants at the lolahesg conference 2018 for their constructive feedback on a previous version of this paper.funding the data collection was funded by the european commission under the horizon 2020 research and innovation programme (grant agreement no. 643476) and part of the compare project (http:// www.compa re-europ e.eu/). s.f.w. himmler receives funding from a marie sklodowska-curie fellowship financed by the european commission (grant agreement no. 721402). conflict of interest the authors declare that they have no conflict of interest.informed consent all individual participants included in this study provided informed consent.open access this article is licensed under a creative commons attribution 4.0 international license, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the creative commons licence, and indicate if changes were made. the images or other third party material in this article are included in the article's creative commons licence, unless indicated otherwise in a credit line to the material. if material is not included in the article's creative commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. to view a copy of this licence, visit http://creat iveco mmons .org/licen ses/by/4.0/. key: cord-016564-od9mf2f2 authors: capodaglio, andrea g.; callegari, arianna title: online monitoring technologies for drinking water systems security date: 2009 journal: risk management of water supply and sanitation systems doi: 10.1007/978-90-481-2365-0_15 sha: doc_id: 16564 cord_uid: od9mf2f2 more stringent constraints placed nowadays on water companies to provide high quality drinking water, increasing water resources scarcity in many areas of the planet, forcing water companies to work on marginal water bodies for supply, and the threat of hostile actions by political extremists and terrorist groups, that may willingly and deliberately cause contamination of an otherwise safe supply, are recent issues that have spurred demand for more efficient and comprehensive online water monitoring technologies. traditionally, quality parameters associated with drinking water provision were monitored using routine grab samples followed by laboratory analysis. this approach only allowed to capture small data sets, mostly unrepresentative of the true variance at the source, and allowed potentially important events to occur undetected. this paper examines state-of-the-art technologies for online monitoring of water quality in supply water systems, and reports some recent application examples. a safe and reliable supply of drinking water is nowadays an issue that bears strategic significance worldwide. in many areas of the globe the demand for high quality freshwater far exceeds the available supply, already; it is estimated, for example (who, 2003) that over 2 million deaths each year are currently caused worldwide by pathogenic bacteria during the consumption of unsanitary water: it is easily foreseeable that in the future the issue of freshwater resource ______ * to whom correspondence should be addressed. andrea g. capodaglio, department of hydraulic & environmental engineering university of pavia, via ferrata 1, 27100 pavia, e-mail: italy capo@unipv.it and drinking water supply scarcity will continue to escalate, driven by population growth and economic development pressure. more stringent constraints placed nowadays on water companies to provide high quality drinking water, increasing water resources scarcity in many areas of the planet, forcing water companies to work on marginal water bodies for supply, and the threat of hostile actions by political extremists and terrorist groups, that may willingly and deliberately cause contamination of an otherwise safe supply, are recent issues that have spurred demand for more efficient and comprehensive online water monitoring technologies. micro-biological contamination of drinking water has an immediate effect on the emergence of infectious diseases. availability of micro-biologically safe drinking water is probably the most effective and economical way to ensure public health. in addition to "traditional" microbiological contamination by human waste, contamination of drinking water sources with pesticides and herbicides as part of the major contaminating factors is a growing problem worldwide. during the last 2 decades, several studies revealed the presence of hazardous contaminants in wastewater effluents, being discharged in rivers and streams due to "normal" anthropic activities, including pesticides (öllers et al., 2001) , natural and synthetic hormones (kolpin et al., 2002) , plasticizers, personal care products and pharmaceuticals compounds (daughton and ternes, 1999; jones et al., 2002) . a recent study estimated that there are 1,415 human pathogens including 217 viruses and prions, 538 bacteria and ricketssiae, 307 fungi, 66 protozoa and 287 helminths; about 12% are considered to be emerging pathogens. their number continues to grow, as evidenced by the relatively recent emergence of severe acute respiratory syndrome (sars), caused by a novel coronavirus. in the past 30-40 years, on average at least one new pathogen has been identified each year, and many more have re-emerged after periods of inactivity or by appearing in areas where they were not previously reported (cunliffe, 2008) . pathogens identified in recent years include a range of organisms that can be transmitted directly from water or by water-related vectors. while a high proportion of the substantial burden of water-borne disease is caused by classical by pathogens such as salmonella typhi and vibrio cholerae, the spectrum of challenges is expanding (table 1) . there are many reasons why water-borne pathogens emerge. in some cases this can be due to advancements in disease diagnosis and detection of the causative organisms: for example, it is likely that cryptosporidium caused diarrhoeal illness before it was diagnosed as a human pathogen in 1976. in other cases true emergence can be caused by factors such as new environments, new technologies and changes in human behaviour. some pathogens have emerged for a combination of reasons. legionella was first detected in 1977 following the 1976 outbreak of legionnaires' disease in philadelphia. however, increased use of water-cooled air conditioning systems and spa pools has contributed to increased incidence of legionnaires' disease. as far as deliberate human hostile actions on urban water supplies, although none has been reported to date, in january, 2002, the fbi circulated a reserved bulletin warning water industry managers that al-qaida terrorists may have been studying american dams and water-supply systems in preparation for new attacks (ionlife, 2002) . traditionally, quality parameters associated with drinking water provision were monitored using routine grab samples followed by laboratory analysis. this approach only allowed to capture small data sets, mostly unrepresentative of the true variance at the source, and allowed potentially important events to occur undetected. it is clear, that, in view of what just reported, this can no longer be considered a satisfactory procedure, and that online monitoring of water supplies, for a larger number of parameters than currently available, is quickly becoming an unavoidable choice. water utilities around the world are already using some form of online monitoring to warn of contamination of drinking water, in anticipation of yetto-be specified regulations: in the united states, turbidity is currently the only quality indicator for which there is a regulatory requirement for continuous online monitoring; in europe, the eu drinking water monitoring regulations (council directive 98/83/ec) do not specifically require online measurements (awwa, 2002). online monitoring is usually defined as the unattended sampling, analysis and reporting of a parameter; it produces a sequence of data at much greater frequency than that permitted by manual (grab) sampling and it also allows real-time feedback for either process control, water quality characterization for operational or regulatory purposes, and alert/alarm purposes. online monitoring can be carried out at onsite as well as remote locations and will deliver measurements at intervals of seconds-to-minutes apart. clearly, online instrumentation must be placed at representative locations in the water system and must be periodically maintained by qualified technical personnel. monitoring requirements can be defined in relationship to: • source water quality: (a) variability, in space and time (very low for groundwater, low for lakes, high for rivers); (b) vulnerability (type and location of possible contaminating activity), time-of-travel of the contaminant to the intake, effectiveness of barriers, control options after an alarm • water treatment: process optimization options and response times, sampling frequency must allow adequate process control • distribution systems: minimization of deterioration of water quality over time and distance, early detection of cross-connections and water losses in addition, it must be considered that online monitors could have different sensitivity and selectivity according to the matrix and range of concentrations analyzed. a multibarrier approach to drinking water quality protection, such as those that are commonly used by facilities worldwide, is based on the concept that contaminants must be subject to as many points of control/treatment (barriers) as possible prior to the tap. the ideal location for control of contaminants is as close to the source as possible. a source water with low vulnerability is therefore characterized by few potential contaminant activities, transit time longer than that required for laboratory analysis, and the presence of multiple physical barriers between contaminating activities and point of intake. in a source water with moderate vulnerability, online monitoring of surrogate parameters (such as toc, doc, uv 254 , ph and conductivity) may be considered to keep track of potential pollution. in a high vulnerability water source, online monitoring of chemical-physical-biological parameters (turbidity, ph, conductivity, redox, fish toxicity) and surrogate parameters in addition to specific indicators (e.g. vocs, phenols and specific toxicity tests) may be preferred. this is summarized in table 2 . some parameters commonly used to monitor in online mode a supply source are listed in table 3 . parameters used to monitor online water treatment are listed in table 4 . both tables list also parameters for which online monitoring technology may not be widely available, nor field proved at the moment, but that may be useful for the given purpose (awwa, 2002) . the availability of real-time analytical information is one of the key benefits of online monitoring instrumentation: this information must however be conveyed to the appropriate user by means of a data collection and transmission system which is often referred to as scada, which consists of the individual online instruments, connected to programmable logic controllers (plcs) or remote telemetry units (rtus), that convert instrument outputs to the desired units, compare them to criteria set by the user, and generate signals for alarm or control to other process equipment. a host computer, that can be used to visualize or store data, or to further utilize them for specific purposes, almost always complements these systems. generally speaking, online monitoring instrumentation can be divided in: • physical monitors (turbidity, particles, color, conductivity, tds, hardness, alkalinity, acidity, streaming current, radioactivity, temperature, redox potential) • inorganic monitors (ph and do, disinfectants, such as chlorines and ozone, metals, fluoride, nutrients, cyanide) • organic monitors (carbon and hydrocarbons, uv adsorption, vocs, pesticides, disinfection by-products) • biological monitors (nonspecific, algae, protozoa, pathogens) • hydraulic monitors (flow, level and pressure) this paper will focus on the four former classes, discussing for each basic operating principles, and evaluation of the technology for online applications in the water distributing system. a wide array of technologies can be used for monitoring physical parameters, among them: light scattering/blocking (turbidity, particles, ss), light absorbance (color), electrochemical (conductivity, hardness, redox), electrophoretic (streaming current), chemical tritration (alkalinity, acidity, hardness) and other (radioactivity, temperature) (table 5) . turbidity refers to the clarity of a water sample, and can be defined as an optical property that causes light to be scattered and absorbed by suspended particulate matter (inorganic or organic) in the sample. it is one of the most commonly monitored parameters in the water industry, and the one that is most amenable to online monitoring. a turbidimeter consists of a light source, a sample cell and a photodetector; commercial instruments differ in the type of light source and the number and location of photodetectors. color is caused by the absorption of visible light by dissolved/colloidal substances or small suspended particles and can be an indication of pollution by disinfection by-products, industrial pollutants and/or metals, although it does not, by itself, represent a health risk. color is in fact regulated as an aesthetic parameter only and is seldom monitored online, except in cases where the water source is subject to industrial effluents and/or storm runoff inputs. color is measured either visually or spectrophotometrically. dissolved organic/inorganic impurities (tds) are compounds of different nature and sources, and constitute the solid residue after all the water evaporates. tds must be determined gravimetrically in the laboratory, however, since these species tend to increase the electrical conductivity of the solution, conductivity is often used as a surrogate measure for tds, especially when in online mode. hardness is defined as the sum of divalent cations in a water sample, and is relevant in drinking water systems for aesthetic reasons and for corrosion concerns. alkalinity is defined as the acid-neutralizing capacity of a solution. both can be determined by chemical tritration (hardness with edta, alkalinity with an acid solution). inorganic monitors are used in online mode to detect influent and effluent water quality, and for treatment process control; the applicable technologies are listed in table 6 . online monitoring of inorganic constituents is still in the early phase for many elements of interest to drinking water concerns. for metals, available technology is an adaptation to automatic operation mode of complex colorimetric methods developed for laboratory applications, and therefore turns out to be expensive and/or complex to operate, nor still suitable for installation in remote or unmanned sites. also, for many metals of interest in drinking water systems (as, cd, pb, hg, se, zn), online monitoring technologies do not exist at all. some promise for future application comes from developments in optode technology, coupled with miniaturized spectrophotometry. the technology for online monitoring of organic compounds includes toc analyzers, uv absorption and differential spectroscopy; it is much more developed than that for inorganics, for this reason, although neither eu nor us regulations require online monitoring of these substances, many drinking water utilities routinely use online organics monitoring to some degree. toc methods measure the carbon content of dissolved and particulate organic matter in the water, without giving information about the nature of this organic substances. table 7 shows the different fractions of carbon measured by an organic carbon analyzer. toc measurement is defined by well established methods and occurs generally according to a four-step process: sample treatment (filtration), inorg. c removal (acidification to ph < 2), oxidation (with uv, catalyzed uv, chemical oxidation, etc.) and co 2 determination (ndir detectors, colorimetry, or conductivity methods). detection limits depend on the specific technique's operating temperature (usually >0.2 mg/l for low temp., >1 mg/l for high temp.). most organic compounds found in water absorb uv radiation: using a uv spectrometer it is therefore possible to estimate the concentration of these compounds. originally, a uv light source with the single wave length of 254 nm was used for such measures, however, in recent years, instrumentation reading the entire uv-vis spectrum (200-750 nm) has been developed and marketed (s-can, 2008) . uv absorption is a well-defined and commonly used methodology; evidence shows strong correlation between these measurements and organic carbon concentration measured with standard methods such as toc or others (figure 1 ). in addition, it has been shown that other parameters can be measured indirectly by correlating their concentration values to uv absorption in the full spectrum ( figure 2) ; several commonly sought organic compounds have typical absorption spectra that make their identification quite easy with appropriate instrumentation (figure 3) . hydrocarbons in general are probably the main class of water pollutants found in surface and ground water (i.e., source water for drinking water systems). methods for online hydrocarbon detection include: fluorometry, reflectivity, light scattering and turbidity measurement, ultrasonic methods, electrical conductivity, spectroscopy, gas-phase detection (after volatilization), resistance-based sensors; some of these methods, however, give just an indication of the presence/absence of oil on the water surface. volatile organic compounds (vocs), including, among the others, aromatic compounds, halogenates and trihalometanes, are compounds that evaporate when exposed to air and can be of health concern when found in drinking water systems (trihalometanes are disinfection by-products -dbps -that can be precursors to the formation of carcinogens). their presence in drinking water can be a symptom of accidental pollution in the source water, of treatment failure/deficiency, or of incorrect disinfection procedures. current online monitoring technologies for vocs include purge-and-trap gaschromatography with flame ionization (fid), electron capture (ecd) or photoionization detectors or mass spectrometry (ms). detection limits for different substances vary according to the detector method. pesticides, including insecticides, fungicides and herbicides comprise triazines and phenlylurea compounds; they are monitored in drinking water systems in order to: detect accidental pollution in source waters, and check the effectiveness of treatment specifically designed to remove such substances. online monitoring of pesticides can be carried out using composite techniques, such as: • high pressure liquid chromatography (hplc)/diode array (da) detection, consists of extraction and enrichment, chromatographic separation and da detection. • gas chromatography (gc), consists of extraction and enrichment, gc separation and mass spectrometer (ms) detection. • liquid chromatography/mass spectrometry, consists in extraction and enrichment, lc separation and ms, thermospray, electrospray or particle beam detection. each technique is capable to optimally detect a group of compounds, for example, hplc/da can be used to analyse; atrazine, chlortoluron, cyanazine, desethylkatrazine, diuron, hexazinone, isoproruton, linuron, metazachlor, methabenzthiszuron, metobrorumon, metolachlor, metoxuron, monolinuron, sebutylazine, simazine and terbutylazine. in theory, any analytical laboratory method can be adapted for use as online measurement, provided that the requirements for consumables and manual intervention can be minimized: current online systems are often a "robotized" adaptation of known offline laboratory procedures, however, not always this solution is the most efficient one. a series of novel technologies, such as optochemical sensors, biosensors, and microbiological sensors are being tested for organics and hydrocarbon analysis. advances already in use include differential uv spectroscopy for dbps detection and microphase solid-phase extraction (spe) for the analysis of semivolatile organics (yongtao et al., 2000) . there are two basic types of biological monitors currently in use: those that use biological species as indicators of the presence of contaminants of concern (e.g. toxic chemicals), and those that screen for the presence of biological species of concern (e.g. nuisance algae, pathogens). in common us terminology, the term biomonitor usually indicates the former, and is in fact used as synonymous with toxicity monitor. in eu terminology, biomonitor refers generally to all types of biologically-based systems. at the present time, many existing biological monitors are quite new and can be considered experimental/unique applications. table 8 shows an overview of the most common types of online biological monitors. sensitivity of test organism to individual compounds must be determined initially. online biological monitors are a very active area of r&d due to increasing regulatory and public demand pressures. while bacterial-based systems show great sensitivity and ease of operation, and development in this area will likely derive from improved fingerprinting of organisms and maintenance cost reduction, most advances can be expected from protozoan monitor technology, with techniques in uv absorption/scattering analysis that may soon allow automated detection of cryptosporidium and giardia. also, molecular techniques initially applied to the recognition of the genomic sequence of specific organisms in clinical applications (bej, 2003) , have also shown great potential for use in the detection of pathogens in water, and are producing extremely interesting results that could lead to widespread online use in the very next future (see also section 5). chemical fingerprinting describes the use of a unique chemical signature, isotopic ratio, mineral species, or pattern analysis to identify different chemicals. optical fingerprinting by uv, vis, and nir absorption spectroscopy can be effectively achieved by low-cost and compact spectrometric devices, that can also be linked to an online diagnostic system, to directly identify some compounds (e.g. benzene) present in the water or to give an indication of the possibility of the presence of related compounds. in optical fingerprinting, a wide portion of the uv, vis and nir spectrum can be monitored simultaneously at high measurement frequency (minutes or fractions thereof); figure 4 shows the spectral fingerprint of a typical municipal wastewater, in the range 230-630 nm, together with three other spectral readings from the same source that were recorded within 18 min from the first. these show clearly different features, indicating a pronounced change in water quality. although this alone will not, in general, indicate the compound or compounds responsible for the change (they will need to be further investigated, if a definite answer is desired), but that can nevertheless trigger an alert to the system's operator, indicating deviation from routine conditions. some relevant applications taken from recent literature, are illustrated in the following sections, as examples of the current state-of-the-art in online monitoring technology. the river trent drains the large, mainly urban, catchment of midlands, with 4 million residents, in the uk; although traditionally it has been considered too polluted to serve as a drinking water supply source, the river has undergone marked improvements in quality until, in 1996, it was targeted as such. water is currently withdrawn from the river into bankside storage lakes, from where is taken to a purification plant. at this point, it became necessary to identify a suitable online monitoring strategy in order to protect this planned development (kirmeyer et al., 2002) . in order to protect water quality and provide operational data to the treatment plant operators, a list of parameters subject to online/laboratory monitoring has been established (table 9 ). online monitors provide an alarm when a preset warning limit is exceeded, prompting immediate additional investigation. when a second, "action" preset limit is exceeded, water extraction into storage is automatically terminated. online monitors include traditional inorganic, organic and oil monitors, as well as an automated liquid chromatography (hplc) system for monitoring organics, originally developed for analysis of triazine and phenylurea in the rhine river, and uniquelly adapted to detect other acid herbicides and phenolic compounds known to be frequently present in the trent, at levels < 1.0 μg/l. vocs are detected by means of a purge-and-trap gc analyzer. maintenance requirements are relatively high (each unit requires 2-3 h of onsite attendance twice a week, in addition to the time required to review data, prepare buffers and standards, etc.). overall, in about 10 years of operation, the system has recorded several incidents of parameter abnormality in the river trent, including ammonia toxicity events and contamination by various herbicides. it has been confirmed that online monitoring gives a far better definition of each recorded event than otherwise available, while spot samples analyzed at the laboratory in parallel have always been in good agreement with the online results. the llobregat river, near barcelona (spain) is characterized by a highly variable flowrate; to ensure sufficient supply during drought periods, dams have been built along its course. in addition, a continuous, automatic system with ten stations along its course has been put in place to monitor critical water quality parameters; these may vary depending on the monitoring location, in order to account for different needs and conditions. there are three types of stations: basic (1, upstream), complete (2, midstream) and special (7, mid-to-downstream); these are described in table 10 . the objectives of the monitoring network are: • to provide warning of pollution incidents, ensuring safety of the water produced by the purification plants • to control instream water quality by monitoring changes in pollution indicators • to provide information for the operators of the water systems and planners through data access with a specifically created scada user interface in the course of several years of operation the automatic water quality monitoring network has recorded various incidents of pollution, including: • repeated occurrences of salinity discharges from potash mines, caused by accidental breakage of collectors or leaching due to rainfall • an event of chromium pollution in a tributary stream, due to industrial source discharges • multiple overflows of drainage and sewer systems causing variations of turbidity, toc, ammonia, uv adsorption and do • highly significant variations of physical and chemical parameters (mainly do, ph) due to photosynthetic activity 4.3. on-line monitoring networks for drinking water security of karst water springs in geological karstic formations are the main source for drinking water supply for about 60% of the austrian population. water quality from these springs shows rapid, occasional instabilities for some parameters, caused by natural events, such as heavy rainfalls. rapid fluctuations of raw water quality, especially concerning turbidity, but also elevated concentrations of dissolved organic substances, and increased bacteria counts can occur in an unpredictable way during storm weather. furthermore, anthropogenic events like accidents and spills in proximity of the springs might also affect the quality of the raw water. the raw water quality is obviously one of the most important factors determining the final quality of the drinking water, in addition, rapid changes of source quality can limit the efficient usage of treatment procedures during drinking water production. as it is impossible to predict the impact of such events, it is vital to enhance drinking water security by monitoring the composition of the raw water continuously. main springs and important locations in the trunk mains had been monitored online since the late 1990s. buildings equipped with power supply, pipe installations and special foundations had to be built for housing cabinet analysers, for monitoring spectral absorption coefficient (uv 254 ), toc, turbidity, electrical conductivity and ph. in year 2000, a novel submersible uv-vis-spectrometer, introduced by s::can messtechnik gmbh, capable of measuring light absorbance in the spectral range of 200 nm to 750 nm, and thus turbidity, organic carbons (e.g. toc, doc) and nitrate without any sample preparation were adopted. this new monitoring equipment provided new opportunities to the waterworks by allowing to monitor the totality of springs, even those without power supply, and by eliminating pumps, filters, membranes and reagents that were used by the old instrumentation, lowering substantially the overall cost of monitoring operations (weingartner and hosftaeder, 2006) . at present, vienna waterworks operates a s::can-based monitoring systems in order to monitor turbidity, sac254, nitrate, toc, doc, temperature and electric conductivity at 22 locations, since monitoring just one parameter would not meet the needs of efficient and safe drinking water supply, as different types of events cause different changes in the composition of the spring water. the results of these monitoring systems are transferred in real time to an early warning system that can be accessed from four central stations. this early warning system manages the raw water sources 24 h a day. whenever current readings exceed limits that are specific to each parameter, the water of the spring of concern will not be used for drinking water production but drained away. oxidation of organic materials for drinking water disinfection is a commonly applied treatment step. during oxidation of natural organic substances in the source water, for example using ozone, large molecules, such as humic and fulvic acids, are cracked into smaller ones. a side-effect of this procedure is the increased availability of microbiologically assimilable organic carbon (aoc): whereas the original large molecules are not readily accessible to microorganisms, the smaller oxidation products can be digested and thus can stimulate bacterial regrowth in the distribution network. aoc is an important parameter especially in drinking water networks where no residual disinfectant is present in the water; its measurement is performed using cell cultures, which is a lengthy procedure. online uv/vis spectroscopy has proven itself as a tool that allows the collection of specific information on the removal efficiency for and subsequent concentrations of (organic) substances in water. the use of two on-line spectrometer instruments, placed before and after a treatment step, and the calculation of the differential spectrum between these two sites could open up a further area in water quality monitoring and process control as it allows calculation and prediction of water quality parameters previously unavailable (van der broeke et al., 2007) . two on-line spectrometers probes were installed in the pilot plant of amsterdam waterworks, weesperkarspel. in this pilot plant, the full treatment train of the waterworks is represented. the source, an artificial lake with high concentrations of natural organic matter, is sequentially treated in an ozone reactor (four bubble columns), pellet softening reactor, biological activated carbon (bac) filtration reactor and finally a slow sand filter. a parallel bac reactor was fed with water from the full scale treatment plant, which uses identical raw water, to be able to verify the effects of the changes in the ozone settings on water quality. the aoc calibration obtained in this way is a surrogate parameter, in the sense that the aoc concentrations in the water are far below the concentrations that can be distinguished using a uv/vis spectrophotometer without sample pre-concentration. the developed calibration, in combination with the on-line spectrometer probe, allows for on-line, in-situ measurement of dissolved ozone concentrations. the use of two instruments simultaneously, required to perform on-line differential measurements, allowed the prediction of the changes in aoc levels in individual treatment steps in real-time. the software ana::larm, for use with online uv spectrometers in a speciallydesigned configuration ( figure 5 ), has been developed specifically for contaminant alarm systems based on thousands of spectra, and is successfully used in several applications in europe. the training of the alarm parameters can be done by the instrument automatically, or is done manually guided by the pc software. it allows for a very simple half-or fully automatic setup and configuration of up to eight spectral alarm parameters within a few minutes. it will react on any type of figure 5 . configuration of online monitoring station for use with the software ana::larm organic contamination that provides an absorption signal in the uv range. the alarm sensitivity for many organic contaminants is between 1 and 500 ppb. at the same time it is most insensitive to any fluctuation of the matrix within the natural "normal" range, and thus keeps false alarms to an unmatched minimum. the approach and methods used are absolutely new and unique, and open a completely new perspective for water monitoring beyond the trending of "classical" concentration parameters (s-can, 2008b) . a contaminant warning system (cws) as an integrated tool that employs in-situ sensors, supervisory control and data acquisition (scada) systems, and water quality event detection systems (eds) to continuously monitor network conditions and warn operations personnel of any potential contamination events, has been studied by the us environmental protection agency (epa) and sandia national laboratories (hasan, et al., 2004; grayman, et al., 2001) . the sensor component can be comprised of various water quality sensing platforms, including contaminant-specific sensors, or existing water quality sensors (e.g., ph, cl, electrical conductivity, etc.) as currently installed in many municipal water distribution systems to provide "surrogate" data to the cws's. it has in fact been proved by experiments conducted in laboratory and pipe test loop systems, that a majority of potential contaminants will change values of at least a surrogate parameter away from normal background levels (byer and carlson, 2005; cook et al., 2005; hall et al., 2007) . monitoring of surrogate parameters can therefore provide information on the presence of contaminants within a distribution system and this information can be transmitted to a central processing location through the scada system. the challenge is to analyze the surrogate parameter signals to accurately identify changes in water quality that are significantly beyond the range of the ambient variability of the background water quality. the recently developed the canary eds software (hart et al., 2007) is an open-source software platform that gathers water quality signal inputs from scada systems and processes the data using one or more event detection algorithms, using a number of statistical models to determine the probability of an anomalous water quality event occurring for a given time step and monitoring location within the distribution network (mckenna and hart, 2008 ). the canary system has not been used in real applications, yet, and offline case studies, based on real hystorical data, have evidentiated some problems in correctly pinpointing alarm causes, as changes in the network hydraulics (such as opening and closing valves, draining/filling of storage tanks and pump operations) can cause significant changes in water quality as waters of different ages and from different sources mix within the network. the existence of escherichia coli (e.coli) in drinking water is an important indicator of faecal pathogens and potential micro biological contamination. currently available detection methods for its presence in drinking water distribution networks are inadequate, for multiple reasons: first of all, current methods are very time consuming meaning that contamination will already have reached end-users before laboratory results are available; secondly, current sampling and analysis procedures lead to detection success rates of only 5% to maximum 25% (with optimized sample taking). research by scientists from the dutch kiwa shows that by using a network of on-line sensors, the success rate can be increased to 80% (koerkamp and van wijlen, 2008) . the core of this labonline system is based on a combination of a concentrator unit and a sensor system using disposable chips, which prevent the sensor system itself from cross-contaminations and guarantee reliable and high quality measurements in time. initially designed for e.coli, the system is theoretically capable of detecting a broad group of microbiological contaminants like bacteria and viruses. industrial production is scheduled for late 2009. the same research group developed a sensor technology for pesticides based on a combination of an integrated optic chip, a biochemical transduction layer (micro)-fluidics, electronics and data acquisition and system control software, called optiqua mobilelab. the optiqua mobilelab sensor for the detection of pesticides will have the following characteristics: low cost per analysis, easy to use, detection of five pesticides (simazine, atrazine, glyphosat, ampa, bam (2,6-dichlorbenzamide), detection of additional pesticides (available in later stages), on site detection, prompt analysis results (minutes), high resolution (e.g. detection limits 0.05 μg/l with a dynamic range of 0.05 -10 μg/l), robust, low maintenance system. knowledge of the sequence of microbial genomes has led to the development of molecular methods for detection of microbial pathogens in clinical specimens as well as water and other environmental samples. a wide array of molecular techniques has therefore been applied to the study of microbiological water quality issues. the application of molecular techniques, such as pcr (polymerase chain reaction) has generated a great deal of valuable information on the occurrence, diversity, and biology of pathogens in water (loge et al., 2002) . in addition, molecular methods demonstrate rapid detection and enhanced specificity compared to other analytical methods. a micro analysis system for water pathogen monitoring consists of a micro polymerase chain reaction (pcr) chip integrated with a continuous-flow microarray that is able to reduce the analysis time from about 24 h to within several hours as compared with the existing epa approved methods was presented by yong (2008) . pathogen samples were successfully detected by the micro analysis system through dna amplification by the micro pcr chip followed by direct transfer of the amplicons to the microarray for detection. in addition to one species monitoring, the system shows potential in direct monitoring of a range of pathogens at the same time through pcr and different probes immobilized on microarrays. comprehensive two-dimensional gas chromatography also referred to as gcxgc, is an analytical technique in which all the eluted compounds from a first column are successively submitted to a new separation in a second column with different selectivity. contrary to gas chromatography (gc) which employs only one chromatographic column, gcxgc uses two chromatographic columns, coupled in series, with a modulator at their junction. developed in 1991, due to its principle, gcxgc offers a much better capacity of separation and a better sensitivity than conventional gas chromatography. thanks to the sensitivity of gcxgc (three-to fivefold higher than gc) some compounds can be detected at the ng/l level. semard et al. (2008) applied this technique to the screening of wastewater and effluents samples. a large range of drugs (antidepressors, antibiotics, anticoagulants…), personal care products (sunscreens, antiseptics, cosmetics…) and carcinogen compounds were found in the raw waste water samples. in addition to the above mentioned micropollutants, a wide variety of nitrogen aliphatic and aromatic structures that could act as dbp (disinfection by-products) precursors, were also uncovered. this technology does not yet have the potential for direct online application. courtois et al. (2008) also propose a molecular technique, based on polymerase chain reactions to detect pathogens. to improve pcr diagnostics for routine analysis purposes, they focus on the processing of the sample, which is crucial for the robustness and the overall performance of the method. their objectives in sample preparation are to increase the concentration of the target organisms to the practical operating range of a given pcr assay; and to produce a purified dna extract that would be representative of the initial water sample and would be free of pcr-inhibitory substances. this can be achieved by means of a twostep ultrafiltration (uf) procedure by using protoptype hollow fiber uf cartridge, and a commercial uf centrifugal concentrator. this paper has overviewed existing instrumentation applicable to water supply online monitoring, and examined a few state-of-the-art application examples. it is clear that technological development in this field is very rapid, and that astonishing advances are anticipated in several areas (fingerprinting, optochemical sensors, biosensors, molecular techniques). software applications, together with new generation sensors, are also contributing to the identification of otherwise difficultly monitored parameters. in spite of the high technology being developed, monitoring costs are bound to become a lesser and lesser part of a water utility budget due to the fact that automation and technological simplification will abate the human cost factor (maintenance and other labour forms) and reduce significantly the complexity of procedures (with those, of reagent requirements, etc.). proper interpretation and use of the growing mass of water quality data that will become available through new technologies will allow better management of water resources, and water treatment and distribution facilities. online monitoring for drinking water utilities molecular based methods for the detection of microbial pathogens in the environment real-time detection of intentional chemical contamination in decision support system for water distribution system monitoring for homeland security universal concentration for large volumes of water samples before molecular detection of waterborne pathogens emerging enteric and potentially waterborne pathogens in the region ternes (1999) pharmaceuticals and personal care products in the environment: agents of subtle change? design of early warning and predictive source water monitoring systems on-line water quality parameters as indicators of distribution system contamination canary: a water quality event detection algorithm development tool safeguarding the security of public water supplies using early warning systems: a brief review case studies of online monitoring systems next generation sensors for detection of pesticides and e.coli in water pharmaceuticals, hormones, and other organic wastewater contaminants in u.s. streams, 1999-2000: a national reconnaissance how safe is our water? the threat of terrorism human pharmaceuticals in the aquatic environment pcr detection of specific pathogens in water: a risk-based analysis on-line identification of adverse water quality events from monitoring of surrogate data: canary software simultaneous quantification of neutral and acidic pharmaceuticals and pesticides at the low-ng/l level in surface waters use of on-line uv/vis-spectrometry in the measurement of dissolved ozone and aoc concentrations in drinking water treatment on-line monitoring networks for drinking water security of karst water. all about karst and water, vienna. who (2003) emerging issues in water and infectious disease. world health organization ) micro analysis system for water pathogen monitoring spe twin-pal. online in situ monitoring of semivolatile organic compounds in water key: cord-195224-7zfq0kxm authors: menda, kunal; becdelievre, jean de; gupta, jayesh k.; kroo, ilan; kochenderfer, mykel j.; manchester, zachary title: scalable identification of partially observed systems with certainty-equivalent em date: 2020-06-20 journal: nan doi: nan sha: doc_id: 195224 cord_uid: 7zfq0kxm system identification is a key step for model-based control, estimator design, and output prediction. this work considers the offline identification of partially observed nonlinear systems. we empirically show that the certainty-equivalent approximation to expectation-maximization can be a reliable and scalable approach for high-dimensional deterministic systems, which are common in robotics. we formulate certainty-equivalent expectation-maximization as block coordinate-ascent, and provide an efficient implementation. the algorithm is tested on a simulated system of coupled lorenz attractors, demonstrating its ability to identify high-dimensional systems that can be intractable for particle-based approaches. our approach is also used to identify the dynamics of an aerobatic helicopter. by augmenting the state with unobserved fluid states, a model is learned that predicts the acceleration of the helicopter better than state-of-the-art approaches. the codebase for this work is available at https://github.com/sisl/ceem. the performance of controllers and state-estimators for non-linear systems depends heavily on the quality of the model of system dynamics (hou & wang, 2013) . systemidentification addresses the problem of learning or calibrating dynamics models from data (ljung, 1999) , which is often a time-history of observations of the system and control inputs. in this work, we address the problem of learning dynamics models of partially observed systems (shown in · · · · · · · · · x txt+1 f θ (x, u, t) g θ (x, u, t) g θ (x, u, t + 1) · · · · · · system identification figure 1. a graphical model representing a partially observed dynamical system. gray-box identification algorithms attempt to search a model class of dynamics and observation models for the model that maximizes the likelihood of the observations. figure 1 ) that are high-dimensional and non-linear. we consider situations in which the system's state cannot be inferred from a single observation, but instead requires inference over time-series of observations. the problem of identifying systems from partial observations arises in robotics (punjani & abbeel, 2015; cory & tedrake, 2008; ordonez et al., 2017) as well as domains such as chemistry (gustavsson, 1975) and biology (sun et al., 2008) . in many robotic settings, we have direct measurements of a robot's pose and velocity, but in many cases we cannot directly observe relevant quantities such as the temperature of actuators, the state the environment around the robot, or the intentions of other agents. for example, abbeel et al. (2010) attempted to map the pose and velocity of an aerobatic helicopter to its acceleration. they found their model to be inaccurate when predicting aggressive maneuvers because of the substantial airflow generated by the helicopter that affected the dynamics. since it is often impossible to directly measure the state of the airflow around a vehicle, identification must be with only partial observability. system identification is a mature field with a rich history (ljung, 1999; 2010) . a variety of techniques have been proposed to learn predictive models from time series arxiv:2006.11615v1 [cs. lg] 20 jun 2020 data. autoregressive approaches directly map a time-history of past inputs to observations, without explicitly reasoning about unobserved states (billings, 2013) , and are the stateof-the-art approach to the aforementioned problem of modeling the aerobatic helicopter (punjani & abbeel, 2015) . in contrast, state-space models (ssm) assume an unobserved state x t that evolves over time and emits observations y t that we measure, as shown in figure 1 . recurrent neural networks (rnns) (bailerjones et al., 1998; zimmermann & neuneier, 2000) are a form of black-box non-linear ssm that can be fit to observation and input time-series, and subspace identification (sid) methods (van overschee & de moor, 1994) can be used to fit linear ssms. however, in many cases prior knowledge can be used to specify structured, parametric models of the system (gupta et al., 2019; in state-space form, commonly refered to as gray-box models. such models can be trained with less data and used with a wider array of control and stateestimation techniques than black-box models (gupta et al., 2019; lutter et al., 2019b; a) . to identify partially observed gray-box models, the unobserved state-trajectory is often considered as a missing data, and techniques based on expectation-maximization (em) are used (dempster et al., 1977; schön et al., 2011; kantas et al., 2015; ghahramani & roweis, 1999) . the smoothing step (e-step) deals with state inference-the current system dynamics estimate is used to infer the distribution of unobserved state-trajectories conditioned on the observations p(x 1:t | y 1:t ). this distribution is sometimes called joint smoothing distribution in the literature, and it is used to estimate the expected log-likelihood of the observations. in the learning step (m-step), the system's dynamics estimate is updated such that it maximizes the expected log-likelihood. the smoothing step can typically be approached with particle approximations of p(x 1:t | y 1:t ), but naive implementations of particle smoothers can be computationally intensive and become rapidly intractable in high dimensions. across various fields and disciplines, numerous methods have been developed to alleviate the computational burden, several of which are discussed in section 2.3. this work is motivated by robotics applications, in which the following assumptions are often valid: • systems evolve nearly deterministically, which implies that the process noise is small and unimodal, and, • the distribution of states conditioned on the observations p(x 1:t | y 1:t ) is unimodal. we study the benefits of making the certainty-equivalent approximation in the e-step of the em procedure, and we refer to this approach as ce-em. specifically, we use non-linear programming to tractably find the maximum-likelihood (ml) point-estimate of the unobserved states, and use it in lieu of a fully characterized approximate distribution. the contributions of this paper are to describe an efficient implementation of ce-em, and to test the approximation against state-of-the-art approaches on a variety of systemidentification problems. we demonstrate on a system of lorenz attractors that: • ce-em can be faster and more reliable than approaches using particle approximations, • ce-em scales to high-dimensional problems, and, • ce-em learns unbiased parameter estimates on deterministic systems with unimodal p(x 1:t | y 1:t ). we also demonstrate the algorithm on the problem of identifying the dynamics of an aerobatic helicopter. we show that a non-linear ssm can be trained with ce-em that outperforms various approaches including the most recent work done on this dataset (punjani & abbeel, 2015) . a codebase implementing ce-em and other supplementary material can be found at our website: https://sites.google. com/stanford.edu/ceem/. this section states the nonlinear system identification problem with partial observations and discusses approaches that use em to solve it. in this work, we assume that we are given a batch of trajectories containing observations y 1:t ∈ r m×t of a dynamical system as it evolves over a time horizon t , possibly forced by some known input sequence u 1:t . we assume that this dynamical system has a state x ∈ r n that evolves and generates observations according to the following equations, where w t is referred to as the process noise and v t as the observation noise. both w t and v t are assumed to be additive for notational simplicity, but this is not a required assumption. 1 without loss of generality, we can drop the dependence on u t , absorbing it into the dependence on t. we further assume that we are provided a class of parameterized models f θ (x, t) and g θ (x, t) for θ ∈ θ that approximate the dynamical system's evolution and observation processes. the goal of our algorithm is to find the parameters θ that maximize the likelihood of the observations. that is, we seek to find: (2) using the graphical model shown in figure 1 , we can rewrite this integral as: (3) finally, using the notation chosen to describe a dynamical system in equation (1), we obtain: the expectation-maximization (em) algorithm has been used in the literature to solve problems of this form. the em algorithm is a two-step procedure that copes with the missing state information by forming an approximation q(θ, θ k ) of the joint state and observation likelihood p θ (x 1:t , y 1:t ) at the kth iteration of the algorithm: q(θ, θ k ) = log p(x 1:t , y 1:t | θ)· p(x 1:t | y 1:t , θ k )dx 1:t classical proofs show that maximizing q(θ, θ k ) with respect to θ results in increasing p θ (x 1:t , y 1:t ) (dempster et al., 1977) . the em algorithm iteratively performs two steps: the evaluation of q(θ, θ k ) in the e-step requires the estimation the p(x 1:t | y 1:t , θ k ), as well as the integration of the likelihood over this distribution. in the linear gaussian case, em can be performed exactly using kalman smoothing (rauch et al., 1965; ghahramani & hinton, 1996) . in the more general case, there is no analytic solution, and previous work on approximating the e-step is summarized in the next section. there are general approaches that are based on particle representations of the joint states-observations likelihood which use sequential monte-carlo (smc) methods such as particle smoothing (ps) (schön et al., 2011; kantas et al., 2015) . with enough particles, ps can handle any system and any joint distribution of states and observations. however, ps suffers from the curse of dimensionality, requiring an intractably large number of particles if the state space is high-dimensional (snyder et al., 2008; kantas et al., 2015) . in the simplest form, both the e-step and the m-step can be quadratic in complexity with respect to the number of particles (schön et al., 2011) . an important body of work has attempted to alleviate some of this burden by using nested smc (naesseth et al., 2015) and forward filtering-backward simulation (ffbsi), (lindsten & schön, 2013) , and conditional particle filtering (lindsten, 2013) in the e-step. ps can also require variance reduction techniques, such as fulladaptation, to perform reliably, and likelihood estimates in the e-step can be noisy . stochastic approximation em can be used to stabilize learning in the presence of this noise (delyon et al., 1999; . another group of methods is based on linearizing the dynamics around the current state estimates to obtain a timedependant linear gaussian dynamical system, to which we can apply kalman smoothing techniques. ghahramani & roweis (1999) use extended kalman smoothing (eks) for a fast e-step and show that, when using radial basis functions to describe the system dynamics, no assumption is required in the m-step. the drawbacks of this approach are that the number of radial basis functions required to accurately represent a function grows exponentially with the input dimension, that the user cannot specify a gray-box ssm, and that eks performance can vary dramatically with the hyperparameters. goodwin & aguero (2005) proposed a method called map-em, which linearizes around the state-trajectory that maximizes joint state and observation likelihood, approximates process and observation noise as gaussian, and performs a local version of em using this linearization. in comparison, the method we consider assumes that the maximum likelihood state-trajectory estimate concentrates all of the probability mass. goodwin & aguero (2005) call this simplification certainty equivalent em (ce-em) and report worse results than their approach on simple examples. this paper shows that ce-em can actually be a fast, simple, and reliable approach. the method we use here is tailored to systems that are not dominated by process noise, in which the state-trajectory distribution is unimodal, and whose high dimensional state-space make other methods intractable. such scenarios are regularly encountered in robotics, but perhaps less so in other applications domains of system identification such as chemistry, biology, or finance. this section introduces the ce-em approximation and presents an efficient algorithm to identify high-dimensional robotic systems. under the certainty-equivalence condition, the distribution of states conditioned on observations is assumed to be a dirac delta function. that is, we assume: in other words, this assumption implies that there is only one state trajectory that satisfies the system dynamics, and is coherent with the data. the e-step can be rewritten as: by observing the maximization of the expression in equation (7) as the e-step, and maximizing the expression in equation (8) as the m-step, we see that ce-em is simply block coordinate-ascent on the single joint-objective: jointly maximizing this objective over x 1:t and θ yields θ ml . though this objective can be optimized as is by a non-linear optimizer, it is not necessarily efficient to do so since θ and x 1:t are highly coupled, leading to inefficient and potentially unstable updates. for this reason, we still opt for the block coordinate-ascent approach similar to em, where θ and x 1:t are each sequentially held constant while the other algorithm 1. ce-em implementation input: observations y 1:t , control inputs u 1:t , stopping criterion tol initialize: model parameters θ initialize: hidden state estimates is optimized. by viewing em as block coordinate-ascent, we may also borrow good practices typically employed when running the algorithm (shi et al., 2016) . in particular, we employ trust-region regularization in order to guarantee convergence in even non-convex settings (grippo & sciandrone, 2000) . at iteration k, we first perform smoothing by holding θ constant and finding the ml point-estimate for x 1:t as follows: (10) where ρ x scales a soft trust-region regularizer similar to damping terms found in levenberg-marquardt methods (levenberg, 1944; marquardt, 1963) . we note that the hessians of this objective with respect to x 1:t are blocksparse, and as a result this step can be efficiently solved with a second-order optimizer in o(n 2 t ). if the cost function j(x 1:t , θ) is a sum of quadratic terms, i.e. if all stochasticity is gaussian, then the smoothing can be solved with a gauss-newton method, where the hessian matrix is approximated using first-order (jacobian) information. in this case, the solution to the smoothing step is equivalent to iteratively performing eks (aravkin et al., 2017) . however, eks typically involves forward and backward passes along each trajectory, performing a riccati-like update at each time step. though square-root versions of the algorithm exist to improve its numerical stability (bierman, 1981) , solving the problem with batch non-linear least-squares as opposed to iteratively performing a forward-backward method can improve stability for long time-series (van dooren, 1981). in the learning step, we hold x 1:t constant and find: again, ρ θ scales a soft trust-region regularizer, and specifying log p(θ) allows us to regularize θ toward a prior. the above optimization problem can be solved using any nonlinear optimizer. we find the nelder-mead (gao & han, 2012) scheme well-suited for small parameter spaces, and first-order schemes such as adam (kingma & ba, 2015) , or quasi-second-order schemes such as l-bfgs (liu & nocedal, 1989 ) suited for larger parameter spaces such as those of neural networks. the routine, summarized in algorithm 1, iterates between the smoothing and learning steps until convergence. it should be noted that making the certainty-equivalent approximation is generally known to bias parameter estimates (celeux & govaert, 1992) , but can yield the correct solutions under the assumptions we make (neal & hinton, 1998) . readers of goodwin & aguero (2005) will likely be left with the incorrect impression that ce-em is an inferior method that would perform poorly in practice. the objective of our experiments is to demonstrate that the ce-em algorithm is capable of identifying high-dimensional nonlinear systems in partially observed settings. we do so in simulation by identifying the parameters of a system of partially observed coupled lorenz attractors, as well as by identifying the dynamics of a real aerobatic helicopter. in the second experiment, we build on previous analysis of the dataset (abbeel et al., 2010; punjani & abbeel, 2015) by attempting to characterize the interaction of the helicopter with the fluid around it, without having any direct observation of the fluid state. instructions for reproducing all experiments are included in the supplementary material. in this experiment, we show that: 1. ce-em learns unbiased parameter estimates of systems that are close to deterministic, and, 2. ce-em scales to high-dimensional problems in which particle-based methods can be intractable. to justify these claims, we use a system that is sufficiently non-linear and partially observable to make particle-based smoothing methods intractable. we choose a system of coupled lorenz attractors for this purpose, owing to their ability to exhibit chaotic behavior and their use in nonlinear atmospheric and fluid flow models (bergé et al., 1984) . arbitrary increases in state dimensionality can be achieved by coupling multiple individual attractors. the state of a system with k coupled lorenz attractors is x ∈ r 3k = {. . . , x 1,k , x 2,k , x 3,k , . . .}. the dynamics of the system are as follows:ẋ where h is an r 3k×3k matrix. we nominally set the parameters (σ k , ρ k , β k ) to the values (10, 28, 8/3), and randomly sample the entries of h from a normal distribution to generate chaotic and coupled behavior between attractors, while avoiding self-coupling. these parameters are estimated during identification. in order to make the system partially observed, the observation y ∈ r (3k−2) is derived from x as follows: where c ∈ r (3k−2)×3k is a known matrix with full rowrank, and v is the observation noise sampled from a gaussian with diagonal covariance σ 2 v i. the entries of c are also randomly sampled from a standard normal distribution. in the following experiments, we simulate the system for t = 128 timesteps at a sample rate of ∆t = 0.04s, and integrate the system using a 4th-order runge-kutta method. initial conditions for each trajectory are sampled such that x 1,k ∼ n (−6, 2.5 2 ), x 2,k ∼ n (−6, 2.5 2 ), x 3,k ∼ n (24, 2.5 2 ). to test the conditions under which ce-em learns unbiased parameter estimates, we simulate a single lorenz system with h = 0 and known c ∈ r 2×3 . we introduce and vary the process noise w ∼ n (0, σ 2 w i), and vary the observation noise coefficient σ v , and then attempt to estimate the parameters (σ, ρ, β). using initial guesses within 10% of the system's true parameter values, we run ce-em on a single sampled trajectory. for each choice of σ w and σ v , we repeat this process for 10 random seeds. table 1 shows the mean and standard errors of parameter estimates for various σ w and σ v . we highlight in red the mean estimates that are not within two standard errors of their true value. we see that σ and ρ are estimated without bias for all scenarios. however, the estimate of β appears to become biased as the process noise is increased, but not as the observation noise is increased. this supports the assumption that the objective used in ce-em is sound when systems evolve close to deterministically, but can be biased if it is not. in section 2, we discussed methods for parameter estimation in state-space systems that are based on particle-filtering and smoothing (kantas et al., 2015) . since in their e-step, these methods approximate the distribution over unobserved state-trajectories as opposed to only their point estimate, such methods can be asymptotically unbiased. however, for a finite number of particles, such methods can result in high-variance estimates. in this experiment, we compare the bias resulting from using ce-em with the variance of using a state-of-the-art particle em algorithm. we attempt to identify the parameters (σ, ρ, β) of the same single lorenz system as in section 4.1.1. however, we introduce process noise w ∼ n (0, 0.1 2 i) and observation noise v ∼ n (0, 0.5 2 i). we use a training dataset of four trajectories sampled with conditions specified in section 4.1. the performance of particle em can vary substantially depending on implementation of the particle filter and smoother in the e-step, and implementation of the mstep. we use a fully-adapted particle filter with systematic and adaptive resampling, following the recommendations of doucet et al. (2000) ; hol et al. (2006) . furthermore, we use the ffbsi algorithm (godsill et al., 2004; lindsten & schön, 2013) in order to generate iid samples of smoothed state-trajectories, while avoiding complexity that is quadratic in the number of particles experienced by the forward filter-backward smoother (ffbsm) approach (doucet et al., 2000) . we then use stochastic approximation em (saem) (delyon et al., 1999) to perform the m-step. 2 we use n p = 100 particles for filtering, and sample n s = 10 smoothed trajectories using ffbsi. figure 2 shows the estimated parameters versus em epoch using ce-em and particle em. we plot learning curves for 10 random seeds, each of which initializes parameter estimates to within 10% of their true value, and uses a different set of training trajectories. we see that ce-em consistently converges to accurate parameter estimates in approximately 5 epochs. estimates of particle em appear to initially diverge but in all but one case converge to a similar accuracy in 50 epochs. furthermore, since the com2 we have found that using ffbsi with saem performs more reliably than ffbsm with the m-step recommended by (schön et al., 2011) , and both implementations can be found in the associated codebase. plexity of ffbsi is o(n p n s ), 3 the runtime per epoch of particle em is 47× more than that of ce-em. 4 since the variance of particle-based methods generally increases with the effective dimension of the system, the bias induced by ce-em may be a worthwhile trade-off for fast and reliable parameter estimation. to demonstrate that ce-em is capable of identifying highdimensional systems, we show that we can estimate the dynamics of an 18 dimensional system of six coupled lorenz attractors. moreover, we test whether ce-em converges to more accurate estimates when more trajectories are provided. to test these claims, we sample 2, 4, and 8 trajectories from a deterministic system with parameters θ true , and σ v = 0.01. we randomly initialize each element of the parameters being optimized (θ = [σ 1:k , ρ 1:k , β 1:k , h]) to within 10% of the their value in θ true . we then run ce-em on each batch, tracking the error in the estimated dynamics as training proceeds. we measure this error, which we call (θ), as follows: in the learning step, we do not regularize θ to a prior and set ρ θ = 0. for comparison, we also run the same particle em implementation as in the previous experiment on this problem. we use the same n p , n s , and hyperparameters as before. figure 3 shows the results of this experiment for four random seeds for each batch size. we can see that, as the number of trajectories used in training increases, the error in the estimated dynamics tends toward zero. furthermore, we see that ce-em convergences monotonically to a local optimum in all cases. in contrast, particle em appears to initially improve but then converges to very poor parameter estimates. the experiments conducted thus far have demonstrated that ce-em can learn unbiased parameter estimates of nearlydeterministic systems, and can scale to high-dimensional problems for which particle-based methods are intractable. in the next experiment, we use ce-em to characterize the effect of unobserved states on the dynamics of an aerobatic helicopter. characterizing the dynamics of a helicopter undergoing aggressive aerobatic maneuvers is widely considered to be a challenging system-identification problem (abbeel et al., 2010; punjani & abbeel, 2015) . the primary challenge is that the forces on the helicopter depend on the induced state of the fluid around it. the state of the fluid cannot be directly observed and its dynamics model is unknown. merely knowing the state of the helicopter and the control commands at a given time does not contain enough information to accurately predict the forces that act on it. in order to address this issue, (punjani & abbeel, 2015) use an approach based on takens theorem, which suggests that a system's state can be reconstructed with a finite number of lagged-observations of it (takens, 1981) . instead of attempting to estimate the unobserved fluid state, they directly learn a mapping from a 0.5 s history of observed state measurements and control commands to the forces acting on the helicopter. this approach is equivalent to considering the past 0.5 s of observations as the system's state. however, it can require a very large number of lagged observations to represent complex phenomena. in reality, the characteristic time of unsteady flows around helicopters can easily be up to tens of seconds. having such a high dimensional state can make the control design and state-estimation more complicated. to avoid large input dimensions, a trade-off between the duration of the history and sample frequency is necessary. this trade-off will either hurt the resolution of low-frequency content or will alias high-frequencies. we attempt to instead explicitly model the unobserved states affecting the system. the objective of this learning problem is to predict y t , the helicopter's acceleration at time t, from an input vector u t containing the current measured state of the helicopter (its velocity and rotation rates) and the control commands. we use data collected by the stanford autonomous helicopter project (abbeel et al., 2010) . trajectories are split into 10 s long chunks and then randomly distributed into train, test, and validation sets according to the established protocol (abbeel et al., 2010; punjani & abbeel, 2015) and summarized in appendix a.1. the train, test, and validation sets respectively contain 466, 100, and 101 trajectories of 500 time-steps each. a simple success metric on a given trajectory is the root mean squared prediction error, where y (measured) t is the measured force from the dataset, y (pred) t is the force predicted by the model, and t is the number of time-steps in each trajectory. naive: we first consider a naive baseline that does not attempt to account for the time-varying nature of the fluidstate. we train a neural-network to map only the current helicopter state and control commands to the accelerations: y t = nn θn (u t ), where nn θn is a neural-network with parameters θ n . we refer to this model as the naive model. we also compare to the work of punjani & abbeel (2015) . they predict y t using a time-history u t−h:t of h = 25 lagged observations of the helicopter's measured state and control commands. this input is passed through a relu-activated neural network with a single hidden-layer combined with what they call a quadratic lag model. as a baseline, we reproduce their performance with a single deep neural network y t = nn θ h (u t−h:t ) with parameters θ h . we call this neural network model the h25 model. both of these models can be trained via stochastic gradient descent to minimize the mean-squared-error (mse) of their predictions for y. the optimization methodology for these models is described in appendix a.2. sid: as a third baseline, we compare with subspaceidentification methods (van overschee & de moor, 1994) . we letỹ t = y t − nn θn (u t ) be the prediction errors of the trained naive model. we use the matlab command n4sid to fit a linear dynamical system of the following form: here, x ∈ r d is the unobserved state with arbitrary dimension d. the learned parameters are θ s = [a s , b s , c s , d s ]. we use a state dimension of 10 and call this model the sid model. the n4sid algorithm scales super-linearly with the amount of data supplied, and thus we train on 10 randomly sampled subsets of 100 trajectories each, and report the distribution in prediction performance. this approach fits a linear system to the residual error of the naive model, therefore the prediction of y t obtained from the sid model is a nonlinear function of the states x t . we also train an lstm (hochreiter & schmidhuber, 1997 ) on the residual time-seriesỹ 1:t and u 1:t . we use ce-em to train a non-linear ssm. similar to the parameterization used for subpace-identification, we fit the prediction errors of the naive model using the following dynamical system: where nnθ nl is a neural network, and θ nl = [a nl , b nl , c nl , d nl ,θ nl ] are the learned parameters. we introduce non-linearity only in the observation function because it is known from koopman theory that a non-linear system can be approximated by a high-dimensional linear system provided the correct non-linear mapping between them (brunton et al., 2016) . while learning, we assume that both process and observation noise are distributed with diagonal gaussian covariance matrices σ w i and σ v i respectively. the values of σ w and σ v are treated as hyperparmeters of ce-em, and are both set to 1. here as well, we use a state dimension of 10 and call this model the nl model. the optimization methodology for this model is described in appendix a.2. it should be noted that the system we learn need not actually correspond to an interpretable model of the fluid-state, but only of time-varying hidden-states that are useful for predicting the accelerations of the helicopter. expert knowledge of helicopter aerodynamics could be used to further inform a gray-box model trained with ce-em. the test rmse of the naive, h25, and lstm models can be evaluated directly on the test trajectories using next-step prediction. however, the sid and nl models require an estimate of the unobserved state before making a prediction. the natural analog of next-step prediction is extended kalman filtering (ekf), during which states are recursively predicted and corrected given observations. at a given timestep, a prediction ofỹ t is made using the current estimate of x t , and is used in the computation of rmse. the stateestimate is then corrected with the measuredỹ t . figure 4a shows the rmse of the compared models on trajectories in the test-set. we see that the nl model is able to consistently predict the accelerations on the helicopter with better accuracy than any of the other models. the naive model performs on average 2.9 times worse than the h25 model, and its results can be found in appendix a.3. the lstm model also performs poorly, on average 1.4 times worse than the h25 model. the sid model notably outperforms the state-of-the-art h25 model, suggesting that a large linear dynamical system can be used to approximate a non-linear and partially observable system (korda & mezić, 2018) . however, introducing non-linearity as in the nl model noticeably improves performance. figure 4b depicts the errors in prediction over a sample trajectory in the test-set. here, we also see that the nl model is able to attenuate the time-varying error present in predictions made by the h25, suggesting that it has accurately characterized the dynamics of unobserved, time-varying states. this experiment validates the effectiveness of ce-em to identify a non-linear dynamical model of unobserved states that affect the forces acting an aerobatic helicopter. this paper presented an algorithm for system identification of non-linear systems given partial state observations. the algorithm optimizes system parameters given a time history of observations by iteratively finding the most likely state-history, and then using it to optimize the system parameters. the approach is particularly well suited for highdimensional and nearly deterministic problems. in simulated experiments on a partially observed system of coupled lorenz attractors, we showed that ce-em can perform identification on a problem that particle-based em methods are ill-suited for. however, we also find that ce-em yields biased parameter estimates in the presence of large process noise. this bias can be partially mitigated by locally approximating the posterior state-marginals as gaussian, as is done by map-em (goodwin & aguero, 2005) . we then used the algorithm to model the timevarying hidden-states that affect the dynamics of an aerobatic helicopter. the model trained with ce-em outperforms state-of-the-art methods because it is able to fit large non-linear models to unobserved states. numerous system-identification problems can be studied using ce-em. recently, there have been tremendous efforts to characterize predictive models for the spread of covid-19 (fanelli & piazza, 2020) . limited capacity for testing the prevalence of the disease makes relevant states partially observed, and thus ce-em may be useful for its modeling. we also hope to apply ce-em to very high-dimensional systems with sparsely coupled dynamics using general-form consensus optimization (boyd et al., 2011) . in this work we use the dataset gathered by abbeel et al. (2010) and available at http://heli.stanford. edu/. a gas-powered helicopter was flown by a professional pilot to collect a large dataset of 6290s of flight. there are four controls: the longitudinal and lateral cyclic pitch, the tail rotor pitch and the collective pitch. the state is measured thanks to an accelerometer, a gyroscope, a magnetometer and vision cameras. abbeel et al. (2010) provide the raw data, as well as states estimates in the earth reference frame obtained with extended kalman smoothing. following punjani & abbeel (2015) , we use the fused sensor data and downsample it from 100hz to 50hz. from the earth frame accelerations provided in the dataset, we compute body frame accelerations (minus gyroscopic terms) which are the prediction targets for our training. using the notations from punjani & abbeel (2015) , we can write the helicopter dynamics in the following form: where s ∈ r 13 is the helicopter state consisting of its position r, quaternion-attitude q, linear velocity v, angular velocity ω, and δ ∈ r 4 to be the control command. c 12 is the rotation-matrix from the body to earth reference frame, and f v and f ω are the linear and angular accelerations caused by aerodynamic forces, and are what we aim to predict. the above notation is related to that used in section 4.2 as follows: • we define u as the concatenation of all inputs to the model, including the relevant state variables v and ω and control commands δ. • we define y as the output predicted, which would correspond to a concatenation of f v and f ω . • we define x as the vector of unobserved flow states to be estimated and is not present in their model. the processed dataset used in our experiments can be found at https://doi.org/10.5281/zenodo. 3662987 (menda et al., 2020) . neural networks in the naive and h25 models have eight hidden layers of size 32 each, and tanh non-linearities. we optimize these models using an adam optimizer (kingma & ba, 2015) with a harmonic learning rate decay, and minibatch size of 512. the neural network in the nl model has two hidden layers of size 32 each, and tanh non-linearity. we train the nl model with ce-em, using ρ x = ρ θ = 0.5, σ w = σ v = 1.0, and use an adam optimizer to optimize equation (11) in the learning step. the learning rate for dynamics parameters in θ nl is 5.0 × 10 −4 and observation parameters in θ nl is 1.0 × 10 −3 . for its relative robustness, we optimize equation (10) using a non-linear least squares optimizer with a trust-region reflective algorithm (jones et al., 2001-) in the smoothing step. this step can be solved very efficiently by providing the solver with the block diagonal sparsity pattern of the jacobian matrix. to evaluate the test metric, running an ekf is required. the output of an ekf depends on several user-provided parameters: • x 0 : value of the initial state • σ 0 : covariance of error on initial state • q: covariance of process noise • r: covariance of observation noise in this work, we assume that q, r and σ 0 are all set to the identity matrix. x 0 is assumed to be 0 on all dimensions. a well-tuned ekf with an inaccurate initial state value converges to accurate estimations in only a few time steps of transient behavior. since the h25 model needs 25 past inputs to predict its first output prediction, we drop the first 25 predictions from the ekf when computing rmse, thereby omitting some of the transient regime. autonomous helicopter aerobatics through apprenticeship learning generalized kalman smoothing: modeling and algorithms a recurrent neural network for modelling dynamical systems order within chaos: towards a deterministic approach to turbulence a new computationally efficient fixedinterval, discrete-time smoother nonlinear system identification: narmax methods in the time, frequency, and spatio-temporal domains distributed optimization and statistical learning via the scalable identification of partially observed systems with ce-em alternating direction method of multipliers. foundations and trends in machine learning koopman invariant subspaces and finite linear representations of nonlinear dynamical systems for control a classification em algorithm for clustering and two stochastic versions experiments in fixed-wing uav perching convergence of a stochastic approximation version of the em algorithm maximum likelihood from incomplete data via the em algorithm on sequential monte carlo sampling methods for bayesian filtering analysis and forecast of covid-19 spreading in china, italy and france implementing the nelder-mead simplex algorithm with adaptive parameters parameter estimation for linear dynamical systems learning nonlinear dynamical systems using an em algorithm monte carlo smoothing for nonlinear time series approximate em algorithms for parameter and state estimation in nonlinear stochastic models on the convergence of the block nonlinear gauss-seidel method under convex constraints a general framework for structured learning of mechanical systems structured mechanical models for robot learning and control survey of applications of identification in chemical and physical processes lstm can solve hard long time lag problems on resampling algorithms for particle filters from model-based control to datadriven control: survey, classification and perspective scipy: open source scientific tools for python on particle methods for parameter estimation in state-space models a method for stochastic optimization linear predictors for nonlinear dynamical systems: koopman operator meets model predictive control a method for the solution of certain nonlinear problems in least squares an efficient stochastic approximation em algorithm using conditional particle filters scalable identification of partially observed systems with ce-em backward simulation methods for monte carlo statistical inference. foundations and trends r in machine learning on the limited memory bfgs method for large scale optimization system identification perspectives on system identification deep lagrangian networks for end-to-end learning of energy-based control for under-actuated systems deep lagrangian networks: using physics as model prior for deep learning an algorithm for least-squares estimation of nonlinear parameters normalized stanford helicopter dataset nested sequential monte carlo methods a view of the em algorithm that justifies incremental, sparse, and other variants learning of skid-steered kinematic and dynamic models for motion planning deep learning helicopter dynamics models maximum likelihood estimates of linear dynamic systems system identification of nonlinear state-space models a primer on coordinate descent algorithms obstacles to high-dimensional particle filtering extended kalman filter for estimation of parameters in nonlinear state-space models of biochemical networks learning dynamical systems with particle stochastic approximation em learning nonlinear state-space models using smooth particle-filterbased likelihood approximations detecting strange attractors in turbulence a generalized eigenvalue approach for solving riccati equations n4sid: subspace algorithms for the identification of combined deterministicstochastic systems modeling dynamical systems by recurrent neural networks this work is supported in part by darpa under agreement number d17ap00032, and by the king abdulaziz city for science and technology (kacst) through the center of excellence in aeronautics and astronautics. the content is solely the responsibility of the authors and does not necessarily represent the official views of darpa or kacst. key: cord-018902-oninjtsn authors: kowalski, wladyslaw title: commercial buildings date: 2009-07-09 journal: ultraviolet germicidal irradiation handbook doi: 10.1007/978-3-642-01999-9_18 sha: doc_id: 18902 cord_uid: oninjtsn applications for uvgi systems in commercial buildings vary with the type of building, but virtually every type of building can benefit from the use of in-duct air disinfection and many buildings can benefit from the use of other types of uv systems. the health hazards and microbiological problems associated with various types of commercial buildings are often unique to the type of facility. the problem of air quality is paramount in commercial office buildings while the problem of biocontamination is of the highest concern in the food industry. other types of buildings have their own microbial concerns and even their own standards. the pharmaceutical industry has the highest aerobiological air quality standards (and lowest airborne microbial levels) while the other extreme, the agricultural industry, has the highest airborne microbial levels and unique aerobiological concerns. the individual problems of these facilities are addressed in the following sections, and industry experience relating to uv applications are discussed, along with recommendations for how uv systems can be applied. the specific types of uvgi systems are described in previous chapters and these should be referred to for detailed information on such applications. applications for uvgi systems in commercial buildings vary with the type of building, but virtually every type of building can benefit from the use of in-duct air disinfection and many buildings can benefit from the use of other types of uv systems. the health hazards and microbiological problems associated with various types of commercial buildings are often unique to the type of facility. the problem of air quality is paramount in commercial office buildings while the problem of biocontamination is of the highest concern in the food industry. other types of buildings have their own microbial concerns and even their own standards. the pharmaceutical industry has the highest aerobiological air quality standards (and lowest airborne microbial levels) while the other extreme, the agricultural industry, has the highest airborne microbial levels and unique aerobiological concerns. the individual problems of these facilities are addressed in the following sections, and industry experience relating to uv applications are discussed, along with recommendations for how uv systems can be applied. the specific types of uvgi systems are described in previous chapters and these should be referred to for detailed information on such applications. commercial buildings are the single largest and most common type of building in the united states today, in terms of total floor area. many common respiratory infections are regularly transmitted inside these structures due to daily occupancy and the extensive interaction of people within office buildings. proximity and duration of exposure are major factors in the transmission of respiratory infections between office workers (lidwell and williams 1961) . the risk of catching the common cold is increased by shared office space (jaakkola and heinonen 1995) . tuberculosis has also been show to transmit in office buildings between co-workers (kenyon et al. 2000 ). an association between respiratory tract symptoms in office workers and exposure to fungal and house dust mite aeroallergens was established by menzies et al. (1998) . the recent appearance of sars virus highlights the susceptibility of office workers to the spread of airborne viruses (yu et al. 2004) . sick building syndrome (sbs), often referred to as building related illness (bri) is a general category for a number of ailments, allergies, and complaints that are due to low levels of pollutants, synthetic irritants, fungi, bacteria or other factors that cause reactions in a certain fraction of building occupants (lundin 1991) . office buildings become contaminated with microbes brought in with the outside air as well as microbes that hail from indoor sources, including the occupants themselves. accumulation of microbes on the cooling coils and ductwork can contribute to indoor air quality problems (fink 1970) . the aerobiology of office buildings can depend on both indoor sources (i.e. occupancy and cleanliness) as well as the composition and concentration of microbes in the outdoor air. environmental microbes can enter via the ventilation system, via occupants (i.e. on clothes and shoes), and infiltration, especially from high-traffic lobbies that are under high negative pressure. office buildings provide an environment that sustains microorganisms long enough for them to transmit infections to new hosts, or sometimes allows them to persist indefinitely, as in the case of fungal spores (kowalski 2006) . the indoor airborne concentrations of bacteria and fungi increase with the presence of occupants, due to the fact that people release bacteria continuously and also because human activity stirs up dust that may contain fungi and bacteria. sessa et al. (2002) measured average office airborne concentrations of 493 cfu/m 3 for bacteria and 858 cfu/m 3 for fungi while levels were about four times lower when there were no occupants. most office buildings use dust filters in the merv 6-8 range to filter outdoor or recirculated air but these have a limited effect on reducing fungal spores, bacteria, and viruses. increasing filtration efficiency is one possible solution but retrofitting merv filters in the 9-13 range, which can be highly effective against most airborne pathogens and allergens, may be difficult in most buildings because of increased pressure losses and possible reduction in airflow (kowalski and bahnfleth 2002) . a suitable alternative to replacing filters is to add uv systems in the ductwork, which will not necessarily have any impact on airflow or pressure drop (see fig. 18 .1). in-duct uvgi is the most economic approach to disinfecting the air of commercial office buildings provided there is space in the ventilation system. the economics of in-duct air disinfection can be greatly improved by locating the uv lamps at or around the cooling coils, thereby disinfecting the coils and improving their heat transfer capabilities. cooling coils can first be steam cleaned to remove biological contamination and then a uvgi system can be added to continuously irradiate the coils. this not only prevents existing microbial growth but will tend to restore cooling coils to their original design operating level of performance, thereby saving energy costs (kelly 1999) . another alternative for improving air quality in office buildings and reducing the incidence of disease transmission between office workers is to locate recirculating uv units or upper room systems around the building to deal with local problems. lower room systems may also be used to control microbial contamination at floor level, especially in hallways or lobbies that see heavy foot traffic. other applications of uvgi in office buildings may include overnight decontamination of kitchens, bathrooms, and storage areas with uv area disinfection units. uvgi may not be a complete solution to aerobiological problems if the cause is due to some other factor like moisture or water damage. for more detailed information on dealing with air quality problems in office buildings see kowalski (2006) . industrial facilities cannot be generalized in terms of the types of respiratory diseases to which workers may be subject because of their wide variety. industrial facilities that handle organic materials have greater microbiological hazards while those that process inorganic products usually have less microbial hazards and more pollution hazards. pollution, however, is a contributing factor in infectious diseases, including respiratory diseases. there are many types of occupational diseases, including asthma, and allergic reactions or hypersensitivity pneumonitis, which are not necessarily due to microbiological causes. air disinfection systems that use uv have little or no effect on non-microbiological contaminants but if pathogens or allergens are the cause of the problem then uv systems may be applied to reduce the hazard. airborne particulate dust in industrial environments may contain organic materials, chemicals, microbes, biological compounds, or inert materials, and form a substrate on which microbes can grow. allergen-bearing airborne particulates are causative agents of lung inflammation via their immunotoxic properties and can induce inflammatory alveolitis (salvaggio 1994) . occupational asthma occurs in 2-6% of the asthmatic population and respiratory infection can be a predisposing factor for occupational asthma (bardana 2003) . workers who clean or enter rarely-used rodent infested structures may be at increased risk of exposure to rodentborne viruses such as hantavirus (armstrong et al. 1995) . industries that process wood or paper, or that make use of paper products, have airborne allergen hazards since cellulose provides a nutrient source for a variety of allergenic fungi. microbiological sampling in furniture factories found the most common airborne microorganisms included corynebacterium, arthrobacter, aspergillus, penicillium, and absidia (krysinska-traczyk et al. 2002) . microbiologial studies of the air in sawmills were conducted by dutkiewicz et al. (1996) , who found that the most common organisms were arthrobacter, corynebacterium, brevibacterium, microbacterium, bacillus spp., gram-negative bacteria (rahnella) and filamentous fungi (aspergillus, penicillium). inhalation anthrax and cutaneous anthrax still occur occasionally in the textile industry. in a mill in the usa where outbreaks had occurred, anthrax levels were measured at up to 300 cfu/m 3 (crook and swan 2001) . q fever, which can result from inhalation or exposure to coxiella burnetti, has been reported among workers in a wool and hair processing plant (sigel et al. 1950) . tuberculosis continues to be an occupational hazard in some parts of the world and is a growing problem elsewhere because of multi-drug resistance. in a study at russian plants by khudushina et al. (1991) the incidence of tuberculosis was highest at a foundry plant at 39.9 per 100,000 workers and in an automatic-assembly plant at 64.4 per 100,000 workers. health care workers, prison guards, and prison inmates may all be at higher risk for tuberculosis. a one-year prospective study of inmates in geneva showed that the prevalence of active and residual tuberculosis is 5-10 times higher among prisoners than in the general population (chevallay et al. 1983) . many prisons in the us incorporate filtration and uvgi systems to help control tuberculosis transmissions. occasional outbreaks of legionnaire's disease have continued to be sporadically reported in the work environment, including a large outbreak among exhibitors at a floral trade show where a whirlpool spa was on display (boshuizen et al. 2001) . water damage in any building increases the risk of workers to allergens if fungal mold growth has occurred. trout et al. (2001) documented extensive fungal contamination, including penicillium, aspergillus, and stachybotrys, in a water-damaged building. control measures for aerobiological contamination in the workplace are similar to those for any buildings in general, including improved air filtration, improved air distribution, and increased outside air combined with energy recovery systems. the use of uvgi for air disinfection can have positive benefits for almost any type of industrial building depending on the nature of the problem. in-duct uv air disinfection systems are likely to be the most economic recourse since they provide centralized control of the air quality, and can simultaneously be applied to the cooling coils (in most applications) to save energy while disinfecting the air. the use of local recirculation units can aid in resolving local microbial contamination problems, and both upper and lower room uvgi systems can be used to control biocontamination. the food industry comprises food and beverage processing facilities, food handling industries, food storage facilities other than agricultural, and kitchens in the restaurant and hospitality industry. there are at least four types of potential health hazards associated with food handling -foodborne human pathogens, spoilage microbes, microbial allergens, and food allergens. foodborne pathogens are generally transmitted by the oral route and almost exclusively cause stomach or intestinal diseases but some foodborne pathogens may be airborne at various stages of processing, storage, cooking, or consumption. the processing and handling of foods may also create opportunities for airborne mold spores to germinate and grow, resulting in secondary inhalation hazards. applications for uvgi in the food industry include both air and surface disinfection, including surface disinfection of packaging materials and food handling equipment. uv has been used for air and surface disinfection in cheese plants, bakeries, breweries, meat plants, for food preservation, and for the decontamination of conveyor surfaces, and packaging containers (koutchma 2008). one of the earliest applications of uv in the food industry involved the use of bare uv lamps to irradiate surfaces in the brewing and cheese making industries to control mold (philips 1985) . liquid food disinfection with uv has been approved by the fda but although uv has been highly effective for water disinfection, successful applications in disinfecting other liquids depends on the transmissivity of the liquid. the direct disinfection of food by uv is generally only effective in cases where only the surface of foodstuffs requires disinfection since uv has limited penetrating ability (fda 2000, yaun and summer 2002) . some foods can be effectively disinfected of certain food spoilage microbes, and uv is most effective on food products that have smooth and clean surfaces (shama 1999 , seiler 1984 . uv irradiation can significantly reduce the mold population on shells of eggs in only 15 min (kuo et al. 1997) . studies have demonstrated that uv can reduce levels of e. coli and salmonella on pork skin and muscle, listeria on chicken meat, and salmonella on poultry (wong et al. 1998 , kim et al. 2001 , wallner-pendleton et al. 1994 . begum et al. (2009) demonstrated that uv irradiation can inactivate food spoilage fungi like aspergillus, penicillium, and eurotium but that the type of surface impacted the degree of disinfection. marquenie et al. (2002) showed that botrytis cinerea and monilinia fructigena, two major post-harvest spoilage fungi of strawberries and cherries, could be reduced 3-4 logs by uv irradiation by doses of about 1000 j/m 2 . stevens et al. (1997) reported that uv could effectively reduce the incidence of storage rot disease on peaches due to monilinia fructicola, reduce green mold (penicillium digitatum) on tangerines, and reduce soft rot due to rhizopus on tomatoes and sweet potatoes. liu et al. (1993) showed that tomato diseases caused by alternaria alternata, botrytis cinerea, and rhizopus stolonifer were effectively reduced by uv treatment. hidaka and kubota (2006) demonstrated a 90% reduction of aspergillus and penicillium species on the surfaces of wheat grain. seiler (1984) reported increases in mold-free shelf life of clear-wrapped bakery products after moderate levels of uv exposure. treatment of baked loaves in uv conveyor belt tunnels resulted in significantly increased shelf life (shama 1999) . valero et al. (2007) found that uv irradiation of harvested grapes could prevent germination of fungi during storage or the dehydration process for raisins, using exposure times of up to 600 s. one specialized application of uv in the food industry involves overhead tank disinfection, in which the airspace at the top of a liquid storage tank is disinfected by uv to control bacteria, yeast, and mold spores. condensation of vapors in the head space of tanks, such as sugar syrup tanks, can produce dilute solutions on the liquid surface that provide ideal conditions for microbial growth. two types of tank top systems are in use -systems which draw the air through a filter and uv system and return sterilized air to the tank space, and systems in which uv lamps are located directly in the head space of the tank and irradiate the liquid surface and internal tank surfaces. see fig. 6 .6 in chap. 6 for an example of an overhead tank uv system. foodborne and waterborne pathogens represent the largest group of microorganisms that present health hazards in the food industry. in general, these do not present inhalation hazards, only ingestion hazards. however, some of these microbes can become airborne during processing and settle on foods, thereby becoming amenable to control by uv air and surface disinfection systems. modern foodborne pathogens are often uniquely virulent and hazardous, like salmonella and shigella, which are contagious and depend on either on excretion in feces or vomiting to facilitate epidemic spread. some agents of food poisoning, like staphylococcus and clostridium, are opportunistic or incidental contaminants of foods. many molds like aspergillus and penicillium are common contaminants of the outdoor and indoor air that can grow on food and although they are not food pathogens they are potential inhalation hazards for food industry workers. foodborne pathogens are predominantly bacteria but one virus has recently emerged and joined this class, norwalk virus. norwalk virus is a waterborne pathogen and has caused outbreaks on cruise ships (marks et al. 2000) . food spoilage microbes are less of a health hazard than they are a nuisance in the food industry because of the damage they can cause to processed food (samson et al. 2000) . a wide variety of yeasts may also be causes of spoilage. table 18 .1 lists the most common foodborne and waterborne pathogens in the food industry along with the type of hazard they present -pathogenic, toxic, allergenic, or spoilage. many of the species listed in table 18 .1 have uv rate constants as given in appendices a, b, and c. virtually all of the fungal spores listed in appendix c are potential contaminants in the food industry. toxins produced by microbes may be endotoxins, exotoxins, or mycotoxins, and these will grow only if the conditions (moisture, temperature, etc.) are right. the key to controlling these toxins is to control microbial growth and to control the concentration of microbes in the surrounding areas. ambient microbial levels can be controlled by food plant sanitation, adequate ventilation, and by various uv air disinfection technologies. most common food spoilage microbes have the capacity to transport in the air and therefore they are controllable to some degree by ventilation and by uv air disinfection systems. evidence exists to suggest listeria monocytogenes, a cause of many recent outbreaks, can settle on foodstuffs via the airborne route ( 1998) . studies indicate that salmonella can survive in air for hours (stersky et al. 1972 ). if air is recirculated through plant ventilation systems, organic material may accumulate inside ductwork and on air handling equipment, where fungi and bacteria may grow ( 1998). air filtration can go along way towards controlling the amount of organic debris that accumulates, but uv is an ideal technology for use in controlling microbial and fungal growth inside air handling units, ductwork, and on cooling coils. concerns about the breakage of bare uv lamps and mercury hazards has led to new lamps that are sealed in an unbreakable plastic coating, such as shown in fig. 18 .2. mold and biofilms can develop on surfaces and equipment in the food and beverage industry, including tanks and vats, cooking equipment, walls and floors, and cooling coils (carpentier and cerf 1993) . in general, standard cleaning and disinfection procedures are adequate to contain these problems but alternatives are available, including antimicrobial coatings like copper. uv irradiation of food processing equipment and surfaces, cooling coil disinfection systems, whole-area uv disinfection, and after-hours irradiation of rooms when personnel are not present are all viable options for maintaining high levels of disinfection in food industry facilities (philips 1985, kowalski and dunn 2002) . uv air disinfection systems may also be useful in controlling airborne hazards that result from hazards are created by industrial food processes that forcibly aerosolize contaminants. pulsed uv light has seen increasing application in the food industry because of its rapid disinfection capabilities -see chap. 16 for more information. educational facilities for children and students are focal points for disease transmission and as such they are ideally suited to uv applications that inhibit the transmission of contagious diseases. the concentration of young people in rooms causes cross-infections when even a single child comes to school with an infection such as a cold or a flu. inevitably children who attend school bring home contagious infections which are then transmitted to family members, thereby continuing the process of epidemic spread in the community. there are basically five types of educational facilities for children and students: day medical schools and colleges have also been subject to outbreaks of mumps and respiratory syncytial virus (rsv). table 18 .2 summarizes many of the various common infections that have been associated with school facilities, with reference sources as indicated. many of the microbes in table 18 .2 are susceptible to uv disinfection in air and on surfaces (see the uv rate constants for these species given in appendix a). fungal spores also accumulate in schools and are responsible for a variety of respiratory illnesses including allergies and asthma. the most frequently encountered fungal taxa identified in a study of dust samples from twelve schools in spain were alternaria, aspergillus, and penicillium (austin 1991) . guidelines for ventilation air in school buildings are provided in ashrae standard 62-2001 (ashrae 2001) but many schools do not meet the minimum recommendations either because the buildings are older or because of attempts to save energy costs due to endless budget cutting. one way to make up for the lack of ventilation air is to provide uv air disinfection systems, including upper room systems and in-duct uv systems. a number of studies have been performed in schools to evaluate the effectiveness of uvgi systems on respiratory infections in schools. all of these studies involved upper room systems (see chap. 9 for a summary of the results). most of the studies produced a reduction of disease transmission with a net average reduction in respiratory infections of approximately 30%, and were effective against measles, mumps, varicella, chickenpox, cold viruses and other respiratory infections. upper room systems can be especially effective in classrooms that have little of no ventilation air and that have high ceilings. there are no studies that have addressed in-duct uvgi systems, which would be likely to prove even more effective if they were adequately designed. materials and books that are kept in museums, libraries, and related archives may be both a source of allergens and a nutrient source for the growth of microorganisms, especially fungi. the growth of fungi and certain bacteria on stored materials contributes to biodeterioration, which is a major concern, and the aerobiology in bahlke (1949) museums and libraries may impact both the occupants as well as the stored materials. most books are made from cellulose and can be degraded by a variety of microbes and insects which may include allergens and a few potential pathogens. over 84 microbial genera, representing 234 species, have been isolated from library materials like books, paper, parchment, feather, textiles, animal and vegetable glues, inks, wax seals, films, magnetic tapes, microfilms, photographs, papyrus, wood, and synthetic materials in books (zyska 1997) . many of the fungi identified in library materials can produce mycotoxins and cause respiratory and other diseases. table 18 .3 summarizes the variety of microbes that have been found in libraries and museums, and indicates the type of materials that they can grow on. the term 'deteriogen' in the table refers to microbes that can cause biodeterioration of materials. libraries often use carpeting to quiet the noise of foot traffic, but carpets tend to accumulate fungi and bacteria over time due to the fact that they settle in air or are brought in from the outdoors on shoes and clothes. in one study of carpeted buildings, carpet dust was found to contain 85,000 cfu/gm of fungi, 12,000,000 cfu/gm of mesophilic bacteria, and 4,500 cfu/gm of thermophilic bacteria (cole et al. 1993) . foot traffic tends to aerosolize microbes and causes them to settle in other locations, including on books and materials. aerosolized microbes can also be inhaled, leading to allergies and asthma. carpets have a high equilibrium moisture content that favors microbial growth (iea 1996) . fabrics kept in museums, especially ancient cloth, are subject to biodeterioration from fungi and bacteria. fungi can grow extensively on cotton fibers at the right humidity and growth rates are highest on 100% cotton (goynes et al. 1995) . studies of biodeterioration caused by fungi that grow on paintings have isolated over one hundred species of fungi, with the most common species being alternaria, cladosporium, fusarium, aspergillus, trichoderma, and penicillium (inoue and koyano 1991) . ultraviolet light is an option for disinfecting surfaces and materials, but it can cause damage and discoloration to pigments and paint and therefore is not necessarily suitable for decontaminating books that are already damaged by microbial growth. uvgi is best used for disinfecting the air and especially for removing mold from air conditioning systems. in-duct uvgi can greatly aid in reducing airborne levels of bacteria and spores and in removing accumulated mold spores from air conditioning cooling coils, which are used extensively to control humidity in libraries and museums. keeping cooling coils disinfected will minimize the spread of spores through a building as well as reducing energy costs (see chap. 10). uvgi systems can be used selectively in areas of museums and libraries that do not contain sensitive materials, such as cafeterias. lower room uv systems may also be ideal for hallways and entryways (where no materials are stored) where outdoor spores may be tracked into buildings. for more detailed information on technologies and techniques for dealing with biocontamination of sensitive materials in libraries and museums see kowalski (2006) . agricultural facilities pose a variety of occupational microbial hazards including infectious diseases from farm animals, allergies from animal dander and foodstuffs, and health threats from mold spores or actinomycetes. animal facilities like barns, poultry houses, swine houses, and kennels can have the highest levels of bioaerosols seen in any indoor environments. animal facilities may also include non-agricultural buildings used to house or service animals such as pet shelters and zoos. many animal pathogens can transmit to humans by direct contact or by the airborne route. the microorganisms of greatest concern are those that can become airborne in animal facilities and these are often respiratory pathogens and allergens. allergens can be produced as a byproduct of animal husbanding or from animal waste, animal feeds, or other farm produce. the actinomycetes are particularly common type of bacteria found in agriculture and can grow on moldy hay. farmers may be routinely exposed to very high concentrations of actinomycetes and may inhale as many as 750,000 spores per minute (lacey and crook 1988 ). farmer's lung represents a group of respiratory problems that often afflict farm workers who receive chronic exposure to high concentrations of actinomycetes (pepys et al. 1963) . table 18 .4 lists some of the most common microorganisms that occur in animal and agricultural environments, and that can transmit to man by various routes. the diseases caused and natural source are identified. for information on the uv susceptibility of these microbes see appendices a, b, and c. most of these microbes can transmit by direct contact or by the airborne. table 18 .5 provides a list of microbes found in sewage, many of which can become airborne. natural ventilation is common in agricultural facilities but mechanical ventilation will generally provide superior control of airborne microorganisms. however, filtration is often necessary to clean both outdoor air and indoor recirculated air. recirculated air in animal facilities can be cleaned more effectively through the use of uvgi combined with filtration, and this approach works well for viruses, which may not filter out easily. other applications for uvgi in animal facilities include upper room systems, and area disinfection systems. after-hours uv disinfection systems are also appropriate for animal facilities provide the animals can be periodically removed from bacteria pathogen the indoor areas in order to disinfect them. such an approach can be more economical than scrubbing with disinfectants, and can enhance the effectiveness of manual scrubbing procedures. for more detailed information on dealing with the complex bioaerosol and dust problems in agricultural and animal facilities see kowalski (2006) . facilities like malls and airports are characterized by large enclosed volumes where large crowds may be concentrated and where heavy cyclical occupancy may occur. places of assembly may include auditoriums, stadiums, theaters, gymnasiums, natatoriums, arenas, town halls, churches, cathedrals, temples, mosques, industrial halls, convention centers, atriums, shopping centers, and other places where large public gatherings may occur indoors. in such buildings infectious diseases may be exchanged by direct contact, indirect contact, or via inhalation, and large numbers of people may be exposed simultaneously. the large volumes of air enclosed in such facilities often ensure good mixing of air, even in naturally ventilated stadiums, and as a result the air quality is often acceptable. however, it is difficult to provide ventilation to all corners and therefore the actual airflow distribution is often uncertain. one of the most famous outbreaks of a respiratory disease in a heavily occupied building was the eponymous legionnaire's disease outbreak at a convention in philadelphia in 1976 that resulted in 29 deaths (spengler et al. 2001) . it was traced to legionella contamination in a cooling tower that apparently wafted from outdoors into a crowded hallway through open doors. an outbreak of measles occurred inside a domed stadium in the minneapolis-st. paul metropolitan area during july 1991 that resulted in sixteen associated cases of measles in seven states (ehresmann et al. 1995) . several tuberculosis outbreaks have occurred in churches and mosques due to a single infectious person. dutt et al. (1995) reports one outbreak of tb in a church where one man exposed 42% of his congregation. a norovirus outbreak at a concert hall was reviewed by evans et al. (2002) in which a concert attendee who vomited in the auditorium and a toilet. gastrointestinal illness occurred among members of school children who attended the following day. transmission was most likely through direct contact with contaminated fomites that remained in the toilet area. large facilities that use 100% outside air provide little or no opportunity for the use of uv systems, since outside air is generally clean and free of airborne pathogens, but facilities that recirculate air may benefit from in-duct uv air disinfection systems. large facilities often have high ceilings and this provides an opportunity to use upper room systems for air disinfection, especially in cases where natural ventilation is employed and there is no other means for disinfecting the air. high power uv systems can be safely used in such applications since the uv lamps can be located far from the occupied floor areas. the use of uv area disinfection systems in public toilets is an appropriate means of dealing with potential fomites, or infectious particles left on surfaces. aircraft, trains, cars, and other compact enclosed environments can pose microbiological hazards from extended exposure due to the fact that risks due to proximity are increased regardless of whether the pathogen transmits through the air or by direct contact. large cruise ships may resemble hotels and apartment buildings in terms of their ventilation systems and health risks but smaller craft like cars and planes create extended opportunities for infectious exchanges due to the close quarters, shared breathing air, potentially extended periods of occupancy, and the limited amount of outside air that may be brought in, especially in cold climates. other microenvironments like elevators and city buses are unlikely to play a major role in the spread of contagious diseases due to their brief occupancy times. aircraft are one of the most crowded environments in which people remain for extended periods of time and the potential for airborne disease transmission is fairly obvious, except perhaps to airline owners who fly in private jets. airplanes are potential vectors for the transmission of airborne diseases between continents and play a role in the global dissemination of epidemic diseases (masterson and green 1991) . airline crews and passengers have a higher risk of contracting infections on long flights (nrc 2002, ungs and sangal 1990) . respiratory pathogens that have been identified onboard airlines include adenovirus, chickenpox, coronavirus, influenza, measles, mumps, mycobacterium tuberculosis, neisseria meningitidis, and sars virus (kowalski 2006) . it has been reported that 85% of newer airplanes have hepa filters (gao 2004) . however, there is a common practice in the industry of referring to merv 14 filters (95% dsp filters) as 'hepa-like' or 'hepa type' filters, which is misleading. merv 14 filters, while being excellent filters for controlling spores and most bacteria, cannot guarantee protection against viruses and smaller bacteria. coupling a merv 14 filter with a uvgi system can, however, can provide superior performance (kowalski and bahnfleth 2002) . furthermore, the energy costs associated with using hepa filters may not be justified when a simple combination of a merv 13-15 filter and an urv 13-15 uvgi system will provide comparable results at lower cost. uvgi systems can be installed in the recirculation ducts of airplanes as well as being located at individual seats (in the overhead 'gaspers'). the transmission of infectious disease aboard ships is a recurring phenomenon, with onboard transmission of some diseases such as norwalk virus and legionnaire's disease being favored. the incidence of respiratory disease aboard military ships increases as ship size decreases (blood and griffith 1990) . a study of a tuberculosis outbreak aboard a navy ship found that although proximity and direct contact played a role in transmission, airborne transmission of droplet nuclei, including via the ventilation system, was responsible for most of the secondary infections (kundsin 1980) . it is common for outbreaks aboard ships to consist of multiple viral and bacterial infections, including diarrheal illness and influenza (ruben and ehreth 2002) . noroviruses are responsible for 23 million cases of illness each year and norwalk viruses have recently caused numerous outbreaks of gastroenteritis on cruise ships. they are largely attributed to fomites on ship surfaces. during 2002, cruise ships with foreign itineraries sailing into us ports reported 21 gastroenteritis outbreaks on 17 cruise ships, of which most were identified as noroviruses (cramer et al. 2003) . in an influenza outbreak aboard a cruise ship from hong kong in 1987, 38% of passengers came down with acute respiratory illnesses (berlingberg et al. 1988) . uv applications for ships include in-duct uv systems to interdict recirculated pathogens and allergens, and surface irradiation systems to control fomites. noroviruses may be particularly susceptible to after hours uv systems placed in hallways, bathrooms, and other locations where occupancy is intermittent. cruise ships can also be irradiated and decontaminated between voyages by portable uv systems. most of the microorganisms associated with waste are either water borne or food borne pathogens and allergens and workers in sewage, wastewater, and waste processing industries are subject to occupational hazards from these microbes. airborne hazards also exist since aerosolization of microbial pathogens, endotoxins, and allergens is an inevitable consequence of the generation and handling of waste material. table 18 .4 lists the most common microorganisms that have been found to grow or to occur in sewage and waste (kowalski 2006) . many of these are human pathogens and they include bacteria, viruses, fungi, and protozoa. for information on the uv susceptibility of these pathogens refer to appendix a, which addresses most of these species. many of these species are potentially airborne and can cause respiratory infections, while most of the remainder are either waterborne or foodborne stomach pathogens. wastewater treatment workers are exposed to a variety of infectious agents and khuder et al. (1998) examined the prevalence of infectious diseases and associated symptoms in wastewater treatment workers over a 12-month period. the wastewater workers exhibited a significantly higher prevalence of gastroenteritis, gastrointestinal symptoms, and headaches over those in a control group but no significant differences were found with regard to respiratory and other symptoms. thorn et al. (2002) , however, found that sewage workers had significantly increased risks for respiratory symptoms, including chronic bronchitis, and toxic pneumonitis, as well as central nervous system problems, over workers in non-sewage industries. reduction of microbial hazards in the sewage and waste industries is probably best approached using source control methods, but there are potential applications for uvgi. in-duct air disinfection can promote healthier breathing air for workers inside plants and both upper room and lower room uvgi systems can provide disinfection of both air and surfaces where mold and bacteria may accumulate. area disinfection systems, like after-hours uv systems, may also provide a means of decontaminating areas during periods when they are not occupied (i.e. overnight). hantavirus pulmonary syndrome associated with entering or cleaning rarely used, rodentinfested structures standard 62: ventilation for acceptable indoor air quality pathogens in the environment effect of ultra-violet irradiation of classrooms on spread of mumps and chickenpox in large rural central schools occupational asthma and allergies rhinovirus infections in nursery school children inactivation of food spoilage fungi by ultraviolet (uvc) irradiation listeria: a practical approach to the organism and its control in foods acute respiratory illness among cruise ship passengers -asia epidemiology of adenovirus-associated virus infection in a nursery population ship size as a factor in illness incidence among u.s. navy vessels subclinical legionella infection in workers near the source of a large outbreak of legionnaires disease prevalence of mycoplasma pneumoniae in children in diyarbakir, the south-east of turkey biofilms and their consequences, with particular reference to hygiene in the food industry epidemiology of pulmonary tuberculosis in the prison environment biocontaminants in carpeted environments outbreaks of gastroenteritis associated with noroviruses on cruise ships -united states bacteria and other bioaerosols in industrial workplaces high pharyngeal carriage rates of streptococcus pyogenes in dunedin school children with a low incidence of rheumatic fever microflora of the air in sawmills as a potential occupational hazard: concentration and composition of microflora and immunologic reactivity of workers to microbial aeroallergens outbreak of tuberculosis in a church an outbreak of measles at an international sporting event with airborne transmission in a domed stadium an outbreak of viral gastroenteritis following environmental contamination at a concert hall ultraviolet radiation for the processing and treatment of food. food and drug administration mold in air conditioner causes pneumonitis in office workers more research needed on the effects of air quality on airliner cabin occupants seroepidemiological studies on the occurrence of common respiratory infections in paediatric student nurses and medical technology students a study of whooping cough epidemic outbreak in castellon biodeterioration of nonwoven fabrics prospective study of respiratory infections at the u.s. naval academy silent passage of echo virus type 20 together with dyspepsia coli strains in a day nursery school study on the sterilization of grain surface using uv radiationdevelopment and evaluation of uv irradiation equipment an outbreak of scarlet fever, impetigo and pharyngitis caused by the same streptococcus pyogenes type t4m4 in a primary school heat and moisture transfer: international energy agency pleurodynia among football players at a high school. an outbreak associated with coxsackievirus b1 fungal contamination of oil paintings in japan shared office space and the risk of the common cold frequency of serological evidence of bordetella infections and mixed infections with other respiratory pathogens in university students with cough illnesses shedding some light on iaq transmission of mycobacterium tuberculosis among employees in a us government office prevalence of infectious diseases and associated symptoms in wastewater treatment workers the optimal system of ambulatory examination of groups with high risk of tuberculosis at a large industrial plant transmission of mycobacterium tuberculosis among high school students in korea effects of uv irradiation on selected pathogens in peptone water and on stainless steel and chicken meat current trends in uvgi air and surface disinfection merv filter models for aerobiological applications aerobiological engineering handbook: a guide to airborne disease control technologies exposure to airborne microorganisms in furniture factories airborne contagion uv irradiation of shell eggs: effect on populations of aerobes, molds, and inoculated salmonella typhimurium fungal and actinomycete spores as pollutants of the workplace and occupational illness the epidemiology of the common cold application of ultraviolet-c light on storage rots and ripening of tomatoes on building-related causes of the sick building syndrome evidence for airborne transmission of norwalk-like virus (nlv) in a hotel restaurant inactivation of conidia of botrytis cinerea and monilinia fructigena using uv-c and heat treatment dissemination of human pathogens by airline travel pharyngitis associated with herpes simplex virus in college students aeroallergens and workrelated respiratory symptoms among office workers illness among schoolchildren during influenza season: effect on school absenteeism, parental absenteeism from work, and secondary illness in families the airliner cabin environment and the health of passengers and crew rheumatic fever-is it still a problem? farmer's lung: thermophilic actinomycetes as a source effect of ultra-violet irradiation of classrooms on the spread of measles in large rural central schools public health management of an outbreak of group c meningococcal disease in university campus residents maritime health: a case for preventing influenza on the high seas inhaled particles and respiratory disease introduction to food and airborne fungi prevalence, rate of persistence and respiratory tract symptoms of chlamydia pneumoniae infection in 1211 kindergarten and school age children preservation of bakery products microbiological indoor air quality in healthy buildings ultraviolet light q fever in a wool and hair processing plant indoor air quality handbook viability of airborne salmonella newbrunswick under various conditions integration of ultraviolet (uv-c) light with yeast treatment for control of post harvest storage rots of fruits and vegetables work related symptoms among sewage workers: a nationwide survey in sweden bioaerosol lung damage in a worker with repeated exposure to fungi in a water-damaged building flight crews with upper respiratory tract infections: epidemiology and failure to seek aeromedical attention effect of germicidal uvc light on fungi isolated from grapes and raisins foreign body prevention, detection and control the use of ultraviolet radiation to reduce salmonella and psychrotrophic bacterial contamination on poultry carcasses tb in leicester: out of control, or just one of those things reduction of escherichia coli and salmonella senftenberg on pork skin and pork muscle using ultraviolet light efficacy of ultraviolet treatments for the inhibition of pathogens on the surface of fresh fruits and vegetables evidence of airborne transmission of the severe acute respiratory syndrome virus strains of parainfluenza virus type 4, isolated in a day nursery fungi isolated from library materials: a review of the literature key: cord-032492-2av9kl1c authors: feldman, sue s.; hikmet, neset; modi, shikha; schooley, benjamin title: impact of provider prior use of hie on system complexity, performance, patient care, quality and system concerns date: 2020-09-23 journal: inf syst front doi: 10.1007/s10796-020-10064-x sha: doc_id: 32492 cord_uid: 2av9kl1c to date, most hie studies have investigated user perceptions of value prior to use. few studies have assessed factors associated with the value of hie through its actual use. this study investigates provider perceptions on hie comparing those who had prior experience vs those who had no experience with it. in so doing, we identify six constructs: prior use, system complexity, system concerns, public/population health, care delivery, and provider performance. this study uses a mixed methods approach to data collection. from 15 interviews of medical community leaders, a survey was constructed and administered to 263 clinicians. descriptive statistics and analysis of variance was used, along with tukey hsd tests for multiple comparisons. results indicated providers whom previously used hie had more positive perceptions about its benefits in terms of system complexity (p = .001), care delivery (p = .000), population health (p = .003), and provider performance (p = .005); women providers were more positive in terms of system concerns (p = .000); patient care (p = .031), and population health (p = .009); providers age 44–55 were more positive than older and younger groups in terms of patient care (p = .032), population health (p = .021), and provider performance (p = .014); while differences also existed across professional license groups (physician, nurse, other license, admin (no license)) for all five constructs (p < .05); and type of organization setting (hospital, ambulatory clinic, medical office, other) for three constructs including system concerns (p = .017), population health (p = .018), and provider performance (p = .018). there were no statistically significant differences found between groups based on a provider’s role in an organization (patient care, administration, teaching/research, other). different provider perspectives about the value derived from hie use exist depending on prior experience with hie, age, gender, license (physician, nurse, other license, admin (no license)), and type of organization setting (hospital, ambulatory clinic, medical office, other). this study draws from the theory of planned behavior to understand factors related to physicians’ perceptions about hie value, serving as a departure point for more detailed investigations of provider perceptions and behavior in regard to future hie use and promoting interoperability. health information exchange (hie) has been described in various ways in the literature, but is generally understood as the act of health information sharing, facilitated by computing infrastructure, that is conducted across affiliated physicians' offices, hospitals, and clinics; or between completely disparate health systems (furukawa et al. 2013) . hie across disparate systems allows clinical information to follow patients as they move across different care settings, whether or not each organization shares an affiliation. this might include a hospital connected to an hie that is, in turn, connected to other forprofit and not-for-profit hospitals, private practices, and clinics. hie is expected to transform the nation's healthcare system through access to patient data from electronic health records to support care provision and coordination and improve care quality and population health (berwick et al. 2008) . health information exchange has also been described in terms of the organizational and technical environment facilitating hie. in this paper, hie is the act of exchanging health information facilitated by a health information exchange organizational and technical environment. while expectations and promises are high, still relatively little is known about the real and perceived value of hie by providers, and how to accomplish large-scale acceptance and use. it has been reported that about two-thirds of hospitals and almost half of physician practices are now engaged in some type of hie with outside organizations (rahurkar et al. 2015) . relatively few of the more than 100 operational u.s. health information exchanges have been the subject of published evaluations (rudin et al. 2014) . after more than a decade of hie hype, utilization by users is still relatively low. a review of hie past research indicated that most studies reported use of hie in 2% to 10% of encounters (rudin et al. 2014 ). further, findings from a review of research on hie sustainability suggest that just one quarter of existing hie organizations consider themselves financially stable (rudin et al. 2014) . a systematic review of hie studies suggests that study stakeholders claim to value hie. yet, the effects on a wide range of outcomes are still unknown (rudin et al. 2014) , and little generalizable evidence currently exists regarding sustainable benefits attributable to hie (rahurkar et al. 2015) . some have noted the potential for widespread hie adoption to reduce the utilization and cost of healthcare services richardson 2001) , though empirical evidence is limited (bailey et al. 2013; fontaine et al. 2010) . continued research is needed to understand the factors associated with adoption and use of such a promising, yet underutilized technology. to date, most hie studies have investigated user perceptions of value prior to use, and the intention to use. few studies have assessed factors associated with the value of hie through its actual use. this study investigates provider perspectives on hie comparing those who had prior experience vs those who have only heard of hie, but not yet had experience with it. the purpose of this study is to investigate provider perceptions about hie, comparing those who have used hie to those who have not used hie and how perceptions differ. the objectives of this study are to explore demographic differences in perceptions across different types of providers, assessing several important factors related to the adoption of health it. literature has determined that factors associated with perceived benefits and challenges of health it adoption and use include: 1) the extent to which users perceive a system to be complex vs easy to use (davis 1989; gadd et al. 2011) , 2) technical standards and business concerns that act as barriers to system use (rudin et al. 2014) , 3) the perceived effects that using a system (and its information) has on public or population health (zech et al. 2015; shapiro et al. 2011; hincapie and warholak 2011; hessler et al. 2009; dobbs et al. 2010) , 4) the perceived effects that using a system (and its information) has on patient care delivery (frisse et al. 2012; furukawa et al. 2013; kaelber and bates 2007) , and 5) the perceived effects that using a system (and its information) has on provider performance (davis 1989) . we review these factors below as well as the literature on prior use of an information system: or the effect of prior use vs nonuse on perceptions and expectations of a system. prior use of an information system has been found to be associated with the intention to use them in the future (jackson et al. 1997; agarwal and prasad 1999) . prior it usage behavior tends to be a significant determinant of future usage intention and/or behavior (jasperson et al. 2005) . prior behavior helps form habit, which induces future behavior in an unthinking, automated, or routinized manner, rather than through a conscious process of cognitive or normative evaluation (eagly and chaiken 1993; triandis 1977) . accordingly, future behavior can be viewed as a joint outcome of behavioral intention and habit. though the indirect effects of prior use have seen little investigation in the literature, preliminary evidence to that effect has been reported (taylor and todd 1995a, b) . these authors argued that as individuals gain familiarity with using a given technology (by virtue of prior use), they tend to focus less attention on the amount of effort needed to use the technology, and more on ways in which the technology can be leveraged to improve their job performance and outcomes. the familiarity with the technology gained from prior usage experience and the knowledge gained from learning-by-doing allows users to form a more accurate and informed opinion of the true performance benefits of the target system and its relevance to their job performance. hence, users' performance expectancy tends to become stronger as their prior use of technology increases. we extend this line of reasoning for business information systems to the case of providers' use of health information technology, or in this case, hie. hie is particularly interesting as a unique and differing context from business due to the multi-organizational, distributed, and shared healthcare information technology setting. perceived system complexity, or the degree to which a person believes that using a particular information system would be difficult vs easy or free of effort, has long been used as a construct to assess user acceptance of information technologies (davis 1989) . studies on ease of use have included a range of health information technologies including electronic health records (ehr) (dünnebeil et al. 2012; hennington and janz 2007) , telemedicine (hu et al. 1999) , clinical information systems (paré et al. 2006) , and others. many studies on hie ease of use have focused on the perceptions of prospective users not currently using hie. in one such study, 74.4% of physicians interested in hie, but not currently engaging in hie, perceived that using hie would be easy . in contrast, 37.1% of physicians not interested in hie perceived that using hie would be easy ). in the past, ease of use of hie has been positively predictive of system adoption and usage , and has shown to impact successful retrieval of patient information that affected patient care (genes et al. 2011) . of significant importance for this study is understanding perceived ease of use from users who have actually used hie vs. those who have not. some past studies have found several concerns that providers have in terms of using hie. a meta-analysis of 38 studies showed that stakeholders consider hie to be valuable, but barriers include technical performance, workflow issues, and privacy concerns (rudin et al. 2014) . concerns also include limits on the amount and type of information that providers want to use , and general fears of information overload from ill designed systems and/or utilization of duplicate or seemingly "competing" information systems (rudin et al. 2014) . for example, in one study, quality assurance (qa) reports generated by a health information exchange for medical practices was reported as the least valued system function due to skepticism about report validity and concerns that reports would reflect negatively on providers . another study found that hie usage was lower in the face of time constraints, questioning whether hie may be considered an information burden rather than a help to users vest and miller 2011) . time constraints, especially in primary and emergency care, have also tended to be a cause for concern in engaging in hie. mixed results from hie evaluations have further raised concerns about its utility. for example, one study found that increased hie adoption has been associated with reduced rates of laboratory testing for primary and specialist providers (ross et al. 2013 ). yet, in the same study, imputed charges for laboratory tests did not shift downward (ross et al. 2013) . this evidence led us to include health information exchange system concerns as an important construct for hie provider perceptions and usage. prior studies have indicated the potential for hie to aid in a range of population health and care coordination activities, care quality, and timely health maintenance and screening. in one study, hie was shown to enable identification of specific patient populations, such as homeless (zech et al. 2015) , and those who have chronic or high risk health conditions ). however, not all studies have showed significant results related to population and patient health outcomes (hincapie and warholak 2011) . the authors in one study concluded that hie usage was unlikely to produce significant direct cost savings, yet also noted that economic benefits of hie may reside instead in other downstream outcomes such as better informed and higher overall quality care delivery (ross et al. 2013) . broader hie impacts include positive relationships with public health agencies (hessler et al. 2009 ), improved public health surveillance (dobbs et al. 2010) , and increased efficiency and quality of public health reporting ). thus, we assess user perceptions of the impact of hie on public/population health. the extent to which hie impacts the delivery of patient care has been addressed in prior studies. more generally, the timely sharing of patient clinical information has been shown to improve the accuracy of diagnoses, reduce the number of duplicative tests, prevent hospital readmissions, and prevent medication errors furukawa et al. 2013; kaelber and bates 2007; yaraghi 2015; eftekhari et al. 2017) . for hie specifically, usage has been associated with decreased odds of diagnostic neuroimaging and increased adherence with evidence-based guidelines (bailey et al. 2013 ). these include timelier access to a broader range of patient interactions with the healthcare system (unertl et al. 2013) , improved coordination of care and patient health outcomes for human immunodeficiency virus patients (shade et al. 2012) , and positive patient perceptions of the impact on care coordination (dimitropoulos et al. 2011) . providers continue to engage in hie with the belief that care delivery will improve (cochran et al. 2015) . further, benefits have been noted for specific care settings. emergency department access to hie has been associated with a cost savings due to reductions in hospital admissions, head and body ct use, and laboratory test ordering . further, hie has been associated with faster outside information access, and faster access was associated with changes in ed care including shorter ed visit length (52.9 min shorter), lower likelihood of imaging (by 2.5, 1.6, and 2.4 percentage points for ct, mri, and radiographs, respectively), lower likelihood of admission (2.4%), and lower average charges ($1187 lower) (p ≤ .001 for all) (everson et al. 2016) . provider perceptions about the positive effects hie has on patient care delivery may be the strongest motivating factor for its adoption. for example, in one study, physicians most agreed that the potential benefits of hie lie in care quality and were least worried about the potential for decreases in revenues resulting from the technology (lee et al. 2012) . a study that looked at perspectives of home healthcare providers indicated a decrease in ed referral rates with hie (vaidya et al. 2012) . in another study, looking up clinical information (test results, clinic notes, and discharge summaries) on a patientby-patient basis was found to be the most valued function for hie users, followed closely by the delivery of test results, for the care of patients ). as such, perceived impact of hie on care delivery is included as a dependent variable. one expected outcome of using hie is that a performance improvement would occur. perceived usefulness is defined here as "the degree to which a person believes that using a particular system would enhance his or her job performance." (davis 1989) in sum, when a system is perceived to be useful, users believe positive performance will result. prior hie studies have shown that certain medical information is perceived to be more useful than others. one prior study found that physicians expressed agreement that hie is useful for pathology and lab reports, medication information, and diagnoses with chief complaints (lee et al. 2012 ). however, they expressed less agreement regarding the need for patient data items, functional test images and charts, care plans at the referring clinic/hospital, or duration of treatment (lee et al. 2012) . different studies reported that providers expected hie data would be useful to improve completeness and accuracy of patients' health records, efficiency with which clinical care is delivered, quality and safety of care, communication with other providers and coordination and continuity of care cochran et al. 2015) . prior studies have also indicated that quality patient information is believed to impact the elimination of duplicated medication as well as lab and imaging tests, prevention of drug-drug interactions, better decision making on the care plan and expedited diagnoses, and better ability to explain care plans to patients (lee et al. 2012) . physicians have noted that data gaps, such as missing notes, adequacy of training (cochran et al. 2015) , and timely availability of information (melvin et al. 2016 ) may pose a significant challenge to future hie usage (rudin et al. 2011) . while these perceptions of usefulness are important relative to expected hie use and resulting provider performance, we see expected and actual system use as scenarios that could potentially result in contrasting viewpoints. perceptions of provider performance that result from actual use of hie may provide a more real-to-life assessment. the objectives of this study are to explore demographic differences in perceptions across different types of providers, assessing several important factors related to the adoption of health it. our hypothesis is that provider age, gender, type of licensure (i.e., doctors, nurses), provider organizational setting (hospital, private practice), role in an organization (administration, patient care), and prior experience using hie are factors that affect provider perceptions about hie. for example, lee and colleagues (lee et al. 2012) found that different physician practice settings significantly influenced individual user perceptions. based on this information, this study is addressing differences in provider perceptions for different hie related constructs described above. the above research question was empirically tested using a field survey of practicing health providers in the state of virginia, usa. the specific technology examined was health information exchange and the action examined was hie. in march 2010, the office of the national coordinator for health it (onc) awarded a state cooperative agreement to the virginia department of health (vdh) to govern statewide hie. in september 2011, community health alliance (cha) was awarded a contract from vdh to build the virginia statewide health information exchange; connectvirginia was subsequently initiated to accomplish this goal. statewide health information exchanges were regarded as an organizational structure to provide a variety of mechanisms to enable hie using standardized technologies, tools, and m e t h o d s . d u r i n g t h e 2 7 -m o n t h s t u d y p e r i o d , connectvirginia designed, tested, developed, and implemented three technical exchange services: connectvirginia direct messaging (a secure messaging system), connectvirginia exchange (the focus of this study), and a public health reporting pathway. connectvirginia exchange is a query/retrieve service in which a deliberate query passively returns one or more standardized continuity of care documents (ccds) that provide a means of sharing standardized health data between organizations on-boarded and connected to connectvirginia. the health information exchange design was based on a secure means to exchange patient information between providers via direct messaging, a secure means for query and retrieval of patient information via exchange, and a secure means for public health reporting. consistent with other health information exchange developments, a standardized product development lifecycle was used to create and implement the system. connectvirginia's direct and exchange protocols were originally established by onc and used as a method to standardize hie of secure messages and ccds. similar to the original nationwide health information exchange (nwhin) connect's exchange, connectvirginia's exchange involves pulling information by providers from an unfamiliar healthcare facility. connectvirginia's direct is a simplified version of the connect software that allows simple exchange of basic information between providers (dimick 2010) . the public health reporting pathway was established using secure file transport protocols. a survey questionnaire was developed and administered to physicians, dentists, nurse practitioners, registered nurses, physician assistants, and nurse midwives in virginia. we evaluated user perceptions of the hie using selected survey items. each clinician was given a summary of the study, the study protocol, consent procedure, and were notified that the research had been approved by the institutional review board of the lead researcher's university. each participant consented to participate and was assured their responses would be anonymous. the survey had three sections: (1) demographics (age, job, gender) and system usage characteristics; (2) familiarity with technology; and (3) user perceptions across an author generated scale inclusive of the following constructs: system complexity, health information exchange system concerns, provider performance, patient care, and population care. a panel of experts reviewed items for face validity. we collected responses for all items on a scale of 1 to 4 from strongly agree (1) to strongly disagree (4). participants could also leave comments in several sections. purposive sampling was used to invite individuals, key informants, and thought leaders in the medical community to participate in 60-min interviews. fifteen interviews were conducted. from these interviews, a survey was created and administered to physicians, dentists, nurse practitioners, registered nurses, physician assistants, and nurse midwives. the sample was achieved from multiple sampling sources including two state medical societies and through a virginia medical providers e-rewards panel. e-rewards, a research now company, is one of the leading providers of online sampling in the u.s. product research industry. surveys were conducted via two channels: telephone and internet. e-rewards surveys were conducted by experienced telephone surveyors. in order to avoid survey bias, online and telephone surveys rotated questions. the goal was to achieve 250 usable responses. over a 4-week period, invitations to participate in the survey and the survey link were distributed in monthly newsletters to qualified members of the medical society of virginia (11,000 members) and the old dominion medical society. old dominion medical society did not disclose the total number of members. this resulted in 125 surveys, of which 113 were usable. e-rewards panel members were called until 150 completed and usable surveys were accomplished. combined, this yielded in 263 usable surveys. ibm spss data collection was used to create and administer the survey online. data were collected from may through june 2012. all surveys collected through telephone were entered into the online survey system as responses were collected. data were analyzed from the online and telephone versions of the survey together. internal reliability of the scale was calculated using cronbach's alpha and used the statistical package ibm spss v. 22 for quantitative analyses. descriptive statistics were compared across demographics and usage characteristics. data were summarized using mean, median, and sd, and a sign test was used to determine if individual subscale items were significantly different from neutral. to determine the effects of our independent variables (prior use, gender, age, professional license, role in the organization, type of organization) on our dependent outcomes, an analysis of variance (anova) was used along with tukey hsd tests for multiple comparisons. the five constructs of interest to this study were health information exchange system complexity, system concerns, public/ population health, care delivery, and provider performance. each construct was measured using multiple-item survey questions, adapted from prior research, and reworded to reflect the current context of providers hie usage. the complete item scales are provided in table 1 . system complexity was measured using four likert-scaled items adapted from davis' perceived usefulness scale (perceived usefulness is also referred to as performance expectancy in the information technology usage literature) (davis 1989 ). perceptions about system concerns were measured using items modified from taylor and todd (taylor and todd 1995a, b) . the effect of hie on patient care delivery, public/population health, and provider performance was measured using investigator developed likert-scaled items guided by literature review. prior hie usage was measured using three items similar to thompson et al. (thompson et al. 1994 ) that asked subjects whether they had previously or currently used the system. we did not have access to actual system-recorded usage data, and thus, self-reported usage data was employed as a proxy for actual recorded usage. since the usage items were in the "yes/ no" format, in contrast to likert scales for other perceptual constructs, common method bias was not expected to be significant. we assess demographic factors including participant gender, age, and professional license (physician, nurse, other, administrative); role in the organization (patient care, administration, teaching/research, other); and type of organization (hospital, ambulatory clinic, medical office, other). these were all determined via selectable items in the instrument. each of the five constructs were tested for their reliability, or the extent to which each represents a consistent measure of a concept. cronbach's alpha was used to measure the strength of consistency. results of validation testing were found to be strong for patient care (0.82), provider performance (.90), and population care (.78); and moderate for system complexity (.62) and system concerns (.64). among responses, 207 physicians indicated they had used hie previously and the 56 remaining physicians indicated they had not yet used hie. respondents represented all clinical specialties, including internal medicine, pediatrics, gynecology, pathology, general surgery, anesthesiology, radiology, neurology, oncology, and cardiology. selected sample demographics, along with the population demographics for all 263 providers at this hospital (obtained directly from the hospital administration), are shown in table 1. a one-way between subject's anova was conducted to compare statistical differences across demographic categories on each previously identified construct. demographic categories included: whether the participant had previously engaged with hie or not, gender, age grouping, professional license, role in the organization, and type of organization. constructs previously identified included health information exchange complexity, health information exchange system concerns, benefits of hie on patient care, population health, and provider performance. results from anova are shown in table 2 . results indicated that there was a statistically significant difference between those that had previously used hie and those that had not used hie for four constructs. these included provider beliefs about the complexity (f(1, 261) = 10.336, table 2 . statistically significant differences between gender groups were found for three constructs including provider system concerns (f(1, 247) = 30.413, p = .000); and perceived benefits on patient care (f(1, 247) = 4.731, p = .031), and on population health (f(1, 247) = 6.989, p = .009). statistically significant differences between age groups were found for three constructs including provider perceived benefits on patient care (f(2, 260) = 3.474, p = .032), population health (f(2, 260) = 3.944, p = .021), and provider performance (f(2, 260) = 4.349, p = .014). statistically significant differences between professional license groups (physician, nurse, other license, admin (no license)) were found for all five constructs, including provider perceptions about system complexity (f(3, 259) = 4.818, p = .003), provider concerns about use (f(3, 259) = 15.927, p = .000), provider perceived benefits on patient care (f(3, 259) = 6.186, p = .000), population health (f(3, 259) = 3.406, p = .018), and provider performance (f(3, 259) = 4.000, p = .008). statistically significant differences between type of organization setting (hospital, ambulatory clinic, medical office, other) were found for three constructs including provider concerns about use (f(3, 259) = 3.438, p = .017), and provider perceived benefits on population health (f(3, 259) = 3.399, p = .018), and on provider performance (f(3, 259) = 3.434, p = .018). there were no statistically significant differences found between groups based on a provider's role in an organization (patient care, administration, teaching/research, other). an anova test is important to assess the significance of results; however, an anova test does not provide information about where the statistically significant differences lie for multiple comparisons (groupings for age, licensure, type of organization). in order to analyze which specific group means are different, tukey's hsd test is conducted for anova results with statistically significant f-values (tukey 1949) . tukey hsd post hoc comparisons indicated that the mean score for participants between 40 to 55 years old (m = 1.62, sd = 0.57) was significantly different from participants over 55 years old (m = 1.82, sd = 0.52) for provider perceived benefits on patient care. these two age groups, respectively, were also significantly different in terms of perceived benefits on population health ( results from tukey hsd post hoc tests on type of organization indicated that participants whose primary work affiliation is with hospitals (m = 2.89, sd = 0.66) were significantly different than those who identified with medical offices/ private practice (m = 2.70, sd = 0.67) in terms of their concerns with using hie. results showed these two groups were .005 (7.992)* .014 (4.349)* .110 (2.577) .008 (4.000)* .914 (.174) .018 (3.434)* *the mean difference is significant at the 0.05 level also significantly different in terms of hie benefits on provider performance ((m = 1.96, sd = 0.57) vs. (m = 2.20, sd = 0.53), respectively). hospital based providers were less concerned and more positive towards performance benefits. results also indicated that ambulatory clinics (m = 1.72, sd = 0.62) were significantly different from medical offices/ private practices (m = 2.10, sd = 0.75) in their beliefs that hie benefits population health. the primary goal of this study was to identify differences in providers' perceptions related to hie based on their demographics and prior use of hie. a field survey was created based on provider interviews and administered to providers from different disciplines. the findings indicate that there were statistically significant differences in most hie perceptions based on demographics including prior hie use, gender, age, professional license, and type of organization. professional role in an organization yielded no statistically significant differences. the providers that had previously used hie showed more positive responses towards hie in each category except for the system concerns category. these results seem to support the business literature indicating that prior use of an information system has been shown to be associated with the intention to use it in the future, regardless of the amount of effort needed to use the technology (jackson et al. 1997; agarwal and prasad 1999) . there was a statistically significant difference in perceptions related to system complexity, system concerns, patient care, and population care between different genders, with females generally showing more positive responses than males for each category. depending on age groups, a statistically significant difference was observed for the patient care, population care, and provider performance constructs. interestingly, the middle group (ages 40 to 55) showed more positive responses than both the younger providers (less than 40 years) and older providers (over 55 years) for each category. while other studies have not looked at age by these constructs, this finding differs from literature suggesting that those 45 years and younger are more likely to adopt ehrs (decker et al. 2012) . it could be that the hie context may be perceived differently than ehrs due to it being connective to and enabling of interoperability between ehrs. it could also be the size of the sample for each age group. in this sample, we have more providers that are in that middle age group compared to those that belong to the younger and older age groups. another potential way to explain this finding could be using the diffusion of innovation theory (kaminski 2011) . younger providers and older providers could be part of the later majority (conservatives) and may be less than enthusiastic at the beginning, as they could be waiting on statistically significant evidence before adopting and implementing new technology. the middle age group could be part of the visionaries or pragmatists as described in the diffusion of innovation theory and may be willing to be the trail blazers or risk takers (kaminski 2011) . statistically significant differences for all five constructs were noted across all professional license groups, which indicates that providers have different perceptions about hie depending on their discipline. in general, nurses reported the lowest scores about perceived complexity of hie, yet the highest scores in terms of system concerns. other licensed professionals reported the highest scores (high perceptions about hie task complexity). nurses and administrators reported the most positive scores on the benefits of hie for patient care, population health, and provider performance. doctors reported the least amount of concern with using hie, while nurses reported the highest amount of concern. statistically significant differences were noted for system concerns, population health, and provider performance constructs between different types of organizational groupings. the hospital based and "other organization" type groupings reported the least concerns about hie use and those working for medical offices reported the highest amount of concern. participants who reported working within ambulatory clinics reported the highest perceived benefit of hie on population health while those working within medical offices/private practice reported the lowest perceived benefit and this is consistent with the literature across a variety of ambulatory settings (haidar et al. 2017) . participants who reported working within hospitals reported the highest perceived benefit of hie on provider performance while those working in medical offices/private practice reported the lowest perceived benefit. the findings of this study should be interpreted in light of its limitations. the first limitation is our measurement of the prior hie usage behavior construct. our self-reported measure of usage was not as accurate, unbiased, or objective as usage data from system logs. we urge future researchers to use system log-based measures of it usage, if available. second, our small sample size, and correspondingly low statistical power, may have contributed to our inability to observe significant effects of prior usage behavior on each construct. we encourage future researchers to consider using larger samples, such as by using pooled observations from two or more hospitals and/or other healthcare facilities. third, there were 14 participants that did not select either male or female gender. thus, the analysis of gender did not include the entire sample. finally, there may be additional factors beyond those examined in this study. our choice of the factors used here was motivated by the hie literature and a first round of interviews with providers. however, there may be other theories, such as innovation diffusion theory or political theory, that may also be relevant to explaining provider perceptions and behavior. future studies can explore those theories for identifying other predictors of provider behavior and/or compare the explanatory ability of those theories discussed herein. the findings of this study have interesting implications for health it practitioners. first, we provide evidence of the perspectives of various types of providers in terms of their beliefs and perceptions about hie. perceptions about system complexity, system concerns, patient care, population health, provider performance, and prior usage have varying effects in terms of influencing provider' perceptions about hie. however, implementing standards of care that incorporate hie is an instance of organizational change, requiring careful planning and orchestrating of change management to influence providers to routinely use the targeted technologies. change management programs designed to enhance provider intentions to use health it should focus on educating users on the expected performance gains from technology usage as well as improving their perceptions of behavioral control by training users to use those technologies appropriately. the significance of prior behavior on future hie usage intention is indicative of the importance of recruiting early adopters to "seed" hie usage in hospitals and healthcare settings. junior practitioners, by virtue of their more recent medical training involving the latest health it, may be viewed as more likely to be such early adopters. however, the results of this study also show that providers age 40 to 55 may provide a strong base of supporters. given their prior usage behavior and correspondingly, higher level of comfort with such technologies, these individuals are likely to continue using hie further in hospital settings, even when other conditions may be less conducive to their usage. with major health it policy efforts focused on interoperability, this study contributes to the perspective that different provider groups may be stronger facilitators of interoperability efforts than others and thus these findings could help managers and policymakers determine strategies for such efforts. this study examined provider perceptions about hie, comparing those who have not used hie with those who have previously used the technology. as such, the role of prior behavior on providers' perceptions and intentions regarding future hie usage provides new insights. one may hypothesize that if individuals use a system that is perceived to provide value, then use of that technology will proliferate and expand throughout the intended user population. findings indicate that this may not always be the case. as systems become more integrated, inter-organizational, and more complex, user perceived value derived from using that system may not be understood by the user. this study contributes to the nascent stage of theorizing in the medical informatics literature by presenting the theory of planned behavior as a referent theory that can not only help us understand providers' usage of hie better, but can also serve as a starting point for more detailed investigations of provider behavior. second, given that the centrality of hie usage to improving healthcare delivery, quality, and outcomes and the uphill battle many states and regions are currently facing to get providers to use hie, our study provides some preliminary suggestions about how providers' behaviors can be influenced using a strong evidence base. we also elaborate the contingent role played by prior hie usage experience in shaping providers' usage patterns. presumably, there may be more such contingent factors that may be the subject of future investigations. in conclusion, we hope that this study will motivate future researchers to examine in further depth provider hie usage behavior and contribute to a cumulative body of research in this area. publisher's note springer nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations. sue feldman, rn, med, phd is professor and director of graduate programs in health informatics in the school of health professions at the university of alabama at birmingham. dr. feldman is also a senior scientist in the informatics institute and senior fellow in the center for the study of community health. her research focuses on health information systemsfrom development to evaluation. dr. feldman also serves on the health informatics accreditation council for the commission on accreditation for health informatics and information management (cahiim), chairs graduate health informatics accreditation site visit teams, and has studied and developed graduate level health informatics curriculum. as a registered nurse (rn) for over 30 years, she brings a unique clinical and informatics blend to everything she does, grounding policy and theory with practice. dr. feldman has published in a variety of top-tier peer-reviewed journals and conference proceedings, led or co-led the development of several information systems that are grounded in research, and has served as program chair for several national forums. her current work involves leading the development of a substance use, abuse, and recovery data collection system for the state of alabama as well as a statewide covid-19 symptom and exposure notification system. dr. feldman has a masters degree in education and a phd in education and also in information systems and technology from claremont graduate university. neşet hikmet is professor of health information technology in the college of engineering and computing, university of south carolina where he also serves as the director of applied sciences at center for applied innovation and advanced analytics. his research expertise includes cloud computing, augmented analytics, health informatics, and healthcare internet of things. as an applied scientist he is heavily involved in design, development, deployment, and maintenance of large-scale computing systems. dr. hikmet has significant experience in leading health data analytics projects within academia, including utilization of a wide range of analytics methods and approaches. his research and projects have been funded by the national science foundation, national institutes of health, and other federal and state agencies and private foundations. shikha modi, mba is a graduate of uab's mba program with an emphasis on healthcare services. ms. modi has a bachelor's degree in biology from university of north alabama. ms. modi's current research projects include health information technology and its impact on outcomes, health information exchange and provider performance, health information exchange and patient and population health, the intersection of health informatics, healthcare quality and safety, and healthcare simulation, patient experience evaluation at dermatology clinics, and identifying and mitigating bias in artificial intelligence systems. benjamin schooley, mba, phd is associate professor of health it in the college of engineering and computing, university of south carolina where he also serves as research director at the health information technology consortium. his research expertise includes human-computer interaction, health informatics, and human factors in the design and application of software systems. as a design scientist, his applied and field research in health, wellness and social-benefit initiatives have been funded by the national science foundation, national institutes of health, the centers for medicaid and medicare services, the u.s. department of labor, social security administration, and other federal and state agencies and private foundations. are individual differences germane to the acceptance of new information technologies? decision sciences does health information exchange reduce unnecessary neuroimaging and improve quality of headache care in the emergency department the triple aim: care, health, and cost health care provider perceptions of a querybased health information exchange: barriers and benefits perceived usefulness, perceived ease of use, and user acceptance of information technology physicians in nonprimary care and small practices and those age 55 and older lag in adopting electronic health record systems nhin direct: onc keeps it simple in effort to jumpstart data exchange public attitudes toward health information exchange: perceived benefits and concerns the northwest public health information exchange's accomplishments in connecting a health information exchange with public health determinants of physicians' technology acceptance for ehealth in ambulatory care the psychology of attitudes do health information exchanges deter repetition of medical services? health information exchange associated with improved emergency department care through faster accessing of patient information from outside organizations systematic review of health information exchange in primary care practices the financial impact of health information exchange on emergency department care the financial impact of health information exchange on emergency department care hospital electronic health information exchange grew substantially in 2008-12 user perspectives on the usability of a regional health information exchange adoption of health information exchange by emergency physicians at three urban academic medical centers association between electronic medical record implementation and otolaryngologist productivity in the ambulatory setting information systems and healthcare xvi: physician adoption of electronic medical records: applying the utaut model in a healthcare context assessing the relationship between health information exchanges and public health agencies the impact of health information exchange on health outcomes examining the technology acceptance model using physician acceptance of telemedicine technology toward an understanding of the behavioral intention to use an information system a comprehensive conceptualization of post-adoptive behaviors associated with information technology enabled work systems health information exchange and patient safety diffusion of innovation theory physicians' perceptions and use of a health information exchange: a pilot program in south korea health information exchange in the ed: what do ed clinicians think? the effects of creating psychological ownership on physicians' acceptance of clinical information systems physicians' potential use and preferences related to health information exchange despite the spread of health information exchange, there is little evidence of its impact on cost, use, and quality of care crossing the quality chasm: a new health system for the 21st century health information exchange in small-to-medium sized family medicine practices: motivators, barriers, and potential facilitators of adoption effects of health information exchange adoption on ambulatory testing rates what affects clinicians' usage of health information exchange usage and effect of health information exchange: a systematic review health information exchange interventions can enhance quality and continuity of hiv care using health information exchange to improve public health assessing it usage: the role of prior experience understanding information technology usage: a test of competing models influence of experience on personal computer utilization: testing a conceptual model interpersonal behavior comparing individual means in the analysis of variance bridging organizational divides in healthcare: an ecological view of health information exchange perceptions of health information exchange in home healthcare the association between health information exchange and measures of patient satisfaction factors motivating and affecting health information exchange usage an empirical analysis of the financial benefits of health information exchange in emergency departments identifying homelessness using health information exchange data key: cord-280459-y0tbvs3t authors: ramvikas, m.; arumugam, m.; chakrabarti, s.r.; jaganathan, k.s. title: nasal vaccine delivery date: 2016-10-07 journal: micro and nanotechnology in vaccine development doi: 10.1016/b978-0-323-39981-4.00015-4 sha: doc_id: 280459 cord_uid: y0tbvs3t the mucosal surfaces represent the major site of entry of many pathogens, and major challenges in vaccine development include safety and stability in a suitable dosage form. microand nanocarrier-based delivery systems as nasal vaccines induce humoral, cellular, and mucosal immunity. the nasal route of vaccination could also offer immunity at several distant mucosal sites (oral, rectal, vaginal, and pulmonary), which is considered a simplified and cost-effective mode of vaccination with enhanced patient compliance. most of the nasal vaccine delivery systems in the form of microparticulates, nanoparticulates, and liposomes are currently under development and prove to offer immunity in animal models. the importance and potential of the nasal route of administration for vaccines is unexplored, and this chapter outlines the opportunities, challenges, and potential delivery solutions to facilitate the development of improved nasal vaccines for infectious diseases. mucosal infections are caused by emerging pathogens such as bacteria and viruses, which are mucosally transmitted and replicate in the mucosal tissues. these pathogens enter the human through single or multiple mucosal sites such as oral, nasal, conjunctival, respiratory, gastrointestinal, and genitourinary tracts, invade the bloodstream, and finally lead to infection. approximately 68% of deaths in children younger than 5 years are due to mucosal infections. 1 bacteria such as streptococcus pneumoniae, 2 staphylococcus aureus, 3 anaerobic bacteria, 4 streptococcus viridans, 5 α-streptococci, enterobacteriaceae, and viruses such as haemophilus influenzae, corona virus, and human adenovirus 6 cause mucosal infections. the mucosal infections are treated by antibiotics, and vaccines are administered through oral, parenteral, and nasal routes. a nasal vaccine induces mucosal immune responses and systemic immunity, which provides better protection against infectious agents. yet developing vaccine delivery systems that induce humoral and cell-mediated response with mucosal immunity has been challenging to date. different mucosal routes are being explored using different delivery systems, and the nasal route is preferred as compared to other mucosal routes. the mucosal route of vaccine delivery is one of the better alternatives to conventional multiple injection vaccines, which employ traumatic procedures and may also lead to the spread of infectious agents via contaminated syringes. mucosal vaccine development has grown extensively, and a multitude of vaccine delivery systems have been developed for application via ocular, nasal, oral, rectal, and vaginal routes. of all the different routes of mucosal vaccination, the oral and nasal routes are most accepted and easily accessible. for many decades, intranasal applications of tobacco snuff, cocaine, and various hallucinogenic and psychotropic agents have been in practice. 7 similarly, for many years synthetic drugs have been administered intranasally for their local effect on the mucosa (eg, antihistamines, antibiotics, and nonpeptide drugs). 8 the first nasal influenza vaccine was introduced in 2001, but it was later withdrawn from the market due to potential toxicity problems. another intranasal vaccine (flumist) was launched in 2003 and was administered using a syringe sprayer. success of a vaccine depends not only on the delivery but also on the route of administration; for example, nasal immunization elicits potent immunoglobulin a (iga) secretion in the respiratory tract. 9 nasal administrations of vaccines have been shown to achieve a better systemic bioavailability and protection from gastric enzymes compared with parenteral and oral administration. nasal delivery of vaccines acts as a "first entry block," that is, blocks the pathogen entry, while invading to the mucosal surface by inducing local microbial-specific immune responses, thus increasing the general efficacy of the vaccine. in addition, vaccine uptake into the blood circulatory system by absorption through mucosa can be relatively fast. 10 it is the most appropriate method of immunization because it is rich in t cells, b cells, and plasma cells and stimulates both antigen-specific systemic and mucosal adaptive immune responses. it provides better patient compliance due to the needle-free delivery. the nasal route is considered an attractive route for vaccine administration with the following advantages: • better patient compliance • numerous microvilli present in the nasal epithelium provide a better absorption surface • mucosal and systemic immune response can be induced • easy immunization of large population groups • nasal immunization does not require needles and syringes many challenges stand in the way of developing nasal vaccines. when nasal vaccines are administered directly on mucosal surface, the antigens may be diluted by mucosal secretions, seized in mucus gels, attacked by proteases and nucleases, and obstructed by epithelial barriers. major challenges in the development of nasal vaccines include: • a relatively large dose of vaccine is required, and it is difficult to administer through the nasal route and also difficult to monitor the actual dose that crosses the mucosa. • costly innovative vaccination strategy. • efficacy of nasal vaccines may be limited due to the including mucociliary clearance and the inefficient uptake of soluble antigens by nasal epithelial cells in the nasal cavity. 11 • nasal delivery may require adjuvants to enhance their immunogenicity and delivery to the mucosal tissues. • lack of multiple human-compatible mucosal adjuvants. • rapid nasal clearance may not allow sufficient retention for antigen to be taken up by antigen-presenting cells (apcs) in the nasal-associated lymphoid tissue (nalt). • several enzymes that are present in the nasal mucosa might affect the stability of drugs. for example, proteins and peptides are subjected to degradation by proteases and amino-peptidase at the mucosal membrane. 12 • delivery volume in the nasal cavity is restricted to 25-200 µl. 13 • high molecular weight compounds cannot be delivered through this route (mass cutoff ∼1 kda). 14 • low antigen entrapment efficiency mode. • normal defense mechanisms like mucociliary clearance and ciliary beating affect the permeability of the drug. 15 the nose is a vital organ in the human body for breathing. the nose has a more complex role as a complete system of defense against inhaled air and air conditioning. the nasal anatomical and physiological structure provides support for nasal immunization against upper respiratory mucosal diseases. nasal anatomy, nasal morphology and physiology, nasal secretions, nasal mucosa, olfactory region, and blood supply to the nasal cavity are described with respect to their link with the vaccine administration route. the nasal cavity is protected by the viscerocranium in the human head. the human nose is divided by the median septum into two symmetrical halves; each half opens to the face through the nostrils and extends posteriorly to the nasopharynx. 16 the nasal cavity and the nasal vestibule are the anterior part, which opens to the face through the nostrils. the atrium is placed in an intermediate region between the vestibule and the respiratory region. the respiratory region, the nasal turbinates, occupies more area of the nasal cavity. it possesses lateral walls that divide it into three sections composed of the superior nasal turbinate (superior region), nasal turbinate (middle region), and inferior turbinate (inferior region). the attachment of the turbinate to the lateral wall is more complex in animals than in humans. these folds provide the nasal cavity with a very high surface area compared to its small volume. the basic functions of the nose are filtration of particles and heating and humidification of inspired air before it reaches the lungs. the olfactory region situated above the superior nasal turbinate possesses specialized ciliated olfactory nerve cells for smell perception. the human nasal cavity has a total volume of 15-20 ml and a total surface area of approximately 150 cm 2 , 17 of which the respiratory region covers about 85%. each nasal cavity can be subdivided into different regions such as nasal vestibule, turbinate (inferior, middle, and superior), olfactory region, frontal sinus, sphenoidal sinus, and cribriform plate of ethmoid bone. nalt is present in the nasal cavity, which is situated in the nasopharynx. the central axon of these nerve cells passes through the cribriform plate of the ethmoid bone and into the olfactory bulb. the nasal vestibule has numerous nasal hair (vibrissae) that filter large airborne particles ( fig. 15.1) . the anterior section of the nasal portion is constituted by a stratified squamous epithelium with sebaceous glands and the posterior section of the nasal portion by pseudostratified columnar cells presenting microvilli. the nasal respiratory mucosa is considered the most important area for delivering drugs systemically, as its made of epithelium, basement membrane, and lamina propria. 18 the nasal respiratory epithelium consists of pseudostratified columnar epithelial cells, goblet cells, basal cells, and mucous and serous glands. 19, 20 most of the epithelial cells on their apical surface with microvilli and the major part have fine projections, called cilia. the nasal secretion, nasal mucosa, olfactory region, and blood supply to the nasal cavity play a major role in nasal physiology. nasal secretions originate mostly from submucosal glands and goblet cells. mucus is composed of water (95%); glycoproteins (2%); albumin, immunogobulins, lysozyme, lactoferrin and other proteins (1%); inorganic salts (1%); and lipids (<1%). 11 glycoproteins are in low proportion, which provides mucus with its characteristic viscoelastic properties. the mucus layer is divided into the low viscosity lower layer of about 5-10 µm thick and the more viscous upper layer of about 0.5-5 µm thick. the human nasal ph is approximately 5-8 with an average baseline of 6.3. the nasal respiratory mucosa consists of epithelium, basement membrane, and lamina propria. the nasal respiratory epithelium contains pseudostratified columnar epithelial cells, goblet cells, basal cells, mucous, and serous glands. many of the epithelial cells are covered on their apical surface with fine projections of microvilli (called cilia), which enhance the respiratory surface area. the nasal epithelium is covered with a thin mucus layer, which is produced by secretory glands and goblet cells. nasal mucus is responsible for several physiological functions, such as humidification and warming of the inhaled air, 21 and offers physical and enzymatic protection. the olfactory region is located in the top of the nasal cavity about 2-10 cm 2 and extends down the septum and lateral wall. neuroepithelium, which is the only part of the central nervous system (cns), is directly exposed to the external environment. the olfactory area is made of pseudostratified columnar epithelium and is composed of supporting cells, basal cells, microvillar cells, and the typical receptor or olfactory cells. 22 olfactory regions also contain olfactory receptors (receptors for smell sensations) and small serous glands (which produce secretions that act as a solvent). the total surface area of the olfactory epithelium is 200-400 mm. the nasal cavity vasculature requires rich blood supply to fulfill the basic physiological functions such as heating, humidification, olfaction, mucociliary clearance, and immunological roles. the nasal vascular bed is intended as such for rapid and easy exchange of fluid and dissolved excipients between blood vessels and nasal tissue. the capillary flow in the nasal mucosa was reported to be 0.5 ml/g/min. 23 antigen uptake in the nose is mostly by two mechanisms: paracellular (aqueous pathway) and transcellular (lipoidal) processes ( fig. 15 .2). the most efficient area for drug absorption is the highly vascularized lateral wall of the nasal cavity: the mucosa lined over the turbinates or conchae. • in the paracellular mechanism, the antigen uptake nasally is through the aqueous route of transport, and this route is slow and passive. the process mainly depends on an inverse log-log correlation between intranasal absorption and the molecular weight of water-soluble compounds/antigens. poor bioavailability is observed for antigens with a molecular weight greater than 1 kda in the paracellular process. 24 an example is chitosan widening the tight junctions between epithelial cells. • in the transcellular mechanism, the antigen uptake nasally is through a lipoidal route and is responsible for the transport of lipophilic antigen based on lipophilicity. other than transcellular and paracellular processes, antigens may also cross cell membranes by an active transport route via passive diffusion (depends on ph of environment and pka of the drug), carrier-mediated means, or transport through the opening of tight junctions in mucosa (ie, through organic cation or amino acid transport), and endocytic process [uptake of antigen mediated by microfold (m) cells]. 25 there are various factors that affect the antigen uptake in the nasal cavity after nasal immunization, but the bioavailability of a nasal delivery vaccine depends on two major factors: nasal physiological factor and physiochemical properties of nasal vaccine formulation. there are different physiological factors that regulate the absorption of antigen. • the permeability of a nasal vaccine depends on the type of cells and number of cells in the nasal cavity. • secretions of nasal mucosa enzymes such as lactate dehydrogenase, 26 oxidative, conjugative enzymes, peptidases, and proteases also act as a barrier and degrade the nasal vaccine. • stimulation of nasal mucosa plays a role in mucosal absorption. parasympathetic stimulates increase permeability of a nasal vaccine. • viscous nasal mucus secretion may retard the uptake of antigen. • permeation of antigen is altered at night (chronokinetics) due to clearance rates and mucosal secretions. • generally, ph of the nasal cavity for adults is 5.5-6.5 and for infants is 5.0-7.0; for better antigenic absorption, the developed nasal formulation ph should be within 4.5-6.5. 27 • mucociliary clearance (mcc) takes about 21 min in the nasal cavity, and increased mcc decreases antigen uptake. 28 • mucociliary functioning may affect nasal mucosa due to diseases such as the common cold, rhinitis, etc. • environmental conditions (ie, increases in temperature and increases in mucus secretion) also affect the mucociliary clearance. • antigen concentration, dose, and volume of administration are three interrelated parameters that affect the performance of the nasal antigen delivery. 29 • ph of nasal formulation should be in the range between 4.5 and 6.5 because lysozymes found in nasal secretions are responsible for destruction of certain bacteria at acidic ph. 30 the lysosomes will be inactivated at ph above 7 and susceptible to microbial infection in nasal tissue. • pharmaceutical dosage form is another important factor to consider while developing a nasal formulation. nasal drops are simpler and more convenient than powder, gel, and suspension sprays. 31 • pharmaceutical excipients used in the formulation such as buffer components, antioxidants, preservatives, surfactants, humactants, solubilizers, and gelling/viscosifying agents also play a crucial role in the delivery of the antigen. nasal clearance is one of the major challenges in the development of a nasal vaccine. in nasal mucosa, mucus acts as a sticky fluid and cilia act as a motivator, which prevents foreign substances, pathogens, and particles from being carried by inhaled air into the lungs. in physiological conditions, mucus is transported at a rate of 5 mm/min, and its transit time in the human nasal cavity is 15-20 min. 32 it depends on the length, density, and beat frequency of cilia as well as the amount and viscoelastic properties of the mucus. the absorption of antigen is influenced by the residence (contact) time between the antigen and the epithelial tissue in mucosa. if the mucociliary clearance increases, the antigen absorption decreases (ie, inversely proportional to each other). nasal mucociliary clearance can also be stimulated or inhibited by the active ingredient (ie, antigen and/or inactive ingredients such as preservatives, absorption enhancers, and/or other excipients), thus affecting the delivery of antigen to the absorption site. a prolonged residence time in the nasal cavity may also be achieved by using bioadhesive polymers or a particulate delivery system or by increasing the viscosity of the formulation. deposition of antigen in an anterior and posterior region of the nose also affects the antigen absorption of nasal formulation. the anterior portion provides a longer nasal residence time for antigen with low permeability, but antigen permeability is higher in the posterior portion of the nose with shorter nasal residence time. an example is that nasal sprays are deposited anteriorly and cleared slowly into the nasal pharynx by mucociliary clearance. nasal drops are deposited posteriorly and removed rapidly into the nasal pharynx. 33 nalt is the primary target site for nasally administered vaccines, and it consists of lymphoid follicles that occur directly beneath the mucosal follicle-associated epithelial (fae) cells. nalt is in the waldeyer's ring, which includes the pharynx and tonsils. the inductive site for nasal immunity is nalt, m cells, b cells, t cells, dendritic cells (dcs), and regional and cervical lymph nodes. nasally immunized vaccine is transported to nalt through the fae that contains specialized villous m cells for process and presentation. villous m cells lack a brush border, which facilitates the binding and delivery of antigens and also does not secrete mucus or any enzymes. nalt is involved in the induction of dc, b cell, and t cell responses following nasal vaccination and also produces local defense against invading pathogens. part of the soluble antigens may migrate to regional draining lymph nodes through the major histocompatibility complex (mhc) to th cells. activation of antigen-specific cd4+ t helper cells (th cells) interacts with b cells, which develop into iga committed (iga+). the iga+ b cells rapidly migrate from the nalt to the draining cervical lymph nodes in the nasal passage where they differentiate into iga-producing plasma in the presence of cytokines such as il-5 and il-6 that are produced by th2 cells and secrete iga in dimers. dimeric iga then becomes s-iga by binding to the polymeric ig receptor, which transports iga to effector sites. s-iga is able to bind toxins, bacteria, or viruses and neutralize their activity, thus preventing entry into the body or reaching the internal organs, and forms a first barrier of defense against invading antigens. intranasally administered vaccines elicit neutralizing iga, preventing colonization of the throat and systemic igg antibodies and facilitating clearance from systemic sites (fig. 15.3) . immune-competent cells in tonsillar lymphoid tissue and ciliated and nonciliated cells also play a role in immunity generation. the production of iga by both the adenoid tissue and the nasal mucosa contributes significantly to immune protection against inhaled bacteria and viruses. 37 mucosa defends against invading pathogens through two types of immune systems: the innate immunity system and adaptive immune system. the key role of innate immunity and adaptive immunity at the mucosal surface are discussed in the following sections. mucosal surfaces of the nasal tract and respiratory tract are adorned with a potential barrier known as epithelial cell lines. epithelial cells in nasal mucosa are active participants in mucosal defense. epithelia and their associated gland produce innate defenses including mucins and antimicrobial proteins. 38 epithelial cells detect the dangerous/foreign microbial components through pattern recognition receptors such as toll-like receptors (tlrs) and send the cytokine and chemokine signals to mucous membrane-associated apcs, such as dcs and macrophages, to trigger nonspecific/innate defenses and stimulate adaptive immune responses. an important characteristic of mucosa is the production and secretion of dimeric iga that is resistant to degradation in the protease-rich surroundings of mucosal surfaces. in humans, production of iga is more comparable to other immunoglobulin isotypes, and high concentrations of iga antibodies (more than 1 mg/ml) are present in the mucosal surface-associated secretions. iga facilitates the entrapment of microbes and foreign bodies into the mucus by avoiding direct contact of pathogens with the mucosal surface, which is known as "immune exclusion." there are numerous mucosal routes of immunization available as an alternative to current parenteral immunization, namely oral, nasal, pulmonary, vaginal, and rectal. the nasal route is more attractive for several reasons. it is a practical site for easy selfadministration, 39 with the use of commercially available wide delivery devices. nasal immunization generally requires much lower doses of antigen compared with the oral or sometimes parenteral route. nasal immunization does not expose antigens to low ph and digestive enzymes like protease and nuclease, and a protective coating of mucus limits access of antigen into the mucosal epithelium. administration of vaccines through the nasal route may predominantly induce potent immune responses. different types of delivery vehicles suitable for the nasal drug delivery systems are being explored; some are being researched, and a few are in clinical phases. they are classified into two major categories: replicating delivery systems and nonreplicating delivery systems. in a replicating delivery system genetically altered live virus acts as a vector; it proliferates in the host tissues after immunization. it is one of the more prevalent and effective presentations of the antigen and more likely to be effective at stimulating an immune response following oral and nasal delivery. there are different virus vector (rhabdo virus, polio virus, influenza virus, cowpea mosaic virus) [40] [41] [42] and bacterial vector (shigella, salmonella, listeria) 43 replicating delivery systems able to induce a ctl response that produces longer-lasting immunity. introduction of multiple foreign genes can replace the nonessential regions of the viral genome, resulting in an immune response against multiple pathogens. this tactic enables the recombinant virus to be used as a vaccine for two or more infectious agents. vaccine priming with hiv-env-expressing influenza virus induced systemic cellular response in mice. 44 a single intranasal dose of shigella vector based hiv-1 vaccine produced cd8+ t cell response comparable to systemic immunization. 45 replicating antigen delivery systems are not preferred due to their complexity of large exploration. nonreplicating delivery systems are those which mimic the antigens to the immune system and cause similar uptake by apcs. different kinds of nonreplicating delivery systems include: • liposomes • micro-and nanoparticulate systems • immune-stimulating complexes (iscoms) • virus-like particles (vlps) • emulsions • bioadhesive delivery systems liposomes are small artificial vesicles made of bilayer lipid molecules such as phospholipid and cholesterol which encapsulate antigens. liposomes are a promising delivery system because of their size, ampiphilic nature, and biocompatibility. these vesicles form spontaneously when an aqueous solution is added to a dried film of the lipid components. the hydrophilic (water-soluble or polar head) portion of the lipid molecule is present toward the aqueous phase, and the hydrophobic (water-insoluble) portion is allied inside the membrane. many of the phospholipids used to make liposomes are from food products (eg, egg yolk or soybeans), 46 so they are nontoxic and safe. the potency of liposomes depends on different factors such as the surface of the lipid layers, electric charge, composition, and method of preparation. 47 liposomes can also act as an adjuvant, 48 which leads to the induction of potential immune response with low antigen payload. they can also convert nonimmunogenic substances into immunogenic forms (eg, by rendering soluble substances particulate in nature). liposomes are taken up by macrophages and by m cells for antigen processing and/or presentation to other lymphoid cells for the induction of immune responses. they can also directly present antigens to lymphoid cells for the induction of immune responses. nasal application of liposomes containing bacterial polysaccharide antigens has been tested in balb/c mice and revealed that enhanced immune responses in pulmonary secretions was observed in mice after immunization of the liposomal antigen in comparison with antigen alone and as an oral immunization. 49 apart from this, many investigations carried out on liposomes using different antigens are described in table 15 .1. a nasal influenza vaccine, based on a liposome (virosomal) formulation of influenza virus subunits, has recently been marketed in europe by the swiss serum institute (berne, switzerland). liposomes could be a promising delivery system for nasal vaccines. particulate carriers have attracted considerable interest as antigen carriers for achieving delivery of antigens at a specific site in the body. nanoparticles range in size from 1 to iga antibody and ctl responses [66] 1000 nm (1 µm), while microparticles range from 1 to 1000 µm; 79 the smaller particle size promotes faster adsorption. other added advantages include a more stable system protecting from the hostile environment of nasal mucosa, prolonged release, bioadhesion properties, and induction of a significant immune response with reduced dose. depending on the method of preparation, microparticles or nanoparticles can be prepared with different properties and release characteristics. the polymers used for the particulate system are biomolecules such as proteins, peptides, polynucleotides, and polysaccharides or synthetic polymers such as polylactide-polyglycolide copolymers, 80 polyacrylates, polyε-caprolacton, 81 and n-trimethyl chitosan-poly(γ-glutamic acid). 82 nano-and microsized particulate carriers are prepared with the antigenic molecule and deliver it to the desired site of action to induce potent and long-lasting immune responses. the mechanisms by which these particulate systems alter the induction of immune responses are size-dependent penetration, depot effect, repetitive antigen display, crosspresentation, and release of soluble mediators such as cytokines that regulate the immune response. 83 many studies carried out on micro-and nanoparticulate-based nasal vaccine delivery systems using different antigens are shown in table 15 .1. hence a polymer-based micro-/nanoparticulate system can be exploited as a viable nasal vaccine delivery system that is capable of delivering a multitude of antigens at the targeted sites and inducing desired immune response. an iscom is a highly versatile and effective antigen presentation system, which is a spherical open cage-like structure (typically 30-40 nm in diameter) 84 that forms spontaneously on mixing cholesterol, a lipid such as phosphatidyl choline, and the mixture of saponins that comprise quillaja saponins a. 85 the antigen-enveloped quillaja saponin acts as a strong inherent adjuvant as well. in 1984 morein and colleagues showed the potential induction of immune responses upon immunization with iscoms containing viral and bacterial membrane glycoproteins. iscoms are the potent inducers of not only humoral (antibody-mediated) immune responses but also cellular (t cell-mediated) immune responses. presentation of antigens unified with the iscom matrix allows their processing via the endogenous and exogenous pathways, resulting in the stimulation of both cd4+ and cd8+ t cells. 86 moreover, adjuvant activity of the quil a moiety induces long-lasting immune responses. the antibody levels stimulated by iscoms are found to be equivalent to those after immunization with conventional adjuvants such as complete freund's adjuvant or alum. 87 some studies revealed that immunization with iscoms induces a wide range of immune responses including all subclasses of igg and cell-mediated immune responses such as delayed-type hypersensitivity (dth) in vivo, antigen-specific proliferative responses and cytokine production in vitro. [88] [89] [90] an additional property of iscoms is their capability to enter the endogenous antigenprocessing pathway and then mhc class i-restricted cytotoxic t cells (ctl). intranasal immunization of echinococcus granulosus surface antigen iscoms evokes higher serum iga titer in relation to igg than the subcutaneous route in mice. 91 several studies on different antigens using iscom-based delivery systems are shown in table 15 .1. hence iscom-based delivery systems may be suitable for nasal vaccine administration. vlps are a promising option for vaccine delivery to the nasal site because they are easy to access, highly vascularized, self adjuvanting, and highly immunostimulatory and have a relatively large surface area and low proteolytic activity. 92 vlps are inert with a 20-100 nm self-assembled empty capsid protein, 93 which contains no dna/rna and shows a similar size and shape as viruses. vlps are efficiently taken up by dcs and induce potent immune responses after nasal immunization, which induces systemic immunity as well as both local and distal mucosal immunity via the common mucosal immune system (cmis). it is even more superior to parenteral administration at eliciting iga at distal mucosal sites. the vlps can be produced within mammalian cells, insect cells, yeast, bacteria, and even plants through recombinant dna techniques. 94, 95 due to these properties, vlps are exploited as the delivery system for different protein/peptide antigens. despite successful usage and potent immunity, vlps as a delivery system for protein antigens are still limited by the relatively complicated genetic modification on protein fusion and the subsequently required structural integrity characterization. vlp technology has been used for many years. an examples includes recombinant hepatitis b surface antigen (hbsag), produced in saccharomyces cerevisiae or pichia pastoris yeasts. 96 the entire recombinant core antigen (hbcag) of the hepatitis b virus (hbv) promotes th1 immunomodulation of the immune response to coadministered antigens, including the hbv surface antigen (hbsag) after nasal administration. this concept also applies to hiv, dengue, and chronic hepatitis c immunotheraphy; details are discussed in table 15 .1. vlps elicit a potent immune response and increase the immunogenicity of poorly immunogenic antigens, including self-proteins. the potential of micro-/nanoemulsion for various routes of administration has continuously been explored for the past two decades. 97 due to low globule size and lipophilic nature, emulsions are widely chosen as a delivery system to enhance uptake across nasal mucosa. emulsions are of various types including oil-in-water (o/w) and water-in-oil (w/o). most of the novel adjuvants are made up of emulsions such as incomplete freund's adjuvant, adjuvant 65, montanide, and mf59. 98 emulsions are isotropic, transparent, thermodynamically stable, and low viscosity colloidal dispersions, which are stabilized by an interfacial film of alternating surfactants and cosurfactant molecules. ease of administration and scale-up is one of the important advantages over other drug delivery systems such as solid dispersion, liposomes, and particulate delivery systems. emulsion formulation contains different inactive ingredients such as oil (soybean oil, sesame oil, isopropyl myristate, etc.), surfactant (tweens, span80, chromophores, peg, lecithin, cetrimide, etc.), cosurfactant (ethanol, propanol, peg, etc.), and aqueous phase. mucoadhesive polymer such as different grades of carbopol (eg, 974p/971p/980p, sodium alginate, pluronics, etc.) is generally incorporated in the composition of an emulsion to prolong the release of antigens. micro-/nanoemulsion provides good sprayability for nasal vaccine delivery compared with other particulate delivery systems. factors to consider during the preparation of a formulation include nature and concentration of oil, surfactant, cosurfactant, and aqueous phase; ratio of oil/ surfactant/cosurfactant; and temperature and ph of the environment. dilutability, particle size, ph, zeta potential, viscosity, and freeze/thaw cycling are critical parameters in the development of emulsion vaccines. nanoemulsion nasal vaccines have been studied for several diseases like hepatitis b, hiv, and influenza. 99 some studies reported that the nasal vaccine immunization produced strong igg and iga antibody levels, which is similar to alum adjuvant-based vaccines. the emulsion-based delivery approach might be promising for low-cost nasal vaccine immunization for the populations of developing and underdeveloped countries. in a bioadhesive delivery system the antigen carrier system adheres to a biological tissue for an extended period. nasal mucosa bioadhesion protects the antigen from mucosal enzymes and increases retention time. bioadhesive-based nasal vaccines are preferred to overcome nasal clearance issue, 100 to facilitate absorption, and for extended antigen delivery. bioadhesive polymers can be used to increase the nasal residence time by slowing mucocilliary clearance, which allows absorption for a longer time with the nasal mucosa and results in a subsequent increased in absorption. different bioadhesive polymers such as chitosan, carrageenan, carbopol, hydroxypropyl methylcellulose (hpmc), k15m and e5, sodium alginate, sodium carboxy methylcellulose, polyvinyl pyrrolidone (pvp) 90, and xanthan gum are used in the formulations. 101 bioadhesion occurs between polymermucin chains through van der waals, hydrogen, hydrophobic, and electrostatic forces. 102 nasal immunization with different antigens such as tetanus toxoid, influenza, pertussis, and diphtheria following encapsulation and administration using different bioadhesive polymers such as pluronic f127, chitosan and its combination, 103, 104 and peg-coated polylactic acid 80 shows enhanced immune response via induction of igg and iga responses. chitosan-based influenza nasal vaccine produced effective immune response in human clinical trials. 105 bioadhesive drug delivery systems may be a potential tool for extended antigen delivery through nasal immunization. intranasal immunizations are simple, easy, convenient, and safer than other routes of administration. the delivery system selection depends upon the antigen being used for the proposed indication, patient type, and marketing preferences. there are different options available to deliver nasal vaccine such as drops, 106 powder, aerosol sprays, and the application of nasal gel. nasal drops are convenient and the most simple method for delivery of nasal vaccines. the nasal drops are administered using a nasal dropper or syringes. the major disadvantage of this system is the lack of dose precision 107 and difficulty for the pediatric population. some studies reported that nasal drops deposit human serum albumin in the nostrils more efficiently than nasal sprays. 108 nasal powder formulations are highly stable compared to liquid formulations. nasal powders can extend the residence time for powder formulations on the nasal mucosa, potentially increasing the local and systemic immune response. 109 however, the production of nasal dry powders is quite complicated with required particle size, particle distribution, and performance characteristics when compared with other dosage forms. the aerosol route of delivery of vaccines is one of the most preferred for nasal administration compared with other nasal dosage forms and also less reactogenic than the subcutaneous route of administration. 110 aerosol vaccination via the lungs targets an epithelium critical to host defense against inhaled pathogens and provides an exciting opportunity in the development of newer and more effective tuberculosis (tb), measles, and influenza vaccines. an aerosol vaccination usually depends on the target pathogen and the sites of the inductive immunity. the aerosols are available in liquid (solution, suspension, and emulsion) and solid forms. in addition to vaccine antigens, carrier/ solvent, emulsifier/surfactants, corrosion inhibitors, and propellant selection and compatability play a critical role in achieving required immunogenicity in infants. aerosol vaccine immunogenecity achievement is based on antigen particle size for prevention of upper respiratory (eg, boredetella pertussis, chlamydia pneumonia) and lower respiratory (eg, streptococcus pneumoniae, bacillus anthracis) bacteria and virus disease. larger particles (∼5 µm) are needed for the aerosol vaccination to prevent upper respiratory tract infection and smaller particles (≤ 3 µm) for lower respiratory tract infection. 111 the dried forms of vaccines with optimum particle size are traditionally prepared by freeze drying or spray drying. the aerosol form of vaccines was administered to many human subjects for a longer period and found to provide excellent protection for diseases such as influenza a and measles. 112, 113 therefore aerosol immunization may be a promising method of vaccination. nasal gels are generally used for colds, allergies, low humidity, or overuse of decongestant. nasal vaccine gels are a high-viscosity solution or suspension in which antigenic molecules are dispersed. the advantages of a nasal gel include the reduction of nasal clearance and anterior leakage due to highly viscous formulation, reduction of irritation by using soothing/emollient excipients, and target delivery to mucosa for better absorption. 114 in addition, it may potentially enhance the immune response, reduce the antigen and/or adjuvant dose, sustain antigen release, and improve antigen uptake with enhanced antigen stability. special application techniques are required for the administration of nasal gel vaccines because of their highly viscous formulation and poor spreading abilities. a wide variety of gelling polymers are available for formulation such as pullulan, deaceylated gellan gum, xantham gum, chitosan, and polyethylene glycol, used to encapsulate vaccine/adjuvant formulation as gel particles. viscosity, sol-gel transition temperature and gelling time, and gel strength and its texture are critical parameters in the development of nasal gel vaccines. recently, pneumococcal surface protein-a nasal gel vaccine, 115 clostridium botulinum type-a neurotoxin bohc/a, and tetanus toxoid 116 were studied in animal models and enhanced both humoral and cellular immunity. nasal gel is an alternative and promising novel dosage delivery system to achieve the immune response. nasal vaccine administration has been considered as an alternative method and found equivalent or superior to parenteral and other mucosal administration. it avoids the risk of transmitting diseases like hepatitis b, hiv, and other agents through improper injection practices and improves patient compliance. the intranasal vaccine delivery system induces both mucosal and systemic immune response, which avoids entry of pathogen in all mucosal routes. multiple delivery systems are being explored (both developmental and clinical phases) and can open the door for practical development of nasal vaccine delivery systems. global regional and national causes of child mortality in 2008: a systematic analysis human nasal challenge with streptococcus pneumoniae is immunising in the absence of carriage staphylococcus aureus binding to human nasal mucin effects of viral and bacterial infection on nasal and sinus mucosa bacterial adherence to nasal mucosal cells diseases of the sinuses: comprehensive textbook of diagnosis and treatment nasal drug delivery in pharmaceutical and biotechnology: present and future. e mucosal penetration enhancers for facilitation of peptide and protein drug absorption design considerations for liposomal vaccines: influence of formulation parameters on antibody and cell-mediated immune responses to liposome associated antigens nasal drug delivery nasal delivery of high molecular weight drugs current challenges in non-invasive insulin delivery systems: a comparative review nasal route: a novelistic approach for targeted drug delivery to cns nasal delivery of insulin using chitosan microspheres nasal mucociliary clearance as a factor in nasal drug delivery anatomy and physiology of the nose anatomy, physiology and function of the nasal cavities in health and disease intranasal drug delivery: how, why and what for? intranasal drug delivery for systemic medications usa: mosby anatomical and histological factors affecting intranasal drug and vaccine delivery morphology of human olfactory epithelium advantagious nasal drug delivery system: a review review on nasal drug delivery system with recent advancement drug absorption studies: in situ, in vitro and in silico models. in: kom d, editor. invitro cellular models for nasal drug absorption studies the isoenzyme pattern of lactate-dehydrogenase in nasal secretions review on nasal drug delivery system the nose and paranasal sinuses physiology and anatomy enhanced bioavailability of drugs via intranasal drug delivery system advances in nasal trans-mucosal drug delivery nasal drug delivery: an approach of drug delivery through nasal route nasal drug delivery: possibilities, problems & solutions review: clinical opportunities provided by the nasal administration of peptides mucosal vaccines: the promise and the challenge nalt-versus peyer's-patch-mediated mucosal immunity recent progress in mucosal vaccine development: potential and limitations the microbial ecology and immunology of the adenoid: implications for otitis media collaboration of epithelial cells with organized mucosal lymphoid tissues advantages of intranasal vaccination and considerations on device selection tracking human antigen-specific memory b cells: a sensitive and generalized elispot system strong cellular and humoral anti-hiv env immune responses induced by a heterologous rhabdoviral prime-boost approach intranasal immunization with a plant virus expressing a peptide from hiv-1gp41 stimulates better mucosal and systemic hiv-1 specific iga and igg than oral immunization immunogenicity of an hiv-1 gag dna vaccine carried by attenuated shigella prime-boost immunization schedules based on influenza virus and vaccinia virus vectors potentiate cellular immune responses against human immunodeficiency virus env protein systemically and in the genitorectal draining lymph nodes vaccination with a shigella dna vaccine vector induces antigen-specific cd8+ t cells and antiviral protective immunity the use of natural and synthetic phospholipids as pharmaceutical excipients liposomes as vaccine delivery systems: a review of the recent advances liposomes act as stronger sub-unit vaccine adjuvants when compared to microspheres adjuvant activity of monophosphoryl lipid a for nasal and oral immunization with soluble or liposome-associated antigen adjuvant activity of monophosphoryl lipid a for nasal and oral immunization with soluble or liposome-associated antigen humans immunized with streptococcus mutans antigens by mucosal routes intranasal immunization with proteoliposomes protects against influenza mucosal immunoadjuvant activity of liposomes: role of alveolar macrophages surface modified liposomes for nasal delivery of dna vaccine intranasal delivery of nanoparticles encapsulating bpi3v proteins induces an early humoral immune response in mice adjuvanted poly(lactic-coglycolic) acid nanoparticle-entrapped inactivated porcine reproductive and respiratory syndrome virus vaccine elicits cross-protective immune response in pigs immunization with biodegradable nanoparticles efficiently induces cellular immunity and protects against influenza virus infection preventative vaccine-loaded mannosylated chitosan nanoparticles intended for nasal mucosal delivery enhance immune responses and potent tumor immunity development and characterization of bipolymer based nanoparticulate carrier system as vaccine adjuvant for effective immunization strong systemic and mucosal immune responses to surface-modified plga microspheres containing recombinant hepatitis b antigen administered intranasally evaluation of mucoadhesive nanoparticle based nasal vaccine chitosan nanoparticle encapsulated hemagglutinin-split influenza virus mucosal vaccine induction of protective immunity against chlamydia trachomatis genital infection by a vaccine based on major outer membrane protein-lipophilic immune response-stimulating complexes intranasal immunisation with influenza-iscom induces strong mucosal as well as systemic antibody and cytotoxic t-lymphocyte responses iscom is an efficient mucosal delivery system for mycoplasma mycoides subsp. mycoides (mmmsc) antigens inducing high mucosal and systemic antibody responses the immunostimulating complex (iscom) is an efficient mucosal delivery system for respiratory syncytial virus (rsv) envelope antigens inducing high local and systemic antibody responses intranasal delivery of norwalk virus-like particles formulated in an in situ gelling, dry powder vaccine a novel intranasal virus-like particle (vlp) vaccine designed to protect against the pandemic 1918 influenza a virus (h1n1) intranasal vaccination with an adjuvanted norwalk virus-like particle vaccine elicits antigen-specific b memory responses in human adult volunteers intranasal immunization with siv virus-like-particles (vlps) elicits systemic and mucosal immunity efficacy, immunogenicity and stability of a novel intranasal nanoemulsion-adjuvanted influenza vaccine in a murine model formulation and characterization of nanoemulsion intranasal adjuvants: effects of surfactant composition on mucoadhesion and immunogenicity nasal immunization with a recombinant hiv gp120 and nanoemulsion adjuvant produces th1 polarized responses and neutralizing antibodies to primary hiv type 1 isolates pre-clinical evaluation of a novel nanoemulsion-based hepatitis b mucosal vaccine a novel bioadhesive intranasal delivery system for inactivated influenza vaccines proteosomes, emulsomes, and cholera toxin b improve nasal immunogenicity of human immunodeficiency virus gp160 in mice: induction of serum, intestinal, vaginal, and lung iga and igg capric acid and hydroxyl propyl methylcellulose increase the immunogenicity of nasally administered peptide vaccines the mucosal adjuvant potential of cross-linked dextran microspheres as dry powder nanoparticles and microparticles for drug and vaccine delivery peg-pla nanoparticles as carriers for nasal vaccine delivery nasal delivery of high molecular weight drugs novel n-trimethyl chitosan-poly(γ-glutamic acid) nanoparticles for mucosal delivery of vaccines applications of nanotechnology for immunology iscom-based vaccines: the second decade iscoms: an adjuvant with multiple functions iscomtm-based vaccines: the second decade immune stimulating complexes as mucosal vaccines the serum antibody response distributed in subclasses and isotypes after intranasal and subcutaneous immunisation with influenza virus immunostimulating complexes immune stimulating complexes containing quil a and protein antigen prime class i mhc-restricted t lymphocytes in vivo and are immunogenic by the oral route effect of iscoms and their adjuvant moiety (matrix) on the initial proliferation and il-2 responses: comparison of spleen cells from mice inoculated with iscoms and/or matrix intranasal immunization of mice with echinococcus granulosus surface antigens iscoms evokes a strong immune response, biased towards glucidic epitopes virus-like particles as immunogens the coming of age of virus-like particles an efficient plant viral expression system generating orally immunogenic norwalk virus-like particles virus-like particle-based human vaccines: quality assessment based on structural and functional properties virus-like particles as vaccine antigens and adjuvants: application to chronic disease, cancer immunotherapy and infectious disease preventive strategies microemulsions for nasal drug delivery system: an overview novel adjuvants & delivery vehicles for vaccines development: a road ahead characterization of stability and nasal delivery systems for immunization with nanoemulsion-based vaccines bioadhesive and formulation parameters affecting nasal absorption in situ gelling, bioadhesive nasal inserts for extended drug delivery: in vitro characterization of a new nasal dosage form bioadhesive polymers for controlled drug delivery projuvant (pluronic f127/chitosan) enhance the immune response to intranasally administered tetanus toxoid chitosan as a novel nasal delivery system for vaccines almeida aj, alpar ho. nasal delivery of vaccines a review on intranasal drug delivery system development of nasal drug delivery: a review dry powder nasal vaccines as an alternative to needle-based delivery a meta-analysis of studies comparing the respiratory route with the subcutaneous route of measles vaccine administration a fast-rotating spectral shadow band radiometer for marine applications feasibility of aerosol vaccination in humans measles aerosol vaccination mucoadhesive hydrogels nanogel-based pneumococcal surface protein a nasal vaccine induces microrna-associated th17 cell responses with neutralizing antibodies against streptococcus pneumoniae in macaques nanogel antigenic proteindelivery system for adjuvant-free intranasal vaccines key: cord-031232-6cv8n2bf authors: de weck, olivier; krob, daniel; lefei, li; lui, pao chuen; rauzy, antoine; zhang, xinguo title: handling the covid‐19 crisis: toward an agile model‐based systems approach date: 2020-08-27 journal: nan doi: 10.1002/sys.21557 sha: doc_id: 31232 cord_uid: 6cv8n2bf the covid‐19 pandemic has caught many nations by surprise and has already caused millions of infections and hundreds of thousands of deaths worldwide. it has also exposed a deep crisis in modeling and exposed a lack of systems thinking by focusing mainly on only the short term and thinking of this event as only a health crisis. in this paper, authors from several of the key countries involved in covid‐19 propose a holistic systems model that views the problem from a perspective of human society including the natural environment, human population, health system, and economic system. we model the crisis theoretically as a feedback control problem with delay, and partial controllability and observability. using a quantitative model of the human population allows us to test different assumptions such as detection threshold, delay to take action, fraction of the population infected, effectiveness and length of confinement strategies, and impact of earlier lifting of social distancing restrictions. each conceptual scenario is subject to 1000+ monte‐carlo simulations and yields both expected and surprising results. for example, we demonstrate through computational experiments that maintaining strict confinement policies for longer than 60 days may indeed be able to suppress lethality below 1% and yield the best health outcomes, but cause economic damages due to lost work that could turn out to be counterproductive in the long term. we conclude by proposing a hierarchical computerized, command, control, and communications (c4) information system and enterprise architecture for covid‐19 with real‐time measurements and control actions taken at each level. f i g u r e 1 confirmed deaths per million people as of july 21, 2020 makes it a systemic crisis and not only a pure health crisis. the closest analog we have at a global scale is the h1n1 influenza pandemic of 1917-1919 ("spanish flu") which killed between 17 and 50 million people worldwide. 6 thus, handling the current covid-19 crisis requires a holistic approach taking into consideration an extremely complex system, ie., society as a whole. another important aspect of the covid-19 crisis is that the pandemic propagation has been very fast, thus demanding rapid decisionmaking. moreover, and we think that this is a structuring feature of this crisis, the incubation time of the disease introduces a delay-that has been estimated as being up to two weeks according to epidemiologists (refs. 7, 8, or 9 )-between the implementation of countermeasures and the observation of their effects. this is compounded by the fact that a significant fraction of the virus carriers appear to be asymptomatic, causing a large difference between the numbers of actual cases and of known or confirmed cases (see refs. 1, 3, or 5) . this explains why the problem of monitoring the covid-19 crisis can be seen as a controltheoretic problem with delay in the feedback loop used to stabilize the situation in addition to the problem of low or only partial observability of the true system states. we shall elaborate further on this point. from a system-theoretic perspective, the above characteristics raise several difficult problems. the first one, which is rather expected, regards scalability: can our current systems engineering and modeling methods (cf. for instance, refs. 10-16, or 17) be extended to a system, or more precisely a system-of-systems (cf. ref. 18 or 19) , as large and as complex as human society as a whole? this question is clearly not easy to solve and appears moreover poorly addressed by the only known models of such scope, ie., the so-called world models, based on generalized volterra equations, that followed the seminal work of forrester in the 1970s (see refs. 20, 21, and 22) . a second problem is caused by the emergence of local and partial solutions which is significant since the covid-19 crisis impacts all sectors of society, including the medical, financial, transportation, manufacturing, and overall economic systems. society therefore needs fast and innovative solutions in order to mitigate as much as possible the consequences of the crisis. time pressure favors local and partial solutions, but also a strong coordination among actors in order to avoid contradictory strategies. a central question is therefore how to favor the emergence of bottom-up local actions while, at the same time, ensuring top-down monitoring and coordination of such actions, with short feedback loops. this calls for an agile approach (see refs. 23, 24, or 25) to the global covid-19 crisis. stating the above problems, we made a clear choice in this paper: we do strongly believe in the use of models, and more precisely of systemic models to think through and manage the crisis. models as we consider them here are, however, not platonic ideals, but observational models which rely on the observation of the reality of the covid-19 crisis, including the effects of the decisions made based on them. such models are intended to capture the systemic nature of the crisis in order to achieve a better understanding of the situation and to allow a better communication among stakeholders. in that respect, models have two main roles: first, the concrete calculation of key performance indicators to support the decision-making process through experiments in silico; and a second more metaphorical one, to help us think better about the dynamic evolution of the systems at stake. the remainder of this paper is organized as follows. in section 2, we discuss which systemic models may support better management of the covid-19 crisis. then, in section 3, we advocate for an agile approach for crisis management. section 4 completes the paper with several recommendations. the general impression which emerges from the large and rapidly expanding literature dedicated to the covid-19 pandemic is that this crisis was first and foremost analyzed as primarily a health crisis (cf. ref. 26 or 2) . economic impacts of the crisis were of course quickly understood, but, as far as we could observe, they were rather considered as an inevitable consequence of the health crisis that has to be managed as a second priority. 27 however, the aggressive mitigation measures that were set up in many countries were and are at the same time quite efficient from a health-preservation point of view (see, for instance, ref. 28 or 29) and highly inefficient from an economical perspective due to their global economic impact on all of society (see ref. 30 or 31) . in this matter, there is-to the best of our knowledge-no rational discussion in the scientific literature on what could be the best tradeoff for jointly minimizing both the health impact and the economic impact of the covid-19 crisis. perhaps the biggest ethical issue around such trade-offs is that it would require placing an explicit economic value on human lives, as discussed for instance in ref. 32 . this is something that no national or regional government in the world has apparently been willing to do. moreover, what shall one do if the health crisis remains endemic in the near future which is one of the possible scenarios (cf. section 2.2.2)? as one can see, thinking from a global rather than a purely local perspective can deeply change the way one addresses the crisis and its consequences. this situation is probably the consequence of the fact that the crisis is mainly observed on daily basis, through for instance the daily covid-19 reports provided by the world health organization, 5 by other institutions, 3 and by each local government, leading to a rather shortterm vision of the crisis. however, changing the time scale of observation gives us immediately a totally different point-of-view on the covid-19 crisis. if we are, for instance, observing the crisis at the time step of a quarter of a year (three months), it becomes almost instantaneous and can be considered as an event-in the classical meaning of synchronous modeling 33 -without any duration. thus, the choice of time step and sampling frequency is critical as it is for any control system. this perspective change forces us to think what could be the next state of the system under observation, ie, human society, which may be on its way toward a deep economic crisis, at least in western countries. continuing the analysis at the same coarse time scale, a possible catastrophic evolution scenario would be a financial crisis result-f i g u r e 2 a possible catastrophic scenario that could result from the initial covid-19 health crisis ing with some delay from an economic crisis initiated by the health crisis, thus generating the specter of a deep and prolonged recession, as pointed out as a possibility by some economists (ref. 27 or 31). moreover, this situation could then also lead to more "classical" health crises in the future (see figure 2 ) due to the two-sided coupled interaction between the public health system and the economic system. in such a catastrophic future scenario, extending the duration of people's confinement in western countries in order to minimize the short-term health impact during the initial crisis could, for instance, result in deeply debilitating the health of more or less the same population in the mid-to long-term future. such a possible paradox is typical in optimal control theory where the optimal trajectory of any nonlinear system can never be obtained through local optimizations alone. 34 in order to take into account and to avoid such paradoxical consequences, one must choose a systems approach to analyze the covid-19 crisis, integrating all existing domains of knowledge into a common understanding of the crisis, in order to obtain a global vision, both in space and time and at different possible observation scales, and thus giving a chance to find the global optimum for human society as a whole. we can thus see that there is another crisis, hidden within the covid-19 crisis, which is a crisis of models. the global community is indeed focusing on short-term health-specific models to better master the crisis, but these models are inadequate as soon as one wants to address the crisis from a longer-term society-wide perspective which requires systemic models. in this matter, let us recall that a model is an abstraction (in the meaning of abstract interpretation theory 35 ) of reality, but not reality itself, as expressed, for instance, by the famous assertion "a map is not the territory it represents, but, if correct, it has a similar structure to the territory, which accounts for its usefulness," popularized by korzybski 36 or the well-known "all models are wrong, some are useful" by box. 37 "models" which are not actually reflecting reality within some error bounds are in fact not models in that observational definition and may even have negative impacts on reality since they will lead to wrong decisions or control actions. these negative impacts of wrong "models" can of course be amplified in the context of a systemic crisis such as covid-19. our point of view is clearly supported by an analysis of the 2020 scientific literature to date. a search of the keyword "covid-19" on google scholar 38 in april 2020, revealed that, at this moment of time, only 10 papers-ie., around 1%-of the first 900 most cited papers on covid-19 were not discussing primarily health issues (health covering here biology, epidemiology, medicine, and health policy and management), but rather focusing on the societal and economic consequences of the crisis. moreover, in terms of citations, most of these 10 papers were poorly cited: two were cited around 20 times, three around 10 times, and the remaining ones less than 5 times, while the average number of citations per paper was 15 in our sample. only very few health-oriented papers, such as ref. 39 , also discuss mixed strategies involving economic or psychological considerations to fight the coronavirus. it seems therefore that the majority of the scientific effort is focused on the short-term, without taking into account what might be the mid-and long-term societal consequences of the covid-19 crisis. one may also notice that there is probably another crisis of medical models that can be observed due to the covid-19 crisis. this other crisis focuses around the merits of hydroxychloroquine and azithromycin as a possible treatment of covid-19, as proposed by raoult and his team. 40 this has since then been shown to be a proposal which was not supported by a rigorous methodological approach according to medical methodologists. 41 however, medical statistical methodology (see ref. 42 for an introduction to this domain) appears also to be questionable from a modeling perspective: the frequency-based models used in methodological medicine usually cannot have probabilistic interpretations due to a lack of large series of experiments required to apply the law of large numbers; 43 hence such frequency-based models can only find correlations between proposed medications and observe effects on structurally limited series due to the high costs of clinical studies. 44 but since correlation is not causation, it is just not possible, without any understanding of the underlying biological mechanisms, to scientifically deduce anything from such studies, as long as we agree on the fact that science deals with causal explanations, which does however not prevent using correlation-based results from a practical perspective as soon as they are established in a sound way. 72 in this analysis, the debate around the rigor of the pragmatic and agile approach followed by raoult may just be a new popperian debate 45 opposing different medical methods for addressing an infectious health crisis, similar to the debates that existed in physics around aristotelian theory in the 16th century 46 or aether theory in the 19th century. to conclude this initial discussion on the crisis of models, we point out that if the scenario that we highlighted in figure 2 comes true, we may also eventually be forced to deal with another crisis of models, namely, the crisis of mathematical models used in finance. these other "models" are not necessarily models in the observational sense that we are using in this paper since they suffer from many well-known issues such as reflexivity, 47 which refers to the fact that mathematical financial models are essentially observing other mathematical financial models, or more deeply the lack of evidence for the market equilibrium hypothesis, 48 which is at the heart of the probabilistic framework used in mathematical finance, but which is in fact rarely observed in practice (see, for instance, ref. 48 or 49), especially in a financial crisis situation where the market is of course highly unbalanced and volatile and therefore out of equilibrium, as pointed out by several researchers. the covid-19 crisis is thus forcing us to open our eyes and to look for the "right" models to use for effectively managing human society. one should use models that are effectively capturing the reality as it is and not as we would like it to be, if we want to make nondominated decisions in the face of a crisis of such magnitude and have a chance to tackle it successfully. as stated above, there is a crucial need for constructing a realistic observational system model of the covid-19 crisis. we shall now present the main ingredients of such a systemic model. taking a systems approach leads us naturally to construct first a systemic framework for modeling the covid-19 crisis. the first step toward that objective is to understand what are the main systems 10 involved in or impacted by the crisis. in that respect, the following ones are quite obvious: • the natural environment from which the coronavirus which initiated the crisis is coming, • the social system, which contains the population that is or can be infected by the coronavirus, • the health system which attempts to cure the people infected by the coronavirus, • the governance system which has to choose the optimal health policy to face the pandemic, • the economic system which may be indirectly impacted by the covid-19 crisis. note that the impact of the covid-19 crisis on the economic system depends of course on the health policy choosen by the governance (political) system. if a health policy recommends or forces-as often done 5 -a large fraction of its population to stay home, it causes a double shock, 31 first on the supply side since economic actors which are lacking a work force must reduce their production and secondly on the demand side since people who are not working anymore are usually paid less or not at all and thus are also consuming less. we can now sketch the first item of our generic covid-19 systemic framework which is the high-level environment 10 that we modeled in figure 3 . this first system view exposes the exchanges of matter, people, information, and money-plus coronavirus here-that exist between the main systems involved in the covid-19 crisis. note that the overall system taken into account here, ie., human society as a whole, including its natural environment, is a closed system on our home planet earth. as a consequence, the only levers to solve the crisis are internal to this global system. using that technique, the point is thus to be able to construct realistic domain-specific lifecycle scenarios for each system involved in the covid-19 environment. we first focus only on the social and economic systems, since we are considering here the situation that occurs after the end of the covid-19 health crisis (see figure 2 ). we can then see that: • the lifecycle of the social system can be analyzed to first order in terms of wealth and health, where these features can be, respectively, in a systems approach, we will thus have to construct the different possible global lifecycle scenarios that can be achieved in this way (see figure 4 for an illustration of this classical process), to evaluate their probabilities and to define means to mitigate the worst consequences. to obtain more detailed models, we shall moreover refine them in terms of space, to capture the geographic dimension of human society, and time, and to make optimal trade-off decisions between the shortand long-term impact of the covid-19 crisis. note also that these lifecycle scenarios are of course highly country-dependent due to the central role of the governance system in the resolution of the covid-19 crisis, as well as the susceptibility of the population which is an initial condition. the last element of our covid-19 systemic framework is finally a mission statement, 10 ie, the core high level requirement regarding human society which expresses the objective that the governance system wants to fulfill. one can indeed understand that the behavior of our system of interest-human society-will be different depending on whether one wants to minimize the impact of the covid-19 crisis on the social, health, or economic system or to find the best balance between the impacts on these three systems. this is a multiobjective optimization problem for which we provide a sample result below, and that we intend to explore more in details in a forthcoming paper. it is therefore of high importance-as system theory tells us (see refs. 10, 14, or 16)-to be able to clearly define the mission to achieve. taking a systems approach to the covid-19 crisis requires instantiating our systemic framework per country. each country has its own specificities, associated with its own history and culture, that one must consider in any systems approach: for instance, chinese traditional medicine and rigorous group behaviors are specific to china, while a centralized governance system and poorly followed health rules are specific to france, while a heterogeneous health system that favors more affluent consumers and differentiated laws and policies by state are specific to the united states of america. these types of compartmental models have significant limitations since they only consider the human population in a macroscopic way, reacting globally in a uniform manner to an epidemic, which is not the case in reality. furthermore, in a classic sird model, eventually 100% of the population is infected, which is never observed in practice. in the covid-19 pandemic, one can also observe clusters where the epidemic seems to recursively focus, 5 which rather suggests a fractal epidemic propagation, as also mentioned in an older paper by jansse et al in 1999 55 which did not seem to have been further explored by the epidemiology community. such fractal behavior is however not at all captured by the classical sird-like compartmental models. note also that, quite surprisingly, we did not find significant scientific papers studying the geometric multiscale structure of the geography of the covid-19 pandemic, which also suggests that this dimension has not yet been analyzed in depth. in order to better integrate geography, which is one of the most important features of the human population system, we choose a as, for instance, in ref. 56 . in such an approach, the human population is modeled as a network, that is to say a nondirected graph, 57 where each node of the network represents an individual or a group of people, eg., a family, and each edge represents a connection between people. for the purpose of our study, we used networks randomly generated according to the barabási-albert model, 58 which is believed to capture the most important features of real social networks. we shall recall that the barabási-albert model generates networks by introducing nodes one by one (after an initial step). a degree d is chosen for each new node, which is then connected to d other nodes chosen at random from the nodes already in the network. to simulate a social network, the average value of the degree d is usually chosen between 2 and 3. the barabási-albert model produces randomized scale-free networks in which most of the nodes have a low degree (below 10), but some may have a very high degree. in order to understand how an epidemic propagates in a population modeled in this way, we used networks with 100 000 nodes and an average degree d for new nodes of 2.1. with these features, the degree of nodes in a social network is typically distributed as shown in table 1 . potential "superspreaders" are individuals with large degree >20. to model the propagation of an epidemic in this network, we discretized a classical sird-like model (see refs. 52 and 54 and figure 5 ) which leads us to represent the evolution of the state of each f i g u r e 6 stochastic state automaton modeling the possible evolution of a node in the social network node of the social network that models the human population by a stochastic finite automaton whose possible transitions are described in typically gaussian-for incubation and sickness times. we, however, think that our experiments can give us a better qualitative understanding of epidemic propagation since we believe that this social-network approach better captures the fundamentals of the social system, compared to the simpler compartment-type models. it may thus be helpful for constructing more realistic epidemic propagation models, even if it would require a very significant amount of data collection and fine tuning. the use of contact tracers in health systems is, for instance, a direct, but laborious, way to reconstruct such social networks to quickly identify infected people and to isolate them before they infect others. 60 our first experiment consisted of simulating increasingly virulent epidemics by assuming increasing values of the probability ρ of infecting somebody (1000 trials were done per value of ρ). our results are described in table 2 . they show a remarkably interesting phenomenon: for all values of the probability ρ, only a tiny fraction π of the population is eventually infected in most of the number ν of simulations (less than 1 of 1000 persons in more than 90% of the cases), or when a significant proportion (greater than 1%) is infected, the fraction of infected people π depends on ρ. in simpler terms, this can be stated as follows: there are a lot of viruses circulating in the population, but only a few of them give rise to epidemic outbreaks. the reasons for which a virus gives rise to an epidemic outbreak are intrinsic to the virus itself, but ta b l e 2 proportion π of the population that is infected, for different values of the propagation probability ρ. also dependent on external factors such as who is infected first, eg, a person with few contacts and low nodal degree or a superspreader with high nodal degree as shown in table 1 , and also the behavior of the population which impacts ρ. this may explain, at least to some extent, why some countries or regions are more stricken than others, which suggests again a fractal interpretation of the geographical scope of an epidemic, as already mentioned above. the second experiment that we shall report on in this section aimed at studying the effects of the deconfinement of a confined population that has been ordered to shelter-in-place. we studied here different proportions τ of the population that becomes sick before the epidemic becomes observable (ie., roughly between day 10 and 20 in figure 5) and different values of the duration γ in terms of days of confinement. we considered that there was a delay δ of 20 days before confinement was put in place and took ρ = 0.015. we also simulated the efficiency of the confinement by reducing the capacity of edges in the social network to propagate the disease by a factor 1 − ε with 0 ≤ ε ≤ 1. this factor represents the degree of adherence of the population to sanitary guidelines for social distancing, wearing face masks, and so forth. at each step of the simulation, representing one day, an infected node has thus probability ρ × (1 − ε) to infect an adjacent healthy node. in our experiment, we took ε = 0.66 (= 2/3). we then reported the computed values of the resulting lethality in table 3 . we assumed that γ is as large as necessary, which is clearly not realistic since confinement cannot be maintained too long for both economic and psychological reasons, but the results give the underlying trend. each measure reported was obtained by means of a monte-carlo simulation of 2000 trials. as expected, the longer the confinement, the fewer deaths. note however that, to be fully efficient, the confinement must be rather long, several months (>90 days) in our virtual experiment. the most interesting part of this experiment comes however from the observation of the total duration of the epidemic outbreak. table 4 shows these durations for the same values of τ and γ as in table 3 . if the confinement is sufficiently long, the lethality drops significantly (in some cases below 1%), but also the total duration of the epidemic outbreak is shortened. if the confinement is not maintained sufficiently long, it is still partially effective, in that it reduces the lethality, but it has a quite paradoxical consequence: the epidemic outbreak lasts longer than if no countermeasures were taken at all. a short confinement does not prevent the disease from significantly propagating: it just slows down the propagation and avoids the sharp peak of infected shown in figure 5 around day 40, which seems to be its main motivation in order not to overwhelm the capacity of the health care system. for this reason, when the population is deconfined too early, the disease is still present and remains endemic. the above experiments do not pretend to fully represent reality, but are just to motivate the use of social-network models for epidemic modeling. as pointed out by stattner and vidot, 56 "network models turn out to be a more realistic approach than simple models like compartment or metapopulation models, since they are more suited to the complexity of real relationships." one of the limitations of existing network models is, however, that they do not distinguish between recurring social links with family members and coworkers and casual links based on one-time encounters such as in public transportation or at large events. they should therefore be further refined and integrated into a model-based agile approach for crisis management, while taking into account their limitations. in this section, we model the potential impact of the epidemic as a function of different actions of the governance system on the economic system (see figure 3 ). in order to do so we must expand the prior analysis by not only considering lethality in terms of deaths (see table 3 ), but also the value of lost economic activity during confinement. reverting back to the simplified sird model in figure 5 , but now accounting for the fraction of population ε actually adhering to confinement during a lockdown of duration γ, which is ordered with some delay δ after a critical cumulative threshold τ of the population has become infected, we run a set of simulations. the baseline run of the model shown in figure 5 is considered as scenario 0 with no countermeasures and it is gradually modified using the one-factor-at-a-time (ofat) technique to test a number of actions by the governance system, including reducing the delay δ to order a lockdown, increasing the level of rigor ε of the confinement, as well as its duration γ . table 5 shows the results of a number of numerical experiments to probe these trade-off in terms of the value of human lives lost, versus productive work lost in the economic system. in order to estimate the economic impact of the epidemic a number of assumptions were made: in the baseline scenario 0, we do not take any countermeasures and the bulk of the $4.38b total loss is due to the deaths of 4% of the population. the $312m in lost work are due to the inability of the infected and sick population to perform work during their illness, which is assumed to last for 14 days. this is the kind of situation we would expect to see in a country with a government that is either unable or unwilling to intervene in the crisis. scenarios 1-3 institute a partial lockdown (ε = 66%) after either 10 or 20 days delay after recognizing the onset of the epidemic and the confinement lasts either 30 or 60 days. the results are not satisfactory, since the total damages exceed the baseline case where no action is taken. this outcome is due to the fact that one-third of the population does not adhere to the confinement and continues to be infected, making the disease endemic. a prolonged partial lockdown for 60 days with only 66% effectiveness as shown in scenario 3 is the worst case and leads to both a high number of deaths (about 4000) as well as high economic damages totaling $6.1b due to the prolonged shutdown, which ultimately is ineffective. this scenario is representative of the overall situation in the united states in mid2020. in scenarios 4-7, we shorten the reaction time to trigger the confinement after only five days (quick government action) and we gradually increase the rigor of the confinement to 90% (strong government enforcement). it turns out that these actions are highly effective, yielding a best-case scenario 7 with only 66 deaths, a short epidemic duration of 61 days and only $740m in damages, mainly due to the strict but short 30-day confinement in which 90% of the population participates. this essentially prevents the epidemic from blossoming and quickly snuffs out the disease. the ta b l e 5 scenario analysis with sird model for assessing total human and economic damages: n, number of daily contacts, ρ, probability of infection, τ, fraction of population infected to trigger confinement, ε, fraction of population adhering to confinement, δ, delay to confinement start, γ, confinement duration, t, duration of epidemic, total number of deaths, lost work in millions $m, and total damages including lost human lives and lost work in billions $b, n = 100,000 population size figure 7 . this may reflect the situation in countries that the economic analysis shows that the initial conditions, speed of response, and rigor of response by the governance system are crucial in determining the outcome. figures 8 and 9 , respectively, show the sharp contrast between the ratio of human loss (deaths) and economic work loss for scenarios 0 (do nothing) and scenario 5 (rapid and strong government response). there is indeed a trade-off between deaths and lost work, as in sce in the previous section, we identified a deep crisis of models that has been exposed by the covid-19 pandemic and proposed to mitigate this issue by constructing a systemic model of the crisis. in this section, we shall deal with some possible solutions to master the crisis using a systems approach. as is well known in any scientific discipline, the solution of a problem highly depends on the clarity and rigor of the way the problem is framed. we will therefore dedicate this short section to the statement of the problem that we need to solve in the context of the covid-19 crisis. a first characteristic of the covid-19 crisis is its global impact on human society. this crisis can thus be considered as a common cause failure-in the meaning of system safety theory 61 -for all main systems forming human society. if we are taking a safety approach, the first problem to solve is thus to mitigate the impacts of the crisis on the vulnerable systems forming human society, that is to say the social, health, and economic systems, as results from the system analysis of section 2.2.1. a second characteristic of the covid-19 crisis comes from the need to take into account strong feedback delays. in this matter, a first type of delay comes from the fact that it is most of the time too late for deploying mitigation actions to limit the epidemic propagation when significant numbers of infections are observed somewhere, since the effects of these actions will only be observable two weeks later. this was clearly shown in table 5 in scenarios 8-10. moreover, a secondtotally different type of delay comes from the fact that focusing on short-term health impacts of the crisis may lead to long-term issues of an economic nature, which forces to arbitrate between short-and longterm consequences of a given action. finally, a last characteristic of the covid-19 crisis is uncertainty. due to the global nature of the crisis and the rather short period of time on which it is concentrated, uncertainty is everywhere. clinical data about the infection are permanently partial, so difficult to interpret. understanding of the real social system network structure is never easy to capture. the exact nature and size of the impact on the economic system are difficult to evaluate. precise data on the capabilities on which to rely may be tricky to obtain. last, but not least, the crisis also results in a massive, heterogeneous and often contradictory amount of data in which the really interesting signals may be either weak or hidden. synthesizing these three features of the crisis, the problem to solve in our context can now be clearly stated: how to optimally mitigate the short-and long-term consequences of the covid-19 pandemic on human society, taking into account delays and uncertainties that are specific to this crisis? one can notice that this statement is a typical control problemin the sense of control theory 62 -integrating here delay and uncertainty, which can be addressed by many existing techniques (see refs. 63 and 64) . consequently, the objective should be to design a new system that can support this controllability objective. based on the closed-loop control principle, which is the only one that allows to achieve a given target behavior along the time axis, 62 such a f i g u r e 1 0 high-level covid-19 environment integrating a specific decision-aid system that has yet to be designed covid-19 decision-aid system (shown as the gray box at the top of figure 10 ) will have to measure the current state of the main systems forming human society in order to provide effective feedback actions on the social system through the governance system, the only legitimate one to make decisions and take control actions. figure 10 depicts how such a decision-aid system could be integrated into the high-level covid-19 environment. there is at least one domain where making decisions under structural uncertainties on an underlying geographic scope is quite well known since a long time in human history, which is the military domain. architecting a covid-19 decision-aid system using the typical architectural pattern of a computerized, command, control, and communications (c4) system (see ref. 65 or 66) , used in the defense area, seems thus quite a natural idea, as it is also quite often used in a system-of-systems engineering context (see ref. 18 or 19) . this leads us to propose an organization for a covid-19 decision-aid system based on the following three hierarchical layers, that correspond to three natural levels of abstraction associated with a given geographic scope (that may be either the international, country, and local levels or country, region, and city levels in practice), exactly like c4 systems are organized: 1. the strategic layer is the place where global situational awareness is required to master the crisis on a given large-scale geographic scope: its mission is to monitor at a high level the crisis and to elaborate strategic decisions based on an overall vision, fed by tactical information; 2. the operation layer is intended to master the crisis on a given medium-scale geographic scope: it is thus a distributed system which has to capture and synthesize tactical information and make operational decisions on their basis in accordance with the upper strategic decisions; 3. the tactical layer is intended to master the crisis on a local geographic scope: it is thus again a distributed system which has to capture and synthetize field information and make tactical decisions on their basis in accordance with the upper operational decisions. note that this architecture shown in figure 11 shall be understood as a hierarchical enterprise architecture, which defines how an organizational system, supported by suitable information systems and systemic models as discussed previously, shall be organized and behave. the main idea underpinning it is the principle of subsidiarity: decisions should be taken as close as possible to the level that is the most appropriate for their resolution. this principle means in particular that an upper level shall avoid to make decisions that are too intrusive at a lower level in order to let each local level take always the more appropriate actions depending on the real local conditions that it can observe, while following at the same time global orientations when locally relevant. this is crucial in the military sphere, but even more so in the context of the covid-19 crisis where speed of decision making is fundamental due to the latency of the epidemic propagation as seen in section 2. note that one shall also capture weak signals of systemic importance at each level of the proposed architecture: to illustrate that point, the fact that a police officer is infected in a certain area is, for instance, a typical weak signal since we may infer from it that there is a certain probability that the whole police force in the concerned area is or will be infected, at least in the near future (since the number of daily contacts or nodal degree of police officers may exceed n > 10, see tables 1 and 5 ). proposing the previous hierarchical architectural pattern is, however, of course not enough to specify how a covid-19 decision-aid system shall work. in this matter, the first point is to organize the systemic model that we sketched out in section 2.2 according to the hierarchy that we just presented and which is used to organize the proposed decision-aid system. hence, such a model shall not be monolast, but not least, the covid-19 decision-aid system that we sketched here shall behave in an agile way, in the meaning of agility in software or industrial development (see refs. 68-70, 71 or 24) . a pending problem is to have a plan, do, check, and act process that can quickly adapt to a quite fast-changing reality. agility allows to solve that issue by structuring in a very rigorous way the analysis, decision, and action processes, while providing a lot of flexibility to all involved actors, which are two mandatory features for addressing a complex crisis like covid-19. in practice, an agile covid-19 decision-aid process has typically to be organized around regular agile rituals-managed in this paper, we draw attention to the core importance of having realistic system models to manage and to mitigate a systemic crisis of the order of magnitude such as the covid-19 crisis. we also sketched out what could be an agile approach to use in this kind of crisis. our purpose was of course not to propose some definitive solution which is probably impossible. we do, however, think that the ideas contained in this paper are valuable contributions that may be of interest in the context of the covid-19 crisis, especially due to the fact the underlying health crisis will probably be endemic for a certain period of time (at least for 200-300 days according to most of our simulation runs) and be coupled with future short-and mid-term economic outcomes. while there are economic impacts due to strong mitigation actions such as mandated confinements (causing lost economic activity), the value loss due to human deaths at an estimated lethality rate of 4% would far exceed the economic losses. we have shown that this depends strongly on the average valuation of a human life, which is in itself a highly controversial issue. there are of course many detailed aspects of the proposed covid-19 decision support system that require further detail and elaboration. we focused on the issue of delay and rigor of action in the overall epidemic control system in this paper. however, as we discover more about the particular nature of this particular coronavirus, the issue of observability of human society (testing) may for instance be an even larger one. the epidemiological characteristics of an outbreak of covid-19) -china report of the who-china joint mission on coronavirus disease 2019 (covid-19). who; 2020 statistics and research -coronavirus pandemic (covid-19). our world in data covid-19) situation reports the site of origin of the 1918 influenza pandemic and its public health implications clinical features of patients infected with 2019 novel coronavirus in wuhan, china evolving epidemiology and transmission dynamics of coronavirus disease 2019 outside hubei province, china: a descriptive and modelling study a novel coronavirus from patients with pneumonia in china cesames systems architecting method -a pocket guide. cesames strategic engineering -designing systems for an uncertain future. mit the art of systems architecting the systems approach: fresh solutions to complex problems through combining science and practical common sense. commons lab| case study series 2 introduction to systems engineering system modeling and simulation-an introduction architecting systems-concepts, principles and practice. college publications theory of modeling and simulation-integrating discrete events and continuous dynamic systems complex system and systems of systems engineering key challenges and opportunities in'system of systems' engineering world dynamics the limit to growth fluctuations in the abundance of a species considered mathematically agile systems engineering. www.incose.org/chapters groups/workinggroups/transformational/agile-systems-se cesam engineering systems agile framework. 10th international conference on complex systems design & management the agile manifesto reworked for systems engineering the global impact of covid-19 and strategies for mitigation and suppression, imperial college covid-19 response team, imperial college the global macroeconomic impacts of covid-19: seven scenarios how will country-based mitigation measures influence the course of the covid-19 epidemic? covid-19 cases per million inhabitants: a comparison. statista confronting the crisis: priorities for the global economy we pay minimum five times as much for a life year with the corona measures than we normal do embedded system design. kluwer adaptive control processes: a guided tour abstract interpretation: a unified lattice model for static analysis of programs by construction or approximation of fixpoints science and sanity-an introduction to non-aristotelian systems and general semantics. the international non-aristotelian library pub all models are wrong, but some are useful the publish or perish book. melbourne: tarma software research pty ltd combining behavioral economics and infectious disease epidemiology to mitigate the covid-19 outbreak hydroxychloroquine and azithromycin as a treatment of covid-19: results of an open-label non-randomized clinical trial statistical review of hydroxychloroquine and azithromycin as a treatment of covid-19: results of an open-label non-randomized clinical trial clinical research methodology and evidence-based medicine: the basics. anshan ltd an introduction to probability theory and its applications. vols. i and ii clinical trial cost is a fraction of the drug development bill, with an average price tag of 19 m$. outsourcing pharma conjectures and refutations: the growth of scientific knowledge. london and new york: basic books galileo studies. branch line reflexivity in credit markets. california institute of technology the flawed foundations of general equilibrium: critical essays on economic theory financial crisis dynamics: attempt to define a market instability indicator a theory of timed automata a contribution to the mathematical theory of epidemics mathematical structure of epidemic systems an introduction to infectious disease modelling lévy-flight spreading of epidemic processes leading to percolating clusters social network analysis in epidemiology: current trends and perspectives the theory of graphs and its applications statistical mechanics of complex networks statistical mechanics of cellular automata cluster-based epidemic control through smartphone-based body area networks system reliability theory-models mathematical control theory: deterministic finite dimensional systems uncertainty and control control of time-delay systems understanding command and control. washington, dc: us department of defense ccrp publication series joint chiefs of staff. doctrine for command, control, communications, and computer (c4) systems support to optimal treatment of an sir epidemic model with time delay agile manifesto a decade of agile methodologies: towards explaining agile software development agile systems engineering national heart institute, national institutes of health, public health service, federal security agency. epidemiological approaches to heart disease: the framingham study. joint session of the epidemiology, health officers, medical care, and statistics sections of the american public health association multiple risk functions for predicting coronary heart disease: the concept, accuracy, and application the authors would like to thank the three anonymous referees for their valuable comments which contributed to a substantial improvement of the paper. we would finally like to stress the fact that systems engineering has an important role to play in the covid-19 context since it can enable the necessary collaboration of the various disciplines-such as biology, economics, engineering, epidemiology, finance, geography, health policy management, immunology, logistics, manufacturing, medicine, safety, sociology, urban systems, and so forth-that are all providing a piece of the complex puzzle posed by the global covid-19 crisis. key: cord-018917-7px75s3c authors: hopkins, richard s.; magnuson, j. a. title: informatics in disease prevention and epidemiology date: 2013-07-29 journal: public health informatics and information systems doi: 10.1007/978-1-4471-4237-9_14 sha: doc_id: 18917 cord_uid: 7px75s3c this chapter provides a description of the components of disease prevention and control programs, and then focuses on information systems designed to support public health surveillance, epidemiologic investigation of cases and outbreaks, and case management. for each such system, we describe sources used to acquire necessary data for use by public health agencies, and the technology used to clean, manage, organize, and display the information. we discuss challenges and successes in sharing information among these various systems, and opportunities presented by emerging technologies. systems to support public health surveillance may support traditional passive case-reporting, as enhanced by electronic laboratory reporting and (emerging) direct reporting from electronic health records, and also a wide variety of different surveillance systems. we address syndromic surveillance and other novel approaches including registries for reporting and follow-up of cases of cancer, birth defects, lead poisoning, hepatitis b, etc., and population-based surveys (such as brfss or prams). systems to support epidemiologic investigation of outbreaks and clusters include generic tools such as excel, sas, spss, and r, and specialized tool-kits for epidemiologic analysis such as epi-info. in addition to supporting outbreak investigation, agencies also need systems to collect and manage summary information about outbreaks, investigations, and responses. systems to support case management, contact tracing, and case-based disease control interventions are often integrated to some degree with surveillance systems. we focus on opportunities and choices in the design and implementation of these systems. systems to support case management, contact tracing, and case-based disease control interventions are often integrated to some degree with surveillance systems. we focus on opportunities and choices in the design and implementation of these systems. public health programs to prevent disease typically have been designed and implemented one disease at a time. each disease has its own patterns of distribution in populations, risk factors, and optimal and practical intervention strategies that are effective in controlling, preventing, or even eliminating cases of the disease. for example, an important strategy to prevent measles is vaccination, the main strategy to prevent gonorrhea is antibiotic treatment of case contacts before they become ill themselves, an important strategy to prevent cervical cancer is screening with pap smears and treatment of preclinical disease, and the main strategy for prevention of neural tube defects is folic acid supplementation of selected foods. still, each disease prevention program's components are drawn from a relatively short list: • planning and evaluation • public health surveillance • outbreak or cluster recognition and response • policy and guidance development • clinical services -screening -immunization -prophylaxis -treatment • laboratory services • case-contact identifi cation and interventions • education and training for clinicians • public education • regulation (for example, of food services, drinking water, child-care centers, hospitals, etc.) • administration and fi nancial management ideally, program managers choose the most effective combination of these program components to prevent or control the disease or diseases they are charged with addressing. however, as this must be done within the constraints imposed by the available funds, cost-effectiveness is the usual criterion for choosing the preferred combination of program components. public health agencies typically are organized both by disease and by function. for example, each disease-specifi c program usually does not have its own laboratory, and a single public health clinical facility and its staff may provide varied services such as immunizations for well children, treatment of people with tuberculosis (tb) and their contacts, and pap smear services. to variable degrees, they may even combine activities in a single patient encounter, for example, testing women for gonorrhea and chlamydia trachomatis infections at the same visit where they get a pap smear, or offering hepatitis b vaccination during a visit for sexually transmitted diseases (std) treatment. as information technology has become more widely used in public health and replaced paper-based systems, it has typically been implemented program area by program area, as resources became available. this has led to the creation of information 'silos.' for example, laboratory information systems usually have developed in isolation from those to support clinical care or public health surveillance. information systems to support clinical operations of public health departments (for example, clinical services for stds, childhood immunizations, hiv/aids, tb, or family planning services) have characteristics similar to those of other electronic health record systems in ambulatory care. however, in some health departments, clinical information systems have been separated by disease or clinic. if one were to design information systems from scratch for a set of disease prevention programs, there would be potential savings and effi ciencies from identifying the ways that one program component depends on information from another, or can serve multiple programs, and then designing the system to provide that information seamlessly. one can identify potential effi ciencies from two perspectives: in reality, it is rare to have an opportunity to design such extensive information systems as a single project. one is dealing with numerous legacy systems that were designed to support program-specifi c workfl ows. so a key challenge for the public health informaticist is to help their agency make decisions about where information system 'integration' will yield substantial benefi ts and where it will not. for example, if it is desired to know (one time) how many people in the jurisdiction have been reported during a particular time interval with both syphilis and hepatitis b, one could do an ad hoc match of information in two independent surveillance information systems. this task might take an analyst a few days or weeks to accomplish -which is almost certainly inexpensive compared to the cost of building a new information system that could do this task almost immediately. for many purposes, it may be useful and suffi cient to be able to display multiple streams of surveillance or programmatic data in the same environment, on the same screen or even in the same chart. in florida, de-identifi ed reportable disease case information and death certifi cate information are imported into the essence analytic environment that was originally designed for syndromic surveillance [ 1 ] , so that trends for similar conditions by age, sex, and geographic area in the two data streams can be easily compared. on the other hand, if it is desired to have real-time information available to the std clinic staff about past diagnoses of hepatitis b, or about past receipt of hepatitis b vaccine, then information systems need to be designed to support this kind of look-up; the usual solution is a shared person index between the two systems. alternatively, a common data repository can be designed in which all information about each person is permanently linked. as mentioned earlier, there are a number of components common to disease control and prevention programs. in this chapter, we will address information systems designed to support the following: • public health surveillance • outbreak or cluster recognition and response • acquisition of laboratory information • case-contact identifi cation and intervention cdc defi nes public health surveillance as "the ongoing, systematic collection, analysis, and interpretation of health data, essential to the planning, implementation, and evaluation of public health practice, closely integrated with the dissemination of these data to those who need to know and linked to prevention and control" [ 2 ] . each word of this defi nition is carefully chosen, and has implications for the design of surveillance information systems. a one-time data collection activity is not surveillance. data collection for research purposes is not surveillance. surveillance data are collected to support public health action, and analyses and recommendations based on these data must be shared with those who provided the data and with others who need to know. objectives of surveillance systems differ at the local, state, and federal levels [ 3 ] . at the local level, immediate response to individual cases is relatively more important, while at the federal level the analysis of larger-scale patterns is the most important function of surveillance. for state health departments, both uses of surveillance data may be important, depending on the disease and the size of the state. public health surveillance systems may be based on data capture from a variety of sources, including case reports, population-based surveys, sentinel providers, electronic health records (including laboratory information management systems for elr and emergency department records for syndromic surveillance), or administrative data (like hospital or physician claims for reimbursement). for some noninfectious diseases, surveillance is carried out through registries (see below). information systems to support reportable disease surveillance contain records representing case reports that currently are, for the most part, entered manually into an application by public health staff, based on information received from doctors, infection control practitioners, hospitals, and laboratories. increasingly, the laboratory information in these records comes from electronic records transmitted by the public health laboratory, hospital laboratories, and commercial laboratories, when there is a positive result meeting certain reporting criteria (like a positive igm antibody test for hepatitis a). these records typically contain a combination of clinical, laboratory, and epidemiologic information about each case. in future, increasing proportions of these case reports will be entered directly into a website by the practitioner creating the case report, or be transmitted electronically from the practitioner's electronic health record (ehr) system. currently almost half the states in the us use the cdc-provided nedss base system (nbs) as their platform for managing case reports. the remainder use either a system developed in-house or one of several commercially-available solutions [ 4 ] . in case-based surveillance practice, there is usually a relatively short list of required elements in the initial case report. for some diseases this is the only information received on all cases. for other diseases, usually of more importance and with lower case numbers, an additional data collection form is initiated by the receiving health department, which gathers information as appropriate from the ill person, the treating physician, and health records. the optimum amount of information to collect in the initial case report, as opposed to the disease-specifi c case report form, is a matter of judgment and may change as technology changes. in a largely manual system, health departments typically desire to minimize barriers to reporting of cases, so the incentive is to keep the initial case report form short. if much of the information desired for the disease-specifi c case report form can in fact be extracted from an electronic medical record with no additional effort by the person making an electronic case report, then the balance changes. careful decisions are needed: for which cases of which diseases are follow-up interviews necessary [ 5 ] ? until very recently, virtually all of the case-based surveillance information used at the federal level was collected initially at the local (or sometimes state) level, where it was used in the fi rst instance for local response. as the case report information passes from the local to the state to the federal level, it is subjected to validation and cleaning: cases not meeting the surveillance case defi nition have been removed from the data submitted to the federal level, missing data have been fi lled in to the extent possible, and cases have been classifi ed as to whether they are confi rmed, probable, or suspected using standard national surveillance case defi nitions (these case defi nitions are developed by the council of state and territorial epidemiologists in consultation with cdc) [ 6 ] . more recently, advances in technology have allowed case reports, and the information on which they are based, to move almost instantaneously from electronic health record systems, maintained by doctors, hospitals, and laboratories, to public health authorities. there are no technical barriers to these data being available at the federal level essentially as early as they are at the local and state levels. this ready availability of unfi ltered clinical information may allow more rapid awareness by public health offi cials at all levels of individual cases of high-priority diseases (like botulism or hemorrhagic fevers like ebola virus infection), and thus lead to more rapid detection and characterization of likely outbreaks. the simultaneous availability of raw data to multiple agencies at different levels of government also presents certain challenges. the user at the local level will have ready access to information from many sources about local conditions and events, and can use this information to interpret local observations. they will be in a position to understand when an apparent anomaly in their surveillance data is due to an artifact or to local conditions that are not a cause for alarm. they will also know whether a problem is already under investigation. a user at a state or federal level will be able to see patterns over a larger area, and thus may be able to identify multijurisdictional outbreaks, patterns, or trends that are not evident at a local level. the fact that several users may be examining the same raw data at the same time requires that these multiple users be in frequent communication about what they are seeing in their data and which apparent anomalies are already explained or need further investigation. there is a danger that users at a higher level may prematurely disseminate or act on information that, while based on facts, is incomplete or misleading. similarly, users at a local level may not realize that what they are seeing is part of a larger phenomenon. in the syndromic surveillance domain, the biosense 2.0 governance group [ 7 ] has adopted a set of etiquette principles which participating jurisdictions will be required to agree to, that spell out the mutual obligations of analysts at each level of the system (scott gordon , association of state and territorial health offi cials, 2013, personal communication). from an information management perspective, an important question is where to put human review of case reports in this information fl ow. for example, it is becoming technically possible for likely cases of reportable diseases to be recognized automatically in health care electronic record systems. some of these could be passed on to public health authorities without human review, in the same way that reportable laboratory results are already passed on in electronic laboratory reporting (elr). for which constellations of fi ndings in the electronic health record would this be appropriate? should some electronic case reports generated by electronic health record systems be passed to state or even federal public health offi cials before they are reviewed and validated at the local or state levels? if so, which ones? as always, there is a tension between the speed of information fl ow and its quality and completeness. there is a need for research to determine which constellations of fi ndings in electronic health records have adequate specifi city and sensitivity to warrant automated identifi cation of a person as being likely to have a case of a reportable disease. the acceptable sensitivity and specifi city will vary by disease. in 2001, cdc published the updated guidelines for evaluating public health surveillance systems [ 8 ] . this document identifi es a set of key attributes of surveillance systems to be assessed during a surveillance system evaluation, including simplicity, fl exibility, data quality, acceptability, sensitivity, predictive value positive, representativeness, timeliness, and stability. these are also useful attributes to consider when designing a surveillance information system [ 9 ] . the relative importance of these attributes will vary depending on the condition under surveillance and the main purposes for surveillance. for example, a surveillance system to detect cases of botulism for immediate public health response puts a high premium on timeliness, and its operators are likely to be willing to accept a modest number of false-positive reports (a lower positive predictive value ) in order to assure that reports are received very quickly. on the other hand, surveillance to support planning of cancer prevention programs and treatment services is less time-sensitive, given the quite long incubation periods for most cancers, and therefore more concerned with diagnostic accuracy of every case report than with speed of reporting. timeliness, positive predictive value, and sensitivity of a public health surveillance system are always in tension with each other; increasing two of these always compromises the third. in systems based on case-reporting from doctors, hospitals, and laboratories, and receipt of electronic health records from these same organizations, records for an individual can in principle be linked with records for that same individual in numerous public health information systems, including those supporting clinical service, immunization registries, case investigation, partner or contact identifi cation, partner or contact notifi cation, and provision of interventions to partners or contacts. sometimes this will be done best by automated messaging of structured data from one system to another, sometimes by supporting real-time look-up capabilities, and sometimes by development of a master person index to underlie some or all of these applications. one key decision is which application to consider as the hub for this information sharing, for example, the surveillance application itself or a clinical application. surveillance systems that are based on sample surveys (such as the behavioral risk factor surveillance system, brfss [ 10 ] ), on sentinel practices (such as ili-net for surveillance of infl uenza-like illness [ 11 ] ) or on syndromic surveillance do not have individual patient identifi ers, and so intrinsically cannot be linked at the individual level to information systems supporting other disease control program components. their data are typically managed in systems built on standard statistical software packages, or other independent systems. syndromic surveillance systems are based on rapid acquisition of unfi ltered, real-time, electronic records without individual identifi ers from hospital emergency rooms [ 12 ] and urgent care centers, and also, increasingly, from outpatient physicians' offi ces and from hospital admissions [ 13 ] . the primary purpose of these systems is to support detection and characterization of community disease outbreaks, as they are refl ected in care received at emergency departments, physicians' offi ces, or hospitals. each visit to an emergency department is assigned to a category or syndrome , based on words and strings contained in the patient's chief complaint and/or the triage nurse's notes. as the records received by the health department do not have individual identifi ers, they cannot be linked to records in other information systems. however, records received by the syndromic surveillance system should contain unique identifi ers that could allow the epidemiologist analyzing the data to work back through the sending facility to an identifi ed clinical record. this traceback might become necessary if the person appeared to have a case of a reportable disease or to be part of a signifi cant outbreak. adding outpatient visits and hospital admissions to the scope of syndromic surveillance is opening up additional uses for this technology, especially in the areas of real-time non-infectious disease surveillance. surveillance for cancers [ 14 ] , stroke [ 15 ] , birth defects [ 16 ] , and some other chronic diseases like amyotrophic lateral sclerosis (als) is carried out through registries. registries are usually established by specifi c legislation, and typically relate to a single topic -for example a registry of records for a disease, or of immunization records. registries may be restricted to a geographic region. a distinctive feature of registries is that individual case reports are kept open for long periods of time, up to several or many years, allowing additional information about treatment, hospitalization, and death or other outcomes to be added. registries thus serve as systems to monitor type, duration, and outcome of treatment for these diseases, in addition to the occurrence of new cases of disease (disease incidence ). they may also support outreach efforts to patients or their families, as a way to document that appropriate steps have been taken to link patients to needed types and sources of care. most cases recorded in state-level cancer registries are acquired from hospitallevel registries, using an electronic case report in a standardized format [ 17 ] . some case abstracts are obtained directly by registry personnel or contractors, when hospitals do not have suitable registries of their own. case reports require extensive review and abstraction of medical records by trained workers. birth defect registries may also be built by active search for cases in hospital and other medical records, and abstraction of those records to make case reports. they also may be built by electronically linking records from vital statistics (birth and death records), centralized hospital discharge record systems, and clinical service providers for children with birth defects (such as state programs for children with special medical needs) [ 18 ] . the latter are much less expensive to develop but cannot be assumed to have captured all cases of the disease under surveillance, or captured them correctly [ 19 ] . a disease outbreak is defi ned as a number of cases greater than the number expected during a particular time interval in a geographic area or population. this term usually is used for events due to infectious diseases, and sometimes for those of toxic origin. a similar increase above expected numbers for a non-infectious disease, such as birth defects or cancer, is usually called a cluster . outbreaks and clusters may be due to diseases for which individual cases are reportable (like shigellosis or breast cancer), or diseases for which they are not (like food poisoning due to staphylococcal or clostridium perfringens toxins in most states, sars when it was new, or multiple sclerosis). surveillance systems are designed to facilitate recognition of outbreaks or clusters by frequent examination of the most current information available. the design of the user interface is particularly important. the interface should allow users to: fl exibly display line lists, bar charts by date of event (epidemic curves), and maps of location of cases; fl exibly select subsets of cases for display; apply appropriate statistical tests to detect improbable increases in case counts; and display multiple streams of data on the same chart. for example, users may want to display the epidemic curve of an infl uenza outbreak for several different regions of a state or for several different age groups, or to display counts of positive infl uenza tests and emergency department visits for infl uenza-like illness on the same graph with different scales for each. syndromic surveillance systems have been leaders in developing and evaluating statistical algorithms for automated detection of anomalies which may, on investigation, turn out to be outbreaks. such algorithms have less frequently been applied for automated detection of possible outbreaks or clusters in reportable disease data streams. most outbreaks and clusters are in fact not recognized by examination of regularly-collected surveillance system data. instead, they are recognized by private citizens (such as the organizer of a social event, a teacher or school nurse, the manager of a child care center, the manager of a food service facility, an employer, or the ill people themselves) or by practicing doctors, and brought to public health attention via a phone call or e-mail or entry on a web site established for the purpose [ 20 ] . public health workers assess the information and make the decision whether or not to do a formal investigation of the outbreak. one part of such an assessment is to look at available streams of surveillance data and determine whether there is information supporting the occurrence of an outbreak. for example, a report of a possible infl uenza outbreak in a high school might prompt closer examination of syndromic surveillance data from nearby hospital emergency departments to determine whether there is a more general increase in visits for infl uenza-like illness. a report of a neighborhood cluster of brain cancers would prompt closer examination of available cancer registry information, which might or might not support an interim conclusion that such a cluster is real and statistically signifi cant. in order to be accountable for the effectiveness of their work, local and state health departments need to track the occurrence of outbreaks and the public health response to those outbreaks. since outbreaks can be due to reportable or nonreportable diseases, this cannot be done only by actions such as identifying some cases in the reportable disease data system as being part of an outbreak. systems to track the occurrence of outbreaks need to document the following: • time and date the fi rst and last cases occurred • total (estimated or counted) number of cases • population group most affected (by age, sex, location) • setting of the outbreak (school, workplace, restaurant, wedding, etc.) • suspected or confi rmed agent • most common clinical presentation • suspected or confi rmed source and mode of spread • methods used to investigate agent, source and mode of spread • control measures recommended • control measures implemented • lessons learned for prevention of future outbreaks and improved investigation and response in future events this information about outbreaks should be stored for ready retrieval, and to serve as a basis for quality improvement efforts. for quality improvement purposes, it is also helpful to document the content of the summary report written about each outbreak. when the outbreak is due to a reportable disease, individual cases in the reportable disease surveillance information system can be linked to the outbreak, for example by having an outbreak identifi er attached to their records. if preliminary information about outbreaks in a jurisdiction is entered into the outbreak information system in real time, as the investigation is proceeding, and if the outbreak database is readily searchable by all communicable disease investigators in the jurisdiction, then local investigators can use the outbreak database to help them with investigations of new illness or outbreak complaints [ 21 ] . for example, if they receive a complaint that illness has occurred in people who consumed a particular food product, they can look in the database and determine whether other recent or current complaints or outbreaks mention the same food product. if they receive a report about a gastroenteritis outbreak in a childcare center, they can determine what agents have been found to be responsible for recent or current similar outbreaks in nearby communities; this can help focus their laboratory testing and initial control strategies. some us states have had long-standing systems to document all outbreaks investigated by local or state personnel, but others have not. a major variable in the design of such systems is the state-local division of responsibilities in each state, including the degree of state oversight of 'routine' local outbreak investigations. the actual investigation of an outbreak or cluster may involve enhanced "active" case-fi nding, use of case-report forms, group surveys, and formal epidemiologic studies. active case-fi nding involves regular solicitation of case reports from doctors, hospitals, and laboratories. managing the reports of possible, probable, and confi rmed cases that are part of the outbreak is an important task. for a reportable disease, the jurisdiction's reportable disease surveillance system may be adequate to manage reported cases. it may be necessary, however, to create a continuouslyupdated line list of possible cases and their current status, which is outside the scope of the standard reportable disease application. outbreak investigation surveys will typically involve interviewing everyone with a possible exposure (like all attendees of a wedding reception), whether they were ill or not. formal studies may involve interviewing selected non-ill people, for example, as part of a case-control study. the investigation may also involve obtaining and sending to a laboratory a large number of specimens from ill persons, and sometimes from exposed non-ill persons and from environmental sources (food, water, air, soil, etc.). managing these disparate types of information is a challenge, especially in a large outbreak or one involving multiple jurisdictions. there is currently no one widely-accepted and satisfactory way to manage data in such settings. each investigation team typically uses the tools it is most familiar with, including some combination of data management tools like ms excel, ms access, or epiinfo [ 22 ] , and standard statistical packages. many health departments maintain libraries of standard questionnaires with associated empty data bases, for use during outbreak investigations. when cdc is involved in a multistate outbreak, the investigation team at the local or state level needs to be able to produce and transmit timely case report and other information in the format desired by cdc. the services of an experienced public health informaticist can be extremely helpful to the investigation team when outbreaks are large and multifocal. an ongoing challenge for cdc and the states is how to make the transition from specialized case reporting during an outbreak of a new disease, such as west nile virus encephalitis or sars, to routine case-based surveillance. if this transition is not well-managed, it is likely to result in the creation of a permanent stand-alone surveillance information system (or silo) for that disease. if the new disease is of national importance, cases should be made nationally notifi able and its surveillance should be incorporated into existing systems. laboratory information is a critical component of disease surveillance and prevention. laboratory data form the foundation of many surveillance systems. there are different types of laboratories involved in the public health data stream. laboratories providing data to public health fall into the general categories of commercial or private industry, hospital or clinical, and public health laboratories. public health laboratory information systems (lis) contain information about test results on specimens submitted for primary diagnosis, for confi rmation of a commercial or hospital laboratory's results, for identifi cation of unusual organisms, or for further characterization of organisms into subgroupings (like serotypes) that are of epidemiologic importance. in some states, all clinical laboratories must submit all isolates of certain organisms to the public health laboratory. many of the results obtained in a public health laboratory turn out to be for diseases that are not reportable and not targets of specifi c prevention programs. some of those results may, however, be for cases of non-reportable diseases that are historically rare in the jurisdiction but of great public health importance, or are new or newly-recognized. the main business of clinical laboratories (located both inside and outside hospitals) is to test specimens for pathogens or groups of pathogens specifi ed by the ordering physician, and return the results to the person who ordered the test. public health agencies have, since the early 1990s, asked or required such laboratories to also identify results meeting certain criteria (indicating the presence of a case of a reportable disease) and send a copy of the results to the public health agency for public health surveillance. initially, case reporting by laboratories was accomplished on paper forms, which were mailed or faxed to public health departments. some laboratories very soon moved to mailing printouts of relevant laboratory results, then to sending diskettes, then to transferring computerized fi les containing laboratory results by direct modem-to-modem transfer, and eventually to transferring such fi les via the internet using standard formats and vocabularies. in some states, public clinics (for example, std clinics) have used contract laboratories for their testing needs. in this situation, the outside laboratory supplies both positive and negative results to the public health agency, increasingly by transfer of electronic results in standard formats. laboratories provide data on reportable conditions to their local or state public health authority. reportable diseases are determined by each state; clinicians, hospitals, and/or laboratories must report to public health when these conditions are identifi ed. some reportable conditions are also nationally notifi able. deidentifi ed cases of these are voluntarily notifi ed by states and territories to cdc, which, in collaboration with the council of state and territorial epidemiologists, maintains a listing of nationally notifi able conditions that includes both infectious (e.g., rabies, tb) and non-infectious (e.g., blood lead, cancer) conditions [ 23 ] . the public health partnership with laboratories has led to the very successful and still increasing implementation of electronic laboratory reporting (elr) in the us. elr refers to the secure, electronic, standards-based reporting of laboratory data to public health. elr implementation has been steadily escalating since its inception around the year 2000, replacing previous reporting systems that relied on slower, more labor-intensive paper reporting. the elr national working group conducted annual surveys from 2004 to 2011 [ 24 ] which gathered data from all 50 states as well as from several territories and large metropolitan areas. these data were supplemented with data for years 2000-2004, retroactively gathered in the 2010 survey. the tracked growth of elr (fig. 14.1 ) illustrates its rapid rise in the us, from the start of early stage planning to fully operational elr [ 25 ] . the expected benefi ts of elr include more rapid reporting of reportable cases to public health departments, allowing faster recognition of priority cases and outbreaks for investigation and response, and thus more effective prevention and control [ 26 ] . elr also is expected to reduce the number of missed cases, as automated systems do not require laboratory staff to actively remember to make case reports, and to improve the item-level completeness and quality of case reports. although experience shows that the expected improvements in timeliness, sensitivity, completeness, and accuracy are generally being realized [ 27 ] , timeliness may not be improved substantially for those diseases where clinicians routinely report based on clinical suspicion without waiting for laboratory confi rmation (for example, meningococcal disease) [ 28 ] . in addition, laboratories (especially referral laboratories) often do not have access in their own information systems to home addresses for people whose specimens they are testing, and have struggled with providing complete demographic information to public health agencies. implementation of an operational elr system is not a trivial undertaking. laboratories must confi gure data into an acceptable message format, most commonly health level seven (hl7 ® ) [ 29 ] . laboratory tests and results should be reported with correlated vocabulary or content codes. two of the most common code systems used for laboratory tests and their associated results are logical observations identifi ers names and codes (loinc ® ) [ 30 ] and systematized nomenclature of medicine (snomed ct ® ) [ 31 ] . neither of these systems is suffi cient by itself to encode all the information needed for public health surveillance. public health jurisdictions have introduced elr to their partner laboratories using one or more of the following approaches: • the "charm" approach -relies on establishing goodwill and collaboration with laboratory partners. while this collegial approach is very appealing, it may be unable to overcome signifi cant barriers such as lack of laboratory funding or resources, and some facilities will supply data only in methods specifi cally required by law. • the incentive approach -involves offering either fi nancial or technical assistance to laboratory partners, assisting them in the startup process of elr. while this approach may be preferred by many laboratories, relatively few jurisdictions have the discretionary funds (or are able to receive federal assistance funds) to implement the approach. • the enforcement or legislative approach -requires reporting rules or legislation that requires laboratories to participate in elr. the most successful enforcement approach will include low-cost options for smaller laboratories, such as web data entry, so that they may benefi t from an elr -"lite" implementation [ 32 ] . the mainstreaming of elr systems in the us has pioneered a clear path forward for public health to begin maximizing its presence in the domain of electronic data interchange. at a local level, case reports for communicable diseases prompt action. although the specifi c action varies by disease, the general approach is the same. it starts with an interview of the ill person (or that person's parents or other surrogates) to determine who or what the person was in contact with in ways that facilitate transmission, both to determine a likely source of infection and to identify other people who may be at risk from exposure to this person. information systems to support contact tracing, partner notifi cation, and postexposure prophylaxis (for stds or tb, for example) contain records about all elicited contacts (exposed persons) for each reported case of the disease in question. these records contain information about each contact, such as whether they were located, whether they received post-exposure prophylaxis, and the results of any additional partner-elicitation interviews or clinical testing that were completed. information systems to support surveillance for other reportable diseases also increasingly contain information about what disease-appropriate action was taken in response to each case; such actions may include identifi cation of contacts, education of household members, vaccination or antibiotic prophylaxis of contacts, isolation of the case (including staying home from work or school), or quarantine of exposed people. std and tb information systems typically capture full locating information for contacts, and can be used both to support fi eld work and to generate statistics on effectiveness of partner notifi cation activities worker by worker and in the aggregate. systems for other reportable diseases may capture only the fact that various interventions were done, and the date that these were initiated. information about the timeliness of initiation of recommended control measures is now required as a performance measure for selected diseases by cdc's public health emergency preparedness cooperative agreement [ 33 ] . in the investigation of a case of meningococcal disease, contacts are people who had very close contact with the original person, for example a household member, boyfriend, or regular playmate. health department staff determines who the close contacts are. each will then be offered specifi c antibiotic treatment to prevent illness. for syphilis, contacts are people who have had sex with the original case. contacts will be examined by a clinician and assessed serologically to see if they are already infected, and offered appropriate prophylactic or curative antibiotic treatment. for measles, contacts may include anyone who spent even a few minutes in the same room as a case. contacts whose exposure was recent enough, and who are not fully immunized already, will receive a dose of measles-containing vaccine, and all contacts will be asked to self-isolate immediately if they develop symptoms of measles. in investigating a common-source outbreak of legionellosis, histoplasmosis, or anthrax, the local health department may want to locate everyone who had a specifi ed exposure to the apparent source of the infection. these exposed people may need antibiotic prophylaxis or may be advised to seek medical care promptly if they become ill. information systems to support this type of work typically have three purposes: 1. serve as a place for workers to record and look up information about people who are or may be contacts, and to track which contacts have and have not yet received needed interventions. 2. serve as a source of information for calculating indices of program or worker timeliness and performance, such as the average number of sexual contacts elicited per syphilis patient interviewed, or the percentage of measles contacts who were identifi ed in a timely way and who received post-exposure measles vaccine prophylaxis. 3. document the workload and effort put in by epidemiology and disease control fi eld staff it seems logical that the surveillance information system should serve as the basis for a system to support fi eld investigation, and this is often the case. the fact that the recommended interventions vary by disease makes designing a single system more complex. existing systems that track fi eld worker activities in detail are much more common for std and tb programs than for others. for general communicable disease fi eldwork, it is currently more common that the system simply documents which interventions were done and when, rather than use the application to track specifi c named contacts or exposed people. the public health informatics institute has published a detailed analysis [ 34 ] of the typical workfl ow involved in surveillance, investigation, and intervention for reportable diseases, and the corresponding information system requirements. the work group that phii convened had representatives of nine different state and local health departments, who were able to identify a large number of processes that were common to all nine jurisdictions, such as case-fi nding, case investigation, data analysis and visualization, monitoring and reporting, case/contact specifi c intervention, and others. these common processes can then serve as a basis for designing information systems to support case-reporting, surveillance, and case-based intervention work that are useable in multiple jurisdictions. consider existing or planned surveillance systems for multiple diseases and conditions. broadly, there are three functions in each of these systems -acquiring the raw data, cleaning and managing the data, and making the data available to users. each of these functions potentially can be integrated, to varying degrees. for example, multiple surveillance systems may benefi t from receiving electronic laboratory reports with a result indicating the presence of a case of a reportable disease. laboratories appreciate having a single set of instructions and a single destination for all their required reports, as this simplifi es their work. the laboratories then benefi t from the ability of the recipient health department to route the reports internally to the right surveillance information system. at the other end of the data pathway, users appreciate having a single interface with which to examine data about multiple conditions or diseases, using the same commands and defi nitions. the users do not have to understand how different surveillance information systems may internally code the same concept in different ways. they also appreciate being able to directly compare information that originally was submitted for the use of different program areas -for example, hepatitis b and gonorrhea in the same chart or table. in the short to medium term, it is not necessary to build a single integrated data repository or a master person index to achieve these goals, even if that is what one would have designed if one were starting from the beginning. however, if one wants to be able to see information about the same person that originates and is stored in multiple systems -for example, so that tb clinicians can see hiv data on their patients and vice versa -then an integrated data repository, or a master person index, or a query system that is extremely accurate in fi nding data on the right person, is needed. modifying existing systems to be able to carry out these functions is time consuming and expensive, so the business case and requirements need to be especially clear. florida's essence system: from syndromic surveillance to routine epidemiologic analysis across syndromic and nonsyndromic data sources (abstract) history of public health surveillance blueprint for a national public health surveillance system for the 21st century status of state electronic disease surveillance systems -united states prioritizing investigations of reported cases of selected enteric infections. paper presented at council of state and territorial epidemiologists nationally notifi able disease surveillance system case defi nitions association of state and territorial health offi cers. biosense 2.0 governance updated guidelines for evaluating public health surveillance systems, recommendations from the guidelines working group design and operation of local and state infectious disease surveillance systems oxford handbook of public health practice behavioral risk factor surveillance system overview of infl uenza surveillance in the united states international society for disease surveillance meaningful use workgroup. final recommendation: core processes and ehr requirements for public health syndromic surveillance electronic syndromic surveillance using hospital in patient and ambulatory clinical care electronic health record data national program of cancer registries coverdell national acute stroke registry surveillance -four states atlanta congenital defects program (macdp) north american association of central cancer registries, inc. (naaccr). implementation guidelines and recommendations report on birth defects in florida a comparison of two surveillance strategies for selected birth defects in florida online food and waterborne illness complaint form biosurveillance plan for human health, version 2.0. atlanta national notifi able diseases surveillance system (nndss), cdc. accessed at http:// wwwn.cdc.gov/nndss/ on available from www.coast2coastin-formatics.com national electronic laboratory reporting (elr) snapshot survey. available from www.coast2coastinformatics.com . cited statewide system of electronic notifi able disease reporting from clinical laboratories: comparing automated reporting with conventional methods a comparison of the completeness and timeliness of automated electronic laboratory reporting and spontaneous reporting of notifi able conditions potential effects of electronic laboratory reporting on improving timeliness of infectious disease notifi cation -florida health level seven (hl7 ® ) homepage. available at logical observation identifi ers names and codes (loinc ® ) systematized nomenclature of medicine-clinical terms (snomed ct ® ) see for example section 64d-3.031(5) of the florida administrative code: notifi cation by laboratories public health emergency preparedness cooperative agreement, budget period 1, performance measures specifi cation and implementation guidance, at-a-glance summary redesigning public health surveillance in an ehealth world on 31 1. what are some of the methods for surveillance besides case-reporting? 2. how are registries different from other surveillance information systems? 3. what are the advantages and disadvantages of building a master person index across surveillance information systems for multiple diseases? 4. what are the expected benefi ts of electronic laboratory reporting as a method to enhance surveillance? 5. what are the advantages and disadvantages of building a system to manage information about case contacts as part of the surveillance information system? 6. who determines for which diseases cases are nationally notifi able? key: cord-026881-57mx3thr authors: neuwirth, rostam j. title: gaia 2048—a ‘glocal agency in anthropocene’: cognitive and institutional change as ‘legal science fiction’ date: 2020-03-28 journal: a post-wto international legal order doi: 10.1007/978-3-030-45428-9_5 sha: doc_id: 26881 cord_uid: 57mx3thr to assess a future scenario of the world without a wto, the present chapter projects the reader into the distant future of the year 2048 by which time the global community is aiming to establish gaia, the so-called “glocal agency in anthropocene”. gaia is designed as the first truly integrated global institution with a universal character with the aim to tackle the complex and multiversal governance challenges of humanity and the planet as a whole. this chapter marks both a legally and a scientifically fictitious account of the years from 2020 until 2048, from a dystopian and a utopian perspective, with the aim of highlighting the importance of cognition for legal and institutional change. the need for cognitive change is driven by changes in the environment, and by the challenges resulting from a perceived acceleration of the pace of change and the unprecedented levels of technological complexity. both change and complexity increase the relevance of cognition, as laws and policies adopted in one area are more likely to affect their success or failure and that of the global governance system as a whole. thus, this chapter predicts that the foremost necessity for law in the future is to build on novel and enhanced modes of human cognition to deal better with complexity and rapid change. why do we remember the past and not the future? 1 we are writing at the end of the year 2048, and marking a period of efforts towards the establishment of a new and comprehensive global governance mechanism, known, for short, as "gaia 2048". gaia stands for "glocal agency in anthropocene", and describes a project for a novel institutional framework to be set up with the objective of tackling humanity's most urgent problems and glocally governing and sustainably developing human affairs in the future. the future is what matters the most right now, as the world is hoping to recover from the devastating effects of the time period known as the "digital dark age". this is the era from 2030 until 2045 when-as the result of an internet blackout (outage)-all prior digital resources, including records and archives, became inaccessible and, because of the wide prevalence of the internet of things (iot), the same happened to most related technical applications and utilities. 2 in short, this dark period wiped out all digitally or electronically stored data. as a consequence, it not only paralysed all technical facilities and caused millions of casualties, but also led to a collective loss of humanity's memory, which, furthermore, caused a loss of identity. many still speculate about what triggered the meltdown of the former information society based on the internet, with the suggested causes ranging from scientific, economic, and political to cognitive and, notably, legal reasons. for instance, scientific explanations range from a cyberattack using malware to a nuclear blackout caused by the detonation of an atomic warhead in outer space, an accident at cern creating a black hole in geneva following a malfunction of the large hadron collider ii, or a failure of the 5g network in combination with a data overload on the world wide web caused by states' geopolitical struggles and the monopolistic tendencies of various tech giants. other people invoke politicoeconomic explanations, simply citing the second global financial crisis of 2029, which was caused by trade and currency wars that, inter alia, eroded the finances needed for the maintenance of the internet's sophisticated infrastructure. environmental factors are also often named, such as floods and rising sea levels, increased volcanic activity and earthquakes, combined with heatwaves and global pandemics, which disrupted or paralysed the energy supply by damaging its essential facilities. politics also played a role, as populism and racism increased, further fragmenting society into ever smaller units, down to its most vulnerable minority, the individual. constitutional democracies came under threat from changes that were, at first, undetectably small but that eventually combined and posed a serious threat. in legal circles, the fragmentation of international law, the rise of norm conflicts due to bad regulation, overregulation and the automation that took over several fields of law 3 are mentioned as the prime causes of the inability of humanity to halt the dangers of the conditions it had created for itself. it again became obvious that the rule of law needed not just legal texts but also stable institutional guarantors at both the national and the global levels. 4 it is more likely that the blackout was due to a combination of interrelated factors in an environment characterized by an increasing complexity and ever faster pace of change. since time immemorial, humanity has struggled with change or, notably, with its perception of change, since this perception may itself have been subject to change. 5 however, since the last millennium many observers have noted an apparent effect of an acceleration of change or so-called "time shrinking". 6 this trend for change to accelerate started to pose a major threat to lawmakers and policymakers and, in particular, to law, as it became more difficult to "preserve its integrity over time, while managing to address the newly emerging circumstances that continually arise throughout our history". 7 another threat to law came from unprecedented levels of technological innovation, which culminated in a situation in which the regulation of specific industries and technologies, such as those of artificial intelligence, nanotechnology, big data and genetics, failed to find a successful balance between their unknown risks and their expected benefits. in short, excessive levels of specialization, not met by an adequate general understanding, led technological progress to spin out of control. 8 thus, by the year 2020 the international legal order had witnessed the disastrous effects of a gradual loss of control caused by decades of neglecting first subtle and later obvious signs of multiple law and policy failures. these policy failures culminated in the failed attempts to reform the most important international institutions, the united nations (un) and the world trade organization (wto), in a holistic and comprehensive manner. there was a failure to bridge the rift that was caused when the international trade organization (ito) did not materialize; the ito would have complemented the institutional balance between the three bretton woods sisters, the ito, the imf and the world bank. in other words, the institutional failure of the ito to materialize also meant that greater coherence, or to integrate trade and non-trade concerns, was renounced as an ideal for global governance. the separation between the un on the one hand and the gatt on the other, can also be regarded as a further strong manifestation of a dualistic mode of thinking based on binary logic. most of all, it was the beginning of a failure of cognitive modes of thinking to keep pace with technological changes in the context so as to provide a stable and coherent global institutional framework for the governance of global affairs. historically, the gatt 1947 was characterised as having found "itself without an inadequate legal and constitutional base and required to fill a vacuum created by the failure of the ito". 9 the situation also meant that attempts to reconcile trade and non-trade concerns in both substantive and institutional terms was postponed to a distant day. this delay later led to the emergence of the "trade linkage debate", by which different pairs of "trade and . . ." problems were supposed to be reconciled. lacking their reconciliation, criticism of the wto also became louder and culminated in the protests accompanying the 1999 seattle wto ministerial conference. 10 since then, the failure to address non-trade concerns and to achieve greater policy coherence continued and provided further momentum to the procedural crisis affecting the wto dispute settlement body. in sum, a lack of political will as much as outdated cognitive modes framed by dualistic thinking led to the law being inadequate to meet the institutional and substantive challenges. to exemplify the impact of cognition on whether the law meets the demands put upon it, the present chapter divides the time between the year 2020 and 2048 into two opposite scenarios. first, sect. 1 traces the lost years by drawing up a dystopian 7 johnson (2007), p. 845. 8 harari (2017 ), p. 51. 9 jackson (1969 ), p. 51. 10 dimatteo (2003 scenario, in which cognition remained largely static and dualistic, such that changes did not keep pace with the speed of technological and scientific innovation. section 2 is more utopian, in the sense that it relies on a newly acquired cognitive mode of thinking as exemplified by synaesthesia, which not only allows for the development of actions as causes of change but also includes a review and evaluation of the consequences. the conclusion finds that an entirely new mode of cognition, triggered by linguistic changes that are reflected in a recent rise of oxymora and paradoxes, opens a new perspective on how lawyers can help to both predict and shape the future. fail' in the world of theory, there are many dichotomies. in the real world, there are many divisions and divides. in the world of power, all too often, these divisions, divides, and dichotomies serve to maintain and reinforce existing imbalanced and skewed power relations between individuals, communities, governments, and nation-states. in the world of power, it is indeed divide and rule. 11 in attempts to describe the evolution of the multilateral trading regime from 1995 until 2048, so-called "digital historians" have argued that 11 december 2019 marked the decisive moment and unofficial date of the decline of the world trade organization. although aggressive regionalism, unilateral measures and resulting trade wars as well as inertia in addressing the trade linkage debate and interinstitutional linkages between the wto and the un had been eroding the multilateral trading regime for some time before, it was on that day that the wto's dispute settlement system became dysfunctional following the expiration of the terms of two of the three remaining members of the appellate body (ab). 12 this left the ab with fewer than the three members required by art. 17.1 dsu to serve on appeal cases. as with many international organizations, nation states and other legal constructs before it, the official decline of the wto and the multilateral trading regime as a whole started slowly and then ended abruptly. the decline was probably the result of a chain of missed opportunities for the reform and adaptation of the relevant system to changes in the context, which notably saw a strong trend towards greater convergence of various industries, technologies and products. 13 this convergence further increased the need for the consideration of non-trade concerns and greater policy coherence. for some time, the system lingered on and ministerial conferences were merely held without achieving tangible results, continuing the deplorable tradition that began with the launch of the doha development round in 2001. most wto members engaged in either a relapse into unilateral measures adopted at the domestic level or a rat race to join numerous regional trade agreements. however, in both cases, the most important challenges faced at that time, in the form of institutional gaps, regulatory fragmentation and overregulation, were not tackled, either nationally or regionally, let alone globally. numerous calls and suggestions for institutional reform to achieve greater policy coherence, under the aegis of the so-called "trade linkage debate" discussing various "trade and . . . problems" or how to better link trade with non-trade issues, were ignored. 14 in this regard, more importantly, the cognitive and conceptual dimension of trade policy was not duly considered, as "transformational change in the institutions and politics of international trade" were found to go "hand in hand with cognitive change" as andrew t. f. lang wrote. 15 he also found a nexus between cognitive change and institutional reform being linked to the criticism of the wto and notably the failure to address the trade linkage debate. 16 as a result, from this time on, the international system, or, more accurately, the remaining "international systemic chaos", 17 was described as having moved "away from an assembly of distinct, territorial, sovereign, legally equal states toward different, more hierarchical, and in many ways more complicated structures". 18 even though an attempt was launched in 2006 to reform the united nations organization in order to streamline the coordination of its many specialized organizations and to enhance the coherence of global policymaking by "delivering as one", 19 the management of this reform eventually proved to be an oxymoron before it failed altogether. 20 for a long time, the un's work in its core areas, from development to the environment, was described as fragmented, and its inefficient governance structure contributed to "policy incoherence, duplication and operational ineffectiveness across the system". 21 what could be framed as a "trade and technology" problem, the earlier us-china trade disputes (2017-2020), also caused frictions in the innovation of new technologies and especially in the realm of telecommunication and information technologies. 22 these frictions led to a further fragmentation of the internet and the market for information and communication technologies (ict). russia initiated the creation of an alternate web, which was later also used by the remaining brics countries, 14 pauwelyn (2005) with other countries following suit. moreover, it was common for the governance gaps and frequent legal inconsistencies between the various fragments of laws governing international trade and commerce to be abused by artificial intelligence and unregulated algorithms, those lacking a kind of lex algoritmica, meaning that global business generally operated in a "black box" inaccessible to consumers and lawmakers. 23 from a commercial perspective, the "world wide web" (www), which was intended to become a "wireless world wide web" (wwww) by virtue of the transition to the 5g network, became further fragmented by national and regional firewalls as well as restrictive measures known as geolocation measures. 24 other conflicts, in the form of regulatory paradoxes framed as dichotomies, such as those of "goods versus services", 25 "free trade in data versus data localization", 26 or "regulatory fragmentation versus technological convergence", 27 were also ignored. in the same way, in other sectors covered by the gatt, gats and trips agreements, the multilateral trading rules were further eroded by trade disputes in which different claims were met by defences made on the basis of national security concerns. 28 known as the "trade and security" problem, questions about the selfjudging nature of the security exceptions widened the institutional gap opened by the split between the un system and the gatt/wto system caused by the failure of the international trade organization (ito) to materialize. this institutional rift had, since 1948, left the world with a conflict of norms in the form of a "catch xxi" or "trade and security" dilemma. 29 after the wto ruling in russia -measures in transit in 2019, in which the panel held that it had jurisdiction to determine whether the requirements of article xxi of the gatt 1994 were satisfied, 30 other countries simply refused to respond to requests for consultations and boycotted the relevant meetings. 31 in the end, the invocation of the security exceptions further eroded belief in the wto and accelerated its demise. the broader conditions in the global economy also grew dimmer and were closely related to the "trade and energy" problem. from as early as 2005, the tipping point in the production of oil was reached, meaning that conventional crude oil production 23 pasquale (2015) . 24 yu (2019), pp. 503-529. 25 smith and woods (2005) , pp. 463-510. 26 burri (2017) was not rising to match increasing demand. 32 the "all-oil peak" meant that, after decades of controversy over its arrival, there was a drastic dampening in economic growth and, actually, the inauguration of an era of global depression. 33 notably, for oil-producing and other resource-rich countries, the so-called "paradox of plenty" aggravated, and the ensuing economic disaster also led to, the collapse of their constitutional system, and also triggered new waves of political violence. since human civilization was a sensitive "complex adaptive system", other countries too, and the globe as a whole, also were drawn into a downward spiral of economic recession and political instability. 34 institutionally, the proliferation and fragmentation of energy organizations and the failure to address the "global energy governance gap" by reforming existing energy agencies or creating a global energy agency, further contributed to the resulting disaster. 35 the complexity of the global economy could also be seen in the connection between trade, the environment and energy. in parallel, numerous efforts towards achieving greater sustainability in economic development based on renewable energies were also unable to offset the devastating effects of the disruptive consequences of the "all-oil peak" and the ensuing global energy crisis on economies and societies around the world. the result was a global energy crisis in 2033 that disrupted most global trade in both goods and services and caused stock crashes through inflationary pressures, and later accelerated the eventual collapse of the international monetary system as it had done during the 1973 oil crisis. renewable energies were also incapable of averting the crisis, even though the impact of the 2015 paris agreement, adopted with the objective of responding to the threat of climate change, had no negative effect on the global economy or on countries' welfare gains. because of the absence of a coherent global regulatory framework, renewable energies remained expensive and investments often did not materialize. in this context, it was also a failure when the wto membership addressed the "trade and environment" problem by, inter alia, reforming the wto subsidies agreement, which resulted in various trade disputes challenging green policies. 36 the lack of global competition rules, as a result of the abandonment of a project in 2004 that was one of four "singapore issues", 37 led to the emergence of various multinational tech giants, which widely abused their dominant positions, explored tax loopholes, and competed unfairly in courts, patent offices and markets. they were also crucial in meddling in elections and were accused of undermining democratic institutions. 38 no efficient dispute settlement system under the multilateral trading regime, and a lack of legal standing for private parties, both natural and legal 32 murray and king (2012) , pp. 433-435. 33 hall and klitgaard (2018) , p. 109. 34 ahmed (2017) . 35 downie (2015) , p. 475. 36 cosbey and mavroidis (2014), pp. 11-47. 37 demedts (2015), pp. 415-416. 38 ginsburg and huq (2018), p. 198. persons, in domestic or supranational courts caused further havoc and greater inequality among global citizens. as early as 2017, inequality was reported to be standing at unprecedented levels, and it was claimed that "just eight men own the same wealth as the poorest half of the world". 39 these inequalities, dividing societies around the world, not only perpetuated themselves but even increased, with devastating effects on the global economy and the environment. another aspect of inequality was found in the "trade and development" link, another complex and cross-cutting "trade and . . ." problem. cutting a long story short, the development discourse continued along the "developing versus developed country" dichotomy, despite calls to the contrary and even its abandonment by the world bank in 2016. 40 even though the definition of "official development assistance" (oda) evolved, it continued to be understood as resource flows from developed to developing countries. 41 with the mindset unchanged, the language use of "developed-developing countries" also remained the same. as a result, the us and the eu continued, on the one hand, to subsidize their agricultural producers and to reap excessive benefits from royalties on various patented and copyrighted products, while on the other, they kept granting support through official development assistance programs to a large number of developing countries. this practice continued to impede the development of local industries in the targeted countries. 42 moreover, the uncertainty surrounding the meaning of "development status" further eroded the wto's status after the us began to challenge it in 2019. 43 at the same time, the trade and development problem was closely tied to the "trade and finance" problem. 44 generally, the trade and finance link suffered from the failure of the ito mentioned above, and the absence of a coherent framework for both trade and finance became manifest in the problem of so-called "currency manipulation", that is, the depreciation of a country's currency relative to other currencies so as to develop a large global and bilateral trade surplus. 45 despite the three organizations, the wto, the imf and the world bank, having pledged to enhance their policy coherence, their inter-institutional dialogues did not yield tangible results and the use of policy coherence was criticized instead as a way to introduce policy conformance. 46 39 an economy for the 99%. oxfam briefing paper, january 2017. https://www-cdn.oxfam.org/ s3fs-public/file_attachments/bp-economy-for-99-percent-160117-en.pdf. 40 neuwirth (2016), pp. 911-925. 41 hynes and scott (2013) . 42 moyo (2009) thomas (2000) , pp. 1249-1278. 45 staiger and sykes (2010) , pp. 583-627. 46 grabel (2007), pp. 335-341. put briefly, the "trade and finance issue" also remained unsolved, and the two respective regulatory regimes of trade and of finance continued to evolve side by side in dramatically different directions in spite of being mutually interdependent systems. 47 this had a serious impact on the world as the regulatory gap between them grew even more intense while the technological reality saw them increasingly converge notably with the emergence of the blockchain technology that underlies cryptocurrencies. cryptocurrencies, from bitcoin to altcoin, proliferated and gradually disrupted business and financial services as well as the global economy. 48 this meant that, in parallel to the third currency war between the world currencies (the dollar, the euro and the yuan), which began in 2010, 49 a digital currency war also began to be waged. in this digital currency war, the traditional world currencies fought against emerging digital currencies backed both by state and non-state actors. the ensuing chaos of currency wars brought about a loss of state control over financial markets and a rise of a global underground economy thriving on tax evasion and criminal activities. 50 it all culminated in a collapse of global trade and finance and ended with the second global financial crisis of the twenty-first century, which began on "black wednesday" of 24 october 2029. together with military conflicts, global health pandemics, like the large-scale outbreak of the novel coronavirus (ncov) infectious disease, and various natural disasters, like large scales rainforest wildfires and bushfires, caused by climate change and other anthropogenic factors, 51 the total disruption of the global economy created a dangerous dynamic, causing a severe migratory wave 52 that increased the number of forcibly displaced people from about 70 million people in 2018 to 1.4 billion less than 15 years later. 53 the crisis of migratory flows was aggravated by national governments and courts, who denied the affected persons the status of climate refugees. 54 at the same time, after an erosion of the rule of law in the majority of countries around the world, ever more restrictive national policies regarding citizenship in general, and dual citizenship in particular, also increased the number of stateless persons, who were thus deprived of all fundamental rights within and across state borders. 55 the european union, paralysed by endless brexit debates and the united kingdom's final exit from the eu on 31 january 2020, made little progress in reforming 47 gadbaw (2010) , p. 552. 48 tapscott and tapscott (2018) . 49 rickards (2011) 55 weissbrodt and collins (2006), pp. 245-276. its institutions, the single market, the single currency or its taxation system. on the contrary, past achievements in the four freedoms were scaled down as the result of nationalist policies in many member states. the eu's so-called "luxembourg treaty", negotiated under the 2029 eu presidency of luxembourg, added nothing that substantively remedied the lost opportunity of creating a constitution for europe in 2001. the eu's failure to become the world's most competitive market (the lisbon strategy) extended the lost decade of the eu, which began in 2000, when, to a lost near-half century. 56 many more regional integration projects worldwide also stagnated. for instance, the brics, representing 42% of the world's population in 2014 and initially set up as a "cooperation and dialogue forum" between brazil, russia, india, china and south africa in order to defy differences and make a difference in global governance, saw no progress, such as by seeking greater policy coherence based on more closely coordinating institutionalizing their cooperation. 57 similarly, the positive effects on infrastructure development based on the belt and road initiative (bri), initiated by the chinese government in 2013, were largely neutralized by opposition from the us and several european countries. the african union also failed to prevent the food security crisis that hit the african continent in 2030 58 and that pushed the achievement of the objectives enshrined in agenda 2063 into an even more distant future. 59 in sum, despite the emergence of a creative economy, which was initially fostered by the potential of new and innovative technologies, the 2015 sustainable development goals were still not realized by 2030. across the globe, political disagreements prevented action for greater regulatory harmonization, while centrifugal forces and fragmentation intensified. legally, a patchwork of isolated areas of law continued to grow, as did the number of regional trade agreements, which were violated or withdrawn from faster than they were negotiated. put simply, the so-called "global governance paradox", or the logical loop that a global platform was needed in order to create a global platform, proved a problem that was too difficult to tackle in conceptual and cognitive terms. with no consistent global legal order, fragmentation prevailed and led to the aggravation of the negative effects of "trade and . . ." problems on global peace and welfare. 56 neuwirth (2020), p. 51. 57 neuwirth et al. (2017) and neuwirth and svetlicinii (2019) . 58 onyutha (2018 onyutha ( ), pp. 1203 onyutha ( -1219 african union (2017) progress report on the implementation of agenda 2063 first ten-year implementation plan. available at: https://archives.au.int/handle/123456789/2618. the limits of my language mean the limits of my world. 60 in 2048, historians are still pondering the reasons for the collapse of the wto. some attribute it to isolationist us trade policies and the political turmoil around brexit, while others tend to focus on the rise of first asian and subsequently african countries. however, a different view identifies this failure as the beginning of the end of "end of . . . stories" 61 or a drastic paradigm shift in cognition. cognition had reached a tipping point in human evolution following two centuries of rapid technological innovations, which "shaped consciousness directly". 62 linguistic changes also confirmed this cognitive shift, mostly through the rise of essentially oxymoronic concepts. 63 as a result of these external factors, epigeneticists found biological and cognitive changes occurring in parallel. new organs of cognition hence emerged, as humans were evolving biologically and consciously. as a visible result, even by 2025 97% of new-born children displayed conditions of synaesthesia, i.e. the ability to better connect stimuli received from different sensory organs, which improved the skills to foresee developments and solve complex problems as well as enhanced their abilities of abstract thought. 64 related changes in educational policies were implemented, with the goal of creating a "world brain", or "an enhanced educational system through the whole body of mankind", designed to "replace our multitude of uncoordinated ganglia, our powerless miscellany of universities, research institutions, literatures with a purpose, national educational systems and the like". 65 this goal was to be achieved by fostering oxymoronic learning methods and giving training in both bivalent and polyvalent logic, complemented by training in multilingualism and oxymoronic thinking. for the trade and health link, the novel coronavirus (ncov) pandemic underscored once more the strong links between international trade, public healthcare and the global economy. as a result, the global healthcare system was integrated with a future trading system and backed by a consistent set of global innovation rules consisting of competition rules, intellectual property laws and fiscal incentives, which prevented price discrimination in pharmaceutical products and secured global access to affordable medicines. additionally, universal healthcare was provided freely by a combination of measures that combined both traditional and conventional medicines. 66 based on further changes in perception, such as through the development of technologies for the visualization of auras, 67 medical diagnosis improved and conventional, traditional and alternative medicines as well as mental health strategies were all integrated into a coherent set of laws and policies. further changes in healthcare were influenced by discoveries in so-called "life after life studies", which transcended the life-death dichotomy as the result of a greater awareness of the missing link in a globally coherent health policy. 68 research on near-death experiences scientifically confirmed popular and religious beliefs about life after life. 69 overall, a new cognitive mindset emerged in parallel with technical tools, leading to a new conception of the areas of economics, politics and law. for instance, the global economy became more sustainable, based on the spirit of "degrowth", which rejected the illusion of endless economic growth by advocating for a "democratically-led shrinking of production and consumption with the aim of achieving social justice and ecological sustainability". 70 at the same time, new and cleaner energy resources were derived from nuclear fusion developed by the iter project, helping to decarbonize the world's energy system. 71 various new and renewable energies complemented this. 72 since a g20 initiative formulated in 2019, the global community embraced an ambitious tax agenda to improve cooperation and transparency on the basis of a strategy for a global taxation system. 73 this initiative originally included a global financial transaction tax to fund the new global governance system. 74 it later included a digital tax on the world's tech giants, which levied taxes in the places where the goods and services were sold rather than the places where the company was based. it also introduced a global minimum tax rate to prevent a company from shifting its sales to a country with lower taxes. finally, a robotic tax, aided by the creation of a single global cryptocurrency (sgcc), 75 covered all activities based on automation to compensate for the disruptive effects of automation on the global labour market. 76 together, these measures helped to contain the outbreak of currency wars and related trade/finance disputes. 77 on the other hand, the changes in the taxation system also helped to reverse the former trend of higher income taxes as opposed to lower corporate taxes. the new system achieved an optimal balance, with around 5% income tax, 25% corporate tax (including on digital activities) and 35% robotic tax, as measures towards greater global tax justice. 78 these and other fiscal policies were introduced in coherence with other incentives to end the poverty trap. 79 the cognitive shift also remodelled the international multilevel governance system towards a "glocal" and holarchic system in which local, largely selfgoverning, entities were complemented by various regional regimes often organized into mega-regional blocks, with those of an inclusive global system enshrined in the gaia charter of 2048. holarchy here means a system in which various selfregulating entities function as autonomous wholes in supra-ordination to their parts but, at the same time, also as dependent parts in subordination to controls on higher levels their local environment. 80 the foundation is a polyvalent logic by which the links between stakeholders at different levels are supported by a dynamic system of subsidiarity and an open method of coordination. 81 it is based on a set of different criteria that the best possible level of regulation is determined without any prior bias towards either the local or the global level of governance. the same method is also applied to territorial questions, such as national sovereignty is no longer fixed but where territorial boundaries are drawn based on a similar set of criteria. thus, like in the quantum world, different political entities, and even national governments' competences, can overlap and even be superimposed without being perceived in conflict. changes in cognition facilitated the adoption of the gaia 2048 charter, as the global community finally found the "common language" 82 to tackle the "global governance paradox" successfully. the paradox was that, in order to create a global legal order, the world community needed a global governance platform that had not previously existed, confirming that linguistic and cognitive changes are quintessential to institutional change. structurally, the gaia 2048 was based on an institutional setting in which every "trade and . . ." problem was coordinated by a "coherence committee", with the competence to avoid conflict between different policy goals. the functioning of the institutional framework was aided by an e-governance system, which used intelligent algorithms to consolidate redundant norms and detect incoherent measures. however, artificial intelligence was considered not only to be hype but also to be an oxymoron, and, in relation to law and policy, artificial intelligence measures were strictly bound by the requirement inherited from the former eu general data protection regulation that they could not be implemented and enforced "without any human intervention". 83 78 brock and pogge (2014), pp. 1-15. 79 thang dao and edenhofer (2018), pp. 253-273. 80 koestler (1967 ), pp. 102-103. 81 neuwirth (2020 halpin and roeben (2009) in substantive terms, existing sources of global law were codified into a single legal document, the gaia code, a global constitutional text that reduced the previous disarray of international laws. in this way, former conflicts between norms that arose because of dichotomies that were too simplistic to account for real complexity, be it between electronic and traditional modes of consumption of goods (gatt) and services (gats), between ip law and competition law, or between international trade law and various subfields of general public international law in the name of "self-contained regimes", were no longer unavoidable and irresolvable. 84 in this context, global competition rules were integrated and enhanced so as to not only guarantee the sustainable economic development of the global market but also "solve social problems ranging from unemployment to income inequality and indeed to improve the functioning of democracy itself". 85 other necessary changes simply came with changes in cognition and understanding, and did not require a change in language as they occurred earlier when a higher court reconsidered its established case law. for instance, in the field of global economic law, the national treatment provisions of gatt article iii, gats article xvii and article 3 of trips were now no longer merely interpreted as prohibiting discrimination between domestic and imported goods or services but also as encompassing "all measures having an equivalent effect on consumers in covered markets". 86 this became necessary to cover personalized price discrimination made possible by the data economy. 87 these cognitive changes, plus a seamless global wireless web, also ended the western failure of the territorial national state. 88 as a result, the "four freedoms" related to the free movement of goods, services, persons and capital, became recognized as ubiquitous civil right and a global reality. 89 the various systemic changes resulting from cognitive changes were supported by, and helped to establish, an effective and inclusive global dispute settlement mechanism, which recognized the right to bring an action and to be heard in court (locus standi) of not only international organizations, states and multinational corporations but also natural persons. 90 it effectively aligned the former wto dispute settlement system with investor-state arbitration. 91 both systems were elevated to a "world trade court" embedded within a fully-fledged global judicial body, the gaia tribunal, that was vested with various constitutional powers to sanction and enforce the rulings it issued. 92 overall, the new institutional design based on these cognitive changes was said to have helped to "improve and eventually overcome the perceived lack of legitimacy of international courts and tribunals in the eyes of the governments, the legal community and civil society". 93 4 conclusion: 'dystopian utopia' or oxymora to predict the future by creating it my project was retarded by laws of nature. the world was not prepared for it. it was too far ahead of time. but the same laws will prevail in the end and make it a triumphal success. 94 a dystopian or a utopian future, does it matter? after all, from the "perspective of the brain, there's a thin line between a good decision and a bad decision". 95 it seems, though, that what matters to everyone is the future, because the future is "where we are all going to spend the rest of our lives". 96 however, perhaps this must also not be the case as, paradoxically, most (or all) dichotomies have a limitation in that they not only trade accuracy for simplicity, 97 but also provide an invisible barrier to a vision of the bigger picture. this is where subtle linguistic changes may gradually trigger cognitive changes and eventually bring in legal and institutional changes, in the same way as dripping water hollows out stone, even though the cognitive habits of binary or dualistic thinking are said to die hard. 98 easy or difficult, it is a source of encouragement for everyone, as we may, à la longue, be able to bring about the change we desire, given that institutional change is possible once a cognitive change has occurred. 99 the recent rise in the number of oxymora and paradoxes, however, seems to indicate this "creative" possibility. for instance, various concepts implicitly reflecting the tensions between prediction and destiny within the confines of past and present, such as science fiction 100 or free will, 101 have been qualified as oxymora. in their context (and possibly in the context of all paradoxes), time therefore appears as the clue, or the key we need to find to unlock the limitations inherent in our current linear perception of time. the fact is that the grammar of many modern languages, conjugating verbs in the "present," "past," and "future" tenses, was said to not be "well-adapted for speaking about the real temporal structure of reality, which is more complex". 102 various "nostalgic visionaries", 93 dimitropoulos (2018) lang (2007) , p. 529. 100 csicsery-ronay jr. (2008), p. 8 ("as its name implies, science fiction is an oxymoron."). 101 crewe (2008) , p. 23. 102 rovelli (2018), p. 111. like herbert g. wells or jules verne, however, transcended the past-future dichotomy in their writings, thereby anticipating many future inventions. 103 lawyers, too, can act in this way, as their work is often similar to that of science fiction authors, given that they can help in translating fiction into legal fact 104 or turning dreams into reality by combining "the law as it is" (lex lata) with "the law as it should be" (de lege ferenda) perspectives. 105 for lawyers to achieve this creative goal and to synthesize the two competing scenarios of the future beyond the wto outlined in this chapter, it will be necessary, first, to work actively towards a cognitive change. applied to legal reasoning, it means to transcend an exclusively dualistic mode of reasoning, which means to solely rely on the "law of the excluded middle" (i.e. "everything must either be or not be."). 106 applied to the regulation of global trade, this kind of "either/or thinking" or that something either belongs to the sphere of trade or is classified as a "non-trade concern" must be complemented by the law of the included middle. the law of the included middle can be achieved through oxymoronic thinking by way of which, for instance, trade and non-trade concerns are not opposed to each other but where their apparent contradiction can be resolved at a higher level of reality or complexity. 107 more concretely, lawyers must be able cognitively to transcend the iron law of binary logic by also being versatile in reasoning based on polyvalent logic-a kind of multivalued logic in which there can be more than two truth values. 108 a good way to remind ourselves of this is given in the following quote: "laws may differ but they do not conflict: the only possible conflict is in the mind of the judge". 109 in this regard, legal education seems the best place to begin. 110 thus, addressing problems by legally expressing them solely using dichotomies is no longer apt to deal with their underlying complexity. it also means that a purely static approach should be complemented with a more dynamic one to ensure that laws are adopted in a way that embraces change ex ante and makes them fit for the future. in institutional terms, a more dynamic approach is also needed, as otherwise we merely observe their initial rise and subsequent demise. 111 in this respect, discursive institutionalism provides an excellent complement, as it regards norms as "dynamic, intersubjective constructs rather than static structures". 112 ultimately, it is submitted here that, based on this cognitive change, novel models of global institutions, like gaia 2048, can emerge. second, the cognitive changes of polyvalent or oxymoronic thinking, when combined with new organs of perception as symbolized by synaesthesia, will enable another important insight, which lies in the realization that the best way to predict the future is by creating (and regulating) it. this insight puts the theory of a selffulfilling prophecy into a new light, one which proves that theory and practice are intimately linked and that sociological predictions are "products of an era, co-determinants of what they assert". 113 when applied to humanity as a whole, predicting the future by creating it (and regulating it accordingly) means that if we plan something and act upon it coherently and persistently, it is more likely to happen, eventually. this, however, first requires a cognitive change in the understanding of humanity as a divided amalgam of different peoples or nations instead of an organic whole united in its diversity. only then can an adequate global governance mechanism capable of putting the necessary global legal order into place be expected to be brought forth. in short, the global governance paradox and other apparent contradictions can only be successfully addressed by expanding our reasoning from bivalent to polyvalent modes of thinking, as reflected in synaesthesia or in new optimized ways to receive and channel information through our different senses or, in legal terms, different institutions. applied to the dichotomy of the past versus the future, a new cognitive mode may one day enable humans to recall the fundamental importance of the present, that is, the magical power of the present to rewrite both the world of yesterday and the world of tomorrow. by the same token, humans can then eventually use the same magic to control the outcome between the two apparently opposite scenarios, the dystopian and the utopian one. for now, the two scenarios outlined in this chapter cannot answer the questions of whether or not, in our perception, linear time exists, or whether or not we have free will or all fate (including the fate of the wto) is already written in a comprehensive book of destiny. this chapter is equally unable to say whether a post-wto legal order will be able to avoid global disaster and humanity's as well as nature's demise. however, the hope is that the chapter shows that the many "scientifically fictitious" developments described in both the dystopian and the utopian scenarios concur in essence or are-if at all-divided by a very thin line, one merely drawn in our minds. nevertheless, this thin line determines how we will deal with the most urgent challenges in the anthropocene, or at any given point in time (when no longer understood as the linear process from the past to the future), because it clearly proves that today we humans cannot say that "we did not know (about the dangers and inherent consequences of our actions)". on the contrary, we can only say "we knew but did not care". 113 neurath (1973), pp. 405-406. g20 (2019) ministerial statement on trade and digital economy should robots pay taxes? tax policy in the age of automation failing states, collapsing systems: biophysical triggers of political violence cryptocurreny & robots: how to tax and pay tax on them artificial intelligence and legal analytics: new tools for law practice in the digital age groping toward grotius: the wto and the international rule of law united nations management -an oxymoron? netherlands interdisciplinary demographic institute, the hague berchin ii et al (2017) climate change and forced migrations: an effort towards recognizing climate refugees logic in reality global tax justice and global justice the governance of data and data flows in trade agreements: the pitfalls of legal adaptation boulder cosbey a, mavroidis pc (2014) a turquoise mess: green subsidies, blue industrial policy and renewable energy: the case for redrafting the subsidies agreement of the wto the common law of international trade and the future of the world trade organization will to self-consummation, and will to crime: a study in criminal motivation the seven beauties of science fiction degrowth: a vocabulary for a new era which future for competition in the global trade system: competition chapters in ftas are we all born synaesthetic? examining the neonatal synaesthesia hypothesis international relations and international law: from competition to complementarity the doha declaration and beyond: giving a voice to non-trade concerns within the wto trade regime investor-state dispute settlement reform and theory of institutional design global energy governance in the g-20: states, coalitions, and crises nuclear energy and sustainability: understanding iter belief in life-after-death, beliefs about the world, and psychiatric symptoms legal fictions systemic regulation of global trade and finance: a tale of two systems how to save a constitutional democracy choice of logic and choice of law policy coherence or conformance? the new world bank-international monetary fund-world trade organization rhetoric on trade and investment in developing countries energy and the wealth of nations: an introduction to biophysical economics theorising the global legal order homo deus: a brief history of tomorrow the age of transition: trajectory of the world-system the evolution of official development assistance: achievements, criticisms and a way forward world trade and the law of gatt. the bobbs merril company irresolvable norm conflicts in international law: the concept of a legal dilemma the culture of time and space 1880-1918 the ghost in the machine fuzzy thinking: the new science of fuzzy logic reflecting on 'linkage': cognitive and institutional change in the international trading system can the trading system survive us-china trade friction? convergence of wto dispute settlement and investor-state arbitration: a closer look at umbrella clauses 2020) the appellate body of the wto and its reform auras in mysticism and synaesthesia: a comparison dead aid: why aid is not working and how there is another way for africa oil's tipping point has passed the civil right we are not ready for: the right of free movement of people on the face of the earth global market integration and the creative economy: the paradox of industry convergence and regulatory divergence global law and sustainable development: change and the "developing-developed country" terminology law in the time of oxymora: a synaesthesia of language, logic and law. routledge on the origin of legal diversity by means of comparative law, or the role of legal education in the solution of legal conflicts international development assistance, china and the brics the european union as an oxymoron: from contest via contradiction to constitution? in: chaisse j (ed) 60 years of european integration: perceptions, interactions and lessons law as a social medicine: enhancing international inter-regime regulatory coopetition as a means for the establishment of a global health governance framework the economic sanctions over the ukraine conflict and the wto: "catch xxi" and the revival of the debate on security exceptions the brics-lawyers' guide to brics texts and materials. the brics-lawyer 2017) the brics-lawyers' guide to global cooperation nuclear fusion and its large potential for the future world energy supply african crop production trends are insufficient to guarantee food security in the sub-saharan region by 2050 owing to persistent poverty the black box society: the secret algorithms that control money and information the sutherland report: a missed opportunity for genuine debate on trade, globalization and reforming the wto the role of the internet of things in network resilience predicting the future: an introduction to the theory of forecasting climate change-induced migration and violent conflict currency wars: the making of the next global crisis (1912) the problems of philosophy discursive institutionalism: the explanatory power of ideas and discourse poverty of stimulus arguments concerning language and folk psychology a distinction without a difference: exploring the boundary between goods and services in the world trade organization and the european union currency manipulation' and world trade the westfailure system blockchain revolution: how the technology behind bitcoin and other cryptocurrencies is changing the world. penguin, new york ten hoopen g et al (1995) auditory isochrony: time shrinking and temporal patterns my inventions and other writings on the fiscal strategies of escaping poverty-environment traps towards sustainable growth balance-of-payments crises in the developing world: balancing trade, finance and development in the new economic order a proposal for international monetary reform big data and personalized price discrimination in eu competition law technology and progress in jules verne, or anticipation in reverse a propos de la lex ferenda the human rights of stateless persons felt time: the psychology of how we perceive time requiem for a paradox: the dubious rise and inevitable fall of hipster antitrust currency wars: offense and defense through systemic thinking a hater's guide to geoblocking key: cord-027700-dezg4155 authors: chehab, khalil; kalboussi, anis; hadj kacem, ahmed title: study of healthcare professionals’ interaction in the patient records based on annotations date: 2020-05-31 journal: the impact of digital technologies on public health in developed and developing countries doi: 10.1007/978-3-030-51517-1_28 sha: doc_id: 27700 cord_uid: dezg4155 the annotation practice is an almost daily activity; it is used by healthcare professionals (phc) to analyze, collaborate, share knowledge and communicate, between them, information present in the healthcare record of patients. these annotations are created in a healthcare cycle that consists of: diagnosis, treatment, advice, follow-up and observation. due to an exponential increase in the number of medical annotation systems that are used by different categories of health professionals, we are faced with a problem of lack of organization of medical annotation systems developed on the basis of formal criteria. as a result, we have a fragmented image of these annotations tools which make the mission of choice of an annotation system by a phc, in a well-defined context (biology, radiology…) and according to their needs to the functionalities offered by these tools, are difficult. in this article we present a classification of thirty annotation tools developed by industry and academia based on 5 generic criteria. we conclude this survey paper with model proposition. the paper annotation practice is very common. indeed, during our reading we are all accustomed to scribble our comments in a margin of document, to highlight, to circle sections, to paste post-it…, which aims to enrich and add value to information [40, 43] . annotation is a central practice in many professions: teachers annotate copies of students; professors exchange annotated documents during their work; engineers co build engines by annotating sketches of plans to make them evolve, doctors comment on patient folder, etc. [39, 44] . annotations thus, take various forms and are used for different functions [28] . moreover, computerization of documents offers us new perspectives to use these annotations (indexation, creation, document, assistance, etc.) which do not exist on paper [41] . assistance is a very important function that can be related to the annotation activity. annotative activity is different when dealing with the professional annotator case. these annotations are created in a specific context, will follow a path, developed for the purpose of determining a specific task etc. the annotation is expanded in a document flow. the latter is a carrier of the ratings. these annotations processed in this context are complementary. they provide us with information scattered over different documents. in fact, if we try to understand each one separately, in other words; if we separate an annotation from its creative context, we find that it does not make sense. in short, the annotation can be understood only in its semantic field. for this reason, the study of annotation in a professional context obliges us to implement an annotation model that describes what actually happens. this model must provide us with the necessary links either at the level of the annotated documents or at the level of the tasks made without forgetting to take into account the specificity of the annotation's production domain. the documents that usually belong to a folder that is made for the purpose of carrying out a task has a definite circuit that repeats itself each time one needs to do this task. the proper understanding of annotations can be done in its creative context. doctors in/after consultation [45] use internet to search information that can help him. based on their annotations it's possible to assist doctors and to gives him automatically pertinent information, from the net, after studying and analyzing their annotations. this paper is organized as follows: sect. 2 gives a classification of these tools based on several criteria; sect. 3 gives a model proposition. finally, sect. 4 concludes this article. in this work, we started with an exhaustive reading for the available papers on medical annotation systems (academic annotation system) and viewing the existing industrial annotation systems in the e-health domain. although the medical annotation systems have already been studied in a variety of contexts, yet when it comes to the phc to choose which system to use it is not a trivial task neither for a researcher to identify future research areas. this is because the annotation systems are so common and many of them share similar objectives. moreover, there are no formal criteria to facilitate the comparison between those systems and to guide phc choice or a researcher. as a result, there is a fragmented picture of these annotation tools. as far as we know, this is the first work to consider the classification of medical annotation system. when we determined the study of these systems we deduced that there are several common criteria which can classify these later. several studies have proposed classifications of annotation systems in several fields [34, 35, 37] or in the field of e-health [42, 43] . we propose a classification based on 5 criteria which are: type of medical annotation object, the medical annotation activity, healthcare professional (practitioner), type of annotation system, type of annotated resource. • cognitive: this annotation is created to be used by a human agent. in this case, the annotation requires a cognitive and intellectual effort to be interpreted. this annotation has a visible visual form on the document [34] . • computational: this annotation is intended to be processed and manipulated by software agents. these annotations are also called meta-data. they allow us to annotate computer resources to facilitate their exploitation by machines. annotation activity begins with the choice of anchor and annotation form in the annotation toolbar related to the annotation software. then, the annotation must complete the properties of the annotation; this process ends with the attachment of the annotation to a well-defined target. based on this process we can classify the annotative activity as: manual, semi-automatic or automatic [38] . • manual: the process already mentioned will be carried out totally by the user himself, who selects the form of the annotation, selects the anchor and creates the annotation. this process is similar to the process of annotation when a paper support is available. • automatic: the annotation process already mentioned is carried out totally by the machine. these annotations are based on either context sensors or pattern recognition techniques, etc. • semi-automatic: in this case, the process will be done from the start by the user. after a while, the system acquires and understands the way the user annotates. it moves to a suggestion of annotations that are automated, based on an annotation model built with rules under development. at this stage, human intervention remains just to validate or not validate and to refine the annotation rules created at a certain level, where there are no corrections and there is complete acceptance of the suggested rules, human intervention is canceled and the process becomes totally automated. it is the annotator that is equipped with an annotation system to use all the functionalities offered by the latter. in our case, the practitioners are healthcare professionals (doctor, nurse, biologist, and radiologist). the healthcare cycle is composed of four phases (diagnostic, treatment, advice, follow up and observation). each practitioner, with a medical annotation system, intervenes in one or many phases, according to their role, to accomplish a specific task in which annotation is made. • application: an application is created to annotate the resources already consulted. these applications offer several functionalities as the types below. • plug-in: these are the expansion modules, an external module that is added to a website or software and which will make it possible to provide annotation functionalities to the latter. • website: these are specialized websites to annotate consulted resources by registered users on the web. knowing the type of annotation system facilitates the development of a patient record model which will be proposed in future research. this model allows communicating with different types of medicals annotations systems. annotated resources can be: word document, pdf, image, text, video, html, audio, etc. table 1 presents a comparative study of the medical annotation systems seen in the bibliographical study using the 5 criteria already explained. table 1 presents a comparative study of thirty medical annotation systems seen in the bibliographical study using the 5 criteria already explained. these annotation systems are ranged on the table according of the chronological order of their publication year. several models are already seen in the literature [33, 35, 36] and [ 37] , these models present many problems, like the inexistence of the modeling of the cycle of care, the consideration of the annotation as an objective, no invocation of the services linked to the annotation, that not allows to use it in the healthcare domain. for this reason and based on the classifications already seen (sect. 2) we propose an annotation model that preserve the semantic of annotative activity in the health domain. our model must reflect the annotation process actually done; it must contain the following concepts: • basic_concept: this group contains the concepts that can exist in each domain. -place: this is the physical place where the annotation is produced by the annotator. -document: it's the annotated document. -part_document: professional documents are usually divided into parts. -element: each part_document consists of a set of elements. -part_element: it is the smallest granularity of document; it is a word, letter etc. in short, it is the annotated passage. • advice: patient education: how to live with this disease, how to act in case of emergency, how to take their drugs… • follow up and observation: this is the last step of the care cycle in which the doctor follows his patient until the stabilization of his condition. in this step, it can also make a strengthening of advice…. • service aspect: this aspect allows us to clarify the semantic of the annotation. it is used to interpret the meaning of the annotation by the annotator himself or by the software agents. -effect: the effect of annotation is the result of web service called from this annotation. the relationships between the concepts of ontology are described through the data model presented in fig. 1 as follows. there are only three type of cardinalities used in this model 1..*, 1 and *. an annotation is created by the phc with a device, at a date and in a place. this annotation is presented by a particular shape. it is pointed to an anchor and related to an annotated content that can be related to another annotated content. this annotated content is a part of the read document. the annotation contains an annotating content that can be related to others annotating contents. an annotating content can have a type. an annotation has an annotated content that is alone, and at the base of this content, annotating content can be created for one or more professionals since this content can be written differently for each destination. each domain has some specific concept. an annotation is related to specifics concept. the annotator, which is a phc, is identified by a name. the place is a location in a medical department especially in a specific service. each service has its own phcs that are classified into categories (medical, paramedic). in the category a phc can have a role and each role is related to a specific authority. a document is an element of a document package. a document package consists of the already known set of documents grouped together to perform a specific task. a document decomposed to parts, each one of it contains a set of elements (text box, radio button…). the annotated content can be an element or a part of an element (word, sentence…). a professional can have a power to launch tasks (medical_care). each healthcare cycle is divided into healthcare phases (diagnostic -treatment -advice -follow up and observation); the latter are linked together so we can fuse them once again when they are completed and we can follow the annotations of a healthcare cycle at the level of its phase. each healthcare phase can be divided itself. a healthcare cycle can also be linked to other healthcare cycle that has a relationship between them. in this paper, we studied annotation systems of the digital health domain available in industrial and research areas in order to propose a unified classification of this kind of system that is omnipresent in hospital information systems. this panoramic view provided is based on the classification of thirty different annotation systems developed in the literature over the past two decades. this organization of annotation tools is built on the basis of five criteria: type of annotation (computational/cognitive); category of annotation system (application/plug-in/website); type of annotative activity (manual/ semi-automatic/automatic); type of annotated resource (text/web page/video/image/ database) and practitioner (biologist/doctor/radiologist/nurse, etc.). this classification based on criteria, already explained in our study, which are transversal organizational criteria, facilitates the identification of limitations and possible challenges in the area of the medical annotation systems. based on this, we proposed an ontology that covers the identified challenges and lead to a more intelligent annotation system. in future research, we try to use the results of this study to create an annotation template for phcs and then try to generalize them to be functional for all professionals in different domains. we are also trying to create the computer services which allow the phc to be assisted throughout the care cycle. open access this chapter is licensed under the terms of the creative commons attribution 4.0 international license (http://creativecommons.org/licenses/by/4.0/), which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the creative commons license and indicate if changes were made. the images or other third party material in this chapter are included in the chapter's creative commons license, unless indicated otherwise in a credit line to the material. if material is not included in the chapter's creative commons license and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. interactive genomic visualization for r/bioconductor odm summary: a tool for automatic structured comparison of multiple medical forms based on semantic annotation with the unified medical language system 3dbionotes v2. 0: a web server for the automatic annotation of macromolecular structures verdant: automated annotation, alignment and phylogenetic analysis of whole chloroplast genomes gidac: a prototype for bioimages annotation and clinical data integration vcf-miner: gui-based application for mining variants and annotations stored in vcf files the biodigital human: a web-based 3d platform for medical visualization and education equipement informatique des annotations et des pratiques d'écriture professionnelles: une étude ancrée pour l'organisation des soins en cancérologie (doctoral dissertation heideltime standalone manual version micropublications: a semantic model for claims, evidence, arguments and annotations in biomedical communications the biological reference repository (bior): a rapid and flexible system for genomics annotation 3d markup of radiological images in epad, a web-based image annotation tool visual interpretation with three-dimensional annotations (vita): three-dimensional image interpretation tool for radiological reporting marky: a lightweight web tracking tool for document annotation medetect: a lod-based system for collective entity annotation in biomedicine the flersa tool: adding semantics to a web content management system smitag: a social network for semantic annotation of medical images mammoapplet: an interactive java applet tool for manual annotation in medical imaging brat: a webbased tool for nlp-assisted text annotation idash: integrating data for analysis, anonymization, and sharing arthemis: annotation software in an integrated capturing and analysis system for colonoscopy les annotations pour supporter la collaboration dans le dossier patient électronique an interactive annotation system to support the learner with web services assistance a formal model of learner's annotations dedicated to web services invocation hospitexte: towards a document-based hypertextual electronic medical record annotative activity as a potential source of web service invocation modèles et outils d'annotations pour une mémoire personnelle de l'enseignant (doctoral dissertation les topic maps sont-ils un bon candidat pour l'ingénierie du web sémantique? comparative study of web annotation systems used by learners to enhance educational practices: features and services quels sites internet utilisent les médecins généralistes en consultation? enquête de pratique auprès de 100 médecins généralistes interoperability in healthcare: benefits, challenges and resolutions towards semantic interoperability in healthcare: ontology mapping from snomed-ct to hl7 version 3 towards data interoperability: practical issues in terminology implementation and mapping study of annotations in e-health domain an annotation model for patient record. health inf modèles et outils logiciels pour l'annotation sémantiquede documentspédagogiques (doctoral dissertation les topic maps sont-ils un bon candidat pour l'ingénierie du web sémantique modèles et outils d'annotations pour une mémoire personnelle de l'enseignant (doctoral dissertation dublin core metadata initiative. dublin core metadata element set, version annotea: an open rdf infrastructure for shared web annotations key: cord-020130-g9p5lgmn authors: ratshidi, lilies; grobbelaar, sara; botha, adele title: categorization of factors influencing community health workers from a socio-technical systems perspective date: 2020-03-10 journal: responsible design, implementation and use of information and communication technology doi: 10.1007/978-3-030-45002-1_8 sha: doc_id: 20130 cord_uid: g9p5lgmn in low-and-middle-income countries (lmics), community health workers (chws) are often seen as a connecting bridge between two dynamic and overlapping systemsthe community and formal health systems. although the importance of chws is acknowledged, there is minimal aggregated evidence contributing towards understanding their position, technological capabilities, barriers and facilitators of their effectiveness in the south african context. despite the widespread enthusiasm around the potential that mobile health (mhealth) technology holds in extending healthcare through chw to underserved communities, an understanding of mhealth’s various implications in a developing world context is imperative to appreciate both the community and health systems context. the chws within this context need to assume multiple roles as they work and live amongst and in the community. the study argues that by examining their multiple roles as part of the healthcare continuum and from within the community setting, appropriating technological solutions can be conceptualized to facilitate and enhance their impact and visibility. this research article then aims to articulate the key conceptual factors which should be considered when implementing technological solutions for chws within the south african context. the aim is operationalized by means of the best-fit framework synthesis method to explore the body of knowledge towards presenting a conceptual understanding through a categorization of factors influencing community health workers from a socio-technical systems perspective. the constitution of south africa enshrines the provision of healthcare access as a basic human right for all its citizens. however, the south african healthcare system is fraught with challenges, some of the major ones include the inadequacy of human and equipment resources [1] , difficulties in synergizing and collaborating policies and lack of legislative commitment to improving the public health sector [2] . according to the world health organization, the shortage of healthcare workers is a major challenge in a country's ability to overhaul its healthcare system and ultimately, achieve universal healthcare coverage [2] . given south africa's limited resources, the need for costeffective strategies is paramount. recent studies [3, 4] identify an adequately competent workforce with multi-faceted roles as having the potential to relieve some of the healthcare system burdens by bridging the healthcare equity gap. several authors [1, 2, 4] have suggested that chws as a workforce could be beneficial in the south african context. in addition, it is noted that chws are considered a dependable vehicle to provide quality contextual health services both in urban and rural settings within the south african context [5] . the south african government has introduced various initiatives to address the historic disparate healthcare system [1] . one such initiative is the national development plan for 2030 that involves building human resources to ensure shared competencies for the health system as part of its plan of action. part of this plan includes a goal of employing and training between 700 000 and 1.3 million chws to implement community-based primary healthcare [5] . regardless of the effort needed to reach this goal quantitively, it can be argued that the potential benefits of chws have not to be realized. this can be attributed to a misalignment in national policies and standards to their work practices, and the significant barriers in training chws to function at the expected level of competence [2, 6] . with the widespread use of mobile technology in sub saharan africa over the past years, there is an estimated increase to 40% of mobile internet penetration [7] . there is mounting evidence that suggests the use of mobile health has the potential to enable chws to mitigate some of the challenges faced [7] . consequently, the utilization of technology as a viable solution for chws has steadily gained significant popularity. despite this, various authors' calls for research specifically focused on investigating mhealth implementation for chws in low-and-medium-income countries (lmics). the definition of lmics adopted was according to the world bank classification with the study focusing more on the sub-saharan region [8] . winters et al. [9] articulate this as a call for more robust evidence on mobile technology implementation strategies as a means of supporting chw practices [9] . granja et al. [10] suggest the successful implementation of technological solutions interventions can be improved through the identification of factors that influences the intervention's outcomes. from these insights, it can be inferred that an in-depth understanding of the healthcare domain and the processes of technology adoption and use by chw are a needed step towards achieving the full potential of mhealth. in this regard, various studies have identified factors influencing chws. these factors include their perceived performance, motivation and job satisfaction [6, 10] . however, verification of how these factors affect the implementation and evaluation of technological solutions for lmics has not sufficiently been documented. this study aims to articulate the key conceptual factors which should be considered when implementing technological solutions for chws within the south african context. the paper outline is as follows: method, descriptive statistics of the results, discussion and construction of the framework, and the conclusion. the study is grounded in social and technical perspectives as it facilitates the duality of the chws' work and community role, further adopts the technique of the "best-fit" framework synthesis method in the exploration. the best-fit framework synthesis is defined in [12] as "a means to test, reinforce and build on an existing model, conceived for a potentially different but relevant population". this method involves creating or employing a framework with priori themes and using it to code the data obtained from the relevant studies as a means to produce a rapid and pragmatic form of synthesis [12] . it advises the use of criteria; one for identifying the models and theories to generate a priori framework, and one for populating the scoping review of primary qualitative research studies. in this study, only one set of the literature search and a study selection was considered for the scoping review as the socio-technical system (sts) framework was used as a priori framework. figure 1 illustrates the methodology approach applied. davis et al. [13] describes the sts framework as a system which considers the people involved with distinct social behaviors and skills, working within a physical infrastructure, using a range of technologies and tools to achieve a set of goals and metrics by following sets of processes and practices under a set of cultural assumptions and norms [12, 13] . sts is defined as an approach to complex work design consisting of technical systems; social systems with an interplay of human agents employing social dependencies that either hold or emerge between them; and finally, organizations that are heterogeneous within unpredictable operational environments, which are autonomous and poorly controllable [14] . the sts theory premises on the combination of social and technical aspects to design a functional work system that can cope with the complexities of the environment within which the system operates in, as well as the dynamics introduced by new technological interventions [15] . hence, to account for the delicate dynamic relationships within the chws' work system, the sts framework was used to diagnose, identify and categorize the literature into the factors and interactions between the social and technical elements, and a summary of the study characteristics was transferred to excel for further synthesis, where they were categorized as either technical or social to generate key inferences regarding the factors which should be considered. the six interrelated elements used are people, infrastructures, goals, technologies, culture, and processes embedded within an external environment [13] . having overviewed the methodology approach the following section outlines the search strategy employed. in [13] , six interrelated elements were presented in the conceptualization of an sts and were used to evaluate the initiatives documented in the literature, from which relevant factors related to each element were identified. a broad literature search was conducted on scopus, google, research gate, and google scholar to identify studies related to chw initiatives and technology implementation previously conducted in lmics. the keywords used for the search were: chws, framework, technology, healthcare innovation ecosystem, social factors, technical factors, socio-technical systems approach in healthcare, and lmics. figure 2 illustrates how the selected inclusion criteria were applied to identify the relevant articles. an iterative process facilitated the addition and removal of studies that were not explicitly addressed by the inclusion criteria. the data extraction process involved recording the full-text articles into publication year, region setting, study type, methodology and key findings after which the six elements were used to identify the relevant factors from the selected literature and categorize them under social or technical perspective [13] . of the 20 articles included for full-text analysis, most of them used qualitative and mixed methods inquiries involving interviews, focus group discussions with chws, healthcare systems stakeholders from government and non-governmental initiatives. the studies were conducted in lmics including south africa, uganda, ethiopia, kenya, mozambique, india, and zimbabwe. the type of publications resulted in 10 articles on reviews, 6 articles on empirical and 4 on analysis studies. the conceptualization of an sts presented in [13] was adapted and applied to the categorization in this study. the following discussion reflects on the analysis and synthesis deduced from the identified literature studies and used to categorize the relevant factors. a study by naimoli et al. [16] posit that the health outcomes achieved through chws programs are a function of a robust, high performing health and community sectoral systems. the study further postulates that the programming activities categorized under social, technical and incentives support functions are influenced by a range of contextual factors in both community and health sectoral systems [16] . however, the narrative presented in the previous studies alludes there is unbalanced attention on the impact of the complex and diverse context-specific nature within which chws work and live in [17] . as a result, de neve et al. [17] propose the need for countries to develop coherent and context-specific approaches to ensure optimal performance by chws through the consideration of the broader context, including demographic, socioeconomic, political, legislative, ecological, sociocultural, and technological factors contributing towards facilitating or inhibiting the success of many chws initiatives [18, 19] . some of the solutions to achieve what is postulated in the studies include coordinating the health system and community system to prioritize factors that inhibit or facilitate the understanding of chws programs' compatibility with community structures, cultural values, and perception, socio-economic context and support system [20] . in addition, integrating and adopting interventions supported by technological solutions, and the sustainability of these interventions should be considered when exploring efforts until the desired health outcomes are achieved to gain a better understanding of chws programs and their roles in lmics [17] . previous research that was focused on chws and their performance placed emphasis on developing frameworks that provide a broad context of the chw's position in a larger environment by describing the interrelations of intrapersonal, family, community, and organizational settings as health professionals [6, 10] . this perspective to a larger extent provides a limited understanding of the impact of the ecological environment on chws [20] . most programs have not been able to effectively address the gap between research evidence and the routine practicality of chws as health professions, hence the poor integration of chws within the healthcare system and an even poorer understanding of their roles within their communities. subsequently, the implication of this postulates the need for a comprehensive approach to plan and design programs that can be integrated with the formal healthcare system's approach to healthcare service delivery through chws roles and organizations [17] . moreover, the chw system requires an interface with the formal healthcare and the community systems involving the political structures, civic groups, faith-based organizations. schneider and lehmann [21] argue that integrating chws into the primary healthcare systems while embedding and supporting them through the community is vital to realize their potential. contrarily, most studies emphasize the need for chws to be integrated within the formal healthcare system whilst placing minimal emphasis on understanding how they are embedded within the community system [21] . moreover, minimal work has been done locally in terms of implementing universal guidelines to guide the integration strategies required to resolve the above-stated implications. previous research has proved that the effectiveness of chws holds the potential to increase access to equitable health in lmics [20] . as a result, chws' understanding of the socio-cultural norms of their communities, their unique intermediary position between communities and the health system places them in a central setting in delivering key health interventions. on an individual level, their effectiveness is influenced by contextual factors, such as socio-cultural factors, gender, traditions and norms, training and supervision, health policies combined with intervention-related factors [22] . furthermore, it is postulated in [16] that support for chws has to be strategic, collaborative and well-coordinated to enhance chws performance between the two overlapping dynamic systems they are expected to function within. among the key challenges presented in the study, the definition and optimization of the impact of the chws' roles are highlighted as an influence on chw performance. also, [3] argues that government and non-governmental institutions are continuously adding functions and tasks to chws, which buttresses the need to inform the type of tasks and position they hold within the healthcare system through competency-based or educational qualification rather than on functionalities. in most lmics such as south africa, the roles and responsibilities of chws with regards to technical and social capital is limited and yet to be understood [11] . khalala et al. [23] state that understanding the nature of the work chws do, enables researchers to explore the relevant technologies that can be exploited to facilitate and support their daily work. this understanding potentially provides information with regards to the choice of technology and how it can be implemented to support the roles of chws. regardless of the evidence on the social factors influencing chws, the exact mechanisms on how to assess the interdependencies of social and technical dynamics' influence on chws outcomes remains understudied. technologies are not neutral or passive objects but rather shape the environment and provoke social dynamics as a result of their existence and necessity for human survival. previous studies reveal that technologies have the potential to influence social, cultural and economic contexts and improve healthcare quality for communities when employed in the healthcare domain [24] . expectedly, the use of mobile technology in south africa in the healthcare system has also increased, particularly among chws [25] . despite chws having limited formal education and training, with poorly defined roles in using technologies within their line of work, it has been emphasized in [4] that most research has focused on the usability and reliability of technologies with minimal emphasis on the users and the important aspect that they are social beings who interact with their immediate and remote communities. iluyemi et al. [26] state that to conceptualize technological solutions and their policy interventions, it is important to start by gaining an understanding of the context of use and needs for the technology from the chws' perspective. in addition, the challenges of technology's usability and supportive structures. existing literature have focused on the reliability, functionality and infrastructure of the technology while paying minimal attention to the end-users perception on the usability and intentions to use the technology, as a result most of the research focus on the technical characteristics whilst neglecting the impact of the social characteristics of both the individual and community setting where the technology is utilized [27] . nonetheless, this implies there is a need to conceptualize the appropriate technologies which can both fit the task they are used to as well as have the capabilities to perform the task. moreover, technology and policy interventions are deemed as necessary developments to enhance technological effectiveness and efficiency, and to ensure sustainability and scalability through the initiatives whilst amplifying their impact [28] . thus, the technology acceptance dimensions are considered in describing the understanding of the technological capabilities of chws, their ability to access and utilize the appropriate technologies applicable to their social backgrounds. there is undeniable evidence from previous studies about the importance of considering the sociotechnical determinants in developing implementation and evaluation conceptual models for technological solutions. determinants including technological appropriateness and socio-cultural sensitivity, political infrastructures, the technology endusers' attributes and variables of ecological settings [29] . in a formative study about the adoption and usage of mhealth by chws in india, kaphle et al. [20] hypothesized that individual characteristics of end-users such as education, experience of care, and demographics hold the potential to influence the uptake of technological adoptions and quality of care. in addition, kim et al. [30] reinforces that the attitude of health professions to use technology influences the behavioral intention to use it. as a result, the relationship between technology and chws' performance is associated with their readiness to align their behavioral intention to use the technology. important to note, technological solutions and processes are not autonomous vehicles, but rather are embedded within systems in a social world where they are used to perform activities which have consequences and influences changes on human behavior, social constructs, and cultural meanings. thus, the effect of this is that not only does the interaction between the technical and social systems exhibit complexity and unpredictable behavior; it inevitably increases the complexities of dynamic and autonomous relationships, which can be detrimental to the system in place. from the above sections, the following is presented in summary to illustrate the factors identified in the discussion using the sts hexagon for illustration as provided in [13] . figure 3 illustrates the social and technical perspectives identified from previous literature according to [13] . from the discussion above, the factors (see fig. 3 ) were categorized into concepts according to their similarities to allow for a reasonable number of concepts to be considered. the approach followed in this study desired that the design activities involve gaining understanding and specification of the context in which the system will be used by referring to the social factors, cultural factors, working practices factors, and the structure of the organization [31] . thus, the terminologies for the concepts were adapted from this standpoint and were cautiously selected by the author through interpretation about the relevant literature as a means to preserve the relevance of the factors. the concepts were grouped under technical and social context depending on the interpretation of the author, from there, they were categorized under the two social dimensions: health and community systems context depending on whether the influence of the factor on the chws was related and reflective of the context in question. this study considers the following fundamental areas under the social system: the individual's needs, humans' social behaviors in work systems, internal and external environmental factors and support systems of the work system under investigation [15] . whilst the technical system focuses on the processes, tasks, infrastructures, and technologies required and used in the work system to achieve the set goals [14] . additionally, the technical dimensions included technology readiness which was concerned with categorizing the factors related to the technological solutions characteristics and compatibility to be used. the second dimension was technology acceptance and utilization which was concerned with factors concerning the chws perspectives and readiness to use and accept the technological solution within the environment they work. finally, the compatibility/fit element was considered as a means to provide the measures required to ensure a balanced joint optimization of both the technical and social aspects [31] . figure 4 illustrates the concepts classed under social and technical context. this research article aimed to articulate the key conceptual factors which should be considered when implementing technological solutions for chws within the south african context. the sts framework was used for the categorization of the factors which were integrated into concepts. for future work, each of these concepts will be evaluated and the categorization refined to present a broader conceptual framework. this study forms part of a dissertation project and the findings in this study form part of the first high-level conceptual framework which will be used in conducting scoping literature review to identify additional components for the secondary-level conceptual framework. in addition, future plans involve exploring the task-technology fit model to identify the relevant technological solutions fit for peri-urban and rural contexts in south africa. challenges of quality improvement in the healthcare of south africa post-apartheid: a critical review community health workers lensed through a south african backdrop of two peri-urban communities in kwazulu-natal who is a community health worker? -a systematic review of definitions use of mobile health (mhealth) technologies and interventions among community health workers globally: a scoping review perceived mhealth barriers and benefits for home-based hiv testing and counseling and other care: qualitative findings from health officials, community health workers, and persons living with hiv in south africa affecting factors on the performance of community health workers in iran's rural areas: a review article systematic review on what works, what does not work and why of implementation of mobile health (mhealth) projects in africa world bank country and lending groups -world bank data help desk using mobile technologies to support the training of community health workers in low-income and middle-income countries: mapping the evidence factors determining the success and failure of ehealth interventions: systematic review of the literature empowering community health workers with technology solutions best fit" framework synthesis: refining the method advancing sociotechnical systems thinking: a call for bravery adaptive socio-technical systems: a requirementsbased approach what encourages community health workers to use mobile technologies for health interventions? emerging lessons from rural rwanda strategic partnering to improve community health worker programming and performance: features of a community-health system integrated approach harmonizing community-based health worker programs for hiv: a narrative review and analytic framework factors associated with community health worker performance differ by task in a multitasked setting in rural zimbabwe which intervention design factors influence performance of community health workers in low-and middle-income countries? a systematic review adoption and usage of mhealth technology on quality and experience of care provided by frontline workers: observations from rural india from community health workers to community health systems: time to widen the horizon? community health workers in rural india: analysing the opportunities and challenges accredited social health activists (ashas) face in realising their multiple roles the roles and needs of community health workers in developing countries: an exploratory case study in south africa technology enabled health" -insights from twitter analytics with a socio-technical perspective adapting mhealth to workflow -a case study in south africa mobile information system, health work and community health workers in less developed countries scoping review assessing the evidence used to support the adoption of mobile health (mhealth) technologies for the education and training of community health workers (chws) in low-income and middle-income countries m-health adoption by healthcare professionals: a systematic review enablers and inhibitors: a review of the situation regarding mhealth adoption in low-and middle-income countries analysis of the factors influencing healthcare professionals' adoption of mobile electronic medical record (emr) using the unified theory of acceptance and use of technology (utaut) in a tertiary hospital socio-technical systems: from design methods to systems engineering key: cord-134378-p83nhiz9 authors: setlur, vidya; kumar, arathi title: sentifiers: interpreting vague intent modifiers in visual analysis using word co-occurrence and sentiment analysis date: 2020-09-26 journal: nan doi: nan sha: doc_id: 134378 cord_uid: p83nhiz9 natural language interaction with data visualization tools often involves the use of vague subjective modifiers in utterances such as"show me the sectors that are performing"and"where is a good neighborhood to buy a house?."interpreting these modifiers is often difficult for these tools because their meanings lack clear semantics and are in part defined by context and personal user preferences. this paper presents a system called system that makes a first step in better understanding these vague predicates. the algorithm employs word co-occurrence and sentiment analysis to determine which data attributes and filters ranges to associate with the vague predicates. the provenance results from the algorithm are exposed to the user as interactive text that can be repaired and refined. we conduct a qualitative evaluation of the sentifiers system that indicates the usefulness of the interface as well as opportunities for better supporting subjective utterances in visual analysis tasks through natural language. understanding user intent in a query has been recognized as an important aspect of any natural language (nl) interaction system [9, 25] . search queries typically consist of keywords and terms called modifiers that imply a diverse set of search intents [23] . while basic keyword matches from users' search queries might elicit a reasonable set of results, interpreting modifiers provides a better understanding of the semantics in the queries [26] . recently, nl interfaces for visual analysis tools have garnered interest in supporting expressive ways for users to interact with their data and see results expressed as visualizations [1-3, 15, 19, 22, 30, 35, 36] . users often employ vague language while formulating natural language queries when exploring data such as "which country has a high number of gold medals?" or "what time of the day do more bird strikes occur?" [21] . there has been some precedence of research to better understand how these simple vague modifiers comprising of superlatives and numerical graded adjectives should be appropriately interpreted [21, 31] . however, users also employ less concrete and often subjective modifiers such as 'best', 'safe', and 'worse' in utterances [21] . the interpretation of such modifiers makes it challenging for natural language interfaces to precisely determine the extensions of such concepts and mapping intent to the analytical functions provided in the visual analysis systems. contribution this paper introduces sentifiers, 1 a system to explore reasonable interpretations and defaults for such subjective vague modifiers in natural language interfaces for visual analysis. the algorithm identifies numerical attributes that can be associated with a modifier using word co-occurrence. sentiment analysis determines the filter ranges applied to the attributes. similar polarities result in associating the top n of data values for an attribute with the modifier, while diverging polarities are mapped to the bottom n. figure 1a indicates that 'unsafe' and the attribute magnitude have similar negative sentiment polarities, defaulting to a higher earthquake magnitude range as seen in the map. the system has the ability to utilize any domain-specific information if available, such as wolframalpha [4] . figure 1b shows diverging polarities for the modifier 'struggling' paired with attributes incomepercapita and lifeexpectancy. lower numerical filter ranges based on the statistical properties of the data are applied to generate the scatterplot. interactive text is displayed to show the provenance of the system's interpretation with clickable portions exposed as widgets that can be refined by the user. an evaluation of the system provides useful insights for future system design of nl input systems for supporting vague concepts in visual analysis. research exploring the semantics of vague concepts for understanding intent transcends three main categories: (1) computational linguistics, (2) intent and modifiers in search systems, and (3) natural language interaction for visual analysis. the notion of vagueness in language has been studied in the computational linguistics community [32] . research has focused on the conceptualization and representation of vague knowledge [8] . the vague system introduces a technique for generating referring expressions that included gradable adjectives [39] . de melo et al. infer adjective grade ordering from large corpora [14] and vegnaduzzo automatically detects subjective adjectives [40] . computational linguists have developed approaches for subjectivity and polarity labeling of word senses [6, 42] . in our work, we draw inspiration from linguistic literature, specifically polarity identification for computing the semantics around vague subjective concepts. search systems have explored techniques to deduce intent in queries during exploratory search. several techniques exist to extract entityoriented search intent to improve query suggestions and recommendations [16] . detecting intent in search systems is also based on query topic classification [33] . bendersky et al. assign weights to terms in a search query based on concept importance [7] . recent work has focused on deriving query intent by fitting queries into templates [5, 25] . li et al. employ semantic and syntactic features to decompose queries into keywords and intent modifiers [25] . researchers have predicted search intent and intentional task types from search behavior [12, 28] . while the goal of our work to interpret intent in queries is similar to that of search tasks, we focus on resolving vague modifiers to generate relevant visualization responses. similar to search systems, natural language interfaces for visual analysis need to understand intent and handle modifiers in the utterances. datatone provides ambiguity widgets to allow a user to update the system's default interpretation [19] . eviza and analyza support simple pragmatics in analytical interaction through contextual inferencing [15, 30] . evizeon [22] and orko [35] extend pragmatics in analytical conversation. none of these systems consider how imprecise modifiers can be interpreted. the ask data system describes the handling of numerical vague concepts such as 'cheap' and 'high' by inferring a range based on the underlying statistical properties of the data [31] . hearst et al. explore appropriate visualization responses to singular and plural superlatives and numerical graded adjectives based on the shape of the data distributions [21] . we extend this work to more vague, subjective modifiers. we introduce a system, sentifiers that interprets vague modifiers such as 'safe' and 'struggling' in a nl interface for visual analysis. the system employs a web-based architecture with the input query processed by an antlr parser with a context-free grammar, similar to parsers described in [22, 30] . a data manager provides information about the data attributes and executes queries to retrieve data. the query upon execution, generates a d3 visualization result [10] . the process for resolving a set of data attributes and their values to a modifier found in the nl input to sentifiers, is outlined as: input: natural language utterance α output: generate visualization response α is the nl input utterance. m is the vague modifier in the utterance α. part-of-speech tagger pos identifies m in α. attrs num is the set of numerical attributes in the dataset d. attrs cnum is the set of co-occurring numerical attributes in d with attrs cnum ∈ attrs num . pmi computes co-occurrence scores w c for m and attrs num . polarity computes sentiment polarities p for m and attrs cnum . 1 invoke pos(α) returning m. 2 compute pmi(m, attrs num ) → w c for each attr i ∈ attrs cnum . 3 compute polarity(m, attrs cnum ) → p. 4 update interface based on w c and p. vague modifiers are gradable adjectives that modify nouns and and are associated with an abstract scale ordered by their semantic intensity [24] . gradable adjectives can be classified into two categories based on their interpretation as measure functions [24] . numerical graded adjectives such as 'large' and 'cheap' are viewed as measurements that are associated with a numerical quantity for size and cost respectively. complex graded adjectives like 'good' and 'healthy' tend to be underspecified for the exact feature being measured. while the interpretation of numerical gradable adjectives has been explored in nl interfaces for visual analysis [21, 30, 31] , this paper specifically focuses on the handling of complex gradable adjectives. sentifiers first applies a commonly used performant partof-speech (pos) tagger during the parsing process to identify these complex gradable adjectives and their referring attributes in the nl utterances [37] . the system can distinguish complex gradable adjectives by checking for the absence of superlative or comparative tags that are used to annotate numerical graded adjectives. the next step maps the vague modifier to a scale based on its semantic intensity so that the modifier can be interpreted as a set of numerical filters for generating a visualization response. we base our approach on linguistic models that represent the subjectivity of complex modifiers as a generalized measure mapping the modifier to numerical attributes in a multidimensional space [18] . for example, the subjectivity of the modifier 'healthy' can be interpreted based on 'weight', 'amount of exercise', and 'hospital visits.' sentifiers computes the semantic relatedness between the modifier and the numerical data attributes using a co-occurrence measure. to have sufficient coverage for co-occurrence, we use an extensive google n-grams 2 corpus [27] . to maximize the chances of cooccurrence, sentifiers considers co-occurrence between all n-gram combinations of the modifier and the attribute names. for example, some of the n-grams for the attribute income per capita are 'income per capita,' 'income per,' 'per capita,' and 'income. ' we employ a pointwise mutual information measure (pmi), an information-theoretic measure that quantifies the probability of how tightly occurring a modifier m and a numerical attribute attr num are to the probability of observing the terms independently [13] . we found this measure to work well and was performant with terse word co-occurrence pairings without requiring sentence embeddings. we consider any numerical attribute attr cnum that has a non-zero pmi score, indicating the presence of a co-occurrence with m. the pmi of modifier n-gram t m with one of the attribute n-grams t attr is: once the modifier is semantically associated with co-occurring numerical attributes, we need to determine a reasonable numerical range to associate with the modifier. sentiment polarity analysis is a linguistic technique that uses positive and negative lexicons to determine the polarity of a phrase [43] . the technique provides the ability to dynamically compute the sentiment of the phrase based on the context in which its terms co-occur rather than pre-tagging the phrase with absolute polarities, which is often not scalable. we determine the individual sentiment scores with a sentiment classification based on a recursive neural tensor network [34] . we choose this technique as its models handle negations and reasonably predict sentiments of terser phrases, characteristic of queries to sentifiers. the sentiments are returned as a 5-class classification: very negative, negative, neutral, positive, and very positive. the values are normalized as [−1, +1], ranging from negative to positive to provide an overall sentiment. we then determine the sentiment polarities of the modifier m and co-occurring attribute attr cnum pair based on their individual sentiments (ignoring the strength of the sentiments) using the following combinatorial logic. we treat neutral sentiment similar to positive sentiment as neutral text tends to lie near the positive boundary of a positive-negative binary classifier [43] . if (sentiment m == positive or sentiment m == neutral) and (sentiment attr cnum == positive or sentiment attr cnum == neutral) then compute topn(attr cnum ). else if (sentiment m == positive or sentiment m == neutral) and sentiment attr cnum == negative then compute bottomn(attr cnum ). else if sentiment m == negative and (sentiment attr cnum == positive or sentiment attr cnum == neutral) then compute bottomn(attr cnum ). else if sentiment m == negative and sentiment attr cnum == negative then compute topn(attr cnum ). end if sentifiers uses sentiment polarities to compute the ranges in two ways: if domain knowledge exists, the system uses the information to determine a default (figure 3a uses the richter scale [4] ). otherwise, the system computes top n to range from [med + mad, max] and bottom n to range from [min, abs(med − mad)] where med, mad, min, and max are the median, median absolute deviation, minimum, and maximum values for attr cnum respectively (see figure 3b ). we choose mad as it tends to be less affected by non-normality [11] . figure 1 shows the sentifiers interface with an input field that accepts text queries. upon execution of the query, range filters for the cooccurring numerical attributes are applied, showing a visualization response. the system interpretation is expressed in the form of interactive text [41] above the visualization (figure 4a ) to help the user understand the provenance of how the modifier was interpreted. positive, negative, and neutral sentiments are shown in blue, red, and yellow respectively (figure 4b) . the text contains widgets that show ranges starting from the highest co-occurring one. similar to other nl systems [19, 30, 31] , we expose system presumptions as widgets (figure 4c ). if domain-specific semantics are used, a link to the source is provided (figure 1a) . to provide easier readability, sentifiers displays up to two widgets. word co-occurrence and sentiment analysis techniques can result in incorrect results. the user has the ability repair the system decisions (figures 4d and f ) and the interface updates to reflect the changes (figure 4e ). these refinements are persistent for the duration of the user session. we conducted a user study of sentifiers with the following goals: (1) collect qualitative feedback on the handling of the modifiers for various visual analysis tasks and (2) identify system limitations. this information would help provide insights as to how the handling of complex vague modifiers could integrate into a more comprehensive nl visual analysis interface. the study was exploratory in nature where we observed the types of vague modifiers people asked and how they responded to the system behavior. because the main goal of our study was to gain qualitative insight in the system behavior, we encouraged participants to think aloud with the experimenter. figure 4 : interactive text response to a query "which countries are booming?". sentifiers provides the ability to refine the system defaults. we recruited ten volunteers (five males, five females, age 24 -65). all were fluent in english and all regularly used some type of nl interface such as google. eight used a visualization tool on a regular basis and the rest considered themselves beginners. each participant was randomly assigned a dataset of earthquakes in the us [38] or the health and wealth of nations [20] with equal number of participants for each. we began with a short introduction of how to use the system. participants were instructed to phrase their queries in whatever way that felt most natural and to tell us whenever the system did something unexpected. we discussed reactions to system behavior throughout the session and concluded with an interview. the study trials were done remotely over a shared videoconference to conform with social distancing protocol due to covid-19. all sessions took approximately 30 minutes. we employed a mixed-methods approach involving qualitative and quantitative analysis, but considered the quantitative analysis mainly as a complement to our qualitative findings. the quantitative analysis consisted of the number of times participants used vague subjective modifiers and interacted with the text response. overall, participants were positive about the system and identified many benefits. several participants were impressed with the ability of the system to understand their queries ("i typed scary to see what it would do, and it understood." [p2]). sentifiers' text feedback was found to be helpful ("i wasn't sure how the system would handle this, but it was pretty clear when i saw the response" [p4]). the participants appreciated the functionality to be able to correct the system's response ("i wanted to tweak the range a bit and it was useful to be able to change the slider and see the result update" [p9]). the number of unique vague modifiers per participant ranged from 3 to 12 (µ = 6.7) with a total of 24 unique complex modifiers overall. the three most common modifiers were 'good', 'bad', 'severe' for the earthquakes dataset and 'prosperous', 'flourishing', 'poor' for the health and wealth of nations dataset. all participants interacted with the text response to understand the system behavior. the most common interaction was updating the data ranges for the attributes (69% of the interactions), followed by adding new attributes (23%), and deleting attributes from the interpreted result (8%). comments relevant to this behavior included, "the range seemed high for me and i changed it. it was nice to see the system remember that" [p10], "i wanted population to be added to the mix and it was easy to just click and do that" [p3], and "i wasn't interested in life expectancy so i just got rid of it" [p1]. the study also revealed several shortcomings and provides opportunities for future nl systems supporting visual analysis tasks: support for more complex interpretations: the current implementation does not support combinations of vague modifiers in the same query. for example, the system was unable to interpret "show me countries that are doing very well and poorly." [p4]. p2 expressed that they wanted flexibility in defining analytical functions such as associating 'unsafe' to the frequency of recently occurring earthquakes with magnitude 5 are greater. sentifiers failed to correctly interpret queries such as "which countries are reasonably doing well," where p7 expected some middle range, though they were able to adjust the ranges after. a comprehensive evaluation with additional datasets would be necessary to ascertain how effective this system would be alongside standard visual analysis tools. handling customization and in-situ curation: the topic of customization of the interpretation behavior came up during the study. for example, p1 said "i typed -show me which countries are affordable and it showed me an income range. i was expecting a response that considered inflation, gdp or have a way for me to define that." the algorithms employed in sentifiers assume that the data attributes are curated with human-readable words and phrases. however, data is often messy with domain-specific terminology. future work should explore mechanisms for users to customize semantics of attributes and interpretations in the flow of their analysis. handling system expectations, biases, and failures: nl algorithms have shown to exhibit socio-economic biases, including gender and racial assumptions often due to the nature of the training data [17] . their use can perpetuate and even amplify cultural stereotypes in nl systems. for example, p7 commented, "i asked for good places to live and the system responded with a high income per capita. to me, that opens up bigger issues such as gentrification and economic segregation." this suggests that there is a responsibility for improved transparency in system behavior; determining appropriate de-biasing methods remains an open research problem. this paper presents a technique to explore how a system can interpret subjective modifiers prevalent in natural language queries during visual analysis. using word co-occurrence and sentiment polarities, we implement sentifiers to map these modifiers to more concrete functions. we expose the provenance of the system's behavior as an interactive text response. an evaluation of the system indicates that participants found the system to be intuitive and appreciated the ability to refine the system choices. feedback from interacting with sentifiers identifies opportunities for handling vagueness in language in the future design of such natural language tools to support data exploration. as bertrand russell stated [29] -"everything is vague to a degree you do not realize till you have tried to make it precise." towards rich query interpretation: walking back and forth for mining query templates improving the impact of subjectivity word sense disambiguation on contextual opinion analysis learning concept importance using a weighted dependence model selected papers -uncertain reasoning track -flairs computational semantics in discourse: underspecification, resolution, and inference d3: data-driven documents doing better statistics in human-computer interaction actively predicting diverse search intent from user browsing behaviors word association norms, mutual information, and lexicography good, great, excellent: global inference of semantic intensities analyza: exploring data with conversation mining coordinated intent representation for entity search and recommendation bias in computer systems multidimensionality in the grammar of gradability data-tone: managing ambiguity in natural language interfaces for data visualization toward interface defaults for vague modifiers in natural language interfaces for visual analysis applying pragmatics principles for interaction with visual analytics determining the user intent of web search engine queries projecting the adjective: the syntax and semantics of gradability and comparison. outstanding dissertations in linguistics. garland understanding the semantic structure of noun phrase queries introduction to information retrieval quantitative analysis of culture using millions of digitized books extracting information seeking intentions for web search sessions the analysis of mind eviza: a natural language interface for visual analysis inferencing underspecified natural language utterances in visual analysis vagueness in context building bridges for web query classification recursive deep models for semantic compositionality over a sentiment treebank orko: facilitating multimodal interaction for visual exploration and analysis of networks articulate: a semi-automated model for translating natural language queries into meaningful visualizations featurerich part-of-speech tagging with a cyclic dependency network usgs. earthquake facts and statistics generating vague descriptions acquisition of subjective adjectives with limited resources explorable explanations creating subjective and objective sentence classifiers from unannotated texts articles: recognizing contextual polarity: an exploration of features for phrase-level sentiment analysis key: cord-007708-hr4smx24 authors: van kampen, antoine h. c.; moerland, perry d. title: taking bioinformatics to systems medicine date: 2015-08-13 journal: systems medicine doi: 10.1007/978-1-4939-3283-2_2 sha: doc_id: 7708 cord_uid: hr4smx24 systems medicine promotes a range of approaches and strategies to study human health and disease at a systems level with the aim of improving the overall well-being of (healthy) individuals, and preventing, diagnosing, or curing disease. in this chapter we discuss how bioinformatics critically contributes to systems medicine. first, we explain the role of bioinformatics in the management and analysis of data. in particular we show the importance of publicly available biological and clinical repositories to support systems medicine studies. second, we discuss how the integration and analysis of multiple types of omics data through integrative bioinformatics may facilitate the determination of more predictive and robust disease signatures, lead to a better understanding of (patho)physiological molecular mechanisms, and facilitate personalized medicine. third, we focus on network analysis and discuss how gene networks can be constructed from omics data and how these networks can be decomposed into smaller modules. we discuss how the resulting modules can be used to generate experimentally testable hypotheses, provide insight into disease mechanisms, and lead to predictive models. throughout, we provide several examples demonstrating how bioinformatics contributes to systems medicine and discuss future challenges in bioinformatics that need to be addressed to enable the advancement of systems medicine. systems medicine fi nds its roots in systems biology, the scientifi c discipline that aims at a systems-level understanding of, for example, biological networks, cells, organs, organisms, and populations. it generally involves a combination of wet-lab experiments and computational (bioinformatics) approaches. systems medicine extends systems biology by focusing on the application of systems-based approaches to clinically relevant applications in order to improve patient health or the overall well-being of (healthy) individuals [ 1 ] . systems medicine is expected to change health care practice in the coming years. it will contribute to new therapeutics through the identifi cation of novel disease genes that provide drug candidates less likely to fail in clinical studies [ 2 , 3 ] . it is also expected to contribute to fundamental insights into networks perturbed by disease, improved prediction of disease progression, stratifi cation of disease subtypes, personalized treatment selection, and prevention of disease. to enable systems medicine it is necessary to characterize the patient at various levels and, consequently, to collect, integrate, and analyze various types of data including not only clinical (phenotype) and molecular data, but also information about cells (e.g., disease-related alterations in organelle morphology), organs (e.g., lung impedance when studying respiratory disorders such as asthma or chronic obstructive pulmonary disease), and even social networks. the full realization of systems medicine therefore requires the integration and analysis of environmental, genetic, physiological, and molecular factors at different temporal and spatial scales, which currently is very challenging. it will require large efforts from various research communities to overcome current experimental, computational, and information management related barriers. in this chapter we show how bioinformatics is an essential part of systems medicine and discuss some of the future challenges that need to be solved. to understand the contribution of bioinformatics to systems medicine, it is helpful to consider the traditional role of bioinformatics in biomedical research, which involves basic and applied (translational) research to augment our understanding of (molecular) processes in health and disease. the term "bioinformatics" was fi rst coined by the dutch theoretical biologist paulien hogeweg in 1970 to refer to the study of information processes in biotic systems [ 4 ] . soon, the fi eld of bioinformatics expanded and bioinformatics efforts accelerated and matured as the fi rst (whole) genome and protein sequences became available. the signifi cance of bioinformatics further increased with the development of highthroughput experimental technologies that allowed wet-lab researchers to perform large-scale measurements. these include determining whole-genome sequences (and gene variants) and genome-wide gene expression with next-generation sequencing technologies (ngs; see table 1 for abbreviations and web links) [ 5 ] , measuring gene expression with dna microarrays [ 6 ] , identifying and quantifying proteins and metabolites with nmr or (lc/ gc-) ms [ 7 ] , measuring epigenetic changes such as methylation and histone modifi cations [ 8 ] , and so on. these, "omics" technologies, are capable of measuring the many molecular building blocks that determine our (patho)physiology. genome-wide measurements have not only signifi cantly advanced our fundamental understanding of the molecular biology of health and disease but table 1 abbreviations and websites have also contributed to new (commercial) diagnostic and prognostic tests [ 9 , 10 ] and the selection and development of (personalized) treatment [ 11 ] . nowadays, bioinformatics is therefore defi ned as "advancing the scientifi c understanding of living systems through computation" (iscb), or more inclusively as "conceptualizing biology in terms of molecules and applying 'informatics techniques' (derived from disciplines such as applied mathematics, computer science and statistics) to understand and organize the information associated with these molecules, on a large scale" [ 12 ] . it is worth noting that solely measuring many molecular components of a biological system does not necessarily result in a deeper understanding of such a system. understanding biological function does indeed require detailed insight into the precise function of these components but, more importantly, it requires a thorough understanding of their static, temporal, and spatial interactions. these interaction networks underlie all (patho)physiological processes, and elucidation of these networks is a major task for bioinformatics and systems medicine . the developments in experimental technologies have led to challenges that require additional expertise and new skills for biomedical researchers: • information management. modern biomedical research projects typically produce large and complex omics data sets , sometimes in the order of hundreds of gigabytes to terabytes of which a large part has become available through public databases [ 13 , 14 ] sometimes even prior to publication (e.g., gtex, icgc, tcga). this not only contributes to knowledge dissemination but also facilitates reanalysis and metaanalysis of data, evaluation of hypotheses that were not considered by the original research group, and development and evaluation of new bioinformatics methods. the use of existing data can in some cases even make new (expensive) experiments superfl uous. alternatively, one can integrate publicly available data with data generated in-house for more comprehensive analyses, or to validate results [ 15 ] . in addition, the obligation of making raw data available may prevent fraud and selective reporting. the management (transfer, storage, annotation, and integration) of data and associated meta-data is one of the main and increasing challenges in bioinformatics that needs attention to safeguard the progression of systems medicine. • data analysis and interpretation . bioinformatics data analysis and interpretation of omics data have become increasingly complex, not only due to the vast volumes and complexity of the data but also as a result of more challenging research questions. bioinformatics covers many types of analyses including nucleotide and protein sequence analysis, elucidation of tertiary protein structures, quality control, pre-processing and statistical analysis of omics data, determination of genotypephenotype relationships, biomarker identifi cation, evolutionary analysis, analysis of gene regulation, reconstruction of biological networks, text mining of literature and electronic patient records, and analysis of imaging data. in addition, bioinformatics has developed approaches to improve experimental design of omics experiments to ensure that the maximum amount of information can be extracted from the data. many of the methods developed in these areas are of direct relevance for systems medicine as exemplifi ed in this chapter. clearly, new experimental technologies have to a large extent turned biomedical research in a data-and compute-intensive endeavor. it has been argued that production of omics data has nowadays become the "easy" part of biomedical research, whereas the real challenges currently comprise information management and bioinformatics analysis. consequently, next to the wet-lab, the computer has become one of the main tools of the biomedical researcher . bioinformatics enables and advances the management and analysis of large omics-based datasets, thereby directly and indirectly contributing to systems medicine in several ways ( fig. 1 3. quality control and pre-processing of omics data. preprocessing typically involves data cleaning (e.g., removal of failed assays) and other steps to obtain quantitative measurements that can be used in downstream data analysis. 4. (statistical) data analysis methods of large and complex omicsbased datasets. this includes methods for the integrative analysis of multiple omics data types (subheading 5 ), and for the elucidation and analysis of biological networks (top-down systems medicine; subheading 6 ). systems medicine comprises top-down and bottom-up approaches. the former represents a specifi c branch of bioinformatics, which distinguishes itself from bottom-up approaches in several ways [ 3 , 19 , 20 ] . top-down approaches use omics data to obtain a holistic view of the components of a biological system and, in general, aim to construct system-wide static functional or physical interaction networks such as gene co-expression networks and protein-protein interaction networks. in contrast, bottom-up approaches aim to develop detailed mechanistic and quantitative mathematical models for sub-systems. these models describe the dynamic and nonlinear behavior of interactions between known components to understand and predict their behavior upon perturbation. however, in contrast to omics-based top-down approaches, these mechanistic models require information about chemical/physical parameters and reaction stoichiometry, which may not be available and require further (experimental) efforts. both the top-down and bottom-up approaches result in testable hypotheses and new wet-lab or in silico experiments that may lead to clinically relevant fi ndings. biomedical research and, consequently, systems medicine are increasingly confronted with the management of continuously growing volumes of molecular and clinical data, results of data analyses and in silico experiments, and mathematical models. due fig. 1 the contribution of bioinformatics ( dark grey boxes ) to systems medicine ( black box ). (omics) experiments, patients, and public repositories provide a wide range of data that is used in bioinformatics and systems medicine studies to policies of scientifi c journals and funding agencies, omics data is often made available to the research community via public databases. in addition, a wide range of databases have been developed, of which more than 1550 are currently listed in the molecular biology database collection [ 14 ] providing a rich source of biomedical information. biological repositories do not merely archive data and models but also serve a range of purposes in systems medicine as illustrated below from a few selected examples. the main repositories are hosted and maintained by the major bioinformatics institutes including ebi, ncbi, and sib that make a major part of the raw experimental omics data available through a number of primary databases including genbank [ 21 ] , geo [ 22 ] , pride [ 23 ] , and metabolights [ 24 ] for sequence, gene expression, ms-based proteomics, and ms-based metabolomics data, respectively. in addition, many secondary databases provide information derived from the processing of primary data, for example pathway databases (e.g., reactome [ 25 ] , kegg [ 26 ] ), protein sequence databases (e.g., uniprotkb [ 27 ] ), and many others. pathway databases provide an important resource to construct mathematical models used to study and further refi ne biological systems [ 28 , 29 ] . other efforts focus on establishing repositories integrating information from multiple public databases. the integration of pathway databases [ 30 -32 ] , and genome browsers that integrate genetic, omics, and other data with whole-genome sequences [ 33 , 34 ] are two examples of this. joint initiatives of the bioinformatics and systems biology communities resulted in repositories such as biomodels, which contains mathematical models of biochemical and cellular systems [ 35 ] , recon 2 that provides a communitydriven, consensus " metabolic reconstruction " of human metabolism suitable for computational modelling [ 36 ] , and seek, which provides a platform designed for the management and exchange of systems biology data and models [ 37 ] . another example of a database that may prove to be of value for systems medicine studies is malacards , an integrated and annotated compendium of about 17,000 human diseases [ 38 ] . malacards integrates 44 disease sources into disease cards and establishes gene-disease associations through integration with the well-known genecards databases [ 39 , 40 ] . integration with genecards and cross-references within malacards enables the construction of networks of related diseases revealing previously unknown interconnections among diseases, which may be used to identify drugs for off-label use. another class of repositories are (expert-curated) knowledge bases containing domain knowledge and data, which aim to provide a single point of entry for a specifi c domain. contents of these knowledge bases are often based on information extracted (either manually or by text mining) from literature or provided by domain experts [ 41 -43 ] . finally, databases are used routinely in the analysis, interpretation, and validation of experimental data. for example, the gene ontology (go) provides a controlled vocabulary of terms for describing gene products, and is often used in gene set analysis to evaluate expression patterns of groups of genes instead of those of individual genes [ 44 ] and has, for example, been applied to investigate hiv-related cognitive disorders [ 45 ] and polycystic kidney disease [ 46 ] . several repositories such as mir2disease [ 47 ] , peroxisomedb [ 41 ] , and mouse genome informatics (mgi) [ 43 ] include associations between genes and disorders, but only provide very limited phenotypic information. phenotype databases are of particular interest to systems medicine. one well-known phenotype repository is the omim database, which primarily describes single-gene (mendelian) disorders [ 48 ] . clinvar is another example and provides an archive of reports and evidence of the relationships among medically important human variations found in patient samples and phenotypes [ 49 ] . clinvar complements dbsnp (for singlenucleotide polymorphisms) [ 50 ] and dbvar (for structural variations) [ 51 ] , which both provide only minimal phenotypic information. the integration of these phenotype repositories with genetic and other molecular information will be a major aim for bioinformatics in the coming decade enabling, for example, the identifi cation of comorbidities, determination of associations between gene (mutations) and disease, and improvement of disease classifi cations [ 52 ] . it will also advance the defi nition of the "human phenome," i.e., the set of phenotypes resulting from genetic variation in the human genome. to increase the quality and (clinical) utility of the phenotype and variant databases as an essential step towards reducing the burden of human genetic disease, the human variome project coordinates efforts in standardization, system development, and (training) infrastructure for the worldwide collection and sharing of genetic variations that affect human health [ 53 , 54 ] . to implement and advance systems medicine to the benefi t of patients' health, it is crucial to integrate and analyze molecular data together with de-identifi ed individual-level clinical data complementing general phenotype descriptions. patient clinical data refers to a wide variety of data including basic patient information (e.g., age, sex, ethnicity), outcomes of physical examinations, patient history, medical diagnoses, treatments, laboratory tests, pathology reports, medical images, and other clinical outcomes. inclusion of clinical data allows the stratifi cation of patient groups into more homogeneous clinical subgroups. availability of clinical data will increase the power of downstream data analysis and modeling to elucidate molecular mechanisms, and to identify molecular biomarkers that predict disease onset or progression, or which guide treatment selection. in biomedical studies clinical information is generally used as part of patient and sample selection, but some omics studies also use clinical data as part of the bioinformatics analysis (e.g., [ 9 , 55 ] ). however, in general, clinical data is unavailable from public resources or only provided on an aggregated level. although good reasons exist for making clinical data available (subheading 2.2 ), ethical and legal issues comprising patient and commercial confi dentiality, and technical issues are the most immediate challenges [ 56 , 57 ] . this potentially hampers the development of systems medicine approaches in a clinical setting since sharing and integration of clinical and nonclinical data is considered a basic requirement [ 1 ] . biobanks [ 58 ] such as bbmri [ 59 ] provide a potential source of biological material and associated (clinical) data but these are, generally, not publicly accessible, although permission to access data may be requested from the biobank provider. clinical trials provide another source of clinical data for systems medicine studies, but these are generally owned by a research group or sponsor and not freely available [ 60 ] although ongoing discussions may change this in the future ( [ 61 ] and references therein). although clinical data is not yet available on a large scale, the bioinformatics and medical informatics communities have been very active in establishing repositories that provide clinical data. one example is the database of genotypes and phenotypes (dbgap) [ 62 ] developed by the ncbi. study metadata, summarylevel (phenotype) data, and documents related to studies are publicly available. access to de-identifi ed individual-level (clinical) data is only granted after approval by an nih data access committee. another example is the cancer genome atlas (tcga) , which also provides individual-level molecular and clinical data through its own portal and the cancer genomics hub (cghub). clinical data from tcga is available without any restrictions but part of the lower level sequencing and microarray data can only be obtained through a formal request managed by dbgap. medical patient records provide an even richer source of phenotypic information , and has already been used to stratify patient groups, discover disease relations and comorbidity, and integrate these records with molecular data to obtain a systems-level view of phenotypes (for a review see [ 63 ] ). on the one hand, this integration facilitates refi nement and analysis of the human phenome to, for example, identify diseases that are clinically uniform but have different underlying molecular mechanisms, or which share a pathogenetic mechanism but with different genetic cause [ 64 ] . on the other hand, using the same data, a phenome-wide association study ( phewas ) [ 65 ] would allow the identifi cation of unrelated phenotypes associated with specifi c shared genetic variant(s), an effect referred to as pleiotropy. moreover, it makes use of information from medical records generated in routine clinical practice and, consequently, has the potential to strengthen the link between biomedical research and clinical practice [ 66 ] . the power of phenome analysis was demonstrated in a study involving 1.5 million patient records, not including genotype information, comprising 161 disorders. in this study it was shown that disease phenotypes form a highly connected network suggesting a shared genetic basis [ 67 ] . indeed, later studies that incorporated genetic data resulted in similar fi ndings and confi rmed a shared genetic basis for a number of different phenotypes. for example, a recent study identifi ed 63 potentially pleiotropic associations through the analysis of 3144 snps that had previously been implicated by genome-wide association studies ( gwas) as mediators of human traits, and 1358 phenotypes derived from patient records of 13,835 individuals [ 68 ] . this demonstrates that phenotypic information extracted manually or through text mining from patient records can help to more precisely defi ne (relations between) diseases. another example comprises the text mining of psychiatric patient records to discover disease correlations [ 52 ] . here, mapping of disease genes from the omim database to information from medical records resulted in protein networks suspected to be involved in psychiatric diseases. integrative bioinformatics comprises the integrative (statistical) analysis of multiple omics data types. many studies demonstrated that using a single omics technology to measure a specifi c molecular level (e.g., dna variation, expression of genes and proteins, metabolite concentrations, epigenetic modifi cations) already provides a wealth of information that can be used for unraveling molecular mechanisms underlying disease. moreover, single-omics disease signatures which combine multiple (e.g., gene expression) markers have been constructed to differentiate between disease subtypes to support diagnosis and prognosis. however, no single technology can reveal the full complexity and details of molecular networks observed in health and disease due to the many interactions across these levels. a systems medicine strategy should ideally aim to understand the functioning of the different levels as a whole by integrating different types of omics data. this is expected to lead to biomarkers with higher predictive value, and novel disease insights that may help to prevent disease and to develop new therapeutic approaches. integrative bioinformatics can also facilitate the prioritization and characterization of genetic variants associated with complex human diseases and traits identifi ed by gwas in which hundreds of thousands to over a million snps are assayed in a large number of individuals. although such studies lack the statistical power to identify all disease-associated loci [ 69 ] , they have been instrumental in identifying loci for many common diseases. however, it remains diffi cult to prioritize the identifi ed variants and to elucidate their effect on downstream pathways ultimately leading to disease [ 70 ] . consequently, methods have been developed to prioritize candidate snps based on integration with other (omics) data such as gene expression, dnase hypersensitive sites, histone modifi cations, and transcription factor-binding sites [ 71 ] . the integration of multiple omics data types is far from trivial and various approaches have been proposed [ 72 -74 ] . one approach is to link different types of omics measurements through common database identifi ers. although this may seem straightforward, in practice this is complicated as a result of technical and standardization issues as well as a lack of biological consensus [ 32 , 75 -77 ] . moreover, the integration of data at the level of the central dogma of molecular biology and, for example, metabolite data is even more challenging due to the indirect relationships between genes, transcripts, and proteins on the one hand and metabolites on the other hand, precluding direct links between the database identifi ers of these molecules. statistical data integration [ 72 ] is a second commonly applied strategy, and various approaches have been applied for the joint analysis of multiple data types (e.g., [ 78 , 79 ] ). one example of statistical data integration is provided by a tcga study that measured various types of omics data to characterize breast cancer [ 80 ] . in this study 466 breast cancer samples were subjected to whole-genome and -exome sequencing, and snp arrays to obtain information about somatic mutations, copy number variations, and chromosomal rearrangements. microarrays and rna-seq were used to determine mrna and microrna expression levels, respectively. reverse-phase protein arrays (rppa) and dna methylation arrays were used to obtain data on protein expression levels and dna methylation, respectively. simultaneous statistical analysis of different data types via a "cluster-of-clusters" approach using consensus clustering on a multi-omics data matrix revealed that four major breast cancer subtypes could be identifi ed. this showed that the intrinsic subtypes (basal, luminal a and b, her2) that had previously been determined using gene expression data only could be largely confi rmed in an integrated analysis of a large number of breast tumors. single-level omics data has extensively been used to identify disease-associated biomarkers such as genes, proteins, and metabolites. in fact, these studies led to more than 150,000 papers documenting thousands of claimed biomarkers, however, it is estimated that fewer than 100 of these are currently used for routine clinical practice [ 81 ] . integration of multiple omics data types is expected to result in more robust and predictive disease profi les since these better refl ect disease biology [ 82 ] . further improvement of these profi les may be obtained through the explicit incorporation of interrelationships between various types of measurements such as microrna-mrna target, or gene methylation-microrna (based on a common target gene). this was demonstrated for the prediction of short-term and long-term survival from serous cystadenocarcinoma tcga data [ 83 ] . according to the recent casym roadmap : "human disease can be perceived as perturbations of complex, integrated genetic, molecular and cellular networks and such complexity necessitates a new approach." [ 84 ] . in this section we discuss how (approximations) to these networks can be constructed from omics data and how these networks can be decomposed in smaller modules. then we discuss how the resulting modules can be used to generate experimentally testable hypotheses, provide insight into disease mechanisms, lead to predictive diagnostic and prognostic models, and help to further subclassify diseases [ 55 , 85 ] (fig. 2 ) network-based approaches will provide medical doctors with molecular level support to make personalized treatment decisions. in a top-down approach the aim of network reconstruction is to infer the connections between the molecules that constitute a biological network. network models can be created using a variety of mathematical and statistical techniques and data types. early approaches for network inference (also called reverse engineering ) used only gene expression data to reconstruct gene networks. here, we discern three types of gene network inference algorithms using methods based on (1) correlation-based approaches, (2) information-theoretic approaches, and (3) bayesian networks [ 86 ] . co-expression networks are an extension of commonly used clustering techniques , in which genes are connected by edges in a network if the amount of correlation of their gene expression profi les exceeds a certain value. co-expression networks have been shown to connect functionally related genes [ 87 ] . note that connections in a co-expression network correspond to either direct (e.g., transcription factor-gene and protein-protein) or indirect (e.g., proteins participating in the same pathway) interactions. in one of the earliest examples of this approach, pair-wise correlations were calculated between gene expression profi les and the level of growth inhibition caused by thousands of tested anticancer agents, for 60 cancer cell lines [ 88 ] . removal of associations weaker than a certain threshold value resulted in networks consisting of highly correlated genes and agents, called relevance networks, which led to targeted hypotheses for potential single-gene determinants of chemotherapeutic susceptibility. information-theoretic approaches have been proposed in order to capture nonlinear dependencies assumed to be present in most biological systems and that cannot be captured by correlation-based distance measures . these approaches often use the concept of mutual information, a generalization of the correlation coeffi cient which quantifi es the degree of statistical (in)dependence. an example of a network inference method that is based on mutual information is aracne, which has been used to reconstruct the human b-cell gene network from a large compendium of human b-cell gene expression profi les [ 89 ] . in order to discover regulatory interactions, aracne removes the majority of putative indirect interactions from the initial mutual information-based gene network using a theorem from information theory, the data processing inequality. this led to the identifi cation of myc as a major hub in the b-cell gene network and a number of novel myc target genes, which were experimentally validated. whether informationtheoretic approaches are more powerful in general than correlationbased approaches is still subject of debate [ 90 ] . bayesian networks allow the description of statistical dependencies between variables in a generic way [ 91 , 92 ] . bayesian networks are directed acyclic networks in which the edges of the network represent conditional dependencies; that is, nodes that are not connected represent variables that are conditionally independent of each other. a major bottleneck in the reconstruction of bayesian networks is their computational complexity. moreover, bayesian networks are acyclic and cannot capture feedback loops that characterize many biological networks. when time-series rather than steady-state data is available, dynamic bayesian networks provide a richer framework in which cyclic networks can be reconstructed [ 93 ] . gene (co-)expression data only offers a partial view on the full complexity of cellular networks. consequently, networks have also been constructed from other types of high-throughput data. for example, physical protein-protein interactions have been measured on a large scale in different organisms including human, using affi nity capture-mass spectrometry or yeast two-hybrid screens, and have been made available in public databases such as biogrid [ 94 ] . regulatory interactions have been probed using chromatin immunoprecipitation sequencing (chip-seq) experiments, for example by the encode consortium [ 95 ] . using probabilistic techniques , heterogeneous types of experimental evidence and prior knowledge have been integrated to construct functional association networks for human [ 96 ] , mouse [ 97 ] , and, most comprehensively, more than 1100 organisms in the string database [ 98 ] . functional association networks can help predict novel pathway components, generate hypotheses for biological functions for a protein of interest, or identify disease-related genes [ 97 ] . prior knowledge required for these approaches is, for example, available in curated biological pathway databases, and via protein associations predicted using text mining based on their cooccurrence in abstracts or even full-text articles. many more integrative network inference methods have been proposed; for a review see [ 99 ] . the integration of gene expression data with chip data [ 100 ] or transcription factor-binding motif data [ 101 ] has shown to be particularly fruitful for inferring transcriptional regulatory networks. recently, li et al. [ 102 ] described the results from a regression-based model that predicts gene expression using encode (chip-seq) and tcga data (mrna expression data complemented with copy number variation, dna methylation, and microrna expression data). this model infers the regulatory activities of expression regulators and their target genes in acute myeloid leukemia samples. eighteen key regulators were identifi ed, whose activities clustered consistently with cytogenetic risk groups. bayesian networks have also been used to integrate multiomics data. the combination of genotypic and gene expression data is particularly powerful, since dna variations represent naturally occurring perturbations that affect gene expression detected as expression quantitative trait loci ( eqtl ). cis -acting eqtls can then be used as constraints in the construction of directed bayesian networks to infer causal relationships between nodes in the network [ 103 ] . large multi-omics datasets consisting of hundreds or sometimes even thousands of samples are available for many commonly occurring human diseases, such as most tumor types (tcga), alzheimer's disease [ 104 ] , and obesity [ 105 ] . however, a major bottleneck for the construction of accurate gene networks is that the number of gene networks that are compatible with the experimental data is several orders of magnitude larger still. in other words, top-down network inference is an underdetermined problem with many possible solutions that explain the data equally well and individual gene-gene interactions are characterized by a high false-positive rate [ 99 ] . most network inference methods therefore try to constrain the number of possible solutions by making certain assumptions about the structure of the network. perhaps the most commonly used strategy to harness the complexity of the gene network inference problem is to analyze experimental data in terms of biological modules, that is, sets of genes that have strong interactions and a common function [ 106 ] . there is considerable evidence that many biological networks are modular [ 107 ] . module-based approaches effectively constrain the number of parameters to estimate and are in general also more robust to the noise that characterizes high-throughput omics measurements. a detailed review of module-based techniques is outside the scope of this chapter (see, for example [ 108 ] ), but we would like to mention a few examples of successful and commonly used modular approaches. weighted gene co-expression network analysis ( wgcna) decomposes a co-expression network into modules using clustering techniques [ 109 ] . modules can be summarized by their module eigengene, a weighted average expression profi le of all gene member of a given module. eigengenes can then be correlated with external sample traits to identify modules that are related with these traits. parikshak et al. [ 110 ] used wgcna to extract modules from a co-expression network constructed using fetal and early postnatal brain development expression data. next, they established that several of these modules were enriched for genes and rare de novo variants implicated in autism spectrum disorder (asd). moreover, the asd-associated modules are also linked at the transcriptional level and 17 transcription factors were found acting as putative co-regulators of asd-associated gene modules during neocortical development. wgcna can also be used when multiple omics data types are available. one example of such an approach involved the integration of transcriptomic and proteomic data from a study investigating the response to sars-cov infection in mice [ 111 ] . in this study wgcna-based gene and protein co-expression modules were constructed and integrated to obtain module-based disease signatures. interestingly, the authors found several cases of identifi er-matched transcripts and proteins that correlated well with the phenotype, but which showed poor or anticorrelation across these two data types. moreover, the highest correlating transcripts and peptides were not the most central ones in the co-expression modules. vice versa , the transcripts and proteins that defi ned the modules were not those with the highest correlation to the phenotype. at the very least this shows that integration of omics data affects the nature of the disease signatures. identifi cation of active modules is another important integrative modular technique . here, experimental data in the form of molecular profi les is projected onto a biological network, for example a protein-protein interaction network. active modules are those subnetworks that show the largest change in expression for a subset of conditions and are likely to contain key drivers or regulators of those processes perturbed in the experiment. active modules have, for example, been used to fi nd a subnetwork that is overexpressed in a particularly aggressive lymphoma subtype [ 112 ] and to detect signifi cantly mutated pathways [ 113 ] . some active module approaches integrate various types of omics data. one example of such an approach is paradigm [ 114 ] , which translates pathways into factor graphs, a class of models that belongs to the same family of models as bayesian networks, and determines sample-specifi c pathway activity from multiple functional genomic datasets. paradigm has been used in several tcga projects, for example, in the integrated analysis of 131 urothelial bladder carcinomas [ 55 ] . paradigm-based analysis of copy number variations and rna-seq gene expression in combination with a propagation-based network analysis algorithm revealed novel associations between mutations and gene expression levels, which subsequently resulted in the identifi cation of pathways altered in bladder cancer. the identifi cation of activating or inhibiting gene mutations in these pathways suggested new targets for treatment. moreover, this effort clearly showed the benefi ts of screening patients for the presence of specifi c mutations to enable personalized treatment strategies. often, published disease signatures cannot be replicated [ 81 ] or provide hardly additional biological insight. also here (modular) network-based approaches have been proposed to alleviate these problems. a common characteristic of most methods is that the molecular activity of a set of genes is summarized on a per sample basis. summarized gene set scores are then used as features in prognostic and predictive models. relevant gene sets can be based on prior knowledge and correspond to canonical pathways, gene ontology categories, or sets of genes sharing common motifs in their promoter regions [ 115 ] . gene set scores can also be determined by projecting molecular data onto a biological network and summarizing scores at the level of subnetworks for each individual sample [ 116 ] . while promising in principle, it is still subject of debate whether gene set-based models outperform gene-based one s [ 117 ] . the comparative analysis of networks across different species is another commonly used approach to constrain the solution space. patterns conserved across species have been shown to be more likely to be true functional interactions [ 107 ] and to harbor useful candidates for human disease genes [ 118 ] . many network alignment methods have been developed in the past decade to identify commonalities between networks. these methods in general combine sequence-based and topological constraints to determine the optimal alignment of two (or more) biological networks. network alignment has, for example, been applied to detect conserved patterns of protein interaction in multiple species [ 107 , 119 ] and to analyze the evolution of co-expression networks between humans and mice [ 120 , 121 ] . network alignment can also be applied to detect diverged patterns [ 120 ] and may thus lead to a better understanding of similarities and differences between animal models and human in health and disease. information from model organisms has also been fruitfully used to identify more robust disease signatures [ 122 -125 ] . sweet-cordero and co-workers [ 122 ] used a gene signature identifi ed in a mouse model of lung adenocarcinoma to uncover an orthologous signature in human lung adenocarcinoma that was not otherwise apparent. bild et al. [ 123 ] defi ned gene expression signatures characterizing several oncogenic pathways of human mammary epithelial cells. they showed that these signatures predicted pathway activity in mouse and human tumors. predictions of pathway activity correlated well with the sensitivity to drugs targeting those pathways and could thus serve as a guide to targeted therapies. a generic approach, pathprint, for the integration of gene expression data across different platforms and species at the level of pathways, networks, and transcriptionally regulated targets was recently described [ 126 ] . the authors used their method to identify four stem cell-related pathways conserved between human and mouse in acute myeloid leukemia, with good prognostic value in four independent clinical studies. we reviewed a wide array of different approaches showing how networks can be used to elucidate integrated genetic, molecular, and cellular networks. however, in general no single approach will be suffi cient and combining different approaches in more complex analysis pipelines will be required. this is fi ttingly illustrated by the diggit (driver-gene inference by genetical-genomics and information theory) algorithm [ 127 ] . in brief, diggit identities candidate master regulators from an aracne gene co-expression network integrated with copy number variations that affect gene expression. this method combines several previously developed computational approaches and was used to identify causal genetic drivers of human disease in general and glioblastoma, breast cancer, and alzheimer's disease in particular. this enabled identifi cation of klhl9 deletions as upstream activators of two previously established master regulators in a specifi c subtype of glioblastoma. systems medicine is one of the steps necessary to make improvements in the prevention and treatment of disease through systems approaches that will (a) elucidate (patho)physiologic mechanisms in much greater detail than currently possible, (b) produce more robust and predictive disease signatures, and (c) enable personalized treatment. in this context, we have shown that bioinformatics has a major role to play. bioinformatics will continue its role in the development, curation, integration, and maintenance of (public) biological and clinical databases to support biomedical research and systems medicine. the bioinformatics community will strengthen its activities in various standardization and curation efforts that already resulted in minimum reporting guidelines [ 128 ] , data capture approaches [ 75 ] , data exchange formats [ 129 ] , and terminology standards for annotation [ 130 ] . one challenge for the future is to remove errors and inconsistencies in data and annotation from databases and prevent new ones from being introduced [ 32 , 76 , 131 -135 ]. an equally important challenge is to establish, improve, and integrate resources containing phenotype and clinical information. to achieve this objective it seems reasonable that bioinformatics and health informatics professionals team up [ 136 -138 ] . traditionally health informatics professionals have focused on hospital information systems (e.g., patient records, pathology reports, medical images) and data exchange standards (e.g., hl7), medical terminology standards (e.g., international classifi cation of disease (icd), snomed), medical image analysis, analysis of clinical data, clinical decision support systems, and so on. while, on the other hand, bioinformatics mainly focused on molecular data, it shares many approaches and methods with health informatics. integration of these disciplines is therefore expected to benefi t systems medicine in various ways [ 139 ] . integrative bioinformatics approaches clearly have added value for systems medicine as they provide a better understanding of biological systems, result in more robust disease markers, and prevent (biological) bias that would possibly occur from using single-omics measurements. however, such studies, and the scientifi c community in general, would benefi t from improved strategies to disseminate and share data which typically will be produced at multiple research centers (e.g., https://www.synapse.org ; [ 140 ] ). integrative studies are expected to increasingly facilitate personalized medicine approaches such as demonstrated by chen and coworkers [ 141 ] . in their study they presented a 14-month "integrative personal omics profi le" (ipop) for a single individual comprising genomic, transcriptomic, proteomic, metabolomic, and autoantibody data. from the whole-genome sequence data an elevated risk for type 2 diabetes (t2d) was detected, and subsequent monitoring of hba1c and glucose levels revealed the onset of t2d, despite the fact that the individual lacked many of the known non-genetic risk factors. subsequent treatment resulted in a gradual return to the normal phenotype. this shows that the genome sequence can be used to determine disease risk in a healthy individual and allows selecting and monitoring specifi c markers that provide information about the actual disease status. network-based approaches will increasingly be used to determine the genetic causes of human diseases. since the effect of a genetic variation is often tissue or cell-type specifi c, a large effort is needed in constructing cell-type-specifi c networks both in health and disease. this can be done using data already available, an approach taken by guan et al. [ 142 ] . the authors proposed 107 tissue-specifi c networks in mouse via their generic approach for constructing functional association networks using lowthroughput, highly reliable tissue-specifi c gene expression information as a constraint. one could also generate new datasets to facilitate the construction of tissue-specifi c networks. examples of such approaches are tcga and the genotype-tissue expression (gtex) project. the aim of gtex is to create a data resource for the systematic study of genetic variation and its effect on gene expression in more than 40 human tissues [ 143 ] . regardless of the way how networks are constructed, it will become more and more important to offer a centralized repository where networks from different cell types and diseases can be stored and accessed. nowadays, these networks are diffi cult to retrieve and are scattered in supplementary fi les with the original papers, links to accompanying web pages, or even not available at all. a resource similar to what the systems biology community has created with the biomodels database would be a great leap forward. there have been some initial attempts in building databases of network models, for example the cellcircuits database [ 123 ] ( http://www.cellcircuits.org ) and the causal biological networks (cbn) database of networks related to lung disease [ 144 ] ( http://causalbionet.com ). however, these are only small-scale initiatives and a much larger and coordinated effort is required. another main bottleneck for the successful application of network inference methods is their validation. most network inference methods to date have been applied to one or a few isolated datasets and were validated using some limited follow-up experiments, for example via gene knockdowns, using prior knowledge from databases and literature as a gold standard, or by generating simulated data from a mathematical model of the underlying network [ 145 , 146 ] . however, strengths and weaknesses of network inference methods across cell types, diseases, and species have hardly been assessed. notable exceptions are collaborative competitions such as the dialogue on reverse engineering assessment and methods (dream) [ 147 ] and industrial methodology for process verifi cation (improver) [ 146 ] . these centralized initiatives propose challenges in which individual research groups can participate and to which they can submit their predictions, which can then be independently validated by the challenge organizers. several dream challenges in the area of network inference have been organized, leading to a better insight into the strengths and weaknesses of individual methods [ 148 ] . another important contribution of dream is that a crowd-based approach integrating predictions from multiple network inference methods was shown to give good and robust performance across diverse data sets [ 149 ] . also in the area of systems medicine challenge-based competitions may offer a framework for independent verifi cation of model predictions. systems medicine promises a more personalized medicine that effectively exploits the growing amount of molecular and clinical data available for individual patients. solid bioinformatics approaches are of crucial importance for the success of systems medicine. however, really delivering the promises of systems medicine will require an overall change of research approach that transcends the current reductionist approach and results in a tighter integration of clinical, wet-lab laboratory, and computational groups adopting a systems-based approach. past, current, and future success of systems medicine will accelerate this change. the road from systems biology to systems medicine participatory medicine: a driving force for revolutionizing healthcare understanding drugs and diseases by systems biology the roots of bioinformatics in theoretical biology sequencing technologies -the next generation exploring the new world of the genome with dna microarrays spectroscopic and statistical techniques for information recovery in metabonomics and metabolomics next-generation technologies and data analytical approaches for epigenomics gene expression profi ling predicts clinical outcome of breast cancer diagnostic tests based on gene expression profi le in breast cancer: from background to clinical use a multigene assay to predict recurrence of tamoxifentreated, node-negative breast cancer what is bioinformatics? a proposed defi nition and overview of the fi eld the importance of biological databases in biological discovery the 2014 nucleic acids research database issue and an updated nar online molecular biology database collection reuse of public genome-wide gene expression data experimental design for gene expression microarrays learning from our gwas mistakes: from experimental design to scientifi c method effi cient experimental design and analysis strategies for the detection of differential expression using rna-sequencing impact of yeast systems biology on industrial biotechnology the nature of systems biology gene expression omnibus: microarray data storage, submission, retrieval, and analysis the proteomics identifi cations (pride) database and associated tools: status in 2013 metabolights--an open-access generalpurpose repository for metabolomics studies and associated meta-data the reactome pathway knowledgebase data, information, knowledge and principle: back to metabolism in kegg activities at the universal protein resource (uniprot) path2models: large-scale generation of computational models from biochemical pathway maps precise generation of systems biology models from kegg pathways pathguide: a pathway resource list pathway commons, a web resource for biological pathway data consensus and confl ict cards for metabolic pathway databases the ucsc genome browser database: 2014 update biomodels database: a repository of mathematical models of biological processes a community-driven global reconstruction of human metabolism the seek: a platform for sharing data and models in systems biology malacards: an integrated compendium for diseases and their annotation genecards version 3: the human gene integrator in-silico human genomics with genecards peroxisomedb 2.0: an integrative view of the global peroxisomal metabolome the mouse age phenome knowledgebase and disease-specifi c inter-species age mapping searching the mouse genome informatics (mgi) resources for information on mouse biology from genotype to phenotype gene-set approach for expression pattern analysis systems analysis of human brain gene expression: mechanisms for hiv-associated neurocognitive impairment and common pathways with alzheimer's disease systems biology approach to identify transcriptome reprogramming and candidate microrna targets during the progression of polycystic kidney disease mir2disease: a manually curated database for microrna deregulation in human disease a new face and new challenges for online mendelian inheritance in man (omim(r)) clinvar: public archive of relationships among sequence variation and human phenotype searching ncbi's dbsnp database dbvar and dgva: public archives for genomic structural variation using electronic patient records to discover disease correlations and stratify patient cohorts on not reinventing the wheel beyond the genomics blueprint: the 4th human variome project meeting comprehensive molecular characterization of urothelial bladder carcinoma open clinical trial data for all? a view from regulators clinical trial data as a public good biobanking for europe whose data set is it anyway? sharing raw data from randomized trials sharing individual participant data from clinical trials: an opinion survey regarding the establishment of a central repository ncbi's database of genotypes and phenotypes: dbgap mining electronic health records: towards better research applications and clinical care phenome connections phewas: demonstrating the feasibility of a phenome-wide scan to discover genedisease associations mining the ultimate phenome repository probing genetic overlap among complex human phenotypes systematic comparison of phenomewide association study of electronic medical record data and genome-wide association study data finding the missing heritability of complex diseases systems genetics: from gwas to disease pathways a review of post-gwas prioritization approaches when one and one gives more than two: challenges and opportunities of integrative omics the model organism as a system: integrating 'omics' data sets principles and methods of integrative genomic analyses in cancer toward interoperable bioscience data critical assessment of human metabolic pathway databases: a stepping stone for future integration the bridgedb framework: standardized access to gene, protein and metabolite identifi er mapping services integration of transcriptomics and metabonomics: improving diagnostics, biomarker identifi cation and phenotyping in ulcerative colitis a multivariate approach to the integration of multi-omics datasets comprehensive molecular portraits of human breast tumours bring on the biomarkers assessing the clinical utility of cancer genomic and proteomic data across tumor types incorporating inter-relationships between different levels of genomic data into cancer clinical outcome prediction the casym roadmap: implementation of systems medicine across europe molecular classifi cation of cancer: class discovery and class prediction by gene expression monitoring how to infer gene networks from expression profi les coexpression analysis of human genes across many microarray data sets discovering functional relationships between rna expression and chemotherapeutic susceptibility using relevance networks reverse engineering of regulatory networks in human b cells comparison of co-expression measures: mutual information, correlation, and model based indices using bayesian networks to analyze expression data probabilistic graphical models: principles and techniques. adaptive computation and machine learning inferring gene networks from time series microarray data using dynamic bayesian networks the biogrid interaction database: 2013 update architecture of the human regulatory network derived from encode data reconstruction of a functional human gene network, with an application for prioritizing positional candidate genes a genomewide functional network for the laboratory mouse string v9.1: protein-protein interaction networks, with increased coverage and integration advantages and limitations of current network inference methods computational discovery of gene modules and regulatory networks a semisupervised method for predicting transcription factor-gene interactions in escherichia coli regression analysis of combined gene expression regulation in acute myeloid leukemia integrating large-scale functional genomic data to dissect the complexity of yeast regulatory networks integrated systems approach identifi es genetic nodes and networks in late-onset alzheimer's disease a survey of the genetics of stomach, liver, and adipose gene expression from a morbidly obese cohort an introduction to systems biology: design principles of biological circuits from signatures to models: understanding cancer using microarrays integrative approaches for fi nding modular structure in biological networks weighted gene coexpression network analysis: state of the art integrative functional genomic analyses implicate specifi c molecular pathways and circuits in autism multi-omic network signatures of disease identifying functional modules in protein-protein interaction networks: an integrated exact approach algorithms for detecting signifi cantly mutated pathways in cancer inference of patient-specifi c pathway activities from multi-dimensional cancer genomics data using paradigm pathway-based personalized analysis of cancer network-based classifi cation of breast cancer metastasis current composite-feature classifi cation methods do not outperform simple singlegenes classifi ers in breast cancer prognosis prediction of human disease genes by humanmouse conserved coexpression analysis a comparison of algorithms for the pairwise alignment of biological networks cross-species analysis of biological networks by bayesian alignment graphalignment: bayesian pairwise alignment of biological networks an oncogenic kras2 expression signature identifi ed by cross-species gene-expression analysis oncogenic pathway signatures in human cancers as a guide to targeted therapies interspecies translation of disease networks increases robustness and predictive accuracy integrated cross-species transcriptional network analysis of metastatic susceptibility pathprinting: an integrative approach to understand the functional basis of disease identifi cation of causal genetic drivers of human disease through systems-level analysis of regulatory networks promoting coherent minimum reporting guidelines for biological and biomedical investigations: the mibbi project data standards for omics data: the basis of data sharing and reuse biomedical ontologies: a functional perspective pdb improvement starts with data deposition what we do not know about sequence analysis and sequence databases annotation error in public databases: misannotation of molecular function in enzyme superfamilies improving the description of metabolic networks: the tca cycle as example more than 1,001 problems with protein domain databases: transmembrane regions, signal peptides and the issue of sequence homology biomedical and health informatics in translational medicine amia board white paper: defi nition of biomedical informatics and specifi cation of core competencies for graduate education in the discipline synergy between medical informatics and bioinformatics: facilitating genomic medicine for future health care elixir: a distributed infrastructure for european biological data enabling transparent and collaborative computational analysis of 12 tumor types within the cancer genome atlas personal omics profi ling reveals dynamic molecular and medical phenotypes tissue-specifi c functional networks for prioritizing phenotype and disease genes the genotype-tissue expression (gtex) project on crowd-verifi cation of biological networks inference and validation of predictive gene networks from biomedical literature and gene expression data verifi cation of systems biology research in the age of collaborative competition dialogue on reverse-engineering assessment and methods: the dream of highthroughput pathway inference revealing strengths and weaknesses of methods for gene network inference wisdom of crowds for robust gene network inference we would like to thank dr. aldo jongejan for his comments that improved the text. key: cord-005078-gr2vioor authors: fedorowicz, jane; gogan, janis l. title: reinvention of interorganizational systems: a case analysis of the diffusion of a bio-terror surveillance system date: 2009-04-03 journal: inf syst front doi: 10.1007/s10796-009-9167-y sha: doc_id: 5078 cord_uid: gr2vioor innovation diffusion theory proposed that adopters—whether individuals or organizations—sometimes reinvent an innovation as they gain experience using it. reinvention can enhance (or impede) the likelihood of an is innovation’s acceptance and further diffusion. this paper reports on a case study of biosense, an interorganizational system that was designed as an early detection tool for bio-terror attacks and subsequently modified to better serve this need as well as to operate as a public health system for pinpointing geographic clusters of dangerous/acute disease outbreaks. by examining the interplay among the political and organizational dynamics and technical properties of the biosense system, we shed light on processes affecting reinvention in an interorganizational context. we discuss our findings in light of theories of the diffusion and reinvention of innovations. we use rogers’ (1995) list of factors supporting reinvention to structure the discussion of the fidelity and uniformity of the innovation within the processes it supports in adopting health services organizations. institutionalization of an innovation is dependent not only on its fit with a variety of user requirements or circumstances, but also on user receptivity toward its implementation processes (goodman et al. 1993; yetton et al. 1999) . "reinvention" refers to the changes or modifications made to an innovation following its adoption and the processes by which the innovation is changed by its adopters (rogers 1995) . complex, process-based innovations that are flexible enough to be reinvented to fit the needs of an adopting organization are more likely to be successfully assimilated into organizational routines. innovative information systems (is) may simplify information processing or analysis tasks, as when an organization adopts a new enterprise system, or the innovative information system may provide access to new data, combinations of data, or new analytical tools, as would be the case when supply chain partners introduce an interorganizational system to share data between buyers and suppliers. the new information system may not necessarily utilize new technologies, but is considered an innovation because it enables changes to extant processes and workflows. since both expected and unexpected changes can take place, care must be taken to align the is innovation with critical organizational tasks, as well as to nurture its adoption and implementation processes (yetton et al. 1999) . the more flexibility inherent in an is-based innovation, the more likely its reinvention will lead to (planned or unplanned) sustainable improvements in organizational work. however, innovation change agents must also ensure that unmonitored or uncontained flexibility does not result in unnecessary organizational churn or user confusion. when the success of the innovation requires buy-in from many organizations as is true with an interorganizational system, the innovation must be malleable enough to fit the requirements, preferences and processes of each partnering organization while preserving the purpose of the collective entity. striking a balance between the ability to customize systems to individuals' or departments' needs (a key benefit of is flexibility) and information-sharing transparency (a key benefit of uniformity) is an ongoing challenge for many organizations. this paper reports on a case study of a public health interorganizational system which was initially designed and promoted as an early detection tool for bio-terror attacks and subsequently was modified to serve a broader and more routine public health purpose: identifying geographic clusters of communicable disease outbreaks. by examining the interplay among emergent political and organizational dynamics and technical properties of the biosense system, we shed some light on the decisions and processes that led to the reinvention of this interorganizational innovation. the paper is organized as follows. first we review prior studies of innovation, with a focus on key findings about reinvention and related processes affecting or resulting from interorganizational system innovations. we explain the methodology employed for the case study, and then describe the biosense initiative, including the context for, design and development of, adoption and early experience with this interorganizational system and reinvention events that took place in the mid-to-late 2000s. we discuss our findings in light of the reviewed literature and offer suggestions for further research on reinvention of interorganizational systems. 2 literature review innovation diffusion theory proposed that adopterswhether individuals or organizations-sometimes adapt or "reinvent" an innovation as they gain experience using it (rogers 1995 ; see also rice and rogers 1980) . herein, we prefer rogers' term "reinvention" to refer to these innovation changes, to distinguish our work from prior is research where the term "adaptation" describes how organizational processes and procedures are changed to accommodate the innovation, without actual changes to the it artifact itself (see for example cooper and zmud 1990) . reinvention usually enhances the likelihood of an innovation's acceptance and further diffusion. reinvention may affect an individual user (as when an individual customizes software developed by someone else to suit his/her specific requirements) or an organization (as when a new scheduling system is introduced to reduce patient waiting time in a medical facility, then modified to better suit the needs of the adopting organization and/or its users). rogers (1995) proposed that reinvention can be spurred by a variety of factors, including: although rogers called for further research on reinvention especially from an organizational perspective (versus user-adopted technologies, which have been extensively studied), few researchers have focused on reinvention. hays (1996) studied how state policies enacted as laws were reinvented as they were diffused into subsequent states, finding that policies were reinvented based on a combination of social learning, political characteristics, and contextual factors. lewis and seibold (1993) consider two components of innovation reinvention, distinguishing between fidelity (how well the reinvention matches the original intent of the design or intended use) and uniformity (referring to the degree of similarity of use across users). they illustrate their framework in a single organization by studying characteristics of adopting individuals. when considering an innovation that is intended to be adopted by a large number of potentially dissimilar organizations, studying both aspects of fidelity and uniformity at the organizational level is likely to increase understanding of the importance and complexity of reinvention in a largescale diffusion (informal) or dissemination (formal) effort. many studies have examined individuals' and businesses' decisions to adopt or not to adopt various is innovations (e.g., chen et al. 2004; plouffe et al. 2001; tan and teo 2000) as well as potential adopters' propensity to innovate (e.g., agarwal and prasad 1998) and their post-adoption attitudes and behavior (e.g., karahanna et al. 1999; parthasarathy and bhattacherjee 1998) . yetton et al. (1999) are careful to distinguish between innovation characteristics that are more likely to impact individual task performance, and implementation processes that are apt to affect group task performance. however, few studies (e.g., tyre and orlikowski 1994) focus on information systems reinvention. one paper, which examined several case studies of the adoption and use of efficient consumer response (ecr) technologies, hinted at reinvention in its conclusion that "each organization will enter into a complex series of interactions with other parties in its industry group … [during which] organizations' knowledge and perceptions of ecr will change, their capabilities will change, and their interactions with industry partners will change." (kurnia and johnston 2000, p. 315) . this study also emphasized the importance of understanding the processes related to interorganizational innovations, and the emergent nature of the system evolution. other studies also suggest that is innovations, as compared with other innovations, are especially susceptible to reinvention. drawing on earlier work on "intellectual technologies" (curley and pyburn 1982 ; see also wildemuth 1999) , lee (1999) notes that, unlike traditional industrial technologieswhich, due to physical limitations, only support a narrow range of functionsinformation technologies are inherently flexible and their uses are constrained primarily by the skills and imagination of designers and users. this flexibility is a central property of is, and lee calls for further research on the implications of this flexibility. if flexibility/adaptability is a central property of is, then reinvention should be a central concern of research in the diffusion-of-innovations stream. however, as noted earlier, most studies in this stream of is research focus on individual users' decisions to adopt new technologies. fewer studies closely examine reinvention processes or outcomes, either in intra-organizational or interorganizational contexts. one review of the extensive diffusion-ofinnovations literature (chin and marcolin 2001) concludes that while much attention has been given to factors affecting potential adopters' attitudes and intentions, further study is needed on "the technological context and interactions such as interface design, data structures, training, and actual usage behavior..." (p. 9, emphasis added). studying the adoption and use of interorganizational systems by collaborating organizations will give researchers insights into the complex world in which both the characteristics of the technology-based innovation and its implementation process combine to determine the role of reinvention in its long term sustainability. although in many ways health care works under a "business" model, its social mission and public nature lead to many different organizational characteristics that are more complex than would be found in a corporate environment. for example, health care professionals must contend with extensive external vigilance (e.g., laws, regulations and government oversight) and unique funding structures (a mix of internal and external sources, the latter usually limited to specific capital projects and loaded with financial and operational restrictions). in addition, unlike the corporate or government sectors, multiple hierarchies exist in the social networks of the medical professions (west et al. 1999; dopson et al. 2002) . doctors have flatter, more informal networks than nurses' hierarchical ones. as a result, doctors are more likely to be effective at influencing peers to adopt or reinvent innovations. these social networks are a dominant means for diffusion of innovation in health care. with their complex organizational structures, tangled regulatory oversight and irregular opportunistic funding for innovations, the health services arena provides a rich and complex background in which to study innovation reinvention and diffusion. an extensive literature exists on the diffusion of innovation in health services delivery (e.g., berwick 2003) . two recent reviews (greenhalgh et al. 2004; fleuren et al. 2004) examined hundreds of published articles; each review proposed a model for enhancing the success of health related innovations and improving the quality of publications in this area. the greenhalgh et al. article concludes with an extensive list of research questions, two of which are particularly relevant to the study related in this paper. they are: "how do innovations in health service organizations arise, and in what circumstances? what mix of factors tends to produce 'adoptable' innovations (e.g. ones that have clear advantages beyond their source organization and low implementation complexity and are readily adaptable to new contexts)?" "how are innovations arising as 'good ideas' in local healthcare systems reinvented as they are transmitted through individual and organizational networks, and how can this process be supported or enhanced?" (p. 617) this paper addresses these questions by reporting on a case study of an innovative interorganizational system (biosense) and its subsequent reinvention by its users. we use rogers' list of factors supporting reinvention to structure the discussion of the fidelity and uniformity of the innovation within the processes it supports in adopting organizations. the biosense case study was part of a larger study of interagency information sharing in egovernment (fedorowicz et al. 2006 & review of other documents available from public sources (journal articles, news accounts, conference presentations). & interviews with three key informants in [2005] [2006] : a statistician at the cdc who played a significant role in designing the biosense system, and two physicians with public health and statistics training who worked in clinical informatics at two participating hospitals who were key players in the diffusion of biosense. a semi-structured interview protocol was utilized, based on a framework that guided all case studies in the larger project. (see appendix.) in interviews lasting one to two hours each, informants were asked to describe their role in biosense and other public health informatics and/or surveillance initiatives and to discuss political, administrative and technical challenges, as well as their thoughts on directions for future interorganizational systems in this domain. the interviews were recorded and professionally transcribed. the authors compared the interview data with the publicly-available sources (from the cdc website, congressional testimony, and other sources) to triangulate on a timeline of events and key facts about biosense and related initiatives. beyond establishing the facts of the biosense case, our analysis of the data utilized an inductive, grounded theory approach. using the constant-comparative method of analysis (see strauss and corbin 1998 ) the authors reviewed the data for themes and sub-themes. analysis started with identification of informants' views regarding political, administrative, and technical aspects, as set forth in our interview guide (appendix). then, consistent with grounded theory, the authors utilized open coding to identify portions of the interview and other data that did not readily fall into the pre-defined categories, along with puzzles and apparent contradictions in the accounts of events and perspectives. two of the initial informants were re-contacted-one via email and the other via several telephone conversationsfor clarification of some of these open issues; they were encouraged to add further comments. a case history was then prepared, which was reviewed by two of our informants. minor changes were then made based on their clarification of events and perspectives. in public health, "surveillance" is the systematic gathering of data about disease outbreaks, so that priorities can be set for dispensing vaccines and medicines, instituting quarantines or taking other measures to contain an outbreak and conduct follow-up work. surveillance is not new; the united states began monitoring cholera, smallpox, plague and yellow fever in 1878. in 1928 all states were required to report on 29 "notifiable" diseases, and in 1961 cdc was given responsibility for aggregating and publishing the states' surveillance data. unfortunately, many diseases are tracked via separate data collection processes and systems, leading to a proliferation of incompatible applications and databases (potts and fraser 2000, p. 5) . realizing that these incompatibilities place constraints on the ability of states to collaborate during widespread outbreaks, many public health experts have called recently for greater coordination in traditional public health surveillance activities. in 1996 the cdc formed a health information and surveillance systems board to coordinate public health surveillance efforts, with broad representation from state and regional public health agencies. in 1999 development of the specifications for a national internet-based health alert network began, and planning soon followed for development of a national electronic disease surveillance system (nedss). cdc announced in 2000: "nedss will electronically integrate and link together a wide variety of surveillance activities and facilitate more accurate and timely reporting of disease information to cdc and state and local health departments. … nedss will include data standards, an internet based communications infrastructure built on industry standards, and policy-level agreements on data access, sharing, burden reduction, and protection of confidentiality." (potts and fraser 2000, p. 7) traditional public health disease surveillance, while vital, operates at a slow pace based on verified outbreaks of notifiable diseases. it is not very effective in responding quickly and effectively to outbreaks of rapid-onset, highly contagious diseases. for example, in 1993 a waterborne parasitic infection in milwaukee sickened 400,000 and killed 100 people (foldy 2004) . this traumatic event sparked several local early detection initiatives in that region, but over the next decade little progress was made on a nationwide basis. according to centers for disease control and prevention director dr. julie gerberding, traditional surveillance procedures, which aim to confirm that a particular disease is involved, emphasize data accuracy and completeness at the expense of timeliness. dr. gerberding stated that during the post-9/11 anthrax attacks in fall of 2001 "the style of not wanting to make a decision until you have all the data gathered and you have the nice tied-up package was a deterrent to effective decision making on a day-to-day basis" (as quoted in altman 2002 ; see also henning 2004; stolberg and miller 2001) . criticism of cdc's problematic response to the 2001 anthrax attacks helped to direct lawmakers' attention to the need for a new kind of surveillance system that would focus on potential bio-terror attacks. in contrast to traditional surveillance which uses data about confirmed diagnoses, near real-time syndromic surveillance aims to "identify illness clusters early, before diagnoses are confirmed and reported to public health agencies, and to mobilize a rapid response…" (henning 2004, p. 7) . as compared with disease surveillance, syndromic surveillance analyzes the symptoms that patients are experiencing (versus confirmed diagnoses, which of necessity come later). with this context in place, we now introduce biosense, followed by a discussion of its reinvention led by its early adopters and proponents. . in the same year, harvard medical school physicians in the health care informatics program at children's hospital medical center in boston began developing a pre-diagnostic syndromic surveillance system. both initiatives involved capturing patient chief complaints data. dr. kenneth mandl recalled that interest in bio-terror surveillance was already rising before september 11, 2001 and accelerated rapidly after the attacks: "i started the bio-surveillance program here in 1999 after discussions with darpa and the hopkins applied physics lab. it was a very early concept at the time. the clinton administration was interested in … protecting the public against bio-terrorist threats [by] … the use of medical and "nontraditional" data sources for purposes of surveillance. [based on a proposal prepared in 1999] we received funding from the agency for health care research and quality in 2000 for a bio-preparedness contract. the work we were doing became very, very popular in 2001. [even before the 9/11 attacks], early in 2001 the federal commitment to bio-surveillance went from $50 million a year to more like $300 million a year. after the attacks in 2001, things really heated up. since we were already on the ground and running, we expanded quite rapidly and i developed a contract with the massachusetts dept of public health to run the massachusetts surveillance system (mss)." thus, before september 11, 2001, work was already underway (in boston and pittsburgh) to develop regional syndromic surveillance systems for rapid detection. at the national level, efforts were initially focused on developing standards and common systems for traditional disease surveillance, but after the 9/11 anthrax attacks the cdc sponsored work on real-time pre-diagnostic syndromic surveillance at the national level, with the biosense system. the biosense project was proposed in 2002 and formally funded in spring 2003, shortly after the beginning of the war in iraq. two other national bio-terrorism initiatives were also funded: biowatch, a network of sensors which capture air samples in key cities to detect known bio-terror agents; and bioshield, which aims to rapidly develop, move and store vaccines and therapeutics such as antibiotics as soon as an outbreak is identified. figure 1 illustrates the relationship of biosense with other u.s. public health activities and systems. nedss is responsible for routine disease surveillance (primarily the traditional but consolidated notifiable disease program, which emphasizes reporting of confirmed disease outbreaks), while bio-sense is for syndromic surveillance, i.e., less precise early detection, based on chief complaints, laboratory orders and other data that help identify symptom clusters (henderson 2003) . table 1 (above) summarizes the evolution of disease surveillance and related work that led to the launching of biosense. with this background on the mission and motivation for developing biosense, we turn now to an examination of how the system and its users adapted to meet a wider spectrum of individual and societal needs. as initially envisioned the focus of the cdc biosense initiative was on developing an early detection tool for bio-terror attacks. the idea was to quickly identify clusters of patients with symptoms related to known biological agents in eleven syndrome groups (fever, respiratory, gastrointestinal, lymphadenitis, specific infection, localized cutaneous lesion, rash, neurologic, botulism-like illness, hemorrhagic illness and severe illness or death; see ma et al. 2004) . biosense was designed to identify a medium to large scale bio-terrorism outbreak rather than a small-scale/ narrow scope attack (such as the anthrax-by-mail attacks that occurred in the aftermath of 9/11) which would likely be picked up by alert clinicians. according to one source, "the principal underlying premise … is that the first signs of a covert biological warfare attack will be clusters of victims who change their behavior because they begin to become symptomatic." . from participating hospitals and clinics, biosense would capture prediagnostic data such as chief complaints (a "chief complaint" is the primary symptom that a patient describes upon arrival at an emergency room or clinic) and laboratory orders (which reveal what evidence the doctor is looking for, as compared with lab test results, which help to confirm the doctor's hunch). chief complaint and lab order data were already being captured and stored in electronic form at many hospitals and clinics. the biosense interorganizational system included tools to aggregate data from multiple locations and to perform statistical analyses that would help to identify abnormal clusters of chief-complaints symptoms, lab orders, and other indicators of bio-terror attacks (loonsk 2004; loonsk et al. 2004; ma et al. 2004; mandl, et al. 2004) . data would be captured in near-real time, aggregated daily, and analyzed once a week unless an unfolding situation warranted a quicker analysis. biosense participants included department of defense (dod) hospital emergency rooms and clinics, veterans affairs (va) emergency departments, va clinics, and lab corp testing locations and patient service centers. when phase i pilot testing began in 2004 (gerberding 2005) the following data were captured: diagnostic codes for chief complaints in dod and va clinics and emergency rooms (up to four codes per patient), several medical procedure codes, laboratory test orders (about 340,000 specimens from lab corp daily), and biowatch sensor data. as of 2008, the biosense system collects data from 432 hospitals, 327 department of defense facilities, and 813 veteran's administration facilities in 10 states. biosense was an is innovation that was reinvented in several ways following its adoption. as discussed in the literature review, rogers (1995) found that reinvention can be spurred by one or more of six factors: changes in adopters' knowledge about what the technology can do, adopters' attempts to simplify innovations that are perceived as overly complex ("simplification"), adopters' need to customize a general-purpose tool ("customization"), adaptation to multiple problems, local "pride of ownership", and encouragement (or pressure) by a change agent. consistent with rogers, we found evidence of reinvention corresponding to many of these factors. recognizing that not all of these factors pertain to the reinvention of a particular innovation, in the following sections we emphasize where and how these factors come into play as we present how biosense was adapted by its users and designers. to begin, we note adaptation-related issues related to the fidelity and uniformity of the biosense innovation, in its dissemination among adopting organizations. when proposed in 2002, biosense was planned as an early detection tool for bio-terror attacks, using "real-time syndromic surveillance." as users learned about how the system worked (signaling changes in adopters' knowledge), some began to doubt the system's ability to achieve its intended goal of real-time detection. several argued that an alert physician was more likely to note an early instance of symptoms indicating a possible bio-terror attack or unusual disease outbreak, as had happened in the 2001 anthrax attacks. so, very early in the biosense rollout, the first shift came in re-articulating the goal of biosense away from noting the first instance of an occurrence of symptoms that might point to an outbreak, to what the designers referred to as "situational awareness": using data to confirm (or disconfirm) an outbreak, to pinpoint where resources are most needed and to direct resources away from localities that do not show clusters of chief complaint symptoms. biosense rapidly delineates geographic clusters of diseases or symptoms, and (even more important, in the view of one informant) helps to verify that a possible cluster is not cause for concern. our informants emphasized also that biosense was designed to spot geographic and time trends in sanitized (or de-identified) data, not to reveal nuances of individual patient data. thus, as adopters gained more knowledge about the system, they were able to adapt its use to be more effectively employed, reflecting in this instance a small shift away from the system's initial mission of "first detection" to "situational awareness." later, its mission and design were broadened further to support more routine disease surveillance activities (such as quickly identifying clusters of patients with symptoms of flu), representing a significant departure from its original intent (illustrating its adaptation to multiple problems). below, we discuss the factors that led to this and other further changes of mission (infidelity) and its use in both bio-terror and natural epidemic detection (reduction in uniformity). public accounts reveal changes in participants' views about biosense. in 2003, joseph henderson, the cdc's director of the office of terrorism preparedness and emergency response, in testimony before a congressional committee stated: "biosense is being developed to support early event detection activities associated with a possible bio-terrorism threat" (henderson 2003) . as time passed and criticism intensified, participants came to realize that the biosense tools that allowed chief complaints data to be shared and analyzed among hospitals could, with some modification also be used to confirm suspicions of naturallyoccurring outbreaks, such as sars or west nile virus, as well as food-or water-borne contamination and other communicable diseases. by 2004, the cdc's cio, jim seligman, was quoted as follows (wolfson 2004) : "we want to make sure the investments we make for terrorism will benefit daily public health, whether we have an event or not. we are trying to avoid building another stovepipe system that would only apply to terrorism and would sit idle 99.9 per cent of the time. the surveillance we are doing for a bio-terrorism event will certainly pick up a naturally occurring event at the same time." seligman's encouragement of reinvention shows how biosense's designers were accepting of changes to its initial mission, in that they promoted flexibility of design (reducing fidelity to the original mission) and use (reducing uniformity of user domains). by doing so, he increased the likelihood of institutionalizing the system by promoting its use in routine situations, and it was no longer reserved for detection of very unlikely events. an article published in 2004 reflected the change in mission for biosense. the article noted that when considering new data sources for the system, the following criteria were employed (per loonsk et al. 2004 earlier presentations about biosense had not listed the "dual-use" criterion. asked about this, an informant explained that in the absence of bio-terror attacks, a consensus had developed among participating hospitals, statisticians and public health officials that they should expand (or customize, in rogers' terms) the system to include symptoms and complaints associated with naturally-occurring outbreaks. with this move to officially condone the dual uses of the system, its designers became supportive change agents for biosense's institutionalization into users' routine processes. the timing was right: no doubt the implications of the sars epidemic abroad was not lost on the biosense participants, and over the next few years the threat of avian flu was also of great concern worldwide. in fall 2006 one interviewee described the change in focus as follows: "for a few years people were saying 'the only thing biosense is any good for spotting is influenza.' now it's like, 'holy cow, this is good for influenza!' there's a lot of federal pressure on biosense to beef up in preparation for monitoring avian flu. last year you could say, 'oh it's just influenza.' this year we're very concerned about influenza." here, the "federal pressure" reflects the encouragement of the funding source acting as a change agent for adapting biosense's mission to cover more routine situations. yet a third potential focus shift was hinted at when one interviewee noted that the statistical tools used in biosense to analyze human syndromes could be applied to outbreaks among animals. if enacted, this would be an even more significant example of adaptation to multiple problems, and would be a more severe example of a uniformity departure than the shift from bio-terrorism to naturally occurring epidemics. thus, while the initial intent was narrowly focused on the design and use of biosense for detecting bio-terror attacks, today biosense has a broader, more routine public health mission, and some participants are exploring a further broadening to include animal outbreaks. biosense has continued to evolve as it becomes more widely adopted and as complementary technologies (chircu and kaufman 2000) become available to support an even more ambitious mission. as can be seen in fig. 2 , the mission of biosense has recently been reinvented yet again in response to changes in the environment. going forward, biosense will aim to "comprehensively monitor the healthcare system of the united states for evidence of acute health threats to the public" by focusing on "early event awareness, health situational awareness, and public health response" ( lenert 2008) . as can be seen in fig. 2 , the cdc is proposing to set up regional collaboratives to work with health information exchanges (hies) and regional health information organizations (rhios), to collect and analyze clinical data identifiable at the facility level. these new organizations represent pressure by the cdc acting as a change agent to adapt biosense to an even broader set of problems, again demonstrating the flexibility of the system to fit the processes designers and users want it to support. this flexibility permits changes to operational processes in addition to technological enablement, as seen in the next section. 6.2 changes to systems and processes yetton et al. (1999) noted that successful adoption at the individual level more frequently involved adjusting innovation characteristics to individual task needs, and at the group level, innovations are significantly impacted by their implementation processes. individuals' reactions to biosense clearly demonstrated the need for simplifying data entry. minimization of manual input was considered especially important. early syndromic surveillance efforts relied on doctors, nurses and administrators to manually fill out checklists of symptoms and other data. this was found to be infeasible; clinicians feel they are simply too busy providing care to take extra time to record information that is not immediately valuable to them. one doctor noted: "if for your bio-surveillance system you require a nurse, administrator or physician to click off new data elements on a daily basis or a per patient basis-even worse!-you're no longer in business." fortunately, in the early years of this century electronic medical records became more pervasive, and in turn more data thus became available in encoded digital form for the biosense effort, enabling adopters to simplify the biosense data capture processes and to increase the likelihood of its success. still, slow progress in health-care standards-setting efforts for free-text clinical descriptions posed an impediment. while much clinical transaction data is now recorded in the national hl7 standard, free-text descriptions of chief complaints are not yet standardized (there is not yet a fully standardized nomenclature for physicians to describe what they hear from their patients). the codes for various procedures and tests are also not yet uniform; while these tend to be homogeneous within a care setting, they are not standardized across settings. thus, the biosense system and its related processes are constrained by the need for changes in health-care processes and systems to yield better and timelier information for rapid analysis. another challenge pertained to incompatibilities between syndromic surveillance based on pre-diagnostic data such as chief complaints, versus traditional public-health processes. this represented an impediment to adopting biosense, as users were concerned about how its use might require changes to existing processes. one interviewee explained: "public health procedures are geared to receiving a single notification of a possible outbreak, during working hours. and, their processes are not designed for rapid reaction to outbreak news. the emphasis is on telephoning individual clinicians, with a focus on individual-case follow-up." designers were called upon to reiterate that "most of our efforts are really not just in counting cases, but in seeing trends and corroborating." users needed this encouragement to be able to distinguish between biosense's syndromic surveillance methods and processes and prior public health efforts in disease surveillance. more recently, biosense designers have begun to take steps toward integrating biosense with the national electronic disease surveillance system (nedss), bringing together syndromic pre-diagnostic (e.g., chief complaints, lab orders) and diagnostic (e.g., lab results, physician diagnoses) data (lenert 2008) . this illustrates the continued pressure by adopters to advance the use and usefulness of biosense by further customizing the tool and expanding its reach beyond its initial mission (infidelity). because it is a public good commodity, biosense designers must consider both these users' needs and the needs of society as a whole in establishing its mission and design, as discussed in the next section. rogers proposed that reinvention sometimes occurs thanks to encouragement by a change agent and this turns out to be critical within health care. early proponents included physicians and others who were recognized nationally for their leadership in health information. these champions took advantage of their considerable professional social networks to advocate for system adoption. however, we note that multiple change agents (cdc, state and local public health officials, the media) exerted both positive and negative political pressures which affected the reinvention of biosense. for example, the addition of the "dual use" criterion in 2004 could well have been motivated by a desire to find common ground with critics. there were also other political issues. as noted earlier, following the 9/11 attacks (and post-9/11 anthrax attacks as well as the iraq war) the total amount of funding for syndromic surveillance increased greatly. in turn, however, this engendered competition as various parties sought to protect their turf. some funds that were previously earmarked for states were transferred to the cdc, which generated some controversy, illustrating the pull of local pride of ownership of a patchwork of systems vs. the added benefits of a national integrated effort. seth foldy, m.d., a spokesperson for the national association of county and city health officials, testifying in 2004 before the u.s. house of representatives articulated the need for improved information sharing between health care providers and public health and safety officials: "in the setting of a communicable disease, a covert bio-terrorism attack, or an environmental emergency, poorly informed decisions by either party result in missed opportunities to prevent injury or illness, sometimes on a massive scale…. improving the timeliness, completeness, and accuracy of information exchange in both directions is a critical goal …" (foldy 2004) . another controversial aspect centered on participants' expectations about the likelihood of future bio-terror attacks. the fall 2001 anthrax attack was only the second deliberate large-scale bio-attack in u.s. history (the first was the contamination of oregon salad bars by a cult in 1984; see mishra, 2001) . as of this writing, the u.s. has seen no large-scale bio-terror attacks since 2001. as time passed, public health officials and informatics coordinators at participating hospitals became concerned that criticism of the initiative might intensify along with the public's perception that bio-terror was unlikely to affect them. by increasing the call for biosense to expand beyond its initial, limited mission, this array of change agents encouraged expansion of the system to cover additional problems, further demonstrating the adaptability of the underlying initiative. foldy also criticized biosense as competing with the broader consolidated public health information network that was far from completed: "biosense … is a worthy, if highly experimental, project for the nation. however, it is essential to remember that it will be local health departments that, when alerted to abnormal disease trends, will do the legwork to validate such suspicions and actually manage the outbreaks. reduced funding for state and local agencies defeats the overall vision. we urge congress and the administration to support instead the larger cdc vision of a public health information network (phin), an enterprise model of information management across local, state, and federal systems, not just a single component. both nationwide projects and local capacity need support, not one at the expense of the other." (foldy, july 2004) as of 2008, the cdc has plans to incorporate regional versions and act as a national "broker" for surveillance data (lenert 2008 ; also see fig. 2 ). this reflects a move to integrate with the myriad of linked public health monitoring systems envisioned by the cdc, congress and the department of homeland security. with these longer range objectives, the collection of surveillance systems with which individuals and organizations interact will employ common, combined and automated data feeds, in effect simplifying input requirements and user effort. the continued reinvention of biosense into a regionally-based and thus customized component of the country's larger public health surveillance system demonstrates the flexibility of the program and its success as a sustainable means to addressing this complex problem. this case study relied on information in publicly available sources, triangulated against information obtained in interviews with three key informants. while these informants were well positioned to provide useful insights, we are certain that much more could have been learned had we been able to interview a greater number of biosense participants. to fully analyze the biosense case, it would be necessary to closely observe participants' behaviors and concerns during its implementation. in contrast, we relied on a limited number of interviews coupled with information found in public accounts. since innovation diffusion is a dynamic process, an ideal research design would be longitudinal. for example, a study reported by tyre and orlikowski (1994) revealed that the process of reinvention (which they termed "technological adaptation") was heavily influenced by timing, with the greatest amount of reinvention taking place shortly after adoption. routinization subsequently led "the technology and its context of use to congeal, often embedding unresolved problems into organizational practice." rather than observing a process of continuous adaptation (as was predicted by many in the literature) tyre and orlikowski observed long periods during which the innovation was not changed, punctuated by occasional short-lived episodes of reinvention. thus, longitudinal studies remain the gold standard for learning about post-adoption behaviors including reinvention. also, organizational and interorganizational is innovations are more likely to be complex than other innovations and thus require a steep post-adoption learning curve (fichman and kemerer 1997; purvis et al. 2001) , which could affect reinvention. furthermore, to derive full value from the adoption of an innovation, an organization may need to invest in complementary technologies and processes (chircu and kauffman 2000; mcafee and brynjolfsson 2008) . so, while the properties of the focal innovation can constrain or enable reinvention, it may well be that the properties of the complementary technologies and aspects of the complementary processes may also affect reinvention. biosense is a good example of a flexible is innovation that users adopted and subsequently reinvented. it illustrates the importance of attending to both users' needs and requirements, and the implementation processes controlled by its designers and other change agents. in health care, initiatives need to balance user viewpoints against benefits to the public good, a role undertaken by government change agents. thus, government support of public health initiatives is necessary to garner the funding for wide-ranging interorganizational initiatives. government involvement also opens access to the vast pools of extant data as well as attracting analysts and other users who would gain from its public good benefits. the biosense system, as initially designed, provided a flexible foundation that supported its subsequent adaptation to other applications. the database, analytical tools and coordination structures were aimed at bio-terror preparedness, yet this foundation proved to be well suited (with modifications) to a much wider range of applications which were adapted over time to support the broader, syndromic surveillance activities for influenza and other common outbreaks. there is no indication in either the interview data or the public record that these subsequent broader applications were anticipated when biosense was first proposed. yet as adopters began to work with the system, and technology emerged that enabled broader and easier application to other societal needs, many individuals worked to expand the mission of the system and proposed its use as a flexible tool for many types of analyses. the biosense case reveals evidence that is consistent with rogers' observations about drivers of innovation reinvention. his six factors contributing to innovation reinvention (adopters' knowledge, simplification, customization, adaptability, pride of ownership, and change agent pressure) were clearly evident in the design and implementation of biosense. there was no single stakeholder that initiated the expanded mission of biosense. rather, knowledgeable adopters reacted to the initial system with suggestions for simpler design and less intrusive processes. they recommended expanding the system to include syndromic surveillance for rapid onset diseases such as influenza and later for an even broader set of medical conditions. meanwhile, some stakeholders balked at replacing locally developed efforts with one imposed on them by a national body. physician opinion leaders promoted and eased implementation efforts among their professional peers. the cdc and other change agents worked to meet both user demands and societal needs for early detection, regardless of the medical origin. the cdc, as initiator of biosense, worked with other change agents to obtain mission-oriented funding. taken together, it is clear that many factors combined to shepherd the system from its origin as a "good idea" to one likely to achieve significant societal benefits. no single concern or reaction determined the reinvention path taken by biosense. instead, many stakeholders contributed to the reinvention through the perspective of all six of rogers' factors, even while recognizing that the system's adaptability was a key contributor to its reinvention. these factors served to reinvent the mission over time, leading to increased flexibility by adapting biosense to a broader scope of problems. biosense continues to be an important tool for use in the (hopefully rare) event of a bio-terror attack, but in addition it is already serving as a useful tool for use by public health officials and clinicians in more common outbreaks of naturally occurring acute diseases. the increase in flexibility reduced fidelity to the initial mission and reduced the uniformity of use by customizing it for other purposes and groups of users. what is the role of reinvention in the diffusion of interorganizational systems? we return to the two questions posed by greenhalgh et al. (2004, p. 617) to highlight biosense findings that would answer this question: "how do innovations in health service organizations arise, and in what circumstances? what mix of factors tends to produce 'adoptable' innovations (e.g. ones that have clear advantages beyond their source organization and low implementation complexity and are readily adaptable to new contexts)?" in this case, we saw a health service innovation arise as a result of the convergence of a political opportunity and a technological solution. on the medical side, we reported on the work of a dedicated set of physician change agents who recognized a gap in an important public health area and saw how information technology could assist in addressing it. they used their extensive professional networks to obtain funding to demonstrate how patient data could be repurposed to detect patterns among symptoms and treatments. at the same time, the government was intent on finding a way to address the homeland security fear of a bio-terror attack, which loosed the purse strings to fund syndromic surveillance projects. the critical events of 9/11 and the anthrax scare plus the availability of data collected in health care is further helped to grown nascent programs. in the case of biosense, we see how political feedback, monitoring of social and technology indicators, or the occurrence of critical events can serve as the impetus for a public sector initiative (kingdon 1995) . designers and supporters of the system were flexible and encouraging in reaction to user requirements and needs. the system itself was adaptable to expanding requirements and user reticence to change processes. every effort was made to not impose additional costs (in time or money) on adopting organizations, and both local and collective benefits accrued from the system's adoption. one very significant "lesson learned" bears note here. the system as originally envisioned was intended to detect infrequent or even highly unlikely events (a bioterror attack). given the low likelihood of such an occurrence, the added value of the system to any one locale could easily be overshadowed by the added "cost" of complying with its extensive data entry requirements. by extending its coverage to more detection of conditions that occur more frequently (such as influenza), the value of the system increased to adopting organizations. the incorporation of a new biosense influenza module for the 2008 flu season is evidence of this transition (lipowicz 2008) . indeed, prior research has found that routine use of a system that also supports emergency situations can greatly increase the acceptance of such a system. for example, the public safety network capwin was initially envisioned to support largescale emergency collaboration. it was only when its value was demonstrated for day-to-day public safety support that it gained a critical mass of adopters (fedorowicz et al. 2006 (fedorowicz et al. , 2007 . adopters and designers of biosense reached a similar conclusion when they added the "dual use" criteria of supporting both bioterror attacks and natural outbreaks. "how are innovations arising as 'good ideas' in local healthcare systems reinvented as they are transmitted through individual and organizational networks, and how can this process be supported or enhanced?" the case also clearly demonstrates the growth of a 'good idea' from its local roots (in massachusetts and pennsylvania) to a national effort (biosense) to the broader network of linked surveillance systems planned for implementation in the next few years. in addition, the case illustrates how users' reactions to features of an innovation can motivate changes, such as the need to repurpose existing data to deal with users' objections to collecting any new data items. the expansion of biosense to cover both bio-terror attacks and natural outbreaks, and from first awareness to situational awareness, and finally from syndrome to disease detection illustrate several stages of reinvention to address multiple problem areas and the addition of other groups of users. because it is a large and complex interorganizational system, the role of both internal and external change agents became clearly evident. the case also shows the important role of physicians' professional social networks in the diffusion process in health care. the involvement of physician change agents, supportive politicians, and thoughtful users was key, as they collectively implemented a system that was minimally intrusive yet provided assistance for a growing set of problem areas. there are still many signs of adaptation, expansion, and integration in the public health surveillance landscape. biosense continues to adapt and expand its operations. for example, in 2008, plans included adding the new influenza module, connecting to health information exchanges and the national health information network, federating with state and local surveillance data bases to create a national system, and integrating lab reporting with nedss. nedss itself is seeking funding to move into twelve remaining states (weiss 2008) . other efforts to detect disease outbreaks continue to develop in parallel with biosense. children's hospital in boston is now promoting its new system healthmap, which scours web sources using rss feeds to detect outbreaks around the world (havenstein 2008) . thus, researchers will continue to be able to study the development of biosurveillance systems over time, to identify patterns of adaptation and better understand the motivations for observed instances of reinvention. furthermore, the domain of emergency response adds a special urgency to this stream of research. in the case of biosense, we are happy to report that our nation has not, at this writing, experienced the sort of bio-terror attack for which it was originally designed and for which it continues to stand at the ready. however, should such an attack occur, there would be great value gained in closely studying how biosense is used in the immediate aftermath and whether such an event would lead the biosense designers and/or users to call for further changes in the system or related processes (such as integration with the biowatch system, which utilizes sensors to capture air samples suggestive of biological or chemical warfare). our results are highly suggestive that the phenomenon of interorganizational systems reinvention is worthy of further, and closer, attention, especially in the domain of emergency response. finally, we concur with lee (1999) , that the flexible adaptability of information systems is an under-studied but important phenomenon. the biosense case leads us to propose that reinvention of information systems is more likely to occur, and the degree of reinvention is likely to be more extensive, than would be true for other innovations. this case illustrates the complexity of reinvention and adaptation within interorganizational systems, particularly the interplay of stakeholder inputs and reactions that can be mapped within the reinvention framework. given the constellation of actors and events affecting systems designed for interorganizational information sharing and collaboration, we believe that in-depth longitudinal case studies offer the most promising methodological avenue for documenting and interpreting the diffusion of interorganizational systems innovation. catalyst prior events/collaborations that provided experience and develop relationships among key participants. events, such as a highly visible disaster or system failure, that triggered the collaboration or propelled it forward. influential supporters within participating organizations or other stakeholder groups. legislative and regulatory requirements that gave rise to a collaboration or constrained its implementation. governance organizing agreements and structures (boards, steering committees, etc.). vision and goals of the collaboration, initially and as the system matures and new participants join. implementation changes required in organizational processes and relationships to support the collaboration. training, staffing, change management. financing funding for the system design and implementation. plan for the long term financial and operational viability. legacy systems previously-installed applications or databases that constrained the design or capabilities of the new system. decisions regarding data sources, definitions, ownership, access rights and restrictions, and stewardship. standards and sourcing criteria decisions regarding de jure or de facto standards data, devices, and interoperability. decisions regarding use of open-source or commercial software for back-end and user-facing technologies. congress authorizes us marine hospital service to monitor cholera, smallpox, plague and yellow fever first summary of notifiable diseases from 19 states all states provide monthly summaries of 29 notifiable diseases cdc assumes responsibility for notifiable disease data collection and publication steering committee on public health information and surveillance system development national electronic disease surveillance system (nedss) project launched. real-time outbreak & disease surveillance (rods) system under development at u. of pittsburgh medical center. real-time syndromic surveillance system proposed by children's nedss architecture v1.0 and public health conceptual data model v1 nedss assessment and planning phase started in 43 locations. nedss architecture v2.0, logical data model overview v1.0, and logical data model data dictionary published cdc recommends that the american college of emergency physicians adopt nedss standards. public health and social services emergency fund provides $1 billion for state and local public health preparedness nedss base system v1.01 released and made available to all states. new $1 billion preparedness award to states and public health agencies, with stipulation regarding phin standards claire broome states that biosense part i is operational (phase i pilot testing) and includes data from an initial set of data providers such as dod and the veterans administration describes the phin functional requirements for systems implemented to collect, integrate, and analyze data from heterogeneous information sources for the early discovery of a potential public health emergency biosense v2.10 released biosense reported collecting data from 432 hospitals, 327 dod facilities, 813 va facilities a conceptual and operational definition of personal innovativeness in the domain of information technology at disease centers, a shift in mission and metabolism. the new york times disseminating innovations in health care consumer acceptance of virtual stores: a theoretical model and critical success factors for virtual stores the future of diffusion research. the data base for advances in information systems limits to value in electronic commerce-related it investments information technology implementation research: a technological diffusion approach intellectual technologies: the key to improving white-collar productivity no more magic targets! changing clinical practice to become more evidence based the egovernment collaboration challenge: lessons from five case studies a collaborative network for first responders the assimilation of software process innovations: an organizational learning perspective statement on behalf of the national association of county and city health officials before the subcommittee on technology, information policy, intergovernmental relations and the census, house committee on government reform. hearing on "health informatics: what is the prescription for success in intergovernmental information sharing and emergency response? determinants of innovation within health care organizations: literature review and delphi study implementing a national biodefense strategy. testimony before the u.s. house of representatives committee on homeland security, subcommittee on prevention of nuclear and biological attack development of level of institutionalization scales for health promotion programs diffusion of innovations in service organizations: systematic review and recommendations. the milbank quarterly infectious disease surveillance 2.0: crawling the net to detect outbreaks influences on reinvention during the diffusion of innovations cdc's disease surveillance systems efforts. testimony before the subcommittee on emergency preparedness and response, select committee on homeland security, united states house of representatives what is syndromic surveillance? information technology adoption across time: a cross-sectional comparison of pre-adoption and post-adoption beliefs agendas, alternatives, and public policies the need for a processual view of interorganizational systems adoption researching mis exchange of health information between public health and clinical care: the vision, challenges, and benefits innovation modification during intraorganizational adoption healthy opportunity: biosense project can bring dollars and prestige to winning contractors the biosense update. presentation biosense: a national initiative for early detection and quantification of public health emergencies implementation of laboratory order data in biosense early event detection and situation awareness system implementing syndromic surveillance: a practical guide informed by the early experience investing in the it that makes a competitive difference a heightened alert for bio-terrorism: detection system is keeping health officials apprised. the boston globe understanding postadoption behavior in the context of online services richness versus parsimony in modeling technology adoption decisions: understanding merchant adoption of a smart card-based payment system national electronic disease surveillance systems stakeholders meeting report. cdc: march the assimilation of knowledge platforms in organizations: an empirical investigation reinvention in the innovation process diffusion of innovations bio-terror role an uneasy fit for the cdc. the new york times basics of qualitative research: grounded theory procedures and techniques factors influencing the adoption of internet banking windows of opportunity: temporal patterns of technological adaptation in organizations syndrome and outbreak detection using chiefcomplaint data: experience of the real-time outbreak and disease surveillance project seven years and counting: national diseasetracking system still unfinished hierarchies and cliques in the social networks of health care professionals: implications for the design of dissemination strategies an ounce of prevention: jim seligman, cio, centers for disease control. cio insight. online at www successful is innovation: the contingent contributions of innovation characteristics and implementation acknowledgements we wish to thank our biosense contacts for their assistance on this case, and the ibm center for the business of government, which provided support. we also thank christine williams, who contributed valuable insights on this case and others in the broader study. interview and analysis guide (from project documents, web sites, and interviews) jane fedorowicz, the rae d. anderson professor of accounting and information systems, holds a joint appointment in the accountancy and information & process management departments at bentley university. dr. fedorowicz earned ms and phd degrees in systems sciences from carnegie mellon university. she is principal investigator of a national science foundation project team studying design issues for police and government agency collaboration using public safety networks. she also served as principal investigator for the bentley invision project, an international research team housed at bentley examining interorganizational information sharing and the coordination infrastructures supporting these relationships in supply chain, government, and health care. dr. fedorowicz has published over 100 articles in refereed journals and conference proceedings. the association for information systems recognized her contributions to the information systems field by naming her an ais fellow in 2006.janis l. gogan holds edm, mba, and dba degrees from harvard university. a member of the information & process management faculty at bentley university she teaches it management courses and conducts field-based research on complex it projects, inter-organizational information sharing under time pressure, and it-enabled innovation in health care. dr. gogan's publications include teaching cases which have been taught in u.s, european, australian and asian schools, and more than 85 papers in refereed conference proceedings and journals (such as communications of the association for information systems, electronic markets, international journal of electronic commerce, and journal of management information systems). key: cord-016912-vnx74hft authors: kornguth, s. title: strategic actionable net-centric biological defense system date: 2005 journal: defense against bioterror doi: 10.1007/1-4020-3384-2_1 sha: doc_id: 16912 cord_uid: vnx74hft technologies required for strategic actionable net-centric biological defense systems consist of : 1) multiplexed multi-array sensors for threat agents and for signatures of the host response to infection; 2) novel vaccines and restricted access antivirals/bacterials to reduce emergence of drug resistant strains preand post-event; 3) telemedicine capabilities to deliver post-event care to 20,000 victims of a biological strike; and 4) communication systems with intelligent software for resource allocation and redundant pathways that survive catastrophic attack. the integrated system must detect all threat agents with minimal false positive/negative events, a seamless integrated broad-band communications capability that enables conversion of data to actionable information, and novel preand post-event treatments. the development of multiplexed multi-array sensors, appropriate vaccines and antibiotics, and integrated communication capabilities are critical to sustaining normal health, commerce, and international activities. the overarching objectives in developing effective countermeasures to biological threats are to protect the defense community and citizenry from such threats, and to develop agile responses to unanticipated events considering that successful terrorists do the unexpected. the need for protection against and responses to biological threats has been strikingly demonstrated by the use of anthrax contaminated letters that were sent through the u.s. mail in october 2001. that attack resulted in human illness, the loss of life, and discontinuity of government operations because of contamination of federal office buildings in washington, dc. a recent report prepared by the center for strategic and international studies (csis) and supported by the defense threat reduction agency (dtra) of the department of defense (dod) came to the conclusion that the u.s. is at present not well prepared for a similar attack using anthrax1. the major problems include a lack of: 1) a clear chain of command, and 2) tools to provide the public with information that permits appropriate responses. the incidence of congo-crimean hemorrhagic fever in afghanistan, an area where coalition forces are being deployed, increases this need. the potential threat posed by emergent disease (e.g., severe acute respiratory syndrome [sars] and west nile fever virus) or from a major release of a contagious biological agent such as smallpox, has been a growing concern at all levels of the international community. this article outlines and discusses a new strategy that is needed if we are to be fully capable of sensing, preventing, and managing biological threats. the current paradigm addresses biological and chemical terrorist threats in a vertical (stove-piped) response. in the arena of developing sensors for the detection of biological agents, the paradigm has been to develop separate detectors for each agent or to develop a platform for detecting 12-24 threat agents using a single probe for each agent. there is a lack of an interactive networked communication system that is capable of managing a devastating emergent disease. to establish a highly networked system that is rapid enough to permit effective protection, it is necessary to evolve from the stove-piped, compartmentalized model currently in use to an integrated, fused, probabilistic, and frequently updated information model. multiplexed multi-array sensor systems, capable of recognizing all bacterial or viral genomic materials related to pathogenicity or of recognizing antigenic domains that are specific indicators of pathogens are one component of a network needed for rapid detection and identification of biological threats. with respect to therapeutics, modern technologies for vaccine and antibiotic production provide decided advantages over older methods. the traditional vaccines require extensive development times before they become available for human use and undesired side effects commonly result from vaccines produced by these protocols. the cost associated with developing and testing vaccines, using traditional technology, approximates 50-100 million dollars per vaccine. the dissemination of antibiotics and antivirals through the world markets has resulted in the appearance of pathogenic bacteria and viruses that are resistant to these drugs. one approach to reduce the development of antibiotic resistance is to restrict the distribution of newly developed antibiotics. such an approach presents ethical and social dilemmas. the consideration of options available for reduction of drug resistance, prior to a threat event, may permit development of a rational policy. a major problem facing our nations in the event of a biological attack or emergent disease is the large numbers of patients that can be anticipated to require medical treatment. although improvements in emergency medical care and hospital equipment have been achieved during the past two decades, the ability of any community to manage an outbreak of infectious disease affecting >10,000 people is lacking. rapid progress has been achieved whereby medical care can be provided to patients at sites that are distant from primary caregivers using telecommunication systems (e.g., the armed services in theater, large scale hmo providers such as kaiser permanente, or the correctional institutions in the u.s.). the funds needed to acquire telecommunication equipment for such distributed medical care delivery are estimated to be less than 100 million dollars for the entire u.s. at the present time such a distributed care system is not readily available. the new paradigm couples a network centric integrated sensor alert system that can detect all threat agents simultaneously, with a seamlessly integrated communication software capability that converts large scale data to actionable information. for this to be effective, the sensor system must yield minimal false positive and false negative results. the new paradigm incorporates large-scale databases on normative values of threat agents in many regions of the world so that significant changes in real time can be detected. the paradigm also includes the development and implementation of novel pre-and post-event treatment capabilities. attention must be paid to the ability of high level decision makers and operators to recognize that a new state of threat has emerged, based upon output of the sensors, data fusion system, and iconographic display. ambiguity of data, lack of an autonomous processing system, and high stress on the operator (e.g., sleep deprivation, lack of training) may all compromise the utility of a highly networked system of systems. what is needed for this new paradigm to succeed? the needs include multiplexed multi-array sensors for biological agents that infect people, livestock, and edible crops. the agents of concern include many on the militarily critical technologies list prepared for the office of the secretary of defense. we need multiplexed multi-array sensor systems with high specificity and selectivity for the rapid detection of host responses to infection. we need a new generation of effective therapeutics, including vaccines, antibiotics, and antivirals. a network centric intelligent communication system that can provide accurate comprehensible information to decision makers (from command officers to unit operators) is required. to minimize morbidity and mortality and optimize containment of disease, a biosurveillance system based on archival health databases, statistical models, and data mining strategies that can provide an early alert to a disease outbreak is required. in many cases the operator may be required to understand the meaning of acquired data in very short time periods (seconds to minutes) if the response is anticipated to change the outcome of an engagement. the tactical decision making under stress (tadmus) program is one example of such a situation. in the biological threat arena, the detection and identification of toxins require rapid analysis and operator comprehension. the large increase in numbers of sensors (for high explosives [hx], biological and chemical agents, meteorological conditions) together with the rapid changes in op tempo required to manage emergence of clinical disease would suggest a need for the development of systems capable of autonomous generation of an alert when threat conditions arise. in the sensors area, the genomes of most biological threat agents have been sequenced and the signatures of toxins described. novel multiplexed multi-array sensor-platform systems utilize the genomic datasets to detect the appearance of threat levels of these agents. in the therapeutics area, researchers are working towards identifying critical antigenic epitopes of these agents. new therapeutics can emerge that have an antigen binding capacity significantly greater than antigen-cell receptor binding, resulting in the potential for agent neutralization. technologies have been developed over the past decade for the development of new drugs and dna based vaccines. restricted access antivirals/antibacterials will need to be developed to reduce the emergence of drug resistant strains pre-and post-event. a significant development in our program at the university of texas at austin (ut-austin)2 has been the novel design and production of an antibody that binds the anthrax pa antigen 1000 times stronger (kd<10-11) than any antibody to date. the antibodies were produced using phage display technology for selection of the antibodies. in tests with experimental rodents in a controlled facility, administering the bacillus anthracis pa antigen to the animals resulted in 100 percent fatalities, whereas the co-administration of the newly developed antibody against the pa antigen resulted in 100 percent survival2. research is also being conducted to determine unique nucleic acid sequences in the genome of pathogenic bacteria and viruses that contribute to the pathogenic properties of the organisms. this information is being used to develop multiplexed assay systems that can detect selected agents simultaneously. by quickly screening for multiple pathogenicity island sequences or pathogenic factors, end-users will have the capability to detect the first signs of emergent disease without requiring screening for each particular organism. in the communications area, researchers are developing 'belief maintenance' software to provide decision makers some estimate of the validity of incoming data. an estimate of information credibility is critical to effective decision-making in crisis situations when one must rely on an 80 percent solution (i.e., 80 percent of needed information is available). waiting for a 100 percent solution could have a catastrophic impact on response effectiveness. in the area of telecommunications, researchers are developing the means to provide effective medical triage to victims in a contaminated hot zone. the hot zone in this case refers to a region experiencing an outbreak of a highly contagious disease that causes death in a significant percentage of infected individuals. advanced telecommunications technology can permit physicians and other medical experts at remote locations to provide medical information and support care delivery to personnel in the hot zone. prior training of these personnel (from physicians to local citizens) is required. an extensive use of local persons will be necessary if it is deemed inadvisable to introduce health care workers and communications experts into the hot zone; the external support teams are required to provide 'back-fill' support to the overburdened local community. biosurveillance systems are being developed to serve as an early warning of emergent disease. a variety of databases are being developed that are health related. examples of these databases include school absenteeism, over-the counter drug sales, hospital emergency clinic visits, and archival records on the incidence of diseases in different geographic regions, conus and oconus. each database must be statistically characterized regarding parameters such as variance, confidence intervals, seasonality, etcetera, and be integrated into validated predictive models. once the reference databases are in place and suitably modeled, statistically significant departures from baseline values can be detected and transmitted in real time to decision makers through intelligent communication systems. a number of critical technology gaps exist that must be addressed if we are to recognize, prevent, and minimize the effect of biological agents. these gaps include: deficiencies in the availability of multiplexed multi-array agent sensors and platforms; critical reagents; capability for large-scale production of effective vaccines, antibiotics and antivirals; ability to treat a large number (10,000) of infected people 24/7 for several weeks in a biological hot zone; archival biosurveillance databases and intelligent and secure communications networks. with the new capabilities and devices anticipated during the next decade, approaches that address these gaps include the use of autonomous (e.g., cell phone-based) microelectronic detectors for the transmission of data on agent exposure, development of novel antibodies, antibiotics and antivirals to manage disease outbreaks, and establishment of global surveillance systems for emergent diseases (e.g., sars, west nile fever, congo-crimean hemorrhagic fever). because of the broad scope of needs for technology to prevent and minimize biological threats, a number of research areas have been identified as critical. these include: the scientific validation that a biological incident has occurred (requisite tools/capabilities include situation awareness systems, sensors and signatures); the availability of medical countermeasures (vaccines, pharmaceuticals, and medical transport); and a highly effective communications network for the secure transmission of data and the conversion of such data to comprehensible information so that decision makers can take appropriate actions. for effective sensors, a variety of materials are being developed that include effective high-affinity binders of biological threat agents. the high affinity binders include antibodies, cdna gene probes, polynucleotide aptamers, and combinatorial chemicals. using phage display methods, antibody fragments can be selected that have a high affinity for agents such as anthrax toxin and brucella. another binding system that has been examined uses polynucleotide aptamers about 31 nucleotides long that have good binding affinity to ricin toxin. these sensor materials require opto/electronic transduction platforms. sensor platform research currently is being focused on micro-electro-mechanical systems (mems) devices, microelectronics technology, microfluidics (laboratory-on-a-chip), dna/protein microarrays, and transduction devices. efforts are also being directed toward the development of multiplexed multi-array systems that detect approximately 100 biological threat agents of concern. for military application, it is essential that sensor systems can detect and identify agents present in samples rapidly, using platforms that are small and have low power requirements. with current approaches, the development-to-market of new vaccines, antibiotics, and antivirals is in the order of 5-10 years. a paradigm shift to newer culture and dna-based technology is needed if we are to have an effective response to a major biological or chemical event. current estimates regarding the time required for developing and fielding new vaccines and antibiotics/antivirals to specific threat agents, using new technology and expedited approval, is in the order of three years. while current computer/informatics research includes the development of telecommunications assets, a critical need in the communications area is the development of seamless integrated communication networks. these network centric systems enable the conversion of data to actionable information. research is being conducted to provide intelligent agent software designs for such communication systems. this will enable an enhanced accuracy in critical decision-making and resource allocations. the integrated system must have redundant pathways that can survive a catastrophic attack. the communication system must be capable of integrating data on an emerging threat in a timely manner, and provide useful information for public safety coordination and perimeter management. telemedicine capabilities can aid in the delivery of post-event care to 10,000-20,000 victims of a biological strike in a densely populated area for 24 hours a day, seven days a week, for several months. in the event of smallpox attack in which 10,000 people develop clinical symptoms of infection within 7-10 days following exposure, local hospitals and medical response capabilities would be overwhelmed if current treatment protocols were used. telemedicine allows physicians at a remote location from the hot zone to provide medical support via telemedicine capabilities (visual, audio) to aid local physicians in treating patients. a treatment level of 50 patients per day per physician would require 200 physicians to provide telemedicine care for 10,000 patients. each physician would require telemedicine devices; hence 200 telemedicine devices would be required at the remote location, and a similar number in the hot zone. a national telemedicine system could include the establishment of approximately eight telemedicine response centers nationally, interconnected via satellite to telepresence and telemedicine/robotic systems. the remote care capability reduces the likelihood of the dissemination of disease to physicians and communities in which the physicians reside. a telemedicine system would also retain health care delivery in communities providing health care back-fill. the end goal is achievement of a rapid appropriate action in response to detection of threat (within minutes of threat identification). in order to meet this time constraint in a real world scenario, it is probable that the man-inthe-loop may have a denial capability rather than an approval function. the tools required for autonomous weighting of data and subsequent reduction of data elements for particular missions remain to be developed and agreed upon. the technologies required for data acquisition, communication, and autonomous processing fundamentally differ from that required for comprehension by an operator. the three technologies are systems that must be developed prior to deployment and will have known probabilities of accuracy and reliability. the fourth element involves the training and state of vigilance/alertness of the operator as well as the development of software (e.g., icons, data mining, and compression) used to display threat conditions. the point of the fourth element is to permit an operator to have rapid comprehension of the state of threat in a rapidly changing environment. because there is a time factor involved in the comprehension of threat conditions by an operator, and in the translation of the information into action, the fourth element must include temporal qualities. since the time dependence (seconds to minutes) of an intense threat situation will differ from that involved in a peacekeeping or supply distribution situation (many minutes to hours on the average), the autonomous weighting factors and data reduction factors can be expected to vary widely. this variability complicates programming of autonomous systems. command teams on the modern information-age medical delivery front face an increasing variety of cognitive stressors: information ambiguity, information overload, fatigue, and fear of contagion and quarantine. there is a requirement for a useful, predictive model of the effect of these stressors on individual and collective cognition within the medical delivery team. a model to quantify stress experienced by the caregiver and to identify countermeasures and mitigators of stress, develop organizational strategies for optimum performance under stress is needed. psychological assessments that can predict individual and team cognitive functioning and physiological markers that can determine quantitatively and objectively the effect of stress experienced on an operators vigilance have been identified ( table 1 ). the identification of specific physiological markers that are predictive of stressinduced changes in complex cognitive functioning will aid in the construction of autonomous weighting systems. lessons from the anthrax attacks: implications for u.s. bioterrorism preparedness. csis funded by the defense threat reduction agency of the department of defense. david heyman investigator protection against anthrax toxin by recombinant antibody fragments correlates with antigen affinity the research reported in this document was performed in connection with contract number daad13-02-c-0079 with the u.s. edgewood chemical biological command. 1. perception/comprehension 2. primary issue of concern 3. full situation awareness is contingent on at least four elements including: 4. large scale acquisition of data (i.e., sigint, masint, humint, sensors) 5. high fidelity communication of data sets to autonomous processing centers 6. data fusion involving weighting of data and marked reduction in data volume to yield information that provides users a common operational picture 7. rapid comprehension of time dependent information by operators facilitated by new iconographic displays, training, measures of vigilance key: cord-012370-omz7kibf authors: dixit, shivani; shrivastava, pankaj; kumawat, r. k.; kaitholia, kamlesh; dash, hirak ranjan; sharma, harsh; choubey, gyaneshwer title: forensic genetic analysis of population of madhya pradesh with powerplex fusion 6c(™) multiplex system date: 2019-02-14 journal: int j legal med doi: 10.1007/s00414-019-02017-0 sha: doc_id: 12370 cord_uid: omz7kibf performance of powerplex fusion 6c kit (pp f6c) was assessed in 374 unrelated individuals belonging to madhya pradesh, an indian state. the study evaluated the forensic parameters for the loci included in pp f6c multiplex system. the combined discrimination power (cpd) and combined exclusion power (cpe) were 1 and 0.999999995, respectively, for all 23 autosomal str loci. se33 showed the greatest power of discrimination (0.990) in the studied population, whereas tpox showed the lowest (0.843). the availability of three y-str loci in the multiplex system is suitable for assessing male contribution and amelogenin deletion in a single multiplex pcr simultaneously. the study also presents the first global report on polymorphism in the indian population on se 33 autosomal str loci and pp fusion 6c multiplex system. the results revealed that the studied str multiplex system is highly polymorphic and suitable for forensic purposes. electronic supplementary material: the online version of this article (10.1007/s00414-019-02017-0) contains supplementary material, which is available to authorized users. description present capillary electrophoresis and pcr-based str analysis using expanded combined dna index system (codis) 20 markers are state of art in forensic dna typing [1] . for performing the identification conclusively and precisely with a significant probability value, an increase in the number of loci is recommended, as the discrimination power of any multiplex system is based on a combination of all the independent loci tested. madhya pradesh is the second largest state in india by area. the population of madhya pradesh is 72,597,565 comprising of 37,612,920 males and 34,984,645 females, contributing 6% to india's total population as per the 2011 census. a few studies have been performed to characterize the population of madhya pradesh on autosomal strs (described in table s2 ) but these studies are based on 15 str markers either using identifiler/identifiler plus (applied biosystem, usa) or powerplex 16/16hs (promega corporation, madison, usa). based on six dyes, pp f6c system (promega corporation, madison, usa) is a 27-locus multiplex that ensures simultaneous detection of 20 autosomal loci in the expanded codis (csf1po, fga, th01, tpox, vwa, d1s1656, d2s1338, d2s441, d3s1358, d5s818, d7s820, d8s1179, d10s1248, d12s391, d13s317, d16s539, d18s51, d19s433, d21s11 , and d22s1045), three more loci for better discrimination (penta d, penta e and se33), amelogenin for sex determination and dys391 and two rapidly mutating y-str loci, dys570 and dys576 for further confirmation of gender. a multi-laboratory evaluation of the pp f6c system has already been published [2] following swgdam guidelines [3] . this study is the first global attempt to work out the efficacy of pp sagar, madhya pradesh, india. the samples included in this study have not been typed by any other multiplex system and this is the first study from india detailing the forensic efficacy of a 27-marker multi-utility str multiplex system. following the code of ethics of the world medical association (i.e., declaration of helsinki) for experiments involving humans, peripheral blood samples of these unrelated individuals were collected. all individuals provided prior written informed consent. no minor was involved in the study. after dna extraction and pcr, analysis was performed on 3500xl genetic analyzer, and statistical evaluation was done as described before [4] . the peak height threshold was 50 rfu for heterozygous and 200 rfu for homozygous alleles. internal laboratory control standards and kit controls were used at all steps of analysis. the authors have passed the proficiency test of the gitad, spain (http://gitad.ugr.es/principal.htm). this article follows the population data publication guidelines formulated by the journal. the allele frequencies and results of the forensic efficiency parameters for the 23 autosomal str loci under study are given in table s1 . the frequency values observed in this study were compared with the previously published data (table s2) on 17 autosomal str markers from india and with recently published data on 22 autosomal str markers [5] . a total of 334 alleles were observed in the population of madhya pradesh, with se 33 possessing the highest (46) number of alleles and tpox with lowest (05) number of alleles (table s1 and fig. 1) . of the 374 samples tested, one sample showed amelogenin deletion, with x-profile on amelogenin marker and deletion on dys576 and dys570 marker with amplification on dys391 marker (fig. s1 ). the studied population followed the hardy-weinberg equilibrium for all loci except for d21s11, d7s820, tpox, d1s1656, d2s441, d10s1248, and fga after the bonferroni correction. the observed and expected heterozygosity in the studied multiplex system ranged from 0.577 to 0.877 and 0.661 to 0.939, respectively. the most polymorphic and discriminatory str loci in the studied population were se 33 with values of 0.94 and 0.990, respectively. all the loci included in the studied multiplex system were found to be polymorphic (pic value = < 0.62 to 0.94). the power of exclusion (pe) ranged from 0.331 (d22s1045) to 0.770 (se 33) with a value of greater than 0.999999995 for the combined power of exclusion (cpe). the power of discrimination (pd) ranged from 0.843 (tpox) to 0.990 (se 33) with a value of one for the combined power of discrimination (cpd). the combined probability of the match (cp m ) and combined paternity index (cpi) for all 23 autosomal str loci were found to be 4.4 × 10 −29 and 1.47 × 10 8 , respectively. therefore, the 23 autosomal str loci exhibited high efficacy and informativeness for forensic investigation and individualization purposes. the data obtained in this study were compared with published indian population data (table s2 ) related to common 15 autosomal str loci. pairwise fst matrix between the studied population (marked as madhya pradesh population) and reference populations at the common 15 loci have been shown in table s2 . nj tree and pca plot are given in fig. s2 and fig. 2 , respectively. the distribution pattern of populations demonstrated that the genetic affinity has existed within ethnic origins and geographically close populations. the studied population was also compared with previously published data of indian population [5] and guangdong population of china [6] at common 22 loci and nj tree was constructed using poptree2 software. the studied population (marked as madhya pradesh population) made a pool with previously published indian population reflecting the same origin and guangdong population of china was found an outlier (fig. 3) . pca plot drawn with the same populations showed that all the compared populations are genetically distant. indian population [5] and the studied population have the same origin but during evolution, they became genetically different which is reflected in pca plot. thus, the nj tree and pca analyses, both were consistent with our observations in the studied population. for the y-linked loci dys391, dys570, and dys576, estimated heterozygosity was observed as 0.32, 0.79, and 0.81 respectively (fig. s3 ). mean genetic diversity and mean genetic distance between individuals based on the cumulative values observed by the analysis of three y-strs included in pp f6c were found to be 0.973 and 5.838, respectively. the study will add to the data bank of various studies conducted on the indian population. with respect to the distribution of alleles at each str locus, the loci were found to be substantially polymorphic in this population indicating good informativeness of all studied autosomal str markers. in addition, the multiplex system is found suitable in deciphering the amelogenin deletion to avoid misinterpretation and/or to assess presence of male dna (if the dna is male mixed). the interested researchers can request the data from the authors. selection and implementation of expanded codis core loci in the united states developmental validation of the powerplex® fusion 6c system validation guidelines for dna analysis methods genetic polymorphism study at 15 autosomal locus in central indian population population genetic analyses and evaluation of 22 autosomal strs in indian populations analysis of genetic polymorphisms and mutations at 23 autosomal str loci in guangdong han the authors gratefully acknowledge the support of director, state forensic science laboratory, sagar (mp) for providing laboratory facility and promega (india) for providing the powerplex fusion 6 c for testing its efficacy on the indian population. conflict of interest the authors declare that they have no competing interest.open access this article is distributed under the terms of the creative comm ons attribution 4.0 international license (http:// creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the creative commons license, and indicate if changes were made.publisher's note springer nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations. key: cord-018336-6fh69mk4 authors: yasnoff, william a.; o'carroll, patrick w.; friede, andrew title: public health informatics and the health information infrastructure date: 2006 journal: biomedical informatics doi: 10.1007/0-387-36278-9_15 sha: doc_id: 18336 cord_uid: 6fh69mk4 what are the three core functions of public health, and how do they help shape the different foci of public health and medicine? what are the current and potential effects of a) the genomics revolution; and b) 9/11 on public health informatics? what were the political, organizational, epidemiological, and technical issues that influenced the development of immunization registries? how do registries promote public health, and how can this model be expanded to other domains (be specific about those domains) ? how might it fail in others?why? what is the vision and purpose of the national health information infrastructure? what kinds of impacts will it have, and in what time periods? why don’t we have one already? what are the political and technical barriers to its implementation? what are the characteristics of any evaluation process that would be used to judge demonstration projects? biomedical informatics includes a wide range of disciplines that span information from the molecular to the population level. this chapter is primarily focused on the population level, which includes informatics applied to public health and to the entire health care system (health information infrastructure). population-level informatics has its own special problems, issues, and considerations. creating information systems at the population level has always been very difficult because of the large number of data elements and individuals that must be included, as well as the need to address data and information issues that affect health in the aggregate (e.g., environmental determinants of health). with faster and cheaper hardware and radically improved software tools, it has become financially and technically feasible to create information systems that will provide the information about individuals and populations necessary for optimized decision-making in medical care and public health. however, much work remains to fully achieve this goal. this chapter deals with public health informatics primarily as it relates to the medical care of populations. however, it should be emphasized that the domain of public health informatics is not limited to the medical care environment. for example, information technology is being applied to automatically detect threats to health from the food supply, water systems, and even driving conditions (such as obstacles on the roadway beyond the reach of visible headlight beams), and to assist in man-made or natural disaster management. monitoring the environment for health risks due to biological, chemical, and radiation exposures (natural and made-made) is of increasing concern to protecting the public's health. for example, systems are now being developed and deployed to rapidly detect airborne bioterror agents. although they do not directly relate to medical care, these applications designed to protect human health should properly be considered within the domain of public health informatics. public health informatics has been defined as the systematic application of information and computer science and technology to public health practice, research, and learning (friede et al., 1995; yasnoff et al., 2000) . public health informatics is distinguished by its focus on populations (versus the individual), its orientation to prevention (rather than diagnosis and treatment), and its governmental context, because public health nearly always involves government agencies. it is a large and complex area that is the focus of another entire textbook in this series (o'carroll et al., 2003) . the differences between public health informatics and other informatics specialty areas relate to the contrast between public health and medical care itself (friede & o'carroll, 1998; yasnoff et al., 2000) . public health focuses on the health of the community, as opposed to that of the individual patient. in the medical care system, individuals with specific diseases or conditions are the primary concern. in public health, issues related to the community as the patient may require "treatment" such as disclosure of the disease status of an individual to prevent further spread of illness or even quarantining some individuals to protect others. environmental factors, especially ones that that affect the health of populations over the long term (e.g. air quality), are also a special focus of the public health domain. public health places a large emphasis on the prevention of disease and injury versus intervention after the problem has already occurred. to the extent that traditional medical care involves prevention, its focus is primarily on delivery of preventive services to individual patients. public health actions are not limited to the clinical encounter. in public health, the nature of a given intervention is not predetermined by professional discipline, but rather by the cost, expediency, and social acceptability of intervening at any potentially effective point in the series of events leading to disease, injury, or disability. public health interventions have included (for example) wastewater treatment and solid waste disposal systems, housing and building codes, fluoridation of municipal water supplies, removal of lead from gasoline, and smoke alarms. contrast this with the modern healthcare system, which generally accomplishes its mission through medical and surgical encounters. public health also generally operates directly or indirectly through government agencies that must be responsive to legislative, regulatory, and policy directives, carefully balance competing priorities, and openly disclose their activities. in addition, certain public health actions involve authority for specific (sometimes coercive) measures to protect the community in an emergency. examples include closing a contaminated pond or a restaurant that fails inspection. community partners to provide such care. though there is great variation across jurisdictions, the fundamental assurance function is unchanged: to assure that all members of the community have adequate access to needed services. the assurance function is not limited to access to clinical care. rather, it refers to assurance of the conditions that allow people to be healthy and free from avoidable threats to health-which includes access to clean water, a safe food supply, well-lighted streets, responsive and effective public safety entities, and so forth. this "core functions" framework has proven to be highly useful in clarifying the fundamental, over-arching responsibilities of public health. but if the core functions describe what public health is for, a more detailed and grounded delineation was needed to describe what public health agencies do. to meet this need, a set of ten essential public health services (table 15 .1) was developed through national and state level deliberations of public health providers and consumers (department of health and human services (dhhs), 1994). it is through these ten services that public health carries out its mission to assure the conditions in which people can be healthy. the core function of assessment, and several of the essential public health services rely heavily on public health surveillance, one of the oldest systematic activities of the public health sector. surveillance in the public health context refers to the ongoing collection, analysis, interpretation, and dissemination of data on health conditions (e.g., breast cancer) and threats to health (e.g., smoking prevalence). surveillance data represent one of the fundamental means by which priorities for public health action are set. surveillance data are useful not only in the short term (e.g., in surveillance for acute infectious diseases such as influenza, measles, and hiv/aids), but also in the longer term, e.g., in determining leading causes of premature death, injury, or disability. in either case, what distinguishes surveillance is that the data are collected for the purposes of action-either to guide a public health response (e.g., an outbreak investigation, or mitigation of a threat to a food or water source) or to help direct public health policy. a recent example of the latter is the surveillance data showing the dramatic rise in obesity in the united states. a tremendous amount of energy and public focus has been brought to bear on this problem-including a major dhhs program, the healthierus initiative-driven largely by compelling surveillance data. 1. monitor the health status of individuals in the community to identify community health problems 2. diagnose and investigate community health problems and community health hazards 3. inform, educate, and empower the community with respect to health issues 4. mobilize community partnerships in identifying and solving community health problems 5. develop policies and plans that support individual and community efforts to improve health 6. enforce laws and rules that protect the public health and ensure safety in accordance with those laws and rules 7. link individuals who have a need for community and personal health services to appropriate community and private providers 8. ensure a competent workforce for the provision of essential public health services 9. research new insights and innovate solutions to community health problems 10. evaluate the effectiveness, accessibility, and quality of personal and population-based health services in a community the fundamental science of public health is epidemiology, which is the study of the prevalence and determinants of disability and disease in populations. hence, most public health information systems have focused on information about aggregate populations. almost all medical information systems focus almost exclusively on identifying information about individuals. for example, almost any clinical laboratory system can quickly find jane smith's culture results. what public health practitioners want to know is the time trend of antibiotic resistance for the population that the clinic serves, or the trend for the population that the clinic actually covers. most health care professionals are surprised to learn that there is no uniform national routine reporting -never mind information system -for most diseases, disabilities, risk factors, or prevention activities in the united states. in contrast, france, great britain, denmark, norway and sweden have comprehensive systems in selected areas, such as occupational injuries, infectious diseases, and cancer; no country, however, has complete reporting for every problem. in fact, it is only births, deaths, and -to a lesser extentfetal deaths that are uniformly and relatively completely reported in the united states by the national vital statistics system, operated by the states and the centers for disease control and prevention (cdc). if you have an angioplasty and survive, nobody at the state or federal level necessarily knows. public health information systems have been designed with special features. for example, they are optimized for retrieval from very large (multi-million) record databases, and to be able to quickly cross-tabulate, study secular trends, and look for patterns. the use of personal identifiers in these systems is very limited, and their use is generally restricted to linking data from different sources (e.g., data from a state laboratory and a disease surveillance form). a few examples of these kinds of populationfocused systems include cdc systems such as the hiv/aids reporting system, which collects millions of observations concerning people infected with the human immunodeficiency virus (hiv) and those diagnosed with acquired immunodeficiency syndrome (aids) and is used to conduct dozens of studies (and which does not collect personal identifiers; individuals are tracked by pseudo-identifiers); the national notifiable disease surveillance system, which state epidemiologists use to report some 60 diseases (the exact number varies as conditions wax and wane) every week to the cdc (and which makes up the center tables in the morbidity and mortality weekly report [mmwr] ). the cdc wonder system (friede et al., 1996) , which contains tens of millions of observations drawn from some 30 databases, explicitly blanks cells with fewer than three to five observations (depending on the dataset), specifically to prevent individuals with unusual characteristics from being identified. if there is no national individual reporting, how are estimates obtained for, say, the trends in teenage smoking or in the incidence of breast cancer? how are epidemics found? data from periodic surveys and special studies, surveillance systems, and disease registries are handled by numerous stand-alone information systems. these systemsusually managed by state health departments and federal health agencies (largely the cdc) or their agents -provide periodic estimates of the incidence and prevalence of diseases and of certain risk factors (for example, smoking and obesity); however, because the data are from population samples, it is usually impossible to obtain estimates at a level of geographic detail finer than a region or state. moreover, many of the behavioral indices are patient self-reported (although extensive validation studies have shown that they are good for trends and sometimes are more reliable than are data obtained from clinical systems). in the case of special surveys, such as cdc's national health and nutrition examination survey (nhanes), there is primary data entry into a cdc system. the data are complete, but the survey costs many millions of dollars, is done only every few years, and it takes years for the data to be made available. there are also disease registries that track -often completely -the incidence of certain conditions, especially cancers, birth defects, and conditions associated with environmental contamination. they tend to focus on one topic or to cover certain diseases for specific time periods. the cdc maintains dozens of surveillance systems that attempt to track completely the incidence of many conditions, including lead poisoning, injuries and deaths in the workplace, and birth defects. (some of these systems use samples or cover only certain states or cities). as discussed above, there is also a list of about 60 notifiable diseases (revised every year) that the state epidemiologists and the cdc have determined are of national significance and warrant routine, complete reporting; however, it is up to providers to report the data, and reporting is still often done by telephone or mail, so the data are incomplete. finally, some states do collect hospital discharge summaries, but now that more care is being delivered in the ambulatory setting, these data capture only a small fraction of medical care. they are also notoriously difficult to access. what all these systems have in common is that they rely on special data collection. it is rare that they are seamlessly linked to ongoing clinical information systems. even clinical data such as hospital infections is reentered. why? all these systems grew up at the same time that information systems were being put in hospitals and clinics. hence, there is duplicate data entry, which can result in the data being shallow, delayed, and subject to input error and recall bias. furthermore, the systems themselves are often unpopular with state agencies and health care providers precisely because they require duplicate data entry (a child with lead poisoning and salmonella needs to be entered in two different cdc systems). the national electronic disease surveillance system (nedss) is a major cdc initiative that addresses this issue by promoting the use of data and information system standards to advance the development of efficient, integrated, and interoperable surveillance systems at federal, state and local levels (see www.cdc.gov/nedss). this activity is designed to facilitate the electronic transfer of appropriate information from clinical information systems in the health care industry to public health departments, reduce provider burden in the provision of information, and enhance both the timeliness and quality of information provided. now that historical and epidemiological forces are making the world smaller and causing lines between medicine and public health to blur, systems will need to be multifunctional, and clinical and public health systems will, of necessity, coalesce. what is needed are systems that can tell us about individuals and the world in which those individuals live. to fill that need, public health and clinical informaticians will need to work closely together to build the tools to study and control new and emerging threats such as bioterror, hiv/aids, sars and its congeners, and the environmental effects of the shrinking ozone layer and greenhouse gases. it can be done. for example, in the late 1990's, columbia presbyterian medical center and the new york city department of health collaborated on the development of a tuberculosis registry for northern manhattan, and the emory university system of health care and the georgia department of public health built a similar system for tuberculosis monitoring and treatment in atlanta. it is not by chance that these two cities each developed tuberculosis systems; rather, tuberculosis is a perfect example of what was once a public health problem (that affected primarily the poor and underserved) coming into the mainstream population as a result of an emerging infectious disease (aids), immigration, increased international travel, multidrug resistance, and our growing prison population. hence, the changing ecology of disease, coupled with revolutionary changes in how health care is managed and paid for, will necessitate information systems that serve both individual medical and public health needs. immunization registries are confidential, population based, computerized information systems that contain data about children and vaccinations (national vaccine advisory committee, 1999). they represent a good example for illustrating the principles of public health informatics. in addition to their orientation to prevention, they can only function properly through continuing interaction with the health care system. they also must exist in a governmental context because there is little incentive (and significant organizational barriers) for the private sector to maintain such registries. although immunization registries are among the largest and most complex public health information systems, the successful implementations show conclusively that it is possible to overcome the challenging informatics problems they present. childhood immunizations have been among the most successful public health interventions, resulting in the near elimination of nine vaccine preventable diseases that historically extracted a major toll in terms of both morbidity and mortality (iom, 2000a) . the need for immunization registries stems from the challenge of assuring complete immunization protection for the approximately 11,000 children born each day in the united states in the context of three complicating factors: the scattering of immunization records among multiple providers; an immunization schedule that has become increasingly complex as the number of vaccines has grown; and the conundrum that the very success of mass immunization has reduced the incidence of disease, lulling parents and providers into a sense of complacency. the 1989-91 u.s. measles outbreak, which resulted in 55,000 cases and 123 preventable deaths (atkinson et al., 1992) , helped stimulate the public health community to expand the limited earlier efforts to develop immunization registries. because cdc was proscribed by congress from creating a single national immunization registry (due to privacy concerns), the robert wood johnson foundation, in cooperation with several other private foundations, established the all kids count (akc) program that awarded funds to 24 states and communities in 1992 to assist in the development of immunization registries. akc funded the best projects through a competitive process, recruited a talented staff to provide technical assistance, and made deliberate efforts to ensure sharing of the lessons learned, such as regular, highly interactive meetings of the grantees. subsequent funding of 13 states by cdc and the woodruff foundation via the information network for public health officials (inpho) project (baker et al., 1995) was greatly augmented by a presidential commitment to immunization registries announced in 1997 (white house, 1997) . this resulted in every state's involvement in registry development. immunization registries must be able to exchange information to ensure that children who relocate receive needed immunizations. to accomplish this, standards were needed to prevent the development of multiple, incompatible immunization transmission formats. beginning in 1995, cdc worked closely with the health level 7 standards development organization (see chapter 7) to define hl7 messages and an implementation guide for immunization record transactions. the initial data standard was approved by hl7 in 1997 and an updated implementation guide was developed in 1999. cdc continues its efforts to encourage the standards-based exchange of immunization records among registries. as more experience accumulated, akc and cdc collaborated to develop an immunization registry development guide (cdc, 1997) that captured the hard-won lessons developed by dozens of projects over many years. by 2000, a consensus on the 12 needed functions of immunization registries had emerged (table 15. 2), codifying years of experience in refining system requirements. cdc also established a measurement system for tracking progress that periodically assesses the percentage of immunization registries that have operationalized each of the 12 functions ( figure 15 1. electronically store data regarding all national vaccine advisory committee-approved core data elements 2. establish a registry record within 6 weeks of birth for each child born in the catchment area 3. enable access to vaccine information from the registry at the time of the encounter 4. receive and process vaccine information within 1 month of vaccine administration 5. protect the confidentiality of medical information 6. protect the security of medical information 7. exchange vaccination records by using health level 7 standards 8. automatically determine the immunization(s) needed when a person is seen by the health care provider for a scheduled vaccination 9. automatically identify persons due or late for vaccinations to enable the production of reminder and recall notices 10. automatically produce vaccine coverage reports by providers, age groups, and geographic areas 11. produce authorized immunization records 12. promote accuracy and completeness of registry data registries, the national healthy people 2010 objectives include the goal of having 95% of all u.s. children covered by fully functioning immunization registries (dhhs, 2000) . the development and implementation of immunization registries presents challenging informatics issues in at least four areas: 1) interdisciplinary communication; 2) organizational and collaborative issues; 3) funding and sustainability; and 4) system design. while the specific manifestations of these issues are unique to immunization registries, these four areas represent the typical domains that must be addressed and overcome in public health informatics projects. interdisciplinary communications is a key challenge in any biomedical informatics project-it is certainly not specific to public health informatics. to be useful, a public health information system must accurately represent and enable the complex concepts and processes that underlie the specific business functions required. information systems represent a highly abstract and complex set of data, processes, and interactions. this complexity needs to be discussed, specified, and understood in detail by a variety of personnel with little or no expertise in the terminology and concepts of information technology. therefore, successful immunization registry implementation requires clear communication among public health specialists, immunization specialists, providers, it specialists, and related disciplines, an effort complicated by the lack of a shared vocabulary and differences in the usage of common terms from the various domains. added to these potential communication problems are the anxieties and concerns inherent in the development of any new information system. change is an inevitable part of such a project-and change is uncomfortable for everyone involved. implementation of information systems. in this context, tensions and anxieties can further degrade communications. to deal with the communications challenges, particularly between it and public health specialists, it is essential to identify an interlocutor who has familiarity with both information technology and public health. the interlocutor should spend sufficient time in the user environment to develop a deep understanding of the information processing context of both the current and proposed systems. it is also important for individuals from all the disciplines related to the project to have representation in the decisionmaking processes. the organizational and collaborative issues involved in developing immunization registries are daunting because of the large number and wide variety of partners. both public and private sector providers and other organizations are likely participants. for the providers, particularly in the private sector, immunization is just one of many concerns. however, it is essential to mobilize private providers to submit immunization information to the registry. in addition to communicating regularly to this group about the goals, plans, and progress of the registry, an invaluable tool to enlist their participation is a technical solution that minimizes their time and expense for registry data entry, while maximizing the benefit in terms of improved information about their patients. it is critical to recognize the constraints of the private provider environment, where income is generated mostly from "piecework" and time is the most precious resource. governance issues are also critical to success. all the key stakeholders need to be represented in the decision-making processes, guided by a mutually acceptable governance mechanism. large information system projects involving multiple partners -such as immunization registries -often require multiple committees to ensure that all parties have a voice in the development process. in particular, all decisions that materially affect a stakeholder should be made in a setting that includes their representation. legislative and regulatory issues must be considered in an informatics context because they impact the likelihood of success of projects. with respect to immunization registries, the specific issues of confidentiality, data submission, and liability are critical. the specific policies with respect to confidentiality must be defined to allow access to those who need it while denying access to others. regulatory or legislative efforts in this domain must also operate within the context of the federal health insurance portability and accountability act (hipaa) that sets national minimum privacy requirements for personal health information. some jurisdictions have enacted regulations requiring providers to submit immunization data to the registry. the effectiveness of such actions on the cooperation of providers must be carefully evaluated. liability of the participating providers and of the registry operation itself may also require legislative and/or regulatory clarification. funding and sustainability are continuing challenges for all immunization registries. in particular, without assurances of ongoing operational funding, it will be difficult to secure the commitments needed for the development work. naturally, an important tool for securing funding is development of a business case that shows the anticipated costs and benefits of the registry. while a substantial amount of information now exists about costs and benefits of immunization registries (horne et al., 2000) , many of the registries that are currently operational had to develop their business cases prior to the availability of good quantitative data. specific benefits associated with registries include preventing duplicative immunizations, eliminating the necessity to review the vaccination records for school and day care entry, and efficiencies in provider offices from the immediate availability of complete immunization history information and patient-specific vaccine schedule recommendations. the careful assessment of costs and benefits of specific immunization registry functions may also be helpful in prioritizing system requirements. as with all information systems, it is important to distinguish "needs" (those things people will pay for) from "wants" (those things people would like to have but are not willing to spend money on) (rubin, 2003). information system "needs" are typically supported by a strong business case, whereas "wants" often are not. system design is also an important factor in the success of immunization registries. difficult design issues include data acquisition, database organization, identification and matching of children, generating immunization recommendations, and access to data, particularly for providers. acquiring immunization data is perhaps the most challenging system design issue. within the context of busy pediatric practices (where the majority of childhood immunizations are given), the data acquisition strategy must of necessity be extremely efficient. ideally, information about immunizations would be extracted from existing electronic medical records or from streams of electronic billing data; either strategy should result in no additional work for participating providers. unfortunately neither of these options is typically available. electronic medical records are currently implemented only in roughly 10-15% of physician practices. while the use of billing records is appealing, it is often difficult to get such records on a timely basis without impinging on their primary function-namely, to generate revenue for the practice. also, data quality, particularly with respect to duplicate records, is often a problem with billing information. a variety of approaches have been used to address this issue, including various forms of direct data entry as well as the use of bar codes (yasnoff, 2003) . database design also must be carefully considered. once the desired functions of an immunization registry are known, the database design must allow efficient implementation of these capabilities. the operational needs for data access and data entry, as well as producing individual assessments of immunization status, often require different approaches to design compared to requirements for population-based immunization assessment, management of vaccine inventory, and generating recall and reminder notices. one particularly important database design decision for immunization registries is whether to represent immunization information by vaccine or by antigen. vaccinebased representations map each available preparation, including those with multiple antigens, into its own specific data element. antigen-based representations translate multi-component vaccines into their individual antigens prior to storage. in some cases, it may be desirable to represent the immunization information both ways. specific consideration of required response times for specific queries must also be factored into key design decisions. identification and matching of individuals within immunization registries is another critical issue. because it is relatively common for a child to receive immunizations from multiple providers, any system must be able to match information from multiple sources to complete an immunization record. in the absence of a national unique patient identifier, most immunization registries will assign an arbitrary number to each child. of course, provisions must be made for the situation where this identification number is lost or unavailable. this requires a matching algorithm, which utilizes multiple items of demographic information to assess the probability that two records are really data from the same person. development of such algorithms and optimization of their parameters has been the subject of active investigation in the context of immunization registries, particularly with respect to deduplication (miller et al., 2001) . another critical design issue is generating vaccine recommendations from a child's prior immunization history, based on guidance from the cdc's advisory committee on immunization practices (acip). as more childhood vaccines have become available, both individually and in various combinations, the immunization schedule has become increasingly complex, especially if any delays occur in receiving doses, a child has a contraindication, or local issues require special consideration. the language used in the written guidelines is sometimes incomplete, not covering every potential situation. in addition, there is often some ambiguity with respect to definitions, e.g., for ages and intervals, making implementation of decision support systems problematic. considering that the recommendations are updated relatively frequently, sometimes several times each year, maintaining software that produces accurate immunization recommendations is a continuing challenge. accordingly, the implementation, testing, and maintenance of decision support systems to produce vaccine recommendations has been the subject of extensive study (yasnoff & miller, 2003) . finally, easy access to the information in an immunization registry is essential. while this may initially seem to be a relatively simple problem, it is complicated by private providers' lack of high-speed connectivity. even if a provider office has the capability for internet access, for example, it may not be immediately available at all times, particularly in the examination room. immunization registries have developed alternative data access methods such as fax-back and telephone query to address this problem. since the primary benefit of the registry to providers is manifest in rapid access to the data, this issue must be addressed. ready access to immunization registry information is a powerful incentive to providers for entering the data from their practice. in the united states, the first major report calling for a health information infrastructure was issued by the institute of medicine of the national academy of sciences in 1991 (iom, 1991) . this report, "the computer-based patient record," was the first in a series of national expert panel reports recommending transformation of the health care system from reliance on paper to electronic information management. in response to the iom report, the computer-based patient record institute (cpri), a private not-for-profit corporation, was formed for the purpose of facilitating the transition to computer-based records. a number of community health information networks (chins) were established around the country in an effort to coalesce the multiple community stakeholders in common efforts towards electronic information exchange. the institute of medicine updated its original report in 1997 (iom, 1997), again emphasizing the urgency to apply information technology to the information intensive field of health care. however, most of the community health information networks were not successful. perhaps the primary reason for this was that the standards and technology were not yet ready for cost-effective community-based electronic health information exchange. another problem was the focus on availability of aggregated health information for secondary users (e.g., policy development), rather than individual information for the direct provision of patient care. also, there was neither a sense of extreme urgency nor were there substantial funds available to pursue these endeavors. however, at least one community, indianapolis, continued to move forward throughout this period and has now emerged as an a national example of the application of information technology to health care both in individual health care settings and throughout the community. the year 2000 brought widespread attention to this issue with the iom report "to err is human" (iom, 2000b) . in this landmark study, the iom documented the accumulating evidence of the high error rate in the medical care system, including an estimated 44,000 to 98,000 preventable deaths each year in hospitals alone. this report has proven to be a milestone in terms of public awareness of the consequences of paperbased information management in health care. along with the follow-up report, "crossing the quality chasm" (iom, 2001) , the systematic inability of the health care system to operate at high degree of reliability has been thoroughly elucidated. the report clearly placed the blame on the system, not the dedicated health care professionals who work in an environment without effective tools to promote quality and minimize errors. several additional national expert panel reports have emphasized the iom findings. in 2001, the president's information technology advisory committee (pitac) issued a report entitled "transforming health care through information technology" (pitac, 2001) . that same year, the computer science and telecommunications board of the national research council (nrc) released "networking health: prescriptions for the internet" (nrc, 2001) which emphasized the potential for using the internet to improve electronic exchange of health care information. finally, the national committee on vital and health statistics (ncvhs) outlined the vision and strategy for building a national health information infrastructure (nhii) in its report, "information for health" (ncvhs, 2001) . ncvhs, a statutory advisory body to dhhs, indicated that federal government leadership was needed to facilitate further development of an nhii. on top of this of bevy of national expert panel reports, there has been continuing attention in both scientific and lay publications to cost, quality, and error issues in the health care system. the anthrax attacks of late 2001 further sensitized the nation to the need for greatly improved disease detection and emergency medical response capabilities. what has followed has been the largest-ever investment in public health information infrastructure in the history of the united states. some local areas, such as indianapolis and pittsburgh, have begun to actively utilize electronic information from the health care system for early detection of bioterrorism and other disease outbreaks. in 2003, separate large national conferences were devoted to both the cdc's public health information network (phin) (cdc, 2003) and the dhhs nhii initiative (dhhs, 2003 yasnoff et al., 2004 . while the discussion here has focused on the development of nhii in the united states, many other countries are involved in similar activities and in fact have progressed further along this road. canada, australia, and a number of european nations have devoted considerable time and resources to their own national health information infrastructures. the united kingdom, for example, has announced its intention to allocate several billion pounds over the next few years to substantially upgrade its health information system capabilities. it should be noted, however, that all of these nations have centralized, government-controlled health care systems. this organizational difference from the multifaceted, mainly private health care system in the u.s. results in a somewhat different set of issues and problems. hopefully, the lessons learned from health information infrastructure development activities across the globe can be effectively shared to ease the difficulties of everyone who is working toward these important goals. the vision of the national health information infrastructure is anytime, anywhere health care information at the point of care. the intent to is to create a distributed system, not a centralized national database. patient information would be collected and stored at each care site. when a patient presented for care, the various existing electronic records would be located, collected, integrated, and immediately delivered to allow the provider to have complete and current information upon which to base clinical decisions. in addition, clinical decision support (see chapter 20) would be integrated with information delivery. in this way, clinicians could receive reminders of the most recent clinical guidelines and research results during the patient care process, thereby avoiding the need for superhuman memory capabilities to assure the effective practice of medicine. the potential benefits of nhii are both numerous and substantial. perhaps most important are error reduction and improved quality of care. numerous studies have shown that the complexity of present-day medical care results in very frequent errors of both omission and commission. this problem was clearly articulated at the 2001 meeting of the institute of medicine: "current practice depends upon the clinical decision making capacity and reliability of autonomous individual practitioners, for classes of problems that routinely exceed the bounds of unaided human cognition" (masys, 2001) . electronic health information systems can contribute significantly to improving this problem by reminding practitioners about recommended actions at the point of care. this can include both notifications of actions that may have been missed, as well as warnings about planned treatments or procedures that may be harmful or unnecessary. literally dozens of research studies have shown that such reminders improve safety and reduce costs (kass, 2001; bates, 2000) . in one such study (bates et al., 1998) , medication errors were reduced by 55%. a more recent study by the rand corporation showed that only 55 % of u.s. adults were receiving recommended care (mcglynn et al., 2003) . the same techniques used to reduce medical errors with electronic health information systems also contribute substantially to ensuring that recommended care is provided. this is becoming increasingly important as the population ages and the prevalence of chronic disease increases. guidelines and reminders also can improve the effectiveness of dissemination of new research results. at present, widespread application of a new research in the clinical setting takes an average of 17 years (balas & boren, 2000) . patient-specific reminders delivered at the point of care highlighting important new research results could substantially increase the adoption rate. another important contribution of nhii to the research domain is improving the efficiency of clinical trials. at present, most clinical trials require creation of a unique information infrastructure to insure protocol compliance and collect essential research data. with nhii, where every practitioner would have access to a fully functional electronic health record, clinical trials could routinely be implemented through the dissemination of guidelines that specify the research protocol. data collection would occur automatically in the course of administering the protocol, reducing time and costs. in addition, there would be substantial value in analyzing deidentified aggregate data from routine patient care to assess the outcomes of various treatments, and monitor the health of the population. another critical function for nhii is early detection of patterns of disease, particularly early detection of possible bioterrorism. our current system of disease surveillance, which depends on alert clinicians diagnosing and reporting unusual conditions, is both slow and potentially unreliable. most disease reporting still occurs using the postal service, and the information is relayed from local to state to national public health authorities. even when fax or phone is employed, the system still depends on the ability of clinicians to accurately recognize rare and unusual diseases. even assuming such capabilities, individual clinicians cannot discern patterns of disease beyond their sphere of practice. these problems are illustrated by the seven unreported cases of cutaneous anthrax in the new york city area two weeks before the so-called "index" case in florida in the fall of 2001 (lipton & johnson, 2001) . since all the patients were seen by different clinicians, the pattern could not have been evident to any of them even if the diagnosis had immediately been made in every case. wagner et al have elucidated nine categories of requirements for surveillance systems for potential bioterrorism outbreaks-several categories must have immediate electronic reporting to insure early detection (wagner et al., 2003) . nhii would allow immediate electronic reporting of both relevant clinical events and laboratory results to public health. not only would this be an invaluable aid in early detection of bioterrorism, it would also serve to improve the detection of the much more frequent naturally occurring disease outbreaks. in fact, early results from a number of electronic reporting demonstration projects show that disease outbreaks can routinely be detected sooner than was ever possible using the current system (overhage et al., 2001) . while early detection has been shown to be a key factor in reducing morbidity and mortality from bioterrorism (kaufmann et al., 1997) , it will also be extremely helpful in reducing the negative consequences from other disease outbreaks. this aspect of nhii is discussed in more detail in section 15.5. finally, nhii can substantially reduce health-care costs. the inefficiencies and duplication in our present paper-based health care system are enormous. recent study showed that the anticipated nationwide savings from implementing advanced computerized provider order entry (cpoe) systems in the outpatient environment would be $44 billion per year (johnston et al., 2003) , while a related study (walker et al., 2004) estimated $78 billion more is savings from health information exchange (for a total of $112 billion per year). substantial additional savings are possible in the inpatient setting-numerous hospitals have reported large net savings from implementation of electronic health records. another example, electronic prescribing, would not only reduce medication errors from transcription, but also drastically decrease the administrative costs of transferring prescription information from provider offices to pharmacies. a more recent analysis concluded that the total efficiency and patient safety savings from nhii would be in range of $142-371 billion each year (hillestad et al., 2005) . while detailed studies of the potential savings from comprehensive implementation of nhii, including both electronic health records and effective exchange of health information, are still ongoing, it is clear that the cost reductions will amount to hundreds of billions of dollars each year. it is important to note that much of the savings depends not just on the widespread implementation of electronic health records, but the effective interchange of this information to insure that the complete medical record for every patient is immediately available in every care setting. there are a number of significant barriers and challenges to the development of nhii. perhaps the most important of these relates to protecting the confidentiality of electronic medical records. the public correctly perceives that all efforts to make medical records more accessible for appropriate and authorized purposes simultaneously carry the risk of increased availability for unscrupulous use. while the implementation of the hipaa privacy and security rules (see chapter 10) has established nationwide policies for access to medical information, maintaining public confidence requires mechanisms that affirmatively prevent privacy and confidentiality breaches before they occur. development, testing, and implementation of such procedures must be an integral part of any nhii strategy. another important barrier to nhii is the misalignment of financial incentives in the health care system. although the benefits of nhii are substantial, they do not accrue equally across all segments of the system. in particular, the benefits are typically not proportional to the required investments for a number of specific stakeholder groups. perhaps most problematic is the situation for individual and small group health care providers, who are being asked to make substantial allocations of resources to electronic health record systems that mostly benefit others. mechanisms must be found to assure the equitable distribution of nhii benefits in proportion to investments made. while this issue is the subject of continuing study, early results indicate that most of the nhii financial benefit accrues to payers of care. therefore, programs and policies must be established to transfer appropriate savings back to those parties who have expended funds to produce them. one consequence of the misaligned financial incentives is that the return on investment for health information technology needed for nhii is relatively uncertain. while a number of health care institutions, particularly large hospitals, have reported substantial cost improvements from electronic medical record systems, the direct financial benefits are by no means a forgone conclusion, especially for smaller organizations. the existing reimbursement system in the united states does not provide ready access to the substantial capital required by many institutions. for health care organizations operating on extremely thin margins, or even in the red, investments in information technology are impractical regardless of the potential return. in addition, certain legal and regulatory barriers prevent the transfer of funds from those who benefit from health information technology to those who need to invest but have neither the means nor the incentive of substantial returns. laws and regulations designed to prevent fraud and abuse, payments for referrals, and private distribution of disguised "profits" from nonprofit organizations are among those needing review. it is important that mechanisms be found to enable appropriate redistribution of savings generated from health information technology without creating loopholes that would allow abusive practices. another key barrier to nhii is that many of the benefits relate to exchanges of information between multiple health care organizations. the lack of interoperable electronic medical record systems that provide for easy transfer of records from one place to another is a substantial obstacle to achieving the advantages of nhii. also, there is a "first mover disadvantage" in such exchange systems. the largest value is generated when all health care organizations in a community participate electronic information exchange. therefore, if only a few organizations begin the effort, their costs may not be offset by the benefits. a number of steps are currently under way to accelerate the progress towards nhii in the united states. these include establishing standards, fostering collaboration, funding demonstration projects in communities that include careful evaluation, and establishing consensus measures of progress. establishing electronic health record standards that would promote interoperability is the most widely recognized need in health information technology at the present time. within institutions that have implemented specific departmental applications, extensive time and energy is spent developing and maintaining interfaces among the various systems. although much progress has been made in this area by organizations such as health level 7, even electronic transactions of specific health care data (such as laboratory results) are often problematic due to differing interpretations of the implementation of existing standards. recently, the u.s. government has made substantial progress in this area. ncvhs, the official advisory body on these matters to dhhs, has been studying the issues of both message and content standards for patient medical record information for several years (ncvhs, 2000) . the consolidated healthcare informatics (chi) initiative recommended five key standards (hl7 version 2.x, loinc, dicom, ieee 1073, and ncpdp script) that were adopted for government-wide use in early 2003, followed by 15 more that were added in 2004. in july, 2003, the federal government licensed the comprehensive medical vocabulary known as snomed (systematized nomenclature of medicine; see chapter 7), making it available to all u.s. users at no charge. this represents a major step forward in the deployment of vocabulary standards for health information systems. unlike message format standards, such as hl7, vocabulary standards are complex and expensive to develop and maintain and therefore require ongoing financial support. deriving the needed funding from end users creates a financial obstacle to deployment of the standard. removing this key barrier to adoption should promote much more widespread use over the next few years. another important project now under way is the joint effort of the institute of medicine and hl7 to develop a detailed functional definition of the electronic health record (ehr). these functional standards will provide a benchmark for comparison of existing and future ehr systems, and also may be utilized as criteria for possible financial incentives that could be provided to individuals and organizations that implement such systems. the elucidation of a consensus functional definition of the ehr also should help prepare the way for its widespread implementation by engaging all the stakeholders in an extended discussion of its desired capabilities. this functional standardization of the ehr is expected to be followed by the development of a formal interchange format standard (ifs) to be added to hl7 version 3. this standard would enable full interoperability of ehr systems through the implementation of an import and export capability to and from the ifs. while it is possible at the present time to exchange complete electronic health records with existing standards, is both difficult and inconvenient. the ifs will greatly simplify the process, making it easy to accomplish the commonly needed operation of transferring an entire electronic medical record from one facility to another. another key standard that is needed involves the representation of guideline recommendations. while the standard known as arden syntax (hl7, 2003; see chapter 7) partially addresses this need, many real-world medical care guidelines are too complex to be represented easily in this format. at the present time, the considerable effort required to translate written guidelines and protocols into computer executable form must be repeated at every health care organization wishing to incorporate them in their ehr. development of an effective guideline interchange standard would allow medical knowledge to be encoded once and then distributed widely, greatly increasing the efficiency of the process (peleg at al., 2003) . collaboration is another important strategy in promoting nhii. to enable the massive changes needed to transform the health care system from its current paper-based operation to the widespread utilization of electronic health information systems, the support of a very large number of organizations and individuals with highly varied agendas is required. gathering and focusing this support requires extensive cooperative efforts and specific mechanisms for insuring that everyone's issues and concerns are expressed, appreciated, and incorporated into the ongoing efforts. this process is greatly aided by a widespread recognition of the serious problems that exist today in the u.s. healthcare system. a number of private collaboration efforts have been established such as the e-health initiative and the national alliance for health information technology (nahit). in the public sector, national health information infrastructure (nhii) has become a focus of activity at dhhs. as part of this effort, the first ever national stakeholders meeting for nhii was convened in mid-2003 to develop a consensus national agenda for moving forward (yasnoff et al., 2004) . these multiple efforts are having the collective effect of both catalyzing and promoting organizational commitment to nhii. for example, many of the key stakeholders are now forming high-level committees to specifically address nhii issues. for some of these organizations, this represents the first formal recognition that this transformational process is underway and will have a major impact on their activities. it is essential to include all stakeholders in this process. in addition to the traditional groups such as providers, payers, hospitals, health plans, health it vendors, and health informatics professionals, representatives of groups such as consumers (e.g., aarp) and the pharmaceutical industry must be brought into the process. the most concrete and visible strategy for promoting nhii is the encouragement of demonstration projects in communities, including the provision of seed funding. by establishing clear examples of the benefits and advantages of comprehensive health information systems in communities, additional support for widespread implementation can be garnered at the same time that concerns of wary citizens and skeptical policymakers are addressed. there are several important reasons for selecting a community-based strategy for nhii implementation. first and foremost, the existing models of health information infrastructures (e.g., indianapolis and spokane, wa) are based in local communities. this provides proof that it is possible to develop comprehensive electronic health care information exchange systems in these environments. in contrast, there is little or no evidence that such systems can be directly developed on a larger scale. furthermore, increasing the size of informatics projects disproportionately increases their complexity and risk of failure. therefore, keeping projects as small as possible is always a good strategy. since nhii can be created by effectively connecting communities that have developed local health information infrastructures (lhiis), it is not necessary to invoke a direct national approach to achieve the desired end result. a good analogy is the telephone network, which is composed of a large number of local exchanges that are then connected to each other to form community and then national and international networks. another important element in the community approach is the need for trust to overcome confidentiality concerns. medical information is extremely sensitive and its exchange requires a high degree of confidence in everyone involved in the process. the level of trust needed seems most likely to be a product of personal relationships developed over time in a local community and motivated by a common desire to improve health care for everyone located in that area. while the technical implementation of information exchange is non-trivial, it pales in comparison to the challenges of establishing the underlying legal agreements and policy changes that must precede it. for example, when indianapolis implemented sharing of patient information in hospital emergency rooms throughout the area, as many as 20 institutional lawyers needed to agree on the same contractual language (overhage, 2002) . the community approach also benefits from the fact that the vast majority of health care is delivered locally. while people do travel extensively, occasionally requiring medical care while away from home, and there are few out-of-town consultations for difficult and unusual medical problems, for the most part people receive their health care in the community in which they reside. the local nature of medical care results in a natural interest of community members in maintaining and improving the quality and efficiency of their local health care system. for the same reasons, it is difficult to motivate interest in improving health care beyond the community level. focusing nhii efforts on one community at a time also keeps the implementation problem more reasonable in its scope. it is much more feasible to enable health information interchange among a few dozen hospitals and a few hundred or even a few thousand providers than to consider such a task for a large region or the whole country. this also allows for customized approaches sensitive to the specific needs of each local community. the problems and issues of medical care in a densely populated urban area are clearly vastly different than in a rural environment. similarly, other demographic and organizational differences as well as the presence of specific highly specialized medical care institutions make each community's health care system unique. a local approach to hii development allows all these complex and varied factors to be considered and addressed, and respects the reality of the american political landscape, which gives high priority to local controls. the community-based approach to hii development also benefits from the establishment of national standards. the same standards that allow effective interchange of information between communities nationwide can also greatly facilitate establishing effective communication of medical information within a community. in fact, by encouraging (and even requiring) communities to utilize national standards in building their own lhiis, the later interconnection of those systems to provide nationwide access to medical care information becomes a much simpler and easier process. demonstration projects also are needed to develop and verify a replicable strategy for lhii development. while there are a small number of existing examples of lhii systems, no organization or group has yet demonstrated the ability to reliably and successfully establish such systems in multiple communities. from the efforts of demonstration projects in numerous communities, it should be possible to define a set of strategies that can be applied repeatedly across the nation. seed funding is essential in the development of lhii systems. while health care in united states is a huge industry, spending approximately $1.5 trillion each year and representing 14% of the gdp, shifting any of the existing funds into substantial it investments is problematic. the beneficiaries of all the existing expenditures seem very likely to strongly oppose any such efforts. on the other hand, once initial investments begin to generate the expected substantial savings, it should be possible to develop mechanisms to channel those savings into expanding and enhancing lhii systems. careful monitoring of the costs and benefits of local health information interchange systems will be needed to verify the practicality of this approach to funding and sustaining these projects. finally, it is important to assess and understand the technical challenges and solutions applied to lhii demonstration projects. while technical obstacles are usually not serious in terms of impeding progress, understanding and disseminating the most effective solutions can result in smoother implementation as experience is gained throughout the nation. the last element in the strategy for promoting a complex and lengthy project such as nhii is careful measurement of progress. the measures used to gauge progress define the end state and therefore must be chosen with care. measures may also be viewed as the initial surrogate for detailed requirements. progress measures should have certain key features. first, they should be sufficiently sensitive so that their values change at a reasonable rate (a measure that only changes value after five years will not be particularly helpful). second, the measures must be comprehensive enough to reflect activities that impact most of the stakeholders and activities needing change. this ensures that efforts in every area will be reflected in improved measures. third, the measures must be meaningful to policymakers. fourth, periodic determinations of the current values of the measures should be easy so that the measurement process does not detract from the actual work. finally, the totality of the measures must reflect the desired end state so that when the goals for all the measures are attained, the project is complete. a number of different types or dimensions of measures for nhii progress are possible. aggregate measures assess nhii progress over the entire nation. examples include the percentage of the population covered by an lhii and the percentage of health care personnel whose training occurs in institutions that utilize electronic health record systems. another type of measure is based on the setting of care. progress in implementation of electronic health record systems in the inpatient, outpatient, long-term care, home, and community environments could clearly be part of an nhii measurement program. yet another dimension is health care functions performed using information systems support, including, for example, registration systems, decision support, cpoe, and community health information exchange. it is also important to assess progress with respect to the semantic encoding of electronic health records. clearly, there is a progression from the electronic exchange of images of documents, where the content is only readable by the end user viewing the image, to fully encoded electronic health records where all the information is indexed and accessible in machine-readable form using standards. finally, progress can also be benchmarked based on usage of electronic health record systems by health care professionals. the transition from paper records to available electronic records to fully used electronic records is an important signal with respect to the success of nhii activities. to illustrate some of the informatics challenges inherent in nhii, the example of its application to homeland security will be used. bioterrorism preparedness in particular is now a key national priority, especially following the anthrax attacks that occurred in the fall of 2001. early detection of bioterrorism is critical to minimize morbidity and mortality. this is because, unlike other terrorist attacks, bioterrorism is usually silent at first. its consequences are usually the first evidence that an attack has occurred. traditional public health surveillance depends on alert clinicians reporting unusual diseases and conditions. however, it is difficult for clinicians to detect rare and unusual diseases since they are neither familiar with their manifestations nor suspicious of the possibility of an attack. also, it is often difficult to differentiate potential bioterrorism from more common and benign manifestations of illness. this is clearly illustrated by the seven cases of cutaneous anthrax that occurred in the new york city area two weeks prior to the "index " case in florida the fall of 2001 (lipton & johnson, 2001) . all these cases presented to different clinicians, none of whom recognized the diagnosis of anthrax with sufficient confidence to notify any public health authority. furthermore, such a pattern involving similar cases presenting to multiple clinicians could not possibly be detected by any of them. it seems likely that had all seven of these patients utilized the same provider, the immediately evident pattern of unusual signs and symptoms alone would have been sufficient to result in an immediate notification of public health authorities even in the absence of any diagnosis. traditional public health surveillance also has significant delays. much routine reporting is still done via postcard and fax to the local health department, and further delays occur before information is collated, analyzed, and reported to state and finally to federal authorities. there is also an obvious need for a carefully coordinated response after a bioterrorism event is detected. health officials, in collaboration with other emergency response agencies, must carefully assess and manage health care assets and ensure rapid deployment of backup resources. also, the substantial increase in workload created from such an incident must be distributed effectively among available hospitals, clinics, and laboratories, often including facilities outside the affected area. the vision for the application of nhii to homeland security involves both early detection of bioterrorism and the response to such an event. clinical information relevant to public health would be reported electronically in near real-time. this would include clinical lab results, emergency room chief complaints, relevant syndromes (e.g., flu-like illness), and unusual signs, symptoms, or diagnoses. by generating these electronic reports automatically from electronic health record systems, the administrative reporting burden currently placed on clinicians would be eliminated. in addition, the specific diseases and conditions reported could be dynamically adjusted in response to an actual incident or even information related to specific threats. this latter capability would be extremely helpful in carefully tracking the development of an event from its early stages. nhii could also provide much more effective medical care resource management in response to events. this could include automatic reporting of all available resources so they could be allocated rapidly and efficiently, immediate operational visibility of all health care assets, and effective balancing of the tremendous surge in demand for medical care services. this would also greatly improve decision making about deployment of backup resources. using nhii for these bioterrorism preparedness functions avoids developing a separate, very expensive infrastructure dedicated to these rare events. as previously stated, the benefits of nhii are substantial and fully justify its creation even without these bioterrorism preparedness capabilities, which would be an added bonus. furthermore, the same infrastructure that serves as an early detection system for bioterrorism also will allow earlier and more sensitive detection of routine naturally occurring disease outbreaks (which are much more common) as well as better management of health care resources in other disaster situations. the application of nhii to homeland security involves a number of difficult informatics challenges. first, this activity requires participation from a very wide range of both public and private organizations. this includes all levels of government and organizations that have not had significant prior interactions with the health care system such as agriculture, police, fire, and animal health. needless to say, these organizations have divergent objectives and cultures that do not necessarily mesh easily. health and law enforcement in particular have a significantly different view of a bioterrorism incident. for example, an item that is considered a "specimen" in the health care system may be regarded as "evidence" by law enforcement. naturally, this wide variety of organizations has incompatible information systems, since for the most part they were designed and deployed without consideration for the issues raised by bioterrorism. not only do they have discordant design objectives, but they lack standardized terminology and messages to facilitate electronic information exchange. furthermore, there are serious policy conflicts among these various organizations, for example, with respect to access to information. in the health care system, access to information is generally regarded as desirable, whereas in law enforcement it must be carefully protected to maintain the integrity of criminal investigations. complicating these organizational, cultural, and information systems issues, bioterrorism preparedness has an ambiguous governance structure. many agencies and organizations have legitimate and overlapping authority and responsibility, so there is often no single clear path to resolve conflicting issues. therefore, a high degree of collaboration and collegiality is required, with extensive pre-event planning so that roles and responsibilities are clarified prior to any emergency. within this complex environment, there is also a need for new types of systems with functions that have never before been performed. bioterrorism preparedness results in new requirements for early disease detection and coordination of the health care system. precisely because these requirements are new, there are few (if any) existing systems that have similar functions. therefore careful consideration to design requirements of bioterrorism preparedness systems is essential to ensure success. most importantly, there is an urgent need for interdisciplinary communication among an even larger number of specialty areas than is typically the case with health information systems. all participants must recognize that each domain has its own specific terminology and operational approaches. as previously mentioned in the public health informatics example, the interlocutor function is vital. since it is highly unlikely that any single person will be able to span all or even most of the varied disciplinary areas, everyone on the team must make a special effort to learn the vocabulary used by others. as a result of these extensive and difficult informatics challenges, there are few operational information systems supporting bioterrorism preparedness. it is interesting to note that all the existing systems developed to date are local. this is most likely a consequence of the same issues previously delineated in the discussion of the advantages of community-based strategies for nhii development. one such system performs automated electronic lab reporting in indianapolis (overhage et al., 2001) . the development of this system was led by the same active informatics group that developed the lhii in the same area. nevertheless, it took several years of persistent and difficult efforts to overcome the technical, organizational, and legal issues involved. for example, even though all laboratories submitted data in "standard" hl7 format, it turned out that many of them were interpreting the standard in such a way that the electronic transactions could not be effectively processed by the recipient system. to address this problem, extensive reworking of the software that generated these transactions was required for many of the participating laboratories. another example of a bioterrorism preparedness system involves emergency room chief complaint reporting in pittsburgh (tsui et al., 2003) . this is a collaborative effort of multiple institutions with existing electronic medical record systems. it has also been led by an active informatics group that has worked long and hard to overcome technical, organizational, and legal challenges. it provides a near real-time "dashboard" for showing the incidence rates of specific types of syndromes, such as gastrointestinal and respiratory. this information is very useful for monitoring the patterns of diseases presenting to the area's emergency departments. note that both of these systems were built upon extensive prior work done by existing informatics groups. they also took advantage of existing local health information infrastructures that provided either available or least accessible electronic data streams. in spite of these advantages, it is clear from these and other efforts that the challenges in building bioterrorism preparedness systems are immense. however, having an existing health information infrastructure appears to be a key prerequisite. such an infrastructure implies the existence of a capable informatics group and available electronic health data in the community. public health informatics may be viewed as the application of biomedical informatics to populations. in a sense, it is the ultimate evolution of biomedical informatics, which has traditionally focused on applications related to individual patients. public health informatics highlights the potential of the health informatics disciplines as a group to integrate information from the molecular to the population level. public health informatics and the development of health information infrastructures are closely related. public health informatics deals with public health applications, whereas health information infrastructures are population-level applications primarily focused on medical care. while the information from these two areas overlaps, the orientation of both is the community rather than the individual. public health and health care have not traditionally interacted as closely as they should. in a larger sense, both really focus on the health of communities-public health does this directly, while the medical care system does it one patient at a time. however, it is now clear that medical care must also focus on the community to integrate the effective delivery of services across all care settings for all individuals. the informatics challenges inherent in both public health informatics and the development of health information infrastructures are immense. they include the challenge of large numbers of different types of organizations including government at all levels. this results in cultural, strategic, and personnel challenges. the legal issues involved in interinstitutional information systems, especially with regard to information sharing, can be daunting. finally, communications challenges are particularly difficult because of the large number of areas of expertise represented, including those that go beyond the health care domain (e.g., law enforcement). to deal with these communication issues, the interlocutor function is particularly critical. however, the effort required to address the challenges of public health informatics and health information infrastructures is worthwhile because the potential benefits are so substantial. effective information systems in these domains can help to assure effective prevention, high-quality care, and minimization of medical errors. in addition to the resultant decreases in both morbidity and mortality, these systems also have the potential to save hundreds of billions of dollars in both direct and indirect costs. it has been previously noted that one of the key differences between public health informatics and other informatics disciplines is that it includes interventions beyond the medical care system, and is not limited to medical and surgical treatments (yasnoff et al., 2000) . so despite the focus of most current public health informatics activities on population-based extensions of the medical care system (leading to the orientation of this chapter), applications beyond this scope are both possible and desirable. indeed, the phenomenal contributions to health made by the hygienic movement of the 19th and early 20th centuries suggest the power of large-scale environmental, legislative, and social changes to promote human health (rosen, 1993) . public health informatics must explore these dimensions as energetically as those associated with prevention and clinical care at the individual level. the effective application of informatics to populations through its use in both public health and the development of health information infrastructures is a key challenge of the 21st century. it is a challenge we must accept, understand, and overcome if we want to create an efficient and effective health care system as well as truly healthy communities for all. questions for further study: while some of the particulars are a little dated, this accessible document shows how public health professionals approach informatics problems can electronic medical record systems transform health care? potential health benefits a consensus action agenda for achieving the national health information infrastructure public health informatics: how information-age technology can strengthen public health public health 101 for informaticians public health informatics and information systems the value of healthcare information exchange and interoperability a consensus action agenda for achieving the national health information infrastructure public health informatics: improving and transforming public health in the information age what are the current and potential effects of a) the genomics revolution; and b) 9/11 on public health informatics? how can the successful model of immunization registries be used in other domains of public health (be specific about those domains)? how might it fail in others? why? fourteen percent of the us gdp is spent on medical care (including public health). how could public health informatics help use those monies more efficiently? or lower the figure absolutely? compare and contrast the database desiderata for clinical versus public health information systems. explain it from non-technical and technical perspectives make the case for and against investing billions in an nhii what organizational options would you consider if you were beginning the development of a local health information infrastructure? what are the pros and cons of each? how would you proceed with making a decision about which one to use? phi) involves the application of information technology in any manner that improves or promotes human health, does this necessarily involve a human "user" that interacts with the phi application? for example, could the information technology underlying anti-lock braking systems be considered a public health informatics application? key: cord-310288-onr700ue authors: sciubba, daniel m.; ehresman, jeff; pennington, zach; lubelski, daniel; feghali, james; bydon, ali; chou, dean; elder, benjamin d.; elsamadicy, aladine a.; goodwin, c. rory; goodwin, matthew l.; harrop, james; klineberg, eric o.; laufer, ilya; lo, sheng-fu l.; neuman, brian j.; passias, peter g.; protopsaltis, themistocles; shin, john h.; theodore, nicholas; witham, timothy f.; benzel, edward c. title: scoring system to triage patients for spine surgery in the setting of limited resources: application to the covid-19 pandemic and beyond date: 2020-05-29 journal: world neurosurg doi: 10.1016/j.wneu.2020.05.233 sha: doc_id: 310288 cord_uid: onr700ue abstract background as of may 04, 2020, the covid-19 pandemic has affected over 3.5 million people and touched every inhabited continent. accordingly, it has stressed health systems the world over leading to the cancellation of elective surgical cases and discussions regarding healthcare resource rationing. it is expected that rationing of surgical resources will continue even after the pandemic peak, and may recur with future pandemics, creating a need for a means of triaging emergent and elective spine surgery patients. methods using a modified delphi technique, a cohort of 16 fellowship-trained spine surgeons from 10 academic medical centers constructed a scoring system for the triage and prioritization of emergent and elective spine surgeries. three separate rounds of videoconferencing and written correspondence were used to reach a final scoring system. sixteen test cases were used to optimize the scoring system so that it could categorize cases as requiring emergent, urgent, high-priority elective, or low-priority elective scheduling. results the devised scoring system included 8 independent components: neurological status, underlying spine stability, presentation of a high-risk post-operative complication, patient medical comorbidities, expected hospital course, expected discharge disposition, facility resource limitations, and local disease burden. the resultant calculator was deployed as a freely-available web-based calculator (https://jhuspine3.shinyapps.io/spineurgencycalculator/). conclusion here we present the first quantitative urgency scoring system for the triage and prioritizing of spine surgery cases in resource-limited settings. we believe that our scoring system, while not all-encompassing, has potential value as a guide for triaging spine surgical cases during the covid pandemic and post-covid period. every inhabited continent. accordingly, it has stressed health systems the world over leading to 4 the cancellation of elective surgical cases and discussions regarding healthcare resource 5 rationing. it is expected that rationing of surgical resources will continue even after the pandemic 6 peak, and may recur with future pandemics, creating a need for a means of triaging emergent and 7 elective spine surgery patients. 8 9 methods 10 using a modified delphi technique, a cohort of 16 fellowship-trained spine surgeons from 10 11 academic medical centers constructed a scoring system for the triage and prioritization of 12 emergent and elective spine surgeries. three separate rounds of videoconferencing and written 13 correspondence were used to reach a final scoring system. sixteen test cases were used to 14 optimize the scoring system so that it could categorize cases as requiring emergent, urgent, high-15 priority elective, or low-priority elective scheduling. 16 17 results the devised scoring system included 8 independent components: neurological status, underlying 19 spine stability, presentation of a high-risk post-operative complication, patient medical 20 comorbidities, expected hospital course, expected discharge disposition, facility resource 21 limitations, and local disease burden. the resultant calculator was deployed as a freely-available 22 web-based calculator (https://jhuspine3.shinyapps.io/spineurgencycalculator/). 23 24 conclusion 25 here we present the first quantitative urgency scoring system for the triage and prioritizing of 26 spine surgery cases in resource-limited settings. we believe that our scoring system, while not 27 all-encompassing, has potential value as a guide for triaging spine surgical cases during the 28 covid pandemic and post-covid period. 29 in acute respiratory distress syndrome (ards) and/or death. 1, 2 since that time it has spread 4 rapidly to affect nearly every country, placing significant stresses on the global healthcare 5 system. 3 in order to mobilize resources to combat this pandemic, the centers for medicare and 6 medicaid services (cms), 4 the centers for disease control and prevention (cdc), 5 and multiple 7 professional organizations 6,7 recommended the cancellation of elective surgical procedures. in 8 spite of this, it was recognized that there were cases, many of them neurosurgical, which required 9 urgent or emergent intervention to minimize patient morbidity and maximize the chances of an 10 optimal outcome. 8 in response, several centers have presented frameworks for the management 11 of neurosurgical patients presenting during the covid-19 pandemic. [8] [9] [10] [11] additionally, a triage 12 scoring system has been previously developed in an attempt to guide spine surgery consults. 12,13 13 however, to date, there has not been a systematic, multi-institutional scoring system that 14 includes resource availability and disease burden to aid in triaging spine surgery patients during 15 this crisis. though certain symptoms referable to chronic spinal conditions may not necessarily 16 be life threatening, these can cause significant pain and disability prompting the challenge of 17 determining who and when to operate in times of crises. 18 19 it is recognized that effective triaging of these cases in the post-covid era will be essential to 20 prevent the healthcare system from being overwhelmed by the backlog of elective spinal cases 21 that have been deferred because of the covid-19 pandemic. [14] [15] [16] recently, a scoring system 22 aimed at triaging such cases has been published in the general surgery literature, 17 however no 23 comparable system has been described for spine patients. here we present an applicable example 24 of such a system assembled based upon input by a multi-institutional collaboration. this scoring 25 system is designed to assist in two ways. first, it may assist spine surgeons and administrators 26 with triaging surgical patients during the covid-19 pandemic. second, the scoring system may 27 help health systems triage elective cases in the post-covid crisis, which is likely to also see a 28 relative shortage of surgical resources and has been described by some as a potential collateral 29 pandemic. 15 30 31 methods 32 scoring system development 33 to generate this scoring system, the first author proposed an a priori scale highlighting those 34 elements thought to be pertinent to the triaging of an operative spine patient in the setting of 35 limited resources. the elements applicable to the spine patient included the patient's current 36 neurological status (rapidity of progressive, severity), the presence of underlying spinal 37 instability, and radiographic evidence of neural element compression. several general elements 38 were added that could be used to triage any surgical patient, including general patient 39 health/comorbidities, expected resource utilization, current resource availability, and local 40 disease burden. medical comorbidities were pulled from the charlson comorbidity index 18 and 41 from previously published series describing comorbidities associated with increased symptom 42 severity in patients infected with the sars-cov-2 virus. 2, [19] [20] [21] [22] [23] after identifying these elements, 43 weights were initially assigned based on input from surgeons at the lead institution using a 44 modified delphi approach that included both neurosurgical and orthopaedic spine surgeons. 45 component weighting of the preliminary scale was tested using ten example spine patients, 46 3 testing the assessed urgency of the patient as determined by the scoring system against the 47 consensus opinion of the group of surgeons. 48 49 after identifying a preliminary scoring system, a multi-institutional group was convened, 50 including neurosurgical and orthopaedic spine surgeons from multiple institutions with varying 51 levels of experience. a modified delphi approach was again used to alter the weights assigned to 52 the categories to refine the preliminary score. three rounds of written communication, polling, 53 and electronic teleconferencing sessions were used to solicit input. example cases were again 54 devised to test the degree of agreement between the scoring system and the consensus opinions 55 regarding the urgency of the hypothetical patient's issue (supplemental data). the final scoring 56 system was then deployed as a freely available, web-based calculator (figure 1 ; 57 https://jhuspine3.shinyapps.io/spineurgencycalculator/). 58 59 details of the multi-institutional panel 60 the study group was comprised of 16 spine surgeons representing 12 the degree of impairment that their deficit causes in ambulation or the ability to perform 75 activities of daily living (adl). 76 77 the scoring system runs from -19 (lowest priority elective case) to 91 (highest priority emergent 78 case) and classifies cases as "emergent," "urgent," "high-priority elective," or "low-priority 79 elective" as identified in table 2 . additionally, in they have not provided an algorithm for the prioritization of such cases in the setting of potential 106 resource shortages. here we present a scoring system devised by a multi-institutional 107 collaboration that aims to assist with these triage issues. the ability to assist with both 108 populations is a strength of this scoring system, which we feel may be a useful tool for health 109 systems both during the covid pandemic and in the post-crisis period, as they struggle to 110 accommodate the large volume of non-emergent surgical cases. additionally, though we hope 111 such a need does not arise, the present scoring system could also have value in the triaging of 112 patients if a "second wave" of the coronavirus pandemic occurs, which may lead to further 113 resource limitations. 26 such a wave occurred during the 1918 spanish influenza pandemic 27 and 114 many experts have speculated that a similar phenomenon could occur during the present 115 pandemic. 26, 28 furthermore, the framework of the proposed scoring system could apply to future 116 pandemics where healthcare resources are similarly stretched as the current covid-19 117 pandemic. 118 119 prior examinations of triaging in neurosurgery 120 there have been several broad descriptions of triage strategies presented in the neurosurgical 121 literature, 29, 30 and guidelines from the american college of surgeons (acs) currently divide 122 surgeries into five levels based upon apparent acuity. 11 however a large proportion of spinal 123 cases require emergent or urgent addressal 29 in addition to a perceived lack of granularity, neurosurgical triage systems published in the pre-130 covid era have predominately focused on emergent surgical issues. triage amongst non-131 emergent cases has been largely overlooked. one exception to this is the "accountability for 132 reasonableness (a4r)" framework described by ibrahim and colleagues 32 to emphasize 133 scheduling fairness and minimize operating room downtime at an academic center seeing a 134 mixture of emergent and elective cases. unlike the present scoring system however, their 135 framework was purely qualitative -triaging was performed by a single stakeholder without an 136 obvious means by which surgical cases were ranked. another exception is the calgary spine 137 severity score proposed by lwu et al. 12 that assessed spine referrals based on the clinical, 138 pathological, and radiological aspects. similar to the a4r framework, however, this score was 139 not intended for implementation in the setting of a crisis or the acute resource shortages that are 140 expected in the post-covid era. 15 constituted an urgent case, namely a surgical issue requiring treatment within 2 weeks that was 155 not identified in the emergency list. elective cases were similarly identified as all cases that did 156 not fall into the above two categories. unlike the system presented here, however, no formalized 157 system was identified for the prioritization of cases within the urgent or elective categories. neuro-oncology (sno) made recommendations to prioritize adjuvant therapies (e.g. 167 chemotherapy and radiotherapy) over earlier surgical intervention for spinal and intracranial 168 malignancies, as this will decrease the risks posed by hospitalizing oncologic patients in the 169 same facility as covid-19-positive patients. 33 however, the groups acknowledge that this is not 170 always possible, and that care deferral may cause some elective cases to progress to the point of 171 requiring urgent operative management. the european association for neurosurgical societies 172 has attempted to address the question of how to prioritize elective neurosurgical cases through an 173 "adapted elective surgery acuity scale." unfortunately, while this scale provides some 174 guidance, the three tiers it employs are quite broad and there are no guidelines for prioritizing 175 cases within a category or a given diagnosis (e.g. "degenerative spinal pathology"). 34 176 consequently, we feel the need for a means of triaging both emergent and elective spine cases 177 remains unmet. 178 179 while there have been several general frameworks highlighting those cranial pathologies 180 requiring emergent management, 8, 10, 11 there has only been one description of a framework for 181 triaging emergent spine surgeries. 25 derived from the experiences at a single italian center tasked 182 with treating cord compression and spinal instability, the framework of giorgi and colleagues is 183 a care pathway intended to expedite the identification, treatment, and safe discharge of patients 184 with spine emergencies. priority within the system was based upon american spinal injury 185 association (asia) grade and radiographic evidence of instability. though good results were 186 described for the 19 patients treated under the framework, the pathway is non-quantitative and 187 seemingly lacks the granularity to prioritize between two or more emergent patients. similarly, it 188 is not equipped to triage non-emergent cases. 189 190 a more quantitative approach was described by jean and colleagues 35 based upon nearly 500 191 respondents to an internet survey, asking respondents to assign an urgency score to each of nine 192 hypothetical cases. the authors found mild-to-moderate agreement regarding the extent of 193 surgical urgency for each case (range 22.8-37.0%), however, their "acuity index" was simplistic 194 in that it was based solely upon the perceived case risk and case urgency assigned to it by 195 respondents. case risk was graded on a 1 to 4 scale ("no risk" and "cannot postpone") and case 196 urgency on a 1 to 5 scale ("leave until after the end of the pandemic" and "case already done"). 197 the scale itself did not incorporate neurological status, patient comorbidities, or local resource 198 limitations, all of which are likely to influence the timing of operative management. because of 199 this lack of granularity, it is unclear that this "acuity index" can be generalized to other case 200 scenarios, thus limiting its potential utility relative to the multidimensional scoring system 201 described here. 202 203 204 limitations 205 as with scoring systems published in other domains of neurosurgery, the present scoring system 206 is not intended to be prescriptive in its guidance. rather, we present it as a potential tool to aid 207 surgeons and healthcare systems when triaging patients in times of national crisis or global 208 resource shortages. as with the triage frameworks presented to date, the present scoring system 209 is derived from expert opinions. consequently, the scoring system is limited by the biases of the 210 surgeons recruited and their respective institutions. we attempted to address this by recruiting 211 surgeons at multiple levels of training, at academic centers spread across a large geographic 212 region subjected to varying covid-19 burdens. furthermore, by only including surgeons into 213 the decision-making process of the urgency of spine patients, there is potential that additional 214 points from the non-surgical and administrative personnel could have altered the final scoring 215 system. additionally, in an effort to maximize the usability of the scoring system, it was 216 necessarily simplified and is consequently not all encompassing. for example, the broad term of 217 "new neurologic deficit" was included under the "high-risk postoperative complication" 218 category, however, this leaves it up to the treating surgeon whether this new deficit is "high-219 risk". therefore, while it can assist in determining surgical priority, final disposition should be 220 based upon the clinical judgment of the treating surgeon and institution. nevertheless, we believe 221 that it can be an effective tool for informing clinical stakeholders as to how each patient's case 222 may be triaged at peer institutions. our scoring system is also limited by the fact that it operates 223 on the assumption that the patient desires surgery at the same time recommended by the treating 224 surgeon. this is not always the case and the ultimate timing of surgery must therefore rely on an 225 in-depth discussion between provider and patient. finally, the present scoring system was 226 devised with the covid-19 pandemic in mind. consequently, it could be argued that it may not 227 be applicable to other resource challenging situations, and future pandemics may limit resources 228 in a manner not assessed in the current work. however, we feel that the modular structure 229 employed could easily be adapted to other crises that cause a shortage of medical resources. 230 therefore, the present system may have utility beyond the present crisis and any "second wave" 231 that may arise. 232 233 conclusion 234 here we present a scoring system for the triaging of spine surgery patients during times of crisis 235 and severe resource scarcity. our system was developed by a multi-institutional panel using a 236 modified delphi technique and has the potential to assist surgeons, hospital administrators, and 237 other clinical stakeholders in assigning priority to both emergent and non-emergent spine surgery 238 patients. while not intended to be prescriptive, this scoring system may prove useful as a guide 239 during both the covid crisis and the post-covid period to help prioritize patients with the 240 greatest surgical needs, though determining the urgency of an individual procedure should be left 241 to the operating surgeon. additionally, we believe the modular structure of the scoring system 242 implies that it may potentially be adapted to other crises resulting in an acute shortage of medical 243 resources. 244 245 tables table 1: spine surgery urgency scoring system table 2 : proposed timeframes for surgical treatment based upon urgency score figure 1 . screenshot of web-based calculator deployed based upon scoring system identified (https://jhuspine3.shinyapps.io/spineurgencycalculator/) 0 key: adl -activity of daily living; asc -ambulatory surgery center; d -day; hrhour; mo -month; snf -skilled nursing facility; wk -week â� whether the complication requires surgical intervention or can be treated with nonoperative management is made at the discretion of the attending surgeon â�¡vital structures include spinal cord, esophagus, trachea, aorta, lung, â§medical comorbidities included: active malignancy, age >65, congestive heart failure, chronic kidney disease, chronic obstructive pulmonary disease, current cigarette or vape use, diabetes mellitus, history of myocardial infarction, interstitial lung disease, moderate-to-severe liver disease. urgent (e.g. within 2 weeks) 10-14 high-priority elective (e.g. within 6 weeks) <10 low-priority elective (e.g. delay until after covid-19 crisis) key: covid-19 -coronavirus disease 2019 a novel coronavirus from patients with pneumonia in china clinical characteristics of coronavirus disease 2019 in china fair allocation of scarce medical resources in the time of covid-19 coronavirus disease 2019 (covid-19): healthcare facility guidance covid-19: recommendations for management of elective surgical procedures. acs: covid-19 and surgery. published 2020. accessed nass guidance document on elective, emergent, and urgent procedures letter: the coronavirus disease 2019 global pandemic: a neurosurgical treatment algorithm neurosurgery in the storm of covid-19: suggestions from the lombardy region, italy (ex malo bonum) academic neurosurgery department response to covid-19 pandemic: the university of miami/jackson memorial hospital model letter: adaptation under fire: two harvard neurosurgical services during the covid-19 pandemic a scoring system for elective triage of referrals: spine severity score validation of the calgary spine severity score how to risk-stratify elective surgery during the covid-19 pandemic? letter: collateral pandemic in face of the present covid-19 pandemic: a neurosurgical perspective editorial. covid-19 and its impact on neurosurgery: our early experience in singapore medically necessary, time-sensitive procedures: scoring system to ethically and efficiently manage resource scarcity and provider risk during the covid-19 pandemic a new method of classifying prognostic comorbidity in longitudinal studies: development and validation covid-19 in critically ill patients in the seattle region -case series clinical characteristics of covid-19 in new york city clinical characteristics of 138 hospitalized patients with 2019 novel coronavirus-infected pneumonia in wuhan, china clinical course and risk factors for mortality of adult inpatients with covid-19 in wuhan, china: a retrospective cohort study clinical course and outcomes of critically ill patients with sars-cov-2 pneumonia in wuhan, china: a single-centered, retrospective, observational study covid-19 and neurosurgical practice: an interim report the management of emergency spinal surgery during the covid-19 pandemic in italy beware of the second wave of covid-19 a year of terror and a century of reflection: perspectives on the great influenza pandemic of 1918-1919 first-wave covid-19 transmissibility and severity in china outside hubei after control measures, and second-wave scenario planning: a modelling impact assessment assessment of a triage protocol for emergent neurosurgical cases at a single institution protocol for urgent and emergent cases at a large academic level 1 trauma center is case triaging a useful tool for emergency surgeries? a review of 106 trauma surgery cases at a level 1 trauma center in south africa priority setting in neurosurgery as exemplified by an everyday challenge inpatient and outpatient case prioritization for patients with neuro-oncologic disease amid the covid-19 pandemic: general guidance for neuro-oncology practitioners from the aans/cns tumor section and society for neuro-oncology triaging non-emergent neurosurgical procedures during the covid-19 outbreak the impact of covid-19 on neurosurgeons and the strategy for triaging non-emergent operations: a global neurosurgery study key: cord-299884-wp4ehemj authors: chen, ray zhuangrui; wong, ming-hung title: integrated wetlands for food production date: 2016-07-31 journal: environmental research doi: 10.1016/j.envres.2016.01.007 sha: doc_id: 299884 cord_uid: wp4ehemj abstract the widespread use of compound pelleted feeds and chemical fertilizers in modern food production contribute to a vast amount of residual nutrients into the production system and adjacent ecosystem are major factors causing eutrophication. furthermore, the extensive development and application of chemical compounds (such as chemical pesticides, disinfectants and hormones used in enhancing productivity) in food production process are hazardous to the ecosystems, as well as human health. these unsustainable food production patterns cannot sustain human living in the long run. wetlands are perceived as self-decontamination ecosystems with high productivities. this review gives an overview about wetlands which are being integrated with food production processes, focusing on aquaculture. in order to increase production and enhance production efficiency in modernized conventional food production systems including aquaculture, high nutrient input, density breeding and assembly line production are usually adopted; with addition of pesticides, disinfectants, antibiotics, vaccines, immune-stimulants, and vitamins. however, efficient management for protecting the adjacent environment is either lacking or insufficient, while these conventional production systems also exert harmful effects to the environment (bunting, 2013; wong et al., 2004) . eutrophication is one of the greatest adverse environmental impact of modern aquaculture due to intensification through increasing use of pelleted feeds and expansion of the aquaculture area (edwards, 2015) . almost two third of the nutrients found in feeds are not consumed by cultured fish during growth and released into the immediate environment, with only one third is assimilated by the fish (edwards, 1993) . the residues originated from uneaten feeds, fecal matters and excreta contributed to most of the nutrients that become pollutants stagnated in the environment. both organic and inorganic matters discharged from aquaculture activities, especially nutrients such as, nitrogen and phosphorus have been involved in stimulating phytoplankton production, and in some instances the development of algal blooms (bunting, 2013; jacobs and harrison, 2014) , including toxic algal blooms which may in turn lead to anoxic zones, fish kills, and loss of biodiversity (anderson, 1989; backer and mcgillicuddy, 2006; hallegraeff, 2014 hallegraeff, , 1993 imai et al., 2014) . food production is increasingly being adversely affected by agriculture and aquaculture, and also domestic, industry discharges. pesticide residues have become nonpoint source pollution in waterways which are threatening the drinking water resources and aquatic ecosystems (dwiyana and mendoza, 2006; krone-davis et al., 2013; matamoros et al., 2008; moore et al., 2009; vymazal and březinová, 2015) . ammonia and nitrite arising from degradation of protein can be of primary toxic to aquatic organisms (bunting, 2013; kadlec and zmarthie, 2010; mook et al., 2012; tyson, 2007) . antimicrobial resistance (amr) bacteria detected in consuming animal food products has become an emerging public health concern, for instance, amr of bacteria was detected in oysters and mussels (rees et al., 2014) , pigs and poultry birds (amaechi et al., 2015) , fishes and prawns and also the ambient soil and water ways (sengeløv et al., 2003; silvester et al., 2015; thiele-bruhn, 2003; vivekanandhan et al., 2002; yohanna et al., 2013) . food safety issue has become increasingly important worldwide (blidariu and grozea, 2011) . efforts have been made to minimize the negative effects of food production systems on our environment. nevertheless, there seems to be an urgent need to develop sustainable aquaculture and agriculture in the near future. (klinger and naylor, 2012; stankus, 2013; turcios and papenbrock, 2014) . however, the food production patterns of currently mainstream worldwide are defective which are wasteful, pollution-carrying and unsustainable. hence, the food industries are facing great challenges. this article aims at reviewing how wetlands could be integrated with food production processes via ecological approach, along with a dual purpose of pollution control and safe and quality production. it is commonly recognized that food security is the greatest challenge to sustain human standard of living in the 21st century (un, 2009) , because of rapid population growth, which the increase in global population has been projected to reach 8.3 billion by 2030 (national intelligence council, 2012) . in addition, more than one billion of the present global population of 6.8 billion is under-nourished (mustafa, 2010) . it is envisaged that global food production will have to increase by at least 40% over the next 2 decades to meet the ever increasing food demand (fao, 2006) . the way to feed the global population equitably and sustainably is in fact a great challenge in the near future (hill and mustafa, 2011) . according to the assessment made by the world wildlife fund, overuse of the planet's natural resources has reached an alarming level, with human demands on natural resources doubled in the last 50 years. the wwf's 'living planet report' states that global consumption of resources is 50% above earth's sustainable ability (wwf, 2010) . furthermore, major pressures are exerted on the environment, due to continued consumerism in developed countries and growing affluence in a number of emerging economies. these led to an even higher demand of natural resources (rockström and karlberg, 2010) . therefore, it is rather difficult to ensure sufficient food and water for all to sustain livelihoods for present and future generations (hill and mustafa, 2011) . climate change also plays a profound role in various food production processes. for example, the impacts of global warming on harmful algal blooms may affect the quality and safety of the aquatic products (hallegraeff, 2014) . besides, extreme changes in climates are observed more frequently in recent years worldwide and aquaculture and agriculture are seriously affected by these extreme weathers, such as severe storms, tornados, salinity intrusions, droughts and floods (sutradhar et al., 2015) . rising sea level can affect aquaculture in coastal areas and cause salinity intrusion into deltas which can also affect freshwater aquaculture (edwards, 2015; sun et al., 2015) . although efforts (such as alternative to the burning of fossil fuels, targets and agreement on reducing the carbon dioxide) have been made by developing technologies to assist sustainable development, the rate of development is insufficient to ease the immediate threats of climate change (hill and mustafa, 2011; woolhouse et al., 2015) . water and soil loss, soil desertification and salinization, water eutrophication and among other factors are threatening agriculture and aquaculture in varying degrees. these environmental pressures are aggravating their effects on food production, and the range of influences has been enlarged by overlapping influences. as a consequence, the normal ecosystem functions and services of our food production systems has been degraded (rockström and karlberg, 2010) . apart from excessive nutrients such as nitrogen and phosphorus, which can cause eutrophication in water, elevated concentrations of a wide range of persistent toxic substances (pts), including heavy metals (such as mercury (hg), chromium (cr), lead (pb)), persistent organic pollutants (pops such as ddt) and certain emerging chemicals of concern (such as flame retardants) found in soil, water and air worldwide during the past decades, are greatly threatening our food production and food safety. despite of the success of many countries and regions of the world (such as the usa and eu) with relatively successful control and management practices for pts, the lack of efficient control and management in this regard, especially in certain rapidly developing countries, making pts easy to spread into environment. this situation has increased the potential poisons to human body by direct ingestion of contaminated water and consumption of food products grown in contaminated fields, and even through skin exposure and breathing air (jane et al., 2000) . for instance, the pearl river delta (prd), one of the most developed regions in china, has become the world's factory for various industries, e.g. textiles, and electrical/electronic products, and rather high concentrations of polychlorinated biphenyls (pcbs), cadmium (cd), pb and hg in fish reared in fish ponds around the area have been frequently reported (cheung et al., 2008; nie et al., 2006; zhou and wong, 2000) . li et al. (2011) reported a soil pollution status of pts in a newly developing industrial area around bohai bay, tianjin in northern china that relatively high concentrations of ddt (varied from nd (less than the limit of quantification) to 2417 ng/g, with a mean value of 73.9 ng/g), hch (varied from nd to 51,299 ng/g, with a mean value of 654 ng/g) and pahs (varied from 68.7 to 43,930 ng/g, with a mean value of 1225 ng/g) compared to most of the earlier studies in china hu et al., 2005; li et al., 2008; zhang et al., , 2009 in and around the industrial area. exposure of pts to the local residents was associated with a higher risk of local residents' health, such as breast cancer (odds ratio: 1.87, 95% confidence interval: 0.21-30.06), stomach cancer (odds ratio: 1.87, 95% confidence interval: 0.26-13.41), dermatitis (odds ratio: 1.72, 95% confidence interval: 1.05-2.80), gastroenteritis (odds ratio: 1.59, 95% confidence interval: 0.94-2.68), and pneumonia (odds ratio: 1.05, 95% confidence interval: 0.58-1.89). most of these pts are non-biodegradable, and could be bioaccumulated and biomagnified in aquatic food chains, which could then subsequently impose health effects on consumers via dietary intake of pts associated with large carnivorous fish (such as swordfish and grouper). it has been observed that trash fish (small fish without high commercial value) used in fish farms as feeds in south china is often contaminated (cheng et al., 2014; liang et al., 2011 liang et al., , 2012 zhou and wong, 2000) . it has been observed that fish meal and oil derived from trash fish served as the major components of feeds for conventional aquaculture are contaminated with ddt, hg, and even dioxins, e.g. in usa and canada (campbell et al., 2005; damsky et al., 2009) . to produce products using contaminated feeds or in contaminated fields will ultimately exert human health risks, especially to sensitive members of the population, such as infants and pregnant women. various studies revealed that pts play a crucial role in the pathogenesis of some health effects, such as pops, hg with male subfertility (hauser et al., 2003; hsu et al., 2014; mcauliffe et al., 2012) , autism in children due to metal overload (bjørklund, 2013; hagmeyer et al., 2015) ; cancers associated with high loadings of pts in human bodies (cohn et al., 2007; li et al., 2011; steinmaus et al., 2013; vogt et al., 2012) , etc. sustainable development involves the integration of cultural, economic, political and environmental factors (hoff, 1998) . however, cultural and political concerns are often given ways to economic development both in the past and at present. there is also a lack of efficient educational and political guidance in developing countries. in addition, unsustainable living and production patterns have also resulted in deterioration of environmental quality for sustainable development. to produce larger quantities of food to satisfy demand of the ever increasing population more effectively, conventional agriculture and aquaculture developed in the last decades focused on intensive production. many food items are produced in monoculture, for cultivating and harvesting crops or animals more easily. high technological farms use compound feeds, chemical fertilizers, pesticides, hormones and confined intensive animal feeding operations, since they are perceived as the most efficient strategies to maximize the output of limited land areas or water bodies. heavy use of antibiotics in confined animal feeding operations contributes to the problem of amr that would extend to affect human health (morrissey et al., 2015; woolhouse et al., 2015) . 175,000 t of antibiotics were estimated to be produced, used and misused per year (laxminarayan et al., 2013) , and under the directional selection by antibiotic residues, the host animals contain amr strains of bacteria may serve as a reservoir for amr genes in other food animals (amaechi et al., 2015; woolhouse et al., 2015) and drive the evolution of other microorganisms (habets and brockhurst, 2012; laxminarayan et al., 2013) . although antimicrobial peptides which have been proposed as a promising new class of antimicrobials and been applied as feed additives (kogut et al., 2013; yu et al., 2013) , the new study provided the first evidence that evolved resistance to a therapeutic antimicrobial peptides can provide cross-resistance to a human immunity peptide (habets and brockhurst, 2012) . amr has become a global environmental problem similar to climate change (woolhouse et al., 2015) . high fertilizer levels applied to enhance production or exploit the productive potential of crops for continuous production had resulted in an imbalanced soil nutrients and heavy use of pesticides (richter et al., 2015; ng et al., 2014) , due to the increased yield is more susceptible to pests, and pesticides are generally used for pest control. on the other hand the natural enemies of pests are also susceptible to pesticides, which in turn, leading to more usage of pesticides. nutrient loss through runoff is also a problem, and it has been reported that non-point source pollution from agriculture accounts for 60-80% of the nutrient loading to rivers and lakes (min and jiao, 2002) , and high nitrate concentrations in drinking water has been associated with methemoglobinemia and consequential cyanosis in infants (fan and steinberg, 1996) . the pesticides used for reducing pests and weeds in conventional farms, together with the surplus of chemical fertilizer have led to negative impacts, causing biodiversity loss and eutrophication (chau et al., 2015; hortle and bush, 2003; richter et al., 2015) , and even threats to human health (hu et al., 2015; jones et al., 2015; lebov et al., 2016; rauh et al., 2011) . there are signs of using less pesticides recently (moore et al., 2009; o'geen et al., 2010; vymazal and březinová, 2015) , but such actions have led to serious crop yield reductions (oerke, 2006; oerke et al., 1994) . compound feeds with high protein contents used to accelerate the growth of food animals without efficient methods to treat the residues are known to cause ecological hazards (edwards, 2015; zhang and fang, 2006) . approximately 5.44 t of nitrogen and 0.86 t of phosphorus (per hectare) are released to the environment, from intensive striped catfish farming (bosma et al., 2009; de silva et al., 2010; phuong et al., 2010) . these high-efficient conventional food production patterns which exploit the potential production, without sufficient concern about the negative impacts on the long-term ecological and human health, are unsustainable to our planet. the more production we achieved, the more natural resources are exploited, leading to higher impacts on ecological and human health. fortunately, some sustainable production techniques are becoming popular to ratify the conventional production systems: sustainable agriculture/ aquaculture, organic farming (paull, 2015) , regenerative agriculture, biodynamics farming and permaculture (koepf, 2014; nandal and kumari, 2015; olsen, 2014; poincelot, 2012; rhodes, 2012; paull, 2015) , all aiming at sustainable production, paying more attention to the balance of nature resources and human activities in food production, utilizing regenerative production resources, and producing safer food with less impacts on the ecosystem. there seems to be an urgent need to produce safe and quality food, which is environmental friendly. to achieve this, it is necessary to explore possibilities by means of technological innovation, and reformation of production patterns, based on the combination of suitable technologies and efficient management methods. in nature, all the materials can be cycled and used. however, over exploitation of resources and the amount and types of waste generated by human beings have disturbed the natural balance. hence, facilitating certain wastes as resources in food production is an essential key to sustainable development. proper treatment and disposal of an ever increasing amount of food wastes generated from densely populated cities such as hong kong is a recent environmental concern. recycling food wastes into feeds to ensure safe and quality fish production seems to be feasible, which could also partially ease disposal pressure and save natural resources (cheng et al., 2014) . water plays a significant role in food production, especially in aquaculture. however, water is also the major medium for diffusion of pts, introducing pts into fish farms, affecting the culture fish, and also discharging pts from farms and impose negative environmental impacts (amaraneni, 2006; an et al., 2006; fatta et al., 2007; rouff et al., 2013) . thus, water quality and management of water are major keys linking to safe and quality fish production, which is environmentally friendly. known as "earth's kidneys" for their purification capacity, wetlands are indispensable components of the earth's ecosystem. they are also among the most important natural habitats that support biodiversity and provide subsistence for humans. despite the fact that wetlands occupy only 6% of the earth's surface, they support approximately 20% of all living organisms on earth (zhao and song, 2004) . with the annual production value of 45-160 times (product value in terms of dollar value) of farmland ecosystems, wetlands are in fact the most productive ecosystem (zhang, 2001) . many wetlands are used for food production, for instance, mariculture, pond aquaculture, and plantation of crops. there exists great potential for integrating different food production systems into wetlands, in order to enhance sustainable food production, taking advantage of its ecological multifunction. these functions includes water conservation, runoff regulation (beutel et al., 2013; lizotte et al., 2012; ludwig and wright, 2015) , peat accumulation, carbon sequestration (kleinen et al., 2012; tuittila et al., 2013) , pollution purification, toxic substance transformation (paing et al., 2015; sudarsan et al., 2015; vymazal and březinová, 2015) , and disaster prevention for both droughts and floods wassens et al., 2011) . some wetlands are used for or linkage to different types of food production, based on their unique characteristics. studies show that wetlands integrated with other systems for food production processes are able to achieve more sustainable food production (please refer to cases below for details). 2.1. cases of integrating wetlands for food production 2.1.1. integrated wetlands with animals wetlands, like paddy fields and cane shoots (zizania latifolia (griseb)) fields are artificial surface flow systems, generally consisting of water on the surface, with cultivated commercial aquatic crops. higher economic benefits have been achieved through ecological approaches via integrating with appropriate animals in these modified flooded fields. rice-duck (wang et al., 2003; yang et al., 2004; zhen et al., 2007) , rice-fish (cruz et al., 1992; suloma and ogata, 2006) , rice-crab (yan et al., 2014) , and rice-prawn (nair et al., 2013) farming commonly practiced in asian countries are recognized as sustainable systems using less fertilizer and pesticide. the paddy fields or cane shoot fields provide shades, shelters and organic matters for the animals, which in turn oxygenate the soil and water, consume pests and favor nutrient recycling. these animals, especially the fish and duck are also viewed as a tool within an integrated pest management (ipm) system to make the agricultural activities more sustainable and environmentally friendly. berg (2001) reported that approximately 65% of pesticides was decreased by the benefit of ipm on rice-fish farms during 3 years, whereas non-ipm farms had increased the amount of pesticides used by 40%. these farming patterns help the farmers to increase the revenue by limited fields. it is reported that net revenue increased from the rice-fish system over the rice monoculture ranged 5%-11% in west africa (ofori et al., 2005) , 47%-66% in indonesia (dwiyana and mendoza, 2006) . rice-fish farming system has been listed by the food and agriculture organization of the united nations (fao) and the united nations educational, scientific and cultural organization (unesco) as one of the globally important agricultural heritage systems (giahs), with qingtian county of zhejiang province of china selected as a pilot conservation site (lu and li, 2006) . rice-fish farming has been practiced more than 1700 years in china and the practices in other countries only began after the mid-nineteenth century (java), mostly in the last century (such as japan, thailand) (little et al., 1996) , and african countries in recent years (such as egypt, nigeria) (ofori et al., 2005; suloma and ogata, 2006) . similar to rice-fish farming, rice-duck, rice-crab, rice-prawn farming makes use of the symbiotic relationship between paddy fields and animals to more effectively utilize nutrients and energy, reduce chemical fertilizer and pesticide inputs, increase food safety and productivity, and also mitigate the pollutants discharge zhang et al., 2001; zhen et al., 2004) . besides, it is reported that co-culture animals in wetland fields, especially organic co-culture patterns, could effectively improve quantity and composition of soil carbohydrates (nair et al., 2013; yan et al., 2014; yang et al., 2004) that would conduce to sustainable use of the soil resources. traditional low input rice-animal farming releases less nitrogen into environment, but the yield is limited. thus, some conventional patterns are invoked to boost yield in most of these practices (edwards, 2015) , such as using chemical fertilizers, pelleted feeds and chemical pesticides. however, increasing feeds or fertilizer use in rice field would also reduce the nitrogen use efficiency. hence, appropriate management of additional input would be necessary. hu et al. (2013) indicated that fish yield can be increased in traditional rice-fish farming system without increasing nitrogen (n) pollution and without decreasing n use efficiency, and rice yields. this could be achieved by managing the ratio of feed-n to fertilizer-n that quantities n input (with a constant input rate of 120 kg/ha). a balance will be reached with 37% fertilizer-n and 63% feed-n, while n not used by rice can be used by phytoplankton or other primary producers in the field that are subsequently consumed by the fish. there are well-developed human-livestock-crop-fish production systems in china, based on efficient waste recycling techniques (delmond et al., 1980; edwards, 2009 ). mulberry-dike-fish pond is one of the giahs (altieri and koohafkan, 2004) and the best known integrated production model. mulberry trees are cultivated on the pond dikes, with leaves fed to silkworms. excreta and pupae of silkworms are added into fishponds serving as fertilizer and fish feeds respectively, and the fertile pond mud consisting of fish excreta, organic matter, and chemical elements is brought up regularly from bottom and applied as fertilizer for the mulberry trees (zhong, 1982; ruddle and zhong, 1988; wong et al., 2004) . these principles of dike-aquaculture have been modified and now widely with many sub-units of ditches and stagnant waters to suit different regional needs (korn, 1996) . accordingly, assorted dike-pond and canal-dike agroecosystems have emerged throughout asia. the types of crops growing on the dikes are often governed by market demand and requirements of pond input. in early days, mulberry dikes, sugarcane dikes, fruit dikes, and miscellaneous crop dikes (such as soy-bean (glycine max), peanut (arachis hypogea), carrot (duucus curotu) were popular in pearl river delta (prd). the principal commercial crops cultivated were mulberry, sugarcane and various fruits (especially litchi (litchi spp.), longan (dimocarpus spp.), and bananas (musa spp.). a wide variety of vegetable crops are intercropping, inter-planting and rotation on the dikes, based on characteristics of different species, and under continuous cultivation. these vegetable crops and also some grasses are planted on all available spare land, including the dike slopes, roadsides, around the settlements, and along the watercourses. those are the fundamental components of a dike-pond system, providing essential food for domestic use as livestock and fish feeds, and vegetables for home consumption and marketing (ruddle et al., 1983) . the fish ponds normally raises several different compatible fish species (mainly of the carp family) at the same time. in this polyculture system, each fish species possesses distinct feeding habits, based on their physiology and morphology, occupying a different trophic level within the pond. there are also other aquatic organisms (such as carps and shrimps) thriving in the same water body. within the same pond, different living organisms are in proximity of each other, connected by nutrient and energy transfer through water. this so called "integrated multitrophic aquaculture (imta)" is in fact a diverse and low-cost approach to waste treatment, combined with efficient ecological management of aquaculture (klinger and naylor, 2012) . the harmonious structure of this system could be achieved through appropriate selection of different fish species and their optimum stocking ratios through the experience gained by fish farmers in the past (turcios and papenbrock, 2014) . in prd, it is common to co-culture bighead, silver carp, grass carp, black carp, mud carp and common carp in the same pond, with grass carp as the principal species, accounting for 50% of the fish. grass carp is the main breeding object which is mainly fed with grasses, sugarcane husks, leafy vegetables and macrophytes grown on dikes or adjacent water bodies and those plant material constitutes 99.6% of fish feed, additional feeds made up food consisting of the fermented residues of soy-bean curd, soy-bean cake, peanut cake and rice bran. the bighead and silver carp are fed by naturally grown plankton which favor the nutrient-rich water. the common and mud carp are detritus feeders which consume excrement, unconsumed food, snails and worms (zhong, 1982) . apart from the typical production model of mulberry-dike-fish pond, there are several secondary production models that coexist with these integrated dike pond systems. different domestic animals, such as rabbits, chickens and goats are reared on the dikes; while ducks on the pond water. furthermore, aquatic plants such as duckweed, water lettuce and water hyacinth are kept in rivulets, canals, and associated bodies of water for feeding fish and pigs. pond mud is used to cultivate mushrooms while the off-season for silkworm in winter and the nutrient-rich mud-bed is also used to fertilize the dike after the final crop of mushrooms have been harvested (zhong, 1982) . the by-products and manures from pigs and chickens are used to generate biogas; and the biogas residues are used as fish feed and fertilizer (jiang and zhao, 2011) . these integrated agriculture-aquaculture systems are excellent examples of sustainable aquaculture, where energy is fully utilized and most if not all elements derived from waste materials and byproducts are efficiently recycled. furthermore, all the living organisms are designed to co-exist harmoniously in this artificial ecosystem and kept balance on the resource utilization and the amount of different species. on the contrary, the conventional fish farming methods are less sustainable in terms of resource utilization. large amounts of nutrient-rich wastewater, containing chemical residues such as antibiotics and disinfectants are produced, together with unconsumed fish feeds and excretion, imposing harmful effects on the receiving environment (klemenčič and bulc, 2015; konnerup et al., 2011; silvester et al., 2015; richter et al., 2015; avant, 2015; tonguthai, 2000) . unfortunately, the area dike-ponds has continued to decline in recent years, given way to factories, residential buildings and other facilities gradually, following the economic reforms of south china started in 1978 (wong et al., 2004) . 2.1.3. "aquaponic" (aquacultureþ hydroponics) system "aquaponic" system is an approach which integrated intensive aquaculture with innovated agriculture technologies, with crops growing in liquid without soil (hydroponic culture). this is basically derived from the recirculating aquaculture system (ras) which recycle water through filters to remove food and fish residues. the name "aquaponic" refers to the combination of 'aquaculture' and 'hydroponic' (chalmers, 2004) , focusing on recycling the aquaculture effluent (which is full of nutrients), through recirculating over trickling filters or other suitable substrates (such as gravel, expanded clay and sand) for growing crops (palm et al., 2014; salam et al., 2015) . based on the principles commonly adopted in constructed wetlands, this technique enhances the purification capacity of the system. as a matter of fact, most of the designs of 'hydroponic' component of "aquaponic" are similar to artificial treatment wetlands, serving as ras for the purification of wastewater, which is connected to a tank for fish culture. some cash crops including fruits, ornamental flowers, culinary or medicinal herbs and vegetables are grown instead of aquatic plants. the basic components of "aquaponics" system consist of a fish culture tank, a settleable component for suspended solids removal, a biofilter, and a hydroponic component, with the latter three usually combined (rakocy and hargreaves, 1993; rakocy, 2012; tyson et al., 2011) . although "aquaponics" system may involve high capital and operating costs, even higher energy inputs than the conventional pond and cage cultures (edwards, 2015) , it is an ideal setup to reduce water pollution and eutrophication derived from aquaculture effluent. it has been noted that integrated use of renewable energy such as solar, wind, and biogas energy could reduce the energy cost which is supposed to be the prime operation cost after the construction of aquaponics system (gigliona, 2015; mohamad et al., 2013; olafs, 2014) . increasing the variety of products and overall productivity of "aquaponics" system could ease the cost and enhance the practicability of this technology. it has been suggested to combine algal culture, aquaculture and aquaponics together (chalmers, 2004; nuttle, 2003) . in this system, quails produce eggs and meats and their manures are used to supply nutrients needed for algal culture. tilapia are used to consume surplus micro-algae and are supplied as food. manure derived from the cultured fish, effluent and algal water serve as fertilizers for the nearby aquaponic crops, without any disease problems. algae are harvested weekly and manufactured for commercial end users. these new economic possibilities are attractive to inland farms in arid regions and water-limited farms in other regions, since it is basically a water re-use system (diver, 2000; graber and junge, 2009; nichols and savidov, 2011) . "aquaponic" has become a fastgrowing industry in australia (lennard, 2004) , as the lack of freshwater is a major limit for aquaculture. constructed treatment wetland (ctw) is an artificial wetland which is able to utilize natural functions of vegetation, soil, and microorganisms for treating wastewater, including municipal or domestic sewage, industrial and agriculture wastewater, landfill leachate, and stormwater runoff, which has been proven to be a well-established and cost-effective method (turcios and papenbrock, 2014; webb et al., 2012) . it is regarded as a low cost, and a low carbon method for wastewater treatment and has been applied in food production processes. for instances: ctws have been used to treat the wastewater from animal husbandry (hammer et al., 1993; lee et al., 2014; niu et al., 2015) and aquaculture (lin et al., 2002a; schwartz and boyd, 1995) , and non-point source pollution from agriculture (o'geen et al., 2010) . it has also been applied to lower heavy metal bioaccumulation (cheng et al., 2002; guittonny-philippe et al., 2014; yadav et al., 2012) , and mitigate the use of hazardous pesticides (kohler et al., 2004; krone-davis et al., 2013; vymazal and březinová, 2015) . a simple integration of ctw into a recirculating hatchery was conducted by buřič et al. (buřič et al., 2015) in the czech republic that different from traditional rass which generally involve specialized technologies, such as microsieve filtration, ozonization, and uv sterilization (azaizeh et al., 2013; good et al., 2011; sharrer et al., 2010; terjesen et al., 2013) . this study indicated that ctw integration has the potential to increase production (potential final biomass with cw use was estimated to be 40% greater than without cw use) of a recirculating hatchery without added operational or environmental costs. for mariculture, webb et al. (2012) demonstrated that ctws planted with salicornia spp. for the treatment of effluents from land-based intensive marine aquaculture farms, and salicornia can be sold as an agricultural crop. however, it requires a relatively extensive area to build ctw systems sufficient for effluent treatment (starkl et al., 2015) . in order to remove major pollutants from catfish, shrimp and milkfish pond effluents, a ctw with the size of 0.7-2.7 times the pond area for treating the polluted fishpond effluents would be needed (lin et al., 2002a; schwartz and boyd, 1995) , with the following hydraulic conditions: a low hydraulic loading rate (hlr) ranging 0.018-0.135 m day à 1 (lin et al., 2002a (lin et al., , 2002b , and a long hydraulic retention time (hrt) ranging 1-12.8 days (sansanayuth et al., 1996; schwartz and boyd, 1995) . according to shpigel et al. (2013) , about 10,000 m 2 of ctw with salicornia are required to remove nitrogen and total suspended solids produced from 900 kg of 45% crude protein fish feed (11 m2 kg à 1 of feed) during one year. nevertheless, most of the food production systems lack spared land to build a sufficiently large ctw. hence, to integrate ctws with food production systems more compact designs would be essential, especially turning ctws into food production fields would be more practical than single function of wastewater treatment. different from the ctws that build on land, agriculture in water is implemented on water surface, using it as a culture medium for agriculture. this method called "chinampa" in the early agriculture in mexico while the aztecs cultivated agriculture islands as early as 1150-1350 bc. plants are cultivated on stationary islands (by staking mud, lake sediment, and decaying vegetation out of the shallow lake bed and fencing in the rectangle with wattle) or movable islands (artificial floating islands, also called "floating gardens") in lake shallows. the build-up layers of vegetation are raised 50 cm above the water level, making the chinampas like sponges of organic earth that achieved the effective use of water surround them (ruiz et al., 2014) . waste materials dredged from canals and surrounding cities were used to irrigate the plants. flood tolerant trees are often planted at the corners to secure the chinampas (turcios and papenbrock, 2014) . fish are raised in the canals forming islands in proximity, and the sedimentary waste from fish is collected to fertilize the crops planted on the islands. these chinampas produce very high crop yields with four or up to seven harvests a year (tuerenhout, 2005; turcios and papenbrock, 2014) . a new floating garden technology is developed in bangladesh (irfanullah et al., 2011) , to allow farmers who live in areas covered by water during wet season to grow food on flooded land. water hyacinth and bamboos are used to construct rafts as floating bodies, soil and cattle manure are placed on the surface of the rafts, with vegetables planted in the soil. new rafts would need to be built every year, and the old ones can be used as fertilizer during the dry season. gourd (lagenaria spp.), okra (abelmoschus spp.), groundnut (arachis spp.) and leafy vegetables are grown organically in these floating gardens and provided vital food and income for the poor people in developing countries. floating island is a new ecological treatment technique for wastewater enriched with nutrients. it can also be used as floating garden but different in the use of modern floating body and culture medium (such as polystyrene, haycite, roseite, etc.) (dai et al., 2006) instead of fertility medium like mud and cow dung. this technique has a variety of forms and is also named as "ecological floating bed", "aquatic grassland", and "artificial floating island" . besides water purification, it is also used as habitat for birds and fish for ecological restoration and protection, and for developing agriculture in water for food production sang and li, 2007) . previous studies showed both terrestrial plants and aquatic plants can be cultured on floating islands as green food or safe feedstuff. these included paddy (oryza sativa, oryza glaberrima), celery (oenanthe javanica), goose grass (eleusine indica), canna (canna indica), barnyard grass (echinochloa crusgali), water spinach (nasturtium officinale), vetiver grass (vetiveria zizanioides), reed (phragmites australis); the yields are generally higher than those cultured on land . it was demonstrated that levels of heavy metals and nitrite contained in crops cultured in an eutrophic lake (shahu lake, wuchang, china) by floating islands were safe for consumption. however, biological concentration of heavy metals and pts would be a public health concern and the quality of products should be strictly controlled. wetlands provide various essential functions to sustain the global ecosystem, and can be integrated into different food production systems mentioned above. there seems to be great potential for producing safe and quality food which is environmentally friendly, by incorporating modern practices with wetlands. this is especially true for enhancing the capabilities of nutrient recycling and water purification of the whole production system, whether it is organic farming or intensive conventional farming. mutual benefits could be achieved by the coexistence of different components and polyculture of different animal species, fully utilizing substances and energy derived from different components. the composition of the whole system is independent of the outside environment. in fact, it is a relatively closed-system, for mitigating the negative effects derived from each of the production system and the environment. the wastes are contained, degraded and utilized, instead of spreading to the outside environment as pollutants (fig. 1) . integrating wetlands with aquaculture, or agriculture and animal farming through adopting different appropriate patterns are the foundation of the whole system, depending on various functions of wetlands which support energy transfer, nutrient cycling and production processes. one of the most potential benefits of wetlands for integrating usages would be contribution to saving water resources. being an indispensable medium for living beings, water is also essential during the production processes of both agriculture and aquaculture (zhong, 1982) . the integration of wetlands with food production systems would greatly enable water storage and would therefore become more self-sustained, with sufficient water supply for production processes. degradation of pollutants in water or wet soil is usually more effective than in dry soil, as microorganisms are more active in wet than dry condition, with a more rapid transfer of nutrients and energy flow. in addition to external interferences, wastes, animal manure and excreta are the main pollutant sources within the production ecosystems, consisting of organic matter, ammonia, nitrite, and pathogenic bacteria. accumulation of these substances is hazardous to both living organisms and the environment, and therefore effective degradation of pollutants in time is vital to prevent negative impacts on the whole ecosystem. the relationship of each component within a well-established and integrated production ecosystem is primarily based on efficient transfers of energy and exchange of substances (zhong, 1982) , with wastes and by-products from one species recycled to become food or fertilizers for another species. the organic wastes are efficiently degraded by microorganisms, earthworms and mollusks into inorganic matter and nutrients. inorganic compounds in wetlands are able to supply the large amount of nutrients required by algae and higher plants (turcios and papenbrock, 2014) . nutrients are recycled within food chains and food webs involving a variety of producers, consumers and decomposers which are supported by suitable habitats and conditions. in the integrated dike-pond system, the pond is the heart of this production system that produce the marketable fish. the organic residues settle to the bottom of the pond as nutrient rich pond mud, and the pond mud is excavated and used to repair and fertilize the upper surface of the dike that produce the essential inputs for the pond that demonstrated the main nutrient cycling. in aquaponics, aquaculture effluent generally contains elevated levels of ammonia, nitrate, nitrite, phosphorus, potassium, and other secondary pollutants, and also micro-nutrients applied to produce hydroponic plants (diver, 2000) . the suitable ratio of farmed animals and plants and their associated components is important to health and stabilization of the ecosystem that determines the efficiency of energy transfer and nutrient cycling which in turn affect productivity. in mulberry-dike-fish pond system, 45% to 55% is the best proportion of dike and ponds, yielding the highest economic benefit (ruddle et al., 1983) . if the dike and pond ratio is not suitable, self-reliance between the needs from fish rearing and dike fertilization could not be achieved, and additional feeds or fertilizer may be needed (zhong, 1982) . it is known that 20 kg of grass can be transformed into 0.5 kg of grass carp and 0.25 kg of bighead carp or silver carp; while 50 kg of sugar-cane leaves can be transformed into 0.5 kg of grass carp (zhong, 1982) . these estimation indicated that 10 t of fish product would require 550 t of cattle manure, 454 t of pig manure or 75 t of duck manure, and consequently the dike-pond system will become a major sink for manure and other organic wastes (bunting, 2013, p. 142) . in general, there is a lack of effective and feasible governance tools to solve various pressing problems facing food production, especially the environmental pressures. this is especially true for environmental problems due to different pts, threating not only food production, but also human health. large predatory fish such as tuna, swordfish and shark containing rather high levels of mercury, hazardous to human health (especially fetus, infants and pregnant mothers), is a typical example (ko et al., 2012) . changes have to be made concerning the patterns of living and production. inevitably, sustainable development is highly dependent on usages of clean/green energy which is pollution-free and renewable, and operations of clean/green industries which are non-polluting without waste generation; in addition to clean/green community and individual life styles which are with high concerns and feasible governance on the ecosystem preservation and individual development (hill and mustafa, 2011) . this can only be achieved by proper and effective management of all input and output, including wastes of individual subsystems such as urban centers, industries and food production fields. therefore, sustainable management of food production is linked to both sustainable agriculture and sustainable aquaculture (hill and mustafa, 2011) . listed below are four principles involved in achieving sustainable food production, focusing on minimal environmental disturbance and safe food production management (fig. 2) . (1) minimize input of hazardous substances. in view of bioaccumulation and biomagnification of various pts (such as hg, pbdes, and ddts) into the food production systems; the amr problem caused by the directional selection of antibiotic; and other problems caused by the residues of pesticides and hormones, that have been threatening the human and ecosystem health, a better control of these inputs to the food production systems is highly essential. integrated farms using strategies such as polyculture and co-culture are more efficient in using resources, with a higher total commercial income than monocultures (nair et al., 2013; ofori et al., 2005; wang et al., 2003; xi and qin, 2009 ). however, serious problems are encountered, if pts are entered into the production systems, and their concentrations are magnified due to bioaccumulation and biomagnification along the aquatic food chains (scholz-starke et al., 2013; windham-myers et al., 2014; zaldívar et al., 2011) . catastrophic loss of crawfish (procambarus clarkii) was reported in an integrated rice-crawfish facility due to the usage of pesticides for the control of rice water weevil (lissorhoptrus oryzophilus) (avault, 2001) . thus, strict management is needed to control the inputs of pts during food production processes. the use of safe feeds (with low and acceptable levels of pts), and bio-degradable pesticides without persistent toxic effects is also recommended. (2) minimize negative impacts on the ecosystem. this is important to maintain a healthy environment adjacent to food production systems, by enhancing the capability of pollutant treatment, especially the wastewater pathway which is the main pathway affecting the adjacent ecosystem. this could be achieved by effective waste treatment, e.g. taking advantages of wetland and biogas techniques, by improving the structure and function of the food production systems, enhancing their capability of water purification and waste management. for instance, inclusion of the components of hydroponic, constructed treatment wetland or floating islands into aquaculture could eliminate potential eutrophication problems (dai et al., 2006; graber and junge, 2009; shutes, 2001; yeh et al., 2015) . (3) recycle waste as resources. there is a need to recycle all fig. 2 . a concept framework for environmental friendly and safe food production management. wastes (e.g., sediment enriched in nutrients could be used as fertilizer) and byproducts (e.g. food processing byproducts such as bone and head of fish could be used as fish meal) generated as resources in food production systems, as much as possible. minimizing inputs of hazardous substances would therefore ensure safer use of these wastes and byproducts. a good management of waste recycling not only minimizes pollution discharges, but also maximizes nutrient utilization, resulting in enhanced yields and economic benefits. for instance, the mud derived from aquaculture is regarded as a good "slow-release" fertilizer in agriculture with high concentrations of organic matter, nitrogen and phosphorus, which has been used to fertilize the dike and cultured mushrooms in the integrated dike-pond system (bergheim et al., 1993; westerman et al., 1993) . (4) select high quality production sites. the selection of high quality food production site is absolutely vital to ensure safety of food products. currently, many sites for food production, and also water sources for food production are polluted, especially in developing countries, where factories are built in the vicinity of food production farms. thus, insulating food production sites from polluted sites would ensure higher quality production of food items, without entry of different toxic chemicals. for instance, 8000 t of red tilapia was killed in the chao phraya river (thailand) in 2007, due to the clandestine and illegal release of untreated factory effluents (pongpao and wipatayotin, 2007) ." in rapidly developing countries, the main safety risks for a food production system include pathophoresis, raw or improperly treated wastewater for irrigation, entry of pesticide residues, heavy metals and other pts into food production systems, from contaminated soils, water, and air, resulting in accumulation of these substances in the cultured products, imposing subsequent health risks, e.g. autism is linked with body metal overload, with children who have high consumption rates of fish contaminated with mercury (ko et al., 2012) . unfortunately, the use of trash fish for feeding carnivorous fish species, and even the commercial pellets containing fishmeal (also made of low quality fish processing waste) is also the major sources of pts, entering into the cultured products (liang et al., 2012) . in addition, the use of food supplements such as hormones and antibiotics for enhancing fish growth should be more strictly regulated and managed (jeong et al., 2010) , but it is difficult to regulate and manage such behaviors". in addition to adopting safe food production management mentioned above to inhibit entry of pts into the food chains, nonchemical methods should be used for controlling pests and diseases, i.e., biological control (resistant cultivars, predators, antagonistic organisms) or physical control (barriers, traps, manipulation of the environment) (chalmers, 2004) . in this regard, farming indoor would require fewer pesticides and fertilizers, which could reduce human health risks associated with high exposure to agrochemicals, comparing to open conventional farming (graff, 2009) . in fact, isolating the production sites from the pollution sources, such as air emission and waste effluent from various industries is absolutely necessary to ensure safe and quality products. for ground-based urban farming, a minimum distance is recommended between production fields and main roads to reduce the contamination of crops by lead and cadmium (säumel et al., 2012) . suitable buffer zones should be considered as one of the components in these production systems in order to minimize potential harmful effects. there have been public health concerns about the potential risks from integrated animal-animal system recently, due to outbreaks of virulent strains such as h5n1 avian flu, when different kinds of animals are reared in close proximity (bunting, 2013) . in view of this, potential mutation of virus within one kind of animal into a more virulent strain should not be overlooked, as it will be more readily be transmitted from human to human. therefore, the type of animals to be included in the integrated system should be carefully chosen, and the possible interaction between different types of animals grown together should be carefully monitored. however, pigs and poultry have been raised together on farms in asia and europe for centuries (edwards et al., 1988) , and there has been no evidence for the transmission of viruses to humans, mediated by such production strategy in the past. however, when compared to the past history, the outbreak of virulent strains has become more frequent. these included h5n1 avian flu, severe acute respiratory syndromes (sars), swine influenza virus (siv) and middle east respiratory syndrome (mers). this is likely caused by the differences in terms of food production patterns between the past and present. rearing livestock in pens, especially under intensive farming, restricts the activity of livestock. continual application of medications and disinfectants to maintain low pathogenic environments may have reduced the immunity of livestock and enhanced the tolerance and infectivity of pathogenic organisms. in addition, a large proportion of human population is having from many types of unhealthy habits (such as eat unhealthily, lack of exercise, irregular schedule of work and rest, live with oversized pressure), resulting a sub-health condition with low resistance, rendering themselves to become more susceptible. there were evidences of correlation between agriculture intensification and biodiversity loss and possible influences on disease emergency (morand et al., 2014; thongsripong et al., 2013) . thus, researches should put more focus on the therapeutic thought of increasing the resistance first, instead of extensive application of antibiotics and disinfectors. considering the recycling of waste produced by an animal species to another as resource, especially the excrement which may contain pathogenic bacteria and viruses, it is suggested to use wetlands for minimizing the risks. wetlands can treat wastewater effectively, converting animal waste to plant biomass, which can be used as feeding materials for rearing animals. wetlands are known for their high efficiency in pathogen removal (wu, 2008) . furthermore, rotating different plant species at certain time intervals is effective on reducing diseases (zhong, 1982) , and ensuring safety of food products (specht et al., 2014) . agriculture and aquaculture are the two main food production patterns. organic production pattern is regarded as a more sustainable farming system than conventional pattern, in terms of conserving soil, water, energy and biodiversity (gabriel et al., 2013; pimentel et al., 2005) , although it is less sustainable in producing the required food for the growing population in order to satisfy the increasing demand for food, conventional production pattern is still indispensable, but improvements to enhance food safety and environmental protection are essential. urgent solutions are needed to address the issue of pts entering into the production systems, and also waste materials (animal wastes and nutrients) escaping the systems, threatening environmental and human health. in principle, wetlands integrated with food production are able to treat inorganic and organic residues, produce safe and quality food, and increase the variety of production by appropriate polyculture that would enhance the overall income (chau et al., 2015) . the relatively closed-systems are more efficient in maximizing usage of resources and minimizing adverse environmental impacts. multiple wetland techniques and functions would be contributing to these strategies which would also be contributing to vertical farming. the strategies of integrated wetlands with food production are illustrated in fig. 3 . the organic combination of agriculture, aquaculture and wetland can form an intensive polyculture production ecosystem. a good management practice is required to assemble various adjacent food production systems (such as aquaculture, animal farming and agriculture), for sharing resources more effectively. in general, functional wetlands are mainly used to treat wastewater, converting organic waste into inorganic matter, and provision of available nutrients for enhancing crop growth. the polyculture practice can maximize total production and economic return per unit area of land in limited areas (ruddle et al., 1983) . the highly polluted conventional production patterns could be integrated with functional wetlands to minimize the adverse environmental impacts, and enhance food production more efficiently. however, hazardous substances for controlling pests should be replaced by less harmful or harmless methods, for instance, pesticides could be replaced by ecological control (such as ipm) or biopesticides, or even some chinese herbs. use prebiotics or probiotics to replace the antimicrobials, as well their mixture, so-called 'synbiotics' (woolhouse et al., 2015) could contribute to enhance the resistance and well-being of their host, but harmless to the ecosystem (marteau and boutron-ruault, 2002; patterson and burkholder, 2003) . although there is a consciousness that we need to produce plentiful food for the growing population (godfray et al., 2010; pinstrup-andersen, 2009 ), the waste of food in most of the developed areas is seriously and still with starvation in some poverty areas (gustavsson et al., 2011; knight and davis, 2010; lundie and peters, 2005) . in fact, how to feed the global population equitably and sustainably is the great challenge in the near future (hill and mustafa, 2011) . in today, how to eat safety and health is becoming the primary concern for most of the peoples. another hand, how to keep our lives sustainable is becoming a big topic, as our environment has been polluted, the normal functions of our ecosystem have been disturbed, and some of the production resources are depleting, such as the non-renewable phosphate rock may be depleted in 50-100 years (cordell et al., 2009) . thus, how to produce safety food via environmentally friendly patterns would be more vital in the coming future. food production systems with more efficient use of resources such as nutrients, energy, land, and water and less interference from hazardous substances are able to generate safe and quality food, which is environmentally friendly. in a properly integrated food production system, nutrients and energy are continuously recycled and utilized within the system, which is also effective, in reducing pollution emissions to the adjacent environment. integrated wetland techniques (such as hydroponic, constructed treatment wetland or floating island) with food production processes, coupled with polyculture of different fish species or other organisms (such as ducks, crabs, shrimps, and etc.), are able to promote ecosystem health, and achieve sustainability, mainly via its wastewater purification and nutrient recycling capability. the integration of agriculture systems with aquaculture systems is one of the important traditional practices. small-scale integrated agriculture-aquaculture systems are still widespread and of considerable importance for rural farming in some countries such as bangladesh, indonesia and vietnam (edwards, 2015) . nevertheless, some of these existing integrated agro-ecosystems are not completely in conformity with safe food production management, or considered as environmentally friendly. some may lack of control on waste and effluent or without proper waste and wastewater treatment, resulting in eutrophication of receiving waters. some may lack of quality control on the additional inputs, such as using feeds contaminated with ddt and hg. moreover, some may lack of suitable management to isolate the production systems from the pollution sources, such as using raw and improperly treated wastewater for irrigation, uptake of pesticide residues, heavy metals and other pts derived from contaminated soils, water, and air. hence, smarter plans for operating the holistic production system would be important and meaningful in order to achieve sustainable living and industrial production patterns. future studies are needed to provide a balanced diet to different species for maintaining a local ecological balance, finding cure to diseases using harmless techniques, raising overall production, and preventing disease dissemination among animals, especially in regions of different climatic conditions. furthermore, the government should formulate political guidance to promote the transform of environmentally unfriendly conventional food production systems into environmentally friendly integrated food production systems and also provide the needed technology and knowledge support. furthermore, both the national governments and relevant departments of the united nations should conduct political guidance to promote the transform of environmentally unfriendly conventional food production and also provide worldwide support of technology and knowledge. for instance, concerns about development of antimicrobial resistance and about transference of antibiotic resistance genes from animal to human microbiota, the european union has forbidden the use of antibiotics in livestock feed in 2006, consequently, relevant guidance was issued by world health organization (who), the food and agricultural organization of the united nations (fao), and the world organization for animal health (oie) (castanon, 2007) . globally important ingenious agricultural heritage systems (giahs): extent, significance, and implications for development plasmid profile and antimicrobial resistance ratings of escherichia coli isolates from pigs and poultry birds in abia state distribution of pesticides, pahs and heavy metals in prawn ponds near kolleru lake wetland residues and distribution character of organochlorine pesticides in stream sediments in southwestern suburb of nanjing toxic algal blooms and red tides: a global perspective catastrophic loss of pond-raised crawfish attributed to rice insecticide constructed wetlands combined with uv disinfection systems for removal of enteric pathogens and wastewater contaminants harmful algal blooms at the interface between coastal oceanography and human health pesticide use in rice and rice-fish farms in the mekong delta treatment and utilization of sludge from landbased farms for salmon fecal coliform removal in a lightly loaded surface-flow constructed treatment wetland polishing agricultural runoff the role of zinc and copper in autism spectrum disorders increasing the economical efficiency and sustainability of indoor fish farming by means of aquaponics -review environmental impact assessment of the pangasius sector in the mekong delta principles of sustainable aquaculture: promoting social, economic and environmental resilience back to the roots: the integration of a constructed wetland into a recirculating hatchery-a case study mercury and other trace elements in a pelagic arctic marine food web (northwater polynya, baffin bay) history of the use of antibiotic as growth promoters in european poultry feeds aquaponics and food safety use of wastewater from striped catfish (pangasianodon hypophthalmus) pond culture for integrated rice-fish-vegetable farming system in the mekong delta efficiency of constructed wetlands in decontamination of water polluted by heavy metals environmental mercury concentrations in cultured low-trophic-level fish using food waste-based diets metal concentrations of common freshwater and marine fish from the pearl river delta, south china. arch. environ ddt and breast cancer in young women: new data on the significance of age at exposure the story of phosphorus: global food security and food for thought ricefish research and development in asia development in the study of man-made floating culture islands' carrier public awareness of mercury in fish: analysis of public awareness and assessment of fish consumption in vermont estimation of nitrogen and phosphorus in effluent from the striped catfish farming sector in the mekong delta a review of integrated livestockfowl farming systems aquaponics-integration of hydroponics with aquaculture comparative productivity, profitability and efficiency of rice monoculture and rice-fish culture systems new technologies in aquaculture: improving production efficiency, quality and environment management aquaculture environment interactions: past, present and likely future trends fish farming and aquaculture environmental issues in integrated agriculture-aquaculture and wastewater-fed fish culture systems health implications of nitrate and nitrite in drinking water: an update on methemoglobinemia occurrence and reproductive and developmental toxicity organochlorine and organophosphoric insecticides, herbicides and heavy metals residue in industrial wastewaters in cyprus food production vs. biodiversity: comparing organic and conventional agriculture implementation of a biogas-system into aquaponics: determination of minimum size of aquaponics and costs per kwh of the produced energy food security: the challenge of feeding 9 billion people the effects of ozonation on performance, health and welfare of rainbow trout oncorhynchus mykiss in low-exchange water recirculation aquaculture systems aquaponic systems: nutrient recycling from fish wastewater by vegetable production a greener revolution: an argument for vertical farming constructed wetlands to reduce metal pollution from industrial catchments in aquatic mediterranean ecosystems: a review to overcome obstacles and suggest potential solutions global food losses and food waste therapeutic antimicrobial peptides may compromise natural immunity effects of trace metal profiles characteristic for autism on synapses in cultured neurons assessment and management of seafood safety and quality: current practices and emerging issues a review of harmful algal blooms and their apparent global increase treating livestock wastewaters with constructed wetlands the relationship between human semen parameters and environmental exposure to polychlorinated biphenyls and p natural resources management and food security in the context of sustainable development sustainable community development: studies in economic, environmental, cultural revitalization consumption in the lower mekong basin as a measure of fish yield. new approaches for improvement of inland capture fishery statistics in the mekong basin airborne persistent organic pollutants and male reproductive health occurrence of trace organic contaminants in bohai bay and its adjacent nanpaiwu river the productivity of traditional rice-fish co-culture can be increased without increasing nitrogen loss to the environment long-and short-term health effects of pesticide exposure: a cohort study from china prediction of toxic algal bloom occurrences and adaptation to toxic blooms to minimize economic loss to the scallop aquaculture industry in hokkaido floating gardening in bangladesh: a means to rebuild lives after devastating flood effects of floating vegetation on denitrification, nitrogen retention, and greenhouse gas production in wetland microcosms moms and pops. environmental working group risk assessment of growth hormones and antimicrobial residues in meat the history retrospect and development trend of integrated fish farming in china incidence of solid tumours among pesticide applicators exposed to the organophosphate insecticide diazinon in the agricultural health study: an updated analysis wetland treatment of leachate from a closed landfill a dynamic model of wetland extent and peat accumulation: results for the holocene the use of vertical constructed wetland and ultrasound in aquaponic systems searching for solutions in aquaculture: charting a sustainable course what a waste! surplus fresh foods research project. food climate research network heavy metal overloads and autism in children from mainland china and hong kong: a preliminary study the biodynamic farm: agriculture in the service of the earth and humanity modulation of chicken intestinal immune gene expression by small cationic peptides as feed additives during the first week posthatch nutrient, metal, and pesticide removal during storm and nonstorm events by a constructed wetland on an urban golf course treatment of fishpond water by recirculating horizontal and vertical flow constructed wetlands in the tropics the dike-pond concept: sustainable agriculture and nutrient recycling in china assessing pesticide reduction in constructed wetlands using a tanks-in-series model within a bayesian framework antibiotic resistance-the need for global solutions pesticide use and risk of end-stage renal disease among licensed pesticide applicators in the agricultural health study nitrogen mass balance in a constructed wetland treating piggery wastewater effluent the potential for aquaponics in australia physiological mechanisms for plant distribution pattern: responses to flooding and drought in three wetland plants from dongting lake environmental pollution by persistent toxic substances and health risk in an industrial area of china investigation and evaluation of ddt residues in soils in the suburbs of beijing studies on aquatic agriculture experiments on lakes the effects of mariculture activities on the adsorption/desorption and chemical fractionations of mercury on sediments the influence of mariculture on mercury distribution in sediments and fish around hong kong and adjacent mainland china waters nutrient removal from aquaculture wastewater using a constructed wetlands system removal of solids and oxygen demand from aquaculture wastewater with a constructed wetland system in the start-up phase fish culture in rainfed rice fields of northeast thailand mitigating agrichemicals from an artificial runoff event using a managed riverine wetland review of rice-fish-farming systems in china -one of the globally important ingenious agricultural heritage systems (giahs) integrated constructed wetlands for treatment of dairy operation runoff in eastern tennessee during first year establishment life cycle assessment of food waste management options nutritional advantages of probiotics and prebiotics organic micropollutant removal in a full-scale surface flow constructed wetland fed with secondary effluent environmental exposure to polychlorinated biphenyls and p,p'-dde and sperm sex-chromosome disomy effects of agricultural non-point source pollution on eutrophication of water body and its control measure development of aquaponic system using solar powered control pump removal of total ammonia nitrogen (tan), nitrate and total organic carbon (toc) from aquaculture wastewater using electrochemical technology: a review mitigation of two pyrethroid insecticides in a mississippi delta constructed wetland infectious diseases and their outbreaks in asia-pacific: biodiversity and its regulation loss matter neonicotinoid contamination of global surface waters and associated risk to aquatic invertebrates: a review seafood security in a changing cimate: adapting fisheries and aquaculture organic rice-prawn farming yields 20% higher revenues adoption of eco-friendly approaches in indian farming system agronomic and stream nitrate load responses to incentives for bioenergy crop cultivation and reductions of carbon emissions and fertilizer use aquaponics: a nutrient and water efficient production system distributions and congener patterns of pcbs in fish from major aquaculture areas in the pearl river delta, south china nitrogen reduction in stormwater from livestock lots by vertical subsurface flow wetlands packed with woodchips the peace technologies-algalculture, aquaculture and aquaponics mitigating nonpoint source pollution in agriculture with constructed and restored wetlands crop losses to pests crop production and crop protection: estimated losses in major food and cash crops rice-fish culture: an option for smallholder sawah rice farmers of the west african lowlands energy conservation by intermittently recirculating and aerating an aquaponics system with an airlift (dissertations and theses) biodynamic agriculture: a valuable alternative to the industrial farming system treatment of high organic content wastewater from food-processing industry with the french vertical flow constructed wetland system significant factors affecting the economic sustainability of closed aquaponic systems. part i: system design, chemo-physical parameters and general aspects application of prebiotics and probiotics in poultry production organic farming: the arrival and uptake of the dissident agriculture meme in australia water quality monitoring in striped catfish (pangasianodom hypophthalmus) farms in the mekong delta, vietnam, college of aquacultrue and fisheries environmental, energetic, and economic comparisons of organic and conventional farming systems food security: definition and measurement toward a more sustainable agriculture toxic river takes huge toll on fish farms aquaponics-integrating fish and plant culture integration of vegetable hydroponics with fish culture: a review seven-year neurodevelopmental scores and prenatal exposure to chlorpyrifos, a common agricultural pesticide occurrence and antimicrobial resistance of escherichia coli in oysters and mussels from atlantic canada feeding and healing the world: through regenerative agriculture and permaculture intensified food production and correlated risks to human health in the greater mekong subregion: a systematic review the quadruple squeeze: defining the safe operating space for freshwater use to achieve a triply green revolution in the anthropocene heavy metal distribution in an urban wetland impacted by combined sewer overflow integrated agriculture-aquaculture in south china: the dike-pond system of the zhujiang delta the mulberry dike-carp pond resource system of the zhujiang (pearl river) delta, people's republic of china: i. environmental context and system overview the chinampa: a sustainable high efficient agrohydrologic system for shallow lacustrine and wetland areas how healthy is urban horticulture in high traffic areas? trace metal concentrations in vegetable crops from plantings within inner city neighbourhoods in feasibility of tomato production in aquaponic system using different substrates discussion on agriculture mode in water based on nitrogen and phosphorus circulation shrimp pond effluent: pollution problems and treatment by constructed wetlands an integrated approach to model the biomagnification of organic pollutants in aquatic food webs of the yangtze three gorges reservoir ecosystem using adapted pollution scenarios constructed wetlands for treatment of channel catfish pond effluents bacterial antibiotic resistance levels in danish farmland as a result of treatment with pig manure slurry the cost and effectiveness of solids thickening technologies for treating backwash and recovering nutrients from intensive aquaculture systems constructed wetland with salicornia as a biofilter for mariculture effluents artificial wetlands and water quality improvement prevalence, antibiotic resistance, virulence and plasmid profiles of vibrio parahaemolyticus from a tropical estuary and adjoining traditional prawn farm along the southwest coast of india urban agriculture of the future: an overview of sustainability aspects of food production in and on buildings integrating biosystems to foster sustainable aquaculture: using black soldier fly larvae as feed in aquaponic systems stakeholder views, financing and policy implications for reuse of wastewater for irrigation: a case from hyderabad drinking water arsenic in northern chile: high cancer risks 40 years after exposure cessation study on treatment of electroplating wastewater using constructed wetland future of rice-fish culture, desert aquaculture and feed development in africa: the case of egypt as the leading country in africa china's coastal wetlands: conservation history, implementation efforts, existing issues and strategies for future improvement a review of good adaptation practices on climate change in bangladesh design, dimensioning, and performance of a research facility for studies on the requirements of fish in ras environments pharmaceutical antibiotic compounds in soils -a review mosquito vector diversity across habitats in central thailand endemic for dengue and other arthropod-borne diseases the use of chemicals in aquaculture in thailand the aztecs: new perspectives. abc-clio ltd wetland chronosequence as a model of peatland development: vegetation succession, peat and carbon accumulation sustainable treatment of aquaculture effluents-what can we learn from the past for the future? sustainability reconciling ph for ammonia biofiltration in a cucumber/tilapia aquaponics system using a perlite medium opportunities and challenges to sustainability in aquaponic systems world population prospects: the 2008 revision population division of the united national antibiotic resistance of aeromonas hydrophila isolated from marketed fish and prawn of south india cancer and non-cancer health effects from food contaminant exposures for children and adults in california: a risk assessment the use of constructed wetlands for removal of pesticides from agricultural runoff and drainage: a review integrated benefits of paddy rice-duck complex ecosystem ying yong sheng tai xue bao¼ the journal of applied ecology/zhongguo sheng tai xue xue hui, zhongguo ke xue yuan shenyang ying yong sheng tai yan jiu suo zhu ban frog breeding in rain-fed wetlands after a period of severe drought: implications for predicting the impacts of climate change halophyte filter beds for treatment of saline wastewater from aquaculture trout manure characterization and nitrogen mineralization rate mercury cycling in agricultural and managed wetlands: a synthesis of methylmercury production, hydrologic export, and bioaccumulation from an integrated field study the dyke-pond systems in south china: past, present and future antimicrobial resistance in humans, livestock and the wider environment integarted vertical-flow constructed wetland emergy evaluation of organic rice-duck mutualism system the removal of heavy metals in wetland microcosms: effects of bed depth, plant species, and metal mobility effect of different rice-crab coculture dodes on soil carbohydrates paddy soil quality of a wetland rice-duck complex ecosystem artificial floating islands for environmental improvement identification of antibiotic resistance cassettes in class 1 integrons in aeromonas spp. strains isolated from fresh fish (cyprinus carpio l.) antimicrobial peptides characteristics and their applications as feed additives in domestic animals an integrated approach for bioaccumulation assessment in mussels: towards the development of environmental quality standards for biota residues of organochlorine pesticides in hong kong soils study on the function and benefit of riceduck agroecosystem guide on implementing ramsar convention in china. china forestry publishing house levels and distribution of hchs and ddts in soils from chengdu economic zone phosphorus accumulation and eutrophication in feedsupply freshwater fishponds wetland utilization and protection in china wetlands ecosystems in asia: function and management ecological control effects of rice-duck integrated farming on main diseases, insects and weeds in paddy fields ecosystem current status and technical prospect of rice-duck mutualistic eco-farming in china the mulberry dike-fish pond complex: a chinese ecosystem of land-water interaction on the pearl river delta mercury accumulation in freshwater fish with emphasis on the dietary influence key: cord-016192-xc4ae7c1 authors: fernando, owen noel newton; rathnayake, vajira sampath; vijaykumar, santosh; lwin, may o.; foo, schubert title: mo-buzz: socially-mediated collaborative platform for ubiquitous location based service date: 2013 journal: human interface and the management of information doi: 10.1007/978-3-642-39215-3_44 sha: doc_id: 16192 cord_uid: xc4ae7c1 this paper describes a middleware platform for user-generated multimedia contents which facilitates visualization and communication of vector-borne diseases (dengue, malaria, etc.). it acts as a community platform, where diverse users from geographically distributed locations can collaborate to seek and contribute multimedia contents of such diseases and related issues (breeding sites, etc.). some of the essential services supported by the system are display of live hotspots, timeline, multimedia and twitter-feed visualization, and location based services for both users and authorities. as a proof-of-concept, dengue disease was selected to build services using this platform to observe its capabilities. dengue is a vector-borne infectious disease that has historically posed continued threats to populations living in both developed and developing countries. dengue affects more than 50 million people in the world every year, in particular countries in the asia-pacific region that share more than 70% of the disease burden [1] . in singapore, there were two serious outbreaks within the past 10 years, affecting 14,032 and 8,287 people in 2005 and 2007 respectively [2] [3] . although various efforts were made to fight dengue in singapore, the country remains vulnerable with an average number of 5, 000 dengue victims reported every year [4] . unfortunately, there is no known vaccination or medicine that can prevent this infection. hence preventing dengue, by destroying breading sites may be the best way to control this disease. lack of information about breading site is one of the major problems in controlling these kinds of diseases. authorities try to be aware about the real situation, but the resources are always a problem, especially in developing nations. several applications have been developed to address this issue by introducing different information gathering and distribution mechanisms. however, one noticeable drawback of these systems is that they are more focusing on a selected subject, hence solutions have its own limitations related to that particular subject. our application stems from a recognition of the growing need to integrate epidemiological practices with health communication interventions -two processes that are traditionally thought to take place complimentary can now do so concurrently. in great measure, this innovation is attributed to the emergence of mobile phones and social media that are transforming the way public health is practiced. to reaffirm the preference for these technological preferences among our populations of interest, we surveyed middle-of-pyramid populations in three asian countries -india, singapore, and vietnam -with a sample size of 1,000 from each country. our survey found that mobile phones are among the top 3 preferred media for seeking health-related information, and that the demand for smartphones is poised to increase in india and vietnam in the coming years. similarly, social media are found to be of immense utility for keeping in touch with people, searching for information and sharing information. these findings, in concert with emerging needs in public health, lead us to identify the need for a system which enables citizens to track disease spread (search for information), contribute to surveillance efforts by engaging with health authorities (share information) and further disseminate health information through members of their social networks (keep in touch with people/share information) using simple mobile phones or smartphones. our solution is an attempt to create a platform that gives authorities awareness about ground situation. the system also creates an interactive channel between the general public and authorities. this also allows interactions with end-users in a structured-hierarchy or a flat structure depend on the situation. hence this solution can be used to collect and distribute large range of data among diverse user groups. instead of asking general public to engage, this platform gives opportunities for them to involve and solve problems themselves. getting citizens more involved in the civic life and health of their communities is more effective in these kinds of situations, especially for resource limited environment. the bidirectional information floors are very important for such issues where it helps to have an equal and better understanding between the authorities and civilians, which will increase the civic engagement [5] in the long run. with the unprecedented growth of the internet and its increasing demand, diversity of standalone applications started to develop as web applications including, health [6] and geographical information systems [7] . in data visualization, map-based visualization is widely used to visualize geospatial data. currently, web-giss are widely used due to its ability to obtain data from geographically distributed locations. hence experiments are continuing to improve the usage of interfaces [8] , and reduce the cost [9] . further, visual health communication [10] is an emerging field because of its ability to help visualize the data in a friendly, interactive manner. therefore, extensive studies have been done in order to create a health based communication platform and use of gis when needed [9] . for fast spreading disease, gis systems have been used to gather information to allow necessary actions to be taken quickly and decisively in a situation like sars [11] . on the other hand, most of the current applications focus on centralized solutions [12] . on the other hand, most of the existing solutions focused on traditional methods, which has limited involvement of the social media or civic engagement [5] . hence the focus of this project is to create a platform for such engagement that can be used for diverse of issues using different channels. building social networks is one of the things that humans always fond of doing [13] . there are large numbers of information flows through social networks under different categories. researchers have been analyzing these contents to have better understanding about the society under different aspects. for instance, twitter is one of the famous social networking sites that can be used for above mentioned surveillance since it has a free and an open network. use of such networks for various domains including heath purposes can be found in [14] [15] [16] . to facilitates the general public to contribute to surveillance efforts in the event of disease outbreaks [17] , we have developed a platform, known as mo-buzz, which is focused on emergence of mobile phones and social media that will help to transform the way public health is practiced. the system is built upon open source technologies and mainly for mobile and web based application which can access through android platform or a browser. android solution helps the main application by running as an agent on mobile devices. the users can report information in various forms (photo, audio, video, text, etc.) using mobile devices. the system also uses sms technologies for feature phones. web based solution is available for everyone; however, it has more features for authorities. the solution consists of two parts, which are interactive system for geospatial visualization and web forms for other details. the solution is developed with the aid of java related technologies, such as richfaces, jpa, javascript, and jquery. server side of this system is supported by apache, tomcat, and mysql. google map api is used for the interactive map while google cloud is used to facilitate android based messaging solution. the overall system modules are shown in the fig. 1 . the system can be divided in to three main modules based on their functionalities, which are content management, visualization, and personalized messages & alerts. each of these modules has set of components that communicate to each other to facilitate attractive features. this component provides the cutting-edge addition to existing pe efforts. the key idea here is to activate the general public to contribute to surveillance efforts in the fig. 1 . system architecture event of disease outbreaks. in this instance, citizens can report breeding sites, mosquito bites and dengue symptoms using their smartphones in image (fig. 2) , video or text (fig. 3) formats. these inputs are automatically reflected in the hotspot maps and can be accessed by health authorities for responding to citizen concerns as well as for initiating preventive actions in specific communities. the process is facilitated rapidly because of two reasons: a) mobile phone-based inputs from citizens are geo-tagged; and b) the mo-buzz system captures geo-spatial coordinates, time and date, and phone number of the contributor. the process of content validation comprises three categories, which are high, moderate, and low. these levels will define the reliability of the contents. based on the reliability level of the data insertion mechanism, the filters are applied to the contents. if the category of the contents is high, then contents are directly available on the system. the content provided by authorities or registered users listed as high-reliable contents, which are fallen in to this category. the contents provided by the unregistered users are considered as low reliable data. such data become available to users that listed as content validates. users under authorities or registered users listed under "content validates" can access this content for verification. based on the content's location, information is pushed to volunteers for verification. after the verification, contents can be fallen into the high or moderate category. if the content is verified by a user under the authority or reliable registered user, the content is ranked as "high" otherwise it will be ranked as "moderate" and will available to appropriate users for further verification. one or more users can verify the same content and all the ranks for the content are recorded in the system. users can voluntarily participate for the content verification and above verification is only applied to some selected categories of content such as photos, videos, and text messages. one main objective of the content ranking is to avoid unwanted, or fraud information reaches the authority. in reality, this will help to optimize the accuracy of the contents and will increase the civic engagement. fig. 3 . report symptom using mobile app visualization mainly consists with maps and forms. the main component of visualization is the interactive map. most of the data in the system are geospatial data, which can be shown through the map. visualization information can be divided into following categories: hotspots are real incidents (ex: reported disease). the interactive map highlights these incidents by circles, which show the incident's housing block (fig.4) . the color of the circle denote by the number of cases reported from each block as explained by the ledger. the visualization of the hotspots is based on the approved data by authorities. the centers of the circles are calculated by using each incident block's postal code. the hotspots are used to provide exact location of the incident to the general public so they can arrange precautions based on their location. this visualization also gives a sense to different authorities about the next emerging incident clusters so they can plan and manage them accordingly. interactive map is encouraging its users (general public) to report about incidents or related information using several channels. users can report this information by sending photos (a of fig. 5 ), or messages (b of fig. 5 ) as shown in fig. 5 . this will help authorities to identify possible important place that need to pay their attention. this will also simulate the reality about different places and trends of the society. system will also allow its users to express their ideas through a forum. application also allows selected user to act as ambassadors of the community or institute. they can also include messages to the system and these messages are considered as verified or reliable information. this system helps to create awareness by listening to the public conversations which are happening in the society. this is another way to get information about the real ground situation. the current version can listen to twitter feeds and filter information based on the user location and keywords. the system will display the associated conversations on the map according to their geographical location as shown in figure 6 . these feeds will offer suggestion to the authorities about the real situation on the ground. the repository of outbreak information based on weather and citizen data is used to disseminate health messages to both, individuals and communities. at the individual level, citizens receive tailored messages based on their input to the system. for instance, a citizen reporting malaria symptoms or mosquito breeding site to mo-buzz will instantly receive a complete information guide on, breeding site or symptoms, and cues to various preventive actions. at the community level, the system will automatically send health education messages to communities/zones (fig. 7) that are highlighted on the maps as possible hotspots. public health surveillance efforts are thus used to generate and deliver health communication messages. at a fundamental level, the system acts as a catalyst between the citizen and the public health system where the contributions of each stand to benefit the other. overall, the intention is to use mo-buzz for efficient and effective risk prevention and outbreak management. in addition to communication modules, the system is capable of sending alerts to citizens living in areas identified as potential hotspots. messages can be alerts, reminders, or any useful information generated by the system or authorized users. personalized messaging is used to disseminate messages to end users according to the message settings. users can select various options in the message registration process which uses by the messaging system. messaging system uses both push and pull techniques according to the selected options. the proposed system always tries to avoid messages broadcasting. instead, it pushes messages to users based on their location, message's priority and other settings of messages and users. also, the client application can pull the messages according its options and user settings. these massages are sending email boxes, to devices through the google cloud or as smss. the developed solution works as a facilitator to manage the information flows between different types of user groups. most important sections of the system are its content, and the validity of its content. since the contents for this system are coming from the ground level and people who voluntarily contribute to the system, the content reflects the ground reality. since dengue is a larger problem at the ground level, there is a better chance to have decent quality content maintained by its users. the civic engagement is one of the important aspects of the proposed system, which is essential when we need to collect large datasets. by going an extra mile, we hope that this engagement will solve some of the small problem that can be ended by themselves (or as a group), without the help of the authorities. system is also encouraging this by maintaining its content as open-content and by providing necessary functionalities. (ex: through social ambassadors, forum, content validates etc.). one of the major challenges of a technology-driven participatory health system enterprise is validating the quality of informational inputs from citizens. selected content of the system must undergo through a verification and filtering (ex: photos and videos) in order to remove unrelated contents. our validation process is consistent in keeping with the core idea of using participatory media and crowd sourcing technologies. in that, we use people (individuals and health systems personnel) as validation experts. if users are self disciplined to provide only related information, then the system can avoid this and get rid of the delay and effort that needs to verify the information. we have created a middleware platform to enable diverse users to collaborate and contribute multimedia contents for vector-borne diseases and related issues. the usage of familiar input channels (such as twitter) for contents collection was made an immense improvement in the system. one of the key points that can observe in the systems is that, except for the hotspots, all other content is from end users. so except for administrative operations, the need of a specific assistant required for the system is minimal. the results obtained show the potential benefit in significantly alleviating the burden of laborious user intervention associated with conventional information gathering. one of the future improvements of the system is to create a better content management system thath will reduce the drawbacks of the current system. the opportunities will consider building a system to prevent users based on various identifications in a misuse of the system. visualization of twitter feeds are also can be enhanced by using an improved filtering mechanism. building on an intelligent content analyzing mechanism will also help to have a better understanding on the ground. another possible future enhancement will be the prediction of hotspot using different parameters. the development of a predictive module will help users to identify danger zones in advance and take the necessary precaution as early as early possible. world health organization, dengue and severe dengue the 2005 dengue epidemic in singapore: epidemiology, prevention and control dengue virus surveillance for early warning singapore scientists find dengue killer in human antibody citizens at the center: a new approach to civic engagement. the case foundation web-based applications in healthcare design and implementation of an internet gis a model-driven approach for designing adaptive web gis interfaces integrating open-source technologies to build low-cost information systems for improved access to public health data mapping as a visual health communication tool: promises and dilemmas a web-gis based urgent medical rescue cscw system for sars disease prevention a framework of web gis based unified public health information visualization platform connected: the surprising power of our social networks and how they shape our lives everyone's an influencer: quantifying influence on twitter social media ownership: using twitter as a window onto current attitudes and beliefs not just for social networking: how brazil is using twitter to better monitor its most destructive disease web-based applications in healthcare key: cord-286858-zbhtl2yn authors: mishra, b. title: tamasomā jyotirgamaya: seeking the self amidst covids’ cytokine cyclones date: 2020-10-22 journal: j indian inst sci doi: 10.1007/s41745-020-00186-1 sha: doc_id: 286858 cord_uid: zbhtl2yn pondering on pandemics and the promise of purification from the plethora of problems that it has spawned, the paper builds on a game-theoretic model of host–pathogen interaction, and... moves beyond. it highlights how quickly this ‘wicked’ problem has led to deceptive nash equilibria of certain information-asymmetric games as well as their sequels of more complex intertwined games at human scale but without an exit strategy in sight. in the absence of clarity (e.g., access to complete information) and yet facing a capricious and complex conspirator, we overview an exemplary solution, created by rxcovea, and examine how it might help. abstract | pondering on pandemics and the promise of purification from the plethora of problems that it has spawned, the paper builds on a game-theoretic model of host-pathogen interaction, and... moves beyond. it highlights how quickly this 'wicked' problem has led to deceptive nash equilibria of certain information-asymmetric games as well as their sequels of more complex intertwined games at human scale but without an exit strategy in sight. in the absence of clarity (e.g., access to complete information) and yet facing a capricious and complex conspirator, we overview an exemplary solution, created by rxcovea, and examine how it might help. biomolecules constitute an important subclass of molecules and are dominated by phosphorous, carbon, nitrogen, oxygen, hydrogen, etc. the biomolecules interact with each other and with water molecules (e.g., via atp hydrolysis reaction process) it is important that the "cellularizing" ensembles of such biomolecules-very likely membrane separated from the environmentremain lively (via a sequence of endergonic and exergonic reactions) and safe (maintaining right ph level and avoiding h 2 o 2 , etc.). because liveliness and safety conferred to the cells can be abstracted by a "utility" function, cells may be abstracted as individual "biomolecular agents" competing with each other in an evolutionary game in which the fittest survives and replicates. these biomolecules are also able to store useful information via concatenation of atplike subunit monomers to form polymers (e.g., rna, dna, microtubules, etc.) and, thus, symbolically encode molecular patterns, each with its own utility. such polymers form the symbolic world, in which they duplicate (or allow transcription and translation into other polymers mapping homomorphic patterns) and propagate the information stored in molecular patterns. if the duplication process permits variations and a utility-dependent replicator dynamics and pattern-specific information propagation is unidirectional, it sets the stage up for a darwinian evolution and amplifies diversity, giving rise to 1 3 j. indian inst. sci. | vol xxx:x | xxx-xxx 2020 | journal.iisc.ernet.in inter-agent information asymmetry. thus, not all possible molecular patterns would be uniformly (or information-symmetrically) represented in the symbolic world; only an infinitesimally small subset of all possible patterns would encode viable genomes (dna), transcriptomes (rna), or translatomes/proteomes (proteins). interactions among biological macro-molecules may be described using a sender-receiver signaling structure, where an expressed macromolecule, such as a protein or rna, constitutes a signal sent on behalf of a sender agent (e.g., cell). the signal may be modeled as the threedimensional conformation and physicochemical properties of the macro-molecule. a receiver agent (e.g., another macro-molecule) may then bind to the signal macro-molecule, which produces an action (such as an enzymatic reaction). the action produces utility for the participating agents, sender and receiver, and thereby-albeit indirectly-a change in overall fitness of the organism, and hence its genome (in evolutionary game theory, utility and fitness are treated as analogous). when there is common interest, the utility is expected to benefit both sender and receiver and their selectivity, thus driving darwinian co-evolution. if there is neither common identity (i.e., they are "strangers") nor common interest (i.e., they pose "dangers"), the sender and receiver may avoid each other in order not to undermine their common fitness. thus, antibody: an important part of the adaptive immune system. it is a protein usually in the blood and destroys or neutralizes bacteria, viruses, or other harmful toxins. antibody production takes place in response to antigen. they are members of a class of proteins known as immunoglobulins, produced by b cells in response to stimulation by an antigen. interferons: a family of proteins (lymphokines), secreted by infected host cells to protect uninfected cells from viral infections. evolution of processes encoding, recognizing, and acting against pamp (pathogen associated molecular patterns) and damp (damage-associated molecular patterns) plays an important role in the evolution of the multicellular immune systems. note that not all strangers are dangerous (e.g., symbionts such as corona virus in bats) and that some non-strangers could be dangerous (e.g., cytokine storms inducing activated white blood cells). however, since the immune systems also play an important role in defining many more higher order evolutionary processes: e.g., hla diversity, mate-selection, cognitive processes, culture, etc., our very human intellectual and cultural journey has also shaped our secular scientific understanding of the physical universe, albeit, it still remains inconsistent and lacks unification (e.g., quantum and classical mechanics). the resulting body of scientific literature, however, forms only a small part of our body of human knowledge that relates us to a tentative, though conventional, picture of a 'natural world,' containing both symbolic and signaling systems. as humans struggle with their understanding of the genesis and mitigation of a surging pandemic, initiated by a single zoonosis event in a remote asian town, it is worthwhile to meditate on these connections among the three worlds-mundus triplex, further subdivided and re-connected by such subdisciplines as virology, immunology, epidemiology, logical/mathematical inference, and economics-going from nanoscale viruses to global-scale human societies. or, "must we wring the neck of a certain system in order to stuff them into [] pigeon holes for the satisfaction of the analogy mongers 1 :" do the worlds divide neatly into bruno's three magics-sciences into three disciplines: physics, mathematics, and divine? or does it just model a biological gödelian self-referential self-seeking, selfishly greedy computation, hopelessly aspiring to halt with stable (sound and complete) turing decidability notion(s) of self(-ves)? can it reach an ne (nash equilibria), in which self separates from non-self, decoy epitopes from neutralizing, susceptible from infected, symptomatic from asymptomatic, essential economic activities from dispensable? replicator dynamics allow the signaling game to be couched in evolutionary terms 6 . replicator dynamics arise from the increased replication of players with higher utility (fitness). thus, if a cytokine: a signaling molecule, acting as a chemical messenger released by white blood cells, including macrophages, monocytes, or lymphocytes. the cytokines include the interferons, the interleukins, tumor necrosis factor, etc. memory t cell: persistent t cells that bear a receptor for a specific antigen that was previously encountered in the course of illness or vaccination. it plays a role of a verifier to ensure safe execution of the signaling game. the human leukocyte antigen (hla) complex is a group of related proteins, encoded by the major histocompatibility complex (mhc) in humans. hla genes are highly polymorphic which means that they have many different alleles, allowing them to fine-tune the adaptive immune system-differently for different individuals. hlas corresponding to mhc class i (a, b, and c) present peptides from inside the cell. foreign antigens presented by mhc class i attract t lymphocytes called killer t cells. figure 1 : molecules, molecular patterns, and (pathogen/damage) associated molecular patterns: who signals who? we depict the three worlds, important to defining a host's self and protected by a complex immune system, but amenable to invasion by a simple deceptive virus, which, in turn, can be deceived by a vaccine. we develop a signaling game framework to study virology, and immunology-while we hope to extend the framework to asymptomatic patients, with misaligned utilities, and impaired by moral hazards in economy. we suggest that they all conspire to make the covid problem a "wicked" (difficult or impossible to solve) problem. courtesy r.x. covea et al. cell has a strategy that results in increased utility, then it will increase in frequency in a population. for a sender cell, this would entail sending a signal that may result in an increased utility, while, for a receiver cell, this would entail undertaking an action that likewise may result in an improved utility (e.g., growth rate) (fig. 2) . as already suggested, these dynamics represent a process analogous to darwinian (adaptive) evolution or positive selection. in other words, as part of the innate immune system, sender agent, a dendritic cell (dc), in a multicellular organism that recognizes an appropriate damp/pamp combination could signal receiver agent, a macrophage (m ), to engulf and digest foreign substances (associated with the molecular patterns, amp) in an action process called phagocytosis. the innate system is said to be engaged in a signaling game, in which dc is the sender; m , the receiver; they signal using cytokines and phagocytosis which is the action taken by the receiver upon receiving the signal. their utility is determined by protection which they confer against non-self (mostly danger, potentially posed by strangers) and tolerance for self. since, many pathogens (e.g., sars-cov-2) can engage in deception (e.g., entering a host cell by targeting host ace2 receptorby binding to a site somewhat distant from the ace2 canonical binding site), innate immune system may need other auxiliary adaptive agents to be "educated" ex tempore, replicate dynamically and retain memory. these cells belonging to the adaptive immune system are not strategic (e.g., do not confer fitness along the germ line), but play an important role as recommenders (b cells)and verifiers (t cells) in taming the deceptive pathogens that can grow and mutate rapidly to frustrate the innate immune system. helper" t-cell, coordinates much of the adaptive immune response. a thymus derived white blood cell that precipitates a variety of cell-mediated immune reactions. three fundamentally different types of t cells are recognized: helper, killer, and suppresser (each has many subdivisions). they can be modeled as verifiers in the signaling game. b-lymphocytes are blood cells derived from the bone marrow and spleen involved in the production of antibodies. b cells produce antibodies, when primed by t cells. b-cell lymphocytes can later differentiate into plasma and memory cells. in our signaling game, they are modeled as recommenders and accelerate convergence to a separating ne based on the past memory of encounters. dendritic cell: an antigenpresenting immune cell. they have elongated, tentacle like branches to trap foreign objects. a large scavenger cell that ingests degenerated cells and secretes messenger proteins (monokines) involved in inflammatory reactions, lymphocyte activation, and acute systemic immune responses. the consumption and destruction of foreign materials by white blood cells like macrophages. however, in an adversarial chase, pathogens can evolve strategies to frustrate the education of t cells (e.g., antigen presentation), but can be remedied by costly signaling via dc's maturation and chemo-taxis to germinal centers. it is no wonder, most immunology jokes end with the punchline 7 , "the immunologist says, 'the thing is, the immune system is very complicated...' and the cardiologist says, 'just shoot me now. " we hope to simplify the model by formulating the innate immune system as a signaling game 4 , 6 , which, though prone to deception, can be critically tamed by the education, surveillance, and memory acquired by the adaptive immune system; the education of the adaptive immune system, though highly costly, can also be hijacked by a deceptively simple virus and points to a need for a better understanding of how the system behaves among various hosts (bats and humans with diverse hla types). signaling games signaling games are multiplayer (usually, two players) games with incomplete information: specifically, one player is informed and the other player is not. the informed player's strategy set consists of signals contingent on information; uninformed player's strategy set consists of actions contingent on signals. game is played as follows 1. player s is assigned a type t ∈ t 2. s send to player r a signal s ∈ m. 3. r takes an action a ∈ a. payoff function the utility functions for sender and receiver are dependent on the type of the sender, signal sent and action carried out by the receiver: equilibrium concept behavior strategies for senders and receivers: describing biomimicry by a virus to fool the immune system: cell can be of two types t c (self) or t d (non-self viral): they can send c signals honestly or by mimicry, respectively, and may be trusted or rejected by the immune system; similarly, they can send d signals erroneously or honestly, respectively, and may be rejected by the immune system. courtesy s. massey et al. j. indian inst. sci. | vol xxx:x | xxx-xxx 2020 | journal.iisc.ernet.in α(s, a) = probability that r takes action a following signal s. we may then say that the receiver may acquire a subjective probability distribution over t: proposition behavior strategies (α * , µ * ) form a nash equilibrium iff for all t ∈ t: signaling games in nature to assume reasonable utility functions, we use two natural maps that relate the sender's private information to receiver's action that would be desirable via rate distortion functions combining i(·, ·) (mutual information or rate) with a distortion d · (·, ·) combined using suitable lagrange parameters · : • mapping types and actions into signals: the model of signaling games the game thus involves two players, namely: a∈a α(s, a) = 1, for all s. . a) . • their roles can be shared; s may only have partial information; and r may be allowed to perform distributed actions. • a notion of type, t private to s, is captured by a random variable t whose support is given by t (known to sender s). π(·) = probability distribution over t is a prior belief of r that the sender's type is t. • note that the distribution of signals received by r is given by the probability distribution π m , where: • • and the distribution of actions produced by r is given by the probability distribution π a , where: • • clearly, π t and π a are probability distributions on t and a, respectively. if π t is the probability distribution on t induced by π a under the function f r , then: • a natural choice of measure of deception may be given by the relative entropy between the probability distributions π t and π t : this definition describes deception from the point of view of the receiver. to get the notion of deception from the point of view of the sender, one needs to play the game several rounds. thus, signaling games may reach a nash equilibrium that is stable but not deception-free (e.g., immune system may reach a homeostasis that may not be ideal in separating self from non-self). (1) π t (·) := π a (f −1 r (·)). (2) • if, on the other hand, the game reaches a pooling equilibrium: all types t send a single signal s * with probability 1. • convention and deception the divergence between the objective probabilities and the subjective probabilities induced by conventional equilibria. • solution costly signaling; credible and noncredible threat; aligned utilities; augmenting with additional non-strategic playersdenoted as: m recommenders + nverifiers the possibility of deception (e.g., biomimicry by sars-cov2) in the nash equilibrium (ne) of the signaling game may be the reason why the innate immune system (with pamp and damp) is not the ultimate solution; the ne must be stable, but also keep up with rapidly evolving pathogens. it leads to a need for an adaptive immune system, training of t cell by dc, chemo-taxis, and maturation processes that dc must go through, cytokine storms, etc. therefore, "it is very complicated.." and more so if we need to create a vaccine that must deceptively mimic the virus, which may have been already deceptively mimicking the host system, and the vaccine must be carefully analyzed and regulated so as not to catalyze autoimmune disorders or antigen-dependent enhancement ade. covid-19 poses a wicked problem. "why does the repeated game it plays-covid game-have long and branching chains, intertwined hierarchies, plenty of room for deception, and explosive growth rates? why not use similar features, perhaps, needed in the counter measures to tame it 1 ?-" a proportionally rapid and rigorous analysis of the game as well as synthesis of hyper-local policies (e.g., strategies) addressing molecular, individual, and community-wide actions. for argument's sake, assuming that it only manifests as a socio-economic question, it remains unfathomable what economic utilities might be ethically and equitably traded off for loss and depreciation of human capital (fig. 3) . many of the unknown unknowns are exacerbated by the impacts of unquantified-or unquantifiable-(1) proportion of asymptomatic patients, (2) comorbidities and vulnerability of various subpopulations, (3) co-evolution of the disease in the presence of others (flu or multiple strains-with unknown cross immunity) , (4) number and combinatorial structure of the network of super-spreaders, (5) individual boundedly rational compliance, (6) spatio-temporal reaction-diffusion nature of the disease (e.g., turing labyrinth for herd immunity), (7) accuracy, frequency, and scaling of biomedical tests (separating asymptomatic from symptomatic, susceptible from infected and infected from immune, etc.), (8) unpredictability of treatment and preventive measures such as socialdistancing, contact-tracing, repeated lock-downs, and quarantines, (9) allocation of resources for patients across vulnerabilities, demographics, genders and races, etc. rxcovea has sought to develop and openly disseminate mathematical and computational tools and theories to address these problems with the help of modelers (sdes, graph-based, hybrid, cellular automata), game theorists (signaling games, minority games), logicians (formal methods, model checkers, belief revision), algorithmicists (multiobjective decision and optimization problems), statisticians (bandit problems, reinforcement learning), and computational systems biologists (in silico vaccine figure 3 : covid poses a wicked problem affecting different human organ, physical, and social systems at different spatio-temporal scales. note: wiki defines "a wicked problem as a problem that is difficult or impossible to solve because of incomplete, contradictory, and changing requirements that are often difficult to recognize. ... another definition is 'a problem whose social complexity means that it has no determinable stopping point.' moreover, because of complex inter-dependencies, the effort to solve one aspect of a wicked problem may reveal or create other problems.' covid problem started as a healthcare problem, but quickly evolved into an economic problem, triggering various issues related to humans' fundamental rights. courtesy: r.x. covea et al. j. indian inst. sci. | vol xxx:x | xxx-xxx 2020 | journal.iisc.ernet.in and drug discovery). detailed game-theoretic analyses in different contexts, namely, germline, somatic, or population evolution, may be found elsewhere 2 , 3 , 5 . (1) 100 nm scale: virology-a task group ("la famiglia"), consisting of salvatore alaimo, eva bischof, jantine broek, ashley duits, alfredo ferro, naomi maria, alfredo pulvienti, and-valentina rapicavoli, has been studying the systems biology of the virus and host-pathogen interactions. the group has been able to replicate in silico-safely, scalably and inexpensively-a large class of in vivo and in vitro experiments to test a substantial number of hypotheses regarding host-pathogen-drug interactions. rigorous statistical analysis, to correct for multiple hypotheses testing, then points to the strategies (possibly involving deceptive biomimicry) that the virus may evolve and how drugs may be re-purposed to enable the host organs, systems, and pathways to counter safely. the group also aims to investigate how sars-cov2 virus achieves a symbiotic existence with other reservoir hosts (e.g., bats and pangolines) and how its genome mutates in order that hosts' immune system may accommodate such a ("pooling") nash equilibria (perhaps, with constrained antigenic drift). another task group ("the gamers") consisting of william casey and steven massey has developed models of biomimicry (especially the ones achieved by sars-cov-2, mimicking polybasic cleavage site, pcs mutations to interact with host furins) that speaks to intelligent and safe vaccine and drug design. charles cantor studies sophisticated multicolor pcr testing methods, tracking the viral genomic mutations. marco antoniotti studies the phylogeny of the genomic strain mutations. tamar schlick with her molecular modeling group studies the structure and dynamics of the fse (frame-shifting element) of the viral rna genome and its screening in silico to discover novel anti-viral therapy to inhibit protein synthesis (fig. 4) . 1 µ m scale: immunology-another task group ("the adaptors") has been studying the host immune system's response to viral figure 4 : target rna residues for anti-viral therapy in the frame-shifting element of sars-cov-2 determined by molecular modeling. a shows the 3d structure of the frame-shifting element of sars-cov-2 as modeled by md (molecular dynamics) with the key residues which are identified by destroying the pseudoknot and/or a stem using graph theory and genetic algorithms. the identified residues are then used in the drug screening algorithm. other identified residues are shown in b and c. courtesy: t. schlick et al. 2 in yacht's simplest incarnation: a user is recommended a pool in which their saliva can be tested within a time interval: if the user selects to be pool-tested and the pool-test result is negative, the user is given a badge (non-counterfeitable plastic wrist tag, encrypted electronic badge or a crypto-coin tag) that lasts a pre-determined period. during that period with the safe badge, the user may be allowed to enter (resp. exit) certain safe (resp. unsafe) locations. if, on the other hand, the user selects to be pool-tested and the pool-test result for the user is positive, then the user is given an unsafe badge-and recommended a uniformly randomly selected pool (hot-potato protocol) and/or exponentially randomly selected time interval (exponential-back-off protocol) for being retested. the user without a safe badge is virtually quarantined from entering a safe region (or leaving unsafe region-which must have sheltering-in-place or hospitals). also, since the subsequent choices of the pools are (by recommendation) independent, if the user is truly negative, they will be determined so (almost surely) no later than a fixed run-length of "(apparent) false-positive" results. if a user fails to be negative after fixed run-length of "(apparent)-true-positive" results, the user must be quarantined. infection: the group consists of james bannon, charles cantor, ashley duits, john conolloy, kelly ganjei, jia han, and mike lotze, and it analyzes longitudinal adaptomic data from patients, using temporal network analysis using advanced combinatorics and formal methods. it is desired, with the availability of adequate resources, to expand the team to study the genomic markers stratifying the probands, who exhibit a wide range of symptoms-asymptomatic vs symptomatic or super-spreaders or kawasaki/misc disease phenotypes. it is conjectured that the immunogenic biomarkers can be characterized by a gwas (genome-wide association study), if certain genomic regions (e.g., p-arm of chr 6) are mapped haplotypically with long-range single-molecule technology such as nanomapping. possible biomarkers clustering around hla, kir, and tlr regions are being studied by a group consisting of marcin imilienski, sanjan kumar, jason reed, and amir toor. vijay chandru and ramesh hariharan are aiming to initiate a similar project in india, which enjoys a more interesting admixture population, driven by endogamy ("the grandest genetic experiment ever performed on man," according to theodosius dobzhansky). (3) 100 m scale: epidemiology/social immunology-rxcovea's third task group ("the labyrinthers") has focused on various epidemiological models and their parametric control using clever (pool-and individual-) testing methods with low latency: the group comprises ved basu, jantine broek, shireshendu chatterjee, inavamsi enaganti, hilary gao, eva guo, mathew jacobs, and kim mishra (for modeling); charles cantor, vijay chandru, manoj gopalakrishnan, inavamsi enaganti, satyajit mahrana, and rohit nandwani (for testing); the two subgroups cross-pollinate intimately. the subgroups started with standard cellular automata (ca) agent-based model for incorporating geographical distributions of infection and easily captures the notion of discrete pairwise interactions, since the new state of a cellular automaton (ca) is based on its current state and the states of its surrounding cas as well as some common evolving rules. the infection spreads from an infected ca to adjacent neighboring cas, while infected patients also recover and become immune to the reinfection. we have assumed the following states for the neutralizing antibody: an antibody that neutralizes (renders harmless) the infectivity of microorganisms, particularly viruses. population exposed, symptomatic, asymptomatic, susceptible, recovered/immunefurther augmented with such other states as: unknown, tested, vaccinated, quarantined, retested, recurrent, etc., with respect to other sars-covs and flu. rxcovea has also implemented ctmc (continuous time markov chain) and realistic sde (stochastic differential equation) models-the later model obtained using turing's reaction-diffusion equation framework and the analysis of its solutions-e.g., turing labyrinths. we conjecture that the formation of turing labyrinths (possibly further stratified by hlas and cov2-strains) will play a role in the formation of herd immunity, vaccination, and social-distancing policies. these pattern formations can be further moulded by rna, antigen, and immunoglobulin tests (yacht, yet another covid-health testing) 2 , as well as contact tracing (with 1 or 2 degrees of separation) both operated at a large scale with a selected periodicity. the original yacht design motivated by the following game-theoretic model: (i) repeated game: repeated saliva pool testing (involving hundreds of individuals in a pool) using rapid and accurate (duplicated) pcr test is feasible with negligible false negatives and manageably low false positive. (ii) private game/ no intermediary: sample collection must remain safe, unsophisticated, ubiquitous, and unbiased (from self-selection) and, thus, may not involve robotics and technicians. (iii)decentrality: data collection and storage must be reliable, private, and yet not centralized. (iv) factual and counter-factual: the analysis is flexible, hypotheses-testable, easyto-communicate, and adaptive in controlling compliance, prevalence, immunization, and resource usage. yacht's resemblance to ethernet's aloha protocol and pagerank's random surfer approach, especially with small/moderate prevalence, adds to our faith in its robustness. we have simulated a model, with promising results, that partition the population into two regions ("home" encountering both safe and unsafe household members and "work" encountering safe non-household members) and assumes that the household statistics differ from country to country (india, afghanistan, usa, and the netherlands). in the near future, we wish to resimulate the system with one additional region: "challenge region," which may be factual or counter factual (e.g., with vir-immunoglobulin: a protein that acts as an antibody to fight off pathogens. there are five classes: igg, iga, igd, igm, and ige. recombinant and pooled immunoglobulins from blood donations are being used to fight off sars-cov-2 infection. tual viral strains and spreading and testing mechanisms) and allow collection of statistical data for hypotheses testing (fig. 5) . (4) 100 km scale: logic, inference, and learning-the fourth task group ("the model checkers") is a data-science group to delineate the causal structures among various pandemic-related events; the group includes: marco antoniotti, jantine broek, daniel cage, will casey, shirshendu chatterjee, vijay chandru, james edmondson, yaron gvili, stella luna, ramon luna, and larry rudolph. the group is preparing for a near future when massive amount of meta-data, (e.g., involving pools, mobility, and geographic occupancy datafrom foursquare) can be combined with machine learning tools augmented with formal methods to hypothesize and test causal relationship and intervention strategies. since our games include complex subgames involving credible and non-credible threats, monitoring human behavior-without assuming perfect rationality-is likely to play a critical role. we plan to use probprog tools (julia/turing 3 ) to understand the disease etiology, and individuals' strategic choices tempered by the knowledge of causality, time, logic and games, privacy, reputation, trust, deception, and cellularization (fig. 6 ). equilibria-the "open sim," group (including ved basu, jantine broek, will casey, shirshendu chatterjee, and yaron gvili) is studying how to connect components of global data to infer effect of stratification of populations based on demographics, age, gender, genomics (e.g., hla), (co-)morbidity, etc. to understand genuine causal structures only using natural experimental data and without getting bogged down by the bias due to multiple hypotheses testing and simpson's effect. the economic roles played by the disrupted interrelationships among different subgroups (and their possible simulation by digital means, when individuals need to be socially distanced) have a strong impact on the v or w shaped economic recovery. the "burpa group" (comprising kose john, samir saadi, and cheryl qi) has designed a novel market microstructure to enable health t helper cells: lymphocytes responsible for assisting other white blood cells in responding to infection, processing antigen, and triggering antibody production (also known as t4 cells and cd4 cells). a major component of cytotoxic lymphocyte response (ctl), responsible for lysing infected or cancerous cells, and t killer cells (not to be confused with natural killer cells) are a subset of cd8+ lymphocytes. insurance for a patient population (social network) interacting with scientists and clinicians (expert network) to create digital healthcare markets that tame its risks by combining multiple spv's (special purpose vehicles) and securitization (fig. 7) . in a more optimistic future, to which we hope to return soon, rxcovea will focus on better algorithms for randomly surfing explainable ais to create a world-wide adaptive immune system to protect humans and things physiologically, physically, and digitally. the adaptive learning recommenders and verifiers will be able to anticipate and create mitigating plans for future zoonotic events. for now, our collective afforts aim to t suppressor cells: t lymphocytes responsible for turning the immune response off after infection is cleared, a subset of cd8+ lymphocytes. yacht's performance on a university campus with five dorms. the student population is simplified to be heterogeneous with two assumed immunogenic biomarkers: e.g., hla types hla' and hla", where agents of type hla' are less infectious than hla" agents. simulation assumes that there is only one viral strain. a observe that increasing pool sizes decreases the total number of infections while increasing quarantining. after a delay, the quarantined time also reduces. b shows the sir plot of the same simulated system with exactly 15 pools per dorm. observe that the prevalence is easily controlled to below 5%. courtesy i. enaganti et al. provide computational models of host-pathogen interactions that will help to define parameters critical to healthcare policies and methods to control the viral spread-"the hammer and the dance." we hope that optimistic future will also include a successful completion of human haplotypic hla genome project, making it unfashionable among immunologists to say, "the thing is, the immune system is very complicated... !". springer nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations. figure 6 : epidemiological model-e.g., compartmental models such as sir with compartments: s (susceptible), i (infected), and r (removed). the diagram shows why the uncertainties in parameters may make the models unpredictable-especially, when population is of finite size. still, the models can be used fruitfully to stress test various plausible policies and affect boundedly rational agent behaviors. courtesy: j. broek et al. joyce' , in finnegans wake: a symposium -exagmination round his incamination of work in progress deception, identity, and security: the game theory of sybil attacks compliance signaling games: toward modeling the deterrence of insider threats evolutionary dynamics of lewis signaling games: signaling systems vs. partial pooling cancer hybrid automata: model, beliefs and therapy signals immunology is where intuition goes to die. the atlantic in mid-march of 2020, spurred by a hypothesis centered around hla-hetero-zygotic advantages of asymptomatic covid patients, we organically converged to form the "cure covid-19 for ever and for all"(rxcovea) group, a rigorous community of scientists, clinicians, ai specialists, mathematical and computational/data modelists, pharmaceutical, public and digital health intelligence representatives from multiple institutions, countries, and scientific training. within the network, self-organized task forces continue to conduct core projects, structured around hypotheses, and minimal viable products (mvp's), mentored, and examined by experienced senior members.special "thank you"s to ashley, charles, larry, lex, and vijay-privately "typed" as friends, mentors, and mentees-who meticulously avoid social-distancing in all the three worlds: the paper has benefited a lot from their signals and actions! the contributions by bm were supported by nsf grants ccf-0836649 and ccf-0926166, nci physical sciences-oncology center grant u54 ca193313-01, and a us army grant w911nf1810427. the contributions by rxc have been voluntary and unsupported. the work of ts (t. schlick) was supported by nsf rapid award 2030377 and nih award r35gm122562 (nigms). the immune system is a host defense system comprising many biomolecular structures and signaling processes within an organism that protects against pathogens, dangerous (and possibly, stranger) to the organism-from viruses to parasitic worms, and distinguish them from the organism's own healthy tissue. it is possible to study the immune systems as a signaling game whose nash equilibria separate two types: self and non-self. the immune system consists of an innate system coupled with an adaptive system: both following replicator dynamics, but innate system selecting over organisms' germ line and adaptive system adapting over organisms' somatic immunological memory. [adapted from https :// morgr idge.org/outre ach/teach ing-resou rces/virol ogy-immun ology /immun e-syste m-gloss ary/]. b. mishra an educator, an inventor, as well as a mentor to technologists, entrepreneurs, and scientists. prof. mishra founded the nyu/courant bioinformatics group, a multidisciplinary group working on research at the interface of computer science, applied mathematics, biology, biomedicine, and bio/ nano-technologies. he is the author of a textbook on algorithmic algebra and more than 200 archived publications. he has advised and mentored more than 35 graduate students and post-docs in the areas of computer science, robotics and control engineering, applied mathematics, finance, biology, and medicine. he holds 21 issued and 23 pending patents in areas ranging over robotics, model checking, intrusion detection, cyber security, emergency response, disaster management, data analysis, biotechnology, nanotechnology, genome mapping and sequencing, mutation calling, cancer biology, fintech, adtech, internet architecture, and linguistics. prof. mishra's pioneering work includes: first application of model checking to hardware verification; first robotics technologies for grasping, reactive grippers, and work holding; first single molecule genotype/haplotype mapping technology (optical mapping); first analysis of copy number variants with a segmentation algorithm, first whole-genome haplotype assembly technology (sutta), first clinical-genomic variant/base calling technology (totalrecaller), first single-molecule single-cell nanomapping technology, etc. prof. mishra has a degree in science from utkal university, in electronics and communication engineering from iit, kharagpur, and ms and phd degrees in computer science from carnegie-mellon university. he is a fellow of ieee, acm, and aaas, eai, a fellow of national academy of inventors (nai), a distinguished alumnus of iit (kharagpur), and an nystar distinguished professor. key: cord-257633-wrem38ex authors: chamola, vinay; kotesh, pavan; agarwal, aayush; naren; gupta, navneet; guizani, mohsen title: a comprehensive review of unmanned aerial vehicle attacks and neutralization techniques date: 2020-10-10 journal: ad hoc netw doi: 10.1016/j.adhoc.2020.102324 sha: doc_id: 257633 cord_uid: wrem38ex unmanned aerial vehicles (uav) have revolutionized the aircraft industry in this decade. uavs are now capable of carrying out remote sensing, remote monitoring, courier delivery, and a lot more. a lot of research is happening on making uavs more robust using energy harvesting techniques to have a better battery lifetime, network performance and to secure against attackers. uav networks are many times used for unmanned missions. there have been many attacks on civilian, military, and industrial targets that were carried out using remotely controlled or automated uavs. this continued misuse has led to research in preventing unauthorized uavs from causing damage to life and property. in this paper, we present a literature review of uavs, uav attacks, and their prevention using anti-uav techniques. we first discuss the different types of uavs, the regulatory laws for uav activities, their use cases, recreational, and military uav incidents. after understanding their operation, various techniques for monitoring and preventing uav attacks are described along with case studies. unmanned aerial vehicles are becoming popular and are being used for various applications at an accelerating rate [1, 2] . uavs are deployed across various sectors including logistics [3, 4] , agriculture [5] , remote sensing [6] , wireless hotspot services [7] , smart city applications [8] and disaster management [9] . notably, uavs are being used for crowd surveillance, public announcements, and sanitizing public places to help in managing the covid-19 pandemic [10] . techniques are also being developed to employ legitimate uavs for tracking and surveillance of suspicious uavs [11] . according to the latest goldman sachs report, by 2020 there would be 7.8 million consumer uav shipments and usd 3.3 billion in revenue, versus only 450,000 shipments and usd 700 million in revenue in 2014 in the commercial uav segment [12] . the market is anticipated to register a compound annual growth rate (cagr) of 56.5% from now till 2025 [13] . uavs are expected to play a huge role in upcoming 5g networks by supporting flexible deployment and backhaul connectivity operations [14, 15] . blockchain and other distributed ledger technologies are expected to enable trusted and transparent sharing of uavs across different commercial enterprises [16, 17, 18, 19, 20] . uavs are also being used for various military applications such as surveillance, target tracking, air-to-ground combat among many others. the us military spending on uavs is estimated to be around usd 17 billion from 2017 to 2021 [12] . for such critical applications, the security of wireless uav-uav and ground-uav communications is a must. security schemes and techniques to ensure essential security features such as mutual authentication and privacy protection, and methods to analyze security vulnerabilities in uav networks are also being developed [21, 22, 23, 24, 25, 26] . there is little doubt that uavs will play a crucial role in our future societies. despite all the advantages of uavs, they are not free from security vulnerabilities. even professional uavs, which are used for critical and sensitive applications such as police operations and enemy surveillance have been shown to possess several security vulnerabilities [27] . when compromised, they can be used by criminals and terrorist organizations for illegal surveillance and unmanned attacks. they may be turned off remotely, hijacked, flown away or stolen. [28] . recent incidents of uav attacks provide an indication of how devastating they can be. take, for example, the attack on saudi arabia's biggest oil refinery, saudi aramco. in this attack, uavs targeted one of saudi aramco's facilities and successfully damaged it declining the production by about 5.7 million barrels of oil a day which is about 5 percent of the global production of oil [29] . all this clearly indicates the need for anti-uav technologies. anti-uav refers to the process of prevention of potential uav attacks by either capturing the uav or jamming its communication channel to disrupt its flight pattern, possibly bring it to a halt on the ground. in this paper, we present a literature review of various anti-uav techniques. this paper is structured such that we first analyze the classification of uavs in order to better understand the methods of attacking uavs. then we present case studies of deliberate uav attacks to get more insights into how the uavs caused damage and how it could have been prevented. next, we present methods of monitoring uavs and successively counterattacking them including jamming their communication networks, neutralizing their autopilot software, and more. an overview of the outline of this paper is shown in figure 1 . the uav industry is extensive with a broad diversity. in this section, we have classified uavs based on various parameters to give the reader an acquaintance with various parameters used for comparing uavs. this will help in giving the reader a perspective through the rest of the paper. they have been classified on the basis of weight, altitude, and range, wings and rotors, and their application. there does not exist a single classification standard throughout the industry. however, we have provided our classification based on the guidelines set by the indian government [30, 31] . • nano: uavs with weight less than 250 gm • micro: uavs with weight greater than 250 gm and less than 2 kg • small: uavs with weight greater than 2 kg and less than 25 kg • medium: uavs with weight greater than 25 kg and less than 150 kg • large: uavs with weight greater than 150 kg • hand-held: uavs that can fly at altitudes of less than 600 m and have a range of less than 2 km. • close: uavs with an altitude of less than 1500 m and range less than 10 km. • nato: uavs with an altitude of less than 3000 m and range less than 50 km. • tactical: uavs with an altitude of less than 5500 m and range less than 160 km. • fixed wing: uavs that resemble an aeroplane design with fixed wings. • single rotor: uavs that resemble a helicopter design with one main rotor and another small one at the tail. • multi-rotor: uavs that have more than one rotors. the most commonly found are tricopters, quadcopters, hexacopters and octacopters. • fixed-wing hybrid vtol: hybrid uavs with longer flight time. they have the stability of fixed-wing uavs as well as the ability to hover, take off and land vertically. here, vtol refers to vertical takeoff and landing. figure 2 illustrates various types of uavs classified based on their wings and rotors and table ii presents an comparison and typical use cases. • personal: used for applications such as videography and entertainment. • commercial: used for applications such as infrastructure monitoring, product delivery, and aerial imaging. as uav technology is advancing, it has become easily approachable and affordable. many recreational and commercial uav pilots are entering the commercial sectors where uavs are extensively used (e.g., real estate, film making, journalism). as the ecosystem of uavs is evolving quickly, many countries have come up with laws to regulate the usage of uavs for various kinds of users. the regulations have evolved with advancements in uav technology. certain countries have established certification authorities to certify the usage of a uav in their air space. in this section, we provide an overview of uav regulations in various countries as of september 2020. it is legal to fly uavs in the united states of america, but one must register their device with the federal aviation authority (faa) on the faadronezone [33] website if the uav weighs over 0.55lbs (250g). to fly a uav for commercial purposes, one must obtain certification from the faa and follow the commercial rules set by the faa which is discussed briefly below. our discussion is limited to a few important rules which are applicable in many states in the usa, but there are specific laws for individual states. pilots are allowed to fly their uavs in class g airspace. according to the united states airspace classification, class g airspace is airspace from ground to 1,200 feet (i.e., 365 m or less), often referred to as uncontrolled airspace. to fly in controlled airspace, one needs to apply for special permission from airspace authorization authorities. if one is flying for hobbies or recreational purposes, they can fly within visual line-of-sight and should not do side jobs or in-kind work. the uav owner is entitled to follow the community-based guidelines programmed by nationwide organizations like the academy of model aeronautics (ama). the weight of the uav cannot exceed 55lbs (25kg), ie., only nano, micro and small uavs as referred in section ii-a, can be used for recreational/hobby purposes. to use medium and large uavs, one needs to get certified by a community-based organization. one cannot fly their uav near emergency response efforts and can fly only in class g airspace. 2) commercial regulations: for commercial flying, one is required to acquire a remote pilot certificate issued by the faa. the uav must fly in class g airspace within visual lineof-sight and weigh less than 55lbs (25kg), including payload, at takeoff. the pilot should ensure that the uav does not cross a speed limit of 100 miles per hour (160 km/h), an altitude of 400 feet (120 m), and the flight should take place only during daylight or civil twilight. the uav should not fly directly over people and should not be operated from a moving vehicle. except for the weight and airspace regulations, there is a provision to waive off the rest of the rules for commercial uav flights by applying for a special waiver. as per european union aviation safety agency regulations [34] , if a uav operator wishes to fly uav in european union, delivery through uavs they must register if the uav weighs more than 250 grams or has an attached camera. every operator who is registering must complete training to receive a certificate, which is valid in other eu countries as well. the registration number needs to be displayed with a sticker on all the uavs you own including those privately built. the pilot should always keep the uav in line of sight, and shouldn't fly higher than 120 meters altitude, close to other objects, airports, or above groups of people. the uav operator cannot intrude anyone's privacy by capturing photographs or videos without their permission. eu has released new rules for uav operators which will be effective from december 31, 2020. as per the new system, the uavs will be categorized under the specified 3 categories (open, specific, and certified). the category is based on the risk of the flight. this risk is determined by the weight of the uav and the location of the flight. depending on the flight category a remote pilot may need certain permits or certificates. it is legal to fly uavs in australia. as per australia's civil aviation safety authority (casa) regulations [35] , the pilot should fly the uav only during the day and keep it within line of sight, i.e., the uav should be visible within the line of sight of the naked eye and not through any device. the pilot should ensure that the uav does not fly above an altitude of 120 m and the uav should always be at 30 m distance from people or objects. one should avoid flying the uav near emergency operations, over gatherings and densely populated areas. a single pilot can fly only one uav at a time and cannot fly over people, including sports events, beaches, etc. if the uav weighs more than 100 g, one must keep at a distance of 5.5km from aviation airfields, commercial airports, and airbases of the military. violating anyone's privacy makes the pilot come under the purview of state laws. casa also released an official mobile application, "can i fly there?" to determine whether one can fly a uav in a particular area for recreational or commercial purposes. 1) commercial regulations: those flying devices weighing over 2kg (4.5 lbs) for commercial purposes, has to inform casa and apply for an aviation reference number and fly within the standard operating conditions. to fly outside the casa listed standard operating conditions which are described in the previous paragraph, one needs to acquire a repl (remote pilot license) and fly with a certified pilot. as per the civil aviation authority of the uk (caa) regulations [36], one should always keep the uav in direct visual sight, to avoid any collision, especially with other aircraft. all uavs, irrespective of their weight, should not fly 122 m above the surface. there are further regulations for those uavs with an attached camera. the pilot shouldn't operate the uav anywhere near populated areas or assembly of 1000 persons by maintaining a minimum distance of 150 m from these areas. the pilot should avoid flying the uav within 50 m of any person or object which is not under his/her control. the pilot should not operate the uav within a 5km radius of an airbase, be it the commercial airport or military airbase. one should not attach any payload that can cause damage by dropping it over anyone or anything on the ground. 1) commercial regulations: to perform commercial uav operations in the uk one needs to take permission from the caa. caa makes it mandatory for all commercial uav operations to have insurance. for foreign operators to carry out commercial work, caa will normally be able to grant permission, with the pre-condition that they are ready to meet the basic safety requirements that are necessary for the regular u.k based uav operators. as per the civil aviation authority of norway (caan) regulations [37] , the uav pilot must maintain visual contact with the aircraft throughout the operation. uavs cannot fly over festivals, near accident areas, military areas, sporting events, and cannot go near the 5 km radius of any airport. they should also avoid flying near traffic, people, and buildings. the pilot should ensure that the uav does not cross an altitude of 394 feet (120 m) above the ground. the pilot is liable for action if found disturbing others' privacy by taking photographs or videos. to perform any commercial uav operations in norway, one should register themselves with the caan. norway has divided the regulations for commercial operators, referred to as ro (remotely piloted aircraft systems operators), into three categories: ro 1, ro 2, and ro 3. table iii shows the regulations for each of these categories. ro 1 and ro 2 regulations apply to nano, micro, and small uavs. ro 3 regulation applies to medium and large uavs. as per india's national aviation authority regulations [38] , foreigners are currently not allowed to fly uavs in their country. india uses the classification of uavs based on the weight mentioned in section ii-a, namely, nano, micro, small, medium, and large during their official registration. except the nano uavs, all others need to acquire unique identification number (uin) by registration. the pilot should always maintain a visual line of sight and shouldn't fly the uav 400 feet (120 m) above ground level. uavs are restricted to fly over no-fly zones like airports, borders, military bases etc. the aviation authority prescribes specific mandatory features for uavs (excluding nano category) like global positioning system (gps), return-to-home (rth), anti-collision light, identification (id plate), a controller with flight data logging, radio frequency identification (rf-id) and subscriber identity module (sim). every uav that fly in india should have these prescribed features. 1) commercial regulations: to fly uav for commercial purposes in india, one should hire an indian entity who can operate their uav and that entity needs to obtain the unique identification number from the directorate general of civil aviation (dgca). except for nano category flown below 50 feet and micro category flown below 200 feet, every commercial operation with a uav in india should be executed only after obtaining a permit from dgca. having registered on [39] platform, all pilots should request permission before every flight through their mobile app. based on the acceptance or rejection from the platform, one can proceed with their flight. uavs find applications in various industries like logistics, structural monitoring, automation, and agriculture [40] . they reduce the cost and provide simpler solutions to problems like generating climate data, monitoring borders, watering of crops, airborne inspection of structures like pipes, etc [12] . some of the major manufacturers of uavs in the field are dji, yuneec, parrot, and sensefly. table iv presents a list of popular uavs and their specifications. unfortunately, unlike military uavs, these solutions do not always undergo vigorous security testing, because the implementation of security features increases the product development time and costs, thereby decreasing revenue for the manufacturing company. additionally, enhanced security measures might make it difficult for general consumers to operate the uav. the internet of things (iot) ecosystem has its own set of security challenges [41, 42, 43] . there is a possibility that uav-iot ecosystems providing aerial iot services [44] might inherit the same security challenges. several security vulnerabilities like the possibility of hijacking, jamming of a uav in flight, disabling the uav, etc. have been reported for many of the popular uavs available in the market [45] . since the vulnerabilities are publicly available, this type of uavs has a high possibility of misuse. cyber attacks on industrial facilities are not very uncommon [46] . with uavs expected to be an integral part of the industrial workforce, it becomes necessary to ensure high levels of security to ensure smooth industrial operations. generally, hobby-level uavs (micro and small uavs, section ii-a) have 0.3 to 2 kg of payload capacity. these uavs can carry a payload, which can be lethal objects like explosives and biological hazards. even the uav itself can be rammed into objects to cause damage. although various countries have set up regulations to prevent such misuses, their enforcement is difficult. in many cases, it may not be possible to identify the owner or uav operator who may have been operating the uav from a remote location. hence, having anti-uav systems, which are discussed in section ix, are very important and need to be widely deployed. the following subsections discuss various malpractices using hobby, commercial, and personal uavs which have been classified as in figure 3 . reference [47] provides a list of incidents and intrusions from around the world using hobby, commercial, and personal uavs. additionally, table v presents the risks posed due to uavs in airports, rescue operations, prisons, offices, residences, and patrols. according to the united states national inter-agency fire center, aerial firefighting efforts were shut down a minimum of nine times, and a minimum of twenty uav incursions had hindered firefighting capabilities within the us from january to october in the year 2019. even though a bill was passed in 2015 by the us legislature, allowing firefighters to neutralize uavs that interrupted their efforts to handle fires, and also imposing and creating penalties for uav pilots who interrupted with firefighters, the uav appearances in such rescue operations have continued to exist [48] . beyond firefighting efforts, uavs are known to interfere with airport traffic. despite being small, uavs they can cause expensive disasters if they come in collision with a large aircraft. collisions with an engine, wing or any part of an airplane can cause severe damage. in 2017, a small uav collided with a commercial airplane flying towards quebec city in canada. although the plane landed safely, this incident introduced concerns about the collisions between unmanned aerial vehicles with commercial aircraft, following which the regulation of uavs has increased [49] . a 2017 federal aviation administration (united states) study [50] found that uavs colliding with large aircraft can cause more damage than birds, a threat which the industry has long faced. unlike the soft mass and tissue of birds, most uavs are made of more rigid materials, which can cause severe damage to the airplane's body, or damage the engine's rotors. unidentified uav sightings frequently halt airport operations [51] . there have been multiple instances where uavs were used to deliver contraband like narcotics, ammunition, and cell phones into prisons by flying over the walls. in march 2019, a uav along with contraband was discovered at the collins bay institution correction facility in ontario, canada. the regional president for the union of canadian correctional officers also stated that similar practices were being carried out at other facilities as well. the countermeasures to prevent these further were not made public to prevent exploitation [52] . there have been similar incidents in london, ohio, new york, and many more. uav detection technologies like audio, video, and radio monitoring, which are discussed in later sections have been deployed across prisons to prevent such incidents [53]. uavs can be modified and additional components can be added to use it as a mobile hacking tool. in [54] , a single board computer with a 3g connection and a wifi packet injector was added to an parrot ar drone to create a botnet. weakly protected wifi networks were detected by the uav, their information was sent to the cloud over a 3g network where they were cracked, and finally packets were injected into the network. another instance was demonstrated in singapore, where researchers added a smartphone as the payload of the uav and used it to intercept printer networks, and hence gain access to important documents in office spaces [55] . many uavs have a high definition camera attached to it for transmitting live footage to the user. this has been misused a lot for several illegal activities. in december 2016 in orem, utah, united states of america a uav was recovered with video captured of several people in their homes [56] . it has also been reported that uavs are used to track law enforcement officials to anticipate their movement, find windows, and plan crimes (especially robberies). similarly, at national borders, they have been used to track border patrols so that they can be avoided while crossing [57] . two uavs carrying explosives were detonated near avenida bol´ıvar in caracas, venezuela where nicolás maduro, the president of venezuela was giving commemorations in the middle of a speech to the bolivarian national guard on august 4, 2018. the uavs were reported to be the dji m600 model loaded with c4 plastic explosives and gun powder. though the president was unharmed, some national guard soldiers were injured. later, a terrorist group made posts on social media claiming responsibility for the attack [58] . this shows that uavs are available without regulation and they can be easily used as tools for terrorism. 2) abqaiq -khurais attack: a group of 10 uavs launched from yemen, attacked saudi aramco oil facilities at abqaiq and khurais on 14th september 2019 in eastern saudi arabia. the uavs attacked the refineries and caused large fires, which were put out several hours later. due to this, both the refineries were shut down and saudi arabia lost half of the oil production (5% of the total supply of oil in the world). due to this shortage, the international crude oil prices rose by more than 12% the following day. a terrorist group claimed responsibility for this attack. samad 1, an autopilot fixed-wing type kamikaze (suicide) uav was used in this attack to carry explosives. saudi arabia's missile defense systems in abqaiq were designed against high flying targets and hence could not detect these uavs which flew at lower altitudes [59] . disruption of operation due to fear of collision between uav and airplanes [49, 51] rescue operations disruption of operation due to fear of collision between uav and helicopters [48] prisons smuggling of contraband by using uavs as carriers [52, 53] offices using uavs as cyber attack penetration node to access wifi networks [54, 55] residences and patrols spying by using uav as a mobile camera [56, 57] v. uavs in military attacks militaries have been using aerial attacks for more than 150 years. such attacks were first observed in 1849 in besieged venice when the austrians attempted to drop bombs over the city by attaching them to balloons with a time fuse. after that, during world war i, the american military developed the earliest prototypes which are modified versions of aeroplane that can hit specific enemy targets. advancement in radio control allowed the uavs to be operated in real-time. american designed curtiss f-5l is the first aircraft [61] controlled remotely which made its successful takeoff, maneuver, and landing in september 1924 completely guided in real-time. using similar radio control technology, u.s navy conducted a torpedo attack using curtiss tg-2 in april 1942 during a test strike on practice warship. in 1970, the ryan aeronautical company produced a new uav named bgm-34a, which was developed based on their early 1960s reconnaissance uav model. this uav became the first unmanned combat aerial vehicle (ucav) [62] which successfully launched air to surface guided missile hitting the target. in the later part of this section, some of the recent attacks using such uavs have been discussed in depth. uavs are used for a wide variety of military applications like reconnaissance/scouting (the military observation of a region to locate or ascertain enemy forces), surveillance, and intelligence. uavs are extensively used in reconnaissance missions because of the high degree of risk involved in manned missions. although traditionally uavs were used only for reconnaissance and surveillance purposes, they are now being used for conducting strikes, rescue missions, and for other miscellaneous applications in the military. table vi shows the details of top military uavs based on payload capacity and weapons on board. with recent improvements in imus (inertial measurement units), cameras, and flight control systems, uavs find a variety of military applications and are fast replacing manned aircraft and satellites [63] . till date, more than ten countries have conducted uav strikes. figure 5 shows a map of various countries that have already conducted uav strikes. many other countries, including saudi arabia, india, and china, among others, maintain armed uavs in their arsenals. figure 4 shows a map of countries that have acquired armed uavs. references [64, 65, 66] provides some insights into military uav attacks conducted on countries like pakistan, afghanistan, yemen, iraq, syria, somalia etc. the usa, uae, israel, uk, italy, and greece were some of the first countries to start developing armed uavs [60] . figure 6 shows various countries that have now developed armed uavs. usa and israel are the largest producers and sellers of uavs. america's leading ucav is the mq-9 reaper (manufactured by general atomics), which has served operations around the world for more than a decade. the mq-1 predator, which served the united states air force for more than 21 years retired in 2018. israel's leading ucav is iai heron and is the largest uav exporter in the world, accounting for 41% of uav exports from 2001 to 2011. along with their popular model ch-3, china also developed the ch-4 and ch-5 models. india is a major uav importer. it accounted for 22.5 percent of all uav imports from 1985 to 2014. also, india has its very own rustom range of uavs. the rustom has 3 variants, namely, rustom-i, rustom-h, rustom-ii (or tapas-bh-201). rustom-i has been tried and tested several times (on at least 15 different occasions) and has had several successful flights. rustom-ii has been designed to conduct reconnaissance and surveillance roles for the indian armed forces. this attack was carried out on jan 13, 2006. it involved four central intelligence agency operated unmanned mq-1l predator uavs that launched agm-114 hellfire missiles into the pakistani village of damadola about 7 km from the afghan border.the attack purportedly targeted the leader of a terrorist group, who was suspected to be in the village [67] . this attack was carried out in 2008 september in miramshah in north waziristan, pakistan. this was targeted at militants and carried out by the united states air force uav named predator b (mq 9-reaper) capable of high-altitude surveillance, air-to-ground missiles, and laser-guided bombs. the missile released by the uav hit two buildings and killed militants [68] . the 2009 makin airstrike is one of the deadliest uav attacks since such operations were started by military forces. it was conducted by a us military uav targeting militants in a funeral procession in the makin city of south waziristan, pakistan, killing several militants [69] . hmeymim air base operated by russia has been under multiple uav attacks. it is located near latakia in hmeimim, latakia governorate, syria, which is very close to the bassel al-assad international airport. on september 27, 2019, a russian defense ministry spokesman said that the air defense and electronic warfare systems deployed at the hmeymim airbase shot down or disabled 118 unmanned uavs during terrorists' attempted attacks on the military facility over the previous two years. the first attack on this airbase was on january 6, 2018 when 13 combat fixed-wing uavs attacked the base. for some of these uavs, the control signals were overpowered and control was obtained, while others had to be destroyed by short-range pantsir-s1 anti-aircraft missiles [70] . radars for detection and a combination of short-range and long-range missiles were used [71] . in this section, we provide a preliminary background of wireless communications necessary to understand uav operations, uav monitoring, and uav neutralization techniques. we begin by discussing spread spectrum which is the concept targeted throughout the rest of the paper. there are several basic modulation techniques such as frequency modulation, amplitude modulation and digital modulation. however, such modulation of the original message signal on a fixed carrier wave makes the resultant signal wave vulnerable to tampering and jamming. such a modulated wave can also be demodulated by anyone to get the original message signal. spread spectrum is a modulation technique which protects the message signal from interference, environmental noise,8cm jamming. it also ensures secure communication and reduces the probability of signal detection. in spread spectrum techniques, the original narrow-band message signal is modulated with an independent wide-band code signal. the resultant signal thus has a higher bandwidth and the original message signal is 'spread' over a wide range of frequencies. at the receiver end, the same wide-band code signal is used to 'despread' the transmitted signal to get back the original narrowband message signal, as shown in figure 7 . in the following discussion, we briefly explain four major variants of spread spectrum techniques which are often used in wireless uav communications: direct sequence spread spectrum, frequency hopping spread spectrum, time hopping spread spectrum and chirp spread spectrum. dsss is the most fundamental form of spread spectrum techniques. in this type, the message or signal wave is spread by modulation using a wide band code known as the pseudo noise (pn) code. this pn code consists of a radio pulse with a very short duration and thus higher bandwidth when compared to the signal wave. figure 8 shows how the signal wave gets spread by the pn code. spreading on the pn code results in the original signal wave getting distributed over a much wider range of frequency bands and causes the modulated signal to have a bandwidth almost identical to the pn code. the smaller the duration of the pn code, the larger the bandwidth over which the data signal gets modulated, and thus higher the system resistance to outside interference. dsss systems generally employ an additional modulation through binary phase shift keying (bpsk) modulation or quadrature phase shift keying (qpsk) modulation. phase shift keying is a modulation technique by which the data bits are expressed as phases in the signal wave. bpsk expresses each 0 or 1 in the data as 0°or 180°phase shifts in the carrier wave, respectively, whereas in qpsk two bits are considered at once and expressed as 0°, 90°, 180°or 270°phase shifts. to summarize, in dsss the data signal first gets modulated with the pn code followed by phase shift keying over a carrier wave. in fhss communication systems the data signal is modulated onto a carrier signal, whose frequency is rapidly switched among different channels. a pseudo-random sequence generator passes a sequence to a frequency table that selects the frequency of the carrier wave. this frequency is then passed to a frequency synthesizer that generates the carrier wave of mentioned frequency, thereby facilitating switching of the carrier wave. this pseudo-random sequence generator is known to both the sender and the receiver. hence, as the carrier wave frequency keeps switching, interference in a particular frequency segment affects the overall transmission for a very short duration. additionally, fhss systems generally employ m-ary frequency shift keying (mfsk) modulation technique. in mfsk, each channel is divided into m tones or smaller segments. in an fhss/mfsk system, log 2 m data bits are used to modulate the carrier frequency which is pseudo-randomly decided. when the carrier wave frequency changes faster than the mfsk tone, it is known as fast fhss and when the carrier wave frequency changes slower than mfsk tone, it known as slow fhss. figure 9 presents a signal flow diagram for fhss/mfsk systems and figure 10 shows how the frequency band is segmented in case of slow fhss. for fhss, it is important that the transmitter and receiver have a synchronized timing so that as the frequency band changes over time, the receiver can hop accordingly. fig. 9 : fhss/mfsk system block diagram. spread spectrum utilizes the entire bandwidth allocated to transmit a signal and hence achieves robust performance against interference. figure 12 shows an upchirp css whose frequency increases linearly with time. in thss, the input signal is not sent continuously. instead, it is broken into fragments and sent in pulses and 2 k discrete pulses are used as carrier signals to transmit k bits/pulse. a transmission window of duration x is divided into n segments and the signal is sent in one of the n segments (which varies with time). figure 11 presents the discrete signal pulses, over a period of time. the resistance to interference is achieved by randomly varying the transmission time by pseudo-randomly changing the carrier pulse period and duty cycle. in a strict sense, time hopping in itself does not introduce any spread spectrum characteristics. hence it is generally used in the hybrid spread spectrum with fhss. the higher bandwidth achieved by the transmitted signal in spread spectrum gives it resistance against interference. the ratio of the bandwidth of the modulated signal to that of the original data signal is referred to as the processing gain (g p ) of the system. this gives a quantifiable measure to the system's resistance to interference. the basic idea behind this is that by increasing the bandwidth of the signal we are making it inefficient for a malicious jammer to interfere with the signal. since there is limited power in the hands of a jammer, it will have to distribute the fixed power for transmission of the interference over a wide range of frequencies, thus introducing very little interference in a particular section of the signal. on the other hand, if the jammer transmits all its power on a particular section of the signal, the rest of the signal remains free of any interference. the level of interference that a spread spectrum system can handle and still be able to perform at par with a specified level of performance is measured with the help of the jamming margin. it depends on the processing gain, system implementation losses and the minimum snr ratio required at the receiver for error-free information transmission. in this section, we present a discussion of the various aspects of uav operation such as the modes, flow of control, systems for navigation, and also review some of the most common commercial transmission protocols. depending upon their mode of operation, uavs can be classified as manual, partially autonomous, and fully autonomous. most traditional applications of uavs involve manual remote radio control over the flight of the uav and its traversed path. in this, the pilot tunes the flight controller parameters to adjust stability and maneuverability. the most common method of doing so is by using a pid (proportional integral derivative) control loop which provides three parameters described in table vii . error refers to the difference between the actual and intended values. applications based on manual operation occasionally involve the transmission of payload signals such as video feedback or positional information. uavs in such scenarios can be detected and neutralized using different rf spectrum jamming techniques which are discussed in section ix. 2) full and partial autonomous: as the complexity of operations performed using uavs increases, many tasks are being offloaded to the onboard flight controller. in the case of a partially autonomous system, the controller takes care of the low-level tasks of the mission such as collision detection and altitude control. the high-level mission constraints and parameters such as way-points are controlled by a human operator. thus, instead of human or manual control, the system uses different sensors such as inertial measurement units, pressure sensors, etc. to determine the current state of the system and maintain porperties such as altitude of the uav. on the other hand, a fully autonomous system application would involve a completely autonomous execution of the mission, without necessarily involving the transmission/reception of rf signals to/from the ground station. a typical example would involve a pre-programmed flight route. in most of these autonomous applications, it is necessary for the uav to track its current location in order to facilitate the higher level mission objectives. this is done with the help of a global navigation satellite system (gnss) receivers such as gps. such systems can be identified and neutralized using gnss/gps jamming. for certain, in the cases of fully autonomous applications, it is possible that there is no transmission of signals with the uav, or it becomes difficult to analyze the rf frequency spectrum reliably. in such cases, radar-based techniques are used to detect unauthorized uavs. major components of an uav include propellers, motors, frame, sensors, speed controllers, flight controller, receiver, and battery pack. besides the uav, the ground station and intermediaries needed for communication also play a vital role. figure 13 shows the various components of uav along with their interaction with each other. the frame of the uav is a simple, lightweight, aerodynamically efficient and stable platform with limited space for on-board electronics. the material and structure of the frame is very important as defects in it can disrupt the uav's operation in several ways. a heavy frame decreases the payload capacity, and a skewed frame will create problems in the stability of its flight. uav frames are constructed mostly using carbon/graphite fibers or thermoplastics such as polyethylene, polystyrene and polyether-ether-ketone (peek). a manual uav receives commands from the pilot through a transmitter and receiver working over various technologies like radio, wi-fi, and zigbee based on various radio protocols like dsmx, a-fhss, etc. after receiving the signal from the radio transmitter, the radio receiver converts them to electrical signals in pulse width modulation (pwm) or pulse position modulation (ppm) format. these signals are interpreted by the flight controller whose commands are converted into specific actions for controlling the uav. for rotor based uavs, the electronic speed controller (esc) takes the signal from the flight controller and power from the battery and adjusts the speed of the motors to control the flight. for fixed-wing uavs, servo motors are used to precisely control the angle of the wings which in turn controls the flight. besides the commands, there is an additional transmitter-receiver pair that transmits crucial information like battery voltage reading and radio signal strength to the ground station, termed as telemetry. a ground station is the control center that facilitates the human control of uavs. an autonomous uav has a predefined flight route based on gnss systems like gps or environment checkpoints which can be detected by the camera. the uav can roughly track its position relative to its initial position by using onboard imu sensors. it corrects its position based on visual checkpoints and gps confirmation. uavs operating in different modes follow different principles for their navigation. manual uavs usually receive signals from the pilot, partially autonomous from the pilot, gnss system, and sensors, whereas completely autonomous ones have capability for flight without pilot intervention. this type of communication method involves a human operating a radio transmitter/receiver, a smartphone, a tablet or a computer as a ground station to control the flight path of the uav. uavs using this type of communication can fly only in the line of sight of the operator, and hence, these types of uavs are generally short-ranged. this type of communication involves uavs communicating with a gnss system. uavs using this type of communication are generally long-ranged. a very common gnss system is gps. gps navigation is provided using more than 30 satellites continuously revolving in their fixed orbits around the earth. each satellite has a stable atomic clock which is synchronized with others and to a base station which updates the time with respect to the ground. for knowing the position accurately, a signal from at least 4 satellites needs to be received. the navigational message includes the almanac data and ephemeris data. the ephemeris data has information regarding the location and time of each satellite in the orbit. almanac message include data regarding the entire constellation of satellites.hence, these messages from satellites are used by the gnss receiver to find its location and time using the trilateration method. in system, another aircraft that is always in a definite range of the uav serves as a relay to give flight control. sometimes, the other aircraft can itself be a uav which is controlled through either satellite navigation or remote control. various sensors such as ultrasonic, lidars and cameras are used for sensing distance from the objects in the environment whereas imus are used to track the orientation of the uav over 9 degrees of freedom. barometers are also used to track altitude of the uav. all of these sensors are used in combination to give feedback to the flight controller for stable flight. these sensors are used to implement localization through simultaneous localization and mapping (slam) algorithms or deep learning approaches [81, 82, 83, 84] . transmission (tx) protocols are implemented in the wireless communication between the transmitter and the receiver. a transmission protocol is defined by the structure of its data packets, and the type of spread spectrum and encryption scheme it uses. some major uav manufacturers implement the same protocols across all of their uavs. these are of utmost concern for the security analysis of a uav. some of the most popular tx protocols include mavlink, digital signal modulation (dsm), automated frequency hopping digital system (afhds) and futaba advanced spread spectrum technology (fasst). table viii presents a list of popular transmission protocols used for uavs. the organisations that use these protocols generally the use the same radio frequency integrated circuit in their implementations. this documentation of these rfics can be looked into for vulnerabilities like default passwords and behavior on disconnection. it is worth noting that mavlink is an open source tx protocol and thus has been thoroughly explained in literature whereas descriptions of other protocols are not readily available. this makes mavlink easier to analyze compared to other protocols. 1) mavlink: the micro aerial vehicle (mav) link protocol is a very lightweight messaging protocol used to carry telemetry, and to command and control multiple uavs. mavlink follows a modern hybrid publish-subscribe and point-to-point design pattern. in the publisher-subscribe model, we have a central broker that manages various topics to which clients either subscribe (listen to messages) or publish (pass messages). when a particular message is published on a topic, all subscribers receive that message. on the other hand, in the point-to-point model, messages are sent directly between the sender and the receiver. therefore, in mavlink, data streams are sent/published as topics whereas configuration sub-protocols such as the mission protocol or parameter protocol are point-to-point protocols with re-transmission. it has two versions: mavlink v1.0 and mavlink 2.0. mavlink v2.0 was widely adopted in 2017 and is the main focus of our discussion. figure 14 presents the packet structure used in mavlink 2.0. the data packet has just 14 bytes of overhead as shown in figure 14 [85] . the start-of-text (stx) marker is used to indicate the beginning of a new packet. the sequence id is used to detect packet loss as successive messages have successive ids. it employs x.25 crc as a checksum to verify data integrity. a checksum is a datum calculated based on a specific block of data for the purpose of detecting errors that may have been introduced during its transmission or storage. the protocol defines 1-byte fields for the source device id and source component id. the target device id and target component id are optional and can be specified in the message payload to indicate where the message should be sent/routed. in the end, there is an optional signature field of 13 bytes to ensure that the link is tamper-proof and ensures endpoint authenticity. the signature has 3 fields linkid, timestamp and a unique sign: • linkid (8 bits) -each link has an id in the mavlink protocol. linkid id this just this id and is usually same as the channel. • timestamp (48 bits) -the timestamp has a unit of 10 microseconds and it monotonically increases for every message on a particular link. • sign (48 bits) -the sign is extracted from the start of the sha-256 hash of the complete packet without the sign itself but including the timestamp appended to the secret key. the secret key is 32 bytes of binary data stored on both ends of a mavlink channel. being a very common protocol, considerable research has been carried out on finding threats and solutions for the mavlink protocol [86] . 2) digital signal modulation 2: the spektrum dsm2 technology operates in the 2.4ghz band and uses wideband dsss. dsm2 uses dualink technology that is based on the use of two channels. the duallink technology assists recovery from polarization blind spots and reflected signal fading. upon switching on, the dsm2 based transmitter scans for two free channels in the 2.4ghz band. on finding them, the transmitter transmits it's distinct globally unique identifier (guid) in these channels. subsequently, on receiving the guid, the receiver locks on to these two free channels [87] . in case one of the channels is noisy, the other channel acts as a backup. this lowers the chance of disconnection of the signal significantly. however, if both channels become unusable, the connection might be lost [88] . hence, the dsm2 signal provides additional resistance to noise, interference and other transmitters transmitting on the same frequency. the spektrum dsmx is based on dsm2 and is a successor to it. dsm2 and dsmx transmitter reciever pairs are compatibile with each other. firstly, an important feature of dsmx, similar to dsm2, is the wide-band signal which compared to the narrow-band signal of other 2.4ghz transmitters, is more resistant to interference and a has a higher snr. secondly, unlike dsm2, dsmx employs frequency hopping spread spectrum. dsmx transmitters have their own unique frequency shift pattern calculated using their guid and each pattern uses just 23 channels in the 2.4ghz spectrum. lastly, it uses duallink technology as well [89] . the flysky automatic frequency hopping digital system (afhds) is a digital protocol that ensures two or more transmitters can operate at the same time without interfering with each other's respective communication. afhds 2a is the second generation of the system that added duplex communication capability and telemetry capabilities [90] . afhds-2a supports up to 14 channels and requires manual-binding. it offers options to change the reception (rx) protocol for the receiver, servo frequency, the link quality indicator output(an indication of the quality of the data packets received by the receiver based on the receiver signal strength), and the fine-tuning of the operational frequency [91] . afhds 3 is the latest generation of the protocol and allows automatic frequency hops in the range of 2.402ghz to 2.481ghz. however, depending on the outcome of the initial transmitter-receiver pairing, the frequencies actually in use and hopping algorithm change. moreover, each time the system is powered on, the frequencies and hopping algorithm change as a security measure. neutralizing or alerting. not all of the various approaches for uav monitoring can efficiently do all the aforementioned operations. approaches for detection, identification and localization are discussed in this section which can be classified as in figure 15 and summarized in table ix at the end of this section. implementation of neutralization as a method for responding is discussed in section ix. radar-based techniques are usable in the case of partial or completely autonomous uavs where no radio communication takes place. for detection by radar, generally, radio waves are transmitted, which on reflection from an object undergo reflection and change in properties such as polarization. based on the properties of received signals like the doppler effect and polarisation of the received radio waves, information about the presence of an object can be calculated. doppler radars are able to discard static objects and specifically track moving objects. micro-doppler radars are able to detect motion, specifically speed differences within moving objects. as uavs have major moving parts like propellers which create a large spectrum of linear speed differences, this is a good approach. millimeter-wave radars, ultra-wideband radars, and non lineof-sight radars are some of the applicable radar technologies [93, 94] . in this method, the rf waves coming from the uav are intercepted. generally, a manually operated uav communicates with the ground station (gs) and a gnss for its operation. both of these may be absent for an autonomous uav working solely based on on-board sensors. methods like fhss are often applied for the communication between the gs and the uav. however, for most commercial uavs, an ai-based classification algorithm can be implemented which analyzes the spectrum using software defined radio (sdr) and based on features such as the hopping sequence, predicts the presence of a uav along with details about it [95, 96, 97] . also, based on the signal strength, it is possible to track and localize the uavs. besides these, the uav may be transmitting information about itself like its coordinates and video feed which can be intercepted and analyzed for localization. these methods have a good detection range depending on the transmit power of the uavs. however, they tend to be expensive. software defined radio is a recent technology that will reduce the cost of this approach [98] . another approach for detecting and monitoring uavs is by using computer vision. video surveillance from strategically placed cameras in an area can be passed though deep learning models trained to detect and track uavs. these deep learning models are trained with datasets of images and videos of uavs from which they eventually extract the features which help in distinguishing and tracking uavs. the models could be based on either identifying the appearance of uavs or their motion. multiple cameras with a varying field of views can be used for faster detection [99, 100] . for situations when the ambient light is low, it is hard for normal systems to carry out detection due to a lack of contrast and features. in such scenarios, thermal imaging can be used [101] . in this approach, the various sounds made by a uav due to its moving parts like motors, propellers, and chassis vibration are analyzed using machine learning and deep learning classification approaches to detect its presence. uav sounds can be recorded in control environments to make datasets that can further be augmented using noisy environments to simulate real environments. with these augmented datasets, various models based on support vector machines (svm) and neural networks can be developed for uav detection [102, 103, 104] . besides these, fundamental audio processing approaches such as audio fingerprinting may also be used [105] . it is also possible to track the position of uavs by using multiple microphones with methods like triangulation. better results can be availed by combining two or more of the aforementioned methods, such as cameras along with microphones assisting each other in their operation. a system can be designed which uses the acoustic sensors to identify uavs by audio fingerprinting along with identifying the direction of the source, after which the camera can rotate to spot the target [106, 107] . once the uav is successfully detected and localized, the next step is to take the appropriate response action. the response action depends on the extent to which it is detected, identified, and localized. based on the mode of operation (i.e., manual, full, and partially autonomous), different situations necessitate different approaches for neutralization. for a manual or semiautonomous uav, the rf radio communication between the ground station and uav happens over various protocols with fixed packet structure. these packets can be intercepted and decoded to obtain the packet architecture and hence a spoofing attack can be launched. rf jammers which transmit large amounts of energy towards the uav can also be used for disrupting communication. this can lead to the uav activating its fail-safe which may be to make a safe landing in its current position, return to a preset base location, or fly haphazardly and crash. for an autonomous uav, the sensor values can be corrupted. the gnss signals specifically can be jammed as well as spoofed. besides these, there are some other physical attacks that are possible as well. figure 16 presents a table listing all the techniques discussed in this section. additionally, a table xii reviews some of the state of the art solutions against uavs avaiable in the industry. in the case of rf radio communication between the uav and the ground control, rf jamming techniques are used to increase the level of noise interference at the rf receiver, in this case, the uav or the uav. this reduces the snr at the receiver. this in turn prevents the receiver from responding to the commands from the sender, and thus, neutralizes its operation. hence, the signals from the pilot or satellite systems can be jammed to disrupt the operation of manual and autonomous uavs that depend on these. however, some uavs that operate solely using other sensors such as imus and cameras are immune to this jamming technique. different uavs may employ different spread spectrum and modulation techniques. to reduce the effectiveness of jamming, various methods have also been developed based on software defined networks [108] . hence, depending upon these situation, different rf jamming techniques will be more efficient for effectively introducing interference at the rf receiver. the different types of rf jamming techniques available in literature [109, 110, 111] are discussed in this section . in addition to these, gnss jamming has been discussed as a subsection due to its relevance to the subject (although it is not a different type of jamming and a specific case of the preceding types). table x presents a comparative analysis of the various jamming techniques discussed. 1) noise jamming: also known as barrage jamming, it is the simplest form of jamming in which a noise signal is applied to a small portion or the entire spectrum of the wideband modulated signal. this form of jamming directly affects and reduces the channel capacity of the system. it reduces the snr value at the receiver, thereby reducing the channel capacity and increasing the information error rate. noise jamming affects fhss systems especially by introducing background interference during clock synchronization and tracking that is required between the sender and the receiver in between every transmission. figure 17 shows a visual representation of how the rf spectrum is jammed using noise jamming. synthetic aperture radar (sar) is a special radar used for two dimensional and three-dimensional spatial mappings and is often employed in uavs to provide autonomy to the uav by using methods such as simultaneous localization and mapping (slam). noise jamming can be used against such systems since the radio interference generated by it is sufficient to hide echoes, thereby rendering the sar system futile [112] . 2) tone jamming: in this type of jamming one or more tones in the spectrum are jammed strategically to introduce interference. the jamming performance depends on the placement of the tones in the spectrum and the transmission power. the transmission power is directly proportional to the interference on the tone. this technique is dependent on being able create sufficient interference in order to overcome the jamming margin, which requires a lot of power as multiple tones are targeted. tone jamming can be of two types: either on a single tone (mono-tone) or on multiple tones (multi-tone). both perform poorly against fhss systems due to the variable nature of the transmission spectrum. monotone jamming performs poorly since the overall performance of the system is not affected by one channel being absent. even in multi-tone jamming, to be able to introduce significant interference, the adversary has to jam enough channels in the spectrum. the performance of single-tone jamming against dsss systems has been analyzed in [113] and compared against narrow-band barrage jamming. with the correct placement of the tone, tone jamming performs fairly well against dsss systems. figure 17 shows a visual representation of how the rf spectrum is jammed using tone jamming. 3) swept jamming: in swept jamming, a relatively narrowband signal, sometimes even a pulse or tone, is scanned in time across the frequency spectrum of interest. at a particular time instant, only a single frequency is targeted. however, over a period of time, the jammer covers a range of frequency bands by sweeping the target frequency. hence, it is a mixture of noise and tone jamming in its application. similar to noise jamming, it focuses on a single tone, but by sweeping, it also emulates tone jamming. unlike noise or tone jamming, sweeping the jamming waveform ensures that it covers the entire set of hop frequencies of the data signal in the entire spectrum. figure 17 shows a visual representation of how the rf spectrum is jammed using swept jamming. swept jamming performs well against dsss systems since the data frequency bands are stationary, unlike fhss systems where the frequency bands hop periodically in a pseudorandom fashion. swept jamming finds most applications against gps enabled systems due to weak strength of the gps signals [114] . ways of safeguarding gnss to swept jamming are also constantly being actively developed and analyzed [115] . 4) follower jamming: a follower jammer always tries to locate the new frequency hop that an fhss system went to and then locates the target signal. once confirmed, it then tries to jam the located frequency. to locate the target fhss system frequency, it requires that the jammer measure the spectrum for energy losses and gains. an energy gain in the spectrum indicates a new signal entering whereas energy loss indicates a signal leaving the band. however, even then it is important to accurately identify whether a particular energy variation corresponds to the target signal or not, for which analysis needs to be carried out [116] . jamming: this type of jamming is applicable when the target signal properties are known previously from sources such as the radio frequency integrated circuit (rfic) specifications. in smart jamming, only the necessary signals are attacked to deny successful communication, and hence they are power efficient and effective. this can be achieved by analyzing the data that is being transmitted and identifying critical points. protocol-aware jamming techniques can be developed using software defined radios. after analyzing the target signal, a jamming signal similar to it in terms such as hopping patterns (fhss systems), pn code data rate (dsss systems), and modulation techniques can be generated. literature on implementations of such systems present the energy-efficient jamming performance as compared to other jamming techniques in terms of bit error rate (ber) introduced in the system. it is found to outperform other techniques in successfully jamming the target signals both in dsss and fhss systems but relies on the analysis of the target signal [117] . 6) gnss jamming: today, almost all uav applications involve an autopilot functionality. that is, they are either partially or fully autonomous in their mode of operation. they house numerous sensors and onboard hardware to estimate the current properties of the uav such as orientation, location, and acceleration. one of these is the gnss module such as gps, which provides signals for localization. gps signals are generally vulnerable in nature due to weak signal strength and are highly susceptible to noise and outside interference [118, 119] . the implementation of gnss jamming is an active field since although it is being used as a technique to disable uavs in this context, the same methods can be used to disable gnss systems in other critical applications such as navigation. different jamming techniques for gps signals have been studied, analyzed, and evaluated based on spectral efficiency, energy efficiency, and complexity. based on this, protocol aware jamming or smart jamming was found to be the most efficient [120] . gps jamming is very prevalent as such and jammers are easily available in the market [121] . a study in 2013 by chronos technology supported by the united kingdom technology strategy board investigated the prevalence of such vectors by setting up several monitoring stations across the uk. results showed that some stations even recorded 5-10 jamming instances in a day [122] . research has also been done to prevent gnss jamming due to the financial and strategic importance that this technology holds for all countries. various methods to mitigate gnss jammming, such as using antenna arrays (which improve the overall snr ratio) have also been proposed [123, 124, 125] . b. spoofing 1) sensor spoofing: the autonomous uav auto-pilot application relies heavily on the onboard sensors such as lidars, 129, 130, 131, 132] sonars and optical flow sensors for localization and navigation. these sensors interact with the environment and provide valuable data for the auto-pilot computer to optimally guide the autonomous uav system. in a sensor spoofing attack, the attacker transmits false sensor values to the flight controller which are in contrast to the actual values. the feedback loops get triggered and in order to correct the destabilized false values, the uav orients itself to stabilize, which in turn destabilizes it. since many sensors share data for actuation, this can even lead to complete loss of control of the system, since one can gain access to the on-board actuator sensors or simulate false values. simulations of sensor spoofing attacks on optical flow sensors focused on uav systems have proven this vulnerability [126] . in order to throttle the rising number of such attacks, control invariants have been introduced into the system which are able to detect differences between the perceived state and actual state. such control invariants are derived from physical dynamics and control systems [127] . for instance, the data of gps and optical flow sensors can be used to validate against other sensor data to detect inconsistencies to detect such attacks [133] . 2) signal spoofing: in contrast to jamming which involves bombarding the receiver with noise signals, spoofing involves the generation of plausible counterfeit input signals with enough power to fool the receiver into believing this to be the original legitimate signal. spoofing of the tx protocol is difficult and requires reverse engineering to understand the structure. gps signals tend to fluctuate in power due to the position of the satellites in the space as well as the weather conditions. most modern-day gps receivers implement an automatic gain control (agc) to compensate for these widespread fluctuations in the gps signal strength. however, this also leaves the system more vulnerable to spoofing attacks [128] . a fake gnss signal can be broadcast in an area making the uav in that area perceive as if it is located in a different position, giving virtual control of the uav to the spoofer [129, 130] similar to meaconing, it is used in systems where the gps signal is protected with cryptography. in this attack, each symbol of the secret code is separately estimated by observing the received signal in the corresponding symbol interval. the symbol estimate is continuously updated by observing the received signal and simultaneously used in the spoofed signal, trying to replicate the secret code as faithfully as possible. then, the spoofed signal is sent with some delay to the original signal. methods such as likelihood ratio testing and its derivatives can help protect against this vector [132, 134] . a. malware 1) maldrone: maldrone is a software malware that overrides the uav's autopilot and allows the attacker to take over control remotely. it can be used against any arm based uav running on linux like ar parrot and dji phantom. it opens a backdoor which gives access to sensors and drivers to the hacker. the developer of maldrone, rahul sasi, tested it on a parrot ar drone in 2015. the parrot ar drone, revealed at the international ces 2010 in las vegas, is a quadcopter that can be controlled by a smartphone or tablet over wifi. a binary named program.elf that manages the ar drone has not been made open-source. however, this could be reverseengineered by using software like ida which can convert asm code to pseudo c code. this gave sufficient information about the uav's operation. maldrone injects itself like a proxy, thus enabling the injection of the desired values for both flight controller and sensor communication [139] . 2) skyjack: skyjack has been developed for searching and hijacking uavs that are in wifi communication range. it gived the skyjack pilot the ability to take control over the hijacked uav and also view the camera. skyjack's developer, samy kamkar, used a raspberry pi with a wifi packet injector installed with skyjack to hack an ar parrot 2 drone in 2015. the ar parrot 2, like its predecessor, works over wifi and can be controlled by a smartphone or tablet. the ar parrot 2 hosts its wireless network which is how the pilot of the uav connects. skyjack first runs aircrack-ng (open source) to run monitor mode to find the uavs and uav owners in its vicinity. the mac addresses of the uavs owned by the parrot company can be detected over any wireless connections from mac addresses as they can be found online in the ieee registration authority's organizationally unique identifiers. after that aireplay-ng (open source) is used to de-authenticate the actual pilot of the uav. once unauthenticated, the attacker can connect as the uav is not connected to any pilot. finally, node-ar-drone is used to control the newly enslaved uav via javascript and node.js [140] . skyjack does have its limitations, however, since it can only select targets within a predefined range of mac addresses on an unsecured network. b. gps spoofing 1) dji matrice 100: a software defined radio along with an open source gps-sdr-sim repository is used to broadcast fake gps signals [141] . the attacker supplies a gps broadcast ephemeris file for specifying the gps satellite constellation. the ephemeris files are used to generate the simulated pseudorange and doppler for the gps satellites in view. after this, the simulated range data is used to generate the digitized i/q samples for the gps signal. further, the sdr converts the i/q to rf waves which are received as stronger signals than the real gps by the uav. almost all uavs that are dependent on gps are vulnerable to this kind of attack owing to the very nature of unencrypted civilian gps signals. 2) iit madras: in february 2020, students at the indian institute of technology, madras designed a uav that can detect other uavs visually, along with a gps spoofing mechanism for neutralizing the operation of gps dependent uavs in the vicinity. it uses a deep learning model to detect motion in order to spot other uavs even in the absence of light. it also uses an sdr approach to broadcast stronger gps signals for gps spoofing. mathematical models of the time differences with the target uav are used to generate the spoofed gps packets. with multiple time differences, eventually the target uav calibrated its clock to the spoofed signals. when there is a large variance in the actual gps position and spoofed position, the target uav's fail-safe is activated that disrupts its operation. this was tested against various civilian gps receivers used in the uav industry such as ublox and dji inhouse gnss. the students were able "to take down the uavs almost instantaneously" [144] . the parrot bebop was tested against various attacks by a research team and they were able to discover three vulnerabilities [142] . the first is a denial of service attack where about 1000 requests were made to the uav rapidly, each trying to connect as controller. the uav's processor and memory could not handle this and therefore, it shut down. this sent the uav into what the team referred to as "an uncontrolled landing". next, a buffer overflow attack where a very large data packet was transmitted to the uav, which exceeded the maximum capacity of the buffer in the system. this also caused the uav to crash. lastly, fake packets claiming the sender to be the uav's controller were sent repeatedly from a laptop. this claim was made by spoofing the ip address. soon the uav accepted the sender as hence its own contact with uav was severed. this led to an emergency landing of the uav. the digi xbee 868lp rf modules provide wireless connectivity to end-point devices in mesh networks. it was used for telemetry between the uav and ground station for a uav, which was exploited by nils rodday and presented in the black hat asia conference 2016 [143] . by referring to the instruction manual of the chip, an api mode was discovered which allowed the user to fabricate their own custom packets for transmission. a broadcast mode was also discovered which allowed pinging all chips in the range and getting their addresses from acknowledgments. the chip also had a feature where its address, used for packet transmission, can be changed remotely by another chip. hence, the attacker introduced another chip in the system besides the ones in the ground station and uav, switched to api mode, and then broadcast to get the addresses of the gs and uav chips. after that, he modified the addresses such that all packet were sent through the attacker chip. hence, he got access to reading and writing information on the channel but was still unaware of the data packet structure. for this, he reverse-engineered the android application in the gs and then used trial and error to list all operation packets. after getting the templates, he could rebroadcast those packet to spoof itself as the uav gs. xi. conclusion uav misuse has led to the formation of regulations and regulatory bodies in various countries. so, in this paper, we highlighted the need for research and implementation of measures to counteract the misuse of uavs. having provided the current situation of uavs from a security perspective, the current global scenario of uavs in the military and commercial usage, existing regulations in various countries, we reviewed and discussed popular transmitter protocols and presented some detecting and neutralizing methods for uavs. we emphasized the rf communication used by the uav as it seems to be the most reliable method which can be deployed on a wide scale, owing to new technologies like sdrs, which reduce the cost dramatically. in the latter part, we presented case studies that specifically talk about how different kinds of commercial uavs can be hacked and thus can be misused. with the growing popularity of uavs in various fields, uavs are soon going to be employed in our day to day life and there is a need for research addressing all the security concerns to accelerate this process securely. unmanned aerial vehicle for internet of everything: opportunities and challenges mac protocols for unmanned aerial vehicle ecosystems: review and challenges application of unmanned aerial vehicles in logistic processes a survey on supply chain security: application areas, security threats, and solution architectures modern agriculture drones the development of smart farmers unmanned aerial vehicles (uavs): a survey on civil applications and key research challenges trajectory optimization of flying energy sources using q-learning to recharge hotspot uavs isdnet: ai-enabled instance segmentation of aerial scenes for smart cities drone applications for supporting disaster management a comprehensive review of the covid-19 pandemic and the role of iot, drones, ai, blockchain, and 5g in managing its impact energy efficient legitimate wireless surveillance of uav communications drones: reporting for work drones market size by product, application and forecast to 2025 flexible and reliable uav-assisted backhaul operation in 5g mmwave cellular networks uav communications for 5g and beyond: recent advances and future trends applications of blockchain in unmanned aerial vehicles: a review odob: one drone one block-based lightweight blockchain architecture for internet of drones a distributed framework for energy trading between uavs and charging stations for critical applications blockchain and cyberphysical systems scheduling drone charging for multi-drone network based on consensus time-stamp and game theory a distributed framework for energy trading between uavs and charging stations for critical applications design and analysis of secure lightweight remote user authentication and key agreement scheme in internet of drones deployment solving the security problem of intelligent transportation system with deep learning parth: a twostage lightweight mutual authentication protocol for uav surveillance networks securing smart city surveillance: a lightweight authentication mechanism for unmanned vehicles treebased attack-defense model for risk assessment in multi-uav networks exploring security vulnerabilities of unmanned aerial vehicles consumer iot: security vulnerability case studies and solutions houthi drone attacks on 2 saudi aramco oil facilities spark fires we enable flight for india's unmanned aircraft types of drones: multi-rotor vs fixed-wing vs single rotor vs hybrid vtol faadronezone webiste european union regulations caan regulations india drone regulations india's digital sky unmanned aerial vehicles (uavs): a survey on civil applications and key research challenges a survey on iot security: application areas, security threats, and solution architectures the rise of ransomware and emerging security challenges in the internet of things mobility enabled security for optimizing iot based intelligent applications low-altitude unmanned aerial vehicles-based internet of things services: comprehensive survey and future perspectives how can drones be hacked? the updated list of vulnerable drones and attack tools industrial control systems: cyberattack trends and countermeasures drone incidents all over the world illegal drones ground water-dropping helicopters at critical moment in maria fire battle drone hits commercial airliner in canada, no injuries uas airborne collision severity evaluation final report flights resume at frankfurt airport after drone sightings halt air traffic correctional officials raise concern over drones smuggling contraband into kingston-area prisons skynet: a 3g-enabled mobile attack drone and stealth botmaster hacking wireless printers with phones on drones orem drone pilot, girlfriend charged with voyeurism criminal intent: fbi details how drones are being used for crime venezuela's maduro says drone blast was bid to kill him, blames colombia how did oil attack breach saudi defences and what will happen next world of drones a brief history of early unmanned aircraft uninhabited combat aerial vehicles: airpower by the people, for the people, but not with the people the new killer drones: understanding the strategic implications of next-generation unmanned combat aerial vehicles uk drone strikes stats another us strike' hits pakistan dozens dead' in us drone strike russia says 13 drones used in attack on its air base, naval facility in syria russia's hmeymim airbase in syria strikes over 100 terrorists' drones over past two years routing protocols for unmanned aerial vehicles intelligent quality of service aware traffic forwarding for software-defined networking/open shortest path first hybrid industrial internet software-defined vehicular networks: architecture, algorithms, and applications: part 1 software-defined vehicular networks: architecture, algorithms, and applications: part 2 interaction of edge-cloud computing based on sdn and nfv for next generation iot guest editorial leveraging machine learning in sdn/nfv-based networks two-phase virtual network function selection and chaining algorithm based on deep learning in sdn/nfv-enabled networks blocksdn: blockchain-as-a-service for software defined networking in smart city applications visionbased slam system for unmanned aerial vehicles 3d lidar slam integration with gps/ins for uavs in urban gps-degraded environments deep neural network for autonomous uav navigation in indoor corridor environments dcnn-ga: a deep neural net architecture for navigation of uav in indoor environment available: https micro air vehicle link (mavlink) in a nutshell: a survey dsm2™ technology dsmx™ technology analysis and decoding of radio signals for remote control of drones detection, localization, and tracking of unauthorized uas and jammers," in detection, localization, and tracking of unauthorized uas and jammers characteristics of ultrawideband radar echoes from a drone security analysis of fhss-type drone controller micro-uav detection and classification from rf fingerprints using machine learning techniques rf-based drone detection and identification using deep learning approaches: an initiative towards a large open source drone database risk assessment of sdr-based attacks with uavs deep learning-based strategies for the detection and tracking of drones using several cameras cost-effective video summarization using deep cnn with hierarchical weighted fusion for iot surveillance networks night-time detection of uavs using thermal infrared camera empirical study of drone sound detection in real-life environment with deep neural networks," in empirical study of drone sound detection in real-life environment with deep neural networks audio based drone detection and identification using deep learning empirical study of drone sound detection in real-life environment with deep neural networks," in empirical study of drone sound detection in real-life environment with deep neural networks drone sound detection drone detection based on an audio-assisted camera array implementation of detection and tracking mechanism for small uas an snrassured anti-jamming routing protocol for reliable communication in industrial wireless sensor networks modern communications jamming principles and techniques proactive eavesdropping via jamming for trajectory tracking of uavs eavesdropping and jamming selection policy for suspicious uavs based on low power consumption over fading channels barrage jamming detection and classification based on convolutional neural network for synthetic aperture radar comparison of jamming methods for underwater acoustic dsss communication systems gnss signal authenticity verifica-information management,communicates,electronic and tion in the presence of structural interference swept gnss jamming mitigation through "capture of uavs through gps spoofing performance analysis "unmanned aircraft capture and control via gps spoofof fll schemes to track swept jammers in an adaptive ing the growing iranian unmanned combat navigationtechnologies and european workshop on aerial vehicle threat needs us action ml-based detection strategy for cryptographic follower jamming rejection in slow fh/mfsk systems gnss anti-spoofing jamming of uav remote control systems gps and optical flow fusion vulnerability of the gps signal security and privacy, ser the threat of gps jamming: the risk to an 10 effective gps jamming techniques for uavs using lowtheir detection on gnss signals keeping uavs under a concept control during gps jamming robust set-membership filtering techniques aharoni, suppression scheme based on antenna array for t a novel array-05980 laser solutions controlling uavs with sensor input spoofing wings kill a drone mid-flight available: http://samy. vehicles: a control invariant approach securing commercial wifi-based uavs from common security attacks," in securing commercial wifi-based uavs from common security attacks hacking a professional drone iit madras designs ai drone that can hack into rogue drones he also worked as a postdoctoral research fellow at the national university of singapore, singapore where he worked in the area of internet of things. he is currently an assistant professor with the department of electrical and electronics engineering working in a software company based out of bangalore in the last 6 years, he has worked and deployed several cryptography projects, websites, and mobile applications he is the author of nine books and more than 500 publications in refereed journals and conferences. he guest edited a number of special issues in ieee journals and magazines. he also served as a member, chair, and general chair of a number of international conferences. throughout his career, he received three teaching awards and four research awards. he also received the 2017 ieee communications society wtc recognition award as well as the 2018 adhoc technical committee recognition award for his contribution to outstanding aayush agarwal naren navneet gupta ☒ the authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper. key: cord-311558-1y6z8qso authors: henry, caitlin title: palliative space-time: expanding and contracting geographies of us health care date: 2020-09-19 journal: soc sci med doi: 10.1016/j.socscimed.2020.113377 sha: doc_id: 311558 cord_uid: 1y6z8qso two important changes are happening in health care in the us. as hospitals close in high numbers, the geographies of health care services are changing. also, the ageing of the population brings about new and complex care needs. these are not discrete trends, as ageing impacts the who, what, and where of care needs, and hospital closures remakes the geographies of where people overall access care. developed out of research on the impacts of hospital restructuring on workers, patients, and communities, this paper aims to understand how health care financing, care needs for the ageing, and new geographies of health services are intertwined. to do so, i look back to 1980s policy changes to medicare, the federal health insurance program for the elderly and disabled. in 1982, congress made two important changes to medicare. the program began covering hospice services, constituting an expansion of care, and the government drastically changed the way it reimburses providers, effectively a contraction of the program. i trace the impacts of these changes over the next decades through analysis of media coverage and secondary research on hospital budgets. drawing on the concept of palliative space-time, i identify a contradictory logic of death at the center of this expansion and contraction of the health care system. this death logic works to destabilize an already uneven geography of health service. yet, this crisis has the potential for more just geographies of health and care. like many global north countries, the us population over 65 is growing, as baby boomers age and americans live longer lives, forcing attention to how societies can support thriving in old age. individuals, governments, families, and institutions are considering new ways of meeting the care needs of elderly people throughout the last decades of their lives, including age-friendly cities, new collective living arrangements, new institutional forms of care, and an increased focus on home care especially (poo 2015) . as activist ai-jen poo (2015, 3) explains, 'people getting older is not a crisis; it's a blessing. we're living longer; the question is how we should live.' the demographic shift comes with a cultural shift around the end-of-life. this is visible through the growing popularity of the death positive movement and caitlin doughty's 'as a mortician' series (the order of the good death n.d.), as well as countless books and podcasts exploring the meanings and practicalities of dying. furthermore, covid-19's disproportionate impact on elderly and people vulnerable due to health conditions, or the strains of systemic oppression, has heightened this conversation. people are asking what a 'good death' could be in a socio-economic system that is threatening to many lives and devalues entire demographics (beech 2020; fraser et al. 2020 ). simultaneously, the health care service landscape has changed dramatically since 2000. hospitals have closed across the country, transforming health care labour markets, how and where people access health care services, and the role of health facilities in communities and the built environment (buchmueller, jacobson, and wold 2006; colliver 2015; henry 2015; alexander 2018) . overall, hospitals are concentrating within and to wealthier urban and suburban centers, meaning both rural and some urban populations are quite far from a hospital. an excellent collaboration on the impacts of facility closures between the milwaukee journal sentinel and pittsburgh post-gazette on the impacts of facility closures found that 'nearly two-thirds of the roughly 230 hospitals opened since 2000 are in wealthier, mostly suburban areas' while the number of urban hospitals has been nearly halved since 1970 (thomas 2014) . rural and disadvantaged communities are greatly impacted and left uncertain what health services will replace the shuttered facility (tribble 2019) . hospitals are closing for multiple reasons. their role in the health system is changing, with more care shifting away from hospitals and towards outpatient and home care (landers batra, betts, and davis 2019) . more importantly, hospitals are increasingly financially unstable and unsustainable institutions (goldsmith and bajner 2017; lovelace jr 2018) . just as aging populations are not exclusive to the us, hospitals are unstable across the west. canada and the uk are also downsizing hospital systems, even while wait times for care are high or increasing and aging populations are requiring more chronic care (howlett and morrow 2014; ewbank, thompson, and mckenna 2017) . every country's health system is unique, but trends in financial stress, care deficits, and inequalities are strikingly consistent across the west. closures raise questions about the built environment when the health care landscape changes because of the large size of hospital properties, the myriad services they house, and their historic role in urban development (henry 2015; day 2016) . this is not to be romantic regarding hospitals but to highlight the importance of hospitals in the social reproduction of individuals, communities, and the health care system (stevens 2007) . it means thinking beyond the impacts of a hospital closure on mortality rates (e.g., joynt et al. 2015) to consider health and health spaces more holistically. this article posits that these two changes are not discrete, as health care is undergoing dramatic spatial and financial changes in the us. thus, the driving question of this article is: what does the convergence of these two simultaneous trends in health care -the remaking of the health service landscape through hospital closures and the growing number of older people needing chronic and eventually end of life care -mean for the wellbeing of the entire health care system? how relational are these two trends? for answers, i turned to medicare and medicaid, the government-provided health insurance for, respectively, people over 65 and low-income people. specifically, i examined the legislative history of medicare to j o u r n a l p r e -p r o o f better understand the role of these programs in hospital financing. in the early 1980s, both programs underwent their biggest changes since implementation, as congress passed the tax equity and fiscal responsibility act of 1982 (tefra) and the social security amendments of 1983 (ssa). this legislation made hospice care a covered service for the first time, and altered the fee structure for reimbursing health care providers. these two moments help explain the current transformations in us health care delivery. the closing of hospitals -the backbone of the health care system -and the ageing of the population are two intertwined trends, tied together through medicare's financial structure. these policies of the 1980s are contradictory. while the hospice benefit expanded coverage, the new payment scheme reduced financial stability. the 1982 and 1983 changes to medicare constituted an expansion and a contraction of medicare and, arguably, the health care system. together, they enacted a contradictory logic that is pervading the current health crises. more specifically, two different approaches to death -a care-full death for individuals accessed through hospice and an abandonment (or the dying) of the health care system via financial destabilization. in turn, these legislative changes, while not the only factor, have helped facilitate the transformation in the health care landscape that is unfolding today. to make sense of these legislative moves, i turn to feminist care ethics and eric cazdyn's (2012) concept of 'palliative time' -a state of existence that foregoes cure and marches towards, but does not always reach, death. i argue that with these structural changes to medicare, death permeates the entire health care system but in different ways. the dying of a health system has radical potential to destroy the system and create something new. in sum, this article contributes to historicizing understandings of the current j o u r n a l p r e -p r o o f transformations in us health care needs and services, adding nuance to understandings of the shape and impacts of hospital closures, care needs, and demographic shifts. this article draws on research from a broader project on the process and impacts of remaking of the hospital landscape in the us. i situate hospital closures and changing needs of an aging population in the historical context of health care funding. i use a textual analysis of media coverage in the 1980s from four major newspapers -wall street journal, new york times, washington post, and the los angeles times -to trace debates about and impacts of these legislative changes. i chose these newspapers because of their wide readership, differing perspectives, and depth of coverage. through lexisnexis, i searched for articles discussing medicare and either hospice or prospective payment from 1980 to 1990, generating over 400 articles. i analyzed the articles in two ways. first, i used them to assemble a timeline of events. second, i analyzed them for how different writers and actors characterized the needs -both financial and care -of medicare and its relationship to the health system more broadly. i paired this news analysis with a review of secondary literature (primarily from health economics and public health) focusing on studies done since 1982 on the impacts of the prospective payment system on hospital financing and the use and cost effectiveness of hospice. in the next section, i turn to feminist care ethics and eric cazdyn's concept of palliative time, which i will later use to understand the implications of these legislative changes. building on these literatures, i propose the concept of palliative space-time (pst) as a useful tool for understanding the contemporary state of health systems. then, after outlining the two changes made in 1982 and 1983, i trace legacies of these changes in the decades since. finally, i apply the idea of palliative space-time to these contradictory j o u r n a l p r e -p r o o f changes, to reveal a death-focused logic at work in us health care today and consider what alternative possibilities exist. i focus primarily on changes made to medicare, the federal program providing health insurance to the elderly, as well as some people with disabilities. when relevant, i mention medicaid, the joint state and federal program providing health insurance to the poor. while these are different programs, they are hardly discrete. policy decisions around one often affect the other, and for patients, these programs sometimes work in tandem to ensure access to care (e.g., medicaid, rather than medicare, covers stays in nursing homes). in the already dead, eric cazdyn (2012) draws on his experiences managing chronic leukaemia to analyse medicine's increasing focus on maintenance over cure. this approach prioritizes important life-maintaining care, cazdyn argues, over lifesaving cures. he describes patients as stuck in palliative time: a time of persistent sickness, hanging on near death. he invokes palliative care, which is comfort care most often delivered when one is near death, to argue that people become trapped in the end-of-life care phase. importantly, this new temporality also describes a political economic condition: the maintenance of a sick and sickening system. cazdyn pairs his critique of medicine with reflections on the impacts of globalization, arguing that globalized capitalism is a broken system, working well for the few and harming the many, that keeps a world lurching along, always near but never reaching death. he draws an analogy between economics and medicine that forces consideration of medicine's approach to chronic care in the political j o u r n a l p r e -p r o o f economic context of the health care system, as palliative time is a descriptor for patients as well as a global economic system. palliative time is not simply a temporal label. it is also an analytical tool that highlights the paradox at the center of the health care system: a near-death sphere of possibility, as cazdyn calls it, referring to the possibility of something new and more just coming out of deadly and also frail system. on the one hand, being stuck in the palliative means forgoing cure. no longer searching for cures, the focus shifts to maintenance. people are expected to live with chronic illnesses both social and medical rather than searching for the end of those illnesses, be they though cure or death. medicine resists death, viewing it as a failure rather than a part of life. for cazdyn, living with sickness means living, but always near death. on the other hand, palliative and hospice care is an ideal practice of care. it is attentive to needs of both individuals and communities, treating death with respect, reverence, and care. it foregrounds the nuances that nursing brings to health care, rather than the colder, more clinical approach of medicine. hospice faces death directly, without losing complexity of human experiences and approaches to death. in death, hospice patients and their loved ones often access the best care they have ever received. thus, palliative time captures a paradox in health care related to care and death, simultaneously holding two different, contradictory philosophies towards the end of life. the paradox describes a deadly system that also promotes, at times and in certain places, very care-full experiences of dying. these contradictory approaches to death offer different ways forward -one that maintains the deadly status quo and another, more progressive way that embraces death and dying as a meaningful part of life that deserves more attention. caring is more about a transformative ethos than an ethical application". care is an ethicopolitical approach to living committed to transforming unjust social organizations of life. it presents a different epistemology of death, life, and care. importantly, the act of care is an inherently spatial matter. as mitchell, marston, and katz (2004, 417) explain, subjects are constituted both 'through time and in space'. this constitution is a care-full and social reproductive process, as 'how we live in space' is how we maintain and reproduce ourselves and communities (2004, 418) . massey's (2005, 10-11) explanation of space is helpful, arguing that space is a 'sphere of possibility' always in process. taken together, space is a sphere of making, caring, reproducing, and creating new. thus, a palliative temporality is also spatial; it can be called palliative space-time (pst). as an ethic of care is both an indictment of present inequalities and a call for a future in which people thrive and are cared for through the entire life course, pst similarly has this dual temporality. by centering possibility, palliative space-time is a tool for evaluating unequal access to the conditions for thriving, as well as offering a different way forward. it focuses attention on ways to better value care and body work, new intergenerational living arrangements, more accessible designs in the built environment, and different forms of institutional care. it also requires attention and correctives to oppressions that foster premature death and means some people are not allowed to thrive or even reach old age. each of these changes center an ethic of care in approaching space-making and design. pst is, therefore, an analytic tool that foregrounds care, death, and futurity in analyses of health systems. it is a descriptor of the workings and implications of actions, systems, policies, and relations. it holds care and death at the centre of the analysis, in relation, tandem, and opposite to each other. thus, pst could be used to understand the implications and workings of a health care system in financial dire straits, as i will show here. or, one could use the concept to consider other aspects of globalised capitalism; after all, ahmed (2014, no page) reminds us that 'racial capitalism is a health system'. as a description of a state of existence, pst is a tool for shedding light on the unevenness of experiences and access to care, and the contradictions that have been built into the health system as it exists now. with the care-full principles of the palliative at its heart, pst centers a commitment to j o u r n a l p r e -p r o o f doing things differently in recognition of the failings of the status quo. this article explores legislation that has helped create those contradictions and fostered the increased unevenness. i employ pst as a descriptor of the impacts of this legislation, and a way to think through the near-death sphere of possibility of accessing health care under these conditions throughout the life course. applying a lens of pst reveals a complex relationship between care and death and decline at work at multiple scales. the qualities of pst are apparent at the national scale, shaping health care systems and access to care for the entire population through changes to the literal spaces that house provisions. death and care operate in contradictory ways at the scale of the community, as hospitals -community anchors -close or shift meaning and role in regions, cities, and rural areas. finally, pst describes the differential access to care individuals experience in life and in death. pst is useful for understanding the implications of those two legislative changes of the early 1980s: the callous palliative state of the health care system, with the care-full service of hospice. while more money is invested in death care, the simultaneous restructuring of medicare financing destabilizes an entire health care system. as a solution, cazdyn (2012, 163) explains that "reclaiming our own deaths, not in a suicidal way, but in terms of our emotional and political consciousness regarding death and dying, therefore is tied to the reclaiming of utopia". facing death as an important part of life that needs care-full attention, systemically, can pave the way for a more just health system. in sum, the health care system has shifted its orientation towards space and time, and this shift is rooted in a new approach to the life course and death. in tracing the impacts of these two changes in the rest of this article, i will show how these possible futures are j o u r n a l p r e -p r o o f animated by a dual logic of death. the legacies of 1982 and 1983 reveal the health care system, both in its care-full and carelessness, working in palliative space-time. expanding medicare: 1982 the two legislative changes i examine here are contradictory. one expanded the services medicare recipients had a right to receive, while the other was a change aimed at reigning in an out of control entitlement. the first change, in 1982, involved hospice care. hospice provides comfort care to the terminally ill, generally wherever the person calls home, be that a house, a nursing home, or a hospital bed (nhpco n.d.) . modern hospice began in the uk in the late 1940s, through the work of physician dame cicely saunders, a nurse-turned-doctor who opened the first modern hospice, st christopher's, in london in 1967 (clark 1998 ). in the 1960s, american nurse florence wald trained with saunders, and established the first hospice in the us in branford, ct in 1974, providing both home hospice and inpatient care (adams 2010) . over this same time, other hospice programs, largely volunteer-driven, began to appear across the country (buck 2011; freudenheim 1983) . hospice is a philosophy of care that foregrounds the patient's goals and values. saunders and other hospice pioneers' goal was for medicine and health care to approach death differently, as a part of life, not as a failure of a body or the medical profession, and to understanding it holistically, involving mind, body, community, and family, and attending to a patient's 'total pain' (clark 2007; livne 2014) . in hospice, the patient is more than themselves, because 'although death [is] a solidary event, hospice advocates insisted it must not be lonely' (abel 2013, 169) . (buck 2011; rich 1983c; 1983a) . in 1986, congress made coverage permanent and expanded hospice funding. the inclusion of hospice allowed medicare patients to access expanded services and benefits, such as prescription drugs not usually covered under the program. though most major insurance companies had some sort of hospice provision by 1982 (torrens 1985) , the inclusion of hospice in medicare was a significant step in standardizing and legitimizing it as a way of care for those at the end of life (quinn 1983 ). this enacted a new ethic of care by establishing hospice as an insurable service. contracting medicare: 1983 j o u r n a l p r e -p r o o f the second legislative change of the early 1980s constituted a contraction of the services, justified by the reagan administration claiming that medicare could be bankrupt by 1990. when created in 1965, medicare needed buy-in from physicians and hospitals in order to succeed. partly to win support from a hostile medical establishment, the program's fee structure paid well. as starr explained, the medical profession quickly learned ' [medicare] was a bonanza' (1982, 370) . the federal government also 'agreed to rules for calculating [reimbursement] costs that were extremely favorable to the hospital industry ' (1982, 375) . for its first 18 years, medicare reimbursed providers retrospectively for the actual cost of care according to a test of 'reasonable cost', meaning medicare and medicaid paid hospitals very well (guterman and dobson 1986; stevens and stevens 2007, 124) . welfare programs have always first served the worthy poor, a population seen by state and society as worthy of assistance, compared to other populations deemed lazy or otherwise unsympathetic, generally according to racialized and gendered norms (garland 2014; piven and cloward 1993) . central to this categorizing, piven and cloward explain, is welfare's role in encouraging people into the workforce as the market demands through denials or granting of benefits, plus the role of employer-provided health insurance (thomasson 2002) . medicare and medicaid are major parts of the welfare state apparatus. they provide patients with vital access to care and funnel financial support to providers through reimbursements. they also operate like other welfare programs: providing (selective) support while disciplining recipients. developed out of decades-long struggles over national health coverage, legislators designed these programs for uneven access (starr 1982; stevens and stevens 2007; quadagno 2004) . by the 1960s, 'the aged could be presumed both needy and deserving, and the contributory nature of social security gave the entire program legitimacy' (starr 1982, 368) . for medicare and medicaid, this includes certain poor j o u r n a l p r e -p r o o f people, people who qualify as disabled, and retired people who already made their societal contributions through a lifetime of work or raising children/future workers. therefore, while many welfare programs regulate people into the workforce, medicare is a benefit for after the workforce. it is a universal program, granted after a lifetime of labor, with age as the only condition for qualifying, and no conditions for kicking one off the rolls. medicare and medicaid in the early 1980s. over this first 15 years of the programs, health care costs grew not only from inflation, but at a rate beyond inflation (pear 1982b; schwartz 1982; waldholz 1982; merry and schorr 1983) . some of this expense came from improvements in health care which includes more expensive procedures and equipment. some of this increased expense, claimed health care economists and legislators, also came from a fee structure that did not encourage cost controls (for example, see coverage by rich 1982) . as stevens and stevens (2007, 117) argue, while medicare grew into a widely accepted program, medicaid remained unable 'to escape the debilitating effect of its welfare parentage'. the introduction of prospective payments for medicare came after reagan's administration had already cut billions from america's suite of welfare programs. while medicare was and remains wildly popular (norton, dijulio, and brodie 2015) , the financial strain on the program made it into a potentially untenable luxury and justified it as a problem that needed solving. as part of tefra, congress implemented some 'interim changes to the medicare reimbursement system' (guterman and dobson 1986) (pear 1982a ). this was a decidedly different purpose for medicare payments. the new payment scheme created set amounts to reimburse providers for every health care product (services, inpatient stays, and physical products). rather than billing for the cost incurred, the pps set per-patient and per-day billing rates as well as a scheme for the reimbursement price for each service or product. rates are set through calculations based on a standardized payment, the area's wage index, and a diagnosis-related group (drg) classification system, plus variations for urban and rural contexts and regional differences. guterman and dobson (1986, 99) identify an important characteristic of pps: "each hospital keeps, or loses, the difference between the payment rate and its cost for that unit of care." in other words, the program expected providers to pick up any slack. this is the same payment structure medicare uses today. from the beginning, these legislative programs contradicted each other, constituting an expansion and contraction in care. the months the federal government spent working out the details of the hospice benefits implementation reveal the challenges of expanding care access in an era overall hostile to public services. the hospice benefit proved contentious immediately after its passage. in the preparations for the hospice benefit, a dispute ensued j o u r n a l p r e -p r o o f between the members of congress who sponsored the bill and congressional budget office (cbo) over the rates at which providers were to be reimbursed. when passing the bill, the house ways and means committee set a payment ceiling of $7600 per patient (rich 1983c stockman, head of the omb, argued that claims that hospice would save money were misleading, as the program would actually cost the government hundreds of millions of dollars in its pilot phase (rich 1983a) , something that would be deeply unpopular given the panic around health care costs (pear 1982b) . lawmakers argued that lowering the rate was a violation of the agreement reached in the initial legislation and would cripple the program (rich 1983c; 1983b) . ultimately, while hhs did set a lower rate than what lawmakers initially wanted, the final payout cap was much closer to the original figure through a series of compromises and amendments. these early months of the program demonstrate not only that the 1980s laws were contradictory, but also reveal the complex financial role of hospice and the trouble with the dominant ways of valuing health care. when health care is seen first and foremost as (too) expensive, the necessity of it takes a back seat. from these rocky early days emerged a landmark program as 'tefra has remained the most central piece of us hospice legislation' (livne 2014, 898) , fueling the expansion and standardization of hospice services. after congress made the hospice benefit permanent in j o u r n a l p r e -p r o o f 1986, funding was expanded in 1989 (nhpco n.d.) . by the mid 1990s when clinton was attempting to reform health care, hospice was taken as an integral part of health care (nhpco n.d.) . since tefra, hospice has become a more standardized set of services with clear criteria for when patients can or should access the care (buck 2011; livne 2014) . for example, carney et al (1989) found that, with the implementation of the medicare hospice benefit, patients received more care from registered nurses, less care from volunteers, and patients accessed care when they were sicker, which they credited to the six-month provision. the standardization-via-medicare took time. uptake of certification was slow in the early years of the program, with only 20% of eligible hospices becoming certified (buck 2011, s41) . at tefra's passage, hundreds of hospices were operating in the us but few received medicare benefits; for facilities to qualify, 'major reorganization will be necessary to conform with the centralized administration required by the congressional act' (los angeles times 1983). as the washington post reported, interviewing john j. mahoney, president of the national hospice organization, "some hospices say medicare rates are too low to justify going through the difficult certification process. others, he said are too small to justify the costs and trouble of certification," and others were waiting to see if the program would exist after 1986 (rich 1985 ; see also buck 2011) . over the 1980s, and especially after the program became permanent part of medicare in 1986, more programs opted to become medicare certified and funding increased (davis 1988; gilinsky 1988) . the numbers of hospices both in general and medicare-certified have grown at a rate of 10-20% each year since tefra (livne 2014, 897-98) . in 1985, hospice became reimbursable under medicaid as well, and now, all states j o u r n a l p r e -p r o o f include the benefit (kaiser family foundation 2019). by the mid 1990s, most commercial health insurance plans included coverage for hospice care (buck 2011, s41) . annually, government spending on the medicare hospice benefit has grown 5-6% annually (medpac 2015, 325) . as of 2016, medicare has become the largest payer in the country, paying for 90% of hospice patient care days. hospice was attractive to lawmakers in 1982 for its potential to save medicare money (bayer 1983; greenberg 1983) . cost effectiveness was based on patients staying at home, in (powers et al. 2015; zuckerman, stearns, and sheingold 2016; medpac 2015) . the fact of savings may be less important than the belief that hospice care is cost effective. as livne's (2014) study of hospice in california shows, the belief in thrifty hospice agencies providing low-cost care is a powerful force shaping how providers care for patients and families. regardless of cost savings, hospice is of growing importance as the population ages. the blunt truth is that a lot of people will die in old age over the next few decades. this is, therefore, an important social and cultural moment in both ageing and the end stages of the j o u r n a l p r e -p r o o f life course. as a significant portion of the population ages and encounters the end stages of life, new questions emerge with urgency: what care is needed at the end of life? how can the system ensure access to such care? as hospice has grown over the past 30 years, more spaces have become palliative. homes take on new caring roles, and new spaces specifically designated for palliative care open (brown 2003; turner et al. 2016) . legislative and financial support for hospice care opened up new possibilities spatially, temporally, politically and economically for relationships to death that foreground care and attention to death. the prospective payment system reflects the other side of the palliative space-time paradox. following 1983, the system expanded over the next two decades to cover physician fees as well as skilled nursing and long-term care facilities (the washington post 1989; wodchis, fries, and hirth 2004) . nearly every state uses a prospective payment system for medicaid reimbursements. recall that financial instability is the major driver behind hospital closures. early on, pps impacted hospital finances (phillips 1984) . in 1984, the first full year of the program, 18% of all hospitals saw a loss of revenue, while 44% of for-profit hospitals saw revenues increase (guterman and dobson 1986) . this means that public and non-profit hospitals, which generally serve deprived communities, felt the impacts of pps more. facilities serving vulnerable populations became more vulnerable. additionally, with pps hospitals found it challenging 'to pass along the costs of free care to the medically indigent' (mclafferty 1986 (mclafferty , 1080 . by its own accounting, the federal government states that pps was partially responsible for many rural hospital closures in the mid-1980s (cbo 1991). as well, facilities serving a high proportion of medicare patients were more vulnerable to closure over the j o u r n a l p r e -p r o o f 1980s (williams, hadley, and pettengill 1992) . pps is not the sole cause of closures, but it is an essential structural factor in their instability. most hospitals operate on break even budgets. approximately one third operate on negative profit margins (aha 2018). hospitals are dependent on a fee structure that does not ensure financial stability. a hospital's revenue comes predominantly from insurance reimbursements. but hospitals serve patients with a mix of insurance -private plans, government-provided insurance, and no coverage. hospitals depend on having a payer mix that earns enough revenue to stay open and provide care to everyone. privately insured patients are key to this, as their plans pay well. a closer look at the breakdown of payments is instructive. over 60% of all care hospitals provide is covered by medicare and medicaid association (aha 2019). while private insurers generally overpay at a rate of 144%, medicare and medicaid generally underpay by 10%, sometimes more (cunningham et al. 2016) . medicare patients are both desirable (they have reliable insurance) and undesirable (medicare does not reimburse well enough). medicare is thus integral to a facility's survival but also risky. then there are the patients who are uninsured and cannot pay for the care they receive. though hospitals try (aggressively) to recoup those costs, they are left to eat the expenses. it is commonly assumed that hospitals cost shift shortfalls from other patients on to private insurers (frakt 2011) . facilities facing financial shortfalls actually do not rely on cost shifting, but instead cut staffing and operating costs per patient (hadley et al. 1987; white and wu 2014) . as white and wu (2014, 28) explain, "over the long run, medicare price cuts do not result in hospitals shifting costs to other payers or more profitable services; they instead constrain overall operations and resource use". without enough insurance reimbursements, and especially from private j o u r n a l p r e -p r o o f insurance, hospitals may not stay open. in addition, not only is a hospital's very existence at risk, but also, quality of care and labor conditions directly bear the brunt of the payment scheme's impacts of a facility's struggle to balance the budget. spaces of care thus change or disappear. pps has implications for access to care that extend beyond hospitals, shaping access to care in multiple ways. for example, long-term care facilities often limit the number of medicare and medicaid patients they will accept, reserving more beds for people who can pay better through private and supplemental insurance (mor et al. 2004; smith et al. 2007 ). medicaid, which uses a pps, is the largest funder of long-term care, creating another fragility in the system providing care for older people and those with chronic health needs. echoing cazdyn, the health care system is stuck in palliative space-time. applying the lens of pst shows a geography of pps impacts as deadly to systems and spaces of care. in the wake of the legislative changes, the health care system is permeated by death and possibility. hospice care is expanding, increasing access to comfort care for dying people and their families. the role of hospitals in the health care system is shifting, from improvements in care, pressures from insurance companies, and increased financial precarity. rather than stabilize medicare, the pps structure maintains the status quo of uneven and precarious access to services and an increasingly uneven geography of services. this continued with the affordable care act, implemented in 2014: importantly, the program j o u r n a l p r e -p r o o f slowed the rise of health care costs and gave people greater access to health care through insurance regulations and expansions to medicaid. yet while hospitals saw net gains, especially in states that expanded medicaid access, the kaiser family foundation (cunningham et al. 2016) has reported that hospitals expect changes to medicaid policy and the high volume of medicaid payments to actually offset some of those gains. increased reliance on a system that reimburses poorly is not a prescription for good financial health. instead, applying a pst lens, the system itself is dying but never allowed to die and not healthy enough to ensure sufficient care for all. this deterioration takes on additional meaning as demographics change. with more people aging, more people will both qualify for medicare and need more care. this means a growing number of medicare patients accessing chronic and hospice care. the demographic and political economic contexts together bring connections between generations, spaces of care, and health care provisions into sharp relief. while hospice is not causing the closure of hospitals, these systems are far from separate, tied together through medicare. this interconnection is apparent when considering intergenerationality in the health care system. the 1980s have afterlives, creating a special relationship between the care of a select population and the care needs of the entire population. a logic of death and dying connect these two concerns. because of the importance of medicare to the financial wellbeing of hospitals, an entire system becomes dependent on a health care system for a single age group. there is an intergenerational dependence (society, health care facilities that everyone uses) on one group of people (seniors) because of their access to a specific program (medicare). a program for some supports an entire system for all; medicare has become the program keeping spaces of health care open and precarious. as the us j o u r n a l p r e -p r o o f population ages, the number of people over 65 -and beneficiaries of medicare -is expected to reach 84 million by 2050 (kaiser family foundation 2017). the financial implications of pps and any changes to it will become even more important. while access to health care in the us has never been universally guaranteed, the death that pervades the health care system destabilizes the entire system. pst helps name the important expansion of care but also the structures that destabilize care for all, holding them together in the same frame. importantly, while the remaking of health care spaces threatens everyone's accesseven those with private insurance -it is most acutely felt by those on the margins: people unable to drive great distances to the next nearest hospital, people without cars whose neighborhoods lack transit, people who suffer heart attacks and wait an hour for the ambulance to take them to the hospital an hour away, people already suffering in an antifeminist, racist, and anti-poor system. the precarity of the system means premature death and logics of pst permeate the health care system. pps makes life precarious until a person reaches 65, when they might have better access to good care at the end of life. the life course, though, unfolds in myriad ways, and people access and need chronic and death care at all ages. depending on a single demographic's program to support an entire system has significant implications beyond that demographic, especially when the general population that more care everywhere in an often-brutal socio-political economic system. the reach of pps is far, with the legislative changes of 1982 and 1983 working in tandem. as livne (2014, 901) hospitals are incentivized to send people home as soon as possible, but also face steep penalties from medicare for quick readmissions (henry 2018) . in contrast, hospices are also paid prospectively, but done so per diem, meaning a longer stay earns more money (increasing self-sufficiency). in other words, "the hospice ethic therefore converges with [hospice's] financial interests, hospital's financial interests, and the overall effort to reduce spending on end-of-life care" (2014, 902), as the last year is typically the most expensive of a person's life. death care is rewarded, while care at other times in life is rolled back and made precarious or harder to access. pps, as understood through pst, is a program that no longer cures ills in the health system, but maintains a fragile and sick system, with communities losing hospitals -anchors of services, economic activity, and built environment. dying is, therefore, at the center of this remaking of health system, spatially, over the life course, and through death itself. instability in the system through financial precarity leads to instability of health care spaces and geographies; this in turn leads to greater instability in the health care system and loss of access to care, as what replaces hospitals is not always clear. importantly, the palliative is political, and placing death at the center of these politics is a contradictory move towards something new. the system is failing people, and as cazdyn argues, addressing the manifestations of palliative space-time head on in a radical way means forcing a change; this might be risky, but the system is already deadly. the care needs and changing approaches to those specific needs are a twin transformation occurring in the health care system. hospice and its increasing importance over the past 40 years, represents the emancipatory and utopian side of palliative space-time -a reclaiming of death and life in a health system that centers death. alternatives and the potential for action are abundant. medicare recipients will grow to become a significant voting block in coming decades, as will the very politically engaged generation z. the sandwich generation of adults taking care of aging parents and children of their own have a stake building a robust and stable health system to support their own care pressures. increasing discontent and attention is turning to hospital closures, with communities protesting such closures, for example, the outrage over the closure of a hospital in brooklyn that culminated in its closure, but also legislation mandating better consideration of community and public health needs before allowing hospitals to shut (frost 2015) . as well, increased media coverage of the impacts of closures is increasing, for example, with kaiser health news's series on the a small kansas town coping with the loss of their hospital (tribble 2019) . furthermore, fed up with the growing care deficit and expense of the health system, public opinion towards universal health care has shifted dramatically in the past ten years. each of these trends have great potential to create more just health systems, making it all the more important to understand the implications of a health program's financial structure. in many ways, these changes to the health care arm of the welfare state do not represent a new process. the hospital system has undergone a series of downsizings since hitting its peak after the passage of the hill-burton act in 1946, the federal funding program that poured money into building more hospitals across the country in the post-war era. geographer sara mclafferty (1986) demonstrated that the wave of hospital closures from the late 1960s to the mid 1980s most impacted smaller facilities serving communities of lower socio-economic status. uneven access is always filtered through difference. this is apparent in the impacts deindustrialization had on public long-term care (winant 2018) . as well, closures of the 1960s-1980s must be understood in the context of integration, or the period when nearly all black hospitals closed (sanford iii 2012) . legacies and patterns of uneven access to care and good health persist. given this history, what is special about the changes in the early 1980s? the changes of 1982 and 1983 signify a movement towards a systematic death drive, towards operating in palliative space-time for the health care system. it is a systemic shift in the system towards a form of care with necropolitical goals -focused on not just making live, letting die, and making die at the level of individual and population, but also at the health care system. palliative space-time names not only a withdrawal of services or an increase of stress on caregivers. it describes a shaking of the entire system through closures of physical locations of care, but with the potential to remake the system out of the ruins of closed facilities. for what replaces a hospital could be something wholly different and just. systems and practices of care change, evolve, improve. this article ultimately is arguing for critically centering the impacts of such changes. what happens in a hospital's wake? where do people receive care? where do workers work? what does the loss of tax dollars, a space of care, and an anchor of community mean for workers, communities, economies, politics, health care systems and provisioning? and how does a payment structure reveal what people actually value? centering these questions might open up a way towards utopia, or at least a more just and equitable health care system. the inevitable hour: a history of caring for dying patients in america dying with dignity in america: the transformational leadership of florence wald feministkilljoys (blog) america's rural hospitals are dangerously fragile trendwatch chartbook 2018: trends affecting hospitals and health systems care of the body: spaces of practice forces of change: the future of health how do you foster 'a good death in a racist society hospice and the spatial paradoxes of terminal care how far to the hospital? policy and the reformation of hospice: lessons from the past for the future of palliative care the impact of reimbursement: the case of hospice the already dead: the new time of politics, culture, and illness. durham and london originating a movement: cicely saunders and the development of st christopher's hospice, 1957-1967 rural hospitals and medicare's prospective payment system understanding medicaid hospital payments and the impact of recent policy changes, issue brief 8884 medicare hospice benefit: early program experiences health care and urban revitalization: a historical overview nhs hospital bed numbers how much do hospitals cost shift? a review of the evidence: how much do hospitals cost shift? ageism and covid-19: what does our society's response say about us? hospices for the dying show dramatic growth ny assembly passes 'lich act' hospital bill the welfare state: a fundamental dimension of modern government hospice concept gains in county 5 ways u.s. hospitals can handle financial losses from medicare patients are we squandering millions on the dying? impact of the medicare prospective payment system for hospitals hospital closures: the sociospatial restructuring of labor and health care five southern ontario hospitals to be closed; replaced with acute-care centre hospital closures had no measurable impact on local hospitalization rates or mortality rates, 2003-11 10 essential facts about medicare's financial outlook medicaid benefits: hospice care the effects of hospice coverage on medicare expenditures the future of home health care: a strategic framework for optimizing value economies of dying: the moralization of economic scarcity in u.s. hospice care compassion in medicare hospital profitability sinks to levels not seen since the financial crisis: moody's for space the geographical restructuring of urban hospitals: spatial dimensions of corporate strategy medicare payment advisory commission (medpac) medicare fund crisis may spur competition in health-care fields life's work: geographies of social reproduction driven to tiers: socioeconomic and racial disparities in the quality of nursing home care medicare and medicaid at 50 hospice use and end-of-life spending trajectories in medicare beneficiaries on hemodialysis us moving to fix uniform fee scale on medicare cases medicare's new limits on hospital payments force wide cost cuts regulating the poor: the functions of public welfare the age of dignity: preparing for the elder boom in a changing america cost savings associated with expanded hospice use in medicare matters of care in technoscience: assembling neglected things why the united states has no national health insurance: stakeholder mobilization against the welfare state hospice care getting official recognition from the government financing end-of-life care in the usa hhs to cut payment rate on new hospice program long-term trends in medicare payments in the last year of life re-thinking the 'balck hospital': race, community, and healing in the jim crow and contemporary eras can the us make hospital costs go down? separate and unequal: racial segregation and disparities in quality across u.s. nursing homes evidence on the cost and cost-effectiveness of palliative care: a literature review the social transformation of american medicine the american hospital as a social institution: past is prologue (1986) anatomy of a dilemma: creating and implementing medicaid, 1965-1970 the order of the good death. n.d medicare and the hospices poor health: poverty and scarce resources in us cities from sickness to health: the twentieth-century development of u.s. health insurance hospice care: what have we learned? dealing with hospital closure, pioneer kansas town asks: what comes next?" kaiser health news (blog) towards a feminist theory of caring caring for a dying spouse at the end of life: 'it's one of the things you volunteer for when you get married': a qualitative study of the oldest carers' experiences hospitals back plan to limit medicare cost how do hospitals cope with sustained slow growth in medicare prices? profits, community role, and hospital closure: an urban and rural analysis a place to die: nursing home abuse and the political economy of the 1970s the effects of medicare's prosepctive payment system on discharge outcomes of skilled nursing facility residents hospice use, hospitalization, and medicare spending at the end of life palliative space-time: expanding and contracting geographies of us health care key: cord-025927-caoklx9l authors: kizza, joseph migga title: introduction to computer network vulnerabilities date: 2020-02-07 journal: guide to computer network security doi: 10.1007/978-3-030-38141-7_4 sha: doc_id: 25927 cord_uid: caoklx9l system vulnerabilities are weaknesses in the software or hardware on a server or a client that can be exploited by a determined intruder to gain access to or shut down a network. vulnerabilities exist not only in the hardware and software that constitute a computer system but also in policies and procedures, especially security policies and procedures, that are used in a computer network system and in users and employees of the computer network systems. vulnerabilities can be found in so many areas in a network system and are indeed anything in a computer network that has the potential to cause or be exploited for an advantage. this chapter explores the sources and assessment of a computer network system’s vulnerabilities. poor security management, incorrect implementation, internet technology vulnerability, the nature of intruder activity, the difficulty of fixing vulnerable systems, the limits of the effectiveness of reactive solutions, and social engineering [2] . the two major components of a computer system, hardware and software, quite often have design flaws. hardware systems are less susceptible to design flaws than their software counterparts owing to less complexity, which makes them easier to test; a limited number of possible inputs and expected outcomes, again making it easy to test and verify; and the long history of hardware engineering. but even with all these factors backing up hardware engineering, because of the complexity in the new computer systems, design flaws are still common. however, the biggest problems in system security vulnerability are due to software design flaws. a number of factors cause software design flaws, including overlooking security issues altogether. however, three major factors contribute a great deal to software design flaws: human factors, software complexity, and trustworthy software sources [3] . in the human factor category, poor software performance can be a result of the following: 1. memory lapses and attentional failures: for example, someone was supposed to have removed or added a line of code, tested, or verified, but did not because of simple forgetfulness. 2. rush to finish: the result of pressure, most often from management, to get the product on the market either to cut development costs or to meet a client deadline, can cause problems. 3. overconfidence and use of nonstandard or untested algorithms: before algorithms are fully tested by peers, they are put into the product line because they seem to have worked on a few test runs. 4. malice: software developers, like any other professionals, have malicious people in their ranks. bugs, viruses, and worms have been known to be embedded and downloaded in software, as is the case with trojan horse software, which boots itself at a timed location. as we will see in sect. 8.4, malice has traditionally been used for vendetta, personal gain (especially monetary), and just irresponsible amusement. although it is possible to safeguard against other types of human errors, it is very difficult to prevent malice. 5. complacency: when either an individual or a software producer has significant experience in software development, it is easy to overlook certain testing and other error control measures in those parts of software that were tested previously in a similar or related product, forgetting that no one software product can conform to all requirements in all environments. both software professionals and nonprofessionals who use software know the differences between software programming and hardware engineering. in these differences underlie many of the causes of software failure and poor performance. consider the following: 1. complexity: unlike hardwired programming in which it is easy to exhaust the possible outcomes on a given set of input sequences, in software programming, a similar program may present billions of possible outcomes on the same input sequence. therefore, in software programming, one can never be sure of all the possibilities on any given input sequence. 2. difficult testing: there will never be a complete set of test programs to check software exhaustively for all bugs for a given input sequence. 3. ease of programming: the fact that software programming is easy to learn encourages many people with little formal training and education in the field to start developing programs, but many are not knowledgeable about good programming practices or able to check for errors. 4. misunderstanding of basic design specifications: this affects the subsequent 5. design phases, including coding, documenting, and testing. it also results in improper and ambiguous specifications of major components of the software and in ill-chosen and poorly defined internal program structures. there are thousands of software sources for the millions of software products on the market today. however, if we were required to name well-known software producers, very few of us would succeed in naming more than a handful. yet we buy software products every day without even ever minding their sources. most importantly, we do not care about the quality of that software, the honesty of the anonymous programmer, and, of course, its reliability as long as it does what we want it to do. even if we want to trace the authorship of the software product, it is impossible because software companies are closed within months of their opening. chances are when a software product is 2 years old, its producer is likely to be out of business. in addition to the difficulties in tracing the producers of software who go out of business as fast as they come in, there is also fear that such software may not even have been tested at all. the growth of the internet and the escalating costs of software production have led many small in-house software developers to use the marketplace as a giant testing laboratory through the use of beta testing, shareware, and freeware. shareware and freeware have a high potential of bringing hostile code into trusted systems. for some strange reason, the more popular the software product gets, the less it is tested. as software products make market inroads, their producers start thinking of producing new versions and releases with little to no testing of current versions. this leads to the growth of what is called a common genesis software product, where all its versions and releases are based on a common code. if such a code has not been fully tested, which is normally the case, then errors are carried through from version to version and from release to release. in the past several years, we have witnessed the growth of the open-source movement. it has been praised as a novel idea to break the monopoly and price gouging by big software producers and, most importantly, as a timely solution to poor software testing. those opposed to the movement have criticized it for being a source of untrusted and many times untested software. despite the wails of the critics, major open-source products such as linux operating system have turned out with few security flaws; still, there are fears that hackers can look at the code and perhaps find a way to cause mischief or steal information. there has been a rise recently in trojan horses inserted into open-source code. in fact, security experts are not recommending running readily available programs such as md5 hashes to ensure that the code has not been altered. using md5 hashes and similar programs such as md4, sha, and sha-1 continually compares codes generated by "healthy" software to hashes of programs in the field, thus exposing the trojans. according to the recent cert advisory, crackers are increasingly inserting trojans into the source code for tcpdump, a utility that monitors network traffic, and libpcap, a packet capture library tool [4] . however, according to the recent study by the aberdeen group, open-source software now accounts for more than half of all security advisories published in the past year by the computer emergency response team (cert). also according to industry study reports, open-source software commonly used in linux, unix, and network routing equipment accounted for 16 of the 29 security advisories during the first 10 months of 2002, and there is an upswing in new virus and trojan horse warnings for unix, linux, mac os x, and open-source software [4] . new developments in software engineering are spearheading new developments such as software reuse and software reengineering. software reuse is the integration and use of software assets from a previously developed system. it is the process in which old or updated software, such as library, component, requirements and design documents, and design patterns, is used along with new software. both software reengineering and reuse are hailed for cutting down on the escalating development and testing costs. they have brought efficiency by reducing time spent designing or coding, popularized standardization, and led to common "lookand-feel" between applications. they have made debugging easier through the use of thoroughly tested designs and code. however, both software techniques have the potential to introduce security flaws in systems. among some of the security flaws that have been introduced into programming is first the mismatch where reused requirement specifications and designs may not completely match the real situation at hand and nonfunctional characteristics of code may not match those of the intended recipient. second, when using object programming, it is important to remember that objects are defined with certain attributes, and any new application using objects defined in terms of the old ones will inherit all their attributes. in chap. 6, we will discuss the many security problems associated with script programming. yet there is now momentum in script programming to bring more dynamism into web programming. scripting suffers from a list of problems, including inadequate searching and/or browsing mechanisms before any interaction between the script code and the server or client software, side effects from software assets that are too large or too small for the projected interface, and undocumented interfaces. security management is both a technical and an administrative security process that involves security policies and controls that the organization decides to put in place to provide the required level of protection. in addition, it also involves security monitoring and evaluation of the effectiveness of those policies. the most effective way to meet these goals is to implement security risk assessment through a security policy and secure access to network resources through the use of firewalls and strong cryptography. these and others offer the security required for the different information systems in the organization in terms of integrity, confidentiality, and availability of that information. security management by itself is a complex process; however, if it is not well organized, it can result in a security nightmare for the organization. poor security management is a result of little control over security implementation, administration, and monitoring. it is a failure in having solid control of the security situation of the organization when the security administrator does not know who is setting the organization's security policy, administering security compliance, and who manages system security configurations and is in charge of security event and incident handling. in addition to the disarray in the security administration, implementation, and monitoring, a poor security administration team may even lack a plan for the wireless component of the network. as we will see in chap. 17, the rapid growth of wireless communication has brought with it serious security problems. there are so many things that can go wrong with security if security administration is poor. unless the organization has a solid security administration team with a sound security policy and secure security implementation, the organization's security may be compromised. an organization's system security is as good as its security policy and its access control policies and procedures and their implementation. good security management is made up of a number of implementable security components that include risk management, information security policies and procedures, standards, guidelines, information classification, security monitoring, and security education. these core components serve to protect the organization's resources: • a risk analysis will identify these assets, discover the threats that put them at risk, and estimate the possible damage and potential loss a company could endure if any of these threats become real. the results of the risk analysis help management construct a budget with the necessary funds to protect the recognized assets from their identified threats and develop applicable security policies that provide direction for security activities. security education takes this information to each and every employee. • security policies and procedures to create, implement, and enforce security issues that may include people and technology. • standards and guidelines to find ways, including automated solution for creating, updating, and tracking compliance of security policies across the organization. • information classification to manage the search, identification, and reduction of system vulnerabilities by establishing security configurations. • security monitoring to prevent and detect intrusions, consolidate event logs for future log and trend analysis, manage security events in real time, manage parameter security including multiple firewall reporting systems, and analyze security events enterprise-wide. • security education to bring security awareness to every employee of the organization and teach them their individual security responsibility. incorrect implantation very often is a result of incompatible interfaces. two product modules can be deployed and work together only if they are compatible. that means that the module must be additive, that is, the environment of the interface needs to remain intact. an incompatible interface, on the other hand, means that the introduction of the module has changed the existing interface in such a way that existing references to the interface can fail or behave incorrectly. this definition means that the things we do on the many system interfaces can result in incompatibility that results in bad or incomplete implementation. for example, ordinary addition of a software or even an addition or removal of an argument to an existing software module may cause an imbalanced interface. this interface sensitivity tells us that it is possible because of interposition that the addition of a simple thing like a symbol or an additional condition can result in an incompatible interface, leading the new symbol or condition to conflict with all applications that have been without problems. to put the interface concept into a wide system framework, consider a systemwide integration of both hardware and software components with differing technologies with no standards. no information system products, whether hardware or software, are based on a standard that the industry has to follow. because of this, manufacturers and consumers must contend with the constant problems of system compatibility. because of the vast number of variables in information systems, especially network systems, involving both hardware and software, it is not possible to test or verify all combinations of hardware and software. consider, for example, that there are no standards in the software industry. software systems involve different models based on platforms and manufacturers. products are heterogeneous both semantically and syntactically. when two or more software modules are to interface one another in the sense that one may feed into the other or one may use the outputs of the other, incompatibility conditions may result from such an interaction. unless there are methodologies and algorithms for checking for interface compatibility, errors are transmitted from one module into another. for example, consider a typical interface created by a method call between software modules. such an interface always makes assumptions about the environment having the necessary availability constraints for the accessibility of local methods to certain states of the module. if such availability constraints are not checked before the modules are allowed to pass parameters via method calls, errors may result. incompatibility in system interfaces may be caused by a variety of conditions usually created by things such as: • too much detail • not enough understanding of the underlying parameters • poor communication during design • selecting the software or hardware modules before understanding the receiving software • ignoring integration issues • error in manual entry many security problems result from the incorrect implementation of both hardware and software. in fact, system reliability in both software and hardware is based on correct implementation, as is the security of the system. in sect. 4.2.1, we discussed design flaws in technology systems as one of the leading causes of system vulnerabilities. in fact, we pointed out that systems are composed of software, hardware, and humanware. there are problems in each one of these components. since the humanware component is influenced by the technology in the software and hardware, we will not discuss this any further. the fact that computer and telecommunication technologies have developed at such an amazing and frightening speed, and people have overwhelmingly embraced both of them, has caused security experts to worry about the side effects of these booming technologies. there were reasons to worry. internet technology has been and continues to be vulnerable. there have been reports of all sorts of loopholes, weaknesses, and gaping holes in both software and hardware technologies. according to the national vulnerability database (nvd), a us government repository of standards-based vulnerability management data using the security content automation protocol (scap), system vulnerabilities have been on the rise ever since system vulnerability data was first captured. the system vulnerability data captured by nvd enables automation of vulnerability management, security measurement, and compliance. nvd includes databases of security checklists, security-related software flaws, misconfigurations, product names, and impact metrics. read more about nvd at https://nvd.nist.gov/home.cfm. there is agreement among security experts that what is reported represents the tip of the iceberg. many vulnerabilities are discovered and, for various reasons, are not reported. because these technologies are used by many who are not security experts (in fact the majority of users are not security literate), one can say that many vulnerabilities are observed and probably not reported because those who observe them do not have the knowledge to classify what has been observed as a vulnerability. even if they do, they may not know how and where to report. no one knows how many of these vulnerabilities there are in both software and hardware. the assumption is that there are thousands. as history has shown us, a few are always discovered every day by hackers. although the list spans both hardware and software, the problem is more prevalent with software. in fact, software vulnerabilities can be put into four categories: • operating system vulnerabilities: operating systems are the main sources of all reported system vulnerabilities. going by the sysadmin, audit, network, and security (sans) institute, a cooperative research and education organization serving security professionals, auditors, system administrators, and network administrators, together with the common weakness enumeration (cwe), a community-developed dictionary of weaknesses of software types, has been issuing lists annually: "cwe/sans top 25 most dangerous software errors." popular operating systems cause many of the vulnerabilities. this is always so because hackers tend to take the easiest route by exploiting the best-known flaws with the most effective and widely known and available attack tools. • port-based vulnerabilities: besides operating systems, network service ports take second place in sourcing system vulnerabilities. for system administrators, knowing the list of most vulnerable ports can go a long way to help enhance system security by blocking those known ports at the firewall. such an operation, though not comprehensive, adds an extra layer of security to the network. in fact, it is advisable that in addition to blocking and deny-everything filtering, security administrators should also monitor all ports, including the blocked ones for intruders who entered the system by some other means. for the most common vulnerable port numbers, the reader is referred to the latest sans at: https://www. sans.org/security-resources/idfaq/which-backdoors-live-on-which-ports/8/4. • application software-based errors. • system protocol software such as client and server browser. in addition to highlighting the need for system administrators to patch the most common vulnerabilities, we hope this will also help many organizations that lack the resources to train security personnel to have a choice of either focusing on the most current or the most persistent vulnerability. one would wonder why a vulnerability would remain among the most common year after year, while there are advisories on it and patches for it. the answer is not very farfetched, but simple: system administrators do not correct many of these flaws because they simply do not know which vulnerabilities are most dangerous; they are too busy to correct them all, or they do not know how to correct them safely. although these vulnerabilities are cited, many of them year after year, as the most common vulnerabilities, there are traditionally thousands of vulnerabilities that hackers often use to attack systems. because they are so numerous and new ones are being discovered every day, many system administrators may be overwhelmed, which may lead to loss of focus on the need to ensure that all systems are protected against the most common attacks. let us take stock of what we have said so far. lots and lots of system vulnerabilities have been observed and documented by sans and cwe in their series, "cwe/ sans top 25 most dangerous software errors." however, there is a stubborn persistence of a number of vulnerabilities making the list year after year. this observation, together with the nature of software, as we have explored in sect. 4.2.1, means it is possible that what has been observed so far is a very small fraction of a potential sea of vulnerabilities; many of them probably will never be discovered because software will ever be subjected to either unexpected input sequences or operated in unexpected environments. besides the inherently embedded vulnerabilities resulting from flawed designs, there are also vulnerabilities introduced in the operating environments as a result of incorrect implementations by operators. the products may not have weaknesses initially, but such weaknesses may be introduced as a result of bad or careless installations. for example, quite often, products are shipped to customers with security features disabled, forcing the technology users to go through the difficult and errorprone process of properly enabling the security features by oneself. it is ironic that as "useful" technology develops so does the "bad" technology. what we call useful technology is the development in all computer and telecommunication technologies that are driving the internet, telecommunication, and the web. "bad" technology is the technology that system intruders are using to attack systems. unfortunately, these technologies are all developing in tandem. in fact, there are times when it looks like hacker technologies are developing faster than the rest of the technologies. one thing is clear, though: hacker technology is flourishing. although it used to take intelligence, determination, enthusiasm, and perseverance to become a hacker, it now requires a good search engine, time, a little bit of knowledge of what to do, and owning a computer. there are thousands of hacker web sites with the latest in script technologies and hundreds of recipe books and sources on how to put together an impact virus or a worm and how to upload it. the ease of availability of these hacker tools; the ability of hackers to disguise their identities and locations; the automation of attack technology which further distances the attacker from the attack; the fact that attackers can go unidentified, limiting the fear of prosecution; and the ease of hacker knowledge acquisition have put a new twist in the art of hacking, making it seem easy and hence attracting more and younger disciples. besides the ease of becoming a hacker and acquiring hacker tools, because of the internet sprawl, hacker impact has become overwhelming, impressive, and more destructive in shorter times than ever before. take, for example, recent virus incidents such as the "i love you," "code red," "slammer," and the "blaster" worms' spread. these worms and viruses probably spread around the world much faster than the human cold virus and the dreaded severe acute respiratory syndrome (sars). what these incidents have demonstrated is that the turnaround time, the time a virus is first launched in the wild and the time it is first cited as affecting the system, is becoming incredibly shorter. both the turnaround time and the speed at which the virus or a worm spreads reduce the response time, the time a security incident is first cited in the system, and the time an effective response to the incident should have been initiated. when the response time is very short, security experts do not have enough time to respond to a security incident effectively. in a broader framework, when the turnaround time is very short, system security experts who develop patches do not have enough time to reverse engineer and analyze the attack in order to produce counter immunization codes. it has been and it is still the case in many security incidents for antivirus companies to take hours and sometimes days, such as in the case of the code red virus, to come up with an effective cure. however, even after a patch is developed, it takes time before it is filtered down to the system managers. meantime, the damage has already been done, and it is multiplying. likewise, system administrators and users have little time to protect their systems. in his testimony to the subcommittee on government efficiency, financial management, and intergovernmental relations of the us house committee on government reform, richard d. pethia, director, cert centers, pointed out the difficulty in fixing known system vulnerabilities as one of the sources of system vulnerabilities. his concern was based on a number of factors, including the everrising number of system vulnerabilities and the ability of system administrators to cope with the number of patches issued for these vulnerabilities. as the number of vulnerabilities rises, system and network administrators face a difficult situation. they are challenged with keeping up with all the systems they have and all the patches released for those systems. patches can be difficult to apply and might even have unexpected side effects as a result of compatibility issues [2] . besides the problem of keeping abreast of the number of vulnerabilities and the corresponding patches, there are also logistic problems between the time at which a vendor releases a security patch and the time at which a system administrator fixes the vulnerable computer system. there are several factors affecting the quick fixing of patches. sometimes, it is the logistics of the distribution of patches. many vendors disseminate the patches on their web sites; others send e-mail alerts. however, sometimes busy system administrators do not get around to these e-mails and security alerts until sometime after. sometimes, it can be months or years before the patches are implemented on a majority of the vulnerable computers. many system administrators are facing the same chronic problems: the neverending system maintenance, limited resources, and highly demanding management. under these conditions, the ever-increasing security system complexity, increasing system vulnerabilities, and the fact that many administrators do not fully understand the security risks, system administrators neither give security a high enough priority nor assign adequate resources. exacerbating the problem is the fact that the demand for skilled system administrators far exceeds the supply [2] . going by daily reports of system attacks and hacks, the number of system attacks is steadily on the rise. however, a small percentage of all attacks are reported, indicating a serious and growing system security problem. as we have pointed out earlier, hacker technology is becoming more readily available, easier to get and assemble, more complex, and their effects more far-reaching. all these indicate that urgent action is needed to find an effective solution to this monstrous problem. the security community, including scrupulous vendors, have come up with various solutions, some good and others not. in fact, in an unexpected reversal of fortunes, one of the new security problems is to find a "good" solution from among thousands of solutions and to find an "expert" security option from the many different views. are we reaching the limits of our efforts, as a community, to come up with a few good and effective solutions to this security problem? there are many signs to support an affirmative answer to this question. it is clear that we are reaching the limits of the effectiveness of our reactive solutions. richard d. pethia gives the following reasons [2] : • the number of vulnerabilities in commercial off-the-shelf software is now at the level that it is virtually impossible for any but the best-resourced organizations to keep up with the vulnerability fixes. • attack technology has now advanced to the point where it is easy for attackers to take advantage of these vulnerable machines and harness them together to launch high-powered attacks. • many attacks are now fully automated, thus reducing the turnaround time even further as they spread around cyberspace. • the attack technology has become increasingly complex and, in some cases, intentionally stealthy, thus reducing the turnaround time and increasing the time it takes to discover and analyze the attack mechanisms in order to produce antidotes. • internet users have become increasingly dependent on the internet and now use it for many critical applications so that a relatively minor attack has the potential to cause huge damages. without being overly pessimistic, these factors, taken together, indicate that there is a high probability that more attacks are likely, and since they are getting more complex and attacking more computers, they are likely to cause significant devastating economic losses and service disruptions. according to john palumbo, social engineering is an outside hacker's use of psychological tricks on legitimate users of a computer system in order to gain the information (usernames and passwords) one needs to gain access to the system [5] . many have classified social engineering as a diversion, in the process of system attack, on people's intelligence to utilize two human weaknesses: first, no one wants to be considered ignorant, and second is human trust. ironically, these are two weaknesses that have made social engineering difficult to fight because no one wants to admit falling for it. this has made social engineering a critical system security hole. many hackers have and continue to use it to get into protected systems. kevin mitnick, the notorious hacker, used it successfully and was arguably one of the most ingenious hackers of our time; he was definitely very gifted with his ability to socially engineer just about anybody [5] . hackers use many approaches to social engineering, including the following [6]: • telephone. this is the most classic approach, in which hackers call up a targeted individual in a position of authority or relevance and initiate a conversation with the goal of gradually pulling information out of the target. this is done mostly to help desks and main telephone switchboards. caller id cannot help because hackers can bypass it through tricks, and the target truly believes that the hacker is actually calling from inside the corporation. • online. hackers are harvesting a boom of vital information online from careless users. the reliance on and excessive use of the internet have resulted in people having several online accounts. currently, an average user has about four to five accounts, including one for home use, one for work, and an additional one or two for social or professional organizations. with many accounts, as probably any reader may concur, one is bound to forget some passwords, especially the least used ones. to overcome this problem, users mistakenly use one password on several accounts. hackers know this, and they regularly target these individuals with clever baits such as telling them they won lotteries or were finalists in sweepstakes where computers select winners or they have won a specific number of prizes in a lotto, where they were computer selected. however, in order to get the award, the user must fill in an online form, usually web-based, and this transmits the password to the hacker. hackers have used hundreds of tricks on unsuspecting users in order for them to surrender their passwords. • dumpster diving is now a growing technique of information theft not only in social engineering but more so in identity theft. the technique, also known as trashing, involves an information thief scavenging through individual and company dumpsters for information. large and critical information can be dug out of dumpsters and trash cans. dumpster diving can recover from dumpsters and trash cans individual social security numbers, bank accounts, individual vital records, and a whole list of personal and work-related information that gives the hackers the exact keys they need to unlock the network. • in person is the oldest of the information-stealing techniques that predates computers. it involves a person physically walking into an organization's offices and casually checking out note boards, trash diving into bathroom trash cans and company hallway dumpsters, and eating lunches together and initiating conversations with employees. in big companies, this can be done only on a few occasions before trusted friendships develop. from such friendships, information can be passed unconsciously. • snail mail is done in several ways and is not limited only to social engineering but has also been used in identity theft and a number of other crimes. it has been in the news recently because of identity theft. it is done in two ways: the hacker picks a victim and goes to the post office and puts in a change of address form to a new box number. this gives the hacker a way to intercept all snail mail of the victim. from the intercepted mail, the hacker can gather a great deal of information that may include the victim's bank and credit card account numbers and access control codes and pins by claiming to have forgotten his or her password or pin and requesting a reissue in the mail. in another form, the hacker drops a bogus survey in the victim's mailbox offering baits of cash awards for completing a "few simple" questions and mailing them in. the questions, in fact, request far more than simple information from an unsuspecting victim. • impersonation is also an old trick played on unsuspecting victims by criminals for a number of goodies. these days the goodies are information. impersonation is generally acting out a victim's character role. it involves the hacker playing a role and passing himself or herself as the victim. in the role, the thief or hacker can then get into legitimate contacts that lead to the needed information. in large organizations with hundreds or thousands of employees scattered around the globe, it is very easy to impersonate a vice president or a chief operations officer. since most employees always want to look good to their bosses, they will end up supplying the requested information to the imposter. overall, social engineering is a cheap but rather threatening security problem that is very difficult to deal with. vulnerability assessment is a process that works on a system to identify, track, and manage the repair of vulnerabilities on the system. the assortment of items that are checked by this process in a system under review varies depending on the organization. it may include all desktops, servers, routers, and firewalls. most vulnerability assessment services will provide system administrators with: • network mapping and system fingerprinting of all known vulnerabilities. • a complete vulnerability analysis and ranking of all exploitable weaknesses based on potential impact and likelihood of occurrence for all services on each host. • prioritized list of misconfigurations. in addition, at the end of the process, a final report is always produced detailing the findings and the best way to go about overcoming such vulnerabilities. this report consists of prioritized recommendations for mitigating or eliminating weaknesses, and based on an organization's operational schedule, it also contains recommendations of further reassessments of the system within given time intervals or on a regular basis. due to the massive growth of the number of companies and organizations owning their own networks, the growth of vulnerability monitoring technologies, the increase in network intrusions and attacks with viruses, and worldwide publicity of such attacks, there is a growing number of companies offering system vulnerability services. these services, targeting the internals and perimeter of the system, web-based applications, and providing a baseline to measure subsequent attacks against, include scanning, assessment and penetration testing, and application assessment. vulnerability scanning services provide a comprehensive security review of the system, including both the perimeter and system internals. the aim of this kind of scanning is to spot critical vulnerabilities and gaps in the system's security practices. comprehensive system scanning usually results in a number of both false positives and negatives. it is the job of the system administrator to find ways of dealing with these false positives and negatives. the final report produced after each scan consists of strategic advice and prioritized recommendations to ensure that critical holes are addressed first. system scanning can be scheduled, depending on the level of the requested scan, by the system user or the service provider, to run automatically and report by either automated or periodic e-mail to a designated user. the scans can also be stored on a secure server for future review. this phase of vulnerability assessment is a hands-on testing of a system for identified and unidentified vulnerabilities. all known hacking techniques and tools are tested during this phase to reproduce real-world attack scenarios. one of the outcomes of these real-life testings is that new and sometimes obscure vulnerabilities are found, processes and procedures of attack are identified, and sources and severity of vulnerabilities are categorized and prioritized based on the user-provided risks. as web applications become more widespread and more entrenched into e-commerce and all other commercial and business areas, applications are slowly becoming the main interface between the user and the network. the increased demands on applications have resulted in new directions in automation and dynamism of these applications. as we saw in chap. 6, scripting in web applications, for example, has opened a new security paradigm in system administration. many organizations have gotten a sense of these dangers and are making substantial progress in protecting their systems from attacks via web-based applications. assessing the security of system applications is, therefore, becoming a special skills requirement needed to secure critical systems. vulnerability online services have many advantages for system administrators. they can, and actually always do, provide and develop signatures and updates for new vulnerabilities and automatically include them in the next scan. this eliminates the need for the system administrator to schedule periodic updates. reports from these services are very detailed not only on the vulnerabilities, sources of vulnerabilities, and existence of false positives, but they also focus on vulnerability identification and provide more information on system configuration that may not be readily available to system administrators. this information alone goes a long way in providing additional security awareness to security experts about additional avenues whereby systems may be attacked. the reports are then encrypted and stored in secure databases accessible only with the proper user credentials. this is because these reports contain critically vital data on the security of the system, and they could, therefore, be a pot of gold for hackers if found. this additional care and awareness adds security to the system. information security: protecting the global enterprise pethia rd information technology-essential but vulnerable: how prepared are we for attacks? ethical and social issues in the information age poster children for security problems social engineering: what is it, why is so little said about it and what can be done? sans part i: hacker tactics probably, the best advantage to an overworked and many times resource-strapped system administrator is the automated and regularly scheduled scan of all network resources. they provide, in addition, a badly needed third-party "security eye," thus helping the administrator to provide an objective yet independent security evaluation of the system. 1. what is a vulnerability? what do you understand by a system vulnerability? 2. discuss four sources of system vulnerabilities. 3 . what are the best ways to identify system vulnerabilities? 4. what is innovative misuse? what role does it play in the search for solutions to system vulnerability? 5. what is incomplete implementation? is it possible to deal with incomplete implementation as a way of dealing with system vulnerabilities? in other words, is it possible to completely deal with incomplete implementation? 6. what is social engineering? why is it such a big issue yet so cheap to perform?is it possible to completely deal with it? why or why not? 7. some have described social engineering as being perpetuated by our internal fears. discuss those fears. 8. what is the role of software security testing in the process of finding solutions to system vulnerabilities? 9. some have sounded an apocalyptic voice as far as finding solutions to system vulnerabilities. should we take them seriously? support your response. 10. what is innovative misuse? what role does it play in the search for solutions to system vulnerabilities? 1. why are vulnerabilities difficult to predict? 2. discuss the sources of system vulnerabilities. 3. is it possible to locate all vulnerabilities in a network? in other words, can one make an authoritative list of those vulnerabilities? defend your response. 4. why are design flaws such a big issue in the study of vulnerability? 5. part of the problem in design flaws involves issues associated with software verification and validation (v&v). what is the role of v&v in system vulnerability? key: cord-290139-b02eghd4 authors: yen, tseng-chang; wang, kuo-hsiung; wu, chia-huang title: reliability-based measure of a retrial machine repair problem with working breakdowns under the f-policy date: 2020-10-06 journal: comput ind eng doi: 10.1016/j.cie.2020.106885 sha: doc_id: 290139 cord_uid: b02eghd4 we study reliability and sensitivity analysis of a retrial machine repair problem with working breakdowns operating under the f-policy. the f-policy studies the most common issue of controlling arrival to a queueing problem and it requires startup time before allowing failed machines to enter the orbit. the server is subject to working breakdowns only when there is at least one failed machine in the system. when the server is busy it works at a fast rate, but when it is subject to working breakdowns, it works at a slow rate. failure and repair times of the server are exponentially distributed. the laplace transform technique is utilized to develop two system performance measures such as system reliability and mean time to system failure (mttf). extensive numerical results are carried out to reveal how performance measures are affected by changing various values of each system parameter. cloud computing is a burgeoning computing paradigm and has gained tremendous popularity in recent years. cloud computing paradigm shifts the deployment of computing infrastructure (such as cpu, network, and storage) from end users to the cloud data center. the provision of cloud computing services is delivered over the internet via virtual machines (vms) and virtual computing resources. cloud services provide scalable and flexible infrastructure for internet of things (iot), machine learning, blockchain, computer-assisted instruction (cai), and etc. in the covid-19 pandemic era, many countries have decided to temporarily close schools to reduce contact and save lives. to avoid disrupting learning and upending lives, teaching is moving online on an untested and unprecedented scale. onlinelearning is an internet-based cai system, which uses internet technology to improve the efficiency of education. by using the benefits of cloud computing, the online-learning cloud system supports the ability to dynamically allocate computing and storage resources for teaching material. to keep the quality of online-learning service and the stability of real-time data transition, the reliability of the cloud system should be investigated. the maintenance of online-learning cloud system can be modelled as a retrial machine repair problem. the retrial machine repair problem has been widely adopted to study many real-world systems, such as call centers, telecommunication systems, cloud computing networks, etc. in this paper, we consider a retrial machine repair problem (rmrp) with working breakdowns that combines f-policy and exponentially startup time before allowing failed machines to enter the retrial system. the server may be subject to working breakdown while serving a failed machine. when a server is busy or subject to working breakdowns, failed machines join the retrial orbit. arriving failed machines which cannot receive service immediately join the retrial orbit for a certain period of time and retry to receive service. maintaining a high level of system reliability is generally a requisite for many real-world repairable systems. for the most comprehensive concepts in retrial queues, we referred the reader to artalejo (1999a artalejo ( , 1999b , artalejo and gomez-corral (2008) , and phung-duc (2019). the literature on retrial systems was very vast and rich. artalejo and falin (2002) provided extensive comparisons of the standard and retrial queueing systems. sherman and kharoufeh (2006) studied an unreliable m/m/1 retrial queue with infinite-capacity orbit and normal queue. the authors derived stability conditions and some important stochastic decomposability results. wang (2006) extended sherman and kharoufeh's work to study an m/g/1/k retrial queue with server breakdowns. efrosinin and winkler (2011) proposed a markovian retrial queue with constant retrial rate and an unreliable server that combines the threshold recovery policy. the authors developed the important results for the waiting time distribution and the optimal threshold level. the authors provided the extensive results of the arrival distribution, the busy period, and the waiting time process. reliability analysis of the retrial queue with server breakdown and repairs was studied by wang et al. (2001) . gharbi and ioualalen (2006) considered finite-source retrial systems with multiple server breakdowns and repairs using generalized stochastic petri nets model. the authors developed steady-state performance and reliability indices. moreover, they also employed sensitivity analysis with numerical illustration. ke et al. (2013) presented the availability analysis of a repairable retrial system with warm standbys. kuo et al. (2014) investigated reliability analysis of a retrial system with mixed standbys. the authors provided sensitivity analysis for the mean time-to-failure as well as the steady-state availability. chen (2018) proposed reliability analysis of retrial machine repair system with working breakdowns and a single repair server with recovery policy. both sensitivity analyses and relative sensitivity analyses of system reliability and mean time to system failure were performed. chen and wang (2018) analyzed the system reliability of the n-policy retrial machine repair problem with a single controllable server. numerical experiments were provided to reveal how performance measures are affected by the change of each system parameter. for f-policy queues, wang et al. (2008) considered the optimum control of a g/m/1/k system combining f-policy and exponentially startup time before starting to allow clients in the system. the authors applied the supplementary variable technique to develop the steady-state probability distribution of the number of clients in the system. wang and yang (2009) examined the f-policy m/m/1/k system with server breakdown. quasi-newton methods and the direct search were adopted to determine the optimal threshold f, the optimal service rate, and the optimal startup rate at minimum cost. an f-policy m/m/1/k system with working vacations and exponentially startup time was explored by yang et al. (2010) . extensive numerical results were carried out to reveal the influence of system parameters on the cost function. for the systematic developments of the control f-policy systems, one can refer to the research papers by jain et al. (2016a jain et al. ( , 2016b and chang et al. (2018) . the fault tolerance problem of repairable redundant system with general retrial times operating under f-policy was analyzed by jain and sanga (2017) . numerical results were provided to study the effects of different system parameters on various performance measures. shekhar et al. (2017) proposed the computational approach to calculate the transient and steady state probabilities of the machine repair problem with an unreliable server operating under f-policy. greedy selection and newton-quasi methods were used to determine the optimal solutions at minimum cost. jain and sanga (2019a) studied f-policy m/m/1/k retrial queueing system with state-dependent rates. a cost function was conducted to determine the optimal service rate at minimum cost. the optimal control fpolicy for m/m/r/k queue with an additional server and balking was investigated by jain and sanga (2019b) . a cost model was framed to determine the optimal threshold parameter and optimal service rate. the finite state dependent queueing model with general retrial times operating under f-policy was explored by jain and sanga (2019c) . sensitivity analysis of the cost function with numerical illustrations were provided. jain and sanga (2020a) provided a state-of-the-art and survey of literature on the state-dependent queueing models operating under f-policy. recently, jain and sanga (2020b) investigated the machine repair system with general retrial times operating under f-policy in fuzzy environment. the signed distance method was used to defuzzify the cost function. the authors applied the genetic algorithm to find the optimal control parameter and corresponding minimum cost. to the best of our knowledge, system reliability and sensitivity analysis of a rmrp with working breakdowns operating under the fpolicy has never been discussed in the literature. this paper differs from previous work in that: (i) queueing problems are broadly divided into two categories, one aims at controlling service category (n-policy) whereas the other aims at controlling arrivals category (fpolicy). the f-policy reliability problem has distinct characteristics which are different from the n-policy reliability problem. (ii) it aims at controlling arrivals to study reliability characteristics of a retrial machine repair problem; (iii) we simultaneously consider the f-policy and working breakdowns which has never been investigated in the existing work. the rest of the paper is organized as follows. section 2 gives the descriptions of the problem. in section 3, we use the laplace transform technique to develop the main results such as system reliability and mean time to system failure. sensitivity and relative sensitivity analysis is also addressed in this section. in section 4, extensive numerical results were carried out to reveal the influence of system parameters on related analysis such as system reliability, mean time to system failure, sensitivity, and relative sensitivity. section 5 concludes the paper. we propose a retrial machine repair problem (rmrp) with identical and   n m s independent machines and a single server in the repair facility. as many as machines can m be operated simultaneously in parallel, the rest of the machines are available as warm-s standby spares. operating machines fails according to independent poisson distribution with parameter similarly, each of the available warm standbys fails independently of the state .  of all the others with poisson rate ( ). whenever one of operating machines fails,  0     it is immediately replaced by a warm standby, if ready. we assume that when a warm standby machine moves into an operating state, its failure characteristics will be that of an operating machine. each failed operating or standby machine is instantly sent to a single server. the server is unreliable and may experience a working breakdown during busy periods. when the server is busy it serves at a fast service rate , but when it is subject to working breakdowns, 1  in place of terminating service completely, it continues at a lower service rate . the 2 1 ( )  service time distribution is assumed to be exponentially distributed. it is assumed that the server may encounter working breakdown at any time with breakdown rate . whenever the server  breaks down, it is immediately repaired at a repair rate . breakdown and repair times of the  server are assumed to be exponentially distributed. when the server recovers from a working breakdown, the service rate is enhanced from to . whenever a machine fails, it is 2  1  immediately sent to the repair facility where repair work is provided in the order of breakdowns; that is, first-come, first-served (fcfs) discipline. arriving failed machines discern that the server is busy and must enter the orbit after a certain period of time and retry to receive service. each failed machine in the orbit repeats its service request with retrial time. retrial times follow the exponential distribution at rate .  furthermore, to control arriving failed machines, an f-policy is implemented. the definition of an f policy is described below: when the number of failed machines in the system reaches full capacity k, no further arriving failed machines are allowed to enter the system until the number of failed machines in orbit decreases to the threshold value f ( ). at 0 1    f k that moment, the server must adopt exponentially start up time with parameter to start  allowing failed machines to join the system. thus, the system operates continuously until the number of failed machines in the system reaches to full capacity k again at which time the above process is repeated over and over. according to the f-policy, to ensure that the server can operate normally, the repair facility prevents new failed machines from entering with probability . on the contrary, when system is full, the entering of new failed machine causes c the server to be unable to complete the repair due to insufficient system resources. the system then moves to the unsafe failure (uf) state with probability , and the occurrence of the 1 c unsafe failure always results in a system fail. a practical situation related to an online-learning cloud system is presented for illustrative purposes. to satisfy the requests of extensive concurrent online-learning connections, an online-learning cloud system is established via a vm cluster on four quad-core servers. since there are total 16 cpu cores (4 4 cores), 16 vms (each vm is assigned with a cpu core) are  deployed to establish a private cloud ( fig. 1 ) which 12 vms served as working online-learning servers, and 4 vms as warm-standby servers. meanwhile, one physical server with linux system and pre-installed cloudstack cloud administration tool is deployed as the vm repair server. besides, a 1tb ceph distributed storage system is installed to improve the i/o efficiency of online-learning system. each vm consists of a kernel program and a 25 gb root filesystem with pre-installed debian linux system and the moodle online-learning tool. an extra 200 gb root filesystem is mounted to the vm repair server to provide storage pool (treated as the orbit) for failed vms. therefore, there are at most 8 (treated as ) failed vms k can be sent to repair server to wait for repair procedure. to promise the quality of service, a failed working vm is replaced with a warm standby vm, and failed vms are sent to the vm repair server, which could check and repair the malfunctioned root filesystem. when a failed vm arrives, it accepts repair procedure immediately according to its system log file if the vm repair server is available. otherwise, the failed vm is transmitted to the storage pool of the repair server to wait for filesystem check and reconfiguration. when the number of failed vms reaches 8, the cloudstack has a 80% (treated as ) chance to prohibit any failed vms from c entering to repair server due to storage space limitation k. the cloudstack may fail to prevent new failed vms from entering the repair server with probability 0.1 and cause an onlinelearning system error due to excessive use of storage space. a faster service rate is provided when the vm repair server is normal. the vm repair server may be subject to failure and provide a slower service rate due to a software or hardware problem, such as abnormal program execution or disk hot swap. in this rmrp, we describe the system states by the pair where denotes the ( , ) i j i system states and represents the number of failed machines in the retrial orbit, j . various values of define system states as follows: 0,1, 2,..., : the server is operating normally but is idle, and arriving/retrial failed machines are 0  i allowed to join the system for service; : the server is operating normally but is busy, one failed machine is being served and 1  i arriving failed machines will be forced to enter the orbit; : the server is operating normally but is busy and the system is blocked, and arriving 2  i failed machines are not allowed to enter the system; : the server is busy during a working breakdown, and failed machines are allowed to 3  i join the system; : the server is busy during a working breakdown, and failed machines are not allowed 4  i to join the system; : the server is idle during a working breakdown, and arriving/retrial failed machines 5  i are allowed to join the system. with the above definition of system states, the state space can be presented as at time t, the probabilities of different states in the system are defined as follows: probability of having failed machines in the retrial orbit when the server is in the mean failure rate and the mean retrial rate are given by the state transition-rate diagram for the rmrp with working breakdowns operating under the f-policy can be shown in figure 2 . figure 2 . reliability of retrial machine repair system with working breakdowns under the f-policy. the differential-difference equations for each state at time t can be derived by figure 2 . for instance, the differential difference equation for the state is implementing the laplace transform and the technique of integration by parts in calculus as similarly, by taking the laplace transform to the differential-difference equation of each system state, the laplace transform equations (1)-(21) are established. (2) * * * 0 0,0 1 1,0 1 2,0 0,0 denotes the transition rate matrix which is also a squared matrix of order . it can be 6 2  k represented by sub-matrices as follows: . ( the sub-matrix is a matrix with only one 1 , , 1 1, ; , 2 1, 1; 0, other , . the sub-matrix and are and matrices defined by the sub-matrix and is a diagonal matrix of order defined by are the probability of that the system has failed at or before time t, the reliability function 2 ( ) uf p t is obtained in the following: , . it is always finite since the integrated function is a monotonic decreasing and bounded function of . thus, it implies that the mean time to system failure (mttf) exists and can be calculated t by (26) consequently, the mttf can be obtained as for each system parameter , differentiating (25) with respect to we obtain the  ,  gradient of system reliability for the sensitivity analysis, shown below similarly, the gradient of mttf can be derived by differentiating (27) moreover, the relative gradient on and mttf can be implemented by ( ) since the derived results and are complex non-linear function of the system ( ) parameters, the explicit closed-form of the associated derivative with respective to parameter is difficult to be obtained. hence, we calculate the gradient numerically. taking as     an example, at point in equation (28) is computed by with a small difference magnitude . this section presents some numerical results to perform several related analyses. in first, as the illustration example of online-learning cloud system provided in section 2.1, the base (initial) parameters are selected as , , , , based on the 0.04 above basic setting, except for parameters m and s (which are always fixed), we change one specific parameter at one time and keep the values of the other parameters. that is, we investigate the effects of each parameter on the system reliability to recognize which parameters are critical and needed to be controlled well. the numerical results of for system ( ) y r t reliability assessment are depicted in figures 3-13 . from these figures, we observe that , ,  c k significantly affect system reliability (see figures 3, 11, 13 ). parameters and affect the  1  system reliability moderately (see figures 5, 6) . parameters affects the system 2 , , ,    f reliability slightly (see figures 7, 8, 9, 12) while and affect the system reliability rarely   (see figures 4, 10) . it shows the managers several possibilities for reliability enhance: (i) enlarge the system capacity; (ii) reduce the mean failure rate or improve the vm stability; (iii) add some necessary mechanisms for successfully blocking the failed vms when the system is full.  figure 11 . system reliabilty affected by . c figure 12 . system reliabilty affected by . f figure 13 . system reliabilty affected by . k in this subsection, some numerical experiments are perfomed to investigat how each system parameter affects the mean time to system failure (mttf extensive numerical results are carried out on sensitivity and relative sensitivity analysis. first, sensitivity analysis on system reliability is provided and depicted in figure 14 . one can see from figure 14 that the order of sensitivity analysis on system reliability affected by each parameter can be ranked below: furthermore, the signs of 1 2 .         c gradient on system reliability with respect to paramters , are negative. it indicates ,  ,    that system reliability increases as these paramters decrease. in constast, the signs of gradient on system reliability with respect to paramters are positive and therefore the , system reliability increases as these paramters increase. in addition, the results show that parameters and have significant effects and therefore they should be carefully estimated  c as accessing the system reliability. therefore, a monitoring system with continuous data collection may be crucial to control them well. figure 14 . sensitivity analysis on reliability function. next, as for sensitivity analysis on mttf affected by each system parameter, table 9 tabulates the gradient of mttf with respect to various parameters. we observe from table 9 , the order of sensitivity analysis on mttf affected by each parameter can be ranked below: besides, the signs of sensitivity on mttf affected by finally, the related sensitivity analyses on system reliability and mttf are also performed. extensive numerical results for relative sensitivity on system reliability and mttf are shown in figure 15 and table 10 , respectively. it appears from figure 15 that the order of relative sensitivity on system reliability affected by each parameter can be ranked below: from in this paper, we introduced a retrial machine repair problem with working breakdowns operating under the probabilistic f-policy. we applied the laplace transform technique and the matrix-analytical method to solve the differential-difference equations of the investigated system. then, the explicit expressions of the system reliability and the mean time to failure were derived. a practical illustration of the online-learning cloud system that the proposed model can be applied to is provided. based on the application case, a reliability-based sensitivity analysis for the effects of system parameters was performed. the associated numerical results were tabulated and displayed graphically to evaluate the impacts of different parameters. the results indicated that the effect of the mean startup rate is ignorable. increasing the prohibit success probability, the mean service rate, or the system capacity can effectively enhance the system reliability. the results also show that the mean arrival rate and the prohibit probability need to be evaluated carefully. once they are changed but the manager does not notice it, the system reliability may become low and cause additional operating cost. in the future, we provide some possible research topics shown below: 1. some random times (e.g. repair times or retrial times) obey general distribution, 2. system availability of the model, 3. server may be subject to unpredictable breakdowns, and 4. the optimal management strategy. we consider a f-policy retrial machine repair problem with working breakdowns.  one practical illustration of the online-learning cloud system is presented.  the differential equations are tabulated and solved by laplace transform technique.  closed-form expressions for the reliability and the mttf are explicitly derived.  reliability sensitivity analysis with respect to system parameters are performed.  several management insights for managers to enhance the reliability are provided. accessible bibliography on retrial queues. mathematical and computer modelling a classified bibliography of research on retrial queues: progress in 1990-1999. top standard and retrial queueing systems: a comparative analysis retrial queueing systems: a computational approach analysis of a standby redundant system with controlled arrival of failed machines reliability analysis of a retrial machine repair problem with warm standbys and a single server with n-policy system reliability analysis of retrial machine repair systems with warm standbys and a single server of working breakdown and recovery policy queueing system with a constant retrial rate, nonreliable server and threshold-based recovery gspn analysis of retrial systems with servers breakdowns and repairs control f-policy for fault tolerance machining system with general retrial attempts f-policy for m/m/1/k retrial queueing model with statedependent rates. performance prediction and analytics of fuzzy, reliability and queuing models optimal control f-policy for m/m/r/k queue with an additional server and balking admission control for finite capacity queueing model with 24 general retrial times and state dependent rates state dependent queueing models under admission control fpolicy: a survey fuzzy cost optimization and admission control for machine interference problem with general retrial control f-policy for markovian retrial queue with server breakdowns. 1 st ieee international conference on power electronics, intelligent control and energy system queueing analysis of machine repair problem with controlled rates and working vacation under f-policy availability of a repairable retrial system with warm standby components reliability-based measures for a retrial system with mixed standby components retrial queueing models: a survey on theory and applications threshold control policy for maintainability of manufacturing system with unreliable workstations an m/m/1 retrial queue with unreliable server reliability analysis m/g/1 queues with general retrial times and server breakdowns reliability analysis of the retrial queue with server breakdowns and repairs a recursive method for the f-policy g/m/1/k queueing system with an exponential startup time controlling arrivals for a queueing system with an unreliable server: newton-quasi method optimization and sensitivity analysis of controlling arrivals in the queueing system with single working vacation key: cord-102383-m5ahicqb authors: romano, alessandra; casazza, marco; gonella, francesco title: energy dynamics for systemic configurations of virus-host co-evolution date: 2020-05-15 journal: biorxiv doi: 10.1101/2020.05.13.092866 sha: doc_id: 102383 cord_uid: m5ahicqb virus cause multiple outbreaks, for which comprehensive tailored therapeutic strategies are still missing. virus and host cell dynamics are strictly connected, and convey in virion assembly to ensure virus spread in the body. study of the systemic behavior of virus-host interaction at the single-cell level is a scientific challenge, considering the difficulties of using experimental approaches and the limited knowledge of the behavior of emerging novel virus as a collectivity. this work focuses on positive-sense, single-stranded rna viruses, like human coronaviruses, in their virus-individual host interaction, studying the changes induced in the host cell bioenergetics. a systems-thinking representation, based on stock-flow diagramming of virus-host interaction at the cellular level, is used here for the first time to simulate the system energy dynamics. we found that reducing the energy flow which fuels virion assembly is the most affordable strategy to limit the virus spread, but its efficacy is mitigated by the contemporary inhibition of other flows relevant for the system. summary positive-single-strand ribonucleic acid ((+)ssrna) viruses can cause multiple outbreaks, for which comprehensive tailored therapeutic strategies are still missing. virus and host cell dynamics are strictly connected, generating a complex dynamics that conveys in virion assembly to ensure virus spread in the body. this work focuses on (+)ssrna viruses in their virus-individual host interaction, studying the changes induced in the host cell bioenergetics. a systems-thinking representation, based on stock-flow diagramming of virus-host interaction at the cellular level, is used here for the first time to simulate the energy dynamics of the system. by means of a computational simulator based on the systemic diagramming, we identifid host protein recycling and folded-protein synthesis as possible new leverage points. these also address different strategies depending on time setting of the therapeutic procedures. reducing the energy flow which fuels virion assembly is addressed as the most affordable strategy to limit the virus spread, but its efficacy is mitigated by the contemporary inhibition of other flows relevant for the system. counterintuitively, targeting rna replication or virion budding does not give rise to relevant systemic effects, and can possibly contribute to further virus spread. the tested combinations of multiple systemic targets are less efficient in minimizing the stock of virions than targeting only the virion assembly process, due to the systemic configuration and its evolution overtime. viral load and early addressing (in the first two days from infection) of leverage points are the most effective strategies on stock dynamics to minimize virion assembly and preserve host-cell bioenergetics. as a whole, our work points out the need for a systemic approach to design effective therapeutic strategies that should take in account the dynamic evolution of the system. virus cause multiple outbreaks, for which comprehensive tailored therapeutic strategies are still missing. virus and host cell dynamics are strictly connected, and convey in virion assembly to ensure virus spread in the body. study of the systemic behavior of virus-host interaction at the single-cell level is a scientific challenge, considering the difficulties of using experimental approaches and the limited knowledge of the behavior of emerging novel virus as a collectivity. this work focuses on positive-sense, single-stranded rna viruses, like human coronaviruses, in their virus-individual host interaction, studying the changes induced in the host cell bioenergetics. a systems-thinking representation, based on stock-flow diagramming of virus-host interaction at the cellular level, is used here for the first time to simulate the system energy dynamics. we found that reducing the energy flow which fuels virion assembly is the most affordable strategy to limit the virus spread, but its efficacy is mitigated by the contemporary inhibition of other flows relevant for the system. positive-single-strand ribonucleic acid ((+)ssrna) viruses can cause multiple outbreaks, for which comprehensive tailored therapeutic strategies are still missing. virus and host cell dynamics are strictly connected, generating a complex dynamics that conveys in virion assembly to ensure virus spread in the body. this work focuses on (+)ssrna viruses in their virus-individual host interaction, studying the changes induced in the host cell bioenergetics. a systems-thinking representation, based on stockflow diagramming of virus-host interaction at the cellular level, is used here for the first time to simulate the energy dynamics of the system. by means of a computational simulator based on the systemic diagramming, we identifid host protein recycling and folded-protein synthesis as possible new leverage points. these also address different strategies depending on time setting of the therapeutic procedures. reducing the energy flow which fuels virion assembly is addressed as the most affordable strategy to limit the virus spread, but its efficacy is mitigated by the contemporary inhibition of other flows relevant for the system. counterintuitively, targeting rna replication or virion budding does not give rise to relevant systemic effects, and can possibly contribute to further virus spread. the tested combinations of multiple systemic targets are less efficient in minimizing the stock of virions than targeting only the virion assembly process, due to the systemic configuration and its evolution overtime. viral load and early addressing (in the first two days from infection) of leverage points are the most effective strategies on stock dynamics to minimize virion assembly and preserve host-cell bioenergetics. as a whole, our work points out the need for a systemic approach to design effective therapeutic strategies that should take in account the dynamic evolution of the system. interaction between a (+)ssrna virus and the host cell and therefore addressing effective intervention strategies. starting from the knowledge of relevant processes in (+ss)rna virus replication, transcription, translation, virions budding and shedding and their energy costs (reported in supplementary methods table 1) , we built up a systems-thinking (st) based energy diagram of the virus-host interaction. figure 1 shows the stock-flow diagram for the system at issue, where each stock was quantified in terms of embedded energy of the corresponding variable. symbols are borrowed from the energy language 51, 52 : shields indicate the stocks, big solid arrows the processes and line arrows the flows, whereas dashed lines indicate the controls exerted by the stocks on the processes. all stocks, flows and processes are expressed in terms of the energy embedded, transmitted and used during the infection. we used atp-equivalents (atp-eq) as energy unit 53 referred to cellular costs, by using the number of atp (or gtp and other atp-equivalents) hydrolysis events as a proxy for energetic cost 53, 54 . the dynamic determination of flows was based on the knowledge of characteristic time-scales of well-established biological processes (for more details see supplementary methods). the output flows j4 and j5 were set to be effective only if the value of the respective stocks q3 and q5 is higher than a threshold, as represented by the two switch symbols in the diagram. the stock q1 represents the embedded energy of resources addressed to protein synthesis in the host cell. the dynamics of allocation for protein synthesis depends on the cell bioenergetics, e.g., the number of mitochondria, ox-phos activity levels, and the cell cycle phase [55] [56] [57] (for more details, see supplementary methods). in the absence of virus, energy flows from q1 (flows j1, j2a and j2b) to produce short-half-life proteins (stock q2a) and long-half-life proteins (stock q2b), whose synthesis, degradation and secretion follow dynamic stationary conditions. in particular, typical of the specialized cells such as those of the pulmonary epithelium, there is a flow of proteins destined for degradation and recycling through the recruitment of autophagic receptors or organelles or proteasomes (identified in the diagram by the flows j21a and j21b, supplementary methods table 1 ). the outflow of folded, fully functional proteins addressed to secretion or surface exposure is first, we investigated the system dynamics under different initial conditions, exploring the possible role of different initial viral loads (figure 2 ). in the configuration of initial null viral load (q3 stock value=0) the value of stocks q1, q2a and q2b were constant and the system behavior was stationary (figure 2a) . assuming a different viral loads (time zero) in the 10-10,000 virions range, we found that there is a threshold in the initial viral load for triggering the progressive reduction of q1, whose amount could in turn trigger the cell death in different ways. apoptosis is a cellular process requiring energy, and a deflection in q1, as shown in figure when a (+)ssrna virus enters the single host cell, the q3 stock is fed, and its proteins can interact with the host proteome to sustain rna replication. based on previous works in the field 59-63 , we identified a time delay of 2 to 6 hours required to record changes in the q5 stock. moreover, the value of q5 varies over the time due to changes occurred at a different timepoints in the stocks q2b, q3 and q4. to be effective, a therapeutic strategy should limit the outflows of virions, j7 and/or j50. however, minimization of j7 seems to be counter-effective in our simulation, due to the increase of q5 as consequence of the feedback action in the virus replication (vr) process. the minimization of j50 prevents the outflow (viral shedding) from q5 without stopping its growth. at the same time, this leads to increased resources diverting from q1 and q2b that could promote the cell death, with consequent spread of the virions in the environment. arising from targeting j5 was mitigated by the combination with reduction of j50 or j21 (figure 3) . the partial reduction of j21, alone or in combination, did not change significantly the dynamic growth of q5, induced increase of q3 at day 5, though it could prevent a reduction of q1, to preserve the cell bioenergetics and preventing the cell death by atp lack. we also simulated the effect of applying the same external inputs at different times: after 1 (extended data figure 4 ), 3 (extended data figure 5 ) or 5 days (extended data figure 6 ) from the initial infection, alone or in combination (figure 4) . application of external forcing factors at day 1, day 3 or day 5 could affect in a non-linear way the system response, with the largest efficacy targeting j5 (figures 4-5) . the combination of external driving forces acting on j5 and j21 did not result to be significantly synergic, while targeting j50 and j21 increased the q5 stock value instead of the expected reduction. it is worth stressing that this kind of behavior is a typical systemic feature, where an intervention on a specific local process may lead to counterintuitive rearrangements in the system dynamics as a whole. late suppression of j5 and j50 at day 5 could reduce only the stock values of q4. only early full reduction of j5 (by day 1) could significantly limit the growth of q5, while the combination of contemporary suppression of j5 and j50 could not prevent q5 growth (figure 5) . thus, application of external driving forces at different timepoints is expected to model additional resilience configurations. in this work, we approached the host-virus interaction dynamics as a systemic problem and, for the first time in the field, we used combined systems thinking tools as a conceptual framework to build up a systemic description of the viral action and host response, critically depending on the existing metabolic environment. the complex dynamic behavior was described in terms of underlying accessible patterns, hierarchical feedback loops, self-organization, and sensitive dependence on external parameters, that were analytically computed by a simulator. the following novelties were addressed: i) the use of energy language as a common quantitative unit for different biological cells and used mass spectrometry to measure protein-protein interactions 6 . with this experimental approach, they identified 332 interactions between viral and host proteins, and noted 69 existing drugs, known to target host proteins or associated pathways, which interact with sars-cov-2, addressing the importance to target the host-virus interaction at the level of rna translation. there are known advantages of in silico modelling the action of therapeutic agents on known diseases through agent-based modelling 74 . however, the literature evidenced some intrinsic limitations on the choice of parameters, like the size of investigated populations 75 , while major problems are related to model validation 75, 76 , also requiring to supplement the models with adequate formal ontologies 77 . thanks to its abstract nature, stock-flow description can be used in a wide range of different fields, realizing the conceptual bridge that connects the language of biological systems to that of ecology. our approach unveils the potential of systems thinking for the study of other diseases or classes of disease, since it appears more and more clear how some incurable pathologies can be described only by adopting a more comprehensive systemic approach, in which the network of relationships between biological elements are treated in quantitative way similar to that applied in this paper. the method used to study the dynamics of the virus-host interaction system is structured in 3 basic steps, namely, the development of a flow-stock diagram, that describes the virus-host interactions, the development of a virus-host systemic simulator, and, finally, its calibration and validation (extended data, figure 1) . a typical systems thinking diagram is formed of stocks, flows and processes. stocks are countable extensive variables qi, i=1,2,...,n, relevant to the study at issue, that constitute an n-ple of numbers that at any time represents a state of the system. a stock may change its value only upon its inflows and/or its outflows, represented by arrows entering or exiting the stock. processes are any occurrence capable to alter -either quantitatively or qualitatively -a flow, by the action of one or more of the system elements. in a stationary state of the system, stocks values are either constant or regularly oscillating. in the dynamics of a system, stocks act as shock absorbers, buffers, and time-delayers. processes are all what happens inside a system that allows the stationarity of its state, or that may perturb the state itself. to occur, a process must be activated by another driver, acting on the flow where the process is located. these interaction flows may be regarded as flows of information, that control the occurring processes and so the value and nature of the matter flows. the pattern of the feedbacks acting in the system configurations is the feature that utlimately defines the systems dynamics. we adopted an energyy approach, where stocks, flows and processes are expressed in terms of the energy embedded, transmitted and used, respectively, during the system operation. the equations, that characterize the flows relevant to the diagram, are typical of dynamic st analysis 51, 78 , and their setting up is linked in many respects to the energy network language 79 . in this approach, each flow depends on the state variables qi by relationships of the kind dqi/dt=kf(qj), i,j=1,...,n, where n is the number of stocks in the system. given a set of proper initial conditions for the stocks (i.e., the initial system state) and a properly chosen set of phenomenological coefficients k, the set of interconnected equations will be treated by standard finite-different method, taking care of choosing a time-step short enough to evidence the possible dynamics of any of the studied processes. the coefficients ki are calculated using data on the dynamics of any single stock, in particular, by estimating the flows and the stocks during the time interval set for the simulation steps (as described in supplementary methods). when different flows co-participate in a process, a single coefficient will gather all the actions that concur to the intensity of the outcoming flow(s). these phenomenological coefficients are not, therefore, related to specific biophysical phenomena or processes, but are set to describe how and how fast any part of the system react to a change in any of the other ones. in general, our model may be then regarded as based on a population-level model (plm) as defined by 80 , which can be run at different scales from organism to sub-cellular ones (see for example 58, 81, 82 ), that already increased the existing knowledge on the life cycle of different infections, like for example the ones generated by hiv and hepatitis c virus 83 . to obtain the simulations, we used the open-source computation software scilab (https://www.scilab.org). the model simulations are developed based on the choice of the initial stock conditions, as well as of the parameters ki (see supplementary information for further details) . a model is valid as far as its output reproduces the reality, and systems modelling must be tested under two aspects, concerned with the capability to correctly describe the system and its dynamics, respectively 84 extended data figure 1 workflow and study design extended data figure 3 effects of targeting leverage points upon the applcation of generic external driving forces d at day 0 interaction of the innate immune system with positive-strand rna virus replication organelles genetic variability and evolution of hepatitis e virus genomic analysis of the emergence, evolution, and spread of human respiratory rna viruses host factors in positive-strand rna virus genome replication antiviral drug targets of single-stranded rna viruses causing chronic human diseases a sars-cov-2 protein interaction map reveals targets for drug repurposing coronaviruses -drug discovery and therapeutic options cytoplasmic viral replication complexes mutational and fitness landscapes of an rna virus revealed through population sequencing the role of mutational robustness in rna virus evolution quasispecies diversity determines pathogenesis through cooperative interactions in a viral population evolvability of an rna virus is determined by its mutational neighbourhood innate immunity induced by composition-dependent rig-i recognition of hepatitis c virus rna middle east respiratory syndrome coronavirus orf4b protein inhibits type i interferon production through both cytoplasmic and nuclear targets resolution of primary severe acute respiratory syndrome-associated coronavirus infection requires stat1 innate immune evasion strategies of dna and rna viruses viral infection switches non-plasmacytoid dendritic cells into high interferon producers cardif is an adaptor protein in the rig-i antiviral pathway and is targeted by hepatitis c virus viral innate immune evasion and the pathogenesis of emerging rna virus infections hla class ii genes determine the natural variance of hepatitis c viral load emerging coronaviruses: genome structure, replication, and pathogenesis a pneumonia outbreak associated with a new coronavirus of probable bat origin genesis of a highly pathogenic and potentially pandemic h5n1 influenza virus in eastern asia isolation of a novel coronavirus from a man with pneumonia in saudi arabia common nodes of virus-host interaction revealed through an integrated network analysis coronaviruses and the human airway: a universal system for virus-host interaction studies generation and comprehensive analysis of host cell interactome of the pa protein of the highly pathogenic h5n1 avian influenza virus in mammalian cells human interactome of the influenza b virus ns1 protein whither systems medicine? chloroquine is a potent inhibitor of sars coronavirus infection and spread in vitro inhibition of severe acute respiratory syndrome coronavirus by chloroquine role of lopinavir/ritonavir in the treatment of sars: initial virological and clinical findings efficacy of chloroquine and hydroxychloroquine in the treatment of covid-19 chloroquine and hydroxychloroquine in the context of covid-19 review: hydroxychloroquine and chloroquine for treatment of sars-cov-2 (covid-19) a rapid systematic review of clinical trials utilizing chloroquine and hydroxychloroquine as a treatment for covid-19 effect of high vs low doses of chloroquine diphosphate as adjunctive therapy for patients hospitalized with severe acute respiratory syndrome coronavirus 2 (sars-cov-2) infection: a randomized clinical trial treating covid-19 with chloroquine hydroxychloroquine, a less toxic derivative of chloroquine, is effective in inhibiting sars-cov-2 infection in vitro in vitro antiviral activity and projection of optimized dosing design of hydroxychloroquine for the treatment of severe acute respiratory syndrome coronavirus 2 (sars-cov-2) chloroquine and hydroxychloroquine as available weapons to fight covid-19 breakthrough: chloroquine phosphate has shown apparent efficacy in treatment of covid-19 associated pneumonia in clinical studies remdesivir and chloroquine effectively inhibit the recently emerged novel coronavirus (2019-ncov) in vitro structural and molecular basis of mismatch correction and ribavirin excision from coronavirus rna thinking in systems : a primer all models are wrong: reflections on becoming a systems scientist modeling for all scales : an introduction to system simulation a picture is worth a thousand words: energy systems language and simulation energetic cost of building a virus the bioenergetic costs of a gene ampk regulates energy expenditure by modulating nad+ metabolism and sirt1 activity the incidence of thromboembolism for lenalidomide versus thalidomide in older patients with newly diagnosed multiple myeloma development of an urban few nexus online analyzer to support urban circular economy strategy planning optimal replication of poliovirus within cells single molecule analysis of rna polymerase elongation reveals uniform kinetic behavior mathematical model of influenza a virus production in large-scale microcarrier culture current estimates for hiv-1 production imply rapid viral clearance in lymphoid tissues viral dynamics in human immunodeficiency virus type 1 infection a complex systems approach to cancer prevention the systemic-evolutionary theory of the origin of cancer (setoc): a new interpretative model of cancer as a complex biological system enhancing implementation science by applying best principles of systems science kinetic modeling of virus growth in cells energy and time determine scaling in biological and computer designs absence of effect of the antiretrovirals duovir and viraday on mitochondrial bioenergetics macrophages exposed to hiv viral protein disrupt lung epithelial cell integrity and mitochondrial bioenergetics via exosomal microrna shuttling approaching complex diseases extremely small energy requirement by poliovirus to proliferate itself is the key to an outbreak of an epidemic an agent-based model for drug-radiation interactions in the tumour microenvironment: hypoxia-activated prodrug sn30000 in multicellular tumour spheroids entrainment and control of bacterial populations: an in silico study over a spatially extended agent based model replicating complex agent based models, a formidable task agent-based modelling of socio-ecological systems: models, projects and ontologies systems and models: complexity, dynamics, evolution, sustainability. (books on demand gmbh explanations of ecological relationships with energy systems concepts combining molecular observations and microbial ecosystem modeling: a practical guide formal aspects of model validity and validation in system dynamics the philosophy and epistemology of simulation: a review competing interests. authors declare no competing financial interests. the author to whom correspondence and material requests should be addressed is:dr. marco casazza key: cord-253548-izya7nws authors: catchpole, ken; bowie, paul; fouquet, sarah; rivera, joy; hignett, sue title: frontiers in human factors: embedding specialists in multi-disciplinary efforts to improve healthcare. date: 2020-09-09 journal: int j qual health care doi: 10.1093/intqhc/mzaa108 sha: doc_id: 253548 cord_uid: izya7nws despite the application of a huge range of human factors (hf) principles in a growing range of care contexts, there is much more that could be done to realize this expertise for patient benefit, staff wellbeing and organizational performance. healthcare has struggled to embrace systems safety approaches, mis-applied or misinterpreted others, and has stuck to a range of outdated and potentially counter-productive myths even has safety science has developed. one consequence of these persistent misunderstandings is that few opportunities exist in clinical settings for qualified hf professionals. instead, hf has been applied by clinicians and others, to highly variable degrees – sometimes great success, but frequently in limited and sometimes counter-productive ways. meanwhile, hf professionals have struggled to make a meaningful impact on frontline care and have had little career structure or support. however, in the last few years, embedded clinical hf practitioners have begun to have considerable success that are now being supported and amplified by professional networks. the recent covid-19 experiences confirm this. closer collaboration between healthcare and hf professionals will result in significant and ultimately beneficial changes to both professions and to clinical care. human factors (hf) perspectives are familiar to many in healthcare, and thousands of people are exploring or benefiting from the value those perspectives bring to a multitude of care contexts. hf has been applied in acute care 1 , primary care 2,3 , emergency care 4 , home care 5 , device design 6 , health it 7 , health systems design 8 , architecture 9 , simulation 10 , education 11, 12 , quality improvement 13 , safety 14 , and reliability 15 . studies have analyzed systems of work 16 , teamwork 17 , decision making 18 , displays 19 , device interactions 20 , risks 21 , threats 22 , performance shaping factors 23 , environmental and organizational approaches 24, 25 , and regulatory influences 26 . the methods of direct observation, interviews, task analysis, usability studies, human reliability analysis, and incident analysis have been frequently, reliability and creatively applied. however, there is much more that could be done to realize the full potential of hf principles and expertise for patient benefit, staff wellbeing and organizational performance 27 . this has raised a number of questions about how hf knowledge and expertise could be integrated better into clinical work. prior to the application in healthcare, hf knowledge and expertise had arisen mostly from either consumer products and software, or heavily engineered industries 28 . in these industries, human actions are often carried out through the technology, with the hf focus on integrating humans, processes and technologies into a 'top-down' work system with known inputs, goals and constraints. clinical systems are not necessarily like those other industries, though there remains a need design for human use. healthcare is more complex, with multiple different goals and outcomes, a huge variation in inputs, work contexts, procedures and necessary variation in process (to account for individual patient needs). new procedures and technologies are continually being introduced. it was never purposely engineered but has grown organically over thousands of years to meet a range of human needs. every healthcare organization is different, and procedures can vary from provider to provider and patient to patient. how the system works is opaque and it can be difficult to understand what is, or what should be, happening. the particular, and arguably unique, constraints and demands in healthcare mean that while the principles of systems safety science in general, and hf in particular, are similar, their application and mechanisms of effect can be very different. while healthcare also contains many different groups who have a strong interest in improving human work (e.g. hr professionals, organisational development specialists, clinical risk, patient safety and quality improvement advisors), the hf professional can provide much-needed 'added-value' to multidisciplinary team efforts to jointly optimise overall organisational performance and human wellbeing. this paper explores the application of hf in relation to those that apply it; what we need to do to be more effective; how this might fundamentally change both healthcare and hf professions; and the opportunities for service transformation it might present. despite much progress in the application of systems thinking to clinical work, there remains a focus on "error" as a clinical-professional issue to be "fixed" rather than signifying on the deeper causes of error, within the clinical system. instead, the psychology, engineering, sociology and other expertise necessary to address the deep systems problems associated with accidental harms has often been simplified to lookalike principles that sadly missed important details 29 . what might be termed, pseudo-hf approaches have arisen 30, 31 where systems-level thinking is eschewed for well-meaning but limited training, or other attempts at direct behavioral change that apply hf tools but do not consider systems complexity. the hf profession focuses on integrating humans and systems, and brings with it knowledge and experience of a range of concepts, principles, standards and methods. these are arguably better suited to understanding and resolving many of the problems and issues routinely experienced in highly complex, dynamic healthcare systems. consequently, hf professionals can spend significant amounts of time and energy challenging the myths and misunderstandings which continue to prevail, particularly amongst key groups and leaders, and trying to persuade some to unlearn what they think they know about hfe. among the widely-held and stubbornly persistent myths are: • the myth of the individual practitioner. instead of clinical outcomes being driven by a single clinician, they are driven by the collaboration of many individuals across a wide number of disciplines. this myth is perpetuated in, for example, physician league tables, despite clear evidence around the importance of teamwork, and organizations in outcomes 32 . • the myth of the perfect system. when harm happens, the tendency is to believe that this was an exception in an otherwise safe system. however, extensive observational evidence demonstrates that clinical systems are far from perfect 33 and require a wide range of work-arounds and trade-offs every day to be successful 34 .. • the myth that reliable care processes lead to safe systems. safety in complex care situations is an emergent property of the whole system, while reliability is a property of individual system components. every system component can function exactly as intended (reliability) but the complex interactions between components can lead to an unsafe system. 35 . • the myth of 'human error'. as people are always implicated in accidents, there has been an assumption that "removing" human error (or humans), would prevent harm. again, multiple safety scientists demonstrate that this a simplistic view of accident causation as it fails to acknowledge the everyday trade-offs humans in the system are required to make. indeed, it is the ability for humans to vary what they do that is responsible for everyday effective systems function. • the myth of standardization and control of variation. a further perturbation of these myths is the need to control individual human variation, and the ubiquitous call for "standardization". however, given that every patient is different, every unit, every specialty, and every organization is also different, variation is not only required, but is an essential part of patient-centered care. • the "zero" myth. though "zero" harms ("never events") may be useful goals, they are, by their nature, unattainable 29 . instead, risks, costs and the number of patients we treat need to be traded against each other. a system where zero harm could be achieved would have such a huge financial burden, and would have such a profound effect on care delivery, that it would fail in a range of other ways. the risk / benefit considerations and outcomes in response to the recent coivd-19 pandemic are an excellent example of how finances and treatment needs need to be carefully balanced with risks to patients and staff, often in the absence of clear data. • the myth of linear determinism. a further fallacious assumption is that process and outcome are directly related; thus a good outcome means everything went well (and no further attention is needed), while bad outcome must mean something went wrong, which requires investigation. again, safety science demonstrates the inadequacy of these assumptions. many things can and do go wrong in the care of a patient, but few affect outcomes, because the people in the system are always adapting to moment-to-moment demands in ways that technologies, policies and processes do not.. instead of understanding healthcare systems as complex and adaptive, where trade-offs judgements are being made between safety, cost and throughput all the time 36 , the pervading myths have led to the rigid application of approach or tools regardless of the context. the use of quality processes derived from highly engineered systems such as fmea and lean/6 sigma suit some contexts and not others, and at least has required adaptation for healthcare 13, 37 . checklists are a complex socio-technical intervention requiring understanding of the tasks, risks, design and use, so evaluating a checklist only on patient outcomes does not help understand the task, team, organizational or clinical specialty variations necessary for it to be adopted and effective 38, 39 . crew resource management or non-technical skills training is often delivered in the absence of appropriate organizational support, and without a sufficient understanding of the underlying engineering that made those approaches successful in aviation. there is no doubt that all are valuable but fail when applied without consideration of the wider systems in which they will be used. learning from other industries benefits from translation, based on an understanding of the mechanism-of-effect and differing constraints. this is where hf expertise can help. seeing hf as a set of 'tools' that can be 'applied' in a rule-based way, or trained in short course will have narrow benefits, and vastly under-appreciates the value that the discipline can bring. hf principles can be applied across highly diverse contexts, organizations and units, informing both improvement in complex systems, and the methods to hypothesize and evaluate them. the expertise of hf is in understanding the potential effects of interventions, and the selection of the appropriate evaluation from a wide range of methods. some clinicians who practice hf have embraced this more nuanced understanding of hf; some have not; and a few have been deeply resistant to this broader systems perspective. we need to think carefully about how to enhance the enthusiasm for hf, while challenging the range of misperceptions that limit its value. the use of hf tools by clinicians is an excellent place to start and should be encouraged, and supported with higher level hf. then, we need to find ways in which hf professionals and clinicians can work together to bring the right hf principles to bear in the right place, at the right time. despite a growing recognition of the need and value of hf in clinical work across the world, one consequence of the persistent misunderstanding of systems safety science is that few opportunities exist in clinical settings for qualified hf professionals, limiting the availability of appropriately sophisticated hf expertise 27 . until the fda mandated hf as part of device design in 2011 40 , hf healthcare work was funded through research, with much of it based at universities rather than hospitals, on a by-project basis rather than being applied full-time alongside and in support of clinicians. given that return-on-investment can be difficult to calculate, and effect on outcomes difficult to measure in a non-linear system, a direct business case is still hard to make. however, this did not prevent the expansion of quality improvement and patient specialists, who are mostly unqualified in their subject matter area, and lack appropriate professional development and regulation, unlike their hf counterparts. while the fda regulatory demands have vastly increased the number of hf experts within the healthcare industry, and there is much that could be learned from this about the development of and spread of hf expertise, device design is often far removed from clinical practice. professional healthcare hf practice outside of device development, has therefore been disparate, ad-hoc and precarious, with no formal career structure. these professional and financial constraints have also limited the ability of hf professionals to understand "work as done", the complexity of healthcare systems, or the new language and approaches required to address clinical needs. this has created a chicken-and-egg problem, where hf professionals have not been employed in care organizations, because there has been a limited understanding of what they can do, no clear and immediate application, no business case, no clear evidence base, and without day-to-day frontline experience of work-as-done they have not always been effective. a "simple checklist" or " training" are easier to package and demonstrate value, no matter how simplistic (and occasionally thoroughly erroneous 41 ), than a multitude of more appropriate approaches to improvement in complex adaptive systems 42 . this is a significant barrier to progress, but it has begun to change. in the last few years, hospitals across the world have begun to employ clinically embedded hf professionals -that is, fully qualified hf practitioners who work alongside clinicians every day to apply hf methods and perspectives. countries that have embedded hf practitioners include the usa, canada, australia, uk, and portugal, and most likely many others. positions have also been appearing in in regulatory roles. effective applied clinical practice thus requires both the technical demands of understanding when and how to apply hf approaches; and the understanding of the complexity of clinical context of a given application. healthcare organizations need to move beyond the underlying fixation on human errors and a lack of systems thinking. they need to know how they can employ hfe specialists and upskill key parts of the workforce through accredited routes at comparative low cost. they also need to know what sort of hf expertise to employ, and when. while there is formal route for hf professional accreditation (through professional societies such as hfes, ciehf, iea), few clinicians, patient safety administrators or quality improvement specialists who practice have any formal qualifications, training or appropriate cpd around risk/safety/hfe when compared with many of their equivalents in other industrial sectors. clinicians who champion hf causes would benefit from appropriate accreditation, and access to and wider support from qualified hf professionals. they may also benefit from understanding the limitations of and adaptions for popular but limited paradigms such as checklists, teamwork training, or non-technical skills. for example, effective teamwork skills in cardiac surgery are likely to be very different from the teamwork skills in robotic surgery 43 . surgeons or anesthesiologists are often well placed to lead this type of approach, but do not always appreciate how these skills fit into the broader clinical systems picture. broader and equally powerful principles -such as safety-ii, user-centered design, task analysis, selection and training, controls and displays, situational awareness, anthropometry -need to be applied in different ways to solve new problems and / or translate to a new context. they are also continually updating as our theoretical and methodological knowledge improves. in order to be effective at a local level, expertise needs to distributed rather than centralized. rather than working independently, we need to support the application of hf locally with national and international networks. often, clinicians and embedded hf practitioners work alone, or in small groups, often on the same problems as others in other organizations (for example, on clabsi, or failure-to-rescue, or retained foreign objects). there may be common themes, but solutions might look very different in different organizations, or even across different units. the ability to share learning and best practices, many of which will not end up in clinical publications, could have a powerful effect in spreading -and especially translating -good practices for different work contexts. such a network would also support embedded hf professionals who may be otherwise isolated from their peers and from new safety science, practice, or methodological development. there have also been a number of successful organizational approaches to support both clinicians applying hf and hf professionals working in healthcare. most notably the clinical human factors group (www.chfg.org) that originally worked to introduce aviation-style approaches but has subsequently begun to embrace the wider systems view. meanwhile, the uk's chartered institute (www.ciehf.org) has been increasingly bringing together clinicians and professionals work on specific projects, and has defined a set of skills, competence levels and training appropriate for clinical practice. in the usa, the human factors and ergonomics society (www.hfes.org) runs an annual conference devoted to healthcare human factors, while also supporting a specific healthcare technical group (www.hctg.hfes.org). meanwhile, the human factors transforming health network (hfth) (www.hfthnetwork.org) has been developed specifically to support embedded clinical hf practice, through a network of practitioners, clinical champions, and organizations aiming to deploy professional hf expertise. we anticipate increasing alignment of professional hf and clinical hf groups as the two fields continue to integrate. thus, despite the challenges of embedding appropriate, professional expertise into frontline clinical work, there is much to be positive about in the future. the rapid response to the covid-19 pandemic has demonstrated this. the spread of covid-19 exposed a range of challenges with the development, design, dissemination, education, assurance and integration of safe, effective healthcare delivery for a range of clinical contexts. this required substantial adaptation based on complex trade-off decisions between care demands, resources, risks and finances, often without clear authoritative evidence, but from continually evolving information. this can be seen as a 'micro systems' problem, where training based on the availability of specific personal protective equipment (ppe) required day-to-day adaptation by staff, based on varying threats and availability; and it can be seen as "macro" systems issue demonstrating close coupling of acute health, population health, society, commerce and politics. at a micro level, the availability of embedded practitioners in acute care demonstrated a range of ways in which hf expertise can be rapidly applied to help clinicians deal with the problem. it was possible to quickly disseminate this information via the hfth professional network. this work has included training in the use of ppe; designing icons to help with donning and doffing; working to support staff new to icu through decision support tools; prone-positioning protocols and checklists; covid-19 specific daily briefing checklists; supporting virtual visits; developing transportation plans; limiting exposure risks from suspected or confirmed patients; developing new protocols for emergency responses. the uk ciehf rapidly produced guidance on the design of ventilators and the design of work procedures for care teams in all sectors (https://covid19.ergonomics.org.uk/). several professionals have taken key leadership roles in the responses in their organizations. others were invited to be involved, including, importantly, in field hospitals where an influx of excess ventilated patients were/are expected. in most cases, it required the hf professional to actively insert themselves into the work, where their perspectives were valued. at a macro level, practices contributed both to the adaptive flexibility required for resilient systems (for example, by taking the load associated with protocol designs off already stretched clinicians), and to the wider understanding of how resilience can be built into future healthcare systems. this includes information sharing; risk perceptions; risk, cost, and throughput trade-offs; the need for adaptive skills rather than invariant process following; cross-training; planning for adaptability; and even the use of social media in the absence of other clinical guidance. indeed, this demonstrated exactly why the linear deterministic view of healthcare systems -where tasks are standardized, variability is controlled, and deviations are "errors" -is a deleterious over-simplification of healthcare work, even in otherwise 'normal' function. it is people that hold complex systems together; and the goal of is to understand and support the people working in the system to be adaptable and effective, rather than seeing them as a hazard 44 . in short, covid-19 demonstrates that the "new normal" has benefitted hugely from embedded hf expertise, but the key question is how to build on and sustain this welcome development beyond a pandemic situation. the ultimate goal of everyone involved in the application of hf principles in healthcare should be to improve the care we deliver to our patients and the working lives of our healthcare professionals. we need to understand and accept that there are multiple routes in, each with different requirements, and each need to be addressed with appropriate professional and practice support. areas where clinicians and others have applied hf techniques have been most notably in teamwork and teamwork training, simulation training, checklists, education and training, hazard identification and risk assessment, safety culture measurement and discussion, and team-based learning from events (table 1 contains links to some practical resources in these areas). however, a focus on these particular interventions are also symptomatic of the limited nature of application and the persistence of debunked safety science myths. hf professionals, on the other hand, have often struggled to bring their expertise to bear for meaningful clinical benefit. however, organizational, financial and evidence-based pressures are bringing about positive practice changes that is enabling embedded hf professional practice, and closer collaboration with clinical professionals, who now have access to appropriate professional certification and supportive professional networks dedicated to the application of healthcare hf. clinical systems offer vast potential for both specific improvement for patient and staff benefit, and as a natural laboratory for systems safety methodological and theoretical innovation. the interaction of hf and clinical work has already changed both professions. healthcare is at the forefront of hf frontiers; and hf is at the forefront of clinical frontiers. this is an exciting possibility that everyone involved is keen to realize. usa) www.hfes.org healthcare technical group www.hctg.hfes.org human factors and ergonomics society of australia www.ergonomics.org.au international ergonomics association www.iea.cc resilience engineering association www.resilience-engineering-association.org clinical support networks clinical human factors group www.chfg.org human factors transforming healthcare network www twelve tips for hf human factors in healthcare white paper systems thinking safety culture discussion cards 2dlb6c2 from safety i to safety ii: a white paper a human factors subsystems approach to trauma care human factors and ergonomics for primary care human factors in general practiceearly thoughts on the educational focus for specialty training and beyond human factors and simulation in emergency medicine health care human factors/ergonomics fieldwork in home and community settings human factors in medical device design: methods, principles, and guidelines a usability and safety analysis of electronic health records: a multi-center study resilient health care: turning patient safety on its head using an integrative mock-up simulation approach for evidence-based evaluation of operating room design prototypes leveraging health care simulation technology for human factors research: closing the gap between lab and bedside a uk perspective on human factors and patient safety education in pharmacy curricula twelve tips for embedding human factors and ergonomics principles in healthcare education human factors and ergonomics and quality improvement science: integrating approaches for safety in healthcare human factors systems approach to healthcare quality and patient safety building high reliability teams: progress and some reflections on teamwork training work systems analysis of sterile processing: decontamination the effect of teamwork training on team performance and clinical outcome in elective orthopaedic surgery: a controlled interrupted time series study impact of robotic surgery on decision making: perspectives of surgical teams evaluation of user-interface alert displays for clinical decision support systems for sepsis the mixed blessings of smart infusion devices and health care it human factors-based risk analysis to improve the safety of doffing enhanced personal protective equipment system factors affecting patient safety in the or: an analysis of safety threats and resiliency a work systems analysis of sterile processing: sterilization and case cart preparation. structural approaches to resilience and resilience engineering in health care enhancing the effectiveness of significant event analysis: exploring personal impact and applying systems thinking in primary care will human factors restore faith in the gmc? human factors in healthcare: welcome progress, but still scratching the surface world war ii and other historical influences on the formation of the still not safe: patient safety and the middle-managing of american medicine the science of human factors: separating fact from fiction spreading human factors expertise in healthcare: untangling the knots in people and systems the relationship between patient safety culture and patient outcomes: a systematic review improving patient safety by identifying latent failures in successful operations learning from positively deviant wards to improve patient safety: an observational study protocol engineering a safer world -systems thinking applied to safety resilient health care: turning patient safety on its head effect of a "lean" intervention to improve safety processes and outcomes on a surgical emergency unit the problem with checklists participatory design of a preliminary safety checklist for general practice applying human factors and usability engineering to medical devices: guidance for industry and food and drug administration staff changes in safety attitude and relationship to decreased postoperative morbidity and mortality following implementation of a checklist-based surgical safety intervention complexity science in healthcare -aspirations, approaches, applications and accomplishments: a white paper integration of robotic surgery into routine practice and impacts on communication, collaboration, and decision making: a realist process evaluation protocol overcoming covid-19: what can human factors and ergonomics offer? key: cord-018723-qd9ps3zb authors: kizza, joseph migga title: introduction to computer network vulnerabilities date: 2017-02-21 journal: guide to computer network security doi: 10.1007/978-3-319-55606-2_4 sha: doc_id: 18723 cord_uid: qd9ps3zb system vulnerabilities are weaknesses in the software or hardware on a server or a client that can be exploited by a determined intruder to gain access to or shut down a network. donald pipkin defines system vulnerability as a condition, a weakness of or an absence of security procedure, or technical, physical, or other controls that could be exploited by a threat [1]. the frequency of attacks in the last several years and the speed and spread of these attacks indicate serious security vulnerability problems in our network systems. there is no definitive list of all possible sources of these system vulnerabilities. many scholars and indeed many security incident reporting agencies, such as bugtraq, the mailing list for vulnerabilities; cert/cc, the us computer emergency response team; ntbugtraq, the mailing list for windows security; rus-cert, the german computer emergency response team; and us doe-ciac, the us department of energy computer incident advisory capability, have called attention to not only one but multiple factors that contribute to these security problems and pose obstacles to the security solutions. among the most frequently mentioned sources of security vulnerability problems in computer networks are design flaws, poor security management, incorrect implementation, internet technology vulnerability, the nature of intruder activity, the difficulty of fixing vulnerable systems, the limits of effectiveness of reactive solutions, and social engineering [2] . the two major components of a computer system, hardware and software, quite often have design flaws. hardware systems are less susceptible to design flaws than their software counterparts owing to less complexity, which makes them easier to test; limited number of possible inputs and expected outcomes, again making it easy to test and verify; and the long history of hardware engineering. but even with all these factors backing up hardware engineering, because of complexity in the new computer systems, design flaws are still common. but the biggest problems in system security vulnerability are due to software design flaws. a number of factors cause software design flaws, including overlooking security issues all together. however, three major factors contribute a great deal to software design flaws: human factors, software complexity, and trustworthy software sources [3] . in the human factor category, poor software performance can be a result of the following: 1. memory lapses and attentional failures: for example, someone was supposed to have removed or added a line of code, tested, or verified, but did not because of simple forgetfulness. 2. rush to finish: the result of pressure, most often from management, to get the product on the market either to cut development costs or to meet a client deadline can cause problems. 3. overconfidence and use of nonstandard or untested algorithms: before algorithms are fully tested by peers, they are put into the product line because they seem to have worked on a few test runs. 4. malice: software developers, like any other professionals, have malicious people in their ranks. bugs, viruses, and worms have been known to be embedded and downloaded in software, as is the case with trojan horse software, which boots itself at a timed location. as we will see in sect. 8.4, malice has traditionally been used for vendetta, personal gain (especially monetary), and just irresponsible amusement. although it is possible to safeguard against other types of human errors, it is very difficult to prevent malice. 5. complacency: when either an individual or a software producer has significant experience in software development, it is easy to overlook certain testing and other error control measures in those parts of software that were tested previously in a similar or related product, forgetting that no one software product can conform to all requirements in all environments. both software professionals and nonprofessionals who use software know the differences between software programming and hardware engineering. in these differences underlie many of the causes of software failure and poor performance. consider the following: 1. complexity: unlike hardwired programming in which it is easy to exhaust the possible outcomes on a given set of input sequences, in software programming a similar program may present billions of possible outcomes on the same input sequence. therefore, in software programming, one can never be sure of all the possibilities on any given input sequence. 2. difficult testing: there will never be a complete set of test programs to check software exhaustively for all bugs for a given input sequence. 3. ease of programming: the fact that software programming is easy to learn encourages many people with little formal training and education in the field to start developing programs, but many are not knowledgeable about good programming practices or able to check for errors. 4. misunderstanding of basic design specifications: this affects the subsequent design phases including coding, documenting, and testing. it also results in improper and ambiguous specifications of major components of the software and in ill-chosen and poorly defined internal program structures. there are thousands of software sources for the millions of software products on the market today. however, if we were required to name well-known software producers, very few of us would succeed in naming more than a handful. yet we buy software products every day without even ever minding their sources. most importantly, we do not care about the quality of that software, the honesty of the anonymous programmer, and of course its reliability as long as it does what we want it to do. even if we want to trace the authorship of the software product, it is impossible because software companies are closed within months of their opening. chances are when a software product is 2 years old, its producer is likely to be out of business. in addition to the difficulties in tracing the producers of software who go out of business as fast as they come in, there is also fear that such software may not even have been tested at all. the growth of the internet and the escalating costs of software production have led many small in-house software developers to use the marketplace as a giant testing laboratory through the use of beta testing, shareware, and freeware. shareware and freeware have a high potential of bringing hostile code into trusted systems. for some strange reason, the more popular the software product gets, the less it is tested. as software products make market inroads, their producers start thinking of producing new versions and releases with little to no testing of current versions. this leads to the growth of what is called a common genesis software product, where all its versions and releases are based on a common code. if such a code has not been fully tested, which is normally the case, then errors are carried through from version to version and from release to release. in the last several years, we have witnessed the growth of the open-source movement. it has been praised as a novel idea to break the monopoly and price gauging by big software producers and most important as a timely solution to poor software testing. those opposed to the movement have criticized it for being a source of untrusted and many times untested software. despite the wails of the critics, major open-source products such as linux operating system have turned out with few security flaws; still there are fears that hackers can look at the code and perhaps find a way to cause mischief or steal information. there has been a rise recently in trojan horses inserted into open-source code. in fact security experts are not recommending running readily available programs such as md5 hashes to ensure that the code hasn't been altered. using md5 hashes and similar programs such as md4, sha, and sha-1 continually compares codes generated by "healthy" software to hashes of programs in the field, thus exposing the trojans. according to the recent cert advisory, crackers are increasingly inserting trojans into the source code for tcpdump, a utility that monitors network traffic, and libpcap, a packet capture library tool [4] . however, according to the recent study by the aberdeen group, open-source software now accounts for more than half of all security advisories published in the past year by the computer emergency response team (cert). also according to industry study reports, open-source software commonly used in linux, unix, and network routing equipment accounted for 16 of the 29 security advisories during the first 10 months of 2002, and there is an upswing in new virus and trojan horse warnings for unix, linux, mac os x, and open-source software [4] . new developments in software engineering are spearheading new developments such as software reuse and software reengineering. software reuse is the integration and use of software assets from a previously developed system. it is the process in which old or updated software such as library, component, requirements and design documents, and design patterns is used along with new software. both software reengineering and reuse are hailed for cutting down on the escalating development and testing costs. they have brought efficiency by reducing time spent designing or coding, popularized standardization, and led to common "look-and-feel" between applications. they have made debugging easier through use of thoroughly tested designs and code. however, both software techniques have the potential to introduce security flaws in systems. among some of the security flaws that have been introduced into programming is first the mismatch where reused requirement specifications and designs may not completely match the real situation at hand and nonfunctional characteristics of code may not match those of the intended recipient. second, when using object programming, it is important to remember that objects are defined with certain attributes, and any new application using objects defined in terms of the old ones will inherit all their attributes. in chap. 6, we will discuss the many security problems associated with script programming. yet there is now momentum in script programming to bring more dynamism into web programming. scripting suffers from a list of problems including inadequate searching and/or browsing mechanisms before any interaction between the script code and the server or client software, side effects from software assets that are too large or too small for the projected interface, and undocumented interfaces. security management is both a technical and an administrative security process that involves security policies and controls that the organization decides to put in place to provide the required level of protection. in addition, it also involves security monitoring and evaluation of the effectiveness of those policies. the most effective way to meet these goals is to implement security risk assessment through a security policy and secure access to network resources through the use of firewalls and strong cryptography. these and others offer the security required for the different information systems in the organization in terms of integrity, confidentiality, and availability of that information. security management by itself is a complex process; however, if it is not well organized, it can result in a security nightmare for the organization. poor security management is a result of little control over security implementation, administration, and monitoring. it is a failure in having solid control of the security situation of the organization when the security administrator does not know who is setting the organization's security policy, administering security compliance, and who manages system security configurations and is in charge of security event and incident handling. in addition to the disarray in the security administration, implementation, and monitoring, a poor security administration team may even lack a plan for the wireless component of the network. as we will see in chap. 17, the rapid growth of wireless communication has brought with it serious security problems. there are so many things that can go wrong with security if security administration is poor. unless the organization has a solid security administration team with a sound security policy and secure security implementation, the organization's security may be compromised. an organization's system security is as good as its security policy and its access control policies and procedures and their implementation. good security management is made up of a number of implementable security components that include risk management, information security policies and procedures, standards, guidelines, information classification, security monitoring, and security education. these core components serve to protect the organization's resources: • a risk analysis will identify these assets, discover the threats that put them at risk, and estimate the possible damage and potential loss a company could endure if any of these threats become real. the results of the risk analysis help management construct a budget with the necessary funds to protect the recognized assets from their identified threats and develop applicable security policies that provide direction for security activities. security education takes this information to each and every employee. • security policies and procedures to create, implement, and enforce security issues that may include people and technology. • standards and guidelines to find ways, including automated solution for creating, updating, and tracking compliance of security policies across the organization. • information classification to manage the search, identification, and reduction of system vulnerabilities by establishing security configurations. • security monitoring to prevent and detect intrusions, consolidate event logs for future log and trend analysis, manage security events in real time, manage parameter security including multiple firewall reporting systems, and analyze security events enterprise-wide. • security education to bring security awareness to every employee of the organization and teach them their individual security responsibility. incorrect implantation very often is a result of incompatible interfaces. two product modules can be deployed and work together only if they are compatible. that means that the module must be additive, that is, the environment of the interface needs to remain intact. an incompatible interface, on the other hand, means that the introduction of the module has changed the existing interface in such a way that existing references to the interface can fail or behave incorrectly. this definition means that the things we do on the many system interfaces can result in incompatibility that results result in bad or incomplete implementation. for example, ordinary addition of a software or even an addition or removal of an argument to an existing software module may cause an imbalanced interface. this interface sensitivity tells us that it is possible because of interposition that the addition of a simple thing like a symbol or an additional condition can result in an incompatible interface, leading the new symbol or condition to conflict with all applications that have been without problems. to put the interface concept into a wide system framework, consider a systemwide integration of both hardware and software components with differing technologies with no standards. no information system products, whether hardware or software, are based on a standard that the industry has to follow. because of this, manufacturers and consumers must contend with the constant problems of system compatibility. because of the vast number of variables in information systems, especially network systems, involving both hardware and software, it is not possible to test or verify all combinations of hardware and software. consider, for example, that there are no standards in the software industry. software systems involve different models based on platforms and manufacturer. products are heterogeneous both semantically and syntactically. when two or more software modules are to interface one another in the sense that one may feed into the other or one may use the outputs of the other, incompatibility conditions may result from such an interaction. unless there are methodologies and algorithms for checking for interface compatibility, errors are transmitted from one module into another. for example, consider a typical interface created by a method call between software modules. such an interface always makes assumptions about the environment having the necessary availability constraints that the accessibility of local methods to certain states of the module. if such availability constraints are not checked before the modules are allowed to pass parameters via method calls, errors may result. incompatibility in system interfaces may be caused by a variety of conditions usually created by things such as: • too much detail • not enough understanding of the underlying parameters • poor communication during design • selecting the software or hardware modules before understanding the receiving software • ignoring integration issues • error in manual entry many security problems result from the incorrect implementation of both hardware and software. in fact, system reliability in both software and hardware is based on correct implementation, as is the security of the system. in sect. 4.2.1, we discussed design flaws in technology systems as one of the leading causes of system vulnerabilities. in fact we pointed out that systems are composed of software, hardware, and humanware. there are problems in each one of these components. since the humanware component is influenced by the technology in the software and hardware, we will not discuss this any further. the fact that computer and telecommunication technologies have developed at such an amazing and frightening speed and people have overwhelmingly embraced both of them has caused security experts to worry about the side effects of these booming technologies. there were reasons to worry. internet technology has been and continues to be vulnerable. there have been reports of all sorts of loopholes, weaknesses, and gaping holes in both software and hardware technologies. according to national vulnerability database (nvd), a us government repository of standards-based vulnerability management data using the security content automation protocol (scap), system vulnerabilities have been on the rise ever since system vulnerability data was first captured. the system vulnerability data captured by nvd enables automation of vulnerability management, security measurement, and compliance. nvd includes databases of security checklists, securityrelated software flaws, misconfigurations, product names, and impact metrics. read more about nvd at https://nvd.nist.gov/home.cfm. there is agreement among security experts that what is reported represents the tip of the iceberg. many vulnerabilities are discovered and, for various reasons, are not reported. because these technologies are used by many who are not security experts (in fact the majority of users are not security literate), one can say that many vulnerabilities are observed and probably not reported because those who observe them do not have the knowledge to classify what has been observed as a vulnerability. even if they do, they may not know how and where to report. no one knows how many of these vulnerabilities are there in both software and hardware. the assumption is that there are thousands. as history has shown us, a few are always discovered every day by hackers. although the list spans both hardware and software, the problem is more prevalent with software. in fact, software vulnerabilities can be put into four categories: • operating system vulnerabilities: operating systems are the main sources of all reported system vulnerabilities. going by the sysadmin, audit, network, and security (sans) institute, a cooperative research and education organization serving security professionals, auditors, system administrators, and network administrators, together with the common weakness enumeration (cwe), a community-developed dictionary of weaknesses of software types, has been issuing lists annually: "cwe/sans top 25 most dangerous software errors." popular operating systems cause many of the vulnerabilities. this is always so because hackers tend to take the easiest route by exploiting the best-known flaws with the most effective and widely known and available attack tools. • port-based vulnerabilities: besides operating systems, network service ports take second place in sourcing system vulnerabilities. for system administrators, knowing the list of most vulnerable ports can go a long way to help enhance system security by blocking those known ports at the firewall. such an operation, though not comprehensive, adds an extra layer of security to the network. in fact it is advisable that in addition to blocking and deny-everything filtering, security administrators should also monitor all ports including the blocked ones for intruders who entered the system by some other means. for the most common vulnerable port numbers, the reader is referred to the latest sans at: https:// www.sans.org/security-resources/idfaq/which-backdoors-live-on-which-ports/ 8/4. • application software-based errors. • system protocol software such as client and server browser. in addition to highlighting the need for system administrators to patch the most common vulnerabilities, we hope this will also help many organizations that lack the resources to train security personnel to have a choice of either focusing on the most current or the most persistent vulnerability. one would wonder why a vulnerability would remain among the most common year after year, while there are advisories on it and patches for it. the answer is not very farfetched, but simple: system administrators do not correct many of these flaws because they simply do not know which vulnerabilities are most dangerous; they are too busy to correct them all or they do not know how to correct them safely. although these vulnerabilities are cited, many of them year after year, as the most common vulnerabilities, there are traditionally thousands of vulnerabilities that hackers often use to attack systems. because they are so numerous and new ones are being discovered every day, many system administrators may be overwhelmed, which may lead to loss of focus on the need to ensure that all systems are protected against the most common attacks. let us take stock of what we have said so far. lots and lots of system vulnerabilities have been observed and documented by sans and cwe in their series, "cwe/sans top 25 most dangerous software errors." however, there is a stubborn persistence of a number of vulnerabilities making the list year after year. this observation, together with the nature of software, as we have explored in sect. 4.2.1, means it is possible that what has been observed so far is a very small fraction of a potential sea of vulnerabilities; many of them probably will never be discovered because software will ever be subjected to either unexpected input sequences or operated in unexpected environments. besides the inherently embedded vulnerabilities resulting from flawed designs, there are also vulnerabilities introduced in the operating environments as a result of incorrect implementations by operators. the products may not have weaknesses initially, but such weaknesses may be introduced as a result of bad or careless installations. for example, quite often products are shipped to customers with security features disabled, forcing the technology users to go through the difficult and error-prone process of properly enabling the security features by oneself. it is ironic that as "useful" technology develops so does the "bad" technology. what we call useful technology is the development in all computer and telecommunication technologies that are driving the internet, telecommunication, and the web. "bad" technology is the technology that system intruders are using to attack systems. unfortunately these technologies are all developing in tandem. in fact, there are times when it looks like hacker technologies are developing faster than the rest of the technologies. one thing is clear, though: hacker technology is flourishing. although it used to take intelligence, determination, enthusiasm, and perseverance to become a hacker, it now requires a good search engine, time, a little bit of knowledge of what to do, and owning a computer. there are thousands of hacker web sites with the latest in script technologies and hundreds of recipe books and sources on how to put together an impact virus or a worm and how to upload it. the ease of availability of these hacker tools; the ability of hackers to disguise their identities and locations; the automation of attack technology which further distances the attacker from the attack; the fact that attackers can go unidentified, limiting the fear of prosecution; and the ease of hacker knowledge acquisition have put a new twist in the art of hacking, making it seem easy and hence attracting more and younger disciples. besides the ease of becoming a hacker and acquiring hacker tools, because of the internet sprawl, hacker impact has become overwhelming, impressive, and more destructive in shorter times than ever before. take, for example, recent virus incidents such as the "i love you," "code red," "slammer," and the "blaster" worms' spread. these worms and viruses probably spread around the world much faster than the human cold virus and the dreaded severe acute respiratory syndrome (sars). what these incidents have demonstrated is that the turnaround time, the time a virus is first launched in the wild and the time it is first cited as affecting the system, is becoming incredibly shorter. both the turnaround time and the speed at which the virus or a worm spreads reduce the response time, the time a security incident is first cited in the system and the time an effective response to the incident should have been initiated. when the response time is very short, security experts do not have enough time to respond to a security incident effectively. in a broader framework, when the turnaround time is very short, system security experts who develop patches do not have enough time to reverse engineer and analyze the attack in order to produce counter immunization codes. it has been and it is still the case in many security incidents for antivirus companies to take hours and sometime days, such as in the case of the code red virus, to come up with an effective cure. however, even after a patch is developed, it takes time before it is filtered down to the system managers. meantime, the damage has already been done, and it is multiplying. likewise, system administrators and users have little time to protect their systems. in his testimony to the subcommittee on government efficiency, financial management, and intergovernmental relations of the us house committee on government reform, richard d. pethia, director, cert centers, pointed out the difficulty in fixing known system vulnerabilities as one of the sources of system vulnerabilities. his concern was based on a number of factors, including the everrising number of system vulnerabilities and the ability of system administrators to cope with the number of patches issued for these vulnerabilities. as the number of vulnerabilities rises, system and network administrators face a difficult situation. they are challenged with keeping up with all the systems they have and all the patches released for those systems. patches can be difficult to apply and might even have unexpected side effects as a result of compatibility issues [2] . besides the problem of keeping abreast of the number of vulnerabilities and the corresponding patches, there are also logistic problems between the time at which a vendor releases a security patch and the time at which a system administrator fixes the vulnerable computer system. there are several factors affecting the quick fixing of patches. sometimes, it is the logistics of the distribution of patches. many vendors disseminate the patches on their web sites; others send e-mail alerts. however, sometimes busy system administrators do not get around to these e-mails and security alerts until sometime after. sometimes, it can be months or years before the patches are implemented on a majority of the vulnerable computers. many system administrators are facing the same chronic problems: the neverending system maintenance, limited resources, and highly demanding management. under these conditions, the ever-increasing security system complexity, increasing system vulnerabilities, and the fact that many administrators do not fully understand the security risks, system administrators neither give security a high enough priority nor assign adequate resources. exacerbating the problem is the fact that the demand for skilled system administrators far exceeds the supply [2] . going by daily reports of system attacks and hacks, the number of system attacks is steadily on the rise. however, a small percentage of all attacks is reported, indicating a serious and growing systems security problem. however, given that just a small percentage of all attacks is reported, this table indicates a serious growing system security problem. as we have pointed out earlier, hacker technology is becoming more readily available, easier to get and assemble, more complex, and their effects more far reaching. all these indicate that urgent action is needed to find an effective solution to this monstrous problem. the security community, including scrupulous vendors, have come up with various solutions, some good and others not. in fact, in an unexpected reversal of fortunes, one of the new security problems is to find a "good" solution from among thousands of solutions and to find an "expert" security option from the many different views. are we reaching the limits of our efforts, as a community, to come up with a few good and effective solutions to this security problem? there are many signs to support an affirmative answer to this question. it is clear that we are reaching the limits of effectiveness of our reactive solutions. richard d. pethia gives the following reasons [2] : • the number of vulnerabilities in commercial off-the-shelf software is now at the level that it is virtually impossible for any but the best resourced organizations to keep up with the vulnerability fixes. • according to world internet usage and population statistics, (http://www. internetworldstats.com/stats.htm), with a 2016 global population of 7,340,093,980, there are currently 3,611,375,813 internet users representing almost half of the global population at 49.2%. this represents a growth of 90.4% since 2000. this is a phenomenal growth and it continues to grow at a rapid pace. at any point in time, there are millions of connected computers and smart mobile devices that are vulnerable to one form of attack or another. • attack technology has now advanced to the point where it is easy for attackers to take advantage of these vulnerable machines and harness them together to launch high-powered attacks. • many attacks are now fully automated, thus reducing the turnaround time even further as they spread around cyberspace. • the attack technology has become increasingly complex and in some cases intentionally stealthy, thus reducing the turnaround time and increasing the time it takes to discover and analyze the attack mechanisms in order to produce antidotes. • internet users have become increasingly dependent on the internet and now use it for many critical applications so that a relatively minor attack has the potential to cause huge damages. without being overly pessimistic, these factors, taken together, indicate that there is a high probability that more attacks are likely and since they are getting more complex and attacking more computers, they are likely to cause significant devastating economic losses and service disruptions. according to john palumbo, social engineering is an outside hacker's use of psychological tricks on legitimate users of a computer system in order to gain the information (usernames and passwords) one needs to gain access to the system [5] . many have classified social engineering as a diversion, in the process of system attack, on people's intelligence to utilize two human weaknesses: first, no one wants to be considered ignorant and second is human trust. ironically, these are two weaknesses that have made social engineering difficult to fight because no one wants to admit falling for it. this has made social engineering a critical system security hole. many hackers have and continue to use it to get into protected systems. kevin mitnick, the notorious hacker, used it successfully and was arguably one of the most ingenious hackers of our time; he was definitely very gifted with his ability to socially engineer just about anybody [5] . hackers use many approaches to social engineering, including the following [6] : • telephone. this is the most classic approach, in which hackers call up a targeted individual in a position of authority or relevance and initiate a conversation with the goal of gradually pulling information out of the target. this is done mostly to help desks and main telephone switch boards. caller id cannot help because hackers can bypass it through tricks and the target truly believes that the hacker is actually calling from inside the corporation. • online. hackers are harvesting a boom of vital information online from careless users. the reliance on and excessive use of the internet have resulted in people having several online accounts. currently an average user has about four to five accounts including one for home use, one for work, and an additional one or two for social or professional organizations. with many accounts, as probably any reader may concur, one is bound to forget some passwords, especially the least used ones. to overcome this problem, users mistakenly use one password on several accounts. hackers know this, and they regularly target these individuals with clever baits such as telling them they won lotteries or were finalists in sweepstakes where computers select winners or they have won a specific number of prizes in a lotto, where they were computer selected. however, in order to get the award, the user must fill in an online form, usually web-based, and this transmits the password to the hacker. hackers have used hundreds of tricks on unsuspecting users in order for them to surrender their passwords. • dumpster diving is now a growing technique of information theft not only in social engineering but more so in identity theft. the technique, also known as trashing, involves an information thief scavenging through individual and company dumpsters for information. large and critical information can be dug out of dumpsters and trash cans. dumpster diving can recover from dumpsters and trash cans individual social security numbers, bank accounts, individual vital records, and a whole list of personal and work-related information that gives the hackers the exact keys they need to unlock the network. • in person is the oldest of the information-stealing techniques that predates computers. it involves a person physically walking into an organization's offices and casually checking out note boards, trash diving into bathroom trash cans and company hallway dumpsters, and eating lunches together and initiating conversations with employees. in big companies, this can be done only on a few occasions before trusted friendships develop. from such friendships, information can be passed unconsciously. • snail mail is done in several ways and is not limited only to social engineering but has also been used in identity theft and a number of other crimes. it has been in the news recently because of identity theft. it is done in two ways: the hacker picks a victim and goes to the post office and puts in a change of address form to a new box number. this gives the hacker a way to intercept all snail mail of the victim. from the intercepted mail, the hacker can gather a great deal of information that may include the victim's bank and credit card account numbers and access control codes and pins by claiming to have forgotten his or her password or pin and requesting a reissue in the mail. in another form, the hacker drops a bogus survey in the victim's mailbox offering baits of cash award for completing a "few simple" questions and mailing them in. the questions, in fact, request far more than simple information from an unsuspecting victim. • impersonation is also an old trick played on unsuspecting victims by criminals for a number of goodies. these days the goodies are information. impersonation is generally acting out a victim's character role. it involves the hacker playing a role and passing himself or herself as the victim. in the role, the thief or hacker can then get into legitimate contacts that lead to the needed information. in large organizations with hundreds or thousands of employees scattered around the globe, it is very easy to impersonate a vice president or a chief operations officer. since most employees always want to look good to their bosses, they will end up supplying the requested information to the imposter. overall, social engineering is a cheap but rather threatening security problem that is very difficult to deal with. vulnerability assessment is a process that works on a system to identify, track, and manage the repair of vulnerabilities on the system. the assortment of items that are checked by this process in a system under review varies depending on the organization. it may include all desktops, servers, routers, and firewalls. most vulnerability assessment services will provide system administrators with: • network mapping and system fingerprinting of all known vulnerabilities • a complete vulnerability analysis and ranking of all exploitable weaknesses based on potential impact and likelihood of occurrence for all services on each host • prioritized list of misconfigurations in addition, at the end of the process, a final report is always produced detailing the findings and the best way to go about overcoming such vulnerabilities. this report consists of prioritized recommendations for mitigating or eliminating weaknesses, and based on an organization's operational schedule, it also contains recommendations of further reassessments of the system within given time intervals or on a regular basis. due to the massive growth of the number of companies and organizations owning their own networks, the growth of vulnerability monitoring technologies, the increase in network intrusions and attacks with viruses, and worldwide publicity of such attacks, there is a growing number of companies offering system vulnerability services. these services, targeting the internals and perimeter of the system, web-based applications, and providing a baseline to measure subsequent attacks against, include scanning, assessment and penetration testing, and application assessment. vulnerability scanning services provide a comprehensive security review of the system, including both the perimeter and system internals. the aim of this kind of scanning is to spot critical vulnerabilities and gaps in the system's security practices. comprehensive system scanning usually results in a number of both false positives and negatives. it is the job of the system administrator to find ways of dealing with these false positives and negatives. the final report produced after each scan consists of strategic advice and prioritized recommendations to ensure that critical holes are addressed first. system scanning can be scheduled, depending on the level of the requested scan, by the system user or the service provider, to run automatically and report by either automated or periodic e-mail to a designated user. the scans can also be stored on a secure server for future review. this phase of vulnerability assessment is a hands-on testing of a system for identified and unidentified vulnerabilities. all known hacking techniques and tools are tested during this phase to reproduce real-world attack scenarios. one of the outcomes of these real-life testings is that new and sometimes obscure vulnerabilities are found, processes and procedures of attack are identified, and sources and severity of vulnerabilities are categorized and prioritized based on the user-provided risks. as web applications become more widespread and more entrenched into e-commerce and all other commercial and business areas, applications are slowly becoming the main interface between the user and the network. the increased demands on applications have resulted into new directions in automation and dynamism of these applications. as we saw in chap. 6, scripting in web applications, for example, has opened a new security paradigm in system administration. many organizations have gotten sense of these dangers and are making substantial progress in protecting their systems from attacks via web-based applications. assessing the security of system applications is, therefore, becoming a special skills requirement needed to secure critical systems. vulnerability online services have many advantages for system administrators. they can, and actually always do, provide and develop signatures and updates for new vulnerabilities and automatically include them in the next scan. this eliminates the need for the system administrator to schedule periodic updates. reports from these services are very detailed not only on the vulnerabilities, sources of vulnerabilities, and existence of false positives, but they also focus on vulnerability identification and provide more information on system configuration that may not be readily available to system administrators. this information alone goes a long way in providing additional security awareness to security experts about additional avenues whereby systems may be attacked. the reports are then encrypted and stored in secure databases accessible only with the proper user credentials. this is because these reports contain critically vital data on the security of the system and they could, therefore, be a pot of gold for hackers if found. this additional care and awareness adds security to the system. probably, the best advantage to an overworked and many times resourcestrapped system administrator is the automated and regularly scheduled scan of all network resources. they provide, in addition, a badly needed third-party "security eye," thus helping the administrator to provide an objective yet independent security evaluation of the system. what is a vulnerability? what do you understand by a system vulnerability? 2. discuss four sources of system vulnerabilities what are the best ways to identify system vulnerabilities? what is innovative misuse? what role does it play in the search for solutions to system vulnerability? what is incomplete implementation? is it possible to deal with incomplete implementation as a way of dealing with system vulnerabilities? what is social engineering? why is it such a big issue yet so cheap to perform? is it possible to completely deal with it? why or why not? some have described social engineering as being perpetuated by our internal fears. discuss those fears what is the role of software security testing in the process of finding solutions to system vulnerabilities? some have sounded an apocalyptic voice as far as finding solutions to system vulnerabilities. should we take them seriously? what is innovative misuse? what role does it play in the search for solutions to system vulnerabilities? is it possible to locate all vulnerabilities in a network? in other words, can one make an authoritative list of those vulnerabilities? defend your response why are design flaws such a big issue in the study of vulnerability? part of the problem in design flaws involves issues associated with software verification and validation (v&v) information security: protecting the global enterprise information technology-essential but vulnerable: how prepared are we for attacks? ethical and social issues in the information age open source and linux: 2002 poster children for security problems social engineering: what is it, why is so little said about it and what can be done? sans social engineering fundamentals, part i: hacker tactics key: cord-015697-j22q89l9 authors: vanderwende, lucy; suzuki, hisami; brockett, chris; nenkova, ani title: beyond sumbasic: task-focused summarization with sentence simplification and lexical expansion date: 2007-04-19 journal: inf process manag doi: 10.1016/j.ipm.2007.01.023 sha: doc_id: 15697 cord_uid: j22q89l9 in recent years, there has been increased interest in topic-focused multi-document summarization. in this task, automatic summaries are produced in response to a specific information request, or topic, stated by the user. the system we have designed to accomplish this task comprises four main components: a generic extractive summarization system, a topic-focusing component, sentence simplification, and lexical expansion of topic words. this paper details each of these components, together with experiments designed to quantify their individual contributions. we include an analysis of our results on two large datasets commonly used to evaluate task-focused summarization, the duc2005 and duc2006 datasets, using automatic metrics. additionally, we include an analysis of our results on the duc2006 task according to human evaluation metrics. in the human evaluation of system summaries compared to human summaries, i.e., the pyramid method, our system ranked first out of 22 systems in terms of overall mean pyramid score; and in the human evaluation of summary responsiveness to the topic, our system ranked third out of 35 systems. in recent years, there has been increased interest in topic-focused multi-document summarization. in this task, automatic summaries are produced in response to a specific information request, or topic, stated by the user. in response to this interest and to stimulate research in this area, the national institute of standards and technology (nist) conducted a series of workshops, the document understanding conference 2 (duc), to provide large-scale common data sets for the evaluation of systems. participants in duc2005 and duc2006 were provided with a topic and a set of relevant documents (newswire articles), and the task was 0306-4573/$ -see front matter ó 2007 elsevier ltd. all rights reserved. doi:10.1016 all rights reserved. doi:10. /j.ipm.2007 to produce an automatic summary of not more than 250 words in length. consider the topic description for clusters d0652g and d0655a, for example: d0652g: identify the world's top banana producers and their levels of production. describe the main markets for imports and relate any major issues in the industry. d0655a: discuss the causes, effects, and treatment of childhood obesity. how widespread is it? in order to evaluate the summaries produced by the participants' systems, called peer summaries, duc provides four human summaries, model summaries, for comparison. several methods for summarization evaluation have been proposed. the automatic metric used in duc is rouge (lin, 2004) specifically, rouge-2, which calculates the overlap in bigrams between the peer and the four model summaries, and rouge-su4, which calculates the bigram overlap, but allowing up to 4 words to be skipped in order to identify a bigram match. automatic metrics are useful as they potentially allow a comparison between different system settings, as we show in section 6.1. however, since we are primarily interested in maximizing the content of our system's summaries, which, due to many issues of semantic realization such as paraphrase, cannot always be captured by measuring bigram overlap with four model summaries, we also evaluate our results with human evaluation metrics. human evaluation is time-consuming and must be conducted carefully, in the same experimental setting, in order to ensure comparison across systems. we participated in duc2006 in order to gain access to a human evaluation of our system, specifically the nist content responsiveness score. in addition, a second human evaluation is coordinated by columbia university, the pyramid method . the pyramid method requires two steps: first, a set of semantic equivalence classes are built for the sets of model summaries, with higher scores assigned to content represented in multiple model summaries, and second, a person identifies the content units in a peer summary that are found in the set of semantic equivalence classes. 3 the scores reported measure the amount of content overlap between the peer summary and the four model summaries. in this paper, we describe the multi-document summarization system we submitted to duc2006. our contribution in duc2006, identified as system 10, builds on an earlier system, sumbasic (nenkova & vanderwende, 2005) which produces generic multi-document summaries; we provide a description of sumbasic in section 2. we then describe each of the remaining three main components that comprise our system: a task-focused extractive summarization system, sentence simplification, and lexical expansion of topic words. we will provide experiments using automatic metrics designed to quantify the contributions of each component. human evaluation metrics, discussed in section 6.2, indicate that this is a relatively successful approach to multi-document summarization; in the pyramid evaluation, our system ranked first out of 22 systems and in the nist metrics for content responsiveness, our system ranked third out of 35 systems. with regard to our system design, it must be noted that this system, similar to almost all multi-document summarization systems, produces summaries by selecting sentences from the document set, either verbatim or with some simplification. using sentence simplification is a step towards generating new summary text, rather than extracting summaries from existing text. there is, however, no consideration for sentence ordering or cohesion other than that sentence ordering is determined exclusively as a result of the sentence selection process (see section 2 for details). sumbasic (nenkova & vanderwende, 2005) is a system that produces generic multi-document summaries. its design is motivated by the observation that words occurring frequently in the document cluster occur with higher probability in the human summaries than words occurring less frequently. specifically, sumbasic uses the following algorithm: step 1. compute the probability distribution over the words w i appearing in the input, p(w i ) for every i; pðw i þ ¼ n n , where n is the number of times the word appeared in the input, and n is the total number of content word tokens in the input. step 2. for each sentence s j in the input, assign a weight equal to the average probability of the words in the sentence, i.e., step 3. pick the best scoring sentence that contains the highest probability word. step 4. for each word w i in the sentence chosen at step 3, update their probability: step 5. if the desired summary length has not been reached, go back to step 2. steps 2 and 3 enforce the desired properties of the summarizer, i.e., that high frequency words from the input are very likely to appear in the human summaries. step 3 ensures that the highest probability word is included in the summary, thus each time a sentence is picked, the word with the highest probability at that point in the summary is also picked. step 4 serves a threefold purpose: 1. it gives the summarizer sensitivity to context. the notion of ''what is most important to include in the summary?'' changes depending on what information has already been included in the summary. in fact, while p old (w i ) can be considered as the probability with which the word w i will be included in the summary, p new (w i ) is an approximation of the probability that the word w i will appear in the summary twice. 2. by updating the probabilities in this intuitive way, we also allow words with initially low probability to have higher impact on the choice of subsequent sentences. 3. the update or word probability gives a natural way to deal with the redundancy in the multidocument input. no further checks for duplication seem to be necessary. the system resembles sum avr as recently described in nenkova, vanderwende, and mckeown (2006) except that the update function in sumbasic uses squaring rather than multiplication by a very small number. comparing sumbasic to luhn (1958) . the idea that simple frequency is indicative of importance in automatic summarization dates back to the seminal work of luhn (1958) . the sumbasic algorithm is distinct from luhn's algorithm, however, in several significant but illustrative ways. luhn first collects the frequencies of words in the document and identifies a subset of significant words, excluding the most frequent (what would now be termed ''stopwords'') and the least frequent (generally, words occurring less than four times). whereas sumbasic uses true initial probabilities and computes the weight of a sentence as equal to the average probability of the words in a sentence, luhn treats all significant words as having equal weight and computes the weight of a sentence as a function of the concentration of significant words in the sentence. this weight is obtained by defining windows as sequences of significant and non-significant words, such that there are no more than four non-significant words between any two significant words in a window; the weight of a sentence is equal to the weight of the highest scoring window, namely, the square root of the number of significant words in the window, divided by the total number of words in the window. finally, since luhn's system is designed to summarize single-documents, there is little need to prevent redundancy, in sharp contrast to multidocument summarization, where the likelihood that several documents might convey highly similar, or even identical, important information necessitates mechanisms to avoid redundancy, a functionality that sumbasic provides by updating the probability of the words on the basis of preceding selected sentences. nenkova and vanderwende (2005) document the impact of frequency on generic multi-document systems, but in the case of task-focused summarization, document frequency alone may not be predictive, since the topic may not be related to the most common information in the document set. thus, in order to incorporate topic constraints in generic summarization, our new system, called sumfocus, captures the information conveyed by the topic description by computing the word probabilities of the topic description. having done so, the weight for each word is computed as a linear combination of the unigram probabilities derived from the topic description, with backoff smoothing to assign words not appearing in the topic a very small probability, and the unigram probabilities from the document, in the following manner (all other aspects of sumbasic remain unchanged): the optimal value of k, 0.9, was empirically determined using the duc2005 corpus, manually optimizing on rouge-2 scores (henceforth r-2). since sentence selection is controlled by choosing the words with the highest weights, it is, in principle, possible for these ''best words'' to come from either the document or from the topic description. in practice, however, the best word is nearly always a word from the topic description due to the high value assigned to k. for duc2005 overall, 618 document words were identified as best on the basis of topic statements and only 22 independently on the basis of frequency alone. for duc2006 overall, all 600 best words were chosen from the topic statements. on the basis of duc2005 data, we added a small list of ''topic stopwords'' (describe, discuss, explain, identify, include, including, involve, involving) that did not receive any weight. the topic statements in duc2006 appear to contain more instructions to the summarizer than in duc2005, suggesting additional words may warrant similar handling (e.g., concerning, note, specify, give, examples, and involved). our goal is to create a summarization system that produces summaries with as much content as possible that satisfies the user, given a set limit on length. 4 since summaries produced by sumfocus alone are extractive, we view sentence simplification (also known as sentence shortening or sentence compression) as a means of creating more space within which to capture important content. the most common approach to sentence simplification for summarization purposes has been to deterministically shorten the sentences selected to be used in the summary. the classy system (conroy, schlesinger, & goldstein stewart, 2005) for example, incorporates a heuristic component for sentence simplification that preprocesses the sentences used in their sentence selection component. columbia university's summarization system uses a syntactic simplification component (siddharthan, nenkova, & mckeown, 2004) the results of which are sent to their sentence clustering component. daumé and marcu (2005a) employ a post-processing approach and report that deleting adverbs and attributive phrases improve rouge scores in the multilingual summarization evaluation, although this post-processing was not found to be useful in duc2005 (daumé & marcu, 2005b) conceivably because the summaries are 250 words long rather than 100 words long. in these approaches, simplification operations apply to all sentences equally, and the core sentence selection component has only either the original or the shortened sentence available to choose from. this may not be optimal, however, because the best simplification strategy is not necessarily the same for all sentences. for example, it might be desirable to delete material x from a sentence only if x is already covered by another sentence in the summary. for this reason, simplification strategies have so far remained conservative, presumably to avoid possible side-effects of oversimplification. an alternative approach to sentence simplification is to provide multiple shortened sentence candidates for the summarization engine to choose from. multi-document trimmer (zajic, dorr, lin, monz, & schwartz, 2005) for instance uses a syntactic simplification engine (dorr, zajic, & schwartz, 2003) initially developed for headline generation, to output multiple simplified versions of the sentences in the document cluster. each of these candidates are submitted to the feature-based sentence selection component, which includes the redundancy score of the sentence given the current state of the summary and the number of trimming operations as features. our approach to sentence simplification most closely resembles that used in the multi-document trimmer (zajic et al., 2005) : we apply a small set of heuristics to a parse tree to create alternatives, after which both the original sentence and (possibly multiple) simplified versions are available for selection. unlike mdt, however, in our system both original and alternative simplified sentences are provided for selection without differentiation, i.e., without retaining any explicit link between them, under the assumption that the sumbasic-based multi-document summarization engine is inherently equipped with the ability to handle redundancy, and the simplified alternatives only add to that redundancy. sumbasic's method for updating the unigram probabilities given the sentences already selected allows the simplified sentence alternatives to be considered independently, while maintaining redundancy at a minimum. 5 given that this approach to sentence simplification allows the sentence selection component to make the optimal decision among alternatives, we are thus freed to pursue more aggressive simplification, since the original non-simplified version is always available for selection. the approach is also extensible to incorporating novel sentence rewrites into a summary, moving in the direction of generative rather than extractive summaries. an example of this is jing and mckeown (2000) who propose a set of operations to edit extracted sentences, not limited to sentence reduction. it is straightforward to integrate such sentence rewrite operations into our framework, as candidate generation works independently of sentence selection, and word probability alone suffices to compute the sentence score. our simplification component consists of heuristic templates for the elimination of syntactic units based on parser output. each sentence in the document cluster is first parsed using a broad-coverage english parser (ringger, moore, charniak, vanderwende, & suzuki, 2004) . we then run a filter on the parse tree that eliminates certain nodes from the parse tree when the node matches the patterns provided heuristically. table 1 lists the syntactic patterns used in our duc2006 system. these patterns are inspired by and similar to those discussed in dunlavy et al. (2003) the principal difference being that extraction makes use of a full-fledged syntactic parser rather than employing a shallow parsing approach. for the first three patterns in table 1 (noun appositive, gerundive clause and non-restrictive relative clause), the parser returns a node label corresponding exactly to these patterns; we simply deleted the nodes with these labels. for the identification of intra-sentential attribution, we added specific conditions for detecting the verbs of attribution (said in table 1 ), its subject (the report), the complementizer (that) and adverbial expressions if any, and deleted the nodes when conditions were matched. in the case of sentence-initial adverbials, we delete only manner and time adverb expressions, the return to whaling will be a sort of homecoming for the makah, whose real name which cannot be written in english means ''people who live by the rocks and the seagulls'' intra-sentential attribution lead adverbials and conjunctions separately, the report said that the murder rate by indians in 1996 was 4 per 100,000, below the national average of 7.9 per 100,000, and less than the white rate of 4.9 per 100,000 using the features returned by the parser. currently, these patterns all apply simultaneously to create maximally one simplified sentence per input, but it would in principle be possible to generate multiple simplified candidates. finally, the punctuation and capitalization of the simplified sentences are cleaned up before the sentences are made available to the selection component along with their original, non-simplified counterparts in the document cluster. the results of applying this simplification filter are discussed in section 6. in addition to sentence simplification, we also investigated the potential for expanding the task terms with synonyms and morphologically-related forms. the use of query expansion has frequently been explored in information retrieval tasks, but without notable success (mitra, singhal, & buckley, 1998) . however, since the purpose of summarization is not to extract entire relevant documents from a large data set, but smaller sentences, it might be hypothesized that individual expansions could have more evident impact. in constructing our system, therefore, we augmented the task terms with lexical expansions supplied by morphological variants (chiefly derived forms) and synonyms or closely-related terms drawn from both static hand-crafted thesauri and dynamically-learned sources extracted from corpora. lexical expansions are applied only at the point where we choose the inventory of ''best words'' with which to determine candidate sentence selection. as already noted in section 3, in duc2006, the ''best words'' are drawn only from the topic statements, and not from the document collection. where a term in the sentence matches a lexical expansion, the formula for computing ''best word'' scores is as follows, where d is the document score of the lexical item in question, and e is the score for each type of matched expansion: the summation p n i e i is the expanded analog of the topicweight in the formula given for unexpanded topics in the discussion of sumfocus in section 3. the k is a uniform default weight applied to the cumulative scores of matches from all expansion types. for the purposes of our duc 2006 submission, the value of k was set at 0.5 after hand inspecting results for r-2 while tuning the system on the duc 2005 dataset. lexical expansion scores were not used to recompute the lexical probabilities of the sentences once they had been selected. morphologically-derived forms were looked up in the version of the american heritage dictionary used by our parser, thereby allowing us to obtain pairs such as develop m development. we also employed a small inventory of paired geographical names and their adjective counterparts, e.g., united states m american, china m chinese, tanzania m tanzanian. expansion scores for these morphological variants were computed on the basis of simple occurrence counts of the number of times the forms were encountered in the task description. thus, if both forms appeared in the topic text, both received a boost. since our implementation did not lemmatize the topic words, only exact matches were considered. a primary objective in investigating lexical expansions was to explore the potential impact of a 65,335-pair synonym list that we automatically acquired from clustered news articles available on the world wide web, the hypothesis here being that a thesaurus derived from news data might be prove more useful, and potentially domain-relevant than static general-domain synonym resources. starting with an initial dataset of 9.5 million sentences in $32,400 pre-clustered news articles, we created a monolingual bitext of $282,600 aligned sentence pairs using a crude heuristic similarity measure based on three criteria, namely, that the sentence pairs should have at least three words in common, have a minimum sentence length ratio of 66.6%, and have a word-based edit distance (i.e,, number of words inserted and deleted) of e 6 12. the reader is referred to quirk, brockett, and dolan (2004) dolan, quirk, and brockett (2004) for further information about the construction of this corpus. this was augmented by a further $20,000 sentence pairs extracted using a support vector machine applying lexical bootstrapping methods described in brockett and dolan (2005) who demonstrate that it is possible to apply giza++ (och & ney, 2003) to this (more or less comparable) corpus to yield an overall word alignment error rate as low as 12.46%. sentence pairs obtained in this fashion typically exhibit substantive semantic overlap, providing a dataset from which a potentially useful thesaurus might be extracted. an example sentence pair is shown in table 2 . the paired sentences were then tokenized, named entities and multiple word expressions were identified, and the words then lemmatized and tagged for part of speech. identical words were deleted from the pairs and the remainder aligned using a log likelihood ratio-based word association technique described in moore (2001) using the formula given in moore (2004) where t and s are variables ranging over the presence (1) or absence (0) of the two words under consideration, and c(t, s) is the observed joint count for their values. the probabilities used are maximum likelihood estimates. after extraction, the word pairs were further filtered to remove typographical errors, mismatches relating to numerical expressions and other artifacts of unnormalized news data. the pairs were then chained up to three steps to expand the dataset, and the association scores coerced into a range between 1.0 and 0.0 in order to generate a distribution that could be utilized by the system. these initial weights were then updated at runtime in the same manner as for morphological variants, by multiplying them by the number of times either member of a pair occurred in the topic description. no attempt was made to discriminate among word senses, in particular, where chaining might have resulted in mismatched terms. (in general, such mismatches would tend to have very low scores.) example synonyms and near synonyms for the word ''virus'' extracted in this manner are shown in table 3 . these range over two major senses, and also are not limited to the same part of speech. on the other hand, the data acquired in this manner can be relatively limited, and does not include, for example, names of major computer viruses, a limitation that in fact appears to have affected results in some newsclusters. in addition to the learned synonyms, we also considered expansions using data extracted from two static thesauri. the first was a list of 125,054 word pairs in the encarta thesaurus (rooney, 2001 ) which we deployed table 2 sample aligned sentence pair used to extract word associations indonesia's electoral commission has formally announced that susilo bambang yudhoyono, a former general and security minister, has won the country's first direct presidential election indonesian election officials today formally declared ex-general susilo bambang yudhoyono as victor in the country's first presidential poll after a final vote tally was completed in the submitted system. heuristic weights were precomputed in the form of a distribution based on number of synonyms for each word, allowing this data to be easily integrated with our acquired word association list. we also experimented with, but did not include in the system submitted to duc 2006, simple undisambiguated synonym expansion using wordnet 2.0 (fellbaum, 1998) no attempt was made to provide weights for the lexical expansions found in wordnet; instead raw occurrence counts were used as with morphological variants, with the uniform k applied in the same manner as to the other resources. table 4 compares the average rouge recall scores for word unigram (r-1), word bigram (r-2) and skip-4 bigram (r-su4) models achieved on duc 2005 data (used in training) and on duc 2006 data by different versions of our system. 6 numbers for sumbasic are presented as baseline. it is difficult to derive meaningful conclusions on the basis of the rouge results in table 4 . on duc 2005 data, sentence simplification consistently appears to improve matters modestly, but on duc 2006 data it paradoxically seems to introduce a small degradation in recall when deployed in conjunction with sumfocus. this appears to be partially offset by the application of lexical expansion. however, the differences in scores consistently fall within the 95% error margins computed by the rouge tool. moreover, p-scores computed using the wilcoxon matched-pairs signed-ranks test indicate that differences over a baseline sumbasic system without simplification were significant (p < 0.05) only on the duc 2005 data used in training. despite the somewhat inconclusive nature of the rouge results, they are suggestive of the relative contributions of different system components. table 5 presents the individual contributions made by various expansion strategies when used in conjunction with sentence simplification. the biggest observable impact comes from morphological expansion (mrf), while other lexical expansions, namely the encarta thesaurus (enc) and word association data (wa) may have contributed to the overall performance of the system to a lesser extent, if at all. mrf + enc + wa corresponds to the system submitted to duc 2006, i.e., sumfocus with lexical expansion and simplification. none of the differences are statistically significant at the level of p < 0.05 on the wilcoxon matched-pairs signed ranks test. table 5 also shows the potential impact of simple synonym expansion using wordnet 2.0 (fellbaum, 1998) which was not included in the submitted system, but which performs at or below baseline level in the table. results obtained from human evaluations are potentially more diagnostic and can inform the direction that future work should take. unfortunately, however, nist or pyramid, as one-time human evaluations, do not lend themselves to comparing system settings. nevertheless, since both nist and pyramid evaluation techniques measure content directly, these are the metrics we focus on, given our primary goal of maximizing summary content. accordingly, we report nist and pyramid metrics only for the system we submitted, which is sumfocus with sentence simplification and with lexical expansion of the topic words. pyramid evaluation. system 10 ranked first in the overall mean pyramid score of the 22 systems that participated in the pyramid evaluation. it must be noted, however, that the maximum pyramid score for each cluster differs since the agreement observed in the model summaries varies from cluster to cluster, and so we find that the average rank is a better method to compare systems, presented in table 6 . system 10 is ranked first for 5 out of 20 clusters, and is in the top 3 for half of the clusters. overall, our percluster mean ranking (5.90) is the best among the 22 systems. on the other hand, our performance across the 6 duc reports rouge numbers that are computed implementing the ''jackknife'' in which each human model summary is successively removed from the evaluation and added to the system (''peer'') summaries. this permits the human summaries to be directly compared with the system summaries using the same metric. for all experiments in this paper, however, we compute rouge without jackknifing, which allows us to use 4 model summaries. as a result, the official duc 2006 scores vary slightly from the numbers reported here; the differences, however, are not significant. clusters, also shown in table 6 , is not evenly distributed; system 10 scores worse than half of the systems for 5 out of 20 clusters. table 6 also presents the nist content responsiveness score, i.e., the human judgment of how well the content responds to the needs expressed in the topic description, on a scale from 1 to 5, where 5 is the highest achievable score. overall, system 10 ranked third on responsiveness. on a per-cluster basis, providing a system ranking according the nist content score is less than instructive since there are only 5 values that can be given. initial investigation showed there is no correlation between pyramid score and nist content score, but we have reached no conclusion yet. it is unexpected that for cluster d0601, system 10 is ranked highest among the peers in the pyramid evaluation, with a relatively high degree of scu overlap, while receiving a poor nist content score for the same cluster. looking at the pyramid analysis a bit more deeply, we also computed the percentage of the number of scus that were scored in system 10 and the number of scus that could be attained within the 250 word limit. we note that for only one cluster are more than half of the attainable scus found in system 10's peer summaries. improving this percentage is a clear direction for future research, though it remains an open question whether improving this percentage will lead to higher content responsiveness scores. nist evaluation. three of the five nist linguistic quality questions are relevant to sentence simplifcation: grammaticality, non-redundancy and referential clarity, along with the responsiveness question. nist evaluated these questions by eliciting human judgments on a five point scale. table 7 shows our duc2006 scores relative to the peers, along with the rank of our system among 35 peer systems. though our system (system 10) implements only a small number of simplification patterns, as described in section 4, its effect was quite extensive. in duc2006, of all the sentences selected in summary, 43.4% of them were the result of simplification. this resulted in adding on average another sentence to the summary: the average number of sentence in a summary increased from 11.32 to 12.52 when we used sentence simplification. the pyramid results suggest that making room for more content and removing redundant material by simplifying sentences is a promising operation for extractive summarization systems. it is also interesting to note that 33.6% of the sentences selected in the summaries were original non-simplified versions, even though a simplified counterpart was also available. manual investigation of the summary for one cluster (d0631d) established that four out of eleven sentences were non-simplified despite the availability of a simplified alternative. these four sentences are shown in table 8 . of these, two were incorrectly parsed as noun appositives, resulting in an unexpectedly large portion of the text being deleted and rendering the simplified version less likely to be selected. the other two sentences present interesting cases where the deleted portion of text (indicated in boldface in table 8 ) included important content, corresponding to summary content units (scus) observed in three or four of the model summaries according to the pyramid evaluation. this manual examination confirms that the best simplification strategy may not be the same in all cases, and that there are benefits in using the summarizer itself to choose the best sentence alternative given the context. sentence simplification undoubtedly contributes to our low score for grammaticality. in part this may be because the parser-based simplification filter can produce ungrammatical sentences and sentences with degraded readability owing to misplaced punctuation marks. however, grammaticality judgments may also be affected by whether or not a system allows incomplete last sentences. since the length limit in duc2006 was 250 words, some systems kept the summaries below 250 words, but permitted only complete sentences, while other systems truncated the summary at 250 words, even if the last sentence was incomplete. sumfocus falls into this latter category. only 2 systems allowing incomplete last sentences scored higher on grammaticality than those with complete last sentences, suggesting that while the low scores for grammaticality may be due to sentence simplification, they may also be a function of system design. referential quality may also be negatively affected by the current simplification filter, since deletion of intrasentential attribution can result in deleting the antecedent of pronouns in the summary. on the other hand, it is encouraging to note that our methods perform well on non-redundancy and content responsiveness. in particular, providing sentence alternatives did not increase redundancy, even though the alternatives were not explicitly linked, indicating that the method of updating unigram weights given context used in sumfocus is robust enough to handle the greater redundancy introduced by providing simplified alternatives. it is difficult to argue on the basis of either human or automated evaluation that lexical expansion significantly enhanced the performance of sumfocus, even when using simplification. we earlier noted the paucity of evidence that thesaurus-based query expansion (as opposed to adding search terms) significantly improves the relevance of ir search results: voorhees (1988) has suggested that use of wordnet may improve results of short queries, but not of longer ones. since the duc2006 tasks constitute moderately extended queries, it is perhaps unsurprising that the expansions had minimal effect. it is likely also that system-related factors mitigated the impact of lexical expansions: for example, it is probable that the means by which the expansion scores were computed may have resulted in many word pairs being scored too low to have any effect, while the use of a uniform expansion k failed to differentiate different expansion types adequately. analysis of rouge scores for individual clusters indicates that depending on the domain and type of task, results were in fact sometimes negatively impacted by the approach taken in sumfocus as opposed to sum-basic. in duc 2006, we observed low individual cluster scores when topic descriptions required the summary to identify a class of items: for example, a description that contains the instruction ''identify computer viruses detected worldwide,'' is expected to yield sentences containing the names of specific computer viruses, such as melissa, love bug and cih, rather than multiple exemplars of the term computer virus. here, it seems that even the use of dynamically-learned word associations was inadequate to this domain-specific task. in the long term, however, acquisition of better domain-relevant resources might be expected to ameliorate system performance in such cases. the pyramid annotation shows us that only rarely does a peer summary match 50% or more of the content in the combined model summaries. a detailed analysis of the percentage of scus per weight must still be table 8 examples of full sentences chosen instead of their simplified counterpart reason: parser error london british aviation authorities on wednesday formally ruled the concorde supersonic airliner unfit to fly unless its manufacturers took steps to prevent the problems that led to last month's fatal air france concorde crash near paris le figaro newspaper on wednesday quoted gayssot, the transport minister, as raising the possibility that the ban on air france concorde flights could remain in place until the accident and inquiry office releases a preliminary report on the crash at the end of august reason: deleted material contains important information paris french investigators looking into the crash last month of an air france concorde said thursday it was probable that a 16-in. piece of metal found on the runway caused a tire to blow out, sending debris from the tire through fuel tanks and triggering a fire that brought down the plane the sleek, needle-nosed aircraft could cross the atlantic at an altitude of 60,000 feet and at 1350 mph, completing the trip from london to new york in less than four hours -half the time of regular jets underlined portion of text was deleted in the simplified sentence. text corresponding to an scu is indicated by boldface. done, but anecdotally, our system matches only half of the high-scoring scus, i.e., those scus that were found in all of the model summaries. clearly, finding methods to model this data more closely offers opportunities for improving the overall content of summaries. one direction will lead us to find more sophisticated methods of modeling the relation between a topic word and any of its lexical expansions, the document words, and the target summaries. and, as we continue to leave purely extractive summarization behind and pursue summarization that generates novel sentences that better capture the information need of the user, we will also expand our system to take full advantage of sentence simplification component. in particular, we plan to include more drastic simplifying and rewriting operations (such as splitting coordinated clauses) and produce multiple candidates per sentence in order to see the full potential for the proposed approach. support vector machines for paraphrase identification and corpus construction classy query-based multi-document summarization bayesian multi-document summarization at mse bayesian summarization at duc and a suggestion for extrinsice evaluation unsupervised construction of large paraphrase corpora: exploiting massively parallel news sources hedge trimmer: a parse-and-trim approach to headline generation performance of a three-stage system for multi-document summarization wordnet: an electronic lexical database cut and past based text summarization rouge: a package for automatic evaluation of summaries the automatic creation of literature abstracts improving automatic query expansion towards a simple and accurate statistical approach to learning translation relationships among words association-based bilingual word alignment the impact of frequency on summarization a compositional context sensitive multidocument summarizer evaluating content selection in summarization: the pyramid method a systematic comparison of various statistical alignment models monolingual machine translation for paraphrase generation using the penn treebank to evaluate non-treebank parsers encarta thesaurus syntactic simplification for improving content selection in multi-document summarization wordnet: an electronic lexical database a sentence-trimming approach to multi-document summarization we are indebted to arul menezes for implementing several features described here. we also thank the nist team that supports duc for the nist evaluation, and we thank the duc community who contributed to the pyramid evaluation. we extend our appreciation to members of the butler-hill group, especially ben gelbart, for their assistance with the pyramid creation and annotation. finally, we are grateful to the anonymous reviewers for their many constructive comments. key: cord-325110-cfo5f99l authors: mirchi, nykan; ledwos, nicole; del maestro, rolando f. title: intelligent tutoring systems: re-envisioning surgical education in response to covid-19 date: 2020-09-10 journal: the canadian journal of neurological sciences. le journal canadien des sciences neurologiques doi: 10.1017/cjn.2020.202 sha: doc_id: 325110 cord_uid: cfo5f99l nan covid-19 has presented a series of unique challenges to surgical education. in the face of the global pandemic, access to traditional training techniques is limited. as health and safety have become top priorities, surgical residents have been reassigned to covid units and most elective surgeries have been either postponed or cancelled. for surgical trainees, these reductions mean the loss of crucial learning opportunities to practice technical psychomotor skills. for residents in their ultimate or pen-ultimate years of residency who are soon to complete their final exams, this is a significant concern. while online resources for operative anatomy are available and training in emergency surgical cases continue, the reduction in elective cases presents an important problem for residents. surgical educators are now faced with the challenge of ensuring their residents receive adequate training during this time. for both residents and surgical educators, it is impossible to predict how long this pandemic will last and what its effects on surgical training will be. advances in high-fidelity simulations provide residents with new opportunities to practice surgical skills and mitigate the challenges faced by current training paradigms including minimal access to standardized and objective feedback. these highfidelity simulations were first extensively employed in the aviation industry, where trainees are required to practice and be examined on simulators before becoming accredited. however, the development and integration of virtual reality simulation in surgical education has been limited, especially in fields involving complex bimanual operative skills such as neurosurgery. this has resulted in a slow adoption of high-fidelity simulation technology paradigms for the assessment, training, and evaluation of trainees in some surgical fields. the current global pandemic provides an opportunity to re-examine surgical education. 1 as many technical skills training programs are temporarily put on hold, residents as well as medical students are concerned about their ability to acquire the necessary skillsets to allow them to provide excellent care to their patients. simulators can be used to help trainees become familiar with specific surgical procedures, analyze 3d anatomical structures, and practice technical skills in risk-free environments. intelligent tutoring systems are automated teaching platforms capable of providing individualized performance feedback to learners using a variety of simulators. these systems, in combination with virtual reality simulators, allow important surgical training to continue even during a pandemic. intelligent tutoring systems integrated with virtual reality simulation allow operators to experience hyper-realistic tool interactions with the anatomical tissue. these systems can harvest the power of large datasets and utilize machine learning to automatically differentiate surgical expertise and provide feedback on operative performance. [2] [3] [4] the virtual operative assistant, an intelligent tutoring system powered by machine learning for neurosurgical simulation, discusses the limitations and challenges of this technology. 5 by deconstructing psychomotor skills into teachable metrics, trainees can obtain personalized feedback on specific factors identified by algorithms which may improve performance. an important factor when re-envisioning surgical education with automated teaching is how to accomplish a smooth integration of intelligent tutoring systems into current technical skills training. dedicated learning centers could be incorporated within active hospital environments, allowing timely and easy access to learners interested in improving surgical psychomotor performance. trainees could practice technical skills on the simulator to the level of mastery and obtain continuous feedback on performance in an iterative manner, with decreasing reliance on educator supervision. this learning-focused environment would provide trainees with an immersive and engaging way to practice and enhance their technical skills, all while practicing social distancing. a prototype for such a center is illustrated in figure 1 . we recognize that the cost associated with such a training center would not be insignificant. however, the value of their development and implementation need to be considered relative to the current toll of technical and procedural errors on patients and society. 6 mistakes in high-risk neurosurgical procedures also have high rates of medico-legal claims further increasing their impact on society. [7] [8] [9] the potential that training in these specialized centers will aid in the understanding and prevention of surgical errors needs to be rigorously explored. recognize that human interaction is vital to learning. rather, we believe that the integration and the wider availability of intelligent tutoring systems may complement present curricula while minimizing the impact of events such as a pandemic on trainees' skills development. surgical education programs will have to adapt in order to smoothly integrate automated and in-person education pedagogy. intelligent tutoring systems can utilize a variety of simulation platforms to provide almost unlimited opportunities for repetitive practice without constraints imposed by the availability of supervision. in these risk-free environments, numerous adaptable and clinically relevant simulations can be tailored to the needs of learners, consistent with best practices for simulationbased education. [10] [11] [12] these systems also increase the surgical educators' armamentarium to help learners achieve mastery levels of surgical performance. several studies are currently underway to compare the effectiveness of intelligent tutoring systems with more traditional formative teaching methods employed by surgical educators. the democratization of data accumulated from these simulation platforms is also important. 13 as simulators become more available across international training centers, it would be important to establish a centralized database of anonymized simulation data. this database should be accessible by research groups across the globe to develop more representative and generalizable models to power intelligent tutoring systems. as we navigate through these unprecedented times, the full impact of covid-19 on surgical education programs is yet to be ascertained. with continued research, increased development, and dissemination of intelligent tutoring systems, we can be better prepared for ever-evolving future challenges. innovations in neurosurgical education during the covid-19 pandemic: is it time to reexamine our neurosurgical training models? machine learning identification of surgical and operative factors associated with surgical expertise in virtual reality simulation artificial intelligence distinguishes surgical training levels in a virtual reality spinal task artificial neural networks to assess virtual reality anterior cervical discectomy performance the virtual operative assistant: an explainable artificial intelligence tool for simulation-based training in surgery and medicine medical errors in neurosurgery rates and characteristics of paid malpractice claims among us physicians by specialty, 1992-2014 malpractice risk according to physician specialty counting the cost of negligence in neurosurgery: lessons to be learned from 10 years of claims in the nhs comparative effectiveness of instructional design features in simulation-based education: systematic review and meta-analysis artificial intelligence in medical education: best practices using machine learning to assess surgical expertise in virtual reality simulation a comparison of visual rating scales and simulated virtual reality metrics in neurosurgical training: a generalizability theory study task force report on artificial intelligence and emerging digital technologies the authors are not advocating for the replacement of the present educational paradigms by automated systems. we this research did not receive any specific grant from funding agencies in the public, commercial, or not-for-profit sectors. nm and rfd conceptualized the manuscript. nm and nl wrote the manuscript with input and feedback from all authors. rfd was in charge of the overall direction and planning. key: cord-125402-9l4k3fle authors: darsena, donatella; gelli, giacinto; iudice, ivan; verde, francesco title: safe and reliable public transportation systems (salutary) in the covid-19 pandemic date: 2020-09-26 journal: nan doi: nan sha: doc_id: 125402 cord_uid: 9l4k3fle the aim of the salutary (safe and reliable public transportation systems) system is to employ modern information and communication technologies (ict) to proactively tackle crowding situations in public transportation (pt) systems,as a consequence of the limitations due to covid-19 pandemic. in particular, it is proposed to adopt in the various segments of the pt system (buses/trams/trains, railway/subway stations, and bus stops) suitable crowd detection techniques based on internet of things (iot) technologies, which measure in real-time the number of users, in order to: (i) monitor and predict crowding events; (ii) adapt in real-time pt system operations, i.e., modifying service frequency, timetables, routes, and so on; (iii) inform the users by electronic displays installed in correspondence of the bus stops/stations and/or by mobile transport applications. the salutary system can be implemented incrementally, as an add-on to the intelligent transportation system (its) solution already in use by major pt companies operating in urban areas. the system is designed as a flexible platform, which can be used to deliver, in addition to the innovative crowd detection/management functionalities, also additional services, such as on-line ticketing, vehicle access control and reservation in severely crowded situations, and evolved crowd-aware route planning. o ne of the most significant repercussions of coronavirus disease (covid-19) pandemic will regard the transport sector and mobility, in particular public transportation (pt) systems in urban areas. a recent survey carried out in china [1] estimated that, as a consequence of the outbreak, the use of private cars will be roughly doubled, increasing from 34% to 66%, whereas the use of public transports (buses/metros) will be more than halved, dropping down from 56% to 24%. furthermore, due to the lack of trust in pt systems, more than manuscript received september xx, 2020; revised xxx yy, 2020; accepted zzzz 2020. date of publication xxx 2020; date of current version yyy 2020. this work was supported in part by..... the associate editor coordinating the review of this manuscript and approving it for publication was dr. x. d. darsena is with the department of engineering, parthenope university, naples i-80143, italy (e-mail: darsena@uniparthenope.it). i. iudice is with italian aerospace research centre (cira), capua i-81043, italy (e-mail: i.iudice@cira.it). g. gelli and f. verde are with the department of electrical engineering and information technology, university federico ii, naples i-80125, italy [e-mail: (gelli,f.verde)@unina.it]. d. darsena, g. gelli, and f. verde are also with the national inter-university consortium for telecommunications (cnit), 80125 naples, italy. digital object identifier xxxx/yyyyyyyy 70% of the surveyed people not owing a car declared their intention to buy a new one, with negative consequences on the environment (landscape and air pollution) in urban areas. in italy, both national and regional authorities issued a number of rules aimed at limiting pt usage as well as avoiding bus and metro overcrowding, which severely limit their capacity to about 60% of the nominal capacity before the outbreak. the effects of such limitations are still not fully perceived, due to the breakthrough of mobility patterns associated to the widespread use of smart working and e-learning practices in education. however, it is expected that they will severely affect the fruition of pt in the recovery phase, when the transport demand will start to increase. moreover, since new outbreaks might be coming in the next months/years, it is likely that some features of pt systems must be adapted to structurally cope with these limitations. in many countries, to counteract the shift to private car usage, governments are favoring the use of individual sustainable mobility and micromobility means, such as bikes, electrical scooters, and segways, and they are planning and deploying the related infrastructures (emergency bike lanes) or powering vehicle sharing services, which can shift to this transport mode a certain percentage of short and medium-distance trips. however, owing to the large number of passengers carried by pt systems in urban areas, it is of utmost importance to adopt measures aimed at assuring safe and reliable pt usage. to this aim, the first adopted measures (during the lockdown) were devoted to minimizing the contagion risk, such as backdoor boarding, cashless operation, frequent sanitization of vehicles and stations, enforcing social distances, requiring the passengers to wear personal protection equipment (e.g., face masks and/or gloves). other medium-term measures are focused on intervening on pt system operations, such as modifying timetables, frequencies, paths, leveraging modal integration, and so on. some of these measures, like increasing pt service frequency to compensate for reduced vehicle capacity, are seen by pt companies as effective [2] , but not sustainable in the long term, due to the limited number of drivers and vehicles. thus, in the re-opening phase, when a capacity-limited pt system must cope with increasing volumes of passengers (especially with the reprise of school and university activities), it will be mandatory to dispose in real-time of reliable and capillary information about the crowding of pt vehicles (e.g., bus, tram, and trains) and of the related access infrastructures (e.g., bus stops and railway stations). such a knowledge will allow one to implement the necessary social distancing measures, while at the same time assuring an adequate level of service. real-time knowledge of crowding data can be used for fast or even proactive adaptation of pt operations (e.g., increasing the service frequency, reallocating vehicles from one line to another, planning alternative routes), in order to cope with spatially and/or temporally localized crowding situations, which cannot be tackled by conventional (statistical) tools used in transportation system design, such as, e.g., analysis of origin-destination flows. a significant performance improvement in crowd avoidance approaches can be ensured by exploiting information gathered by intelligent transportation systems (itss). in a modern its, commonly available data are automated vehicle location (avl) -via global positioning system (gps) -and automatic passenger count (apc) or automatic fare collection (afc). in [3] , optimization and planning strategies based on such data are surveyed, at four different levels: strategic, tactical, operational, and real-time. in particular, it is mentioned that the lack of passenger arrival information, especially in realtime, is a limiting factor for accurate studies. it is also stated [4] that "the availability of real-time passenger demand data can significantly improve the performance of control models in case of overcrowding". to overcome such limitations, and cope with covid-19related issues, a fundamental role can be played by information and communication technologies (ict) and internet of things (iot) technologies, some of which are already employed in state-of-the-art its. in particular, the fifth generation (5g) of cellular networks is potentially able to support massive iot connections, where billions of smart devices can be connected to the internet. in [5] , the use of new technologies, such as iot, unmanned aerial vehicles (uavs), artificial intelligence (ai), blockchain and 5g, for managing the impact of covid-19 in health applications has been considered. in [6] , [7] a review of technologies for social distancing has been provided, with emphasis on wireless technologies for positioning, including crowd detection and people density estimation. to the best of our knowledge, the application of ict and iot, particularly crowd-detection techniques, to cope with covid-19 effects in a pt system has not been considered yet. in this paper, we propose a new system concept, called salu-tary (safe and reliable public transportation system), aimed at integrating the legacy its, which is already available in many pt systems in urban areas, for fleet management and travel information purposes. specifically, the salutary system is built upon a distributed iot subsystem, composed by a network of capillary active/passive sensor, aimed at measuring passenger crowding in the different segments of the pt system (buses, trams, trains, bus stops, and railway/metro stations). the acquired measures are transmitted in real-time by a communication infrastructure to a control subsystem, which performs optimized and proactive management of the pt system. the crowding data can also be reported (in aggregated form, for privacy concerns) to the pt users, by means of ad hoc mobile applications (apps) or by integrating it in the existing mobile transport apps (e.g., moovit or proprietary operator applications). since crowding on pt systems has a big impact on travel satisfaction [8] , we are confident that the added features will be of interest for improving the quality of service also when the covid-19 limitations will be finally removed. the paper is organized as follows. in section ii, related work on crowd analysis, detection, and prediction is discussed. an overview of the salutary system concept and its architecture is given in section iii. various sensing solutions are presented in section iv for different indoor and outdoor scenarios of interest. section v is devoted to highlight innovations and advantages provided by the salutary system. finally, conclusions are drawn in section vi. estimating and predicting some features of a "crowd" in indoor and outdoor locations is an active research topic, with many applications, including surveillance and security, situation awareness, emergencies, and crowd management. in our application, the feature of interest is the number of components of the crowd and/or its density. a review of crowd analysis techniques for urban applications, including transportation systems, is provided in [9] , where different data sources are compared and discussed, including information fusion approaches. when dedicated crowd detection infrastructures are not available, crowdsourcing or participatory sensing 1 applications have been developed to acquire such information [10] , [11] . applications of crowd analysis to pt systems is described in [12] and [13] . in [12] , in particular, it is described a lowcost hardware/software solution to estimate the current number of passengers in a vehicle, by counting the number of probe requests, i.e., medium access control (mac) addresses, sent by wifi-equipped (ieee 802.11) devices in the vehicle. in [13] , such a passenger counting technique is incorporated in a bus navigation system capable of giving crowd-aware route recommendations: such a system has been tested in the municipal bus infrastructure of madrid, spain. one of the problem inherent to the use of techniques based on radio frequency (rf) for crowd sensing is the ability to distinguish between people outside the bus and actual passengers. this issue was tackled in [12] , [13] by filtering the probes by a sliding window, aimed at removing mac addresses that were not detected over a longer period of time. the system was able to detect only around 20% of the passengers, since several users may have turned off the wifi interface. moreover, the system is only tailored at crowd analysis in buses, and do not take into account other scenarios (trains, bus stops, and railway stations). in [14] , a crowdsensing-based pt information service is described, called trafficinfo, which exploits mobile or participatory sensing to enrich the static travel information without requiring deployment of a vehicle tracking infrastructure. a solution to count the people in a queue, based on bluetooth low-energy (ble) devices, is described in [15] . the system is completely passive and estimates the number of persons in the queue by analyzing the mean and variance of the received signal strength indicator (rssi) values between a ble beacon and a receiver covering a certain area. in general, rssibased approaches for crowd analysis exhibit good performance only for small monitored environments, where the propagation channel variation is mainly dominated by the attenuation caused by the present people. on the other hand, in a rich scattering environment, crowd analysis based on channel state information (csi) provides a more reliable people counting. in this respect, the received wifi and long term evolution (lte) downlink control signals are processed in [16] and [17] , respectively, to extract the changes of the propagation channel induced by the presence of different number of people. crowd prediction algorithms can be classified on the basis of the prediction horizon into short-term (less than 60 minutes) and long-term ones. they usually rely on three different types of information: (i) real-time load station data, (ii) real-time train load data, and (iii) historical data. the latter data aim to improve prediction by exploiting possible cyclic daily, weekly, or seasonal variations in passenger loads. prediction approaches can be further classified, on the basis of the prediction methodology, into model-based methods or data-driven ones. model-based methods include time-series analysis, regression modeling or kalman filtering models. in [18] , e.g., a traffic flow prediction algorithm based on timeseries is employed, and many variants have been subsequently proposed to improve the prediction accuracy. due to the highly nonlinear and random nature of traffic flow, datadriven approaches have recently gained significant attention, including k-nearest neighbor (k-nn) methods and artificial neural networks techniques (see, e.g., [19] , [20] and references therein). in some cases, simulation approaches, which employ traffic simulation tools to predict traffic flow, have been considered as well. most of these techniques are mainly devoted to congestion management in urban transportation and can exploit information from a multitude of sensor sources, including inductive loops, radars, cameras, gps, social media, etc. passenger load prediction for pt systems, instead, is less common: recent studies are [21] , [22] , [23] . the aim of the salutary system is to integrate/augment the its system already available in a pt urban system with innovative functionalities, aimed at smart and proactive control and reduction of passenger crowding. it is based on the integration of heterogenous sensing and communication technologies, depending on the operation scenario and the ict infrastructure available in the urban area where the system must be implemented. detailed system design involves strong interdisciplinary skills, including transportation engineering, telecommunications, computer science, electronics, data analyis, artificial intelligence and machine learning (ml). the salutary system is composed (fig. 1 ) by three subsystems: 1) the sensing and actuator subsystem (sass); 2) the communication subsystem (css); 3) the monitoring, prediction and control subsystem (mpcss). the proposed system introduces new data flows (marked in blue in fig. 1 ) compared to existing data exchanges (in violet) found in state-of-the-art its systems for pt services. the core and main innovative part of the system is the sass, with particular reference to the iot sensing technologies, whereas the actuator part mainly encompasses the flow of information toward the users, such as audio speakers, variable-message panels or displays, which are typically already present in the pt system, or can be readily installed at the bus stops or in the railway station (and even inside the vehicles). due to its centrality in our concept, we focus attention on such a subsystem in section iv. the characteristics of the css are strongly dependent on the communications infrastructure available in the urban area of interest, or the communication network owned by the pt service operator. in general, they encompass public wireless networks (such as cellular networks) and/or private wired and/or wireless networks owned by the operator, such as e.g. global system for mobile railway (gsm-r) or lte for railway (lte-r). to cope with this heterogeneity, it is envisioned that, at the protocol level stack, the css can be readily interfaced with the other subsystems by using standard interfaces and/or by means of simple adaptation layers. the mpcss performs data collection and real-time crowding prediction, also by means of ai and ml techniques. based on such predictions, modifications to the transport services can be implemented in real-time, the related control data are sent to service operators (drivers, supervisors, etc.), whereas service information is sent to the passengers by means of displays and/or mobile transport apps. this information could be notified by the same applications to all the users of the pt, so as to discourage the access to overcrowded stations and/or bus stops, and propose alternative travel solutions. in our concept, the mpcss is strongly integrated (and typically colocated with) the its control system of the pt service operator. the transportation scenarios to be considered for the sass are essentially of three types: 1) train and bus/tram (indoor scenario); 2) railway/metro station (indoor/outdoor scenario); 3) bus station (mainly outdoor scenario). in general, the sensing solutions to be adopted belong to the crowd detection family introduced in sec. ii-a. they can be classified [24] as visual-based (vb) solutions, based on still or moving images acquired by optical, thermal, or laser cameras, and non-visual based (nvb), which utilize techniques not based on optical sensors (mainly rf devices). the main characteristics of the available sensing solutions for the different scenarios are summarized in table i and will be discussed in the forthcoming subsections. thanks to recent advances in ai, traditional optical, thermal and laser vb sensors are becoming "smart" and can detect, recognize, and identify persons. optical cameras are widely used in private and public spaces for surveillance and security. thermal cameras can detect people in low-light environments, complete darkness, or other challenging conditions, such as smoke-filled and dusty environments. another option to detect and track persons is represented by laser imaging detection and ranging (lidar) sensors, especially in environments where there are several interacting people. however, these systems do not allow to estimate the number of present people with sufficient accuracy, due to possible obstructions, clutter, and poor light/weather conditions, and involve high deployment costs in complex environments. moreover, they require electrical power supply and may involve privacy problems. the main advantage of vb technologies is that they do not require cooperation/participation of the users. among nvb technologies, sensing systems based on mobile devices (also called crowdsensing or mobile sensing [25] , [26] ) represent an interesting solution, due to the diffusion of smartphones and other portable/wearable devices, such as pedometers, smart watches, or biometrical sensors. on one hand, compared to vb solutions, data collected by means of device-aided nvb systems can be used not only to count persons, but also to gather additional information, e.g., planned routes, passengers using off-peak hours group ticket, and so on. on the other hand, a problem inherent to deviceaided systems is that they usually require the cooperation of the people who must agree to share their data (so called participatory or collaborative sensing). to motivate participation, it is sometimes needed to introduce incentive or reward mechanisms, or introduce radical modifications in the procedures to access the pt service, such as an authentication phase to use the transportation service. although this issue could raise significant privacy concerns, it could also be useful as a means to increase the overall safety of the pt systems, by reducing the risk that infected people can access the system or the execution of possible crimes from people already known to the police. when device-aided nvb techniques cannot be used for crowd characterization, due to, e.g., lack of user cooperation and/or security issues, rf non-device aided approaches can be pursued, by analyzing the propagation channel variations of wireless signals (e.g. wifi or lte), which are induced by the people present in a given spatial area. the impact of the monitored people on the rf signal can be assessed either using traditional radar methodologies (range and doppler analysis) or by analyzing features extracted by channel quality measurements, such as rssi and csi. for vehicular scenarios, straightforward nvb solutions for passenger counting are passive infrared sensors (ir). in this case, a couple of ir emitter and receiver are mounted in correspondence of each gate of the vehicle. a passive ir (pir) sensor can detect variations of the ir signal, depending on the temperature and the characteristics of the surface interrupting the beam. more generally, many technologies are available to solve such a "crowd counting" problem in scenarios characterized by a well delimited space with a limited number of accesses. in the transportation community, apc systems [27] for rail and road vehicles are widespread and mainly based on simple technologies, such as counting from the number of afc or by using independent sensors, typically infrared (active) or pyroelectric (passive) sensors, or combinations thereof, aimed at detecting the passage of people at the gates. other techniques can be based on load sensors placed on the ground or on the suspensions, or pressure-sensitive switches ("treadle mats") placed on the steps, or weigh-in-motion (wim) systems, which estimate the number of passengers by the ground loading of the vehicles detected before and after the stops. more sophisticated techniques can also be employed, such as camera-based systems or combined sensing (such as ir plus rf or ir plus ultrasound). as indicated in table i , even though more sophisticated nvb technologies may be used as well, their usage is not expected to lead to significant innovations in this scenario, compared to the aforementioned simpler solutions and systems (i.e., pir, apc, afc, and wim). for this scenario, both vb and nvb technologies can be applied, as reported in table i . among the latter ones, near field communication (nfc) represents a viable solution that does not need to deploy special hardware [28] . nowadays, android and ios phones/tablets support this technology, by allowing mobile devices to act as a nfc reader/scanner. when a device with nfc functionality appears in the reader's working range, which can be placed at the gates or at any other fixed access point, it "wakes up" and sends a signal containing encoded data. the reader receives and decodes such data, and sends them to a management entity. another interesting solution for such a scenario can be to install 802.11 (wifi) access points in the stations, and the number of persons entering the stations can be estimated on the basis of the number of connections established by the user devices with the access points. in this case, the uplink signals transmitted by the user devices are sniffed to capture their network accesses and, thus, it can be categorized as a device-aided solution. in contrast, without requiring aid to the monitored devices, the number of users served by a wifi access point can be estimated by sniffing the downlink control information transmitted by the access point and relying on the fact that, as expected, there is a strong correlation between the number of people in the considered area and the variations of some features (e.g., rssi and csi) of the signal received by a dedicated device designed as a people counter. similarly to wifi networks, lte signals can be used for device-aided and non-device aided crowd counting, thanks to their ubiquitous availability and good penetration in indoor environments. for instance, they could be available in areas where the wifi coverage is not present, such as remote and small railways/metro stations. this scenario is by far the most difficult to manage, since bus stops are usually located in outdoor space not well delimited by fixed gates. also in this case, both vb and nvb systems can be used, but it is imperative to adopt costeffective, rugged and low-power solutions, in order to reduce the maintenance cost, as well as solutions that do not require significant infrastructures, since in many cases the bus stops are not equipped with shelters and are indicated by simple poles. bluetooth can represent an efficient solution: recently, a low-energy consumption version of the standard, called ble, has been introduced, which assures better communication performances with a limited power consumption. so-called bluetooth beacons, namely, small transmitting devices that transmit ble signals to compatible devices, can be easily installed at the bus stations, since they can be battery-powered and are characterized by simpler flexibility and simplified installation compared to wifi. this solution relies on the same technology introduced by google and apple in recent versions of smartphone operating systems, and is used by many national contact-tracing apps (e.g., immuni for italy [29] ). lte and 5g signals can also be exploited for counting people in bus stop environments. in particular, the deployment of 5g wireless technology over the coming decade is expected to rely on the introdution of many small cells working in the millimeter-wave (mmw) band. from the viewpoint of crowd analysis, the placement of small cells allows one to more precisely monitor spatially limited areas, like bus stops, with and without the aid of user devices. in addition, at bus stops, microwave sar tomography techniques [30] , [31] could provide specific images from which more detailed crowd information can be extracted, by using complex image classification algorithms (i.e., count, distribution, mobility, etc.); model issues induced by the intrinsic near-field scenario (e.g., typical for bus stops) could be overcome by using specialized algorithms [32] . the salutary system can provide original and innovative functionalities that are not present in the legacy its: proactive control of station access: in railway/subway applications, on the basis of the knowledge of the number of passengers aboard the arriving trains and the prediction of those getting off at the station, it will be possible to program the number of station accesses with a low error margin and in real-time. this number can be displayed at the users (by displays at the station gates or by the mobile transport apps) and can be used by the security operators to filter passengers at the turnstiles. priority policies can be envisioned, such as taking into account the time already spent in queue, or the trip motivations (e.g., a priority could be assured to health workers, disabled or elder users, law enforcement, and teachers/students). vehicle access reservation: in bus/tram trips, a vehicle access reservation system can be implemented, wherein a sensor at the bus stop detects the user presence and exchanges information with its device (i.e, the smartphone), so as to grant him/her the access to board the first arriving bus (a virtual queuing system) or putting him/her in an overbooking list (with priority) to allow him/her to board the next one. the application can generate an e-ticket with the access grant (e.g., a qr-code) that can be validated on board at the ticket machine. crowding information dissemination: users receive information related to capacity (in terms of number of seats or in percentage) of the bus/trains in arrival and/or the crowding at the bus stops/stations by means of displays installed in correspondence of the bus stops or at the entrance of the stations, or by alerts issued by mobile transport apps, so as to avoid unnecessary waiting and crowding and possibly reschedule their movements. crowd-aware route planning: users can plan their trips on the mobile transport app, by taking into account not only geographical information and traveling times (static data), but also traffic and crowding information about vehicles and bus stops/stations during the trip (dynamic data). the app can suggest not necessarily the shortest route, but the least crowded one, taking into account also crowding levels measured during the trip. such a feature not only help reduce crowds and the consequent infection risk, but also distribute more efficiently the load on the transportation network. at first sight, the salutary system can be seen as a technique aimed at enforcing social distancing measures. however, its scope is wider, since at the same time it tries to optimize the performance of the pt system. in practice, the aim of salutary is to increase the flexibility and adaptability of the pt system, to partially recover the drawbacks and inefficiencies due to the adoption of rigid social distancing measures, and to avoid the use of private cars with the obvious negative impact on the air quality in urban areas. the main advantages of salutary system are summarized in tab. ii. in addition, the salutary system can also be used at the end of the pandemic, allowing for more efficient planning and real-time control of the operation of the pt system, compared to static methods, like traditional survey-based compilation of origin-destination matrix flows. the large amount of generated data can be used by ai and ml algorithms to better understand and plan a series of aspects generally associated with improvements of the quality of life in urban areas and smart cities. the salutary system can also be useful to allow schools and universities to open as safely and quickly as possible for in-person learning. indeed, to enable schools and universities to open and remain open, it is important to adopt and implement actions to slow down the spread of covid-19, not only inside the school, but also in the pt system, by optimally and dynamically adjusting transport timetables. we presented in this paper the main functionalities and some potential applications of the salutary system concept. in particular, system-level considerations were reported to corroborate the feasibility of integrating state-of-the-art wireless iot technologies into the different segments of the pt infrastructure. the proposed system is based on some basic components and subsystems, to be used as building blocks to implement an evolved its, capable of real-time monitoring and predicting crowd situations, as well as disseminating useful information to users at the bus stops/stations and/or through mobile transport apps. some features of the salutary system are similar to those of a contact tracing system, which can be implemented more easily if based on user cooperation. in this sense, the crowd detection functionalities can be incorporated in a more complex system, which can implement, besides the typical mobile transport app functionalities (like moovit) also the possibility to buy tickets, to reserve the access to the vehicles, in conditions of particular crowding. this could represent a decisive incentive to the use of the system. however, the more appropriate crowd sensing solution must be singled out caseby-case, in dependence on the ict infrastructure owned by the pt operator, of the socio-economic context, and the costbenefits ratio. the potentials of the salutary system go beyond the scope of dealing with typical social distancing problems, by potentially allowing real-time optimization and management of the pt system. for instance, the success in preventing the introduction and subsequent transmission of covid-19 in schools and universities is strongly dependent on preventing disease transmission in the pt system used by students, families, teachers, school staff, and all community members. impact of coronavirus to new car purchase in china evolution of mobility sector during and beyond covid-19 emergency: a viewpoint of industry consultancies and public transport companies combining its and optimization in public transportation planning: state of the art and future research paths real-time holding control for high-frequency transit with dynamics a comprehensive review of the covid-19 pandemic and the role of iot, drones, ai, blockchain, and 5g in managing its impact a comprehensive survey of enabling and emerging technologies for social distancingã¢è�å¤part i: fundamentals and enabling technologies a comprehensive survey of enabling and emerging technologies for social distancing ã¢è�å¤ part ii: emerging technologies and open issues examining the factors that impact upon public transport commuting stress advances in crowd analysis for urban applications through urban event detection cooperative transit tracking using smart-phones how long to wait? predicting bus arrival time with mobile phone based participatory sensing crowd density estimation for public transport vehicles an internet-of-things enabled connected navigation system for urban bus riders crowdsending based public transport information service in smart cities cutiqueue: people counting in waiting lines using bluetooth low energy based passive presence detection device-free people counting in iot environments: new insights, results, and open challenges lte signals for device-free crowd density estimation through csi secant set and svd short-term prediction of traffic volume in urban arterials dynamic near-term traffic flow prediction: system-oriented approach based on past experiences supervised weighting-online learning algorithm for short-term traffic flow prediction a mesoscopic transit assignment model including real-time predictive information on crowding a real-time passenger flow estimation and prediction method for urban bus transit systems data-driven metro train crowding prediction based on real-time load data crowd analysis using visual and non-visual sensors, a survey a survey on mobile crowdsensing systems: challenges, solutions, and opportunities a review of mobile crowdsourcing architectures and challenges: toward crowd-empowered internet-of-things automatic passenger counting and vehicle load monitoring near field communication immuni documentation extended wavenumber-domain synthetic aperture radar focusing with integrated motion compensation sar tomography with optimized constellation and its application to forested scenes fast imaging of vegetation on gpus based on non-uniform ffts the authors would like to thank the ieee comsoc/vts italian chapter for supporting this work by awarding the first prize to the salutary proposal within the call for ideas in response to covid-19 outbreak in italy. key: cord-304070-jw1lxwyd authors: lapinsky, stephen e; wax, randy; showalter, randy; martinez-motta, j carlos; hallett, david; mehta, sangeeta; burry, lisa; stewart, thomas e title: prospective evaluation of an internet-linked handheld computer critical care knowledge access system date: 2004-10-14 journal: crit care doi: 10.1186/cc2967 sha: doc_id: 304070 cord_uid: jw1lxwyd introduction: critical care physicians may benefit from immediate access to medical reference material. we evaluated the feasibility and potential benefits of a handheld computer based knowledge access system linking a central academic intensive care unit (icu) to multiple community-based icus. methods: four community hospital icus with 17 physicians participated in this prospective interventional study. following training in the use of an internet-linked, updateable handheld computer knowledge access system, the physicians used the handheld devices in their clinical environment for a 12-month intervention period. feasibility of the system was evaluated by tracking use of the handheld computer and by conducting surveys and focus group discussions. before and after the intervention period, participants underwent simulated patient care scenarios designed to evaluate the information sources they accessed, as well as the speed and quality of their decision making. participants generated admission orders during each scenario, which were scored by blinded evaluators. results: ten physicians (59%) used the system regularly, predominantly for nonmedical applications (median 32.8/month, interquartile range [iqr] 28.3–126.8), with medical software accessed less often (median 9/month, iqr 3.7–13.7). eight out of 13 physicians (62%) who completed the final scenarios chose to use the handheld computer for information access. the median time to access information on the handheld handheld computer was 19 s (iqr 15–40 s). this group exhibited a significant improvement in admission order score as compared with those who used other resources (p = 0.018). benefits and barriers to use of this technology were identified. conclusion: an updateable handheld computer system is feasible as a means of point-of-care access to medical reference material and may improve clinical decision making. however, during the study, acceptance of the system was variable. improved training and new technology may overcome some of the barriers we identified. the rate of expansion of medical knowledge is increasing rapidly, and it is frequently difficult for clinicians to keep abreast of important new literature. for example, several recently published randomized controlled trials in critical care have demonstrated mortality benefits [1] [2] [3] [4] [5] , but uptake of new knowledge into clinical practice is often delayed [6] [7] [8] . improving access to this knowledge base at the point of care may lead to better clinical decision making, which could improve patient outcome, reduce costs and optimize bed utilization [9] . in critical care, rapid access to medical reference information may be particularly important in facilitating timely management decisions and avoiding errors [10] . computing technology can allow point-of-care access to upto-date medical reference material [11] . a study evaluating a mobile computerized cart to make evidence available to clinicians in an internal medicine setting [12] demonstrated that evidence-based medicine was more likely to be incorporated into patient care when the computerized system was used. because of their portability, handheld devices may be more practical tools for disseminating knowledge to the point of care. despite the popularity of handheld devices in medicine, few studies have evaluated the usefulness of this technology [13] . before widespread dissemination of this type of technology can be encouraged, its impact must be thoroughly evaluated [14] . in the present study we evaluated whether it would be feasible and effective to provide updateable reference information from a central academic centre to handheld computers used by critical care specialists in community hospitals. a total of 17 intensivists at four community hospital intensive care units (icus) in the greater toronto area participated in the present prospective interventional study. after training, each physician was equipped with a handheld computing device (palm m505; palm inc., milpitas, ca, usa) loaded with medical reference material pertinent to the critical care physician. this information included a customized critical care information handbook ('critical care'), which was previously developed for use by residents and physicians at our centre (additional file 1). commercially available medical reference software was also incorporated, namely pepid ed (pepid llc, skokie, il, usa) and medcalc http://med calc.med-ia.net/. the handheld devices were able to receive literature updates on a regular basis, using customized software (iqsync; infiniq software, mississauga, ontario, canada), which accessed an internet-based server using either a connection via desktop computer or infrared data transfer to a telephone modem (fig. 1 ). new information was sent to the handheld devices and appeared in a file called 'what's new'. these updates, provided every 2-3 weeks, comprised brief reviews of relevant new literature including a short summary, a commentary and the article abstract. all handheld devices were equipped with backup software that allowed the content to be rapidly restored in the event of a hardware failure (backupbuddy vfs; blue nomad software, redwood city, ca, usa). the devices were also equipped with software capable of generating a log of the applications used (appusage; benc software production, slavonski brod, croatia). between september and november 2002 the handheld devices were distributed to participating physicians, at which time they each received a 1-hour training session on the use of the handheld device and the internet link (fig. 2) . after training, the participants were able to utilize the devices in clinical practice for 12 months. we provided 24-hour support by telephone and e-mail, with a website for independent review. feasibility feasibility of the system was assessed by tracking physicians' use of the handheld device and tracking their access of the individual handheld applications during the study period. physicians who updated their handheld computers at least once a month for 6 months were identified as 'regular users'. a qualitative assessment of the system was achieved through surveys and focus group methodology. participants completed surveys at baseline to identify their prior familiarity with handheld devices, and at the end of the study period to evaluate subjectively the handheld reference system and the individual handheld applications. survey data were scored on a 7-point scale, in which 'poor' scored 1 and 'excellent' scored 7. an independent company (the nrc+picker group, markham, canada) conducted the focus group evaluations at the end of the intervention period, to determine the perceived utility of the information system. each hospital physician group participated in one focus group meeting. information sources that physicians accessed to make clinical decisions were evaluated during simulated patient care scenarios, completed in the physicians' own icu utilizing a computerized patient simulator (simman; laerdal medical corporation, wappingers falls, ny, usa). each physician completed one scenario before the handheld device was introduced (baseline scenario) and one at the end of the intervention period (final scenario), when the handheld device could be used (fig. 2) . a small pool of five scenarios with equivalent complexity was developed, such that physicians would likely need to access information sources in order to make management decisions. the scenarios involved unusual but important conditions, namely thyroid storm, myasthenia gravis, methanol toxicity, malaria and methemoglobinaemia. they were allocated to study participants in such a way as to avoid participants from the same site receiving the same scenario at the same time point, and to avoid repetition of scenarios among individual participants. each scenario concluded with the physician writing admission orders for the simulated patient. during the scenarios we tracked all medical reference sources utilized by the physicians, who were encouraged to use a 'think the internet-based data transfer system the internet-based data transfer system. updated information is downloaded to the handheld device from a study server. connection to the internet can take place via hardwire synchronization with a desktop computer or using infrared (ir) data transmission to a dial-up modem. isp, internet service provider. the study time course the study time course. aloud' process [15] . an audiovisual recording was made of the scenarios for later analysis, and when the handheld was used real-time screen capture was incorporated into the recording (additional file 2). this allowed us to document which handheld applications were accessed, the time taken to access information and the time taken to complete the scenario. we developed an objective scoring system for the admission orders generated at each scenario. the admission orders were assigned a score (range 0-100) by a critical care physician (sm) and critical care pharmacist (lb), who were blinded as to whether the physician used the handheld device. the scenario-specific scoring system allocated points for all necessary diagnostic and therapeutic interventions, weighted according to relative importance. negative points were given for potentially harmful orders. data are presented as median and interquartile range (iqr), and permutation tests were used for comparisons because numbers were small and not normally distributed. the differences between the final and baseline admission order scores and the time to completion of scenarios were calculated for each participant. a two-sample permutation test was used to compare these differences between the group of physicians who chose to use the handheld in the final scenario and those who did not use the device. admission order scores obtained for each of the five scenarios were compared. outcomes were considered statistically significant at α < 0.05. the sas system for window version 8.2 (sas institute, inc., cary, nc, usa) was used for all analyses. focus groups were recorded, transcribed verbatim and subsequently analyzed. themes were identified and unique perspectives on key issues noted [16] . the handheld information system functioned well during the study period. tracking of the deployment of handhelds identified 10 regular users (59%), four physicians (23%) who used the system variably and three physicians (18%) who never used their handheld device. the regular users accessed the personal information management applications more commonly (median 32.8 times/month, iqr 28.3-126.8) than the medical software (median 9/month, iqr 3.7-13.7; p = 0.028), although significant variation was noted (table 1) . baseline survey data identified that, of the 17 critical care physicians participating, 12 (71%) had previous experience with handheld devices (nine had used the palm operating system, and three had used windows ce) for a median duration of 1 year (range 1 month to 3.8 years). seven participants (41%) reported using handhelds for accessing medical information before the study. of the 16 final survey respondents, seven (44%) felt that the handheld system had had a positive impact on their clinical practice. the handheld medical applications (critical care, what's new, medcalc and pepid) received similar ratings, with overall evaluation scores ranging from 4.1 to 5.3 on the 7-point scale. four focus group meetings, involving a total of 13 participants (76%), identified the benefits and barriers to use of handhelds for information access, and made suggestions for improvement ( table 2 ). the overall impression of participants was that there is a role for handhelds for mobile information access, but that in situations away from the bedside other electronic media such as desktop computers were preferable. not all study physicians were able to participate in the simulated clinical scenarios on the pre-assigned day. fourteen physicians (82.3%) participated in the baseline scenarios and 13 (76.5%) in the final scenarios. information sources utilized during the baseline scenarios included the internet (50% of participants; e.g. medline searches and electronic textbooks), textbooks (43%), telephoning colleagues, the icu pharmacist or poison control centre (71%), and other sources such as pocket guides (21%). in the final scenarios, the handheld device was used as the primary source of information by eight participants (62%; table 3 ). of 14 information searches on the handheld device, 11 searches (79%) were successful and the median time to access information was 19 s (iqr 15-40 s). the information sources of those participants not using the handheld device were similar to those in the baseline surveys ( fig. 3 ). when scores recorded for each of the five clinical scenarios were compared, no significant difference was noted, reducing the likelihood that scenario assignment influenced outcomes. this study demonstrates the feasibility of using an electronic knowledge translation system to provide high quality, regularly updated medical reference information from a central academic centre to multiple peripheral users. user acceptance of this technology was not uniform, with just over half of the participants using their handheld devices to access information on a regular basis. nevertheless, the availability of point-ofcare access to information may have improved the quality of clinical decision-making. although mobile computing devices have potential beneficial roles to play in clinical medicine, few publications describe formal evaluation of this technology [13] . because the present study was an early hypothesis-generating evaluation of this technology, multiple quantitative and qualitative outcomes were measured. we generated novel data on the use of handheld devices in a clinical situation, but the study has several limitations. the number of physicians involved was relatively small, with a significant proportion not utilizing the technology. the allocation of clinical scenarios was not randomized, because they were allocated predominantly to avoid using the same scenario at the same site and time point. however, the analysis performed compared participants who used the handheld with those who did not; because it was not known which participant would use the handheld at the time of allocation of scenarios, potential bias was minimized. furthermore, the scenarios appeared to be equivalent in difficulty because no difference was noted when scores for the individual scenarios were compared. a confounding factor in the study was the outbreak of sars (severe acute respiratory syndrome) from march to may 2003, which had a significant impact on the study icus [17] . participants were advised to avoid using their handhelds during patient contact because of the potential to transmit infection, and this affected continuity of the study. had we not encountered this event, utilization might have been higher. the lack of universal acceptance of this technology is not surprising and may be due to a number of factors, including inadequate training and the lack of familiarity with the technology [18] . training is essential when introducing handheld computing technology [19, 20] and, although all users underwent a training programme, the surveys and focus groups indicated a need for improvement. familiarity with handhelds is increasing, with 33% of all canadian physicians and 53% of under 35year-olds using these devices in 2003, but these levels of utilization remain relatively low when compared with use of the internet, at 88% [21] . increasing familiarity with the technology will probably increase acceptance of such a system. other potential barriers to use of the handheld system may be addressed by the rapidly developing technology, including improved screen resolution, ease of data entry and wireless connectivity. acceptance may be increased through the development of an all-in-one package on the handheld, allowing additional functionality such as decision support, billing, electronic prescribing and communication. the study demonstrated the potential role of an updateable handheld information system for knowledge translation in critical care. rapid access to current clinical guidelines may be a valuable component of a comprehensive solution to reducing error and improving efficiency. information access may be most beneficial in areas without full-time critical care physicians, particularly given the current imbalance between demand and supply with critical care physicians, which is expected to worsen [9, 10] . recent recommendations highlight the importance of leveraging information technology to standardize practice and promote efficiency in critical care [10] . handheld information access alone is unlikely to change clinical practice, but it should be considered a component of an electronic knowledge translation system. in many situations other media, such as desktop or tablet computers, may be preferable for information access. although the study was carried out in a critical care environment, such a system is probably applicable to other specialties in which clinicians are mobile and may not have ready access to a desktop computer (for example, anaesthesia, emergency medicine, home care). this study provides insight into the potential impact of this technology in improving health care outcomes [14] . nevertheless, further study that builds on our findings is essential to determine how these new technologies can best be incorporated into the patient care setting. a handheld computer system is feasible as a means of providing point-of-care access to medical reference material in the icu. during this study acceptance of this system was variable, and improved training and more advanced technology may be required to overcome some of the barriers we identified. in clinical simulations, use of such a system appeared to improve clinical decision-making. the acute respiratory distress network: ventilation with lower tidal volumes as compared with traditional tidal volumes for acute lung injury and the acute respiratory distress syndrome efficacy and safety of recombinant human activated protein c for severe sepsis bouillon r: intensive insulin therapy in critically ill patients effect of treatment with low doses of hydrocortisone and fludrocortisone on mortality in patients with septic shock early goal-directed therapy collaborative group: early goal-directed therapy in the treatment of severe sepsis and septic shock publication of study results does not increase use of lung protective ventilation in patients with acute lung injury introduction of sedative, analgesic, and neuromuscular blocking agent guidelines in a medical intensive care unit: physician and nurse adherence dreyfuss d: ventilator circuit and secretion management strategies: a franco-canadian survey physician staffing patterns and clinical outcomes in critically ill patients the critical care crisis in the united states: a report from the profession improving safety with information technology finding and applying evidence during clinical rounds: the 'evidence cart handheld computing in medicine what next for electronic communication and health care? analysis of complex decision-making processes in health care: cognitive approaches to health informatics qualitative evaluation and research methods 2nd edition critically ill patients with severe acute respiratory syndrome (sars) doctors' experience with handheld computers in clinical practice: qualitative study introduction of handheld computing to a family practice residency program surgical procedure logging with use of a handheld computer more than half of mds under 35 age now using pdas we acknowledge the contributions of the intensive care physicians from william osler health centre (brampton memorial campus), scarborough general hospital, north york general hospital and trillium health centre (mississauga). this study would not have been possible without the financial support of the change foundation of the ontario hospital association (grant 01011) and bayer canada inc. we acknowledge the technical support provided by infiniq software (http://www.infiniq.com; mississauga, ontario, canada) and would like to thank dr arthur slutsky and dr allan detsky for reviewing the manuscript and providing valuable comments. stephen lapinsky, randy wax and thomas stewart were responsible for study design. stephen lapinsky, randy wax, randy showalter and carlos martinez implemented the handheld system and collected study data. sangeeta mehta and lisa burry were responsible for data collection and interpretation. stephen lapinsky and david hallet analyzed the data. the manuscript was written by stephen lapinsky, randy showalter and thomas stewart, with all authors participating in revisions and giving approval to the final draft for submission for publication. the author(s) declare that they have no competing intrests. • this study demonstrated that an updateable handheld computer information resource is a feasible means for providing point-of-care access to medical reference information in the icu.• acceptance of this system was variable and may be improved by enhanced training and newer technological innovations.• in clinical simulations, this system appeared to improve clinical decision making. key: cord-326220-uosjmht0 authors: gaskell, jen; stoker, gerry; jennings, will; devine, daniel title: covid‐19 and the blunders of our governments: long‐run system failings aggravated by political choices date: 2020-08-11 journal: polit q doi: 10.1111/1467-923x.12894 sha: doc_id: 326220 cord_uid: uosjmht0 more urgently than ever we need an answer to the question posed by the late mick moran in the political quarterly nearly two decades ago: ‘if government now invests huge resources in trying to be smart why does it often act so dumb?’. we reflect on this question in the context of governmental responses to covid‐19 in four steps. first, we argue that blunders occur because of systemic weaknesses that stimulate poor policy choices. second, we review and assess the performance of governments on covid‐19 across a range of advanced democracies. third, in the light of these comparisons we argue that the uk system of governance has proved itself vulnerable to failure at the time when its citizens most needed it. finally, we outline an agenda of reform that seeks to rectify structural weaknesses of that governance capacity. the covid-19 pandemic has confronted every government in the world with a wide-ranging set of urgent challenges and policy dilemmas. some governments are considered to have handled the crisis better than others, prompting debate over the reasons for variation in performance. this article does not claim to provide a definitive answer to why some countries have fared better (so far) than others during the crisis and has no crystal ball as to what the future might hold for the virus. rather, it argues that to assess the performance of governments we should focus on how the structural features of governance systems interact with, and amplify, missteps in policy choices made by political leaders and their advisors. to develop our argument, we proceed as follows. firstly, we develop the idea that governance failures reflect not only policy choice errors but also structural features of governance systems that make shortcomings more likely. secondly, we briefly assess the performance of national governments on covid-19 in a range of european democracies and show that other comparable countries, with similar or less warning of the dangers of the virus than uk, achieved better outcomes. we argue that outcome was possible in part because those countries had governance systems with a greater depth and spread of decision-making points, resources, and the capacity to use them. no governance system is perfect, but some perform better than others and some are better able to mitigate the ever-present possibilities of deficient political choicesespecially in times of crisis. the uk population has suffered an excess mortality rate owing to the covid-19 pandemic in the first half of 2020 that was among the highest for comparable developed nations. collectively, those deaths represent a governance failure that demands we break from a history where policy disasters, failure, catastrophes, fiascos, and blunders have been a recurrent theme in assessments of british government. 1 our evidence and argument might be used to apportion blame, but it would be better deployed to encourage thinking and action about a radical reform of our system of governance the political quarterly and we conclude with suggestions for that process. a framework for understanding governance failure we need an answer to the question posed by the late mick moran in his essay in the political quarterly nearly two decades ago: 'if government now invests huge resources in trying to be smart why does it often act so dumb?'. 2 a useful framework based on an extensive reading of the comparative literature on policy failure is provided by jennings, lodge and ryan. 3 they identify factors that account for the propensity of governments to commit policy blunders and draw a distinction between those based on the intentional choices of individuals or groups of individuals and factors that reflect underlying structural features of systems of governance. blunders often occur because politicians and other policy makers take the wrong decisions (for example through over-or under-reaction to problems, or owing to preoccupation with ideological and symbolic concerns) or because the wrong policy tool is adopted to meet the challenge. sometimes policy choices are made with good faith and in the light of the limited evidence available, but turn out with the benefit of hindsight to have been mistaken. sometimes decision makers persist with courses of action at odds with available evidence and advice, against even their own interests, in the hope of turning failure into success. these choice-based causes of failure are widely recognised and often the subject of formal inquiries or media investigations, and are a focus for a politics of blame or, more positively, accountability at the ballot box. the focus on structures, however, is equally important and may hold the key to answering the question as to why blunders seem to be repeated regularly. how could structures or systems of governance affect the propensity to blunder? structures of governance frame the way that decision makers work and operate, what they can see and hear, what tools they think they have available to them, what resources they view as at their disposal, and how they view other actors in the system. different kinds of institutions allow individuals to think different kinds of thoughts and may indeed construct 'thought styles' that are self-reinforcing, blocking out potential challenges or collaborations. 4 all governance systems are multilevel and involve a mix of institutions and a distribution of power that can be either more centralised or decentralised. there is no perfect system of governance, but failure is more likely to occur when a structural weakness is compounded by a thought style that is problematic. table 1 highlights some prominent causes of governance failure that reflect these connected dysfunctional elements that in turn lead to policy blunders. let us examine the dynamic of each of these causes in more detail. there is a structural difference between governance arrangements that decentralise power within the system and those that concentrate power at the centre, as the uk tends to do. governance systems can be highly centralised and one advantage of this, it is claimed, is that they can make decisive and timely interventions. strong coordinating capacity is highlighted by many as essential to managing the covid-19 crisis. yet if coordination is replaced by either over-confident or ineffectual central direction these systems can be prone to blunders when combined with decision making affected by group think and defensiveness when challenged. other problems include over-confidence of governing elites driven by a lack of understanding of operational issues and limited access to operational know-how. these issues can be compounded by losses of institutional memory caused by perpetual administrative reorganisations and shrinkage of the central state machinery, which creates a recipe for policy blunders. a second structural feature rests on the distinction made by hooghe and marks between 'type i' and 'type ii' multilevel governance. 5 the first type reflects federalist thinking and disperses authority to a limited number of discrete, multipurpose governing units. the second type has different kinds of decentralised units constructed around functions and tasks, creating a complex mix of responsibilities and overlapping jurisdictions. most governance systems involve some mix of type i and type ii divisions. the emerging post-devolution settlement system in the uk creates an especially complex hybrid of these two multilevel governance models, with a strong emphasis on functional division combined with territorial autonomy granted in different forms to scotland, wales and northern ireland. such systems are characterised by functional divisions where some powers are 'devolved' (differently across the three nations in the case of the uk) while some are 'reserved' at the centre. what are the strengths and weaknesses of the two intergovernmental systems? type i systems, given the power vested in autonomous decentralised units, can be vulnerable to inertia and gridlock owing to obstruction by 'veto players' at different levels of government. pride in local, regional, or national autonomy, and a desire to express difference is the thought style commonly associated with type i systems. but a relatively small number of decentralised units and potentially effective integration does offer capacity for dialogue and negotiation between government levels, if participants are willing. type ii systems can be flexible and provide an opportunity for matching task to scale in a more varied way. the comparative advantage of these institutions is their technical expertise and concentrated focus. but the system in which they operate can suffer from complexity and proliferation of the number of institutions that might be involved in a cross-cutting task. type ii systems tend to encourage silo thinking in that they are set up to operate within their functional domain rather than to reach out beyond it. coordination problems and weak communication between different parts of government are common in type ii systems. hybrid systems such as that of the uk are even more vulnerable to failure where policy issues cut across both devolved and reserved functions, such as brexit for example and the covid-19 crisis. 6 connected to this last point is the argument that all systems of governance need effective joining up, and a third structural cause of failure is attributable to deficiencies in the connecting of separate spheres of governance. the division essential in any governance system is best when matched with collaborative capacities to share and incorporate knowledge relevant to policy making. 7 but collaboration is not easy and requires both partnership institutions, resources, and commitment and capacity for learning and adaptability. it also requires trust between levels of government as well as between government and citizens and sectoral interests such as businesses or wider civil society organisations. the relevant competences and technical capacities to support collaboration require shared values and mutual respect. failures of collaboration can arise because of breakdowns in information sharing and can be compounded by a thinking style reflecting a lack of trust and respect between actors, all of which can lead to governance failure and blunders. the fourth and final structural weakness is weak capacity for policy experimentation and adaption in response to heterogeneous local contexts. decision making benefits from a diverse mix of participants, thereby reducing group think. lu hong and page use mathematical modelling to show that when facing complex challenges, the best strategy is to not to rely on a team of 'the brightest and the best', but instead pick as diverse a group of problem solvers as possible. 8 why? again, the reason has to do with the way that institutions and contexts make people think. a centralised collection of similar, talented people will generate ideas and strategies of quality, but will tend to believe that one-size-fits-all and be prone to group think. a more diverse group will use different heuristics depending on their backgrounds, search more widely and with greater variety of outcomes for solutions, and will build their searches on the back of local knowledge and understanding that is not available to a group of the brightest and best. more institutionally focussed studies consistently demonstrate the advantages of power sharing and what the nobel prize winner elinor ostrom refers to as the advantages of decentralised systems nested in a polycentric system. for ostrom these advantages, established through painstaking research, include use of local and disaggregated knowledge, inclusion of trustworthy participants, and lower enforcement costs. 9 a capacity for local experimentation and a willingness to celebrate difference and diversity would appear to be a system-enhancing feature and their absence a potential cause of system failure. having established a framework for thinking about the causes of governance failure, we need to establish some assessment of the performance of different systems in the unfolding of the covid-19 crisis during the first half of 2020. we start, in figure 1 (a), with data relating to the outbreak in the uk and fifteen other west european countries (france, germany, italy, spain, austria, the netherlands, ireland, portugal, finland, norway, greece, belgium, denmark, sweden, switzerland), with the uk's trajectory indicated by the black line and the other countries shown in grey (since our focus is on the comparative performance of the uk). we choose these countries as advanced democracies that are geographically proximate to the uk and broadly comparable in terms of the level of economic development and the public health systems. the figure plots the number of reported cases of covid-19 in each country from january to june 2020. this reveals that while the outbreak in the uk initially lagged behind three other countries (italy, spain and france), by mid-may it had overtaken its european counterparts-with the slope of its curve steeper at the end of the period, indicating that the growth of cases was declining at the slowest rate. it is also possible to compare the timelines and restrictiveness of the containment policies put in place by the governments of these countries, using the oxford covid-19 government response tracker (oxcgrt). this systematically collects information on common policy responses and calculates a standardised scale of the stringency of government measures. the index is plotted in figure 1 (b) and highlights the different timings and levels of stringency of containment measures across western europe. this tells us how quickly the country shut down, in the context of the severity of the health impact of the virus. what is most striking from this graph is that, with the exception of sweden, the uk was the last major european country to introduce significant restrictions on social and economic life-waiting until 23 march to do so, when other countries had already enacted substantial containment policies by 18 march at the latest. this delay is more notable if one considers that even by 18 march the uk was sixth in terms of cases (1,950) and fourth in terms of deaths (81). it also highlights that it began to ease its shutdown around the same time as many other countries, despite having a considerably higher number of cases. in broad comparative terms, this evidence suggests that the uk has performed badly during the pandemic, an observation supported by a death toll from covid-19 that is at the top of those recorded by developed nations, notwithstanding some differences in counting practices. a quick look at other cases suggests how things might have been different, without suggesting that any country met the challenges without problems or stumbles. with one of the largest ageing population in europe and a healthcare infrastructure weakened by a decade of austerity, portugal took the threat posed by the virus very seriously and moved quickly to close schools and impose a more general lockdown on 16 march when it had recorded just 245 cases, although an outbreak of cases from late may onwards has damaged its reputation as a 'safe destination' for visitors to some degree. one commentator suggested that strong central direction was crucial for achieving this outcome: 'although the epidemic is concentrated in the north, around the city of porto, portugal's centralized system of government allowed for rapid nationwide measures rather than the piecemeal regional action adopted elsewhere.' 10 throughout the crisis, the portuguese government applauded citizens' 'civic spirit' in complying with strict containment measures that were clearly and consistently communicated to them, including the early days of easing restrictions on a regional basis in light of differing requirements. the government also benefitted from the collaborative approach taken by the opposition, with social democratic party (spd) member of parliament ricardo baptista leite observing: 'we're no longer dealing with the socialist government; we are dealing with the government of portugal', further noting 'we are having very candid but supportive meetings to present constructive criticism and also solutions through back channels, showing a sense of unity for the national interest.' 11 in denmark, prime minister metter fredriksen delivered her lockdown address to the nation on 11 march when the country had around 500 recorded cases, closing schools against the recommendation of its health authorities. 12 denmark has a well-developed multilevel governance system with decentralised power to regional and local authorities. in this case, strong central direction was combined with a reflective, consensual style of leadership. in her announcement, fredriksen urged citizens to 'stand together by keeping apart', a message that struck a chord of national solidarity among danes. she also acknowledged that the country was entering uncharted territory and took responsibility for 'undoubtedly making mistakes along the way'. 13 neighbouring sweden is an interesting case because its light-touch response has been at odds with other european countries. indeed, its schools and industry have remained largely open throughout the pandemic, with central government providing support to furloughed workers and healthcare provisions delivered by regional and local authorities. in this way, sweden offers a model of coherent strategic direction combined with a coordinating approach by the centre, strongly influenced by its chief epidemiologist anders tegnell. however, in contrast to other scandinavian countries its covid-related fatalities passed the 5,000 mark in mid-june, and its strategy has been widely criticised as having failed to manage down the number of cases while experiencing the same economic downturn as countries that pursued stricter lockdown measures. while jon pierre argues that the strategy was premised on erroneous assumptions regarding asymptomatic transmission, he also describes a failure of governance arrangements in the lack of capacity at regional and local levels in terms of healthcare staff training and the absence of communication channels between different governance levels which were traditionally perceived to be successful within their functional remits. 14 when this perceived competence broke down in the crisis, the lack of mechanisms for collaboration and mutual learning, according to pierre, resulted in 'poor performance of nursing homes and other elements of elderly care' which only became apparent to the government months into the pandemic. 15 some countries who did not benefit from a strong central direction and its perceived ability to provide swift, decisive actions were able to leverage other aspects of their more decentralised systems. germany for example suffered early delays in implementing any nationwide policy. unlike denmark and portugal, which implemented strict measures before any covid-related deaths, germany only closed schools three days after its third death was recorded on 12 march. it then suspended public events and closed its land borders four and eight days later, respectively. 16 germany's relative success so far has been credited to two main factors: chancellor angela merkel's leadership style and the country's decentralised response to the crisis. 17 the country's leading coronavirus expert has stated that besides being extremely well-informed, merkel's successful steering of competing regional and local interests through the crisis can be attributed to her leadership style of 'thoughtfulness and ability to reassure', as well as not seeking to use the crisis as a political opportunity. 18 a spokesperson for the german federal government also attributed the country's low mortality rate to 'a confluence of many other factors. these include the country's federal system of government, which means there are hundreds of health officials overseeing the pandemic response across the 16 states, rather than one centralized response from the country's national health ministry'. 19 switzerland suffered similar delays to germany at the very start of the crisis as a result of its highly decentralised federal system. however, as the pandemic developed, it benefitted from inclusion of a broader range of stakeholders in decision making. indeed, all three levels of governance-the federal, cantonal (regional) and communal (equivalent to county or city councils)-mobilised resources to tackle the outbreak. each commune has the power to issue its own specific guidance in line with federal and cantonal directives. according to the swiss press, communes are at the frontline of the response effort. 20 in switzerland, mutual learning is institutionalised in policy making at the cantonal and communal levels which continuously informs and is integrated into central decision making. during the crisis, these consultation mechanisms were accelerated rather than discarded, taking place weekly and feeding into crisis decision making. this seemed to have been further strengthened by a commitment to different approaches and providing local governance centres with the freedom and resources to undertake what they feel is needed for their communities. the commune of bovernier, for instance, a small village with 900 inhabitants in the canton of valais, decided to call each of the 110 households with a resident over the age of sixty-five to arrange food and medicine deliveries. 21 in geneva, orca's (organisation in case of catastrophe and extraordinary situation) immediate focus was on job security and support for commercial organisations. in this way, different approaches are encouraged for their localised capacity to innovate rapid, relevant responses, as the realisation emerged among various stakeholders that 'one-size might not fit all'. these examples highlight several points. firstly, it is not necessarily the formal constitutional arrangements of a governance system (federal or unitary) that matter as the capacity to work through and with the system in place-an argument behind the framework put forward by jennings, lodge and ryan. secondly, it is not only that some countries adopted better tools than others, but also the case that some proved better at implementation. crucially, countries needed to avoid the drivers of governance failure identified in table 1 : not relying too much on central direction or allowing rigidity in structures to undermine dialogue. they need also needed to be capable of effective collaboration and local adaption to circumstances. most countries partially failed at least one of those hurdles. the uk, as we shall see, failed all four. in this section we outline how structural failings and dysfunctional policy choices have combined to produce such a flawed response to the covid-19 crisis in the uk. following the framework presented in table 1 , the uk suffered in part because of over-weening and ineffectual central direction. an initial list of where these issues were to the fore would include: a sluggish approach to implementing lockdown measures; catastrophic shortage of personal protective equipment (ppe) for frontline medical staff; failure of the government's 'ventilator challenge' to deliver new machines to meet expected increases in demand; confusing communications that have led to a chaotic experience of easing lockdown; no clear plan in place for the reopening of schools to all pupils in england; and the continued failure to deliver an operational 'test, track and trace' system that is widely deemed essential for safe easing of lockdown measures. the centre of british government lacks operational understanding or links to learn from those at street level and in other centres of government. perhaps the most consequential decision reflecting that structural weakness at the early stages of the crisis was the discharging of elderly patients from hospitals to care homes without mandatory covid-19 tests-until the requirement was introduced on 15 april. in march, nhs england and nhs improvement had advised hospital trusts to expand critical care capacity (a major concern based on the italian experience) by freeing up hospital beds-which contributed to increased discharge of patients to care homes who were potentially carrying the disease. between 9 march and 13 april, one in five care homes (around 3,500 in total) reported at least one outbreak of covid-19 to public health england (phe). there were also signs of over-confidence and group think at the heart of british government. on 3 march, as italy reached seventy-nine deaths, the prime minister reported 'our country remains extremely well prepared', and (contrary to the guidance from scientific advisors) declared 'i'm shaking hands'. this proved symbolic of the casual approach taken by the government (which may have been shaken when multiple members of the government fell ill with the virus), and covid-19 was subsequently optimistically characterised by the prime minister as 'overwhelmingly a disease that is moderate in its effect'. 22 the group think problem was arguably evident in three key areas: an over-reliance on data-driven modelling in making key policy decisions; a reluctance by the scientific advisory group for emergencies (sage) to consider lockdown measures deemed politically out of scope; and the consistent drive to centralise implementation. the government's reliance on epidemiological modelling to inform its strategy meant it was only on 16 march, after new data suggested that critical care capacity in the nhs could be overwhelmed, that sage advised more stringent restrictions be introduced as soon as possible. this was despite evidence being available from italy at the start of march that one in ten people infected with the virus required intensive care. well before that, the unusually rapid construction of sixteen temporary hospitals in wuhan in january should have provided a clear warning of the intense pressures that health services would soon face. even at this point-before the uk's shutdown was implemented-it was known that authorities in south korea, hong kong, singapore, and taiwan had successfully brought the virus under control (with effective test, track and trace operations, and widespread wearing of masks by citizens), yet this did not figure prominently in deliberations of policy responses among uk experts. the 'data-driven' approach taken by sage, on behalf of the government, meant that these information signals from more obvious sources were ignored. coordination problems and low-grade conflicts between levels of government were also constant issues. the devolved governments complained about lack of consultation, as did other public bodies including local authorities, hospitals, and gp services. a constant flow of changing centrally-driven directives to agencies of all types created a sense of confusion and frustration during the initial phase of lockdown. this issue is further highlighted by the way containment measures have been eased across england. after the government announced a relaxation of guidelines on 2 june, the mayors of greater manchester and liverpool held a joint press conference urging a revision of guidance based on localised r estimates. 23 these examples suggest a lack of commitment to sustained consultation, with key stakeholders seemingly excluded from the planning phases of both these strategies. the government's plan that schools would reopen again for all pupils on 1 june offers a notable example, when it was scrapped following opposition from local councils and teachers' unions who argued it to be impossible while maintaining social distancing guidelines. this was further compounded by the ensuing blame game between government on the one hand, and local authorities and teachers' unions on the other, over who was at fault for the failure to get pupils back to the classroom before the summer holidays. throughout its response, the uk government has been plagued by inconsistent (and some might say incoherent) messaging and implementation. much of this is the result of a mixture of conflictual relationships at different governance levels, which systematically results in a lack of integration, collaboration and mutual learning, and a weak capacity to experiment and tailor policy to meet the diverse needs of different locations with different demographic makeups, cultures and dominant industries. the ways in which these structural elements of the uk's governance system hampered an effective response to the pandemic were apparent as soon as lockdown guidelines were issued. for a time in march, the nhs 111 service provided conflicting messages to uk travellers returning from italy to that of ministers. also, the government guidelines for the construction sector were published and then immediately withdrawn as they were deemed unworkable on construction sites by industry specialists. 24 more directly, early tracking of the spread of the virus encountered difficulties, as despite a reliable test having been developed for covid-19, phe had limited capacity to carry out the testing (and that capacity was initially assigned to tests for seasonal flu), and was slow to allow the expansion of testing through labs in hospital trusts, universities and private settings. 25 these issues were compounded by a strong preference for onesize-fits-all solutions which is often contrasted to the successful and decentralised german approach to testing. the uk's governance system's weak capacity to experiment and tailor policy to meet diversity is illustrated in its failure to stockpile ppe for health workers, instead sticking to 'just in time' procurement principles until april-leaving staff in hospitals and care homes to work without protection. 26 little exemplifies more the confluence of structural failures which led to a national inability to track and trace covid-19 cases than ceredigion council's development of its own in-house test, track and trace system, which contributed to one of the lowest death rates across the whole of the uk. 27 this example shows that local centres of governance can possess a large amount of knowledge and resourcefulness which, if leveraged, can enable responses that benefit local communities and highlight best practice or innovative measures that could be replicated elsewhere. these have been neglected in the uk's response to covid-19. so far, the uk experience has seen little attempt at allowing local capacity and resources to energise and innovate in responding to the pandemic. local authorities and the primary health care teams based around gp surgeries have been largely overlooked, despite their local knowledge and contacts, exemplifying the uk's tendency to centralised, one-sizefits-all policy solutions. we have provided only a partial explanation of the weaknesses of the uk's policy response to covid-19 because we want to focus attention not so much on the complexity of policy choices made in the context of high uncertainty and crisis, but how failings reflect broader structural weaknesses and associated thought styles within the uk system of governance. there is a more detailed, nuanced, and complicated narrative to tell about what went wrong (and perhaps what went right) in the uk government's handling of covid-19. certainly, how the government's response was influenced by its ideological instincts and interests, and the nature of scientific advice that it received, seem important factors to understand. but our primary objective has been to illustrate from the experience of other countries that there are better ways to govern (and be governed). notably, the chief advisor to the prime minister has previously made a similar observation about the profound failings of our governing system: one of the most extraordinary aspects of english politics and education is the lack of structured, disciplined thought about what works and doesn't work, and how to build reliable systems that allow improvement in performance . . . most activity in whitehall occurs without asking 'who, somewhere in the world, has already solved this problem? ' 28 maybe it is time to also think about the deep-rooted structural imperfections of our governing system, and the tendency of successive generations of our political class to bandage over them with piecemeal reforms rather than thinking more holistically about why our patchwork of governance lacks coherence and continues to hoard power and resources at the centre. we think, contrary to the arguments of the chancellor of the duchy of lancaster, that neither relocation of the civil service to dispersed geographical locations, nor the fostering of greater intellectual diversity in its makeup by recruiting scientists, data scientists, or mathematicians, provide the answer (though there is merit in both proposals). 29 it is, rather, another example of mick moran's charge that by focussing on quick-fix solutions to make government smart (past solutions have included performance management and contracting-out) we run the risk of making it dumber in new ways. the covid-19 experience already hints at the limits of data science in the context of the absence of good, up-to-date data and subject to conditions of considerable uncertainty. our agenda for reform, outlined in table 2 , is both more prosaic and more radical. we identify short-and long-term mitigation strategies. different approaches and choices by political leaders could provide more consensual leadership, a willingness to share ownership for problems and insights, greater trust, and mutual respect between levels of government and a wider openness to local learning and diversity. these would all represent improvements in the governance arrangements of the uk. longer-term we need structural reforms that shift both powers and responsibilities, and clearly define and enshrine them on a constitutional basis, rather than muddling through as has long been the british political tradition. we do not offer detailed proposals, but rather map out a general direction of travel. we need more decentralised government with a matching redistribution of powers and resources. that decentralisation must be consistent and comprehensive, rather than uneven, unequal and inchoate. we need much more effective institutions for mutual exchange and consultation between levels and types of government. we need to know much more about what good practice is, what drives it and how it can be replicated. parallel to that we need to abandon centralised one-size-fits-all thinking and instead recognise the need for local tailored solutions and experimentation. policy disasters: explaining the uk's record not steering but drowning: policy catastrophes and the regulatory state not steering but drowning comparing blunders in government how institutions think unraveling the central state, but how? types of multilevel governance intergovernmental relations in the uk: time for a radical overhaul?', the political quarterly collaborative governance in theory and practice groups of diverse problem solvers can outperform groups of high-ability problem solvers 281-2; and for further carefully constructed evidence on the advantages of power sharing see p. norris, driving democracy. do powersharing institutions work? how portugal became europe's coronavirus exception first to close-first to reopen: denmark's gain from virus response', financial times statsminister mette frederiksens indledning p a pressemøde i statsministeriet om corona-virus den 11 nudges against pandemics: sweden's covid-19 containment strategy in perspective how portugal became europe's coronavirus exception the inquiry: why does germany have such a low number of deaths from covid-19? for many, i'm the evil guy crippling the economy"', the guardian germany's coronavirus response: separating fact from fiction les communes en premi ere ligne de la pand emie prime minister's statement on coronavirus (covid-19) north west mayors want to empower communities with weekly publication of r number, to encourage local decision making construction confusion as uk working instructions published then withdrawn how the uk got coronavirus testing wrong', financial times government ignored warning to stockpile ppe as covid-19 spread', the guardian the area of wales that missed coronavirus-and the simple system it set up the hollow men ii: some reflections on westminster and whitehall dysfunction', dominic cummings's blog the privilege of public service', ditchley annual lecture given by michael gove this article is part of the work the three-year trustgov project (https://trustgov.net/) funded by the uk's economic and social research council (es/s009809/1). key: cord-018038-gqdylj6n authors: snyder, william m.; wenger, etienne title: our world as a learning system: a communities-of-practice approach date: 2010 journal: social learning systems and communities of practice doi: 10.1007/978-1-84996-133-2_7 sha: doc_id: 18038 cord_uid: gqdylj6n we live in a small world, where a rural chinese butcher who contracts a new type of deadly flu virus can infect a visiting international traveller, who later infects attendees at a conference in a hong kong hotel, who within weeks spread the disease to vietnam, singapore, canada, and ireland. fortunately, the virulence of the severe acute respiratory syndrome (sars) was matched by the passion and skill of a worldwide community of scientists, health care workers, and institutional leaders who stewarded a highly successful campaign to quarantine and treat those who were infected while identifying the causes of the disease and ways to prevent its spread. in such a world, we depend on expert practitioners to connect and collaborate on a global scale to solve problems like this one – and to prevent future ones. as global citizens. what does it mean to 'think globally and act locally'? does global stewardship primarily imply building international organisations that address social and environmental issues to compensate for the economic focus of global corporations? is such a global perspective sufficient to address issues that are essentially local? how can we connect the power and accessibility of local civic engagement with active stewardship at national and international levels? what are the design criteria for such a system and what might it look like? we believe there are three fundamental design criteria that help specify essential characteristics of a world learning system capable of addressing the scope and scale of the global challenges we face today. problems such as overpopulation, world hunger, poverty, illiteracy, armed conflict, inequity, disease, and environmental degradation are inextricably interconnected. moreover, they are complex, dynamic, and globally distributed. to address such challenges, we must increase our global intelligence along several dimensions: cognitive, behavioural, and moral. we must increase, by orders of magnitude, our societal capacity for inquiry; our ability to continuously create, adapt, and transfer solutions (churchman, 1971) . a world learning system that can match the challenges we face must meet three basic specifications: • action-learning capacity to address problems while continuously reflecting on what approaches are working and why -and then using these insights to guide future actions. • cross-boundary representation that includes participants from all sectors -private, public, and nonprofit -and from a sufficient range of demographic constituencies and professional disciplines to match the complexity of factors and stakeholders driving the problem. • cross-level linkages that connect learning-system activities at local, national, and global levels -wherever civic problems and opportunities arise. civic development is essentially a social process of action learning, in which practitioners from diverse sectors, disciplines, and organisations work together to share ideas and best practices, create new approaches, and build new capabilities. the full potential of this learning process is only realised when it connects all the players at various levels who can contribute to it. there are a number of organisations -including the united nations, the world bank, and an array of nongovernmental organisations such as doctors without borders, the world council of churches, oxfam international, major foundations, and many others -whose mission is to address worldwide problems. but these organisations typically focus on solving the manifestations of problems -eliminating land mines from war-torn regions or reducing the incidence of aids, for instance. given the urgency of these problems, it is understandable that these organisations do not focus on the underlying learning capacity of a city or country. while it is essential to address these and other urgent problems on their own terms, our society's longterm capacity to solve them at both local and global levels will nevertheless require step-change increases in our foundational capacity for intelligent social action. what is the nature of large-scale learning systems that can operate at local and global levels? how can we take steps to create such learning systems? to what extent can they be designed and what does design even mean in such a context? these learning challenges are among our world's most urgent as we find ourselves today in a race between learning and self-destruction. fortunately, we have examples of transformative, inquiry-oriented learning systems in hundreds of private-sector organisations, with a growing number in public and nonprofit organisations as well -at both organisational and interorganisational levels. strong, broad-based secular forces are driving this movement. most organisations today, including domestic firms as well as multinationals, have been forced to confront large-scale learning issues to compete in the knowledge economy. there is much we can learn from the experience of organisations about how to increase our society's collective intelligence. the most salient lesson is that managing strategic capabilities primarily entails supporting self-organising groups of practitioners who have the required knowledge, use it, and need it. we call these groups 'communities of practice' to reflect the principle that practitioners themselves -in active collaboration with stakeholders -are in the best position to steward knowledge assets related to their work. a well-known private-sector example of such practitioner stewardship is the network of 'tech clubs' that chrysler engineers formed in the early 1990s (see wenger et al., 2002, chapter 1) . the company had just reorganised its product-development unit into 'car platforms' focused on vehicle types (small cars, large cars, minivans, etc.). design engineers with specialties related to the various vehicle components -such as brakes, interior, and windshield wipers -organised communities of practice to foster knowledge sharing across car platforms. the cross-boundary sharing of these communities was a critical success factor for the reorganisation. we are now seeing a proliferation of organisations fostering the development of communities of practice across industry sectors, geographic locations, and various elements of the value chain. communities of practice are not new. they have existed since homo sapiens evolved 50,000 years ago, 1 but organisations have now become increasingly explicit about cultivating these communities. distinctive competencies in today's markets depend on knowledge-based structures that are not restricted by formal affiliation and accountability structures. the most distinctive, valuable knowledge in organisations is difficult or impossible to codify and is tightly associated with a professional's personal identity. developing and disseminating such knowledge depends on informal learning much more than formal -on conversation, storytelling, mentorships, and lessons learned through experience. this informal learning, in turn, depends on collegial relationships with those you trust and who are willing to help when you ask. informal learning activities and personal relationships among colleagues are the hallmarks of communities of practice. hence, we see an increasing focus on informal community structures whose aggregate purpose is to steward the learning of an organisation and its invaluable knowledge assets. communities of practice have three basic dimensions: domain, community, and practice. a community's effectiveness as a social learning system depends on its strength in all three structural dimensions. • domain. a community of practice focuses on a specific 'domain,' which defines its identity and what it cares about -whether it is designing brakes, reducing gun violence, or upgrading urban slums. passion for the domain is crucial. members' passion for a domain is not an abstract, disinterested experience. it is often a deep part of their personal identity and a means to express what their life's work is about. • community. the second element is the community itself and the quality of the relationships that bind members. optimally, the membership mirrors the diversity of perspectives and approaches relevant to leading-edge innovation efforts in the domain. leadership by an effective 'community coordinator' and core group is a key success factor. the feeling of community is essential. it provides a strong foundation for learning and collaboration among diverse members. • practice. each community develops its practice by sharing and developing the knowledge of practitioners in its domain. elements of a practice include its repertoire of tools, frameworks, methods, and stories -as well as activities related to learning and innovation. the activities of a community of practice differ along several dimensionsface-to-face to virtual; formal to informal; public to private. further, activities are orchestrated according to various rhythms -for instance, in one community, listserv announcements come weekly, teleconferences monthly or bi-monthly, projects and visits occur when an opportunity presents itself, back-channel e-mails and phone calls are ongoing; and the whole group gathers once or twice a year face-to-face (see fig. 7 .1). these activities form an ecology of interactions that provide value on multiple levels. beyond their instrumental purpose of creating and sharing knowledge, they increase the community's 'presence' in members' lives and reinforce the sense of belonging and identity that are the foundation for collective learning and collaborative activities. communities of practice do not replace more formal organisational structures such as teams and business units. on the one hand, the purpose of formal units, such as functional departments or cross-functional teams, is to deliver a product or service and to be accountable for quality, cost, and customer service. communities, on the other hand, help ensure that learning and innovation activities occur across formal structural boundaries. indeed, a salient benefit of communities is to bridge established organisational boundaries in order to increase the collective knowledge, skills, and professional trust of those who serve in these formal units. for instance, at daimlerchrysler, brake engineers have their primary affiliation with the car platform where they design vehicles. yet they also belong to a community of practice where they share ideas, lessons learned, and tricks of the trade. by belonging to both types of structure, they can bring the learning of their team to the community so that it is shared through the organisation, and, conversely, they can apply the learning of their community to the work of their team. pioneering, knowledge-intensive organisations have recognised that beyond the formal structures designed to run the business lies a learning system whose building blocks are communities of practice that cannot be designed in the same manner as formal, hierarchical structures. communities of practice function well when they are based on the voluntary engagement of members. they flourish when they build on the passions of their members and allow this passion to guide the community's development. in this sense, communities of practice are fundamentally self-governed. our experience suggests, however, that while communities do best with internal leadership and initiative, there is much that organisations can do to cultivate new communities and help current ones thrive. the intentional and systematic cultivation of communities cannot be defined simply in terms of conventional strategy development or organisational design. rather, sponsors and community leaders must be ready to engage in an evolutionary design process whereby the organisation fosters the development of communities among practitioners, creates structures that provide support and sponsorship for these communities, and finds ways to involve them in the conduct of the business. the design of knowledge organisations entails the active integration of these two systems -the formal system that is accountable for delivering products and services at specified levels of quality and cost, and the community-based learning system that focuses on building and diffusing the capabilities necessary for formal systems to meet performance objectives. it is crucial for organisational sponsors as well as community leaders to recognise the distinct roles of these two systems while ensuring that they function in tandem to promote sustained performance. the fundamental learning challenges and nature of responses in business and civic contexts are very similar. the size, scope, and assets of many businesses create management challenges that rival those of large cities, or even small countries. in both cases, one needs to connect practitioners across distance, boundaries, and interests in order to solve large-scale problems. organisations have found that communities of practice are extremely versatile in complementing formal structures. they are known for their ability to divide and subdivide to address hundreds of domains within and across organisations; they lend themselves to applications where scalability, broad scope, and the need for extensive, complex linkages are relevant. hence there is much we can learn from the early, highly developed business examples. the approaches for building largescale learning systems in organisations -by combining both formal and informal structures -provide a blueprint for thinking about how to build such systems in the messy world of civil society. communities of practice already exist in the civic domain, where they complement place-based communities as well as the ecology of formal organisations, including businesses, schools, churches, and nonprofits. in the civic arena as well as in organisations, our challenge is not to create communities of practice so much as to foster them more systematically. our analysis of societal learning systems -whether at local, national, or international levels -focuses on cities (which we define as an entire metropolitan region) as highleverage points of entry for a number of reasons. for one, as of the year 2000, there are more humans on the planet living in cities than outside them. in 2002, there were twenty megalopolises in the world with more than 10 million people, and by 2015 there will be nearly forty. cities have always been the font of new ideas, new applications of technologies, new cultural movements, and social change. they constitute natural nodes in a network for disseminating innovations. in the problems they face and the opportunities they offer, they also provide a microcosm of the world. finally, cities possess an organisational infrastructure and established leadership groups with the potential to see the value and to sponsor the design of a local learning system. in many cities, multisector coalitions or alliances are formed to take on a pressing issue such as improving urban schools, increasing access to low-income housing, cleaning up a business district, or building a stadium, park, or cultural facility (see grogan and proscio, 2000) . these coalitions, however, generally do not take sustained responsibility for stewarding a civic domain or for bringing together the full array of stakeholder constituencies to identify and address short-and long-term priorities. one way to assess the level of civic stewardship in any city or region is to map the prevalence, inclusiveness, and effectiveness of civic communities of practice (also known as coalitions, associations, partnerships, and alliances, among other terms) who take responsibility for clusters of issues related to particular civic domains, such as education, economic development, health, housing, public safety, infrastructure, culture, recreation, and the environment. the reality is that in many cities these domains have no explicit stewardship, or they are left to public agencies the city as a learning system: stewarding the 'whole round' of civic domains or to a menagerie of disparate, often competitive and conflicting organisations that carve out small pieces of the puzzle -regarding housing availability, for examplebut do not coordinate efforts or leverage a common base of expertise and resources. the city, re-imagined as a learning system, consists of a constellation of crosssector groups that provide stewardship for the whole round of civic domains (see fig. 7.2) . cultivating a learning system at the city level means taking stock of the current stewardship capacity in the city and accounting for the array of civic disciplines and the quality of active communities of practice stewarding them. this city-level assessment provides a template for what a nation can do. at the nation level, leaders might evaluate a representative sample of major cities and regions as a baseline assessment of its civic stewardship capacity. by extension, an evaluation of the top 500 strategic cities in the world could provide a benchmark for our civic learning capacity at a global level. at the national and global levels, the analysis also considers the strength and quality of linkages across cities both within and across nations. of course, even at the city level, there are subsectors and neighbourhoods that are fractal elements of the city, each with its own whole round of civic practices, and among which neighbourhood-to-neighbourhood linkages are as instrumental as ones that connect cities and nations. a city-based initiative to promote economic development in chicago provides an example of an effort designed to leverage communities as agents of civic development. 2 in 1999, the city of chicago established a cross-sector coalition, the mayor's council of technology advisors, to create 40,000 new high-tech jobs in the chicagoland region. the coalition leaders began by pulling together a group of forty-five civic leaders to brainstorm ways to achieve this goal. according to a study commissioned before the group met, the greatest challenge they faced was encouraging business development in high-tech industries such as telecommunications and biotech. a related challenge was cultivating local sources of seed capital for start-ups in these industries. the result of the group's first meeting was a slate of long-and short-term initiatives -including the introduction of technology in schools; encouraging young women and minorities to explore technology careers; and building a stronger digital infrastructure in the city, especially in underserved areas. several of the groups focused on initiatives specific to the industry sectors identified in the initial study: telecommunications, software development, biotech, venture capital, and emerging areas such as nanotechnology. the industry groups were particularly successful in this initiative, largely because they were able to coalesce communities specific to development challenges in each industry sector. the civic leaders in chicago understood that coalescing communities of practice -in this case, along industry lines -was the foundation for building relationships, generating ideas, and catalysing business initiatives. as one leader put it, 'our first objective was to create communities, period. the technology industries were fragmented without a sense of commonality. for example, we have more software developers than in silicon valley, but here it's only 9% of the workforce. so we started getting people connected and networked and building a sense of community in our high-tech sectors.' the chicago biotech network (cbn) is one of the more mature high-tech communities in chicago and provides an illustration of the influence and stewardship such a community can have over time. cbn started as a grassroots group that held about five seminars a year for diverse constituents interested in biotech developments. at first, it was more for individuals interested in life sciences. then companies (such as abbott laboratories and baxter, two fortune 500 pharmaceuticals located in the chicago area), started to attend the meetings as well, and they brought different perspectives. over time, the community came to include scientists, university deans, lawyers, venture capitalists, angel investors, city and state business development staff, and others. anywhere from twenty-five to two hundred people showed up at the meetings, which were held at various places and sponsored by members. these gatherings provided an opportunity for members to discuss science and industry trends and build relationships. one of the leaders summarised the community's evolution: 'early on, people mostly came for the personal value of networking and discussing ideas. now the domain of the community is to promote science and business development in the biotech sector in the chicago area. we focus on science ideas, business development know-how, and knowledge transfer processes.' offshoots of community activities include targeted events that link scientists, angel investors, and large pharmaceuticals to fund biotech startups that can commercialise promising innovations coming out of university labs. on a broader level, the community has helped increase biotech lab space in the city, lobbied at state and federal levels for increased research funding, and recruited biotech companies to locate in chicagoland. the leader of the chicago-based biotech community estimated the value of the community's activities for generating start-ups and, by extension, job creation in the region: 'i can't point to anything specific, but our events have brought structure to the interface between r&d scientists and the venture community; and we've gone from very little venture funding to the point where we now have $50 million coming to various biotech companies this year.' the chicago biotech network illustrates how an industry-based community of practice can serve as a powerful force for civic development. in this case, the focus was on economic development, but the key point is that strong stewardship of civic issues, even in the hard-nosed area of industry development, depends on vital communities of practice. the purpose of the communities was not only to provide professional development and networking opportunities but also to cultivate thriving high-tech industries in chicagoland. these communities advocated for their domain as a strategic focus for the city, built relationships among community members from various backgrounds, and shared know-how among practitioners. finally, as one community leader stated, they worked to serve the city they loved, and ultimately their children, who would inherit their civic legacy. communities of practice can also provide powerful stewardship for civic issues at the national level by connecting innovative civic groups across cities. the safecities community, for example, was organised in march 1999 by vice president al gore's reinventing government initiative to reduce gun violence in the united states. the announcement of the safecities community coincided with publication of the fbi's crime-rate statistics, which showed significant variation across cities in injuries and fatalities caused by gun violence. senior executives in the national partnership for reinventing government (npr) office began by convening officials from relevant agencies and developing a shared vision for what the network would be about and how they would work together. they sent out an invitation to cities and regions nationwide and selected ten coalitions to participate in the safecities communitybased on criteria that included multisector collaboration, a track record of innovation, and commitment to improved results. these local coalitions provided stewardship for public safety issues in their cities as did the industry-focused communities in chicago. a striking characteristic of the initiative was that it offered participants no funding -the value of participation was to get connected, to learn, and to enhance the capacity to reduce gun violence. the scale of the initiative was also distinctiveconnecting civic coalitions from across the nation for the purpose of sharing ideas, collaborating on innovation initiatives, and helping to shape policy at local, state, and federal levels. 3 the safecities community can be described in terms of the three structural dimensions of communities of practice. each of the coalition members was focused, broadly speaking, on issues related to the domain of public safety. their specific domain targeted a subdomain within this area -defined as reducing injuries and fatalities due to gun violence. the specificity of this domain was crucial for coalescing a community with overlapping interests, focusing its learning activities, and attracting sponsors. the community was composed of members at local and national levels and from various disciplines and constituencies, such as officials from the fbi, the bureau of alcohol, tobacco, firearms and explosives, and an assortment of divisions within the justice department at the national level; and mayors, police chiefs, faith leaders, hospital and social workers, school principals, neighbourhood activists, and district attorneys at the local level. finally, the practice of safecities members included community policing strategies, after-school programs, crimemapping methods, prosecutorial strategies, the design of local gun-possession laws, and ways to improve the interaction between at-risk youth and law-enforcement professionals. after a couple of preliminary teleconferences, safecities was launched at a faceto-face meeting in washington, d.c., explicitly billed as a community-of-practice launch. the sponsors and community coordinating team (based in the npr office) posed three basic questions for the group to address during the 2-day conference: what is safecities about (domain)? who is part of safecities (community)? what does safecities do (practice)? the conference included opportunities for members to meet informally, including an evening reception and knowledge-sharing 'fair'. a nationally renowned police chief from highpoint, north carolina, gave a talk about his city's success at reducing gun violence through both rehabilitation and enforcement efforts that focused on the city's most violent individuals. (he was so impressed with the gathering that he asked npr officials if his coalition could join, and they agreed to make his group an honorary member.) during the conference, members outlined a design for how they would learn together -including teleconferences, visits, a website, and other activities. the issues they identified became topics for their biweekly teleconferences. the conference was instrumental in coalescing members around a shared agenda and building trust and reciprocity. the safecities teleconferences subsequently became more active and members were more forthcoming about selecting topics and offering to speak to the group about their experiences. fostering 'community' -a sense of mutual trust, shared identity, and belonging -took on more prominence as an important structural element that made safecities successful. one of the outcomes of the initial conference illustrates the value of network participation for members. after hearing the highpoint police chief talk about his success, groups from ft. wayne, indiana and inkster, michigan -including police chiefs, mayors, and faith leaders from both cities -visited highpoint and observed programs in action. both coalitions then adapted the highpoint model for their own locales with coaching from highpoint. safecities operated successfully from march 1999 until june 2002, spanning the transition from a democratic to a republican administration. political appointees from both parties, as well as senior civil servants in the justice and treasury departments (where the sponsorship was primarily based) believed in the crosslevel, cross-sector approach that safecities embodied. sponsors were impressed to see such active participation on the part of senior civic leaders, even though they received no government funding for participating. these local leaders felt strongly about the value safecities provided -in terms of ideas, access to expertise, and opportunities for national visibility and influence based on local success. agency sponsors ultimately decided to close the safecities community in favour of a more conventional federal program. the decision confused many of the participants, given the minimal federal costs associated with the initiative, principally the cost of funding the community's full-time coordinator (a junior staff person, albeit a talented leader) and intermittent attention by agency champions. the coordinator's role was particularly important -arranging speakers for teleconferences, documenting insights on the website, arranging peer-to-peer visits, and coordinating with state and federal officials. the loss of the coordinator and agency attention was a fatal blow to the community. in its place, the us justice department enacted a new program, called safeneighborhoods, which provided funding for local initiatives such as after-school programs. the program managers intended to build on the safecities foundation, but they did not appreciate the distinctive characteristics of the community -opportunities for peer-to-peer learning and collaboration across cities, sectors, and levels of government. while safecities members were glad that the government was providing new funds to support local initiatives, they passionately argued that such funding could never substitute or compensate for the value of the safecities community. the safecities story thus validates the power of cross-city communities of practice while highlighting a key challenge: how to educate senior leaders with the power to sponsor such initiatives -from public, private, or nonprofit sectors (including foundations). these and other questions about starting, sustaining, and scaling such initiatives must be addressed for communities to succeed at local, national, and international levels. at the international level there are a myriad of professional groups and organisations that focus on global civic issues. in recent years a number of these have developed a stronger emphasis on peer-to-peer learning and innovation among members from diverse disciplines. the ayuda urbana initiative was started in conversations about developing municipal capabilities between world bank urban specialists and several mayors of capital cities in the central american and caribbean region. they recognised the value of connecting with peers across borders to address problems and challenges that all cities in the region face. a group of ten cities decided to participate in the initiative: guatemala city, havana, managua, mexico city, panama city, san josé, san juan, san salvador, santo domingo, and tegucigalpa. the people involved in the project include the mayors and their staff in each of the ten cities. additional partners include the world bank, which provides overall coordination, some regional organisations to provide local legitimacy, and the british and dutch governments to provide funding. 4 the project was to create a constellation of communities of practice that would take advantage of the knowledge available in the participating cities. the domains would focus on a challenge of urban development and management the cities shared, including e-government, urban upgrading, environmental sanitation, municipal finances, urban transportation, renovation of historical city centres and poverty alleviation, and disaster prevention and management. the communities would consist of urban specialists in each domain from the participating cities and from local organisations. together they would build their practice by comparing experiences and sharing resources across cities, with input from world bank experts about what had been learned elsewhere. the communities of practice were officially launched through a series of 2-day workshops, each focused on one of the topics. each workshop brought together specialists from the participating cities as well as a few world bank experts. the purpose of the workshops was to • create an initial forum to develop relationships and trust through face-to-face interactions among participants • provide an opportunity for each participating city to share its experience • engage participants in a discussion of lessons learned based on presentations by world bank experts • establish a prioritised list of the most pressing issues and most frequently asked questions • introduce web-based tools for use in facilitating an ongoing learning process and train participants to use the system • choose a person to coordinate the collection of resources to be shared via e-mail and the web site. the project has created an interactive website, available to the public, which serves as a repository for the various communities of practice. the site includes a library of resources, downloadable manuals, bibliographic references, and proceedings of meetings. in addition, the site hosts an online forum to give participants the opportunity to discuss issues, ask questions, share relevant information, and stay in touch. for example, a community member asked how to price waste management services. another member from san salvador responded with a posting that explained how his city determined the price of such services. the ayuda urbana initiative illustrates the value of collaboration across borders to address urgent issues in urban development, and it raises salient issues common to international communities. creating communities of practice among cities from different countries is not all that different from similar efforts within a country, but there is additional complexity. the regional focus of ayuda urbana meant that participants spoke the same language and shared a cultural context. the situation would have been more complex if the project had expanded beyond the region. another issue is the role of the convener when members do not share the same national government. sponsorship has to come from an organisation like the world bank, which can appreciate the vision of cross-border communities and the subtleties involved in cultivating such communities. indeed, ayuda urbana represents the latest development of a broader initiative at the world bank to focus on knowledge as a key lever in the fight against poverty. the bank started an initiative in 1998 to support the development of communities internally, and since then the number of communities has grown from twenty-five to more than a hundred -and the influence of several has been considerable. an external study of the communities found that they were the 'heart and soul' of the bank's new strategy to serve its clients as a 'knowledge bank.' the ayuda urbana initiative highlights the importance of a skilled convener who is committed to a community-based approach as a way to address societal challenges. in this case, the world bank is applying the same knowledge strategy with client countries that it has been applying internally. indeed, the bank's experience in cultivating communities of practice was critical to the success of the ayuda urbana project. the result is a new model for facilitating knowledge development among countries. experts at the bank consider it their task not just to provide their knowledge to clients but to build communities of practice among them as a way to develop their capabilities. the bank experts still have a role to play, but not in a one-way transfer. instead, their contribution takes place in the context of a community of practice that emphasises peer-to-peer learning. this approach models a shift in the traditional relationship between sources and recipients of knowledge. cultivating civic learning systems involves many of the challenges that organisations face in cultivating internal learning systems, but many of these become amplified in the civic context. the domains are especially complex; the communities tend to be very diverse; and the practices involve different disciplines, varied local conditions, and less well-defined opportunities to work together on projects. but perhaps the greatest challenge is the scale required for civic learning systems to leverage their full potential and match the scale of the problems they address. how do you significantly increase the scale of a community-based learning system without losing core elements of its success -identification with a well-defined domain, close personal relationships, and direct access to practitioners for mutual learning? the principle to apply is that of a fractal structure (see gleich, 1987; wheatley, 1994) . in such a structure, each level of substructure shares the characteristics of the other levels. applying such a design principle, it is possible to preserve a small-community feeling while extending a system from the local to the international level. local coalitions such as the chicago biotech network and each of the safecities partners created a local focus of engagement that made it possible for members to participate in broader networks at national and international levels. the idea is to grow a 'community of communities' in which each level of sub-communities shares basic characteristics: focal issues, values, and a practice repertoire. each dimension of a community of practice provides opportunities for the constitution of a fractal learning system. fractal domain. in many cases, domains may start more broadly and eventually subdivide as members discover nuances and opportunities to focus on different subtopics or to apply a topic to different localities. ayuda urbana, for example, is spawning subdomains related to particular civic practices and engaging members with particular expertise and interest in those areas. the city-based coalitions of safecities focused on the same issues but within the context of their situations. all these subdivisions retain a global coherence that gives the entire system a recognisable identity and allows members to see themselves as belonging to an overall community even as they focus on local issues. fractal community. topical and geographic subgroups help create local intimacy, but they must be connected in ways that strengthen the overall fabric of the network. a key to this process is multimembership. members such as those in the safecities network join at the local level but end up participating in multiple communities in ways that help interweave relationships in the broader community. as a result, they become brokers of relationships between levels in equivalent types of communities. this works because trust relationships have a transitive character: i trust people trusted by those i trust. the police chief in highpoint, for example, had developed strong relationships with fbi officials, which in turn encouraged his peers to work more closely with federal agents. fractal practice. useful knowledge is not of the cookie-cutter variety. local conditions require adaptability and intelligent application. a fractal community is useful in this regard because it allows people to explore the principles that underlie a successful local practice and discuss ideas and methods in ways that make them relevant to circumstances elsewhere. a fractal community can create a shared repertoire and develop global principles while remaining true to local knowledge and idiosyncrasies. moreover, if one locality has a problem or an idea, the broader community provides an extraordinary learning laboratory to test proposals in practice with motivated sites. in the safecities community, local coalition members were ready and willing to share results quickly and convincingly with peers and then translate these into action. a safecities member from michigan reported, for example, that a visit to meet with innovating colleagues in highpoint 'added ideas and motivation to an initiative that we had been planning for a year. once our mayor visited, he wanted to do it.' highpoint members then helped the michigan coalition adapt their model successfully. each locality constitutes a local learning experiment that benefits from and contributes to the overall learning system. the key insight of a fractal structure is that crucial features of communities of practice can be maintained no matter how many participants join -as long as the basic configuration, organising principles, and opportunities for local engagement are the same. at scale, in fact, the learning potential of the overall network and the influence at local levels can increase significantly. the key challenge of a large-scale learning system is not whether people can learn from each other without direct contact but whether they can trust a broader community of communities to serve their local goals as well as a global purpose. this depends on the communities at all levels -local, state, national, and international -to establish a culture of trust, reciprocity, and shared values. developing this social capital across all levels is the critical success factor for going to scale. the evolution of a learning system must therefore be paced at the time-scale of social relationships, not according to an externally imposed objective to achieve short-term results. organisers need be careful not to scale up too fast. they need to establish trust and shared values at different levels of aggregation through various mechanisms, including a network of trusted brokers across localities. in the civic domain, the institutional context can be fragmented and the issues politically charged. this presents particular challenges for finding sponsorship, organising support, and managing potentially conflicting constituencies. sponsorship. all three communities depended on sponsorship from executives such as the mayor of chicago, the vice president of the united states, or representatives of the world bank and funding governments. sponsorship is especially important for large-scale learning systems that will require additional activities to connect localities. it can be difficult, however, to identify the 'client' who benefits when a learning system is so dispersed. when you try to engage a city to sponsor a constellation of cross-sector communities of practice to address an array of civic domains, where do you start? a civic community of practice is such an innovative approach that leaders typically do not have enough context to see its value. sustained sponsorship, furthermore, requires community members to make the value visible enough to demonstrate the payoff of sponsor and stakeholder investments. finally, the legitimacy of sponsorship can be contested in a politically fractious context, where the role of institutions such as the world bank or the federal government in orchestrating local affairs is not universally welcome. support. process support was key to the communities we have described. they needed help with local event planning, finding resources, coordinating projects across levels, finding others to connect with, and designing ways to connect. all three communities needed facilitation at meetings, and safecities and ayuda urbana both required moderation for their online interactions. a challenge for civic learning systems is that there may be no clearly defined institutional context or financing model for process support. the ayuda urbana experience also suggests that one must be ready to provide a lot of support at the start to help develop members' local capabilities and prepare the group to operate more independently. civic communities of practice also need help to build a technology infrastructure for communicating across geographies and time zones, and for building accessible knowledge repositories. this can be particularly difficult when communities span multiple organisational contexts. conflict management and collaborative inquiry. civic communities of practice organised around contentious issues such as housing, education, and health will face considerable obstacles from formal and informal groups with entrenched and opposing views and interests. there are good reasons these basic conflicts have been so intractable: views and values are divergent and trust among players is often low. moreover, businesses, nonprofits, governments, and universities have reasons to resist the development of communities of practice. these formal organisations and their leaders have developed established, privileged positions in society, and changes initiated by community members may not be welcomed. inevitable mistakes early on could further diminish low trust levels and reduce the low-to-medium public readiness to invest time in these unfamiliar social commitments. communities that face such tensions will have to develop expertise in collaborative inquiry and conflict management and learn to build trust over time through activities that enable members to find common ground. there is an emerging, global zeitgeist about community and learning. these issues have become commonplace in multinational organisations -private, public, and nonprofit. still, when one looks at the learning requirements of the world, the complexity of the required learning system may seem so overwhelming as to discourage action. but the advantage of a community-of-practice approach is that it can be evolutionary -starting small and building up progressively, one community at a time. it is not necessary to have broad alignment of the kind required for designing or changing formal structures. we can start wherever there is opportunity, energy, and existing connections. we can build on what already exists. indeed, we have found successful examples of initiatives to cultivate learning systems: within cities, across cities at a national level, and across cities internationally. taken together, these early examples paint a picture of what a mature world learning system may look like, and they give some indication of what it will take to cultivate such a system. we now need to develop frameworks for describing the organisational nature of civil society as a community-based action-learning system -and tools and methods for cultivating such systems. this chapter is thus not only a call to action and a proposal for what is possible. it also calls for a new discipline. a discipline that expands the field of organisation design and applies analogous principles at the world level. a discipline that promotes the development of strategic social learning systems to steward civic practices at local, national, and global levels. a discipline whose scope is the world and whose focus is our ability to design the world as a learning system -a discipline of world design. this chapter is only a beginning. there are many established and emerging disciplines -political science, economic sociology, social network analysis -that can inform the work in this domain. a community-based approach to world design is not a silver bullet for solving the problems of the world. while the emphasis here has been on community, a complete discipline of world design would address how the power of communities can be most fully realised by aligning community activities within a broader ecology of formal and informal structures -institutions, cultural groups, laws, and social networks. to steward such a discipline, we need a community of practice ourselvesor indeed a constellation of communities on the topic of world design, at local, national, and global levels. for instance, a small group of people passionate about civic development may gather to outline an approach to cultivating the city as a learning system. they might connect with various civic leaders and extant initiatives, and organise a gathering for the purpose of assessing the implicit structure of the city today as a practice-based learning system. which practices have active stewardship? what groups are providing it with what sorts of initiatives and results? who is represented? where is the focus of sponsors -such as local government, corporations, universities, the media, and foundations? to what degree is there a shared language and understanding across constituencies of the nature of crosssector civic governance and how to participate effectively? these questions become the concerns of 'meta-communities' at various levels, which can link together -as a community of meta-communities -and build their own practice to support the development, effectiveness, and influence of civic communities at all levels. the complexity and intelligence of such a social learning system must match the complexity of world-design challenges and the knowledge requirements associated with them. the messy problems of civil society require a commensurate capacity for learning, innovation, and collaboration across diverse constituencies and levels. the challenge to intentionally and systematically design and develop the world as a learning system must be a global, diverse, interwoven social movement. this social movement is not simply about advocacy; nor is it a political revolution. rather, it is about the transformation of civic consciousness -a way of thinking about governance as an action-learning process, as a role for civic actors across sectors, as a process that links the local and global in clear and concrete ways. and it depends, fundamentally, on individuals finding a way to participate locally -whether that means a community of place or practice, or both -a way that gives them access to the entire learning system. let us begin. the division of labor in society chaos: making a new science organizing for economic development in chicago: a case study of strategy communities of practice: a new tool for managers,' ibm endowment for the business of government our world as a learning system: a communities-of-practice approach ayuda urbana: a constellation of communities of practice focused on urban issues and challenges in central america, mexico, and the caribbean region. written for the beep project of the european union cultivating communities of practice leadership and the new science key: cord-325445-80p6wthw authors: goh, ong sing; fung, chun che; wong, kok wai title: query based intelligent web interaction with real world knowledge date: 2008-03-14 journal: new gener comput doi: 10.1007/s00354-007-0031-7 sha: doc_id: 325445 cord_uid: 80p6wthw this paper describes an integrated system based on open-domain and domain-specific knowledge for the purpose of providing query-based intelligent web interaction. it is understood that general purpose conversational agents are not able to answer questions on specific domain subject. on the other hand, domain specific systems lack the flexibility to handle common sense questions. to overcome the above limitations, this paper proposed an integrated system comprises of an artificial intelligent conversation software robot or chatterbot, called artificial intelligence natural-language identity (hereafter, aini), and an automated knowledge extraction agent (akea) for the acquisition of real world knowledge from the internet. the objective of akea is to retrieve real world knowledge or information from trustworthy websites. aini is the mechanism used to manage the knowledge and to provide appropriate answer to the user. in this paper, we compare the performance of the proposed system against two popular search engines, two question answering systems and two other conversational systems. traditional media such as television, radio, newspapers and magazines play an important role in reporting and providing the latest information on current events. however, nearly all of these media are linear and unidirectional in nature. in other words, they do not provide interactive communication nor can they answer any query from the users. most of these traditional means of media also do not link the answers to the source or reference of the information. hence, viewers or readers may require additional effort in locating or verifying the information. on the other hand, the internet, in particular, the world-wide-web (www), coupled with multimedia, browser and web service technologies, presents a powerful way of communication. if the system is designed properly, it could provide high level of interactivity between the users and the computer. this is clearly a major step in the advancement over the traditional means of communication. one of the possible applications of such system is to provide answers to queries on specific topics or knowledge domains. consider situations such as outbreak of diseases, natural disasters and terrorist attacks, they have caused much miseries, fear and confusion around the world. examples of such crisis are the severe acute respiratory syndrome (sars), bird flu, september 11, earthquakes and tsunamis. in such times, a lot of people will be eager to know as much information as possible. this is where the traditional media may be found inefficient as they are limited with constraints such as page number and air-time. they may also be unable to source all the information from around the world. in addition, people such as managers and decision-makers, frontline specialists or emergency services personnel, and concerned citizens who are directly or indirectly involved in the situations, require to be better informed of the situation and the development. to this end, the internet could have a major role to play as an essential communication channel in providing an intelligent interactive interface for the users. a form of a "global crisis communication system" could be used to provide a natural language query-and-answer system. this system should be capable of providing relevant responses to queries from the users in a conversational manner. in this paper, we describe part of a project which is currently under development. the aim of the project is to develop an intelligent conversation agent called aini to answer domain specific questions as well as open-domain (or common sense) questions. in this report, we have used the subject -bird flu as the domain knowledge of interest to demonstrate the feasibility of the developed system. the key contribution described in this paper is the integration of the common sense knowledge and domain specific knowledge in the form of a "knowledge matrix." the system is based on a layered and modular design, and the answers for the queries are searched from these modules. the proposed system is based on an intelligent conversation agent called aini and an akea. the objective of akea is to retrieve real world knowledge or information from trustworthy websites whereas aini is the mechanism used to manage the knowledge and to provide appropriate answer to the user. descriptions of the system are given in the subsequent sections. the prototype system is compared to two popular search engines -google and yahoo!, two question answering systems -start and askjeeves and two conversational systems -eliza and alice. while it is understood that the purpose of those systems are different, the objective of the comparison is to demonstrate the need of the proposed system described in this paper. this also shows that the proposed system is capable to provide appropriate answers by integrating open domain and domain specific knowledge into one conversation agent architecture. §2 aini's conversational agent architecture the aini architecture is shown in fig.1 . the architecture has been reported in previous publications by the authors. 19, 20) basically, the aini engine comprises a number of knowledge modules in the data layer. it has the ability to communicate with three layers: the application layer, the data layer and the client layer. the client layer is capable to communicate with the user via different channels such as web browser, mobile browser, wap browser and gsm interface. it can carry on multiple independent conversations at the same time. aini's knowledge bases are located within the data layer. there are a number of modules supporting the application layer which governs the manipulation and searching of the answers. the modules use plug-in principles that can quickly be augmented with domain knowledge for specific purposes. originally, this research project involves the establishment of an embodied conversational agent (eca) based on an aini architecture. 20) the prototype system is designed specifically for the web and mobile technology as shown in the client layer in figure 1 . the complete software agent can be considered as a multi-domain knowledge system with multimodal human-computer communication interface. the query and answer between the user and the computer are communicated via the common protocol tcp/ip. aini is designed to engage the user with focus on the chosen subject topic. in this particular application, the topic is on the possible pandemic virus, h5n1. aini communicates with the user in natural language via typed messages. the system is also capable to reply in text-prompts or text-to-speech synthesis together with appropriate facial-expressions on the displayed object which can be an animated avatar or a human face. as illustrated in fig.1 , aini employs an internet three-tier, thin-client architecture that may be configured to work with any web application. it comprises a client layer, an application layer and a data server layer. the hybrid architecture provides features of multimodal interface (client layer), multilevel natural language query (application layer) and multiple knowledge bases (data layer). the process of communication and answering is as follows. given a question, aini first performs a question analysis by extracting pertinent information to be used in query formulation, such as the noun phrases (nps) and verb phrases (vps) using the minipar parser. 26) minipar is a broad-coverage parser for the english language. an evaluation with the susanne corpus shows that minipar achieves about 88% precision and 80% recall with respect to dependency relationships. in our experiment by using corpus extracted by automated knowledge extraction agent (akea), 18) minipar parser is capable to parses nearly 500 words per second on a dell precision pws380 server 3gh with 1gb memory. the user interface resides in the thin-client layer and it supports webbased and mobile service interface. for the web-based interface, the system is based on multimodal agent markup language (maml) interpreter in order to handle the user interface. maml is a prototype multimodal markup language based on xml that enables animated presentation agents or avatars. it involves a virtual lifelike talking 3d agent character designed to carry out a more natural conversation with the user. for mobile devices, due to their small screens, there are limitations on the amount of information that can be presented at one time. reading large amounts of information from such devices can require excessive amount of scrolling and concentration. to reduce distraction, interactions, and potential information overload, a better way of presenting information might be through multilevel or hierarchical mechanisms. 9) a chat mode interface will suit the multilevel mechanism nicely and this will be a better solution for mobile service. in addition, current wireless network service vendors are now providing a wide bandwidth telephone network, known as 3g communication. 23) this development has led to the increasing adoption of smartphone as a client in the traditional distributed systems. on such system, the proposed system will require the mobile flash player. 2) for wap services, the application was embedded wap browsers from vendors such as openwave * 1 and nokia. * 2 all communication with aini takes place through typed text messages and they are processed based on natural language understanding and reasoning. aini's engine implements its decision making network based on the information it encounters in the six levels of knowledge modules. the input and output of each module is an xml-encoded data structure that keeps track of the current computational state. the knowledge modules can be considered as transformations over this xml data structure. the system accepts questions or queries from the users and it processes the queries based on the information contained in aini's knowledge bases. the system is implemented by open-source architecture employing a kannel mobile gateway, php, perl scripting language, apache server and knowledge base stored in a mysql server. in this server, it handles the process of the queries. here, one or more application servers are configured to compute the dialogue logic through the multilevel natural language query algorithm. in this layer, it is based on a goal-driven or top-down natural language query (nl-query) approach which is similar to the way that humans process their language. as indicated by literature in the field of natural language processing (nlp), the top-down approach is by far the best approach. as shown in fig. 1 , the top-down nl-query approach consists of six levels of queries, namely spell checker (level 0), natural language understanding and reasoning (nlur) (level 1), faqchat (level 2), metadata index search (level 3), pattern matching and case-based reasoning (pmcbr) (level 4) and supervised learning (level 5). all these have been discussed in reference. 17) the data layer serves as storage for data and knowledge required by the system. this is where the aini bot's conversational knowledge bases are stored. in the proposed approach, the architecture can be considered as a domain matrix knowledge model 16) for the support of the conversational system. it is well understood that true intelligent action requires large quantities of knowledge. such reservoir of knowledge is harvested from the internet and deployed in the domain matrix knowledge bases architecture. this form the basis for the construction of large-scale knowledge bases to be used as the engine for the intelligent conversation systems. these databases established so far are dictionary, domain-specific knowledge bases, open domain knowledge bases and chatlog (conversation logs and unanswered question logs). as mentioned previously, the first step taken by aini is to perform a spell check to eliminate possible wrong spellings. the spell checker is located at the application layer and the dictionary contains the database of words. the spell checker used in this study is called ispell. ispell is an open-source program that is used to correct spelling and typographical errors in a file. ispell was first run on tops-20 systems at the mit-ai lab. many people have contributed dictionaries to allow ispell to be used with their native languages. at least 50 dictionaries have already been established. as far as this project is concerned, only english is used. however, it can be anticipated that aini has the potential to be used as a true intelligent web interaction with users who are using different languages. combining the dictionary and the spell checker in the application layer, this combination can be considered as the level 0 knowledge. in terms of the domain-specific knowledge databases, it can be observed in fig. 1 that the domain specific knowledge modules are made up of a natural language corpus and a faq (frequent asked questions) module. the natural language understanding & reasoning (nlur) module in the application layer is used to gather data to be stored in the natural language corpus. in addition, the akea is used to extract information from the web and in particular trusted web sites. the architecture of akea has been reported previously and the details can be found in reference. 18) a description of its operation will also be given in the subsequent section. this forms the level 1 knowledge. the faq (frequent asked questions) module is connected to the faqchat module in the application layer. it is used to provide information based on the faq sections from websites and from the actual chat logs. in other words, the conversations and answers given by the user to the system are also used to enhance the information in the faq module. this forms the level 2 knowledge. in this study, the bulk of the information was collected during the height of the sars epidemic in 2003 19) and the bird flu pandemic crisis in 2005. 21) however, in this paper, the domain specific knowledge is only focused on bird flu. the open-domain knowledge modules in the data layer are based on trec corpus, minipixel and the open-domain knowledge base (aaa). they are further supported by the conversation logs which store the previous conversation for extraction of answers from the unanswered questions. it is assumed that such answers could be used in future interaction with the users. details of these open-domain knowledge modules are described in the section 3.1 and they form the subsequent levels of knowledge in the application layer. §3 domain knowledge matrix model in this paper, bird flu pandemic is the domain-specific knowledge used as an example to illustrate the application of this proposal. research and information on h5n1 pandemic have become increasingly important as the pandemic may have dire global implications. wall street journal online 7) predicted that this pandemic could be worse than the one in 1918 which killed at least 20 million people. in addition, the world health organization estimates the h5n1 virus could infect up to 30 percent of the world's population. shigeru omi, a who official, also warned that an estimation of 2 to 7 million deaths are "conservative" and that the maximum figure could be as high as 50 million. 29) the aini's domain knowledge matrix model incorporates several knowledge subjects. this is analogous to the consultation of expertise knowledge from multiple experts. for example, a sales domain knowledge should contain expertise or knowledge on how to improve sales. however, as a sales person, he or she is expected to have a wide range of common sense knowledge and the ability to engage the potential customer. hence, the intelligent system should also incorporate open-domain knowledge to handle general or generic questions. in here, the open-domain knowledge is not necessary the common sense knowledge which is assumed to be possessed by everyone. the open-domain refers to the accessibility of the knowledge. hence, they are still categorized according to knowledge domain. by including multiple domain knowledge bases with aini's single domain knowledge, the proposed aini will be able to hold "meaningful" conversations with the users. in this proposed system, the open-domain and domain-specific knowledge are pre-defined in the data layer. these modules are used to support the various knowledge levels at the application layer. depending on the user's input, the agent will respond or switch from one level to another. while the system is capable to communicate with the user beyond the domain knowledge, there are cases that the system will exhaust its capability to answer the queries. in such case, the system will attempt to divert the focus back to the current topic of interest by responding with some predefined random statements. the purpose is to direct the users' back to the system's domain-specific state. hence aini will attempt to "cycle" between the 6 levels of information processing within the application layer supported by the various knowledge modules in the data layer. a way to view the proposed domain knowledge matrix model is given in fig. 2 . in this approach, the knowledge base of the aini can be considered as a collection of specific conversation domain units. each unit handles a specific body of knowledge used during the conversation between aini and the user. the knowledge can be seen as arranged in the vertical columns making up the open domain or domain-specific knowledge. in addition, specific subjects are shown in the horizontal rows. for example, in the open domain knowledge, the subject units will cover topics such as personality, business, biology, computer, etc. in this report, our focus is on the medical subject and in particular, the bird flu pandemic, therefore there are additional modules being incorporated. the domain knowledge model plays a major role in conversational systems. such systems normally are comprised with two subcategories: the traditional or narrow domain, and, the open domain. in the traditional domain, systems attempt conversational fluency based on limited domains of expertise. eliza 34) for example simulates a rogerian psychotherapist, and this implementation is commonly known as doctor. the rogerian psychotherapist knowledge base attempts to engage the patient by repeating the patient's statements, and by encouraging the patient to continue talking. terry winograd's shrdlu 37) is another program simulating a robot which is able to interact within a simple world which consists of colored building blocks and boxes on a flat surface. shrdlu has knowledge about its world and it can answer questions in natural language. three decades have passed since eliza was created. computers have it is assumed that the human brain contains a world of knowledge, but it has limitations on memory retrieval. hence, knowledge needs to be specified. in the proposed system, the domain knowledge matrix model is designed along this line. the system uses custom domain-oriented knowledge bases and existing knowledge bases from online documents and training corpora. it contains a spectrum of possible solutions from queries on specific domains to general conversation questions. it can also be considered as a xml-like metadata model. this approach does not attempt to predict possible inputs from the user. instead, the system aims to handle conversations within a specified domain or focus on 'domain-specific' conversations. in this project, the novel contribution is the development of the "domain knowledge plug-in components." with this arrangement, the domain-specific knowledge could become portable, scalable and incorporated easily with other applications. this approach will allow future improvements to encourage collaborative contribution to the specific knowledge domain. the proposed system will enable the development of a wide range of information sources. open-domain conversational systems need to deal with questions about nearly any topic. it is very difficult to rely on ontological information due to the absence of wide and yet detailed banks of world knowledge. on the other hand, these systems have much more information and data to be used in the process of answering the queries then any domain specific systems. in aini's conversation system, information from the large-scale mass collaboration mindpixel 30) and training data sets from the text retrieval conference's (trec) training corpus 32) are used. aini also uses alice annotated aiml (aaa), 5) the loebner prize winner 28) and the chatterbox challenge winner 10) hand crafted knowledge bases. these are illustrated in the data layer in figure 1 and under the open-domain columns in fig. 2 . mindpixel is a common sense knowledge component and it is similar to openmind * 3 and cyc. * 4 the system accepts public contributions. however, cyc model and openmind had bottlenecks which prevent truly large-scale collaboration 33) . the first is the fact that knowledge does not grow by itself. every new rule or axiom has to be entered manually and the process takes a lot of patience and time. furthermore, information has to be input with the cycl programming language and to follow the rules of the system. the second drawback is the complexity of cyc. it will need months to install and implement a system that is based on the knowledge base of cyc. on the other hand, mindpixel started collecting their propositions privately via email in 1994 and then evolved to online mass collaboration. to date, the project's user base of nearly fifty thousand people has contributed more than one million propositions and recorded almost ten million individual propositional response measurements. ainis use only 10% of the mindpixel propositions. in practice, 10% of the training corpus is held back from training to act as a generalization test to ensure the system does not simply memorize the corpus. passing this generalization test would be the basis for claiming that the system is able to replicate human-level intelligence in a machine. although a lot of knowledge has been collected, it is recognized that the system is still less than the uncountable "pieces" of common sense knowledge that are estimated to be involved with human intelligence. 31) a second common sense knowledge component deployed by aini is a training corpus from trec as shown in table 1 . trec, organized each year by the national institute of standards and technology (nist), has offered a specific track to evaluate large-scale open domain question-answering (qa) systems since 1999. finding textual answers to open domain questions in large text collections is a difficult problem. in our system, we only extracted factoid questions to be incorporated in the aini's engine. our concern is the types of questions that could be answered in more than one way. we have tried to avoid such questions. in conversational systems, factoid questions should have only single factual answers. 3, 12, 13, 36) these are considered as a good stimulus-response type of knowledge unit. examples of such questions are, "who is the author of the book, the iron lady: a biography's of margaret thatcher? " "what was the name of the first russian astronaut to do a spacewalk? " or "when was the telegraph invented? " trec's corpus has a considerably lower rate of answer redundancy than the web and thus, it is easier to answer a question by simply extracting the answers from the matching text. to gather this data, we automatically classified questions in the trec 8 through trec 10 test sets by their 'wh'-word and then manually distinguished factoid questions, which represented * 3 www.openmind.org * 4 www.cyc.com around half of the initial corpus as shown in table 1 . the third knowledge base in the aini's open domain knowledge model is obtained from hand-crafted annotated alice aiml (aaa), * 5 a loebner prize winning 28) conversation system knowledge base. aaa is a free and opensource software package based on xml specifications. it is a set of artificial intelligence markup language (aiml) scripts and this is the backbone of the award winning conversation system. aaa is specifically reorganized to facilitate conversational system developers to clone the 'brain' of the conversation system and to enable the creation of customized conversation agent personalities. the approach has reduced the need to invest huge efforts in editing the original aaa content. aaa's knowledge bases covered a wide range of subject domains based on the conversation agent's "personality." example subjects include ai, games, emotion, economics, film, books, sport, science, epistemology and metaphysics. these subjects are shown in fig.2 as part of the domain knowledge matrix model. in order to illustrate the ability of alice in handling common sense or open domain queries, alice has won the 2000, 2001 and 2004 loebner prize for being the most lifelike machine. the competition is based on the turing test 1) which aims to determine whether the responses from a computer can convince a human into thinking that the computer is a real person. in the competition, alice used a library of over 30,000 stimulus-response pairs written in aiml to answer the queries. the development of alice is based on the fact that the distribution of the sentences in conversations tends to follow zipf's law. 25) it is indicated that the number of "first words" is only limited to about two thousand. the frequency of any word is roughly inversely proportional to its rank in the frequency table. the most frequently used word will occur approximately twice as often as the second most frequent word. it in turn occurs twice as often as the third most frequent word, and so forth. questions starting with "what is" tend to have zipf-like distributions. this type of analysis can now be accomplished in a few milliseconds of computer time. while the possibilities * 5 www.alicebot.org/aiml/aaa/ of what can be said are infinite, the range of what is actually said in conversation in most cases is surprisingly small. specifically, 1800 words cover 95% of all the first words input. it is this principle that aini is operating on which enables it to be able to respond in an efficient and mostly accurate manner. at present, the world-wide web provides a distributed hypermedia interface to a vast amount of information available online. for instance, google 14) currently has a training corpus of more than one trillion words (1,024,908,267,229) from public web pages. this is valuable for many types of research. the web is a potentially unlimited source of knowledge repository; however, commercial search engines may not be the best way to gather answers from queries due to the overwhelming number of results from a search. before the rise of domain-oriented conversational systems using on natural language understanding and reasoning, evaluation was never a problem, as information retrieval-based metrics were readily available for use. however, when conversation systems begin to become more domain specific, evaluation becomes a real issue. 15) this is especially true when natural language processing (nlp) is required to cater for a wider variety of questions and, at the same time, required to achieve high quality responses. as shown in fig. 1 and 2 , aini's domain-specific knowledge bases consist of natural language corpus and frequently asked questions (faq). both components are extracted from the online documents using the akea as described in reference. 18) another significant aspect of this paper is the objective of aini to deliver essential information from trusted sources while capable of interacting with the users. a discussion on the selection of the trusted websites is given below. as the web that we know today becomes increasingly chaotic, overpowering and untrustworthy, selection of trusted web pages is becoming an important factor contributing to its long-term survival as a useful global information repository. in our experiment, the selection of the trusted websites is based on pagerank tm . 8) pagerank tm is a system for ranking web pages developed by larry page and sergey brin at stanford university. pagerank tm relies on the uniquely democratic nature of the web by using its vast link structure as an indicator of an individual page's value and high-quality sites always receive a higher pagerank tm . in our study, the selection of trustworthy websites started with the initial six seed words: bird, flu, avian, influenza, pandemic and h5n 1. these seeds are supposed to be representative of the domain under investigation. the seed terms are randomly combined and each combination is used in google api * 6 and bootcat tool 6) for bootstrapping corpora and terms from the web. we used the seeds to perform a set of google searches, asking google to return a maximum of 20 urls per query and then we collected the corpus. after visual inspection of the corpus, we used the top 40 seeds extracted from token frequencies for the second run. finally, we retrieved 1,428 urls out of 1,500 urls related to the domain being investigated. the reduction in number is due to the duplicated and broken link urls being removed. based on the 1,428 urls, we sent a query to google's pagerank tm directory using parameter tool 11) to determine their rankings. figure 3 shows the results of the top 10 site based on the pagerank tm scale. the pagerank tm scale goes from 1 to 10. a less important site is the one with a pagerank (pr) of 1. the most referenced and supposedly important sites are those with a pr of between 7 and 10. the final set of urls was further culled to include only selected sites which are attributed to a regulated authority (such as a governmental or educational institution) that controls the content of the sites. once the seed set is determined, each url's page is further examined and rated as either reliable or reputable. this selection is reviewed, rated and tested for connectivity with the trusted seed pages. from this exercise, whitehouse.gov, pandemicflu.org, cdc.gov and who.int were selected due to their pagerank tm scale scores being above 7. the most important factors in determining the "reliable authority" of a site is based on its history and the number of back-links to the governmental and international organization links. the more established and relevantly linked the more it will be considered as "stronger" or "more reliable." this effectively gives the linked site a measure of "trust" and "credential." the selected urls are then used as the source knowledge base for akea to extract the contents on bird flu so as to build aini's domain-specific knowledge base. a discussion of the extracted information is given in the following section. in aini's domain knowledge matrix model, the unit domains in the natural language corpus component consist of knowledge and information harvested from or expressed in ontologies, gazetteers, named entities and wordnet. these have been implemented as domain-dependent modular components. the named entity module identifies named locations, named persons, named organization, dates, times and key measures in text formats. the information is obtained by akea. for example, information for diseases is based on symptoms, causes, diagnoses, vaccination locations, persons and organizations. in order to identify these entities, our system uses rules to specify the named entities' structure in terms of text tokens and information from the source such as tagger, morphosyntactic analyzer and knowledge bases of names, clue words and abbreviations. the web knowledge base is then continuously updated with facts extracted from online pandemic news using information extraction (ie) by akea. ie is the task of extracting relevant fragments of text from larger documents and to allow the fragments to be processed further in an automated manner. an example of an application of akea is to prepare an answer for a user's query. the ontology and gazetteer have been implemented as domain-dependent modular components which will allow future improvements in the domain knowledge. in aini's faq component, the unit domain consists of information concerning diseases, symptoms, causes, diagnoses, vaccinations, etc. the selection of faq trusted web pages has been carried out using pagerank tm as discussed above. but at this stage, each of the selected websites was evaluated in order to find the more suitable and reliable faq pages. from this experiment, the answers.pandemicflu.gov and who.int/csr/disease/avian influenza/avian faq pages have been selected as the source of information for akea to build aini's faq knowledge base. based on the proposed approach, the quality of the results returned from aini's engine using the faq knowledge base are either similar to or better than those generated by search engines such as google. aini's sql engine uses the most significant words as keywords or phrase. it attempts to find the longest pattern to match without using any linguistic tools or nlp analysis. in this component, aini does not need a linguistic knowledge unit and relies on just an sql query. all questions and answers can be extracted from the database which was built by akea after applying a filtering process to remove unnecessary tags. example results from the system are illustrated in the next section. table 2 , aini's open domain knowledge base currently has more than 150,000 entries in the common sense stimulus-response categories. out of these, 100,000 came from mindpixel, 997 factoid questions from the trec training corpus and 45,318 categories from aaa knowledge bases. on the domain-specific knowledge base, aini has more then 1,000 online documents extracted by akea. this makes up 10,000 stimulus-response items in total. aini also has 158 faq pairs of questions and answers which have been updated using akea. in addition, aini has also collected more than 382,623 utterances in conversations with online users since 2005. these utterances will be integrated into aini's knowledge bases through supervised learning by domain experts. at present, aini has learnt 50,000 categories from conversations with online users. all of this combined knowledge has made up the total of 206,473 stimulus response categories in aini's knowledge bases. in comparison to other system, the original conversational programs such as eliza, 35) written by professor joseph weizenbaum of mit, have only 200 stimulus response categories. alice silver edition was ranked the "most human" computer, and has about 120,000 categories which include 80,000 taken from mindpixel. in this experiment, three types of systems are compared. they are search engine, question answering system and conversational system. for each system, we compared two different engines against aini. the two search engines compared are google and yahoo. for the question answering engines, askjeeves and start are used. they are supposed to use natural language processing for their queries. for the conversational engines, eliza and alice are selected. in particular, alice was ranked as the "most human computer" in the turing test competition. 28) google is a well known search engine which determines relevancy of information primarily on their pagerank algorithm. 24) in our experiment, we developed a search engine interface using google soap search api service 22) and yahoo!. 38) for the question answering system, the idea behind ask jeeves and start is to allow users to get answers for questions posed in natural language. ask jeeves is the first commercially question answering system available on the internet. start 27) is the world's first web-based question answering system which commenced operation since december, 1993. eliza is a well known program in the discipline of artificial intelligence and it is also the oldest system in a way, aini can also be considered as an enhancement of alice with the inclusion of the pandemic domain-specific knowledge base which was extracted by akea, and it also has parsing capabilities based on a full natural language understanding engine for multilevel natural language query. 17) in the present study, appropriate or accurate domain responses are expected to be important. the control question set was submitted to the seven urls where the seven systems were located. the responses of the queries are then collected and displayed as shown in fig. 4 in the course of the present study, a question was raised on how to compare the performance of the proposed system. it was decided that the performance of the system would be based on the keywords instead of complex phrases. for example, if the question "what is bird flu?" is asked, responses from the search engines, question-answering engines and conversational engines are summarized in table 3 , 4 and 5 respectively. the responses are noted as follows: • search engines -the systems identify the key words and return documents that are relevant to the query. the results could be tens of thousand or even millions of hits as shown in table 3 . • natural language question-answering systems -they generate responses using nlp and return fewer possible answers. the answer is table 3 responses from popular search engines -google and yahoo table 4 responses from popular question answering engines table 5 respohnses from conversational engines possibly within the context of the document. the sample response is shown in table 4 . • conversation engines -provide quick response from the stimulus. simple pattern matching and substitution are used based on a surprisingly small number of pre-defined rules. the example is shown in table 5 . §6 discussion in this example, eliza responded with "does that question interest you? " it is observed that eliza tries to ask another question, instead of giving an answer. the objective is to encourage the user to continue with the conversation. on the other hand, alice attempts to convince the user by generating randomly answer from aiml knowledge base. alice's response does not need a grammatical parser as her knowledge base contains the pattern "what is bird flu?" and the witty reply is "xfind *" with an aiml categories. by using "xfind *" pattern, alice will randomly generated responses such as "is there only one," "let me think about it," "have you tried a web search," "i haven't heard of bird flu," etc. the pattern matching language used in alice permits only one wild-card ('*') match character per pattern. therefore, al-ice responds with a variety of inputs from the users. alice does not concern whether it really "understands" the input. it aims to provide a coherent response to the client in order to convey the impression that the system understands the client's intention. for the eliza and alice systems, they are not able to handle questions that demand specific answers. they are simply not designed for such purpose. the three possible ways to handle these types of questions are: (a) analyze the problems with nlp and then provide an appropriate answer, (b) rely on human to review the conversation logs and continually improves the knowledge base, or, (c) treat the query as impossible and then choose a pre-defined random answer. for the aini chatterbot, the response was "avian influenza, or "bird flu," is a contagious disease of animals caused by viruses. . . ". the answer was generated from the domain-specific knowledge base using natural language understanding parsing from level 1. in this query, the answers were discovered by aini from the trusted sites such as who. in addition, the response is based on the natural language understanding and reasoning. the reasoning mechanism of the aini is based on answer discovery in layer-oriented knowledge base. currently ongoing work includes quantitative measurement and assessment of the results and performance of these different systems. although the systems used in this study were built with different objectives in mind, the purpose of this study is to show that there is a need of the proposed system to handle domain specific applications. at the same time, this study also shown that the proposed conversation agent architecture can achieve the expected objectives. §7 conclusion in this paper, we have reported the use of an aini conversation agent architecture to develop a domain specific intelligent web interaction. aini consists of a knowledge acquisition tool called akea for the gathering of conversation and domain-related knowledge. the proposed system has the potential to be used in a domain specific application area. the study has demonstrated with one particular area of domain expertise -bird flu. in this paper, we only worked on selected pandemic crisis websites where we performed knowledge extraction through akea for the domain-specific knowledge databases on the server. it is believed that the approach would be useful and applicable for other domains. we have also compared and tested the flexibility of aini against other popular search engines, question answering systems and conversation systems. furthermore, we have found that domain-specific knowledge base has higher response than the corresponding conversational-style responses. further work will be done on expanding the sources of knowledge and to provide quantitative measurements of the quality of responses from the aini. computing machinery and intelligence mobile and devices analysis of factoid questions for effective relation extraction artificial linguistic internet computer entity the annotated a.l.i.c.e. aiml bootcat: bootstrapping corpora and terms from the web just how deadly is bird flu? it depends on whom you ask inside pagerank overcoming the lack of screen spaces on mobile computers alice winner of chatterbox challenge the effect of document retrieval quality on factoid question-answering performance factoid question answering over unstructured and structured web content all our n-gram are belong to you a black-box approach for response quality evaluation conversational agent system domain matrix knowledge model for embodied conversation agents top-down natural language query approach for embodied conversational agent automated knowledge extraction from internet for a crisis communication portal domain knowledge model for embodied conversation agent intelligent agents for an internet-based global crisis communication system embodied conversational agents for h5n1 pandemic crisis google soap search api (beta) 3gsm statistics google and yahoo search engine technology comparison zipf's law dependency-based evaluation of minipar building a reusable test collection for question answering loebner prize is asian bird flu the next pandemic? common sense in humans text retrieval conference (trec) building large knowledge bases by mass collaboration computer power and human reason eliza -a computer program for the study of natural language communication between man and machine monolingual web-based factoid question answering in chinese, swedish, english and japanese understanding natural language he was associate professor of faculty of information technology and communication, university technical malaysia melaka. his research interest is in the development of intelligent agent and conversational agents to facilitate graceful human-computer interactions. he is a member of ieee we would like to thank the anonymous referees for the constructive and helpful suggestions. this research project is funded by murdoch university research excellence grant scheme (regs), 2006. key: cord-302848-a246wl7f authors: lawler, j. j.; spencer, b.; olden, j. d.; kim, s.-h.; lowe, c.; bolton, s.; beamon, b. m.; thompson, l.; voss, j. g. title: 4.25 mitigation and adaptation strategies to reduce climate vulnerabilities and maintain ecosystem services date: 2013-12-31 journal: climate vulnerability doi: 10.1016/b978-0-12-384703-4.00436-6 sha: doc_id: 302848 cord_uid: a246wl7f abstract increasing temperatures and altered precipitation regimes associated with human-caused changes in the earth s climate are having substantial impacts on ecological systems and human well-being. maintaining functioning ecosystems, the provision of ecosystem services, and healthy human populations into the future will require integrating adaptation and mitigation strategies. adaptation strategies are actions that help human and natural systems accommodate changes. mitigation strategies are actions that reduce anthropogenic influences on climate. here, we provide an overview of what will likely be some of the most effective and most important mitigation and adaptation strategies for addressing climate change. in addition to describing the ways in which these strategies can address impacts to natural and human systems, we discuss the social considerations that we believe must be incorporated into the development and application of mitigation or adaptation strategies to address political situations, cultural differences, and economic limitations. adaptation adjustment in natural or human systems in response to actual or expected climatic stimuli or their effects, which moderates harm or exploits beneficial opportunities. various types of adaptation can be distinguished, including anticipatory and reactive adaptation, private and public adaptation, and autonomous and planned adaptation (ipcc 2001) . climate change a significant and lasting change in the statistical distribution of weather patterns over periods ranging from decades to millions of years. we focus largely on changes in temperature and precipitation, as they are changing in many regions and have direct impacts on ecosystems and human well-being. mitigation actions that reduce the effects of humans on the climate. resilience amount of change a system can undergo without changing state (ipcc 2001) . sensitivity it is the degree to which a system is affected, either adversely or beneficially, by climate-related stimuli. the effect may be direct (e.g., a change in crop yield in response to a change in the mean, range, or variability of temperature) or indirect (e.g., damages caused by an increase in the frequency of coastal flooding due to sealevel rise) (ipcc 2001) . vulnerability the degree to which a system is susceptible to, or unable to cope with, adverse effects of climate change, including climate variability and extremes. vulnerability is a function of the character, magnitude, and rate of climate variation to which a system is exposed, its sensitivity, and its adaptive capacity (ipcc 2001) . virtually all regions of the earth are experiencing rapid changes in temperature, precipitation, and/or other aspects of climate. human-caused changes in the earth's climate are greatly altering ecological systems, with consequences for human well-being. for example, recent studies have documented shifts in the distribution of plants and animals, advances in the timing of key ecological processes, and extinctions of wildlife populations and species that are likely linked to recent increases in temperature (parmesan 2006) . such changes are creating regional combinations of environmental conditions that, within the next 50-100 years, may have no current-day analog. the resulting changes in ecosystem function will undoubtedly affect the provision of ecosystem services for human communities. maintaining or increasing ecosystem services into the future will require integrating adaptation strategies (actions that help human and natural systems accommodate changes) and mitigation strategies (actions that reduce anthropogenic influences on climate) ( figure 1 ). mitigation strategies are those that reduce the magnitude of climate change that will occur. the vast majority of these strategies involve either reducing greenhouse gas (ghg) emissions or sequestering carbon, although they can address any human climate forcing. by contrast, adaptation can be defined as alterations to human or natural systems that are designed to reduce the negative impacts ofor exploit the opportunities created byclimate change (ipcc 2007a) . adaptation strategies are much more varied than mitigation strategies, as they are more often specific to particular systems or locations. mitigation and adaptation strategies designed to address changes in climate may have additional positive or negative effects on ecosystem functioning, ecosystem services, and human well-being. for example, replacing fossil fuels with renewable energy sources reduces ghg emissions while simultaneously reducing air pollution, acid rain, and smog, as well as asthma and respiratory ailments of humans. conversely, coastal armoring, the building of seawalls, breakwaters, and jetties, may reduce damage to built structures from the combined effects of sea-level rise and storm surges but such actions can negatively affect coastal ecosystems. in this chapter, we provide an overview of what will likely be some of the most effective and most important mitigation and adaptation strategies for addressing changes to the climate system stemming from increased ghg emissions. we attempt to cover the entire spectrum of life, from plants and animals to humans, as well as the geophysical properties of our planet that support these organisms. in relation to human populations, we focus primarily on human health as an index of well-being. we also introduce concepts of climate justice. many of our examples are taken from the united states, although we also include other regions of the world in our analysis as appropriate. we discuss various ways in which mitigation and adaptation strategies can help reduce the magnitude and the impacts of the changes we are likely to experience, as well as improve human health and directly or indirectly affect ecosystem functions and services. finally, although our analysis sets priorities and orders of importance for an ideal world unimpeded by political considerations, cultural differences, or economic limitations, we include a discussion of the social considerations that we believe must be incorporated into the development and application of mitigation or adaptation strategies. in the following sections, we briefly discuss what will likely be some of the most effective mitigation strategies for addressing climate change, largely focusing on strategies to reduce co 2 and other ghg emissions ( table 1) . we have grouped these strategies into five categories: transportation, shelter, food, energy, and carbon storage and bioengineering. although we focus on mitigation strategies that will reduce the magnitude of future climatic changes, we also discuss how these strategies can improve ecosystem functioning and, in some cases, human health. transportation, the movement of people and goods, significantly impacts climate through the emission of ghgs. in 2004, transportation accounted for 23% of the world's energy-related ghg emissions and road transportation represented 74% of the total carbon dioxide (co 2 ) emissions from transportation (ipcc 2007b) . in addition to being a major source of emissions, transportation also significantly affects ecosystem functions and services. pollution from vehicles affects ecosystem services such as clean air and water directly, and indirectly through impacts on the plants and animals that regulate ecosystem function. as species are lost from or move into ecosystems the functioning of those systems can change, resulting in altered ecosystem services. human health is likewise affected by pollution from vehicles (e.g., through chronic lung disease). transportation networks and moving vehicles also fragment landscapes, reducing the flows of energy and biota resulting in isolated populations and potentially making these populations vulnerable to extirpation and impairing ecosystem functions. mitigation strategies that address transportation can be placed into three basic categories: those that increase the efficiency of particular transportation approaches, those that result in a shift in the mode of transportation, and those that reduce the amount of transportation that is needed. here, we briefly table 1 examples of mitigation strategies aimed at reducing ghg emissions or, in the case of the last category, sequestering carbon to reduce co 2 concentrations in the atmosphere mitigation strategies barriers to implementation a large reduction in ghg emissions can be accomplished by increasing the fuel efficiency of vehicles. for example, increasing the efficiency of light-duty vehicles (cars and small trucks) has the potential to reduce their carbon emissions by up to 50% by 2030 (ipcc 2007b) . such efficiencies can be attained by a combination of reducing vehicle weights, increasing aerodynamics, increasing engine efficiency (particularly diesel engine efficiency), and increasing the use of biofuels and electric and hybrid vehicles. in 2006, the international energy association projected that the proportion of transportation energy supplied by biofuels could be increased to 10% (iea 2006a) . although there is good evidence of successful biofuel markets and programs (e.g., ethanol from sugarcane in brazil, goldemberg 2007) , there are also negative consequences of increased biofuel production for biodiversity and carbon storage (fargione et al. 2008; fletcher et al. 2010) . one opportunity for transportation mitigation strategy is the promotion of behavioral changes of drivers. efficiency of road vehicles could be improved by 5-20% by changes in driving styles, improved maintenance, more efficient tires, reduced idling, and traffic design and maintenance (ipcc 2007b). a second approach to reducing emissions and many of the other effects of transportation involves changing the ways in which people and goods are moved. one such approach is to increase the use of public and human-powered transportation. increasing the amount of walking, biking, and use of public transit will depend largely on the available infrastructure and the capacity to provide it. supporting the development of 'walkable' cities and neighborhoods, bike-friendly roadways and bike paths, carpooling and car-sharing programs, and efficient and safe mass transit are all approaches for shifting the mode of urban transport. present transportation alternatives vary by country, with europe, japan, and many developing countries having the highest levels of public transportation use, and the united states having the greatest use of personal automobiles. in some developing countries, such as vietnam or china, personal automobile use is a recent phenomenon, and personal motorized transport is a high aspiration because it conveys increased social status. this disparity in the historical development of the transportation sector will mean that mitigation strategies, barriers, and solutions will have to be location specific and may be dynamic over time. in addition to the reduction in emissions and the other environmental benefits of shifts to more human-powered transportation, such changes also have clear human health benefits resulting from increased exercise, weight management, and decreases in air pollution (nemet et al. 2010) . simultaneously improving the quality of public transport and leapfrogging transport-intensive stages of economic growth is needed in many countries to increase higher levels of mass transit use and human-powered transportation (wright and fulton 2005) . further reductions in emissions can be made by changing modes of transport for other transportation such as moving more freight by train and ship and less by truck. the third way to reduce transportation-related ghg emissions is to reduce the amount of transportation that is needed. decreasing the demand for transportation largely involves changes in human behavior, urban and landscape planning, and changes in technology. sprawling, low-density urban neighborhoods, such as those found in the united states, perpetuate automobile dependency and transform ecologically functional landscapes into subdivisions. compact, highdensity communities with local urban centers allow residents to work and live in close proximity, and help build social capital among residents and protect natural areas. in conjunction with human-centered transportation infrastructure such as sidewalks, bike lanes, and public transportation, compact neighborhoods reduce car dependency while simultaneously improving the physical fitness of residents. public green space and urban forests sequester carbon while improving air quality and mental well-being. increased vegetation, shade trees, and less asphalt combat urban heat-island effects and reduce energy demand for cooling buildings. advances in communication technologies also have the potential to reduce the need for transportation. the internet, online meeting, document sharing, and conference tools have led to an increase in telecommuting and web-based meetings. similarly, online meetings have begun to replace some inperson meetings and have the potential to dramatically reduce air travel, which can account for a significant portion of an individual's carbon footprint (fox et al. 2009 ). the most significant barriers to implementing mitigation strategies in the transportation sector are long-established personal transportation behaviors and the lack of government interest or capacity to invest in new technologies. changing the way and the amount that people move around will be very difficult. in the united states and elsewhere, people's relationships with the automobile involve issues of convenience, independence, safety, and social status. changing these relationships will involve making forms of public transportation more convenient, more effective, and more appealing. rising fuel costs and commute times may provide some opportunity for implementing transportation-based mitigation strategies. failing automobile industries and economic crises may provide another. similar to the projects undertaken by the civilian conservation corps in the united states in the depression of the 1930s, modern-day work relief programs could be used to transform transportation systems. short of such crises, major changes in transportation will take a combination of regulatory action, education, and incentive programs. housing is one of the most idiosyncratic and regionally variable aspects of human culture. the design of structures is dependent on local climate, land access, available materials, and regional architectural histories. much of the recent human-caused change in climate stems, in large part, from the way we design, construct, and inhabit the built environment. the following section focuses on a representative selection of design strategies that mitigate the built environment's contribution to carbon emissions and, in many cases, provide cobenefits to human health and local/regional ecological resilience. building material extraction can have profound impacts on ecosystems and ecosystem services. mining degrades natural landscapes, destroys habitat, pollutes freshwater environments, and can expose human populations to toxins. poorly managed lumber extraction leads to deforestation, eliminates suitable habitats, erodes soils, and diminishes the earth's carbon sequestration capacity. mines that observe best management practices such as land reclamation have fewer negative impacts on climate, local ecosystems, and human health. sustainably harvested lumber can act as a long-term carbon sink and contribute to the productive and ecological resilience of forests. rapidly renewable lumber alternatives such as bamboo can offset the demand for slow-growing trees species, rebuild soils, and set the stage for reforestation. however, as with any renewable resource, expanding the production of bamboo in monocultures has the potential to eliminate and/or degrade existing ecosystems. eight percent of annual carbon emissions worldwide can be attributed to building material extraction, production, transportation, and installation (mazria and kerschner 2008) . choosing building materials with low embodied energy (the cumulative energy of extraction, production, transport, installation, and disposal) and/or that use alternative energy sources reduces the building sector's carbon footprint. locally sourced materials produce fewer carbon emissions than those transported long distances and both durable materials and those with recycled content help reduce demand for virgin materials. fully recyclable materials can be reprocessed and repurposed with low energy inputs indefinitely. removing toxic compounds from material production processes and building products facilitates the recycling or biodegradation of building components. it also helps prevent air and waterborne industrial pollution and associated human illness. in some cases, it may be possible to choose building materials that actually sequester more carbon than is emitted in their production. the production of portland cement, the most commonly used cement in construction, is a relatively large source of co 2 emissions. magnesium oxide-based cement is as strong as normal portland cement, but can be manufactured at low temperatures using renewable energy sources. magnesium oxide is an abundant mineral and is easily recyclable (smith 2005) . as it cures, it absorbs more co 2 than is released during its production, resulting in a net negative carbon budget. the average lifespan of buildings in the united states is less than 50 years (o'connor 2004) . design for adaptationdesign that easily accommodates changes in configuration and useextends the life of buildings and stems demand for new construction and virgin materials. when buildings are ultimately decommissioned, design for disassembly facilitates the breakdown and reuse of their constituent components. both design for adaptation and design for disassembly act upstream from recycling as part of a multicyclic system that repositions well-weathered buildings and building materials as resources rather than wastes, reducing carbon emissions in the process. building operations such as heating, cooling, and lighting account for 42% of electricity consumption in the united states (mazria and kerschner 2008) . passive design strategies can drastically reduce a building's dependence on carbon-intensive energy production. in cold climates, airtight, well-insulated building envelopes can work in tandem with building orientation, thermal mass, and passive solar radiation to provide thermal comfort and reduce heating bills. in hot climates, external shading, light colored/reflective roofs and walls, and passive ventilation strategies can reduce cooling loads. daylighting strategies such as light shelves and shallow floor plans bring natural light into interior spaces, reducing the demand for electrical lighting while, at the same time, contributing to the well-being and productivity of building occupants. similar to the use of building materials to sequester more carbon than they produce, buildings can generate more energy than they consume. buildings equipped with photovoltaics, wind turbines, and fuel cells are some of the energy producing technologies that make this possible. recent advances in computer-aided design software help designers address building performance issues early in the design process and optimize building design for energy use and production. improvements in digital monitoring and analysis allow occupants to scrutinize building performance and optimize energy use once buildings are operational. in the future, smart buildings and systems will likely respond dynamically to human occupation, weather, daily cycles, and seasonal change, further reducing their energy footprint. compact communities (discussed in the transportation section, above), land-use planning, and high-density zoning have the potential to reduce co 2 emissions, reduce sensible and latent heat fluxes, and improve human health and ecosystem function. centralized infrastructure and zoning laws that segregate industrial, commercial, agricultural, and residential land use can be beneficial to public health within localities. however, these practices also create a cognitive disconnect between human action and its environmental consequences. they facilitate the transfer of local environmental burdens to regional and global scales, contribute to climate change, and undermine the health of vulnerable populations. integrated land use and responsibly managed distributed ecological infrastructure have the potential to safeguard public health without the transference of environmental burdens. synthesizing commercial, industrial, agricultural, and residential precincts, reimagining landscape as infrastructure, and bringing utilities into the public realm raises awareness about the causal relationships that link human activities, environmental degradation, and human health. urban agriculture, for example, reduces dependence on carbonintensive industrial food production, minimizes the carbon footprint of food transport, teaches urbanites how to grow their own food, and provides nutrition to communities. carbon-conscious materials, regenerative buildings, compact communities, and integrated landscapes have the potential to mitigate the worst effects of climate change. their success will depend on the ability to implement them not only in developed countries but also in the burgeoning cites of the global south. we must adapt these strategies to diverse climates, contexts, and cultures and pursue them in tandem with issues of economic development and social equity. in modern history, land distribution and regulation have always been the prerogative of governments. for this reason, national, regional, and local governments have a large role to play in housing mitigation strategies. governments control land-use planning, zoning, and building-code regulations. the ability to act on the mitigation strategies identified above, therefore, is largely dependent upon the regulatory and enforcement environment in a region. in a democratic society, these will in turn be dependent upon the inclination and ability of the state to assume progressive and proactive attitudes toward mitigation. agriculture releases considerable amounts of ghgs into the atmosphere. in 2005, it accounted for 10-12% of the total global anthropogenic ghgs including approximately 60% of nitrous oxide (n 2 o) and 50% of methane (ch 4 ) of the anthropogenic totals (rosenzweig and tubiello 2007; smith et al. 2008; burney et al. 2010) . land-use change, nitrogenous fertilizer applications, livestock production, rice farming, and biomass burning are among the major direct sources of ghg emissions. indirect sources of ghgs from agriculture include the production and/or applications of fertilizers and pesticides, the operation of farm machinery, and the transportation of agricultural products (burney et al. 2010 ). there are multiple opportunities for mitigating climate change impacts through modifications in agricultural practices and food consumption. with respect to agricultural production, mitigation strategies include soil carbon sequestration and the reduction of ghg emissions through altered production methods. with respect to food consumption, society can mitigate the impacts of climate change through changes in diet and by reducing the amount of transportation needed to deliver food (e.g., increasing local food production). below, we discuss several of these mitigation strategies. the topic of increasing local food production is covered in previous sections on transportation and shelter. unlike housing, food is a sector that is potentially much more amenable to individual decision-making and personal choice. this will depend on the relative proportion of food in a household budget. while americans spend only 5% of their incomes on average on food, the figure for indonesia is 30%. this indicates that people from wealthy countries would have greater flexibility to support alternative and nonindustrial agriculture through consumer preferences than those from countries where food is a larger part of household budgets. agricultural and degraded soils can act as large carbon sinks with the potential to sequester 55-78 gt of carbon globally (lal 2004) . soil organic carbon (soc) is the major component (62%) of this large soil carbon pool (lal 2004) . soc accumulation is facilitated by adding biomass to the soil and minimizing soil disturbances. the increased soc improves the physical, chemical, and biological properties of the soil and in turn enhances crop productivity, water holding capacity, and land sustainability under a low-input cropping system. several agricultural practices facilitate accumulation and retention of soc in cropland soils, e.g., conservation tillage, mulches, cover crops, manuring, crop rotations, and agroforestry (lal 2004) . the benefits of these agricultural practices go beyond carbon sequestration. they have the potential to reduce erosion, limit positive feedbacks in drought cycles, and benefit human health. over the past 40 years, 30% of the world arable land has been taken out of production as a result of erosion (pimentel 2006) . conventional agricultural practices involving frequent tillage result in soil erosion, which in turn increases concentrations of airborne particulates. these particles can carry pathogens causing infectious diseases and can provide positive feedbacks to drought conditions. recent climate modeling has demonstrated a positive feedback in the 'dust bowl' drought of the 1930s in the united states (schubert et al. 2004; cook et al. 2009 ). cook et al. (2009) discovered that a reduction in vegetation cover and the addition of eroded soil dusts to the air resulted in a high temperature anomaly over the northern united states and intensified the drought. as a result of the dust bowl, more than 50% of the surveyed farms comprising 8.7 million acres were seriously eroded, and the economic loss was valued close to us$200 million (aaas 1936) . a combination of warming, drought, and conventional agricultural practices together have the potential to re-create the dust bowl of the 1930s in the great plains of the united states (rosenzweig and hillel 1993) . the livestock industry is a main contributor to three major climate-related problems associated with global food systems: ghg emissions, reactive nitrogen mobilization, and plant biomass appropriation (pelletier and tyedmers 2010) . increases in livestock production and meat consumption are also blamed for the evolution of new and virulent emergent diseases. any improvement in the environmental sustainability of livestock production will concurrently have a benefit for human health. pelletier and tyedmers (2010) estimated that globally the livestock sector contributed 14% of anthropogenic ghg emissions, 63% of reactive nitrogen mobilization, and consumed 58% of human-appropriated biomass as of 2000. reduction in ghg emissions in agriculture can be achieved by developing strategies involving improved production efficiency, land-use changes, cropland and rangeland management, and livestock and manure management (rosenzweig and tubiello 2007; smith et al. 2008; burney et al. 2010; pelletier and tyedmers 2010) . for example, enhancing production efficiency through agricultural intensification has been credited with the avoidance of 161 gt of carbon emission globally since 1961 (burney et al. 2010 ). this figure highlights that the investment in agricultural research to improve the environmental sustainability of global food production systems (i.e., livestock, crop, and aquatic systems) should be a prioritized mitigation strategy (lobell et al. 2008; burney et al. 2010; steinfeld and gerber 2010) . another mitigation strategy to reduce emissions involves shifting livestock production regimes from ruminants to more efficient and lower impact monogastric species (e.g., poultry), well-managed fisheries and aquaculture, and promoting plant-based protein sources (pelletier and tyedmers 2010; steinfeld and gerber 2010) . changing livestock production from ruminants to monogastric animals and reducing livestock production overall both require significant changes in diet in many parts of the world. this is, of course, where tradition and culture play an important role, yet it is not impossible to induce dietary changes. initiatives that reduce the consumption of meat could have dramatic effects on ghg emissions particularly in developed countries (mcmichael et al. 2007) . such policies would also reduce impacts on ecosystems and promote other ecosystem services such as a reduction in the clearing of amazonian rain forests, reduced nitrogen deposition, and a reduction in the application of high-phosphate fertilizers used to grow feed. reduced meat consumption could also improve human health in developed countries particularly when coupled with incentive structures and educational measures to replace high-fat, sugar-rich foods with more complex diets based on plant proteins. reduced red meat consumption will likely lower the risk of obesity, diabetes, and several types of cancer (and specifically colorectal cancer), and may reduce the risk of ischemic heart disease (mcmichael et al. 2007) . a shift from meat-based proteins to plant-based proteins has the potential to substantially increase the total food calories available for consumption worldwide. worldwide educational efforts, altered trade policies, shifts in farm subsidies, public health campaigns and new environmental stewardship programs will be needed to achieve a fundamental change in the consumption of meat to achieve large-scale cobenefits for the environment and human health. energy consumption and production affect carbon emissions, which in turn affect human health and ecosystem functioning and services. these effects can be divided into the influence of energy consumption and production. while human well-being is in many ways positively affected by the increased availability of energy, there are also negative health effects resulting from the energy consumption and production. below, we describe potential mitigation strategies for the energy sector. the us energy information administration reports on energy consumption in the united states in four sectors: residential, commercial, transportation, and industrial. in 2009, consumption across the four sectors accounted for 22, 19, 29, and 30% of overall energy consumption, respectively (us energy information administration 2010). overall energy consumption, as well as co 2 emissions from energy consumption, continues to rise. total us co 2 emissions from energy consumption were 1390 million metric tons of carbon in 1990 and 1586 million metric tons in 2008 (research and innovation technology administration 2011). reducing ghg emissions through declining energy consumption can be accomplished through a combination of technological advances and changes in human behavior (including the adoption of new or alternative technologies). several mitigation strategies for addressing energy consumption have been provided in the previous sections on transportation and shelter. in addition to those strategies, increasing the efficiency of appliances and lighting is likely to provide significant reductions in ghg emissions. the most efficient appliances in use today use half to one-fifth of their most inefficient counterparts currently in use (ipcc 2007b) . in developed countries, substituting smaller and more efficient refrigerators, washing machines, and other appliances for less efficient ones can substantially reduce energy use. in addition, in developed countries, improvements in the efficiency of electronic devices with inefficient standby modes and power supplies (or more simply unplugging these devices when not in use) can significantly reduce energy consumption. in developing countries where biomass (e.g., wood, dung, and charcoal) is used for cooking fuel, substantial reductions in ghg emissions and improvements in human health may be attained by increasing the efficiency of biomass stoves, fostering a shift to cleaner burning liquid and gaseous fuels, and improving access to electricity (ipcc 2007b). significant reductions in emissions can also be made through the use of more efficient lighting. the emissions from electric lighting worldwide are equivalent to roughly 70% of those generated by light passenger vehicles (iea 2006b). substituting more efficient lighting technology (e.g., compact fluorescent bulbs and occupancy detectors) can reduce residential energy use by a factor of four or five (ipcc 2007b). programs designed to increase the energy efficiency of appliances, vehicles, and lighting (e.g., the energy star and vehicle fuel economy (cafã�)) also have the potential to reduce ghg emissions. although such programs have not decreased energy use in the united states, they may be partially responsible for halting the growth of per capita energy consumption (us energy information administration 2009). methods for reducing the impacts of energy production on atmospheric co 2 concentrations, human health, and the environment focus primarily on improvements in an increased generation of renewable energy. renewable energy sources include those based on waves and tides, biofuels, solar, wind, and hydropower. a shift to renewable energy sources has great potential to reduce ghg emissions, reduce impacts on ecosystems and ecosystem services, and improve human health. nonetheless, the production of renewable energy will have adverse effects on some ecosystems. below, we discuss some of the benefits and potential side effects of some sources of renewable energy. in regions of large tides and tidal currents, such as the united kingdom, tidal energy could supply as much as 10% of the country's energy supply (blunden and bahaj 2007) . two types of systems are in use or are proposed, the tidal barrage and tidal turbines. tidal barrages allow water to flow into a bay or estuary during flood tide, then they release the water back during ebb tide. as water is released it flows through turbines generating energy. the impact of barrages on fish may be substantial (aprahamian et al. 2010) . power can also be extracted in the marine environment by turbines in regions of high wave and current energy. however, the ecological impact of the extraction of energy in highly energetic environments is not clear (shields et al. 2011) . the effects of such installations include alteration of benthic (ocean bottom) environments through changes in sediment transport and detrimental impacts on intertidal species through altered currents and nutrient flows. biofuels (discussed briefly in section 4.25.2.1 transportation) include corn-and sugarcane-based ethanol, woodbased methanol, biodiesel from soy, rapeseed oil, and switchgrass. biomass is also used in cofiring (combining with coal or other fossil fuels in the burning process) or burned alone as a fuel. crops can be grown explicitly for biofuels, or residues from farming or forestry can be used as biofuels. biofuels can also be converted into gasses and then used to fuel gas engines. nonetheless, many biofuels require significant amounts of land and water to grow them, which brings fuel production into conflict with food production as well as potentially result in a climate effect from the alteration in the surface fluxes of heat and moisture into the atmosphere. in addition, fuel plants may not be as effective as others at absorbing ghgs, and more natural lands converted to agriculture for biofuels will result in increased erosion, decreased water quality, and a loss of wildlife habitat and biodiversity. solar energy can be used to generate electricity, heat water, and heat buildings and can be a highly effective form of renewable energy in some regions. although photovoltaic power generation only accounted for 0.004% of world power production as of 2007, expansion is occurring at roughly 30% per year, largely in developing countries (ipcc 2007b) . hot water production is the second single largest use of energy in residences in both the united states and china. solar water heating is a cost-effective alternative to other fuel sources in many locations. similarly, passive solar heating of buildings (mentioned in the section 4.25.2.2 shelter) is an effective heating measure in many regions. finally, solar energy can be used to generate power with solar thermal electric plants that focus solar rays to heat a liquid, which is then used to generate electricity. passive solar space heating and hot water production likely have the lowest environmental costs. decentralized photovoltaic use on residences and buildings incurs environmental costs in terms of the production process, but likely have few other negative effects. conversely, photovoltaic fields and solar thermal electric plants can have large footprints, adversely affecting plant and animal habitat. like solar power, wind-generated power is more effective in some areas than others. wind produced 0.5% of global electricity in 2004 but has been growing at a rate of approximately 28% per year since 2000 (ipcc 2007b ). there are potential adverse effects on birds, through collision, displacement due to disturbance, barrier effects, and habitat loss, with more research needed in all areas (drewitt and langston 2006; fox et al. 2006 ). in addition, the impact of wind turbines on views is perceived to be a substantial barrier to implementation in some communities (wolsink 2007) . as of 2004, hydropower accounted for 16% of global electricity (ipcc 2007b) . together, large hydropower plants and small and micro hydropower systems can supply power to large municipalities and isolated rural communities with relatively few emissions. hydropower is not carbon neutral, however, and some studies have estimated significant emissions from hydropower reservoirs (ipcc 2007b) . in addition, dams and reservoirs fragment river networks, alter natural flows of water and sediments, and change stream temperatures. these effects are discussed in section 4.25.3.5 human-dominated systems. there are several other major renewable energy sources including nuclear, hydrogen, and geothermal. as with the renewable sources described above, there are both benefits and costs to using these other renewable energies. most of the mitigation strategies discussed above are focused on reducing ghg emissions. additional strategies can be used to increase carbon sequestration, removing co 2 from the atmosphere. below, we discuss a few strategies that have been proposed for increasing carbon sequestration and storage as well as much riskier geoengineering proposals. deforestation is one of the largest sources of emission accounting for over 8 gt co 2 per year in 2000 (stern 2006) . although there are many other uses of natural resources that result in carbon emissions, here we focus on mitigation strategies that focus on deforestation. given the slow rate at which trees absorb carbon dioxide, simply planting new trees in place of those removed does not immediately offset the removal of a tree. a more rapid approach to mitigation is to reduce deforestation. unlike the majority of emissions that emanate from industrialized countries, most emissions from deforestation are generated by developing countries. deforestation also itself alters the climate system (pielke et al. 2007 ). reduced emissions from deforestation and forest degradation (reddã¾) is a program of the united nations that seeks to mitigate climate change by providing incentives to conserve forest cover. given recognized challenges in redd (dickson et al. 2009 )including country by country differences in who owns the forest, who should receive the monetary benefits of preserving the forest, and the reliance of various indigenous groups on forests for livelihood and cultural identitythe program was modified to reddã¾ in 2010 to minimize negative consequences and respect the rights of indigenous peoples and members of local communities (kanninen et al. 2010) . assumed cobenefits include the suite of ecosystem services such as watershed protection; reduction of soil erosion; flood control; provision of fuel, timber, and foods; and cultural and spiritual amenities for local communities. biodiversity is presumably also enhanced by protecting natural systems. it is possible that forest-dependent communities will be able to maintain access to forests through this initiative. potential coharms are increased demand for conversion of other ecosystems if forests are protected, replacement of mixed stands with plantations, and benefits accruing to those not responsible for care and maintenance of the forests. prevention of deforestation mitigates atmospheric co 2 concentrations by preventing the release of stored carbon that occurs when forests are harvested and protecting forest stands that absorb carbon, as well as limits changes in surface energy and water fluxes. as of 2011, south america and africa continue to have the largest net loss of forests and large decreases in carbon storage (fao 2011) . although tropical forests have been the focus of early redd efforts, recent evidence indicates that a large amount of carbon can be stored in mangrove forests (donato et al. 2011) , peatlands (dise 2009) , and boreal forests (carlson et al. 2010) , making these potential targets for conservation efforts. urban forests have also been largely neglected as a potential carbon sink. escobedo et al. (2011) review the services and disservices of urban forests combined with human health and pollution mitigation as well as carbon sequestration. in some locations, urban forests are on par with other techniques for carbon sequestration and reduction. in addition to capturing carbon in plants, it may be possible to increase carbon storage in the water and in the earth's crust with various methods. these proposed approaches are still on the scientific fringe and considered to be highly unproven and/or potentially quite risky. one such approach is to capture carbon in the oceans through iron fertilization. in regions of the ocean where there are ample nutrients to drive primary production (photosynthesis) but observations show that chlorophyll is relatively low, the lack of iron is thought to limit the productivity. over the last two decades, several experiments have attempted to determine whether the addition of elemental iron into the water could cause an increase in algal production and ultimately, as photosynthesis fixes carbon into organic molecules that then sink, the sequestration of carbon in deep ocean sediments. however, the results of these experiments are mixed (de baar et al. 2005) , with the export of carbon being difficult to assess and likely quite modest. at the same time, the impacts on local ecosystems and thus ultimately fisheries in potentially highly productive regions of the subpolar oceans are not yet known. other carbon sequestration approaches have also been proposed including pumping air down into the oceans, injecting carbon into the ground, and incorporating biochar into soils. given the magnitude of projected changes in climate and the difficulty in implementing meaningful mitigation measures, some scientists have argued for large-scale efforts to reduce the amount of incoming solar radiation. these approaches are collectively referred to as geoengineering. at present, these approaches are unproven, risky, and, at best, will only address some of the impacts of the human influence on climate. one such geoengineering approach would inject sulfate aerosols into the stratosphere. these aerosols would reflect incoming solar rays and reduce warming. although such an approach might reduce average global temperatures, it would also likely decrease average global precipitation (bala et al. 2008 ) as well as alter atmospheric and ocean circulation patterns. for a given change in radiative forcing, solar forcing is more effective at changing the hydrological cycle than forcing from carbon dioxide owing to differences in the surface latent heat (and thus evaporation) response. while the impact of carbon dioxide fertilization could increase net primary production in terrestrial ecosystems (govindasamy et al. 2002) , ecosystems could continue to be vulnerable due to changes in water resources. in addition, solar radiation management would not address the potentially significant problem of ocean acidification to marine ecosystems. the majority of both natural and human systems will have to adapt to climate change in some form or the other. a broad array of adaptation strategies has been proposed for reducing the impacts of climate change in both more natural systems and human communities (ipcc 2007a; heller and zavaleta 2009) ( table 2) . many of these strategies are either general concepts or strategies that apply to most ecosystems and regions. we begin by describing general concepts and strategies. we then describe some of the strategies that have been proposed for four broad types of ecosystems including freshwater, terrestrial, coastal, and human-dominated systems. for the freshwater, terrestrial, and coastal systems, we focus on strategies that would lessen negative impacts of changing climate on ecosystem functioning and ecosystem services. for the human-dominated systems, we discuss the ways that some of the most often discussed strategies for climate adaptation in these systems will impact (either negatively or positively) ecosystem functioning and the provision of ecosystem services in these and in surrounding areas. developing adaptation strategies to address future shifts in climate will require expanding the temporal and spatial scales of plans, policies, and management (kareiva et al. 2008; lawler three-year, 5-year, and even 10-year plans will fail to address many climate impacts. future planning will need to address both short-term and long-term changes in systems. to address long-term changes, plans and policies will need to look at least 20 years into the future and will be more useful if they look ahead 50 or more years for many ecosystems and ecosystem services. it will often also be necessary to expand the spatial scale at which systems are managed and policies and plans are developed. as species move and ecosystems change, it will be necessary to think well beyond neighborhood, park, refuge, state, or even national boundaries. planning will need to occur at multiple, integrated scales and will need to involve local, regional, and sometimes national participants (kareiva et al. 2008) . while expanding the temporal and spatial scale of climate adaptation strategies, it will be necessary to keep in view local histories, politics, and economies. scale and locality also play a formative role for issues of 'environmental' or 'climate' justice. environmental justice describes the tendency for poor communities to be more vulnerable to environmental degradation. poor communities tend to bear not only the unequal burden of polluting, destructive, and destabilizing industries but also a disproportionate share of the blame for environmental destruction and responsibility for ameliorating environmental change. for example, polluting industries are often located in poor communities due to cheap land values and the inability of poor communities to politically resist their presence, or a desire to develop jobs at any cost. for instance, swidden agriculturalists in southeast asia have been blamed for forest fires despite satellite data pinning large fires to governmentsponsored oil palm plantations (harwell 2000) and southeast asian fishers living near toxic riparian runoff from mining have been blamed for polluting local waters by cyanide fishing (lowe 2006) . the broad spatial scales that are required to address the effects of a changing climate necessitate cooperation and coordination among managers, planners, and policy makers from different jurisdictions, agencies, and management units (kareiva et al. 2008) . they require agencies with diverse mandates and cultures to work together in ways that they have not done in the past. as an example, the us climate change science program is a federal organization that reaches across federal agencies. the broad scale of climate impacts also requires cooperation across sectors of society and different types of organizations including nongovernmental organizations, local and regional governments, citizen groups, and industry. the us fish and wildlife service's landscape conservation cooperatives are an example of a system designed to bring together diverse groups of scientists, stakeholders, policy makers, and managers to, in part, address climate change. in addition, as emissions from one locale affect the climate in other locales, addressing the global nature of climate change will involve cooperation at an international level (hannah 2010) . although, as the above examples demonstrate, some regional, national, and international efforts have been put in place to foster collaboration, more such efforts will need to be made to adequately address climate change. in addition to cooperation among scientific and political leaders, local and regional stakeholders need to work together as a unified force for social and environmental change. representatives from the global north need to appreciate perspectives emanating from the global south and vice versa. climate adaptation can be brought into many other environmental and social justice agendas among community advocacy groups and should not remain the purview of the community of knowledge leaders with scientific expertise. there are three fundamental approaches to managing systems in a changing climatepromoting resistance, resilience, and transition. resistance-based adaptation strategies focus on maintaining the current state of a system in the face of climate change or climate-driven changes in the environment. building a seawall to prevent erosion as sea levels rise and storm surges intensify is an example of a resistance-based adaptation strategy. resilience can be defined as the ability of a species or system to return to its current state following a perturbation (e.g., holling 1973) . resilient systems will be able to maintain ecosystem functions and processes and avoid a transition to a new state as climates change. most of the discussion of climate adaptation to date has focused on managing ecosystems for resistance or resilience. most of the general adaptation strategies discussed below and the more specific strategies discussed for each of the four types of ecosystems are intended to increase resilience. the magnitude and rate of projected climatic changes over the coming century will likely make both resistance-and resilience-based strategies insufficient or ineffective in the long run. managing many systems will require promoting change to a new state. strategies for promoting change in human-dominated systems may include planting new crops, redesigning road networks, and relocating people. strategies for fostering change in more natural systems include assisted colonization of threatened plants and animals (see below) and shifting management efforts from one species to another. the outcomes of these more aggressive and forward-looking strategies will be more uncertain, and their implementation will be more controversial. change is especially hard in social contexts where it is uncomfortable, disadvantageous, expensive, or where the old system is remunerative or beneficial to elites, decision makers, or other small influential groups. adaptive management is a flexible approach to managing a system in which the management actions are treated as largescale experiments (holling 1978; walters and hilborn 1978) . it is an iterative process in which the system is monitored following the initial management action and new actions (again treated as experiments) are designed and implemented in response to the results of the initial actions. adaptive management was conceived for managing highly uncertain systems, and thus it has great potential for managing systems in a changing climate (arvai et al. 2006) . adaptive management approaches designed to address climate change will likely have four basic steps (kareiva et al. 2008) : (1) assessing the potential impacts of climate change on the system in question, (2) designing management actionsin the form of experiments with testable hypotheses or predictionsto address the potential impacts, (3) monitoring the system for climatic changes and system responses, and (4) using the results of the monitoring to evaluate the effectiveness of the management actions and redesign them as necessary before repeating the four steps. it is generally accepted that more diverse systems are more resilient to environmental change than less diverse systems. thus, restoring or protecting the biological diversity of a system will likely increase resilience of that system to climate change (glick et al. 2009 ). this diversity can be in the form of species diversity, environmental diversity, or genetic diversity (schindler et al. 2010) . a second, general approach to increasing resilience of species and systems involves reducing the impacts of other factors that reduce the overall resilience of the species or system. for individual species, this might mean reducing harvest, restoring or protecting habitat, eliminating diseases, or reducing human interactions. for ecosystems, this may mean reducing pollutants, restoring vegetation, reducing human use, and controlling invasive species. as climates change, many species need to move to track suitable climatic conditions and changing habitats. in some cases, it may be possible to facilitate movement by increasing connectivity of freshwater and terrestrial landscapes (see sections 4.25.3.2 freshwater systems and 4.25.3.3 terrestrial systems). however, many species with limited dispersal abilities or that face significant barriers to dispersal will be unable to respond to changing climates. assisted colonization is one strategy that has been proposed for facilitating climate-driven range shifts (hunter 2007; mclachlan et al. 2007 ); also referred to as managed relocation and assisted migrationassisted colonization is the translocation of species outside their native range to facilitate movement in response to climate change. the concept is controversial for both ethical and ecological reasons. some researchers have highlighted the potential for negative, ecological, evolutionary, and economic impacts, as well as ethical concerns (ricciardi and simberloff 2009; sax et al. 2009 ). advocates argue that assisted colonization will be essential for cases in which species will have to move to find suitable environments but will be unable to do so, that many of the potential risks are overblown, and that guidance can be developed for the strategic and informed use of assisted colonization. several decision-making frameworks and prioritization strategies for applying assisted colonization have been proposed (hoegh-guldberg et al. 2008; richardson et al. 2009 ). although assisted colonization will likely be a useful strategy in many systems, there will be specific factors and risks that will need to be considered for particular systems (e.g., in freshwater systems, olden et al. 2011 ). virtually all freshwater systems are already impacted by human activities (e.g., vã¶rã¶smarty et al. 2010) . under current climate projections, virtually all freshwater ecosystems will face significant impacts by the middle of this century. most of these impacts will be detrimental from the perspective of existing freshwater ecosystems and the human livelihoods and communities that depend upon them (poff et al. 2002) . discussions have broadly focused on rising water and air temperatures, increasing and decreasing precipitation, increasing evapotranspiration, and changing regimes of groundwater and surface runoff. there are four basic types of adaptation strategies that have been proposed for addressing these impending impacts. we define these as ecological water management, connectivity, protected areas, and restoration strategies. below, we briefly describe some of the specific strategies that have been proposed in each of these areas. increased water scarcity associated with decreased precipitation and increased warming in some regions is likely to prompt the modification of dams and reservoirs to improve storage capacity (see section 4.25.3.5.3 managed aquatic systems). traditional water management has generally sought to dampen the natural variability of river flows to attain steady and dependable water supplies for domestic and industrial uses, irrigation, navigation, and hydropower, and to moderate extreme water conditions such as floods and droughts (postel and richter 2003) . however, the extraordinary species richness and productivity characteristic of freshwater ecosystems is strongly dependent upon, and attributable to, the natural variability of their hydrologic conditions. thus, large changes to natural flow regimes can have large impacts on species, ecosystems, and society (postel and carpenter 1997; iucn 2000; wcd 2000) . the water needs of humans and natural ecosystems are commonly viewed as competing. however, water managers and political leaders increasingly recognize that there are limits to the amount of water that can be withdrawn from freshwater systems before their natural functioning and productivity, native species, and the array of goods and services they provide are severely degraded ). this has led to a growing realization that society derives substantial benefits both from out-of-stream extractions of water as well as by maintaining adequate flows of water within freshwater ecosystems. in recent years, the water needs of river ecosystems are receiving increasing attention in water-supply planning, offering hope that many rivers can be protected before their health and ability to provide important goods and services is seriously compromised by water development. restoring or protecting some semblance of the natural river flow conditions necessary to support ecosystem function (called environmental flows) into the future is one of the most important climate change adaptation strategies for flowing waters. recent regulatory mandates and policy decisions calling for restoring and preserving environmental flows have led to a reexamination of dam operations for better protection of river health (watts et al. 2011) . for example, the south african national water act (1998) calls for the creation of a reserve of water in each river basin to meet both basic human needs and protect river ecosystem health. similarly, other national directives and international agreements such as the water framework directive of the european union are providing mechanisms for river protection, including the provision of adequate environmental flows (postel and richter 2003) . maintenance of environmental flows (flows that sustain river ecosystems) is likely to be the highest priority adaptation response for freshwater ecosystems in most contexts. this will require policies and implementation mechanisms to protect (and, if necessary, restore) flows now, and to continue to provide environmental flow regimes under changing patterns of runoff associated with climate change. as air and water temperatures warm, many freshwater species will be forced to rapidly adapt to their new environments, to migrate to more suitable habitats, or face extinction (heino et al. 2009 ). responses of aquatic organisms to climate change will be constrained because they are limited to dispersal along pathways of connected water. consequently, the linear nature of river systems makes them highly prone to fragmentation from waterfalls, dams, and water withdrawals. such fragmentation can not only limit dispersal and the ability of species to track climate change but also disrupt the completion of life cycles for many freshwater organisms. for many streams, especially in arid and semiarid ecosystems, there is concern that more frequent and severe droughts will lead to the building of more dams and water diversions, further fragmenting stream networks (seager et al. 2007) . increasing the connectivity of freshwater systems is one key way to help aquatic systems adapt to climate change. species responses to climate change will be limited by human-engineered structures, including hundreds of thousands of dams, diversions, and impassable road culverts that exist globally (nilsson and schopfhauser 1995) . in the united states alone, there are over 80 000 large dams and an estimated 2.5 million or more small impoundments that disrupt the dispersal of aquatic organisms. in recent decades, however, new water laws and programs have helped to increase habitat connectivity by removing or modifying human barriers to movement in river systems. increasingly, small dams and diversions are being removed for a number of ecological, social, and economic reasons, including the enhancement of longitudinal connectivity for fish migration (stanley and doyle 2003) . similarly, temporary barriers such as impassable culverts under roadways are being modified to allow for easier recolonization and movement of fishes (roni et al. 2008) . although this management strategy may promote the adaptive capacity of species to respond to climate change, the removal of such obstructions can simultaneously increase the risk of invasion by nonnative species and associated diseases, which are major threats to ecosystem structure and function. in fact, the conversion of culverts into permanent barriers has been a common management approach to protect isolated native populations from invasive species spreading upstream (fausch et al. 2009 ). given the complexities of promoting the movement of native species while concurrently limiting the spread of invasive species, it is unlikely that the removal of barriers to enhance habitat connectivity will be a successful adaptation strategy in all areas for reducing the impacts of climate change on ecosystem function. in addition to restoring or protecting environmental flows and increasing connectivity within stream networks, it may be necessary to protect the species themselves to allow them to move through or persist in more intensely managed landscapes. in aquatic ecosystems, this may involve increasing the size of existing reserves or creating new protected areas that encompass important freshwater resources and allow for native species and assemblages to persist into the future (abell et al. 2007) . such acquisitions and agreements ideally would capture a large portion of the geographical, ecological, and geophysical range of multiple species, thus increasing opportunities for organisms to adapt and evolve to changing environmental conditions. protected areas have historically been a cornerstone of terrestrial conservationand are discussed in much more detail in that section, below. nonetheless, protected areas will likely play a major role in protecting aquatic species as climate change and areas could easily be prioritized to provide protection for both terrestrial and aquatic systems in a changing climate (abell et al. 2007 ). projected increases in air temperatures, combined with reduced snowpack, earlier onset of spring peak flows, and lower summer baseflows, will have direct implications for the thermal regimes of streams and rivers (poff et al. 2002) . the ecological implications of stream warming are significant. water temperature directly influences the metabolic rates, physiology, and life histories of aquatic species and helps determine rates of important ecological processes such as nutrient cycling and productivity. in general, warming will produce a general shift in species distribution poleward with extinctions and extirpations of cold-water species at lower latitudes and range expansion of warm-water (often invasive species) and cool-water species into higher latitudes. restoring and protecting riparian vegetationthe trees and shrubs that grow along the banks of rivers and streamshas the potential to reduce stream temperatures in certain places. recent modeling suggests that riparian habitat restoration can play an important role in offsetting the effects of climate change. battin et al. (2007) used a series of linked models of climate, land cover, hydrology, and salmon population dynamics to investigate the impacts of altered stream flow and temperature on the effectiveness of proposed habitat restoration efforts designed to recover chinook salmon populations in the pacific northwest of the united states. this study showed that in the absence of habitat restoration, basin-wide spawning populations would decline by an average of 20-40% by 2050. by contrast, they found large increases in juvenile rearing capacity associated with proposed riparian habitat restoration, which limited climate-induced declines to 5% according to one climate model and increased salmon abundance by 19% according to another model. however, the model results showed that not all expected climate impacts could be mitigated entirely. in relatively narrow streams, reforestation was predicted to decrease water temperatures by increasing shading, but in wide, main-stem reaches where most chinook salmon spawn, riparian vegetation had a minor effect on water temperature. in addition to restoring riparian areas, targeted protection of forested riparian buffers using fencing to stop livestock from entering the stream and trampling/grazing vegetation or conservation easements to eliminate farming activities may also help to reduce stream temperatures. the many adaptation strategies that have been proposed for addressing climate change in terrestrial systems can be loosely grouped into three categoriesthose that involve protected lands and conservation planning, those that address connectivity, and those that are more species or site specific in nature. protecting land is arguably one of the most effective ways of conserving biodiversity and terrestrial ecosystem services. it is generally assumed that lands that are selected to protect today's species and ecological systems will protect those species and systems into the future. climate change brings this assumption into question. as climates change and species distributions shift, species will move into and out of reserves. changes in species composition, the timing of ecological events, and the rates of ecosystem processes will result in changes in the functioning of ecosystems and the services they provide. many proposed adaptation strategies address these challenges by augmenting current protected areas with additional reserves, parks, and preserves (heller and zavaleta 2009; lawler 2009 ). these strategies range from broad calls for more protected lands to more sophisticated recommendations about how to select new lands or design new reserves to better protect biodiversity and ecosystem services as climates change. a number of strategies have been proposed for determining where to put new protected lands to best address the effects of climate change. some of the simplest strategies involve placing reserves at the poleward or elevational boundaries of the range limits of specific species (peters and darling 1985) , at major transitions between vegetation formations (halpin 1997) , or at the core of species environmental distributions (araãºjo et al. 2004) . others have proposed placing reserves between existing reservesin the hopes that those new reserves will act as stepping stones allowing species to better move from reserve to reserve. yet another recommendation involves placing reserves across strong environmental gradients. reserves that span many different environments will potentially allow species to shift their distributions without leaving the reserve. in addition to these simple rules of thumb for placing reserves to address climate change, many more sophisticated approaches to selecting areas for new reserves have been proposed. for example, modeled climate-driven shifts in species distributions can be used to select new reserves that will protect biodiversity today and into the future (hannah et al. 2007; vos et al. 2008; hole et al. 2009 ). although this approach may prove useful for some species and systems, care must be taken in the use of the models developed to project species range shifts. there are many uncertainties associated with the models themselves and the climate projections on which they rely. another proposed approach to building a reserve network that will be resilient to climate change involves protecting environments in lieu of protecting species and systems. this approach has been referred to as 'protecting the ecological stage.' this analogy implies that the species and the ecological systems are players and that the soils and topography of a region form the stage on which they interact and evolve. by protecting a suite of diverse environments, it is assumed that one will protect the areas where new ecological systems can develop as climates change (anderson and ferree 2010; beier and brost 2010) . similarly, others have suggested that more resilient reserve networks will be those that include a diversity of current climates (pyke and fischer 2005) . although climates will change, many of the climate gradients generated by latitudinal and topographic patterns will remain. thus, a set of reserves that protects a diversity of climates today will likely protect a diversity of climates in the future. at least one recent study has applied the concept of protecting environments (topographies, soils, and climate gradients) to a reserve-selection problem (schloss et al. 2011) . one last approach to building a resilient reserve network in a changing climate involves placing reserves in areas that are likely to serve as climate refugia. refugia are areas that are projected to experience minimal climatic changes and/or have cooler microclimates (saxon et al. 2005; hansen et al. 2010; shoo et al. 2011) . one potential opportunity for climate change adaptation is to consider people as an essential component of species conservation and climate adaptation. while reserve and park systems have a long history of alienating local people, using violence and other coercive measures to separate people and wildlife, in this case, local livelihoods are also threatened by climate change. this provides an opening for a new approach to conservation and a new discourse on the relationship between local people and wildlife. as for freshwater systems, improving the connectivity of the terrestrial landscape will be critical for addressing climate change. in response to historical climatic changes, many species shifted their rangesmoving toward the equator as ice sheets advanced and moving poleward as the ice sheets retreated. today's species will have a much harder time responding to current and impending climatic changes. present-day landscapesunlike the landscapes of the distant pastare fragmented by roads, agricultural fields, and residential development. given that many species will have trouble moving across these human-dominated landscapes, it will be essential to reconnect fragmented landscapes to allow species to respond to shifting climates. not surprisingly, improving landscape connectivity was the most often recommended climate change adaptation strategy in a recent review of the literature (heller and zavaleta 2009 ). many of the recommendations for increasing connectivity involve designing corridors that connect current protected lands. traditionally, such corridors have been designed for particular focal species, and they have connected reserves or remaining fragments of habitat. however, corridors that facilitate species ranges shifts and the reshuffling of ecosystems will need to connect what is habitat for a species today with an area that may become habitat in the future. in this way, corridor design will need to evolve to address climate change. one such approach to developing corridors for addressing climate change involves basing the corridors on environmental gradients instead of on the current vegetation (which is often used as a measure of habitat) (beier and brost 2010) . another approach uses projected changes in climate to orient corridors (ackerly et al. 2010) . a third method involves using modeled shifts in species distributions to identify potential routes that species would need to take to move from currently suitable climates to places where climates will likely be suitable in the future (williams et al. 2005; rose and burton 2009) . these three approaches are based on different assumptions and have different levels of associated uncertainty. in general, the uncertainty increases with the intensity of modeling required to identify the corridor, and thus the first of the three approaches has the least inherent uncertainty and the last of the three has the most. although corridors may be useful for some species, they will not be feasible or effective in all places or for all species. in addition to designing corridors to facilitate species movements, it will likely be necessary to improve the permeability of the landscape in general (franklin and lindenmayer 2009 ). increasing landscape permeability will involve changing the way we use and manage the landscape. changes in forest harvest practices from clear-cuts to selective, retention harvests can increase the connectivity of forested landscapes. landscape planning and zoning for residential development can reduce landscape fragmentation. windbreaks, fencerows, and crop choices can be used to increase the connectivity of agricultural lands. at a finer scale, choices of landscaping plants, road placement, and wildlife over-and underpasses can increase landscape permeability. protected lands and connectivity increase species ability to adapt to changing climates at relatively large spatial scales. in general, they address species movements. adaptation strategies are also needed at finer scales. in particular, strategies are needed that address individual species and ecosystems. many of these finer scale approaches tend to involve reducing temperatures or otherwise offsetting the exposure to the changes in climate or climate-driven changes in environmental conditions. for example, it may be possible to reduce temperatures by restoring vegetation to provide shade (wilby et al. 1998; wilby and perry 2006) . it may be possible to reduce the impacts of changing fire regimes through forest management practices. fire suppression policies, may, for example, have particularly severe ramifications in a climate in which fires will generally be larger and more frequent. it may also be feasible to provide supplemental water for particularly vulnerable plants or animals in times of drought or during heat waves. similarly, shade shelters could be provided to help some animals weather heat waves in extreme climates. although some of these strategies may prove useful in the short term, many will be neither cost-effective nor long-term solutions. another system-specific approach to addressing climate change involves altering species composition or community structure in a way that allows a system to better cope with climatic changes. for a forested system, this may mean planting new species or planting seeds of the same species that have a different genetic makeup (millar et al. 2007; glick et al. 2009 ). likewise, for grasslands and shrublands, this may mean choosing different species or strains for restoration purposes. moreover, it may mean facilitating the transformation of an ecosystemfor example from a shrubland to a woodland or from a shrubland to a grassland. in many cases, the first species to move into these transitioning systems may be invasive species. facilitating the transition to a new state using species native to the region may, in many cases be preferable to having new systems dominated by invasive species. these extreme cases will require ex situ conservation strategies, including seed banking and captive breeding to ensure the long-term survival of the species. the interface between the land, sea, and air found in coastal areas is a high-energy system subject to storms, winds, and tides. it is also where the majority of protein comes from in many regions of the world. many vital ecosystem services are provided along coastal margins of continents and islands. fringing coral reefs and mangrove forests buffer wave energy, which reduces erosion along the coast. these systems also provide critical habitat for many species, especially nursery areas for provisional services such as fisheries. in many areas, sea-level rise and associated storm surges will make it difficult to protect these systems and the services they provide. nonetheless, there are two main approaches that will likely prove most useful in coastal systems: building resilience through protection and restoration and facilitating the transition to new ecosystems. one of the more beneficial ways, from both an environmental and economic standpoint, to adapt to predicted changes from climate change is by protecting and restoring natural systems that provide multiple ecosystems services. this would include protection of human infrastructure from sea-level rise and increased inundation. protection and restoration of natural buffers such as barrier islands, mangroves, and coral reefs not only require little financial construction and maintenance but also maintain the wealth of ecosystem services associated with these systems, which include nursery and rearing habitat for many commercial seafood stocks such as fish and shrimp, recreational and cultural opportunities for humans, and protection from inundation. some natural protection features such as sea ice cannot be maintained in the face of increasing temperatures. in alaska, the loss of sea ice and melting of permafrost due to warming ocean and air temperatures has exposed the shore to wave energy that used to be absorbed or damped by sea ice or frozen soils. undoubtedly, ecosystem services that depended on that sea ice protection have also been lost. many regions have seen a huge decrease in the area covered by mangroves as coastal areas are drained and mangroves removed for aquaculture. worldwide, it is estimated that more than 50% of mangrove forests have been destroyed or converted to other uses. on a local scale, many areas have lost all of their mangroves. this loss destroys a wide range of ecosystem services provided by these forests, which range from carbon sequestration to flood protection, spawning, and rearing grounds for many fish and other marine species. restoring mangrove forests results in increased storm surge protection for natural and human coastal systems, provides nursery habitat for many commercially important fisheries, and stores sediments that may damage offshore coral reefs. worldwide, corals exhibit well-documented environmental adaptability over time scales of weeks to thousands of years. corals are actually two creatures that live together in a symbiotic relationship. zooxanthellae are the algal component of corals that provide nutrients generated from photosynthesis to the animal component. different zooxanthellae species and different species of corals in general have differential environmental tolerances. for example, some corals near the arabian peninsula exist under conditions of salinity and temperature that are greater than near-term predicted future average salinities and temperatures resulting from climate change. these adaptive changes likely operate on a decadal time scale. thus, in some cases, it may be possible to identify particular species or zooxanthellae and algal combinations that are more resilient to warming. protecting coral reefs from other stressors such as excess sediment and/or nutrient and pollutant inputs from terrestrial sources and unregulated diving can increase the coral's ability to adapt to increased water temperatures. technological adaptation measures include using sprinklers to create roughened sea surface to decrease light penetration or covering reefs with shade cloth to limit bleaching of corals. as sea levels rise, many coastal systems will need to migrate inland. when there are extensive human uses of coastal space or urban buildup, space for ecosystem recovery and migration of species will not be available. providing space for the migration of biotic elements of the coastal system at the same time as when human populations are forced to move would provide cobenefits for coastal life and human communities. ecosystems will usually adjust to changing salinities and water levels, and the services provided by the system will be maintained where conditions are suitable. for example, in 1996, a subdivision east of houston in baytown, texas, was bought out, the structures removed, and the entire area restored to coastal wetlands. a more extreme example of planned retreat is the maldive islands. their current plan is to exploit the current coastal amenities, primarily beaches and coral reefs; secure capital with which to train younger citizens; and prepare them for relocation to other countries such as india, sri lanka, and australia. in the short term, there are negative consequences to ecosystem services due to commercial and touristic exploitation, but in the long run, by abandoning the islands, the system will be able to come to a new ecological state. in some cases, removing human infrastructure will be a necessary but insufficient means of fostering landward shifts in coastal systems. in such cases, it may be necessary to facilitate the shift in natural systems and stabilize soils by planting more coastal species inland as water levels rise. such actions have been taken as part of the alligator river climate change adaptation project on the north carolina coast in the southeastern united states poulter 2005) . here we describe some of the adaptation strategies that have been proposed for built environments, agricultural systems, and managed freshwater systems. we explicitly focus on how the most commonly proposed strategies could affect associated ecosystems. the severity of climate change impacts will depend, in large part, on our ability to transform the built environment in response to changing conditions. the following section discusses some of the climate change adaptation strategies that will likely take place in built environments and the effects that these strategies may have on ecosystem functions and services. we briefly discuss human migrations, flood control, sea-level rise, increased water storage, and urban heat-island effects. perhaps the most basic form of climate change adaptation involving the built environment is human migration. vulnerable populations will be forced to abandon their homes, relocate, and rebuild. they will displace resident populations and open up the possibility of conflict in the process. the bulk of climate change-related human migration that is already occurring involves movement from rural areas to cities in developing countries. unplanned urban growth will fuel the expansion of informal urban settlements, and by 2050, 3 ã� 10 9 people, one-third of the world's population, may inhabit urban slums. in both rural and urban environments, proactive planning and resettlement strategies that identify populations at risk and relocate them stand to drastically reduce long-term human hardship stemming from climate change. they can also protect vital ecosystems from the degradation associated with unplanned development. however, resettlement can have profound negative impacts. poorly executed, it can degrade indigenous culture, undermine traditional livelihoods, destroy social networks, and deepen poverty by distancing poor communities from employment opportunities. given these dangers, in some cases, it is preferable to fortify communities against climate change impacts in place. this approach introduces improvements to infrastructure and housing, builds upon the prior efforts of communities, and preserves social networks. in many regions, climate change will result in increased rainfall and flooding. conventional approaches to storm water management rely on defensive hard structures. water runs from impermeable concrete and asphalt surfaces into storm drains, culverts, and concrete embankments. rivers are straightened, channelized, and isolated from their natural floodplains to accommodate human settlement. as flood events become more intense and frequent with climate change, storm water management strategies must evolve. rather than take a defensive stance, they should promote the coexistence of humans, habitat, and water. soft infrastructure or low-impact development strategies, such as permeable paving, green roofs, green walls, rain gardens, bioswales, and constructed wetlands offer a promising alternative to conventional storm water infrastructure. they promote groundwater recharge, reduce flooding, and improve water quality with downstream benefits to aquatic habitats. in areas of severe flood risk, resettling residents on higher ground and reintroducing rivers to their floodplains protects human populations, dissipates flood energy, and creates wetland habitat. through temporal landuse practices such as intermittent farming, floods become an asset rather than a threat, depositing sediments that enrich agricultural lands. by 2050, rising sea levels may displace as many as 162 million people (myers 2002) . like conventional storm water infrastructure, conventional seawall infrastructure relies on defensive hard structures. as sea levels rise, existing levees will become obsolete. here again, soft-infrastructure strategies offer a promising alternative. graduated transitions from sea to land including manmade offshore islands, expansive shallows, and coastal marshes act as buffers for storm surge better equipped to absorb the impacts of extreme weather events. they provide habitat for marine species and, in coastal cities, public space for recreation when conditions permit. in places like coastal peru and sub-saharan africa, disappearing glaciers and reduced precipitation will increase the frequency, duration, and impacts of drought, undermining food and water security. a number of interventions in the built environment can help communities cope with water scarcity. increasing water storage at scales ranging from dams to urban reservoirs and household rainwater barrels helps bolster water supplies during prolonged dry periods. in coastal areas, desalination plants and, in select localities of south america and africa, fog harvesting may prove to be viable alternative water sources. ecological sanitation technologies such as dry toilets and gray water wetlands conserve potable water for drinking and, as part of a network of integrated ecological infrastructure systems, irrigate and provide nutrients for urban farms and forests. they also help prevent contamination of local water bodies, preserving their viability as sources of potable water and ecological integrity. further impacts of, and opportunities associated with, these various adaptation strategies are discussed in the sections 4. 25.3.5.2 and 4.25.3.5.3. cities in developed countries are on average 1-3 c hotter than the surrounding countryside (un human settlements programme 2011). higher urban temperatures increase energy use, degrade urban air quality, raise the temperature of urban storm water runoff, and contribute to general discomfort, respiratory illnesses, heat stroke, and other heat-related illnesses. warming in conjunction with increased urbanization will amplify urban heat-island effects and their associated impacts. several strategies can help reduce urban temperatures. at the scale of the site, urban trees with high, dense canopies, strategically planted around buildings, in parking lots, and along streets permit the passage of air, shade heat-absorbing surfaces, and cool the atmosphere through evapotranspiration. they also reduce the temperature of runoff water. green roofs perform a similar function on roof surfaces. buildings and landscapes constructed from materials with high solar reflectance or albedo absorb less heat and reduce urban temperatures. at a larger scale, streets oriented parallel (or up to 30 ) to prevailing winds, permeable networks of public open space, strategically situated low-rise buildings, and gaps between high-rise structures increase the air permeability of urban environments. increased temperatures and changes in precipitation regimes are likely to have negative impacts on global food security by worsening the existing regional disparities between developing and developed countries (easterling et al. 2007; rosenzweig and tubiello 2007) . this is supported by the prediction that a moderate warming accompanied with elevated co 2 concentrations is likely to lead to an increase in crop productivity of developed countries in temperate mid and high latitudes, whereas even a slight increase in temperature could damage crops that are already growing at supraoptimal temperatures in countries located in tropical, low latitudes (easterling et al. 2007; easterling 2010) . as in other systems, even with significant mitigation efforts, adaptation strategies will be necessary to address the impacts of climate change on agriculture. adaptation strategies for more resilient agricultural systems may include altering the timing and location of cropping, switching crop species and cultivars, selecting livestock breeds, crop rotations, and efficiently using and managing water resources. these types of adaptation strategies have been referred to as autonomous strategies (easterling 2010) . implementing some of these strategies, such as changing the type of livestock that is grazed or changing crop rotations could have profound effects on ecosystem functioning. however, in general, these types of adaptation strategies will have environmental impacts smaller than more intensive strategies that are designed to transform systems. for many agricultural systems, the magnitude of climatic changes will necessitate more intensive planned adaptation strategies that foster significant transformations of systems or infrastructures (easterling 2010) . such intensive strategies will be particularly important for sub-saharan africa and south asia where climate will have major impacts on agriculture (lobell et al. 2008; nelson et al. 2009 ). these planned adaptation options include developing new infrastructure (e.g., irrigation infrastructure, transport and storage infrastructure, and efficient water use and reuse technologies), investing in new technical and management options (e.g., crop and livestock improvement, enhancing germplasm), shifts in the distribution of agriculture (e.g., abandoning some areas and farming new lands), and building the capacity to make continuing adjustments. here, we discuss two of these strategiesimproving crop performance and water conservationin more detail. stresses on crops due to extreme heat and pest and pathogen pressures are likely to increase with climate change (deutsch et al. 2008; battisti and naylor 2009) . multiple adaptation options are available for improving crop performance in the face of such climate-driven stresses. first, in conjunction with conventional breeding, crop improvement through genetic engineering is seen as an effective adaptation tool in a changing climatealthough public opinion is often divided on the use and consequences of genetically engineered crops (macdiarmid 2007; jones 2011) . a second approach involves maintaining crop genetic diversity by, for example, collecting and storing the diversity of current cultivars or local landraces. a third adaptation strategy involves exploiting symbiotic relationships between crops and other organisms. for example, it has been shown that numerous fungal and bacterial endophytes confer stress tolerance to heat, drought, salinity, and pathogen attacks (rodriguez et al. 2008; rodriguez et al. 2009 ), and others promote growth and fix atmospheric nitrogen xin et al. 2009 ). other strategies include the application of kaolin particle films to reduce heat stress and photoinhibition and to mitigate pest damage (glenn et al. 1999; glenn 2009 ) as well as the incorporation of biochar into soils to confer numerous agronomic benefits that enhance crop performance and stress tolerance (marris 2006) . irrigated agriculture accounts for about 70% of the world's freshwater withdrawals (rosegrant et al. 2009 ). with increasing global population, the demand for irrigated agriculture will continue to increase globally because realized crop yields normally increase considerably with irrigation. adaptation strategies that foster more efficient use of water in agriculture will be critical for achieving global food security while conserving water in a changing climate. these strategies will include institutional water management reform, economic incentives for efficient water use, and infrastructure investment for efficient irrigation and water delivery systems (rosegrant et al. 2009 ). the 'virtual water' trade through crop products from water-abundant to water-deficit areas should be considered as one of the key adaptation options to climate change (rosegrant et al. 2009; easterling 2010) . improving crops for higher water-use efficiency (i.e., greater yield with less water use) should be a goal for genetic, ecological, and agronomic options of adaptation discussed above. alternative strategies of increasing water storage capacity with dams and increasing the number of stream diversions for agriculture will, in contrast, have significant impacts on aquatic ecosystems (see the section 4.25.3.5.3 managed aquatic systems). as discussed above, changes in temperature and precipitation are likely to lead to changes in the demand for water. projected climatic changes will likely lead to increased demands for residential use, irrigated agriculture, hydropower production, and carbon capture and storage technologies. in some regions, current water management may not adequately cope with the impacts of changing precipitation regimes and increased evaporation on the reliability of water supply, flood risk, health, agriculture, energy generation, and freshwater ecosystems (palmer 2008) . adaptation strategies designed to address water management for human uses will either directly or indirectly affect ecosystem functioning and ecosystem services. in addition to the adaptation strategies for addressing water demands for agriculture and residential use (addressed to some extent in sections 4.25.3.5.1 and 4.25.3.5.2), other important adaptation strategies for managed aquatic systems that will impact ecosystem functions and services include increased extraction of groundwater, improving storage capacity by building or modifying dams and reservoirs, desalination of seawater, and expansion of rainwater storage and water transfer (bates et al. 2008; hirji and davis 2009) . improving storage capacity by building or modifying dams and reservoirs is likely to be one of the most common regional responses to decreased water availability and increased demand for water. the great utility of running water has resulted in the extensive exploitation of streams and rivers by humans throughout the world, a process greatly facilitated by the construction of hundreds of thousands of dams globally (nilsson et al. 2005) . by capturing high river flows and releasing the water in a carefully controlled manner, dam managers can deliver steady and dependable water supplies to downstream areas, protect settlements from floods, or generate power. although human manipulation of the world's river flows has provided many societal benefits, it has also caused considerable ecological damage and the loss of important ecosystem services valued by society (baron et al. 2002) . flow regulation for hydropower and storage, diversion for human use, and extraction of groundwater connected to stream systems often significantly affect the natural seasonal and interannual variation in river flow, commonly referred to as the natural flow regime (poff et al. 1997) . a natural flow regime is needed to sustain the complex riverine and riparian ecological functions and processes that support native biodiversity and provide myriad ecological services including maintaining water quality, recharging groundwater, and nutrient cycling (naiman et al. 2002) . facing an ominous future of increasingly severe watersupply shortages in many areas of the world, social planners and government leaders are exploring strategies for ensuring that enough water of sufficient quality is available for use by future generations. human demands for water are expected to increase under a warmer climate, exacerbating current management problems. increasing demands for irrigation and industrial cooling water could conflict with the increasing demands for municipal water supplies resulting from urban growth. climate-induced changes to water availability and increasing human demand for water are likely to prompt the construction or modification of dams and reservoirs to improve storage capacity and enhance water security (wcd 2000; palmer 2008 ). for instance, by storing water in reservoirs, water managers capture high flows during wet years or seasons to supplement water supplies at drier times, thereby maximizing the reliability of water supplies and certain economic benefits each year. for regions where increases in runoff variability or more prolonged drought conditions are associated with climate change, the pressure to build watersupply reservoirs will be great. although the construction of additional dams and reservoirs will undoubtedly provide many societal benefits, a rich body of scientific knowledge has shown that river regulation also causes considerable ecological damage and the loss of important ecosystem services valued by society (baron et al. 2002; postel and richter 2003) . river ecosystem health deteriorates when natural flows of water, sediments, and organic materials through a river system are substantially disrupted or modified by human activities (poff et al. 1997; richter et al. 2003) . river damming and associated environmental alteration is now widely recognized as a leading cause of declines in freshwater biodiversity globally (bunn and arthington 2002) . dams have also been implicated in the loss of commercial fisheries in many estuaries and coastal areas, and in the degradation of other natural ecosystem products and services worldwide (wcd 2000) . in addition, reservoirs increase the loss of water due to evaporation. finally, as mentioned above, reservoirs emit substantial quantities of carbon dioxide and methane into the atmosphere (st. louis et al. 2000) , and therefore, the creation of additional reservoirs is likely to further promote climate change. in many regions, impending water shortages will also lead to greater interest in desalination as a technique for tapping into the vast and infinitely tempting water supplies of the sea. recent improvements in reverse osmosis technology, coupled with the rising cost and increasing unreliability of traditional water supplies both currently and projected in the future, are making desalination a water-supply option with major plants in operation, in planning, or under consideration in europe, north africa, north america, australia, china, and india. like most large-scale industrial processes, making water has a number of actual or potential impacts on aquatic goods and services (wwf 2007) . emerging issues include significant cost and pollution emitted by desalination plants and the mostly unknown, but potentially substantially, effects on freshwater and coastal ecosystems. intake pipes can remove larvae and small marine organisms, and discharge from desalination plants can elevate levels of salt and other constituents of seawater such as boron; dead sea life, which consumes oxygen while decomposing; chemicals added to change the composition of the water for processing, as well as reducing contamination and clogging of filters and membranes; corrosion byproducts; and the heat added for or during processing. finally, water manufacturing is an energy-intensive process. although increasing ghg concentrations and the resulting increasing temperatures are global phenomena, the globe will often be an inappropriate scale for developing mitigation and adaptation strategies. human activities generate differential risks and vulnerabilities that vary with location due to differences in social, political, cultural, and economic conditions (beck 1992) . although, in one sense, all people are vulnerable to catastrophic climate change, at another more fundamental level, the risk of climate change impacts (along with blame and responsibility) are unevenly distributed. we can begin to think of the unequal distribution of the vulnerability to climate change as a scale issue. mitigation and adaptation strategies can be applied at multiple, and often overlapping, scales. at the finest scale, the development of mitigation strategies will depend on local leadership and the attitudes of local populations and individuals toward climate change. at national and international levels, mitigation efforts will be organized through large and formal programs and institutions (e.g., un-redd). although some adaptation strategies will be national and international in nature, much adaptation will take place at finer scales of communities and regions. a hallmark of climate change as a global phenomenon is that leadership and coordination across local, national, and international scales is important for ameliorating its effects. in turn, coordination across scales will entail understanding local climate risks and local and regional understandings of the causes of climate change and the costs and benefits of different mitigation and adaptation strategies. the effectiveness of any mitigation or adaptation strategy (building codes, population planning, dietary changes, etc.) depends on its acceptance in local, regional, and national communities. social impediments to adoption of climate change strategies include ideological outlooks, structural constraints, and cultural barriers. for example, free-market ideologies can impede the ability of governments to invest in alternative energies; agricultural subsidies and corporate monopolies on agricultural inputs may create structural barriers to alternative agriculture; and cultural norms for relative water and energy use, family size, or the flavor and texture of food can inhibit conservation of resources and changes in diet. there is a clear disparity between societies that have contributed the most to ghg emissions and those that will likely suffer the direst consequences of climate change. at the international level, efforts to shift blame from the activities of the rich to the actions of the poor have been used to shirk the responsibility to mitigate climate change and to impede the development of global mitigation strategies. the dispute between the global north and south over who caused climate change and who must now take responsibility for mitigation is by now well known. countries in the global south argue that northern countries have already developed their economies relying on fossil fuel consumption and now, when it is their turn to develop, they are asked to put restrictions on their energy consumption and natural resource use. meanwhile, the united states has used the reluctance of developing countries to cap emissions as an excuse for inaction on its part. mitigation and adaptation activities that do not take globally inequalities in wealth, resources, and adaptive capacity into account, or insist on 'equality' in action in a demonstrably unequal world cannot be effective. addressing population growth clearly plays a key role in both mitigation and adaptation strategies. however, focusing on population size while ignoring global patterns in the consumption of resources and the distribution of wealth will result in failed policies and strategies. the world's human population is approaching 7 ã� 10 9 , and a growing population inevitably means greater demand for the numerous goods and services supported by terrestrial and aquatic ecosystems. while the availability of resources has seemingly stretched beyond the direst predictions of several decades ago, the crisis of overconsumptionnamely, of coal and other fossil fuels that produce ghgs and contribute to climate changeis simultaneously becoming clearer. while trends over the past several decades suggest that population growth is a significant driver of co 2 emissions (bongaarts 1992; dietz and rosa 1997) , it is also true that urbanization, aging, and changes in household size similarly affect energy use and emissions (mackeller et al. 1995; cole and neumayer 2004) . particular point sources of ghg emissions from energy consumption in developed countries, like the internationally managed oil fields of nigeria or emissions from the recent iraq war, which since 2003 has released over 140 million metric tons of carbon dioxide, the equivalent of adding 25 million new cars to american roads (reisch and kretzmann 2008) , should bear at least as much scrutiny as the desires of third world women and men to bear children. an overarching emphasis on population detracts from a nuanced understanding of the multiple drivers of ghg emissions. it will be ineffective to focus on the effects of climate change on ecosystems without considering the concurrent effects on human well-being and vice versa. disparities in climate change risk and human health often overlap, and human health and environmental health are intricately intertwined. although diseases are often approached as singular phenomena, good models exist that demonstrate the interactive effects of multiple morbidity factors on health outcomes and disease trajectories. for example, interactions between acquired immune deficiency syndrome and tuberculosis, or diabetes and severe acute respiratory syndrome, have synergistic impacts on patients that must be understood together. likewise, social attributes, like poverty or exposure to racism, interact with disease conditions to increase the damaging effects of disease. such interactions have the potential to influence the effects of climate change on disease movement and clustering, disease interactions, and the impact of climate change on global health (baer and singer 2008) . human action and human vulnerability to such interactions between climate, disease, and the environment are structured by social and economic inequality, and thus mitigation and adaptation strategies will need to take these inequalities into account. what is clear from a social and political perspective on mitigation and adaptation is that no one set of mitigation or adaptation strategies will be applicable to all people in all places. suggested approaches to either mitigation or adaptation will involve costs and benefits that are unequally distributed across nations, populations, and societies. there will, for example, always be those who can escape pollution with air filters and private vehicles, use air conditioning to survive heat waves, have access to global food markets and multiple water sources, and those who cannot. the most effective mitigation and adaptation strategies, therefore, will address both climate change and environmental justice. the dust bowl area unlocking the potential of protected areas for freshwaters the geography of climate change: implications for conservation biogeography conserving the stage: climate change and the geophysical underpinnings of species diversity climate change and the green energy paradox: the consequences for twaite shad alosa fallax from the river severn would climate change drive species out of reserves? an assessment of existing reserveselection methods adaptive management of the global climate problem: bridging the gap between climate research and climate policy global warming and the political ecology of health impact of geoengineering schemes on the global hydrological cycle meeting ecological and societal needs for freshwater climate change and water projected impacts of climate change on salmon habitat restoration historical warnings of future food insecurity with unprecedented seasonal heat risk society: toward a new modernity use of land facets to plan for climate change: conserving the arenas, not the actors tidal energy resource assessment for tidal stream generators population growth and global warming basic principles and ecological consequences of altered flow regimes for aquatic biodiversity greenhouse gas mitigation by agricultural intensification. proc. natl. acad. sci. usa maintaining the role of canada's forests and peatlands in climate regulation examining the impact of demographic factors on air pollution amplification of the north american 'dust bowl' drought through human-induced land degradation synthesis of iron fertilization experiments: from the iron age in the age of enlightenment impacts of climate warming on terrestrial ectotherms across latitude carbon markets and forest conservation: a review of the environmental benefits of redd mechanisms effects of population and affluence on co 2 emissions peatland response to global change mangroves among the most carbon-rich forests in the tropics diazotrophic endophytes of native black cottonwood and willow assessing the impacts of wind farms on birds guidelines for adapting agriculture to climate change. handbook of climate change and agroecosystems: impacts, adaptation, and mitigation climate change 2007: impacts, adaptation and vulnerability. contribution of working group ii to the fourth assessment report of the intergovernmental panel on climate change urban forests and pollution mitigation: analyzing ecosystem services and disservices state of the world's forests land clearing and the biofuel carbon debt invasion versus isolation: trade-offs in managing native salmonids with barriers to upstream movement biodiversity conservation in the era of biofuels: risks and opportunities information needs to support environmental impact assessment of the effects of european marine offshore wind farms on birds why do we fly? ecologists' sins of emission importance of matrix habitats in maintaining biological diversity particle film mechanisms of action that reduce the effect of environmental stress in 'empire' apple hydrophobic particle films: a new paradigm for suppression of arthropod pests and plant diseases a new era for conservation: review of climate change adaptation literature. page 69 ethanol for a sustainable energy future impact of geoengineering schemes on the terrestrial biosphere global climate change and natural-area protection: management responses and research directions 2010: a global conservation system for climate-change adaptation protected area needs in a changing climate designing climate-smart conservation: guidance and case studies remote sensibilities: discourses of technology and the making of indonesia's natural disaster climate change and freshwater biodiversity: detected patterns, future trends and adaptations in northern regions biodiversity management in the face of climate change: a review of 22 years of recommendations environmental flows in water resources policies, plans, and projects: findings and recommendations assisted colonization and rapid climate change projected impacts of climate change on a continent-wide protected area network resilience and stability of ecological systems climate change and moving species: furthering the debate on assisted colonization light's labour's lost. policies for energy efficient lighting climate change 2007: impacts, adaptation and vulnerability. contribution of working group ii to the fourth assessment report of the intergovernmental panel on climate change climate change 2007: mitigation. contribution of working group iii to the fourth assessment report of the intergovernmental panel on climate change vision for water and nature: a world strategy for conservation and sustainable management of water resources in the 21st century. international union for the conservation of nature why genetically modified crops? forests and society -responding to global drivers of change synthesis and conclusions. a report by the u.s. climate change science program and the subcommittee on global change research. preliminary review of adaptation options for climate-sensitive ecosystems and soil carbon sequestration impacts on global climate change and food security climate change adaptation strategies for resource management and conservation planning resource management in a changing and uncertain climate prioritizing climate change adaptation needs for food security in 2030 wild profusion: biodiversity conservation in an indonesian archipelago genetically modified crop plants: science versus society? a perspective population, households, and co 2 emissions putting the carbon back: black is the new green the 2030 blueprint, solving climate change saves millions a framework for debate of assisted migration in an era of climate change food, livestock production, energy, climate change, and health climate change and forests of the future: managing in the face of uncertainty environmental refugees, a growing phenomenon of the 21st century legitimizing fluvial ecosystems as users of water: an overview climate change: impact on agriculture and costs of adaptation. international food policy research institute. number of implications of incorporating air-quality co-benefits into climate change policymaking fragmentation and flow regulation of the world's large river systems the carbon sequestration potential of a global afforestation program survey on actual service lives of north american buildings. woodframe housing durability and disaster issues conference challenges and opportunities in implementing managed relocation for conservation of freshwater species climate change and the world's river basins: anticipating management options ecological and evolutionary responses to recent climate change managing for future change on the albemarle sound adapting coastal lowlands to rising seas. principles of conservation biology forecasting potential global environmental costs of livestock production 2000-2050 the greenhouse effect and nature reserves: global warming would diminish biological diversity by causing extinctions among reserve species an overview of regional land use and land cover impacts on rainfall soil erosion: a food and environmental threat the natural flow regime: a paradigm for river conservation and restoration aquatic ecosystems and global climate change: potential impacts on inland freshwater and coastal wetland ecosystems in the united states freshwater ecosystem services rivers for life: managing water for people and nature selection of bioclimatically representative biological reserve systems under climate change research and innovation technology administration, cited 2011: national transportation statistics assisted colonization is not a viable conservation strategy multidimensional evaluation of managed relocation ecologically sustainable water management: managing river flows for river integrity stress tolerance in plants via habitatadapted symbiosis fungal endophytes: diversity and functional roles global review of the physical and biological effectiveness of stream habitat rehabilitation techniques using bioclimatic envelopes to identify temporal corridors in support of conservation planning in a changing climate water for agriculture: maintaining food security under growing scarcity the dust bowl of the 1930s -analog of greenhouse effect in the great plains adaptation and mitigation strategies in agriculture: an analysis of potential synergies managed relocation: a nuanced evaluation is needed mapping environments at risk under different global climate change scenarios 2010: population diversity and the portfolio effect in an exploited species systematic conservation planning in the face of climate change: bet-hedging on the columbia plateau on the cause of the 1930s dust bowl model projections of an imminent transition to a more arid climate in southwestern north america marine renewable energy: the ecological implications of altering the hydrodynamics of the marine environment targeted protection and restoration to conserve tropical biodiversity in a warming world greenhouse gas mitigation in agriculture architecture in a climate of change reservoir surfaces as sources of greenhouse gases to the atmosphere: a global estimate trading off: the ecological effects of dam removal livestock production and the global environment: consume less or produce better? stern review: economics of climate change. hm treasury. un human settlements programme energy information administration global threats to human water security and river biodiversity adapting landscapes to climate change: examples of climateproof ecosystem networks and priority adaptation zones ecological optimization and adaptive management dam reoperation in an era of climate change dams and development: a new framework for decision-making climate change, biodiversity and the urban environment: a critical review based on london statistical downscaling of general circulation model output: a comparison of methods planning for climate change: identifying minimum-dispersal corridors for the cape proteaceae wind power implementation: the nature of public attitudes: equity and fairness instead of 'backyard motives climate change mitigation and transport in developing nations making water -desalination: option or distraction for a thirsty world? world wildlife fund a diazotrophic, indole-3-acetic acid-producing endophyte from wild cottonwood we thank the global health and environment faculty fellows at the university of washington as well as thomas hinckley, richard fenske, judith wasserheit, and tikvah weiner for valuable discussions and support. key: cord-021081-yqu1ykc9 authors: nan title: early warning systems a state of the art analysis and future directions date: 2012-11-02 journal: nan doi: 10.1016/j.envdev.2012.09.004 sha: doc_id: 21081 cord_uid: yqu1ykc9 nan united nations' international strategy for disaster reduction (isdr), it integrates (united nations, 2006) : 1. risk knowledge: risk assessment provides essential information to set priorities for mitigation and prevention strategies and designing early warning systems. 2. monitoring and predicting: systems with monitoring and predicting capabilities provide timely estimates of the potential risk faced by communities, economies and the environment. 3. disseminating information: communication systems are needed for delivering warning messages to the potentially affected locations to alert local and regional governmental agencies. the messages need to be reliable, synthetic and simple to be understood by authorities and the public. 4. response: coordination, good governance and appropriate action plans are key points in effective early warning. likewise, public awareness and education are critical aspects of disaster mitigation. failure of any part of the system will imply failure of the whole system. for example, accurate warnings will have no impact if the population is not prepared or if the alerts are received but not disseminated by the agencies receiving the messages. the basic idea behind early warning is that the earlier and more accurately we are able to predict short-and long term potential risks associated with natural and human induced hazards, the more likely we will be able to manage and mitigate a disaster's impact on society, economies, and environment. environmental hazards can be associated with: ongoing and rapid/sudden-onset threats and slow-onset (or ''creeping'') threats: 1. ongoing and rapid/sudden-onset: these include such hazards as follows: accidental oil spills, nuclear plant failures, and chemical plant accidents -such as inadvertent chemical releases to the air or into rivers and water bodies -geological hazards and hydro-meteorological hazards (except droughts). 2. slow-onset (or ''creeping''): incremental but long-term and cumulative environmental changes that usually receive little attention in their early phases but which, over time, may cause serious crises. these include such issues as deteriorating air and water quality, soil pollution, acid rain, climate change, desertification processes (including soil erosion and land degradation), drought, ecosystems change, deforestation and forest fragmentation, loss of biodiversity and habitats, nitrogen overloading, radioactive waste, coastal erosion, pressures on living marine resources, rapid and unplanned urban growth, environment and health issues (emerging and re-emerging infectious diseases and links to environmental change), land cover/land changes, and environmental impacts of conflict, among others. such creeping changes are often left unaddressed as policy-makers choose or need to cope with immediate crises. eventually, neglected creeping changes may become urgent crises that are more costly to deal with. slow-onset threats can be classified into location-specific environmental threats, new emerging science and contemporary environmental threats (table 1) . rapid/sudden-onset and slow-onset events will provide different amounts of available warning time. fig. 1 shows warning times for climatic hazards. early warning systems may provide seconds of available warning time for earthquakes to months of warning for droughts, which are the quickest and slowest onset hazards, respectively. specifically, early warning systems provide tens of seconds of warning for earthquakes, days to hours for volcanic eruptions, and hours for tsunamis. tornado warnings provide minutes of lead-time for response. hurricane warning time varies from weeks to hours. the warning time provided by warning systems, increases to years or even decades of leadtime available for slow-onset threats (such as el niñ o, global warming etc., as shown in fig. 1 ). drought warning time is in the range of months to weeks. slow-onset (or creeping) changes may cause serious problems to environment and society, if preventive measures are not taken when needed. such creeping environmental changes require effective early warning technologies due to the high potential impact of incremental cumulative changes on society and the environment. (golnaraghi, 2005) . the graph shows the timeliness of early warning systems for hydrometeorological hazards and the area of impact (by specifying the diameter of the spherical area) for climatic hazards. early warning systems help to reduce economic losses and mitigate the number of injuries or deaths from a disaster, by providing information that allows individuals and communities to protect their lives and property. early warning information empowers people to take action prior to a disaster. if well integrated with risk assessment studies and communication and action plans, early warning systems can lead to substantive benefits. effective early warning systems embrace the following aspects: risk analysis; monitoring and predicting location and intensity of the disaster; communicating alerts to authorities and to those potentially affected; and responding to the disaster. the early warning system has to address all aspects. monitoring and predicting is only one part of the early warning process. this step provides the input information for the early warning process that needs to be disseminated to those whose responsibility is to respond (fig. 2) . early warnings may be disseminated to targeted users (local early warning applications) or broadly to communities, regions or to media (regional or global early warning applications). this information gives the possibility of taking action to initiate mitigation or security measures before a catastrophic event occurs. when monitoring and predicting systems are associated with communication systems and response plans, they are considered early warning systems (glantz, 2003) . commonly, however, early warning systems lack one or more elements. in fact, a review of existing early warning systems shows that in most cases communication systems and adequate response plans are missing. to be effective, warnings also must be timely so as to provide enough lead-time for responding; reliable, so that those responsible for responding to the warning will feel confident in taking action; and simple, so as to be understood. timeliness often conflicts with the desire to have reliable predictions, which become more accurate as more observations are collected from the monitoring system . thus, there is an inevitable trade-off between the amount of warning time available and the reliability of the predictions provided by the ews. an initial alert signal may be sent to give the maximum amount of warning time when a minimum level of prediction accuracy has been reached. however, the prediction accuracy for the location and size of the event will continue to improve as more data are collected by the monitoring system part of the ews network. it must be understood that every prediction, by its very nature, is associated with uncertainty. because of the uncertainties associated with the predicted parameters that characterize the incoming disaster, it is possible that a wrong decision may be made. two kinds of wrong decisions may occur : missed alarm (or false negative), when the mitigation action is not taken when it should have been or false alarm (or false positive), when the mitigation action is taken when it should not have been. finally, the message should communicate the level of uncertainty and expected cost of taking action but also be stated in simple language so as to be understood by those who receive it. most often, there is a communication gap between ew specialists who use technical and engineering language and the ews users, who are generally outside of the scientific community. to avoid this, these early warnings need to be reported concisely, in layman's terms and without scientific jargon. an effective early warning system needs an effective communication system. early warning communication systems have two main components (second international conference on early warning (ewcii), 2003): communication infrastructure hardware that must be reliable and robust, especially during the disaster; and appropriate and effective interactions among the main actors of the early warning process, such as the scientific community, stakeholders, decision-makers, the public, and the media. redundancy of communication systems is essential for disaster management, while emergency power supplies and back-up systems are critical in order to avoid the collapse of communication systems after disasters occur. in addition, to ensure the communication systems operate reliably and effectively during and after a disaster occurs, and to avoid network congestion, frequencies and channels must be reserved and dedicated to disaster relief operations. many communication tools are currently available for warning dissemination, such as short message service (sms) (cellular phone text messaging), e-mail, radio, tv and web service. information and communication technology (ict) is a key element in early warning, which plays an important role in disaster communication and disseminating information to organizations in charge of responding to warnings and to the public during and after a disaster (tubtiang, 2005) . today, the decentralization of information and data through the world wide web makes it possible for millions of people worldwide to have easy, instantaneous access to a vast amount of diverse online information. this powerful communication medium has spread rapidly to interconnect our world, enabling near-real-time communication and data exchanges worldwide. according to the internet world stats database, as of december 2011, global documented internet usage was 2.3 billion people. thus, the internet has become an important medium to access and deliver information worldwide in a very timely fashion. in addition, remote sensing satellites now provide a continuous stream of data. they are capable of rapidly and effectively detecting hazards, such as transboundary air pollutants, wildfires, deforestation, changes in water levels, and natural hazards. with rapid advances in data collection, analysis, visualization and dissemination, including technologies such as remote sensing, geographical information systems (gis), web mapping, sensor webs, telecommunications and ever-growing internet connectivity, it is now feasible to deliver relevant information on a regular basis to a worldwide audience relatively inexpensively. in recent years, commercial companies such as google, yahoo, and microsoft have started incorporating maps and satellite imagery into their products and services, delivering compelling visual images and providing easy tools that everyone can use to add to their geographic knowledge. ews: decision making procedure based on cost-benefit analysis. to improve the performance of ews, a performance based decision making procedure needs to be based on the expected consequences of taking action, in terms of the probability of a false and missed alarm. an innovative approach sets the threshold based on the acceptable probability of false (missed) alarms, from a cost-benefit analysis . consider the case of a ews decision making strategy based on raising the alarm if a critical severity level, a, is predicted to be exceeded at a site. the decision of whether to activate the alarm or not is based on the predicted severity of the event. a decision model that takes into account the uncertainty of the prediction and the consequences of taking action will be capable of controlling and reducing the incidence of false and missed alerts. the proposed decision making procedure intends to fill this gap. the ews will provide the user with a real-time prediction of the severity of the event,ŝðtþ, and its error, e tot ðtþ. during the course of the event, the increase in available data will improve prediction accuracy. the prediction and its uncertainty are updated as more data come in. the actual severity of the event, e tot , is unknown and may be defined by adding the prediction error to the predicted value,ŝ. the potential probability of false (missed) alarm is given by the probability of being less (greater) than the critical threshold; it becomes an actual probability of false (missed) alarm if the alarm is (not) raised: referring to the principle of maximum entropy (jaynes, 2003) , the prediction error is modelled by gaussian distribution, representing the most uninformative distribution possible due to lack of information. hence, at time t, the actual severity of the event, s, may be modelled with a gaussian distribution, having mean equal to the predictionŝðtþ and uncertainty equal to s tot ðtþ, that is the standard deviation of the prediction error e tot ðtþ. eqs. (1) and (2) may be written as : where f represents the gaussian cumulative distribution function. the tolerable level at which mitigation action should be taken can be determined from a cost-benefit analysis by minimizing the cost of taking action: where c save are the savings due to mitigation actions and c fa is the cost of false alert. note that the tolerable levels a and b sum up to one which directly exhibits the trade-off between the threshold probabilities that are tolerable for false and missed alarms. the methodology offers an effective approach for decision making under uncertainty focusing on user requirements in terms of reliability and cost of action. information is now available in a near-real-time mode from a variety of sources at global and local levels. in the coming years, the multi-scaled global information network will greatly improve thanks to new technological advances that facilitate the global distribution of data and information at all levels. globalization and rapid communication provides an unprecedented opportunity to catalyse effective action at every level by rapidly providing authorities and the general public with high-quality and scientifically credible information in a timely fashion. the dissemination of warnings often follows a cascade process, which starts at the international or national level and then moves outwards or downwards in scale to regional and community levels (twigg, 2003) . early warnings may activate other early warnings at different authoritative levels, flowing down in responsibility roles, although all are equally necessary for effective early warning. standard protocols play a fundamental role in addressing the challenge of effective coordination and data exchange among the actors in the early warning process and it aids in the process for warning communication and dissemination. the common alerting protocol (cap), really simple syndication (rss) and extensible markup language (xml) are examples of standard data interchange formats for structured information that can be applied to warning messages for a broad range of information management and warning dissemination systems. the advantage of standard format alerts is that they are compatible with all information systems, warning systems, media, and most importantly, with new technologies such as web services. cap, for example, defines a single standard message format for all hazards, which can activate multiple warning systems at the same time and with a single input. this guarantees consistency of warning messages and would easily replace specific application-oriented messages with a single multihazard message format. cap is compatible with all types of information systems and public alerting systems (including broadcast radio and television), public and private data networks, multi-lingual warning systems and emerging technologies such as internet web services and existing systems such as the us national emergency alert system and the national oceanic and atmospheric organization (noaa) weather radio. cap uses extensible markup language (xml), which contains information about the alert message, the specific hazard event, and appropriate responses, including the urgency of action to be taken, severity of the event, and certainty of the information. for early warning systems to be effective, it is essential that they be integrated into policies for disaster mitigation. good governance priorities include protecting the public from disasters through the implementation of disaster risk reduction policies. it is clear that natural phenomena cannot be prevented, but their human, socio-economic and environmental impacts can and should be minimized through appropriate measures, including risk and vulnerability reduction strategies, early warning, and appropriate action plans. most often, these problems are given attention during or immediately after a disaster. disaster risk reduction measures require long term plans and early warning should be seen as a strategy to effectively reduce the growing vulnerability of communities and assets. the information provided by early warning systems enables authorities and institutions at various levels to immediately and effectively respond to a disaster. it is crucial that local government, local institutions, and communities be involved in the entire policy-making process, so they are fully aware and prepared to respond with short and long-term action plans. the early warning process, as previously described, is composed of 4 main stages: risk assessment, monitoring and predicting, disseminating and communicating warnings, and response. within this framework, the first phase, when short-and long-term actions plans are laid out based on risk assessment analysis, is the realm of institutional and political actors. then ew acquires a technical dimension in the monitoring and predicting phase, while in the communication phase, ew involves both technical and institutional responsibility. the response phase then involves many more sectors, such as national and local institutions, non-governmental organizations, communities, and individuals. below is a summary of recommendations for effective decision making within the early warning process (sarevitz et al., 2000) . prediction efforts by the scientific community alone are insufficient for decision making. the scientific community and policy-makers should outline the strategy for effective and timely decision making by indicating what information is needed by decision-makers, how predictions will be used, how reliable the prediction must be to produce an effective response, and how to communicate this information and the tolerable prediction uncertainty so that the information can be received and understood by authorities and public. a miscommunicated or misused prediction can result in costs to society. prediction, communication, and use of the information are necessary factors in effective decision making within the early warning process. wishing not to appear ''alarmist'' or to avoid criticism, local and national governments have sometimes kept the public in the dark when receiving technical information regarding imminent threats. the lack of clear and easy-to-use information can sometimes confuse people and undermine their confidence in public officials. conversely, there are quite a few cases where the public may have refused to respond to early warnings from authorities, and have therefore exposed themselves to danger or forced governments to impose removal measures. in any case, clear and balanced information is critical, even when some level of uncertainty remains. for this reason, the information's uncertainty level must be communicated to users together with the early warning . resources must be allocated wisely and priorities should be set, based on risk assessment, for long-and short-term decision making, such as investing in local early warning systems, education, or enhanced monitoring and observational systems. in addition, decision-makers need to be able to set priorities for timely and effective response to a disaster when it occurs based on the information received from the early warning system. decision-makers should receive necessary training on how to use the information received when an alert is issued and what that information means. institutional networks should be developed with clear responsibilities. complex problems such as disaster mitigation and response require multidisciplinary research, multi-sector policy and planning, multi-stakeholder participation, and networking involving all the participants of the process, such as the scientific research community (including social sciences aspects), land use planning, environment, finance, development, education, health, energy, communications, transportation, labour, and social security and national defence. decentralization in the decision making process could lead to optimal solutions by clarifying local government and community responsibilities. collaboration will improve efficiency, credibility, accountability, trust, and costeffectiveness. this collaboration consists of joint research projects, sharing information and participatory strategic planning and programming. because there are numerous actors involved in early warning response plans (such as governing authorities, municipalities, townships, and local communities), the decision making and legal framework of responsibilities should be set up in advance in order to be prepared when a disaster occurs. hurricane katrina in 2005 showed gaps in the legal frameworks and definition of responsibilities that exacerbated the disaster. such ineffective decision making must be dealt with to avoid future disasters such as the one in new orleans. earth observation (eo), through measuring and monitoring, provides an insight and understanding into earth's complex processes and changes. eo includes measurements that can be made directly or by sensors in-situ or remotely (i.e. satellite remote sensing, aerial surveys, land or oceanbased monitoring systems, fig. 3 ), to provide key information to models or other tools to support decision making processes. eo assists governments and civil society to identify and shape corrective and new measures to achieve sustainable development through original, scientifically valid assessments and early warning information on the recent and potential long-term consequences of human activities on the biosphere. at a time when the world community is striving to identify the impacts of human actions on the planet's life support system, time sequenced satellite images help to determine these impacts and provide unique, visible and scientifically convincing evidence that human actions are causing substantial changes to the earth's environment and natural resource base (i.e. ecosystems changes, urban growth, transboundary pollutants, loss of wetlands, etc.). by enhancing the visualization of scientific information on environmental change, satellite imagery will enhance environmental management and raise the awareness of emerging environmental threats. eo provides the opportunity to explore, to discover, and to understand the world in which we live from the unique vantage point of space. the following section discusses the potential role of eo for each type of environmental threat. 2.1. ongoing and rapid/sudden-onset environmental threats 2.1.1. oil spills earth observation is increasingly used to detect illegal marine discharges and oil spills. infra-red (ir) video and photography from airborne platforms, thermal infrared imaging, airborne laser fluorosensors, airborne and satellite optical sensors, as well as airborne and satellite synthetic aperture radar (sar) are used for this purpose. sar has the advantage of also providing data during cloud cover conditions and darkness, unlike optical sensors. in addition, optical-sensor techniques applied to oil spills detection are associated to a high number of false alarms, more often cloud shadows, sun glint, and other conditions such as precipitation, fog, and the amounts of daylight present also may be erroneously associated with oil spills. for this reason, sar is preferred over optical sensors, especially when spills cover vast areas of the marine environment, and when the oil cannot be seen or discriminated against the background. sar detects changes in sea-surface roughness patterns modified by oil spills. the largest shortcoming of oil spills detection using sar images is accurate discrimination between oil spills and natural films (brekke and soldberg, 2005) . to date, operational application of satellite imagery for oil spill detection still remains a challenge due to limited spatial and temporal resolution. in addition, processing times are often too long for operational purposes, and it is still not possible to measure the thickness of the oil spill (mansor et al., 2007; us fig. 3 . illustration of multiple observing systems in use on the ground, at sea, in the atmosphere and from space for monitoring and researching the climate system (wmo, 2011). interior, minerals management service, 2007) . existing applications are presented in the section 3. chemical and nuclear accidents may have disastrous consequences, such as the 1984 accident in bhopal, india, which killed more than 2000 and injured about 150 000, and the 1986 explosion of the reactors of the nuclear power plant in chernobyl, ukraine, which was the worst such accident to date, affecting part of the soviet union, eastern europe, scandinavia, and later, western europe. meteorological factors such as wind speed and direction, turbulence, stability layers, humidity, cloudiness, precipitation and topographical features, influence the impact of chemical and nuclear accidents and have to be taken into account in decision models. in some cases, emergencies are localized while in others, transport processes are most important. eo provides key data for monitoring and forecasting the dispersion and spread of the substance. geohazards associated with geological processes such as earthquakes, landslides, and volcanic eruptions are mainly controlled by ground deformation. eo data allows monitoring of key physical parameters associated with geohazards, such as deformation, plate movements, seismic monitoring, baseline topographic, and geoscience mapping. eo products are useful for detection and mitigation before the event, and for damage assessment during the aftermath. for geohazards, stereo optical and radar interferometry associated with ground-based global positioning system (gps) and seismic networks are used. for volcanic eruptions additional parameters are observed such as temperature and gas emissions. ground based measurements have the advantage of being continuous in time but have limited spatial extent, while satellite observations cover wide areas but are not continuous in time. these data need to be integrated for an improved and more comprehensive approach (committee on earth observation satellites (ceos), 2002; integrated global observing strategy (igos-p), 2003). earthquakes are due to a sudden release of stresses accumulated around the faults in the earth's crust. this energy is released through seismic waves that travel from the origin zone, which cause the ground to shake. severe earthquakes can affect buildings and populations. the level of damage depends on many factors, such as the intensity and depth of the earthquake, and the vulnerability of structures and their distance from the earthquake's origin. for earthquakes, information on the location and magnitude of the event first needs to be conveyed to responsible authorities. this information is used by seismic early warning systems to activate security measures within seconds after the earthquake's origin and before strong shaking occurs at the site. shakemaps generated within five minutes provide essential information to assess the intensity of ground shaking and the damaged areas. the combination of data from seismic networks and gps may help to increase reliability and timeliness of this information. earthquake frequency and probability shakemaps based on historical seismicity and base maps (geological, soil type, active faults, hydrological, and dems), assist in the earthquake mitigation phase and need to be included in the building code design process for improved land use and building practices. for responses, additional data are needed, such as seismicity, intensity, strain, dems, soil type, moisture conditions, infrastructure and population, to produce post-event damage maps. thermal information needs to continuously be monitored. this is obtained from low/medium resolution ir imagery from polar and geostationary satellites for thermal background characterization (advanced very high resolution radiometer (avhrr), atsr, modis and goes) together with deformation from edm and/or gps network; borehole strainmetres; and sar interferometry. landslides are displacements of earth, rock, and debris caused by heavy rains, floods, earthquakes, volcanoes, and wildfires. useful information for landslides and ground instability include the following: hazard zonation maps (landslides, debris flows, rockfalls, subsidence, and ground instability scenarios) during the mitigation phase, associated with landlside inventories, dem, deformation (gps network; sar interferometry; other surveys such as leveling, laser scanning, aerial, etc.), hydrology, geology, soil, geophysical, geotechnical, climatic, seismic zonation maps, land cover, land use, and historical archives. forecasting the location and extent of ground instability or landslides is quite challenging. landslides can be preceded by cracks, accelerating movement, and rock fall activity. real-time monitoring of key parameters thus becomes essential. the observed acceleration, deformation or displacement, exceeding a theoretical pre-fixed threshold is the trigger for issuing an alert signal. an alternative approach is based on hydrologic forecasting. it should be said that for large areas site-specific monitoring is not feasible. in this case, hazard mapping associated with monitoring of high risk zones remains the best option for warning. local rapid mapping of affected areas, updated scenarios and real-time monitoring (deformation, seismic data, and weather forecasts) assist during the response phase. a tsunami is a series of ocean waves generated by sudden displacements in the sea floor, landslides, or volcanic activity. although a tsunami cannot be prevented, the impact of a tsunami can be mitigated through community preparedness, timely warnings, and effective response. observations of seismic activity, sea floor bathymetry, topography, sea level data (tide gauge observations of sea height; real-time tsunami warning buoy data; deep ocean assessment and reporting of tsunamis (dart) buoys and sea-level variations from the topex/poseidon and jason, the european space agency's envisat, and the us navy's geosat follow-on), are used in combination with tsunami models to create inundation and evacuation maps and to issue tsunami watches and warnings. volcanic eruptions may be mild, releasing steam and gases or lava flows, or they can be violent explosions that release ashes and gases into the atmosphere. volcanic eruptions can destroy land and communities living in their path, affect air quality, and even influence the earth's climate. volcanic ash can impact aviation and communications. data needs for volcanic eruptions include hazard zonation maps, real-time seismic, deformation (electronic distance measurement (edm) and/or gps network; leveling and tilt networks; borehole strainmeters; gravity surveys; sar interferometry), thermal (landsat, aster, geostationary operational environmental satellites (goes), modis); air borne ir cameras; medium-high resolution heat flux imagery and gas emissions (cospec, licor surveys); satellite imagery (i.e., aster) and digital elevation maps (dem). as soon as the volcanic unrest initiates, information needs to be timely and relatively high-resolution. once the eruption starts, the flow of information has to speed up. seismic behaviour and deformation patterns need to be observed throughout the eruption especially to detect a change of eruption site (3-6 seismometers ideally with 3-directional sensors; a regional network). hydro-meteorological hazards include the wide variety of meteorological, hydrological and climate phenomena that can pose a threat to life, property and the environment. these types of hazards are monitored using the meteorological, or weather, satellite programs, beginning in the early 1960s. in the united states, nasa, noaa, and the department of defense (dod) have all been involved with developing and operating weather satellites. in europe, esa and eumetsat (european organisation for the exploitation of meteorological satellites) operate the meteorological satellite system (us centennial of flight commission). data from geostationary satellite and polar microwave derived products (goes) and polar orbiters (microwave data from the defense meteorological satellite program (dmsp), special sensor microwave/imager (ssm/i), noaa/advanced microwave sounding unit (amsu), and tropical rainfall measuring mission (trmm)) are key in weather analysis and forecasting. goes has the capability of observing the atmosphere and its cloud cover from the global scale down to the storm scale, frequently and at high resolution. microwave data are available on only an intermittent basis, but are strongly related to cloud and atmospheric properties. is key for monitoring meteorological processes from the global scale to the synoptic scale to the mesoscale and finally to the storm scale. (scofield et al., 2002) . goes and poes weather satellites provide useful information on precipitation, moisture, temperature, winds and soil wetness, which is combined with ground observation. floods are often triggered by severe storms, tropical cyclones, and tornadoes. the number of floods has continued to rise steadily; together with droughts, they have become the most deadly disasters over the past decades. the increase in losses from floods is also due to climate variability, which has caused increased precipitation in parts of the northern hemisphere (natural hazards working group, 2005) . floods can be deadly, particularly when they arrive without warning. in particular, polar orbital and geostationary satellite data are used for flood observation. polar orbital satellites include optical low (avhrr), medium (landsat, spot, irs) and high resolution (ikonos) and microwave sensors (high (sar-radarsat, jers and ers) and low resolution passive sensors (ssmi). meteorological satellites include goes 8 and 10, meteosat, gms, the indian insat and the russian goms; and polar orbitals such as noaa (noaa 15) and ssmi. for storms, additional parameters are monitored, such as sea surface temperature, air humidity, surface wind speed, rain estimates (from dmsp/ssmi, trmm, ers, quikscat, avhrr, radarsat). trmm offers unique opportunities to examine tropical cyclones. with trmm, scientists are able to make extremely precise radar measurements of tropical storms over the oceans and identify their intensity variations, providing invaluable insights into the dynamics of tropical storms and rainfall. epidemics such as malaria and meningitis are linked to environmental factors. satellite data can provide essential information on these factors and help to better understand diseases. as an example, the esa epidemio project, launched in 2004, utilizes data from esa's envisat or the french space agency's spot, and field data to gather information on the spread of epidemics, helping to better prepare for epidemic outbreaks. geo, with who and other partners, are working together on the meningitis environmental risk information technologies (merit) project to better understand the relationship between meningitis and environmental factors using remote sensing. wildfires pose a threat to lives and properties and are often connected to secondary effects such as landslides, erosion, and changes in water quality. wildfires may be natural processes, human induced for agriculture purposes, or just the result of human negligence. wildfire detection using satellite technologies is possible thanks to significant temperature difference between the earth's surface (usually not exceeding 10-25 1c) and the heat of fire (300-900 1c), which results in a thousand times difference in heat radiation generated by these objects. noaa (avhrr radiometer with 1100 m spatial resolution and 3000 km swath width) and earth observing satellites (eos) (terra and aqua satellites with modis radiometer installed on them with 250, 500 and 1000 m spatial resolution and 2330 km swath width) are the most widely used modern satellites for operative fire monitoring (klaver et al., 1998) . high-resolution sensors, such as the landsat thematic mapper, spot multispectral scanner, or national oceanic and atmospheric administration's avhrr or modis, are used for fire potential definition. sensors used for fire detection and monitoring include avhrr, which has a thermal sensor and daily overflights, the defense meteorological satellite program's optical linescan system (ols) sensor, which has daily overflights and operationally collects visible images during its nighttime pass, and the modis land rapid response system. avhrr and higher resolution images (spot, landsat, and radar) can be used to assess the extent and impact of the fire. smog is the product of human and natural activities, such as industry, transportation, wildfires, volcanic eruptions, etc. and can have serious effects on human health and the environment. a variety of eo tools are available to monitor air quality. the national aeronautics and space administration (nasa) and the european space agency (esa) both have instruments to monitor air quality. the canadian mopitt (measurements of pollution in the troposphere) aboard the terra satellite monitors the lower atmosphere to observe how it interacts with the land and ocean biospheres, distribution, transport, sources, and sinks of carbon monoxide and methane in the troposphere. the total ozone mapping spectrometer (toms) instrument measures the total amount of ozone in a column of atmosphere as well as cloud cover over the entire globe. additionally, toms measures the amount of solar radiation escaping from the top of the atmosphere to accurately estimate the amount of ultraviolet radiation that reaches the earth's surface. the ozone monitoring instrument (omi) on aura will continue the toms record for total ozone and other atmospheric parameters related to ozone chemistry and climate. the omi instrument distinguishes between aerosol types, such as smoke, dust, and sulphates, and can measure cloud pressure and coverage. esa's schiamachy (scanning imaging absorption spectro-meter for atmospheric chartography) maps atmosphere over a very wide wavelength range (240-2380 nm), which allows detection of trace gases, ozone and related gases, clouds and dust particles throughout the atmosphere (athena global, 2005) . the moderate resolution imaging spectroradiometer (modis) sensor measures the relative amount of aerosols and the relative size of aerosol particles-solid or liquid particles suspended in the atmosphere. examples of such aerosols include dust, sea salts, volcanic ash, and smoke. the modis aerosol optical depth product is a measure of how much light airborne particles prevent from passing through a column of atmosphere. new technologies are also being explored for monitoring air quality, such as mobile phones equipped with simple sensors to empower citizens to collect and share real-time air quality measurements. this technology is being developed by a consortium called urban atmospheres. the traditional methods of monitoring coastal water quality require scientists to use boats to gather water samples, typically on a monthly basis because of the high costs of these surveys. this method captures episodic events affecting water quality, such as the seasonal freshwater runoff, but is not able to monitor and detect fast changes. satellite data provide measures of key indicators of water qualityturbidity and water clarity -to help monitor fast changes in factors that affect water quality, such as winds, tides and human influences including pollution and runoff. geoeye's sea-viewing wide field-ofview sensor (seawifs) instrument, launched aboard the orbview-2 satellite in 1997, collects ocean colour data used to determine factors affecting global change, particularly ocean ecology and chemistry. modis sensor, launched aboard the aqua satellite in 2002, together with its counterpart instrument aboard the terra satellite, collects measurements from the entire earth surface every 1-2 days and can also provide measurements of turbidity (bjorn-hansen, 2007) . overall, air and water quality monitoring coverage still appears to be irregular and adequate and available in real-time only for some contaminants (global earth observation system of systems, 2005). 2.2.3.1. droughts. noaa's national weather service (nws) defines a drought as ''a period of abnormally dry weather sufficiently prolonged for the lack of water to cause serious hydrologic imbalance in the affected area.'' drought can be classified by using 4 different definitions: meteorological (deviation from normal precipitation); agricultural (abnormal soil moisture conditions); hydrological (related to abnormal water resources); and socio-economic (when water shortage impacts people's lives and economies). a comprehensive and integrated approach is required to monitor droughts, due to the complex nature of the problem. although all types of droughts originate from a precipitation deficiency, it is insufficient to monitor solely this parameter to assess severity and resultant impacts (world meteorological organization, 2006) . effective drought early warning systems must integrate precipitation and other climatic parameters with water information such as streamflow, snow pack, groundwater levels, reservoir and lake levels, and soil moisture, into a comprehensive assessment of current and future drought and water supply conditions (svoboda et al., 2002) . in particular, there are 6 key parameters that are used in a composite product developed from a rich information stream, including climate indices, numerical models, and the input of regional and local experts. these are as follows: 1) palmer drought severity index (based on precipitation data, temperature data, division constants (water capacity of the soil, etc.) and previous history of the indices). 2) soil moisture model percentile (calculated through a hydrological model that takes observed precipitation and temperature and calculates soil moisture, evaporation and runoff. the potential evaporation is estimated from observed temperature). 3) daily stream flow percentiles. 4) percent of normal precipitation. 5) standardized precipitation index, and 6) remotely sensed vegetation health index. additional indicators may include the palmer crop moisture index, keetch-byram drought index, fire danger index, evaporation-related observations such as relative humidity and temperature departure from normal, reservoir and lake levels, groundwater levels, field observations of surface soil moisture, and snowpack observations. some of these indices and indicators are computed for point locations, and others are computed for climate divisions, drainage (hydrological) basins, or other geographical regions (svoboda et al., 2002) . a complete list of drought products can be found on noaa's national environmental satellite, data, & information service (noaanesdis) web page. 2.2.3.2. desertification. desertification refers to the degradation of land in arid, semi-arid, and dry sub-humid areas due to climatic variations or human activity. desertification can occur due to inappropriate land use, overgrazing, deforestation, and over-exploitation. land degradation affects many countries worldwide and has its greatest impact in africa. in spite of the potential benefits of eo information, the lack of awareness of the value and availability of information, inadequate institutional resources and financial problems are the most frequent challenges to overcome in detecting desertification (sarmap, 2003) . in 2004, through a project called desertwatch, esa has developed a set of indicators based principally on land surface parameters retrieved from satellite observations for monitoring land degradation and desertification. desertwatch is being tested and applied in mozambique, portugal, and brazil. the un food and agriculture organization's global information and early warning system (giews). these provide information on food availability, market prices and livelihoods. the observations of climate-related variables on a global scale have made it possible to document and analyse the behaviour of earth's climate, made available through programs as follows: the ioc-wmo-unep-icsu global ocean observing system (goos); the fao-wmo-unesco-unepicsu global terrestrial observing system (gtos); the wmo global observing system (gos) and global atmosphere watch (gaw); the research observing systems and observing systems research of the wmo-ioc-icsu world climate research programme (wcrp) and other climate relevant international programs; and wmo-unesco-icsuioc-unep global climate observing system (gcos). the intergovernmental panel on climate change (ipcc) periodically reviews and assesses the most recent scientific, technical and socio-economic information produced worldwide relevant to the understanding of climate change. hundreds of scientists worldwide contribute to the preparation and review of these reports. according to the recent ipcc report, the atmospheric buildup of greenhouse gases is already shaping the earth's climate and ecosystems from the poles to the tropics, which face inevitable, possibly profound, alteration. the ipcc has predicted widening droughts in southern europe and the middle east, sub-saharan africa, the american southwest and mexico, and flooding that could imperil low-lying islands and the crowded river deltas of southern asia. it stressed that many of the regions facing the greatest risks are among the world's poorest. information about the impacts of climate variability impact information is needed by communities and resource managers to adapt and prepare for larger fluctuations as global climate change becomes more evident. this information includes evidence of changes occurring due to climate variability, such as loss of ecosystems, ice melting, coastal degradation, and severe droughts. such information will provide policy-makers scientifically valid assessment and early warning information on the current and potential long-term consequences of human activities on the environment. (i.e., ecosystem changes, loss of biodiversity and habitats, land cover/land changes, coastal erosion, urban growth, etc.). landsat satellites (series 1-7) are extensively used to monitor location-specific environmental changes. they have the great advantage of providing repetitive, synoptic, global coverage of high-resolution multi-spectral imagery (fadhil, 2006) . landsat can be used for change detection applications to identify differences in the state of an object or phenomenon by comparing the satellite imagery at different times. change detection is key in natural resources management (singh, 1989) . central to this theme is the characterization, monitoring and understanding of land cover and land use change, since they have a major impact on sustainable land use, biodiversity, conservation, biogeochemical cycles, as well as land-atmosphere interactions affecting climate and they are indicators of climate change, especially at a regional level (igos-p, 2004) . the united nations environment programme's (unep) bestselling publication one planet, many people: atlas of our changing environment, which shows before and after satellite photos to document changes to the earth's surface over the past 30 years, proves the importance and impact of visual evidence of environmental change in hotspots. the atlas contains some remarkable landsat satellite imagery and illustrates the alarming rate of environmental destruction. through the innovative use of some 271 satellite images, 215 ground photos and 66 maps, the atlas provides visual proof of global environmental changes -both positive and negative -resulting from natural processes and human activities. case studies include themes such as atmosphere, coastal areas, waters, forests, croplands, grasslands, urban areas, and tundra and polar regions. the atlas demonstrates how our growing numbers and our consumption patterns are shrinking our natural resource base. the aim of this report is to identify current gaps and future needs of early warning systems through the analysis of the state of the art of existing early warning and monitoring systems for environmental hazards. among existing early warning/monitoring systems, only systems that provide publicly accessible information and products have been included in the analysis. for the present study, several sources have been used, such as the global survey of early warning systems (united nations, 2006) together with the online inventory of early warning systems on isdr's platform for the promotion of early warning (ppew) website, and several additional online sources, technical reports and scientific articles listed in the references. for each hazard type, a gap analysis has been carried out to identify critical aspects and future needs of ews, considering aspects such as geographical coverage, and essential ews elements such as monitoring and prediction capability, communication systems and application of early warning information in responses. below is the outcome of the review of existing early warning/monitoring systems for each hazard type. details of all systems, organized in tables by hazard type, are listed in the appendix. the current gaps identified for each hazard type could be related to technological, organizational, communication or geographical coverage aspects. to assess the geographical coverage of existing systems for each hazard type, the existing systems have been imposed on the hazard's risk map. for this analysis, the maps of risks of mortality and economic loss were taken from natural disaster hotspots: a global risk analysis, a report from the world bank (dilley et al., 2005) . to detect operational oil spills, satellite overpasses and aerial surveillance flights need to be used in an integrated manner. in many countries in northern europe, the ksat manual approach is currently used to identify oil spills from the satellite images. ksat has provided this operational service since 1996, and in europe, use of satellites for oil spill detection is well established and well integrated within the national and regional oil pollution surveillance and response chains. operational algorithms utilizing satellite-borne c-band sar instruments (radarsat-1, envisat, radarsat-2) are also being developed for oil-spill detection in the baltic sea area. releases of a hazardous substance from industrial accidents can have immediate adverse effects on human and animal life or the environment. wmo together with iaea provides specialized meteorological support to environmental emergency response related to nuclear accidents and radiological emergencies. the wmo network of eight specialized numerical modelling centres called regional specialized meteorological centres (rsmcs) provides predictions of the movement of contaminants in the atmosphere. the inter-agency committee on the response to nuclear accidents (iacrna) of the iaea, coordinates the international intergovernmental organizations responding to nuclear and radiological emergencies. iacrna members are: the european commission (ec), the european police office (europol), the food and agriculture organization of the united nations (fao), iaea, the international civil aviation organization (icao), the international criminal police organization (interpol), the nuclear energy agency of the organization for economic co-operation and development (oecd/nea), the pan american health organization (paho), unep, the united nations office for the co-ordination of humanitarian affairs (un-ocha), the united nations office for outer space affairs (unoosa), the world health organization (who), and wmo. the agency's goal is to provide support during incidents or emergencies by providing near real-time reporting of information through the following: the incident and emergency centre (iec), which maintains a 24 h oncall system for rapid initial assessment, and if needed, triggers response operations; the emergency notification and assistance convention website (enac), which allows the exchange of information on nuclear accidents or radiological emergencies; and the nuclear event webbased system (news), which provides information on all significant events in nuclear power plants, research reactors, nuclear fuel cycle facilities and occurrences involving radiation sources or the transport of radioactive material. the global chemical incident alert and response system of the international programme on chemical safety, which is part of who, focuses on disease outbreaks from chemical releases and also provides technical assistance to member states for response to chemical incidents and emergencies. formal and informal sources are used to collect information and if necessary, additional information and verification is sought through official channels: national authorities, who offices, who collaborating centres, other united nations agencies, and members of the communicable disease global outbreak alert and response network (goarn), internet-based resources, particularly the global public health intelligence network (gphin) and promed-mail. based on this information, a risk assessment is carried out to determine the potential impact and if assistance needs to be offered to member states. 3.1.3. geological hazards 3.1.3.1. earthquakes. earthquake early warning systems are a relatively new approach to seismic risk reduction. they provide a rapid estimate of seismic parameters such as magnitude and location associated with a seismic event based on the first seconds of seismic data registered at the epicentre. this information can then be used to predict ground motion parameters of engineering interest including peak ground acceleration and spectral acceleration. earthquake warning systems are currently operational in mexico, japan, romania, taiwan and turkey (espinosa aranda et al., 1995; wu et al., 1998; wu and teng, 2002; odaka et al., 2003; kamigaichi, 2004; nakamura, 2004; horiuchi et al., 2005) . systems are under development for seismic risk mitigation in california and italy. local and national scale seismic early warning systems, which provide seismic information between a few seconds and tens of seconds before shaking occurs at the target site, are used for a variety of applications such as shutting down power plants, stopping trains, evacuating buildings, closing gas valves, and alerting wide segments of the population through the tv, among others. on the global scale, multi-national initiatives, such as the us geological survey (usgs) and geo-forschungs netz (geofon), operate global seismic networks for seismic monitoring but do not provide seismic early warning information. today, the usgs in cooperation with incorporated research institutions for seismology (iris) operates the global seismic networks (gsn), which comprises more than 100 stations providing free, real-time, open access data. geofon collects information from several networks and makes this information available to the public online. usgs earthquake notification service (ens) provides publicly available e-mail notification for earthquakes worldwide within 5minutes for earthquakes in us and within 30 min for events worldwide. usgs also provides near-real-time maps of ground motion and shaking intensity following significant earthquakes. this product, called shakemap, is being used for post-earthquake response and recovery, public and scientific information, as well as for preparedness exercises and disaster planning. effective early warning technologies for earthquakes are much more challenging to develop than for other natural hazards because warning times range from only a few seconds in the area close to a rupturing fault to a minute or so (heaton, 1985; allen and kanamori, 2003; kanamori, 2005) . several local and regional applications exist worldwide but no global system exists or could possibly exist for seismic early warning at global scale, due to timing constraints. earthquake early warning systems applications must be designed at the local or regional level. although various early warning systems exist worldwide at the local or regional scale, there are still high seismic risk areas that lack early warning applications, such as peru, chile, iran, pakistan, and india. the international consortium on landslides (icl), created at the kyoto symposium in january 2002, is an international non-governmental and non-profit scientific organization, which is supported by the united nations educational, scientific and cultural organization (unesco), the wmo, fao, and the united nations international strategy for disaster reduction (un/isdr). icl's mission is to promote landslide research for the benefit of society and the environment and promote a global, multidisciplinary programme regarding landslides. icl provides information about current landslides on its website, streaming this information from various sources such as the geological survey of canada. this information does not provide any early warning since it is based on news reports after the events have occurred. enhancing icl's existing organizational infrastructure by improving landslide prediction capability would allow icl to provide early warning to authorities and populations. technologies for slopes monitoring has greatly improved, but currently only few slopes are being monitored at a global scale. the use of these technologies would be greatly beneficial for mitigating losses from landslides worldwide. 3.1.3.3. tsunamis. the indian ocean tsunami of december 2004 killed 220 000 people and left 1.5 million homeless. it highlighted gaps and deficiencies in existing tsunami warning systems. in response to this disaster, in june 2005 the intergovernmental oceanographic commission (ioc) secretariat was mandated by its member states to coordinate the implementation of a tsunami warning system for the indian ocean, the northeast atlantic and mediterranean, and the caribbean. efforts to develop these systems are ongoing. since march 2011, the indonesian meteorological, climatological and geophysical agency has been operating the german-indonesian tsunami early warning system for the indian ocean. milestones, such as the development of the automatic data processing software and underwater communication for the transmission of pressure data from the ocean floor to a warning centre, have been reached. these systems will be part of the global ocean observing system (goos), which will be part of geoss. the pacific basin is monitored by the pacific tsunami warning system (ptws), which was established by 26 member states and is operated by the pacific tsunami warning center (ptwc), located near honolulu, hawaii. ptwc monitors stations throughout the pacific basin to issue tsunami warnings to member states, serving as the regional centre for hawaii and as a national and international tsunami information centre. it is part of the ptws effort. noaa national weather service operates ptwc and the alaska tsunami warning center (atwc) in palmer, alaska, which serves as the regional tsunami warning center for alaska, british columbia, washington, oregon, and california. ptws monitors seismic stations operated by ptwc, usgs and atwc to detect potentially tsunamigenic earthquakes. such earthquakes meet specific criteria for generation of a tsunami in terms of location, depth, and magnitude. ptws issues tsunami warnings to potentially affected areas, by providing estimates of tsunami arrival times and areas potentially most affected. if a significant tsunami is detected, the tsunami warning is extended to the pacific basin. the international tsunami information center (itic), under the auspices of ioc, aims to mitigate tsunami risk by providing guidance and assistance to improve education and preparedness. itic also provides a complete list of tsunami events worldwide. official tsunami bulletins are released by ptwc, atwc, and the japan meteorological agency (jma). regional and national tsunami information centres exist worldwide; the complete list is available from ioc. currently, no global tsunami warning system is in place. in addition, fully operational tsunami early warning systems are needed for the indian ocean and the caribbean. initial steps have been taken in this direction. in 2010, noaa established the caribbean tsunami warning program as the first step towards the development of a caribbean tsunami warning center. since 2005, steps have been taken to develop an indian ocean tsunami system, such as establishing 26 tsunami information centres and deploying 23 real-time sea level stations and 3 deep ocean buoys in countries bordering indian ocean. in 2005, the united states agency for international development (usaid) launched the us indian ocean tsunami warning systems program as the us government's direct contribution to the international effort led by the ioc. since then, there are ongoing activities, such as germany's 5-year german-indonesia tsunami early warning system program with indonesia, the tsunami regional trust fund established in 2005, and the united kingdom's tsunami funds reserved for early warning capacity building. nevertheless, on 17 july 2006, only one month after the announcement that the indian ocean's tsunami warning system was operational, a tsunami in java, indonesia, killed hundreds of people. on that day, tsunami warnings were issued to alert jakarta but there was not enough time to alert the coastal areas. the july 2006 tsunami disaster illustrates that there are still operational gaps to be solved in the indian ocean tsunami early warning system, notably in warning coastal communities on time. 3.1.3.4. volcanic eruptions. volcanic eruptions are always anticipated by precursor activities. in fact, seismic monitoring, ground deformation monitoring, gas monitoring, visual observations, and surveying are used to monitor volcanic activity. volcano observatories are distributed worldwide. a complete list of volcano observatories is available at the world organization of volcanic observatories (wovo) website. however, there is still a divide between developed and developing countries. in particular, a large number of observatories and research centres monitor volcanoes in japan and the united states very well. most central and south american countries (mexico, guatemala, el salvador, nicaragua, costa rica, colombia, ecuador, peru, chile, trinidad, and the antilles) have volcano observatories that provide public access to volcanic activity information. in africa, only two countries (congo and cameroon) have volcano monitoring observatories and they do not provide public access to information. only a small number, probably fewer than 50, of the world's volcanoes are well monitored, mostly due to inadequate resources in poor countries (national hazards working group, 2005) . there is a need to fill this gap by increasing the coverage of volcanic observatories. currently, there is no global early warning system for volcanic eruptions except for aviation safety. global volcanic activity information is provided by the smithsonian institution, which partners with the usgs under the global volcanism program to provide online access to volcanic activity information, collected from volcano observatories worldwide. reports and warnings are available on a daily basis. weekly and monthly summary reports are also available, but these only report changes in volcanic activity level, ash advisories, and news reports. the information is also available through google earth. this information is essential for the aviation sector, which must be alerted to ash-producing eruptions. there are several ash advisory centres distributed worldwide, in london, toulouse, anchorage, washington, montreal, darwin, wellington, tokyo, and buenos aires. however, there is a need to coordinate interaction and data sharing among the approximately 80 volcano observatories that make up wovo. esa is developing globvolcano, an information system to provide earth observations for volcanic risk monitoring. 3.1.3.5. wildfires. early warning methodologies for wildfires are based on the prediction of precursors, such as fuel loads and lightning danger. these parameters are relevant for triggering prediction, but once the fire has begun, fire behaviour and pattern modelling are fundamental for estimating fire propagation patterns. most industrial countries have ew capabilities in place, while most developing countries have neither fire early warning nor monitoring systems in place (goldammer et al., 2003) . local and regional scale fire monitoring systems are available for canada, south america, mexico and south africa. an interactive mapping service based on google maps and eo imagery from inpe, the brazilian space research institute, has been available since september 2008. individuals can contribute with information from the ground; in only 3 months the service has received 41 million reports on forest fires and illegal logging, making it one of the most successful web sites in brazil; it has had real impact through follow up legal initiatives and parliamentary enquiries. wildfire information is available worldwide through the global fire monitoring center (gfmc), a global portal for fire data products, information, and monitoring. this information is accessible to the public through the gfmc website but is not actively disseminated. the gfmc provides global fire products through a worldwide network of cooperating institutions. gfmc fire products include the following: fire danger maps and forecasts, which provide assessment of fire onset risk; near realtime fire events information; an archive of global fire information; and assistance and support in the case of a fire emergency. global fire weather forecasts are provided by the experimental climate prediction center (ecpc), which also provides national and regional scale forecasts. noaa provides experimental, potential fire products based on estimated intensity and duration of vegetation stress, which can be used as a proxy for assessment of potential fire danger. the webfire mapper, part of fao's global fire information management system (gfims), initially developed by the university of maryland and financially supported by nasa, provides near real-time information on active fires worldwide, detected by the modis rapid response system. the webfire mapper integrates satellite data with gis technologies for active fire information. this information is available to the public through the website and e-mail alerts. the european forest fire information system also provides information on current fire situations and forecasts for europe and the mediterranean area. although global scale fire monitoring systems exist, an internationally standardized approach is required to create a globally comprehensive early fire warning system. integration of existing fire monitoring systems could significantly improve fire monitoring and early warning capabilities. an information network must be developed to disseminate early warnings about wild-fire danger at both the global and local levels, to quickly detect and report fires, and to enhance rapid fire detection and classification capabilities at national and regional levels. the global early warning system for wild-fires, which is under development as part of the global earth observation system of systems (geoss) effort, will address these issues. 3.1.4.1. floods. among natural hazards that are currently increasing in frequency, floods are the deadliest. this study shows there is inadequate coverage of flood warning and monitoring systems, especially in developing or least developed countries such as china, india, bangladesh, nepal, west africa, and brazil. at the local scale, there are several stand-alone warning systems, for example, in guatemala, honduras, el salvador, nicaragua, zimbabwe, south africa, belize, czech republic, and germany. however, they do not provide public access to information. the european flood alert system (efas), which is an initiative by ec-jrc, provides information on the possibility of river flooding occurring within the next three days. efas also provides an overview of current floods based on information received from the national hydrological services and the global runoff data center in germany. floods are monitored worldwide from the dartmouth flood observatory, which provides public access to major flood information, satellite images and estimated discharge. orbital remote sensing (advanced scanning microradiometer (amsr-e and quickscat)) is used to detect and map major floods worldwide. satellite microwave sensors can monitor, at a global scale and on a daily basis, increases of floodplain water surface without cloud interference. the dartmouth flood observatory provides estimated discharge and satellite images of major floods worldwide but does not provide forecasts of flood conditions or precipitation amounts that could allow flood warnings to be issued days in advance of events. noaa provides observed hydrologic conditions of major us river basins and predicted values of precipitation for rivers in the united states. noaa also provides information on excessive rainfall that could lead to flash-flooding and if necessary warnings are issued within 6 h in advance. ifnet global flood alert system (gfas) uses global satellite precipitation estimates for flood forecasting and warning. the gfas website publishes useful public information for flood forecasting and warning, such as precipitation probability estimates, but the system is currently running on a trial basis. at a global scale, flood monitoring systems are more developed than flood early warning systems. for this reason, existing technologies for flood monitoring must be improved to increase prediction capabilities and flood warning lead times. 3.1.4.2. severe weather, storms and tropical cyclones. at the global level, the world weather watch (www) and hydrology and water resources programmes coordinated by wmo provide global collection, analysis and distribution of weather observations, forecasts and warnings. the www is composed of the global observing system (gos), which provides the observed meteorological data; the global telecommunications system (gts), which reports observations, forecasts and other products and the global data processing system (gdps), which provides weather analyses, forecasts and other products. the www is an operational framework of coordinated national systems, operated by national governments. the tropical cyclone programme (tcp) is also part of the www. tcp is in charge of issuing tropical cyclones and hurricanes forecasts, warnings and advisories, and seeks to promote and coordinate efforts to mitigate risks associated with tropical cyclones. tcp has established tropical cyclone committees that extend across regional bodies (regional specialized meteorological centres (rsmc)), which, together with national meteorological and hydrological services (nmhss), monitor tropical cyclones globally and issue official warnings to the regional meteorological services of countries at risk. regional bodies worldwide have adopted standardized wmo-tcp operational plans and manuals, which promote internationally accepted procedures in terms of units, terminology, data and information exchange, operational procedures, and telecommunication of cyclone information. each member of a regional body is normally responsible for its land and coastal waters warnings. a complete list of wmo members and rsmcs is available on the wmo-tcp website. wmo then collects cyclone information and visualizes it on world maps. the university of hawaii collects information from wmo and provides online information on cyclone categories, wind speed, and current and predicted courses. although comprehensive coverage of early warning systems for storms and tropical cyclones is available, recent disasters such as hurricane katrina of 2005 have highlighted inadequacies in early warning system technologies for enabling effective and timely emergency response. there is a pressing need to improve communication between the sectors involved by strengthening the links between scientific research, organizations responsible for issuing warnings, and authorities in charge of responding to these warnings. while the www is an efficient framework of existing rsmc, nmhss and networks, national capacities in most developing countries need improvements in order to effectively issue and manage early warnings. action plans must also be improved. epidemics pose a significant threat worldwide, particularly in those areas that are already affected by other serious hazards, poverty, or under-development. epidemics spread easily across country borders. globalization increases the potential of a catastrophic disease outbreak: there is the risk that millions of people worldwide could potentially be affected. a global disease outbreak early warning system is urgently needed. who is already working in this field through the epidemic and pandemic alert and response, which provides real-time information on disease outbreaks, and goarn. the 192 who member countries, disease experts, institutions, agencies, and laboratories, part of an outbreak verification list, are constantly informed of rumoured and confirmed outbreaks. the who constantly monitors anthrax, avian influenza, crimean-congo hemorrhagic fever (cchf), dengue haemorrhagic fever, ebola haemorrhagic fever, hepatitis, influenza, lassa fever, marburg haemorrhagic fever, meningococcal disease, plague, rift valley fever, severe acute respiratory syndrome (sars), tularaemia, and yellow fever. a global early warning system for animal diseases transmissible to humans was formally launched in july 2006 by the fao, the world organization for animal health (oie), and who. the global early warning and response system for major animal diseases, including zoonoses (glews) monitors outbreaks of major animal diseases worldwide. a malaria early warning system is not yet available and the need for system development is pressing, especially in sub-saharan africa where malaria causes more than one million deaths every year. the iri institute at columbia university provides malaria risk maps based on rainfall anomaly, which is one of the factors influencing malaria outbreak and distribution, but no warning is disseminated to the potentially affected population. in addition, the malaria atlas project (map) supported by the wellcome trust, the global fund to fight aids, tuberculosis and malaria, the university of oxford-li ka shing foundation global health programme and others, aims to disseminate free, accurate and up-to-date information on malaria. the map is a joint effort of researchers from around the globe working in different fields (from public health to mathematics, geography and epidemiology). map produces and makes available a range of maps and estimates to support effective planning of malaria control at national and international scales. air pollution affects developing and developed countries without exception. for this reason, air quality monitoring and early warning systems are in place in most countries worldwide. nevertheless, there is still a technological divide between developed and developing countries; in fact, these systems are most developed in the united states, canada, and europe. there are several successful cases to mention in asia (taiwan, china, hong kong, korea, japan, and thailand), a few in latin america (argentina, brazil, and mexico city) and only one in africa (cape town, south africa). most of the existing systems focus on real-time air quality monitoring by collecting and analysing pollutant concentration measurements from ground stations. satellite observation is extremely useful for aviation and tropospheric ozone monitoring, which is done by nasa and esa. air quality information is communicated mainly through web services. the us environmental protection agency (epa) provides an e-mail alert service (epa airnow) only available in the us and the ministry of environment of ontario, canada, also provides e-mail alerts. the epa airnow notification service provides air quality information in real-time to subscribers via e-mail, cell phone or pager, allowing them to take steps to protect their health in critical situations. while current air quality information is provided by each of the air quality monitoring systems listed in the appendix, few sources provide forecasts. the following agencies provide forecasts, which are fundamental for early warning: us epa, esa, prev'air, and the environmental agencies of belgium, germany, and canada (see the appendix). prediction capability is an essential component of the early warning process. existing air quality monitoring systems need to be improved in order to provide predictions to users days in advance so they can act when unhealthy air quality conditions occur. 3.2.2.1. drought. drought early warning systems are the least developed systems due its complex processes and environmental and social impacts. the study of existing drought early warning systems shows that only a few such systems exist worldwide. on a regional scale, the fews net for eastern africa, afghanistan, and central america reports on current famine conditions, including droughts, by providing monthly bulletins that are accessible on the fews net web page. for the united states, the us drought monitor (svoboda et al., 2002) provides current drought conditions at the national and state level through an interactive map available on the website accompanied by a narrative on current drought impacts and a brief description of forecasts for the following week. the us drought monitor, a joint effort between the us department of agriculture (usda), noaa, the climate prediction center, the university of nebraska lincoln and others, has become the best available product for droughts (svoboda et al., 2002) . it has a unique approach that integrates multiple drought indicators with field information and expert input, and provides information through a single easy-to-read map of current drought conditions and short notes on drought forecast conditions. for china, the beijing climate center (bcc) of the china meteorological administration (cma) monitors drought development. based on precipitation and soil moisture monitoring from an agricultural meteorological station network and remote-sensing-based monitoring from cma's national satellite meteorological center, a drought report and a map on current drought conditions are produced daily and made available on their website. the european commission joint research center (ec-jrc) provides publicly available drought-relevant information through the following real-time online maps: daily soil moisture maps of europe; daily soil moisture anomaly maps of europe; and daily maps of the forecasted top soil moisture development in europe (7-day trend). at a global scale, two institutions (fao's global information and early warning system on food and agriculture (giews) and benfield hazard research center of the university college london) provide some information on major droughts occurring worldwide. the fao-giews provides information on countries facing food insecurity through monthly briefing reports on crop prospects and food situations, including drought information, together with an interactive map of countries in crisis, available through the fao website. benfield hazard research center uses various data to produce a monthly map of current drought conditions accompanied by a short description for each country. in addition, the wmo provides useful global meteorological information, such as precipitation levels, cloudiness, and weather forecasts, which are visualized on a clickable map on the wmo website. existing approaches for drought early warning must be improved. due to the complex nature of droughts, a comprehensive and integrated approach (such as the one adopted by the us drought monitor) that would consider numerous drought indicators is required for drought monitoring and early warning. in addition, for large parts of the world suffering from severe droughts, early warning systems are not yet in place, such as in western and southern africa, and in eastern africa where fews net is available but no drought forecast is provided. parts of europe (spain, parts of france, southern sweden, and northern poland) are characterized by high drought risk but have no system in place. india, parts of thailand, turkey, iran, iraq, eastern china, areas of ecuador, colombia, and the south-eastern and western parts of australia also require a drought warning system. 3.2.2.2. desertification. the united nations convention to combat desertification (unccd), signed by 110 governments in 1994, aims to promote local action programs and international activities. national action programmes at the regional or sub-regional levels are key instruments for implementing the convention and are often supported by action programmes at sub-regional and regional levels. these programs lay out regional and local action plans and strategies to combat desertification. the unccd website provides a desertification map together with documentation, reports, and briefing notes on the implementation of action programs for each country worldwide. currently no desertification early warning system is fully implemented, despite their potential to mitigate desertification. 3.2.2.3. food security. the fao's giews supports policy-makers by delivering periodic reports through the giews web page and an e-mail service. giews also promotes collaboration and data exchange with other organizations and governments. the frequency of briefs and reports -which are released monthly or bimonthly -may not be adequate for early warning purposes. the wfp is also involved in disseminating reports and news on famine crises through its web service. no active dissemination is provided by wfp. another service is fews net, a collaborative effort of the usgs, united states agency for international development (usaid), nasa, and noaa, which reports on food insecurity conditions and issues watches and warnings to decision-makers. these bulletins are also available on their website. food security prediction estimates and maps would be extremely useful for emergency response, resources allocation, and early warning. the food security and nutrition working group (fsnwg) serves as a platform to promote the disaster risk reduction agenda in the region and provides monthly updates on food security through maps and reports available on its website. fewsnet and fsnwg were instrumental in predicting the food crisis in 2010-2011 in the east african region in a timely manner. nevertheless these early warnings did not lead to early action to address the food crisis. if they had been used adequately, the impacts of the serious humanitarian crisis in the horn of africa could have been partially mitigated (ververs, 2011) . nearly all efforts to cope with climate change or variability focus on either mitigation to reduce emissions or on adaptation to adjust to changes in climate. although it is imperative to continue with these efforts, the on-going pace of climate change and the slow international response suggests that a third option is becoming increasingly important: to protect the population against the immediate threat and consequences of climate-related extreme events, including heat waves, forest fires, floods and droughts, by providing it with timely, reliable and actionable warnings. although great strides have been made in developing climate-related warning systems over the past few years, current systems only deal with some aspects of climate related risks or hazards, and have large gaps in geographic coverage. information exists for melting glaciers, lake water level, sea height and sea surface temperature anomalies, el nino and la nina. the national snow and ice data center (nsidc)-ice concentration and snow extent provides near real-time data on daily global ice concentration and snow coverage. the usda, in cooperation with the nasa and the university of maryland, routinely monitors lake and reservoir height variations for approximately 100 lakes worldwide and provides online public access to lake water level data. information on sea height anomaly (sha) and significant wave height data are available from altimeter jason-1, topex, ers-2, envisat and gfo on a near-real time basis with an average 2-day delay. this information is provided by noaa. additionally, near real-time sea surface temperature (sst) products are available from noaa's goes and poes, as well as nasa's eos, aqua and terra. the international research institute (iri) for climate and society provides a monthly summary of the el nino and la nina southern oscillation, providing forecast summary, probabilistic forecasts, and a sea surface temperature index. however, these systems are still far from providing the coverage and scope that is needed and technically feasible. large parts of the world's most vulnerable regions are still not covered by a comprehensive early warning system. most systems only deal with one aspect of climate-related risks or hazards, e.g., heat waves or drought. finally, most systems do not cover the entire early warning landscape from collection of meteorological data to delivery and response of users. recently, the world meteorological organization proposed a global framework for climate services, which aims to strengthen the global cooperative system for collecting, processing and exchanging observations and for using climate-related information (wmo, 2011). early warning technologies appear to be mature in certain fields but not yet in others. considerable progress has been made, thanks to advances in scientific research and in communication and information technologies. nevertheless, a significant amount of work remains to fill existing technological, communication, and geographical coverage gaps. early warning technologies are now available for almost all types of hazards, although for some hazards (such as droughts and landslides) these technologies are still less developed. most countries appear to have early warning systems for disaster risk reduction. however, there is still a technological and national capacity divide between developed and developing countries. from an operational point of view, some elements of the early warning process are not yet mature. in particular, it is essential to strengthen the links between sectors involved in early warning, including organizations responsible for issuing warnings and the authorities in charge of responding to these warnings, as well as promoting good governance and appropriate action plans. it is generally recognized that it is fundamental to establish effective early warning systems to better identify the risk and occurrence of hazards and to better monitor the population's level of vulnerability. although several early warning systems are in place at the global scale in most countries for most hazard types, there is the need ''to work expeditiously towards the establishment of a worldwide early warning system for all natural hazards with regional nodes, building on existing national and regional capacity such as the newly established indian ocean tsunami warning and mitigation system'' (2005 un world summit outcome). by building upon ongoing efforts to promote early warning, a multi-hazard early warning system will have a critical role in preventing hazardous events from turning into disasters. a globally comprehensive early warning system can be built, based on the many existing systems and capacities. this will not be a single, centrally planned and commanded system, but a networked and coordinated assemblage of nationally owned and operated systems. it will make use of existing observation networks, warning centres, modelling and forecasting capacities, telecommunication networks, and preparedness and response capacities (united nations, 2006) . a global approach to early warning will also guarantee consistency of warning messages and mitigation approaches globally thus improving coordination at a multi-level and multi-sector scale among the different national actors such as the technical agencies, the academic community, disaster managers, civil society, and the international community. the next section provides an analysis of existing global early warning/monitoring systems that aggregate multi-hazard information. this section presents the results of a comparative analysis of multi-hazard global monitoring/ early warning systems. the aim of this analysis is to assess the effectiveness of existing global scale multi-hazard systems and define the set of needs suggested by comparing existing services. it assesses existing monitoring/early warning systems (singh and grasso, 2007) , chosen to be multi-hazard with global coverage, such as wfp's (the un food aid agency's) hews; alertnet, the humanitarian information alert service by reuters; reliefweb, the humanitarian information alert service by un-ocha; gdacs (global disaster alert and coordination system), a joint initiative of the united nations office for the coordination of humanitarian affairs (un-ocha) and the ec-jrc; and usgs ens. these systems have been analysed for the type of events covered, variety of output/ communication forms and range of users served. these systems cover a range of hazards and communicate results using a variety of methods. usgs ens only provides information on earthquakes and volcanic eruptions through the volcano hazards program (vhp) in collaboration with smithsonian institution. reliefweb focuses on natural hazards (earthquakes, tsunamis, severe weather, volcanic eruptions, storms, floods, droughts, cyclones, insect infestation, fires, and technological hazards) and health; alertnet additionally provides information on food insecurity and conflicts. gdacs provides timely information on natural hazards (earthquakes, tsunamis, volcanic eruptions, floods, and cyclones). hews informs users on earthquakes, severe weather, volcanic eruptions, floods, and locusts. existing systems, such as hews, post the information on a website, provide mobile phone and rss services, and gdacs sends emails, sms and faxes to users. the gdacs notification service mostly addresses humanitarian organizations, rescue teams or aid agencies. alertnet provides information to users through a web service, e-mail, sms and reports. reliefweb uses web services, e-mail and reports to disseminate information to users. reliefweb and alertnet also use new communications tools (facebook and twitter). ocha's virtual on-site operations coordination centre (virtual-osocc) enables real-time information exchange by all actors of the international disaster response community during the early phases following disasters. this service has been integrated within gdacs but is restricted to disaster managers. the only natural event notification provided by usgs e-mail service is earthquakes. hews offers no e-mail notifications for natural events. alertnet and reliefweb inform users on natural hazards and on health, food and security issues. usgs, reliefweb, alertnet and gdacs serve a wide a variety of users such as international organizations, humanitarian aid, policy/decision-makers, and civil society (fig. 4) . the optimal global coverage multi-hazard system has to be as comprehensive as possible in terms of content, output and range of users. it will enhance existing systems by streaming data and information from existing sources and it will deliver this information in a variety of user-friendly formats to reach the widest range of users. by building on existing systems the multi-hazard system will inherit both the technological and geographical coverage gaps and limitations of existing early warning systems. the review analysis performed in the section ''inventory of early warning systems'' has shown that for some hazards (such as droughts and landslides) these technologies are still less developed and for tsunamis these systems are still under development for areas at risk. the analysis has shown that there is still a technological and national capacity divide between developed and developing countries. from an operational point of view, the links and communication networks between all sectors involved (organizations responsible for issuing warnings and the authorities in charge of responding to these warnings) need improvement. similarly, good governance and appropriate action plans need to be promoted. overcoming these gaps and enhancing, integrating, and coordinating existing systems is the first priority for the development of a global scale multi-hazard early warning system . early warning technologies have greatly benefited from recent advances in communication and information technologies and an improved knowledge on natural hazards and the underlying science. nevertheless many gaps still exist in early warning technologies and capacities, especially in the developing world, and a lot more has to be done to develop a global scale multi-hazard system. operational gaps need to be filled for slow-onset hazards both in monitoring, communication and response phases. effective and timely decision making is needed for slow onset hazards. below are some recommendations: (1) fill existing gaps: the section 3 identified the weaknesses and gaps in existing early warning systems. technological, geographical coverage, and capacity gaps exist, in addition to operational gaps for slow-onset hazards. in particular, actions need to be taken to improve prediction capabilities for landslides hazard aimed at developing a landslides early warning system. likewise, there is a pressing need to improve existing prediction capabilities for droughts. a global early fire warning system is not yet in place, the tsunami early warning systems for the indian ocean and the caribbean are not yet fully operational and a desertification early warning system has not been developed yet. there are ongoing efforts to develop these systems, such as the gfmc effort for the global fire ews, the indian ocean tsunami warning system operated by indonesia and a noaa led effort in the caribbean. a malaria early warning system is mandatory for africa, where one million deaths occur every year due to malaria. climate variability impacts need to be monitored within a global and coordinated effort, and the global framework for climate services needs to be further elaborated and operationalized. local earthquake early warning systems applications are needed in high seismic risk areas, where early warning systems are not yet in place. air quality and flood systems require improvements in prediction capabilities. dust storms and transboundary early warning systems do not yet exist. a coordinated volcanic early warning system that would integrate existing resources is also needed as well as an increase in coverage of volcanic observatories. particular attention should be paid to fill gaps in decision making processes for slow-onset hazards. their extent and impact are challenging to quantify. for this reason, actions and response are far more difficult tasks for slow-onset hazards than they are for other natural hazards. an institutional mechanism to regularly monitor and communicate slow-onset changes is needed to keep changes under review and to enable rational and timely decisions to be taken based on improved information. (2) build capacity: the evaluation study of existing early warning systems (the section 3) highlighted that a technological divide between developed and developing countries still exists. it is critical to develop basic early warning infrastructures and capacities in parts of the developing world most affected by disasters; it is also important to promote education programs on disaster mitigation and preparedness and integrate disaster mitigation plans into the broader development context. poor countries suffer greater economic losses from disasters than rich countries. development plays a key role and has a significant impact on disaster risk. almost 85 percent of the people exposed to the deadliest hazards, earthquakes, floods, cyclones and droughts live in the developing world. the impact of disasters is mostly influenced by previous development choices. by integrating disaster mitigation strategies into planning and policies, the effects of disasters can be sensibly reduced and managed. ''disaster risk is not inevitable, but on the contrary can be managed and reduced through appropriate development actions'' (united nations development programme-undp, 2004) . it is through ''risk-sensitive development planning that countries can help reduce disaster risks''. key targets for capacity building include the following: 1. developing national research, monitoring and assessment capacity, including training in assessment and early warning. 2. supporting national and regional institutions in data collection, analysis and monitoring of natural and man-made hazards. 3. providing access to scientific and technological information, including information on stateof-the-art technologies. 4. education and awareness-raising, including networking among universities with programmes of excellence in the field of the emergency management. 5. organizing of training courses for local decision-makers and communities. 6. bridging the gap between emergency relief and long-term development. (3) bridge the gaps between science and decision making, and strengthen coordination and communication links: scientific and technological advances in modelling, monitoring and predicting capabilities could bring immense benefits to early warning if science were effectively translated into disaster management actions. bridging the gap between scientific research and decision making will make it possible to fully exploit capacities of early warning technologies for societal benefit. the major challenge is to ensure that early warnings result in prompt responses by governments and potentially the international community. this requires that information be effectively disseminated in an accessible form down to the end user. this is achievable by adopting standard formats and easy-to-use tools for information dissemination, such as interactive maps, emails, sms, etc. the adoption of standard formats (such as the common alerting protocol cap) for disseminating and exchanging information has to be promoted. the advantage of standard format alerts is their compatibility with all information systems, warning systems, media, and most importantly, with new technologies such as web services. the adoption of standard formats guarantees consistency of warning messages and is compatible with all types of information systems and public alerting systems, including broadcast radio and television as well as public and private data networks, with multi-lingual warning systems and emerging technologies. this would easily replace specific application oriented messages and will allow the merging of warning messages from several early warning systems into a single multihazard message format. finally, it is critical to strengthen coordination and communication links by defining responsibility mechanisms and appropriate action plans. more often, time-sequenced warning messages are released in early warning processes, implying a decrease in warning times available for action and in reliability of the information. this trade-off needs to be addressed. the content and views expressed in this publication are those of the authors and do not necessarily reflect the views or policies, or carry the endorsement of the contributory organizations or the united nations environment programme (unep). the designations employed and the presentation of material in this publication do not imply the expression of any opinion whatsoever on the part of unep concerning the legal status of any country, territory or city or its authorities, or concerning the delimitation of its frontiers and boundaries. reference to a commercial company or product in this publication does not imply the endorsement of unep. & maps, photos, and illustrations as specified. this publication may be reproduced in whole or in part and in any form for educational or non-profit purposes without special permission from the copyright holder, provided acknowledgement of the source is made. unep would appreciate receiving a copy of any publication that uses this publication as a source. no use of this publication may be made for resale or any other commercial purpose whatsoever without prior permission in writing from unep. applications for such permission, with a statement of purpose and intent of the reproduction, should be addressed to the director, division of communications and public information (dcpi), unep, p.o. box 30552, nairobi 00100, kenya. the use of information from this publication concerning proprietary products for publicity or advertising is not permitted. citation: unep (2012 adaptation: (1) actions taken to help communities and ecosystems cope with changing climate conditions (unfccc). (2) genetically determined characteristic that enhances the ability of an organism to cope with its environment (cbd). aerosols: a collection of airborne solid or liquid particles, with a typical size between 0.01 and 10 mm, that resides in the atmosphere for at least several hours. aerosols may be of either natural or anthropogenic origin. air quality: smog is the product of human and natural activities such as industry, transportation, wild-fires, volcanic eruptions, etc. and can have serious effects on human health and the environment. us epa uses and air quality index (aqi) which is calculated on five major air pollutants regulated by the clean air act: ground-level ozone, particle pollution (also known as particulate matter), carbon monoxide, sulfur dioxide, and nitrogen dioxide. for each of these pollutants, epa has established national air quality standards to protect public health. groundlevel ozone and airborne particles are the two pollutants that pose the greatest threat to human health in this country. biodiversity: the variety of life on earth, including diversity at the genetic level, among species and among ecosystems and habitats. it includes diversity in abundance, distribution and in behaviour. biodiversity also incorporates human cultural diversity, which can both be affected by the same drivers as biodiversity, and itself has impacts on the diversity of genes, other species and ecosystems. biofuels: fuel produced from dry organic matter or combustible oils from plants, such as alcohol from fermented sugar, black liquor from the paper manufacturing process, wood and soybean oil. biomass: organic material, both above ground and below ground, and both living and dead, such as trees, crops, grasses, tree litter and roots. capacity: the combination of all the strengths, attributes and resources available within a community, society or organization that can be used to achieve agreed goals. comment: capacity may include infrastructure and physical means, institutions, societal coping abilities, as well as human knowledge, skills and collective attributes such as social relationships, leadership and management. capacity also may be described as capability. capacity assessment is a term for the process by which the capacity of a group is reviewed against desired goals, and the capacity gaps are identified for further action. capacity building: process of developing the technical skills, institutional capability, and personnel. climate change: change of climate, which is attributed directly or indirectly to human activity that alters the composition of the global atmosphere and which is in addition to natural climate variability observed over comparable time periods. climate variability: variations in the mean state and other statistics (such as standard deviations and the occurrence of extremes) of the climate on all temporal and spatial scales beyond that of individual weather events. variability may be due to natural internal processes in the climate system (internal variability), or to variations in natural or anthropogenic external forcing (external variability). common alerting protocol: the common alerting protocol (cap) provides an open, nonproprietary digital message format for all types of alerts and notifications. it does not address any particular application or telecommunications method. the cap format is compatible with emerging techniques, such as web services, as well as existing formats including the specific area message encoding (same) used for the united states' national oceanic and atmospheric administration (noaa) weather radio and the emergency alert system (eas). cost-benefit analysis: a technique designed to determine the feasibility of a project or plan by quantifying its costs and benefits. cyclone: an atmospheric closed circulation rotating counter clockwise in the northern hemisphere and clockwise in the southern hemisphere. deforestation: the direct human-induced conversion of forested land to non-forested land. desertification: degradation of land in arid, semi-arid and dry sub-humid areas, resulting from various factors, including climatic variations and human activities. disaster: a serious disruption of the functioning of a community or a society involving widespread human, material, economic or environmental losses and impacts, which exceeds the ability of the affected community or society to cope using its own resources. comment: disasters are often described as a result of the combination of: the exposure to a hazard; the conditions of vulnerability that are present; and insufficient capacity or measures to reduce or cope with the potential negative consequences. disaster impacts may include loss of life, injury, disease and other negative effects on human physical, mental and social well-being, together with damage to property, destruction of assets, loss of services, social and economic disruption and environmental degradation. disaster risk: the potential disaster losses, in lives, health status, livelihoods, assets and services, which could occur to a particular community or a society over some specified future time period. comment: the definition of disaster risk reflects the concept of disasters as the outcome of continuously present conditions of risk. disaster risk comprises different types of potential losses which are often difficult to quantify. nevertheless, with knowledge of the prevailing hazards and the patterns of population and socio-economic development, disaster risks can be assessed and mapped, in broad terms at least. disaster risk reduction: the concept and practice of reducing disaster risks through systematic efforts to analyse and manage the causal factors of disasters, including through reduced exposure to hazards, lessened vulnerability of people and property, wise management of land and the environment, and improved preparedness for adverse events. comment: a comprehensive approach to reduce disaster risks is set out in the united nations-endorsed hyogo framework for action, adopted in 2005, whose expected outcome is ''the substantial reduction of disaster losses, in lives and the social, economic and environmental assets of communities and countries.'' the international strategy for disaster reduction (isdr) system provides a vehicle for cooperation among governments, organisations and civil society actors to assist in the implementation of the framework. note that while the term ''disaster reduction'' is sometimes used, the term ''disaster risk reduction'' provides a better recognition of the ongoing nature of disaster risks and the ongoing potential to reduce these risks. droughts: a period of abnormally dry weather sufficiently prolonged for the lack of water to cause serious hydrologic imbalance in the affected area. early warning system: the set of capacities needed to generate and disseminate timely and meaningful warning information to enable individuals, communities and organizations threatened by a hazard to prepare and to act appropriately and in sufficient time to reduce the possibility of harm or loss. comment: this definition encompasses the range of factors necessary to achieve effective responses to warnings. a people-centred early warning system necessarily comprises four key elements: knowledge of the risks; monitoring, analysis and forecasting of the hazards; communication or dissemination of alerts and warnings; and local capabilities to respond to the warnings received. the expression ''end-to-end warning system'' is also used to emphasize that warning systems need to span all steps from hazard detection through to community response. earth observation: earth observation, through measuring and monitoring, provides an insight and understanding into earth's complex processes and changes. eo include measurements that can be made directly or by sensors in-situ or remotely (i.e. satellite remote sensing, aerial surveys, land or ocean-based monitoring systems, fig. 3 ), to provide key information to models or other tools to support decision making processes. earthquakes: earthquakes are due to a sudden release of stresses accumulated around the faults in the earth's crust. this energy is released through seismic waves that travel from the origin zone, which cause the ground to shake. severe earthquakes can affect buildings and populations. the level of damage depends on many factors such as intensity of the earthquake, depth, vulnerability of the structures, and distance from the earthquake origin. ecosystem: dynamic complex of plant, animal, microorganism communities and their non-living environment, interacting as a functional unit. ecosystems are irrespective of political boundaries. el niñ o-southern oscillation: a complex interaction of the tropical pacific ocean and the global atmosphere that results in irregularly occurring episodes of changed ocean and weather patterns in many parts of the world, often with significant impacts over many months, such as altered marine habitats, rainfall changes, floods, droughts, and changes in storm patterns. comment: the el nino part of the el nino-southern oscillation (enso) phenomenon refers to the well-above average ocean temperatures that occur along the coasts of ecuador, peru and northern chile and across the eastern equatorial pacific ocean, while la nina part refers to the opposite circumstances when well-below-average ocean temperatures occur. the southern oscillation refers to the accompanying changes in the global air pressure patterns that are associated with the changed weather patterns experienced in different parts of the world. emergency management: the organization and management of resources and responsibilities for addressing all aspects of emergencies, in particular preparedness, response and initial recovery steps. comment: a crisis or emergency is a threatening condition that requires urgent action. effective emergency action can avoid the escalation of an event into a disaster. emergency management involves plans and institutional arrangements to engage and guide the efforts of government, non-government, voluntary and private agencies in comprehensive and coordinated ways to respond to the entire spectrum of emergency needs. the expression ''disaster management'' is sometimes used instead of emergency management. extensible markup language: a markup language that defines a set of rules for encoding documents in a format that is both human-readable and machine-readable. e-waste: a generic term encompassing various forms of electrical and electronic equipment that has ceased to be of value and is disposed of. a practical definition of e-waste is ''any electrically powered appliance that fails to satisfy the current owner for its originally intended purpose.'' false alarm: in the context of early warning systems, a false alarm is defined as the situation in which an alarm is activated when it should not have been. fine particle: particulate matter suspended in the atmosphere less than 2.5 mm in size (pm2.5). floods: an overflow of water onto normally dry land. theinundation of a normally dry area caused by rising water in an existing waterway, such as a river, stream, or drainage ditch. ponding of water at or near the point where the rain fell. flooding is a longer term event than flash flooding: it may last days or weeks. floods are often triggered by severe storms, tropical cyclones, and tornadoes. food security: when all people at all times have access to sufficient, safe, nutritious food to maintain a healthy and active life. forecast: definite statement or statistical estimate of the likely occurrence of a future event or conditions for a specific area. comment: in meteorology a forecast refers to a future condition, whereas a warning refers to a potentially dangerous future condition. forest: land spanning more than 0.5 ha with trees higher than 5 m and a canopy cover of more than 10 percent, or trees able to reach these thresholds in situ. it does not include land that is predominantly under agricultural or urban land use. gaussian distribution: the gaussian (normal) distribution was historically called the law of errors. it was used by gauss to model errors in astronomical observations, which is why it is usually referred to as the gaussian distribution. geological hazard: geological process or phenomenon that may cause loss of life, injury or other health impacts, property damage, loss of livelihoods and services, social and economic disruption, or environmental damage. comment: geological hazards include internal earth processes, such as earthquakes, volcanic activity and emissions, and related geophysical processes such as mass movements, landslides, rockslides, surface collapses, and debris or mud flows. hydrometeorological factors are important contributors to some of these processes. tsunamis are difficult to categorize; although they are triggered by undersea earthquakes and other geological events, they are essentially an oceanic process that is manifested as a coastal water-related hazard. within this report, tsunamis are included in the geological hazards group. geographic information system: a computerized system organizing data sets through a geographical referencing of all data included in its collections. the potential for earthquake early warning in southern california oregon department of environmental quality. water quality credit trading in oregon: a case study report disaster management support group (dmsg) ceos disaster management support group report. the use of earth observing satellites for hazard support: assessments & scenarios. final report of the ceos disaster management support group natural disaster hotspots: a global risk analysis mexico city seismic alert system environmental change monitoring by geoinformation technology for baghdad and its neighboring areas geoss 10-year implementation plan reference document early warning systems early warning of wildland fires usable science: early warning systems: do's and don'ts automated decision procedure for earthquake early warning automated decision procedure for earthquake early warning a model for a seismic computerized alert network an automatic processing system for broadcasting earthquake alarms global observations of the land, a proposed theme to the igos-partnership-version 2 probability theory: the logic of science jma earthquake early warning real-time seismology and earthquake damage mitigation global forest fire watch: wildfire potential, detection, monitoring and assessment oil spill detection and monitoring from satellite image on a rational strong motion index compared with other various indices the role of a new method of quickly estimating epicentral distance and magnitude from a single seismic record prediction, science, decision making and the future of nature university of valencia,eos.d2c. chinese academy of forestry effective early warning -use of hazard maps as a tool for effective risk communication among policy makers and communities -session organized by the government of japan global environmental alert service digital change detection techniques using remotely sensed data the drought monitor ict in pre-disaster awareness and preparedness. presented at the apt-itu joint meeting for ict role in disaster management early warning systems for natural disasters reduction undp-united nations development programme bureau for crisis prevention and recovery united nations convention to combat desertification the east african food crisis: did regional early warning systems function climate knowledge for action: a global framework for climate services-empowering the most vulnerable quick and reliable determination of magnitude for seismic early warning a virtual sub-network approach to earthquake early warning key: cord-005191-a70eedna authors: cohen, irun r. title: informational landscapes in art, science, and evolution date: 2006-06-08 journal: bull math biol doi: 10.1007/s11538-006-9118-4 sha: doc_id: 5191 cord_uid: a70eedna an informational landscape refers to an array of information related to a particular theme or function. the internet is an example of an informational landscape designed by humans for purposes of communication. once it exists, however, any informational landscape may be exploited to serve a new purpose. listening post is the name of a dynamic multimedia work of art that exploits the informational landscape of the internet to produce a visual and auditory environment. here, i use listening post as a prototypic example for considering the creative role of informational landscapes in the processes that beget evolution and science. invites us to explore it-physically or in reverie. a landscape of associations weaves a narrative. an informational landscape denotes an array of information that, like a natural landscape, invites exploration; the informational landscape too holds a narrative. here, i shall use listening post as an allegory to explore two other systems that deal with informational landscapes: biologic evolution and human understanding. waddington has used the term epigenetic landscape as a metaphor to describe the interactions between genes and environment that take place during embryonic development (waddington, 1940) . an informational landscape is quite another matter; this landscape represents the maze of information available for potential exploitation by a suitable system. as we shall see below, the informational landscape is a substrate for system-making. let's start by seeing how listening post exploits information to organize a work of art. listening post is formed by two components: an overt visual-auditory display designed by artist rubin and a covert process designed by mathematician hansen. the display is composed of a suspended rectangular grid of over 200 brick-sized electronic monitors and a set of audio installations (fig. 1) . the monitors pulsate with fragments of texts and compositions of light and the sound tracks pulsate with musical passages and artificial speech. the display triggers living associations: "a sense of cycles in life, day and night, the seasons. . .the information. . .lighting up as if the world is awakening from sleep and later changing to large sweeps." (eleanor rubin, personal communication; see, http//www.ellyrubin.com). the covert process that produces listening post's art is an algorithm developed by hansen, then a mathematician at bell laboratories. the algorithm randomly samples, in real time, the many thousands of chats, bulletin boards, and bits of message that flow dynamically through the cyberspace of the internet. this simultaneous mélange of signals, in the aggregate, is meaningless noise. the algorithm, by seeking key words and patterns of activity, artfully exploits this raw information to construct patterns of light, sound, and words that please human minds. the substrate of information flowing over the internet is in constant flux so the patterns presented by listening post are unpredictable at the fine microscopic scale; but at the macroscopic scale of sensible experience, listening post is manifestly pleasing. the patterned flow of sights, sounds, and words (seen and heard) arouses associations, memories, and feelings in the mind of the viewer-which is what we call art: art, whether visual, auditory, tactile, gustatory, or verbal, is an artifact made to arouse associations, memories, and feelings. listening post is an artful representation of the complexity of information flowing through the internet; listening post transforms the internet's massive informational landscape into a comprehensible miniature. two attributes of listening post illustrate our theme: the work feeds on information designed for other purposes and it survives by engaging our minds. above, i have used four words-information, signal, noise, and meaning-that usually need no definition; we use them every day. but they are important to this discussion, so i will define them. i use the word information in the sense defined by shannon (1948) : information is a just-so arrangement-a defined structure-as opposed to randomness (cohen, 2000) . information becomes a signal if you respond to the information. the meaning of the signal is how you respond to it (atlan and cohen, 1998) . for example, a chinese character bears information (it is arranged in a characteristic form), but the character is not a signal unless you know how to read chinese. what the chinese character means is the way the community of chinese readers use the character and respond to it. information, then, is an intrinsic property of form. in biology, form is expressed by the characteristic structures of molecules, cells, organs, organisms, populations, and societies. informational structures are not limited to material entities: organizations and processes through their defining regularities also fashion informational structures. an informational landscape encompasses a totality of information-material and organizational. information expressed in biological structures is essential to life, but structure alone is not sufficient for living. biological information is collective and reproducible-the structural information repeats itself at various scales, from molecules through societies (and beyond into human culture). the information encoded in biological structures persists and cycles; we call it development, physiology, and reproduction. but most importantly, biological structures perform functions-biological information bears meaning (neuman, 2004) . meaning, in contrast to information, is not an intrinsic property of an entity (a word or a molecule, for example); the meaning of an entity emerges from the interactions of the test entity (the word or molecule) with other entities (for example, words move people, and molecules interact with their receptors, ligands, enzymes, etc.). interactions mark all manifestations of biological structure-molecular through social. meaning can thus be viewed as the impact of information-what a word means is how people use it; what a molecule means is how the organism uses it; what an organism means is what it does to others and how others respond to it; and so on over the scales life-meaning is generated by interaction (cohen, 2000) . in summary, we can connect the three conceptsinformation, signal, and meaning thusly: a signal is information with meaning. in other words, signalling is the ability of information to elicit a response, which is the meaning of the information. note that a signal, like information in general, is free of intentions; astrophysicists, for example, receive signals from distant galaxies, which have no interest in communicating with an astrophysicist; it is the response of the astrophysicist to the galactic radiation (meaning-making) that transforms the structure of the radiation (its information) into a signal. noise is more varied than signal. noise can refer to a number of different situations: randomness as opposed to structured information (a scribble versus a chinese character); information without meaning (a greek conversation overheard by one who does not understand greek); meaningful information in a meaningless context (across-the-room cocktail-party chatter). noise in the generic sense proposed by shannon is the unstructured; but noise can also refer to meaningless information that might be meaningful in other circumstances. for example, a dna sequence composing an immunoglobulin v gene element has little meaning until the sequence is recombined with other dna sequences to form a functioning antibody gene (cohen, 2000) -that recombination charges the dna sequence with meaning. whether cyberspace transmits noise or signal, as we have defined the terms, depends on who you are and what you seek. if you are the recipient of a specific bit of information-a chat or an email directed to you, for example-then you perceive signal. but if you don't understand the language, or if you do understand the language but see intruding spam, or experience a profusion of many messages at once, then you hear only noise. (noise too can have meaning if it makes you leave the room). the difference then between signal and noise, one might claim, is having the right reception. or, to put it another way, the same information can be either noise or signal, depending on how you perceive it-or transmit it. combatants may attempt to evade detection by disguising signals as noise; for example, humans encrypt secret transmissions (kahn, 1996) and infectious agents scramble antigens (cohen, 2000; cohen, 2003) . the transformation of noise back into signal is part of the game; counter-intelligence can learn to break the enemy's linguistic codes; an immune system can learn to decipher a pathogen's molecular codes. in informational terms, listening post is a machine that transforms noisy cyberspace information into a new narrative by selecting and recombining fragments of the flux. listening post dynamically self-organizes, similar to a living organism (atlan, 1987; weisbuch and solomon, 2002) . the internet created a new informational landscape, a new niche, that could be sampled and exploited by hansen and rubin to enhance their fitness as artists in the wilds of the manhattan art world (fig. 2) . biological evolution traditionally is described in terms of fitness: species evolve because the fittest survive to pass on their genes, while the unfit die with their genes (darwin, 1859). survival-of-the-fittest thus selects the "best" genetic variants from among the phenotypes in the breeding population (plotkin, 1994) . the process leads, with time, to creatures that are better adapted and improved. improved fitness is the aim of evolution, so say some experts. but there are at least two problems with the concept of fitness: first, fitness is difficult to define; most definitions involve a circular argument-what survives is fit, by definition (gould and lewontin, 1979) . this amounts to a tautology: fit is fit. attempts have been made to define fitness in terms of reproductive success (hoffman et al., 2004) . but different species survive well despite vastly different numbers of surviving offspring: compare the reproductive strategy of the elephant with that of the gnat that rides on its back; compare the whale with the sardine; each to its own reproductive profligacy or dearth. second, evolution does not rest with fit creatures; evolution assembles increasingly complex creatures. accumulating complexity is manifest in the evolutionary tree. creatures higher in the evolutionary tree-more recent creatures-tend to be more complex than the creatures that preceded them: eukaryotic cells deploy more genes and house more organelles than do prokaryotic cells; mammals have more organs and express more behaviors than do the trees, round worms, or insects that preceded them. quite simply, evolution generates complexity (fig. 3) . now, one might argue that the more complex species is the more fit species; if that is true, then the quest for fitness alone should generate increasing complexity. but is that true; does fitness itself drive complexity onward? what is complexity? complexity is a relative term: more complex entities compared to simpler entities incorporate more component parts and integrate more diverse interactions between their component parts. moreover, the single component parts of complex living systems usually participate in a variety of different functions (pleiotropism). complexity thus can be defined in terms of information; complex systems sense, store, and deploy more information than do simple systems. complexity presents two aspects: intrinsic and extrinsic. a complex system such as a cell, brain, organism, or society is complex intrinsically because of the way its parts interact and hold the system together. a complex system is also complex extrinsically because we who study it have difficulty understanding the properties that emerge from it (the biological perspective); we also have trouble fixing it (the medical perspective). evolution. the environment constitutes an informational landscape of considerable complexity (designated by the bundles of arrows) that in the aggregate is not readily useful (note the puzzled face, ?). however, a creature with a suitable physiology can extract useful information (a limited bundle of information) and the creature's new phenotype is subject to selection. survival can lead to a new species (new meaning) and so the complexity of the informational landscape is amplified and enriched through positive feedback. the new species too becomes an informational landscape for further exploitation by parasites and yet newer species. fitness, unlike complexity, does not relate to the way a system is put together or the way the system is understood by those who study it. fitness relates to the success of a system in thriving in its own world. we can conclude, therefore, that fitness and complexity describe independent attributes. so there is no necessary correlation between complexity and fitness: no dinosaurs have survived irrespective of how complex they were (and some were very complex indeed). in fact, it seems that the dinosaurs were unfit because they were too complex to survive the environmental disruptions brought about by the earth's collision with a comet (morrison, 2003) . primitive bacteria have survived such calamities despite their relative simplicity (probably because of their relative simplicity). indeed, the lowest and simplest bacteria will probably prove to be more fit for survival than we more complex humans if we both have to face a really severe change in the world environment. extremely complex systems, like us, are extremely fragile, and so they are less fit in certain situations. the bottom line is that the quest for fitness cannot explain the rise of complexity. but why then is complexity usually-but not always-more evident the higher one climbs up the evolutionary tree? it has been possible to demonstrate the evolution of complexity mathematically (chaitin, 1998; wolfram, 2002; lenski et al., 2003) . but evolution on a computer (in silico) is not evolution in the world of nature (in mundo). if complex systems tend to be fragile, why does evolution persist in devising them? the accumulation of complexity during evolution can be explained, despite fragility, by a principle of self-organization; the principle, formulated by atlan, is that existing information tends automatically to breed additional new information (atlan, 1987 ). atlan's argument goes like this: existing information first generates surplus copies of itself, which happens regularly in reproducing biological systems. the surplus copies can then safely undergo mutations, and so create modified (new), added information without destroying the untouched copies of the old information. the system thus becomes enriched; it now contains the new information along with the old information. indeed, it appears that the complexity of vertebrate evolution was preceded and made possible by a seminal duplication of the ancestral genome (dehal and boore, 2005 ). atlan's formulation implies that the more structures (the more information) a system encompasses, the greater the likelihood of variation in that system (discussed in cohen, 2000) . hence, the amount of existing information composing a system (previously evolved structures) breeds a commensurate amount of new information (variant structures). note that the constant variation of existing information is guaranteed by the second law of thermodynamics-order tends automatically to dissipate into variant structures; hence, any information will mutate over time (fig. 4) . information, in other words, feeds back on itself in a positive way; a great amount of information, through its variation, leads to even more information. and as information varies it increases, and so does complexity. once a relatively stable, nonrandom structure (an informational entity) comes into being, it will be subject to variation. all genes, for example, mutate; proteins assume different conformations and functions; minds get new ideas. repositories of information like genes, proteins, minds, cultures, and so forth, vary to generate new genes, proteins, minds, or cultures that then get exploited for additional uses. a human is manyfold more complex than is a round worm, yet the human bears less than twice the number of genes borne by the worm (about 35,000 human genes compared to about 20,000 analogous worm genes). the human species has accumulated its characteristic complexity by using its set of gene products in much more complicated ways than does the worm. humans exploit a more complex informational landscape than do round worms. humans are also more fragile: the columbia space shuttle that disintegrated upon re-entry into the atmosphere in 2003 carried both humans and round worms; only the round worms survived. the difference between classical darwinian evolution and the idea of evolving informational landscapes is highlighted by the difference between selection and exploitation. darwin, familiar with the art of animal husbandry, applied to nature the atlan (1987) , the net amount of information in a system will increase only if two conditions are satisfied: the original information duplicates to produce surplus copies and the surplus copies mutate to produce variant new information. system x, for example, has not duplicated its information (triangle); even if the triangle mutates, there will be no net increase in information, since the original information is merely replaced (n = n). system y, in contrast, has duplicated its original information (both triangle and box), and so mutation of each surplus copy generates a net increase in information. also note that information accelerates logarithmically (log2): in system y, two mutations can give rise to n × 4 complexity. a system that starts out with more information (system y) will generate new information faster than does a system that starts with less information (system x). wisdom of artificial selection; the wise breeder selects for propagation from among the variant household creatures those individuals most fitting market or household needs. likewise, nature has selected for propagation those creatures most fit for survival-natural selection recapitulates artificial selection (darwin, 1859). my point here is that the natural informational landscape, in contrast to the 19th century english manor, does not merely provide grounds for selecting what fits the market or the whims of the landlord; the natural informational landscape provides grounds for extravagant exploitation. any organism, simple or complex, that manages to mine the landscape for enough energy and information to create meaning (through productive interactions) might manage to survive there. exploitation, as i use the term here, refers to the modification of information for new uses. listening post arose by exploiting the new informational landscape provided by the internet, which itself arose by exploiting other informational landscapeslanguage, computers, society, culture. let us extend the listening post metaphor and say that biologic evolution proceeds through the inevitable exploitation of new informational landscapes by new or variant creatures. evolving and evolved creatures themselves become part of an enriched informational landscape, continuously available for further exploitation by other creatures. the dynamics of evolution are the dynamics of information. like the algorithm of listening post, an evolving species creates new meaning by exploiting information flowing through its environment-its cyberspace. that in a nutshell is the informational view of evolution. information exploits information and compounds information to generate new meanings; life is constant ferment. the escalation of complexity is evident medically. consider parasitism: there exists no creature that is not exploited by some other creature. hosts are informational landscapes for raising parasites. indeed, the biosphere is one great food chain; each species makes a living by exploiting the information (structures and processes) of other creatures. the generative informational landscape includes even the artifacts of cultural evolution, and not only the natural products of chemical and cellular evolution. the human brain generated language; language generated culture; and culture is changing the world. the evolution of human air travel-a complex of machines, logistics, economics, and habits of mind designed for anything but parasites-contributed to the informational landscapes that generated the spread of hiv and west nile virus; poultry farming gave rise to sars and avian influenza; air conditioning systems provided a landscape for legionnaires' disease. information, because of positive feedback, breeds more information, and complexity results. note that positive feedback will accelerate any process; witness the accelerating complexity that marks our world: 100,000 years of hunting and gathering was replaced by 10,000 years of agriculture and animal husbandry that generated a thousand years of accelerated urbanization, 200 years of industrialization, a few decades of informational civilization, and an emerging global economic culture. unfortunately, positive feedback, unless regulated by negative feedback or other kinds of control, accelerates into instability, chaos, and disaster (robertson, 1991; segel and bar-or, 1999) . information that breeds ever more complex systems is dangerous-like the dinosaurs, an overly complex system collapses of its own inherent fragility, and complexity has to again re-start its evolution (fig. 5) . consider an example closer to home: your personal computer crashes when you try to run too many programs simultaneously; the more complex your program, the more often you have to push restart. (being smarter than dinosaurs, we might use our foresight to prevent global warming, over-population, collective terror, and who knows what else; let us avoid having to restart a world.) in summary, we can say that complexity is managed throughout evolution by a balance between two opposing forces: autocatalysis and fragility. on the one hand, complexity inexorably increases in time through the autocatalytic force of increasing information. on the other hand, catastrophic extinctions mark the fragility of large complex ecosystems; on a lesser scale, complexity may also be held in check or reduced by the day-to-day survival of the fittest creature that may be the less complex creature. darwin's concept of natural selection, including survival of the fittest, does play a critical role in the process of evolution, but mostly after a new or variant species has begun to exploit an informational landscape. quite simply, the species has to survive long enough to maintain itself. indeed, the informational landscape might include other species competing for the same sources of information and energy; fig. 4 ) over evolutionary time (t) leads to intrinsically fragile complex systems (c) that are susceptible to crash when challenged by severe environmental perturbations. complexity then has to restart its accumulation from a lower level. the scales and the form of the curve shown here are hypothetical. a species' fitness may be quantified as the measure of time occupied by that species from its origin to its extinction. this formulation avoids the difficulties of identifying factors common to all the various species with their vastly different strategies for survival (reproduction rate, lifespan, efficiency of energy use, etc.); to quantify the fitness of a species, we merely measure its survival time. in the hypothetical figure, we compare the fitness of a hypothetical bacterium that continues to survive for some 5 × 10 8 years compared to a hypothetical dinosaur species that survived for some 10 7 years till its extinction about 65 × 10 6 years ago. the human species arose some 10 5 years ago, and who knows how long it will last. the figure suggests that there is no positive correlation between complexity (c) and fitness; the opposite might be the case. in that case, survival of the fittest is an apt description of the conflict between the competitors. but much of evolution occurs without competition. in fact it is clear that many products of evolution are neutral; they manifest no selective advantage over other phenotypes (kimura, 1983) . neutral evolution simply expresses the exploitation of an informational landscape: survival of the fittest is not an explanation for neutral evolution. fitness, then, is only another word for survival (so, as i said, survival of the fittest is a tautology). the measure of a species' fitness can be assessed most clearly by hindsight, by observing the species' past history of success. a species that manages to last only a short time in its informational landscape is manifestly less fit than is a species that lasts a longer time in its informational landscape. thus the fitness of a species is commensurate with the amount of time the species has persisted from its inception to its extinction, its crash. fitness can be measured quantitatively by the amount of time needed by the environment to eradicate the species-by the elapsed time to unfitness, the time to extinction (fig. 5) . this notion of fitness will be elaborated elsewhere; the point i want to make here is that fitness is merely the temporary absence of unfitness. fitness, in other words, can be reduced to mere survival. the offspring of fitness is taught classically to be improvement. listening post shows us that the evolution of new arrangements of information (new complexity) may not necessarily lead to improvement. hansen and rubin use new information to create a new art; is listening post an improvement over rembrandt's old art? homo sapiens is certainly more artistically pleasing than is e. coli or c. elegans, but hardly better adapted (cohen, 2000) . indeed, in the world of art, fitness has always been measured by survival time; it's only natural. rembrandt's paintings have thrived for hundreds of years and will certainly continue to live with us. influenced by the beauty of newtonian mechanics, biologists have long embraced the hope that if we could inspect the ultimate parts of an organism, we would be able to reduce the complexity of the organism to simple principles-the aim of ultimate understanding. the organism just had to be some sort of machine, however complicated. now we have almost achieved our wish and are close to seeing all the parts of the organism (the genome, the proteome) out on the table. but to our dismay, the organism is not just a complicated clock. even solving the genome project has not graced us with ultimate understanding (cohen and atlan, 2003) . the organism clearly is not a collection of wheels and cogs; the organism is more akin to cyberspace. in place of electromagnetic codes generated by computer networks, the information flowing within and through the cell-life's subunit-is encoded in molecules. but the informational structure of both networks, cell and internet, is similar: each molecule in a cell, like a chat box signal, emerges from a specific origin, bears an address, and carries a message. our problem is that the cell's molecules are not addressed to our minds, so we don't understand them. the mind exists on a different scale than does the cell; the mind and the cell live in different informational landscapes. we are unable to directly see molecular information; we have to translate the cell's molecules and processes into abstract representations: words, numbers, and pictures. the cell looks to us like a seething swarm of molecules, large and small, that appear redundant, pleiotropic, and degenerate (cohen, 2000) . every ligand signals more than one receptor, every receptor binds more than one ligand; every species of molecule has more than one function; and every function seems to be carried out redundantly by different agents. causes cannot be reduced to simple one-to-one relationships between molecules; biologic causality seems more like a complex pattern or web of interactions. the flowing patterns of subunit molecules in the cell, like aggregates of internet signals, make no obvious sense to us, the outside observers. we stand before the cyberspace of the cell and the maelstrom of information that confronts us becomes noise (fig. 6) . the more information we gather, the more confused we become. the flow of information generated by the living cell, viewed from our scale, is turbulence. how can we understand living matter when its complexity exceeds the ability of our minds to remember and process the mass of accumulating information? intrinsic complexity (the organism) leads to extrinsic complexity (our confusion). the informational landscape of the cell-organism-species-society is like the informational landscape of the internet; viewed in the aggregate it is incomprehensible noise. true, minds can be helped by computers. but understanding a cell is not to be had merely by reproducing in silico the complexity of a real cell, even if that were possible. cataloguing the molecules and their connections to simulate the cell on a computer is a useful way to begin. but precise specification is not enough. human understanding is not mere representation-linguistic, mathematical, visual, or auditory; understanding is the exercise of proficiency (cohen, 2005; efroni et al., 2005) . we understand a thing when we know how to interact with it and use it well. thus, we understand complex information by transforming it, as does listening post, into a meaningful signal. we understand complexity by learning to respond to it in new and productive ways; information triggers new thoughts. human understanding is not a static state of knowledge; human understanding is a creative process of interaction with the world (fig. 6) . the information present in the world is a fertile landscape for growing ideas. this functional view of understanding fits the definition of science proposed by the scientist and educator james b. conant, president of harvard university from 1933 to 1953 is "an interconnected series of concepts and conceptual schemes that have developed as the result of experimentation and observation and are fruitful for further experimentation and observation" (conant, 1951) . science, according to conant, is a self-perpetuating process: scientific understanding amounts to doing good experiments that lead to more ideas for better experiments (and leading, in the case of needs, to better solutions). so to understand the cell means to turn its complexity into a metaphor that productively stimulates our minds to devise better experiments, ideas, and treatments. classically, we have been taught that science is driven by the formulation of hypotheses and by experiments designed to discredit them (popper, 2002) . a hypothesis that has been falsified experimentally is then replaced by a modified hypothesis. the new, modified hypothesis too is tested by experimentation and its falsification leads to a third hypothesis. thus, science advances ideally toward the truth by continuously adjusting its hypotheses through experimentation. unfortunately, this description of science may be suitable for certain fundamental aspects of physics and chemistry, but the study of biology and other complex systems doesn't really progress that way. living systems are just too complex to be described adequately by simple hypotheses (pennisi, 2003) . we seem to learn much more by tinkering with them than we do by hypothecating about them. but biology aims for more than mere tinkering; we don't want merely to accumulate data; we want to comprehend essential principles about life. how can masses of complex data be transformed into comprehension? biologists today (and in time to come) are joining forces with information scientists to develop ways to answer that question. computers are helpful. the most primitive of computers puts our memory to shame; our conscious minds are no match for advanced parallel processing. nevertheless, we are endowed with a unique gift. our cognitive advantage over the computer is our ability to see associations, to create and use metaphors. the computer's memory is composed of precisely retrievable lists; our memory is a web of associations-our memory is not the computer's ram. but we, unlike computers, can create art and science, and we can respond to art and science. in closing, i will describe one example, most familiar to me, of the new synthesis between biology and information science-between mind and computer. my colleagues and i have begun to consider ways we might achieve two objectives: to record and catalogue complex scientific data in a precise format amenable to computer-assisted simulation and testing; and to have the data themselves construct representations that stimulate human minds productively. we have termed this two-tiered approach reactive animation (ra). ra emerged from our simulation of the complex development of a key class of cells in the adaptive immune system-t cells (efroni et al., 2003) . in the first tier, we recorded basic information about t-cell development culled from some 400 research papers using the visual language of statecharts to convert the data to a precise computer format (harel, 1987) ; statecharts had been developed by david harel and his colleagues more than 20 years earlier for building and analyzing complex man-made systems (actually, statecharts grew out of the need to coordinate the planning of a new fighter aircraft). statecharts seems suitable to deal with biologically evolved systems and not only with systems generated by human minds; objects such as molecules, cells, and organs can be described in terms of their component parts, interactions, and transitions from state to state, which is the way we actually do our experiments and record the data. moreover, the statecharts formalism is modular; we can easily add new objects, interactions, and states as we accumulate information. and most importantly, we can run the system dynamically on a computer and see the outcomes in statecharts format of any stimulation or modification of the system we care to study. sol efroni, at the time a student jointly supervised by david harel and me, then added a second tier to the representation; he devised a way for the statecharts simulation to produce and direct an animation of the action showing the t cells and other cells moving, interacting, multiplying, differentiating, or dying in the course of development in the organ called the thymus (efroni et al., 2003) . ra makes it possible for us to cross scales-to zoom into individual cells and their component molecules and to zoom out to view thousands of cells forming the thymus as an organ, as we please. ra allows us to experiment with the animated system in silico. best of all, ra shows us the emergence of properties in t-cell development we never dreamed were there; the animation arm of ra reveals to our eyes aspects of the data hidden from intuition. the experiments motivated by ra in silico lead to new laboratory experiments in mundo; the results produce new data for improved statecharts, and the cycle described by conant continues (fig. 7) . the details of ra and of t cells and thymus are well beyond the scope of the present article; but know that ra, like listening post provides us with a representational analogue of how we might approach seemingly incomprehensible complexity; the complexity is reduced to representations that engage the mind. ra transforms the seeming noise of a complex system into a useful informational landscape (fig. 6) . listening post exemplifies how transformations of informational landscapes are at the roots of the tree of life-a tree whose arborizations include biologic evolution and human understanding. look again at figs. 2, 3, and 6; they represent the same processes in essence-only the labels vary. art and science flourish when they transform old information into a narrative of new ideas that engage ever more human minds. this issue of the bulletin for mathematical biology is composed of a series of papers written in memory of our colleague lee segel, professor of applied mathematics at the weizmann institute of science, whose recent death has touched us all. lee's child-like curiosity led him to apply math as an experimental tool to probe the wonder of living systems. he was always asking questions; he always wanted to see the latest experimental results; he always had an interpretation and a new question. lee loved to teach as he loved to learn. the pleasure of his company was grounded in curiosity and wonder. for me, lee was a central influence in a landscape of information that led me to explore ideas beyond the raw data of my particular field of research-cellular immunology. for me, lee transformed experimental information into a narrative of new ideas that engaged my mind in ever evolving ways. he was my teacher as well as my friend. he lives on in all my work. fig. 7 reactive animation. the translation of experimental data into the language of statecharts converts complex masses of basic facts into a format suitable for simulation in silico. reactive animation (ra) empowers the statecharts simulation to generate a realistic animation of the system and allows one to experiment with the representation in silico. the animation in silico reveals emergent properties of the system and stimulates the experimenter to undertake new experiments in mundo. the new data enter the cycle of animated representation and improved experimentation. ra represents the data in a way that engages the mind. self-creation of meaning immune information, self-organization and meaning the limits of mathematics, the unknowable, exploring randomness, conversations with a mathematician tending adam's garden: evolving the cognitive immune self regen und auferstehung: talmud und naturwissenschaft im dialog mit der welt limits to genetic explanations impose limits on the human genome project hiv. escape artist par excellence science and common sense two rounds of whole genome duplication in the ancestral vertebrate toward rigorous comprehension of biological complexity: modeling, execution, and visualization of thymic t-cell maturation a theory for complex systems: reactive animation poised between old and new. the gallery. the wall street journal the spandrels of san marco and the panglossian paradigm: a critique of the adaptationist programme statecharts: a visual formalism for complex systems exploring the relationship between parental relatedness and male reproductive success in the antarctic fur seal arctocephalus gazella the codebreakers: the comprehensive history of secret communication from ancient times to the internet, rev the neutral theory of molecular evolution the evolutionary origin of complex features impacts and evolution: future prospects meaning-making in the immune system tracing life's circuitry darwin machines and the nature of knowledge the logic of scientific discovery, 15th edn on the role of feedback in promoting conflicting goals of the adaptive immune system a mathematical theory of communication. bell syst. tech. j. 30, 50. waddington, c.h., 1940. organizers and genes a new kind of science i am the mauerberger professor of immunology at the weizmann institute of science, the director of the center for the study of emerging diseases and a member of the steering committee of the center for complexity science, jerusalem, and the director of the national institute for biotechnology in the negev, ben-gurion university of the negev. this paper has emerged from discussions with henri atlan, yonatan cohen, sol efroni, david harel, uri hershberg, dan mishmar, ohad parnes, ben rubin, david rubin, eleanor rubin, eitan rubin, lee segel, and sorin solomon. key: cord-018947-d4im0p9e authors: helbing, dirk title: challenges in economics date: 2012-02-10 journal: social self-organization doi: 10.1007/978-3-642-24004-1_16 sha: doc_id: 18947 cord_uid: d4im0p9e in the same way as the hilbert program was a response to the foundational crisis of mathematics [1], this article tries to formulate a research program for the socio-economic sciences. the aim of this contribution is to stimulate research in order to close serious knowledge gaps in mainstream economics that the recent financial and economic crisis has revealed. by identifying weak points of conventional approaches in economics, we identify the scientific problems which need to be addressed. we expect that solving these questions will bring scientists in a position to give better decision support and policy advice. we also indicate, what kinds of insights can be contributed by scientists from other research fields such as physics, biology, computer and social science. in order to make a quick progress and gain a systemic understanding of the whole interconnected socio-economic-environmental system, using the data, information and computer systems available today and in the near future, we suggest a multi-disciplinary collaboration as most promising research approach. static where the world was dynamic, it assumed competitive markets where few existed, it assumed rationality when we knew full well that economic agents were not rational . . . economics had no way of dealing with changing tastes and technology . . . econometrics was equally plagued with intractable problems: economic observations are never randomly drawn and seldom independent, the number of excluded variables is always unmanageably large, the degrees of freedom unacceptably small, the stability of significance tests seldom unequivocably established, the errors in measurement too large to yield meaningful results . . . " [5] . in the following, we will try to identify the scientific challenges that must be addressed to come up with better theories in the near future. this comprises practical challenges, i.e. the real-life problems that must be faced (see sect. 16 .2), and fundamental challenges, i.e. the methodological advances that are required to solve these problems (see sect. 16.3) . after this, we will discuss, which contribution can be made by related scientific disciplines such as econophysics and the social sciences. the intention of this contribution is constructive. it tries to stimulate a fruitful scientific exchange, in order to find the best way out of the crisis. according to our perception, the economic challenges we are currently facing can only be mastered by large-scale, multi-disciplinary efforts and by innovative approaches [6] . we fully recognize the large variety of non-mainstream approaches that has been developed by "heterodox economists". however, the research traditions in economics seem to be so powerful that these are not paid much attention to. besides, there is no agreement on which of the alternative modeling approaches would be the most promising ones, i.e. the heterogeneity of alternatives is one of the problems, which slows down their success. this situation clearly implies institutional challenges as well, but these go beyond the scope of this contribution and will therefore be addressed in the future. since decades, if not since hundreds of years, the world is facing a number of recurrent socio-economic problems, which are obviously hard to solve. before addressing related fundamental scientific challenges in economics, we will therefore point out practical challenges one needs to pay attention to. this basically requires to classify the multitude of problems into packages of interrelated problems. probably, such classification attempts are subjective to a certain extent. at least, the list presented below differs from the one elaborated by lomborg et al. [7] , who identified the following top ten problems: air pollution, security/conflict, disease control, education, climate change, hunger/malnutrition, water sanitation, barriers to migration and trade, transnational terrorism and, finally, women and development. the following (non-ranked) list, in contrast, is more focused on socio-economic factors rather than resource and engineering issues, and it is more oriented at the roots of problems rather than their symptoms: 1. demographic change of the population structure (change of birth rate, migration, integration. . . ) 2. financial and economic (in)stability (government debts, taxation, and inflation/ deflation; sustainability of social benefit systems; consumption and investment behavior. . . ) 3. social, economic and political participation and inclusion (of people of different gender, age, health, education, income, religion, culture, language, preferences; reduction of unemployment. . . ) 4. balance of power in a multi-polar world (between different countries and economic centers; also between individual and collective rights, political and company power; avoidance of monopolies; formation of coalitions; protection of pluralism, individual freedoms, minorities. . . ) 5. collective social behavior and opinion dynamics (abrupt changes in consumer behavior; social contagion, extremism, hooliganism, changing values; breakdown of cooperation, trust, compliance, solidarity. . . ) 6. security and peace (organized crime, terrorism, social unrest, independence movements, conflict, war. . . ) 7. institutional design (intellectual property rights; over-regulation; corruption; balance between global and local, central and decentral control. . . ) 8. sustainable use of resources and environment (consumption habits, travel behavior, sustainable and efficient use of energy and other resources, participation in recycling efforts, environmental protection. . . ) 9. information management (cyber risks, misuse of sensitive data, espionage, violation of privacy; data deluge, spam; education and inheritance of culture. . . ) 10 . public health (food safety; spreading of epidemics [flu, sars, h1n1, hiv], obesity, smoking, or unhealthy diets. . . ) some of these challenges are interdependent. in the following, we will try to identify the fundamental theoretical challenges that need to be addressed in order to understand the above practical problems and to draw conclusions regarding possible solutions. the most difficult part of scientific research is often not to find the right answer. the problem is to ask the right questions. in this context it can be a problem that people are trained to think in certain ways. it is not easy to leave these ways and see the problem from a new angle, thereby revealing a previously unnoticed solution. three factors contribute to this: 1. we may overlook the relevant facts because we have not learned to see them, i.e. we do not pay attention to them. the issue is known from internalized norms, which prevent people from considering possible alternatives. 2. we know the stylized facts, but may not have the right tools at hand to interpret them. it is often difficult to make sense of patterns detected in data. turning data into knowledge is quite challenging. 3. we know the stylized facts and can interpret them, but may not take them seriously enough, as we underestimate their implications. this may result from misjudgements or from herding effects, i.e. from a tendency to follow traditions and majority opinions. in fact, most of the issues discussed below have been pointed out before, but it seems that this did not have an effect on mainstream economics so far or on what decision-makers know about economics. this is probably because mainstream theory has become a norm [8] , and alternative approaches are sanctioned as norm-deviant behavior [9, 10] . as we will try to explain, the following fundamental issues are not just a matter of approximations (which often lead to the right understanding, but wrong numbers). rather they concern fundamental errors in the sense that certain conclusions following from them are seriously misleading. as the recent financial crisis has demonstrated, such errors can be very costly. however, it is not trivial to see what dramatic consequences factors such as dynamics, spatial interactions, randomness, non-linearity, network effects, differentiation and heterogeneity, irreversibility or irrationality can have. despite of criticisms by several nobel prize winners such as reinhard selten (1994), joseph stiglitz and george akerlof (2001) , or daniel kahneman (2002) , the paradigm of the homo economicus, i.e. of the "perfect egoist", is still the dominating approach in economics. it assumes that people would have quasi-infinite memory and processing capacities and would determine the best one among all possible alternative behaviors by strategic thinking (systematic utility optimization), and would implement it into practice without mistakes. the nobel prize winner of 1976, milton friedman, supported the hypothesis of homo economicus by the following argument: "irrational agents will lose money and will be driven out of the market by rational agents" [11] . more recently, robert e. lucas jr., the nobel prize winner of 1995, used the rationality hypothesis to narrow down the class of empirically relevant equilibria [12] . the rational agent hypothesis is very charming, as its implications are clear and it is possible to derive beautiful and powerful economic theorems and theories from it. the best way to illustrate homo economicus is maybe a company that is run by using optimization methods from operation research, applying supercomputers. another example are professional chess players, who are trying to anticipate the possible future moves of their opponents. obviously, in both examples, the future course of actions can not be fully predicted, even if there are no random effects and mistakes. it is, therefore, no wonder that people have repeatedly expressed doubts regarding the realism of the rational agent approach [13, 14] . bertrand russell, for example, claimed: "most people would rather die than think". while this seems to be a rather extreme opinion, the following scientific arguments must be taken seriously: 1. human cognitive capacities are bounded [16, 17] . already phone calls or conversations can reduce people's attention to events in the environment a lot. also, the abilities to memorize facts and to perform complicated logical analyses are clearly limited. 2. in case of np-hard optimization problems, even supercomputers are facing limits, i.e. optimization jobs cannot be performed in real-time anymore. therefore, approximations or simplifications such as the application of heuristics may be necessary. in fact, psychologists have identified a number of heuristics, which people use when making decisions [18] . 3. people perform strategic thinking mainly in important new situations. in normal, everyday situation, however, they seem to pursue a satisficing rather than optimizing strategy [17] . meeting a certain aspiration level rather than finding the optimal strategy can save time and energy spent on problem solving. in many situation, people even seem to perform routine choices [14] , for example, when evading other pedestrians in counterflows. 4. there is a long list of cognitive biases which question rational behavior [19] . for example, individuals are favorable of taking small risks (which are preceived as "chances", as the participation in lotteries shows), but they avoid large risks [20] . furthermore, non-exponential temporal discounting may lead to paradoxical behaviors [21] and requires one to rethink, how future expectations must be modeled. 5. most individuals have a tendency towards other-regarding behavior and fairness [22, 23] . for example, the dictator game [24] and other experiments [25] show that people tend to share, even if there is no reason for this. leaving a tip for the waiter in a restaurant that people visit only once is a typical example (particularly in countries where tipping is not common) [26] . such behavior has often been interpreted as sign of social norms. while social norms can certainly change the payoff structure, it has been found that the overall payoffs resulting from them do not need to create a user or system optimum [27] [28] [29] . this suggests that behavioral choices may be irrational in the sense of non-optimal. a typical example is the existence of unfavorable norms, which are supported by people although nobody likes them [30] . 6. certain optimization problems can have an infinite number of local optima or nash equilibria, which makes it impossible to decide what is the best strategy [31] . 7. convergence towards the optimal solution may require such a huge amount of time that the folk theorem becomes useless. this can make it practically impossible to play the best response strategy [32] . 8. the optimal strategy may be deterministically chaotic, i.e. sensitive to arbitrarily small details of the initial condition, which makes the dynamic solution unpredictable on the long run ("butterfly effect") [33, 34] . this fundamental limit of predictability also implies a limit of control-two circumstances that are even more true for non-deterministic systems with a certain degree of randomness. in conclusion, although the rational agent paradigm (the paradigm of homo economicus) is theoretically powerful and appealing, there are a number of empirical and theoretical facts, which suggest deficiencies. in fact, most methods used in financial trading (such as technical analysis) are not well compatible with the rational agent approach. even if an optimal solution exists, it may be undecidable for practical reasons or for theoretical ones [35, 36] . this is also relevant for the following challenges, as boundedly rational agents may react inefficently and with delays, which questions the efficient market hypothesis, the equilibrium paradigm, and other fundamental concepts, calling for the consideration of spatial, network, and time-dependencies, heterogeneity and correlations etc. it will be shown that these points can have dramatic implications regarding the predictions of economic models. the efficient market hypothesis (emh) was first developed by eugene fama [37] in his ph.d. thesis and rapidly spread among leading economists, who used it as an argument to promote laissez-faire policies. the emh states that current prices reflect all publicly available information and (in its stronger formulation) that prices instantly change to reflect new public information. the idea of self-regulating markets goes back to adam smith [38] , who believed that "the free market, while appearing chaotic and unrestrained, is actually guided to produce the right amount and variety of goods by a so-called "invisible hand"." furthermore, "by pursuing his own interest, [the individual] frequently promotes that of the society more effectually than when he intends to promote it" [39] . for this reason, adam smith is often considered to be the father of free market economics. curiously enough, however, he also wrote a book on "the theory of moral sentiments" [40] . "his goal in writing the work was to explain the source of mankind's ability to form moral judgements, in spite of man's natural inclinations towards self-interest. smith proposes a theory of sympathy, in which the act of observing others makes people aware of themselves and the morality of their own behavior . . . [and] seek the approval of the "impartial spectator" as a result of a natural desire to have outside observers sympathize with them" [38] . such a reputation-based concept would be considered today as indirect reciprocity [41] . of course, there are criticisms of the efficient market hypothesis [42] , and the nobel prize winner of 2001, joseph stiglitz, even believes that "there is not invisible hand" [43] . the following list gives a number of empirical and theoretical arguments questioning the efficient market hypothesis: 1. examples of market failures are well-known and can result, for example, in cases of monopolies or oligopolies, if there is not enough liquidity or if information symmetry is not given. 2. while the concept of the "invisible hand" assumes something like an optimal self-organization [44] , it is well-known that this requires certain conditions, such as symmetrical interactions. in general, however, self-organization does not necessarily imply system-optimal solutions. stop-and-go traffic [45] or crowd disasters [46] are two obvious examples for systems, in which individuals competitively try to reach individually optimal outcomes, but where the optimal solution is dynamically unstable. 3. the limited processing capacity of boundedly rational individuals implies potential delays in their responses to sensorial inputs, which can cause such instabilities [47] . for example, a delayed adaptation in production systems may contribute to the occurrence of business cycles [48] . the same applies to the labor market of specially skilled people, which cannot adjust on short time scales. even without delayed reactions, however, the competitive optimization of individuals can lead to suboptimal individual results, as the "tragedy of the commons" in public goods dilemmas demonstrates [49, 50] . 4. bubbles and crashes, or more generally, extreme events in financial markets should not occur, if the efficient market hypothesis was correct (see next subsection). 5. collective social behavior such as "herding effects" as well as deviations of human behavior from what is expected from rational agents can lead to such bubbles and crashes [51] , or can further increase their size through feedback effects [52] . cyclical feedbacks leading to oscillations are also known from the beer game [53] or from business cycles [48] . the efficient market paradigm implies the equilibrium paradigm. this becomes clear, if we split it up into its underlying hypotheses: 1. the market can be in equilibrium, i.e. there exists an equilibrium. 2. there is one and only one equilibrium. 3. the equilibrium is stable, i.e. any deviations from the equilibrium due to "fluctuations" or "perturbations" tend to disappear eventually. 4. the relaxation to the equilibrium occurs at an infinite rate. note that, in order to act like an "invisible hand", the stable equilibrium (nash equilibrium) furthermore needs to be a system optimum, i.e. to maximize the average utility. this is true for coordination games, when interactions are well-mixed and exploration behavior as well as transaction costs can be neglected [54] . however, it is not fulfilled by so-called social dilemmas [49] . let us discuss the evidence for the validity of the above hypotheses one by one: 1. a market is a system of extremely many dynamically coupled variables. theoretically, it is not obvious that such a system would have a stationary solution. for example, the system could behave periodic, quasi-periodic, chaotic, or turbulent [81-83, 85-87, 94] . in all these cases, there would be no convergence to a stationary solution. 2. if a stationary solution exists, it is not clear that there are no further stationary solutions. if many variables are non-linearly coupled, the phenomenon of multistability can easily occur [55] . that is, the solution to which the system converges may not only depend on the model parameters, but also on the initial condition, history, or perturbation size. such facts are known as path-dependencies or hysteresis effects and are usually visualized by so-called phase diagrams [56] . 3. in systems of non-linearly interacting variables, the existence of a stationary solution does not necessarily imply that it is stable, i.e. that the system will converge to this solution. for example, the stationary solution could be a focal point with orbiting solutions (as for the classical lotka-volterra equations [57] ), or it could be unstable and give rise to a limit cycle [58] or a chaotic solution [33] , for example (see also item 1). in fact, experimental results suggest that volatility clusters in financial markets may be a result of over-reactions to deviations from the fundamental value [59] . 4. an infinite relaxation rate is rather unusual, as most decisions and related implemenations take time [15, 60] . the points listed in the beginning of this subsection are also questioned by empirical evidence. in this connection, one may mention the existence of business cycles [48] or unstable orders and deliveries observed in the experimental beer game [53] . moreover, bubbles and crashes have been found in financial market games [61] . today, there seems to be more evidence against than for the equilibrium paradigm. in the past, however, most economists assumed that bubbles and crashes would not exist (and many of them still do). the following quotes are quite typical for this kind of thinking (from [62] ): in 2004, the federal reserve chairman of the u.s., alan greenspan, stated that the rise in house values was "not enough in our judgment to raise major concerns". in july 2005 when asked about the possibility of a housing bubble and the potential for this to lead to a recession in the future, the present u.s. federal reserve chairman ben bernanke (then chairman of the council of economic advisors) said: "it's a pretty unlikely possibility. we've never had a decline in housing prices on a nationwide basis. so, what i think is more likely is that house prices will slow, maybe stabilize, might slow consumption spending a bit. i don't think it's going to drive the economy too far from it's full path though." as late as may 2007 bernanke stated that the federal reserve "do not expect significant spillovers from the subprime market to the rest of the economy". according to the classical interpretation, sudden changes in stock prices result from new information, e.g. from innovations ("technological shocks"). the dynamics in such systems has, for example, been described by the method of comparative statics (i.e. a series of snapshots). here, the system is assumed to be in equilibrium in each moment, but the equilibrium changes adiabatically (i.e. without delay), as the system parameters change (e.g. through new facts). such a treatment of system dynamics, however, has certain deficiencies: 1. the approach cannot explain changes in or of the system, such as phase transitions ("systemic shifts"), when the system is at a critical point ("tipping point"). 2. it does not allow one to understand innovations and other changes as results of an endogeneous system dynamics. 3. it cannot describe effects of delays or instabilities, such as overshooting, self-organization, emergence, systemic breakdowns or extreme events (see sect. 16.3.4). 4. it does not allow one to study effects of different time scales. for example, when there are fast autocatalytic (self-reinfocing) effects and slow inhibitory effects, this may lead to pattern formation phenomena in space and time [63, 64] . the formation of settlements, where people agglomerate in space, may serve as an example [65, 66] . 5. it ignores long-term correlations such as memory effects. 6. it neglects frictional effects, which are often proportional to change ("speed") and occur in most complex systems. without friction, however, it is difficult to understand entropy and other path-dependent effects, in particular irreversibility (i.e. the fact that the system may not be able to get back to the previous state) [67] . for example, the unemployment rate has the property that it does not go back to the previous level in most countries after a business cycle [68] . comparative statics is, of course, not the only method used in economics to describe the dynamics of the system under consideration. as in physics and other fields, one may use a linear approximation around a stationary solution to study the response of the system to fluctuations or perturbations [69] . such a linear stability analysis allows one to study, whether the system will return to the stationary solution (which is the case for a stable [nash] equilibrium) or not (which implies that the system will eventually be driven into a new state or regime). in fact, the great majority of statistical analyses use linear models to fit empirical data (also when they do not involve time-dependencies). it is know, however, that linear models have special features, which are not representative for the rich variety of possible functional dependencies, dynamics, and outcomes. therefore, the neglection of non-linearity has serious consequences: 1. as it was mentioned before, phenomena like multiple equilibria, chaos or turbulence cannot be understood by linear models. the same is true for selforganization phenomena or emergence. additionally, in non-linearly coupled systems, usually "more is different", i.e. the system may change its behavior fundamentally as it grows beyond a certain size. furthermore, the system is often hard to predict and difficult to control (see sect. 16.3.8). 2. linear modeling tends to overlook that a strong coupling of variables, which would show a normally distributed behavior in separation, often leads to fat tail distributions (such as "power laws") [70, 71] . this implies that extreme events are much more frequent than expected according to a gaussian distribution. for example, when additive noise is replaced by multiplicative noise, a number of surprising phenomena may result, including noise-induced transitions [72] or directed random walks ("ratchet effects") [73] . 3. phenomena such as catastrophes [74] or phase transition ("system shifts") [75] cannot be well understood within a linear modeling framework. the same applies to the phenomenon of "self-organized criticality" [79] (where the system drives itself to a critical state, typically with power-law characteristics) or cascading effects, which can result from network interactions (overcritically challenged network nodes or links) [77, 78] . it should be added that the relevance of network effects resulting from the on-going globalization is often underestimated. for example, "the stock market crash of 1987, began with a small drop in prices which triggered an avalanche of sell orders in computerized trading programs, causing a further price decline that triggered more automatic sales." [80] therefore, while linear models have the advantage of being analytically solvable, they are often unrealistic. studying non-linear behavior, in contrast, often requires numerical computational approaches. it is likely that most of today's unsolved economic puzzles cannot be well understood through linear models, no matter how complicated they may be (in terms of the number of variables and parameters) [81] [82] [83] [84] [85] [86] [87] [88] [89] [90] [91] [92] [93] [94] . the following list mentions some areas, where the importance of non-linear interdependencies is most likely underestimated: â�¢ collective opinions, such as trends, fashions, or herding effects. â�¢ the success of new (and old) technologies, products, etc. â�¢ cultural or opinion shifts, e.g. regarding nuclear power, genetically manipulated food, etc. â�¢ the "fitness" or competitiveness of a product, value, quality perceptions, etc. â�¢ the respect for copyrights. â�¢ social capital (trust, cooperation, compliance, solidarity, . . . ). â�¢ booms and recessions, bubbles and crashes. â�¢ bank panics. â�¢ community, cluster, or group formation. â�¢ relationships between different countries, including war (or trade war) and peace. another common simplification in economic modeling is the representative agent approach, which is known in physics as mean field approximation. within this framework, time-dependencies and non-linear dependencies are often considered, but it is assumed that the interaction with other agents (e.g. of one company with all the other companies) can be treated as if this agent would interact with an average agent, the "representative agent". let us illustrate this with the example of the public goods dilemma. here, everyone can decide whether to make an individual contribution to the public good or not. the sum of all contributions is multiplied by a synergy factor, reflecting the benefit of cooperation, and the resulting value is equally shared among all people. the prediction of the representative agent approach is that, due to the selfishness of agents, a "tragedy of the commons" would result [49] . according to this, everybody should free-ride, i.e. nobody should make a contribution to the public good and nobody would gain anything. however, if everybody would contribute, everybody could multiply his or her contribution by the synergy factor. this example is particularly relevant as society is facing a lot of public goods problems and would not work without cooperation. everything from the creation of public infrastructures (streets, theaters, universities, libraries, schools, the world wide web, wikipedia etc.) over the use of environmental resources (water, forests, air, etc.) or of social benefit systems (such as a public health insurance), maybe even the creation and maintainance of a commonly shared language and culture are public goods problems (although the last examples are often viewed as coordination problems). even the process of creating public goods is a public good [95] . while it is a well-known problem that people tend to make unfair contributions to public goods or try to get a bigger share of them, individuals cooperate much more than one would expect according to the representative agent approach. if they would not, society could simply not exist. in economics, one tries to solve the problem by introducing taxes (i.e. another incentive structure) or a "shadow of the future" (i.e. a strategic optimization over infinite time horizons in accordance with the rational agent approach) [96, 97] . both comes down to changing the payoff structure in a way that transforms the public good problem into another one that does not constitute a social dilemma [98] . however, there are other solutions of the problem. when the realm of the mean-field approximation underlying the representative agent approach is left and spatial or network interactions or the heterogeneity among agents are considered, a miracle occurs: cooperation can survive or even thrive through correlations and co-evolutionary effects [99] [100] [101] . a similar result is found for the public goods game with costly punishment. here, the representative agent model predicts that individuals avoid to invest into punishment, so that punishment efforts eventually disappear (and, as a consequence, cooperation as well). however, this "second-order free-rider problem" is naturally resolved and cooperation can spread, if one discards the mean-field approximation and considers the fact that interactions take place in space or social networks [56] . societies can overcome the tragedy of the commons even without transforming the incentive structure through taxes. for example, social norms as well as group dynamical and reputation effects can do so [102] . the representative agent approach implies just the opposite conclusion and cannot well explain the mechanisms on which society is built. it is worth pointing out that the relevance of public goods dilemmas is probably underestimated in economics. partially related to adam smith's belief in an "invisible hand", one often assumes underlying coordination games and that they would automatically create a harmony between an individually and system optimal state in the course of time [54] . however, running a stable financial system and economy is most likely a public goods problem. considering unemployment, recessions always go along with a breakdown of solidarity and cooperation. efficient production clearly requires mutual cooperation (as the counter-example of countries with many strikes illustrates). the failure of the interbank market when banks stop lending to each other, is a good example for the breakdown of both, trust and cooperation. we must be aware that there are many other systems that would not work anymore, if people would lose their trust: electronic banking, e-mail and internet use, facebook, ebusiness and egovernance, for example. money itself would not work without trust, as bank panics and hyperinflation scenarios show. similarly, cheating customers by selling low-quality products or selling products at overrated prices, or by manipulating their choices by advertisements rather than informing them objectively and when they want, may create profits on the short run, but it affects the trust of customers (and their willingness to invest). the failure of the immunization campaign during the swine flu pandemics may serve as an example. furthermore, people would probably spend more money, if the products of competing companies were better compatible with each other. therefore, on the long run, more cooperation among companies and with the customers would pay off and create additional value. besides providing a misleading picture of how cooperation comes about, there are a number of other deficiencies of the representative agent approach, which are listed below: 1. correlations between variables are neglected, which is acceptable only for "well-mixing" systems. according to what is known from critical phenomena in physics, this approximation is valid only, when the interactions take place in high-dimensional spaces or if the system elements are well connected. (however, as the example of the public goods dilemma showed, this case does not necessarily have beneficial consequences. well-mixed interactions could rather cause a breakdown of social or economic institutions, and it is conceivable that this played a role in the recent financial crisis.) 2. percolation phenomena, describing how far an idea, innovation, technology, or (computer) virus spreads through a social or business network, are not well reproduced, as they depend on details of the network structure, not just on the average node degree [103] . 3. the heterogeneity of agents is ignored. for this reason, factors underlying economic exchange, perturbations, or systemic robustness [104] cannot be well described. moreover, as socio-economic differentiation and specialization imply heterogeneity, they cannot be understood as emergent phenomena within a representative agent approach. finally, it is not possible to grasp innovation without the consideration of variability. in fact, according to evolutionary theory, the innovation rate would be zero, if the variability was zero [105] . furthermore, in order to explain innovation in modern societies, schumpeter introduced the concept of the "political entrepreneur" [106] , an extra-ordinarily gifted person capable of creating disruptive change and innovation. such an extraordinary individual can, by definition, not be modeled by a "representative agent". one of the most important drawbacks of the representative agent approach is that it cannot explain the fundamental fact of economic exchange, since it requires one to assume a heterogeneity in resources or production costs, or to consider a variation in the value of goods among individuals. ken arrow, nobel prize winner in 1972, formulated this point as follows [107] : "one of the things that microeconomics teaches you is that individuals are not alike. there is heterogeneity, and probably the most important heterogeneity here is heterogeneity of expectations. if we didn't have heterogeneity, there would be no trade." we close this section by mentioning that economic approaches, which go beyond the representative agent approach, can be found in refs. [108, 109] . another deficiency of economic theory that needs to be mentioned is the lack of a link between micro-and macroeconomics. neoclassical economics implicitly assumes that individuals make their decisions in isolation, using only the information received from static market signals. within this oversimplified framework, macro-aggregates are just projections of some representative agent behavior, instead of the outcome of complex interactions with asymmetric information among a myriad of heterogeneous agents. in principle, it should be understandable how the macroeconomic dynamics results from the microscopic decisions and interactions on the level of producers and consumers [81, 110] (as it was possible in the past to derive micro-macro links for other systems with a complex dynamical behavior such as interactive vehicle traffic [111] ). it should also be comprehensible how the macroscopic level (the aggregate econonomic situation) feeds back on the microscopic level (the behavior of consumers and producers), and to understand the economy as a complex, adaptive, self-organizing system [112, 113] . concepts from evolutionary theory [114] and ecology [115] appear to be particularly promising [116] . this, however, requires a recognition of the importance of heterogeneity for the system (see the the previous subsection). the lack of ecological thinking implies not only that the sensitive network interdependencies between the various agents in an economic system (as well as minority solutions) are not properly valued. it also causes deficiencies in the development and implementation of a sustainable economic approach based on recycling and renewable resources. today, forestry science is probably the best developed scientific discipline concerning sustainability concepts [117] . economic growth to maintain social welfare is a serious misconception. from other scientific disciplines, it is well known that stable pattern formation is also possible for a constant (and potentially sustainable) inflow of energy [69, 118] . one of the great achievements of economics is that it has developed a multitude of methods to use scarce resources efficiently. a conventional approach to this is optimization. in principle, there is nothing wrong about this approach. nevertheless, there are a number of problems with the way it is usually applied: 1. one can only optimize for one goal at a time, while usually, one needs to meet several objectives. this is mostly addressed by weighting the different goals (objectives), by executing a hierarchy of optimization steps (through ranking and prioritization), or by applying a satisficing strategy (requiring a minimum performance for each goal) [119, 120] . however, when different optimization goals are in conflict with each other (such as maximizing the throughput and minimizing the queue length in a production system), a sophisticated timedependent strategy may be needed [121] . high profit? best customer satisfaction? large throughput? competitive advantage? resilience? [122] in fact, the choice of the optimization function is arbitrary to a certain extent and, therefore, the result of optimization may vary largely. goal selection requires strategic decisions, which may involve normative or moral factors (as in politics). in fact, one can often observe that, in the course of time, different goal functions are chosen. moreover, note that the maximization of certain objectives such as resilience or "fitness" depends not only on factors that are under the control of a company. resilience and "fitness" are functions of the whole system, in particularly, they also depend on the competitors and the strategies chosen by them. 3. the best solution may be the combination of two bad solutions and may, therefore, be overlooked. in other words, there are "evolutionary dead ends", so that gradual optimization may not work. (this problem can be partially overcome by the application of evolutionary mechanisms [120] ). 4. in certain systems (such as many transport, logistic, or production systems), optimization tends to drive the system towards instability, since the point of maximum efficiency is often in the neighborhood or even identical with the point of breakdown of performance. such breakdowns in capacity or performance can result from inefficiencies due to dynamic interaction effects. for example, when traffic flow reaches its maximum capacity, sooner or later it breaks down. as a consequence, the road capacity tends to drop during the time period where it is most urgently needed, namely during the rush hour [45, 123] . 5 . optimization often eliminates reduncancies in the system and, thereby, increases the vulnerability to perturbations, i.e. it decreases robustness and resilience. 6. optimization tends to eliminate heterogeneity in the system [80] , while heterogeneity frequently supports adaptability and resilience. 7. optimization is often performed with centralized concepts (e.g. by using supercomputers that process information collected all over the system). such centralized systems are vulnerable to disturbances or failures of the central control unit. they are also sensitive to information overload, wrong selection of control parameters, and delays in adaptive feedback control. in contrast, decentralized control (with a certain degree of autonomy of local control units) may perform better, when the system is complex and composed of many heterogeneous elements, when the optimization problem is np hard, the degree of fluctuations is large, and predictability is restricted to short time periods [77, 124] . under such conditions, decentralized control strategies can perform well by adaptation to the actual local conditions, while being robust to perturbations. urban traffic light control is a good example for this [121, 125] . 8. further on, today's concept of quality control appears to be awkward. it leads to a never-ending contest, requiring people and organizations to fulfil permanently increasing standards. this leads to over-emphasizing measured performance criteria, while non-measured success factors are neglected. the engagement into non-rewarded activities is discouraged, and innovation may be suppressed (e.g. when evaluating scientists by means of their h-index, which requires them to focus on a big research field that generates many citations in a short time). while so-called "beauty contests" are considered to produce the best results, they will eventually absorb more and more resources for this contest, while less and less time remains for the work that is actually to be performed, when the contest is won. besides, a large number of competitors have to waste considerable resources for these contests which, of course, have to be paid by someone. in this way, private and public sectors (from physicians over hospitals, administrations, up to schools and universities) are aching under the evaluationrelated administrative load, while little time remains to perform the work that the corresponding experts have been trained for. it seems naã¯ve to believe that this would not waste resources. rather than making use of individual strengths, which are highly heterogeneous, today's way of evaluating performance enforces a large degree of conformity. there are also some problems with parameter fitting, a method based on optimization as well. in this case, the goal function is typically an error function or a likelihood function. not only are calibration methods often "blindly" applied in practice (by people who are not experts in statistics), which can lead to overfitting (the fitting of meaningless "noise"), to the neglection of collinearities (implying largely variable parameter values), or to inaccurate and problematic parameter determinations (when the data set is insufficient in size, for example, when large portfolios are to be optimized [126] ). as estimates for past data are not necessarily indicative for the future, making predictions with interpolation approaches can be quite problematic (see also sect. 16.3.3 for the challenge of time dependence). moreover, classical calibration methods do not reveal inappropriate model specifications (e.g. linear ones, when non-linear models would be needed, or unsuitable choices of model variables). finally, they do not identify unknown unknowns (i.e. relevant explanatory variables, which have been overlooked in the modeling process). managing economic systems is a particular challenge, not only for the reasons discussed in the previous section. as large economic systems belong to the class of complex systems, they are hard or even impossible to manage with classical control approaches [76, 77] . complex systems are characterized by a large number of system elements (e.g. individuals, companies, countries, . . . ), which have non-linear or network interactions causing mutual dependencies and responses. such systems tend to behave dynamic rather than static and probabilistic rather than deterministic. they usually show a rich, hardly predictable, and sometimes paradoxical system behavior. therefore, they challenge our way of thinking [127] , and their controllability is often overestimated (which is sometimes paraphrased as "illusion of control") [80, 128, 129] . in particular, causes and effects are typically not proportional to each other, which makes it difficult to predict the impact of a control attempt. a complex system may be unresponsive to a control attempt, or the latter may lead to unexpected, large changes in the system behavior (so-called "phase transitions", "regime shifts", or "catastrophes") [75] . the unresponsiveness is known as principle of le chatelier or goodhart's law [130] , according to which a complex system tends to counteract external control attempts. however, regime shifts can occur, when the system gets close to so-called "critical points" (also known as "tipping points"). examples are sudden changes in public opinion (e.g. from pro to anti-war mood, from a smoking tolerance to a public smoking ban, or from buying energy-hungry sport utilities vehicles (suvs) to buying environmentally-friendly cars). particularly in case of network interactions, big changes may have small, no, or unexpected effects. feedback loops, unwanted side effects, and circuli vitiosi are quite typical. delays may cause unstable system behavior (such as bull-whip effects) [53] , and over-critical perturbations can create cascading failures [78] . systemic breakdowns (such as large-scale blackouts, bankruptcy cascades, etc.) are often a result of such domino or avalanche effects [77] , and their probability of occurrence as well as their resulting damage are usually underestimated. further examples are epidemic spreading phenomena or disasters with an impact on the socio-economic system. a more detailed discussion is given in refs. [76, 77] . other factors contributing to the difficulty to manage economic systems are the large heterogeneity of system elements and the considerable level of randomness as well as the possibility of a chaotic or turbulent dynamics (see sect. 16.3.4) . furthermore, the agents in economic systems are responsive to information, which can create self-fulfilling or self-destroying prophecy effects. inflation may be viewed as example of such an effect. interestingly, in some cases one even does not know in advance, which of these effects will occur. it is also not obvious that the control mechanisms are well designed from a cybernetic perspective, i.e. that we have sufficient information about the system and suitable control variables to make control feasible. for example, central banks do not have terribly many options to influence the economic system. among them are performing open-market operations (to control money supply), adjustments in fractional-reserve banking (keeping only a limited deposit, while lending a large part of the assets to others), or adaptations in the discount rate (the interest rate charged to banks for borrowing short-term funds directly from a central bank). nevertheless, the central banks are asked to meet multiple goals such as: â�¢ to guarantee well-functioning and robust financial markets. â�¢ to support economic growth. â�¢ to balance between inflation and unemployment. â�¢ to keep exchange rates within reasonable limits. furthermore, the one-dimensional variable of "money" is also used to influence individual behavior via taxes (by changing behavioral incentives). it is questionable, whether money can optimally meet all these goals at the same time (see sect. 16.3.7) . we believe that a computer, good food, friendship, social status, love, fairness, and knowledge can only to a certain extent be replaced by and traded against each other. probably for this reason, social exchange comprises more than just material exchange [131] [132] [133] . it is conceivable that financial markets as well are trying to meet too many goals at the same time. this includes: â�¢ to match supply and demand. â�¢ to discover a fair price. â�¢ to raise the foreign direct investment (fdi). â�¢ to couple local economies with the international system. â�¢ to facilitate large-scale investments. â�¢ to boost development. â�¢ to share risk. â�¢ to support a robust economy, and â�¢ to create opportunities (to gamble, to become rich, etc.). therefore, it would be worth stuyding the system from a cybernetic control perspective. maybe, it would work better to separate some of these functions from each other rather than mixing them. another aspect that tends to be overlooked in mainstream economics is the relevance of psychological and social factors such as emotions, creativity, social norms, herding effects, etc. it would probably be wrong to interpret these effects just as a result of perception biases (see sect. 16.3.1) . most likely, these human factors serve certain functions such as supporting the creation of public goods [102] or collective intelligence [134, 135] . as bruno frey has pointed out, economics should be seen from a social science perspective [136] . in particular, research on happiness has revealed that there are more incentives than just financial ones that motivate people to work hard [133] . interestingly, there are quite a number of factors which promote volunteering [132] . it would also be misleading to judge emotions from the perspective of irrational behavior. they are a quite universal and a relatively energy-consuming way of signalling. therefore, they are probably more reliable than non-emotional signals. moreover, they create empathy and, consequently, stimulate mutual support and a readiness for compromises. it is quite likely that this creates a higher degree of cooperativeness in social dilemma situations and, thereby, a higher payoff on average as compared to emotionless decisions, which often have drawbacks later on. finally, there is no good theory that would allow one to assess the relevance of information in economic systems. most economic models do not consider information as an explanatory variable, although information is actually a stronger driving force of urban growth and social dynamics than energy [137] . while we have an information theory to determine the number of bits required to encode a message, we are lacking a theory, which would allow us to assess what kind of information is relevant or important, or what kind of information will change the social or economic world, or history. this may actually be largely dependent on the perception of pieces of information, and on normative or moral issues filtering or weighting information. moreover, we lack theories describing what will happen in cases of coincidence or contradiction of several pieces of information. when pieces of information interact, this can change their interpretation and, thereby, the decisions and behaviors resulting from them. that is one of the reasons why socio-economic systems are so hard to predict: "unknown unknowns", structural instabilities, and innovations cause emergent results and create a dynamics of surprise [138] . the problems discussed in the previous two sections pose interesting practical and fundamental challenges for economists, but also other disciplines interested in understanding economic systems. econophysics, for example, pursues a physical approach to economic systems, applying methods from statistical physics [81] , network theory [139, 140] , and the theory of complex systems [85, 87] . a contribution of physics appears quite natural, in fact, not only because of its tradition in detecting and modeling regularities in large data sets [141] . physics also has a lot of experience how to theoretically deal with problems such as time-dependence, fluctuations, friction, entropy, non-linearity, strong interactions, correlations, heterogeneity, and many-particle simulations (which can be easily extended towards multi-agent simulations). in fact, physics has influenced economic modeling already in the past. macroeconomic models, for example, were inspired by thermodynamics. more recent examples of relevant contributions by physicists concern models of self-organizing conventions [54] , of geographic agglomeration [65] , of innovation spreading [142] , or of financial markets [143] , to mention just a few examples. one can probably say that physicists have been among the pioneers calling for new approaches in economics [81, 87, [143] [144] [145] [146] [147] . a particularly visionary book beside wolfgang weidlich's work was the "introduction to quantitative aspects of social phenomena" by elliott w. montroll and wade w. badger, which addressed by mathematical and empirical analysis subjects as diverse as population dynamics, the arms race, speculation patterns in stock markets, congestion in vehicular traffic as well as the problems of atmospheric pollution, city growth and developing countries already in 1974 [148] . unfortunately, it is impossible in our paper to reflect the numerous contributions of the field of econophysics in any adequate way. the richness of scientific contributions is probably reflected best by the econophysics forum run by yi-cheng zhang [149] . many econophysics solutions are interesting, but so far they are not broad and mighty enough to replace the rational agent paradigm with its large body of implications and applications. nevertheless, considering the relatively small number of econophysicists, there have been many promising results. the probably largest fraction of publications in econophysics in the last years had a data-driven or computer modeling approach to financial markets [143] . but econophyics has more to offer than the analysis of financial data (such as fluctuations in stock and foreign currency exchange markets), the creation of interaction models for stock markets, or the development of risk management strategies. other scientists have focused on statistical laws underlying income and wealth distributions, nonlinear market dynamics, macroeconomic production functions and conditions for economic growth or agglomeration, sustainable economic systems, business cycles, microeconomic interaction models, network models, the growth of companies, supply and production systems, logistic and transport networks, or innovation dynamics and diffusion. an overview of subjects is given, for example, by ref. [152] and the contributions to annual spring workshop of the physics of socio-economic systems division of the dpg [153] . to the dissatisfaction of many econophysicists, the transfer of knowledge often did not work very well or, if so, has not been well recognized [150] . besides scepticism on the side of many economists with regard to novel approaches introduced by "outsiders", the limited resonance and level of interdisciplinary exchange in the past was also caused in part by econophysicists. in many cases, questions have been answered, which no economist asked, rather than addressing puzzles economists are interested in. apart from this, the econophysics work was not always presented in a way that linked it to the traditions of economics and pointed out deficiencies of existing models, highlighting the relevance of the new approach well. typical responses are: why has this model been proposed and not another one? why has this simplification been used (e.g. an ising model of interacting spins rather than a rational agent model)? why are existing models not good enough to describe the same facts? what is the relevance of the work compared to previous publications? what practical implications does the finding have? what kind of paradigm shift does the approach imply? can existing models be modified or extended in a way that solves the problem without requiring a paradigm shift? correspondingly, there have been criticisms not only by mainstream economists, but also by colleagues, who are open to new approaches [151] . therefore, we would like to suggest to study the various economic subjects from the perspective of the above-mentioned fundamental challenges, and to contrast econophysics models with traditional economic models, showing that the latter leave out important features. it is important to demonstrate what properties of economic systems cannot be understood for fundamental reasons within the mainstream framework (i.e. cannot be dealt with by additional terms within the modeling class that is conventionally used). in other words, one needs to show why a paradigm shift is unavoidable, and this requires careful argumentation. we are not claiming that this has not been done in the past, but it certainly takes an additional effort to explain the essence of the econophysics approach in the language of economics, particularly as mainstream economics may not always provide suitable terms and frameworks to do this. this is particularly important, as the number of econophysicists is small compared to the number of economists, i.e. a minority wants to convince an established majority. to be taken seriously, one must also demonstrate a solid knowledge of related previous work of economists, to prevent the stereotypical reaction that the subject of the paper has been studied already long time ago (tacitly implying that it does not require another paper or model to address what has already been looked at before). a reasonable and promising strategy to address the above fundamental and practical challenges is to set up multi-disciplinary collaborations in order to combine the best of all relevant scientific methods and knowledge. it seems plausible that this will generate better models and higher impact than working in separation, and it will stimulate scientific innovation. physicists can contribute with their experience in handling large data sets, in creating and simulating mathematical models, in developing useful approximations, in setting up laboratory experiments and measurement concepts. current research activities in economics do not seem to put enough focus on: â�¢ modeling approaches for complex systems [154] . â�¢ computational modeling of what is not analytically tractable anymore, e.g. by agent-based models [155] [156] [157] . â�¢ testable predictions and their empirical or experimental validation [164] . â�¢ managing complexity and systems engineering approaches to identify alternative ways of organizing financial markets and economic systems [91, 93, 165] , and â�¢ an advance testing of the effectiveness, efficiency, safety, and systemic impact (side effects) of innovations, before they are implemented in economic systems. this is in sharp contrast to mechanical, electrical, nuclear, chemical and medical drug engineering, for example. expanding the scope of economic thinking and paying more attention to these natural, computer and engineering science aspects will certainly help to address the theoretical and practical challenges posed by economic systems. besides physics, we anticipate that also evolutionary biology, ecology, psychology, neuroscience, and artificial intelligence will be able to make significant contributions to the understanding of the roots of economic problems and how to solve them. in conclusion, there are interesting scientific times ahead. it is a good question, whether answering the above list of fundamental challenges will sooner or later solve the practical problems as well. we think, this is a precondition, but it takes more, namely the consideration of social factors. in particular, the following questions need to be answered: 8. how do costly punishment, antisocial punishment, and discrimination come about? 9. how can the formation of social norms and conventions, social roles and socialization, conformity and integration be understood? 10. how do language and culture evolve? 11. how to comprehend the formation of group identity and group dynamics? what are the laws of coalition formation, crowd behavior, and social movements? 12. how to understand social networks, social structure, stratification, organizations and institutions? 13. how do social differentiation, specialization, inequality and segregation come about? 14. how to model deviance and crime, conflicts, violence, and wars? 15. how to understand social exchange, trading, and market dynamics? we think that, despite the large amount of research performed on these subjects, they are still not fully understood. the ultimate goal would be to formulate mathematical models, which would allow one to understand these issues as emergent phenomena based on first principles, e.g. as a result of (co-)evolutionary processes. such first principles would be the basic facts of human capabilities and the kinds of interactions resulting from them, namely: 1. birth, death, and reproduction. 2. the need of and competition for resources (such as food and water). 3. the ability to observe their environment (with different senses). 4. the capability to memorize, learn, and imitate. 5. empathy and emotions. 6. signaling and communication abilities. 7. constructive (e.g. tool-making) and destructive (e.g. fighting) abilities. 8. mobility and (limited) carrying capacity. 9. the possibility of social and economic exchange. such features can, in principle, be implemented in agent-based models [158] [159] [160] [161] [162] [163] . computer simulations of many interacting agents would allow one to study the phenomena emerging in the resulting (artificial or) model societies, and to compare them with stylized facts [163, 168, 169] . the main challenge, however, is not to program a seemingly realistic computer game. we are looking for scientific models, i.e. the underlying assumptions need to be validated, and this requires to link computer simulations with empirical and experimental research [170] , and with massive (but privacy-respecting) mining of social interaction data [141] . in the ideal case, there would also be an analytical understanding in the end, as it has been recently gained for interactive driver behavior [111] . as well as for inspiring discussions during a visioneer workshop in zurich from january 13 how to understand human decision-making? how to explain deviations from rational choice theory and the decision-theoretical paradoxes? why are people risk averse? 2. how does consciousness and self-consciousness come about? how to understand creativity and innovation? 4. how to explain homophily, i.e. the fact that individuals tend to agglomerate, interact with and imitate similar others? how to explain social influence, collective decision making, opinion dynamics and voting behavior? why do individuals often cooperate in social dilemma situations? how do indirect reciprocity, trust and reputation evolve? references 1 how did economists get it so wrong? the financial crisis and the systemic failure of academic economics (dahlem report address of the governor of the central bank of barbados on the futurict knowledge accelerator: unleashing the power of information for a sustainable future global solutions: costs and benefits economics as religion: from samuelson to chicago and beyond the counter revolution of science the case of flexible exchange rates adaptive behavior and economic theory bounded rationality. the adaptive toolbox the bounds of reason: game theory and the unification of the behavioral sciences on information efficiency and financial stability simon models of man simple heuristics that make us smart judgment under uncertainty: heuristics and biases physics of risk and uncertainty in quantum decision making a theory of fairness, competition, and cooperation foundations of human sociality: economic experiments and ethnographic evidence from fifteen small-scale societies social distance and other-regarding behavior in dictator games measuring social value orientation economics and restaurant gratuities: determining tip rates the cement of society: a study of social order the rewards of punishment. a relational theory of norm enforcement the emergence of homogeneous norms in heterogeneous populations the emperor's dilemma: a computational model of selfenforcing norms non-explanatory equilibria: an extremely simple game with (mostly) unattainable fixed points the myth of the folktheorem deterministic chaos for example, three-body planetary motion has deterministic chaotic solutions, although it is a problem in classical mechanics, where the equations of motion optimize a lagrangian functional on formally undecidable propositions of principia mathematica and related systems on computable numbers, with an application to the entscheidungsproblem the behavior of stock market prices an inquiry into the nature and causes of the wealth of nations the theory of moral sentiments (1759) evolution of indirect reciprocity revisiting market efficiency: the stock market as a complex adaptive system there is no invisible hand. the guardian optimal self-organization traffic and related self-driven many-particle systems from crowd dynamics to crowd safety: a video-based analysis stability and stabilization of time-delay systems network-induced oscillatory behavior in material flow networks and irregular business cycles the tragedy of the commons altruistic punishment in humans switching phenomena in a system with no switches animal spirits: how human psychology drives the economy, and why it matters for global capitalism testing behavioral simulation models by direct experiment a mathematical model for behavioral changes by pair interactions multistability in a dynamic cournot game with three oligopolists evolutionary establishment of moral and double moral standards through spatial interactions abzweigungen einer periodischen lã¶sung von einer stationã¤ren lã¶sung eines differentialgleichungssystems dynamic decision behavior and optimal guidance through information services: models and experiments the dynamics of general equilibrium modeling the stylized facts in finance through simple nonlinear adaptive systems predicting moments of crisis in physics and finance" during the workshop "windows to complexity the chemical basis of morphogenesis lectures on nonlinear differential equation-models in biology settlement formation ii: numerical simulation self-organization in space and induced by fluctuations stokes integral of economic growth: calculus and the solow model hysteresis and the european unemployment problem pattern formation and dynamics in nonequilibrium systems critical phenomena in natural sciences: chaos, fractals, selforganization and disorder the science of disasters noise-induced transitions: theory and applications in physics, chemistry, and biology brownian motors: noisy transport far from equilibrium catastrophe theory introduction to phase transitions and critical phenomena managing complexity: an introduction. pages 1-16 in systemic risks in society and economics systemic risk in a unifying framework for cascading processes on networks how nature works: the science of self-organized criticality why economists failed to predict the financial crisis physics and social science-the approach of synergetics nonlinear economic dynamics the self-organizing economy self-organization of complex structures: from individual to collective dynamics complex economic dynamics sociodynamics: a systematic approach to mathematical modelling in the social sciences business dynamics: systems thinking and modeling for a complex world growth theory, non-linear dynamics and economic modelling complexity hints for economic policy coping with the complexity of economics handbook of research on complexity the rise and decline of nations: economic growth, stagflation, and social rigidities the evolution of cooperation a general theory of equilibrium selection phase transitions to cooperation in the prisoner's dilemma evolutionary games and spatial chaos social diversity promotes the emergence of cooperation in public goods games the outbreak of cooperation among success-driven individuals under noisy conditions governing the commons. the evolution of institutions for collective action topological traps control flow on real networks: the case of coordination failures modelling supply networks and business cycles as unstable transport phenomena the selforganization of matter and the evolution of biological macromolecules the theory of economic development the changing face of economics. conversations with cutting edge economists beyond the representative agent economics with heterogeneous interacting agents modeling aggregate behavior and fluctuations in economics collection of papers on an analytical theory of traffic flows in the economy as an evolving complex system ii the economy as an evolving complex system iii evolutionary economics ecology for bankers origin of wealth: evolution, complexity, and the radical remaking of economics introduction to forest ecosystem science and management models of biological pattern formation multi-objective management in freight logistics: increasing capacity, service level and safety with optimization algorithms handbook of research on nature-inspired computing for economics and management self-control of traffic lights and vehicle flows in urban road networks biologistics and the struggle for efficiency: concepts and perspectives analytical calculation of critical perturbation amplitudes and critical densities by non-linear stability analysis of a simple traffic flow model complexity cube for the characterization of complex production systems verfahren zur koordination konkurrierender prozesse oder zur steuerung des transports von mobilen einheiten innerhalb eines netzwerkes (method for coordination of concurrent processes for noise sensitivity of portfolio selection under various risk measures the logic of failure: recognizing and avoiding error in complex situations complexity and the enterprise: the illusion of control nurturing breakthroughs: lessons from complexity theory monetary relationships: a view from threadneedle street. (papers in monetary economics, reserve bank of australia, 1975); for applications of le chatelier's principle to economics see also p. a. samuelson, foundations of economic analysis structures of social life: the four elementary forms of human relations understanding and assessing the motivations of volunteers: a functional approach happiness: a revolution in economics the wisdom of crowds: why the many are smarter than the few and how collective wisdom shapes business swarm intelligence. introduction and applications economics as a science of human behaviour: towards a new social science paradigm growth, innovation, scaling and the pace of life in cities uncertainty and surprise in complex systems scale-free networks: a decade and beyond economic networks: the new challenges hyperselection and innovation described by a stochastic model of technological evolution introduction to econophysics: correlations and complexity in finance minority games: interacting agents in financial markets economics needs a scientific revolution the economy needs agent-based modelling meltdown modelling introduction to quantitative aspects of social phenomena econophysics forum fifteen years of econophysics: worries, hopes and prospects worrying trends in econophysics econophysics and sociophysics: trends and perspectives aims and scope of the physics of socio-economic systems division of the german physical society pluralistic modeling of complex systems handbook of computational economics simulation modeling in organizational and management research developing theory through simulation methods understanding complex social dynamics: a plea for cellular automata based modelling platforms and methods for agent-based modeling from factors to actors: computational sociology and agent-based modeling artificial societies. multiagent systems and the micro-macro link in sociological theory generative social science: studies in agent-based computational modeling the handbook of experimental economics managing complexity: concepts, insights, applications engineering economy statistical physics of social dynamics the future of social experimenting the authors are grateful for partial financial support by the eth competence center "coping with crises in complex socio-economic systems" (ccss) through eth research grant ch1-01 08-2 and by the future and emerging technologies programme fp7-cosi-ict of the european commission through the project visioneer (grant no.: 248438). they would like to thank for feedbacks on the manuscript by kenett dror, tobias preis and gabriele tedeschi key: cord-013377-d4tbf05a authors: ungurean, ioan; gaitan, nicoleta cristina title: a software architecture for the industrial internet of things—a conceptual model date: 2020-09-30 journal: sensors (basel) doi: 10.3390/s20195603 sha: doc_id: 13377 cord_uid: d4tbf05a the internet of things (iot) is an emerging concept that has revolutionized the use of new technologies in everyday life. the economic impact of iot becoming very important, and it began to be used in the industrial environment under the name of the industrial internet of things (iiot) concept, which is a sub-domain of iot. the iiot changes the way industrial processes are controlled and monitored, increasing operating efficiency. this article proposes a software architecture for iiot that has a low degree of abstraction compared to the reference architectures presented in the literature. the architecture is organized on four-layer and it integrates the latest concepts related to fog and edge computing. these concepts are activated through the use of fog/edge/gateway nodes, where the processing of data acquired from things is performed and it is the place where things interact with each other in the virtual environment. the main contributions of this paper are the proposal and description of a complete iiot software architecture, the use of a unified address space, and the use of the computing platform based on soc (system on chip) with specialized co-processors in order to be able to execute in real-time certain time-critical operations specific to the industrial environment. the internet of things (iot) [1, 2] is an emerging concept that changes the interactions of people with things in everyday life. iot [3] allows the connection of ubiquitous objects/things to the internet in order to provide innovative services that can lead to time-and money-saving in the daily activity of the individuals and that can increase the quality of life [4] . the iot concept is applied in a wide variety of applications such as smart building [5, 6] smart transport [7] , smart cities [8] , smart healthcare [9, 10] , and smart living with the aim to provide new services and an efficiency of the operating costs. in the design and development of iot applications, existing technologies can be used to activate the iot concept, but new technologies can be developed to help develop the specific applications. initially, the iot concept uses only wireless technologies for connection of the things to the internet but now it can use any available technologies, wired and wireless. in fact, iot can reuse wired or wireless communication technologies that have been used in other types of application in which various devices can connect to a computing system directly or through a gateway. the things/objects form everyday life are brought into the virtual environment through sensors or other methods of acquiring real-data from their environment, such as human-machine interfaces. acquired data or virtual things are transmitted to computing platforms where they are processed, they interact between them, and decisions are made, decisions that can be transmitted to the execution sensors 2020, 20, 5603 3 of 19 in the design and development of iiot solutions, the main issues and challenges are: • the largest number and great diversity of fieldbuses and devices used in the industrial environment; • the compliance with real-time requirements specific to the industrial applications; • interoperability between fieldbuses and devices; • interoperability between iiot systems. in the specialized literature, several reference architectures for iot and iiot are proposed, but these are abstract models which do not deal with how to integrate things from the industrial environment, especially as in this environment are used communication systems (fieldbuses) with specific capabilities such as real-time monitoring and control of time-critical operations. practical examples are also presented in the specialized literature, but these solutions are designed and developed for a certain type of network and a small number of device types. this paper proposes a software architecture for iiot that integrates the concepts of fog/edge computing and fieldbuses specific to the industrial environment. the proposed architecture is organized in layers and it exploits the performances of the data processing at the edge of the network to reduce the bandwidth used for the cloud connection but also to obtain a shorter response time when dealing with time-critical operations. fog/edge computing can integrate features such as low latency, predictability, hard real-time that are very important for monitoring and controlling time-critical operations specific to the industrial environment. furthermore, the architecture contains a virtual environment used for interaction and data exchange between the virtual and/or real objects/things. unlike the iiot architectures presented in the specialized literature, the proposed iiot architecture aims to integrate many fieldbuses into the system. in addition, by using a device description language, they can be added to the system through the plug and play method. the proposal and description of complete iiot software represents the main contribution of this paper. the conceptual model for the iiot system contains some operational and implementation details that are useful in developing an iiot solution based on the proposed architecture. the strengths of the proposed architecture are: • versatility-it can be adapted to the requirements and configuration of different industrial environments; • real-time capabilities-using system on chip (soc) systems with specialized processors for the design and development of the fog nodes; • the integration of several fieldbuses at the same time; • interoperability between different fieldbuses by building a unified address space; • interoperability between iiot systems by using a standardized middleware system. this paper is structured as follows: section 2 presents some iiot architectures presented in the specialized literature and some companies, and the motivation of the proposed iiot architecture. section 3 describes the proposed iiot architecture with some discussions related the implementation. section 4 presents some discussions related to the security issues addressed by the proposed iiot architecture. section 5 presents a comparison between proposed architecture and the architectures analyzed in section 2. the conclusions are drawn in section 6. an iiot reference architecture is the abstractization of an iiot architecture that allows the identification of general components and implementation challenges. in the specialized literature, more reference architectures are proposed for iiot. in june 2019, the industrial internet consortium (iic) released version 1.9 of the reference architecture for the iiot [24] . it is focused on different viewpoints (business, usage, functional, and implementation viewpoints). the implementation viewpoint presents the system components and the technologies that can be used to implement the functionalities described in the functional viewpoint. furthermore, the implementation viewpoint contains a technical description of the architecture components, including interfaces, protocols, behaviors, and other properties [24] , but it does not contain information related to the integration of the fieldbuses and devices from the industrial environment. another important iiot reference architecture is the reference architectural model industry 4.0 (rami 4.0) [25, 26] . this is based on a 3-d model where the axes are live cycle, value stream, and hierarchy levels, and it is service-oriented architecture. the hierarchy level of the architecture is organized in the following levels: product, field devices, control devices, station, work centers, enterprise, and connected world. the value stream level of the architecture is related to the different component functionalities, it includes a communication layer and it is organized the following layers with a high level of abstraction: asset, integration, communication, information, functional, and business. other iot reference architectures are organized on layers, such that defined by the international telecommunication union (itu) that consists of four layers: devices, network, service support and application support, and application [27] . the device layer includes the device capabilities for interaction for the communication network and gateway capabilities for supporting multiple communication networks. the network layer includes networking and transport capabilities, service support, and the application support layer includes the generic and specific support for the application layer. in [28] , the authors proposed a case study on the growth of big data in iiot systems, a classification of key concepts, and a presentation of key frameworks and continued with the presentation of future technologies, opportunities, and challenges. these researches concluded that augmented reality, iot devices, cyber-physical systems and industry 4.0 big data and analytics (bda) platforms are at an early stage in iiot systems and that solutions should be found in developing new standards that allow interoperability between various platforms but also processing capability of the end-to-end applications for concentric computing systems. a solution to improve the blockchain scalability of iiot systems by guaranteeing system security, latency but also decentralization, that was proposed in the article [29] have led to performance optimization with a deep reinforcement learning technique (drl). the authors obtained results that demonstrated they can obtain a better efficiency compared to the basic parameters of the system. in another research paper [19] , the authors concluded based on conducted researches that the success of iiot can be hindered for different reasons such as challenging collaboration between various heterogeneous iiot systems, efficient data management, large, solid, and flexible data technologies, iiot protocols, operating systems, reliable iiot systems but also the coexistence of wireless technologies. in addition to a survey of existing definitions of iiot in [30] , the authors also present a proposal for a new definition of what the iiot means. the authors also present an analysis of a framework for iiot devices based on security-related issues surrounding iiot but as well as an analysis of the relationships between cyber-physical systems and industry 4.0. a secure fog-based iiot architecture by suitably plugging a number of security features that reduce the trust and burden on the cloud and resource-constrained devices, but also that reduce latency in decision making that improves the performance, is presented in another research paper from the specialized literature [31] . also, the authors demonstrated that by offloading several computationally intensive tasks to the fog nodes, the battery life of the resource-constrained of end devices is greatly saved. the validation of the architecture was demonstrated through theoretical analyses, practical experiments, but also through simulation and testbed implementation. in [32] , an iiot application for a sewage treatment plant is proposed. this iiot solution uses control station systems based on the stmicroelectronics stm32 microcontrollers to update the automation system and to activate the iiot concept. basically, the stm32-based control stations act as gateways between field devices connected to fieldbuses and their connection within the local network and further to the internet for remote monitoring and cloud connections. the monitoring operations can be performed remotely from pcs or mobile devices such as smartphones and tablets. the authors concluded that this solution provide the real-time performances and reliability required for monitoring and controlling the sewage treatment plant. in [33] , the authors define an architecture organized on four layers: sensing layer, networking layer, service layer, and interface layer. in [34] an architecture is proposed that contains the physical layer, transport layer middleware layer, and application. an architecture with three layers (perception/sensing layer, network layer, and service/application layer) is proposed in [35, 36] . all these reference architectures have a high level of abstraction and do not provide details related to the development of the iiot solutions based on these reference architectures. a trust framework for automated guided vehicles (agvs) in smart factories is presented in [37] . the authors defined a trust measure for avg, designed the trust framework, and presented a set of experiments based on this framework, but they did not provide details related to the communication with the avgs and how the data are acquired from the avgs and integrated into the proposed framework. a software architecture for iiot is presented in [38] and updated in [39] . the proposed architecture is organized on 4 levels: things, data provider, middleware, and applications. the interaction between things is performed at the application level, the upper level in the architecture. in this paper, we propose an evolution of the architecture presented in [38, 39] by including new technologies such as fog/edge computing concepts. furthermore, now things interact with each other at a lower layer, and at the application/service layer the services necessary for the development of applications are provided, such as monitoring and control specific to the industrial environment or applications for manufacturing tracing and monitoring. in addition, the proposed architecture provides implementation details, not being an abstract architecture as are most of reference architectures from the specialized literature. regarding soc systems that can be used for iot and iiot, in [40] the authors present a comparison between several single-board computers that can be used for iiot solutions, namely raspberry pi 3, beaglebone black, banana pi, and odroid-c1. of these, the most popular system is the raspberry pi, but in terms of the peripherals provided, the beaglebone black is the best. in [41] , the authors propose a soc architecture for the industrial internet of things. the soc architecture is implemented on an field programmable gate array (fpga) system with two arm processors that execute the linux operating system. the fpga gates are used to implement real-time operations specific to the industrial environment. a detailed survey related to the security in iiot is presented in [42] , where the authors performed a systematic review of the literature and identified the security requirements for iiot. these security requirements are related to the confidentiality, integrity, availability (cia) triad, authentication, access control, maintainability, resilience, data security and data sharing, security monitoring, and network security. additionally, the authors included a description of how fog computing can address these requirements and identify some research opportunity for the use of the fog computing for iiot security, namely fog-enabled authentication, fog-enabled access control, fog-enabled maintainability, fog-enabled resilience, and fog-enabled data security and data sharing. a survey for the security of the iiot protocols is presented in [43] . they focused on the main 33 protocols and standards used in iiot (from middleware systems to fieldbuses) and identify the security vulnerability with the common vulnerability scoring system (cvss). they proposed a vulnerability analysis framework (vaf) that can be used to analyze 1363 vulnerabilities for iiot systems. data security in edge-based iiot is investigated in [44] . authors identify four main challenges reliable data storage, convenient data usage, efficient data search, and secure data deletion, and they proposed a cloud-fog-edge device storage framework for iiot that addresses the challenges identified. in this section, we propose an iiot software architecture that can be used in the development of the practical iiot solutions. before describing the proposed architecture, we will discuss the main challenges in the design and development of the iiot architectures. the main requirements for the development of an iiot architecture are represented by modularity, scalability, and interoperability between the different technologies used in the industrial environment. while iot is human-centered, iiot is machine-oriented [4] and is based on existing technologies and devices for reliability. the iiot systems are a special class of iot systems that have additional requirements related to latency, real-time capabilities, security, reliability, and safety. these requirements result from the specific nature of the industrial environment where time-critical activities can be carried out. perhaps the most important challenge in developing an iiot architecture is data security because most data in the industrial environment are not public. for this reason, there must be a clear policy regarding restricted access to data only from trusted entities. another challenge in developing an iiot system is the large number of industrial networks (fieldbuses) used in the industrial environment, each with its own characteristics related to the latency, real-time capabilities, transmission rate, and line protocol. all these fieldbuses should coexist in an iiot system. furthermore, in the industrial environment, there are many heterogeneous systems (ex. devices, sensors, programmable logic controllers, and human-machine interface) that must collaborate within an iiot system. the heterogeneous nature of the systems results from the data source data, data stream, data storage, and processing requirements. another challenge is the performance related to soft and hard real-time capabilities. usually, in the industrial environment, critical activities are carried out for missions and safety with imposed requirements regarding timing and reliability. the iiot architecture proposed in this article addresses all these challenges. the proposed architecture is organized on layers and includes the edge/fog computing paradigm applied for the iiot and support for the real-time and low latency requirements specific to the industrial field. figure 1 presents the proposed software architecture organized on four layers: sensing/things, data provider, fog computing, and applications/service. in addition, fog computing and application/service layers can be connected to a cloud server to retrieve and save data. each layer includes services for management and security through which the user can configure the functionality and security implementation at the layer-level. in figure 1 the management and security capabilities are presented vertically because they transcend all layers of architecture. sensors 2020, 20, x for peer review 6 of 18 development of an iiot architecture are represented by modularity, scalability, and interoperability between the different technologies used in the industrial environment. while iot is human-centered, iiot is machine-oriented [4] and is based on existing technologies and devices for reliability. the iiot systems are a special class of iot systems that have additional requirements related to latency, real-time capabilities, security, reliability, and safety. these requirements result from the specific nature of the industrial environment where time-critical activities can be carried out. perhaps the most important challenge in developing an iiot architecture is data security because most data in the industrial environment are not public. for this reason, there must be a clear policy regarding restricted access to data only from trusted entities. another challenge in developing an iiot system is the large number of industrial networks (fieldbuses) used in the industrial environment, each with its own characteristics related to the latency, real-time capabilities, transmission rate, and line protocol. all these fieldbuses should coexist in an iiot system. furthermore, in the industrial environment, there are many heterogeneous systems (ex. devices, sensors, programmable logic controllers, and human-machine interface) that must collaborate within an iiot system. the heterogeneous nature of the systems results from the data source data, data stream, data storage, and processing requirements. another challenge is the performance related to soft and hard real-time capabilities. usually, in the industrial environment, critical activities are carried out for missions and safety with imposed requirements regarding timing and reliability. the iiot architecture proposed in this article addresses all these challenges. the proposed architecture is organized on layers and includes the edge/fog computing paradigm applied for the iiot and support for the real-time and low latency requirements specific to the industrial field. figure 1 presents the proposed software architecture organized on four layers: sensing/things, data provider, fog computing, and applications/service. in addition, fog computing and application/service layers can be connected to a cloud server to retrieve and save data. each layer includes services for management and security through which the user can configure the functionality and security implementation at the layer-level. in figure 1 the management and security capabilities are presented vertically because they transcend all layers of architecture. an important aspect of the proposed architecture is the integration of fog/edge computing concepts. thus, in the proposed architecture there are fog/edge/gateway nodes that integrate the fog/edge and data provider layers presented in figure 1 . there may be more such nodes in the iiot architecture implementation. in this context of fog/edge/gateway nodes, another perspective of the proposed architecture is presented in figure 2 (without application/service layer). in the iiot architecture, there can be several nodes, each node connects to one or more fieldbuses, and these nodes implement the fog and data provider layers for these fieldbuses. these layers are software packages implemented for a specific device based on a microprocessor or a soc (system on chip) that runs on a high-level operating system such as windows, linux, or android. these devices have peripherals such as usb (universal serial bus), can (controller area network) bus, uart (universal asynchronous receiver-transmitter), spi (serial peripheral interface), and i2c (inter-integrated circuit) that allows the interfacing with fieldbuses used in the industrial environment. for example, through usb we can connect to canopen fieldbus using a usb-can interface, the can bus can be used to connect to canopen fieldbus, the uart can be used to connect to profibus or modbus fieldbus via a uart-rs485 interface, and spi/i2c can be used to connect to canopen fieldbus via an spi-can or i2c-can interface. proposed architecture is presented in figure 2 (without application/service layer). in the iiot architecture, there can be several nodes, each node connects to one or more fieldbuses, and these nodes implement the fog and data provider layers for these fieldbuses. these layers are software packages implemented for a specific device based on a microprocessor or a soc (system on chip) that runs on a high-level operating system such as windows, linux, or android. these devices have peripherals such as usb (universal serial bus), can (controller area network) bus, uart (universal asynchronous receiver-transmitter), spi (serial peripheral interface), and i2c (inter-integrated circuit) that allows the interfacing with fieldbuses used in the industrial environment. for example, through usb we can connect to canopen fieldbus using a usb-can interface, the can bus can be used to connect to canopen fieldbus, the uart can be used to connect to profibus or modbus fieldbus via a uart-rs485 interface, and spi/i2c can be used to connect to canopen fieldbus via an spi-can or i2c-can interface. these fog/edge/gateway nodes can be implemented on various computing systems such as beaglebone ai, raspberry pi 4, industrial pcs or other types of computing system that allow connection to industrial networks and sufficient computing power for data processing. for the internet connection, 5g modules can also be used to have a high bandwidth regardless of the physical location of the node. the 5g is a technology that is beginning to be adopted by most gsm (global system for mobile communications) service providers. the 5g modules are the hardware support for the internet connection and, for this reason, we will not insist on this aspect. from the software point of view, how the connection to the internet is made is transparent. the following sections will be described in detail for each layer of the four layers in which the proposed architecture is organized. these fog/edge/gateway nodes can be implemented on various computing systems such as beaglebone ai, raspberry pi 4, industrial pcs or other types of computing system that allow connection to industrial networks and sufficient computing power for data processing. for the internet connection, 5g modules can also be used to have a high bandwidth regardless of the physical location of the node. the 5g is a technology that is beginning to be adopted by most gsm (global system for mobile communications) service providers. the 5g modules are the hardware support for the internet connection and, for this reason, we will not insist on this aspect. from the software point of view, how the connection to the internet is made is transparent. the following sections will be described in detail for each layer of the four layers in which the proposed architecture is organized. at this level, there are physical devices, sensors, actuators, and plcs (programmable logic controllers) specific to the industrial environment. these devices are connected to a specific fieldbus and can send and/or receive data from this fieldbus. examples of such fieldbuses are canopen, modbus, profibus, profinet, ethercat, and wirelesshart. at the upper layer, there must be different adapters/interfaces for communication with devices connected to fieldbuses. for example, usb-can interfaces or soc-based systems with a can port can be used for a canopen fieldbus. also, at the upper layer, there must be extensions through which the devices connected to the network can be configured, depending on the characteristics of each fieldbus. due to the nature of the industrial environment, the complexity, and the requirements for the execution of time-critical activities, some configuration operations of the field devices must be performed manually. once this operation is performed, the fieldbus can operate without major configuration changes. the purpose of this architecture is not to go into detail, it is considered that the networks are configured and at the top layer drivers specific to each fieldbus and the soc on which the fog/edge/gateway node is implemented can be designed and developed. this layer is defined in the proposed iiot architecture only from the hardware point of view, from the software point of view, at the upper level there must be modules through which the fieldbuses and devices connected to these fieldbuses can be configured. the purpose of this layer is to acquire data from devices connected to fieldbuses and store it in a buffer memory in order to be transmitted to the next layer or to send data from the memory buffer to the fieldbuses when data is received from the upper layer. it must be specified that the data flow is bidirectional, in the sense that the values from different sensors/devices can be acquired from the fieldbuses or commands that can be sent to the actuators connected on the fieldbuses. as we specified previously, this layer together with the fog/edge layer is designed and developed to be executed on fog/edge nodes. these nodes can be represented by pcs or soc-based computing systems that have features and can help to achieve real-time and reliable communication with fieldbuses. a system with sufficient computing power to process the data and peripherals needed to connect to the fieldbus that is monitored by the fog node must be chosen. the software architecture of a fog/edge/gateway node is presented in figure 3 . it can be observed from figure 3 that the data provider level connects directly to the fieldbuses, and a fog/edge node can connect to one or more fieldbuses depending on the iiot application and connection capabilities of the fog/edge node. due to the diversity of fieldbuses that can be used in the industrial environment, this layer contains a driver for each type of fieldbus that can be integrated into the proposed iiot architecture. these drivers implement the fieldbus protocol stacks for which it is designed and developed, and the connection method/way for fieldbuses must be considered, using an interface or directly throughout a peripheral (e.g., for a canopen fieldbus we can use a can port, a can-ethernet interface, or a can-usb interface, depending on the connectivity provided by the soc system used). from figure 3 , we can see that the data provider layer includes the drivers for the fieldbuses. a node can instantiate one or more drivers depending on the capabilities of the fog system and the configuration of the industrial environment that is included in the virtual industrial environment internet of things. we can design and develop drivers for mature networks that exist in the industrial environment for a long time or on new communication systems such as narrowband iot or lte cat-m based on 5g technology. in order to meet the real-time requirements specific to the industrial environment, the fog nodes can be designed and developed on soc systems with specialized coprocessors for the communication implementation with fieldbuses. for example, we can use soc systems that have one or more cores based on the arm cortex ax architecture for the execution of the complex operations and specialized processors (e.g., based on arm cortex mx architectures or other optimized architecture as proposed in [45] ) for the communication in real-time with the fieldbuses. thus, the tasks with specific real-time requirements for time-critical operations specific to the industrial environment are executed on the specialized coprocessor where these requirements can be fulfilled and guaranteed. the basic idea is to use multiprocessor soc systems that have processors on which linux embedded can be executed for the execution of non-critical operations in the best-effort (soft real-time) manner and specialized coprocessors on which a real-time operating system [46] can be executed for the implementation of the protocol stack and the execution of critical time operations. with this solution, low and predictable latencies can be achieved at the occurrence of events or at the execution of the critical operations by decoupling the software part with real-time requirements from the software part that does not have strict real-time requirements. in the case of these soc systems, the drivers have a part that runs on specialized coprocessors (real-time cores) and a part that runs on the main processors (application cores). the software part from the specialized coprocessors implements the protocol stack, and the software part from the main processors works as a wrapper that saves the unit data in a buffer memory from where it will be taken and processed by other software modules. in terms of implementation, this solution involves the development of software for asymmetric multi-processing, and great care must be taken at the implementation of the communication between processors in order not to introduce performance bottlenecks. fog nodes can also be developed on computing systems without specialized co-processors, but in this case, we can obtain at most soft real-time capabilities due to the limitations of windows and linux operating systems [47] (including real-time extensions such as rtai (real-time application interface) or preempt_rt). the specialized co-processors provide support for the development of small software modules with strict hard real-time capabilities and predictable latencies. each fieldbus will have an associated address space that defines the things connected to the fieldbus. one of the main goals of the drivers is to build the address space in a unitary way, hiding the specific details of each fieldbus and connected devices, such as the addressing mode or the in order to meet the real-time requirements specific to the industrial environment, the fog nodes can be designed and developed on soc systems with specialized coprocessors for the communication implementation with fieldbuses. for example, we can use soc systems that have one or more cores based on the arm cortex ax architecture for the execution of the complex operations and specialized processors (e.g., based on arm cortex mx architectures or other optimized architecture as proposed in [45] ) for the communication in real-time with the fieldbuses. thus, the tasks with specific real-time requirements for time-critical operations specific to the industrial environment are executed on the specialized coprocessor where these requirements can be fulfilled and guaranteed. the basic idea is to use multiprocessor soc systems that have processors on which linux embedded can be executed for the execution of non-critical operations in the best-effort (soft real-time) manner and specialized coprocessors on which a real-time operating system [46] can be executed for the implementation of the protocol stack and the execution of critical time operations. with this solution, low and predictable latencies can be achieved at the occurrence of events or at the execution of the critical operations by decoupling the software part with real-time requirements from the software part that does not have strict real-time requirements. in the case of these soc systems, the drivers have a part that runs on specialized coprocessors (real-time cores) and a part that runs on the main processors (application cores). the software part from the specialized coprocessors implements the protocol stack, and the software part from the main processors works as a wrapper that saves the unit data in a buffer memory from where it will be taken and processed by other software modules. in terms of implementation, this solution involves the development of software for asymmetric multi-processing, and great care must be taken at the implementation of the communication between processors in order not to introduce performance bottlenecks. fog nodes can also be developed on computing systems without specialized co-processors, but in this case, we can obtain at most soft real-time capabilities due to the limitations of windows and linux operating systems [47] (including real-time extensions such as rtai (real-time application interface) or preempt_rt). the specialized co-processors provide support for the development of small software modules with strict hard real-time capabilities and predictable latencies. each fieldbus will have an associated address space that defines the things connected to the fieldbus. one of the main goals of the drivers is to build the address space in a unitary way, hiding the specific details of each fieldbus and connected devices, such as the addressing mode or the message format. thus, it is possible to obtain the aggregation and processing of the data acquired from different fieldbuses to which the fog node is connected through a unique software interface, ensuring interoperability between different fieldbuses. the structure of this address space is organized in a tree and it is stored in extensible markup language (xml) files that are loaded when the software module that implements the data provider layer is instantiated. it is considered that a fieldbus can have several devices/sensors/actuators connected, and each device can have several things such as a temperature, a pressure, a level, and so on. each thing is characterized by several attributes such as data type, quality, timestamp, and access type. the data type can be numeric, string, and logic, and the quality can be good or bad. the timestamp represents the time and date of the last update of the things, and the access type can be read-only, write-only, and read-write. a sensor will have the read-only access type and an actuator can have write-only or write-read access type. an example of a space address for a device is presented in figure 4 . thus, we have a device that has n analog inputs and n digital outputs. analog inputs can be connected to sensors such as thermocouples, a resistance thermometer, pressure transducers, etc. and digital outputs that can be connected to execution elements via relays. the address space for this device built in the fog node contains n things for the n analog inputs and n things for the n digital outputs, as illustrated in figure 4 . sensors 2020, 20, x for peer review 10 of 18 message format. thus, it is possible to obtain the aggregation and processing of the data acquired from different fieldbuses to which the fog node is connected through a unique software interface, ensuring interoperability between different fieldbuses. the structure of this address space is organized in a tree and it is stored in extensible markup language (xml) files that are loaded when the software module that implements the data provider layer is instantiated. it is considered that a fieldbus can have several devices/sensors/actuators connected, and each device can have several things such as a temperature, a pressure, a level, and so on. each thing is characterized by several attributes such as data type, quality, timestamp, and access type. the data type can be numeric, string, and logic, and the quality can be good or bad. the timestamp represents the time and date of the last update of the things, and the access type can be read-only, write-only, and read-write. a sensor will have the read-only access type and an actuator can have write-only or write-read access type. an example of a space address for a device is presented in figure 4 . thus, we have a device that has n analog inputs and n digital outputs. analog inputs can be connected to sensors such as thermocouples, a resistance thermometer, pressure transducers, etc. and digital outputs that can be connected to execution elements via relays. the address space for this device built in the fog node contains n things for the n analog inputs and n things for the n digital outputs, as illustrated in figure 4 . the data acquired from the fieldbuses are saved in a buffer memory, where they will be retrieved and transmitted to the upper layer. the buffer memory is allocated according to the definition found in the xml configuration files associated with each fieldbus. the user has the possibility to configure the communication parameters on the network as well as the introduction or elimination of new devices. this address space is used by the upper layer to read/transmit data to the thing. a thing can be accessed through a path in the form of "network name. device name. thing name". each device will have associated an xml file that defines all the things and attributes of the things it makes available. these files contain generic names for things that can be replaced with aliases in the network setup process. each fieldbus will have an associated xml file in which network-specific configurations (such as communication speed) and the list of devices connected to the fieldbus along with the associated xml files are stored. a fog/edge node has an associated xml file in which the list of fieldbuses to which it connects with the associated xml files are stored. these files are automatically updated during the configuration of fieldbuses to which the fog node connects and loaded when the software module that implements the functionalities of the fog node is executed. the data acquired from the fieldbuses are saved in a buffer memory, where they will be retrieved and transmitted to the upper layer. the buffer memory is allocated according to the definition found in the xml configuration files associated with each fieldbus. the user has the possibility to configure the communication parameters on the network as well as the introduction or elimination of new devices. this address space is used by the upper layer to read/transmit data to the thing. a thing can be accessed through a path in the form of "network name. device name. thing name". each device will have associated an xml file that defines all the things and attributes of the things it makes available. these files contain generic names for things that can be replaced with aliases in the network setup process. each fieldbus will have an associated xml file in which network-specific configurations (such as communication speed) and the list of devices connected to the fieldbus along with the associated xml files are stored. a fog/edge node has an associated xml file in which the list of fieldbuses to which it connects with the associated xml files are stored. these files are automatically updated during the configuration of fieldbuses to which the fog node connects and loaded when the software module that implements the functionalities of the fog node is executed. an important part of the xml file associated with a device is the part that describes how the value of a thing is read or written. this is translated into messages that can be transmitted and read on the fieldbuses, depending on the characteristics of each network. this means that the xml file must also be interpreted by the driver to take over the commands/messages specific to each thing. depending on the type of network, an acquisition cycle can be defined by which data in the buffer are updated periodically and prioritized depending on the criticality of the monitored data and the user configuration. an example of such an acquisition cycle is presented in [48] . at this layer, the local processing of the data acquired from the industrial fieldbuses is performed and it is the place where things interact with each other. practically at this level, the operations specific to the edge and fog computing concepts are performed. thus, in order to interact between the things, it is proposed to use the publisher-subscriber paradigm, in the sense that a thing publishes its value periodically or when an event occurs, and other things subscribe to this value. there are two types of thing: physical things that are associated with things defined at the data provider layer and virtual things that can be defined at this level and represent the result of processing data from physical and/or virtual things. the virtual things subscribe to one or more values published by the other things, perform certain processing on the read values (mathematical operations, mathematical functions, aggregation functions, etc.) and the obtained result is published as a new value. from the functional point of view, no distinction is made between virtual things and physical things. things can connect to one or more things in a virtual communication environment that is based on the publish-subscribe paradigm and that performs some processing on data and can publish its value. a graphical representation of the virtual publishing-subscriber environment is shown in figure 5 . some things only publish data, for example, things associated with a sensor such as temperature sensor, pressure sensor, and things that subscribe to one or more values but do not publish any values, such as things that are associated with actuators from the industrial environment. internally, things perform a wide range of data processing tasks such as mathematical operations, mediation, aggregation, or other mathematical functions. there may be cloud things that subscribe to certain data and save it to a remote or local cloud server, or things that connect to data and save it to a remote or local database. these types of thing perform certain cloud services on the fog node, such as storage services. the interaction between things can be undertaken at the level of a fog/edge node, but we also want the interaction between things from different fog/edge nodes. in order to achieve requirements related to the interaction between things, a middleware system that provides support for publish-subscribe communication can be used. at the moment, there are several middleware systems that operate on the publisher-subscriber paradigm, such as the advanced message-queuing protocol (amqp) [49] , message-queuing telemetry transport (mqtt) [50] , and data distribution service (dds) for real-time systems [51] . we focused on middleware systems that are based on the publish-subscribe paradigm because the iiot system will be much more versatile. there are other important middleware systems that are widely used in the development of iot systems [52] such as xmpp (extensible messaging and presence protocol) or coap (constrained application protocol) but these middleware systems have not been considered because they are not based on the publish-subscribe paradigm. from the middleware systems that are based on the publish-subscribe paradigm, we suggest the use of dds for real-time systems is chosen because it was designed specifically for real-time systems. in order to argue for the selection of the dds protocol, the study by [52] can be analyzed, where the http (s), coap, mqtt, amqp, xmpp, and dds are identified as the main middleware systems based on messages that can be used in the development of the iot systems. from these middleware systems, mqtt and amqp use a broker that can be a bottleneck point. the dds is the only one that uses a bus-like architecture (mqtt uses tree architecture and amqt uses star architecture), has 23 quality of service (qos) levels (mqtt and amqp uses 3 qos levels), is data-centric, and implements a security mechanism like tls, dtls, and dds security. also, the data distribution is very versatile with the possibility to achieve 1 to 1, 1 to n, and n to 1 [52] . by using a standard middleware system, interoperability can be ensured between the iiot systems and with other systems could take data from the iiot system. shown in figure 5 . some things only publish data, for example, things associated with a sensor such as temperature sensor, pressure sensor, and things that subscribe to one or more values but do not publish any values, such as things that are associated with actuators from the industrial environment. internally, things perform a wide range of data processing tasks such as mathematical operations, mediation, aggregation, or other mathematical functions. there may be cloud things that subscribe to certain data and save it to a remote or local cloud server, or things that connect to data and save it to a remote or local database. these types of thing perform certain cloud services on the fog node, such as storage services. thus, a dds domain is defined for the architecture, which is divided into several dds partitions, a dds partition corresponding to a fog/edge node. dds defines a unit of information as a topic. thus, for each thing defined at the data provider level, a topic will be associated, which will have the unique name of the access path defined at the previous level. in addition to these things, virtual things are also defined, things that can subscribe to different dds topics, and different processing is performed on the received data. all these things are created by the user in the configuration process based on predefined things that are instantiated and that subscribe to one or more topics. perhaps the most important predefined virtual thing is the expression thing in which certain mathematical operations and functions can be applied to the data with which it is subscribed. thus, the user can create virtual things according to his needs and in accordance with the application of the industrial environment that is integrated into the iiot architecture. due to the solution adopted for the interaction between things, the architecture is very versatile, being able to be used in a wide range of industrial applications. in the case of using the dds middleware system, the publishing-subscription environment from figure 5 can be represented as in figure 6 . the connections between things are not made only locally, but in the dds domain, which is defined at the level of the iiot system, things from the dds domain can be from the same node or from remote fog/edge nodes in a local network or on the internet. as can be seen from figure 6 , in the case of dds middleware, each topic is associated with different qos policies. these policies can be related to data availability, data delivery, data timeliness, and resources. for data availability, we can control how data is available within a domain through the durability, lifespan, and history policies. for data delivery, we can control how data is delivered and exclusive rights for data through the presentation, reliability, partition, destination_order, and ownership policies. for data timeliness, we can control the timeliness for the data through the deadline, latency_budget, and transport_priority policies. through the resources policies, we can control the computing and network resources. all of these policies are described in the dds specifications and they are included in the implementations of these specifications [51] [52] [53] . as can be seen from figure 6 , in the case of dds middleware, each topic is associated with different qos policies. these policies can be related to data availability, data delivery, data timeliness, and resources. for data availability, we can control how data is available within a domain through the durability, lifespan, and history policies. for data delivery, we can control how data is delivered and exclusive rights for data through the presentation, reliability, partition, destination_order, and ownership policies. for data timeliness, we can control the timeliness for the data through the deadline, latency_budget, and transport_priority policies. through the resources policies, we can control the computing and network resources. all of these policies are described in the dds specifications and they are included in the implementations of these specifications [51] [52] [53] . the application/services layer is the place where industry-specific applications can be developed, such as those for remote monitoring and control. we can develop a supervisory control and data acquisition (scada) type application by subscribing and publishing in the dds domain where the things interact with each other. at this layer, it can be designed and developed software solutions/applications that can monitor things in the industrial environment and send commands to these things. also, based on the data saved on the fog nodes or in the cloud, different reports and graphical representations can be made regarding the evolution of the data over time and of the industrial process. at this layer and depending on the capabilities of the fog nodes, on each fog node a web server can be developed that allows the visualization of the values published by the fog node in the dds domain. the web server can also have an extension that provides the facility to configure the fog/edge node remotely, especially since fog/edge nodes can operate without the graphical interface. at this layer, an hmi application can be designed and developed like that presented in [39] and which can be used to graphically visualization of data acquired from the industrial environment and send commands in the industrial environment through the graphical interface. the application/services layer is the place where industry-specific applications can be developed, such as those for remote monitoring and control. we can develop a supervisory control and data acquisition (scada) type application by subscribing and publishing in the dds domain where the things interact with each other. at this layer, it can be designed and developed software solutions/applications that can monitor things in the industrial environment and send commands to these things. also, based on the data saved on the fog nodes or in the cloud, different reports and graphical representations can be made regarding the evolution of the data over time and of the industrial process. at this layer and depending on the capabilities of the fog nodes, on each fog node a web server can be developed that allows the visualization of the values published by the fog node in the dds domain. the web server can also have an extension that provides the facility to configure the fog/edge node remotely, especially since fog/edge nodes can operate without the graphical interface. at this layer, an hmi application can be designed and developed like that presented in [39] and which can be used to graphically visualization of data acquired from the industrial environment and send commands in the industrial environment through the graphical interface. security is an important issue in any application distributed over the internet. in the case of iiot, it is even more important because non-public data is transmitted over the internet, and access to this data must be granted on the basis of clear security policies. at the level of things/sensing, security is ensured by the capabilities of each fieldbus. usually, in these fieldbuses, the security mechanisms are not implemented because not everyone has physical access to them. from a security point of view, the risks at this level are small, especially if physical access to them is restricted. in addition, devices connected to networks can have different security mechanisms in place. from the point of view of the current iiot architecture, the security at this level depends on the configuration made at the level of each fieldbus and filed device used, and no new security mechanisms are implemented because this level does not involve the development of software modules. at higher layers, the security mechanisms must be implemented at the fog node level. remote access to fog nodes must be restricted, thus avoiding any security holes that may occur. the operating system must also be kept up to date with the latest security updates. configuration xml files can be encrypted. this does not degrade the performance because they are decrypted when starting the software modules to implement the data provider and fog/edge computing layers, and they are saved in encrypted mode following the configuration operations performed by the user. the biggest security issue can be when publishing data in the publisher-subscriber environment provided by the dds domain defined for the current iiot architecture because those messages can be intercepted when they are transmitted over the internet. in 2018, the latest security specifications for the dds protocol were published. these specifications are also implemented in the opendds implementation, an open-source implementation that can be used for the design and development of fog/edge nodes. the specifications provide the plugins for authentication, access control, logging, data tagging, cryptography, and certificates [54] . through these facilities, the data are transmitted encrypted over the internet ensuring a high degree of security regarding confidentiality, integrity, and availability. table 1 presents a comparison between the proposed architecture and those described in the related work section. a more detailed quantitative or qualitative analysis between the proposed iiot architecture and the existing ones requires more information than those presented in the literature. moreover, we propose a conceptual model and the experimental results will be implemented and presented in future research articles. for example, we can use beaglebone black for implementation. this is based on the sitara™ am335x soc with 2 arm cortex a8 processors and two programmable real-time unit subsystem and industrial communication subsystems (pru-icss). the arm cortex a8 processors use a linux-based operating system. one pru-icss can be used to design and implement a driver for the modbus rtu (remote terminal unit) fieldbus and the other pru-icss can be used to design and implement a driver for the canopen fieldbus. the data provider, fog computing, and applications/service layers can be designed and implemented to be executed on the arm cortex a8 processors under linux. by using many of beaglebone black systems, we can build a complex iiot system that can be geographically distributed. the proposed iiot software architecture the use of the soc-based computing platform with specialized co-processors in order for fog/edge nodes. four layers: sensing/things, data provider, fog/edge, applications/services fog/edge nodes yes. it is proposed an unified address space and a driver for each field bus integrated this paper proposed a conceptual model software architecture for the iiot. the proposed architecture is a conceptual model with a low level of abstraction, being oriented towards the design and development of real-time applications for the iiot. among the strengths of the proposed architecture, we can mention: the integration in the system of many fieldbuses used in the industrial environment, addressing the specific requirements of the industrial environment such as real-time and low-latency capabilities, and the integration in the architecture of the concepts of fog and edge computing. also, the use of the soc-based computing platform with specialized co-processors is proposed in order to be able to execute in real time certain time-critical operations specific to the industrial environment. the specialized co-processors provide support for the development of small software modules with strict hard real-time capabilities and predictable latencies while the main processors provide support for the development of software modules with soft real-time capabilities. we can conclude that the conceptual model is feasible for a combination of hard and soft real-time systems with hard real-time constraints provided by the specialized co-processors. the fog/edge computing can integrate features such as low latency, predictability, and hard real time that are very important for monitoring and controlling time-critical operations specific to the industrial environment. furthermore, the architecture contains a virtual environment used for the interaction and data exchange between the virtual and/or real objects/things located on the same fog / edge node or on different fog/edge nodes. as future work, we intend to implement the proposed architecture on beaglebone black and beaglebone ai systems. privacy enhancing technologies in the internet of things: perspectives and challenges internet of things forensics: recent advances, taxonomy, requirements, and open challenges industrial internet of things: challenges, opportunities, and directions energy efficiency in smart buildings: iot approaches monitoring and control system for smart buildings based on opc ua specifications technology selection for iot-based smart transportation systems smart city iot services creation through large-scale collaboration an internet of things framework for remote monitoring of the healthcare parameters speech analysis for medical predictions based on cell broadband engine making internet of things real the growth in connected iot devices is expected to generate 79.4zb of data in 2025 a smart manufacturing service system based on edge computing, fog computing, and cloud computing edge computing in the industrial internet of things environment: software-defined-networks-based edge-cloud interplay structuring reference architectures for the industrial internet of things customer expectation from industrial internet of things (iiot) deploying fog computing in industrial internet of things and industry 4.0 literature review of industry 4.0 and related technologies industrial internet of things: recent advances, enabling technologies and open challenges industrial-iot-iiot-market-worth-263-4-billion-by-2027-exclusive-report-covering-pre-and-post-covid-19-market-analysis-and-forecasts-by-meticulous-research.html the industrial information technology handbook industrial communication systems and their future challenges: next-generation ethernet, iiot, and 5g narrowband internet of things: a comprehensive study analysis of the cyber-security of industry 4.0 technologies based on rami 4.0 and identification of requirements an architecture based on rami 4.0 to discover equipment to process operations required by products internet protocol aspects and next-generation networks, next generation networks-frameworks and functional architecture models, overview of the internet of things the role of big data analytics in industrial internet of things performance optimization for blockchain-enabled industrial internet of things (iiot) systems: a deep reinforcement learning approach the industrial internet of things (iiot): an analysis framework das bit, s. a secure fog based architecture for industrial internet of things and industry 4.0 renovation of automation system based on industrial internet of things: a case study of a sewage treatment plant internet of things in industries: a survey efficient naming, addressing and profile services in internet-of-things sensory environments. ad hoc netw rfid technology and its applications in internet of things (iot) an overview of the internet of things for people with disabilities a trust-based team formation framework for mobile intelligence in smart factories an iot architecture for things from industrial environment a middleware based architecture for the industrial internet of things, ksii transactions on internet & information systems analysis of single-board computers for iot and iiot solutions in embedded control systems smart sensor: soc architecture for the industrial internet of things a systematic survey of industrial internet of things security: requirements and fog computing opportunities a survey of iiot protocols toward data security in edge intelligent iiot improving interrupt handling in the nmpra timing comparison of the real-time operating systems for small microcontrollers hard real-time computing systems a flexible acquisition cycle for incompletely defined fieldbus protocols industrial shop-floor integration based on amqp protocol in an iot environment vulnerabilities and limitations of mqtt protocol used between iot devices dds-based protocol-compatible communication platform for mining power system the infrastructure used for this work was partially supported by the project "integrated center for research, development and innovation in advanced materials, nanotechnologies, and distributed systems for fabrication and control", contract no. 671/09.04.2015, sectoral operational program for increase of the economic competitiveness co-funded from the european regional development fund. the authors declare no conflict of interest. the funders had no role in the design of the study; in the collection, analyses, or interpretation of data; in the writing of the manuscript, or in the decision to publish the results. key: cord-005384-204jch3h authors: gao, jun; zeng, lingjie; cao, changsheng; ye, wei; zhang, xu title: multi-objective optimization for sensor placement against suddenly released contaminant in air duct system date: 2017-05-16 journal: build simul doi: 10.1007/s12273-017-0374-z sha: doc_id: 5384 cord_uid: 204jch3h when a chemical or biological agent is suddenly released into a ventilation system, its dispersion needs to be promptly and accurately detected. in this work, an optimization method for sensors layout in air ductwork was presented. three optimal objectives were defined, i.e. the minimum detection time, minimum contaminant exposure, and minimum probability of undetected pollution events. genetic algorithm (ga) method was used to obtain the non-dominated solutions of multiobjectives optimization problem and the global optimal solution was selected among all of the non-dominated solutions by ordering solutions method. since the biochemical attack occurred in a ventilation system was a random process, two releasing scenarios were proposed, i.e. the uniform and the air volume-based probability distribution. it was found that such a probability distribution affected the results of optimal sensors layout and also resulted in different detect time and different probability of undetected events. it was discussed how the objective functions are being compatible and competitive with each other, and how sensor quantity affect the optimal results and computational load. the impact of changes on other parameters was given, i.e. the deposition coefficient, the air volume distribution and the manual releasing. this work presents an angle of air ductwork design for indoor environment protection and expects to help in realizing the optimized sensor system design for sudden contaminant releasing within ventilation systems. buildings are particularly vulnerable to chemical and biological warfare (cbw) agent contamination since ventilation system and the central air-conditioning serves as a natural carrier for spreading the released contaminant from an artificial source in the ductwork or indoor environment. such kind of contaminant can spread out to the entire indoor environment within a short period of time (xue and zhai 2017; das et al. 2014; chen et al. 2014; zhang et al. 2013) . particular examples of cbw agent contamination include a revelation that terrorists planned to put cyanide into airconditioning system of public buildings across the united states in 2011 (pearson 2011) and an investigation that the sars virus spread through ventilation system and resulted more people in disease in 2003 (kowalski et al. 2003) . such intentional events have highlighted the potential exposure of indoor occupants to hazardous chemical and biological substances. bank (2008, 2010) indicated that the most potentially dangerous scenario for a bioterrorism attack on a building involved introducing an aerosolized agent into a building's ventilation system. as in the case of the cbw agents, rapid detection could help isolate the spread of the contaminants before a large number of people were exposed through the ventilation operation (bastani et al. 2012) . therefore, early detection and warning of cbw agents released in air ductwork play important roles in protecting the occupants and minimizing the adverse consequences when facing such sudden events. a previous study (zhou et al. 2015) has considered sensor design for monitoring contaminant dispersion in indoor environments, but it has ignored the agent transportation in ventilation system. glover (2002) presented an idea that the most efficient points to locate such detection sensors would be the fresh air intake build simul (2018 ) 11: 139-153 https://doi.org/10.1007 list of symbols a ij priority order number obtained by x i and x j comparing for all objectives. c u m agent concentration at upstream main duct of node m (mg/m 3 ) c dm−1 agent concentration at downstream main duct of node m−1 (mg/m 3 ) e i (x) inequality constraint e k total contaminant exposure for releasing at the k-th node (mg) exp (m, t) contaminant exposure for air outlet m at time t (mg) j det the minimum detection time (s) j pro the minimum probability of undetected pollution events j rel the minimum contaminant exposure (mg) n number of potential releasing locations p k probability for contaminant releasing at the k-th node q k air volume at the k-th node (m 3 /h) q m air volume at air outlet m (m 3 /h) r j (x) equality constraint s total number of air outlets t det-k the shortest detection time among all the distributed sensors for contaminant releasing at the k-th node (s) t m−1 time at the node m−1 (s) u m−1 air velocity along the duct between node m−1 and m (m/s) u k u k = 0 if the k-th contamination event is detected by sensors, otherwise, u k = 1 x m−1 distance between source and node m−1 (m) μ near peak time of agent concentration at the nearest sensor node (s) σ near population variance at the nearest sensor node (s) ε x agent eddy diffusivity (m 2 /s)  better performance evaluation of an objective function or the mixing box in the ventilation system, but this idea of sensor placement had no specific optimization for the ventilation system. a research group at uc berkeley (usa) and lawrence berkeley national laboratory (lbnl) did a plenty of seminal studies on incorporating such sensors into a monitoring system designed to protect building occupants (sreedharan et al. 2006 (sreedharan et al. , 2007 sohn and lorenzetti 2011) . sreedharan et al. (2007 sreedharan et al. ( , 2011 ) explored a bayesian interpretation approach to characterize an indoor release using threshold sensor data and applied this approach to assess the effects of various sensor characteristics, such as response time and accuracy. walter et al. (2012) presented a probabilistic approach to design an indoor sensor network for detecting an accidental or intentional chemical or biological release, and demonstrated it by optimizing networks against uncertain release locations, source terms and sensor characteristics. the group's work has greatly developed the monitoring system in buildings to minimize the adverse impact when facing biochemical attacks. however, the calculation model used in their study cannot simulate a sudden pollution occurring in the ventilation system. due to the effect of convection, contaminant spreads much faster in air ductworks than in rooms. it is of significant importance to devise a monitoring system in air ductwork that can detect, locate, and characterize accidental cbw agents. in this work, an optimization method for sensors layout in the air duct system is presented. it can serve as a supplementary study of the lbnl's work and can provide an optimization approach considering both the rooms and the ventilation systems in buildings. to achieve the maximum protection of occupants, sensor design for the air duct system needs to be carefully considered. chen and wen (2008) used both detection time and total occupant exposure as optimization objective functions for the contaminant sensor system design in buildings. genetic algorithm (ga) method was applied to optimize the sensor sensitivity, location and quantity in order to obtain the best sensor layout while reducing the total system cost. in their work, they assumed that the agent source was located at the indoor area and each attack scenario had the same probability of occurrence. however, this assumption is not sufficient when the terrorists choose to attack the ventilation system directly, because sensors located at indoor zones cannot detect the agent timely until it spreads into rooms through air outlets. thus, to design a sensor layout for ventilation system is at least as important as for the indoor environment. in order to resolve the problem for optimal sensor layout in the air ductwork, the airflow pattern and contaminant dispersion need to be known. the airflow pattern in a ductwork is relatively easy to be obtained due to the air volume distribution determined by the ventilation system design and operational commissioning. as for the contaminant dispersion in ductwork after the releasing of a cbw agent, you and wan (2014) derived new models for predicting the concentration dynamics in the straight duct section after a particle resuspension or puff injection. then, an analytical solution method for species transport through the air duct system was derived by gao et al. (2016) and was adopted in this study. sensor layout design is a multi-objective optimization problem (eliades et al. 2013; zhang et al. 2007; ostfeld and salomons 2004; ahmad et al. 2016; chen and wen 2012) . it is discussed widely in water distribution field. watson et al. (2009) showed the ability of robust sensor placement for municipal water distribution networks in reducing the amount and magnitude of 9/11-style attacks. bazargan-lari (2014) presented a new methodology to identify the best layout of water quality monitoring stations for drinking water distribution systems threatened by deliberate contamination events. in this study, the evidential reasoning (er) approach was applied to rank the obtained feasible sensor placements and a simulation model (epanet) was embedded to predict the contaminant transport. in indoor air field, chen and wen (2010) utilized a multi-zone modeling approach to design systematic sensor system in buildings. it had been proved that sensor systems designed using data from simpler airflow models could perform as well as those designed using complex cfd model. arvelo et al. (2002) described the enhancements made to the contam multi-zone model and for determining the optimal placement of cbw sensors in a canonical facility representative of an office building. liu and zhai (2009) introduced an adjoint probability method to assist in optimizing high-performance iaq sensor placement and identifying potential contaminant source locations for a residential dwelling based on limited sensor outputs. more recently, fontanini et al. (2016) presented a data driven sensor placement algorithm based on dynamical systems approach. in this approach finite dimensional perron-frobenius (pf) concept was applied. the pf operator was used to construct an observability gramian that naturally incorporated sensor accuracy, location and sensing constraints. the method presented in their work could determine the response times, sensor coverage maps, and the number of sensors needed for an enclosed environment. the aforementioned studies provided several optimization methods on sensor placement and design in indoor environment and water distribution system. however, few studies have been carried out to consider the optimal sensor layout in the ventilation duct system. various methods can be used to solve the sensors layout optimization problem. the probabilistic approach based on the multi-zone contaminant transport model was applied to indoor monitoring system design (sreedharan et al. 2007; walter et al. 2012) . as far as we know that multi-objective optimization problem has competing constraints and objectives, which can produce numerous non-dominated solutions (carr et al. 2006) . to obtain the optimal solution from a large number of feasible solutions a robust algorithm is required. the ga method is one such algorithm which can address this issue (shastri and diwekar 2006) . meier and barkdol (2000) utilized ga to optimize the locations of flow measurement needed for the calibration of the water supply network. preis and ostfeld (2008) proposed the use of ga to study a multi-objective model for optimal sensor placement in the water distribution system, but the limitation was that they assumed the contaminant source was known. walter et.al (2012) doubted that the probability of release points must be considered as part of the sensor network optimization for indoor environment and this type of uncertainty is difficult to consider using ga. however, since the air duct system had more similarities with the water distribution system in both contaminants transport and hydraulic characteristics than indoor environment, we draw on the experience of sensor network design for the water distribution system for which ga method was used as an optimization tool. whenever sensors are too expensive to deploy widely throughout the whole ventilation system, the ga method may help balance the competing design constraints and the objectives of the sensor network. developing a sensor system for a specific ventilation system involves the type and number of individual sensors, their placement, the optimization objectives and the methodology of solving multi-objectives problem. this work focused on the optimal sensors layout in the entire ductwork, and took into account three optimized objective functions, i.e., minimum detection time, minimum contaminant exposure and minimum probability of undetected pollution events. the ga method was used to obtain the non-dominated solutions of multi-objectives optimization problem and the global optimal solution was selected among all of the non-dominated solutions by ordering solutions method. this work was to present an optimization method for sensors placement in the air duct system. it was also to check the required number of sensors and the independence of objective functions during the sensor layout optimization. this section is to present an optimization method for sensor placement in an air duct system in the puff-releasing scenarios. to minimize the adverse impact on the building environment served by a ventilation system, three optimal objective functions for sensor placement were selected, minimum detection time f 1 , minimum contaminant exposure f 2 , and minimum probability of undetected pollution events f 3 . ga method was used to obtain the non-dominated solutions of sensor layouts based on the calculated contaminant data. the global optimal solution for sensor layout was selected among all of the non-dominated solutions by ordering solutions method. when the basic information about air and pollutant transport in ductwork is available, the main task for optimal sensor layout is to choose a good optimization approach. to minimize detection time and occupant exposure in an artificial puffreleasing scenario, several objective functions may exist and need to be extensively defined in section2.2. here, it is confirmed that the optimization problem for this study is actually to determine the vector of decision variables that satisfies a series of constraints and optimizes a vector function whose elements represent the objective functions (chang et al. 2012; preis and ostfeld 2008) . consequently, such kind of multi-objective optimization problem can be generally expressed as follows: is vector of decision variables. s means the number of inequality constraints. l denotes the number of equality constraints. the optimization goal for sensor layout is to find one set of solution which yield optimal values with respect to all the defined objective functions from all sets of solutions satisfying eqs. (2) and (3). this group of solutions is entitled as the non-dominated solution set. each solution x in the non-dominated solution set is optimal in the sense that it is not possible to improve one objective without making at least one of the others worse (deb and kalyanmoy 2001) . any two solutions x 1 and x 2 are compared based on domination, where a solution x 1 dominates x 2 if the following conditions hold. if x 1 is strictly better than x 2 in at least one objective, then if x 1 is not worse than x 2 in all objectives, then where, "  " indicates a better performance evaluation of an objective function, m is the number of objective functions which will be determined in section 2.2. the contaminant dispersion process within the ductwork is a complicated non-linear process and the contaminant source identification is a multi-objective optimization problem. figure 1 describes the procedure of the method applied. we generate n random solutions to serve as the initial population and fitness of each solution is calculated and fig. 1 flow chart of the proposed optimization methodology applied for the sensor layout with multi-objectives in air duct system compared for all objectives. the non-dominated solutions are obtained if the conditions in eqs. (4) or (5) are met. the ordering solutions method is used to rank the nondominated solutions, and the best solution in the current population is stored in the procedure. then a new population is generated by ga operator (selection, crossover and mutation) and the information of the previous generation is retained in the new population. once a new population is generated, the aforementioned steps are repeated again. finally we obtain the global optimal solution if the process repeats for a predefined number of generations or the value of each objective tends to be constant. the objective functions for designing sensor systems for building protection purpose should be able to detect contaminants within a period of time that poses the least threat to building occupants across multiple releasing locations (chen and wen 2010) . this paper introduced three optimal objective functions, two recommended in the indoor environment by chen and wen (2008) and an extra one applied in the field of water pollution krause et al. 2008 ). the first objective function used is to minimize the detection time j det , as follows: where, p k is the probability for contaminant releasing at the k-th node. t det-k is defined as the shortest detection time among all the distributed sensors in the ductwork when the contaminant is released at the k-th node. n is the number of releasing locations. to obtain the required detection time t det-k , the corresponding time when the particle concentration occurs at the k-th node needs to be introduced. in the present work, we firstly determined a steady-state air mass distribution in the ductwork and then calculated the dynamic particle concentration following a sudden release. we took into consideration both the turbulent diffusion and deposition effect to derive the theoretical solution for the concentration dynamics in the ducts. mass balance for the contaminant in the ductwork is shown as follows (gao et al. 2016; zhao et al. 2003; feustel 1999; zhou et al. 2011 where, c is the particle concentration which is the function of location x and time t. ε x is the particle eddy diffusivity. k is the deposition coefficient. the solution for eq. (7) with puff releasing boundary could be expressed as follows (gao et al. 2016) : obviously, eq. (8) only describes the particle dispersion in main duct and has no regard for branches and outlets. however, the main and branch ducts or outlets in the ventilation system are usually inter-connected through junctions as tee or cross to form a whole ductwork. thus, we develop a general model that contains branches and outlets to extend advection-diffusion main duct flow to network flow. particle concentration at outlet m is expressed as where, u m c is the particle concentration at outlet m. u m−1 is air velocity along the duct prior to outlet m. d 1 m cis the particle concentration at downstream duct of outlet m−1. t m and t m−1 denote the time at the center of outlets m and m−1. x m and x m−1 denote the distance between the source and outlets. equation (9) has some similarities with normal distribution with parameters (μ, σ 2 ), where μ is the population mean and σ 2 is the population variance. based on the theory of statistics, we defined  and assumed the deposition coefficient k=0. then, eq. (9) could be adapted as following: according to eq. (10), if we assume that , the peak concentration at outlet m and the corresponding time when it occurs can be obtained as follows, respectively: as shown in fig. 2 , since and μ = t p m , the area beneath the dynamic concentration curve within [μ−3σ, μ+3σ] accounts for 99.73% of total area, i.e. the particle concentration outside the time range from μ−3σ to μ+3σ can be neglected. then, the onset time of particle concentration at the k-th node is obtained as (gao et al. 2016 ): here we introduced a kind of ideal sensor whose lower limit of detection is equal to the agent concentration at time μ−3σ. consequently, t det-k can be derived as the onset time of agent concentration at the nearest sensor node, i.e. the shortest detection time among all the distributed sensors in the ductwork. where, μ near is the peak time of particle concentration at the nearest sensor node. σ near is the population variance at the nearest sensor node. it should be noted that the derivation of eqs. (8)-(14) is feasible and reliable, based on the experiments using dehs (diethylhexyl sebacate) aerosols as the representative pollution (gao et al. 2016 ). the second objective function is to minimize the contaminant exposure j rel (chen and wen 2008) . total contaminant exposure e k , for releasing at the k-th node is defined as where, exp(m, t) is the contaminant exposure for the m-th air outlet at time t, and it can be calculated by eq. (16). s is the number of air outlets. for each air outlet, the exposure is approximately defined as the total contaminant mass outflow at the m-th air outlet after a puff release of contaminant. where, q m is the air volume at outlet m. the definition of other parameters in eq. (16) is the same as the one described above. then, for all n potential releasing nodes, the objective function based on the total exposure load j rel , is defined as the performance of an early warning system depends on the number of successful detections, i.e., the undetected pollution events should be as few as possible. to further increase the reliability of sensor layouts, an extra objective function was introduced to minimize the probability of undetected pollution events in this study. where, u k = 0 if the k-th contamination event is detected by the sensors. otherwise, u k = 1. n s is the total number of pollution events specified in the study. a ventilation system installed in a five-star hotel (suzhou, china) was selected as the prototype ductwork for this study. as shown in fig. 3 , the system is an all-fresh air system without air return and serves a banquet hall through air diffusers. therefore, there is a single-direction plug-flow pattern along the air ductwork in both air-conditioning and ventilation mode. it is reasonable to extract this system from the building for the case study of multi objective sensor layout optimization. fig. 3 layout of an air ductwork system applied for the optimal sensor placement, which is a full-fresh air-conditioning system serving a banquet hall in a five-star hotel. the main ducts are labelled with red as shown in fig. 4 , this prototype ductwork can be divided into 6 main ducts, 50 branch ducts and 52 nodes, where the total supply air volume at fan is 24210 m 3 /h. thirty air supply outlets are arranged along the ductwork. the spacing between the nodes is set as 4 m in case studies. air supply rate of each outlet is basically equivalent through the hydraulic commissioning before system operation. the main parameters of this ventilation system were provided in table 1 . since the biochemical attack occurred in the ventilation system is a random process, it is of practical significance to decide the typical releasing probability at the different fig. 4 prototype ductwork serving a banquet hall extracted from a real hotel building. the number together with the node is the location identifier within the air duct system. the main ducts are labelled with red and the number together with the duct is the main duct identifier nodes along the ductwork reasonably. to represent the typical releasing scenarios in the air ductwork, two modes of probability distribution throughout the releasing nodes are discussed in this study. the first mode is the average probability distribution which assumes uniform probability for each releasing node. the other one is defined as that the probability for each releasing node depends on the air volume, which means the attack should be much elaborately planned by someone familiar with the ventilation system. the two modes of probability distribution are achieved by mathematical definition of p k , the probability for contaminant releasing at k-th node. for the uniform probability distribution, where, n is the total number of concerned releasing nodes. for the air-volume based probability distribution, p k is where, q k is the air volume at the k-th node. this function means that the releasing probability at the node near the fresh air inlet will be the highest. on the basis of the two modes of releasing probability, the cases are defined in table 2 . to achieve the fitness of the three objective functions f 1 , f 2 and f 3 through the ga optimization, a contamination matrix as described by ostfeld et al. (2008) as shown in eq. (21), it is specified in this study that the number of releasing location is one, while the potential location could be at each node. the releasing time is at the system initial time and the total contaminant released is assumed 100 mg. in this case, the releasing probability for each potential node in the ventilation system as fig. 3 is equal to 1/52 and the sensor number was three. through the ga optimization, the total number of non-dominated solutions in this case is 108 when three sensors are specified. figure 5 presents the detailed sensor layout patterns for four distinct nondominated solutions in the case of uniform releasing probability. it is shown that sensors are placed at the main ducts in solutions 1.1 and 1.2, which meets the objectives of f 1 and f 2 , respectively. however, in solution 1.3 the three sensors are set at the ends of the ductwork to meet the objective of f 3 . finally, in the case of uniform probability distribution of contaminant releasing at all potential nodes, an optimal solution 1.4 is obtained based on all the three objectives. to further analyze how the three objectives mutually link or compete, the tradeoff relationship between the objectives of f 1 and f 3 and between the objectives of f 1 and f 2 are generalized in figs. 6 and 7. as shown in fig. 6 , with the tradeoff between f 1 and f 3 , the detection time increases while the probability of undetected pollution events decrease. it takes only 9.2 s for solution 1.1 to detect the agent with the objective of f 1 . however, 35.8 s more are required for figure 7 presents the tradeoff relationship between the objective of f 1 and f 2 . it is observed that the detection time increases while the contaminant exposure increases. it takes 10.5 s for solution 1.2 to detect the agent, only 1.3 s later than solution 1.1. meanwhile, particle outflows through air outlet are almost the same in the two optimal solutions. it is noted that the objectives of f 1 and f 2 are compatible during optimization, which has not been clearly discussed in the previous studies wen 2010, 2012) . this convergent result can give rise to reconsideration of the objective functions. figure 8 shows the tradeoff behavior of the objective values among all 108 non-dominated solutions with the three objective functions and also presents the global optimal solution, solution 1.4, obtained by the ordering solutions method (deb and kalyanmoy 2001) . the derivation of the ordering solutions method could be express as follows then, the priority order number can be obtained by , 1,2 , , , where, x i and x j are the non-dominated solutions with objective set i. if we compare all the priority order numbers for each non-dominated solutions, the biggest priority order number can be selected and the corresponding solution is the global optimal solution. it is observed from fig. 8 that the optimal solution is characterized by relatively low detection time and contaminant exposure but relatively high probability of undetected pollution events. according to eq. (22), if the objectives of f 1 and f 2 both approach minimum, the priority order number tends to be bigger than the situation when the objective of f 3 tends to be minimum. it implies that the optimal solution is more determined by the objectives of f 1 and f 2 . as shown in table 3 , this overall optimal solution actually takes on 20% more probability of undetected pollution events compared with the lowest one by solution 1.3 to preferentially ensure the fast detection and less exposure. fig. 9 the probability pk for the releasing at each potential node based on air volume-based releasing probability when the objective f 1 or f 2 is dominated and to be placed at the end of the ventilation system when the objective f 3 is preferred. such placement pattern is much similar to that of case 1. finally, the global optimal solution 2.4 is obtained based on all of the three objectives. tradeoff relationship between the objective of f 1 and f 3 and the one between the objective of f 1 and f 2 were also generalized for case 2. as shown in figs. 11 and 12 , it is observed again in this case that the objectives of f 1 and f 3 were incompatible, while the objectives of f 1 and f 2 are compatible during the optimization. figure 13 shows the tradeoff behavior of the objective values among all nondominated solutions with the three objective functions, and it also presents the final optimal solution, solution 2.4, for case 2. it is observed again that the optimal solution is characteristic of relatively low detection time and contaminant exposure but relatively high probability of undetected pollution events. compared with case 1, the minimum detection time is 3.6 s earlier, and the minimum contaminant exposure in case 2 is 20.5 mg less (see table 4 ). it can be explained that there is higher releasing probability at nodes close to the fresh air inlet in case 2, which will shorten the detection time and reduce the contaminant exposure as well. meanwhile, in the global optimal result for case 2, the probability of undetected pollution events is 17.1%, much lower than that in case 1. this is caused by the much lower releasing probability at the end of the ductwork in this case. in this section, firstly, the comparison of optimal sensor layouts based on different sensor numbers was discussed. secondly, the simplification of optimal objectives was investigated by consideration of the compatibility observed in section 3.2. thirdly, the impact of the assumed deposition coefficient k on optimal results was studied. finally, we take into consideration both the effect of various airflow distribution and manual releasing. sensor quantity affects the detection time, contaminant exposure and the probability of undetected pollution events. it is of importance to investigate the optimization results based on different number of sensors. figure 14 presents the global optimal sensor locations with the air volume-based releasing probability with one, three and five sensors. it is observed that the layout pattern tends to be more dispersive along the ductwork with increased sensor quantity. figure 15 describes the tradeoff relationship between objective functions of f 1 and f 3 and the optimal results when using different number of sensors. from all the nondominated solutions, the objective function values tend to decrease as the sensor quantity increases in the air duct system. it is found that one sensor can never achieve a probability of undetected pollution events less than 12% nor obtain the detection time shorter than 19.0 s, while the global optimal solution of one sensor layout shows that the values of f 1 and f 3 are 21.0 s and 26.2%, respectively. compared with the global optimal results, the minimum detection time is fig. 15 tradeoff curves of non-dominated solutions between the objective f1 and f3, and the optimal solutions with different number of sensors almost the same when the number of sensors was increased from 3 to 5 while the probability of undetected pollution events is reduced by 8.1%. it is also found the convergence generation of ga optimization is increased from 1227 to 6014 when the number of sensors is increased from 3 to 5. if the sensor cost (portable sensor, $7500 each) and computational load are considered, the optimal sensor quantity for this specific ventilation system can be reasonably recommended as 3. it means that if the total sensor quantity is chosen less than this recommended sensor quantity, the minimum detection time will increase a lot. generally, in order to maximize building protection, both specific sensor quantity and optimal sensor placement are required. it should be noted that different sensor quantity needs to be considered to obtain the optimal sensor layouts. meanwhile, there should be a balance between the desired optimal results and the cost of the sensor system. as there is compatibility between the objectives of f 1 and f 2 described in figs. 7 and 12, the purpose of minimizing detection time is actually coupled with the minimum contaminant exposure, and the objectives required sensors are to be set close to the potential releasing location. thus, it is necessary to verify the "fairness" of competition between the objectives and to discuss the feasibility of objective simplification. tables 5 and 6 presents the comparison between three and two objective functions on convergence generations, sensor node locations and the final values of the objective functions, while the minimum contaminant exposure is neglected. the results indicate that the integration of objective f 1 and f 2 do not significantly shorten the detection time, reduce the contaminant exposure nor decrease the probability of undetected pollution events. such integration leads to very similar placement of sensors, but results in much convergence load. from this work, it is feasible to reduce one of the two objectives f 1 and f 2 . the deposition coefficient k in eq. (7) was calculated by k=pv d /a (you and wan 2014) , where a is the cross sectional area of the duct. p is the cross sectional perimeter of duct and v d is the particle deposition velocity. the reasonable range of v d was indicated by zhao and wu (2006) . in the aforementioned studies, the deposition coefficient k was assumed to be 0 since we didn't conduct any experiment on the particle deposition velocity v d . however, it does not mean that the deposition coefficient k has no effect on the optimization results. further, we take consideration of the k to calculate the results of objective functions. according to eq. (18), the probability of undetected pollution events only relates to the probability for contaminant releasing p k . thus, the k has no effect on the result of f 3 . according to eq. (9), if the deposition coefficient k≠0, then eq. (10) can be rewritten as follows: equation (25) the corresponding time of the peak concentration when it occurs is equal to the description in eq. (12). we substituted eqs. (12) and (25) into eqs. (13) and (16). it was noted that the deposition coefficient k had no effect on the prediction of the sensor detection time. it only affected the result of the total contaminant exposure (f 2 ). the change in k is proportional to the change in the result of f 2 . thus, if the total contaminant exposure of the sensor placement i when k is neglected is minimum, the sensor placement i also has the minimum f 2 when k is considered. figure 16 describes the total contaminant mass entering the chamber on the condition of various particle deposition velocity v d based on the uniform probability distribution in case 1. it can be observed that the v d increases while the total contaminant exposure decreases. therefore, it is beneficial for sensor network design when the deposition coefficient k is involved. table 7 , the fan frequency was adjusted to alter the air speed of each duct, so that the air volume distribution changed in the ventilation system. the sensor detection time t det-k and the total contaminant exposure e k both changed according to eqs. (13) and (16). figure 17 presents the objective function values of f 1 and f 2 based on various air volume distributions. the results indicate that the air speed of each duct also reduces. the sensor detection time t det-k increases with the same sensor placement and releasing probability assumption based on eq. (13). meanwhile, the time increase is inversely proportional to the air speed reduction. for the total contaminant exposure e k , according to eqs. (15) and (16), t det-k increases while the air volume at outlet q m decreases as the air speed reduces. however, we cannot give qualitative judgments on the total contaminant exposure due to the uncertainty of instantaneous concentration at air outlet. it is found that the increase of e k is less than 4.0% with different airflow distribution. fig. 17 the impact of various flow distributions on both the sensor detection time and the total contaminant exposure therefore, although the required detection time increases, the variation of total contaminant entering the room is small due to the reduction of the air speed. such results mean that the sensor placement optimization based on the designed air volume distribution can be adapted to the altered flow distribution. the optimal solutions of sensor placement are significantly influenced by the source location. actually, the biochemical attack occurred in the ventilation system is a random process and we cannot ascertain in advance where the attacker may release the source. consider an ideal situation, we manually place a contaminant source at the fresh air inlet (see the node 1 in fig. 4) , in this case, if no sensor is arranged in the ventilation system, the pollutants will follow the airflow and transport through the whole ductwork, causing the affected indoor area reaching the maximum. meanwhile, almost all of the contaminant will eventually enter the room, which may lead to numerous casualties. however, since we know that the source of contamination is at the node 1, only one sensor is enough at the node 2 which is nearest to the source location to obtain the optimal sensor layout in the air duct system. then, the pollutant can be detected within the shortest time after releasing. if we shut down the fan immediately, pollutants can't even reach the air outlets. pollutants spread very slowly without the convection effect of air supply, which makes time for evacuation of indoor occupants. it should be pointed out that the sensor optimization in this ideal case is a local optimization process. in fact, each sensor layout optimization resulted from the manually placed source is a local optimization. such a sensor placement cannot adapt the early warning situations because the source location may change. thus, two modes of probability distribution are proposed, covering all nodes in the ventilation system. we reasonably turn the manually placed source into the probabilistic source, which makes the local optimization become a global optimization. the objective function values such as the detection time and total contaminant exposure resulted from the manually placed source are preferable to the values obtained from the probabilistic source. however, the optimal sensor layout based on the assumption of the probability distribution of the source is appropriate for the entire air duct system, while the assumption of manually placed source considers only one node. obviously, the aforementioned case 1 is the most unfavorable case since the attack is a completely random process. compared with the case 1, it is assumed in case 2 that the source releasing probability is based on the air volume, which means that the attackers know well of the ventilation system. namely, the description of attacker's behavior is included in case 2. thus, the objective function values resulted from case2 are better than that from case1. in this work, an optimization method for sensor placement in an air duct system was presented. three optimal objectives were applied and ga method was utilized to obtain the non-dominated solutions of multi-objectives optimization problem. the global optimal solution was selected among all of the non-dominated solutions by the ordering solutions method. the main conclusions are provided as follows.  optimal sensor layouts are sensitized to the contaminant releasing probability distribution. it is found that the optimal sensor layout with higher releasing probability specified at nodes which is close to the fresh air inlet (i.e., volume-based releasing scenario) resulted in shorter detection time, smaller contaminant exposure and lower probability of pollution events. such result means that a pertinent strategy of sensor placement is possible to well respond to an elaborated releasing event.  some of the objective functions have conflicting goals. the results of optimal sensor layouts against suddenly released contaminant in the ventilation system of this study are generally characterized by relatively low detection time and contaminant exposure and conversely relatively high probability of undetected pollution events, due to the predominant effect of objectives of f 1 and f 2 . the reason can be explained that the solution mainly determined by the objectives of f 1 and f 2 has the biggest priority order number among all non-dominated solutions, which meet the selection criteria of optimal solution in the ordering solutions method.  sensor quantity required for a ventilation system is affected by the objective competition, sensor cost and computational load. the increase of sensor number leads to shorter detection time, smaller contaminant exposure and lower probability of undetected pollution events. with the required values for objectives, it is able to derive the sensor quantity and design an optimal sensor system for a specific ventilation system. future work should be further studied on the impact of sensor type, sensitivity, accuracy and other characteristics on the sensor system design for ventilation system. a design approach for sensor system considering various potential contaminant releasing scenarios should also be studied. computational intelligence techniques for hvac systems: a review. building simulation an enhanced multizone model and its application to optimum placement of cbw sensors contaminant source identification within a building: toward design of immune buildings an evidential reasoning approach to optimal monitoring of drinking water distribution systems for detecting deliberate contamination events robust optimization of contaminant sensor placement for community water systems optimal sensor deployment in a large-scale complex drinking water network: comparisons between a rule-based decision support system and optimization models predicting transient particle transport in enclosed environments with the combined cfd and markov chain method sensor system design for building indoor air protection comparison of sensor systems designed using multizone, zonal, and cfd data for protection of indoor environments the selection of the most appropriate airflow model for designing indoor air sensor systems using probabilistic sampling-based sensitivity analyses for indoor air quality modelling multi-objective optimization using evolutionary algorithms security-oriented sensor placement in intelligent buildings comis-an international multizone air-flow and contaminant transport model a methodology for optimal placement of sensors in enclosed environments: a dynamical systems approach solution for sudden contamination transport through air duct system: under a puff release countering chemical and biological terrorism modeling immune building systems for bioterrorism defense efficient sensor placement optimization for securing large water distribution networks protecting a whole building from critical indoor contamination with optimal sensor network design and source identification methods sampling design for network model calibration using genetic algorithms optimal layout of early warning detection stations for water distribution systems security the battle of the water sensor networks (bwsn): a design challenge for engineers and algorithms bioterrorism preparedness: the united kingdom approach multiobjective contaminant sensor network design for water distribution systems sensor placement in water networks: a stochastic programming approach siting bio-samplers in buildings rapidly locating and characterizing pollutant releases in buildings systems approach to evaluating sensor characteristics for real-time monitoring of high-risk indoor contaminant releases influence of indoor transport and mixing time scales on the performance of sensor systems for characterizing contaminant releases towards improved characterization of high-risk releases using heterogeneous indoor sensor systems survey of bioterrorism risk in buildings use of system dynamics as a decisionmaking tool in building design and operation siting samplers to minimize expected time to detection formulation and optimization of robust sensor placement problems for drinking water contamination warning system inverse identification of multiple outdoor pollutant sources with a mobile sensor particle concentration dynamics in the ventilation duct after an artificial release: for countering potential bioterrorist attack optimal sensor placement for airborne contaminant detection in an aircraft cabin an inverse method based on cfd to quantify the temporal release rate of a continuously released pollutant source a simplified system for indoor airflow simulation modeling particle deposition onto rough walls in ventilation duct how particle resuspension from inner surfaces of ventilation ducts affects indoor air quality-a modeling analysis wireless sensor network based monitoring system for a large-scale indoor space: data process and supply air allocation optimization this work was supported by the national natural science foundation of china (no. 51278370). key: cord-262719-onjxtixk authors: williams, richard a. title: user experiences using flame: a case study modelling conflict in large enterprise system implementations date: 2020-09-19 journal: simul model pract theory doi: 10.1016/j.simpat.2020.102196 sha: doc_id: 262719 cord_uid: onjxtixk the complexity of systems now under consideration (be they biological, physical, chemical, social, etc), together with the technicalities of experimentation in the real-world and the non-linear nature of system dynamics, means that computational modelling is indispensible in the pursuit of furthering our understanding of complex systems. agent-based modelling and simulation is rapidly increasing in its popularity, in part due to the increased appreciation of the paradigm by the non-computer science community, but also due to the increase in the usability, sophistication and number of modelling frameworks that use the approach. the flexible large-scale agent-based modelling environment (flame) is a relatively recent addition to the list. flame was designed and developed from the outset to deal with massive simulations, and to ensure that the simulation code is portable across different scales of computing and across different operating systems. in this study, we report our experiences when using flame to model the development and propagation of conflict within large multi-partner enterprise system implementations, which acts as an example of a complex dynamical social system. we believe flame is an excellent choice for experienced modellers, who will be able to fully harness the capabilities that it has to offer, and also be competent in diagnosing and solving any limitations that are encountered. conversely, because flame requires considerable development of instrumentation tools, along with development of statistical analysis scripts, we believe that it is not suitable for the novice modeller, who may be better suited to using a graphical user interface driven framework until their experience with modelling and competence in programming increases. over the past two decades, the use of computational approaches to investigate social systems, including organizational and behavioural research has progressed from mere quantitative data analysis to one that complements traditional social science techniques through the use of advanced techniques 5 in modelling and simulation. the techniques used within computational social science have evolved from mathematical approaches that utilize deterministic or stochastic equations, through to computational modelling approaches such as agent-based modelling and simulation [1, 2] . an advantage of these computational modelling approaches is the ability to fully control 10 the underlying mechanistic component interactions of the model, thus once a computational model is established, quick studies of the effects of changes to the elements and associated parameter values can be seen through equationsolving or simulation. as such, simulation-based experimental results from a well engineered model are directly related to the level of abstraction and 15 assumptions made during model design and development. this provides a firm basis for testing hypotheses on the mechanisms behind social behaviours and dynamics than traditional approaches such as participant observations or action research, where the unseen variables/factors can introduce additional complexity and uncertainty, which limits our ability to translate interpreta20 tions of observational/qualitative results to the real-world system. real-world systems are complex, with a range of system behaviours, properties and characteristics that emerge from the low-level interactions of a highly connected set of system components that function through temporal and spatial dimensions. one of the main strengths of the systems approach to 25 investigating complex systems, is that it focuses on three key properties when modelling the systems: 1) system structures, 2) system dynamics, and 3) system control [3] . indeed, previous discussions around agent-based modelling and simulation (abms) advise that it can be used to test working theories of the underlying mechanics of component interactions within systems and their 30 resulting dynamics [4, 5] . as such, we believe that abms is the preferred approach to facilitate the micro-to-macro mapping of complex dynamical systems, in particular, social systems [6] . this is performed through altering the way in which agent interactions occur, or relaxing of assumptions at the individual agent-level, in order to investigate the emergence of system-level 35 behaviours at the system-level. we therefore believe that abms provides a low-cost computational approach for developing hypotheses of the real-world system. with abms having recently become accepted as a scientific instrument for investigation [7] , it is now imperative that agent-based models (abms) adhere to the scientific method through a principled approach to 40 their design, development and use, to ensure they are fit-for-purpose [4] . technology advances in hardware architecture, data storage and processing speed over the past twenty years has encouraged the development of new simulation frameworks that can handle increasingly complex computational models. with respect to abms, this has resulted in the development 45 of a number of frameworks aimed at either subject matter experts or novice modellers, which use graphical user interfaces, or experienced computational modellers that are comfortable with command line terminals, a range of basic programming languages and shell scripting. a relatively new abms framework that falls in the latter category is the flexible large-scale agent-based 50 modelling environment (flame). within this study we have used flame to model the development and propagation of conflict within a large multipartner enterprise system implementation. this manuscript provides an account of our experiences in using flame, with particular reference to a number of it's strengths and weaknesses with 55 respect to: abm design; abm development; constraints due to its underlying technical architecture; and performance. in particular, we highlight: the ease of model design for experienced computational modellers who are familiar with object-oriented programming and the unified modelling language (uml); and the ease of model development for experienced computational 60 modellers who are comfortable with the logic behind uml state transition diagrams. in addition, we discuss a number of significant constraints of flame due to the underlying architecture, such as: issues when setting the pseudo-random number generator seed value; the inability to send messages between simulation time-steps; the inability to use global mutable parameters; performance challenges due to the i/o rate-limiting characteristics and the need for significant fast storage allocation to accommodate realistic simulations of social systems. the manuscript is structured as follows: section 2 provides an overview of the major concepts that contribute to the theory behind our study; section 3 discusses the case study; section 70 4 provides an account of our user experiences when using flame to model the case study; section 5 is the discussion section; and section 6 provides our conclusions. this section provides an overview of the major concepts that contribute 75 to the theory behind our study. social networks and the various dynamics and emergent behaviours that develop from interactions between individual human agents are complex, meaning that they often display non-linear dynamics. computational social 80 scientists often model these social dynamics through equation-based mathematical approaches (e.g., system dynamics or differential equations) or abms. these computational modelling approaches facilitate the analysis and investigation of complex social systems that would otherwise be intractable. this may be due to direct experimentation of the system being either unethical or 85 impossible across the scale and hierarchies of the social system; difficulties in observing system dynamics due to extremely short or indeed large timescales; difficulties in observing the whole population due to the magnitude of the system, or to the location or complexity of the system (e.g., virtual team). importantly, it is frequently apparent that a significant amount of data 90 generated through observations, participant interviews or surveys in the social sciences, accumulates and is collected in a manner comparable to the object-oriented paradigm for design of computational systems. historically, computational modellers have taken inspiration from the natural and physical sciences and utilized the reductionist approach, which advocates that 95 systems should be investigated through their decomposition to their smallest indivisible unit, which we believe is analogous to looking for 'objects' within nature. as such, we believe that an object-oriented approach to computational modelling, through the use of a bottom-up approach for design and development of the models is a useful formalism for modellers in general, 100 and those interested in computational social systems in particular. abms intuitively expands upon the object-oriented approach to model design and development, and significantly benefits through the principal enhancement that an agent is active instead of passive, and secondarily, that abms is not bound by a central computational mechanism of control, but instead has multiple threads of control. macal and north [8] provide a tutorial on abms, which describes this by stating that the "fundamental feature of an agent is the capacity of the component to make independent decisions". in addition, jennings [9] further extends the notion of an agent by advising that they are asynchronous components of a computational model that interact 110 with the environment. as such, agents have the capability to sense their environment, and through processing the input(s) that they receive from the environment, may act upon it. the origins of abms are in the modelling and simulation of human societies, systems, and networks and the associated dynamics and relationships 115 within these hierarchical structures [10], with particular connection to the fields of: complex systems, computer science, and management science [8] . in fact, the agent-based approach was developed to allow investigation of complex social systems at the level of individual actors/people, so borrowed the reductionist perspective from the natural sciences. the belief 120 here, which was also borrowed from engineering, is that complex systems are built from the ground up, which was a marked contrast to the beliefs of systems thinkers who took a top-down view. since its inception, the agentbased approach has gained the attention of researchers from fields far away from its origins within the social sciences. examples of the use of abms 125 to model complex dynamical social systems include: the seemingly inocuous renting and sales patterns of houseowners/tennants within a reimplementation of shelling's bounded neighbourhood model [11] ; the most recent banking crisis associated with the british banking sector [12] ; the way that virtual project team performance is affected by communication technologies 130 [13] ; and the recent covid-19 pandemic [14] . the behaviour of individual agents is dictated by rules for their behaviour(s) and interactions. the interactions between compatible agents gives rise to behaviour(s) and dynamics at the system-level. this population-level behaviour, develops through the aggregated dynamics of individual agents, 135 and is consequently deemed an emergent behaviour because it is not defined in the technical design of the computational model. epstein [6] advises that this study of the emergent system-level behaviours is one of the key advantages of the abm approach, and the ability to investigate the effects of the heterogeneous and autonomous individual agents has led to its increased 140 adoption over the past decade [8, 10] . moreover, an agent-based approach allows the modeller to design and develop the model by explicitly taking the individual agent's location into consideration, which results in the model more accurately reflecting the spatial aspects of the complex social system, and in particular the network of social interactions within the system. the engineering of viable computational models of social networks is not a straightforward process. this is due to the real-world system containing multiple feedback signals and non-linear dynamics, along with the model requiring the use of numerous parameters whose values have a degree of uncertainty (or may even be unknown) from observations/participant inter-150 views. this uncertainty is further compounded through the introduction of randomness at key decision points/system events to provide the heterogeneity in behaviours that are seen across a population of human actors within the system [15] . abms equips the model developer with a set of techniques to integrate multiple data types gained from empirical data collection and 155 knowledge themes gained from secondary data sources. furthermore, one of the key strengths of the agent-based approach is that it allows us to model the complex non-deterministic and heterogeneous behaviours, which allows us to investigate the role of differences in chracteristics (e.g., demographic or behavioural) of individuals within social systems and develop testable hy-160 potheses for new empirical research. see williams [4] for lessons learned on development and application of abms of complex dynamical systems. the work of nikolai and madey [16] has reviewed the main agent-based modelling frameworks available, and discussed their different computational underpinnings; examples include mason [17] , repast [18] , and netlogo 165 [19] . the commonality between all of these frameworks, is that they represent the individual agent as having a specific state at any particular point in time. consequently, representations of each individual agent may be considered as a state machine, where the combination of current and previous inputs (previous states), along with the logic/functions associated with the agent-170 type, determines the output (next state) of the agent. increasing progress in hardware architecture, storage and speed over the past twenty years has encouraged the development of new simulation frameworks that can handle increasingly complex computational models, which simulate at magnitudes and dimensions that are much more closely aligned to 175 real-world societal populations. for example, the flexible large-scale agentbased modelling environment (flame 1 ) was developed [20] to provide an intuitive development framework for large-scale abms. flame was developed for use over the full range of computer hardware, which allows for the early coding and unit/system testing on a personal computer, before be-180 ing moved to high-performance computing infrastructure for full-scale testing and simulation. flame has been used by researchers across a variety of domains, ranging from biology [21], to economics [22] , to human resources [23] , to transport and logistics [24] . the flame simulation framework comprises a collection of application programming interfaces (apis), templates to define the agent-based model, and compilation and parsing routines to create the c code for running simulations. due to flame's conceptual architecture being based on communicating stream x-machines, templates are used to define the rule-based 190 functions (written in c code) of agents, with the agent types being defined through xml templates, and the instantiation of the initial iteration of a simulation run using another xml template that defines the attributes and internal agent states for the simulation run. flame requires installation and configuration of the message passing interface (mpi) to develop mes-195 sages that communicate interactions between specific agents and changes within the system's environment. these agent interactions are simulated through transition functions that specify the rule-based logic of the abm, with all agents that transition to a new state having their internal memory updated accordingly. following specification of the model in the appropriate 200 templates (e.g., c and xml), the xparser tool is used to generate simulation code (see figure 1 ). the xparser is written in c with the use of standard libraries, and is actually a collection of template files and header files that are stored in the desired directory of the computer (i.e., laptop, desktop, or hpc) and compiled using the freely available gcc compiler. furthermore, 205 through the use of the mpi communication framework, the generated simulation code is also portable to hpc platforms that use parallel architectures, thus enabling efficient communication between agents that may be across different compute nodes, and therefore remain in sync [25] . an x-machine is a formalized specification developed by eilenberg [27] 210 that has the capability to model both the system's data and the specification method (function) for controlling the system. they were originally introduced in 1974, but did not receive widespread acknowledgement in the modelling community until 1988 [28] when the approach was advocated as figure 1 : flame relies on the definition of models using the xml and c templates, and their parsing and subsequent compilation into simulation code. this simulation code is then run, through linking the main executable file to an initial starting parameters file (0.xml). due to flame utilizing the discrete-event approach, individual simulation runs generate separate output files for each time-step increment within the simulation, with each output file acting as the input file for the subsequent simulation time-step (after [26] ). the basis for a formal model specification language. subsequently, this was 215 built upon in 1992 through the concept of stream x-machines, which utilized sets of input and output symbols that were read through in a stream. from a conceptual modelling perspective, x-machines employ both a diagrammatic approach and formal notation to model the system, where the x-machine contains infinite internal memory, a set of functions, and a current state. the 220 current state of control (the function defined in the specification method) and the current state of the memory, is processed alongside an input symbol from the input stream to determine the next state of the x-machine, update it's memory state, and calculate the output symbol, which becomes part of the output stream that is used for communicating to other x-machines. this can be summarized in that the system's new state is a product of it's current state (using memory and reference to a list of input symbols) and the relevant function [29] . a communicating stream x-machine model is a formalized specification that builds upon that of x-machines to introduce additional modelling cap-230 abilities. in particular, communicating stream x-machines can be used to compute the functional behaviour of the system at a lower-level of granularity (i.e., individual agents), whose individual dynamics may be aggregated up to the overall system level in order to generate the emergent behaviour of the system as a whole. the formal notation of the communicating stream x-machine specification utilizes a 10-tuple notation, with c x i representing the i th communicating stream x-machine component, which is defined by stamatopoulou [30] as: 240 -î£ and î� are respectively the input and output alphabets. -q is the finite set of real-world system states. -m is the (possibly) infinite set that relates to memory. -î¦, the type of the x-machine x, is a set of partial functions ï� that translates a specific input and a specific memory state to a specific output and a possibly different memory state, ï� : î£ x m â�� î� x m. -f is the next state partial function, f : q x î¦ â�� q, which given a state and a function from the type î¦ determines the next state. f is often described as a state transition diagram. -q 0 and m 0 are respectively the initial state and initial memory. -iî¦ i is the communication interface for the input messages. -oî¦ i is the communication interface for the output messages. an x-machine is defined by holcombe [28] as a system that has internal memory and an internal computational state, which dependent on environmental input and their current internal state, can transition to another computational state (see figure 2 ). the additional functionality incorporated into 245 communicating stream x-machines means that they are able to model in a single process specification, the functional behaviours and dynamics of an agent, as well as the intrinsic system data that it is modelling. a communication matrix using mpi facilitates communication (and thus interactions) between individual communicating stream x-machines. this communica-250 tion matrix is essentially a simulation-level message board that facilitates the reading and writing of information between every communicating stream x-machine. the gartner group devised the term enterprise resource planning (erp) 255 in 1990 [32] to define the software that provided functionality for organizations to use to manage their core administrative (back office) functions such as finance, payroll and human resources. a number of leading software vendors (e.g., oracle, jd edwards, peoplesoft and sap) offered erp software packages to store the organizational data and provide assistance to 260 facilitate compliance to a standardized set of business processes [33] . with the expansion of information systems (is) and information technology (it) throughout the late twentieth century, the individual software modules that focused on the different organizational business functions were integrated together. these larger software systems, which were termed enterprise sys-265 tems, not only integrated the individual erp modules, but also provided additional functionality, such as business intelligence, advanced planning, and automatically processing data from external supplier/customer relationships. enterprise systems are now commonly used to help organizations (of all sizes) manage their human, financial and physical resources (e.g., staff, 270 money, and products) along with key external relationships (e.g., customers and suppliers) more effectively [34] . enterprise systems require considerable time and resource commitments in order to be implemented properly within an organization. these implementation projects within large organizations are usually implemented by 275 third-party service providers, with the largest implementations using the consultancy services of both the vendor (e.g., sap or oracle) and professional services firms (e.g., accenture, ibm, capgemini, etc.). the various customer, vendor, and professional services personnel are usually structured into project teams that relate to the functional modules within the enterprise 280 system, such as finanicals, payroll and human resources, which gives rise to a wider implementation programme. whitty [35] has postulated that the increasing complexity and scale of reach into the business by these enterprise system implementation programmes, contributes to them exhibiting similar behaviours and traits to those of com-285 plex systems. we have previously discussed that the emergent behaviour generated within the social network of these large implementation programmes, may, to a large degree, be due to the complexity stemming from the large number of team members, and the growing trend of using multiple thirdparty providers to implement the individual projects that combine to form 290 the programme. in addition, we discovered that the individual project teams may have competing priorities and objectives, and that these may lead to various forms of conflict propagating throughout the wider programme, which we view as a social network of formal workplace relationships [36]. conflict within group situations, such as the project teams that implement functional modules of enterprise system software, has been defined as interpersonal incompatibilities or the holding of divergent views/perspectives amidst the members/participants, which may be individuals within a single group (intragroup conflict) or between different groups (intergroup conflict) 300 [37] . conflict has been reasoned to be an intrinsic part of group [38] and project team dynamics, which can propagate throughout the network of group members as a shared affect [36] . conflict develops during a variety of circumstances relating to the implementation of group-and team-based activities, and in three main forms: task, process, and relationship conflict [39] . 305 enterprise system implementations at large organizations, often require the personnel, expertise and resources of multiple third-party organizations, which may have different, and often incompatible, business objectives and commercial drivers. as discussed above, the programme-wide implementation of the enterprise system is routinely divided into discrete project teams 310 that map on to the corresponding functional modules within the enterprise system software (e.g., finance, payroll, hr). our recent study [36] showed that within large multi-partner enterprise system implementations, conflict (be that task, process or relationship) can develop between members of a particular project team, or between members of different project teams, and once 315 developed, can propagate throughout the social network of the multi-partner enterprise system implementation. furthermore, this study conceptualized the propagation of conflict within large multi-partner enterprise system implementations as conflict propagating between team members of an individual project team, and also between team members that are situated within dif-320 ferent project teams (see figure 3 ). in addition, we built upon the work of gamero et al. [40] who discovered that conflict is dynamic and can transition over time, by hypothesizing that task or process conflict can transition to relationship conflict, and that relationship conflict (being directly related to shared affect), is the most common form of conflict to propagate through 325 the social network of enterprise system implementations. we therefore hypothesize that the conflict, which initially developed between a subset of team members from two different project teams, has the potential to propagate throughout the social network of the wider enterprise system structure, and may negatively affect implementation of the enterprise system as a whole. the case study relates to a large uk-based organization that was implementing an enterprise system along with associated it hardware as part of a major strategic modernization initiative. this is/it change programme was underpinned by the need to integrate the enterprise system with multiple, 335 existing, third-party legacy systems, to facilitate significant cost savings and promote more efficient business processes. the client selected a large enterprise applications vendor to implement the installation and configuration of the enterprise system, and to lead the design and assist in the development of the technical architecture that the enterprise system was embedded within. in addition, two different professional service providers were also contracted to act as domain expert consultants for the configuration and extension of functional modules within the enterprise system, along with a third professional service provider who consulted on the hardware and middleware requirements for hosting the enterprise system. such an environment is best described as a multi-partner enterprise system programme implementation. the resulting social network was structured into project teams that aligned to the functional modules within the enterprise system, along with a programme management office (pmo) and project teams that focused on developing technical extensions (e.g., cus-350 tom forms, reports, and database triggers), training material, and installing and configuring the technical architecture that hosted the enterprise system. overall, there were 159 resources from these five organizations, with 972 undirected workplace relationships between them, which formed the basis of the social network map (see figure 4 ). the conceptual framework presen-355 ted in [36], acted as our conceptual model for the abm, by providing the phenomenological behaviours that are the basis of the emergent behaviours generated from the abm (see figure 3 ), alongside the detailed person-level data and social network map that formed the basis for the individual agents within our computational model, and the rules that dictate which agents can 360 interact with each other. the case therefore constitutes a large multi-partner enterprise system implementation, which utilizes is/it consulting personnel from four external organizations. these external resources were combined with client resources, to form a number of project teams that align to the underlying functional modules of the enterprise system. the resources worked together within their respective project team, with a number of them also acting as bridgers to interact with other project teams, which is diagrammatically represented in figure 4 as the programme-wide interfirm social network. this multi-partner social network, introduces a risk that conflict may develop due to the differing 370 backgrounds of project team personnel (e.g., different professional identities, cultural backgrounds, education, and normalized behaviours), along with their constrasting drivers and motives for being at their chosen employer (and occupation), and thus on the implementation programme. it was discussed in our previous work [36] , that conflict is to all intents and purposes inherent 375 within large multi-partner enterprise system implementations, and as such is almost always going to occur at some point during the lifecycle of the programme-wide implementation, or within one or more of its constituent project teams. our abm has been designed to model this case. it allows us to simulate the development and propagation of conflict throughout the 380 social network of the workplace relationships within the individual project teams and the wider programme as a whole. the abm [41] was designed and developed to reflect findings from our previous qualitative studies [36, 42] . in the subsections below, we discuss our 385 experiences using flame following the design, development, and simulationbased experimentation of an abm of conflict development and propagation within our case study. our discussion focuses on four main areas: the ease of model design having made the decision to use flame; the ease of model development using flame; constraints due to the underlying architecture 390 of flame; and performance of flame in running simulations. as discussed above, the flame simulation framework utilizes the concept of communicating stream x-machines to define the logical entities and the rule-based logic of their interactions within the agent-based model. the con-395 ceptual framework [36, 42] , acted as the functional specification for our abm, which in turn was used as the basis for developing our technical specification. when developing the technical specification, we were cognizant of the underlying architecture of flame, and made explicit reference to: the c programming language and the xml markup language, which were used for 400 coding agent interaction functionality and defining agents; the communicating stream x-machine architecture for defining agents and their associated behaviours and dynamics; along with the use of the centralized message board to facilitate communication between agents. briefly, our technical specification consisted of a two-dimensional, undirected network of 159 agents that represented the individual team members from the case study. communication between individual members followed the rules specified in the adjacency matrix and resulting social network map (figure 4), and this communication within the abm is modelled as a process with no velocity (i.e., link between two agents, but no speed). specification of the agents was straightforward because the communicating stream x-machine architecture facilitates easy compliance to an object-oriented approach to design. in addition, flame is flexible with respect to defining the physical boundary of the model, which allowed us to design a 2-dimensional physical environment for our abm, and the use of cartesian co-ordinates 415 to position individual agents within the environment. this allowed us to group agents together in 2-dimensional space to represent the clustering of resources into their respective project team, and to disperse individual teams within an open-plan office, another office in the building, or potentially in another location. system-level behaviours corresponded to those depicted in our rich picture (figure 3), with the main agent attributes that facilitate simulation of conflict development and propagation relating to: project team (e.g., programme, hr, financials, etc.); organization (e.g., client, vendor, consulting firm); role type (e.g., management, functional, technical, training); conflict quotient for each type of conflict; stress quotient for each type of conflict; and formal authority quotient, which indicates how much formal authority/power the agent has over others. with respect to communication between agents, although flame utilizes a centralized message board, we were able to design restrictions into the communication mechanism to ensure 430 agent communication corresponded to the workplace social interactions, as defined within the adjacency matrix from our case study (i.e., the 972 workplace relationships between agents). this was facilitated through adding an attribute to the agent definition that explicitly defined the other agents that it is able to communicate and interact with. one point to note is that when diagrammatically modelling an x-machine as part of the technical specification, the x-machine state is associated with the computational system state transitions and does not correlate to the social (domain specific) states. these internal x-machine state transitions are achieved through the use of transition functions, which encode the logic that 440 changes the agents memory, and communicates with other agents through the use of messages sent to (using output port) and received from (using input port) the centralized communication matrix. for those familiar with uml, an abm developed using x-machines can be specified (from a system behavioural perspective) as a set of connected state transition diagrams. for those comfortable with integrated development environments (to write code), the command line terminal (to run simulations and submit batch jobs to hpc architecture), and the use of shell scripts (to submit jobs to a server, link to scripts for post-processing of output files, and link to analysis scripts [such as r or matlab] to generate descriptive statistics and graphs), the flame simulation framework is an intuitive and very friendly tool for developing agent-based models using xml and c. unfortunately, the converse of this holds for the novice modeller, and we conjecture that a more user friendly agent-based modelling framework that uses a graphical user 455 interface, such as netlogo, would be suitable as a first step to developing competence in model development and simulation-based experimentation. flame provides a simulation engine that parses the abm specification files (xml agent definition files and c functions files), manages the execution of simulations (on either a single node or over parallel architecture), 460 and manages potential interactions between individual x-machine agents. within flame simulation runs, the notion of time is modelled as discrete time-steps. each time-step therefore involves the individual x-machines to iterate through their internal computational states, which may result in updates to their real-world social states following interactions with either the 465 environment or other x-machine agents. furthermore, for those familiar with object-oriented design and development, flame, being underpinned by the concept of communicating stream x-machines, allows the various functions within the abm to be constructed in a modular fashion. consequently, flame ensures that each type of x-machine agent can be designed in an 470 object-oriented way, which allows their construction and modification to be performed without the risk of affecting the functionality of other agent types as long as a standard interface is used for communication and interactions between agents. the underlying process related to the technical architecture of flame 475 is the requirement for synchronization, through a simulation-level time-step after all agents within the simulation run have completed one internal computational transition function. the effect of this technical processing constraint, is that the end and start of computational transition functions, incorporate the ability to synchronize communication and internal computational state 480 transitions. with this in mind, care is required to ensure that agent behaviours do not contain any loops, which would incur the risk of undecideability problems, and that the interactions between agents (through use of the centralized message board) does not result in causal loops, which could impact upon the step-by-step updating of the agent internal states. flame simulation framework that "when communicating x-machines are used to represent agents in an [abm], communication is [often] restricted to interactions with neighbouring agents that are [located] close to one another" [20] . within our social system model, there are two broad categories of 490 messages used within the simulation runs to communicate an x-machine agents' current location and their potential to initiate or be part of a social interaction. the location message is sent from individual x-machines to the communication matrix to broadcast their current location so that other x-machine agents can calculate whether they are within the appropriate spatial 495 range for initiating an interaction, or alternatively to determine whether they are connected within the social network. with specific reference to the latter, the agents will elucidate whether they are connected (as defined in the adjacency matrix), and once [a] suitable agent(s) is/are identified, messaging to the centralized communication matrix would then be used to 500 initiate interactions between the relevant agents. finally, one point to be aware of, is that although development of an abm is one of the principal essential activities, in isolation, it will not enable simulation-based experimentation. in order to run simulations and analyze the resultant data, a number of additional computational tools need to be de-505 veloped (termed instruments). for our research, we developed the following instrumentation tools alongside the development of the abm itself: unix shell (specifically, bash) and ruby scripts to facilitate a semi-automated submission of simulation runs to the hpc resources; python scripts to facilitate a semi-automated mechanism to generate the initial starting parameters 510 files (using xml templates) for initiating simulation runs; scripts for the processing and transformation of xml output files to csv files (python and matlab scripts); scripts to automatically generate graphs (in matlab and r) for analyzing the data; a visualization tool to provide a graphical and animated view of the simulation run's system dynamics over time (separate 515 package provided by the flame development team, that can be configured to the abm); and various statistical analysis scripts (developed in matlab and python) to analyze the simulation output data for statistical significance using the kolmogorov-smirnov test [43] , and effect magnitude using the a-test [44] . these instrumentation tools (apart from the flame visualizer) 520 were developed using the appropriate scripting/programming language, so in order to ensure that abms developed in flame can be robustly tested and analyzed, the developer requires a working knowledge of various statistical techniques along with competence in various scripting/programming languages. with that in mind, we do not believe that flame is appropriate 525 for the novice modeller or subject matter expert. with flame being designed and developed from the outset to utilize parallel processing hardware architectures, the notion of a global mutable parameter unfortunately does not exist within the simulation framework. this is because such functionality would introduce issues around concurrency control and potentially lead to dead-locking. for instance, an individual compute node within the hpc architecture may be processing the simulation data for a particular x-machine agent that is dependent on the current value of a global parameter, but the global parameter is currently 535 being processed by another compute node. to mitigate risks around using outdated parameter values, the compute nodes would be required to wait until they confirmed that the global parameter was not being processed. when amplified up to the level of the overall simulation run, the cumulative waiting would incur a significant overhead regarding the wall-clock time 540 of simulation runs, which would significantly offset the benefits of parallel processing. consequently, we were unable to utilize a global counter to keep track of system-level quantities or to set system-level chracteristics, such as a pseudo-random number generator seed value. pseudo-random number generators (prngs) allow us to introduce probabilistic behaviour into our computational models, such as the interactions between x-machine agents, or between the agents and the system environment. during the calibration exercise, and later more formally during the verification process, we ensure that the computational model is not over-550 tuned through running multiple replicate simulations that utilize different prng seed values and performing statistical analysis to ascertain the variance in simulation output data. the principle behind explicitly setting the seed value for a prng is that you set once, and use multiple times [45] . a significant constraint that we discovered with flame is that it does not in-555 clude a mechanism for you to do this very easily, because mutable parameter values can only be defined and set within the definition of an agents' logic (the c functions file). as discussed in the previous paragraph, the design decisions taken when flame was first developed means there is no opportunity to set a global constant within the simulation environment setup of a 560 figure 5 : the levels within a simulation run at which the prng seed value can be set when using flame. although the principle of using prng seed values is that you set once per simulation and use multiple times, flame's explicit exclusion of global mutable parameters means that this is not straightforward. due to flame requiring all functionality to be written at the level of agents, this provides us with the ability to experiment to see whether any of the levels allow us to develop a technical workaround, for instance through setting at the level of agent-type, where any function would be called once per agent-type per iteration, or at the level of individual agents, where any function would be called once for each agent and for each iteration. simulation run due to the inefficiencies involved with it propagating across the multiple nodes within the hpc architecture. with that in mind, not only are global variables, such as total counts, unable to be updated, but the prng seed value cannot be set at the global level. this introduces the constraint of either not being able to set your own prng seed value, and 565 therefore being forced to use the system clock to derive the seed value, or to develop a technical workaround where the prng seed value is explicitly set within an individual agent's definition and logic (see figure 5) . when setting the prng seed value at the level of agent-type, you unfortunately encounter the issue of the seed value being reset during each 570 simulation time-step, and thus do not gain the full benefit of using a prng to simulate stochastic behaviours within the computaional model. through explicitly setting the prng seed value a list of random numbers is generated, albeit in a determinstic manner so that you can reproduce simulation runs by using the same seed value (hence the use of the prefix pseudo), to be used 575 within probabilistic functions [46] . the resetting of the prng seed with the same value and at the level of agent-type, results in the same deterministic list of pseudo-random numbers being generated as per the previous simulation time-step, which reduces the benefits of using a prng. this can be succinctly illustrated within our case study: if you set the seed value at the 580 level of an individual agent within our abm that contains 159 agents (e.g., separate programme team members) over a 2,000 time-step simulation, you in effect repeat the resetting of the seed value 318,000 times. likewise, if you set the seed value at the level of agent-type, you in effect repeat the resetting of the seed value 2,000 times (assuming a single high-level agent type of team member and 2,000 time-step simulation run). although the overall effects at this level are orders of magnitude smaller (e.g., far fewer resets of the prng seed value), it still significantly affects our ability to incorporate stochasticity into the abm and associated simulation runs. one approach for incorporating stochasticity into abms developed using 590 flame, and therefore gaining variance between replicate simulations is to run simulations using the built-in production mode within flame, which derives the seed value from the system clock. unfortunately, this approach does not enable exact reproduction of individual simulation runs because the system clock is continuously updating with the progression of time, which 595 seriously impacts our ability to repeat simulation-based experiments that have interesting dynamics, or to help resolve issues during the debugging activities in model development. fortunately, a technical workaround was identified that resolved this issue, which involved the creation of a dummy agent whose sole purpose is to set the prng seed value in the first iteration 600 of an individual simulation run. this utilized an agent-level counter (to count the simulation time-step), and logic to only set the prng seed value when the counter equals zero. in addition, and to keep the simulator tidy of computational artefacts, the logic also ensures that this dummy agent is removed from the simulation once the simulation time-step counter reaches 605 a pre-defined number. explicitly set memory allocation additional constraints, although minor in relation to the above, are that a single simulation time-step is taken as a standalone run of a simulation, and 610 that memory allocation for the agents and messages requires a continuous block size of memory. with respect to the single simulation time-step, this means that upon completion of all functions in that time-step, the global message board is emptied of all messages generated by agents during that time-step. simulation modellers therefore have to take this constraint into 615 account when designing and developing the abm, because messages cannot be sent between time-steps, so pertinent information (e.g., temporary parameter values or communication from specific neighbours) will need to be stored within the agent's memory (i.e., within an agent attribute). conversely, with respect to the block size, this is important for parallelization 620 when using mpi, because the simulator needs to know how to package up data that is sent across nodes, so the need to explicitly define the size of the agent memory and messages (both in bytes) is crucial. during the calibration process, initial simulations were run using a desktop 625 pc that had quad-core processor, 8gb ram and used the windows operating system. this meant that three separate simulation runs could comfortably be performed simultaneously in distributed mode across three out of the four cores in the processor. we found the performance of flame to be very promising during initial evaluation when running a single simulation 630 on a single compute node across multiple hardware platforms. both cpu time and wall-clock time were found to be linear following increases in the number of agents within a simulation, and also linear when the simulation length increased up to 50,000 time-steps (the maximum needed in our simulation), which both indicate that flame scales very well. however, further 635 investigation identified that the rate-limiting processes and tasks associated with simulation runs was the input/output ([i/o], e.g., read and write) speed for writing and reading the large number of xml files that are created as part of the simulation output. the cause for this relates to the underlying design principles of flame, with its conceptual architecture being based 640 on communicating stream x-machines, which require the generation of individual xml files for each simulation time-step (these xml files contain parameter values and states for every x-machine agent that is instantiated). due to the output xml file for a specific simulation time-step, becoming the input xml file for the next simulation time-step, a performance bottleneck 645 is encountered with respect to the speed of reading from and writing to, the storage disk. for example, our abm contained 159 separate agents that corresponded to the individual team members in the case study, and resulted in each xml output file being approximately 50kb in size. a 50,000 iteration simulation 650 run, would therefore generate 50,000 individual simulation time-step output files, that contain the parameter values and states associated with each of the 159 agents at the respective simulation time-step, which in our case approximated to 2.5gb of output data per simulation run. furthermore, following aleatory uncertainty analysis, we discovered that 75 replicates of each 655 simulation run (using different prng seed values) are required to stabilize the median averages of system dynamics [47] , which results in approximately 190gb of simulation output being generated for each set of simulation runs. as such, complex abms that incorporate a large number of agents, can quickly generate significant volumes of simulation output data, which could 660 significantly impact the ability to perform simulation-based experimentation if large capacity storage is not available on the computing hardware. our relatively simple abm took approximately 25min to run on the windows pc, along with an extra 10min to process and transform the xml simulation output files (using python scripts) to csv files, and then subsequently 665 transforming these into a single csv file that contained the median average dynamics of each agent over the duration of the simulation. through using a pipeline of scripts in this way, we were able to ensure that the large volume of output data spent the minimum of time on the storage facility. to confirm i/o rate-limiting characteristics of flame, we performed an 670 identical set of simulation runs using the same windows pc, but for this experiment, the output files were written to an external usb hard drive. we found the overall wall-clock time increased due to the flow of data across the usb port being slower than that to the onboard hard disk. we built upon these initial findings through using another desktop setup, which this time 675 encompassed an apple mac mini with 8gb ram and solid state disk (ssd), along with a high-performance computing facility run by the northern eight universities consortium (n8) (based within the north of england). the mac mini with ssd was 28% faster (with respect to wall-clock time) than the windows pc with internal disk, and 38% faster than that setup when using 680 an external usb hard drive, resulting in the overall wall-clock time of a 50,000 time-step simulation run being reduced down to 18min. likewise, the n8 hpc was 85% faster than the setup using mac mini with ssd, bringing the wall-clock time for simulation runs down to just under 3min (see figure 6 ). access to fast storage (of suitable large capacity) is therefore crucial for any moderately complicated abm that requires replicate simulations to be run in order to account for the aleatory uncertainty within the model. an additional observation on performance relates to the communication figure 6 : the performance of flame was found to be linear with respect to the number of iterations within a simulation run and the wall clock time. flame was found to be input-output rate-limiting by running a simulation on various hardware setups. for instance, we ran the simulation on: a windows pc with internal hdd and on an external hdd, a mac mini with ssd, and a high-performance cluster with dedicated fast lustre storage. between agents over parallel architectures, and the resultant sequence of messages being sent and read. such communication dependencies between agents 690 requires a synchronization block between the cores (on a laptop/desktop) or nodes (on a hpc) to ensure that messages arrive in time to meet the dependency, i.e., functions being performed in the specific time-step of the simulation do not try to read a message before the node/core has received it from another node/core. these synchronization blocks are a consider-695 able time bottle neck, which introduce performance losses into simulations. as such, the fewer synchronization blocks introduced into a simulation, the greater the performance of the overall simulation run. real-world social systems are complex, with sets of behaviours, charac-700 teristics and dynamics that emerge through the individual relationships that function through time and space. one of the major benefits of the agentbased modelling paradigm, is the emphasis on three principal characteristics of complex systems: 1) system structures, 2) system dynamics, and 3) system control [3] . one of the advantages of the agent-based approach is that the 705 simulations aim to replicate the dynamics of the real-world system, in order to ensure the validity of the underlying assumptions behind the computational model can be tested. however, in order for these abms to be successful in performing their role as scientific instruments that act as credible abstractions of complex social systems, it is crucial that the preferred modelling 710 and simulation framework is able: to realistically represent system structure and dynamics; is modular, so that the abm can be incrementally updated with new functionality, without the need to re-engineer the entire model; can expand with reference to the hierarchical-scale of the real-world system, e.g., individual team members, to discrete project teams, to the programme-wide 715 network as a whole; and is amenable to a thorough validation and verification process, including stringent statistical analysis of simulation output data. within this study, we used flame to develop an abm that was calibrated to the quantitative and qualitative data from our case study around the development and propagation of conflict within multi-partner enterprise 720 system implementations. as discussed above, the flame modelling and simulation framework utilizes the conceptual and technical architectures associated with communicating stream x-machines to facilitate agent-based models in a discrete-event manner (the simulation time-steps are the discrete events). it has been reported to provide very significant improvements 725 in performance over more traditional abm frameworks [48] . flame's overarching purpose is to deal with massive simulations, allowing for modelling abstraction levels that cater for large scope with respect to the real-world system of interest, and hundreds of thousands, to millions, of x-machine agents. through being designed and developed to comply with the mpi 730 communication framework, flame code is also deployable on to parallel hardware platforms. our study has identified a number of strengths and weaknesses for using flame to model complex social systems. firstly, with respect to the strengths, and for modellers who are familiar with the object-oriented approach to design of computational models, we have identified that flame is an intuitive framework for designing abms of complex social systems due to its template-driven approach. similarly, for modellers who are comfortable with command line terminal, basic programming languages, and shell scripts, we have identified that flame is an intuitive framework for de-740 veloping abms of complex social systems. conversely, we have shown that flame suffers with a number of constraints that introduced technical issues into the development process for our abm. the four major constraints were: the lack of built-in functionality to set the prng seed value at the simulation level; the inability to utilize functionality associated with global 745 mutable parameters; the inability to communicate between simulation timesteps/iterations; and the lack of instrumentation to analyze simulation output data, thus requiring the development of various statistical and data transformation scripts, which is fine for experienced modellers, but is a real constraint for novice modellers or subject matter experts. these constraints are unfortunately a direct consequence of the design decisions taken during the initial creation of flame, because the underlying premise was that it would harness the power of parallel processing to run individual simulations across hpc architectures. a number of technical challenges were introduced by these constraints, but fortunately, we were able 755 to develop workarounds to resolve them. as such, we want to inform future users of the flame simulation framework that they need to be cognizant of these constraints, especially due to the fact that it has been proposed as suitable for subject matter experts within the real-world domain of interest, who may have limited experience and/or competence in computational mod-760 elling or programming. with this in mind, we advocate that this message be moderated, because subject matter experts may not have the technical competence or modelling experience to fully investigate, analyze, diagnose and resolve the full variety of problems and constraints that we have experienced during this study. 765 furthermore, and of more significance is that we discovered a major limitation when using flame, is that models that incorporate large numbers of individual agents are computationally expensive with respect to wall-clock time, the i/o load, the need for hpc architecture to run multiple replications in distributed mode, and the consequent very large size of output simulation 770 data. this computational expense is compounded when the abm is increased in scale to reproduce results consistent with system-level dynamics from the real-world domain. due to flame's communicating stream x-machine architecture, which relies on input and output streams of messages to facilitate communication, we unexpectedly discovered that comparatively small simu-775 lations (with respect to total x-machine agent numbers, the complexity of interaction logic, and the total time-steps within the simulation) could gen-erate a considerable number of xml data files, which directly correlates to the volume of simulation output data produced. the wall-clock time for single simulation runs was found during diagnostic tests, to grow linearly in 780 accordance with the number of time-steps within simulation runs. diagnostic tests also indicated the input-output rate-limited nature of flame (as opposed to being rate-limited through the central processing unit), which is due to separate xml output files being generated for each time-step within simulation runs. to put this into further context, the communicating stream x-machine nature of flame requires that individual xml output files are generated at the end of each time-step to record the parameter values for internal computational and simulated real-world states along with internal memory values for each x-machine agent; these are then used as the xml input file at the 790 beginning of the next time-step to set the corresponding starting parameter values, states, and memory values for the next time-step of the simulation run. a bottleneck is therefore experienced regarding computational performance, which results from the speed of reading from and writing to, the onboard or peripheral storage disk. the results from aleatory uncertainty analysis further compounded this performance issue by identifying that a minimum of 75 simulation replicates were required to achieve stable median dynamics, which resulted in approximately 3,750,000 total xml output files, which as disucssed, amounts to 190gb for our relatively small abm. there can often exist a delicate balance with respect to computational 800 efficiency and performance during aleatory uncertainty analysis, which is introduced from the desire to achieve stable simulation results by using a high number of replicates for calculating the median average simulation results, whilst being cognizant of the computational resources required (e.g., wall-clock time, number of cpu cores on desktop or nodes on hpc, and access to 805 fast storage disk). the number of simulation replicates chosen to calculate the median average dynamics is therefore usually a compromise between minimizing the impact of aleatory uncertainty versus the acceptable costs in computational resources for the project. our findings therefore indicate that average, everyday desktop computing resources (such as a windows pc or 810 apple mac mini) are unsuitable for running simulations that require large numbers of replicates to achieve stable average dynamics to act as a baseline for simulation-based experimentation. we therefore conjecture that access to very fast multi-core desktops with significant storage capacity (such as fast ssd raid capabilities), or to hpc architecture is critical for ensuring that our abm of conflict development and propagation within a complex social network can be used for simulation-based experimentation. the above discussion has focused on our contributions to the computational modelling community through out user experiences when using flame 820 to model a complex dynamical social system. specifically, the discussion has focused on the strengths and weaknesses that we identified when using flame to model the development and propagation of conflict within the social network of large multi-partner enterprise system implementations. we found the design of our model to be straightforward due to flame's un-825 derlying architecture using communicating stream x-machines. the objectoriented nature of communicating stream x-machines, which are in effect, computational instantiations of uml state transition diagrams, makes the design and subsequent development relatively intuitive. in addition, the use of xml to define agents and c to code the agent communication and in-830 teraction rules, allows for a truly modular approach to abm development. conversely, however, the limitations of flame are also due to the underlying conceptual and technical architecture of flame. we discovered four main constraints relating to the lack of in-built functionality to set prng seed values, the inability to use global mutable parameters, the inability to 835 communicate between time-steps, and the lack of instrumentation to analyze simulation output data. in addition, we identified a significant limitation with respect to performance, which is a direct consequence of the communicating stream x-machine architecture, in that the output file for a simulation time-step acts as the input file for the subsequent time-step. our analysis 840 indicated that this not only leads flame to be i/o rate-limiting, but also means that large simulations (with respect to either total number of agents or total number of simulation time-steps) quickly generate prohibitively large amounts of simulation output data. we were lucky that our abm of the case study is relatively simple and had only 159 agents within the social network, 845 but even still, following aleatory uncertainty analysis we were required to run 75 replicates of simulations in order to mitigate the effects of aleatory uncertainty, and produced 190gb. as such, we conjecture that for large, complex, abms, the large volumes of simulation output data generated by flame may mean that it is rejected as a potential simulation framework if access to 850 very large capacity and high-speed storage capabilities cannot be achieved. to conclude, we believe flame is an excellent choice for experienced modellers, who will be able to fully harness the capabilities that it has to offer, and also be competent in diagnosing and solving any limitations that are encountered. conversely, because flame requires considerable develop-855 ment of instrumentation tools, along with development of statistical analysis scripts, we believe that it is not suitable for the novice modeller, who may be better suited to using a graphical user interface driven framework until their experience with modelling and competence in programming increases. in our opinion, flame's major strength is its flexibility, in that once the model 860 definition (along with any technical workarounds) has been developed using the xml and c templates (e.g., the social network topology, communication rules, rules for conflict development, and functionality to explicitly set the prng seed value), the augmentation of the abm with new functionality is comparatively easy due to the template-driven nature of flame facilitat-865 ing its modular approach to design and development. in addition, we have found flame to be excellent for quickly developing abms of complex dynamical systems in both our case study presented here, and indeed in previous work into complex dynamical biological systems [21] . we do however believe that it requires significant computational modelling skills in order to develop 870 workarounds to some of the constraints that have been imposed due to the underlying technical architecture, and to develop a pipeline of scripts (e.g., ruby, unix shell, ms dos, and python) to semi-automate the submission of jobs to a hpc and to transform and analyze simulation output data to reduce the storage load when large numbers of replicate simulations are required to 875 account for the aleatory uncertainty within the model. finally, we are aware that a version of flame has been developed that harnesses the parallel processing power of graphics processing units (gpu) [49] , which has recently been extended [50]), and could prove beneficial, because a number of the limitations that we discovered (e.g., no global mutable parameter and the i/o 880 resource intensiveness) may be resolved through the difference in processing architecture. one final point to highlight on this however, is that flame gpu, being based on cuda, requires nvidia graphics cards, which could prove problematic for those developers who are using apple mac computers because of the current issues with drivers and mac mojave/catalina operab-885 ility, and the need to use egpu and various scripts to get the thunderbolt connectivity working with older macs -we are aware that newer macs have thunderbolt 3 connectivity and that mac os high sierra supports nvidia drivers. to this end, we are in the process of trialling flame gpu on such a setup. distributed mason: a scalable distributed multi-agent simulation environment a new approach to decoding life: systems biology lessons learned on development and application of 910 agent-based models of complex dynamical systems distributed simulation optimization and paramter exploration framework for the cloud agent-based computational models and generative social science engineering simulations as scientific instruments: a pattern language tutorial on agent-based modeling and simulation on agent-based software engineering agent-based modelling: methods and techniques for simulating human systems using the cos-mos approach to study schelling's bounded neighbourhood model proceedings of the 2004 workshop on complex systems modelling and simulation a tipping point in 300 years of banking? a conceptual simulation of the british banking system understanding the impact of communication technologies on virtual team performance: an agentbased simulation model how simulation modelling can help reduce he impact of covid-19 reverse engineering of biological complexity tools of the trade: a survey of various agent based modeling platforms mason: a multi-agent simulation environment repast orgainzation for architecture and design netlogo, the center for connected learning and 955 computer-based modeling using x-machines as a formal basis for describing agents in agent-based modelling investigating ikk dynamics in the nf-îºb signalling pathway using x-machines large-scale modeling of economic systems an agent based modeling approach in the strategic human resource management, including endogen-970 ous and exogenous factors road network simulation using flame gpu proceedings of the 21st international conference on parallel and distributed computing designing and building parallel programs automata languages and machines x-machines as a basis for dynamic system specification using x-machines to model and test discrete event simulation programs modelling the dynamics structure of biological state-based systems rosen's (m,r) system as an x-machine erp: a vision of the next-generation mrp ii, scenario s-300-339 enterprise resource planning (erp) -a brief history beyond critical success factors: a dynamic 1000 model of enterprise system innovation and then came complex project management (revised) conflict propagation within large technology and software engineering programmes: a multi-partner enterprise system implementation as case study conflict and defense a multimethod examination of the benefits and detriments of intragroup conflict the dynamic nature of conflict: a longitudinal study of intragroup conflict and group performance the influence of intra-team conflict on work teams' affective climate: a longitudinal study modelling conflict within the social networks of 1020 large multi-vendor software projects using communicating stream x-machines cybernetics of conflict within multi-partner technology and software engineering programmes the kolmogorov-smirnov test for goodness of fit a critique and improvement of the cl common language effect size statistics of mcgraw and wong how much impact does the choice of a random number generator really have? gambling with randomness: the use 1035 of pseudo-random number generators in gis techniques for grounding agent-based simulations in the real domain: a case study in experimental autoimmune encephalomyelitis, mathematical and computer 1040 modelling of proceedings of the 9th international conference on autonomous agents and multiagent systems cellular level agent-based modelling on the graphics processing unit simulating heterogeneous behaviours in complex systems on gpus key: cord-330503-w1m1ci4i authors: yamin, mohammad title: it applications in healthcare management: a survey date: 2018-05-31 journal: int j inf technol doi: 10.1007/s41870-018-0203-3 sha: doc_id: 330503 cord_uid: w1m1ci4i healthcare management is currently undergoing substantial changes, and reshaping our perception of the medical field. one spectrum is that of the considerable changes that we see in surgical machines and equipment, and the way the procedures are performed. computing power, internet and associated technologies are transforming surgical operations into model based procedures. the other spectrum is the management side of healthcare, which is equally critical to the medical profession. in particular, recent advances in the field of information technology (it) is assisting in better management of health appointments and record management. with the proliferation of it and management, data is now playing a vital role in diagnostics, drug administration and management of healthcare services. with the advancement in data processing, large amounts of medical data collected by medical centres and providers, can now be mined and analysed to assist in planning and making appropriate decisions. in this article, we shall provide an overview of the role of it that have been reshaping the healthcare management, hospital, health profession and industry. internet and web 2.0 have been instrumental in evolving most of the information technology (it) applications that we are now accustomed to. barely 20 years ago, we had not imagined the technological advancements, which are now implemented into our lives. technological advancement, riding on the wave of disruptive technologies of the last quarter of a century, is reshaping and redefining the world's social order and norms. the medical field is part and parcel of our lives and is perhaps one that we care the most about. in the recent times, this field has undergone vast changes. the medical profession today has evolved greatly since the beginning of this century. not only the medical procedures, the medical data flow and management has also undergone considerable improvement. advance data transfer and management techniques have made improvements in disease diagnostic and have been a critical role in national health planning and efficient record keeping. in particular, the medical profession has undergone substantial changes through the capabilities of database management, which has given rise to the healthcare information systems (his). medical data collected from different institutions can now be mined and analysed in time using big data analytics tools. this article shall provide an overview of the involvement, importance and benefits of it and how data management is contributing to the medical science field. in particular, the following will be discussed: 5. patient and data management 6. big medical data analytics 7. privacy and security of healthcare data 2 artificial intelligence and robotics in healthcare artificial intelligence (ai) is a field of computer science which has its roots in logic (mathematics), psychology, philosophy, linguistics, arts, science and management among others. many ai-dominant tools and applications can be found in games, auto spare parts, heavy machinery and various medical instrumentations. according to [1] , many programs are developed with the help of ai to perform specific tasks which make use of many activities including medical diagnostic, time sharing, interactive interpreters, graphical user interfaces and the computer mouse, rapid development environments, the linked listdata structure, automatic storage management, symbolic, functional, dynamic, and object-oriented programming. here are some other examples. pharmaceutical developments involve extensive clinical trials in real environment and may take many years, sometimes more than a decade. this process requires a considerable amount of resources and can cost billions of dollars. in order to save lives and money, it is desirable to expedite this time consuming process to make healthcare cheaper. an urgency of this nature was witnessed during the ebola crisis in some west african nations [2] . an urgency of this nature was witnessed during the ebola crisis in some west african nations [2] , where a program powered by ai was used to scan existing medicines that could be redesigned to fight the disease. nowadays medical consultation, with the help of an online illness database, can take place without the direct interaction of a medical practitioner. an example of such a practice can be found in [2] . advantages of such consultations lies in the fact that the symptoms presented online by the patients are matched with those of about ten thousand known diseases [3] , whereas doctors can only match the symptoms against a fraction of the known diseases which they are familiar without the intervention of an it database. likewise virtual nursing to support patients online is also available from [4] . yet another app [5] developed by boston children hospital provides basic health information and advice for parents of ill children. since the introduction in 1960s, robots have been used in many scientific and social fields. their use in fields like nuclear arsenal, production of automobiles, and manipulations in space has been widespread. in comparison, the medical science field has somewhat been gradual to take advantage of this technology. nevertheless, robots are now days being utilised to assist in or perform many complex medical procedures, which has given rise to the term robotic surgery. a comprehensive discussion of robotic surgery is available in [6] . indeed there are many advantages of using robots in healthcare management. for example, developing pharmaceuticals through clinical trials can take many years and cost may rise to billions of dollars. making this process faster and more affordable could change the world. robots can play a critical role in such trials. robots are also very effective in medico training where simulations are achieved. use of robots in abdominal surgery [7] is innovative in comparison to the conventional laparoscopy procedures, and has the potential to eliminate the existing shortcomings and drawbacks. according to [8] , the most promising procedures are those in which the robot enables a laparoscopic approach where open surgery is usually required. likewise robotic surgery for gynaecology related ailments [9] can also be very effective. the conventional laparoscopy, the surgeon would have limited degree of freedom with a 2d vision, whereas the robotic system would provide a 3d view with intuitive motion and enable additional degrees of freedom. prostatic surgery, being complex in the way of the degree of freedom, is another area where robots are very effective [10] . nosocomial infections pose a major problem hospitals and clinics around the world. this becomes worst in case of large and intense gatherings of people like in hajj [11] . to keep the environment free of these viruses and bacteria, the environments needs to be cleaned efficiently and regularly. however some viruses like ebola [2] and mers coronavirus [12] are highly contagious and cannot be cleaned by humans without having protective gear, which can be costly and may or may not be effective. cleaning the hospital with chlorine is an effective way. however, there are many drawbacks of this method including a high risk for cleaners to be infected themselves. a sophisticated approach was implemented in us hospitals by using robots [13] to purify the space. however, some of the robots used are not motorized and may require a human to place them in the infected room or area, posing the risk of infection to humans. it is not known as to which substance or matter can effectively eliminate deadly viruses like ebola and mers coronavirus. however, according to [14] , beta-coronaviruses, which is one of the four strands of mers coronavirus, can be effectively eliminated by uv light. for such a deadly and highly contagious virus clearing, a robot that can be remote controlled is needed, which can move forward, backward and sideways, and is able to clean itself. concept and related technology of virtual reality (vr), augmented reality (ar) and mixed reality (mr), which originate from image processing and make extensive use of ai, have been in use for decades in games and movies. for some time now, the vr, ar and mr tools and technology are assisting the medical field in a significant way. virtual reality has been used in robotic surgery to develop simulators to train surgeons in a stress free environment using realistic models and surgical simulation, without adversely affecting operating time or patient safety [15] . an account of ar and vr technologies, including the advantages and disadvantages can be found in [16] . the ar and vr technology can be very useful in medical and healthcare education, an account of which can be found in [17] . applications of vr, ar and mr can also be very helpful in urology [18] . healthcare management information systems (hmis) is the generic name for many kinds of information systems used in healthcare industry. the use of hmis is widespread in various units of hospitals, clinics, and insurance companies. a doctor records patient data (habits, complaints, prescriptions, vital signs, etc.) in one such system they use. a number of hospital/clinic units like radiology, pathology, and pharmacy store patient data in these systems for later retrieval and usage by different stakeholders as shown in fig. 1 . appointment system itself can also be part of a hmis or a standalone system. modern healthcare without hmis cannot be comprehended. an information system or electronic system dealing includes a database system, sometimes known as the backend of the main system. a database is a collection of data, tailored to the information needs of an organisation for a specific purpose. it can grow bigger or shrink to a smaller size depending on the needs of different times. a database in itself would be of no use unless it was coupled with a software system known as the database management system (dbms), which facilitates various functions and processes like data entry and storage, retrieval, and update, data redundancy, data sharing, data consistency, data integrity, concurrency, and data privacy & security. more information about database systems can be found in [19] . data in an information system can be organised in several different ways. classical representation of data [20] is tabular. most tables can be regarded as mathematical relations, for this reason, a dbms with tabular data organisation is known as relational. relational databases have by far been dominant in the business organisations until recently. in the eighties and nineties of the last century, object-oriented and semantic data models were also introduced. in particular, object-oriented databases [20] became very popular for managing audio-visual data. semantic database technology [21] , although very efficient for search queries, did not reach its expected peak, perhaps because of its complex data model dealing with ontologies. a comparative study of relational, object oriented and semantic database systems is carried out in [21] . although the evolution of information technologies began much earlier, however, its implementation by businesses and corporations occurred in the second half of the last century. the healthcare industry a bit slow to take the advantage of the technology. earlier, medical records and systems were paper based. thus the first phase of the usage of information technology and systems in hospital and healthcare management was to transform paper based records to database systems. not only did it change paper based systems to electronic ones but also allowed data manipulation. as a result, more data could be processed in a very short time, which was critical in making timely decisions. a discussion of early stages of this transformation can be found in [22] . consolidation of this technology resulted in healthcare management information systems (hmis). timely decision is critical in all walks of life but it assumes a much greater urgency in some healthcare cases. hmis is capable to transferring data from one location or application to another within split of a second, as opposed to days without the help of electronic systems. this allows users to access, use and manipulate data instantly and concurrently. as the technology continues to evolve and refine, the performance of information systems operations, in particular those of hmis, is bound to improve. ubiquitous health system is meant to provide healthcare services remotely. according to [23] , ubiquitous healthcare system means the environment that users can receive the medical treatment regardless of the location and time. essentially, a ubiquitous healthcare system would monitor daily or periodic activities of patients and alert the patients or health workers of problems, if any. a discussion of national healthcare systems in korea, including the concept of u-hospitals, is discussed in [24] . a rich picture of a ubiquitous healthcare system is shown in fig. 2. internet of things (iot) is an emerging technology. data in organisations, including hospitals, clinics, and insurance companies, is increasing every day. to process this large data, advanced data processing techniques are combined into what is now known as the big data analytics. internet of things (iot) is a general paradigm. when dealing with medical environment, it is known as internet of medical of things (iomt), which is same as the medical internet of things. a discussion of iomt can be found in [25] . the fundamental objective of iot is to provide ubiquitous access to numerous devices and machines on service providers (sps) [26] , covering many areas such as location based services (lbs), smart home, smart city, e-health [27, 28] . these tools, devices and machines cover service providers in many areas of location based services. a few other could be smart home, street or city, elements of ubiquitous health systems, tools for electronic learning and electronic business, the number which is increasing day by day and it may reach fifty billions in 2020 [29, 30] . we have conceptualised this in fig. 3 . the iot applications invariably use cloud storage coupled with fog computing [31] . data in organisations is growing at a rapid rate. some organisations have collected huge pile from their operations. organisational data needs to be mined and analysed on a regular basis to uncover hidden patterns, unknown correlations, market trends, customers, patients and treatment records to help organizations to plan and make more informed decisions. mining and analysis of big data captures the immense wealth of intelligence hidden in organisational data, makes information transparent and usable at much higher frequency, and enhance performance. all of these contribute to providing higher value in efficient decision making. when data grows, it cannot be handled manually, often big data organisations use robotic arm to perform data loading and unloading operations. medical data is also growing at a rapid rate in many hospitals, clinics and insurance companies. mining and analysis of healthcare data is even more critical as it relates to the wellbeing of the society. data centric research is highly desirable to find answers to questions on many chronical diseases, and to plan and manage national and international health programs. potential befits of big data analytics in healthcare are discussed in [32] . in particular, authors have provided five strategies for success with big data. these are (1) implementing big data governance, (2) developing an information sharing culture, (3) training key personnel to use big data analytics, (4) incorporating cloud computing into the organization's big data analytics, and (5) generating new business ideas from big data analytics. a survey of big data analytics in healthcare and government is conducted in [33] , where the need for the analytics is linked to the provision and improvement of patient centric services, detection of diseases before they spread, monitoring the quality of services in hospitals, improving methods of treatment, and provision of quality medical education. authors in [34] identify medical signals as a major source of big data, discuss analytical techniques of such data. some of the well known tools used for big data analytics are apache tm hadoop ã� is highly scalable storage platform. it provides cost-effective storage solution for large data volumes. it doesn't require any particular format. apache spark is an open source, in-memory processing machine. its performance is much faster than that of hadoop. mapreduce is used for iterative algorithms or interactive data mining. there are many other big data analytics tools, a list of them can be found at http://bigdata-madesimple.com/top-30-big-data-tools-data-analysis/. there are always security and privacy concerns associated with data. protection of privacy and security is a responsibility of data holding organisations. with medical data these concerns assumes higher pronounce and priority. medical data can be very sensitive, and may be linked to life and death of the patients. often socio political issues are linked with medical data. it is well known that india was divided into two countries giving rise to pakistan in 1947. mohammad ali jinnah, very clever and shrewd, was the leader of pakistan campaign, larry collins and dominique lapierre [35] argue that the partition of india could have been avoided if ''the most closely guarded secret in india'' had become known. jinnah was suffering from tuberculosis which was slowly but surely killing him. in [28, 36, 37] , privacy issues of data are discussed. healthcare procedures and management are now greatly relying on it applications, which are providing realtime access to data and utilities. without these applications, healthcare will be limited, compromised and prone to major problems. many of the medical equipments, which are used in procedures, are highly dependent on technology. ai, robots, vr, ar, mr, iomt, ubiquitous medical services, and big data analytics are all directly or indirectly related to it. hmis is critical for efficient management of records, appointment systems, diagnostics and their needs of medical centres. as the technology advances, the healthcare is likely to further improve. artificial intelligence: a modern approach perspectives on west africa ebola virus disease outbreak intelligent machines, the artificially intelligent doctor will hear you now meet molly-the virtual nurse boston children's hospital launches cloud-based education on amazon alexa-enabled devices robotic surgery-a personal view of the past, present and future robotic abdominal surgery status of robotic assistance-a less traumatic and more accurate minimally invasive surgery? robotic surgery in gynaecology a randomised controlled trial of robotic vs. open radical prostatectomy: early outcomes health management in crowded events: hajj and kumbh. bvicam's middle east respiratory syndrome coronavirus (mers-cov) origin and animal reservoir the iowa disinfection cleaning project: opportunities, successes, and challenges of a structured intervention program in 56 hospitals inactivation of middle east respiratory syndrome-coronavirus in human plasma using amotosalen and ultraviolet a light impact of virtual reality simulators in training of robotic surgery augmented and virtual reality in surgery-the digital surgical environment: applications, limitations and legal pitfalls preliminary study of vr and ar applications in medical and healthcare education application of virtual, augmented, and mixed reality to urology database systems. a practical approach to design, implementation and management, 5th edn a relational model of data for large shared data banks semantic, relational and object oriented systems: a comparative study health care in the information society: a prognosis for the year 2013 unified ubiquitous healthcare system architecture with collaborative model national health information system in korea medical internet of things and big data in healthcare information analytics for healthcare service discovery intelligent human activity recognition scheme for e-health applications preserving privacy in internet of things-a survey internet of things: converging. technologies for smart environments and integrated ecosystems security of the internet of things: perspectives and challenges a survey on fog computing: properties, roles and challenges big data analytics: understanding its capabilities and potential benefits for healthcare organizations a survey of big data analytics in healthcare and government. 2nd international symposium on big data and cloud computing (isbcc'15) big data analytics in healthcare freedom at midnight: the epic drama of india's stuggle for independence improving privacy and security of user data in location based services big data security and privacy in healthcare: a review key: cord-024343-mrri46oh authors: buldakov, nikolay; khalilev, timur; distefano, salvatore; mazzara, manuel title: an open source solution for smart contract-based parking management date: 2020-05-05 journal: open source systems doi: 10.1007/978-3-030-47240-5_6 sha: doc_id: 24343 cord_uid: mrri46oh this paper discusses an open-source solution for smart-parking in highly urbanized areas. we have conducted interviews with domain experts, defined user stories and proposed a system architecture with a case study. our solution allows integration of independent owners of parking space into one unified system, that facilitates parking in a smart city. the adoption of such a system raises trust issues among the stakeholders involved in the management of the parking. in order to tackle such issues, we propose a smart contract-based solution encapsulating sensitive information, agreements and processes into transparent and distributed smart contracts . it has been estimated that, with the current trend, 60% of people will end up living in urban areas [24] . the increasing pace of the urbanization process made the management of city services more challenging among such services transportation has strategic importance since the increase of population leads to increased traffic congestion and pollution, which significantly impacts on the citizens' lives. several approaches have been adopted to address these issues, and in 1970s some countries decided to widen the roads and create new ones. for example, in hague, netherlands, many canals were drained and covered for this purpose [1] . more modern approaches focus on the factors affecting congestion and operate on them. this trend has led to smart management of streetlights [18] , and the introduction of payed parking lots and roundabouts. several studies have also shown that searching for a free parking stall drastically affects the traffic as vehicles spend some time going around looking for parking [12] . smart parking solutions can, therefore, significantly improve traffic conditions. there are several open issues and challenges on the current parking management. first of all, in every district, there are slots of unused land, owned either by private or public entities. providing easy means of renting out such properties for parking can simplify the process of creating new parking lots. moreover, collection and aggregation of information about the current occupancy of all parking lots can significantly simplify the process of finding a free stall, thus decreasing traffic congestion. another significant issue is the trustworthiness of a potential parking system. with the current solutions on the market, such as [27] sensitive information is stored in a centralised way, which makes it vulnerable and simplifies malicious intrusion. finally, various parking providers need aggregation under one universal system, that will guarantee the unbiased treatment of all parking lots. such strategy will allow potential seekers of parking lots to observe the entire situation in the city instead of seeing only partial information. this work aims at creating an open-source solution in order to tackle the aforementioned problems. every city has governing authorities: city hall, municipality and it can be divided up into districts with local councils. it can also be organized in terms of smart governance [25] . we will further refer to these authorities as an administrator. the administrator is responsible for authorising processes in the city. there are also people and companies possessing pieces of land that are suitable for parking. we refer to them as landlords. a landlord can be a juridical person or a natural person; it does not matter for the system. a landlord wants to provide their land for parking in order to profit from parking fees. besides, there are entities called tenants. a tenant is a company or a person who will to whom a landlord rents out a subset of their parking land-based on some conditions. now that all the roles are defined, one can observe the relations connection different roles in fig. 1 . in order to provide their land for parking, a landlord must first sign a contract with the administrator. there is always only one administrator for each area in the city. the process starting from a request to create such contract (from the landlord's side) up until its signing by both parties will be called landlord registration. this process involves checking whether the landlord is eligible for renting out their property, and negotiation on the details of the contract. eventually, the signed contract contains such details as the tax which the landlord guarantees to pay, information about the registered land, duration of the contract and others. both parties can further amend the contract, given that they are satisfied with the amendment. the landlord, in turn, can not only provide their land for parking but also they can rent it out to tenants. in this case, the income from parking will not go directly to the landlord, however, they receive guaranteed rent payments and, possibly, a percentage from each parking transaction. similarly, another contract preserved the agreement between a tenant and a landlord. this contract describes all the details of the rent. such details are the duration of the contract, the rent fee, the frequency of payments, the landlord's share in each parking transaction and, most importantly, the exact borders of the area rented to each tenant. again, all these details can be amended later, given the consensus of both parties. there is a set of preconditions, which this study assumes parking lots to hold. first of all, the parking areas have to be divided up into a set of spots. we refer to these spots as parking stalls. this partition can be represented by road marking. moreover, there has to be a tracking device which will make the parking lot fully observable. a particular example can be a camera with an iot-powered box, produced by park smart [2] as described in their patent [26] . this device has to run image processing algorithms in spot in order to tell at each moment which parking stalls are occupied (a particular implementation is provided in [5] ) and what the plate numbers of the parked cars are. a visualisation of these assumptions can be found in fig. 2 . a real example could be a mall with a big parking zone. stalls can be partitioned among the businesses that are present in the mall. big companies, like ikea could rent the closest stalls in order to ease transportation of goods from the store to cars. restaurants could want to offer reduced fares to their visitors for parking. there can also be stalls that are not rented by any company. in this case, a car parked there pays according to the pricing policy of the landlord, i.e. the mall. the proposed solution presented in this paper aims to tackle the problems described above by using blockchain smart contracts. it assumes a set of roles to be present is the city. one of them is a city hall, that will be responsible for the deployment of the system as well as setting several policies following the local legislation. another role is a landlord who possesses a piece of land. a landlord can be a juridical person or a natural person, it does not matter for the system as long as their land satisfies the constraints describer in the assumptions section. such situations are common: a landlord does not want to maintain the land fully themselves and rents its parts out, in this case the proposed system allows a landlord to partition their land as they desire and set complex paying policies, that will ensure the agreed-upon conditions at the time of payment for parking. the last role is a driver interested in parking their car. they can observe free spots available at every parking lot and the pricing policy and choose the most suitable one. payment is also covered conducted through the system, using a micro-payment channel, that makes it possible to enforce and guarantee the distribution of money among the landlords, the city hall and the tenants. by the time of writing, various smart parking systems exist in the literature, that focus on various aspects of parking. in principle, we can divide them into two groups: centralized solutions and decentralised solutions. there is a garden variety of solutions present both in the market and research, which attempt to prove a framework to manage smart parking. however, the major drawback of most of them is their centralised nature. many of them rely on a particular supreme entity, their "source of truth". in [17] a parking system is proposed that relies on iot devices and exchanges data by means of tcp/ip protocol. however, the application resides on only one server, that makes it vulnerable to issues with security, trustability and single point of failure. other works propose architectures that utilise the blockchain solutions as their backbone. all centralised solutions share a common set of problems that make them undesirable for practical use. the most important of such issues is trustworthiness. all participants of such systems have to rely on a particular entity, that will possess the power of accessing, chaining and removing their data, with no guarantee at the time of signing an agreement or registering that the owner of the system will obey to the promised rules. lack of transparency is another significant shortcoming. when the implementation is not exposed to the endusers, they cannot validate that a particular solution is guaranteed to function in accordance with their needs and that it does not have any back-doors. finally, considering the ongoing competition on any market, including parking, a centralised solution can be unfair to some participants if the owner is biased towards some companies. in [6] a system is proposed for incorporating blockchain into regular parking scenarios. this paper presents a layered architecture that standardizes the typical architecture of an integrated smart parking system. it also describes the major components of the system as well as provides an overview of possible work-flows of the solution. in this system, there are three major fig. 3 . overview of integrated smart parking system, taken from [6] participants: parking service provider, blockchain network, and user. the network enjoys the standard blockchain characteristics: a consensus algorithm, a shared public ledger, running a local copy of the ledger at each parking area. the overview schema of the system can be seen in fig. 3 . the architecture in the described solution consists of four layers: application layer, network layer, transaction layer, and physical layer. this layout of technologies can be considered the current state of the art and is adapted in the solution, proposed in this thesis work. finally, the authors of the paper describe two main workflows supported by their system: 1. search and request of a parking spot and 2. parking provider operations in the system. even though the paper describes a useful basic concept, it supports a minimal number of scenarios, and thus, one cannot consider it as realistic. another blockchain-powered system is presented in [15] . it aims to take into account the process of setting up the parking space for the land-owner. for this they a landlord has to go through government validated process and procedures such as land registry and government mandate external system. the external control is conducted by presenting another entity, named oracle. in addition to that, they tackle the problem of leasing lands to the so-called contractors by distributing non-fungible tokens among them for a particular period. these tokens are issued to the certified landlords by the government. the critical advantage of this solution is that it describes in great details peculiarities related to how governing authorities and lease of land can be integrated into a blockchain system. however, many processes are supposed to happen off-chain, which is a serious limitation for the true decentralisation of the system. [16] presents registration, search, rent, payment modules built by the means of the bcos smart contracts framework. the main novelty of this system is the use of one-time pad mechanism and a group-signature mechanism together with a union of privileged nodes in order to conceal the details of each transaction and thus preserve users' privacy while the privileged nodes can still access users' data. one-time padding means that instead of receiving a real address for each transaction, users, instead, receive a newly generated address each time. this way, the users' addresses are not exposed. a group signature mechanism allows signing a transaction on behalf of a group, which hides the mapping between transactions and users. other concepts implemented in this system are relatively similar to the works mentioned above, so the privacy mechanisms are the main beneficial feature of the paper. finally [11] introduces a gamification approach that simplifies the process of finding a parking lot. it encourages users to take part in the reporting process, by using the data history in order to calculate which places are more likely to be empty and by providing a framework that does not assume or require any particular installation of infrastructure. in this system, the concept of mobile crowdsensing plays a pivotal role. in order to ensure the involvement of drivers, the system assigns individual points to each driver for their collaboration. examples of such collaborations are the installation of a special beacon or reporting on the number of free parking spaces in the street. pedestrians can also participate by reporting through a mobile app. these points can be exchanged for free parking minutes. the authors claim their approach to bring a variety of benefits including a faster search for parking (leading to lower co2 emissions), easy deployment of the solution as it does not require much an existing infrastructure. although their reasoning is valid, there is almost no information in the paper how the blockchain technology was incorporated. although the works mentioned in this literature review present a variety of parking solutions, a significant limitation is in the fact that all of them lack flexibility for the end users. there is a variety of possible configurations of the system present, for example with a governing agency involved [15] or without [6] . however, none of them allows the community to choose what type of configuration they need. the same issue arises with the pricing policy. all papers consider some pricing policy present and even fluctuating depending on some factors as time and demand [6] . however, the details are omitted of how exactly the pricing is organized as well as how shares are distributed among different entities in the system. finally, the topic of leasing or renting out the land is not thoroughly defined, in particular, how exactly it can be done through the system. in [15] a token system is proposed but the details of how exactly the partitioning of a land will be tackled in the system are not presented. the objective of this work is to take into account drawbacks of the current centralised parking solutions, as well as limitations of the decentralised parking systems and to design and build a system that would address those issues. the system will utilise smart contracts and take into consideration some realistic requirements gathered by interviewing people from the industry. any proper system-development commences with gathering important requirements of the system. furthermore, as [13] states "one might use questionnaires or interviews to explore what problems members see as most important, how members place themselves in various classification schemes, etc." thus, an interview has been conducted with a representative of park smart [2] . park smart is a smart parking solution company founded in italy, messina in 2014 and its main aim is to implement a software-based technology able to analyse, in real-time, the availability of space in in-street parking. the company has already been in the field for a few years. therefore, was valuable to question them about their experience and to learn what are the main requirements for a parking system. the interview has shown some essential aspects of the modern smart parking system and it has also revealed some common shortcomings of the existing system. for instance, if a company wants to use this solution for paid parking, they can provide their end point to park smart in order to receive money through the system. this approach has inspired the idea of service providers in our system. moreover, the interviewee has confirmed our concern about the centralisation of current systems. park smart itself has a patent about blockchain architecture for their system, however, it has not been implemented yet. besides, the importance of flexible pricing policies has been highlighted. companies have different rates at a different time of the day, therefore, this part of functionally is to be flexible. finally, we found out what particular information about users and processes is needed for such a system, and this has affected our decisions of what to store in the proposed solution. in order to comprehend the needs of various user roles in the system, user stories have been gathered. the roles that we considered are the following: administrator, landlord, tenant, driver, and service provider. the user stories have revealed a particular set of features that every role needed. here we extract the key functionality from the user stories. first of all, both driver and parking providers (landlords and tenants) require a safe, trustworthy way of conducting payments in such a way that both parties are guaranteed that the amount of funds transferred is equivalent to the provided service. besides, landlords and tenants want to ensure that both parties respect the renting contracts they make among them and that such contracts can be extended or amended over time. parking providers also want the system to postulate equal treatment for all parking lots so that there are no privileged ones, promoted by that system. finally, the administrative authority requires to have privileged over the system (bounded by conditions of contracts made with other parties). in total, the backlog is constituted by more than 30 user stories, which were considered during the design and implementation phases. there are several participants in the system: landlord, tenant, driver or car, iot box installed in each parking system, administrator and as a governing authority. the parking system consists of thin clients (such as a mobile or a web app), and each participant can access their part of functionality through the thin client. every thin client is connected to a local evm (etherium virtual machine) node, which in turn is connected to the rest of the etherium network. the local node has an instance of the ledger, shared by all nodes, and also hosts smart contracts. whenever a participant wants to perform an action (e.g. initiate parking or update number of free parking sports), the thin client tells the evm to invoke a corresponding method of some smart contract. this action is performed in the form of a transaction on the blockchain. the blockchain network contains a public ledger and updates the public ledger with the valid transactions only. a consensus mechanism is used to verify the transactions. once the new transaction is verified, the public ledger is updated, and the local copies of the ledger in each node are synchronised and all participants can observe the new state of the parking system. this overview is visualised in fig. 4 . smart contracts are the back-end part of the system. the logic is distributed among several contracts, which correspond to logical components of the system. each contract encapsulates its sensitive data and controls access to its functions. this way, only authorised entities can perform actions in the system, even though the contracts are publicly exposed. actors of the system deploy some of the contracts, other contracts are deployed automatically in existing contracts. the decision of whether a contract is deployed from the outside of the system or within contracts depends on two questions: 1. is the nature of the contract static or dynamic? 2. should the system validate and control the deployment process of a contract? the nature of a contract matters because some contracts are deployed once and remain active in the system for a long time, whereas others are needed only for a short amount of time, and after their expiration, they cannot be used inside the system anymore. an example of a static contract could be the parking system contract, which is deployed by the authorities and the life cycle of this contract defines the life cycle of the system itself. on the other hand, an example of a contract of a more dynamic nature could be a payment channel. whenever a new parking process commences, the system automatically generates a payment channel. such a contract has a short lifetime and cannot be reused. thus the burden of its creation lies upon the shoulders of a static contract -parking provider. the latter question tackles the problem of validation. contracts can be inherited, and their behaviour can be changed by other parties. for example, the parking lot contract can be modified to surpass the tax obligation. that is why one requests the system to deploy such a contract. the system verifies all information related to the contract and only after that deploys it. for other contracts, on the contrary, extensions are allowed. for instance, payment policy defines a set of methods to be implemented to calculate the price of particular parking, however, it is up to a parking provider how exactly the price will be calculated. thus, a parking provider is entitled to extend and deploy their payment policy and supply the address of this policy to the system. parking system is the first contract to be deployed to launch the system. it is deployed by an administrative authority (e.g. a city hall) and it implements the main managing functionality of the system. the parking system contract is responsible for the registration of new parking lots and cars as well as for storing addresses of all other entities in the system. whenever some request cannot be fulfilled automatically by the contract, it saves the request and pends administration's approval. moreover, this contact provides a means of control and management of the whole system. thus, the system cannot exists without this contract. parking provider is an abstract contract that cannot be used on its own but should be extended in order to be used. it implements the logic of the parking process and stores information about what parking stalls it possesses and what cars are parked at the stalls. in other words, whenever a car wants to park, the code of this contract is used. in order to park a car sends funds to this contract. it also specifies a stall number and till what time it wants to be parked. the contract calculates the price, checks if the funds sent are sufficient and whether a parking stall is free and after that creates a payment channel. optionally parking provider can also have a set a service providers, in this case a car can specify which service provider it is using and a fraction of the payments will go to the service provider. this contract preserves general information about a car. this information includes its plate number, account address of its owner and the rating of the car in the system. in addition to that, it stores information about parking. that includes whether the car is parked now or not, the address of the payment channel, parking history. if one needs some information about a car, this is the contract to address. parking lot extends parking provider contract and is deployed by the parking system contracts at a request of a landlord and with approval of the administrator. this contract adds functionality to partition the parking lot among several tenants. in order to do so, a tenant creates a request to this contract. if the landlord approves the request, the contract creates a renting contract between the landlord and the tenant. it also contains general information about the parking lot, such as its rating, address and so on. tenant also extends parking provider contract. unlike parking lot contract, this contract does not need permission from the system to be deployed as it interacts only with a parking lot. a tenant can set its payment policy and it also stores the address of its contract with a parking lot. conceptually a service provider is a third-party entity that collaborates with a parking provider in order to place information about the parking provider's stall on their platform. thus, the contract only facilitates the interaction between a service provider and a parking provider. it contains information about a service provider and about parking provider with whom it collaborates. renting contract implements a relationship between a parking lot and a tenant in such a way that both parties are guaranteed that their agreement will be respected in the system. the contract is deployed when the parking lot contract approves registration of a new tenant. after that, the tenant reviews the contract and if conditions are satisfactory, the renting process begins. the contract also contains logic for penalties in case of delayed payment. finally, it supports the introduction of amendments from both parties. in the case of mutual agreement, such amendments can modify the behaviour of the contract. payment channel implements the logic for micropayments between two parties, in such a way that both parties are guaranteed to receive the promised funds. one party opens the contract and sends funds to it. after that, the funds are locked in the contract, and neither of the parties can access them. micropayments are conducted off-chain, therefore, they are not part of this contract. once the payment process is completed, the receiving party sends the last encrypted message to the contract, and the contract uses encryption algorithms to verify that the party is entitled to receive the funds. if that is the case, funds are sent to the receiver's address. the detailed description of the principles behind this contract can be found in sect. 3. payment policy is a protocol that defines two methods: get payment rate at a particular time and get a total price for parking given the start and the end time. it is up to parking providers to define their parking policies, depending on their business goals. a particular implementation of this protocol is provided. it implements a policy that can have a specific rate depending on an hour and a day of the week. the front-end part of the parking system is a distributed application in the form of web, mobile or embedded software installed on mobile phones and computers of the administrator, landlords, tenants and drivers as well as in iot boxes at parking lots. in future they will be referred to as think clients. every thin client provides a user interface or an api for interaction with the system. user interfaces are used by landlords, administrator, tenants and drivers. the api is used by the iot box or a smart car. the front-end part is responsible for translating users' actions into requests to the smart contracts. in order for the front-end distributed application to communicate with the evm, there has to be a specific data-interchange format. this format is called json-rpc [14] . it is utilised through a javascript library called web3.js 1 , which allows us to interact with a local or remote ethereum node using a http or ipc connection [4] . as depicted in fig. 5 every front-end module of the distributed system communicates mostly with their own smart contract and this contract, in turn, is responsible for the interaction with the parking system module and other modules. whenever a car gets parked at a parking lot, there is a mutual agreement between the parking lot and the car: the car receives a service and transfers money for parking. in a traditional system, the payment is conducted in advance for a particular period, [19] . such system lacks trustworthiness, as a car has to blindly trust the parking provider, that the service will be provided sufficiently and will not be interrupted earlier, the parking provider, in turn, has to ensure that the car is not parked for longer than the paid period, this requires additional control that brings an additional cost. finally, parking is not a discrete process, unlike the pricing policy in traditional parking systems, which results in drivers oftentimes paying more than they should. this problem can be solved by implementing a micro-payment channel through a smart contract and direct signed messages. the high-level picture of such payment includes three steps: 1. driver deploys a contract with locked funds of some amount that exceeds any possible parking period. 2. driver emits messages directly to the service provider. the messages are the "microtransactions" containing how the amount is transferred. the parking lot can check the validity of the messages as they are signed by car. 3. after the parking process is over, the service provider can use the last message to retrieve the claimed amount of funds from the contract. the remained of the locked funds are sent back to the car. it is essential to mention that in this scenario only steps 1 and 3 happen on the blockchain and the most intense process of micro-payments is conducted offchain through a peer-to-peer communication [3] . thus, the parties do not have to conduct costly blockchain transactions (gas in etherium terminology [8] ) for each fig. 6 . sequence diagram of micro-payments over blockchain payment. only two transactions are required: to deploy the contract (by the car) and to claim the end of the payment period (by the parking lot). moreover, the transaction fee will be paid by the service provider, thus removing any additional fees from the car. this process is visualised in fig. 6. a significant amount of research has been conducted on smart parking. in this paper, we have examined some of the recent work related to this topic and propose a solution. interviews have been conducted with domain experts, user stories defined and a system architecture has been proposed with a case study. our solution allows independent owners of parking space to be integrated into one unified system, that facilitates the parking situation in a smart city. the utilization of such a system raises the issues of trust and transparency among several actors of the parking process. in order to tackle those, we propose a smart contract-based solution, that brings in trust by encapsulating sensitive relations and processes into transparent and distributed smart contracts. from the architecture point of view services and, in particular, microservices [7, 9, 22] and their composition, orchestration [20, 23] , reconfiguration [21] and recovery [10] have not been discussed. all these are open issues that need to be investigated in future. mvrdv unveils plans to reopen the hague's forgotten canals the peerjs library documentation real-time image-based parking occupancy detection using deep learning a blockchain-based architecture for integrated smart parking systems microservices, science and engineering introducing ethereum and solidity microservices: yesterday, today, and tomorrow a formal semantics for the ws-bpel recovery framework a blockchain and gamification approach for smart parking a novel architecture of parking management for smart cities techniques for requirements elicitation parkchain: a blockchain powered parking solution for smart cities parking management: a blockchain-based privacy-preserving system iot based smart parking system a traffic-aware street lighting scheme for smart cities using autonomous networked sensors parking pricing implementation guidelines how more efficient parking pricing can help solve parking and traffic problems, increase revenue, and achieve other planning objectives towards abstractions for web services composition design, modelling and analysis of a workflow reconfiguration size matters: microservices research and applications. microservices a case study of web services orchestration urbanisation concepts and trends governing the smart city: a review of the literature on smart urban governance allocating an area to a vehicle smart parking service based on wireless sensor networks key: cord-301117-egd1gxby authors: barh, debmalya; chaitankar, vijender; yiannakopoulou, eugenia ch; salawu, emmanuel o.; chowbina, sudhir; ghosh, preetam; azevedo, vasco title: in silico models: from simple networks to complex diseases date: 2013-11-15 journal: animal biotechnology doi: 10.1016/b978-0-12-416002-6.00021-3 sha: doc_id: 301117 cord_uid: egd1gxby in this chapter, we consider in silico modeling of diseases starting from some simple to some complex (and mathematical) concepts. examples and applications of in silico modeling for some important categories of diseases (such as for cancers, infectious diseases, and neuronal diseases) are also given. mathematically and computed models are established. these in silico models encode and test hypotheses about mechanisms underlying the function of cells, the pathogenesis and pathophysiology of disease, and contribute to identification of new drug targets and drug design. the development of in silico models is facilitated by rapidly advancing experimental and analytical tools that generate information-rich, high-throughput biological data. bioinformatics provides tools for pattern recognition, machine learning, statistical modeling, and data extraction from databases that contribute to in silico modeling. dynamical systems theory is the natural language for investigating complex biological systems that demonstrate nonlinear spatio-temporal behavior. most in silico models aim to complement (and not replace) experimental research. experimental data are needed for parameterization, calibration, and validation of in silico models. typical examples in biology are models for molecular networks, where the behavior of cells is expressed in terms of quantitative changes in the levels of transcripts and gene products, as well as models of cell cycle. in medicine, in silico models of cancer, immunological disease, lung disease, and infectious diseases complement conventional research with in vitro models, animal models, and clinical trials. this chapter presents basic concepts of bioinformatics, systems biology, their applications in in silico modeling, and also reviews applications in biology and disease. biotechnology will be the most promising life science frontier for the next decade. together with informatics, biotechnology is leading revolutionary changes in our society and economy. this genomic revolution is global, and is creating new prospects in all biological sciences, including medicine, human health, disease, and nutrition, agronomy, and animal biotechnology. animal biotechnology is a source of innovation in production and processing, profoundly impacting the animal husbandry sector, which seeks to improve animal product quality, health, and well-being. biotechnological research products, such as vaccines, diagnostics, in vitro fertilization, transgenic animals, stem cells, and a number of other therapeutic recombinant products, are now commercially available. in view of the immense potential of biotechnology in the livestock and poultry sectors, interest in animal biotechnology has increased over the years. the fundamental requirement for modern biotechnology projects is the ability to gather, store, classify, analyze, and distribute biological information derived from genomics projects. bioinformatics deals with methods for storing, retrieving, and analyzing biological data and protein sequences, structures, functions, pathways, and networks, and recently, in silico disease modeling and simulation using systems biology. bioinformatics encompasses both conceptual and practical tools for the propagation, generation, processing, and understanding of scientific ideas and biological information. genomics is the scientific study of structure, function, and interrelationships of both individual genes and the genome. lately, genomics research has played an important role in uncovering the building blocks of biology and complete genome mapping of various living organisms. this has enabled researchers to decipher fundamental cellular functions at the dna level, such as gene regulation or protein-protein interactions, and thus to discover molecular signatures (clusters of genes, proteins, metabolites, etc.), which are characteristic of a biological process or of a specific phenotype. bioinformatics methods and databases can be developed to provide solutions to challenges of handling massive amounts of data. the history of animal biotechnology with bioinformatics is to make a strong research community that will build the resources and support veterinary and agricultural research. there are some technologies that were used dating back to 5,000 b.c. many of these techniques are still being used today. for example, hybridizing animals by crossing specific strains of animals to create greater genetic varieties is still in practice. the offspring of some of these crosses are selectively bred afterward to produce the most desirable traits in those specific animals. there has been significant interest in the complete analysis of the genome sequence of farm animals such as chickens, pigs, cattle, sheep, fish, and rabbits. the genomes of farm animals have been altered to search for preferred phenotypic traits, and then selected for better-quality animals to continue into the next generation. access to these sequences has given rise to genome array chips and a number of web-based mapping tools and bioinformatics tools required to make sense of the data. in addition, organization of gigabytes of sequence data requires efficient bioinformatics databases. fadiel et al. (2005) provides a nice overview of resources related to farm animal bioinformatics and genome projects. with farm animals consuming large amounts of genetically modified crops, such as modified corn and soybean crops, it is good to question the effect this will have on their meat. some of the benefits of this technology are that what once took many years of trial and error is now completed in just months. the meats that are being produced are coming from animals that are better nourished by the use of biotechnology. biotechnology and conventional approaches are benefiting both poultry and livestock producers. this will give a more wholesome affordable product that will meet growing population demands. moreover, bioinformatics methods devoted to investigating the genomes of farm animals can bring eventual economic benefits, such as ensuring food safety and better food quality in the case of beef. recent advances in highthroughput dna sequencing techniques, microarray technology, and proteomics have led to effective research in bovine muscle physiology to improve beef quality, either by breeding or rearing factors. bioinformatics is a key tool to analyze the huge datasets obtained from these techniques. the computational analysis of global gene expression profiling at the mrna or protein level has shown that previously unsuspected genes may be associated either with muscle development or growth, and may lead to the development of new molecular indicators of tenderness. gene expression profiling has been used to document changes in gene expression; for example, following infection by pathological organisms, during the metabolic changes imposed by lactation in dairy cows, in cloned bovine embryos, and in various other models. bioinformatics enrichment tools are playing an important role in facilitating the functional analysis of large gene lists from various high-throughput biological studies. huang et al. discusses 68 bioinformatics enrichment tools, which helps us understand their algorithms and the details of a particular tool. however, in biology genes do not act independently, but in a highly coordinated and interdependent manner. in order to understand the biological meaning, one needs to map these genes into gene-ontology (go) categories or metabolic and regulatory pathways. different bioinformatics approaches and tools are employed for this task, starting form go-ranking methods, pathway mappings, and biological network analysis (werner, 2008) . the awareness of these resources and methods is essential to make the best choices for particular research interests. knowledge of bioinformatics tools will facilitate their wide application in the field of animal biotechnology. bioinformatics is the computational data management discipline that helps us gather, analyze, and represent this information in order to educate ourselves, understand biological processes in healthy and diseased states, and to facilitate discovery of better animal products. continued efforts are required to develop cost-effective and efficient computational platforms that can retrieve, integrate, and interpret the knowledge behind the genome sequences. the application of bioinformatics tools for biotechnology research will have significant implications in the life sciences and for the betterment of human lives. bioinformatics is being adopted worldwide by academic groups, companies, and national and international research groups, and it should be thought of as an important pillar of current and future biotechnology, without which rapid progress in the field would not be possible. systems approaches in combination with genomics, proteomics, metabolomics, and kinomics data have tremendous potential for providing insights into various biological mechanisms, including the most important human diseases. we are witnessing the birth of a new era in biology. the ability to uncover the genetic code of living organisms has dramatically changed the biological and biomedical sciences approach towards research. these new approaches have also brought in newer challenges. one such challenge is that recent and novel technologies produce biological datasets of ever-increasing size, including genomic sequences, rna and protein abundances, their interactions with each other, and the identity and abundance of other biological molecules. the storage and compilation of such quantities of biological data is a challenge: the human genome, for example, contains 3 billion chemical units of dna, whereas a protozoan genome has 670 billion units of dna. data management and interpretation requires development of newly sophisticated computational methods based on research in biology, medicine, pharmacology, and agricultural studies and using methods from computer science and mathematics -in other words, the multi-disciplinary subject of bioinformatics. bioinformatics enables researchers to store large datasets in a standard computer database format and provides tools and algorithms scientists use to extract integrated information from the databases and use it to create hypotheses and models. bioinformatics is a growth area because almost every experiment now involves multiple sources of data, requiring the ability to handle those data and to draw out inferences and knowledge. after 15 years of rapid evolution, the subject is now quite ubiquitous. another challenge lies in deciphering the complex interactions in biological systems, known as systems biology. systems biology can be described as a biology-based interdisciplinary field of study that focuses on complex interactions of biological systems. those in the field claim that it represents a shift in perspective towards holism instead of reductionism. systems biology has great potential to facilitate development of drugs to treat specific diseases. the drugs currently on the market can target only those proteins that are known to cause disease. however, with the human genome now completely mapped, we can target the interaction of genes and proteins at a systems biology level. this will enable the pharmaceutical industry to design drugs that will only target those genes that are diseased, improving healthcare in the united states. like two organs in one body, systems analysis and bioinformatics are separate but interdependent. computational methods take an interdisciplinary approach, involving mathematicians, chemists, biologists, biochemists, and biomedical engineers. the robustness of datasets related to gene interaction and co-operation at a systems level requires multifaceted approaches to create a hypothesis that can be tested. two approaches are used to understand the network interactions in systems biology, namely experimental and theoretical and modeling techniques (choi, 2007) . in the following sections is a detailed overview of the different computational or bioinformatics methods in modern systems biology. experimental methods utilize real situations to test the hypothesis of mined data sets. as such, living organisms are used whereby various aspects of genome-wide measurements and interactions are monitored. specific examples on this point include: protein-protein interaction predictions are methods used to predict the outcome of pairs or groups of protein interactions. these predictions are done in vivo, and various methods can be used to carry out the predictions. interaction prediction is important as it helps researchers make inferences of the outcomes of ppi. ppi can be studied by phylogenetic profiling, identifying structural patterns and homologous pairs, intracellular localization, and post-translational modifications, among others. a survey of available tools and web servers for analysis of protein-protein interactions is provided by tuncbag et al., 2009. within biological systems, several activities involving the basic units of a gene take place. such processes as dna replication, and rna translation and transcription into proteins must be controlled; otherwise, the systems could yield numerous destructive or useless gene products. transcriptional control networks, also called gene regulatory networks, are segments within the dna that govern the rate and product of each gene. bioinformatics have devised methods to look for destroyed, dormant, or unresponsive control networks. the discovery of such networks helps in corrective therapy, hence the ability to control some diseases resulting from such control network breakdowns. there has also been rapid progress in the development of computational methods for the genome-wide "reverse engineering" of such networks. aracne is an algorithm to identify direct transcriptional interactions in mammalian cellular networks, and promises to enhance the ability to use microarray data to elucidate cellular processes and to identify molecular targets of pharmacological drugs in mammalian cellular networks. in addition to methods like aracne, systems biology approaches are needed that incorporate heterogeneous data sources, such as genome sequence and protein-dna interaction data. the development of such computational modeling techniques to include diverse types of molecular biological information clearly supports the gene regulatory network inference process and enables the modeling of the dynamics of gene regulatory systems. one such technique is the template-based method to construct networks. an overview of the method is shown in flow chart 21.1. the template-based transcriptional control network reconstruction method exploits the principle that orthologous proteins regulate orthologous target genes. given a genome of interest (goi), the first step is to select the template genome (tg) and known regulatory interactions (i.e. template network, tn) in this genome. in step 2, for every protein (p) in tn, a blast search is performed against goi to obtain the best hit sequences (px). in step three these px are then used as a query to perform a blast search against tg. if the best hit using px as a query happens to be p, then both p and px are selected as orthologous proteins in step four. if orthologs were detected for an interacting p and target gene then the interaction is transferred in goi in the final step. note that this automated way of detecting orthologs can infer false positives. signal transduction is how cells communicate with each other. signal transduction pathways involve interactions between proteins, micro-and macro-molecules, and dna. a breakdown in signal transduction pathways could lead flow chart 21.1 template-based method for regulatory network reconstruction. to detrimental consequences within the system due to lack of integrated communication. correction of broken signal transduction pathways is a therapeutic approach researched for use in many areas of medicine. high-throughput and multiplex techniques for quantifying signaling and cellular responses are becoming increasingly available and affordable. a high-throughput quantitative multiplex kinase assay, mass spectrometrybased proteomics, and single-cell proteomics are a few of the experimental methods used to elucidate signal transduction mechanisms of cells. these large-scale experiments are generating large data sets on protein abundance and signaling activity. data-driven modeling approaches such as clustering, principal components analysis, and partial least squares need to be developed to derive biological hypotheses. the potential of data-driven models to study large-scale data sets quantitatively and comprehensively will make sure that these methods will emerge as standard tools for understanding signal-transduction networks. the systems biology and mathematical biology fields focus on modeling biological systems. computational systems biology aims to develop computational models of biological systems. specifically, it focuses on developing and using efficient algorithms, data structures, visualization tools, and communication tools. a mathematical model can provide new insights into a biological model of interest and help in generating testable predictions. modeling or simulation can be viewed as a way of creating an artificial biological system in vitro whose properties can be changed or made dynamic. by externally controlling the model, new datasets can be created and implemented at a systems level to create novel insights into treating generelated problems. in modeling and simulation, sets of differential equations and logic clauses are used to create a dynamic systems environment that can be tested. mathematical models of biochemical networks (signal transduction cascades, metabolic pathways, gene regulatory networks) are a central component of modern systems biology. the development of formal methods adopted from theoretical computing science is essential for the modeling and simulation of these complex networks. the computational methods that are being employed in mathematical biology and bioinformatics are the following: (a) directed graphs, (b) bayesian networks, (c) boolean networks and their generalizations, (d) ordinary and partial differential equations, (e) qualitative differential equations, (f) stochastic equations, and (g) rule-based formalisms. below are a few specific examples of the applications of these methods. mathematical models can be used to investigate the effects of drugs under a given set of perturbations based on specific tumor properties. this integration can help in the development of tools that aid in diagnosis and prognosis, and thus improve treatment outcome in patients with cancer. for example, breast cancer, being a well-studied disease over the last decade, serves as a model disease. one can thus apply the principles of molecular biology and pathology in designing new predictive mathematical frameworks that can unravel the dynamic nature of the disease. genetic mutations of brca1, brca2, tp53, and pten significantly affect disease prognosis and increase the likelihood of adverse reactions to certain therapies. these mutations enable normal cells to become self-sufficient in survival in a stepwise process. enderling et al. (2006) modeled this mutation and expansion process by assuming that mutations in two tumor-suppressor genes are sufficient to give rise to a cancer. they modified enderling's earlier model, which was based on an established partial differential equation model of solid tumor growth and invasion. the stepwise mutations from a normal breast stem cell to a tumor cell have been described using a model consisting of four differential equations. recently, woolf et al. (2005) applied a novel graphical modeling methodology known as bayesian network analysis to model discovery and model selection for signaling events that direct mouse embryonic stem cells (an important preliminary step in hypothesis testing) in protein signaling networks. the model predicts bidirectional dependence between the two molecules erk and fak. it is interesting to appreciate that the apparent complexity of these dynamic erk-fak interactions is quite likely responsible for the difficulty in determining clear "upstream" versus "downstream" influence relationships by means of standard molecular cell biology methods. bayesian networks determine the relative probability of statistical dependence models of arbitrary complexity for a given set of data. this method offers further clues to apply bayesian approaches to cancer biology problems. cell cycle is a process in which cells proliferate while collectively performing a series of coordinated actions. cell-cycle models also have an impact on drug discovery. chassagnole et al. (2006) used a mathematical model to simulate and unravel the effect of multi-target kinase inhibitors of cyclin-dependent kinases (cdks). they quantitatively predict the cytotoxicity of a set of kinase inhibitors based on the in vitro ic 50 measurement values. finally, they assess the pharmaceutical value of these inhibitors as anticancer therapeutics. in cancer, avascular tumor growth is characterized by localized, benign tumor growth where the nearby tissues consume most of the nutrients. mathematical modeling of avascular tumor growth is important to understanding the advanced stages of cancer. kiran et al. (2009) have developed a spatial-temporal mathematical model classified as a different zone model (dzm) for avascular tumor growth based on diffusion of nutrients and their consumption, and it includes key mechanisms in the tumor. the diffusion and nutrient consumption are represented using partial differential equations. this model predicts that onset of necrosis occurs when the concentrations of vital nutrients are below critical values, and also the overall tumor growth based on the size effects of the proliferation zone, quiescent zone, and necrotic zone. the mathematical approaches towards modeling the three natural scales of interest (subcellular, cellular, and tissue) are discussed above. developing models that can predict the effects across biological scales is a challenge. the long-term goal is to build a "virtual human made up of mathematical models with connections at the different biological scales (from genes to tissue to organ)." a model is an optimal mix of hypotheses, evidence, and abstraction to explain a phenomenon. hypothesis is a tentative explanation for an observation, phenomenon, or scientific problem that can be tested by further investigation. evidence describes information (i.e. experimental data) that helps in forming a conclusion or judgment. abstraction is an act of filtering out the required information to focus on a specific property only. for example, archiving books based on the year of publication, irrespective of the author name, would be an example of abstraction. in this process, some detail is lost and some gained. predictions are made through modeling that can be tested by experiment. a model may be simple (e.g. the logistic equation describing how a population of bacteria grows) or complicated. models may be mathematical or statistical. mathematical models make predictions, whereas statistical models enable us to draw statistical inferences about the probable properties of a system. in other words, models can be deductive or inductive. if the prediction is necessarily true given that the model is also true, then the model is a deductive model. on the other hand, if the prediction is statistically inferred from observations, then the model is inductive. deductive models contain a mathematical description; for example, the reaction-diffusion equation that makes predictions about reality. if these predictions do not agree with experiment, then the validity of the entire model may be questioned. mathematical models are commonly applied in physical sciences. on the other hand, inductive models are mostly applied in the biological sciences. in biology, models are used to describe, simulate, analyze, and predict the behavior of biological systems. modeling in biology provides a framework that enables description and understanding of biological systems through building equations that express biological knowledge. modeling enables the simulation of the behavior of a biological system by performing in silico experiments (i.e. numerically solving the equations or rules that describe the model). the results of these in silico experiments become the input for further analysis; for example, identification of key parameters or mechanisms, interpretation of data, or comparison of the ability of different mechanisms to generate observed data. in particular, systems biology employs an integrative approach to characterizing biological systems in which interactions among all components in a system are described mathematically to establish a computable model. these in silico models complement traditional in vivo animal models and can be applied to quantitatively study the behavior of a system of interacting components. the term "in silico" is poorly defined, with several researchers claiming their role in its origination (ekins et al., 2007) . sieburg (1990) and (danchin et al. 1991) were two of the earliest published works that used this term. specifically, in silico models gained much interest in the early stages by various imaging studies (chakroborty et al., 2003) . as an example, microarray analysis that enabled measurement of genome-scale expression levels of genes provided a method to investigate regulatory networks. years of regulatory network studies (that included microarray-based investigations) led to the development of some well-characterized regulatory networks such as e. coli and yeast regulatory networks. these networks are available in the genenetweaver (gnw) tool. gnw is an open-source tool for in silico benchmark generation and performance profiling of network inference methods. thus, the advent of high-throughput experimental tools has allowed for the simultaneous measurement of thousands of biomolecules, opening the way for in silico model construction of increasingly large and diverse biological systems. integrating heterogeneous dynamic data into quantitative predictive models holds great promise for significantly increasing our ability to understand and rationally intervene in disease-perturbed biological systems. this promise -particularly with regards to personalized medicine and medical intervention -has motivated the development of new methods for systems analysis of human biology and disease. such approaches offer the possibility of gaining new insights into the behavior of biological systems, of providing new frameworks for organizing and storing data and performing statistical analyses, suggesting new hypotheses and new experiments, and even of offering a "virtual laboratory" to supplement in vivo and in vitro work. however, in silico modeling in the life sciences is far from straightforward, and suffers from a number of potential pitfalls. thus, mathematically sophisticated but biologically useless models often arise because of a lack of biological input, leading to models that are biologically unrealistic, or that address a question of little biological importance. on the other hand, models may be biologically realistic but mathematically intractable. this problem usually arises because biologists unfamiliar with the limitations of mathematical analysis want to include every known biological effect in the model. even if it were possible to produce such models, they would be of little use since their behavior would be as complex to investigate as the experimental situation. these problems can be avoided by formulating clear explicit biological goals before attempting to construct a model. this will ensure that the resulting model is biologically sound, can be experimentally verified, and will generate biological insight or new biological hypotheses. the aim of a model should not simply be to reproduce biological data. indeed, often the most useful models are those that exhibit discrepancies from experiment. such deviations will typically stimulate new experiments or hypotheses. an iterative approach has been proposed, starting with a biological problem, developing a mathematical model, and then feeding back into the biology. once established, this collaborative loop can be traversed many times, leading to ever increasing understanding. the ultimate goal of in silico modeling in biology is the detailed understanding of the function of molecular networks as they appear in metabolism, gene regulation, or signal transduction. this is achieved by using a level of mathematical abstraction that needs a minimum of biological information to capture all physiologically relevant features of a cellular network. for example, ideally, for in silico modeling of a molecular network, knowledge of the network structure, all reaction rates, concentrations, and spatial distributions of molecules at any time point is needed. unfortunately, such information is unavailable even for the best-studied systems. in silico simulations thus always have to use a level of mathematical abstraction, which is dictated by the extent of our biological knowledge, by molecular details of the network, and by the specific questions that are addressed. understanding the complexity of the disease and its biological significance in health can be achieved by integrating data from the different functional genomics experiments with medical, physiological, and environmental factor information, and computing mathematically. the advantage of mathematical modeling of disease lies in the fact that such models not only shed light on how a complex process works, which could be very difficult for inferring an understanding of each component of this process, but also predict what may follow as time evolves or as the characteristics of particular system components are modified. mathematical models have generally been utilized in association with an increased understanding of what models can offer in terms of prediction and insight. the two distinct roles of models are prediction and understanding the accuracy, transparency, and flexibility of model properties. prediction of the models should be accurate, including all the complexities and population-level heterogeneity that have an additional use as a statistical tool. it also provides the understanding of how the disease spreads in the real world and how the complexity affects the dynamics. model understanding aids in developing sophisticated predictive models, along with gathering more relevant epidemiological data. a model should be as simple as possible and should have balance in accuracy, transparency, and flexibility; in other words, a model should be well suited for its purpose. the model should be helpful in understanding the behavior of the disease and able to simplify the other disease condition. several projects are proceeding along these lines, such as e-cell (tomita, 2001) and simulations of biochemical pathways. whole cell modeling integrates information from metabolic pathways, gene regulation, and gene expression. three elements are needed for constructing of a good cell model: precise knowledge of the phenomenon, an accurate mathematical representation, and a good simulation tool. a cell represents a dynamic environment of interaction among nucleic acids, proteins, carbohydrates, ions, ph, temperature, pressure, and electrical signals. many cells with similar functionality form tissue. in addition, each type of tissue uses a subset of this cellular inventory to accomplish a particular function. for example, in neurons, electro-chemical phenomena take precedence over cell division, whereas, cell division is a fundamental function of skin, lymphocytes, and bone marrow cells. thus, an ideal virtual cell not only represents all the information, but also exhibits the potential to differentiate into neuronal or epithelial cells. the first step in creating a whole cell model is to divide the entire network into pathways, and pathways into individual reactions. any two reactions belong to a pathway if they share a common intermediate. in silico modeling consists not only of decomposing events into manageable units, but also of assembling these units into a unified framework. in other words, mathematical modeling is the art of converting biology into numbers. for whole cell modeling, a checklist of biological phenomena that call for mathematical representation is needed. biological phenomena taken into account for in silico modeling of whole cells are the following: 1. dna replication and repair 2. translation 3. transcription and regulation of transcription 4. energy metabolism 5. cell division 6. chromatin modeling 7. signaling pathways 8. membrane transport (ion channels, pump, nutrients) 9. intracellular molecular trafficking 10. cell membrane dynamics 11. metabolic pathways the whole cell metabolism includes enzymatic and nonenzymatic processes. enzymatic processes cover most of the metabolic events, while non-enzymatic processes include gene expression and regulation, signal transduction, and diffusion. in silico modeling of whole cells not only requires precise qualitative and quantitative data, but also an appropriate mathematical representation of each event. for metabolic modeling, the data input consists of kinetics of individual reactions and also effects of cofactors, ph, and ions on the model. the key step in modeling is to choose an appropriate assumption. for example, a metabolic pathway may be a mix of forward and reverse reactions. furthermore, inhibitors that are part of the pathway may influence some reactions. at every step, enzymatic equations are needed that best describe the process. in silico models are built because they are easy to understand, controllable, and can store and analyze large amounts of information. a well-built model has diagnostic and predictive abilities. a cell by itself is a complete biochemical reactor that contains all the information one needs to understand life. whole cell modeling enables investigation of the cell cycle, physiology, spatial organization, and cell-cell communication. sequential actions in whole cell modeling are the following: 1. catalog all the substances that make up a cell. substances (for qualitative modeling). 5. add rate constants, concentration of substances, and strength of inhibition. 6. assume appropriate mathematical representations for individual reactions. 7. simulate reactions with suitable simulation software. 8. diagnose the system with system analysis software. 9. perturb the system and correlate its behavior to an underlying genetic and/or biochemical factor. 10. predict phenomenon using a hypothesis generator. in silico modeling of disease combines the advantages of both in vivo and in vitro experimentation. unlike in vitro experiments, which exist in isolation, in silico models provide the ability to include a virtually unlimited array of parameters, which render the results more applicable to the organism as a whole. in silico modeling allows us to examine the workings of biological processes such as homeostasis, reproduction, evolution, etc. for example, one can explore the processes of darwinian evolution through in silico modeling, which are not practical to study in real time. in silico modeling of disease is quite challenging. attempting to incorporate every single known interaction rapidly leads to an unmanageable model. furthermore, parameter determination in such models can be a frightening experience. estimates come from diverse experiments, which may be elegantly designed and well executed but can still give rise to widely differing values for parameters. data can come from both in vivo and in vitro experiments, and results that hold in one medium may not always hold in the other. furthermore, despite the many similarities between mammalian systems, significant differences do exist, and so results obtained from experiments using animal and human tissue may not always be consistent. also there are many considerations that cannot be applied. for example, one cannot investigate the role of stochastic fluctuations by removing them from the system, or one cannot directly explore the process that gave rise to current organisms. in silico modeling has been applied in cancer, systemic inflammatory response syndrome, immune diseases, neuronal diseases, and infectious diseases (among others). in silico models of disease can contribute to a better understanding of the pathophysiology of the disease, suggest new treatment strategies, and provide insight into the design of experimental and clinical trials for the investigation of new treatment modalities. in silico modeling of cancer has become an interesting alternative approach to traditional cancer research. in silico models of cancer are expected to predict the complexity of cancer at multiple temporal and spatial resolutions, with the aim of supplementing diagnosis and treatment by helping plan more focused and effective therapy via surgical resection, standard and targeted chemotherapy, and novel treatments. in silico models of cancer include: (a) statistical models of cancer, such as molecular signatures of perturbed genes and molecular pathways, and statistically-inferred reaction networks; (b) models that represent biochemical, metabolic, and signaling reaction networks important in oncogenesis, including constraint-based and dynamic approaches for the reconstruction of such networks; and (c) models of the tumor microenvironment and tissue-level interactions (edelman et al., 2010) . statistical models of cancer can be broadly divided into those that employ unbiased statistical inference, and those that also incorporate a priori constraints of specific biological interactions from data. statistical models of cancer biology at the genetic, chromosomal, transcriptomic, and pathway levels provide insight about molecular etiology and consequences of malignant transformation despite incomplete knowledge of underlying biological interactions. these models are able to identify molecular signatures that can inform diagnosis and treatment selection, for example with molecular targeted therapies such as imatinib (gleevec) (edelman et al., 2010) . however, in order to characterize specific biomolecular mechanisms that drive oncogenesis, genetic and transcriptional activity must be considered in the context of cellular networks that ultimately drive cellular behavior. in microbial cells, network inference tools have been developed and applied for the modeling of diverse biochemical, signaling, and gene expression networks. however, due to the much larger size of the human genome compared to microbes, and the substantially increased complexity of eukaryotic genetic regulation, inference of transcriptional regulatory networks in cancer presents increased practical and theoretical challenges. biochemical reaction networks are constructed to represent explicitly the mechanistic relationships between genes, proteins, and the chemical inter-conversion of metabolites within a biological system. in these models, network links are based on pre-established biomolecular interactions rather than statistical associations; significant experimental characterization is thus needed to reconstruct biochemical reaction networks in human cells. these biochemical reaction networks require, at a minimum, knowledge of the stoichiometry of the participating reactions. additional information such as thermodynamics, enzyme capacity constraints, time-series concentration profiles, and kinetic rate constants can be incorporated to compose more detailed dynamic models (edelman et al., 2010) . microenvironment-tissue level models of cancer apply an "engineering" approach that views tumor lesions as complex micro-structured materials, where three-dimensional tissue architecture ("morphology") and dynamics are coupled in complex ways to cell phenotype, which in turn is influenced by factors in the microenvironment. computational approaches of in silico cancer research include continuum models, discrete models, and hybrid models. in continuum models, extracellular parameters can be represented as continuously distributed variables to mathematically model cell-cell or cell-environment interactions in the context of cancers and the tumor microenvironment. systems of partial differential equations have been used to simulate the magnitude of interaction between these factors. continuum models are suitable for describing the individual cell migration, change of cancer cell density, diffusion of chemo-attractants, heat transfer in hyperthermia treatment for skin cancer, cell adhesion, and the molecular network of a cancer cell as an entire entity. however, these types of in silico models have limited ability for investigating singlecell behavior and cell-cell interaction. on the other hand, "discrete" models (i.e. cellular automata models) represent cancer cells as discrete entities of defined location and scale, interacting with one another and external factors in discrete time intervals according to predefined rules. agent-based models expand the cellular automata paradigm to include entities of divergent functionalities interacting together in a single spatial representation, including different cell types, genetic elements, and environmental factors. agent-based models have been used for modeling three-dimensional tumor cell patterning, immune system surveillance, angiogenesis, and the kinetics of cell motility. hybrid models have been created which incorporate both continuum and agent-based variables in a modular approach. hybrid models are ideal for examining direct interactions between individual cells and between the cells and their microenvironment, but they also allow us to analyze the emergent properties of complex multi-cellular systems (such as cancer). hybrid models are often multi-scale by definition, integrating processes on different temporal and spatial scales, such as gene expression, intracellular pathways, intercellular signaling, and cell growth or migration. there are two general classes of hybrid models, those that are defined upon a lattice and those that are off-lattice. the classification of hybrid models on these two classes depends on the number of cells these models can handle and the included details of each individual cell structure, i.e. models dealing with large cell populations but with simplified cell geometry, and those that model small colonies of fully deformable cells. for example, a hybrid model investigated the invasion of healthy tissue by a solid tumor. the model focused on four key parameters implicated in the invasion process; tumor cells, host tissue (extracellular matrix), matrix-degradative enzymes, and oxygen. the model is actually hybrid, wherein the tumor cells were considered to be discrete (in terms of concentrations), and the remaining variables were in the continuous domain in terms of concentrations. this hybrid model can make predictions on the effects of individual-based cell interactions (both between individuals and the matrix) on tumor shape. the model of zhang et al. (2007) incorporated a continuous model of a receptor signaling pathway, an intracellular transcriptional regulatory network, cell-cycle kinetics, and three-dimensional cell migration in an integrated, agent-based simulation of solid brain tumor development. the interactions between cellular and microenvironment states have also been considered in a multi-scale model that predicts tumor morphology and phenotypic evolution in response to such extracellular pressures. the biological context in which cancers develop is taken into consideration in in silico models of the tumor microenvironment. such complex tumor microenvironments may integrate multiple factors including extracellular biomolecules, vasculature, and the immune system. however, rarely have these methods been integrated with a large cell-cell communication network in a complex tumor microenvironment. recently, an interesting effort of in silico modeling was described in which the investigators integrated all the intercellular signaling pathways known to date for human glioma and generated a dynamic cell-cell communication network associated with the glioma microenvironment. then they applied evolutionary population dynamics and the hill functions to interrogate this intercellular signaling network and execute an in silico tumor microenvironment development. the observed results revealed a profound influence of the micro-environmental factors on tumor initiation and growth, and suggested new options for glioma treatment by targeting cells or soluble mediators in the tumor microenvironment (wu et al., 2012) . trauma and infection can cause acute inflammatory responses, the degree of which may have several pathological manifestations like systemic inflammatory response syndrome (sirs), sepsis, and multiple organ failure (mof). however, an appropriate management of these states requires further investigation. translating the results of basic science research to effective therapeutic regimes has been a longstanding issue due in part to the failure to account for the complex nonlinear nature of the inflammatory process wherein sirs/mof represent a disordered state. hence, the in silico modeling approach can be a promising research direction in this area. indeed, in silico modeling of inflammation has been applied in an effort to bridge the gap between basic science and clinical trials. specifically, both agent-based modeling and equation-based modeling have been utilized . equation-based modeling encompasses primarily ordinary differential equations (ode) and partial differential equations (pde). initial modeling studies were focused on the pathophysiology of the acute inflammatory response to stress, and these studies suggested common underlying processes generated in response to infection, injury, and shock. later, mathematical models included the recovery phase of injury and gave insight into the link between the initial inflammatory response and subsequent healing process. the first mathematical models of wound healing dates back to the 1980s and early 1990s. these models and others developed in the 1990s investigated epidermal healing, repair of the dermal extracellular matrix, wound contraction, and wound angiogenesis. most of these models were deterministic and formulated using differential equations. in addition, recent models have been formulated using differential equations to analyze different strategies for improved healing, including wound vacs, commercially engineered skin substitutes, and hyperbaric oxygen. in addition, agent-based models have been used in wound healing research. for example, mi et al. (2007) developed an agent-based model to analyze different treatment strategies with wound debridement and topical administration of growth factors. their model produced the expected results of healing when analyzing for different treatment strategies including debridement, release of pdgf, reduction in tumor necrosis factor-î±, and increase of tgf-î²1. the investigators suggested that a drug company should use a mathematical model to test a new drug before going through the expensive process of basic science testing, toxicology and clinical trials. indeed, clinical trial design can be improved by prior in silico modeling. for example, in silico modeling has led to the knowledge that patients who suffered from the immune-suppressed phenotype of late-stage multiple organ failure, and were susceptible to usually trivial nosocomial infections, demonstrated sustained elevated markers of tissue damage and inflammation through two weeks of simulated time. however, anti-cytokine drug trials with treatment protocols of only one dose or one day had not incorporated this knowledge into their design, with subsequent failure of candidate treatments. by now the reader is expected to be familiar with the meaning and the basics of in silico modeling. in this section we discuss the application of in silico modeling in the understanding of infectious diseases and in the proposition/ development of better treatments for infectious diseases. in fact, the applications of in silico modeling can help far beyond just the understanding of the dynamics (and sometimes, statistics) of infectious diseases, and far beyond the proposition/development of better treatments for infectious diseases. the modeling can be helpful even in the understanding of better prevention of infectious diseases. the level of pathogen within the host defines the process of infection; such pathogen levels are determined by the growth rate of the pathogen and its interaction with the host's immune response system. initially, no pathogen is present, but just a low-level, nonspecific immunity within the host. on infection, the parasite grows abundantly over time with the potential to transmit the infection to other susceptible individuals. to comprehensively understand in silico modeling in the domains of infectious diseases, one should first understand the "triad of infectious diseases," and the characteristics of "infectious agent," "host," and "environment" on which the models are always based. in fact, modeling of infectious diseases is just impossible without this triad; after all, the model would be built on some parameters (also called variables in more general language), and those parameters always have their origin from the so-called "triad of infectious diseases." at this point, a good question would be: what is a "triad of infectious diseases?" "triad of infectious diseases" means the interactions between (1) agent, which is the disease causing organism (the pathogen); (2) host, which is the infected organism, or in the case of pre-infection, the organism to be infected is the host (thus in this case the host is the animal the agent infects); and (3) environment, which is a kind of link between the agent and the host, and is essentially an umbrella word for the entirety of the possible media through which the agent reaches the host. now that we have an idea on what in silico modeling of infectious diseases are generally based on, we will outline a better understanding of the parameters that are considered in most in silico disease models. to discuss the parameters in an orderly manner, we just categorize them under each of the three components of the "triad of infectious diseases," and summarize them in the next subsection. it must be emphasized at this point that (1) even though all the possible parameters for in silico modeling of infectious diseases can be successfully categorized under the characteristics of one of any of the three components of the "triad of infectious diseases" (agent, host, and environment), (2) the parameters discussed in the next sub-section are by no means the entirety of all the possible parameters that can be included in in silico modeling of infectious diseases. in fact, several parameters exist, and this section cannot possibly enumerate them all. that is why we have discussed the parameters using a categorical approach. some of the parameters for in silico modeling of infectious diseases are essentially a measure of infectivity (ability to enter the host), pathogenicity (ability to cause divergence from homeostasis/disease), virulence (degree of divergence from homeostasis caused/ability to cause death), antigenicity (ability to bind to mediators of the host's adaptive immune system), and immunogenicity (ability to trigger adaptive immune response) of the concerned infectious agent. the exact measure (and thus the units) used can vary markedly depending on the intentions for which the in silico infectious diseases model is built, as well as the assumptions on which the in silico disease model is based. from the knowledge of the agent's characteristics, one should know that unlike parameters related to the other characteristics of the agent, the parameters related to infectivity find their most important use only in the modeling of the preinfection stage in infectious disease modeling. finally, some of the agent-related parameters of great importance in in silico modeling of infectious diseases are concentration of the agent's antigen-host antibody complex, case fatality rate, strain of the agent, other genetic information of the agent, etc. the parameters originating from characteristics of the host can also be diversified and based on the intentions for which the in silico infectious diseases model are built and the assumptions on which the in silico disease model are based; however, the parameters could then be grouped and explained under the host's genotype (the allele at the host's specified genetic locus), immunity/health status (biological defenses to avoid infection), nutritional status (feeding habits/food intake characteristics), gender (often categorized as male or female), age, and behavior (the host's behaviors that affect its resistance to homeostasis disruptors). typical examples of host-related parameters are the alleles at some specifically targeted genetic loci; the total white blood cell counts; differential white blood cell counts, and/or much more sophisticated counts of specific blood cell types; blood levels of some specific cytokines, hormones, and/or neurotransmitters; daily calories, protein, and/or fat intake; daily amount of energy expended and/or duration of exercise; etc. at first parameters originating from the environment might seem irrelevant to the in silico modeling of infectious diseases, but they are relevant. even after the pre-infection stage, the environment still modulates the host-agent intersections. for example, the ability (and thus the related parameters) of the agent to multiply and/or harm the host are continually influenced by the host's environmental conditions, and in a similar way the hosts defense against the adverse effects of the agents are modulated by the host's environmental conditions. but somehow, not so many of these parameters have been included in in silico infectious disease models in the recent past. a few examples of these parameters are the host's ambient temperature, the host's ambient atmospheric humidity, altitude, the host's light-dark cycle, etc. now that we know the parameters for in silico infectious disease modeling, the next reasonable question would be "what form does a typical in silico infectious disease model take?" so, this sub-section attempts to answer this very important question. let us view the in silico model as a system of wellintegrated functional equations or formulae. such well-integrated functional equations can be viewed or approximated as a single, albeit more complex, functional equation/formula. it is hence possible to vary any (or a combination) of the variables contained in this equation by running numerical simulations on a computer depending on the kind of prediction one wants to make. such in silico models can hence investigate several (maybe close to infinite) possible data points within reasonable limits that one sets depending on the nature of the variables considered. so the equations behind a typical infectious disease in silico model could take the form (equation 21.1): where h is the output from a smaller equation that is based on host parameters; î² is a constant; f and g are link functions which may be the same as or different from each other and other link functions in this system of equations; a is the output from a smaller equation that is based on agent parameters; g is a link function which may be the same or different from other link functions in this system of equations; e is the output from a smaller equation that is based on environment parameters; and æ� is a random error parameter. readers should know that we use the term "link function" to refer to any of the various possible forms of mathematical operations or functions. this means that based on the complexity of the model, a particular "link function" might be as simple as a mere addition or as complex as several combinations of operators with high degree polynomials. where î² a is a constant; f a1 , f a2 , â�¦ f ax are link functions that may be same or different (individually) from (every) other link function in this system of equations; a 1 , a 2 , â�¦ a x are a set of the agent's parameters (e.g. case fatality rate, agent's genotype, etc); and æ� is a random error parameter. where î² e is a constant; f e1 , f e2 , â�¦f ex are link functions which may be the same or different (individually) from (every) other link function in this system of equations; e 1 , e 2 , â�¦ e x are a set of environmental parameters (e.g. host's ambient temperature, host's ambient atmospheric humidity, etc.); and æ� is a random error parameter. muã±oz-elã­as et al. (2005) documented (through their paper "replication dynamics of mycobacterium tuberculosis in chronically infected mice") a successful in silico modeling of infectious diseases (specifically, tuberculosis). in their in silico modeling of tuberculosis in mice, the researchers investigated both the static and dynamic host-pathogen/agent equilibrium (i.e. mice-mycobacterium tuberculosis static and dynamic equilibrium). the rationale behind their study was that a better understanding of host-pathogen/agent interactions would make possible the development of better anti-microbial drugs for the treatment of tuberculosis (as well as provide similar understanding for the cases of other chronic infectious diseases). they modeled different types of host-pathogen/ agent equilibriums (ranging from completely static equilibrium, all the way through semi-dynamic, down to completely dynamic scenarios) by varying the rate of multiplication/growth and the rate of death of the pathogen/ agent (mycobacterium tuberculosis) during the infection's chronic phase. through their in silico study (which was also verified experimentally), they documented a number of remarkable findings. for example, they established that "viable bacterial counts and total bacterial counts in the lungs of chronically infected mice do not diverge over time," and they explained that "rapid degradation of dead bacteria is unlikely to account for the stability of total counts in the lungs over time because treatment of mice with isoniazid for 8 weeks led to a marked reduction in viable counts without reducing the total count. readers who are interested in further details on the generation of this in silico model for the dynamics of mycobacterium tuberculosis infection, as well as the complete details of the parameters/variables considered, and the comprehensive findings of the study, should refer to the article of ernesto et al. published in infection and immunity. another one of the many notable works in the domain of infectious disease in silico modeling is the study by navratil et al. (2011) . using protein-protein interaction data that the authors obtained from available literature and public databases, they (after first curating and validating the data) computationally (in silico) re-examined the virus-human protein interactome. interestingly, the authors were able to show that the onset and pathogenesis of some disease conditions (especially chronic disease conditions) often believed to be of genetic, lifestyle, or environmental origin, are, in fact, modulated by infectious agents. models have been constructed to simulate bacterial dynamics, such as growth under various nutritional and chemical conditions, chemotactic response, and interaction with host immunity. clinically important models of bacterial dynamics relating to peritoneal dialysis, pulmonary infections, and particularly of antibiotic treatment and bacterial resisitance, have also been developed. baccam et al. (2006) utilized a series of mathematical models of increasing complexity that incorporated target cell limitation and the innate interferon response. the models were applied to examine influenza a virus kinetics in the upper respiratory tracts of experimentally infected adults. they showed the models to be applicable for improving the understanding of influenza in a virus infection, and estimated that during an upper respiratory tract infection, the influenza virus initially spreads rapidly with one cell, infecting (on average) about 20 others (daun and clermont, 2007) . model parameter and spread of disease: model parameters are one of the main challenges in mathematical modeling since all models do not have a physiological meaning. sensitivity analysis and bifurcation analysis give us the opportunity to understand how model outcome and model parameters are correlated, how the sensitivity of the system is with respect to certain parameters, and the uncertainty in the model outcome yielded by the uncertainties in the parameter values. uncertainty and sensitivity analysis was used to evaluate the input parameters play in the basic productive rate (ro) of severe acute respiratory syndrome (sars) and tuberculosis. control of the outbreak depends on identifying the disease parameters that are likely to lead to a reduction in r. difficulty in finding the most appropriate set of parameters for in silico modeling of infectious diseases is often a challenge. it is hoped this challenge will subside with the advancement in infectonomics and high-throughput technology. however, another important challenge lies in the understanding (and the provision of reasonable interpretations for) the results from all the complex interactions of parameters considered. in this sub-section we focus on the application of in silico modeling to improve knowledge of neuronal diseases, and thus improve the applications of neurological knowledge for solving neuronal health problems. it is not an overstatement to say that one of the many aspects of life sciences where in silico disease modeling would have the biggest applications is in the better understanding of the pathophysiology of nervous system (neuronal) diseases. this is basically because of the inherent delicate nature of the nervous system and the usual extra need to be sure of how to proceed prior to attempting to treat neuronal disease conditions. by this we mean that the need to first model neuronal disease conditions in silico prior to deciding on or suggesting (for example) a treatment plan is, in fact, rising. this is not unexpected; after all, it is better to be sure of what would work (say, through in silico modeling) than to try what would not work. obtaining appropriate parameters for the in silico modeling of a nervous system (neuronal) disease is rooted in a good understanding of the pathophysiology of such neuronal disease. since comprehensive details of pathophysiology of neuronal diseases is beyond the scope of this book, we only present the basic idea that would allow the reader to understand how in silico modeling of a nervous system (neuronal) disease can be done. to give a generalized explanation and still concisely present the basic ideas underlying the pathophysiology of neuronal diseases, we proceed by systematically categorizing the mediators of neuronal disease pathophysiology: (1) nerve cell characteristics, (2) signaling chemicals and body electrolytes, (3) host/organism factors, and (4) environmental factors. readers need to see all these categories as being highly integrated pathophysiologically rather than as separate entities, and also that we have only grouped them this way to make simpler the explanation of how the parameters for in silico modeling of neuronal diseases are generated. when something goes wrong with (or there is a marked deviation from equilibrium in) a component of any of the four categories above, the other components (within and/ or outside the same category) try hard to make adjustments so as to annul/compensate for the undesired change. for example, if the secretion of a chemical signal suddenly becomes abnormally low, the target cells for the chemical signal may develop mechanisms to use the signaling chemical more efficiently, and the degradation rate of the signaling chemical may be reduced considerably. through these, the potentially detrimental effects of reduced secretion of the chemical signal are annulled via compensation from the other components. this is just a simple example; much more complex regulatory and homeostatic mechanisms exist in the neuronal system. despite the robustness of those mechanisms, things still get out of hand sometimes, and disease conditions result. the exploration of what happens in (and to) each and all of the components of this giant system of disease conditions is called the pathophysiology of neuronal disease, and it this pathophysiology that "provides" parameters for the in silico modeling of neuronal diseases. some of the important parameters (that are of nerve cell origin) for a typical in silico modeling of a neuronal disease (say, alzheimer's disease) are the population (or relative population) of specific neuronal cells (such as glial cells: microglia, astrocytes, etc.), motion of specific neuronal cells (e.g. microglia), amyloid production, aggregation and removal of amyloid, morphology of specific neuronal cells, status of neuronal cell receptors, generation/regeneration/ degeneration rate of neuronal cells, status of ion neuronal cell channels, etc. based on their relevance to the pathophysiology of the neuronal disease being studied, many of these parameters are often considered in the in silico modeling of the neuronal disease. more importantly, their spatiotemporal dynamics are often seriously considered. the importance of signaling chemicals and electrolytes in the nervous system makes parameters related to them very important. the secretion, uptake, degradation, and diffusion rates of various neurotransmitters and cytokines are often important parameters in the in silico modeling of neurodiseases. other important parameters are the concentration gradients of the various neurotransmitters and cytokines, the availability and concentration of second messengers, and the electrolyte status/balance of the cells/systems. the spatiotemporal dynamics of all of these are also often seriously considered. the parameters under host/organism factors can be highly varied depending on the intentions and the assumptions governing the in silico disease modeling. nonetheless, one could basically group and list the parameters collectively under genotype (based on the allele at a specified genetic locus), nutritional status (feeding habits/food intake characteristics; e.g. daily calories, protein intake, etc.), gender (male or female), age, and behavior (host's behaviors/lifestyle that influences homeostasis and/or responses to stimuli). a few examples of these parameters are ambient temperature, altitude, light-dark cycle, social network, type of influences from people in the network, etc. just like other in silico models, a neuronal disease in silico model is also based on what could be viewed as a single giant functional equation, which is composed of highly integrated simpler functional equations. so the equations behind a typical neuronal disease in silico model could take the form (equation 21.5): where n could be a parameter that is a direct measure of the disease manifestation; î² is a constant; f, g, j, and k are link functions which may be the same or different from other link functions in this system of equations; c, s, h, and e are the outputs from smaller equations that are based on parameters from neuronal cell characteristics, signaling molecule and electrolyte parameters, host parameters, and environment parameters, respectively; and æ� is a random error parameter. the reader should know that each of n, c, s, h and e could have resulted from smaller equations that could take forms similar to those (equations 21.2 to 21.4) described under in silico modeling of infectious diseases (previous sub-section). in silico models edelstein-keshet and spiros (2002) used in silico modeling to study the mechanism and/formation of alzheimer's disease. the target of their in silico modeling was to explore and demystify how various parts implicated in the etiology and pathophysiology of alzheimer's disease work together as a whole. employing the strength of in silico modeling, the researchers were able to transcend the difficulty of identifying detailed disease progression scenarios, and they were able to test a wide variety of hypothetical mechanisms at various levels of detail. readers interested in the complete details of the assumptions that govern in silico modeling of alzheimer's disease, the various other aspects of the model, and more detailed accounts of the findings should look at the article by edelstein-keshet and spiros. several other interesting studies have applied in silico modeling techniques to investigate various neuronal diseases. a few examples include the work of altmann and boyton (2004) , who investigated multiple sclerosis (a very common disease resulting from demyelination in the central nervous system) using in silico modeling techniques; lewis et al. (2010) , who used in silico modeling to study the metabolic interactions between multiple cell types in alzheimer's disease; and raichura et al. (2006) , who applied in silico modeling techniques to dynamically model alpha-synuclein processing in normal and parkinson's disease states. a more specific example of a molecular level in silico alzheimer's disease model can be found in ghosh et al. (2010) . among the amyloid proteins, amyloid-î² (aî²) peptides (aî²42 and aî²40) are known to form aggregates that deposit as senile plaques in the brains of alzheimer's disease patients. the process of aî²-aggregation is strongly nucleation-dependent, and is inferred by the occurrence of a "lag-phase" prior to fibril growth that shows a sigmoidal pattern. ghosh et al. (2010) dissected the growth curve into three biophysically distinct sections to simplify modeling and to allow the data to be experimentally verifiable. stage i is where the pre-nucleation events occur whose mechanism is largely unknown. the pre-nucleation stage is extremely important in dictating the overall aggregation process where critical events such as conformation change and concomitant aggregation take place, and it is also the most experimentally challenging to decipher. in addition to mechanistic reasons, this stage is also physiologically important as lowmolecular-weight (lmw) species are implicated in ad pathology. the rate-limiting step of nucleation is followed by growth. the overall growth kinetics and structure and shape of the fibrils are mainly determined by the structure of the nucleating species. an important intermediate along the aggregation pathway, called "protofibrils," have been isolated and characterized that have propensities to both elongate (by monomer addition) as well as to laterally associate (protofibril-protofibril association) to grow into mature fibrils (stage iii in the growth curve). aggregation ghosh et al. (2010) generated an ode-based molecular simulation (using mass-kinetics methodology) of this fibril growth process to estimate the rate constants involved in the entire pathway. the dynamics involved in the protofibril elongation stage of the aggregation (stage iii of the process) were estimated and validated by in vitro biophysical analysis. ghosh et al. (2010) next used the rate constants identified from stage iii to create a complete aggregation pathway simulation (combining stages i, ii, and iii) to approximately identify the nucleation mass involved in aî²-aggregation. in order to model the aî²-system, one needs to estimate the rate constants involved in the complete pathway and the nucleation mass itself. it is difficult to iterate through different values for each of these variables to get close to the experimental plots (fibril growth curves measured via fluorescence measurements with time) due to the large solution space; also, finding the nucleation phase cannot be done independently without estimating the rate constants alongside. however, having separately estimated the post-nucleation stage rate constants (as mentioned above) reduces the overall parameter estimation complexity. the complete pathway simulation was used to study the lag times associated with the aggregation pathway, and hence predict possible estimates of the nucleation mass. the following strategy was used: estimate the pre-nucleation rate constants that give the maximum lag times for each possible estimate of the nucleation mass. this led to four distinctly different regimes of possible nucleation masses corresponding to four different pairs of rate constants for the pre-nucleation phase (regime 1, where n = 7, 8, 9, 10, 11; regime 2, where n = 12, 13, 14; regime 3, where n = 15, 16, 17; and regime 4, where n = 18, 19, 20, 21) . however, it was experimentally observed that the semi-log plot of the lag times against initial concentration of aî² is linear, and this characteristic was used to figure out what values of nucleation mass are most feasible for the aî²42-aggregation pathway. the simulated plots show a more stable relationship between the lag times and the initial concentrations, and the best predictions for the nucleation mass were reported to be in the range 10-16. such molecular pathway level studies are extremely useful in understanding the pathogenesis of ad in general, and can motivate drug development exercises in the future. for example, characterization of the nucleation mass is important as it has been observed that various fatty acid interfaces can arrest the fibril growth process (by stopping the reactions beyond the pre-nucleation stage). such in depth modeling of the aggregation pathway can suggest what concentrations of fatty acid interfaces should be used (under a given aî² concentration in the brain) to arrest the fibril formation process leading to direct drug dosage and interval prediction for ad patients. despite the fact that we have mentioned several possible parameters for in silico modeling of neuro-diseases, it is noteworthy that finding a set of the most reasonable parameters for the modeling is in fact a big challenge. on the other hand, understanding (and thus finding reasonable biological interpretations for) the results from the complex interaction of all parameters considered is also a big challenge. in addition, a number of assumptions that models are sometimes based on still have controversial issues. accurately modeling spatio-temporal dynamics of neurons and neurotransmitters (and other chemicals/ligands) also constitutes a huge challenge. understanding the complex systems involved in a disease will make it possible to develop smarter therapeutic strategies. treatments for existing tumors will use multiple drugs to target the pathways or perturbed networks that show an altered state of activity. in addition, models can effectively form the basis for translational research and personalized medicine. biological function arises as the result of processes interacting across a range of spatiotemporal scales. the ultimate goal of the applications of bioinformatics in systems biology is to aid in the development of individualized therapy protocols to minimize patient suffering while maximizing treatment effectiveness. it is now being increasingly recognized that multi-scale mathematical and computational tools are necessary if we are going to be able to fully understand these complex interactions (e.g. in cancer and heart diseases). with the bioinformatics tools, computational theories, and mathematical models introduced in this article, readers should be able to dive into the exhilarating area of formal computational systems biology. investigating these models and confirming their findings by experimental and clinical observations is a way to bring together molecular reductionism with quantitative holistic approaches to create an integrated mathematical view of disease progression. we hope to have shown that there are many interesting challenges yet to be solved, and that a structured and principled approach is essential for tackling them. systems biology is an emerging field that aims to understand biological systems at the systems level with a high degree of mathematical and statistical modeling. in silico modeling of infectious diseases is a rich and growing field focused on modeling the spread and containment of infections with model designs being flexible and enabling adaptation to new data types. the advantages of avoiding animal testing have often been seen as one of the advantages offered by in silico modeling; the biggest advantage is that there are no ethical issues in performing in silico experiments as they don't require any animals or live cells. furthermore, as the entire modeling and analysis are based on computational approaches, we can obtain the results of such analysis even within an hour. this saves huge amounts of time and reduces costs, two major factors associated with in vitro studies. however, a key issue that needs to be considered is whether in silico testing will ever be as accurate as in vitro or in vivo testing, or whether in silico results will always require non-simulated experimental confirmation. tracqui et al. (1995) successfully developed a glioma model to show how chemo-resistant tumor sub-populations cause treatment failure. similarly, a computational model of tumor invasion by frieboes et al. (2006) is able to demonstrate that the growth of a tumor depends on the microenvironmental nutrient status, the pressure of the tissue, and the applied chemotherapeutic drugs. the 3d spatio-temporal simulation model of a tumor by dionysiou et al. (2004) was able to repopulate, expand, and shrink tumor cells, thus providing a computational approach for assessment of radiotherapy outcomes. the glioblastoma model of kirby et al. (2007) is able to predict survival outcome post-radiotherapy. wu et al. (2012) has also developed an in silico glioma microenvironment that demonstrates that targeting the microenvironmental components could be a potential anti-tumor therapeutic approach. the in silico model-based systems biology approach to skin sensitization (tnf-alpha production in the epidermis) and risk of skin allergy assessment has been successfully carried out; it can replace well known in vitro assays, such as the mouse local lymph node assay (llna) used for the same purpose (by maxwell and mackay (2008) at the unilever safety and environmental assurance centre). similarly, davies et al. (2011) effectively demonstrated an in silico skin permeation assay based on time course data for application in skin sensitization risk assessment. kovatchev et al. (2012) showed how the in silico model of alcohol dependence can provide virtual clues for classifying the physiology and behavior of patients so that personalized therapy can be developed. pharmacokinetics and pharmacodynamics are used to study absorption, distribution, metabolism, and excretion (adme) of administered drugs. in silico models have tremendous efficacy in early estimation of various adme properties. quantitative structure-activity relationship (qsar) and quantitative structure-property relationship (qspr) models have been commonly used for several decades to predict adme properties of a drug at early phases of development. there are several in silico models applied in adme analysis, and readers are encouraged to read the review by van de waterbeemd and gifford (2003) . gastroplusâ�¢, developed at simulations plus (www.simulations-plus.com), is highly advanced, physiologically based rapid pharmacokinetic (pbpk) simulation software that can generate results within 5 seconds, thus saving huge amounts of time and cost in clinical studies. the software is an essential tool to formulation scientists for in vitro dose disintegration and dissolution studies. towards next-generation treatment of spinal cord injuries, novartis (www.novartis. com) is working to model the human spinal cord and its surrounding tissues in silico to check the feasibility of monoclonal antibody-based drug administration and their pharmacokinetics and pharmacodynamics study results. the in silico "drug re-purposing" approach by bisson et al. (2007) demonstrated how phenothiazine derivative antipsychotic drugs such as acetophenazine can cause endocrine side effects. recently aguda et al. (2011) reported a computational model for sarcoidosis dynamics that is useful for pre-clinical therapeutic studies for assessment of dose optimization of targeted drugs used to treat sarcoidosis. towards designing personalized therapy of larynx injury leading to acute vocal fold damage, li et al. (2008) developed agent-based computational models. in a further advancement, entelos â® (www.entelos.com) has developed "virtual patients," in silico mechanistic models of type-2 diabetes, rheumatoid arthritis, hypertension, and atherosclerosis for identification of biomarkers, drug targets, development of therapeutics, and clinical trial design, and patient stratification. entelos' virtual idd9 mouse (nod mouse) can replace diabetes resistance type-1 diabetes live mice for various in vivo experiments. apart from diseases, systems level modeling of basic biological phenomena and their applications in disease have also been reported. an in silico model to mimic the in vitro rolling, activation, and adhesion of individual leukocytes has been developed by tang et al. (2007) . developing virtual mitochondria, cree et al. (2008) vi p r ( h t t p : / / w w w. v i p r b r c . o rg / b r c / h o m e . d o ? decorator=vipr) is one of the five bioinformatics resource centers (brcs) funded by the national institute of allergy and infectious diseases (niaid). this website provides a publicly available database and a number of computational analysis tools to search and analyze data for virus pathogens. some of the tools available at vipr are the following: 1. gatu (genome annotation transfer utility), a tool to transfer annotations from a previously annotated reference to a new, closely related target genome. 2. pcr premier design, a tool for designing pcr primers. 3. a sequence format conversion tool. 4. a tool to identify short peptides in proteins. a meta-driven comparative analysis tool. as there are many different kinds of tools available the tools on the website are organized by the virus family. the rat genome database (rgd) the rat genome database (http://rgd.mcw.edu/wg/ home) is funded by the national heart, lung, and blood institute (nhlbi) of the national institutes of health (nih). the goal of this project is to consolidate research work from various institutes to generate and maintain a rat genomic database (and make it available to the scientific community). the website provides a variety of tools to analyze data. influenza resource centers (brcs) funded by the national institute of allergy and infectious diseases (niaid). this website provides a publicly available database and a number of computational analysis tools to search and analyze data for influenza virus. this website provides many of the same tools that are provided at vibr. there are numerous other tools such as models of infectious disease agent study (midas), which is an in silico model for assessing infectious disease dynamics. midas assists in preparing, detecting, and responding to infectious disease threats. the wellcome trust sanger institute the sanger institute (http://www.sanger.ac.uk/) investigates genomes in the study of diseases that have an impact on global health. the sanger institute has made a significant contribution to genomic research and developing a new understanding of genomes and their role in biology. the website provides sequence genomes for various bacterial, viral, and model organisms such as zebrafish, mouse, gorilla, etc. a number of open source software tools for visualizing and analyzing data sets are available at the sanger institute website. an in silico modeling approach to understanding the dynamics of sarcoidosis models of multiple sclerosis. autoimmune diseases kinetics of influenza a virus infection in humans discovery of antiandrogen activity of nonsteroidal scaffolds of marketed drugs using a mammalian cell cycle simulation to interpret differential kinase inhibition in antitumour pharmaceutical development in silico models for cellular and molecular immunology: successes, promises and challenges introduction to systems biology a reduction of mitochondrial dna molecules during embryogenesis explains the rapid segregation of genotypes from data banks to data bases in silico modeling in infectious disease determining epidermal disposition kinetics for use in an integrated nonanimal approach to skin sensitization risk assessment a four-dimensional simulation model of tumour response to radiotherapy in vivo: parametric validation considering radiosensitivity, genetic profile and fractionation in silico models of cancer mathematical modeling of radiotherapy strategies for early breast cancer exploring the formation of alzheimer's disease senile plaques in silico in silico pharmacology for drug discovery: methods for virtual ligand screening and profiling farm animal genomics and informatics: an update an integrated computational/experimental model of tumor invasion dynamics of protofibril elongation and association involved in abeta42 peptide aggregation in alzheimer's disease bioinformatics enrichment tools: paths toward the comprehensive functional analysis of large gene lists mathematical modeling of avascular tumour growth based on diffusion of nutrients and its validation in silico models of alcohol dependence and treatment large-scale in silico modeling of metabolic interactions between cell types in the human brain a patient-specific in silico model of inflammation and healing tested in acute vocal fold injury application of a systems biology approach to skin allergy risk assessment agent-based model of inflammation and wound healing: insights into diabetic foot ulcer pathology and the role of transforming growth factor-î²1. wound repair regeneration replication dynamics of mycobacterium tuberculosis in chronically infected mice when the human viral infectome and diseasome networks collide: towards a systems biology platform for the aetiology of human diseases dynamic modeling of alphasynuclein aggregation for the sporadic and genetic forms of parkinson's disease physiological studies in silico computational and experimental models of ca2+-dependent arrhythmias dynamics of in silico leukocyte rolling, activation, and adhesion whole-cell simulation: a grand challenge of the 21st century a mathematical model of glioma growth: the effect of chemotherapy on spatio-temporal growth computational cardiology: the heart of the matter a survey of available tools and web servers for analysis of protein-protein interactions and interfaces admet in silico modelling: towards prediction paradise? translational systems biology of inflammation bioinformatics applications for pathway analysis of microarray data bayesian analysis of signaling networks governing embryonic stem cell fate decisions in silico experimentation of glioma microenvironment development and anti-tumor therapy development of a three-dimensional multiscale agent-based tumor model: simulating gene-protein interaction profiles, cell phenotypes and multicellular patterns in brain cancer further reading silico' simulation of biological processes silico toxicology: principles and applications multiscale cancer modeling silico immunology silico: 3d animation and simulation of cell biology with maya and mel algorithm any well-defined computational procedure that takes some values, or set of values, as input, and produces some value, or set of values, as output. bioinformatics bioinformatics is the application of statistics and computer science to the field of molecular biology. biotechnology the exploitation of biological processes for industrial and other purposes. data structures a way to store and organize data on a computer in order to facilitate access and modifications. genome the complete set of genetic material of an organism. genomics the branch of molecular biology concerned with the structure, function, evolution, and mapping of genomes. gene ontology a major bioinformatics initiative to unify the representation of gene and gene product attributes across all species. informatics the science of processing data for storage and retrieval; information science. in silico in silico is an expression used to mean "performed on a computer or via computer simulation." in vivo in microbiology in vivo is often used to refer to experimentation done in live isolated cells rather than in a whole organism. in vitro in vitro studies in experimental biology are those that are conducted using components of an organism that have been isolated from their usual biological surroundings in order to permit a more detailed or more convenient analysis than can be done with whole organisms. kinomics kinomics is the study of kinase signaling within cellular or tissue lysates. oncogenesis the progression of cytological, genetic, and cellular changes that culminate in a malignant tumor. pathophysiology the disordered physiological processes associated with disease or injury. proteomics the branch of genetics that studies the full set of proteins encoded by a genome. sequencing the process of determining the precise order of nucleotides within a dna molecule. systems biology an inter-disciplinary field of study that focuses on complex interactions within biological systems by using a more holistic perspective. 1. the template-based transcriptional control network reconstruction method exploits the principle that orthologous proteins regulate orthologous target genes. in this approach, regulatory interactions are transferred from a genome (such as a genome of a model organism or well studied organism) to the new genome. 2. the ultimate goal of in silico modeling in biology is the detailed understanding of the function of molecular networks as they appear in metabolism, gene regulation, or signal transduction. there are two major challenges in modeling infectious diseases: a. difficulty in finding the most appropriate set of parameters for the in silico modeling of infectious diseases is often a challenge. b. understanding the results from all the complex interactions of parameters considered. 4. there are three types of cancer models. continuum models: in these models extracellular parameters can be represented as continuously distributed variables to mathematically model cell-cell or cell-environment interactions in the context of cancers and the tumor microenvironment. discrete models: these models represent cancer cells as discrete entities of defined location and scale, interacting with one another and external factors in discrete time intervals according to predefined rules. hybrid models: these models incorporate both continuum and discrete variables in a modular approach. there are three types of parameters considered for in silico modeling of infectious diseases: a. parameters derived from characteristics of agent: examples: concentration of the agent's antigen-host antibody complex; case fatality rate; strain of the agent; other genetic information of the agent; etc. examples: the total white blood cell counts; differential white blood cell counts, and/or much more sophisticated counts of specific blood cell types; blood levels of some specific cytokines, hormones, and/or neurotransmitters; daily calories, protein, and/or fat intake; daily amount of energy expended and/or duration of exercise; etc. c. parameters derived from characteristics of environment: examples: host's ambient temperature; host's ambient atmospheric humidity; altitude; host's lightdark cycle; etc. key: cord-103435-yufvt44t authors: van aalst, marvin; ebenhöh, oliver; matuszyńska, anna title: constructing and analysing dynamic models with modelbase v1.0 a software update date: 2020-10-02 journal: biorxiv doi: 10.1101/2020.09.30.321380 sha: doc_id: 103435 cord_uid: yufvt44t background computational mathematical models of biological and biomedical systems have been successfully applied to advance our understanding of various regulatory processes, metabolic fluxes, effects of drug therapies and disease evolution or transmission. unfortunately, despite community efforts leading to the development of sbml or the biomodels database, many published models have not been fully exploited, largely due to lack of proper documentation or the dependence on proprietary software. to facilitate synergies within the emerging research fields of systems biology and medicine by reusing and further developing existing models, an open-source toolbox that makes the overall process of model construction more consistent, understandable, transparent and reproducible is desired. results and discussion we provide here the update on the development of modelbase, a free expandable python package for constructing and analysing ordinary differential equation-based mathematical models of dynamic systems. it provides intuitive and unified methods to construct and solve these systems. significantly expanded visualisation methods allow convenient analyses of structural and dynamic properties of the models. specifying reaction stoichiometries and rate equations, the system of differential equations is assembled automatically. a newly provided library of common kinetic rate laws highly reduces the repetitiveness of the computer programming code, and provides full sbml compatibility. previous versions provided functions for automatic construction of networks for isotope labelling studies. using user-provided label maps, modelbase v1.0 streamlines the expansion of classic models to their isotope-specific versions. finally, the library of previously published models implemented in modelbase is continuously growing. ranging from photosynthesis over tumour cell growth to viral infection evolution, all models are available now in a transparent, reusable and unified format using modelbase. conclusion with the small price of learning a new software package, which is written in python, currently one of the most popular programming languages, the user can develop new models and actively profit from the work of others, repeating and reproducing models in a consistent, tractable and expandable manner. moreover, the expansion of models to their label specific versions enables simulating label propagation, thus providing quantitative information regarding network topology and metabolic fluxes. mathematical models are accepted as valuable tools in advancing biological and medical research [1, 2] . in particular, models based on ordinary differential equations (ode) found their application in a variety of fields. most recently, deterministic models simulating the dynamics of infectious diseases gained the interest of the general public during our combat of the covid-19 pandemic, when a large number of ode based mathematical models has been developed and discussed even in nonscientific journals (see for example [3] [4] [5] ). such focus on mathematical modelling is not surprising, because computational models allow for methodical investigations of complex systems under fixed, controlled and reproducible conditions. hence, the effect of various perturbations of the systems in silico can be inspected systematically. importantly, long before exploring their predictive power, the model building process itself plays an important role in integrating and systematising vast amounts of available information [6] . properly designed and verified computational models can be used to develop hypotheses to guide the design of new research experiments (e.g., in immunology to study lymphoid tissue formation [7] ), support metabolic engineering efforts (e.g., identification of enzymes to enhance essential oil production in peppermint [8] ), contribute to tailoring medical treatment to the individual patient in the spirit of precision medicine (e.g., in oncology [2] ), or guide political decision making and governmental strategies (see the review on the impact of modelling for european union policy [9] ). considering their potential impact, it is crucial that models are openly accessible so that they can be verified and corrected, if necessary. in many publications, modelling efforts are justified by the emergence of extraordinary amounts of data provided by new experimental techniques. however, arguing for the necessity of model construction only because a certain type or amount of data exists, ignores several important aspects. firstly, computational models are generally a result of months, if not years, of intense research, which involves gathering and sorting information, simplifying numerous details and distilling out the essentials, implementing the mathematical description in computer code, carrying out performance tests and, finally, validation of the simulation results. our understanding of many phenomena could become deeper if instead of constructing yet another first-generation model, we could efficiently build on the knowledge that was systematically collected in previously developed models. secondly, the invaluable knowledge generated during the model construction process is often lost, mainly because of the main developer leaves the research team, but also due to unfavourable funding strategies. it is easier to obtain research funds for the construction of novel, even if perfunctory models, than to support a long-term maintenance of existing ones. preservation of the information collected in the form of a computational model has became an important quest in systems biology, and has been to some extend addressed by the community. development of the systems biology markup language (sbml) [10] for unified communication and storage of biomedical computational models and the existence of the biomodels repository [11] already ensured the survival of constructed models beyond the academic lifetime of their developers or the lifetime of the software used to create them. but a completed model in the sbml format does not allow to follow the logic of model construction and the knowledge generated by the building process is not preserved. such knowledge loss can be prevented by providing simple-to-use toolboxes enforcing a universal and readable form of constructing models. we have therefore decided to develop modelbase [12] , a python package that encourages the user to actively engage in the model building process. on the one hand we fix the core of the model construction process, while on the other hand the software does not make the definitions too strict, and fully integrates the model construction process into the python programming language. this differentiates our software from 2/13 many available python-based modelling tools (such as scrumpy [13] or pysces [14] ) and other mathematical modelling languages (recently reviewed from a software engineering perspective by schölzel and colleagues [15] ). we report here new features in modelbase v1.0, developed over the last two years. we have significantly improved the interface to make model construction easier and more intuitive. the accompanying repository of re-implemented, published models has been considerably expanded, and now includes a diverse selection of biomedical models. this diversity highlights the general applicability of our software. essentially, every dynamic process that can be described by a system of odes can be implemented with modelbase. implementation modelbase is a python package to facilitate construction and analysis of ode based mathematical models of biological systems. version 1.0 introduces changes not compatible with the previous official release 0.2.5 published in [12] . all api changes are summarised in the official documentation hosted by readthedocs. the model building process starts by creating a modelling object in the dedicated python class model and adding to it the chemical compounds of the system. then, following the intuition of connecting the compounds, you construct the network by adding the reactions one by one. each reaction requires stoichiometric coefficients and a kinetic rate law. the latter can be provided either as a custom function or selecting from the newly provided library of rate laws. the usage of this library (ratelaws) reduces the repetitiveness by avoiding boilerplate code. it requires the user to explicitly define reaction properties, such as directionality. this contributes to a systematic and understandable construction process, following the second guideline from the zen of python: "explicit is better than implicit". from this, modelbase automatically assembles the system of odes. it also provides numerous methods to conveniently retrieve information about the constructed model. in particular, the get * methods allow inspecting all components of the model, and calculate reaction rates for given concentration values. these functions have multiple variants to return all common data structures (array, dictionary, data frames). after the model building process is completed, simulation and analyses of the model are performed with the simulator class. currently, we offer interfaces to two integrators to solve stiff and non-stiff ode systems. provided you have installed the assimulo package [16] , as recommended in our installation guide, modelbase will be using cvode, a variable-order, variable-step multi-step algorithm. the cvode class provides a direct connection to sundials, the suite of nonlinear and differential/algebraic equation solvers [17] which is a powerful industrial solver and robust time integrator, with a high computing performance. in case when assimulo is not available, the software will automatically switch to the scipy library [18] using lsoda as an integrator, which in our experience showed a lower computing performance. sensitivity analysis provides a theoretical foundation to systematically quantify effects of small parameter perturbations on the global system behaviour. in particular, metabolic control analysis (mca), initially developed to study metabolic systems, is an important and widely used framework providing quantitative information about the response of the system to perturbations [19, 20] . this new version of modelbase now has a full suite of methods to calculate response coefficients and elasticises, and plotting them as a heat-map, giving a clear and intuitive colour-coded visualisation of the results. an example of such visualisation, for a re-implemented toy model of the upper part of glycolysis (section 3.1.2 [21] ), can be found in figure 1 . many of the available relevant software packages for building computational models restrict the users by providing unmodifiable plotting routines with predefined settings that may not suit your personal preferences. with modelbase v1.0 we constructed our plotting functions allowing the user to pass optional keyword-arguments (often abbreviated as **kwargs), so you still access and change all plot elements, providing a transparent and flexible interface to the commonly used matplotlib library [22] . the easy access functions to visualise the results of simulations were expanded from the previous version. they now include plotting selections of compounds or fluxes, phase-plane analysis and the results of mca. models for isotope tracing modelbase has additionally been developed to aid the in silico analyses of label propagation during isotopic studies. in order to simulate the dynamic distribution of isotopes all possible labelling patterns for all intermediates need to be created. by providing an atom transition map in the form of a list or a tuple, all 2 n isotope-specific versions of a chemical compound are created automatically, where n denotes the number of possibly labelled atoms. changing the name of previous function carbonmap to labelmap in v1.0 acknowledges the diversity of possible labelling experiments that can be reproduced with models built using our software. sokol and portais derived the theory of dynamic label propagation under stationary assumption [23] . in steady state the space of possible solutions is reduced and the labelling dynamics can be represented by a set of linear differential equations. we have used this theory and implemented an additional class linearlabelmodel which allows figure 2 . labelling curves in a linear non-reversible pathway. example of label propagation curves for a linear non-reversible pathway of 5 randomly sized metabolite pools, as proposed in the paper by sokol and portais [23] . circles mark the position at which the first derivative of each labelling curve reaches maximum. in the original paper this information has been used to analyse the label shock wave (lsw) propagation. to reproduce these results run the jupyter notebook from the additional file 2. rapid calculation of the label propagation given the steady state concentrations and fluxes of the metabolites [23] . modelbase will automatically build the linear label model after providing the label maps. such a model is provided in figure 2 , where we simulate label propagation in a linear non-reversible pathway, as in figure 1 in [23] . the linear label models are constructed using modelbase rate laws and hence can be fully exported as sbml file. many models loose their readability due to the inconsistent, intractable or misguided naming of their components (an example is a model with reactions named as v1 -v10, without referencing them properly). by providing meta data for any modelbase object, you can abbreviate component names in a personally meaningful manner and then supply additional annotation information in accordance with standards such as miriam [24] via the newly developed meta data interface. this interface can also be used to supply additional important information compulsorily shared in a publication but not necessarily inside the code, such like the unit of a parameter. with the newly implemented changes our package becomes more versatile and user friendly. as argued before, its strength lies in its flexibility and applicability to virtually any biological system, with dynamics that can be described using an ode system. there exist countless mathematical models of biological and biomedical systems derived using odes. many of them are rarely re-used, at least not to the extent that could be reached, if models were shared in a readable, understandable and reusable way [15] . as our 5/13 package can be efficiently used both for the development of new models, as well as the reconstruction of existing ones, as long as they are published with all kinetic information required for the reconstruction, we hope that modelbase will in particular support users with limited modelling experience in re-constructing already existing work, serving as a starting point for their further exploration and development. we have previously demonstrated the versatility of modelbase by re-implementing mathematical models previously published without the source code: two models of biochemical processes in plants [25, 26] , and a model of the non-oxidative pentose phosphate pathway of human erythrocytes [27, 28] . to present how the software can be applied to study medical systems, we used modelbase to re-implement various models, not published by our group, and reproduced key results of the original manuscripts. it was beyond our focus to verify the scientific accuracy of the corresponding model assumptions. we have selected them to show that despite describing different processes, they all share a unified construct. this highlights that by learning how to build a dynamic model with modelbase, you in fact do not learn how to build a one-purpose model, but expand your toolbox to be capable of reproducing any given ode based model. all examples are available as jupyter notebooks and listed in the additional files. for the purpose of this paper, we surveyed available computational models and subjectively selected a relatively old publication of significant impact, based on the number of citations, published without providing the computational source code, nor details regarding the numerical integration. we have chosen a four compartment model of hiv immunology that investigates the interaction of single virus population with the immune system described only by the cd4 + t cells, commonly known as t helper cells [29] . we have implemented the four odes describing the dynamics of uninfected (t), latently infected (l) and actively infected cd4 + t cells (a) and infectious hiv population (v). we have reproduced the results from figure 3 from the original paper [29] showing the decrease in the overall population of cd4+ t-cell (uninfected + latently infected + actively infected cd4+) over time, depending on the number of infectious particles produced per actively infected cell (n). to reproduce these results run the jupyter notebook from the additional file 3. in the figure 3 , we reproduce the results from fig. 3 from the original paper, where by changing the number of infectious particles produced per actively infected cell (n) we follow the dynamics of the overall t cell population (t+l+a) over a period of 10 years. the model has been further used to explore the effect of azidothymidine, an antiretroviral medication, by decreasing the value of n after 3 years by 25% or 75%, mimicking the blocking of the viral replication of hiv. a more detailed description of the timedependent drug concentration in the body is often achieved with pharmacokinetic models. mathematical models based on a system of differential equations that link the dosing regimen with the dynamics of a disease are called pharmacokinetic-pharmacodynamic (pk-pd) models [30] and with the next example we explore how modelbase can be used to develop such models. the technological advances forced a paradigm shift in many fields of our life, including medicine, making more personalised healthcare not only a possibility but a necessity. a pivotal role in the success of precision medicine will be to correctly determine dosing regimes for drugs [31] and pk-pd models provide a quantitative tool to support this [32] . pk-pd models have proved quite successful in many fields, including oncology [33] and here we used the classical tumour growth model by simeoni and colleagues, originally implemented using the industry standard software winnonlin [34] . as the pharmacokinetic model has not been fully described we reproduced only the highly simplified case, where we assume a single drug administration and investigate the effect of drug potency (k 2 ) on simulated tumour growth curves. in figure 4 we plot the simulation results of the modelbase implementation of the system of four odes over the period of 18 days where we systematically changed the value of k 2 , assuming a single drug administration on day 9. with the mca suite available with our software we can calculate the change of the system in response to perturbation of all other system parameters. such quantitative description of the systems response to local parameter perturbation provides support in further studies of the rational design of combined drug therapy or discovery of new drug targets, as described in the review by cascante and colleagues [35] . finally, compartmental models based on ode systems have a long history of application in mathematical epidemiology [36] . many of them, including numerous recent publications studying the spread of coronavirus, are based on the classic epidemic susceptible-infected-recovered (sir) model, originating from the theories developed by kermack and mckendrick at the beginning of last century [37] . one of the most critical information searched for while simulating the dynamics of infectious disease is the existence of disease free or endemic equilibrium and assessment of its stability [38] . indeed periodic oscillations have been observed for several infectious diseases, including measles, influenza and smallpox [36] . to provide an overview of more modelbase functionalities we have implemented a relatively simple sir model based on the recently published autonomous model for smallpox [39] . we have generated damped oscillations and visualised them using the built-in function plot phase plane (see figure 5 ). in the attached jupyter notebook we present how quickly and efficiently in terms of lines of code, the sir model is built and how to add and remove new reactions and/or compounds to construct further variants of this model, such as a seir (e-exposed) or sird (d-deceased) model. figure 4 . compartmental pharmacokinetic-pharmacodynamic model of tumour growth after anticancer therapy. we have reproduced the simplified version of the pk-pd model of tumour growth, where pk part is reduced to a single input and simulated the effect of drug potency (k 2 ) on tumour growth curves. the system of four odes describing the dynamics of the system visualised on a scheme above is integrated over the period of 18 days. we systematically changed the value of k 2 , assuming a single drug administration on day 9. we have obtained the same results as in the figure 3 in the original paper [34] . to reproduce these results run the jupyter notebook from the additional file 4. sir model with vital dynamics including birth rate has been adapted based on the autonomous model to simulate periodicity of chicken pox outbreak in hida, japan [39] . to reproduce these results run the jupyter notebook from the additional file 5. we are presenting here updates of our modelling software that has been developed to simplify the building process of mathematical models based on odes. modelbase is fully embedded in the python programming language. it facilities a systematic construction of new models, and reproducing models in a consistent, tractable and expandable manner. as odes provide a core method to describe the dynamical systems, we hope that our software will serve as the base for deterministic modelling, hence it's name. with the smoothed interface and clearer description of how the software can be used for medical purposes, such as simulation of possible drug regimens for precision medicine, we expect to broaden our user community. we envisage that by providing the mca functionality, also users new to mathematical modelling will adapt a working scheme where such sensitivity analyses become an integral part of the model development and study. the value of sensitivity analyses is demonstrated by considering how results of such analyses have given rise to new potential targets for drug discovery [35] . we especially anticipate that the capability of modelbase to automatically generate labelspecific models will prove useful in predicting fluxes and label propagation dynamics through various metabolic networks. in emerging fields such as computational oncology, such models will be useful to, e.g., predict the appearance of labels in cancer cells. if you have any questions regarding modelbase, you are very welcome to ask them. it is our mission to enable reproducible science and to help putting the theory into action. project name: modelbase project home page: https://pypi.org/project/modelbase/ operating system(s): platform independent programming language: python other requirements: none licence: gnu general public license (gpl), version 3 any restrictions to use by non-academics: none computational systems biology computational oncology -mathematical modelling of drug regimens for precision medicine effective containment explains subexponential growth in recent confirmed covid-19 cases in china an updated estimation of the risk of transmission of the novel coronavirus (2019-ncov) covid-19 outbreak on the diamond princess cruise ship: estimating the epidemic potential and effectiveness of public health countermeasures how computational models can help unlock biological systems model-driven experimentation: a new approach to understand mechanisms of tertiary lymphoid tissue formation, function, and therapeutic resolution mathematical modeling-guided evaluation of biochemical, developmental, environmental, and genotypic determinants of essential oil composition and yield in peppermint leaves modelling for eu policy support: impact assessments the systems biology markup language (sbml): a medium for representation and exchange of biochemical network models biomodels database: a repository of mathematical models of biological processes building mathematical models of biological systems with modelbase scrumpy : metabolic modelling with python modelling cellular systems with pysces required characteristics for modeling languages in systems biology: a software engineering perspective. biorxiv sciencedirect assimulo: a unified framework for ode solvers sundials : suite of nonlinear and differential / algebraic equation solvers algorithms for scientific computing in python the control of flux: 2 1 a linear steady-state treatment of enzymatic chains. general properties, control and effector strength systems biology: a textbook matplotlib: a 2d graphics environment theoretical basis for dynamic label propagation in stationary metabolic networks under step and periodic inputs minimum information requested in the annotation of biochemical models (miriam) short-term acclimation of the photosynthetic electron transfer chain to changing light: a mathematical model a mathematical model of the calvin photosynthesis cycle comparison of computer simulations of the f-type and l-type non-oxidative hexose monophosphate shunts with 31p-nmr experimental data from human erythrocytes 13c n.m.r. isotopomer and computersimulation studies of the non-oxidative pentose phosphate pathway of human erythrocytes dynamics of hiv infection of cd4+ t cells modeling of pharmacokinetic/pharmacodynamic (pk/pd) relationships: concepts and perspectives precision medicine: an opportunity for a paradigm shift in veterinary medicine hhs public access precision dosing in clinical medicine: present and future different ode models of tumor growth can deliver similar results predictive pharmacokinetic-pharmacodynamic modeling of tumor growth kinetics in xenograft models after administration of anticancer agents metabolic control analysis in drug discovery and disease the mathematics of infectious diseases a contribution to the mathematical theory of epidemics qualitative and bifurcation analysis using an sir model with a saturated treatment function emergence of oscillations in a simple epidemic model with demographic data the authors declare that they have no competing interests. jupyter notebook with the modelbaseimplementation of the minimal pharmacokineticpharmacodynamic (pk-pd)model linking that linking the dosing regimen of an anticancer agent to the tumour growth, proposed by simeoni and colleagues [34] . jupyter notebook with the modelbase implementation of the classic epidemic susceptible-infected-recovered (sir) model parametrised as the autonomous model used to simulate periodicity of chicken pox outbreak in hida, japan [39] . the official documentation is hosted on readthedocs. key: cord-323251-yd29gk7q authors: li, kin fun title: smart home technology for telemedicine and emergency management date: 2012-05-11 journal: j ambient intell humaniz comput doi: 10.1007/s12652-012-0129-8 sha: doc_id: 323251 cord_uid: yd29gk7q with the ageing population, mobility is an important issue and it deters the elderlies to visit health clinics on a regular basis. individuals with disabilities also face the same obstacles for their out-of-home medical visits. in addition, people living in remote areas often do not get the needed health care attention unless they are willing to spend the time, effort and cost to travel. advances in information and telecommunication technologies have made telemedicine possible. using the latest sensor technologies, a person’s vital data can be collected in a smart home environment. the bio-information can then be transferred wirelessly or via the internet to medical databases and the healthcare professionals. using the appropriate sensing apparatus at a smart home setting, patients, elderlies and people with disabilities can have their health signals and information examined on a real-time and archival basis. recovery process can be charted on a regular basis. remote emergency alerts can be intercepted and responded quickly. health deterioration can be monitored closely enabling corrective actions. medical practitioners can therefore provide the necessary health-related services to more people. this paper surveys and compiles the state-of-the-art smart home technologies and telemedicine systems. in the past decade, one of the fastest growing multidisciplinary research areas is telemedicine. many definitions of this term exist depending on the specific context. other terms that often used interchangeably, in conjunction with, or under the umbrella of telemedicine, include telehealthcare, telemonitoring, e-health, e-care, ambient assisted living, smart homecare etc. all these terminologies, though, have the same objectives of delivering remote healthcare to an individual's home and enabling continuous health monitoring and emergency management. the increasing attention given to telemedicine is due to many factors. with unaccommodating transportation, and crowed hospitals and clinics in most cities, it is difficult for the elderlies and physically challenged to venture out of their homes to meet with health professionals for the needed treatment or for their routine clinical check-ups. similarly, healthcare delivery in remote rural areas is a demanding endeavour. having the capability of delivering healthcare services at the patients' home not only saves a great deal in monetary and human resources, but it also reduces response time in emergency situations. this is especially beneficial in the case where a large number of patients can be dealt with via communication technologies quickly from a centralized location without sending medical personnel to their individual homes. telemedicine has drawn worldwide attention in the 2000s as modern technologies have made remote healthcare delivery a reality. within the european community, there were the hhh (home or hospital in heart failure) trials of a low-cost, self-managed home monitoring system for patients with chronic heart failures (pinna et al. 2003) . it was found that monthly home telemonitoring of cardiorespiratory signals being sent to a centralized interactive voice responding system is feasible and patients' compliance is high. the sm4all (smart homes for all) project initiated by the european commission aims to provide a middleware platform for pervasive embedded service in a smart environment (baldoni 2009 ). the objectives of sm4all are to provide dependability, scalability, privacy and security to users with disabilities. the brazilian family health program was initiated for preventive medicine delivered to the lower income population living in remote regions (correia et al. 2008) . the associated borboleta system enables healthcare professionals to use pdas (personal digital assistants) and mobile communication technologies for providing on-site home healthcare and improving the quality of public health services. in the usa, healthcare is a big financial burden for government, employers, and citizens. white et al. (2006) discussed major challenges to improve healthcare quality and concluded that a distributed diagnosis and home healthcare paradigm is the best approach for affordability and quality. germany has an initiative to provide encrypted health data for patients during emergency using electronic health card (dunnebeil et al. 2011) , though there is resistance by some medical professionals mainly due to privacy issues. telemedicine is a multidisciplinary research and application area using advanced technologies in information processing, telecommunication, bio-sensing, and artificial intelligence. specifically, smart environment and technologies play important roles in making home telemedicine feasible. one can view sensors being the foundation and communication networks as the pillars of a building, supporting various telemedicine applications under the roof to facilitate and provide a smart home environment to individuals, as shown in fig. 1 . this work presents a survey of the latest advances in smart environment and home telemedicine. it is an extensively updated version of an earlier work on home telemedicine review (li and li 2011) . in addition, the current focus is on smart environment and emergency management. there exist numerous works in the literature and therefore only representative, technologically innovative, and interesting systems and approaches are presented. to properly introduce smart environment in telemedicine and emergency management, the fundamental technologies, potential applications, their evaluations, and future directions are presented in a sequential coherent perspective. section 2 introduces smart environment enabling technologies including communication, sensor and sensor network. applications of smart telemedicine technologies for specific diseases, emergency management, and the elderly and physically challenged, are presented in sect. 3. target user groups' concerns and healthcare professionals' evaluation of telemedicine and smart homecare systems are discussed in sect. 4. in the concluding sect. 5, challenges, concerns and issues related to smart homecare are considered, and future trends of smart homecare environment are identified. in order to provide telemedicine effectively, various techniques and methodologies from different areas have to be integrated seamlessly into a smart system. this section examines the primary state-of-the-art enabling technologies for smart environment within a homecare setting for telemedicine: communication and associated telehealth standards, sensors, and sensor networks. many telemedicine systems leverage the latest mobile and wireless communication technologies as well as the widely available internet infrastructure to deliver quality services to home patients (castro et al. 2001; bonacina and masseroli 2006; li et al. 2008) . a home patient monitoring system proposed by figueredo and dias (2004) uses a simple rs232 serial interface to connect a mobile phone to patient monitoring devices, and transmits vital signs over the internet to the hospital. vnurse is a system developed by rehunathan et al. (2011) that uses the smart phone platform to provide secure and remote patient monitoring. bio-data in vnurse are collected from a wireless body network and transmitted using mobile networks with ip (internet protocol) connection. multi-agent systems are commonly found in smart telehealth applications. one of the earliest telemedicine projects, the independent lifestyle assistant, incorporates techniques in sensing model, distress situation detection, network response planning and machine learning (haigh et al. 2002) . the assistant is implemented as a multi-agent oriented assistive living architecture. liao et al. (2009) presented a telemonitoring architecture based on mobile agents and they also addressed the issues of reliability, security, and manageability of this platform. bramanti et al. (2010) employed gis (geographical information system) technology to identify the optimal locations of clinics that could provide neurological telemedicine services to patients. a remote-client, servicecenter architecture was proposed that uses 3g/cdma network and the internet to transfer vital sign information and medical analysis reports (zhang et al. 2010) . the web is a common and well-developed medium for communicating monitored and emergency data. raad and yang (2009) used the web for reporting real-time status and actions of a home bound elderly. voice over ip (voip) is another family of technologies and communication protocols that can be used effectively in smart environment and telehealthcare. menkens and kurschl (2010) investigated and evaluated voip enabled communication platforms and proposed a system for regular monitoring as well as providing responsive actions in emergency situations. citing decision and notification delays in typical telemedicine homecare settings, as a result of the large volume of information occupying the limited communication resources and consuming valuable computation resources on the remote server, chen (2010) team designed a smart gateway bridging a home-based wireless senor network and a public communication network. implemented as a standalone embedded system, the gateway is also capable of tracking a patient's health state and providing fast response in emergencies. communication is the foundation of providing telehealthcare in the smart home environment. there are many existing and emerging standards such as the ihe (integrating the healthcare enterprise) recommendations for information exchange (ihe 2012) , and the very popular zigbee (zigbee alliance 2012) that have been used in many smart environment projects (lin and sheng 2008; fraile et al. 2010) . de toledo et al. (2006a, b) argued that in order for e-health to progress, the use of standards in providing plugand-play devices and interoperable modules is necessary. iso/iec 29341 universal plug and play (upnp) device architecture (iso 2012a) is a standard for plug-and-play. lin and sheng (2008) carried out a study of using upnp network protocols to facilitate services in a residential network. park et al. (2011) examined the iso/ieee 11073 point-of-care medical device communication (iso 2012b) and its compatibility to legacy devices. one of the important works related to standards is anagnostaki et al. (2002) development of a codification scheme for vital signs in health information interchange. they also discussed the practical aspect of integrating the proposed scheme into medical devices. jang et al. (2007) provided a noteworthy comparison of the key features, including power, complexity, number of nodes, latency, range, expandability, data rate, security, and audio connection, among the short-range communication protocols: wi-fi ieee 802.11b standard (ieee 2012), zigbee (zigbee alliance 2012), and bluetooth (bluetooth 2012). ultra-wideband radio has also been proven to be effective in a smart home environment (bonato 2010). many research projects have drafted an exhaustive list of sensors to be used in their prototype work such as the tafeta (technology assisted friendly environment for the third age) group's implementation (arcelus et al. 2007 ). tafeta has shown how various sensors can be used effectively for different purposes in a smart home: magnetic switches on doors to monitor entry and exit, thermistor to track temperature, accelerometers on chairs and flooring to measure impact which is indicative of a falling condition, rfid (radio-frequency identification) to assist people with alzheimer or dementia, infrared motion sensor to detect mobility and presence, microphone array to detect abnormal noises and cries for help, smart grab bars with pressure sensors embedded to measure strength and balance trend, and pressure sensitive mats under bed to monitor deviated entry and exit patterns. in a typical smart home telemedicine system, there are two major types of data that need to be collected. the occupant's health state and the environmental information are important parameters to monitor and track. different types of sensors can be used to detect vital signs such as ecg (electrocardiogram) for heart rate, emg (electromyogram) for muscle activities, and blood pressure monitor for hypertension, while accelerometer, microphone, light sensor, air pressure recorder, and humidity indicator can be used to detect motion and location (fraile et al. 2010 ). in addition to signals obtained from sensors that are attached to the body or physical measurement devices in the home, there are other media that could be monitored such as video and sound. though, sound is the preferred medium due to privacy concerns. smart home technology 537 istrate et al. (2006) team developed a sound analysis system to detect and classify alarming sounds in a noisy environment. using wavelet technology to process sound signals, their system achieves good performance comparable to other systems using different monitoring technologies. laydrus et al. (2007) investigated the use of frequency domain features in an automated sound analysis system and was able to achieve over 90 % accuracy in classifying 19 real-world sounds. vacher et al. (2009) presented a sound classification and speech recognition system with microphones placed in the home to detect short sentences or words that may indicate a distress situation. though, fleury et al. (2008) found that screams and distress sentences are difficult to differentiate due to distress sentences are similar to short words. hollosi et al. (2010) devised an acoustic event detection and classification system for ambient assistive living environment and interpreted coughing as an indicator of upcoming illness. pressure sensors have been used extensively in various parts of a smart home. arcelus et al. (2009) installed pressure sensors under the bed and on the floor next to the bed. their collected data include sitting pressure, standing pressure, and the time from sit to stand, which provide valuable information on a patient's mobility and other health-related data. leusmann et al. (2011) used arrays of piezo sensors to implement a sensor floor to track the whereabouts of the home occupants. moreover, data collected can be used to analyze impact patterns in case of fall or other emergency situations. similarly, miao et al. (2009) proposed a fall detection approach based on head tracking using two video cameras. they constructed three-dimensional head position models with unimportant objects and background filtered out. location tracking is an important aspect of providing a smart home environment. global positioning system (gps) has limitation operating indoor. various techniques have been used for indoor position location including the use of cascade correlation network and neural network modeling (chen et al. 2011 ). helal et al. (2003 at the university of florida has been working on the house of matilda project for the past several years. matilda is an in-laboratory mock up house to experiment various innovative ideas for smart home. one of their focuses is location tracking of the elderly by the use of ultrasonic sensor technology. wearable devices have been research extensively in the past decade. with the advances in device miniaturization and communication techniques, wearable devices can serve many different roles in telehealthcare, for instance, as a standalone monitoring device or as a node in a sensor network (hung et al. 2004; axisa et al. 2005; kang et al. 2006) . chaczko et al. (2008) developed a small wearable device that incorporates data storage, display screen, speaker, microphone, gps, rfid and accelerometers. the ability to monitor and locate makes this an ideal device to be installed in various places in the home and on the patient. wang et al. (2007) presented a body piconet based on smart sensor nodes for ecg. their work uses bluetooth technology for in-home data transfer and the internet for network data transfer to a hospital. nag and sharma (2006) presented their work on a wireless smart clothing system that is capable of acquiring ecg, pulse oximetry, body motion/tilt and skin temperature data. bonato (2010) gave an extensive survey and review of the major approaches to implement wearable systems. enabling technologies discussed in that work include miniaturization, lower-power radio, and dedicated operating system, which make body sensor network a reality. instead of focusing on specific or a few signals, multimodal systems are gaining popularity. medjahed et al. (2009) presented a fuzzy logic system utilising a wide range of sensors to recognize activities in a home environment. these sensors include physiological types (cardiac frequency, activity, agitation, posture and fall detection), microphones, infrared, water debit and statechange in appliances. many innovative and non-intrusive approaches have been developed in the past several years. ichihashi and sankai (2007) developed a small 'smart telecom' unit that integrates sensing circuit, digital signal processor, and wireless communication into a device of size 25 by 37 mm. such small devices can monitor one's vital signs without being invasive. kim et al. (2007) developed an integrated home health monitor using a chair as a non-invasive platform. this all-in-one monitor can record ecg, estimate blood pressure, measure blood glucose level and ear temperature. these sampled signals are then transmitted to a home server using zigbee technology, which in turn are transmitted to a healthcare center via the internet. ho et al. (2009) presented a very interesting initiative in an attempt to harvest body temperature and vibrations in the house to power implantable glucose sensors within a wireless home network. ping et al. (2009) home healthcare research project places heavy emphasis on recognizing a patient's emotional state in addition to physical state. facial, vocal, eye-movement, and physiological signals are tracked and analyzed to provide the psychological aspects of a patient's health. hussain and colleagues (2009) explored the integration of rfid and wireless sensor network in a smart home environment. their idea is to use the wireless sensor network for tracking the movement and location of individuals while the identification of a person can be accomplished by the wearing of rfid tags. the separation of these events allows for quick decision-making and responses to various situations. this section presents the major application areas that smart telemedicine plays a dominant role. unlike face-to-face medical consultation, useful information must be derived from the raw data in telemedicine. it is almost impossible for humans to examine this large volume of data manually and to detect any changes. therefore, techniques have to be developed in order to automate the diagnostics and abnormality detection process. this section introduces smart telehealth systems developed for specific illnesses. back in 2000, finkelstein and friedman (2000) presented a home asthma telemonitoring system that has the capability of assisting asthma patients in self-care plans as well as alerting healthcare personnel when distress situation is detected. lau et al. (2002) implemented a multimedia system that employs bio-sensor data, messages and video transmission to allow physicians and patients with shoulder replacement surgery to communicate and monitor the progress. chun et al. (2003) developed a home telemedicine system for cardiac and respiratory disease, diabetes, and hypertension. using artificial intelligence techniques, this system is capable of providing automated diagnostic and consultation. performance evaluation has shown that the implemented system is a viable alternative to personal monitoring and consultation. rudi and celler (2006) presented an expert telemedicine system that is capable of storing various data and measurement related to diabetes management. in addition to present to medical professionals records associated with individual patients, the system is also capable of recommending insulin dose adjustment to patients. jiang and xie (2009) proposed a telehealth monitoring system that uses data mining techniques to deal with the large volume of biological data. they also utilized association rules to recommend actions to be taken for hypertension. zhang et al. (2009) developed a chronic disease management system using a real-time knowledge base and the case based reasoning approach, together with a web patient monitoring system, to improve the diagnostic of diabetes and hypertension. fergus et al. (2009) developed a body area sensor network intended for physiotherapist's use. this system collects and stores motion data of the home patient, and provides some quantitative assessments of the patient's progress. gobbi et al. (2009) designed a system that addresses issues related to real-time data processing, network architecture, and web-based data management and services. as an automated home monitoring system for lung obstruction, that system was shown to be reliable and efficient. silva et al. (2010) developed an internet-based system for home monitoring of patients with respiratory muscle disorders. they concluded that their system would be a useful tool for the evaluation of inspiratory muscle in telemedicine services, thus reducing the costs of handling these patients. a smart home with technologies to enhance the quality of life of quadriplegia patient was proposed by uzunay and bicakci (2007) . they placed heavy emphasis on the security aspect of their voice activated smart home facilities. to monitor blood flow velocity in a wireless home environment, wu et al. (2011) presented a self-monitoring device to measure blood flow velocity that is integrated in a labview (labview 2012) mobile environment. smart home telemedicine technology is ideal for patient selfmonitoring and quality of life improvement, which are the subjects in the next section. the segments of our population that need quality healthcare the most are the elderly and physically impaired. home telemedicine and telehealthcare systems are the solution to deliver low-cost yet quality services for these individuals. in 2006, the united nation estimated that at least 650 million people have disabilities worldwide (united nation 2006) . there are various types and degrees of disabilities. according to the massachusetts department of elementary and secondary education, the impairments can be classified into communication, emotional, health, intellectual, neurological, physical, and sensory (mdese 2000) . andrich et al. (2006) presented their smart home project with case studies involving people with disabilities in different categories: hemiplegia, paraplegia, quadriplegia, motor impairment, and cognitive impairment. disabled person with the specific type of impairment would require specialised care and tailored smart home technologies. many telehealth and assistive systems have been proposed and are in place for the elderlies. meng et al. (2000) has been working on teleoperated mobile robots via the internet to provide quality healthcare to the elderly. they have also designed robotic pets that are capable of physiological data collection and transmission, as well as simple healthcare tasks (ling et al. 2005) . briere and colleagues (2009) presented a teleoperated mobile robot with videoconferencing capability for in-home telerehabilitation services. vergados et al. (2008 vergados et al. ( , 2010 discussed their inhome project which goal is to provide intelligent ambient assistive living services to elderly people at home for independent living. lim et al. (2010) presented a home healthcare set-top box specially designed for the elderlies. their work focused on standard compatibility and smart home technology 539 adherence to the iso/ieee 11073 personal health data standard. vinjumur et al. (2010) implemented a web-based medicine management system that uses rfid tags to monitor medication intake for the elderly at home. with multimodal signal sampling, it is imperative to incorporate data fusion techniques in telehealth monitoring and decision-making. virone et al. (2003) investigated data fusion of collected video and sound signals in the monitoring of elderly patients at home. medjahed's group focused on data fusion of multi-senor inputs to provide medical recommendation using a rule based approach (medjahed et al. 2011) . artificial intelligence techniques play a major role in smart home systems. papamatthaiakis and colleagues (2010) presented an indoor activity recognition system for the elderlies. using association rules and allen's temporal relations, they claimed a 100 % recognition rate in identifying everyday activity such as bathing, preparing meals, doing laundry, etc. the mavhome project aims to provide a smart and adaptive environment for inhabitants (jakkula et al. 2007) . in order to meet the goals of comfort and efficiency in mavhome, health trend monitoring and prediction are made using support vector machine and forecasting tools based on time series data collected in a sensor network. huo et al. (2009) presented a healthcare environment for the elderlies using a home sensor network and a body sensor network. the interesting aspects of this work lie in the functionalities provided, including outdoor monitoring, and emergency decision and alarms. various communication techniques, such as automated telephone call, sms and email, are integrated into the system, transmitting emergency signals to caregivers and family members. smart decision-making using hidden markov model is proposed to speed up the decision process, increase the accuracy of event detection, minimise measurement errors, and correct transmission errors. franco et al. (2010) proposed the use of passive infrared sensors placed in the living quarters of an elderly person to detect abnormal changes in behavior. this system would allow early admission of dependent care for those who show a shift in daily routine, for example, individuals with alzheimer disease. the monitoring of behavioural changes is an area of interest in telehealth since the onset of certain alarming situations could be detected and healthcare professionals could then be notified. the next section focuses on using smart healthcare system to handle emergency. emergency planning and management with respect to inhome healthcare has been a vigorously research topic. rosen et al. (2002) proposed the cybercare system to handle national scale disasters. taking a military approach to map strategy, operations and technology to the healthcare realm of policy, functionality and network infrastructure technologies, they argued the necessity of a nation-wide support information infrastructure to cope with disaster, and of incorporating surge-capacity into a national disaster response system. citing the fact that the patients who receive critical emergency services often are not the actual victims of the disaster or outbreak such as sars (severe acute respiratory syndrome), joseph et al. (2005) developed a home healthcare disaster planning, recovery, and management system to facilitate treatment of the actual victims. by integrating in-home intelligent devices to provide timely measured information to public offices such as red cross, local police etc., they reasoned that the medical personnel can provide homecare remotely and deal with a mass-scale epidemic and natural disasters appropriately. smart environment not only can be used in a home environment but can also be used outdoor. smalls et al. (2009) devised a health monitoring system for use in mass causality emergency situations. by placing a health monitoring device on the body of a victim and integrating this node to a wireless ad-hoc network, vital signs of the victim can be transmitted to regional and national emergency response institutions. this is particular useful in cases where infrastructures are non-operational due to catastrophic disaster or when communication channels are overwhelmed by emergency requests. advances in wireless technology have driven down the cost of ad-hoc networks, and with fewer attending field personnel, smalls et al.'s approach may prove to be cost effective in the very near future. wouhaybi et al. (2010) also proposed a system with similar objectives but they focused on the reduction of false alarm using a rule-based decision system. a disaster management framework proposed by bessis et al. (2010) integrates various technologies including web services, grid and cloud computing, ad-hoc mobile networks, and wearable and body sensors. their aim is to provide coherent and collective resource utilization and decisionmaking for multiple parties in emergency situations. jang et al. (2007) proposed a system using multiple sensor technologies including bio-sensors to monitor an elderly's daily physiological status. many services are provided to the patient including early warning due to a change of health status, advice for health improvement, and appointment with specific medical practitioners. in addition to sensors, an active robot that provides services to human is part of a smart assistive environment proposed by lin et al. (2009) . using multimodal observations to recognize voice and other events, their project shows that health predictions, evaluations, and decisions become more reasonable in the evaluation of emergency level and the assistance required in critical events. in one of the early studies, de lunsignan et al. (2000) examined the effectiveness of a home cardiopulmonary monitoring system. this system collects various vital measures via sensors attached to the body and transmits the data wirelessly to a nearby unit at the patient's home. the data are then transferred to a centralised monitoring station. they found the system is acceptable to patients, functionally satisfactory in the home environment, and very reliable in the collection of objective data. capomolla et al. (2004) monitored patients with chf (chronic heart failure) either under usual care or telemonitoring care. their study showed that home telemonitoring is more effective than usual care in reducing healthcare requirements and can improve outcome in chf patients. raju et al. (2004) presented their study on the cost effectiveness of mobile telemedicine applications, and the quality of care and medical error reduction. they reported on their extensive literature search and felt that the findings are inconclusive and well-designed protocols are necessary to conduct further large-scale investigations. zhang and bai (2005) used a queueing model to evaluate the performance of a home ecg and blood pressure monitoring system based on trial data over a 4-month period. their objective was to study the traffic load, response time, and scalability of the system. the results are more than satisfactory and show great potential of the examined monitoring system. de toledo et al. (2006a) reported their experience in using a telemedicine homecare system for patients with chronic obstructive pulmonary disease. their results suggest that home telemedicine services provide good support to the healthcare professionals, improve patients' condition, and incur low costs. jasemian (2008) argued that a successful telemedicine application depends on the patient's compliance, trust and comfort in such home-based systems. an experiment was carried out in 2008 with the patients using ecg monitoring device with a real-time wireless system continuously for a long period of time. over half of the participants found the system user friendly, reliable, and usable with acceptable performance. though they found the ecg device heavy and not user-friendly. some have argued that the evaluation of home telehealth information systems should not be assessed simply on grounds of technical innovation but should use a holistic interpretive approach. in 2008, kaldoudi and colleagues (2008) proposed a framework for evaluating home telehealth intervention, together with its application for peritoneal dialysis at home. ojasalo et al. (2010) conducted a study on smart home technologies and services for intellectually disabled. they found that safety technologies are very well received, and the balance of safety and privacy can be addressed by the appropriate technologies. a 2011 study conducted by ziefle et al. (2011) on the acceptance of video monitoring in smart home environment for elderly and disabled reveals that acceptance and users' needs and wants are the main issues in a successful deployment of such home medical monitoring technology. the study also shows that there are serious concerns that data may be altered, illegally accessed, and deleted intentionally or due to system failure or virus attack. beer and takayama (2011) performed a study on elderlies who used a mobile remote presence (mrp) system. this study reveals many interesting issues. benefits of mrp identified by the elderlies include being able to see and be seen with a mrp system, reduction in travel costs and hassles, and a decrease in isolation in a social context. however, concerns raised include mrp usage etiquette, personal privacy, possible overuse, and reliance on the system. though the elderlies in the study are not technologically inclined, they prefer to operate the mrp system by themselves. smart home healthcare and telemedicine systems are here to stay, but there are still many challenges and issues to be resolved. innovation is important to improve the efficiency and effectiveness, and hence acceptance, of these systems. security is major concern in the acceptance of homecare with modern technologies. many research groups have focused on one of the most important issue in telemedicine, that of the integrity and security of transmitted data. simpson et al. (2006) posed several challenging issues regarding continuous home monitoring: who should receive information as the patient, family members, caregivers and medical professionals all have interest; what information should each person receive without violating any data privacy and security concerns; how should information be presented since each party may prefer different mode of communication such as telephone, computer, etc. proposed solutions to the security issue include cryptography (mantas et al. 2009 ) and context-aware access control rules (guennoun and el-khatib 2009). due to the sensitive nature of health information, privacy is an important issue in data transmission and storage in telehealth systems. without proper and established protocols and regulations in place, telemedicine would not be used widely as many people are concerned with the smart home technology 541 privacy of their health information. kotz and colleagues (2009) compared existing privacy frameworks, identified a set of desirable privacy properties in mobile healthcare systems, and used their findings to propose a privacy framework. they also raised several privacy issues and questions that need to be addressed by technical people, governmental agencies and regulatory organisations. reliability is another critical factor in the implementation of smart homecare. wireless technologies make telehealth care possible, however, such networks that operate in hostile outdoor environment have many reliability issues. using rural china as a case study, fong and pecht (2010) identified and reviewed the factors that can impact on the reliability of telehealth care networks: physical obstacles, atmospheric absorption, inadequate fade margin and system failure. gaddam et al. (2008) studied extensively the various issues related to the implementation of sensor networks for home healthcare and emergency. they have identified potential challenges including interoperability and interference, real-time data acquisition and processing, reliability and robustness, energy conservation, data management, data privacy and security, and comfort and unobtrusive operation. in addition, design issues for wireless networks considered are deployment of sensor nodes, mobility of wireless sensor, cost and size of the wireless node, infrastructure of the network, network coverage, network size, power management, life-time of the sensor networks, and quality of service requirements in such networks. since the second-half of the 2000s, there have been numerous reported work and innovation in smart healthcare systems. taylor and dajani (2008) gave a sound argument that the future home healthcare systems should take into consideration a well-balanced implementation infrastructure based on the web, mobile, and broadband technologies. moritz et al. (2009) emphasized the importance of various fundamental issues in using interoperable devices in home healthcare. these issues include energy consumption, power supply, memory, computing power, and bandwidth. koufi and colleagues (2010) implemented a grid portal that provides services to people who need medical advice at their homes. the portal is an integrated system of wireless personal digital assistants (pdas) and grid technology, with heavy design emphasis placed on security access and storage of data. for telehealth, the monitoring of an individual creates a continuous stream of data that must be stored either locally at home or at a centralised database. lasierra et al. (2010 lasierra et al. ( , 2011 used an ontology approach to arrange patient data and records in a formal structure so as to support health information interoperability. invasive devices are being recognised as one of the prime reasons at-home patients are hesitant to use telehealth care monitoring systems. motoi et al. (2010) demonstrated the possibility of using non-invasive technologies for home healthcare. devices are installed on toilets, bathtubs, and beds to measure various vital signals. they deployed this prototyping system for subjects with cardiovascular disease or sleep disorder successfully. modifying and conditioning patient's behaviour is another innovative trend that is gaining attention these days. evers and krose (2010) developed a monitoring system tracking the physical and emotional condition via a sensor network. patients are provided with feedback to promote activities and behaviour with positive health impact. also, the integration of wearable technology and robots is a promising avenue to facilitate therapeutic intervention for chronic conditions, which potentially can reap benefits from these combined technologies (bonato 2010). point-of-care testing is another developing trend in enhancing home healthcare. many innovative devices are being investigated that potentially shorten the time between testing and diagnostic, and are especially useful in rural settings. beyette et al. (2011) provided a comprehensive survey and a special volume on these innovative handheld and home-based devices. these devices include sensors for multispectral imaging, tomography, cardiac, eeg, chlamydia trachomatis, bladder pressure, respiratory impairment, as well as for detection and analysis of macula swelling, pathogen detection, metabolic syndrome prediction, energy metabolism, chagas disease, vascular tree identification, parkinson's disease, etc. back in 2005, stankovic et al. (2005) at the university of virginia identified three critical development issues in wireless sensor networks for in-home healthcare. the first issue is the enabling technologies for future medical devices: interoperability, real-time data acquisition and analysis, reliability and robustness, and new node architectures. embedded, real-time, networked system infrastructure is the other critical development area which includes patient and object tracking, communication, interference, multimodal collaboration, energy conservation, and multi-tiered data management. the third issue concerns medical practice-driven models and requirements including role-base access control and delegation in realtime, unobtrusive operation, and records and data privacy and security. the critical development areas of concern, as suggested by stankovic's team, have not yet been fully addressed with today's technology. there are ample opportunities for research and development in improving telemedicine systems as they are still at an infancy stage. some of the challenges and issues in building a smart home environment are shown in fig. 2 . without a doubt, smart home telemedicine systems will be deployed in an increasingly rapid pace in the years to come. anagnostaki a et al (2002) your floor knows where you are: sensing and acquisition of movement data a survy on home telemedice home healthcare platform based on wireless sensor networks an extensible telemonitoring architecture based on mobile agent method home healthcare set top-box for senior chronic care using iso/ieee 11073 phd standard using osgi upnp and zigbee to provide a wireless ubiquitous home healthcare environment decision making in assistive environments using multimodal observations a study of integrating digital health network with upnp in an elderly nursing home robotic pet based interactive home healthcare system integrity mechanism for ehealth tele-monitoring system in smart home environment massachusetts department of elementary and secondary education human activities of daily living recognition using fuzzy logic for elderly home monitoring a pervasive multi-sensor data fusion for smart home healthcare monitoring e-service robot in home healthcare voip based telehomecare application kiosk fall detection in the elderly by head tracking web services to improve interoperability of home healthcare devices development and clinical evaluation of a home healthcare system measuring in toilet, bathtub and bed without attachment of any biological sensors wireless e-jacket for multiparameter biophysical monitoring and telemedicine applications better technologies and services for smart homes of disabled people: empirical findings from an explorative study among intellectually disabled monitoring and modeling simple everyday activities of the elderly at home /ieee 11073 phd standardization of legacy healthcare device for home healthcare services designing an emotional majormodo in smart home healthcare home telemonitoring of respiratory activity and heart rate variability in chronic heart failure patients: the challenge of the home or hospital in heart failure project a ubiquitous smart home for the elderly status of mobile computing in health care: an evidence study vnurse: using virtualisation on mobile phones for remote health monitoring the future of command and control for disaster response design and implementation of experttelemedicine system for diabetes management at home an internet-based system for home monitoring of respiratory muscle disorders plans and planning in smart homes in lncs in computer science health monitoring systems for massive emergency situations the future of homecare systems in the context of the ubiquitous web and its related mobile technologies convention on the rights of persons with disabilities sha: a secure voice activated smart home for quadriplegia patients speech recognition in a smart home: some experiments for telemonitoring intelligent services for assisting independent living of elderly people at home service personalization for assistive living in a mobile ambient healthcare-networked environment web based medicine intake tracking application smart home technology 545 first steps in data fusion between a multichannel audio acquisition and an information system for home healthcare body piconet for the telemedicine system based on home networks improving healthcare quality through distributed diagnosis and home healthcare (d2h2) a context-management framework for telemedicine: an emergency medicine case study. wireless health home self-monitoring of blood flow velocity based on labview mobile environment research on home health care telemedicine service system concerned with the improvement of medical resources utilization rate and medical conditions performance analysis of a home telemonitoring system agent-based web healthcare systems for realtime chronic disease medical technology in smart homes: exploring the user's perspective on privacy, intimacy and trust key: cord-336621-0w3rroir authors: kim, kyoung ok title: a first step toward understanding patient safety date: 2016-07-25 journal: korean j anesthesiol doi: 10.4097/kjae.2016.69.5.429 sha: doc_id: 336621 cord_uid: 0w3rroir patient safety has become an important policy agenda in healthcare systems since publication of the 1999 report entitled "to err is human." the paradigm has changed from blaming the individual for the error to identifying the weakness in the system that led to the adverse events. anesthesia is one of the first healthcare specialties to adopt techniques and lessons from the aviation industry. the widespread use of simulation programs and the application of human factors engineering to clinical practice are the influences of the aviation industry. despite holding relatively advanced medical technology and comparable safety records, the korean health industry has little understanding of the systems approach to patient safety. because implementation of the existing system and program requires time, dedication, and financial support, the korean healthcare industry is in urgent need of developing patient safety policies and putting them into practice to improve patient safety before it is too late. vol. 69, no. 5, october 2016 patient safety human error is inevitable even in highly reliable industries, such as the aviation, nuclear, and health industries [5] . we need to focus on analyzing the various processes in the system that lead to errors rather than consuming valuable resources holding individuals responsible for their actions. in this paper, two approaches will be introduced to deal with the problem of human error. the general concepts of patient safety in anesthesiology will then be discussed, followed by a discussion of the cultural influence on patient safety and ways to improve the patient safety culture in the korean healthcare industry. in 1999, "to err is human" reported a high frequency of human errors and adverse events in healthcare institutions [6] . according to several retrospective studies, 1 in 10 hospital inpatients likely suffers the consequence of a human error during their hospital stay [7, 8] . as a result, patient safety issues in healthcare have become of interest to researchers. reason [5] , the renowned human error expert who has studied human error for 25 years, presented a theory suggesting that the human error problem can be evaluated using a person approach and a systems approach. the person approach focuses on the errors of individuals. individuals who commit an error should be held responsible for their delinquency, and the delinquency is normally addressed with disciplinary measures. this paradigm remains the mainstream belief in medicine and elsewhere [5] . however, the person approach has been challenged because people do not choose to make mistakes; bad things happen to good people [9] . people in the healthcare industry have lost jobs, suffered serious stress, or taken their own life after being blamed for errors [10, 11] . indeed, maintaining this tradition is likely to victimize hardworking innocent people and delay the development of a safer healthcare environment [5] . the systems model is based on the idea that humans are prone to err and that human errors are inevitable, even in wellorganized institutions. errors occur when a series of failures in the system occur, which is well illustrated by the swiss cheese model [5] . this model likens human systems to multiple slices of swiss cheese with holes, stacked side by side. although highly technological systems have many defensive layers, such as guidelines and protocols, to prevent errors, each layer also has its own deficiencies like the holes in swiss cheese. the presence of holes in any one "slice" does not allow a risk to materialize, as another layer of defense is in place. however, when the holes in many layers momentarily line up, a mistake passes through the holes in all of the slices, resulting in an unwanted incident. the systems approach advocates that a medical error is the result of a system flaw, not a character flaw [5] , which makes it easier for an individual to report an error promptly and honestly. this kind of robust reporting helps identify errors and strengthens problematic areas so that the same error does not occur again [9] . the airline pilot martin bromiley lost his wife to an anesthesia incident and wanted to change the way in which medical errors were handled in the healthcare industry. after his wife's tragic death, he expected an investigation by an independent body. the air accidents investigation branch in the airline industry routinely investigates all accidents [12] . he was surprised when he was told that no such investigation would take place unless he sued or filed a formal complaint. an independent investigation was conducted, but mr. bromiley did not file charges against individuals and only hoped that some lesson might be taught to others as a result of a fair investigation. as he wished, all professionals involved in the case remained to serve and help the system to avoid similar errors. implementing the systems approach is vital to improving patient safety because it enables medical professionals to learn lessons from their errors [8, 9] . a main premise of the systems approach is that a completely error-free system is impossible and emphasizes learning from errors and preventing recurrence [8, 9] . one way to learn from errors is to establish a reporting system. the purpose of a reporting system is to analyze the data and provide a remedy and information that leads to a change in the system that affects all healthcare organizations [13, 14] . several studies have reported that healthcare workers often do not report adverse events for fear of punitive action, prosecution, or being ostracized at the workplace [15, 16] . anonymity, confidentiality, or protection from punishment is essential for potential reporters to overcome the barrier of fear [17, 18] . the aviation industry recognized the importance of data and introduced an internal confidential error reporting system in 1982 [19] . this confidential reporting system guarantees immunity from prosecution and encourages individuals to report errors without fear of punishment, enabling frank and accurate discussions about errors and system weaknesses. this benefit is equally applicable to the practice of clinical medicine and is korean j anesthesiol kyoung ok kim essential for patient safety [19] . medical error cases are often so complex and debatable that honest and detailed reporting is key to identifying the reasons for the accident. an error reporting system is currently being used in the usa, uk, denmark, and several other countries. the danish parliament passed the act on patient safety in 2003, following a study reporting that 9% of patients admitted to a danish hospital were involved in an adverse event [20] . this is the first legislation in the world that has sought to improve patient safety by ensuring that all adverse events are reported and that the national board of health will disseminate the results nationally [21] . the us senate passed the patient safety and quality improvement act (psqia) in 2005 [22] . the psqia introduced a voluntary reporting system to facilitate assessment of data to improve patient safety and healthcare quality. the uk's national patient safety agency created the national reporting and learning system in 2003, which is a nationwide voluntary event-reporting system for patient safety [23] . japanese healthcare workers are also familiar with event-reporting systems because all japanese hospitals established an in-house, legally bound event-reporting system in 2002 [24] . the patient safety act, which includes a voluntary reporting system, will go into effect in july 2016 in korea [25] . recent developments in practice and technology have made anesthesia much safer than before, and anesthesiology is the leading medical specialty addressing the patient safety issue [26] . clinicians working in anesthesiology became interested in patient safety partly because administering anesthesia can cause adverse events but has no therapeutic benefit of its own. several technological innovations have contributed substantially to patient safety [26] . one has been to introduce real-time patient monitoring systems, such as electrocardiography, pulse oximetry, and capnography. the laryngeal mask airway and video laryngoscope have had a huge effect on managing patients with a difficult airway. another strategy adopted by anesthesiologists to improve patient safety has been the establishment of guidelines and standards to provide guidance in specific clinical situations [26] . in particular, guidelines for managing difficult intubation and for regional anesthesia in patients taking antithrombotic agents have important implications for patient safety. the aviation industry first acknowledged that human factors dominate the risks in aviation safety and that errors are inevitable as a result of human physiological and cognitive limitations [4, 19] . consequently, the aviation industry introduced a team training model called crew resource management (crm) more than two decades ago to improve individual nontechnical skills [27] . crm is a set of training procedures used in environments where human errors can have devastating effects. crm consists of four categories: teaching situational awareness, improved communication, appropriate task distribution, and optimal teamwork. anesthesia is one of the first healthcare specialties that have adopted techniques and lessons from the aviation industry. the widespread use of simulation programs and applying human factor engineering to clinical practice are influences of the aviation industry. the anesthesia crisis resource management training modules [28] and the anesthetists' non-technical skills system (ants) for assessing behavioral markers [29] were developed while adopting the human factors engineering model. the ants skills framework includes four skill categories of situational awareness, decision-making, task management, and team working, each with example behaviors. the ants system has satisfactory levels of validity, reliability, and usability, provided raters receive adequate training [30] . in addition to these academic successes, anesthesiology contributed to the institutionalization of patient safety as a subject of professional interest. the anesthesia patient safety foundation (apsf) is a pioneer organization dedicated to patient safety that was launched in late 1985 as an independent nonprofit organization with the vision that "no patient shall be harmed by anesthesia" [31] . the apsf publishes a quarterly newsletter on anesthesia and patient safety and has funded many important projects that would probably never have been financed by a "traditional" organization. the most outstanding achievement of the apsf program has been helping to create a cadre of experts who devote their careers to patient safety by financially supporting them [26] . the american medical association, influenced by the huge success of the apsf, created the national patient safety foundation decades later [32]. despite relatively advanced medical technology and comparable safety records, the korean healthcare industry has little understanding of human factors engineering or the systems approach to patient safety, and anesthesia is not the exception. the quality of equipment, procedural techniques, and anesthetic medicine is quite standardized among developed countries, partly due to the tremendous marketing effort of multinational pharmaceutical and device companies. technologic developments for patient safety can be adopted quickly because no time or sophisticated knowledge is required. furthermore, outcomes are easily recognizable. however, creating the system and applying a training program, such as ants, are taxing and require time and commitment. considerable interest in the use of ants has been generated worldwide, and ants is currently vol. 69, no. 5, october 2016 patient safety used in anesthetic simulation training in many countries, such as canada, spain, sweden, and the netherlands [30] . unfortunately, no attempt has been made by korean anesthesia societies to introduce a crm-style training course or establish a simulation center. in addition, no patient safety committee has been created by the korean society of anesthesiologists. cultural traditions may be influencing patient safety in korea. in a study investigating the characteristics of the patient safety culture in japan, taiwan, and the us, the authors emphasized the need for open communication in taiwan [33] . a lack of open communication is not unique to taiwan but is deeply seated in the confucian culture, including korea. koreans are more reluctant to mention another individual's behavior for fear of offending them; if the individual is senior, such action is often regarded as challenging authority. feedback and communication about errors are not prevalent in the korean healthcare industry because people believe that this kind of discussion may humiliate coworkers, and they are more concerned with punitive measures [34, 35] . this confucian culture also plays an important role in communication among healthcare providers [36] . even in western culture, it is difficult for junior staff to question seniors about their actions. this is even more difficult in east asian cultures, including korea, where younger people are expected to show their respect to their elders and superiors due to the long influence of confucianism. for example, korean air had more plane crashes than almost any other airline in the world by the end of the 1990s. gladwell blamed those crashes on crew members whose cultural legacy made them too deferential to communicate clearly about a problem that could cause the plane to crash [37] . this hierarchical communication also exists in the korean medical profession and is one of the main causes of adverse events. because medical service is delivered by a team, communication with other team members is important during patient care. strong verbal communication skills in the context of a complex medical system is at the core of patient safety and effective teamwork. following is a typical case that shows how important communication is to patient safety. a 4-year-old child (height, 106 cm and weight, 16.7 kg) was admitted to the hospital for excision of a preauricular cyst. a physician-in-training (resident) in the ear-nose-throat (ent) department telephoned an anesthesia resident about a sedative dose of ketamine. the anesthesia resident said, "10 milli would be enough. " the anesthesia resident interpreted the word "milli" as short for milligram. however, the ent resident considered that "milli" was short for milliliter and wrote the order as follows: inject 500 mg/10 cc of ketamine over 1 min. this was 50 times the therapeutic dose. a nurse was concerned about the dose and asked the resident "is this right?" to verify the order. the resident approved the medication by answering the nurse, "yes, give it. " the nurse executed the order. this accident was discovered when the parent reported that the child had not woken up from sedation for several hours after finishing the computed tomography scan. fortunately, the child recovered fully without complications. the quality improvement committee reviewed the case and a remedy was implemented. the computerized order system was supplemented with dose-adjustment calculations based on weight and prohibited anesthetic prescriptions to be written by a non-anesthesiologist so that such an error would never happen again. the committee also recommended that nurses should confer with the attending physician through a senior nurse if they have an issue with a resident regarding an inappropriate subscription order. the committee made an effort to improve the system, but it did not address the communication problem properly and failed to suggest a remedy. the above-described tragedy began when two residents failed to communicate properly. the anesthesia resident assumed that the ent resident understood that "milli" meant "milligram. " this so-called "receiver-orientated" style is more prevalent in oriental culture, as the korean saying goes, gaetteoggat-i malhaedo chaltteoggat-i al-adeul-eola (i.e., "you must get it down to perfec tion, even though i speak gibberish") [38] . this type of interaction reflects the typical authoritarian culture in korea. higher-ranking individuals generally speak and then expect subordinates, who are usually listeners, to understand. if they fail, then it is the listener's fault. in contrast, western languages are typically "sender orientated, " meaning it is the speaker who has the responsibility to communicate clearly and unambiguously [38] . this style emphasizes the speaker's role in transmitting the information correctly from the beginning, which is obviously more reasonable [36] . in this way, we can reduce the possibility of miscommunication. there has been growing concern for improving verbal communication in clinical practice. therefore, many practical strategies to enhance medical communication already exist. one suggested method, which may have been helpful in this case, is the repeat-back method [39] . this means repeating what was said back to the speaker to confirm mutual understanding (e.g., the listener repeats an order from a speaker: "â�¦okay, so that is 10 milliliters of ketamine"). communication failure can arise for a number of reasons, but above all, a dysfunctional hierarchical system exists in healthcare professions. although authoritarian culture permeates all of korean j anesthesiol kyoung ok kim healthcare, physicians dominate the culture and influence others [40] . nurses traditionally provide care within the ordering scope of physicians. this traditional role plus a sense of privilege and status has led some physicians to believe that they are superior to nurses. this dysfunctional culture has created a barrier to the open communication and feedback that are essential for patient care. even an experienced nurse in the previous ent case would not challenge the novice resident. teamwork is another casualty of authoritarian behavior [40] . expecting deferential treatment from a subordinate or lacking respect for colleagues are detrimental to teamwork, which is the cornerstone of safe practice. as a team, physicians should treat other healthcare workers as their colleagues and not as subor-dinates. building healthy relationships with mutual respect is essential to the well-being of patients and healthcare workers. patient safety has become an important policy agenda in the healthcare industries of developed countries for the past decade, and numerous changes have been made to improve patient safety. however, the korean healthcare industry is well behind in patient safety for many reasons, including culture and policy. the korean healthcare industry and healthcare authorities should work together to develop and put policies into practice to improve patient safety. konkuk university respiratory disease epidemiological investigation and follow-up action plan jsp?menuids=home001-mnu1154-mnu0005-mnu0011&cid=66463 korean ) epidemiological findings and future plan jsp?menuids=home001-mnu1154-mnu0005-mnu0011&cid=66441 korean audit on risk management and prevention of mers outbreak human factors in anaesthesia: lessons from aviation human error: models and management to err is human: building a safer health incidence of adverse events and negligence in hospitalized patients. results of the harvard medical practice study i adverse events in british hospitals: preliminary retrospective record review understanding adverse events: human factors the emotional impact of medical error involvement on physicians: a call for leadership and organisational accountability taking their own lives --the high rate of physician suicide we need to change the culture in the nhs guardian news and media critical incident reporting and learning a review of medical error reporting system design considerations and a proposed cross-level systems research framework barriers to incident reporting are hospital incidents being reported? design elements for a primary care medical error reporting system reporting of adverse events confidential reporting: from aviation to clinical medicine incidence of adverse events in hospitals. a retrospective study of medical records the danish patient safety experience: the act on patient safety in the danish health care system department of health & human services. patient safety and quality improvement act of 2005 statute and rule department of health & human services patient safety 23. national patient safety agency (gb) gb): national patient safety agency healthcare safety committee in japan: mandatory accountability reporting system and punishment a legal framework for improving patient safety in korea anaesthesiology as a model for patient safety in health care development of the notechs (non-technical skills) system for assessing pilots' crm skills simulation-based training in anaesthesia crisis resource management (acrm): a decade of experience anaesthetists' non-technical skills (ants): evaluation of a behavioural marker system anaesthetists' non-technical skills foundation history national patient safety foundation (us) national patient safety foundation the characteristics of patient safety culture in japan, taiwan and the united states nurses' perception of error reporting and patient safety culture in korea experience and perception on patient safety culture of employees in hospitals improving verbal communication in critical care medicine new york, little, brown and company intercultural communication in cognitive values: americans and koreans medical team training: applying crew resource management in the veterans health administration perspective: a culture of respect, part 1: the nature and causes of disrespectful behavior by physicians key: cord-351490-2fx0w30u authors: russell, clark d.; baillie, j. kenneth title: treatable traits and therapeutic targets: goals for systems biology in infectious disease date: 2017-04-27 journal: curr opin syst biol doi: 10.1016/j.coisb.2017.04.003 sha: doc_id: 351490 cord_uid: 2fx0w30u among the many medical applications of systems biology, we contend that infectious disease is one of the most important and tractable targets. we take the view that the complexity of the immune system is an inevitable consequence of its evolution, and this complexity has frustrated reductionist efforts to develop host-directed therapies for infection. however, since hosts vary widely in susceptibility and tolerance to infection, host-directed therapies are likely to be effective, by altering the biology of a susceptible host to induce a response more similar to a host who survives. such therapies should exert minimal selection pressure on organisms, thus greatly decreasing the probability of pathogen resistance developing. a systems medicine approach to infection has the potential to provide new solutions to old problems: to identify host traits that are potentially amenable to therapeutic intervention, and the host immune factors that could be targeted by host-directed therapies. furthermore, undiscovered sub-groups with different responses to treatment are almost certain to exist among patients presenting with life-threatening infection, since this population is markedly clinically heterogeneous. a major driving force behind high-throughput clinical phenotyping studies is the aspiration that these subgroups, hitherto opaque to observation, may be observed in the data generated by new technologies. subgroups of patients are unlikely to be static – serial clinical and biological phenotyping may reveal different trajectories through the pathophysiology of disease, in which different therapeutic approaches are required. we suggest there are two major goals for systems biology in infection medicine: (1) to identify subgroups of patients that share treatable features; and, (2) to integrate high-throughput data from clinical and in vitro sources in order to predict tractable therapeutic targets with the potential to alter disease trajectories for individual patients. infection is the largest single cause of death in humans worldwide and many infectious agents provide relevant in vitro model systems that are both amenable to study with high-throughput techniques, and recapitulate key events in disease pathogenesis. in this review, we consider how systems biology approaches may be leveraged to address the major unmet needs in infection medicine in the 21st century, with the aim of improving outcomes for patients with infection. in clinical practice we are unable to therapeutically modulate the host immune response to infection, largely due to its inevitable complexity. despite this, we contend that host-directed therapies have a high probability of success, since there is already considerable innate variation in host responses to infectious disease, ranging from extreme susceptibility, to complete resistance, and tolerance. infectious diseases are survivable if you have the right genetics. the challenge is to make the same diseases survivable for patients who would otherwise succumb. a systems medicine approach to infection has the potential to combine and integrate relevant signals from clinical, genomic, transcriptomic, proteomic and pathogen biology data to draw inferences about disease pathogenesis. below we discuss examples of aspects of this approach applied to various infectious diseases, and suggest future goals for the application of systems biology to infection medicine. more than 70 years after the discovery of penicillin [1] , this same drug is still a prominent weapon in our antibacterial armamentarium. more broadly, the concept underlying this therapeutic approach -attempting to eradicate the pathogen from a patient's body using antimicrobial drugs -remains the only effective treatment. although spectacularly successful, the focus on the pathogen has two limitations. firstly, death frequently occurs in infectious disease despite effective antimicrobial therapy. alongside the direct effects of microbial virulence factors, tissue damage is also caused by the host immune response. immune-mediated damage leading to respiratory, cardiovascular and renal failure (sepsis) continues even after eradication of the pathogen [2] . at present, no treatments exist to modify these deleterious aspects of the host immune response. secondly, antimicrobial resistance threatens to liberate pathogens from the range of our solitary weapon against them. unless something changes, deaths from infection are predicted to soar, overtaking malignant disease even in developed countries by 2050 [3] . therapies to modulate the host response to infection would have the theoretical advantage that, in addition to promoting survival in the presence of effective antimicrobials, a host-targeted therapy may exert a less powerful selection pressure on pathogens, and may be more difficult for a pathogen to evolve to overcome. in our view, the development of such therapies is wellsuited to the application of systems approaches. inevitable complexity of the immune system the human immune system is arguably the most complicated organ system in the body, encompassing numerous effectors, inter-related feedback loops and extensive redundancy. this complexity is unsurprising when considering that our immune system has evolved in the face of microbial virulence factors that directly interfere with regulatory and effector mechanisms. examples of microbial interference with host immune mechanisms are numerous and diverse. for example, one of the first innate immune mechanisms encountered by many pathogens is phagocytosis, which serves to both prime the adaptive response and eliminate invading pathogens by intracellular killing. pathogenic yersinia species, a group of facultative intracellular pathogens, encode a type three secretion system to directly inject effector proteins into the host cell cytoplasm, modulating the cytoskeleton to prevent phagocytosis, and inducing apoptosis of immune cells and blocking the mapk and nf-kb pathways to reduce cytokine production [4] . another bacterium, pseudomonas aeruginosa, secretes a protease that cleaves a host protein (corticosteroid-binding globulin) to release the corticosteroid hormone cortisol at the site of initial infection, incapacitating the local innate immune response [5] . even the relatively tiny genome of the influenza a virus encodes a protein (ns1) which is nonessential for replication and seems to be dedicated to interfering with both the induction and action of the host antiviral interferon response by sequestering viral dsrna, preventing activation of rig-1 signalling and inhibiting protein kinase r and oas/rnase l [6] . the adaptive response, mediated by t and b lymphocytes, is also a target. the human immunodeficiency virus encodes three proteins that each down-regulate cell surface mhc-1 expression by distinct mechanisms, preventing mhc-i signalling to activate the cytotoxic t-cell response to virus-infected cells [7] . to prevent b-cells mounting an antibody response to infection, the staphylococcus aureus surface protein a binds to the fc-g portion of antibodies [8] . these examples cover a few of the mechanisms pathogens have evolved to extensively interfere with the host immune response. the animal innate immune system is thought to have evolved over 1000 million years, starting with amebae able to phagocytose external material for nutrition [9] . the adaptive immune system in mammals is thought to have arisen 500 million years ago in fish [10] . since these initial events, the immune system in each host species has participated in a genetic arms race, evolving alongside relentless exposure to these microbial immune interference strategies from innumerable pathogens. furthermore, the immune system must successfully distinguish self from non-self antigens, with deleterious consequences arising (i.e. autoimmune disease) when this fails. the requirement to overcome these microbial immune interference strategies whilst preserving the recognition of self-antigens has provided the necessary pressure to drive the human immune system to evolve into a hugely complex organ system. in the context of this inevitable complexity, it is no surprise that reductionist approaches to development of hosttargeted therapies in infectious disease have largely a subgroup within a population of patients who are distinguished by a shared disease process. treatable trait the pathophysiological feature (or, in a looser sense, a biomarker or group of biomarkers for that feature) that determines whether a given therapy will improve a given patient's outcome. the same trait may be present in many different clinical syndromes or disease processes. it is reasonable to expect that as-yet undiscovered therapeutic interventions could alter the host immune response to promote survival. we infer this from the simple fact that some hosts do better than others when confronted with the same pathogen: the host response to infection is, in all cases that we know to have been studied, heterogeneous. furthermore, much of this variation is heritable [11] . we conclude that host factors exist that promote survival from specific infections, and that these must vary between individuals, and hence that it should be possible to identify and utilise these factors therapeutically to alter the biology of a susceptible host to induce a response more similar to a host who survives. the scale of the challenge of finding these targets is such that it is hard to imagine a solution being found without the power of systems approaches [12] . conceptually, this could involve promoting resistance to (suppressing pathogen replication), or tolerance of (preventing damage associated with immune response to pathogen), an infecting pathogen [13] . although theoretically attractive as a tool to limit damage to the host [14] , inducing tolerance is not without potential dangers: a sustained high pathogen load could facilitate transmission (iatrogenic super-shedders) and, perhaps more worryingly in emerging zoonotic infections, provide the time required for the selection of mutants with better host adaptation. due to their reliable and broad acting anti-inflammatory effects, corticosteroids represent an intuitively attractive strategy to reduce inflammation during infection. indeed, a survival benefit has been demonstrated in a small number of uncommon infections (bacterial meningitis, tuberculous meningitis and pericarditis, hypoxaemic pneumocystis jiroveci pneumonia) [15] . in contrast, there is uncertainty over safety and benefit in other infections (e.g. rsv bronchiolitis) and clear evidence of harm in others (viral hepatitis, cerebral malaria, influenza virus, sars coronavirus, hiv-associated cryptococcal meningitis) [15e19]. a more specific host-directed therapy, recombinant human activated protein c (rhapc), was licensed for treatment of severe sepsis based on the results of a single clinical trial [20] . rhapc has anti-inflammatory and anti-thrombotic properties, and circulating levels are low in patients with sepsis [21] . sadly, a subsequent trial in an overlapping patient group showed a trend towards increased mortality [22] . a third trial, mandated by the regulatory authorities, did not detect any mortality benefit, and the drug was quickly withdrawn from the market [23] . these negative trial results in sepsis are not necessarily conclusive. the heterogeneity of the host response, and the diverse range of microbes involved, means that amongst all patients with sepsis (itself an unrealistically broad syndrome) there are likely to be numerous biologically-different sub-groups, with distinct immune responses and more importantly, different risk: benefit balance for a given therapy. corticosteroids, for example, may save some patients but harm others. our current understanding of infection does not allow clinical differentiation of these sub-groups, so potentially beneficial therapies may be falsely rejected in clinical trials. we believe that a major output of systems approaches to infection will be the elucidation of previously hidden therapeutically-important subgroups of patients who share a 'treatable trait' (i.e. response to therapy). we have discussed two central problems in infection medicine. firstly, the development of therapies to modulate the host immune response is impeded by the inevitable complexity of the human immune system. secondly, the range and degree of characterisation of clinical syndromes in medicine is constrained by the range of observations that are available; we likely fail to identify sub-groups of patients with treatable traits due to a paucity of relevant observations. the promise of systems approaches to infection medicine is that new technologies may provide solutions to these old problems. large-scale biological data-sets from an increasing range of high-throughput technologies are becoming available, including (but not limited to) high-resolution transcriptional profiling [24] , mass spectrometry-based proteomics [25] and metabolomics, genome-scale crispr-cas9 knockout screening [26] , and whole genome sequencing/genome-wide association studies. these modalities could provide new, biologically important observations that have not previously been observed in patients. it is very likely that some of these observations will be directly relevant to clinical management e the challenge will be to identify the ones that matter. there are three broad categories of clinical utility for these new data sources in infection medicine: identifying treatable traits and therapeuticallyrelevant subgroups; identifying new therapeutic targets in the immune response; and, improving prognostication. the value of prognostication in current clinical practice is, in our view, limited by the range of therapeutic options. put simply, it is only useful to predict the future if you have some capacity to change it. particularly in the case of acute and immediately lifethreatening infection, our range of therapeutic options is very limited, regardless of the degree of certainty attached to the prognosis, therefore we suggest the first two applications should be prioritised. in contrast, the identification of syndromes (collections of clinical observations that tend to occur in patients suffering the same disease) has been the primary mechanism for progress in the understanding of human disease since long before the time of hippocrates [27] . finding a syndrome is the first step towards identifying the common biological processes that define a disease, and ultimately to identifying effective treatments. we hope that the application of systems technologies will help us define treatable traits in infection, thus providing a starting point for new therapeutic interventions ( figure 1 ). importantly, a systems model of the disease process need not predict every transcript and metabolite in the massively multi-dimensional datasets generated by new technologies. the primary challenge is instead to identify those components of inter-host variation that are amenable to intervention; the evidence of disease processes that we can change. this is agusti's concept of "treatable traits" [28] , a term coined in the field of chronic obstructive lung disease but no less relevant here. a computational model of infection could therefore include not only traditional measures of pathogen burden and evidence of systemic injury [29] , but also independent components of the immune response or the metabolic consequences, detected using highthroughput technologies. the behaviour of the whole system may be impenetrably complex, but the components required to predict the effect of an intervention may be far simpler. the trajectory followed by each patient along an informative set of vectors may reveal different groups of patients that appear clinically similar, and may even have similar outcomes, but different disease processes ( figure 2 ). by mapping the "flight path" of each patient through disease, we anticipate that summary of a systems medicine approach to infection. a wide range of data sources can be combined using various methods (see text) to achieve two fundamental goalsclinically-informative phenotyping of patients, and identification of therapeutic targets. important similarities and differences in immune response will become apparent [29] . our ability to identify groups of patients sharing therapeutically-relevant similarities is dependent on measuring the relevant biological signal that determines classification. this is the fundamental attraction of high-throughput technologies e the probability of measuring important signals is greater if more signals are measured. that such groupings of patients, or disease endotypes, exist is already clear: therapeuticallyimportant sub-classifications have recently been discovered that redefine the clinical syndromes of asthma [30, 31] , ards [32] , and acute mountain sickness [33] . in two related autoimmune conditions, anca-associated vasculitis (aav) and systemic lupus erythematosus (sle), a t-cell gene expression signature clearly delineates two distinct endotypes [34] . subsequent work elucidated the immunological process underlying this sub-classification, cd8 t-cell exhaustion [35] . this process is associated with better outcomes in autoimmune disease, but poor clearance of viral infection. in the future it may be possible to manipulate this pathway therapeutically in patients with aav or sle, to prevent relapse, or in the opposite direction in patients with chronic viral infection, to promote clearance. even in the best-case scenario, the distance between the identification of a tractable therapeutic target and successful exploitation in clinical practice is substantial, so it is no surprise that potential host-directed therapies discovered through high-dimensionality analytics have not yet been proven to be effective in clinical trials. nonetheless there are some promising leads, a few of which are described here. these exemplify different approaches: integrating cell culture, animal and human data sequentially to identify a host anti-viral factor (influenza virus); and using computational predictions from transcriptional data with proteomic and genetically modified animal studies to identify host pathways involved in pathogenesis (sars coronavirus). viruses are obligate parasites and undergo exclusively intracellular replication; properties that make them ideal for study in cell culture where host factors that affect viral replication can be expected to do the same in vivo. sirna screening has been used as a genomewide approach to identify such host factors for influenza virus infection. ifitm3 was identified as a novel host anti-viral factor by sirna screening with confirmatory in vitro work (including exogenous interferon administration and stable ifitm3 expression) demonstrating it is required for an effective interferon response to inhibit influenza virus replication [36] . work in ifitm3e/e mice was then undertaken, confirming that in vivo, influenza virus-infected mice suffer fatal viral pneumonia, even when infected with a low-pathogenicity virus [37] . the genisis/mosaic groups identified a single nucleotide polymorphism hypothetical trajectories of two groups of patients through multidimensional space. each line indicates the path taken by a single patient, with periods of organ failure highlighted in red. a superficially similar group of patients may appear clinically indistinguishable (a), but different trajectories through illness are revealed by informative vectors derived from high-throughput data (b). it is reasonable to expect that such biological differences in disease process will underlie different responses to host-directed therapies. (rs12252-c) within the ifitm3 coding region in humans that was strongly over-represented in patients hospitalised with influenza virus infection [37] , the majority of whom required invasive ventilation. metaanalysis of genomic studies of ifitm3 and influenza virus infection has confirmed this association between snp rs12252-c and increased susceptibility to infection in humans [38] . work is now underway in many groups to investigate the impact of ifitm proteins in antiviral defence, with a view to generating hosttargeted antiviral therapies. this example highlights the potential of a systems-wide approach, integrating and cross-validating results from various experimental modalities (high-throughput screening in cell culture, followed by targeted studies in mice and humans) to converge from large-scale data onto a single critical host immune factor. acute lung injury (ali) and progression to acute respiratory distress syndrome are major features of the pathophysiology of sars coronavirus (and indeed other respiratory virus) infection. pathological changes in mice are very similar to humans and there is a dose-response relationship between viral inoculum and the severity of ali. host responses to the virus that result in ali are poorly understood. to investigate host responses associated with more severe acute lung injury in a murine model of sars coronavirus (sars-cov) infection, transcriptomic profiles of mice infected with lethal and sub-lethal doses of virus were compared and correlated with pathological data at multiple time points, then subject to bioinformatic network analysis [39] . a module of genes involved in cell adhesion, extracellular matrix remodelling and wound healing was significantly up-regulated in the lethal infection model, and components of the urokinase pathway were found to be the most enriched and differentially regulated. massspectrometry proteomic analysis was then used to explore this transcriptional data further, demonstrating that sars-cov infection resulted in increased expression of fibrin b and g chains, factor viii and cytokeratins (all components of hyaline membranes), and reduced expression of surfactant proteins in the lung. these data are consistent with histological post-mortem findings in sars-cov infected humans, where extensive fibrin exudate, extensive hyaline membrane formation and alveolar collapse have been observed [40] . serpine1 is part of the urokinase pathway and contributes to ecm remodelling. to confirm this finding from transcriptional and proteomic studies, serpine1e/e knockout mice were infected with sars-cov and found to have a worse outcome compared to wild type mice. this systemsbased approach to sars-cov, sequentially applying transcriptional, proteomic then genetic modification techniques, demonstrates that the urokinase pathway contributes to ali, thus identifying a target for future experimental medicine work to determine if it can be therapeutically altered to benefit the host. these examples give us confidence that we can expect more progress along similar lines as the potential of systems medicine approaches becomes more widely appreciated, as expertise in computational methodologies grows, and as the cost of generating relevant data falls. physicians have long made progress by recognising patterns of similar observations in groups of patients, and by determining which biological features of disease are amenable to therapy. what has changed is the unprecedented rate of advance in new resources and tools with which to tackle the ancient problems of diagnosis and therapy in infectious disease. our responsibility is to ensure a similar acceleration in clinical progress. . sorensen ti, nielsen gg, andersen pk, teasdale tw: genetic and environmental influences on premature death in adult adoptees. n engl j med 1988, 318:727-732. this prospective cohort study of adoptees demonstrated that genetic background contributes more to the relative risk of death due to infection (i.e. biologic parent also died of infection) than for death due to cancer, vascular disease or natural causes, thus affirming that susceptibility to infection is a strongly heritable trait. papers of particular interest, published within the period of review, have been highlighted as: of special interest of outstanding interest first clinical use of penicillin severe sepsis and septic shock review on antimicrobial resistance. tackling drug-resistant infections globally: final report and recommendations the yersinia ysc-yop 'type iii' weaponry pseudomonas aeruginosa elastase disrupts the cortisol-binding activity of corticosteroidbinding globulin the multifunctional ns1 protein of influenza a viruses mechanisms of hiv-1 to escape from the host immune surveillance role of protein a in the evasion of host adaptive immune responses by staphylococcus aureus evolution of innate immunity: clues from invertebrates via fish to mammals origin and evolution of the adaptive immune system: genetic events and selective pressures disentangling genetic variation for resistance and tolerance to infectious diseases in animals this study utilises a murine malaria model to demonstrate that resistance to and tolerance of infection represent genetically distinct host defence mechanisms disease tolerance as a defense strategy use of corticosteroids in treating infectious diseases adjunct prednisone therapy for patients with communityacquired pneumonia: a multicentre, double-blind, randomised, placebo-controlled trial adjunctive dexamethasone in hiv-associated cryptococcal meningitis corticosteroids as adjunctive therapy in the treatment of influenza sars: systematic review of treatment effects efficacy and safety of recombinant human activated protein c for severe sepsis role of activated protein c in the pathophysiology of severe sepsis drotrecogin alfa (activated) in severe sepsis drotrecogin alfa (activated) in adults with septic shock gateways to the fantom5 promoter level mammalian expression atlas mass spectrometry-based proteomics genome-scale crispr-cas9 knockout screening in human cells genome editing using the crispr-cas9 system can be applied to genome-wide knockout screening to identify genes with an important phenotype in an in vitro model, as exemplified in this study of genes required for cell viability in cancer and genes implicated in susceptibility to a therapeutic raf inhibitor used in melanoma hippocrates on ancient medicine phenotypes and disease characterization in chronic obstructive pulmonary disease. toward the extinction of phenotypes? health trajectories reveal the dynamic contributions of host genetic resistance and tolerance to infection outcome multidimensional endotypes of asthma: topological data analysis of crosssectional clinical, pathological, and immunological data asthma endotypes: a new approach to classification of disease entities within the asthma syndrome subphenotypes in acute respiratory distress syndrome: latent class analysis of data from two randomised controlled trials network analysis reveals distinct clinical syndromes underlying acute mountain sickness a systematic network analysis (using software originally designed for analysing microarray expression data) of symptoms associated with acute mountain sickness revealed three distinct sub-groups within the syndrome, resulting in a revision of international diagnostic criteria for a cd8+ t cell transcription signature predicts prognosis in autoimmune disease transcriptional profiling identified two distinct sub-groups (related to t-cell survival and memory) which could then be differentiated on the basis of only three genes, providing a useful biomarker for clinical trials of immunosuppressant therapies and focussing further studies on these pathways t-cell exhaustion, co-stimulation and clinical outcome in autoimmunity and infection the ifitm proteins mediate cellular resistance to influenza a h1n1 virus, west nile virus, and dengue virus ifitm3 restricts the morbidity and mortality associated with influenza data was integrated from cell culture work, a murine infection model, human genotyping and patient clinical variables to validate ifitm3 as a critical host defence against influenza virus infection and thus a tractable therapeutic target interferon-inducible transmembrane protein 3 genetic variant rs12252 and influenza susceptibility and severity: a meta-analysis mechanisms of severe acute respiratory syndrome coronavirus-induced acute lung injury multiple organ infection and the pathogenesis of sars key: cord-318119-h0vnfcuq authors: lee, s. h.; lee, w. l. title: site verification and modeling of desiccant-based system as an alternative to conventional air-conditioning systems for wet markets date: 2013-06-15 journal: energy doi: 10.1016/j.energy.2013.04.029 sha: doc_id: 318119 cord_uid: h0vnfcuq abstract desiccant cooling system for active humidity control and to conserve energy has been in commercial applications for over two decades. however, its use in humid wet markets has never been examined. a gas-fired desiccant cooling system has been installed in a wet market in hong kong. in this study, the annual energy saving in conjunction with the use of desiccant cooling system was investigated by in-situ measurements, site surveys and simulations. the verified computer model was used for further simulations. it was found that for the use of a minimum ventilation rate of 10.3 l/s/person, the use of desiccant cooling system as compared to conventional system saved 4% of the energy and could achieve the desired space conditions. a parametric study under various ventilation rates indicated that use of desiccant cooling system in wet markets in hot and humid hong kong would lead to energy and energy cost savings, as well as co2 emission reduction amounting from 1% to 13%. the savings were more evident when wet markets were designed for a ventilation rate of 20 l/s/person. furthermore, the actual occupancy profile, and lighting and small power densities determined in this study would be useful for future studies on wet market. in most commercial and residential applications, the space relative humidity is not controlled because conventional air-conditioning systems rely on operating the cold coil at very low effective temperatures to enable the removal of moisture by condensation. as such, the desired space relative humidity can only be achieved by matching the equipment (sensible heat ratio) shr with the building shr, but a perfect match rarely happens. this divergence leads to excessive cooling which increases energy use, and moisture problems which accelerate microbial growth, particularly for subtropical climates like hong kong with very humid outdoor air. in solving the excessive cooling and moisture problems, previous studies [1e3] advocate decoupling of dehumidification from cooling for better humidity control and energy performance. desiccant cooling system, which can actively control the humidity level, is often used for achieving the dehumidification objective. recent studies on desiccant cooling system have been done on optimizing the operating variables for better energy performance [4e7]; developing empirical model for investigation of the system performance [8] ; improving the control and operation by experimental studies [9, 10] ; and widening the application to different types of buildings [11e14] . however, it is noted that they are either restricted to applications where ventilation requirements are set to remove heat generated by people, lighting and equipment, or where moisture removal is not a design intent [13, 14] . as for applications where ventilation requirements are set for moisture and contaminates removal, little has been found in extant literature. the recent occurrence of sars (severe acute respiratory syndrome), avian flu and anthracnose in some countries makes environmental health one of the most important elements for an airconditioning system [15] . in achieving the environmental health objective, ventilation requirement for some premises is not just for heat removal, but for moisture and contaminant removal. the wetmarket in hong kong is one typical example. modernized wet markets in hong kong are housed in airconditioned buildings. they traditionally sell fresh meat, fruits and vegetables, and live animals. live animals include fish, shellfish, and poultry. because water is used to wash the floors, keep the fruits and vegetables fresh, and keep animals alive, wet markets are an extremely damp place. for moisture and contaminant removal, wet markets often require very high ventilation rate and this, combined with the humid outdoor air, increases the latent load and thus a huge amount of energy will be consumed if conventional air conditioning system is used. a gas-fired desiccant cooling system has been installed in a wet market in hong kong which may probably be the first installation of its kind in the world. the actual operating data of the pilot installation will be used to validate simulation predictions. upon successful validation, further simulations will be conducted to evaluate the overall performance of desiccant cooling system as compared to a conventional air-conditioning system. given designers are free to design the ventilation rates they deem proper for wet markets, the influence of ventilation rates on the energy and energy cost savings, as well as co 2 emission reduction of desiccant cooling system will further be investigated. desiccant dehumidification differs from conventional airconditioning system in terms of the thermodynamics of the process air. instead of cooling the air below its dew point to condense its moisture, desiccants attract moisture from the air. desiccant has a vapor pressure lower than the air in its active state. this vapor pressure differential drives the water vapor from air onto the desiccant [16] . figs. 1 and 2 show the psychrometric processes of the two systems. a conventional system typically admits (outdoor air) oa at state 0 to mix with return air at state r to become mixed air (state 1). the mixed air will subsequently be cooled to state 2 followed by a reheating process to become supply air (state 3) for achieving the desired space temperature and relative humidity (state r). for the removal of moisture by condensation, state 2 is often conditioned to a very low temperature (10e12 c dry-bulb) and is close to saturation (90e95% relative humidity). in a desiccant cooling system, the latent cooling is performed by desiccant dehumidification whilst the sensible cooling is treated by a cold-coil air handler. the oa at state 0 is first brought into the gas-fired desiccant dehumidifier and is dehumidified adiabatically to state 1. the dried and hot oa is then pre-cooled by the exhaust air at state r through an air-to-air heat exchanger to state 2. the pre-cooled oa is subsequently mixed with the return air to state 3 followed by a cooling process at the cold-coil air-handler (state 4) to become supply air conditions for achieving the desired space conditions (state r). the exhaust air leaving the air-to-air heat exchanger at state 5 is heated up at the gas burner to become hot air (state 6) for regeneration of the desiccant dehumidifier. warm and humid air at state 7 will finally be exhausted to the environment. in the regeneration process of the desiccant dehumidifier, the desiccant wheel rotates slowly into the stream of gas-fired hot air (state 6), which raises the vapor pressure of the desiccant above that of the surrounding air. with the vapor pressure differential reversed, water vapor moves from the desiccant to the regeneration air, which carries the moisture away from the system. the schematic of the air flow of the desiccant cooling system is shown in fig. 3 . this study evaluates the overall performance of a gas desiccant cooling system as compared to a conventional air-conditioning for application in a case study wet market. the influence of ventilation requirements on the energy and energy cost savings, as well as co 2 emission reduction of desiccant cooling system will also be evaluated. evaluations were based on in-situ measurements, site surveys and energy simulations. in-situ measurements results were retrieved from the remote monitoring system. the retrieved results include the space air conditions and the year round energy use derived from fans (process and regeneration), chilled water circulating pumps, chillers and burners of the gas-fired dehumidifiers. given key parameters like occupancy, and installed lighting and small power densities are not available; site surveys have been conducted to ascertain these parameters. the in-situ measurements and site surveys results were used for simulation validation to facilitate further simulations. a simulation program, energyplus was adopted to simulate the energy use of the major equipments of the desiccant cooling system for energy analysis. a hypothetical wet market (not the studied wet market) was designed to represent typical modernized wet markets in hong kong to facilitate the evaluations. it was formulated after an the case study market is housed on the ground floor of a 5storey carport in a public housing estate. car parking spaces are provided from 1/f to 4/f with a playground on the roof. the total floor area of the market is 2380 m 2 (53.0 m â 44.9 m). the headroom is 3.5 m high. it is a typical wet market in hong kong which sells all sorts of fresh produce and live seafood and poultry. the desiccant cooling system installed in this market consists of two parallel systems: a dehumidification system comprises of three units of gas-fired desiccant dehumidifiers installed on the roof dedicated to dehumidify oa, and a chilled water system. the chilled water system is not different from a conventional air-conditioning system which consists of two chillers each of 915 kw cooling capacity and the associated circulation pumps. the capacity is sufficient to cover the sensible and latent loads of the three air-handlers for treatment of mixed air with and without the operation of the desiccant dehumidifiers. this serves as a standby provision to ensure relative humidity of the wet market can always be met. to satisfy the minimum ventilation requirement for non-domestic buildings in hong kong [17] , the market is designed for a ventilation rate of 5 air change per hour (ach). the building, design and system characteristics of the case study wet market are summarized in table 1 . in-situ measurements and site surveys, with details as discussed below, were adopted to collect data for simulation validation. a remote monitoring system is deployed to monitor the performance of the desiccant cooling system. it can relay commands signals to carry out remote control of the dehumidifiers. the operation of the system can be viewed either locally on site or in the remote office through the internet. the records (air states in hypothesis are with reference to fig. 2) can be retrieved by this system which includes space air temperature and relative humidity (state r), dried and hot outdoor air temperature and relative humidity (state 1), hourly operation schedule and energy use of major equipments. the monitoring system also enables the set points of relative humidity to be adjusted actively in order to achieve individual needs at a different zone. the system configuration is shown in fig. 4 . the indoor and outdoor air conditions and the energy use of major equipments were retrieved from the remote monitoring system for comparison with simulation results. exemplary results in one summer period are shown in fig. 5 . the average indoor temperature and rh were 25.2 c and 65.8% respectively. in the site surveys, the wet market was divided into three zones. each zone was assigned with a responsible person to record to the number of customers as well as stall retailers in every 60 min intervals, and to ascertain the installed lighting and small power densities. occupancy flow measurements were taken on three consecutive days to cover weekdays and weekends during which the occupancy levels were recorded from 7:00 to 19:00. the hourly average values are summarized in table 2 . the (installed lighting power density) ilpd was ascertained by counting the numbers and types of luminaires installed in the market. there were altogether four types of luminaires including 60 w florescent lamps, 48 w fluorescent lamps, 400 w spot lights and 200 w light bulbs. the surveyed ilpd is summarized in table 3 . the (installed small power density) ispd was determined by a walk-through survey on the power ratings of the fixed and portable appliances including refrigerators, boilers, cooking devices, and ventilation fans. the surveyed ispd is also summarized in table 3 . energy simulation based on the site survey results (including occupancy, lighting and small power densities and occupation patterns), average space air-conditions as indoor set-point conditions (25.2 c dry-bulb and 65.8% rh), building envelop details (table 1) , and physical geometry of the wet-market were input to energyplus for simulating the annual energy use of the major equipments. the power requirement of the chiller was calculated based on the doe-2 model. the doe-2 model is an empirical model and its mathematical expressions are shown in equations (1)e(3), in which capft is the cooling capacity factor; eirft is the energy input to cooling output factor; and eirfplr is the energy input to cooling output factor at part load condition [18e20]. where t cw,s is the supply chilled water temperature; t a,e is the condenser air entering temperature, which is the hourly weather conditions of hong kong in 1995 (the typical meteorological year for hong kong [21] ); plr refers to the chiller's part load ratio; a 1 to f 1 , a 2 to f 2 and a 3 to c 3 are the default coefficients in energyplus. capft, eirft and eirfplr equal to 1 (dimensionless) at reference condition. chiller capacity control is to maintain the designed t cw,s (¼7 c) at constant flow condition. the energy consumption of the desiccant dehumidifier was simulated based on a default performance model in energyplus. it is a generic model for determining the leaving dry bulb temperature (t l , c) and humidity ratio (w l , kg/kg dry air) of the process air and the specific regeneration energy (e r, j/kg h 2 o) and regeneration velocity (v r , m/s) of the desiccant wheel shown mathematically in equation (4). where x is the modeled parameter (i.e. t l , w l , e r and v r ); t e and w e are the process air entering dry bulb temperature ( c) and humidity ratio (kg/kg dry air), which follow the hourly weather conditions of hong kong in 1995 (the typical meteorological year for hong kong [21] ); v is the process air velocity (m/s); and c1 to c16 are the default coefficients in energyplus which correspond to the parameter modeled. equation (4) is valid for the following range of process inlet conditions: dry-bulb temperatures of 1.67 ce48.9 c and humidity ratios of 0.002857 kg/kg to 0.02857 kg/kg. the output of desiccant dehumidifier is to maintain the leaving process air staying at the designed outlet conditions, which in this analysis is moisture content below 0.004 kg/kg dry air. in the simulations, a perfect control and feedback system was assumed such that the output of the equipments would match perfectly with the system cooling demand. table 4 compares the actual and simulated energy use results, illustrating that the difference between the two sets of results is between à3.3% and þ5.8%. the difference is considered acceptable considering that the best achievable uncertainty in the measured energy use as estimated based on the kline and mcclintock's equation [22, 23] is ae0.6% (assuming all instruments conform to ashrae standard 114-1986). the comparison of energy breakdown of major equipments in table 5 also shows that their difference is ae2%. to check consistency of the two sets of energy breakdown, bivariate correlation was conducted. it was found that the correlation coefficient was 0.996 to indicate correlation was significant at the 0.05 level (2-tailed). to compare the actual and simulated indoor conditions, their standard errors with the set-point conditions in one summer week period (1st to 7th july) are compared in table 6 . their standard errors are shown to be comparable, illustrating the simulated results agree well with the actual conditions. given the acceptable range of the difference and the fundamental consistency in the two sets of data, validations are regarded as acceptable to conclude that it is reasonable to adopt simulations for further investigations. further simulations were conducted to compare the energy performance of desiccant cooling system with the conventional system. in this comparison study, the actual occupancy (table 2) , the measured lighting and small power densities (table 3) , and the installed equipment characteristics of the studied wet market were used in the simulation, but not the space air conditions and ventilation rate for which the ashrae recommendations could not be fulfilled [24] . according to ashrae [25] , the indoor air condition in summer should have a wet bulb temperature below 20 c and dry bulb temperature between 22.5 c and 27 c. thus for the actual space air temperature of 25.2 c, the corresponding relative humidity should be set at 50%. they were therefore set as indoor set-point conditions in the simulation. whilst for ventilation rate, although there has been no guideline for wet markets, it is noted that the actual rate of 5 ach (corresponds to approximately 3.3 l/s/person) is far lower than the ashrae recommended rate for supermarkets of 7.6 l/s/person [24] . given wet markets are much more humid than supermarkets, to avoid the possible microbial growth, ashrae's highest recommended rate for the same air class, i.e. 10.3 l/s/person, has been adopted in the simulation. with regards to the equipment characteristics, the desiccant cooling system was assumed provided with the actual installed equipments ( table 1 ), only that our pilot simulation indicated that table 3 installed lighting power density (ilpd) and installed small power density (ispd) in the wet market. with lowering the space relative humidity, the moisture removal rate of the desiccant dehumidifier was too small and thus an electric heater was added to increase the temperature of the regeneration air for supplementing the dehumidification requirements. electric heater was chosen for their common use in buildings in hong kong [26] . the electric heater capacity was 173 kw determined using energyplus auto size option. the conventional air-conditioning system has been assumed identical to the installed chilled water system (table 1) . however, to enable a side-by-side comparison of the energy performance of the two systems, despite the fact that conventional air-conditioning systems control only the space air temperature, it has been assumed in the simulation that chilled water flow rate of the conventional system can be varied to cool and dehumidify the supply air to a level for achieving the desired space air temperature as well as relative humidity; whilst overcooling is assumed offset by electric reheating to maintain the space air temperature. the electric heater capacity was again determined using energyplus auto size option. through hour-by-hour simulations, the year-round space air temperatures and relative humidities achieved by the use of the desiccant cooling and conventional systems were found. the standard error between the simulated results and the set-point conditions for the two systems is summarized in table 7 . it can be seen that their standard errors are comparable to confirm that the two systems have been side-by-side compared based on same space air conditions. energy use of the conventional and desiccant cooling systems for achieving the same space air conditions is compared in fig. 6 , in which gas energy is used for the burner and the other equipments are powered by electricity. the desiccant cooling system is shown to be 4% more energy efficient than the conventional system. note that this is on the basis that gas is used for regeneration of the desiccant dehumidifier. should "free" thermal energy (for example, waste heat from co-generators or solar energy) is available; the total saving can be increased up to 22% which is comparable to results of a similar installation for supermarket applications [14] . further benefits can also be achieved for the difference in energy cost and (effective emission factor) eef between electricity generation and gas burning. the associated energy cost and environmental benefits will be discussed in the later sections. the consumption breakdowns of major equipments of the two systems are compared in table 8 . bivariate correlation was again conducted. it was found that the correlation coefficient was 0.91 to indicate correlation was significant at the 0.05 level (2-tailed). the results indicate that chillers are the dominant energy consumer for the two systems, accounting for more than 40% of the total energy use. the evaluations confirm that the simulation results are reasonable, and small energy saving can be achieved by the use of desiccant cooling system. the parametric study is to enable the study of the influence of ventilation requirements on the energy performance, co 2 emission level, and energy cost saving of desiccant cooling system. to provide a basis for the parametric study, a hypothetical wet market representing typical wet markets in hong kong was formulated. a survey of physical characteristics of 19 modernized wet markets in hong kong was conducted. it was found that their areas were ranging from 800 m 2 to 18,650 m 2 , with a mean of 1440 m 2 ; the aspect ratios (ratio of width to length) were between 0.4 and 0.8, with a mean of 0.625; and the headrooms were between 3 m and 4.5 m, with a mean of 4 m. considering the collected data are of equal importance and should be given equal weight, mean values were used to set physical dimensions (w â l â h) of the hypothetical wet market which is 30 m â 48 m â 4 m. given it is common to see humid environment is provided with 100% outdoor air for control of airborne infectious diseases [27] , the range of variations has been set between 10 l/s/person at 10 l/s intervals and complete outdoor air flow rate corresponding to 58 l/s/person. in the parametric study, the equipment capacities were determined using energyplus auto size option; only that the rated performance of major equipments including chillers, fans, pumps, burners and desiccant dehumidifiers were assumed identical to those installed at the studied wet market. fig. 7 indicates the energy use comparison between the two systems under various ventilation rate conditions. it is noted that the energy use of both systems is comparable when the ventilation rate is at 10 l/s/person which is consistent with the comparison study result in the earlier section. this again confirms the reliability of the parametric study results. parametric study results revealed that further increase in ventilation rate lead to a steady increase in energy use. however, the rate of rise for the conventional system was higher than that of the desiccant cooling system when the ventilation rate was increased from 10 l/s/person to 20 l/s/person. the results indicate that at a ventilation rate of 20 l/s per person, energy saving of 8% can be achieved by the use of desiccant cooling system. the significant saving can be explained by the fact that the increase in ventilation rate can offset part of the space cooling load in mild season, but further increase in ventilation rate leads to a sharp increase in space cooling load as shown in fig. 8 . based on the energy consumptions of the conventional (e cr , kwh) and desiccant cooling (e dc , kwh) systems; and the energy split between gas (e dc,gas ) and electricity (e dc,elc ) of desiccant cooling system, the percentage energy cost saving and emission reduction (p) can be predicted based on the use of equation (5) derived as follows: and: where c is the energy cost saving/co 2 emission reduction between the use of desiccant cooling system and conventional system. using equations (5) and (6) becomes: where: w ¼ the ratio of gas consumption to total energy consumption of according to clp and towngas, the electricity and town gas tariffs are $0.86/kwh and $0.6/kwh respectively, and thus (m) in equation (3) is 0.698 (¼0.6/0.86). fig. 9 compares the annual energy costs of the two systems. it is noted that there is larger energy cost saving as compared to energy saving for the desiccant cooling system because gas tariff is lower. the saving ranged between 4% and 10%, and the optimal saving, as expected, occurred when the ventilation rate was at 20 l/s/person. the environmental impact is assessed based on (carbon dioxide) co 2 saving because the amount of co 2 in the atmosphere accounts for about 50% of the global warming effect [28] . the (effective emission factor) eef for electricity generation in hong kong based upon the use of different fuels is estimated to be 0.615 kg/kwh, and that for gas is 0.238 kg/kwh [29] . to estimate the amount of co 2 saving, (m) in equation (3) is 0.387 (¼0.238/0.615). fig. 10 compares the annual co 2 emission level of the two systems. it is noted that similar to energy cost saving, there is larger emission reduction for the desiccant cooling system as compared to energy saving because the effective emission factor for gas is much lower. the reduction ranged between 6% and 13%, and again, the optimal reduction occurred when the ventilation rate was at 20 l/s/person. a desiccant cooling system was installed in a wet market in hong kong. in this study, the actual energy performance of the desiccant cooling system was evaluated. evaluations indicated that use of gas desiccant cooling system was an effective and flexible mean for providing active humidity control. further evaluations based on in-situ measurements, site survey and simulation studies were conducted. in-situ measurements provided the actual energy use data for comparison with simulated results and for simulation validation. further simulations based on actual system, equipment and operating characteristics of the studied wet market were conducted for prediction of energy benefits of desiccant cooling system as compared to conventional air-conditioning system, which was found to be 4%. to evaluate the influence of ventilation rates on the associated energy and energy cost savings as well as co 2 emission reduction of desiccant cooling system, a parametric study under various ventilation rates was performed. the parametric study was conducted based on a hypothetical wet market formulated to represent typical physical characteristics of wet markets in hong kong. the study results revealed that use of desiccant cooling system in wet markets in hot and humid hong kong would lead to energy, energy cost and co 2 emission reduction up to 13% when the ventilation rate is 20 l/s/person. the results of this study would be useful for establishing essential criteria for the use of supplementary energy source with respect to air-conditioning system design. hvac dehumidification systems for thermal comfort: a critical review energy and peak power savings potential of radiant cooling systems dehumidification equipment advances energy savings potential of chilled-ceiling combined with desiccant cooling in hot and humid climates performance improvement of a gas turbine cycle by using a desiccant-based evaporative cooling system experimental analysis on the dehumidification and thermal performance of a desiccant wheel desiccant wheel regenerated by thermal energy from a microcogenerator: experimental assessment of the performances experimental validation of constant efficiency models for the subsystems of an unconventional desiccant-based air handling unit and investigation of its performance parametric analysis of variables that affect the performance of a desiccant dehumidification system integration of microturbine with single effect exhaust driven absorption chiller and solid wheel desiccant system desiccant cooling for swedish office buildings the impact of desiccant dehumidification on classroom humidity levels modelling and experimental verification of a solarpowered liquid desiccant cooling system for greenhouse food production in hot climates hybrid hvac systems with chemical dehumidification for supermarket applications energy consumption analysis on a dedicated outdoor air system with rotary desiccant wheel hvac systems and equipment, chapter 22 desiccant dehumidification and pressure drying equipment practice note for authorized persons, registered structural engineers and registered geotechnical engineer pnap adm-2. buildings department. hong kong sar government an evaluation of empirically-based models for predicting energy performance of vapor-compression water chillers evaluation of the suitability of empirically-based models for predicting energy performance of centrifugal water chillers with variable chilled water flow energyplus energy simulation software. eere, us department of energy generation of a typical meteorological year for hong kong measurement systems application and design experimental methods for engineers ashrae standard 62. ventilation for acceptable indoor air quality guidelines on performance-based building energy code. electrical and mechanical services department control of airborne infectious diseases in ventilated spaces benchmarking the energy efficiency and greenhouse gases emissions of school buildings in central argentina greenhouse gas emissions in hong kong annual co 2 emission comparison under different ventilation rates the authors wish to thank the commercial & industrial marketing department of the hong kong & china gas company limited for their assistance in preparing this paper. key: cord-311868-40bri19f authors: fattahi, a.; sijm, j.; faaij, a. title: a systemic approach to analyze integrated energy system modeling tools: a review of national models date: 2020-11-30 journal: renewable and sustainable energy reviews doi: 10.1016/j.rser.2020.110195 sha: doc_id: 311868 cord_uid: 40bri19f we reviewed the literature focusing on nineteen integrated energy system models (esms) to: (i) identify the capabilities and shortcomings of current esms to analyze adequately the transition towards a low-carbon energy system; (ii) assess the performance of the selected models by means of the derived criteria, and (iii) discuss some potential solutions to address the esm gaps. this paper delivers three main outcomes. first, we identify key criteria for analyzing current esms and we describe seven current and future low-carbon energy system modeling challenges: the increasing need for flexibility, further electrification, emergence of new technologies, technological learning and efficiency improvements, decentralization, macroeconomic interactions, and the role of social behavior in the energy system transition. these criteria are then translated into required modeling capabilities such as the need for hourly temporal resolution, sectoral coupling technologies (e.g., p2x), technological learning, flexibility technologies, stakeholder behavior, cross border trade, and linking with macroeconomic models. second, a multi-criteria analysis (mca) is used as a framework to identify modeling gaps while clarifying high modeling capabilities of markal, times, remix, primes, and metis. third, to bridge major energy modeling gaps, two conceptual modeling suites are suggested, based on both optimization and simulation methodologies, in which the integrated esm is hard-linked with a regional model and an energy market model and soft-linked with a macroeconomic model. the long-term energy strategy of the eu is aimed at a 80-95% reduction of greenhouse gas (ghg) emissions by 2050, relative to 1990. reaching this goal requires a number of key actions to make a transition from a conventional energy system to a low-carbon energy system [1] . as a result, low-carbon energy system models (esms) have been developed to guide decision-makers on taking long-term robust policy decisions towards energy system transition. however, every esm has been developed to answer specific policy questions, due to the complexity of the energy system and limited computational power. as a result, each model comes with specific capabilities and shortcomings. a large growing body of the literature has listed and classified esms with different aims and scopes. connolly et al. provided a comprehensive overview of suitable esms addressing issues related to renewable energy integration [2] . similarly, bhattacharyya et al. compared energy models to identify their suitability for developing countries [3] . aiming to find the prevalent modeling approaches for the u.k., hall et al. classified and compared esms based on their structure, technological detail, and mathematical approach [4] . to find trends in energy system modeling, lopion et al. reviewed esms in a temporal manner [5] . some reviews have emphasized the role of policy strategies and the corresponding modeling challenges. by grouping energy models into four categories, pfenniger et al. examined the policy challenges they face in each group [6] . horschig et al. reviewed esms to provide a framework for identifying a suitable methodology for the evaluation of renewable energy policies [7] . while savvidis et al. identified the gaps between low-carbon energy policy challenges and modeling capabilities with a focus on electricity market models [8] , ringkjøb et al. classified esms with a focus on the electricity sector [9] . lastly, li et al. reviewed socio-technical models emphasizing societal dynamics [10] . the increasing share of variable renewable energy sources (vres) has caused the low-carbon energy system transition to face several major challenges, such as the increasing need for flexibility, further electrification, emergence of new technologies, technological learning, efficiency improvements, decentralization, macroeconomic interactions, and the deeper involvement of stakeholders in the energy system transition. additionally, some policy questions at the macro level, such as the impact of the energy transition on the macroeconomic indicators (e.g., economic growth and employment), require more indepth integrated analysis, i.e., analyzing the whole energy system consisting of technical, microeconomic, and macroeconomic aspects. however, current esms lack specific capabilities for adequately addressing low-carbon energy system changes that can cause debated conclusions. for instance, one study found no feasible way to achieve a 100% renewable power system by 2050 [11] , while another study claims that a 100% renewable eu power system scenario would require 30% higher annual costs [12] . separate analyses suggested that a 100% renewable eu energy system can be achieved by 2050 with only 12% higher annual energy system costs [13] , while neglecting significant parameters such as electricity grid costs, location of renewables, key technological detail, and flexible electricity demand. brouwer et al. provided a detailed analysis of the west european power sector with high shares of renewables, while neglecting the heat and transport sectors [14] . brown et al. analyzed the cross-sectoral and cross-border integration of renewables in europe, while assuming no national transmission costs, limited efficiency measures, and limited technology options [15] . social aspects of the energy system transition are usually neglected in esms. to this end, some studies included actors' behavior in the energy system from the demand perspective, for example, the thermal demand transition [16] or the efficiency of adaptation measures in households [17] . analyzing each of the major changes in the energy system can be challenging for conventional esms as they need further capabilities such as fine technological detail, high temporal and spatial resolutions, and the presence of stakeholders' behavior. this study concentrates on the energy modeling challenges which result from the increasing share of vres, complexity, and system integration. the transition towards a decarbonized energy system involves other policies such as the higher energy efficiency and change in energy demand, the use of nuclear power supply, and using carbon capture utilization and storage (ccus) technologies. due to the diversity of esms, two major limitations were imposed in this review. first, we focused on energy models at the national level. therefore, the reviewed models were designed for national analysis (or they can be used for national assessments, e.g., primes). second, the models covered all energy system sectors (i.e., residential, services, agriculture, transport, and industrial sectors). the overarching research question of this study is "what are the potential solutions to address the shortcomings of current esms considering current and future low-carbon energy system challenges?". to answer this question, we first describe the current and future lowcarbon energy system modeling challenges. based on these modeling challenges, we identify the required modeling capabilities, such as the need for hourly temporal resolution, sectoral coupling technologies (i.e., p2x), technological learning, flexibility and storage technologies, human behavior, cross border trade, and linking with market and macroeconomic models. the required capabilities were then translated into assessment criteria to be used in the multi-criteria analysis (mca). finally, potential model development solutions are discussed and a modeling suite is proposed as a model-linking solution to address the energy modeling challenges. (fig. 1 ) seven major low-carbon energy system modeling challenges were identified. the challenges were translated into a number of required energy system modeling capabilities and criteria. nineteen models were selected from other reviews ( [2, 4] ). primary inclusion criteria for the selected models (see table 1 ) were: (1) being used at national level, and (2) covering the whole energy system (i.e., integrated energy system models). all the information from the selected models was gathered from officially published documents that may be incomplete or outdated (notably when this paper is published), as models are continuously developed. for each model, a brief description was provided in the appendix section. ahp multi criteria analysis was used as a transparent framework to analyze diverse esms from different perspectives. mca is a methodology that evaluates complex choices (i.e., various criteria, objectives, and indicators), which has been used extensively to analyze energy transition policies [40] . the major advantage of mca is that it provides a rational structure of complex alternatives that presents substantial elements for identifying the desired choice [41] . although mca may have different purposes, we were particularly interested in: first, breaking down complicated energy models into key criteria; and second, identifying the importance or relative weight of each criterion for each alternative. models were ranked based on known criteria, but this did not mean one model was superior to others. therefore, the intention was not to compare models but to identify modeling capabilities and gaps when used for structuring a low-carbon energy system modeling framework. based on the identified modeling gaps, a conceptual modeling suite was proposed to address future low-carbon energy system modeling challenges. the proposed suite included a core integrated energy system model that was hard-linked with a regional model and soft-linked with both an energy market model and a macroeconomic model. energy policies are designed to meet three key objectives, which are providing energy reliability (i.e., supply security), affordability (i.e., economics and job creation), and sustainability (i.e., environment and climate) [42] . with the aim of reviewing electricity market models, savvidis et al. [8] clustered twelve energy policy questions as a basis to quantify the gap between models and policy questions. based on the literature and experts' opinions, we divided energy modeling related policy questions into four categories as follows: 1. technical questions, such as a lack of insights in higher share of intermittent renewables, role of new technologies, and further electrification of the energy system. 2. microeconomic questions, such as a lack of insights in decentralization, human behavior, and liberalized energy markets. 3. macroeconomic questions, such as a lack of insights on economic growth and jobs due to the energy transition. 4. a mix of the above questions, such as lack of insights on the effect of further electrification on energy markets. providing a solution for each policy inquiry can be a challenge for energy system modeling. these challenges can alter the choice of modeling methodology and parameters. in this section, energy modeling challenges and the corresponding modeling parameters are described. some sources of renewable energy such as wind and solar energies have an intermittent characteristic i.e., they are (highly) variable and less predictable [43] . the power generation from intermittent renewables is directly dependent on weather conditions [44] . as wind and solar power generation technologies are becoming more competitive [45] , it is expected that wind and solar power generation will cover up to 30% and 20% of the eu's electricity demand by 2030, respectively ( [46, 47] ). hence, a high share of intermittent renewables in the electricity generation sector is imminent. technically, the power system needs to be in balance at all temporal instances and geographical locations. therefore, the electricity sector should be structured in a way to ensure the balancing of demand and supply. the higher share of intermittent renewables entails variability on the power system balance [48] . solutions to deal with power balance variabilities are called flexibility options (fos) as they provide flexibility to the power system against the variable and uncertain residual load profiles. traditionally, conventional power supplies and grid ancillary services were primary sources of flexibility. however, the power system needs further fos as the share of intermittent renewables in the power generation increases while the share of conventional power supplies -i. e., notably dispatchable gas-fired power plants -decreases. several review papers can be used as a starting point for a review on fos ( [49, 50] ). lund et al. [51] listed different fos as demand side management (dsm), storage, power to x, electricity market designs, conventional supply, grid ancillary services, and infrastructure (e.g., smart grids and microgrids). further, sijm et al. [24] investigated fos by suggesting three fig. 1 . proposed modeling suite approach. the reviewed models and their corresponding developers. causes of the demand for flexibility as the variability of the residual load, the uncertainty of the residual load, and the congestion of the grid. michaelis et al. [52] divided fos based on the residual load in three groups: downward, upward, and shifting flexibility. due to high detail and complications regarding each fo, some studies focused mainly on one or a few technologies. to name a few examples: blanco et al. investigated the cost-optimal share of power to methane in the eu energy transition [53] . the potential of power to heat and power to ammonia in the dutch energy system was investigated by hers et al. [54] and ispt [55] , respectively. some other studies followed an integrated approach that included several fos in different sectors; however, they made several assumptions as the computational capacity was limited (e. g., see ref. [15] ). flexibility options can be divided into five main groups, i.e., storage, demand response (dr), vre curtailment, conventional generation, and cross border trade. instead of analyzing the pros and cons of each option, we were interested in identifying key energy modeling issues regarding each flexibility option. (fig. 2) from a temporal perspective, storage fos can be divided into daily and seasonal storage options. on the one hand, solid-state and flow batteries, such as li-ion, ni-cd, nas, icb, vrb, and znbr batteries, provide high ramp rate with limited capacity, which is suitable for diurnal power storage. modeling these batteries with diurnal temporal resolution can be different to hourly temporal resolution (htr) or hourly time-slices (i.e., grouping hours featuring similar characteristics [56] ). improvements in temporal resolution can have a significant impact on modeling results considering the high share of intermittent renewables (e.g., see ref. [43, 57, 58] ). on the supply side, the uncertainty regarding weather forecasts needs to be implemented in the model as weather conditions have a significant impact on intermittent renewables' generation (e.g., see ref. [59] [60] [61] ). on the other hand, technology options, such as pumped-hydro energy storage (phes), thermal energy storage (tes), large-scale hydrogen storage (lshs), and compressed air energy storage (caes), provide huge capacities that makes them suitable for seasonal energy storage. modeling seasonal storage options requires the inclusion of chronological order (cho) of the temporal parameter together with a fine temporal resolution, as the chronological order of time from summer to winter (and vice versa) determines the charge/discharge of seasonal storage options. dr refers to a set of schemes to shift the demand in a certain time period (e.g., an hour) to another time period of the day, week, or month, either forward or backward [24] . currently, electricity comprises around 22% of eu final energy consumption. power to x (p2x) technology options can provide further dr potentials by linking energy sectors and energy carriers together through converting electricity to other forms of energy, services, or products. in its latest report, the world energy council suggests that p2x will be a key element for the transition to a low-carbon energy system [62] . due to high detail and complications regarding each technology option, several studies focus mainly on one or a few options. at eu level, blanco et al. investigated the cost-optimal share of p2g in the eu energy transition [53] . at the national level, the potential of p2heat [54] and p2ammonia [55] in the dutch energy system was examined. there is a huge potential for demand response in the built environment sector as it is responsible for 40% of energy consumption and 36% of co2 emissions in the eu. while individuals can passively participate in either price-based 1 or incentive-based 2 demand response schemes [63] , proactive participation of consumers can increase market efficiency and reduce price volatility [64] . as heating demand averages 80% of eu household energy consumption, the dr potential can be realized by coupling electricity and heat demands. dr in the built environment can consist of three main components including p2heat technologies (e.g., heat pumps and electric boilers), storage (e.g., thermal tank storage and thermally activate building), and smart controllers (that consider market participation, consumer behavior, and weather forecast) [65] . as p2x technology options bridge two different energy sectors or carriers, analysis of these options requires multi-sectoral modeling, and preferably, integrated energy system modeling. moreover, the hourly temporal resolution of the power sector should be maintained. table 2 summarizes key modeling capabilities and concerning energy sectors and carriers for each p2x technology option. vre curtailment and conventional generation options have been used as fos in the power sector. modeling these options is relatively straightforward, as they do not involve other sectors or energy carriers. still, the hourly temporal resolution remains the key modeling capability for these options. from the energy security perspective, modeling conventional generation may require modeling capacity mechanisms, 3 preferably in combination with cross border power trade [66] . the eu is promoting an internal single electricity market by removing obstacles and trade barriers (see e.g., com/2016/0864 final -2016/0380). "the objective is to ensure a functioning market with fair market access and a high level of consumer protection, as well as adequate levels of interconnection and generation capacity" [67] . one of the products of an internal eu electricity market is the potential to offer flexibility in the power system, as the load can be distributed among a larger group of producers and consumers. sijm et al. identified the cross-border power trade as the largest flexibility potential for the netherlands [68] . similar to other flexibility options, one of the key modeling capabilities here is the hourly temporal resolution. table 2 summarizes the required key modeling capabilities for representing and analyzing flexibility options in esms. the main requirement is the inclusion of (at least) an hourly temporal resolution. models' capabilities can improve substantially by adding seasonal storage options, which require the inclusion of chronological order and different energy carriers. moreover, the inclusion of cross border trade can play an important role in the optimal portfolio of flexibility options, especially in eu countries. higher shares of intermittent renewables affect the reliability of power generation and distribution as residual loads become less predictable. for instance, the prediction accuracy of a single wind turbine generation decreases from 5-7%-20% mean absolute error for the hour ahead and day-ahead forecasts, respectively [69] . the increased uncertainty of the power generation due to higher shares of vre sources requires models to include short-term weather forecast and balancing mechanisms in their calculations. uncertainty analyses gain more importance for long-term esms as they model the energy system for several decades in an uncertain future that can get affected by parameters outside the energy system boundaries. energy system optimization models use four main uncertainty analysis methods, which are monte carlo simulation (mcs), stochastic programming (sp), robust optimization (ro), and modeling to generate alternatives (mga) [70] . in 2017, almost 22% of the eu final energy demand was satisfied by electricity, while heat consumption and transport accounted for the rest. current heating and cooling production in the eu come from fossil fuel sources, as renewable energy sources have a 19.5% share of gross heating and cooling consumption. the transport sector is highly dependent on fossil fuels, with only 7.5% of final energy consumption from renewables. therefore, decarbonization of the heat and transport sectors is getting more attention as it has a higher ghg emissions reduction potential. the eu commission suggests electricity as an alternative fuel for urban and suburban driving in its report entitled clean power for transport [71] . further electrification of heating, cooling, and transport sectors may contribute to ghg reduction, assuming the electricity is generated from renewables rather than fossil fuels [72] . due to the high seasonal variation of heating and cooling demand profiles (mainly in the built environment), further electrification of this sector requires huge seasonal storage capacities or other flexible supply options. currently, there are four main high capacity seasonal storage options, which are pumped hydro energy storage (phes), compressed air energy storage (caes), thermal energy storage (tes), and hydrogen energy storage (hes). by using tes technologies, hourly heat and power demand profiles can be decoupled resulting in a higher potential for dr flexibility option [73] . tes technologies can be divided into three main groups based on their thermodynamic method of storing heat energy: sensible, latent, and chemical heat [74] . sensible heat storage (shs) technologies stock the heat by the difference in the materials' temperature, for example by warming up water or molten salt tank. latent heat storage (lhs) technologies make use of phase-change materials (pcm) in a constant-temperature process to absorb or release thermal energy. chemical heat storage (chs) technologies make use of thermo-chemical materials (tcm) in a reversible endothermic or exothermic (i.e., a chemical reaction in which the heat is absorbed or released, respectively) process, for example, the reversible ammonia dissociation process (i.e., 2nh 3 =n 2 +3h 2 ). xu et al. [75] provided an extensive review of current seasonal thermal energy storage technology options. further electrification of the energy system, which is expected to account for 36-39% of final eu energy consumption by 2050 [76] , generates higher interdependencies between energy sectors. single sector models, which are not able to capture sector coupling effects, may provide misleading conclusions by neglecting these interdependencies. as more sources of intermittent renewables are deployed in the energy system, the further electrification implies further volatility of the energy system that highlights the higher demand for flexibility options. moreover, analyzing sector coupling technologies such as evs (p2mobility), heat pumps and electric boilers (p2heat), and electrolyzers (p2gas) become more important. inclusion of sector coupling options in the esm requires modeling of electricity, transport, and heat sectors simultaneously. due to high variations in the electricity supply, a fine temporal resolution should also be employed in the transport and heat sectors in order to adequately address the flexibility issues of sector coupling. development of new technologies and technological change are key drivers of the transition to a low-carbon energy system and are at the core of most energy-climate policies worldwide [77] . for instance, the price decline of pv cells from 3.37 usd/w to 0.48 usd/w in the last 10 years [78] has made solar energy an economic option independent of subsidies. development of new technologies have made additional renewable energy supply sources available such as advanced biofuels, blue hydrogen, deep geothermal, wave, and seaweed. it also provides innovative opportunities for the further integration of the energy system by implementing p2x technologies, which mainly consist of p2heat, p2g, p2h 2 , p2l, 4 and p2mobility technology options. the seasonal variation of wind and solar increases the need for seasonal storage options such as thermal energy storage and caes. ccs and ccu technologies can be considered as alternative solutions for conventional ghg emission emitters. deep decarbonization of the industrial sector can be achieved by the development of new industrial processes while considering the whole value chain [79] . the development of zero energy buildings [80] and formation of energy-neutral neighborhoods [81] can contribute to substantial energy savings in the built environment. esms currently represent technological learning either exogenously or endogenously [82] . technological change is prevalently expressed as a log-linear equation relating technology cost to its cumulative production units. this one-factor equation provides the learning rate, which is the cost reduction that results from doubling the cumulative produced units of the concerned technology [83] . the prominent alternative is the two-factor equation that incorporates both cumulative produced units and r&d investments [84] . endogenous technological learning (etl) is widely used in long-term esm analysis (e.g., see ref. [85] [86] [87] ). etl can be further elaborated as multi-cluster learning (mcl) and multi-regional learning (mrl) [88] . mcl (or so-called compound learning) describes a cluster of technologies, which share the same component and learn together (e.g. see ref. [89] ). mrl differentiates between region-specific technological learning and global technological learning. the consideration of new technologies and technological learning can greatly affect the energy system modeling results, particularly in long-term models. for instance, heuberger et al. [90] . concluded that the presence of global etl will result in 50% more economically optimal offshore wind capacity by 2050. as part of the clean energy for all europeans package, the eu sets binding targets of at least 32.5% energy efficiency improvement by 2030, relative to the business as usual scenario [91] . this policy emphasizes particularly the built environment as the largest energy consumer in europe. although energy-efficient technologies provide financial and environmental costs reduction, they are not widely adopted by energy consumers. this "energy efficiency gap" can be a consequence of market failures, consumer behavior, and modeling and measurement errors [92] . energy efficiency policies may induce the rebound effect (or backfire), in which energy efficiency improvements lead to an increase in energy use. the rebound effect may have a direct decreasing impact in energy consumption (e.g., a decrease in residential energy consumption), while having an indirect increasing impact (e.g., an increase in energy use by expansion of energy-intensive industries) [93] . providing an accurate estimate of the magnitude of the rebound effect can be challenging ( [94] , while the existence of this effect is a matter of discussion in the literature [95] . although energy-efficient technologies can play an effective role in energy system transition, modeling and analyzing its direct and indirect effects is challenging. energy infrastructure has a key role in the low-carbon energy system transition by facilitating sectoral coupling, integrating renewable energies, improving efficiency, and enabling demand-side management. however, analyzing energy infrastructure can come up with some challenges, such as the complexities of distributing costs and benefits of investments and allocation of risk between investors and consumers [96] . conventional energy infrastructure facilities are usually managed by a monopoly as public goods are not traded in a market. therefore, the clear disaggregation of costs and benefits of infrastructure changes due to energy transition needs further evaluation [97] . long-term infrastructure investment and risk profiles of investors and consumers can be highly diverse as energy infrastructures can undergo drastic changes. moreover, social acceptance of energy infrastructure plays a key role in energy transition, particularly in decentralized infrastructures such as ccus networks, transmission lines, district heating, and local energy storage. modeling the social acceptance of energy infrastructure requires a combination of qualitative and quantitative datasets which can be highly locally dependent [98] . assuming the above-mentioned datasets are available, esms would require specific capabilities to analyze energy infrastructure. the esm should be geographically resolved, as energy infrastructure can have both local and national scales. moreover, there is a need for gis-based geographical resolution of esms as costs and benefits of energy infrastructures can change drastically by their geographical location. over the past decades, energy used to be supplied by large power plants and then transmitted across to the consumers. by emerging renewable energy supplies, a new alternative concept of the energy system has been developed. the decentralized energy system, as the name suggests, is comprised of a large number of small-scale energy suppliers and consumers. a transition from a centralized fossil-fuel and nuclear-based energy system to a decentralized energy system based on intermittent renewable energy sources can be a cost-effective solution for europe [99] . the local energy supply reduces transmission costs, transmission risks, environmental emissions, and to some extent promotes energy security for a sustainable society, with a competitive energy market. on the other hand, it can increase generation costs capacity investment, distribution, and energy reliability. therefore, there is a need to determine the optimal role of energy system decentralization by carefully analyzing costs and opportunities. conventional energy modeling tools were based on the centralized energy system and they have faced difficulties fulfilling the decentralized energy system demands. in conventional energy models, the location of the power plants does not play an important role, while spatial detail may be critically important for renewables. for instance, economic potentials, solar potentials, generation costs, environmental and social impacts, network constraints, and energy storage potentials are some location-dependent factors that can vary greatly across different regions. some other factors such as wind potential and infrastructural costs can vary greatly even with little dislocation. therefore, a fine spatial resolution is required in order to assess the role of locationdependent parameters in the energy system. esms can use national, regional, or gis (geographical information system) based spatial resolution. using a fine spatial resolution can be limited by the available computational power and spatial data. therefore, the choice of spatial resolution is the compromise between these two parameters. due to the huge computational load of gis-based esms, they are usually applied at urban level rather than national level. gisbased models can be used in a preprocessing phase in order to provide spatially resolved datasets for national esms. for instance, the global onshore wind energy potential dataset is produced at approximately 1 km resolution [100] . assuming the availability of spatial data, the computational limitation can be addressed by linking a coarse resolution energy model with a spatial modeling tool such as arcgis (e.g., see ref. [101] ). conventional energy models neglected social stakeholders as the energy system was managed and controlled by central decision-makers. in order to reach a sustainable low-carbon energy system, technical and social insights should be integrated into these models [102, 103] . according to the technology review of the u.s. department of energy, the balance of energy supply and demand is affected as much by individual choices, preferences, and behavior as by technical performance [104] . the reliability of energy models is often low because they are excessively sensitive to cost analysis while ignoring major energy policy drivers such as social equity, politics, and human behavior [105] . several studies have recently indicated the role of social sciences in energy research [106, 107] . social parameters are usually difficult to quantify, and consequently, are usually neglected in quantitative energy models. however, there are practical methods of integrating human aspects into technical energy models, such as the inclusion of prosumers and agent-based modeling. originally coined by alvin toffler in his 1980 book the third wave [108] , prosumer is the mixture of the words producer and consumer, explaining the active role of energy consumers in the production process. the conventional energy grid was dependent on the interaction between supplier and distributor, while consumers play an active role in the decentralized energy system. an important element of this new system is the role of prosumers i.e., consumers who also produce and share surplus of renewable energy generated with the grid and/or other consumers in the community [109] . with the emerging renewable energies at the microscale, prosumers are not only an important stakeholder of the future smart grids but may also play a vital role in peak demand management [110] . however, social acceptance of the decentralized energy system faces several drivers and barriers [111] that need quantification in order to be imported into energy models. the emergence of prosumers has increased the diffusion of social sciences in energy system modeling (e.g., see refs. [112] [113] [114] [115] ). in order to grasp an adequate knowledge of the decentralized energy system, the behavior of the prosumers on energy grid should thus be considered alongside the techno-economical characteristics (e.g., see ref. [116] [117] [118] ). based on the position of the decision-maker, esms can be divided into two main categories. the common approach is the assumption of a system planner who optimizes the single objective function (e.g., system cost minimization). contrary, agent-based models practice decentralized decision making by assuming autonomous agents who decide based on their own objectives. agent-based modeling has been proposed as a suitable modeling approach for complex socio-technical problems [119] and it is used in modeling the wholesale electricity market considering human complexities [120] . ringler et al. reviewed agent-based models considering demand response, distributed generation, and other smart grids paradigms [121] . the term "agent" can be used to describe different types of players in the energy system such as prosumers, power generators, storage operators, or policy makers. agents can optimize their own objective function, which can be based on economic (e.g., capital, npv, and tariffs), technical (e.g., efficiency, emissions, and maximum capacity), and social (e.g., bounded rationality, neighborhood effect, and heterogeneity) factors. including techno-economic factors in the objective function is relatively easier due to the quantitative nature of these parameters, while integrating qualitative social parameters remains a complicated task. qualitative parameters such as the perceived weight of environmental costs and impacts, expected utilities, social networks, and communication can be estimated by socio-demographic factors and behavior curves (e.g., see ref. [122] [123] [124] ). macroeconomic models follow a top-down analytical approach compared to techno-economic esms that use a bottom-up approach. the analytical approach is the way to break a system into elementary elements in order to understand the type of interactions that exist between them. this system reduction may be done in different ways. based on the reduction approach, esms are usually differentiated into three main groups: top-down, bottom-up, and hybrid models. top-down (td) models describe the energy-economy system as a whole to assess the energy and/or climate change policies in monetary units [125] . these models mainly describe the relations between the energy system and the variations in macroeconomic and environmental factors such as economic growth, demographics, employment rate, global warming, and ghg emissions. consequently, top-down models lack detail on current and future technological options which may be relevant for an appropriate assessment of energy policy proposals [126] . macroeconomic equilibrium models are an example of top-down models. bottom-up (bu) models, provide a higher degree of technological detail (in comparison to top-down models). characterized by a rich description of the current and prospective energy supply and end-use technologies, bottom-up models describe energy systems evolutions as resulting from a myriad of decisions on technology adoption [127] . they can compute the least-cost solution of meeting energy balances subject to various systems constraints, such as exogenous emission reduction targets [128] . hybrid models (i.e., linking td and bu models) can be a solution to integrate top-down consistency while maintaining bottom-up detail. the major advantage of top-down models is their consistency with welfare, market, economic growth, and other macroeconomic indicators that leads to a comprehensive understanding of energy policy impacts on the economy of a nation or a region. on the other hand, they lack an appropriate indication of technological progress, energy efficiency developments, non-economical boundaries of the system, and other technical details. instead, bottom-up models describe the energy system with detailed technological properties. moreover, bottom-up models lack feedback from the macro-effects of the technical changes in the overall economy. therefore, closing the gap between top-down and bottom-up energy models results in more consistent modeling outcomes (e.g., see ref. [129] [130] [131] [132] ). model linking is not an exclusive solution for td and bu models. hourcade et al. [133] argued that the three main dimensions of an energy-economy system are: technological explicitness, microeconomic realism, and macroeconomic completeness. the main advantage of model linking (i.e., modeling suite) is the ability to provide consistent results while considering two or all three dimensions. each of these dimensions can be modeled with a number of different models depending on the complexity of the problem. the model linking approach can be classified into three subcategories, based on the level of linking [135] . first, individual stand-alone models are linked together manually meaning that the processing and transferring of the information between models are controlled by the user, preferably in an iterative manner (i.e., soft-linking). second, a reduced version of one model exchanges data with the master model while both running at the same time (i.e., hard-linking). third, a combined methodology features an integrated model through a unified mathematical approach (e.g., mixed complementarity problems [136] ). (fig. 3) helgesen [134] used another classification based on the linking type of models and the terminology proposed by wene (i.e., soft-linking and hard-linking) [137] . the advantages of soft-linking can be summarized as practicality, transparency, and learning, while the advantages of hard-linking are efficiency, scalability, and control [138] . the above discussion of the main challenges of the present and future esms identified several required modeling capabilities, which are summarized in table 3 . in order to review models based on mentioned challenges, required capabilities are grouped into several model assessment criteria in table 4 . it should be noted that the integrated energy system analysis capability is not mentioned further as all reviewed models were integrated. moreover, the linking esms with td models' capability is discussed further in section 5. apart from the criteria that results from emergent challenges of future esms, three additional criteria are considered in table 4 , namely: (i) the underlying methodology of the model to separate calculator models from non-calculator ones; (ii) the source of the model's datasets to measure input-data quality; and (iii) the accessibility and the number of the model's applications to determine the models' use and acceptance. considering the criteria regarding the future low-carbon energy systems and available models, it can be concluded that no perfect model exists. models can be assessed based on the list of criteria such as temporal resolution, spatial resolution, the social aspect, data source quality, accessibility, and application ( table 4 ). the capability of the model in each criterion is given a score from five (highest) to one (lowest) as presented in table 5 . the results are highly dependent on the scores and weights, which are both -to some extent -subjective. readers can alter the results by incorporating new criteria or changing the perspective weights. in the following sections, these modeling capabilities and the corresponding scores are explained. there are two parameters that differ across integrated esms, which are the inclusion of fos and the inclusion of technological learning. therefore, models can be grouped into three groups: (i) no flexibility option and no technological learning that would score one; (ii) the inclusion of either flexibility options or technological learning that would score three; and (iii) the inclusion of flexibility options and technological learning that would score five. esms usually balance the supply and demand on a yearly basis or a limited amount of (hourly) time-slices per year. nevertheless, some models have a higher temporal resolution and balance the system on an hourly basis. reviewed models can be categorized in three groups: (i) temporal resolution on yearly basis that would score one; (ii) time-slice approach that would score three; and (iii) hourly temporal resolution that would score five. some models have the capability to model the regions inside a country. this ability can provide regional insights on energy system policies and vice versa. although the limited computational capacity and the lack of data make it difficult to perform a detailed regional analysis, some models balance the system in different regions inside the country based on different capacities and properties of the regions (e.g., esme in the uk). reviewed models are divided in three groups: (i) models without regional depth that would score one; (ii) models which consider regions that would score four, since it is a considerable improvement; and (iii) models which consider gis data that would score five. the role of social analysis in techno-economic models is usually negligible. however, some modeling tools practice multi-agent programming in order to model qualitative aspects of stakeholders' decision-making practice on energy systems. models are categorized into two groups: (i) models capturing socio-economic parameters only based on demand curves that would score one, and (ii) agent-based models considering a set of decision-making rules for different stakeholders in the energy system that would score five. reviewed models practice a different set of methodologies. in this review, the main categorization between methodologies can be made between the calculator and non-calculator methodologies. therefore, models can be grouped into two groups: (i) calculator models that would score one, and (ii) non-calculator models that would score five. the depth of technical detail and the quality of the data play a crucial role in providing accurate insights into the energy system with regard to new technologies and sectoral coupling. moreover, data access is the first limitation of energy system research as databases are rather private. models can then be divided into five groups: (i) models not indicating a data source that would score one; (ii) models using generalized opensource data that would score two; (iii) models using limited countryspecific data that would score three; (iv) models using detailed opensource data that would score four; and (v) models using detailed country-specific datasets, possibly in combination with global datasets that would score five. open-access models provide an opportunity to test the model and provide feedback. these models are divided into five groups: (i) models which provide no access that would score one; (ii) models which provide limited access that would score two; (iii) models which are commercial that would score three; (iv) models which are open-source but need permission that would score four, and (v) models which are completely open-source and accessible on internet that would score five. a model with more applications and users makes it easier to disseminate and discuss results. models are grouped in five sets: (i) models with no publication yet that would score one; (ii) models applied in one country that would score two; (iii) models applied in two countries that would score three; (iv) models applied across eu countries that would score four, and (v) models which applied in many countries and are well-known that would score five. table 6 demonstrates the mca analysis with equal weight for all criteria. to calculate the score of each model for each criterion, the weighted percentage of that criteria in the model's total score is demonstrated. this percentage is calculated endogenously, as explained by (equation (1) . it indicates the share of the models' score in each criterion out of the models' total score. primes would score high mainly due to the inclusion of social parameters, while the high score of remix is due to its high spatial resolution. these models merely demonstrate improved capabilities compared to others; therefore, it does not mean that these models are "best" models. moreover, some features of the models are not reflected in this table. for instance, metis works complementary to long-term esms as it only simulates one specific year. besides, the mca results can be changed considerably by assigning slightly different scores to various criteria as total scores are relatively close. models such as the markal family and metis demonstrate high scores mainly due to their high granularity; however, they lack the inclusion of social parameters. ensysi includes social parameters while lacking spatial resolution and application. addressing all the policy-induced challenges of the energy system requires a comprehensive esm that is not yet available. therefore, a compromise should be made based on the challenges that the model is designed to address. consequently, a weighted decision matrix can be formed by using the ahp method [139] . in this method, the criteria are rated against each other with respect to the challenges. a consistency ratio (cr) is calculated to indicate the reliability of the weight table. saaty suggests that the crs of more than 0.1 are too inconsistent to be considered reliable [139] . here the challenges are divided into two main groups, first: intermittency, flexibility, and further electrification; and second: human behavior and decentralization. the first group of challenges puts emphasis on the technological detail, the high temporal and spatial resolution; while the second group emphasizes the inclusion of social parameters and high spatial resolution. the pair-wise weighted decision matrix for the first group of challenges is formed in table 7 . in each row, the importance of a criterion compared to other criteria is given a number in this order: 1 would be equal importance, 3 would be fairly important, and 5 would be extremely important. it should be noted that these numbers are entirely subjective, thus, the user can make his own decision matrix. then, the table is normalized and the sum of each row is reported as the weight of the criterion. to calculate the cr, each column of the pair-wise table is multiplied by the corresponding weights. then, the sum of each row is divided by the corresponding weight to get λ values. the cr is then calculated by equation (2), in which n is the number of criteria. repeating the same procedure for the second group of challenges leads to table 8 . in both cases, the low cr indicates low levels of inconsistency in the assigned weights. using the weights in table 8 to update the mca will lead to a slightly different result, presented in table 9 . for the first group, it is expected that models with high scores in technological detail, temporal resolution, and spatial resolution will get higher total scores. the remix model gets a high total score mainly due to the inclusion of high spatial resolution with the use of gis data and the inclusion of key flexibility and storage technologies with the exogenous technological learning. the metis model provides lower technological detail by neglecting technological learning while incorporating hourly temporal resolution and gis-based spatial resolution. for the second group, the inclusion of social parameters and fine spatial resolution gains importance. models with the inclusion of social parameters such as primes and ensysi get higher scores. although the metis model does not include social parameters, it keeps a high score due to its fine spatial resolution. irrespective of the assigned weights, we find four models at the top of the mca table which are remix, primes, metis, and the markal family models. these models had high scores in nearly all criteria, while a low score in one criterion (for instance, lack of social parameters for remix) is compensated with a high score in another criteria (high temporal and spatial resolution). these four models were either developed recently (e.g., remix and metis) or are under constant development (e.g., markal family and primes). it shows how integrated energy system modeling points towards the models with improved capabilities in all the criteria. other models keep the same ranking position except for iwes, ensysi, and energyplan, which changed their position considerably (i. e., more than two steps change). this position change can be explained by the asymmetry of these models' scores in the mca table. for instance, the iwes model gets a high score in the first four criteria while getting a low score in the last four criteria. the mca represents an overview of the current state of esms with regard to low-carbon energy system modeling challenges. however, there is a need for adding new capabilities to current esms in order to address future modeling challenges. in the next section, we discuss two potential modeling solutions based on our observation from the current state. the overall solution is to expand single models and/or to link different models. it is not practical to decide on the best model that addresses challenges regarding low-carbon energy systems, as each model has specific pros and cons. from a techno-economic point of view, the mca indicates that for modeling the low-carbon energy system, current models require specific capabilities such as hourly temporal resolution, regional spatial resolution, inclusion of sectoral coupling technologies, technological learning, and inclusion of social parameters. there are major gaps between policy questions and modeling capabilities in the criteria, which were used to assess the models' performance. however, these criteria mainly focus on the technical policy questions rather than the entire technical, microeconomic, and macroeconomic aspects. although techno-economic models are rich in detail, they lack the capability to answer microeconomic and macroeconomic policy questions. therefore, specific models, such as energy market models and general equilibrium models, have been developed. due to the strong interconnection between energy and economy, mixed policy questions arise that require analyzing the technical, microeconomic, and note: percentages may not add up to 100 due to rounding. macroeconomic aspects of the energy-economy system. such analysis can be conducted either by developing single models or by combining different models (i.e., soft-linking, hard-linking, or integrating). current single models can be developed and/or extended by incorporating further capabilities up to acceptable computational limits. considering the limitations, the modeler makes choices and/or tradeoffs on extensions to the model. developing a single model that can cover all the mentioned gaps could face limitations, such as complicated mathematical methodology and limited computational capacities (except generic limitations such as high data needs and lack of transparency). some common energy system modeling methodologies are optimization, simulation, accounting, multi-agent, and equilibrium. each mathematical methodology can be developed to answer specific energy modeling questions. integrating two different methodologies can be mathematically very complicated (e.g., mixed complementarity problems in which the optimization and equilibrium formulations are mixed) or not feasible (e.g., mixing optimization and simulation formulations). therefore, single esms are naturally limited by their underlying methodology. one of the main limitations for improving the temporal and geographical resolution of esms is the computational capacity. the computational limitation can be addressed either by hardware or software development. (fig. 4) hardware development follows an exponential growth and relates to improvements in the number of transistors, clock frequency, and power consumption of processors. on the other hand, software methods can be divided into solver-related and modelspecific methods to improve computing times of linear optimization esms [140] . solver-related methods focus on improving the solving methodology by using different off-the-shelf algorithms, such as lindo, cplex, gurubi, and mosek, or by practicing customized algorithms, such as bender's decomposition 5 and parallelization. 6 model-specific methods relate to heuristic methods, such as clustering, model reduction, decomposition, and parallelization. hardware-related developments proceed at a specific pace that is usually not affected by energy system modelers as users. solver-related developments are followed by a few energy system modelers (e.g., see the beam-me project [184]), while the rest of the energy system modeling community follows model reduction and clustering methods that can be applied on temporal resolution, spatial resolution, and technological detail (e.g., see [185] ). depending on the research questions, energy system modelers reduce or coarsen the resolution of the model in order to provide an answer in an adequate timeframe. therefore, a trade-off should be made between different modeling capabilities by making smart assumptions. an alternative approach to overcome the limitations of single model development is to form a modeling suite. model linking can be done between any set of desired models in order to enhance modeling capabilities. however, two types of energy model linking are more frequent in the literature: (1) linking bu and td models such as optimization energy system models (oesms) linked with cge models, and (2) linking two bu models such as oesms combined with energy market models (i. e., unit commitment or dispatch models). although linking models provides further modeling capabilities, it comes with certain challenges [141] such as the identification of connection points in soft-linking (e.g., see ref. [142] ), convergent solution in soft-linking (e.g., see ref. [143] ), and mathematical formulation for integrated linking (e.g., see ref. [144, 145] ). in summary, linking models can be resource-intensive as it requires the knowledge of different modeling frameworks. each model has its own set of assumptions and methodologies, which makes it complicated to maintain the harmonization of modeling assumptions in all steps of linking. the lack of harmonization in assumptions may result in inconsistent results from linked models. although this process seems straightforward, it is rather a puzzling procedure as esms are moderately complex. therefore, having an overview of different energy models and their capabilities is essential to provide the desired modeling suite. a linking approach is proposed for addressing current energy system modeling gaps. table 10 provides an overview of identified energy modeling gaps and corresponding linking suggestions. these suggestions can form a modeling suite that involves four different models, namely, the energy system model (esm), the energy market model (emm), the macroeconomic model (mem), and the socio-spatial model (ssm). the suggestions in table 10 can be framed in two separate modeling suites based on the methodology of the core esm. (fig. 6) the first modeling suite can be formed around an optimization esm (oesm) that provides the cost-optimal state of the energy system assuming a fully rational central social welfare planner. the second modeling suite uses a socio-technical esm (stesm) that demonstrates a more realistic state of the energy system by assuming profit maximizer agents who consider social decision-making parameters in their decision-making process. such parameters include behavioral economics, bounded rationality, neighborhood effect, and technology diffusion curve. the core component of the suggested modeling suite is the presence of a central techno-economic esm as an information processor hub that exchanges the outputs with different models. based on the current state of the energy system and future scenarios, the esm can determine the technology and energy mix, commodity and energy prices, amount and price of emissions, and total energy system cost. however, this standalone analysis is based on specific scenario assumptions of demand profiles, energy import and export profiles, decentralized energy supply prospects, and macroeconomic expectations. it is suggested to use linear relations (i.e., linear optimization methodology) to keep the computational load manageable. while the optimization framework determines the theoretically optimal state of the energy system, the simulation methodology can demonstrate feasible pathways to reach the optimal state. therefore, by comparing the results of the optimization and simulation frameworks, the gap between the optimal solution and the feasible solution (symbolically demonstrated in fig. 5 ) can be identified. several policy parameters can affect the width of this gap by bringing the feasible solution close to the optimal one. therefore, the analysis of the simulation and the optimization methodologies can provide a better understanding of the role of each parameter in reaching the optimal state of the energy system policy targets. based on the review and the mca, several optimization esms such as times and remix can be used as the core esm of the modeling suite, due to their fine temporal resolution and ample technological detail. agentbased simulation esms are not as common as optimization esms; therefore, from the reviewed models, only ensysi and primes can be selected as simulation core esms. current esms lack the capability to model the regional implications of the energy system such as decentralized supply and demand, infrastructure costs and benefits, land use, and resource allocation. although some local energy system models such as energis [146] and gisa sol [147] provide geographically resolved energy system analysis, they lack the interaction with other country regions. as regional variations of the energy system can have drastic effects on the system itself, it is suggested to hard-link the regional model into the core esm. the geographical resolution of esms can be improved depending on the research questions and available resources. for instance, after identifying spatially sensitive parameters of the energy system, such as heat supply location, renewable power production, transmission capacity expansion, and storage infrastructure, sahoo et al. provided a framework to integrate them into an esm (i.e., the opera model) [148] . focusing on infrastructure, van den broek et al. clustered the co 2 source regions using the arcgis software and then incorporated the spatially resolved data into the markal-nl-uu as the optimization-based esm [149] . for well-connected countries, it is suggested to hard-link an emm with the core esm to capture the flexibility potential of the cross-border energy trade, albeit some studies that use a soft-linking approach (e.g., see ref. [150, 151] ). in particular, for eu countries, this hard-linking is necessary as the interconnection flexibility option (fo) can be in direct competition with domestic fos such as demand response or storage. emms usually use the milp underlying methodology in order to model unit commitment; therefore, the inclusion of emm inside esm can be computationally intensive. it is suggested to use a linear optimization methodology in accordance with the core esm to reduce computational load, while reaching a fair estimate of the energy import and export flows, particularly for electricity. assuming the regional and interconnection capabilities are integrated into the core esm, in order to capture consistent economic analysis, one soft-linking loop is suggested as follows. this loop incorporates a macroeconomic model, which keeps demand and supply of commodities in equilibrium based on the statistical economic data such as capital stocks and investments, demographics, labor market, and trade and taxes. the esm outputs, such as energy prices, energy mix, and emissions are fed into the mem to update the supply and demand and price data of energy and fuel commodities. the mem provides the equilibrium demographics, gdp and income, monetary flows between economic sectors, trade, and employment rate. this loop can be performed once or it can continue until the results reach a convergence criterion, which is a user-defined criterion that determines the maximum gap between the results of two models. moreover, mem outputs can feed into an abm simulation esm in table 10 model development and model linking suggestions based on the identified energy modeling gaps. lack of sectoral coupling technologies between electricity, heat, and transport sectors. developing a long-term planning optimization energy system model (esm) that involves all energy sectors, hourly temporal resolution, regional spatial resolution, seasonal storage options, and technological learning lack of new seasonal storage technology options such as tes and hes. lack of endogenous technological learning rates. lack of hourly temporal resolution for capturing intermittent renewables and corresponding potentials. lack of regional spatial resolution for analyzing energy flows between regions across a country. hard-linking esm with a regional energy system model (resm) that involves resolved spatial resolution, land use analysis, and infrastructure analysis lack of fine geographical resolution options such as gis, fine mesh, and clustering for analyzing decentralized intermittent supply and infrastructure costs and benefits lack of spatially resolved datasets such as infrastructure and local storage. simplistic modeling of human behavior in the current abms. developing an abm simulation socio-technical energy model (stesm) that involves stakeholders' behavior, local and neighborhood effects, bounded rationality, and perceived environmental values. the focus of current datasets is only on technological detail, rather than stakeholders' behavior. high dependence of esms on consumer load profiles. lack of national energy modeling consistency with a european (or an international) energy market. hard-linking esm with an international (or european) energy market model (emm) that involves an optimal dispatch electricity market, the gas and oil market, hourly temporal resolution, regional spatial resolution, and a detailed generation database lack of energy modeling consistency with macroeconomic indicators which consumer demand profiles are generated based on demographics, income, and employment (e.g., see ref. [152] ). the stesm analyzes the social aspects of the energy system such as stakeholders' behavior, bounded rationality, imperfect communication, and environmental perceived value. the choice of models, connection points, and scenarios are dependent on the aims of the energy system modeling, available expertise and resources, and access to models and datasets. in summary, we identified the capabilities and shortcomings of current esms to analyze adequately the transition towards a low-carbon energy system. in this regard, we described seven current and future low-carbon energy system modeling challenges. finally, to bridge major energy modeling gaps, two conceptual modeling suites are suggested, based on both optimization and simulation methodologies, in which the integrated esm is hard-linked with a regional model and an energy market model and soft-linked with a macroeconomic model. model development and linking choices can be affected by major changes in the energy system outlook. for instance, the current covid-19 situation can have major impacts on economic activities, indicating the importance of soft-linking with macroeconomic models. a limitation of this study is that all the information about models has been gathered from published documents, which might be outdated as models are constantly under development. therefore, this review provides a rather static view on esms. only a limited number of energy system models was presented in this review, which is mainly due to limited time, resources, and access to modeling databases. other challenges of low-carbon energy systems modeling not included here are the need for energy policy harmonization, energy market design, business models of new technologies, legislation and legal aspect of the energy transition, and social acceptance implications of the energy transition. another limitation of this study is the subjective assignation of scores with the mca, however, a clear explanation for the assignation of scores was provided. furthermore, the mca only considered single esms while in practice a combination of models can be analyzed. a more comprehensive mca would consider the capabilities and limitations of suites of models. the authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper the authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper. the revised renewable energy directive: clean energy for all europeans a review of computer tools for analysing the integration of renewable energy into various energy systems a review of energy system models a review of energy systems models in the uk: prevalent usage and categorisation a review of current challenges and trends in energy systems modeling energy systems modeling for twenty-first century energy challenges are decisions well supported for the energy transition? a review on modeling approaches for renewable energy policy evaluation the gap between energy policy challenges and model capabilities a review of modelling tools for energy and electricity systems with large shares of variable renewables a review of socio-technical energy transition (stet) models burden of proof: a comprehensive review of the feasibility of 100% renewable-electricity systems broek mv d. is a 100% renewable european power system feasible by 2050? smart energy europe: the technical and economic impact of one potential 100% renewable energy scenario for the european union least-cost options for integrating intermittent renewables in low-carbon power systems synergies of sector coupling and transmission reinforcement in a cost-optimised, highly renewable european energy system agent-based modeling of a thermal energy transition in the built environment adoption of energy efficient technologies by households -barriers, policies and agent-based modelling studies dynemo: model documentation. london: ucl energy institute dynemo: a dynamic energy model for the exploration of energy, society, and environment metis technical note t5: metis software introduction and architecture. european commission. brussels: directorate-general for energy australian energy projections to 2049-50 the national energy modeling system. an over view. energy information administration the supply of flexibility for the power system in the netherlands a simulation model for the dutch energy system. the hague: pbl netherlands environmental assessment agency osemosys: the open source energy modeling system an introduction to its ethos, structure and development modelling low-carbon energy system designs with the eti esme model. eti poles-jrc model documentation. luxembourg: publications office of the european union primes model, version 6. national technical university of athens a time step energy process model for germany -model structure and results scenario analysis: electrification of commercial urban road transportation and impacts on the energy system future security of power supply in germany-the role of stochastic power plant outages and intermittent generation analysis of alternative uk heat decarbonisation pathways. imperial college london for the committee on climate change institute for sustainable solutions and innovations (isusi) stockholm environment institute system analysis: dtu, department of management engineering documentation for the markal family of models documentation for the times model, part i. international energy agency a review of multi criteria decision making (mcdm) towards sustainable renewable energy development sensitivity analysis of multicriteria decision making methodology developed for selection of typologies of earth-retaining walls in an urban highway the goals of energy policy: professional perspectives on energy security, economics, and the environment impact of the level of temporal and operational detail in energy-system planning models large-scale variability of weather dependent renewable energy sources. management of weather and climate risk in the energy industry 10 trends reshaping climate and energy the european political strategy centre wind energy in europe: scenarios for 2030 an industrial policy for solar in europe. solarpower europe the potential of intermittent renewables to meet electric power demand: current methods and emerging analytical techniques flexibility requirements of renewable energy based electricity systems -a review of research results and methodologies a comprehensive survey of flexibility options for supporting the low-carbon energy future review of energy system flexibility measures to enable high levels of variable renewable electricity comparison of the techno-economic characteristics of different flexibility options in the european energy system potential of power-to-methane in the eu energy transition to a low carbon system using cost optimization potential for power-to-heat in the netherlands power to ammonia: feasibility study for the value chains and business cases to produce co2-free ammonia suitable for various market applications. institute for sustainable process technology the importance of high temporal resolution in modeling renewable energy penetration scenarios. 9th conference on applied infrastructure research implications of temporal resolution for modeling renewables-based power systems high-resolution modeling framework for planning electricity systems with high penetration of renewables the effect of weather uncertainty on the financial risk of green electricity producers under various renewable policies weather forecasting error in solar energy forecasting impact of wind power uncertainty forecasting on the market integration of wind energy in spain international aspects of a power-to-x roadmap benefits of demand response in electricity markets and recommendations for achieving them from passive demand response to proactive demand participation decentralised storage and demand response: impact on renewable share in grids and buildings. world sustainable energy days (wsed) capacity mechanisms for electricity. eprs | european parliamentary research service internal energy market. fact sheets on the european union the demand for flexibility of the power system in the netherlands wind power myths debunked common questions and misconceptions a review of approaches to uncertainty assessment in energy system optimization models clean transport -support to the member states for the implementation of the directive on the deployment of alternative fuels infrastructure: good practice examples gas and the electrification of heating & transport: scenarios for 2050 heat electrification: the latest research in europe a comprehensive review of thermal energy storage a review of available technologies for seasonal thermal energy storage luxembourg: publications office of the european union lessons on technological learning for policy makers and industry. technological learning in the energy sector, lessons for policy. industry and science the transition of energy intensive processing industries towards deep decarbonization: characteristics and implications for future research the role of nearly-zero energy buildings in the transition towards post-carbon cities an assessment methodology of sustainable energy transition scenarios for realizing energy neutral neighborhoods technological learning in energy-environment-economy modelling: a survey a review of learning rates for electricity supply technologies a review of uncertainties in technology experience curves long-term payoffs of near-term low-carbon deployment policies decarbonising road transport with hydrogen and electricity: long term global technology learning scenarios modelling the evolutionary paths of multiple carbon-free energy technologies with policy incentives the role of technology diffusion in a decarbonizing world to limit global warming to well below 2 • c: an assessment with application of global times model multi-cluster technology learning in times: a transport sector case study with tiam-ucl. informing energy and climate policies using energy systems models power capacity expansion planning considering endogenous technology cost learning clean energy for all europeans. luxembourg: publications office of the european union assessing the energy-efficiency gap residential energy efficiency policies: costs, emissions and rebound effects the rebound effect and energy efficiency policy energy efficiency and consumption -the rebound effect -a survey infrastructure, investment and the low carbon transition. new challenges in energy security infrastructure transformation as a socio-technical processimplications for the governance of energy distribution networks in the uk a conceptual framework for understanding the social acceptance of energy infrastructure: insights from energy storage decentralized energy systems. european parliament's committee on industry temporally-explicit and spatially-resolved global onshore wind energy potentials designing a cost-effective co2 storage infrastructure using a gis based linear optimization energy model energy research and the contributions of the social sciences: a retrospective examination energy research and the contributions of the social sciences: a contemporary examination integrating social science in energy research what kind of socio-technical research for what sort of influence on energy policy? the reality of cross-disciplinary energy research in the united kingdom: a social science perspective the third wave. united states: bantam books a novel prosumer-based energy sharing and management (pesm) approach for cooperative demand side management (dsm) in smart grid prosumer based energy management and sharing in smart grid distributed energy systems on a neighborhood scale: reviewing drivers of and barriers to social acceptance the flexible prosumer: measuring the willingness to co-create distributed flexibility self-sustainable community of electricity prosumers in the emerging distribution system an optimized energy system planning and operation on distribution grid level-the decentralized market agent as a novel approach incentivizing prosumer coalitions with energy management using cooperative game theory socio-technical transitions and policy changeadvocacy coalitions in swiss energy policy transformative policy mixes in socio-technical scenarios: the case of the low-carbon transition of the german electricity system socio-technical evolution of decentralized energy systems: a critical review and implications for urban planning and policy agent-based modeling: methods and techniques for simulating human systems99. national academy of sciences agent-based modeling and simulation of competitive wholesale electricity markets. handbook of power systems ii agent-based modelling and simulation of smart electricity grids and markets -a literature review towards circular economy implementation: an agent-based simulation approach for business model changes agent-based modeling of energy technology adoption: empirical integration of social, behavioral, economic, and environmental factors70. environmental modelling & software multi-agent based distributed control architecture for microgrid energy management and optimization introduction to energy systems modelling combining bottom-up and top-down reconciling topdown and bottom-up energy/economy models: a case of tiam-fr and imaclim-r. chaire modélisation prospective au service du développement technological change in economic models of environmental policy: a survey advances in techno-economic energy modeling: costs, dynamics and hybrid aspects useful models for simulating policies to induce technological change comparison of top-down and bottom-up estimates of sectoral and regional greenhouse gas emission reduction potentials closing the gap? top-down versus bottom-up projections of china's regional energy use and co2 emissions hybrid modeling: new answers to old challenges introduction to the special issue of top-down and bottom-up: combining energy system models and macroeconomic general equilibrium models integrated assessment of energy policies: decomposing top-down and bottom-up extension of gams for complementarity problems arising in applied economic analysis energy-economy analysis: linking the macroeconomic and systems engineering approaches hybrid modelling: linking and integrating top-down and bottom-up models. the h2020 project. set-nav mathematical models for decision support methods to improve computing times in linear energy system optimization models integrating short term variations of the power system into integrated energy system models: a methodological review challenges in top-down and bottom-up soft-linking: lessons from linking a swedish energy system model with a cge model energy-economy analysis: linking the macroeconomic and systems engineering approaches bridging the gap using energy services: demonstrating a novel framework for soft linking top-down and bottom-up models from linking to integration of energy system models and computational general equilibrium models -effects on equilibria and convergence energis: a geographical information based system for the evaluation of integrated energy conversion systems in urban areas a gisbased decision support tool for renewable energy management and planning in semi-arid rural environments of northeast of brazil electricity sector flexibility analysis: the case of the north of the netherlands feasibility of storing co2 in the utsira formation as part of a long term dutch ccs strategy: an evaluation based on a gis/markal toolbox modelling flexible power demand and supply in the eu power system: soft-linking between jrc-eu-times and the open-source dispa-set model. institute of thermal technology soft-linking exercises between times, power system models and housing stock models linking agent-based energy market with computable general equilibrium model: an integrated approach to climate-economy-energy system the authors would like to thank paul koutstaal, pascal wissink, joost van stralen, somadutta sahoo, ö zge ö zdemir, bert daniels, german morales-españa, and other team members of the estrac project and other anonymous reviewers and the editor for their constructive comments, conversations, and feedback. the authors wish to acknowledge the support given by the estrac integrated energy system analysis project financed by the new energy coalition (finance code: 656039). the views expressed here are those of the authors alone and do not necessarily reflect the views of the project partners or the policies of the funding partners. supplementary data to this article can be found online at https://doi. org/10.1016/j.rser.2020.110195. optimization-based or simulation-based conceptual model linking framework for the low-carbon energy system modeling suite. key: cord-310872-2z1wnj63 authors: spellberg, brad title: alignment with market forces: the “re-whithering” of infectious diseases date: 2020-06-20 journal: open forum infect dis doi: 10.1093/ofid/ofaa245 sha: doc_id: 310872 cord_uid: 2z1wnj63 given constant emergence of new infectious threats, infectious diseases (id) should be one of the most attractive medical specialties to students and trainees. yet, id fellowship programs continue to not fill in the match, and id remains among the lowest paid specialties. approximately 35 years after dr. petersdorf first asked the question, we find ourselves once again wondering, “whither infectious diseases?” to answer this question, and align with predominant us market forces, id experts should push for the following: (1) restrictions regarding utilization of id diagnostics and antimicrobial agents; (2) pay-for-performance measures regarding antimicrobial prescribing rates; and (3) healthcare reform as called for by the american college of physicians to move away from fee-for-service medicine. einstein said, “continuing to do the same thing over and over and expecting a different result is the definition of insanity.” we must move towards alignment with market forces, to benefit our patients, society, and our colleagues. the last several decades have continually reminded the world of how important infectious diseases (id) remain to the health and welfare of people all over the planet. anthrax scared americans in 2001. severe acute respiratory syndrome (sars) terrified the world in 2003. in 2004, west nile virus became a global problem, from seemingly out of nowhere. in the first decade of the 21st century, id experts brought forth astounding advances in diagnostics and therapeutics that converted human immunodeficiency virus (hiv)/acquired immune deficiency syndrome from a death sentence to a manageable, chronic disease. meanwhile, the world was in the midst of a burgeoning crisis of rising antibiotic resistance and a collapse of new antibiotic development. in the years that followed, the 2009 h1n1 influenza pandemic struck. new therapies became available that made hepatitis c virus curable. ebola once again terrified the world in 2014, followed by the zika scare in 2015. moreover, years of intensive id effort on the antibiotic resistance front have begun to pay off, with promising very recent declines in antibiotic resistance rates, combined with a surge of new antibiotics becoming available [1] [2] [3] . and then of course, the once-ina-century global coronavirus 2019 (covid-19) pandemic struck [4] [5] [6] . within weeks, diagnostics became available for sars coronavirus 2, within months therapeutics began to become available, and we anticipate a vaccine within 1 to 2 years. infectious diseases experts once again became media celebrities. history has shown us that new infectious threats continually emerge, and successes have continually followed. surely then, the id community must be now basking in the glow of recognition and the public need for top talent to come into the id field. indeed, some might speculate that the covid-19 pandemic alone would lure top talent into the field, as hiv did 30 years earlier-or not. after decades of failed efforts to change, id remains very near the bottom of the renumeration ladder amongst physicians [7] . this poor remuneration is in an era of ever-worsening, crushing medical student debt, the average of which has doubled since 2000 [8] . indeed, id practitioners are 1 of only 2 subspecialties remunerated below general internists in the united states [8] , raising the obvious questionwhy should top talent choose to spend 2 or more additional years in training to end up making less money? whether students and residents are interested in the concepts of id or not, the ratio of debt to income for id relative to other specialties is a substantial deterrent to top talent entering the field [9] . in 2019, despite years of effort to reverse the trend, more than one third of us id fellowships did not fill, and 20% did not match any candidates at all [10] . what has gone wrong? the answer is very simple. the field of id has never adapted to the reality of market forces. in 1977, dr. robert petersdorf, legendary id physician and luminary of internal medicine, published an article in the new england journal of medicine in which he predicted the end of id as a specialty. his exact quote was, "even with my personal loyalty to infectious diseases, i cannot conceive of the need for 309 more infectious disease experts [ie, graduating fellows] unless they spend their time culturing each other" [11] . nearly a decade later, dr. petersdorf expanded on this theme in an incredibly prescient keynote lecture at the annual meeting of the infectious diseases society of america titled, "whither infectious diseases? memories, manpower, and money" [12] . he said, "in a fee-for-service environment…infectious disease practitioners have difficulty in making a living. there are few or no procedures. there is a lot of uncompensated phone time, and there is the need to visit several hospitals and spend a good deal of time traveling. in academic medical centers, infectious disease divisions are almost invariably loss leaders, and i know of few that are not heavily supported by university salaries, hospital salaries, and grants. infectious disease divisions do not earn enough in practice to make a go of it and also require subsidies from the higher-earning divisions in their parent departments" [12] . these realities called out by dr. pedersdorf 34 years ago remain just as true today, leading us to again ask in 2020, whither infectious diseases? some years ago, drs. liise-anne pirofski and arturo casadevall pointed out to me another flash of insight. one of the predominant handicaps of the id clinical specialty is that there is nothing we do that no one else can do. only oncologists prescribe cancer chemotherapy. only cardiologists do cardiac caths. only surgeons take patients to the operating room. what is it that only id practitioners do? we know antibiotics better than anyone, but anyone, up to and including the nurse practitioner at the walk-in clinic in the pharmacy on the corner, can prescribe them. we understand how to establish a microbial etiology of infections, and distinguish them from colonization, but there is little financial pressure on health systems to value this skill. we know hiv care better than anyone, but hiv fellows can be generalists, not subspecialists. viral pandemics may be infections, but they hit emergency medicine, hospitalists, critical care doctors, and primary care doctors harder than they hit the id specialists. there are no diagnostic tests or therapeutic options that only we can prescribe. add to this realization the fact that in a fee-for-service environment, the operating budgets of hospitals are highly dependent on high margin procedures [13] [14] [15] , not cognitive encounters. infectious diseases practitioners may be better at diagnosing and treating infections than those who practice other specialties, but to what financial advantage to healthcare systems that hang on by their fingernails with operating margins under 2% [16, 17] ? health systems have little room for expansion of costs into low return-on-investment service lines. how can the advantages id experts bring be monetized to justify higher id salaries? i am the chief medical officer of a large public hospital. like my colleagues in the c suite, i am responsible for ensuring that our hospital stays as close to within budget as is humanly possible [18] . running deeply in the red threatens hospital closure, harming the hundreds of thousands of patients per year we serve, and putting out of work 10 000 people. so, even though i am an id expert and love my clinical and research work, is it realistic to think that i can artificially inflate id salaries at my hospital above the level the market will bear? no, it is not. and if you cannot convince me to do that, good luck convincing anyone else. infectious diseases practitioners simply do not bring in revenue in a way that makes them a priority to health system operating margins in a fee-for-service environment. the encounter-based, fee-for-service healthcare payment model inherently cripples cognitive specialties' remuneration relative to proceduralists. cognitive services will never compete with the value that procedural encounters bring in a fee-for-service healthcare system. market forces are intrinsically against us. dr. petersdorf understood this nearly 35 years ago. for years, id representatives have lobbied for better reimbursement for id practitioners through the medicare relative value scale (rvs) update committee (ruc). these efforts are meritorious and should and will continue. nevertheless, they would be likely much more effective if the lobbing efforts were backed by and aligned with market forces. for example, only an orthopedic surgeon can implant an artificial hip; they have a monopoly, with no competition. if their reimbursement fell such that the specialty became less attractive, and the number of trained orthopedic surgeons fell, patients would demand access, and the imbalance of supply-demand for hip surgeries would intrinsically drive up reimbursement. however, all licensed practitioners function as competition for id. if the number of id specialists decline, we may argue that quality of care will decline, but there will be plenty of other practitioners who can do what the id practitioners can do. there is no market-forces alignment with lobbying for higher rvu reimbursement. it is just an argument of merit and quality, with no economic market force behind it. are there data that support this assertion? absolutely. a recent analysis underscored how few id specialists there are across the united states to handle the covid-19 pandemic [19] . years of lobbying for rvu increases have not led to changes in relative reimbursement or reversed the decline in fellowship applicants and trained id physicians. in the absence of id specialists, many other types of providers care for patients with covid-19. the covid-19 pandemic has not led to reversal of the problem, but rather it further exposed this inequity. what we must do is change the interaction between our specialty and the healthcare system such that id work becomes aligned with market forces. there are practical solutions at hand, but they will require legal and/or regulatory changes. therefore, they will require a shift in emphasis of our specialty lobbying efforts. these solutions would allow our specialty to work in concert with market forces rather than against them. first, id practitioners have unique expertise in the diagnosis and treatment of infections, which can lead to less antimicrobial resistance and superinfections, better outcomes, and lower cost for patients and health systems. unfortunately, that unique expertise is hard to monetize. anyone can read the result of a diagnostic or antimicrobial susceptibility study in the electronic medical record. in addition, anyone can prescribe an antimicrobial agent. however, only an expert understands whether further diagnostics (eg, molecular studies) are necessary to benefit the patient and/or public health. it also takes an expert to know specifically what to prescribe and, perhaps more importantly, when not too. nevertheless, nothing stops nonexperts from doing this work now, leaving id practitioners unable to monetize that hard-earned expertise. granting the ability to any licensed practitioner, with no specialty training, to interpret complex diagnostic or susceptibility results, or prescribe powerful, new antimicrobial agents, seems antithetical to health of the public [20] . only those who have undergone specialized id training, whether by accredited training or certification course, should be allowed (eg, via law, regulation, or medical staff credentialing) to interpret diagnostic/ susceptibility results or prescribe newly approved antimicrobial agents [20] [21] [22] . this change would have the potential to improve prescriptions, decrease antibiotic abuse, diminish selective pressure driving antibiotic resistance, and decrease cost. all of this would be of great advantage to our patients and to public health. it would also have the effect of finally granting to id practitioners alignment with market forces, by providing us something that only we can do, much as oncologists and proceduralists have long had. second, we must push to mandate public reporting of antimicrobial prescriptions at the system level and linking pay for performance measures to such reporting [21, 22] . systems that use at the highest end of antimicrobial agents, adjusted for disease severity, should receive payment penalties, whereas systems at the lower end receive payment bonuses. this construct is built upon the success of infection prevention, in which public reporting and pay-for-performance measures have successfully driven down hospital-acquired infection rates. as national-level financial penalties and rewards come into play, they will motivate health system c-suites to prioritize hiring and funding id experts to lead and staff antibiotic stewardship programs to improve the financial situation of the health systems [18] . we would finally achieve true alignment with id expertise and market forces governing health system operating margins. moreover, society would benefit from diminished selective pressure driving resistance, prolonging the efficacy of life-saving therapies (including future biological therapies such as phage or immune modulatory therapy). third, we should be pushing for true reform to the payment structure of our healthcare system. the us healthcare system is by far the most expensive in the world, by any measure, and for that cost it delivers bad outcomes, including higher mortality rates and shorter life spans than peer nations [23] . the fee-forservice basis of payment is a core driver of this high cost and bad value. paying for each episode of care, and in particular paying more for expensive, potentially dangerous procedures, encourages excess care delivery, which drives the operating margins of healthcare delivery entities [23] . expensive, high volume, dangerous procedural-based, fee-for-service encounters may be bankrupting the united states as a whole, but they make money for hospitals and health systems. that market force discrepancy between societal and health system advantage is not in the best interests of patients, doctors, or society as a whole. in contrast, payments that are made for population-based healthcare favor judicious use of resources to maximize benefit to society. cognitive specialists become the gatekeepers of precious limited resources available for healthcare in the latter model. therefore, in the reformed model, cognitive specialists who keep people from becoming sick and minimize waste and harm become more valuable to healthcare payers than those who conduct expensive procedures on patients who are already sick. the american college of physicians has recently called for fundamental healthcare reform in the united states based on just this principle [24] [25] [26] [27] . our specialty has been unfortunately silent in the aftermath of this call for change, to our own detriment. infectious diseases will always plague the world. the question is, how will our subspecialty evolve over time when our work and remuneration are in opposition to the predominant feefor-service market forces that govern us healthcare? years of pressing for incremental tweaks to physician reimbursement in this system have thus far not worked to change the equation. after so many years of effort, continuing to lobby without alignment with market forces seems unlikely to change reimbursement. if we want to fundamentally change the equation of id practitioner value in the us healthcare system, we need to change id work and remuneration to become aligned with market forces. this situation can be remedied with advocacy and lobbying, and it must be. furthermore, once having mustered the political will to do so, the case should be easy to make to the public, because it is clearly in the public's and our patients' best interest to have id experts as the stewards of infectious-related diagnostic and therapeutic modalities. we can limit waste and improve precision, which improve patient and population outcomes, and at lower cost to the system at large. however, thus far, we have lacked the will to make this case. it is time to do so. ensuring sustainability of needed antibiotics: aiming for the dart board sustainable discovery and development of antibiotics -is a nonprofit approach the future? multidrug-resistant bacterial infections in u.s. hospitalized patients covid-19-new insights on a rapidly changing epidemic coronavirus infections-more than just the common cold covid-19 -navigating the uncharted average student loan debt for medical school charting the future of infectious disease: anticipating and addressing the supply and demand mismatch id-fellowship-match-results-slight-declinesfrom-last-year/id the doctors' dilemma whither infectious diseases? memories, manpower, and money sb170-operating-room-procedures-united-states-2011. pdf. accessed 23 the list is in: hospitals' most profitable specialties factors of u.s. hospitals associated with improved profit margins: an observational study operating margins stabilize, but not-for-profit hospitals still vulnerable moody's finds how to pitch an antibiotic stewardship program to the hospital c-suite where is the id in covid-19? new societal approaches to empowering antibiotic stewardship the future of antibiotics and resistance seven ways to preserve the miracle of antibiotics bankrupt, and dying: how to solve the great american healthcare rip-off the american college of physicians' endorsement of single-payer reform: a sea change for the medical profession medical practice and quality committee of the american college of physicians. envisioning a better u.s. health care system for all: health care delivery and payment system reforms health and public policy committee and medical practice and quality committee of the american college of physicians. envisioning a better u.s. health care system for all: a call to action by the american college of physicians engel ls; health and public policy committee of the american college of physicians. envisioning a better u.s. health care system for all: coverage and cost of care key: cord-335166-60lfjfvs authors: hanney, stephen r.; kanya, lucy; pokhrel, subhash; jones, teresa h.; boaz, annette title: how to strengthen a health research system: who’s review, whose literature and who is providing leadership? date: 2020-06-23 journal: health res policy syst doi: 10.1186/s12961-020-00581-1 sha: doc_id: 335166 cord_uid: 60lfjfvs background: health research is important for the achievement of the sustainable development goals. however, there are many challenges facing health research, including securing sufficient funds, building capacity, producing research findings and using both local and global evidence, and avoiding waste. a who initiative addressed these challenges by developing a conceptual framework with four functions to guide the development of national health research systems. despite some progress, more is needed before health research systems can meet their full potential of improving health systems. the who regional office for europe commissioned an evidence synthesis of the systems-level literature. this opinion piece considers its findings before reflecting on the vast additional literature available on the range of specific health research system functions related to the various challenges. finally, it considers who should lead research system strengthening. main text: the evidence synthesis identifies two main approaches for strengthening national health research systems, namely implementing comprehensive and coherent strategies and participation in partnerships. the literature describing these approaches at the systems level also provides data on ways to strengthen each of the four functions of governance, securing financing, capacity-building, and production and use of research. countries effectively implementing strategies include england, ireland and rwanda, whereas west africa experienced effective partnerships. recommended policy approaches for system strengthening are context specific. the vast literature on each function and the ever-growing evidence-base are illustrated by considering papers in just one key journal, health research policy and systems, and analysing the contribution of two national studies. a review of the functions of the iranian system identifies over 200 relevant and mostly national records; an analysis of the creation of the english national institute for health research describes the key leadership role played by the health department. furthermore, who is playing leadership roles in helping coordinate partnerships within and across health research systems that have been attempting to tackle the covid-19 crisis. conclusions: the evidence synthesis provides a firm basis for decision-making by policy-makers and research leaders looking to strengthen national health research systems within their own national context. it identifies five crucial policy approaches — conducting situation analysis, sustaining a comprehensive strategy, engaging stakeholders, evaluating impacts on health systems, and partnership participation. the vast and ever-growing additional literature could provide further perspectives, including on crucial leadership roles for health ministries. keywords: biomedical research, capacity-building, evidence-based practice, health ministries, health research systems, health services research, policy-making, priority-setting, research utilisation, sustainable development goals, translational medical research background interest in strengthening health research systems has intensified following increasing recognition of the importance of research in achieving key goals such as universal health coverage [1] and the sustainable development goals (sdgs) [2] . however, achieving progress in health research faces many challenges, including securing sufficient funds [3] [4] [5] [6] [7] [8] [9] , building and retaining capacity [3, 7, [10] [11] [12] [13] [14] , producing research findings, and using both local and global evidence [1, [15] [16] [17] [18] [19] [20] . chalmers and glasziou [21] dramatically highlighted the extent of the challenges facing health research by claiming, in 2009, that even where there was funding and capacity, up to 85% of all biomedical research was wasted because it asked the wrong questions, was poorly designed, or was either not published or poorly reported, with only about 50% of studies being published in full. many of these challenges have long been recognised and the adoption of a systems approach advocated. in 2000, the bangkok declaration on health research for development promoted the importance of a systems approach, following consideration of how a health research system could "be integrated with a nation's health development plan" [15] . it suggested that establishing and strengthening an effective health research system needed coherent and coordinated health research strategies [15] . national strategies should have specific combinations of various health research system components, tailored to the country's circumstances. the who's knowledge for better health initiative involved further work on these issues [3, 16] . the mexico statement on health research, issued in 2004 by a ministerial summit, called for nations to take actions to strengthen their national health research systems (nhrss). it was endorsed in 2005 by the fifty-eighth world health assembly in a resolution committing its member states to strengthening their nhrss as a pathway to improve their overall health system [22] . as part of the initiative, pang et al. [3] developed a conceptual framework to guide the analysis and strengthening of health research systems, including development of a health research strategy. while this can be used for planning, monitoring and evaluation of health research systems, it did not claim to provide a precise blueprint. the framework defined a health research system as "the people, institutions, and activities whose primary purpose in relation to research is to generate high-quality knowledge that can be used to promote, restore, and/or maintain the health status of populations; it should include the mechanisms adopted to encourage the utilization of research" [3] . the framework indicates the range of constituent components and how they can best be brought together into a coherent system. it identified four main functions for an effective system, namely stewardship, financing, capacity-building (or creating and sustaining resources), and producing and using research [3] . each function is defined by operational components and consists of one or more of a total of nine such components. since then, progress is evidenced by analyses of developments in individual countries, including the national institute for health research (nihr) in england [23] [24] [25] , and in repeat surveys conducted in various who regions, including africa [4, 26, 27] and the pan-american health organization (paho) [28] . however, as reported by those surveys and other publications, many challenges remain. for example, in february 2020 a new analysis by the who global observatory on health r&d examined health research funding, concluding that "neglected diseases such as those on the who list of neglected tropical diseases remain very neglected in terms of r&d investments" [29] . nevertheless, there are various initiatives underway, including in who's regional office for europe, which commissioned an evidence synthesis on the topic as part of its action plan to strengthen the use of evidence, information and research for policy-making in the who european region [18] . the synthesis is published in the who region's health evidence network (hen) report series and consists of a scoping review addressing the question "what is the evidence on policies, interventions and tools for establishing and/or strengthening nhrss and their effectiveness?" [30] . the evidence synthesis focuses on the systems level and so primarily includes publications taking a systems approach at either the national or multi-national level. not surprisingly, health research policy and systems (harps) is the single largest source of papers included in the hen report. these were papers directly identified in the review's search or papers included in the hen report to illustrate a key point because they had been cited in one of the who reports or other systems-level collations of papers included in the synthesis. while the system level papers did provide considerable data about each function, limited resources to conduct the scoping review meant that we had to exclude papers focusing solely on one specific function of a health research system or on just one field of health research. as acknowledged in the hen report's agenda for further research, there is a large number of publications (papers and grey literature) covering each function [30] . therefore, reviewing all of these publications would be a major task but some exploration of the extent of the task, and the nature of such literature, could be informative. furthermore, additional papers are continuously emerging, including from the various initiatives that are ongoing or just underway, for example, the european health research network [31] . the three sections of this paper sequentially address the question of how to strengthen a health research system by: 1. describing key points and conclusions from who's hen report. 2. illustrating the nature of the ever-widening literature available on each function, or component, of a health research system by examining two sources in particular. first, the full range of papers published in harps in the 30 months up to february 2020. second, the range of data gathered from publications or interviews that is included in detailed studies of the national health research systems in two countries -iran [32] and england [33] ; between them, these two papers also illustrate diverse aspects of the additional material that could be drawn upon. 3. considering a key question in the analysis of the current and future initiatives, namely who is going to steer the development of health research systems? here, information and insights from the hen about this sometimes-controversial issue, along with wider continuing analysis, are drawn on in the more flexible and speculative way that can be undertaken in an opinion piece compared to a formal evidence synthesis. who's review, whose literature and who is providing leadership? who's review the evidence synthesis described by the hen report [30] starts by describing the importance of nhrss in helping to achieve universal health coverage [1] and the sdgs [2] . it goes on to analyse the challenges facing health research and describes how issues remain unresolved despite the development and application of a systems approach including who's framework for health research systems [3] . many countries do not have comprehensive national health research policies or strategies that would facilitate the introduction of a systems approach. therefore, challenges remain around two key and overlapping sets of issues. first, how to develop a systems approach to maximise the benefits from the research resources availablethis can be a challenge even in high-income countries with considerable research funding. second, how best to strengthen each specific function and component of a health research system [30] . the hen identifies two main systems-level approaches to strengthening nhrss. the first is comprehensive and coherent strategies, which can be contained in either policy documents, such as those from the english nihr [34], the irish health research board (hrb) [35] and the rwandan ministry of health [14] , or in specific legislation as in the philippines [36] . the second systems-level approach involves partnerships and multicountry initiatives, especially with international organisations. two initiatives from the west african health organization (waho) are particularly important examples [5, 37] . here, the ministries of health of the 15 west african member countries worked together in a joint initiative covering all the countries and with funding and expertise from a range of partners, including the council on health research for development (cohred), the canadian international research centre, the special programme for research and training in tropical diseases, and the wellcome trust. all who regions have seen multi-country activities by who and/or cohred to strengthen nhrss, including the repeat surveys that identify areas for action [4, 26, 28] . then, broadly using the who framework as the structure [3] , the hen identifies key points from systems-level literature on each of the four functions and nine components. the components of the stewardship and governance function include defining a vision, ethical review, research priority-setting, and appropriate monitoring and evaluation [3] . consultation with health system stakeholders should enhance the relevance of the research priorities to the healthcare system, with examples of extensive priority-setting engagement activities sometimes being seen as a key aspect of building the nhrs as in brazil [38] . evaluating the impact of research on policy and practice should help researchers to focus on achieving such impact and was therefore promoted in the world health report 2013 [1] . securing finance can involve obtaining funding from sources within the country and from external donors or multi-national organisations [30] . targets for research expenditure, such as the 2% of national health expenditure set by the 1990 commission on health research for development [39] , can usefully be brought into health research system strategies as in rwanda [14] . major health research strategies from countries within the european union can highlight the importance of european union funding as in france [40] , ireland [35] and malta [41] . requests for funding can be more effective when linked to other parts of the overall strategy, including identified priorities that need supporting through donor funding [42] and assessments of the benefits obtained from previous funding such as in england [24] . capacity-building involves building, strengthening and sustaining the human and physical capacity to conduct, absorb and utilise health research [3] . in 2016, santoro et al. [43] identified the generally low levels of research production in 17 countries of the former soviet union and south-eastern europe and made recommendations for the sustained investment in training and career development of researchers, which should go beyond scholarships for training abroad and involve comprehensive strategies to ensure clear career structures. strategies such as that from inserm in france set out comprehensive plans for capacity-building [40] and strategies in both england and south africa addressed priority gaps identified in the research capacity within the healthcare professions [34, 44] . donors can play an important part in building capacity but, recognising the need to avoid donor domination, often do so through partnerships. these can take diverse forms ranging from multi-country initiatives, such as that by waho, which included an initiative focusing on the challenges of postconflict countries but was unable to meet all the needs [37] , to accounts that focus on the partnership to address a broad range of capacity issues in a single country such as malawi [7] , to partnerships between individual institutions. examples of the latter can feature particular challengesthe james cook university in australia worked with the atoifi adventist hospital in malaita, the most populous province of the solomon islands, to start establishing health research system capacity on the island using an inclusive, participatory approach [45] . increasingly, there are also south-south partnerships, for example, an account of the panamanian health research system described how the country's first doctoral programme in biotechnology was established with support from acharya nagarjuna university in india [46] . the rwandan strategy described plans to tackle the 'brain drain' through making the country an appealing place to conduct health research in terms of job requirements and providing opportunities for career advancement [14] . the three mutually reinforcing components of the producing and using research function encourage the production of scientifically valid findings that are relevant for users and communicated to them in an effective manner [30] . major research funding bodies increasingly seek to address the waste issues raised by chalmers and glasziou [21] by working together in the ensuring value in research (evir) funders' collaboration and development forum. it issued a consensus statement committing the organisations signing it to "require robust research design, conduct and analysis" [47] . the forum is convened by the english nihr, the netherlands organization for health research and development, and the patient-centered outcomes research institute (united states) with the active support of major research funding organisations from australia, ireland (hrb), italy, sweden and wales, plus the special programme for research and training in tropical diseases [48] . the first waho intervention also worked to boost research publications, including by creating a regional peerreviewed, multilingual journal [5] . how research is produced can increase the chance that the evidence will be used in the health system, for example, the english nihr strategy noted that leading medical centres with substantial funding to conduct translational research can act as "early adopters of new insights in technologies, techniques and treatments for improving health" [34] . fostering the use of research requires specific knowledge translation and management approaches that draw on both locally produced and globally available evidence. various health research strategies promote the role of cochrane, including in england, where a unified knowledge management system to meet the needs of various stakeholders, including patients and their carers, involves funding both cochrane and a review centre focusing on the needs of the national health system [34]. in ireland, the hrb strategy facilitated evidence-informed decisions through promoting access to the cochrane library and supporting training in conducting high-quality cochrane reviews [35] . south africa cochrane featured as an important element in the nhrs [44] . the rwandan strategy stated that "the government of rwanda is committed to using research findings to make evidencebased decisions that will improve health in rwanda" [14] . it aimed to orientate various functions, including agenda-setting, monitoring and evaluation, and capacitybuilding, towards facilitating this challenging aim. the world health report 2013 highlighted various mechanisms that health research systems could adopt, including evipnet (evidence-informed policy network), to promote the use of research [1, 49] . the review also considers the effectiveness of approaches to strengthening nhrss. several reviews identified the effectiveness of the comprehensive approach taken by professor dame sally davies in creating the english nhrs [23, 25, 50] . the title of one analysis, 'nihr at 10: 100 examples, 10 themes, 1 transformation', emphasises that the success of the nihr depended on a range of elements being brought together in one transformation [25, 50] . one of the 10 themes was the involvement of patients in decisions about research priorities and processes and, based on this, another recent analysis highlighted england and alberta (canada) as having health research systems that had made important progress [51] . davies herself reflected on the success of the nihr and stated: "what we envisaged was integrating a health research system into the health care delivery system so that the two would become interdependent and synergistic" [24] . who's regional office for africa drew on their series of surveys of the performance of countries in building nhrss and analysed the data from the 2014 and 2018 surveys using the nhrs barometer that they developed to score progress on a range of items linked to the list of nhrs functions [11, 26] . in the 2014 survey, the rwandan system was identified as the best performing and it, along with the majority of systems, was reported to have further improved in the 2018 survey; by then, south africa was reported to have the best performance in africa. the surveys also illustrate how the multi-country approach makes a useful contribution to strengthening nhrss by helping to target action. furthermore, the waho interventions made some progress but, while the evaluations identified the importance of political will and leadership provided by waho's parent organisation of west african states, they also emphasised that building capacity for a whole nhrs is a significant task requiring commitment over the long-term [17, 37] . the hen review collated a range of examples of tools for nhrs strengthening. these were identified from the systems level discussions of nhrs strategies and partnerships and/or the major reports calling for nhrs strengthening such as the world health report 2013 [1] . the hen lists these in an annex [30] . the discussion in the hen draws on the literature that was included to identify five key policies that those responsible for strengthening nhrss could consider [30] , namely conduct context, or situational, analyses to inform strengthening activities [5, 34, 35, 37, [52] [53] [54] , develop a comprehensive and coherent strategy [14, [34] [35] [36] , engage stakeholders in the development and operation of the strategy [7, 23, 34, 35, 38, 41, 44, 51, [55] [56] [57] [58] [59] , adopt monitoring and evaluation tools that focus on the objectives of the nhrs, including health improvement [1, 14, 24, 60, 61] , and develop partnerships [5, 11, 28, 37, 62] . examples of the evidence to support or illustrate each policy are given in table 1 . in summary, therefore, this section shows that the who evidence synthesis, published as a hen report [30] , provides a firm basis for decision-making by policy-makers and research leaders looking to strengthen the health research system in their country. it analyses, in turn, the individual functions and components within a system and identifies a series of tools that can be used for strengthening many of them. finally, this section highlights the five crucial policy approaches that the hen report suggests can be applied as appropriate to the context of the country (table 1) . as noted above, the hen was a scoping review and focused on the literature at the systems level rather than on publications (papers and grey literature) related solely to specific functions, types or fields of research [30] . therefore, there is scope for further work to incorporate an even wider range of publications than the 112 included in the hen review [30] . the discussion in the hen suggests that further research could usefully take the form of a series of reviews on the extensive literature on each of the nhrs functions or components, which could then be collated [30] . just two of the many available sources illustrate the nature of the vast literature available on each function, or component, of a health research system and the way the literature on that, and the system level developments, is ever-widening. first, we can examine the papers published in harps, the specialist journal in the field of building nhrss. second, we can focus on two very different but detailed studies of individual nhrssone conducted for a phd thesis to show the 50 year history of the development of all the functions in the iranian health research system [32] and the other an interview-based study to understand the factors behind the creation of the nihr with its new strategy [33] . in terms of further reviews of the literature on specific functions or components, harps would probably be a key source. in the summer of 2017, an analysis by the retiring editors of the papers published in the journal from its inception in 2002 identified many papers that had been published on each of the functions or components of a health research system [63] . while this editors' analysis was included in the hen review because it organised its discussion of the papers at the systems level, the individual papers in it were, in general, only included in the hen review if they, too, adopted a systems approach at the national or partnership level, or were also cited in a report such as the world health report 2013 [1] . examples of such papers include viergever et al. on priority-setting [59] , bates et al. on capacity-building [64] , and lavis et al. on the support tools for evidence-informed policy-making [65] . therefore, many additional papers related to specific functions (or fields) could be consulted, in a formal review or otherwise, in any future series of reviews, each with a narrow focus on strengthening a specific function. to further inform this current opinion piece, a quick 'hand-search' was conducted of the papers published in harps in the 30 months since the previous analysis in mid-2017 [63] . this again identified a wide range of papers on specific components, especially priority-setting, evaluation of research impacts, capacity-building and the translation of research (or knowledge mobilisation). various papers linked the final two points and discussed capacity-building and knowledge translation [13, 66] . such a focus is entirely consistent with the aim described by the incoming editors in autumn 2017 of bringing "all elements of the research-policy world togethersuch that the research which is done is useful and that it is used" [67] . in this more recent phase of harps, there have also been important papers on issues related to the policies 'recommended' at the end of the hen and listed above, including the contribution of stakeholder engagement in research [68] . the more recent papers could sometimes provide useful further tools on specific functions. their narrow focus meant they had not been directly included through the hen search and, further, they had not been included in any of the major reports also used as sources for tools such as the world health report 2013 [1] . in some instances, this was because they were too recent, for example, the isria statement by adam et al. [69] describing the ten-point guidelines for an effective process of research impact assessment prepared by the international school on research impact assessment (isria). even more recently, the intervention scalability assessment tool, developed by milat et al. [70] , was proposed for use not only by health policy-makers and practitioners for selecting interventions to scale up but also to help design research to fill evidence gaps. this analysis of the papers from just one journal reinforces the message that there is likely to be a plentiful supply of literature for a future review on any of the main specific components. this message is further reinforced by a more detailed analysis of the papers in harps in the first 2 months of 2020. articles on the main components of a nhrs were supplemented by some important papers on topics that are highly relevant but which feature less frequently in harps. these include a study aimed at reducing the research waste that arises from disproportionate regulation by examining the practices for exempting low-risk research from ethics review in four high-income countries [71] , the global observatory's paper on research funding described earlier [29] , a study on the governance of national health research funding institutions [72] , and one on a more recent topic of growing significancean analysis of attempts to boost gender equality in health research [73] . additionally, some of the papers on specific components, such as impact evaluation or use of evidence, are extending the analysis. examples include consideration of how research impact assessments are conduct context or situational analyses of current national position to inform strengthening activities cohred, in particular, has developed tools to assist countries in conducting situational analyses as part of wider advice [52] and this approach was an important element in the waho interventions being successful to the extent that they were [5, 37, 53, 54] . strategies informed by analyses of their current situation include those for the english nihr [34] and the irish hrb [35] develop a comprehensive and coherent nhrs strategy comprehensive and coherent strategies with at least some degree of success (as seen in progress on some or all of the nihr functions) had set out how they intended to take action on the range of health research system functions and components, even if not necessarily explicitly using the who framework [3] ; examples include the strategies for the english nihr [34], the irish hrb [35] , and in the philippines [36] and rwanda [14] engage stakeholders in the development and operation of the nhrs strategy strategy documents such as those for the nihr [34] and hrb [35] , plus ones in british columbia [55] , malta [41] and new zealand [56] , describe the importance and/or range of stakeholders engaged in developing the strategy. articles describing the approach in south africa [44] and zambia [57] also highlighted the importance of wide stakeholder engagement. an analysis of stakeholder engagement in the creation and operation of the nihr identified it as making a key contribution to its success [23] . there is increasing support for the engagement of stakeholders in setting the priorities for research as well as in research processes and translation [7, 38, 51, 58, 59] adopt monitoring and evaluation tools that focus on the objectives of the nhrs, including health system improvement a range of documents, including ones on the nihr [24] , hrb [60] and rwandan strategies [14] , and the world health report 2013 [1] , demonstrate the importance of adopting monitoring and evaluation approaches that include a focus on assessing the impacts of research on health polices/practice and the economy, e.g. through application of the payback framework [60, 61] develop/participate in partnerships across regions, bilaterally or within the nhrs examples of progress made by partnerships between countries, sometimes along with international organisations and donors, include the waho interventions [5, 37, 53, 54] and the work of who regional offices for africa [11, 26] implemented in practice within research organisations [74] and how evidence is used in decision-making in crisis zones [75] . to illustrate the volume of studies being produced, there has been a flurry of studies, in the first 2 months of 2020 alone, on the collaboration and coproduction of health research. the titles include 'building an integrated knowledge translation (ikt) evidence base: colloquium proceedings and research direction' [76] , 'using a 'rich picture' to facilitate systems thinking in research coproduction' [77] , 'exploring the evolution of engagement between academic public health researchers and decision-makers: from initiation to dissolution' [78] , 'research co-design in health: a rapid overview of reviews' [79] , and 'conceptualising the initiation of researcher and research user partnerships: a metanarrative review' [80] . finally, another article in may 2020 presented a new conceptual model for health research systems to strengthen health inequalities research [81] . here, we have focused on just one journal, harps, because it was the largest single source of papers in the hen report, which totalled 140 publications (additional publications were included to the 112 in the review to help set the background, provide examples of key tools, etc). however, even with the review's focus on the system level, harps only provided 22% (31 out of 140) of the publications; 31% (43 of 140) came from other journals and 47% (66 of 140) were other types of publication. if the focus was shifted to including papers on specific functions it is highly likely that there would be a higher proportion of papers from other journals. the authors of two single-country papers on the development of the health research system, mansoori [32] about iran and atkinson et al. [33] on the creation of the nihr in england, both highlight the importance of context but also claim their findings could have wider application. examining these two papers is also informative because of the differences between the studies, including one being located in a low-or middle-income country, and the other not. mansoori's narrative review of studies addressing the health research system of iran included 204 relevant and mostly national records, categorised using an approach informed by the functions and components of who's nhrs framework [32] . the papers and grey literature documents included were all available in english or persian, and mostly published in journals other than harps, and illustrate the vast literature available at a global level on the various components of a nhrs. they informed an impressively detailed account of the various nhrs components and the attempts to strengthen them. for example, the account of the development of the national level ethical overview includes a fully documented chronology of the progress over 25 years and some insightful analysis of how the progress was facilitated by the pivotal role of professor bagher larijani, who was a prominent medical practitioner, leading researcher and founder of the medical ethics research centre in iran. he was able to "use the confidence that iranian authorities had in him as an opportunity" [32] . while mansoori's review was included in the hen review, only a tiny fraction of the available data about iran could be included, primarily in a brief description of the system's effectiveness [30] . however, the full paper could usefully inform the approach of researchers and/or policy-makers planning a detailed analysis of their own nhrs prior to embarking on exercises to strengthen it, and "[t]he findings emphasized that improvement of hrs functions requires addressing context-specific problems" [32] . as an illustration, mansoori's review identified a need for "a more systematic, inclusive" approach to research priority-setting [32] and, in the same stream of research, she co-led just such a priority-setting exercise to help address the knowledge gaps related to achieving both iran's national health policies and the sdgs [82] . atkinson et al. examined the creation of what might be viewed as the most successful attempt to strengthen a health research system in their paper ''all the stars were aligned'? the origins of england's national institute for health research' [33] . compared with mansoori, the authors adopted a different but equally detailed approach in their analysis, which was conducted principally through interviews and a witness seminar but also drew on the existing literature and documents [33] . they showed how the formation of the nihr was led from the department of health by a key group driven by sally davies. they aimed to improve patient care through both the strengthening of evidence-based medicine and through boosting the infrastructure to facilitate pharmaceutical clinical trials that would also meet wider industrial and economic goals. as with mansoori's study, consideration was given to how the full analysis could be informative to any planned detailed study or reforms in any other country. the key observations were similar to the recommendations from the hen report with a focus on stakeholder engagement and building support: "[t]wo measures likely to contribute to political support are to place the greatest emphasis on 'problem' rather than 'investigation' research, and to devote attention to measuring and reporting research 'payback'" [33] . atkinson et al.'s paper is also a link to the other main source considered here because it was a recent paper published in harps. in summary, if further analysis and research beyond that in the who evidence synthesis [30] is thought to be relevant in the particular country looking to strengthen its health research system, this opinion piece indicates some of the types of additional sources of information that are available and how they might be organised. the vast literature on each function and the ever-growing evidence base are illustrated by considering papers in just one key journal, harps, and analysing the contribution of two national studies. a review of the functions of the iranian system identifies over 200 relevant, mostly national, records and an analysis of the creation of the english nihr describes the key leadership role played from the health department. who is providing leadership? the above analysis demonstrates that there is no shortage of useful material on which to draw when strengthening health research systems. however, key questions remain as to who might best lead or steer attempts to strengthen such a system. the papers by both mansoori [32] and atkinson et al. [33] illustrate that, where a key committed individual has the capacity and opportunity to provide leadership, this can be a vital element in making progress. however, the institutional factors are also crucial. the hen developed the argument that a department or ministry of health will have a particular interest and perhaps experience in promoting research agendas that meet the needs of the healthcare system and in helping to develop mechanisms to use the findings from such research, where appropriate, to inform local policy and practice [30] . the health ministry or a research council responsible to it played an important role in the various systems identified above as being effective, as was also the case in the waho initiative [30] . in some cases, as with zambia, more progress was made once the ministry of health elected to play a more important role, sometimes in place of other stakeholders [57] . examples of the important role that health ministries can play were described in the 2013 world health report, including on paraguay: "the support of the minister of health backed by the president of paraguay has been a key factor in the development of a national health research system" [1] . additionally, naturally enough, the activities of the various who regional offices in boosting nhrss tend to focus on working with the national ministries of health, including work in europe [31] and by paho [28] . conversely, several analyses illustrate that progress in strengthening the nhrs might be limited where key parts of the ministry of health, for whatever reason, do not provide support [9, 83] . nevertheless, some disadvantages or dangers were identified when the ministry of health plays the leading role. first, in england prior to the creation of the nihr as well as in some other countries, the research funds controlled by the health ministry were sometimes appropriated by other parts of the health system when they were under particular pressure for resources [84] . similarly, there have been a few reports that health research funding lost out when donor funds that had previously been allocated specifically for health research programmes were replaced by donations of funds to be allocated by the nation's own health system according to its own priorities [85, 86] . one way of attempting to mitigate the danger is, as undertaken by the nihr and described by atkinson, by building support for health research through measuring and reporting the payback from research [24, 33] . the second danger arises because, traditionally, many researchers argued that the best science came when they had the freedom to identify the key research topics, rather than having priorities set by others [84] . therefore, they argued, the responsibility for funding and organising health research should be left to organisations that are part of the research system and independent of the health system [84] . furthermore, despite the growth of interest in coproduction approaches noted above, there have also been recent doubts raised about the assumption that coproduction is always the most appropriate approach [87] . this issue clearly requires sensitive handling. indeed, atkinson et al. [33] argue that one of the great successes of the nihr is that this issue has been so skilfully handled by the nihr that external input, or stakeholder engagement, in setting agendas has become widely accepted and the structures created give ministers a sense of ownership without sacrificing scientific independence. the efforts of waho [5, 37] and the who regional offices for africa and paho [11, 26, 28, 62] indicate that partnerships can be helpful. in europe, the who regional office worked with member states to create the european health research network, which is intended to help nations with limited nhrss who wish to make more progress [31] . partnerships can provide important support and encouragement, but the evidence suggests there must be strong political will somewhere within the political and/ or health systems for a health research system to be fully strengthened. the central asian countries in who's european region seem to provide an illustration of this point. a cohred collaborative initiative successfully resulted in situation analyses being produced in each country and then jointly discussed as the basis for action [88] , but according to the analysis by santoro et al. [43] , limited progress seems to have been made in the subsequent years. the importance of partnerships and collaboration in focusing research efforts in an extreme crisis, with a leadership role for the who, has been seen in the race to find treatments for covid-19 and vaccines against severe acute respiratory syndrome coronavirus 2 (sars-cov-2), which causes the covid-19 disease [89] . in many nhrss across the globe, including in the philippines, scientists are coming together to participate in who's solidarity trial, which will test the safety and effectiveness of various possible therapies for treating covid-19 [90] . sarah gilbert, leader of oxford university's jenner institute's work on developing one of the leading vaccine candidates explained that cooperation was vital for tackling the crisis: "work is continuing at a very fast pace, and i am in no doubt that we will see an unprecedented spirit of collaboration and cooperation, convened by who, as we move towards a shared global goal of covid-19 prevention through vaccination" [91] . a key issue going forward is how such cooperation can be built on in strengthening nhrss into the future. for now, it is recommended that a prospective study be conducted to analyse all that is being done in different nhrss to speed up research during the pandemic, with a view to taking lessons about cooperation, partnerships and other matters into strengthening nhrss in the future [89] . the who evidence synthesis, published as a hen report [30] , provides a firm basis for decision-making by policy-makers and research leaders looking to strengthen the health research system in their country. it identifies five crucial policy approaches that can be applied as appropriate to the context of the countryconducting situation analyses, sustaining a comprehensive strategy, engaging stakeholders, evaluating impacts on health policies and practices, and partnership participation. it also analyses, in turn, the individual functions and components within a system and identifies a series of tools that can be used for strengthening many of them. if further analysis and research is thought to be relevant in the particular country looking to strengthen its health research system, this opinion piece indicates some of the types of additional sources of information that are available. the opinion piece also discusses aspects of the sometimes-controversial question of who should lead or steer attempts to strengthen nhrss. again, the context of the particular nation will be crucial in determining the most appropriate course to take, as emphasised by both mansoori [32] and atkinson et al. [33] , but at least some involvement of the ministry of health is likely to be beneficial; additionally, sometimes, key individuals can play a crucial leadership role in strengthening the whole system or one component. in countries with a less developed tradition of conducting health research, partnerships with other countries and/ or with international organisations can help lead the progress and learning for all partners. the valuable role that international organisations, such as who, can play in leading partnerships and cooperation to strengthen health research systems is being highlighted during the covid-19 crisis. overall, therefore, the full who hen report not only provides a detailed analysis of nhrs strengthening, it also provides a structure within which an even wider and ongoing literature can be considered. additionally, it contains a perhaps more nuanced account, on which this paper builds, of some aspects of the literature around the issue of who should provide leadership in developing nhrss and identifies the importance of ministry of health involvement. world health organization. the world health report 2013: research for universal health coverage transforming our world: the 2030 agenda for sustainable development. general assembly resolution 70/1 knowledge for better health: a conceptual framework and foundation for health research systems national health research systems in the who african region: current status and the way forward the west african health organization's experience in improving the health research environment in the ecowas region donor funding health policy and systems research in low-and middle-income countries: how much, from where and to whom implementing a national health research for development platform in a low-income country: a review of malawi's health research capacity strengthening initiative why do case studies on national health research systems matter? identifying common challenges in low-and middleincome countries the palestinian health research system: who orchestrates the system, how and based on what? a qualitatitve assessment health research systems in six pacific island countries and territories developing the african national health research systems barometer national research for health system mapping in 5 countries in the eastern mediterranean region and perspectives on strengthening the systems institutional capacity to generate and use evidence in lmics: current state and opportunities for hpsr kigali: rwanda ministry of health international organizing committee world report on knowledge for better health: strengthening health systems. geneva: who world health organization. national health research systems: report of an international workshop. international workshop on national health research systems, cha-am action plan to strengthen the use of evidence, information and research for policy-making in the who european region. regional committee for europe 66th session evidence map of knowledge translation strategies, outcomes, facilitators and barriers in african health systems review of published evidence on knowledge translation capacity, practice and support among researchers and research institutions in low-and middle-income countries avoidable waste in the production and reporting of research evidence geneva: who who needs what from a national health research system: lessons from reforms to the english department of health's r&d system ten years of the nihr: achievements and challenges for the next decade the national institute for health at 10 years: an impact synthesis. summary report strengthening national health research systems in the who african region: progress towards universal health coverage towards universal health coverage: can health research systems deliver contextualised evidence to guide progress in africa? national research for health systems in latin america and the caribbean: moving towards the right direction? resource allocation for biomedical research: analysis of investments by major funders what is the evidence on policies, interventions and tools for establishing and/or strengthening national health research systems and their effectiveness? health evidence network (hen) synthesis report 69. copenhagen: who regional office for europe strengthening national health research systems: implementation of the action plan to strengthen the use of evidence, information and research for policymaking in the who european region evolution of iran's health research system over the past 50 years: a narrative review all the stars were aligned'? the origins of england's national institute for health research best research for best health: a new national health research strategy. the nhs contribution to health research in england. london: department of health action. health research board strategy republic act no. 10532. manilla: republic of the philippines evaluation of regional project to strengthen national health research systems in four countries in west africa: lessons learned defining and implementing a national policy for science, technology, and innovation in health: lessons from the brazilian experience commission on health research for development. health research: essential link to equity and development institut national de la santé et de la recherche médicale draft national strategy for health research and innovation. il-kalkara: malta council for science and technology research for health and health system strengthening in africa mapping health research capacity in 17 countries of the former soviet union and southeastern europe: an exploratory study south african health review 2012/13. durban: health systems trust research workshop to research work: initial steps in establishing health research systems on malaita, solomon islands the panamanian health research system: a baseline analysis for the construction of a new phase ensuring value in health-related research ensuring value in research (evir) funders' collaboration and development forum copenhagen: who regional office for europe building health research systems: who is generating global perspectives, and who's celebrating national successes? public involvement and health research system governance: a qualitative study priority setting for research for health: a management process for countries. geneva: council on health research for development priorities and policies in national research for health systems in west africa. geneva: council on health research for development the state of the research for health environment in the ministries of health of the economic community of the west african states (ecowas) vancouver: michael smith foundation for health research wellington: ministry of business, innovation and employment/ministry of health developing a national health research system: participatory approaches to legislative, institutional and networking dimensions in zambia public involvement in health research systems: a governance framework a checklist for health research priority setting: nine common themes of good practice health research: making an impact. the economic and social benefits of hrbfunded research. dublin: health research board how can payback from health services research be assessed? cross-sectional study on the utilization of the pan american health organization/world health organization policy on research for health knowledge for better health' revisited: the increasing significance of health research systems -a review by departing editors-in-chief indicators of sustainable capacity building for health research: analysis of four african case studies support tools for evidenceinformed health policymaking (stp) policymakers' experience of a capacity-building intervention designed to increase their use of research: a realist process evaluation building a bright, evidence-informed future: a conversation starter from the incoming editors engaging stakeholders in implementation research: lessons from the future health systems research programme experience isria statement: ten-point guidelines for an effective process of research impact assessment intervention scalability assessment tool: a decision support tool for health policy makers and implementers exempting low-risk health and medical research from ethics reviews: comparing australia, the united kingdom, the united states and the netherlands governance of health research funding institutions: an integrated conceptual framework and actionable functions of governance understanding the athena swan award scheme for gender equality as a complex social intervention in a complex system: analysis of silver award action plans in a comparative european perspective how do organisations implement research impact assessment (ria) principles and good practice? a narrative review and exploratory study of four international research funding and administrative organisations supporting the use of research evidence in decision-making in crisis zones in low-and middle-income countries: a critical interpretive synthesis building an integrated knowledge translation (ikt) evidence base: colloquium proceedings and research direction using a 'rich picture' to facilitate systems thinking in research coproduction exploring the evolution of engagement between academic public health researchers and decisionmakers: from initiation to dissolution research co-design in health: a rapid overview of reviews conceptualising the initiation of researcher and research user partnerships: a meta-narrative review a novel conceptual model and heuristic tool to strengthen understanding and capacities for health inequalities research the iranian chnri health research priority setting group, et al. setting research priorities to achieve long-term health targets in iran the political undertones of building national health research systems: reflections from the gambia freedom and need: the evolution of public strategy for biomedical and health research in england towards fair and effective north-south collaboration: realizing a programme for demand-driven and locally led research bringing health research forward the dark side of coproduction: do the costs outweigh the benefits for health research? strengthening health research systems in central asia: a system mapping and consultative process from covid-19 research to vaccine application: why might it take 17 months not 17 years and what are the wider lessons who western pacific: philippines. ph solidarity trial for covid-19 treatments receives green light from ethics review body. press release. who. 22 carving a path towards a covid-19 vaccine publisher's note springer nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations authors' contributions sp, lk and sh planned the original phase of the who evidence synthesis, including the search strategy. lk led the original literature search and contributed article selection and data extraction and analysis. ab and sh planned the second phase of the evidence synthesis. tj led the second phase of the literature search and contributed to the article selection and data extraction. ab contributed to the final version of the health evidence network report. sh led the data extraction and analysis and drafting of the report. sh conducted the additional analysis of the literature and initial drafting for this opinion piece. all authors commented on the opinion piece and approved the final version. key: cord-311651-v2ff33jd authors: long, nathaniel; wolpaw, daniel r.; boothe, david; caldwell, catherine; dillon, peter; gottshall, lauren; koetter, paige; pooshpas, pardis; wolpaw, terry; gonzalo, jed d. title: contributions of health professions students to health system needs during the covid-19 pandemic: potential strategies and process for u.s. medical schools date: 2020-07-15 journal: acad med doi: 10.1097/acm.0000000000003611 sha: doc_id: 311651 cord_uid: v2ff33jd the covid-19 pandemic poses an unprecedented challenge to u.s. health systems, particularly academic health centers (ahcs) that lead in providing advanced clinical care and medical education. no phase of ahc efforts is untouched by the crisis, and medical schools, prioritizing learner welfare, are in the throes of adjusting to suspended clinical activities and virtual classrooms. while health professions students are currently limited in their contributions to direct clinical care, they remain the same smart, innovative, and motivated individuals who chose a career in health care and who are passionate about contributing to the needs of people in troubled times. the groundwork for operationalizing their commitment has already been established through the identification of value-added, participatory roles that support learning and professional development in health systems science (hss) and clinical skills. this pandemic, with rapidly expanding workforce and patient care needs, has prompted a new look at how students can contribute. at the penn state college of medicine, staff and student leaders formed the covid-19 response team to prioritize and align student work with health system needs. starting in mid-march 2020, the authors used qualitative methods and content analysis of data collated from several sources to identify 4 categories for student contributions: the community, the health care delivery system, the workforce, and the medical school. the authors describe a nimble coproduction process that brings together all stakeholders to facilitate work. the learning agenda for these roles maps to hss competencies, an evolving requirement for all students. the covid-19 pandemic has provided a unique opportunity to harness the capability of students to improve health. other ahcs may find this operational framework useful both during the covid-19 pandemic and as a blueprint for responding to future challenges that disrupt systems of education and health care in the united states. is a global pandemic, threatening individuals and disrupting communities and economies. 1 the impact on all u.s. health systems continues to escalate, and health systems are undergoing rapid operational change to meet the demands. 2 as leaders in complex care, research, and medical education, u.s. academic health centers (ahcs) are in many ways at the epicenter of this storm, which means that our medical students are fellow travelers, and their professional pathways are profoundly affected. [3] [4] [5] no aspect of the tripartite mission of ahcs-clinical care, education, and research-has been untouched by the crisis. clinical operations are pivoting to covid-19 preparation; research labs are closing as part of mitigation efforts; and medical schools, prioritizing learner welfare, are in the throes of adjusting to suspended clinical activities and remote learning. 6 the overall impact on medical education programs is unprecedented and rapidly evolving. but while normal learning activities are disrupted and schools are scrambling to adjust, the passion of our students to contribute to the needs of people and health systems in this crisis remains very much alive and well. [7] [8] [9] although many faculty find teaching to be rewarding and validating, traditional curricular designs have not used medical students' potential to cocreate and offer value to the health system. as a result, faculty often consider teaching medical students in the first few years of their curriculum as extra work, added time, and decreased efficiency. 10 substantive learner contributions are often viewed as coming in year 4 or during residency. recent advances in medical education, however, have focused on developing the concept of value-added medical education. 10, 11 this is defined as "experiential roles that have the potential to positively impact individuals and population health outcomes, cost of care, or other processes within the healthcare system, while also enhancing student knowledge, attitudes, and skills in the clinical or health systems sciences." 12 examples of value-added roles include student patient navigators, clinical care extenders, advocates, and resource managers. [12] [13] [14] [15] [16] necessity is the mother of invention, and currently, u.s. ahcs have the unique opportunity to operationalize the concept of value-added medical education in this escalating crisis, exploring how medical and other health professions students can contribute to the acute and overwhelming needs of health systems and the populations they serve. in the face of the evolving covid-19 pandemic, our team used qualitative methods and a content analysis of data collated from several sources-a literature review, a modified crowdsourcing method used to gather student perspectives, discussions with health system leaders, and the covid-19 pandemic poses an unprecedented challenge to u.s. health systems, particularly academic health centers (ahcs) that lead in providing advanced clinical care and medical education. no phase of ahc efforts is untouched by the crisis, and medical schools, prioritizing learner welfare, are in the throes of adjusting to suspended clinical activities and virtual classrooms. while health professions students are currently limited in their contributions to direct clinical care, they remain the same smart, innovative, and motivated individuals who chose a career in health care and who are passionate about contributing to the needs of people in troubled times. the groundwork for operationalizing their commitment has already been established through the identification of value-added, participatory roles that support learning and professional development in health systems science (hss) and clinical skills. this pandemic, with rapidly expanding workforce and patient care needs, has prompted a new look at how students can contribute. at the penn state college of medicine, staff and student leaders formed the covid-19 response team to prioritize and align student work with health system needs. starting in mid-march 2020, the authors used qualitative methods and content analysis of data collated from several sources to identify 4 categories for student contributions: the community, the health care delivery system, the workforce, and the medical school. the authors describe a nimble coproduction process that brings together all stakeholders to facilitate work. the learning agenda for these roles maps to hss competencies, an evolving requirement for all students. the covid-19 pandemic has provided a unique opportunity to harness the capability of students to improve health. other ahcs may find this operational framework useful both during the covid-19 pandemic and as a blueprint for responding to future challenges that disrupt systems of education and health care in the united states. health system needs during the covid-19 pandemic: potential strategies and process for u.s. medical schools communication with frontline physician educators-to develop strategies for leveraging student capabilities and contributions. in this article, we (1) articulate 4 categories in which medical students can contribute to the work of u.s. ahcs and the wellness of their communities; (2) describe the educational benefits for learners and the alignment with health systems science (hss) competencies; and (3) highlight a process for coproduction between students, medical schools, and the health system. our goal is to develop an operational framework that other ahcs can use in this crisis and to provide a blueprint for responding to future challenges that disrupt our systems of education and health care. in mid-march 2020, anticipating the arrival of the covid-19 pandemic in south central pennsylvania, education and student leaders from the penn state college of medicine (pscom) collaborated with penn state health system leaders on a process for strategically identifying avenues for meaningful student contributions to meeting workforce needs. primed by a well-established curriculum in patient navigation and hss and encouraged by an invested faculty, over 400 students (primarily medical students with contributions from students in public health [mph and drph], nurse practitioner, and physician assistant programs) made commitments to contribute to the pandemic within the first 6 weeks. the initial planning phase, which was rapid and nimble, was guided by the goal of safely integrating students into supervised interprofessional work projects that would contribute directly to the needs of penn state health and the community, while also providing educational benefits. a covid-19 response team was quickly established with student codirectors. to capture the energy of the moment, several early projects were piloted while a concurrent intentional analysis of needs was performed. we sought to collect a broad and diverse range of ideas from multiple stakeholders, including students, educators, and system leaders. we then performed a focused literature review, inclusive of our prior works related to value-added medical education. [12] [13] [14] [15] [16] our team employed a modified crowdsourcing method, which included soliciting open-ended suggestions for activities from all pscom medical students in google docs, on social media platforms, and via personal email communications. 17 simultaneously, we asked both administrative leaders and clinical faculty within our health system to identify vital system needs. a lead author (j.d.g.) also personally contacted physician educators at several u.s. ahcs to explore their perspectives on possible needs. lastly, we reviewed information on social media platforms to gather ideas from other medical schools. using this dataset, we performed a content analysis, collapsed ideas into similar roles, and grouped all tasks into overarching categories. 18, 19 our underlying assumptions were that students bring maturity and commitment, medical and health care delivery awareness, technological competency, creativity in problem solving, and community understanding to the current challenge, making them uniquely positioned to provide valuable contributions. on the basis of our analysis, we identified 4 categories of projects for our health professions students. below we describe each of these 4 categories, and table 1 lists potential and active tasks (n = 38) with descriptions for each. we identified various roles in which students could contribute to increasing the understanding of the pandemic in the neighborhoods and communities in the region surrounding pscom. these roles leverage students' knowledge of the communities' strengths and weaknesses to promote large-scale awareness and strategies to remain safe and flatten the curve of the pandemic (e.g., social distancing, handwashing, etc.). several projects and tasks focus on expanding the workforce for emerging pandemic-specific needs (e.g., telehealth encounters, follow-up check-ins, patient rescheduling, etc.) as well as the ongoing work of caring for non-covid-19 patients (discharge navigation, etc.). in addition, health system leadership identified the need for an evidencebased medicine team to assist them in answering questions. this team creates daily succinct summaries of emerging information regarding covid-19 from a breadth of perspectives to facilitate strategic and clinical decision making within the ahc. we identified several projects and tasks that allow students to support the needs of caregivers and staff-the workforce of the health system. ensuring frontline clinicians and staff can remain in their jobs requires assisting them in their life responsibilities outside of work, including childcare, maintaining a food supply, running errands, and attending to personal well-being. the modification of clerkships and in-person educational experiences has forced medical schools into uncharted territory. the curriculum has transitioned exclusively to an online format. some traditional activities can be replicated or approximated, but many will require creative and innovative changes that necessitate active student engagement. the students are key stakeholders and contributors, offering technological expertise and piloting innovative ideas for curriculum development and implementation. after identifying a comprehensive list of projects and tasks, we developed a process for prioritizing activities by working with students and medical school and health system leadership. our first action was to collaborate with the legal department to ensure appropriate safety and regulatory steps, based on principles that the medical school and health system leadership had created and agreed upon, were in place. most notably, it was evident that activities students engaged in would need to be accomplished remotely, without direct patient or team contact, and under adequate supervision of penn state faculty or staff. in addition, an important distinction emerged between efforts that students developed independently and operated outside of our health system, such as regional blood banking programs, and efforts that the health system solicited from students and cocreated with them by using their ongoing feedback and input (e.g., covid-19 follow-up calls, contact tracing, etc.). while participation in all activities is completely voluntary, projects officially approved by the health system and medical school require a standard of recognition that adheres to the guiding principles and includes either curricular credit or monetary compensation. regardless of training year, each student participating in a task force is enrolled for a time-variable elective credit (e.g., 1 credit for each 10 hours, maximum 4 credits), which can fulfill 1 elective requirement for postclerkship students; the associate dean for health systems education (j.d.g.) provides formal oversight for all electives. see table 1 for a description of projects and designation of key features. we quickly learned the importance of a formal organizational structure featuring coproduction, well-defined roles, clear workflow, and regular communication. figure 1 depicts our covid-19 response team, the relationship of students to pscom and penn state health leadership, and a workflow designed to consider student and faculty ideas while ensuring collaboration and prioritized task force assignments. with a rapidly evolving landscape of system and patient needs, student efforts must be channeled thoughtfully to maximize value and safety. this structure was established to ensure that new tasks and ideas continue to meet legal requirements and are free a since the creation of the task forces at the penn state college of medicine in mid-march 2020, the number of students involved in task forces has been dynamic. six weeks into the program (mid-to late april), the total number of students on task forces or awaiting placement = 418; students onboarded to functioning task forces = 348 (medical students = 323, public health students = 13, nurse practitioner students = 8, physician assistant students = 4). at that date, the breakdown by category was as follows: category 1 = 167 (contact tracing = 106), category 2 = 144 (patient navigator discharge = 37), category 3 = 0, category 4 = 37 (medical school and logistics planning = 33). priorities were assigned to the task forces on the basis of how many of the 3 key features (3 right-hand columns) they have. b educational value relates to learning in systems-based practice/health systems science (e.g., identification of health system gaps and improvement processes, application of core systems principles to the pandemic, contribution to system needs), interpersonal and communication skills, interprofessional collaboration, knowledge for practice, and professionalism. of ethical or coercive conflicts, formally approved by pscom, and systematically address the most pressing needs of the health system. a developmental cascade starting with principles and workflow allowed for further clarification and prioritization of potential opportunities (see table 1 ). many of these tasks were variations of roles that had already existed in our curriculum (e.g., students as patient navigators), and others emerged from system needs (e.g., evidence-based medicine consultants, telehealth extenders, etc.). [12] [13] [14] [15] [16] several tasks were proposed by faculty members who were shifting their research and clinical work to meet the needs of the pandemic and sought collaboration to extend the workforce to perform these tasks (e.g., an internal medicine physician leading contact tracing, a geriatrician leading long-term care facility outreach). the willingness of leaders and faculty to invest time and energy in this system highlights the value-added nature of the task forces. in mid-to late april 2020, within 1 month of discussion and planning, we launched 17 task forces, and the work has substantially benefitted our health system and community. for example, by the end of april, the contact tracing task force has onboarded 100+ students who have called over 900 patients with covid-19 and their contacts to inform and educate them on infection control measures. we fully anticipate the work of these task forces will change, blend figure 1 depiction of the covid-19 response team workflow in identifying, prioritizing, and establishing task forces for student contributions during the pandemic. ideas from the health system, medical school, and students are reviewed and prioritized by the covid-19 response team, composed of health system leadership (executive vice president/chief clinical officer and/or his or her designee) and medical school leadership (vice dean for educational affairs, associate dean), and 5 student leaders. the covid-19 response team chooses ideas for task forces that meet legal guidelines, are deemed of high need to the health system, and are of educational value (see table 1 ). the covid-19 response team establishes each task force by designating a student and a faculty leader, who work together to create goals, collaborate with entities needed for specific tasks (e.g., department of health), and oversee the recruitment/preparedness of students. faculty leaders include a diverse group of physicians (e.g., surgery, internal medicine, family and community medicine), nurse practitioners, and social workers. once a process has been developed and the number of students needed has been determined, students are onboarded via voluntary sign-ups and formally enrolled in the elective. the faculty member and student lead ensure appropriate training for each task and assist with assessing student progress, logging of hours, compliance, and contributions to the task force. huddle groups between various levels of the covid-19 response team allow for collaboration and feedback to ensure the dynamic needs of the task forces, health system, and medical school are met. with other groups, or phase out as the pandemic continues to evolve and affect our communities as well as our personal and professional lives. however, this coproduction process can provide the longitudinal and sustainable methods for work through the pandemic, even as health systems face unprecedented adaptations. this model for the development and implementation of task forces with interprofessional stakeholders provides an efficient and effective process to adapt and respond to changing health care needs, even if legal parameters and hospital policies change. such changes may include the most extreme hypotheticals, such as having students rejoin the workforce on the frontlines. 20 we learned the following lessons, which other medical schools may want to consider. hss, with roots in george engel's biopsychosocial model and the systembased practice competency domain of the accreditation council for graduate medical education, has emerged as a major focus in medical education and includes competencies in health care delivery, social determinants of health, high-value care, change management, and systems thinking. [21] [22] [23] [24] [25] [26] this growth has mirrored an increased recognition of the importance of hss in the care of patients and populations and reflects the needs of health systems that are undergoing transformation. educators and students increasingly understand how the contexts of our patients and care environments are critical to learning and health. at the same time, health system leaders are increasingly looking for the hss perspective and skill set in their workforce. 27 seven years ago, pscom began a comprehensive curricular program of classroom and experiential learning in hss. 28 those students who developed an evolved skill set-serving as patient navigators for individuals in need, obtaining white and yellow belt certifications in operational excellence, 29 and honing a nascent systems-based perspective toward health care practice-are the same students who have been organizing themselves to perform similar and additional roles during the covid-19 pandemic. their formal studies have been interrupted, but their professional path, the way they see themselves as collaborators and contributors in health care, is thriving. at the same time that education and experience in hss have primed our students to contribute in this pandemic, the crisis is setting up a unique opportunity for students to deepen and extend their learning in hss, as well as interpersonal communication, knowledge for practice, and professionalism. recognizing this, our medical school has been able to accelerate the approval of a health systems and covid-19 response elective for students across all phases of the curriculum. in line with an outcomesbased educational model, task force activities and reflection assignments map to our hss competencies, and continual feedback ensures a growth mindset for all parties involved (formal course proposal available upon request). we believe the covid-19 pandemic is an ideal opportunity for students to contribute to the needs of the health system while also helping them develop systems-based competencies and the mindset necessary for forming an evolved professional identity. [30] [31] [32] [33] "systems citizens" are physicians who not only care for individual patients but also know how to perform in developing systems of care, embodying the motivation to grow and contribute to the evolution of the health care system itself. continually improving our systems of care-in response to pandemics and beyond-will require all physicians to have this skill set and professional identity. [34] [35] [36] [37] coproduction between students, medical school, and health system the convergence of education and system needs around hss described above has created a unique opportunity to realize coproduction between key stakeholders. 38 while the potential for this collaboration has been present for some time, there has been no urgent motivation to make it happen. there have been important initiatives, but there has also been a tendency for stakeholders on all sides to function independently. the covid-19 pandemic has upended many businessas-usual perspectives and activities and has vividly and tragically brought the potential for coproduction into high relief. the table has been set by health systems and education programs, and now we have passionate, skilled students sitting at the table with a wide range of stakeholders and leaders to think and plan together. coproduction is no longer just a possibility or an idea; it is playing out in our programs and likely in medical centers all over the country. the covid-19 pandemic is challenging our ahcs and our local, national, and global communities in tragic and unprecedented ways. confronting this crisis is prompting widespread reexamination of what we thought we knew, how we act, and how we respond to adversity. as we are distracted by events and responses, we are also opening to new opportunities. it has been easy to talk about students as our junior colleagues and the future of our profession, but those words do not always translate into what we do and how we do it. it is comfortable to envision a steady progression of professional development through incremental biomedical coursework, clinical training, and responsibility. but the reality is that for several years, hss and associated added-value roles have been opening up a distinct, equally important thread within students' professional development. the feasibility, validity, and impact of these approaches have been well documented and widely disseminated. [12] [13] [14] [15] [16] and we know that health systems value and very much need an hss professional development pathway for their future workforce. many educators and system leaders have recognized this need, but work toward achieving the goals of authentic codevelopment of roles and the invitation to contribute has been slow. in this tragic time of covid-19, nothing is slow. many things, too many bad but some good, have been brought into stark relief. one that stands out clearly on the positive side is how we can come together-the health system and education leaders, faculty and staff, and students-to make a difference. our hope is that the program of aligning missions and employing coproduction to create value-added participatory roles for students can serve as a blueprint for other medical schools as we continue to navigate these uncharted waters. world health organization american hospital association. updates and resources on novel coronavirus (covid-19) the evolving academic health center: strategies and priorities at emory university the transformation of academic health centers: meeting the challenges of healthcare's changing landscape shifting academic health centers from a culture of community service to community engagement and integration association of american medical colleges. coronavirus resources for the academic medicine community medical students, sidelined for now, find new ways to fight coronavirus. the new york times value-added medical education: engaging future doctors to transform health care delivery today meaningful roles for medical students in the provision of longitudinal patient care compensation to a department of medicine and its faculty members for the teaching of medical students and house staff transforming from centers of learning to learning health systems: the challenge for academic health centers a constructive reframing of student roles and systems learning in medical education using a communities of practice lens value-added clinical systems learning roles for medical students that transform education and health: a guide for building partnerships between medical schools and health systems adding value to the health care system: identifying value-added systems roles for medical students how can medical students add value? identifying roles, barriers, and strategies to advance the value of undergraduate medical education to patient care and the health system a practical guide for implementing and maintaining value-added clinical systems learning roles for medical students using a diffusion of innovations framework crowdsourcing as a method of transdisciplinary research-tapping the full potential of participants transforming qualitative information: thematic analysis and code development research design: qualitative, quantitative, and mixed methods approaches coronavirus cases rise rapidly, but cuomo sees progress. the new york times health systems science curricula in undergraduate medical education: identifying and defining a potential curricular framework ama education consortium. health systems science domain of competence: systems-based practice systemsbased practice: the sixth core competency systems-based practice defined: taxonomy development and role identification for competency assessment of residents health systems science in medical education: unifying the components to catalyze transformation aligning education with health care transformation: identifying a shared mental model of "new" faculty competencies for academic faculty educating for the 21 st -century health care system: an interdependent framework of basic, clinical, and systems sciences implementing six sigma and lean toward a common taxonomy of competency domains for the health professions and competencies for physicians reframing medical education to support professional identity formation moving beyond nostalgia and motives: towards a complexity science view of medical professionalism physicians' professional responsibility to improve the quality of care the fifth discipline: the art and practice of the learning organization. rev and updated ed systems citizenship: the leadership mandate for this millennium educating patient-centered, systems-aware physicians: a qualitative analysis of medical student perceptions of value-added clinical systems learning roles curricular transformation in health systems science: the need for global change coproduction of healthcare service acknowledgments: the authors would like to express their deepest gratitude to all the health professions students at penn state college of medicine who have dedicated time to these initiatives to improve patient health and strengthen our community. key: cord-337275-phgfpzbt authors: andrew, jones; nikolay, strigul title: is spread of covid-19 a chaotic epidemic? date: 2020-10-20 journal: chaos solitons fractals doi: 10.1016/j.chaos.2020.110376 sha: doc_id: 337275 cord_uid: phgfpzbt the covid-19 epidemic challenges humanity in 2020. it has already taken an enormous number of human lives and had a substantial negative economic impact. traditional compartmental epidemiological models demonstrated limited ability to predict the scale and dynamics of covid-19 epidemic in different countries. in order to gain a deeper understanding of its behavior, we turn to chaotic dynamics, which proved fruitful in analyzing previous diseases such as measles. we hypothesize that the unpredictability of the pandemic could be a fundamental property if the disease spread is a chaotic dynamical system. our mathematical examination of covid-19 epidemic data in different countries reveals similarity of this dynamic to the chaotic behavior of many dynamics systems, such as logistic maps. we conclude that the data does suggest that the covid-19 epidemic demonstrates chaotic behavior, which should be taken into account by public policy makers. furthermore, the scale and behavior of the epidemic may be essentially unpredictable due to the properties of chaotic systems, rather than due to the limited data available for model parameterization. the developing covid-19 pandemic challenges humanity in 2020. according to aggregated worldwide data posted at the international worldometer.info website, more than 15 million were infected by covid-19 and more than 600,000 people died in relation to this disease (as of july 20, 2020) . the pandemic has already caused a huge economic loss due to national lock-downs, travel restrictions, and global distractions of trade and manufacturing chains. as the pandemic developed around the globe, severe border control and travel limitations were implemented that led to unprecedented national isolation. different countries, while being isolated, implemented various strategies to limit epidemic spread: many countries implemented extended national-level lock-downs including closure of businesses, schools and stay-at-home orders (for example france, italy, germany, austria, hungary, south korea, and more), some introduced practically no such lock-down measures (for example, sweden, belarus, and brazil). many countries implemented only regional lock-downs (for example, china and the usa). the overall challenge for the development of scientifically-based covid-19 containment strategy is the apparent unpredictability of this pandemic [5] . traditional compartmental epidemiological models demonstrated quite limited ability to predict the scale and dynamics of this epidemic in different countries [5, 25, 11] . this apparent unpredictability of the covid-19 pandemic creates an additional challenge for governments around the globe, who need more accurate predictions to develop a reasonable containment strategies [16, 9] . this limitation of traditional modeling approaches can be partially explained by the novelty of the virus and limited relevant epidemiological data necessary for model parameterization [28] as well as statistical problems in model calibration [25] . in particular, until recently, we did not know the rate and mechanisms of the transmission of covid-19 virus as well as its biology and ability to survive and spread outside host organisms [19, 11] . in a previous study, [4] demonstrated that the coronavirus raw data in china's first two months of the disease suggest chaotic growth, similar to other epidemics like h1n1 and measles. mathematical chaos theory originated from studying deterministic systems represented as differential equations where initial conditions dictate their behavior. in these systems sensitivity to the initial conditions was so large that the systems were practically unpredictable. this was originally studied by poincaré in the 1880s in relation to the three-body problem in astronomy, an example of which is the earth-moon-sun celestial system [23] . as poincaré critically stated, "it may happen that small differences in the initial conditions produce very great ones in the final phenomena" [23] . in physical systems described by differential equations, systems which experience this effect are described as having "high sensitivity" to initial conditions. one example which demonstrates this concept is a bowling ball; imagine a very small spin on the ball at the beginning of its roll. this small spin in combination with the oiled bowling lane could cause it to move wildly off-course, making it "highly-sensitive" to initial conditions. due to the effect poincaré found, "measurements made on the state of a system at a given time may not allow us to predict the future situation" [3] . these systems are now termed "chaotic." unpredictability due to highly-sensitive reliance on initial conditions inspired the term "deterministic chaos." after poincaré's studies, the deterministic chaotic behavior was discovered in numerous dynamical systems and confirmed experimentally [15, 6, 2, 20] . the spread and "fade out" cycle of epidemic diseases often exhibits deterministic chaos. qualitative analysis of that deterministic chaos can offer more information than a "multifactorial stochastic paradigm of causation" [21] . in the late 80s, scientists noticed that measles outbreaks exhibited deterministic behavior. by studying this they hoped to quantify conditions for the maintenance of [an] infection in human populations" [7] . in 1991, grenfell published his study of the chaotic measles outbreaks in the paper "chance and chaos in measles dynamics." that study found that certain parameters in the chaotic disease system created deterministic behavior. for example, applying his model to the city of copenhagen and mapping with poincaré's methods produced drastic results: one example is that doubling the immigration rate into the city could completely eliminate the fade-out of measles [7] . similar findings in relation to covid-19 could have wide-ranging societal implications. in this work, we consider the covid-19 epidemic within the framework of complex dynamic systems [14] . this framework is broadly applied in modern ecology, social and medical sciences [21, 13, 6, 2, 20] . in particular, the covid-19 pandemic can be considered as a complex phenomena developing simultaneously at multiple temporal and spatial scales varying from individual level to the global scale. within this framework, particular disease dynamics patterns result from major self-organisation mechanisms within the system. the chaotic behavior is a common phenomenon in complex systems, and we hypothesise that the unpredictability of the epidemic scope in different countries is a fundamental property of this dynamic system which demonstrates chaotic behavior. in order to examine this hypothesis we have analysed covid-19 epidemic data from different countries. in particular, we examine if the covid-19 epidemic demonstrates a chaotic regime based on the analysis of observed data. we do not aim to derive or validate a correct dynamical system model. instead we consider epidemiological data collected in different isolated countries as an independent observations of the same dynamical system. in order to evaluate whether or not the spread of coronavirus is chaotic, we employ the following criteria, derived from poincaré's definition of chaos [23, 4] [23] . in the study, we find extremely high variation between countries, even when normalizing by patient-zero date and total population of each country. 2. sensitive -does the growth of the virus experience large regions of activity? are there real reasons for regions of activity, specifically changes in the physical system? this criterion has precedent in the aforementioned university of florida / wuhan university paper [4] . later in the study, we present multiple examples of high regions of sensitivity in the spread of covid-19, as revealed by the derivatives of the system. 3. numerically unpredictable -can the behavior of the system change unexpectedly from one point to another? this criterion was also introduced in the university of florida / wuhan university paper [4] . we found numerous examples of sets of countries which exemplified unpredictability by behaving similarly and then suddenly diverging, which we discuss later in the paper. 4. deterministic -what causes activity in the system? is the system random, or is it determined by some factors of the physical system, as required in order to be chaotic? this is a qualitative criterion derived from poincaré's definition of chaos [23] . we present cases where real-world changes such as mandated lockdowns had a profound impact on the spread of the disease, suggesting deterministic behavior. the study concluded that the spread of covid-19 exhibits the major qualitative characteristics of chaotic systems. most countries show a roughly logistic growth curve, but activity in the second derivative revealed great variability in system behavior. when examined under the context that infectious epidemics in the past have exhibited chaotic behavior, the study concluded that the spread of covid-19 suggests that the epidemic is a chaotic system. johns hopkins university has continuously collected data on the coronavirus epidemic from various sources such as the world health organization, including daily cases per country, which is the data source used for this study [10] . the data is organized by country and cumulative cases by date. a sample with the first five days of one country is shown in table 1 . we considered the data from the beginning of the global data set, january 22nd, 2020, to may 30th, 2020. each data-point tells the number of confirmed covid-19 cases in that country by that day. in the example shown in table 1 , the united states had five confirmed cases by january 26th, 2020. with almost 35,000 data-points corresponding to 267 countries or territories, the study required a dynamic analysis tool instead of analyzing static charts. in order to investigate deeper, a web-based interface was designed and developed to allow for close inspection of the data through interactive charts. the evolution of the covid-19 epidemic was mapped over time, in terms of total number of confirmed cases per country per day. data was adjusted to account for country population and date of first-confirmed-case in order to accurately compare the spread behavior between countries. the raw data was extracted as an csv file and analysed using original javascript software [12] , the graphing utility plotly [22] was employed to generate all the graphs. figure 1 demonstrated the raw data as percentages of each country's population. since the data only pertains to the total number of confirmed cases, not accounting for decreases like recovery and death, the total number of cases in every country/region increases or remains constant at all times. highlighted in figure 2 , almost all regions show 1 of 3 possible parts of the beginning of a logistic graph: a curved ramp-up (russia), or a curved ramp-up into roughly a line (us), or an upward curve into a line which curves toward horizontal (spain), which is a logistic curve. this is sensible for an infectious disease with a maximum number of infections, that maximum being the population of each country. per capita.png so far, these patterns don't directly suggest chaos: it still seems possible there might be some parameters which control the shape of the curves predictably, potentially population density, weather patterns, etc. figure 3 demonstrates the case data shifted to show the percent of the population that had been infected as a function of the number of days since the first case in each country, instead of the absolute date. this graph is more revealing about the relative behavior of the epidemic in each country. it is clear the virus ran its course faster in some countries, where the growth seems to have flattened in fewer days. for example, only 55 days after the first case in iceland, the country had almost completely stopped the spread. meanwhile, the us at 130 days in was still seeing almost linear positive spread. comparison of iceland and the us these differences confirm that there are a large number of potential solutions or equilibrium and varying behavior between countries over time. another key observation from this graph is that countries which seemed to follow similar growth curves can very rapidly diverge. for example, as shown in figure 5 , in the first 40 days of the epidemic in ireland, the netherlands, and turkey, the percentage of the population that was infected grew almost identically in each country. however, between days 40 and 42, the three countries' curves diverged greatly, to such an extent that by day 60, ireland had almost three times the infected population percentage as turkey, and turkey and the netherlands are 25% apart as well. this example exhibits unpredictability because, by poincaré's definition, the behavior following day 40 cannot be predicted by the state at day 40, since there are clearly multiple possible behaviors. this is one example confirming unpredictability in the spread of covid-19. it is also important to note that these three countries are not alone in displaying unpredictability after formerly-similar spread of covid-19. another example is kuwait, france, and sweden, shown in figure 6 . until approximately day 70, the infected percentage in all three countries had grew with similar shaped curves. however, within days after that point, the infected percentage in kuwait spiked drastically, while it entered linear growth in france and sweden. by 10 days later, even france and sweden diverged, with sweden's infected percent still growing linearly while the spread slowed down in france. by poincaré's definition of unpredictability, knowing the spread behavior in sweden from day 60 to 120 would not provide any accurate prediction of the behavior in kuwait or france, suggesting a chaotic nature.yet another example of unpredictability is cabo verde and new zealand, shown in figure 7 . in cabo verde and new zealand, the disease spread almost identically until day 25, at which point it continued to grow similarly in each country until day 40, and then the behaviors sharply diverge: cases grow steeply linearly in cabo verde, but level off almost entirely in new zealand. this example further demonstrate that spread of covid-19 is unpredictable, because at any given point there are multiple possible behaviors in the time following that point. the observation of unpredictability fulfills one of poincaré's key criteria for choatic systems. we looked at the rate of spread of covid-19, the derivative of the number of cases, adjusted similarly to figure 3 . this data, shown in figure 8 shows the spread rate as a function of the number of days since the first patient. negative derivatives represent decreasing numbers of cases, which shows corrections to data, since the data set does not account for real decreases in active cases (by recovery or death). while the raw data shows mostly similar shaped, roughly logistic trajectories with some small bumps and jumps, the daily rate of growth shows a lot of variation in behavior. for example, in the us from day 92 to day 99, just one week, the daily growth rate decreases by 30% and increases back to almost the same starting point (shown in figure 9 ). we also see drastically different behavior between different regions, unlike the roughly logistic graphs of the raw data. one example is the comparison of the spread rates of the us and spain. by their respective day 30s, the spread rate in spain started to decline, while the us spread rate was just starting to increase, as shown in figure 10 . the amount of unpredictable variation within a country and different possible models for each country suggest chaos. however, most countries still follow a very rough pattern of increasing and then leveling-off and decreasing spread rates. the second derivative helps to measure this sensitivity ( figure 11 ) because its behavior shows activity in the system [4] . the rate of change of the spread rate in each region fluctuates greatly. the us ( figure 12 ) seems to oscillate between roughly two values, spain and italy ( figure 13 ) appear to exhibit a single heart-beat-like pulse surrounded by ramp-up and ramp-down, and russia ( figure 14) and germany show clusters of high activity. in summary, the results reveal the following: 1. large number of solutions -while most countries have a roughly logistic curve of covid-19 growth, there is a huge amount of variation in: the time it takes to reach the same point in that logistic curve, the highest percent of the population which the logistic curve reaches, and the degree of curvature of the logistic model. 2. sensitive -the second derivative shows clusters, spikes, and oscillations, revealing that the system is highly sensitive due to the high amount of activity in the system instead of steady or constant behavior. 3. numerically unpredictable -while the raw data shows roughly predictable shapes, the extreme variation first and second derivatives of different countries reveal a high amount of variability, making the growth of the system unpredictable. furthermore, there are examples of growth curves appearing identical and then suddenly diverging. we may predict that spread in the us will level off logistically, but we cannot tell when that will occur based on the us data or other countries' curves. one important question remaining is what causes the activity: is the system random, or is it determined by some factors of the physical system, as required in order to be chaotic? qualitative investigation is required to answer that, and even then we cannot definitively determine the factors contributing to every change in every country. however, we investigated some of the countries and found good examples suggesting the system is deterministic. new cases take up to 14 days to show symptoms and therefore be reported [18] . in iceland, lockdown was initiated on march 24th [29] , day 25 when shifted. exactly two weeks after that change, there are few new cases reported ( figure 15 ) and almost all activity shown by the second derivative ceases (figure 16). this is also the day with the sharpest increase in testing in ireland [8] , which would explain the jump in the data and higher activity following that day. in bahrain, re-opening efforts began on april 9th [1] , which is the likely cause of a the immediately following spikes in number of cases ( figure 19) . these examples suggest that real changes in the physical conditions of the system are causing changes in the spread of the virus. the outcome, amount and rate of spread, is determined by these real factors. this suggests the behavior of the system is deterministic, not random, a key qualifier for a chaotic system. complex natural systems often demonstrate chaotic behavior. however, rigorous proof of chaos from empirical data or experimental observations alone is a substantial challenge. in this work, rather than attempt a rigorous mathematical proof, we investigate the hypothesis that the covid-19 pandemic exhibits chaotic behavior by mapping the disease over time from available epidemiological data. our results suggest that the covid-19 epidemic exhibits deterministic chaos. the overall predictions of the sir model demonstrate a typical sigmoidal curve. this functional response is characterised by an exponential growth stage, inflection point and slowdown growth phase towards a horizontal asymptote. the first wave of covid-19 pandemics in different countries demonstrates similar behavior ( figure 3 ). empirical sigmoidal models such as the logistic curve are also broadly employed for modeling of covid-19 and other epidemics [27, 26, 24] . the discrete counterpart of the logistic curve is a well-known logistic map model that demonstrates a chaotic behavior [17] . our examination of the epidemic dynamics in different countries reveals amazing similarity to the chaotic behavior known in this and many other dynamics systems. we conclude that the scale of the epidemic is essentially unpredictable due to fundamental reasons rather than due to the limited data available for model parameterization. we find that the chaotic behavior in the spread of covid-19 suggests that it is a deterministic chaotic system, which should be taken into account by public policy makers. through use of an interactive data map, it was shown that the spread of covid-19 exhibits the major characteristics of chaotic systems, namely, determinism, high sensitivity, large number of equilibria, and unpredictability. when examined under the context that infectious epidemics in the past have exhibited chaotic behavior, we conclude that spread of covid-19 is likely a chaotic system. we may be able to gain some insights into its behavior, such as the common logistic pattern, but we cannot assume that it will follow a logistic path in any one country and we cannot numerically predict the behavior of a particular logistic curve. ☒ the authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper. ☐the authors declare the following financial interests/personal relationships which may be considered as potential competing interests: claration of interest statement coronavirus: bahrain one of first nations to ease lockdown as malls reopen complex population dynamics: nonlinear modeling in ecology deterministic chaos theory: basic concepts first two months of the 2019 coronavirus disease (covid-19) epidemic in china: real-time surveillance and evaluation with a second derivative model when will the coronavirus outbreak peak non-linear dynamics for clinicians: chaos theory, fractals, and complexity at the bedside. the lancet chance and chaos in measles dynamics covid-19: 28 more deaths, 500 further cases offline: covid-19 and the nhs-"a national scandal novel coronavirus (covid-19) cases data predictive mathematical models of the covid-19 pandemic: underlying principles and value of projections original software: covid-19 graphs: javascript ecosystems and the biosphere as complex adaptive systems complex adaptive systems: exploring the known, the unknown and the unknowable deterministic nonperiodic flow effective containment explains subexponential growth in recent confirmed covid-19 cases in china simple mathematical models with very complicated dynamics covid-19 information hub: what you need to know a systematic review of covid-19 epidemiology based on current evidence complexity, simplicity, and epidemiology chaos, population biology, and epidemiology: some research implications plotly graphing libraries: javascript sur leséquations de la dynamique et le probleme des trois corps estimation of covid-19 dynamics "on a back-of-envelope": does the simplest sir model provide quantitative parameters and predictions? chaos why is it difficult to accurately predict the covid-19 epidemic? real-time forecasts of the covid-19 epidemic in china from february 5th to february 24th covid-19 epidemic in italy: evolution, projections and impact of government measures modeling the epidemic dynamics and control of covid-19 outbreak in china hert samkomubann: ekki fleiri en 20 mega koma saman key: cord-332432-q7u943k6 authors: hofkirchner, wolfgang title: a paradigm shift for the great bifurcation date: 2020-06-30 journal: biosystems doi: 10.1016/j.biosystems.2020.104193 sha: doc_id: 332432 cord_uid: q7u943k6 this paper is an attempt to achieve an understanding of the situation the evolution of humanity is confronted with in the age of global challenges. since global challenges are problems of unprecedented complexity, it is argued that a secular paradigm shift is required away from the overemphasis on allegedly neutral standpoints, on a mechanistic picture of the world and on deductive logics towards accounts of emergence, of systemicity, informationality and conviviality, building upon each other and providing together a transdisciplinary edifice of the sciences, in the end, for, and by the inclusion of, citizens. viewed from such a combined perspective, the current social evolution is punctuated by a great bifurcation similar to bifurcations other emergent systems have been facing. on the one hand, humankind is on the brink of extinction. it is the world occurrence of the enclosure of commons that is detrimental to sharing the systemic synergy effects and thus to the cohesion of social systems. on the other hand, humanity is on the threshold of a planetary society. another leap in integration would be the appropriate response to the complexity confronted with. humans and their social systems are informational agents and, as such, they are able to generate requisite information and use it to catch up with the complex challenges. they can establish convivial rules of living together in that they disclose the commons world-wide. by doing so, they would accomplish another evolutionary step in anthroposociogenesis. the concept of the global sustainable information society describes the framework of necessary conditions of conviviality under the new circumstances. the seemingly disruptive advent of the covid-19 pandemic outshone the climate change that has been gaining obvious momentum since the last fifty years to an extent that it threatens with a much more decisive rupture if science, technology and society are not unwilling to learn from the pandemic that there is nonlinear growth with complex challenges be they small or large and that human actors are not completely doomed to helplessness though. for such a lesson to learn, a secular shift in thinking and acting throughout sciences and everyday life is required because human actors need to be capacitated to cope with complex challenges such as the global problems. a shift is already underway though not yet hegemonic. this shift has to overcome three prejudices of conventional science: • the outdated ideology of value-free scientific research. the absence of values would make science distinct from biased everyday thought. but that's not the distinction. any research is driven by societal interests even if mediated by personal curiosity. any research implies particular values, reflected or not if not even camouflaged. of course, these values must not divert the findings of research, quite the contrary, they shall stimulate evidence-based research -and that's the distinction from biased opinion of everyday. science can critique opinions. in the last two decades, several labels have become aspirational for scientists: research shall be responsible, university research shall be aware of its third mission, namely, to serve the common good, applied research shall be replaced by use-inspired basic research, research shall become practically transdisciplinary in that it transcends science towards, and include in science, the values of everyday people that are affected by the results of research, best by letting them participate in research. these are attempts in the right direction: the acceptance that there is a limited controllability of what can be done. as everyday-thinking and -acting, science has limited controllability over its impact on society but within that certain limit it is capable of controlling and thus it must aim at doing so in a precautious way. neither phantasies of omnipotence nor of impotence are called for but a deliberate activism is (hofkirchner, 2013a, 62-71) . not everything that might be feasible is also desirable and not everything that might be desirable is also feasible. the feasible and the desirable need to be made compatible with each other. • the outdated mechanistic picture of the world. cause-effect relationships are fancied as if pertaining to a machine constructed by humans. cause and effect would obey laws of strict determinacy such that the effect would follow necessarily from the cause. but that is true only for a small subdivision of effective causality -causes can have different effects and effects can be brought about by different causes -, let alone final, material and formal causality (hofkirchner, 2013a, 86-95) . laws of nature and other parts of the world are not given for eternity. the late karl raimund popper called those laws propensities -an asymmetrical, contingent behaviour of the universe (1990) . this shows the right direction. strict determinism as well as indeterminism are false alternatives. less-than-strict determinism avoids both fallacies. • the outdated preponderance of methodologies that are based on deductive logics. deductivism is an attempt to deduce that which shall be explained or predicted from premises such that the phenomenon can be subsumed under a proposition of the form of a universal implication that covers the phenomenon. the premises suffice by definition for the conclusion, the phenomenon is thus reduced to a sufficient condition. but, in fact, those conditions are rarely sufficient. there is the search for alternative logics such as the hype for abduction or claims for a trans-classical logic (günther, 1990) . and there is the logic in reality of joseph brenner (2008) who grounds logic in reality, that is, he gives primacy to how reality works in principle when postulating logical principles. anyway, it needs to be accepted that explanation and prediction are incomplete and that they should focus on the adjacent necessary instead, that is, a necessary condition that might rarely be sufficient but should form a basis of understanding as close as possible to the phenomenon that shall be explained or predicted (hofkirchner, 2013a, 131-139; 1998) . neither deductivism nor irrationalism -for which anything would go -can convince but a reflexive rationalism that accepts incomplete deducibility with an ascendance from the abstract to the concrete where by each step a new assumption is introduced without deduction. the build-up of such a specification hierarchy is important for the transdisciplinarity in its theoretical sense -the consideration of as many facets of the phenomena as possible in order to achieve a unified understanding. the removal of those impediments for the progress of science are the milestones that the paradigm shift has to master. only if they will have been achieved, humanity will be ready to confront the global challenges in a way that safeguards mankind against man-made extinction. the next sections substantiate how, on the three pillars of deliberate activism, less-than-strict determinism and reflexive rationalism, a new understanding of the current situation of world society can be erected and how it can make the disciplines of the whole edifice of science responsive to that task. the sections will proceed from a systemic level to an informational level to a convivialist level. 1 emergentist systemism 'emergentist systemism' is a term introduced by poe yu-ze wan (2011) in the context of social theory, after it had been used in the field of social work in switzerland (e.g., obrecht, 2005) to characterise the approach of philosopher of science, mario bunge. bunge himself was rather used to terms such as 'emergentist materialism' to signify, e.g., his position in the field of the mind-body problem. however, he defined systemism in a broader sense, namely as 'ontology: everything is either a system or a component of some system. epistemology: every piece of knowledge is or ought to become a member of a conceptual system, such as a theory. axiology: every value is or ought to become a component of a system of interrelated values ' (2012, 189) . and he defined emergence as '[…] advent of qualitative novelty. a property of systems ' (2012, 185) . thus, emergentism is 'a world view or an approach' that focuses on emergence (2003, 38) . 'systemism, or emergentism', as he said (2003, 38-39) , 'is seem to subsume four general but one-sided approaches: 1. each of these four views holds a grain of truth. in putting them together, systemism (or emergentism) helps avoid four common fallacies.' this is by and large the sense in which emergentist systemism is understood here (hofkirchner, 2017a ) -as weltanschauung, that is, a world view that is not value-free (a german term by which mark davidson 1983 summed up ludwig von bertalanffy's general system theory and which will be subsumed here under the term praxiology), as conception of the world (ontology) and as way of thinking to generate knowledge about the world (epistemology). in a nutshell, emergent systemism means that, practically, humans intervene as a rule for the betterment of social life in real-world systems they conceive of by patterns they have already identified and that these systems are emergent from the co-operation of other systems that become or are their elements. this is called selforganisation. since the idea of emergent systems implies a kind of evolution, these systems are also known by the term evolutionary systems. evolutionary systems theory -a term coined by ervin laszlo (1987) , vilmos csanyi (1989) and susantha goonatilake (1991) but extended here to cover the meaning it received after the seminar held at the konrad lorenz institute for evolution and cognition research in vienna 1995 (van de vijver et al., 1998) -is the proper theory of self-organisation, a merger of systems theory and evolutionary theory by which the first was enabled to include more than ideas of maintaining systems only and the latter could emancipate from mechanistic interpretations of the darwinian model. self-organisation is characterised by evolvability and systemicity. that means that matter, nature, real-world events or entities evolve such that systems materialise as organisation of components (hofkirchner, 2013a, 58-59) . applying emergentist systemism to the edifice of science(s) brings a profound change (hofkirchner, 2017a) . how is the old paradigm's view of that edifice? let's start with philosophy, composed of epistemology, ontology and praxiology (ethics, aesthetics and else) as the most abstract enterprise and put it in the background. before that background, you have the three categories of formal sciences, real world sciences and applied sciences in juxtaposition. the formal sciences include logics and mathematics as disciplines. the real-world sciences comprise disciplines that investigate nature, on the one hand, and were called typically physics, chemistry, biology and else, and disciplines that investigate the social world, on the other, nowadays summarised under the term social and human sciences including sociology, cultural, political, and economic sciences and else. applied sciences assemble engineering, management, arts and else. every discipline is divided by sub-disciplines and sub-sub-disciplines besides all having their own legitimation as basic research, formal sciences are known for providing instruments for gaining knowledge in the real-world sciences, real-world sciences are needed for the provision of evidence for developing technologies, organisation, pieces of art. however, what makes the co-operation of sciences difficult is that they are siloed against each other by impermeable boundaries. connection between those mono-disciplines can be attempted only by heaping some of them together in a multi-disciplinary approach, which is no connection at all, or by peripheral exchanges in an interdisciplinary approach, which does not admit internal changes and keeps the disciplines as alien to each other as they have been before. driven by the confrontation with complex problems, things have begun to change already in the direction of semi-permeability of disciplinary boundaries, which, in the long run, paves the way for the establishment of new stable relations between them. emergentist systemism is not another discipline that just adds to the picture of the old disciplines. it causes rather a paradigm shift that has the potential to transform the whole edifice of science(s). philosophy that was deprived of fruitful relations to the disciplines of science in what had become normal science turns into systems philosophy now; formal sciences turn into formal as well as non-formal systems methodology; realworld sciences turn into sciences of real-world systems, that is, material, living or social systems; and, finally, applied sciences turn into a science that makes artefacts by designing systems and, in doing so, integrates them with social systems. so, at one blow, connectedness is unveiled between all inhabitants of the edifice. transgressions from one scientific endeavour to another can be mediated via jumping forth and back over shared levels of scientific knowledge. those levels form now a specification hierarchy. jumping from one specific level to a more general level allows comparison and adjustment of both levels. it allows the initial level to instigate knowledge adaptations on the target level or adoption of knowledge on re-entry from the target level. in addition, a more general level works as bridge for jumping up the ladder to even higher levels or down to different lower levels so as to help understand that their knowledge is just another specification of the knowledge they share at a higher level. this makes the sciences of evolutionary systems a transdiscipline and its inherent emergentist systemism makes the edifice of disciplines a transdisciplinary, common endeavour of all science. semi-permeability does not lift the boundaries. relative autonomy of disciplines is maintained in the overall transdisciplinary network. paraphrasing bunge, informationism is a term used here to denote a praxiological perspective on, an ontological conception of, and an epistemological way of thinking about, information, which takes centre stage in this tenet. for the sake of consistence, information is set to be based upon, and concretise further, systems, in particular, emergent information shall easily relate to emergent systems. this can be achieved through the assumption of informational agents -agents being emergent systems. thus, the generation of information is enshrined in the self-organisation of systems. any time a system self-organises, its agency brings forth information. an evolutionary system can be defined as 'a collection of (1) elements e that interact such that (2) relations r emerge that -because of providing synergistic effects -dominate their interaction in (3) a dynamics d. this yields a distinction between micro-level (e) and macro-level (r) and a process (d) that links both levels in a feedback loop' (hofkirchner, 2013a, 105) . with reference to, but in modification of, a triadic semiotics after charles sanders peirce (2000) , laying emphasis on the intrinsic connection of self-organisation with negentropy after edgar morin (1992, 350 and 368) and by usage of the term 'perturbation' introduced by humberto maturana and francesco varela (1980), information can be defined as 'relation such that (1) the order o built up spontaneously (signans; the sign) (2) reflects some perturbation p (signandum/signatum; (to-be-)signified) (3) in the negentropic perspective of an evolutionary system s e (signator; the signmaker' (hofkirchner, 2013a, 171) . 'information is generated if self-organising systems relate to some external perturbation by the spontaneous build-up of order they execute when exposed to this perturbation. in the terms of triadic semiotics, the self-organising systems, by doing so, assign a signification to the order and make it a sign which stands for the so signified perturbation' (hofkirchner, 2013a, 172) . this is the approach of a unified theory of information (hofkirchner, 2013) . it is worth noting that those assumptions attribute information generability to emergent systems according to the evolutionary type they represent. not only social systems and their inhabitants are qualified as informational agents (this would import the acceptance of umberto eco's threshold of semiosis applicable to the realm of human culture exclusively), not only biotic systems (this is the threshold of biosemiotics) but also physical systems in so far as they are able to self-organise are qualified to generate information in shades -as far as the respective evolutionary stages allow. emergent systems of any kind produce emergent information. as to the new scientific edifice, informationism is mounting systemism. systems philosophy becomes a systemic philosophy of information; systems methodology becomes a systemic information methodology; the sciences of real-world systems become sciences of information of real-world systems, that is, of material information, living information and social information; and the science of designing artificial systems becomes the science of designing information in artificial systems. all that information is emergent information. convivialism is a term denoting a social perspective (praxiology), a conception of the social world (ontology) and a social-scientific way of thinking (epistemology) for which conviviality is key. conviviality as term was introduced by the austrian-american writer, ivan illich, who published a book with the title tools for conviviality (1973) . it contained a philosophy of technology according to which technology should be socially controlled so as to reclaim personal freedom that is restricted by uncontrolled technological development. conviviality -illich was familiar with the spanish term convivialidad -has latin origins and means the quality of living together in the manner of dining together (convivor) of hosts (convivator) and guests (conviva) at common feasts (convivium). in the last decade, that term gained new attention when mainly about fourty french intellectuals -among them serge latouche, edgar morin or chantal mouffe -opened the discussion on a political manifesto for the redesign of social relations. the first manifesto was followed by a second, up-dated one in 2020 (internationale convivialiste). according to the latter, convivialism 'is the name given to everything that in doctrines and wisdom, existing or past, secular or religious, contributes to the search for principles that allow human beings to compete without massacring each other in order to cooperate better, and to advance us as human beings in a full awareness of the finiteness of natural resources and in a shared concern for the care of the world. philosophy of the art of living together, it is not a new doctrine that would replace others by claiming to cancel them or radically overcome them. it is the movement of their mutual questioning based on a sense of extreme urgency in the face of multiple threats to the future of humanity. it intends to retain the most precious principles enshrined in the doctrines and wisdom which were handed down to us.' convivialism is emergentist if seen in the context of emergent systems and emergent information. social systems are here considered as evolutionary systems, which is in stark contrast to how german sociologist niklas luhmann (1995) considered them (wan, 2011). though luhmann originally claimed to start with general system theory when elaborating his theory of social systems, a revisiting of bertalanffy would lead to different conclusions . such an approach has been pursued not only by bunge but also by members of ervin laszlo's general evolution research group, among them robert artigiani (1991) , by representatives of critical realism, in particular margaret s. archer (1995; 2010; and her project group on social morphogenesis at the centre for social ontology, and workers departing from us sociologist walter f. buckley (1967) , including the economist tony lawson (2013) and the relational sociologist pierpaolo donati (donati, 2011; donati and archer, 2015) . of course, many other sociologists are worth mentioning; even if they do not explicitly share a systems approach, they have nevertheless contributed with important insights to such a framework (giddens, 1984; alexander, 1995; mouzelis, 1995; reckwitz 1997) . social systems are the evolutionary product of living systems but contain living systems that get a social shape. the elements of social systems as conceived here are social agents -humans called actors -and their organisational relations are social relations -called structure. actors inhabit the micro-level of social systems, while the macro-level is where the structure is located. the structure is produced by the actors and it exerts a downward causation on the actors and their agency (hofkirchner, 2013b, 136; lawson, 2013 ). thus, social systems self-organise as living and material systems do, but they differ from living and material systems as to their mode of self-organisation. 'social self-organisation goes beyond biotic self-organisation, which, in turn, goes beyond physicochemical self-organisation' (hofkirchner, 2014, 120) . social self-organisation does so in that it transcends, re-invents, creates the social systems through the action, interaction and co-action of their actors, who -as informational agents -cognise, communicate and co-operate mindfully when reproducing and transforming their social systems, which, in turn, can be considered as higher-level informational agents. social self-organisation is not conceivable without the generation of specific social information. the triple-c model postulates a hierarchy of information processes such that cognition is the necessary condition for the functioning of communication and communication the necessary condition for the functioning of co-operative information, in short, co-operation (hofkirchner, 2013) . psychic functions such as thought and others, the ability to speak and the ability to devise and manufacture artefacts, in particular, tools, are characteristic of humans. all of them are knit together in social information: 'human thought is part of human cognition […]; human language is part of communication […] ; human tools are part of work that belongs to human co-operation' (hofkirchner, 2016, 286) . starting with work at the top (which refers to the structure on the system's macro-level), it is about constituting common goals and instituting common goals. work is consensual. co-operation involves finding and building consensus. what is needed here, are common intentions. common intentionality provides the perspective of the whole we, the perspective of the social system. consensualisation, in turn, presupposes a certain collaboration that designs specific tasks for reaching the shared goals and assigns these tasks to certain actors. that is done on the social information level below, on the level of language (which refers to the network of interactions the actors form on the system's micro-level). communication functions as the means to realise that kind of collaboration that is needed for the upper level. that is, taking the perspective of the other facilitates collaboration. however, taking the perspective of the other is promoted by taking the perspective of the whole in which one's own and the others' roles are included. what is required here, is readiness for a dialogue with sense for consilience. collaboration, in turn, presupposes a certain co-ordination that devises certain operations for fulfilling the tasks and supervises certain actors in performing the operations. that is worked out on the lowermost level, on the level of thought (which refers to the actions of the individual actors who are also located on the system's micro-level). cognition allows the actors to understand what kind of co-ordination is needed by the upper level. it enables the actors to reflect upon the relationship between operations, tasks and goals. what is necessitated here, is reflexivity, the capacity to reflect the social context in which the cognising actor is embedded (archer, 2010) , and conceptuality, the capacity to use concepts, all of which are influenced by verbal language (logan, 2007; . the rationale of every complex system is synergy (corning, 1983; . agents produce synergetic effects when co-operating systemically -effects they could not produce when in isolation. in social systems, synergy 'takes on the form of some social good. actors contribute together to the good and are common beneficiaries of that good -the good is a common good, it is a commons' (hofkirchner, 2014, 121) . the social relations are commoning relations. conviviality then, as a feature of emergent social systems with emergent social information, can be determined as the historical-concrete shape of the commoning relations. it is a social value that is highly esteemed, it is a theoretical conceptualisation of a social practice, and it is a measurand the value of which can be estimated by empirical studies -it is all of them in one because it is an expression of the quality of the commoning relations. it expresses how just those relations are constructed, how equitable, free and solidary, and to which degree they enclose or disclose the commons. conviviality is visionary and longs for actualisation. its actualisation would 'make the social systems inclusive through the disclosing of the enclosed commons and, by doing so, […] warrant eudaimonia, a good life in a good society, the flourishing of happy individuals in convivial social relations' (hofkirchner, 2017b, 286) . having defined the commons as social synergy and conviviality as measure of the actualisation of envisioned commoning relations, the critical theory perspective becomes apparent (hofkirchner, 2015, 97-99) -the perspective of a critical social systems theory as part of the social systems sciences in the new edifice of disciplines. conviviality is emergent. it develops over time and changes its forms in a contingent way. referring to michael tomasello's shared intentionality hypothesis and his interdependence hypothesis (tomasello et al., 2012; tomasello, 2014; , there have been two key steps in anthroposociogenesis (the becoming of humans and society) so far and, following the new systemic, informational and convivialist paradigm, a possible third one is imminent. the next subsections discuss those steps. leaps in quality emerge in systems as novel organisation due to a change in the organisational relations. thus, changes on the top-most levels of information generation and usage are decisive. all of them are shifts in co-operation. if work, language and thought build the human/social hierarchical levels of information from a synchronic point of view, then, from the diachronic point of view, it may well be assumed 'that it is conditions of co-operation that made the difference in evolution. evolutionary pressure unfolded a ratchet effect that yielded ever higher complex co-operation' (hofkirchner, 2016, 287) . the state of co-operation in the ancestors of humans is the origin of anthroposociogenesis. self-reinforcing processes came about. changes in the state of co-operation proliferated down to provoke changes on the level of communication -the development of human language -in order to propel co-operation and changes in the state of communication proliferated down to provoke changes on the cognition level -the development of thinking -in order to propel communication. in the beginning, so tomasello, there was a shift from individual to joint intentionality. individual intentionality of common ancestors of chimpanzees and humans was the point of departure about six million years ago. as living together was driven by self-interest of animal monads, there was no need for taking in consideration common goals, no need for thinking on a level beyond the actual egocentric perspective (tomasello, 2014, 4, 30) . early humans began to speciate only when they took advantage of going beyond individual intentionality and adopted 'more complex forms of cooperative sociality' (31). a first step occurred 'in the context of collaborative foraging' (33), that is, the hunting of large game and gathering of plant foods, around 2 million years ago. this step culminated about 400.000 years ago, when joint intentionality emerged. hunters and gatherers developed dyadic cooperations driven by a 'second-person morality ' (2016, 6) . hence a need for acknowledging a common goal, that is, an understanding that the partner shares the goal and both are committed to act according to its achievement. multiple and vanishing dyadic relationships formed in which early humans shared a joint goal. in order to support the negotiation of joint goals and the coordination of collaboration, human communication originated with 'a commitment to informing others of things honestly and accurately, that is, truthfully ' (2014, 51) . cognitively, 'when early humans began engaging in obligate collaborative foraging, they schematized a cognitive model of the dual-level collaborative structure comprising a joint goal with individual roles and joint attention with individual perspectives' (69). this was a premature state of conviviality. dyadic co-operation guaranteed the common good for the included actors. the shift from individual to joint intentionality was followed by a shift from joint to collective intentionality. collective intentionality emerged with early humans about 150.000 to 100.000 years ago. this shift occurred with the advent of culture, that is, of separate and distinct cultural groups, the interdependence that caused co-operation reigned 'not just at the level of the collaborating dyad, and not just in the domain of foraging, but at the level of the entire cultural group, and in all domains of life' (tomasello, 2016, 85) . this step created objective morality (6). co-operation became triadic. since then a need for group-thinking has become characteristic of humanity, that is, knowing that any person belonging to the same group culture can be expected to share the same values and norms -by constructing a meta-level such that any group member can imagine the whole of the group, the roles taken, her own as well as others' replaceability. in line with that, communication was to start with discourses about 'objective' facts in need of compelling arguments and cognition had to turn into fullblown human reasoning; 'the individual no longer contrasted her own perspective with that of a specific other […]; rather, she contrasted her own perspective with some kind of generic perspective of anyone and everyone about things that were objectively real, true and right from any perspective whatsoever ' (2014, 122) . cognition involved a new feature of generalisation capacity. this was the next step of conviviality. the third of the triad is relations of society that relate individuals to each other with respect to the common good -even if the concrete content of the common good became a matter of disputation and conflict. and today, a third step of anthroposociogenesis can be hypothesised. there might be a shift from collective intentionality to one that is shared universally, that is, on a planetary scale. that would be the transition to another convivial regime -an extension of the triad to the whole of humanity, an omniad. this extension would be necessary because the conflict over the commons has reached an extension that endangers conviviality at all and the curbing of the extension of the conflict over the commons by extending the triad to an omniad can be considered possible, which is discussed in the sub-subsections to follow. another step is necessary, given that it is agreed that there shall be a human future, which is tantamount with a humane future (hofkirchner 2017b ). in the course of evolution, complex systems move on trajectories on which bifurcations occur. they occur if and when the provision of synergy effects becomes problematic. bifurcations force the systems to change their trajectory. the old one cannot be continued any more. it bifurcates into a variety of possible future trajectories. there are two of them that span the whole variety in the possibility space between two extremes: systems might be able to achieve a leap from the previous level of evolution on which they could enjoy a steady state onto a higher level which forms part of a successful mega-evolution (haefner, 1992, 314; oeser, 1992 , 103-104) -a breakthrough to a path that transforms the systems into what is called meta-system (the metasystem transition) or supra-system -or they might even not be in the position to avert devolution -a path that leads to the breakdown of the systems. amplified fluctuations of parameters indicate the crossroads that demand taking one path or another. the nonlinear dynamics of complex systems make the crossroads appear in one as windows of opportunity to spiral upwards and as tipping points that let the systems spiral downwards into oblivion (laszlo 2001; 2006) . complex systems that can be observed today are those that could manage so far to harness synergy. the evolution of social systems is no exception. 'today, enclosures of the commons have been aggravated to such a degree that all of them morphed into global challenges. global challenges drive an accumulation of crises that mark a decisive bifurcation' (hofkirchner, 2017a, 10) . not only do global challenges cause a multi-crisis in the tension between, and among, social systems from the granularity of today's nation states down to the granularity of the smallest units made up by individualised actors, cutting across all social areas such as the cultural, the political, the economic, the ecological (eco-social) and the technological (techno-social) area and affect so humanity as a whole, but they also threaten, for the first time in human evolution, with the ultimate impact for humanity -with extinction. thus, that decisive bifurcation, in which a branch is much sought after to lead out of a dead-end branch, is called here the great bifurcation. that term resembles karl polanyi's term of the great transformation (1944) in that it embeds the conflict of market capitalism with democracy -the point that was of utmost importance to polanyi -in the complex systems context of anthroposociogenesis. 'either the social systems that together constitute mankind undergo a metasystem transition onto a higher level of organisation that allows the continuation of social evolution on earth or they, eventually, fall apart and discontinue anthropogenesis; either they succeed in rising their complexity such that they break through to a new step in the mega-evolution of humanity or there is a decline in complexity, a breakdown and devolution; either their differences can be integrated or they disintegrate themselves' (hofkirchner, 2020a, 1) . another step is not only necessary for a surviving and flourishing humankind. it is also possible and the reason for that is not only that such a step is grounded objectively in the possibility space given by the bifurcation. moreover, humans can be conceded the subjective potency to find the right way out of the crossroads, in particular, since the problems they come across are of anthropogenic origin and can be solved by a proper re-organisation of the social systems. they can view the evolution of humanity from the inside, explore and anticipate the way out and, finally, intervene accordingly (laszlo, 1987; 2001; 2006) . they belong to the first species on earth that can overcome self-made problems in the sharing of synergy. any emergent system can boost emergent information to catch up with the complexity of the challenges. 'if there is a mismatch between the complexity of a system and the complexity of the problems faced by the system, that system can catch up. […] intelligence is the capability of selforganising systems to generate that information which contributes in the best way to solving problems. the better their collective intelligence, that is, the better their problem-solving capacity and the better their capability to generate information, the better their handling of the crisis and the order they can reach. higher complexity not only signifies a higher degree of differentiation. at least as importantly, it signifies a new quality of integration. only a new level of integration can deal with an intensification of differentiation' (hofkirchner, 2017a, 10) . this can be called the law of requisite information (hofkirchner, 2020a, 3) that is elaborated on the basis of w. ross ashby's law of requisite variety (ashby, 1956) . according to the latter, a system is said to be able to steer another system, if the variety it disposes of corresponds, if not surpasses, the variety of the system to be steered. by departing from the narrow cyberneticist view through connecting variety with complexity and complexity with information and extending the reach of that which is to be steered from the outside to the inside, it can be concluded: 'requisite information is that appropriate information a system has about the complexity of the exterior and interior environment. requisite information safeguards the functioning of the system' (hofkirchner, 2020a, 3) . humanity entered the great bifurcation because 'the social relations of any partition of humanity are based on the principle of othering of partitions that are considered outside of them, thus not doing justice to legitimate self-interests of the rest of the partitions. frictions […] are caused by the lack of relations that would be valid for all partitions from a bird's eye view, that is, from a meta-level perspective. the establishment of such relations would mean the abolition of those frictions by a new supra-system in which all existing systems take part and shape according to the new relations on a higher level, following the application of the subsidiary principle as a basis for the preservation of diversity and autonomous agency" (hofkirchner et al., 2019, 455) . despite some literature based on biases due to biologism unable to imagine a transgression of the conceptual framework of the nationstate we, transnational relations have been taking shape. there is empirical evidence of co-operation between culturally homogeneous groups several tens of thousands of years ago, between cities around five thousand years ago, and between modern states since the seventeenth century (messner and weinlich, 2016; neumann, 2016; grimalda, 2016) . 'this co-operation between collective actors like groups, cities and states has already been paving the way for co-operation among the whole of humankind in the same way that dyadic, interpersonal co-operation between individual actors opened up the space of possibilities for triadic, societal co-operation' (hofkirchner et al., 2019, 455) . the term information society is gaining a new meaning. it does not mean a society that is only informatised, that is, penetrated by information technology as a report to france's president originally insinuated (nora and minc, 1978) . it means an informatised society only if that society uses its informatisation for becoming informational in a non-technical sense. becoming informational entails becoming sustainable and becoming sustainable entails, in turn, becoming global. such is information society on the point of obtaining another meaning in the context of critical social systems theory, which crystallises as critical information society theory. it points towards the global sustainable information society as a framework (hofkirchner, 2017c) : • informationality of the global sustainable information society means 'the envisioned state of informedness of informational actors and systems in which they will catch up with the complexity they are challenged by the great bifurcation to such an extent that […] they will dispose of the capacity to recognise the causes of the global challenges in order to accordingly re-organise human life on earth to master those challenges' (hofkirchner, 2020a, 2) . informationalisation signifies the provision of social information for the installation of safeguards against the deprivation of commons world-wide and thus a new step in the evolution of conviviality. • the provision of such safeguards, in turn, is the process of executing sustainability. sustainability in the framework of the global sustainable information society does so receive a new meaning too. it means 'the envisioned state of the world system that will be shaped and shaping the social relationships between all parts, and throughout any part, of humanity pursuant to the commoning relations on the higher level' such that 'dysfunctions in the working of the organisation of the social system are kept below a threshold the transgression of which would discontinue social evolution' (hofkirchner, 2020a , 2). • the higher level on which the commons shall be provided, is 'the envisioned state of world society as an integrated meta-and supra-system in which social relationships will connect all parts of humanity in all fields of human life', which, eventually, conveys a new meaning to globality in the context of the global sustainable information society. 'these commoning relations need to be lifted onto the planetary level, and the emerging superordinate system will nest all actors and systems according to the new expanded commoning relations. by that, global governance is carried out' (hofkirchner, 2020a, 2) . globalisation signifies the provision of the commons worldwide. the notion of the global sustainable information society is far from a blueprint of the future society but describes which necessary conditions need to be met if the great bifurcation shall be successfully passed. there are three imperatives of social information that must be obeyed so as to enable actors to take that next step in the evolution of convivial humanity. on the co-operative level, normative, valueladen information must become hyper-commonist, that is, it must orient the consciousness and conscience of the actors towards the reclaiming of the commons in a universal manner; on the communicative level, dialogical information must become all-inclusive, that is, it must not exclude any actor in a universal conversation about the common good; on the cognitive level, reflexive information must become meta-reflexive, that is, it must be concerned about changes of the meta-level that is a universe for all actors (hofkirchner, 2017c) . in order to accomplish that third step in conviviality, those imperatives, investigated by social sciences and humanities, need to be provided to civil society by translational sciences, all of them integrated and implemented by the new paradigm shift as transdisciplinary basis. scientific thinking as well as everyday thinking need to support each other in the comprehension and tackling of the next step. thus, emergentist systemism, informationism and convivialism, shifting research to a remedy for the global challenges, to a reconciliation of determinacy and indeterminacy, and to a logic of emergence, are no academic exercise of no avail. also, common sense is, in principle, capable of understanding those issues of the paradigm shift as well as becoming activist on that premises. the step will be an unprecedented revolutionary one. revolutionary thinking 'needs to focus on future social relations that are not yet actualised. it needs to anticipate them ideationally on a new meta-level, it needs to anticipate the meta-/suprasystem transition of the social systems.' and, taking up an idea of ernst bloch (1967), it 'does not only need to anticipate what is desirable but needs to explore which desirable is also possible in the here and now. only what is potential can be actualised. thus, it looks in the space of possibilities now for the foreshadowing of something that might become a future third' (hofkirchner, 2020b, 4) . the conclusion is that the current state of human evolution has been reached as emergent response to requirements of co-operation through two steps in anthroposociogenesis, namely, from the living together of individual monads towards a joint interaction in dyads and from that to a collective working together that was mediated by social relations -which are the social system's relations of the organisation of the commons -such that a triad has taken over the co-action of humans: a meta-level was constructed as a third that relates the interaction of the group members as a second and any action of a member as a first. now that global conditions require global co-operation, the third needs to be extended to another level ushering in a new phase. current discourses on whether or not our time shall be called anthropocene or whether or not we can stop the climate change or prevent pandemics like the covid-19 one, are dominated by pejorative connotations and negative imaginaries of the future. they lack a focus on the real potentialities of humanity that is just on the point of going through a possible next step of social evolution. extermination is the risk of the crisis. meta-reflexive global citizens, engaging in a global dialogue can kick off the emergence of global governance and thus solve the crisis. fin de siecle social theory -relativism, reduction, and the problem of reason realist social theory -the morphogenetic approach structure, agency and the internal conversation making our way through the world -human reflexivity and social mobility the reflexive imperative in late modernity social evolution: a nonequilibrium systems model an introduction to cybernetics sociology and modern systems theory the synergism hypothesis -a theory of progressive evolution nature's magic -synergy in evolution and the fate of humankind evolutionary systems and society -a general theory of life, mind and culture uncommon sense. tarcher relational sociology -a new paradigm for the social sciences the possibilities of global we-identities die tradition der logik und das konzept einer transklassischen rationalität emergence and the logic of explanation emergent information -a unified theory of information framework self-organisation as the mechanism of development and evolution on the validity of describing 'morphogenic society' as a system and justifyability of thinking about it as a social formation generative mechanisms transforming the social order ethics from systems -origin, development and current state of normativity transdisciplinarity needs systemism. systems 5, 15 creating common good -the global sustainable information society as the good society information for a global sustainable information society social relations -building on ludwig von bertalanffy intelligence, artificial intelligence and wisdom in the global sustainable information society taking the perspective of the third -a contribution to the origins of systems thinking icts connecting global citizens, global dialogue and global governance -a call for needful designs seconde manifeste convivialiste -pour une monde post-néolibéral macroshift -navigating the transformation to a sustainable world emergence and morphogenesis -causal reduction and downward causation the extended mind. the origin of language and culture what is information? propagating organisation in the biosphere, symbolosphere, technosphere and econosphere social systems the evolution of human cooperation the nature of nature sociological theory: what went wrong? routledge global cooperation and the human factor in international relations l'informatisation de la société mega-evolution of information processing systems semiotische schriften, 3 vols the great transformation -the political and economic origins of our time struktur -zur sozialwissenschaftlichen analyse von regeln und regelmäßigkeiten the direction of evolution -the rise of cooperative organization the metasystem transition a natural history of human thinking two key steps in the evolution of human cooperation -the interdependence hypothesis evolutionary systems -biological and epistemological perspectives on selection and self-organization such an account can be reached by a paradigm shift towards emergentist systemism, on the basis of which emergentist informationism is elaborated, on the basis of which, in turn, emergentist convivialism is elaborated. from that perspective, the great bifurcation can be regarded as a problem of coming-of-age of humanity. by accomplishing that evolutionary step, the rise of co-operative organisation would enable 'the emergence of a coordinated and integrated global entity' (stewart, 2014, 35) not seen before. this research did not receive any specific grant from funding agencies in the public, commercial, or not-for-profit sectors. this research did not receive any specific grant from funding agencies in the public, commercial, or not-for-profit sectors. key: cord-349581-o320ogmg authors: robertson, lindsay j. title: the technological 'exposure' of populations; characterisation and future reduction date: 2020-05-25 journal: futures doi: 10.1016/j.futures.2020.102584 sha: doc_id: 349581 cord_uid: o320ogmg the nature and level of individuals' exposure to technological systems has been explored previously and is briefly restated here. this paper demonstrates how the concept of technological exposure can be extended to generic needs of individuals, and further to the needs of populations of individuals and even as far as “existential threats” to humanity. technological categories that incur high levels of population exposure are explored, and categories are described. a theoretical basis for reducing population exposure is developed from the basic concepts of technological exposure. technological developments that potentially enable less centralised societies having lower levels of population exposure, are considered for practicality and effectiveness as are the factors that could allow and cause transition to a less technologically centralised model. some conclusions regarding practicality, triggers, and issues arising from a decentralised society are considered and include the key conclusion that a higher level of decentralisation and exposure reduction is both desirable and possible. the fundamental impossibility of never-ending growth have been recognised for a long time (e.g. daly 1990 , henrique et al. 2013 ) and similarly authors (e.g. rao 1975) have recognised the danger of corporates of transnational size. this paper considers an associated but distinct issue, the growing technological vulnerability of populations. increasingly, essential goods and services are only accessible via technological systems that are both sophisticated, and also centralized. in this situation, end-user technological vulnerability becomes significant, but quantifying the extent and nature of such vulnerabilities have been hindered by the complexity of the analysis (haimes & jiang the supply of essential goods and services will involve heterogeneous systems (i.e. systems where goods/services are created by a progression of steps, rather than a system in which completed goods are simply transported/distributed), that involve an arbitrary number of linked operations each of which requires inputs, executes some transformation process, and produces an output that is received by a subsequent process. four essential principles have been proposed (author 2017) to allow and justify the development of a metric that evaluates the contribution of a heterogeneous technological system, to the vulnerability of an individual (author 2017a). these principles are: 1) the metric is applicable to an individual end-user: when an individual user is considered, not only is the performance of the supply system readily defined, but the relevant system description is clearer. 2) the metric acknowledges that events external to a technology system only threaten the output of the technology system if the external events align with a weakness in the technology system. if a hazard does not align with a weakness then it has no significance. conversely if a weakness exists within a technological system and has not been identified, then hazards that can align with the weakness are also unlikely to be recognised. if the configuration of a particular technology system is changed, weaknesses may be removed while other weaknesses may be added. 3) the metric depends upon the observation that although some hazards occur randomly and can be assessed statistically, over a sufficiently long period the probability of these occurring approaches 1.0. the metric also depends upon the observation that intelligently (mis)guided hazards (i.e. arising when a person j o u r n a l p r e -p r o o f intentionally seeks a weakness in order to create a hazard) do not occur randomly. the effect of a guided hazard upon a risk assessment is qualitatively different from the effect of a random hazard. the guided hazard will occur every time the perpetrator elects to cause the hazard and therefore such a hazard has a probability of 1.0. 4) the metric depends upon the observation that it is possible to not only describe goods or services that are delivered to the individual (end-user), but also to define a service level at which the specified goods or services either are or are-not delivered. this approach allows the output of a technological system to be expressed as a boolean variable (true/false), and allows the effect of the configuration of a technological system to be measured against a single performance criterion. applying these principles, the arbitrary heterogeneous technological system supplying goods/services at a defined level to an individual, can be described by a configured system of notional and/or/not functions. having represented a specific technological system using a boolean algebraic expression, a 'truth table' can be constructed to display all permutations of process and stream availabilities as inputs, and technological system output as a single true or false value. from the truth table, a count of the cases in which a single input failure will cause output failure, and assign that total to the variable "e1". a count of the cases where two input failures (exclusive of inputs whose failure will alone cause output failure) cause output failure, and assign that total value to e2. a further count of the cases in which three input failures cause output failure (and where neither single nor double input failures within that triple combination would alone cause output failure) and assign that total value to the variable "e3" and similarly for further "e" values. the exposure metric, {e1, e2, e3…}, for a defined system supplying specific goods/services, can be shown to measure the level of vulnerability of the individual to that technological system (author 2017). for many practical cases a generic service could be considered -e.g. "basic food" rather than "fresh milk", and vulnerability assessed by nominating multiple specific services as alternative suppliers (an "or" gate) of a nominated generic service. the exposure of the individual to lack of secure access to the necessities of life, can thus be assessed using exactly the same approach used to assess exposure to nonavailability of any specific need. illustrative examples of generic services could include "all needs for maslow's physiological level". conceptually this would include ((food_a or food_b or food_c…) and warmth and water and shelter) or possibly "all needs to occupy apartment on month-by-month basis". (sewage services and water supply and power supply). the concept of an individual's exposure may be yet further modified and extended to consider the quantification of a population's exposure, in regard to supply of either a particular, or a generic service. it is common for some "population" to need the same services/goods, and obtain these via an identical technological system. the "technological system" that supplies such a population may not be identical to that which supplies an individual: consider a case in which four individuals each rely on their cars to commute to work; for the output "arrive at work", each of their individually-owned cars contribute to the e1 exposure of each of the 4 individuals. if they reach a car-pooling agreement, the "car" then contributes to the e4 exposure for j o u r n a l p r e -p r o o f "arrive at work" of each individual, since any of the 4 cars can supply that functionality. a population exposure is developed by modifying an individual exposure evaluation (for a population using a functionally identical service) by considering each functional element used by more than one of the population, and whether each such functional element can be made accessible to more than one. if an exposure level (metric) is then nominated, it is possible in principle to establish the largest population which is subject to this level of exposure. for example, we might identify the largest population using a functionally identical service or supply of goods, that has a non-zero e1 exposure (i.e. has at least on single point of failure): should a city discover that the progressive centralisation of services had resulted in a situation where the whole population of the city had a non-zero e1 value for an essential service, some urgent action would be strongly suggested. conversely, if the supply of generic service to even small suburbs had low exposure values, then a good level of robustness would have been demonstrated for that generic service, at the suburb level. extrapolating these principles, where a "population" is actually the totality of the homo-sapiens species, and the "service" is essential for every individual's survival, then a definition of "existential threat" as proposed by bostrom (2002) , is approached. the analysis of exposure considers loci of failure and hence intentionally avoids consideration of specific hazards or their probabilities. nevertheless, hazards that affect various populations certainly exist, and a short list will serve to illustrate the significance of evaluating population exposure levels. the following list makes no attempt to be comprehensive, but illustrates hazards relevant to large corporates, j o u r n a l p r e -p r o o f apartment-dwellers, members of isolated communities and users of electronic communications.  a large group could be criminally held to ransom by malicious encryption of essential data (pathak 2016) .  a large group could be effectively excluded from access to a service because of conditions imposed by a (single, national) owner (tanczer, et al. 2016 , deibert 2003 , sandywell 2006 .  an isolated group (on antarctica, moon or perhaps mars) could cease to be viable if they cannot import supplies that cannot be generated locally (p?kalski & sznajd the occupiers of an apartment block could encounter a situation in where there was minimal (financial or other) pressure on the provider of sewage removal services to restore service, but where the occupiers faced major consequences (need to de-camp) in the absence of this service. similarly, (author 2017a) has noted that a failure to provide financial transaction services to a small retailer may incur negligible financial cost to a bank, but may cause the retailer's business to fail. these are examples of asymmetric consequences.  the whole of humanity exists on the planet earth: a failure of the natural environment can doom either a regional or possibly a global population (diamond 2005) , and can be certainly be considered to be an "existential threat", as defined by bostrom (2002) .  persons born early in the 20th century were very familiar with the possibility of highly unjust treatments (e.g. loss of employment), if private information became known -and they acted accordingly. for many born early in this century, such concerns seem remote. personal information privacy issues have thus failed to gain a j o u r n a l p r e -p r o o f high visibility, and we have perhaps become blasé about the costs of lack of privacy. nevertheless, the ubiquity of surveillance (miller 2014 , citron & gray 2013 , the increasing frequency of major data breaches (edwards et al. 2016) , and the recent rise in algorithmic decision-making (moses 2018) on such matters as health insurance, credit worthiness, validity of job application and security targeting are bringing this issue to increased prominence.  the recent revelation of hardware vulnerabilities in general processors (eckersley & portnoy 2017 , ermolov & goryachy 2017 ) demonstrated the significance of unappreciated weaknesses, even when overall operation of a system has been reliable. other publications (author 2010 , martin 1996 have noted society's vulnerability to technological systems and considered the relationship between time-todisaster and time-to-repair: that analysis was not an exposure analysis, but did consider the range of services corresponding to a broad category of human needs and effectively considered the significance of the exposure categories noted above. later works (author 2017a) have analysed the individual exposure associated with a broad range of goods and services. those works also noted that the analysed values for these specific examples could be reasonably extrapolated to many other similar goods and services. the general analyses of (author 2010) did attempt to cover the scope of individual needs and as such identified a range of services (or goods-supply needs) that were considered to incur high levels of vulnerability. the more detailed exposure analyses of (author 2017a) were indexed to the exposure of an individual, and although a limited number of examples were studied, these were considered to be representative of the high-exposure items relevant to j o u r n a l p r e -p r o o f individuals living in urbanised settings in the 21st century. the assessment of the population exposure of those exemplar technological systems shows several distinctive changes from the analysis indexed to the individual. primarily, the e1 items that arise close to the final point of use by the individual lose significance and for many cases cease to be significant at the e3 level (arbitrarily chosen as the cut-off point for consideration). the components that remain significant are those that genuinely represent exposure values for the population under consideration. the analysis of population exposure of a broad spectrum of needs showed that it was possible to identify some high exposure technological fields, specifically complex components, complex artificial substances, finance, communications, energy and information, these are considered more fully as follows;  complex components: complex components may be distinguished from large components (such as a building, hydroelectric dam, or road-bridge), and qualitatively described as being beyond the capacity of a skilled individual craftsperson to fashion in reasonable time and to required tolerances. under this category we might consider such items as a carburettor body for an internal combustion engine, food processing equipment and construction machinery (crane). for many consumer items within the "complex component" category, the population exposure is significantly lower than the individual exposure, since numerous examples of item exist (either owned by others, or stockpiled for sale). the level of centralisation may however be very high, with (for example) perhaps only one worldwide source of a carburettor body for a specific vehicle.  complex artificial substances: complex artificial substances includes advanced metallurgical alloys, advanced plastics and composites, drugs, and vaccines: these j o u r n a l p r e -p r o o f are distinguished by the complexity of composition, rather than complexity of form. in many cases the centralisation that causes high exposure levels for the production of complex substances, has resulted primarily from available economies of scale, and only secondarily from the substances' complexity. some complex substances (notably pharmaceuticals) have patent protection, which creates centralisation at up to global level.  finance: as the range of goods and services, and the geographical scope of supply of those goods and services has increased, so has the need for the facility to exchange value in return for goods and services: this has inevitably led to elaborate mechanisms for secure exchange of value. recognising that the exchange of value can also facilitate illegal activities, state actors have also enacted significant surveillance and control of financial transactions. technologies associated with the exchange of value have acquired high large levels of exposure (colloquially, many things that can go wrong) and high levels of centralisation, in the process of meetings these demands.  communications: the ubiquity of the internet has become a remarkable feature of the last 20 years: although there are exceptions, most areas of the earth and a very large proportion of total population can communicate via internet. although cellphone use is common, it is evolving as a mobile access connection, with the internet carrying the data. internet communications has been made possible by wellestablished protocols, however high level systems for routing continue to be centralised, and while problems are rare, nation-states have occasionally decided to discontinue connection to the internet, showing that high levels of centralisation exist (howard et al. 2011) . although the feasibility of internet communications can be attributed to open source protocols, the practicality of current connectivity has been j o u r n a l p r e -p r o o f largely enabled by the very large data rates possible via fibre-optic cables, yet this capacity has also introduced a high level of exposure for both individuals and populations. duplication of undersea fibre-optic cables has somewhat reduced the level of population exposure, yet the trend for dependence on high-data-rates has increased at similar pace (market forces have driven the need for additional cables), and communications via internet carry a high level of population exposure. recent reports (rose 2017 , clark 2016 have noted that the disruption of a very small number of fibre-optic cable would lead to unacceptable slowdowns, and trends to ownership of undersea cables by corporate entities further contributes to e1 values of exposure and high population exposure. the numbers of cables in service show that internet communications is centralised at a small-nation level.  energy: energy has been noted as key to civilisation (smil 2017) . coke allowed the smelting of iron, and oil enabled almost all current transportation. coal, nuclear and geothermal heat and hydro storage allow electricity generation on-demand. national power transmission systems generally have high reliability and many have some level of design redundancy. nevertheless, the generation and distribution of electric power incurs significant individual and population exposure: large power stations are bespoke designs as are large transformers (sobczak & behr 2015) , and transmission networks may face significant "resilience" issues (carvajal et al. 2013 , sidhu 2010 . liquid fuels can be stockpiled at national level, and to a lesser extent locally, however the level of stockpiling is limited -and even the higher levels of stockpiling are likely to be small compared to the time to rebuild a major production plant (terminal, or refinery). at the consumer and industrial user-level, although solar pv and wind can produce power at $/kwh rates close to thermal generation, but currently no economically viable technology that allows storage of megawatt-hours  information: information, whether medical reference information, contractual records or engineering design information, is not a coincidental by-product of a technological society, it is information that fundamentally allows the design and construction of technological systems and to the full operation of society (dartnell 2014 , shapiro 2009 , van den heuvel 2017 ). yet while it has become possible to generate and transmit enormous quantities of information, the information storage remains a particularly high-exposure issue (bergeron 2001) . currently the ascii codes largely standardise information representation, and protocols for transmission are also largely standardised but a gap in the standardisation of information storage (including writing and recovery) contributes to a high exposure for the final delivery of information to users. hard-disk drives are still the most common storage technology, yet these have short lives, and use proprietary data storage formats plus proprietary approaches to the writing and recovery of data. this issue has been wellreported, authors such as (cerf 2015) have predicted a "digital dark age", i.e. a future society that cannot recover/read most of the information generated in the current era. this describes a situation of high individual and population exposure, and since there j o u r n a l p r e -p r o o f are few of manufacturers of hdd's, information storage can also be noted to illustrate centralisation at multi-nation level. several technologies allowing longterm storage have been proposed (kazansky et al 2016, longnow foundation; nanoarchival™ technology) but these are currently expensive and lack the integration to allow them to truly offer changes to population exposure. by contrast, some fields have generally low population exposure: basic building materials, foodstuffs, natural fabrics and clothing are commonly supplied via relatively simple technological systems in which technological design redundancies are commonly large, and for which population exposure is therefore low. technologies such as creation of ceramics and glassblowing may require skill but are also not dependent on high technological sophistication and so contribute low technological exposure. similarly, the collection and storage of rainwater is feasible with low technological exposure for even densely populated urban populations. in addition to identifying high exposure fields, it is also possible to identify categories of exposure contribution that commonly apply across a range of technological fields. these are proposed to include initial resource availability, complex unit operations, lack of buffering, single points of failure (spof), contributory systems, highly centralised processes and "practical unavailability", and are described more fully as follows: initial resource availability: all technological systems producing goods and services for users, ultimately depend upon raw materials and viable environmental conditions. where raw materials cease to be available, or environmental conditions change permanently, services to users will inevitably be affected. raw material supplies and acceptable environmental conditions must therefore be identified (diamond 2005) j o u r n a l p r e -p r o o f as sources of exposure and hence vulnerability to users. complex unit operations: we use the descriptor "complex" as a characteristic of a process whose internal operation is practically unknowable to the user and cannot realistically be repaired by the user. personal computers, routers and related equipment are examples. it is also possible to consider situations where a critical application has been compiled from an outdated programming language and runs on a computer for which no spare hardware is available (hignet 2018). another example might consider critical information held on a very old storage medium (teja 1985) . these examples illustrate three categories of complex processes: in the first case, while the inner workings of a pc may be exceedingly complex, the format of incoming data (tcp/ip packets) and protocols (www, email etc.) are in the public domain (fall & stevens 2011 ) and so it is not only possible but practical for alternative machines to offer the same services. in the second case, assuming the functional specifications of the application processes are known, the application can be re-coded (and fully documented) using a language for which larger numbers of maintenance programmers exist, and on a more common platform. the third case of data encoded on old storage medium, illustrates a subcategory where the internal details of the storage are proprietary (not in public domain), alternative equipment is unavailable, and creation of replica equipment for reading the data is probably impractical, leading some authors e.g. (cerf 2015) to express fears of a "digital dark age". lack of buffering: for the supply of long-life products, it is both possible and practical to provide buffer stocks at various points in the process. by contrast, since ac power (for example) is not readily storable, all processes involving uses of ac power will fail immediately if the power supply fails. single points of failure (spof): all single points-of-failure (spof) contribute to e1 values and so make a primary contribution to users' vulnerability. three subcategories of spof are noted: the first is j o u r n a l p r e -p r o o f where delivery of services to users involves some processes immediately adjacent to the user, known as "last mile" services in the telecommunications field. the second subcategory of spof is illustrated by considering a small rural town whose eftpos, landline phone service, cell-phone service and internet connection have all been progressively migrated to data services, carried by a single fibre-optic cable and thus have inadvertently created a spof. the third is where a particular failure will inevitably cause failure of other components that are not functionally connected -a cascading failure. finally it is noted that contributory systems are a common source of exposure: whenever a system is made dependent upon another, the contributory system's exposures are reflected in the total exposure to the user. a common example is where a simple user service is made dependent upon internet access; the mandatory internet access may add huge levels of exposure to a system that would otherwise incur low levels of vulnerability. some specific technologies including artificial intelligence, nuclear weapons and asteroid strikes have been examined, and authors such as baum (2018) have pondered their potential to incur existential threats by threatening multiple systems. others including baum (2015) and green (2016) have considered approaches to limiting the scope of such hazards. the above categories of exposure may apply to any technological process; there are additionally several categories that are specifically relevant to the study of "population exposure", these include highly centralised processes, for example, the evacuation and treatment of sewage requires a network of pipes and pumps to collect sewage and deliver it to the treatment station. this is an example of a centralised system that is large but technologically simple. other examples of large centralised systems include financial transaction systems (o'mahony et al. 1997) , and the international data transmission systems of undersea fibre-optic cables (clark 2016) . such systems tend to j o u r n a l p r e -p r o o f be monopolies and are commonly controlled by entities that have little if any obligation to provide service or to negotiate terms acceptable to individual users. authors such as li et al. (2019) have shown that highly interconnected systems have similar characteristics to centralised systems, and it is well-recognised that the most "centralised" system is earth's natural environment, because the natural environment (including air and water) are essential to all life. practical unavailability: consider the hypothetical case where a user wishes to communicate sensitive information, but only has access to one data transmission facility that is known to be under surveillance. although technically operational, the inevitable absence of privacy associated with that data transmission facility has made the facility practically unavailable. for technological systems that are highly centralised and near-monopoly, practical unavailability is a significant possibility. the en metric has been explained earlier in terms of its significance for measuring vulnerability, however a wider and more future-oriented applicability of the metric itself can also be demonstrated in several ways. the assumption that there is an average cost of defending any vulnerability could be challenged but across a broad enough spread of examples, it is workable. under that broad assumption, whereas the cost of defence for an e3 exposurecontributor is precisely the same as the cost of defence for a contributor to an e1 vulnerability (it is the cost of protecting one only vulnerability, since the e3 contributor requires successful attack of all three vulnerabilities), the cost of mounting a successful attack on a e3 vulnerability is 3 times greater than the cost of attacking a vulnerability j o u r n a l p r e -p r o o f contributing to the e1 value, since the attacker needs to identify and successfully attack all 3 e3-contributory nodes simultaneously. in addition to its value for measuring vulnerability, the exposure metric is therefore also broadly significant for planning the reduction in vulnerability of a system. considering the generation of an exposure metric, the process itself provides valuable insights into options for reduction of the final values. the process of generating an exposure metric must be started from the delivery point, and follow a systematic redrawing and track-back process until a justifiable end-point is reached. the selection of a justifiable end-point has been addressed elsewhere, and could be associated with "no further contributors to an en level" criterion. the process of achieving a final representation of the system will very likely require a progressive redrawing of stream and process relationships; an example of a common re-drawing is presented in figure 1 . in a practical process of building an exposure metric, subsystems (which may have been analysed separately) commonly contribute goods or services to "higher" systems. if the exposure metric of a subsystem is known then there is little value in recreating a truth table for a higher level super-system, that re-explores all of the inputs to every sub-system. the more-effective approach is to consider the point at which the subsystem output contributes to the input of a gate within the higher level system, and how the subsystem exposure metric is transmitted/accumulated by the upstream gate of the super-system. we may generalise this process by considering that each input to a gate (boolean and or or operation) has an exposure vector, and developing the principles by which the gate output can be calculated from these inputs. this is illustrated in figure 2 . for the and gate, the contributory exposure vectors are simply added. for the or gate, the issue is more complex, but the higher levels of exposure are quickly avoided. the process of generating the metric will itself highlight sources of large exposure, and hence specific options for reduction. the detailed process for analysis of exposure has illustrated how the e values are accumulated and how specific exposure reduction options may be identified. generalised approaches to the reduction of the exposure of a population can also be identified. the process of redrawing shows that even where alternative subsystems can supply higher systems, if there is a locus of weakness that is common to alternative subsystems, the exposure analysis will ensure that such facts are preserved and the e1 contribution will actually appear in the exposure value for the whole target consumer (group). if the "o" ring seal failure had been identified as a contributor to the e1 j o u r n a l p r e -p r o o f exposure of the thiokol solid booster rocket then the elimination of all e1 values for the parent system that was the challenger space shuttle, could not have been achieved unless the "o" ring weakness were addressed. the use of an exposure metric therefore potentially addresses the colloquial saying "it's always the smallest things that get you". one of the learnings from the analysis of the "challenger" tragedy was that even known subsystem weaknesses could become lost as analyses were progressively summarised. when genuinely independent, alternative sources are available, their effect on the next-highest systems (process, consumer or group) is illustrated by the operation of an "or" gate. if 4 genuinely independent/alternative subsystems that each have exposure vectors with non-zero values of e1, are combined via an "or" gate, the higher system does not see any exposure at above the e4 level from that source. this principle might be qualitatively or intuitively perceived, but the effect upon an analysis of exposure provides a starkly quantitative analysis. it may also be observed that while reducing the exposure of each subsystem would require separately addressing each contributor to the subsystems e1, e2 and e3 values (potentially a major task), if 4 genuinely alternative subsystems exist then the combined "or" exposure has no nonzero contributor more serious than e4 and may warrant no further action. whereas many 20th century devices were designed for a single purpose and high throughput (mass production), some recent trends have been to devices that can be repurposed -and the pinnacle of flexibility is the human being! for any case where a piece of equipment contributes to the e1 value (is a single point-of-failure), if the capability of that equipment were able to be undertaken by multiple options (e.g. j o u r n a l p r e -p r o o f alternative human operators), the exposure contribution may be reduced to the en level, where n is the number of alternatives or persons capable of undertaking the equipment's function. an illustration may help to clarify this principle: if a sensor provides input that is required by an upstream function, that upstream function and all higher dependencies are exposed to the capability of the sensor to provide input: if 'n' humans are able to provide their best assessment of the sensor's input, then the exposure of the higher system to that functionality is reduced to the en level. considering the high-exposure technological fields that were identified earlier, generalised approaches to reducing the accrued actual exposure include standardisation of specifications that allow competitive supply of complex components and complex substances, avoiding the large exposure of some contributory systems, retention of genuine alternatives (e.g. cash/gold as well as electronic transactions). there is an intuitive appreciation that it is undesirable to have high vulnerability levels for systems that affect large numbers of persons. some may also intuitively appreciate that trends to centralisation, driven by economies of scale, increases the technological vulnerability of large population groups. the analysis of population exposure and the principles of exposure analysis therefore provide a quantitative approach that can be used not only to assess the level of exposure of current systems, but more importantly to show quite widely applicable principles for exposure reduction. specifically the analysis shows that centralisation of production almost inevitably j o u r n a l p r e -p r o o f creates higher population exposure values, and provides a sound theoretical basis for promoting decentralisation of production as an approach for the reduction of population exposure. it is important to consider the practicality of population exposure reduction by decentralisation; this is reviewed within each of the fields previously identified as incurring high exposure: a) complex components. in order for this functionality to be genuinely available at a significantly decentralised level, we can consider the sub-systems that are required, and the current level of maturity of technology options within each of those subsystems. for a complex component, relevant subsystems include those associated with the supply of materials and the creation of complex shapes from basic material stocks. for many cases, assembly of components is likely to be straightforward but we must also consider cases where assembly itself requires specialised equipment. for the majority of cases, the composition of the complex component can be assumed to be uniform, however cases where composition is not uniform (e.g. multilayer circuit board) must also be acknowledged. equipment for additive and subtractive manufacturing is available; specifically 3d printing equipment is readily available at small scale, and large scale implementations (bassoli et al. 2007 , kraft et al. 2013 ) have been tested. a moderately standardised format for 3d printer design information exists as iso 14649-1:2003, and iso 10303:2014 (many sections) , and instructions for operating additive and subtractive manufacturing equipment are such that high skill levels are un-necessary. a significant body of designs (pinshape™,  complex substances. in order for complex substance synthesis to be genuinely available at a significantly decentralised level, we can consider the types of complex substances that are of interest, the sub-systems that are required for each, and the current level of maturity of technology options within each of those subsystems. broadly, the complex substances could be categorised as: complex alloys, complex inorganic liquids (oils, detergents, etc.), complex organic materials (pharmaceuticals, insecticides, herbicides), complex substances derived from living organisms -yeasts, vaccines, fermentation bacteria, and polymers. for a complex molecular substance, it is currently common for a range of supply chains to bring raw materials to a synthesis plant. a sequence of synthesis steps (including unit operations of heating and cooling, separation, reaction, precipitation and dissolution) are carried out to generate a complex substance. for "organic" compounds, temperatures are generally limited to below 120ºc. for a complex metallic component, it is currently common for granulated pure metals or metal-compounds to be melted together, sometimes in vacuum or inert gas, and then a controlled set of cooling/holding/reheating steps are used to generate the final material. considering whether these syntheses could be practically decentralised, drexler's seminal paper (drexler 1981) considered the options and practicality issues associated with general-purpose synthesis of complex substances. since 2010, the "engineering and physical sciences research council" (epsrc) have been funding a "dial-a-molecule" challenge which runs parallel to the work of others (crow 2014 , peplow 2014 , and commercial ventures such as "mattersift™" (manipulating matter at the molecular scale) have demonstrated the progress towards this goal. even where general-purpose synthesis capabilities are not available, the availability of knowledge does in principle allow relatively complex syntheses e.g. daraprim™, to be undertaken by small groups (hunjan 2016) . for j o u r n a l p r e -p r o o f inorganic materials, progress has been made with solar pyrometallurgy (neelameggham 2008) and although much development is needed, there would seem to be no fundamental reason why the required temperatures and composition constraints could not be achieved on small scale and with limited equipment. published proposals (crow 2014 , peplow 2014 ) have proposed that it is possible to build a standardised facility capable of carrying out an arbitrary sequence of unit operations required to make any organic compound. while the technologically maturity of decentralised synthesis of complex materials is lower than the decentralised production of complex components, many of the processes are actually feasible at present, and others are rapidly maturing.  finance: decentralised tokens of wealth (tokens of exchange) have existed for as long as societies have existed. in order for a token of exchange to continue to avoid the large exposure of current centralised financial systems, a token must retain the qualities of ubiquitous acceptance, transparent and irrevocable transactions; this is currently feasible. blockchain technology (swan 2015) has recently offered another decentralised system for secure and irrevocable transfer of wealth, allowing broad acceptance and thus meets the criteria for acceptability. blockchain-based currencies are however reliant on a communications system that is currently highly centralised, and so fall short of the security expected and exist in numbers of as-yet-incompatible forms. the difficulties with current blockchain technologies seem to be solvable, and this technology offers a promising approach to decentralised exchange of value. current, high-security banknotes and bullion fulfil many of the requirements for a decentralised approach to transactions, and do not incur the exposure that is inherent with blockchain-based currencies, but do incur a high risk of theft and the exposure of a physical transmission system if the transaction is to span significant distance. j o u r n a l p r e -p r o o f  communications: a communications system could be considered decentralised (within the population size envisaged) when it has no e1 value above zero, and is capable of communicating with any other (decentralised) population. secure encryption is currently possible (schneier 2015) , and despite some practical difficulties, mature approaches such as one-time-pads, seem to be proof against even projected (de wolf 2017) technologies. radio transmission on shortwave bands (using ionospheric reflection) of encrypted material can be received globally. assuming a 10 mhz "open" band, and a very narrow bandwidth (and very slow transmission rate), many channels would be available in principle. this is a low level of practicality, but is must be noted that completely decentralised communication is inherently feasible. massively-redundant fibre-optic cable systems with decentralised routing systems are also technically feasible, and it is even feasible to consider significantly higher levels of design redundancy for undersea cables. while both feasible and practical for land-based systems, massive design redundancy appears to be feasible for undersea routes but not practical for the exposure level sought. selfdiscovering radio-based mesh communications for land-based systems are (author 2007) feasible at present and are likely to be more practical and economical than massively redundant fibre-optic systems for land-based communications. hybrid approaches, e.g. using self-discovering mesh for communications within a single land mass, and allowing access to a significant number of undersea cables, could meet the population levels and exposure levels to allow a claim of feasible decentralisation using current technology. suggests that fundamental breakthroughs may not be likely. so-called "ultracapacitor" technology (de rosa et al. 2017 ) may or may-not overtake battery storage for smaller power levels in the future. the decentralised production of biofuel using macro-algae (zev et al. 2017 , chen et al. 2017 ) is also immature, but the capability to treat sewage and create storable hydrocarbon fuel with high energy-density is promising and is perhaps the decentralised heating-energy storage mode that is closest to technical maturity at present. information. the information storage requirements for a community could include significant medical information, all data required for manufacture of complex components and synthesis of complex substances in addition to contractual, financial, genealogical etc. data. human-readable information storage (books, tablets) have very low exposure and high decentralisation currently. a decentralised and low-exposure approach for storage of machine readable information does j o u r n a l p r e -p r o o f however pose a non-trivial technological challenge. existing computer hard-disk drive storage technology is mature, but this approach has a very high technological exposure since the format of storage and the recovery of data is via a complex and proprietary system, and the actual storage medium is not user-accessible. a criterion of e1<1 could in principle be achieved by massive redundancy, but in practice the use of identical operating systems and software make it likely that residual exposure will remain. technologies such as the 5d glass storage approach (kazansky et al. 2016) or proposals by the longnow foundation; or organisations such as "nanoarchival™ technology" avoid the reliance upon proprietary data retrieval systems and provide very long life -but still lack a mature and decentralised datawriting approach, and a mature and decentralised approach for reading stored data back into a machine-readable form. the advances needed in order to achieve a durable, low-exposure storage medium are immature but are technologically achievable. other: it is useful to note many other fields in which significant capabilities can be achieved with simple components: while far short of laboratory quality, the principles of spectrography and chromatography (lichtenegger et al. 2017 , ghobadian et al. 2017 are actually accessible at a domestic level, and microscope (prakash et al. 2017) (jones 1979) and mars explorer spacecraft. organisations such as the longnow corporation have considered the requirements for durable engineering, and plan items such as a "10,000 year clock". in the context of this paper and earlier studies (author 2010), we can consider that it is practical to design at least some classes of components and equipment for a usable timeframe that is demonstrably longer than the time required to re-develop a production facility to recreate them; this criterion is a valid test of low exposure practicality. in summary: low exposure and decentralised options for a range of technologies have been examined: some are mature and some have a low level of technological maturity. it is likely that some could mature rapidly (e.g. additive/subtractive manufacturing, self-discovering communications networks, information storage), while others such as energy storage have absorbed enormous r&d effort already with limited progress. this does not mean that a trend to decentralisation cannot begin, simply that a decentralised society may have more sophisticated capabilities in some fields than others. the ability to form fixed-duration, ad-hoc associations to enable some largescale development are not only possible for a society comprising decentralised groups, but are considered to be essential. this conclusion differs from the assertion by jebari & jebari (2015) that "…isolated, self-sufficient, and continuously manned underground refuges…" are appropriate, rather proposing intentionally independent population units who control their specific interactions with other units, achieving reduced vulnerability but without forfeiting the capability to aggregate selected resources for mutual gain. smaller-scale concepts such a crowdsourcing approaches are already demonstrating somewhat similar options for shorter-duration, ad-hoc design advances. situations such as ships travelling in convoy are common and perhaps form a close analogy, demonstrating both the independence of each and the possibility of cooperation. this paper focussed on broad issues of technological vulnerability, but implementation details (resource usage efficiency, waste creation/treatment, gross greenhouse gas emissions and many other issues are also acknowledged. sociological/anthropological research (dunbar 2010) has indicated that quite small populations provide a sufficient sociological group size for most, and this conclusion seems to remain broadly valid even where high levels of electronic connection are possible (maccarron 2016). the concept of a technological system's exposure provides both a tool and a metric, which can be applied to either individuals or to populations of individuals, and can supply useful data to the forecasting process. population exposure is found to be high for many categories of current technological systems, and the current trend is to increased levels of population exposure. the categories that have been considered as typical, span and affect many goods and services that would be considered essential. items as diverse as internet usage and financial transactions already have multi-national levels of population exposure (and hence high levels of centralisation) and although various authors (diamond 2005) j o u r n a l p r e -p r o o f does not use the term "exposure" nor precisely describe the concept, he explains that environmental damage actually has a global exposure level. population exposure is a topic that does generate intuitive awareness of vulnerability: it can be observed that awareness of technological vulnerability exists at both national level (see j critical infrastructure protection, pub elsevier) and at individual level (slovic 2016 , martin 1996 , huddleston 2017 , kabel & chmidling 2014 , reiderer 2018 . since a measure of exposure correlates closely with the effort required to protect a system, a continued trend to centralisation and increased population exposure is very likely to lead to progressively herculean efforts to ensure that vulnerable loci are not attacked; such efforts are likely to include progressively wider surveillance to identify potential threats, and progressively stronger efforts to control and monitor access -each of which are themselves factors that can serve to make the service practically unavailable to individuals. if no value were attached to high and rising levels of population exposure, then highly centralised options are likely to continue to be preferred -but the consequences of the high and rising population exposure are demonstrated by reference to a metric of exposure, are also intuitively understood and are illustrated by the "continued centralisation" option in figure 3 . if there were indeed no technological options for reducing the level of population exposure without major reductions in the levels of technological sophistication available, then large populations are indeed exposed to the danger of losing access to services. that scenario would indeed result in a catastrophic situation where a major reduction in sophistication of services occurs, but the remnant j o u r n a l p r e -p r o o f exposure is also reduced. that scenario has been labelled as the "apocalyptic survivor" scenario in figure 3 . the third alternative shown in figure 3 , is for progress towards a significantly decentralised society, with a comparatively low population exposure and a level of sophistication (in terms of technological services and goods available) that does not decrease and actually has the real possibility of advancing with time. in this paper, the description of "forecastable options" has been considered under each of the categories of high population exposure, and it has been noted that for each of them technological developments that enable significantly reduced population exposure exist. the current level of technological maturity of these options vary, but none are infeasible and complement the proposals advanced by a number of authors including gillies (2015) and blühdorn (2017) . j o u r n a l p r e -p r o o f in the course of considering decentralisation options, this paper has identified a small number of technological capabilities that would facilitate a more decentralised option, but which are currently at a low level of technological maturity. these include:  further advances on general purpose chemical synthesis  a durable, open-source machine-accessible information storage system that can be created and read in a decentralised context (requiring only minor development)  an self-discovering network using an open-source approach to allow information transmission (requiring some development)  a large-capacity, durable and economically accessible energy storage technology (requiring significant development)  further development of trustworthy and decentralised financial transaction systems (requiring some development).  a non-technological system to allow ad-hoc cooperation between decentralised groups, allowing resource aggregation without long-term centralisation (requiring significant development). despite the inevitable variations in technological maturity across a broad range of technologies, the analyses have concluded firstly that centralisation and high population exposure result in severe and increasing vulnerabilities for large numbers of persons, and secondly that the combination of maturing decentralised technological capabilities and the storage of knowledge allows a transition to a "sophisticated decentralisation" model to be considered as a serious option. while not the primary topic of this paper, it is noted that even in the presence of technological options, change may not occur until some triggering event occurs; events that could trigger a more rapid transition to a "sophisticated decentralisation" could j o u r n a l p r e -p r o o f include a truly major and long-term disruption of some highly centralised technology such as undersea cables or an irremediable malfunction of international financial systems, or a pandemic requiring high levels of population isolation 1 . the analysis of technological exposure and reduced exposure options have concluded that practical options for substantial decentralisation exist, or could be reasonably forecast as possible. it has also been proposed that there are substantive and immediate reasons to consider a qualitatively distinct "fork" from the high population exposure of current centralised society to a more decentralised model. any selection of a decentralised technological model might be triggered by some event which crystallised the exposure of a highly centralised model: the drivers that had produced the centralised model will still remain however, and will arguably tend to cause a re-centralisation without ongoing efforts. this issue is outside the scope of this paper, but is noted as a topic requiring further research. the capability for decentralised and machine-accessible storage of knowledge and the creation of complex components and substances has recently, or will soon, create a cusp at which local equipment is capable of reproducing itself. sufficient computation facilities at local scale are already able to make genuine advances, and with equipment capable of replication, is sufficient to allow fully sustainable, sophisticated and decentralised communities to diverge from current trends. declarations of interest: none consumer preferences for household-level battery energy storage on some recent definitions and analysis frameworks for risk, vulnerability, and resilience 3d printing technique applied to rapid casting confronting future catastrophic threats to humanity dark ages ii: when the digital data die eco-political hopes beyond sustainability. global discourse existential risks: analyzing human extinction scenarios and related hazards calling dunbar's numbers colombian ancillary services and international connections: current weaknesses and policy challenges digital vellum macroalgae for biofuels production: progress and perspectives undersea cables and the future of submarine competition addressing the harm of total surveillance: a reply to professor neil richards mechanics of turn-milling operations the anything factory sustainable growth-an impossibility theorem the knowledge: how to rebuild civilization in the aftermath of a cataclysm black code: censorship, surveillance, and the militarisation of cyberspace collapse: how societies choose to fail or succeed cascading blackout overall structure and some implications for sampling and mitigation molecular engineering: an approach to the development of general capabilities for molecular manipulation how many friends does one person need?: dunbar's number and other evolutionary quirks intel's management engine is a security hazard, and users need a way to disable it hype and heavy tails: a closer look at data breaches how to hack a turned off computer, or running unsigned code in intel management engine the protocols kinematic self-replicating machines an innovative homemade instrument for the determination of doxylamine succinate based on the electrochemiluminescence of ru(bpy)2+ 3 paul mason's postcapitalism emerging technologies, catastrophic risks, and ethics: three strategies for reducing risk a critical review of cascading failure analysis and modelling of power system in: near-net shape manufacturing of miniature spur gears by wire spark erosion machining. materials forming, machining and tribology leontief based model of risk in complex interconnected infrastructures weaving the web: otlet's visualizations of a global information society and his concept of a universal civilization long-lost satellite tech is so old nasa can't read it. tech & science. nasa image. observer. nasa satellite lost for 13 years recovered by amateur astronomer when do states disconnect their digital networks? regime responses to the political uses of social media. the communication review conceptual design and dimensional synthesis for a 3-dof module of the trivariant-a novel 5-dof reconfigurable hybrid robot prepper" as resilient citizen. responses to disasters and climate change daraprim) drug's key ingredient recreated by high school students in sydney industrial automation systems and integration --physical device control --data model for computerized numerical controllers automation systems and integration -product data representation and exchange. it is known informally as "step existential risks: exploring a robust risk reduction strategy automatic fault protection in the voyager spacecraft disaster prepper: health, identity, and american survivalist culture eternal 5d data storage via ultrafast-laser writing in glass nasa tests limits of 3-d printing with powerful rocket engine check. nasa engineers prepare to hot-fire test a 3-d printed rocket part on test stand 115 at the marshall center recent progress on cascading failures and recovery in interdependent networks visible light spectral domain optical coherence microscopy system for ex vivo imaging longnow foundation total surveillance, big data, and predictive crime technology: privacy's perfect storm is your algorithm dangerous? solar pyrometallurgy-an historic review electronic payment systems. artech house organic synthesis: the robo-chemist. the race is on to build a machine that can synthesize any organic compound. it could transform chemistry permanent archival solution: nanoarchival provides ultra-long term archival solutions to enterprise customers, based on its proprietary a dangerous trend of cybercrime: ransomware growing challenge population dynamics with and without selection optical microscope 3d printing based on imaging data: review of medical applications doomsday goes mainstream high performance spiro ammonium electrolyte for electric double layer capacitors emerging threats: outer space, cyberspace, and undersea cables von neumann machine. encyclopedia of computer science. 4th. pages 1841-1842 monsters in cyberspace cyberphobia and cultural panic in the information age. information applied cryptography, second edition: protocols, algorthms, and source code in c, 20th anniversary edition isbn information. online isbn: 9781119183471. print isbn a new rationale for returning to the moon? protecting civilization with a sanctuary a thesis submitted to faculty of graduate studies and research, mcgill university in partial fulfilment of the requirements of the degree of the perception of risk energy and civilization; a history hulking transformers prove a challenge for vulnerable grid. e&e 2015-11-24 and see energy news digest for blockchain: blueprint for a new economy censorship and surveillance in the digital age: the technological challenges for academics the designer's guide to disk drives the potential impact of quantum computers on society. ethics and information technology high-yield bio-oil production from macroalgae (saccharina japonica) in supercritical ethanol and its combustion behaviour key: cord-304013-nzigx0k0 authors: lipinski, tom; ahmad, darem; serey, nicolas; jouhara, hussam title: review of ventilation strategies to reduce the risk of disease transmission in high occupancy buildings date: 2020-09-13 journal: nan doi: 10.1016/j.ijft.2020.100045 sha: doc_id: 304013 cord_uid: nzigx0k0 an unforeseen pandemic is facing the world caused by a corona virus known as sars-cov-2. numerous measures are being put in place to try and reduce the spread of this deadly disease, with the most effective response to the outbreak being mass quarantines, a public health technique borrowed from the middle ages. the widely accepted main transmission mechanism is through droplet borne pathways. however, many researchers and studies are considering that this virus can also spread via the airborne route and remain for up to three hours in the air. this is leading to questions as to whether enough is being done regarding ventilation to reduce the risk of the spread of this or other diseases that may be air borne. ventilation and air conditioning systems are the main focus when it comes to the transmission of such deadly pathogens and should be appropriately designed and operated. this paper reviews and critically evaluates the current ventilation strategies used in buildings to assess the state of the art and elaborates if there is room for further development, especially for high occupancy buildings, to reduce or eradicate the risk of pathogen transmission and adapt ventilation measures to new threats posed by pandemics. an unprecedented viral disease has brought our globe to a halt, impacting most of mankind's activities. at the time of writing, more than 620,000 people are dead worldwide, the global economy is on the verge of an unprecedented depression, with the covid-19 pandemic still raging and a second wave predicted as inevitable. covid-19 belongs to the group of coronavirus, also known as severe acute respiratory syndrome corona virus 2 (sars-cov-2) [1] . this virus has already surpassed the number of infections of two other epidemics in this century [2] . current measures introduced worldwide, and designed to control the spread of the virus include lockdowns, selfisolation, social distancing, use of face masks and the recommendation to wash hands as frequently as possible, [3] . n95 masks have been recommended by who and have been known to help prevent infected individuals from spreading the virus if not necessarily preventing healthy individuals from contracting it from others [4] , [1] . covid-19 is one of the most contagious viruses that mankind has experienced, spreading across most of china in only 14 days [2] , then worldwide within a couple of months. the widely accepted mechanism of covid-19 transmission is by droplet and contact methods as backed-up by the who, but the possible air transmission route has been broadly documented by new scientific research [5] and the who is not ruling out this possibility. as individuals are infected with the respiratory disease, the rate of expiratory events increases which in turn increases the generation and dispersion of droplets containing the virus. such expiratory events include not only coughing and sneezing but also talking [6] . considering how fast this disease has spread across the world, many researchers [7] state that an additional mode of transmission, nuclei borne by air droplets, plays an important role in the spread of this virus. flow dynamics of air particles can be complex and include turbulent jets, droplet evaporation, airmucous interaction and particle sedimentation amongst others [5] . these flow dynamics and particle interaction with air is at the core of transmission of the covid-19 virus. as mentioned previously, a variety of physical containment methods have been introduced, such as wearing the recommended personal protective equipment (ppe) and improving personal hygiene. however, since the vast majority of infections occur indoors [8] , it has been noted that ventilation strategies can play a vital role in controlling or at least reducing the risk of respiratory infections [9] . droplet nuclei are fine air particles that remain airborne for a considerable length of time. any air particles below 5 micrometres are classified as being able to be airborne. sars-cov-2 is yet to be officially classified as an airborne disease by the who, however, a weight of emerging evidence is being established for aerosol driven infections [10] [11] [12] [13] . the chartered institution of building services engineers, cibse, recently provided guidance on using ventilation as a way of diluting airborne pathogens. it is stated that: "there is good evidence that demonstrates room occupants are more at risk of catching an illness in a poorly ventilated room than in a well-ventilated room." besides this, new evidence that has been found shows high rates of infection in poorly ventilated spaces [14] . since sars-cov-2 has spread around the world at an unprecedented pace, infecting millions of people, and further aerosol driven infections are highly likely to emerge, ventilation plays a key role in efforts to limit the transmission rate of this and other diseases. this paper will discuss the factors affecting air particle properties in-terms of flow dynamics and critically analyse current ventilation strategies and mechanisms and identify areas for improvement in the search for the reduction of indoor infections. thoughtful modifications to the built environment -to how schools, offices or health-care facilities are designed, operated and maintained -could help curb the spread of infectious disease, reducing the toll of future outbreaks as well as the current covid-19 pandemic. this section will review the current state-of-the-art covering characteristics of respiratory particles, the ability of viruses to spread as well as the mechanisms for airborne transmission and how current ventilation strategies can affect the risk of transmission. respiratory particles are formed during any respiratory activity such as coughing, sneezing, talking and breathing. once the particles are released into the surroundings, the mechanisms in-which they flow and settle depend on the fluid dynamics of the particles and the conditions in the vicinity. fluid dynamics can characterise the evaporation rate of the particles which allows the determination of the prevalence of small droplets and nuclei droplet transport [5] . covid-19 belongs to the betacovs pathogen group and has an approximate diameter of 60-140 nm. the shape of the virus can be spherical or ellipsoidal [15] . particle trajectories depend primarily on their size and the balance of various forces acting on the particle in the air. gravitational and aerodynamic forces are the two primary forces acting on such particles, where the latter force dictates the flow behaviour of the particle. figure 1 shows the trajectories for particles of various sizes ranging from 0.01 µm to 100 µm [8] . figure 1 : trajectories of particles with various sizes [8] . stokes law explains the frictional force relation exerted on spherical objects with very small reynolds numbers in viscous fluid. to help explain the trajectories of air particles, the aerodynamic drag coefficient (cd), shown in equation 1 of a spherical particle relative to reynolds number, shown in equation 2, is not constant. reynolds number is a ratio between inertial and viscous forces that originate from interactions between a body and a fluid. the equation for reynolds number is shown below. where fd is the aerodynamic drag force, and a is the frontal area. using reynolds equation, for natural ventilation systems with air velocity of 0.3 m/s and a particle size of 140 nm, the reynolds number is calculated at 0.003. this shows that for such air flow environment, the flow is laminar whereby the viscous forces prevail. therefore, drag coefficient for viscous non-separated flow around a sphere can be defined by equation 3. however, this equation implies that drag force is proportional to the diameter which is not entirely accurate. the resistance coefficient may decrease with increasing diameter, but the drag force increases linearly with the diameter. however, since the gravitational force increases with the mass, it increases with diameter cubed, thus, more rapidly than the drag force as the diameter increases. therefore, larger particles have the tendency to settle [16] . this phenomenon helps to explain why smaller particles are more likely to be airborne. the drag coefficient for a sphere as a function of reynolds number can be plotted to show the influence of changing flow regime on the drag coefficient. this is shown in figure 2 , whereby a sphere exhibits a reduction in drag coefficient until the flow regime becomes turbulent causing a sudden dip. furthermore, when the boundary layer on the leading surface becomes turbulent, the separation occurs farther round the sphere and the drag coefficient decreases. a rough surface, such as on a golf ball, promotes an earlier transition to turbulence. the numbers on figure 2 refer to various flow regimes and their corresponding changes in the drag coefficient. flow regime 2 on the figure refers to stokes flow and steady separated flow, whereby the reynolds number is less than 1 which is typical for fluid velocities are very slow and the viscosities are very high. flow regime 3 on the figure refers to separated, unsteady flow, whereby a laminar flow boundary is formed upstream of the separation and producing vortex street. flow regime 4 refers to separated unsteady flow with a laminar boundary layer at the upstream side and chaotic turbulent wake downstream of the sphere. regime 5 refers to a turbulent boundary layer [16] . a graph of aerodynamic drag coefficient against reynolds number [16] . various parameters affect the transmission of droplet borne infections, including density, initial velocity and the size distribution of the droplets that are released during respiratory activities, as well as indoor air velocity and direction. many research studies have been carried out to measure these characteristics [17] . sneezing is the respiratory activity that ejects the most droplets, in the range of 10 4 or more. the droplets from sneezing have initial velocities in the range of 20 m/s. other respiratory activities release droplets at a significantly lower density and lower initial velocities. table 1 shows the droplet densities and velocities for the common respiratory activities. the terminal velocity at which any spherical particle settles due to gravity in a fluid can be explained by stokes' law for fine particle size and newton's law for coarse particles. both equations have been shown below. where = gravitational acceleration = density of solid and fluid respectively d = particle diameter = drag coefficient μ = fluid viscosity the terminal velocity equations shown above have been used to generate graphical representations of the expected particle speeds at various conditions as shown below in figure 3 . since covid-19 has an approximate diameter of 60-140 nm [15] , stokes' law applies for ventilation in buildings whereby the air flow exhibits a laminar behaviour. figure 3 shows that at very small diameter particles of less than 0.1 µm, the terminal velocity is almost negligible which further amplifies the fact that if covid-19 was proven to be airborne then the virus and its potent materials could be airborne for long durations especially if enclosed environments are not ventilated adequately. a mathematical model by c.p cummins et al. shows that particles with a diameter smaller than 70 µm (stoke's number ≤ 8.5 × 10 −5 ) are almost unaffected by the gravity and air extraction is very efficient at removing these fine particles [19] . this is explained by the major impact of the drag forces on the particle's movement for this diameter. the settling velocity calculated by stokes' law (equation 3) is proportional with the diameter squared. particle terminal velocities remain elusive due to a vast number of factors, such as droplet diameter, droplet density, contributing to this phenomenon and primarily due to the difficulty in making measurements of such parameters [18, 20, 21 ]. pathogen transmission can occur through different routes: direct contact, indirect contact, droplet borne or airborne such as shown in figure 4 [22] [23] [24] . the direct contact route covers any contact between a contagious person and a susceptible person: touching, kissing, sexual contact, contact with skin lesions or oral secretion. this route is well documented and outside the scope for this paper. in the indirect contact transmission mode, the contagious person touches or expels contaminated droplets containing infectious organisms which settle on an inanimate object, such as a doorknob or an elevator button, called "fomite". the person being infected touches this fomite and then an area where the pathogen can enter the body such as the eyes, nose, or mouth [7] . droplet transmission can occur when infective large droplets expelled from a contagious person reach an uninfected person. airborne transmission occurs when very small particles (5-10 µm in diameter) or small droplets evaporating to a small enough size [26] , are expelled by coughing, sneezing, speaking, or breathing. these droplets are small enough to remain airborne for long periods of time (up to several hours), until they are inhaled by or land on the uninfected person. airborne transmission is not yet widely accepted for various transmissible respiratory diseases. in the case of sars-cov-2, the world health organisation only recognizes the risk for airborne transmission under certain medical procedures producing large amounts of infective respiratory particles [22, 23, [27] [28] [29] . there is now growing pressure on the who from the scientific community to relax the dogmatic and outdated division between aerosols and small droplets and acknowledge the mounting evidence for the indoor spreading of the covid-19 infection through the air [30] . in the case of sars-cov-1 and mers-cov, retrospection on the different outbreaks and multiple studies make a strong case for the route as an opportunistic transmission, meaning that the virus will transmit preferably through other routes but can potentially infect by means of respiratory particles when conditions are met. another factor explaining the elusive validation of airborne transmission is the dilution of small particles after they are emitted. indeed, people standing farther away from a contagious emitter see an exponentially decreasing concentration of droplet nuclei. the potential transmission over larger distances are by nature more difficult to identify especially in the context of an outbreak or a pandemic when the sources are multiple. furthermore, when the transmission happens close to an identified source, the transmission is indiscernible from that due to droplets or indirect transmission [27, 29] . in order for viruses to be transmitted through the airborne route, some conditions must be satisfied: the virus must be able to remain viable outside of the host, withstand the external conditions and be transported to a susceptible area of a new host. the effect of evaporation, light, humidity and temperature on the concentration and virality (capacity to infect and reproduce) of the pathogen need to be further researched to make conclusions on the threat level of the airborne route [31] . there are already multiple pathogens for which the airborne route is acknowledged. such viruses including adenovirus, influenza, measles, meningococcal disease, mumps, pertussis, parvovirus b19, respiratory syncitial virus, rubella, tuberculosis and varicella, can spread by droplet nuclei [32] . measles and tuberculosis for example are proven to be preferentially airborne transmitted diseases [29] . but the fact that these diseases have been controlled with widely available drugs and vaccines resulted in a decrease in research effort into airborne transmission mechanisms. recent sars-cov-1 and mers-cov outbreak have given a new impetus to research on this subject. indeed, sars-cov-1 is thought to be transmissible by direct, indirect, and droplet contact, the amoy garden case identifies a strong possibility of airborne transmission. during the outbreak, the virus spread from a faulty dried-out bathroom drainage system and rose to a extraction fan, then was carried by the wind into the adjacent building infecting several occupants [7, 16, 25, 28, 31, 33] . ribonucleic acid, rna, and even viable viruses have been detected in aerosols in healthcare facilities for some respiratory viruses such as seasonal and avian influenzas virus, mers-cov and respiratory syncytial virus [34] . in the case of mers-cov, samples taken in hospitals and isolation wards in south korea confirmed the airborne transmission. some patients of the same ward were infected, even though they were standing more than two meters away from the source. air samples and surrounding areas, including regularly disinfected accessible and inaccessible surfaces, were all contaminated by mers-cov [35] . this experimental data challenges the previously recognized transmission routes, acknowledging the high possibility of airborne transmission as an opportunistic route at close range and even long range when the conditions were favourable and concentrations at the source were high. sars-cov-2 shares the same modes of transmission as the sars cov-1 and, although the viability and virality of an air sample has not been proven at the time of writing, it cannot be ruled out. the airborne route, being opportunistic [25] , is by its nature difficult to interpret. air samples take time before being tested after which the concentration might be too low, through dilution, to establish with confidence the presence of viable virus on droplet nuclei. rna detection is also not usually enough to interpret the risk of airborne transmission [34] . a number of studies indicate airborne or droplet borne transmission [14] , and to the authors' knowledge, there is no study yet demonstrating the lack of infection in a situation where only airborne transmission is permitted (and with particles in high concentration). this would be the only definitive way to disprove the mode of transmission [36] . recent superspreading events also support the argument for airborne transmission. during a choir rehearsal in mount vernon (washington), although precautionary care was taken to suppress direct contacts and keep distance between the singers, 53 out of the 61 participants caught the disease [28] and at least two people were reported to have died [37] . the aerosol stability of sars-cov-2 was recently tested in laboratory conditions. a three-jet collison nebulizer created contaminated aerosols (< 5µm) fed into a goldberg drum. in this environment, the sars-cov-2 remained viable with a half-life between 1.1 and 1.2 hours (95% credible interval 0.64 to 2.64h) as shown in figure 5 . the researchers compared the results with sars-cov-1 and found similar half-life results (95% credible interval 0.78 to 2.43h). the reduction in infectious concentration (titer) during the 3-hour long tests was from 10 3.5 to 10 2.7 tcid50 (fifty-percent tissue culture infective dose) per litre of air for sars-cov-2. this reduction was similar to that observed with sars-cov-1, from 10 4.3 to 10 3.5 tcid50 per litre of air [27] . in order to better understand how to combat airborne transmission, one must understand the relation between the particle emission and deposition mechanisms. droplets can be generated from different places in the respiratory tract of the infected person with various resulting diameters. once deposited on the uninfected individual they then infect the conjunctiva or mucous membranes in the upper respiratory tract of the new host [13, 26, 38] . small particles can be generated during speech in the alveoli of the lungs and vocal cord vibration by a fluid film burst mechanism [7, 39] . it was reported that vocalization emits 10 times more particles than breathing, and some speech super-emitters can (for reasons unclear yet) expel 10 times more particles than average [6] . this could be an explanation to the mount vernon choir outbreak: the higher concentration of infected particles added to the need for extra deep breathing required for singing facilitates airborne transmission. coughing and sneezing also generate large numbers of particles, the majority of which are inhalable (< 100μm in size): 78.6-96.0% for coughing and 98.9% for sneezing. furthermore, between 7.1-46.7% of cough expelled particles and 18.8% of particles produced by sneezing were under 4μm in diameter [38] . these smaller particles penetrate deeper and can deposit further in the respiratory tract. generally, particles above 10 µm do not reach the alveoli whereas particles above 20 µm deposit in the upper respiratory tract as shown in figure 6 [5, 13, 26, 28, 34, 38] . [29] . transmission that occurs in the lower respiratory tract, shown in figure 6 due to smaller particles potentially leads to aggravated symptoms and a higher mortality rate, whereas deposition in the upper respiratory track by larger aerosols necessitates a larger number of viruses for the host to develop symptoms [29] . this is due to the fact that the nasal and tracheobronchial regions have an additional defence system. the nasal tracts are covered by a mucus layer that entraps deposited particulates. the continuous movement of cilia pushes the captive infected particles up to the gastrointestinal track where they cannot infect the host [5, 38] . research studies have shown that small particles still present a substantial risk of infection [26] , and it was reported that for some respiratory diseases, a single virus can cause illness. in the case of sars-cov-2, a minimum required viral load has not yet been confirmed [5, 7, 29] . once the virus finds a susceptible cell, it can infect individuals through binding its pike proteins to the cell wall then uses the cell to replicate before bursting it. thereafter, it releases more viruses that can either contaminate other cells, be destroyed by the immune system or be expelled from the body and potentially transmit to a new host [23] . studies have shown that that the ensuing incubation period is on average 5.1 days, but can range from 2 to 10 days [23] . the virus remains detectable for a median of 20 days [40, 41] . a critical specificity of sars-cov-2 is the serial interval (or the time lapse) between an infection of a host and the transmission to a susceptible uninfected person. an average of 5.8 days, with a 95% credible interval between 4.8 and 6.8 days, and median of 5.2 days, with credible interval between 4.1 and 6.4 days, were reported [41] . the small-time delay and possible overlap between the end of the incubation period and the secondary infection shows that pre-symptomatic transmission is very likely. it is believed that the infectiousness starts 2.5 days before the first symptoms of the disease and peaks when the symptoms are starting [5, 41] . this would be the major cause of the rapid spread of the pandemic. the speed of the spread is represented by the basic reproduction, ro, number which is the average number of susceptible persons infected by a single host [24] . for sars-cov-2 ro is estimated to range between 1.4 to 6.49 (mean, 3.23; median, 2.79) [23] . furthermore, the ro is rapidly evolving and other studies have found values of ro between 1 and 4 before intervention to limit the spread [42] . the basic reproduction number corresponding to pre-symptomatic transmissions alone is estimated to be 0.9, with 95% credible interval between 0.2 and 1.1. a ro value superior to 1 means the virus will spread exponentially, indicating these transmissions are almost sufficient to sustain the pandemic [24] . at the beginning of the wuhan outbreak, the contribution of pre-symptomatic cases accounted for 46% of all infections, with 10% for asymptomatic cases, and it is now widely accepted that seemingly healthy people can spread the virus, though uncertainty remains over how much they have contributed to the pandemic. though estimates vary, models using data from hong kong, singapore and china suggest that 30 to 60 percent of spreading occurs when people have no symptoms [24, 41, 43] . as the body starts to build antibodies against the virus, the concentration begins to decrease, and the infectiousness of the disease declines significantly after 10 days [24] . when a significant portion of the population stops being susceptible to the virus, the ro falls below 1 and the number of new infections drops below the sustaining rate. this can be achieved through vaccination or by building immunity after recovering from the disease -thus the state of herd immunity may be achieved [24] . although mutation from the virus and decay of the immune system memory could still pose a challenge to achieving this goal. in order to supress or slow the progression of the pandemic, efforts must be made to reduce ro. precautionary measures must consider the entire population since pre-symptomatic and asymptomatic transmissions are a critical factor in the spread of sars-cov-2. if the virus can spread from seemingly healthy carriers or people who had not yet developed symptoms, the publicawareness campaigns, airport screening and 'stay-home-if-you're sick' policies might not stop it. more targeted measures are required including a considered re-design of indoor environments, especially aspects handling the air buildings' ventilation systems [44] . both the airborne and or droplet borne routes cannot be ignored. pre-symptomatic and asymptomatic hosts do not cough or sneeze extensively [5, 7, 40] . when doctors in wuhan, china, where the new virus first emerged, studied 138 early cases, they concluded that 41 percent of patients had most likely contracted the disease in the hospital [40] . therefore, the hypothesis stating that infection through small particles plays a more important role in the transmission along with the direct and indirect (fomites) routes can be used. thus, ventilation plays an important role in reducing the risk of transmission through dilution and removal of the infected particles within the indoor environment [25] . preparedness to fight airborne disease is essential and sars-cov-2 offers the chance to research and apply mitigating ventilation solutions which could prove life-saving now and in the future when another virulent and deadly pandemic arises [29] . ashrae standard 62.1:2019 defines acceptable indoor air quality as "air in which there are no known contaminants at harmful concentrations, as determined by cognizant authorities, and with which a substantial majority (80% or more) of the people exposed do not express dissatisfaction". [45] if airborne infectious particles are to be counted as harmful even in small concentrations, as explained in the previous section, either the amount of fresh air supplied to a room needs to be dramatically increased or the ventilation strategy needs to be reconsidered to protect the occupants. this paper focuses on high occupancy buildings such as schools or office spaces where the occupants are static most of the time. current standards already have predetermined values to meet the acceptable indoor air quality. for example, the ashrae standards dictate a minimum ventilation rate of 5l/s per person or 0.9 l/s.m 2 in educational facilities. for office buildings, the minimum values are 2.5l/s per person or 0.06 l/s.m 2 [45] . having said that, due to variability in ventilation methodologies, similar ventilation rates may translate to significantly different indoor transmission outcomes. fresh air can be provided in a number of ways, relying on many different technologies and having different extraction, dilution and air distribution effectiveness. the british standards bs en 16798-3:2017 defines 4 basic types of ventilation systems: [44] . system type fan assisted air volume flow in only one direction. this system is balanced by air transfer devices within the building envelope. fan assisted system operates in supply and exhaust directions. bidirectional ventilation system natural ventilation relies on natural driving forces. natural ventilation system hybrid systems rely on natural and mechanical mechanisms. any combination of the ventilation mechanisms can be used depending on the situation. several factors play a role after a ventilation solution is selected that affect the final system performance and indoor air quality. the poor initial design of a selected ventilation solution could be due to various reasons such as error in sizing, reduced performance due to lack of maintenance, lack of operator knowledge or the intentional reduced use in order to save energy or reduce noise and all can be detrimental to the occupants' health. several studies have demonstrated that in classrooms, the ventilation rates have often failed to reach the minimum standard required. the peak co 2 concentration, which can be used as an indicator of the ventilation rate in occupied spaces, often exceeded the recommended levels. in a study, the reported co 2 measurements in several thousand classrooms has shown that all classroom averages exceeded 1000 ppm (0.1%) which is an indicator of a ventilation rate lower than 7l/s/person at default occupancy. in many instances, the average co 2 levels were above 2000 ppm with peak concentrations between 3000 and 6000 ppm [46] . this discrepancy between the building standards and reality demonstrated the need for a thorough reconsideration of ventilation design and ventilation systems specified in high occupancy buildings well before the impact of coronavirus. in the light of sars-cov-2 pandemic there is an even more desperate need to address the ventilation design and effectiveness. improved ventilation has been noted to deliver a positive health impact with a noticeable reduction in illnesses and absences. in particular, reduced respiratory health effects, such as mucosal and allergy symptoms are significantly reduced with increased ventilation rates [46] . a study based on californian schools demonstrated that an increase in the ventilation rate by 1l/s/person resulted in a 1.6% reduction in illness related absences [47] . poor ventilation has also been linked to many adverse health effects: transmission of infectious diseases, acute respiratory symptoms and impaired cognition performance [48] [49] [50] . it expresses the dilution of the said pollutant as a function of the ventilation rate effectiveness which can be engineered to suit the room, occupant type, and the risk at hand. in the case of infected particles, the recommendations from rehva, the federation of european heating, ventilation and air conditioning associations, are to supply as much outside air as possible. according to rehva, mechanical ventilation should be activated more often (24/7 when possible with lower rates during quiet times) and at least to start ventilating before and after busy hours while the density of occupancy needs to be decreased when possible this will increase the distance between people and lower the pollutant emission rate. with or without mechanical ventilation, window airing should be used to boost the air exchange rate. toilet windows on the other hand need to remain closed and mechanical extraction activated at all times to create negative pressure and prevent contaminated particles from entering other parts of the building though doors or by an unforeseen route through nearby open windows [25] . prior to covid-19, densely packed open-plan offices were already suspected of making employees sick [52] . viruses and other pathogens are not the typical pollutants and even small and temporary exposure has been proven to lead to infections. studies of viral infections spread through indoor spaces document clearly that mechanically induced, mixing airflow can pose a greater risk of infection spread as it pushes turbulent air deep into rooms, possibly picking up infected droplets along the way [53] . further research by the university of oregon demonstrated how air conditioning or hybrid ventilation can spread the pathogens much farther than 6 feet, even when the host is positioned a long distance from the fan driven system [53, 54] . it appears that it is not just the rate of supply of fresh air that needs to be considered but also the air flow dynamics and air distribution pathways through occupied spaces that urgently need review, which include the type of airflow, velocity, its turbulence and its direction. keeping indoor environments virus-free plays a key part in reducing or slowing the transmission of various airborne infections. since viruses have an approximate diameter of 150 nm, they can be easily carried by aerosol droplets in the air and linger afloat for many minutes and sometimes hours. an inappropriate or inadequate ventilation strategy can dramatically increase the risk of disease transmission. a research study conducted by a team of scientists at the defence science and technology laboratory on the aerosol survival of sars-cov-2 in artificial saliva and tissue culture media and high humidity found that covid-19 could be transmitted via airborne droplets in addition to physical contact with droplets deposited onto surfaces [55] . the study used the sars-cov-2 england variant which was suspended in the air using tissue culture media at medium and high relative humidity, 40-60% and 68-88%. the outcome of the study has shown that the virus was still detectable after 90 minutes. taking the above-mentioned publications into account, ventilation and comfort strategies have been categorised by airflow characteristics and their potential impact on pathogen spread through occupied spaces in the next section. considering the dynamics of droplet and aerosol spread indoors, various ventilation and air conditioning strategies can be grouped into three main categories:  recirculating ventilation (frequently called mixing or hybrid ventilation) and conditioning systems that either move the indoor air around (typical split ac or vrf system or even a ceiling fan) or mix indoor air with outdoor air before pumping it into the room (such as hybrid or 'heat recycling' ventilation systems that have been lately installed in many schools). this ventilation method generally produces turbulent air flows with stale air either partly or fully recirculated back into the affected rooms.  mixing ventilation systems that are designed to distribute fresh air throughout the occupied space ensuring all occupants experience similar air quality, with air supplied through specific ventilation outlets or diffusers. they are predominantly mechanical systems such as large, centralised air handling units (ahu) or smaller, localised mechanical ventilation systems with heat recovery (mvhr). this ventilation method generally produces turbulent, mixing air flows within rooms.  displacement ventilation systems that remove contaminated indoor air and supply fresh outside air in a predominantly even, buoyancy assisted fashion, effectively displacing it with no or little disruption. they are primarily passive systems such as natural ventilation cowls, façade louvres or automatically opening windows. this ventilation method generally produces a laminar airflow within rooms (outside of very windy conditions). typical ventilation measures are listed and grouped in the table below, complete with a brief description: building integrated measures designed to displace stale air and supply fresh air using buoyancy including elements such as windows, passive stacks or solar chimneys. natural ventilation systems natural ventilation products or systems utilising buoyancy in their operation including roof cowls, wall mounted iaq responsive louvres or iaq controlled window openers. the recirculating approach has been popular choice for new schools in recent years for both ventilation and comfort provision, mainly due to its low capital cost and simplicity. some recirculating systems are used for ventilation with the recirculated stale air mixed with fresh, outside air, in order to increase the temperature of the air supplied to the classroom, necessary to reduce discomfort in absence of a heat exchanger. the diagram, shown in figure 7 , illustrates the concept of hybrid ventilation: the mixed, partly recirculated, air is fan driven in such a way as to reach far into the classroom up to 8m from the system and to supply enough fresh air, mixed with stale air for comfort, to maintain co 2 levels below an average of 1000ppm. as this requires fresh air flows rate in a range of 150 l/s, when combined with stale air it may reach a volume flow rate of 300 l/s or higher which, considering the small size of the air diffusers, can generate substantial air velocities in occupied spaces -leading to high air turbulence. according to a recent study and modelling conducted by the university of oregon, this type of airflow has the potential for high spread of coronavirus infected droplets within densely occupied spaces, even with just one person exhaling the virus droplets [53, 54] . apart from the recirculation itself, the transmission appears to be facilitated by the type and velocity of turbulent airflow designed to reach deep into the occupied space as shown in figure 8 . this covers most of the air conditioning systems used commercially with the mode of operation similar to the hybrid ventilation systems mentioned above with the exception of containing no fresh air in its supply path -all is constantly recirculated. air is pulled into the system from the room, conditioned, either cooled or heated, and then supplied back into the room at high enough velocity to reach to the end of the occupied space. since the systems are designed to ensure that conditioned air gets to every area of the room, providing comfort, the pathogens picked up by the circulating air can travel much further than 2m [53, 54, 58] . a typical ac airflow can be illustrated by the computational fluid dynamics picture in figure 9 : ceiling fan is another recirculating comfort system and is designed to either reduce air stratification in the room, bringing the warm air down from the ceiling, or to introduce enough air movement to cool the occupants through wind chill effect. both actions are designed to improve comfort while displacing the need for more power-hungry ac or excess space heating. as it can be seen in the cfd snapshot in figure 10 , ceiling fans also ensure that air within the room is fully mixed. as much as there can be various iterations of the above approach, the unifying factor is that ceiling fans continuously mix air within spaces where they serve, such as classrooms, lecture theatres or offices. consequently, any contaminated droplets and aerosols sneezed, coughed or exhaled can travel significant distances within the rooms and reach occupants who are much farther away from the infected host than 2 metres [59] . the research carried out by jaakkola et al. [61] shows the importance of the introduction of fresh air into any occupied room, with recirculation of the air posing a significant risk with many of the air borne diseases not just being carried from person to person by the airflow but also entering the ventilation system itself, possibly being trapped in the air filter. however, many of the pathogens may escape filters causing further infections and droplets can be carried for long distances across spaces by fan induced turbulent airflow without having to enter the recirculating system. since the vast majority of air conditioners in high occupancy buildings utilise air recirculation, it raises the question over their safety and indicates the need for further research into such comfort, ventilation and indoor air quality provision so that the occupant safety can be improved, especially in the light of the covid-19 pandemic [61] . air handling units are generally located in the basement or lower floors of a building such as office blocks (us) or on top of the building (uk). ahus are tasked to supply hundreds of m 3 of air per second across the whole building and generally dehumidify, heat and cool the incoming fresh air as required. the air is supplied through floor or ceiling grills generating positive pressure within the building, it then moves across the ventilated space mixing with existing, stale air along the way and is exhausted through various building fabric openings, exhaust ducts or atria created for that purpose as shown in figure 11 . in some cases, buildings can utilise a hybrid solution combining both natural ventilation principles (even natural free cooling) and mechanical assistance. figure 11 : typical ahu ventilation arrangement with supply ducts located in ceiling voids [62] . a cfd study of a simplified room, with one simulated occupant, using a positive pressure mixing ventilation system can be seen in figure 12 . however, as the fresh air is supplied, a significant degree of mixing will occur within the occupied space. the general design of ahu ductwork, as well as deep floor plan offices exacerbate the fan induced mixing throughout occupied spaces leading to increased risk of long-distance droplet movement. these balanced ventilation systems are generally localised and mounted within the ceiling void of the room or classroom they serve, extracting air from the occupied space, passing it through the air to air heat exchanger to recover the heat and warm up the incoming fresh air, as shown in figure 13 . as much as both air flows are channelled through the same box, there is generally no mixing between the two paths. these systems allow for higher thermal efficiencies due to heat recovery but with an electrical penalty of two fans running constantly. a typical classroom mvhr ventilation arrangement can be seen in figure 14 constructed through simulation software, whereby an air plume generated by supply airflow in façade mounted mvhr system. mvhr systems have been documented to deliver adequate indoor air quality as well as expected comfort levels. their design is to deliver fresh, tempered air to every area of the ventilated space, as it can be seen on a cfd of the façade mounded mvhr below, which, in case of the risk of infection, appears to have similar airflow flow characteristics as recirculating ventilation modelled by the university of oregon. more turbulent air flow at higher velocity is much more likely to mix with existing air and carry larger droplets further into the room, possibly spreading virus contaminated droplets around the room and leading to a higher risk of infection spread. as this is specific to air delivery design rather than the system characteristics, it may be possible to reduce the risk of infection by a careful redesign of the ductwork, its size as well as inlet and outlet location and sizing. it may also be theoretically possible to use mvhr as displacement ventilation in some cases if a complete change of installation design is possible. as much as this is an important area for further research, specific ventilation ductwork design or ventilation design guidance are outside the scope of this paper. piv is very similar in its airflow characteristics to positive pressure air handling units described beforehand with the main difference being the local placement of the fan powered unit or system. the unit supplies the air into an occupied area with enough velocity to evenly distribute it throughout the space. due to its design, it has similar air mixing characteristics as ahus. although mechanical ventilation usually comes with better controllability, it can increase both capital and operating costs which are some of the drawbacks. the need for higher maintenance and the loss of performance when not effectively managed also need to be factored in. it was demonstrated that the severity of symptoms associated with sick building syndromes can be linked with the cleanness of the air filters and hvac system. the occupants' symptoms were recorded by questionnaire before and after cleaning a part of the hvac system and changing the filters. in the renovated section, the severity of the symptoms decreased while they remained identical in the untouched section. when using dirty filters, which is often the case in many high occupancy buildings equipped with such mechanical ventilation, the emissions from the used filters were found to increase with the outdoor airflow rate. increasing the ventilation did not improved the air quality, while raising the operation costs [66] . the main difference between mixing and displacement ventilation can be seen in figure 15 . as much as the risk of recirculation is significantly lower or negligible within the ductwork, virus contaminated droplets can still be carried for long distances across occupied spaces by fan induced turbulent airflow. it may be theoretically possible, however, to design the air distribution ductwork and room diffusers in such a way as to reduce in-room mixing and thus the risk of airflow induced infection spread. furthermore, there is even a possibility of adapting existing building services with covid-19 specific office floor alterations. further research into the possible development of such comfort, ventilation and indoor air quality provision is urgently needed so that designers, engineers and facilities managers can ensure occupant safety, especially in the light of the covid-19 pandemic. displacement ventilation systems can be broadly divided into mechanical and natural. mechanical displacement systems can service the entire building, often using a simplified version of air handling units with centralised extract systems or with specific rooms, office floors or classrooms using localised extract systems. continuous extract ventilation is more frequently used in domestic buildings and is currently used predominantly in bathroom areas in high occupancy buildings. natural displacement ventilation approaches can include whole building integrated systems, room specific systems, ventilation products, such as passive ventilation cowls or specific wall integrated louvres. they can also rely on windows, either automated or manually operated, in which case they may be placed at the opposing ends of the room and at different heights with the exhaust located at a high level close to the ceiling, providing maximum displacement ventilation benefit. brief examples of each approach are listed below: conventionally, the most economical way to provide ventilation was to rely on natural forces acting on air, taking advantage of atmospheric pressure differentials such as wind pressure moving air sideways or making use of the buoyancy of warmer air moving upwards. figure the warmed stale air floats to the exhaust opening faster while drawing air from all connected floors, increasing the ventilation rate [67] . even though natural ventilation methods, including operable windows that are either manual or automatic, are one of the simplest methods of providing ventilation they frequently suffered the most from drawbacks such as bad design and implementation. main design issues included calculating full window area as an opening, which in reality is often less than 1/10 th of the window, locating windows in the wrong area or at the wrong height. as much as varied height cross ventilation can be effective, a row of short windows at mid height will generate almost no air movement, no solar shading and window related overheating, cold draughts, noise, incompatibility with interiors such as blinds or with the user behaviour. the study by the university of oregon [54, 58] observed that natural ventilation with a plentiful supply of fresh air dilutes and removes contaminated air much more effectively than fan driven, recirculated air movement, significantly reducing the risk of infection, as shown in figure 17 . however, ventilation design that requires the stale air to move across the entire floor plan, or through common areas such as hallways and staircases before being exhausted from the building, is understandably more likely to spread infection than when the stale air is exhausted at the source, directly to the outside. considering the research conducted so far, if designed and implemented appropriately, natural ventilation measures, or a combination of localised mechanical exhaust and large cross section natural inlets, can provide an adequate displacement ventilation solution, significantly reducing the risk of infection. [68] . a frequently quoted drawback of natural ventilation measures is the perceived lack of control and dependence on external factors such as wind speed and air temperature to provide fresh air. moreover, the need to reduce the internal airflow resistance and maintain large openings and air pathways in large, building-integrated natural solutions reduces the possibilities for noise dampening or provision of adequate temperature management. in noisy, hot or cold environments, this ventilation strategy is often rejected in favour of mechanical ventilation solutions which are simpler to design and do not require familiarity with building physics. as opposed to building integrated ventilation methods or measures such as opening windows, the natural ventilation systems are products that can include roof mounted cowls and/or façade integrated elements. they can be designed or sized specifically for the application, be it a classroom or a sports hall. the systems rely on either the roof cowls to both supply fresh and extract stale air or on façade elements entirely, in which case, they would be placed at the top and bottom of each space to maximise the stack effect. some natural ventilation products can include heat recovery or comfort cooling. most of these systems can ensure displacement ventilation with little contaminated air mixing, as long as used with appropriate internal air dividers, as illustrated in figure 18 . various examples are listed below: roof mounted natural ventilation systems: these have been available for many decades and successfully used in schools and other low-rise high occupancy buildings. their use has declined in recent years due to comparatively higher capital costs than simple mixing systems and the lack of a heat recovery function, although overall energy consumption is reduced due to the lack of fans and the associated electricity use and heat recovery systems have recently become available. these systems are available from several uk manufacturers. roof mounted natural ventilation systems with heat recovery: these are relatively new additions to the natural ventilation product range, even though the addition of heat recovery to natural ventilation has been researched considerably with several academic publications considering heat transfer with heat pipes or thermal wheels in order to improve natural ventilation whilst saving energy. their operation is broadly similar to the standard roof cowl systems in terms of air movement, and can also include façade integrated ventilation for boost, with the addition of heat recovery capability which reduces ventilation related heat loss, further increasing energy efficiency. an example of this mechanism is shown in figure 19 : ventive windhive (natural ventilation with heat recovery) systems. figure 19 . numerous academic papers covered the use of natural ventilation systems, both with and without heat recovery, and demonstrated that, when appropriately sized and designed, they work very effectively [69] [70] [71] . façade mounted natural ventilation systems, these could be stand alone or have additional heating: these are generally automatically actuated façade openings with a room matching grille on the inside and a weather louvre on the outside, located at both high and low level in the room. depending on co 2 concentrations, one or both dampers would open automatically to provide the required, buoyancy driven airflow. in some cases, these systems may include a heating coil, to reduce the cold drafts that could be generated during winter in the absence of a heat recovery function. if required, they can also include a bank of acoustic attenuators to reduce external noise transfer to the indoors as shown in figure 20 . intelligent façade mounted systems with heat pump: latest technology developments include the addition of a heat pump to façade mounted natural ventilation systems to provide both the heat recovery feature. this feature is implemented by transferring heat from the exhaust vent to the supply vent with the help from a low power compressor as well as heat pump driven summer cooling as shown in figure 21 . the general operation is the same as for the twin façade system noted above -fresh air is supplied at low level with the exhaust mounted at high level. however, unlike the above, the incoming air is tempered to improve occupant comfort using the exhausted air energyeither warmed in winter or cooled in the summer. the usual electrical consumption of the compressor is, in part, offset by the reduced electrical consumption due to the absence of fans. the other potential benefit of the system is that the summer cooling tends to boost natural ventilation by heating the exhaust air above the ambient temperature enabling the natural ventilation equipment to maintain adequate air flows all year round. a school in grong in norway implemented hybrid bidirectional ventilation, shown in figure 22 , for the provision of the required indoor air quality based on a balance between supply and exhaust air. underground ducts connect the basement of the building to the wind tower. the mechanism in which this ventilation strategy works relies on air entering the tower further down the school field either naturally or using a fan, depending on the available buoyancy. the air is then driven under the school through a duct entering the rooms through the lower level then rising due to buoyancy, leaving the building through the chimney-like stack. the ventilation rate for each room is controlled by the degree of opening of the exhaust damper and the optional extraction fan. this type of system is simple mechanically, and offers fresh air either naturally, during winter, or using fans when buoyancy is too weak to drive the airflow, with compressor driven heat recovery that utilises a centralised concept for ventilation [72] . [72] . a study in a school in new-zealand highlights the positive effects of a better ventilation strategy on airborne particles. two adjacent classrooms were monitored and compared, one with continuous mechanical extraction ventilation solutions and the other where the occupants were relied on to open windows. the levels of inorganic airborne particles were monitored to show how they were removed from the breathing space. the first room was equipped with a unidirectional ventilation system consisting of a fan assisted solar roof prototype with a double-layer roof made of a polycarbonate layer over a steel corrugated north-facing roof, where outdoor air was passed between the two layers, while the other classroom had no specific ventilation system with manually operable windows as the only means of ventilation. the goal of the study was to assess the air quality and the effect of ventilation on reducing respiratory and cardiovascular diseases associated with poor air quality. air samples were measured to identify the number of inorganic particles sizing under 10 μm. the exhaled airborne particle concentration and dilution provided by the ventilation system follow the same trend, since, at this size, aerodynamic forces prevail over gravitational forces as explained in section 2.1. the ventilated system provided 32.3 l/s of tempered outside air, or approximately 0.6 air change per hour, for a classroom volume of 200 m 3 . the unidirectional ventilation system reduced the average co 2 concentrations by 27 %, from 1345 ppm to 980ppm, and average moisture content by 12%, from 9.08 g of water per kg of dry air to 7.98 g of water per kg of dry air. the actively ventilated classroom had on average a 66% lower concentration of airborne particles sizing under 10 μm than the unventilated classroom as shown in figure 23 [73] . [73] . displacement ventilation systems, such as natural ventilation, naturally assisted extraction ventilation and continuous mechanical extraction ventilation, appear to be the most promising ventilation solution in terms of reducing the spread of viruses and other pathogens indoors this is mainly due to the lower air flow velocity, non-turbulent air flow characteristics and a much lower likelihood of air mixing occurring within the ventilated spaces. this observation is investigated further using computational fluid dynamics in the next section. two of the above listed displacement ventilation systems have been selected for further analysis of room specific airflow dynamics and its potential impact on exhaled droplet and pathogen distribution through occupied spaces to assess their impact on the potential spread of infection: 1) roof mounted natural ventilation with heat recovery and 2) the façade mounted naturally intelligent ventilation system with heat pump. due to their similarity to each group of the natural ventilation systems mentioned above, we consider the results of the cfd modelling to be representative across the roof mounted and façade natural ventilation range. both of the selected systems appear thermally and physically well designed to be able to passively ventilate occupied spaces whilst recovering heat through either passive or active methods, using wind speed, wind pressure and air buoyancy to drive the air flow. under normal circumstances, a natural ventilation strategy has many benefits over mechanical or fan-powered systems, including lower carbon emissions, reduced operating costs, and ease of installation. according to the research mentioned above, it also appears to have measurable advantages when it comes to reducing the risk of infection spread indoors -this is further investigated below. to aid in the assessment of expected airflow and classroom comfort levels achieved in-situ for two displacement systems, a computational fluid dynamics study has been carried out. the cfd study aims to quantify the steady state performance within a school classroom for the following two natural ventilation systems with heat recovery: ventive windhive and ventive active. the overall ventilation performance will be quantified in terms of the simulated co 2 concentration levels within the classroom alongside more specific modelling results of the temperature and, most importantly, air velocity and airflow characteristics. the global setting for the analysis of the two ventilation systems is that of a fully enclosed classroom, with dimensions 3000h x 7000d x 8000w (mm), where: h is height, d is depth and w is width. the classroom is occupied by 30 seated students with corresponding chairs and tables as shown in figure 24 for the ventive windhive system. to enable a true representation of the natural ventilation system's working environment an additional region is included to represent the outside ambient with dimensions 10,000h x 10,000d x 10,000w (mm). although the two ventilation systems will be modelled in the same working environment, the configuration and set-up of each unit is different, and therefore each will be presented separately for clarity. the windhive system, shown in figure 24 , is a roof-mounted unit, with the indoor diffuser located at an arbitrary central location such that it is not positioned directly above any person. the ventive active system, shown in figure 25 , has a different buoyancy driving set-up utilising the external wall of the classroom with the supply unit located no more than 200mm from the floor level and the extract unit located at the top of the room (preferably 2.4m from the finished floor level or higher). following the finalisation of the geometry an idealised room specific 3d model of each set-up was created using cad software before being imported into star-ccm+ v13.06. to help reduce the computational expense of the simulations in terms of mesh and physics several modelling assumptions were made, these are listed below:  simplified geometry for the children and desks to act as the sole obstacles in the classroom  u value of 0.5 w/m 2 /k applied to the walls to account for an average heat flux  each child assumed to act as a heat source producing 70w (equating to a total heat source of 2100w)  wind, solar gain and equipment loads including lighting and airtightness are ignored  grill losses located on the invent active and passive are accounted for through specification of a porous medium with an effective opening of 85% and a porous inertial (α) and viscous (β) resistance of 1.77 and 1.71 m/s, respectively  to achieve a realistic outside ambient a small constant velocity field ([0.1] m/s) is active across the length of the domain, in order to account for natural wind and to help initiate a flow solution. this is adopted also for the additional ambient region within the invent passive simulation to account for the open window.  ambient and initial temperature field is equal to 10 °c  initial classroom co 2 level is set at 450ppm whilst the ambient/environment is equal to 400ppm  each child is presumed to provide a co 2 release rate of 0.035 m 3 /h equating to a bulk class co 2 supply of 1.05 m 3 /h the aim is to complete two independent cfd simulations, one for each of the natural ventilation systems. each simulation will operate with a multi-component gas as the working fluid, separated such that the co 2 and other components of air are modelled independently. the chosen software package for all simulations is that of the commercial cfd package, starccm+ v 13.06, supplied by siemens. this is a well validated cfd code that is widely used throughout both industry and academia. to provide a solution domain suitable for the cfd solver the 3d model must be discretised into a series of smaller fluid volumes by successively splitting nominally hexahedral cells until a desired cell size is achieved. the accuracy of a given simulation is largely controlled by the size and quality of the cells and therefore a number of refinement regions in areas of interest were created. it should also be noted that a grid independent solution is achievable, at this point further reduction of the cells will only account for a higher computational cost instead of improved accuracy. all meshes presented here have been generated using a trimmed cell mesh with local refinement regions and prism layers to account for the near wall effects. each mesh is illustrated below with the total number of cells for each specified. the total number of cells used for the windhive model is 9.5 million. an image of the mesh for windhive is shown in figure 26 . the total number of cells used for the invent active model is 7.7 million. an image of the mesh for windhive is shown in figure 27 . figure 28 shows the velocity scenes for the two cases for vertical and horizontal cross-sections. for both systems it can be seen that the greatest flow speeds are experienced at the inlet and outlet locations, with both the ventive windhive and ventive active showing the greatest velocities regarding the inlet stream. it also provides a top down projection of a 1m velocity slice, directly inline with the heads of the seated students. this image shows more clearly the locations and magnitude of the incoming air stream of the windhive. it must be noted that the ventive active's inlet is below this point. table 4 indicates the mass and volume flow rates calculated at the inlets and outlets of the two respective systems. the greatest flow rate is provided by the windhive (150 l/s) even without the wall purge opening whilst the active is somewhat lower in the fully passive operation mode (113 l/s). the stated flow rates relate to values of 4.8l/s (ventive windhive), and 3.6l/s (ventive active) per person with reference to 30 children and 1 teacher. an important factor aiding in achieving a desired level of co 2 within a room is the total rate of air change in one hour. based on a classroom area of 165m 3 the estimated number of air changes per hour for the two configurations (based on the inlet flow rates provided in table 4 ) are calculated as: • windhive = 3.3 air changes per hour • ventive active = 2.5 air changes per hour stagnation regions are possible for such systems according to the model whereby figure 28 shows the velocity profiles. however, people within such environment tend to stand up and move around slightly which will induce further air movement within the stagnated air regions. the cen report cr 1752 ventilation for buildings states that if seated occupants are the only source of pollution then the co 2 concertation should be below 800ppm for category a, 1000ppm for category b and 1500 for category c, where the outdoor ambient level is quoted as 350ppm. category a represents a high level of expectation, recommended for fragile and sensitive persons like young children, elderly, sick or handicapped persons. category b is the normal level of expectation that should be considered for new buildings or renovations. category c represents moderate level of expectation that may be used for existing buildings [74] . expectedly the windhive configuration provides the most air changes over a given period due to its higher flow rates, and this correlates to a more favourable mass fraction of co 2 , as shown in figure 29figure 28. although it should be noted that despite having a lower rate of air change, the ventive active reduces the levels of co 2 comparably to the windhive. both these systems appear to be able to maintain the initial co 2 conditions through the regular exchange and circulation of air throughout the room. the distinct airflow profiles of both natural ventilation systems shown above are of great interest in relation to spread of coronavirus and other pathogens indoors. the supplied fresh air is observed to distribute evenly at low level (well below the breathing, and especially exhaling, area) at a higher volume but lower velocity than fan induced ventilation (thus is much less likely to pick up heavier droplets already dropping to the floor) while the stale air appears to move steadily upwards before being exhausted out of the building, with no observed mixing indoors. this, in combination with the low observed co 2 levels (which are a good proxy for contaminated, exhaled air) as well as the prior research listed above (where, unlike mechanical and recirculating ventilation, the cloud of droplets and particles is not pushed around the room but instead travels in a fairly direct line from window to the exhaust vent, resulting in fewer people being exposed to it), indicates that natural ventilation has a much lower risk of spreading the droplet and aerosol borne infection indoors than other ventilation methods. this displacement and stacking effect in interesting also since it protects the occupant regardless of the configuration off the room: for example, younger children often sit around large table instead of rows. by moving the stale air upward the concentration at the breathing level is reduced as well as the infection risk. airborne disease transmission is highly contagious in enclosed environments and especially in buildings with inadequate or inappropriate ventilation systems. the theory shows that small droplets of less than 10 µm in diameter have the potential to remain in the air for hours since their terminal velocities would be at 3mm/s, depending on various characteristics and conditions at the time. with the unprecedented pandemic of the covid-19 spreading through the society at an extremely fast pace, many academics and industry professionals are raising the question of whether the current ventilation strategies are outdated and inadequate for such contagious diseases. even though the widely accepted mechanism of sars-cov-2 transmission to the date of writing this article has been through droplet borne pathways, the who is accepting the possibility that the deadly virus could be transmitted through the air. many research studies have shown that with appropriate ventilation, the risk of transmission is greatly reduced, further emphasizing the need for research into and adaptation of current ventilation methodologies. furthermore, an insufficient ventilation rate and inappropriate ventilation strategy (mixing of in-room or recycled air, poor mechanical ventilation maintenance) have been linked to degraded health outcomes for the users of high occupancy buildings. this includes facilitated airborne transmission of diseases, sick building syndrome, increased sickness absence and reduced cognition. this paper has reviewed widely used ventilation strategies adopted in high occupancy buildings such as schools and offices. the currently accepted approach to reducing airborne disease transmission recommended by various industry associations (ashrae, cibse) as well as the world health organisation is to increase the ventilation rate using plentiful fresh air to dilute the contaminant to a safe level. in many schools and other high occupancy buildings, the target ventilation rates are rarely met. the benefits of better health and attendance and resultant economic outcomes largely outweighed the capital investment of installing, renovating, or retrofitting appropriate ventilation solutions, even before the covid-19 pandemic. as can be seen above, the most commonly used ventilation approaches are inadequate when it comes to lowering airborne transmission risks. different strategies were reviewed with cfd examples to consider their impact on the pathogen propagation indoors. it seems beyond doubt that recirculating ventilation strategies should be avoided as they limit and prevent the dilution of harmful particles while also facilitating the distribution of stale, possibly contaminated air throughout occupied spaces. as the occupancy of the room increases (expressed in time and number of people), so does the exposure. mixing ventilation approaches are also disadvantageous as they may increase the range of infectious particles within the room and the range of sizes of particle that can sustainably remain airborne. displacement ventilation with a generously sized natural inlet is preferred as it can move stale, contaminated air directly to the exhaust of the room in a laminar fashion whilst the concentration of small droplets and airborne particles in the indoor air is significantly reduced. the mode of ventilation can be achieved by either fully natural ventilation or natural supply with a mechanical extraction strategy. natural displacement ventilation offers many other advantages such as reduced power consumption and low maintenance costs. on the other hand, some natural ventilation systems may lack controllability or a heat recovery, both of which can be addressed through careful system selection (ensuring sensor-based responses as well as automatic actuators for the supply and exhaust openings) and good design practice. mechanical solutions have an energy consumption penalty due to the use of fans and may require larger capital investment and maintenance costs. if balanced mechanical systems are to be considered, a significant, research based, re-design effort is required with larger ductwork and openings to avoid high air velocity and in-room mixing ventilation dynamics which currently can defeat the purpose of ventilation by increasing the concentrations and the range of the infectious particles. it has also been demonstrated that the use of filters can prove detrimental when not properly maintained and regularly cleaned or changed. on the other hand, the use of mouth and nose covering, such as 3 ply masks for example, directly impact the quantity of droplets and particles emitted by the occupants in the indoor volume. in practice, the constant use of these masks can reduce the comfort level of the occupants and may be difficult to enforce in high occupancy buildings since it relies on the cooperation of each individual and following procedures. whereas, the implementation of a natural displacement ventilation systems passively offers an additional form of transmission risk reduction. two ventive natural displacement ventilation systems were chosen for study using cfd simulations. the large air volume combined with low airflow speed allow sufficient quantities of fresh air to be supplied into the space, distributing evenly through the bottom of the room and gradually displacing the stale air to the top of the space thanks to the buoyancy-driven stack effect. the advantages of this type of ventilation strategy is that it can significantly reduce the risk of airborne disease transmission. it provides large volume ventilation at low air speed, which facilitates a stratification effect. this stratification effect along with the appropriate placement of air inlets and outlets allow a natural and almost universal upward flow of air to the top of the room and out through the appropriate exhaust vents. this restricts the horizontal movement of airborne particles which contain pathogens that are produced when infected individuals breath, speak or cough, capturing the smaller droplets to migrate almost upwards, while allowing larger droplets to fall out of breathable level air, vastly reducing the risk of disease transmission via indoor air. the large volume of fresh air supplied into rooms lowers the concentration of other contaminants in the room and increases the iaq to desired levels. as an added benefit, in both closely studied systems, the heat recovery capability and dynamic, connected controls both increase comfort levels, making the high air refresh rate more bearable for occupants, and enable remote adaptation of ventilation provision, which improves the response of facilities managers to risk levels. building services of high occupancy buildings must be better adapted as a matter of urgency to facilitate the reduction of disease transmission resulting from inappropriate or inadequate ventilation. the covid-19 pandemic has exposed areas requiring urgent development to protect both our health, wellbeing and the economy by providing safe indoor environments for employees or students. this paper has demonstrated possible routes for indoor disease transmission, the mechanisms in which diseases can spread, facilitated by conventional ventilation systems, the gaps in current knowledge and technologies and areas of interest for future research and development. although many cases of disease transmission can be reduced by social distancing or wearing the recommended ppe, the air surrounding us indoors requires much better management to safely remove air borne pathogens. many of the current ventilation strategies that rely on centralised air distribution and ceiling level supply or recirculation can provide the optimum conditions for rapid disease spread in high occupancy buildings. on the other hand, displacement ventilation strategies, such as the natural ventilation or naturally assisted ventilation explored above, can provide an effective starting point for reclaiming our buildings for safe use. however, significant gaps remain in the knowledge needed for the development of more personalised ventilation mechanisms at an economic level to allow for adaptation of already installed ventilation systems to help mitigate the risk of infections in existing buildings. none world health organization, coronavirus disease (covid-2019) situation reports epidemiological characteristics and transmission model of corona virus disease 2019 in china mass masking in the covid-19 epidemic: people need guidance advice-on-the-use-of-masks the flow physics of covid-19 size distribution and sites of origin of droplets expelled from the human respiratory tract during expiratory activities the coronavirus pandemic and aerosols: does covid-19 transmit via expiratory particles? an analysis of the transmission modes of covid-19 in light of the concepts of indoor air quality factors involved in the aerosol transmission of infection and control of ventilation in healthcare premises role of mechanical ventilation in the airborne transmission of infectious agents in buildings role of air distribution in sars transmission during the largest nosocomial outbreak in hong kong evidence of airborne transmission of the severe acute respiratory syndrome virus nosocomial transmission of emerging viruses via aerosol-generating medical procedures cibse covid-19 ventilation guidance features, evaluation and treatment coronavirus (covid-19)., in: treasure island (fl) roles of sunlight and natural ventilation for controlling infection: historical and current perspectives the size and the duration of air-carriage of respiratory droplets and droplet-nuclei characterizations of particle size distribution of the droplets exhaled by sneeze the dispersion of spherical droplets in source-sink flows and their relevance to the covid-19 pandemic characterization of expiration air jets and droplet size distributions immediately at the mouth opening airborne transmission of sars-cov-2: the world should face the reality world health organization, modes of transmission of virus causing covid-19 epidemiology, virology, and clinical features of severe acute respiratory syndrome -coronavirus-2, 2 (2011) 1-23 quantifying sars-cov-2 transmission suggests epidemic control with digital contact tracing, science (80-. ) how to operate and use building services in order to prevent the spread of the coronavirus disease (covid-19) virus (sars-cov-2) in workplaces toward understanding the risk of secondary airborne infection: emission of respirable pathogens aerosol and surface stability of sars-cov-2 as compared with sars-cov-1 airborne route and bad use of ventilation systems as non-negligible factors in sars-cov-2 transmission airborne transmission of communicable infection -the elusive pathway 239 experts with one big claim: the coronavirus is airborne natural ventilation for infection control in health-care settings airborne infectious diseases, in: patty's ind. hyg ashrae position document on airborne infectious diseases by ashrae board of directors controversy around airborne versus droplet transmission of respiratory viruses: implication for infection prevention extensive viable middle east respiratory syndrome (mers) coronavirus contamination in air and surrounding environment in mers isolation wards viruses and indoor air pollution how exactly do you catch covid-19? there is a growing consensus particle size and pathogenicity in the respiratory tract modality of human expired aerosol size distributions temporal dynamics in viral shedding and transmissibility of covid-19 changes in contact patterns shape the dynamics of the covid-19 outbreak in china, science estimating the generation interval for coronavirus disease (covid-19) based on symptom onset data transmission of 2019-ncov infection from an asymptomatic contact in germany ashrae standing standard projecrt committee 62.1, ansi/ashrae standard 62.1-2019 ventilation for acceptable indoor air quality the ventilation problem in schools: literature review, indoor air association of classroom ventilation with reduced illness absence: a prospective study in california elementary schools role of ventilation in airborne transmission of infectious agents in the built environment ? a multidisciplinary systematic review association of ventilation rates and co2 concentrations with health andother responses in commercial and institutional buildings, indoor air ventilation rates and health: multidisciplinary review of the scientific literature guidelines for ventilation requirements in buildings office design's impact on sick leave rates covid-19 outbreak associated with air conditioning in restaurant novel coronavirus (covid-19) pandemic: built environment considerations to reduce transmission experimental aerosol survival of sars-cov-2 in artificial saliva and tissue culture media at medium and high humidity coronavirus: can we stay safe as lockdown eases? -bbc news novel coronavirus (covid-19) pandemic: built environment considerations to reduce transmission indoor airflow simulation inside lecture room: a cfd approach cfd modeling approach for turbomachinery using mrf model | learncax air recirculation and sick building syndrome: a blinded crossover trial grounding hvac motor shafts: protecting bearings and lowering repair costs -construction canada mixing ventilation i simscale mechanical ventilation simulation and optimization with sav systems ventilation filters and indoor air quality: a review of research from the international centre for indoor environment and energy, indoor air sustainable ventilation strategies in buildings: cfd research performance of a natural ventilation system with heat recovery in uk classrooms: an experimental study makris-makridis, s. consultant, passive ventilation with heat recovery in an urban school: performance in use performance of a natural ventilation system with heat recovery in uk classrooms: an experimental study towards sustainable, energyefficient and healthy ventilation strategies in buildings: a review the effect of ventilation on air particulate matter in school classrooms on the definition of ventilation requirements in iaq standards -a method based on emission rates of pollutants on the definition of ventilation requirements in iaq standards -a method based on emission rates of pollutants rua luis reis santos key: cord-120017-vsoc9v85 authors: jiang, helen; senge, erwen title: usable security for ml systems in mental health: a framework date: 2020-08-18 journal: nan doi: nan sha: doc_id: 120017 cord_uid: vsoc9v85 while the applications and demands of machine learning (ml) systems in mental health are growing, there is little discussion nor consensus regarding a uniquely challenging aspect: building security methods and requirements into these ml systems, and keep the ml system usable for end-users. this question of usable security is very important, because the lack of consideration in either security or usability would hinder large-scale user adoption and active usage of ml systems in mental health applications. in this short paper, we introduce a framework of four pillars, and a set of desired properties which can be used to systematically guide and evaluate security-related designs, implementations, and deployments of ml systems for mental health. we aim to weave together threads from different domains, incorporate existing views, and propose new principles and requirements, in an effort to lay out a clear framework where criteria and expectations are established, and are used to make security mechanisms usable for end-users of those ml systems in mental health. together with this framework, we present several concrete scenarios where different usable security cases and profiles in ml-systems in mental health applications are examined and evaluated. with a mental health crisis looming large and many ml systems being built for mental health use cases, it is challenging to trace, analyze, and compare all the designs and implementations of such systems. so far, there is a lack of well-defined framework that describes properties relating to the security of such ml systems in mental health, and even less considerations are given to how such security mechanisms can be usable for those systems' end users. however, without usable security, undiscovered, undisclosed, and ill-considered limitations and properties of security decisions would hold back large-scale adoption and usage [2] of ml systems in mental health use cases. for more detailed and nuanced discussions, see our treatment at section 4.3. the goal of this framework is to establish discussions in communities of mental health, ml, and security, so we can build a common ground for directions and expectations for usable security in ml systems used in mental health scenarios. moreover, this framework serves to raise awareness, so that both ml and mental health communities will heed this critical aspect of usable security in ml systems for mental health. we hope that this new, interdisciplinary framework would allow researchers and practitioners to systematically compare usable security attributes across ml systems for mental health, meanwhile to identify potential limitations of particular approaches and trade-offs in different scenarios. in this short paper, we propose that ml systems in mental health use cases, beyond the privacy and security requirements already mandated by legislation's and regulations -for example, health insurance portability and accountability act (hippa) [38, 43, 64] in united states, and general data protection regulation (gdpr) in european union and its member states' national laws [11, 12] -should consider properties of usable security proposed by this framework's four pillars, and be evaluated on their (1)context models, (2)functionality criteria, (3)trustworthiness requirements, and (4)recovery principles across their life cycles. this work presents our effort to generate discussions and consensus for a common framework in a naturally interdisciplinary area. we built our research on the foundation of computer security research, which has a rich history and long tradition of devising criteria and evaluation rubrics for system designs and implementations. we also incorporated important and recent literature from human-computer interaction (hci), usable security, and fairness, accountability, and transparency (fat) research of ml. weaving these interdisciplinary threads together, we hope that our framework will benefit both researchers and practitioners working on ml systems in mental health. there is a long and distinguished tradition in computer security research: presciently define evaluation criteria and structure assessment frameworks, while research communities were still in their early stages of formation. from this tradition, many remarkable security research outcomes have flourished, and guided the design and building of systems and infrastructure we rely on today [21, 22, 30, 31, 37, 51, 67] . however, while the pioneers of security research laid down "psychological acceptability" of users as a key principle for secure system design and implementations [49] , this principle has not been actively researched within the security community until much later while security measures keep confusing even experts [3, 10, 54, 68, 71] . moreover, the "psychological acceptability" principle is often doubted as incompatible with the goal of "security" [7, 15, 44, 47, 56, 63, 69, 71] , and much of usable security research has traditionally been done in the hci community, and usable security is still a small community [41, 45, 65] compared to other areas of security research. while "psychological acceptability" principle is first identified as the meaning of "usable" in "usable security" [49, 71] , there are other efforts trying to precisely define "usability" especially in hci contexts, based on the "human-centered" attribute of interactive systems. a prominent example is iso 9241-210 [23] : "usability" is "the extent to which a product can be used by specified users to achieve specified goals with effectiveness, efficiency, and satisfaction in a specified context of use". built on this definition, [62] of nist proposed measurements on usability evaluations. however, as [62, 71] both point out, measurement of usable security can be highly diverse and context-dependent, meanwhile, such measurements and evaluations focus on the system and its interactions with targeted users, often done with small groups in controlled environments [32, 42] , with security as the users' top concern. while the "security is top priority" assumption can be very reasonable for use cases such as national and corporate security, the same assumption likely does not stand when we are evaluating ml systems in mental health use cases, in which users have diverse top priorities. this complicates the already fragmented landscape [26] of usable security, and while ml applications in mental health and fat ml research are booming [53, 57] , they still do not take usability and security into serious consideration. our framework evaluates usable security of ml systems in mental health based on four pillars. each pillar, in turn, serves as the top concern for each major phase of the computer system life cycle, which can be summarized as: (1)design and implementation; (2)deployment; (3)mass adoption and usage; and finally, (4)maintenance and/or disposal [39, 40, 46] . (1) context: this pillar considers the intended operational environment of the ml system, and how it is designed and built to interact with different types of users with varying purposes, goals, and maliciousness. this pillar is most important during the design and implementation phase of ml systems for mental health. (2) functionality: this pillar tackles the well-known securityfunctionality trade-off [4, 25, 27, 35, 71] . keeping ml systems functional while making security usable, it is imperative to ask questions about the complexity and resourceintensity of security methods within the already complex and often resource-intensive ml system, the flexibility of chosen methods to accommodate future security requirements, and how they influence user interactions with the ml system. this pillar is most crucial in the deployment phase of ml systems, especially in the initial stage, when such system is in limited use, without users' significant investment of trust and time. (3) trustworthiness: this pillar is by nature user-centered. many non-expert, lay users are already distrustful and leery of ml, and this set of requirements show that on the matter of security and usability, ml systems may still induce users' trust in the sensitive context of mental health. this pillar is the most critical in achieving active usage and large-scale adoption [2] of secure ml systems for mental health. (4) recovery: this pillar handles perhaps one of the toughest challenges in both security and usability: what happens, should a security incident (e.g. a breach, a compromise, or a previously undiscovered vulnerability) happens? what are we going to do with the system and users, now and later? how do we account for the incident this time, to minimize the chance that it would happen again? this pillar is the top priority in maintenance and/or disposal phase of the computer system life cycle. the list can help ask the right questions for designing and building usable security [51] into ml systems for mental health: it determines what and how much "usability" to be considered in a security environment, and move from the more general security threat models, to specific cases of user interactions in mental health scenarios, and also to weigh in negative use cases. the properties below are agnostic to programming languages, software stacks, deployment platforms, and hardware specifications, so they are also flexible enough to accommodate a large class of usable security scenarios for ml systems in mental health. c1 asset audit. ml systems in mental health almost inevitably acquire information assets while in use, for example, it may include users' locations, device types etc., as well as patients' functional status information, providers' notes, and organization's intervention plans. understandably, existing regulations mostly focus on these acquired assets. however, in ml systems, "asset" is not only acquired, but also native to the system itself: its algorithms and models, ground truths, datasets, decision-making logic, and result evaluations, etc. therefore, identifying both native and acquired assets of the ml system is critical for usable security. c2 target user profiling. ml systems can be utilized by different stakeholders in mental health: from patients, providers, to government officials, they use the system to achieve different goals. profiling the system's targeted users is the basis to make concrete observations and reasonable estimations, which are then incorporated into design and implementation requirements. knowing the targeted users and what they use the ml system for, this is usable security's positive case: legitimate users can establish trusted paths and use the system without being hindered by its security requirements. usable security's negative case is given in c4. c3 behaviors categorization. behaviors of targeted legitimated users described in c2 can be either expected by the system, or unexpected and cause the system to fail, error out, or even trigger security incidents. while it is not possible to iterate through all unexpected behaviors from legitimate users, unexpected user behaviors raise two key components of usable security, and need to be addressed in design and implementation: (1) motivating users to behave in a secure manner so to minimize the systems' failures, errors, and security exposures, because users are not the enemy [3] ; (2) when such motivations fail, follow the "fail-safe" principle [49] , meanwhile deliver warning messages about security and failures with usability in mind [5, 50, 58] . this property is interdependent with f5, where we discuss robustness. c4 threat modeling. once the assets are audited and target users and behaviors profiled, threat modeling is essential for security, as threat modeling is a well-studied and used subject in computer security [17, 33, 48, 55] . there are three main components to consider: (1) assets the ml system needs to protect, (2) scope of interactions between system and user based on c3; and (3) malicious actors and their actions the systems need to defend against. in contrast to c2, malicious actors are usable security's negative case: malicious users are stopped or slowed down by the system's security measures. the following properties are most useful when seen from a deployment perspective. they describe how a ml system works with inplace security requirements while interacting with users. f1 complexity. most, if not all ml systems and applications have at least one of the three constraints: time, memory, and computational power. therefore, any security measures should consider these constraints and its impact on how well the ml system serves the end users. for example, in a high concurrency event where many users are utilizing the same ml system, if a given security method uses negligible computational power resource on users' end but consumers a lot of system resources, we should consider alternatives for this security method. to measure such complexity, we can use either formal algorithmic complexity notions (e.g. big o, little o), or empirical evaluations. for example, in 10-user, 100-user, 1,000-user concurrency scenarios, what is the average computational overhead or latency for specific sets of security requirements, with other software and hardware constraints stay the same. f2 availability. for large-scale ml systems, e.g. mental health use cases with multiple targeted user groups, security measures also need to scale. availability evaluates how well security methods can generalize to cover a ml system's targeted users and behaviors (c2, c3) without hindering their access to the ml system. a quantitative heuristic for availability of security measures is estimated user adoption rates across user groups, as well as among the genera user base. notice that the availability criteria is a trade-off to the "least common mechanism" principle for secure system design [49] , and the relative importance between the two are dependent on results of context modeling, in particular c4. regardless of which one of the two weighs more heavily in specific scenarios, the security mechanism in question must be carefully designed, judiciously implemented, and rigorously tested before real user runs. f3 flexibility. retrofitting security to usability is usually a bad idea and doesn't work well [7, 69] , therefore it is important to not only prioritize usable security when designing and building ml systems for mental health, but also to not let current implementations become roadblocks to additional security requirements or system capabilities. having flexibility accommodates future changes in the system and shifting user base, and is a long-term commitment to the system's usable security traits. f4 experience validation. to ascertain that security measures did not hold back users, it is crucial to validate real user interactions and experience with the system, regardless of the methods: ideal controlled environments, synthetic experiments, or random sampling. for positive case of usable security (c2) that makes the system more secure but not harder for legitimate users, conducting user studies to evaluate their experiences, interactions, effectiveness, and satisfactions with the system [29, 60] would be indispensable evidence for the ml system's real-world usability. f5 robustness. robustness is well-researched in computer system [1, 6, 18, 36, 59] , and recent interests in adversarial ml [8, 9, 16] has early roots in ml robustness [61] . in our consideration, robustness is also related to recovery principles in section 3.4, and has two angles: (1) for security, to tolerate and withstand certain errors and faults from the ml layer, the system layer, and user interaction layer; and (2) for usability, to communicates to users clearly and timely, when trusted paths cannot be established because of scenarios exceeding (1)'s robustness levels. interdependent with this criterion is c3 for unexpected user behavior categorization. many non-experts are suspicious and distrustful of ml, because of ml's "blackbox magic" reputation. moreover, the technical nature of fat ml methods has not endeared lay users towards machine learning either. now, suppose that another layer of hard-to-use and hard-to-navigate security measures and designs is added to an ml system, such distrust is perhaps only going to grow more intense and open. while the users' sentiment of distrust is understandable, the need for good mental health is agnostic about one's feelings towards machine learning and usability of security designs. therefore, to enable active usage and large-scale adoption [2] of secure ml systems in mental health cases, it is important to first induce users' trust in the ml systems used, before their active utilization of such ml systems. the trustworthiness requirement suggests how ml systems in mental health may still earn users' trust, through its security and usability, by well-designed user interactions and communications. t1 clarity. articulating relevant security mechanisms, and their intents, impacts, and implications to users, is fundamental to trustbuilding. we identified three clarity aspects: (1)clarity of ml, where certain artifacts of the ml system's decision-making logic and process (e.g. summary statistics, explanations for classification labels) are exposed and explained to user in non-technical manners; (2)clarity of security, where user-facing security mechanisms (e.g. trusted path establishment, or revocation of access delegation), and these mechanisms' intents and purposes, are disclosed before users engage in these security mechanisms and take actions, preferably in non-technical terms; and (3)clarity of failure modes, where recovery (section 3.4) plan in case of security incidents, is summarized and communicated to users in non-technical terminology. t2 constraints. complementary to t1, whose focus is on positive cases -i.e. what can be and is done -this requirement focuses mostly on negative cases. while providing clarity, ml systems need to draw boundaries and limitations on their capabilities and responsibilities, and then communicate such information. when determining the scope of usable security and communicating to users, we suggest three main factors: (1) limitations, emphasizing what the system cannot do (e.g. delegating access without explicit user actions from a trusted path), is not authorized to do (e.g. sharing chatbot history with unknown third parties), or unwilling to do (e.g. exposing ml models' features and parameters) for technical and non-technical reasons; (2) boundaries, concerning what the user's actions cannot accomplish; and (3) expectations, dealing with interactions between users and systems, on what users' expectations for the systems should not be. this requirement may seem counterintuitive, but it is founded on the "fail-safe" principle of computer security [14, 49] : the default situation is lack of access -that is, by default, actions and operations are constrained and not allowed to execute. t3 consistency and stability. for similar user behaviors under similar contextual conditions, ideally, usability-and security-related experience and interactions should be: (1) similar, within fixed ml systems (data, algorithm, procedure, parameters, input), and (2) comparable, across different ml systems capable to cover the same contextual conditions in their use cases. we name it "consistency" property. conversely, for the same usability and security methods, when provided with the same user behavior inputs, should respond with similar user experience and interaction. we call this "stability" property. there properties can help users build their own mental models for how security mechanisms and the general ml system work, and align their expectations with the system's responses. note that we controlled the variables ("similar", "fixed", "same") while describing consistency and stability, therefore consistency is not constancy, and stability is not staleness. in fact, the dynamic nature of usable security and the user expectation-system behavior alignment model are both well-known [24] . the goal of alignment, is to motivate secure user behavior and raise user's trust level in the system, and consistency and stability are inroads to alignment. t4 reciprocity. leveraging the human tendency to return favors, ml systems in mental health can elicit actions of trust from users, and motivate their secure behaviors and active engagements, as hci research showed [10, 19, 20] : after users receive helpful information from a computer system, they are more likely to provide useful information or actions back to the system. for reciprocity schemes in ml systems in mental health, we identify two stages: (1)initial exchange of reciprocity, where after volunteering helpful information to users, the system prompts user for desirable information or behavior input; and (2)continuous engagement, meaning that after the initial round, if the user reciprocates, the system should aim to maintain exchanges with users, when user behaviors and other contextual conditions warrant so. depending on specific areas where the ml system needs to induce trust and motivate behaviors (e.g. having users enable security features, or actively use ml capacities), details of the interaction mechanisms, from the initial offer of help to ongoing engagement patterns, will vary. because reciprocity largely depends on user interactions with the system, it naturally focuses on usability, and has different tradeoff with security for different context models. therefore, any reciprocity schemes must be designed, implemented, and validated judiciously to defend against reciprocity attacks [70] . good security needs failure modes, and usable security is no exception. with a variety of assets to protect c1, many functionalities to perform, and user trust to gain and maintain, ml systems in mental health must have a concrete plan for security failures. these principles lay out a foundation to consider the immediate and long-term aftermath of security incidents and their responses, so ml systems in mental health can retain usable security attributes and rebuild trust with users (t4). r1 response. previous research [28, 62] surveyed security incidents such as user data leaks, but did not address more complex security challenges to ml systems in mental health, whose sensitive and diverse assets, both native and acquired, make juicy targets. therefore, ml systems must have protocols and procedures in place, timely reviewed and revised, and ready to respond to security incidents, to achieve three goals: (1) evaluate scope and impact of incident, (2) minimize damages to impacted assets, (3) investigate and attribute sources of incident, and most importantly, (4) rebuild trust in users for the system. (1) through (3) address immediate actions, while (4) is a long-term process that ensures ml systems can maintain its stay with user bases in mental health. this principle is related to c1, c4, and trustworthiness requirements. r2 provenance and chronology. the usability of security, in its failure mode, entails that security failures can be traced, examined, analyzed, and inform future security decisions, and such need is satisfied by post-incident provenance and chronology. in ml systems for mental health, provenance and chronology should not only supply (1) a time ordering of system events, technical vulnerabilities or disadvantages, procedural limitations, uncovered edge cases, user interactions, statistics, and likely warning signals leading up to the incident, but also (2) records of any changes (e.g. content, metadata, mode, appearance) in impacted assets (e.g. manipulated ml model parameter, altered user interface, leaked health history), from when the incident happened, to when it is uncovered. both provenance and chronology can be considered for user-facing purposes as a tool for repair (r3) and to rebuild trustworthiness. r3 repair. post-security-incident repair has two aspects: (1)repairing the system itself, and (2)repairing users' trust in the system. (1) is the direct logical next step of r1 and r2 with immediate impact and results, while (2) tends to be long-term, and is more difficult -it needs all the building blocks of trustworthiness to repair users' trust in ml systems impacted by security incidents, especially when incidents concern user data, user-system interaction, or even users' offline behaviors. repairing trust needs to address additional psychological barriers of users, hence harder than building trust at first, but it is still possible when t2 and t4 are emphasized and utilized in the repair process. our framework is a suggestion, an encouragement, a proposal, and an invitation to the community to start acknowledging and researching usability and security in ml systems for mental health. while our framework is not a standardized rubric, we realize that it may become a foundation for future standards, guidelines, or recommendations by organizations such as nist, iso, or ieee, for usable security in generic interactive ml systems, or specifically in mental health applications. previously, standards were issued on transparency and autonomy in autonomous systems [52] , and we are sanguine about a general consensus on usable security in ml systems, especially for mental health use cases. we intentionally crafted this framework to be agnostic to ml techniques: hence, we can focus on providing a unified structure that is not only comprehensive enough to cover the current interdisciplinary area between traditional computer security, hci, and ml, but is also flexible enough to accommodate future changes and progresses in these areas. we hope this framework can enable researchers and practitioners to: (1) identify gaps in security and usability between their theoretical capacities, design variances, actual implementations, and real-world usage patterns; and (2) quickly appraise properties of particular security and usability methods to decide on the most appropriate mechanism for their desired use cases. in addition, our evaluation framework can be used as a reporting rubric targeting regulators, government officials, and policy makers, so they can quickly get all information in one place, in a clear, structured, and comparable manner. when we speak of "practitioners" in the section above, in the specific context of ml systems for mental health, there are broadly two categories that we target: (1) security practitioners: in general system security contexts, security mechanisms and policies are researched, designed, implemented, tested, maintained, and improved by security professionals. (2) ml practitioners: in general ml system contexts, ml practitioners research, apply, curate, train, validate, test, maintain, improve ml models, algorithms, and date . yet, as we discuss usable security in ml systems for mental health, the matter gets more complex: there are more stakeholders, both on the system builders' side, and on the system users' side. and on each side, there are multiple considerations, interests, and mental models that come into play. table 1 below shows the different stakeholders when we build security to be usable into ml systems for mental health. comparing it with figure 1 , the critical differences between the building usable security into general system versus into ml systems for mental health can be clearly discerned. to summarize: there are more stakeholders on the users' side who deserve usable security for their more diverse needs of the ml system for mental health, and there are more stakeholders on the builders' side who have distinct desires for what they want do with, and how they wish such system to behave. for example, while security and ml practitioners desire different ideal attributes from the system and those attributes are not necessarily at odds or contradict with each other, there are tradeoffs to make. between "strong defense" with implications for privacy on patient information and "collect data" for training when in general, more data is usually better, the builders within themselves need to reach a delicate balance first. on the other end, instead of the cohesive and more-or-less predictable and uniform sets of actions normally expected from user models built for general software or ml systems, we now have a diverse set of potential users, with various sets of actions and behaviors that are not usually taken into account for in those general purpose software or ml systems. in those general purpose systems, figure 1 shows the path of how security mechanism and experience are delivered to end users. behaviors, actions, expectations, and use scenarios of these end users would be captured in user models, and security practitioners would design, build, and deploy security measures and experiences according to those user models. but because of the diverse and varying expectations and actions from distinct groups of end users 1 , such user models would be too narrow and missing out on legitimate use actions and behaviors. this is a major reason that we crafted this framework: to properly account for and appreciate the diversity and variety of users and their actions in ml systems for mental health, with the end goal to bring a usable and secure experience to all. as described within sections for each of those sub-attributes, those attributes are not mutually exclusive nor completely independent from each other. instead, there are rich and dynamic interactions between these sub-attributes, both within a single pillar and across different pillars. four major types of interactions are list below with short examples. (1) inter-dependence: f5 and c3 are interdependent. in this case, without behavior categorization, robustness is next to impossible to plan for or implement; and without robustness measures tested and used in real-life, it would be very hard to validate if the behavior categorizations are reasonable or sufficient. (2) trade-offs: f2 is a trade-off to the "least common mechanism" principle for secure system design as articulated in [49] : for security measures to generalize to diverse sets of targeted users and behaviors, commonality increases and distinctions decline. (3) prioritization: in many scenarios, prioritizing particular principles before others is the most reasonable and sensible course of action. for example, a large-scale online platform delivering automated conversational therapy may prioritize f2 and f5, at the same time de-prioritize trustworthiness requirements based on the assumption that people seeking online automated services generally have greater trust in ml systems and technology, and are likely to be technically proficient enough to navigate security designs built in place. (4) complements: t1 and t2 are complementary: they consider opposite sides of the same issue, and from there, create a comprehensive view and enables balanced and holistic decisions for usable security designs and implementations. these dynamic and interactive relationships carry deep implications for usable security in ml systems for mental health, and we will explore some examples that showcase these interactive and dynamic relationships between the properties in section 5. we will now apply the four pillared framework, and share several tangible use cases of ml systems for mental health where we evaluate and examine their usable security needs and profiles. this way, we can concretely demonstrate the practicality of our framework, illustrate the dynamic and interactive relationships between the pillars and their corresponding sub-attributes, and showcase the complex and distinct stakeholder demands for usable security that warrant such a framework. we will elaborate on example 1 with brief comparisons and contrasts to the three other examples, and leave examples 2 to 4 for readers' exercise. (1) chatbot providing conversation-based therapy to young adults with mental disorders (2) auto-diagnosis algorithms of neuro-images for psychiatrists (3) personalized matching for providers & patients (4) ml system analyzing facial and verbal expressions during tele-therapy sessions in 1, the chatbot's context is an online automated services, and hence is more likely to experience high concurrency requests from many different users with existing mental disorders, and such online large-scale services may also be needed in times of distress (e.g. quarantine during covid-19 global pandemic) to parts of the general population. therefore, its usable security mechanisms need to prioritize being available and robust enough to handle more users, and wider ranges behaviors and actions of legitimate users. at the same time, inducing users' trust may not be as important, because we may consider people willing to use online chatbot services are more trusting towards ml and technology in general. although, soon we would see that if security mechanisms and designs are not usable or robust enough, such assumption of trust may not be warranted, and if there were any, would be drastically diminished. the assets it needs to protect are not only the security of its general software infrastructure, but also its ml algorithms that generate live conversation responses to users: recall the infamous incident of microsoft's chatbot tay on twitter [66] , caution must be taken to ensure that legitimate users who need the therapy service could easily and readily access it without much hassle, and that malicious users could be fended off so they could not manipulate the algorithms. while this may seem simple at first, we must properly categorize behaviors of our potential legitimate users, who have existing mental disorders. take attention-deficit/hyperactivity disorder (adhd) for instance. suppose that the chatbot implements classes of captcha or recaptcha methods -which may include text, image, and sound recognition, as well as text and image matching -to defend against its threat model actors that include bots and malicious users trying to poison its algorithms. while these methods may be effective to defend against these threats, legitimate users with adhd, whose attention spans are usually shorter than the general population [34] , may be unlikely to complete the captchas, especially when there are several ones that come one after another. when security defense designs turn legitimate users away, these users may leave with the idea that such mechanisms built to trick them, and the system behind it has no genuine intention to provide them help, and hence their implied trust in the chatbot when they first approached this online automated service, may likely diminish. hence, it would be advisable to also take trustworthiness requirement seriously, especially the constraint property. one way to demonstrate it, is using clear language or visual images to inform users of failures. for instance, when a user's attention span is too short to successfully finish a series of recaptcha challenges, a message displays: "sorry we could not tell if you are a human or bot. do you want to try another way?" this or similar messages could communicate to legitimate users that the system genuinely intends to provide services, the recaptchas are there because it is a security design, not a farce or trick to turn them away, and there are certain things that these recaptchas are not capable of doing. further thoughts bring more usable security considerations to the discussion on example 1. should the chatbot store any records of its user interactions, so that human providers and caregivers (e.g. parents or legal guardians) of these young adults could monitor their progress, provide better diagnosis, treatment, and care, then we are onto more complex scenarios. there are now two more groups of users to consider, and how to provide usable security for them is a crucial challenge. moreover, because now there are stored user records, there is an additional asset to protect, and the recovery principles need to be elevated to higher priorities, especially repairing users' trust in the service in the event of a breach or leak. this is where flexibility also comes into the picture: if builders of the chatbot had not originally considered this sharing services, and only later decided to add it, flexibility of previous security capabilities to accommodate the additional security requirements that come with sharing user records, is extremely important. in a base case, even when the chatbot is originally built to not only converse with patients, but also store their records and allows them to share their records with their providers and caregivers, clarity of usable security designs that are informed by behavior categorization would be integral. for instance, some young adult patients may decide to simply share their passwords with their providers and caretakers for the latter groups to look at their chat records. however, if these patients are in the u.s., this simple act may land their providers and caretakers in legal trouble: because a shared password, even a voluntarily shared one, counts as unauthorized access by the computer fraud and abuse act [13] . preempting such behaviors would greatly inform usable security decisions when designing and building this chatbot. for example, builders may decide to use methods other than passwords to check for user authorization and authentication status; to utilize the constraint criteria, and outwardly warn users to not share their passwords even with trusted providers and caregivers; or to add particular terms in the end-user licensing agreement, security & privacy policies, or terms & services documents, and specify cases where the patient could share passwords; or to build a security measure so that patients can delegate access to their records to authenticated providers and caregivers. to choose the most suitable usable security mechanisms or combinations of such mechanisms, builders of the chatbot would need to deliberate on which contexts, and especially which threat models they decide to focus on. in comparison, example 2 has a very specific and focused profile of targeted users (psychiatrists), so the inter-dependent properties of behavior categorization and robustness would be straightforward to analyze and design, and the assets and threat models are also relatively clearly defined and direct. meanwhile, because such system is used for medical diagnosis, all attributes related to trustworthiness need to be prioritized: the builders cannot assume that psychiatrists are trusting the ml system's decisions. moreover, while the threat models are relatively simple compared to example 1 and 4, the system still needs to induce trust from the psychiatrists: how do they know it is them, instead of malicious attackers described in the threat models, who see the images, patients' information, and the algorithm outputs? to address these issues, some security designs may include: ml explanation options that accompany each diagnosis; a side bar that shows access activities; or a device-based two-factor authentication check. again, it is up to both the ml and security practitioners who build this system, to decide on the specific contexts and threats they would like to prioritize. example 3 also has a straightforward profile for behaviors as example 2, but the threat models could be tricky: because depending on what the builders of the system decide to gather from both the patients and providers for the match, assets that the system needs to protect could swing a rather wide range. meanwhile, because of the usual one-on-one nature of patient-provider relationships, in contrast to example 1, de-prioritizing the availability of security measures to large numbers of online users could be sensible, and the system might even be able to afford using more time-, memory-, or computationally-complex security mechanisms that are nonetheless usable for both providers and patients of the service. for example, incorporating reciprocity into the human-system interaction process, by engaging users in short q&a games about secure behaviors -which by the way, could also induces trust from users about the system's security, and fulfill part of the trustworthiness requirement. but on the ml front, trustworthiness here is similar to the premise of 1 but the spirit of example 2. while patients and providers who choose to use a ml-powered matching service could be assumed to have a greater degree of general trust in ml and technology, the same level of trust could not be assumed in the specific matching decisions: "how and why did this black-box know that i would be a good fit for this patient/provider?" would be the question to answer for every patient or provider who uses the service. example 4 involves more diverse user groups (patients, providers, likely other caregivers, and potentially policy-makers). hence, the assets need to be protected are more varied and diverse, the threat models more complex, the recovery scenarios more important, and the usable security mechanisms may need to make trade-offs between availability and flexibility while still being functional when processing live audio and video data, which is another subtle constraint on the complexity of usable security mechanisms. similar to examples 2 and 3, the inevitable question of trustworthiness would arise on the ml system's decision rationales and explanations, and there is also an incentive on the builders' end to assure that algorithms and models powering the system are not being manipulated. because the information being processed and analyzed by the system is largely private and sensitive, convincing different users of the effectiveness and strength of the security mechanisms is also an important task. comparing it to example 1 where there are clearer priorities, this system poses a set of full-on challenge for usable security design, implementations, and evaluations. in our work, we presented four categories of desired properties -based on context, functionality, trustworthiness, and recovery -to systematically frame and evaluate usable security in ml system for mental health. we discussed those properties' intents, rationales, and sources in the intersection of security, usability, ml, and mental health. we propose that ml systems in mental health be evaluated by the way of this framework for security and usability, in different phases of the computer system life cycle. we have analyzed, structured, and presented several examples of ml systems in mental health in this framework, and for next steps, we plan to evaluate more real-life ml systems in mental health, preferably similar to the four described examples, so we can test, validate, and improve our framework and criteria. simultaneously, we also plan to interview builders of these ml systems in mental health, to understand their awareness of, thought processes behind, and decision rationales of usable security in the systems they designed and built. because the framework covers the computer system life cycle, while we prefer already deployed, large-scale systems, we are also happy to examine systems in early stages of the cycle. we plan to publish results on websites where this interdisciplinary community can also submit their own framework evaluation results. in a deeper dive, our future work will explore a tiered approach to usable security for ml systems, inspired by classic security literature [37] , meanwhile further examine interactions -e.g. trade-offs, enhancements, overlaps from different perspectives, complements, and interdependence -between desirable usability and security properties for ml systems in mental health. ieee standard glossary of software engineering terminology inclusive persuasion for security software adoption users are not the enemy improving information security awareness and behaviour through dialogue, participation and collective reflection. an intervention study usable security: revealing end-users comprehensions on security warnings on the assessment of robustness in search of usable security: five lessons from the field aleksander madry, and alexey kurakin. 2019. on evaluating adversarial robustness towards evaluating the robustness of neural networks even experts deserve usable security: design guidelines for security management systems overview of the national laws on electronic health records in the eu member states european commission. 2019. data protection in the eu vulnerability, sharing, and privacy: analyzing art therapy for older adults with dementia cybersecurity and united states infrastructure security agency (cisa), department of homeland security is usable security an oxymoron? interactions enhancing performance prediction robustness by combining analytical modeling and machine learning a modelbased approach for robustness testing how users reciprocate to computers: an experiment that demonstrates behavior change persuasive technology: using computers to change what we think and do european union agency for cybersecurity information technology ã¢ä�å¤ trusted platform module library ã¢ä�å¤ part 1: architecture ergonomics of human-system interaction ã¢ä�å¤ part 210: human-centred design for interactive systems practical unix and internet security effects on employees' information security abilities by e-learning consolidating principles and patterns for human-centred usable security research and development security and usability: analysis and evaluation data breaches: user comprehension, expectations, and concerns with handling exposed data towards robust experimental design for user studies in security and privacy formal models for computer security computer security online privacy and aging of digital artifacts threat modeling as a basis for security requirements attention-deficit/hyperactivity disorder approximating saml using similarity based imprecision network security & robustness trusted computer system evaluation criteria hipaa security rule crosswalk to nist cybersecurity framework the department of justice systems development life cycle guidance document usable cybersecurity framework for improving critical infrastructure cybersecurity hci and security systems a brief introduction to usable security an empirical study of the cobb-douglas production function properties of software development effort from usability to secure computing and back again toward a secure system engineering methodolgy the protection of information in computer systems transforming the ã¢ä�å¸weakest linkã¢ä�å¹ã¢ä�å¤a human/computer interaction approach to usable and effective security if a1 is the answer, what was the question? an edgy naif's retrospective on promulgating the trusted computer systems evaluation criteria ieee standard reviewã¢ä�å¤ethically aligned design: a vision for prioritizing human wellbeing with artificial intelligence and autonomous systems machine learning in mental health: a scoping review of methods and applications why johnny still canã¢ä�å¹t encrypt: evaluating the usability of email encryption software threat modeling: designing for security usable security: oxymoron or challenge explainability fact sheets: a framework for systematic assessment of explainable approaches on the challenges in usable security lab studies: lessons learned from replicating a study on ssl warnings building robust systems an essay evaluating usercomputer interaction: a framework building robust learning systems by combining induction and optimization usable security is usable security an oxymoron? united states department of health and human services usenix. 2015. usenix soups 2020 conference page microsoft created a twitter bot to learn from users. it quickly became a racist jerk security controls for computer systems: report of defense science board task force on computer security aligning security and usability reciprocity attacks user-centered security key: cord-020896-yrocw53j authors: agarwal, mansi; leekha, maitree; sawhney, ramit; ratn shah, rajiv; kumar yadav, rajesh; kumar vishwakarma, dinesh title: memis: multimodal emergency management information system date: 2020-03-17 journal: advances in information retrieval doi: 10.1007/978-3-030-45439-5_32 sha: doc_id: 20896 cord_uid: yrocw53j the recent upsurge in the usage of social media and the multimedia data generated therein has attracted many researchers for analyzing and decoding the information to automate decision-making in several fields. this work focuses on one such application: disaster management in times of crises and calamities. the existing research on disaster damage analysis has primarily taken only unimodal information in the form of text or image into account. these unimodal systems, although useful, fail to model the relationship between the various modalities. different modalities often present supporting facts about the task, and therefore, learning them together can enhance performance. we present memis, a system that can be used in emergencies like disasters to identify and analyze the damage indicated by user-generated multimodal social media posts, thereby helping the disaster management groups in making informed decisions. our leave-one-disaster-out experiments on a multimodal dataset suggest that not only does fusing information in different media forms improves performance, but that our system can also generalize well to new disaster categories. further qualitative analysis reveals that the system is responsive and computationally efficient. the amount of data generated every day is colossal [10] . it is produced in many different ways and many different media forms. analyzing and utilizing this data to drive the decision-making process in various fields intelligently has been the primary focus of the research community [22] . disaster response management is one such area. natural calamities occur frequently, and in times of such crisis, if the large amount of data being generated across different platforms is harnessed m. agarwal and m. leekha-the authors contributed equally, and wish that they be regarded as joint first authors. rajiv ratn shah is partly supported by the infosys center for ai, iiit delhi. well, the relief groups will be able to make effective decisions that have the potential to enhance the response outcomes in the affected areas. to design an executable plan, disaster management and relief groups should combine information from different sources and in different forms. however, at present, the only primary source of information is the textual reports which describe the disaster's location, severity, etc. and may contain statistics of the number of victims, infrastructural loss, etc. motivated by the cause of humanitarian aid in times of crises and disasters, we propose a novel system that leverages both textual and visual cues from the mass of user-uploaded information on social media to identify damage and assess the level of damage incurred. in essence, we propose memis, a system that aims to pave the way to automate a vast multitude of problems ranging from automated emergency management, community rehabilitation via better planning from the cues and patterns observed in such data and improve the quality of such social media data to further the cause of immediate response, improving situational awareness and propagating actionable information. using a real-world dataset, crisismmd, created by alam et al. [1] , which is the first publicly available dataset of its kind, we present the case for a novel multimodal system, and through our results report its efficiency, effectiveness, and generalizability. in this section, we briefly discuss the disaster detection techniques of the current literature, along with their strengths and weaknesses. we also highlight how our approach overcomes the issues present in the existing ones, thereby emphasizing the effectiveness of our system for disaster management. chaudhuri et al. [7] examined the images from earthquake-hit urban environments by employing a simple cnn architecture. however, recent research has revealed that often fine-tuning pre-trained architectures for downstream tasks outperform simpler models trained from scratch [18] . we build on this by employing transfer learning with several successful models from the imagenet [9] , and observed significant improvements in the performance of our disaster detection and analysis models, in comparison to a simple cnn model. sreenivasulu et al. [24] investigated microblog text messages for identifying those which were informative, and therefore, could be used for further damage assessment. they employed a convolutional neural network (cnn) for modeling the text classification problem, using the dataset curated by alam et al. [1] . extending on their work on crisismmd, we experimented with several other state-of-the-art architectures and observed that adding recurrent layers improved the text modeling. although researchers in the past have designed and experimented with unimodal disaster assessment systems [2, 3] , realizing that multimodal systems may outperform unimodal frameworks [16] , the focus has now shifted to leveraging information in different media forms for disaster management [20] . in addition to using several different media forms and feature extraction techniques, several researchers have also employed various methods to combine the information obtained from these modalities, to make a final decision [19] . yang et al. [28] developed a multimodal system-madis which leverages both text and image modalities, using hand-crafted features such as tf-idf vectors, and low-level color features. although their contribution was a step towards advancing damage assessment systems, the features used were relatively simple and weak, as opposed to the deep neural network models, where each layer captures complex information about the modality [17] . therefore, we utilize the latent representation of text and image modalities, extracted from their respective deep learning models, as features to our system. another characteristic that is essential for a damage assessment system is generalizability. however, most of the work carried out so far did not discuss this practical perspective. furthermore, to the best of our knowledge, so far no work has been done on developing an end-to-end multimodal damage identification and assessment system. to this end, we propose memis, a multimodal system capable of extracting information from social media, and employs both images and text for identifying damage and its severity in real-time (refer sect. 3). through extensive quantitative experimentation in the leave-one-disaster-out training setting and qualitative analysis, we report the system's efficiency, effectiveness, and generalizability. our results show how combining features from different modalities improves the system's performance over unimodal frameworks. in this section, we describe the different modules of our proposed system in greater detail. the architecture for the system is shown in fig. 1 . the internal methodological details of the individual modules are in the next section. the tweet streaming module uses the twitter streaming api 1 to scrap realtime tweets. as input to the api, the user can enter filtering rules based on the available information like hashtags, keywords, phrases, and location. the module outputs all the tweets that match these defined cases as soon as they are live on social media. multiple rules can be defined to extract tweets for several disasters at the same time. data from any social media platform can be used as input to the proposed framework. however, in this work, we consume disaster-related posts on twitter. furthermore, although the proposed system is explicitly for multimodal tweets having both images and text, we let the streaming module filter both unimodal and multimodal disaster tweets. we discuss in sect. 5.5 how our pipeline can be generalized to process unimodal tweets as well, making it more robust. a large proportion of the tweets obtained using the streaming module may be retweets that have already been processed by the system. therefore, to avoid overheads, we maintain a list of identifiers (ids) of all tweets that have been processed by the system. in case an incoming tweet is a retweet that has already been processed by the system before, we discard it. furthermore, some tweets may also have location or geographic information. this information is also stored to maintain a list of places where relief groups are already providing services currently. if a streamed geo-tagged tweet is from a location where the relief groups are already providing aid, the tweet is not processed further. a substantial number of tweets streamed from the social media platforms are likely to be irrelevant for disaster response and management. furthermore, different relief groups have varying criteria for what is relevant to them for responding to the situation. for instance, a particular relief group could be interested only in reaching out to the injured victims, while another provides resources for infrastructural damages. therefore, for them to make proper use of information from social media platforms, the relevant information must be filtered. we propose two sub-modules for filtering: (i) the first filters the informative tweets, i.e., the tweets that provide information relevant to a disaster, which could be useful to a relief group, (ii) the second filter is specific to the relief group, based on the type of damage response they provide. to demonstrate the system, in this work, we filter tweets that indicate infrastructural damage or physical damage in buildings and other structures. finally, once the relevant tweets have been filtered, we analyze them for the severity of the damage indicated. the system categorizes the severity of infrastructural damage into three levels: high, medium and low. based on the damage severity assessment by the system, the relief group can provide resources and services to a particular location. this information must further be updated in the database storing the information about all the places where the group is providing aid currently. furthermore, although not shown in the system diagram, we must also remove a location from the database once the relief group's activity is over, and it is no longer actively providing service there. this ensures that if there is an incoming request from that location after it was removed from the database, it can be entertained. in this section, we discuss the implementation details of the two main modules of the system for relevance filtering and severity analysis. we begin by describing the data pre-processing required for the multimodal tweets, followed by the deep learning-based models that we use for the modules. image pre-processing: the images are resized to 299 × 299 for the transfer learning model [29] and then normalized in the range [0, 1] across all channels (rgb). text pre-processing: all http urls, retweet headers of the form rt, punctuation marks, and twitter user handles specified as @username are removed. the tweets are then lemmatized and transformed into a stream of tokens that can be fed as input to the models used in the downstream modules. these tokens act as indices to an embedding matrix, which stores the vector representation for tokens corresponding to all the words maintained in the vocabulary. in this work, we use 100 dimensional fasttext word-embeddings [6] , trained on the cri-sismmd dataset [1] that has been used in this work. the system as a whole, however, is independent of the choice of vector representation. for the proposed pipeline, we use recurrent convolutional neural network (rcnn) [14] as the text classification model. it adds a recurrent structure to the convolutional block, thereby capturing contextual information with long term dependencies and the phrases which play a vital role at the same time. furthermore, we use the inception-v3 model [25] , pre-trained on the imagenet dataset [9] for modelling the image modality. the same underlying architectures, for both text and image respectively, are used to filter the tweets that convey useful information regarding the presence of infrastructural damage in the relevance filtering modules, and the analysis of damage in the severity analysis module. therefore, we effectively have three models for each modality: first for filtering the informative tweets, then for those pertaining to the infrastructural damage (or any other category related to the relief group), and finally for assessing the severity of damage present. in this subsection, we describe how we combine the unimodal predictions from the text and image models for different modules. we also discuss in each case about how the system would treat a unimodal text or image only input tweet. gated approach for relevance filtering. for the two modules within relevance filtering, we use a simplistic approach of combining the outputs from the text and image models by using the or function (⊕). technically speaking, we conclude that the combined output is positive if at least one of the unimodal models predicts so. therefore, if a tweet is predicted as informative by either the text, or the image, or both the models, the system predicts the tweet as informative, and it is considered for further processing in the pipeline. similarly, if at least one of the text and the image modality predicts an informative tweet as containing infrastructural damage, the tweet undergoes severity analysis. this simple technique helps avoid missing any tweet that might have even the slightest hint of damage, in either or both the modalities. any false positive can also be easily handled in this approach. if, say, a non-informative tweet is predicted as informative in the first step at relevance filtering, it might still be the case that in the second step, the tweet is predicted as not containing any infrastructural damage. furthermore, in case a tweet is unimodal and has just the text or the image, then the system can take the default prediction of the missing modality as negative (or false for a boolean or function), which is the identity for the or operation. in that case, the prediction based on the available modality will guide the analysis (fig. 2) . attention fusion for severity analysis. the availability of data from different media sources has encouraged researchers to explore and leverage the potential boost in performance by combining unimodal classifiers trained on individual modalities [5, 27] . here, we use attention fusion to combine the feature interpretations from the text and image modalities for the severity analysis module [12, 26] . the idea of attention fusion is to attend particular input features as compared to others while predicting the output class. the features, i.e., the outputs of the penultimate layer or the layer before the softmax, of the text and image models are concatenated. this is followed by a softmax layer to learn the attention weights for each feature dimension, i.e., the attention weight α i for a feature x i is given by: therefore, the input feature after applying the attention weights is, where, i, j ∈ 1, 2, .., p, and p is the total number of dimensions in the multimodal concatenated feature vector. w is the weight matrix learned by the model. this vector of attended features is then used to classify the given multimodal input. with this type of fusion, we can also analyze how the different modalities are interacting with each other employing their attention weights. moving from the relevance filtering to the severity analysis module, we strengthen our fusion technique by using attention mechanism. this is required since human resources are almost always scarce, and it is necessary to correctly assess the requirements at different locations based on the severity of the damage. as opposed to an or function, using attention, we are able to combine the most important information as seen by the different modalities to together analyze the damage severity. in this case, the treatment of unimodal tweets is not that straightforward, since the final prediction using attention fusion occurs after concatenation of the latent feature vectors of the individual modalities. therefore, in case the text or image is missing, we use the unimodal model for the available modality. in other words, we use attention mechanism only when both the modalities are present to analyze damage severity, else we use the unimodal models. recently, several datasets on crisis damage analysis have been released to foster research in the area [21] . in this work, we have used the first multimodal, labeled, publicly available damage related to the twitter dataset, crisismmd, created by alam et al. [1] . it was collected by crawling the blogs posted by users during seven natural disasters, which can be grouped into 4 disaster categories, namely-floods, hurricanes, wildfires and earthquakes. crisismmd introduces three hierarchical tasks: 1. informativeness. this initial task classifies each multimodal post as informative or non-informative. alam et al. define a multimodal post as informative if it serves to be useful in identifying areas where damage has occurred due to disaster. it is therefore a binary classification problem, with the two classes being informative and non-informative. 2. infrastructural damage. the damage in an informative tweet may be of many different kinds [1, 4] . crisismmd identifies several categories for the type of damage, namely-infrastructure and utility damage, vehicle damage, affected individuals, missing or found people, other relevant information, none. alam et al. [1] also noted that the tweets which signify physical damage in structures, where people could be stuck, are especially beneficial for the rescue operation groups to provide aid. out of the above-listed categories, the tweets having infrastructure and utility damage are therefore identified in this task. this again is modelled as a classification problem with two classesinfrastructural and non-infrastructural damage. 3. damage severity analysis. this final task uses the text and image modalities together to analyze the severity of infrastructural damage in a tweet ashigh, medium, or low. we add another label, no-damage, to support the pipeline framework that can handle false positives as well. specifically, if a tweet having no infrastructural damage is predicted as positive, it can be detected here as having no damage. this is modelled as a multi-class classification problem. the individual modules of the proposed pipeline essentially model the above three tasks of crisismmd. specifically, the two relevance filtering modules model the first and the second tasks, respectively, whereas the severity analysis module models the third task (table 1) . to evaluate how well our system can generalize to new disaster categories, we train our models for all the three tasks in a leave-one-disaster-out (lodo) training paradigm. therefore, we train on 3 disaster categories and evaluate the performance on the left-out disaster. to handle the class imbalance, we also used smote [8] with the word embeddings of the training fold samples for linguistic baselines. we used adam optimizer with an initial learning rate of 0.001, the values of β1 and β2 as 0.9 and 0.999, respectively, and a batch size of 64 to train our models. we use f1-score as the metric to compare the model performance. all the models were trained on a geforce gtx 1080 ti gpu with a memory speed of 11 gbps. to demonstrate the effectiveness of the proposed system for multimodal damage assessment on social media, we perform an ablation study, the results for which have been described below. design choices. we tried different statistical and deep learning techniques for modelling text-tf-idf features with svm, naive bayes (nb) and logistic regression (lr); and in the latter category, cnn [13] , hierarchical attention model (hattn), bidirectional lstm (bilstm) and rcnn [14] . as input to the deep learning models, we use 100-dimensional fasttext word embeddings [6] trained on the dataset. by operating at the character n-gram level, fasttext tends to capture the morphological structure well. thus, helping the otherwise out of vocabulary words (such as hash-tags) to share semantically similar embeddings with its component words. as shown in table 2 , the rcnn model performed the best on all three tasks of the relevance filtering and severity analysis modules. specifically, the average lodo f1-scores of rcnn on the three tasks are 0.82, 0.76, and 0.79, respectively. furthermore, the architecture considerably reduces the effect of noise in social media posts [14] . for images, we fine-tuned the vgg-16 [23] , resnet-50 [11] and incep-tionv3 [25] models, pre-trained on the imagenet dataset [9] . we also trained a cnn model from scratch. experimental results in table 2 reveal that incep-tionv3 performed the best, and the average f1-score with lodo training for the three tasks are 0.74, 0.77, and 0.79, respectively. the architecture employs multiple sized filters to get a thick rather than a deep architecture, as very deep networks are prone to over-fitting. such a design makes the network computationally less expensive, which is a prime concern for our system as we want to minimize latency to give quick service to the disaster relief groups. table 3 highlights the results of an ablation study over the best linguistic and vision models, along with the results obtained when the predictions by these individual models are combined as discussed in sect. 4.3. the results for all the modules demonstrate the effectiveness of multimodal damage assessment models. specifically, we observe that for each disaster category in the lodo training paradigm, the f1-score for the multimodal model is always better than or compares with those of the text and image unimodal models. in this section, we analyze some specific samples to understand the shortcomings of using unimodal systems, and to demonstrate the effectiveness of our proposed multimodal system. table 4 records these sample tweets along with their predictions as given by the different modules. in green are the correct predictions, whereas the incorrect ones are shown in red they have been discussed below in order: 1. the image in the first sample portrays the city landscape from the top, damaged by the calamity. due to the visual noise, the image does not give much information about the intensity of damage present, and therefore, the image model incorrectly predicts the tweet as mildly damaged. on the other hand, the text model can identify the severe damage indicated by phrases like 'hit hard'. combining the two predictions by using attention fusion, therefore, helps in overcoming the unimodal misclassifications. in this tweet, the text uses several keywords, such as 'damaged' and 'earthquake', which misleads the text model in predicting it as severely damaged. however, the image does not hold the same perspective. by combining the feature representations, attention fusion can correctly predict the tweet as having mild damage. the given tweet is informative and therefore, it is considered for damage analysis. however, the text classifier, despite the presence of words like 'killed' and 'destroyed', incorrectly classifies it to the non-infrastructural damage class. the image classifier correctly identifies the presence of damage, and therefore, the overall prediction for the tweet is infrastructural damage, which is correct. furthermore, both the text and image models are unable to identify the severity of damage present, but the proposed system can detect the presence of severe damage using attention fusion. the sample shows how the severity analysis module combines the text and visual cues by identifying and attending to more important features than others. this helps in modelling the dependency between the two modalities, even when both, individually give incorrect predictions. the image in the tweet shows some hurricane destroyed structures, depicting severe damage. however, the text talks about 'raising funds and rebuilding', which does not indicate severe damage. the multimodal system learns to attend the text features more and correctly classifies the sample as having no damage, even though both the individual models predicted incorrectly. furthermore, in this particular example, even by using the or function, the system could not correctly classify it as not having infrastructural damage. yet, the damage severity analysis module identifies this false positive and correctly classifies it. in this section, we discuss some of the practical and deployment aspects of our system, as well as some of its limitations. we simulate an experiment to analyze the computational efficiency of the individual modules in terms of the time they take to process a tweet, i.e., the latency. we are particularly interested in analyzing the relevance filtering and severity analysis modules. we developed a simulator program to act as the tweet streaming module that publishes tweets at different load rates (number of tweets in 1 second) to be processed by the downstream modules. the modules also process the incoming tweets at the same rate. we calculate the average time for processing a tweet by a particular module as the total processing time divided by the total number of tweets used in the experiment. we used 15, 000 multimodal tweets from crisismmd, streamed at varying rates. the performance of the two relevance filtering modules and the severity analysis module as we gradually increase the load rate is shown in the fig. 3 . as a whole, including all the modules, we observed that on an average, the system can process 80 tweets in 1 minute. this experiment was done using an intel i7-8550u cpu having 16 gb ram. one can expect to see an improvement if a gpu is used over a cpu. generalization. the proposed system is also general and robust, especially in three aspects. firstly, the results of our lodo experiments indicate that the system can perform well in case it is used for analyzing new disasters, which were not used for training the system. this makes it suitable for real-world deployment where circumstance with new disaster categories cannot be foreseen. furthermore, we also saw how the two main modules of the system work seamlessly, even when one of the modalities is missing. this ensures that the system can utilize all the information that is available on the media platforms to analyze the disaster. finally, the second module in relevance filtering can be trained to suit the needs of several relief groups that target different types of damage, and therefore, the system is capable of being utilized for many different response activities. limitations. although the proposed system is robust and efficient, some limitations must be considered before it can be used in real-time. firstly, the system is contingent on the credibility i.e., the veracity of the content shared by users on social media platforms. it may so happen that false information is spread by some users to create panic amongst others [15] . in this work, we have not evaluated the content for veracity, and therefore, it will not be able to differentiate such false news media. another aspect that is also critical to all systems that utilize data generated on social media is the socio-economic and geographic bias. specifically, the system will only be able to get information about the areas where people have access to social media, mostly the urban cities, whereas damage in the rural locations may go unnoticed since it did not appear on twitter or any other platform. one way to overcome this is to make use of aerial images, that can provide a top view of such locations as the rural lands. however, this again has a drawback as to utilize aerial images effectively, a bulk load of data would have to be gathered and processed. identifying damage and human casualties in real-time from social media posts is critical to providing prompt and suitable resources and medical attention, to save as many lives as possible. with millions of social media users continuously posting content, an opportunity is present to utilize this data and learn a damage recognition system. in this work, we propose memis, a novel multimodal emergency management information system for identifying and analyzing the level of damage severity in social media posts with the scope for betterment in disaster management and planning. the system leverages both textual and visual cues to automate the process of damage identification and assessment from social media data. our results show how the proposed multimodal system outperforms the state-of-the-art unimodal frameworks. we also report the system's responsiveness through extensive system analysis. the leave-one-disaster-out training setting proves the system is generic and can be deployed for any new unseen disaster. crisismmd: multimodal twitter datasets from natural disasters processing social media images by combining human and machine computing during crises crisisdps: crisis data processing services a twitter tale of three hurricanes: harvey, irma, and maria. arxiv multimodal vehicle detection: fusing 3d-lidar and color camera data enriching word vectors with subword information application of image analytics for disaster response in smart cities smote: synthetic minority over-sampling technique imagenet: a large-scale hierarchical image database how much data do we create every day? the mind-blowing stats everyone should read deep residual learning for image recognition an attention-based decision fusion scheme for multimedia information retrieval convolutional neural networks for sentence classification recurrent convolutional neural networks for text classification from chirps to whistles: discovering eventspecific informative content from twitter damage identification in social media posts using multimodal deep learning handcrafted vs. non-handcrafted features for computer vision classification a survey on transfer learning multimodal deep learning based on multiple correspondence analysis for disaster management a computationally efficient multimodal classification approach of disaster-related twitter images natural disasters detection in social media and satellite imagery: a survey multimodal analysis of user-generated multimedia content very deep convolutional networks for large-scale image recognition detecting informative tweets during disaster using deep neural networks rethinking the inception architecture for computer vision attention is all you need multimodal fusion of eeg and fmri for epilepsy detection madis: a multimedia-aided disaster information integration system for emergency management how transferable are features in deep neural networks? in: advances in neural information processing systems key: cord-324656-6xq5rs0u authors: bellika, johan gustav; hasvold, toralf; hartvigsen, gunnar title: propagation of program control: a tool for distributed disease surveillance date: 2006-04-18 journal: int j med inform doi: 10.1016/j.ijmedinf.2006.02.007 sha: doc_id: 324656 cord_uid: 6xq5rs0u purpose: the purpose of the study was (1) to identify the requirements for syndromic, disease surveillance and epidemiology systems arising from events such as the sars outbreak in march 2003, and the deliberate spread of bacillus anthracis, or anthrax, in the us in 2001; and (2) to use these specifications as input to the construction of a system intended to meet these requirements. an important goal was to provide information about the diffusion of a communicable disease without being dependent on centralised storage of information about individual patients or revealing patient-identifiable information. methods: the method applied is rooted in the engineering paradigm involving phases of analysis, system specification, design, implementation, and testing. the requirements were established from earlier projects’ conclusions and analysis of disease outbreaks. the requirements were validated by a literature study of syndromic and disease surveillance systems. the system was tested on simulated ehr databases generated from microbiology laboratory data. results: a requirements list that a syndromic and disease surveillance system should meet, and an open source system, “the snow agent system”, has been developed. the snow agent system is a distributed system for monitoring the status of a population's health by distributing processes to, and extracting epidemiological data directly from, the electronic health records (ehr) system in a geographic area. conclusions: syndromic and disease surveillance tools should be able to operate at all levels in the health systems and across national borders. such systems should avoid transferring patient identifiable data, support two-way communications and be able to define and incorporate new and unknown diseases and syndrome definitions that should be reported by the system. the initial tests of the snow agent system shows that it will easily scale to national level in norway. the severe acute respiratory syndrome (sars) outbreak in 2003 showed that existing electronic health record (ehr) syndromic or disease surveillance system should therefore serve all levels, from the local healthcare service to the national and global level [2, 3] . syndromic or disease surveillance has two distinct phases: a detection phase, the goal of which must be to detect an outbreak as early as possible, and a monitoring phase, where the actual disease is known and the goal is to prevent the disease from spreading. in the latter case, it is important to detect new cases and to track existing cases of the disease. to enable early detection of disease outbreaks and the monitoring of the diffusion of a disease through the identification of abnormal disease patterns, "sensors" in the form of a software system should be distributed to all the points in the healthcare system where patients present, or where they leave traces of evidence of illness [4] . lober et al. [5] provide a good description of the information sources that can be monitored to detect an outbreak as early as possible. where affected people present may depend on the type of disease and on the organisation of the healthcare service. in norway, which has a system of personal general practitioners (gps) with gatekeeper functions to secondary and tertiary levels of care, patients will typically consult their gp or the casualty clinic in the first instance. patients are only likely to present at a hospital emergency unit if they are acutely ill (for example, if they have been injured in an accident, or referred to the casualty clinic by the gp). a telephone survey in the usa, in new york, showed that 29.1% of persons with influenzalike illness (ili) visited a physician, 21.4% called a physician for advice, 8.8% visited the emergency department, and 3.8% called a nurse or health hotline [6] . the ehr systems used by gps are therefore likely to be very good sources for detecting disease outbreaks in a healthcare context where most people normally consult their gp in the first instance if they get ill. however, using patient data stored in the ehr system is difficult because of the privacy issues involved in exporting data from the ehr systems [7] [8] [9] [10] [11] . the ehr systems used by gps in norway use the international classification of primary care (icpc), which is more specifically symptom-related than icd-9 and icd-10, making it more applicable for syndromic and disease surveillance use. unfortunately, there is no messaging standard such as hl7 available for primary care services or hospitals in norway, which could enable the use of an available open source solution for syndromic or disease surveillance [2, 11, 12] . syndromic or disease surveillance is relatively easy to implement for all well-defined and existing diseases. if a new disease appears, as in the case of sars, or as result of a deliberate spread, disease surveillance may become more difficult. first, we do not have codes for specifying confirmed, probable or possible cases of the new disease. this may easily be solved if it is sufficient merely to update the coding system. how fast such an update can be performed depends on a number of factors: how fast can the coding system be updated, how fast can the new version be disseminated, how fast can the updated code-set be employed, etc. updating a disease classification is normally done very infrequently and the reliability of such classifications is therefore problematic when the goal is to count occurrences of a new disease. the creation of an additional coding system for contagious diseases, which enables quick updating, may provide a solution to this prob-lem. however, this is also problematic, because duplication of coding systems adds to the quantity of information, which must be entered into the ehr systems. basing surveillance of new diseases on existing classification categories is therefore problematic and adds to the complexity of the problem. however, without such disease codes we cannot automatically count the number of cases and get a picture of the geographical diffusion of the disease. another solution may be to use a list of prodromes or early symptoms (syndrome definition) as basis for definitions of possible and probable cases, but we cannot guarantee that a new disease will be covered by such predefined lists of symptoms. conditions with similar symptom patterns may be interpreted as possible or probable cases, thereby polluting the diffusion pattern. in the case of sars, a visit or contact with a person who had visited a geographic area where there were reported cases was a main indicator of probable or possible cases. such conditions are not known beforehand [13] . in the case of sars, the definition of possible and probable cases evolved over the weeks following the outbreak from initially being very vague to becoming a very specific set of symptoms. it may therefore be necessary to distribute new and updated syndrome definitions if local classification of cases based on the syndrome definition is required. this dynamic behaviour of a disease or syndrome definition makes it hard to build software that automatically identifies and distinguishes between possible, probable and confirmed cases. there are at least two approaches to building a surveillance system: a centralised and a distributed approach. all of the systems reported by lober et al. [5] and kun et al. [14] are based on the centralised storage and analysis of data. these systems have been classified as using first-or second-generation architectures for data integration [9] . the real-time outbreak and disease surveillance system (rods) from the university of pittsburgh [12] also uses a centralised approach. the emergency departments covered by the system send hl7 messages containing icd-9 codes in real time to a centralised inference system where outbreaks are detected. when a disease outbreak is detected by rods, the appropriate authorities are informed about the situation. the outbreak detection process is run periodically on the data available to the system. the bio-surveillance, analysis, feedback, evaluation and response (b-safer) system [2, 15] is based on a federated approach involving the open source openemed infrastructure. openemed [16] (formerly known as telemed [17] ) is based on the clinical observation access service (coas) [18] and patient identification data service (pids) [19] corbamed specification of the object management group. b-safer and openemed take a more interoperable approach that demonstrates the value of the federated approach to syndromic surveillance, which makes it possible to view and manage information at several levels ranging from local to regional, national and global levels [2] . we have constructed a system, which we have named "the snow agent system", after the famous dr. john snow. in the snow agent system we also take a distributed approach, where all contributors of data to the system also have the opportunity to access information about the current situation in a geographic area directly from the system. in that sense, the snow agent system is a true "peer-to-peer" net-work among general practitioners. according to lober's classification [9] , this system would be classified as using thirdgeneration data integration. the snow agent system does not depend on the centralised storage of patient information, and may therefore be used by any group of health institutions or general practitioners that want to share data about the health of the population, without exposing any patientsensitive information. it is designed for everyday use by gps who want to share information about contagious diseases and disease outbreaks in their local community. it also avoids the problematic privacy issues, because no patient-identifiable information ever leaves the ehr system. it also supports the requirement of regular usage, which is important in being prepared for an emergency [3, 20] . the snow agent system has a number of other applications besides syndromic or disease surveillance and epidemiology. most important is the extraction, collection and visualisation of a patient's health record from distributed ehr systems for authorised persons. the system also makes it possible to generate statistics about the activity in the health institutions covered by the system. the messaging system used by the snow agent system may also be used for transferring any kind of data between health institutions, which is a valuable spin-off. however, in this paper we focus on the requirements that we argue syndromic, disease surveillance and epidemiology systems may need to meet when facing a local, national or a pandemic outbreak of a communicable disease. some of these requirements also correspond to the requirements for extraction, collection and visualisation of a patient's health record. the paper presents the core principle utilised in building a distributed system for monitoring and interrogating the status of the population's health, and discusses the results of the initial system tests. the work reported here has been conceived within the engineering paradigm. the processes involved in building a system can be divided into the distinctive phases of analysis, system specification, design, implementation and testing. the system described here has gone through several iterations of analysis, system specification, design, implementation, and testing. the requirements listed in the section below are derived from the following sources: (1) a project that evaluated the needs and conditions for access to patient information from geographically distributed electronic health records [21] ; (2) an analysis of the sars outbreak in march 2003; (3) an analysis of a whooping cough outbreak in troms county in north norway in october/november 2003. the requirements were validated and augmented by a literature study of syndromic and disease surveillance systems and solutions directed towards early detections of bio-terrorist attacks [2] [3] [4] [5] [8] [9] [10] [11] [12] [13] [14] [15] 20, [22] [23] [24] [25] [26] [27] [28] [29] [30] [31] [32] [33] [34] [35] [36] [37] [38] [39] [40] . the snow agent system is a redesign and a complete reimplementation of the virtual secretary system [41] in terms of the requirements reported here. the initial virtual secretary system was developed at the department of computer science at the university of tromsø. the current version is built on top of the jabber instant messaging and presence system. (see details in section 4, "propagation of program control" below.) to test the system components, we implemented a distributed epidemiology service which extracts epidemiological data from simulated ehr databases. the test data used for constructing the ehr databases is described in the next section. the hardware and software used for system testing is described in section 2.2 below. getting access to production data and system is problematic from both a privacy and resource point of view. we therefore chose to create fictitious ehr databases based on data from the microbiology laboratory at the university hospital of north norway. this laboratory serves a large geographical area and many gp clinics, and therefore has data that is well suited to our needs. the data used in the system test was laboratory test data from the microbiology lab. from the pertussis lab dataset we constructed a very limited ehr database by using a number of assumptions: 1. we assume that it takes 1 day to deliver the test sample to the hospital laboratory. the consultation date with the gp is therefore test received date minus 1 day, and consultation time equals test received time. 2. we assume that all gps have the same behaviour regarding entering data into the electronic patient record (ehr) system. 3. they all create a consultation entry in the ehr system using the date and time values specified above. 4. they all do a test from the posterior nasopharynx using darcon or calcium alginate swabs, which they send to the hospital laboratory and order a "b. pertussis pcr" (bordertella pertussis) laboratory test (icpc2 process code-33 microbiological/immunological test) (code 6232 and 6192 in the lab system). 5. if the lab report is negative, the gp does nothing. if the lab test is positive, the doctor creates a telephone consultation with the patient using "r71 whooping cough" as the diagnosis, and prescribes erythromycin. five days later the patient is assumed not to be contagious to others. (pertussis is most contagious during the first week. three weeks after the onset of symptoms, the patient is seldom contagious.) 6. the "x days since onset" value for the whooping cough patients will probably vary widely. we assume the distribution of x is gaussian. we assume that most people will not see their gp before the second stage of the disease has started (7-14 days after onset of symptoms), when the severe cough with whooping and vomiting begins. this value may also vary with the age and gender of the patient. we assume it is likely that babies under the age of one (below 1 year in the test dataset) probably see their doctor earlier than older children do. we also assume male patients wait longer to see their gp than female patients do. knowledge about an outbreak probably also affects how early a patient sees their gp. we assume that children <2 years see their gp 2-5 days after onset of symptoms, children ≥2, <8 years see their gp 3-7 days after onset, and that children ≥8, <18 years see their gp 5-10 days after onset. female ≥18 years see their gp 10-15 days after onset and male >18 years see their gp 12-20 days after onset. from negative laboratory reports, the following ehr documents were created: first consultation, lab request and lab report. from positive laboratory reports, the following ehr documents were created: first consultation, lab request, lab report, second consultation (telephone), drug prescription (erythromycin). the dataset contained 7939 rows of lab results from 3997 different patients. the dataset contained 6463 negative lab result records and 198 lab result records with "pos" results, 794 patients with test result = "neg" (non-negative blood sample test results) and 194 patients with a "pos" test result. the dataset contained 487 test requesters, including hospital internal requesters. of these test requesters, 459 were from outside the hospital and 28 from inside the hospital. the dataset covered 372 postcodes, 153 postcodes with test result = "neg" (non-negative blood sample test results) and 54 postcodes with test result = "pos". the dataset contained patients from 138 municipalities, where 62 municipalities had test result = "neg" (non-negative blood sample test results) and 25 municipalities with test result = "pos". there were 4528 female patients and 3411 male patients in the data set, including 103 female patients and 95 male patients with a "pos" test result, and 779 female patients and 697 male patients with test result = "neg" (non-negative blood sample test results). in the system test, we only used confirmed cases as test data (test result = "pos"). in addition to the laboratory dataset, a map was needed, on which to plot the data. the map was created from bitmaps made available in the norgeshelsa system [42] . the bitmaps were first converted to svg [43] polygons. then the map polygons were inserted into a mysql database [44] in a convenient format for map generation, and coded with geographical information. in our experiment, we wanted to determine the performance of the system in a close to reality configuration of the system. the results of the analysis described in section 2 above identified the requirements for the tools to be constructed for distributed syndromic or disease surveillance. we have divided the requirements which distributed surveillance and epidemiology systems should fulfil, are shown below. while hospitals can afford a real-time connection to a centralised system such as rods [12] , this solution is not very suitable for all gp clinics. for some gp clinics a more suitable solution may be connection to a shared interconnecting network on a regular basis for reading emails, receiving electronic lab results and electronic hospital discharge letters. this has been the most common solution for gp clinics connected to the north norwegian regional health network. however, the gp clinics are now migrating to an alternative solution, in which they are constantly connected to the health network. to be able to cover all connection alternatives, a distributed solution for epidemiology and healthcare surveillance must support periodic connection to a shared interconnecting network, minimise communication costs, and tolerate narrow bandwidth. coverage is perhaps the most important aspect of a distributed epidemiology and surveillance system. the best approach to achieving coverage is through developing a free and opensource solution that has a minimum of hardware and software costs. the rods [12] system and b-safer [2] system, and several other tools used in outbreak detection, use this approach. the heterogeneity of ehr solutions for general practices is problematic because of the range of different ways in which they store medical data. existing systems may have problems converting information stored in the internal system to a standardised format [9, [47] [48] [49] . they may also lack important information items such as the onset date of a communicable disease. a distributed epidemiology and disease surveillance system would benefit from using a standardised data format internally. a standardised data format will make querying of data easier. a syndromic or disease surveillance system must be automatic (independent of manual data entry for surveillance purposes) to be sustainable [22, 37] . the primary task of healthcare workers is to treat patients, not to perform computer system configuration or system maintenance. however, many institutions in primary care are small, and do not normally employ professional system administrators. in some areas, there is a shortage of available staff with the skills for system administration. a system should therefore ideally require zero configuration and maintenance. this requirement may be hard to meet because most systems need at least a minimum of configuration and maintenance to operate smoothly. this can be difficult to achieve because of the need for manually specifying and ensuring a security policy (see section 4.7 below). a system intended to enable the computation of global epidemiological queries must be asynchronous. it is difficult to anticipate how a globally distributed computation can be performed while a user is waiting for a system response. if we allow participating servers to connect periodically to a shared network, we must ensure that the computations performed on the servers are independent of the computation requester being available and online. asynchronous computations are also more autonomous. a surveillance system must be dynamic in terms of when and how often calculations are performed. the need for reporting frequency may span from running a query every week to several times a day. if an outbreak is detected, the frequency of calculating the diffusion of the disease may need to be increased. the responsibility for switching a gp clinic system into online mode (constantly connected to the shared interconnecting network) may need to be external to the gp clinic. this decision should only be made after a local authentication and authorisation of the request. to be able to meet our objective of not revealing patient identification, and of not being dependent on centralised storage of information about individual patients, the system needs to be distributed. classification of cases into disease or syndrome groups must therefore be done locally, within each ehr sys-tem. many of the syndromic surveillance systems in the usa utilise the 'chief complaints' free text field, and icd-9 or icd-10 codes in the hl7 messages to perform classification of events into syndrome groups. in these systems, the classification of cases against disease and syndrome groups is done centrally, which makes it easy to define and put into use new disease definitions. in norway and several other european countries, the gps use the icpc classification. icpc is more directly related to symptom recording than either icd-9 and icd-10, which makes it more applicable for syndromic and disease surveillance. however, recent travel to affected areas or contact with infected persons is not part of the classification. nor are explicit values of biometrical measurements such as body temperature >38.4 • c. these features were among the criteria for identifying a possible sars case. the most difficult requirement for existing ehr systems to meet is therefore the capacity to incorporate new syndrome definitions that need to be reported. it is important that these syndrome definitions are distributed and automatically incorporated into the ehr system, because of the limitations which paper-based forms have in an outbreak situation, as identified by foldy [13] . for some ehr systems, a new disease or syndrome definition may require that new software is added to the existing ehr system. an example that illustrates this issue is the use of biometrical measurements such as body temperature, pulse, or blood pressure as part of the disease classification. this problem does not apply to all ehr systems, as we will see below. constructing, distributing and updating ehr system software are all time-consuming activities that preferably should not be necessary at the point when an outbreak of a new and dangerous disease is detected. in the case of sars, we saw that the definition of the disease evolved and became more specific over time. as new knowledge about a disease becomes available, it needs to be disseminated to all systems participating in surveillance. this may require that the ehr system is revised to include the new definition of the disease. experience from milwaukee, wisconsin [13] shows that manual routines for updating screening forms are problematic. the "counting" part of a distributed disease surveillance system also needs to be highly dynamic and to allow for inclusion of new disease definitions "on the fly". to be able to monitor the diffusion of a new disease, we argue that we need systems able to include new definitions of diseases in real time. communication in such systems therefore must be two-way, enabling the flow of information about diseases or syndrome definitions in one direction, and information about occurrences and diffusion of the condition in the other direction. the requirements above seem to present a perfect case for mobile code, also known as mobile software agent solutions [50, 51] . many implementations of mobile software agent solutions already exist. mobile software agent technology makes it possible to build highly dynamic distributed systems that support asynchronous and autonomous computations. new functionality in ehr systems may be defined and disseminated as source code to the target systems. however, are mobile-code-based solutions imaginable in healthcare? the risks of mobile code are that it has the capacity to bring down the ehr system in a gp's office due to programming errors, deliberately or unintentionally dump patient data on the internet, take all computing resources away from the gp, etc. even if the source code is certified, it should be asked whether it is appropriate to trust software providers which assert that they "will not be held responsible", if patient data finds its way onto the internet. yes, indeed, it may be difficult to accept mobile-code-based solutions in healthcare information management. is the mobile-code-based solution the only solution able to meet the requirements? mobile code is not suitable, because the physician responsible for the patient data should be able to know exactly what the software that accesses the patient data do. if not, it is unlikely that a physician will authorise the use of such a system. authorisation is closely connected to the tasks performed. if the physician does not know beforehand what the software does, it will be inappropriate to provide authorisation. instead of moving code, we can get some of the flexibility inherent in mobile-code-based approaches by moving program control and data. propagation of program control allows program control to be moved between hosts, in the same manner as mobile code, by transferring a task specification and data between the hosts. the principle is located somewhere between process migration and remote procedure call [52] . instead of letting a process or software agent wander autonomously at its own will between hosts, we ensure that the software agent is under the control of several authorities at all times. these authorities may terminate the software agent at any time. by applying this principle, we gain control over the code executed, but lose some of the flexibility of mobile-code-based solutions. as described, the snow agent system would not appear to provide a simple solution to the hard requirements outlined above. the system may however be used as a dissemination tool for technology that is able to meet the hard requirements above. we will come back to this issue in section 4.8, below. the current version of the snow agent system is a redesign and a complete reimplementation of the virtual secretary system developed at the department of computer science at the university of tromsø [41] in 1993-1998. the first version of the system was initially a mobile-code-based solution for mobile software agents. however, due to the security issues with mobile code [53] [54] [55] , we have moved away from this approach. the current version of the snow agent system does not support mobile code, but allows program control to propagate between hosts using two daemon processes as the mobility enabling system services. these two services are the agent daemon and the mission controller (mc). their tasks and responsibilities are explained in more detail below. the agent daemon and the mission controller are built as extensions to the jabber extensible open source server version 1.4.2 [56] , as shown in fig. 1 . jabber implements the extensible messaging and presence protocol (xmpp) (ietf rfcs) [57, 58] , which allows users to show their presence, chat, and exchange instant messages (xmpp messages). the xmpp messages are expressed using xml. the agent daemon, the mission controller, snow agents and users of the snow agent system also communicate using xmpp messages. the jabber server communicates with jabber clients using the "c2s" (client-to-server) component. jabber talks to other jabber servers using the "s2s" (server-toserver) component. we define an agent as a software entity that performs a series of operations on one or more host computers on behalf of a user. an agent may be mobile and able to migrate or propagate between host computers. an agent has a mission that it seeks to accomplish. this mission is specified by the user (or "mission requester") and is expressed as a mission specification using xml. we use the term "mission agent" for the incarnation of an agent that runs on a single computer. the mission agent is instantiated by an agent daemon that creates the mission agent processes. an agent (or agent mission) may therefore consist of several mission agents that run in parallel on multiple hosts or serially as a relay. a mission agent may also employ sub-missions by sending a mission specification to a mission controller. the agent daemon has more or less the same responsibility as traditional server technology and middleware such as corba, j2ee etc., and can easily be replaced by such technology. the agent daemon manages local resources such as processor, disk, network, and agent applications. its main responsibility is to instantiate, evict and restore mission agents. it negotiates agent missions with remote mcs and performs authentication of instantiated mission agents. in fig. 1 , above, the topmost agent daemon has instantiated two mission agents. these mission agents may have been requested by agent 1 or by a user using a jabber client. the agent daemon also provides information about running mission agents to system administrators, local and remote agents and users if allowed by the local system administrator and each agent. the mission controller functions as an intermediary between the user client and the agent daemon which instantiate the processes based on the specification provided by the user. by performing mission control on behalf of the user, the user becomes independent of the agent execution and the agent becomes more autonomous and independent of the user. the mc knows the set of applications (or mission types) available for each user based on user identity. the mc performs mission control by receiving mission specifications from end users, negotiating mission agent instantiation with remote agent daemons, keeps track of the whereabouts of the agent and notifies the mission requester when a mission is finished, when mission results are available, or if an error occurred. the mission controller performs the migration service for agents executing on a host by receiving the mission specification from the local agent. the mission controller also provides mission control to agents that want to use sub missions on remote hosts. a mission agent is a process that is instantiated by the agent daemon based on a specification received from a mc. the mission specification is expressed in xml and contains the name and version of the application to run, user identity, certificates and signatures proving the identity and authorisation of the user, public keys for encryption of mission results, time and mobility constraints, list of hosts to visit and data used during the mission. the code used for constructing the mission agent must exist on the target host before the mission agent can be instantiated. the system owner needs to consent to supporting the agent type before the agent type can be instantiated. a snow agent has the capability to communicate with any entity using xmpp messages. it can ask either the mission controller or the agent daemon, or both, for a list of local or remote agents. it can participate in a conference with users or other agents or even obtain presence information about local or remote agents and users. the agents may send mission results directly to the mission requester using ordinary xmpp messages with encrypted or unencrypted contents, or to any other valid recipient. the mission controller is able to control two kinds of agent distribution schemes: spread mode missions and jump mode missions, as shown in fig. 2 . in fig. 2 an agent, labelled the "parent agent", requests a sub-mission to three hosts by sending a mission specification to the local mc (not shown). in fig. 2a , the mission specification specifies a spread mode mission, in fig. 2b it specifies a jump mode mission. fig. 3 shows the phases of the mission agent's life cycle on the hosts in a spread mode mission. in spread mode missions, the mission controller forwards the mission specification to the remote agent daemons, indicated by the arrows labelled "1" in fig. 2a . the mission controller negotiates with several remote agent daemons simultaneously. this is illustrated by the negotiation phase in fig. 3 . the agent daemons may respond with a waiting time for execution. this is illustrated by the waiting phase shown in fig. 3 . after the potential waiting phase, the mission agents are instantiated by the agent daemon and the mission specification is transferred (through the connection to the c2s component). this stage is shown as the activation phase in fig. 3 . when the complete mission specification (including the task specification and data) is transferred, the mission agents become operational and start performing their tasks. this is illustrated by the working phase in fig. 3 . mission agents are potentially instantiated on each host covered by the mission and run in parallel until the mission has ended. when a mission agent has finished its task, the mission result can be transferred to the mission requester as illustrated by the arrows labelled "2" in fig. 2a . in jump mode (fig. 2b) , the mission controller sends the mission specification to the agent daemon on the first target host. this is illustrated by the arrow labelled "1" in fig. 2b . a negotiation phase may follow while the agent daemon and the mission controller try to reach an agreement on the mission specification. this is illustrated by the negotiation phase (t1-t2) in fig. 4 . after the negotiation phase, a waiting, activation and working phase may follow, as in the spread mode case. when execution on that site has finished, the mission agent asks the local mission controller for migration service to a new host. this is illustrated by the migration phase (t5-t6) shown in fig. 4 . the local mission controller negotiates the migration with the agent daemon on a remote site by transferring and negotiating the contents of the mission specification. the main mission controller (on the initiating host in fig. 2b ) regains control when the agent has migrated to the new host. the first migration is illustrated by the arrow labelled "2" in fig. 2b . this scheme continues until all hosts in the agent's itinerary have been visited as illustrated by the arrows labelled "3" and "4" in fig. 2b. the users of the snow agent system may use any jabber client that supports sending raw xml messages or specialised plugins that produces the mission specifications. jabber clients such as exodus [59] and jeti [60] provide a plug-in interface. specialised graphical user interfaces (gui) to the snow agent system applications may be implemented as exodus or jeti plug-ins and provide gui for specifying missions and interacting with the snow agent system. the snow agent system clients communicate with mission controllers, create missions, obtain mission results, terminate, restore and remove agent missions, and show incoming or stored mission results. snow agent system missions are not dependent on the jeti or exodus jabber clients. any program or jabber client (or even telnet) capable of communicating with a jabber server may be used to deploy snow agent missions and receive mission results. in this section we describe how the snow agent system meets the requirements stated in section 3 above. the snow agent system may use several connection or organisation models. peer-to-peer model where the snow agent servers must be constantly connected and available for mission requests. each snow agent server is responsible for one gp clinic or patient clinic. specialised snow agent servers, supporting agent types that coordinate large agent missions, may also be used. communication between the servers is done using the standard "s2s" jabber component. in this model, one snow agent server serves as post office for a number of snow agent servers that poll the post office for messages and processing requests. a post office may serve all gps in a county or a single municipality. if the ehr installation needs to be protected by a firewall that blocks incoming connections from the network into the ehr system, the hierarchical model is needed. the snow agent system uses two additional jabber extensions to enable periodical connections to the centralised snow agent server (jabber + snow agent system extensions). in this organisation, the snow agent servers have two distinct roles, the "poller" and the "post office" roles. the "post office" server provides offline storage and relay of xmpp messages on behalf of the "poller" which is the ehr installation. the snow agent server behind the firewall, the poller, must take the initiative to connect to the post office (outgoing connection). when the connection is established, and authentication is performed, the poller will receive all messages and processing requests stored by the post office server while the poller was offline (see fig. 1 above.) the poller may also choose to stay constantly connected to the post office. these two components make it possible to treat a hierarchically organised network as a peerto-peer network. the hierarchical model enables gp clinics protected by firewalls to periodically receive computing requests (in the form of a mission specification). it also satisfies the norwegian data inspectorate's requirements for how small health institutions and gp clinics can connect to a common health network [61] . these requirements state that it should be impossible to open connections from the network into the ehr system protected by the firewall. the connection frequency of the poller component is configurable. if an outbreak is detected, an external authority may request the component to stay permanently connected. the poller component is also capable of connecting to several post office servers, which enables participation in several organisation models or "networks" simultaneously. the snow agent system is designed to be an automatic epidemiology and surveillance service. as long as healthcare workers continue to enter data into the ehr system, the system will be able to collect statistical data, given that the necessary consent for collecting the data is obtained. contents and collection frequency are however dependent on the configuration of the participating hosts. the system can only collect information about diseases explicitly supported by the gps. the reporting frequency depends on the connection model supported by the gp clinics. if all gp clinics are constantly connected (using the peer-to-peer or hierarchical model), then the system supports a high reporting frequency. if the gp clinics connect periodically, then the gp clinic with the lowest connection frequency decides the maximum reporting frequency if 100% coverage and up-to-date data is necessary. the snow agent system epidemiology service does however cache epidemiological data (mission results) locally and on the "post office" servers, which makes it possible to have a higher reporting frequency if the data "freshness" requirement can be relaxed. the computations performed by the mission agents within the snow agent system operate independently of its requester. the requester may however terminate or query the state of the mission agent at any time. the important issue is that the computation is independent of an online mission requester. a snow agent mission may run for days or weeks without the requester being available and online. a snow agent system server capable of connecting to and extracting data from an ehr system needs to operate under a security policy. the security policy states which agent applications may be allowed to run by which user. snow agent applications necessarily generate network traffic and the associated communication costs. such issues may also influence the policy for hosting snow agent applications. in terms of the hard requirements outlined above, it may be necessary to distribute new disease or syndrome definitions to be able to automatically count occurrences of possible, probable and confirmed cases of a new disease. such definitions may be built using archetypes as used in the openehr approach [62] . archetypes are explicit definitions of the structure and content of clinical data and of the constraints on the data. the archetypes may be built by an epidemiologist using an archetype editor. the archetype (disease or syndrome definition) may be read by a form generator component to build a graphical user interface for the input of data according to the structure, contents and constraints in the archetype. the screen representation may also be built manually, as in the proper system [63] , which is based on openemed [16] . the data produced by the gui may be validated by a validator component that uses the archetype definition as input, to ensure that the data stored by the input system is valid. to count cases of a new disease such as sars, specialised archetypes may be used to define concepts like "possible sars", "probable sars" and "confirmed sars" cases. the counting of cases may be done by evaluating stored data against these archetypes. the specialisation mechanisms in the openehr archetype definition language (adl) [64] allow for revision of a disease definition and may ensure that the definition of a new disease evolves without losing the cases defined by earlier definitions of the disease. the snow agent system and the openehr or the proper approaches fit perfectly together because the snow agent system is able to distribute a disease or syndrome definition on a global scale. the definition, represented by explicit archetypes, can be used to: (1) generate gui for data input, (2) validate user-supplied data, and (3) provide definitions or screening forms of concepts such as "possible disease x", "probable disease x" and "confirmed disease x". these definitions may be used by snow agents monitoring the diffusion of disease x in a geographical area. a first version of the system has been developed at the norwegian centre for telemedicine and at the distributed system technology centre (dstc) at the university of queensland, in brisbane, australia. the major parts of the system will be open source and available for download and use from the project web page at http://www.telemed.no/opensource/snow/ or http://mit.cs.uit.no/snow/. to learn more about the strengths, weaknesses and performance of a system based on propagation of program control, we performed a series of tests and experiments to see how fast a distributed epidemiology and surveillance system can provide useful information to the system's users. this question is interesting because we expect that a distributed system based on propagation of program control would need to have considerably slower response time than a centralised system. the reason for this is that such a system is more complex and may involve low-performance servers using low-bandwidth network connections. we also wanted to test the scalability of the mission controller component. this is necessary because mc is the component that handles most of the communication in the system, mainly because of its role as an intermediary between users, agents and agent daemons. the task for the epidemiology system was to plot the pertussis occurrences on an interactive map implemented in svg [43] . the pertussis occurrences were extracted from a set of simulated ehr databases. the ehr databases were generated from microbiology laboratory data as described in section 2.1 above. the map polygons were created from bitmaps made available in the norgeshelsa system [42] and converted to svg polygons. then the map polygons were inserted into a mysql database [44] in a convenient format for map generation and coded with geographical information. the interactive map was based on the vienna example provided by carto.net [65] . in our experiments, we wanted to determine the performance of the system in a close to reality configuration of the system. in norway, this means that we needed to use the hierarchical connection model with one post office server and several gp installations polling for processing requests. we used two mission agents, the "main-epidemio" mission agent, which ran on the post office server, collecting and merging epidemiological data from the participating hosts, and the "ehr-epidemio" agent, which computed on our gp clinics servers. the ehrepidemio mission agent queried the local ehr database for pertussis cases in the given time period, and reported the result to the main-epidemio agent. in our experimental setup, a computer located in tromsø, norway, acted as post office server for the gp clinic snow agent servers. the poller components in the five gp clinic servers were configured with a permanent connection to the post office. this configuration provides the fastest system response possible. (see section 2 above for description of the hardware and software used on the snow agent servers.) in our experiments we first tested the computation time for each participating host. in this case the hosts acted as both post offices and gp clinic servers. the databases and mission specification were identical for all hosts. we measured the amount of time used from receiving the mission or submission request until the mission result notification message was sent by the mission controller. our results are shown in table 2 . the table shows the average processing time from 10 execution runs on each target. in fig. 5 we have plotted the processing time for an identical mission specification on each of our target hosts (shown as points on the line), which shows that the processing time is dependent on processor speed. the time difference between the ehr agent and the main agent (line labelled diff) consists of processing time used to prepare the sub-mission specification and process sub-mission result (from ehr-epidemio agents) and producing the final svg file (113 kb in this case). in our next experiment, we wanted to see how the cost of administrating several target hosts in the sub-mission affected the total mission duration. to test this, we deployed the main-epidemio agent on our post office server. we deployed the ehr-epidemio mission to an increasing number of target hosts, starting with the slowest host first. again, we averaged the processing time over ten consecutive runs. we used the same database and mission specification as in experiment 1. this should make the computation needs for the sub fig. 5 -processing time as a function of processor speed. missions similar to experiment 1. however, the administration cost of the sub mission has increased because the mission specification was transferred over the internet to an increasing number of participating hosts. this is likely to cause an increased delay. in table 3 , we see that the increase of total processing time, compared to that in table 2 , stems from an increase in the ehr mission processing time measured by the mission controller on host 1. we believe the variations in processing time result from varying network conditions, as the participating hosts were spread around the globe. there is an increase in processing needs required by the mission controller to administrate the sub mission, but these processing costs are probably less than the variation in network conditions we experienced. some evidence for this may be found in the standard deviation columns for the processing time. the good news from the second experiment is that the total processing time seems to be minimally affected by increasing the geographical area covered by the epidemiological query. this result is according to our expectations. however, we have only tested the system on a minimal number of hosts. the processing time reported in table 3 shows a wait of about 25 s for getting updated information directly from the ehr systems. a 25-s wait may be a bit too long for busy gps. with the expectation of slow response times in mind, we designed the epidemiology service with a cache. use of the cache reduces the processing time to an average of 1.53 s (over 10 execution runs with s.d. 0.57) when interrogating the server in tromsø, norway from brisbane, australia. by relaxing the requirement for freshness of data, and scheduling periodic epidemiology missions to slow hosts, it seems to be possible to achieve an acceptable response time from the epidemiology service even if servers with 1997 technology (server 6) are used. in fig. 6 we see that the processing times involving six hosts are shorter than those involving five hosts. this effect is explained by the varying bandwidth conditions on the internet during the experiments, and represents a worst-case scenario as intended in the design of the experiment. in fig. 7 we see the uml sequence diagram for an agent mission involving one target ehr host. the diagram shows that the mission controller is the component, which handles most messages. the scalability of the system is therefore partly dependent on the ability of this component to scale when the number of target hosts in a spread mode mission grows. in the second experiment the "service provider host" and the "post office" was the same host, which means that a single mission controller handled both agent missions shown in the sequence diagram. to establish a rough estimate of the scalability of the snow agent server and the mission controller we computed the processing requirements and then performed two tests: (1) message throughput test for the snow agent server and, (2) mission controller and the snow agent server's ability to send outgoing messages. in norway, there are approximately 1800 gp clinics [66] . the distribution of gp clinics by county is shown in table 4 . a hypothetical deployment of the snow agent system could be configured with one snow agent post office server in each county, to which all gp clinics in that county connect. in this configuration, the snow agent post office servers need to be able to support 180-200 ehr system connections simultaneously. the mission specification used in the above experiment is about 1 k of data for the ehr epidemiology mission. for each additional host participating in the mission, the mission specification grows by about 100 b. the output requirement for the mission controller therefore has an n squared growth, or o n 2 in big-oh notation. the output requirement is shown in fig. 8 . the input requirement, shown in fig. 9 , is linear. to achieve this we had to modify the data format returned by the ehr agent running on the ehr hosts, about 2 kb for 15 postcodes for a period of 34 days. the system internal messages shown in fig. 7 , the "ok", "alive" and "finished" status messages require 179, 179 and 180 b, respectively. first, we tested the message throughput of the snow agent server. in this test we used server 2 (a pentium 4, 2.4 ghz processor) and 3 (a pentium iii 800 mhz processor) connected through a 100 mbit/s network. we measured the time taken to transmit messages (4 kb) from a client program on server 3 over the network to server 2 and back to a second client on server 3. because server 3 had a slower processor than server 2, we only observed a 30% load of the snow agent server running on server 2. the throughput in the test stabilised at 0.8 mb/s or 197 messages per second. this throughput may actually reflect the maximum performance of server 3, which both sent and received the messages. in the second test we measured how fast the mission controller and the jabber server were able to output messages. the mission controller on server 2 sent 1000 messages to a client on server 3. we measured the time from when the mission controller sent the message, until the client received the message (using synchronized clocks). because of limitations in the test software, the maximum message size was 4 kb. the test showed that the mission controller can send approximately 400 messages per second. however, a 4 kb mission specification will cover 31 hosts. to cover 200 hosts, the corresponding mission specification will be 20 k, and the total output would need to be 4 mb. in the test, the first 200 messages were sent by server 2 and received on server 3 after 0.6 s. four megabytes was transmitted and received after 1.6 s (output rate 2.5 mb/s). how fast a real system would be able to distribute the mission specifications depends on many parameters, which makes it difficult to predict a realistic estimate for a deployed system. however, our results show that it is realistic to support 200 ehr host per post office server. the theoretical bandwidth of a server connected to a 100 mbit/s network is an output of 12.5 mb/s, which corresponds to a mission with approximately 350 participating hosts. with utilisation of about 70% of the network adaptor, the server can support 295 hosts (8.6 mb mission controller output) in 1 s. for the scalability of the input, it would be beneficial to distribute the activation of the sub mission agents over time, because this will distribute the load on the network and the receiving server over time. the input is also dependent on how many cases of infected persons are identified in a single query. in a situation with many infected persons, the size of the mission result will increase towards the maximum of all postcodes covered by the gp clinic. the realistic capacity requirement of the system can therefore be estimated by counting the number of postcodes covered by the gp clinic and calculating the maximum throughput required for the system. the goal of syndromic and disease surveillance systems is to detect disease outbreaks as early as possible. however, syndromic surveillance systems has not yet proved its capacity to discover outbreaks earlier than traditional surveillance methods [24, 29, 38] . also, the reliance on the ability of "astute clinicians" to detect disease outbreaks in time has been questioned [32] . in the future, syndromic surveillance may be based on data provided by human sensors, as described bẙ arsand et al. [67] . the use of human sensors may enable earlier detection of disease outbreaks than what is possible today. a study of healthcare seeking behaviour in new york showed that in that healthcare context, only 8.8% of patients with ili symptoms attend an emergency department. about 50% of the patients consulted a physician (gp) [6] . because most patients in norway see a gp as their first point of contact with the healthcare system, we should provide the general practitioners with the necessary tools to discover local disease outbreaks. a syndromic surveillance system should therefore include data from the ehr system used by doctors to increase the coverage of the system. in norway this implies using ehr data from gps for surveillance purpose. this is feasible, because norway has close to 100% coverage of ehr systems among gps. most gp surgeries are now becoming connected to the national health network. the current disease surveillance system in norway (msis) [40] is based on reporting both from laboratories and from gps. the gps report the cases on pre-printed paper-based forms provided by the laboratory that performed the diseaseconfirming lab test. in addition to the mandatory reporting undertaken by all gps, approximately 200 gp clinics report weekly, during the winter, about the occurrence of a selection of diseases (including ili). achieving good coverage and high accuracy has been a problem in norway as in other countries [39, [68] [69] [70] [71] and is also limited by the question of cost. previous attempts to report the number of cases of selected diseases on pre-printed postcards proved too expensive to be sustainable. the norwegian institute of public health produces a weekly report (the msis report) containing the number of occurrences at a national and county level. the reporting frequency has been too slow and the geographic level too coarse-grained to be of any practical use in triggering general practitioners' awareness of disease outbreaks. however, the norwegian institute of public health is changing its reporting system to a web-based interface that supports a higher level of geographical granularity. the inclusion of data from a large number of ehr systems used by gps raises the need to rethink what technology might be appropriate for this purpose. current middleware approaches seem to be focused on the idea of a centralised, highly available and powerful server (or pool of servers), which utilises the request reply call semantics for distributing the computations between clients and servers or tiers. in contrast, our need is to have distributed autonomous computations on many ehr system servers, with potentially periodic connectivity and narrow bandwidth that cannot support the request reply semantics for security reasons. from our point of view, a peer-to-peer system among gps within the local communities seems to be the appropriate model on which to base a tool for discovering local disease outbreaks. such a system could also avoid the privacy issues associated with the use of ehr data. however, building a distributed and decentralised system for syndromic or disease surveillance raise new challenges not present in centralised systems such as rods [12] . distributed and decentralised surveillance systems need to address scalability, system maintenance and the need for two-way communication, as outlined in the requirements section above. we have described the requirements for such a system, and found that an ideal surveillance system is one that: (1) enables epidemiologists to define the disease to be monitored, including codes, symptom lists and constraints on biometrical measurements, (2) enables distribution of a disease definition to all systems participating in disease surveillance, (3) enables clinicians to start reporting cases in their normal working tool, the ehr system, immediately after receiving the disease definition, (4) immediately enables collection of statistical data after dissemination of a disease definition, and, (5) supports revision of the disease definition as more knowledge becomes available about the disease. this procedure needs to be achievable in a minimum of time. the human factor should be the main time consumer. the surveillance system needs to support flexible connection methods, enabling automatic, independent and dynamic reporting mechanisms. it must support asynchronous (and autonomous) computations and must provide adequate security mechanisms. syndromic and disease surveillance systems traditionally report events. an event is typically a positive laboratory test that confirms a diagnosis. in our system, it seems more natural to report the number of cases affected by the disease or syndrome classification used. this is natural because the ehr system is constantly updated with new electronic lab reports. it also provides the opportunity to review the development of cases retrospectively, because historical data is also available in the ehr system. the real-time outbreak and disease surveillance system (rods) [12] uses a centralised approach to perform syndromic surveillance. an interesting feature of rods is the use of a health system resident component (hsrc) that runs within the health institutions' firewall. this component has much more data available because it can operate on patient-identifiable clinical data. hsrc is able to link and use laboratory and radiology data in its inference and is able to achieve much higher specificity of patient categorization through access to more information. these benefits also apply to our system. the sars outbreak showed that a disease outbreak can easily cross borders. this represents a problem when an outbreak occurs on the border between two disease surveillance systems. the use of a common disease surveillance system can help to overcome such problems. the decentralised approach used in the snow agent system has the potential to solve the problem of cross-border disease surveillance because it removes the need to export patient related information to a centralised storage for processing. from our survey of available literature, we have not discovered any systems that are able to update the coding system or symptom list as a feature of the disease surveillance system. however, the centralised inference and detection system could easily be updated to use new diagnosis codes or syndrome definitions. in distributed and decentralised surveillance systems, this issue can be solved by enabling two-way communication, passing disease or syndrome definitions in one direction and data about the distribution of cases in the other direction. while rods is a monitoring system that feeds data from many sources into a centralised inference system, the snow agent system is more a "peer-to-peer" approach aimed at helping clinicians to discover ongoing outbreaks in the local community and aiding the diagnosis process on a daily basis. we believe such use of the system will have a positive effect on data quality in primary care. the snow agent system approach has the advantage that it is capable of accessing high quality data that precedes disease outbreak detection in a laboratory by many hours. the time advantage may sometimes be as much as a day, depending on geography and sample delivery routines. if the system counts the occurrences of prodromes or even just the rate of lab test requests, it can raise the awareness of local gps about an outbreak long before the laboratory sees enough cases to issue an alarm. the snow agent system also has the advantage that it can count cases that are diagnosed using epidemiological techniques, for instance in situations where a whole family is affected. such cases may never be confirmed by sending a sample to a laboratory. these two features are unique advantages using the peer-to-peer approach between gps. our performance test shows that the system will scale to national level in norway. the test shows that a normal desktop computer running the linux operating system can distribute a computing request to all gp clinics within any norwegian county within a reasonable time. the results from the epidemiology processes running on all ehr system installations can be merged, and may be depicted on an interactive map by an end user anywhere, using a normal web browser with a svg viewer component installed. if caching of computing results is performed, the response time may be reduced dramatically. by scheduling epidemiological queries at regular intervals, it is possible to achieve an acceptable level of responsiveness by the system. the asynchronous nature of the system, together with the autonomy of the mobile agents, makes it possible to compensate for the use of old servers with narrow bandwidth and periodic connectivity. we believe that the system can scale further, by adding more levels to the hierarchical organisation implemented by the system. the mission controller component is the critical component regarding the scalability of the system. the jabber server supports several server configurations that enable the server to handle many server connections. this feature makes it possible to scale up the processing capacity of a snow agent server dramatically. the snow agent system's epidemiology service is not yet sufficiently developed to qualify as an ideal surveillance system. however, it has some benefits over existing approaches. most important, it avoids the privacy problems associated with using ehr data. the two-way communication supported by the snow agent system's epidemiology service makes it possible to update the computer systems that feed the disease surveillance system with new kinds of data. the system is designed for everyday use by general practitioners who need epidemiological information in their daily work, which is beneficial for surveillance systems [3] . the system may also be used as a feeder system to a centralised outbreak detection system such as rods. in this paper we have presented the requirements that we propose distributed surveillance and epidemiology systems must meet when facing a local, national or a pandemic outbreak of a communicable disease. the surveillance systems should make use of the ehr systems used by gp clinics, because most patients consult their gp as the first point of contact with the health service when they get ill. from our point of view, such systems should be distributed and decentralised to provide doctors with an appropriate tool to discover local disease outbreaks, and to avoid the associated privacy issues. such a system must support two-way communication, to enable the participating ehr systems to incorporate new disease or syndrome definitions. the snow agent system makes it possible to collect epidemiological data directly from the ehr system in a geographical area based on a task specification. the core principle utilised to achieve this is propagation of program control, which lets computing processes or programs migrate between hosts in the same manner as a mobile-code-based system. in our approach, we do not move code, because of the security and authorisation problems inherent in mobile-code-based systems. instead, we propagate program control by moving a task specification and data between the hosts. we have tested our approach in realistic settings to learn about the efficiency and scalability of a system based on the propagation of program control principle. in our test, we placed servers around the globe. the results of our test show little or no performance penalty associated with covering a larger geographical area in an epidemiology query. the response time of the slowest server limits the response time of the epidemiological service. by applying a cache, we reduced the response time of the system from about 25-1.5 s, to produce an interactive map containing the epidemiological data. our scalability tests indicate that the system can easily scale to national level in norway. we would like to thank gudleif arnosen at the microbiology laboratory at the university hospital of north norway, for providing lab data for our system; ivar ness for donating time, bandwidth and processing cycles on his server in spain to the project; andreas neumann for letting us use the javascript source code from the vienna svg map example; and dstc, in particular dr. hoylen sue and the titanium project members, for all their help and technological insight which they have so willingly shared. we are also very grateful for the help from hilde pettersen and eva henriksen with proofreading of this manuscript. what was known before the study: the utility of syndromic surveillance systems, compared to traditional surveillance systems, has so far not been demonstrated [24, 29, 38] . syndromic surveillance systems utilise many different information sources and data from emergency departments is an important one. a telephone survey from new york showed that about 50% of patients with influenza like illness consult a physician, about 9% visit an emergency department and about 4% called a nurse or a health hotline [6] . however, very few of these systems utilise data from general practitioners, gpsurv from auckland, new zealand is one exception [72] . syndromic and disease surveillance systems should complement, rather than replace the "astute clinician" [26, 30] . protection of patient privacy is an important issue to address for disease surveillance systems [7] [8] [9] [10] [11] . what this study contributes: the propagation of program control principle provides a platform for mobile autonomous agent solutions that can be applied in the healthcare environment. this platform have been used to build an epidemiology service that may be expanded to include a distributed syndromic and/or disease surveillance system. distributed syndromic and/or disease surveillance systems can be implemented without being dependent of transferring patient identifiable data which eliminates the privacy issue. a surveillance system built using the propagation of program control principle can easily scale to national level in norway and is able to provide up to date data from the participating source systems within 25 s, even if server technology from 1997 is used. distributed syndromic and/or disease surveillance systems should provide two-way communication to enable distribution and update of syndrome and disease definitions for use by the ehr systems contributing data. investigating public health emergency response information system initiatives in china setting standards for improved syndromic surveillance linking better surveillance to better outcomes value of icd-9 coded chief complaints for detection of epidemics roundtable on bioterrorism detection: information system-based surveillance how many illnesses does one emergency department visit represent? using a population-based telephone survey to estimate the syndromic multiplier review of the 2003 national syndromic surveillance conference-lessons learned and questions to be answered health information privacy and syndromic surveillance systems information system architectures for syndromic surveillance biosense-a national initiative for early detection and quantification of public health emergencies removing a barrier to computer-based outbreak and disease surveillance-the rods open source project technical description of rods: a real-time public health surveillance system sars surveillance project-internet-enabled multiregion surveillance for rapidly emerging disease building dual-or multiple-use infrastructures is the task at hand for state and local health departments results from the fielding of the bio-surveillance analysis, feedback, evaluation and response (b-safer) system in albuquerque an international collaboratory based on virtual patient records on site: past and future emergency response information systems prerequisites for developing and deploying a system for ubiquitous access to patient information what is syndromic surveillance? national bioterrorism syndromic surveillance demonstration program if syndromic surveillance is the answer, what is the question? knowledge-based bioterrorism surveillance on the threshold information technology in the age of emergency public health response. the framework for an integrated disease surveillance system for rapid detection, tracking, and managing of public health threats the economic impact of a bioterrorist attack: are prevention and postattack intervention programs justifiable? emerg syndromic surveillance and bioterrorism-related epidemics disease outbreak detection system using syndromic data in the greater washington dc area using automated medical records for rapid identification of illness syndromes (syndromic surveillance): the example of lower respiratory infection syndromic surveillance syndromic surveillance systems hospital admissions syndromic surveillance-connecticut syndromic surveillance at hospital emergency departments-southeastern virginia the informatics response in disaster, terrorism, and war three years of emergency department gastrointestinal syndromic surveillance in new york city: what have we found? a system for electronic disease reporting and management. determining the extent/spread of problems and minimizing consequences through rapid reporting and dissemination of critical information the norwegian institute of public health. surveillance of communicable diseases in norway the virtual library secretary-a user model based software agent the norwegian institute of public health scalable vector graphics (svg) specification w3c recommendation bogomips mini-howto importing clinical data into electronic health records: lessons learnt from the first australian gehr trials seda: sentrale data fra allmennlegetjenesten -teknisk dokumentasjon sentrale data fra allmennlegetjenesten. sluttrapport fra pilotprosjektet an introduction to the tacoma distributed system version 1.0 mobile agents implementing remote procedure calls security in the ajanta mobile agent system mole concepts of a mobile agent system mobile agents: are they a good idea? jabber software foundation. jabber technology overview exodus -escape from proprietary im systen the data inspectorate. veiledning i informasjonssikkerhet for kommuner og fylker ehr design principles archetypes: the proper way archetype definition language carto:net using blood glucose data as an indicator for epidemic disease outbreaks timeliness of case reporting in the swedish statutory surveillance of communicable diseases notification of infectious diseases by general practitioners: a quantitative and qualitative study mandatory notification of communicable diseases: what physicians think reasons for under-reporting of notifiable conditions evaluation of an electronic general-practitioner-based syndromic surveillance system-auckland this work has been funded by the norwegian research council, grant no. 142162/320 and by the norwegian centre for telemedicine. many individuals have contributed to its realisation, and their help is very much appreciated. in particular, key: cord-314275-twjaq5do authors: diwekar, u.; amekudzi-kennedy, a.; bakshi, b.; baumgartner, r.; boumans, r.; burger, p.; cabezas, h.; egler, m.; farley, j.; fath, b.; gleason, t.; huang, y.; karunanithi, a.; khanna, v.; mangan, a.; mayer, a.l.; mukherjee, r.; mullally, g.; rico-ramirez, v.; shonnard, d.; svanström, m.; theis, t. title: a perspective on the role of uncertainty in sustainability science and engineering date: 2020-09-09 journal: resour conserv recycl doi: 10.1016/j.resconrec.2020.105140 sha: doc_id: 314275 cord_uid: twjaq5do the trans-atlantic research and development interchange on sustainability workshop (tardis) is a meeting on scientific topics related to sustainability. the 2019 workshop theme was "on the role of uncertainty in managing the earth for global sustainability." this paper presents the perspectives on this topic derived from talks and discussions at the 2019 tardis workshop. there are four kinds of uncertainties encountered in sustainability ranging from clear enough futures to true surprises. the current state-of-the-art in assessing and mitigating these uncertainties is discussed. the demands of humans on the earth have grown considerably with the human population and the increase in per capita consumption of all kinds (fuels and energy, food, water, minerals, etc.). consider that while the earth and its energy budget from the sun have remained roughly constant over the past 4000 years, the human population has risen from approximately 7 million around 4000 bce to more than 7 billion (worldmeters, 2020) at present. the value of the global economic activity, as measured by the world gross product, a proxy for consumption, had risen from approximately us$1 billion around 4000 bce to about us$78 trillion (wikipedia 2020; worldbank 2015) by 2014, both in 1990 international us dollars. about 94% of the population increase and almost 100% of the rise in the world gross product have happened since 1500 bce. these conditions create an unprecedented planet-wide situation where humanity has the imperative of managing the earth within very tight constraints with little margin for error. this requires that humans be able to: (1) accurately assess the current condition of the earth, (2) forecast future trends as accurately as possible, and (3) develop effective management strategies. uncertainty poses significant difficulties for all three efforts. the concept of sustainability is closely related to that of sustainable development, but the two are different. sustainability is about ensuring that the earth can meet the material and energy needs to support complex systems including humanity over the long term. this has economic dimensions because the rate of resource consumption heavily depends on economics, and it has social dimensions because the ultimate goal is to support human society. sustainable development provides a vision of how human society could develop sustainably, and it adds a stronger focus on social dimensions such as intergenerational equity. sustainability is, therefore, a basic concept oriented to making the survival of civilized human existence on earth feasible. the focus of tardis is mostly on sustainability. however, we did not attempt to create our own formal definition of sustainability. we merely used the aforementioned framework as a working definition of sustainability to focus on discussions. the trans-atlantic research and development interchange on sustainability (tardis) workshop is a three-day study session on scientific topics related to sustainability, stimulated by presentations, and discussion. the workshop involves participants from the united states, europe, and other parts of the world in the multidisciplinary field of sustainability. the 2019 tardis group included the participation of experts from engineering, mathematical sciences, physics, ecology, economics, decision sciences, and political science fields. the theme of the 2019 workshop was "on the role of uncertainty in managing the earth for global sustainability." this paper presents the summary perspectives on this topic derived from talks and discussions at the 2019 tardis. sustainability and uncertainty: the problems involved in understanding, modeling, assessing, and managing for sustainability, fall under what rittel and weber (1973) call "wicked problems". for example, a recent study by the world bank (damania et al., 2019; steffen et al., 2015) that focuses on threats to global water quality concludes that the uncontrolled release of reactive nitrogen (nr) into rivers, lakes, and estuaries presents us with the "largest global externality" that threatens human health and ecosystem stability at a scale that is not fully recognized by the governments of the world. as often happens with such "wicked" problems, solutions are confounded by interconnected positive and negative impacts that are difficult or impossible to unravel. for instance, reactive nitrogen in the environment is intertwined with several of the un sustainable development goals: zero hunger (2), good health and well-being (3), clean water and sanitation (6), affordable and clean energy (7), industry, innovation, and infrastructure (9), sustainable cities and communities (11), responsible consumption and production (12), climate action (13), life below water (14), and life on land (15). actions taken to address the role of nr in any one of these goals may exacerbate nrrelated impacts in the others. multiple stakeholders and their conflicting perspectives also generate wicked sustainability-related problems. one of the major challenges in the sustainability system, which results in wicked problems is the problem of uncertainties. some examples of uncertainties in sustainability include definition and quantification of various objectives, impact assessment methods and models, forecasting future, and unexpected events. in a broad sense, uncertainty may be defined simply as limited knowledge about future, past, or current events (walker et al., 2003) . fig. 1 shows four different quadrants of information leading to uncertainty. this diagram is the focal point in business strategy and planning literature (marshall et al., 2019) . sustainability systems and issues extend to all four quadrants of information, whether the problem is of community sustainability, manufacturing sustainability, sustainable economics, regional or global sustainability, as described in the various sections below. the paper is divided into the following sections. section 2 presents types of uncertainties followed by a section on systemic risks, precautionary principle, and normative risks. section 4 talks about the effect of uncertainties on the environmental policy, followed by a section related to sustainable economics and uncertainty. section 6 is devoted to community sustainability and uncertainty. sustainable manufacturing and sustainability assessment sections follow that. decision making under deep uncertainty is section 9, followed by global sustainability and stochastic processes. section 11 is devoted to the communication of uncertainties in sustainability education. the summary section is last. the literature related to uncertainty defines two kinds of uncertainties, namely epistemic uncertainty and aleatory uncertainty. epistemic uncertainty is a lack of knowledge on underlying fundamentals and characterized by alternative models. aleatory uncertainty refers to the inherent uncertainty due to the probabilistic variability. these uncertainties are characterized by a probability distribution. sustainability problems are difficult because they involve deep uncertainties. a recent open book on decision making under deep uncertainty divides uncertainties into four types. • level 1 uncertainty represents situations in which one admits that one is not absolutely certain, but one does not see the need for or is not able to measure the degree of uncertainty in any explicit way (hillier and lieberman 2001) . these are generally short-term uncertainties (clear enough futures) where historical data can be used to predict the future. sensitivity analysis can be used to address these types of uncertainties. • level 2 uncertainty can be described in terms of probability distributions, probability bounds, interval methods, or fuzzy information. these uncertainties are useful where system models are reasonably closer to the real world. most of the manufacturing uncertainties fall into these categories. • level 1 and 2 uncertainties are observed for known unknowns. according to knight (1921) , levels 1 and 2 uncertainties come under the category of "risks" and are often associated with optimality, unlike levels 3 & 4, which involve "robustness." • level 3 uncertainties involve situations in which there are a limited set of plausible futures, system models, outcomes, or weights, and types of information: known-knowns are known to exist, and information is available, known-unknowns are known to exist, but no information is available, unknown-knowns are not included in the analysis but the information is possible if they did, and unknown-unknowns are not known to exist, and no information is available if they did (marshall et al., 2019) . probabilities cannot be assigned to them-so the tools of neither level 1 nor level 2 are appropriate. in these cases, traditional scenario analysis is usually used. long-term assessment of sustainability (also comes under level 4 uncertainty) comes under this category. • level 4 uncertainty represents the deepest level of recognized uncertainty. this is called deep uncertainty. there are two types of deep uncertainties: the uncertainty where there are many possible futures and outcomes, and there are unknown futures and unknown outcomes. deep uncertainties are related to decreasing confidence in our ability to anticipate correctly future technological, economic, and social developments, future changes in the system we are trying to improve, or the multiplicity and time-varying preferences of stakeholders regarding the system's outcomes, or handling low probability but high impact events (black swans (taleb 2010) ) like a natural disaster, a pandemic, a financial crisis, a terrorist attack, or truly novel events for which there is no historical experience. (unknown knowns and unknown unknowns, true surprises). systemic risk, ontological uncertainty, and time-dependent uncertainties come under this category of level 4 uncertainty. dealing with these kinds of uncertainties involve monitoring the present and adaptations over time. the precautionary principle and options theory are two widely used methods to deal with these uncertainties. uncertainty, as well as the precautionary principle, take a prominent place within the scientific debate on sustainable development in general and on managing systemic risks specifically (renn 2008; becker 2014) . against the backdrop of the many meanings of systemic risks within the scientific discourse, we argue that the precautionary principle is only a negative or threshold criterion and that it is worthwhile to add positive normative criteria to inform management of systemic risks. however, we introduce the concept of normative uncertainty adding up to epistemic uncertainty in dealing with systemic risk. systemic risks are a relatively new type of risk concept (renn, 2016) . according to predominant understanding, the occurrence of an event would not simply lead to harm but to a breakdown of the system (kaufman and scott 2003; smaga, 2014) . in other words, the system would go beyond its resilience level and would no longer be able to execute its functions. with some probability the "event" climate change can, for example, lead to a breakdown of the earth's life-supporting functions; or a global financial crisis can be accompanied by major banks going bankrupt which in turn would severely undermine their functions supporting the economy potentially leading to a breakdown of the economy. such risks display new characteristics in comparison to traditional risks such as droughts, hailstorms, a car traveling, or smoking. whereas the latter are computable, systemic risks are not. it is neither possible to estimate the probability of occurrence of the triggering events, nor is it possible to assess the related harm (and to bring that into a cost-benefit analysis). these come under level 3 and level 4 types of uncertainties. risk assessments are always accompanied by epistemic uncertainties. the results of measuring frequencies of events or modeling the complexity of systems together with assessing potential harm are normally given within ranges expressing possible limitations. however, uncertainties related to systemic risks do not express a given range of likelihood. they represent the mode "we do not know," due to its character as not being computable. we can, for example, not give a reasonable likelihood estimation for whether the financial crisis in 2008 and the thread of big banks potentially collapsing would have led to a breakdown of the economic system. the issues are just too complex to come up with estimations. the possibility that it could and would bring about a huge amount of harm by a breakdown of the economy was seen as sufficiently reasonable to take action. the motivating principle, according to actions, is known as the precautionary principle (gardiner 2006) . it does not claim that you should not take risks. it rather says (in case of the financial crisis in 2008): we do not know whether the collapse of system-relevant banks will lead to a breakdown of the economic system. however, the level of risk resulting from such a breakdown is sufficient to intervene and not to let those banks go bankrupt. the precautionary principle is an action-guiding principle where the occurrence of events could have the effect that a threshold (or resilience) level of a major system supporting societal functions will trespass. however, the precautionary principle only indicates not to take a specific path. in contrast, it cannot inform positively decision making for "doing x," e.g., deciding that saving exposed banks whose activities brought about the crisis is the best decision to take. additional criteria are needed in order to make a positive decision. according to relevant theories in political economy and philosophy (renn 2009; stirling 2007; kaul and mendoza 2003) , public decisions should (ideally) primarily be motivated by taking public or common goods as well as the overall goal of human well-being into account. hence, the negatively oriented precautionary principle should be accompanied by a positive criterion on public goods and human wellbeing. together they build a sound basis for decision making regarding systemic risks, nowadays prominently expressed by the agenda 2030 and its 17 sustainable development goals. when tackling systemic risks from a perspective of societal goals, alternative options to deal with, as well as different outcomes of the crisis, come into the picture. specifically, it substantiates and rationalizes so-called 'pro-active intervention strategies' to systemic risks, such as adaptation and transformation (irgc 2018), scenario discovery approach (bryant and lampert, 2010) , etc. however, although such a positive approach offers a productive evaluative basis for assessing first what systemic risks are and what goods have to be taken into account, in strategies for dealing with (avoiding) systemic risks, this opens up a broad space of interpretation. this happens because there is not one single understanding of what constitutes public goods or belongs to human well-being. this points to another field of uncertainties, i.e., normative uncertainty (e.g., mazouz 2003) . it is by far not the case that anything goes when talking about justice or sustainable development. philosophy, social sciences, and economics provide a number of well-established approaches to frame justice (cf. miller 2017). we have a variety of options producing a range of normative claims and assessments (cf. burger 2018 for types of sustainability conceptions). as we have to learn to deal with epistemic uncertainties, the sciences additionally need to improve their understanding of normative uncertainties when dealing with systemic risks like climate change. lastly, in this overview of uncertainty types, we consider the role of ontological uncertainty (nielsen and ulanowicz 2011) , which refers to the emergence of new unpredictable or un-"pre-statable" types (kauffman 2019). any system that exhibits requisite complexity will have novel behaviors from the interactions of the agents within the system. for example, simple combinatorics tells us that the interactions of 80 distinguishable components will offer approximately 80! =~7 × 10, 118 possible configurations. yet, given the age of the universe, estimates are that there have only been~10, 110 quantum level events (elsasser and staude 1978) . in other words, there will be surprise events and other events that will never materialize. this level of uncertainty, which challenges the old school determinism, has manifest itself in many 20th century theories as given in table 1 (examples are taken from nielsen et al., 2020, p. 30) . the realization of ontic uncertainty is not inherently negatively consequential, but rather has benefits as it implies ontic openness that potentially brings new opportunities to any complex adaptive system, including humans striving for sustainable development. of course, this also reinforces the importance of applying the precautionary principle as prudent guidance into an unknown future. as in most areas of policy, environmental and sustainability policy must often contend with scientific uncertainty when prescribing actions and identifying targets (bradshaw and borchers 2000) . depending on the problem, scientific uncertainties in policymaking can be of any one of the four types described earlier. policymakers often grant implementing agencies considerable discretion to modify their actions in response to new information or understandings, but there are limits to how much discretion can be accommodated before policies must be rewritten (woods and morey 2008; lind-riehl et al., 2016) . at some point, when the language and concepts used in laws, regulations, and policies no longer relate to the state of knowledge in the field, these prescriptions must be amended. the us endangered species act or esa is one such example of science moving beyond the legal terminology. as implied by its title, the esa is a law that protects species from extinction. enacted in 1973, the esa is one of the most stringent environmental laws in the world, saving thousands of species from extinction and enabling the recovery of dozens of well-known and unique species (suckling et al., 2016) . when faced with a species in decline, the two enforcement agencies, the us fish and wildlife service and the national marine fisheries service, must first determine whether the species meets the esa's criteria for being listable. one of these criteria includes the distinctiveness of that species, subspecies, or population from other more common species. advancements in genetic technology and taxonomic science have complicated the boundaries between species, making it more difficult to use the esa to protect biodiversity from threats faced by land-use change, pollution, invasive species, and climate change (ritchie et al., 2018 ). an increasing number of lawsuits and petitions call on wildlife agencies to delist or downlist species from the esa, based on new information that calls the species' distinctiveness into question. one such example is the coastal california gnatcatcher (polioptila californica), a small, nonmigratory songbird that inhabits the rapidly urbanizing region of southern california and northern baja california peninsula in mexico (mayer, forthcoming) . the protection of this northern-most subspecies of california gnatcatcher has been constantly challenged by development industries in california, mainly over the scientific uncertainty of its distinctiveness from other subspecies. although ecological and morphological evidence continues to indicate that the coastal california gnatcatcher is substantially different from california gnatcatchers further south in mexico, genetic evidence has been more equivocal and contentious (mccormack and maley 2015; patten 2015; zink et al., 2013 zink et al., , 2016 . difficulties in determining the clarity of boundaries between species and subspecies are not unique to the california gnatcatcher, but rather are inherent in the concept of a species (zachos 2018) . species are continuously evolving over time and can and do hybridize with closely related species as they evolve, blurring their genetic and morphological distinctiveness. the difficulty of species delineation, and ongoing disagreements over concepts and methods among scientists, translates into difficulty for policy decision-making, even with considerable discretion for implementing agencies. insight into the roles of ambiguity, uncertainty, and discretion in the interaction between the scientific process and policy process can improve policy implementation and outcomes (doremus 1997; bradshaw and borchers 2000; cairney et al., 2016; wilhere 2017) . perhaps the first work applying financial decision-making models to environmental issues relevant to sustainability was by weisbrod (1964) . weisbrod considered the case of the sequoia national park, for which demand is infrequent and uncertain (i.e., many people consider going, but few actually go). he imagines a private owner who is able to price discriminate in charging entrance fees and thus capture the entire consumer surplus for himself. even under these conditions, however, entrance fees do not cover the full costs of running the park. ignoring all non-recreation benefits of the ecosystem in question, a rational owner might choose to irreversibly convert the area to other more profitable uses (e.g., chop down the forests and build condominiums). however, when demand is infrequent and uncertain, there is a certain value people give to retaining the option to visit the park even if they never do so. this value is a pure public good: as long as the park is there, the option itself is non-excludable and non-rival, and there are no non-coercive mechanisms for charging consumers for this option value. if enough people benefit from this option value, it would be socially efficient to keep the park open even if privately inefficient. the solution may be public ownership or subsidies. cicchetti and freeman (1971) have shown that weisbrod's option value will be positive when there is uncertainty in either demand or supply, and it has the same effect on decisions as risk aversion. arrow and fischer (1974) examined a slightly different situation, in which there is an opportunity to irreversibly convert an ecosystem to some other use, such as damming a river or converting a forest to housing developments. they look at the situation in which decisions this period change the expected values of conversion and/or non-conversion in the next period. for example, society might learn about a valuable service provided by the ecosystem, or a new medicinal plant, if it is left unconverted. they concluded that these unknown values created a quasi-option value that again increases the value of avoiding irreversible outcomes, acting the same as risk aversion. conrad (1980) later showed that the quasi-option value is equivalent to the expected value of information, while the option value is equivalent to the expected value of perfect information. however, in financial decision making, and in the models described above, unknown outcomes are of a very restricted variety: possible outcomes are known, as are the probabilities of each. knight (1921) referred to this type of unknown outcome as a risk. in the highly complex ecological-economic systems that humans want to sustain, it is often impossible to make objective assessments of the probabilities of different outcomes, and therefore impossible to model expected values. in many cases, we cannot even guess at possible outcomes. for example, human activities change ecosystems in ways that generate new evolutionary pressures on the species within them. humans react to their self-created changes through cultural evolution, including the development of new technologies. both evolution and technological advances can lead to entirely unpredictable outcomes, in which not even possible outcomes can be objectively assessed. this type of unknown outcome has been referred to as surprise (schneider et al., 1998; casti, 1994; faber et al., 1992; kates, 1985) novelty (faber and proops,1998) and ignorance (faber et al., 1992; daly and farley, 2003) . how to deal with ignorance and uncertainty in modeling sustainability is a serious challenge. this is within the area of deep uncertainty. in reality, due to the aforementioned uncertainties, we suffer table 1 examples of uncertainty and indeterminacy in the modern understanding of nature (nielsen et al., 2020) . creative destruction as part of the cyclic process jørgensen heisenberg extended to ecosystems kauffman continuous evolution into the possible adjacent states u. diwekar, et al. resources, conservation & recycling 164 (2021) 105140 profound ignorance about the most serious problems we currently face in our rapidly evolving system. it is a characteristic of complex systems that if we ramp up the value of a critical parameter beyond some critical point, the system can "flip" into an alternate regime about which we know very little and from which it can be difficult or impossible to return to the previous state (hughes et al., 2013; lenton et al., 2008; lenton and williams, 2013; pearce, 2007) . we are currently ramping up the value of innumerable critical parameters, both ecological and economic, simultaneously. climate change, biodiversity loss, nutrient flows, and land-use change are a few of the most egregious ecological examples (rockstrom et al., 2009; steffen et al., 2015) . financial systems regularly experience explosive growth followed by crisis. stocks are now held on average for some 30 s, down from 8 years in the 1960s (hudson 2011) . over half the value of stocks in the us are owned by the richest 1% of the population (wigglesworth 2020). the s&p 500 increased by 260% from 2009 to 2019, vs. a 35% increase in gdp, and by more than 12 times as much as gdp in 2019, contributing to an explosive increase in inequality. since over the counter foreign exchange trading first appeared in the early 1980s, transactions have soared to $6.6 trillion per day nearly 30 times greater than global gdp, with purchases also held for only seconds on average (hudson 2011) . we are often uncertain precisely which parameters are critical, can only guess when they will reach critical thresholds, and remain profoundly ignorant about new regimes into which our system may flip. as has been eloquently (taleb 2010) pointed out, the most consequential events in history have been black swans: extremely rare events with very high impacts that could not have been predicted ahead of time. the previous experience provides little to no guidance in predicting novel events. another major source of uncertainty is the reflexive nature of social systems and social science (soros, 2013) . societies develop beliefs and norms that affect their members' behavior. for example, social scientists develop theories to explain how people behave and how social systems function, but it is widely believed, the theories themselves can affect human behavior. theories can be self-fulfilling or self-negating. for example, even prior to the covid-19 crisis, many pundits were predicting a recession. if producers believe predictions, they will stop investing in new productive capacity, reducing the creation of new jobs if consumers believe that as well, they will start saving their money, reducing aggregate demand, leading producers to lay off workers, who respond by saving even more money in a positive feedback loop. these are the actions that can trigger a recession. in contrast, before the financial crisis that exploded in 2007, ben bernanke and other influential economists claimed we had achieved a 'great moderation' and no longer had to fear financial crisis (bernanke 2004; stock and watson 2002) . this very likely stimulated excessive risk-taking, and that precipitated the crisis, as predicted by minsky's financial instability hypothesis (minsky 1977) . several community environmental quality problems fall under wicked problems because depending on the stakes one has, the problem formulation -different characterizations of the problem that emerge when viewed from the perspectives of different stakeholders -can be very different: industry stakeholders whose primary objective is usually economic optimization within environmental regulation standards; communities that seek to balance socioeconomic welfare through access to jobs while reducing associated environmental contaminants -if they are dependent on jobs from the contaminating industries; more affluent communities that would rather have polluting industries removed or distanced from their communities to optimize environmental quality and public health, and government agencies whose primary objective is to administer regulations at the nexus of these competing demands. the triple bottom line does come into play here, but the relative importance of the different types of capital pertaining to the problem situation can be very different -sometimes reflected in long drawn out battles over community environmental contamination issues (brackett, 2019) . these multiple objectives bring in uncertainties as some objectives are well defined and can be quantified reasonably as others cannot be quantified accurately. use of multi-objective programming (mop) under uncertainty (fu et al., 2001; diwekar, 2012) or many objective robust decision making (mordm) (kasprzyk et al., 2013; singh et al., 2015) methods are useful in this context. the methods are suitable for level 2 as well as level 3 & 4 types of uncertainties. environmental contamination in local communities is a pervasive problem that affects sustainable development and public health at the local level, judging from the news and social media reporting. the popular media has intermittently reported on environmental contamination in various communities around the country where residents have been unaware of such contamination and its impact on their health for extended periods of time. examples include ethylene oxide emissions in the states of georgia and illinois, and the flint water crisis in michigan (goodman and miller, 2019; lutz, 2019; 11 alive news, 2019; abc-7 chicago, 2019; scalia, 2019; bowles, 2018) . communities that have been unknowingly exposed to environmental contaminants for relatively extended periods tend to develop a distrust of the public officials responsible for environmental quality (goodman and miller, 2019; lutz, 2019; 11 alive news, 2019; abc-7 chicago, 2019) . notable uncertainty exists concerning community exposure to environmental contaminants in the air, water, and soil, and there are several synthetic compounds in use today in various consumer products that have not been reviewed for safety by any government agency (asce, 2006) . therefore, uncertainties abound with local environmental contamination, both known unknowns and unknown unknowns. the emergence of lower-cost environmental quality sensors offers a growing opportunity for communities to engage in crowd science, partnering with universities and government officials, to develop a better understanding of their local environmental risks and begin to monitor and contribute to managing the most critical of them. how can emerging sensor technologies be adapted to monitor various critical contaminants in a community's risk profile in a cost-effective manner, and what is the feasibility and cost of large-scale deployments? the news media report that thousands of people around the country are beginning to measure air quality as climate change reports become increasingly dire, wildfires tear across the american west, and trust in the federal government's air quality oversight fades (un, 2015) . the purpleair map (2020), for example, is available to the public via the internet, and air quality monitoring network built on a new generation of "internet of things" sensors being used by individuals and communities. developed on the concern that local air contamination was higher than indicated, purpleair monitors are now being used by several individuals and communities around the world with air particulate data being fed in real-time over wi-fi to a public visual display online. coupling standard public domain reporting with organized crowd science efforts to monitor critical environmental contaminants, such technology can allow communities to initiate a new level of data-driven engagement with public officials. this can go a long way in fostering bottom-up approaches to balance the top-down approaches to environmental quality management at the local level. wicked problems that have persisted across years, decades, and centuries may have possible solution pathways that begin with the education of local communities to understand the profile of risks they are contending within their communities. it can then continue with communities partnering with universities, government officials, and other stakeholders to address such long-standing problems -leveraging emerging technologies and gathering relevant data to engage in smart governance. a recent case study in colorado presents a stake-holder-focused approach to local government sustainability planning to explicitly link u. diwekar, et al. resources, conservation & recycling 164 (2021) 105140 bottom-up and top-down approaches to environmental management (hopton et al., 2010; dubinsky and karunanithi, 2017a, b) . the deliberative process was intertwined with different types of uncertainties. during the stakeholder engagement phase, social values, competing interests, and points of view, trade-offs and choices would constitute different forms of uncertainties. the participation of community members introduced local context and local knowledge, which provided clear roadmaps for the construction of robust, meaningful, and realistic scenarios. it also facilitated the knowledge transfer process and ensured that locally defined sustainability benchmarks would be emphasized. it is highly recommended that the stakeholder-centric methodology be used in any future local governance sustainability projects. in a second case study, a multidisciplinary team evaluated a suite of six metrics of urban performance (ecological footprint, green net product, net energy, human wellbeing index, fisher information, emergy) linked to an agent-based simulation model to explore current conditions and trends in greater chicago. it was found as cronin (1991) suggested, that chicago and other urban systems are not static but constantly evolving. this poses the question of evolving uncertainties. the question for sustainability then is not whether an urban system (or any system) is sustainable at a given point in time, but rather whether that system has the capability and capacity to successfully evolve and adapt to the conditions that it will encounter in the future. manufacturing sustainability has become a global issue, especially in developed and fast-developing countries. an improved corporate sustainability performance does not automatically lead to an improved sustainability performance of the systems within which a company is embedded. a comprehensive assessment framework is needed to analyze whether an improved corporate sustainability performance can positively contribute to sustainable development on a global level. this includes first-order and second-order performance measurements (baumgartner, r. j., & korhonen, j., 2010; baumgartner and rauter, 2017) . the former assesses the direct sustainability impacts and usually focuses on narrow issues of efficiency, while second-order sustainability performance focuses on systemic effectiveness and covers sustainability impacts on society and nature in total, i.e., the entire system. this problem is usually posed as a multi-objective optimization problem (moradi and huang 2016 , liu and huang 2012 , 2013 , z. 2015 . however, there are not only uncertainties associated with each performance measure, but deciding the weights for different objectives presents significant uncertainties in solutions. there are both the aleatory and epistemic uncertainties that appear in various types of manufacturing sustainability problems. the uncertainties in secondorder performance measures are much higher than in first-order measures, and this needs to be balanced. this is because of the long-term nature of the performance objectives. this is obvious in the example of plastics (plastics europe, 2017; plastics europe 2020; geyer et al., 2017; shonnard et al., 2019; benavides et al., 2017; gracida, 2019; closed loop partners, 2019), and environmental impacts by the disposal of plastic from municipal solid waste. landfilling and leakage to the environment (as opposed to recycling or re-use) are thought to be the largest end-of-life fates for plastics that are used in packaging, which is the most common use of plastics. plastic packaging exhibits the shortest lifetime between its production and appearing at its end-of-life as waste in need of management. a circular economy for these polymeric materials, rather than a linear economy, is proposed as a solution to the responsible management of waste plastics. however, large-scale implementation of a circular economy for plastics is not well-understood with respect to economic costs and benefits, environmental impacts, and societal effects relative to business-as-usual linear management. a systems analysis for sustainability for a circular economy of plastics is difficult because of several problems related to uncertainties related to the definition of goals and knowledge gaps on current and future recycling processes. sustainable manufacturing often uses life cycle analysis for sustainability assessment. given the importance of life cycle assessment in sustainability, we have devoted a special section below on life cycle assessment and uncertainty. increasing awareness of environmental degradation and resource depletion has led to the incorporation of environmental sustainability considerations in engineering design and analysis. one such widely popular technique is the environmental life cycle assessment (graedel and allenby, 2019) , which aims to model and quantify the environmental impacts of products and systems over their entire life cycle (rebitzer et al., 2004) . by assessing the entire life cycle of the system of interest, one can evaluate the environmental impact of released emissions or of materials and energy used during various stages (material extraction, manufacturing, usage, disposal) of the product or system of interest. lca methods tend to focus heavily on emissions and their impact and resource use. it is also important to include ecosystem goods and services as well as societal benefits and impacts in lca to move towards sustainable development. however, ecosystem services and societal implications are either not accounted for or not well represented in most life cycle methods. some recent lca approaches have been developed for the purpose of assessing the role of ecosystem services in process and input-output life cycles (eco-lca) (zhang et al., 2010) . however, these have been limited in their scope, and placeholders exist for many ecosystem goods and serviced. these placeholders in lca (known unknowns) can lead to wrong policies. in "frames in toxicity controversy," arnold tucker (tucker, 1999) presents a case study where researchers had to advise the dutch government about the choice of incineration technology. however, lca at that time lacked fate modeling and could not provide any certainty about the choice. the researchers used the precautionary principle and asked for more research. however, the politicians did not have time or money to carry out the research, and it resulted in the wrong policy. these placeholders represent known unknowns and are part of level 3 uncertainties. as an example, consider the critical ecosystem service of pollination provided by insects. a lack of this service would be a significant detriment to not only ecosystem biodiversity and function, but also to various industrial sectors, agriculture, and the world economy (potts et al., 2016; klein et al., 2007) . in the united states, insectmediated pollination of crops accounts for between us$ 14-23 billion of pollination-dependent crop production alone, making this ecosystem service a highly valuable asset to the nutritional and economic welfare of the agricultural sector (chopra et al., 2015) . in addition, there are upstream and downstream industry sectors that rely upon pollinationdependent crop production (fertilizers, pesticides) as well as nonagricultural industrial sectors (pharmaceutical, fuel) that share linkages with crop production and agricultural sectors, leading to intricate indirect dependence upon pollination service mediated by insects. although this example is specific to insect pollination service, there are numerous other ecosystem goods and services that are being exploited at unsustainable rates and facing severe degradation (carpenter et al., 2006) . while the economic valuation of ecosystem goods and services provides an elegant framework highlighting their importance for society and human welfare, there is a need to explicitly account for their contribution when designing and developing products and services. data and models are needed at multiple scales to account for the contribution of ecosystem goods and services in environmental sustainability assessments and engineering design. at the process scale, recent work has attempted to account for the role of nature and specific ecosystem goods and services for the sustainable design of engineered u. diwekar, et al. resources, conservation & recycling 164 (2021) 105140 systems (carpenter et al., 2006; gopalakrishnan et al., 2016; charles et al., 2020) . from the lca perspective, new environmental impact categories will need to be developed for a comprehensive accounting of ecosystem goods and services. for example, the use of process-based models such as invest (sharp et al., 2018) could be integrated into life cycle frameworks. environmentally-extended input-output models have shown promise for an accounting of ecosystem goods and services, but they are also fraught with uncertainty associated with data and aggregation, which can hinder detailed assessments (zhang et al., 2010) . some of these challenges could be addressed with hybrid approaches (suh et al., 2004) . a more accurate valuation of ecosystem goods and services and their inclusion in human and engineering decision-making has the potential to motivate and guide conservation, revitalization efforts, and policy decision-making. recent efforts have focused on explicitly accounting for the role of ecosystems in supporting human activities by the framework of techno-ecological synergy (tes) . the goal of this framework is to determine the extent of ecological overshoot from specific activities for each ecosystem service and to encourage mutually beneficial designs of technological and ecological systems. several case studies indicate the potential environmental and economic benefits of establishing such synergies (bakshi, 2019; hanes et al., 2017; r.j. 2018; gopalakrishnan et al., 2016 gopalakrishnan et al., ,2017 gopalakrishnan and bakshi, 2018; martinez-hernandez et al., 2017; hernandez et al., 2019) . however, these studies are based on simplified models that do not account for the dynamics of technological and ecological systems, which can be quite different. technological systems are usually designed to have low variability and predictable behavior. thus, such systems tend to stay around a "set point" and exhibit homeostasis. in contrast, ecosystems tend to be intermittent and are not predictable. they exhibit "homeorhesis." benefiting from tes requires designs that combine the desired homeostasis of technologies and human-designed systems with the homeorhesis of nature. our hypothesis is that tes designs are more resilient than conventional techno-centric designs. exploring this hypothesis requires approaches for quantifying resilience. various approaches may be relevant for this task, depending on the nature of information available about the designed systems. ecological network analysis relies on information about the flow between nodes in a network. it relies on information-theoretic metrics that are useful for understanding the character of ecological and economic systems (ulanowicz et al., 2009) . if time-series data from the system are available, methods such as fisher information (eason et al., 2014 ) may be used. if more details such as dynamic models of the system, then mathematical approaches based on viability theory (béné, and doyen, 2018 ) may be applied. various approaches have been developed to account for the effect of uncertainties in network analysis (guesnet et al., 2015; hines et al., 2018) the effect of uncertainties may be included in the stochastic models used in these methods (de lara et al., 2009 ) and by the use of robust and stochastic optimization methods (sahinidis, 2004; diwekar, 2010) . the merriam-webster dictionary defines decision making as the act or process of deciding something, especially with a group of people. decision making under uncertainty methods are around for some time and is the topic of several books (e.g., morgan and henrion 1990; bedford and cooke 2001) . however, these methods do not deal with deep uncertainties that are present in regional as well as global sustainability models. dmdu methods are a topic of intense research in recent literature. dmdu methods are not based on the 'predict-then-act" paradigm, but that aims to prepare and adapt. according to a recent book on dmdu , there are four methods to deal with decision making under deep uncertainties. these include robust decision making (rdm), dynamic adaptive planning (dap), dynamic adaptive policy pathways (dapp), and engineering options analysis (eoa). while the first three involves hybrid approaches and described briefly below, eoa is quantitative in nature and is discussed in next section. for details of these methods, please refer to marchau et al., 2019. rdm draws from scenario analysis the concept of organizing information about the future into a small number of distinct cases that help people engage with, explore, and communicate deep uncertainty. however, rdm is different from scenario analysis that it uses computer simulations to generate large ensembles of future states of world as compared to a small number of cases in scenario analysis. rdm involves four steps 1 consider diverse plausible futures 2 seek strategies which are robust across a wide range of plausible futures 3 employ adaptive strategies to achieve robustness 4 use humans and computers alternatively to test each other's conclusions about futures and strategies. dap is a dmdu approach for designing a plan that explicitly includes provisions for adaptation as conditions change and knowledge is gained. dap approach involves 1 specifying a set of objectives and constraints 2 designing short-term actions initial plan 3 establish a framework to guide future (contingent) actions. dapp explicitly includes decision making over time. it is proactive and dynamic planning. it involves adaption pathways for multiple futures. dapp supports the design of a dynamic adaptive strategy that includes initial actions, long-term options, and adaptation signals to identify when to implement the long-term options or revisit decisions. also, central to the approach is the concept of adaptation tipping points. the adaptation tipping point (atp) approach was developed in the netherlands in response to a desire of the national government for a planning approach less dependent on any particular set of scenarios. dmdu approaches have been used for regional sustainability assessment and policy analysis. for example, molina-perez et al. (2019) studied how to design adaptive plans (rdm) for water supply infrastructure under deep uncertainty to ensure sustainable development in montreal, mexico. groves et al. (2019) presented a case study of the application of rdm to long-term water resources planning for the colorado river basin. vincent et al. (2019) used dap to derive innovative traffic safety technology in the netherlands. in a chapter, lawrence et al. (2019) describes how flood risk managers at a regional level in new zealand applied the dapp approach to managing deep uncertainty around flood frequency associated with changing climate, and examines the lessons learned from taking dapp theory into practice. hamarat et al. (2014) applied multi-objective simulation-based optimization with rdm to study how the european union can achieve its carbon reduction target under deep uncertainty. trindade et al. (2019) proposed deeply uncertain pathways for multicity regional water supply infrastructure investment and portfolio management. shi et al. (2019) proposed two adaptive screening procedures to identify when comprehensive decision analysis methods, such as dmdu methods introduced here, should be implemented. mathematical models featuring the critical components of a real ecosystem can aid in the formal study of sustainability. the first such comprehensive model for studying global sustainability is the model from the club of rome (meadows et al., 1972; meadows, 2014) called the world model. this model has components like the human u. diwekar, et al. resources, conservation & recycling 164 (2021) 105140 population, natural resources, pollution, industrial production, and agricultural land use. the results of this model established growth patterns and limits for the first time. models accounting for processes such as resource extraction, waste assimilation, recycling, and pollution in an integrated ecological, economic framework have also been proposed in the literature (van den bergh, 1996) . in recent years, the united global metamodel of the biosphere (gumbo) (boumans et al., 2002) was developed to include dynamic feedbacks among human technology, economic production and welfare, and ecosystem goods and services. the model was calibrated using time series data for 14 key variables from 1900 to 2000. recently, the model predictions were compared with data from 2000 to 2018 (boumans, 2019) . there are a number of planetary integrated assessment models available in the literature (wilson et al., 2017) to study global phenomena such as global climate change. a model that deals with food-energy nexus for global sustainability is recently proposed by kotecha et al. (2013) . since these models are dealing with long-term sustainability, they face the problem of time-dependent uncertainties (deep uncertainty). for example, fig. 2 shows how the global temperature change over the time span of 400 years. we can see that there is significant uncertainty in global warming and is changing with time. we cannot describe these uncertainties using static probability distributions. these are time-dependent uncertainty. financial literature abounds with such uncertainties and hence, as a result, provided a way to deal with such uncertainties using real options theory. real options theory uses stochastic processes to represent the time-dependent uncertainties. a stochastic process is one that evolves over time in an uncertain way. stochastic processes do not have time derivatives in the conventional sense, and, as a result, they cannot be manipulated using the ordinary rules of calculus as needed to solve the stochastic optimal control problems. ito (1951 ito ( , 1974 provided a way around this by defining a particular kind of uncertainty representation based on the wiener process as a building block. the wiener process, also known as brownian motion, is a continuous limit of the random walk and is a continuous-time stochastic process. a wiener process (continuous or discrete) can be used as a building block to model an extremely broad range of variables that vary continuously and stochastically through time. examples of ito processes are simple brownian motion, geometric brownian motion, mean-reverting process, etc. in the real options analysis (roa), dixit and pindyck (1994) used stochastic calculus (ito's lemma) coupled with dynamic programming to study investment decisions under uncertainty. this approach uses ito's lemma, which is called the fundamental theorem of stochastic calculus and allows us to differentiate and to integrate functions of stochastic processes. however, for deep uncertainties involved in sustainability, we need to consider the engineering options analysis. the difference between eoa and roa is that eoa considers multiple options with a range of possibilities. however, the mathematical methods used for roa and eoa are similar. recently, diwekar (diwekar, 2008; rico-ramirez et al., 2003; rico-ramirez and diwekar, 2004) presented basic concepts for dealing with time-dependent uncertainties modeling. these concepts are derived from the financial and economics literature and engineering optimal control theory. these concepts could be extended to studying sustainability as presented in the recent literature (shastri and diwekar, 2008; shastri et al., 2008a,b; diwekar and shastri, 2010; diwekar, 2012b diwekar, , 2015 doshi et al., 2015 , rodriguez-gonzalez et al., 2019 . although the tardis workshop of 2019 focused on the role of uncertainty in sustainability science and engineering, it was also discussed whether attempts to accurately assess the current condition of the earth and forecast future trends as accurately as possible are in fact in themselves symptoms of the underlying problem and reflects an anthropocentric view of human exemption, dominance, and omnipotence. effective management strategies might, therefore, focus also on fostering new approaches that reflect a more humble and flexible human-nature relationship. a recent article in pnas (steffen et al., 2018) argued that the "social and technological trends and decisions occurring over the next decade or so could significantly influence the trajectory of the earth system for tens to hundreds of thousands of years and potentially lead to conditions inhospitable to current human societies and to many other contemporary species." despite the best efforts of scientists to qualify their interventions and acknowledge that their scenarios are "mediating tools to communicate vulnerabilities, perceptions of risk or possible consequences, they often become taken as statements of the future" (yusoff and gabrys, 2011) . transformation is a way of reflecting on existing practices and imagining new ways of doing and knowing (duncan et al., 2018) , the capacity to collectively envision and meaningfully debate realistic and desirable futures (milkoreit, 2017) . transformations, however, involve "systems change and because of the political nature of change are subject to contestation" (hebinck et al., 2018) . norgaard (norgaard, 2018) suggests that while we have made significant progress in developing an ecological imagination through advances in climate science, we have made much less progress in actually changing course. the gap between responses to climate change and the socio-political realities of contemporary life have led to inaction, but the further problem is that we do not know and cannot know what changes are in motion (nightingale et al., 2019). beck's world risk society idea and its attendant uncertainty capture the sense that the speed and complexity of interactions blur cause and effect, creating the escalating potential for cascading catastrophic risks and a vision of the future characterized by chaos (rickards et al., 2014) . living with uncertainty is characterized "as the continuous emergency of its own emergence" (rickards et al., 2014) captured in the idea of the 'long emergency' (kunstler, 2005) . there is growing experimentation with participative and deliberative designs adapted from innovative governance mechanisms, e.g., citizens assemblies for national dialogues on climate change as a method for engaged research. the focus is very much on facilitating the active participation of local communities and civil society actors to explore the relationship between probable, preferable, and plausible futures (ellyard, 2011) . faced by growing societal demand for urgent action on climate change, e.g., extinction rebellion and the global climate strikes inspired by greta thunberg, there is an opportunity for more meaningful engagement between science and society. yet this is not straightforward and poses very particular challenges in preparing u. diwekar, et al. resources, conservation & recycling 164 (2021) 105140 present and future generations of researchers and professionals with the tools that are fit for the purpose of addressing these challenges. it is increasingly claimed that we are living in a world that is so complex, interconnected and uncertain, and that many of the situations that professionals, for example, engineers, have to deal with should be seen and treated as 'wicked problems' (an early description of such problems was provided by rittel & webber in 1973 . andersson et al. (2014 talk about societal systems as wicked, featuring properties that make them a combination of complicated and complex, using their terminology. farrell & hooker (2013) talk about all realworld science and engineering problems as wicked and describe three sources of wickedness: (1) finitude of cognitive abilities, (2) complexity of the problems, and (3) normativity in problem understanding and resolution. again, we can take the example of reactive nitrogen to describe the wickedness of problems. reactive nitrogen encompasses biologically and radiatively active, and chemically reactive nitrogen compounds. on a global scale, human activities now create approximately two-fold more nr than natural continental ecosystems. in the united states, nr creation by human activity is about 5-fold larger than natural processes by: (1) the haber-bosch process to generate ammonia (nh3) for synthetic nitrogen fertilizer and industrial feedstocks, (2) the enhancement of biological nitrogen fixation (bnf) in crop cultivation (e.g., legumes), and (3) the combustion of fossil fuels. the first two anthropogenic activities form nr on purpose; the last one forms nr as an unwanted pollutant. comparing these three sources, haber-bosch nr is by far the largest (epa et al., 2011) . anthropogenic creation of nr provides essential benefits for humans-first and foremost, in meeting human dietary needs. a large fraction of the human population of the earth could not be sustained if fertilizers containing haber-bosch (nr) did not augment food production significantly. there are, however, costs associated with these benefits. essentially all of the nr created by human activities is lost to the environment, often with negative, unintended consequences. there it circulates between, and accumulates within, environmental systems and contributes to a number of adverse public health and environmental effects. in an educational context, the collaboration of haber (a chemist) and bosch (a chemical engineer) is often presented as a triumph of science, and the ideal model for technological advancement. few at the time could foresee the unintended consequences of nr proliferation; indeed, perhaps these consequences were not foreseeable-the fields of environmental science and engineering, ecology, and environmental chemistry were at best immature, and the widely perceived vastness of earth lent it an aura of immutability. as hardin points out in his seminal essay "the tragedy of the commons" (hardin, 1968) , the concept of "waste" is relatively modern; humankind evolved on earth where almost all natural cycles were closed-loop systems-wastes simply did not exist. in many ways, our education paradigm is just emerging from its siloed past in which insular disciplinary approaches reigned supreme. the need for a new paradigm of convergent education is now widely recognized (nsf, 2017) . so, what approaches are more useful for dealing with wicked problems? porter & córdoba (2009) describe three different approaches to systems thinking. functionalist approaches are made from a scientific, systems analytic perspective and with a reductionist worldview. interpretive approaches, on the other hand, view systems as "mental constructs of observers" and explicitly explore normative assumptions behind the problem definition. complex adaptive systems approach instead recognize systems as not only complex, but also "adaptive," characterized by self-organization, emergence, and bottom-up change and thereby suggest that conflicts may be ultimately unresolvable and that there may not be definitive solutions to sustainability problems -or even a "best way of getting things done." if real-world problems are actually wicked, the third approach should be more useful, and it is then a problem that professionals are not properly trained to deal with such contexts. jonassen (2000) , for example, argues that engineering students are primarily trained to solve "story problems", using functionalist approaches. this is likely a result of the predominant culture in science and engineering based on objectivism, positivism, and reductionism and with a lack of recognition and discussions of the usefulness of alternative views. in an attempt to guide engineering education towards approaches that recognize the wickedness of real-world problems, lönngren et al. (2017; ) developed a description of and educational elements for developing such approaches. the description contains five 'structural' aspects related to the ability to deal with wicked problems, that focus on the interconnection between problems parts, improvement measures and secondary problems, but also five 'referential' aspects that focus on local contexts and stakeholders and their spheres of influence as well as the importance of lack of information and uncertainties. these latter five try to capture a new type of relationship to problem-solving, or rather to the management of situations, as wicked problems do not really have solutions. our relationship to what we know -and more importantly, what we do not know -requires mechanisms and tools that prepare researchers and professionals to foster adaptive relationships to the dynamics of complex socio-ecological systems. the challenges addressed here are akin to those identified by bai et al. " to find ways to live and act without knowing the future…science needs to have closer and different relations with practice, …where science not only informs practice but also learns from practice" (bai et al., 2016) . at its most basic core, sustainable development is an effort by humans to make institutions and choices to ensure that the earth can support our continued presence for the indefinite future. this is a difficult challenge given the size of the human population, the level of human activity, and the finite size of the earth. this brings humanity to a situation where it has to learn to manage the earth out of simple necessity. this is a difficult challenge because: (1) there are multiple aspects that must be considered and acted on, including policy, economics, manufacturing, environmental impacts, and education to name but a few, and (2) the knowledge necessary to effectively conduct the management action comes with substantial uncertainty which can be specific to some aspect or systemic and is often time-dependent. further, the challenges to the earth system can fall into any of the four known categories, where the most difficult one is that unknown unknowns, i.e., issues that are not even known to exists. here we have explored the challenges and made an effort to frame approaches that can be reasonably expected to lead to effective solutions or least management approaches. the authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper. the vision for civil engineering in 2025. based on the summit on the future of civil engineering -2025 sustainable engineering: principles and practice techno-ecological synergy: a framework for sustainable engineering strategic perspectives of corporate sustainability management to develop a sustainable organization sustainability science: managing risk and resilience for sustainable development probabilistic risk analysis: foundations and methods from resistance to transformation: a generic metric of resilience through viability life-cycle analysis of fuels from post-use non-recycled plastics the great moderation modeling the dynamics of the integrated earth system and the value of global ecosystem services using the gumbo model revising gumbo model flint water crises -five years later: there's still no trust uncertainty as information: narrowing the sciencepolicy gap thinking inside the box: a participatory, computerassisted approach to scenario discovery sustainability, sustainability assessment, and the place of fiscal sustainability to bridge the divide between evidence and policy: reduce ambiguity as much as uncertainty millennium ecosystem assessment: research needs complexification: explaining a paradoxical world through the science of surprise connecting air quality regulating ecosystem services with beneficiaries through quantitative serviceshed analysis economic dependence of us industrial sectors on animal-mediated pollination service option demand and consumer surplus: further comment quasi-option value and the expected value of information nature's metropolis: chicago and the great west ecological economics: principles and applications quality unknown: the invisible water crisis. the world bank group multi-criteria dynamic decision under uncertainty: a stochastic viability analysis and an application to sustainable fishery management introduction to applied optimization optimization under uncertainty in process systems engineering a systems analysis perspective of green design green engineering and sustainability: a systems analysis perspective. sustainability: multidisciplinary perspectives 273 perspective on pursuit of sustainability: challenges for engineering community green process design, green energy, and sustainability: a systems analysis perspective investment under uncertainty listing decisions under the endangered species act: why better science isn't always better policy maximizing sustainability of ecosystem model through socio-economic policies derived from multivariable optimal control theory greenhouse gas accounting of rural agrarian regions: the case of san luis valley transformation is 'experienced, not delivered': insights from grounding the discourse in practice to inform policy and theory managing for resilience: early detection of regime shifts in complex systems on the polarization of young stellar objects global sustainable society environmental protection agency science advisory board. reactive nitrogen in the united states: an analysis of inputs, flows, consequences, and management options -a report of the epa science advisory board humankind and the environment: an anatomy of surprise and ignorance evolution, time, production and the environment design, science and wicked problems a core precautionary principle production, use, and fate of all plastics ever made assessing the capacity of local ecosystems to meet industrial demand for ecosystem services ecosystems as unit operations for local techno-ecological synergy: integrated process design with treatment wetlands biosolids management with net-zero co2 emissions: a techno-ecological synergy design towards sustainable production of chemicals and fuels from the fast pyrolysis of waste polyolefin plastics, phd dissertation in: robust decision making (rdm): application to water planningand climate policy in decision making under deep uncertainties an exploratory approach for adaptive policymaking by using multi-objective robust optimization synergies and trade-offs in renewable energy landscapes: balancing energy production with economics and ecosystem services including nature in the food-energywater nexus can improve sustainability across multiple ecosystem services the tragedy of the commons imagining transformative futures: participatory foresight for food systems change techno-ecological synergies of solar energy for global sustainability introduction to operations research uncertainty analyses for ecological network analysis enable stronger inferences development of a multidisciplinary approach to assess regional sustainability higher taxes on top 1% equals higher productivity. the real news network multiscale regime shifts and planetary boundaries guidelines for the governance of systemic risks on stochastic differential equations on stochastic differentials toward a design theory of problem solving success, strain, and surprise a world beyond physics: the emergence and evolution of life what is systemic risk, and do bank regulators retard or contribute to it? providing global public goods: managing globalization importance of pollinators in changing landscapes for world crops risk, uncertainty, and profit model based approach to study the impact of biofuels on the sustainability of an integrated system many objective robust decision making for complex environmental systems undergoing change the long emergency: surviving the converging catastrophes of the 21st century dynamic adaptive policy pathways (dapp): from theory to practice in decision making under deep uncertainties tipping elements in the earth's climate system on the origin of planetary-scale tipping points hybridization, agency discretion, and implementation of the u.s. endangered species act sustainability enhancement under uncertainty: a monte carlo based simulation and system optimization method sustainable distributed biodiesel manufacturing under uncertainty: an interval-parameter-programming-based approach technology evaluation and decision making for sustainability enhancement of industrial systems under uncertainty avoid, control, succumb, or balance: engineering students' approaches to a wicked sustainability problem scaffolding strategies in a rubric-based intervention to promote engineering students' ability to address wicked problems residents, officials outraged over toxic emissions from cobb plant forthcoming. the bird and the bulldozer unsicherheit der normativität und normativität der unsicherheit in den diskursfeldern "globaler wandel" und "nachhaltigkeit decision making under deep uncertainty: from theory to practice forecasting unknownunknowns by boosting the risk radar within the risk intelligent organisation a framework for modeling local production systems with techno-ecological interactions interpreting negative results with taxonomic and conservation implications: another look at the distinctness of coastal california gnatcatchers the limits to growth limits to growth: the 30-year update imaginary politics: climate change and making the future the financial instability hypothesis: an interpretation of keynes and an alternative to "standard developing a robust water strategy for monterrey, mexico: diversification and adaptation for coping with climate, economic, and technological uncertainties multistage optimization for chemical process sustainability enhancement under uncertainty uncertainty: a guide to dealing with uncertainty in quantitative risk and policy analysis ontic openness: an absolute necessity for all developmental processes 2020. a new ecology: systems perspective the sociological imagination in a time of climate change subspecies and the philosophy of science with speed and violence: why scientists fear tipping points in climate change three views of systems theories and their implications for sustainability education summary for policymakers of the assessment report of the intergovernmental science-policy platform on biodiversity and ecosystem services on pollinators, pollination and food production risk governance. coping with uncertainty in a complex world precaution and the governance of risk systemic risks: the new kid on the block, environment: science and policy for sustainable development opening and closing the future: climate change, adaptation, and scenario planning real option theory from finance to batch distillation stochastic maximum principle for optimal control under uncertainty species definitions shape policy dilemmas in a general theory of planning a new approach to solving stochastic optimal control problems life cycle assessment: part 1: framework, goal and scope definition, inventory analysis, and applications a safe operating space for humanity optimization under uncertainty: state-of-the-art and opportunities imaginable surprise in global change science invest: integrated valuation of ecosystem services and trade-offs optimal control of lake ph for mercury bioaccumulation control optimal control theory for sustainable environmental management is sustainability achievable? exploring the limits of sustainability with model systems when can decision analysis improve climate adaptation planning? two procedures to match analysis approaches with adaptation problems systems analysis for pet and olefin polymers in a circular economy many-objective robust decision making for managing an ecosystem with a deeply uncertain threshold response the concept of systemic risk fallibility, reflexivity, and the human uncertainty principle planetary boundaries: guiding human development on a changing planet trajectories of the earth system in the anthropocene deliberate futures: precaution and progress in social choice of sustainable technology eradication of tephritid fruit fly pest populations: outcomes and prospects the black swan: the impact of the highly improbable deeply uncertain pathways: integrated multicity regional water supply infrastructure investment and portfolio management frames in the toxicity controversy. risk assessment and policy analysis related to the dutch chlorine debate and the swedish pvc debate quantifying sustainability: resilience, efficiency and the return of information theory in: ecological economics and sustainable development, chapter a multisectoral growth model with materials flows and economic-ecological interactions dynamic adaptive planning (dap): the case of intelligent speed adaptation. in decision making under deep uncertainties defining uncertainty: a conceptual basis for uncertainty management in model based decision support collective consumption services of individual consumption goods the role of scientists in statutory interpretation of the u.s. endangered species act in: evaluating process-based integrated assessment models of climate change mitigation. laxenbur, austria. international institute for applied systems analysis working paper uncertainty and the endangered species act the world bank databank climate change and the imagination new) species concepts, species delimitation and the inherent limitations of taxonomy accounting for ecosystem services in life cycle assessment, part ii: toward an ecologically based lca phylogeography of the california gnatcatcher (polioptila californica) using multilocus dna sequences and ecological niche modeling: implications for conservation geographic variation, null hypotheses, and subspecies limits in the california gnatcatcher: a response to mccormack and maley foundation under the grant cbet 1854519. key: cord-347241-9jn5agir authors: shahzad, arfan; hassan, rohail; aremu, adejare yusuff; hussain, arsalan; lodhi, rab nawaz title: effects of covid-19 in e-learning on higher education institution students: the group comparison between male and female date: 2020-08-04 journal: qual quant doi: 10.1007/s11135-020-01028-z sha: doc_id: 347241 cord_uid: 9jn5agir in response to the emerging and ever solution to the covid-19 outbreak. this study proposes a theoretical framework based on literature and model to determined e-learning portal success. the study compared males and females to e-learning portal usage. the study objective is to check the difference between male and female e-learning portals’ accessibility among the students’ perspective. the study included service quality, system quality, information quality, user satisfaction, system use, and e-learning portal success. the empirical data of 280 students participated from the different universities of malaysia through google surveys analyzed using the partial least squares structural equation modelling. the study further divided the full model into two domains, which are female and male. in the male model, information quality and system quality have direct relationships with user satisfaction. information quality also supported the relationship with system use. at the same time, there is a positive relationship between user satisfaction and e-learning portals. likewise, in the female model, e-service quality and information quality both are supported by system use and user satisfaction. similarly, system quality has a positive relationship with user satisfaction, and user satisfaction has a positive relationship with e-learning portals. the study will be further helpful for the malaysian universities policy-makers such as top management, ministry of higher education, malaysian universities union in designing the policies and programs on e-learning portal success in the country. the findings of the study reveal that males and females have a different level of in terms of usage of towards e-learning portals in malaysian universities. in the 21st century, at the end of 2019 in wuhan, the high technology business hubs of china experience an epidemic of an entirely distinctive coronavirus appeared that had killed a few thousand chinese within the fifty days of spreads and thousands of other citizens are suffered. the novel virus was nominated as covid-19 novel coronavirus by the chinese scientists (shereen et al. 2020) . later on, in a shorter period, this covid-2019 spread worldwide. several country's economies are severely affected due to covid-2019. further, the outbreak has changed the operating conditions all over the globe within a month. the consequences of a pandemic are unstoppable and uncontrollable for many industries of the world. later on, almost 120 countries have stopped face-to-face learning; approximately a billion students' education is effected worldwide with covid-19. most of the higher education system is operating through the e-learning (azzi-huck and shmis 2020; shahzad et al. 2020a, b) . meanwhile, to tackle the covid-19 pandemic, almost all the world, and including malaysian higher education ministry, has issued the ordered to close the public school and higher education closure as an emergency measure to stop spreading the infection. technologies have changed the traditional way of education to the modern way of learning, like artificial intelligence (di vaio et al. 2020a) . thus e-learning is covered under a larger term of technology-based learning through websites, learning portals, video conferencing, youtube, mobile apps, and thousand types of free available websites for blended learning tools. currently, e-learning is enhancing students' knowledge, even the academic staff and professional and industry people skills through the internet (adams et al. 2018; chopra et al. 2019 ). most of the higher education universities are providing online courses for their students within and off campuses. in malaysia, the government is providing many resources to higher education. based on the news reports, the malaysian universities, colleges, polytechnics are using massive open online courses (moocs). the growth of the online education market is expected 16.4% annually over the forecast period, 2016-2023. with the massive growth of the internet, maybe university teaching and learning models will be changed in 10 to 15 years. thus, it based on male and female students on the use of the e-learning portal. this study focuses on comparisons between male and female counterparts on e-learning portal usage among university students during the covid-2019 period. globally, due to covid-19 outbreak universities closed and lockdown, most teachers and students are happy by the move online education. the faculty members of worldrenowned universities have begun to get online instructor certifications to deliver online teaching to their students. at the same time, faculty and staff members are learning how to use online learning platforms. previous, they are using only the delivery through face-toface teaching. however, the shift to online mode has raised many queries on the quality of education (sahu 2020) . furthermore, the quality of education and excellent infrastructures such as computers and it modern equipment reception are now in massive demand and universities are changing their teaching models with the use of intellectual capital (alvino et al. 2020; di vaio et al. 2020b ). thus, an unexpected shift from face-to-face learning to online, there are few difficulties faced by students and lectures. moreover, most of the countries significant issues with technological infrastructure in rural areas; thus, the standard of online education may be a critical issue that needs essential focus. therefore, based on the above-said issues, this study tries to investigate the impact of male and female students on the use of the e-learning portal. in previous literature, the d&m model is tested on the overall population, like banks and other financial sectors. in the current study, the whole population is divided into males and female categories to hoped different theoretical contributions by having a division of the population. consequently, universities in malaysia are offering an online course to the students on campus and off-campus. the current study focuses on the male and female user satisfaction and e-learning system use toward the e-learning portal success of the malaysian universities. based on many researchers (cidral et al. 2018; selvaraj 2019) claimed that user satisfaction and e-learning system use have a significant impact on success. this study will conduct on the students how are enrolled with malaysian universities and using the e-learning portal for their learning. based on the argument so far, there is a still gap in literature the e-learning system among universities after the spread of the covid-19 outbreak on higher education closure. the purpose of the present study to investigate the effect of information quality, system quality, and service quality toward user satisfaction and e-learning system use impact on the e-learning portal success. therefore, the study focuses on group comparison between male and female students on the e-learning portal uses. this paper contains five major sections. section 2 briefly described the compiling review of previous literature that how the learning curve has shifted towards online portals by using delone and mclean model. in sect. 3, a description of the research method and the overall process of data collection and analysis were the main focus. section 4 relates to the multi-group analysis (mga) and the interpretation of the result. lastly, sect. 5 concluded with a holistic discussion related to the male and female groups of the study. toward the end of february, as alerts sounded on the increasing spread of the covid-19 infection, the world bank built up a multi-sectoral worldwide task force team to help nation reaction and adapting measures. at that point, just china and some other influenced nations were upholding social distance through the closure of schools. in the meantime, following fourteen days after the fact, 120 countries have closed schools impacting almost a billion students across the world that have experience closures of their schools for the period (azzi-huck and shmis 2020). in this light, the covid-19 pandemic has forced the universities to close face-to-face education and send students home. this force the universities to introduce courses through online portals. also, education industries are adopting the technologies available such as digital video conferencing platforms like zoom, microsoft platform, and webex blackboard and google classroom (larry 2020). therefore, this will be enhancing e-learning globally (chen 2010; yengin et al. 2011; larry 2020) . therefore, the current study concentrating on the comparison between male and female on e-learning. in this light, applying remote learning and education resources to mitigate the loss of learning: in web-based technology, electron learning is well-knowns as well as the earliest application (azhari and ming 2015) . in today the e-learning is getting very popular worldwide. e-learning is described as the delivery of learning through technology and the internet (gros et al. 2016; hong et al. 2017; aljawarneh 2020) . almost all the universities and colleges have developed the e-learning portal of their students and faculties (moore et al. 2011 ). in the 21st century, the e-learning creates a more significant impact on all types of the student, much as the part-time and full-time or distance learning student in the higher education institution (azhari and ming 2015) . nowadays, the majority of the postgraduate students are registered as a part-time student, because they are working in the companies. e-learning helps them a lot because of their time constrain. the advancement in e-learning has been started through massive open online courses (moocs) for students, society, and the industry as well since 2012 (calisir et al. 2014; margaryan et al. 2015) . moocs are recognized as a significant development in higher education million of the peoples and student are taking the benefits and uplifting the existing skill (gupta and gupta 2020) . moreover, in recent decades, several malaysian universities have adopted the e-learning portals (hussin et al. 2009; paechter and maier 2010) . based on the research of azhari and ming (2015) highlighted several issues related to the learning management system (lms) of the malaysian universities such as the lack of trained lectures, slow down of the internet speed, wifi coverage, infrastructure, the interface of design, quality of content, system use and students' adoption. in the present research, the comparison between male and female students is measured based on e-learning portal success. meanwhile, the researchers will find out the importance of e-learning tools' success in terms of male and female malaysian student perspectives. d & m model iss success model has gained significant attention from researchers in the field of information systems. in 1992, this model was initially developed by delone and mclean to measure the dependent construct of is success (delone and mclean 1992) was primarily based on following three aspects: (shannon and weaver 1949 ) study on communications, taxonomy of (mason 1978) measuring information output and research work on information system during that period. there are three levels of communication per (shannon and weaver 1949) first level: technical: (accuracy of information system) second level: semantic (success of right information conveyed to the right receiver) third level: effectiveness (influence of information on the receiver). the information success model (1992) has discussed the six dimensions, such as information quality, system quality, system use, user satisfaction, and organizational impact. after one decade, the author modified the original model by adding service quality dimension and the end replaced the individual impact and organizational impact with the net benefits. figure 1 showed the original d&m model having six interdependent dimensions (delone and mclean 1992; mohammadi 2015) . in this model, the "system quality" construct depicts "technical success." in contrast, the "information quality" variable demonstrate "semantic success," while the other four elements "use," "user satisfaction," "individual impact," and "organizational impact" show "effectiveness success." therefore, this study focus on male students' comparison with female students on the e-learning portal. as time passed, the researcher used d&m model to check the success of the information communication system. many studies suggested to add the new dimensions of d&m model, and some have recommended to include the such as "service quality," "workgroup impact," "consultant/vendor quality" in the d & m model (gable et al. 2003; ifinedo 2006) . while the few researchers criticize d & m model "use" and "user satisfaction," dimensions. many studies have changed the number of aspects according to their context, but no study considers organization capabilities mediating role in the relation between alone impact and organizational impact. in this light, a comparison of males and females among university students to determine the e-learning portal. the study of ifinedo (2011) highlights the importance of the system quality creates a great impact on the e-learning portal. the system quality of the e-learning will generate query results more quickly. moreover, system quality will increase the interest of the enduser. also, the user-friendly interface and modern graphical interface increase the level of user satisfaction. the study of petter et al. (2013) highlighted that service provider should adopt the new changes and modify time to time the system. also, the system quality construct has been used in the context of the e-learning system, which depicts whether the user is satisfied with the quality of the e-learning portal. "perceived ease of use" is the most used measure of "system quality" (davis, 1989) . furthermore, sq has been identified as ease of learning, ease of use, the convenience of access, the usefulness of system features, system complexity, system features, and response time of information system (beheshti and beheshti 2010) . furthermore, as suggested by sedera et al. (2013) , sq means ease of learning, efficient, consistent, modifiable, efficient, personalizable, meets the requirements, ease of use, and reliable. in addition, (delone and mclean 2016) acknowledged system quality by measures: ease of use, availability, flexibility, reliability, usefulness, and response time. there are certain modifications in standards of system quality that occurs over time. however, some measures remain the same and consistently being applied and validated, which are as follows: the luxury of use, the comfort of learning, reliability, personalizabality, reply-time, availability, system interactivity, and system security. moreover, the study concentrates on the male and female comparison. the iq depicts the precision as well as the accuracy of the information provided by the e-learning system; timeliness is another important indicator of information quality that information should be generated within time and latest. so, that higher management can make quick decisions, sufficiency is another characteristic of information quality that it should not be insufficient and must contain all information required to the user. understandability is a very effective characteristic of the iq construct that it should be easy to understand and should not be complex that difficult to grasp. conciseness is another vital part of information quality produced by the e-learning system, system in any organization (petter et al. 2013; cai and zhu 2015; chiang et al. 2019) . additionally, information quality demonstrates the output characteristics of the information system that it is proving the timely information to all the departments of the organization; the information should be relevant to the particular user or the department. the required information is available at the right time to the right person; the data' provided by the information system should be understandable to the users (ifinedo, 2014; muda and erlina 2019; pham et al. cr55) . therefore, the comparison of male and female students on the e-learning portal is the target of this study. furthermore, the main purpose of information quality is to provide users online knowledge with correct relevant information on 24/7. so, it must be considered active for the e-learning portal success. information quality in past literature considered being part of user satisfaction measurement. it is not treated as a separate variable further argued that information quality measures vary depending upon the type of information system success going to be evaluated. however, there are consistent measures of information quality for e-learning success as follows: relevance, usefulness, understandability, accuracy, reliability, currency, completeness, timeliness. based on the comparison of male and female university students. service quality refers to "responsiveness," which is a very important indicator. it means how efficiently the technical department responds to the queries of users. also, empathy is another characteristic of service quality of how attentively they are helping the users (petter et al. 2013; haryaka et al. 2017; xu and du 2018) . moreover, in the d&m model, service quality was neglected and updated in d&m (2013). is success model. many items have been stated to measure e-learning system service quality but mostly cited instrument developed by authors (parasuraman et al. 1986; parasuraman et al. 1988; parasuraman et al. 2002) . initially, ten dimensions were used to measure service quality, which later transformed into five named; tangibles, reliability, responsibility, assurance, and empathy. this updated model of d&m model found very useful for evaluating the success of different types of technologies related application. most of the studies used this model of an information system, erp system success, e-procurement application, e-government application on the user perspective, e-banking application use success, and also several other online application successes business (hsu et al. 2015; almarashdeh 2016; aparicio et al. 2017; ojo 2017; cidral et al. 2018; aparicio et al. 2019 ). e-learning involves the usage of modern technology to impart learning; thus, the present study needs to investigate how the end-user accepts much e-learning portal success. therefore, the present study highlighted the comparison between male and female students on the e-learning portal. thus, fig. 2 has shown the research framework of the present study and hypothesis. h 1 e-service quality has a significant positive impact between males and females on the system use. h 2 e-service quality has a significant positive impact between males and females on user satisfaction. based on previous research, the current study has developed a survey instrument. the questionnaire was adopted/adapted and reworded in the form of e-learning. all items used a 5point likert scale ranging from 1 to 5 from "strongly disagree" to "strongly agree." the questionnaire items of system quality, information quality, system usage, satisfaction user was adopted from (mcgill, hobbs and klobas, 2003; rai, lang and welker, 2002) . the e-learning portal success survey instrument items were adopted from (freeze et al. 2010) . all items of e-learning portal success were used a 5-point likert scale ranging from 1-"poor," 2-"fair," 3-"satisfactory," 4-"very good" to 5-" excellent." the research used a quantitative approach; the survey was conducted through the google form; the links were shared with the student through the whatsapp group of lecturers. all the malaysian university student is participating in the e-learning portal survey. the convenience sampling technique was used. the study employed a cross-sectional survey method. in table 1 , the students of undergraduate 212 (75.7%) and postgraduate 68 (24.3%) have participated in the online survey conducted from august to september 2019. in table 2 , in the demographic analysis, the 'gender' factor showed that female participants have a more response rate of 67.9% as compared to 32.1% who were male. in malaysian universities is more enrolment of female as compared to male. regarding the 'age group' of respondents, data revealed that the majority was having the age-group of respondents 21-30 years 212 (75.7%). also, regarding the "experience using the e-learning portal," data showed that most of the students who participated in the survey they have experienced using the e-learning portal is more than two years. to achieve the research objectives, the study has employed partial least squares (pls) version 3.3.0 to facilitate data analysis. based on the context of inferential analyses, partial least squares-structural equation modelling (pls-sem) application has been applied in several disciplines (hair et al. 2016 ). these developments contribute to the growth of pls-sem that is generally used as a research instrument in the field of management information systems as well as the social sciences (hair et al. 2016) . also, confirm the importance of pls's ability to analyze variables in complex models, simultaneously. smart pls evaluates two models, mainly, which are measurement and structural (hair et al. 2016 ). internal consistency mentions the degree to which all indicators are different then each other (sub) scale is evaluating an equivalent concept (hair et al. 2016) . inline thereupon, the composite reliability score value must be higher than 0.70, and the ave score value to quite 0.50 (hair et al. 2013 (hair et al. , 2016 . therefore, it explained in table 3 below, all the constructs included in the present study have ave, and composite reliability (cr) is above the criteria above said, which may be a suggestion of measurement model reliability. while table 3 stated that the average variance extracted (ave) and cr values of all variables are in an appropriate range. it shows that all the constructs of the female and male groups have ave's and cr value are above the threshold values, which proved the reliability of the measurement model (figs. 3, 4 , 5). in the present study, also check the discriminant validity criterion, which measures the degree to which a variable is not equivalent to other constructs (hair et al. 2016 ). according to fornell & lacker criterion explained that the higher level of discriminant validity proposes that constructs are differents than the respective variables and not explaining some phenomena. the present study performed the discriminant validity taking the square root of ave of the constructs. thus, the values are higher than the correlations among latent constructs (hwang and min 2015) . therefore, in table 4 , the present study of male and female and full model having no issues with discriminant validity (naala et al. 2017 ). the measure two sets of university students, male and female, the study applies an invariance test. at the same, it is vital to conduct the invariance test before conducting a multi-group analysis. thus, the purpose is to determine "whether, under different conditions of observing and studying phenomena, measurement models yield measures of the same attribute" (henseler et al. 2016 ). afterward, the study follows three steps, namely, configural invariance, compositional invariance, and equality of composite means values and variances to test measurement invariance (henseler et al. 2016) . firstly, since the measurement models have the same number of constructs for both groups, therefore the male and female group data is established for configural invariance (see tables 3, 4 ). secondly, compositional invariance was measured engaging a permutation test. this assures that the composite scores are the same between the groups. lastly, equality of composite variances and means values of groups were assessed. thus, the difference of the composite's mean and variance ratio results, as shown in table 5 , must fall within the 95% confidence interval. as indicated in table 5 , the result reveals that each of the composite constructs has non-significant differences regarding the composite mean and variances ratio. moreover, in table 5 , full measurement invariance is depicted for male and female. therefore, it is often deduced that the various model estimations of male and female groups students are not distinct in terms of content or usage of the e-learning portal. in the current study, the researchers have applied pls-mga in order to calculate the differences by using welch-satterthwait test on male and female groups (sarstedt et al. 2011) . furthermore, table 6 is depicting the path coefficient and difference of the composite's means. meanwhile, several paths are found to be different in terms of male and female data sets and significantly different. the present study found the e-service quality toward the system use male and female have a difference. on the otherwise information quality toward the system use having a difference between the male and female. as per the female group assessment concerned in table 6 , hypothesis 1 predicted that esq is positively significantly related to system use with path coefficient of (0.285**), while (p value > 0.05) not supporting hypothesis 1. thus, hypothesis 2, demonstrate the significant positive association among esq and us (β = 0.503, p value < 0.05) hypothesis 2 supported. hypothesis 3 articulated that information quality is significant positively linked with system use (β = 0.388, p value > 0.05), not supporting hypothesis 3. equally, hypothesis 4, demonstrates a significant positive association with iq and us (β = 0.287, p value < 0.05) supporting hypothesis 4. furthermore, hypothesis 5 predicted that system quality is insignificant negatively associated with system use (β = 0.063, p value > 0.05), not supporting hypothesis 5. results of hypothesis 6 demonstrate a significant positive association between system quality and user satisfaction (β = 0.181, p value > 0.05), not supporting hypothesis 6. hence, hypothesis 7 articulated that system use is significant positively linked with e-learning portal success (β = 0.008, p value > 0.05), not supporting hypothesis 7. finally, hypothesis 8, demonstrates an insignificant negative association with user satisfaction and e-learning portal success (β = 0.701, p value > 0.05), not supporting hypothesis 8. therefore, based on the above evidence, it can be concluded that female students more understand the important usage of e-learning portal success in the content of the university. this implies that female students hold the dominant positions on the usage of the e-learning portal. regarding male group assessment, table 6 hypothesized that esq is negatively insignificantly related to system use with (β = 0.165, p value > 0.05) not supporting hypothesis 1. next, with hypothesis 2, that demonstrates an insignificant negative association between esq and us (β = 0.204, p value < 0.05) hypothesis 2 supporting. while hypothesis 3 articulated that iq is significant positively linked with system use (β = 0.354, p value > 0.05), not supporting hypothesis 3. additionally, hypothesis 4, demonstrates a significant positive association with information quality and user satisfaction (β = 0.551, p value < 0.05) supporting hypothesis 4. consequently, hypothesis 5 revealed that system quality is insignificant negatively associated with system use (β = 0.074, p value > 0.05), not supporting hypothesis 5. results of hypothesis 6 demonstrate a significantly positive association between sq and us (β = 0.220, p value > 0.05), not supporting hypothesis 6. thus, hypothesis 7 articulated that system use is significant positively linked with e-learning portal success (β = 0.017, p value > 0.05) not (table 7) and (figs. 6, 7, 8) . in regard to the hypothesis testing on the female group, six (6) variables were, directly and indirectly, significant and supported to e-learning portal success. in the female students model, e-service quality and information quality both are supported with system use and user satisfaction. similarly, system quality has a positive relationship with user satisfaction, and user satisfaction has a positive relationship with e-learning portals. on the other hand, in male students, four (4) variables are significant. more precisely, information quality and system quality have direct relationships with user satisfaction. information quality also supported the relationship with system use. at the same time, there is a positive relationship between user satisfaction and e-learning portals. in the full model, user satisfaction is significant in terms of information quality and service quality. consequently, user satisfaction is also significant in terms of e-learning portals. thus, e-learning portal usage is more towards female students in malaysian universities. regarding theoretical implication, the adoption studies are numerously studied phenomena both at an individual (customer) as well as organizational (management) levels. although, technology adoption studies like e-learning portals success among students is a rarely researched phenomenon, particularly in malaysian universities. the present study is considered a pioneer in terms of e-portals implementation in universities to test empirical, theoretical relationships in terms of males and females. mainly, it elaborated on the relationship between the variables such as e-learning portal success, esq, iq, sq, su has a significant impact on us. this present study proposes and examined the factors that affect the e-learning portal by extending d & m theory to incorporate elements that are more associated with the is success and e-learning among university students. additionally, d & m model is taken into account because the is tangible factors that would help universities strengthen and enhance their services. the significant contribution of the present study is the behavior of model changes if we divide the full model into parts (male and females). the proposed that d and m models provide misleading out in terms of the full model. thus, its theoretical relationship changes as the model divided into different subgroups. this confirms the effectiveness of the e-learning portals in different for male and female students of malaysian universities. the current study, in the context of malaysian as well as other countries' universities, has numerous implications related to e-learning portals technology. the study will be further helpful for the malaysian government and university policy-makers such as top management, ministry of higher education, malaysian universities union in designing the policies and programs on e-learning portal success in the country. at the same time, university top management/dean of faculty and hod of the department need to concentrate on the importance of enhancing university quality education. the study further implies that females students of malaysian universities are more focused on the e-learning portal as compare to male students. in the covid-19 outbreak, the operational habits have changed around the globe within a short period, and mainly the education sector affected worldwide. in the future, most of the universities will be offering online courses to the students. the covid-19 will create a long-term impact on higher education institutions. if the pandemic remains longer, it might change education from face to face to online. based on that, the quality of the e-learning system, quality of information that will create an impact on user satisfaction and system use that will lead toward the e-learning portals. it will decrease the education cost, and education will reach outside the border as well. education will be borderless in 5 to 10 years. this study recommends some of the suggestion to higher education institution such accessibility of the e-learning portal 24/7, error-free information, quality of information, content quality, the robustness of the server, training module materials related to e-learning portal use for new users, updated information, well-organized data, userfriendly design of the portal, and time to time feedback from the user will increase the durability and acceptability of the e-learning portal. this study focuses on comparisons between male and female counterparts on e-learning portal usage among university students. e-learning portal services adaptability in the among the student perspective of male and female. the present study highlight difference between the male and female success-fully use on the e-learning portal in malaysia. while future studies will have to consider some variables like (technology infrastructure support, system change perceived of use, perceived usefulness, user's perception, and technical expertise), moreover, the further studies can be conducted by applying longitudinal design to empirically test the theoretical constructs by analyzing other countries' universities. in the end, top management of universities is provided with recommendations for developing an understanding of the implication of e-service quality, information quality, system quality, system use, and user satisfaction concerning the e-learning portal success. e-learning readiness among students of diverse backgrounds in a leading malaysian higher education institution sharing instructors' experience of learning management system: a technology perspective of user satisfaction in distance learning course reviewing and exploring innovative ubiquitous learning tools in higher education intellectual capital and sustainable development: a systematic literature review grit in the path to e-learning success gamification: a key determinant of massive open online course (mooc) success review of e-learning practice at the tertiary education level in malaysia managing the impact of covid-19 on education systems around the world: how countries are preparing, coping, and planning for recovery improving productivity and firm performance with enterprise resource planning the challenges of data quality and data quality assessment in the big data era predicting the intention to use a webbased learning system: perceived content quality, anxiety, perceived system quality, image, and the technology acceptance model linking employees'e-learning system use to their overall job outcomes: an empirical study based on the is success model the investigation of e-learning system design quality on usage intention effectiveness of e-learning portal from students' perspective: a structural equation model (sem) approach. interactive technology and smart education e-learning success determinants: brazilian empirical study perceived usefulness, perceived ease of use, and user acceptance of information technology information systems success: the quest for the dependent variable the delone and mclean model of information systems success: a ten-year update information systems success measurement artificial intelligence in the agri-food system: rethinking sustainable business models in the covid-19 scenario human resources disclosure in the eu directive 2014/95/ eu perspective: a systematic literature review is success model in e-learning context based on students' perceptions enterprise systems success: a measurement model future trends in the design strategies and technological affordances of e-learning. learning, design, and technology: an international compendium of theory technology and e-learning in higher education partial least squares structural equation modeling (pls-sem) an emerging tool in business research partial least squares structural equation modeling: rigorous applications, better results and higher acceptance. long range plan user satisfaction model for e-learning using smartphone using pls path modeling in new technology research: updated guidelines. industrial management & data systems internet cognitive failure relevant to users' satisfaction with content and interface design to reflect continuance intention to use a government e-learning system assessing erp post-implementation success at the individual level: revisiting the role of service quality instructional design and e-learning: examining learners' perspective in malaysian institutions of higher learning. campus-wide information systems identifying the drivers of enterprise resource planning and assessing its impacts on supply chain performances extending the gable et al enterprise systems success measurement model: a preliminary study an empirical analysis of factors influencing internet/e-business technologies adoption by smes in canada information systems security policy compliance: an empirical study of the effects of socialisation, influence, and cognition instructional quality of massive open online courses (moocs) measuring information output: a communication systems approach user-developed applications and information systems success: a test of delone and mclean's model investigating users' perspectives on e-learning: an integration of tam and is success model e-learning, online learning, and distance learning environments: are they the same? influence of human resources to the effect of system quality and information quality on the user satisfaction of accrual-based accounting system innovation capability and firm performance relationship: a study of pls-structural equation modeling (pls-sem) validation of the delone and mclean information systems success model online or face-to-face? students' experiences and preferences in e-learning. the internet and higher education refinement and reassessment of the servqual scale servqual: a multiple-item scale for measuring consumer perceptions of service quality servqual: a multiple-item scale for measuring consumer perc information systems success: the quest for the independent variables does e-learning service quality influence e-learning student satisfaction and loyalty evidence from vietnam assessing the validity of is success models: an empirical test and theoretical analysis closure of universities due to coronavirus disease 2019 (covid-19): impact on education and mental health of students and academic staff multigroup analysis in partial least squares (pls) path modeling: alternative methods and empirical results success of e-learning systems in management education in chennai city-using user's satisfaction approach malaysian smes performance and the use of e-commerce: a multi-group analysis of click-and-mortar and pureplay e-retailers covid-19 impact on e-commerce usage: an empirical evidence from malaysian healthcare industry the mathematical theory of communication covid-19 infection: origin, transmission, and characteristics of human coronaviruses factors influencing users' satisfaction and loyalty to digital libraries in chinese universities e-learning success model for instructors' satisfactions in perspective of interaction and usability outcomes are we there yet? a step closer to theorizing information systems success publisher's note springer nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations acknowledgements the authors would like to thank the anonymous referees for providing helpful comments and suggestions which lead to the improvement of the paper.funding this research received no external funding. the authors declare no conflicts of interest. arfan shahzad 1 · rohail hassan 1 · adejare yusuff aremu 1 · arsalan hussain 1 · rab nawaz lodhi 2 key: cord-289775-40bi87iz authors: haines, david e.; beheiry, salwa; akar, joseph g.; baker, janice l.; beinborn, doug; beshai, john f.; brysiewicz, neil; chiu-man, christine; collins, kathryn k.; dare, matthew; fetterly, kenneth; fisher, john d.; hongo, richard; irefin, samuel; lopez, john; miller, john m.; perry, james c.; slotwiner, david j.; tomassoni, gery f.; weiss, esther title: heart rhythm society expert consensus statement on electrophysiology laboratory standards: process, protocols, equipment, personnel, and safety date: 2014-05-07 journal: heart rhythm doi: 10.1016/j.hrthm.2014.03.042 sha: doc_id: 289775 cord_uid: 40bi87iz nan the modern electrophysiology (ep) laboratory is a complex environment providing an array of interventions for the diagnosis and treatment of heart rhythm disorders and is a result of many transformations over the last three decades. the ep field has witnessed rapid expansion in the number of therapeutic procedures treating a wide range of arrhythmias and in the new technologies available to perform these procedures. because of the increasing complexity of equipment and procedures and an ever-expanding knowledge base, it was concluded that the field would benefit from a consensus document that would define the critical components and processes of a modern ep laboratory. to this end, the heart rhythm society (hrs) convened a multidisciplinary team to review ep laboratory design, ergonomics, personnel, equipment, occupational hazards, and patient safety, as well as clinical and ethical issues related to diagnostic and therapeutic ep procedures. the goal is to provide physicians, administrators, and regulatory personnel with the recommended requirements for building, staffing, and running a modern ep laboratory to optimize patient outcomes, minimize patient risk, and provide a safe and positive environment for physicians and staff. the writing committee was formed by the scientific and clinical documents committee of the hrs, with approval by the president of the hrs and the hrs executive committee. the composition of the committee was meant to represent the range of stakeholders in the ep laboratory. the choice of the writing committee members was in accordance with the hrs relationships with industry policy. 1 all members of the writing committee were required to fully disclose all potential conflicts of interest (see appendix 1). relatively little published literature addresses the ep laboratory environment, staffing, and processes. therefore, many of the statements in this document are the product of expert consensus by the writing committee and reviewers. for cases in which there were divergent opinions on a statement, a vote among writing committee members was taken, and if a two-third majority supported the statement, it was adopted in the document. the sections pertaining to pediatric and adult congenital heart disease were reviewed and approved by the pediatric and congenital electrophysiology society (paces), a nonprofit organization dedicated to the treatment of arrhythmia disorders in children and individuals with congenital heart disease (chd). the final document was approved by the board of trustees of the hrs. this document is directed to all health care professionals who design, manage, and/or work in the ep laboratory environment. the field of clinical cardiac electrophysiology (ccep) has grown from its origin as a field of clinical research for arrhythmogenesis to its present-day incarnation as an important specialty offering advanced therapies for a wide variety of disorders. clinical ep laboratories emerged in the late 1960s, and by the early 1970s, formal fellowships had been established and ep laboratories were taking shape. firstgeneration ep laboratories often shared space with cardiac catheterization laboratories and were typically subordinate to coronary angiographic and hemodynamic procedures. when a space was dedicated for electrophysiological testing, it was often small, and fluoroscopy was delivered with portable carm units. these laboratories were sufficient for diagnostic ep studies and electropharmacological testing. secondgeneration ep laboratories developed in the 1980s with the introduction of catheter ablation and cardiac implantable electronic devices (cieds) to the electrophysiologist's armamentarium. pacemaker implantation was shifting from the domain of surgery to that of cardiac ep. with increasingly complex procedures being performed in ep laboratories, more space was allocated to new dedicated laboratories and fluoroscopy equipment began to be upgraded to systems commensurate with those used in cardiac catheterization laboratories. the third generation of interventional cardiac ep has been driven by the success of catheter ablation and advanced device therapy. the precise anatomy and physiology of a wide variety of arrhythmias has been elucidated through the development of advanced mapping systems and improvements in ablation catheter technologies. modern device therapy incorporates multimodal multisite pacing, sophisticated therapies for tachyarrhythmias, and advanced diagnostics. with the increasing complexity of ep procedures and equipment has come increasing sophistication of laboratory processes and greater demands on laboratory personnel. the cost and complexity of the modern ep laboratory now demands that standards are developed to ensure a high level of care. highly complex procedures or procedures on patients with certain conditions and comorbidities that are associated with higher procedural risk should not be performed in a freestanding laboratory (i.e., an ep laboratory that is not physically attached to a hospital). emergency cardiovascular surgical support should be immediately available in case of life-threatening bleeding complications from the extraction of chronic device leads and complex mapping/ablation procedures, particularly those requiring pericardial access. high-risk procedures in critically ill patients, such as ablation of ventricular tachycardia in patients requiring extracorporeal hemodynamic support, can only be safely performed in institutions offering comprehensive programs with active engagement from electrophysiologists, surgeons, intensivists, and anesthesiologists. there are multiple options and practice settings for performing ep and implantable device procedures. medical centers may adopt one or more of the following laboratory operations for their practice. the choice among the following options involves a trade-off between increasing capability for procedure complexity and increasing construction and operating costs. in a dedicated ep laboratory, the staff space and procedure room space are separate from the cardiac catheterization laboratory and/or radiology laboratory, although the staff space and procedure room space often exist within a common area. the preparatory and recovery rooms are often shared with other subspecialties. procedures that can be performed in this laboratory setting include diagnostic ep studies, ablation procedures, use of cardiac implantable devices, implantable device extractions, use of temporary pacemakers, threedimensional (3d) mapping, intracardiac echocardiography (ice), and use of robotics. the advantages of using a dedicated ep laboratory include greater availability of more highly trained allied personnel, room equipment dedicated to only ep procedures, and decreased overall equipment costs per room. a shared procedural laboratory program is usually in association with a cardiac catheterization laboratory program, but can also be shared with an interventional radiology program. a shared room allows for two or more practices to share common equipment that includes fluoroscopic equipment, recording systems, emergency equipment, and anesthesia equipment, as well as the space. this is helpful in circumstances of low overall volumes when sharing a room allows for flexibility in patient care while controlling costs and space requirements. these types of procedure rooms have been created at largevolume institutions that can support a procedure room dedicated only to cied surgery. the procedures performed in this type of room include the use of pacemakers and defibrillators that are single chamber, dual chamber, or biventricular in operation. other procedures can include the use of temporary pacemakers, the use of implantable loop recorders, and lead and device extractions. device and lead extractions may also be performed in a surgical operating room (or) on the basis of the patient's condition or on the standard agreed on by the institution. advanced mapping and ep recording systems are not required, and the costs of equipping this type of laboratory are lower, which is the key advantage of this type of room. device-only laboratories are appropriate for high-volume centers that already have one or more fully outfitted ep laboratories. these procedure rooms are designed to the rigorous standards of ors (positive airflow, medical gas availability, surgical lighting, and substerile scrub area) but have high-quality fixed fluoroscopy and a full complement of ep and/or cardiac catheterization equipment. these rooms are ideal for procedures that may be combined with open or minimally invasive cardiac surgery and for lead extraction procedures. when not being used for hybrid surgical procedures, these laboratories can function either as fully functional ors or as fully functional ep/catheterization suites. procedures that can be performed include complex ablation procedures that involve ep and surgical components, left atrial appendage occlusion or clipping, epicardial lead placement, and minimally invasive valve replacement. some organizations incorporate special noninvasive rooms into their practice to accommodate patient care that does not require fluoroscopy or other specialty equipment. these rooms are often used to perform minor procedures such as cardioversions, tilt table studies, and noninvasive programmed stimulation defibrillation threshold testing. autonomic testing with head-up tilt table testing requires a procedure table that has the capability for 70º head-up tilt, an electrocardiogram (ecg) monitor, noninvasive blood pressure monitor, supplemental oxygen, and basic supplies. equipping these rooms is much less expensive than equipping a full procedural laboratory and can help improve patient flow and volume through a busy ep department. the room and equipment standards for pediatric ep procedures are similar to those for adult ep procedures, except for the availability of pediatric resuscitation equipment and drug doses as well as a wider inventory of smaller catheters. pediatric and congenital ep patients can require a combined procedure of ep and the need for cardiac catheterization, including angiography and possible intervention. thus, it is optimal (although not a necessity) for a pediatric/congenital ep laboratory to meet all the standards of a pediatric catheterization laboratory. pediatric ep procedures in young children should be performed in pediatric hospitals or hospitals that have a pediatric cardiology and ep service. an ep laboratory that is not physically attached to a hospital is considered a freestanding laboratory. freestanding ep laboratories can be privately owned, and when owned by physicians, there may be concerns about conflicts of interest (as discussed in section 12) . this arrangement presents challenges that stem from the separation of the laboratory from vital hospital services. in the event of a life-threatening complication, such as pericardial tamponade 2 or endovascular tear during lead extractions, 3 an emergency response from certain hospital-based services such as cardiothoracic surgery can become necessary, and even possibly lifesaving. performing ep procedures in freestanding ep laboratories on patients with clinical conditions that confer increased risk are relatively contraindicated. these include preexisting advanced heart failure and severe left ventricular dysfunction 4 ; recent myocardial infarction, recent stroke, chronic kidney disease, severe chronic obstructive pulmonary disease, pulmonary hypertension, and severe/morbid obesity 5 ; and severe valvular dysfunction or prosthetic heart valve, chd (including atrial septal defect repair), active oral anticoagulation, advanced age, and pediatric age. procedures that necessitate lesion creation close to coronary arteries, such as aortic cusp ablation 6 and epicardial ablation, 7 carry a higher risk of intraprocedural myocardial infarction and should not be performed outside a hospital. as part of the consent process, patients should be informed that the procedure is being performed without on-site surgical backup. in order to ensure the safety of a patient undergoing a procedure in a freestanding ep laboratory, a functional and tested system must be in place to quickly transfer patients to a hospital with immediate surgical support in case of an unanticipated complication. the receiving program should be familiar with complications unique to the ep laboratory. there must be a standing agreement between the laboratory and the receiving hospital so that there is no unnecessary delay in the transfer process. the hospital environment plays an important role in shaping the structure and function of the ep laboratory. a "closed ep laboratory" is commonly present in academic institutions and limits physician practice to faculty members of the particular institution or university. in contrast, "open ep laboratories" allow credentialing and the participation of multiple physician groups, including those who do not hold faculty level appointments. such laboratory structuring is common in community and private institutions and is also present in some academic settings. whether an ep laboratory is open or closed is determined by the institution's leadership on the basis of economic, historical, political, and geographical factors that are often beyond physician control. an inherent difficulty in the open ep laboratory format lies in procedure scheduling for multiple physicians; a centralized scheduling structure that can arrange scheduling while organizing and prioritizing procedures on the basis of urgency and acuity is important to avoid conflicts and optimize patient care. the complexity and degree of invasiveness of ep procedures is dependent on the level of support provided by the hospital or other health care organization in terms of personnel, facilities, and equipment. anesthesia support is desirable for the safe performance of potentially lengthy and complex procedures. the role of anesthesia services in the ep laboratory is detailed in section 6. surgery backup must be immediately present for lead extraction procedures in which a lead to be removed is older than 1 year (or require tools other than a standard stylet to be removed if younger than 1 year from implantation) 8 , and mapping/ablation procedures require pericardial access. complex ablation procedures, such as atrial fibrillation and ventricular tachycardia (vt) ablation, should be performed only in hospitals equipped and prepared to manage these types of emergencies, with access to emergency surgical support when required. finally, high-risk procedures in critically ill patients, such as ablation of vt in patients requiring hemodynamic support with extracorporeal membrane oxygenation, can only be safely performed in institutions offering comprehensive programs with active engagement from electrophysiologists, surgeons, and anesthesiologists. although such collaborations were limited to advanced tertiary care institutions in the past, the increasing availability of institutional resources and support has expanded the range of facilities in which complex procedures are performed to include private institutions. federal guidelines for the construction and retrofitting of health care facilities have been influenced by recent catastrophic events, such as the northridge earthquake of 1994, hurricane katrina in 2005, and the f5 tornado that made a direct hit on a hospital in joplin, mo, in 2011. in the mid-1990s, three formerly competing code writing agencies united to form the international code council. their mission was to develop a national construction code that, among other entities, would regulate the construction of health care facilities to mitigate the risk of damage due to seismic, wind, and flood dangers. known as the international building code, one of its versions has been adopted by every state. in addition, the federal emergency management agency, a branch of the department of homeland security, published revised guidelines for improving hospital safety in earthquakes, floods, and high winds. the primary legislative avenues for controlling the dissemination of expensive health care services are certificate of need (con) laws. as of 2009, 39 states still have a con process, law, or set of requirements. in most cases, the approval of con is based on the actual or projected volume of services provided in the procedural laboratories. as procedural volumes for percutaneous coronary arterial interventions have diminished at most tertiary referral hospitals, many hospitals have shifted some coronary interventional laboratory cons to ep laboratories. once an ep laboratory is established, the primary government body overseeing its operations, policies, and procedures is the joint commission (tjc). the guidelines for design and construction of hospitals and health care facilities published by the american institute of architects and the facility guidelines institute provide space and functionality standards for ep laboratories with a goal to improve work flow in the ep environment. (specific recommendations not derived from this document are based on the consensus opinion of the writing committee.) the minimal procedural area of a complete ep laboratory (not including control room space) is 350 sq ft of clear floor area. current electrical system regulations for health care facilities should follow article 517 of the national electrical code (nec) handbook. an uninterruptible power supply for all computer equipment is required. the air flow/heating, ventilation, and air conditioning design should comply with the guidelines for environmental infection control in the american institute of architects and the facility guidelines institute regularly publish the guidelines for design and construction of hospitals and health care facilities. 9 this document is recognized by federal and state authorities, and recently this document has included ep laboratories. it provides defined standards in terms of the space and functionality of ep laboratories with a goal to specifically improve work flow in the ep environment, acknowledging that the ep laboratory requires more space than an angiographic/interventional laboratory for supporting equipment and supplies. traditionally, however, the construction of an ep laboratory had no specific guidelines because of its special applications. the typical layout is generally derived from a cardiac catheterization laboratory, 9 which is not ideal for the performance of the full range of ep procedures. the limitations of direct adaptation of an angiography suite design to the practice of cardiac ep include space constraints relative to the special equipment used in ep procedures, the necessity to work on either side of the patient table, and the requirement to access the patient's upper chest for device implantation. ep laboratory plans should take into account not only the available space within the procedure room but also its location relative to pertinent services such as the patient prep area, recovery area, or, intensive care unit, the ward, and specialized resources such as an adjacent magnetic resonance imaging (mri) suite that might permit real-time mri imaging during procedures in the future. the rationale is to consider the proximity of all needed services in the overall design during the planning stage so that enhanced patient flow can be achieved. the aim of the planning committee should be to build a consensus on a minimum set of specifications that will meet the needs of the clinicians and support staff, and enable them to provide optimal patient care, while maintaining occupational safety for the staff. the ep laboratory needs as much space as is practical to ensure the freedom of movement of the operator and staff, to accommodate all equipment used, and to facilitate movement of staff in emergency situations. the recommended procedural area of a complete ep laboratory (not including control room space) is 500 sq ft or greater of clear floor area, although 350 sq ft is the absolute minimum requirement. there should be a minimum of 8 ft of clear space between the wall and the edges of each side of the patient table when it is positioned at the isocenter. enough clearance at the head of the bed should be allocated for anesthesia equipment on either side and sterile access to jugular vein entry sites, if employed, while allowing for free range of movement of the fluoroscopy c-arm. the ceiling height is dependent on the requirements of the x-ray/fluoroscopic equipment 9 (figure 1 ). preexisting laboratories that are being renovated where it is impossible to expand the gross area because of building and location constraints should follow federal and state code requirements, but due caution should be taken to meet suggested recommendations. the fluoroscopic equipment plays a major role in determining the amount of ideal space in the procedural area and could serve as the reference point. equipment can be either mounted on the floor or suspended from the ceiling. the latter configuration allows for the floor to be optimally cleaned; however, because of the amount of equipment that would need to be suspended from the ceiling (monitors, surgical lights, x-ray barriers, equipment racks, and anesthesia gas supply), a floor-mounted configuration may be more practical in some laboratories. it is best if x-ray generators and tanks are located in a space separate from the procedure and control rooms. the size and portability of the fluoroscopy unit is important in planning room size, especially when cabinetry and other fixtures are planned for installation on the walls within the procedural area. installation of cabinetry at the head of the bed is discouraged because it further limits space to allow free movement of the x-ray arm, anesthesia supply cart, and life support equipment. cabinetry for supplies frequently used during cases should be positioned on the side walls for easy access. the room should be wide enough to accommodate the cabinet and open door swing without impinging on the sterile field and traffic flow through the laboratory. most peripheral equipment such as recording systems, stimulators, and radiofrequency (rf) generators are made from multiple components, some of which need to be in a control room and others in the laboratory itself. it is strongly recommended that none of the modules sit on the floor. this can reduce sterility and cleanliness as well as put the equipment at risk of being damaged by fluids. a ceilingmounted boom removes all equipment from the floor and reduces damage to cables by allowing them to remain connected at all times. by placing the recording system amplifier, the rf generator, the mapping system amplifier, the stimulator amplifier and router, and other peripheral equipment together on a ceiling-mounted equipment boom, all cabling will be permanently placed and connected, reducing cable wear. the removal of rolling equipment carts from the room improves staff access to the patient. removing cables and equipment from the floor reduces the tripping hazard to the staff and risk of equipment damage. because additional portable ep equipment is often employed during a procedure, it is necessary to have ample power outlets installed to accommodate such needs. anesthesia gases are best supplied via a ceiling-mounted anesthesia boom, which should include two oxygen lines, one nitrous oxide line, one medical air line, two vacuum lines, and one waste anesthetic gas disposal line. 10 it should be equipped with at least one slide clamp for vacuum canister placement, which should allow the canisters to be located within 4 in. of the floor for ease of removal when full. the anesthesia boom should have a minimum of six electrical outlets, at least some of which should be on emergency (red plug) circuits in case of general power outage during a procedure. a mounted light controlled independently from the room lighting for charting in a dark room is a useful option. video can be routed from the anesthesia boom to display data from an anesthesia cart to monitors placed around the room. the hybrid laboratory has all the requirements of a full ep laboratory but has added features that allow it to serve as a fully functional operating suite. these laboratories are often larger and have the fluoroscopy equipment on a track so that it can be entirely removed from the surgical field. it is typically located within or contiguous to the other ors and has a full substerile scrub and supply area. the use of a hybrid laboratory for ep procedures is evolving. hybrid laboratories in which ep procedures are performed need to be outfitted with the appropriate ep-specific equipment, including ep recording systems, mapping systems, and programmed stimulators. procedures that might benefit from performance in this setting would include those where surgical intervention or extracorporeal hemodynamic support might be required, such as lead extractions, vt ablation procedures in patients with structural heart disease, and hybrid atrial fibrillation ablation procedures. although some ep laboratories house all the monitoring and stimulating equipment in the procedure room, it may be preferable to have a contiguous control room with an interposed leaded wall and large viewing window so that members of the team (apart from the primary operator, the circulating nurse, and the anesthesia professional) can work without exposure to ionizing radiation. the control rooms can be shared among two or more laboratories. a separate control room demands a full duplex intercom system so that there is no barrier to communication. the space required for a control room is not inclusive of the procedural area measurements. adequate ventilation should be supplied to account for excess heat production from the electronics. the counters should be at least 30 in. deep so that the monitors can be 20 in. away from the user. at least 160 in. of desk space is suggested for a laboratory with a singleplane fluoroscopy system and 180 in. of desk space for a biplane fluoroscopy system to allow for fluoroscopy monitors, a mapping system, a recording system, and a stimulator. an additional 45 in. of desk space is suggested for a two-monitor reading station or a single-monitor workstation ( figure 2 ). the participation of an ergonomics expert in the planning should be considered as a measure to comply with occupational safety and health administration standards. the ideal design for an ep suite should be similar to that of an or, including a substerile entrance with scrub sinks (dedicated or common). patient transport from the prep area to the ep laboratory and vice versa should be limited to a common egress that connects to hallways leading to the hospital wards and other areas. if the ep laboratories are placed in existing space that does not allow for or-quality substerile entrances and hallways, every effort should be made to prevent through traffic flow past the entrance to the ep laboratories where sterile procedures are being performed. ep suites require special consideration from electrical design engineers because there are multiple high load and electrically sensitive pieces of equipment in this wet environment. conduits used as wireways should follow the specifications of articles 376, 378, and 392 of the nec handbook. 11 the ep laboratory setup primarily involves the data and power cabling layout that connects equipment between the control room and the procedural area, and the following requirements should suffice, considering the few cables that need to be run in these enclosures. for rooms that are not equipped with ceiling-mounted equipment booms, the conduits should be at least two runs of 4-in.-diameter tubes that connect the procedure room to the control room through the floor, dedicated solely to ep equipment cabling (separate from x-ray equipment and power receptacle requirements). this conduit should be conductive and bonded to equipotential grounding. floor openings or ports should be concealed by an enclosure that should be fluid tight with protective grommets that will prevent cable insulation damage. the length/reach is dependent on the location of each cable termination linking the equipment, as specified by the ep representative who oversees the room project and design. for rooms equipped with ceiling-mounted equipment booms, cabling runs through ceiling trays connecting the control room to the procedure room boom. the trays can be used in conjunction with other equipment that terminates at the equipment boom as long as there is enough separation between power lines and data transmission lines to prevent electromagnetic interference (emi) induced by adjacent power lines running in parallel. open trays are preferable for ease of access above the ceiling and should be conductive and bonded to equipotential grounding. the length/reach is dependent on the location of each cable termination linking the equipment, as specified by the ep representative who oversees the room project and design. backup temporary cabling should be available in case of failure of conduit cabling during a case. current regulations for health care facilities should follow article 517 of the nec handbook. because the ep procedure room is classified as a "wet procedure location," the installation of an isolated power system with line isolation monitoring is required, which provides a layer of protection from the hazards of electric shock with the added benefit of line noise isolation because of its design. 9 in addition, all computer equipment directly related to the ongoing monitoring and treatment of a patient must have an uninterruptible power supply (ups). the ups may be integrated into the power for the entire suite, or individual ups may be placed in line for each central processing unit. the main purpose of the ups is to prevent the ep system, mapping system, or other critical imaging or monitoring system from going through a hard shutdown and full reboot procedure in case of a transient power outage or surge. other important electrical components of the laboratory, such as the imaging train, should be connected to emergency backup power so that cases can be completed even if line power is lost. power lines and data lines should be run separately and isolated from each other in different conduits to prevent emi from power line wiring induced through data line wiring that could affect optimal performance of the ep equipment. if open cable trays are used above the ceiling, careful consideration should be given to the placement of power lines and other fixtures that can be sources of emi. although power lines used on these runs do not necessarily involve enough energy to induce heating, it is still a good rule to follow the specifications of article 300.20 of the nec handbook as a reference. 11 adequate spacing of ep laboratory equipment in the procedural area should be followed. interface cables between the patient and the equipment (e.g., ecg cables and intracardiac catheter cables) should not dangle by the x-ray tube and should be kept neatly arranged by the side of the patient to provide easy access for troubleshooting purposes during the procedure. air flow should be of or quality. the design should comply with the guidelines for environmental infection control in health-care facilities recommendations from the centers for disease control and prevention and sections 5 and 6 of the healthcare infection control practices advisory committee document. 10 emphasis should be placed on the use of in-line filters or mechanical smoke evacuation systems to prevent airborne infective and toxic particles from the plume produced by electrocautery and similar equipment. the temperature control should support effective configuration for temperatures as low as 60ºf. this allows comfort for practitioners who are wearing sterile gowns, hats, and masks on top of lead aprons during long procedures. patient comfort should also be addressed, particularly as they are fully draped and may be only lightly sedated. the patient table should be flanked by large lighting squares or the equivalent to flood the main procedure area with light. appropriate grounding is required to prevent emi from these lights. the lighting squares should be tied to an x-ray pedal switch that can be turned on and off at will by the x-ray operator. additional spotlights that are dimmable from a distant wall switch are also recommended for procedures that require a darker environment to optimize glare reduction and visualization of display systems in front of the operator. there should be at least one overhead or light of surgical quality mounted on an articulating arm, strategically placed to be accessible for use on the left shoulder, right shoulder, or abdomen at either side of the patient. there should be sufficient range of motion to be able to focus light intensity at a steeper angle toward and into the implant pocket. two lights are optimal for reducing shadows. the preferred or light is mounted on a boom that extends from the ceiling and has free range of movement to focus the beam at the angles and distance optimal to adequately light the surgical field and device implant pockets. anesthesia and/or nursing should have a light over their workspace that is independent of the room lighting on either side of the patient table, which should be oblique at a distance from the x-ray c-arm. 9 for laboratory designs that employ a separate control room, there may be difficulties with the use of communication systems that link the operator in the procedure room to the control room staff. because critical processes such as timing of ablation onset and offset require close coordination between the bedside and the control room, the importance of good two-way communication for patient safety and quality of care cannot be overstated. the ideal equipment is capable of a always-on, two-way system because of the constant and instantaneous need to communicate. the ideal system is an always-on, full-duplex, two-way intercom system, with a toggle to silence unnecessary chatter from the control room. this requires electronic noise cancellation to prevent acoustical feedback and has variable effectiveness depending on room acoustics. a simpler solution is a oneway push-to-talk intercom, but this does not allow spontaneous back-and-forth communication. the use of wireless headsets is a favorable solution, which broadcasts spoken words directly to the headphone users, with simultaneous talk paths open as needed. whatever system is selected should be high fidelity, spectrum friendly, and encrypted to prevent eavesdropping and potential hipaa violations, making it a more expensive solution. procedural charting and operative reports should be part of the institution's electronic medical record. the network cabling and hardware should have a minimum capability of support for gigabit ethernet speed. 9 the data demands of imaging systems, including 3d electroanatomic mapping systems, are great and require larger storage repositories in comparison with the compressed images of major imaging equipment such as ultrasound and x-ray radiograph systems. there is an increased use of imaging created by computed tomography (ct) and mri, which are 3d in nature, necessitating high transfer speeds between the picture archiving and communication system (pacs) and the ep laboratory environment. collaboration with the information technology (it) department and its infrastructure within the institution is necessary in this venture. ep systems gather information in digitized format for patient records and review at a later time. it will be important for industry to develop a better and unified standard for storing and retrieving cardiac electrogram information. waveform information in ep is constructively different from image information and needs to be handled in a different manner. the complexity involved in translating the files without losing the ability to utilize the tool sets needed during review, and to scroll through the whole ep study, is a challenge. the digital imaging and communications in medicine standard is a more robust model to follow and should be the preferred method, when feasible. 12 for current equipment standards and needs, the recommendation is to involve the it department in the safekeeping of digital records of patient information. storing information in an enterprise-wide network repository managed by the health care it staff within the institution is recommended, as they are adequately equipped to comply with policies governing hospital data. data storage must be hipaa compliant 13 and must be maintained according to the laws of each individual state-typically 5-7 years for adults and 5-7 years past the age of maturity for pediatric patients. practically, the duration of data storage should be longer than the minimum requirement, because old invasive study data are often important in the management of patients decades later. electronic storage of all ep laboratory information could require 5-10 terabytes of space annually; therefore, the it department must anticipate commitment of these resources for this process. regardless of the equipment's capability to store to the network, the it department should be involved as long as they comply with the ep equipment manufactureres' recommendations. both single-plane and biplane fluoroscopic systems are suitable for the modern ep laboratory. a basic ep laboratory should be equipped with a monitoring system that includes 12-lead surface ecg and 24 intracardiac electrogram channels; advanced laboratories (e.g., those performing complex ablation procedures) require ep systems with 64-128-channel capabilities. a biphasic external defibrillator is required in each ep laboratory, with a backup defibrillator immediately accessible. an anesthesia cart that contains endotracheal intubation equipment, as well as sedative, paralytic, and anesthetic agents, should be readily accessible for all ep procedures. emergency trays should be immediately available for pericardiocentesis, thoracentesis, and thoracotomy. programmable electrical stimulators must provide reliable, accurate, and effective electrical stimulation. it is recommended that all ep laboratory personnel using the ablation systems are able to demonstrate familiarity and proficiency with the setup, operation, and characteristics of all ablation system(s) employed at their site. advanced mapping systems should be available for complex ablation procedures. ice may be useful as an adjunctive imaging modality during complex procedures. transthoracic echocardiography and transesophageal echocardiography should be readily available for emergency use and for adjunctive imaging in selected cases. integrated data display systems provide flexibility and efficiency in data display; it is advisable to have separate backup monitors in case of failure. patient safety and comfort are the most important considerations for the modern ep laboratory table. the ability to support a heavy patient is one of the most important features of the modern ep procedure table, with tables capable of supporting more than 200 kg being commercially available. the length and width of the table are also important considerations. although standard table lengths are usually sufficient to accommodate most patients, there is growing need for the increased width provided by bariatric surgical tables. motorized tables with adjustable height and a tilting capacity of up to 20º have become standard. tilting into the trendelenburg position may be helpful in cases of difficult subclavian venous access or internal jugular venous access in ablation and device procedures. reverse trendelenburg positioning can be helpful for patients unable to lie flat because of musculoskeletal or respiratory difficulties. table rotation up to 180º facilitates patient transport but more importantly provides better access to the table head in cases of emergency. this feature, as well as the ability to tilt sideways, may also be helpful for maximizing surgical exposure in hybrid or laboratories. given the need to perform both right-and left-sided procedures, having rails on both sides of the table is particularly useful for mounting equipment and tableside controls. finally, given the length of some ep procedures, in which patients may lay supine for several hours, a comfortable and supportive ep table pad is important. foam material is commonly used in ep table pads, but other materials are also available. although fluoroscopy remains the mainstay of ep procedures, it is imperative to reduce ionizing radiation exposure to patients, operators, and staff as best possible. specific issues related to radiation and limiting exposure are detailed in section 11. the complexity of procedures performed in the laboratory is the primary determinant of the specific fluoroscopy features needed. both single-and biplane fluoroscopic systems are suitable for the modern ep laboratory, and the choice of the system is dictated by the specific needs of the laboratory. in basic ep laboratories designed primarily for device implantation, a single-plane system is usually sufficient. biplane systems are often preferred in more advanced laboratories where ablation is performed, as these biplane systems can be converted to single-plane units for device insertion; however, the advent of 3d mapping technology has diminished operator reliance on biplane fluoroscopy. the introduction of digital imaging has been the most important recent change in fluoroscopic imaging. digital flat panel detectors permit reduction in radiation and provide excellent image quality with a physically smaller and thinner detector. these systems allow greater temporal resolution and contrast ratio with less image distortion and veiling glare and allow the acquisition of high-quality still images. the latter feature is particularly useful for procedures depending on the imaging of vascular structures such as coronary arteries, the coronary sinus, and its branches. floor-and ceiling-mounted units are available depending on the exact specifications and setup of the laboratory space. some digital fluoroscopic systems offer advanced imaging capabilities, which may be useful in ep procedures including rotational angiography, rotational ct imaging, and multimodality integration of 3d magnetic resonance and ct images. these features are generally more suited for advanced laboratories performing complex ablation procedures. three-dimensional reconstructed images from ct, mri, and rotational fluoroscopy can guide ablation planning, catheter navigation, and catheter ablation. 14 the pattern of myocardial scarring defined by delayed enhancement mri scanning can influence the method of access (endocardial vs. epicardial), catheter type, and type of mapping technology. 15 in the setting of atrial fibrillation ablation, a preprocedural 3d image can be helpful in cases of unusual atrial or pulmonary vein anatomy. creation of a 3d map during the procedure using a mapping system can obviate the need for a preprocedural 3d image. an ep system refers to the hardware and software programs that allow clinicians to record, display, store, and review data acquired during ep procedures. the monitoring system includes a computer workstation with both local and bedside high-resolution color display monitors, a recorder, amplifiers and filters for signal acquisition and processing, a printer, and device interface cables. the workstation contains an integrated computer that uses data processing software with amplifiers and adjustable filters to process and display electrogram signals and waveforms. at a minimum, the system should contain 12-lead surface ecg and 24 intracardiac electrogram channels, which is sufficient for the basic ep laboratory. advanced laboratories performing complex ablation procedures require ep systems with 64-128-channel capabilities to simultaneously record signals from different multipolar catheters and display hemodynamic data from arterial and/or left atrial pressure transducers. useful features for ep systems include a triggered sweep, template matching, and capability to save fluoroscopic images. these data are displayed on color monitors that include both real-time and review screens for visualization and analysis of electrogram signals during mapping and ablation. the number of available channels displayed on color monitors is configurable and differs among the various ep systems. storage capabilities are often included in ep systems with various hard disk capacities and digital media for archival purposes and retrieval of data. ideally, data should be stored in a central repository and be available to any workstation over the network. integration and interfacing with rf-generating devices, fluoroscopy, mapping, and ablation systems are also important components of the system. finally, the systems should be capable of communicating with institutional information systems and electronic medical records. resuscitation equipment is mandatory, given the potential for induction of malignant arrhythmias. a biphasic external defibrillator is required in each ep laboratory, with a backup defibrillator immediately accessible. routine preventative maintenance of external defibrillators should be performed, according to u.s. food and drug administration (fda) guidelines and manufacturer recommendations. 16 a crash cart containing standard advanced cardiac life support (acls) medications must be available to assist with the management of tachy-and bradyarrhythmias. standard acls medications should be available, including, but not limited to, epinephrine, atropine, dopamine, vasopressin, adenosine, amiodarone, and lidocaine, in addition to magnesium sulfate, calcium chloride, potassium chloride, and sodium bicarbonate. sedative reversal agents should also be available, including flumazenil and naloxone. it is essential that the laboratory be stocked with appropriate long needles, guide wires, and catheters for emergency pericardiocentesis and that all operators and staff are familiar with the use of this equipment. given the increasing complexity of ep procedures and the potential need for general anesthesia, an anesthesia cart that contains endotracheal intubation equipment as well as sedative, paralytic, and anesthetic agents is highly recommended. this includes a resuscitator bag and mask, a nonrebreather mask, suction equipment, and arterial blood gas kits. such a cart should also contain a separate monitoring system for ecg and hemodynamics, including a pressure transducer and end-tidal carbon dioxide monitor, and should be available even in cases not staffed by an anesthesiologist. finally, all modern ep laboratories should possess high-flow oxygen and vacuum for suctioning as detailed in section 9. programmable electrical stimulators are the mainstay of ep studies and must provide reliable, accurate, and effective electrical stimulation. modern programmable electrical stimulators have multiple output channels, usually ranging from two to four channels. it is important for these channels to be independent and isolated and to accurately provide stimuli of adjustable amplitude and pulse duration. burst pacing and delivery of one or more premature extrastimuli are standard features of all stimulators. in addition, some modern stimulators are fully automated and have the capacity of delivering several types of preprogrammed stimulation protocols to assess physiological parameters such as thresholds, sinus node recovery times, refractory periods, and wenckebach periods. in order to perform catheter ablation of cardiac arrhythmias, an ablation system is required in the ep laboratory. ablation systems generally consist of a generator, cables, and catheters for the delivery of energy and may or may not include a ground patch, depending on the energy source. the ablation systems should interface with ep monitoring and electroanatomic mapping systems. energy sources can be in the form of rf ablation, cryoablation, ultrasound ablation, microwave ablation, and laser ablation. rf and cryotherapy sources are the most widely clinically utilized, and a discussion of the other sources is beyond the scope of this document. rf ablation as a therapeutic modality is the most commonly used and has been proven to be highly effective and safe for the treatment of a wide array of arrhythmias. 17 irrigated rf energy ablation systems require an irrigation pump to infuse saline in either a closed-or an open-irrigated tip catheter. cryoablation systems consist of a cryocatheter, a refrigeration console with nitrous oxide, a coaxial tube for the delivery of nitrous oxide, and an electrical cable. during cryoablation, heat is removed from the tissue by using a refrigerant (nitrous oxide) in a closedirrigated tip catheter. cryoablation can be delivered at a single site (catheter based) or over a larger tissue area (balloon device). the selection of ablation modality depends on operator preference, patient size, 18 and ablation target. rf energy remains the most established modality for ablation. cooled rf technologies are generally employed where deep and/or transmural lesions are required, such as with vt ablation. either irrigated rf energy or the cyrothermic balloon ablation system is commonly used for atrial fibrillation ablation procedures, depending on operator preference. it is desirable for an ep laboratory to have more than one type of ablation system, but the selection of an ablation system and energy type is entirely discretionary. different catheters have different handling characteristics, and different ablation systems have different strengths and weaknesses. it is recommended that all ep laboratory personnel using the ablation systems are able to demonstrate familiarity and proficiency with the setup, operation, and characteristics of all ablation system(s) employed at their site. three-dimensional electroanatomic mapping systems are commonly used in the ep laboratory for the acquisition of accurate and reproducible electrical and anatomic information and display in 3d. reconstruction of complex cardiac geometry with direct nonfluoroscopic catheter visualization is combined with endocardial electrogram data to create a 3d map of the cardiac chamber. advanced signal processing can present acquired electrophysiological data in a variety of formats to direct the operator to optimal ablation targets. in addition, standard fluoroscopy, ct, mri, and intracardiac ultrasound images can be integrated with electroanatomic mapping systems to link electrogram information with anatomical structures. this allows nonfluoroscopic catheter localization, reducing radiation exposure during catheter ablation procedures. 19 mapping systems consist of a workstation computer, local and bedside monitors, fiber-optic media converter with a fiber-optic cable, an amplifier, diagnostic and ablative catheters, and a patient interface unit that provides the central connection of the computer system to catheters, cables, and the amplifier. the system can interface with recording systems and integrate with ultrasound, fluoroscopy, and ct/mri systems. the system consists of a workstation computer, local and bedside monitors, an amplifier, fiber-optic media converter with a fiber-optic cable, and a multielectrode array catheter. ice is often useful as an adjunctive imaging modality during complex procedures. it has the potential to improve both the safety and the efficacy of a procedure. dynamic visualization of intracardiac structures, catheters, and other procedural devices is possible using ice. the ability to use this modality in real time is an advantage that improves the work flow of the procedure compared with using other pre-or postprocedural augmentative imaging modalities. using ice to directly visualize and confirm the proper position of the transseptal needle on the atrial septum can minimize procedural complications, such as cardiac perforation. pulmonary vein stenosis can be avoided by using ice to confirm an ostial position of the lasso catheter during pulmonary vein isolation. 20 early detection of complications, such as pericardial effusion or intracardiac thrombus formation, can lead to earlier and more effective interventions. 21 fluoroscopic exposure and its associated risks can be minimized when navigation of catheters and procedural devices are guided by using ice. 22 the success of a procedure can depend on the recognition and successful navigation of challenging anatomy that can be detectable through ice, such as a prominent eustachian ridge during atrial flutter ablation, a crista terminalis ectopic tachycardia focus, or a ventricular arrhythmia involving the papillary muscles or aortic cusps. 23 contact of the ablation catheter with tissue can be verified before the delivery of ablative energy, and ablative effects on the tissue can be monitored by assessing morphological changes, including tissue swelling and increased tissue echogenicity. presently, two different types of ice systems are available: systems using a linear phased array transducer that produces a 90º image longitudinal to the catheter and systems that use a rotational transducer to display a 360º image perpendicular to the catheter. each system has relative advantages and disadvantages, and their selection is based on operator preference. some ultrasound catheters can work with 3d electroanatomic mapping systems and can import 2d ultrasound images to augment 3d electroanatomic mapping. 24 despite the potential value of ice, reviewed in detail above, it is important to recognize that clinical trials are not available to demonstrate that the use of ice improves the outcomes or safety of ablation procedures. although some operators and centers depend heavily on ice, many others use it only in selective situations. ice substantially increases procedure costs, requires an additional site for vascular access, and requires extensive training in order to accurately interpret the images. 25 catheter movement can be performed using robotic navigation systems, allowing for reproducible complex catheter manipulation, improved tissue contact and stability, and the potential for more efficient and efficacious lesion formation. because of the automated nature of catheter navigation using 3d anatomic mapping systems, fluoroscopic exposure may be reduced, especially for the primary operator, who typically performs the ablation procedure seated in the control room. this may also translate into less orthopedic strain from the use of lead aprons. two distinctly different types of robotic navigation systems are currently available. robotic arm systems use steerable sheaths to direct catheter movement. these systems can use a full array of conventional catheters, including irrigated ablation catheters. the rigidity of the sheath and the lack of tactile feedback increase the risk of cardiac perforation and pericardial tamponade. 26 pressure sensor technology is used to assess appropriate tissue contact and to avoid perforation, but can be confounded by indirect forces and tortuous catheter positions. a simpler robotic approach to control the catheter movement involves the use of a robotic arm to remotely manipulate a steerable ablation catheter exactly as an operator would manipulate the catheter directly. 27 although the operator sacrifices the tactile feel of catheter manipulation with this system, it allows the operator to move to a radiationfree space and to perform the ablation from a seated position. magnetic systems use two large banks of external magnets to manipulate a magnetized catheter. these magnets can be either solid magnets that are physically moved or electromagnets using electromagnetic field manipulation. specialized ablation catheters for these systems are available, including open-irrigated tip catheters. because the body of the catheter has no rigidity and the catheters are directed solely by a limited low-intensity magnetic field, the risk of cardiac perforation is virtually eliminated. 28 the constant magnetic force holds the catheter in contact with tissue, even during cardiac and respiratory motion, translating to potentially more precise and efficacious lesions. 29 the use of robotic navigation systems takes the primary operator away from the patient's side during the procedure; thus, subtle changes in clinical status that are usually noticed in close proximity to the patient or the tactile sensation of a steam pop may no longer be detectable. hence, close monitoring by an anesthesiologist and the nursing staff is of paramount importance when robotic navigation is being used. as the breadth of technologies in the modern ep laboratory has grown, so too has the challenge of displaying information in a meaningful and useful way. the model using a fixed number of separate monitors, each displaying a single signal, is not well suited for laboratories using multiple systems and performing complex procedures. modern advanced laboratories have increasingly taken advantage of integrated data display systems (iddss). these iddss replace the multiple fixed monitors with a single large screen that displays multiple signals, thereby allowing the physician and laboratory staff to display as many images as required in whatever layout they choose. not only do iddss enhance flexibility, they also diminish the physical requirements for monitoring, thereby liberating space within the ep laboratory. the drawback of iddss is the addition of another layer between the operator and the source systems that may be susceptible to image distortion or complete failure that would affect all signals. thus, it is necessary to have separate backup monitors for critical functions in case of failure. lastly, iddss should have a simple, intuitive user interface; otherwise, any benefit they provide would be outweighed by issues relating to the complexity of use. telemedicine has grown in many areas of medicine over the past decade, and ep is no exception. in fact, ep is better suited than most specialties to leverage this growing trend, thanks in part to the integration of many laboratory systems into a single interface and to advances in remote catheter navigation systems. remote diagnostics are already a reality because of the growth of several networks that link various laboratories and facilities together. physicians from a number of institutions can broadcast live and prerecorded procedures and perform real-time consultations with other participating facilities. remote surgery has been demonstrated using the current generation of remote catheter navigation technologies and has been further bolstered by the addition of newer laboratory integration systems. while the requirements for remote surgery are similar to those of remote diagnostics, there should be much less tolerance for latency and system responsiveness as well as enhanced fail-safe measures and the ability for local override. significant gaps in state, federal, and international regulations will need to be addressed before telemedicine can reach its full potential in this field. medical staff credentialing committees should be familiar with the training and credentialing standards for specialists in cardiac arrhythmias. staff physicians must have prerequisite training and appropriate credentialing reflecting expertise in the management and treatment of cardiac arrhythmias. because of the complexity of the ep procedures, patient safety and positive outcomes are critically dependent on the skill levels of the staff. additional staff is needed as the complexity of the case increases and more equipment is required. it is desirable that anesthesia services be an integral part of clinical practice in the ep laboratory. advanced practice nurses (apns) and physician assistants (pas) should be used in areas where they will have a maximum impact on patient care and where they can assume roles and responsibilities unique to their training and certification. at least one registered nurse should be present for every invasive procedure in the ep laboratory. industry representatives should function according to clear policies under the direction of the laboratory manager, staff, or physician. staff physicians must have prerequisite training and appropriate credentialing reflecting expertise in the management and treatment of cardiac arrhythmias. training requirements and guidelines for pacemaker/icd selection, implantation, and follow-up as well as catheter ablation procedures have been addressed by the american heart association (aha), american college of cardiology (acc), and hrs [30] [31] [32] [33] [34] and are addressed in section 7. physicians performing procedures in the ep laboratory often supervise the administration of intravenous sedatives given by the nursing personnel in the laboratory. therefore, all physicians in the laboratory should demonstrate proficiency in sedation pharmacology, patient monitoring, and airway management. there should be a credentialing process in the institution that establishes a standard for conscious sedation management. the ep laboratory medical director must be an expert in ccep and satisfy the above requirements, in addition to carrying out important administrative duties that include physician leadership, patient care clinical leadership, quality of care, and education. as a physician leader, the medical director is responsible for providing overall medical direction and supervision within the ep laboratory. the roles and responsibilities of the other ep staff physicians must be specifically outlined by the director so that there are clear measures by which the ep staff physicians are evaluated. ensuring staff members are appropriately credentialed and that they are maintaining cognitive and procedural competency is important for maintaining up-to-date health care provider standards. the laboratory director should work with the institution's leadership to establish specific trainingand volume-based credentialing and recredentialing criteria based on published clinical care guidelines (when available). those criteria should be understood and adhered to by all. the medical director must develop and implement quality measures that result in fewer complications, reduced cost, and successful patient outcomes. working closely with administrative staff to develop policies, procedures, and practice guidelines impacts accountability measures used by accreditation authorities, including tjc and the national committee for quality assurance. additional responsibilities may include planning or coordinating ongoing educational opportunities for all ep personnel, championing the ep service line, identifying budgetary savings and efficiencies, participating in or initiating purchasing of capital items that keep the service line current, and assisting as requested with the development and review of ep-related policies and procedures. policies should be compatible with other areas with which the ep service interacts, such as the prep and recovery areas, anesthesia, surgery, and the cardiac catheterization laboratory. faculty physicians typically work in a teaching hospital or affiliate institution. they must satisfy the same qualifications as above, in addition to those set forth by the accreditation council for graduate medical education (acgme). these requirements are quite rigorous, and failure to adhere to requirements may result in the program being placed on probation or loss of accreditation. although certain components of the procedure can be delegated to a trainee or other secondary operator, the laboratory's attending physician of record is ultimately responsible for all activities within the laboratory and for patient welfare. it is important for the staff physician to recognize that patient safety and successful outcomes depend greatly on effective communication in the ep laboratory. this communication should include preoperative discussions with all members of the team before the case is underway regarding specific patient needs. the physician should review the diagnosis, indications for the procedure, anticipated equipment needed, and potential findings of the procedure. the patient should have a clear understanding of what to expect postprocedure in order to minimize anxiety. after the procedure, clear communication of the procedure findings, postprocedure orders, and recommendations should be exchanged with the treatment team, including physicians, apns, pas, and nurses. secondary operators are those physicians assisting with a procedure who might or might not participate in certain aspects of ep procedures and who might bill separately for an area of expertise not provided by the primary physician in the laboratory (table 1) . their role is planned and limited to nonemergency procedures. the patient should be informed before the procedure of any secondary operators expected to be assisting with the case. the role of the fellow can be variable and dependent on the attending physician present in the laboratory. there are specific requirements that each fellow in training must satisfy in order to successfully complete his or her training and be eligible for the american board of internal medicine (abim) certification examination (or american board of osteopathic medicine for those individuals following the osteopathic route). the fellow should begin under the direct supervision of a key clinical faculty member from the training program. with ongoing evaluation and feedback, the fellow is given graduating responsibility. varying levels of supervision are appropriate depending on skill level and level of training. it is appropriate for fellows to perform components of the procedure without direct supervision (such as vascular access, catheter placement, device pocket incisions, and pocket closures), but the attending physician must be available to intervene promptly if any issues arise. it is desirable that anesthesia services be an integral part of clinical practice in the ep laboratory. an anesthesia group composed of anesthesiologists and certified registered nurse anesthetists (crnas) can provide a high level of perioperative/periprocedural care to patients undergoing ep procedures. having anesthesia services readily available for the ep service is advantageous. the anesthesia service can provide important educational assistance to nonanesthesia staff administering conscious sedation, such as training on the use of various sedation agents, and the use of special monitoring techniques such as capnography. patients undergoing ep procedures present special challenges related to sedation. it is imperative that sedation/anesthesia personnel function collaboratively with the electrophysiologist in the management of these patients during procedures. procedural issues relating to anesthesia management are discussed further in section 8. to ensure optimal safety and efficacy of interventional ep, it is important to emphasize the necessity of a multidisciplinary team approach. in this respect, the term allied professionals has been employed. allied professionals are defined as all nonphysician members of the health care team involved with the care of the patient in the ep laboratory. this includes, but is not limited to, registered nurses (rns), ep technologists, radiological technologists, certified nurse practitioners (nps), pas, crnas, patient prep and recovery staff, and or staff. other key personnel that are important for the safe and efficient function of the laboratory include quality assurance (qa) staff; information technologists; biomedical engineers; scheduling coordinators; purchasing, inventory, and supply personnel; and housekeeping. based on evidence-based practice and best practice patterns, it is important to acknowledge that there is limited published research regarding the roles and responsibilities inherent in ep. recommendations as to how these positions may be filled by any one of the several categories of personnel are discussed below. apns and pas can play major roles and serve many functions in the ep laboratory, as determined by the director of the laboratory. they should be placed in those areas where they will have maximum impact on patient care and assume roles and responsibilities unique to their training and certification. apns are often placed in clinic settings where they may evaluate and treat arrhythmia or device-related issues. they can make rounds on inpatients, make assessments, develop plans for care, write histories and physical exams, and admit and discharge patients. they can perform pre-and postprocedural evaluations and follow-up. particularly in nonacademic institutions or practices, an apn or pa may function as the most experienced or skilled nonphysician practitioner in the laboratory setting and thus function as a first assistant for many technical aspects of the procedure. each institution should have established policies defining the role of the apn and/or pa in the care of hospital patients. an rn should be present for every invasive procedure in the ep laboratory. the nurse must be familiar with the overall function of the laboratory as well as coordinate with the physician operator and the other team members. the nurse (either rn or crna) is the primary individual responsible for the direct observation, sedation, and nursing care of the patient during the ep procedure and must be prepared to respond to any emergency. the number and type of nursing personnel required in the ep laboratory will vary depending on the type of procedure, equipment used, and additional support staff assigned to the procedure. 35 ep procedures are complex by their nature, and it is essential that the nursing staff participating in such procedures provide safe, evidence-based care. in institutions where nurses are responsible for the administration of intraprocedural sedation, they are to follow institutional training and guidelines for the care of the patient. when a nurse is administering deep sedation, his or her focus should be only on monitoring patient status, vital signs, oxygenation, and level of sedation. however, during moderate or light sedation, this individual may assist with minor, interruptible tasks once the patient's level of sedation/analgesia and vital signs have stabilized, provided that adequate monitoring of the patient's level of sedation is maintained. 36 the nurse can also manage point-of-care testing for activated clotting times (acts), oxygen saturation, and blood gas measurements. in most states, only rns may administer medications and blood products. the nurse optimizes patient safety by adhering to policies, protocols, and procedures, such as completing the "active time-out" preprocedure and ensuring that the proper airway assessments are completed before the administration of sedation. keeping a record or charting during the procedure is generally the responsibility of nurses. in addition, training on the use of stimulators, infusers, and ablation generators is recommended so that nurses are able to function in multiple roles. overall, nurses are coordinators of all patient care in the laboratory and they oversee the care other allied professional-ep personnel are providing. because of the extremely complex and technical aspects of many ep procedures, there should be at least one additional person involved in the more complex procedures, in addition to the nurse who provides direct patient care. depending on the complexity of the procedure, there may need to be more than one additional person. in this arena, specific training, experience, and certifications may determine which team member occupies each role. for example, a nurse and a technologist may be equally capable of performing a certain duty or responsibility but economics, staffing availability, and the simultaneous performance of multiple duties can dictate who does each job in the laboratory. because of the multiplicity of roles, it is useful for members of the ep team to be cross-trained and be able to function in multiple roles and situations. there is a wide array of additional equipment that requires training to operate. this includes, but is not limited to, lasers, energy source generators, electroanatomic mapping systems, robotic and magnetic catheter navigation systems, echocardiography (transesophageal and intracardiac), and ct and mri imaging. in many laboratories, it is the technologist or nurse who monitors and operates the recording system. this activity requires a thorough understanding and knowledge of the electrophysiological properties of the heart as well as pacing protocols and ablation. the operator must be able to troubleshoot pacing problems and remain calm and functional in emergency situations. all technologists must have basic cardiac life support certification, and acls certification is preferred. in the pediatric laboratory, pediatric cardiac life support certification is required. as with the nurse, the technologist should have the ability to review, understand, and synthesize into practice new knowledge and practices. ep technologists perform as essential team members. they may be a first assistant, which requires in-depth knowledge of percutaneous procedures, catheters, sterile technique, energy generators, and integrated noninvasive imaging. they should be trained in the use, maintenance, and troubleshooting of all the equipment. ep technologists should be skilled in sterile technique, passing sterile supplies, and obtaining and performing point-of-care testing on blood samples. they are often the person who assists on device implant cases and lead extractions -roles that require fastidious adherence to sterile technique and an in-depth understanding of the implant process along with its risks and goals. a technologist or nurse may serve as the first assistant for an invasive case. the circulator is typically a nurse, but this role can be filled by a technologist, depending on the staffing mix in the laboratory, the scope of practice in this job description, and the institutional requirements. at least one department member should be a certified radiological technologist or equivalent technologist with expertise in the operation of fluoroscopic equipment as well as expertise in radiographic and angiographic imaging principles and techniques. requirements for the participation of a radiological technologist in fluoroscopic procedures vary from state to state. in conjunction with a qualified medical physicist, the radiological technologist should monitor radiation safety techniques for patients and laboratory personnel. in many states, the nuclear regulatory commission has specific regulations for who may operate ionizing radiation equipment and under what circumstances. it is imperative that these regulations are understood and followed in the laboratory for the protection of patients and staff. device programmers, mapping and recording systems, and some ablation systems may sometimes be operated by industry representatives. industry representatives must function according to clear policies under the direction of the laboratory manager, staff, or physician. they are often required to provide the institution with evidence of appropriate immunizations, competency documentation, and endorsement from their company before being allowed in the laboratory. they are generally allowed to have patient contact only under direct staff supervision. 25 during device implants or other device-related procedures, a clinical industry representative may be present under the direct supervision of the attending physician. they may bring device equipment to the laboratory, provide intraprocedural programming and testing, and may even be asked to participate in data collection related to device registries. a member of the ep laboratory staff, however, should be assigned the ultimate responsibility for the accurate and complete submission of data to national device registries. these industry representatives are often excellent sources of information and education for the regular ep laboratory staff. they may assist in the provision of formal training and education on device-related issues. 25 to ensure optimal safety and efficacy of interventional ep, it is important to emphasize the necessity of a multidisciplinary team approach. ep procedures are complex and include diagnostic, interventional, and therapeutic measures and should be performed by experienced personnel. because of the complexity of the ep procedures, patient safety and positive outcomes are highly dependent on the skill levels of the staff (table 2) . therefore, personnel dedicated to ep laboratory procedures are recommended. additional personnel are needed as the complexity of the case increases, and more equipment is required. the staffing mix may be influenced by regulations, regional practice patterns, type of institution (academic vs. nonacademic), credentialing bodies, and economics. cross-training of staff within the ep department maximizes staffing flexibility and is strongly recommended. the role of the ep department administrator is typically held by someone with broad knowledge of the field of ep. depending on the size and volume of the laboratory, the administrator may have no clinical obligations or may serve as the head nurse of the laboratory. the responsibilities can include, but are not limited to, the following: strategic planning in association with the medical director, managing operational issues, capital planning, budgeting, hiring, planning orientations and training programs for allied professionals, and other general administration duties. a nurse or a cardiovascular technologist, preferably with some business training or experience, is best suited for this role. in shared or combined cardiac catheterization laboratories and ep laboratories, there may be a common administrator overseeing both areas. in many departments, the manager is a nurse. the responsibilities of a nurse manager include an overall understanding of the day-to-day operations of the laboratory, management of preand postprocedural care areas, and direct participation in the observation and care of patients undergoing ep procedures. additional areas of responsibility include application of institutional guidelines for patient monitoring, medication administration, procedural sedation, and patient safety. staff competencies and proficiency in performing tasks required before, during, and after the procedure must be developed, updated, and reviewed on a regular basis. the nurse manager will collaborate with anesthesia, pharmacy, biomedical engineering, purchasing, equipment vendors, and housekeeping to coordinate the operation of the ep laboratory. all clinicians working in the ep laboratory have a responsibility to achieve and maintain the recommended credentials and continue medical education to optimize patient care. it is recommended that any non-ccep-certified physician who wants privileges for implantable cardioverter-defibrillator implantation should complete formal training in this field as defined by the cocats criteria, achieve certification of competency in cardiac rhythm device therapy for the physician (ccds) from the international board of heart rhythm examiners (ibhre), and maintain an adequate volume of device implants to meet hospital-based credentialing criteria. for the benefit of patients, it is paramount that physicians be held to a high performance standard and that remediation, withholding recredentialing, or revocation of privileges occurs if criteria are not met. 31 all ccep board-certified physicians have completed at least 1 year of comprehensive subspecialty training in ccep at an acgme-approved training program 30 or had substantial experience and a career focus in ccep if they trained in an era before the development of formal ccep training programs. the majority of programs encourage a second year non-acgme advanced fellowship. the 2013 guidelines for advanced training in pediatric and congenital ep represent procedural requirements for those completing training. 37 the clinical competence statement on invasive electrophysiology studies, catheter ablation, and cardioversion, 34 supplemented by expert consensus statements on crna ¼ certified registered nurse anesthetist; cv ¼ cardiovascular; ep ¼ electrophysiology; md ¼ physician; np ¼ nurse practitioner; pa ¼ physician assistant; tee ¼ transesophageal echocardiography. * in procedures performed with deep sedation/analgesia, the crna or nurse administering sedation/anesthesia should have no responsibilities other than monitoring the patient. a second nurse or technologist must be available to circulate and document. however, in procedures performed with moderate or light sedation, this individual may assist with minor, interruptible tasks once the patient's level of sedation/analgesia and vital signs have stabilized, provided that adequate monitoring for the patient's level of sedation is maintained. 52 transvenous lead extraction, 8 catheter and surgical ablation of atrial fibrillation, 38 and catheter ablation of ventricular arrhythmias, 39 provides guidelines for appropriate training in ccep. this document is scheduled to be updated in the near future. as training standards evolve, these minimum requirements will be updated regularly. a successful passage of the abim ccep board examination is required to receive the certificate of added qualification in ccep. a similar but alternate pathway is available for doctors of osteopathy through the american board of osteopathic medicine. physicians for whom these pathways are unavailable because of international training or pathway choices and who are actively involved in the clinical management of ep patients may choose to certify through the ibhre with the certification examination for competency in cardiac electrophysiology. physicians performing complex catheter ablation procedures, such as atrial fibrillation/complex atrial tachycardia ablation and vt ablation, should treat at least 25 cases of each with an experienced mentor before becoming independent. alternatively, they should perform these procedures during their ccep training program, as recommended by the 2012 hrs/ehra/ecas expert consensus statement on catheter and surgical ablation of atrial fibrillation 38 and the 2009 ehra/hrs expert consensus on catheter ablation of ventricular arrhythmias. 39 a number of training pathways can lead to the practice of implanting cieds. although physicians who are board certified in ccep have met minimum training standards for cied implantation, some cardiovascular diseases (cvd) board-certified physicians and some american board of surgery-certified cardiac, thoracic, and general surgeons also devote a substantial portion of their practice to the prescription, implantation, and follow-up of cieds. the criteria established by the abim program requirements 28 and the recommendations for training in adult cardiovascular medicine core cardiology training document 33 strongly recommend that any non-ccep-certified physician who wants privileges for icd implantation in adults completes formal training in this field. achieving ccds from the ibhre for cied prescription, implantation, and follow-up is strongly recommended. credentialing is ultimately the responsibility of the credentialing committee of the individual hospital, who should be familiar with the training and credentialing standards for specialists in cardiac arrhythmias. although most major centers already follow the guidelines above, some centers allow less qualified practitioners to perform these ep laboratory procedures. unfortunately, hospital credentialing committees have substantial conflicts of interest that could lead to granting privileges to physicians without appropriate board certification and experience, as refusal of these credentials usually results in a loss of patients and revenues to competing institutions. ultimately, the patient pays the price when inappropriate credentials are allowed. all ep laboratory physicians should be subject to periodic peer review and recredentialing. important components in the recredentialing process should include a review of abim board certification and ibhre ccds status, case volume, patient outcomes, peer evaluation, and continuing medical education (cme). the specific criteria for recredentialing are determined by each individual hospital, but should generally parallel the following recommendations: abim ccep board certification and ibhre certification are limited to 10 years; to stay current for ccep, the physician must complete a series of cme and/or practice improvement activities 9 ; recertification examination for ccep and ccds are each required at 10-year intervals; to ensure that cognitive and technical skills are maintained, the physician's clinical competence must be evaluated and documented on a regular basis; it is the responsibility of the medical staff credentialing committee to ensure that physicians perform the necessary number of evaluations and procedures needed to maintain their expertise 31 and also that they participate in regular cme activities. the ep laboratory should have a robust qa process (see section 9), and physician outcomes should be compared with national benchmarks derived from the literature or databases such as the national cardiovascular data registry (ncdr) on a regular basis; regular 360º evaluations, including evaluations from physician coworkers, fellows, nursing staff, technical staff, and patients, should be considered as part of the recredentialing review process. for the benefit of patients, it is paramount that physicians be held to a high performance standard and that remediation, withholding recredentialing, or revocation of privileges occurs if criteria are not met. the physician leaders must be committed to working aggressively to maintain the highest standards of patient care in their laboratories. 40 7.1.3. pediatric training and credentialing paces, working in conjunction with hrs, has developed guidelines for advanced training in ep focused on pediatric patients and patients of any age with chd. the original guidelines, endorsed by the hrs and published in 2005, required at least 12 months of specialized training, and were followed by a 2008 document focused on the implantation of pacemakers and defibrillators in these populations. 41, 42 there are currently no third-tier board examinations for diplomates of the pediatric cardiology subboard of the american board of pediatrics. however, pediatric electrophysiologists are eligible to take the ibhre examination for physicians with special competency in ep, as that examination includes a pediatric module. paces has recently written a competency statement for training in this field. 43 that competency statement strongly recommended that all graduating fellows and active pediatric ep clinicians take the ibhre ep examination. the field of adult congenital heart disease (achd) is an important and expanding clinical domain that is typically staffed by clinicians who are competent in both pediatrics and internal medicine. cardiologists providing invasive ep care for this unique patient population can enter the field from either specialty, but a portion of their formal training must be focused on the complex anatomy and unique ep of achd. further recommendations on the expertise necessary to care for this patient group can be found in a recent consensus statement supported by aha/acc/hrs. 44 a board certification process for achd is being developed and is scheduled for implementation by 2015. nursing licensure, credentialing, recredentialing, continuing education, and laboratory training are affected by the requirements of multiple agencies, including federal and state governments, the health care organization, and occupational safety and health administration. the standards of professional practice for nurses employed in the ep laboratory environment have been defined. 33 all ep nurses should have a critical care or a strong cardiology background, acls certification, and, in the pediatric ep laboratory, pediatric acls. an extensive knowledge of cardiac anatomy and physiology, electrocardiography, pharmacology, and training in sterile technique is also required. nurses need to have a thorough understanding of catheter-based interventions and surgical procedures, cardioversion, arrhythmia discrimination, and emergency treatment of life-threatening arrhythmias and complications/emergencies. familiarity with fluoroscopic, electroanatomic, and echocardiographic imaging is a required skill set. annual or biannual competencies required for nurses working in heart rhythm service operations should include basic life support, acls, infection control, emergency and tjc preparedness, training in conscious sedation, charting, and patient safety. demonstration of competency in radiation safety, sterile technique, external defibrillator operation, unitspecific nursing protocols, act operation, and temporary pacemaker operation should also be mandatory. depending on the roles and responsibilities assumed by rns, competencies may be needed in the ep recording system and programmed stimulator operation, ablation, generator operation, mobile laboratory operations, sheath insertion/ removal, operation of vascular ultrasound, 3d mapping operation, and ice operation. certification offered by the (ibhre is an integral part of heart rhythm education, training expectations, and requirements. in addition to the standard certification requirements for being an rn, the ibhre offers two certification examinations for allied professionals designed to demonstrate a mastery of knowledge in cardiac rhythm management. continuing education requirements are highly variable by state and nurse specialty. nurses need to review requirements for the state in which they are practicing to ensure proper compliance and maintenance of certification, as requirements can range from no required continuing education to as many as 30 hours every 2 years. 45 the certification examination for competency in cardiac rhythm device therapy and the certification examination for competency in cardiac electrophysiology are required at 10-year intervals. the apn (usually a np) is educated through a certified graduate level np program, meets the requirements of the state credentialing bodies, and practices according to the american nurses association consensus model. 46 an np is trained to perform preprocedural evaluation, order and interpret diagnostic tests, and conduct postprocedural follow-up. after on-the-job training, nps may assist in diagnostic ep studies, catheter ablation procedures, and device implants but cannot serve as a primary operator. nps working in heart rhythm services should be certified in basic and advanced cardiac life support and have knowledge of radiation safety, sterile technique, external defibrillator operation, ice operation, and temporary pacemaker operation. among laboratories represented by writing committee members, nps (or pas) are involved in pre-and postprocedural care in more than 75% of the centers but only 19% of the centers employ these individuals for assistance in the procedural laboratory. there are no uniform recredentialing criteria for nps in the ep laboratory. nps are expected to maintain certification and licensure as per certification and state guidelines. the institution should establish volume criteria for the maintenance of procedural competencies. individuals with a variety of backgrounds and qualifications can work in an ep laboratory as a cardiovascular technologist or technician. there is no professional regulatory body for cardiac technologists, although efforts are underway in some regions to achieve this goal. most technologists have postsecondary education (university degree or college diploma) with extensive on-the-job training. industry-sponsored courses often provide supplemental education specific to technologies used in the ep laboratory environment. some cardiovascular technologist programs offer ep as a component within a cardiovascular technology program; there are also certificate programs available at some accredited colleges. 47 cardiovascular technologists can be credentialed as a registered cardiovascular invasive specialist, registered cardiology technologist, registered cardiopulmonary technologist, or registered cardiac electrophysiology specialist through an accredited association such as cardiovascular credentialing international. 46 advanced ep specialty certification is achieved through the ibhre. satisfactory institutional performance appraisals (e.g., through 360º assessments of skills, competency, professional development, decision making, and leadership) are recommended. the individual should treat a sufficient volume of cases to maintain competency. if the technologist is ibhre certified, then the maintenance of certification through continuing education or certification examination is required. the pa is an advanced practice professional who is trained through a graduate level university program to perform many tasks, including preprocedural evaluation (history and physical examination and diagnostic tests) and postprocedural follow-up under the direct supervision of the physician. 48, 49 after on-the-job training, pas may assist in diagnostic ep studies, 3d mapping, catheter ablation procedures, and device implants, but cannot serve as a primary operator. institutional internal certification, minimum volume of annual cases to maintain competency in invasive ep, performance appraisal, and maintenance of continuing education should be a requirement for a pa practicing in the clinical ep laboratory. there are no uniform recredentialing criteria for pas. they should perform a minimum number of procedures as determined by the ep laboratory director and ep laboratory manager and demonstrate current competence on the basis of the results of ongoing professional practice evaluation and outcomes. industry employed allied professionals (ieaps) are hired employees of a medical device company who may serve as assistants in the ep laboratory. the hrs published a statement on the clinical role of ieaps in 2008. ieaps should provide technical assistance only on the manufacturer-specific products they represent, and they must work under the direct supervision of the responsible physician. 25 although ieaps may contribute substantially to patient care in some settings, overreliance on their service may lead to a lack of continuity of care, suboptimal patient education and counseling, and issues with liability and accountability. among laboratories represented by writing committee members, approximately 90% of the laboratories use ieap support in most or all device implant cases, and two thirds of the laboratories use ieap support in most or all 3d mapping cases. preparation for ep procedures requires a preprocedural history and physical examination by a physician, np, or pa. as many management strategies for arrhythmias require chronic and/or periprocedural anticoagulation, careful evaluation, assessment, and planning are needed. in patients undergoing pacemaker or defibrillator lead extraction, or who require pericardial access for epicardial ablation or left atrial ablation ligation, additional preparation may be required on a case-by-case basis, such as typing and crossmatching of blood products in select patients and immediate availability of thoracic surgical backup. in most diagnostic and ablation cases, rhythm active drugs (including β-blockers and calcium-channel blockers) are discontinued five half-lives before the procedure to allow the target arrhythmia to be induced, mapped, and ablated. a complete description of the procedure, including the anticipated success rates and possible complications, is best delivered in the outpatient setting before the ep procedure. a "time-out" must be performed immediately before the initiation of the procedure when all key personnel are present. health care facilities should insist that clinicians administering or supervising the administration of moderate sedation meet the requirements of the american society of anesthesiologists. anticoagulation is necessary for all left heart procedures with heparin (activated clotting time z250-350 seconds) or with bivalirudin in patients allergic to heparin. it is important to achieve the lowest possible noise signal with all recording systems. all physicians and staff are required to be familiar with identifying all potential procedural complications and to understand their role in managing them. the decision for patient discharge takes into account procedural detail, patient age and health status, potential for complications (such as blood loss), and the ability of the patient (or caregivers) to evaluate signs of concern. the procedure report should include, at minimum, all the following: the primary and secondary operators, the indication for the procedure, names and doses of any medications administered, catheter/pacing/icd lead model and serial numbers, insertion sites and intracardiac destinations, findings and procedure performed, complications encountered, and fluoroscopic exposure (fluoroscopy time, radiation dose, and the dose-area product) by an advanced cardiac life support (acls)/pediatric advanced life support (pals)-certified nurse. preparation for ep procedures requires a careful preprocedural history and physical examination by a physician, np, or pa to confirm the reason for the procedure that day and identify all comorbidities that could adversely impact procedure outcome. a thorough medication history, including allergies, must be gathered. the patient needs to be evaluated for factors that will impact anesthesia management (adequacy of airway, history of anesthesia experiences, obstructive sleep apnea, and physical indicators for difficult intubation). all adult patients should have recent (usually within 2 weeks) laboratory work, including electrolytes, blood urea nitrogen, creatinine, complete blood count, and, if taking anticoagulants, prothrombin time. all women of childbearing potential, including girls older than 12 years, should have serum or urine pregnancy testing within 2 weeks before the procedure. the need for a preprocedure laboratory exam in healthy children undergoing elective electrophysiological testing is not clear and is not common practice. as many management strategies for arrhythmias require chronic and/or periprocedural anticoagulation, careful evaluation, assessment, and planning are needed. among the considerations are the agent used, thromboembolic risk, bleeding risk, comorbidities, laboratory values, and availability of reversal agents or blood products such as fresh frozen plasma. consideration should be given to performing additional preprocedural transesophageal echocardiograms or the use of intracardiac ultrasound to reduce the risk of complications. in patients undergoing pacemaker or defibrillator lead extraction or who require pericardial access for epicardial ablation or left atrial ablation ligation, additional preparations may be required, including typing and crossmatching of blood products, availability of thoracic surgical backup and/or or, and, in some cases, intraprocedural transesophageal echocardiogram. many patients are taking one or more medications to control the heart rate and/or rhythm at the time of an ep procedure. in most cases, rhythm active drugs (including β-blockers and calciumchannel blockers) are discontinued five half-lives before the procedure to allow the target arrhythmia to be induced, mapped, and ablated. in patients undergoing anatomically based ablation, withholding these drugs may not be necessary. for most patients, the ep laboratory is an unfamiliar and intimidating environment, one in which an equally unfamiliar procedure is about to be performed. a complete description of the anticipated events is best delivered in the outpatient setting before the procedure day. education as to the planned agenda, the other participants (nurses, technologists, ep doctors, and anesthetists/anesthesiologists), and the nature of some of the equipment in the laboratory is important to ease the patient's anxiety and aid in their cooperation during the procedure. many of the technical terms used to describe the procedure are foreign to the patient; the staff must take care to use lay language in their descriptions, to evaluate the patient's ability to learn and preference how to learn, and to assess the patient's comprehension. this role is usually filled by an rn familiar with the procedure. the requisite process for informed consent is detailed in section 12. critical components include ensuring patient understanding, full disclosure of the risks and alternatives to the planned procedure, and the opportunity for the patients to ask questions and fully discuss their concerns. the patient education and consent process must be completed before the administration of any sedative or anxiolytic agents. in the case of pediatric patients or adult patients with cognitive impairment, education must be given and consent requested of the patient and the legal guardian. a time-out must be performed immediately before the initiation of the procedure when all key personnel are present. all members of the team are to cease their activities while one member recites two patient identifiers (i.e., name, date of birth, and medical record number), the type and laterality of the procedure, the name of the operator, and any known allergies. all members of the team must agree on all points before the procedure can commence. 50 the goal of analgesia and anesthesia in the ep laboratory should be to provide a safe, nontraumatic experience for the patient. the administration of anesthesia varies among case types and among institutions, from anxiolysis or moderate procedural sedation by an advanced cardiac life support (acls)/pediatric advanced life support (pals)-certified nurse under the supervision of the cardiac electrophysiologist 51,52 to monitored anesthesia care or general anesthesia administered by an anesthesiologist or crna under the supervision of the anesthesiologist. the health care institution must require those administering or supervising moderate sedation who are not anesthesiologists to meet the requirements of the american society of anesthesiologists to obtain privileges. 53 credentialing for this privilege must be periodically renewed. if intravenous procedural sedation will be used, the physician must establish an american society of anesthesiologists classification and mallampati score for the patient before the procedure. 36 it is necessary to have these assessments done before the procedure so that, if necessary, alternate plans for sedation may be arranged to optimize patient safety and minimize procedural delays. medications typically employed include etomidate, propofol, ketamine, fentanyl, midazolam, methohexital, and inhalational agents. individual states regulate what medications can be administered by nonanesthesiologists. as the provision of sedation is a continuum and the depth of sedation may vary, best practice is that all patients receiving moderate or deep sedation be evaluated by continual observation of qualitative clinical signs, pulse oximetry, noninvasive blood pressure monitoring, heart rate and rhythm, and monitoring for the presence of exhaled carbon dioxide to ensure the adequacy of ventilation unless precluded or invalidated by the nature of the patient, procedure, or equipment. 51 monitoring equipment should be in working order and have appropriate audible alarms. in pediatric cases, factors to be considered for the choice of sedation or general anesthesia include young age, preexisting medical conditions, presence of chd, airway issues, physician or family choice, and the length and complexity of the procedure. the 2002 naspe position statement on pediatric ablation delineated the types of anesthesia (conscious sedation, moderate sedation, and general anesthesia), and these remain applicable. 54 regardless of whether the administration of sedative agents is under the control of the electrophysiologist or another caregiver, the electrophysiologist must have a working knowledge of the effects of the agents used and how they might impact the electrical aspects of the procedure (such as arrhythmia inducibility and effects on blood pressure) or interact with other medications that may be given. deep sedation or general anesthesia can minimize patient discomfort, can benefit the ep procedure by preventing patient movement, and is necessary during defibrillation threshold testing. an immobile patient facilitates accurate and precise 3d mapping and reduces risk during transseptal puncture, pericardial access, or ablation in close proximity to critical structures. note that in cases where the assessment of phrenic nerve function is important for a favorable case outcome (such as placement of a coronary vein branch pacing lead or ablation within the right superior pulmonary vein), paralytic dosing should be reduced or eliminated. improved safety, efficacy, and procedure times have been shown with the use of general anesthesia with certain procedures such as atrial fibrillation ablation. 55 using high-frequency ventilation can further minimize respiration-related cardiac movement during ablation. 56 although adequate sedation should be administered to ensure patient comfort because certain arrhythmias such as atrial tachycardias and outflow tract vts can be dependent on adrenergic tone, excessive sedation can result in the inability to induce the clinical arrhythmia. in cases in which an adrenergicdependent arrhythmia is suspected, sedation must be minimized until the clinical arrhythmia is induced and mapped. deep sedation is usually administered while performing ablation to prevent patient movement. when assessing the end point in these cases, care must be used to differentiate the effect of sedation from the actual elimination of the arrhythmia. although the risk of infection is extremely low with ep catheter procedures, 57 appropriate sterile techniques should be maintained. this includes sterile preparation of all access sites, such as the groin and neck. if there is the potential for pericardial access, the subxiphoid region, and possibly the parasternal and apical regions, should also be prepped and draped. in cases with a higher risk of cardiac tamponade, sterile preparation of the subxiphoid region can be considered at the onset of the procedure. catheters with a smaller french size and fewer electrodes are more flexible, exert lower axial force, and may carry a lower risk of perforation, 58 but should perhaps be avoided because of difficulty maintaining stable catheter position. catheters with more electrodes can facilitate rapid recognition of arrhythmia activation patterns and are particularly beneficial at sites such as the coronary sinus. smaller electrodes and narrower interelectrode spacing detect a more local activation signal and can provide more precise activation mapping, but may be less maneuverable. activation confined to a small structure or circuit, such as the his bundle or an accessory pathway, however, may be difficult to localize with narrow electrode spacing. the field of signal detection can be increased either by changing to a catheter with wider electrode spacing or by reconfiguring the electrode pairing. with this wider field of view, anatomical localization is decreased. some diagnostic catheters are designed with electrodes in a specialized spatial configuration, such as circular/ring, basket, or star-shaped catheters. these catheters can enable rapid deciphering of an activation pattern even with a paucity of arrhythmia. the number of catheters and recording sites should be adequate to achieve the desired end points of the procedure, but not so many that vascular damage or obstruction or intracardiac entanglement could occur. anticoagulation is necessary for all left heart procedures with heparin or bivalirudin in patients allergic to heparin. 38, 39 even in patients with therapeutic international normalized ratio on warfarin, heparin must be administered for left heart procedures, though typically in lower doses than in patients not taking warfarin. for right heart procedures, there is no evidence favoring routine use of anticoagulation; in cases where there is concern that the patient is at an increased risk for thromboembolic complications (prolonged procedure, known or discovered patent foramen ovale), some centers administer heparin for anticoagulation. selection of the mode and catheter for catheter ablation is operator dependent. catheter factors such as torque delivery, axial stiffness, steerability, and introducer diameter affect device selection. ablation modes, including rf, cooled rf, laser, and cryothermy all have their strengths and weaknesses. the goal is to achieve a therapeutic ablation by identifying the ablation target, maneuvering to that site, and then destroying enough tissue to prevent arrhythmia initiation/propagation while minimizing the risk of collateral injury. the use of open-irrigated tip catheter ablation may result in the infusion of 2-3 l of volume during the case, which can precipitate heart failure in susceptible patients. 59 multielectrode or "single-shot" ablation systems are being developed for the treatment of complex substrates such as atrial fibrillation. experience with many of these technologies is limited. a preferred device for these applications may emerge in the future as our experience increases. selecting the appropriate ablation catheter is a process that involves a correct interpretation of the arrhythmia mechanism, a firm understanding of the advantages and disadvantages of the different catheters and energy sources, and the need for the responsible use of resources. 60 8.2.6. optimizing signal recording bipolar intracardiac recordings are standard in most laboratories because they theoretically detect only near-field signals, unlike unipolar recordings that incorporate both near and far-field components. 61, 62 far-field signals can still be detected in bipolar recordings but are typically a lower amplitude and frequency. unipolar recordings can be helpful in mapping sites of focal activation, such as ventricular insertion sites of accessory pathways in preexcitation syndromes and idiopathic outflow tract ventricular arrhythmias, in which deep sharp qs configurations signify a site from which activation emanates (i.e., site of the earliest ventricular activation). the unipolar signal can help clarify the content of the bipolar electrogram (near vs. far-field), the timing of actual local depolarization (intrinsicoid deflection of the unipolar signal), and the relative proximity of the tip vs. the ring electrode to the ablation target. the accurate interpretation of potential ablation target sites involves correctly differentiating local from distant activation as well as from electrical noise. noise troubleshooting is a complex issue and involves many variables. it is necessary to be familiar with the basics of signal acquisition provided in appendix 1 to correct noise issues. an inadequate signal-to-noise ratio will result in physiological signals being obscured by ambient noise with loss of critical information. at the onset of the procedure, steps should be taken to ensure that the signal quality is optimized for successful mapping. this should include the following: (1) choosing the appropriate electrode spacing, (2) setting the high-pass filter high enough to exclude low-frequency artifacts, such as respiratory drift, (3) setting the low-pass filter low enough to exclude high-frequency noise artifacts, (4) turning on the notch filter that excludes the 50-60-hz bandwidth typical of electrical interference, and (5) gaining the signal appropriately to visualize low-amplitude signals of interest while minimizing the magnification of noise artifacts. it should be noted that using the notch filter on bipolar intracardiac signals can introduce ringing to sharp simple signals, making them appear fractionated. this is a particular concern when targeting fractionated potentials in cases of vt and atrial fibrillation. if the laboratory and equipment are properly grounded and the electricity in the laboratory is conditioned, there should be no 50-60 hz noise on the intracardiac signals. lower gain recording should be employed if the electrogram signals exceed the recording range of the amplifier. efforts should be made, working with the facility's biomedical engineering personnel, to achieve the lowest noise signals possible. steps toward this goal include appropriate equipment grounding and shielding of cables, scheduled maintenance of connecting cables with replacement if contact plugs lose continuity, maintaining the shortest distance traveled by all electrical cables, ensuring that cables are off the floor and removed from potential hazards such as wheeled carts and cleaning solutions, and maintaining separation between high-voltage lines, such as power cables, and low-voltage lines used for transmitting the patient's electrical signals. dealing with complications in the ep laboratory has several components: avoidance, recognition, and response. to the extent possible, complications should be avoided by adhering to standard techniques and practices. when a complication occurs, the outcome for the patient hinges on how quickly a problem is recognized and appropriately evaluated as well as how quickly and appropriately the response to the incident rectifies the situation. even the most careful and skilled operator will have occasional unavoidable complications (table 3) one of the most important risks in device implantation procedures is infection. the device implantation laboratory should be regarded as an or, with the same attention to sterile technique. hats, masks, and shoe covers should be worn in the procedure room when the sterile field is exposed. efforts should be made to restrict traffic in and out of the procedure rooms and minimize the number of personnel in the room. studies have demonstrated that microbial counts increase significantly in unoccupied ors when the door is left open to the hallway. 69 after explanation of a device from an infected pocket, the room should undergo cleaning according to standard procedures employed in the or for contaminated cases. the use of preoperative antibiotics has been conclusively proven to reduce cied infection. 70 administration of an antibiotic, usually a first-generation cephalosporin, 1 hour before implantation is required. in light of the prevalence of the colonization with methicillin-resistant strains of staphylococcus, some operators choose vancomycin in patients at higher risk of infection, although data are lacking to support this practice. vancomycin is a suitable choice for patients with penicillin allergies. if vancomycin is selected, it should be administered within 2 hours of the procedure. 71 the instruments and components used in the device implant laboratory must be opened in a clean air environment. by doing so, bacterial contamination of these instruments and components will be kept to a minimum. all personnel in the room must wear a cap and mask at all times, and all who are in contact with the sterile field must perform a complete surgical scrub and must be gowned and gloved. the operative site(s) should be prepared with an antiseptic agent. although these agents eliminate the immediate bacterial count on the skin surface at the operative site, hair follicles may prevent complete sterilization of the skin. hair clipping close to the skin in the prep room, rather than shaving, is recommended because bacteria on the skin surface begin to recolonize within 30 minutes in the presence of hair follicles despite complete sterilization. 72 some centers instruct patients to use preprocedural home cleansing kits. site preparation with alcohol-based solutions should be allowed to dry completely before draping. the combination of evaporating alcohol, supplemental oxygen, and electrocautery poses a significant fire risk in the surgical field. in some cases, additional femoral venous access for the placement of a temporary pacing catheter may be warranted (e.g., lead extraction and pacemaker-dependent patients with an inadequate escape rhythm who are undergoing generator change). 8 large-bore venous sheaths are useful for rapid volume resuscitation in the event of vascular tears during lead extraction, and arterial access facilitates beat-to-beat monitoring of blood pressure. (table 4) complications associated with acute cied implantation are often technique related. attention to detail will minimize the risk of difficulties related to the implant procedure. venous sheaths may be removed at the end of the procedure if no anticoagulant has been administered, with pressure held for 10-20 minutes. if heparin has been administered, waiting until the act is more than 175 seconds (o250 seconds if the patient is receiving therapeutic warfarin) before sheaths are removed decreases the likelihood of bleeding and hematoma formation. reversal of heparin effect with roentgen (r) is the unit of radiation exposure in air. the total charge produced in air per unit mass by ionizing radiation is easily measurable with survey instruments. gray (gy) is the international system of units (si) of absorbed radiation dose of ionizing radiation, which has replaced the term radiation absorbed dose (in rad). one gray is the absorption of 1 j of ionizing radiation by 1 kg of matter (equivalent to 100 rad), and it assesses the potential biological risk to that tissue. the u. s. unit for absorbed dose is rad. 1 gy ¼ 100 rad. equivalent dose sievert (sv) is a measure of equivalent dose, and is also an si unit. it takes into account the different probability of effects that occur when the same amount of absorbed dose is delivered by different types of radiation (protons vs. x-rays). it is equal to the absorbed dose in gy multiplied by the radiation weighting factor, w r , and other modifying factors. the w r is 1 for x-rays. the equivalent dose can be used to assess radiation risk if the person's whole body is uniformly irradiated. for partial body exposure, such as cardiac electrophysiology, additional correction is necessary to assess the radiation risk. effective dose is used to assess the risk when only a part of the body absorbs energy from radiation. since some organs in the body are more sensitive to radiation effects than others, the equivalent dose is multiplied by the appropriate tissue weighting factors. this terminology is used to assess the risk of radiationinduced cancer and hereditary effects. for example, the effective dose for a typical chest x-ray radiograph is 0.0001 sv (0.1 msv). the u.s. unit for sievert (sv) is rem. 1 sv ¼ 100 rem. as low as reasonably achievable (alara) standard is a system for limiting the amount of radiation a person receives. radiation exposure should be justified on the basis of the assumption that there is no threshold below which ionizing radiation is free from harmful biological effects and that shielding from radiation exposure is needed, no matter how low the dose. kerma is an acronym for kinetic energy released in the material. kerma is measured in gy. kerma-area product (p ka ) is the integral of air kerma (absorbed dose to air) across the entire x-ray beam emitted from the x-ray tube. p ka is a surrogate measurement for the entire amount of energy delivered to the patient by the beam. p ka is measured in gy á cm 2 . another term for this is the dose-area product (in gy á cm 2 ). reference air kerma (k a,r ) is the kerma-area product at a specific point in space relative to the fluoroscopic gantry (the interventional reference point) during a procedure. it is measured in gy. peak skin dose is the highest dose of radiation exposure on any portion of a patient's skin during a procedure. protamine is employed in many laboratories to more rapidly reverse heparin effect and allow almost immediate sheath removal, although one must be prepared to treat uncommon but sometimes severe protamine reactions. 64 vascular closure devices are uncommonly used in ep, 64, 65 but are an appropriate choice for arterial closure. after atrial fibrillation ablation, the reestablishment of therapeutic anticoagulation soon after sheath removal is desirable to lessen periprocedural stroke risk but there is no consensus on the optimal regimen or timing. when mild anesthesia is used, vital signs and oxygen saturation should be monitored continuously until the patient is conscious and communicative. access sites, cardiac rhythm, and neurological state should be assessed every 15 minutes during the first hour and then periodically thereafter. late complications, such as access site hematomas and hemodynamically significant pericardial effusion, can develop after the patient leaves the postprocedural recovery area. if a patient received midazolam during the procedure and a dose of its antidote (romazicon/flumazenil) was administered, the patient must be monitored for a rebound effect of midazolam. if general anesthesia was used, patients usually recover in a postanesthesia care unit. procedural complications that can arise after the patient leaves the laboratory area (or even after hospital discharge) are listed in table 4 . a process for tracking postprocedural complications should be in place as part of the laboratory's qa process (see section 9). postprocedure anticoagulation is recommended in patients who are at high risk of stroke on the basis of evaluation tools such as chads 2 or cha 2 ds 2 -vasc scores. in many laboratories, chronic warfarin therapy is not interrupted during either device implantation or ablation procedures in patients who are taking it for stroke prevention in the setting of atrial fibrillation or mechanical heart valves; the procedure is safe, with the international normalized ratio ranging from 2.0 to 3.5. 35, 64 in patients with insulin-dependent diabetes, the morning insulin dose is typically halved on the day of the procedure and glucose is periodically monitored during the procedure and the patient is treated accordingly. the setting for ep procedures may be outpatient, 23-hour observation, or inpatient. the decision for discharge takes into account procedural detail, patient age, and health status, the potential for complications (such as blood loss), and the ability of the patient (or caregivers) to evaluate signs of concern. 67 this is a medical decision and should be determined irrespective of reimbursement issues. the procedure report should include, at a minimum, the following: the primary and secondary operators; the indication for the procedure; names and doses of any medications administered; intake, output, and estimated blood loss; catheter/pacing/icd lead model numbers, serial numbers, insertion sites, and intracardiac destinations; findings and procedure performed; complications encountered; and fluoroscopic exposure (minutes; mgy; dose-area product). patients who receive excessive radiation exposure during a procedure (typically 43000 mgy, but requirements vary by state) must be notified and followed up for evidence of skin damage. ideally, this information is stored in a database for qa purposes. recordings made during the procedure (electrograms and fluoroscopic and mapping system images) should be archived on digital media (ideally on a network, or alternatively on a cd or dvd) for future reference, if needed. 64 pediatric ep procedures on small and young children should be performed in centers where there is pediatric surgical backup. procedures on adult patients with chd can be performed in pediatric or adult facilities by physicians who have expertise in the area of chd and the potential arrhythmia substrates of patients with chd. there are special considerations for performing pediatric ep procedures, including unusual arrhythmia mechanisms, small patient size, and the effect on future patient growth. issues that pertain to ep laboratory standards and practice for pediatric ep and patients with pediatric and adult chd with rhythm abnormalities differ from those pertaining to adults and are not confined to issues of patient and cardiac size compared with the adult patient. the decision-making process for interventions has implications for patient quality of life and development. success, failure, and procedural factors related to intervention therefore span many decades. a significant factor in treating cases involving young patients in the ep laboratory is the need for age-appropriate supportive care. arrhythmia mechanisms in young patients vary by age 72 and influence decision making. patient size can dictate the use of and expertise with smaller ablation catheter sizes or the use of esophageal pacing if vascular access is limited, such as is the case in neonates. knowledge of ablation lesion formation and potential expansion in an immature myocardium is critical to the care of pediatric patients. 73 children and young adults will experience decades of device-related issues compared with the typical adult patient. these issues should affect decision making in terms of timing and location of cardiac ep devices, accounting for growth, potential need for multiple extractions and replacements, and issues of venous occlusion. for patients with chd, these factors are complex and affect the decision to intervene. surgical interventions for all forms of chd have resulted in improved survival rates, 72 and the details of these surgical interventions are critical in the analysis of rhythm substrates in the ep laboratory. more complex pediatric chd survivors comprise an increasing percentage of the adult chd population. 72 it is recommended that procedures in pediatrics and patients with chd be performed by (1) pediatric cardiologists, (2) a collaboration of adult and pediatric cardiologists, or (3) an adult cardiologist with established interest and expertise in adult chd. the indications for catheter ablation in the pediatric population derived from an understanding of the natural history of arrhythmias in young patients, likely rates of procedural success and complications, and the risk of recurrence and have been reviewed in prior publications. 54, 74 the guidelines for the assessment of the asymptomatic young patient with wolff-parkinson-white syndrome are published as a joint paces/ hrs statement. 75 in the pediatric population, the presence of chd affects the expected results of ablation and recommendations for intervention. the guidelines for the implantation of cardiac rhythm devices in young patients and patients with chd were last updated in 2008. 76 epicardial pacing is used for those in whom transvenous pacing is contraindicated, such as prosthetic tricuspid valves, right-to-left intracardiac shunts, and small patient size, or for those undergoing concomitant heart surgery. the majority of icds are implanted via the transvenous route, but this may not be possible in some individuals because of anatomical constraints. because icd leads are larger and prone to fibrosis, patient size limitations for transvenous systems are considerable. although established in adults, the indications for cardiac resynchronization therapy in children are less certain and are based on retrospective reviews, rather than randomized trials. 77,78 because younger patients may have a longer life span after ep procedures than do adults, the lifetime risks of malignancy and birth defects (stochastic risks) are higher. adult patients with chd incur increased radiation exposure resulting from the electroanatomic complexity of cases and the need for multiple procedures. strategies for radiation dose reduction as detailed in section 11 should be aggressively implemented. the most significant complications in small children include pericardial effusion, pneumothorax, atrioventricular block, and death. 79, 80 animal and clinical studies have shown the potential expansion of scar tissue with maturation 70 and the risk of late coronary artery injury 79 potentially related to ablation location. the risk of atrioventricular block is increased by small patient size and septal pathways, presumably because of the smaller anatomical dimensions of structures. smaller tip catheters, lowered rf energy, shorter lesion duration, and the use of apnea and pacing techniques may diminish risk. 81 cryoablation is perceived as being safer, but may have a higher recurrence rate compared with rf lesions. 82 it is recommended that ep laboratories conducting procedures on pediatric patients have cryoablation capability. the procedure setting for invasive ep studies and ablation for pediatric and congenital ep patients may be outpatient or inpatient. the decision for discharge takes into account procedural detail, patient size, potential for complications, and the ability of the parents to evaluate signs of concern. the protocol for ep device placement or revision is mostly less than 1 day of observation, but generator changes in older patients may not require more than 6 hours. young patients with new devices are monitored overnight to administer periprocedural antibiotics, evaluate for pneumothorax or hemothorax, evaluate the device parameters, check lead location, and manage pain. the goal of sedation in the pediatric and congenital ep laboratory should be to provide a safe, nontraumatic experience for the patient by considering young age, preexisting conditions, presence of chd, airway issues, family choice, and complexity of the procedure. the 2002 naspe position statement on pediatric ablation delineated the types of anesthesia (e.g., conscious sedation, moderate sedation, and general anesthesia), and these remain applicable. 54 personnel responsible for sedation, anesthesia, and administration of medications must be experienced with pediatric and congenital ep patients, and pals and acls certification should be maintained. all physicians performing ep procedures should be knowledgeable regarding sedation, monitoring, and airway management. allied health professionals (e.g., nurses, apns, and pas) can be involved with sedation of a patient, if directly supervised by a physician. the room and equipment standards for pediatric ep procedures are similar to those for adult ep procedures (see sections 4 and 5) but must have a cardiac defibrillator specifically for use with children, a code cart meeting pediatric needs, and age-appropriate anesthesia equipment. pediatric and congenital ep patients may require a combined procedure of ep and hemodynamic catheterization, including angiography and possible intervention. thus, it is desirable that a pediatric/chd ep laboratory meet the same standards as a pediatric catheterization laboratory. general recommendations for ep laboratory staffing are detailed in section 6 and table 2 . pediatric and congenital ep guidelines recommend that the facility and laboratory staff should be appropriate for the patient population and pediatric and chd interventional and surgical experts be urgently available during laboratory procedures. 42 all members of the team should be trained in pals (when treating a child/infant) and acls (if treating an adult). training and board certification pathways employed by physicians who perform ep procedures on pediatric and congenital ep patients include the pediatrics and internal medicine pathways (see sections 6 and 7). recommended staffing for pediatric cases is detailed in table 3 . while laboratory staff roles are similar to those outlined in section 6 for pediatric cases, these staff should have expertise in performing cardiac procedures in pediatric and congenital ep patients. pediatric ep procedures should be performed (earlier than the teenage years) in centers where there is pediatric cardiovascular surgical availability. for procedures performed on patients in their teenage years or procedures on adult patients with chd that are performed in adult or mixed pediatric and adult institutions, a cv surgical plan should be formalized to accommodate on-site emergencies (notably, cardiac perforation) and a rapid transfer made to a pediatric cardiovascular center should the anatomy of the patient require this expertise. the postprocedural care can be performed in a separate periprocedural area, the general cardiology floor, or the intensive care unit. continuous telemetry should be available for the evaluation of heart rhythm. the environment for postprocedural care should be appropriate for patient age and development. the nursing and physician staff should be experienced in the care of pediatric and congenital ep patients. a process for tracking postprocedural complications should be in place as part of the laboratory's qa process. an essential component of a successful ep laboratory is to have an internal qa/quality improvement (qi) process in place, in addition to public reporting requirements. components of the qa/qi process should include national requirements for tracking (e.g., device implants), minimum acceptable complication rates (e.g., infections), and compliance with national registries, including the ncdr icd registry. it is the responsibility of each institution to ensure that staff credentialing, maintenance of certification, and necessary continuing medical education requirements are met. procedure outcomes, including success rates and complications, should be documented and recorded. data acquired from the ep laboratory qa process should be used to benchmark the complication rates and outcomes of both individual practitioners and the overall ep laboratory. physicians should participate in regularly scheduled qi and/or peer review meetings to maintain privileges and evaluate procedural appropriateness. a quarterly ep laboratory morbidity and mortality conference should be mandatory, with attendance documented. a qa process for the equipment should be established that provides a mechanism to demonstrate optimal function and operation of the equipment and that offers staff training in equipment maintenance, setup, and operation. given the often poorly defined relationship between case volumes and outcomes, a more appropriate measure is to ensure that all major complications are reviewed by the qa committee and handled as described in the previous section. high-quality, consistent care delivery in a busy cardiac ep laboratory requires standard protocols for procedures, communication channels, and documentation. an essential component of a successful ep laboratory is to have an internal qa/qi process in place, in addition to public reporting requirements. this requires a commitment from facility administrators to provide adequate staffing, including a committee chair, staff coordinator, and funding for collecting and managing data. the goal of the program should be to motivate and encourage physicians and staff to participate and to take initiative in the qa/qi process and overall success of the laboratory. it is acknowledged that there is little published data on the qa/qi process in the ep laboratory and that expert consensus is the primary basis for our recommendations. further research and development of quality metrics specific to the practice of cardiac ep is ongoing. to begin the qa/qi process, its components should be identified. these components include national requirements for tracking (e.g., device implants), minimum acceptable complication rates (e.g., infections), and compliance with national registries, including the ncdr icd registry. a qa/qi program must ensure that key data are collected prospectively and systematically. when the qa/qi team is identifying potential quality metrics, either strong scientific evidence or expert consensus must support the metric. the metric must measure areas important to patient care, and it is best if it covers an aspect of practice where there is a gap in patient care. the data must be available in a usable format for future analysis. these data allow the laboratory to benchmark its performance and provides a reference by which appropriate changes can be made. provider qualifications are typically well established by national guidelines and certification bodies. it is the responsibility of each institution to ensure staff credentialing, maintenance of certification, and necessary cme requirements are met (see section 7) . practitioners should be expected to adhere to published practice guidelines unless the reason for deviation is documented. clinical situations not directly addressed by the guidelines inevitably arise and require judgment and skill to address. periodic peer review of cases that fall outside the guidelines is recommended to ensure appropriate delivery of care. the guidelines evolve and, by definition, require a consensus of data before they can be written and revised. the writing group recognizes and encourages the development of new clinical pathways and tools. this development is best achieved through research protocols that adequately capture patient demographic characteristics, procedure characteristics, and outcomes. the research consent process ensures that patients are aware that the treatment being offered is beyond the current recommendations of the practice guidelines. procedure outcomes, including success rates and complications (and ideally including 30-day outcomes), should be documented and recorded. the writing group recognizes that success for some procedures requires clinical follow-up (particularly atrial fibrillation and vt ablation procedures). in these instances, an acute end point for the procedure should be specified (e.g., pulmonary vein isolation, noninducibility for vt, or creation of a planned rf ablation lesion set based on the results of an endocardial voltage scar map) and documentation should indicate if the end point was achieved. an assessment of freedom from arrhythmia recurrence after 1 year of followup should be performed. risk-adjusted models are not well developed by which the relative frequency of complications and successful outcomes among different patient populations can be interpreted and physician results compared. therefore, the interpretation of success rates and complication rates requires judgment by peers. physicians should participate in regularly scheduled qi and/or peer review meetings to maintain privileges and evaluate procedural appropriateness. a quarterly ep laboratory morbidity and mortality conference (stand-alone or as a component of another conference) should be mandatory, with attendance documented. the modern cardiac ep laboratory depends on many complex hemodynamic and physiological recording systems, advanced imaging systems, advanced mapping systems, and multiple ablation systems. rigorous processes must be established to ensure that (1) a qa process for equipment is established; (2) equipment is tested and demonstrated to be functioning appropriately, both on a routine basis and immediately before a case in which the specific item will be used; verification of equipment function should be included in the time-out procedure; (3) ep laboratory staff are appropriately trained in the maintenance, setup, and operation of the equipment; and/or (4) representatives from the vendor are available, qualified, and cleared by administration and occupational health to participate in the operation of the equipment before, during, and after the procedures. competencies in clinical skills, acls, pals (when appropriate), sterile technique, radiation safety, and fire safety should be assessed on a regular basis. the ep laboratory requires processes be in place to ensure proper communication within the ep laboratory and with other hospital services. within the laboratory, protocols for emergency situations (such as tamponade or ventricular fibrillation refractory to defibrillation) can make the difference between an organized, streamlined, successful resuscitation effort and a chaotic effort during which leadership is absent, team members duplicate some activities and neglect others, and ultimately a tragic but potentially avoidable outcome occurs. beyond the boundaries of the ep laboratory, communication between the ep team and other health care professionals is essential, particularly during handoffs from one care team to another and at the time of patient discharge. it is the role of the qa/qi committee to oversee that excellent communication processes are developed, maintained, and adhered to by the staff. data acquired from the ep laboratory qa process should be used to benchmark the complication rates and outcomes of both individual practitioners and the overall ep laboratory. for practitioners with complication rates above the benchmark, an objective unbiased peer review of the relevant cases is critical to determining whether a deviation in the standard of care occurred. because event rates are low and riskadjusted models are not well developed for the ep laboratory, peer review is particularly important. practitioners should not be penalized for accepting higher risk and/or more challenging cases. however, if reckless behavior or inadequate skills or knowledge is deemed to be present and a deviation in care occurs, verbal and written communication by the chair of qa is imperative. this communication should include a clear plan for corrective action and documentation of potential future actions if corrective action is not successful. a link between operator case volume, skills, and outcomes has been documented in some but not all areas of cardiac ep, yet controversy and conflicting data remain. specific case volumes for training are outlined by the abim 29 (table 3) , and for clinical competency they are available in the hrs clinical competency statements. 8, 17, 33, 38, 39, 42 given the often poorly defined relationship between case volumes and outcomes, a more appropriate measure is to ensure that all major complications (see table 3 ) are reviewed by the qa committee and handled as described in the previous section. a prospective plan to acquire data in an accessible and functional database is an essential building block for any qa/qi process. without objective, reliable data to measure outcomes, no meaningful effort at qi can be undertaken. minimum data that should be recorded in a searchable aggregate form include patient demographic characteristics, relevant history of present illness, medications, cied product information data, and data on outcome and complications from any invasive procedures. patients with cied should be identifiable by the device that has been implanted to permit rapid identification of patients who may have received defective hardware in the event of a recall or other notification from the manufacturer or fda. the field of medical informatics is on the verge of exponential growth. registries such as the acc/ncdr icd registry have identified specific data fields that should be captured for participation in the registry and compliance with payer mandates. 83 beyond these basic requirements, recommendations for more detailed database fields are included in clinical guidance documents for the management of patients with atrial fibrillation and for the management of patients with vt. 38, 39 these standard data elements provide an opportunity for ep laboratories invested in research to aggregate and/or compare their data with that of other laboratories working in the same field. to date, qa efforts in pediatric and congenital ep have centered on the creation of large ep procedural registries. 84, 85 in late 2010, a paces taskforce began to develop and implement a selfsustaining multicenter qi registry known as map-it. presently, the map-it taskforce is creating a registry of patient-centered late outcome measures of catheter ablation procedures. for the first time, an empirical and data-derived method of risk/complexity adjustment for pediatric and chd ep procedures, known as the compass score, has been developed. the future of qa efforts for procedure-based subspecialties will require the benchmarking and reporting of risk-adjusted "patient-centered" outcome measures. widespread implementation of the map-it initiative within paces should satisfy this need. fluoroscopy equipment should report three parameters: fluoroscopy time; radiation dose (air kerma, in gy), a measure of deterministic injury potential; and the dose-area product (in cgy á cm 2 ), a measure of stochastic injury potential. a minimum of 0.5-mm leadequivalent protective apron, thyroid shield, and eye protection should be used by ep laboratory personnel. all lead should be tested at 6-month intervals to check for cracks or leaks. risks of acquisition of infectious diseases by health care workers can be minimized by adherence to current infection control guidelines. the field of cardiac ep is greatly dependent on fluoroscopic imaging for the placement of catheters and device leads into the heart. this results in significant radiation exposure to the patient, the operator, and the laboratory staff. while this exposure cannot be eliminated in most cases, attention to fluoroscopic technique can minimize radiation dose. competencies in radiation safety should be completed yearly by the ep laboratory staff. some states mandate fluoroscopy licenses be obtained by all personnel using fluoroscopy and renewed periodically, including physicians and radiation technologists. nonionizing radiation, such as microwave or infrared radiation, can cause heating but not molecular damage to cells. ionizing radiation, such as β, γ, and x radiation, strips electrons from atoms and causes molecular injury to dna. ionizing radiation has great potential for damage to tissue, including burns and malignancy. understanding the basic definitions and terminology used to describe ionizing radiation is helpful when trying to understand the potential effects on the human body (see table 4 ). 86 11.1.2. biological risks from radiation exposure radiation effects are described by their deterministic and stochastic effects. 87 deterministic effects are harmful tissue reactions that are determined by an absorbed threshold dose. radiation-induced skin burns are an example of deterministic effects. 88 stochastic effects include malignancy and heritable effects and are not determined directly by the dose, but a higher dose increases the probability of an adverse outcome. human tissue radiosensitivity varies directly with the rate of cellular proliferation and number of future divisions and indirectly with the degree of morphological and functional differentiation. the most sensitive tissues include the bone marrow, spermatocytes, and intestinal crypt cells. 87 local skin injury is the most commonly encountered deterministic effect in cardiovascular medicine, with changes noted at doses above 2 gy. findings often do not appear until weeks after exposure. radiation exposure increases lifetime risk of fatal malignancy. the as low as reasonably achievable standard was derived from the biological effects of ionizing radiation vii report. 89 this report makes the assumption that cancer risk increases proportionally with radiation exposure and that there is no radiation dose that is without risk. all personnel working in the laboratory must be aware of the as low as reasonably achievable standard. a skin threshold dose of 2 sv should not be exceeded. because the prevalence of fatal malignancies continues to increase over time after radiation exposure, children and young adults are more susceptible to these complications in their lifetimes. adult patients with chd incur increased radiation exposure resulting from the electroanatomic complexity of cases and the need for multiple procedures. a normalized x-ray dose at a specific kilovolt peak 1 m from the source, total fluoroscopic time, backscatter correction factor, and source-to-skin distance can be used to estimate radiation exposure. 90 a direct measurement of radiation doses at multiple sites can be performed with lithium fluoride thermoluminescent dosimeter sensors and optically stimulated luminescence. the highest exposure to patients has been observed with a median skin entrance dose of 7.26 rem (range 0.31-135.7 rem) at the ninth vertebral body. this dose is predicted to be associated with a lifetime excess risk of malignancy to the female breast, active bone marrow, and lung of greater than 700 cases per million undergoing routine catheter ablation. operator exposure was highest at the left hand, waist, and left maxilla. 91 fluoroscopy equipment should report three parameters: fluoroscopy time; radiation dose (air kerma, in gy), a measure of deterministic injury potential; and the dose-area product (in cgy á cm 2 ), a measure of stochastic injury potential. radiation exposure is a superior metric to fluoroscopy time; reliance on fluoroscopy time is discouraged. the united states nuclear regulatory commission annual dose limits for radiation are 0.50 sv for skin, arms, and legs; 0.15 sv for eyes; and 0.05 sv for the whole body. the fluoroscopy dose for each case should be recorded in the medical record and accessible to patients. the predominant strategy for minimizing radiation exposure to patients is to minimize the radiation dose. 86, 92 the most effective approach is to minimize fluoroscopy pedal time. operators should develop the habit of tapping the fluoroscopy pedal rather than standing on the pedal for a long period of time. if the eyes stray from the fluoroscopy screen, the foot should come off the pedal immediately. decreasing the fluoroscopy pulse rate will significantly reduce dose at the cost of temporal resolution of the image. supplementation of fluoroscopic imaging with nonfluoroscopic electroanatomic guidance systems by using stored fluoroscopy loops rather than cine loops and using pulsed fluoroscopy will reduce the total procedural dose. as the x-ray tube gets closer to the patient, x-rays at the skin entry point increase and the risk of deterministic injury also increases; thus, the operator should position the table at a comfortable height with some distance between the tube and the patient. if the image intensifier is not positioned as close to the patient as possible, the image will be magnified but the radiation dose will be much higher. to magnify the image, use the appropriate magnification mode. both geometric and electronic magnification increases dose to the patient. doses may be limited effectively by collimation, limiting the field of view with shutters as much as possible. for example, reducing the diameter of the field of view by 29% will reduce the radiation dose by half. steeply angulated projections should be avoided, and if used, the c-arm should be repositioned somewhat throughout the procedure to avoid delivering radiation to the identical skin entry site. depending on which procedure is being performed, local shielding of the patient's thyroid and gonads can be employed. 93 since the stochastic effects of radiation exposure are cumulative, the caregiver should be sensitive to the lifetime exposure of the patient to ionizing radiation. there has been active discussion among regulators on implementing a system for lifetime tracking of radiation exposure to patients, but the tools for this type of system are not available, and therefore it is not presently mandated. 94 it is important to emphasize that radiation exposure is dependent on the age and condition of the equipment. thus, aggressive limitation of fluoroscopy pedal time is desirable, but those efforts may be futile if the dose rate is high because of a high frame rate or employment of an old imaging train. it is now an accepted standard that fluoroscopy dose, not only fluoroscopy time, is entered in the permanent medical record for each fluoroscopic procedure. ideally, the peak skin dose, the reference air kerma, the kermaarea product, and the fluoroscopy time should be recorded for every case. 95 a review of fluoroscopy use by individual operators should be part of every laboratory's qa process. the primary approaches used to reduce radiation exposure to the operator and laboratory staff are increasing distance from the source, scatter reduction, and dose limitation. radiation dissipates in proportion to the square of the distance from the source, and so even a modest effort to move away from the tube will significantly reduce exposure. radiation scatter occurs as radiation from the generator tube enters the patient and is partially reflected or refracted by body tissues. scatter from the patient is the main source of radiation exposure to the patient outside the imaging field and to the operator. the operator and laboratory personnel must be protected from exposure to the scatter radiation with shielding. a minimum of 0.5-mm lead-equivalent protective apron, thyroid shield, and eye protection should be used. proper table shielding can dramatically reduce the scatter radiation escaping into the environment. scattered radiation exits the body at all angles but is greatest on the same side of the patient as the x-ray source because only 1%-5% of radiation completely penetrates the patient's body and exits on the other side. 91 therefore, proper undertable shielding is paramount. shield extensions above the table rail and a contoured ceilingmounted shield in contact with the patient's torso will substantially reduce operator exposure. since radiation doses decrease with the square of the distance from the source, the operator should perform the procedure as far from the radiation tube as is practical. barium-impregnated drapes can further reduce radiation scatter in the procedure field. 96 all recommendations described above for limiting total fluoroscopy dose will also reduce the operator and laboratory staff radiation exposure. radiation exposure to a pregnant ep laboratory worker is a special situation and should be resolved on a case-by-case basis. it is recommended that radiation exposure to the pregnant staff member, as measured by a waist dosimeter (under the lead apron), should not exceed 0.05 rem/mo, or 0.5 rem for the entire pregnancy. 35 additional layers of lead can be worn over the abdomen to further protect the fetus. the fda regulates fluoroscopy equipment manufacturing and has dose limits for systems with automatic exposure control. 97 states regulate the safe use and operation of radiationproducing machines, such as fluoroscopic imaging systems. tjc's sentinel event is an unexpected occurrence involving death or serious injury. prolonged fluoroscopy that exceeds doses of 1500 rad (15 gy) to the skin is a reportable sentinel event and requires an institutional response. a qualified medical physicist should perform initial acceptance testing and annual testing to ensure optimal image quality and radiation dose. unfortunately, assuming that fluoroscopic equipment is functioning properly, the most important factor in radiation exposure to patients and staff is operator knowledge and behavior. tjc recommended (but did not mandate) credentialing standards for fluoroscopists, 98 and the recommended curriculum was endorsed by hrs, american college of cardiology foundation, aha, and the society for cardiovascular angiography and interventions (scai). 86 the training of ep physicians and staff in radiation physics, radiation biology, and technological developments in x-ray imaging systems and x-ray dose management is highly variable, and physician credentialing and recredentialing has no requirement regarding knowledge of radiation safety. it is therefore incumbent on the hospital leaders to establish high local standards and to track fluoroscopy use and behavior. lead-equivalent aprons required for radiation protection of staff are heavy and present a substantial physical burden to the interventional cardiologist. 99 historically, lead aprons were made using 0.5-mm lead-equivalent materials, with weight per unit area of these garments being 7 kg/m 2 . 98 an increased risk of cervical spondylosis is a known consequence for cardiologists who wear protective garments while standing for long hours performing procedures. 87 orthopedic problems and user fatigue associated with the continued use of heavy aprons contributed to the development of lowerweight lead-equivalent materials that are commonly used for protective garments today. modern nonlead 0.5-mm leadequivalent protective garments have a weight reduction of 30% or more than do traditional lead aprons. 98 that modern aprons are lighter might be expected to lessen, but not eliminate, orthopedic discomfort and injury. it is important to emphasize that all lead should be tested at 6-month intervals to check for cracks or leaks. along with weight, there are several other physical and ergonomic aspects of radiation protection garments that should be considered. to minimize discomfort, garments must fit correctly and should be tightened around the midsection to shift weight from the shoulders to the hips as much as possible. there are several garment designs that are customized for various uses. many interventional cardiologists choose a two-piece garment consisting of a skirt and vest (including internal frame), both of which should be snugly tightened around the midsection. such garments are frequently designed to "wraparound" the wearer and typically provide the fully specified lead-equivalent protection from radiation only in the front where the garment overlaps. a thyroid shield should always be used, 100 and leaded glasses should be worn to minimize the risk of developing cataracts. 101 tableside alternatives to lead-equivalent garments include a floor-mounted radiation protection cabin and a ceiling-or gantry-mounted suspended radiation protection system. 99, 102, 103 both these systems are designed to remove the weight burden of radiation protection while allowing tableside access in a sterile working environment. because the weight of the shielding is not borne by the operator, thicker, heavier materials can be used. this results in a 16-78-fold decrease in radiation exposure to the operator. 103 the disadvantages of these systems are that they restrict the motion of the operator to some degree, and they increase the equipment that is present in an already crowded procedure laboratory environment. robotic manipulation of ablation catheters can be accomplished using external magnetic fields to guide magnetic tipped catheters or robotic armatures that actuate sheath and catheter movement remotely. these systems allow electrophysiologists to perform most of the procedure behind fixed radiation barriers, thereby eliminating exposure to scattered radiation for the operator (but not the patient or in-laboratory staff) and eliminating the need to wear protective lead. 26, 104, 105 although there is a learning curve associated with these technologies, they appear to yield results equivalent to conventional manual catheter procedures. recent developments in electroanatomic mapping and ice have been used to perform catheter ablation of atrial fibrillation 22 with minimal use of x-ray imaging. future developments of these and other imaging technologies that do not use ionizing radiation may obviate the need for radiation protection garments in the ep laboratory of the future. the physical stresses associated with working in the ep/ interventional laboratory have been identified as a high prevalence of orthopedic problems, particularly those related to the spine, hips, knees, and ankles. 102, 106 the primary contributor to orthopedic problems is wearing personal radiation protection apparel. other factors may be those related to ergonomic design, increasing complexity and duration of interventional procedures, falls, and lengthy careers. a multi-specialty occupational health group has been formed to evaluate risks and hazards and advocate for efforts to reduce these hazards. 107 table 5 provides a list of measures to be considered in laboratory design and procedural processes to reduce the incidence of ergonomic stresses on the ep laboratory physicians and staff. preventing the transmission of infectious agents to ensure the safety of the operator and other staff in the ep laboratory as much as possible involves, first and foremost, adherence to standardized uniformly applied universal precautions in every aspect of patient care. risks of acquisition of infectious diseases by health care workers can be minimized by adherence to current infection control guidelines. 108 institutions have policies on required annual or biannual staff inoculations. these may include inoculations such as hepatitis b, influenza, pertussis, and rubeola (measles). such inoculations can provide immunity from certain highly transmissible diseases, enabling staff to care for these patients without inordinate risk. for example, health care workers not immune to chicken pox should not be required to care for patients with chicken pox. 109 because of the specialization of staff in the ep laboratory, it can be difficult to provide adequate staffing if multiple concessions have to be made for noncompliant staff. the interval for regular testing of staff for tuberculosis is defined by every institution. in special situations, staff may be fitted for n95 filter masks, which are particulate respirators that are used to prevent inhalation of small infectious airborne particles transmitted by patients with active tuberculosis. institutional policies should be followed closely in these situations. the major features of universal precautions and body substance isolation are incorporated in standard precautions and are used universally to prevent transmission of highly contagious or virulent infectious agents that may be spread by air and/or contact. the basic principle is that all blood, body fluids, secretions, excretions (except sweat), nonintact skin, and mucous membranes may contain transmissible infectious agents. since it is impossible to identify all sources of all infectious agents, the same precautions are applied to every patient at every encounter. each institution has a detailed description of how standard precautions are implemented. the following text highlights certain aspects particularly pertinent to ep laboratory processes. hand hygiene is the single most important factor in controlling and preventing the transmission of germs. additional protection is offered by the appropriate use of medical gloves; however, the unnecessary and inappropriate use of gloves results in a waste of resources and may increase the risk of germ transmission. 110 hands are washed each time after gloves are removed. hand-washing supplies should be readily accessible in the ep laboratory and environments. sterile gloves are indicated for any surgical procedure, invasive radiologic procedure, and procedures involving vascular access (central lines). examination gloves are indicated in clinical situations where there is potential for touching blood, body fluids, secretions, excretions, and items visibly soiled by body fluids. splash protection with masks and eye shields is recommended when working with arterial and venous catheterization. gloves are not indicated (except for contact precautions) for tasks involving direct patient exposure, including taking blood pressure, pulse, and temperature; administering intramuscular and subcutaneous injections; bathing and dressing the patient; transporting the patient; and any vascular line manipulation in the absence of blood leakage, or with indirect patient exposure, including using the phone, writing in the chart, removing and replacing linen for the patient's bed, and placing noninvasive ventilation equipment and oxygen cannula. 110 safe injection practices, including the use of needleless injection systems and proper disposal of any sharps and other equipment used for invasive procedures, are paramount. single-dose vials are preferable to multiple-dose vials, particularly if the vial will be used for multiple patients. transmission-based precautions are as described in each institution's infection control policies. contact precautions require the use of gowns and gloves when there is any contact with the patient or the surrounding area. droplet precautions are invoked when infectious respiratory droplets can travel directly from the patient's respiratory tract to the recipient, generally over short distances. this necessitates facial protection, such as surgical mask, eye protection, and/ or face shield. personal eyeglasses and contact lenses are not considered adequate eye protection. 110 historically, the area of defined risk has been a distance of 3 ft or more around the patient, although depending on the size of the droplets (smallpox and sars are examples of small droplets), the infective area may be up to 6 ft from the patient. droplets do not remain infectious over long periods of time and therefore do not require special air handling and ventilation. airborne infection isolation precautions prevent transmission of infectious agents that remain infectious over long distances when suspended in the air (e.g., rubeola virus [measles], varicella virus [chicken pox], mycobacterium tuberculosis, and possibly sars coronavirus). if at all possible, ep procedures should be deferred in these patients unless a negative airflow procedure room is available. if an ep procedure is unavoidable, a regular positive pressure procedure room with airflow similar to an or and a portable highefficiency particulate air filtration unit may be used. 110, 111 the patient should wear a surgical mask, and the staff should wear n95 filter masks. the procedure should be the last case of the day, and the room left empty until adequate air exchange has taken place to clear the room. protocols and regulations as established by the institution for the disposal or processing of infectious material, drapes, and fluids should be followed. care should be taken to avoid shaking drapes or linens, which can aerosolize contaminants. all sharps that are to be reprocessed should be placed in puncture-resistant containers, leak proof on all sides and bottom, and labeled with a biohazard label. blood, suction material, or other contaminated liquids can be converted to a solid by the addition of a gel-forming substance to avoid risk of fluid leaking out of containers. "clean" and "dirty" areas of the laboratory should be maintained during procedures. personnel touching a contaminated patient/wound/area should try to avoid touching surrounding objects before removing gloves and washing hands. after procedures, all touched surfaces need to be cleaned with a hospital-approved disinfectant, leaving the surfaces wet for the amount of time as directed on the bottle. this cleaning includes monitoring cables, intravenous pumps, transfer equipment, and all nondisposable objects within 3-6 ft of a patient on droplet precautions and within 6 ft if the patient has been coughing. catheter reprocessing is being adopted by many ep laboratories nationwide, which is aimed at reducing the cost of expensive single-use device and to lessen the environmental impact from discarded supplies. with the passage and enactment of the medical device user fee and modernization act of 2002, the reprocessing of single-use devices is now supported in federal law. physicians and the entire ep team must come to an agreement on whether reprocessing is going to positively impact their clinical operation and patient care from the financial, legal, and/or ethical point of view. ablation catheters are classified by the fda as class iii devices; therefore, they cannot be reprocessed at this time. there are a few third-party fda-approved reprocessors used by many ep and catheterization laboratories. in choosing a vendor for reprocessing, each team should consider not only the price of the reprocessed catheters but also the particular vendor's turnaround time, catheter collection logistics, and reprocessing success rate. when a patient must be transported to other units or departments, the referring area should communicate the patient's isolation needs to the receiving area before transporting the patient. all precautions should be followed throughout the transport process. gloves and other appropriate barriers should be worn by the patient and/or health care worker during all transfers. protective equipment is then to be removed, the hands washed, and the patient is to be transported to or from the laboratory. patients on droplet precautions must have their mouths and/or tracheostomy covered with a surgical mask. transporting a patient on airborne precautions should be discouraged, but if absolutely necessary, consult with the infection control officer as to specific transportation methods. obtaining an informed consent is necessary for all procedures. obtaining an informed consent is a process whereby communication between a patient and a physician results in the patient authorizing the medical procedure. this is an integral procedure that safeguards patient autonomy and provides the patient with information about the procedure that allows them to understand and agree to the procedure being recommended. this process is not only a legal requirement but also an ethical obligation. to ensure appropriate communication, sufficient time should be allocated to this process. a physician, apn, or pa that is part of the ep team should obtain the informed consent, and it should be made clear if trainees, pas, or apns will be operators. the process must take place before premedication with sedation so that the patient is able to understand the discussion and communicate a decision. the discussion should take place out of the procedure room and be in a language that the patient adequately understands, with a qualified interpreter when needed. common risks, even if not considered serious, should be discussed, as should serious risks that are potentially life-threatening, even if exceedingly rare. all aspects of the procedure that can be reasonably anticipated should be included in the discussion. informed consent in the majority of pediatric cases will be granted by the parent or guardian acting on the patient's behalf. adult patients with chd are similar to other adults except in cases of cognitive handicaps, as in some genetic syndromes. a minor should be invited to participate in the process, but the consent should be obtained from a legal guardian. consent/assent forms for young patients older than 12 years are frequently used. serum or urine pregnancy testing is generally applied for females older than 12 years within 2 weeks before the procedure. the physician should be aware that they can have a substantial influence on their patient's decision. as an expert, the patient looks to the physician for guidance. it is an ethical obligation for the physician to present a rational argument for undergoing the procedure. in contrast, any form of coercion with the use of verbal or nonverbal threat or manipulation through the incomplete or nontruthful presentation of information is always unethical. patients should be given ample opportunity to discuss their concerns about the procedure and to have their questions answered satisfactorily. if the patient is incapacitated, every effort should be made to seek out and obtain consent from an appropriate surrogate. teaching invasive procedural skills is a necessary part of maintaining the availability of widespread medical expertise to patients. the use of simulators can be a helpful adjunct to teaching invasive procedures but cannot replace the experience gained from actual patient procedures. 112 although teaching is the charge of institutions with postgraduate training programs and affiliations with medical schools, the teaching of invasive procedures can occur in any type of institution. it is the ethical duty of physicians to communicate to patients when and in what way trainees will be involved in their procedures. when asked before the procedure, the majority of patients consent to allowing trainees to practice their skills. 113 most teaching institutions have patients sign a form during the admission process that acknowledges their implicit consent to allow trainees to perform procedures under the supervision of senior physicians. although this may be sufficient communication to patients for minor procedures such as venipuncture or acquisition of a surface ecg, in more invasive procedures it is mandatory that a separate discussion takes places during the informed consent process that clearly states the participation and the role of the trainee. allowing a trainee to practice on a patient under general anesthesia without the patient's explicit consent does not respect the patient's autonomy and compromises the trainee's moral integrity. trainees should be invited to participate in the procedure only as an integral part of their training, not for mere curiosity. it is the responsibility of the supervising physician to allow the trainee to participate in the capacity that they are capable. the trainee should not be left performing the procedure unsupervised. without clinical research, promising potentially lifesaving technologies and therapies cannot be safely made available for patients. our understanding of disease processes progresses because of ongoing research. clinical research, however, exposes patients to some risk without proven direct benefit. as a researcher, the operator's primary responsibility is generating knowledge, whereas as a physician, the operator's primary responsibility is the well-being of the patient. when there is a conflict between these two roles, the role of the physician must override the role of the researcher. patients should be considered for a research protocol if they are looking for improved outcomes compared with those of current therapies. recruitment of patients should be carried out in a manner that does not coerce participation. similarly, the opportunity for participation in available investigation protocols should be presented to all patients fulfilling inclusion and exclusion criteria even if the attending physician does not believe that the patient is an ideal candidate. a necessary amount of time should be given to the informed consent process in sufficient advance of the procedure so that the patient is not unduly pressured into making a decision. when children or adult patients unable to provide an informed consent are being considered for a research protocol, it may be helpful to have an independent advocate ensure that parents or legally authorized representatives are making a rational decision exposing the subject to risk. it is not enough to obtain a patient's consent to participate in a study. it is the ethical duty of an investigator to ensure that the research protocol does not place the participants at unreasonable risk that is disproportionate to the expected benefits of the research study. the protocol must have scientific merit with an adequate likelihood of success as well as an expectation of social benefit. all research protocols performed in the ep laboratory must be reviewed and approved by an independent group, such as an institutional review board, to ensure that the study is ethically acceptable. results must be reported honestly, not only to honor the risk that research participants took but also to protect future recipients of the technology and therapies made available through research. industry can partner with physicians to promote medical knowledge and improve patient care through examples such as supporting cme programs, physician training, and clinical research. although physicians and industry may share a common goal of advancing medical care, the primary interest of the physician is the well-being of the patient whereas that of the industry is profitability. any interaction with industry, therefore, has the potential to influence a physician's decision regarding a company's product or service and a physician must stay vigilant to avoid being biased by these interactions. 1 any gift or amenity that is offered has the potential to exert influence. 114 institutions and physician groups may define policies regarding the acceptance of industry gifts, but anyone approached by industry must be able to judge whether accepting the gift carries the danger of biasing a medical decision and compromising patient care. it is unethical to accept any gift that is predicated on using a particular product or service. it is imperative that the conflict of interest disclosures to employers, editors, professional organizations, and audiences be accurate and up to date. the conflict of interest policies of each physician's institution and professional organization must be followed. physicians who are on product review and new product introduction committees for their hospital or professional organization and who have consulting or other relationships with industry must disclose those relationships and recuse themselves from product decisions in those cases. guidance for the heart rhythm society pertaining to interactions with industry endorsed by the heart rhythm society on outcomes of cardiac perforation complicating catheter ablation of ventricular arrhythmias extraction of implantable cardiac electronic devices irrigated radiofrequency catheter ablation guided by electroanatomic mapping for recurrent ventricular tachycardia after myocardial infarction: the multicenter thermocool ventricular tachycardia ablation trial impact of pulmonary hypertension on the outcomes of noncardiac surgery: predictors of perioperative morbidity and mortality ventricular tachycardia originating from the aortic sinus cusp in patients with idiopathic dilated cardiomyopathy epicardial ventricular tachycardia ablation: a multicenter safety study transvenous lead extraction: heart rhythm society expert consensus on facilities, training, indications, and patient management guidelines for design and construction of health care facilities guidelines for environmental infection control in health-care facilities. center for disease control and the healthcare infection control practices advisory committee (hicpac) ma: national fire protection association medical imaging & technology alliance. the dicom standard: digital imaging and communications in medicine delayed-enhanced mr. scar imaging and intraprocedural registration into an electroanatomical mapping system in postinfarction patients isolated septal substrate for ventricular tachycardia in nonischemic dilated cardiomyopathy: incidence, characterization, and implications strategies for clinical and biomedical engineers to maintain readiness of external defibrilators naspe policy statement on catheter ablation: personnel, policy, procedures, and therapeutic recommendations cryoablation for atrioventricular nodal reentrant tachycardia in young patients: predictors of recurrence reconstruction of endocardial potentials and activation sequences from intracavitary probe measurements. localization of pacing sites and effects of myocardial structure intracardiac echocardiography in complex cardiac catheter ablation procedures left atrial thrombus associated with ablation for atrial fibrillation: identification with intracardiac echocardiography catheter ablation of atrial fibrillation without fluoroscopy using intracardiac echocardiography and electroanatomic mapping predictors of successful catheter ablation of ventricular arrhythmias arising from the papillary muscles on the use of cartosound for left atrial navigation heart rhythm society policy statement update: recommendations on the role of industry employed allied professionals (ieaps) worldwide experience with the robotic navigation system in catheter ablation of atrial fibrillation: methodology, efficacy and safety a novel remote catheter system, demonstrates safety and efficacy magnetic guidance system for cardiac electrophysiology: a prospective trial of safety and efficacy in humans remote magnetic navigation to map and ablate left coronary cusp ventricular tachycardia american board of internal medicine: accreditation coucil for graduate medical education. program requirements for graduate medical education in clinical cardiac electrophysiology american board of internal medicine. policies and procedures for certification task force 1: training in clinical cardiology task force 6: training in specialized electrophysiology, cardiac pacing, and arrhythmia management endorsed by the heart rhythm society american college of cardiology/american heart association 2006 update of the clinical competence statement on invasive electrophysiology studies, catheter ablation, and cardioversion: a report of the american college of cardiology/american heart association/american college of physicians task force on clinical competence and training developed in collaboration with the heart rhythm society american college of cardiology foundation/society for cardiovascular angiography and interventions expert consensus document on cardiac catheterization laboratory standards update a report of the american college of cardiology foundation task force on expert consensus documents american society of anesthesiologists task force on sedation and analgesia by non-anesthesiologist. practice guidelines for sedation and analgesia by nonanesthesiologists recommendations for advanced fellowship training in clinical pediatric and congenital electrophysiology: a report from the training and credentialing committee of the pediatric and congenital electrophysiology society hrs/ehra/ecas expert consensus statement on catheter and surgical ablation of atrial fibrillation: recommendations for patient selection, procedural techniques, patient management and follow-up, definitions, endpoints, and research trial design ehra/hrs expert consensus on catheter ablation of ventricular arrhythmias: developed in a partnership with the european heart rhythm association (ehra), a registered branch of the european society of cardiology (esc), and the heart rhythm society (hrs); in collaboration with the american college of cardiology (acc) and the american heart association (aha) frequently asked questions: clinical cardiac electrophysiology: review committee for internal medicine accf/aha/aap recommendations for training in pediatric cardiology. task force 4: recommendations for training guidelines in pediatric cardiac electrophysiology endorsed by the heart rhythm society heart rhythm society/pediatric and congenital electrophysiology society clinical competency statement: training pathways for implantation of cardioverter-defibrillators and cardiac resynchronization therapy devices in pediatric and congenital heart patients recommendations for advanced fellowship training in clinical pediatric and congenital electrophysiology: a report from the training and credentialing committee of the pediatric and congenital electrophysiology society guidelines for the management of adults with congenital heart disease: a report of the american college of cardiology/american heart association task force on practice guidelines (writing committee to develop guidelines on the management of adults with congenital heart disease) national council of state boards of nursing. model for aprn regulation: licensure, accreditation, certification and education commission on accreditation of allied health education programs. profession description & certficaition information physician assistants in the united states defining the physician assistant role in ontario: ontario physician assistant scope of practice statement and ontario physician assistant competency profile. healthforce ontario the joint commission. national patient safety goals: hospital accreditation program standards for basic anesthetic monitoring a retrospective analysis of proceduralist-directed, nurse-administered propofol sedation for implantable cardioverter-defibrillator procedures statement on granting privileges for administration of moderate sedation to practitioners who are not anesthesia professionals naspe expert consensus conference: radiofrequency catheter ablation in children with and without congenital heart disease. report of the writing committee. north american society of pacing and electrophysiology general anesthesia reduces the prevalence of pulmonary vein reconnection during repeat ablation when compared with conscious sedation: results from a randomized study anesthetic management of patients undergoing pulmonary vein isolation for treatment of atrial fibrillation using high-frequency jet ventilation complications during catheter ablation of atrial fibrillation: identification and prevention assessment of catheter tip contact force resulting in cardiac perforation in swine atria using force sensing technology ablation of atrial fibrillation using an irrigated-tip catheter: open or closed? efficacy and safety of cryoballoon ablation for atrial fibrillation: a systematic review of published studies signals and signal processing for the electrophysiologist: part i: electrogram acquisition signals and signal processing for the electrophysiologist: part ii: signal processing and artifact hospital infection control practices advisory committee american college of cardiology/ society for cardiac angiography and interventions clinical expert consensus document on cardiac catheterization laboratory standards. a report of the american college of cardiology task force on clinical expert consensus documents consensus guidelines for periprocedural management of coagulation status and hemostasis risk in percutaneous imageguided interventions evaluation of a new standardized protocol for the perioperative management of chronically anticoagulated patients receiving implantable cardiac arrhythmia devices implantable cardioverter-defibrillator registry risk score models for acute procedural complications or death after implantable cardioverter-defibrillator implantation left atrial catheter ablation and ischemic stroke risk factors for particulate and microbial contamination of air in operating theatres update on cardiovascular implantable electronic device infections and their management: a scientific statement from the american heart association prophylactic use of vancomycin in adult cardiology and cardiac surgery sudden cardiac death and malignant arrhythmias: the scope of the problem in adult congenital heart patients late enlargement of radiofrequency lesions in infant lambs. implications for ablation procedures in small children radiofrequency catheter ablation: indications and complications paces/hrs expert consensus statement on the management of the asymptomatic young patient with a wolff-parkinson-white (wpw, ventricular preexcitation) electrocardiographic pattern: developed in partnership between the pediatric and congenital electrophysiology society (paces) and the heart rhythm society (hrs) guidelines for device-based therapy of cardiac rhythm abnormalities: a report of the american college of cardiology/american heart association task force on practice guidelines (writing committee to revise the acc/aha/ naspe 2002 guideline update for implantation of cardiac pacemakers and antiarrhythmia devices): developed in collaboration with the american association for thoracic surgery and society of thoracic surgeons cardiac resynchronization therapy in pediatric and congenital heart disease cardiac resynchronization therapy (and multisite pacing) in pediatrics and congenital heart disease: five years experience in a single institution pediatric radiofrequency catheter ablation registry success, fluoroscopy time, and complication rate for supraventricular tachycardia: comparison of early and recent eras radiofrequency catheter ablation in infants o 18 months old: when is it done and how do they fare? short-term data from the pediatric ablation registry techniques to avoid atrioventricular block during radiofrequency catheter ablation of septal tachycardia substrates in young patients cryoablation for atrioventricular nodal reentrant tachycardia in young patients: predictors of recurrence acc/aha/hrs 2006 key data elements and definitions for electrophysiological studies and procedures: a report of the american college of cardiology/american heart association task force on clinical data standards (acc/aha/hrs writing committee to develop data standards on electrophysiology) use of cryoablation for treatment of tachyarrhythmias in 2010: survey of current practices of pediatric electrophysiologists prospective assessment after pediatric cardiac ablation: fate of intracardiac structure and function, as assessed by serial echocardiography accf/aha/hrs/scai clinical competence statement on physician knowledge to optimize patient safety and image quality in fluoroscopically guided invasive cardiovascular procedures. a report of the american college of cardiology foundation/american heart association/american college of physicians task force on clinical competence and training icrp publication 113. education and training in radiological protection for diagnostic and interventional procedures skin injury following prolonged fluoroscopy: early and late appearances committee to assess health risks from exposure to low levels of ionizing radiation national research council. health risks from exposure to low levels of ionizing radiation: beir vii phase predictors of fluoroscopy time and estimated radiation exposure during radiofrequency catheter ablation procedures radiation exposure during radiofrequency catheter ablation of accessory atrioventricular connections radiation dose to patients from cardiac diagnostic imaging nrc: annual dose limits for radiation workers ionizing radiation in cardiac imaging: a science advisory from the american heart association committee on cardiac imaging of the council on clinical cardiology and committee on cardiovascular imaging and intervention of the council on cardiovascular radiology and intervention quality improvement guidelines for recording patient radiation dose in the medical record for fluoroscopically guided procedures a novel radiation protection drape reduces radiation exposure during fluoroscopy-guided electrophysiology procedures cfr-code of federal regualtions title 21 prevalence and risk factors for cervical and lumbar spondylosis in interventional electrophysiologists cardiac catheterization: impact of face and neck shielding on new estimates of effective dose interventional cardiologists and risk of radiation-induced cataract: results of a french multicenter observational study prevalence of spinal disc disease among interventional cardiologists evaluation of a radiation protection cabin for invasive electrophysiological procedures ablation of atrial fibrillation utilizing robotic catheter navigation in comparison to manual navigation and ablation: single-center experience robotic magnetic navigation for atrial fibrillation ablation occupational hazards of interventional cardiologists: prevalence of orthopedic health problems in contemporary practice occupational health hazards in the interventional laboratory: time for a safer environment lessons learned: protection of healthcare workers from infectious disease risks guideline for isolation precautions: preventing transmission of infectious agents in health care settings glove use information leaflet new ventilation guidelines for health-care facilities intravenous catheter training system: computerbased education versus traditional learning methods requesting consent for an invasive procedure in newly deceased adults physicians' behavior and their interactions with drug companies. a controlled study of physicians who requested additions to a hospital drug formulary see tables a1 and a2 . key: cord-324507-w32pe2pz authors: dubé, mirette; kaba, alyshah; cronin, theresa; barnes, sue; fuselli, tara; grant, vincent title: covid-19 pandemic preparation: using simulation for systems-based learning to prepare the largest healthcare workforce and system in canada date: 2020-08-18 journal: adv simul (lond) doi: 10.1186/s41077-020-00138-w sha: doc_id: 324507 cord_uid: w32pe2pz healthcare resources have been strained to previously unforeseeable limits as a result of the covid-19 pandemic of 2020. this has prompted the emergence of critical just-in-time covid-19 education, including rapid simulation preparedness, evaluation and training across all healthcare sectors. simulation has been proven to be pivotal for both healthcare provider learning and systems integration in the context of testing and integrating new processes, workflows, and rapid changes to practice (e.g., new cognitive aids, checklists, protocols) and changes to the delivery of clinical care. the individual, team, and systems learnings generated from proactive simulation training is occurring at unprecedented volume and speed in our healthcare system. establishing a clear process to collect and report simulation outcomes has never been more important for staff and patient safety to reduce preventable harm. our provincial simulation program in the province of alberta, canada (population = 4.37 million; geographic area = 661,848 km(2)), has rapidly responded to this need by leading the intake, design, development, planning, and co-facilitation of over 400 acute care simulations across our province in both urban and rural emergency departments, intensive care units, operating rooms, labor and delivery units, urgent care centers, diagnostic imaging and in-patient units over a 5-week period to an estimated 30,000 learners of real frontline team members. unfortunately, the speed at which the covid-19 pandemic has emerged in canada may prevent healthcare sectors in both urban and rural settings to have an opportunity for healthcare teams to participate in just-in-time in situ simulation-based learning prior to a potential surge of covid-19 patients. our coordinated approach and infrastructure have enabled organizational learnings and the ability to theme and categorize a mass volume of simulation outcome data, primarily from acute care settings to help all sectors further anticipate and plan. the goal of this paper is to share the unique features and advantages of using a centralized provincial simulation response team, preparedness using learning and systems integration methods, and to share the highest risk and highest frequency outcomes from analyzing a mass volume of covid-19 simulation data across the largest health authority in canada. with the emergence of the covid-19 pandemic of 2020, healthcare resources have been strained to unforeseeable capacities, promoting the need for rapid, effective, and efficient preparedness. this has prompted the emergence of just-in-time preparedness strategies, including simulation for systems evaluation and healthcare provider (hcp) learning to support planning across healthcare sectors [1] [2] [3] [4] [5] [6] [7] [8] [9] . simulation has been pivotal for systems testing and integrating new and improved components such as novel workflows, protocols, and cognitive aids with rapid changes to practice and care delivery. finding clear approaches to rapidly collect and report what is working well and what needs to change is urgently required for hcp and patient safety to ultimately reduce preventable harm. the provincial simulation program, named esim (educate, simulate, innovate, motivate) in the province of alberta, canada (population = 4.37 million; geographic area = 661,848 km 2 , [10] ), has rapidly responded to preparedness training by establishing a central esim provincial covid response team (similar to an emergency command center operations team for simulation) to facilitate a large-scale project managing the initiation, planning, execution, and performance/monitoring of a provincial covid-19 simulation response [11] . over just 5 weeks, this response team enabled a harmonized intake process, design, and development of a robust covid-19 simulation curriculum, mobilized a data collection/outcome reporting team, and a response plan to facilitate over 400 acute care simulation session requests across alberta's broad geographical zones. this coordinated approach and infrastructure enabled an integrated provincial multi-site simulation response, allowing the ability to rapidly theme and categorize a mass number of simulation findings (over 2,500 systems issues) from over 30,000 learners (hcp) on the frontline. the simulation "command center" served to disseminate the outcomes across a large single health authority to further support ongoing planning, develop and refine the curriculum, and remain a specialized contact team of simulation experts for all health sectors (primarily acute care; urban and rural). the goal of this paper is to share the unique features and advantages of using a centralized provincial simulation response team, preparedness using learning and systems integration methods, and to share the highest risk and highest frequency outcomes from analyzing a mass volume of covid-19 simulation data across the largest health authority in canada. alberta health services: largest integrated healthcare system in canada alberta health services (ahs) has a centralized approach to its leadership and core operational functions (e.g., human resources and information technology) and provides medical care at over 650 facilities across the province, including 106 acute care hospitals and 25,653 continuing care beds/spaces, five stand-alone psychiatric facilities, 2723 addiction and mental health beds and 243 community palliative and hospice beds. ahs has over 125,000 employees and over 10,000 physicians and service accountability zones are divided into five geographical areas: north, edmonton, central, calgary, and south [10] . there are few organizations in the world, which are comparable to ahs' integrated healthcare system relative to its size and capacity [12] , making it a unique opportunity to glean insights and lessons learned from covid-19 pandemic preparedness. while disaster planning has become core content within some training curricula, the majority of health professionals remain ill prepared to deal with large scale disasters and will not encounter these critical scenarios in practice [13, 14] . simulation is one method that has been identified to improve readiness and preparedness in times of disaster through deliberate practice and exposure to rare situations [15] [16] [17] [18] . simulation-based learning has historically focused on individual and team training of practicing hcps [19] [20] [21] [22] [23] [24] [25] , although it has evolved to include just-in-time in situ training within the actual clinical environment [26] [27] [28] including simulation for systems integration (sis), targeting the testing and integration of systems and processes (e.g., workflows, care pathways) that are uniquely important to disaster preparedness [29] [30] [31] [32] . the esim covid-19 response team has been involved in pioneering work in sis [11, 32, 33] systems integration, an engineering term defined as bringing many subsystems together into one better functioning system, has the potential to improve the safety and quality of care through re-engineering of the processes and systems in which hcp work [34] . the system engineering initiative for patient safety (seips) 2.0 model provides a framework outlining how the work system components (e.g., tools/technology, tasks, environment, people/teams) provide a matrix for thematically categorizing systemfocused debriefing (sfd) outcomes in a complex adaptive healthcare system [35] . sis/sfd allows testing of new processes within healthcare systems to inform design and utility, and to identify system issues proactively to prevent harm [15, [19] [20] [21] . the rapid onset of the covid-19 crisis is having a profound impact on global health, increasing demand, and risks on healthcare systems due to rapidly changing and unpredictable circumstances. the use of in situ simulation as a proactive risk mitigation strategy to prepare healthcare organizations for pandemic planning is well supported in the literature [8, 9, [26] [27] [28] . pandemics place high demands on clinical care and potential risk of contamination for hcp, which increases the fear of spread to others [1, 36, 37] . deliberate practice through simulation can reduce the cognitive load of hcp involved in direct frontline patient care, potentially mitigating latent safety threats (lsts) (e.g., errors in design, organization, and/or training that may have a significant impact on patient safety) in times of extended pressure, exhaustion, and burnout [38] . as recently illustrated in both italy [2] and singapore [3] , simulation is key to preparedness by optimizing work flow structures, developing new processes, managing staffing levels, procuring equipment, bed management, and enforcing consistency of medical management of patients. in those ways, simulation can be used as both a learning and evaluation tool (e.g., sis/sfd) [32] . diekmann et al. also described the potential of using simulation to improve hospital responses to the covid-19 crisis [6] . recently published covid-19 simulation papers [8, 9] share lessons learned in an attempt to support organizations that may seek to use simulation for diagnosing, testing, and embedding these approaches within pandemic constraints. while these papers do not provide specific outcomes from their system simulations, they share practical tips, tools, scenarios, debriefing questions, and resources which can be used to analyze the current needs and responses to potentially mitigate the negative impacts of the covid-19 crisis. similarly, chan and nickson [7] , published an example of practical considerations for organizations in the development for just-in-time simulation training, including the scenario development, prebriefing, and debriefing, using system and process simulation for the testing of airway management for suspected/confirmed cases of covid-19. while many of the findings in the emergent literature above glean insights into the use of simulation as both a learning and evaluation tool to prepare for covid-19, a key gap identified is that many of these outcomes are unique to the experience of one specific site or one unique institution, which limit the generalizability and scalability of the findings. this is especially important when using simulation for pandemic preparedness to rapidly test systems and processes and prepare frontline teams to care for potential covid-19 patients. therefore, an identified need in the literature is the ability to proactively identify systems issues, while testing new pandemic processes in real time, and sharing these organizational learnings and system-level outcomes on a mass scale to both anticipate and plan for covid-19. this is an invaluable opportunity to support healthcare organizations' preparedness during a global pandemic. our provincial simulation program rapidly mobilized to respond to our organization's need by establishing a central simulation covid response team and a large-scale evaluation project to manage the initiation, planning, execution, and performance/monitoring for all simulation-based requests. the following sections will summarize our project evaluation approach based on common project phases [39] : (a) project initiation and planning; (b) project execution; (c) project performance and monitoring; (d) organizational learning; and (e) reporting of outcomes. rapidly designating the esim covid-19 response team allowed a centralized contact and triage system across the province for all simulation-based requests coming from hundreds of centers, allowing coordination, planning, and resource oversight for simulation training. team members included a team lead, geographically dispersed esim consultants (clinicians whose workplace role is to lead the design, delivery, faculty development, and evaluation of simulation methodologies and projects at ahs) and a designated data and outcomes team solely responsible for theming and categorizing thousands of simulation data points from covid-19 simulations. the simulation infrastructure in place in ahs prior to covid-19 (e.g., esim consultants positioned in every zone (rural, urban, and mobile program) and > 1300 esim-trained simulation educators across ahs) ensured every geographical zone was supported. the intake process was maintained through a central spreadsheet accessible to the entire provincial team and a needs assessment facilitated by an esim consultant (additional file 1 intake form) to ensure the needs and objectives aligned with established simulation-based methodologies and capacity. requests were prioritized as they were processed and whenever possible, prior relationships with simulation-trained educators comfortable with the use of simulation-based methods were leveraged to help facilitate the large number of sessions. esim consultants either fully led the sessions, initiated sessions while mentoring others to replicate delivery on an on-going basis (especially when large numbers of hcp were involved), and/or shared resources with faculty who had the capacity and experience to lead sessions independently. the first wave of intake requests (and patients) in alberta, organically flooded from emergency departments, followed by intensive care units, operating rooms, labor and delivery units, and neonatal icus in calgary. simulations were triaged for covid-19 training and preparedness for the highest risk locations and activities across acute care. a second wave of requests included diagnostic imaging and inpatient medical units (including newly created inpatient units for covid-19, among others). a similar pattern of requests followed from all of the five geographic zones in alberta in concordance with the number of covid-19 cases starting to move through the system. daily response team meetings ensured strategic planning with esim consultants to then specifically target and reach out to acute care sites with no simulation trained champion, those who had not made any requests, and to ensure simulation support and curriculum resources were shared across all zones. a novel part of our approach was the creation of a robust, centrally located simulation curriculum, which was rapidly developed and mobilized over a 10-day period at the onset of the covid-19 simulation response. curriculum content was vetted through medical experts, clinical leaders, infection prevention and control consultants, and the organization's emergency command center to ensure accuracy, alignment, and validity before application. new emerging curriculum resources for covid-19 were reviewed daily to ensure the most relevant up-to-date information was reflected within current best practice recommendations. the curriculum included covid-19 simulation scenarios for all sectors, and included a prebriefing script, debriefing tools [32] , cognitive aids, "how-to" guides and shared webinars. curricular materials were shared over the 5-week period with over 600 teams across ahs. objectives for the covid-19 simulation scenarios were based on the highest risk and highest impact system issues identified and prioritized by integrated interprofessional teams of clinicians, managers, and educators-based on their units/departments greatest needs. table 1 provides samples of frequently used scenario objectives across all simulations and their related work system categories [35] . central to our unique approach was the planning and execution of three common simulation-based methods to prepare teams and the healthcare system during covid-19 preparedness (table 2 ). these methods include (1) surge planning and table top debriefing; (2) process walkthrough and environmental scans; and (3) rapid cycle simulation and debriefing. both learnerfocused and system-focused debriefing approaches were used across these three methods [22, 32] . in some instances, esim consultants and human factors (hf) specialists led the co-facilitation and debriefing of table top surge planning exercises. these exercises allowed for proactive pandemic surge planning assessment of facility, departments, programs, and services related to covid-19 patient flow. esim and hf were then utilized for designing decision algorithms, observation of new work processes and work environments, and identifying further improvement opportunities. early preparatory work focused on using a systems approach to co-design new covid-19 processes and spaces. these physical walkthrough simulations were based on day-to-day movement, case-specific patient presentations, and workflows that would be impacted by covid-19. they were also used to train multiple hcps to orientate them to the new covid-19 response for their areas (see additional file 2 for key points to consider in a process walkthrough environmental scan planning exercise). the primary focus was on identifying lsts prior to the first patient experience which allowed for application of a new processes in a plan, do, study, act (pdsa) cycle [40] . the team moved through a department/clinical area, doing an environmental scan for barriers, identification of missing equipment, testing of communication pathways, and identification of new tasks/roles/responsibilities. any changes to the space and processes were actioned to leadership. once final covid-19 processes and workflows were established, departments/clinical areas progressed to rapid cycle simulation training followed by debriefing [41] . this involved rapid training sessions (average 20min scenario duration followed by 20-min debriefings) to ensure small groups of interprofessional team members were able to apply new processes in the live clinical environment prior to use with actual patients. all questions related to medical management, infection, prevention and control (ipac) and process were also debriefed during these sessions. a data outcomes team was designated to rapidly process and analyze data collected from all provincial covid-19 simulations. figure 1 outlines a systematic iterative process developed for intake and entry of data. an initial coding scheme for categorization of data was created. data analysis included the theming of system-focused debriefing outcomes [32] based on the seips 2.0 system categories (e.g., tools/technology, tasks, environment, people/teams) [35] . all outcome data were analyzed to determine convergence of themes and repeated expression of reoccurring constructs. discrepancies in the codes and labeling of themes were discussed by the core project team regularly and resolved; with themes simplified and altered accordingly until complete agreement was reached. saturation of emergent themes was reached across > 400 simulation sessions provincially. the number of learners/participants and simulation sessions was collected using a standardized intake spreadsheet and was collated biweekly. when using a simulation for systems integration approach, the collection and sharing of systems issues and learnings is key to informing a parent organization. the esim program's infrastructure and approach was critical in a pandemic situation, as the information and learnings from the sis were used to iteratively make improvements and help teams across the organization anticipate and plan for issue mitigation and process improvement. the timely analysis of large volumes of data that was being collated and themed in real time by the esim response team proactively informed and enabled scaling up of quality improvement activities across sites, departments, and zones. our organizational learning included broad sharing of weekly dashboard updates of key outcomes, webinars (local and international webinars shared across the entire ahs organization and beyond resulting in over 4000 unique views; recording shared with > 100, 000 email addresses) [4, 42] , and key covid-19 resources developed for simulation. highest impact and highest frequency outcomes the provincial esim covid-19 simulation response team facilitated simulation session requests by leading the intake, design, development, planning, and facilitation of over 400 acute care simulation sessions across all sectors of our provincial health program. in under a 5-week period, an estimated 30,000 learners participated in simulation in both rural (17%) and urban (83%) centers across the province (fig. 2) . there was a wide variance in the number of sessions/ learners per request ranging from 1-3 days of simulation training and 10-400 people requiring training per request. well over 2500 systems issues, including lsts, have been proactively identified and mitigated through > 400 simulation sessions. systems issues across all sessions were categorized into processes (61%), tools and technology (14%), persons and tasks (14%), environment (7%), and organization (4%). table 3 highlights the highest priority themes discussed in the debriefings (impact) and the highest reported outcomes (frequency) that emerged from all covid-19 simulations. in analyzing the nine themes, it is clear that the rapid knowledge translation of best practices, new guidelines, and processes following system simulation events can potentially serve other organizations that may seek to learn from our centralized, coordinated approach to using system integration simulation for pandemic planning and preparedness. in synthesizing the learnings from the highest impact and highest frequency outcomes of the 9 themes reported in table 3 , there are several reasons on why these salient themes came together based on our unique context and organizational level approach to sis. each of these themes informed our organization as to what was working well at one site, department, and zone and what needed to be improved and scaled up across the organization. for example, although we had an ipac team, and multiple related resources and cognitive aids designed to support ipac processes; it became clear, as identified by the frontline through simulation, that more needed to be done to address the nuances and unique clinical practices of ipac specific to the pandemic. while hundreds of learners continued to struggle with safe doffing, simulation was able to inform the development of emerging best practices surrounding 1:1 doffing, what questions were repeatedly being discussed; why breaches were happening; and ensuring one person guided and supported individuals during doffing. observing the trending of process-related systems issues enabled our team to share this finding broadly and focus our simulation tools and approaches to further address systems issues that impacted not just one unit, site or department enabling the learnings identified through simulation to be scaffolded and shared across the entire province. this led to further development of a systemsbased process walk through approach, with debriefing, to ensure a systematic identification of process issues. repeated sis debriefings where frontline team members asked about personal items such as watches, earrings, and identification badges in covid rooms allowed us to share rapidly evolving strategies to address proper maintenance of isolation environments and the prevention of contamination. for teams who were struggling with effective ways to communicate outside of covid rooms, we were able to rapidly share innovations on a large scale to any other teams who were in the same position in preparing their unit and staff to respond to the evolving nature of the global pandemic. this involved designing, through simulation, innovative approaches necessary to keep staff and patients safe by communicating using dry erase markers on glass between rooms, as one example. as protected covid-19 intubations were identified as a high anxiety and high-risk time for healthcare workers, as shown by repeated simulation requests from healthcare staff targeting these objectives, having the ability to test these processes in simulation and refine a critical care medicine cognitive aid for pre-intubation and then share these findings on such a large scale was essential to successful preparation and safety of staff. as a result of multiple simulations on intubation, a covid-19 cognitive aid was developed, which was then contextualized for rural sites and then broadly shared across the province using our coordinated system. our use of simulation for systems integration involved taking a project approach to the initiation, planning, execution, and reporting [39] (e.g., shared learnings) to enable proactive improvement work for safer, more efficient, and reliable care processes for patients and healthcare teams [43] [44] [45] [46] [47] [48] [49] [50] . this paper adds to the emerging literature on using simulation for pandemic planning and preparedness [8, 9] as it describes a highly coordinated covid-19 pandemic simulation response using a centralized team, robust valid curriculum, and data outcomes team to analyze and rapidly share an unprecedented volume of simulation data for a large-scale sis project across the largest single health authority in canada. unfortunately, the speed at which the covid-19 pandemic has emerged in canada may prevent every acute care center in both urban and rural settings in having healthcare teams participate in "just-in-time" in situ simulation-based learning and systems/process. the ability to test new pandemic processes, and share organizational learnings to be disseminated on a mass scale has been pivotal in preparing our healthcare system to respond to the emergent threat of covid-19. the importance of using simulation as a "first choice" strategy for ensuring individual, team, and system readiness in times of crisis is supported by multiple publications in the literature [5, [51] [52] [53] , and highlights that a sustained investment in simulation programs will have immeasurable impacts across healthcare systems following the pandemic. many of these papers [3, [6] [7] [8] [9] ] indicate a single site or unit approach to pandemic preparation, although do not specifically highlight using a coordinated and centralized simulation team, development of robust valid curriculum, and real time data analysis of emerging themes from hundreds of simulations. although "more" may not necessarily always be better, we advocate that during times of pandemic where time sensitivity and reliable information are of utmost importance, a coordinated organizational-wide simulation table 3 highest impact and highest frequency outcomes response approach allows for broader and faster sharing, scalability, and generalizability of the findings. the validation of these 9 themes from simulations across multiple sites and teams outweighs the findings at only one. one of the emerging findings that differed our project from other covid-19 system simulations experiences both nationally and globally in using simulation as both a learning and evaluation tool to prepare [3, [6] [7] [8] [9] , was recognizing the critical importance of embedding simulation as a central part of the organizational learning, and the overall pandemic preparedness strategy. similar to an operational "emergency command center," simulation programs (regardless of the size) need to situate themselves to be members "at the table" with key programs informing decision-making, and influencing organizational planning and pandemic use of trigger scripts on pagers to signal a priority response. scripts like "covid airway" or "covid transport" to alert a team and get the right people and the right equipment to the right place. preparedness from the beginning. many factors influence the ability of simulation programs to "take this seat" including how the program is established prior to pandemic and how well it may be recognized by hospital administration staff in informing the organization of the needs and challenges coming directly from the frontline care providers. we situated ourselves as key informants to leaders and the organization's administration, through planning and executing a large-scale simulation for systems integration project. our outcomes, which identified over 2500 systems issues, including lsts, had meaning to the organization because they were widespread and converged data from several sites including both urban and rural centers and across the continuum of care. in addition, we could equally share and reach all sites within our organizational structure for the new emerging themes we were uncovering in real time. we informed our organization on multiple levels through our webinars, dashboards, and relationships built across a large health authority. queueing ipac team members of ongoing concerns with best practices for safe doffing to enable targeted approaches; sharing weekly questions still arising from the frontline to the ipac team; and then supporting the development of a "doffing buddy" program are a few examples. this example highlights that while the emerging literature on covid-19 simulations [3, [6] [7] [8] [9] differ in their approach and scale of sis, there is overlapping outcomes and triangulation of findings specific to ipac across the different systems, countries, and jurisdictions. essentially, this underlines the validity and robustness of the use of simulation for systems integration methodology for organizational learning. we realized that process-related systems issues were widespread and the requests for environmental scans with debriefing prompted hundreds of teams to start redesigning their covid care spaces after one of our "shared learnings" webinars. seeing the rapid uptake of teams developing checklists and cognitive aids in isolation of each other, and with limited human factors experience, led us to establish collaborative human factors tools on how to develop an effective cognitive aid and principles of safe use. we believe all of these developments were the result of a swift and purposeful largescale centralized approach. recognizing that not all health authorities have opportunity to coordinate or operationally support a centralized team and curriculum across sites; we recommend the explicit effort of simulation programs to align with other programs in meaningful ways to analyze and share emerging data in real-time to support validation for broader sharing and scalability when possible. the simulation methods and outcomes used in the covid-19 response have had a profound impact on our teams, processes, and system functioning. our experience offers other organizations' learnings to glean and consider the importance of establishing a centralized simulation response team for scale, spread and speed of knowledge translation, implementation, and change management effectiveness. with on-going use of sis sessions informing cycles of improvement, the simulation will continue to be a key organizational tool we will use to further manage this evolving crisis, as well as future needs of our healthcare organization. our pandemic preparedness highlighted the essential use of simulation as both an evaluation tool capable of testing systems and processes, and identifying and mitigating lsts, as well as an education tool capable of rapidly preparing frontline teams in terms of the changes identified above. this project has identified how the dissemination and broadcasting of curriculum and lessons learned (e.g., emerging themes, innovations, systems-based approaches) from simulation can rapidly help a large organization over a large geographic area be adequately prepared for an evolving situation like the covid-19 pandemic of 2020. supporting the health care workforce during the covid-19 global epidemic critical care utilization for the covid-19 outbreak in lombardy, italy: early experience and forecast during an emergency response preparing for a covid-19 pandemic: a review of operating room outbreak response measures in a large tertiary hospital in singapore covid 19 simulation shared learning and resource review preparing and responding to 2019 novel coronavirus with simulation and technology-enhanced learning for healthcare professionals: challenges and opportunities in china the use of simulation to prepare and improve responses to infectious disease outbreaks like covid-19: practical tips and resources from norway, denmark, and the uk covid-19 airway management: better care through simulation • litfl [internet]. life in the fast lane • litfl • medical blog translational simulation for rapid transformation of health services, using the example of the covid-19 pandemic preparation lessons learned in preparing for and responding to the early stages of the covid-19 pandemic: one simulation's program experience adapting to the new normal our story: building the largest geographical provincial simulation program in canada. accepted article in medical training magazine,. medical training magazine goals, recommendations, and the how-to strategies for developing and facilitating patient safety and system integration simulations a multiphase disaster training exercise for emergency medicine residents: opportunity knocks precepting at the time of a natural disaster impact of repeated simulation on learning curve characteristics of residents exposed to rare life threatening situations simulation as a critical resource in the response to ebola virus disease use of simulation to gauge preparedness for ebola at a free-standing children's hospital an active shooter in your hospital: a novel method to develop a response policy using in situ simulation and video framework analysis see one, do one, teach one: is it enough? no: trainee focus the value proposition of simulation-based education collaborative practice in action: building interprofessional competencies through simulation based education and novel approaches to team training promoting excellence and reflective learning in simulation (pearls): development and rationale for a blended approach to health care simulation debriefing in situ simulation: detection of safety threats and teamwork training in a high risk emergency department in situ simulation in continuing education for the health care professions: a systematic review simulation in the clinical setting: towards a standard lexicon connecting simulation and quality improvement: how can healthcare simulation really improve patient care? adapting form to function: can simulation serve our healthcare system and educational needs? crash testing the dummy: a review of in situ trauma simulation at a canadian tertiary centre translational simulation: not 'where?' but 'why?' a functional view of in situ simulation reducing door-to-needle times in stroke thrombolysis to 13 min through protocol revision and simulation training: a quality improvement project in a norwegian stroke centre priorities related to improving healthcare safety through simulation pearls for systems integration: a modified pearls framework for debriefing systemsfocused simulations commissioning simulations to test new healthcare facilities: a proactive and innovative approach to healthcare system safety agency for healthcare research and quality seips 2.0: a human factors framework for studying and improving the work of healthcare professionals and patients covid-19: protecting worker health. ann work expo health what factors might have led to the emergence of ebola in west africa? beyond the clinical team: evaluating the human factors-oriented training of non-clinical professionals working in healthcare contexts basics of quality improvement in health care systematic review of the application of the plan-do-study-act method to improve quality in healthcare pediatric resident resuscitation skills improve after "rapid cycle deliberate practice covid 19 simulations simulation canada variation and adaptation: learning from success in patient safety-oriented simulation training systems integration, human factors, and simulation comprehensive healthcare simulation simulation to assess the safety of new healthcare teams and new facilities use of simulation to test systems and prepare staff for a new hospital transition using in situ simulation to evaluate operational readiness of a children's hospital-based obstetrics unit the use of in situ simulation to detect latent safety threats in paediatrics: a cross-sectional survey work system design for patient safety: the seips model using in situ simulation to identify and resolve latent environmental threats to patient safety: case study involving a labor and delivery ward using briefing, simulation and debriefing to improve human and system performance rapid cycle deliberate practice in medical education -a systematic review considerations for psychological safety with system-focused debriefings springer nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations the authors would like to recognize the many partnerships and people that enabled an impressive simulation response to the covid-19 pandemic. this project, considering its scope and scale, would not have been possible without the efforts of many team members from the alberta health services' esim provincial simulation program, kidsim pediatric simulation program, and covenant health authority. the authors would like to acknowledge the following people for their contributions to the project curriculum design, delivery, data entry, manuscript review, and input throughout the covid supplementary information accompanies this paper at https://doi.org/10. 1186/s41077-020-00138-w. authors' information md is a masters certified critical care registered respiratory therapist with 24 years of experience in healthcare. she has over thirteen years' experience in simulation and debriefing; training hundreds of interprofessional teams, faculty development, simulation for systems integration and debriefing, undergraduate education, global health, human factors, design thinking, research, project and change management, former provincial director of quality and patient safety education, and leading provincial simulation and quality improvement projects. she is passionate about patient safety, improving healthcare systems, and has multiple peer-reviewed publications on interprofessional collaboration, team training, and using simulation and debriefing for systems improvement. during the covid-19 pandemic of 2020, md was the esim provincial simulation response team lead for the province of alberta, canada. no funding was required for the development of this manuscript. data sharing is not applicable to this article as no datasets were generated or analyzed.ethics approval and consent to participate no ethics approval or consent was needed for this manuscript. the authors declare that they have no competing interests.author details 1 esim provincial simulation program, alberta health services, alberta health services, 1403 29th street nw, calgary, alberta t2n 2 t9, canada. key: cord-337325-h9l7yy5z authors: bruzzone, francesco; scorrano, mariangela; nocera, silvio title: the combination of e-bike-sharing and demand-responsive transport systems in rural areas: a case study of velenje date: 2020-09-29 journal: nan doi: 10.1016/j.rtbm.2020.100570 sha: doc_id: 337325 cord_uid: h9l7yy5z an analysis of the operational characteristics of the transit system serving the town of velenje (slovenia) revealed poor performance and the need for improvements. this paper describes the potential integration of an electric bike-sharing system and a semi-flexible demand-responsive transport system to effectively solve this issue. additionally, general guidance is provided for transit systems with low travel demand. appropriate transport system schedules are proposed to facilitate customers' use and thus to move demand shares away from private motorized transport. focus group interviews, implemented to directly involve local stakeholders, revealed an overall positive perception of the proposed transport system. furthermore, the cost analysis demonstrated that the costs of the new system would not be much higher for the municipality than those currently incurred, making it an important performance improvement achieved at low cost. the considerable growth of mobility in urban centers over the last century has been accompanied by issues of sustainability. estimates show that while the world population has grown fourfold over the past century, passenger-kilometers in passenger transport and tons-kilometers in freight transport have increased by about 100 times during the same period (ksenofontov & milyakin, 2018; mulrow & derrible, 2020) . although this massive travel development has had numerous positive effects, the intensity and extent of the negative local and global consequences on the environment are clearly evident, especially in terms of air and noise pollution, soil consumption, and land degradation (benintendi, merino gòmez, de mare, nesticò, & balsamo, 2020) . the current global strategy has thus become reducing the overall consequences while still keeping performance efficiency high. public transport is a key piece of the sustainable mobility puzzle. it can help move more people with lower energy consumption per passenger, provides a more affordable alternative for getting around, has a track record of fewer accidents, has significant potential for electrification, reduces congestion, improves air quality, and makes cities more livable and inclusive. outside of these benefits, however, public transport service is plagued with many challenges that reduce its attractiveness, including long waits at transfer points, insufficient coverage of more dispersed areas, insufficient integration with green modes such as cycling, and a sometimes negative perception or attitude towards public transport. this is especially true in suburban areas. these are larger than traditional cities and have significantly lower densities, implying greater travel distances for most trips, fewer origins and destinations within walking distance of any single route, and more kilometers travelled to reach activities. conventional public transport in such areas is often unable to meet accessibility needs and requirements of different user groups, resulting in large portions of the population relying on private motorized transport, high operational costs, and thus increased fares and low revenues. this leads to a vicious circle ( fig. 1 ) of service cuts, further ridership fall, even lower revenues, and so on, to the point where services are discontinued (dirks, frank, & walther, 2019 ; organization for economic co-operation and development [oecd] & international transport forum [itf], 2015; velaga, nelson, wright, & farrington, 2012) . to tackle these challenges, transit planning must respond with appropriate services and policies to allow public transport to compete with private automobiles. there are two possible solutions. first, transit planning can utilize more flexible bus service, thus minimizing travel time by ensuring well-timed connections and providing these connections as effortlessly as possible with short walk distances, tight scheduling, and appropriate frequencies. demand-responsive transport (drt) is an effective method for instituting such a service. second, https://doi.org/10.1016/j.rtbm.2020.100570 transit planning can offer better sustainable accessibility by connecting public transit with other transport modes, such as bicycles. despite the well-known potential of flexible transport systems (fts; mounce, wright, emele, zeng, & nelson, 2018) in increasing the use of transit and accessibility of rural areas, the operational, technical, and-especially-market feasibility of such systems remains an issue. studies have shown that operational sustainability of fts is not always granted; funding is often an open issue; and in a number of cases, a need for high subsidies has emerged (jokinen, sihvola, & mladenovic, 2019; oecd & itf, 2015) . other detected barriers to the appeal, implementation, and use of fts are poor understanding of mobility needs, the lack of integration with other modes, the difficulty in framing demand and user behavior, and the lack of communication between users and agency (brake, mulley, nelson, & wright, 2007; te morsche, puello, & geurs, 2019; velaga et al., 2012) . the last big issue to consider is related to the lack of viable options to cover the "last mile" between public transport stops and people's final destinations. to the best of our knowledge, no experiment has yet been carried out specifically on the integration between a demand responsive transport system (drts) and an electric bike-sharing system (e-bss), with the latter simultaneously acting as feeder and complement to the bus network. 1 thus, this paper investigates the potential of the integration of a semi-flexible drts and an e-bss to effectively solve the problem of low transit travel demand. to achieve this aim, we analyzed the slovenian town of velenje as a case study. together with the municipality of velenje and through a consultation process with stakeholders and citizens, we developed a feasible and fundable drts proposal that integrates an extended version of the existing bss. we find that within the funding possibilities of the municipality and in full compliance with the objectives and targets of ongoing european union (eu)-funded programs, numerous progresses can be gained by benchmarking the proposal against the existing traditional bus system. this paper is structured as follows. section 2 details the current kirchhoff (1995) . velenje's location. 1 the integration of bike sharing (including e-bikes) and traditional transit systems has been studied previously and intensively. regarding the role of biking within modal integration, félix, cambra, and moura (2020) recently considered the effects of cycling infrastructure and the implementation of an e-bss in lisbon. they found that the combined effect of both interventions was a game changer for the city's cycling maturity. ma, zhang, li, wang, and zhao (2019) showed that demand patterns of commuters strongly impact the relationship between shared bikes and public transportation. their data showed that the transactions between shared bikes and buses had evident commuting characteristics during weekdays, while on weekends, travelers preferred to use shared bikes as a substitute for public transport. a similar result was achieved by sun, chen, and jiao (2019) , who considered how loyal members use a bss more for commuting while non-members use them more for recreation. fyhri and fearnley (2015) found that e-bikes, compared to traditional bikes, increased the number of trips and trip length for all age groups, regardless of trip reason (leisure or work), and are a practical and accepted solution for everyday travel. in a successive study, fyhri and beate sundfør (2020) found that e-bikes resulted in a greater shift away from cars compared to traditional bikes and argued that such mode shifts are long term rather than temporarily induced by good meteorological conditions or novelty. bronsvoort (2019) and bronsvoort, alonso-gonzález, van oort, molin, and hoogendoorn (2020) studied the possible upgrades for conventional pt services in rural settings, considering both the integration with a bss and the substitution with a drt, and found that time and cost are more important to users than reliability and flexibility. in a recent (footnote continued) study, sun et al. (2020) found that e-bikes prominently substituted car use for both commuting and shopping, and that those living in rural areas were among the most likely to forgo their cars in favor of e-bikes. guo and he (2020) studied the integration between bss and conventional public transport, concluding that areas with lower density of public transport stations have higher incidence of integrated use. from a drts planning perspective, studies agree that multimodality is a key driver for service sustainability and success (dirks et al., 2019; nelson & phonphitakchai, 2012) . features of the transit system in velenje. section 3 describes the method that we propose to improve the service, including an economic analysis. section 4 discusses the results obtained. finally, section 5 concludes the paper. velenje is slovenia's sixth largest town and is located 75 km (by road) northeast of the capital city ljubljana, 124 km south of the austrian city of graz, and 136 km northwest of zagreb (fig. 2) . in 2020, the slovenian municipality of velenje 2 was home to 33,330 inhabitants (table 1 ) distributed into 25 local communities (sistat, 2020). its morphological and demographic characteristics make it a typical example of a dispersed area: it lies in a relatively wide lake basin surrounded by a hilly and rural landscape, throughout which are scattered settlements (fig. 3) . to the west of the main urban core, the paka river valley leads towards central slovenia, hosting the railway towards ljubljana and a major road. also on the railway, the town of šoštanj lies on the lakeshore opposite velenje. the modal split in velenje is unbalanced towards the use of private cars, despite a car ownership rate of 500 cars/1000 inhabitants, which is less than the slovenian average of 541 cars/1000 inhabitants (european environmental agency, 2020; halilović, 2019) . comparing data collected in the 2016 and 2019 municipality surveys, 3 it is evident that the decrease in the modal share of private cars (61% vs 51%) has been fully compensated by an increase in the share of active modes (walking and cycling increased from 29% in 2016 to 39% in 2019). the role of public transport is therefore marginal (with a share of 10% and 8% respectively). the 2019 survey also investigated commuting distances, finding that a vast majority of respondents (78%) worked within 5 km of their home (table 2) , a distance that the slovenian ministry of transport considered to be cyclable in the 2017 guidelines for building pedestrian infrastructure (as cited in halilović, 2019) . these data demonstrate how the use of private cars exceeds expectations, which may be explained by the fact that public transport is currently not a daily commute option for those living outside the velenje city center, as schedules do not fit traditional office and business hours. to overcome the transport issues for those rural settlements lying in the northern, eastern, and southern sectors of the municipality of velenje, which are not served by rail or interurban buses, and to improve the modal share of public transport and active mobility, the city has established three suburban bus routes. the routes operate on weekdays only as part of a five-route local network (fig. 4) , and they have an inaugurated bss. the bus service, which cost €348,700 to run in 2019, is currently free of charge for users (municipality of velenje, 2020). in 2017, velenje's bus service transported 420,000 passengers, mostly aboard the yellow line in the city center (municipality of velenje, 2018). as shown in fig. 4 , three suburban routes-the blue, green, and orange lines-are currently in operation. the yellow line provides frequent service within the urban core, and the red line is a "virtual" line comprised of a longer interurban route. at peak times (e.g., school hours), the orange, blue, and green lines do not run, and specific students-only services are operated (municipality of velenje, 2020b). in total, up to four high-floor midi-buses are used for velenje's local services: three on the yellow route (two off-peak) and one alternately serving the blue, orange, and green lines (table 3 ). the red line does not have dedicated vehicles because it is shared between several longer routes; however, the service is subsidized by the municipality of velenje within the contract for the local bus system and has thus been included in the analysis. table 3 shows the route lengths and frequencies of the different lines. currently, the velenje bus system covers 272,480 km/year; thus, the value of the subsidy for public transport operations is 1.28 €/km. even if subsidized consistently by the municipality, the current public transport offer in velenje is underutilized. with the exception of the yellow line, it cannot be used to commute on a daily basis, as it does not operate during the morning rush hour and it offers a limited number of daily connections. its only, clear imprinting is that of facilitating weak categories such as the elderly and those impaired to drive in getting to the city center and making use of its functions, a common approach in slovenia's recent policy (gabrovec & bole, 2009 ). the yellow line, in contrast, provides relatively frequent service within central velenje. in short, velenje's public transit is characterized by a low frequency of service, lack of time coordination, low load factors, and a lack of ambition on the part of the municipality to fund it further. the only ways out of the vicious cycle in which deteriorating quality, tariff increases, and/or reduced routes lead to a lack of service are consistent resource investments from the authority in charge or a change in the service provision. this paper addresses these issues by proposing a different, integrated transportation concept for velenje and its suburbs that aims to increase the accessibility and ridership of its public transport, reducing the perceived distances between velenje and its nearby settlements while allowing the municipality to keep investments in public transport affordable. in this paper, a different strategy for velenje's mobility patterns that is based on a combination of a semi-flexible drts and an e-bss is discussed and evaluated through citizens' involvement (focus groups) and a preliminary cost analysis. the municipality of velenje has been successfully involved in eu-funded projects aimed at empowering cycling, walking, and public transport as a means to reduce externalities from the transport sector, enhance sustainability of commuting patterns, and increase accessibility throughout the region (recent and ongoing projects include chestnut, smile, and smart commuting). a discussion on possible options for changing the current underutilized public transport system began within the municipal offices and was later expanded as part of the smart commuting project. this paper is framed within that context and aims to design a financially sustainable transit network to best serve velenje's settlements. the combination of drts and e-bss has been identified as a promising solution through the analysis of the existing literature; this combination was refined thanks to a productive dialogue with the municipality of velenje and its citizens. relevant stakeholders and citizens were involved in this research through the eu-funded smart commuting project (which includes various engagement activities within the drafting of a sustainable urban mobility plan-sump-for the area) and through an interview campaign developed specifically as part of this research and carried out in the form of focus groups in march 2020. a preliminary part of this work aimed at identifying daily mobility issues that people living in velenje face and exploring how citizens may perceive a change in the transit system. focus group interviews were hence implemented to examine citizens' willingness to use the new transport system and the trade-off between travel modes. the results have guided the service design and policymaking. transportation researchers often use focus groups to collect data (e.g., hwang, li, stough, lee, & turnbull, 2020; levin, 2019; naznin, currie, & logan, 2017; pudāne et al., 2019) . focus group interviews have several advantages over face-to-face interviews or questionnaires (hwang et al., 2020) . they allow for direct interaction with participants, which can help the researcher to get better insight into their needs and opinions than asking them to fill out a questionnaire. in fact, participants can exchange anecdotes, views, and experiences as well as express ideas that otherwise may have gone unheard. moreover, this qualitative method is a cost-effective way of collecting data because group interviews take less time than individual interviews. we made a cautious effort to select a balanced mix of individuals in terms of age, gender, professional status, ownership and/or availability of a car, and area of residence. participants were recruited among residents through snowball sampling, a nonprobability approach that is often used in qualitative research and in which the researcher recruits a few volunteers who, in turn, recruit other volunteers. people were recruited if they lived in velenje, spoke english, were 18 years old or older, and had internet access. the authors acted as moderators for the focus groups. older people, who are usually retired and may need to use public transport the most, could be less confident speaking english or using the interview technology. thus, to prevent them from being overlooked in the focus groups, we invited their grandchildren/family members to help them. for those who did not have english-speaking friends or relatives, we made an interpreter available to ask questions in their native language of slovenian, allowing them to feel more comfortable. the participants were invited to the focus groups by e-mail or phone. we informed them about the focus group arrangements and asked for their consent to audio record the sessions and to use the collected data for scientific purpose. we guaranteed them that the data would be anonymized for confidentiality reasons. all participants agreed and gave permission for their quotes to be used in research publications. a total of 30 people were recruited. we organized three focus groups of 10 participants each. the sample size and group size choices were in line with the best practice literature (fern, 1982; guest, namey, & mckenna, 2017) . the groups had to be small enough for everyone to have the opportunity to share insights and yet large enough to provide diversity of perceptions, as suggested by krueger and casey (2015) . grouping people with similar characteristics increases the likelihood of shared experiences and fosters more comfortable conversation within each group (hesse-biber & leavy, 2011). we thus clustered the participants into three groups based on the area of residence (the suburban areas vs. the urban center of velenje) and, for residents in suburban areas, on age and professional status. since residents of less-served suburban areas might be more aware of the limits of the existing system and would more likely be the main potential users of the newly proposed public transport system, they were overrepresented with respect to people living in the city center. we further divided them into two groups since differences in the choice of transport mode may exist among younger working people and older retired ones. the interviews were conducted during the last two weeks of march 2020 via the skype and ms teams platforms. the covid-19 emergency prevented us from personally meeting the participants. however, as confirmed by the respondents, receiving a video call at home-an environment that is comfortable and familiar-decreased stress and made them feel more confident. moreover, it lowered time costs for the participants, allowing us to involve participants who would not have been willing to travel to reach the meeting place. fortunately, we did not encounter any technical issues, and everyone was able to easily connect to the platform. in alignment with the previous literature (krueger & casey, 2015; simons et al., 2014) , the interviews lasted approximately 50 min in order to avoid exhausting the respondents. long meetings often lead to decreased attention spans. the timeframe allowed us to avoid this while still having enough time to develop the main topics. before each focus group, all participants were asked to complete a short questionnaire to collect data about their sociodemographic characteristics, including gender, age, education, annual income, work status, and the number of cars owned by the family in order to identify users who are more likely to choose the new transport system. table 1 in appendix a summarizes this information. the overall sample was balanced between men and women. the majority of the respondents (43%) were between 25 and 44 years old, 33% were between 45 and 70 years old, and 23% were under 25. additionally, 53% had a full-time job, and 50% had a high school diploma. for half of the sample, the family owned only one car and had children. finally, 70% of the respondents stated that their annual family income was between €30,000 and €50,000. we then used semi-structured guided questions for the interviews. a full list of guided questions can be found in appendix b. the interview began by asking participants their current daily travel mode and the main determinants of their choice (i.e., why they do or do not use the existing public transport system). thus, we encouraged them to put forward any problems with the current system and to suggest improvements. a scenario-type approach was then employed in which participants were presented with the hypothetical new transit system that we had developed together with the municipality based on public data about the existing system (e.g., routes, areas served, and number of buses) and from previous literature. the newly proposed system f. bruzzone, et al. research in transportation business & management xxx (xxxx) xxxx accounted for many of the issues that emerged in the first part of the focus groups. we showed participants the velenje map and explained our draft proposal for serving the area, which features a fixed schedule "backbone" line, a number of on-demand stops to be served upon reservation, and an e-bss that would be promoted alongside the drts (see fig. 5 ). the use of the map enabled the participants to have a deeper understanding of the new system and to provide answers in a more systematic way. data analysis allowed us to identify four main themes: current mode choice, attitude towards buses, attitude towards cycling, and evaluation of the drts and e-bss. table 4 summarizes the main results. the focus groups pointed out noteworthy differences between citizens living in the urban center (group 1) and in the suburban areas (groups 2 and 3) of velenje. nearly 60% of the participants living in suburban areas owned at least two cars (70% for younger/working people vs. 50% for older/retired participants), whereas 80% of urban participants owned only one car. this difference is mainly explained by the poor accessibility and frequency of public transport in peripheral areas forcing respondents of these areas to choose a private car as their primary transportation mode. long distances, flexibility, and shorter travel times were the reasons suburban citizens primarily travelled by car. most of the respondents living in the urban area moved around the city center on foot or by personal bike. the short distance from their workplace to home was the main determinant of their choice. we observed differences between citizens in urban and peripheral areas in terms of their attitude towards using the bus. respondents living in suburban areas stated that they would like to use their cars less and switch to using the bus, but the limitations of the existing system prevented this. they stressed their need to walk more than 1 km to reach the nearest bus station and complained about long waiting times at stations. they pointed out the low frequency of buses, the mismatch between bus schedules and working hours, and inefficient bus routes (to reach as many areas as possible, the routes are long and only go in one direction, thus lengthening the time it takes to reach the city center). one participant (a 45-year-old male) stated the following: there are only four buses a day, and they ride a not convenient route for me; they go in the opposite direction than what i need. they travel a longer route and take 20/25 min to get me to work. with my car i reach the workplace in only 4 min. time matters. another woman (35 years old) complained "my working hours do not match with those of the bus, therefore i am forced to take the car." a retired woman claimed "i would take the bus to go downtown, but there are very few routes, and the risk is to wait too long to go home." for the respondents living in urban areas, they forgo using the bus to move around the city center, preferring active mobility facilitated by the short distances. unlike the people living in more peripheral areas, they were quite satisfied with the existing public transport in terms of frequency, daily coverage, and costs (buses are free of charge for velenje residents). the long distances from the city center and the hilly landscape of the suburban areas meant that most people living in such areas did not utilize bicycles to reach the city center. other factors, such as travelling with a child or having a lot to carry, also decreased the likelihood of cycling. younger people, however, stated that they made use of bikes, but only for recreational purposes. replies such as, "i have a traditional bike, but i am not fit enough to cycle uphill for a round trip" or "i am too lazy to cycle" were common among older respondents. citizens in urban areas, on the other hand, proved more inclined to use bicycles because of the flat nature of the route and the shorter distances. most of them, however, stated that they would only use bikes if the weather was nice. we asked respondents if they felt that the proposed system could overcome the issues previously mentioned and if they would be willing f. bruzzone, et al. research in transportation business & management xxx (xxxx) xxxx to switch to this new system. we also investigated affordable tickets, expected booking time, reservation and payment methods, and whether they agreed that this type of service could improve the level of transport service, especially in less-served suburban areas. we used the collected information and suggestions to better define the design and policymaking of the drts. overall, we recorded a positive perception of the new transport service, which we feel is even more significant given the pandemic period in which the interviews were conducted. as for the drts system, there was enthusiasm, especially among participants living in the suburbs. they stated that if such a service guaranteed that there would be bus stops located closer to their homes and destinations as well as more frequent buses during working hours, they would consider the use of public transit as their primary daily travel mode. this implies that drts is a promising transit service in this area. the e-bss, in contrast, was deemed useful, but less so for daily trips: i would not ride a bike, even an e-bike, to get the city center or to reach the nearest bus stop. i often carry bags; i play sax, and i often carry it with me, so it would be inconvenient for me to ride a bike. it would be different to use this service on weekends. in that case, yes, i would gladly take the bike. (23-year-old female). the area where i live is not flat, then the distance from the center is relevant … using the bike at my age and in these conditions (up and down) is not the best! but the electric bike could be a great solution. i could consider it, but only to have fun during a sunny day. (63year-old female). great the possibility to rent the electric bike and not have to buy it. you take it only when you need it, and if you shop and have bags with you, you go back by bus. i think it's a good idea. it is also a way of doing some physical exercise. (45-year-old female). the discussion with participants also generated topics that the moderator had not anticipated, such as environmental aspects. some participants were particularly aware of environmental issues and wanted to contribute to environmental protection in some way, such as by leaving their car at home. thus, they were happy with the proposal and hoped it would be implemented. regarding the economic aspects, even though the existing public transport is free of charge, all of the respondents from the suburban areas claimed that they would be willing to pay for a ticket if the new service guaranteed more flexibility in terms of transit times and coverage. most of the respondents, in fact, seemed to be well-aware of the costs incurred from operating a car (fuel economy, parking fees, maintenance, etc.) and were therefore willing to consider the trade-off with the new system. people in urban areas were also willing to pay a ticket, but on average at a lower price with respect to those in suburban areas. many citizens suggested keeping the price low and considering the same fare for on-demand bus and e-bike sharing services. the reservation requirement was not negatively perceived by respondents as a barrier that would restrict flexible travel. younger respondents were enthusiastic about the app as a reservation tool but at the same time expressed doubts about the effective accessibility of such technology for older people or for people who are reluctant towards new technologies. for such users, they suggested also allowing phone reservations. we registered the same attitude among older respondents. as previously mentioned, on-demand services can provide transit to more users at lower costs by efficiently employing economic resources in terms of vehicles, staff, and fuel, thus improving services without a significant cost increase (rahimi, amirgholy, & gonzales, 2018) . depending on the characteristics of the areas to be served, the elements of flexibility can vary and may include scheduling, type of operations, type of vehicle, and area of operations (oecd & itf, 2015) . based on the features of the operations, four settings in particular were identified by nocera and tsakarestos (2004) : line operation (one to one), band operation (few to one), sector operation (many to one), and area operation (many to many; fig. 6 ). a geographic information systembased analysis of current public transport operations and the distribution of households in the velenje area, together with some productive dialogue with the municipality of velenje, allowed us to identify the most relevant corridors and areas for public transport. these and other relevant settlements that are currently isolated will be included in the new drts and e-bss. a band type semi-flexible drts setting 4 was selected among various possibilities as the most suitable for serving the area. this setting features a fixed schedule "backbone" line together with a limited number of on-demand stops served on reservation. a second sector-type drt line would also be introduced to serve selected areas and connect them to the train station. to enhance system accessibility, the improvement of the bss and an increased number of available e-bikes is promoted alongside the drts. fig. 7 intuitively shows the functioning of a drt band-operation bus line, showing how the schedule may adjust for some intermediate stops to allow for efficient and reliable service in all situations, independently from the bus taking detours to serve on-demand stops or not. at selected intermediate stops, the schedule provides allowance for waiting in case the bus is requested to perform the longest route, thus allowing it to continue onwards within a given schedule allowance. this reduces the effectiveness of the service by lowering its commercial speed. however, it also cuts down on time fluctuations (which are generally not well-tolerated among customers), thus making up for some possible accumulated delay. as mentioned above, a semi-flexible drts was introduced for velenje. the draft discussed in the focus groups included two bus routes (to be operated by the existing fleet; see fig. 8 ), an extended bss (compatible with the existing system; fig. 8 ), and suggested a growth of the role played by buses and train stations as modal interchange hubs. during the early stages of the proposal development, a reduced bus coverage was considered, to be flanked by the additional provision of a collective taxi service to reach residents who live in rural communities and are unable to cycle. this option, however, was later scrapped due to excessive costs and low interest expressed by the municipality and key stakeholders, who stated their preference for a "proper" bus system. 5 for these reasons, the proposed drts has been expanded to virtually any location reachable by bus within the municipality of velenje, thus providing a sensible increase in public transport offering. another reason of concern while defining the new transit system for velenje was the substitution of the yellow line with an on-demand service. it is interesting to stress that the change arising with the demand shifting from buses to other modes might not be fully absorbed by active transport (coutinho et al., 2020) . however, in this particular situation, this might not be the case for one should also consider that the yellow line provides a useful yet inefficient service for those unable or unwilling to walk or cycle within the urban core. as explained in section 2 and as made evident by the focus groups' outcomes ("it is faster to walk [than to use the yellow line, ed]"), the yellow line mostly serves inelastic demand. moreover, velenje's city center is not an easily drivable area due to the many restrictions enforced. for these reasons, we believe that our proposed drt and e-bss could solve the issue presented by coutinho et al. for it aims at satisfying the inelastic demand that needs a pt service and it attracts new users currently relying on walking or driving to parking facilities and then walking the last mile. the architecture of the proposed drts for the velenje area is reported in fig. 9 , which shows the new bus lines (in blue and red) and their relation to new and existing bike sharing and bike parking facilities (grey with green and black borders, respectively). fig. 10 details the location of the bus stops and numbers them according to the schedule proposals reported in tables 4 and 5. the "backbone" line 1, represented in blue, would run on weekdays every 30 min from 6:00 to 20:00. it would operate as a fixed-schedule line with a limited number of on-demand diversions (band operations) and would perform 29 daily departures. diversions are designed to serve on-demand rural communities, often located uphill, thus easing access and regress to pt service while keeping costs and trip time low. the line would link the stadium (stop 1), where a parking lot is located, with the train station and the gorenje factory (stop 4), the main bus station (stop 7), and some of the most inhabited suburbs of the city (laze, bevče, pirešica, vinska gora, and prelska). line 2, shown in red, would operate as an on-demand feeder to line 1 along the red line. it would start at the train station (stop 4) and offer a 60-min headway between 6:00 and 19:00, with peak-time reinforcements. its scheduled departures, performed only if requested, would guarantee a quick interchange with line 1 at the train or bus stations. the line links velenje city center with škale (stops 29 and 30) and pesje (stops 32 and 33) before ending at the velenje-pesje train station. the details of the drts operations are reported in tables 5 and 6 . both lines would be operated according to a repetitive, easy-to-remember schedule. line 1 is 9.1 km one way (excluding on-demand diversions) and takes 23 min. including layovers, two vehicles would be (footnote continued) public transport. the balance between subsidy and fare revenue is at times also worse for collective taxi systems. the uk government statistical office (2020) declared a net subsidy per passenger trip of £0.628 in 2018/19, in line with values shown in the last decade, for rural pt services in england. a different government study (uk commission for integrated transport, 2008), instead, shows that collective taxi services in the uk and in mainland europe require a subsidy per passenger journey between £1 and £7, rising over £10 in smaller systems. needed to guarantee a 30-minute headway. line 2 is comprised of three different subsections-one serving škale (8 km long), one serving pesje (6.2 km long), and a loop serving the velenje city center and bus station (6.1 km long)-all of which would be operated on demand only and would terminate at the train station. one vehicle could guarantee a 60minute headway on all sections, as shown in table 5 . stops of both lines have been chosen in full accordance with the municipality of velenje, and existing stops (when present) have been maintained and reassigned to the new system. moreover, as mentioned already, a gis-based analysis has allowed to identify the distribution of households and population in the area. reservations would be made by phone or using a web application, with expected pickup times of a few minutes. the use of recent, realtime operations technological innovations would contribute to making fts a more inclusive, reliable, and attractive format compared to the current service (dimitrakopoulos, uden, & varlamis, 2020; mohamed, rye, & fonzone, 2019). current information communication technologies allow for real-time scheduling, which is managed by on-board a bss was recently developed in velenje as part of the european project known as chestnut (municipality of velenje, 2020c). the system has since been expanded and now offers 57 bikes distributed among 12 stations in velenje (including three e-bikes; see fig. 7 ) and 25 bikes among five stations in the nearby town of šoštanj (municipality of velenje, 2020d). since both the implementation and maintenance phases have been fully funded by eu funds, the existing bss should not be included among current costs for public and shared mobility (bicy, 2020) . this paper proposes a reinforcement of the bss outside the city's main core for use as a feeder to both bus lines, with a specific focus on the modal interchange between both shared and private bikes and the fixed-operations buses of line 1. the integration of drt and bike sharing is, to the authors' knowledge, an innovative solution in rural contexts, and literature on the topic is not very extensive. however, benefits from "hardware" and "software" integration between multiple transport modes, which contribute to the mobility as a service concept, have been significant in a variety of studies as highlighted in ambrosino, nelson, boero, and pettinelli (2016) . the expansion of velenje's bss would include the provision of 28 additional docking stations, six of which would be located at bus stops, and 96 e-bikes. despite increasing investments and maintenance costs, the choice of ebikes as a complement to the existing bss is necessary due to the hilly geography of the area. 6 the weather in velenje is known to be mild, with cold but dry winters compared to ljubljana and to the slovenian average (weatherbase, 2020) . 7 the valley surrounding the city is sometimes advertised as the "valley of the sun". the literature has demonstrated that even with typical north-european weather, the use of e-bikes is consistent (fyhri & beate sundfør, 2020; sun, feng, kemperman, & spahn, 2020) ; the favorable climate in velenje has aided the success of the existing bss and encouraged future expansions, such as the one proposed in this paper. stations located in proximity to bus stops would be similar than existing ones, with eight to 10 docks, a standard rotation of five e-bikes, and racks for safe storage of personal bikes, per european guidelines (obis-iee, 2011). stations located in rural settlements would be smaller, with an endowment of three e-bikes and five docks, in an effort to contain costs while enhancing the accessibility of the drts via bss or private bike. fig. 7 shows existing (grey with black border) and new (grey with green border) bss locations. moreover, a digital tool will be made available for desktop and mobile use. the tool will manage all aspects related to service registration, bookings, payments, and information for both the bss and the drts. access to services will be granted by phone for users not acquainted with web technology. an additional or alternative measure to the development of the e-bss would be the provision of financial incentives to support private citizens in the purchase of their own (e)-bike. this measure was however not included in this paper as, unlike the expansion of the bss, it is not a structural solution and it is also not fundable through eu funds. the municipality of velenje acknowledged that the possibility of obtaining regional/national funds for this policy is scant under the current regional and national administrations, and that they had no political interest in such a solution. indeed, the outcomes of a plan of this sort are uncertain and, more importantly, it does not provide the city with bss infrastructure. the combination of semi-flexible drts and e-bss would increase the number of daily connections in velenje's settlements. in particular, residents of škale and pesje would benefit from a maximum of 14 daily rides compared to the current four and eight, respectively. residents of settlements located to the east of velenje, currently served by the green and orange lines, would benefit from a maximum of 29 daily rides compared to the existing four. e-bikes and personal bike storage at bus stops would allow residents of rural communities to access the drts, thus increasing the potential user base. the integrated strategy as a whole is expected to generate a modal shift at the expense of private motorized transport (cass & faulconbridge, 2016) by expanding the number of potential users thanks to better coverage (both spatial and temporal, introducing peak time services and modal integration), thus allowing for a reduction in external costs generated by mobility in the area. the diverse elements of the proposed integrated strategy-the new, semi-flexible drt; the expansion of the bss with the provision of 96 e-bikes and 28 additional stations; and the development of a digital tool to manage the system-were subject to a cost analysis, which was compared with the current public transport and bike sharing offer. the cost analysis is reported in the following section. as discussed in the previous sections, the new transport system would prove particularly useful for satisfying velenje citizens' mobility needs. the project, however, must be economically feasible in order for the municipality to implement it. this section aims to verify whether the proposed transport system is economically advantageous compared to the existing one. for this purpose, we considered all the costs related to the implementation of a drts, an e-bss, and their integration. according to rahimi et al. (2018) , the costs for operating a bus public transport system typically include capital costs for vehicle purchases as well as variable operational costs related to fleet size (e.g., taxes, registration costs, and vehicle insurance), vehicle hours travelled (e.g., total wages, fringe costs, and overhead costs), and vehicle miles travelled (e.g., fuel, tires, and body repair). the total operating costs are usually shown in terms of operational hours and kilometers. the municipality of velenje awarded the public transport contract to a local contractor and currently pays an annual service contribution of €348,731, which covers an average annual distance equal to 272,480 km; thus, the service has an all-inclusive cost of 1.28 €/km, of which 53% goes to operating costs and 47% to drivers' wages (municipality of velenje, 2020). the proposed integrated system would optimize the overall routes by both lowering the minimum number of vehicles required to meet the demand without violating the time window constraint (thus decreasing the number of drivers) and by reducing the overall annual distance travelled. the latter varies between 137,228 km if no citizens reserve the on-demand bus service, and therefore the bus does not deviate from the main route, and 226,200 km if all of the on-demand stops must be served, thus considering both detour and line-haul travels. consequently, considering the current cost per km, the annual costs for operating the bus service range between €175,652 and €289,536. a monte carlo algorithm allowed us to use the process of repeated random sampling to make numerical estimations of such parameter. we thus estimated an annual distance travelled of 177,516 km and an annual cost for operating the integrated system equal to €227,220. an e-bss is both a flexible addition/complement and an alternative to the public transport system. this system thus represents an opportunity for public transport operators to increase the attractiveness of their services because bikes can be used independently of timetables. the main costs to implement such a service from an operational point of view can be divided into two categories: infrastructure and start of the 6 e-bikes have proven to be particularly useful when trip distances are important and when vulnerabilities-both in terms of the lay of the urban land (for example in the case of hilly streets) and individual physical conditions (for example, when considering people with health difficulties and physical impairments)-make traditional bicycles unsuitable. an e-bike reduces the effort required while saving travel time. thus, an e-bss can enhance connectivity for cities that are not bike friendly and have underdeveloped public transportation systems (ji, cherry, han, & jordan, 2014; langford, cherry, yoon, worley, & smith, 2013 service as well as operating costs. the costs of implementing large-scale systems (solar or grid-powered stations) would vary from €2500-3000 per bicycle (obis-iee, 2011), depending on the system configuration. this range includes both the hardware (bicycles, stations, workshop, etc.) and software (system back end and customer interfaces). the operating costs would be between €1500-2500 8 per bicycle per year, though this would vary according to the size of the system and the usage rates. the greater the bicycle usage, the greater the maintenance costs, customer support needs, and redistribution interventions. hence, the cost per bicycle would increase. overall, the total cost for implementing such a service is estimated at €288,600 (purchase of e-bikes and stalls) and €144,000/year (maintenance). the integration between the drt and e-bss with the existing bss is an important aspect that should be taken into consideration. such an integration must take place on three levels: integration of information for facilitating intermodality, physical integration in order to integrate the bss with public transport during peak hours or in areas where public transport cannot satisfy all mobility requests, and integration of technological methods of access to services and tariffs. to this aim, we propose e-bike sharing stations located near bus stops and outside the city center for use in combination with public transport. regarding access and rates, taking into account the suggestions collected during the focus groups, we propose a fully integrated fare and ticketing system. such a change would allow citizens to use public transport and rent bikes/e-bikes for bike sharing with a single card or take advantage of special discounts, such as a single daily rate or a special discount for using bike sharing and other mobility services. these integrations would inevitably rely on mobile technology and thus require management software and an app. however, this would enhance public transport use and facilitate operational planning. among the most significant its applications for the modernization and rationalization of the public transport sector are the avl/avm systems for real-time tracking and localization of vehicles; user information systems for providing real-time waiting time at stops and/or on personal devices (smartphones/tablets); and multimodal and multicarrier platforms for mobile payments (based on microchip smartcards, contactless smartcards, short message service, mobile apps, nearfield communication, the internet, etc.). the overall costs of implementing such software would depend on its complexity. we estimate that it would cost €40,000, of which €20,000 would be used to equip each bus with an avl/avm system for real-time tracking and localization of vehicles, and the other €20,000 would go to the trip planning and the payment app (osservatorio nazionale sulle politiche del trasporto pubblico locale, 2015). the advantages derived from the application of its will be significant both for citizens (in terms of the regularity and reliability of the improved service) as well as for the services company and programmers. the mere extension of the avl/ avm system to the entire circulating fleet could lead to relevant savings in terms of the regularity of the vehicles, fuel savings, optimization of the use of vehicles, and driver shifts. as summarized in table 7 , considering the overall costs of the individual systems and their integration, we estimate an initial investment equal to €328,600 and an annual operating cost equal to €371,220 (the latter only €22,500 higher than the costs of the current system). although the new system requires an initial investment and annual operating costs that are overall higher than the annual contribution currently paid by the municipality, we believe that the public will find it attractive and economically advantageous for at least three reasons. firstly, the new system would allow for a reduction in social costs by promoting a modal shift from private transport to bicycles and/or public transport. this would contribute not only to lower urban air and noise pollution but also increase citizens' well-being through the use of table 7 cost analysis. bruzzone, et al. research in transportation business & management xxx (xxxx) xxxx active transportation modes. the latter is both a direct effect given by new users of bike sharing and an indirect effect given by the greater diffusion of cycling that the presence of bike sharing typically induces. additionally, the presence of personal bike stands alongside those dedicated to bike sharing (which are monitored) reduces the risk of theft of individuals' personal bikes. in this sense, the bike sharing could contribute to bicycle marketing by giving it a positive and smart aspect and, more generally, to the development of an advanced and attractive image of the city that does not undervalue equity considerations. secondly, the integration of bike sharing with drts and the existing transport services (in terms of registration, payments, and unique smart cards for accessing services) would allow users to combine multiple modes of transport and thus contribute to making travel more convenient and efficient. for this reason, we expect an increase in the demand for such a system with respect to the existing one (equal to 35,000 passengers per month). however, even if the demand remained unchanged with respect to the current system, if the service required a fee (an integrated ticket) equal to €1 per ride, the municipality could obtain an annual revenue of €420,000, which would completely recover the initial investment in just one year. if the municipality instead chose to institute the new service free of charge (no registration and/or use fees), it could cover the annual imbalance with respect to the status quo by relying on advertising contracts, sponsorships (for the entire service or for individual components, stations, and/or bicycles), or revenue generated by parking charges or congestion charges. thirdly, the proposed system might help reducing the marginalization processes that typically characterize rural areas (daniels & mulley, 2012; vitale brovarone & cotella, 2020) . the low density and the peculiar geomorphological characteristics of such areas, generally combined with considerable distances from the city center where basic services and work and leisure opportunities are located, make these territories highly car-dependent. thus, the poor transit quality of velenje's surroundings definitely leads to reduced mobility of those who, due to age, economic, or cultural barriers, have no permanent access to a car. the proposed system, by providing a widespread and equitable mobility offer (not only to the elderly, but also to young people), might help not only to increase active mobility, alleviate congestion, and reduce environmental impacts, but also to counteract transport-related social exclusion. the resulting improved accessibility will thus contribute to a sustainable development of velenje. in this paper, we analyzed an unattractive and uncoordinated transit system in a slovenian town and proposed a new system organization based on the combination of a semi-flexible drt and an e-bss in order to solve an evident performance problem and promote the local population's use of public transport. this combined service would incorporate coordinated schedules for an easy-to-remember headway and to provide users with real-time information. the purpose is to minimize waiting times and operating costs while introducing a series of elements capable of increasing the perceived quality and thus shifting demand shares from private transport (chakrabarti, 2017) . in any pilot project that is based on the identification of the mobility needs of the populations concerned and on shortcomings in the current transport offer, the aim is to offer a system of transport services that can meet demand needs at an adequate level of service with minimal social cost for the community (liu & xu, 2018) . in such cases, the trial phase should be followed by regular assessments in order to correctly calibrate significant parameters. our results demonstrate that the proposed system would provide better quality service for the transit customers in velenje while using approximately the same amount of financial resources for its operation. compared to the existing bus system, the proposed combined drt and e-bss provides all-day service to a wider percentage of the local population, thus enhancing social inclusion and empowering those who are currently unable to reach the city center and train and bus stations autonomously. contextually, this system may also enable the local municipality to generate resources to cover the up-front costs through the introduction of low-price tickets and seasonal tickets. evidence from the focus group survey demonstrated a moderate consensus among the sample of possible customers, who expressed a substantial willingness to pay for a drt service that offers a high level of satisfaction. other results demonstrated the possibility of actually cutting down some external costs due to reductions in car use. some relevant gains were also apparent in the variation in the customers' average travel time: the performance of the integrated drts and e-bss would be supported by the application of an its solution for conveying information to customers. in interpreting these results, we have made some conservative assumptions to bolster our argument. first of all, the discussion of the results has not included any possible increase in travel demand, even though it seems legitimate to expect a customer gain due to the increased attractiveness of the transit supply. this, in turn, can generate positive effects related to (possible) consistently higher revenues, which could be the object of a specific assessment. the same holds true for the analysis of travel times: by its refinement, it could also be possible to see some positive time gains and consequent financial savings. another aspect to consider is the possibility to improve the level of customer satisfaction through specific actions, such as the creation of dedicated interchange facilities. in our proposal, we have tried to contain the expense figures and hence did not consider such a possibility in an earlier phase. however, because the proposed system is based on the train and bus stations, this lays the groundwork for further integrations in the future. furthermore, we did not consider the option of covering the initial infrastructural costs through revenues not already available to the municipality (e.g., those possibly deriving from the acquisition of a future eu-funded project, outstanding state loans, or contributions by foundations). however, the involvement of other key stakeholders could also bring advantages to the integrated system project. the methodology presented here is generalizable to other similar contexts, which is a significant advantage and a relevant benefit for policy purposes. future research could involve the use of optimization models to improve operational efficiency of the transport service (iliopoulou & kepaptsoglou, 2019; verma, kumari, tahlyan, & hosapujari, 2017; wei et al., 2017) or to tackle service and energy costs more specifically (batarce & galilea, 2018; brown, 2018; cavallaro, danielis, nocera, & rotaris, 2018; tong, hendrickson, biehler, jaramillo, & seki, 2017) . future work could also examine additional customer-oriented smart services because interconnected transit solutions foster the possibility of offering further specific services to organizations and transit users (nocera, fabio, & cavallaro, 2020) . finally, some heuristic meta-strategies could be used to improve the design of the bus routes (bräysy, dullaert, & nakari, 2009; suman & bolia, 2019) . sustainable mobility practices can be fostered through the promotion and diffusion of the right technologies as well as by offering combinations of transportation modes that are favorable to the population. ad-hoc measures aimed at increasing the availability and quality of public transport services can rebalance the preferred mode of transport in favor of sustainable transportation and reduce the number of vehicles on the road (lah, 2019) . the goal is to create a mobility system that strengthens the competitiveness of the territories through high quality services while also ensuring a more effective use of resources. among the key issues is the better integration of modal networks through increasingly connected systems and multimodal connection platforms for passengers. successful, environmentally friendly solutions include smart mobility (lyons, 2018) , carpooling and carsharing (bulteau, feuillet, & dantan, 2019) , enhancement of local transport (mctigue, rye, & monios, 2020) , integrated planning of transport modes (holz-rau & scheiner, 2019), apps and systems for infomobility (catalano & migliore, 2014) , the construction of new cycle paths (mayakuntla & verma, 2018) , toll and pricing policies (cavallaro, giaretta, & nocera, 2018) , and electric mobility (lemme, arruda, & bahiense, 2019) . in this context, any pilot projects that foster integrated planning into this process would have particular value. in this paper, we described the integration of an e-bss and a drts to solve the issue of low demand for public transport in the slovenian town of velenje. in general terms, placing a pilot action in real contexts means involving local stakeholders and policymakers from the very first stages of planning. additionally, the plan must consider how differences in infrastructure, geography, orography, settlement and urban planning, and travel demand (including non-systematic mobility) may influence the definition of the key aspects of the system. in the case of system combinations, a main transport system along the primary routes must coexist with one or more adduction systems supporting it (le pira, ignaccolo, inturri, pluchino, & rapisarda, 2016) . our results show that with funding levels comparable to the existing conventional bus system, a combination of drts and e-bss could be set up to offer broader service hours to a greater share of municipal citizens. unlike most literature on the topic, in this paper, we developed and evaluated a preliminary cost analysis of this fundable system and concluded that even if there was no increase in users and no application of fares, the municipality of velenje would be able to finance the proposed system with its own resources or by participation in eu-funded sustainable mobility projects. the proposed integrated mobility system presented here would not optimally solve transport issues in velenje's suburban areas; however, it would increase the number of settlements with daily and frequent access to the train and bus stations and to public functions downtown, thus allowing citizens to access public transit and sharing services independently and to choose them for their daily commute. thus, the proposed system would allow for more equitable distribution of opportunities and accessibility throughout the municipality, making a small step towards transport universality. people and authorities of the 21st century tend to take for granted freedom of movement. however, awareness is growing regarding the environmental and social costs of travelling as well as the need for fair, inclusive, and accessible transport systems. some concerns about individual responsibilities for containing primary pollution are also emerging at both the local level (zhou & lin, 2019) and on a global scale as more people are now conscious of and concerned by climate change (nocera, ruiz-alarcón quintero, & cavallaro, 2018; nocera & tonin, 2014; nocera, tonin, & cavallaro, 2015) . to some extent, the ongoing coronavirus disease 2019 (covid-19) crisis has shown that individual behavior can be substantially modified and travel substituted through technological innovations. in many cases, however, there is still no substitute for direct contact, which requires physical displacement and the subsequent creation of certain social costs. however, these costs can be contained through the provision of efficient and lowimpact transport systems. the provision of such services must be a central consideration in order to guarantee sustainable future development. however, considering the complexity of modal split mechanisms, striving for an efficient and low impact service may not be sufficient to reduce the negative effects of the mobility system as a whole. this research has been developed under the common responsibility of all authors. (continued on next page) − welcome and thank the participants. − introduce yourself and provide a brief description of the research. − review the ground rules: everyone's ideas are important, and everyone will be given an opportunity to speak. there are no right or wrong answers; even negative comments are useful in gaining insight about the topic under discussion. − remind everyone that the session will be recorded. − remind everyone that the whole process is confidential: anonymity will be kept; all the audio recordings and transcriptions will be used solely for research purposes. − provide the researchers' contact details. − ask if there are any initial questions before the focus group starts. − ask everybody to introduce themselves: discuss the existing public transport in velenje (20 min): − which mode of transport do you use most often to go to the city center? which are the main determinants of your choice? − why do you (or do not) use public transport? • let people share their thoughts and experiences. − do you have a bike? do you usually ride a bike to go to the city center? why? • let people share their thoughts and experiences. introduce a scenario-type approach in which the focus group participants are presented the drts + e-bss (15 min): − explain that this is our draft scheme based on previous contacts with the municipality and on the literature. • are you interested in this service? why or why not? • would you be willing to pay a ticket for this new service? if so, how much would you be willing to pay? • is 30-min notice enough time to book the bus service? • booking and payment would be done via app. would this be acceptable? − thank all the focus group participants for their time and effort. − remind them that the use of all information collected will be confidential. − ask the participants whether they would like to receive a follow up (to be generally informed about the conclusions of the study). enabling intermodal urban transport through complementary services: from flexible mobility services to the shared use mobility agency: workshop 4. developing inter-modal transport systems cost and fare estimation for the bus transit system of santiago energy, environment and sustainable development of the belt and road initiative: the chinese scenario and western contributions available online at key lessons learned from recent experience with flexible transport services the potential of optimization in communal routing problems: case studies from finland exploring alternative public transport in rural areas preferences towards bus alternatives in rural areas of the netherlands: a stated f. bruzzone, et al. research in transportation business & management xxx (xxxx) xxxx choice experiment fair fares? how flat and variable fares affect transit equity in los angeles carpooling and carsharing for commuting in the paris region: a comprehensive exploration of the individual and contextual correlates of their uses commuting practices: new insights into modal shift from theories of social practice a stackelberg-game approach to support the design of logistic terminals should bevs be subsidized or taxed? a european perspective based on the economic value of co2 emissions the potential of road pricing schemes to reduce carbon emissions how can public transit get people out of their cars? an analysis of transit mode choice for commute trips in los angeles impacts of replacing a fixed public transport line by a demand responsive transport system: case study of a rural area in amsterdam flexible transport services: overcoming barriers to implementation in low-density urban areas. urban policy and research intelligent transport systems and smart mobility. the future of intelligent transport systems designing intermodal transportation systems in rural areas available online at a survey on planning semiflexible transit systems: methodological issues and a unifying framework build it and give 'em bikes, and they will come: the effects of cycling infrastructure and bike-sharing system in lisbon. case studies on transport policy the use of focus groups for idea generation: the effects of group size, acquaintanceship, and moderator on response quantity and quality do people who buy e-bikes cycle more? effects of e-bikes on bicycle use and mode share adapting transport related innovations to rural needs: smart mobility and the example of the heinsberg region how many focus groups are enough? building an evidence base for nonprobability sample sizes built environment effects on the integration of dockless bikesharing and the metro master's thesisuniverza v ljubljani, filozofska fakulteta, oddelek za geografijo the practice of qualitative research land-use and transport planning -a field of complex cause-impact relationships. thoughts on transport growth, greenhouse gas emissions and the built environment a focus group study on the potential of autonomous vehicles as a viable transportation option: perspectives from people with disabilities and public transit agencies integrated transit route network design and infrastructure planning for on-line electric vehicles electric bike sharing: simulation of user demand and system availability policy lessons from the flexible transport service pilot kutsuplus in the helsinki capital region public transit research and development in germany focus groups: a practical guide for applied research the automobilization process and its determining factors in the past, present, and future sustainable urban mobility in action. sustainable urban, mobility pathways north america's first e-bikeshare: a year of experience modelling stakeholder participation in transport planning optimization model to assess electric vehicles as an alternative for fleet composition in station-based car sharing systems how may public transport influence the practice of everyday life among younger and older people and how may their practices influence public transport? feeder transit services: choosing between fixed and demand responsive policy integrated multilevel measures for the transformation to a transit metropolis: the successful and unsuccessful practices in beijing getting smart about urban mobility -aligning the paradigms of smart and sustainable impacts of free-floating bikesharing system on public transit ridership a novel methodology for construction of driving cycles for indian cities identifying barriers to implementation of local transport policy -lessons learned from case studies on bus policy implementation in great britain operational and policy implications of ridesourcing services: a case of uber in london a tool to aid redesign of flexible transport services to increase efficiency in rural transport service provision is slower more sustainable? the role of speed in achieving environmental goals sustainable transport in velenje podatki poslovanje v3, 29.01.2020. municipality of velenje. chestnut project web page bicy bike sharing web page key challenges in tram/streetcar driving from the tram driver's perspective-a qualitative study an evaluation of the user characteristics of an open access drt service the adoption of grid transit networks in nonmetropolitan contexts assessing carbon emissions from road transport through traffic flow estimators a joint probability density function for reducing the uncertainty of marginal social cost of carbon evaluation in transport planning carbon estimation and urban mobility plans: opportunities in a context of austerity demand responsive transport systems for rural areas in germany optimizing bike sharing in european cities: a handbook international experiences on public transport provision in rural areas how will automated vehicles shape users' daily activities? insights from focus groups with commuters in the netherlands demi-flexible operating policies to promote the performance of public transit in low-demand areas system modeling of demand responsive transportation services: evaluating cost efficiency of service and coordinated taxi usage why do young adults choose different transport modes? a focus group study selected data on municipalities improvement in direct bus services through route planning research in transportation business & management xxx (xxxx) xxxx promoting public bike-sharing: a lesson from the unsuccessful pronto system modal shift implications of e-bike use in the netherlands: moving towards sustainability? potential uptake of adaptive transport services: an exploration of service attributes and attitudes life cycle ownership cost and environmental externality of alternative fuel options for transit buses a new approach to rural public transport the potential role of flexible transport services in enhancing rural public transport provision development of hub and spoke model for improving operational efficiency of bus transit network of bangalore city improving rural accessibility: a multilayer approach rainy days in ljubljana and velenje evaluating public transit services for operational efficiency and access equity spatial-temporal heterogeneity of air pollution: the relationship between built environment and on-road pm2.5 at micro scale research in transportation business & management xxx (xxxx) xxxx key: cord-268193-xwptzgvl authors: wang, tzong-luen; jang, tsrang-neng; huang, chien-hsien; kao, shang-jyh; lin, chor-ming; lee, fang-niarn; liu, cheng-yao; chong, chee-fah; lin, chu-mei; dorji, harnod; teng, hsueh-ju; chang, hang title: establishing a clinical decision rule of severe acute respiratory syndrome at the emergency department() date: 2003-12-29 journal: ann emerg med doi: 10.1016/j.annemergmed.2003.08.002 sha: doc_id: 268193 cord_uid: xwptzgvl study objective: in the absence of reliable rapid confirmatory tests during severe acute respiratory syndrome (sars) endemics, we designed a 2-phase cohort study to establish a scoring system for sars and to evaluate whether it could improve the sensitivity and specificity of the world health organization (who) criteria. methods: according to the clinical characteristics and initial laboratory findings of 175 suspected cases defined by the who criteria (20 confirmed as cases of sars) in 3 university teaching hospitals in taipei between march 1 and april 20, 2003, the scoring system for sars was designed by multivariate analysis and stepwise logistic regression as the simple arithmetic sum of point values assigned to 7 parameters. we thereafter applied the scoring system for sars to the consecutive 232 patients (the validation group) who met the who criteria of suspected cases from april 21 to may 22, 2003. final diagnosis of sars was determined by the results of real-time polymerase chain reaction and paired serum. results: the scoring system for sars was defined as radiographic findings of multilobar or bilateral infiltrates (3 points), sputum monocyte predominance (3 points), lymphocytopenia (2 points), history of exposure (1 point), lactate dehydrogenase more than 450 u/l (1 point), c-reactive protein more than 5.0 mg/dl (1 point), and activated partial prothrombin time more than 40 seconds (1 point). of the validation group, 60 patients (group a) were confirmed as having cases of sars, and the other 172 (group b) patients tested negative for sars. the total points of the scoring system for sars at initial presentation were significantly higher in the sars group (median 9; range 6 to 11) than in the non-sars group (median 4; range 3 to 7; p<.001). at the cutoff value of 6 points, the sensitivity and specificity of the scoring system for sars in diagnosing sars were 100% and 93%, respectively. the positive and negative predictive values of the scoring system for sars were 83% and 100%, respectively. conclusion: the scoring system for sars can provide a rapid and reliable clinical decision to help emergency physicians detect cases of sars more accurately in the endemic area. study objective: in the absence of reliable rapid confirmatory tests during severe acute respiratory syndrome (sars) endemics, we designed a 2-phase cohort study to establish a scoring system for sars and to evaluate whether it could improve the sensitivity and specificity of the world health organization (who) criteria. methods: according to the clinical characteristics and initial laboratory findings of 175 suspected cases defined by the who criteria (20 confirmed as cases of sars) in 3 university teaching hospitals in taipei between march 1 and april 20, 2003, the scoring system for sars was designed by multivariate analysis and stepwise logistic regression as the simple arithmetic sum of point values assigned to 7 parameters. we thereafter applied the scoring system for sars to the consecutive 232 patients (the validation group) who met the who criteria of suspected cases from april 21 to may 22, 2003. final diagnosis of sars was determined by the results of real-time polymerase chain reaction and paired serum. results: the scoring system for sars was defined as radiographic findings of multilobar or bilateral infiltrates (3 points), sputum monocyte predominance (3 points), lymphocytopenia (2 points), history of exposure (1 point), lactate dehydrogenase more than 450 u/l (1 point), c-reactive protein more than 5.0 mg/dl (1 point), and activated partial prothrombin time more than 40 seconds (1 point). of the validation group, 60 patients (group a) were confirmed as having cases of sars, and the other 172 (group b) patients tested negative for sars. the total points of the scoring system for sars at initial presentation were significantly higher in the sars group (median 9; range 6 to 11) than in the non-sars group (median 4; range 3 to 7; p<.001). at the cutoff value of 6 points, the sensitivity and specificity of the scoring system for sars in diagnosing sars were 100% and 93%, respectively. the positive and negative predictive values of the scoring system for sars were 83% and 100%, respectively. conclusion: the scoring system for sars can provide a rapid and reliable clinical decision to help emergency physicians detect cases of sars more accurately in the endemic area. the possibly low sensitivity of the who criteria, we reported a 2-phase study that included developing a scoring system for sars in a cohort population and validating it in a second cohort. the febrile patients who consulted our institutes (3 university teaching hospitals, accounting for a population of 700,000 in taipei, taiwan) and met with the who criteria of suspected sars were prospectively enrolled in this study beginning march 2003. the history of exposure and associated symptoms such as cough, dyspnea, myalgia, diarrhea, and rigor were recorded. all patients were completely evaluated in isolated facilities at an emergency department (ed) within 3 hours and underwent cbc count (with a differential count), clotting profiles (prothrombin time, activated partial-thromboplastin time, international normalized ratio), and biochemical measurements. in addition, chest radiographs were obtained. throat swab, sputum, or both were collected for gram's stain and screening tests for common viruses, notably influenza a and b and respiratory syncytial virus. legionella and pneumococcal urinary antigen testing were also examined. final diagnosis of sars was documented by the center for disease control in taiwan after polymerase chain reaction and paired serum for coronavirus antibody were measured. with positive polymerase chain reaction, positive paired serum, or both, the patients were confirmed as having sars. patients considered probable cases according to the who criteria were admitted, and the others were disposed to be isolated at home for 10 days. convalescent serum was obtained during admission for the former patients and at 14 days at our clinics for the latter patients. anyone who had clinical deterioration during home isolation was immediately transported back to the hospital by ambulance. the protocol was approved by our institutional review board, and informed consent was obtained. performance of the multivariate analysis and derivation of the risk score were based on the derivation patients with complete data at presentation. univariate relationships between baseline characteristics (including clinical symptoms and signs and radiologic and laboratory examinations mentioned previously) and the diagnosis of sars were assessed by logistic regression analysis. independent predictors of sars were identisevere acute respiratory syndrome (sars) is a disease manifested by atypical pneumonia and rapid progression to respiratory distress. [1] [2] [3] [4] it has been proven to be caused by the coronavirus. [5] [6] [7] according to the definition of the world health organization (who), 8 characteristics of a suspected case are a documented fever (body temperature >38°c [>100.3°f]), lower respiratory symptoms, and contact with index patients. a patient with chest radiographic findings of pneumonia, acute respiratory distress syndrome, or unexplained respiratory disease resulting in death with autopsy results demonstrating the pathology comparable with sars is considered a probable case. although the who has provided the guidelines for sars control and some diagnostic tools such as polymerase chain reaction, 6, 7, 9 indirect fluorescent antibody, or enzyme-linked immunosorbent assay antibody are being developed, 9 there are still many clinical difficulties in diagnosing the disease quickly. for example, the sensitivity of polymerase chain reaction is still uncertain, and the antibody titer may be detectable at the 10th to 21st day after the onset of sars. 9 it is a difficult issue for emergency physicians to detect patients with sars specifically if there is neither a reliable history of exposure nor a rapid diagnostic tool. recent epidemiologic studies in hong kong 1,2 and canada 3,10 demonstrated the clinical characteristics and important laboratory findings of sars. to improve no tests or predictive models exist to establish a diagnosis of severe acute respiratory syndrome (sars) in the emergency department (ed) setting. a sars diagnostic scoring system using information available in the ed was developed from 175 suspected sars cases and validated in 232 subsequent cases. a scoring system based on radiographic infiltrates, history of exposure, and 5 laboratory tests was helpful in discriminating true sars cases. although this scoring system may not perform as well in areas with different sars epidemiology, it is an important first step in developing diagnostic strategies for this new illness. significant predictors of sars (table 2 ). in addition, the 7-variable regression model demonstrated a good discriminatory capacity with a c statistic of 0.750. these 7 characteristics accounted for 98% of the predictive capacity of the multivariate model and were selected for inclusion in the scoring system for sars. accordingly, the scoring system for sars was defined as radiographic findings of multilobar or bilateral infiltrates (3 points), monocyte predominance on sputum gram's stain (which was defined as the monocyte: polymorphs more than 1:1, 3 points), peripheral lymphocytes less than 1.0×10 9 /l (2 points), history of exposure to index patients (1 point), lactate dehydrogenase more than 450 u/l (upper normal limit 225 u/l; 1 point), c-reactive protein more than 5.0 mg/dl (upper normal limit 0.5 mg/dl; immage immunochemistry system, beckman coulter, fullerton, ca ; 1 point), and activated partial prothrombin time more than 40 seconds (1 point). we have prospectively tested the diagnostic accuracy of the scoring system in sars since april 21, 2003. a total of 232 patients with suspected sars were enrolled to test the predictive capacity of the scoring system in fied by stepwise logistic regression. all variables at presentation entered the initial model and were maintained if the p value was less than .05. selection of independent predictors for inclusion in the scoring system for sars was based on their relative predictive contribution in the full logistic regression model. variables were ranked by z score, and those with the least contribution were sequentially removed from the model until 7 variables that captured 98% of the overall prognostic information from the full multivariate model (evaluated as a ratio of the global 2 statistic from the reduced compared with full model) were reached. for each patient, the scoring system for sars was calculated as the simple arithmetic sum of point values assigned to each risk factor according to the multivariate-adjusted risk relationship: 1 point for an odds ratio (or) of 1.0 to less than 2.0, 2 points for an or of 2.0 to 3.0, and 3 points for an or of more than 3.0. for evaluation of the risk score in the suspected cases, missing variables contributed a point value of 0 to the total score. the discriminatory capacity of the risk score was assessed by using the area under the receiver operating characteristic (roc) curve as an index of model performance in both derivation and validation phases. the roc curve reflects the concordance of predictions with actual outcomes in rank order, with an roc curve of 1. in the derivation phase, we studied 175 patients enrolled between march 1, 2003, and april 20, 2003 , to develop the scoring system for sars. these patients consulted the ed because they met the who criteria of suspected sars. twenty of the patients were confirmed as having cases of sars, whereas the other 155 patients tested negative for sars. each of the initial clinical characteristics was evaluated by univariate analysis (table 1 ). when all of the candidate variables were assessed simultaneously by multivariate analysis, 7 remained table 1 . tims in group a had a scoring system for sars of at least 6 points. twelve patients of group b had 6 points, and the remaining 160 had a scoring system for sars less than or equal to 5 points. at this cutoff value of 6 points, the sensitivity and specificity of the scoring system for sars were 100% and 93%, respectively. the positive and negative predictive values were 83% and 100%, respectively. in contrast, the sensitivity of the who criteria for suspected cases was only 26%. the sensitivity and specificity of the who criteria for probable cases were 96% and 57%, respectively, and its positive and negative predictive values were 44% and 98%, respectively. there are still some limitations in our study. first, the coronavirus that is the pathogen of sars has been reported to have many genetic variations. whether the genetic variations can produce different clinical manifestations remains to be elucidated. therefore, the scoring system for sars may be continuously modified to maintain its diagnostic reliability. second, there were 2 patients who had coronavirus and bacterial infections (acinetobacter sp and s pneumoniae). mixed infection may produce confusing clinical pictures that reduce the accuracy of the scoring system for sars in diagnosing sars. third, the scoring system for sars is a clinical decision rule for suspected cases of sars instead of a in the sars group, the most common presentations at the early stage were the presence of abnormal chest radiograph results (97%; 58/60), followed by monocyte-predominant sputum smear (80% of the patients with sars, or 100% of the sputum smears for 48 patients whose sputum samples could be obtained), lymphocytopenia (67%, 40/60), elevated lactate dehydrogenase level (53%; 32/60) and abnormal c-reactive protein level (50%; 30/60; table 3 ). the criteria of prolonged partial prothrombin time, thrombocytopenia, abnormal creatine kinase level, and alanine transaminase depicted in table 1 were met in 41% (25/60), 33% (20/60), 23% (14/60), and 23% (14/60) of patients. in contrast, the patients in the non-sars group had a significantly lower incidence of these manifestations ( table 3) . the total points of the scoring system for sars at initial presentation were significantly higher in the sars group (median 9; range 6 to 11) than in the non-sars group (median 4; range 3 to 7; p<.001). all of the vicseemed relatively high but still not high enough. the 3% missing rate could become a pitfall in surveillance and possibly cause a devastating event in the endemic area. the only perfect clinical decision rule for such infectious diseases should have 100% sensitivity, as the scoring system for sars provided. the use of the algorithm might also partially account for the absence of hospital staff infection in our institute during this event. our data demonstrated monocyte predominance on sputum smear presented as a critical finding in the early stage of sars. although the patients might have only dry cough and scanty sputum that resulted in relatively low availability of the data (80% in our series), we still strongly recommend examining sputum smear because of its strong positive predictive value for sars and low cost. to prompt early diagnosis, our study has focused on the early manifestations of sars instead of its fullblown characteristics. in our series, patients with sars had similar clinical manifestations during hospitalization, as investigators in hong kong reported. 2 of 60 patients with sars in this study, 100% had a temperature higher than 38°c (>100.3°f), 67% had cough, and 63% had myalgia along the whole course. the overall laboratory findings during hospitalization included lymphocytopenia (73%), thrombocytopenia (47%), elevated lactate dehydrogenase (>450 u/l; 77%), increased c-reactive protein (>5 mg/dl; 63%) and prolonged activated partial thromboplastin time (>40 seconds; 53%). the full-blown manifestations were different from the initial presentations that the emergency physicians always observed. in summary, in the absence of available diagnostic tools that can provide confirmatory results at an early stage, the scoring system for sars can help emergency physicians make rapid and accurate diagnosis of sars quickly and cost-efficiently. author contributions: all authors jointly collected clinical data of the patients. tlw and ch designed the study and revised the manuscript. tnj, chh, and sjk assisted with constructing the analytic data file. dh and cml were responsible for statistical analysis. tlw takes responsibility for the paper as a whole. screening tool for the general population. the sensitivity and specificity of the scoring may be expected to be different in other patient populations with a different mixture of comorbidities or in the setting of outbreaks of other respiratory diseases, such as influenza. there has been a major global outbreak of sars. [1] [2] [3] [4] 10, 11 although confirmatory tests such as polymerase chain reaction and measurements of coronavirus antibody have been conducted in many laboratories, 9 they still cannot provide instant and correct information for clinicians initially. the who criteria may help screen suspected and probable cases, 8 but the low specificity may indicate the lack of cost-effectiveness in an endemic area. this study demonstrated the scoring system for sars as a simple and reliable clinical decision rule to help emergency clinicians detect patients with sars quickly and cost-efficiently. during the endemic times, there was usually chaos when the isolated facilities were not sufficient and the who criteria could not discriminate definitely between victims of sars and non-sars febrile patients. our report revealed that the who criteria for probable cases had only 44% specificity for patients who met the who criteria for suspected cases. this means there ought to be at least 2.3-fold reservation of isolated facilities and medical costs if all probable cases were admitted to hospitals. the average expenditure for each case to complete the workup for the scoring system for sars was approximately us$200 in our series, whereas the medical cost during admission was us$1,100 per day. although the total expenditure of completing the scoring system for sars for our 232 validating patients was approximately us$46,400, the method saved at least 72 unnecessary admissions or us$1,108,800 (if the average duration of admission was 14 days). in addition, the rapid screening tests for common viruses and legionella and pneumococcal urinary antigen testing in our protocol were mainly for confirmation of the final diagnosis to establish the validity of the scoring system for sars. our primary objective was to develop a scoring system that used only common laboratory and radiographic examinations available at most eds instead of other rapid tests. in other words, if no rapid screening tests were available, the scoring system still worked well. the sensitivity of the who criteria for suspected cases was 26%, whereas that of the who criteria for probable cases was 97%. the sensitivity of the latter revision received august 6 a cluster of cases of severe respiratory syndrome in hong kong a major outbreak of severe acute respiratory syndrome in hong kong identification of severe acute respiratory syndrome in canada update: outbreak of severe acute respiratory syndrome: worldwide coronavirus as a possible cause of severe acute respiratory syndrome a novel coronavirus associated with severe acute respiratory syndrome identification of a novel coronavirus in patients with severe acute respiratory syndrome world health organization. case definitions for surveillance of severe acute respiratory syndrome (sars) severe acute respiratory syndrome (sars): laboratory diagnostic tests clinical features and short-term outcomes of 144 patients with sars in the greater toronto area world health organization. cumulative number of reported probable cases of severe acute respiratory syndrome (sars) key: cord-028866-ci3ypbq8 authors: reindl, andrea; meier, hans; niemetz, michael title: scalable, decentralized battery management system based on self-organizing nodes date: 2020-06-12 journal: architecture of computing systems arcs 2020 doi: 10.1007/978-3-030-52794-5_13 sha: doc_id: 28866 cord_uid: ci3ypbq8 due to the transition to renewable energy sources and the increasing share of electric vehicles and smart grids, batteries are gaining in importance. battery management systems (bmss) are required for optimal, reliable operation. in this paper, existing bms topologies are presented and evaluated in terms of reliability, scalability and flexibility. the decentralisation of bmss and associated advantages are shown. a scalable, reconfigurable bms based on a distributed architecture of self-organized, locally controlled nodes is proposed. for distributed system control, producers, batteries and consumers each are equipped with a local microcontroller based control unit, which monitors and controls the local parameters with its own computing and communication resources. features, advantages and challenges to overcome of the proposed approach are described. with an increasing share of renewable energy sources and electric vehicles, batteries are one of the most utilized energy storage media [1] . battery use is essential for maintaining the energy balance and for improving the quality as well as the reliability of power supply in renewable energy systems [2] . a critical challenge facing the widespread adoption of battery technology is to ensure uninterrupted, fail-safe power supply and safe, optimal battery operation to extend battery life. battery management systems (bmss) are used for these purposes and provide the interfaces between energy producers, consumers and batteries ( fig. 1 ). they administer system control and management with regard to energy storage and transmission. main functions of the bms include charge and discharge control, balancing, input/output current and voltage monitoring, temperature control, battery protection, fault diagnosis and evaluation [3] . for this purpose, the following functional requirements are relevant for a bms: -current, voltage and temperature measurement -state of charge (soc) and state of health (soh) determination -communication -robustness against electromagnetic interference (emi) -redundancy of the system in terms of functional safety -electrical isolation of the functional systems -balancing [4, 5] besides the bms unit, which includes data acquisition, status monitoring and control, the topology of the bms is crucial for large-scale battery management. the topology covers the electrical connection of the individual batteries or battery cells, the control structure and the communication architecture. it directly influences costs, ease of installation, maintenance, measurement accuracy and above all the reliability of the system. this paper first describes existing bms topologies together with relevant literature and outlines their benefits and limitations. the proposed classification divides the bms topologies into -centralized, -modularized, -distributed and -decentralized. the identified trend towards the decentralization of bmss is shown: centralized bmss with a single control unit [6] [7] [8] are increasingly replaced by a decentralized management, whereby sensor, control and computing resources are distributed [9] [10] [11] [12] [13] . the characteristics of the control strategies are therefore analysed and compared. an approach for a fully decentralized, distributed bms based on autonomous, locally operating units is proposed. the characteristics and advantages of the proposed approach are described. the requirements, particularly in terms of system control and management, are analysed and challenges to be overcome are identified. the aim is to provide a holistic overview of the features of the proposed bms and the resulting system requirements. in centralized bmss, the entire functionality is integrated into a single module, which is connected to the batteries or battery cells via several wires (see fig. 2 ) [14] . the centralized bms provides single cell voltage, string current and temperature measurement. a centralized bms is described in [15] based on a single chip. the protective function is divided into two stages. the first stage monitors voltage, current, temperature and coordinates the balancing function. another approach for a centralized bms is provided in [16] . advantages of centralized bms include costeffectiveness as well as maintenance and repair. if only a single integrated circuit is used, costs are reduced and errors are easily detected. another advantage is the accuracy, as centralized bms use the same offsets for all cells. the clearly defined coordination structure provides effective system control. disadvantages include the large number of long cable connections, which considerably increase the risk of short circuits. furthermore, inputs can easily be mixed up and incorrectly connected and connections can become loose, which increases the susceptibility to errors. another disadvantage is the lack of scalability and flexibility of the system architecture. in central master-slave bmss, the maximum number of batteries is strictly predefined. during system development, the number of actively used batteries is fixed and can usually only be changed afterwards by changing the wiring. adding additional cells is not possible at all if all input connectors are used or vice versa, some inputs might remain unused. in addition, only predefined, mostly single battery technologies are supported and combinations thereof are not feasible. furthermore, the master controller is a single point of failure. the entire system control depends on the error-free function of the master controller. in case of failure or malfunction of the master controller, the entire system operation is endangered. this is a significant disadvantage, especially with regard to a reliable, uninterruptible power supply. modularized bmss are characterized by several identical modules, which are connected to the individual batteries or battery cells via cables, similar to centralized bms (fig. 3) . the bms modules provide data acquisition (single cell voltage, current, temperature) and communication interfaces to the other bms modules. often one of the modules is assigned to the role of master or a separate module serves as master. the master module controls the entire battery pack and communicates with the rest of the system, while the other modules merely record the measured data and transmit it to the master. a modularized bms with the aim of improving the performance of bms to provide a safe, reliable and cost-efficient solution for smart grids and electric vehicles is proposed in [3] . the modularized bms for electric vehicles presented in [17] focuses on effective single cell monitoring and balancing for a large number of battery cells with comparatively small size and complexity. an advantage of modularized bmss is the improved manageability. the modules are placed close to the batteries, which avoids long cables. to improve functional safety, the function of the bms can be easily replicated on the individual modules. the scalability is also increased compared to centralized bmss. if the battery pack is extended by further cells, another bms module is simply appended. the number of inputs of the bms modules is still fixed and under certain circumstances, inputs may remain unused. in addition, the costs of modularized bmss are higher. compared to centralized bms, the failure of one bms module does not endanger the entire battery operation. defective battery cells or batteries are simply removed from the system, reducing capacity but maintaining operation. in distributed bmss, each cell string or cell is equipped with its own bms module. the cell bms modules provide measurement of operating parameters, balancing and communication. the bms controller handles the calculation and communication (fig. 4) . a distributed bms divided into a master and several battery modules for real-time monitoring and reporting of battery operating conditions is proposed in [18] . this approach combines central control management and distributed data collection. in order to reduce costs and time-to-market and to increase flexibility, scalability and adaptability, a distributed bms with smart battery cell monitoring is presented in [19] . the smart battery cell monitoring consists of electronics for monitoring and a data transmission interface for bidirectional communication with the superordinate bms. the bms functions as the master and controls energy storage at system level. the distributed bms simultaneously offers a high level of reliability and robustness as well as a cost-efficient development process, allowing a significant reduction in the cost of the final battery pack. the advantages of distributed bmss compared to centralized and modularized topologies are scalability and flexibility. no maximum number of inputs is defined and cells can be added or removed even after installation. this allows easy hardware integration for homogeneous modules. scaling the battery pack to the size required for different applications does not require any changes to the hardware or software of the modules-only additional battery cell modules have to be assembled or removed. furthermore, the single point of failure of centralized approaches is avoided. local control of each cell additionally increases safety. sensor information only needs to be processed for the local cell and mandatory actions can be triggered immediately. a further advantage is the high measurement accuracy, which is achieved by the specialization of the battery cell module. furthermore, shorter connecting wires enable more accurate voltage measurement and better interference immunity. maintenance or replacement of defective parts is facilitated by the modular, distributed architecture. disadvantageous are the increased costs for the bms, as a separate bms module is required for each cell and for most applications also an additional master module. the decentralization of bmss is a possible solution to overcome the disadvantages of central control structures. decentralized bmss consist of several equal units, which provide the entire functionality locally and autonomously. each of the individual bms units is able to operate independently of the remaining ones. communication lines between the units enable information exchange and task coordination between the units. they are used in several decentralized bms (fig. 5) . while this architecture offers advantages like scalability, minimal integration effort and increased functional safety, the development requires new methods. decentralized bmss are further subdivided into communication-less, wireless and wired communication based topologies. a decentralized bms without communication requirements is proposed in [20] . the smart cells work locally and autonomously, which increases safety and reliability. a decentralized bms based on the droop control for a series connection of battery cells is presented in [21] . droop control is applied to ensure power sharing among connected components. droop characteristics are used for the power distribution, which correspond to v-i characteristics in voltage droop control. they determine the required output/input current according to the actual voltage deviation. physically the droop control behaves like an output resistance. therefore the droop characteristic is also called virtual resistance. [22, 23] droop control offers high reliability due to the decentralized architecture and the communication-less control. a drawback of the droop-based control is the imprecise control [24] . with the consideration of line resistance in a droop-controlled system, the output voltage of each converter cannot be exactly the same. therefore, the output current sharing accuracy is affected. in addition, the voltage deviation increases with the load due to the droop characteristic [25] . due to the possibility of cable breaks in wired communication systems like can or i 2 c, bms approaches based on wireless communication are developed [26] . as a possible solution, [26] proposes a distributed and decentralized wireless bms based on an internet of things (iot) network. in [27] , a fully decentralized bms is proposed, whereby the entire bms functionality is integrated into the cell management units. one cell management unit per cell is used, providing local sensing and management capabilities autonomously and system-level functionality by coordination via communication. a can bus is used for wired communication, which enables broadcast communication between the cells. the major advantage of decentralized bmss is the absence of a central control unit, on which error-free function the entire operation depends. furthermore, the scalability and flexibility are advantageous. the number of inputs is not fixed and can be extended/reduced even after installation. a challenging feature is the distributed system control based on the equal, parallel-operating and autonomous nodes. in addition, it has to be ensured that the single point of failure is not only shifted but eliminated. for a reliable system, a holistic approach is required. the decentralization of the bms topology results in functionality distributed to several individual units. the functional units are closer to the battery/battery cell and more elaborately equipped to work independently. operation is becoming increasingly independent of a central coordination unit and the failure of individual functional units has a minor impact on the system function. as a result, the reliability of the system is improved. the scalability increases with rising decentralisation. the number of batteries/battery cells is not limited by pre-defined inputs but is variable even after the initial layout. individual batteries/battery cells can be added or removed. a variable number of batteries results in enhanced flexibility. the bms is adaptable to the requirements of a wide range of applications. table 1 summarizes the evaluation of existing bms topologies in terms of reliability, scalability and flexibility. compliance with the criteria is evaluated, where ++ means full compliance, + partial compliance, 0 neutral, -partially not satisfied, and --not satisfied at all. the proposed system is fully decentralized and consists, in contrary to the proposed approaches, of three types of modules: renewable energy producers, batteries and consumers. all components are connected together with a common power line and at least one global communication bus (fig. 6 ). for distributed, autonomous control, each battery, producer and consumer is equipped with its own local control unit (lcu). the lcu includes: -current, voltage and temperature measurement to record actual operating parameters, -a communication interface for data exchange between the components. -a microcontroller for calculation, data management and evaluation, -a dc/dc converter with target current and target voltage values which are adjustable during operation, and -a relay which is opened in case of failures to avoid safety critical voltage levels or for maintenance purposes. producers and consumers use the lcu to provide their operating parameters for load/generation forecasts and for voltage control. batteries provide the ability to absorb excess power or deliver missing power and thus are able to control the system. therefore, additional algorithms for system control and leader election are implemented on the lcus of the batteries. the implemented software for system control manages both the actual operating data such as current, voltage and temperature and the system states resulting from previous measurements. the soc and the soh are determined. in addition, the battery fitness (bf) is defined. the bf is a numerical value based primarily on soc, soh, number of charge cycles, time of last charge/discharge, the system-wide normalized capacity and the actual operating parameters. taking into account the optimum operating range of the respective battery technology, the battery condition is evaluated. the criteria, e.g. soc or temperature, are weighted. the criteria weighting can be adjusted depending on the battery technology and the system status. the adjustment of the weighting provides the basis for system optimization according to various criteria such as cost minimization, maximum safety or availability. the bf enables a system-wide definite evaluation of different battery technologies. in turn, this enables the combination of different battery technologies in a single system. the combination of different battery technologies offers advantages including optimization of the system control, extending battery life and increasing system reliability [28] . additionally, it offers a second life application to a wide range of batteries [29, 30] . the bf is also a decision criterion for the leader election. the participating nodes work autonomously and locally and control the system in a collaborative manner. highly parallel computer systems exist for solving complicated mathematical problems. in contrast, the challenging task in the context of the proposed approach is to structure, intelligently equip and network the nodes to such an extent that the overall system and its control interact harmoniously. the lcus are interacting in the physical domain in their control task while communication latency for negotiations is high compared to the control requirements. in addition, in reality the nodes do not work perfectly synchronized but asynchronously [31] . therefore, the development of a system control consisting of decentralized, autonomous, distributed, asynchronous nodes is a non-trivial, challenging task. the target of the decentralized control structure is to make the system independent of the error-free function of a component. this can be achieved if the role of the central control unit is not permanently assigned to a single component. therefore, instead of the decentralized system control being distributed to all nodes, the approach of the system control coordinated by a temporary master which gets reassigned on a regular basis was chosen. one lcu of the batteries is chosen as the temporary central control unit applying a leader election algorithm. the temporary central control unit determines the required charge/discharge power of the remaining battery nodes, taking into account their bf. in case of failure, malfunction or changes in control capability, one of the battery nodes is selected as the new central control unit. as a result, the single point of failure of existing centralised approaches is avoided. communication between the peer nodes is the key to the autonomous, local control of the decentralized bms. for autonomous decision making and system control, the nodes communicate their operating parameters and work on a system-wide consistent database. a suitable communication methodology is required to enable fast and energy-efficient communication between the nodes. furthermore, a robust communication architecture is required to withstand the harsh environments of e.g. automotive applications. in addition, establishing a secure communication protocol between the individual nodes is essential for the safe operation of the bms. therefore a well-proven, robust, noise-free, fast and reliable communication technology is required. to achieve a minimum of integration effort, an architecture with minimal wiring harness is required for the distributed topology. a bus-based communication architecture achieves higher bandwidth and enables broadcast communication between the nodes, which is advantageous for the leader election and system control. controller area network (can) is a robust bus-based broadcast communication technology. it is particularly suitable for applications with a small amount of information to be exchanged. furthermore, can is a message-based network and each node is equipped with a filtering mechanism that filters messages based on their identifiers. thus, only messages relevant to the node are considered. due to its characteristics can is chosen as communication technology for the decentralized bms. for first implementations a communication based on a single can bus is used. for future developments dual can, can in combination with optical data transmission via polymer optical fiber (pof) and can combined with ethernet are conceivable approaches providing diverse redundancy to increase system reliability and availability. the decentralized bms is able to support active balancing. on the one hand, weaker batteries are protected by taking the bf and thus also the soc into account when setting the target value for individual energy delivery. in addition, batteries with higher soc are set to higher target currents during discharge while those with lower soc absorb higher charging currents. on the other hand, the controllable relays allow individual batteries to be disconnected from the power line. an additional power line between the batteries could additionally enable effective, active balancing by connecting the batteries to be balanced (fig. 7) . this architecture enables one-to-one, one-to-many and manyto-many balancing at a voltage level controlled by the dc/dc converter [32] . taking into account the bf, the system-wide standardised nominal capacity and the soc, the more powerful batteries supply the weaker ones. the number of inputs and thus of participants is not fixed in the proposed decentralized bms. a minimum of two batteries is recommended for a reliable supply. adding and removing nodes is possible after installation and during operation. both hardware and software are designed for effective integration [33] . the variable number of participants, which can be adjusted and changed during operation, allows the system to be adapted to requirements changing over its lifetime. optimizations in terms of e.g. cost efficiency, safety or maximum service life can be implemented or changed. the reconfigurable architecture increases reliability, performance and flexibility of the proposed bms [34] . the variable number of participants and the possibility to use and combine different battery technologies increases the flexibility of the system. existing fig. 7 . additional lines and individually controllable relays enable one-to-one, one-tomany and many-to-many active balancing approaches tend to specialize in a single battery technology [35, 36] . in order to improve the performance and energy density, new battery technologies are constantly being developed [37] [38] [39] . the bms is flexible and effective in adapting to changing conditions for optimal and safe battery operation. the software is effectively expandable and software updates during operation support the effective integration and potentially necessary software adjustments supporting new battery technologies [40] . the flexible, scalable, reconfigurable architecture opens up various fields of application including uninterruptible power supply, electric vehicles, (islanded) dc microgrids, grid support for peak load shaving or load management. the applications result in different requirements for the bms. for electric vehicles, for instance, high availability, safety and energy density with minimum size and weight are required. for islanded micro grids, the relevant criteria include effective service lifetime, cost efficiency, reliability and resistance to environmental effects. in addition, various battery technologies and combinations thereof are supported. the combination of different battery technologies improves the system control as well as the battery life of various applications [41] . furthermore, second life and second use applications are possible for a large number of batteries [30] . in this paper, existing bms topologies were presented and discussed in terms of scalability, flexibility and reliability (cf. table 1) . a decentralized, distributed bms based on self-organized and locally operating nodes was proposed. the system control is distributed among the lcus, which record operating parameters and provide their own computing and communication capacities. possible approaches for the coordination of a control system based on a manymicrocomputer system were suggested. communication requirements were analysed and suitable technologies were selected. the resulting flexible architecture allows optimized system configurations for a wide range of applications, adaptability to newly developed battery technologies and multi-criteria optimizations. future developments will further optimize the reliability and fault tolerance of the system. several communication technologies are combined to achieve various redundancies. as a fallback strategy in case of communication failure, the implementation of a droop-based control is planed. it is avoided to move only the single point of failure. the goal is to avoid a single point of failure holistically on the system. additionally a strategy for active charge balancing during operation under consideration of the bf, which does not require additional hardware, will be developed. the importance of energy storage in renewable power generation: a review review of battery storage optimization in distributed generation battery management system: an overview of its application in the smart grid and electric vehicles battery management system hardware concepts: an overview battery management algorithm for electric vehicles a cost optimized battery management system with active cell balancing for lithium ion battery stacks research of battery management system for integrated power supply design and implementation of an optimal battery management system for hybrid electric vehicles modularized battery management for large lithium ion cells decentralized master-slave communication and control architecture of a battery swapping station decentralized battery management system a multi-agent decentralized energy management system based on distributed intelligence for the design and control of autonomous polygeneration microgrids decentralized power management of a pv/battery hybrid unit in a droop-controlled islanded microgrid battery management systems for large lithium-ion battery packs the smart battery management system multicell 36-v to 48-v battery management system reference design a modularized charge equalizer using a battery monitoring ic for series-connected li-ion battery strings in electric vehicles research on dynamic equalization for lithium battery management system smart battery cell monitoring with contactless data transmission completely decentralized active balancing battery management system a novel battery management system using a duality of the adaptive droop control theory improved droop control of parallel inverter system in standalone microgrid impact of dc line voltage drops on power flow of mtdc using droop control adaptive droop control strategy for load sharing and circulating current minimization in low-voltage standalone dc microgrid an improved droop control method for dc microgrids based on low bandwidth communication with dc bus voltage restoration and enhanced current sharing accuracy an internet of things (iot)-based network for dispersed and decentralized wireless battery management systems smart cells for embedded battery management optimization of an off-grid hybrid power supply system based on battery aging models for different battery technologies planning and operation of isolated microgrids based on repurposed electric vehicle batteries sustainable business model archetypes for the electric vehicle battery second use industry: towards a conceptual framework a formal architecture pattern for real-time distributed systems distributed reconfigurable battery system management architectures software framework for the simulation of a decentralized battery management system consisting of intelligent battery cells comparative analysis of reconfiguration assisted management of battery storage systems annual conference of the ieee industrial electronics society battery management system for li-ion battery functional safety analysis and design of bms for lithium-ion battery energy storage system recent progress in rechargeable sodium-ion batteries: toward highpower applications advanced li-sexsy battery system: electrodes and electrolytes. elsevier aqueous organic and redox-mediated redox flow batteries: a review software update of a decentralized, intelligent battery management system based on multi-microcomputers energy storage technologies and real life applications -a state of the art review the authors thank n. balbierer and m. farmbauer for helpful discussions and t. singer for developing a test environment to validate dc/dc converters. key: cord-326210-216atclj authors: sturmberg, joachim p.; martin, carmel m. title: covid‐19 – how a pandemic reveals that everything is connected to everything else date: 2020-07-06 journal: j eval clin pract doi: 10.1111/jep.13419 sha: doc_id: 326210 cord_uid: 216atclj nan entrained ways of thinking and acting. in our view, it is a classic example that demonstrates how suddenly changing dynamics can destabilize a system and tip it into an unstable state. covid-19-rather than something else-turned out to be what we colloquially call the last straw that broke the camel's back or, put in system dynamics terms, what pushed our societal systems over a tipping point. when a system suddenly tips over, the linkages between most of its agents break, and a chaotic situation ensues. chaotic states entail a high degree of uncertainty, a state in which previously proven interventions no longer maintain the status quo. the uncertainties triggered by covid19 have not only shown the fragility of health and national systems but also highlighted the intrinsic and tacit dynamics underpinning them. most notable are the markedly different responses at the policy and community level. china drastically clamped down on all societal activities and rapidly built large new hospitals to deal with those fallen ill. iceland rapidly tested every potential case. sweden implemented limited social distancing measures. italy hospitalized many mild cases in an environment of limited hospital resources, and the united states, for several weeks, denied that there is a significant problem. each of these approaches has its own dynamics affecting individuals, communities, health systems, the economy, and the nation as a whole-new patterns emerge that become understandable with increasing knowledge (figure 1 ). however, the long-term outcomes and effects on the system as a whole will only become evident over the next months and years. 2 cass contain many different agents that interact in non-linear dynamic ways. they are open systems that constantly interact with their environment. within given constraints, the self-organizing properties of a cas makes it resilient to most perturbations to any of its constituent parts. nevertheless, a cas may become unexpectedly unstable triggered by a small-often unforeseen/unforeseeableevent. the emerging dynamics then shift the system to a new-stable or unstable-state, where the relationships and the interactions between the systems' agents have permanently changed within the context of a different set of constraints. 3 when system perturbations result in unexpected outcomes, we become aware of the ever-present uncertainties in our lives. these experiences easily result in over-exaggerated fear, which in turn results in ad hoc responses that invariably perpetuate and/or exacerbate the underlying system dysfunctions. in general, we are not good at seeing and comprehending the complexities in issues, and we have great difficulties in managing their underlying dynamics into the future. the human brain has not evolved to keep all components of a problem in mind and to appreciate their changing dynamics more than two or three steps ahead. 4 at the physiological level, the experiences of failing to manage a complex problem creates cognitive dissonance and emotional distress-we experience anxiety and physical symptoms such as palpitations, sweating, and tremors. having to solve problems with high levels of unknowns often results in interventions that are ad hoc focussed on what appears to be the most obvious without considering the wider consequences. dörner's studies 4 in the 1980s demonstrated how people of all walks of life handle unexpected contextual problems. most of us succumb to the logic of failure; we over-respond and, when realizing the consequences, promptly react with an over-response in the opposite direction and so forth. few among us use the approach of first closely analysing the problem, second responding by introducing small interventions, and finally taking time to observe what happens. in dynamic systems, the true effects of an action are only evident after a time delay. one has to observe and evaluate a cas' feedback to guide responses; invariably infrequent small tweaks-rather than rapid and dramatic actions-achieve a stabilization of the situation and ultimately provide the necessary space for a (re)solution to emerge. covid-19 is a wicked problem 1 -there are many known unknowns, unknown knowns, and unknown unknowns. we yet do not know enough about the virus (but we are painfully learning)-the true mode and speed of infectivity, its population dynamics, and treatment; we do not know enough about the risk to the population as a whole and on any subpopulation-age groups, behavioural risk takers, and those with pre-existing morbidities; we did not have ready-to-go pandemic such problems are known as vuca problems-they entail volatility, uncertainty, complexity, and ambiguity. resolving such problems requires vuca leadership-vision, understanding, clarity, and agility. 5 vuca problems place health and political leaders in a difficult position as any decision will have difficult-to-foresee consequences. for example, imposing community-wide (self-) isolation entails that "almost all activities stop", destroying the economy and resulting in high unemployment, poverty, and increasing disease burden, while implementing strategies to slow down the spread of infection will not guarantee that we will not overwhelm health systems or stabilize the pandemic. as all parts of a cas at every level of organization are connected to everything else, a change in any part of the system has reverberations across the system as a whole. two processes control the dynamics of a cas-top-down causation transfers information from higher system levels to lower ones, which constrains the work that lower system levels can do, that is, it limits the system's bottom-up emergent possibilities. 6 if top-down constraints are too tight, they can bring the system to a standstill. system stability also depends on the law of requisite variety, meaning that a system must have a sufficient repertoire of responses (capacity for self-organization) that is at least as nuanced (and numerous) as the problems arising within its environment. 7 if the possible ways of responding are fewer than what is demanded from the system, it will fail in its entirety. both these constraints feature in the covid-19 crisis. figure 2 brings these complexities and interdependencies into a single view. it demonstrates the top-down causation in cas and particularly emphasizes to policy-makers the importance not to limit system constraints f i g u r e 1 emergent patterns resulting from the system dynamics triggered by covid-19. while we as yet have no clear understanding of the mechanisms of covid-19 on people, some common features emerged for those developing severe disease and high fatality outcomes. moderate severe disease may be associated with increasing age and potential, otherwise innocent, genetic factors. as the development of immunity to sars-cov-2 is uncertain, previously infected people may continue to spread the disease and/or remain susceptible to reinfection f i g u r e 2 a complex adaptive system appreciation of the covid-19 crisis. the virus triggered unexpected and competing challenges at the policy level with direct and indirect effects on public health policy, the political economy, and individuals (the red tension arrows). each of these domains has its own circular feedback loops-all link back to the policy level. ongoing research into the disease, its treatment, and prevention potentially provides "external input" into the systems, which may modify doctors' disease management and alter patients' health outcomes. as described elsewhere, 8 individual health arises at the interface between the environmental and biological domains and is-at times-supported by health care interventions. the detrimental effects of the covid-19 pandemic on the economy, besides that of the disease fears, increases the dysregulation of the physiological stress responses that, in turn, result in the dysregulation of upstream metabolic pathways, which have longterm health consequences far beyond the direct effects of the pandemic to such a degree that the agents at other levels cannot do the work that needs to be done. it also points to the lack of requisite variety at multiple levels, which ultimately limit the system's options to self-organize back to its prior stable state. these deficiencies can be identified at all three key system levels-policy, service delivery, and biology. while this representation is obviously a simplification, it is useful as it helps to maintain the all-important focus on the system as a whole. 9 all systems require a central focus-for a health system, it ought to be the person. 10 health ultimately emerges from hierarchical network interactions between the external environment and internal physiology. 8 pandemic planning should provide ready-to-go policy, principles and highlevel strategic plans that provide an epidemiology informed framework to guide population-based actions, allow health professionals to locally adapt overarching policy decisions, and ensure required resources are in place. in return, health professionals need to have systems to reliably provide accurate data about the spread of the disease, disease burden, resource utilization, and staffing needs to inform policy decision makers. these data must be contextual-they always need to have a meaningful denominator, such as whole/affected population size or socio-economic/age demography, and acknowledge cluster sizes and rates of spread in different environments to enable meaningful comparisons and aid informed decision making. 12 most importantly, all observations must be communicated in a transparent fashion to ensure maximum adherence to guidelines and directives aimed to achieve pandemic control. covid-19 creates many biomedical research challenges addressing genomic, cell, organ, and whole-of-person level questions. an understanding of the virus and its disease mechanisms is necessary to provide clinicians with the best evidence-informed treatment options. 13 at the same time, these basic understandings are the necessary stepping stones to develop disease-specific drugs to cure the disease, as well as a vaccine to stop the pandemic and prevent future recurrences. the uncertainties created by covid-19 require analysis that provides the deep understanding needed to formulate and implement necessary interventions. the cynefin framework 14 however, certain responses can legitimately operate in known and knowable domains based on context. 15 understanding the multifaceted dynamics of the covid-19 pandemic requires interdisciplinary and transdisciplinary approaches. 16 as outlined above, there are many contradictions and tensions within the policy community; they are unavoidable, but people must ultimately make sense of the multiple dynamics in and between the different biological, social, environmental, and politico-economic domains. 17 sensemaking (or sense-making) is the process of people giving meaning to their experiences. at the collective level, a transdisciplinary process involving, among others, mathematics, biology, philosophy, sociology and cognitive science, communication studies, and complexity sciences offers the best way to understand the covid-19 pandemic. while these discourses cannot deliver certainty, 18 they offer the best change to allow the emergence of best adapted solutions that can ultimately resolve such problems. 16 one approach to reaching best adapted solutions involves mapping out the problem to visualize its agents and their interdependencies. mapping is the basis for multi-stakeholder modelling of a problem; it allows the testing out of many different possible interventions and the comparison of their potential outcomes on the system as a whole. 19 however, modelling is not a panacea. as rosen pointed out, model outputs only reflect an anticipation of a future state of a system. 20 models are a mental representation of reality; they are not reality itself. 9 models are never entirely valid but are useful if they can "recreate" a reasonably accurate "current state" of the system based on available in time, real-world feedback will provide more complete data about the dynamic behaviours of the pandemic, which will result in refined models that better anticipate the future-desirable or undesirableoutcomes. well-designed contextual models can provide fairly accurate projections of the to-be-expected real-world outcomes of the pandemic on the health system, social activities, and the economy. however, non-modelled interactions can have significant unintended consequences, for example, the focus on surge capacity of icu for covid-19 patients can reduce resources for other services and lead to greater morbidity and mortality of aged care residents or those affected by multiple morbidities. f i g u r e 3 -sensemaking dynamics of complex adaptive problems (adapted from martin 15 ). this cynefin-based model outlines the issues arising from the covid-19 pandemic in relation to different knowledge domains. it provides a starting point to design an anticipatory system model. each knowledge domain has its own dynamics and strength and weaknesses in understanding the pandemic as a whole. note the nature of the strategies required to move from one domain to its neighbouring ones. this understanding is crucial as it has major implications for decision makers (for more detail on the cynefin model, see kurtz and snowden 14 ) social determinants-poverty, social isolation and support, housing, food security. what can we take away at this stage of the journey? 23 dynamic interactions always keep social systems in a state close to instability (or dys-equilibrium). if political and economic constraints on our societal system are too tight and lack the necessary redundancies, the system cannot adapt to the disruptions of a pandemic like covid-19. 24 health systems, and particularly their public health divisions, have been constrained by the neglect of pandemic risk planning and inadequate resourcing. health services are constrained by a lack of surge capacity. 25 the lack of consistent population health surveillance and health-related information systems minimize the ability to collect and utilize vital clinical and public health data in their proper context. 12 inconsistent or non-transparent communication hinders the collective deliberation needed to make decisions in an environment of uncertainty and competing demands. 26 the longterm consequences of the hit-and-miss efforts in this crisis often remain unrecognized and thereby perpetuate socio-economic disadvantage and health inequities for future generations. 27 we all face the challenge of adapting to the inevitable "new norms" of the emergent new societal systems characterized by different structures and dynamics. the "new norms" should emerge from our shared values and our humanitarian ability of sensemaking, which will take us forward on this quest. what we-collectively-need is a better and more widespread understanding of the sciences of cas-they are wholes that cannot be understood by the nature and behaviour of its constituent parts; they are self-organizing and emergent in light of challenges and changing contexts. we also need to acknowledge and mediate our "natural tendencies" to respond to unexpected complex problems in ad hoc-knee-jerk-ways. these understandings enable different approaches to manage the chaos of this (and other) unexpected crises. in addition, it, one, supports a far more nuanced communication approach to convey the scientific insights into the virus and its dynamics and, two, dampens the heightened anxieties associated with the uncertainties inherent in the unknowns of this continually emerging pandemic. dilemmas in a general theory of planning policy sciences how the pandemic will end handbook of systems and complexity in health the logic of failure: recognizing and avoiding error in complex situations chapel hill, nc: unc kenan-flagler business school top-down causation and emergence: some comments on mechanisms requisite variety and its implications for the control of complex systems health and diseaseemergent states resulting from adaptive social and biological network interactions empirical model building and response surfaces health system redesign. how to make health care person-centered, equitable, and sustainable understanding health care delivery as a complex system coronavirus disease 2019: the harms of exaggerated information and non-evidence-based measures pharmacologic treatments for coronavirus disease 2019 (covid-19): a review the new dynamics of strategy: sense-making in a complex and complicated world distortions, belief and sense making in complex adaptive systems for health making sense of polarities in health organizations for policy and leadership unclear? accurate . . . inaccurate? objective . . . subjective? research . . . practice? why polarities impede the research, practice and design of information systems and how sense-making methodology attempts to bridge the gaps. part 1 the illusion of certainty -a deluded perception handbook of systems and complexity in health transcript of a videotaped interview leadership without easy answers knowledge translation in healthcare -towards understanding its true complexities; comment on "using complexity and network concepts to inform healthcare knowledge translation perturbing ongoing conversations about systems and complexity in health services and systems covid-19: time for paradigm shift in the nexus between local, national and global health health care reform -the need for a complex adaptive systems approach complexity perspectives on clinical decision making in an intensive care unit the health gap: doctors and the social determinants of health key: cord-309379-ml75kvl5 authors: kanger, laur; sillak, silver title: emergence, consolidation and dominance of meta-regimes: exploring the historical evolution of mass production (1765–1972) from the deep transitions perspective date: 2020-09-09 journal: technol soc doi: 10.1016/j.techsoc.2020.101393 sha: doc_id: 309379 cord_uid: ml75kvl5 grand environmental and societal challenges have drawn increasing attention to system innovation and socio-technical transitions. a recent deep transitions framework has provided a comprehensive theory of the co-evolutionary patterns of multiple socio-technical systems over the last 250 years. however, so far the framework has not been subjected to systematic empirical exploration. in this paper we address this gap by exploring the co-evolutionary model linking niche-level dynamics, transitions in single systems and ‘great surges of development’, as conceptualized by schot and kanger (2018) [1]. for this purpose, we conduct a case study on the historical evolution of mass production in the transatlantic region from 1765 to 1972. instead of focusing on dominant technologies or common practices the development of mass production is understood as the emergence of a meta-regime, i.e. a set of mutually aligned rules guiding production activities in multiple socio-technical systems. the results broadly confirm the overall model but also enable to extend the deep transitions framework by uncovering new mechanisms and patterns in the variation, diffusion and contestation of meta-regimes. the world is confronted by a socio-ecological emergency brought on by climate change, resource depletion and loss of biodiversity. solving these crises requires rapid and deep decarbonization of a broad range of socio-technical systems including energy, housing, food, water and mobility. these challenges have drawn increasing attention to system innovation in the sustainability transitions field, including co-evolutionary dynamics extending beyond single systems. although so far the stream of studies on multi-regime interaction has remained fairly small [2] [3] [4] [5] [6] , lately the importance of the topic has been increasingly recognized in the sustainability transitions research network agenda as well as the debates around it. the need for 'zooming out' has been advocated [7, p. 6] in order to shift "emphasis away from within system dynamics that have received considerable attention to the interconnected and multi-scalar qualities of socio-technical systems that are less well understood" [8, p. 4] . a recently developed deep transitions (dt) framework [1, 9] attempts to go beyond the analytical limitations of studies focused on single systems. synthesizing insights from sustainability transitions studies, long wave theory and industrialization literature dt theorizes how interactions between socio-technical systems produce 40-60 year long 'great surges of development' [10] and how successive surges, in turn, accumulate into a set of principles driving every industrial societyan industrial modernity. the authors of the dt framework argue that the co-evolution of single systems, interconnected systems and industrial modernity -the first deep transition -has created a fundamentally unsustainable trajectory of environmental degradation while not being able to solve recurring problems of social inequality. altering the situation requires a shift of comparable magnitude -the second deep transition. dt aspires to offer a comprehensive conceptual framework of the co-evolutionary patterns of multiple socio-technical systems over the last 250 years. however, beyond the provision of selected illustrative historical examples in the original outline, the framework has yet to be subjected to systematic empirical assessment. in this paper we address this gap by exploring the co-evolutionary model outlined in schot and kanger [1] which links niche-level dynamics, transitions in single systems and great surges of development through the emergence, consolidation and alignment of rules. for this purpose, we conduct a historical case study of the development of mass production in the transatlantic region from 1765 to 1972. the case of mass production has been chosen because of its centrality to the 4 th long wave beginning at the early 20 th century [10, 11] making it both a 'most-likely' case for the dt framework and an influential case with considerable environmental impact. our main research question is: how does the historical development of mass production correspond to the co-evolutionary patterns as proposed by the deep transitions framework? section 2 defines the central concepts of the framework -rules, meta-rules, regimes, meta-regimes -and outlines a model of the co-evolutionary dynamics of rules. section 3 describes our methodological approach. section 4 presents a stylized narrative of the historical evolution of mass production. section 5 assesses the extent to which the observed dynamics match the theoretical expectations and situates our findings in broader literature on socio-technical change. similarly to early case studies exemplifying the multi-level perspective on socio-technical transitions [12] [13] [14] [15] we also use our results to modify and extend the dt framework. section 6 concludes. the dt framework [1] was developed to address two gaps in existing knowledge on multi-system co-evolution. first, to theorize how niche-regime dynamics and transitions in single systems [13, 16] relate to long waves [10, 11] . second, to understand how successive long waves accumulate into major historical continuities characterizing the overall industrialization process [9] . dt focuses on rules -"humanly devised constraints that structure human action, leading to regular patterns of practice" [1, p. 1053] -as a central coordination mechanism. by being embedded in the very structure of socio-technical systems rules shape the behaviour of actors and provide systems with long-lasting directionality. rules differ in terms of their scope and degree of alignment to other rules, resulting in a four-fold classification: 1. rule is a single prescription for action present in a single system. for example, a principle 'design for modularity' has its origins in the american housing system around 1920s-1930s, where the use of modular components was seen as means to lower construction costs, reduce waste and increase efficiency [17] . 2. meta-rule is a single rule present in multiple systems. for example, russell [17] describes how metaphors such as 'architecture', 'throughput' and 'modularity' started to appear in the vocabulary of american computer engineers in the 1950s. this indicates that the 'design for modularity' rule had crossed a boundary between housing and data processing systems. 3. regime is a set of aligned rules present in a single system. for example, zuboff [18] analyses how google invented and perfected interrelated principles constituting what she calls 'surveillance capitalism'. this involved treating human experience as a free raw material, using this 'behavioural surplus' for the fabrication of prediction products and trading these predictions in behavioural futures markets [18, p. 8] . from the early 2000s this regime became paradigmatic in the communication system and was adopted by major players like microsoft or facebook. 4. meta-regime is a rule-set present in multiple systems. for example, by treating usergenerated data from self-driving vehicles as a commodity to be sold to advertisers [19, 20] the regime of surveillance capitalism might be currently expanding from the communication system to the mobility system. drawing on these concepts, schot and kanger [1] connected processes on niche-and system-level as described by the multi-level perspective on socio-technical transitions [13, 16] , to successive 'great surges of development' as conceptualized by carlota perez. perez [10] argues that since the late 18 th century there have been five 40-60 year surges 1 , each characterized by particular phases: 1) gestation (pre-surge period); 2) installation period (first half of the surge, lasting 20-30 years), further divided into irruption and frenzy phases; 3) turning point; 4) deployment period (second half of the surge, lasting 20-30 years), further divided into synergy and maturity phases. the model by schot and kanger [1] relates the emergence, consolidation and alignment of rules to different phases of each surge, resulting in a specific pattern: 1. a protracted gestation period is characterized by the emergence of new rules rules in separate niches, i.e. spaces governed by specific selection criteria such as military applications prioritizing performance over cost. in some niches some of these rules may be aligned to each other. in exceptional instances, exogenous macro-level 'landscape' pressures might destabilize dominant systems, opening up a window of opportunity for niches and resulting in regime-shifts, i.e. transitions in single systems [13] . 2. each surge starts with the irruption phase where emerging and incumbent rules come to compete against each other, resulting in further transitions or transition failures. early interactions between some systems might occur and some rules may turn into meta-rules as a result. overall these interactions as well as their outcomes remain ad hoc, nonstandardized and accidental in nature: at this point no lasting connections between systems are created, and thus no clear directionality in multi-system co-evolution is established. 3. at the beginning of the frenzy phase, many rules increasingly start to cross the boundaries of a single system, generating widespread enthusiasm about the prospects of emerging rules and associated technological opportunities but also major concerns about their anticipated societal impacts. partial alignment between different meta-rules starts to occur leading to the gradual emergence of structural and functional couplings between systems, e.g. different systems relying on the same infrastructure or forming input-output relations [22] . this process is further amplified by the purposeful aggregation work of transnational organizations aiming to homogenize and standardize within-and between-system practices. as a result, a clearer new directionality becomes visible, yet in this phase there will still be competing options (alternative directions of evolution) available. 4. the competition between meta-rules and their various combinations is resolved at the turning point. major crises such as wars provide an impetus for the alignment of expectations, enabling powerful actors to tilt the playing field in favour of a particular ruleset. therefore, from this point forward one can start talking about the existence of the dominant meta-regime providing directionality across many socio-technical systems. 5. during the synergy phase the dominant meta-regime acts as a selection mechanism, favouring niches compatible with its logic and rejecting non-compatible ones [23] . it continues to diffuse from one system to another, leading to the increasing take-up of its principles in various systems. because of its expansive nature the meta-regime now also starts to shape landscape structure and dynamics. 6. in the maturity phase new problems start to appear which cannot be fully resolved within the confines of the dominant meta-regime. the scene is set for yet another surge with new niches and systems becoming the main loci of radical innovation. as the former metaregime has now become part of the landscape, it continues to structure new niche-regimemeta-regime interactions. figure 1 presents a visual summary of the model. j o u r n a l p r e -p r o o f as the above model focuses on developments distinctive to each phase, it implies but does not really make explicit the accumulation of phase-specific developments into long-term outcomes. an alternative way to look at the process of meta-regime building would be to separate the overall coevolutionary dynamics of rules into different sub-trends. such an analytical exercise potentially enables to observe different patterns and mechanisms related to various dimensions of meta-regime evolution that might otherwise remain unnoticed. although we recognize the difficulties with making clear-cut conceptual distinctions, we suggest that broadly speaking the co-evolutionary model of schot and kanger [1] can be seen as a result of three interacting sub-trends: 1. variation: emergence of new rules or the alignment of existing rules into rule-sets (regimes/ meta-regimes) with novel qualities. from the evolutionary perspective we expect gestation, irruption and frenzy phases to be dominated by high but gradually decreasing variety, reaching its low point during the synergy phase. this would reflect a move from radical to incremental innovation and the increasing alignment of rules leaving less possibilities for the emergence of major novelty. however, towards the end of the maturity phase we expect variety to increase again as the deepening crisis of the dominant meta-regime provides an opportunity for radical niches. 2. diffusion: imitation and selective adoption of rules and rule-sets in different systems and various countries. although we assume the process of diffusion to occur in a piecemeal manner involving various setbacks and failures, as an aggregate trend we expect the emerging meta-regime to spread to new systems and locations throughout the surge. 3. contestation: debates, conflicts and struggles around emerging rules and rule-sets concerning their application, their changing relations with existing modes of production and their broader societal effects. here the implied pattern is somewhat more complex. during irruption and frenzy phases we expect that the increasing public visibility of the emerging meta-regime and the attempts to apply it in different systems would prompt increasing societal contestation up to the turning point when large-scale conflicts are resolved. during the following phases we expect the dynamics to be reversed: low contestation during the synergy phase followed by renewed contestation during the maturity phase, reflecting a gradual shift from the dominance of the meta-regime to its crisis. these phase-specific and cross-phase trends constitute a set of expectations against which empirical data on the historical development of mass production will be assessed. this will be done in sections 5.1 and 5.2 respectively. the research is based on a single longitudinal case study design [24] , adopting a 'most-likely' case selection strategy. the literature on case selection stresses the symmetrical strengths of 'most-likely' and 'least-likely' cases for assessing the validity of an existing theory. whereas 'most-likely' cases cannot offer strong proof for a theory (as the case is expected to conform closely to theoretical propositions) they can strongly undermine it if the case does not fit the theory (because if the theory does not even explain the closest expected match it will probably perform even worse on less likely cases). conversely, 'least-likely' cases can offer strong proof for a theory (demonstrating that the theory explains even less likely cases) but they cannot strongly undermine it if the case does not fit the theory (as the case is not expected to conform strongly to theoretical propositions in the first place) [25, p. 121] . where multiple theories exist for explaining a given phenomenon, the strongest supporting evidence can be obtained by choosing a case least-likely for a given theory and mostlikely for competing theories (all of which also make different predictions about the outcome of interest). the strongest weakening evidence can be obtained by choosing a most-likely case for a given theory as well as all competing theories making similar predictions, and failing to find supporting evidence for it [25, p. 121] . arguments like this [26] [27] [28] implicitly assume a rather mature field of research characterized by considerable knowledge base (established through numerous prior case studies) and the existence of a number of competing theories for a given phenomenon. although this might be an accurate description of the authors' own disciplines (usually political science), neither assumption holds for the current framework which establishes a new phenomenon -deep transitions -through a synthesis of existing theories (multi-level perspective and techno-economic paradigms). in this way dt goes beyond mlp and tep by design and cannot be argued to offer strictly competing explanations to either of the two frameworks. this is well evidenced by geels's seminal analysis of the transformation of factory production from the mlp perspective [15] . not only is the study geographically and temporally restricted (usa, 1850 (usa, -1930 , it also focuses on the breakthrough of mass production where impacts exceeding the mobility system and emerging connections with other systems are largely excluded. the most complete long-wave account of mass production [11, pp. 257-300] , on the other hand, largely focuses on a few leading countries (primarily usa) during the 4 th wave only, neglecting the role niche-system dynamics in generating the surge. in contrast, the use of the dt framework necessitates the observation of interactions between niche, regime, metaregime and landscape dynamics over a much longer time-frame and broader geographical scope (see below). we suggest that in the context of scarce knowledge of the focal phenomenon (deep transitions) and the lack of competing theories offering alternative explanations, a 'least-likely' case would constitute a far too strong first empirical test of the framework. a similar reasoning, although not explicitly spelled out as such, seems to have been pursued in seminal works on socio-technical transitions which initially focused on transport, sanitation and production [12] [13] [14] [15] . it is very difficult to see what would have been gained if mlp had started out from a 'least-likely' case such as rock'n'roll instead [2] . therefore, in our view a 'most-likely' case constitutes the best option for a first empirical test of a nascent theory. mass production was chosen as a case for three reasons. first, as the tep framework considers mass production a central element of the 4 th surge it can be considered a 'most-likely' case for the dt framework. this means that we expect the evolution of mass production to mirror the conceptual model of schot and kanger [1] . a failure to detect a close match with the theoretical propositions, on the other hand, would cast serious doubt on the overall validity of the dt framework. second, during the past century mass production has become increasingly central to modern lifestyles in developed and developing countries, thereby exacerbating environmental problems such as pollution, waste or resource depletion [29] [30] [31] . because of its global impacts mass production constitutes an influential case the co-evolutionary dynamics of which are crucial to understand in its own right. third, practical considerations such as the availability of a broad array of secondary historical literature on the topic also played a role in the case selection. conducting indepth qualitative work with primary sources on the scale required for exploring the dt framework would have simply fallen beyond the scope of the study. the case is bounded as follows. temporally, we focus on the events between 1765 and 1972. this covers the long gestation period of mass production (1765-1907), its installation during the 4 th surge (including irruption, 1908-1919, and frenzy phases, 1920-1938), turning point (1939) (1940) (1941) (1942) (1943) (1944) (1945) as well as the deployment period (including synergy, 1946-1959, and maturity phases, 1960-1972) . geographically, we focus on the transatlantic region. this enables us to trace the varying origin and j o u r n a l p r e -p r o o f circulation of rules across national boundaries, including their regional adaptations. thematically, we focus primarily on the engineering-related dimension (production organization and labour control), largely excluding the dimensions of economy, policy, everyday life and culture. while the latter choice has been largely made for feasibility concerns we also recognize the resulting limitations of our study (see below). following an established tradition in transitions studies our research employs a stylized narrative explanation based on an interpretive analysis and synthesis of a broad range of secondary historical literature. as geels and schot argue, "the strength of a narrative is that it can capture complex interactions between agency and changing contexts, time, event sequences, making moves in games, and identities" [32, p. 97] . although narrative-based approach is versatile, flexible and attentive to complexity, it has also been criticized for the subjectivity of researcher choices or difficulties with assessing the extent to which the results conform to expected patterns [33, 34] . a similar sentiment is echoed in nuvolari's assessment of freeman and louçã (2001) as both the most articulated treatment of this [long wave] approach but also highly impressionistic and descriptive [35, pp. 34, 42] . given our aim -to provide an early empirical assessment of a complex theoretical framework of broad scope -we argue that the strengths of a narrative-based approach outweigh its weaknesses. to address the shortcomings of the narrative approach we try to provide a clear assessment about the possible biases of our study (see below), to substantiate each statement on a general trend with empirical support (section 4) and to establish clear connections between empirical data and theoretical abstractions (section 5). nevertheless we readily acknowledge that ultimately our selection, presentation and interpretation of data contains a certain degree of subjectivity. data collection and analysis was conducted in four stages, involving a great degree of iteration between different steps: 1. beginning from authoritative works [36] [37] [38] and literature search from various databases we used a snowball sampling approach to assemble a corpus of secondary literature on the history of mass production. given the analytical dimensions of interest (see section 2) and the case bounding (see above) we aimed at a systematic coverage. 2. based on the literature we then constructed a genealogy of mass production, consisting of different rules, combinations of these rules (rule-sets) as well as several historical dead-ends (see figure 2 ). this stage involved interpretive work, whereby various technologies, practices and principles described in the sources were transformed into rules (see box 1). the emerging interpretations were discussed with two historians of technology with expertise in mass and specialty production to further improve the genealogy. 3. we then compiled a stylized narrative explanation of the development of the rules underlying mass production. upon detecting gaps in the narrative, additional literature search was conducted to cover the missing dimensions and to update the genealogy. 4. in the final stage, we used the pattern-matching technique [24, pp. 224-225 ] to compare the patterns that emerged from the stylized empirical narrative with the expected ones derived from theory. in doing so we assessed overlaps and deviations and, where necessary, developed further conceptual extensions. the pattern analysis was twofold. first, the phasespecific patterns of every period were matched to the six propositions (gestation, irruption, frenzy, turning point, synergy, maturity) described in subsection 2.1. the results of this exercise are presented in subsection 5.1. second, the cross-phase patterns evident throughout the periods were matched to the three overall trends (variation, diffusion, contestation) described in subsection 2.2. these results are presented in subsection 5.2. the limitations of our approach should be noted. firstly, as our study draws on works published in english the narrative might suffer from source bias. to address this issue we attempted to locate studies on different countries or studies with an international dimension [38] [39] [40] . secondly, given our focus on the engineering dimension, the narrative might somewhat overstate the prominence of usa while downplaying the contribution of different countries to the correlates of mass production in other dimensions (economy, policy, everyday life, culture), e.g. interwar era soviet experiments with centralized planning as a precursor for large-scale state intervention in the entire transatlantic region after world war ii. it appears, therefore, that conveyors and gravity slides were adopted either immediately before the assembly line experiments or resulted from the "work in motion" principle brought to life by the assembly line [36, pp. 238-239, on ford's experiments]. mass production of automobiles, as developed by henry ford in the half-dozen years before the first world war, depended on three basic principles: the standardisation of the product, the use of special-purpose equipment, and the elimination of skilled labour in direct production [41, p. 153] . "thorough elimination of wasteful practices" is the basic concept of the toyota production system. this concept is supported by the following two fundamental principles: 1. "right on time"; 2. "automation" [42, p. 87] . use hand tools for making the products the following section presents a narrative overview of the historical evolution of mass production between 1765 to 1972. in order to establish clearer connections with the theoretical framework (see section 2) the results have been divided in different phases, with each phase focusing on three trends -variation, diffusion and contestation. note, however, that because of continuous interactions (e.g. new rule-sets emerging and starting to compete with each other) the following narrative cannot always maintain as neat analytical separation of the three trends as implied by the respective titles of each sub-section. before the 20 th century flexible specialty production was a dominant mode in the entire transatlantic region. it relied on one worker performing all tasks, the use of customizable specialpurpose machinery, the imperative to compete on novelty and/or quality (rather than price), making systematic use of shop-floor worker knowledge for production improvements, and maintaining personal and informal relations inside the enterprise and between different enterprises [43] . from the 18 th century experiments in various niches started to challenge these principles. the precursors of mass production can be broken down to four broad streams: standardization, continuous movement, electrification and efficiency. the first stream began with the ideal of interchangeable components in armsmaking that emerged in the french military from 1765 and was realized with hand tools in 1790 [44] . during the first half of the 19 th century us state armouries (harpers ferry, springfield) added two important rules: using special-purpose equipment to perform one function only and placing machines in a sequence according to the work process [36, 45] . by mid-19 th century the alignment of these three principles constituted a rule-set of distinctive character, named american system of production by contemporary observers [36] . closely related to the interchangeability of components was also the emerging idea of product standardization which allowed to minimize changes in design and to avoid expensive and time-consuming retooling [38, pp. 27-28] . another stream was related to experiments with continuous movement and flow production in various applications such as flourmilling, bakeries, breweries, cigarette-making, canning, oil and chemical industries, foundries and bicycle production. other important antecedents were the disassembly lines used in meat-processing in cincinnati (from the 1860s, see figure 3 ) and chicago (from the 1880s) as well as edison's iron mining facility in ogdenburg, which was visited by henry ford [36,38, p. 26;40] . a third strand entailed the electrification of factories. electric motor was first introduced in the 1880s, first as an add-on to the central steam engine but gradually moving toward unit drive, i.e. each machine having a separate electric motor. before that the central power source largely dictated the placement of the machines and also limited the precision that could be achieved. additional bonuses of electrification included improved lighting and ventilation enabling increases in precision and working time but also better health for the workers [15,38, p. 27] . the fourth stream aimed to increase the efficiency of labour and resource use. this included a division of production into specific tasks to be carried out by a specific worker, introduction of a clear separation between engineering, management and executive roles, and replacing workers' rules of thumb with ones devised by experts. these principles were central to the thinking of frederick winslow taylor whose work experience in steelmaking gradually led him to formulate a system of 'scientific management' [47, 48] . as part of a wider efficiency movement gaining strength in industrial nations from the late 19 th century other ideas on systems management and factory organization gradually led to an idea to optimize the work process as a whole [46, 49] . similar concerns also directed attention to the need to minimize waste of resources and, where possible, reuse waste materials in the production process. the precursors of mass production originated from both europe and usa. interchangeability had a french origin but the ideas were transferred to the usa by thomas jefferson, french military experts and books. the idea to sequence machines according to the work process was developed by thomas blanchard at the us springfield armoury around the late 1810s but the idea built on the british example of wooden blockmaking from the 1790s [36, pp. 25-27, 41] . the benefits of subdivision of work into specific tasks had been famously described by adam smith in 1776 but the idea was taken to another level by taylor's scientific management more than a century later. in terms of sectoral origin there was even greater variety: armsmaking, mobility, food, energy and chemical industry all contained niches for experimenting with new principles of production. the initial barrier to the adoption of the american system of production was cost-related: achieving full interchangeability turned out to be more expensive than hand-fitting [36, pp. 48-49) . however, over the second half of the 19 th century a number of broader factors in usa started to favour increasing mechanization of production. these included a shortage of skilled labour (thereby providing incentives for investing in machinery), road and rail network enabling nation-wide markets, fast population growth coupled with rising purchasing power, the emergence and growth of large-scale corporations able to capture various economies of scale, the rise of the engineers as a new and increasingly influential social group but also the american cultural values favouring the development of a highly accelerated society [15,38, pp. 5-10;46] . during the second half of the 19 th century the american system spread in a gradual and piecemeal fashion to various industries such as private armsmaking, clockmacking, sewing machines and typewriters [36] . perhaps the most notable example of pre-fordist large-scale production was the pope manufacturing company where the automation of work on a moving assembly line, interchangeable parts across a wide variety of bicycle models and specialization of work process management in functional departments were combined for bicycle production [50] . interchangeability and standardized parts were first introduced to the automobile industry by the cadillac company in 1908 [46, p. 298 ]. since the precursors of mass production were dispersed in various niches interactions between these rules and specialty production remained fairly sporadic. enterprises relying on flexible production did occasionally employ new principles when it suited them, e.g. printing industry in new york being early adopters of electric drive systems [43, p. 129 ], but overall this did not lead to a fundamental overhaul of the mode of production itself. occasionally, new rules were explicitly contested. for example, french experiments with new modes of weapon production were terminated for political reasons at the beginning of the 19 th century [44] . another example concerns taylor's attempts to rationalize production at bethelem steel company, leading to stiffened worker resistance and culminating in his dismissal in 1901 [47, p. 195 ]. from 1908 henry ford and his team of engineers undertook a wave of experimentation to mechanize car production, turning simultaneous attention to gauging, fixtures, machine tool design, materials handling, factory layout and many other areas [36, p. 219 ]. in so doing the team made use of various principles pioneered during the gestation phase (see figure 2 and 4.1.1) but often realized them in a novel manner. 'move work to worker' rule is a good example: whereas the 19 th century bicycle industry had used runners to deliver parts to stationary machinists ford's engineers developed a moving assembly line for the same purpose; whereas meat-processing had used a sequential approach to disassemble the corpse, ford reversed the process. the success of ford motor company also exposed a crucial difference between 'fordism' and 'taylorism'. although both approaches offered solutions for increasing the volume of production and were seen by contemporary observers as similar and complementary, taylor focused on task optimization while ford directed his attention to the work process as a whole. the supremacy of the latter approach was clearly demonstrated early on: in 1914 ford's production exceeded the 'taylorist' packard factory by 30 times [38, p. 250 ]. despite taylor's search for 'one best way' turning out to be a historical dead end as a rule-set for production organization, his legacy proved to be much more enduring for labour management [48, 53] . . wwi also provided a stimulus to extend the new regime to the defence system, e.g. citroën using mass production techniques to produce artillery shells (ibid.). sending men and horses to the front facilitated the mechanization of agriculture, reflected in the introduction of the fordson tractor in 1917. however, not all attempts to apply mass production were successful, e.g. ford's failure to deliver submarine patrol boats as quickly as anticipated [55] . high degree of task fragmentation and the accompanying routineness of work imposed severe demands on labour who were de-skilled and had very little control over the work process. as such mass production was contested from the outset. in 1912, ford broke a strike in buffalo plant by closing it and sending the machinery to detroit; his right-hand man, charles sorensen, had to go to britain to break the metal workers union [54, p. 45] . nevertheless, high labour turnover remained a pressing problem, reaching 380% in 1913 [36, p. 11] . in order to counter this ford famously introduced a 5-dollar day in 1914, prompting immediate discussions about the societal impacts of this move. whereas american socialists and radical press celebrated the rise in worker income, conservative voices in industry, media and church worried that ford's high wage policy may aggravate worker inequality and drive out other businesses thus leading to social unrest and overall decrease in local prosperity [38, pp. 98-102] . whereas ford's mass production was starting to push aside its competitors (e.g. taylorism) in the mobility system, specialty production continued to thrive elsewhere. in usa it contributed roughly about 1/3 of the value added in 1909 [43, pp. 13-16] whereas in europe the craft tradition remained largely unchallenged. in many areas such as jewellery or furniture quickly changing consumer demand made mass production techniques non-viable. however, whereas the gestation period had been characterized by loose borrowing of selected principles, some specialty producers now started to establish more symbiotic relationships with mass producers, e.g. machine shops supplying specialized single-purpose machinery for early automobile manufacturers [43, p. 218 ]. (1920-1938) whereas ford had inaugurated a new regime of mass production his continued focus on productivity maximization and price-cutting of a single product made him less responsive to variations in demand. from the 1920s general motors had begun to seek for alternatives in order to balance the economies of scale offered by mass production with diversity in demand characteristic of market economies. this led gm to introduce many business-related innovations such as the installation of multi-divisional decentralized management structure, use of consumer credit or the facilitation of used-car trade-ins. however, gm's strategy also implied changes in production leading to a new variation, 'flexible mass production' [36] . as a guiding rule production had to be designed for annual model change, often involving minor stylistic changes. the combination of uniformity and variety was achieved by using similar components across different car models. the flexibility of production was further increased by the customization of general-purpose machinery (vs. highly specialized single-purpose machines that had characterized ford's production). the overall effectiveness of increased flexibility was reflected in the fact that in 1927 gm's chevrolet outsold ford in the american market for the first time [54, p. 69 ]. from the 1920s mass production consolidated its foothold in the systems of mobility (increasing diffusion of cars, especially in usa), defence (mechanization of war as an anticipated direction of evolution) and food (continued mechanization of agriculture, new techniques of fruit canning and baking, introduction of toasters). new consumer durables often acted as vehicles for introducing mass production to new systems, e.g. communication (radio sets) and housing (washing machines, vacuum cleaners, electric irons) [38, p. 51] . the take-up of mass production often resulted in three types of outcomes: 1) consolidating existing links within a system, e.g. oil production and car diffusion in the mobility system; 2) acting as one of the building blocks for system transitions: for example, in the food system mechanization was increasingly combined with the principles of chemicalization (e.g. the use of nitrogen fertilizers and pesticides) and selection of plants and animals suitable for standardized production, gradually paving way to the intensive agriculture regime [56, pp. 375-376]; 3) consolidating or creating new links between systems, e.g. drive-in cinemas linking mobility and communication systems or electrical appliances in the housing system intensifying production in the energy system. mass production also started to become an increasingly "powerful general concept. it quickly shed its technical and precise meaning and started to move toward an abstract ideal of standardization, mechanization and repetitive production, allied to implications of order, rationality and universality" [37, p. xvii] . this was reflected in using the term as a loose metaphor to describe various technical and social activities (energy production, education, artistic performances), the influence of machine aesthetic on industrial designers and artists (e. at the same time attempts to extend mass production through direct imitation often resulted in failures. us automotive dealers largely resisted ford's efforts to introduce routinized work procedures, division of labour, specialized machinery and progressive layout of equipment [57] . in both europe and usa various attempts were made (and abandoned) to produce houses on the assembly line. american mechanical engineers also advocated the increasing use of mass production in woodworking and furniture industry on the grounds of increased efficiency, largely neglecting consumer preferences favouring diversity [ during the 1920s-1930s the contestation of mass production gradually changed from an individual worker strategy of 'voting with one's feet' to organized resistance. added to the familiar concerns about repetitiveness and de-skilling were the various tactics of the american automobile industry for increasing the pace of work without a corresponding rise in wages, e.g. increase of line speed after installing new machinery or setting special daily production goals [64] . until the end of the 1920s the industry's high wages had generally neutralized the need for worker organization. however, with the great depression the power of management over labour was further increased as worker job security turned ever more fragile. this situation eventually led to the establishment of the united automobile workers union in 1935 and the introduction of sit-down strikes [38, pp. 116-120] . although the automotive industry started making gradual concessions to workers, the conflict was not entirely resolved by the end of the 1930s: the link between mass production and labour organization was to be consolidated only after wwii. the relationship between mass and specialty production was characterized by important regional differences. in european countries with a strong craft tradition, mass production was claimed to be an american phenomenon that, because of smaller markets, lower incomes and diversified consumer preferences, could not be copied [65] . in usa, however, a shift from symbiosis to competition between mass and specialty production was taking place. for example, in the early 1920s several clothing manufacturers installed two or more assembly lines, one for standardized and the other for customized production [66] . however, by the end of the decade the prevalent rhetoric of 'simplification', advocated by president hoover, engineering societies and managers, started to associate industrial diversity with inefficiency and waste. the federal trade commission and department of justice also countered attempts at regulating competition through price coordination and exclusive sales agreements. this effectively forced specialty producers to compete on price, making them lose one of their distinctive characteristics, and initiating the gradual waning of flexible production in usa [43] . increasing public visibility of mass production raised wildly ranging hopes and fears about its societal effects. for example, ford's waste handling practices were initially argued to contribute to the preservation of america's timber resources [67, p. 151 ]. an influential advocate of fordism argued that the abundance enabled by mass production would lead to the "abandonment of all class thinking and the substitution of fact-finding for tradition" [68, p. 1]. mass production was also associated with the possibility of shorter working hours leading, in turn, to worries about future technological unemployment. these utopian promises initially made mass production attractive to radical political forces and totalitarian governments [69] . however, by 1930s widespread optimism had given way to two streams of critique. the great depression had problematized the necessity of connections between mass production, high employment, rising wages and increasing living standard. especially in europe mass production was argued to lead to the homogenization of products, consumers, personality and culture [38, pp. 88-93] . for example, a french satirical film "à nous la liberté" from 1931 drew parallels between industrial working conditions and life in prison (see figure 5 ). existential threats posed by wartime conditions prompted several experiments to improve the performance of weapons and to reduce the number of defects. strategies for seeking trade-offs between product quality and quantity led to differing national strategies: whereas german and british managers preferred increasing quality and constant modifications, american and soviet industrialists stressed quantity and refrained from changes preventing the full deployment of assembly line techniques [70, p. 358 ]. the management of this balance led to experiments with continuous improvement and just-in-time strategy in airplane production [71] . german concentration camps were also experimenting with flexibility in uniform production combining the use of female prisoners with no special skills or training needed, state-of-the-art sewing machines and electric motors [38, p. 132 ]. however, these experiments largely failed to spill over to the civil sector after the war when both usa and europe largely reverted to better established techniques of mass production. war requirements greatly increased the need for standardized products, including clothing, weaponry and food. government procurement, investments in factory equipment and other subsidies directly facilitated mass production [72] as did cooperation needs between allies. especially important was the lend lease act (1941) through which usa began to provide supplies to allies, e.g. trucks, boots and food for the soviet red army or car parts to britain for the manufacturing of jeeps [38, p. 135;70, p. 354] . whereas wwi had provided a stimulus to extend the nascent mass production regime to the defence system, wwii helped to consolidate its centrality for war in the entire region. in the american food system, where the adoption of mass produced vehicles was well underway, wwii proved to be a literal turning point (see figure 6 ). the war also demonstrated possibilities for extending the meta-regime from one application to another. for example, the construction tycoon henry j. kaiser established the california shipbuilding corporation in 1941 for the purpose of mass producing military ships and although he had no prior experience in the field, the enterprise became the country's leading shipbuilder [73] . another example is the hiring of henry ford's chief engineer to design the willow run production line for assembling b-24 heavy bombers [74] . the established infrastructure often facilitated dual use: in the usa aircraft engines were produced in converted automobile factories [75] whereas in germany wartime aircraft production led to the development of a strong supply network forming a basis for post-war automobile industries [76] . wartime conditions heightened the need for 'mass loyalty' which meant putting labour conflict on hold and mobilizing the entire population in the name of cooperation and shared sacrifice [38, pp. 128-131;78] . in this way the combination of direct and indirect effects of wwii paved way for the post-war dominance of mass production. the lack of demand for specialty production together with state investments in mass production created a productive capacity that could be converted for civil purposes after the war. the forced savings of a substantial amount of soldiers [72] created a possibility for post-war mass consumption. the devastation of war also raised pressing concerns for which mass production seemed to provide appropriate solution. the food system is a case in point. although organic farming had started to gain public visibility in britain shortly before wwii, severe post-war food shortages provided strong incentives for industries and newly founded international organizations such as fao to favour intensive agriculture instead [79, pp. 115-120] . (1946-1959) the post-war era can be described as the societal embedding of mass production: connecting it to horizontally integrated and multi-divisional big enterprises in the economic dimension, social contract between employers, labour and policymakers within the framework of a nationallybounded welfare state in the political dimension, individualized private mass consumption in everyday life and the ideology of material and social progress in the cultural dimension [11, 80] . the combination of these features put a foundation to long-lasting economic growth in various regions of the world, enabling them to catch up with the usa. mass production continued to develop in two directions. in usa, labour unrest had prompted a managerial vision of a fully automated factory. underlying this vision were two principles: embedding the task sequences of skilled workmen in machine tools (initially through 'record and playback' systems) and using the idea of feedback to increase the flexibility of machine tools. the full realization of these principles was envisioned to result in computer-controlled assembly lines staffed with robots that would enable to replace not only semi-skilled blue-collar workers but also skilled blue-collar and white-collar employees [38, pp. 157-161] . whereas the rhetoric of automation was also embraced in post-war japan, experiments undertaken in toyota from 1948 focused much more on improvements in product quality, reorganization of work and increasing labour involvement. responding to constraints that had made europeans largely reject mass production in the interwar era relative lack of capital, smaller volumes of production and higher variety of models toyota started to introduce novel production principles. these included an imperative to design production for continuous product and process improvement (vs. the fairly limited flexibility of the american approach), using worker expertise as a constant input for production improvement (vs. restricting workers to the role of task execution), allowing workers to stop the production process upon spotting an error (vs. having to succumb to the dictate of the assembly line) and 'just-in-time' provision of an exact amount of supplies (vs. 'just-in-case' buffering of stocks) [42, [82] [83] [84] (see figure 7) . similarly to ford's early activities these rules emerged in a piecemeal fashion: by the end of the 1950s toyota's practices had not been even formally codified yet [85] . post-war decades were characterized by the continued deepening of mass production in systems where it had been previously employed, reflected in a move from vehicles and consumer durables to other system components. for example, in order to employ picking machines in the american food system, a standardized tomato with a thicker skin had to be bred, picked green, chemically ripened and 'enhanced' with salt and sugar to compensate for the loss of flavour [86, pp. 291-293) . another way of deepening involved a shift from previously attempted full-scale imitation to more selective adoption. for example, whereas the 'move work to worker' principle had been abandoned in the housing system, the use of standardized components and architecture still enabled a rapid construction of blocks of identical-looking houses [38, pp. 149-151] . as a result couplings between systems became consolidated in a manner that would stimulate the further deployment of mass production. this was the case for us mobility and housing systems: mass produced cars enabled to live further from the city centre facilitating urban sprawl, whereas the creation of new suburbs without efficiently functioning public transport amplified the need for vehicles. figure 7 . 'provide supplies exactly where/when required' and 'anyone should be able to stop production upon spotting an error' rules contrasted to traditional mass production [42, p. 92] . note the use of the word 'automation' for describing the latter. cold war provided another stimulus for the take-up of mass production as the competition between two superpowers also involved claims about living standard. through channels such as the marshall plan, the productivity mission, fulbright program and united states information agency mass production techniques, corporate organization structures, management styles, advertising, and consumer goods started to flood the transatlantic region. although the adoption of mass production remained selective and uneven [87] in many ways european countries started to become more similar to usa [60] . these influences extended beyond the iron curtain, e.g. the idea of mass produced people's car à la volkswagen beetle was present in east germany, soviet union and czechoslovakia [88] . however, as the socialist bloc prioritized the needs of the military-industrial complex both the availability and quality of consumer goods remained notably lower [89] . as a new direction automation was highly visible. by 1958, usa had 16,000 'automatic assembly machines' applied to the production of automobiles, electric motors, tv-s and radios. the role of automation in post-war industrial reconstruction was also stressed in germany, france, ussr and japan [38, pp. 161, 166-167] . in contrast, very little was known about japanese experiments in the west. toyota's engineers themselves, however, acquired information from diverse sources, including us factory visits and thorough studies of various production practices. a strive towards continuous improvements was realized through the deployment of statistical methods of quality control which had been pioneered in usa during wwii but largely abandoned thereafter. just-intime supply was derived from the supply chain practices of american supermarkets, whereas the self-stoppage mechanisms of toyota's pre-war era automatic looms inspired an idea to let the workers stop the assembly line upon spotting an error [38, pp. 187-201;42] . although the post-war era was initially characterized by the immediate eruption of major strike waves in the entire region [90] , a general trend was the gradual institutionalization of labour conflict in the entire region. on one hand unions came to be acknowledged as a legitimate part of statecapital-labour nexus; on the other hand their activities were increasingly restricted by law (e.g. the introduction of taft-hartley act in usa in 1947). continued but decreasing level of strikes was thus accompanied by the increasingly routine and ritual nature of these activities [91] . the increasing acceptance of mass production was also evident in its relation to possible alternatives such as specialty production. instead of asking whether to adopt mass production, different countries outside usa now focused on the question of how to make best use of these techniques in given national conditions [38, pp.135-140;60] . in terms of societal effects mass production was heavily propagated as a basis for postwar reconstruction with rising productivity linked to higher living standards and social progress. the general theme of prosperity was now dominant in the entire region. however, the cultural critique of mass production also started to spread in a similar manner. increasingly influential authors of the frankfurt school argued that alienation was a consequence of the concentration of production into large-scale monopolistic corporations, the division of labour between mental and manual work and the increasing routineness and monotony of work tasks [48] . importantly, the sources of alienation were not seen as specific to particular economic or political systems but rather the underlying features of all modern industrial societies. for example, fromm claimed that both capitalist and communist countries were developing into managerial societies that "make machines that act like men and produce men who act like machines" [92, p. 33 ]. (1960-1972) in the 1960s the excesses of mass production started to provoke radical responses from niches. counterculture activists increasingly experimented with food cooperatives and small-scale enterprises under local ownership, emphasizing home-made goods, recycling, organic food, smallscale technologies, and do-it-yourself furniture, food, energy and shelter [38, pp. 175-177] . in parallel, with the initiation of border industrialization program in 1965 some american corporations started to shift manufacturing jobs to northern mexico [93] . this enabled to sustain profitability by cutting the labour costs while also decoupling production in one location from end use in another. although the broader implications were not fully understood at the time, the expansion and knock-on effects of offshoring -de-industrializing the west and putting an end to the post-war welfare state compromise -would come to exhibit significant global impacts over the following decades [94, 95] . the american ideal of computer-aided design and manufacturing continued to hold sway as reflected, for example, in the development of numerically-controlled machine tools [96] . although influential experts such as norbert wiener expected full factory automation to occur by the 1960s, numerous technical difficulties resulted in far more modest outcomes: by mid-1970s the number of robots in us enterprises was still around 6,000 [38, pp. 159-161] . continuing large investments in r&d and various experiments in the field of information technologies nevertheless helped to establish a foundation to the 5 th great surge of development, unfolding from the early 1970s [10, 11] . whereas automation had run into difficulties in usa, the japanese alternative was gradually maturing to take the world by surprise in the 1970s. novel principles included a strive to implement changeover as quickly as possible (e.g. shigeo shingo's single-minute exchange dies), designing products so that they would be easy to manufacture, organizing work in teams of multi-skilled workers and maintaining close and cooperative relations throughout the entire supply chain [82, 83, 97, 98] . in the mid-1960s the new approach was formally documented for the first time as the toyota production system [85] . further consolidation of mass production in different systems and countries continued to be the dominant theme in the 1960s, reflected in the diffusion of various consumer goods such as automobiles, refrigerators or tv sets (see figure 8 ). the logic of mass production and consumption had also pervaded the socialist bloc: for example, the soviet seven-year plan for 1958-1965 promised to match the usa in housing and consumer durables. the resulting 'consumer socialism' [99] attempted to emulate the west in terms of product quality and availability. as a result, one could observe the homogenization of energy and materials usage profiles in the region: whereas the consumerist west was producing increasing amounts of waste, the more limited consumption in the east was amply offset by the deployment of inefficient production technologies [100] and general disregard of environmental impacts. the situation with american and japanese approaches to mass production continued to be similar: high international visibility of the automation discourse whereas toyota's experiments remained largely unknown outside japan. notably, the increasing reach of mass production stimulated niche responses on the same scale, e.g. the internationalization of organic farming [79, pp. 156-183] and appropriate technology movement [47, pp. 453-459] . by 1960s two broad trends in the contestation of mass production had become visible: geographical homogenization and broadening of resistance. as european countries increasingly followed the us lead in adopting mass production, the societal critique associating mass production with cultural homogenization, loss of individuality, creativity and spontaneity was now increasingly present in the usa. by the end of the decade, the contestation of mass production had escalated into an all-out attack on industrial society and its underlying values of efficiency, standardization and businessdrivenness [38, pp. 168-170] . these sentiments were expressed in many influential works by herbert marcuse, jacques ellul, theodore roszak and lewis mumford [102] [103] [104] [105] . a novel strand of critique [106, 107] attacked the western lifestyle for its high use of energy, materials, food and water, increasing production of waste, throw-away consumer culture, and the environmental consequences of these practices. the different tensions culminated in 1968 as mass anti-establishment protests spread over the us and europe, most notably in france and czechoslovakia, involving a wide range of people from workers to students. in 1970 the first earth day celebration in usa brought together 20 million people, including students and housewives' organizations, further demonstrating the power of citizen activism [108, p. 264 ]. the analysis in this section follows the structure of section 2 where we describe our theoretical framework. we begin by comparing the empirical patterns evident in every period to the phasespecific model of dt consisting of six propositions (gestation, irruption, frenzy, turning point, synergy, maturity). we then move to comparing the patterns that we detected throughout the periods to the three overall trends prescribed by the dt framework (variation, diffusion, contestation). for all six phases we cover main trends in niches and single systems (rules/regimes), interconnected systems (meta-rules/meta-regimes) and on the landscape level. this yields 18 cells presented in table 2. each cell has been colour coded to facilitate obtaining a quick visual overview of the main results. green was used when the authors agreed that the development of mass production was yellow was used when the authors either failed to find empirical support for a particular proposition or when unexpected findings were detected. these assessments reflect the authors' interpretive consensus as decisions whether the findings are 'largely in line' or 'sufficiently deviant' entail an unavoidable and irreducible amount of subjectivity. although below we try to substantiate each theoretical claim with empirical illustrations, the reader is referred back to section 4 for more empirical detail backing up our conclusions. shortage, road and rail networks, population growth, big corporations, rise of engineering, cultural values) set the stage for the emergence of mass production 1908-1919 rules/regimes: alignment of various rules into mass production regime in the mobility system (ford, usa) meta-rules/meta-regimes: early experiments with mass production in food and defence systems (tractors, artillery shells), including some initial setbacks (submarine patrol boats) landscape: wwi stimulates the extension of mass production to new systems and locations (europe) 1920-1938 rules/regimes: 'flexible mass production' emerges in the mobility system (general motors, usa) meta-rules/meta-regimes: competition between rulesets (fordist vs. flexible mass production), selective adoption of us rules in europe resulting in regional variations (bataism, bedaux system); emergence and consolidation of links within and between systems (e.g. energy-housing, mobility-communication) landscape: great depression decreases the utopian promises of mass production, while stimulating increasingly organized worker resistance 1939-1945 rules/regimes: experiments with flexible and just-intime production of airplanes (usa and europe) meta-rules/meta-regimes: consolidation of mass production in some systems (food) and extension in others (ships and airplanes in the defence system); factories as structural couplings (e.g. between mobility and defence systems) landscape: direct and indirect effects of wwii (creation of productive capacity, putting the resolution of labour conflict on hold, forced savings, acute shortages) increasingly favour mass production 1946-1959 rules/regimes: experiments with automation (usa), and constant improvement (toyota, japan) meta-rules/meta-regimes: societal embedding of mass production, further consolidation in systems and 'americanization' of the transatlantic region landscape: facilitated by cold war, mass production starts to contribute to long-lasting post-war economic growth in the entire region rules/regimes: further development of variations of mass production (usa, japan), resurgence of radical alternatives to mass production in niches (organic farming, local production, appropriate technology) meta-rules/meta-regimes: dominant meta-regime further optimized (start of offshoring, usa) landscape: mass production increasingly associated with broad societal fears (cultural homogenization, alienation) and severe environmental impacts (high resource use, waste, pollution) in general we assess that the development of mass production largely matches the theoretical expectations in 12 cells out of 18. furthermore, apart from frenzy, all phases contained minimally a 2/3 match with the expected dynamics. the frenzy phase was also the only one where yellow colour was partly used because of our inability to find supporting evidence. namely, whereas the theory stressed the role of transnational organizations in meta-regime building, our evidence pointed to the role of national organizations instead (e.g. bhpc in uk, rkw in germany). this might reflect a source bias of our study as historians of technology have fairly recently started to focus on transnational organizations [40, 109] . in all other instances yellow colour denotes unexpected findings that indicate the need to extend the dt framework. first, based on our analysis of meta-regime building in the frenzy phase we propose that in addition to structural and functional couplings systems also link to each other through shared discourses or 'rhetorical couplings'. these couplings manifest themselves in three ways: 1) claims about the paradigmatic system as a direct source of imitation, e.g. interwar era beliefs that assembly line techniques from car production can be applied to prefabricated housing; 2) downplaying the difficulties associated with cross-system or cross-regional knowledge transfer, e.g. ford's belief that mass production can be adopted anywhere with minimal changes; 3) abstracting the technical principles of the meta-regime into more general concepts, e.g. the use of mass production as a loose mobilizing metaphor or part of an overall societal philosophy. whereas the first two strategies often led to setbacks and failures when attempting to move from rhetoric to practice, the diffuseness, openness and flexibility of 'generalized fordism' enabled to connect different systems on a rhetorical level in the entire transatlantic region. somewhat paradoxically these findings suggest that, at least in early stages, rhetorical couplings facilitate meta-regime building because of the high degree of abstraction of claims involved, not because of their specificity. the rest of the deviations in each phase (from irruption to maturity) relate to the effect of landscape dynamics on the evolution of rules and rule-sets throughout the entire surge. based on our data we distinguish between three types of landscape impacts: 1) stimulating new rules and rule-sets, e.g. the set of various broad factors in usa facilitating the precursors of mass production (gestation phase); 2) amplifying existing rules and rule-sets, e.g. the role of wwi (irruption phase) and the cold war (synergy and maturity phases) in extending mass production to new systems and locations; 3) closing off alternative directions, e.g. the role of great depression in detaching mass production from its utopian promises of societal overhaul (frenzy phase). importantly, as a turning point event wwii exerted all three types of effects, e.g. experiments with flexible production of airplanes, extension of mass production in defence and food systems, and the reduction of possibilities for alternative practices such as organic farming. our findings complement the theoretical model of dt that restricts the importance of landscape impacts largely to early stages and the turning point. they also call for more reflections on the role of the landscape in the study of single system transitions where exogenous pressures are often relegated to the role of early enablers [13, 32] . moreover, the results call into question conceptualizations in long wave literature claiming surges to be driven by an internal logic, e.g. the shifting balance between production and finance capital in perez's [10] model. this suggests two possibilities: either these literatures have so far paid insufficient attention to landscape dynamics or meta-regimes are somehow distinctive in requiring continuous landscape support. in any case, further theorization of the stimulating, amplifying and terminating effects of landscape dynamics is warranted. we now move forward to assessing the cross-phase trends related to the variation, diffusion and contestation of rules. the main results are presented on figure 9 . note that similarly to interpretive accounts in long wave and transitions literature [10, p. 30; 13, p. 83 ] the curves are meant as a visual aid for the reader and do not reflect the results of a strict quantitative measurement. as with the previous section, the assessments rather reflect the authors' interpretive consensus. the results largely confirm the expected pattern in variation: gestation was characterized by high variety in different niches, irruption to synergy by the emergence, gradual improvements and societal embedding of mass production, and maturity by the resurgence of radical alternatives to mass production. however, we also found that major experiments with mass production continued in the post-war era, e.g. automation in usa and constant improvement of production in japan. this suggests that the existing dt model has somewhat overstated the incremental nature of innovation during the synergy phase. the dt framework thereby downplays the possibility that niche experiments directed at improving the existing meta-regime might eventually accumulate into a significant transformation of this meta-regime. this is precisely what happened in japan where toyota's experiments in the late 1940s gradually grew into a distinctively novel 'lean' version of mass production that started to reshape global manufacturing from the 1970s [85] . more research is thus required to find out what conditions facilitate the emergence of radical internal variations of meta-regimes and what factors enable to scale up these variations. the general trend was also confirmed for diffusion: despite various setbacks, mass production spread through selective adoption to successive systems and countries throughout the entire surge. however, our analysis also revealed specific channels of early diffusion: 1) mass production was more likely to move from one location to another when the underlying systems were similar, e.g. the early adoption of us techniques by french car producers; 2) mass production was more likely to move from one system to another in a given location, e.g. an early extension of mass production to the food system in usa; 3) in different systems mass production was first more likely to be applied to similar technologies, e.g. the use of mechanized vehicles in the american food system preceding the standardization of tomatoes. these findings are in line with evolutionary economic geography which has demonstrated the role of different forms of proximity (cognitive, organizational, social, institutional and geographical) in facilitating learning and innovation [110] [111] [112] . however, explanations relying on differing forms of proximity fail to explain why radical movements and totalitarian governments were initially most receptive to mass production [69] . in our view this observation might be better explained by the mechanism of social differentiation as used in sociological and marketing research [113, 114] . works in these disciplines show that consumers use new products to express individual identity, to highlight their group affiliation and to signal their distinctiveness from other social groups. mcmeekin and southerton [115] have applied this idea to socio-technical systems, arguing that contrasting group definitions of users can either support or hinder transitions. our findings suggest that the mechanism of social differentiation can be further extended to entire societies and to the diffusion of meta-regimes. this is in line with recent historical literature, highlighting the central role of fordism in the attempts of nazi germany and soviet union to create a societal alternative to western liberal capitalism [62, 116] . in terms of contestation our findings confirmed that irruption and synergy were characterized by lower degrees of conflict than frenzy and maturity phases. however, the specific forms of contestation turned out to be quite different before and after wwii. during irruption and frenzy phases we observed a high degree of initial uncertainty about the societal impacts of mass production (e.g. celebration of and worries about the 5-dollar day in usa) and individualized worker resistance being reduced to fewer but regionally varying themes by the 1930s (organized worker resistance in usa where mass production was being adopted, cultural critique in europe where mass production was resisted). during synergy and frenzy phases we first observed the homogenization of themes of contestation across the entire region (e.g. institutionalization of labour conflict in western europe, cultural critique of mass production being taken up in usa). this was followed by the generalization of contestation to broader societal and environmental themes of j o u r n a l p r e -p r o o f which mass production was only a part (e.g. revolutions of 1968, earth day in 1970). we suggest that the homogenization of contestation mirrors the transatlantic diffusion of rules (i.e. equalization of both experienced issues and anticipated fears), whereas the generalization of critique reflects the impacts of the meta-regime on the socio-material landscape. in this paper we have provided the first systematic empirical assessment of the co-evolutionary rulebased model of deep transitions [1] by studying the development of mass production in the transatlantic region between 1765 to 1972. our main findings are as follows: 1. in general the historical evolution of mass production matches the expected phase-specific patterns of the model. unexpected findings include the lack of empirical evidence of the role transnational actors in the frenzy phase, the importance of landscape dynamics throughout the entire surge and the role of rhetorical couplings in connecting different systems. 2. expected cross-phase patterns were also largely detected in the variation, diffusion and contestation of rules and rule-sets. unexpected findings include the emergence of alternative versions of the dominant meta-regime in niches even during synergy and maturity phases, the role of various forms of proximity and social differentiation in facilitating the diffusion of meta-regimes, and contestation mirroring the increasing scale, scope and societal embeddedness of the meta-regime. 3. landscape dynamics were found to support the evolution of the emerging meta-regime by a) stimulating new rules and rule-sets; b) amplifying existing rules and rule-sets; and; c) closing off alternative directions. the turning point event exerts all three types of impacts. 4. meta-regime building is facilitated by three mechanisms: rhetorical couplings, proximity and social differentiation. interestingly, we found that the optimistic expectations maintained by rhetoric and utopian promises of societal differentiation can counterbalance the considerable difficulties of implementing the meta-regime in practice. although our results have largely confirmed the dt model, the support offered by an exploratory qualitative case study remains tentative. the next step is to move from solely qualitative assessments to mixed method approaches, combining historical narratives with text mining techniques. this enables to quantify the emergence and alignment of rules but also to interpret the findings in a historically informed manner [blinded, forthcoming]. also, mass production constituted a case most likely to follow the theoretical propositions. further work is required to find out whether the model applies to more recent meta-regimes such as digitalization or circular economy. these cases also offer opportunities to look into the operation of mechanisms uncovered by our study, e.g. the fourth industrial revolution [117] as a rhetorical coupling connecting the digitalization of industry to the promise of sustainability, or societal differentiation as a mechanism connecting big data, ai, facial recognition software and social credit schemes to the chinese vision of 'harmonized' society [118, 119] . one can also analyse the role of landscape events in shaping these meta-regimes, e.g. the role of 9/11 or the current coronavirus pandemic in facilitating the societal embedding of digitalization towards increased citizen monitoring and surveillance in usa and china respectively [18, 120] . similarly to the early applications of the multi-level perspective the dt framework has so far primarily focused on the identification of broad patterns. although empirical work has begun on the role of transnational actors in shaping emerging meta-regimes [121] , van der vleuten [122] has argued for the need to broaden the analysis to 'system entanglers', producing cross-regional similarities and differences between systems. another theme which has not been tackled by transitions studies, long wave literature or the dt framework itself, is the continued evolution and transformation of mature meta-regimes during new surges. the interactions of mass production, it deep transitions: emergence, acceleration, stabilization and directionality analysing the breakthrough of rock'n'roll (1930-1970): multi-regime interaction and reconfiguration in the multi-level perspective boundary crossing innovations: case studies from the energy domain system interactions in socio-technical transitions: extending the multi-level perspective conceptualising multi-regime interactions: the role of the agriculture sector in renewable energy transitions governance of interactions between infrastructure sectors: the making of smart grids in the uk an agenda for sustainability transitions research: state of the art and future directions engaging with multi-system interactions in sustainability transitions: a comment on the transitions research agenda deep transitions: theorizing the long-term patterns of socio-technical change technological revolutions and financial capital: the dynamics of bubbles and golden ages as time goes by: from the industrial revolutions to the information revolution technological transitions as evolutionary reconfiguration processes: a multi-level perspective and a case-study technological transitions and system innovations: a co-evolutionary and socio-technical analysis co-evolution of technology and society: the transition in water supply and personal hygiene in the netherlands (1850-1930) -a case study in multi-level perspective major system change through stepwise reconfiguration: a multi-level analysis of the transformation of american factory production (1850-1930) transitions to sustainable development: new directions in the study of long term transformative change modularity: an interdisciplinary history of an ordering concept the age of surveillance capitalism new service opportunities to create new customer benefits eyes on the road: surveillance logics in the autonomous vehicle economy technological revolutions and techno-economic paradigms multi-regime dynamics in the analysis of sectoral transformation potential: evidence from german utility sectors finance and technical change: a long-term view case study research and applications: design and methods, 6 th edition case studies and theory development in the social sciences case study research. principles and practices 2010. rethinking social inquiry. diverse tools multi-method social science a new green history of the world: the environment and the collapse of great civilizations the industrial revolution in world history, 4 th edition contributions of socio-metabolic research to sustainability science the dynamics of socio-technical transitions: a socio-technical perspective rethinking the multi-level perspective of technological transitions explaining sociotechnical transitions: a critical realist perspective understanding successive industrial revolutions: a "development block from the american system to mass production, 1800-1932: the development of manufacturing technology in the united states the rise and fall of mass production, 2 volumes america's assembly line the emergence of modern business enterprise in france building europe on expertise: innovators, organizers between fordism and flexibility: the automobile industry and its workers past, present and future how the toyota production system was created endless novelty: specialty production and american industrialization innovation and amnesia: engineering rationality and the fate of interchangeable parts manufacturing in france harpers ferry armory and the new technology: the challenge of change the control revolution: technological and economic origins of the information society american genesis. a century of invention and technological enthusiasm managerial control of american workers: methods and technology from the 1880s to today the engineered factory popeism and fordism: examining the roots of mass production pioneers, engineers, and scoundrels: the dawn of the automobile in america the future of automobile from mass production to flexible/agile production the automobile: a chronology of its antecedents, development, and impact ford eagle boats and mass production during world war i a history of world agriculture: from the neolithic age to the current crisis the failure of fordism: reform of the automobile repair scientific american. 02.1939. dated chimpanzees -by enterprises and b phases: the overseas expansion of u.s. auto companies in the 1920s and japanese auto companies in the 1980s economic culture and its transfer: americanization and european enterprise the american connection: ideology and the transfer of american technology to the soviet union transnational fordism. ford motor company, nazi germany, and the soviet union in the interwar years. doctoral dissertation adolf hitler vs. henry ford: the volkswagen, the role of america as a model, and the failure of a nazi consumer society the speedup: the focal point of workers grievances, 1919-1941 the reichskuratorium für wirtschaftlichkeit: fordism and organized capitalism in germany redesigning the workplace: the north american factory in the interwar period ford principles and practice at river rouge. xiii -manufacturing and assembling body parts in collaboration with wood, c. w. 1931. successful living in this machine age between taylorism and technocracy: european ideologies and the vision of industrial productivity in the 1920s the pursuit of power: technology, armed force, and society since a.d. 1000 flexibility and mass production at war: aircraft manufacture in britain, the united states, and germany, 1939-1945 the rise and fall of american growth made in the usa: the rise and retreat of american manufacturing world war ii manufacturing and the postwar southern economy industrial innovation and productive structures: the creation of america's 'arsenal of democracy supplier networks in the german aircraft industry during world war ii and their long-term effects on west germany's automobile industry during the 'wirtschaftswunder' reshaping the landscape: the impact and diffusion of the tractor in american agriculture, 1910-1960 the role of war in deep transitions: exploring mechanisms, imprints and rules in sociotechnical systems the global history of organic farming empire of things. how we became a world of consumers, from the fifteenth century to the twenty-first europe's golden age what makes the toyota production system really tick how toyota shortened supply lot production time, waiting time and conveyance time from the ford system to the just-in-time production system. a historical study of international shifts in automobile production systems, their connection and their transformation the genealogy of lean production the machine in america: a social history of technology americanization and its limits: reworking us technology and management in post-war europe and japan the socialist people's car. automobiles, shortages and consent in the czechoslovak road to mass production the socialist system: the political economy of communism world-scale patterns of labor-capital conflict: labor unrest, long waves, and cycles of world hegemony labour unrest in the united states the present human condition an analysis of mexico's border industrialization program the new international division of labour: structural unemployment in industrialized countries and industrialization in developing countries global inequality: a new approach for the age of globalization social choice in machine design: the case of automatically controlled machine tools and a challenge for labor beyond mass production: the social organization of production and innovation in japan triumph of lean production system domesticating the cold war: household consumption as propaganda in marshall plan germany the metabolic transition of a planned economy: material flows in the ussr and the russian federation the chat dataset one-dimensional man: studies in the ideology of advanced industrial society the technological society the making of a counter culture: reflection on the technocratic society and its youthful opposition the myth of the machine, 2 volumes silent spring the limits to growth: a report for club of rome's project on the predicament of mankind consumers, tinkerers, rebels: the people who shaped europe writing the rules for europe. cartels, experts and international organizations proximity and innovation: a critical assessment proximity and innovation: from statics to dynamics proximity, knowledge base and the innovation process: towards an integrated framework distinction: a social critique of the judgement of taste an investigation into the social context of early adoption behavior sustainability transitions and final consumption: practices and sociotechnical systems forging global fordism: soviet russia, nazi germany, and the contest over the industrial order constructing and data-driven society: china's social credit system as a state surveillance infrastructure china's "politics of harmony" and the quest for soft power in international politics the new normal': china's excessive coronavirus public monitoring could be here to stay adopting and diffusing the circular economy as a policy concept: the case of the european union radical change and deep transitions: lessons from europe's infrastructure transition 1815-2015 this article has been discussed with various colleagues at the international sustainability transitions 2018 conference and in a series of workshops, including the deep transitions research team and invited guests. in particular we would like to thank johan schot, ed steinmueller, carlota perez, david nye and phil scranton for their informative comments. this work was supported by james anderson and baillie gifford & co.j o u r n a l p r e -p r o o f key: cord-354941-0ocsf255 authors: amorin‐woods, deisy; fraenkel, peter; mosconi, andrea; nisse, martine; munoz, susana title: family therapy and covid‐19: international reflections during the pandemic from systemic therapists across the globe date: 2020-06-08 journal: aust n z j fam ther doi: 10.1002/anzf.1416 sha: doc_id: 354941 cord_uid: 0ocsf255 the covid‐19 pandemic has convulsed human communities across the globe like no previous event in history. family therapists, paradoxically, given the core of their work is with systems, are also experiencing upheaval in professional and personal lives, trying to work amidst a society in chaos. this paper offers a collection of reflections by systemic and family therapists from diverse cultures and contexts penned in the midst of the pandemic. the main intention in distilling these narratives is to preserve the ‘cultural diversity’ and ‘ecological position’ of the contributors, guided by phenomenology, cultural ecology, and systemic worldviews of ‘experiencing.’ the second intention is to ‘unite’ promoting solidarity in this isolating situation by bringing each story together, creating its own metaphor of a family: united, connected, stronger. as a cross‐cultural family practitioner, with a strong mission for collaboration, the lead author acknowledges the importance of context – the nation and location of the experience; culture – the manner in which culture impacts on experience; collaboration – enhancing partnership, enriching knowledge, and mapping the journey’s direction; and connectedness – combating isolation while enhancing unity. since the key transmission of culture is through language, raw reflections were sought initially in the practitioners’ own language, which were translated for an english‐speaking readership. these narratives are honest and rich descriptions of the authors’ lived experiences, diverse and distinctive. the contributors trust colleagues will find these reflections helpful, validating and acknowledging the challenges of this unique period in history. deisy amorin-woods 1 this compilation of reflections, while not a research project per se, is written in an auto-ethnographic style (ellis & bochner, 2000; rhodes, 2018) by five systemic therapists from varied cultures and contexts about the covid-19 pandemic. their narratives, experiences, perceptions, ponderings, and feelings were composed as they were living the experience; unique in their meaning making and in the way they contribute to this collection. as the lead author, i live and practice in perth, australia. reflecting my international and collective soul, my curiosity has given me the impetus to facilitate global exchange and initiate cross-cultural conversations. peter fraenkel, in new york, united states, incorporates his buddhist insight and psycho-musical intervention to provide a message of connection and hope to help others preserve a capacity for joy. andrea mosconi, from padua, italy, provides students and colleagues with a gentle message of wisdom, and insights to stimulate critical thinking and opportunities for new ways forward. martine nisse, from paris, france, delivers a 'mood note' which introduces a sense of 'lightness' in the midst of her complex and challenging work with domestic violence and incest. susana munoz from santiago, chile, with a background as a midwife, uses the metaphor of labour and birth to demonstrate survival. this 'pandemic project' initially arose mid-march as i reached out to colleagues abroad to check how they were navigating through the pandemic chaos. through our 'cross-cultural' conversations, the sense of feeling understood through shared experience and empathic means led to strengthened connection. this gave me the impetus to ask myself: wouldn't it be helpful if we could write something together to find some healing, whilst sharing our stories with others, in order for them to also find healing? i realised this was a global crisis; a collective narrative unfolding daily which has left no one untouched, and we as family therapists had important and individual stories to tell. time was of the essence, the view from this window was unique and so i drafted a brief. given my strong interest in collaboration and collaborative practice, over the years i had fostered relationships and developed partnerships including with family therapists abroad (amorin-woods, 2019). thus, i reached out to this wide network of colleagues and invited them to join me in telling our stories. most i invited responded enthusiastically to this initiative almost immediately. regrettably, due to the tight deadline, a few were understandably overwhelmed with other priorities. as a cross-cultural family therapist, my mission is to be diverse and inclusive when meeting the varied cultural needs of my clients, developing programs, delivering services, and working with families. this project was no different. as i heard the international collective voices it was thus vital to me that contributions were garnered from a cross-section of nations and continents as i wanted to honour different cultural backgrounds and language representation. i believed that by tackling phase 1sharing our experiences, we could capture each of our cultural and ecological worldviews and thus unite in this process. then, we could contemplate stepping into phase 2regenerative process in order to rebuild our own lives post-pandemic. after we are given the space to process the raw and organic heaviness of our own experience, we are able to support our clients to come up with practical responses, but not before. it was critical to put the stories together before the 'immediacy' of the raw accounts was lost. we are in the midst of the experience, in 'situ,' in confinement, confused, hypervigilant. we live in a world where we seem to 'rush' to come up with labels, treatments, and solutions to human distress, often bypassing our lived experience of suffering, loss, and isolation. i felt as if these early collections merged into one, represent the 'holding, healing space' that we as therapists need, before we dare consider what life may look like on the 'other side.' without this, how could we support our clients in putting themselves, their families, and their lives back together, in even contemplating what a world of 'functionality' or 'normality' may look like? while family therapists are aware of the importance of 'unmasking' to remain authentic to our clients and to self-disclose to deepen connection and trust, therapists do not often have the chance to share their accounts as they too travel through challenging life experiences. these reflections provide that opportunity. finally, any conversation about culture must include the element of language. since the key transmission of culture is through language, organic reflections were sought in the practitioner's primary language. as in therapy, authenticity of feelings and vulnerability cannot be truly transmitted except in the primary language (amorin-woods, 2020). however, since the main demographic of this journal speak english as their primary language, these reflections are translated and offered in english. i am deeply appreciative to each contributor for responding as i reached out. from the bottom of my heart, thank you andrea, peter, martine, and susana. deisy amorin-woods, perth, australia this pandemic is a collective trauma event and its multi-faceted impact cannot be underestimated. this leads us to first acknowledge and respect the interconnectedness and relationship between systems, within our body, between us and our environment, and between one another. this also causes us to question the notion of permanence versus impermanence and certainty versus uncertainty. as a cross-cultural family and systemic therapist, i have developed a thirst for collaboration within programs, across organisations, within communities, and across the globe (lee, thompson, & amorin-woods, 2009 ). as a migrant to australia from peru, i have always felt drawn to the idea of collectivism (amorin-woods, 2020), a sense of 'being together' and of 'joining with the other,' and that notion has never left me. whether working in government, non-government organisations (ngos), in private practice, or in academia, i have ensured i bring my 'collective soul' into my profession in the way i practice and in my teaching. i try to connect with the issues that impact on the lives of our families regardless of where they are located or where they originate (amorin-woods, 2016a , 2016b . the issues are varied and the systems diverse, whether a couple facing grief, families with intergenerational trauma, or refugees who have fled persecution. when the news broke in early 2020 about a possible epidemic, i had not conceptualised either the extent or the utter devastation that was to follow. i was due to travel to basel, switzerland in early march to present at the world congress of family global family therapists reflect on covid-19 ª 2020 australian association of family therapy therapy under the international family therapy association (ifta). ironically, my presentation was titled, therapeutic conversations and trauma informed systemic practice: acknowledging meaning making in the backdrop of relentless fear and unpredictability. two days before my departure, the conference was cancelled due to the pandemic, which was rapidly spreading around the globe. in the weeks that followed, the whole world convulsed through a rapid period of change. the need to find refuge from this potent, yet invisible, force left us vulnerable, confused, distressed, fearful, and ambivalent. in my own space, due to the need to isolate, and the closure of state borders, i have not been able to see my daughters or embrace my little 'grandies.' this has left me profoundly sad and nostalgic. having family, friends, and colleagues around the world, many located in the countries worst hit, i was driven to connect, to check-in on how they were navigating through the crisis. i heard directly their accounts of anguish, dread, panic, powerlessness, loss, and grief. this virus was burning, just like the recent ignition of highly combustible dry eucalyptus trees in the australian bush, spreading rapidly like a wildfire, loud and forceful, the echoed thoughts keeping alive the nagging fear. people wondering, will i be next? will i be a carrier and infect my loved ones? families losing parents, grandparents . . . a whole generation seemingly vanishing as in a puff of smoke. listening to these raw and distressing stories became rather confronting. i found myself holding their collective pain in my arms and with a virtual embrace. i was attempting to ease their suffering. personally, these stories transported me back to a similar experience as a child living in peru. this was a time of 'sendero luminoso' (shining path) a guerrilla group, along with 'sinchis' an anti-subversive 'police sub-group,' both of whom were responsible for committing human rights violations, terrorising and decimating over 70,000 people, most of whom were indigenous. this led to cases of intergenerational trauma, the stories of fear, sorrow, and confusion passed down as through the 'mother's breast milk' for generations. these painful memories accumulate in one's body becoming a historical site which is transported through time (rueda, 2015) . i remember my mother distraught as the situation evolved given my sibling was studying at the university at the epicentre. while not directly impacted by the war and the trauma, i felt them indirectly, yet latently, through the suffering of my mother and the stories told by my sibling who witnessed young students being murdered. this brings me back to the present time and leaves me concerned wondering whether we will see stories of pain and sorrow passed down as a result of the covid-19 war? will this generation transmit trauma to those to follow? professionally, as the epidemic evolved, so did the need to transition my practice from face-to-face to purely tele-health. while australia has been fortunate not having been impacted as heavily as other countries, i have observed among my clients signs of anxiety and hypervigilance related to the fear of transmission, as well as new manifestations of suicidal ideation previously absent. these signs are connected to a number of issues: the stress related to job loss and financial uncertainty; the sadness and loneliness linked to the inability to see family and friends; grandparents' sorrow not seeing their grandchildren, an embrace appearing so distant. children unsettled, mothers feeling stressed and exhausted juggling extra roles, including home-schooling. while some people see isolation ('iso') as simply confinement, others see it as imprisonment, fearing the threat of imminent danger if leaving their safe and familiar bubble. for others the uncertainty, the not knowing, the unpredictable re-organisation of families and communities, is too much to bear and leads to heightened levels of suicidal ideation. people ask themselves: will there be an end to this? are we facing the end of the world? there is documented concern for heightened risk of suicide among communities continuing past the pandemic (gunnell et al., 2020) . further to this, given my extensive experience working with people with acute mental health issues and with complex trauma, i am aware that people who have experienced trauma are highly impacted by threats about the safety and stability of the world (brown, 2009; james & mackinnon, 2012) . i have also observed how vulnerable families (such as families with intergenerational trauma and child abuse survivors) are particularly at high risk, and consequently presenting with clear signs of re-traumatisation. throughout this pandemic transition, i have noticed various community stances rapidly forming. one that blames, with xenophobic undertones, pointing to race and culture as the original cause of the virus, discriminating against certain cultural groups. another that labels, describing people as 'paranoid' or 'hysterical' as people frantically accumulate food supplies or simply voluntarily decide to self-isolate. a third, with traces of denial and avoidance about the existence or magnitude of the situation, that judges and ridicules those taking a more conservative approach. within this stance, there is an underlying expectation and pressure from strangers or loved ones to 'move on' rapidly through the stages of grief (kubler-ross & kessler, 2009) from shock and denial to acceptance, without recognition that people need to be allowed to 'feel their feelings.' on the other hand, i have also seen demonstrations of kindness, generosity, and solidarity for fellow community members, much of which has been led by children. teddies perched in trees, messages of encouragement and hope written around neighbourhoods to lighten the tone and brighten people's days. boxes of fresh produce or non-perishables left to help others financially disadvantaged. i have observed some people viewing this pandemic as an opportunity to start over and live in a healthier, humble, more acknowledging and respectful way towards self, towards family and their environment. i have seen occasions when cut-off families have re-connected, and couples considering separation have learned to appreciate and not take each other for granted. i have been mindful of being available for my clients, to provide the holding space to acknowledge and validate their feelings and allow them to process their experiences. the need for self-care and self-compassion takes on a new meaning in its recognition of our shared humanity. i thus ponder: are we able to provide clients with a holding space to process things if we are not connecting with ourselves? . . . even though we too are living amidst the chaos. we do not have to abnegate ourselves or our experiences and feelings to show we are capable practitioners, because when we abnegate ourselves we tell our clients to let go of parts of self in order to function, in order to accept themselves, in order to relate. we need to 'be,' before we can 'do.' remaining authentic is key in therapy. i often say to my students and supervisees, authenticity is to a therapist what breath is to life. we need to be authentic with the families who come to us with hope and trust and this includes possessing heightened awareness of ourselves, hence the need to uncover the 'self'. i recall a similar description often during my fellowship at the accademia di psicoterapia della famiglia in rome. andolfi would often make reference to ridding self of the mask and being fully self, fully human (andolfi, 2012) . it is only through such a process that we arrive to a place of recognition of what is ours and what is theirs. it is crucial that we are able to name and process our experiences in order to support our global family therapists reflect on covid-19 ª 2020 australian association of family therapy clients to do the same, in order to provide the holding space they need, and joining-in with them (minuchin, 1974) . this then allows us to welcome the rich exchange between each other in order to develop an empathic connection and trusting relationship with them (aponte, 1992) , because how can we join in with families and trust be elicited without this important element? this also helps not to transfer or project our experiences onto our clients (lum, 2002) . as systemic therapists we are in a privileged position as collaborators and partners influencing the family system and the environment and context where families are placed. this gives us the opportunity to awaken deeper knowledge and understanding within a given system, within the family who puts their trust in us. we become responsible in co-creating and co-constructing the therapeutic reality and in eliciting change (von foerster, 1981) . we influence the environment whether we bring empathy, healing, anxiety, or fear. understanding the self and understanding each other becomes the impetus for healing. around the globe, we are all living the experience of the covid-19 pandemic as a collective trauma. the chaos is real to us all. we are dealing with challenges that are comparable yet distinct. while we are all impacted, the impact is different depending on where we are located, in our culture, our ecological system, and the politics of our day. a frequently used expression suggests we are all in the same boat; however, i would like to use the metaphor, while we may be sailing the same storm, we are in different boats. how robust our ship is, and how we manoeuvre it, may determine how we survive the storm. in writing this i am mindful of families as they navigate through this pandemic. i am interested in supporting them to acknowledge their being 'human,' while rejecting reductionist ideas of 'experiencing' that rush them to numb or bypass their experience. instead, it is time to pause and connect with the basics, our relationships. the lesson from gregory bateson was the importance of the interconnectedness of living things to their natural environment (bateson, 1979) . this is so relevant to our current situation. we hear loudly the desire of communities to go back to 'normal' . . . however, do we really want to go back to the normal that we knew? or do we want to look at this as an opportunity for social change? for a change in direction? in the way we do things, in the way we relate to self, to other people, to other cultures, to our environment. instead let's nurture families and preserve relationships. let's propose a paradigm shift about the way we think of ourselves not as passive closed-off beings, but as active authors of our life, insightful, creative, purposeful, and connecting. let us use this time to co-create a space for healing and holding the human spirit. coping with covid-19: a time to focus on the simple gifts of life peter fraenkel 2 , new york, united states as news of the covid-19 pandemic quickly grew in march of this year, i immediately found myself re-immersed in the traumatic disorientation of 11 september, 2001. terrorist planes crashing into the world trade center launched new york city and the united states more generally, into what i termed 'the new normal' (fraenkel, 2001b) . yet this is different: an invisible virus with an unpredictable course, unseen yet life threatening. where to go? what to do? the answers to those questions quickly were determined in march 2020 by the governor of new york with guidance from federal and state health experts: go home, work at home. on 13 march, i delivered the last in-person workshop at the ackerman institute for the family. attendees spread out across the room as a safety precaution to decrease the likelihood of communicating the virus among us. none of the usual handshakes between newlymet colleagues, none of the usual hugs between old friends, my former students, and professional acquaintances. we carried on, focusing on effective techniques in couple therapy. but we all had a nagging sense, mostly at the periphery of our consciousness, that the world outside was growing increasingly fearsome, and that we were better off in here, held by the warm embrace of what mary pipher has called the 'shelter of each other.' as i wrote in another article after 9/11, we therapists were fortunate to be helped by the act of helping othersto belong to a community of care (fraenkel, 2002) . i've specialised for many years in work with families traumatised by incest, domestic violence, and homelessness. in my private practice, i've specialised in what i've called 'last chance couples'those who've often seen one or more couple therapists, without much improvement, and who are now on the brink of divorce or its nonmarried equivalent of relationship dissolution (fraenkel, 2019) . these two areas of my work now overlap: distressed, disengaged partners already strongly considering separating, or already living apart, now stuck together with kids and sometimes their elders and even other families. precipitous layoffs and loss of income, infection with the virus, and in some cases, the known death of relatives, friends, or colleagues, have led to the kind of traumatic effects of living through a tsunami, gathering force every day with no end in sight. at the same time, accurate information, guidance, and leadership from the federal government is sorely lacking, alarming in its own wayno comfort there. my clients are experiencing all the usual symptoms of psychological trauma: intrusive thoughts and nightmares, edginess and hypervigilance, sleeplessness, anxiety, and depression, as well as relational trauma and ruptures between couple partners, and among family members. the factor most clearly correlated with resilience in the face of trauma and other disruptive experiencessocial supportis in short supply, despite inventive use of social media and online meetings, including teletherapy. i have occasionally ventured into a nearby park with my percussion instruments to record myself playing along with inspiring songs about social justice and love, posting these videos on facebook and instagram. the response has been overwhelmingly one of gratitude and unexpected pleasure, and those responses have gratefully been received by me, giving me a slight sense that through my 'out-of-the-box' psycho-musical 'intervention,' i've helped others preserve their energy, hope, and capacity for joy. but i then return to my apartment, to be alone once again, as my kids are now attending college in europe. we're in close touch, as i am with friends and colleagues, but i surely feel isolated; and when i occasionally venture out and on to the streets to shop or bank, there's the uneasy, unreal sense of being on another planet, or at least, a familiar but altered landscape, with fearful masked neighbours strolling six feet apart. coincidentally, just a few weeks prior to news about the coronavirus, i had started writing a book for the general public about lessons to be learned from taoism about love and other relationships (fraenkel & akambe, 2020) . as a long-time buddhist and taoist, i've practiced noticing the little, familiar things and accustomed events of life that can bring unexpected joy once brought into the gentle gaze of the 'now.' for decades, i've tried to live my life as if inside a haiku poem, taking pleasure just from being sensate, breathing, and alive. musing on the nature of existence, the creator of taoism, 6th-century b.c. court librarian, philosopher, and social critic lao tzu, writes in the first passage of his classic text, the way of life (6th century bc): existence is beyond the power of words to define terms may be used but are none of them absolute . . . if name be needed, wonder names them both from wonder into wonder existence opens i've been advising all my clientsindividuals, couples, and familiesto use this disorienting time to reflect upon the following taoist suggestions, to look for opportunities to experience wonder, just as i have as a fellow traveller through this time. specifically, i've suggested they: look for pleasure and joy in small, everyday experiences. i took a break from writing this article, went to the kitchen window in my fifth-floor apartment, and watched the rain falling upon the black-painted fire escape. it was beautiful. a few hours later, i took another break, and saw my two kittens, whom i adopted from a shelter in october, cuddled together in my furry chair, sleeping contentedly. it was beautiful. it calmed me down. slow down. our usual life pace in nyc is frenetic, our lives overstuffed with aspiration, achievement, a never-ending quest for riches, possessions, and when we've accumulated enough of those, the relentless search for new experiences (which i've termed 'experience greed'), for unique travel destinations and novel experiences to empty our constantly refilling 'bucket list.' lao tzu (6th century bc) writes: gravity is the root of grace, the mainstay of all speed . . . what lord of countless chariots would ride them in vain, would make himself fool of the realm, with pace beyond rein, speed beyond helm? it's a time to stop, and look closely at what and who we already have in our lives. it's a time to breathe, walk, eat, and talk slowly, compassionately, and patiently with loved onesthose living with us or those whom the virus has separated from us. and while slowed down, take a good long look at your partner, your child, your parent, your friend, your neighbour, your colleague (online, most likely!)listen to their voices, and enjoy their unique aliveness. in the timeless words of songwriter cole porter and a song made famous by frank sinatra ('i get a kick out of you'), let yourselves 'get a kick' out of them (fraenkel, 2001a) . look at them like there's no tomorrow for them, or for you. let go of control. on a related note, we cannot definitively avoid illness, and we cannot insure through our hand-washing and social distancing and mask wearing that we will survive this pandemic, or at least, leave it unscathed health-wise, financially, or emotionally. nor can we avoid the suffering of learning that a family member, a neighbour, a student, a mentor, or a colleague is sick, or has died. radical humility is called for in this troubling, unpredictable time. take precautions, yes, but recognise that this virus is nature, and nature has its way of overcoming even the best-laid plans of mice and men. learn from persons who inhabit oppressed social locations. lao tzu (6th century bc) advocated wisdom over knowledge: leave off find learning! end the nuisance of saying yes to this and perhaps to that, what slightest use are they! if one man leads, another must follow, how silly that is and how false! communities that have experienced generations of oppression due to race, ethnicity, geographic region/country, culture, and economic status have much wisdom to share about surviving and even thriving under hardship, and about how to make do with few resources. one hysterically funny video that circulated around the internet early in the emergence of the coronavirus came from the philippines, in response to privileged westerners' panic about running out of toilet paper. it was a lively music video, with dancing women behind the male lead singer, demonstrating how to use a little colourful plastic pot called the 'tabo' to wash up after defecating. it's high time we erase the terms 'first world' and 'third world' and the power and knowledge hierarchy between the countries and cultures belonging to each politically constructed group. the world must come together to defeat this virus, and to survive, as a unified, mutually respectful people in harmony with the earth and nature itself. my mother mimi bialostolsky fraenkel, who died of cancer in 2001 just before 9/11, believed the world might eventually come together in a time she called 'creative chaos.' i think of her now, and whether this worldwide pandemic is enough to unite us in a common effort to live on. i'd like to hope so . . . but i'm not sure. what we are going through is not an ordinary crisis, this is an epochal crisis! it seems to me that it is the most obvious consideration which echoes with many. the question then may be the systemic one: what do we do in each situation for every person who presents to us with a problem? as i often say, a kind of systemic mantra: perfection, what does this allow me to learn? it is now clear that this virus has three aspects that makes it scary: 1) it is new and we cannot be immunised; 2) it also passes through healthy carriers who, even if they have a lower viral load, are still capable of contagion and therefore cannot be easily controlled; and 3) it is not a flu virus like most flu viruses. this virus directly attacks the lungs and therefore gives viral pneumonia; it also attacks many other global family therapists reflect on covid-19 ª 2020 australian association of family therapy systems in our body. from this point of view, respect for the required government measures is essential as the only defence, while there is no effective drug or vaccine. the alternative is to build herd immunity, through a selection of the species. only those with resistance will survive. on the level of interaction between this virus and the social system, the question is: how long will it last? if we take into account the date of government measures, the fact that the need to tighten up has often been felt, and also the fact that it is always difficult both to impose and to accept strict rules and that the incubation time is 15 days, it is not difficult to hypothesise that the so-called 'peak' of the infections is still to be reached. from there, it takes time for the sick to heal and for additional time to pass before the virus is eliminated by the carriers. by putting all this together, i believe that we must accept that the current situation will last a long time. epidemiologists seem to be moving increasingly in this direction, obviously, barring unforeseen events for the better and for the worse. let's have patience! however, let us look wider, at the macrosystem where all this happens. if we take into consideration the relationship between us, 'homo sapiens,' and the natural system into which we are inserted, this story changes some rules of the relationship. it allows us to realise that we are not omnipotent, but that we are part of a system that can destroy us in a short time if we do not take into account the feedback that comes from the interaction with the other elements of the system itself. its 'liberation' is part of this process of inappropriate invasion by us 'sapiens' of natural contexts not coordinated with the human system. from this point of view, this virus is a warning, it is almost a homeostatic mechanism that is produced in a system that is already at the limit of its range of possible interactions and is reaching a possible 'bifurcation point' (prigogine, 2001 p. 160) . this is a test of a general catastrophe, a global crisis. basically there are other 'covid' around which we do not want to be aware of and which can equally undermine our survival in a global way: global warming, pollution, deforestation, desertification, water consumption, economic imbalances within societies and between different parts of the world. just like the case of 'covid,' we pretend not to see them yet they advance in a hidden and creeping way until they explode. just like covid-19! the 'good' thing we can say about the virus is that, unlike wars which can be taken out on someone who is considered guilty, the same cannot happen as this virus does not discriminate; it is a common enemy of the whole system. the virus applies equally to everyone, just like in the family systems with which we want to work. just as the members of a family system at the end of a session should not be able to say who is right or who is wrong (selvini palazzoli, boscolo, cecchin, & prata, 1980) , so as humans we are driven to look at each other, to confront each other, to even try to understand and help those, who until yesterday, rejected each other. yes, there are those who, not understanding, continue to wave the flag of personal interest, or hunt the culprit, or act in their own defence, but for now they are exposed and forced to admit or to try to make amends. for now, solidarity prevails, and there is admiration for the commitment and generosity of those on the front line, a sense of being a community. and we see the increasingly clear signs that, where man takes a step back, the natural system of which we are part takes a breath, pollution decreases, and everything seems to show us what we must consider for development and the future. a great opportunity, don't you think? but several times i have said: for now . . . the real question is: will we be able to take this into account? this brings another consideration regarding the social system. in recent years, perhaps blinded by the race for well-being or perhaps because well-being itself had allowed many to take advantage of it incorrectly even in public structures, the focus has been rebalancing national budgets by destroying the network of structures that had functioned as the skeleton, blood, and neurological system of society by supporting and bringing food throughout the social system. i am talking about healthcare, public education, welfare . . . let's consider what it means not to have continued to be aware of the value of these 'elements' of the social system. i say this both for those who have dishonestly taken advantage of it from within and for those who, from their own ideology, have fought them. here, this crisis can help us rearrange the scale of values regarding what is most important to preserve in a social system, counterbalancing the excessive importance given to the production system, to take into due consideration what is at the basis of a possible safe and civil coexistence. even regarding the enormous development of the computer network that is becoming more and more the neuronal network of the planet: on the one hand it allows us to accelerate the feedback between parts of the system facilitating the possibility of co-building solutions; on the other hand tomorrow it will put us in the face of problems such as the enormous possibility of controlling, even more than now, individual lives and determining who and how to manage the enormous power that all this enters . . . and in whose hands? of course, it is always the two polarities of the life of a system that must be balanced: the competitive system and the collaborative one. the same goes for natural selection which is the survival of the fittest and the other towards the idea that the average good of the most is better than the maximum good of the few. but here the question is: will we remember it later? and what does all this say about our systems of daily interaction, to our closest systems? of course, stopping puts us in a position to change our position in the systems of which we are a part and the relationship rules change. we feel distant from those who were close to us, communicating virtually with those we used to touch, look in the face, caress, shake hands, pat on the shoulder, take by the arm. in contrast, we find ourselves living with those who we did not have close to us for so many hours or who we were even in conflict with, seeing him/her every day and maybe having to talk to each other. we can no longer take advantage of dissipative structures, as they are called, which diluted and differentiated, distributed the tensions of the systems: work, travel, school, various activities. maybe if we choose to use the avoidance mechanism that is so useful to delude ourselves that there is a balance in relationships? no, we can't back off now, we're in touch! and then? this is an opportunity to stop and listen to ourselves and others, try to look at them with different eyes, re-appreciate the small gestures, let things arise from building together and patiently look for solutions, not problems. so, in the infinite space we can perhaps rediscover the value of everyday life, of silence, of simple things, of doing together, of allowing our imagination to bring to mind an idea. sometimes it's simpler than it seems! and with our patients . . . 'clients?' here too our position changes. from time to time we may find ourselves inventing different ways of making ourselves feel close, exploring new and different tools for staying in relationship and offering help, believing that, beyond the tool used, the relationship is what matters. cecchin's words echo on us: we all need to feel seen, and to feel seen we are willing to invent all the colors! (cecchin, 1987 p. 410) . and then of course: hypothesis, circularity, and neutrality! (selvini palazzoli et al., 1980) . so, our patients will feel us present, even if in a different way, and they will appreciate our sincere and attentive commitment. as milton erickson (erickson & rossi, 1979, p. 87) said: there are no difficult or incurable patients, there are only therapists who are able or unable to find a way to communicate with them. another thing comes to mind. let's see how a small virus, which affects the system at a specific point, achieves great effects. this can be a teaching for our therapy. the 'saltology' mentioned by the milan team in the first pages of paradox and counterparadox comes to mind: the results confirmed that, when it is possible to discover and change a fundamental rule of a system, pathological behavior can quickly disappear. this leads us to accept rabkin's proposed idea: that in nature events of radical importance can happen suddenly when a fundamental rule of a system is changed. rabkin proposes the term "saltology", that is saltology (from the latin saltus) for the discipline that should study these phenomena. this finds its correspondence in the general theory of systems whose theorists speak of "p.s." as of that point of the system on which the maximum number of essential functions converges to a system, changing when obtaining the maximum change with a minimum of energy expenditure. (selvini palazzoli, boscolo, cecchin, & prata, 1975, p. 12) so, these are my reflections these days which i wanted to share. all this 'allows us to learn' and perhaps finally arrive. it is an opportunity to become aware what this covid virus . . . offers us as systemic therapists. let's go ahead, let's build better times together! small mood note from a french family therapist in times of pandemic (petit billet d'humeur d'une th erapeute familiale franc ßaise en temps de pand emie) the family therapy sessions i conduct are very rarely by videoconference. when they are, they involve my patients who, for one reason or another, have gone abroad to settle temporarily or permanently. but since the arrival of the pandemic in france, one of my patients, herself a psychotherapist and familiar with consultations by videoconference, has pushed me towards this mode of therapy. two days after the announcement of the confinement, i was equipped, and i launched the invitations to my patients. after researching what angle of view for my webcam would symbolically give an open message about the future, i decided to turn my back to my patio, so that the view behind me would lead them towards the sun and the plants. the weather is extraordinarily nice, the pollution in paris has dropped dramatically, the sun is shining without any halo of pollution. birds are heckling in the city. i half draw two curtains, in the colour of my therapy room, which has been abandoned since the confinement, and adjust lights towards my face to gently counterbalance the brightness of the outside. it is, finally, a kind of 'chiaroscuro' (light/dark) that makes my first affected and surprised patients say, keep going . . . it reminds us of vermeer . . . the surprises are mutual, i arrive directly in their living room, or their office, even if they have passed through my 'virtual waiting room' of zoom. i note that some have asked their spouse to use their one-hour right of exit during the consultation to be quiet, others are gathered on the living room sofa, a cup of tea in hand with their pet. sometimes a decorative detail jumps out at me, so 'connected' with the patient's problem that i cannot avoid referring to it. one of these surprised patients says to me, do you want me to show you where i live? i pose and respond, no thanks. or . . . maybe . . . yes, you could ask all your patients to show you their home, you could learn a lot from that? . . . don't you think so? . . . mmm, no, i don't think so. holding the structure is one of the signatures of family therapy. most of my patients have been exposed to violence that has broken into their psyche (nisse, 2020; nisse & sabourin, 2004) . re-establishing boundaries or maintaining distance from others requires an ongoing effort. it requires constant therapeutic adjustment. i find that they are grateful for the availability of their therapist. i find that after the first few sessions, i feel as if i am regenerated. despite the shock of this epidemic, i have not forgotten anything about my way of being a therapist with each of them, nor their family history. my abilities as a therapist seem to be naturally at my disposal. the new and artificial proximity of the screen requires me to be attentive to maintain a therapeutic atmosphere (nisse, 2020) , in conformity with the pre-existing one: that is to say, intimate and respectful at the same time. i also note that since the beginning of the pandemic their psychological work seems to be more productive between sessions. some patients have refused videoconference sessions. they cannot imagine hearing themselves or me talking at home about the violence, especially sexual violence, for which they have consulted; they don't have housing where it's possible to really isolate themselves, or they are single women raising their children. sometimes, a patient who has not responded to the offer checks in with her therapist with a fear of illness. some of them tested positive for covid-19. it is nothing too serious, but a great deal of fatigue and fear for the impact on others. i don't have a pre-established bilateral agreement for the therapeutic meeting by videoconferencethis bothers me a little, but i know my patients, i trust them. it will be possible to establish it afterwards for the next sessions. the french or european family therapy societies provide support, stimulate reflection on this subject, offer platforms for exchange with family therapists, or platforms for helping families stressed by covid-19 or affected by it and also affected by sudden bereavement. a large part of our patient population is made up of children who have been placed in care by the juvenile judges. they come from all over france, and half of the country is currently in the 'red zone,' which means that children are not allowed to go to school or to travel more than 100 kilometres to go to court. as for each of the people living in this 'red zone,' sometimes, as i know from the supervisions i give by videoconference, a certain number of them paradoxically relax knowing they will not receive any more parental visits, mediated at the same time . . . recently, the status of social workers has changed. they are now considered during this time of pandemic as health professionals, and as such they have the right to travel to meet with children. the idea emerges to organise family network therapy sessions by videoconference before the end of the confinement. the centre des buttes-chaumont is again in demand. the conventions established with each of the participants in the network session and the therapeutic tandem usefully frame the new disturbing context for the most vulnerable among them, by calming the fears of abandonment, or on the contrary of dictatorial control through this means of communication. a spontaneous energy flow appears as the homage 'at the windows' is paid every evening at 8 pm to the nursing staff. i too hit something, a shell casing from world war i (empty!)it reminds me of the spanish flu pandemicand as a drumstick, a saharan jewel offeredwell, well, well!by one of my former patients who went to the sahel to offer solidarity to women. it makes a rather high-pitched bell sound, somewhat close to the bells of the tibetan monks . . . everything blends into this positive energy. a neighbour who is ill with cancer is the most alert to beat the call of the neighbours . . . her care continues during this time. the calmness is conducive of reflection. what do i want to change in my life as a therapist? . . . nothing, other than taking more time for myself. sports, baking, tidying up, painting, talking . . . i miss my friends, but the family exchange is nicely intensifying. confinement slogans tirelessly spread their message, one week, two weeks, three weeks, we don't count the weeks anymore, time has changed value, the pace has slowed down. look, why don't i take the time to check my pension rights? no, i'd rather watch a good series . . . humanity in times of pandemic (humanidad en pandemia) therapists and patients living through times of pandemic have evolved in a context of threat and uncertainty. due to the regulations to avoid contagion, family members have been forced to stay in the same space for indefinite times. the personal and group impact of this dynamic unfolds in multiple dimensions and has an unsuspected scope. the experience of physical space has evolved as hours, days, weeks have passed. although at first it could be perceived as a break from the whirlwind of everyday life, the 'forced' stay has turned into a kind of narrowing of the limits; of the physical space, of the psychic/corporal world, relational, bonding. no matter how many people inhabit a place, the members resent the isolation and helplessness on many occasions. in some way, previous forms of relationship, ties and family, group, and social functioning have pointed out the dominant styles of behaviour in this type of closure (muñoz, 2019) . without external compensatory systems, which operate as sedatives of the sense and deniers of finitude, we observe how the first attachment style appears in a dynamic that goes through different levels depending on the global context and the verbal and non-verbal information that emerges from authority (bowlby, 1993) . groups with primarily disorganised attachments generate contexts where friction, punishment, and violence emerge quickly as a way to relieve anxiety, fear, and tension. when the primary attachments are associated with anxiety and ambivalence, the group is submerged in fear that spreads through networks infiltrating the psychocorporal world, facilitating extreme care behaviours alternated with reckless risk behaviours that increase fear and anxiety. systems with predominantly anxious-avoidant attachment tend to focus on demand and performance by amplifying effective control systems at the expense of body and emotions. these are subject to the dominance of reason, dissociating body and its messages controlling fear, anxietiy, and uncertainty. on the contrary, groups with predominantly secure attachments creatively and adaptively go through this turbulence at the pole of action, creating and recreating new realities and ways of living. then, the isolation gradually infiltrates the family system, so fear and emptiness take over the bonds, as a melody that silences with consumer products. therefore, an impossible gap associated with the absence of meaning and a deep fear of damage and death is attempted to be filled. so, time becomes a waiting time. simultaneously, we witness an institutional and organisational crisis with the ensuing collapse of credibility and trust. a massive disconnection of people who in this paralysis have lost their jobs confirms those premises about the hope and credibility violated, and although cognitively it is explainable emotionally and affectively, the experience is overwhelming. uncertainty, fear, and the experience of injustice increase in a context that is impossible to decode. on the other hand, teleworking has been a way of maintaining continuity of work and staying on the move. however, in many organisations, this system has forced workers to be permanently available online. for executive women workers, the demand has increased exponentially due to the exercise of multiple roles that overlap and require time, effort, and dedication. uncertainty in an emotional, relational, social, global context reduces security for people; groups, institutions, when faced with the threat, exacerbate control and defence mechanisms. they bring solutions that only increase the problem, generating fear and pain expressed in different ways. they deny mourning in the face of loss at all levels: stability, power, status, the lives of loved ones, and their own lives (sluzki, 1993) . and the body? often forgotten and uninhabited due to the predominance of the image, it becomes the repository of emotions that, given the context, are impossible to symbolise and integrate. raising these emotions to consciousness reminds us that we hurt ourselves in bonds and heal in them, so that it is possible to agree to feel enough fear for self-care and care for others. holding the bond of intimacy that the therapeutic space provides in this transit that emerges as a new context from the face-to-face to virtual, implies an opening to newness. in turn, group networks that met in transit rituals establishing contact, providing support, direction, and meaning when assisting life and death, have also been impacted and injured. however, the voice and face have enhanced as a contact image and company (sluzki, 1993) . the question of my being a therapist . . . being also a midwife refers me to processes and learning; my history and its multiple resonances weaved into a systemic psycho-dramatic tapestry that includes myself and humanity. i feel that we cross a threshold similar to labour, being delivered and giving birth, simultaneously, in a channel whose timelessness is felt in our bodies. we are leaving a womb that could no longer contain us. today by force of contractions, we remain at times with fear, compressed by narrow walls that adhere to the personal, group, social and human body, with fears of harm and death. simultaneously and in another polarity, with an unknown force and with the survival instinct to the maximum, we open the virtual space in search of the exit. ª 2020 australian association of family therapy where are we going? just like before we were born, it is a mystery; however, from another perspective i know that we will look to another territory, with keys, codes, and ways of survival different from what is known. a place where we will need to put into operation new approaches that, probably without much awareness, we have developed in this previous gestation process. these are bodily, personal, group, social, and of humanity. i feel that, as in any process, we may reach the other side crying, it is also possible that we remain detained in a space 'in-between,' without being able to advance. so, as humanity we are at risk; it is the essential trust, the conscience and bonding that sustains us to arrive someday at that 'other side. ' nowadays, for me to be a therapist is to be a midwife, creating contexts that, in the intimacy of psychotherapy, allow me to accompany in uncertainty, in fear, in the pain of losses, in silence and respect for the expression of grief. i trust that the strength of the bonds will allow us to be born to other unimaginable dimensions from the prisms in which we contact today. i only miss hugging my son and daughter. i carry with me the nostalgia and smell of their bodies. the loneliness of the therapist . . . my being a therapist cuts across the many roles i carry out. with maturity and conscience, the person of the therapist talks and integrates with my entire person. on one level, i feel lonely, but, on another, it is a joy to feel the strength of the bonds of systemic therapists around the world to reach out. this unique practice collection offers readers a glimpse into the professional and personal experiences and reflections of an international group of family and systemic therapists across the globe as they experience the first phase of the covid-19 pandemic. the world is navigating through unpredictable times. therapists need to 'be,' before they can 'do.' systemic therapists are able to heal and in turn support the healing of the families, couples, and individuals they work with through the process of reaching out to fellow therapists in shared experience. it is only then they can contemplate stepping into a next phase; the 'regenerative process' where they can rebuild their lives post-pandemic. there needs to be time to pose and consider; whether the familiar pre-pandemic 'normal' is the 'normal' that is desperately sought . . . or in fact whether this calls for the creation of new opportunities for social change. the pandemic illustrates the reality that society remains at its essence 'collectivist.' we are all in this together, as a collective humanity. it is evident humans are inter-dependent on one another. there is an inescapable inter-connectedness and relationship between systems, within the body, between one another; humanity cannot separate from the environment, just as therapists cannot separate from their families or fellow therapists. this crisis may assist therapists to rearrange the scale of values regarding what is important. beyond the interventions used, preservation of the relationship is what really matters. it seems ironic that an enforced need to 'stay apart' from one another (in order to stay alive), has birthed an invitation to be more human (in order to stay emotionally and relationally alive), and be closer to each other than ever before. this collection illustrates now more than ever the importance of looking at the 'macro' issues presenting for people and society from a systemic perspective. the more complex the issues, the more important they be considered and addressed through a systemic lens. approaching these complexities from a sequestered, individual perspective is reductionist, invalidating, unrealistic, but also disrespectful to other cultures. this present challenge also causes a questioning of the notion of permanence and certainty to give room to impermanence and uncertainty; while distressing, and unsettling, this provides opportunities. this is an unpredictable and crucial time. if there has ever been a need for systemic therapists and the 'world of systems' to advocate for systems change, this is the time. humanity is part of a system that can destroy it in a short time if it does not listen to the feedback that comes from the interaction with the other elements of that system. as the pathway to the 'other side' is navigated, there is a need to value context, culture, collaboration, and connectedness in order to combat isolation and trauma while enhancing unity. together we can begin to think about some of the implications and recommendations for family therapy practice and research in relation to covid-19. first, family and systemic therapists are in a key position to advise stakeholders such as governments and health departments in developing and implementing a response to the covid-19 pandemic. second, there are numerous advantages to understanding the effects of the pandemic through a systemic lens. it is unrealistic, illogical, and unscientific to imagine that complex issues like covid-19 can be considered simply by focusing on individuals. third, this suggests an integrated approach to the management of pandemic trauma and suicide prevention utilising 'systemic thinking' as a foundation. future collaborative research could focus on: the collective nature of trauma to consider the consequences of traumatic events shared by a 'social collective,' and how this may differ from 'interpersonal trauma'; mental health consequences that take into account the impact of pre-existing and co-existing mental health issues; the relational consequences of covid-19 in exploring whether collective traumas create greater resilience given the collective shared experience; further rigorous qualitative and phenomenological studies to capture the experiences of family therapists honouring different cultural backgrounds and languages. associate professor of psychology, subprogram in clinical psychology, department of psychology director of the paduan center of family therapy, academic of the milanese center of family therapy. email: mosconia1@gmail.com member ipscan (international society for prevention of child abuse and neglect il multi-linguaggio e il multi-tempo dell'amore: il lavoro con le coppie interculturali (the multi-language and the multi-time of love: work with intercultural couples). paper presented at the convegno residenziale apf 'il processo terapeutico. tempi e fasi della terapia familiare my story, your story: the role of culture and language in emotion expression of cross-cultural couples. the mi culture model reflection on aaft family therapy conference getting together with like minded people': a conference edition habla mi idioma? an exploratory review of working systemically with people from diverse cultures: an australian perspective children in the margins training the person of the therapist in structural family therapy mind and nature: a necessary unity el apego (attachment). barcelona: paid os treating complex traumatic stress disorders: an evidence-based guide revisione dei concetti di ipotizzazione, circolarit a, neutralit a: un invito alla curiosit a (hypothesizing, circularity, and neutrality revisited: an invitation to curiosity) autoethnography, personal narrative, reflexivity: researcher as subject hypnotherapy: an exploratory casework getting a kick out of you: the jazz taoist key to love the new normal: living with a transformed reality the helpers and the helped: viewing the mental health profession through the lens of love in action: an integrative approach to last chance couple therapy the tao of love: life lessons learned from laotzu and the way of life suicide risk and prevention during the covid-19 pandemic integrating a trauma lens into a family therapy framework: ten principles for family therapists the five stages of grief the way of life one service, many voices: enhancing consumer participation in a primary health service for multicultural women the use of self of the therapist families and family therapy v ınculo, percepci on y conciencia en la coordinaci on grupal: la persona del coordinador (link, perception and consciousness in group coordination: the person of the coordinator) g enogramme et inceste: tempo th erapeutique et tempo judiciaire, in i les g enogrammes aujourd'hui la clinique syst emique en mouvement quand la famille marche sur la tête: inceste, p edophilie, maltraitance (when the family walks on your head: incest, pedophilia, abuse) la fin des certitudes. temps, chaos et lois de la nature (the end of certainties. time, cahos and the laws of nature) how family therapy stole my interiority and was then rescued by open dialogue memory, trauma, and phantasmagoria in claudia llosa's 'la teta asustada paradosso e controparadosso. un nuovo modello nella terapia della famiglia a transazione schizofrenica (paradox and counterparadox. a new model in schizophrenic transaction family therapy) ipotizzazione, circolarit a, neutralit a: tre direttive per la conduzione della seduta (hypothesizing, circularity, neutrality: three guidelines for the conductor of the session) la red social: frontera de la pr actica sist emica observing systems the authors look forward to further systemic themed papers on the family therapy response to covid-19 such as focusing on the regenerative phase of the pandemic and the reporting of practice and practical responses. lyndon amorin-woods for assistance in preparation of the draft manuscript. key: cord-029880-mhmvc0kq authors: sy, charlle; bernardo, ezekiel; miguel, angelimarie; san juan, jayne lois; mayol, andres philip; ching, phoebe mae; culaba, alvin; ubando, aristotle; mutuc, jose edgar title: policy development for pandemic response using system dynamics: a case study on covid-19 date: 2020-07-29 journal: process integr optim sustain doi: 10.1007/s41660-020-00130-x sha: doc_id: 29880 cord_uid: mhmvc0kq the coronavirus disease 2019 (covid-19) outbreak has burdened several countries. its high transmissibility and mortality rate have caused devastating impacts on human lives. this has led countries to implement control strategies, such as social distancing, travel bans, and community lockdowns, with varying levels of success. however, a disease outbreak can cause significant economic disruption from business closures and risk avoidance behaviors. this paper raises policy recommendations through a system dynamics modeling approach. the developed model captures relationships, feedbacks, and delays present in a disease transmission system. the dynamics of several policies are analyzed and assessed based on effectiveness in mitigating infection and the resulting economic strain. the ongoing covid-19 pandemic is raising unprecedented global health concerns because of its high infection and mortality potential. since the first case reported on december 30, 2019, the number of confirmed cases worldwide has grown at an alarming exponential rate (huang et al. 2020) , reaching hundreds of thousands being infected in different countries. as such, the world health organization (who) declares covid-19 a public health emergency at an international level . this spurred immediate action to identify, prevent, and control the transmission of this virus. similar precautions and strategies are suggested by experts for combatting covid-19. sohrabi et al. (2020) propose immediate diagnosis and isolation for patients with contact to the virus. xiao and torok (2020) similarly suggest limiting human-human interaction, identifying and isolating persons with exposure, and providing immediate care to those who have been infected. globally, each country has put up their own efforts against covid-19. china, as well as many other countries, imposed travel bans and community lockdowns, especially to and from the epicenter of the outbreak wuhan, china (hua and shaw 2020) . their strict governance has allowed them to "flatten the curve"; however, a second wave is expected because of their premature lifting of the lockdowns and travel ban . nonetheless, late detection allowed their people to travel internationally, leading to a global spread of the virus. singapore and south korea, although the latter initially saw a significant spike in cases because of virus transmission during religious gatherings, effectively address this issue through strict screening, contact tracing, and quarantine measures (gilbert et al. 2020 ). on the other hand, europe has become a hotspot for the covid-19 virus; infection and mortality cases soar in italy, spain, germany, and france, with case counts reaching hundreds of thousands per country. this may be attributed to delayed response, lack of rapid testing, and the high mobility of tourists around europe (the lancet 2020). for the same reasons, the united states of america (usa) has the most number of cases worldwide, having reached over 300,000 cases as of april 7, 2020 (who 2020). in south east asia, the philippines has the second most number of cases at 3660 confirmed cases as of april 7, 2020, closely following malaysia. as a response to covid-19, the philippine government implemented a travel ban of foreigners and a gradual implementation of enhanced community quarantine (comparable with lockdowns implemented in other countries). there have also been efforts to increase testing and quarantine capacities (department of health 2020). although vaccination is seen as an effective pharmaceutical-based mitigation strategy, existing vaccines may be ineffectual against novel virus strains (araz 2013) , such as the case in covid-19. unfortunately, effective vaccines and treatment for this virus are still under research and development . the persistence of an influenza pandemic can have a significant negative impact on an economy's productive capacity because of its crippling effect on labor supply depending on its morbidity and mortality rate. fundamental economic theory establishes that labor is required in conjunction with capital and natural resources for the production of valuable goods. additionally, this triggers a domino effect, wherein the loss in productivity of one sector would inevitably result in the succeeding customer sector to fail to meet their targets and orders. moreover, government policies, such as school closures and discouraging workers to attend their workplace, can increase worker absenteeism. however, this could be offset by the availability of informal care arrangements and the ability of the parents to work from home (keogh-brown 2014). due to the novelty of the situation, there had yet to be any formal evaluation of the success of online platforms for work in maintaining productivity. based on the performance of global supply chains, it is apparent that online platforms will fail to sustain the volume of commercial activity prior to the outbreak (ivanov 2020) . variations in the methods for delivering online education also indicate that universities have yet to converge on a best approach for this scenario, with some universities suspending classes completely and others persisting with online lectures. prior studies on using technology to deliver the online lectures cite difficulties due to lack of reliable internet connection, promoting student initiative and autonomy in learning, and the learning delays of adjusting to the technology (jowsey et al. 2020; cuaca dharma et al. 2017) . these indicate that, although quarantine is necessary to some extent to control the situation, extended quarantine is not an appropriate solution. furthermore, despite its unpredictability, there is evidence that suggests externalities such as fear-driven behavioral changes could potentially be the largest contributor to the economic costs of disease outbreaks. from the onset of an outbreak until a considerable period after events stabilize, people are likely to take precautionary actions on top of lockdowns and quarantines imposed, such as avoiding public transportation and international travels, avoiding congregative and entertainment events, and limiting shopping to essentials that would all aggregate to an increase in gdp loss in the first year of about 2% of gdp (keogh-brown 2014). thus, although public health is of highest priority, there is also the need to minimize the negative economic impact of pandemic outbreak responses (xiao and torok 2020) . mathematical and statistical models have been used to gain a better understanding of disease outbreaks and aid in emergency preparedness and response decision-making. these models have been integral in providing insights necessary for developing risk management strategies that minimize the transmission of disease outbreaks and other negative impacts, such as shortages in essential resources and economic declines. one particular methodology, system dynamics (sd) has been employed in disease outbreaks such as influenza. it is a simulation and modeling technique that is established in the application of strategic planning. it is a comprehensive systems perspective approach to map relationships present within complex systems and capturing any nonlinearities, feedback loops, and delays. all of this may provide useful insights towards designing and developing policy interventions (edaibat et al. 2017) . safarishahrbijari et al. (2017) combined system dynamics with particle filtering approach to predict future influenza outbreaks. their results outlined the improved accuracy and robustness of the prediction of future influenza outbreak, thus proposing the development of standardized guidelines to control and regulate the potential outbreak. araz (2013) proposed an integrated framework joining system dynamics with analytic hierarchy process (ahp) a decision-making tool for the assessment of public health strategies in an influenza outbreak capturing multiple stakeholder preferences. the results are aimed to aid the decision-makers on health strategies in the event that an influenza pandemic outbreak. vincenot and moriya (2011) adopted an integrated concept of system dynamics and individual-based modeling on the impact of resurgence of influenza pandemic. the results highlighted the behavior of spread over different scenarios outlining the significance of spatial disaggregation during outbreaks. currently, no studies have been found in the application of system dynamics on the covid-19 outbreak. to address this gap, this research is aimed to demonstrate the viability of system dynamics as a framework to understand and develop response strategies for disease pandemics such as covid-19. the general objective of the study is to develop a system dynamics model of covid-19 for different scenarios aiming to equip decision-makers with evidence-based judgment in the control of the outbreak. alternatives to the extended community quarantine are proposed using the sd framework. healthcare capacity, community control policies, and individual human behavior, among the other factors to be considered in this study, vary greatly in the magnitude and nature of their impact to the situation. yet, it is evident they are critical to pandemic response. under the sd framework, the relationships between these factors are organized into feedback loops, then are simulated as stocks and flows. however, it is important to note that the numbers or values obtained from sd are not predictive. instead, sd is used to demonstrate the trend and behavior of problem variables over time as influenced by feedbacks present in the system. the sd model developed in this study builds upon the basic susceptible-infectious-recovered (sir) model originally proposed by grassly and fraser (2008) . capturing the natural infection process, the number of infected persons, divided into the asymptomatic and symptomatic, will increase the amount of people exposed as shown in the causal loop diagram presented in fig. 1 . the values of these variables are influenced by actions taken to control the situation, such as through the quarantine of those infected, social distancing, travel bans, and personal isolation and protection strategies. conversely, the magnitude of the problem at various points in time will also influence the magnitude of the response to control the situation. likewise, a tendency for relaxation is encouraged by negative economic performance. 1 hypothetical and literature-based data were used during model implementation, especially because data is not always available and accurate to represent soft variables and highlevel relationships. sd framework was followed, wherein the model is validated against a reference model for the problem variable. the sd model is used to model and simulate the impact of various policies. as a preliminary study, effectiveness will be evaluated in terms of the number of infected people in a population, as this roughly reflects the duration that a country is experiencing a pandemic. figure 2 a and b show some of the common responses to the pandemic. the construction of additional hospitals and quarantine centers is one of the primary responses to a pandemic. while some increase in healthcare capacity is necessary, this will only address infection by a limited extent, and is infeasible for developing countries. another common policy is to encourage social distancing through either education, publicity, or the closure of establishments (fig. 2b) . this not only reduces the peak levels of infection but also extends the duration that a country is affected by the pandemic. some consideration may need to be given for the economy or out of necessity, by allowing for certain industries to continue their operations. even if we yield to economic pressures, overall infection under social distancing will be less than the baseline (fig. 2c) . similarly, by relaxing the social distancing measures for low-risk demographics, overall infection will still be less than the baseline (fig. 2d) . community quarantine is intended to starve the virus, by preventing the spread of the disease between households. depending on the duration of the quarantine period, this can merely delay the peak of the pandemic without significantly lowering overall infection (fig. 2e) . gradual quarantine lifting, i.e., reducing the stringency of quarantine over time, gives an opportunity for the economy to recover faster while keeping the potential for infection under control (fig. 2f) . as a final note, the policies may be implemented together with varying degrees of potency according to the resources and disposition of a particular community. the sd framework made it possible to see the interactions between policies in elemental form. this gives the policymaker more room to modify and improve the pre-existing methods for handling pandemics. first, it answers the question of which policies can be implemented together efficiently. some policies are complementary. precautionary measures, for instance, will reduce the chance for infection without introducing new consequences to the economy. there are also policies that are inefficient when implemented in basic form, yet have great potential after being modified. in particular, the gradual lifting of quarantine limits infection and also reduces the impairment of the economy resulting from complete quarantines. rather than investing much in a single policy, multiple policies can leverage on one another to improve the state of a country under the pandemic. from this point, further analysis can be conducted to identify the best parameters for implementation. impactful parameters include quarantine duration, additional healthcare capacity, and decisions on the quantity and characteristics of low-risk citizens, among other specifics that need to be defined when implementing a policy. by understanding the dynamics of the system, it becomes possible to fine-tune the solution through the parameters, without being too dependent on these values for impact. the covid-19 outbreak has proven to be a complex problem with effects rooted in public health and economics among some. it is crucial that deeper analysis and assessment frameworks are developed to support decision-making for risk management strategies and transmission control interventions that account for the interactions between participating sectors. based on initial modeling, the most effective strategies focus on avoiding exposure to the virus from happening in the first place; focusing on increasing healthcare capacities only delays the inevitable system collapse as its effectiveness assumes people getting infected first. system dynamics may also be utilized to gain better understanding of other aspects of the system, such as the management of healthcare operations in the face of an influenza pandemic. furthermore, other modeling techniques, such as optimization modeling, may be employed for resource allocation, especially in the face of scarce essential commodities and medical supplies. it may also be used to select between feasible policy alternatives and to operationalize their implementation based on various, and even conflicting, stakeholder objectives. conflict of interest the authors declare that they have no conflicts of interest. integrating complex system dynamics of pandemic influenza with a multi-criteria decision making model for evaluating public health strategies basic japanese grammar and conversation e-learning through skype and zoom online application 2020) covid daily testing capacity increases, numbers to improve further to 10k system dynamics simulation modeling of health information exchange (hie) adoption and policy intervention: a case study in the state of maryland preparedness and vulnerability of african countries against importations of covid-19: a modelling study mathematical models of infectious disease transmission infodemic" and emerging issues through a data lens: the case of china clinical features of patients infected with 2019 novel coronavirus in predicting the impacts of epidemic outbreaks on global supply chains: a simulation-based analysis on the coronavirus outbreak (covid-19/sars-cov-2) case blended learning via distance in pre-registration nursing education: a scoping review macroeconomic effect of infectious disease outbreaks. ency health econ transmission dynamics and evolutionary history of 2019-ncov predictive accuracy of particle filtering in dynamic models supporting outbreak projections world health organization declares global emergency: a review of the 2019 novel coronavirus (covid-19) covid-19: learning from experience impact of the topology of metapopulations on the resurgence of epidemics rendered by a new multiscale hybrid modeling approach world health organization (2020) coronavirus disease 2019 (covid-19) situation report taking the right measures to control covid-19 beware of the second wave of covid-19 publisher's note springer nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations key: cord-333599-hl11ln2r authors: tulchinsky, theodore h.; varavikova, elena a. title: planning and managing health systems date: 2014-10-10 journal: the new public health doi: 10.1016/b978-0-12-415766-8.00012-4 sha: doc_id: 333599 cord_uid: hl11ln2r health systems are complex organizations. they are often the largest single employer in a country, with expenditures of public and private money of 4–17 percent of gross domestic product. overall and individual facility management requires mission statements, objectives, targets, budgets, activities planning, human interaction, services delivery, and quality assurance. health organization involves a vast complex of stakeholders and participants, suppliers and purchasers, regulators and direct providers, and individual patients, and their decision-making. these include pyramidal and network organizations and ethical decision-making based on public interest, resource allocations, priority selection, and assurance of certain codes of law and ethical conduct. this chapter discusses how complex organizations work, with potential for application in health, and the motivations of workers and of the population being served. organization theory helps in devising methods to integrate relevant factors to become more effective in defining and achieving goals and missions. health systems are complex organizations and their management is an important concept in the new public health. health is a major sector of any economy and often employs more people in the industrialized countries than any other industry. health has complex networks of services and provider agencies, including funding through public or private insurance or through national health service systems. whether insurance is provided by the state or through private and public sources combined, skilled management is required at the macro-or national and the micro-or local level, including the many institutions that make up the system. management training of public health professionals and clinical services personnel is a requisite and not a luxury. planning and management are changing in the era of the new public health with advances in prevention and treatment of disease, population health needs, innovative technologies such as genetic engineering, new immunizations that prevent cancers and infectious diseases, prevention of non-communicable diseases, environmental and nutritional health, and health promotion to reduce risk factors and improve healthful living for the individual and the community. modern and successful public health also must address social, economic, and community determinants of health and the promotion of public policies and individual behaviors for health and well-being. the social capital and norms that promote cooperation among people are the basis of a "civil society" (i.e., the totality of voluntary, civic, and social organizations and institutions of a functioning society alongside the structures of governmental and commercial institutions). health systems are ideally knowledge-and evidence-based in using technologies available in medicine and the environment to promote the health and well-being of a population, including security against the effects of threatened terrorism, growing social isolation, and inequities in health. management in health can learn much from concepts of business management that have evolved to address the economic and human resource aspects of a health system at the macrolevel or an individual unit of service at the microlevel. the new public health is not contained within one organization, but rather reflects the collective efforts of national, state, regional, and local governments, many organizations in the public and non-governmental sectors, and finally efforts of individual or group advocates and providers and the public itself. the political level is crucial for adequate funding, legislation, and promotion of health-oriented policy positions and in public health management. the responsibility for health management is shared across all parts of society, including individuals, communities, business, and all levels of government. the new public health identifies and addresses community health risks and needs. planning is critical to the process of keeping a health system sustainable and adaptable and in creating adequate responses to new health threats. monitoring, measurement, and documentation of health needs are vital to design and adapt an effective program and to measure impact. data on the targeted issues must be accessible while protecting individual privacy. health is a hugely expensive and expansive complex of services, facilities, and programs provided by a wide range of professional and support service personnel making up one of the largest employers of any sector in a developed country. services are increasingly delivered by organized groups of providers. but all health systems operate in an environment of economic constraints, imposing a need to seek efficiency in the use of resources. how organizations function is of great importance not only for their economic survival, but also, and equally important, for the well-being of the clients and providers of care. an organization is two or more people working together to achieve a common goal. management is the process of defining the goals and making effective use of an organization to attain those goals. even very small units of a human organization require management. management of human resources is vital to the success of an organization, whether in a production or service industry. health systems may chapter 12 vary from a single structure to a network of many organizations. no matter how organizations are financed or operated, they require management. management in health care has much to learn from approaches to management in other industries. elements of theories and practices of profit-oriented sector management can be applied to health services even if they are operated as non-profit enterprises. physicians, nurses, and other health professionals will very likely be involved in the management of some part of the health care system, whether a hospital department, a managed care system, a clinic, or even a small health care team. at every level, management always means working with people, using resources, providing services, and working towards common objectives. health providers require preparation in the theory and practice of management. a management orientation can help providers to understand the wider implications of clinical decisions and their role in helping the health care system to achieve goals and targets. students and practitioners of public health need preparation in order to recognize that a health care system is more complex than the direct provision of individual services. similarly, policy and management personnel need to be familiar with both individual and population health needs and related care issues. health has evolved from an individual one-on-one service to complex systems organized within financing arrangements, mostly under government auspices. as a governmental priority, health may be influenced by political ideology, sometimes reflecting societal attitudes of the party in power and sometimes apparently at odds with its general social policy. following bismarck's introduction in germany in 1881 of national health insurance for workers and their families, funded by both workers and their employers, most countries in the industrialized world introduced variants of this national health plan. usually, this has been at the initiative of socialist or liberal political leadership, but conservative political parties have preserved national health programs once implemented. despite the new conservatism since the 1990s with its pre-eminent ideology of market forces, the growing roles for national, state, and local authorities in health have led to a predominantly government role in financing and overall responsibility for health care, even where there is no universal national health system, as in the usa. the uk's national health service (nhs), initiated by a labour government in 1948, has survived through many changes of government, including the conservative margaret thatcher period in which many national industries and services were privatized. health policy is a function of national (government) responsibility overall for health, but implementation is formulated and met at state, local, or institutional levels. the division of responsibilities is not always clear cut but needs to be addressed and revised both professionally and politically within constitutional, legal, and financial constraints. selection of the direction to be taken in organizing health services is usually based on a mix of factors, including the political view of the government, public opinion, and rational assessment of needs as indicated through epidemiological data, cost-benefit analysis, the experience of "good public health practice" from leading countries, and recommendations by expert groups. lobbying on the part of professional or lay groups for particular interests they wish to promote is part of the process of policy formulation and has an important role in the planning and management of health care systems. there are always competing interests for limited resources of funding, by personnel within the health field itself and in competition with other demands outside the health sector. the political level is vitally involved in health management in establishing and maintaining national health systems, and in determining the place of health care as a percentage of total governmental budgetary expenditures, in allocating funds among the competing priorities. these competing priorities for government expenditures include defense, roads, education, and many others, as well as those within the health sector itself. traditionally, there are competing priorities between the hospital and medical sector and the public health and community programs sector. a political commitment to health must be accompanied by allocation of resources adequate to the scope of the task. thus, health policy is largely determined by societal priorities and is not a prerogative of government, health care providers, or any institution alone. as a result of long struggles by trade unions, advocacy groups, and political action, well-developed market economies have come to accept health as a national obligation and essential to an economically successful and well-ordered society. this realization has led to the implementation of universal access systems in most of the industrialized countries. once initiated, national health systems require high levels of resources, because the health system is labor intensive with relatively high salaries for health care professionals. in these countries, health expenditures consume between 7 and nearly 18 percent of gross domestic product (gdp). some industrialized countries, notably those in the former soviet bloc, lacking mechanisms for advocacy, including consumer and professional opinion, tended to view health with a political objective of social benefits, and also as a "non-productive" consumer of resources rather than a producer of new wealth. as a result, budget allocations and total expenditures for health as a percentage of gdp were well below those of other industrialized countries (figure 12 .1). salaries for health personnel in the semashko system were low compared to industrial workers in the "productive" sectors. furthermore, industrial policy did not promote modern health-related industries, compared to the military or heavy industrial sectors. the former socialist countries of eastern europe which have joined the european union (eu) have gradually increased allocation to health from 5.44 percent of gdp in 1995 to 7.1 percent in 2010, while the pre-2004 members of the eu increased their expenditures from 8.5 percent of gdp to 10.6 percent. the average spend in the commonwealth of independent states (russia, ukraine, and others) increased from 5.6 percent in 1995 to 5.74 percent in 2010, and in the central asian republics (kazakhstan, uzbekistan, and others) from 4.8 percent in 1995 to 5.2 percent in 2010 (who health for all database, january 2013). however, russian health expenditure in 2011 was still only 6.2 percent of gdp and there is a lingering idea of health being a non-productive investment. the developing countries generally spend under 4 percent of gnp on health, because health is addressed as a relatively low political priority, and they depend very much on international donors for even the most basic of public health programs such as immunization. financing of health care and resource allocation requires a balance among primary, secondary, and tertiary care. economic assessment, monitoring, and evaluation are part of determining the health needs of the population. regulatory agencies are responsible for defining goals, priorities, and objectives for resulting services. targets and methods of achieving them provide the basis for implementation and evaluation strategies. planning requires written plans that include a statement of vision, mission objectives, target strategies, methods, and coordination during the implementation. designation and evaluation of responsibilities, resources to be committed, and participants and partners in the procedure are part of the continuous process of management. the dangers of taking a "wrong" direction may be severe, not only in terms of financial costs, but also in terms of high levels of preventable morbidity and mortality. health policy is often as imprecise a science as medicine itself. the difference is that inappropriate policy can affect the lives and well-being of very large numbers of people, as opposed to an individual being harmed by the mistake of one doctor. there may be no "correct" answer, and there are numerous controversies along the path. health policy remains more an "art" than the more quantitative and seemingly precise field of health economics. societal, economic, and cultural factors as well as personal habits have long been accepted as having an important impact on vulnerability to coronary heart disease. but other factors such as the degree of control over one's life, as suggested in studies of british civil servants, religiosity, and the effects of migration on families left behind are part of the social gradients and inequalities seen in many disease entities, with consequent excess morbidity and mortality in some contexts, such as in russia and ukraine. health policy, planning, and management are interrelated and interdependent. any set goal should be accompanied by planning how to attain it. a policy should state the values on which it is based, as well as specify sources of funding, planning, and management arrangements for its implementation. examination of the costs and benefits of alternative forms of health care helps in making decisions as to the structure and the content of health care services, both internal structures (within one organization) and external linkages (intersectoral cooperation with other organizations). the methods chosen to attain the goals become the applied health policy. the world health organization's (who's) 1977 health for all strategy was directed at the political level and intended to increase governmental awareness of health as a key component of overall development. to some degree it succeeded despite its expansive aspirations, and even after nearly 40 years, its objectives remain worthwhile even in well-developed health systems. within health, primary care was stressed as the most effective investment to improve the health status of the population. in 1993, the world bank's world development report adopted the health for all strategy and promoted the view that health is an important investment sector for general economic and social development. however, economic policies promoting privatization and deregulation in the health sector threaten to undermine this larger goal in countries with national health systems. in the usa, major steps are being taken to increase coverage of health insurance for all as the number of uninsured americans declined from 50 million people uninsured in 2010 to 48.6 million in 2011, edging down from 16.3 to 15.7 percent of the total population. further decline in the uninsured population is expected as the patient protection and affordable care act (ppaca, or "obamacare") comes into effect in the coming years, bringing many millions of americans into health insurance and meeting federal standards of fair practices such as eliminating exclusion for preexisting conditions by private insurers. the ppaca comes into effect on 1 january 2014 and will guarantee coverage for pre-existing conditions, and ensure that premiums cannot vary based on gender or medical history. it will subsidize the cost of coverage, and new state-based health insurance exchanges will help consumers to find suitable policies. it will introduce many preventive care measures into public and private insurance plans, and will promote efficiencies in the health systems including reduction in fraudulent claims and wasteful funding systems. all of this will require skilled management in the components of the health system (see chapters 10 and 13). in the new public health, health promotion, preventive care, and clinical care are all part of public health because the well-being of the individual and the community requires a coordinated effort from all elements of the health spectrum. establishing and achieving national health goals require planning, management, and coordination at all levels. the achievement of health advances depends on organizations and structured efforts to reach health goals such as those defined above, and more recently by the united nations (un) in the millennium development goals (mdgs) (see chapter 2), and requires some understanding of organizations and how they work. the study of organizations developed within sociology, but has gradually become a multidisciplinary activity involving many other professional fields, such as economics, anthropology, individual and group psychology, political science, human resources management, and engineering. organizations, whether in the public or private sector, exist within an external environment, and utilize their own structure, participants, and technology to achieve goals. for an organization to survive and thrive, it must adapt to the physical, social, cultural, and economic environment. organizations participating in health care establish the connection between service providers and consumers, with the goal of better health for the individual and the community. the factors for this include legislation, regulation, professionalism, instrumentation, medications, vaccines, education, and other modalities of intervention for prevention and treatment. the social structure of an organization may be formal (structured stability), natural (groupings reflecting common interests), or open (loosely coupled, interacting, and self-adjusting systems to achieve goals). formal systems are deliberately structured for the purposes of the organization. natural systems are less formal structures where participants work together collaboratively to achieve common goals defined by the organization. open systems relate elements of the organization to coalitions of partners in the external environment to achieve mutually desirable goals. in the health system, structures should focus on prevention and treatment of disease and improvement in health and well-being of society. the social structure of an organization includes values, norms, and roles governing the behavior of its participants. government, business, or service organizations, including health systems, require organizational structures, with a defined mission and set of values, in order to function. an organizational structure needs to be tailored to the size and complexity of the entity and the goals it wishes to achieve. the structure of an organization is the way in which it divides its labor into distinct tasks and coordinates them. the major organizational models, which are not mutually exclusive and may indeed be complementary, are the pyramidal (bureaucratic) and network structures. the bureaucratic model is based on a hierarchical chain of command with clearly defined roles. in contrast, the matrix or network organization brings together professional or technical people to work on specific programs, projects, or tasks. both are vital to most organizations to meet ongoing responsibilities and to address special challenges. some classic organization theory concepts help to set the base for modern management ideas as applied to the health sector. scientific management was pioneered by frederick winslow taylor . his work was pragmatic and based on empirical engineering, developed in observational studies carried out for the purpose of increasing worker, and therefore system, efficiency. taylor's industrial engineering studies of scientific management were based on the concept that the best way to improve worker productivity was by designing improved techniques or methods used by workers. this theory viewed workers as instruments to be manipulated by management, and assumed that efficient, rationally planned methods would produce better industrial results and industrial peace as the tasks of managers and workers would be better defined. time and motion studies analyzed work tasks to seek more efficient methods of work in factories. motivation of workers was seen to be related to payment by piecework and economic self-interest to maximize productivity. taylor sought to improve the productivity of each worker and to make management more efficient in order to increase earnings of employers and workers. he found that the worker was more efficient and productive if the worker was goal oriented rather than task oriented. this approach dominated organization theory during the early decades of the twentieth century. resistance to taylor's ideas came from both management and labor; the former because it seemed to interfere with managerial prerogatives and the latter because it expected the worker to function at top efficiency at all times. however, taylor's work had a lasting influence on the theory of work and organizations. the traditional pyramidal bureaucratic organization is classically seen in the military and civil services, but also in large-scale industry, where discipline, obedience, and loyalty to the organization are demanded, and individuality is minimized. this form of organization was analyzed by sociologist max weber between 1904 and 1924. leadership is assigned by higher authority, and is presumed to have greater knowledge than members lower down in the organization. this form of organization is effective when the external and internal environments, the technology, and functions are relatively well defined, routine, and stable. the pyramidal system (figure 12 .2) has an apex of policy and executive functions, a middle level of management personnel and support staff, and a base of the people who produce the output of the organization. the flow of information is generally one way, from the bottom to the top level, where decisions are made for the detailed performance of duties at all levels. lateralizing the information systems so that essential data can be shared to help staff at the middle and field or factory-floor levels of management is generally discouraged because this may promote decentralized rather than centralized management. even these types of organization have increasingly come to emphasize small-group loyalty, leadership initiative, and self-reliance. the bureaucratic organization has the following characteristics: l there is a fixed division of labor with a clear jurisdiction and based on assignments, which are subject to change by the leader. l there is a hierarchy of offices, with each lower functionary controlled and supervised by a higher one. employment is viewed as a tenured career for officials, after an initial trial period. the bureaucratic system, based on formal rationality, structure, and discipline, is widely used in production, service, and governmental agencies, including military and civilian departments and agencies. health systems, like other organizations, are dynamic and require continuous management, adjustment, and systems control. continuous monitoring and feedback, evaluation, and revision help to meet individual and community needs. the input-process-output model (figure 12 .3) depends on feedback systems to make the administrative or educational changes needed to keep moving towards the selected objectives and targets. organizations use resources or inputs that are processed to achieve desired results or outputs. the resource inputs are money, personnel, information, and supplies. process is the accumulation of all activities taken to achieve the results intended. output, or outcome, is the product, its marketing, its reputation and quality, and profit. in a service sector such as health, output or impact can be measured in terms of reduced morbidity and/or mortality, improved health, or number of successfully treated and satisfied patients at affordable costs. the management system provides the resources and organizes the process by which it hopes to achieve the established goals. program implementation requires systematic feedback for the process to work effectively. when targets are set and strategy is defined, resources, whether new or existing, are placed at the service of the new program. management is then responsible for using the resources to achieve the intended targets. the results are the outcome or output measures, which are evaluated and fed back to the input and process levels. health systems consist of many subsystems, each with an organization, leaders, goals, targets, and internal information systems. subsystems need to communicate within themselves, with peer organizations, and with the macro (health) system. leadership style is central to this process. the surgeon as the leader of the team in the operating room depends on the support and judgment of other crucial people on the team, such as anesthesiologists, operating room nurses, pathologists, radiologists, and laboratory services, all of whom lead their own teams. hospital and public health directors cannot function without a high degree of decentralized responsibility and a creative team approach to quality development of the facility. health systems management includes analysis of service policy, budget, decision-making in policy, as well as operation, regulation, supervision, provision, maintenance, ethical standards, and legislation. policy formulation involves a set of decisions made in pursuit of a course of action for achieving selected health targets, such as those in the mdgs or continuing to update healthy people 2020 health targets in the usa (see chapter 2). cybernetics, a term coined by norbert wiener, refers to systems or organizations which are dependent on each other to function, and whose interdependence requires flexibility of response. cybernetics gained wide credence in engineering in the early 1950s, and feedback systems became part of standard practice of all modern management systems. its later transformations appeared in operating service systems, as information for management. application of this concept is entering the health sector. rapid advances in computer technology, by which personal computers have access to internet systems and large amounts of data, have already enhanced this process. in mechanistic systems, the behavior of each unit or part is constrained and limited; in organic systems, there is more interaction between parts of the system. the example used in figure 12 .4 is the use of a thermostat to control the temperature and function of a heater according to conditions in the room. this is also described as a feedback system. cybernetics opens up new vistas on the use of health information for managing the operation of health systems. a database for each health district would allow assessment of current epidemiological patterns, with appropriate comparisons to neighboring districts or regional, state, and national patterns. data would need to be processed at state or national levels in comparable forms for a broad range of health status indicators. furthermore, the data should be prepared for online availability to local districts in the form of current health profiles. thus, data can be aggregated and disaggregated to meet the management needs of the service, and may be used to generate real targets and measure progress towards meeting them. a geographic information system may demonstrate high rates of a disease in a region due to local population risk factors, and thus become the basis for an intervention program. in the health field, the development of reporting systems based on specific diseases or categories has been handicapped by a lack of integrative systems and a geographic reporting approach. the technology of computers and the internet should be used to process data systems in real time and in a more user-friendly manner. this would enable local health authorities and providers to respond to actual health problems of the communities. health is a knowledge-based service industry, so that knowledge management and information technology are extremely important parts of the new public health, not only in patient care systems in hospitals, but also in public health delivery systems in the community, school, place of work, and home. mobilization of evidence and experience of best practices for policies and management decisionmaking is a fundamental responsibility of health leaders. the gap between information and action is wide and presents an ethical as well as a political challenge. regions with the most severe health problems lack trained personnel in assessment and exploitation of current state-of-the-art practices and technology in many practical public health fields, including immunization policy and in management of risk factors for stroke. knowledge and evidence are continuously evolving, but the capacity to access and interpret information is commonly poorly implemented in many countries so that very large numbers of people die of preventable diseases even when there are, overall, sufficient resources to address the challenges. international guidelines are vital to help countries to adopt current standards and make use of the available knowledge for public policy. political support and openness to international norms are crucial to this process of technology diffusion and building the physical and human resource infrastructure needed to achieve better population health with current best practices. development of health standards in low-income countries is progressing but is seriously handicapped by low levels of funding, lack of emphasis on training sufficient and appropriate human resource personnel and administrative support to promote measures which can save millions of lives. in high-income countries, the slow adoption of best international health standards can have harsh effects on population health, such as in the long delay in adopting national health insurance in the usa. in the european context, the eu has failed to adopt a harmonized recommended immunization program, which is badly needed for the new and potential members, as well as the older member countries. in countries of the former socialist bloc, mortality rates from stroke and coronary heart disease are slowly declining but remain two to four times higher than in countries of western europe (see chapter 5). systems management requires access to and the use of knowledge to bridge these gaps. adoption and adaptation of knowledge to address local problems are essential in a globalized world, if only to prevent the international spread of threatened pandemics or adoption of unhealthy lifestyles (diet, smoking, and lack of exercise) to middle-income countries, which are developing a growing middle class alongside massive poverty. the application of knowledge and experience that has been successful in leading countries can foster innovation and create experience that may generate a local renewal process. management is crucial to address the complex "strategy areas for improving performance of health organizations: standards and guidelines, organizational design, education and training, improved process, technology and tool development, incentives, organizational culture, and leadership and management" (bradley et al., 2012) . managing a knowledge-based service industry or facility relies on leadership, collaboration to realize the potential of technology, professional skills, and social capital to the address the health problems faced by all countries. the management of resources to achieve productivity and measurable success has been characterized and accompanied by the development of systems of organizing people to create solutions to problems or to innovate towards defined objectives. operations research is a concept developed by british scientists and military personnel in search of solutions for specific problems of warfare during world wars i and ii. the approach was based on the development of multidisciplinary teams of scientists and personnel. the development of the anti-submarine detection investigation committee for underwater detection of submarines during world war i characterized and pioneered this form of research. the famous bletchley park enigma code-breaking success in britain and the manhattan project, in which the usa assembled a powerful research and development team which produced the atomic bomb, are prime world war ii examples. team-and goal-oriented work was very effective in problem solving under the enormous pressure of wartime needs. it also influenced postwar approaches to developmental needs in terms of applied science in such areas as the aerospace and computer industries. the computer hardware and software industries are characterized by innovation conceived and developed through informal working groups with a high level of individual competence, peer group dynamism, and commitment to problem solving. thus, the "nerds" of macintosh and microsoft beat the "suits" of ibm in innovation and introduction of the personal computer. similar startup groups, such as google and facebook, successfully took the internet to startling new levels of global applications, showing the capacity of innovation from california's silicon valley and its counterparts in other places in the usa and worldwide. in the health field, innovation in organization developed prepaid group practice which became the health maintenance organization (hmo), and later the managed care organization (mco), now a major, if controversial, factor in health care provision in the usa. other examples may be found in multidisciplinary research teams working on vaccines or pharmaceutical research, and in the increasingly multidisciplinary function of hospital departments and especially highly interdependent intensive care or home care teams. the business concept of management by objectives (mbo), pioneered in the 1960s, has become a common theme in health management. mbo is a process whereby managers of an enterprise jointly identify its goals, define each individual's areas of responsibility in terms of the results expected, and use these measures as guides for operating the unit and assessing the contributions of its members. the common goals and then the individual unit goals must be established, as well as the organizational structure developed to help achieve these goals. the goals may be established in terms of outcome variables, such as defined targets for reduction of infant or maternal mortality rates. goals may also be set in terms of intervening or process variables, such as achieving 95 percent immunization coverage, prenatal care attendance, or screening for breast cancer and mammography. achievements are measured in terms of relevancy, efficiency, impact, and effectiveness. the mbo approach has been subject to criticism in the field of business management because of its stress on mechanical application of quantitative outcome measures and because it ignores the issue of quality. this approach had great influence on the adoption of the objective of "health for all" by the who, and on the us department of health and human services' 1979 health targets for the year 2000, later as healthy people 2010, and now, based on these experiences and new evidence, renewed as healthy people 2020. targeting diseases for eradication may contribute to institution building by developing experience and technical competence to broaden the organizational capacity. however, categorical programs or target-oriented programs can detract from the development of more comprehensive systems approaches. addressing the mdgs of reducing child and maternal mortality is at odds to some extent with targeting poliomyelitis for eradication and reliance on national immunization days, which distract planning and resource allocation for the buildup of the essential public health infrastructure for the basic immunization system so fundamental to child health. immunization and human immunodeficiency virus (hiv) control draw the major part of donor resources in developing countries, while education for strengthening human resources and infrastructure draw less donor attention. a balance between comprehensive and categorical approaches requires very skilled management. the mdgs agreed to by the un in 2001 as targets for the year 2015 provide a set of measurable objectives and a formula for international aid and for national development planning to help the poorest nations, with the wealthy nations providing aid, education, debt relief, and economic development through fairer trade practices. they are now being reviewed for extension to 2020 based on experience to date, with successes and failures, and recognizing the vital importance of non-communicable diseases as central to the health burden of low-and middleincome countries. management is the activity of coordinating and integrating organizational resources, including people, money, materials, time, and space. the purpose is to achieve defined/ stated objectives as effectively and efficiently as possible. whether in terms of producing goods and profits or in delivering services effectively, management deals with human motivation and behavior because workers are the key to achieving goals. knowledge and motivation of the individual client and the community are also essential for achieving good health. thus, management must take into account the knowledge, attitudes, beliefs, and practices of the consumer as much as or more than those of the people working within the system, as well as the general cultural and knowledge level in the society, as reflected in the media, political opinions, and organizations addressing the issues. management, like medicine, is both a science and an art. the application of scientific knowledge and technology in medicine involves both theory and practice. similarly, management practice involves elements of organizational theory, which, in turn, draws on the behavioral and social sciences and quantitative methodologies. sociology, psychology, anthropology, political science, history, and ethics contribute to the understanding of psychosocial systems, motivation, status, group dynamics, influence, power, authority, and leadership. quantitative methods including statistics, epidemiology, survey methods, and economic theory are also basic to development of systems concepts. comparative institutional analysis helps principles of organization and management to develop, while philosophy, ethics, and law are part of understanding individual and group value systems. organizational theory, a relatively new discipline in health, as an academic study of organizations, addresses health-related issues using the methods of economics, sociology, political science, anthropology, and psychology. the application of organizational theory in health care has evolved and become an integral part of training for, and the practice of, health administration. related practical disciplines include human resources, and industrial and organizational psychology. translation of organizational theory into management practice requires knowledge, planning, organization, mobilization of professional and other staff support for evidence-based best practices, assembly of resources, motivation, monitoring and control. health organizations have become more complex and costly over time, especially in their mix of specializations in science, technology, and professional services. organization and management are particularly crucial for successful application of the principles of the new public health, as it involves integration of traditionally separate health services. delegation of responsibilities in health systems, such as in intensive care units, is fundamental to success in patient care, with nurses taking increasing responsibility for the management of the severely ill patient suffering from multiple system failure. delegation or devolution of health care responsibilities to non-medical practitioners has been an ongoing development affecting nurse practitioners, physician assistants, paramedics, community health workers and others, as discussed in chapter 14. it is a vital process to provide needs not met by physicians because of shortages and inappropriate location or specialty preferences that leave primary care or other medical specialties unable to meet community and patient needs. elton mayo of the harvard school of business carried out a series of observational studies at the hawthorne, illinois, plant of the western electric company between 1927 and 1932. mayo and his industrial engineer, along with psychologist colleagues, made a major contribution to the development of management theory. mayo began with industrial engineering studies of the effect of increased lighting on production at an assembly line. this was followed by other improvements in working conditions, including reduced length of the working day, longer rest periods, better illumination, color schemes, background music, and other factors in the physical environment. these studies showed that production increased with each of these changes and improvements. however, the researchers discovered, to their surprise, that production continued to increase when the improvements were withdrawn. furthermore, in a control group where conditions remained the same, productivity also grew during the study period. these results led mayo to conclude that the performance of workers improved because of a sense that management was interested in them, and that worker participation contributes to improved production. traditionally, industrial management viewed employees as mechanistic components of a production system. previous theory was that productivity was a function of working conditions and monetary incentives. what came to be known as the hawthorne effect showed the importance of social and psychological factors on productivity. formal and informal social organizations among management and employees were recognized as key elements in productivity, now called industrial humanism. research methods adapted from the behavioral sciences contributed to scientific studies in industrial management. traditional theories of the bureaucratic model of organization and management were modified by the behavioral sciences. this led to the emergence of the systems approach, or scientific analysis to analyze complex structures or organizations, taking into account the mutually interdependent elements of activities, interactions, and interpersonal relationships between management and workers. some revisits to the hawthorne studies suggest that the data do not support the conclusions, and offer a different interpretation. one is that informal groups such as workers on a production line themselves set standards for work which assert an informal social control outside the authority system of the organization. the informal cohesive group can thus control the norms of the amount of work acceptable to the group, i.e., not "too much" and not "too little". others point out that the effects were temporary and that there were extraneous factors, but the added value of the hawthorne effect remains part of the history of and had a culturechanging effect on management theory. the hawthorne effect in management is in some ways comparable to the placebo effect in clinical research and health care practice. it is also applied to clinical practice, whereby medical care provided by doctors is measured for specific "tracer conditions" to assess completeness of care according to current clinical guidelines. review of clinical records has been shown to be a factor in improving performance by doctors in practice, such as in treatment of acute myocardial infarction, management of hypertension, or completeness of carrying out preventive procedures such as screening for cancer of the cervix, breast, or colon (see chapters 3 and 15). awareness of being studied is a factor in improved performance or response to an intervention. studies of clinical practice-based research or public health interventions need to consider whether different types of studies and outcomes are more or less susceptible to the hawthorne effect (fernald et al., 2012 ). abraham maslow's hierarchy of human needs made an important contribution to management theory. maslow was an american psychologist, considered "the father of humanism" in psychology. maslow defined a prioritization of human needs (figure 12 .5), starting with those of basic physical survival; at higher levels, human needs include social affiliation, self-esteem, and self-fulfillment. others in the hierarchy include socialization and self-realization; later revisions include cognitive needs. the survival needs of an employee include a base salary and benefits, including health insurance and pension; the safety and security needs include protection from injury, toxic exposure or excess stress; social needs at work include an identity, pride, friendships, union solidarity, company social activities and benefits; esteem and recognition include job titles, awards, and financial rewards for achievement by individuals, groups, or all employees; and self-actualization includes promotion to more challenging jobs with benefits, both financial and in terms of recognition. this concept is important in terms of management because it identifies human needs beyond those of physical and economic well-being. it relates them to the social context of the work environment with needs of recognition, satisfaction, self-esteem, and self-fulfillment. maslow's conclusions opened many positive areas of management research, not only in the motivation of workers in production and service industries, but also in the motivation of consumers. maslow's hierarchy of human needs contributed to the idea that workers' sense of well-being is important to management. his theories played an important role in application of sociological theory to client behavior, just as the topic of personal lifestyle in health became a central part of public health and clinical management of many conditions, such as in risk factor reduction for cardiovascular diseases. this concept fits well with the epidemiological studies referred to in the introduction, such as those showing strong relationships with sociopolitical factors as well as socioeconomic conditions. theory x-theory y (table 12 .1), developed by clinical psychologist and professor of management douglas mcgregor in the 1960s, examined two extremes in management assumptions about human nature that ultimately affect the operations of organizations. organizations with centralized decision-making, a hierarchical pyramid, and external control are based on certain concepts of human nature and motivation. mcgregor's theory, drawing on maslow's hierarchy of needs, describes an alternative set of assumptions that credit most people with the capacity for self-direction. traditional approaches to organization and management stress direction and external control. theory x assumes that workers are lazy, unambitious, uncreative, and motivated only by basic physiological needs or fear. theory y places stress on integration and self-control. this model provides a more optimistic leadership model, emphasizing management development programs and promoting human potential, assuming that, if properly motivated, people can be self-directed and creative at work, and that the role of management is to unleash this potential in workers with performance appraisal. many other theories of motivation and management have been developed to explain human behavior and how to utilize inherent skills to produce a more creative work environment, reduce resistance to change, reduce unnecessary disputes, and ultimately create a more effective organization. variants of the human motivation approach in management carried the concept further by examining industrial organization to determine the effects of management practices on individual behavior and personal growth within the work environment. they describe two contrasting models of workforce motivation. theory x assumes that management produces immature responses on the part of the worker: passivity, dependence, erratically shallow interests, shortterm perspective, subordination, and lack of self-awareness. in contrast, at the other end of the immaturity-maturity spectrum was the mature worker, with an active approach, an independent mind capable of a broad range of responses, deeper and stronger interests, a long-term perspective, and a high level of awareness and self-control. this model has been tested in a variety of industrial settings, showing that giving workers the opportunity to grow and mature on the job helps them to satisfy more than basic survival needs and allows them to use more of their potential in accomplishing organizational goals. this model became widely influential in human resource management theory of organizational behavior, organizational communication, and organizational development, and in the practical management of business and service enterprises. in the motivation to work (1959) , us clinical psychologist frederick herzberg wrote of his motivationhygiene theory. he developed this theory after extensive studies of engineers and accountants, examining what he called hygiene factors (i.e., administrative, supervisory, monetary, security, and status issues in work settings). his motivating factors included achievement, recognition of accomplishment, challenging work, and increased responsibility with personal and collective growth and development. he proved that the motivating factors had a substantial positive effect on job satisfaction. these human resource theories of management helped to change industrial approaches to motivation from "job enrichment" to a more fundamental and deliberate upgrading of responsibility, scope, and challenge of work, by letting workers develop their own ways of achieving objectives. even when the theories were applied to apparently unskilled workers, such as plant janitors, the workers changed from an apathetic, poorly performing group into a cohesive, productive team, taking pride in their work and appearance. this approach gave members of the team the opportunity to meet their human self-actualization needs by taking greater responsibility for problem solving, and it resulted in less absenteeism, higher morale, and greater productivity with improved quality. rensis likert, with mcdougal and herzberg, helped to pioneer the "human relations school" in the 1960s, applying human resource theory to management systems and styles. likert classified his theory into four different systems, as follows. l system 1 -management has no confidence or trust in subordinates, and avoids involving them in decisions and goal setting, which are made from the top down. management is task oriented, highly structured, and authoritarian. fear, punishment, threats, and occasional rewards are the principal methods of motivation. worker-management interaction is based on fear and mistrust. informal organizations within the system often develop that lead to passive resistance of management and are destructive to the goals of the formal organization. l system 2 -management has a condescending relationship with subordinates, with some degree of trust and confidence. most decisions are centralized, but some decentralization is permitted. rewards and punishments are used for motivation. informal organizations become more important in the overall structure. l system 3 -management places a greater degree of trust and confidence in subordinates, who are given a greater degree of decision-making powers. broad policy remains a centralized function. l system 4 -management is seen as having complete confidence in subordinates. decision-making is dispersed, and communication flows upward, downward, and laterally. economic rewards are associated with achieving goals and improving methods. relationships between management and subordinates are frequent and friendly, with a sense of teamwork and a high degree of mutual respect. case studies showed that a shift in management from likert system 1 towards system 4 radically changed the performance of production, cut manufacturing costs, reduced staff turnover, and increased staff morale. furthermore, workers and managers both shared a concern for the quality of the product or service and the competitiveness and success of their business. the health industry includes highly trained professionals and paraprofessional workers who function as a team with a high degree of cohesion, mutual dependence, and autonomy, such as a surgical or an emergency department team. the network, or task-oriented working group, is basically a more democratic and participatory form of organization meant to elicit free interchange of concerns and ideas. this is a more organic form of organization, best suited to be effective for adaptation when the environment is complex and dynamic, when the workforce is largely professional, and when the technology and system functions change rapidly. complexities and technological change require information, expertise, flexibility, and innovation, strengths best promoted in free exchange of ideas in a mutually stimulating environment. in a network organization, leadership may be formal or informal, assigned to a particular function, which may be temporary, medium term, or permanent, to achieve a single defined task or develop an intersectoral program. the task force is usually for a short-term specific assignment; a working group, often for a medium-term project, such as integrating services of a region; and a committee for permanent tasks such as monitoring an immunization program. significant advantages of this form of organization are the challenge and the sharing of information and responsibility, which give professionals responsibility and job satisfaction by providing the opportunity to demonstrate their creativity. members of the task force may each report within their own pyramidal structure, but as a group they work to achieve the assigned objective. they may also be interdisciplinary or interagency working groups to review the state of the art in this particular issue as documented in reports and professional literature, and to coordinate activities, review previous work, or plan common future activities. an ongoing network organization may be a government cabinet committee to coordinate government policy and the work of various government departments, or a joint chiefs of staff to coordinate the various armed services. this approach is commonly used for task groups wherein interdisciplinary teams of professionals meet to coordinate functions of a department in a hospital, or where a multidisciplinary group of experts is established with the specified task of a technical nature. network organizational activity is part of the regular functions of a health professional. informal networking is a day-to-day activity of a physician in consultations with colleagues and also a part of more formalized network groups. the hospital department must, to a large extent, function as a network organization with different professionals working as a team more effectively than would be possible in a strictly authoritarian pyramidal model. a ministry of health may need to develop a joint working group with the ministry of transport, the police, and those responsible for standards of motor vehicles to seek ways to reduce road accident deaths and injuries. if a measles eradication project is envisioned, a multidisciplinary and multiorganizational team, or a network, should be established to plan and carry out the complex of tasks needed to achieve the target (figure 12.6) . in a public health context, a task group to determine how to reduce obesity rates in school-aged children, or to eradicate measles locally, might be chaired by the deputy chief medical officer or senior health promotion person; if the project is reduction of obesity among school children, the lead agency may be the department of education, perhaps jointly with the local department of health; if reduction in road traffic deaths is the topic, the lead may be the police department with participation of emergency transportation and hospital emergency room lead personnel. members may include the chief district nurse, an administrative and budget officer, a pharmacist, the chief of the pediatric department of the district hospital, a primary school administrator, a health educator, a medical association representative, the director of laboratories, the director of the supply department, a representative of the department of education, representatives of voluntary organizations interested in the topic, and others as appropriate. most organizational structures are mixed, combining elements of both the formal pyramidal and the less structured network structure with a task-oriented mandate. it is often difficult for a rigid pyramidal structure to deal with parallel bodies in a structured way, so the network approach is necessary to establish working relations with outside bodies to achieve common goals. a network is a democratic functional grouping of those professionals and organizations needed to achieve a defined target, sometimes involving people from many different organizations. the terms of reference of the working group are crucial to its function as well as its composition, time-frame, and access to relevant information. the application of this concept is increasingly central in health care organization as multilevel health systems evolve in the form of managed care or district health systems. these are vertically integrated management systems involving highly professional teams and units whose interdependence for patient care and financial responsibility are central elements of the new public health. in the usa during world war ii, w. edwards deming, a physicist and statistician, developed a system of economic and statistical methods of quality control in production industries. following the war, deming was invited to teach in japan and moved from the university to the level of industrial management. japanese industrialists adopted his principles of management and introduced quality management into all industries, with astonishingly successful results within a decade. the concept, later called total quality management (tqm), has since been adopted widely in production and service industries. in the deming approach to company management, quality is the top priority and is the key responsibility of management, not of the workers. if management sets the tone and involves the workers, quality goes up, costs come down, and both customer satisfaction and loyalty increase. having their ideas listened to, and avoiding a punitive inspection approach, enhances the pride of the workers. it is the responsibility of leadership to remove fear and build mutual participation and common interest. training is one of the most important investments of the organization. the differences between traditional management and the tqm approach are shown in boxes 12.1 and 12.2. in societies with growing economies, the role of an educated workforce becomes greater as information technology and services, such as health, become larger parts of the economy and require professionalism and self-motivating workers. the tqm approach integrates the scientific management and human relations approaches by giving workers credit for intellectual capacity and expects them to use it to analyze and improve the tasks they perform. even more, this approach expects workers at all levels to contribute to better quality in the process of design, manufacture, and even marketing of the product or the service. the tqm ideas were revolutionary and successful when applied in business management in production industries. the tqm concept is much in discussion in the service industries. the who has adapted tqm to a model called continuous quality improvement (cqi), with the stress on mutual responsibilities throughout a health system for quality of care. the application of tqm and cqi approaches is discussed in chapter 15, including the external regulatory and self-development tqm approaches. in the health sector, issues such as prevention of health facility-acquired infections require staff dedicated to promoting a culture of cleaning, frequent and thorough hand washing, sterilization, isolation techniques, intravenous and intratracheal catheter and tube care technique, and immunization of hospital personnel. these and many other crossdisciplinary measures promote patient safety and prevent the costly and frequently deadly effects of serious respiratory or urinary tract injection acquired in hospitals or other health care facilities. human behavior is individual but takes place in a social context. changes to individual behavior are needed to reduce risk factors for many diseases. change can be threatening; it requires alteration, substitution, transformation, or modification of purposes, procedures, methods, or style. the implementation of plans usually requires some change, which often meets resistance. the resistance to change may be professional, technical, psychological, political, emotional, or a mix of all of these. the manager of a health facility or service has to cope with change and gather the support of those involved to participate in creating or implementing the change effectively. the behavior of the worker in a production or service industry is vital to the success of the organization. equally important is the behavior of the purchaser or consumer of the product or service. diagnosing organizational problems is an important skill to bring to leadership in health systems. even more important is the ability to identify and alter the variables that require change and adaptation to improve the performance of the organization. high expectations are essential to produce high performance and improved standards of service or productivity. conversely, low expectations not only lead to low performance, but produce a downward spiraling effect. this applies not only within the organization, but to the individuals and community served, l judgment, punishment, and reward for above-or belowaverage performance destroy teamwork essential for quality production. l work with suppliers to improve quality and costs. l profits are generated by loyal customers -running a company for profit alone is like driving a car by looking in the rearview mirror. whether in terms of purchase of goods produced or in terms of health-related behavior. people often resist change because of fear of the unknown. participation in the process of defining problems, formulating objectives, and identifying alternatives is needed to bring about changes. change in organizational performance is complex, and this is the test of leadership. similarly, change at the individual level is essential to achieve the goals of the group, whether this is in terms of the functioning of a health care service unit, such as a hospital, or whether it is an individual's decision to change from smoking to non-smoking status. the health of both an individual and a population depends on the individual health team member's motivation and experience. the behavior of the individual is important to his or her personal and community health. even small steps in the direction of a desirable change in behavior should be rewarded as soon as possible (i.e., reinforcing positive performance in increments). behavior modification is based on the concept that change of behavior starts with the feelings and attitudes within the individual, but can be influenced by knowledge, peer pressure, media coverage, and legislative standards. change involves a number of elements to define a current or previous starting point: change in behavior is vital in the health field: in the organization, in the community, in individual behavior, and in societal regulation and norms. the health belief model (chapter 2) is widely influential in psychology and health promotion. the belief intervention approach involves programs meant to reduce risk factors for a public health problem. it may require change in the law and in organizational behavior, with involvement and feedback to the people who determine policy, those who manage services, and the community being served. obesity in school-aged children is being fought by many measures including healthier menus and banning the sale of high sugar drinks on school property. high cholesterol is being fought on many fronts including dietary change and banning the use of transfats in food processing. deaths from bulimia are not uncommon and may stem from teenage identification of beauty with ultrathin body image. banning television and modeling agencies from using models with a very low body mass index is an intervention in advertising which encourages harmful practices that are a danger to health and life. banning cigarette advertising and smoking in public places promotes behavioral change, as does raising the taxes on cigarettes. gun control laws are meant to prevent disturbed individuals or political fanatics having easy access to firearms to commit mass murder. strict enforcement of drinking and driving laws can prevent drunk driving and reduce road traffic deaths (see chapter 15). in the 1980s, major industries in the usa were unable to compete successfully with the japanese in the consumer electronics and automobile industries. management theory began to place greater emphasis on empowerment as a management tool. the tqm approach stresses teamwork and involvement of the worker in order to achieve better quality of production. comparatively, empowerment went further to involve the worker in operation, quality assessment, and even planning of the design and production process. results in production industries were remarkable, with increased efficiency, less absenteeism, and greater searching for ideas to improve quality and quantity of production, with the worker as a participant in the management and production process. the concept of empowerment entered the service industries with the same rationale. the rationale is that improvements in quality and effectiveness of service require the active physical and emotional participation of the worker. participation in decision-making is the key to empowerment. this requires management to adopt new methods that allow the worker, whether professional or manual, to be an active participant. successful application of the empowerment principles in health care extends to the patient, the family, and the community, emphasizing patients' rights to informed participation in decisions affecting their medical care, and the protection of privacy and dignity. diffusion of powers occurs when management of services is decentralized. delegation of powers to professional groups, non-governmental organizations (ngos), and advocacy organizations is part of empowerment in health care organizations. governmental powers to govern or promote areas such as licensure, accreditation, training, research, and service can be devolved to local authorities or ngos by delegation of authority or transfer of funds. organizational change may involve decentralization. institutional changes such as amalgamation of hospitals, long-term care facilities, home care programs, day surgery, ambulatory care, and public health services are needed to produce a more effective use of resources. integration of services under community leadership and management should encourage transfer of funds within a district health network from institutional care to community-based care. such changes are a test of leadership skills to achieve cultural change within an organization, which requires behavioral change and involvement of health workers in policy and management of the change process. strategic management emphasizes the importance of positioning the organization in its environment in relation to its mission, resources, consumers, and competitors. it requires development of a plan of action or implementation of a strategy to achieve the mission or goal of the organization within acceptable ethical and legal guidelines. articulation of these is a key role of the management level of an organization. defining the mission and goals of the organization must take into account the external and internal environment, resources, and operational needs to implement and evaluate the adequacy of the outcomes. the strategy of the organization matches its internal approach with external factors, such as consumer attitudes and competing organizations. strategy is a set of methods and skills of the health care manager to attain the objectives of a health organization, including: policy is the formulation of objectives and priorities. strategy refers to long-range plans to achieve stated objectives, indicating the problems to be expected and how to deal with them. strategy does not identify all actions to be taken, but it includes evaluation of progress made towards a stated goal. while the term has traditionally been used in a military context, it has become an essential concept in management, whether of industry, business, or health care. tactics are the methods used to fulfill the strategy. thus, strategic mbo is applicable to the health system, incorporating definitions of goals and targets, and the methods to achieve them (box 12.3). change in health organizations may involve a substantial alteration in the size or relationships between existing, well-established facilities and programs (table 12. 2). a strategic plan for health reform in response to the need for cost containment, redefined health targets, or dissatisfaction with the status quo requires a model or a vision for the future and a well-managed program. opposition to change may occur for psychological, social, and economic reasons, or because of fear of loss of jobs or changes in assignments, salary, authority, benefits, or status. downsizing in the hospital sector, with buildup of community health services, is one of the major issues in health reforms in many countries. it can be accomplished over time by naturally occurring vacancies or attrition due to retirement, or by retraining and reassignment, all of which require skilled leadership. the introduction of new categories of health workers in hospitals such as phlebotomists, hospitalist doctors, and technicians of all kinds has improved hospital efficiency and safety. community health has benefited from home care and in many situations community health workers to assist and supervise patient care in remote rural villages and in urban centers, even in high-income countries, with health guides trained to help people to function with chronic illnesses and dementias (see chapter 14). the new public health is an integration or coordination of many participating health care facilities and health-promoting programs. it is evolving in various forms in different places as networks with administrative and financial interaction between participating elements. each organization provides its own specific services or groups of services. how they function internally and how they interact functionally and financially are important aspects of the management and outcomes of health systems. the health system functions as a network with formal and informal relationships; it may be very broad and loosely connected as in a highly decentralized system, with many lines of communication, payment, regulation, standards setting, and levels of authority. the relationship and interchange between different health care providers have functional and economic elements. as an example, an educated adult woman is more likely than an uneducated woman to prepare herself for the requirements of pregnancy by smoking and alcohol or drug cessation, folic acid intake, healthful diet, and attending professional antenatal care. a pregnant woman who is healthy and prepared for pregnancy physically and emotionally, and who receives comprehensive prenatal care, is less likely than a woman whose health is neglected to develop complications and require prolonged hospital care as a result of childbirth. the cost of good prenatal care is a fraction of the economic cost of treating the potential complications and damage to her health or that of the newborn. a health system is responsible for ensuring that a woman of reproductive age takes folic acid tablets orally before becoming pregnant, has had access to family planning services so that the pregnancy is a desired one, ensures that the space between pregnancies is adequate for her health and that of her baby, and receives adequate prenatal care. an obstetrics department should be involved in assuring or providing the prenatal care, especially for high-risk cases, and delivery should be in hygienic and professionally supervised settings. similarly, for children and elderly people, there is a wide range of public health and personal care services that make up an adequate and cost-effective set of services and programs. the economic burden of caring for the sick child falls on the hospital. when there is a per capita grant to a district, the hospital and the primary care service have a mutual interest in reducing morbidity and hence mortality. this is the principle of the hmos and district health systems discussed elsewhere. it is also a fundamental principle of the new public health. health care organizations differ according to size, complexity, ownership, affiliations, types of services, and location. traditionally, a health care organization provides a single type of service, such as an acute care hospital providing episodic inpatient care, or a home health care agency. in present-day health reforms, health care organizations, such as an hmo or a district health system, provide a populationbased, comprehensive service program. each organization must have or develop a structure suited to meet its goals, in both the internal and external environments. the common elements that each organization must deal with include governance of policy, production or service, maintenance, financing, relating to the external environment, and adapting to changing conditions. a functional model of an organization perhaps best suited to the smaller hospital is the division of labor into specific functional departments; for example, medical, nursing, administration, pharmacy, maintenance, and dietary, each reporting through a single chain of command to the chief executive officer (ceo) (figure 12.7) . the governing agency, which may be a local non-profit board or a national health system, has overall legal responsibility for the operation and financial status of the hospital, as well as raising capital for improvements. the medical staff may be in private practice and work in the hospital with their own patients by application for this right as "attending physician", according to their professional qualifications, or the medical staff may be employed by the hospital in a similar way to the rest of the staff. salaried medical staff may include physicians in administration, pathology, anesthesia, and radiology, so that even in a private practice market system many medical staff members are hospital employees. increasingly, hospitals are employing "hospitalists", who are full-or part-time physicians whose work is in the health facility, to provide continuity of inpatient and emergency department services, augmenting the services of senior or attending staff or private practice physicians. this shift is in part related to the increasing numbers of female physicians who run their homes and families as well as practice medicine and who find this mode of work more attractive than full-time private practice. this model is the common arrangement in north american hospitals. the governing board of a "voluntary", nongovernmental, not-for-profit organization with municipal and community representatives may be appointed by a sponsoring religious, municipal, or fraternal organization. the corporate model in health care organization (figure 12.8) is often used in larger hospitals or where mergers with other hospitals or health facilities are taking place. the ceo delegates responsibility to other members of the senior management team who have operational responsibility for major sectors of the hospital's functioning. a variation of the corporate model is the divisional model of a health care organization based on the individual service divisions allowing middle management a high degree of autonomy (figure 12.9 ). there is often departmental budgeting for each service, which operates as an economic unit; that is, balancing income and expenditures. each division is responsible for its own performance, with powers of strategic and operational decision-making authority. this model is used widely in private corporations, and in many hospitals in the usa. with increasing complexity of services, it is also employed in corporate health systems in the usa, with regional divisions. the matrix model of a health care organization is based on a combination of pyramidal and network organization. this model is suited to a public health department in a state, county, or city. individual staff people report in the pyramidal chain of command, but also function in multidisciplinary teams to work on specific programs or projects. a nutritionist in the geriatric department is responsible to the chief of nutrition services but is functionally a member of the team on the geriatric unit. in a laterally integrated health maintenance organization or district health system, specialized staff may serve in both institutional (i.e., hospital) and community health roles (figure 12.10) . the organizational structure appropriate to one set of circumstances may not be suitable for all. whether the payment system is by norm (i.e., by predetermined numbers of staff, their salaries, and fixed costs for all services), per diem (i.e., payment of a daily rate times the number of days of stay), historical budget, or per capita in a regional or district health system structure (see chapters 10 and 11), the internal operation of a hospital will require a model of organization appropriate to it. hospitals need to modify their organizational structure as they evolve, and as the economics of health care change. leadership in an organization requires the ability to define the goals or mission of the organization and to develop a strategy and define steps needed to achieve these goals. it requires an ability to motivate and engender enthusiasm for this vision by working with others to gain their ideas, their support, and their participation in the effort. in health care as in other organizations, it is easier to formulate plans than to implement them. change requires the ability not only to formulate the concept of change, but also to modify the organizational structure, the budgeted resources, the operational policies and, perhaps most importantly, the corporate culture of the organization. management involves skills that are not automatically part of a health professional's training. skilled clinicians often move into positions requiring management skills in order to build and develop the health care infrastructure. in some countries, hospital managers must be physicians, often senior surgeons. clinical capability does not transfer automatically into management skills to deal with personnel, budgets, and resources. therefore, training in management is vital for the health professional. the manager needs training for investigations and factfinding as well as the ability to evaluate personnel, programs, and issues, and set priorities for dealing with the short-and long-term issues. negotiating with staff and outside agencies is a constant activity of the manager, ranging from the trivial to major decisions with wide implications. perhaps the most crucial skill of the manager is communication: the ability to convey verbal, written, or unwritten messages that are received and understood and to assess the responses as an equal part of the exchange. interpersonal skills are a part of management practice. the capable manager can relate to personnel at all levels in an open and equal manner. this skill is essential to help foster a sense of pride and involvement of all personnel in working towards the same goals and objectives, and to show that each member of the team is important to meeting the objectives of the organization. at the same time, the manager needs to communicate information, especially as to how the organization is doing in achieving its objectives. the manager is responsible for organizing, planning, controlling, directing, and motivating. managers assume multiple roles. a role is an organized set of behaviors. henry mintzberg described the roles needed by all managers: informational, interpersonal, and decisional roles. robert katz (1974) identified three managerial skills that are essential to successful management: technical, human, and conceptual: "technical skill involves process or technique knowledge and proficiency. managers use the processes, techniques and tools of a specific area. human skill involves the ability to interact effectively with people. managers interact and cooperate with employees. conceptual skill involves the formulation of ideas. managers understand abstract relationships, develop ideas, and solve problems creatively". technical skill deals with things, human skill concerns people, and conceptual skill has to do with ideas. the distribution of these skills between the levels of management is shown in figure 12 .11. hospital directors in the past were often senior physicians, often called superintendents, without training in health management. the business manager ceo has become common in hospital management in the usa. during the 1950s, the ceo was called an administrator, and worked under the direction of a board of trustees who raised funds, set policies, and were often involved in internal administration. where the ceo was a non-physician, the usual case in north american hospitals, a conflict often existed with the clinical staff of the hospital. in some settings, this led to appointment of a parallel structure with a full-time chief of medical staff with a focus on clinical and qualitative matters. in european hospitals, the ceo is usually a physician, often by law, and the integration of the management function with the role of clinical chief is the prevalent model. over time, as the cost and complexity of the health system have increased, the ceo role has changed to one of a "coordinator". the ceo is now more involved in external relations and less in the day-to-day operation of the facility. the ceo is a leader/partner but primus inter pares, or first among equals, in a management team that shares information and works to define objectives and solve problems. this de-emphasizes the authoritarian role and stresses the integrative function. the ceo is responsible for the financial management of operational and capital budgets of the facility, which is integral to the planning and future development of the facility. budgets include four main factors: income, fixed or regular overhead, variable or unpredictable overhead, and capital or development costs, all essential to the survival and development of the organization. the key role of top management is to develop a vision, goals, and targets for the institution, to maintain an atmosphere and systems to promote the quality of care, financial solidity, and to represent the institution to the public. the overall responsibility for the function and well-being of the program is with the ceo and the governing board of directors. community participation in management of health facilities has a long-standing and constructive tradition. the traditional hospital board has served as a mechanism for community participation and leadership in promoting health facility development and management at the community level. the role of hospital boards evolved from primarily a philanthropic and fund-raising one to a greater overall responsibility for policy and planning function working closely with management and senior professional staff. this change occurred as operational costs increased rapidly, as government insurance schemes were implemented, and as court decisions defined the liability of hospitals and reinforced the broadened role of governing boards in malpractice cases and quality assurance. centrally developed health systems such as the uk's nhs have promoted district and county health systems with high degrees of community participation and management, both at the district level and for services or facilities. the role of local authorities, as well as state and national governments, is crucial to the functioning of public health in its traditional issues such as safe water supply, sanitation, business licensing, social welfare, and many others, as discussed in chapter 10. these functions have not diminished with the greater roles of state and federal or national governments in health. in healthful living environments the local authority functions are of continuing and indeed expanding importance, as in urban planning and transportation, promoting easy access to commercial facilities for shopping and healthy food sources for poorer sections as well as those available to prosperous members of the community. advocacy has always been an important part of public health. an illustration of this is seen in box 12.4 in changing the law banning birth control in massachusetts in the 1960s. the issue of birth control still casts a heavy burden on women globally owing to religious objections, so this example from the 1960s is still relevant as a political issue both in the usa and in many other countries. community participation can be crucial to the success of an intervention to promote community health. sensitivity to local, religious, or ethnic concerns is part of planning any study or intervention in public health. this does not mean that the national, state, and local health authorities must continuously canvass public opinion, but there is advantage in holding referenda on some issues compared to governmental fiat. the usa has higher rates of fluoridation than most countries, and this is implemented after referenda in each municipality (see chapter 7). in portland, oregon, the city council profluoridation vote in 2012 (new york times, 12 september 2012) was later rejected in the public referendum. portland is the only major american city without fluoridation (portland tribune, 21 may 2013). rationalization of health facilities increasingly means organizational linkages between previously independent facilities. mergers of health facilities are common events in many health systems. in the usa, there are frequent mergers between hospitals, or between facilities linked to hmos or managed care systems. health reform in many countries is based on similar linkages. governmental approval and alteration to financing systems are needed to promote linkages between services to achieve greater efficiency and improve patient care (see chapters 10 and 11). lateral integration is the term used for amalgamation among similar facilities. like a chain of hotels, in health care this involves two or more hospitals, usually meant to achieve cost savings, improve financing and efficiency, and reduce duplication of services. urban hospitals, both notfor-profit as well as for-profit, often respond to competition by purchasing or amalgamating with other hospitals to increase market share in competitive environments. this is often easier for hospital-oriented ceos and staff to comprehend and manage, but it avoids the issues of downsizing and integration with community-based services. vertical integration describes organizational linkages between different kinds of health care facilities to form integrated, comprehensive health service networks. this permits a shift of emphasis and resources from inpatient care to long-term, home, and ambulatory care, and is known as the managed care or district health system model. community interest is a factor in promoting change to integrate services, which can be a major change for the management culture, especially of the hospital. the survival of a health care facility may depend on integration with appropriate changes in concepts of management. in the 1990s, a large majority of california residents moved to managed care programs because of the high cost of fee-for-service indemnity health insurance and because of federal waivers to promote managed care for medicare and medicaid beneficiaries. independent community hospitals without a strong connection to managed care organizations (mcos) were in danger of losing their financial base. hospital bed supplies were reduced in the usa however, the article served to stimulate the legislature to revisit the law, leading to its repeal in 1966, thus allowing use of all methods of birth control. the controversy subsided and women were free to control their own fertility as a result of this advocacy. by diagnosis-related group (drg), rather than on a per diem basis. similar trends are seen in european countries, although in the commonwealth of independent states the number of hospital beds declined between 1990 and 2005-2011 but stabilized at high and inefficient levels (8 beds per 1000 population) compared to the number in western europe, which fell from 5 beds per 1000 in 1990 to 3.4 in 2011, and in some countries to 2 per 1000 population despite increased longevity and aging of the population. there was a shift to stronger ambulatory care, as occurred throughout the industrialized countries despite an aging of the population. these trends were largely due to greater emphasis on ambulatory surgery and other care, and major medical centers responded with strategic plans to purchase community hospitals and develop affiliated medical groups and contract relationships with managed care organizations to strengthen their "market share" service population base for the future. the new payment environment and managed care also promoted hospital mergers (lateral integration) and linkages between different levels of service, such as teaching hospitals with community hospitals and primary community care services (vertical integration). vertical integration not only is important in urban areas, but can serve as a basis for developing rural health care in both developed and developing countries. the district hospital and primary care center operating as an integrated program can provide a high-quality program. hospitalcentered health care, common in industrialized countries, has traditionally channeled a high percentage of total health expenditures into hospital services. over recent years, there has been a reduction in hospital bed supply in most industrialized countries, with shorter length of stay, more emphasis on ambulatory care, improved diagnostic facilities, and improved outcomes of care (see chapter 3). expenditures on the hospital component of care have come down to between 40 and 45 percent of total health expenditures in many countries, with a growing proportion going to ambulatory and primary care, and increased percentages to public health. this shift in priorities has been an evolutionary process that will continue, but requires skilled management leadership, grounded in health systems management training and epidemiological knowledge, and skilled negotiating skills to foster primary care and health promotion approaches both within the organization and in relation to outside services, especially preventive services. this shift in policy direction will be fostered in implementation of the ppaca (obamacare), discussed in chapters 10 and 13. managed care systems or accountable care organizations (acos) will integrate hospital and community care and try to limit hospital care by strengthening ambulatory and primary care, and especially preventive care. this will have both economic and epidemiological benefits, but will depend on skilled management to understand and lead in their implementation. much of the rationale for these changes is discussed in the literature and summarized in a 2012 report from the us institute of medicine, entitled "best care at lower cost". this report calls for overhauling the health system in a continuous evolution based on evidence and lessons learned from decades of innovative care systems and research into their workings. the health system needs to relate to other community services with a shared population orientation (institute of medicine, 2012). norms are useful to promote efficient use of resources and promote high standards of care, if based on empirical standards proved by experience, trial and error, and scientific observation. norms may be needed even without adequate evidence, but should be tested in the reality of observation, experience, and experiment. this process requires data for selected health indicators and trained observers free to examine, report, and publish their findings for open discussion among colleagues and peers in proceedings open to the media and the general public. normative standards of planning are the determination of a number per unit of population that is deemed to be suitable for population needs; for example, the number of beds or doctors per 1000 population or length of stay in hospital. many organizations based on the bureaucratic model used norms as the basis for planning and allocation of resources including funding (see chapter 11). this led to payment systems which encouraged greater use of that resource. if a factory is paid by the number of workers and not the number and quality of the cars produced, then management will have no incentive to introduce efficiency or quality improvement measures. if a district or a hospital is paid by the number of beds, or by days of care in the hospital, there is no incentive to introduce alternative services such as same-day or outpatient surgery and home care. performance indicators are measures of completion of specific functions of preventive care such as immunization, mammography, pap smears, and diabetes and hypertension screening. they are indirect measures of economy, efficiency, and effectiveness of a service and are being adopted as better methods of monitoring and paying for a service, such as by paying a premium. general practitioners in the uk receive additional payments for full immunization coverage of the children registered in their practices. a block grant or per capita sum may be tied to indicators that reflect good standards of care or prevention, such as low infant, child, and maternal mortality. incentive payments to hospitals can promote ambulatory services as alternatives to admissions and reduce lengths of stay. limitations of financial resources in the industrialized countries and even more so in the developing countries make the use of appropriate performance indicators of great importance in the management of resources. pay-for-performance is a system of paying for health services developed in the uk for paying general practitioners, with apparently satisfactory results. it is now widely used in the usa. it is defined as "a strategy to improve health care delivery that relies on the use of market or purchaser power. agency for healthcare research and quality (ahrq) resources on pay for performance (p4p), depending on the context, refers to financial incentives that reward providers for the achievement of a range of payer objectives, including delivery efficiencies, submission of data and measures to payer, and improved quality and patient safety" (agency for healthcare research and quality, 2012) . more than half of commercial hmos are using pay-for-performance. recent legislation requires the medicare and medicaid programs to adopt this approach for beneficiaries and providers. as commercial programs have evolved during the past 5 years, the categories of providers (clinicians, hospitals, and other health care facilities), number of measures, and dollar amounts at risk have increased. this method of payment is likely to be promoted in the affordable care act implementation to improve quality and control cost increases in us health care (see chapters 10, 11, and 13). payfor-performance has also been adopted in other countries trying to improve quality of care, such as macedonia (lazarevik and kasapinov, 2012) . social marketing is the systematic application of marketing alongside other concepts and techniques to achieve specific behavioral goals for a social good. initially focused on commercial goals in the 1970s, the concept became part of health promotion activities to address health issues where there was no current biomedical approach, such as in smoking reduction and in safe sex practices to prevent the spread of hiv. social marketing was based initially on commercial marketing techniques but now integrates a full range of social sciences and social policy approaches using the strong customer understanding and insight approach to inform and guide effective policy and strategy development. it has become part of public health practice and policy setting to achieve both strategic and operational targets. a classic example of the success is seen with tobacco reduction strategies in many countries using education, taxation, and legislative restrictions. other challenges in this field include risk behavior such as alcohol abuse through binge drinking, unsafe sex practices, and dietary practices harmful to health. philanthropy and volunteerism have long been important elements of health systems through building hospitals, mission houses, and food provision, and other prototype initiatives on a demonstration basis. this approach has been instrumental in such areas as improved care and prevention of hiv, immunization in underdeveloped countries, global health strategies, and maternal and child health services. during the late twentieth and early twenty-first centuries, a new "social entrepreneurship" was initiated and developed by prominent reform-minded former us president bill clinton, microsoft's bill gates, and the open society institute of george soros. the rotary club international has been a major factor in funding and promoting the global campaign to eradicate poliomyelitis. this has promoted integration and consortia for the promotion of acquired immunodeficiency syndrome (aids) prevention and malaria control in many developing countries. the global alliance for vaccine and immunization (gavi) is a us-based organization which links international public and private organizations and resources to extend access to immunization globally. it includes the united nations children's fund (unicef), who, bilateral donor countries, the vaccine industry, the gates foundation, and other major donors. gavi has made an important contribution to advancing vaccine coverage and adding important new vaccines in many developing countries and regions. these organizations focus funds and activities on promoting improved care and prevention of hiv, tuberculosis, and malaria, along with improved vaccination for children, reproductive health, global health strategies, technologies, and advocacy. these programs generate publicity and raise consciousness at political levels where resource allocations are made. a central feature of these programs is the promotion of "civil society" as active partners in a globalized world of free trade, democracy, and peace. specific initiatives included promoting improved largescale marketing of antiretroviral drugs for the treatment of hiv infection, including price reduction so that developing countries can offer antiretroviral treatment, especially to reduce mother-to-infant transmission. programs have branched out into the distribution of malaria-preventing bed nets, provision of low-cost pharmaceuticals, marketing drugs for the poor, desalination plants, solar roof units, lowcost small loans, and cell phones, mainly in africa. another form of social entrepreneurship that has gained support in the private sector is proactiveness in environmental consciousness to address issues raised by the environmental movement, and public interest for environmental accountability. the automobile industry is facing both public concern and federal legal mandates for improved gas mileage as opposed to public demand for larger cars. hybrid cars using less fuel have been successfully introduced into the market for low-emission, fuel-efficient cars, and electric cars are gradually entering the field. public opinion is showing signs of moving towards promoting environmentally friendly design, marketing, and purchasing practices in energy consumption, conservation practices, and public policy. public opinion and the price of fuel will play a major part in driving governments to legislate energy and conservation policies to address global warming and damage to the environment, with their many negative health consequences. however, such changes must work with public opinion because of the sensitivity of consumers to the price of fuel. in addition, when food crops, such as corn, are used to produce ethanol for energy to replace oil, then food prices rise and consumers suffer and respond vigorously. corporations adopt policies of environmental responsibility in part because of public relations and partly because of potential liability claims. much of the planning and financial costs of offshore petroleum and gas drilling is spent on safety measures to protect the environment. the explosion in 2010 at a british petroleum site in the gulf of mexico, off the coast of texas and louisiana, caused massive pollution and environmental damage, and resulted in the us government being awarded us$4.5 billion against bp for cleanup and damages. the reputation of the corporation suffered and some executive officers lost their positions. thus, corporate social responsibility can be seen as self-interest. new models of health care organization are emerging and developing rapidly in many countries. this is partly a result of a search for more economical methods of delivering health care and partly the result of the target-oriented approach to health planning that seeks the best way to define and achieve health objectives. the developed countries seek ways to restrain cost increases, and the developing countries seek effective ways to quickly and inexpensively raise health standards for their populations. new organizational models that try to meet these objectives include district health systems, managed care organizations (mcos) and accountable care organizations (acos), described in greater detail in chapter 11. critical and basic elements of a health system organization are shown in figure 12 .12. new initiatives are part of the growth and development of any organization or health service system, as needs, technologies, resources, and public demand change. identification of issues and decisions to launch new endeavors or projects to advance the state of the art, to address unmet needs, or to meet competition are part of organizational responsibility, in the public sector to meet needs, and in the private sector to remain competitive. in developing and developed countries, many ngos provide funding from abroad for essential services that a government may be unable to provide. such projects focus on issues directed from the head offices in the usa or europe of the funding source or management offices for specific vertical programs which are often not fully integrated with national priorities and programs. however, these need coordination and approval by the local national government agency responsible for that sector of public service. new projects run by ngos may run in parallel to each other, or to state health services as uncoordinated activities. governmental public health agencies have responsibility for oversight of health systems and can play a leadership and regulatory role in coordinating activities and directing new programs to areas of greatest national need. the public health agency may also seek funding to launch new pilot or specific needs programs. the agency may introduce a new vaccine into a routine immunization program in phases, pending government approval and funding to incorporate it as a routine immunization program based on evaluation of the initial phase. an example is the introduction of haemophilus influenzae type b vaccine in albania in 2006, which was funded by gavi for 5 years based on a study and proposal including a cost-effectiveness study (bino s, ginsberg g, personal communication, 2007) . proposals for health projects by ngos or private agencies need to be prepared in keeping with the vision, mission, and objectives of the responsible governmental agency, with ethics review and community participation. a project proposal should include why the project is important, its specific goals and objectives, available or new resources, and the time-frame required to achieve success (box 12.5). it should describe the means proposed to accomplish the goals, and how the proposed program will impact the community, providing recommendations for follow-up and/or further action. the introduction of the project proposal outlines the current state of the problem and the case for action. it should describe existing programs which address that issue, with proposed collaboration, and expansion or improvement of programs, but avoiding duplication of services. background information needs to relate the project to the priorities of the prospective funding organization. the objectives should follow the acronym "smart": specific, measurable, achievable, relevant, and time-based. this term, originally used for computer disc self-management, has been adapted as a current form of mbo from the 1950s and 1960s. the project objectives should be feasible and the expected results of the project should be based on the stated objectives. organizations: behavior, structure, process. new york: mcgraw-hill/ irwin; 2003. the proposed funding agency expects convincing evidence of how this program will be effective, efficient, practical, and realistic. this information is presented in the activities section, which also needs to address the resources that will be needed to implement the program such as the budget for staff, supervison, training, management, materials (vaccines, syringes, equipment, ongoing supplies and others), transportation, and costs of premises. after completing the activities section, a realistic and achievable work plan and time-frame are required. well-planned projects have monitoring and evaluation criteria. monitoring follows the performance of the program, documenting successes, failures, and lessons learned, as well as expenditures. evaluation guidelines of the program define the methods used to assess the impact of the project and whether the project was carried out in an effective and efficient manner, and may be required periodically throughout the life of the project. the most difficult issue is sustainability. a project funded by an ngo is usually time limited to 3-4 years and the survival of the program usually depends on its acceptability and the capacity of government to continue it. thus, evaluation becomes even more crucial for the follow-up of even successful short-term projects. harm reduction programs include tackling hiv in drug users, reducing maternal-child hiv transmission, tobacco control programs, and reducing levels of obesity in schoolchildren. sustainability and diffusion of positive findings to wider application are important challenges, especially to global health. even in high-income countries, diffusion of best practices is often slow and fraught with controversy and inertia. examples of this slow or non-diffusion of evidence-based public health include the failure of most european countries to harmonize salt fortification with iodine or total indifference to flour fortification with folic acid to prevent neural tube defects (see chapters 6 and 8). public health work within departments or ministries of health or local health authorities operates at a disadvantage in comparison with other health activities, especially hospitals, pharmaceuticals, diagnostics, and medical care. the competition for resources in a centrally funded system is intense, and the political and bureaucratic battles for funds may pit new immunization agents or health promotion programs against new cancer treatment drugs or scanners, and this is very often a difficult struggle. the presentation of program proposals for new public health interventions requires skill, professionalism, good timing, and the help of informed public and professional opinion. allocation of resources is decided at the political level in a tax-based universal system, while even in a social security (bismarckian) system where funding is through an employee-employer payroll deduction, additional funding from government is essential to keep up with the continuing flow of new modalities of treatment or prevention. public health is handicapped in portraying the costs and benefits of important interventions, leaving new programs with insufficient resources, including the staffing and administrative costs (e.g., office space, phone service, transportation costs), which are essential parts of any public health program. portraying the cost of the new proposed program should be based on the total population served, not just the specific target population for a new program; that is, it should be represented as a per capita cost. similarly, projected benefits should extrapolate the results from other areas, such as pandemic or avian flu or severe acute respiratory syndrome (sars), and the likely impact on the target geographic area and its population. public health has prime responsibility for monitoring the health status of the population as well as in preventing infectious and non-communicable diseases and injuries, preparing for disasters, and many other functions. this role requires an adequate multidisciplinary workforce with high levels of competencies. this topic is discussed extensively in chapter 14. canada's experience with the sars epidemic in 2003 led to a reappraisal of public health preparedness and standards. this, in turn, led to the establishment of the national public health agency of canada, which is mandated to develop standards and practices to raise the quality of public health in the country and especially to prepare for possible pandemics. the agency issued standards of competency for public health personnel and fostered the development of regional laboratories, and schools of public health were developed across canada. core competencies for program planning implementation and evaluation are seen in box 12.7. health care systems throughout the world are being scrutinized because of their growing costs in relation to national wealth. at the same time, techniques for evaluating health care with respect to appropriateness, quality, and resource allocation are being developed. these techniques are multifactorial since they must relate to all aspects of health care, including the characteristics of the population being served; available health care resources; measures of the process and utilization of care; measures of health care outcomes; peer review, including quality assessment of health care providers; consumer attitudes, knowledge, and compliance; care provided for "tracer" or sample conditions; and economic cost-benefit studies. evaluation in health care assumes that a health care system and the providers of health care within that system are responsible and accountable for the health status of the population. it must, however, recognize that health services are not the sole determinants of health status; social, economic, and cultural factors also play key roles. a comprehensive approach to evaluation in health care is described in chapter 3. many of the components that are available in health care systems exist, while others that remain to be developed are discussed. evaluation is an integral part of a comprehensive health care system, in that the components of evaluation must be built into any national system. as long as rationality is expected of health care, evaluation is an essential element of the overall system (tulchinsky, 1982) (see chapter 3). the purpose of management in health is the improvement of health, and not merely the maintenance of an institution. separate management of a variety of health facilities serving a community has derived from different historical development and funding systems. in competition for public attention and political support, public health suffers in comparison to hospitals, new technology and drugs, and other competitors for limited resources. the experience of successes in reducing mortality from both non-infectious and infectious conditions comes largely from public health interventions. medical care is also an essential part of public health, so that management and resource allocation within the total health sector are interactive and mutually dependent. the new public health looks at all services as part of a network of interdependent services, each contributing to health needs, whether in hospital care or in enforcing public health law regarding; for example, motor vehicle safety and smoking restriction in public places. separate management and budgeting of a complex of services results in disproportionate funds, staff, and attention being directed towards high-cost services such as hospitals, and fails to redirect resources to more cost-effective and patient-sensitive kinds of services, such as home and preventive care. however, reducing the supply of hospital beds and implementing payment systems with resources for early diagnosis and incentives for short stays have changed this situation quite dramatically in recent decades. the effects of incentives and disincentives built into funding systems are central issues in determining how management approaches problem solving and program planning, and are therefore important considerations in promoting health. the management approach to resolving this dilemma is professional vision and leadership to promote the broader new public health. thus, managers of hospitals and other health facilities need broad-based training in a new public health in order to understand the interrelationships of services, funding, and population health. managers who continue to work with an obsolescent paradigm with the traditional emphasis, regardless of the larger picture, may find the hospital non-competitive in a new climate where economic incentives promote downsizing institutions and upgrading health promotion. defensive, internalized management will become obsolete, while forward-looking management will be the pioneers of the new public health. this may be seen as a systems approach to improve population and individual health, based on strategic planning for immediate needs and adaptation of health systems in the longer term issues in health. examples of national planning that cut across health and social services include national insurance policies and the provision of new services to meet rising needs, as shown for alzheimer's disease, in france since 2001 (box 12.8) and in the usa since 2011 (box 12.9). health care is one of the largest and most important industries in any country, consuming anywhere from 3 to nearly 18 percent of gnp, and still growing. it is a service, not a production industry, and is vital to the health and well-being box 12.7 core competencies for program planning, implementation and evaluation management, from policy to operational management of a production or a service system. creative management of health systems is vital to the functioning of the system at the macrolevel, as well as in the individual department or service. this implies effective use of resources to achieve objectives, and community, provider, and consumer satisfaction. these are formidable challenges, not only when money is available in abundance, but even more so when resources are limited and difficult choices need to be made. modern management includes knowledge and skills in identifying and measuring community health needs and health risks. critical needs are addressed in strategic planning with measurable impacts and targets. public health managers should have skills gained in marketing, networking, data management, managing human resources and finance, engaging community partners, and communicating public health messages. many of the methods of management and organization theory developed as part of the business world have become part of public health. these include defining the mission, values and objectives of the organization, strategic planning and management, mbo, human resource management (recognizing individual and professional values), incentives-disincentives, regulation, education, and economic resources. the ultimate mission of public health is the saving of human life and improving its quality, and achieving this efficiently with high standards of professionalism and community involvement. the scope of the new public health is broad. it includes the traditional public health programs, but equally must concern itself with managing and planning comprehensive service systems and measuring their function. the selection of targets and priorities is often determined by the feasible rather than the ideal. the health manager, either at the macrolevel of health or managing a local clinic, needs to be able to conceptualize the possibilities of improving the health of individuals and the population in his or her service responsibility with current and appropriate methods. good management means designing objectives based on a balance between the feasible and the desirable. public health has benefited greatly from its work with the social sciences and assistance from management and systems sciences to adapt and absorb the new challenges and technologies in applied public health. the new public health is not only a concept; it is a management approach to improve the health of individuals and the population. for a complete bibliography and guidance for student reviews and expected competencies please see companion web site at http://booksite.elsevier.com/9780124157668 bibliography electronic resources glossary of managed care terms national association of public hospitals and health systems world health organization, the health manager's website pay for performance (p4p): ahrq resources dr. deming: the american who taught the japanese about quality achieving a high performance health care system with universal access: what the united states can learn from other countries improving the effectiveness of health care and public health: a multi-scale complex systems analysis framework for program evaluation in public health biological and chemical terrorism: strategic plan for preparedness and response. recommendations of the cdc strategic planning workgroup a framework for program evaluation. office of the associate director for program -program evaluation developing leadership skills patterns of ambulatory health care in five different delivery systems capacity planning in health care: a review of the international experience. who, on behalf of the european observatory on health systems and policies strategic management of health care organizations social media engagement and public health communication: implications for public health organizations being truly "social crossing the quality chasm: a new health system for the twenty-first century future of the public's health in the 21st century best care at lower cost: the path to continuously learning health care in america behavioral interventions to reduce risk for sexual transmission of hiv among men who have sex with men cd001230. available at the wisdom of teams: creating the high-performance organization social marketing: influencing behaviors for good the interaction of public health and primary care: functional roles and organizational models that bridge individual and population perspectives the powers and pitfalls of the payment for performance three skills every 21st-century manager needs total quality management as competitive advantage: a review and empirical study making the best of hospital pay for performance the public health approach to eliminating disparities in health public health systems and services research: building the evidence base to improve public health practice united states innovations in healthcare delivery a call to action: lowering the cost of health care reduced mortality with hospital pay for performance in england practical challenges of systems thinking and modeling in public health public health: essentials of public health health united states the funding organization will want to know what will be the expected product of the program in measurable process (e.g., immunization coverage) or outcome indicators (e.g., reduced child mortality). projections will be based on the intended activities and known outcomes of other past programs with similar goals in the same or other countries (environmental scan), and should be supported by a review of local and international literature on the topic. the activities section of a proposal should include a timeline of the intended actions and a description of activities based on best practices. the expected outcomes, monitoring and evaluation, and justification are all part of the presentation (box 12.6). the following utility standards ensure that an evaluation will serve the information needs of intended users: l identify and engage stakeholders, including relevant government agencies, people or communities involved in or affected by the evaluation, so that their needs and concerns can be addressed. l develop and describe the program. l focus the evaluation design with ethical standards and review requirements respected.l gather credible evidence -the people conducting the evaluation should be trustworthy and competent in performing the evaluation for findings to achieve maximum credibility and acceptance. information collected should address pertinent questions regarding the program and be responsive to the needs and interests of clients and other specified stakeholders.l justify the conclusions -the perspectives, procedures, and rationale used to interpret the findings should be carefully described so that the bases for value judgments are clear. l ensure sharing and use of information and lessons learned -evaluation reports should clearly describe the program being evaluated, including its context and the purposes, procedures, and findings of the evaluation so that essential information is provided and easily understood. substantial interim findings and evaluation reports should be disseminated to intended users so that they can be used in a timely fashion to encourage follow-through by stakeholders, to increase the likelihood of the evaluation being used.l standards of a project should focus on scientific justification, utility, feasibility, propriety, and accuracy. l a program in this context includes: -direct service interventions -community mobilization efforts -research initiatives -surveillance systems -policy development activities -outbreak investigations -laboratory diagnostics -communication campaigns -infrastructure building projects -training and education services -administrative systems and others. title page -name of project; principal people and implementing organizations; contact person(s); timeframe; country (state, region); target group of project; estimated project cost; date of submission.l introduction -provides project background including the health issue(s) to be addressed, a situational analysis of the health problem, the at-risk and target populations, and existing programs in the community; includes an international and national literature review of the topic with references. budget -estimated cost of expenditures, including human resources, activities, running costs, and overheads for project and evaluation. l monitoring and evaluation -what evidence will be used to indicate how the program has performed? what plan is recommended for periodic follow-up of project activities (including timeline and measures) to implement lessons learned from positive or negative outcomes, and use of resources? how efficient and effective is the project? l conclusions -what conclusions regarding program performance may be drawn? what conclusions regarding program performance are justified by comparing the available evidence to the selected standards? l reporting -report the project to the key stakeholders and public bodies; publication in peer-reviewed journal if possible.l justification -why is this project important and timely, and how will implementation benefit health of the community?core competencies are essential knowledge, skills, and attitudes necessary for the practice of public health. they transcend the boundaries of specific disciplines and are independent of program and topic. they are the building blocks for effective public health practice, and the use of an overall public health approach.generic core competencies provide a baseline for what is required to fulfill public health system core functions. these include population health assessment, surveillance, disease and injury prevention, health promotion, and health protection.the core competencies are needed to effectively choose options, and to plan, implement, and evaluate policies and/ or programs in public health, including the management of incidents such as outbreaks and emergencies.a public health practitioner is able to: l describe selected policy and program options to address a specific public health issue l describe the implications of each option, especially as they apply to the determinants of health and recommend or decide on a course of action l develop a plan to implement a course of action taking into account relevant evidence, legislation, emergency planning procedures, regulations, and policies l implement a policy or program and/or take appropriate action to address a specific public health issue l demonstrate the ability to implement effective practice guidelines l evaluate an action, a policy, or a program l demonstrate an ability to set and follow priorities, to maximize outcomes based on available resources l demonstrate the ability to fulfill functional roles in response to a public health emergency. of the individual, the population, and the economy. because health care employs large numbers of skilled professionals and many unskilled people, it is often vital to the economic survival of small communities, as well as for a sense of community well-being.management includes planning, leading, controlling, organizing, motivating, and decision-making. it is the application of resources and personnel towards achieving targets. therefore, it involves the study of the use of resources, and the motivation and function of the people involved, including the producer or provider of service, and the customer, client, or patient. this cannot take place in a vacuum, but is based on the continuous monitoring of information and its communication to all parties involved. these functions are applicable at all levels of an estimated 600,000 french people lived with dementia; half were diagnosed and one-third were receiving treatment; 75 percent of people with alzheimer's disease were living at home; 50 percent of all nursing home residents lived with some form of dementia; a day's care cost €60 while full-time residency in a nursing home ranged between €3000 and €4600. l identify the early symptoms of dementia and refer people to specialists. l create a network of "memory centers" to enable earlier diagnosis. "for millions of americans, the heartbreak of watching a loved one struggle with alzheimer's disease is a pain they know all too well. alzheimer's disease burdens an increasing number of our nation's elders and their families, and it is essential that we confront the challenge it poses to our public health. " on 4 january 2011, president barack obama signed into law the national alzheimer's project act (napa), requiring the secretary of the us department of health and human services (hhs) to establish the national alzheimer's project to: l create and maintain an integrated national plan to overcome alzheimer's disease (ad). l coordinate alzheimer's disease research and services across all federal agencies. l accelerate the development of treatments to prevent, halt, or reverse the course of ad. l improve early diagnosis and coordination of care and treatment of ad. l improve outcomes for ethnic and racial minority populations that are at higher risk for ad. l coordinate with international bodies to fight ad globally. the law also establishes the advisory council on alzheimer's research, care, and services and requires the secretary of hhs, in collaboration with the advisory council, to create and maintain a national plan to overcome ad. research funds are being allocated towards that end. education for health providers, strengthening of the workforce, for direct care and for public health guidelines for management of ad, education and support for caring families, addressing special housing needs for ad patients and many other initiatives are proposed in this comprehensive approach to a growing public health problem. enhancing public awareness is crucial to achieve the goals set out in this plan. key: cord-319828-9ru9lh0c authors: shi, shuyun; he, debiao; li, li; kumar, neeraj; khan, muhammad khurram; choo, kim-kwang raymond title: applications of blockchain in ensuring the security and privacy of electronic health record systems: a survey date: 2020-07-15 journal: comput secur doi: 10.1016/j.cose.2020.101966 sha: doc_id: 319828 cord_uid: 9ru9lh0c due to the popularity of blockchain, there have been many proposed applications of blockchain in the healthcare sector, such as electronic health record (ehr) systems. therefore, in this paper we perform a systematic literature review of blockchain approaches designed for ehr systems, focusing only on the security and privacy aspects. as part of the review, we introduce relevant background knowledge relating to both ehr systems and blockchain, prior to investigating the (potential) applications of blockchain in ehr systems. we also identify a number of research challenges and opportunities. there is an increasing interest in digitalizing healthcare systems by governments and related industry sectors, partly evidenced by various initiatives taking place in different countries and sectors. for example, the then u.s. president signed into law the health information technology for economic and clinical health (hitech) act of 2009, as 5 part of the american recovery and reinvestment act of 2009. hitech is designed to encourage broader adoption of electronic health records (ehrs), with the ultimate aim of benefiting patients and society. the potential benefits associated with ehr systems (e.g. public healthcare management, online patient access, and patients medical data sharing) have also attracted the interest of the research community [1, 2, 3, 4, 5, 6, 7, 8, 9] . the 10 potential of ehrs is also evidenced by the recent 2019 novel coronavirus (also referred to as 2019-ncov and covid-2019) pandemic, where remote patient monitoring and other healthcare deliveries are increasingly used in order to contain the situation. as with any maturing consumer technologies, there are a number of research and operational challenges. for example, many existing ehr systems use a centralized server 15 model, and hence such deployments inherit security and privacy limitations associated with the centralized server model (e.g. single point of failure and performance bottleneck). in addition, as ehr systems become more commonplace and the increasing understanding of the importance of data (particularly healthcare data), honest but curious servers may surreptitiously collect personal information of users while carrying out 20 their normal activities. in recent times, there is an increasing trend in deploying blockchain in a broad range of applications, including healthcare (e.g. public healthcare management, counterfeit drug prevention, and clinical trial) [10, 11, 12 ]. this is not surprising, since blockchain is an immutable, transparent and decentralized distributed database [13] that can be 25 leveraged to provide a secure and trusty value chain. an architecture of blockchain-based healthcare systems is shown in fig. 1 . blockchain is a distributed ledger database on a peer-to-peer (p2p) network that comprises a list of ordered blocks chronologically. in other words, this is a decentralized and trustworthy distributed system (without relying on any third party). trust relation among 30 distributed nodes is established by mathematical methods and cryptography technologies instead of semi-trusted central institutions. blockchain-based systems can mitigate the limitation of the single point of failure. besides, since data is recorded in the public ledger, and all of nodes in the blockchain network have ledger backups and can access these data anytime and anywhere, such a system ensures data transparency and helps to 35 build trust among distributed nodes. it also facilitates data audit and accountability by having the capability to trace tamper-resistant historical record in the ledger. depending on the actual deployment, data in the ledger can be stored in the encrypted form using different cryptographic techniques; hence, preserving data privacy. users can also protect their real identities in the sense of pseudo-anonymity. to enhance robustness, 40 we can introduce smart contracts (i.e. a kind of self-executing program deployed on the distributed blockchain network) to support diverse functions for different application scenarios. specifically, the terms of smart contract can be preset by users and the smart contract will only be executed if the terms are fulfilled. hence, this hands over control to the owner of the data. there are a (small) number of real-world blockchain-based 45 healthcare systems, such as gem, guardtime and healthbank [14] . hence, in this paper we focus on blockchain-based healthcare systems. specifically, we will comprehensively review some existing work, and identify existing and emerging challenges and potential research opportunities. prior to presenting the results of our re-3 view, we will first introduce ehr system and blockchain architecture in the next section. 50 then, in section 3, we will review the extant literature and provide a comparative summary of some existing systems. in section 4, we identify a number of potential research opportunities. finally, we conclude the paper in the last section. in a centralized architecture, such as those that underpin a conventional ehr system, 55 a central institution is tasked with managing, coordinating and controlling of the entire network. however, in a distributed architecture, all nodes are maintained without relying on a central authority. now, we will briefly explain the ehr system and blockchain technology. the electronic health record (ehr) is generally defined to be the collection of patients' electronic health information (e.g. in the form of electronic medical records -emrs). emrs can serve as a data source for ehr mainly from healthcare providers in the medical institutions. the personal health record (phr) contains personal healthcare information, such as those obtained from wearable devices owned and controlled by 65 patients. information collected as part of phrs can be available to healthcare providers, by users (patients). in theory, ehr systems should ensure the confidentiality, integrity and availability of the stored data, and data can be shared securely among authorized users (e.g. medical practitioners with the right need to access particular patient's data to facilitate diagno-70 sis). in addition, such a system if implemented well, can reduce data replication and the risk of lost record, and so on. however, the challenge of securing data in such systems, whether in-transit or at-rest, is compounded by the increasing connectivity to these systems (e.g. more potential attack vectors). for example, mobile devices that can sync with the ehr system is a potential attack vector that can be targeted (e.g. an attacker 75 can seek to exploit a known vulnerability in the hospital-issued mobile devices and install malware to facilitate covert exfiltration of sensitive data (e.g. phrs)). one of the key benefits of ehr systems is the availability of large volumes of data, which can be used to facilitate data analysis and machine learning, for example to inform other medical research efforts such as disease forecasting (e.g. the 2019 novel 80 coronavirus). furthermore, wearable and other internet of things (iot) devices can collect and upload relevant information, including those relating to phrs, to the ehr systems, which can facilitate healthcare monitoring and personalized health services. blockchain is made popular by the success of bitcoin [15] , and can be used to facilitate 85 trustworthy and secure transactions across an untrusted network without relying on any centralized third party. we will now introduce the fundamental building blocks in the blockchain [16, 17, 18] . blockchain is a chronological sequence of blocks including a list of complete and valid transaction record. blocks are linked to the previous block by a reference (hash value), 90 and thus forming a chain. the block preceding a given block is called its parent block, and the first block is known as the genesis block. a block [15] consists of the block header the block header contains: • block version: block validation rules; 95 • previous block hash: hash value of the previous block; • timestamp: the creation time of the current block; • nonce: a 4-byte random field that miners adjust for every hash calculation to solve a pow mining puzzle (see also section 2.2.2); • body root hash: hash value of the merkle tree root built by transactions in the 100 block body; • target hash: target threshold of hash value of a new valid block. the target hash is used to determine the difficulty of the pow puzzle (see also section 2.2.2). merkle tree is used to store all the valid transactions, in which every leaf node is a 105 transaction and every non-leaf node is the hash value of its two concatenated child nodes. such a tree structure is efficient for the verification of the transaction's existence and integrity, since any node can confirm the validation of any transaction by the hash value of the corresponding branches rather than entire merkle tree. meanwhile, any modification on the transaction will generate a new hash value in the upper layer and 110 this will result in a falsified root hash. besides, the maximum number of transactions that a block can contain depends on the size of each transaction and the block size. these blocks are then chained together using cryptographic hash function in an append-only structure. that means new data is only appended in the form of additional blocks chained with previous blocks since altering and deleting previously confirmed data 115 is impossible. as previously discussed, any modification of one of the blocks will generate a different hash value and different link relation. hence, achieving immutability and security. digital signature based on asymmetric cryptography is generally used for transaction 120 authentication in an untrustworthy environment [19, 20] . blockchain uses asymmetric 6 cryptography mechanism to send transactions and verify the authentication of transacotherwise, it will be discarded in this process. only valid transactions can be stored in the new block of blockchain network. we will take the coin transfer as an example (see fig. 3 ). alice transfers a certain amount of coins to bob. in step 1, she initiates a transaction signed by her private key. the transaction can be easily verified by others using alice's public key. in step 2, the 135 transaction is broadcasted to other nodes through the p2p network. in step 3, each node will verify the transaction by predefined rules. in step 4, each validated transaction will be packed chronologically and appended to a new block once a miner solves the puzzle. finally, every node will update and back up the new block. in the blockchain network, there is no trusted central authority. thus, reaching a consensus for these transactions among untrustworthy nodes in a distributed network is an important issue, which is a transformation of the byzantine generals (bg) problem proposed in [22] . the bg problem is that a group of generals command the byzantine army to circle the city, and they have no chance of winning the war unless all of them 145 attack at the same time. however, they are not sure whether there are traitors who might retreat in a distributed environment. thus, they have to reach an agreement to attack or retreat. it is the same challenge for the blockchain network. a number of protocols have been designed to reach consensus among all the distributed nodes before a new block is linked into blockchain [23] , such as the following: • pow (proof of work) is the consensus mechanism used in bitcoin. if the miner node who has certain computing (hashing) power wishes to obtain some rewards, the miner must perform the laborious task of mining to prove that he is not malicious. the task requires that the node repeatedly performs hash computations to find an eligible nonce value that satisfies the requirement that a hashed block 155 head must be less than (or equal to) the target hash value. the nonce is difficult to generate but easy for other nodes to validate. the task is costly (in terms of computing resources) due to the number of difficult calculations. a 51% attack is a potential attack in the blockchain network, where if a miner or a group of miners can control more than 51% of the computing power, they could interfere with the 160 generation of new blocks and create fraudulent transaction records beneficial for the attackers. • pos (proof of stake) is an improved and energy-saving mechanism of pow. it is believed that nodes with the largest number of stakes (e.g. currency) would be less likely to attack the network. however, the selection based on account balance is 165 unfair because the richest node is more likely to be dominant in the network, which would be similar to a centralized system gradually. blockchain systems are divided into three types based on permissions given to network 210 nodes: • public blockchain. the public blockchain is open to anyone who wants to join anytime and acts as a simple node or as a miner for economic rewards. bitcoin [15] and ethereum [25] are two well-known public blockchain platforms. • private blockchain. the private blockchain network works based on access control, 215 in which participants must obtain an invitation or permissions to join. gemos [26] and multichain [27] are both typical private blockchain platforms. • consortium blockchain. the consortium blockchain is "semi-private" sitting on the fence between public and private blockchains. it is granted to a group of approved organizations commonly associated with enterprise use to improve business. hy-220 perledger fabric [28] is a business consortium blockchain framework. ethereum also supports for building consortium blockchains. generally, ehrs mainly contain patient medical history, personal statistics (e.g. age and weight), laboratory test results and so on. hence, it is crucial to ensure the security 225 and privacy of these data. in addition, hospitals in countries such as u.s. are subject to exacting regulatory oversight. there are also a number of challenges in deploying and implementing healthcare systems in practice. for example, centralized server models are vulnerable to the single-point attack limitations and malicious insider attacks, as previously discussed. users (e.g. patients) whose data is outsourced or stored in these 230 ehr systems generally lose control of their data, and have no way of knowing who is accessing their data and for what kind of purposes (i.e. violation of personal privacy). such information may also be at risk of being leaked by malicious insiders to another organization, for example an insurance company may deny insurance coverage to the particular patient based on leaked medical history. meanwhile, data sharing is increasingly crucial particularly as our society and population become more mobile. by leveraging the interconnectivity between different healthcare entities, shared data can improve medical service delivery, and so on. overcoming the "information and resource island" (information silo) will be challenging, for example due to privacy concerns and regulations. the information silo also contributes to 240 unnecessary data redundancy and red-tape. in this case, the health insurance portability and accountability act (hipaa) was • unique identifiers rule. only the national provider identifier (npi) identifies covered entities in the standard transactions to protect the patient identity information. • enforcement rule. investigation and penalties for violating hipaa rules. there is another common framework for audit trails for ehrs, called iso 27789, to keep personal health information auditable across systems and domains. secure audit record must be created each time any operation is triggered via the system complying with iso 27789. hence, we posit the importance of a collaborative and transparent data 260 sharing system, which also facilitates audit and post-incident investigation or forensics in the event of an alleged misconduct (e.g. data leakage). such a notion (forensic-by-design) is also emphasized by forensic researchers [29, 30] . as a regulatory response to security concerns about managing the distribution, storage and retrieval of health record by medical industry, title 21 cfr part 11 places 265 requirements on medical systems, including measures such as document encryption and the use of digital signature standards to ensure the authenticity, integrity and confidentiality of record. we summarize the following requirements that should be met based on these relevant standards above when implementing the next generation secure ehr systems: • accuracy and integrity of data (e.g. any unauthorized modification of data is not allowed, and can be detected); • security and privacy of data; • efficient data sharing mechanism (e.g. [31] ); • mechanism to return the control of ehrs back to the patients (e.g. patients can 275 monitor their record and receive notification for loss or unauthorized acquisition); • audit and accountability of data (e.g. forensic-by-design [29, 30] ). the above properties can be achieved using blockchain, as explained below: • decentralization. compared with the centralized mode, blockchain no longer needs to rely on the semi-trusted third party. • security. it is resilient to single point of failure and insider attacks in the blockchainbased decentralized system. • pseudonymity. each node is bound with a public pseudonymous address to protect its real identity. • immutability. it is computationally hard to delete or modify any record of any 285 block included in the blockchain by one-way cryptographic hash function. • autonomy. patients hold the rights of their own data and share their data flexibly by the settings of special items in the smart contract. • incentive mechanism. incentive mechanism of blockchain can stimulate the cooperation and sharing of competitive institutions to promote the development of 290 medical services and research. • auditability. it is easy to keep trace of any operation since any historical transaction is recorded in the blockchain. hence, if blockchain is applied correctly in the ehr systems, it can help to ensure the security of ehr systems, enhance the integrity and privacy of data, encourage orga-295 nizations and individuals to share data, and facilitate both audit and accountability. based on the requirements of a new version of secure ehr systems and the characteristics of blockchain discussed in the preceding section 2.3, we will now describe the key goals in the implementation of secure blockchain-based ehr systems as follows: • privacy: individual data will be used privately and only authorized parties can access the requested data. • security: in the sense of confidentiality, integrity and availability (cia): 1. confidentiality: only authorized users can access the data. integrity: data must be accurate in transit and not be altered by unauthorized 305 entity(ies). 3. availability: legitimate user's access to information and resources is not improperly denied. • accountability: an individual or an organization will be audited and be responsible for misbehavior. • authenticity: capability to validate the identities of requestors before allowing access to sensitive data. • anonymity: entities have no visible identifier for privacy. complete anonymity is challenging, and pseudo-anonymity is more common (i.e. users are identified by something other than their actual identities). in order to satisfy the above goals, existing blockchain-based research in the healthcare domain includes the following main aspects: • data storage. blockchain serves as a trusted ledger database to store a broad range of private healthcare data. data privacy should be guaranteed when secure storage is achieved. however, healthcare data volume tends to be large and complex in practice. hence, a corresponding challenge is how to deal with big data storage without having an adverse impact on the performance of blockchain network. • data sharing. in most existing healthcare systems, service providers usually maintain primary stewardship of data. with the notion of self-sovereignty, it is a trend to return the ownership of healthcare data back to the user who is capable of sharing (or not sharing) his personal data at will. it is also necessary to achieve secure data sharing across different organizations and domains. • data audit. audit logs can serve as proofs to hold requestors accountable for their interactions with ehrs when disputes arise. some systems utilize blockchain and smart contract to keep trace for auditability purpose. any operation or request will be recorded in the blockchain ledger, and can be retrieved at any time. • identity manager. the legitimacy of each user's identity needs to be guaranteed in 335 the system. in other words, only legitimate users can make the relevant requests to ensure system security and avoid malicious attacks. in the remaining of this section, we will review existing approaches to achieve data storage, data sharing, data audit, and identity manager (see sections 3.1 to 3.4). according to section 2.3, one of the solutions to ensure greater security in the ehr system is the use of blockchain technology. however, there are potential privacy problems for all of raw/encrypted data in the public ledger, since blockchain as a public database has the risk of sensitive data being exposed under the statistical attack. some measures should be taken to enhance the privacy protection of sensitive health record in the blockchain-based ehr systems. in generally, privacy preserving approaches can be classified into cryptographic and non-cryptographic approaches, including encryption, anonymisation and access control mechanism respectively. encryption scheme is a relatively common method, such as public key encryption 350 (pke), symmetric key encryption (ske), secure multi-party computation (mpc) [33] and so on. al. [35] proposed that sensors data will be uploaded using a pair of unique private and public keys in the blockchain network to protect the privacy and security of biometric information. zheng et al. [36] proposed that data will be encrypted before being uploaded to cloud servers by symmetric key scheme (i.e. rijndael aes [37] ) with threshold encryption 360 scheme. the symmetric key will be split into multiple shares distributed among different key keepers by shamir's secret sharing scheme [38] . only if data requestor gets enough key shares, he can decrypt the ciphertext. compromising of some key keepers(less than threshold) would not lead to data leakage. yue et al. [39] designed an app on smartphones based on blockchain with mpc tech-365 nique, called healthcare data gateway (hdg). the system allows to run computations of encrypted data directly on the private blockchain cloud and obtain the final results without revealing the raw data. besides, guo et al. [40] proposed an attribute-based signature scheme with multiple authorities (ma-abs) in the healthcare blockchain. the signature of this scheme attests 370 not to the identity of the patient who endorses a message, instead to a claim (like access policy) regarding the attributes delegated from some authorities he possesses. meanwhile, the system has the ability to resist collusion attack by sharing the secret pseudorandom function (prf) seeds among authorities. in order to resist malicious attacks (e.g. statistical attack), healthcare systems have 375 to change the encryption keys frequently of general methods. it will bring the cost for storage and management of a large amount of historical keys since these historical keys must be stored well to decrypt some historical data in future, then the storage cost will be greater, especially for limited computational resource and storage devices. to address this problem, zhao et al. [41] designed a lightweight backup and effi-380 cient recovery key management scheme for body senor networks (bsns) to protect the privacy of sensor data from human body and greatly reduce the storage cost of secret keys. fuzzy vault technology is applied for the generation, backup and recovery of keys without storing any encryption key, and the recovery of the key is executed by bsns. the adversary hardly decrypts sensor data without symmetric key since sensor data is 385 encrypted by symmetric encryption technology (i.e. aes or 3des). we compare and analyse some systems above, shown in table 1 and 2. most systems use cryptographic technology to enhance the security and privacy of healthcare data in the blockchain. however, encryption technique is not absolutely secure. the computational cost of encryption is high for some limited devices. transaction record may 390 also reveal user behaviors and identity because of the fixed account address. malicious attackers may break the ciphertext stored in the public ledger by some means. 2. all of data will be exposed once the corresponding symmetric key is lost table 2 : systems requirements that have been met in table 1 paper security privacy anonymity integrity authentication controllability auditability accountability [34] [35] [36] [39] [40] [42] [41] meanwhile, another important issue is key management. it is the foundation of entire data field safety that private keys do not reveal. the loss of private key means that the holder would have no ability to control the corresponding data. once the 395 private/symmetric key is compromised, all of data may be exposed by attackers. so, both encryption technique and key management should be considered when developers design a secure ehr system. additionally, it must guarantee that only authorized legitimate users can access private data to enhance security. non-cryptographic approaches mainly use access control 400 mechanism for security and preserving privacy. with regard to the security goals, access control mechanism is a kind of security technique that performs identification authenti-19 cation and authorization for entities. it is a tool widely used in the secure data sharing with minimal risk of data leakage. we will discuss this mechanism in details in the next section 3.2.2. the ehr systems can upload medical record and other information in the blockchain. if these data is stored directly in the blockchain network, it will increase computational overhead and storage burden due to the fixed and limited block size. what's more, these data would also suffer from privacy leakage. to solve these problems, most relevant research and applications [36, 42, 43, 44] yue et al. [39] proposed that a simple unified indicator centric schema (ics) could organize all kinds of personal healthcare data easily in one simple "table". in this system, data is uploaded once and retrieved many times. they designed multi-level index and most systems in the previous sections are adopted third-party database architecture. the third-party services (such as cloud computing) in the far-end assist the users to improve quality of service (qos) of the applications by providing data storage and computation power, but with a transmission latency. such a storage system has gained common acceptance depending on a trusted third table (dht). nguyen et al. [48] designed a system that integrates smart contract with ipfs to improve decentralized cloud storage and controlled data sharing for better user access management. rifi et al. [49] also adopted ipfs as the candidate for off-chain database to store large amounts of sensor personal data. 475 wang et al. [50] designed a system that utilizes ipfs to store the encrypted file. the encryption key of the file is first encrypted using abe algorithm, then encrypted with other information (file location hash ciphertext) using aes algorithm. only when the attributes set of the requestor meets the access policy predefined by data owner, the requestor can obtain the clue from blockchain, then download and decrypt the files from 480 ipfs. 22 table 4 : systems requirements that have been met in table 3 paper security privacy anonymity integrity authentication controllability auditability accountability [44] [47] [42] [45] [36] [43] [48] [49] [50] according to table 3 and 4, the common architecture for data storage in the ehr system is shown in fig. 5 . the advantages of integrating off-line storage into blockchain systems are as follows. first, detailed medical record is not allowed to access directly for patient's data privacy preserving. second, it helps to reduce the throughput require-485 ment significantly, since only transaction record and a few metadata are stored in the blockchain. besides, data pointers stored in the block can be linked to the location of raw data in the off-chain database for data integrity. however, it is difficult to fully trust the third parties to store these sensitive data. meanwhile, it may also contradict the idea of decentralization. further research is needed 490 to accelerate the acceptance of distributed storage systems in practice, like ipfs. also, the next step should be to improve the storage architecture of blockchain for high storage capacity. healthcare industry relies on multiple sources of information recorded in different sys-495 tems, such as hospitals, clinics, laboratories and so on. healthcare data should be stored, retrieved and manipulated by different healthcare providers for medical purposes. however, such a sharing approach of medical data is challenging due to heterogeneous data structures among different organizations. it is necessary to consider interoperability of 25 figure 5 : common architecture for data storage in the ehr system data among different organizations before sharing data. we will introduce interoperabil-500 ity first. interoperability of ehr is the degree to which ehr is understood and used by multiple different providers as they read each other's data. interoperability can be used to standardize and optimize the quality of health care. interoperability can mainly be 505 classified into three levels: • syntactic interoperability: one ehr system can communicate with another system through compatible formats. • semantic interoperability: data can be exchanged and accurately interpreted at the data field level between different systems. the lack of unified interoperability standards has been a major barrier in the highperformance data sharing between different entities. according to the study [51] , there in some studies [10, 52, 53] , they adopted the health level seven international (fhir) as data specification and standard formats for data exchange between different organi-520 zations. the criterion was created by hl7 healthcare standards organization. the system in [10] bahga et al. [56] proposed that cloud health information systems technology architecture (chistar) achieves semantic interoperability, defines a general purpose set of data structures and attributes and allows to aggregate healthcare data from disparate 545 data sources. besides, it can support security features and address the key requirements of hipaa. chen et al. [57] designed a secure interoperable cloud-based ehr service with continuity of care document (ccd). they provided self-protecting security for health documents with support for embedding and user-friendly control. in a word, interoperability is the basic ability for different information systems to communicate, exchange and use data in the healthcare context. ehr systems following international standards can achieve interoperability and support for data sharing between multiple healthcare providers and organizations. we will discuss data sharing in detail next. it is obviously inconvenient and inefficient to transfer paper medical record between different hospitals by patients themselves.sharing healthcare data is considered to be a critical approach to improve the quality of healthcare service and reduce medical costs. though current ehr systems bring much convenience, many obstacles still exist in 560 the healthcare information systems in practice, hinder secure and scalable data sharing across multiple organizations and thus limit the development of medical decision-making and research. as mentioned above, there are risks of the single-point attack and data leakage in a centralized system. besides, patients cannot preserve the ownership of their own private 565 data to share with someone who they trust. it may result in unauthorized use of private data by curious organizations. furthermore, different competing organizations lacking of trust partnerships are not willing to share data, which would also hinder the development of data sharing. in this case, it is necessary to ensure security and privacy-protection and return the 570 control right of data back to users in order to encourage data sharing. it is relatively simply to deal with security and privacy issues when data resides in a single organisa-tion, but it will be challenging in the case of secure health information exchange across different domains. meanwhile, it also needs to consider further how to encourage efficient collaboration in the medical industry. secure access control mechanism as one of common approaches requires that only authorized entities can access sharing data. this mechanism includes access policy commonly consisting of access control list (acl) associated with data owner. acl is a list of requestors who can access data, and related permissions (read, write, update) to specific data. authorization is a function of granting permission to authenticated users in order to access the protected resources following predefined access policies. the authentication process always comes before the authorization process. access policies of this mechanism mainly focus on who is performing which action on what data object for which purposes. traditional access control approaches for ehrs 585 sharing are deployed, managed and run by third parties. users always assume that third parties (e.g. cloud servers) perform authentication and access requests on data usage honestly. however, in fact, the server is honest but curious. it is promising that combining blockchain with access control mechanism is to build a trustworthy system. users can realize secure self-management of their own data and 590 keep shared data private. in this new model, patients can predefine access permissions (authorize, refuse, revoke), operation (read, write, update, delete) and duration to share their data by smart contracts on the blockchain without the loss of control right. smart contracts can be triggered on the blockchain once all of preconditions are met and can provide audit mechanism for any request recorded in the ledger as well. there 595 are many existing studies and applications applying smart contract for secure healthcare data sharing. peterson et al. [10] proposed that patients can authorize access to their record only under predefined conditions (research of a certain type, and for a given time range). smart contract placed directly on the blockchain verifies whether data requestors meet 600 these conditions to access the specified data. if the requestor does not have the access rights, the system will abort the session. similarly, smart contracts in [58] can be used for granting and revocation of access right and notifying the updated information as smart contract in most systems includes predefined access policies depending on requestors' role/purposes and based-role/based-purpose privileges. however, it is inflexible to handle unplanned or dynamic events and may lead to potential security threats [60]. another mechanism, attribute-based access control (abac), has been applied in the secure systems to handle remaining issues in the extensions of rbac and enhance the security in some specific cases. the systems based on access control mechanism record any operation about access policies by logging. however, it is vulnerable to malicious tampering without the assurance of integrity of these logs in the traditional systems. blockchain and smart contract can perform access authorization automatically in a secure container and make sure the integrity of policies and operations. thus, access control mechanism integrated with blockchain can provide secure data sharing. the diversified forms of access control can be applied into different situations depending on the demands for system security. audit-based access control aims to enhance the 670 reliability of posteriori verification [64] . organization-based access control (orbac) [65] can be expressed dynamically based on hierarchical structure, including organization, role, activity, view and context. 1. user's identity may be exposed without de-identification mechanism table 6 : systems requirements that have been met in table 5 paper security privacy anonymity integrity authentication controllability auditability accountability [10] [58] [44] [49] [48] [59] [39] [62] 36 table 6 : systems requirements that have been met in table 5 paper security privacy anonymity integrity authentication controllability auditability accountability [61] [66] [67] [68] [50] [69] [42] [70] [71] based on the information in the table 5 we can also use cryptography technology to enhance secure data sharing and the security of access control mechanism in most ehr systems. dubovitskaya et al. [66] proposed a framework to manage and share emrs for cancer patient care based on symmetric encryption. patients can generate symmetric encryption keys to encrypt/decrypt the sharing data with doctors. if the symmetric key is 685 compromised, proxy re-encryption algorithm on the data stored in the trusty cloud can be performed and then a new key will be shared with clinicians according to predefined access policies. only the patients can share symmetric keys and set up the access policies by smart contract to enhance the security of sharing data. xia et al. [67] designed a system that allows users to get access to requested data from 690 a shared sensitive data repository after both their identities and issuing keys are verified. in this system, user-issuer protocol is designed to create membership verification key and transaction key. user-verifier protocol is used for membership verification, then only valid users can send data request to the system. ramani et al. [68] utilized lightweight public key cryptographic operations to enhance 695 the security of permissioned requests (append, retrieve). nobody can change the patients' data without sending a notification to patients, since the requested transaction will be checked whether it has signed by the patient before being stored on a private blockchain. wang et al. [50] designed a system that combines ethereum with attribute-based encryption (abe) technology to achieve fine-grained access control over data in the de-700 centralized storage system without trusted private key generator (pkg). the encryption key of the file is stored on the blockchain in the encrypted format using aes algorithm. requestors whose attributes meet the access policies can decrypt the file encryption key and then download the encrypted file from ipfs. besides, the keyword search implemented by smart contract can avoid dishonest behavior of cloud servers. liu et al. [42] proposed blockchain-based privacy-preserving data sharing scheme for emr called bpds. the system adopted content extraction signature (ces) [73] 715 which can remove sensitive information of emrs, support for selective sharing data and generate valid extraction signatures to reduce the risk of data privacy leakage and help enhance the security of access control policies. besides, users can use different public keys for different transactions to keep anonymous. huang et al. [70] designed a blockchain-based data sharing scheme in the cloud com-720 puting environment to solve the trust issue among different groups using group signature and ensure the reliability of the data from other organizations. requestors can verify the as shown in table 5 and 6, cryptography technology can protect sensitive data directly and improve the traditional access control mechanism to meet the demand for security and privacy. however, public key encryption has high computational overhead 735 and trusted pki is necessary for authentication. the similar problem exists in a trusted pkg as one of important components of abe. besides, how to transmit the shared key securely should be addressed in the symmetric encryption. as mentioned before, mpc may not be suitable for wearable devices in the iot context due to high computational cost. it is necessary to improve these algorithms to adapt devices/sensors with limited 740 resource. above all, blockchain as a secure, immutable and decentralized framework makes the 39 control right of data return to patients themselves in the healthcare industry. as shown in fig. 6 , the combination of access control mechanism by smart contract with cryptography technology on sensitive data can be achieved secure data sharing among different 745 individuals and institutions. meanwhile, all of record is included in the immutable public ledger to ensure the integrity and reliability of data and minimize the risk of raw data leakage. concerning potential dishonest behavior or wrong results of third parties (cloud servers) holding large amounts of raw/encrypted data, blockchain offers immutable his-750 torical record for traceability and accountability, sometimes with cryptography technique (such as group signature). next we discuss about secure audit to enhance the security of ehr systems further. healthcare systems also rely on audit log management as security mechanism since 755 some exceptions may have resulted from the misuse of access privileges or dishonest behavior by third parties or data requestors. audit log can serve as proofs when disputes arise to hold users accountable for their interactions with patient record. immutable public ledger and smart contract in the blockchain can provide immutable record for all of access requests to achieve traceability and accountability. audit log mainly contains vital and understandable information: • timestamp of logged event • user id which requests the data • data owner id whose data is accessed • action type (create, delete, query, update) • the validation result of the request qi et al. [74] designed a data sharing model with the ability to effectively track the dishonest behaviour of sharing data as well as revoke access right to violated permissions and malicious access. the system provides provenance, audit and medical data sharing among cloud service providers with minimal risk of data privacy. the similar system in [67] provides auditable and accountable access control for shared cloud repositories among big data entities in a trust-less environment. azaria et al. [53] also provided auditability via comprehensive log. they mentioned that obfuscation for privacy needs further exploration while preserving auditability in the public ledger. fernandez et al. [75] designed a blockchain-based system called auditchain to manto improve quality of research by better reproducibility, the timestamped statistical analysis on clinical trials ensures traceability and integrity of each samples metadata in [77] based on blockchain which allows to store proofs of existence of data. the related analytical code to process the data must be timestamped in order that data is checked 790 and analysis is reproducible. timestamp in the blockchain will provide for better version control than git. the above-mentioned studies indicate that blockchain plays an important role in auditing and accountability. users can not only hold the control right of their own data, but also monitor all request operations for data audit and accountability when disputes 795 occur. above all, audit log provides reliable evidence for anomalous and potentially malicious behavior to improve the security of access control models. meanwhile, it brings benefits to the adjustment of healthcare service by gaining insight into personnel interactions and workflows in hospitals. store and process. currently, audit log data does not contain required and representative information reliably, which would be difficult to interpret or hardly access. it would get worse in the collaboration of multiple ehr organizations. in this case, it is necessary to consider how to achieve interoperable and well-formatted audit log standard for the 805 support of secure data exchange among different healthcare institutions. membership verification is the first step to ensure the security of any system before getting access to any resource. in the access control mechanism mentioned before, identity authentication is always first performed to make sure that specific rights are granted 810 to data requestors with legal identity before sharing data. common types of user authentication have pass-through authentication, biometric authentication and identity verification based on public key cryptography algorithms. public key infrastructure (pki) is commonly used, which relies on trusted third parties to provide membership management services. identity registration is performed in [44] with registrar smart contract to map valid string form of identity information to a unique ethereum address via public key cryptography. it can employe a dns-like implementation to allow the mapping of regulate existing forms of id. 835 zhang et al. [69] established secure links for wireless body area network (wban) area and wireless body area network (psn) area after authentication and key establishment through an improved ieee 802.15.6 display authenticated association protocol [78] . the protocol can protect collected data through human body channels (hbcs) and reduce computational load on the sensors. 840 xia et al. [67] designed an efficient and secure identity-based authentication and key agreement protocol for membership authentication with anonymity in a permissioned blockchain. the process of verification is a challenge-response dialog to prove whether the sender is authentic when the verifier receives a verification request from a user using shared key. most blockchain-based systems use pseudonyms to hide the real identity for privacy. however, there is conflict between privacy preserving and authenticity. that means how to verify the identity without exposing the information of real identity. in addition, adversaries or curious third parties can guess the real identity and relevant behavior pattern through inference attacks, such as transaction graph analysis. 850 shae et al. [79] designed an anonymous identity authentication mechanism based on zero-knowledge technology [80] , which can address two conflicting requirements: maintain the identity anonymous and verify the legitimacy of user identity as well as iot devices. sun et al. [45] proposed a decentralizing attribute-based signature (called dabs) scheme to provide effective verification of signer's attributes without his identity infor-855 mation leakage. multiple authorities can issue valid signature keys according to user's attributes rather than real identity and provide privacy-preserving verification service. other nodes can verify whether the data owner is qualified by verification key corresponding to satisfied attributes without revealing owner identity. hardjono et al. [81] designed an anonymous but verifiable identity scheme, called 860 chainachor, using the epid zero-knowledge proof scheme. these anonymous identities can achieve unlinkable transactions using different public key in the blockchain when 43 nodes execute zero-knowledge proof protocol successfully. they also provide optional disclosure of the real identity when disputes occur. biometric authentication is also widely used, such as face and voice pattern identifi-865 cation, retinal pattern analysis, hand characteristics and automated fingerprint analysis based on pattern recognition. lee et al. [35] proposed that human nails can be used for identity authentication since nails have the high degree of uniqueness. the system uses histogram of oriented gradients (hog) and local binary pattern (lbp) feature to extract the biometric identification 870 signature, then svm and convolutional neural network are utilized for authentication with high accuracy. this identity verification technology with dynamic identity rather than regular real identity information ensures user anonymity and privacy. the main goal of identity management is to ensure that only authenticated users can be authorized to access the specified resource. currently, most systems rely on 875 membership service component or similar providers for identity authentication. traditional authentication process mainly adopts password authentication and even transmit user account in the clear text. anyone can eavesdrop on the external connection to intercept user account. in this case, attackers or curious third parties may impersonate compromised users to gain access to sensitive data. it is difficult to find and rely on such a trustworthy third membership service party that validates user identity and accomplishes complex cross-heterogeneous domains authentication honestly without potential risk of real identity leakage. besides, typical blockchain systems cannot provide privacy-preserving verification due to public transaction record including pseudonyms and related behavior. in this case, curious third 885 servers or network nodes may collect large amounts of data to infer the real identity by statistical analysis. blockchain can also allow rollback models storage if false predication rate is high. blockchain stores the pointers of relevant data of retrained models in a secure and immutable manner. juneja et al. [43] proposed that retraining models indexed by pointers 930 in the blockchain can increase the accuracies for continuous remote systems in the context of irregular arrhythmia alarm rate. additionally, artificial intelligence can be applied to design automatic generation of smart contact to enhance secure and flexible operations. in the context of iot, the locations of products can be tracked at each step with radio-frequency identification (rfid), sensors or gps tags. individual healthy situation can be monitored at home via sensor devices and shared on the cloud environment where physical providers can access to provide on-time medical supports. however, as the use of sensors is experiencing exponential growth in various environ-940 ments, the security level of sensitive data of these sensors has to be improved. currently, a few studies focus on solving the above mentioned problems. related 980 research mainly focuses on the improvement of consensus algorithm, block size design [67] and so on. croman et al. [89] mainly improved the scalability of blockchain on latency, throughput and other parameters. the experiments showed that block size and generation inter-val in bitcoin are the first step toward throughput improvements and latency reduction without threat to system decentralization. new challenges for two data types in the blockchain-based system are throughput and fairness. two fairness-based packing algorithms are designed to improve the throughput 1000 and fairness of system among users. in the practical application scenario, how to encourage miners to participate in the network is important for the maintenance of trustworthy and stable blockchain. azaria et al. [44] proposed an incentive mechanism to encourage medical researchers and healthcare authorities as miners and create data economics by awarding for big data on hospital 1005 records to researchers. yang et al. [92] proposed a selection method in the incentive mechanism. providers have less significance (means the efforts that providers have been made on network maintenance and new blocks generation) with higher probabilities of being selected to carry out the task of new block generation and will be granted significance as bonus to reduce 1010 the selected probability in future. pham et al. [93] made further improvements on gas prices of blockchain, which can boost the priority in the processing transaction queue by automatically adjusting the gas price and then trigger an emergency contact to providers for on-time treatment immediately. meanwhile, it should be noted that all transactions can be "seen" by any node in the blockchain network. homomorphic encryption and zero knowledge proofs could be utilized to prevent data forensics by inference, maintain the privacy of individual information and allow computations to be performed without the leakage of input and output of computations. as the above statement, blockchain still has many limitations and more aggressive extensions will require fundamental protocol redesign. so it is urgent to be towards to the improvement of underlying architecture of blockchain for better service. in the context of iot, personal healthcare data streams collected from wearable devices are high in volume and at fast rate. large amounts of data can support for big 1025 data and machine learning to increase the quality of data and provide more intelligent health service. however, it may lead to high network latency due to the physical distance to mobile devices and traffic congestion on the cloud servers. besides, the mining process and some encryption algorithms may cost high computational power on resource-limited devices 1030 and restrict the use of blockchain. a new trend is increasingly moving from the function of clouds towards network edge with low network latency. it is mainly required by time-sensitive applications, like healthcare monitor applications. combining with edge computing, blockchain is broadened to a wide range of services from pure data storage, such as device configuration 1035 and governance, sensor data storage and management, and multi-access payments. if new technologies enter the market without some form of vetting, they should be adopted with care for example based on a cost-benefit-analysis. hence, to improve compliance, security, interoperability and other factors, we need to develop uniform stan-1040 dards, policies and regulations (e.g. those relating to data security and privacy, and blockchain ecosystem). for example, we would likely need different independent and trusted mechanisms to evaluate different blockchain solutions for different applications and context, in terms of privacy, security, throughput, latency, capacity, etc. we would also need to be able to police and enforce penalty for misbehavior and/or violations (e.g. non-compliance or not delivering as agreed in the contract). blockchain has shown great potential in transforming the conventional healthcare industry, as demonstrated in this paper. there, however, remain a number of research and operational challenges, when attempting to fully integrate blockchain technology 1050 with existing ehr systems. in this paper, we reviewed and discussed some of these challenges. then, we identified a number of potential research opportunities, for example relating to iot, big data, machine learning and edge computing. we hope this review will contribute to further insight into the development and implementation of the next generation ehr systems, which will benefit our (ageing) society. healthcare professionals organisational barriers to health information technologiesa lit-1065 erature review maturity models of healthcare information systems and technologies: a literature review security and privacy in electronic health records: a systematic literature review implementing electronic health records in hospitals: a systematic literature review electronic health record use by nurses in mental health settings: a literature review personal electronic healthcare records: what influences consumers to engage with their clinical data online? a literature review methodologies for designing healthcare analytics solutions: a literature analysis opportunities and challenges in healthcare information systems research: caring for patients with chronic conditions visualization of blockchain data: a systematic review a blockchain-based approach to health information exchange networks blockchain in healthcare applications: research challenges and opportunities blockchain: a panacea for healthcare cloud-based data security and privacy? 2017 ieee technology & engineering management conference (temscon) blockchain technology in healthcare: the revolution starts here bitcoin: a peer-to-peer electronic cash system dcap: a secure and efficient decentralized 1100 conditional anonymous payment system based on blockchain an efficient nizk scheme for privacy-preserving transactions over account-model blockchain a survey on privacy protection in blockchain system secure and efficient two-party signing protocol for the identity-based signature scheme in the ieee p1363 standard for public key cryptography multi-party signing protocol for the identitybased signature scheme in ieee p1363 standard the byzantine generals problem a survey on consensus mechanisms and mining strategy management in blockchain networks practical byzantine fault tolerance ethereum: blockchain app platforms multichain: open platform for building blockchains forensic-by-design framework for cyberphysical cloud systems medical cyber-physical systems development: a forensics-driven approach sdte: a secure blockchain-based data trading ecosystem class: cloud log assuring soundness and secrecy scheme for cloud forensics enigma: decentralized computation platform with guaranteed privacy medibchain: a blockchain based privacy preserving platform for healthcare data fingernail analysis management system using microscopy sensor and blockchain technology ordieres-mere, blockchain-based personal health data sharing system using cloud storage the design of rijndael: aes -the advanced encryption standard. 1150 [38] s. vanstone, a. menezes, p. v. oorschot, handbook of applied cryptography healthcare data gateways: found healthcare intelligence on blockchain with novel privacy risk control secure attribute-based signature scheme with multiple authorities for blockchain in electronic health records systems lightweight backup and efficient recovery scheme for health blockchain keys bpds: a blockchain based privacy-preserving data sharing for electronic medical records leveraging blockchain for retraining deep learning architecture in patientspecific arrhythmia classification medrec: using blockchain for medical data access 1165 and permission management a decentralizing attribute-based signature for healthcare blockchain using java to generate globally unique identifiers for dicom objects a framework for secure and decentralized sharing of medical imaging data via blockchain consensus blockchain for secure ehrs sharing of 1175 mobile cloud based e-health systems towards using blockchain technology for ehealth data access management a blockchain-based framework for data sharing with fine-grained 1180 access control in decentralized storage systems an overview of interoperability standards for electronic health records, usa: society for design and process science 2018 ieee international conference on bioinformatics and biomedicine medrec: using blockchain for medical data access and permission management fhirchain: applying blockchain 1190 to securely and scalably share clinical data applying software patterns to address interoperability in blockchain-based healthcare apps a cloud-based approach for interoperable electronic health records 1195 (ehrs) design for a secure interoperable cloud-based personal health record service how distributed ledgers can improve provider data management and support 1200 interoperabilityhttps integrating blockchain for data sharing and collaboration in mobile healthcare applications security and privacy in electronic health records: a systematic literature review blockchain for access control in e-health scenarios blockchain based access control servicesdoi blockchain based delegatable access control scheme for a collaborative e-health environment audit-based access control with a distributed ledger: applications to healthcare organizations organization based access control secure and trustable electronic medical records sharing using blockchain bbds: blockchain-based data sharing for electronic medical records in cloud environments secure and efficient data accessibility in blockchain based healthcare systems a secure system for pervasive social network-based healthcare blockchain-based multiple groups data sharing with anonymity and traceability privacy-preserving attribute-based access control model for xml-based electronic health record system dynamic access control policy based on blockchain and machine learning for the internet of things content extraction signatures medshare: trust-less medical data sharing among cloud service providers via blockchain security and privacy in electronic health records: a systematic literature review improving data transparency in clinical trials using blockchain smart contracts blockchain technology for improving clinical research quality blockchain distributed ledger technologies for biomedical 1250 and health care applications on the design of a blockchain platform for clinical trial and precision medicine non-interactive zero-knowledge and its applications verifiable anonymous identities and access control in permissioned blockchains big data: are biomedical and health informatics training programs ready? privacy preserving in blockchain based on partial homomorphic encryption system for ai applications a fully homomorphic encryption scheme an architecture and protocol for smart continuous ehealth monitoring using 5g 5g-smart diabetes: toward personalized diabetes 1270 diagnosis with healthcare big data clouds permissioned blockchain and edge computing empowered privacy-preserving smart grid networks integrated blockchain and edge computing systems: a survey, some research issues and challenges blochie: a blockchain-based platform for healthcare information exchange a design of blockchain-based architecture for the security of electronic health record (ehr) systems a secure remote healthcare system for hospital using blockchain smart contract shuyun shi received the bachelor degree in 2019, from the school of computer 1290 she is currently working toward a master degree at the key laboratory of aerospace information security and trusted computing ministry of education he is currently a professor of the key laboratory of aerospace information security and trusted computing, ministry of education, school of cyber science and engineering, wuhan uni-1300 versity, wuhan 430072, china. his main research interests include cryptography and information security li li received her ph.d degree in computer science from computer school she is currently an associate professor at school of software, wuhan univer-1305 sity. her research interests include data security and privacy, applied cryptography and security protocols his research is focused on mobile computing, parallel/distributed computing, multi-agent systems, service oriented computing, routing and security issues in mobile ad hoc, sensor and mesh networks. he has more than 100 technical research papers in leading journals such as-ieee tii his research is supported from dst, tcs and ugc. he has guided many students leading to m.e. and ph.d australia day achievement medallion, and british computer society's wilkes award in 2008. he is also a fellow of the australian computer society digital rights management for multimedia interest group we thank the anonymous reviewers for their valuable comments and suggestions which helped us to improve the content and presentation of this paper. the authors declare that they have no conflicts of interest. 61 key: cord-338512-vz0jos3f authors: katz, rebecca; may, larissa; baker, julia; test, elisa title: redefining syndromic surveillance date: 2011-08-18 journal: j epidemiol glob health doi: 10.1016/j.jegh.2011.06.003 sha: doc_id: 338512 cord_uid: vz0jos3f with growing concerns about international spread of disease and expanding use of early disease detection surveillance methods, the field of syndromic surveillance has received increased attention over the last decade. the purpose of this article is to clarify the various meanings that have been assigned to the term syndromic surveillance and to propose a refined categorization of the characteristics of these systems. existing literature and conference proceedings were examined on syndromic surveillance from 1998 to 2010, focusing on lowand middle-income settings. based on the 36 unique definitions of syndromic surveillance found in the literature, five commonly accepted principles of syndromic surveillance systems were identified, as well as two fundamental categories: specific and non-specific disease detection. ultimately, the proposed categorization of syndromic surveillance distinguishes between systems that focus on detecting defined syndromes or outcomes of interest and those that aim to uncover non-specific trends that suggest an outbreak may be occurring. by providing an accurate and comprehensive picture of this field’s capabilities, and differentiating among system types, a unified understanding of the syndromic surveillance field can be developed, encouraging the adoption, investment in, and implementation of these systems in settings that need bolstered surveillance capacity, particularly lowand middle-income countries. abstract with growing concerns about international spread of disease and expanding use of early disease detection surveillance methods, the field of syndromic surveillance has received increased attention over the last decade. the purpose of this article is to clarify the various meanings that have been assigned to the term syndromic surveillance and to propose a refined categorization of the characteristics of these systems. existing literature and conference proceedings were examined on syndromic surveillance from 1998 to 2010, focusing on low-and middle-income settings. based on the 36 unique definitions of syndromic surveillance found in the literature, five commonly accepted principles of syndromic surveillance systems were identified, as well as two fundamental categories: specific and non-specific disease detection. ultimately, the proposed categorization of syndromic surveillance distinguishes between systems that focus on detecting defined syndromes or outcomes of interest and those that aim to uncover non-specific trends that suggest an outbreak may be occurring. by providing an accurate and comprehensive picture of this fieldõs capabilities, and differentiating among system types, a unified understanding of the syndromic surveillance field can be developed, encouraging the adoption, investment in, and implementation of these systems in settings that need bolstered surveillance capacity, particularly low-and middle-income countries. ª 2011 the field of syndromic surveillance is best understood through the context of global efforts to respond and adapt to modern-day surveillance challenges and disease threats. globalization and the ease of international spread of disease require improved global surveillance capacity in order to rapidly detect and contain public health emergencies. recognition of this need has led to increased efforts to enhance disease surveillance and demands examination of all available tools-one of which is syndromic surveillance. such thinking is exemplified by the world health organizationõs (who) decision to revise the international health regulations (ihr). as part of the 10-year ihr revision process, who sponsored a pilot study in 22 countries from 1997 to 1999 to evaluate syndromic reporting. it was concluded that ''syndromic reporting, although valuable within a national system, was not appropriate for use in the context of a regulatory framework'' [1] . the final negotiated ihr (2005) regulates detection, reporting and response within a more adaptive category of ''events that may constitute a public health emergency of international concern'' [2] . the ihr (2005) also obligates every member state of the who to build national core competency for disease surveillance. however, the regulations do not prescribe exactly how nations are to meet this core capacity. certain low-and middleincome countries-particularly those facing the need to rapidly strengthen disease surveillance and overall public health infrastructure to meet their ihr (2005) obligations-may be looking to syndromic surveillance options and opportunities. a report from the ihr (2005) negotiations stresses this point: ''because areas with the highest needs for surveillance of communicable diseases have often the poorest surveillance systems, new surveillance approaches, such as the surveillance of syndromes, adapted to poor laboratory infrastructure should be developed to respond to the challenge of development gaps'' [3] . in addition to the increased attention to syndromic surveillance in the negotiations of ihr (2005), syndromic surveillance has gained importance for national governments and has become widely used at the country level, particularly in high-income countries. examples include a syndromic surveillance system in the united kingdom based on data from the national telehealth system (nhs direct) and a system in denmark that utilizes ambulance dispatch records [4] . in the united states (us), state and local syndromic surveillance systems are widespread [5] , as evidenced by a recent survey that concluded, ''populations covered by health departments that reported conducting syndromic surveillance account for 72% of the us population'' [6] . spurred by a series of reports from the us government accountability office [7] and other organizations [8] , the us federal government has recently begun re-examining how to best ensure effective and efficient disease surveillance capacity. in this context, the centers for disease control and prevention (cdc) must re-evaluate biosurveillance for human health [9] . the united states agency for international developmentõs (usaid) predict program aims to integrate human and animal disease surveillance, primarily focused on the implementation of programs in developing nations [10] . researchers in the field of syndromic surveillance have similarly turned toward translating syndromic surveillance for use in lower resource settings [11] [12] [13] . a 2007 disease surveillance workshop held in bangkok, thailand, sponsored by the department of defense global emerging infections surveillance and response system (dod-geis) and johns hopkins university/applied physics laboratory (jhu/apl), focused on the adaptation of electronic surveillance tools to low-and middle-income settings [11] . in order to reenergize these discussions (which were begun by 13 countries and other stakeholders during the 2007 workshop), clarification of the definition, functions, and challenges of syndromic surveillance is needed. doing so would help ensure that syndromic surveillance will be adopted in all places where it would be of benefit. since syndromic surveillance systems began being used in the 1990s and became widespread in early 2000, vast applications of these systems have demonstrated many capabilities and uses. there are numerous variations among syndromic surveillance system definitions, objectives, and surveillance methodologies, which is why there is a need for a comprehensive characterization of the breadth of the term ''syndromic surveillance.'' common themes across the literature have emerged, suggesting general agreement among those in the field. the commonly accepted principles of syndromic surveillance include: • early detection and response: most articles on syndromic surveillance discuss the value of these systems in signaling the presence of an abnormal trend with ''sufficient probability'' to warrant further investigation (without necessarily providing definitive detection) [14] [15] [16] [17] [18] [19] [20] . • use of ''continuously acquired'' pre-diagnostic information: by focusing on data collected prior to clinical diagnosis or laboratory confirmation, syndromic surveillance uses non-traditional health indicator data [17, 21, 22] . • possible situational awareness use: chretien and his co-authors describe situational awareness as ''monitoring the effectiveness of epidemic responses and characterizing affected populations'' [12] . by providing a tool for following the course of an outbreak, syndromic surveillance has value besides merely initial detection in augmenting public health surveillance as well as outbreak response [6, 11, 15, [23] [24] [25] . • providing reassurance that an outbreak is not actually occurring: by monitoring outbreak thresholds, as well as collecting data from a variety of sources, a syndromic surveillance system can provide information to public health authorities confirming or refuting the occurrence of an outbreak [17, 19, 24, 26] . • augments traditional public health surveillance: in order to improve outbreak detection [13, 17, 19] , several definitions of syndromic surveillance emphasize that its goal is to ''enhance, rather than replace, traditional approaches to epidemic detection'' [14] . despite these points of agreement and the popularity of syndromic surveillance systems in many countries, there is still little or no consensus regarding a standard definition encompassing the full scope of the term ''syndromic surveillance.'' according to mostashari and hartman, the term syndromic surveillance is ''imprecise and potentially misleading'' [27] . the first point of confusion is the fact that ''many of the systems under discussion do not monitor well-defined constellations of signs and symptoms (syndromes), but instead target non-specific indicators of health, such as a patient with a chief complaint of ôcoughõ or the sale of over-the-counter cold medication; conversely, many systems that do monitor syndromes (e.g., acute flaccid paralysis, reyeõs syndrome, or carpal tunnel syndrome) are not included in these discussions'' [27] . second, some refer to the term ''syndrome'' as a specific, clinically defined phenomenon, such as severe acute respiratory syndrome (sars) or acquired immune deficiency syndrome (aids), and others use it more loosely and non-specifically as simply a group of symptoms [28] . several researchers have proposed alternative names to differentiate the forms of syndromic surveillance; however, these suggestions for clarified terminology have not yet taken hold. ten other names that have been proposed in the literature include: outbreak detection systems, early warning systems, health indicator surveillance, prodromal/ ic surveillance, information system-based sentinel surveillance, pre-diagnosis surveillance, nontraditional surveillance, enhanced surveillance, drop-in surveillance, and biosurveillance [12, 19, 27, 28] . problematically, these terms overlap, contradict, or are inconsistently applied, maintaining the terminological confusion. further, several terms frequently applied to syndromic surveillance are not adequately descriptive to convey the type of system being referred to or do not distinguish between types of systems, and thus may not be appropriate as general, overarching terms. given the challenge of developing clearer terminology, the potentially confusing term ''syndromic surveillance'' is still being used [27] . due to the significant increase in applications of syndromic surveillance, and the new technologies and expanded potential of the tool-demonstrated by the evolution of the proceedings of the international society for disease surveillance (isds) conferences on syndromic surveillance [29] -it is evident that the field has expanded over the last decade. thus, there is now an even greater need for a consensus about what syndromic surveillance means. additionally, as plans to translate syndromic surveillance systems to lower resource settings proceed, a proper conceptualization of the systems could increase their acceptance and efficient use. the development and communication of a unified understanding of the field may encourage governments or localities to adopt, invest in, and implement syndromic surveillance, where appropriate, and thus enhance compliance with ihr (2005); this adoption in middle-and low-income nations is being explored in the ongoing work to identify exemplary case studies of successful utilization of syndromic surveillance [30] . the purpose of this article is to clarify the various meanings that have been assigned to the term syndromic surveillance and to propose a refined categorization of the characteristics of the systems. in an effort to capture the variety of definitions and explanations of syndromic surveillance in the literature, medline, scopus, google scholar, proceedings from all isds conferences, and previous literature reviews and reference lists related to this topic were searched from 1998 to 2010. search terms included ''syndromic surveillance'' and the 10 other terms mentioned above, which are considered synonymous with syndromic surveillance. in addition to general overview articles, the set of articles pertaining to country-and region-specific systems were narrowed by including the terms ''low-income'' and ''developing country'' to fit the emphasis on the translation of these systems to lower resource settings. through a review of titles, abstracts, and fulllength articles, 81 general articles were identified describing and evaluating syndromic surveillance systems along with several hundred articles delineating surveillance systems implemented in specific countries or regions. within the articles collected, those that defined syndromic surveillance or provided a description of fundamental aspects of these systems were selected and compiled for comparison. in total, 43 separate articles defined syndromic surveillance, of which 36 provided unique definitions. a majority of the unique definitions found came from overview of articles of syndromic surveillance, rather than countryspecific articles. however, the country-specific articles provided distinguishing examples of systems being implemented in various settings. table 1 contains the 36 unique definitions of syndromic surveillance found in the literature. the five general points of agreement among those in the field and mentioned above are frequently noted in the definitions and are designated in the set of columns on the far right. the two shaded columns indicate that there are two fundamental categories of syndromic surveillance systems conveyed by the collection of definitions. these two categories involve the same investigational approach, and may even be components of the same system, but monitor two distinct outcome types: specific and non-specific outcomes. within the two fundamental categories of syndromic surveillance (defined further below), all five principles are relevant, with the principles of early detection and response and the use of pre-diagnostic information being the most commonly referred to across the definitions. the literature review yielded multiple articles that noted the distinction between specific and non-specific surveillance systems. in a foundational article introducing the field of syndromic surveillance to a wider audience, sosin explains that indicators of a disease outbreak can either be suggestive of ''highly specific syndrome[s]'' or ''non-specific expressions of the target diseases that occur before a diagnosis would routinely be made'' [18] . the following year, an article based on recommendations from a cdc working group contrasted surveillance of a syndrome that ''is relatively specific for the condition of interest'' (such as acute flaccid paralysis as ''a syndromic marker'' in the detection of poliomyelitis) to surveillance with a broader purpose, such as ''sexually transmitted disease detection and control'' [35] . more recently, fricker differentiates ''well-defined'' data that are ''linked to specific types of outbreaks'' to data that are ''vaguely defined and perhaps only weakly linked to specific types of outbreaks, such as over-the-counter sales [14] x x x x x x centers for disease control and prevention [33] x x x pavlin [21] x x x reingold [34] x x x smolinski et al. [28] x x x x sosin [18] x x x x sosin [19] x x x x x x buehler [35] x x x x mmwr editors [36] x x henning [26] x x lombardo et al. [37] x x mandl et al. [22] x x x stoto et al. [16] x x x ang et al. [38] berger et al. [17] x x x x stoto et al. [39] x x x chaves and pascual [40] x morse [41] x x buehler et al. [6] x x x x x centers for disease control and prevention [20] x x chretien et al. [12] x x x x x chretien et al. [11] x x x fearnley [42] x x fricker [43] x x x fricker et al. [15] x x x x x jefferson et al. [44] x x x nordin et al. [39] tsui et al. [46] x buehler et al. [23] x x x gault et al. [47] x may et al. [13] x x sintchenko and gallego [24] x x x zhang et al. [48] x x x josseran et al. [49] x of cough and cold medication or absenteeism rates'' [43] . fricker continues his summary of the types of syndromic surveillance by observing that the meaning of the term ''syndrome'' has evolved in the context of syndromic surveillance: ''a syndrome is ôa set of symptoms or conditions that occur together and suggest the presence of a certain disease or an increased chance of developing the diseaseõ. in the context of syndromic surveillance, a syndrome is a set of non-specific pre-diagnosis medical and other information that may indicate the release of a bioterrorism agent or natural disease outbreak'' [emphasis ours] [43] . clearly this term ''syndromic'' as it is narrowly defined imperfectly captures the full range of these systems. an adjustment of the term, while maintaining the root of its meaning to ensure continuity in the field, can help strengthen and expand understanding of this field. table 2 outlines the two categories of syndromic surveillance system types-specific and non-specific. as explained above and demonstrated in the literature, the purposes of each syndromic surveil-lance category are different. whereas the ''specific disease/syndrome detection'' category focuses on detecting defined syndromes or a defined outcome of interest, the ''non-specific disease detection'' category aims to monitor or uncover non-specific indicators/trends that suggest an outbreak may be occurring. based on this distinction, an alteration of the term ''syndromic surveillance'' to ''syndrome-based'' surveillance (sbs) referring to the more specific type of surveillance and ''syndromic-non-specific'' surveillance (sns) referring to the now more common, non-specific category of disease detection is proposed. the categorization of sbs and sns is confirmed by the examined literature and will be used throughout the remainder of the article to refer to these categories of surveillance. because of the terminological confusion with syndromic surveillance, this proposed categorization is necessary. newcomers to the field of syndromic surveillance frequently have a narrow perspective of what these systems entail. initial misconceptions include the false belief that it is surveillance based solely on syndromes (such as acute flaccid paralysis) or that this type of surveillance requires significant information technology (it) capacity. the former point is immediately early symptoms (such as gastrointestinal complaints, influenza-like illness) example: an increase in gastrointestinal illness cases indicating a water-borne outbreak data sources ''constellations of medical signs and symptoms in persons seen in various clinical settings'' [35] , such as icd-9 codes in addition to the types of data sources at left, unusual patterns in health-related behaviors (e.g., over-the-counter and health product purchases, such as cough medicine; and absenteeism from work or school) example ''the syndromes to be notified where an outbreak is of urgent international public health importance are: acute hemorrhagic fever syndrome, acute respiratory syndrome, acute diarrheal syndrome, acute jaundice syndrome, [and] acute neurological syndrome. in addition, any other syndrome of severe illness not included in the above should be notified if an outbreak is of urgent international public health importance'' [31] ''[in developing countries,] syndromic surveillance can identify outbreaks that do not fall into pre-established diagnostic categories, a capability essential for prompt control of new or changing diseases'' [12] southeast asiaõs early warning outbreak recognition system (ewors) provides surveillance of 29 non-specific signs and symptoms, which are not grouped into specific syndromes [50] clarified by separating the definition of syndromic surveillance into two separate terms: sbs and sns. the latter point concerning technological capacity will be discussed later in the text where the characteristic of technological dependence is described as a gradation within the two larger categories. other attempts to categorize syndromic surveillance systems have also divided the field into two separate components; however, these categorizations do not encompass the full range of systems and most critical distinctions evident in the literature. one attempt separated systems into those based on a ''data collection system that is dedicated to the purpose of this public health surveillance'' (e.g., during a specific event) and those for the day-to-day monitoring of ''data that are routinely collected for other purposes'' [27] . another categorization divided systems by data source: those data sources based on the use of health-care services and those based on health-related behaviors [6] . while these are useful distinctions to make when examining syndromic surveillance systems, they are not the most fundamental. the proposed categorization within the scope of this research addresses the most critical distinction among syndromic surveillance systems, which is the level of specificity of the outcome under surveillance. the distinction between data sources as sub-categories within each of the two larger categories (specific and non-specific detection) is taken into consideration because there is a fair amount of overlap. as noted in table 2 , the data sources used in sns systems can include those data sources used in sbs systems, but frequently focus primarily on pre-clinical data. data sources for public health surveillance have been traditionally divided into three levels: ''preclinical data, clinical pre-diagnostic data, and diagnostic data. syndromic surveillance usually uses two types of data sources: pre-clinical and clinical pre-diagnostic data; traditional surveillance generally focuses on diagnostic data'' [17] . these levels can be further divided among the two categories of syndromic surveillance, with the sns category being largely comprised of pre-clinical data, whereas the sbs category is focused on clinical pre-diagnostic data. the 36 definitions are rife with examples of each level of data source. frequently cited clinical prediagnostic data sources for the sbs systems include: patient chief complaints or icd-9 coded health information from clinical records (outpa-tient, emergency department, and hospital), billing databases, and emergency department triage and discharge data. though icd-9 coded health information is a form of diagnostic information, experts in the field of syndromic surveillance have suggested that general groupings of icd-9 codes can be considered early diagnostic data [33, 35, 38, 43] . pre-clinical data used in sns systems are often pulled from existing databases intended for other purposes [21, 22, 24, 27] and are therefore ''weakly linked'' to the target disease [43] . examples include ''. . . ôindicatorõ (pre-diagnostic) data (e.g., syndromes, medication sales, absenteeism, patient chief complaints)'' [12] , pharmacy records, telephone health advice/consultation, poison control centers, 911 calls, ''phone calls to or internet use of a health-care information site'' [18] , laboratory test requests/orders [35] , veterinary health records, health department requests for influenza testing [48] , health care utilization patterns [24] , ambulance services, and number of hospital admissions. environmental data sources and water utility complaint lines [17] are similarly non-specific. within the two overarching categories of sbs and sns, three additional sub-classifications became evident through the literature review, specifically surveillance to detect influenza-like illness and possible bioterrorism events, as well as the gradations in technological capacity. one common use of syndromic surveillance is for the monitoring and detection of influenza-like illness (ili), which is unique in that it falls within both specific and non-specific surveillance categories. as a specific syndrome, ili can be used for monitoring known strains: the ili syndrome is useful for ''clarify[ing] the timing and characteristics of annual influenza outbreaks'' [18] . conversely, in sns, an increased number of cases of respiratory symptoms and fever could indicate a bioterrorism-related event or a new strain of a virus with pandemic potential. as previously mentioned, syndromic surveillance has evolved from specific disease detection to encompassing more non-specific disease detection. in line with this change, and in response to the anthrax event in 2001, syndromic surveillance came to be seen as potentially useful for the detection of bioterrorism outbreaks. its utilization of data from a variety of sources makes it a valuable addition to traditional surveillance methods [22] . while bioterrorism detection is not typically the primary use of syndromic surveillance today, non-specific disease detection continues to be thought of and investigated as a biosecurity tool [20] . those in the bioterrorism field explain that syndromic surveillance, when applied to large, concentrated events, can ''detect the early manifestations of illness that may occur during a bioterrorism-related epidemic. . . [such as] the prodromes of bioterrorism-related disease. . . [but] other uses of syndromic surveillance include detecting naturally occurring epidemics'' [14] , and it is more widely applicable than as merely a tool for bioterrorism detection [18] . the incorporation of biosecurity concepts into the syndromic surveillance field has contributed to the broadening of the five syndrome categories defined by who in 1998 (''acute hemorrhagic fever syndrome, acute respiratory syndrome, acute diarrheal syndrome, acute jaundice syndrome, and acute neurological syndrome'') [31, 51] to include additional categories of symptoms that can be monitored through non-specific surveillance. a cdc-led, multi-agency workgroup identified 11 ''syndrome categories to be monitored that were indicative of the clinical presentations of several critical bioterrorism-associated conditions,'' including several of the who syndrome categories as well as more non-specific symptoms such as ''rash'' and ''fever'' [33, 5, 6 ]. within each of the two categories-specific (sbs) and non-specific (sns)-there are varying degrees of technology that can be used for the syndromic surveillance system. depending on the data sources available and the outcome of interest, some systems require significant it and electronic capabilities [6] . however, there are also examples of less it-dependent systems that monitor specific syndromes and/or non-specific disease indicators [44] . thus, the distinction between high and low it dependence is considered a sub-category within each of the two larger categories. the literature review revealed that highly automated systems tend to be used in more developed countries, for large catchment areas, and when there is a focus on bioterrorism. typically, these systems involve electronic collection and analysis of data [49] , potentially utilizing the ''automated extraction of data from electronic medical records'' [38] . on the other hand, less automated, less it-dependent systems are more frequently seen in developing countries and often incorporate some element of manual data entry, extraction, or analysis, or the involvement of fax or mobile technology [52] , resulting in detection that is ''near real-time'' as opposed to ''real-time'' [53] . in a basic form, syndromic surveillance ''is a feasible and effective tool for surveillance in developing countries'' and should be supported [13] . given the significant applicability of syndromic surveillance systems to lowand medium-resource countries, it is critical that the definition of syndromic surveillance not be limited to highly it-dependent, strictly automated systems. clearly, though, where infrastructure allows, ''automation of the full cycle of surveillance'' allows for more real-time results [6] . early syndromic surveillance systems, including those part of the 1997-1999 who pilot study and as described in the 1998 update on the revision of the ihr, were largely focused on monitoring health events ''for which the case definition is based on a syndrome. . . e.g., acute hemorrhagic fever syndrome, acute respiratory syndrome'' [32] . over time, the systems have transitioned to monitoring less specific outcomes [43] . morse summarized this transition well: syndromic surveillance ''once referred to the use of clinical syndromes as criteria for reporting. now, it usually means data collected from automated non-diagnostic systems such as pharmacy records, ambulance call categories, personnel absences, or emergency department chief complaints'' [41] . of the 36 unique definitions found in the literature review, several appeared to be overly narrow and might contribute to the confusion ascribed to this term. most of these narrow definitions suggested that syndromic surveillance is limited to highly it-dependent systems, require automation or immediate analysis, or are limited to one function, such as bioterrorism [16, 26, 39, 45] . the literature review makes evident that many systems that are considered ''syndromic surveillance'' are less it-dependent, may include some manual component, and have much broader applicability. the importance of taking an all-inclusive view to the field of syndromic surveillance is put best by fricker: ''a myopic focus only on early event detection for bioterrorism in syndromic surveillance systems misses other important benefits electronic biosurveillance can provide, particularly the potential to significantly advance and modernize the practice of public health surveillance'' [43] . an important contribution of a syndromic surveillance system is that it can be established in countries of any resource level. a broad definition, accounting for all of the purposes of syndromic surveillance-and acknowledging the flexibility of the infrastructure requirements-will facilitate its introduction and use in a variety of settings. a recent review described 10 syndromic surveillance systems in developing countries, demonstrating the ''feasibility of ôlow-techõ syndromic surveillance in low resource countries'' [13] , ewors being one commonly cited example in southeast asia [52] . in developing countries, data sources not traditionally employed in surveillance can be useful, such as environmental sources assisting the detection of vector-borne and neglected tropical diseases, monitoring indoor resting densities of vectors, climate and land use data, and satellite imagery [40] . surveillance of sexually transmitted infections could also be augmented by syndromic surveillance. according to who, syndromic surveillance of non-specific symptoms, including ''urethral discharge and genital ulcer, are potentially useful for monitoring trends in std incidence'' [54] . as the field has expanded, the truly broad nature of these surveillance systems has become apparent. today, the term ''syndromic surveillance'' imperfectly describes all forms of syndromic surveillance systems. fearnley suggests, ''this terminological instability reflects an underlying ontological and normative instability,'' and without a generalized consensus of the definition of syndromic surveillance, ''designers and users [may] continue to dispute what syndromic systems can and should do'' [42] . based on the results of the literature review, and in order to improve the conceptualization of this term, it was necessary to categorize syndromic surveillance into sbs and sns, based on the fundamentals of specific and non-specific disease detection. the sub-categorization of the systems by data source and it-capacity required is based on the broad range of features that constitute syndromic surveillance systems. prior categorizations, described above, have not sufficiently encapsulated all that syndromic surveillance can entail. recognizing that syndromic surveillance systems comprise these two categories with two different purposes helps clarify the added value of this kind of surveillance and may reduce ontological instability. it is recognized, as mentioned above, that in practice, the distinction between specific and non-specific syndromic surveillance categories can be lost, since many of these systems-particularly those in the united states-incorporate both categories within the same system. these dual-function systems (specific and non-specific detection) collect data from several sources-both the pre-clinical and clinical non-diagnostic types. nevertheless, the acceptance and application of improved terminology regarding these systems can reduce ambiguity in the field and increase adoption of syndromic surveillance systems where appropriate. future research must explore the combination of the sbs and sns systems in more depth. these ongoing questions highlight the importance of incorporating robust system evaluation into future syndromic surveillance implementation efforts. empirical, quantifiable evidence about the utility of these systems for improved surveillance and detection must be established. such evidence will be essential for decision makers contemplating investing in syndromic surveillance to help meet ihr (2005) obligations. despite early concerns about the benefits of the syndromic approach to surveillance [34, 42] and the continued need for further research, this approach has been proven successful in a wide variety of settings [5, 17, 18, 30] . this paper has attempted to take a broad perspective on the field of syndromic surveillance, acknowledging the numerous syndromic surveillance systems that have been making important contributions to public health for over a decade, and summarizes the termõs many definitions. by providing an accurate and comprehensive picture of this fieldõs capabilities, and differentiating between sbs and sns, it is hoped that syndromic surveillance will be seen more widely as a tool that can help any nation (high, middle, or low income) build comprehensive disease surveillance capacity. all authors contributed to this manuscript. rebecca katz and larissa may conceptualized the project, directed the research, and reviewed and edited drafts of the manuscript. elisa test and julia baker conducted the literature review and drafted the manuscript. all authors approved the final article. this work was supported by a grant from the george washington university research facilitating funds. the sponsor had no role in the study beyond providing financial support for researchersõ time. revision of the international health regulations: progress report epidemiological surveillance and international health regulations syndromic surveillance -a european perspective. the international society for disease surveillance: global outreach committee enewsletter evaluation challenges for syndromic surveillance-making incremental progress syndromic surveillance practice in the united states: findings from a survey of state, territorial, and selected local health departments biosurveillance: efforts to develop a national biosurveillance capability need a national strategy and a designated leader international disease surveillance: united states government goals and paths forward concept plan for the implementation of the national biosurveillance strategy for human health emerging pandemic threats -program overview electronic public health surveillance in developing settings: meeting summary syndromic surveillance: adapting innovations to developing settings beyond traditional surveillance: applying syndromic surveillance to developing settings-opportunities and challenges syndromic surveillance and bioterrorism-related epidemics comparing syndromic surveillance detection methods: earsõ versus a cusumbased methodology syndromic surveillance: is it worth the effort? review of syndromic surveillance: implications for waterborne disease detection syndromic surveillance: the case for skillful investment draft framework for evaluating syndromic surveillance systems syndromic surveillance: an applied approach to outbreak detection investigation of disease outbreaks detected by ''syndromic'' surveillance systems implementing syndromic surveillance: a practical guide informed by the early experience situational uses of syndromic surveillance laboratory-guided detection of disease outbreaks: three generations of surveillance systems utilization of syndromic surveillance with multiple data sources to enhance public health response what is syndromic surveillance? syndromic surveillance: a local perspective (editorial) microbial threats to health: emergence, detection, and response applications of syndromic surveillance in resource poor settings: a series of case studies revision of the international health regulations -regional committee document who, department of communicable disease surveillance and response. who recommended surveillance standards syndrome definitions for diseases associated with critical bioterrorism-associated agents if syndromic surveillance is the answer, what is the question? framework for evaluating public health surveillance systems for early detection of outbreaks. recommendations from the cdc working group preface to syndromic surveillance: reports from a national conference essence ii and the framework for evaluating syndromic surveillance systems an assessment of electronically captured data in the patient care enhancement system (paces) for syndromic surveillance statistical methods in counterterrorism: game theory, modeling, syndromic surveillance, and biometric authentication comparing models for early warning systems of neglected tropical diseases global infectious disease surveillance and health intelligence signals come and go: syndromic surveillance and styles of biosecurity syndromic surveillance evaluation of syndromic surveillance for the early detection of outbreaks among military personnel in a tropical country bioterrorism surveillance and privacy: intersection of hipaa, the common rule, and public health law a review of healthcare, public health, and syndromic surveillance performance of a syndromic system for influenza based on the activity of general practitioners automatic online news monitoring and classification for syndromic surveillance assessment of a syndromic surveillance system based on morbidity data: results from the oscour network during a heat wave ewors: using a syndromic-based surveillance tool for disease outbreak detection in indonesia who, fifty-first world health assembly. revision of the international health regulations: progress report by the director-general using nfc-enabled mobile phones for public health in developing countries surveillance for emerging infection epidemics in developing countries guidelines for surveillance of sexually transmitted diseases none declared. key: cord-347566-8x4vwarn authors: klerkx, laurens; begemann, stephanie title: supporting food systems transformation: the what, why, who, where and how of mission-oriented agricultural innovation systems date: 2020-08-05 journal: agric syst doi: 10.1016/j.agsy.2020.102901 sha: doc_id: 347566 cord_uid: 8x4vwarn agricultural innovation systems has become a popular approach to understand and facilitate agricultural innovation. however, there is often no explicit reflection on the role of agricultural innovation systems in food systems transformation and how they relate to transformative concepts and visions (e.g. agroecology, digital agriculture, agriculture 4.0, agtech and foodtech, vertical agriculture, protein transitions). to support such reflection we elaborate on the importance of a mission-oriented perspective on agricultural innovation systems. we review pertinent literature from innovation, transition and policy sciences, and argue that a mission-oriented agricultural innovation systems (mais) approach can help understand how agricultural innovation systems at different geographical scales develop to enable food systems transformation, in terms of forces, catalysts, and barriers in transformative food systems change. focus points can be in the mapping of missions and sub-missions of mais within and across countries, or understanding the drivers, networks, governance, theories of change, evolution and impacts of mais. future work is needed on further conceptual and empirical development of mais and its connections with existing food systems transformation frameworks. also, we argue that agricultural systems scholars and practitioners need to reflect on how the technologies and concepts they work on relate to mais, how these represent a particular directionality in innovation, and whether these also may support exnovation. agricultural systems transformation and more broadly the food systems transformation to which this contributes, has been the subject of debate for several decades already (dentoni et al., 2017; el bilali, 2019a , 2019b elzen et al., 2012; weber et al., 2020; klerkx et al., 2010; lamine, 2011) . however, recent years show a rise in concerns of how to manage future food security and sustainability without compromising food safety (vågsholm et al., 2020) . this has generated a social momentum for change regarding food systems development with studies concluding that the food system is 'broken' (oliver et al., 2018) and needs to be made 'planet-proof' (fraser and campbell, 2019; rockström et al., 2020; willett et al., 2019) , 'circular', 'regenerative' and 'inclusive' (gosnell et al., 2019; pouw et al., 2019; van zanten et al., 2019) . 1 kanter and boza, 2020; stephens et al., 2020) . as a response to these calls to transformative action, there is increasing scientific reflection on how to shape future food systems and the technologies and concepts that would underpin future food systems (herrero et al., 2020; kirova et al., 2019; klerkx and rose, 2020; mier et al., 2018; tittonell, 2014) . 2 in order to realize future food systems, there is a key role for agricultural innovation systems (ais) . 3 an ais is concerned with the networks of actors from science, business, civil society, and government that co-produce the suite of technological, social, and institutional innovations that co-shape these future food systems (gaitán-cremaschi et al., 2019; herrero et al., 2020; mier et al., 2018) . many of the future food systems envision novel forms of agriculture (e.g. urban farming, vertical agriculture, cellular agriculture), which deviate substantially from the current systems which are usually land-based and in rural areas. therefore, an ais increasingly incorporates actors from other sectors (e.g. information technology, energy, construction) and may employ a suite of generic technologies with applications in different sectors (e.g. solar energy generation, satellite technology, robot technology, nature based and ecologically regenerative solutions) frantzeskaki, 2019; laibach et al., 2019; pigford et al., 2018; tittonell, 2020) . also, it has been argued that agricultural innovation systems, in view of them supporting particular transition pathways towards future food systems, need to become 'mission-oriented' (klerkx and rose, 2020; pigford et al., 2018) . these missions need to tackle grand societal and planetary challenges, such as ecosystems integrity, biodiversity, climate change mitigation and adaptation as also comprised by the sustainable development goals (mazzucato et al., 2020) . the idea of mission orientation in innovation has become increasingly popular, and this has inspired concepts such as 'challenge-led innovation' and ' mission-oriented innovation policy' (boon and edler, 2018; mazzucato, 2016; rathenau institute, 2020) . mission-oriented innovation policy brings together elements of innovation policy, which traditionally aims to create economic growth, and transition policy, which principally aims to create change that is beneficial for society at large (alkemade et al., 2011; schot and steinmueller, 2018b; rathenau institute, 2020) . in this way, missionoriented innovation policy serves a clear transformative purpose and aspiration in view of realizing public goods such as a clean environment, biodiversity, animal welfare, and social justice (hall and dijkman, 2019; pigford et al., 2018; schlaile et al., 2017; timmermann, 2021) . instead of focusing on single technological fields or disciplines, mission-oriented policies are targeting a concrete problem/challenge, often cross-disciplinary, with a large impact and a well-defined time frame (wittmann et al., 2020) . also, there is a much stronger emphasis on directionality, to articulate the role of innovation and its contribution to a societal or environmental end (boon and edler, 2018; janssen et al., 2020; pigford et al., 2018; schlaile et al., 2017; rathenau institute, 2020) . this requires a more active and guiding role of the state with comprehensive innovation policies to govern innovation towards this end mazzucato, 2016) . moreover, state involvement moves from fixing market failure to shaping markets for societally relevant innovation and fixing directionality failures (robinson and mazzucato, 2019) . in the mission-oriented innovation policy framework, roughly four basic principles are maintained (mazzucato, 2016) the ideas on mission-oriented innovation are not completely new (robinson and mazzucato, 2019) , and have been inspired by earlier mission-oriented programs such as those from nasa. in agriculture, missions have also played a role, such as those underpinning productivity growth and modernisation in view of the green revolution (spiertz and kropff, 2011; wright, 2012) . however, these missions were often seen from an agricultural science system perspective rather than an ais perspective. 6 combining the idea of mission-oriented innovation policy with the innovation systems perspective introduced the concept of 'mission-oriented innovation systems', an idea alluded to earlier also for ais by pigford et al. (2018) . a missionoriented innovation system is defined as "the network of agents and set of institutions that contribute to the development and diffusion of innovative solutions with the aim to define, pursue and complete a societal mission" (hekkert et al., 2020:77) . steered by a clear directionality, it can be seen as "a temporary innovation system in which policy makers and other actors aim to coordinate innovation activities, with the objective of developing a coherent set of technological, institutional and behavioural solutions" (hekkert et al., 2020: 78) . the concept of mission-oriented innovation systems can be used to critically reflect upon how mission-oriented policies are nationally and supra-nationally embedded and how this loops back on the process of 2 also, it is the subject of debate of policy initiatives and think tanks such as the transforming food systems under a changing climate initiative led by ccafs (https://www.transformingfoodsystems.com/), the global alliance for the future of food (https://futureoffood.org/), the food systems dialogues (https://foodsystemsdialogues.org/), the commission on sustainable agriculture intensification (https://wle.cgiar.org/cosai/), the international panel of experts on sustainable food systems (ipes-food-http: //www.ipesfood.org/) and food tank (https://foodtank.com/), part of policy strategies such as the eu farm2fork strategy as part of the european union's green deal (europeanunion, 2020), the focus of technology events, incubators and accelerators such as evoke ag, food & farm 4.0, f&a next, and thought for food (https://www.farm-and-food.com/en/, https://www.fanext.com/, https:// evokeag.com/, https://thoughtforfood.org/), high level events such as the un food systems summit 2021 (https://www.un.org/sustainabledevelopment/ food-systems-summit-2021/), and steering the agenda of science organizations such as the australian future foods crc (https://www. futurefoodsystems.com.au/), oxford university (https://www.oxfordmartin. ox.ac.uk/food/) in the united kingdom, and utrecht university (https:// www.uu.nl/en/research/sustainability/research/future-food-utrecht) and wageningen university and research (https://www.wur.nl/en/about-wageningen/strategic-plan.htm) in the netherlands. 3 for an extensive explanation of emergence of the ais concept see (hall et al., 2006) . ais have been variedly conceptualised and studied at a national or regional, sectoral (e.g. dairy, horticulture), and technology level (e.g. pest management, water harvesting) (kebebe et al., 2015; kruger, 2017; minh, 2019; sixt et al., 2018; turner et al., 2016) . see (klerkx et al., 2012) for an explanation how the different boundaries of ais are cross-cutting (e.g. an ais at a country level may contemplate several sectoral and technological ais, which in turn may have global characteristics). in the european union context, ais are generally referred to as agricultural knowledge and innovation systems (akis). 4 for a broader explanation, see also the insightful position paper of janssen et al. (2020) . see also the informative overview of key elements of missiondriven innovation policy by rathenau institute (2020), whose piece provided inspiration for the title of this paper, and also refers to the dutch circular agriculture mission (see further footnote 8). 5 lock-in implies that a (food) system becomes inert to change in view of the interplay of established infrastructures, institutional frameworks (rules, regulations, values), and sunk investments. it has been argued that many current food systems have such a lock-in (i.e. 'food regimes') (gaitán-cremaschi et al., 2019; leach et al., 2020) , but it may also happen that an emerging alternative system becomes locked-in before it has fulfilled the envisioned goal. 6 though earlier mission-oriented programmes incorporated ais principles such as transforum in the netherlands (fischer et al., 2012) , and the our land and water science challenge in new zealand ( innovation and system change. we argue that studying ais as a 'mission-oriented agricultural innovation system' (mais) would enable a better understanding of forces, interactions, catalysts, barriers in transformative food systems change, as so far studies on ais have rarely engaged with issues such as directionality, power, and the diversity of food systems futures (hall and dijkman, 2019; pigford et al., 2018; mier et al., 2018) . the purpose of this perspective is to get a debate and reflection started on this topic among agricultural systems and food systems scholars. to enact research on mais, there are different sorts of questions to be asked, in terms of what, why, who, where and how, on which we will elaborate in section 2 (see also table 1 ). as mazzucato (2016) argues, missions are about setting concrete directions and comprise a strategic portfolio of innovation projects (technological, social, institutional innovation), i.e. what meynard et al. (2017) have called 'coupled innovations'. the scope of these portfolios may vary and be driven by different expectations, narratives, assumptions, policy rationales and theories of change thornton et al., 2017) ). they may be science and discovery based and technology-driven or may be driven by visions for broad systemic change (polt, 2019; rathenau institute, 2020) . 7 to achieve food systems transformation, missions may include concepts such as agroecology, circular or regenerative agriculture, digital transformation of agriculture, bioeconomy, or protein transitions (gosnell et al., 2019; mockshell and kamanda, 2018; shepherd et al., 2018; tziva et al., 2020; van zanten et al., 2019; wezel et al., 2009) , or a combination of concepts (kristensen et al., 2016; laibach et al., 2019; mockshell and kamanda, 2018) . different existing and emerging agriculture and food technologies may support the former approaches (herrero et al., 2020) . these rather broad conceptual missions for food systems transformation may be underpinned by different types of 'sub-missions'. for example the broad mission of 'circular agriculture' may be achieved by submissions emphasizing either a place-based local model with closing cycles on farms, high in-field diversity and short supply chains, or a regional model in which different sorts of monoculture agriculture are connected within wider rural-urban interactions and material and energy flows (garrett et al., 2020; van zanten et al., 2019) . hence, these sub-missions represent diversity in possible future food systems transition pathways and supporting technologies (gaitán-cremaschi et al., 2020) . hence, it important to characterize what sort of mission a mais represents, how broad or narrow it is, how explicit it is made, and possibly what sub-missions may exist. finally, just as mais may have a layering in its mission, there may also be several competing, co-existing or converging mais (e.g. agroecology and digital transformation), in view of the diversity argument and portfolio approach (mazzucato, 2016) . 8 a variety of competing or co-existing sub-missions has already been observed for concepts such as agroecology and agriculture 4.0 (plumecocq et al., 2018; klerkx and rose, 2020; bellwood-howard and ripoll, 2020) . studying this diversity of mais can illuminate aligning or competing interests and infrastructures that have the potential to facilitate or constrain food systems change. as already alluded to in the previous point, it is important to determine where missions come from, as mission-oriented policy is a product of the system which it is supposed to change. sometimes, a mission may involve a truly novel focus, and sometimes it is a 'reframing' or 'rebranding' of ongoing efforts . missions may arise from a supply-push (e.g. by policy, science, or business), or rather from a demand-pull (raised by social movements and worried citizens and consumers) or a combination of these forces. they may arise 'pro-actively' in view of horizon scanning and scenario building exercises of future food systems (csirofutures, 2017; de wilde, 2016; hebinck et al., 2018; manners et al., 2020; rutter, 2012; van der weele et al., 2019; worldbank, 2019) , or rather 'reactively' when sectors or countries are faced with intractable problems such as intensifying droughts. in terms of the mission set up, it is important to consider if the missions provide strong steering and close down innovation options or consider a wide range of solutions that allow to be tinkered with, as this will result in different operationalization strategies of the missions . it is also important to contemplate whether mais can be considered as an 'alternative' ais or emerge from within the dominant ais of a country or sector. it has for example been argued that an ais in a country may on the one hand still be focused on improving 'business-asusual', while on the other hand may have the characteristics of a mais with highly transformative ambitions (pigford et al., 2018) . this would require what has been referred to as 'ambidexterity' (turner et al., 2017; rathenau institute, 2020) , i.e. the capacity of an ais to simultaneously manage existing networks and policies to support (incremental) innovation in current food systems, and create new networks and policies to overcome lock-in and support radical innovation and food systems transformation. perhaps paradoxically, food systems transformation thus also implies deconstruction of existing ais systems or 'exnovation', by for example phasing out research investments in a non-sustainable technology or practice (david and gross, 2019; kivimaa and kern, 2016; krüger and pellicer-sifres, 2020) . beyond questioning environmental sustainability of production systems and supply chain set-up, exnovation may also include questioning prevalent economic paradigms (e.g. neoliberal capitalism, economic growth) (feola, 2020; ghisellini et al., 2016; mier et al., 2018; giuliani, 2018) . this requires scrutinizing prevalent policy frames, goals, and policy instruments (candel and biesbroek, 2016; galli et al., 2020; janssen et al., 2020) . understanding the dynamics of mais emergence and its set up may as such shed light on particular policy framings, directionalities, and prevailing lock-in scenario's that influence the innovation and exnovation capacity of mais. 9 7 polt defines 4 types of missions: science missions (e.g. us cancer moonshotfundamental/basic research with high uncertainty), technological missions (e.g. concorde, apollo mission -specific goal with a strong focus on technological/ scientific solutions), transformative missions (e.g. german energiewendeaiming at systemic change), umbrella missions (e.g. german high tech strategy -comprehensive long-term policy frame). as an elaboration on this, wittmann et al. (2020) distinguish accelerator and transformer missions, the former more science or technology based, and the latter more broadly based on societal changes and economic and system infrastructure change. 8 for example, the policy agenda from the dutch ministry of agriculture emphasizes circular agriculture (ministry of agriculture, 2019), which according to ploegmakers et al. (2020) can already be divided in four narratives (footnote continued) (which may possibly underpin different sub-missions). the knowledge and innovation agenda of the dutch topsector approach for agri-food harbours six missions (https://kia-landbouwwatervoedsel.nl/). 9 innovation capacity is built through the practices, routines or processes used to mobilise, create and reconfigure arrangements of resources and capabilities in order to innovate (turner et al., 2017) . exnovation as a concept has had limited application in agriculture and food systems, but there is ongoing work, see e.g. the project of laura van oers (https://www.uu.nl/staff/lmvanoers/ research). to drive missions, mazzucato (2016) emphasizes direction setting by policymakers and highlights the role of the public sector. borrás and edler (2020) have refined these ideas and show that states can combine roles as observer, warner, mitigator, opportunist, facilitator, lead-user, enabler of societal engagement, gatekeeper, promoter, moderator, initiator, guarantor and watchdog. as we argued in the previous sections and following , missions may also arise from and be shaped by different actors beyond the state. while it can already be challenging to get 'conventional' ais actors to co-innovate (meynard et al., 2017) , mais may include food economy actors previously not considered to a large degree in agricultural transition and ais studies (weber et al., 2020) , such as agtech and foodtech start-ups, think tanks, and social enterprises. given this diversity of actors and values, there are processes of negotiation and contestation in mais. the greater complexity or 'wickedness' of the grand challenge, the greater the negotiation and contestation generally is wanzenböck et al., 2019) . 10 therefore it is important who is included in a mais, and who is excluded (herrero et al., 2020; leach et al., 2020; pigford et al., 2018) (see 2.1). and also, whether those excluded in one mais may be served by another mais which enacts transformative missions in a different way, thus accommodating a diversity of farmers and food system actors in terms of for example farm size, farming style, value chain model (klerkx and rose, 2020; stringer et al., 2020) . furthermore, the question is not only 'who' drives mais, but also 'what'. it is increasingly argued that also nature itself and biological and ecological agents (e.g. plants, animals, ecosystems) need to be seen as drivers in innovation systems in view of ideas on ecological feedback loops and biomimicry and the 'more-than-human' debate in rural sociology, as well as technologies such as robotics, digital twins and artificial intelligence (andersen and wicken, 2020; darnhofer, 2020; klerkx et al., 2019; pigford et al., 2018; van der jagt et al., 2020) . many proposed food systems transformation concepts transcend national, sectoral and technological boundaries. they are developed in many countries simultaneously, and are connected to supra-national or even global transformative policy narratives, and flows of technologies and capital (wanzenböck and frenken, 2020) . also, the spaces where missions are defined (see footnote 2) are highly globalized since they consider actors from different countries, or are shaped in virtual spaces (e.g. social media). hence, a mais may have the traits of a global innovation system (binz and truffer, 2017) and at the same time may be connected to specific national or even sector contexts. hence, the geographical boundaries of a mais are fluid janssen et al., 2020; pigford et al., 2018) . missions may 'travel' geographically and a mais may have a different pace of development in different countries. this is due to differences in state governance, and between cultural and regulatory contexts (e.g. the common agricultural policy and novel food legislation of the european union) that for example may determine consumer attitude (bekker et al., 2017; gupta et al., 2013) . such travel may be through multinational companies who introduce products in different countries (e.g. plant based protein products), through large international innovation programs and efforts from countries to 'export' missions (minkman and van buuren, 2019) , or through social movements (e.g. agroecology) (mier et al., 2018; wezel et al., 2018) . to understand mais, it is this important to unravel global linkages in enacting missions. also, one can compare mais development between countries, in terms of what missions are espoused by a country, the stage of development of missions and whether technologies and practices have reached certain stages of readiness to go to scale (herrero et al., 2020; sartas et al., 2020) . it has been argued that transformative change in agriculture is generally a process in which innovative processes in several 'innovation niches' gradually change incumbent systems or 'regimes' (el bilali, 2019a; geels et al., 2016; geels and schot, 2007; leach et al., 2012; olsson et al., 2014; pigford et al., 2018) , though it is increasingly recognized it may also come from within incumbent agricultural systems (gaitán-cremaschi et al., 2019; runhaar et al., 2020) . transformation is then the outcome of many 'small wins' instead of a sudden radical change (termeer and dewulf, 2019; termeer and metze, 2019) . equally, a mais does not establish overnight, but the network of actors connected to the mais, their interactions and the funding and innovation policies that support the mais will emerge gradually (eastwood et al., 2017) . though the mission-oriented innovation approach presupposes a degree of steering, some parts of a mais (being a complex adaptive system) will be self-organized without the support of central steering mechanisms. given the geographical fluidity of a mais, system complexity and degree of self-organization, the analytical boundaries of a mais not easy to establish. this affects mais characterization, as well as the accomplishment of a mission by a mais (or for that matter-several co-existing or competing mais). janssen et al. (2020) mais analysts, drawing on generic innovation system theories, can utilize some of the existing innovation system analysis frameworks such as functional-structural analysis, innovation policy mix analysis, or social network analysis to analyse mais (hermans et al., 2019; lamprinopoulou et al., 2014; spielman et al., 2011; wieczorek and hekkert, 2012; schut et al., 2015) . this can be combined with agricultural and food systems assessment and modelling approaches focusing for example on resilience (meuwissen et al., 2019; tittonell, 2020) , and drawing on recent frameworks to analyse different aspects of food systems transformation (gaitán-cremaschi et al., 2019; hebinck et al., 2018; kanter et al., 2015; termeer et al., 2018; zurek et al., 2018) . such analysis may make use of data science or citizen science driven approaches bringing together large datasets of biophysical and socioeconomic data to track mission evolution (beza et al., 2017; fielke et al., 2020; guo et al., 2020; klerkx et al., 2019; sauermann et al., 2020) . since mais are often global with national or regional 'branches', 10 a relevant consideration is where the boundaries of mais lie: from a food systems perspective, consumers are for example highly important in view of food preference and dietary change as well as food choice environments (retail and domestic environments) and food waste prevention and recycling willett et al., 2019) . new agtech and foodtech inspired food systems (e.g. urban agriculture, vertical agriculture) also bring new players into ais (pigford et al., 2018) . also, food systems may have a 'blue' component, based on food captured or cultivated in water bodies tezzo et al., 2020) so can aquaculture also be considered a form of agriculture and are 'aquaculture innovation systems' (joffre et al., 2018) also part of mais? one can even question whether mais is the correct term, if the mission is food systems transformation. for example, the concept of a 'food innovation system' (leach et al., 2020) may be then perhaps more appropriate. initiatives like the food systems dashboard (fanzo et al., 2020) , the cgiar platform for big data in agriculture, the agricultural science and technology indicators network (asti), and capacity development for agricultural innovation systems project (cdais) could play roles in enabling or hosting 'mais mapping'. 11 in this perspective paper, we have elaborated on the emerging concept of mais. we think its relevance lies in better understanding what drives transformative change, and create insights for policy makers and other ais actors on innovation system directionality and innovation policy mixes for enhancing food systems transformation. this may spur reflection on subnational, national and supranational innovation activities and the adequacy of mais for supporting transformative change (wanzenböck and frenken, 2020) . table 1 summarizes some of the possible actions that may be undertaken to investigate mais and do mais mapping. besides introducing a mais perspective to understand food systems transformation, this paper aims to stimulate agricultural systems scholars and field level practitioners to critically reflect upon their own positionality within a mais. 12 this includes asking questions on what mission or sub-missions they contribute to and what type of contribution they make, e.g. systems (re)-design, modelling and scenario building to support mission formulation, or monitoring and analysing effects of technologies and interventions in missions. 13 furthermore, in view of the need to create coupled innovations (meynard et al., 2017) as well as exnovation, it requires scrutiny of the actor networks and institutional structures they are part of and whether and how these align with a mais. this also includes asking questions with whom they need to engage to support the formulation and development of missions and what kind of incentive systems are needed to support these interactions. we conclude this paper by inviting the agricultural systems community as well as other natural and social scientists to progress the analysis of mais and develop it further as a concept and approach to understand and support agricultural innovation in relation to food systems transformation. the authors declare that there are no conflicts of interest related to this paper. questions and possible actions to further conceptual development and analysis of mais. map the diversity of food systems transformation missions, and the sub-missions that underpin them, including the contribution of different sorts of science (e.g., biophysical science, engineering, social and policy science) and the role of trans-disciplinarity why do mais emerge or are set-up? identify the (policy) drivers for missions and their emergence and evolution map the networks of actors driving missions and constituting mais, and the governance and power dynamics within mais where do mais play out? map mais at a (sub) country level, how they are linked supranationally, and what are the connections with other sectors how do mais evolve and how do we study this process? further develop methodologies to capture mais and connect them to ongoing agricultural innovation and food systems analysis 11 see https://cdais.net/home/, https://www.asti.cgiar.org/, https://bigdata. cgiar.org/. furthermore, generic observatories and programs on transformative innovation policy could be connected to, such as the mission-oriented innovation policy observatory https://www.uu.nl/en/research/copernicusinstitute-of-sustainable-development/mission-oriented-innovation-policyobservatory and the transformative innovation policy consortium http://www.tipconsortium.net/ (schot and steinmueller, 2018a) . 12 it has been argued that food systems transformation also has implications for advisory systems (klerkx, 2020) . but many scholars by being engaged in trans-disciplinary research and co-innovation are also field-level practitioners. 13 this also requires scrutiny of agricultural systems scholar on how instrumental or non-instrumental their knowledge is in view of missions and what perspective they take, e.g. knowledge as a private good or common good (basu et al., 2017a (basu et al., , 2017b , and in what way they engage with transition processes (schut et al., 2014; wittmayer and schäpke, 2014) . transition policy and innovation policy: friends or foes? the present and future sustainability of clothing and textiles in the united kingdom making sense of how the natural environment shapes innovation, industry dynamics, and sustainability challenges. innovation and development the role of inter-sectoral dynamics in sustainability transitions: a comment on the transitions research agenda beyond the dichotomy of instrumentality and non-instrumentality of knowledge production: the case of generation challenge programme the emergence of a hybrid mode of knowledge production in the generation challenge programme rice research network (gcp-rrn) in india: exploring the concept of commons-based peer production (cbpp) fixing our global agricultural system to prevent the next covid-19 meet meat: an explorative study on meat and cultured meat as seen by chinese, ethiopians and dutch divergent understandings of agroecology in the era of the african green revolution sustainable commoditization of seafood what are the prospects for citizen science in agriculture? evidence from three continents on motivation and mobile telephone use of resource-poor farmers global innovation systems-a conceptual framework for innovation dynamics in transnational contexts post covid 19 and food pathways to sustainable transformation making sense of new trends in innovation policy toward a processual understanding of policy integration waste couture: environmental impact of the clothing industry food and agribusiness a roadmap for unlocking value-adding growth opportunities for australia farming from a process-relational perspective: making openings for change visible futurizing politics and the sustainability of real-world experiments: what role for innovation and exnovation in the german energy transition? the future of technology in agriculture. stt netherlands study centre for technology trends pathways of transformation in global food and agricultural systems: implications from a large systems change theory perspective transformation is 'experienced, not delivered': insights from grounding the discourse in practice to inform policy and theory dynamics and distribution of public and private research and extension roles for technological innovation and diffusion: case studies of the implementation and adaptation of precision farming technologies the multi-level perspective in research on sustainability transitions in agriculture and food systems: a systematic review research on agro-food sustainability transitions: a systematic review of research themes and an analysis of research gaps farming systems research into the 21st century: the new dynamic farm to fork strategy. in: for a fair, healthy and environmentally-friendly food system the food systems dashboard is a new tool to inform better food policy capitalism in sustainability transitions research: time for a critical turn? digitalisation of agricultural knowledge and advice networks: a state-of-the-art review transforum system innovation towards sustainable food. a review. agronomy for sustainable development seven lessons for planning nature-based solutions in cities agriculture 5.0: reconciling production with planetary health characterizing diversity of food systems in view of sustainability transitions. a review sustainability transition pathways through ecological intensification: an assessment of vegetable food systems in chile drivers of decoupling and recoupling of crop and livestock systems at farm and territorial scales typology of sociotechnical transition pathways the enactment of socio-technical transition pathways: a reformulated typology and a comparative multi-level analysis of the german and uk low-carbon electricity transitions a review on circular economy: the expected transition to a balanced interplay of environmental and economic systems regulating global capitalism amid rampant corporate wrongdoing-reply to "three frames for innovation policy transformational adaptation on the farm: processes of change and persistence in transitions to 'climate-smart' regenerative agriculture big earth data science: an information framework for a sustainable planet expert views on societal responses to different applications of nanotechnology: a comparative analysis of experts in countries with different economic and regulatory environments public agricultural research and development in an era of transformation independent science council of the cgiar and commonwealth scientific and industrial research organisation enhancing agricultural innovation: how to go beyond the strenghthening of research systems imagining transformative futures: participatory foresight for food systems change public-private partnerships as systemic agricultural innovation policy instruments -assessing their contribution to innovation system function dynamics innovation can accelerate the transition towards a sustainable food system position paper 'mission-oriented innovation policy observatory aquaculture innovation system analysis of transition to sustainable intensification in shrimp farming giving science innovation systems a 'nudge strengthening local food systems in times of concomitant global crises: reflections from chile a conceptual framework for understanding the impacts of agriculture and food system policies on nutrition and health understanding socio-economic and policy constraints to dairy development in ethiopia: a coupled functional-structural innovation systems analysis research for agri committee -megatrends in the agri-food sector: global overview and possible policy response from an eu perspective creative destruction or mere niche support? innovation policy mixes for sustainability transitions advisory services and transformation, plurality and disruption of agriculture and food systems: towards a new research agenda for agricultural education and extension studies dealing with the game-changing technologies of agriculture 4.0: how do we manage diversity and responsibility in food system transition pathways? global adaptive management in agricultural innovation systems: the interactions between innovation networks and their environment evolution of systems approaches to agricultural innovation: concepts, analysis and interventions a review of social science on digital agriculture, smart farming and agriculture 4.0: new contributions and a future research agenda enabling sustainable agro-food futures: exploring fault lines and synergies between the integrated territorial paradigm, rural eco-economy and circular economy creating an enabling environment for industry-driven pest suppression: the case of suppressing queensland fruit fly through area-wide management from innovations to exnovations. conflicts, (de-) politicization processes, and power relations are key in analysing the ecological crisis exploring the future of the bioeconomy: an expert-based scoping study examining key enabling technology fields with potential to foster the transition toward a bio-based economy transition pathways towards a robust ecologization of agriculture and the need for system redesign. cases from organic farming and ipm application of an integrated systemic framework for analysing agricultural innovation systems and informing innovation policies: comparing the dutch and scottish agrifood sectors transforming innovation for sustainability food politics and development transitioning european protein-rich food consumption and production towards more sustainable patterns-strategies and policy suggestions from market fixing to market-creating: a new framework for innovation policy challenge-driven innovation policy: towards a new policy toolkit a framework to assess the resilience of farming systems designing coupled innovations for the sustainability transition of agrifood systems bringing agroecology to scale: key drivers and emblematic cases unpacking the systemic problems and blocking mechanisms of a regional agricultural innovation system: an integrated regional-functional-structural analysis branding in policy translation: how the dutch delta approach became an international brand beyond the agroecological and sustainable agricultural intensification debate: is blended sustainability the way forward? overcoming undesirable resilience in the global food system sustainability transformations: a resilience perspective beyond agricultural innovation systems? exploring an agricultural innovation ecosystems approach for niche design and development in sustainability transitions analyse debat kringlooplandbouw the plurality of values in sustainable agriculture models: diverse lock-in and coevolution patterns re-orienting sti policies towards mission-oriented innovation policies -where do we stand? where are we heading? joint cstp/ ciie workshop session 2: reorienting sti policies and funding to tackle societal challenges editorial overview: towards an inclusive food systems approach for sustainable food and nutrition security mission-driven innovation policy: what, how, why? rathenau institute the evolution of mission-oriented policies: exploring changing market creating policies in the us and european space sector planet-proofing the global food system endogenous regime change: lessons from transition pathways in dutch dairy farming a 'smart' future for ruminant livestock production? scaling readiness: science and practice of an approach to enhance impact of research for development citizen science and sustainability transitions innovation systems for transformations towards sustainability? taking the normative dimension seriously new directions for innovation studies: missions and transformations three frames for innovation policy: r&d, systems of innovation and transformative change raais: rapid appraisal of agricultural innovation systems (part i). a diagnostic tool for integrated analysis of complex problems and innovation capacity towards dynamic research configurations: a framework for reflection on the contribution of research to policy and innovation processes priorities for science to overcome hurdles thwarting the full promise of the 'digital agriculture' revolution transitions in water harvesting practices in jordan's rainfed agricultural systems: systemic problems and blocking mechanisms in an emerging technological innovation system rural innovation systems and networks: findings from a study of ethiopian smallholders adaptation of knowledge systems to changes in agriculture and society: the case of the netherlands editorial: impacts of covid-19 on agricultural and food systems worldwide and on progress to the sustainable development goals adaptation and development pathways for different types of farmers a small wins framework to overcome the evaluation paradox of governing wicked problems a diagnostic framework for food system governance arrangements: the case of south africa more than peanuts: transformation towards a circular economy through a small-wins governance framework food system perspective on fisheries and aquaculture development in asia responding to global change: a theory of change approach to making agricultural research for development outcome-based social justice and agricultural innovation ecological intensification of agriculture-sustainable by nature assessing resilience and adaptability in agroecological transitions systemic problems affecting co-innovation in the new zealand agricultural innovation system: identification of blocking mechanisms and underlying institutional logics unpacking systemic innovation capacity as strategic ambidexterity: how projects dynamically configure capabilities for agricultural innovation understanding the protein transition: the rise of plant-based meat substitutes food security, safety, and sustainability-getting the trade-offs right the role of farm animals in a circular food system changing diets and transforming food systems the subsidiarity principle in innovation policy for societal challenges a framework for mission-oriented innovation policy: alternative pathways through the problemsolution space what are the ingredients for food systems change towards sustainability? -insights from the literature agroecology as a science, a movement and a practice. a review agroecology in europe: research, education, collective action networks, and alternative food systems systemic instruments for systemic innovation problems: a framework for policy makers and innovation scholars developing a typology for mission-oriented innovation policies action, research and participation: roles of researchers in sustainability transitions future of food harnessing digital technologies to improve food system outcomes grand missions of agricultural innovation assessing sustainable food and nutrition security of the eu food system-an integrated approach the authors wish to thank the postdoc programme of the department of social sciences at wageningen university, which enabled the contribution of stephanie begemann to this paper. interactions with xianping jia (research institute of agricultural economics at northwest a & f university, china), jos van den broek, isabelle van elzakker and laurens hessels (rathenau institute, the netherlands), marko hekkert, wouter boon, matthijs janssen, simona negro, joeri wesseling, and giuseppe feola (innovation studies group, utrecht university, the netherlands), and barbara van mierlo (knowledge, technology and innovation group, wageningen university, the netherlands) were helpful in shaping the thinking which led to this paper. constructive comments by the reviewers and editor guillaume martin were very useful in finetuning the paper. key: cord-264974-hspek930 authors: timmis, kenneth; brüssow, harald title: the covid‐19 pandemic: some lessons learned about crisis preparedness and management, and the need for international benchmarking to reduce deficits date: 2020-05-03 journal: environ microbiol doi: 10.1111/1462-2920.15029 sha: doc_id: 264974 cord_uid: hspek930 nan if, despite the explicit warning of the world health organization in 2011 that 'the world is ill-prepared to respond to a severe influenza pandemic or to any similarly global, sustained and threatening public-health emergency' (https://apps.who.int/gb/ebwha/pdf_files/wha64/a64_10en.pdf), it was not apparent to those in charge, and to the general public-i.e., those suffering from covid-19 infections and the funders of health services (tax/insurance payers)-that existing health systems had inherent vulnerabilities which could prove to be devastating when seriously stressed, the sars-cov-2 pandemic (e.g., see brüssow, 2020 ) has brutally exposed it now. in some countries, preparedness, despite being officially considered to be of strong operational readiness against health emergencies (kandel et al., 2020) , was inadequate at multiple levels (e.g., horton, 2020) . similarly, a fundamental lack of preparedness is the case for a number of impending non-health crises (e.g., global warming, poverty, the soil crisis, etc.). once we are over the covid-19 pandemic, important questions will be: what have we learned/can we learn and how can we improve our systems? below, we argue for the necessity for major realignment of crisis responsiveness, and indeed of health system operationality, based on international benchmarking and adequately funded preparedness. international benchmarking is mandatory, because it has become clear that there is a wide range of effectiveness in the ability of different countries with developed economies to respond to this crisis (and probably others), and the tax-paying public has no compelling reason to tolerate perpetuation of factors underlying poor responses to crises. disparity in country/regional responses to sars-cov-2 leaving aside for the moment decisions about whether to robustly contain the outbreak-to kill it by throttling-the classical strategy of infection control, but which leaves most of the population susceptible to a new outbreak, or attempt to manage an outbreak to achieve herd immunity and a population unsusceptible to a repeat outbreak, it is obviously imperative to know how the outbreak is spreading and how effective are any containment measures that are instituted, so that a change in course of action remains an option. widespread testing for the viral pathogen, with correct sampling and analysis procedures, is thus essential. this enables, inter alia, calculation of reliable mortality (case fatality) rates, 1 which ordinarily play a key role in determining crisis response policy, calculation of the basic reproduction number, r 0 , and reliable modelling of transmission and mortality trends. there have been enormous differences in testing coverages among countries. early predictions of transmission and mortality trends are only as good as the adequacy of the information fed into models so, with inadequate testing, prediction of trends are unreliable. mortality-based policy formulation for sars-cov-2 responses in some countries was based on incomplete information. • diagnostics and widespread testing are the basis of informed policy development for crisis management of infectious disease epi/pandemics and must become its centrepiece the disparity of sars-cov-2 testing coverage in different countries is much discussed but the reasons are sometimes rather opaque. there are many tests available (e.g., see https://www.finddx.org/covid-19/pipeline/), so one bottleneck would appear to be a limitation in authorized testing facilities. scaling up testing in existing official centres requires not only acquisition of the appropriate instrumentation and reagents, but also of competent personnel disposing of the necessary expertise, which may constitute a bottleneck. however, it is abundantly clear that the instrumentation and expertise needed to carry out sars-cov-2 testing are widespread in academic research laboratories, as is the eagerness of many research groups to contribute to efforts to combat the pandemic. while issues of safety, quality control, logistics, data reporting and security and so on, need to be addressed, a failure in some countries to harness early in the crisis the expertise and enthusiasm of young researchers to fulfil a key need and, with it, the opportunity to acquire data that could have resulted in responses that saved lives, is regrettable. paradoxically, while there have been frantic efforts to open new hospital facilities to accommodate covid-19 patients, and to recruit the health professionals needed to operate them, there has been an indiscriminate closing of research institutes capable of carrying out diagnostic work, and thus of identifying infected and, importantly for frontline health professionals, non-infected individuals. • rapidly developing pandemics necessitate rapid responses. getting diagnostics and testing facilities up and running that are able to handle large numbers of samples are key to efforts to manage the disease. crisis preparedness demands not only formulation of strategies to enable rapid scale up official facilities, but also advance identification of relevant available resources outside of the health system, and strategies of how to promptly and effectively harness them. widespread testing for viral rna must be complemented by widespread testing, or at least the testing of sentinels of the population, for anti-viral antibodies. this is essential for relating the dynamics of infection to virus shedding (being infectious = infective for others) and to symptom development. and if (and this is a big if) the presence of antiviral antibodies reflects protective immunity, antibody testing is essential for herd immunity policy scenarios, to provide the data needed for monitoring and modelling immune population densities/granularity, and also for identification of those in the population who are in principle protected and hence able to return to normality and spearhead safe exit strategies from lockdown measures. • contingency planning requires identification of facilities or alliances able to promptly develop, produce in quantity and distribute easy-to-use antiviral antibody tests. patients having contracted covid-19 are grouped into three categories: in the china outbreak, 81% experienced mild, 14% severe and 5% critical infections (wu and mcgoogan, 2020) . for those requiring hospitalization, and those with the most acute symptoms requiring intensive care, two key variables in treatment capacity seem to be bed availability generally, and in intensive care units particularly (images of patients lying on the floors of some hospitals made this abundantly clear to the entire world), on one hand, and ventilator/intubation tube availability for patients needing intubation, on the other. there are enormous differences between countries in terms the availability of hospital beds/1000 population (e.g., japan 13.1, germany 8.0, france 6.0, switzerland 4.5, italy 3.2, uk/canada/denmark 2.5, india 0.5: https:// data.oecd.org/healtheqt/hospital-beds.htm), icu beds/1000 population, and numbers of available ventilators/intubation tubes (here, ventilators can be considered a proxy for any other clinical device that may be needed in a health crisis). an insufficiency of beds (and often health professionals) also resulted in 'non-critical' interventions 2 being postponed in some countries, and who, among critically ill patients suffering from different ailments, should be given an icu bed. there are different reasons for insufficient beds and clinical devices. but the fact remains: some countries manage better than others; some countries do not have enough beds for even small increases representing normal fluctuations in patient needs and anticipated seasonal variations, let alone exceptional demands made by epidemics. • it is essential to increase bed, especially icu bed, capacities in many countries, in order to reduce stress situations where patients cannot receive required treatment, and to prevent hospitals from being overwhelmed in times of crisis, when there is a spike in patients requiring hospitalization. one new development triggered by the covid-19 pandemic was the creation of so-called 'fangcang shelter hospitals'-rapidly constructed, large scale, low cost, temporary hospitals, by converting existing public venues into healthcare facilities in wuhan to manage the rapidly increasing covid-19 patient numbers (chen et al., 2020) . instead of being delivered to regular hospitals, covid-19 patients were sent to shelter hospitals, where they were isolated and received basic medical care and frequent monitoring. patients whose mild disease state transitioned to severe were then transferred to regular hospitals where they received intensive care. although new emergency hospitals have been created in other countries, the purpose of these is generally to receive spillover from traditional hospitals, when these become overwhelmed by patient numbers. the purpose of fangcang shelter hospitals is, in contrast, to centralize clinical management of the epi/pandemic outside of the traditional hospital system. covid-19 patients are channelled to shelter hospitals, thereby minimizing viral ingress into hospitals and infection of health professionals, and helping to maintain staffing levels and normal functioning of outpatient and inpatient facilities. it is important to note that shelter hospitals constitute low-cost clinical settings-they require fewer health professionals and diagnostic-treatment infrastructure than normal hospitals, because all patients have the same clinical issue and most have only mild-to-moderate disease-and thereby relieve pressure on traditional hospitals with sophisticated infrastructure in limited capacity that constitute high-cost clinical settings (the creation of parallel low-cost clinical settings to relieve pressure on limited capacity high cost clinical settings is more broadly applicable over and above epi/pandemic response situations: see timmis and timmis, 2017, for an example in primary healthcare). • shelter hospitals should be incorporated into pandemic planning as the primary destination for pandemic patients, to allow traditional hospitals to continue functioning as normal as possible (or normally as long as possible). it may seem so trivially obvious to say, but obviously needs saying because it was not apparent from the health system responses of a number of countries: those most at risk of infection are those in contact with the infected, i.e., front-line doctors and nurses. and as they become infected, the numbers of available health professionals left to treat patients goes down as patient numbers go up. and, of course, infected health professionals become transmitters of infection among one another, and to uninfected patients, since in the hectic reality of emergencies, they may not always be able to practice adequate physical distancing. this obviously means that the greatest protection from infection must be accorded the front-line professionals. however, there were substantive differences between countries in terms of the availability and use of best practice protective clothing (personal protective equipment, ppe) in the early days of the covid-19 crisis; these differences were mainly in different degrees of deficiencies. the incomplete protection of front-line health professionals that occurred in a number of countries in the early days of the crisis, and that resulted in many infections and some deaths, is an unacceptable deficit in their health systems, particularly since the covid-19 outbreak was, from end of january 2020, a predictable disaster of international magnitude. then there are those one might designate accessory front-line professionals: those who transport infected individuals, like ambulance drivers, non-medical workers in hospitals, and so forth, carers ministering to people in care homes or in private homes, and others like some pharmacy and supermarket staff who, because of the nature of their work, come into physical contact with many people and cannot always achieve prescribed physical distancing. these are also particularly vulnerable to infection and to becoming infection transmitters. since the people they care for are, because of their ages and underlying morbidities, often themselves particularly vulnerable to severe outcomes, infected carers may, unknowingly and unwillingly, become 'angels of death'. accessory front-line professionals thus also require best ppe. there are wide regional and occupational differences in the availability and use of such clothing by these professionals. in addition to the issue of ppe, there is the issue of hygiene in the workplace-the surfaces that become contaminated and sources of infection. while traditionally these have been cleaned by auxiliary staff, such people are themselves at considerable risk of being infected in such environments and, as a result, there may be an insufficient number to continue carrying out this task, thereby raising infection risk. robots are in principle able to carry out various mechanical operations, so might take an increasing share in disinfection of high-risk, high touch areas (e.g., robot-controlled noncontact ultraviolet surface disinfection), and indeed other hospital tasks, such as delivering medications and food, diagnostic sample collection and transport, and so forth, (yang et al., 2020) , that may reduce both the work burden of overstretched staff and their infection risk. • the incorporation of robots into appropriate hospital operations should be energetically explored leadership in times of crisis is crucial to ongoing damage limitation and outcome severity, quite apart from its importance in planning crisis preparedness. although we need to look back when all this is over, and take stock of what went right, what was wrong, and what went wrong (i.e., to perform a gap analysis), at this point it seems that most countries were on their own, acting largely independently of others during the sars-cov-2 outbreak, at least in the early days. however, a pandemic is by definition an international crisis, requiring an international response (national-self-interest-policies may even be counterproductive in times of pandemics). extensive and effective cooperation, coordination and sharing of resources were not evident (e.g., see herszenhorn and wheaton, 2020) . leadership quality and effectiveness varies significantly among countries and among relevant international agencies. where leadership is suboptimal, dissemination of misinformation flourishes, and people are subjected to unnecessary levels of uncertainty and associated stress. • effective and decisive, biomedical science-guided, national and international leadership and coordination is absolutely crucial in pandemics, to prevent-hindermanage-minimize damage, acquire-integrate-learn from collective experience, make recommendations for crisis management, publish best practice procedures and standards. there is significant room for improvement. it is well known that experts have been warning of impending deadly epi/pandemics, including coronavirus outbreaks, for a long time (e.g., turinici and danchin, 2007; ge et al., 2013; menachery et al., 2015; https:// www.ted.com/talks/bill_gates_the_next_outbreak_we_re_ not_ready?language=en; editorial (2016) predicting pandemics, lancet doi: https://doi.org/10.1016/s0140-6736 (16)32578-8; https://apps.who.int/gpmb/assets/annual_ report/gpmb_annualreport_2019.pdf; https://www. weforum.org/agenda/2020/03/a-visual-history-ofpandemics/). now while the nature, evolution, timing and source of novel emerging infectious agents is uncertain, pandemics are always counteracted by the same timehonoured strategy: interruption of infection chains and anticipation of a surge in need for treatment of acute disease (here, we are nearly in the same situation as in the world confronted by spanish flu in 1918). we, therefore, only need one epidemic preparedness. despite this, the sars-cov-2 outbreak has clearly exposed how unprepared we were. there are multiple reasons for this, including. contingency planning is long term, lacks immediacy and 'wow factor' and so may not always enjoy high political priority, and thus is often neglected a primordial responsibility of government is to protect its citizens. this includes effective contingency planning for pandemics. however, due to the global nature of pandemics, coordination with neighbours (and factoring in potential flashpoints located more distantly, such as refugee camps in greece and elsewhere, which could become, if not cared for, sources of a second wave of infection when the first is over), and intergovernmental cooperation is essential. adequate contingency planning for deadly and devastating infectious disease outbreaks is not an optional policy, and the public have the right to insist on it, even if it becomes politically or economically expedient to neglect. for the public-the key stakeholders in this-transparency is essential and it must have access to information on the current state of preparedness, and future plans of government, and those of different political parties during election campaigns. trusted biomedical science organizations must support the public in this by providing expert scrutiny and assessment. governments must become accountable for the efforts they make to protect us. • governments must engage the public in issues of crisis preparedness and publish their contingency plans for scrutiny. • scientific organizations should have press/web groups that become trusted sources for evidencebased information for the public. catastrophe prediction/management expertise is not always at the heart of government, and thus able to inform and influence policy governments establish the presence of experts in key posts for topics they consider to be vital for informed policy and legislative activities. such experts exert an influence in policy development by providing input that is upto-the-minute in a changing world. while some governments contain epidemic/catastrophe experts, others do not. without such expertise, responses to catastrophes will generally be slow, ad hoc and inadequate, as has been the case in some countries in responding to the sars-cov-2 outbreak. for governments to fulfil their responsibilities to protect their citizens, it is essential that they have expert-informed contingency planning. learned societies and academies also have a major responsibility to seek to inform and influence government. the royal society, uk, and the american society for microbiology exemplify strategic influencing of national and international policy; other learned societies could be more proactive. • expert scientists must be embedded in the heart of government to enable development of evidencebased informed policies contingency planning involves inter alia the acquisition and maintenance of resources, such as beds, icu capacity, stocks of ventilators, protective clothing, and so forth, in the case of pandemics (e.g., kain and fowler, 2019) , that are by definition surplus to day-to-day requirements, and that will only be used if and when the catastrophe occurs. it also includes the development of generic platforms for rapid responses; in the case of pandemics, the development and testing of diagnostics, vaccine candidates, and effective treatments (see also below). this entails a significant recurring budgetary commitment. political and economic viewpoints that such costs are not cost-effective are fundamentally flawed because they generally only take into account the immediate cost elements, not the potential overall cost of the crisis and all its knock-on effects. these are being brutally revealed by the unfolding sars-cov-2 outbreak which, at this still early stage, is involving governmental support of national economies amounting collectively to trillions of dollars. and this is only the tip of the economic iceberg. bankruptcies, loss of employment, recession, loss of tax revenues, large scale deterioration of existing medical conditions in populations, potentially wide-scale deterioration of mental health, and so forth, and the economic costs of these, also need to be taken into account when reflecting on the cost of the contingency planning insurance policy. as an illustration of knock-on effects, global economic estimates of the benefits of vaccination have also shown that they extend well beyond those estimated from prevention of the specific disease in vaccinated individuals (bloom, 2015) . it is also worth comparing crisis preparedness costs with military expenditures. the latter are indeed budgetary commitments for preparedness for another type of crisis, namely a military conflict (excepting countries that use their military for internal affairs). and, as is the case in epi/pandemic preparedness, a considerable fraction of military resources is dedicated to surveillance operations. while accepting that military expenditures are also justified in terms of deterrence of hostile actions, and a multitude of non-combat roles armed forces may undertake, it is not self-evident that future military conflicts may result in losses of life and economic damage as high as the current covid-19 pandemic. in any case, in terms of protecting citizens, it should be abundantly clear that effective contingency preparedness for pandemics, and other crises, 3 should be equated with military preparedness, and budgeted accordingly. • the principle of citizen protection demands that governments budget for adequate crisis preparedness in the same way that they budget for military preparedness. it is simply one of several essential 'insurance premiums' to which the state must commit. from earlier infectious disease outbreaks, we can assume that the most probable source of a new pandemic will be an animal virus, probably a coronavirus, whose natural host is a wild animal, possibly a bat (e.g., see brüssow, 2012) , that mutates and, as a result, becomes infectious for humans, or for an intermediate host, from which it subsequently jumps to humans. close contacts between humans and the animal host provide the opportunities for transmission. reducing such close contacts will reduce the probability of spillover and thus of an outbreak. close contacts between wild animals and humans occur in wet markets in asia, small-scale mixed farming activities with ducks and pigs, and so forth, or when humans encroach into wildlife habitats, e.g., through ecotourism, or destroy wildlife habitats for economic activities, forcing wildlife to enter human habitats (e.g., the destruction of rainforest for palm oil cultivation appears to have catalyzed a nipah virus outbreak; brüssow, 2012) . in any case, although pathogen:host interactions underlying disease are generally well studied, current knowledge about the ecology of infectious agentswhere pathogens are and what they are doing prior to infection of humans, especially those having alternative hosts, and how they are circulating and evolving new pathogenic and host-range potentialis inadequate. in order to transit from response mode to pro-active ecological measures to prevent outbreaks from occurring, there needs to be a major research effort to obtain a fundamental understanding of pathogen ecology (see e.g. timmis, 2001) . • greater efforts are needed to reduce human:wildlife contacts and habitat overlaps, in order to decrease the probability of viral pandemics • effective outbreak prevention measures require acquisition of fundamental knowledge about pathogen ecology contingency planning and the public memory. it is human nature that, once this crisis is over, people, except those who lost loved ones, employment, and so forth, will generally want to forget it as quickly as possible and get back to normal. the number of individuals who try to keep it in the forefront of memory, in order to institute new measures that adequately protect us from the next crisis, and there will undoubtedly be new crises (see above), will be few and far between. some, not all, leading politicians who now (often for the first time) insist that their responses are being guided by the best scientific evidence and advice, as though it were the most natural thing in the world, will quietly shed themselves of their scientific credentials and revert to business as usual, even when unpleasant issues like global warming, the antibiotic resistance crisis, our vulnerability to terrorist and cyber-attacks, 4 again come to the fore. in order that our collective memory retains the crucial need for crisis preparedness, it is essential that each year governments publish updated and independently audited contingency plans. literacy. and the public-the central stakeholders in, and funders of, government policy/actions-must be able to understand the issues and personally evaluate the sometimes vague policy statements they hear. to do this, society must become knowledgeable about/literate in such things. in the case of infectious disease crises, such as the one currently ravaging humanity, and the contingency plans necessary for these, literacy in relevant microbiology topics is, as we have previously argued, essential (timmis et al., 2019) . interrupting the transmission chain in a pandemic may require lock-down, which imposes major personal sacrifices on the public, including confinement: loss of freedom of movement/social activities/family visits; closure of workplaces/loss of employment and income, resulting in economic hardship/increases in debt; closure of schools/ places of worship/hospitality venues/fitness studios/clubs of all sorts; restrictions on shopping; and elevated stress/ worsening of psychiatric conditions. it is, therefore, crucial that such measures are accepted and supported by the public. for this, people must be engaged and presented with coherent lock-down plans that are convincingly justified, in order to solicit compliance, solidarity and sharing of responsibilities. federal structures, like those in the usa, germany and switzerland may lead to uncoordinated actions in different parts of the country that are unsettling and unconvincing, because the public perceives them as arbitrary. such countries require coherent national plans that are consistent for the entire country. of course, all people in lock-down want an exit as soon as possible, and it is essential for governments to develop and communicate as soon as possible their exit strategy, and the determining parameters and assumptions upon which it is based. interestingly, some members of the public favour staggered exit plans, which implies a willingness to accept an infection risk. it will, therefore, be important for the government to have a public discussion on different risk scenarios, to obtain, present and discuss human/economic cost:benefit estimates (e.g., human lives against cost in loss of income /economic prosperity underlying the lock-down versus herd immunity approach-how much unemployment averts how many deaths or years of productive life when considering the age structure of death). and this discussion needs to take place in the context of the probabilities of loss of life through other adverse causes, such as annual influenza epidemics. family-friend contacts with terminally ill patients. one of the most shocking aspects of the covid-19 pandemic is the daily reporting of relatives of terminally ill patients who are unable to be with their loved ones at the end, and to pay last respects before burial. while this may be understandable in the context of patient isolation, social distancing, and the unbelievable hectic in overwhelmed icus, serious effort should be made to find a solution, perhaps moving terminally ill patients to an environment that permits both end-of-life patient care and limited safe contact with loved ones. • governments must publish annual audited overviews of the national state of crisis preparedness, with critical analyses of its strengths and weaknesses and plans to address the weaknesses • governments and education ministries must raise public awareness of crisis potential and promote understanding of key elements of crisis management, inter alia through investing in school curricula changes and public information campaigns that increase literacy in topics such as microbiology and public health • governments should involve civil society in discussing restrictive measures because this increases compliance and the solidarity to shoulder the consequences. achievement of optimal preparedness for, and operational responses to, a pandemic demands two things: international benchmarking and transparency/accountability in health systems (and of those who regulate and finance them). this includes chains of command and shared administrative responsibilities, procurement services, reliance on external suppliers of essential materials, and so forth. the disparities in responses we have listed above, that demonstrate significant differences between countries in the ability to respond to pandemics, are not justifiable in terms of operational efficiency, protection of frontline professionals, clinical outcome, and so forth, and cannot be allowed to persist, to be manifested again in future crises. health systems worldwide largely operate within narrow national perspectives, with little interest in better systems elsewhere. we urgently need objective and transparent benchmarking, and automatic mandating of adoption of the best practices in the world, where feasible. transparency and convincing justification for failure to adopt the benchmark must become the norm. of course, different health systems operate in different frameworks-payers, insurance, authorization and recommendation agencies, and so forth-but the tail cannot be allowed to wag the dog. existing frameworks can no longer be accepted as default excuses not to improve. they must be adapted to allow adoption of the benchmarks, where possible, not the other way round. in the final analysis, there are only two elements relevant: the person in the icu, who pays tax/insurance, and hence for the health system, and the government, which is responsible for health system functioning/evolution and protection of its citizens. both of their goals are in principle aligned, so there should be no controversy: provision of the best achievable health system that is adequately prepared for catastrophic pandemics. • governments and health systems must subject national health systems, and national health system crisis preparedness, to international benchmark scrutiny, and transparently strive for attainment of best international standards. it is the responsibility of government to protect its citizens and the role of industry to innovate and create commercial products and services. these two goals are not always aligned for current clinical exigences. but to provide a vital health system, government and industry must align and form alliances that create synergies. there are, of course, many successful examples of such beneficial alliances. however, there is sometimes an unrealistic perception of the role of industry, particularly by some governments when confronted with a crisis for which they are not prepared, as articulated in the generic cry: why do not we have a vaccine for this, why do not we have a drug for that? for example, regulatory and payment hurdles incentivize industry to develop cancer drugs rather than antimicrobials, so it is irrational and unwarranted to complain about the poor state of pipelines for new antivirals in the time of covid-19, of antimicrobials in the time of the antimicrobial resistance crisis. if industry is to realign its research priorities towards current clinical priorities, it needs incentives to do so, e.g., through adequately funded creative governmentindustry-academia-clinical-regulatory strategic alliances. we have previously proposed a mechanism to create novel pipelines for accelerated discovery of new drugs and diagnostics (timmis et al., 2014 ; and, simultaneously, to promote long-term revival of struggling economies, interestingly in response to a financial crisis-that of 2008-which the sars-cov-2 pandemic will again unleash with considerable severity). this proposal calls for the use of infrastructure budgets (not overstretched research-education-health budgets) to be targeted to the creation of new strategic national/ regional alliances between (i) cell biology and microbial diversity research groups, to discover and develop new diagnostics, drug targets and assays, and new drug leads from new microbes, (ii) biochemical engineers, chemists and pharma, to produce, evaluate and develop drug candidates, (iii) pharma, clinical research and regulatory agencies to assess clinical efficacy and safety of, and develop new drug candidates. in the context of the sars-cov-2 pandemic, an alliance between virology, cell biology, microbial diversity, and synthetic microbiology groups in upstream discovery would accelerate new antiviral discovery and populate antiviral drug pipelines, but also pipelines of new antimicrobials urgently needed for the treatment of bacterial superinfections responsible for some of the covid-19 mortalities. and: while advanced age, underlying co-morbidities and infection dose are identified as predisposing factors for development of severe covid-19 disease, deaths among young healthy individuals also occur for reasons currently unknown. once predisposing factors for this group have been elucidated, diagnostics to identify young people at risk, especially those most exposed to sars-cov-2, will be needed in order to reduce their exposure. vaccines, despite their proven value in protecting against disease and their much-heralded pivotal importance for lockdown exit and herd immunity, are the cinderellas of clinical practice and, in normal times, not only attract little interest from governments but also are controversial, due to negative publicity from vociferous anti-vaccine groups propagating unfounded claims. the development and use of a number of current vaccines/vaccine candidates are orchestrated and funded, not by industry and public health systems, but by philanthropic organizations, like the gates foundation, working with agencies like cepi (coalition for epidemic preparedness innovations) and gavi (global alliance for vaccines and immunizations). indeed, the gates foundation is also playing a leading role in the search for, and development of, a vaccine against covid-19 (https://www.gatesfoundation.org/ media-center/press-releases/2020/02/bill-and-melinda-gates-foundation-dedicates-additional-funding-to-the-novel-coronavirus-response). however, it is the duty of national governments and international organizations, as part of their pandemic preparedness, to finance vaccine development platforms that are able to rapidly create new vaccines in response to an outbreak. once a new vaccine candidate is shown to be safe and protective, its rapid large-scale production and distribution requires the infrastructure of large pharmaceutical companies. as epidemics cannot be planned, industrial managers cannot be expected to promote projects without a market. governments must therefore intervene to maintain the interest and technical capacity of industry in developing vaccines and antibiotics (a smouldering fire) by creating a market in form of governmental orders. assessments of value-for-money of these strategic alliances must be made in the context of the global costs of pandemics like that of sars-cov-2. • pandemic preparedness requires rapid creation, production and distribution of effective materials for diagnosis, prophylaxis and therapy. this necessitates significant long-term investment in research and development involving unconventional alliances of disparate academic science and medical research groups, industry, philanthropic foundations, vaccine enabling coalitions, and crisis preparedness taskforces. there is great diversity in stress resilience (e.g., the ability to deal with peaks of illness) of different health systems, with some being at least regionally overwhelmed during the winter influenza season. the less resilient systems will generally be the first to become overwhelmed in a health crisis. while there are numerous parameters involved in health system resilience, and experts know most of the pinch points and solutions that can deal with these (but also what is uncertain and what needs to be understood before effective 'solutions' can be formulated), three elements worth consideration in efforts to increase health system resilience are discussed here. healthcare systems are by and large extremely large, complex, heavily bureaucratic and fragmented. the often system-wide, multi-level consultations, decisions and responses needed in times of emergencies are challenging and often slow, usually slower than crisis development, which means that healthcare systems follow and react to events, rather than managing them. crises are in some ways analogous to wars, and bureaucracies are not designed to manage wars, which is the job of the military. in crises, we need crisis strategy-tactics specialists, a taskforce with short, well defined and effective chains of command, tasked with overriding normal procedures and taking charge of supply chains and requisitioning of assets, (re)deployment of personnel, organization and prioritization of allocation of infrastructure, managing logistics, and so forth. these could be specially trained taskforces of existing staff within healthcare systems, external taskforces or combinations of both. of course, for taskforces to operate optimally, they, together with the best available scientific minds, must also plan in advance the required resources, supply chains, personnel, strategic options, and so forth. they must also organize regular 'infection games'/public health manoeurvres (https://www.ted.com/talks/bill_gates_the_ next_outbreak_we_re_not_ready?language=en) = crisis 'fire drills', to train nationally and transnationally, refresh skills and explore and anticipate unexpected events to ensure preparedness, so that appropriate responses can be rolled out rapidly anywhere, independently of national borders. 5 another, mandatory, task for the taskforce would be to conduct regular 'stress tests' of healthcare system resilience, as have been instituted for banks to ensure that they have adequate resources (= resilience) to withstand crises. such stress tests should be designed by health experts, epidemiologists-modellers, procurement agencies, representatives of the diagnostics-vaccine-drug industry, and so forth, and the design and implementation of the stress tests overseen by the taskforce. • national crisis task forces consisting of dedicated strategy-tactics specialists need to be established to plan crisis preparedness, make recommendations to improve health system resilience, and carry out regular crisis "fire drills" and stress tests. an important aspect of the sars-cov-2 outbreak is that, in most countries, it has become more difficult to obtain consultations with primary healthcare clinics/physicians, because of social distancing practices, illness or involvement in crisis management (e.g., see keesara et al., 2020) . as time goes on, the inability to access many primary healthcare services leads to progressive worsening of existing and new conditions in some individuals. access to primary healthcare, which in some countries was already unsatisfactory before covid-19, is becoming a new crisis. this has resulted in the 'flight to the web' for information (sometimes obtaining disinformation in the process): the web is becoming a substitute for clinical consultations, in terms of obtaining information relating to symptoms experienced. this will ultimately have a significant impact on how the public views the computer as a facilitator-mediator of primary healthcare. while classical telemedicine-the ad hoc consultation of a remote, unknown physician who can advise on the symptoms presented-may be helpful in times of inadequate access to regular primary healthcare facilities, it cannot replace clinical advice informed by patient case histories and personal knowledge of the patient. reduced access to primary healthcare below a certain threshold constitutes itself a significant health hazard and is counter to a government's duty to protect its citizens. what to do to increase resilience of primary health care and increase access? one important contribution will be the 'digital healthcare revolution' (keesara et al., 2020) , i.e., some traditional one-on-one meetings between patient and doctor being replaced by web-based consultations. but also imagine teleconsultations based on (i) complete personal case histories, combined with (ii) up-to-date population epidemiological information, combined with (iii) individual patient best practice recommendations based on precision medicine analyses/predictions: welcome to the national clinical informatics centre (ncic; timmis and timmis, 2017) , informing in real time a virtual doctor, a clinically-programed, ai-evolving server. this doctor, interfacing with both the patient and ncic, diagnoses according to detailed case history and patient symptom input via computer (and aided, where necessary, by diagnostic information obtained through in-home patient self-diagnosis with apparatus/diagnostic materials promptly delivered by a medical logistics service), and makes treatment recommendations (timmis, 2020) . in some countries/regions, access to primary healthcare already involves significant waiting periods. the additional restrictions on access to primary healthcare resulting from the sars-cov-2 outbreak are resulting in further suffering and frustration that will surely make the prospect of a consultation with a virtual doctor providing personalized medicine, who is instantly available 24/7, an increasingly attractive future possibility. of course, many health issues cannot be handled remotely via the web (though the proportion will increase steadily with the development of informatic infrastructure and easy-to-use home diagnostics), and will result in referral to a clinician. but, web-based consultations can significantly reduce numbers of patients requiring clinician consultations and the associated stress on the health system. • it is essential that health systems urgently develop centralized, secure informatic infrastructure needed to underpin web-based machine learning-facilitated precision medicine, and evolve web-based consultations, available on demand 24/7, as an integral mainstream component of primary healthcare services. the current sars-cov-2 outbreak has brutally exposed the current vulnerability of society to pandemics, even those that have been long predicted and anticipated (ge et al., 2013; menachery et al., 2015) . most healthcare systems have not evolved for resilience in times of catastrophe, nor for effective rapid responses to pandemics. a key principle steering evolution has been value-for-money within a fixed budget; contingency planning within this framework (outlays for materials that may never be used) may be considered to be a nuisance that diminishes what can otherwise be done with limited funds, and so to a greater or lesser extent may be postponed. for this reason, it is crucial that budgets for contingency planning are separate from health system budgets. equally important, it has emphasized the fact that some healthcare systems have for a long time been on the edge of the cliff, just waiting for an event to push them over. their adaptation to changing needs has often been through a 'sticking plaster' response. evolution has been ad hoc, via responses to new developments and challenges, and often led to fragmentation rather than coherence. the lessons to be learned are thus not only to take scientifically-founded pandemic predictions seriously into account in policy elaboration, but also to streamline and institute changes in healthcare systems that impose an evolutionary trajectory that increases coherence, efficiency and preparedness, and the necessary mechanisms to maintain these as new exigencies arise (e.g., see timmis and timmis, 2017) . and, especially because this crisis has revealed enormous disparities in responsiveness, effectiveness and the quality of responses in different countries, both preparedness for pandemics and the general improvement of healthcare mandate international benchmarking for contingency planning and the evolution of healthcare systems. comparisons/benchmarking within countries-within single systems-is no longer acceptable. many healthcare systems need substantive improvements through strategic investments, in most cases targeted to system changes, not just extra funding of existing services. and above all, they need crisis taskforces embedded in them that can prepare for, and take charge in times of, impending catastrophes. another lesson learned is that the sars-cov-2 outbreak has revealed new synergy potentials, such as the manufacture of ventilators by engineering companies not normally active in the manufacture of medical devices. it is not unreasonable to assume that new innovations can and will emerge from new interactions between creative engineers and clinicians. for example, best practice for breathing difficulty and poor blood oxygenation is intubation and ventilation. the paucity of ventilators is a 'critical control point' for best treatment practice in some hospitals, which has been discussed above. anecdotal evidence suggests that, of those individuals who die, despite best treatment practice involving intubation, the cause of death is often due to superinfection by antibiotic resistant bacteria (vincent et al., 2020) . the cause of this may indeed be intubation, causing perturbation of normal lung physiology and creating susceptibility to superinfection. there are, however, less invasive means of increasing blood oxygen levels. perhaps engineers, together with clinicians, will devise new or improved non-invasive approaches to blood oxygenation. and once creative engineers from the non-medical field start to expertly scrutinize current medical devices, perhaps we will see new approaches and new designs that significantly advance medical practice. but perhaps the most important lesson learned is about our frontline health professionals ministering to covid-19 patients, especially those with severe disease. these clinicians and nurses who willingly and selflessly work long, sometimes multiple shifts to the point of utter exhaustion, often not able to see their families for long periods because of the danger of infecting them, always under unbelievable stress working in what are essentially war zones with the accompanying horrors (e.g. see http:// www.sixthtone.com/news/1005474/i-spent-seven-weeksin-a-wuhan-icu.-heres-what-i-learned?utm_source=sfmc& utm_medium=email&utm_campaign=2716680_agenda_ weekly-17april2020&utm_term=&emailtype=newsletter), sometimes without adequate protective clothing and always in danger of contracting covid-19, sometimes becoming infected, and sometimes paying the ultimate price. these are the heroes of the pandemic, the faces of resilience of covid-19 healthcare, exceptional citizens demonstrating exceptional fortitude, personal sacrifice and professional dedication: they are our role models of the 21st century. real estimates of mortality following covid-19 infection valuing vaccines: deficiencies and remedies on viruses, bats and men: a natural history of food-borne viral infections the novel coronavirus-a snapshot of current knowledge fangcang shelter hospitals: a novel concept for responding to public health emergencies isolation and characterisation of a bat sars-like coronavirus that uses the ace2 receptor offline: covid-19 and the nhs: "a national scandal preparing intensive care for the next pandemic influenza health security capacities in the context of covid-19 outbreak: an analysis of international health regulations annual report data from 182 countries covid-19 and health care's digital revolution a sars-like cluster of circulating bat coronaviruses shows potential for human emergence estimating case fatality rates of covid-19 the diy digital medical centre biological land mines: bioterrorism underscores major knowledge deficits in the ecology of infectious agents the home clinic or all in a day's work of dr the urgent need for microbiology literacy in society pipelines for new chemicals: a strategy to create new value chains and stimulate innovation-based economic revival in southern european countries the sars case study. an alarm clock prevalence and outcomes of infection among patients in intensive care units in 2017 characteristics of and important lessons from the coronavirus disease 2019 (covid-19) outbreak in china combatting covid-19-the role of robotics in managing public health and infectious diseases key: cord-332583-5enha3g9 authors: bodine, erin n.; panoff, robert m.; voit, eberhard o.; weisstein, anton e. title: agent-based modeling and simulation in mathematics and biology education date: 2020-07-28 journal: bull math biol doi: 10.1007/s11538-020-00778-z sha: doc_id: 332583 cord_uid: 5enha3g9 with advances in computing, agent-based models (abms) have become a feasible and appealing tool to study biological systems. abms are seeing increased incorporation into both the biology and mathematics classrooms as powerful modeling tools to study processes involving substantial amounts of stochasticity, nonlinear interactions, and/or heterogeneous spatial structures. here we present a brief synopsis of the agent-based modeling approach with an emphasis on its use to simulate biological systems, and provide a discussion of its role and limitations in both the biology and mathematics classrooms. agent-based models (abms) are computational structures in which system-level (macro) behavior is generated by the (micro) behavior of individual agents, which may be persons, cells, molecules or any other discrete quantities. typical abms contain three elements: agents, an environment, and rules governing each agent's behavior and its local interactions with other agents and with the environment. decades of advancement in computer power has made agent-based modeling a feasible and appealing tool to study a variety of complex and dynamic systems, especially within the life sciences. as the use of abms in research has grown, so too has the inclusion of abms in life science and mathematical modeling courses as a means of exploring and predicting how individual-level behavior and interactions among individuals lead to system-level observable patterns. abms are now one of the many types of models students studying the life sciences or applied mathematics should encounter in their undergraduate education. prior to the introduction of abms into biological and applied mathematics curricula, the clear model format of choice was the ordinary differential equation (ode), or maybe a pair of them; occasionally, discrete difference equations and/or matrix equations would also be introduced. exponential growth and decay were ready examples, paving the way for extensions of the exponential growth process toward a carrying capacity in the form of the logistic growth process (voit 2020) . this logistic process was easily generalized to two populations, which were at first independent, but then allowed to interact. depending on these interactions, the result was a pair of two populations competing for the same resource or a simple predator-prey model in the format of a two-variable lotka-volterra system. although odes and any other types of "diff-e-qs" are a priori dreaded by almost all but mathematicians and physicists, the concept of an ode, if adequately explained, becomes quite intuitive. for instance, one may ease a novice into the world of odes by considering changes in the water level w of a lake over time (ayalew 2019) . whereas these dynamics are difficult to formulate as an explicit function w (t), newcomers readily understand that changes in the water level depend on influxes from tributaries, rain, and other sources on the supply side, and on effluxes, evaporation and water utilization on the side of reducing the amount of water. just putting these components into an equation leads directly to a differential equation of the system (weisstein 2011 ). on the left side, one finds the change over time as dw /dt, and this change is driven, on the right-hand side, by a sum of augmenting and diminishing processes. there is hardly a limit to what can be achieved with odes in biology, with the very important exception of processes that have genuine spatial features. and while it is not difficult to ease a biology undergraduate into ordinary differential equations, the same is not necessarily true for partial differential equations (pdes). however, spatial phenomena in biology seldom occur in homogeneous conditions. as examples, consider the formation of tumors with angiogenesis and necrosis; the local patterns of cell-to-cell signaling that governs the embryonic development; the spread of the red fire ant (solenopsis invicta) from mobile, al, its alleged port of entry into the usa, all along the gulf and east coasts; or the population size and dynamics of the santa cruz island fox (urocyon littoralis santacruzae) being driven by territory size which in turn depends on local vegetation (scott 2019) . until relatively recently, the conundrum of space was often dealt with in the final chapter of mathematical modeling in biology. a sea change came with the development of abms, which are natural formats for both stochasticity and spatial phenomena. by their nature, these models are computationally expensive, which initially prevented their use in most classrooms. however, this situation has obviously changed. as the bio2010 report (2003) stated: "computer use is a fact of life of all modern life scientists. exposure during the early years of their undergraduate careers will help life science students use current computer methods and learn how to exploit emerging computer technologies as they arise." classroom use of abms has thus become not just logistically feasible, but also very appealing for demonstrating spatial dynamics in a wide range of biological systems (kottonau 2011; triulzi & pyka 2011; shiflet 2013; pinder 2013) . supporting this appeal is a repertoire of software tools, such as simbio and netlogo (see sect. 3), that contain predefined examples and require minimal computer coding skills for model analysis. here, we present a brief synopsis and history of this modeling approach with emphasis on life science applications (sect. 2), describe some of the software tools most frequently used in the classroom (sect. 3), and then focus on some of its roles and limitations in the classroom (sect. 4). the origins of agent-based modeling. the true origins of any method or procedure are seldom identifiable in an unambiguous manner. in the case of agent-based modeling, one could think of craig reynolds' 1987 seminal article on the formation of bird flocks (with the agents denoted as boids, short for "bird-oid object"), which he was able to represent with just three rules of behavior: (1) avoid collisions with nearby birds; (2) attempt to match the velocity of nearby birds; and (3) attempt to stay close to nearby birds in the flock (reynolds 1987; gooding 2019) . the result of simulations with this simple abm was very realistic-looking flocking behavior. particularly intriguing in this study was the fact that there was no leader or a global organizing principle. instead, the virtual birds were truly individual agents that self-organized locally, thereby generating a globally coherent flight pattern. while reynolds' work was a milestone, key concepts leading to modern abms can be found much earlier. one notable contributor of ideas was nobel laureate enrico fermi, who used mechanical addition machines to generate probabilities for stochastic models with which he solved otherwise unwieldy problems (gooding 2019) . this procedure was an early form of the method of a monte carlo simulation, which was later independently developed and published by stanislav ulam, like fermi a member of the manhattan project (metropolis & ulam 1949; metropolis 1987) . another very important contribution to the budding development of abms was the turing machine (turing 1936) , which is a mathematical model of computation that uses a set of rules to manipulate symbols in discrete cells on an infinite tape. much closer to abms were ideas of ulam, who was fascinated by the "automatic" emergence of patterns in two-dimensional games with very simple rules (ulam 1950; metropolis 1987) . together with the new concept of game theory (von neumann & morgenstern 1944) , all these ideas were developed into the concept of cellular automata, which are direct predecessors of abms (ulam 1950; ulametal 1947; von neumann & morgenstern 1944) . a very appealing implementation of a cellular automaton was john conway's famous game of life (gardner 1970) . the social sciences adopted computational methods in the 1960s for microanalytic simulations or, simply, micro-simulations (gilbert & troitzsch 2005; gooding 2019 ). in contrast to today's abms, which use simple rules to recreate observed or unknown patterns, the agents in the original microsimulations acted according to empirical data (bae 2016). the advantage of this strategy is that model analysis can reveal systemic behaviors under different realistic scenarios (gooding 2019) . a seminal paper in this context described generic segregation processes (schelling 1971) . other agent-based modeling work in sociology and economics was gleaned from the biological abm work of smith (1982) , who formulated darwin's ideas of evolution as a computer simulation. this idea inspired nelson & winter to apply similar concepts and implementations to market studies, where firms were modeled like animals that followed specific routines. in particular, the firms were in competition, and the market weeded out bad routines while rewarding the fittest. nelson & winter's influential book an evolutionary theory of economic change (nelson & winter 1982) strongly proposed the use of computer simulations, which today would fall within the scope of agentbased modeling. their ideas led to a school of thought called evolutionary economics (hanappi 2017 ). an early and particularly influential paper in this context tried to shed light on the stock market (palmer 1994) . initially, abms of artificial life simulated simple homogeneous agents that acted like huge colonies of ants that could just move and eat in the pursuit of food. somewhat more sophisticated, economic simulations used as the main agent homo economicus, a consistently rational human pursuing the optimization of some economic goal or utility with exclusive self-interest (persky 1995) . based on these humble beginnings, sophistication in computing soon permitted heterogeneous agents and much more complicated landscapes than before. the successes in economics were so tantalizing that simulation studies eventually reached the most prestigious journals of economics and the social sciences geanakoplos 2012; hanappi 2017) . modern abms in economics are capable of capturing much of the complexity of macroeconomic systems (e.g., caiani (2016) ). following directly the principles of cellular automata, kauffman studied large grids with elements that changed features in a binary fashion (kauffman 1993) . for instance, a white agent could turn black, and this shift occurred according to boolean rules that usually involved some or all neighboring grid points. kauffman was able to demonstrate the emergence of complex patterns, such as oscillations and percolation. starting in the 1980, wolfram performed systematic studies of cellular automata, which led to his influential 2002 book a new kind of science (wolfram 2002 ) that assigns cellular automata a wide range of applications in a variety of fields. biological applications of agent-based modeling. abms have been constructed to study a wide range of biological phenomenon. numerous reviews and research efforts using abms have focused on specific biomedical systems. issues of gene expression were modeled by thomas (2019) . morphogenetic and developmental processes were discussed in grant (2006) , robertson (2007) , , tang (2011), and glen (2019) . models of tissue mechanics and microvasculature are captured in and van liedekerke (2015) . inflammation, wound healing and immune responses were addressed in , ), chavali (2008 , and castiglione & celada (2015) . other authors (wang 2015; segovia 2004; cisse 2013 ) used abms to model cancer growth, tuberculosis, and schistosomiasis, respectively. lardon and colleagues (lardon 2011) used abms to analyze biofilm dynamics. butler and colleagues described the use of abms in pharmacology (butler 2015) . abms studying multicellular systems provide a unique capability to examine interactions and feedback loops across the different hierarchies of the system (hellweger 2016) . reviews of abms in the context of ecology, environmental management, and land use include (bousquet 2004; matthews 2007; grimm & railsback 2005; caplat 2008 ; deangelis & diaz 2019). in some applications, interventions or treatments were addressed and therefore required the adaptation of agents to changing scenarios (berry 2002) . abms have also been used to simulate epidemics with analyses examining the impact of implemented or potential intervention measures (e.g., quarantining/physical distancing, mask wearing, and vaccination) (mniszewski 2013; perez & dragicevic 2009; tracy 2018) . visual representations of epidemiological abms have even been used by news outlets during the covid-19 pandemic to help explain to the public how various intervention methods change the shape of an epidemic (i.e., "flatten the curve) or the basic reproduction number (r 0 ) of an epidemic; see, for example, fox (2020) and stevens (2020). rationale & pitfalls of abms. the two most frequent goals of an abm analysis are (1) the elucidation and explanation of emergent behaviors of a complex system and (2) the inference of rules that govern the actions of the agents and lead to these emerging system behaviors. this type of inference is based on large numbers of simulations, i.e., replicate experiments with the abm using the same assumptions and parameters, and different experiments over which assumptions or parameter values are systematically changed. simulations of abms essentially always yield different outcomes, because movements, actions and interactions of agents with each other or with the environment are stochastic events. the inference of rules from simulation results is an abductive process (voit 2019) that is challenging, because one can easily demonstrate that different rule sets may lead to the emergence of the same systemic behaviors, and because even numerous simulations seldom cover the entire repertoire of a system's possible responses. in fact, hanappi (2017) warned: "assumptions on microagents that play the role of axioms from which the aggregate patterns are derived need not-and indeed never should-be the end of abm research." arguably the greatest appeal of abms, and at the same time a treacherous pitfall, is their enormous flexibility, which is attributatble to the fact that any number of rules can be imposed on the agents, and that the environment may be very simple but can also be exceedingly complicated. for instance, the environment may exhibit gradients or even different individually programmable patches (barth 2012; gooding 2019) including importing geographic information systems (gis) data to define the characteristics of each patch (see scott (2019) for an example with a detailed explanation of how gis data are incorporated into an abm). in particular, the repeated addition of new elements to a model can quickly increase the complexity of the model, thereby possibly distracting from the core drivers of the system's behavior, obscuring the importance of each of the rules the agents must follow, and generally making interpretations of results more difficult. related to this option of adding elements is the critique that abms can be 'tuned' by researchers to create outcomes that support the researcher's narrative (gooding 2019) . to counteract increases in complexity, some authors have begun to develop methods of model reduction that retain the core features of a model, but eliminate unnecessary details (zou 2012). another critique of abms is that simulations are not readily reproducible, because the rules can be complicated and stochasticity rarely repeats the same model trajectories. a strategy to increase the reproducibility of abms was the establishment of protocols for the standardized design and analysis of abms (lorek & sonnenschein 1999; grimm 2006 grimm , 2010 heard 2015) . (2017). in a slight extension of abms, lattilä (2010) and cisse (2013) described hybrid simulations involving abms and dynamic systems, and heard (2015) discussed statistical methods of abm analysis. these introductory tutorials and reviews, however, are typically not designed for undergraduates with limited mathematical or computational modeling experience. the scientific community has also worked hard on facilitating the use of abms by offering software like netlogo, swarm, repast, and mason. summaries and evaluations of some of the currently pertinent software are available in berryman (2008) and abar (2017) , and netlogo is described more fully in sect. 3. a variety of software tools can be used to construct, simulate, and analyze abms. when abms are taught or used in biology or mathematics courses, software should be chosen to align with pedagogical objectives. note that the pedagogy of abms in life science and mathematical modeling courses is discussed in more detail in sect. 4. in this section, we highlight some of the most used software packages in an educational setting. ecobeaker & simbio. ecobeaker was developed by eli meir and first released in 1996 as software that ran simulated experiments designed to explore ecological principles. in 1998, simbio (meir 1998 ) was founded (then called beakerware) for the release of the second version of ecobeaker and has since grown to include simulated experiments in evolution, cell biology, genetics, and neurobiology. many of the simulated experiments in the simbio virtual labs software are agent-based simulations. in a simbio virtual lab, the user interacts with a graphical interface portraying the agents (individuals in the experiment) and sometimes their environment, which can be manipulated in various ways for different experimental designs. the graphical interface also contains relevant graphs which are updated as the simulated experiment runs. ecobeaker and simbio virtual labs are examples of software where the focus is on experimental design and the use of simulation to understand biological concepts. the user never interfaces with the code and does not need to understand the underlying algorithms which produce the simulation. simbio virtual labs are used in many biology classrooms across the united states. the software is priced per student (currently $6/lab/student or $49/student for unlimited labs). netlogo. the agent-based modeling environment netlogo (wilensky 1999) was developed by uri wilensky and first released in 1999. netlogo is free and continues to be improved and updated (the software is currently on version 6). additionally, a simplified version of netlogo can be run through a web browser at http://netlogoweb. org/. netlogo is a programming platform allowing the implementation of any abm a user might design. as such, its user interface includes a tab where abm code is written in the netlogo programming language, and a tab for the user to view a visualization of the abm and user specified outputs as the abm is simulated. textbooks by railsback & grimm (2012) and wilensky & rand (2015) provide introductions to the netlogo prgramming languages as well as providing a thorough overview of the algorithmic structures of abms. since its initial development, netlogo has built up an extensive model library of abms. additionally, over the years, faculty at various institutions have developed abm modules through netlogo that allow students to explore a variety of biological phenomenon. for example, the virtual biology lab (jones 2016) has created 20 different virtual laboratory modules using netlogo through a web browser for exploring topics in ecology, evolution, and cell biology. the virtual biology labs are similar in scope to the ecobeaker and simbio labs. another example is infections on networks (iontw) which provides an abm framework and teaching modules for examining aspects of disease dynamics on various network structures (just 2015a, b, c) . one of the responses to the bio2010 report (2003) has been a push to create biocalculus courses or to insert more biological application examples and projects within traditional calculus courses. indeed, studies have shown that including applications from the life sciences in classic math courses like calculus leads to students gaining equivalent or better conceptual knowledge than from similar courses without life science applications (comar 2008; . however, many mathematics and biology educators have pointed out that the subset of mathematics applicable to biology extends well beyond calculus, and undergraduates (especially those majoring in biology) should be exposed to a variety of mathematical models and methods of analysis across biology and mathematics courses (bressoud 2004; gross 2004; robeva & laubenbacher 2009 ). the only prerequisites for analyzing the simulations of an abm are a basic understanding of the underlying biology and, in some instances, knowledge of how to perform basic statistical calculations or generate graphical representations of results. many pre-built abms in simbio virtual labs and netlogo generate relevant graphs and/or produce spreadsheets or text files of the relevant data. thus, a student can utilize abms in life science courses without having to learn how to implement (by writing code) an abm. the prerequisites for learning how to implement abms are not as extensive as for other forms of mathematical models. the most essential prerequisite is some exposure to the fundamentals of computer programming such as understanding loop structures and conditional statements, implementing stochastic processes, and understanding how the order of executed operations within a program impacts the program's output. agent-based modeling software like netlogo (wilensky 1999 ) keeps model implementation and analysis relatively simple by providing built-in model visualization tools and automatically randomizing the order in which agents execute programmed operations. due to their appealing visual representation, abms can easily be used in the classroom to demonstrate biological processes ranging from chemical reactions to interactions among species, as if the model were simply an animation. however, using abms in this way is much like using a cell phone to hammer nails (q.v., theobald (2004) ): it may work in the desired fashion, but represents an utter waste of the tool's real potential. adding just one more step, the collection of model-generated data, transforms a passive learning experience into an active one. students can be asked to calculate means and variances, graph relationships between variables, discuss the sample size needed for reliable results, and generate quantitative predictions under different hypotheses, all in the context of a specific biological question. this can be done even in a large classroom with only a single, instructor-controlled computer, bridging the gap between lecture and lab. if students have access to individual computers, much more is possible. either individually or in small groups, students can use abms to collect and analyze their own data. free file-sharing resources such as googledocs make it easy to pool data across many groups, thereby crowd-sourcing problems that would be too large for any one group to handle on their own. in smaller classes and lab sections, individuals or groups can be assigned to model different scenarios (e.g., the interaction effects between different parameters), prompting discussions of the most appropriate parameter values and model settings. such models can even be extended into miniature research projects. for example, in a unit on community ecology, students might be assigned a question about two interacting species, then use online resources to find relevant information and parameter estimates, design and conduct a series of model runs, analyze their data using simple statistical techniques, and present their findings to the class. although abms can be used to simulate almost any biological process, meaningful exploration of a model typically requires a substantial commitment of class time and instructor engagement. as a result, except in modeling courses, it is seldom practical to incorporate abms into every lesson plan. in our experience, their educational value is highest for studying processes involving (a) substantial amounts of stochasticity, (b) nonlinear interactions, and/or (c) a defined spatial structure. the inclusion of abms in math courses generally comes in two different modes: (1) abms are taught as one modeling technique in a course covering multiple modeling techniques, and (2) the construction and analysis of abms are taught in a course where abms are the only type of modeling being used. however, due to the minimal prerequisites for learning agent-based modeling, both types of courses can potentially be offered with one or no prerequisite courses. bodine (2018) provides an example of a course where abms are taught as one type of discrete-time modeling technique in an undergraduate course designed for biology, mathematics, biomathematics, and environmental science majors that has no prerequisites beyond high-school algebra. an example of a course where abms are the only type of modeling being used is given by bodine (2019); this course has only a single prerequisite: either a prior math modeling course which introduces basic computer programming or an introduction to computer science course. the use of biological applications in teaching mathematical modeling (including modeling with abms) is often viewed as having a lower entry point with less new vocabulary overhead than other types of applications (e.g., those from physics, chemistry, or economics). in particular, models of population dynamics, even those involving interactions between multiple subpopulations or different species, usually do not require any new vocabulary for most students, which allows for a more immediate focus on mechanisms of population change and impact of interactions between individuals within the populations. video game vs. scientific process. in our experience, students sometimes react to an abm's many controls and visual output by treating the model as a video game, clicking buttons at random to see what entertaining patterns they can create. other students prefer to complete the exercise as quickly as possible by blindly following the prescribed instructions. neither of these approaches substantially engages students in thinking about the question(s) underlying the model, different strategies for collecting and analyzing data, or the model's limitations. to foster these higher-order cognitive skills, use of the abm should be explicitly framed as an example of the scientific process. this approach begins with a set of initial observations and a specific biological question. for example, what management practices would be most effective in controlling the invasive brown tree snake? after familiarizing themselves with the biological system, students propose hypotheses about the factors contributing to the snake's success on guam, suggest management strategies, and set model parameters that reflect their chosen strategy. finally, they run the model multiple times to collect data that allow them to measure their strategy's effectiveness. under this pedagogical approach, abms become a vehicle for design-ing and conducting a miniature research project, enabling experiments that would not otherwise be practical due to cost, logistics, or ethical considerations. the modeling exercise can also reinforce lessons on how scientific knowledge is constructed and tested (e.g., the three p's of science, namely, problem posing, problem solving, and peer persuasion (watkins 1992) ). as part of this exercise, students should engage in the process of deciding how best to collect, analyze, and present their data. for example, as part of the brown tree snake project, students might be asked to explore the practical steps that other pacific islands could take to prevent invasions or eradicate invaders. one group of students decides to focus on two different control measures: cargo checks of all inbound flights and deployment of poison baits around airstrips. following an overview of different statistical approaches, the students determine that a multiple regression analysis would best allow them to address their question. allowed only a limited 'budget' of 30 model runs, students settle on a factorial design using three treatment levels of cargo checks, three levels of baiting, and three replicates of each combination. the students set up a spreadsheet to record the data from each model run, graph their data in a scatter plot, and use software such as microsoft excel's analysis toolpak to conduct their analysis. a model as a caricature of the real world. students at early stages of their academic career often envision science as a collection of factual information and fixed procedures. students with this mindset may dismiss as useless any model that does not incorporate every detail of a particular biological system. by contrast, scientists recognize that models, whether mathematical, physical, or conceptual, are deliberate simplifications that attempt to capture certain properties of a biological system while ignoring others (dahlquist 2017) . for example, the standard epidemiological sir model (diagrammed in fig. 1a ) divides a population in three subpopulations (susceptible, infectious, and removed) while ignoring any potential heterogeneity within each subpopulation (e.g., age, treatment status, groups at higher risk for transmission). students will need to engage in activities that frame the abm as a hypothesis about the organization and function of a specific biological system (weisstein 2011) . after a description (and possible exploration) of the basic model, students can work in groups to suggest additional processes and variables that seem relevant to understanding the system. they can then choose one or two of the factors that they consider most important to addressing the question being asked. finally, they should consider how to modify the model to incorporate the chosen features. for example, a standard epidemiological model divides the host population into susceptible, infectious, and removed subpopulations, and studies the movement of individuals among these subpopulations (fig. 1a) . a group of students decides to modify this model to track a malarial epidemic. after discussing mortality rates, prevention and treatment options, and genetic and age-related variation in host susceptibility, the students decide to focus on incorporating vector transmission into their model. through guided discussion with the instructor, they realize that transmission now occurs in two directions: from infected vectors to susceptible hosts and from infected hosts to uninfected vectors. they therefore develop a schematic model (fig. 1b) that depicts these revised rules for each agent in the abm. even if the students do not actually build the corresponding computational model, this exercise in extending a model to reflect specific biological assumptions helps students understand the iterative process by which models are developed and the utility of even simple models to clarify key features of the system's behavior. algorithms vs. equations. the concept of an equation is introduced fairly early in mathematics education. in the united states, children can encounter simple algebraic equations in elementary school (common core 2019) and then continue to see increasingly complex equations in math classes through college. because of this long exposure to equations, the use of functions and systems of equations to model systems in the natural world feels "natural" or logical to students when they first encounter differential equation models or matrix models. abms, on the other hand, can seem confusing to students because they lack the ability to be expressed as an equation or set of equations. an abm is constructed as an algorithm describing when and how each agent interacts with their local environment (which may include other agents). often these interactions are governed by stochastic processes, and thus "decisions" by agents are made through the generation of random numbers. when first introducing students to abms, it can be helpful to teach students how to read and construct computer program flowcharts and to create a visual representation of what is occurring within an algorithm or portion of an algorithm (see bodine (2019) for example assignments that utilize program flowchart construction). in life science classes where abms are being analyzed but not constructed and implemented, a flow diagram can be a useful tool for conveying the order processes occur in the model. class discussions can question whether the order of processes make biological sense, and whether there are alternatives. in math modeling classes, the construction of flowcharts, even for simple abms, can help students elucidate where decision points are within the code, and what procedures are repeated through loop structures. the construction of flowcharts as students progress to more complicated abms can help students rectify the order of events in their implemented algorithm against the order in which events should be occurring biologically. whether students are working with abms in life science or math modeling classes, it is helpful for them to learn how to read and understand flow diagrams as they are often included in research publications that use agent-based modeling. describing an abm. much to the alarm of many math students beginning to develop abms, the formal description of an abm requires more than just writing the computer code. the standard method for describing abms in scientific publications, referred to as the overview, design concepts, and details (odd) protocol (grimm 2006 (grimm , 2010 railsback & grimm 2012) , often requires more than one page to fully describe even a simple abm. this can make abms seem overwhelming to students as they first begin to explore abms. in courses which teach the implementation and description of abms, instructors should take care not to introduce the computer code implementation simultaneous to the model description via the odd protocol. note that the introductory text on agent-based modeling by railsback & grimm (2012) does not introduce the concept of the odd protocol until chapter 3, which comes after the introduction and implementation (in netlogo) of a simple abm in chapter 2. in the course materials by bodine (2019) , the concept of the odd protocol is introduced prior to project 2, but the students are not required to write their own odd protocol description until the final project, once they have seen odd descriptions for multiple models. model implementation vs. the modeling cycle. courses that aim to teach methods in mathematical modeling often start with a discussion of the modeling cycle, which is typically presented as a flow diagram showing the loop of taking a real world question, representing it as a mathematical model, analyzing the model to address the question, and then using the results to ask the next question or refine the original real world question. figure 2 shows an example of a modeling cycle diagram. in courses where the mathematical models are encapsulated in one or a small handful of equations, the time spent on representing the real world as a mathematical model (the green box in fig. 2) is relatively short. the construction of abms, however, can be a fairly lengthy process, as abms are designed to simulate interactions between individuals and the local environment. when students are in the middle of constructing their first few abms, they often lose sight of where they are in the modeling cycle because the model implementation becomes a cycle of its own; a cycle of writing bits of code, testing the code to see if it runs and produces reasonable results, and repeating this process to slowly add all the components needed for the full algorithm of the abm. as students are first learning agent-based modeling, they need to be reminded often to pull back and view where they are in the modeling cycle; to see the flock for the boids, as it were. model validation. within the modeling cycle, there is a smaller cycle of model validation (see dashed line in fig. 2) . in a course where students are first introduced to the classic lotka-volterra predator-prey model, the students are usually first introduced to a predator-prey data set (like the 200-year data set of canadian lynx and snowshoe hare pelts purchased by the hudson bay company (maclulich 1937; elton & nicholson 1942) ), which shows the oscillating population densities of the predator and prey populations. when the students then simulate the lotka-volterra model for various parameter sets, they find that they are able to produce the same oscillating behavior of the predator and prey populations. this is a form of model validation, and a model which did not display this distinctive trend seen in the data would be considered invalid for that data set. a similar process must occur for validating abms against observed biological patterns. however, in order to engage in this validation process for abms, students must first understand how decisions of individual agents and interactions between neighboring agents can lead to system-level observable patterns, a phenomenon referred to as "emergence" or the "emergent properties" of an abm. the classic abm example for easily identifying an emergent property is a flocking example (a stock example of flocking exists in the netlogo models library, and is explored in chapter 8 of the abm textbook by railsback & grimm (2012) ). the concept of an emergent property can take a little while for students to fully understand. by definition, it is an observable outcome that the system was not specifically programmed to produced. in particular, it is not a summation of individual-level characteristics, and is typically not easily predicted from the behaviors and characteristics of the agents. for example, a variation of the reynolds (1987) flocking model is included in the netlogo library and is explored in railsback & grimm (2012, chapter 8 ). in the model, each agent moves based on three rules: 1. separate: maintain a minimum distance from nearby agents 2. align: move in the same direction as nearby agents 3. cohere: move closer to nearby agents where all agents move at the same speed and different schemes for determining who is a nearby agent can be used. additionally, there are model parameters for the minimum distance to be maintained between agents, and the degree to which an agent can turn left or right in a single time step in order to align, cohere, and separate. it is not immediately evident from this set of rules that individual agents might be able to form flocks (or swarm together), and indeed that system-level behavior does not emerge for all parameter sets. however, certain parameter sets do lead to the agents forming one or more flocks that move together through the model landscape. students observations: algorithms for pattern recognition. one of the most exciting moments for students when they first begin running simulations of an abm is seeing systemlevel patterns emerge before their eyes. one of the challenges for students (and agentbased modelers, in general) is to develop algorithms to identify and/or quantify the patterns we can easily identify by sight. for example, in the flocking abm discussed above, an observer watching the locations of individual agents change at each time step can easily see the formation of flocks. if however, the observer wanted to systematically explore the parameter space of the flocking abm and determine the regions of the parameter space under which flocking occurs (a process which might involve running hundreds or thousands of simulations), it would be a tedious and time-consuming task to physically watch each simulation and record whether the agents formed a flock or not. instead, the observer must choose the criteria that indicate the formation of a flock, and then determine a measure or an algorithm for determining whether a flock (or multiple flocks) have formed. in a course designed to teach the construction and analysis of abms, this is a point where students should be encouraged to be both creative and methodical about developing such measures and algorithms. the development of these observational measures and algorithms also provides a great opportunity for collaboration between students. it is especially helpful if students with a diversity of academic backgrounds can be brought together to brainstorm ideas; for instance, mixing students with various levels of exposure in mathematics, computer science, and biology can be very beneficial. rapid advances in computing power over the past decades have made agent-based modeling a feasible and appealing tool to study biological systems. in undergraduate mathematical biology education, there are multiple modes by which abms are utilized and taught in the classroom. in biology classrooms, abms can be used to engage students in hypothesis testing and in the experimental design and data collection processes of otherwise infeasible experiments, and to enable students to utilize models as a part of the scientific process. all of this can be done without students having to learn a programming language. by contrast, students who have had some exposure to computer programming can learn the construction, implementation, and analysis of agent-based models in a math or computer science modeling class. biological applications are ideal systems for first attempts at agent-based models as they typically do not necessitate learning extensive new vocabulary and theory to understand the basic components that need to be included in the model. throughout this article, we endeavored to articulate the benefits and challenges of including abms in undergraduate life science and math modeling courses. consistent with the bio2010 report (2003), we recommend that undergraduate biology and life science curricula be structured to ensure that all students have some exposure to mathematical modeling. we additionally recommend that this includes agent-based modeling. while not every student necessarily needs to take a course exclusively focused on agent-based modeling, every undergraduate biology student should have the opportunity to utilize an abm to perform experiments and to collect and analyze data. as we educate the next-generation of life scientists, let us empower them with the ability to utilize abms to simulate and better understand our complex and dynamic world. agent based modelling and simulation tools: a review of the state-of-art software in silico experiments of existing and hypotheticalcytokine-directed clinical trials using agentbased modeling agent-based models in translational systems biology aligning simulation models: a case study and results integration of biology, mathematics and computing in the classroom through the creation and repeated use of transdisciplinary modules. primus, in revision bae jw multi-cell agent-based simulation of the microvasculature to study the dynamics of circulating inflammatory cell trafficking typical pitfalls of simulation modeling-lessons learned from armed forces and business adaptive agents, intelligence, and emergent human organization: capturing complexity through agent-based modeling land operations division dsto, defence science and technology organisation, po box 1500 agent-based modeling: methods and techniques for simulating human systems multi-agent simulations and ecosystem management: a review the changing face of calculus: first-and second-semester calculus as college courses mathematics for the life sciences discrete math modeling with biological applications (course materials) agent-based modeling course materials agentbased modeling in systems pharmacology agent based-stock flow consistent macroeconomics: towards a benchmark model symmetric competition causes population oscillations in an individualbased model of forest dynamics immune system modelling and simulation characterizing emergent properties of immunological systems with multicellular rule-based computational modeling assessing the spatial impact on an agent-based modeling of epidemic control: case of schistosomiasis the integration of biology into calculus courses common core state standards initiatives an invitation to modeling: building a community with shared explicit practices decision-making in agent-based modeling: a current review and future prospectus the case for biocalculus: design, retention, and student performance the ten-year cycle in numbers of the lynx in canada a (2020) how epidemics like covid-19 end (and how to end them faster) mathematical games-the fantastic combinations of john conway's new solitaire game getting at systemic risk via an agent-based model of the housing market review: agent-based modeling of morphogenetic systemsadvantages and challenges agent-based model history and development. in: economics for a fairer society-going back to basics using agent-based models simulation of biological cell sorting using a two-dimensional extended potts model simulating properties of in vitro epithelial cell morphogenesis a standard protocol for describing individual-based and agent-based models the odd protocol: a review and update individual-based modeling and ecology interdisciplinarity and the undergraduate biology curriculum: finding a balance a systematic review of agent-based modelling and simulation applications in the higher education domain agent-based modelling. history, essence, future the history, philosophy, and practive of agent-based modeling and the development of the conceptual model for simulation diagram. doctoral dissertation a survey of agent-based modeling practices agent-based models and microsimulation advancing microbial sciences by individual-based modelling cellular potts modeling of complex multicellular behaviors in tissue morphogenesis virtual biology lab: an inquiry-based learning environment transmission of infectious diseases: data, models, and simulations disease transmission dynamics on networks: network structure versus disease dynamics an interactive computer model for improved student understanding of random particle motion and osmosis idynomics: next-generation individual-based modelling of biofilms hybrid simulation models-when, why, how? modelling and simulation software to support individual-oriented ecological modelling tutorial on agent-based modelling and simulation fluctuations in the numbers of the varying hare (lepus americanus) agent-based land-use models: a review of applications the beginning of the monte carlo method the monte carlo method episims: large-scale agent-based modeling of the spread of disease bio2010: transforming undergraduate education for future research biologists. the national academies press an evolutionary theory of economic change agent-based computing from multi-agent systems to agent-based models: a visual survey artificial economic life: a simple model of a stockmarket retrospectives: the ethology of homo economicus an agent-based approach for modeling dynamics of contagious disease spread an active learning exercise for introducing agent-based modeling agent-based and individual-based modeling: a practical introduction flocks, herds and schools: a distributed behaviour model multiscale computational analysis of xenopus laevis morphogenesis reveals key insights of systems-level behavior mathematical biology education: beyond calculus identifying control mechanisms of granuloma formation during m. tuberculosis infection using an agent-based model dynamic models of segregation an agent-based model of the spatial distribution and density of the santa cruz island fox. integrated population biology and modeling part b undergraduate module on computational modeling: introducing modeling the cane toad invasion why outbreaks like coronavirus spread exponentially, and how to "flatten the curve phenotypic transition maps of 3d breast acini obtained by imaging-guided agent-based modeling 29+ evidences for macroevolution intrinsic and extrinsic noise of gene expression in lineage trees agent-based modeling of multicell morphogenic processes during development agent-based modeling in public health: current applications and future directions learning by modeling: insights from an agent-based model of university-industry relationships intelligent machinery a heretical theory statistical methods in neutron diffusion ulam s (1950) random processes and transformations simulating tissue mechanics with agent-based models: concepts, perspectives and some novel results perspective: dimensions of the scientific method press) von neumann j, morgenstern o (1944) theory of games and economic behaviour multi-scale modeling in morphogenesis: a critical analysis of the cellular potts model teaching students to think like scientists: software enables experiments to be conducted that would be impossible in a laboratory simulating cancer growth with multiscale agent-based modeling building mathematical models and biological insights in an introductory biology course an introduction to agent-based modeling: modeling natural, social, and engineers complex systems with netlogo agent-based modeling: an introduction and primer model reduction for agent-based social simulation: coarse-graining a civil violence model publisher's note springer nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations key: cord-346920-3wvo7fs3 authors: bajželj, bojana; quested, thomas e.; röös, elin; swannell, richard p.j. title: the role of reducing food waste for resilient food systems date: 2020-07-31 journal: ecosyst serv doi: 10.1016/j.ecoser.2020.101140 sha: doc_id: 346920 cord_uid: 3wvo7fs3 food waste undermines long-term resilience of the global food system by aggravating ecosystem damage. the global community must therefore work to reduce the amount of food that gets wasted. however, we should be mindful of some potential conflicts between food waste reduction and food system resilience. over-production and over-supply are a contributing cause of waste, yet they also provide resilience in the form of redundancy. in this paper, we examine individual interventions designed to minimise food waste by scoring their impact on different aspects of resilience. we find that there are strong synergistic elements and interventions that support shortand long-term resilience, such as improved storage, which reduces the need to provide a constant flow of ‘surplus food’ and replaces it with a stock of ‘spare’ food. some interventions carry a risk of trade-offs due to possible losses of redundancy, and investment lock-in that may reduce the ability of farmers to adapt by changing what and where they farm. trade-offs do not mean that those interventions should not be pursuit, but they should be recognised so that can be adequately addressed with complimentary actions. this review underlines the necessity of food-systems thinking and joined-up policy. food systems around the world face multiple challenges, and multiple objectives as response to those challenges. two of such separate objectives are (i) to increase the resilience of food system -the ability of the food system to withstand and to recover from shocks, (sustainable development goal 2.4), and (ii) to reduce food waste in the food system (sustainable development goal 12.3). the underlying reason for improving resilience is to ensure disturbances do not reduce food security, and the underlying reason for reducing food waste is to improve the environmental sustainability of food production. there are a few possible interpretations of the relationship between 'sustainability' and 'resilience' (roostaie et al., 2019) . the two concepts can be seen as one and the same, or largely overlapping (maleksaeidi and karami, 2013) . tendall et al. (2015) see food system resilience as a pre-requisite to its sustainability, as food systems cannot 'sustainably' continue provisioning food unless they are able to overcome disturbances. however, due to the magnitude of the impacts that food systems have on the environment and ecosystem services that support the food production itself (foley et al., 2005 , costanza et al., 2014 we argue that food system sustainability (aimed at reducing those impacts) is also a prerequisite for long-term food system resilience. this paper therefore takes the view that sustainability and resilience are separate concepts, but the achievement of one is largely required to achieve the other. but herein lies a tension: while food sustainability and resilience are pre-requisites of each-other, some of their building blocks are opposing. the concept of redundancy for example, which is considered as one of key principles of resilience (biggs et al., 2012; tendall et al., 2015) , can in some manifestations be in conflict with increasing efficiency, including reductions of waste, which are key strategies to achieve food sustainability (godfray and garnett, 2014; bajželj et al., 2014) . how can we still achieve the goals of resilience and sustainability simultaneously? are there food waste reduction interventions that are synergetic with resilience that we could prioritise, and others that are opposing and that we should therefore de-prioritise or re-consider? what trade-offs should we be mindful of? to answer these questions, this papers sets out to analyse linkages between food waste reductions and food system resilience. t food waste reduction, impacts on other areas of concern -in this case the resilience of the overall food system. with this paper, we aim to cover all major stages of the supply chain, and develop concepts broad and universal enough to cover different geographies, traditional and modern food systems, low income and high-income settings. often, food systems span across different geographies and income settings and modes of production (for example, farmers in kenya growing green beans for markets in the european union along with subsistence crops). while the need for more resilient food system is most acutely felt in the low-income setting, the opportunities to improve both food waste and resilience exist across all types of food system, its stages and geographies. while the challenges of some actors in the food supply chains, e.g. small-holder farmers, are unique, many concepts apply to several stages of supply chain. for example, both a farmer and a household provider may struggle with matching food 'supply and demand'. for a farmer this may mean planting just the right amount to meet his contract without wasting seeds, fertiliser, time and energy. for a provider this may mean buying just the right amount to feed the family for the next week. food system resilience, the ability of the food system to withstand and to recover from shocks, is of increasing interest due to the increase in climatic and price variability, political instability. adding to this it the recent experience of the threats to the food system from pandemics, threatening to further increase the increasing number of undernourished people globally (fao, ifad, unicef, wfp and who, 2018) . existing definitions of food system resilience (schipanski et al., 2016; tendall et al., 2015; urruty et al., 2016; biggs et al., 2012) refer to the ability to withstand shocks and external pressures while maintaining the system's basic structure, processes, and functions, through buffering capacity, adaptability and ability to recover from disturbances. food system resilience is closely related to ecosystem services, as the production related disturbances we worry about (crop failures due to e.g. floods, droughts and disease) are often related to the failure or insufficient provision of supporting and regulating ecosystem services. the stocks of natural capital that provide these services continue to shrink (costanza et al., 2017 (costanza et al., , 2014 , driven by anthropogenic climate and global environmental change. there are multiple causes of this, but one of the largest driver is actually food production and its expansion itself (foley et al., 2005) . meaning that food production activities undermine their own resilience, which is exacerbated by over-production of food caused by much of the food being wasted. threats to food system resilience are compounded by a number of other factors, including the increasing homogenization of crops and agricultural landscapes. food resilience is also related to food security (adequate access to safe and nutritious food by all people), but here we treat them as separate concepts. for the purpose of this paper, we consider the stability and recovery from disturbances as main attributes of resilience, which is one of the important conditions to achieving stable food supply and therefore food security. other conditions, most notably sufficient social security and income levels, are additional factors needed to ensure food security. in this paper we use the term 'food waste' as a general term to refer to material which was produced with the intention to be consumed by people, but ultimately exited the food supply chain. we do not separate the terms 'food loss' (often used to describe losses earlier in the supply chain, or those related mostly to the lack of infrastructure and access to market), and the term 'food waste' (often used to describe losses later in the supply chain, or those related to the behaviour of different actors) (chaboud and daviron, 2017) , as often multiple factors -a combination of structural and behavioural ones -are at play regardless of where in the supply chain food is lost. the intervention to address food loss and food waste are often hard to distinguish as well; however, the consequences of the interventions may be different (see the discussion). the un fao estimated that roughly one-third, or about 1.3 billion tonnes per year, of the edible parts of food produced globally for human consumption is lost or wasted (gustavsson et al., 2011) . this global estimate is based on extrapolation and is in need of an update, but while it is possible that the wastage is somewhat higher or lower, it is undoubtedly significant. food waste across supply chain has been more robustly measured by a small number of individual countries, roughly arriving at similar estimates (wrap, 2019). the causes of food waste are complex and varied. in this paper, we put forward the following framework on causes of food waste. as a simplification, each instance of food waste could be explained as a 'rational' economic decision based on comparing the cost of time and materials (infrastructure, appropriate storage etc.) needed to prevent wasting food, with the value of food at risk of waste. this decisionmaking process by actors in the supply chain is however further complicated by the following issues: 1. in low-income settings, access to capital and infrastructure present barriers, for example a farmer in kenya may be aware that investing in a cooler would pay back quickly through all the milk saved from spoilage, but they may still not be able to afford to buy a cooler in the first place; see (gromko and abdurasulova, 2018) . 2. issues related to these decisions are being made on the basis of perceived costs (of both time and resources needed on one side and food on the other side), which are often different to market costs (value of food on the market), which themselves are different to true costs (costs also including externalities). people are often unaware of the cumulative financial value of the food that they waste (this is true for both consumers, as well as factory managers and even farmers (wrap, 2017)). furthermore, the financial cost of food is typically much below its true cost, i.e. one that would price in all externalities such as ghg emissions, the cost of pollution generated, the economic losses associated with biodiversity losses etc. (chen et al., 2014; national research council, 2012) . the market therefore tends to under-price and under-value food (benton and bailey, 2019) . as a consequence, the decisions that lead to food waste may be rational for that individual and the market, but are not rational at all from a societal or whole-system perspective. 3. many decisions leading to food waste may not be rational at all, as we are not very good at rationally considering everyday decisions, which are often done habitually on a subconscious level (kahneman, 2012) . it has been suggested that an oversupply of 30% over the nutritional need is desired to ensure adequate food supply (papargyropoulou et al., 2014; smil, 2004) . it has therefore been suggested, that only waste over 30% (as is likely the case in most high income countries, but not low income countries) is excessive -the rest is a natural result of the overproduction needed for food security, in other words, short-term resilience. on the other hand, a significant food waste reduction has been suggested as one of several key systemic changes that could significantly reduce the unsustainable use of ecosystem services and natural capital, including a significant reduction in ghg emission from the food system, which are key for long-term resilience. kummu et al. (2012) show the total land, water and fertiliser footprint of global food loss and waste are significant. bajželj et al. (2014) , röös et al. (2017) and springmann et al. (2018) have all shown, on the basis of modelling approaches, that low-waste food systems (food systems where current wastage rates are halved) would require significantly less land, water and reduce ghg emissions associated with the food system in the future in scenarios for 2050. according to these studies, food sustainability goals seem out of reach without a significant reduction of food waste; such findings has also led to the formulation of sdg 12.3 of halving food loss and waste. stopping the unsustainable use of natural capital is important for food system resilience in the long term, as food system resilience is already at risk from climate change and increasing scarcity of suitable agricultural land and water reserves and decline in many ecosystem services crucial for food production. however, as the concept of the 30% safety margin exemplifies, food waste reduction may run contrary to some aspects of resilience. the complex direct and indirect, shortterm and long-term relationships between resilience and food waste reduction have not yet been explored. in this paper we examine the main linkages, positive and negative, between low waste and high resilience food systems. a significant reduction of food waste seems essential to establish long-term resilience, but could lead to short-term trade-offs. we therefore also examine individual interventions aimed at food waste reduction through the lens of food system resilience. we discuss in more detail the interventions that appeared most synergistic for both food system objectives, and suggest how policy could benefit. we studied the literature that describe the principles, policies, strategies and characteristics to achieve either food waste reduction or increased resilience, and mapped reinforcing and opposing linkages between these two food system objectives. the literature describes different 'characteristics' or 'principles' of a resilient food system. for food waste, the literature mostly discusses 'policies' (e.g. food waste measurements and reporting), and environmental and social 'costs' of the current food waste levels. in this paper, we suggest a novel approach to group and differentiate these concepts in a way that facilitates identifying linkages between the two objectives. we propose a separation between: 1. interventions, which are defined as concrete, practical mechanisms to achieve fostering conditions. for example, putting irrigation in place is a resilience intervention that provides stability. provision of dryers improves overall storage capacity and is an example of a food waste reduction intervention. 2. fostering conditions are conditions that will make reaching desired food system objectives more likely (some of which we can directly influence by policy or interventions); 3. objectives are key characteristics that describe the status of the food system that were the overall goal of the interventions; and 4. other outcomes are those that are also likely to occur as a consequence of the reaching the objective that are of concern for sustainability or resilience (desired or undesired). we reviewed a wide range of academic and grey literature to characterise food waste reduction interventions, fostering conditions, objectives and outcomes, all described below. food waste reduction interventions were defined as specific activities and changes to the food system that lead to less food being lost or wasted across the supply chain. these include changes to infrastructure and operations alongside changes that target human behaviour (e.g. through training or campaigns) along the supply chain. these need to be specific enough to have a clear mechanism through which they work. to ensure we were comprehensive, we searched in scopus using the following combinations of terms: either "food waste" or "food loss" and either "intervention" or "behaviour change" for any year. a range of articles related to food waste policy were also reviewed. the majority of articles related to the household consumption stage. we augmented the academic literature review with case studies from grey literature and media. fostering conditions were one of the following: broader system characteristics that multiple interventions aimed to achieve; broader policy context; or a reversal of the key drivers for waste, drawn from the same literature as above, most importantly from the following papers and reports: parfitt et al., (2010) , quested et al. (2013) , papargyropoulou et al. (2014) , hlpe (2014), schanes et al. (2018) , wrap (2017) and flanagan et al. (2019) . these agree that higher awareness of food waste as an issue and widespread measurement and reporting are the basic condition to reduce the levels of food waste across all stages of the supply chain. some of these authors and organisation however also emphasise that this in itself in not enough. better storage capacity/infrastructure as well as reduced rate of spoilage (either through improved conditions or skills) of food when it is either in storage and transit, are commonly mentioned. matching of supply (production) and demand, both in quantity, quality and time is another broad condition that would prevent waste in most stages of supply chain (either through planning, forecasting or communication). increasing the value of food (e.g. with priced-in externalities) or cost of wasting is highlighted by fao (2018) and benton and bailey (2019) . the objectives of a low waste food system were defined as a significant reduction in the flow of food material leaving the food system, and a significant increase in the ratio between food consumed and food grown. other outcomes. according to economic theory (rutten, 2013) , reducing food waste may lead to two outcomes (providing we avoid shifting waste from one stage of supply chain to another): (i) increase food consumption or (ii) reduced food production. in the absence of other changes in the food system, the occurrence of one over the other depends on the elasticity of supply and demand, through a downward pressure on food prices. a combination of both may also occur. policy can be used to encourage one of these outcomes over the other. both of these outcomes have consequences for resilience, through different mechanisms. where food security is an issue, increased consumption of (nutritious) foods can be a positive development and directly improve resilience. elsewhere, and when it comes to less nutritious foods, it would be more beneficial from a sustainability perspective to steer the system towards reducing over-production (rather than increasing consumption) and therefore reducing the use of resources including land, water, fertiliser, pesticides and seeds, reducing ghg emissions and pollution and maximising ecosystem services. an additional reduction in ghg emissions and pollutions comes from reduced waste management requirements (e.g. less waste in landfill). however, reduced overproduction also means that there is a reduced surplus flow of food in the system. we characterised food system resilience based on the following literature: biggs et al., 2012; ingram, 2011; schipanski et al., 2016; tendall et al., 2015; urruty et al., 2016; . from these papers, we pooled together what authors described as characteristics, principles or mechanisms of food system resilience, and categorised them according to the conceptual framework designed here. resilience interventions -concrete and specific activities implemented to improve food production that recovers quickly from disruptions -were taken as examples from the literature listed above. unlike for food waste we did not attempt to provide an exhaustive list of the interventions, as the main focus of this paper is to examine the role of food waste reduction on resilience (not vice-versa; although we also touch on that in the initial step -conceptual mapping). as fostering conditions for resilience we considered: • stability, mainly reduced likelihood and magnitude of disruption events themselves, such as extreme weather events leading to crop failures, and the ongoing increase in mean temperatures affecting crops, livestock, pest and pollinators in various ways, water scarcity, pest and disease outbreaks (ipcc, 2019), and also shocks to supply chains including disrupted access to labour and markets, as the ongoing (2020) disruptions to the food systems due to the new corona virus epidemic exemplifies. • diversity of crops, livestock, production systems, inputs, landscapes, income streams, customers and suppliers increases resilience according to biggs et al., 2012 and schipanski et al., 2016) . the principle of resilience in diversity is that individual crops, communities or products will carry different responses to the disruptive condition. diversity of crops and landscape may also for example prevent or slow down the spread of pest and disease outbreaks (schipanski et al., 2016 ). • redundancy, which can mean either interchangeability (according to biggs et al. (2012) , however we will consider this under flexibility) or spare capacity (of food itself, but also land, water, income, inputs (e.g. fertilisers and fuels), supporting and regulating ecosystem services), according to tendall et al. (2015) . spare food capacity emerges as a critical issue with apparent trade-offs between resilience and low waste/efficiency. • flexibility and interchangeability, being able to use the food that you have for several purposes (biggs et al., 2012) , swap one food for another, or derive income in different ways from different sources. e.g. pigs were traditionally valued as domestic animals that could take a variety of different feeds, but modern livestock systems use breeds and systems that are specialised, increasing efficiency at the expense of flexibility. today food supply chains are quite inflexible through pursuit of efficiency. inflexibility (e.g. strict cosmetic specification, strict contracting in time and quantity) can also lead to waste on farm levels. • adaptability is related to flexibility, but refers to the relative ease of making significant changes, i.e. adaptation to the way food is produced and procured. for example, farmers changing crops, calendars, locations. transformational (i.e. abrupt) adaptation may be needed due to the pace of climate change and change in weather patterns -not just step-wise adaptation to the signals as they come, but a pre-emptive, fundamental shift in practices and locations . adaptation also includes technical innovations such as more resilient breeds. • connectivity in food system includes trade, communication along the supply chain, as well as the connection between different kinds b. bajželj, et al. ecosystem services 45 (2020) 101140 of ecosystem services, for example the proximity of pollinator habitats to crops requiring pollination (biggs et al., 2012) . the downside of high connectivity is that diseases and other disturbances can spread faster. the objectives are defined as stable supply in the face of disruption and quick recovery from disruption. other outcomes. improved food security and stable food prices are two of the outcomes that are desired from a resilient food system. however, redundancy, flexibility and diversity may lead to reduced efficiency. diversity typically reduces economies of scale associated with specialisation (compare a large farm growing few crops in rotation with specialised equipment to a farm that grow many crops), while redundancy may lead to not all food produced being eaten. we mapped the interlinkages between the objectives, outcomes, and fostering conditions of the two desired system states on a long-term food systems level, by laying them out and examining where the causes and interactions (both synergetic and opposing) might occur between the systems, and represented them graphically on fig. 1 using a similar technique also used to produce causal loop diagrams (e.g. foresight, 2015) . to further untangle potential synergies and trade-offs, and move from abstract to concrete examples, we assessed how individual food waste interventions influence short-term resilience, as represented by fostering condition of the resilience (as described above in conceptual framework). the list of food waste interventions and resilience conditions were obtained from the literature as explained above. we grouped the interventions by different stages of the supply chain in three tables: primary production, then grouping together processing, manufacturing, distribution, retail and food service, and finally the consumer stage. in the tables we considered how a large-scale implementation of each intervention would impact on food system resilience, looking at each fostering condition at the time. three of the four co-authors, giving a good spread of expertise across food waste along the supply chain, and food system resilience, scored the influences individually and independently, by posing themselves the question: would the system be more or less resilient after the intervention in question, and through what mechanisms. scores were there compared, and we had a discussion, focusing particularly on any scores that diverged, agreeing the final scores. we scored the impact with a '+' if the intervention would impact positively on that fostering condition for resilience, a '-' if it was negative, a '0′ if it had little or no effect and a '+/−' if it had an mixed effect. if we all felt that the effect was unclear, we marked it with a '?'. in regard to stability, as we have already established that successful food waste prevention will have positive effects on climate stability, and the supply of ecosystem goods and services including water, we have focused on more short-term aspects such as their effect on stable supply and demand (otherwise all interventions would be scored positively for stability). 3.1. linkages between food waste reduction and food system resilience on a system level fig. 1 shows the interlinkages between the objectives, outcomes, and fostering conditions of the two desired system states. solid lines show reinforcing interactions, and dashed lines show opposing interactions. the magnitudes of the links are difficult to quantify, although it is clear that some of the links are more significant and more direct than others. one link that has been quantified in the literature is between the reduced over-production and reduced ghg emissions, land and water use. bajželj et al. (2014) and springmann et al. (2018) for example quantified these for hypothetical, global future scenarios, and most recently by philippidis et al. (2019) for current situation in the european union. through this analysis we concluded that food waste reduction is essential for long-term food system resilience (through substantial reduction of resource use and ghg emissions, both of which are critical for long-term resilience), but that the issue of 'redundancy' was a hotspot where both trade-offs and synergies between food waste reduction and resilience in the short-term may occur. this is further illustrated with fig. 2 . actors in the food chain cannot exactly predict how much food they will produce and/or require. under-supply typically has graver shortterm consequences compared to over-supply, leading to actors, who want to achieve resilience, systemically over-producing and over-supplying. high levels of waste are, in part, a symptom of such resiliencemotivated over-production, at the expense of long-term resilience, as it depletes natural capital, reduces supply of ecosystem services and contributes to de-stabilisation of climate. another direct link between food waste reduction and resilience occurs when increased food consumption is needed and positively affects consumers, directly increasing food security. in these cases, food waste reduction (most often through prevention of spoilage) directly improves resilience. food waste reduction can also help with food affordability if prices rise. this is partially what we understand happened in the uk between 2007 and 2012, when the food waste reduction information campaign (love food hate waste) in combination with economic downturn and rising food prices, helped reduce household food waste by estimated 24% whilst also helping people to save money and maintain the quality of their food purchases (britton et al., 2014; wrap, 2019) . we can assume a similar effect in low-income settings as it follows from economic theory (rutten, 2013) . however, we lack empirical data to test this hypothesis. table 1 shows the result of assessing individual interventions in primary production, focused mostly on farms with some interventions that also apply to other types of primary production, e.g. fishing. primary production is a hotspot for both waste and resilience, as it is exposed to two sources of variability and uncertainty: 1) the variability of natural systems and growing conditions and therefore yields and timing, and 2) the variability of the social system, reflected in dashed line signifies that over-production is often a result of seeking resilience. b. bajželj, et al. ecosystem services 45 (2020) 101140 table 1 the interactions between food waste reduction interventions and resilience at primary production level. for definitions and description of the resilience conditions, see the methodology section. '+' marks positive impact of the intervention on that fostering condition for resilience, '-' a negative impact, '0′ little or no effect, '+/−' mixed effect, and '?' if effects are unclear (bradford et al., 2018; cbi, 2014; gitonga et al., 2013; morante et al., 2010; neff et al., 2015; springer et al., 2013) . fluctuations in the demand quantities, prices on the market and demand timing. some of the interventions are aimed at reducing the variability (e.g. robust varieties), or help farmers match the levels of production and demand (forecasting) or bridge them in time (storage). these interventions generally help reduce both food waste and improve resilience, provided that they are done well and are resilient themselves (for example, storage could be sensitive to disturbances e.g. floods or disruptions in energy supply). overall, we have found that even in the short-term, most food waste interventions at primary production level have positive, synergetic influences on various aspects of resilience, particularly stability and flexibility, and a few also for connectivity. the implementation of these farm-level interventions, particularly if aimed at small-holders, will require provision of financing, equipment and training. as expected, redundancy was one aspect for which the scores were mixed. some interventions, mostly those directly or indirectly related to storage and food longevity, were scored as positive, while others that reduce the amount of surplus without increasing longevity scored negatively. interventions that reduce over-production without compensating for redundancy in another way may pose some risk in reducing short-term resilience, but may still be worthwhile doing to improve long term resilience. for farmers, losses of crops due to something unexpected can be catastrophic for their business and livelihood. the surplus has comparatively small effect, even if wasted (feeback, 2016; johnson et al. 2012) . it is therefore farmer's need for resilience that leads to this type of 'systemic over-production' and waste. however, this 'need for redundancy' could be reduced if the risks associated with variability were more evenly distributed along the supply chain (e.g. by wholesalers and retailers giving more flexibility to their suppliers), though improved insurance, forecasting and storage. interventions that directly target resilience, such as increasing income diversity and improving water supply, can also remove some of the farmer's need to over-plant and therefore reduce waste. adaptability was the other aspect that could be negatively affected by food waste interventions, when the intervention created a lock-in to certain location and crop. for example, a construction of silo or an immobile drying unit can further lock the farmer to a specific crop and location, depending on how crop-specific and mobile the intervention is. we have found that the food waste interventions seemed neutral for the diversity of crops, breeds and foods (and only linked to landscape diversity in the long-term through land resource savings). we grouped the intermediate stages in the supply chain because some of the interventions are common, for example those that refer to a seller and buyer relationship. another reason is that, compared to primary production and the end-consumer, the rest of supply chain receives relatively little coverage in the academic literature and grey literature, and therefore the number of suggested interventions and case studies is relatively low. similarly, existing food resilience literature mainly considers primary production as the stage that is most exposed to natural variability, or consumers and their availability to access safe and nutritious food. many interactions were given a neutral score, especially for diversity and adaptability. we did not find as much synergistic potential with improving stability as we did for the primary production. however, connectivity stood out as the resilience condition that would benefit from many food waste interventions in the middle of the supply chain. similar to primary production, some food waste interventions were scored as positive, some negative and some as mixed for redundancy. interventions targeted at household waste have the highest potential when it comes to freeing-up resources and reducing food waste related ghg emissions, and therefore, long-term resilience through stability, for two reasons: in high and middle-income settings, the volume of waste is the largest at the consumption stage, and secondly, this food has accumulated more impact as it processed through the supply chain (for example, it has been transported, stored, perhaps processed and pre-prepared). there are many different interventions targeted at households. they broadly fall in two categories: a) behavioural interventions -promoting behaviours that lead to lower wastage of food; aimed at householders themselves, such as storing and using up leftover food, including incentives to reduce overbuying, improved understanding of date labels, correct portioning when cooking, use of leftovers, optimal storage (including keeping fridges at a temperature below 5 degrees celsius), freezing of food that will not be used in time, and use of long shelf life products (canned and dried foods) (quested and luzecka, 2014; reynolds et al., 2019; schanes et al., 2018) b) technical interventions -changes to products themselves, for example a change in packaging that increases shelf-life and therefore increases the likelihood that the food will get eaten before it spoils, including changes in pack-sizes and pricing structures to discourage over-buying; increasing shelf-life and open-life through innovative packing, changes to food itself (e.g. using different bacteria cultures in cheese or yoghurt), or reducing the time it takes to get food to market (lee et al., 2015) ; changing the date labelling to avoid overly-cautious discarding of food; improving storage and freezing guidance on packs (wrap et al., 2017) . consumer behaviour can affect the stability of demand and cause spikes and dips in food demand. in the uk for example, foods such as burgers, strawberries and lettuce are typical foods that will sell very well in sunny weather and spike around sporting events, whilst demand plummets in rainy weather. growers and producers aim to meet surges in demand, however if expected demand does not materialize produce remains in the field (wrap, 2017) or is wasted elsewhere. some of this waste could be prevented with better forecasting, however the consumption habits could also adapt; for example, by aiming to match consumption to the growing season. most interventions investigated were scored positively for improving stability. the scores were mixed again for redundancy and also more mixed for flexibility and adaptability, with some interventions potentially reducing the number of options households have in any given moment when it comes to food. there were more links with diversity than in other stages of supply chain -as some interventions promote specific types of foods, typically long-life products, which either add or reduce diversity, but very little identified impact on connectivity. key interventions that emerged as synergistic for food waste and are improved storage, transport, surplus valorisation, increased shelf life, and promotion of long shelf life products. food storage can take place in many stages of the supply chain and can take many forms, for example grain stores, which can be operated from national to village or individual farm scales. food is stored in many other ways as well (not necessarily labelled as storage): on farms, in warehouses, cold stores and industrial freezers, in supermarkets, while in transport on ships and trucks, and also individual households' cupboards, fridges and freezers. each one of these stages adds up to the overall storage capacity of the food system, and each could potentially be improved. (see tables 1-3 for examples) . preparing and storing food optimally creates a buffering capacity different to a continuous surplus production by creating a reserve. in systems dynamics terminology, better storage replaces a need for a constant additional 'flow' with increasing the 'stock' (from which occasionally an additional flow can be drawn). for example, families could rely more on having well-stocked cupboards with long-life cupboard essentials that can also be made into a nutritious meal, rather than constantly buy more perishable foods than they use. surplus production could be diverted to become a reserve by extending shelf life through canning and dehydration of fruit, meat, dairy and vegetable products, to be used in times of scarcity and disruptions in food supply due to a variety of different reasons from droughts to epidemics. this would be a much more resource-efficient way of providing resilience compared to over-production year on year. improved storage is not necessarily about only the size of storage (warehouse, fridge etc.), but table 2 the interactions between food waste reduction interventions and resilience at intermediate steps in the food supply chain (blanke, 2014; wrap, 2013a; cohen et al., 2014; dora et al., 2019; freedman and brochado, 2010; kallbekken and saelen, 2013; parfitt et al., 2016; refed, 2016; stöckli et al., 2018; wansink and van ittersum, 2013; williamson et al., 2016; wrap, 2013b) . the interactions between food waste reduction interventions and resilience at consumer level (devaney and davies, 2017; fisher and whittaker, 2018; ganglbauer et al., 2013) . conditions (e.g. temperature, air moisture) and preparation of food stored (drying, pre-cooling, mould-free) . it should be noted that there are some challenges associated with having a higher proportion of food as reserves. stock management in both the supply chain and in homes may need to be improved to ensure that the food in storage is circulated correctly. secondly, on a foodsystem basis, having a larger proportion of overall food in storage may mean that on average, the time between harvest and consumption increases, highlighting the need to monitor not only food safety, but also food quality and nutritional value. thirdly, there are trade-offs between increasing some types of storage capacity, especially cooling and freezing, and energy use -increased energy use leading to increases in ghg emissions and cost. and lastly, the resilience of storage against same of different kinds of disturbances that effect food production should also be considered (e.g. energy supply, susceptibility to pests etc.). improved storage should reduce price volatility. at harvest time, especially in good years, the price of produce (e.g. grains, vegetables) on the market is low, but farmers without safe storage options are forced to sell despite the low prices they receive. similarly, consumers without storage or access to food with longer shelf-life cannot bulk-buy and store foods when prices are low. if neither producers of consumers can store, prices are more likely to spike outside main harvest seasons. higher prices in one year can attract many newcomer producers in the next season, leading to over-production the next year, therefore low prices and high waste when price does not even justify harvest costs (which are significant, johnson et al. 2012 ) perpetuating a boom and bust cycle. price volatility is therefore not only a sign of a system with low resilience, but it can also lead to waste. in a market economy, changing prices are unavoidable, and are valuable signals about scarcity and over-production, and distorting them through some market mechanism may lead to unintended consequences. however improved storage, preparation of food for storage, diversification into longer-life products and innovation in insurance could reduce some of the high demand volatility and make optimal production decisions easier for farmers and other actors in the food supply chain. reducing food waste in most cases leads to a good return on investment (wrap, 2015; hanson and mitchell, 2017; gromko and abdurasulova, 2018) , improving the profitability and income of the people involved in the point of the stage of supply chain where food waste is being reduced. as such, food waste reduction leads to increased resilience of that actor in the chain, say a farmer or manufacturer. this also applies to consumers. for example, data in the uk (britton et al., 2014) suggests that in the context of reducing disposable income and rising food prices, people that reduced the food that they wasted were able to continue buying the same quality of food they were buying before. reduced food waste can also present an opportunity to increase added value in the supply chain. when customers save money but not over-buying in quantity, they are often happy to spend more on 'quality' (britton et al., 2014) . however, assuming food waste reduction at consumer level does lead to financial savings, these could result in rebound effects if they are spent on other polluting activities, diminishing some of the environmental gains (martinez-sanchez et al., 2016; salemdeeb et al., 2017) . in this paper we focused on reducing food waste 'at source' (i.e. food waste prevention). however, there are other mitigating actions that also count as food waste reduction: for example, feeding the surplus food material or food scraps to livestock (zu ermgassen et al., 2016) and using it as feedstocks in bio-economy, for example extracting limonene from waste orange peel (wrap, 2015) . these 'surplus valorisation activities' too can lead to significant resource savings, although typically less than prevention. zu ermgassen et al. (2016) for example calculated that feeding food scraps to pigs through advanced centralised swill operations similar to those operating in japan and south korea could also lead to significant land-use savings of 1.8 million hectares in europe, compared to 6.0 million hectares calculated for food waste prevention by philippidis et al. (2019) . they can also potentially contribute to increased resilience if the flows of material out of the food system can be used to feed humans during times of crisis, and should be pursued in addition to food waste prevention to reduce pressure on resources. some surprising linkages between food waste and resilience emerged through our investigation. some food waste can be seen as a consequence of actors wanting to achieve resilience through redundancy: over-production and over-purchasing, so that they call on this surplus if something unexpected happens; these 'unexpected events' could be a grave issue such as a farmer experiencing a low yield, or relatively small things like consumers entertaining unexpected guests or not finding the time to go do your weekly shop at the regular time. however, some wasted food did not provide resilience at any point in time. generally, if the 'food at risk of becoming waste' can be tapped into relatively quickly (for example, by relaxing cosmetic standards), then that quantity does provide redundancy and therefore some shortterm resilience. but if there are no means of preventing the spoilage and waste, then waste food never provided any resilience. some of the types of food waste that provide short-term resilience are also some of the easiest to address to reduce over-production. interventions such as relaxing cosmetic standards and reducing overpurchasing through better planning are examples of changes that can be implemented fairly easily, and therefore also get implemented when something unexpected occurs, i.e. in time of scarcity. for example, in 2012 untypical weather resulted in poor yields of fruits and vegetables, in response to which uk's supermarkets relaxed their rules on their cosmetic appearance (vidal, 2012) , evening the supply. the relative ease of intervention implementation provided redundancy. however 'saving' these interventions for times of scarcity comes with cost a of diminishing natural capital and contribution to climate change from over-production. therefore, the recommendation of the authors is not that the interventions that reduce redundancy are not pursued (they could be considered as a priority due to their ease of implementation), but that they are balanced with complementary interventions that compensate for lost redundancy, most notably improved storage. this investigation of food waste and resilience led us to a new proposal to conceptualise wasted food, depending on whether it represents mainly a) a loss of needed nutrition (a direct loss of food security) or b) a loss of resources used to produce it (leading to indirect, long-term loss of resilience). in practice it may be difficult to allocate a loss of an individual item of wasted food between the two, as the mechanisms and causes of loss may be identical, but the consequences are not. they also depend on the context, particularly the socio-economic status of the actor. as actors improve their socio-economic status, they often seek to improve their food security through increasing over-production, rather than through reducing food loss though interventions, for a variety of different reasons. therefore, the nutrition loss shifts to resource loss, even though the causes and mechanisms (the actual process of food becoming inedible) may remain unchanged. the short-term resilience of the actor improves, but the long-term resilience of the whole food systems diminishes through increased depletion of natural capital and reduced stability. as progress is made towards halving food waste, it would be helpful to track the nutrition loss and the resource loss (over-production) separately at a large/global scale. for this, better production and consumption statistics would be needed. to complement food waste reduction, and also to encourage it, policy should carefully consider which of the two outcomes of reduced food waste are preferable for the circumstances: reduced production or increased consumption. at national food policy levels, food waste reduction targets are rarely linked to other outcomes. (if anything, they are vaguely linked to improving import-export balance). this lack of systemic food policy represents a missed opportunity to strengthen other desirable food system transitions, including increased short-term or long-term resilience. long-term resilience could be doubly-encouraged for example, by gradual internalisation of externalities so that food price would be stable despite downwards pressure from food waste reduction (benton and bailey, 2019; rutten, 2013) . this should lead to reduction of over-production, and therefore reduction of ghg emissions and increase in other ecosystem services that can be provided by spared land. the delivery of specific ecosystem services, many of which can again be conductive to long-term resilience, can be then further encouraged with compatible financial incentives, and would, through competition for resources, further reduce food over-production. the opportunities include: farmers being able to build in more resilienceinducing features that protect soils, pollinators and other resources on the spared land (for example: buffer zones, hedgerows, stony habitats, pollinator habitat enhancement and lagoons, see gardner et al., 2019) ; reduced use of fertiliser (and its negative impacts) not only through reduced production but also reduced fertiliser subsidies; water reservoirs and aquifers re-filling (or being used less intensely), increasing the resilience of water supply in the future; healthier ecosystems on and surrounding the agricultural lands. reduction in food loss and waste combined with an ecosystem services approach can therefore steer the food system to a more resilient and sustainable future. on the other hand, in circumstance where an increase in consumption is preferred to directly improve food security, prices can be allowed to decrease through food waste reduction interventions. this is particularly appropriate for highly nutritious foods that are underconsumed in the population. however, an increase in consumption is not helpful in all circumstances and for all types of food. particularly for food with high energy and poor nutrient values amongst populations with an obesity problem, without taking a systemic approach pursuing food waste reduction in isolation may contribute to exacerbating the problems. the lack of systemic approach also reduces the chances of food waste reduction incentives succeeding. the first signal from a significant reduction of food waste at one point in the supply chain at either demand or supply side is bound to be a reduction in price (rutten, 2013) . this can encourage waste at another stage in supply chain, or at another geography. for this reason, it is important that food waste reduction is being pursued on a global scale and in all stages of supply chain. good monitoring systems are needed to be put in place to ensure that waste is not pushed from one end of the supply chain to another, and from one place to another. global monitoring frameworks are currently being set up by un fao and unep, but the challenges associated with the monitoring of food waste should not be underestimated. in the future, however, it would be beneficial to monitor other separate indices at critical points in transition to high-resilience, low waste system, particularly the total storage capacity and overproduction as indicators of food system resilience and efficiency. this could mean for example an introduction of food reserves targets and monitoring on national, business or even household level (% of total food reserves, % of households with access to safe food storage options in kg/capita). the discourse on food waste for the past ten years have been quite simplistic: food waste is big, bad, and it must be reduced. this was justifiable given the low awareness surrounding food waste, its scale and missed opportunities it embodies. now that awareness has increased, at least amongst policy makers, researchers and businesses (flanagan et al., 2019) , it is time we enter the next stage in food waste policy and discourse: one that is more nuanced and realistic, and tackles both synergies and trade-offs with other food system sustainability goals more explicitly, achieving better outcomes for all goals. in the eu, food waste reduction measures on farm are currently not considered as a part of circular economy package (european commission, 2019), however it could be something that is encouraged through the common agricultural policy. common agricultural policy has a potential to increase farm resilience by encouraging resource efficiency, establishment of resilience features and encourage supporting and regulating ecosystem services. the parallel pursuit of food waste reduction should help free up the resources needed to do this, without sacrificing productivity. in this paper, we used a theoretical approach to try to structure and characterise complex interactions between different parts of and changes to the food system, focusing interactions between interventions aimed at food waste and resilience. this approach was largely based on the experiences and knowledge of the experts involved. where possible we tried to base the judgements on potential causes and effects on observed data; however, this was not possible for all identified linkages. furthermore, while we strived to be comprehensive by taking a systemic approach, it is possible that some important linkages were missed entirely, which could be improved in the future by involving a wider range of stakeholders in the system mapping process. nonetheless, we believe this approach and its conclusions have value, and will hopefully provide a basis for future research on these questions fundamental to food sustainability. there are strong linkages between reducing food waste and food system resilience, that through the analysis in this paper we separated into: a) long-term linkages: food waste prevention interventions influence long-term resilience of the food system though its sustainability, by reducing ghg emissions and therefore climate change and variability, and potential to increase natural capital and improve supporting and regulating ecosystem services. these interactions are overwhelmingly positive for resilience and significant in strength and scale. b) short term-linkages where food waste interventions interact with stability, diversity, redundancy, flexibility, adaptability and connectivity of the system through interventions that lead to changes in livelihoods, storage capacities, variability in supply and demand etc. these interactions can be positive or negative. they form two clusters: one around redundancy -where trade-offs are most apparent, and one around stability and reduced volatility (of supply, demand and price), where synergies are most apparent. the scale and strength of these linkages are not yet known. individual food waste prevention interventions that focus on reducing over-production, for example the relaxation of cosmetic standards, do reduce redundancy and therefore short-term resilience. however, these interventions should not be discouraged as they present a vital opportunity to improve resilience in the long-term. the trade-offs should be recognised so that they can be adequately addressed and compensated for. one example is by improved storage, which by reducing the variability of supply and demand, smoothing prices provides redundancy not in the form of surplus, but a reserve. policy recommendations and implications of this work are the following: 1. replace surplus with reserves. current approach to food resilience relies on over-production. this is exemplified by a notion that 30% of food over-supply is required for adequate food supply and security. however, such reliance on over-production undermines food system resilience in the long-term. the approach to food system resilience needs to shift from over-production to reserves (storage and extension of shelf life e.g. though dehydration), with accompanying storage-related incentives and targets. food waste reduction should be easier to achieve when it is recognised that for many actors, the need to provide resilience is the driver for over-production and therefore waste. the actors need to be reassured that the food waste reduction activity is going to improve rather than reduce their resilience. 3. increasing resilience can indirectly reduce food waste. overproduction for the sake of resilience can be replaced through other resilience conditions, e.g. through increased diversity, by installing irrigation or alternative producer income opportunities related to other ecosystem services. for that purpose, it is important to have an understanding of ways to support resilience alternative to redundancy. 4. approach food policy systemically. food waste reduction policy should consider two of its possible outcomes: reduced production and increased consumption, and actively steer towards the one that is considered more desirable depending a range of circumstances ranging from food resilience, security, public health, state of the environment and ecosystem services. food waste reduction would both facilitate and be facilitated by pricing-in of externalities into food production as well as diversifying the income from other ecosystem services. reducing food waste on its own is less likely to succeed and result in positive food system changes, but such combination of strategies can steer the food system to a more resilient and sustainable future. food and agricultural policy should not overlook this opportunity. the covid-19 epidemic (ongoing at the time of writing) highlights the importance of food system resilience, as it is disrupting food supply chains in many different ways though e.g. order cancelations by the entire hospitality sector, creation of supply chain choke-points (such as the closure of slaughter-houses in the us), harvest-labour shortages (in india and europe). all of this seems to be leading to a large increase in waste food -so not only does waste lead to lower resilience, low resilience may also leads to waste. the amount of food that is wasted instead of being supplied to people is particularly tragic, as at the same time, people are also becoming increasingly food insecure primarily due to slumps in incomes (world food programme, 2020). the interventions that we recommended in this paper, such as improved storage options and strategic well-managed stocks of long-life foods at household, business and regional/national level would help to alleviate such pressures. tom quested, richard swannell and bojana bajzelj are associated with wrap, a uk-based charity with a longstanding history of working on food waste reduction. no other potential conflict of interest is declared. importance of food-demand management for climate mitigation the paradox of productivity: agricultural productivity promotes food system inefficiency toward principles for enhancing the resilience of ecosystem services reducing ethylene levels along the food supply chain: a key to reducing food waste? the dry chain: reducing postharvest losses and improving food safety in humid climates econometric modelling and household food waste product competitiveness for dried mangoes food losses and waste: navigating the inconsistencies assessment of agri-environmental externalities at regional levels in finland impact of the new u.s. department of agriculture school meal standards on food selection, consumption, and waste changes in the global value of ecosystem services twenty years of ecosystem services: how far have we come and how far do we still need to go? disrupting household food consumption through experimental homelabs: outcomes, connections, contexts the added value of a food systems approach in policy brief strategic working group on food systems importance of sustainable operations in food loss: evidence from the belgian food processing industry report on the implementation of the circular economy action plan (com(2019) 190 final), archives of the roentgen ray the future of food and agriculture alternative pathways to 2050. licence: cc by-nc-sa 3.0 igo rome, fao building climate resilience for food security and nutrition 10 causes of food waste in international supply chain opportunities to reduce waste along the journey of milk, from dairy to home reducing food loss and waste: setting a global action agenda global consequences of land use tackling obesities: future choices-obesity system atlas reducing portion size reduces food intake and plate waste negotiating food waste 2019. agricultural resilience: perspectives from ecology and economics impact of metal silos on households' maize storage, storage losses and food security: an application of a propensity score matching food security and sustainable intensification climate change mitigation and food loss and waste reduction: exploring the business case global food losses and food waste food losses and waste in the context of sustainable food systems the business case for reducing food loss and waste a food systems approach to researching food security and its interactions with global environmental change climate change and land: an ipcc special report on climate change, desertification, land degradation, sustainable land management, food security, and greenhouse gas fluxes in terrestrial ecosystems field measurement in vegetable crops indicates need for reevaluation of on-farm food loss estimates in north america thinking, fast and slow nudging" hotel guests to reduce food waste as a win-win environmental measure lost food, wasted resources: global food supply chain losses and their impacts on freshwater, cropland, and fertiliser use reducing food waste by extending product life social-ecological resilience and sustainable agriculture under water scarcity life-cycle costing of food waste management in denmark: importance of indirect effects tolerance to postharvest physiological deterioration in cassava roots reducing food loss and waste while improving the public's health. health aff the food waste hierarchy as a framework for the management of food surplus and food waste food waste within food supply chains: quantification and potential for change to 2050 quantification of food surplus, waste and related materials in the grocery supply chain waste not, want not: a bioeconomic impact assessment of household food waste reductions in the eu household food and drink waste: a people focus spaghetti soup: the complex world of food waste behaviours a roadmap to reduce u.s. food waste by 20 percent consumptionstage food waste reduction interventions -what works and how to design better interventions greedy or needy? land use and climate impacts of food in 2050 under different livestock futures integrated sustainability and resilience assessment framework: from theory to practice what economic theory tells us about the impacts of reducing food losses and/or waste: implications for research, policy and practice. agric. food secur. 2 a holistic approach to the environmental evaluation of food waste prevention. waste manage food waste matters -a systematic review of household food waste practices and their policy implications realizing resilient food systems improving efficiency and reducing waste in our food system losses in the field: an opportunity ripe for harvesting options for keeping the food system within environmental limits call for testing interventions to prevent consumer food waste food system resilience: defining the concept food systems and natural resources. a report of the working group on food systems of the international resource panel stability, robustness, vulnerability and resilience of agricultural systems. a review transformation in practice: a review of empirical cases of transformational adaptation in agriculture under climate change ugly" fruit and veg make the grade on uk supermarket shelves | environment | the guardian portion size me: plate-size induced consumption norms and win-win solutions for reducing food intake and waste of waste and waists: the effect of plate material on food consumption and waste covid-19 will double number of people facing food crises unless swift action is taken understanding out of home consumer food waste food waste in primary production -a preliminary study on strawberries and lettuces. banbury, uk. wrap, 2019. food surplus and waste in the uk. banbury, uk. wrap, fsa, defra, 2017. labelling guidance key: cord-322511-hnvqvajx authors: speerin, robyn; needs, christopher; chua, jason; woodhouse, linda j.; nordin, margareta; mcglasson, rhona; briggs, andrew m. title: implementing models of care for musculoskeletal conditions in health systems to support value-based care date: 2020-07-25 journal: best pract res clin rheumatol doi: 10.1016/j.berh.2020.101548 sha: doc_id: 322511 cord_uid: hnvqvajx models of care (mocs), and their local models of service delivery, for people with musculoskeletal conditions are becoming an acceptable way of supporting effective implementation of value-based care. mocs can support the quadruple aim of value-based care through providing people with musculoskeletal disease improved access to health services, better health outcomes and satisfactory experience of their healthcare; ensure the health professionals involved are experiencing satisfaction in delivering such care and health system resources are better utilised. implementation of mocs is relevant at the levels of clinical practice (micro), service delivery organisations (meso) and health system (macro) levels. the development, implementation and evaluation of mocs has evolved over the last decade to more purposively engage people with lived experience of their condition, to operationalise the chronic care model and to employ innovative solutions. this paper explores how mocs have evolved and are supporting the delivery of value-based care in health systems. models of care (mocs), and their local models of service delivery, for people with musculoskeletal conditions are becoming an acceptable way of supporting effective implementation of valuebased care. mocs can support the quadruple aim of value-based care through providing people with musculoskeletal disease improved access to health services, better health outcomes and satisfactory experience of their healthcare; ensure the health professionals involved are experiencing satisfaction in delivering such care and health system resources are better utilised. implementation of mocs is relevant at the levels of clinical practice (micro), service delivery organisations (meso) and health system (macro) levels. the development, implementation and evaluation of mocs has evolved over the last decade to more purposively value-based care was first discussed by berwick in 2008 as a triple aim of measuring health outcomes, improved patient experience and better use of health resources [21] . porter added to the concept by including health professionals' experiences, seen as an important factor in achieving valuebased care. value-based care can be viewed as a continuum from high to low. 'high-value' care refers to care for which evidence suggests it confers benefit on patients, or probability of benefit exceeds possible harm, or, more broadly, the added costs of the intervention provides proportional added benefits relative to alternatives [22] . are mocs supporting the shift to value-based care across the quadruple aim? does the evolution of mocs for people living with musculoskeletal conditions support the achievement of the quadruple aim? the paper will reflect on the changing attitudes towards healthcare by all stakeholders in the health system. consideration of some of the workforce requirements to deliver the health outcomes will be discussed, along with progress of consumer and clinical experiences. are better health outcomes being achieved? evolution of contemporary models of care for musculoskeletal conditions mocs were borne from frustration that traditional clinical pathways developed from evidence-informed healthcare were not being implemented for individuals, let alone across health settings and systems, resulting in unacceptable care variation and inequity in access to care [23e25] . evolving population health states, changing health requirements and expectations of the patient and their families, and changing approaches to care, mandated that a different approach to care planning, delivery and participation be taken. mocs were conceived to combine best practice healthcare and other services that are important to the person, population group or patient cohort across the continuum of a condition, injury or health event. mocs aim to ensure people get the right care, at the right time, by the right team, in the right place, with the right resources [26e28]. mocs for long-term conditions represent a shift from episodic provision of care to meet crisis situations, to care delivery across the disease continuum and life course [29, 30] . the chronic care approach and the biopsychosocial model while the chronic care model proposed by wagner et al. in the 1990s [31] and the biopsychosocial model proposed by engel in 1977 [32] are not new, the acceptance and integration of these personcentred and multidimensional approaches into care planning and delivery continues to evolve beyond the historic reductionist biomedical model. for the majority of long-term musculoskeletal conditions, application of the reductionist biomedical model has not been effective in improving outcomes [33e35] . thus, the chronic care approach and the biopsychosocial model are now central foci for mocs for long-term conditions. they are particularly relevant to musculoskeletal mocs, where target conditions are typically life-long and associated with persistent pain and functional impairment, and require interaction with a range of health services over decades from primary care to specialist medical and surgical services, even if intermittent. in chronic care, self-management support remains a fundamental component of effective care. operationalising these approaches within mocs requires consideration of the person's lived experience (incorporating biological, psychological and social sequelae), person-centred health outcomes and their experiences of care. recent debate in chronic pain care, for example, suggests that the biopsychosocial model of pain care is inadequately fit for purpose and needs to evolve further towards a sociopsychobiological moc [34] . the inclusion of the person's lived experience of a condition(s), including the psychosocial sequelae, is now accepted as fundamental to designing, implementing and evaluating mocs. in order to understand and listen to those who know the condition best e the person living with the condition and their family/carer e several elements have been added to the development, delivery and evaluation of mocs in recent years [18] . this a clear shift from mocs developed prior to the past decade or so. this shift has seen health teams, organisations and systems working to incorporate these concepts within musculoskeletal mocs through the use of multi-stakeholder networks, such as those in western australia and new south wales, australia (see https://ww2.health.wa.gov.au/articles/j_m/ musculoskeletal-health-network and https://www.aci.health.nsw.gov.au/networks/musculoskeletal). while efforts were made in the past to include the lived experience, recent thinking and evidence has revealed that an even higher level of partnership between stakeholders is required to develop health services that meet the needs of those who access them as recipients of care [36] . therefore, methodologies, such as clinical redesign [37] , shared decision-making [38] and co-design [30] , have been used in recent years to facilitate this in-depth level of partnership. incorporating advice borne from experience can richly inform 'what' and 'how' care should be delivered in a local system. with the voice of the lived experience augmenting clinical experience and healthcare evidence concerning a particular condition, the resulting care is more likely to be acceptable to all involved and better reflect care that is high value [39e42]. critically, satisfaction of receivers of care is more likely to be associated with effective and active participation in their healthcare requirements [43, 44] . measuring what matters to people: patient-reported measures patient-reported measures (prms) have evolved as a mechanism to report on and incorporate the patient voice in mocs. prms enable evaluation of healthcare, and can inform higher value-oriented changes to health services and health systems [45, 46] . prms are commonly classified as two separate outcome measures: patient-reported outcome measures (proms) and patient-reported experience measures (prems). studies have shown that inclusion of proms enhances treatment decisions, patient satisfaction and subsequent adherence to agreed treatments [47, 48] . systematic review evidence points to the likely benefit of using proms in healthcare [49] . prems indicate the patient's perspective on issues, such as access to care, how they were approached and included in their treatment decisions [49e51]. while satisfaction surveys have long been a part of mocs, prems are relatively new with few tools to support their inclusion in clinical practice with local teams often developing their own. proms have the ability to identify issues that may not be elucidated during clinical assessments. there is appreciation now that background psychological issues, such as depression, anxiety and an individual's health beliefs and expectations, may impact upon recovery and their participation in care. this has led to specific proms, such as those that assess important person-centred impacts of living with a condition (e.g. in the management of people with back pain [52] ) and others that focus more on generic issues that occur across healthcare needs, inclusive of social requirements [53] . as such, proms have the dual capability of informing clinicians as to the whole of health impact of disease while at the same time providing a conduit via which previously unstated patient concerns may be addressed. proms and prems are important components to the quadruple aim of value-based care. innovation in development and implementation of models of care and service delivery as our understanding of the requisite components and practices for developing mocs and mosds evolve, innovation in development practices similarly evolves. like many other countries, early and appropriate conservative care for osteoarthritis (oa) remains underutilised in new zealand (nz). currently, nz has an outcomes framework for service development and commissioning for long-term conditions [54] , including specific strategies for diabetes [55] and healthy ageing [56] . however, no frameworks or policies specifically exist for musculoskeletal healthcare. in response to this gap, in 2015, the nz government allocated nzd 6 million over 3 years to trial and evaluate local healthcare programmes that aimed to improve access to early community-based interventions that provided contemporary clinical interventions and self-management support of people with musculoskeletal conditions e the mobility action programme' [57] . an additional $44 million (new zealand $) was allocated to address access issues to joint replacement surgery [58] . the aim of the mobility action programme is to trial, evaluate and commission effective and sustainable programmes for broader dissemination across nz in the future. why a co-design approach was needed in new zealand for an oa model of service delivery despite these investments, a national framework to align the health system with best practice recommendations for musculoskeletal conditions does not exist prompting a call to action for an oa moc for nz [59] . furthermore, there is currently no guidance for health service delivery for decision-makers, such as planners, coordinators or funders, about which interventions for oa offer the greatest perceived value in the nz health system. for oa, this decision can be particularly challenging given that there are many interventions to choose or recommend in a national-level service model, and because implementing recommended oa care is typically complex and influenced by many interdependent barriers and facilitators across the health system [12, 60] . for these reasons, adopting the principle of co-design with stakeholders from across the sector in reform efforts is essential, including the perspectives of people with a lived experience of the condition and vulnerable groups, to ensure appropriate consideration of context' and fit' [18, 36] . strong evidence points towards incorporating context, that is, the environmental conditions which influence the barriers and enablers of implementation [61] ) into the decision to adopt or commission an intervention for implementation [12, 60] . in particular, establishing the fit' of established or emerging interventions within the nz context could help to enhance implementation of oa care by more closely aligning intervention performance with what stakeholders want. alignment of recommendations for oa care will potentially reduce healthcare waste [36] . for example, interventions that align closely with decision-makers' decisionmaking criteria, such as intervention cost, accessibility and effectiveness, and evidence about interventions' performances on these criteria could enhance implementation efforts and better align with system policy priorities. developing national recommendations for oa care is a complex endeavour and should ideally represent the views and opinions of the people most relevant to oa care in a particular context. in nz, this context includes not only people across the community living with oa, but also specifically m aori healthcare advocates. they need to work in tandem with healthcare providers, policy-makers and oa research and clinical experts working in the various care settings. however, these eclectic stakeholders typically make complex decisions involving many considerations, or criteria, which often compete. weighing up these different criteria to reach a decision should ideally occur through a transparent, trustworthy and fair process. approaches for reaching consensus include, for example, verbal agreement, delphi surveys, nominal group technique and consensus development panels (e.g. deliberative dialogues) [36] . some limitations of these methods include: (1) decision-making criteria may not be explicit and decisions may be made without appropriate time for reflection; (2) decision-makers' preferences or values are typically implicit; (3) new evidence, ideas or alternative choices may invalidate previously reached consensus and (4) engagement can be limited, particularly among those with a lived experience of a health condition. multi-criteria decision analysis (mcda) is a systematic, transparent and fair approach to decision-making that can address these important considerations [62] . mcda structures decision-making by incorporating subjective and objective data in a systematic and transparent process that identifies and weighs multiple evaluation criteria in order to prioritise different healthcare interventions, policy options or alternatives [63] . this structured, systematic approach is a defining characteristic of mcda and helps to overcome mental shortcuts (often employed in complex decisions, e.g. gut feeling') which can lead to systematic mistakes, poor decisions, and ultimately, poor decision-making credibility [64] . the advantages of mcda include: i) explicit evaluation criteria enforces transparency, accountability and fairness; ii) scalability e the ability to prioritise new or emerging interventions without capturing stakeholders' preferences for these interventions each time and iii) efficiencies in design and deployment streamline decision-making allow for broader and more meaningful engagement with a diverse range of stakeholders. in recent years, mcda has become widespread in healthcare research [65, 66] . for example, it has been used to explore the preferences of people with oa concerning physical activity [67] , patients' preferences for the use of medicines [36] , healthcare providers' treatment choices for people with oa [68] and policy-makers' and clinical decision-makers' preferences for intervention choice [69] . it has not been applied previously to inform a cross-sector, consensusbased mosd for people with oa. how has mcda been applied in the nz context for oa care? through a qualitative study in 2016e2017, multidisciplinary and cross-sectoral stakeholders in nz (people with the lived experience of oa, healthcare providers, policymakers and oa clinical and research experts) identified nine criteria for selecting or recommending oa interventions in the nz public health system: accessibility, active versus passive interventions, appropriateness, cost, duration, effectiveness, quality of evidence, recommendation and risk of harm [70] . criteria were organised according to the guidance for mcda [71] , for example, selecting nonredundant and non-overlapping criteria. the criteria were then categorised into levels' describing their performance. for example, the criterion effectiveness' was categorised into high, moderate and low levels of performance using the standardised mean difference. choicebased surveys were used to quantify people's preferences for the criteria, that is, their relative weights. interventions were then rated on the performance levels of the criteria, sourced from clinical guidelines [72] , local data and a nationally representative panel of experts in oa management in nz. the final step involved combining the preference weights and intervention ratings together to calculate a total performance score for each intervention. digital and electronic health solutions digital health systems now offer unprecedented capabilities to health systems and consumers in data collection, delivery of information and access to health services. access is enhanced through realtime access to care (e.g. through telehealth) and digitally based tailored care (e.g. through adaptive mhealth platforms). the healthcare opportunities offered by digital systems and the ubiquitous use of digital platforms globally, such as mobile phones, rationalise digital health systems as a key strategy for health system strengthening [73] . these technologies have appeal, particularly for younger people [74] , and also provide opportunities to close care disparity gaps that exist due to geography and socioeconomic circumstance, and allow for electronic recording of care access and delivery over time. while there has been an uptake in telehealth and mhealth solutions for some chronic conditions in the past decade, including opportunities for musculoskeletal and pain care [75] , system-wide adoption in musculoskeletal care outside hospitals has been slow, despite promising opportunities. many reasons can be attributable to the slow uptake, in particular the major gap between innovation and testing and appropriate scale-up into systems [76] . there is an opportunity to close innovation-adoption gaps by better integrating digital solutions into the design of mocs. in the case of telehealth, one reason is the belief that physical clinical assessment is paramount to diagnosing and monitoring a musculoskeletal condition. perhaps this will change following the covid-19 pandemic, which forced clinicians to adopt these tools as part of routine care. in the case of electronic medical recording, the required allocation of significant resources in settings (out of hospital acute care) that have traditionally not been seen as important is limiting uptake. however, these systems can add value to better utilisation of clinical time, ease of recording of prms and greatly assist in evaluation efforts [77] . how well are models of care accepted in health systems and their services? it has long been accepted and expected that a health system uses structures and processes to create an integrated care environment that is appropriate for the population it serves [78, 79] . mocs, when developed as described in this paper, have been a fundamental means of enabling this to happen in some jurisdictions and care settings [80, 81] . for example, national health policies for nonfindings from this activity could be used to inform recommendations in a national mosd for oa, which outlines those interventions that offer the greatest perceived value to nz stakeholders. the mcda process enabled the views and perspectives of all stakeholder groups, particularly people living with oa and m aori advocates, to be respected and equally considered. the development, implementation and evaluation of mocs for musculoskeletal conditions requires equal partnerships between those with lived experience of the disease/condition and their carers, health professionals across all disciplines, service resource personnel, researchers and policy-makers in jurisdictions. enhanced decision-making methods, such as multi-criteria decision analysis, can be used to inform more robust and trustworthy health policy decision-making through broader and more inclusive engagement of people with lived experience; this could help policy-makers identify better value-based care options. there is an urgent need for further uptake of digital technologies to support implementation of mocs. communicable diseases refer to the need to develop and implement mocs to drive health system strengthening [82] . the national health service in the united kingdom is a good example where mocs have been the main drivers of change including in the development of strategy to turn attention to value-based healthcare [83, 84] . acceptability of mocs by stakeholders from across the health system in australia was validated in a large qualitative study [25] . informants identified the value of mocs in translating evidence to policy and practice and supporting system-wide health transformation. implementing models of care to support value-based care when mocs are developed as described in this paper and a quality improvement cycle is used, such as an established framework for evaluation [18] , they can provide clear evidence for health systems and policy-makers when making decisions regarding equitable use of resources that will optimise health system outcomes across the quadruple aim for value-based care. low back pain, identified as the single greatest contributor to the global burden of disability since 1990 [1] represents an important focus for health systems to realign to deliver value-based care. currently, health systems deliver too much low-value care and too little high-value care for low back pain. recent reviews have identified key challenges for health systems in delivery of high-value care for low back pain and pain care in general [33, 34, 85, 86] . these include the financial interests of pharmaceutical and other companies; inflexible payment systems that favour medical care over patients' self-management; and deep-rooted biomedical traditions and beliefs about pain among physicians and the community at large. authors have argued for system-level reform strategies, such as the implementation of mocs, to shift resources from unnecessary, low-to high-value care and that such endeavours could be cost-neutral and have widespread impact. evidence is building concerning whether mocs can support value-based care. health systems are now using mocs to inform strategy concerning value-based care [87, 88] . for example, musculoskeletal mocs for people living with osteoarthritis and those experiencing fragility fractures are included in the suite of services for value-based care initiatives in australia [89] , while in canada, a national approach to hip fracture care has been developed [90] . the new south wales health system in australia is now into the fourth year of formally implementing value-based care, with mocs remaining at the forefront of decision-making on each tranche of implementation [91, 92] . multidisciplinary team-based care as an enabler to implementing musculoskeletal models of care healthcare transformation, designed to improve access and quality of care while containing cost, is a worldwide priority [93e95], aligned with value-based care and enabled through mocs. many countries have responded by enabling existing healthcare professionals to work to their maximum scope (advanced scope of practice [96e101], which has also been articulated as an enabler to implementing mocs [102] . historically, this approach has worked to address shortages in available healthcare practitioners in remote and rural areas and on the front line to triage injured soldiers during wartime. more recently, advanced practice roles have been implemented as part of new interprofessional models of team-based musculoskeletal care. these innovative mocs require close collaboration and communication among healthcare providers, integration across care sectors (private/ public, hospital/primary/community based) and funding models that enable and promote evidencebased care across the full continuum of care [103] . while people with musculoskeletal conditions access a variety of medical and surgical specialties, there is a need for other non-medical health professionals to be involved in order to access high-value care; for example, supporting the person's understanding of and addressing issues, such as psychological needs, co-morbidities, weight loss and physical activity in osteoarthritis care. nurses and allied health professionals, such as physiotherapists, dieticians and others, are key members of a true multidisciplinary musculoskeletal care team. not surprisingly, the high cost and volume of people, who require improved access to musculoskeletal care in the primary care sector, has resulted in the emergence of the new inter-professional service models inclusive of health professionals with advanced scope of practice skills such as nurse practitioners and physiotherapists who have had advanced training in musculoskeletal healthcare [98,100,104e108] . as demand for musculoskeletal health services increase, mocs will increasingly recommend alternate and innovative workforce models to ensure timely access to care and high-value service delivery. in high-income settings, these examples of new cadres who are educated and authorised to function autonomously and collaboratively in advanced and extended clinical roles may help to achieve some of the quadruple aims of value-based care. these may include reducing wait times in emergency departments, triaging to surgical consult for total knee or hip joint replacement, re-fracture prevention assessments and investigation, and performing as the lead health professional in supporting people presenting with acute low back pain [109e121]. ongoing evaluation of clinical, economic and patient experience outcomes will be important to maintain momentum in optimising workforce configurations. in lower resourced settings, building capacity among appropriately skilled and trained community health workers will be important to implementing mocs and avoiding catastrophic out of pocket expenditure for citizens, such as integrated care services for older people [122] and communitybased spine care [123] . an important aspect of value-based care and involvement of multidisciplinary teams is health professional experience. to understand their experience, there is a real need to develop a standardised way of measuring elements that are meaningful to health professionals in the delivery of care. an example is the national health service in the uk that could be developed for use across jurisdictional borders. see at: https://www.nhsstaffsurveys.com/caches/files/st19_core%20questionnaire_final_ 20190705.pdf. as the acceptance of mocs as levers for system reform becomes more widespread, there will be an increasing need for implementation support within health systems and sharing of experiences. implementation support can occur at different levels: from local support for an mosd, to sub-national, to national support for system wide implementation efforts, through to global-level support for multinational implementation. along this continuum, the level and focus of support may vary from comprehensive local support (e.g. organisational site visits) through to principles or guiding actions for countries to work towards and self-evaluate. local and sub-national implementation support initiatives typically involve assisting organisations across a health system to implement an moc through an operational-level mosd, usually adapted to the local context. for example, in new south wales, australia since 2009, there has been increasing support for implementation of several mocs for the healthcare needs of people with musculoskeletal conditions, including osteoarthritis, secondary fragility fracture prevention, hip fracture care, acute low back pain, and children and adolescents living with rheumatologic conditions. this has been achieved initially through a clinical network working with government in supporting local organisations to develop business cases for implementing the mocs and the provision of seed funds to enable pilot implementation initiatives with common evaluation frameworks. implementation scale-up across the there is an urgent need to develop a tool that measures health professional experience across the domains of job satisfaction, burnout, how adverse events are handled, how engagement up and down the hierarchy occurs, their ability to provide care that incorporates all the impacting issues that a person presents for care is experiencing across biopsychosocial areas, and their capacity to influence change within the health service. funding and implementation of team-based mocs that allow for local adaptation in rural and remote as well as urban areas that can be scaled and spread are essential. ongoing evaluation of advanced practice nursing and allied health professional mocs will be important to maintain and optimise workforce configurations. system was achieved through integration of the mocs for osteoarthritis and secondary fragility fracture prevention within a larger whole-of-system reform initiative e the 'leading better value care' programme: https://www.health.nsw.gov.au/value/lbvc/pages/default.aspx [89] . the south west england mocs support network is an example of an online community of practice formed to support implementation of mocs across primary care in the south west of england and beyond (https://modelsofcare.co.uk/). multiple other sub-national implementation support programmes also likely exist, although currently no repository exists to share implementation experiences. international efforts to support the implementation of mocs and mosds across countries focus on specific service models or offer guidance that is transferable across conditions. examples of specific service models include: the global fragility fracture network (https://www.fragilityfracturenetwork.org/) supports implementation of best practice and localised mocs for people with fragility fractures across the continuum of care through formal education opportunities and peer mentoring. this includes efforts for health professionals working with the patient cohort at presentation with fracture to peri-operative care, to rehabilitation and to secondary fracture prevention. the good life with osteoarthritis in denmark (gla:dâ®) (https://www.glaid.dk/english.html), now implemented in denmark, canada, china and australia, with further countries joining (switzerland, new zealand and austria) [124] . the mosd provides specific implementation guidance for physiotherapist-led exercise and education for people with hip and/or knee osteoarthritis. the international osteoporosis foundation capture the fractureâ® programme (https://www. capturethefracture.org/) [125] facilitates the implementation of coordinated, multi-disciplinary service models for secondary fragility fracture prevention. the programme offers multiple implementation resources to support countries implement the programme and evaluate the scale of implementation achieved (https://www.capturethefracture.org/resources). the global spine care initiative (https://www.globalspinecareinitiative.org/) [123] supports the implementation of local spine care services in low-and middle-income countries. it is based on eight principles that consider implementation from the micro to macro contexts. the clinical and health economic outcomes of the model are yet to be established. a framework (the 'framework'; fig. 2 ) to support implementation of any musculoskeletal moc was developed in 2016, empirically derived with input from 93 stakeholders across 30 countries [126] . the framework guides those tasked with implementation in a health system to consider and evaluate the following domains: -implementation readiness -best practice approaches to support implementation -evaluation. the framework is now endorsed by 54 peak international organisations and has been used internationally to support development, implementation and evaluation of mocs (see fig. 2 ). the majority of public-facing mocs for musculoskeletal conditions have been developed and supported for implementation in high-income countries. while there are some initiatives occurring in low-and middle-income countries [127, 128] , there remains a need to support development and implementation of musculoskeletal mocs in low-and middle-income countries that supports, rather than threatens, fragile health systems [129, 130] . recent research, for example, has confirmed the suitability of the chronic care model for low-and middle-income settings, but identified that some adaptation and expansion is required for low-resource settings [131] . the who integrated care for older people (icope) model supports countries at all stages of maturity to consider actions required to realign health systems towards the needs of older people, including their mobility and musculoskeletal health [122] . implementation tools are now available to support the implementation of icope internationally in health services and health systems [132] . systematic reviews have reported a range of factors that have been identified as barriers and enablers to implementation of mocs in health services and health systems [12,133e135] . as outlined in the framework (fig. 2) , consideration of these factors and others that are relevant to the local context are essential in approaching any implementation effort. system-level mocs and their operational-level mosds are one of many tools that support the translation of evidence into system (macro)-level (e.g. policy), service (meso)-level (e.g. programme implementation) and clinical (micro)-level (e.g. practice) changes [14] . however, all too often evidence translation initiatives end with short-term and non-recurrent resourcing, resulting in multiple shortterm trial reforms, programmes or pilots. these then often lead to reform fatigue among those tasked with implementation and monitoring [137] . on this background, appropriate evaluation of mocs is an essential component of supporting long-term implementation and sustained reform and quality improvements efforts in health systems. local system evaluation is critical [138] , recognising that outcomes between settings may not necessarily be comparable; for example, between high-and lowincome economies, or even at the sub-national level between jurisdictions with geographic differences that can impede access to services and/or expert services. evaluation of mocs must be pragmatic, flexible to a dynamic health system, respectful of local contextual issues and meaningful to intended end users. the parameters of evaluation should be codesigned with decision-makers and end users to ensure the outcomes are meaningful, useable and contemporary [139] . the perspectives of people with lived experience of a health condition are also critical to ensure meaningful concepts are measured [36] . evaluation approaches need to consider design and methods, target levels of the health system and outcomes. while 'effectiveness' evaluation (research) remains essential to advancing health innovation, identifying high-value care and minimising health waste, this mode of evaluation may be less relevant and feasible at the stage of implementing mocs in health systems. by definition, the components of an moc should be evidence based [14] ; suggesting that evaluation of effectiveness is less of a priority for the downstream evidence translation phase of implementation. the necessary structured and rigid design of gold standard effectiveness research designs, such as the randomised controlled trial (rct), may collide with the dynamic nature of health systems and health services and inadequately reflect outcomes of the broader population health state. in particular, rcts may often exclude people with multi-morbid health states, which now reflect the norm rather than a subgroup [140] . researchers are it is timely to now consider multi-morbidity in models of care (mocs), given the prevalence of multi-morbidity of non-communicable diseases with musculoskeletal conditions commonly being prevalent. evolution of future mocs needs to more explicitly consider multi-morbidity and how condition-specific or multi-morbidity mocs can support delivery of high-value care. while there are some examples in recent years to support the development and implementation of some musculoskeletal mocs in low-and middle-income countries, for example, fragility fracture and spine care, there is a dire need to adapt and support implementation of moc in a manner that is suitable for fragile health systems. more research is required concerning health and economic outcomes achievable through implementation of mocs in order to drive further health system improvements globally. the approach proposed by jessup and colleagues is a good starting point [136] . starting to apply more innovative trial designs to deal with this challenge and focus specifically on implementation trial designs [141] , although more experience and work is required to apply such methods across services or across a system. the incompatibility between rcts and health systems research is particularly relevant in the context of mocs with a strong ehealth component [142] , where evaluation approaches other than effectiveness designs are recommended and supported by guiding frameworks [76,143e146] . these evolving frameworks could feasibly be applied beyond ehealth, with many of the guiding principles already reflected in a framework developed to specifically guide evaluation of musculoskeletal mocs [126] . collectively, the framework advocates for continuous cycles of mixed-methods evaluation (or formative evaluations), aligned with a process evaluation approach to iteratively understand how implementation could be optimised in a given context and be responsive to changing circumstances [147] , before initiating a summative (impact) evaluation. the framework also suggests that evaluation should not be based on single outcomes, but consider a multidimensional approach to defining 'success', 'performance' or 'benefit', achieved through mixed-methods approaches that consider outcomes relevant to the consumer (e.g. proms and prems) and the health system [126, 145] . the importance of emphasising qualitative research exploring stakeholders' perceptions and attitudes towards implementation feasibility, acceptability and sustainability is widely proposed [36, 148] . evaluation of mocs or downstream mosds can be targeted at different levels of the health system. an evaluation hierarchy, or pyramid, has been proposed, which suggests evaluation can be targeted at one or more of three levels (see fig. 3 ) [14] : 1) specific components of an moc or mosd (e.g. a specific clinical behaviour, work cadre or self-management strategy); 2) at the programme level (e.g. where a specific health improvement programme is implemented, such as the gla:dâ® programme [124] or capture the fractureâ® initiative [125] ) or 3) at the whole-of-system level (e.g. the system wide impact of implementing an moc or multiple service models across a whole system such as the who icope approach [122] ). most implementation and evaluation efforts occur at the base of the pyramid, while the least occurs at the system level [149, 150] , likely reflecting the challenges associated with whole-of-system evaluation endeavours. evaluation at the system level, however, remains of critical importance and underscores the need for appropriate health surveillance infrastructure and systems [33, 82, 122, 151] , particularly in low-and middle-income countries [130] . as implementation of mocs evolve and evaluation systems become more sophisticated, the need for a pyramid inversion will become greater. indeed, many jurisdictions are now emphasising the need to reorient evaluation towards a whole-ofsystem lens in order to build a better picture of overall system functioning and performance against reform targets, inclusive of an approach that integrates patient, clinician and system performance outcomes [89, 152] . while system-and service-level integration of proms and prems is challenging, as identified in a recent systematic review of barriers to implementation [153] , recent data from new south wales, australia, point to the feasibility of measuring proms across a system though electronic platforms [154] . in order to achieve integrated measurement of patient and clinical outcomes (micro level), service-level (meso) outcomes and system-level (macro) outcomes, co-design with end users of the evaluation (typically policy-makers, health administrators/mangers and funders) is needed on a background of a lens to healthcare planning and delivery that prioritises 'value' over 'volume' [89, 139] . infrastructure and governance to enable consistent and systematic measurement is essential and enabled through registries to capture proms and prems, agreed performance indicators for health services [155, 156] and linked data systems [89] . in the context of mocs specifically, the recent evaluation framework guides measurement of 'success' of a musculoskeletal moc in a health system and recommends a range of outcome domains should be considered (fig. 2) [18] . this paper has revealed that in the past decade, many positive influences have supported the concept of utilising mocs to support value-based musculoskeletal care. the evidence is clear that the development, implementation and evaluation of mocs must rely more on the involvement of those with the lived experience. their involvement must be in collaboration with those who provide care, their managers and funders. collectively, the advice from those intimately involved whether through personal experience of musculoskeletal diseases or conditions, or clinical practice, or research of the conditions, or through management and funding leads to all elements of the quadruple aim being achieved, as described by porter in 2010 [19] . however, challenges remain that hinder widespread implementation in many areas of the globe, with many being directly attributable to some specific embedded practices [85,157e159] . fig. 4 summarises challenges commonly experienced at the various levels of the health system. for mocs to be even more successful in guiding clinical practice global strategies such as choosing wisely need to be encouraged and supported by governments internationally [160e162]. in low-to middle-income countries, there is a policy swing that now acknowledges the contribution from non-communicable diseases to the burden on population health [163] . for example, osteoporotic fractures in asia are predicted to be higher than seen elsewhere in the next decade [164] . future mocs for healthcare will need to be incorporated into government policy concerning housing, education and employment. the momentum for such change is evidenced by the recent us document encouraging the addition of social care to all healthcare considerations [165] . evaluation of mocs is a critical enabler to supporting sustainable implementation and resourcing of mocs. shifting from a reliance on effectiveness' evaluations to evaluation approaches that better align with multiple domains relevant to implementation is important. outcomes should be meaningful and useable to intended end users and decision-makers, informed through purposive co-design. evaluation at the system level, rather than at the programme level, may better support implementation at scale. in summary mocs are 'adopting evidence-based clinical pathways and protocols, aligning incentives, effectively managing resource, continuously monitoring and improving performance, and investing in supporting information technologies' [166] . their use is to encouraged more and more across the globe. this paper was developed without specific funding for any of the authors. the authors declare no conflicts of interest. all involved in the development, implementation and evaluation of musculoskeletal mocs should remain alert to opportunities for whole of government approaches to managing musculoskeletal conditions such as partnering with housing, education and employment sectors. global, regional, and national incidence, prevalence, and years lived with disability for 354 diseases and injuries for 195 countries and territories, 1990-2017: a systematic analysis for the global burden of disease study lost productive life years caused by chronic conditions in australians aged 45-64 years effective treatment options for musculoskeletal pain in primary care: a systematic overview of current evidence what does best practice care for musculoskeletal pain look like? eleven consistent recommendations from high-quality clinical practice guidelines: systematic review the quality of health care delivered to adults in the united states caretrack: assessing the appropriateness of health care delivery in australia prevention and treatment of low back pain: evidence, challenges, and promising directions do physical therapists follow evidence-based guidelines when managing musculoskeletal conditions? systematic review professional interventions for general practitioners on the management of musculoskeletal conditions effectiveness of multifaceted implementation strategies for the implementation of back and neck pain guidelines in health care: a systematic review factors influencing the implementation of clinical guidelines for health care professionals: a systematic meta-review achieving change in primary care-causes of the evidence to practice gap: systematic reviews of reviews physician-reported barriers to using evidence-based recommendations for low back pain in clinical practice: a systematic review and synthesis of qualitative studies using the theoretical domains framework models of care for musculoskeletal health: moving towards meaningful implementation and evaluation across conditions and care settings moving from evidence to practice: models of care for the prevention and management of musculoskeletal conditions models of care for musculoskeletal health in australia: now more than ever to drive evidence into health policy and practice american college of rheumatology/arthritis foundation guideline for the management of osteoarthritis of the hand, hip, and knee supporting the evaluation and implementation of musculoskeletal models of care: a globally informed framework for judging readiness and success what is value in health care? the quadruple aim: care, health, cost and meaning in work the triple aim: care, health, and cost levers for addressing medical underuse and overuse: achieving high-value health care factors influencing the implementation of clinical guidelines for health care professionals: a systematic meta-review applying a health network approach to translate evidence-informed policy into practice: a review and case study on musculoskeletal health models of care for musculoskeletal health: a cross-sectional qualitative study of australian stakeholders' perspectives on relevance and standardised evaluation right care, right time, right place, every time western australian government. implementation of models of care and frameworks e progress report 2015. department of health the right care, in the right place, at the right time -every time integrated care models: an overview using experience-based co-design to improve the quality of healthcare: mapping where we are now and establishing future directions. london: united kingdom: king's college organizing care for patients with chronic illness the need for a new medical model: a challenge for biomedicine system strengthening to support value-based care and healthy ageing for people with chronic pain flipping the pain care model: a sociopsychobiological approach to high-value chronic pain care is it time to reframe how we care for people with non-traumatic musculoskeletal pain? practical guidance for engaging patients in health research, treatment guidelines and regulatory processes: results of an expert group meeting organized by the world health organization (who) and the best practice & research clinical rheumatology xxx (xxxx) xxx european society for clinical and economic aspects of osteoporosis, osteoarthritis and musculoskeletal diseases (esceo) patient journeys: the process of clinical redesign interventions for increasing the use of shared decision making by healthcare professionals patients' perceived needs of health care providers for low back pain management: a systematic scoping review patients' perceived needs of osteoarthritis health information: a systematic scoping review systematic scoping review of patients' perceived needs of health services for osteoporosis patients' perceived health information needs in inflammatory arthritis: a systematic review do patients treated with dignity report higher satisfaction, adherence, and receipt of preventive care? practical guidance for engaging patients in health research, treatment guidelines and regulatory processes: results of an expert group meeting organized by the world health organization (who) and the european society for clinical and economic aspects of osteoporosis implementing patient-reported outcome measures into clinical practice across nsw: mixed methods evaluation of the first year standardizing and personalizing the treat to target (t2t) approach for rheumatoid arthritis using the patient-reported outcomes measurement information system (promis): baseline findings on patient-centred treatment priorities patient-reported outcomes in ra care improve patient communication, decisionmaking, satisfaction and confidence: qualitative results the use of patient reported outcome measures (proms) in clinical assessment a systematic review of randomised controlled trials evaluating the use of patientreported outcome measures (proms) treat-to-target in rheumatoid arthritis: evaluating the patient perspective using the patient opinion real-time anonymous liaison system: the ra t2t portal study patient-reported outcomes use during orthopaedic surgery clinic visits improves the patient experience a primary care back pain screening tool: identifying patient subgroups for initial treatment performance of the patient-reported outcomes measurement information system-29 in scleroderma: a scleroderma patient-centered intervention network cohort study long-term conditions: work programme. ministry of health ministry of social development. new zealand disability strategy 2016-2026 associate minister of health. healthy ageing strategy. in: health mo. wellington: ministry of health the mobility action programme. ministry of health; [updated mapping new zealand bridging the evidence-practice gap for people with painful musculoskeletal conditions an osteoarthritis model of care should be a national priority for new zealand barriers and facilitators to implementing evidence-based interventions among third sector organisations: a systematic review fostering implementation of health services research findings into practice: a consolidated framework for advancing implementation science multiple criteria decision analysis : an integrated approach making good decisions in healthcare with multi-criteria decision analysis: the use, current research and future development of mcda multiple criteria decision analysis for health care decision making-emerging good practices: report 2 of the ispor mcda emerging good practices task force discrete choice experiments in health economics: a review of the literature discrete choice experiments in health economics: a review of the literature preferences for physical activity: a conjoint analysis involving people with chronic knee pain how do physicians weigh benefits and risks associated with treatments in patients with osteoarthritis in the united kingdom? which criteria are considered in healthcare decisions? insights from an international survey of policy and clinical decision makers choosing interventions for hip or knee osteoarthritis -what matters to stakeholders? a mixed-methods study multi-criteria decision analysis (mcda) in healthcare decision-making royal australian college of general practitioners. guideline for the management of knee and hip osteoarthritis world health organization. who guideline: recommendations on digital interventions for health system strengthening young people's experiences of persistent musculoskeletal pain, needs, gaps and perceptions about the role of digital technologies to support their co-care: a qualitative study use of ehealth technologies to enable implementation of musculoskeletal models of care: the evidence and practice beyond adoption: a new framework for theorizing and evaluating nonadoption, abandonment, and challenges to the scale-up, spread, and sustainability of health and care technologies deepening our understanding of quality in australia (duqua): an overview of a nation-wide, multi-level analysis of relationships between quality management systems and patient factors in 32 hospitals health systems integration: state of the evidence the future of health systems to 2030: a roadmap for global progress and sustainability implementing new models of care: lessons from the new care models programme in england implementation of musculoskeletal models of care in primary care settings: theory, practice, evaluation and outcomes for musculoskeletal health in high-income economies integrated prevention and management of non-communicable diseases, including musculoskeletal health: a systematic policy analysis among oecd countries developing a blueprint for the future of nhs and care services united kingdom united kingdom: south west academic health science network low back pain: a call for action care for low back pain: can health systems deliver? the value transformation framework: an approach to value-based care in federally qualified health centers acute care for elders (ace) team model of care: a clinical overview implementing value-based health care at scale: the nsw experience national hip fracture toolkit. toronto: bone and joint canada leading better value care implementing value-based health care at scale: the nsw experience canada: health system review health care spending in the united states and other high-income countries health system performance assessment landscape at the eu level: a structured synthesis of actors and actions advanced practice physiotherapy in patients with musculoskeletal disorders: a systematic review the efficacy of an extended scope physiotherapy clinic in paediatric orthopaedics a framework for the development and implementation of an advanced practice role for physiotherapists that improves access and quality of care for patients advanced physiotherapy practice: a qualitative study on the potential challenges and barriers to implementation in ghana ministry of health and long-term care. wait time strategy: better access to care. public information advanced musculoskeletal physiotherapy practice in ireland: a national survey victorian model of care for osteoarthritis of the hip and knee. melbourne: musculoskeletal australia building multidisciplinary health workforce capacity to support the implementation of integrated, people-centred models of care for musculoskeletal health a framework for the development and implementation of an advanced practice role for physiotherapists that improves access and quality of care for patients health force ontario, new roles in health care a framework for the introduction and evaluation of advanced practice nursing roles utilization of nurse practitioners to increase patient access to primary healthcare in canada-thinking outside the box an advanced clinician practitioner in arthritis care can improve access to rheumatology care in community-based practice development, implementation and evaluation of an advanced practice in continence and women's health physiotherapy model of care physiotherapists in emergency departments: responsibilities, accountability and education implementation of an expanded-scope-of-practice physiotherapist role in a regional hospital emergency department patients are satisfied with advanced practice physiotherapists in a role traditionally performed by orthopaedic surgeons the physical therapist as a musculoskeletal specialist in the emergency department concordance between physiotherapists and physicians for care of patients with musculoskeletal disorders presenting to the emergency department evaluation of performance quality of an advanced scope physiotherapy role in a hospital emergency department reducing hip and knee replacement wait times: an expanded role for physiotherapists in orthopedic surgical clinics role of the advanced practice physiotherapist in decreasing surgical wait times a model of care for osteoarthritis of the hip and knee: development of a system-wide plan for the health sector in victoria compliance to the joint commission proposed core measure set on osteoporosis-associated fracture: review of different secondary fracture prevention programs in an open medical system from screening, assessment, and treatment of osteoporosis for the nurse practitioner: key questions and answers for clinical practice-a canadian perspective advanced musculoskeletal physiotherapists are effective and safe in managing patients with acute low back pain presenting to emergency departments actions required to implement integrated care for older people in the community using the world health organization's icope approach: a global delphi consensus study the global spine care initiative: model of care and implementation gla:d to have a high-value option for patients with knee and hip arthritis across four continents: good life with osteoarthritis from denmark capture the fracture: a best practice framework and global campaign to break the fragility fracture cycle a framework to evaluate musculoskeletal models of care. cornwall: global alliance for musculoskeletal health of the bone and joint decade secondary prevention of osteoporotic fractures-an "optimal" model of care from singapore fragility fracture network (ffn)-china successfully held forum to support ffn global call to action to improve the care of people with fragility fractures a time for action: opportunities for preventing the growing burden and disability from musculoskeletal conditions in low-and middle-income countries health system strengthening is needed to respond to the burden of pain in low-and middle-income countries and to support healthy ageing models of care for chronic conditions in low/middle-income countries: a 'best fit' framework synthesis world health organization. integrated care for older people (icope) implementation framework: guidance for systems and services effectiveness of chronic care models: opportunities for improving healthcare practice and health outcomes: a systematic review facilitators and barriers of implementing the chronic care model in primary care: a systematic review best practice & research clinical rheumatology xxx (xxxx) xxx implementing models of care for musculoskeletal conditions in health systems to support value-based care understanding the attributes of implementation frameworks to guide the implementation of a model of community-based integrated health care for older adults with complex chronic conditions: a metanarrative review alternative service models for delivery of healthcare services in high-income countries: a scoping review of systematic reviews leading health system transformation to the next level estimating the excess costs for patients with incident fractures, prevalent fractures, and nonfracture osteoporosis achieving research impact through co-creation in community-based health services: literature review and case study epidemiology of multimorbidity and implications for health care, research, and medical education: a cross-sectional study oarsi clinical trials recommendations: design and conduct of implementation trials of interventions for osteoarthritis beyond the randomized controlled trial: a review of alternatives in mhealth clinical trial methods a holistic framework to improve the uptake and impact of ehealth technologies why do evaluations of ehealth programs fail? an alternative set of guiding principles digital health benefits evaluation frameworks: building the evidence to support australia's national digital health strategy understanding program evaluation. an aci framework. sydney: nsw agency for clinical innovation process evaluation of complex interventions: medical research council guidance qualitative evidence to improve guidelines and health decision-making elements of integrated care approaches for older people: a review of reviews integrated care for older populations and its implementation facilitators and barriers: a rapid scoping review models of care for musculoskeletal pain conditions: driving change to improve outcomes combining patient, clinical and system perspectives in assessing performance in healthcare: an integrated measurement framework the facilitators and barriers to implementing patient reported outcome measures in organisations delivering health related services: a systematic review of reviews implementing patient-reported outcome measures into clinical practice across nsw: mixed methods evaluation of the first year development of system-level performance measures for evaluation of models of care for inflammatory arthritis in canada development of key performance indicators to evaluate centralized intake for patients with osteoarthritis and rheumatoid arthritis a global call to action to improve the care of people with fragility fractures three steps to changing the narrative about knee osteoarthritis care: a call to action musculoskeletal health conditions represent a global threat to healthy aging: a report for the 2015 world health organization world report on ageing and health de-implementing wisely: developing the evidence base to reduce low-value care measuring 21 low-value hospital procedures: claims analysis of australian private health insurance data the effectiveness of inpatient rehabilitation after uncomplicated total hip arthroplasty: a propensity score matched cohort reducing the global burden of musculoskeletal conditions quality improvement initiatives in fragility fracture care and prevention consensus study report: integrating social care into the delivery of health care: moving upstream to improve the nation's health quality measurement and improvement in rheumatology: rheumatoid arthritis as a case study key: cord-025337-lkv75bgf authors: vakkuri, ville; kemell, kai-kristian; jantunen, marianna; abrahamsson, pekka title: “this is just a prototype”: how ethics are ignored in software startup-like environments date: 2020-05-06 journal: agile processes in software engineering and extreme programming doi: 10.1007/978-3-030-49392-9_13 sha: doc_id: 25337 cord_uid: lkv75bgf artificial intelligence (ai) solutions are becoming increasingly common in software development endeavors, and consequently exert a growing societal influence as well. due to their unique nature, ai based systems influence a wide range of stakeholders with or without their consent, and thus the development of these systems necessitates a higher degree of ethical consideration than is currently carried out in most cases. various practical examples of ai failures have also highlighted this need. however, there is only limited research on methods and tools for implementing ai ethics in software development, and we currently have little knowledge of the state of practice. in this study, we explore the state of the art in startup-like environments where majority of the ai software today gets developed. based on a multiple case study, we discuss the current state of practice and highlight issues. the cases underline the complete ignorance of ethical consideration in ai endeavors. we also outline existing good practices that can already support the implementation of ai ethics, such as documentation and error handling. ai systems have become increasingly common in software engineering projects [1] . while much of the media attention is on flashier systems such as autonomous vehicles, less high-profile ai systems such as decision-making support systems have become increasingly widespread in various organizations. ai systems often operate under the surface in the form of e.g. recommendation algorithms, making the high-profile systems in the middle of the media hype only the tip of the iceberg. over the last two decades, progress on ai has been accelerating rapidly. ai systems are now widely used in various areas and for various purposes. examples include medical systems [2] , law enforcement [3] , and manufacturing industries and industry 4.0 [4] , among numerous others. as the field progresses, the already impressive potential of ai systems becomes even larger, including applications such as general ai systems, the likes of which are already being developed by the technology giants such as alphabet. it is exactly because of this impressive potential and impact of these systems, especially in the future, that their potential negative impacts should also discussed more. ai systems are ultimately still software. they are affected by largely the same requirements as any other software system. ai development projects are still for the most part conventional software engineering, with machine learning related tasks only comprising a small portion of these projects [5] . however, ai systems are unique in terms of their effects on various stakeholders to the point where they can even exert society-wide influence. moreover, these stakeholders often have little power in opting out of using these systems. e.g. it is difficult to avoid having a firm filter your job application using ai or trying to avoid being monitored using ai-based surveillance systems if such systems are in place in the area. various system failures have already highlighted some of the potential issues these systems can have in practice. past incidents that have received global media coverage, even smaller incidents can be costly for the affected organization(s). for example, the national finnish broadcasting company, yle 1 , utilized ai for moderation purposes in its services. having already changed its processes to suit the automation of the moderation, the organization ultimately ran into problems with the ai moderator system. though the software was working fine on the technical level, the socio-ethical issues forced the organization to revert back to human moderators. many of these issues are ultimately rooted in ethics. ai ethics has thus become a new non-functional requirement to address; an -ility among the likes of quality, maintainability, and scalability. existing methods have focused on tackling these functional and non-functional requirements. however, no such methods currently exist for ai ethics [6] , with the existing tools and methods largely being technical and limited to narrow contexts in ml as opposed to being project-level methods. in the absence of methods, how are ethics currently implemented? much of the current literature in the area has been theoretical, and our understanding of the state of practice in ai ethics is currently lacking. [6] ai ethics literature discusses various aspects of ai ethics that should be taken into account, but bridging the gap between research and practice in the area remains an on-going challenge [7, 8] . guidelines for implementing ai ethics exist, but their effect on the start of practice remains unknown. thus, to begin bridging this gap in the area, we conduct an empirical study to help us understand the current state of practice. we do so by means of a multiple case study of three projects focusing on healthcare systems. the goal of this study is two-fold: (1) to help us understand the current state of practice in ai ethics; and (2) to discover existing good practices that might help in implementing ai ethics. out of these two goals, the first is a theoretical contribution while the second one is a practical one. the specific research question of the paper is as follows: rq: how are ai ethics taken into consideration in software engineering projects when they are not formally considered? ethics in software development and interactive systems design in general has a history of over 30 years. for example, bynum [9] introduced the idea of adapting human values in design before the rise of human computer interaction and other human-centric paradigms. theoretically grounded approaches such as value sensitive design (vsd) and its variants have provided tools to design technology that takes into account human values in the design process [10, 11] . as more progress is made in the field of ai systems, old theoretical scenarios in ai ethics are slowly becoming reality. this calls for new methods to manage the ethical issues arising from these new systems [7, 12] . indeed, vallach and allen [12] argue that ai and ai-based systems produce new requirements to consider. specifically, they propose that designers implicitly embed values in the technologies they create [12] . ai and other complex systems force designers to consider what kind of values are embedded in the technologies and also how the practical implementation of these values could be carried out and how these systems could be governed [13] . yet, little is currently known about software development practices and methods in the context of ai ethics, as empirical studies in the area are scarce. our results from an existing study suggest that ai ethics are seldom formally implemented in se projects, [14] . similarly, there are currently no project-level methods that could aid in implementing ai ethics [6] . on the other hand, various tools that can support specific elements of ai ethics do exist, such as tools for managing machine learning [6] . however, they do not help developers implement ai ethics in general. in this light, it can be said that ai ethics has hardly been incorporated into mainstream se literature yet. the reason why ai ethics has received little attention in the prior engineering literature is three-fold: 1) prior research has been predominantly philosophical, 2) the field has not sensed the need to address ethical concerns and 3) thus it has not been part of the education system. though some practice-focused research does exist (e.g. [15] ), most of the research on ai ethics has been conceptual and theoretical in nature. these studies have e.g. focused on defining ai ethics in a practical manner through various constructs in the form of values. for the time being, this discussion on defining ai ethics has come to center around four values: transparency [16, 17] , accountability [8, 16] , responsibility [16] , and fairness (e.g. [18] ). not all four of these values are universally agreed to form the core of ai ethics, however, as we discuss in the following section while presenting our research framework. following various real-life incidents out on the field (e.g. amazon's biased recruitment ai 2 ), ai ethics has also begun to spawn public discussion. this has led to governments, standardization institutions, and practitioner organizations reacting by producing their own demands and guidelines for involving ethics into ai development, with many standards and regulations in the works. countries such as france [19] and germany [20] have emphasized the role of ethics in ai, and on an international level the eu began to draft its own ai ethics guidelines which were presented in april 2019 [21] . moreover, iso has founded its own ethical, trustworthy ai in iso/iec jtc 1/sc 42 artificial intelligence subcommittee [22] . finally, some larger practitioner organizations have also presented their own guidelines concerning ethics in ai (e.g. google [23] and microsoft [24] guidelines). thus far, these various attempts to bring this on-going academic discussion out on the field have been primarily made in the form of guidelines and principles. out of these guidelines, perhaps the most prominent ones up until now have been the ieee guidelines for ethically aligned design (ead), born from the ieee global initiative on ethics of autonomous and intelligent systems alongside its ieee p7000™ standards working groups, which were branded under the concept of ead [8] . existing literature has shown us that guidelines and principles in the field of ict ethics do not seem to be effective. mittelstadt [25] argue that ai developers lack the professional norms and methods to translate principles into practice in successful way. to this end, mcnamara et al. [26] also argue based on empirical data that the acm ethical guidelines 3 had ultimately had very little impact on developers, who had not changed their ways of working at all. in this light, this is likely to be the case with the aforementioned ai ethics guidelines as well, as mittelstadt suggest [25] . this notion is further supported by morley et al. [6] who argue that developers focused on practicality are unlikely to adopt them when the competitive advantage of ead is unclear. to assist in the data collection and analysis in this study, we devised a research framework based on prominent literature in the area. this research framework and the justifications behind it are further discussed in an existing paper [27] (fig. 1 ). as the basis of the framework, we utilized the art principles of dignum [16] , which consist of accountability, responsibility, and transparency. these have been central constructs in the area, having also been featured in the ead guidelines of ieee. transparency is required for accountability and responsibility (line 1.c), as we must understand why the system acts in a certain fashion, as well as who made what decisions during development in order to establish accountability [17] . whereas accountability can be considered to be externally motivated, closely related but separate construct responsibility is internally motivated. the concept of accountability holds a key role in aiming to prevent misuse of ai and in supporting wellbeing through ai [8] . accountability refers to determining who is accountable or liable for the decisions made by the ai. dignum [16] in their recent works defines accountability to be the explanation and justification of one's decisions and one's actions to the relevant stakeholders. in the context of this research framework, accountability is used not only in the context of systems, but also in a more general sense. we consider, e.g., how various accountability issues (legal, social) were considered during development. dignum [16] defines responsibility as a chain of responsibility that links the actions of the systems to all the decisions made by the stakeholders. we consider it to be the least accurately defined part of the art model, and thus have taken a more comprehensive approach to it in our research framework. according to the ead guidelines, responsibility can be considered to be an attitude or a moral obligation for acting responsibly [8] a simplified way of approaching responsibility would be for a developer to ask oneself e.g. "would i be fine with using my own system?". in addition to the art principles, we utilized the three ai ethics categories presented by dignum [28] to make these constructs more practical. dignum suggests that ai ethics can be divided into: • ethics by design (integration of ethical reasoning capabilities as a part of the behaviour of artificial autonomous system, e.g. ethical robots); • ethics in design (the regulatory and engineering methods supporting ethical implications of ai systems); and • ethics for design: (codes of conduct, standards, and certification processes that ensure the integrity of developers and users) [28] . in this paper, we focus on the ethically aligned development process, and therefore the last two categories were included into the research framework. finally, aspects of commitment were utilized in the framework to aid data analysis. specifically, we utilized the commitment net model of abrahamsson [29] to approach the implementation of ethics into practice and have an explaining theoretical framework to examine ethics role to developers. from this model, we focused on concerns and actions. concerns were analyzed to understand what ethical issues were of interest to the developers. actions were then studied to understand how these concerns were actually tackled, or whether they were tackled at all. in commitment net model, actions are connected to concerns because when actions are taken, they are always driven from concerns [29] . on the other hand, however, concerns can exist without any actions taken to address them. the dynamic between actions and concerns was considered a tangible way to approach the focus of this study: practices for implementing ai ethics. developers actions could be likened to practices that were taking during the development. on the other hand, analyzing the concerns that developers have opens a view to understanding e.g. whether the developers perhaps wanted to implement ethics but were unable to do so. this section is split into three subsections. first, we discuss the cases of the case study. in the second and third ones we discuss the data collection and analysis, respectively. we conducted a multiple case study featuring three case projects. in all of the case projects, ai systems were being developed for the healthcare sector. these cases are outlined in the table below (table 1) . we chose to utilize a qualitative case study approach due to the exploratory nature of the topic, as the research area is novel as far as empirical studies are concerned. healthcare cases were selected due to the assumption that ethical consideration would be more common in healthcare-related projects due to the nature of the area in closely dealing with human well-being (e.g. the tradition of bio and medical ethics). indeed, healthcare systems can, for example, influence the decisions made by doctors or their patients related to the health of the patients. moreover, due to the emphasis on taxfunded public healthcare in finland, where the cases were from, the area is particularly regulated. these regulations impose some ethical requirements on software systems as well, especially in relation to handling patient data, which is considered particularly sensitive data from a legal point of view. in the paper title, we characterize these case projects as being startup-like because the projects shared various characteristics typically associated with software startups. first, agile methods were commonly utilized in the projects. secondly, the projects were all characterized by notable time pressure. thirdly, the projects operated with scarce resources. fourthly, the cases were centered around the development of functional prototypes, which were intended to as proof-of-concept type artifacts. however, the prototypes were being developed with real customers and tested in practice. finally, the projects exhibited exploratory approaches that focused on experimentation. currently, much of the on-going ai development is happening in startups [1] , even if the multinational organizations receive much media coverage in relation to ai. in characterizing them as startup-like, we consider them to be representative of the current ai development projects. data from the cases were collected using semi-structured interviews [30] . this interview strategy enabled the interviews to be conducted in a way that allowed for flexibility from the interview questions, but without steering too far from the topic. the interview instrument used in the interviews can be found externally as a reference 4 . all interviews were conducted as f2f interviews and the audio was recorded for transcription. the analysis was conducted using the transcripts. the interviews were conducted in finnish. this was done so that the respondents would not give shorter responses due to being uncomfortable with communicating in english, especially while being on record. the respondents from the cases were either developers or managers. as we wanted to focus on development practices and project issues, we focused on the personnel directly involved with the practical development issues in the projects. the respondents are outlined in the table in the previous subsection. in terms of experience, respondents 4, 5, 7, and 8 were junior developers. respondents 3 and 6, on the other hand, were senior developers. respondent 1 was a junior data scientist. we analyzed the data in two phases. first, we utilized a grounded theory (heath [31] ) inspired approach to code the transcripts quote by quote for each interview. this process was carried out iteratively as the list of codes was updated during the process. this approach was chosen due to the lack of existing studies on the current state of practice in the area. in the second phase, we utilized the commitment net model of abrahamsson [29] to then further analyze and categorize the coded content. we utilized the model by focusing on the concerns and actions of the developers. the concerns and actions of each respondent were compared across cases in search of recurring concerns and actions between cases and respondents. by evaluating the relationships between the actions taken in development the development process and the concerns of the developers, we could better understand the motivation behind the actions. similarly, we could also see which concerns did not lead to any actions, pointing to a lack of commitment towards tackling those concerns. the data were then compared with the research framework again to evaluate how ai ethics were implemented in each project. actions were the emphasis here, as the focus of this study was on tangible implementation of ai ethics and how it was carried out in terms of tools, practices, or methods. however, we also highlighted interesting findings in relation to the mere concerns related to ai ethics. this section is split into four subsections. the first three feature the analysis split between the accountability, responsibility and transparency constructs. the final subsection summarizes the analysis. we highlight our findings as primary empirical conclusions (pecs). during the analysis, we use quotes from the interviews to elaborate on the topic at hand. however, it should be noted that the conclusions are not drawn merely based on these individual citations. the concerns of the developers related to responsibility were varied, but ultimately detached from practice as far as concerns related to ai ethics were considered. the concerns the developers discussed in relation to responsibility were simply very practical concerns related to internal project matters or delivering a high quality product: "responsibility on reporting and keeping the project on schedule" (r6) pec1. developers feel most responsibility towards tackling problems related to software development, such as finding bugs, meeting project goals. on the other hand, as the interviews progressed, the developers did also express some concerns towards various ethical issues. however, these concerns were detached from their current work. they did not affect the way they worked, and the developers felt that these types of concerns were not relevant during development. the presence of concerns in the absence of actions to address those concerns pointed towards a lack of commitment on this front. "it is just a prototype" (r8) "i do my best" (r5) "but this is a prototype, an experiment, just to show people that you can do this type of thing. this doesn't really have any responsibility issues in it." (r1) pec2. on a personal level, developers are concerned about the ethical aspects of product development. however, little is done to tackle these concerns. furthermore, it was evident that in none of the cases had the hypothetical effects of the system on the stakeholders been discussed. to give a practical example, a system potentially affecting memory illness diagnoses clearly has various effects on its potential users, especially when the test can be taken without supervision. yet, the developers of this particular tool also felt that their users would not be curious about the workings of the system. they considered it sufficient if the responsibility was outsourced to the user and it was underlined that the system does not make the diagnosis but simply advises doctors. the developers did not consider the potential harm of the system past the tangible, physical harm potential of the systems. for example, stress or other negative effects on users and other stakeholders were not considered. in all three cases, the respondents did not consider the system to have had any potential of causing physical harm, and thus did not consider the system to have any notable harm potential at all. "nobody wants to listen to ethics-related technical stuff. no five hour lectures about it. it's not relevant to the users" (r5) "i don't really understand what it [responsibility] has to do with product development. we developers are all responsible." (r7) "what could it affect… the distribution of funds in a region, or it could result in a school taking useless action… it does have its own risks, but no one is going to die because of it" (r1) pec3. responsibility of developers is unclear. case a highlighted the potential importance of mathematical expertise. the team had internal mathematical capabilities that allowed them to develop their own algorithms, as well as to better understand third party components, in order to have achieve a higher standard of transparency. they utilized algorithms they were familiar with and which they understood on an in-depth level. thus, the team considered themselves to be able to understand why the system made certain decisions in certain situations. this underlines the importance of mathematical skills in preventing the birth of black boxes in ai development. "in that sense it's not really a black box as we can understand what's going on in there just fine, and we can show the nodes and what affects them. it's a very transparent algorithm." (r3) the other two cases utilized existing ai solutions. they did not have an in-depth understanding of the technologies they were utilizing, which resulted in their systems being (partially) black boxes. they understood any components created by the team but did not have a full understanding of the third party components they had used as a base. this presents problems for feature traceability. even though transparency of algorithms and data was not present in two of the cases, the developers in case b nonetheless acknowledged its potential importance however, as it was not considered a formal requirement in the projects, the managers did not devote resources towards pursuing it. even in case a, transparency was not produced as a result of ethical goals but out of business reasons. "we have talked about the risks of decision-making support systems but it doesn't really affect what we do" (r5) pec5. developers recognize transparency as a goal, but it is not formally pursued. on the other hand, in relation to transparency of systems development, all three cases displayed transparency. by having formal decision-making strategies, they were able to keep track of higher-level decisions related to the system. through proper documentation, they were able to keep track of decisions made on the code level. version control also assisted in this regard, making it clear who made what changes and when in retrospect. there were thus various existing practices that produced transparency of systems development. two of the cases also acknowledged the effects of team size on transparency of systems development. they noted that, in addition to documentation practices, the small team size itself made it easy to keep track of the actions of individual developers even in an ad hoc manner. established se practices, such as code documentation and code review, support transparency of systems development. some aspects of accountability were clear points of focus in the projects, namely ones related to security in terms of general information security as well as data management. the respondents were aware of being in possession of personal data, given that they developed healthcare solutions, and were concerned with keeping it secure. they mentioned taking measures to keep the data secure from potentially malicious actors, and they were aware that they would have to take measures to act in accordance with laws and regulations in the area. however, in some cases they had not done so yet. "it's really important how you handle any kind of data, that you preserve it correctly, among researchers, and don't hand it out to any government actors. for example, many of the data packages have kind of interesting data and it can't get into the wrong hands. i personally can't see any way to harm anyone with the data we have though" (r2). "we haven't really paid much attention to the [data] safety aspects yet… it hasn't really been a main focus. there's probably a lot of things we have to take into account [eventually]" (r5). the ethical concerns they had in relation to accountability were in general largely related to existing areas of focus in software development. for example, error handling was one aspect of accountability the respondents were particularly concerned with. this was tied with their goal of making quality software, which they considered their responsibility as professionals. the respondents could, to this end, discuss what tangible practices they utilized to deal with error handling. pec7. developers feel accountable for error handling and have the means to deal with it. however, error handling was largely considered from the point of view of writing code and testing it in a laboratory setting. i.e. the system was considered error free if there were no red lines in the code in the ide during development. only case company b discussed measures they had taken to monitor errors in use. furthermore, potential misuse (e.g. a prankster drawing a horizontal white line on the pavement to intentionally confuse autonomous vehicles) and error scenarios during the operational life of the system had not been actively considered in any of the case projects. "the calculations are made in the algorithms, so it doesn't really make mistakes" (r2) pec8. product misuse and error scenarios are only considered during development. they are not considered in terms of the future operational life of the system out on the field. due to the nature of machine learning, ai systems learn as they are taught with new data or as they collect it themselves while operating out on the field. from this arises the potential issue of unexpected behavior as a result of machine learning. none of the respondents had made plans to tackle potential unexpected behavior during the operational life of their system, should such behavior arise. in only one of the projects was the possibility directly acknowledged: "we just put it up for end-users to test and note that this is still being developed" (r7). pec9. developers do not have plans to deal with unexpected behavior of the system resulting from e.g. machine learning or the future expansion of the use context of the system. past the art constructs, we highlight some commonalities between the cases on a more general level while summarizing our findings. in none of the cases were ethics implemented by following a formal method or tool, nor were ethical issues considered directly as ethical issues. rather, any ethical issues tackled in the projects were tackled for practical reasons (e.g. error free software is beneficial from the point of view of customer relations). nonetheless, some of the ethical issues such as error handling and transparency of systems development were tackled in a systematic manner through existing software engineering practices such as code documentation and version control. on the other hand, though ethics were not taken into consideration on a project level, the respondents still exhibited some concern towards the potential socio-ethical issues in the systems. when prompted, they were able to come up with various negative effects the systems could have on different stakeholders. they considered these to be potential real issues, but did not have a way to address these concerns in the absence of tools, practices, and methods for doing so. moreover, they seemed to realize these potential issues only after being directly asked about them in the interviews. this also points to a lack of tools to aid in ethical analyses. in this section, we have collected all the primary empirical conclusions (pec) outlined in preceding analysis section into table 2 . we relate each of these findings to existing literature and discuss their implications in this section. we classify each of these pecs based on their contribution into either novel findings, findings that (empirically) validated existing literature, or findings that contradict existing literature. many of our findings underline a gap between research and practice in the area. whereas research on ai ethics alongside various guidelines devised by researchers [8] and practitioners [23, 24] alike has discussed various ethical goals for ai systems, these goals have not been widely adopted out on the field. in this sense, we consider some of our findings (pecs 4, 5, 8, and 9) to contradict existing literature. for example, extant literature has highlighted the importance of transparency of algorithms and data [15] [16] [17] . without understanding how the system works, it is impossible to establish why it malfunctioned in a certain situation, which may e.g. be pivotal in understanding the causes of an accident that resulted in material damage [15] . our findings point towards transparency being largely ignored as a goal (pec5). existing system components are utilized as black boxes, and developers do not see this as a notable problem (pec4). we consider pec5 to contradict existing literature in that existing literature has, on multiple occasions, highlighted the importance of transparency in ai systems. yet, out on the field, this importance does not seem to be recognized to the point where it would result in changing development practices. the situation is similar for tackling potential misuse of the systems, error handling during system operations, and handling unexpected system behavior (pec8-9). these goals are included into the ieee ead guidelines [8] . however, none of the case companies took any measures to address these potential issues. on a further note of transparency, however, the lack of emphasis placed on it is also curious in relation to feature traceability in se. for decades, understanding the inner workings of the system was considered key in any se endeavor. yet, in the context of ai systems, the long-standing goal of feature traceability seems to be waning. our findings point towards this being at least partially a result of a lack of mathematical understanding, as the one case company that considered their system to be fully transparent also noted that they fully understood the mathematics behind the algorithms they utilized. in using existing components in their systems, developers may not always understand the algorithms in these components. indeed, in this vein, [32] noted that simply seeing the code is not enough if the algorithm is not understood, or the system is not understood as a whole. though we discovered various examples of ethics not being implemented, we also discovered that various existing and established se practices can be used to implement ai ethics. documentation, version control, and project management practices such as meeting transcripts produce transparency of systems development by tracking actions and decision-making (pec6). similarly, software quality practices help in error handling also in the context of ai ethics (pec7), although they do not specifically account for the errors autonomous systems may face while operating out on the field. while discussing responsibility with the respondents, we also discovered that most of their responsibility was related to producing quality software and meeting project requirements. this validates existing literature in the area of spi (e.g. unterkalmsteiner, [33] ). notably, we also discovered that the developers had ethical concerns towards their systems, which is a novel finding in this context (pec2). little is currently known about the state of practice out on the field, although a recent version of the ead guidelines speculated about a gap in the area, which our findings support in relation to most aspects of ai ethics. despite ai ethics largely not being implemented, our findings point towards it partially being a result of a lack of formal methods and tools to implement it. in our data, the reason given by multiple respondents for not actively considering ethical issues was that they were developing a prototype. however, prototypes do influence the final product or service developed based by them, as shown by existing studies [34] . ai ethical issues should be tackled during earlier stages of development as well, seeing as many of them are higher-level design decisions (such as how to carry out machine learning in the system [15] ), which can be difficult to undo later. following this study, as well as a past case study [14] , we suggest that future research seek to tackle the lack of methods and tooling in the area. though developers may be concerned about ethical issues, they lack the means to address these concerns. on the other hand, methods can also raise the awareness of developers in relation to ai ethics, creating concerns where there now are none. in creating these methods, we suggest exploring existing practices that can be used as is or tailored to implement ai ethics, as we have discussed here. given the amount of activity in ai ethics currently, with many governmental actors drafting their own ai ethics guidelines, likely followed by regulations, methods and tools will likely have practical demand in the future. thus, even if one barrier to implementing ai ethics is currently the fact that it is seldom considered a requirement on a project level, regulations and laws can force organizations to take ethics into account. this would inevitably result in a demand for methods in this area, as well as the birth of various in-house ones. finally, in terms of limitations, the most notable limitations of the study stem from the data and the research approach. the qualitative multiple case study approach always poses problems for the generalizability of the data. we acknowledge this as a limitation, although we also refer to eisenhardt [35] in arguing in favor of qualitative case studies, especially in the case of novel research areas. ai ethics, as far as empirical data goes, is a novel area of research. moreover, the multiple case study approach adds some further validity to the data, as we do not base our arguments on a single case. nonetheless, another limitation in the data is also that all the cases were based on finland. for example, the implementation of ai ethics can be more of a focus in us-based companies, as much of the current discussion on ai ethics also originates from the us. one other limitation in the data is that the interviews were conducted in finnish. the constructs such as transparency may not carry the same connotations in finnish as they do in english. this is especially the case with accountability and responsibility, which may not translate in a straightforward manner. however, during the interviews, we sought to clear any misunderstandings related to the constructs with the respondents. the research framework can also be argued to be a limitation. as ai ethics is a currently active field in terms of theoretical discussion, the constructs in the area are constantly evolving. the art principles and ead chosen as a basis of the framework were, at the time of writing, some of the most prominent works in the area. the framework ultimately presents but one way of perceiving ai ethics. this paper furthers our understanding of the current state of practice in the field of ai ethics. by means of a multiple case study, we studied the way ai ethics is currently implemented in practice, if it is implemented at all, when it is not formally or systematically implemented in software engineering projects. our findings can be summarized through the following two key takeaways: • even when ethics are not particularly considered, some currently commonly used software development practices, such as documentation, support ead. this is also the case with focusing on information security. • while the developers speculate potential socioethical impacts of the resulting system, they do not have means to address them. thus, from the point of view of software engineering methods and practices, this highlights a gap in the area. while some of the existing common practices support the implementation of some aspects of ai ethics, there are no methods or practices that help implement it on a project-level. further studies on the topic should seek to assist in the practical implementation of ai ethics. singular practices and especially project-level methods are needed to bridge the gap between research and practice in the area. this lack of higher-level methods was also highlighted in a review of tools and methods in the area [6] . open access this chapter is licensed under the terms of the creative commons attribution 4.0 international license (http://creativecommons.org/licenses/by/4.0/), which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the creative commons license and indicate if changes were made. the images or other third party material in this chapter are included in the chapter's creative commons license, unless indicated otherwise in a credit line to the material. if material is not included in the chapter's creative commons license and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. ai 50: america's most promising artificial intelligence companies artificial intelligence in medicine artificial intelligence for law enforcement: challenges and opportunities industrial artificial intelligence for industry 4.0-based manufacturing systems hidden technical debt in machine learning systems from what to how: an initial review of publicly available ai ethics tools, methods and research to translate principles in-to practices towards moral autonomous systems ethically aligned design: a vision for prioritizing human well-being with autonomous and intelligent systems, first edition flourishing ethics value-sensitive design value sensitive design: applications, adaptations, and critiques. in: van den hoven why machine ethics? incorporating ethics into artificial intelligence ethically aligned design of autonomous systems: industry viewpoint and an empirical study stop explaining black box machine learning models for high stakes decisions and use interpretable models instead the ethics of information transparency false positives, false negatives, and false analyses: a rejoinder to "machine bias: there's software used across the country to predict future criminals, and it's biased against blacks for a meaningful artificial intelligence: towards a french and european strategy german federal ministry of transport and digital infrastructure: automated and con-nected driving ethics guidelines for trustworthy ai iso/iec jtc 1/sc 42 artificial intelligence responsible bots: 10 guidelines for developers of conversational ai principles alone cannot guarantee ethical ai does acm's code of ethics change ethical decision making in software development? ai ethics in industry: a research framework ethics in artificial intelligence: introduction to the special issue commitment nets in software process improvement mastering the semi-structured interview and beyond: from research design to analysis and publication developing a grounded theory approach: a comparison of glaser and strauss seeing without knowing: limitations of the transparency ideal and its application to algorithmic accountability evaluation and measurement of software process improvementa systematic literature review minimum viable product or multiple facet product? the role of mvp in software startups building theories from case study research. acad key: cord-318509-zwb758cg authors: nenonen, suvi; storbacka, kaj title: don't adapt, shape! use the crisis to shape your minimum viable system – and the wider market date: 2020-07-31 journal: industrial marketing management doi: 10.1016/j.indmarman.2020.05.022 sha: doc_id: 318509 cord_uid: zwb758cg abstract in addition to being resilient and adaptive, firms should also utilize shocks such as covid-19 to generate new business opportunities. two processes make markets and other economic systems more malleable during times of crises: (1) as the stasis of the market system is interrupted, it forces the system “into movement” and it requires less effort to nudge an already moving system in a specific direction; and (2) as deeply-rooted mental models are challenged during crises, any market-shaping initiative which promises a credible end to current instability with a new equilibrium will appeal to the natural human craving for stability. this malleability, in turn, creates multiple opportunities for firms to shape their markets and hence drive the market's development in favorable directions. we outline a generic process of market-shaping, comprising eight steps: (1) determining whether to act now or actively wait, (2) deciding whether to be a shaper or a supporter, (3) developing a scalable vision for the future market, (4) recognizing the minimum viable system linked to this vision, (5) driving changes in market-level properties, (6) securing that value is quantified and shared, (7) inviting actor engagement for implementation, and (8) defending against possible retaliations from threatened market systems. disruptions and crises are particularly suitable times for shaping markets and other economic systems. market systems which are turbulent, discontinuous, or unstable are more malleable and, hence, more shapeable. there are two process at play: (1) as the stasis of the market system is interrupted, it forces the system "into movement" -and it requires less effort to nudge an already moving system in a specific direction; and (2) as deeply-rooted mental models are challenged during crises, actors are more prone to engage in higher-level learning, suggesting that any market-shaping initiative which promises a credible end to current instability with a new equilibrium will appeal to the natural human craving for stability. market systems are plastic and differ in their capacity to change (take form) and to remain stable (retain form) during different points of time (nenonen et al., 2014) . viable market systems have several mechanisms that stabilize them, ranging from high levels of compatibility between the system elements to various feedback loops (peters, nenonen, polese, frow, & payne, 2020) . due to these mechanisms, stable market systems are more likely to resist intentional attempts to shape them than market systems that are undergoing changes. one can argue that market systems function like physical systems in that the static coefficient of friction is much larger than the kinetic one. consequently, nudging an already-disrupted market into a specific favored direction is far easier than stirring a static one into motion in any direction (nenonen & storbacka, 2018) . the niche construction theorists acknowledge, in a similar vein, that ungoverned occurrences such as environmental or financial crises, give rise to deliberate shaping by individual actors or collectives (luksha, 2008) . therefore, it seems obvious that market shapers should time their efforts to coincide with periods of instability or discontinuity (mcgrath, 2019; nenonen & storbacka, 2018) . what this also suggests is that actors with less clout have a greater chance to change their market if they time their shaping efforts to periods in which this system is already experiencing turmoil and institutional conflict. as argued by economic sociology (callon, 1998; granovetter, 1992) , markets can be viewed as socially constructed human artifacts, implying that markets in the objective sense do not exist; i.e., there are no markets that exist independently from us humans. markets are what we -market actors -make them to be (kjellberg et al., 2012) . particularly, shared cognitive structures emerge among market actors over time, stabilizing the market (rosa, porac, runser-spanjol, & saxon, 1999) . in other words: as market actors start to view various elements of the market as stable, the market becomes increasingly so. however, this link between the shared cognitive structures and the markets' stability works in the other direction, too. crises in the business environment, whether man-or virus-made, challenge the stabilitypromoting mental models, i.e., dominant logics (prahalad, 2004) or industry recipes (spender, 1989 ) that cause active inertia (sull, 1999) for firms and markets alike. a crisis is a discontinuous event that creates a disorienting dilemma (mezirow, 1991) , which decreases the level of inertia around established roles and shared frames of reference. therefore, it often acts as a "trigger" (storbacka & nenonen, 2015) for higher-level learning, i.e., learning beyond adaptation and beyond the extant learning boundary. this type of learning has been called transformative (mezirow, 1991) , expansive (engeström, 2001) or doubleloop (argyris & schön, 1978) learning. such learning requires proactive unlearning of key organizational processes and the questioning of an organization's assumptions about itself and its environment (morgan & berthon, 2008) . this leads to breakdowns in the flow of normal actions and forces actors to question their current practices (cope, 2003) . the consequence is simple: a crisis both forces and enables highlevel learning in which both human and organizational actors are ready to question current mental models and managerial practices and engage in developing new ones. in this section we synthesize the current notions in the emerging literature on market-shaping and shaping strategies (cf., nenonen & storbacka, 2018; patvardhan & ramachandran, 2020) into a marketshaping process, comprising eight steps: (1) determining whether to act now or actively wait, (2) deciding whether to be a shaper or a supporter, (3) developing a scalable vision for the future market, (4) recognizing the minimum viable system linked to this vision, (5) driving changes in market-level properties, (6) securing that value is quantified and shared, (7) inviting actor engagement for implementation, and (8) defending against possible retaliations from threatened market systems. this process -leading to a shaped market -is summarized in fig. 1 . strategy research and many strategy practitioners have an implicit bias for action. however, to have a genuine strategy is also about saying "no" and being clear about the opportunities that firm will not pursue (cf., rumelt, 2011; magretta, 2011) . this applies to market shaping strategies, too. when contemplating the possibility of shaping markets, companies should pause and consider critically whether it is in their best interests -here and now -to start influencing the development path of their market (nenonen & storbacka, 2018) . thus, the prospective market-shaper first needs to determine whether the right time to act is now -or whether it makes sense to wait further. as discussed above, crises lead to malleability in markets, which in turn leads to shapeability of these markets. however, the fact that a market is shapeable now does not mean that it would not become even more shapeable in the near future. perhaps there is another major economic stimulus package in preparation, and the market-shaping endeavor is better timed only after its launch. alternatively, maybe the market is now at its most malleable, and waiting further would unduly increase the chances of someone else nudging the market onto a path that is detrimental to our aspirations. the likely future "shape" of the market being shaped gives some indications about this now vs. later timing decision. if the future market is likely to exhibit network effects, as many platform-based markets do (mcintyre & srinivasan, 2017) , then there may be some early-mover advantages. however, if the functioning of the future market is dependent on complementary assets and resources that are not available at all, or not available in sufficient quantities, then it is advisable to wait before commencing market-shaping (adner, 2012; adner & kapoor, 2010) . in case the decision is to wait, then it does not indicate doing nothing. quite to the contrary, firms aspiring to shape their markets should engage in what management scholars have called 'strategy as active waiting' (sull, 2005) , i.e., making all necessary preparations but waiting for the market to be ready to be influenced, be it a golden opportunity or a sudden-death threat. even though most of the popular press portrays covid-19 as a negative shock to economies globally, it may also be a golden opportunity for many organizations. regardless of the optimal timing decision discussed above, firms need to determine whether they should be leading the eventual marketshaping initiative -or to play a supporting role. a decision is needed whether to innovate or imitate, and whether to adopt the role of a first mover, a fast follower or a late entrant; (cf., katila & chen, 2008; markides & geroski, 2004; posen, lee, & yi, 2013) . the extant research has investigated these strategic choices in the context of market entry (barczak, 1995; chiu, chen, shyu, & tzeng, 2006) and innovation success (katila & chen, 2008; posen et al., 2013) . entering an existing market as a new company or with a new product is, however, a different strategic challenge from shaping a market, and hence we cannot rely solely on these studies for definite guidance. again, the likely future "shape" of the market provides insights to this decision. in case the market is likely to be reliant on a single keystone organization (iansiti & levien, 2004) , orchestrating the market and holding a key leadership role in it, then this keystone organization should assume the role of the market shaper. under other circumstances, when the playing field is more even, multiple firms may consider assuming the role of the leading market-shaper. even though the everyday terms and sayings such as "trailblazer" and "playing second fiddle" elevate the role of the shaper, marketshaping is very much a collective endeavor. collective action is found out to be crucial to shaping existing markets (baker & nenonen, 2020; maciel & fischer, 2020) and creating new ones (lee, struben, & bingham, 2018; struben et al., 2020) . therefore, supporter firms -by being part of this necessary collective action -play an instrumental part in market-shaping strategies. this supporter role may well allow the firm to spend less resources in the market-shaping process, but still benefit from the shaped market, and be able to influence how exactly the market-shaping unfolds. thus, also supporter firms should have a thorough understanding of all the steps of the market-shaping processes. finally, it would be too simplistic to assume that market-shaping roles -that of a shaper and a supporter -would remain unchanged during a long-term, systemic change process. instead, these roles are often fluid: a shaper may turn into a supporter later on -and vice versa. hence, having sufficient strategic flexibility to revisit this decision over the course of the market-shaping process is not only possible, but often necessary. the starting point for many market-shaping journeys is a deep dissatisfaction with the current state of things, leading the individual or the collective to question the status quo (patvardhan & ramachandran, 2020) . to use the crisis as an opportunity for higher level system-based learning, a focal actors needs to engage key individuals both in its own organization and in the wider market system to analyze the questions raised by the crisis and critically consider and, if needed, reject some of the accepted practices and 'wisdoms' of the existing situation. however, mere frustration with the status quo is not enough; it is also necessary to imagine an alternate state (nenonen & storbacka, 2018; patvardhan & ramachandran, 2020) , by modeling possible and alternative ways (sometimes called 'protovisions') to solve the identified problem. a key skill for a focal actor is the authoring of meanings (weick, 1995) , which can take the form of articulating simplified versions of the model through some medium that enables boundary crossing between market actors. such market-shaping vision usually is based on a deep understanding of customers' and other stakeholders' value drivers, an ability to comprehend the entire market system outside the firm-customer dyad, an ability to reframe phenomena creatively or to change the reference points, and prioritizing the (long-term) value creation for customers and other stakeholders over the (shortterm) value capture for the market-shaping firm (nenonen, storbacka, & windahl, 2019) . when developing and evaluating possible visions for the future market, managers should pay special attention to the scalability of this vision: it is relatively easy to achieve even radical changes on a small scale, but the viability and durability of market-shaping is oftentimes dependent on achieving critical mass (laland, matthews, & feldman, 2016; patvardhan & ramachandran, 2020) . developing a scalable vision needs to be initiated by the shaping actor, but it will require dialogue with other actors in order to be moved from a protovision to a believable declaration of intent that can guide further strategy development for many market actors. one of the challenges of (business) marketing and management is that it offers limited granularity to the "operating environment" surrounding the firm (möller et al., 2020) . to put it bluntly: there is the firm, its immediate customers and suppliers -and then the elusive (cue your favorite term) industry / market / operating environment / landscape. however, to implement -or even conceive -market-shaping strategies, managers need to appreciate the different layers of their operating environment. for market-shaping strategies, the lower meso layer (möller et al., 2020) , or the strategic network or ecosystem is of particular importance. this layer consists of the "multilateral set of partners that need to interact in order for a focal value proposition to materialize" (adner, 2017, p. 40) . in a similar vein, niche construction theorists propose that organisms and organizations can become "landscape shapers" if they focus their shaping efforts to the niche they allowing customers to pick-and-choose the individual services included in previously bundled logistics solutions to lower the perceived risk of overpaying property rights being exchanged (e.g., ownership vs. access) moving from leasing ict equipment to be used in offices to selling ict equipment to be used in home offices price or pricing pricing logic/price carrier moving from "per person/year" to "per trip" pricing in corporate travel insurance to acknowledge the current drop (and likely future increase) in travel volumes price point increasing the price point considerably to signal the move from a mass service to more restricted one (that is less prone to infections) channels connecting providers and customers changing agricultural auctions from physical ones to fully online transaction mechanism (how providers and customers are matched) moving from online auctions to "everyone gets x% of their requested volume at the average bid price" to manage an acute shortage of critical supplies actor network supply network number of participants in the supply network diversifying the raw material provision by increasing the number of providers types of participants in the supply network sourcing some components from local manufacturers to make the global supply chain more resilient work division in the supply network (e.g., out-vs. insourcing) insourcing assembly operations to reduce the reliance on far-away partners new customer groups broadening the target customer definition of online grocery shopping from only families and professional couples to also elderly people customers' competences to use the product / service tutorials on how to use videoconferencing services to their full potential customers' value perceptions (utility sought) demonstrating that leadership development can be delivered more effectively and efficiently online than in a traditional classroom setting work division between firm and customers (e.g., self-service vs. full service) grocery shops launching services with ready-made food plans & recipes for the week infrastructure supporting customers' use processes lobbying the government to invest in high-speed internet network in rural areas to support access to remote work & education provision number of competing providers (directly or indirectly) using the liquidity crisis to buy a competitor that has been competing with environmentally unsustainable business practices how competing providers interact or cooperate in order to service customers commercial landlords collective developing a process for business tenants to apply for temporary rent reductions representations terminology used labelling online delivery services "contactless" or "contact-free" to highlight their safety how media portrays the market encouraging media to run stories promoting local recreation and tourism market research and statistics convincing gartner to develop market research on the available remote workforce management tools key events and/or awards portraying the market pivot the 'auctioneer of the year' award and the related event to 'online auctioneer of the year' industry associations (e.g., businesses represented, themes promoted) technical standards (e.g., specifications, industry self-regulation) developing industry self-regulation related to safe restaurant practices formal rules and laws (regional, national or international) promoting more strict national self-sufficiency laws related to agriculture as well as pharmaceutical products and equipment social norms (e.g., societal values, industry conventions) augmenting and solidifying the transition to remote / flexible working arrangements in contexts that have had mandatory office presence occupy instead of their entire external environment (cf., laland, odling-smee, & feldman, 2000; luksha, 2008) . this minimum viable system is the smallest and simplest system that can support any market-shaping vision (cf., konietzko, bocken, & hultink, 2020) . the boundaries of a niche can be depicted by the division of labor and distribution of relevant knowledge (brittain, 1994) , dependence on common resources (hannan & freeman, 1989) and common rules of the game (luksha, 2008) . hence, the second step in market-shaping process is to outline one's "niche", which we label a minimum viable system: which actors are needed in order to realize the market-shaping vision in its most streamlined form? when analyzing the role of higher-level learning in a marketshaping context, storbacka and nenonen (2015) argued for a need to expand the view beyond organizational learning. for the learning to have the ability to shape markets, the learning processes need to involve other market actors that are central to the focal actor, i.e., the minimum viable system. therefore, the target is not only learning within the system, but learning as a system; i.e., groups of organizations coming together to learn as a group (knight & pye, 2005) . this clearly indicates that a firm taking a supporter role needs to secure a role in the minimum viable system, and actively participate in the on-going learning process. in addition to creating a market-shaping vision and involving the actors in the minimum viable system, for market-shaping to happen, the market has to be shaped: changes must occur in market-level properties such as market structures, market practices, and market agendas (storbacka & nenonen, 2015) . the, again, will require participation of both the shaper and the supporter(s). in practice, this is usually best done in two phases: first outlining the required changes to the minimum viable system and after that the required changes to the broader market. for a shaper firm this implies a need to start by hypothesizing potential changes needed in the minimum viable system for the vision to become real. a long list of possible changes can be categorized under three themes: changes linked to (a) exchange, typically related to modifying business models: developing the offering, adjusting price or pricing, and modifying matching methods for supply and demand, (b) the actor network, associated to modifying the focal firm's own supply network, modifying various customer-side features, and modifying provision, i.e., the availability of competing offerings, and (c) the institutional transmitters related to the market (e.g., representations that symbolize the market as well as the formal and informal norms guiding actor behaviors) (nenonen, storbacka, & frethey-bentham, 2019; nenonen, storbacka, & windahl, 2019) . a summary of the possible changes, and illustrative examples related to the covid-19 crisis, can be found in table 1 . nenonen, storbacka, and windahl (2019) found that successful market-shapers had applied idiosyncratic patterns of shaping efforts, as the "elements are interconnected and contingent on each other" (p. 626). hence, it appears that it is not possible to induce a market-level change by influencing only one of the aspects outlined in table 1 . instead, market-shaping actors drive changes in several (but not all) market elements, either simultaneously or sequentially. finally, the term 'hypothesize' also carries a special meaning in this step. as market-shaping is a highly complex task, entailing reconfiguring both firm's business model as well as the market system, these strategies are often implemented through on-going iteration and experimentation (nenonen, storbacka, & windahl, 2019) . this iterative nature of implementing market-shaping strategies elevates the role of supporter firms: they will have their own responsibilities to drive changes in certain market elements, and to contribute to the collective learning process. success in shaping markets requires that other actors than the shaper also change the way they operate. hence, it is not surprising that even the earliest works on market-shaping recognized value propositions as key components of these strategies (cf., kindström et al., 2018; kumar, scheer, & kotler, 2000; storbacka & nenonen, 2011) . these value propositions, summarizing the benefits of the future market to the particular actor, should be developed to every actor included in the minimum viable system. furthermore, these value propositions should be "win-win-win": the future market system should leave everyone involved in the minimum viable system better off than -or at least as well off as -the current market (nenonen & storbacka, 2018; nenonen, storbacka, sklyar, frow, & payne, 2020) . a detailed analysis of market-shaping value propositions reveals further two additional desirable features. first, value propositions that include quantification of the promised value and/or are verified by objective thirds parties tend to be more believable . second, involving other actors in a collaborative process of developing and communicating the value proposition supports the acceptance of this value proposition . hence, firm choosing supporter roles play a critical role in securing an appropriate approach to value creation and value appropriation in the minimum viable system. all strategies make or break at the point of implementation, and market-shaping strategies are no exception to this rule. however, implementing market-shaping strategies requires managers to embrace a much more collaborative approach than competitive strategies (clarke & fuller, 2010; ketchen jr, ireland, & snow, 2007) . internally, this implies cross-functional coordination and alignment, as no functional area is likely to command all resources or expertise related to marketshaping (nenonen, storbacka, & windahl, 2019) . externally, collaborative approach to implementing market-shaping strategies requires engaging other actors to join the common change process. this interorganizational collaboration is likely to focus on actors supporting the minimum viable system, and it is can be supported by helping other actors to operate in the new market and to de-risk or incentivize them to change their ways (baker & nenonen, 2020; nenonen, storbacka, & windahl, 2019) . engaging actors in the minimum viable system can be difficult as the shaping firm does not have any formal authority over other organizations in the system. to achieve "impact without authority," market shapers need to orchestrate (sull & ruelas-gossi, 2010) collaborative efforts by providing platforms or events that allow for emergence, by creating room for interactions, and building interdependence among actors in the minimum viable system. orchestration of resources and activities in the minimum viable system requires a switch from a selfcentric, firm-based view to an allocentric ("othercentered") view in which value is created and shared in the minimum viable system. the allocentric view allows firms to recognize and seize opportunities that can only be created by collaboration between organizations rather than by an individual firm, no matter how powerful (nenonen & storbacka, 2018) . many organizational processes can be designed in order to invite extensive collaboration. one example of this is open innovation processes (chesbrough & appleyard, 2007) involving a multitude of actors across functional and corporate boundaries. a market-shaping firm can often focus primarily on the platform that enables collaboration, because in our information-saturated age, various supporting organizations already have all the data needed for combinatorial innovation (yoo, boland jr, lyytinen, & majchrzak, 2012) . finally, the inherent complexity and unpredictability of marketshaping strategies require that managers empower others -both inside their own organization as well as external supporting firms -to make decisions and adjust their activities as they learn (storbacka & nenonen, 2015) . one powerful way of fostering such autonomous, but still coherent, decision-making under complexity are simple rules: a limited number of guidelines or heuristics designed to guide everyday decision-making in a way that balances concrete guidance with the freedom to exercise discretion and creativity (bingham & eisenhardt, 2011; eisenhardt & sull, 2001) . even though market-shaping value propositions should benefit everyone in the minimum viable system, market-shaping strategies are not free from competition, quests for dominance and even outright retaliation. systems theory suggests that harmonious interactions (i.e., 'resonance') between system elements and different systems is needed for long-term viability (barile & polese, 2010; wieland, polese, vargo, & lusch, 2012) . as well-designed market-shaping strategies ensure win-win-win outcomes for the actors within the particular minimum viable system, the competitive pressures are more likely to come from "external" market systems -especially if these associated market systems face reduced growth, profit or power as a result of the market-shaping strategy. hence, market-shapers should avoid -or actively managepossible retaliation by actors occupying negatively affected market systems (lawlor & kavanagh, 2015; peters et al., 2020) . this will require the allocation of resources and securing the support of various institutions in the market (such as industry associations). in this process both shapers and supporters play an important role. we appreciate that making a dramatic strategic change during a time of crisis is a daunting prospect -and developing the required capabilities when nations are in lockdown is even more so. however, it can be done. we are currently collaborating with a global company to support them to develop and implement their market-shaping strategy, spanning multiple regions and product lines. all of the work -analyzing the current state of organizational capabilities, developing tools and learning modules, as well as providing support to teams when they are going through the process outlined above -has happened online. only time will tell whether this organization will be successful in shaping their market, but their experiences hopefully show that it is possible to go after shaping strategies under these unusual circumstances. in addition to market-shaping being possible during this covid-19 crisis, it may also be essential. the array of challenges facing the globesuch as poverty, inequality, natural resource depletion, and climate change -are going to require the collective action of governments, civil society and businesses. scholars are increasingly in agreement that corporate responsibility and philanthropic approaches these sustainability challenges are inadequate (cf., haigh & hoffman, 2014; ritala, huotari, bocken, albareda, & puumalainen, 2018) . instead, what is needed are changes at the core of the commercial business models (bocken, short, rana, & evans, 2014 ) and a large-scale transition to the circular economy (geissdoerfer, savaget, bocken, & hultink, 2017) . what would be a better time to ramp up these efforts than a global crisis, commanding the attention of every business? our hope it is that the market-shaping process outlined in this paper helps businesses to rise to this challenge, whether they assume a shaper or a supporter role. this period of disruption is also a major call-to-action for the researcher community. there are still a lot of unknowns when it comes to market dynamics and market-shaping strategies. for example, what kind of indicators should managers use to assess the malleability of their markets -and hence to time their market-shaping actions? how fast do market systems "cool off", closing the window of opportunity for proactive market-shaping? is it possible to design markets to become 'antifragile' (taleb, 2012) , so that they would not only withstand but benefit from external shocks and turbulence? do different external shock vary in terms of their market-level effects -and hence are the opportunities for market-shaping different when faced with environmental (e.g., earthquake, flood), economical (e.g., deep recession), political (e.g., fall of communist states in the late 20th century), or biological (e.g., pandemic) shocks? if we are asking the managers to rise to the occasion, be agile and turn the current crisis into a major opportunity, we researchers should do the same too. the wide lens: a new strategy for innovation ecosystem as structure: an actionable construct for strategy value creation in innovation ecosystems: how the structure of technological interdependence affects firm performance in new technology generations organizational learning: a theory of action perspective collaborating to shape markets: emergent collective market work markets changing, changing markets: institutional work as market shaping new product strategy, structure, process, and performance in the telecommunications industry smart service systems and viable service systems rational heuristics: the "simple rules" that strategists learn from process experience a literature and practice review to develop sustainable business model archetypes exploring proactive market strategies. industrial marketing management density-independent selection and community evolution introduction: the embeddedness of economic markets in economics open innovation and strategy an evaluation model of new product launch strategy collaborative strategic management: strategy formulation and implementation by multi-organizational cross-sector social partnerships entrepreneurial learning and critical reflection: discontinuous events as triggers for "higher-level" learning exaptation and niche construction: behavioral insights for an evolutionary theory strategy as simple rules expansive learning at work: toward an activity theoretical reconceptualization searching, shaping, and the quest for superior performance the circular economy-a new sustainability paradigm economic institutions as social constructions the new heretics: hybrid organizations and the challenges they present to corporate sustainability organizational ecology strategy as ecology successful market innovation effects of search timing on innovation: the value of not being in sync with rivals strategic entrepreneurship, collaborative innovation, and wealth creation unraveling firm-level activities for shaping markets market innovation processes: balancing stability and change market futures/future markets: commentary on future research directions in the study of markets network learning: an empirically derived model of learning by groups of organizations circular ecosystem innovation: an initial set of principles from market driven to market driving an introduction to niche construction theory niche construction, biological evolution and cultural change infighting and fitting in: following innovation in the stent actor-network. industrial marketing management collective action and market formation: an integrative framework niche construction: the process of opportunity creation in the environment collaborative market driving: how peer firms can develop markets through collective action understanding michael porter: the essential guide to competition and strategy fast second: how smart companies bypass radical innovation to enter and dominate new markets seeing around corners: how to spot inflection points in business before they happen networks, platforms, and strategy: emerging views and next steps transformative dimensions of adult learning reinvigorating business marketing: navigating the nested, multimodal and transitionary business environment market orientation, generative learning, innovation strategy and business performance inter-relationships in bioscience firms a new perspective on market dynamics: market plasticity and the stability-fluidity dialectics. marketing theory smash: using market shaping to design new strategies for innovation, value creation, and growth is your industrial marketing work working? developing a composite index of market change value propositions as market-shaping devices: a qualitative comparative analysis. industrial marketing management capabilities for market-shaping: triggering and facilitating increased value creation shaping the future: strategy making as artificial evolution viability mechanisms in market emergence: prerequisites for market-shaping the power of imperfect imitation the blinders of dominant logic toward reimagining strategy research: retrospection and prospection on the 2011 amr decade award article sustainable business model adoption among s&p 500 firms: a longitudinal content analysis study sociocognitive dynamics in a product market good strategy/bad strategy: the difference and why it matters an inquiry into the nature and sources of managerial judgment actor engagement, value creation and market innovation scripting markets: from value propositions to market propositions learning with the market: facilitating market innovation collective action problems during market formation: the role of resource allocation strategic orchestration the dynamics of standing still: firestone tire & rubber and the radial revolution antifragile: things that gain from disorder managing risk and resilience sensemaking in organizations toward a service (eco)systems perspective on value co-creation strategic design: orchestrating and leveraging market-shaping capabilities organizing for innovation in the digitized world this research is funded by a marsden grant (uoa1333) from the royal society of new zealand. key: cord-021248-ui1di3qa authors: jung, kwangho; lee, sabinne title: a systematic review of rfid applications and diffusion: key areas and public policy issues date: 2015-09-04 journal: nan doi: 10.1186/s40852-015-0010-z sha: doc_id: 21248 cord_uid: ui1di3qa rfid applicants called as e-id, smart tag, and contactless smart card are being applied to numerous areas in our daily life, including tracking manufactured goods, currency, and patients to payments systems. to review these various applications of rfid is important to exploring not only ongoing e-governance issues such as digital identification, delivery process, and governance but also business oriented application areas like supply chain. through a systematic review methodology from 111 previous studies about rfid technology for public sector, we found six key areas of rfid applications: defense and security, identification, environmental applications, transportation, healthcare and welfare, and agriculture-livestock. we also suggest that the diffusion and applications of rfid can involve unexpected disadvantages including technological deficiency, uncertain benefits, dubious transparency, uncomfortable privacy issue, and unequal distribution of digital power and literacy. further research on rfid impact includes not only various theoretical issues of but also legal and managerial problems. rigorous research is required to explore what factors are critical to adopt and implement new rfid applications in terms of technology governance and digital literacy. massive data driven research is also expected to identify rfid performance in government agencies and various industry sectors. background rfid technology has been widely implemented all over the world and its impact on our daily life is very diverse and massive wyld, 2005) . those diverse areas of rfid application include logistical tracking, monitoring and maintenance of products, product safety and information, and payment process. today many governments around the world in both developed 1 and developing 2 countries are trying to apply it for various areas from tracking manufactured goods, currency, and patients to securing sagety of payments systems. massive rfid applications around all the industry sectors and countries are expected to generate a huge potential benefits for sustainable efficient energy infrastructure, transportation safety, and health care. over the past 50 years, rfid technology went through innovations and progressions to become a more efficient and effective gadget for human beings as well as effective solutions of technical and organizational problems in various industry sectors. however, key issues of appropriate ict technology, governing networks among rfid domains, standardization requirement, and privacy still remain unsolved 3 . we review previous literature about rfid technology used in public sectors in order to identify what has been done and found to suggest policy implications and further research agenda. more specifically, we discuss four aspects regarding rfid research issues and policy implications. first, we examine various competing concepts of rfid use by governments all over the world. second, we categorize numerous applications of rfid technology through analyzing previous literature. third, we try to figure out technological issues and governance problems that rfid technology faces today. last, we draw key public issues and suggest future research agenda. a brief history of rfid technology rfid technology was emerged as frederick hertz found existence of radio frequency during his experiment in 1886 (wyld, 2005) and developed for the purpose of defense during the second world war 4 . during 1970s and 1980s, the rfid system attracted plenty of scholars and innovators, so efforts to register patents progressed (takahashi, 2004) . researchers like charles walton had registered a patent to use rfid. in the 1980s, many us and european companies recognized the importance of developing rfid technology and started to manufacture rfid tags. soon scholars at mit university opened an auto-id center to promote the use and implementation of rfid technology. but most of the scholars report that the first commercialization of rfid technology was done by wal-mart as they launched rfid based material identifying system in 2005 (shahram and manish 2005) . wal-mart is now tracking merchandise including food, apparels, and electronic items with rfid technology in their supply chain. 5 . rfid technology is a brand new policy tool that can ensure high transparency, efficiency and effectiveness not only in industrial areas but also in government service delivery. table 1 describes a brief history of how rfid technology was developed and diffused. we searched online data base and expert based information to identify rfid publications between 2003 and 2015. we categorized rfid applications and analyzed issues and concerns that rfid faces today by systematically reviewing published literature. we have the idea of using radio frequency to reflect waves from objects was started from frederick hertz's experiment. american navy research laboratories developed a system known as iff (identify friend or foe). the first application of rfid consisted of identifying allied or enemy planes during ww2 through the use of iff system. charles walton, a former ibm researcher registered patent using rfid technology, a radio-operated door lock. [1980] [1981] [1982] [1983] [1984] [1985] [1986] [1987] [1988] [1989] [1990] many us and european companies started to manufacture rfid tags. the auto-id center for mit became epc global, an organization whose objective is to promote the use and adoption of rfid technology. wal-mart launched and rfid pilot. collected literature we use for systematic review from two different resources. first, most of the studies are found by searching the e-database. we could access electronic databases, such as google scholar, world web of science (wws), proquest central, and science direct through seoul national university's main library homepage. we had set 'rfid technology' , 'rfid government, ' 'rfid application, and 'rfid issue' as keywords for searching literature. we found most of the research through this method of searching. the second method we used for collecting data was having discussions with experts. to do this, we first made a list of experts who specialize in it technology, science technology, and public administration. five experts agreed to help us and recommended some research papers that were known for their fluent flow of logic and plentiful contents. we chose relevant research papers from among experts' recommendations. in sum we had used previous literature collected from two methods we discussed above, searching e-database and asking experts, as our resource of searching. [ figure 1 ] shows analytical frame that we use for this study. we have determined the literature for systematic review according to three stages shown on the flow chart. first, the original total number of studies we have found from the e-database was 4260. also 185 research papers were found from experts' recommendations and previous public papers. a total of 4,445 studies were chosen through the first stage. second, we excluded 4,121 following general eligibility criteria by screening title and abstract. more specifically, we excluded rfid studies only with one of the following criteria: 1) studies focusing on private sector; 2) studies without considering how public sector implemented rfid technology; 3) studies that did not discuss any social scientific implications; and 4) studies that only deal with rfid technology from pure scientific and engineering points of view. in sum, we included only 324 papers that discussed rfid issues and their implications in public sector. third, we removed further 213 studies fig. 1 analytical frame too much focusing on private sector or rfid technology itself, rather on its applications in our society and social scientific implications. finally, 111 articles were chosen for our systematic review. [ figure 2 ] below showed descriptive statistics of collected literatures by published year. it shows 22 studies were published in 2007 among 111 literatures. as we already described above in history of rfid section, the popularization and commercialization of rfid technology was started in 2005 with wal-mart's adoption. it seems that after wal-mart's innovative footsteps hit the world, many scholars were started to recognize the potential of new technology and tried to understand and develop rfid technology. besides some governments from all over the world implemented new way of public service delivery using rfid technology. consequently, 49 literatures were publishedbetween 2006 and 2008 and it forms almost 45 % of our collected studies for this study, we categorized governments' way of using rfid technology in 6 areas; agriculture and livestock, defense and security, environmental applications, healthcare and welfare, identification, and transportation. [ figure 3 ] shows descriptive statistics of collected literatures categorized by applications. we categorized studies that did not focus on specific sector and analyze and introduce rfid technology from the general perspectives as 'rfid general'. 'rfid general' studies usually deal with various ways of using rfid technology in diverse sectors simultaneously. as we can see from [fig. 3] , rfid general area had 42 papers. that means still lots of rfid studies could not be fully specialized and remained in status of generally introducing rfid technology. identification sector scores secondly highest number of published literatures among areas. this result seems natural because e-id card or e-passport have most powerful force that can hurt privacy, one of the most serious and notorious issues that rfid technology face today key applied areas of rfid as we show in [table 1 ], the history of rfid technology was started from the need for ensuring national security. almost 60 years have passed since us army developed rfid based identification system to identify allies and enemies, rfid technology is still used for protecting people. for instance, weinstein (2005) and konsysnki & smith (2003) reported how the us army and navy implement rfid technology in cargo containers to identify materials. the us army and navy implement rfid not only to identify us troops' own weapons and containers but also to identify enemies in battle (tien 2004) 6 . rfid systems are also important in terms of airport and port security. after the 9/11 terror attack on the united states, president george w. bush let all the airport and port in us adopt identification systems based on rfid technology to protect its nation from additional terrorist attacks (werb and sereiko 2002) 7 . in 2012, the taiwanese government decided to implement an rfid based e-seal system to increase security and efficiency (tsai and huang 2012) . in addition, rfid technology can be used effectively in prison management 8 and child protection. in some countries like japan and republic of korea, the rfid tag is implemented in child protection monitoring (table 2 ). 9 electronic passports like 'e-passports' were adopted electrically after the 9/11 attack. after the terrible tragedy broke heart of united states, the american government became aware of the importance of checking visas and passports correctly. the us department of state soon let people who wanted to enter us to use rfid tag embedded electronic passports instead of traditional barcode based passports 10 . the european union also endorsed the inclusion of biological information in e-passports. the eu justice and home affairs council decided to include fingerprints as a second mandatory identifier on passports in 2004. 11 in addition, rfid can be used in e-id cards in various countries. for example, in the united kingdom, prime minister tony blair and his labor party convinced the nation to adopt biometrically-enhanced national identification cards (ezovski and watkins 2007) . tony blair's administration announced its will to implement rfid tag embedded national identification card in late 2004. china is another case where the e-id card is used today. as a matter of fact, china is the country where e-id card is widely and largely adopted today. the beijing olympics held in 2008 lit the fuse of adoption. the largest smart card project was implemented as a part of preparing the most prominent international sports event. in (table 3) . rfid technology can be widely applied in environmental applications. adopting an rfid system in waste management is the most prominent way of using rfid to ensure efficient, eco-friendly waste management among lots of countries in the world. payt (pay-as-you-throw) program done by european union (eu) is the pioneer of this field. payt is an rfid based waste pricing model that allows each individuals or each household to pay for the tag along with the total amount of waste they throw. since each household and individual has a waste box in which rfid tags are embedded, the exact volume of waste can be calculated. in europe this incentive based system has been proven to be a powerful policy tool for reducing the total amount of waste and for encouraging recycling (schindler et al. 2012) . similar systems are broadly implemented in us (ransford et al. 2012) . in south korea, the ministry of environment introduced it to industry and urged them to use an rfid based waste management system, especially in medical waste management. rfid technology is implemented in waste management in developed and developing countries, but the purpose of adoption is somewhat different from europe to the us. india, the second-most populated country in the world, has adopted rfid technology to cope with the rapid increase of volume and types of waste (infotech 2013) . similarly in 2010, what china faced were the world expo and huge amounts of construction waste that comprised 30 % to 40 % of the total urban waste. shanghai was chosen for a pilot project using an rfid based waste management system. all the waste dumping trucks had an embed rfid tag and volume of waste they carry was checked by the local government (ruan and hu 2011) . another interesting case of environmental application emerges from south korea. the south korean government operates u-street trees systems through which the exact location and status of street trees can be monitored. information about location and status of street trees are collected by an rfid tag that is attached to each tree is saved in a web information system, so trees can be managed effectively. kim et al. (2006) claim that this web based information system could manage information remotely with an interactive system (table 4) . public transportation is another popular sector for rfid technology applications. rfid based electronic toll collection technology is one of the oldest and widespread rfid implementation (ulatowski 2007) . as soon as an rfid tag embedded car arrives at a toll booth, the rfid reader scans and reads the information that the rfid tag contains. the driver will pay debits according to the price that electronic reader suggests. in the us, electronic toll collection is thought as efficient and effective method that eliminates long lines of traffic at toll booth (ulatowski 2007) . rfid based toll collection is also adopted in criminal cases because it enables prosecutors to identify the exact location (smith 2006) . in south korea, the korean government has set credit card-linked electronic toll collection system called 'hypass' especially for collecting transportation tolls on express ways. if an rfid tag is embedded on their cars, drivers can pass the tollbooth without stopping the car because rfid reader scan the data immediately and handle the whole payment process in about 5 s (kim 2008) . hong kong launched similar public transportation toll collection system in 1997 and the 'octopus card' is now internationally famous for its convenience. this system is able to handle 10 million transactions per day and includes all modes of public transport (kovavisaruch and suntharasaj 2007) . south korea has set credit card-linked electronic toll collection system called 'hypass' especially for collecting transportation tolls on express ways. rfid technology is also implemented in railroad toll collection in india, where railroads are the most widely used form of public transportation. if an rfid tag is embedded on their cars, drivers can pass the tollbooth without stopping the car because rfid reader scan the data immediately and handle the whole payment process in about 5 s (kim, 2008) . in addition, rfid has been used as a critical technology to promote efficiency and transparency for public transportation system in developing countries. for instance, the mexican government runs "creating traffic knowledge in mexico: applying rfid to prevent vandalism" and one of the purposes of this innovative project is to develop a transportation information system to acquire more subtle data necessary for government decision making (prado et al. 2010 ). analogous to mexican case, in bangladesh where brta (bangladesh road transport authority) was started in 2003, the technology is operated mainly for control and supervision of the road transport systems ). rfid technology is also implemented in railroad toll collection in india, where railroads are the most widely used form of public transportation (table 5) . rfid enables hospitals to manage their equipment more easily and save expenses in public health areas 12 . the us government agencies like fda have also already used rfid tag in monitoring drug industry 13 . since american hospitals handle almost 4,000 (table 6) . rfid technology can be an effective tool for securing food safety and managing agriculture and livestock. another major advantage to this system is that animal disease tracking can be realized through innovative technology like rfid (hossain and quaddus 2009) . with the government support, researchers have developed the navigation system for appropriate pesticide use as a basic system for risk management in agriculture (nanseki et al. 2005) . rfid technology in agriculture was first introduced by the european union (eu) in the late 1990s and shortly thereafter many countries, such as australia, japan and south korea, adopted the innovation. among those countries, the australian government was the most passionate in implementing rfid 16 . for instance, all the livestock in australia have rfid embedded tags on their bodies immediately after they are born; information that enables farmers to identify each entity and its health status is registered in national livestock identification system (nlis). rfid technology in japan has been also adopted in agriculture especially to secure food safety and agricultural risk management that can occur by abusing pesticides (nanseki et al. 2005 , sugahara 2009 ). the japanese government planned to make a food traceability system by 2010 as a part of the "e-japan" plan . the united states is another case that applies mandatory rfid based identification system in managing livestock. according to rfid gazette (2006), the usda is pushing for rfid tagging of cattle to make tracing of disease patterns easier. with the formation of national institute for animal agriculture (niaa) in 2002, the plan for setting the national animal identification system was started. what the us taiwan government fulfilled through this program was "to be able to identify all animals and premises that have had contact with a foreign or domestic animal disease of concern within 48 h" (wyld, 2005) because "the sooner animal health officials can identify infected and exposed animals and premises, the sooner they can contain the disease and stop its spread (usda-aphis 2005)" (table 7) . public policy issues from rfid diffusion rfid applications and diffusion generate complex policy and governance problems. we address public policy issues such as technological gap and uncertainty of expecting potential benefits and costs from a rapid and massive rfid diffusion. uneasy governing issues in transparency, digital identification and power distribution are arising from inappropriate rfid applications. we discuss governance issues such as corruption, privacy problem, and digital monopoly and literacy in the following. technology is not still enough to satisfy all the elements that rfid is trying to perform various operational mechanisms. rfid technology deficiencies inevitably occur with the application of technology because there is niche space still left. for instance, rfid technology does not have a unified frequency standard yet. since there are no internationally agreed upon frequencies for rfid operations, permitted scanner/reader powers also differ between countries. there are still significant differences between the frequencies from the eu and the usa ). in addition, reichenhach (2008) pointed out the lack of storage capacity. in the eu, where rfid based waste management is common, there are technological barriers like a shortage of storage capacity 17 . ema and fujigaki (2011) draw implications from a child monitoring case done in japan that being informed of children's exact location cannot guarantee their actual safety, but rfid tags often lead to that cherished illusion. vining (2005) warned about another possibility of niche space. according to his study about port security in the us, stealing goods without damaging rfid tag is possible because at ports, the container can be drilled into and contents can be removed. the rfid tag does not have to endure any damage through this whole process. in the us, as a response to continued pressure from various stakeholders, the us government even adopted the 'faraday cage' for privacy protection 18 (table 8) . 19 . in reality, the rfid tag is much more expensive than a barcode, which was very popular in identifying materials before the rise of rfid technology (becker 2004) . purchasing rfid devices, hardware, and tags is not sufficient to drive system relevantly. to guarantee a better quality of service, the rfid system needs more additional things such as "circular process mechanism, the richness of consultant, project manager, programmers and plentiful project labors" . these elements for a better rfid performance may involve considerable costs. kuo and chen (2008) reported that rfid technology consumers and government should pay the extra hidden cost in the healthcare industry (table 9) . rfid technology is expected to increase transparency and monitor corruption. however, rfid technology cannot ensure a high level of transparency than expected. as a matter of fact, rfid tags can be cloned and manipulated quite easily, and this kind of tag corruption can occur at every stage of rfid implementation. there are various examples to show an inappropriate use of rfid technology. for instance, armknecht et al. (2010a, b) warned the possibility of tag corruption. lee et al. (2012) pointed out reader corruption of the rfid technology. an existing security model mainly focuses on the possibility of tag corruption, but reader corruption can hurt consumers' privacy as seriously as tag corruption can. jules (2006) reported one of the tag corruption cases that observed in united states. one of the staff members who worked in a dupyu store, an unscrupulous retailer, attached a cloned tag to counterfeit drugs. avoine et al. (2010) argued that internet based databases can also be directly attacked and emphasized the possibility of reader corruption. there are also unethical behaviors to avoid rfid monitoring process. in the eu where an rfid based waste management system is aggressively implemented, some people disposed waste that came from their house at work places in order to avoid exact calculation through the rfid system. not only this, bilitewski (2008) reported that some conscienceless people are burning or transferring (table 10) . one of the most serious issues that rfid technology faces today is whether rfid technology is secure enough to protect privacy. privacy is the most important concern rfid users have to deal with (perakslis and wolk 2005) . rfid tag embedded chips often contain important personal information and usually this kind of private information can hurt one's privacy seriously if leaked. to prevent leakage of private information, engineers developed cryptography, but there remains criticism 20 . the reason why these sorts of privacy concerns arise is because of the lack of security protection capacity of modern rfid technology. as we discussed above in the technological issues section, rfid technology today is not developed to secure perfect privacy. the technology itself has lots of deficiencies and people are smart enough to find niche spaces that can destroy the rfid security process. rfid itself can involve not only various hidden costs 21 but also induces a serious privacy problem 22 . however, despite these possibilities of attacks on privacy, there are lots of stakeholders and scholars who advocate the potential benefits of rfid. they claim that tracking and profiling consumers is solely for implementing rfid chips more effectively. eaward rerisi, one of the producers of early implementation of rfid technology argued that, "an rfid reader can read the number on a tag, but without knowing what the number means, there is no way to access personal information. the idea that the tags can be read by just anybody-that's pretty impossible" (murray 2003) (table 11) . unequal distribution of rfid technology can generate unequal distribution of various resources such as information and digital literacy. especially in developing countries, active tag, which is more safe for securing privacy than passive tag is more expensive than passive tag the combination of unbalanced power distribution between stakeholders and a low level of digital literacy can cause serious problems. ketprom et al. (2007) emphasized that in developing countries like thailand 23 , governments should provide education and training on how to use brand new technology to poor farmers whose digital literacy remains relatively low. but poor farmers in thailand are not the only stakeholders who are suffering from a lack of digital literacy. in bangladesh, where rfid toll collection is common, traffic policies have no interest in using rfid technology for managing public transportation systems. rather, they prefer traditional ways of toll collecting to information based technology . prasanth et al. (2009) found that the lack of digital literacy among the indian people hampered an effective process of railroad toll collection in india. another problem developing countries face is an unbalanced power distribution due to lack of democratic value embedded governance. chen et al. (2008) criticized the taiwanese government because it monopolizes most of the information collected by rfid technology. as we already discussed above, when rfid tag scanned, information saved in rfid tag is scanned by reader and then transmitted to an internet based database. if that data were available to the public, individuals and industry could make more reasonable decisions by analyzing them. we find another unbalanced power distribution case in china's waste management system. according to ruan and hu (2011) , the chinese government benefits most from the rfid system 24 (table 12) . we found, relying on a systematic review from 111 rfid studies, six key areas of rfid applications. specifically in the defense and security section, we addressed how military and airports/ports manage rfid systems to ensure security. we also found that rfid is effectively implemented in prison management and child protection programs. numerous governments have introduced rfid identification tools such as e-passport and e-id. rfid systems for waste management and street tree management are widely used from rich to poor countries. in healthcare and welfare delivery, rfid based smart cards have turned out to be very efficient. rfid is now being used to monitor counterfeit drugs. rfid has been applied to delivering service for the impaired and to trace infection. however, despite potential benefits from rfid applications, various unexpected problems arise. rfid can still involve technological deficiencies, especially in securing cryptography techniques, international standards of frequency, and storage capacity. rfid technology is not still enough to be efficient and effective in some areas (becker 2004 , jensen et al. 2007 ). tag and reader corruption can hurt transparency and security. privacy issues are still the most serious issues that rfid faces today (naumann and hogben 2008) . rfid itself can generate new unequal digital literacy and power distribution, especially in developing countries such as thailand and bangladesh. even the most latest innovative technologies, like rfid, do not have perfect answers to securing efficiency, effectiveness, convenience, and transparency. rather, rfid technology itself creates unexpected problems. it should be noted that democratic governance and trust is still important to technological innovation and policy issues arising from a rapid rfid diffusion. our systematic review is incomplete to discuss all of the rfid issues from technology, market and management, e-government, and legal aspects. further research on rfid diffusion and impact include not only various theoretical issues of but also legal and managerial problems. for instance, both qualitative and quantitative research is required to explore what factors are critical to adopt and implement new rfid technology in terms of governance and digital literacy. both micro and macro approaches with massive data are also required to identify how rfid improve not only organizational performance in government agencies and various industry sectors but also quality of our life. endnotes 1 for example, after serious attack by osama bin laden on 9/11, the american government decided to implement an rfid tag embedded e-passport and visa waiver program. the us government asked their member countries to implement e-passport by it is essential to understanding how a rapid diffusion and massive applications of rfid generate conflict or harmony among human behaviors, digital literacy, institutional rules, and technology. 4 us army and its allies could not only manage weapons and soldiers but also identify who was the enemy or not (castro and wamba, 2007) . this whole project of developing an rfid based identifying system was known as iff (identify friend of foe the new york city government also started an rfid e-seal pilot project in the new jersey port. once rfid read and scan the tag, it can identify the contents of the container box. also, the port of tacoma and seattle planned to adopt e-seals, made of metal bolts with embedded rfid devices to ensure its security (konsynski and smith 2003) . 8 calpatria prison, located in los angeles, adopted a prisoner monitoring system using rfid chips in 2000 as the very first pilot using rfid in prison management in united states (kim 2008) . according to regulations of calpatria prison, all the prison inmates were issued bracelets in which rfid chips were embedded. since the pilot project at the calpatria prison was successful, the local government let other prisons in los angeles adopt the innovative bracelet. the la county prison started to use a brand new bracelet in response to the state government's order; it is reported that through its adoption the prison could increase efficiency and effectiveness and decrease crimes occurred between inmates simultaneously (nicholas 2008). 9 for instance, city governments in the gifu and osaka prefecture provided rfid tags that can be attached in students' schoolbags to public elementary schools (ema and fujigaki, 2011) . similarly, in haewoondae beach, one of the most famous vacation spots in south korea, busan metropolitan city provides for parents rfid embedded bracelet that enables tracking exact location of their child by a smart phone (http://news.naver.com/ main/read.nhn?mode=lsd&mid=sec&sid1=102&oid=001&aid=0003353674). 10 this project began in 2002, but it took 3 years to fully implement for all 16 us passports (meingast et al. 2007) . the appearance of the e-passport is very similar to old passports, but woven into the paper of the passport, there is rfid tag that information about owner of the passport is included. information about nationality, sex, age, and so on is scanned, as airport staff members scan the passport through rfid reader (lorenc 2007 (kuo et al. 2004 ) with strong government support. 15 one peculiar characteristic of this system as compared to other systems is that visually impaired people are both the reader carrier and the service beneficiaries, simultaneously. generally in government service delivery using rfid technology, the service provider usually carries rfid tag readers and service beneficiaries usually act more passive roles by attaching rfid tags. but in this pakistani case, the service beneficiaries can identify objects around them by operating rfid tag reader they have. 16 the australian government passed legislation on mandatory use of rfid tags in the livestock industry, so hossain and quaddus (2014) categorized australian case as a very rare and special adoption case. according to trevarthen and michael (2007) 's case study, one of the australian farms where the rfid tag is implemented, farmers not only track the exact location of the cows but also check the condition, identify cows and even feed the new born cows automatically by using an rfid system. 17 usually people throw waste in various places. they may throw it away at their houses or at work places, like an office. since the rfid tag is only attached to a garbage can in house, it is impossible to track all types of waste throwing behaviors. inevitably, this shortage of storage capacity leads to selective waste collection monitoring. 18 the faraday cage is an object in metal; proponents of this device argue that faraday cage can prevent hacker's attack because electronic devices are prevented from passing through the object (ezovski et al. 2007) . but speculation about stability of this technology still remains. lorenc (2007) reported that if there is no additional technology, the faraday cage cannot preserve sensitive security. 19 as a matter of fact, there are two kinds of rfid tags, the passive tag and the active tag. these two tags provide owners with different benefits and liabilities. active tags are implemented by a power source, such as small battery. active tags are more efficient and safe in protecting privacy than passive tags, so sensitive organizations like military prefer active tags to passive tags. but ordinary consumers have less accessibility to active tags because active tags are much more expensive than passive ones (jensen et al. 2007 ). 20 according to laurie (2007) hwang et al. (2009) , technological deficiency enables hackers to engage in cloning, eavesdropping, replay attack, denial of service, forward security, tag tracing, individual data privacy, and data forging. specifically, the hacker can read the tag and then clone the tag (cloning), surreptitiously listen to all the communications between and the tag (eavesdropping), repeat or delay the message (replay attack), send large amount of message to break down rfid system (denial of service), compromise a tag (forward security), trace the exact location of the tag (tag tracing), find out shopping trend of the consumer (individual data privacy), and modify information saved on an rfid tag (data forging). in addition, numann and hogben (2008) categorized the privacy attacking cases more briefly. according to their research, the hacker can attack rfid tag in some ways. first, the attacker can open a connection to the chip and can steal the data inside (skimming). second, the attacker can intercept the communication between tag and reader (eavesdropping). last, the attacker can track the exact location of the tag or the person. 23 in thailand, most of the farms are trying to adopt an rfid system in farm management, but rfid technology is widening the gap between poor and rich farmers. poor farmers are usually less educated people who have hardly had any experience using digital technology like rfid. on the other hand, well-educated wealthy farmers face low entry barriers and easily adopt the technology. rich farmers armed with innovative technology not only make enormous fortunes by increasing efficiency but also by replacing poor labors with rfid embedded devices. according to the thailand ministry of labor (2006), most farm labors are afraid of being replaced. unfortunately, this phenomenon eventually correlates to a serious gap between rich and poor. as readers, tags, hardware, and so on, but in the long run cost of management will decrease. on the other hand, the waste industry could carry a very heavy debt. the waste contractors have to deal with expensive rfid tag rental and as well as the cost of construction simultaneously. although this situation is totally unfavorable for them, the waste management industry cannot resist to this policy because the chinese government is the entity which has made the use of rfid policy and set prices. the industry has no other choice but consent. anonymous authentication for rfid systems on rfid privacy with mutual authentication and tag corruption time measurement threatens privacy-friendly rfid authentication protocols rfid and corporate responsibility: hidden costs in rfid implementation a new game of leapfrog? rfid is rapidly changing the product-tracking process. some say the technologyonce costs drop-could displace bar-coding from traditional to modern fee systems an inside look at rfid technology using rfid technology in food produce traceability rfid-based hospital real-time patient management system how far can child surveillance go?: assessing the parental perceptions of an rfid child monitoring system in japan the electronic passport and the future of government-issued rfid-based identification exploring the perceived measures of privacy: rfid in public applications an adoption-diffusion model for rfid applications in bangladesh designing and implementing rfid technology for vehicle tracking in bangladesh developing and validating a hierarchical model of external responsiveness: a study on rfid technology privacy and security requirements for rfid applications the impact of government trust perception on privacy risk perceptions and consumer acceptance of residual rfid technologies mitigating consumer perceptions of privacy and security risks with the use of residual rfid technologies through governmental trust rfid security and privacy: a research survey. selected areas rfid based waste management system closing digital gap on rfid usage for better farm management a divide-and-conquer technique for throughput enhancement of rfid anti-collision protocol a management system of street trees by using rfid. in: web and wireless geographical information systems developments in practice x: radio frequency identification (rfid)-an internet for physical objects converging technology in society: opportunity for radio frequency identification (rfid) in thailand's transportation system the critical issues about deploying rfid in healthcare industry by service perspective the development of rfid in healthcare in taiwan. bejing: iceb practical attacks against rfid a state-aware rfid privacy model with reader corruption radio frequency identification technology: applications, technical challenges and strategies mark of the beast: us government use of rfid in government-issued documents embedded rfid and everyday things: a case study of the security and privacy risks of the us e-passport rfaide-an rfid based navigation and object recognition assistant for visually impaired people privacy concerns mount over retail use of rfid technology a navigation system for appropriate pesticide use: design and implementation privacy features of european eid card specifications social acceptance of rfid as a biometric security method creating traffic knowledge system in mexico: applying rfid to prevent the vandalism. the 15th international business information management association conference ticketing solutions for indian railways using rfid technology mementos: system support for long-running computation on rfid-scale devices status and prospects of pay-as-you-throw in europe-a review of pilot research and implementation studies gaining deeper insights into rfid adoption in hospital pharmacies application of an rfid-based system for construction waste transport: a case in shanghai rfid hybrid tech: combining gps for location tracking smart trash -study on rfid tags and the recycling industry, technical report. tr-1283-ec you can run, but you can't hide: protecting privacy from radio frequency identification technology traceability system for agricultural productsbased on rfid and mobile technology rfid tags should track inventory, not people the father of rfid beyond mere compliance of rfid regulations by the farming community: a case study of the cochrane dairy farm cost-benefit analysis of implementing rfid system in port of kaohsiung recent developments in rfid technology: weighing utility against potential privacy concerns national animal identification system: animal identification number(ain) rfid alone can't resolve cargo container security issues rfid: a technical overview and its application to the enterprise. it professional more than just tracking radio frequency identification applications in hospital environments delta airlines tags baggage with rfid. re:id magazine the right frequency for government. washington dc: ibm center for the business of government. ibm center zhang r. a transportation security system applying rfid and gps the authors declare that they have no competing interests.authors' contributions kj and sl carried out a systematic literature review not only from not only technological point of view but also from social scientific point of view. both authors read and approved the final manuscript. key: cord-356353-e6jb0sex authors: fourcade, marion; johns, fleur title: loops, ladders and links: the recursivity of social and machine learning date: 2020-08-26 journal: theory soc doi: 10.1007/s11186-020-09409-x sha: doc_id: 356353 cord_uid: e6jb0sex machine learning algorithms reshape how people communicate, exchange, and associate; how institutions sort them and slot them into social positions; and how they experience life, down to the most ordinary and intimate aspects. in this article, we draw on examples from the field of social media to review the commonalities, interactions, and contradictions between the dispositions of people and those of machines as they learn from and make sense of each other. a fundamental intuition of actor-network theory holds that what we call "the social" is assembled from heterogeneous collections of human and non-human "actants." this may include human-made physical objects (e.g., a seat belt), mathematical formulas (e.g., financial derivatives), or elements from the natural world-such as plants, microbes, or scallops (callon 1984; latour 1989 latour , 1993 . in the words of bruno latour (2005, p . 5) sociology is nothing but the "tracing of associations." "tracing," however, is a rather capacious concept: socio-technical associations, including those involving non-human "actants," always crystallize in concrete places, structural positions, or social collectives. for instance, men are more likely to "associate" with video games than women (bulut 2020) . furthermore, since the connection between men and video games is known, men, women and institutions might develop strategies around, against, and through it. in other words, techno-social mediations are always both objective and subjective. they "exist … in things and in minds … outside and inside of agents" (wacquant 1989, p. 43) . this is why people think, relate, and fight over them, with them, and through them. all of this makes digital technologies a particularly rich terrain for sociologists to study. what, we may wonder, is the glue that holds things together at the automated interface of online and offline lives? what kind of subjectivities and relations manifest on and around social network sites, for instance? and how do the specific mediations these sites rely upon-be it hardware, software, human labor-concretely matter for the nature and shape of associations, including the most mundane? in this article, we are especially concerned with one particular kind of associative practice: a branch of artificial intelligence called machine learning. machine learning is ubiquitous on social media platforms and applications, where it is routinely deployed to automate, predict, and intervene in human and non-human behavior. generally speaking, machine learning refers to the practice of automating the discovery of rules and patterns from data, however dispersed and heterogeneous it may be, and drawing inferences from those patterns, without explicit programming. 1 using examples drawn from social media, we seek to understand the kinds of social dispositions that machine learning techniques tend to elicit or reinforce; how these social dispositions, in turn, help to support 1 according to pedro domingos's account, approaches to machine learning may be broken down into five "tribes." symbolists proceed through inverse deduction, starting with received premises or known facts and working backwards from those to identify rules that would allow those premises or facts to be inferred. the algorithm of choice for the symbolist is the decision tree. connectionists model machine learning on the brain, devising multilayered neural networks. their preferred algorithm is backpropagation, or the iterative adjustment of network parameters (initially set randomly) to try to bring that network's output closer and closer to a desired result (that is, towards satisfactory performance of an assigned task). evolutionaries canvas entire "populations" of hypotheses and devise computer programs to combine and swap these randomly, repeatedly assessing these combinations' "fitness" by comparing output to training data. their preferred kind of algorithm is the so-called genetic algorithm designed to simulate the biological process of evolution. bayesians are concerned with navigating uncertainty, which they do through probabilistic inference. bayesian models start with an estimate of the probability of certain outcomes (or a series of such estimates comprising one or more hypothetical bayesian network(s)) and then update these estimates as they encounter and process more data. analogizers focus on recognizing similarities within data and inferring other similarities on that basis. two of their go-to algorithms are the nearest-neighbor classifier and the support vector machine. the first makes predictions about how to classify unseen data by finding labeled data most similar to that unseen data (pattern matching). the second classifies unseen data into sets by plotting the coordinates of available or observed data according to their similarity to one another and inferring a decision boundary that would enable their distinction. machine learning implementations; and what kinds of social formations these interactions give rise to-all of these, indicatively rather than exhaustively. our arguments are fourfold. in the first two sections below, we argue that the accretive effects of social and machine learning are fostering an ever-more-prevalent hunger for data, and searching dispositions responsive to this hunger -"loops" in this paper's title. we then show how interactions between those so disposed and machine learning systems are producing new orders of stratification and association, or "ladders" and "links", and new stakes in the struggle in and around these orders. the penultimate section contends that such interactions, through social and mechanical infrastructures of machine learning, tend to engineer competition and psycho-social and economic dependencies conducive to evermore intensive data production, and hence to the redoubling of machine-learned stratification. finally, before concluding, we argue that machine learning implementations are inclined, in many respects, towards the degradation of sociality. consequently, new implementations are been called upon to judge and test the kind of solidaristic associations that machine learned systems have themselves produced, as a sort of second order learning process. our conclusion is a call to action: to renew, at the social and machine learning interface, fundamental questions of how to live and act together. the things that feel natural to us are not natural at all. they are the result of long processes of inculcation, exposure, and training that fall under the broad concept of "socialization" or "social learning." because the term "social learning" helps us better draw the parallel with "machine learning," we use it here to refer to the range of processes by which societies and their constituent elements (individuals, institutions, and so on) iteratively and interactively take on certain characteristics, and exhibit change-or not-over time. historically, the concept is perhaps most strongly associated with theories of how individuals, and specifically children, learn to feel, act, think, and relate to the world and to each other. theories of social learning and socialization have explained how people come to assume behaviors and attitudes in ways not well captured by a focus on internal motivation or conscious deliberation (miller and dollard 1941; bandura 1962; mauss 1979; elias 2000) . empirical studies have explored, for instance, how children learn speech and social grammar through a combination of direct experience (trying things out and experiencing rewarding or punishing consequences) and modeling (observing and imitating others, especially primary associates) (gopnik 2009 ). berger and luckmann (1966) , relying on the work of george herbert mead, discuss the learning process of socialization as one involving two stages: in the primary stage, children form a self by internalizing the attitudes of those others with whom they entertain an emotional relationship (typically their parents); in the secondary stage, persons-in-becoming learn to play appropriate roles in institutionalized subworlds, such as work or school. pierre bourdieu offers a similar approach to the formation of habitus. as a system of dispositions that "generates meaningful practices and meaning-giving perceptions," habitus takes shape through at least two types of social learning: "early, imperceptible learning" (as in the family) and "scholastic...methodical learning" (within educational and other institutions) (bourdieu 1984, pp. 170, 66) . organizations and collective entities also learn. for instance, scholars have used the concept of social learning to understand how states, institutions, and communities (at various scales) acquire distinguishing characteristics and assemble what appear to be convictions-in-common. ludwik fleck (2012 fleck ( [1935 ) and later thomas kuhn (1970) famously argued that science normally works through adherence to common ways of thinking about and puzzling over problems. relying explicitly on kuhn, hall (1993) makes a similar argument about elites and experts being socialized into long lasting political and policy positions. collective socialization into policy paradigms is one of the main drivers of institutional path dependency, as it makes it difficult for people to imagine alternatives. for our purposes, social learning encapsulates all those social processes-material, institutional, embodied, and symbolic-through which particular ways of knowing, acting, and relating to one another as aggregate and individuated actants are encoded and reproduced, or by which "[e]ach society [gains and sustains] its own special habits" (mauss 1979, p. 99) . "learning" in this context implies much more than the acquisition of skills and knowledge. it extends to adoption through imitation, stylistic borrowing, riffing, meme-making, sampling, acculturation, identification, modeling, prioritization, valuation, and the propagation and practice of informal pedagogies of many kinds. understood in this way, "learning" does not hinge decisively upon the embodied capacities and needs of human individuals because those capacities and needs are only ever realized relationally or through "ecological interaction," including through interaction with machines (foster 2018) . it is not hard to see why digital domains, online interactions, and social media networks have become a privileged site of observation for such processes (e.g., greenhow and robelia 2009 ), all the more so since socialization there often starts in childhood. this suggests that (contra dreyfus 1974) social and machine learning must be analyzed as co-productive of, rather than antithetical to, one another. machine learning is, similarly, a catch-all term-one encompassing a range of ways of programming computers or computing systems to undertake certain tasks (and satisfy certain performance thresholds) without explicitly directing the machines in question how to do so. instead, machine learning is aimed at having computers learn (more or less autonomously) from preexisting data, including the data output from prior attempts to undertake the tasks in question, and devise their own ways of both tackling those tasks and iteratively improving at them (alpaydin 2014) . implementations of machine learning now span all areas of social and economic life. machine learning "has been turning up everywhere, driven by exponentially growing mountains of [digital] data" (domingos 2015) . in this article, we take social media as one domain in which machine learning has been widely implemented. we do so recognizing that not all data analysis in which social media platforms engage is automated, and that those aspects that are automated do not necessarily involve machine learning. two points are important for our purposes: most "machines" must be trained, cleaned, and tested by humans in order to "learn." in implementations of machine learning on social media platforms, for instance, humans are everywhere "in the loop"-an immense, poorly paid, and crowdsourced workforce that relentlessly labels, rates, and expunges the "content" to be consumed (gillespie 2018; gray and suri 2019) . and yet, both supervised and unsupervised machines generate new patterns of interpretation, new ways of reading the social world and of intervening in it. any reference to machine learning throughout this article should be taken to encapsulate these "more-thanhuman" and "more-than-machine" qualities of machine learning. cybernetic feedback, data hunger, and meaning accretion analogies between human (or social) learning and machine-based learning are at least as old as artificial intelligence itself. the transdisciplinary search for common properties among physical systems, biological systems, and social systems, for instance, was an impetus for the macy foundation conferences on "circular causal and feedback mechanisms in biology and social systems" in the early days of cybernetics (1946) (1947) (1948) (1949) (1950) (1951) (1952) (1953) . in the analytical model developed by norbert wiener, "the concept of feedback provides the basis for the theoretical elimination of the frontier between the living and the non-living" (lafontaine 2007, p. 31) . just as the knowing and feeling person is dynamically produced through communication and interactions with others, the ideal cybernetic system continuously enriches itself from the reactions it causes. in both cases, life is irrelevant: what matters, for both living and inanimate objects, is that information circulates in an everrenewed loop. put another way, information/computation are "substrate independent" (tegmark 2017, p. 67 ). wiener's ambitions (and even more, the exaggerated claims of his posthumanist descendants, see, e.g., kurzweil 2000) were immediately met with criticism. starting in the 1960s, philosopher hubert dreyfus arose as one of the main critics of the claim that artificial intelligence would ever approach its human equivalent. likening the field to "alchemy" (1965), he argued that machines would never be able to replicate the unconscious processes necessary for the understanding of context and the acquisition of tacit skills (1974, 1979 )-the fact that, to quote michael polanyi (1966) , "we know more than we can tell." in other words, machines cannot develop anything like the embodied intuition that characterizes humans. furthermore, machines are poorly equipped to deal with the fact that all human learning is cultural, that is, anchored not in individual psyches but in collective systems of meaning and in sedimented, relational histories (vygotsky 1980; bourdieu 1990; durkheim 2001; hasse 2019) . is this starting to change today when machines successfully recognize images, translate texts, answer the phone, and write news briefs? some social and computational scientists believe that we are on the verge of a real revolution, where machine learning tools will help decode tacit knowledge, make sense of cultural repertoires, and understand micro-dynamics at the individual level (foster 2018 ). our concern is not, however, with confirming or refuting predictive claims about what computation can and cannot do to advance scholars' understanding of social life. rather, we are interested in how social and computational learning already interact. not only may social and machine learning usefully be compared, but they are reinforcing and shaping one another in practice. in those jurisdictions in which a large proportion of the population is interacting, communicating, and transacting ubiquitously online, social learning and machine learning share certain tendencies and dependencies. both practices rely upon and reinforce a pervasive appetite for digital input or feedback that we characterize as "data hunger." they also share a propensity to assemble insight and make meaning accretively-a propensity that we denote here as "world or meaning accretion." throughout this article, we probe the dynamic interaction of social and machine learning by drawing examples from one genre of online social contention and connection in which the pervasive influence of machine learning is evident: namely, that which occurs across social media channels and platforms. below we explain first how data hunger is fostered by both social and computing systems and techniques, and then how world or meaning accretion manifests in social and machine learning practices. these explanations set the stage for our subsequent discussion of how these interlocking dynamics operate to constitute and distribute power. data hunger: searching as a natural attitude as suggested earlier, the human person is the product of a long, dynamic, and never settled process of socialization. it is through this process of sustained exposure that the self (or the habitus, in pierre bourdieu's vocabulary) becomes adjusted to its specific social world. as bourdieu puts it, "when habitus encounters a social world of which it is the product, it is like a 'fish in water': it does not feel the weight of the water, and it takes the world about itself for granted" (bourdieu in wacquant 1989, p. 43) . the socialized self is a constantly learning self. the richer the process-the more varied and intense the interactions-the more "information" about different parts of the social world will be internalized and the more socially versatile-and socially effective, possibly-the outcome. (this is why, for instance, parents with means often seek to offer "all-round" training to their offspring [lareau 2011]) . machine learning, like social learning, is data hungry. "learning" in this context entails a computing system acquiring capacity to generalize beyond the range of data with which it has been presented in the training phase. "learning" is therefore contingent upon continuous access to data-which, in the kinds of cases that preoccupy us, means continuous access to output from individuals, groups, and "bots" designed to mimic individuals and groups. at the outset, access to data in enough volume and variety must be ensured to enable a particular learnermodel combination to attain desired accuracy and confidence levels. thereafter, data of even greater volume and variety is typically (though not universally) required if machine learning is to deliver continuous improvement, or at least maintain performance, on assigned tasks. the data hunger of machine learning interacts with that of social learning in important ways. engineers, particularly in the social media sector, have structured machine learning technologies not only to take advantage of vast quantities of behavioral traces that people leave behind when they interact with digital artefacts, but also to solicit more through playful or addictive designs and cybernetic feedback loops. the machine-learning self is not only encouraged to respond more, interact more, and volunteer more, but also primed to develop a new attitude toward the acquisition of information (andrejevic 2019, p.53). with the world's knowledge at her fingertips, she understands that she must "do her own research" about everything-be it religion, politics, vaccines, or cooking. her responsibility as a citizen is not only to learn the collective norms, but also to know how to search and learn so as to make her own opinion "for herself," or figure out where she belongs, or gain new skills. the development of searching as a "natural attitude" (schutz 1970 ) is an eminently social process of course: it often means finding the right people to follow or emulate (pariser 2011) , using the right keywords so that the search process yields results consistent with expectations (tripodi 2018) , or implicitly soliciting feedback from others in the form of likes and comments. the social media user also must extend this searching disposition to her own person: through cybernetic feedback, algorithms habituate her to search for herself in the data. this involves looking reflexively at her own past behavior so as to inform her future behavior. surrounded by digital devices, some of which she owns, she internalizes the all-seeing eye and learns to watch herself and respond to algorithmic demands (brubaker 2020 ). data hunger transmutes into self-hunger: an imperative to be digitally discernible in order to be present as a subject. this, of course, exacts a kind of self-producing discipline that may be eerily familiar to those populations that have always been under heavy institutional surveillance, such as the poor, felons, migrants, racial minorities (browne 2015; benjamin 2019) , or the citizens of authoritarian countries. it may also be increasingly familiar to users of health or car insurance, people living in a smart home, or anyone being "tracked" by their employer or school by virtue of simply using institutionally licensed it infrastructure. but the productive nature of the process is not a simple extension of what michel foucault called "disciplinary power" nor of the self-governance characteristic of "governmentality." rather than simply adjusting herself to algorithmic demands, the user internalizes the injunction to produce herself through the machine-learning-driven process itself. in that sense the machine-learnable self is altogether different from the socially learning, self-surveilling, or self-improving self. the point for her is not simply to track herself so she can conform or become a better version of herself; it is, instead, about the productive reorganization of her own experience and self-understanding. as such, it is generative of a new sense of selfhood-a sense of discovering and crafting oneself through digital means that is quite different from the "analog" means of self-cultivation through training and introspection. when one is learning from a machine, and in the process making oneself learnable by it, mundane activities undergo a subtle redefinition. hydrating regularly or taking a stroll are not only imperatives to be followed or coerced into. their actual phenomenology morphs into the practice of feeding or assembling longitudinal databases and keeping track of one's performance: "step counting" and its counterparts (schüll 2016; adams 2019) . likewise, what makes friendships real and defines their true nature is what the machine sees: usually, frequency of online interaction. for instance, snapchat has perfected the art of classifying-and ranking-relationships that way, so people are constantly presented with an ever-changing picture of their own dyadic connections, ranked from most to least important. no longer, contra foucault (1988) , is "permanent self-examination" crucial to self-crafting so much as attention to data-productive practices capable of making the self learnable and sustaining its searching process. to ensure one's learnability-and thereby one's selfhood-one must both feed and reproduce a hunger for data on and around the self. human learning is not only about constant, dynamic social exposure and world hunger, it is also about what we might call world or meaning accretion. 2 the self is constantly both unsettled (by new experiences) and settling (as a result of past experiences). people take on well institutionalized social roles (berger and luckmann 1966) . they develop habits, styles, personalities-a "system of dispositions" in bourdieu's vocabulary-by which they become adjusted to their social world. this system is made accretively, through the conscious and unconscious sedimentation of social experiences and interactions that are specific to the individual, and variable in quality and form. accretion here refers to a process, like the incremental build-up of sediment on a riverbank, involving the gradual accumulation of additional layers or matter. even when change occurs rapidly and unexpectedly, the ongoing process of learning how to constitute and comport oneself and perform as a social agent requires one to grapple with and mobilize social legacies, social memory, and pre-established social norms (goffman 1983) . the habitus, bourdieu would say, is both structured and structuring, historical and generative. social processes of impression formation offer a good illustration of how social learning depends upon accreting data at volume, irrespective of the value of any particular datum. the popular insight that first impressions matter and tend to endure is broadly supported by research in social psychology and social cognition (uleman and kressel 2013) . it is clear that impressions are formed cumulatively and that early-acquired information tends to structure and inform the interpretation of information later acquired about persons and groups encountered in social life (hamilton and sherman 1996) . this has also been shown to be the case in online environments (marlow et al. 2013) . in other words, social impressions are constituted by the incremental build-up of a variegated mass of data. machine learning produces insight in a somewhat comparable way-that is, accretively. insofar as machine learning yields outputs that may be regarded as meaningful (which is often taken to mean "useful" for the task assigned), then that "meaning" is assembled through the accumulation of "experience" or from iterative exposure to available data in sufficient volume, whether in the form of a stream or in a succession of batches. machine learning, like social learning, never produces insight entirely ab initio or independently of preexisting data. to say that meaning is made accretively in machine learning is not to say that machine learning programs are inflexible or inattentive to the unpredictable; far from it. all machine learning provides for the handling of the unforeseen; indeed, capacity to extend from the known to the unknown is what qualifies machine learning as "learning." moreover, a number of techniques are available to make machine learning systems robust in the face of "unknown unknowns" (that is, rare events not manifest in training data). nonetheless, machine learning does entail giving far greater weight to experience than to the event. 3 the more data that has been ingested by a machine learning system, the less revolutionary, reconfigurative force might be borne by any adventitious datum that it encounters. if, paraphrasing marx, one considers that people make their own history, but not in circumstances they choose for themselves, but rather in present circumstances given and inherited, then the social-machine learning interface emphasizes the preponderance of the "given and inherited" in present circumstances, far more than the potentiality for "mak[ing]" that may lie within them (marx 1996 (marx [1852 ). one example of the compound effect of social and automated meaning accretion in the exemplary setting to which we return throughout this articlesocial media-is the durability of negative reputation across interlocking platforms. for instance, people experience considerable difficulty in countering the effects of "revenge porn" online, reversing the harms of identity theft, or managing spoiled identities once they are digitally archived (lageson and maruna 2018) . as langlois and slane have observed, "[w]hen somebody is publicly shamed online, that shaming becomes a live archive, stored on servers and circulating through information networks via search, instant messaging, sharing, liking, copying, and pasting" (langlois and slane 2017) . in such settings, the data accretion upon which machine learning depends for the development of granular insights-and, on social media platforms, associated auctioning and targeting of advertising-compounds the cumulative, sedimentary effect of social data, making negative impressions generated by "revenge porn," or by one's online identity having been fraudulently coopted, hard to displace or renew. the truth value of later, positive data may be irrelevant if enough negative data has accumulated in the meantime. data hunger and the accretive making of meaning are two aspects of the embedded sociality of machine learning and of the "mechanical" dimensions of social learning. together, they suggest modes of social relation, conflict, and action that machine learning systems may nourish among people on whom those systems bear, knowingly or unknowingly. this has significant implications for social and economic inequality, as we explore below. what are the social consequences of machine learning's signature hunger for diverse, continuous, ever more detailed and "meaningful" data and the tendency of many automated systems to hoard historic data from which to learn? in this section, we discuss three observable consequences of data hunger and meaning accretion. we show how these establish certain non-negotiable preconditions for social inclusion; we highlight how they fuel the production of digitally-based forms of social stratification and association; and we specify some recurrent modes of relation fostered thereby. all three ordering effects entail the uneven distribution of power and resources and all three play a role in sustaining intersecting hierarchies of race, class, gender, and other modes of domination and axes of inequality. machine learning's data appetite and the "digestive" or computational abilities that attend it are often sold as tools for the increased organizational efficiency, responsiveness, and inclusiveness of societies and social institutions. with the help of machine learning, the argument goes, governments and non-governmental organizations develop an ability to render visible and classify populations that are traditionally unseen by standard data infrastructures. moreover, those who have historically been seen may be seen at a greater resolution, or in a more finelygrained, timely, and difference-attentive way. among international organizations, too, there is much hope that enhanced learning along these lines might result from the further utilization of machine learning capacities (johns 2019) . for instance, machine learning, deployed in fingerprint, iris, or facial recognition, or to nourish sophisticated forms of online identification, is increasingly replacing older, document-based ones (torpey 2018 )-and transforming the very concept of citizenship in the process (cheney-lippold 2016). whatever the pluses and minuses of "inclusiveness" in this mode, it entails a major infrastructural shift in the way that social learning takes place at the state and inter-state level, or how governments come to "know" their polities. governments around the world are exploring possibilities for gathering and analysing digital data algorithmically, to supplement-and eventually, perhaps, supersede-household surveys, telephone surveys, field site visits, and other traditional data collection methods. this devolves the process of assembling and representing a polity, and understanding its social and economic condition, down to agents outside the scope of public administration: commercial satellite operators (capturing satellite image data being used to assess a range of conditions, including agricultural yield and poverty), supermarkets (gathering scanner data, now widely used in cpi generation), and social media platforms. if official statistics (and associated data gathering infrastructures and labor forces) have been key to producing the modern polity, governmental embrace of machine learning capacities signals a change in ownership of that means of production. social media has become a key site for public and private parties-police departments, immigration agencies, schools, employers and insurers among others-to gather intelligence about the social networks of individuals, their health habits, their propensity to take risks or the danger they might represent to the public, to an organization's bottom line or to its reputation (trottier 2012; omand 2017; bousquet 2018; amoore 2020; stark 2020) . informational and power asymmetries characteristic of these institutions are often intensified in the process. this is notwithstanding the fact that automated systems' effects may be tempered by manual work-arounds and other modes of resistance within bureaucracies, such as the practices of frontline welfare workers intervening in automated systems in the interests of their clients, and strategies of foot-dragging and data obfuscation by legal professionals confronting predictive technologies in criminal justice (raso 2017; brayne and christin 2020) . the deployment of machine learning to the ends outlined in the foregoing paragraph furthers the centrality of data hungry social media platforms to the distribution of all sorts of economic and social opportunities and scarce public resources. at every scale, machine-learning-powered corporations are becoming indispensable mediators of relations between the governing and the governed (a transition process sharply accelerated by the covid-19 pandemic). this invests them with power of a specific sort: the power of "translating the images and concerns of one world into that of another, and then disciplining or maintaining that translation in order to stabilize a powerful network" and their own influential position within it (star 1990, p. 32) . the "powerful network" in question is society, but it is heterogeneous, comprising living and non-living, automated and organic elements: a composite to which we can give the name "society" only with impropriety (that is, without adherence to conventional, anthropocentric understandings of the term). for all practical purposes, much of social life already is digital. this insertion of new translators, or repositioning of old translators, within the circuits of society is an important socio-economic transformation in its own right. and the social consequences of this new "inclusion" are uneven in ways commonly conceived in terms of bias, but not well captured by that term. socially disadvantaged populations are most at risk of being surveilled in this way and profiled into new kinds of "measurable types" (cheney-lippold 2017). in addition, social media user samples are known to be non-representative, which might further unbalance the burden of surveillant attention. (twitter users, for instance, are skewed towards young, urban, minority individuals (murthy et al. 2016 ).) consequently, satisfaction of data hunger and practices of automated meaning accretion may come at the cost of increased social distrust, fostering strategies of posturing, evasion, and resistance among those targeted by such practices. these reactions, in turn, may undermine the capacity of state agents to tap into social data-gathering practices, further compounding existing power and information asymmetries (harkin 2015) . for instance, sarah brayne (2014) finds that government surveillance via social media and other means encourages marginalized communities to engage in "system avoidance," jeopardizing their access to valuable social services in the process. finally, people accustomed to being surveilled will not hesitate to instrumentalize social media to reverse monitor their relationships with surveilling institutions, for instance by taping public interactions with police officers or with social workers and sharing them online (byrne et al. 2019) . while this kind of resistance might further draw a wedge between vulnerable populations and those formally in charge of assisting and protecting them, it has also become a powerful aspect of grassroots mobilization in and around machine learning and techno-social approaches to institutional reform (benjamin 2019) . in all the foregoing settings, aspirations for greater inclusiveness, timeliness, and accuracy of data representation-upon which machine learning is predicated and which underlie its data hunger-produce newly actionable social divisions. the remainder of this article analyzes some recurrent types of social division that machine learning generates, and types of social action and experience elicited thereby. there is, of course, no society without ordering-and no computing either. social order, like computing order, comes in many shapes and varieties but generally "the gap between computation and human problem solving may be much smaller than we think" (foster 2018, p. 152) . in what follows, we cut through the complexity of this socialcomputational interface by distinguishing between two main ideal types of classification: ordinal (organized by judgments of positionality, priority, probability or value along one particular dimension) and nominal (organized by judgments of difference and similarity) (fourcade 2016) . social processes of ordinalization in the analog world might include exams, tests, or sports competitions: every level allows one to compete for the next level and be ranked accordingly. in the digital world, ordinal scoring might take the form of predictive analytics-which, in the case of social media, typically means the algorithmic optimization of online verification and visibility. by contrast, processes of nominalization include, in the analog world, various forms of homophily (the tendency of people to associate with others who are similar to them in various ways) and institutional sorting by category. translated for the digital world, these find an echo in clustering technologies-for instance a recommendation algorithm that works by finding the "nearest neighbors" whose taste is similar to one's own, or one that matches people based on some physical characteristic or career trajectory. the difference between ordinal systems and nominal systems maps well onto the difference between bayesian and analogical approaches to machine learning, to reference pedro domingos's (2015) useful typology. it is, however, only at the output or interface stage that these socially ubiquitous machine learning orderings become accessible to experience. what does it mean, and what does it feel like, to live in a society that is regulated through machine learning systems-or rather, where machine learning systems are interacting productively with social ordering systems of an ordinal and nominal kind? in this section, we identify some new, or newly manifest, drivers of social structure that emerge in machine learning-dominated environments. let us begin with the ordinal effects of these technologies (remembering that machine learning systems comprise human as well as non-human elements). as machine learning systems become more universal, the benefits of inclusion now depend less on access itself, and more on one's performance within each system and according to its rules. for instance, visibility on social media depends on "engagement," or how important each individual is to the activity of the platform. if one does not post frequently and consistently, comment or message others on facebook or instagram, or if others do not interact with one's posts, one's visibility to them diminishes quickly. if one is not active on the dating app tinder, one cannot expect one's profile to be shown to prospective suitors. similarly, uber drivers and riders rank one another on punctuality, friendliness, and the like, but uber (the company) ranks both drivers and riders on their behavior within the system, from canceling too many rides to failing to provide feedback. uber egypt states on its website: "the rating system is designed to give mutual feedback. if you never rate your drivers, you may see your own rating fall." even for those willing to incur the social costs of disengagement, opting out of machine learning may not be an option. failure to respond to someone's tag, or to like their photo, or otherwise maintain data productivity, and one might be dropped from their network, consciously or unconsciously, a dangerous proposition in a world where self-worth has become closely associated with measures of network centrality or social influence. as bucher has observed, "abstaining from using a digital device for one week does not result in disconnection, or less data production, but more digital data points … to an algorithm, … absence provides important pieces of information" (bucher 2020, p. 2) . engagement can also be forced on non-participants by the actions of other users-through tagging, rating, commenting, and endorsing, for instance (casemajor et al. 2015) . note that none of this is a scandal or a gross misuse of the technology. on the contrary, this is what any system looking for efficiency and relevance is bound to look like. but any ordering system that acts on people will generate social learning, including action directed at itself in return. engagement, to feed data hunger and enable the accretion of "meaningful" data from noise, is not neutral, socially or psychologically. the constant monitoring and management of one's social connections, interactions, and interpellations places a nontrivial burden on one's life. the first strategy of engagement is simply massive time investment, to manage the seemingly ever-growing myriad of online relationships (boyd 2015). to help with the process, social media platforms now bombard their users constantly with notifications, making it difficult to stay away and orienting users' behavior toward mindless and unproductive "grinding" (for instance, repetitively "liking" every post in their feed). but even this intensive "nudging" is often not enough. otherwise, how can we explain the fact that a whole industry of social media derivatives has popped up, to help people optimize their behavior vis-a-vis the algorithm, manage their following, and gain an edge so that they can climb the priority order over other, less savvy users? now users need to manage two systems (if not more): the primary one and the (often multiple) analytics apps that help improve and adjust their conduct in it. in these ways, interaction with machine learning systems tends to encourage continuous effort towards ordinal self-optimization. however, efforts of ordinal optimization, too, may soon become useless: as marilyn strathern (citing british economist charles goodhardt) put it, "when a measure becomes a target, it ceases to be a good measure" (strathern 1997, p. 308) . machine learning systems do not reward time spent on engagement without regard to the impact of that engagement across the network as a whole. now, in desperation, those with the disposable income to do so may turn to money as the next saving grace to satisfy the imperative to produce "good" data at volume and without interruption, and reap social rewards for doing so. the demand for maximizing one's data productivity and machine learning measurability is there, so the market is happy to oblige. with a monthly subscription to a social media platform, or even a social media marketing service, users can render themselves more visible. this possibility, and the payoffs of visibility, are learned socially, both through the observation and mimicry of models (influencers, for instance) or through explicit instruction (from the numerous online and offline guides to maximizing "personal brand"). one can buy oneself instagram or twitter followers. social media scheduling tools, such as tweetdeck and post planner, help one to plan ahead to try to maximize engagement with one's postings, including by strategically managing their release across time zones. a paying account on linkedin dramatically improves a user's chance of being seen by other users. the same is true of tinder. if a user cannot afford the premium subscription, the site still offers them one-off "boosts" for $1.99 that will send their profile near the top of their potential matches' swiping queue for 30 min. finally, wealthier users can completely outsource the process of online profile management to someone else (perhaps recruiting a freelance social media manager through an online platform like upwork, the interface of which exhibits ordinal features like client ratings and job success scores). in all the foregoing ways, the inclusionary promise of machine learning has shifted toward more familiar sociological terrain, where money and other vectors of domination determine outcomes. in addition to economic capital, distributions of social and cultural capital, as well as traditional ascriptive characteristics, such as race or gender, play an outsized role in determining likeability and other outcomes of socially learned modes of engagement with machine learning systems. for instance, experiments with mechanical turkers have shown that being attractive increases the likelihood of appearing trustworthy on twitter, but being black creates a contrarian negative effect (groggel et al. 2019) . in another example, empirical studies of social media use among those bilingual in hindi and english have observed that positive modes of social media engagement tend to be expressed in english, with negative emotions and profanity more commonly voiced in hindi. one speculative explanation for this is that english is the language of "aspiration" in india or offers greater prospects for accumulating social and cultural capital on social media than hindi (rudra et al. 2016) . in short, wellestablished off-platform distinctions and social hierarchies shape the extent to which on-platform identities and forms of materialized labor will be defined as valuable and value-generating in the field of social media. in summary, ordinality is a necessary feature of all online socio-technical systems and it demands a relentless catering to one's digital doppelgängers' interactions with others and with algorithms. to be sure, design features tend to make systems addictive and feed this sentiment of oppression (boyd 2015). what really fuels both, however, is the work of social ordering and the generation of ordinal salience by the algorithm. in the social world, any type of scoring, whether implicit or explicit, produces tremendous amounts of status anxiety and often leads to productive resources (time and money) being diverted in an effort to better one's odds (espeland and sauder 2016; mau 2019) . 4 those who are short on both presumably fare worse, not only because that makes them less desirable in the real world, but also because they cannot afford the effort and expense needed to overcome their disadvantage in the online world. the very act of ranking thus both recycles old forms of social inequality and also creates new categories of undesirables. as every teenager knows, those who have a high ratio of following to followers exhibit low social status or look "desperate." in this light, jeff bezos may be the perfect illustration of intertwining between asymmetries of real world and virtual world power: the founder and ceo of amazon and currently the richest man in the world has 1.4 million followers on twitter, but follows only one person: his ex-wife. ordinalization has implications not just for hierarchical positioning, but also for belonging-an important dimension of all social systems (simmel 1910) . ordinal stigma (the shame of being perceived as inferior) often translates into nominal stigma, or the shame of non-belonging. not obtaining recognition (in the form of "likes" or "followers"), in return for one's appreciation of other people, can be a painful experience, all the more since it is public. concern to lessen the sting of this kind of algorithmic cruelty is indeed why, presumably, tinder has moved from a simple elo or desirability score (which depends on who has swiped to indicate liking for the person in question, and their own scores, an ordinal measure) to a system that relies more heavily on type matching (a nominal logic), where people are connected based on taste similarity as expressed through swiping, sound, and image features (carman 2019) . in addition to employing machine learning to rank users, most social media platforms also use forms of clustering and type matching, which allow them to group users according to some underlying similarity (analogical machine learning in domingos's terms). this kind of computing is just as hungry for data as those we discuss above, but its social consequences are different. now the aim is trying to figure a person out or at least to amplify and reinforce a version of that person that appears in some confluence of data exhaust within the system in question. that is, in part, the aim of the algorithm (or rather, of the socio-technical system from which the algorithm emanates) behind facebook's news feed (cooper 2020) . typically, the more data one feeds the algorithm, the better its prediction, the more focused the offering, and the more homogeneous the network of associations forged through receipt and onward sharing of similar offerings. homogenous networks may, in turn, nourish better-and more saleablemachine learning programs. the more predictable one is, the better the chances that one will be seen-and engaged-by relevant audiences. being inconsistent or too frequently turning against type in data-generative behaviors can make it harder for a machine learning system to place and connect a person associatively. in both offline and online social worlds (not that the two can easily be disentangled), deviations from those expectations that data correlations tend to yield are often harshly punished by verbal abuse, dis-association, or both. experiences of being so punished, alongside experiences of being rewarded by a machine learning interface for having found a comfortable group (or a group within which one has strong correlations), can lead to some form of social closure, a desire to "play to type." as one heavy social media user told us, "you want to mimic the behavior [and the style] of the people who are worthy of your likes" in the hope that they will like you in return. that's why social media have been variously accused of generating "online echo chambers" and "filter bubbles," and of fueling polarization (e.g., pariser 2011). on the other hand, being visible to the wrong group is often a recipe for being ostracized, "woke-shamed," "called-out," or even "canceled" (yar and bromwich 2019) . in these and other ways, implementations of machine learning in social media complement and reinforce certain predilections widely learned socially. in many physical, familial, political, legal, cultural, and institutional environments, people learn socially to feel suspicious of those they experience as unfamiliar or fundamentally different from themselves. there is an extensive body of scholarly work investigating social rules and procedures through which people learn to recognize, deal with, and distance themselves from bodies that they read as strange and ultimately align themselves with and against pre-existing nominal social groupings and identities (ahmed 2013; goffman 1963) . this is vital to the operation of the genre of algorithm known as a recommendation algorithm, a feature of all social media platforms. on facebook, such an algorithm generates a list of "people you may know" and on twitter, a "who to follow" list. recommendation algorithms derive value from this social learning of homophily (mcpherson et al. 2001) . for one, it makes reactions to automated recommendations more predictable. recommendation algorithms also reinforce this social learning by minimizing social media encounters with identities likely to be read as strange or nonassimilable, which in turn improves the likelihood of their recommendations being actioned. accordingly, it has been observed that the profile pictures of accounts recommended on tiktok tend to exhibit similarities-physical and racial-to the profile image of the initial account holder to whom those recommendations are presented (heilweil 2020) . in that sense, part of what digital technologies do is organize the online migration of existing offline associations. but it would be an error to think that machine learning only reinforces patterns that exist otherwise in the social world. first, growing awareness that extreme type consistency may lead to online boredom, claustrophobia, and insularity (crawford 2009 ) has led platforms to experiment with and implement various kinds of exploratory features. second, people willfully sort themselves online in all sorts of non-overlapping ways: through twitter hashtags, group signups, click and purchasing behavior, social networks, and much more. the abundance of data, which is a product of the sheer compulsion that people feel to self-index and classify others (harcourt 2015; brubaker 2020) , might be repurposed to revisit common off-line classifications. categories like marriage or citizenship can now be algorithmically parsed and tested in ways that wield power over people. for instance, advertisers' appetite for information about major life events has spurred the application of predictive analytics to personal relationships. speech recognition, browsing patterns, and email and text messages can be mined for information about, for instance, the likelihood of relationships enduring or breaking up (dickson 2019) . similarly, the us national security agency measures people's national allegiance from how they search on the internet, redefining rights in the process (cheney-lippold 2016). even age-virtual rather than chronological-can be calculated according to standards of mental and physical fitness and vary widely depending on daily performance (cheney-lippold 2017, p. 97). quantitatively measured identities-algorithmic gender, ethnicity, or sexuality-do not have to correspond to discrete nominal types anymore. they can be fully ordinalized along a continuum of intensity (fourcade 2016) . the question now is: how much of a us citizen are you, really? how latinx? how gay? 5 in a machine learning world, where each individual can be represented as a bundle of vectors, everyone is ultimately a unique combination, a category of one, however "precisely inaccurate" that category's digital content may be (mcfarland and mcfarland 2015) . changes in market research from the 1970s to the 1990s, aimed at tracking consumer mobility and aspiration through attention to "psychographic variables," constitute a pre-history, of sorts, for contemporary machine learning practices in commercial settings (arvidsson 2004; gandy 1993; fourcade and healy 2017; lauer 2017) . however, the volume and variety of variables now digitally discernible mean that the latter have outstripped the former exponentially. machine learning techniques have the potential to reveal unlikely associations, no matter how small, that may have been invisible, or muted, in the physically constraining geography of the offline world. repurposed for intervention, disparate data can be assembled to form new, meaningful types and social entities. paraphrasing donald mackenzie (2006), machine learning is an "engine, not a camera." christopher wylie, a former lead scientist at the defunct firm cambridge analytica-which famously matched fraudulently obtained facebook data with consumer data bought from us data brokers and weaponized them in the context of the 2016 us presidential election-recalls the experience of searching for-and discovering-incongruous social universes: "[we] spent hours exploring random and weird combinations of attributes.… one day we found ourselves wondering whether there were donors to anti-gay churches who also shopped at organic food stores. we did a search of the consumer data sets we had acquired for the pilot and i found a handful of people whose data showed that they did both. i instantly wanted to meet one of these mythical creatures." after identifying a potential target in fairfax county, he discovered a real person who wore yoga pants, drank kombucha, and held fire-andbrimstone views on religion and sexuality. "how the hell would a pollster classify this woman?" only with the benefit of machine learning-and associated predictive analytics-could wylie and his colleagues claim capacity to microtarget such anomalous, alloyed types, and monetize that capacity (wylie 2019, pp. 72-74) . to summarize, optimization makes social hierarchies, including new ones, and pattern recognition makes measurable types and social groupings, including new ones. in practice, ordinality and nominality often work in concert, both in the offline and in the online worlds (fourcade 2016 ). as we have seen, old categories (e.g., race and gender) may reassert themselves through new, machine-learned hierarchies, and new, machine-learned categories may gain purchase in all sorts of offline hierarchies (micheli et al. 2018; madden et al. 2017) . this is why people strive to raise their digital profiles and to belong to those categories that are most valued there (for instance "verified" badges or recognition as a social media "influencer"). conversely, patternmatching can be a strategy of optimization, too: people will carefully manage their affiliations, for instance, so as to raise their score-aligning themselves with the visible and disassociating themselves from the underperforming. we examine these complex interconnections below and discuss the dispositions and sentiments that they foster and nourish. it should be clear by now that, paraphrasing latour (2013, p. 307), we can expect little from the "social explanation" of machine learning; machine learning is "its own explanation." the social does not lie "behind" it, any more than machine learning algorithms lie "behind" contemporary social life. social relations fostered by the automated instantiation of stratification and association-including in social mediaare diverse, algorithmic predictability notwithstanding. also, they are continually shifting and unfolding. just as latour (2013, p. 221) reminds us not to confuse technology with the objects it leaves in its wake, it is important not to presume the "social" of social media to be fixed by its automated operations. we can, nevertheless, observe certain modes of social relation and patterns of experience that tend to be engineered into the ordinal and nominal orders that machine learning (re)produces. in this section, we specify some of these modes of relation, before showing how machine learning can both reify and ramify them. our argument here is with accounts of machine learning that envisage social and political stakes and conflicts as exogenous to the practice-considerations to be addressed through ex ante ethics-by-design initiatives or ex post audits or certifications-rather than fundamental to machine learning structures and operations. machine learning is social learning, as we highlighted above. in this section, we examine further the kinds of sociality that machine learning makes-specifically those of competitive struggle and dependency-before turning to prospects for their change. social scientists' accounts of modes of sociality online are often rendered in terms of the antagonism between competition and cooperation immanent in capitalism (e.g., fuchs 2007 ). this is not without justification. after all, social media platforms are sites of social struggle, where people seek recognition: to be seen, first and foremost, but also to see-to be a voyeur of themselves and of others (harcourt 2015; brubaker 2020) . in that sense, platforms may be likened to fields in the bourdieusian sense, where people who invest in platform-specific stakes and rules of the game 6 are best positioned to accumulate platform-specific forms of capital (e.g., likes, followers, views, retweets, etc.) (levina and arriaga 2014) . some of this capital may transfer to other platforms through built-in technological bridges (e.g., between facebook and instagram), or undergo a process of "conversion" when made efficacious and profitable in other fields (bourdieu 2011; fourcade and healy 2017) . for instance, as social status built online becomes a path to economic accumulation in its own right (by allowing payment in the form of advertising, sponsorships, or fans' gifts), new career aspirations are attached to social media platforms. according to a recent and well-publicized survey, "vlogger/youtuber" has replaced "astronaut" as the most enviable job for american and british children (berger 2019) . in a more mundane manner, college admissions offices or prospective employers increasingly expect one's presentation of self to include the careful management of one's online personality-often referred to as one's "brand" (e.g., sweetwood 2017) . similarly, private services will aggregate and score any potentially relevant information (and highlight "red flags") about individuals across platforms and throughout the web, for a fee. in this real-life competition, digitally produced ordinal positions (e.g., popularity, visibility, influence, social network location) and nominal associations (e.g., matches to advertised products, educational institutions, jobs) may be relevant. machine learning algorithms within social media both depend on and reinforce competitive striving within ordinal registers of the kind highlighted above-or in bourdieu's terms, competitive struggles over field-specific forms of capital. as georg simmel observed, the practice of competing socializes people to compete; it "compels the competitor" (simmel 2008 (simmel [1903 ). socially learned habits of competition are essential to maintain data-productive engagement with social media platforms. for instance, empirical studies suggest that motives for "friending" and following others on social media include upward and downward social comparison (ouwerkerk and johnson 2016; vogel et al. 2014) . social media platforms' interfaces then reinforce these social habits of comparison by making visible and comparable public tallies of the affirmative attention that particular profiles and posts have garnered: "[b]eing social in social media means accumulating accolades: likes, comments, and above all, friends or followers" (gehl 2015, p. 7) . in this competitive "[l]ike economy," "user interactions are instantly transformed into comparable forms of data and presented to other users in a way that generates more traffic and engagement" (gerlitz and helmond 2013, p. 1349 )-engagement from which algorithms can continuously learn in order to enhance their own predictive capacity and its monetization through sales of advertising. at the same time, the distributed structure of social media (that is, its multinodal and cumulative composition) also fosters forms of cooperation, gift exchange, redistribution, and reciprocity. redistributive behavior on social media platforms manifests primarily in a philanthropic mode rather than in the equitypromoting mode characteristic of, for instance, progressive taxation. 7 examples include practices like the #followfriday or #ff hashtag on twitter, a spontaneous form of redistributive behavior that emerged in 2009 whereby "micro-influencers" started actively encouraging their own followers to follow others. 8 insofar as those so recommended are themselves able to monetize their growing follower base through product endorsement and content creation for advertisers, this redistribution of social capital serves, at least potentially, as a redistribution of economic capital. even so, to the extent that purportedly "free" gifts, in the digital economy and elsewhere, tend to be reciprocated (fourcade and kluttz 2020) , such generosity might amount to little more than an effective strategy of burnishing one's social media "brand," enlarging one's follower base, and thereby increasing one's store of accumulated social (and potentially economic) capital. 9 far from being antithetical to competitive relations on social media, redistributive practices in a gift-giving mode often complement them (mauss 1990) . social media cooperation can also be explicitly anti-social, even violent (e.g., patton et al. 2019) . in these and other ways, digitized sociality is often at once competitive and cooperative, connective and divisive (zukin and papadantonakis 2017) . whether it is enacted in competitive, redistributive or other modes, sociality on social media is nonetheless emergent and dynamic. no wonder that bruno latour was the social theorist of choice when we started this investigation. but-as latour (2012) himself pointed out-gabriel tarde might have been a better choice. what makes social forms cohere are behaviors of imitation, counter-7 an exception to this observation would be social media campaigns directed at equitable goals, such as campaigns to increase the prominence and influence of previously under-represented groups-womenalsoknowstuff and pocalsoknowstuff twitter handles, hashtags, and feeds, for example. 8 recommendation in this mode has been shown to increase recommended users' chance of being followed by a factor of roughly two or three compared to a recommendation-free scenario (garcia gavilanes et al. 2013). 9 for instance, lewis (2018, p. 5) reports that "how-to manuals for building influence on youtube often list collaborations as one of the most effective strategies." imitation, and influence (tarde 1903) . social media, powered by trends and virality, mimicry and applause, parody and mockery, mindless "grinding" and tagging, looks quintessentially tardian. even so, social media does not amount simply to transfering online practices of imitation naturally occuring offline. the properties of machine learning highlighted above-cybernetic feedback; data hunger; accretive meaning-making; ordinal and nominal ordering-lend social media platforms and interfaces a distinctive, compulsive, and calculating quality-engineering a relentlessly "participatory subjectivity" (bucher 2020, p.88; boyd 2015) . how one feels and how one acts when on social media is not just an effect of subjective perceptions and predispositions. it is also an effect of the software and hardware that mediate the imitative (or counter-imitative) process itself-and of the economic rationale behind their implementation. we cannot understand the structural features and phenomenological nature of digital technologies in general, and of social media in particular, if we do not understand the purposes for which they were designed. the simple answer, of course, is that data hunger and meaning accretion are essential to the generation of profit (zuboff 2019), whether profit accrues from a saleable power to target advertising, commercializable developments in artificial intelligence, or by other comparable means. strategies for producing continuous and usable data flows to profit-making ends vary, but tend to leverage precisely the social-machine learning interface that we highlighted above. social media interfaces tend to exhibit design features at both the back-and front-end that support user dependency and enable its monetization. for example, the "infinite scroll," which allows users to swipe down a page endlessly (without clicking or refreshing) rapidly became a staple of social media apps after its invention in 2006, giving them an almost hypnotic feel and maximizing the "time on device" and hence users' availability to advertisers (andersson 2018) . similarly, youtube's recommendation algorithm was famously optimized to maximize users' time on site, so as to serve them more advertisements (levin 2017; roose 2019) . social media platforms also employ psycho-social strategies to this end, including campaigns to draw people in by drumming up reciprocity and participation-the notifications, the singling out of trends, the introduction of "challenges"-and more generally the formation of habits through gamification. prominent critics of social media, such as tristan harris (originally from google) and sandy parakilas (originally from facebook), have denounced apps that look like "slot machines" and use a wide range of intermittent rewards to keep users hooked and in the (instagram, tiktok, facebook, …) zone, addicted "by design" (schüll 2012; fourcade 2017) . importantly, this dependency has broader social ramifications than may be captured by a focus on individual unfreedom. worries about the "psychic numbing" of the liberal subject (zuboff 2019) , or the demise of the sovereign consumer, do not preoccupy us so much as the ongoing immiseration of the many who "toil on the invisible margins of the social factory" (morozov 2019) or whose data traces make them the targets of particularly punitive extractive processes. dependencies engineered into social media interfaces help, in combination with a range of other structural factors, to sustain broader economic dependencies, the burdens and benefits of which land very differently across the globe (see, e.g., taylor and broeders 2015) . in this light, the question of how amenable these dynamics may be to social change becomes salient for many. recent advances in digital technology are often characterized as revolutionary. however, as well as being addictive, the combined effect of machine learning and social learning may be as conducive to social inertia as it is to social change. data hunger on the part of mechanisms of both social learning and machine learning, together with their dependence on data accretion to make meaning, encourage replication of interface features and usage practices known to foster continuous, data-productive engagement. significant shifts in interface design-and in the social learning that has accreted around use of a particular interface-risk negatively impacting data-productive engagement. one study of users' reactions to changes in the facebook timeline suggested that "major interface changes induce psychological stress as well as technology-related stress" (wisniewski et al. 2014) . in recognition of these sensitivities, those responsible for social media platforms' interfaces tend to approach their redesign incrementally, so as to promote continuity rather than discontinuity in user behaviour. the emphasis placed on continuity in social media platform design may foster tentativeness in other respects as well, as we discuss in the next section. at the same time, social learning and machine learning, in combination, are not necessarily inimical to social change. machine learning's associative design and propensity to virality have the potential to loosen or unsettle social orders rapidly. and much as the built environment of the new urban economy can be structured to foster otherwise unlikely encounters (hanson and hillier 1987; zukin 2020) , so digital space can be structured to similar effect. for example, the popular chinese social media platform wechat has three features, enabled by machine learning, that encourage openended, opportunistic interactions between random users-shake, drift bottle, and people nearby-albeit, in the case of people nearby, random users within one's immediate geographic vicinity. (these are distinct from the more narrow, instrumental range of encounters among strangers occasioned by platforms like tinder, the sexual tenor of which are clearly established in advance, with machine learning parameters set accordingly.) qualitative investigation of wechat use and its impact on chinese social practices has suggested that wechat challenges some existing social practices, while reinforcing others. it may also foster the establishment of new social practices, some defiant of prevailing social order. for instance, people report interacting with strangers via wechat in ways they normally would not, including shifting to horizontallystructured interactions atypical of chinese social structures offline (wang et al. 2016 ). this is not necessarily unique to wechat. the kinds of ruptures and reorderings engineered through machine learning do not, however, create equal opportunities for value creation and accumulation, any more than they are inherently liberating or democratizing. social media channels have been shown to serve autocratic goals of "regime entrenchment" quite effectively (gunitsky 2015) . 10 likewise, they serve economic goals of data accumulation and concentration (zuboff 2019) . machine-learned sociality lives on corporate servers and must be 10 with regard to wechat in china and vkontakte in russia, as well as to government initiatives in egypt, the ukraine, and elsewhere, seva gunitsky (2015) highlights a number of reasons why, and means by which, nondemocratic regimes have proactively sought (with mixed success) to co-opt social media, rather than simply trying to suppress it, in order to try to ensure central government regimes' durability. meticulously "programmed" (bucher 2018) to meet specific economic objectives. as such, it is both an extremely lucrative proposition for some and (we have seen) a socially dangerous one for many. it favors certain companies, their shareholders and executives, while compounding conditions of social dependency and economic precarity for most other people. finally, with its content sanitized by underground armies of ghost workers (gray and suri 2019), it is artificial in both a technical and literal sense-"artificially artificial," in the words of jeff bezos (casilli and posada 2019). we have already suggested that machine-learned sociality, as it manifests on social media, tends to be competitive and individualizing (in its ordinal dimension) and algorithmic and emergent (in its nominal dimension). although resistance to algorithms is growing, those who are classified in ways they find detrimental (on either dimension) may be more likely to try to work on themselves or navigate algorithmic workarounds than to contest the classificatory instrument itself (ziewitz 2019) . furthermore, we know that people who work under distributed, algorithmically managed conditions (e.g., mechanical turk workers, uber drivers) find it difficult to communicate amongst themselves and organize (irani and silberman 2013; lehdonvirta 2016; dubal 2017) . these features of the growing entanglement of social and machine learning may imply dire prospects for collective action-and beyond it, for the achievement of any sort of broad-based, solidaristic project. in this section, we tentatively review possibilities for solidarity and mobilization as they present themselves in the field of social media. machine learning systems' capacity to ingest and represent immense quantities of data does increase the chances that those with common experiences will find one another, at least insofar as those experiences are shared online. machine-learned types thereby become potentially important determinants of solidarity, displacing or supplementing the traditional forces of geography, ascribed identities, and voluntary association. those dimensions of social life that social media algorithms have determined people really care about often help give rise to, or supercharge, amorphous but effective forms of offline action, if only because the broadcasting costs are close to zero. examples may include the viral amplification of videos and messages, the spontaneity of flash mobs (molnár 2014) , the leaderless, networked protests of the arab spring (tufekci 2016) , or of the french gilets jaunes (haynes 2019), and the #metoo movement's reliance on public disclosures on social media platforms. nonetheless, the thinness, fleeting character, and relative randomness of the affiliations summoned in those ways (based on segmented versions of the self, which may or may not overlap) might make social recognition and commonality of purpose difficult to sustain in the long run. more significant, perhaps, is the emergence of modes of collective action that are specifically designed not only to fit the online medium, but also to capitalize on its technical features. many of these strategies were first implemented to stigmatize or sow division, although there is no fatality that this is their only possible use. examples include the anti-semitic (((echo))) tagging on twitter-originally devised to facilitate trolling by online mobs (weisman 2018) but later repurposed by non-jews as an expression of solidarity; the in-the-wild training of a microsoft chatter bot, literally "taught" by well-organized users to tweet inflammatory comments; the artificial manipulation of conversations and trends through robotic accounts; or the effective delegation, by the trump 2020 campaign, of the management of its ad-buying activities to facebook's algorithms, optimized on the likelihood that users will take certain campaign-relevant actions-"signing up for a rally, buying a hat, giving up a phone number" (bogost and madrigal 2020) . 11 the exploitation of algorithms for divisive purposes often spurs its own reactions, from organized counter-mobilizations to institutional interventions by platforms themselves. during the 2020 black lives matter protests, for instance, kpop fans flooded rightwing hashtags on instagram and twitter with fancams and memes in order to overwhelm racist messaging. even so, often the work of "civilizing" the social media public sphere is left to algorithms, supported by human decision-makers working through rules and protocols (and replacing them in especially sensitive cases). social media companies ban millions of accounts every month for inappropriate language or astroturfing (coordinated operations on social media that masquerade as a grassroot movement): algorithms have been trained to detect and exclude certain types of coalitions on the basis of a combination of social structure and content. in 2020, the british far right movement "britain first" moved to tiktok after being expelled from facebook, twitter, instagram, and youtube-and then over to vkontakte or vk, a russian platform, after being banned from tiktok (usa news 2020). chastised in the offline world for stirring discord and hate, the economic engines that gave the movement a megaphone have relegated it to their margins with embarrassment. the episode goes to show that there is nothing inherently inclusive in the kind of group solidarity that machine learning enables, and thus it has to be constantly put to the (machine learning) test. in the end, platforms' ideal of collective action may resemble the tardean, imitative but atomized crowd, nimble but lacking in endurance and capacity (tufekci 2016) . mimetic expressions of solidarity, such as photo filters (e.g., rainbow), the "blacking out" of one's newsfeed, or the much-bemoaned superficiality of "clicktivism" may be effective at raising consciousness or the profile of an issue, but they may be insufficient to support broader-based social and political transformations. in fact, social media might actually crowd out other solidaristic institutions by also serving as a (feeble, often) palliative for their failures. for example, crowdsourced campaigns, now commonly used to finance healthcare costs, loss of employment, or educational expenses, perform a privatized solidarity that is a far cry from the universal logic of public welfare institutions. up to this point, our emphasis has been on the kinds of sociality that machine learning implementations tend to engender on the social media field, in both vertical (ordinal) and horizontal (nominal) configurations. we have, in a sense, been "reassembling the social" afresh, with an eye, especially, to its computational components and chains of reference (latour 2005) . throughout, we have stressed, nonetheless, that machine learning and other applications of artificial intelligence must be understood as forces internal to social life-both subject to and integral to its contingent properties-not forces external to it or determinative of it. accordingly, it is just as important to engage in efforts to reassemble "the machine"-that is, to revisit and put once more into contention the associative preconditions for machine learning taking the form that it currently does, in social media platforms for instance. and if we seek to reassemble the machine, paraphrasing latour (2005, p. 233) , "it's necessary, aside from the circulation and formatting of traditionally conceived [socio-technical] ties, to detect other circulating entities." so what could be some "other circulating entities" within the socio-technical complex of machine learning, or how could we envisage its elements circulating, and associating, otherwise? on some level, our analysis suggests that the world has changed very little. like every society, machine-learned society is powered by two fundamental, sometimes contradictory forces: stratification and association, vertical and horizontal difference. to be sure, preexisting social divisions and inequalities are still very much part of its operations. but the forces of ordinality and nominality have also been materialized and formatted in new ways, of which for-profit social media offer a particularly stark illustration. the machinelearnable manifestations of these forces in social media: these are among the "other circulating entities" now traceable. recursive dynamics between social and machine learning arise where social structures, economic relations and computational systems intersect. central to these dynamics in the social media field are the development of a searching disposition to match the searchability of the environment, the learnability of the self through quantified measurement, the role of scores in the processing of social positions and hierarchies, the decategorization and recategorization of associational identities, automated feedback that fosters compulsive habits and competitive social dispositions, and strategic interactions between users and platforms around the manipulation of algorithms. what, then, of prospects for reassembly of existing configurations? notwithstanding the lofty claims of the it industry, there is nothing inherently democratizing or solidaristic about the kinds of social inclusiveness that machine learning brings about. the effects of individuals and groups' social lives being rendered algorithmically learnable are ambivalent and uneven. in fact, they may be as divisive and hierarchizing as they may be connective and flattening. moreover, the conditions for entry into struggle in the social media field are set by a remarkably small number of corporate entities and "great men of tech" with global reach and influence (grewal 2008) . a level playing field this most definitely is not. rather, it has been carved up and crenellated by those who happen to have accumulated greatest access to the data processing and storage capacity that machine learning systems require, together with the real property, intellectual property, and personal property rights, and the network of political and regulatory lobbyists that ensure that exclusivity of access is maintained (cohen 2019) . power in this field is, accordingly, unlikely to be reconfigured or redistributed organically, or through generalized exhortation to commit to equity or ethics (many versions of which are self-serving on the part of major players). instead, political action aimed at building or rebuilding social solidarities across such hierarchies and among such clusters must work with and through them, in ways attentive to the specifics of their instantiation in particular techno-social settings. to open to meaningful political negotiation those allocations and configurations of power that machine learning systems help to inscribe in public and private life-this demands more than encompassing a greater proportion of people within existing practices of ruling and being ruled, and more than tinkering around the edges of existing rules. the greater the change in sociality and social relations-and machine learning is transforming both, as we have recounted-the more arrant and urgent the need for social, political and regulatory action specifically attuned to that change and to the possibility of further changes. social and political action must be organized around the inequalities and nominal embattlements axiomatic to the field of social media, and to all fields shaped in large part by machine learning. and these inequalities and embattlements must be approached not as minor deviations from a prevailing norm of equality (that is, something that can be corrected after the fact or addressed through incremental, technical fixes), but as constitutive of the field itself. this cannot, moreover, be left up to the few whose interests and investments have most shaped the field to date. it is not our aim to set out a program for this here so much as to elucidate some of the social and automated conditions under which such action may be advanced. that, we must recognize, is a task for society, in all its heterogeneity. it is up to society, in other words, to reassemble the machine. how the reification of merit breeds inequality: theory and experimental evidence step-counting in the "health-society strange encounters:embodied others in post-coloniality introduction to machine learning cloud ethics: algorithms and the attributes of ourselves and others social media apps are "deliberately" addictive to users on the 'pre-history of the panoptic sort': mobility in market research social learning through imitation race after technology. abolitionist tools for the new jim code. cambridge: polity american kids would much rather be youtubers than astronauts. ars technica the social construction of reality: a treatise in the sociology of knowledge how facebook works for trump distinction: a social critique of the judgement of taste (r the logic of practice the field of cultural production the forms of capital mining social media data for policing, the ethical way. government technology surveillance and system avoidance: criminal justice contact and institutional attachment technologies of crime prediction: the reception of algorithms in policing and criminal courts dark matters: on the surveillance of blackness digital hyperconnectivity and the self if ... then: algorithmic power and politics nothing to disconnect from? being singular plural in an age of machine learning a precarious game: the illusion of dream jobs in the video game industry social media surveillance in social work: practice realities and ethical implications some elements of a sociology of translation: domestication of the scallops and the fishermen of st brieuc bay tinder says it no longer uses a "desirability" score to rank people. the verge non-participation in digital media: toward a framework of mediated political action: media the platformization of labor and society jus algoritmi: how the national security agency remade citizenship we are data. algorithms and the making of our digital selves between truth and power: the legal constructions of informational capitalism how the facebook algorithm works in 2020 and how to work with it following you: disciplines of listening in social media can alexa and facebook predict the end of your relationship accessed 9 the master algorithm: how the quest for the ultimate learning machine will remake our world alchemy and artificial intelligence. rand corporation artificial intelligence the drive to precarity: a political history of work, regulation, & labor advocacy in san francisco's taxi & uber economics the elementary forms of religious life the civilizing process:sociogenetic and psychogenetic investigations engines of anxiety: academic rankings, reputation, and accountability genesis and development of a scientific fact culture and computation: steps to a probably approximately correct theory of culture technologies of the self. lectures at university of vermont the fly and the cookie: alignment and unhingement in 21st-century capitalism seeing like a market a maussian bargain: accumulation by gift in the digital economy internet and society: social theory in the information age the panoptic sort: a political economy of personal information follow my friends this friday! an analysis of human-generated friendship recommendations the case for alternative social media the like economy: social buttons and the data-intensive web custodians of the internet: platforms, content moderation, and the hidden decisions that shape social media stigma: notes on the management of spoiled identity the interaction order the philosophical baby: what children's minds tell us about truth, love, and the meaning of life ghost work. how to stop sillicon valley from building a new underclass old communication, new literacies: social network sites as social learning resources network power. the social dynamics of globalization race and the beauty premium: mechanical turk workers' evaluations of twitter accounts. information corrupting the cyber-commons: social media as a tool of autocratic stability policy paradigms, social learning, and the state: the case of economic policymaking in britain perceiving persons and groups the architecture of community: some new proposals on the social consequences of architectural and planning decisions exposed: desire and disobedience in the digital age simmel, the police form and the limits of democratic policing posthuman learning: ai from novice to expert? gilets jaunes and the two faces of facebook our weird behavior during the pandemic is messing with ai models there's something strange about tiktok recommendations turkopticon: interrupting worker invisibility in amazon mechanical turk from planning to prototypes: new ways of seeing like a state the structure of scientific revolutions the age of spiritual machines: when computers exceed human intelligence the cybernetic matrix of`french theory digital degradation: stigma management in the internet age economies of reputation: the case of revenge porn unequal childhoods: class, race, and family life the moral dilemmas of a safety belt the pasteurization of france reassembling the social: an introduction to actor-network-theory gabriel tarde and the end of the social an inquiry into modes of existence: an anthropology of the moderns creditworthy: a history of consumer surveillance and financial identity in america algorithms that divide and unite: delocalisation, identity and collective action in 'microwork google to hire thousands of moderators after outcry over youtube abuse videos | technology. the guardian distinction and status production on user-generated content platforms: using bourdieu's theory of cultural production to understand social dynamics in online fields alternative influence: broadcasting the reactionary right on youtube an engine, not a camera: how financial models shape markets privacy, poverty, and big data: a matrix of vulnerabilities for poor americans impression formation in online peer production: activity traces and personal profiles in github the eighteenth brumaire of louis bonaparte the metric society: on the quantification of the social techniques of the body the gift. the form and reason of exchange in archaic societies big data and the danger of being precisely inaccurate birds of a feather: homophily in social networks digital footprints: an emerging dimension of digital inequality social learning and imitation (pp. xiv, 341) reframing public space through digital mobilization: flash mobs and contemporary urban youth culture capitalism's new clothes. the baffler urban social media demographics: an exploration of twitter use in major american cities the palgrave handbook of security, risk and intelligence motives for online friending and following: the dark side of social network site connections the filter bubble: what the internet is hiding from you when twitter fingers turn to trigger fingers: a qualitative study of social media-related gang violence the tacit dimension displacement as regulation: new regulatory technologies and front-line decision-making in ontario works the making of a youtube radical understanding language preference for expression of opinion and sentiment: what do hindi-english speakers do on twitter? addiction by design: machine gambling in las vegas data for life: wearable technology and the design of self-care alfred schutz on phenomenology and social relations how is society possible? sociology of competition power, technology and the phenomenology of conventions: on being allergic to onions testing and being tested in pandemic times improving ratings': audit in the british university system 10 social media tips for students to improve their college admission chances the laws of imitation in the name of development: power, profit and the datafication of the global south life 3.0: being human in the age of artificial intelligence the invention of the passport: surveillance, citizenship, and the state analyzing scriptural inference in conservative news practices policing social media twitter and tear gas: the power and fragility of networked protest a brief history of theory and research on impression formation far-right activists tommy robinson and britain first turn to russia's vk after being banned from tiktok and every big social platform social comparison, social media, and selfesteem mind in society: the development of higher psychological processes towards a reflexive sociology: a workshop with pierre bourdieu. sociological theory space collapse: reinforcing, reconfiguring and enhancing chinese social practices through wechat. conference on web and social media (icwsm 2016) semitism))): being jewish in america in the age of trump understanding user adaptation strategies for the launching of facebook timeline mindf*ck. cambridge analytica and the plot to break america tales from the teenage cancel culture. the new york times rethinking gaming. the ethical work of optimization in web search engines the age of surveillance capitalism. the fight for a human future at the new frontier of power the innovation complex: cities, tech and the new economy hackathons as co-optation ritual: socializing workers and institutionalizing innovation in the publisher's note springer nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations as well as numerous articles that explore and theorize national variations in political mores, valuation cultures, economic policy, and economic knowledge. more recently, she has written extensively on the political economy of digitality, looking specifically at the changing nature of inequality and stratification in the digital era since 2015, she has been conducting fieldwork on the role of digital technology and digital data in development, humanitarian aid, and disaster relief-work funded, since 2018, by the australian research council. relevant publications include "global governance through the pairing of list and algorithm" (environment and planning d: society and space 2015); "data, detection, and the redistribution of the sensible acknowledgments we are grateful to kieran healy, etienne ollion, john torpey, wayne wobcke, and sharon zukin for helpful comments and suggestions. we also thank the institute for advanced study for institutional support. an earlier version of this article was presented at the "social and ethical challenges of machine learning" workshop at the institute for advanced study, princeton, november 2019. key: cord-330560-amqs5lb6 authors: rana, sourav; bhattacharya, sabyasachi; pal, joydeep; n’guérékata, gaston m.; chattopadhyay, joydev title: paradox of enrichment: a fractional differential approach with memory date: 2013-09-01 journal: physica a doi: 10.1016/j.physa.2013.03.061 sha: doc_id: 330560 cord_uid: amqs5lb6 the paradox of enrichment (poe) proposed by rosenzweig [m. rosenzweig, the paradox of enrichment, science 171 (1971) 385–387] is still a fundamental problem in ecology. most of the solutions have been proposed at an individual species level of organization and solutions at community level are lacking. knowledge of how learning and memory modify behavioral responses to species is a key factor in making a crucial link between species and community levels. poe resolution via these two organizational levels can be interpreted as a microscopicand macroscopic-level solution. fractional derivatives provide an excellent tool for describing this memory and the hereditary properties of various materials and processes. the derivatives can be physically interpreted via two time scales that are considered simultaneously: the ideal, equably flowing homogeneous local time, and the cosmic (inhomogeneous) non-local time. several mechanisms and theories have been proposed to resolve the poe problem, but a universally accepted theory is still lacking because most studies have focused on local effects and ignored non-local effects, which capture memory. here we formulate the fractional counterpart of the rosenzweig model and analyze the stability behavior of a system. we conclude that there is a threshold for the memory effect parameter beyond which the rosenzweig model is stable and may be used as a potential agent to resolve poe from a new perspective via fractional differential equations. the paradox of enrichment (poe) phenomenon in population ecology was first proposed by michael rosenzweig in 1971 [1] . rosenzweig observed that enrichment may destroy the steady state of a predator-prey system. he formulated his model using ordinary differential equations (odes) and considered enrichment by increasing the prey-carrying capacity. he observed that this gradually leads to destabilization of the system, usually into a limit cycle. this cyclical pattern was further studied by may [2] and gilpin and rosenzweig [3] . enrichment leads to destabilization of the equilibrium of the rosenzweig and many other models [4] ; huge amplitudes occur that frequently drive population densities to very low values and this may result in extinction. the word ''paradox'' is used because enrichment leads to extinction. since the 1990s, the word ''paradox'' has also been associated with the fact that destabilization is not always (or rarely) observed in natural environments, as discussed by jensen and ginzburg [5] . they reviewed the literature on experimental attempts to demonstrate poe and found an insufficient number of studies in favor of poe. some commonly misinterpreted examples and experiments (artificial lake eutrophication, didinium-paramecium systems [6, 7] , rotifer-algae systems [8] ) addressing the question of whether poe exists need further discussion. several theoretical and empirical studies have been carried out in attempts to explain the poe phenomenon. roy and chattopadhyay provide a detailed review of the studies and a brief overview of the proposed solutions [4] . they described some reasonable mechanisms, such as the presence of inedible prey, invulnerable prey, unpalatable prey, ratio-dependent functional responses, spatiotemporal chaos, inducible defense, density-dependent predator mortality and toxic food, to resolve the paradoxical outcome of ecosystem enrichment. several additional mechanisms have also been proposed. for example, abrams and roth proposed refuges and immigration as a possible solution [9] . more recently, mougi and nishimura showed that rapid trait evolution of predator-prey species can stabilize the system [10] . an imperfect optimal foraging mechanism was proposed by mougi and nishimura [11] . rall et al. predicted that a sigmoidal or predator inference functional response can dampen population oscillations due to enrichment that resolves poe [12] . disease in ecological systems has been recognized as an essential mechanism in different ecological issues. sharp and pastor recently considered a densitydependent susceptible deer population, an infected population and an environmental pool of prions that infect the deer population [13] . they showed that the disease population has the ability to dampen population oscillations. solutions to the poe problem have been established by several authors from diverse perspectives and dimensions, but most of the solutions have some common factors associated with the behavioral aspects of predator-prey species at the individual level and inducible or morphological defense mechanisms of the prey. however, solutions that consider community-level organization are still lacking. knowledge of how learning and memory modify behavioral responses to species is a key factor in making a crucial link between the species and community levels [14] . resolution of poe via these two organizational levels can be interpreted as a microscopic-and macroscopic-level solution. in statistical physics the connection between microscopic dynamics in a stochastic system and a macroscopic description of their behavior as a whole is very attractive [15] . it has been already established that the macroscopic behavior of stochastic systems contains a manifestation of microscopic dynamics due to memory effects [15] . memory in the biological domain in physical systems is manifest as a series of events that induce episodic memory [16, 17] containing information about past and present events and can help in making future decisions [18] . biological systems are also enriched with ecological memory [19] . memory relates to historic events and requires an entire long-term record for analysis. for example, seasonal succession of phytoplankton in lakes responds to small changes in trophic structure and natural fluctuations of gradients [19] . aquatic and phytoplankton systems possess a remarkable memory of climatic events [20] . apart from such climatic phenomena, aquatic zooplankton exhibit memory-based phenomenological responses: small changes in the environment induce morphological defense organs such as helmet formation and the development of spines through cyclomorphosis [21] . these changes are due to a mixed seasonal and genetically inherited reaction [22] induced over long time scales, suggesting a phenotypic change through memory-based actions. prey refuge, group hunting and prey swarming are all well-coordinated actions based on memories of past events that lead to cultural learning [14] . experience accrued through learning may direct anti-predator responses [23] [24] [25] . in aquatic systems, learning of chemical cues and the production of counter responses are well documented among zooplankton [26] [27] [28] [29] [30] [31] . westerlund remarked that, surprisingly, even ''dead matter has memory'' [32] . a recent study by zheng et al. [33] revealed that discrete earthquake events are related to seismic memory. fractional derivatives provide an excellent tool for describing memory and the hereditary properties of various materials and processes [34, 35] . in other words, fractional dynamic systems in applications can adequately represent some long-term memory and non-local effects that are typical for many anomalous processes [36] . in this calculus, a caputo derivative implies a memory effect via convolution between an integer-order derivative and a power of time [37] . note that a fractionalorder system is more stable than its integer counterpart since the stability domain in the complex space of eigenvalues of the linearized system for fractional differential equations (fdes) remains contained and is larger than the corresponding domain for ordinary differential equations [38] . fdes also help in reducing errors arising from neglected parameters in modeling of real-life phenomena [39] . ahmed and elgazzar used fdes to study non-local epidemics synergistic with other complex adaptive systems such as severe acute respiratory syndrome, avian flu, and foot-and-mouth disease [40] . local and non-local interactions were widely observed in recent outbreaks of the above diseases and the model proposed by ahmed and elgazzar captured these interactions. arafa et al. also used fdes to elegantly model childhood diseases with constant vaccination and obtained an approximate analytic solution through the homotopy analysis method [41] . immune systems include both antigen-specific and systematic response and involve memory. a fractional-order example of two immune effectors attacking an antigen was proposed by hashish and ahmed [42] . apart from ecology and epidemiology, fdes have interdisciplinary applications in various fields. for example, nonlinear earthquake oscillations can be modeled using fractional derivatives [41] and a fluiddynamics traffic model with fractional derivatives can eliminate the efficiency arising from assumption of continuous traffic flow [41] . gomez et al. used a fractional temporal operator in rc equivalent circuits to modify the standard kirchhoff law in an fde domain [43] . many researchers have tried to resolve poe from different perspectives but to the best of our knowledge there have been no studies involving fdes. here we formulate a fractional counterpart of the rosenzweig model and analyze the stability of the model. we infer that there is a critical threshold of the memory effect parameter beyond which the rosenzweig system is stable and we thus resolve poe from a new perspective. the remainder of the paper is organized as follows. we describe local and non-local kernels in section 2. in section 3 we present our mathematical model with highlight the existence and uniqueness of its solution. in section 4 we investigate the model behavior around ecologically feasible equilibria. the paper ends with results and conclusions. it is well known that integer-order derivatives have local kernels with clear physical interpretations, which significantly simplifies their use for solving applied problems in various fields of science. however, this is not true in the case of fractionalorder integrals and derivatives, which have non-local kernels. nevertheless, there is rapidly growing interest in both theory and applications to real world problems [44] . the physical interpretation of fractional integration and differentiation is based on cosmic time and individual time [45] . the fractional integral of a function is generally interpreted as the real distance passed by a moving object, for which the recorded local values of its speed represent individual speed and the local values of time represent individual time. the relationship between the locally recorded time (which is considered as flowing equably and is represented as t) and cosmic time (which is considered as flowing non-equably) τ is given by a known mathematical function g t (τ ). the function g t (τ ) describes the inhomogeneous time scale, which depends on both τ and t, which here is the last value measured for the individual time of the moving object. when t changes, the entire preceding cosmic time interval changes as well. the derivative of this distance based on local time, which is also a function of non-local time, is interpreted as the fractional derivate [45] . in other words, the homogeneous time scale is just an ideal notion and is necessary for developing mathematical models describing inhomogeneous cosmic time and its change. thus, the ideal model of equably flowing homogeneous time can be considered as a rough approximation of cosmic time, which is non-local [45] . fractional dynamical systems adequately represent some long-term memory and these non-local effects that are typical for many anomalous processes [36] . such non-local effects can be captured through the fractional differential operator, which has a power law form [46] . several mechanisms have been proposed to resolve the poe problem. this universally accepted theory is still an unachieved goal because most researchers consider only local effects and ignore non-local effects, which capture memory. there are many definitions of fractional derivatives. those most frequently used are the grunwald-letnikov, riemann-liouville (rl), and caputo definitions [44, [47] [48] [49] . note that the zero singularity inherent to the rl derivative may pose challenges and difficulties. the caputo derivative needs more regularity as it uses the first derivative of the function in question, whereas the rl derivative does not require as much smoothness. in the caputo case the derivative of a constant is zero and we can properly define the initial conditions for fdes so that they can be handled analogously to the classical integer case. the caputo derivative implies a memory effect by means of a convolution between the integer-order derivative and a power of time [44, 48, 49] . we consider the model proposed by rosenzweig [1, model 4] and explicitly studied by gilpin [3] . the model is as follows: where n is prey density and p is predator density. k is the prey carrying capacity. the predator isocline is at n = j. the other parameters of (3.1) influence the shape of the prey zero isocline and the rate of system response [3] . we assume that all parameters are positive. now we introduce fractional order into model (3.1) . the fractional rosenzweig model is obtained from the classical equations by replacing the first-order time derivatives by fractional derivatives of order α, where 0 < α < 1. the fractional relaxation equations [50] [51] [52] contain an integral operator with a slowly decaying power law kernel so that the process is not markovian and depends on the prehistory of the system [46, 53] . the new system is described by the following set of fdes: where d α y(x), α ∈ (0, 1] is the fractional-order derivative of y in the sense of caputo, defined as is the value of α rounded up to the nearest integer, y m (x) is the usual mth derivative of y(x), is the rl integral operator of order β > 0, and γ (β) is the gamma function [44, [47] [48] [49] 54] . we are now in a position to discuss the existence and uniqueness of the solution of (3.2). consider the fractional-order rosenzweig predator-prey system (3.2) with initial conditions n(0) = c 1 , p(0) = c 2 . this can be written as , t < ∞, and let c 2 (i) be the class of all continuous column vector functions let l 2 1 (i) be the class of all lebesgue-integrable column vector functions on i with norm now we consider the following assumptions [55] . condition (2) implies that the functions f i satisfy the lipschitz condition (1) and (2) with m 1 = max{b, rb, akcb, ak}. now we have the following theorem. let assumptions (1) and (2) be satisfied. then the initial value problem (3.3) has a unique solution x ∈ c 2 (i), x ′ ∈ c 2 (i σ ), and x ′ ∈ l 2 1 (i). proof. the proof follows from [55, theorem 1 ]. an approximate analytic solution of system (3.2) obtained using the homotopy analysis method [56] is presented in appendix a. the equilibrium points of system (3.2) the jacobian matrix at e 0 (0, 0) is the eigenvalues corresponding to e 0 are r, ak( e −cj − 1), so e 0 is unstable for all α ∈ (0, 1]. the jacobian matrix at e 1 (k , 0) is  . the eigenvalues corresponding to e 1 are −r, ak( e −cj − e −ck ), so e 1 is locally asymptotically stable if k < j. the jacobian matrix at e the eigenvalues corresponding to e are a sufficient condition for the local asymptotic stability of the equilibrium point e is (4.1) the major review of empirical and theoretical poe studies presented by roy and chattopadhyay [4] provides a number of plausible solutions and partial resolution of the problem. the experimental evidence was challenged by jensen and ginzburg [5] , who showed that most examples proposed as evidence of poe are insufficient to justify the phenomenon in natural systems. a common example of poe is the process of lake eutrophication. enrichment of aquatic systems appears to increase the carrying capacity of producers and leads to a bloom that covers the lake. poe may be suggested as a cause of this phenomenon, but in fact it is the limiting dissolved oxygen that is responsible for system collapse in this case, irrespective of the enrichment response. other empirical studies using the didinium-paramecium system were carried out by luckinbill [6] and veilleux [7] . both authors showed that the system proposed by gause [57] could be modified to allow coexistence of predator and prey. addition of methyl cellulose prevented the extinction of any of the species in the system and provided an artificial refuge for prey species. similarly, a study of rotifer-algae by fussmann et al. showed that predator extinction resulted from enrichment [8] . similar to the experiments of luckinbill and veilleux, the results showed that reducing nutrient input can bring the system from a region of consistent predator extinction to a region of coexistence. capture of this ubiquitous dynamic behavior involved in poe remains an open problem from both theoretical and ecological perspectives. here we considered the rosenzweig model of poe to provide a plausible solution from a new perspective that considers the ecological memory that exists for most species. most of the solutions proposed so far involve species-level organization and ignore the community level. as a consequence, the impact of memory on system dynamics has been neglected. this memory can be well captured by a fractional derivative consisting of non-local operators. the memory effect can also be incorporated using other frameworks such as volterra integral equations and delay differential equations [58] . volterra integral equations describe systems with continuously distributed memory over all past events. for example, the spread of epidemics with a general (not necessarily exponential) distribution of infection times can table 1 parameter values used to solve (3.2) (taken from [3] ). be modeled by volterra integral equations. in another example, the evolution of a set of competing biological species with memory can be modeled through a system of integro-differential equations, which can be reduced to a system of volterra integral equations [58] . besides population dynamics, the evolution of capital stock under an investment strategy can be described by a volterra integral equation. in an ode system in ecology, the representation of a process by a function is often based on experiments. the response of the function chosen can be fitted to experimental data. this functional response should not be derived mechanistically but phenomenologically [59] . the functional response may be prey-and/or predator-dependent according to experimental results. naturally, these types of experiment can have various time lengths and integrate memory. note that even if an ode describes an instantaneous process, the notion of instant depends on the time scale considered, whereas fdes have the property of fading memory and depend on the range of α (0 < α < 1). such memories can describe current events with the collective information from preceding events, while events in the far past can often be neglected compared to contributions from the near past [60] . volterra defined the notion of fading memory as ''the principle of dissipation of hereditary action'' [61] . podlubny [44] called this the ''short memory principle''. in this study we used fdes to capture the memory effect in an ecological framework. naturally we expect that our modified rosenzweig model (3.2) with memory characterized by α ∈ (0, 1) is more stable than the classical rosenzweig model with integer α(=1). note that α plays a key role in understanding memory effects on species. the system sensitivity with respect to α is an important aspect to be discussed. we analyzed the sensitivity of model (3.2) with input parameter α and output the range of k up to which the system is stable (appendix c). we observe that when the value of α decreases, the system has a high elasticity index, that is, the system is more sensitive with respect to α. since most fdes do not have exact analytic solutions, approximation and numerical techniques must be used. several analytical and numerical methods exist for solving fdes. for numerical solution of (3.2), we used the fde12 package in matlab which is an implementation of the adams-bashforth-moulton predictor-corrector method [62] [63] [64] [65] . the parameter values listed in table 1 were used. fig. 1(a) shows that the classical rosenzweig model with no memory is stable for a carrying capacity of k = 48. when we enrich the prey carrying capacity (k = 60, k = 80) and keep the other parameter fixed, the system destabilizes ( fig. 1(b) and (c) ). however, when we incorporate the memory effect in the classical model with α ∈ (0, 1), the system no longer shows a pattern of destabilization on enrichment (figs. 2 and 3 ). fig. 4 shows that the system has a memory threshold (α ≈ 0.2332) below which it is always stable, even if we further increase the carrying capacity without restriction. mathematically this intermediate value of α can be interpreted as a filter with partial memory that falls between two extreme filters with complete memory and with no memory [66] . the individual-level organization discussed above is mainly associated with the behavioral aspects of predator-prey relationships and inducible or morphological prey mechanisms. learning and memory can modify these anti-predator behavioral responses, which is key to making the link between individual-level processes and higher-level organization in ecological systems [14] . for example, prey refuge is one of the most manifest and well-coordinated actions based on memories of past events that lead to cultural learning [14] . to illustrate this issue, we incorporated a refuge parameter in the fractional-order rosenzweig conditions for which the modified rosenzweig model is stable are given in appendix b. the region of stability for the corresponding enrichment parameter (k ) and refuge parameter (m) expands if we gradually increase the memory effect in comparison to a system with no memory (fig. 5) . estimation of the model parameters for such a system is an important task from a data analysis perspective. the total least squares approach is suitable for this purpose and we applied this method to illustrate model (3.2) and fitted an estimated curve for a simulated data set (appendix c). this memory phenomenon is clearly an approximate tradeoff between fractional differentiation and stability. against this backdrop, we conclude that ecological memories can protect a system against destabilization and may represent a potential agent for resolving poe from a new perspective using fdes. we are grateful to the reviewers for their comments and suggestions on an earlier version of this paper. our research work is supported by the council of scientific and industrial research (csir), human resource development group, new delhi. in this appendix we apply the homotopy analysis method [56] to obtain a numerical analytical solution for fractional system (3.2). we choose the base function as {t (n+m)α |n, m ≥ 0}, so the solutions are of the form a n,m t (n+m)α where a n,m , b n,m are the coefficients. it is straightforward to choose n 0 (t) = c 1 , p 0 (t) = c 2 as our initial approximation of n(t) and p(t), and the linear operator is then where a is the integration constant, which is determined by the initial conditions. if q ∈ [0, 1] and h indicate the embedding and non-zero auxiliary parameters, respectively, then the zero-order deformation problems are of the following form: cn(t;q) ). for q = 0 and q = 1, zero-order (a.3) have the solutionŝ n(t; 0) = n 0 (t),p(t; 0) = p 0 (t) andn (t; 1) = n(t),p(t; 1) = p(t). when q increases from 0 to 1, thenn(t; q) andp(t; q) vary from n 0 (t) and p 0 (t) to n(t) and p(t), respectively. expandingn andp in taylor series with respect to q, we havê we take the mth-order homotopy derivative of the zero-order (a.3) and use [67, to obtain the m-order deformation equations with the initial conditions if m > 1 . in this way, it is easy to solve the linear non-homogeneous equations (a.7) with initial conditions (a.8) for all m ≥ 1 and we obtain proceeding similarly, the pth term of the approximate solutions of (3.2) is (a.10) this appendix provides the conditions for which our fractional rosenzweig model is stable. . the corresponding variational matrix is where λ 1 , λ 2 are the eigenvalues of the jacobian matrix v and α (0 < α ≤ 1) is the order of the fractional derivative. we estimated the parameters of our fractional rosenzweig model and analyzed its sensitivity with respect to α. to estimate the parameters we used the total least squares method [68, 69] , also known as orthogonal distance fitting (odf), which has significant advantages [70] in the case of several spatial dimensions. odf uses perpendicular (orthogonal) distances between given points and the fitting curve. the sum of orthogonal distances between two sets of points is described by where n is the number of experimental points and the parameters α 1 , α 2 , . . . , α n influence the function f , which is called the fitting curve or the set of fitting points; d represents the distance from given experimental points, with coordinates (x i , y i ), to the fitting curve f . we used the fmincon optimization algorithm in matlab for data fitting of model (3.2) as described by skovranek et al. [71] . the coordinates of the points to be fitted in the state space are generated randomly in 2d space. we generated an initial data set of 30 points from model (3.2) with the parameter values listed in table 1 , along with k = 20 and α = 0.75. the final data set was generated by adding gaussian noise to the data set for each predator-prey population with variance of 0.1 and 0.6 for prey and predator populations, respectively. fig. 6 shows the results, in which the green points are randomly generated and the red line represents the estimated curve. the estimated model parameters and error of fit using the fmincon procedure are given in table 2 . we conducted a sensitivity analysis of model (3.2) with respect to α. we used the sensitivity coefficient proposed by loucks et al. for our analysis [72] . this measures the magnitude of change in an output variable q per unit change in the magnitude of an input parameter value α from its base value α 0 . we used the sensitivity coefficient [73] s α = max{|(q 0 −q i )/ (α 0 − α i )|, |(q 0 − q j )/(α 0 − α j )|} and calculated the elasticity index [72, 73] e α =  α 0 q (α 0 )  s α , which is a dimensionless sensitivity measure of the relative change in output q for a relative change in input α. here the index i represent a decrease and j an increase in the parameter from its base value α 0 . for model (3.2) we calculated the elasticity index with respect to input variable α. the output(q ) is the range of k up to which the system is stable. we calculated the output (q ) for values of α from 0.4 to 1 and then calculated the elasticity index and plotted this as a function of α in fig. 7 . it is clear that the elasticity index increases as α decreases. it should be noted that enrichment does not hold for values less than a threshold of α = 0.2332, so the range α ∈ [0.4, 1] is used in fig. 7 . the paradox of enrichment limit cycles in predator-prey communities enriched predator-prey systems: theoretical stability the stability of ecosystems: a brief overview of the paradox of enrichment paradoxes or theoretical failures? the jury is still out coexistence in laboratory populations of paramecium aurelia and its predator didinium nasutum an analysis of the predatory interaction between paramecium and didinium crossing the hopf bifurcation in a live predator-prey system the effects of enrichment of three-species food chain with nonlinear functional responses the paradox of enrichment in an adaptive world imperfect optimal foraging and the paradox of enrichment food-web connectance and predator interference dampen the paradox of enrichment stable limit cycles and the paradox of enrichment in a model of chronic wasting disease learning, memory and predator avoidance by freshwater snails: effects of experience on predator recognition and defensive strategy memory effects and macroscopic manifestation of randomness episodic-like memory during cache recovery by scrub jays scrub jays (aphelocoma coerulescens) remember the relative time of caching as well as the location and content of their caches elements of episodic-like memory in animals seasonal succession of phytoplankton in a large shallow lake (balaton, hungary) -a dynamic approach to ecological memory, its possible role and mechanisms meteorological forcing of plankton dynamics in a large and deep continental european lake what is cyclomorphosis? freshwater biol predator-induced reaction norms predator identity and consumer behavior: differential effects of fish and crayfish on the habitat use of a freshwater snail antipredator responses and defensive behavior: ecological and ethological approaches for the neurosciences morphological and behavioral plasticity of larval anurans in response to different predators non-visual communication in freshwater benthos: an overview chemical alarm signalling in aquatic predator-prey systems: a review and prospectus the scent of death: chemosensory assessment of predation risk by prey animals acquired recognition of chemical stimuli from an unfamiliar predator: associative learning by adult newts, notophthalmus viridescens predator-recognition training enhances survival of brook trout: evidence from laboratory and field enclosure studies anti-predator behavior of naive compared with experienced juvenile roach dead matter has memory! scaling of seismic memory with earthquake size a new dissipation model based on memory mechanism synchronization of chaotic fractional-order systems via linear control complex oscillations and limit cycles in autonomous two-component incommensurate fractional dynamical systems fractional mechanical oscillators equilibrium points, stability and numerical solutions of fractional-order predator-prey and rabies models fractional order seir model with density dependent death rate on fractional order differential equations model for nonlocal epidemics solutions of fractional order model of childhood diseases with constant vaccination strategy towards understanding the immune system modeling and simulation of equivalent circuits in description of biological systems -a fractional calculus approach fractional differential equations geometric and physical interpretation of fractional integration and fractional differentiation fractional integrals and derivatives: theory and applications theory and applications of fractional differential equations an introduction to the fractional calculus and fractional differential equations the fractional calculus: theory and applications of differentiation and integration to arbitrary order cole-davidson dielectric relaxation as a self-similar relaxation process the justified data-curve fitting approach: recognition of the new type of kinetic equations in fractional derivatives from analysis of raw dielectric data anomalous diffusion and dielectric relaxation in an n-fold cosine potential memory regeneration phenomenon in dielectrics: the fractional derivative approach topics in fractional differential equations on the fractional-order games with non-uniform interaction rate and asymmetric games beyond perturbation: introduction to the homotopy analysis method the struggle for existence optimal control of impulsive volterra equations with variable impulse times predator functional responses: discriminating between handling and digesting prey fractional dynamics of globally slow transcription and its impact on deterministic genetic oscillation theory of functionals and of integral and integro-differential equations on linear stability of predictor-corrector algorithms for fractional differential equations efficient solution of multi-term fractional differential equations using p(ec)me methods detailed error analysis for a fractional adams method fast numerical solution of nonlinear volterra convolution equations physical and geometrical interpretation of fractional operators homotopy analysis method for solving fractional lorenz system an analysis of the total least squares problem analysis and solution of the nongeneric total least squares problem state space description of national economies: the v4 countries data fitting using solutions of differential equations: fractional-order model versus integer-order model water resources systems planning and management: an introduction to methods, models and applications hydrologic modelling: statistical methods and applications key: cord-349831-0u9y35qo authors: ellis, george f. r. title: the causal closure of physics in real world contexts date: 2020-08-18 journal: found phys doi: 10.1007/s10701-020-00366-0 sha: doc_id: 349831 cord_uid: 0u9y35qo the causal closure of physics is usually discussed in a context free way. here i discuss it in the context of engineering systems and biology, where strong emergence takes place due to a combination of upwards emergence and downwards causation (ellis, emergence in solid state physics and biology, 2020, arxiv:2004.13591). firstly, i show that causal closure is strictly limited in terms of spatial interactions because these are cases that are of necessity strongly interacting with the environment. effective spatial closure holds ceteris parabus, and can be violated by black swan events. secondly, i show that causal closure in the hierarchy of emergence is a strictly interlevel affair, and in the cases of engineering and biology encompasses all levels from the social level to the particle physics level. however effective causal closure can usefully be defined for a restricted set of levels, and one can experimentally determine effective theories that hold at each level. this does not however imply those effective theories are causally complete by themselves. in particular, the particle physics level is not causally complete by itself in the contexts of solid state physics (because of interlevel wave–particle duality), digital computers (where algorithms determine outcomes), or biology (because of time dependent constraints). furthermore inextricably intertwined levels occur in all these contexts. it is often supposed that causal closure occurs at the micro level in physical systems, and hence prevents the occurrence of strong emergence because the macrostate supervenes on the microstate [81, 82] . this is discussed in [27, 68, 89, 54] . in contrast, [20] shows by careful philosophical argument that one can have emergence with reduction, as well as without it; and emergence without supervenience, as well as with it. here i want to examine the issue in a different way, by dealing in some detail with the hierarchical nature of emergence in real world contexts: the cases of engineering, based in the underlying solid state physics, and biology, based in the underlying molecular biology, in turn based in the underlying physics. the context is my paper [41] that establishes that strong emergence does indeed take place in both those cases, so that the argument against strong emergence has to be wrong in those contexts. reference [41] examines the issue of strong emergence of properties p of macrodynamics m out of the underlying microdynamics m in the context of condensed matter physics and biology. following anderson's lead [4, 5] that symmetry breaking is at the heart of emergence, its method was to identify five different kinds of symmetry breaking occurring in different contexts ( [41] , sect. 2.3] and then to trace how broken symmetry states at the macro and micro levels interact with each other. • the microscale dynamics m, based in the laws of physics l, obeys symmetries s: s(m)=m. spontaneous symmetry breaking ssb(m) leads to symmetry breaking of the macro scale dynamics m through the emergence process e whereby m emerges from the microscale dynamics m. this is weak emergence ∶ → ∶ ( ) ≠ . • this spontaneously broken macro state m reaches down to create quasiparticles such as phonons at the micro level, which play a key dynamical role at that level. 1 the base microdynamics m is altered to produce an effective microdynamics m ′ which breaks the symmetry of the underlying physical laws l. thus → � ∶ ( � ) ≠ . • to derive correctly the properties of macro dynamics m from the micro dynamical level, you must coarse grain the effective theory m ′ rather than m. • thus strong emergence takes place in this case: you cannot even in principle derive the macrodynamics m from the microdynamics m in a strictly bottom up way, because m satisfies the symmetry s and m does not. 2 as a consequence [41] : sect. 4.4, in the case of solid state physics, the underlying microphysics m cannot be causally complete, because by itself it cannot lead to the emergence of known properties of solids such as electrical conductivity. the lower level physics only gives the correct outcome when modified by inclusion of terms a(m) arising from the higher level state m (so ( ) ≠ ). the same is true for living systems. that is, in both these cases, causal completeness is only attained by considering both the low level properties m and the higher level properties m (which lead to the alteration → ′ ) together. the real causally closed system comprises both those levels. the aim of this paper is to extend that result by investigating causal closure of physics in terms of determining dynamic properties 3 p(d) of entities in engineering and biological contexts (i use the word closure rather than completeness for reasons that become apparent below as the theme develops). a separate very interesting project would extend this to considering the dynamic emergence of entities e(d) over time, see e.g. [24] . section 2 sets the context for the discussion, which is the hierarchies of emergence in the cases of engineering and the life sciences respectively (table 1) . it introduce the idea of an effective theory at each level l, and discusses bottom up and top down causation in the hierarchy of emergence (sect. 2.3). section 3 introduces the idea of a domain of interest (doi), and the concept of effective spatial closure (sect. 3.3) . it is shown that in terms of spatial interactions, causal closure in engineering and biology only holds ceteris parabus. yes of course philosophers know that this is the case; the point is that it has real consequences in real world contexts. section 4 defines levels of interest (loi) and sect. 4.4 introduces the need for restricted domains of interest. section 5 introduces interlevel causal closure in the case of biology (sect. 5.3), sect. 6 extends this to the case of digital computers and physics. section 7 summarizes the main results of this paper, emphasizes that unavoidable unpredictability also undermines causal closure, and comments on ways people ignore the issues raised in this paper. the novel concepts introduced are effective theories the context of the discussion is the hierarchy of emergence. as stated by [4] , at each level of complexity, entirely new properties appear ... at each stage entirely new laws, concepts, and generalizations are necessary. the emergent hierarchy is shown in table 1 for the cases of engineering on the left and life sciences, in particular the case of humanity, on the right. the left hand side represent the sciences of the artificial as discussed by [126] . the right hand side represents the structures and processes of biology, as discussed by [23] . this hierarchy has important aspects. • the bottom three levels l1-l3 are the same on both sides. this is one of the great discoveries science has made: inanimate matter and living matter are made of the same stuff at the bottom. electrons are at level l3, interacting with the nucleus. • the atomic level l3 is where new properties emerge out of the underlying physics, as characterised by the periodic table of the elements, another great discovery. • the components enabling complexity to arise occur at level l4. both semiconductors and metals are crystals, and they are the key components of machines. 4 solid state physics covers levels l3-l4. biomolecules such as dna, rna, and proteins are the foundations of biological emergence. • level l5 is where the basic units of complexity arise, showing functional emergent properties, and being the basis for building complex entities. on the machines side, these are devices such as transistors, light emitting diodes, photodetectors, and lasers. on the life sciences side, they are cells: the basic building block of life, which come in many different types. this is the lowest level where the processes of life occur, entailing metabolism and information processing hartwell et al. [61] . • level l6 is where on the machine side, devices are integrated into functional units, such as the central processing unit in a microprocessor, which is itself a device. on the life sciences side, organs comprising physiological systems occur. • level l7 is where functional units occur that have an integrity of their own: they are effectively causally closed systems imbedded in a larger environment. on the engineering side, they are machines built to carry out some purpose, such as aircraft or digital computers or particle colliders. on the life sciences side, they are individuals with autonomy of action. • level l8 is the same on both sides. both machines and individual human beings exist in the context of a society with social, economic, and political aspects, which sets the stage for their existence and functioning. • finally, level l9 is again the same on both sides. it reflects the fact that each society exists in a natural environment with both ecological and geophysical aspects. note that this table has chosen a particular set of levels to represent causation all the way from the particle physics level l1 to the environmental level l9. however most scientific studies will be interested in a much more restricted sets of levels: the levels of interest (lois) discussed in sect. 4. given such a choice, one will in general use a more fine-grained set of levels than represented in table 1 . thus for example if the loi is (l4-l6), one might divide that range into a finer set of sublevels. it is a common belief that the lower levels are more real than the higher levels, because bottom up causation from the lower to higher levels is the source of higher level properties. arthur eddington in on the nature of the physical world ( [34] , pp. 5-12) muses on the dual (solid macroscopic/atomic microscopic) nature of his writing desk, and concludes (p. 10) that because of the scientific world view, the external world of physics has thus become a world of shadows. in removing our illusions we have removed the substance, for indeed we have seen that substance is one of our great illusions. however this view is subject to dispute. richard feynman in his book the character of physical law (50] , pp. 125-126) considers whether one level or another is more fundamental, and using a religious metaphor, argues that 'the fundamental laws are no nearer to god than emergent laws'. 5 phil anderson arguably had a similar view. sylvan schweber commented as follows [124] : anderson believes in emergent laws. he holds the view that each level has its own "fundamental" laws and its own ontology. translated into the language of particle physicists, anderson would say each level has its effective lagrangian and its set of quasistable particles. in each level the effective lagrangian -the "fundamental" description at that level -is the best we can do. thus this does not recognize any level as more fundamental than any other. recently, denis noble has proposed a "principle of biological relativity" [105] : all levels one deals with in studying emergence in biology are equally valid, there is no privileged level of causation. effective theories a good way to express this is that there is a valid effective theory 6 (et) at each level. elena castellani gives this definition [25] : an effective theory (et) is a theory which 'effectively' captures what is physically relevant in a given domain, where 'theory' is a set of fundamental equations (or simply some lagrangian) for describing some entities, their behaviour and interactions... more precisely, an et is an appropriate description of the important (relevant) physics in a given region of the parameter space of the physical world. in parallel to the way the functioning of the laws of physics was sketched in [41] , one can characterise an effective theory valid at some level l as follows; an effective theory at a level l is a reliable relation between initial conditions described by effective variables v ∈ and outcomes o ∈ : in a reliable way, whether is an exact or statistical law. it is important to note that an effective theory may have a randomisation element r: where r might for example produce a gaussian distribution. equal causal validity in terms of effective theories for emergent properties p(d), noble's principle [105] as extended in [41] can be restated: table 1 ) represents an effective theory , so each level is equally valid in a causal sense. this implies no level is a fundamental level with priority over the others, and particularly there is not a primary one at the bottom level. this is just as well, because there is no well-established bottom-most physical level to which physics can be reduced [102] . every emergent level equally represents an effective theory. 7 equality of validity of effective theories at every level is possible because causation is not just bottom-up. rather higher level properties p(d) are linked to lower levels by a combination of upwards and downwards causation [39, 41, 105] , which enables emergence of effective laws at each level. upwards emergence this has two different aspects ( [41] , §1.1). 7 while luu and meißner are critical of my claims on emergence [87] , they agree on this point. 6 not to be confused with an effective field theory (eft), see [19, 25, 60] , which is a special case of an et. note that efts such as in [87] cannot deal with emergence in solid state physics, as they do not allow for symmetry breaking. first there is the emergence e of a macro system from its components. in terms of levels, this corresponds to creation of a higher level ln from a lower level ln: that is, → , > . this may lead to topological non-trivial states emerging such as networks, or quantum entanglement may take place. the issue of phase transitions is important here. first order phase transitions occur when spontaneous symmetry breaking ssb occurs leading to the emergent level et having lower symmetries than the underlying et. in terms of the associated micro dynamics m and macrodynamics m, if s is the symmetry set of m, then second there is emergence p of properties of the emergent level ln out of properties of the underlying constituent level ln once ln has come into existence. this corresponds to emergence of a higher level out of a lower level one. some form of coarse graining c of properties may suffice if the higher and lower levels have the same symmetries s, but not if their symmetries are different due to ssb (see [41] ). downward causation a classification of different types of downward causation was given in [37, 39] . here i will rather approach the issue from an viewpoint. there are essentially two kinds of downwards effects that can happen: downward alteration of lower level dynamics l via either constraints or effective potentials, and downward alteration of dynamics at level l by altering the set of lower level variables. constraints and effective potentials the way downward causation by constraints works is that the outcomes p(d) at level l depend on constraints c at the level l arising from conditions at a level of influence . thus when interlevel interactions are taken into account, relation (1) is modified (see [41, (30) essentially the same holds if the effect of the level of influence li on the level l is expressed in terms of an effective potential v(v ) at level l (see [41, (9) ]). thus constraints act as causes [76] . the constraints c may be time independent: c ∕ t = 0 in which case they are structural constraints; or they may be time dependent: c = c (t), c ∕ t ≠ 0, in which case they are signalling or controlling constraints. an important case is feedback control (engineering), which is essentially the same as homeostasis (biology). then the constraints c (t) depend on goals g valid at level l but set at the level of influence li. similarly the potential v(v ) may depend on time-dependent variin both cases the level l is no longer causally complete on its own; at a minimum, only the combination {l,{li} of levels can be causally complete. altered variables the causal effect due to the level of influence li may rather be due to changes in the variables v at level l due to variables v at the higher level li: where the new set {v � } of effective variables at level l may be smaller, larger, or altered. they are smaller if they are changed by deleting lower level elements. this occurs when downward causation by adaptive selection takes place, altering or deleting selected lower level elements according to some selection criterion c. this enables alteration of structures and functions at level l so as to meet new challenges at level li. this plays an important role in enabling by organisms to have agency and choice, enabled by stochasticity, as explained in [106] : choice in the behavior of organisms involves novelty, which may be unpredictable. yet in retrospect, we can usually provide a rationale for the choice. a deterministic view of life cannot explain this. the solution to this paradox is that organisms can harness stochasticity through which they can generate many possible solutions to environmental challenges. they must then employ a comparator to find the solution that fits the challenge. what therefore is unpredictable in prospect can become comprehensible in retrospect. harnessing stochastic and/or chaotic processes is essential to the ability of organisms to have agency and to make choices. they are larger if for example one has downward creation of quasiparticles such as phonons via interlevel wave-particle duality ( [41] and sect. 6.3), which underlies the properties of metals and semi-conductors. this is what carl gillett calls a "foundational determinative relation" [55] . the are altered if the number is the same but the properties of an element changes. when they are bound in an emergent complex their own properties may change (for example, neutrons decay in 11 min when free but last for billions of years when bound in a nucleus), or their interactions with external entities may change (for example electrons bound in an atom interact with light quite differently than a free electron does). downward causation is related to aristotle's formal cause, see [130] , but i will not follow that strand here. to give these rather abstract statements flesh, see many examples given in [39, 104] . downward causation in relation to the key physics-chemistry link is discussed in [86] . multiple realisability of higher level variables at lower levels plays a key role in downward causation [93] . any particular higher level state can be realised in a multiplicity of ways in terms of lower level states. in an engineering or biological cases, a high level need determines the high level effective function that needs to be realised and thus the high level structure that fulfills it. this higher structure and function is then realised by suitable lower level structures and functions, but there are billions of ways this can happen it does not matter which of the equivalence class of lower level realisations is used to fulfill the higher level need, as long as it is indeed fulfilled. consequently you cannot even express the dynamics driving what is happening in a sensible way at a lower level. the issue is not just the huge number lower level entities involved in realising a higher level systems, as characterised by avagadro's number it is the huge different numbers of ways combinations of lower level entities can represent a single higher level variable. any one of the entire equivalence class at the lower level will do. thus it is not the individual variables at the lower level that are the key to what is going on: it is the equivalence class to which they belong. but that whole equivalence class can be describer by a single variable at the macro level, so that is the real effective variable in the dynamics that is going on. this is a kind of interlevel duality: where e − (v − ) is the equivalence class of variables v − at level − corresponding to the one variable v at level l. the effective law at level l for the (possibly vectorial or matrix) variables v at that level is equivalent to a law for an entire equivalence class e − (v − ) of variables at level l-1. it does not translate into an effective law for natural variables v − per se at level l-1. it is important to note the following: one establishes the validity of an for some chosen level l by doing experiments or making observations on phenomena occurring at that level. this involves the experimenter intervening at the level l, hence it is an interlevel interaction. for example a particle physics experiment considers effective laws at level l1 but involves scientists at level l7 and organisations at level l8 acting down to affect things at level l1. consequently, one can make the following important observation: existence and functioning of effective theories at level l does not necessarily imply causal closure of level l. the issue is what determines constraints c, potentials v, and effective variables v ′ that may occur at that level. they may be influenced by other levels. that is what sects. 5 and 6 are about. determining an effective law at level l involves other levels then l. looking in more detail at the hierarchy, it is a hierarchy made of modules (this section) which form networks (next section). it is a modular hierarchy for very good reasons. five principles of complex structure ([16] , §1.3) gives five principles of complex structure, developing from [126] , starting from the idea the role of decomposition: the technique of mastering complexity has been known since ancient times: divide et impera (divide and rule). the five principles, applicable to both engineering and biology, are stated by him to be, (1) modules modularity is the property of a system that has been decomposed into a set of cohesive and loosely coupled modules ( [16] , p. 56). they can be represented by abstractions, where "an abstraction denotes the essential characteristics of an object that distinguish it from all other kinds of objects and thus provide crisply defined conceptual boundaries, relative to the perspective of the viewer. an abstraction focuses on the outside view of an object and so serves to separate an object's essential behavior from its implementation" ( [16] , pp. [44] [45] [46] [47] [48] [49] [50] . they involve encapsulation ( [16] , pp. [50] [51] [52] [53] , that is, the internal details of the module's workings are hidden from the external world), and multiple realisability: the required module functioning can be fulfilled in many ways by its internal structure and variables. hierarchy is a ranking or ordering of abstractions ( [16] , p. 58). a feature of particular interest is that emergent systems may give rise to effective theories that involve topological constraints. indeed this happens quite often because emergent complexity in both engineering and biology often involves interaction networks, and a key feature of such networks is their topological connectivity, described by graph theory. thus for example arthur peacocke points out that in electrical circuit theory there are certain topological constraints, the boundary conditions that one element imposes on another ( [112] , p. 74). they obviously have strongly emergent properties: their functioning does not follow from any local characteristics of the elements that make up the circuit. the electric light won't work until you change its open circuit topology (isomorphic to an open interval) when the switch is off, to a closed topology (isomorphic to a circle) when the switch is on. this macro event than reaches done to alter the flow of billions of electrons at the micro level. networks can be physical networks, or interaction networks. physical networks physical networks are embodied in physical links between nodes, which constrain what interactions can take place by dictating what nodes can interact with what other nodes. thus physical networks in fact create interaction networks by constraining interactions between links. this is the key structure-function relationship of engineering and biology. examples in engineering are computer architecture [132] , computer networks [83] , and artificial neural networks [73] . the case of importance in biology is the nervous system [59] and neural networks [63] . interaction networks interaction networks occur due to the presence of a variety of reagents that selectively interact with each other. this requires firstly a container that keeps the reagents within interaction distance of each other, rather than just diffusing away, and second the presence of an appropriate set of reagent that do indeed interact with each other. a key role is then played by selectively letting specific reagents enter or exit the container so as to control their interaction densities. on a large scale, examples of importance in engineering are purification plants, chemical engineering reaction vessels, water treatment plants, sewage treatment plants. in biology, they arguably are the endocrine system, controlling signalling, and the digestive system, controlling metabolism at the systems level [59] , and on a larger scale, ecological networks [75] . on a small scale, there are many interaction networks in cell biology [18] . these are crucially dependent on the existence of cells bounded by cell walls, that serve as the necessary reaction containers. they have ion channels imbedded in those walls that control movement of ions in and out of the cells, and molecular channels controlling movement of molecules in and out. they include • gene regulatory networks [24, 75, 139] , also known as transcription networks [3] , • metabolic networks [18, 75, 139] , • cell signalling networks [11, 18] , • protein interaction networks [18, 75] , • signal transduction networks [18, 75] . these networks are the heart of cell biology [85] and underlie how information flows and logic underlie biological functioning as emphasized by [30, 45, 109] . networks and hierarchy networks may have a hierarchical character in that subnetworks can often be identified within an overall network, and so define levels within the network [111, 121, 142] . this is an interesting topic i will not develop further here except to remark that firstly, subnetworks include network motifs [3, 94] , which are small subnetworks of particular functional significance. for example they include the autoregulation motif, which is nothing other than feedback control ( [3] , pp. [27] [28] [29] [30] [31] [32] [33] [34] [35] [36] [37] [38] [39] [40] and the feed-forward loop motif ( [3] , pp. . they may contain higher-dimensional interactions characterised by clique complexes [118] . networks may also contain hubs, central nodes of importance [75] . their nature is highly dynamic [32] . causation because interaction networks are directed graphs (i.e. the edges between nodes have orientations), they represent causal effects, where causation is defined as [113, 114] . reference [67] shows how such diagrams can be used to exemplify causal entailment in a diverse range of processes: enzyme action, construction of automata, and ribosomal polypeptide synthesis through the genetic code. their causal effects can be tested by experiment, where this is possible (vary conditions at one node and show that, ceteris parabus (i.e. conditions at other links to the node are unchanged) this results in a reliable change at another node. when this is not possible, one can use counterfactual arguments: demonstrating that as a result of the nature of the network links this should indeed be the outcome if one were to make such a change. this is the kind of argument i will use to claim that both upward and downward relations between levels are also causal ( table 3) . networks and strong emergence because of their systemic properties, biochemical networks display strong emergence [17] . in examining the issue of causal closure of properties p, one must have the context clearly in mind. to do so, it is useful to define the domain of interest (doi) of such study. this has three quite different aspects. first, there will a specific topic of interest (toi) one wishes to investigate. for example, it might be physics or engineering or chemistry or biology. in physics, one might have in mind atomic physics or condensed matter physics or plasma physics; in biology, molecular biology or physiology or neuroscience or population evolution. or one might want to investigate relations between various of these topics. in this paper, the interest is the nature of causal closure in the relation between physics, engineering, and biology. second, given a choice of topic of interest, the domain of interest doi of a system of interest s consists firstly of interaction limits for s with its surroundings, and secondly of time limits on the duration when we are interested in the behaviour of s. together these comprise spacetime limitations (sect. 3.1), leading to effectively isolated systems in the case of physics (sect. 3.2) and effective spatial closure in the case of biology and engineering (sect. 3.3). thirdly, there will be a choice of levels of interest (loi). the issue of lois is the focus of this paper, and is discussed in the following sect. 4. to be of physical interest, s must be spatially limited. although they are often talked about, systems of infinite extent do not occur in the real universe [46] . 8 spacetime boundaries define the time and spatial domain we are interested in in relation to s. from a spacetime viewpoint, this is a world tube of finite radius r that surrounds s, large enough to contain s and all the elements strongly interacting with it, bounded by an initial time t i and final time t f defined in a suitable way. this governs the kinds of interactions it can have with other systems. time limitations we may be interested in short or long timescales characterised by the starting time t i and ending time t f , depending on what we wish to study. we may be interested in, • evolutionary processes e whereby the family of systems of similar type to s came into existence over long timescales via reproduction with variation followed by selection; • developmental processes e whereby a specific system s came into existence through developmental or manufacturing processes, or perhaps by self assembly; • functional processes whereby the properties p of the system s considered over short timescales emerge from the underlying physics. this is the focus of this paper. each involves very different choices of the timescale δt ∶= t f − t i relevant to our study. isolated systems causal closure of a system s cannot happen if uncontrolled influences arrive from the surroundings: as these influences vary with time, they will cause changes in the the state of the system with time that cannot be predicted from a knowledge of the properties of the system alone. the system is not causally closed. physics deals with this by introducing the idea of an isolated system. this is usually expressed by giving limits on any incoming influences "at infinity", for example such conditions are imposed in studying electromagnetic and gravitational radiation. however, as just stated, infinity is not a valid physics concept. one should instead refer to finite infinity i [36] : a world tube of finite radius r i ≫ r chosen so that incoming radiation and matter will not seriously interfere with s. 9 the dynamics of the system will then be autonomous except for small perturbations due to incoming matter and radiation crossing i, which can be treated as small effects. the dynamics of s can be treated as an autonomous system, affected by small incoming perturbations over i. however, there are two problems with this idea: one to do with physics, and one to do with engineering and biology. causal domains the first is that famously, in general relativity, causal domains are determined by null cones rather than timelike tubes [62] . why have i not defined the causal limits, which are basic in term of causal closure, in terms of null surfaces rather than a timelike world tube? the answer is that on astronomical scales, effective causal limits are indeed given by timelike world tubes rather than null surfaces. on astronomical scales at recent times, the dynamic effects of radiation are very small compared with those of matter. we get a very usable i by choosing r i to be about 1 megaparsec in comoving coordinates [47] . nothing outside there has had a significant effect on the history of our galaxy or the solar system. yes some radiation and matter is coming in, but it is negligible compared to the energies involved in daily life. the one form of radiation of significance for the world is light from the sun, which comes from well within those limits: 1 astronomical unit might indeed suffice for local physics. the radiative energy coming from greater distances has negligible dynamical effects on earth. a timelike world tube of radius 1 au will do just fine in terms of considering causal closure of the solar system. isolated systems: laboratories however physics practice works in a different way: the key concept is an isolated system in a laboratory. it's a system that is in fact interacting strongly with the environment (sect. 3.3), but that interaction is strictly controlled so that it is highly predictable. the system is shielded from influences outside the laboratory as far as possible. this then enables the results of experiments to also be highly predictable. and that is what enables the determination of the effective theories that hold at a level l. examples are the expensive isolation and cooling systems underlying the success of quantum optics experiments. engineering and biology as open systems the real problem is different. it is that no biological system can be closed: they have by their nature to be open systems. and the same is true for engineering systems. in these cases, the 'isolated system' paradigm is simply wrong. life cannot exist as an isolated system. biological systems are inherently open systems interacting with the environment [65, 112, pp. 10-11] ) states, biological organisations can only maintain themselves in existence if there is a flow of energy and this flow requires that the system not be in equilibrium and therefore spatially inhomogeneous. 10 the effect of the outside world is not negligible. on the contrary, it is essential to biological functioning. it cannot be treated as a perturbation. the biosphere experiences incoming high grade radiation from the sun and radiates outgoing low grade heat to the dark sky, and this is its energy source enabling it to function thermodynamically [115] . organisms need a flow of material in, and, because of the second law of thermodynamics (essentially: as time progresses, matter and energy will be transformed from usable to unusable forms), need to dispose of waste matter and heat resulting from internal non-equilibrium metabolic processes. a living system s must take in materials and energy from the surrounding environment e and dispose of waste matter and energy to e: in summary, living systems are essentially interacting systems. the same is true for engineering systems, because they do work of some kind. reliable interactions they must therefore interact strongly with an environment that is stable enough that the interactions with the environment are reliable and reasonably constant so they do not disrupt the dynamics of the system over time. in physics this is the concept of a heath bath or thermal reservoir. you are in contact with an environment but don't need to take its dynamics into account because it is in a state usually assumed to be static, characterised only by a constant temperature t; and it is so large that the system s has negligible influence on it state. 11 life: interaction limits how then does one limit those interactions to those that will enable life to sustain itself? humberto maturana and francisco varela essentially dealt with this by introducing the idea of autopoiesis [91] , which inter alia expresses the idea of causal closure in terms of system interaction with its environment. the key point is how does one define the boundary of a system in this context. instead of choosing a spacetime tube of some chosen radius r as in the astronomy case, one chooses a system boundary b that characterizes the system as being effectively autonomous. this underlies the meaning of level l8 in the hierarchy of emergence (table 1 , sect. 2.1). a person has a skin that is her physical boundary with the outside world, but still allows interaction with it. energy and matter transfer takes place across the boundary. a machine similarly has a boundary that defines its limits, but will have some form of energy input enabling it to do work, and in many cases complex cooling devices to get waste heat out (paradoxically, they may consume large amounts of energy). but this idea extends down to other levels, for example it holds also at the device level/cellular level l5 and the component level/organ level l6. for example, a cell is the core of biology. it exists as an entity with its own integrity, characterised by the cell wall which allows controlled ingress and egress of materials and energy, yet interacts strongly with the environment. how then does life handle functioning in this context? the environment must be sufficiently stable so as to allow effective predictability. this is the case when the system is not causally closed but has predictable interactions with the environment that makes its own functions predictable. it has an environment that can be treated as predictable up to perturbations. the environment may change with time, but if so, slowly enough to allow adaptation to the changing situation. this is often the case, and is what on the one hand allows living systems to flourish, and on the other allows biology to be a genuine scientific subject [23] . the exceptions but that is not always the case: take covid-19 as an example. that started at the social level (l9) in one house, then spread worldwide via aircraft affecting life world wide and thereby affecting in a downward way all the biomolecules (l4) and electrons (l1) in the bodies of doctors and nurses and patients effected. but additionally it affected the engineering side by closing down thousands of flights across the world, thereby reaching down to affect all the billions of atoms (l3) and particles (l1) that make up those aircraft: a classic example of the interlevel causation i turn to next. as far as predictability is concerned, the system s has reliable interactions with the environment and so is predictable most of the time, except when we this is not the case. predictability holds when all things are equal, but there is no guarantee this will be the situation. and that is the best we can do. 12 elaborating, investopaedia states this concept as follows: 12 and no this could not be predicted in principle in a strictly bottom up way if we knew the detailed positions and momenta of all the particles in our causal past to utmost precision, for multiple reasons [41, 42] . these are strongly emergent phenomena, unpredictable even in principle. ceteris paribus is a latin phrase that generally means 'all other things being equal.' in economics, it acts as a shorthand indication of the effect one economic variable has on another, provided all other variables remain the same. ... ceteris paribus assumptions help transform an otherwise deductive social science into a methodologically positive 'hard' science. it creates an imaginary system of rules and conditions from which economists can pursue a specific end. put another way; it helps the economist circumvent human nature and the problems of limited knowledge'. in other words, effective spatial closure works except when it doesn't. unpredictability happens when black swan events take place, possible when there is a fattailed rather than a gaussian distribution [131] , and with major significance at the macro level. and when it doesn't work, the effects chain all the way down from level l8 to the atomic level l3 and particle level l1 in table 1 , as in the case of the coronavirus pandemic. setting this aside, the key conceptual issue i will deal with in this paper is the relation of the causal closure of emergent properties p to levels in the hierarchy of emergence. effective theories and existence of levels is discussed in sect (sect. 2.2) is a set of variables and equations representing interactions and constraints at a particular level l, such that initial data implies a reliable outcome at that level. it is the possibility of existence of an effective theory (1) at each level l (the dynamics at that level is determined at that level) that underlies the very concept of levels in the first place. as commented by peacocke ([112] , p. 10), following from [126] natural hierarchies are more often than not 'nearly decomposable' -that is, the interactions among the sub-systems (the 'parts') are relatively weak compared withe the interactions among the subsystems, a property which simplifies there behaviour and description. the fact that such levels exist is a consequence of the nature of the underlying physical laws and the values of the constants of nature [136] . it allows the existence of the modular hierarchical structures that are the core foundation of complexity (sects. 2.1, 2.6). reference [126] defines a hierarchical system as follows, quoted in ( [112] , p. 249): 'a hierarchical system: a system of composed of inter-related subsystems, each of the latter being in turn, hierarchical in structure until we reach some lowest level of elementary subsystem (the choice of this lowest level he regarded as arbitrary). this is what i have taken for granted above. while it has all the levels shown in table 1 (sect. 2.1), we usually do not want to consider them all at once. reference [15] says it thus: thus we take it as a given that when a portion of the universe is selected for study, be it a gas or a galaxy, we are allowed to blissfully ignore what is going on at scales that are much larger, or indeed much smaller, than the one we are considering. that is what i am formalizing by defining the concept of levels of interest (loi). they are defined as follows: a loi is defined by its top level tl and its bottom level bl, thus where the union sign " ∪ " means include all levels between tl and bl. some studies are unilevel: tl = bl, and some are explicitly interlevel: > • one can validly define such a loi regardless of what levels you choose, because the levels are equally causally valid (sect. 2.2). • your choice will depend on your topic of interest (toi) (see sect. 3). it is helpful if the choice of levels is made explicit, e.g. loi(3-5) covers levels 3 to 5. • then one can for the purposes of studying the dynamics at those levels legitimately ignore higher and lower levels, in the sense to be explored below. • this is possible because the levels effectively decouple in the sense of allowing valid effective theories at each level l. • however this does not give you the right to deny the validity of levels that lie outside your levels of interest. • there is no guarantee that causal completion will occur by including only those levels characterised by your choice of loi. that is the topic of interlevel causal closure, which i discuss below (sect. 5). are there limits on the lois one can choose? one can choose to investigate any desired lois, not investigating or take for granted the interactions that will inevitably occur from higher and lower levels. this is done to establish s. for example noble [103] "modeling the heart-from genes to cells to the whole organ" chooses to investigate the range of levels stated, namely levels l5 to l8 in the hierarchy of emergence (table 1) , and not for example on the one hand the interactions between protons and electrons that make this possible, and on the other hand the mental and social influences that will inevitably be having an effect on how the heart is functioning. what he does do is investigate the interlevel relations within the levels loi(5-8) he has chosen, because that is the domain of physiology. other examples are shown in table 2 . physics right down to level l1 always underlies what is happening, even if it lies outside the levels of interest to you. what happens in the real world right down to the physics levels l1-l3 is always influenced by what happens at higher levels including l9, even if that is not what interests you in your particular studies. what is a sensible doi depends on conditions. the suitable choice of lois will follow. this is the context within which "causal completeness of physics" must be evaluated. strongly interacting levels there is a basic problem with lois, however. that is the fact that, as mentioned above, every level interacts with every other level! the choices one makes relates inter alia as to whether one wants to answer how questions, why questions, or both (sect. 4.5). there is a practical way out that i discuss in sect. 5.3 by introducing the idea of effective causal closure. in order to examine the emergence of machines on the engineering side and organisms on the biology side, all one needs is newton's laws of motion, maxwell's equations, galilean gravity, and maybe the schrödinger equation ( [84] , §4.1); in [41] . that is, level l3 is an adequate base. all engineering and biology emerges from this level, it is the lowest level engineers and biologists need to study. as explained in [41] and sect. 2.3, conversely engineering and biology reach down to shape outcomes at level l3 via time dependent potentials or constraints. however levels l3 and l2 emerge from the quantum field theory and the standard model of particle physics at level l1 [110] . thus l1 is a deeper foundation of emergence. however it is essentially decoupled from everyday life [4, 84] . nevertheless outcomes at level l1 too must also be shaped in a downward way by engineering and biological variables, via outcomes at level l3. but this is not the bottom. underlying l1 is some theory of fundamental physics at level l0, a "theory of everything" (toe) [141] : maybe string theory/m theory, maybe not. there are variety of competing theories on offer [102] . this is the ultimate source of the emergence of dynamic properties p(d) in engineering and biology (using the notation of ( [41] , §2.2). it affects us every day. these lie outside the lois of engineers and biologists. that is just as well, as we do not know what the answer is at level l0, even though it underlies all physical emergence. as explained above, it suffices to deal with effective theories that hold at higher levels. at the top: cosmology and existence e consider isaac newton seeing an apple drop. this occurs for a variety of reasons: the law of gravity acting on the apple, the light rays that convey this image to his retina, the analysis of the image by his brain, and so on. but there are far deeper underlying issues. why does the apple exist? why does the earth exist? why does the solar system and the galaxy exist? why does the universe exist and have the nature it does? these are all the background reasons why the apple fell, and why isaac newton existed for that matter. these questions of how everything came into being (e) is the domain of cosmology [117] . it deals with issues such as, where do elements such as helium and carbon come from? how did the galaxy, the sun, and the earth arise? the philosophy of cosmology [38] considers issues such as why is physics of such a nature as to allow life to exist? reference [128] . particularly: why are the constants of nature [136] of such a character as to allow the hierarchical structure in table 1 to emerge? thus cosmology affects us every day by underlying our existence. everyday effects of cosmology p there are more immediate issues as well, in the relation of cosmology to everyday life [36] : why is the sky dark at night, serving as a heat sink for the earth's waste energy? this is crucial to the functioning of the biosphere ( [115] , pp. 411-417). why is there an arrow of time? references [29, 44, 115] ? this is crucial to all macro level physics, biology, and chemistry. both are due to the cosmological context. thus cosmology affects the emergence of properties p in engineering and biology today. the point then is that while it does indeed have a major causal effect on daily life [125] , this is a rock solid relation that does not change with time. it is a fixed unchanging background that does not alter effective laws as time passes. it is thus not a case of ceteris parabus 13 (c.f. sect. 3.3) and so can be taken for granted and not considered further when investigating causal closure in engineering, biology, and physics. an issue in choosing lois is if one is interested in how questions or why questions. how questions consider physical interactions on the one hand, and mechanisms on the other. thus they will relate to levels l1-l7 in table 1 , including l7 because that relates to the integration of systems to produce the organism as a who [120, 122] . why questions relate to motivation, meaning, and philosophical issues. thus they will relate to levels l7-l8 in table 1 , including l7 because this is the level where as well as philosophy, psychology and motivation come in [33, 79] and l8 where the causal power of social structures enters [35] . there is of course a trend for some strong reductionists to deny that the why questions are valid or have any real meaning. from the viewpoint of this paper, that simply means that they themselves have a restricted set of lois that excludes those higher levels. because of the equal validity of levels espoused in [41, 105] , and in this paper, that restricted set of interests does not provide a justification for denying the validity of the levels with effective theories outside their particular set of interests. the crucial concept in this paper is that of interlevel causal closure of properties p(d). i first consider causation and causal closure (sect. 5.1) and the nature of biology (sect. 5.2). then i consider interlevel causal closure in the case of biology (sect. 5.3), because this is where it is clearest and has been discussed most. i introduce here the key concept of effective causal closure. a stronger relation is the idea of inextricably intertwined levels(iils) which i discuss in sect. 5.4. 13 unless we live in a false vacuum in which case local physics could suddenly change in a way that might wipe out all life [133] . causation in order to consider causal closure, one must first have a view on how one justifies claims of causation. this has been laid out in depth by pearl [113, 114] . causal inference is based in causal models (directed graphs) validated by experimental intervention, or when that is not possible, by counterfactual arguments. causal models here one consider causal models of the influences at work . in effect, the diagram of the hierarchy of levels table 1 in sect. 2 is such a (very simplified) model, when one introduces the arrows of both upward emergence (left) and downward constraint or control (right). to develop this approach more fully, one needs to expand table 3 to a hierarchical diagram that represents the modular nature of the hierarchy (sect. 2.6). this is a very worthwhile project, but i will not attempt it here. it is roughly indicated in ( [112] , pp. [8] [9] [10] [11] [247] [248] , and examples are in ( [18] , pp. 6, 10-11, 22, 95, 110, 132, 160) . intervention here one actually intervenes at level li and reliably observes a resultant change at level lf. this has been done both for effective theories at each level l, and in many case for both upwards and downwards interlevel effects. one can do this also using digital computer models; for example [51, 105] have done this to show downwards causation occurring in computer models of heart function. counterfactual views [48] here one considers what would happen if one intervened at level li, and plausibly argues that this will cause an actual difference at level lf, when upward causation takes place: > (left column bu) or when downward causation takes place: > (right column td). this has been used to establish that downward causal effects exist, e.g. [22, 43, 45] . causal closureconsider a multilevel system s (which could have only one level). note that this includes the physicalist idea of causal closure where all follows from a single lowest physical level ll, for that is the case = , chosen as l1 in table 1 . causal closure requires effective predictability on the one hand (sect. 3.3), and an effectively causally closed set of levels on the other, which concept i now consider. the issue: two opposing strands • there are no isolated sets of levels, as just discussed (sect. 4.4). causal closure as just defined is an ideal that does not occur in practice unless one takes ll = l1, hl = l9: you are giving data for the whole thing. • however there are in practice preferred restricted sets levels with a special integrity in terms of causal closure (see the comments just after table 1 ). how do we deal with this tension? the clearest domain in which to tackle this is biology (sect. 5.3). the lessons from there carry over to engineering (sect. 6.2), and physics (sect. 6.4). biological organisms have purpose, as stated by nobel prize winning biologist hartwell et al. [61] 14 : although living systems obey the laws of physics and chemistry, the notion of function or purpose differentiates biology from other natural sciences. organisms exist to reproduce, whereas, outside religious belief, rocks and stars have no purpose. selection for function has produced the living cell, with a unique set of properties that distinguish it from inanimate systems of interacting molecules. cells exist far from thermal equilibrium by harvesting energy from their environment. they are composed of thousands of different types of molecule. they contain information for their survival and reproduction, in the form of their dna. their interactions with the environment depend in a byzantine fashion on this information, and the information and the machinery that interprets it are replicated by reproducing the cell. many biological concepts and language are often sui generis and not reducible to physics and chemistry, certainly not in the form to which they apply to simpler and restricted atomic and molecular systems. in the case of biology, unless the concepts considered include purpose and function, it will miss the essence of what is going on, as pointed out by [61] . you also need to introduce the concepts "alive" and "dead", which do not occur at any lower level than the cellular level in biology, and do not occur at any physics level. without this concept you cannot for example discuss the theory of natural selection [92] . upward and downward causation as just stated, all biological entities have purpose or function, and that controls in a top-down way what happens at lower levels [105] reaching down to the underlying physical levels [45] . the physics does not control the higher levels, rather-without any violation of the laws of physics-it does what the biology asks it do. this functioning occurs via a combinations of upwards and downwards causation [104, 105] , for example gene regulation taking place on the basis of the state of the heart [51] or the brain [78] . this dynamic reaches down to the molecular level and then the underlying electron level. the enabling factors are black boxing [7] to get higher level logic out of lower level logic, together with time dependent constraints at the lower level that are regulated by higher level biological variables [45] . together they underlie the emergent effective laws at each level l in biology. preferred levels the cellular level l5 is a key level in biology: cells have an organisation and integrity of their own, and are living integral entities that are the basic units of life. they interact with other cells at the same level, and react to their environment in appropriate ways. in multicellular organisms they depend on higher levels for nutrition, materials, waste disposal, and signals as to what to do. but they can be treated as modules (sect. 2.6) with an integrity of their own that responds to inputs and produces outputs. the associated set of biological levels, taking the underlying physics for granted, is the set of levels l4-l5. similarly the level of individual organisms l7 again represents a level of emergent integrity. individuals are entities that can be treated as autonomous entities that respond to environmental cues (from the levels above) and other individuals (at the same level). the associated set of biological levels, taking the underlying physics for granted, is the set of levels l4-l7. so the issue is, how does this kind of autonomy emerge at these particular levels, given that all levels are interacting? what characterizes these special sets of levels? the key point as to what occurs is organisational closure in biological organisms [100, 101] : the central aim of this paper consists in arguing that biological organisms realize a specific kind of causal regime that we call 'organisational closure'; i.e., a distinct level of causation, operating in addition to physical laws, generated by the action of material structures acting as constraints. we argue that organisational closure constitutes a fundamental property of biological systems since even its minimal instances are likely to possess at least some of the typical features of biological organisation as exhibited by more complex organisms. this is a distinct causal regime, as explained in [98] : in biological systems, closure refers to a holistic feature such that their constitutive processes, operations and transformations (1) depend on each other for their production and maintenance and (2) collectively contribute to determine the conditions at which the whole organization can exist. according to several theoretical biologists, the concept of closure captures one of the central features of biological organization since it constitutes, as well as evolution by natural selection, an emergent and distinctively biological causal regime. this is developed further in [95] , identifying biological organisation as closure of constraints we propose a conceptual and formal characterisation of biological organisation as a closure of constraints. we first establish a distinction between two causal regimes at work in biological systems: processes, which refer to the whole set of changes occurring in non-equilibrium open thermodynamic conditions; and constraints, those entities which, while acting upon the processes, exhibit some form of conservation (symmetry) at the relevant time scales. we then argue that, in biological systems, constraints realise closure, i.e. mutual dependence such that they both depend on and contribute to maintaining each other. thus biological organisation is an interlevel affair, involving downward causation as well as upwards emergence, thus enabling teleology [12, 99] . from the viewpoint of this paper, these authors are identifying specific sets of levels where effective interlevel causal closure occurs: the topmost level links to the bottom-most level to close the dynamic loop that leads to biological emergence. i will quote three more papers that have essentially the same view. reference [66] emphasizes this property in the case of the cell: [the] property of self-fabrication is the most basic expression of biological anticipation and of life itself. self-fabricating systems must be closed to efficient causation... i identify the classes of efficient biochemical causes in the cell and show how they are organized in a hierarchical cycle, the hallmark of a system closed to efficient causation. broadly speaking, the three classes of efficient causes are the enzyme catalysts of covalent metabolic chemistry, the intracellular milieu that drives the supramolecular processes of chaperoneassisted folding and self-assembly of polypeptides and nucleic acids into functional catalysts and transporters, and the membrane transporters that maintain the intracellular milieu, in particular its electrolyte composition. you need all these components and levels for the thing to work. reference [49] emphasizes that multi-level homeostasis is part of the mix: two broad features are jointly necessary for autonomous agency: organisational closure and the embodiment of an objective-function providing a 'goal': so far only organisms demonstrate both. organisational closure has been studied (mostly in abstract), especially as cell autopoiesis and the cybernetic prin-ciples of autonomy, but the role of an internalised 'goal' and how it is instantiated by cell signalling and the functioning of nervous systems has received less attention. here i add some biological 'flesh' to the cybernetic theory and trace the evolutionary development of step-changes in autonomy: (1) homeostasis of organisationally closed systems; (2) perception-action systems; (3) action selection systems; (4) cognitive systems; (5) memory supporting a selfmodel able to anticipate and evaluate actions and consequences. each stage is characterised by the number of nested goal-directed control-loops embodied by the organism, summarised as will-nestedness. finally [107] argue for circular causality: we argue that (1) emergent phenomena are real and important; (2) for many of these, causality in their development and maintenance is necessarily circular; (3) the circularity occurs between levels of organization; (4) although the forms of causation can be different at different levels, there is no privileged level of causation a priori: the forms and roles of causation are open to experimental investigation; (5) the upward and downward forms of causation do not occur in sequence, they occur in parallel (i.e. simultaneously); (6) there is therefore no privileged direction of emergence -the upper levels constrain the events at the lower levels just as much as the lower levels are necessary for those upper-level constraints to exist. modern biology has confirmed [...] that organisms harness stochasticity at low levels to generate their functionality. this example shows in fine detail why higher-level causality can, in many cases, be seen to be more important than lower-level processes. this is closely related to the idea of autonomous systems, characterized by their organizational and operational closure [138] , where [135] organizational closure refers to the self-referential (circular and recursive) network of relations that defines the system as a unity, and operational closure to the re-entrant and recurrent dynamics of such a system which is just the idea above. this discussion is related to the idea of autopoiesisa system capable of reproducing and maintaining itself-mentioned above, and to the idea of autocatalytic sets [69, 70] . however i will not develop those links here. rather my purpose is to claim that exactly the same applies in engineering systems in general, and even in physics in some cases. that is what i develop below. given that it is understood i am considering causal closure in terms of levels and lois, i can summarise as follows 15 : [131] that lie outside the normal operating environment. thus this characterizes the set of levels needed for an entity (a cell or an organism) to function successfully. there is then no preferred level enabling the system to function: they all equally enable this to happen [107] . reference [57] argue that in such cases, while the effective theories are contained in the range {bl-tl}, one needs different models at each level: no single mathematical model can account for behaviors at all spatial and temporal scales, and the modeler must therefore combine different mathematical models relying on different boundary conditions. these are the different s for each level. but note that one then needs data d for each level l too. thus the set of levels where data is given has to be the same as the set of levels where ecc occurs. thus in the definition of effective causal closure just given, one should set {bl-tl} = {ll-hl}. reference [57] give the examples of epithelial sheets and mechanical modeling of gastrulation. to be clear: one is free to work with an effective theory , with appropriate data for that level, at any level chosen l in the range {bl-tl}; but one only gets effective causal closure by including that full set of levels and data. when does this occur in biology? there are two cases where ecc occurs in biology. • cells the cellular level is the lowest level showing all the attributes of life. it is a case of ecc involving levels l4-l5. • individuals the organism level is the major coherent emergent level in life, assuming it is a multicellular organism such as a human being. this is a case of ecc involving levels l4-l7. however there is an interesting different view: that human beings are essentially social beings, so that in fact it is a mistake to view them as being capable of living on their own, as is implied by that categorisation, thus berger and luckmann [10] wrote about the social construction of reality: our worldview-an inescapable part of our nature shaping our actions-is crucially shaped by the society in which we live. merlin donald's book a mind so rare [33] essentially agrees, as does andy clark's book supersizing the mind [26] and [108] . in short, top-down effects from society so crucially shape our being that they are not just perturbations of independent existence: they are essential, and that characterisation is wrong. the correct ecc statement is • social human beings human beings are essentially social, and are in fact a case of ecc involving levels l4-l8. ignoring the lower levelsa further key comment regards the other end of the scale: why is it legitimate to ignore levels l1-l3 here? the answer is the existence of quantum and classical protectorates that are governed by emergent rules and are insensitive to microscopics [84] . this is another way of affirming the causal efficacy of the effective theories at each emergent level l. however the effectively causally closed levels will reach down to determine what happens at those levels via time dependent constraints (4) [45] . in summary: interlevel effective causal closure as identified here is a key feature of biological functioning, emphasized in [8] and [96] . [24] (where it is not identified as such, but is there) and [123] , where the relation is made explicit. setting the data one final issue remains: we don't in practice set data at all the levels ll-hl required in a particular context. there is no way we could in fact set the data at the lower levels. and there is no need for us to do so. this comment applies both in theory and in practice. that is, it is a statement both about epistemology (what we can know) and ontology (what we can do). a higher level may be essential to a lower level an important possibility is that the properties p of two levels {bl,tl} may have an essential relationship with each other: each level cannot function without the other, as in some cases of symbiosis. consider an individual human being. it is no surprise that the level l7 of the individual cannot exist without the level l5 of cells, for human beings are made out of cells and depend on them for their existence and physiological functioning. but the fact is that the converse is also true: the cells cannot exist and function without the existence of the body that they comprise. the reason is that cells have specialised for specific functions, and cannot survive on their own. they are supplied with oxygen-laden blood by the lungs, heart, and indeed the entire circulatory system, without which they die in a matter of minutes (as happens if a heart attack occurs). thus levels l5 and l6 are inextricably intertwined. but organs are part of the individual and won't function without systemic integration at that level. hence levels {l5-l7} are in fact inextricably intertwined. now an interesting issue arises: ecc occurs for levels l5-l7. should i have included l4 in the inextricably intertwined levels? certainly level l4 is required in order that cells exist at level l5, but is the other way round true also? i believe one can claim it is, because the gene regulatory networks that control production of proteins at level l4 are at level l5, and they would not exist if it were not for their functioning. thus the real inextricably intertwined set of levels is {l4-l7}: the same as the ecc set of levels. there is however this difference: the ecc relation is ceteris parabus, as explained above. the iil relation is not, it is essential, whatever happens at other levels, these levels are crucially dependent on each other. the discussion in the last two sections makes clear a set of principles that apply equally to engineering, and that is what i will show in this section. to make the discussion concrete, i will consider the case of digital computers. but it will apply equally to other branches of engineering: automobiles, aircraft, chemical plant, water supply systems, sewerage systems, and so on. i consider the nature of digital computers (sect. 6.1), where interlevel effective causal closure again occurs (sect. 6.2). inextricably intertwined levels again occur in computers (sect. 6.3), and in physics and chemistry (sect. 6.4). the relevant hierarchy [43, 132] 16 is shown in table 4 . upward and downward causation the dynamics of a computer is driven by the algorithms encoded in the programs loaded, together with the data used by those programs. these control the flow of electrons through gates at the transistor level via a combinations of upward and downward causation ( [39] , chap. 2, [43] ). this enables the emergent effective laws at each level l. different algorithms result in different flows of electrons, as can be demonstrated by running different computer programs which produce different patterns of electron flows through transistors at level l2, and cause major effects at social levels l8 and l9 [88] . the bigger picture the fact that the higher levels {l8,l9} reach down to affect what happens at the lower levels {l0-l6} is stated in [43] as follows: causal closure in the case of computers: in the real world, it is only the combination of physics with its logical, social, psychological, and engineering contexts (which includes the values guiding policy) that can be causally complete, because it is this whole that determines what computer programs will be written and what data utilised, hence what electron flows will take place in integrated circuits, as per the discussion in this paper. this is in parallel to the interlevel causal closure that takes place in biology, as discussed in sect. 5.3. reference [43] gives a specific example: as a specific example: the amount of money that can be dispersed to you from an atm will be limited by an agreement you have reached with your bank. the program used to control the atm will take into account the existence of such limits, and the specific amount you are able to take out in a given time period will be limited by a logical and operation linking this agreed amount to the amount of money in your account. thus these abstract variables will control electron flows in both the bank computers and the atm dispenser mechanism. every relevant abstract variable has physical counterparts; in other words, it?s realized by some physical properties on some relevant physical substrate. but crucially there is much more than this: there is the whole issue of the purposes computers are used for in society, from controlling manufacturing to enabling the internet, cell phones, and social media, and they way that this whole enterprise is shaped by the values of those that control the system. the book coders [134] considers "the morality and politics of code, including its implications for civic life and the economy. programmers shape our everyday behavior: when they make something easy to do, we do more of it. when they make it hard or impossible, we do less of it." all this is expressed in the flows of electrons through gates at the digital levels l2-l3. nevertheless, just as in the case of biology, effective causal closure can occur when interlevel causation results in a high degree of autonomy of operation. analogously to the case of biology, one can state to an autonomous system that do not destroy its autonomy, and (ii) it is a ceteris parabus relation, as discussed in §3.3. it can be destroyed by black swan events that lie outside the normal operating environment. the two emergent levels with their own causal integrity emerging through ecc are the integrated circuit level (l4), the equivalent of the cell in biology, with ecc given by levels l2-l4; and the computer level (l5), the equivalent of the individual in biology, with ecc given by levels l2-l5. but just as in the case of biology one can make a case that one should really include the societal level, the same applies here too. the quotes above suggest that ecc for computers really only occurs for levels l2-l9, including the highest level because of the effect of the world wide web. engineering and applied physics the same kind of considerations apply to all branches of engineering, and equally to all branches of applied physics. the applications (socially determined at level l8) determine what physical effects occur (levels l1-l3). inextricably intertwined levels (iils) do these occur in this case too, as they did in biology? here there is a major difference: the transistors do not depend on the computer for their continued existence, whereas cells depend on the organism for their existence. while in biology iils link the individual as a whole to the molecular level, here they also occur, but only at the levels l0-l1 in table 4 . the reason for that relation is that downward emergence of key properties at the electron level l0 takes place, due to properties of the crystal level l1, as explained in detail in [41] . this is called a "foundational determinative relation" (fdr) by carl gillett, see [55] . in more detail, quasiparticles such as phonons exist due to the broken symmetries of the emergent lattice structure. they come into being as effective particles at the lower level l0 because they are dynamically equivalent to collective oscillations of a level l1 structure (the crystal lattice) ( [127] , pp. [82] [83] . this is an essentially quantum theory phenomenon. one can think of it as an interlevel wave(macro)-particle(micro) duality. reference [52] say it this way: phonons [are] quasi-particles that have some claim to be emergent, not least because the way in which they relate to the underlying crystal is almost precisely analogous to the way in which quantum particles relate to the underlying quantum field theory. stephen blundell states the key point thus ( [15] , p. 244): so now we come to the key question: are these emergent particles real? from the perspective of quantum field theory, the answer is a resounding yes. each of these particles emerges from a wave-like description in a manner that is entirely analogous to that of photons. these emergent particles behave like particles: you can scatter other particles off them. electrons will scatter off phonons, an interaction that is involved in superconductivity. neutrons can be used to study the dispersion relation of both phonons and magnons using inelastic scattering techniques. yes, they are the result of a collective excitation of an underlying substrate. but so are 'ordinary' electrons and photons, which are excitations of quantum field modes. as a consequence, the levels {l0, l1} are inextricably intertwined. another way of stating this is the way that bloch's theorem [14] shows how the crystal structure causes the electron wave functions to have a basis consisting of bloch eigenstates with the same periodicity as the crystal. this is a proof that the solid state physics occurring in digital computers is a case where causal closure is impossible at the micro level l0 alone. it also shows that in general the set of iils is not the same as the eccs. while these considerations apply to digital computers, of course they also apply in particular to the solid state physics itself that underlies their operation, due to the nature of crystals. for the reasons just discussed in solid state physics, as a consequence of interlevel wave-particle duality, levels {l1,l4} in table 1 are inextricably intertwined levels. however the iil phenomenon is not confined to this case. the laser is another example ( [119] , §2) the laser involves a kind of 'circular' causality which occurs in the continuous interplay between macrolevel resonances in the cavity guiding, and being reinforced by, self-organization of the molecular behaviour. a quite different example is resonance energy transfer (ret) in the transport of electronic energy from one atom or molecule to another [74] . as described in that paper, the individual electrons do not migrate between molecules during the transfer process, since the molecular orbitals (the wavefunctions) do not overlap, but instead move between individual electronic states within the molecules. ... energy transfer, through dipole coupling between molecules, mostly depends on two important quantities: spectral overlap and intermolecular distance. this lead to a r −6 distance-dependence for the resonance energy transfer rate in the short-distance regime. the behaviour results from interwining between the electron level l1 and the molecular level l4. because of these interactions, second-order perturbation theory is the minimal needed to describe ret. while this paper refers to 'molecules', ret occurs in atoms, chromophores, particles and carbon nanotubes. its applications include nanosensors and photodynamic therapy. this raises the issue that whenever molecular physics [21] is concerned, there is inextricable intertwining between the molecular structure at level l4, bound by electrons, and the motions of the electrons at level l1, controlled by that structure. this is manifest in binding energy ( [21] , pp. 17-19, [90] , p. 569) and the existence of covalent bonds between atoms ( [21] , pp. [17] [18] [19] . molecular physics and quantum chemistry also exhibit iils between levels l1 and l4, which is why chemistry is a classic example of downward causation [86] . the group theory underlying this intertwining is discussed in [13] . it is believed by many that because the bottom-most physics level is causally complete, and only upward causation takes place, higher levels are purely derivative: they have no real causal validity. in this paper and its companion [41] , i argue against that position. it is invalid because it treats physics in a way that ignores context, whereas physics outcomes always depend on context. in fact downward effects imply the opposite: in real world contexts, the bottom-most physics level is not by itself causally complete. in this section, i look at the contextual nature of causal closure of physics (sect. 7.1), the way that unpredictability undermines causal closure of physics per se (sect. 7.2), and comment on ways people ignore the issues discussed in this paper (sect. 7.3). we can consider physics per se, or in relation to the natural world, or in relation to biology, or in relation to engineering. within physics, the issue of causal closure depends on what aspects we are considering: particle physics, nuclear physics, condensed matter physics, cosmology for example. firstly, we have no reliable tested toe at the very bottom level l0. we don't try to reduce to that most fundamental physical level (sect. 4.4). rather we reduce to a level that is convenient. that that can work is due to the existence of quantum protectorates, as explained in [84] . but then it is common to assume that level l1 (particle physics) is causally complete. is that indeed so? i have argued that this is not the case in the contexts of physics and biology (sect. 5.3); physics and engineering, as exemplified by digital computers (sect. 6.2); solid state physics (sect. 6.3); and physics and chemistry (sect. 6.4). in each case the real causal closure that takes place is an interlevel affair, as emphasized in particular in the case of biology by many perceptive writers (sect. 5.3). effective causal closure in real world contexts spans many levels, in the case of biology reaching down from the level of the organism to the underlying physics via time dependent constraints. this implies how it works in terms of physics in relation to society. the causal effects of the coronavirus pandemic at the social level reaches down to cause major changes at the physical levels l1-l3 through a complex interaction between social behaviours, virology, and microbiology that for example has temporarily destroyed international air travel and so the trajectories of the billions of particles that make up aircraft. causal closure only occurs when we take all these factors and levels into account. considering only disembodied physical laws seriously misleads about the nature of causation and causal closure in real world contexts. in summary, this is formalized by the concepts of effective causal closure (sects. 5.3, 6.2), and inextricably intertwined levels (sects. 5.4, 6.3). within solid state physics itself, the lower levels l1-l3 are not causally closed by themselves because of interlevel particle-wave duality between the particle level l1 where electrons and phonons live, and the crystal level l4 where lattice vibrations take place (sect. 6.3). properties of level l4 decouple from the lower physics levels. as stated by [84] , the crystalline state is the simplest known example of a quantum protectorate, a stable state of matter whose generic low-energy properties are determined by a higher organizing principle and nothing else. ignoring interlevel issues, if by causal closure one means that data specified as precisely as possible leads to unique outcomes, then unavoidable unpredictability undermines the possibility of physics per se (whether quantum or classical) being causally closed. quantum physics quantum effects doubly cause uncertainty in outcomes. firstly, the heisenberg uncertainty principle states that the standard deviations of position x and momentum p obeys so one cannot even in principle apply laplace's dream of setting initial data precisely at level l1. consequently, outcomes are also uncertain. secondly, collapse of the wave function introduces an irreducible uncertainty in classical outcomes at this level when interactions take place [53] . this can reach up to macro levels through various amplifiers such as photon multipliers and ccds. in the engineering case it causes predictability issues at macro scales in terms of digital computer reliability because cosmic rays cause errors in computer memories [56, 143] , and the emission of a cosmic ray by an excited atom is a quantum event that is (10) x p ≥ ℏ∕2 unpredictable even in principle. as regards biology, cosmic rays have had a significant effect on evolutionary history by causing genetic mutations [116] . the classical case uncertainty of outcomes occurs in this case too, because one can't set initial data to infinite precision [31] . this is an outcome of the fact that infinity never occurs in physical reality [46] . thus physics is not causally closed in the classical case at higher levels because of chaotic dynamics (the butterfly effect), together with the impossibility of specifying initial data to infinite accuracy. this occurs for instance at the level at which fluid motion is determined. reference [6] puts it this way: a fluid dynamicist when studying the chaotic outcome of convection in a benard cell knows to a gnat's eyelash the equations of motion of his fluid but also knows, through the operation of those equations of motion, that the details of the outcome are fundamentally unpredictable, although he hopes to get to understand the gross behaviour. this aspect is an example of a very general reality: the existence of universal law does not, in general, produce deterministic, cause-and-effect behaviour. this fundamentally undermines the concept of a causally closed physical levels in the case of classical physics. as was already known to poincare, this occurs even in the 3-body gravitational case. microbiology in the case of microbiology, interactions take place in the context of what hoffmann [65] has called "the molecular storm". molecular machines use ratchet-like mechanisms to harness energy from that storm, and organisms use it to provide an ensemble of options from which they can choose preferred lower level states and so attain biological objectives [106] (see the quote in sect. 2.3). one has the opposite of the calm relation between initial data and outcomes supposed by laplace. here are some ways that the nature of causal closure as discussed in this paper is avoided. partial reduction this is very common. francis crick in the astonishing hypothesis [28] states, you, your joys and your sorrows, your memories and your ambitions, your sense of personal identity and free will, are in fact no more than the behavior of a vast assembly of nerve cells and their associated molecules. in other words, he is reducing l7 to {l5-l4}. now my physics colleagues who believe that all that matters is the particle interactions at level l1 will just laugh and say, cells at level l4 and molecules at l5 are nothing but particles interacting with each other. thus crick believes in the reality and effectiveness of causality at levels l4 and l5 that for example [71, 58] , who believe that all causality resides at level l1, clearly must deny. so why did crick emphasize causality at those levels? the answer of course is that those were the levels at which he worked-and experienced the effectiveness of causality in terms of the interactions between entities (molecules, neurons) at those levels. from a strictly reductionist viewpoint, this is an illegitimate move. it is however fine if you accept noble's principle of biological relativity [105] , as extended in [41] and this paper: then causality is real at the levels {l4, l5} he studies. but that removes crick's justification for denying the reality of causation at level l7. ignoring context crucial contextual effects are simply ignored by some writers. for example [58, 71] . the view is "you are nothing but a bag of particles, it's just a matter of particles interacting via a known set of forces". context has nothing to do with it. the physicists holding this view all come from the particle physics/cosmology side, where this is to some extent true. physicists from the solid state physics side (the largest section of the physics community) do not hold this view, see e.g. [5, 127] . this leads to the large divide in the physics community between these two groups, as discussed by schweber [124] . denying top-down causation there is a frequent denial of the possibility of top-down causation, even though it occurs in physics and cosmology. in the latter case it occurs in the context of primordial nucleosynthesis in the early universe ( [117] , §4.3) and structure formation in the later universe ( [117] , §5), which are both dependent on the cosmological context at level l10. that is the reason that primordial element abundances on the one hand and matter power spectra and cosmic background radiation angular power spectra on the other can be used to place strong limits on the background model parameters [1] , as discussed in ( [39] , pp. 275-277). if there were not such downward causation, this would not be possible. top down causation clearly occurs in biology [22, 51, 104] . a recent example is [119] : top-down approaches focus on system-wide states as causal actors in models and on the computational (or optimality) principles governing global system dynamics. planck 2018 results. vi. cosmological parameters an introduction to systems biology: design principles of biological circuits more is different: broken symmetry and the nature of the hierarchical structure of science a career in theoretical science: a 'dappled world' or a 'seamless web'? design for a brain: the origin of adaptive behaviour biological mechanisms: organized to maintain autonomy invitation to sociology: a humanistic perspective the social construction of reality: a treatise in the sociology of knowledge cell signalling biology glycemia regulation: from feedback loops to organizational closure contextual emergence of physical properties über die quantenmechanik der elektronen in kristallgittern phase transitions, broken symmetry and the renormalization group object oriented analysis and design with application emergence and its place in nature: a case study of biochemical networks networks in cell biology an introduction to effective field theory emergence, reduction and supervenience: a varied landscape molecular physics downward causation in hierarchically organised biological systems endless forms most beautiful: the new science of evo devo reductionism, emergence, and effective field theories supersizing the mind: embodiment, action, and cognitive extension the emergentist hypothesis from science to religion the astonishing hypothesis: the scientific search for the soul the arrow of time the demon in the machine: how hidden webs of information are solving the mystery of life physics without determinism: alternative interpretations of classical physics principles of dynamical modularity in biological regulatory networks a mind so rare: the evolution of human consciousness the nature of the physical world the causal power of social structures cosmology and local physics top-down causation and emergence: some comments on mechanisms on the philosophy of cosmology how can physics underlie the mind? top-down causation in the human context top-down effects in the brain emergence in solid state physics and biology does physics deny free will? aeon essay how downwards causation occurs in digital computers emergence of time the dynamical emergence of biology from physics the physics of infinity the evolution of our local cosmic domain: effective causal limits the functional theory of counterfactual thinking how organisms gained causal independence and how it might be quantified the character of physical law noble model emergence without limits: the case of phonons sneaking a look at god's cards: unraveling the mysteries of quantum mechanics the routledge handbook of emergence. routledge emergence, downward causation and its alternatives: critically surveying a foundational issue field testing for cosmic ray soft errors in semiconductor memories biology meets physics: reductionism and multi-scale modeling of morphogenesis until the end of time: mind, matter, and our search for meaning in an evolving universe basic human physiology: normal functions and mechanisms of disease effective field theories, reductionism and scientific explanation from molecular to modular cell biology the large scale structure of spacetime neural networks: a comprehensive foundation conceptual physics life's ratchet: how molecular machines extract order from chaos basic biological anticipation causation, constructors and codes being reduced: new essays on reduction, explanation, and causation autocatalytic sets: from the origin of life to the economy autocatalytic networks at the basis of life? origin and organization the case for strong emergence global warming: the complete briefing artificial neural networks: a tutorial resonance energy transfer: from fundamental theory to recent applications analysis of biological networks dynamics in action: intentional behavior as a complex system a new intellectual framework for psychiatry the molecular biology of memory storage: a dialogue between genes and synapses the age of insight: the quest to understand the unconscious in art, mind, and brain, from vienna 1900 to the present. penguin random house principles of neural science mind in a physical world supervenient properties and micro-based properties: a reply to noordhof computer networking: a top-down approach featuring the internet the theory of everything the touchstone of life: molecular information, cell communication and the foundations of life emergence in chemistry: chemistry as the embodiment of emergence on the topic of emergence from an effective field theory perspective nine algorithms that changed the future emergence in mind quantum chemistry. university science books autopoiesis and cognition: the realization of the living what evolution is the causal efficacy of mental states network motifs: simple building blocks of complex networks biological organisation as closure of constraints biological autonomy: a philosophical and theoretical enquiry on nature and normativity: normativity, teleology, and mechanism in biological explanation closure, causal what makes biological organisation teleological? organisational closure in biological organisms an organizational account of biological functions foundations of space and time: reflections on quantum gravity modeling the heart-from genes to cells to the whole organ the music of life: biology beyond genes a theory of biological relativity: no privileged level of causation harnessing stochasticity: how do organisms make choices? chaos interdiscip a-mergence of biological systems biological relativity requires circular causality but not symmetry of causation: so, where, what and when are the boundaries? life, logic and information the theory of almost everything: the standard model, the unsung triumph of modern physics hierarchical thinking in network biology: the unbiased modularization of biochemical networks an introduction to the physical chemistry of biological organization causality: models, reasoning, and inference the book of why: the new science of cause and effect the emperor's new mind schrödinger's quantum cat primordial cosmology homological scaffolds of brain functional networks top-down models in biology: explanation and control of complex living systems above the molecular level eckert animal physiology: mechanisms and adaptations hierarchical organization of modularity in metabolic networks autonomy in evolution: from minimal to complex life physics, community, and the crisis in physical theory the unity of the universe the sciences of the artificial the oxford solid state basics fine tuning in the physical universe the major transitions in evolution the metaphysics of downward causation: rediscovering the formal cause the black swan: the impact of the highly improbable structured computer organisation, 5th edn is a doomsday catastrophe likely? coders: the making of a new tribe and the remaking of the world. penguin mind in life: biology, phenomenology, and the sciences of mind. harvard university press the fundamental constants and their variation: observational and theoretical status from biological determination to entangled causation enactive autonomy in computational systems arrival of the fittest: solving evolution's greatest puzzle. penguin dreams of a final theory: the scientist's search for the ultimate laws of nature. penguin random house the architecture of biological networks effect of cosmic rays on computer memories that paper gives fascinating examples of downward causation from morphogenesis and regenerative medicine.downward causation is obvious in subjects other than particle physics and cosmology, and in particular in the functioning of the brain [39, 40, 77, 79] . for example, our minds are shaped by society, and shape society, including material outcomes in terms of architecture, engineering, art, and so on. society does not exist without mind, and mind does not exist without society [9, 10, 33] . that is the nature of interlevel causal closure. key: cord-337218-risqto89 authors: chu, ellen w.; karr, james r. title: environmental impact, concept and measurement of date: 2013-02-05 journal: encyclopedia of biodiversity doi: 10.1016/b978-0-12-384719-5.00253-7 sha: doc_id: 337218 cord_uid: risqto89 environments on earth are always changing, and living systems evolve within them. for most of their history, human beings did the same. but in the last two centuries, humans have become the planet's dominant species, changing and impoverishing the environment for all life on earth and even decimating humans' own cultural diversity. contemporary cultural worldviews that have severed humans' ancient connections with the natural world, along with consumption and population growth, have deepened this impoverishment. understanding, measuring, and managing human environmental impacts – the most important of which is the impoverishment of living systems – is the 21st century's greatest challenge. all organisms change their environment as they live, grow, and reproduce. over millennia, organisms evolve to contend with changes in their environment. those that do not adapt go extinct. those that survive are molded by evolution and biogeography as the environment changes. even unusual or seemingly catastrophic events, like tidal waves from earthquakes, are an integral part of the ecological contexts to which organisms adapt over long time spans. some organisms, like beavers and elephants, change their surroundings so dramatically that they have been called ecosystem engineers. beaver dams alter the flow of rivers, increase dissolved oxygen in downstream waters, create wetlands, and modify streamside zones. african elephants convert wooded savanna to open grassland by toppling trees as they browse. change brought about by living things, including ecosystem engineers, has been slow and incremental in evolutionary terms. ecosystem engineers evolve along with other inhabitants of their environments, developing ways to coexist; like other environmental components, ecosystem engineers, and their effects are part of an evolving ecological context. in contrast, human effects since the industrial revolutionincluding many that may be invisible to a casual observeroutpace the capacity of living systems to respond. in evolutionary terms, these effects are recent and outside the experience of most organisms. over the past two centuriesbarely more than two human lifetimes -humans have profoundly altered living and nonliving systems everywhere. for the first time in the earth's history, the environmental impact of one species, homo sapiens, is the principal agent of global change. understanding the environmental impacts of human actions is one of modern science's greatest challenges. understanding the consequences of those impacts, and managing them to protect the well-being of human society and other life on earth, is humanity's greatest challenge. the human evolutionary line began in africa about 7 million years ago (ma). it took some 5 or 6 million years (my) for protohumans to spread from africa to asia and then to europe. these early humans, like other primates, made their living by seeking food and shelter from their environment, gathering plant foods, and hunting easy-to-kill prey. sometimes, they also experienced threats from their environment, including accidents, droughts, vector-borne diseases, and attacks from predators. at this stage, with relatively low population densities and limited technologies, humans were not ecosystem engineers. by some 50,000 years ago, however, humans had learned to use fire; developed complex tools, weapons, and language; and created art. on local scales, these modern humans were very much ecosystem engineers. sometimes their enhanced abilities to make a living outstripped their local environment's capacity to provide that living, and they caused local ecological disruptions. on several continents, for example, humans hunted large mammals to the point where many, such as the marsupial lion of australia, went extinct. as humans became more efficient at exploiting their local environments, they spread farther. by 13,000 years ago, modern humans had spread to all continents and many islands across the globe. then, about 10,000 years ago, people began to domesticate plants and animals. instead of searching for food, they began to produce food. food production changed the course of human and environmental history. domestication of plants and animals enabled people to adopt a sedentary lifestyle. as detailed by geographer and ecologist diamond (1997 diamond ( , 2002 , populations grew as agriculture developed, because larger sedentary populations both demanded and enabled more food production. local ecological disruptions became more numerous and widespread and more intense. with animal domestication, contagious diseases of pets and livestock adapted to new, human hosts. diseases spread more quickly in crowded conditions; inadequate sanitation compounded the effects. from agriculture, civilization followed and with it, cities, writing, advanced technology, and political empires. in just 10,000 years, these developments led to nearly 7 billion people on earth, industrial societies, and a global economy founded on complicated technologies and fossil fuels. humans have emerged as ecosystem engineers on a global scale. the ecological disruptions we cause are no longer just local or regional but global, and we have become the principal threat to the environment. yet despite today's advanced technologies, humans are as dependent on their environments as other organisms are. history, not just ecology, has been very clear on this point. from the old kingdom of egypt more than 4000 years ago to the culture that created the huge stone monoliths on easter island between 1000 and ad 1550 to the 1930s dust bowl of north america, civilizations or ways of life have prospered and failed by using and (mostly unwittingly) abusing natural resources. in old kingdom egypt, the resource was the valley of the nile, richly fertilized with sediment at each flooding of the river, laced with canals and side streams, blessed with a luxuriant delta. agriculture flourished and populations swelled, until unusually severe droughts brought on the civilization's collapse. on easter island, the resource was trees, which gave polynesians colonizing the island the means to build shelter, canoes for fishing the open waters around the island, and log rollers for moving the ceremonial stone monuments for which the island is famous. deforestation not only eliminated the humans' source of wood, but also further deprived the already poor soil of nutrients and made it impossible to sustain the agriculture that had sustained the island's civilization. on the dry great plains of north america, settlers were convinced that rain would follow the plow, and so they plowed homestead after homestead, only to watch their homesteads' soils literally blow away in the wind. in these cases and many others, human civilizations damaged their environments, and their actions also worsened the effects on their civilizations of climatic or other natural cycles. yet in each case, a human culture was operating precisely the way it had evolved to operate: the culture of old kingdom egypt enabled its people to prosper on the nile's natural bounty, but prolonged, unprecedented drought brought starvation and political disorder. easter islanders thrived and populated the island until its resources were exhausted. dust bowl farmers lived out their culture's view of dominating and exploiting the land for all it was worth. the inevitable outcome for all three cases was a catastrophe for the immediate environment and the people it supported -not only because the people were unprepared to cope with dramatic natural changes in their environments but because their own actions magnified the disastrous effects of those changes. quoting an apt bit of cynical graffiti, historical philosopher wright (2004, p. 107 ) sums up what he calls the hazards of human progress this way: ''each time history repeats itself, the price goes up.'' indeed, as the second decade of the 21st century begins, humans are ecosystem engineers on a planetary scale, and our global civilization threatens the life-sustaining capacity of all of earth's environmental ''spheres'': • geosphere (lithosphere): earth's crust and upper mantle, containing nonrenewable fossil fuels, minerals, and nutrients that plants require. the activities of plants, animals, and microorganisms weather mineral soils and rocks, create organic soils, and alter erosion and sedimentation rates. humans mine minerals, metals, and gems; extract fossil fuels including coal, oil, and natural gas; and increase erosion and sedimentation by removing or altering natural plant cover through agriculture, logging, and urbanization. • atmosphere: the thin envelope of gases encircling the planet. living systems modify the atmosphere, its temperature, and the amount of water it contains by continually generating oxygen and consuming carbon dioxide through photosynthesis and affecting the amount and forms of other gases. humans release toxic chemicals into the air and alter the climate by raising the atmospheric concentration of greenhouse gases, such as carbon dioxide and methane, through the burning of fossil fuels in motor vehicles, ships, airplanes, and power plants. • hydrosphere: earth's liquid surface and underground water; its polar ice caps, oceanic icebergs, and terrestrial permafrost; and its atmospheric water vapor. living systems alter the water cycle by modifying the earth's temperature and the amount of water plants send into the atmosphere through a process called evapotranspiration. humans build dams, irrigation canals, drinking-water delivery systems, and wastewater treatment plants. they use water to generate electricity; they mine groundwater from dwindling underground aquifers for farming as well as drinking; they alter the flows of surface waters for everything from transportation to the mining of gold; they drain wetlands to gain land area and abate waterborne diseases. modern human interference in global climate is likely to disrupt the entire planetary water cycle. • biosphere: the totality of earth's living systems, that part of the earth inhabited by living organisms. life on earth emerged 3.9 billion years ago and has sustained itself through changes in form, diversity, and detail since then. no planet yet discovered supports complex life as we know it on earth. as predators, humans have decimated or eliminated wild animal populations worldwide. as domesticators of animals and plants, humans have massively reshaped landscapes by cutting forests, burning and plowing grasslands, building cities, desertifying vast areas, and overharvesting fish and shellfish. human actions have precipitated a spasm of extinctions that today rivals five previous mass extinctions caused by astronomical or geological forces, each of which eliminated more than 70% of species then existing. humans themselves may be thought of as a sphere within the greater biosphere: the ethnosphere, or the sum total of all thoughts and intuitions, myths and beliefs, ideas and inspirations brought into being by the human imagination since the dawn of consciousness. in the words of anthropologist davis (2009, p. 2) , who coined and defined the term in 2002, ''the ethnosphere is humanity's greatest legacy. it is the product of our dreams, the embodiment of our hopes, the symbol of all we are and all that we, as a wildly inquisitive and astonishingly adaptive species, have created.'' but, davis notes, just as the biosphere is being severely eroded, so too is the ethnosphere, but at a much faster pace. today, the scientific consensus is that h. sapiens -a single species -rivals astronomical and geological forces in its impact on life on earth. the first step in dealing with the present impact of human activity is to correctly identify the nature of humanity's environmental impact, concept and measurement of relationship with the environment and how human actions affect that relationship. many people still see the environment as something people must overcome, or they regard environmental needs as something that ought to be balanced against human needs (for example, jobs vs. the environment). most people still regard the environment as a provider of commodities or a receptacle for waste. when asked to name humanity's primary environmental challenges, people typically think of running out of nonrenewable raw materials and energy or about water and air pollution. environmental research and development institutions focus on ways technology can help solve each problem, such as fuel cells to provide clean, potentially renewable energy or scrubbers to curb smokestack pollution. even when people worry about biodiversity loss, they are concerned primarily with stopping the extinction of species, rather than with understanding the underlying losses leading up to species extinctions or the broader biological crisis that extinctions signal. these perspectives miss a crucial point: the reason pollution, energy use, extinction, and dozens of other human impacts are important is their larger impact on the biosphere. ecosystems, particularly their living components, have always provided the capital to fuel human economies. when populations were small, humans making a living from nature's wealth caused no more disruption than other species. but with nearly 7 billion people occupying or using resources from every place on earth, humans are overwhelming the ability of other life-forms to make a living and depleting the planet's natural wealth. at this point in the planet's history, one species is compromising earth's ability to support the living systems that evolved here over millions of years. the systematic reduction in earth's capacity to support life -which woodwell (1990) termed 'biotic impoverishment' -is thus the most important human-caused environmental impact. at best, the ethics of this impact are questionable; at worst, it is jeopardizing our own survival. the connection between biotic impoverishment and extinction is intuitively obvious. by overharvesting fish, overcutting forests, overgrazing grasslands, or paving over land for cities, we are clearly killing other organisms outright or eliminating their habitats, thereby driving species to extinction and impoverishing the diversity of life. but biotic impoverishment takes many forms besides extinction. it encompasses three categories of human impacts on the biosphere: (1) indirect depletion of living systems through alterations in physical and chemical systems, (2) direct depletion of nonhuman life, and (3) direct degradation of human life (table 1) . identifying and understanding the biological significance of our actions -their effects on living systems, including our own social and economic systems -is the key to developing effective ways to manage our impacts. humans affect virtually all the physical and chemical systems life depends on: water, soils, air, and the biogeochemical cycles linking them. some human-driven physical and chemical changes have no repercussions on the biota; others do, becoming agents of biotic impoverishment. humans probably spend more energy, money, and time trying to control the movement and availability of water than to manage any other natural resource. in the process, we contaminate water, move water across and out of natural basins, deplete surface and groundwater; modify the timing and amount of flow in rivers, straighten or build dikes to constrain rivers, and alter natural flood patterns. we change the amount, timing, and chemistry of fresh water reaching coastal regions, and we dry up wetlands, lakes, and inland seas. our demands are outrunning supplies of this nonrenewable resource, and the scale of our transformations risks altering the planetary water cycle. physical alterations of the earth's waters, combined with massive industrial, agricultural, and residential pollution, have taken a heavy toll on nonhuman aquatic life. by 2005 as much as one-fifth of the world's coral reefs had been destroyed, and only 30% remained healthy. globally, the number of oceanic dead zones, where little or no dissolved oxygen exists, tripled during the last 30 years of the 20th century. the biota of freshwater systems has fared no better. a 4-year survey of the freshwater fishes inhabiting malaysian rivers in the late 1980s found only 46% of 266 known malaysian species. some 40% of north america's freshwater fishes are at risk of extinction; two-thirds of freshwater mussels and crayfishes and one-third of amphibians that depend on aquatic habitats in the united states are rare or imperiled. humans use at least 54% of the earth's accessible water runoff, a figure that is likely to grow to 70% by 2025. by then, more than a third of the world's population could suffer shortages of fresh water for drinking and irrigation. groundwater aquifers in many of the world's most important cropproducing regions are being drained faster than they can be replenished: a study published in 2010 found that the rate of groundwater depletion worldwide had more than doubled from 1960 to 2000. natural flood regimes, as in the nile river basin, no longer spread nutrient-rich silt across floodplains to nourish agriculture; indeed, the high dam at aswan traps so much silt behind it that the nile delta, essential to egypt's present-day economy, is slumping into the sea. whole inland seas, such as the aral sea in uzbekistan, are drying up because the streams feeding them contain so little water. in addition to eliminating habitat for resident organisms, the sea's drying is bringing diseases to surrounding human populations. indeed, diseases caused by waterborne pathogens are making a comeback even in industrialized nations. in the past five or six decades, the number of large dams on the world's rivers grew more than seven times, to more than 40,000 today. the mammoth three gorges dam across china's yangtze river, completed in 2006, created a 660-km-long serpentine lake behind it. the dam displaced more than 1 million people and may force the relocation of another 4 million from the reservoir region, which, at 58,000 km 2 , is larger than switzerland. the dam has greatly altered ecosystems on the yangtze's middle reaches, compounding perils already faced by prized and endemic fish and aquatic mammals. the sheer weight of the water and silt behind the concrete dam raises the risk of landslides and strains the region's geological structure, while water released from the dam eats away at downstream banks and scours the bottom. and by slowing the flow of the yangtze and nearby tributaries, the dam drains the river's ability to flush out and detoxify pollutants from upstream industries. not just dirt, soil is a living system that makes it possible for raw elements from air, water, and bedrock to be physically and chemically assembled, disassembled, and reassembled with the aid of living macro-and microorganisms into the green cloak of life above ground. accumulated over thousands of years, soil cannot be renewed in any time frame useful to humans alive today, or even to their great-grandchildren. humans degrade soils when they compact it, erode it, disrupt its organic and inorganic structure, turn it too salty for life, and cause desertification. urbanization, logging, mining, overgrazing, alterations in soil moisture, air pollution, fires, chemical pollution, and leaching out of minerals all damage or destroy soils. thanks to removal of vegetative cover, mining, agriculture, and other activities, the world's topsoils are eroded by wind and water ten to hundreds of times faster than they are renewed (at roughly 10 tons ha à1 year à1 ). soils constitute the foundation of human agriculture, yet agriculture, including livestock raising, is the worst culprit in degrading soils. agricultural practices have eroded or degraded more than 40% of present cropland. over the last half century, some 24,000 villages in northern and western china have been overrun by the drifting sands of desertification. besides topsoil erosion, the damage includes salting and saturation of poorly managed irrigated lands; compaction by heavy machinery and the hooves of livestock; and pollution from excessive fertilizers, animal wastes, and pesticides. living, dead, and decomposing organic matter is the key to soil structure and fertility. soil depleted of organic matter is less permeable to water and air and thus less able to support either aboveground plants or soil organisms. the linkages between soil's inorganic components and the soil biotanaturalist wilson's (1987) ''little things that run the world''are what give soil its life-sustaining capacity. a clear-cut forest patch whose soil biota has been damaged beyond recovery can no longer sustain trees, no matter how many are planted; another clear-cut patch whose soil community is intact -especially the close associations among fungi and tree rootswill support new tree growth. destroying soil biota unleashes a whole series of impoverishing biotic effects both below and above ground. in 1962, rachel carson's landmark book, silent spring, alerted the world to the pervasiveness of synthetic chemicals produced since world war ii. as many as 100,000 synthetic chemicals are in use today. true to one company's slogan, many of these have brought ''better living through chemistry,'' providing new fabrics and lighter manufacturing materials, antibiotics, and life-saving drugs. but industrial nations have carelessly pumped chemicals into every medium. chemicals -as varied as prescription drugs flowing out of sewage plants, pesticides, heavy metals, and cancer-causing by-products of countless manufacturing processes -now lace the world's water, soil, and air and the bodies of all living things, including humans. chemicals directly poison organisms; they accumulate in physical surroundings and are passed through and, in many cases, concentrated within portions of the food web. chemicals cause cancer, interfere with hormonal systems, provoke asthma, and impair the functioning of immune systems. they have intergenerational effects, such as intellectual impairment in children whose mothers have eaten contaminated fish. what's more, over half a century of pesticide and antibiotic overuse has bred resistance to these chemicals among insects, plants, and microbes, giving rise to new and reemerging scourges. many chemicals travel oceanic and atmospheric currents to sites far from their source. sulfur emissions from the us midwest, for example, fall to earth again as acid rain in europe, killing forests and so acidifying streams and lakes that they, too, effectively die. china's burning of soft coal sends air pollution all the way to northwestern north america; the heavy haze hanging over china's chief farming regions may be cutting agricultural production by a third. chlorofluorocarbons (cfcs), once widely used as refrigerants, have damaged the atmospheric ozone layer, which moderates how much ultraviolet radiation reaches the earth, and opened ozone holes over the arctic and antarctic. but even more alarming is an unprecedented acidification of the oceans that has only recently attracted the attention of major scientific research consortiums. acid added to the world ocean by human activity has lowered the ocean's ph; it is lower now than it has been in 20 my, which translates into a 30% increase in sea-surface acidity since industrialization began. the future of marine life in an ocean acidifying at this speed and intensity looks bleak. as the concentration of hydrogen ions rises, the calcium carbonate in the shells or skeletons of organisms such as tropical corals, microscopic foraminifera, and mollusks begins to dissolve; further, more hydrogen ions combine with the calcium carbonate building blocks the organisms need, making it harder for them to extract this compound from the water and build their shells in the first place. although many of the most obviously deadly chemicals were banned in the 1970s, they continue to impoverish the biosphere. polychlorinated biphenyls -stable, nonflammable compounds once used in electrical transformers and many other industrial and household applications -remain in the environment for long periods, cycling among air, water, and soils and persisting in the food web. they are found in polar bears and arctic villagers; they are implicated in reproductive disorders, particularly in such animals as marine mammals, whose long lives, thick fat layers where chemicals concentrate, and position as top predators make them especially vulnerable. the agricultural pesticide ddt, sprayed with abandon in the 1940s and 1950s, even directly on children, had severely thinned wild birds' eggshells by the time it was banned in the united states. populations of birds such as the brown pelican and bald eagle had dropped precipitously by the 1970s, although they have recovered enough for the species to be taken off the us endangered species list, the bald eagle in 2007 and the brown pelican in 2009. reproduction of central california populations of the california condor, in contrast, continues to be threatened by ddt breakdown products, which, 40 years after the pesticide was banned, are still found in the sea lion carcasses the birds sometimes feed on. carson's book revealed the real danger of chemical pollutants: they have not simply perturbed the chemistry of water, soil, and air but harmed the biosphere as well. the list of chemicals' effects on living things is so long that chemical pollution equals humans' environmental impact in most people's minds, yet it is just one form of biotic impoverishment. all the substances found in living things -such as water, carbon, nitrogen, phosphorus, and sulfur -cycle through ecosystems in biogeochemical cycles. human activities modify or have the potential to modify all these cycles. sometimes the results stem from changing the amount or the precise chemistry of the cycled substance; in other cases, humans change biogeochemical cycles by changing the biota itself. freshwater use, dams, and other engineering ventures affect the amount and rate of river flow to the oceans and increase evaporation rates, directly affecting the water cycle and indirectly impoverishing aquatic life. direct human modifications of living systems also alter the water cycle. in south africa, european settlers supplemented the treeless native scrub, or fynbos, with such trees as pines and australian acacias from similar mediterranean climates. but because these trees are larger and thirstier than the native scrub, regional water tables have fallen sharply. human activity has disrupted the global nitrogen cycle by greatly increasing the amount of nitrogen fixed from the atmosphere (combined into compounds usable by living things). the increase comes mostly from deliberate addition of nitrogen to soils as fertilizer but also as a by-product of the burning of fossil fuels. agriculture, livestock raising, and residential yard maintenance chronically add tons of excess nutrients, including nitrogen and phosphorus, to soils and water. the additions are often invisible; their biological impacts are often dramatic. increased nutrients in coastal waters, for example, trigger blooms of toxic dinoflagellates -the algae that cause red tides, fish kills, and tumors and other diseases in varied sea creatures. when huge blooms of algae die, they fall to the seafloor, where their decomposition so robs the water of oxygen that fish and other marine organisms can no longer live there. with nitrogen concentrations in the mississippi river two to three times as high as they were 50 years ago, a gigantic dead zone forms in the gulf of mexico every summer; in summer 2010 this dead zone covered 20,000 km 2 . the burning of fossil fuels is transforming the carbon cycle, primarily by raising the atmospheric concentration of carbon dioxide. with other greenhouse gases, such as methane and oxides of nitrogen, carbon dioxide helps keep earth's surface at a livable temperature and drives plant photosynthesis, but since the industrial revolution, atmospheric carbon dioxide concentrations have risen nearly 40% and are now widely thought to be disrupting the planet's climate. in addition, the effects of catastrophic oil spills like the one that followed the april 2010 explosion of the deepwater horizon drilling rig in the gulf of mexico -and the effects of the chemicals used to disperse the resulting plumes of oil -may reverberate for decades. in its 2007 report, written and reviewed by more than 3500 scientists, the intergovernmental panel on climate change (ipcc) concluded that climate warming was ''unequivocal'' (p. 2), that most of the average global warming over the previous 50 years was ''very likely due to anthropogenic ghg [greenhouse gas] increases'' (p. 5), and that this warming has likely had ''a discernible influence at the global scale on observed changes in many physical and biological systems'' (p. 6). the concentrations of heat-trapping gases in the atmosphere are at their highest level in more than 200,000 years. the 20th century in the northern hemisphere was the warmest of the past millennium; the first decade of the 21st first century, and the first 7 months of 2010, were the warmest on record. higher global temperatures set in motion a whole series of effects, making the study of climate change, and of humans' role in it, complex and controversial. spring arrives at least 1 week earlier in the northern hemisphere. polar glaciers and ice sheets are receding. the arctic is warming twice as fast as the rest of the planet, and arctic sea ice melted at a near-record pace in 2010. with the sun heating the newly open water, winter refreezing will take longer, and the resulting thinner ice will melt more easily the following summer. the large-scale circulation of global air masses is changing and, with them, the large-scale cycles in ocean currents, including the periodic warming and cooling in the tropical pacific ocean known as el niñ o and la niñ a. as a result, the distribution, timing, and amount of rain and snow are also changing, making the weather seem more unpredictable than ever. unusually warm or cold winters, massive hurricanes like those that devastated the us gulf coast in 2005, and weatherrelated damage to human life and property are all predicted to increase with global warming. in the united states in 2005, weather-related damage totaled nearly $97 billion. where other nutrients are not limiting, rising carbon dioxide concentrations may enhance plant photosynthesis and growth. rising temperatures may shift the ranges of many plants and animals -both wild and domestic -rearranging the composition and distribution of the world's biomes, as well as those of agricultural systems. the resulting displacements will have far-reaching implications not only for the displaced plants and animals but also for the goods and services humans depend on from these living systems. from their beginnings as hunter-gatherers, humans have become highly efficient, machine-aided ecosystem engineers and predators. we transform the land so it produces what we need or want; we harvest the oceans in addition to reaping our own fields; we cover the land, even agricultural land, with sprawling cities. all these activities directly affect the ability of other life-forms to survive and reproduce. we deplete nonhuman life by eliminating some forms and favoring others; the result is a loss of genetic, population, and species diversity. we are irreversibly homogenizing life on earth, in effect exercising an unnatural selection that is erasing the diversity generated by millions of years of evolution by natural selection. one species is now determining which other species will survive, reproduce, and thereby contribute the raw material for future evolution. in the 1930s, so many sardines were scooped from the waters off monterey's cannery row in california that the population collapsed, taking other sea creatures and human livelihoods with it; after rebounding somewhat in the first decade of the 2000s, the species has still not recovered fully. according to the us national marine fisheries service, nearly 80% of commercially valuable fish of known status were overfished or fished to their full potential by 1993. atlantic commercial fish species at their lowest levels in history include tuna, marlin, cod, and swordfish. overfishing not only depletes target species but restructures entire marine food webs. marine mammals, including whales, seals, sea lions, manatees, and sea otters, were so badly depleted by human hunters that one species, steller's sea cow (hydrodamalis gigas), went extinct; many other species almost disappeared. in the 19th century, russian fur traders wiped out sea otters (enhydra lutris) along the central california coast. with the otters gone, their principal prey, purple sea urchins (stronglyocentrotus purpuratus), overran the offshore forests of giant kelp (macrocystis pyrifera), decimating the kelp fronds and the habitat they provided for countless other marine creatures, including commercially harvested fishes. thanks to five decades of protection, marine mammal populations began slowly recovering -only to face food shortages as regional marine food webs continue to unravel because of fishing, changing oceanic conditions, and contamination. timber harvest has stripped vegetation from the amazonian rainforest to mountainsides on all continents, diminishing and fragmenting habitat for innumerable forest and stream organisms, eroding soils, worsening floods, and contributing significantly to global carbon dioxide emissions. in the northern hemisphere, 10% or less remains of old-growth temperate rainforests. the uniform stands of trees usually replanted after logging do not replace the diversity lost with the native forest, any more than monocultures of corn replace the diversity within native tallgrass prairies. a great deal of human ecosystem engineering not only alters or damages the habitats of other living things but often destroys those habitats. satellite-mounted remote-sensing instruments have revealed transformations of terrestrial landscapes on a scale unimaginable in centuries past. together, cropland and pastures occupy 40% of earth's land surface. estimates of the share of land wholly transformed or degraded by humans hover around 50%. our roads, farms, cities, feedlots, and ranches either fragment or destroy the habitats of most large carnivorous mammals. mining and oil drilling damage the soil, remove vegetation, and pollute marine areas. grazing compacts soil and sends silt and manure into streams, where they harm stream life. landscapes that have not been entirely converted to human use have been cut into fragments. in song of the dodo, writer quammen (1996) likens our actions to starting with a fine persian carpet and then slicing it neatly into 36 equal pieces; even if we had the same square footage, we would not have 36 nice persian rugs -only ragged, nonfunctional fragments. and in fact, we do not even have the original square footage because we have destroyed an enormous fraction of it. such habitat destruction is not limited to terrestrial environments. human channelization of rivers may remove whole segments of riverbed. in the kissimmee river of the us state of florida, for example, channelization in the 1960s transformed 165 km of free-flowing river into 90 km of canal, effectively removing 35 km of river channel and drastically altering the orphaned river meanders left behind. wetlands worldwide continue to disappear, drained to create shoreline communities for people and filled to increase cropland. the lower 48 united states lost 53% of their wetlands between the 1700s and mid-1980s. such losses destroy major fish and shellfish nurseries, natural flood and pollution control, and habitat for countless plants and animals. the mosaic of habitats in, on, or near the seafloor -home to 98% of all marine species -is also being decimated. like clear-cutting of an old-growth forest, the use of large, heavy trawls dragged along the sea bottom to catch groundfish and other species flattens and simplifies complex, structured habitats such as gravels, coral reefs, crevices, and boulders and drastically reduces biodiversity. studies reported on by the national research council of the us national academy of sciences have shown that a single tow can injure or destroy upward of two-thirds of certain bottom-dwelling species, which may still not have recovered after a year or more of no trawling. habitat fragmentation and destruction, whether on land or in freshwater and marine environments, may lead directly to extinction or isolate organisms in ways that make them extremely vulnerable to natural disturbances, climate change, or further human disturbance. ''the one process ongoing y that will take millions of years to correct,' ' wilson (1994, p. 355) admonishes us, ''is the loss of genetic and species diversity by the destruction of natural habitats. this is the folly our descendants are least likely to forgive us.'' both deliberately and unwittingly, humans are rearranging earth's living components, reducing diversity and homogenizing biotas around the world. the present, continuing loss of genetic diversity, of populations, and of species vastly exceeds background rates. at the same time, our global economy is transporting species worldwide at unprecedented scales. the globe is now experiencing its sixth mass extinction, the largest since the dinosaurs vanished 65 ma; present extinction rates are thought to be on the order of 100-1000 times those before people dominated earth. according to the millennium ecosystem assessment, a 5-year project begun in 2001 to assess the world's ecosystems, an estimated 10-15% of the world's species will be committed to extinction over the next 30 years. approximately 20% of all vertebrates, including 33% of sharks and rays, are at risk of extinction. at least one of every eight plant species is also threatened with extinction. although mammals and birds typically receive the most attention, massive extinctions of plants, which form the basis of the biosphere's food webs, undermine lifesupport foundations. the mutualistic relationships between animals and plants, particularly evident in tropical forests, mean that extinctions in one group have cascading effects in other groups. plants reliant on animals for pollination or seed dispersal, for example, are themselves threatened by the extinction of the animal species they depend on. not surprisingly, some scientists view extinction as the worst biological tragedy, but extinction is just another symptom of global biotic impoverishment. ever since they began to spread over the globe, people have transported other organisms with them, sometimes for food, sometimes for aesthetic reasons, and most often inadvertently. with the mobility of modern societies and today's especially speedy globalization of trade, the introduction of alien species has reached epidemic proportions, causing some scientists to label it biological pollution. aliens -zebra mussels (dreissena polymorpha) and tamarisks, or saltcedar (tamarix spp.), in north america; the red sea sea jelly rhopilema nomadica and the common aquarium alga caulerpa taxifolia now choking the mediterranean sea; and leidy's comb jelly (mnemiopsis leidyi) of northeastern america in the black sea, to name just a few -are present everywhere, and they usually thrive and spread at the expense of native species. on many islands, for example, more than half the plant species are not native, and in many continental areas the figure reaches 20% or more. the costs of such invasions, in both economic and ecological terms, are high. in the united states, for example, annual economic losses due to damage by invasive species or the costs of controlling them exceed $137 billion per year -$40 billion more than the nation's losses from weather-related damage in 2005. furthermore, alien invasions cause extinctions and, when added to other extinctions and the deliberate monocultures of agricultural crops, homogenize biotas still more. introduced species are fast catching up with habitat fragmentation and destruction as the major engines of ecological deterioration. humans have been manipulating their crop plants and domesticated animals for 10,000 years or so -selecting seeds or individuals and breeding and cross-breeding them. the goal was something better, bigger, tastier, hardier, or all of the above; success was sometimes elusive, but the result was crop homogenization. of the myriad strains of potatoes domesticated by south american cultures, for example, only one was accepted and cultivated when potatoes first made it to europe. the new crop made it possible to feed more people from an equivalent area of land and initially staved off malnutrition. but the strain succumbed to a fungal potato blight in the 1800s. had more than one strain been cultivated, the tragic irish potato famines might have been averted. in the last few decades of the 20th century, people began to manipulate genes directly using the tools of molecular biotechnology, even cloning sheep and cows from adult body cells. us farmers routinely plant their fields with corn whose genetic material incorporates a bacterial gene resistant to certain pathogens. more than 40 genetically altered crops have been approved for sale to us farmers since 1992, with genes borrowed from bacteria, viruses, and insects. the united states accounts for nearly two-thirds of biotechnology crops planted globally. worldwide in 2003, 67.6 million hectares in 18 countries on six continents were planted with genetically modified crops, as compared with 1.7 million hectares in six countries in 1996 -a 40-fold expansion in 8 years. biotechnologists focus on the potential of this newmillennium green revolution to feed the growing world population, which has added almost 1 billion people in the past decade alone. but other scientists worry about unknown human and ecological health risks; these concerns have stirred deep scientific and public debate, especially in europe, akin to the debate over pesticides in rachel carson's time. one worrisome practice is plant genetic engineers' technique of attaching the genes they want to introduce into plants to an antibiotic-resistant gene. they can then easily select plants that have acquired the desired genes by treating them with the antibiotic, which kills any nonresistant plants. critics worry that the antibiotic-resistant genes could spread to human pathogens and worsen an already growing antibioticresistance problem. another concern arises from allergies humans might have or develop in response to genetically modified foods. although supporters of genetic engineering believe that genetically altered crops pose few ecological risks, ecologists have raised a variety of concerns. studies in the late 1990s indicated that pollen from genetically engineered bt corn can kill monarch butterfly caterpillars. bt is a strain of bacterium that has been used since the 1980s as a pesticidal spray; its genes have also been inserted directly into corn and other crops. ecologists have long worried that genetically engineered plants could escape from fields and cross-breed with wild relatives. studies in radishes, sorghum, canola, and sunflowers found that genes from an engineered plant could jump to wild relatives through interbreeding. the fear is that a gene conferring insect or herbicide resistance might spread through wild plants, creating invasive superweeds that could potentially lower crop yields and further disturb natural ecosystems. in fact, herbicide-resistant turf grass tested in oregon in 2006 did escape and spread, and transgenic canola has been appearing throughout the us state of north dakota, which has tens of thousands of hectares in conventional and genetically modified canola. according to the scientists who discovered the transgenic escapees growing in north dakota -far from any canola field -the plants are likely to be cross-pollinating in the wild and swapping introduced genes; the plants' novel gene combinations indicate that the transgenic traits are stable and evolving outside of cultivation. genetically engineered crops do confer some economic and environmental benefits: for farmers, higher yields, lower costs, savings in management time and gains in flexibility; for the environment, indirect benefits from using fewer pesticides and herbicides. but it is still an open question whether such benefits outweigh the potential ecological risks or whether the public will embrace having genetically modified foods as staples in their diet. human biotic impacts are not confined to other species; human cultures themselves have suffered from the widening circles of indirect and direct effects humans have imposed on the rest of nature. over the past hundred years, human technology has cut both ways with regard to public health. wonder drugs controlled common pathogens at the same time that natural selection strengthened those pathogens' ability to resist the drugs. reservoirs in the tropics made water supplies more reliable for humans but also created ideal environments for human parasites. industrialization exposed human society to a remarkable array of toxic substances. although man's inhumanity to man has been both fact and the subject of discourse for thousands of years, the discussions have mostly been removed from any environmental context. few people today regard social ills as environmental impacts or humans as part of a biota. but diminished societal well-being -whether manifest in high death rates or poor quality of life -shares many of its roots with diminished nonhuman life as a form of biotic impoverishment. the intersection of the environment and human health is the core of the discipline known as environmental health. among the environmental challenges to public health are the direct effects of toxic chemicals; occupational health threats, including exposures to hazardous materials on the job; and sanitation and disposal of hazardous wastes. exploitation of nonrenewable natural resources -including coal mining, rock quarrying or other mining operations, and petroleum extraction and refining -often chronically impairs workers' health and shortens their lives. farmworkers around the world suffer long-term ills from high exposures to pesticides and herbicides. partly because of increased air pollution, asthma rates are rising, particularly in big cities. synthetic volatile solvents are used in products from shoes to semiconductors, producing lung diseases and toxic wastes. nuclear weapons production starting in world war ii, and associated contamination, have been linked to a variety of illnesses, including syndromes neither recognized nor understood at the time and whose causes were not diagnosed until decades afterward. the grayish metal beryllium, for example, was used in weapons production and was found decades later to scar the lungs of workers and people living near toxic waste sites. infectious diseases have challenged human populations throughout history, playing a significant role in their evolution and cultural development over the past 10,000 years, with 75% of human diseases linked to wildlife or domestic animals. the 20th century brought major successes in eradicating such infectious diseases as smallpox, polio, and many waterborne illnesses. but toward the century's end, emerging and reemerging diseases were again reaching pandemic proportions. infectious diseases thought to be on the wane -including tuberculosis, malaria, cholera, diptheria, leptospirosis, encephalitis, and dengue fever -have begun a resurgence. in addition, seemingly new scourges -ebola virus, hantavirus, hiv/aids, lyme disease, and west nile fever -are also spreading, often, it appears, from wild animal hosts to humans as people encroach further upon previously undisturbed regions. a number of studies over the decade before 2010 show complex connections between biodiversity and disease: biodiversity loss often increases disease transmission, as in lyme disease and west nile fever, but diverse ecosystems also serve as sources of pathogens. overall, however, the studies indicate that preserving intact ecosystems and their endemic biodiversity often reduces the prevalence of infectious diseases. human migrations -including their modern incarnation through air travel -also accelerate pathogen traffic and launch global pandemics, such as the 2003 outbreak of severe acute respiratory syndrome and the 2009 swine flu outbreak caused by the h1n1 virus. even something as simple and apparently benign as lighting can become an indirect agent of disease. artificial lighting, especially in the tropics, for example, can alter human and insect behavior in ways that speed transmission of insect-borne diseases, such as chagas's disease, malaria, and leishmaniasis. in addition, especially in highly developed countries such as the united states, diseases of affluence and overconsumption are taking a toll. heart disease is the number one cause of death in the united states; overnutrition, obesity, and diabetes due to sedentary, technology-driven lifestyles, particularly among children, are chronic and rising. one estimate put the share of us children considered overweight or obese at one in three. this rise in obesity rates has been stunningly rapid. as recently as 1980, just 15% of adults were obese; by 2008 the rate had hit 34%, and two-thirds of americans are now considered either overweight or obese. although not conventionally regarded as elements of biodiversity, human languages, customs, agricultural systems, technologies, and political systems have evolved out of specific regional environments. like other organisms' adaptive traits and behaviors, these elements of human culture constitute unique natural histories adapted, like any natural history, to the biogeographical context in which they arose. yet modern technology, transportation, and trade have pushed the world into a globalized culture, thereby reducing human biological and cultural diversity. linguists, for example, are predicting that at least half of the 7000 languages spoken today will become extinct in the 21st century. with the spread of euro-american culture, unique indigenous human cultures, with their knowledge of local medicines and geographically specialized economies, are disappearing even more rapidly than the natural systems that nurtured them. this loss of human biodiversity is in every way as troubling as the loss of nonhuman biodiversity. the effects of environmental degradation on human quality of life are another symptom of biotic impoverishment. food availability, which depends on environmental conditions, is a basic determinant of quality of life. yet according to the world health organization, nearly half the world's population suffers from one of two forms of poor nutrition: undernutrition or overnutrition. a big belly is now a symptom shared by malnourished children, who lack calories and protein, and overweight residents of the developed world, who suffer clogged arteries and heart disease from eating too much. independent of race or economic class, declining quality of life in today's world is manifest in symptoms such as increased asthma in the united states caused by environmental contaminants and the high disease rates in the former soviet bloc after decades of unregulated pollution. even with explicit legal requirements that industries release information on their toxic emissions, many people throughout the world still lack both information and the decision-making power that would give them any control over the quality of their lives. aggrieved about the degraded environment and resulting quality of life in his homeland, ogoni activist ken saro-wiwa issued a statement shortly before he was executed by the nigerian government in 1995 saying, ''the environment is man's first right. without a safe environment, man cannot exist to claim other rights, be they political, social, or economic.'' kenyan maathai (2009, p. 249) , 2004 winner of the nobel peace prize, has also written, ''[i]f we destroy it, we will undermine our own ways of life and ultimately kill ourselves. this is why the environment needs to be at the center of domestic and international policy and practice. if it is not, we don't stand a chance of alleviating poverty in any significant way.'' having ignored this kind of advice for decades, nations are seeing a new kind of refugee attempting to escape environmental degradation and desperate living conditions; the number of international environmental refugees exceeded the number of political refugees around the world for the first time in 1999. environmental refugees flee homelands devastated by flooding from dam building, extraction of mineral resources, desertification, and unjust policies of national and international institutions. such degradation preempts many fundamental human rights, including the rights to health, livelihood, culture, privacy, and property. people have long recognized that human activities that degrade environmental conditions threaten not only the biosphere but also humans' own quality of life. as early as 4500 years ago in mesopotamia and south asia, writings revealed an awareness of biodiversity, of natural order among living things, and of consequences of disrupting the biosphere. throughout history, even as civilization grew increasingly divorced from its natural underpinnings, writers, thinkers, activists, and people from all walks of life have continued to see and extol the benefits of nature to humans' quality of life. contemporary society still has the chance to relearn how important the environment is to quality of life. it is encouraging that the united steelworkers of america in 1990 released a report recognizing that protecting steelworker jobs could not be done by ignoring environmental problems and that the destruction of the environment may pose the greatest threat to their children's future. it is also encouraging that the 2007 nobel peace prize was awarded to a political figure and a group of scientists for their work on climate change. making a living from nature's wealth has consistently opened gaps between haves and have-nots, between those who bear the brunt of environmental damage to their home places and those who do not, and between the rights of people alive now and those of future generations; these disparities too are part of biotic impoverishment. inequitable access to ''man's first right'' -a healthy local environment -has come to be known as environmental injustice. environmental injustices, such as institutional racism, occur in industrial and nonindustrial nations. injustice can be overt, as when land-use planning sites landfills, incinerators, and hazardous waste facilities in minority communities, or when environmental agencies levy fines for hazardous waste violations that are lower in minority communities than in white communities. less overt, but no less unjust, is the harm done to one community when unsound environmental practices benefit another, as when clear-cut logging in the highlands of northwestern north america benefits logging communities while damaging the livelihoods of lowland fishing communities subjected to debris flows, sedimentation, and downstream flooding. the plight of the working poor and the disparities between rich and poor are also examples of biotic impoverishment within the human community. according to the united nations research institute for social development, the collective wealth of the world's 358 billionaires equaled the combined income of the poorest 2.4 billion people in 1994. forbes magazine put the number of billionaires in early 2010 at 1011, with a total worth of $3.6 trillion. in the united states during the last decade of the 20th century, the incomes of poor and middle-class families stagnated or fell, despite a booming stock market. the center on budget and policy priorities and the economic policy institute reported that between 1988 and 1998, earnings of the poorest fifth of american families rose less than 1%, while earnings of the richest fifth jumped 15%. by the middle of the first decade of the 21st century, americans' income inequality had become the widest among industrialized nations, with the wealthiest 20% of the population holding 85% of the wealth. the wealthiest americans continued to prosper even during the global recession late in that decade, while the less well-off kept losing ground. but perhaps the grossest example of human and environmental domination leading to continued injustice is the creation of a so-called third world to supply raw materials and labor to the dominant european civilization after 1500 and the resulting schism between today's developed and developing nations. developing regions throughout the world held tremendous stores of natural wealth, some of it -like petroleum -having obvious monetary value in the dominant economies and some having a value invisible to those economies -like vast intact ecosystems. a 2010 united nations study (teeb) estimated that even today, earth's ecosystems account for roughly half to 90% of the source of livelihoods for rural and forest-dwelling peoples; the study calls this value the gross domestic product (gdp) of the poor. dominant european civilizations unabashedly exploited this natural wealth and colonized or enslaved the people in whose homelands the wealth was found. but the dominant civilizations also exported their ways of thinking and their economic models to the developing world, not only colonizing places but also effecting what wangari maathai has called a colonization of the mind. although dominant 21st century society tends to dismiss ancient wisdom as irrelevant in the modern world, perhaps the cruelest impoverishment of all is the cultural and spiritual deracination experienced by exploited peoples worldwide. exploitation of poor nations and their citizens by richer, consumer countries -and in many cases by the same governments that fought for independence from the colonists while adopting the colonists' attitudes and economic models -persists today in agriculture, wild materials harvesting, and textile and other manufacturing sweatshops. in the mid-1990s, industrial countries consumed 86% of the globe's aluminum, 81% of its paper, 80% of its iron and steel, 75% of its energy, and 61% of its meat; they are thus responsible for most of the environmental degradation associated with producing these goods. most of the actual degradation, however, still takes place in developing nations. as a result, continuing environmental and social injusticeenvironmental and social impoverishment perpetrated by outsiders and insiders alike -pervades developing nations. such impoverishment can take the form of wrenching physical dislocation like the massive displacements enforced by china's three gorges dam. it can appear as environmental devastation of homelands and murder of the people who fought to keep their lands, as in the nigerian government-backed exploitation of ogoniland's oil reserves by the shell petroleum development corporation. after saro-wiwa's execution, the ogoni were left, without a voice, to deal with a scarred and oilpolluted landscape. despite great advances in the welfare of women and children over the past century, poverty still plagues both groups. children from impoverished communities, even in affluent nations, suffer from the lethargy and impaired physical and intellectual development known as failure to thrive. poverty forces many children to work the land or in industrial sweatshops; lack of education prevents them from attaining their intellectual potential. this impoverishment in the lives of women and children is as much a symptom of biotic impoverishment as are deforestation, invasive alien organisms, or species extinctions. little by little, community-based conservation and development initiatives are being mounted by local citizens to combat this impoverishment: witness maathai's green belt movement, which began with tree planting to restore community landscapes and provide livelihoods for residents, and the rise of ecotourism and microlending (small loans made to individuals, especially women, to start independent businesses) as ways to bring monetary benefits directly to local people without further damaging their environments. ultimately, one could see all efforts to protect the ethnosphere and the biosphere as a fight for the rights of future generations to an environment that can support them. only during the last two decades of the 20th century did environmental issues find a place on international diplomatic agendas, as scholars began calling attention to -and governments began to see -irreversible connections between environmental degradation and national security. british scholar myers (1993) , noting that environmental problems were likely to become predominant causes of conflict in the decades ahead, was one of the first to define a new concept of environmental security. national security threatened by unprecedented environmental changes irrespective of political boundaries will require unprecedented responses altogether different from military actions, he warned. nations cannot deploy their armies to hold back advancing deserts, rising seas, or the greenhouse effect. canadian scholar homer-dixon (1999) showed that environmental scarcities -whether created by ecological constraints or sociopolitical factors including growing populations, depletion of renewable resources such as fish or timber, and environmental injustice perpetrated by one segment of a population on another -were fast becoming a permanent, independent cause of civil strife and ethnic violence. he found that such scarcity was helping to drive societies into a self-reinforcing spiral of dysfunction and violence, including terrorism. environmental and economic injustices worldwide leave no country immune to this type of threat. typically, diplomacy has stalled in conflicts over natural resources: arguments over water rights have more than once held up israeli-palestinian peace agreements; fights over fish erupted between canada and the united states, spain, and portugal. in contrast, in adopting the montreal protocol on substances that deplete the ozone layer in 1987, governments, nongovernmental organizations, and industry successfully worked together to safeguard part of the environmental commons. the treaty requires signatory nations to curb their use of cfcs and other ozone-destroying chemicals and has been, according to former united nations secretary general kofi annan, perhaps the most successful international agreement to date. if scientists have learned anything about the factors leading to biotic impoverishment, they have learned that the factors' cumulative effects can take on surprising dimensions. as scholars like fagan (1999) and diamond (2005) have chronicled, the multiple stresses of global climatic cycles such as el niñ o, natural events like droughts or floods, resource depletion, and social upheaval have shaped the fates of civilizations. societies as far-flung as ancient egypt, peru, the american southwest, and easter island prospered and collapsed because of unwise management of their environments. the city of ubar, built on desert sands in what is now southern oman, literally disappeared into the sinkhole created by drawing too much water out of its great well. in modern sahelian africa, a combination of well digging and improved medical care and sanitation led to a threefold population increase; sedentary ways, heavy taxes imposed by a colonial government, and an impoverished people took the place of a nomadic culture evolved within the desert's realities. during the first decade of the 21st century, numerous natural disasters befell nations around the world: wildfires in australia, bolivia, canada, and russia; flooding in china, india, romania, and west africa; devastating hurricanes and typhoons in the caribbean, philippines, taiwan, and southeastern united states; catastrophic landslides and floods in china, guatemala, pakistan, and portugal; and destructive earthquakes in chile, china, haiti, indonesia, and pakistan. neither the rains nor the earthquakes were caused by human activity, but the cumulative effects of human land uses and management practices -from dikes separating the mississippi from its floodplain to deforestation in haiti -made the losses of human life and property much worse than they might have been otherwise. the ultimate cause of humans' massive environmental impact is our individual and collective consumptive and reproductive behavior, which has given us spectacular success as a species. but the very things that enabled humans to thrive in nearly every environment have magnified our impacts on those environments, and the technological and political steps we take to mitigate our impacts often aggravate them. too many of us simply take too much from the natural world and ask it to absorb too much waste. for most of human history, people remained tied to their natural surroundings. even as agriculture, writing, and technology advanced, barriers of geography, language, and culture kept humans a diverse lot, each group depending on mostly local and regional knowledge about where and when to find resources necessary for survival. their worldviews, and resulting economies, reflected this dependency. for example, in northwestern north america starting about 3000 years ago, a native economy centered on the abundance of salmon. at its core was the concept of the gift and a belief system that treated all parts of the earth -animate and inanimate -as equal members of a community. in this and other ancient gift economies, a gift was not a possession that could be owned; rather, it had to be passed on, creating a cycle of obligatory returns. individuals or tribes gained prestige through the size of their gifts, not the amount of wealth they accumulated. this system coevolved with the migratory habits of the salmon, which moved en masse upriver to spawn each year. because the indians viewed salmon as equals to themselves, killing salmon represented a gift of food from salmon to people. fishers were obligated to treat salmon with respect or risk losing this vital gift. the exchange of gifts between salmon and humans -food for respectful treatment -minimized waste and overharvest and ensured a continuous supply of food. further, the perennial trading of gifts among the people effectively redistributed the natural wealth brought each year by fluctuating populations of migrating fish, leveling out the boom-and-bust cycles that usually accompany reliance on an uncertain resource. in modern times, the gift economy along with the egalitarian worldview that accompanied it, has been eclipsed by a redistributive economy tied not to an exchange of gifts with nature but to the exploitation of nature and to the technologies that enhance that exploitation. nature became a resource for humans, rather than an equal to humans. in economic terms, natural resources fell under the heading of 'land' in an economic trinity comprising three factors of production: land, labor, and capital. land and resources, including crops, became commodities -expendable or easily substitutable forms of capital -whose value was determined solely by their value in the human marketplace. in 1776 adam smith published his famous inquiry into the nature and causes of the wealth of nations, in which he argued that society is merely the sum of its individuals, that the social good is the sum of individual wants, and that the market (the so-called invisible hand) automatically guides individual behavior to the common good. crucial to his theories were division of labor and the idea that all the factors of production were freely mobile. his mechanistic views created an economic rationale for no longer regarding individuals as members of a community linked by ethical, social, and ecological bonds. about the same time, fueling and fueled by the beginnings of the industrial revolution, the study of the natural world was morphing into modern physics, chemistry, geology, and biology. before the mid-19 century, those who studied the natural world -early 19 century german biogeographer baron alexander von humboldt and his disciple charles darwin among them -took an integrated view of science and nature, including humans. both scientists regarded the understanding of the complex interdependencies among living things as the noblest and most important result of scientific inquiry. but this integrated natural philosophy was soon supplanted by more atomistic views, which fit better with industrialization. mass production of new machines relied on division of labor and interchangeable parts. like automobiles on an assembly line, natural phenomena too were broken down into their supposed component parts in a reductionism that has dominated science ever since. rushing to gain indepth, specialized knowledge, science and society lost sight of the need to tie the knowledge together. disciplinary specialization replaced integrative scholarship. neoclassical economics, which arose around 1870, ushered in the economic worldview that rules today. a good's value was no longer tied to the labor required to make it but derived instead from its scarcity. a good's price was determined only by the interaction of supply and demand. as part of 'land,' natural resources therefore became part of the human economy, rather than the material foundation making the human economy possible. because of its doctrine of infinite substitutability, neoclassical economics rejects any limits on growth; forgotten are the classical economic thinkers and contemporaries of von humboldt, including thomas malthus and john stuart mill, who saw limits to the growth of human population and material well-being. consequently, the 19th and 20th centuries saw the rise to dominance of economic indicators that fostered the economic invisibility of nature, misleading society about the relevance of earth's living systems to human well-being. among the worst offenders in this regard are gross national product (gnp) and its cousin, gdp. gnp measures the value of goods and services produced by a nation's citizens or companies, regardless of their location around the globe. gdp, in contrast, measures the value of goods and services produced within a country's borders, regardless of who generates those goods and services. in effect, both gnp and gdp measure money changing hands, no matter what the money pays for; they make no distinction between what is desirable and undesirable, between costs and benefits. both indicators ignore important aspects of the economy like unpaid work or nonmonetary contributions to human fulfillment -parenting, volunteering, checking books out of the library. worse, the indicators also omit social and environmental costs, such as pollution, illness, or resource depletion; they only add and do not subtract. gdp math adds in the value of paid daycare or a stay in the hospital and ignores the value of unpaid parenting or being cared for at home by friends. it adds in the value of timber sold, but fails to subtract the losses in biodiversity, watershed protection, or climate regulation that come when a forest is cut. efforts have been made in the past few decades to create less blinkered economic indicators. social scientists herman daly and john cobb in 1989 developed an index of sustainable economic welfare, which adjusts the united states' gnp by adding in environmental good things and subtracting environmental bad things. public expenditures on education, for example, are weighted as ''goods'' while costs of pollution cleanup, depletion of natural resources, and treating environment-related illnesses are counted as 'bads.' unlike the soaring gdp of recent decades, this index of sustainable economic welfare remained nearly unchanged over the same period. still other work aims to reveal nature's worth in monetary terms by assigning dollar values to ecological goods and services. a 1997 study by ecologist david pimentel and colleagues calculated separate values for specific biological services, such as soil formation, crop breeding, or pollination; by summing these figures, these researchers estimated the total economic benefits of biodiversity for the united states at $319 billion -5% of us gdp at the time -and for the world at $2928 billion. a 2000 analysis by pimentel and colleagues reported that the approximately 50,000 nonnative species in the united states cause major environmental damage and reparation costs amounting to $137 billion a year. as part of the united nations international year of biodiversity in 2010, several studies have translated the value of the world's ecosystems into dollar values. one report estimated the worth of crucial ecosystem services delivered to humans by living systems at $21-72 trillion per year -comparable to a world gross national income of $58 trillion in 2008. another study reported, among other things, that as many as 500 million people worldwide depend on coral reefs -valued between $30 billion and $172 billion a year -for fisheries, tourism, and protection from ocean storms and high waves, services threatened by warmer and more acidic seas. although a monetary approach does not create a comprehensive indicator of environmental condition, it certainly points out that ecological values ignored by the global economy are enormous. the us census bureau predicts that by 2012, the global human population will top 7 billion -having grown by nearly 1 billion people during the decade between the first and second editions of this encyclopedia. from the appearance of h. sapiens about 200,000 years ago, it took humans until 1804 to reach their first billion, 123 years to double to 2 billion, and 33 years to achieve 3 billion. human population doubled again from 3 to 6 billion in about 40 years -before most post-world war ii baby boomers reached retirement age. even with fertility rates declining in developed countries, china, and some developing countries where women are gaining education and economic power, and with pandemics like aids claiming more lives, the census bureau predicts that world population will reach 9 billion by 2044. humans appropriate about 40% of global plant production, 54% of earth's freshwater runoff, and enough of the ocean's bounty to have depleted 63% of assessed marine fish stocks. in energy terms, one person's food consumption amounts to 2500-3000 calories a day, about the same as that of a common dolphin. but with all the other energy and materials humans use, global per capita energy and material consumption have soared even faster than population growth over the past 50 years. now, instead of coevolving with a natural economy, global society is consuming the foundations of that economy, impoverishing earth's living systems, and undermining the foundations of its own existence (figure 1 ). for most of the 20th century, environmental measurements, or indicators, tracked primarily two classes of information: counts of activities directed at environmental protection and the supply of products to people. regulatory agencies are typically preoccupied with legislation, permitting, or enforcement, such as the numbers of environmental laws passed, permits issued, enforcement actions taken, or treatment plants constructed. resource protection agencies concentrate on resource harvest and allocation. water managers, for example, measure water quantity; they allocate water to domestic, industrial, and agricultural uses but seldom make it a priority to reserve supplies for sustaining aquatic life, to protect scenic and recreational values, or simply to maintain the water cycle. foresters, farmers, and fishers count board-feet of timber, bushels of grain, and tons of fish harvested. governmental and nongovernmental organizations charged with protecting biological resources also keep counts of threatened and endangered species. as in the parable of the three blind men and the elephanteach of whom thinks the elephant looks like the one body part he can touch -these or similar indicators measure only one aspect of environmental quality. counting bureaucratic achievements focuses on actions rather than on information about real ecological status and trends. measurements of resource supply keep track of commodity production, not necessarily a system's capacity to continue supplying that commodity. and measuring only what we remove from natural systems, as if we were taking out the interest on a savings account, overlooks the fact that we are usually depleting principal as well. even biologists' counts of threatened and endangered species -which would seem to measure biotic impoverishment directly -still focus narrowly on biological parts, not ecological wholes. enumerating threatened and endangered species is just like counting any other commodity. it brings our attention to a system already in trouble, perhaps too late. and it subtly reinforces our view that we know which parts of the biota are most important. society needs to rethink its use of available environmental indicators, and it needs to develop new indicators that represent current conditions and trends in the systems humans depend on ( table 2) . it particularly needs objective measures more directly tied to the condition, or health, of the environment so that people can judge whether their actions are compromising that condition. such measures should be quantitative, yet easy to understand and communicate; they should be cost-effective and applicable in many circumstances. unlike narrow criteria tracking only administrative, commodity, or endangered species numbers, they should provide reliable signals about status and trends in ecological systems. ideally, effective indicators should describe the present condition of a place, aid in diagnosing the underlying causes of that condition, and make predictions about future trends. they should reveal not only risks from present activities but also potential benefits from alternative management decisions. most important, these indicators should, either singly or in combination, give information explicitly about living systems. measurements of physical or chemical factors can sometimes act as surrogates for direct biological measurements, but only when the connection between those measures and living systems is clearly understood. too often we make assumptionswhen water managers assume that chemically clean water equals a healthy aquatic biota, for example -that turn out to be wrong and fail to protect living systems. as environmental concerns have become more urgent -and governmental and nongovernmental organizations have struggled to define and implement the concept of sustainable development -the effort has grown to create indicator systems that explicitly direct the public's and policymakers' attention to the value of living things. moving well past solely economic indexes like gdp, several indexes now integrate ecological, social, and economic well-being. the index of environmental trends for nine industrialized countries, developed by the nonprofit national center for economic and security alternatives, incorporates ratings of air, land, and water quality; chemical and waste generation; and social system economic system natural system figure 1 relationships among the natural, social, and economic systems on earth. human economies may be thought of as icing atop a two-layer cake. the economic icing is eroding the human social and natural layers beneath it, threatening the foundation and sustainability of all three systems. modified from karr jr and chu ew (1995) ecological integrity: reclaiming lost connections. in: westra l and lemons j (eds.) perspectives in ecological integrity, pp 34-48. dordrecht, netherlands: kluwer academic. energy use since 1970. by its 1995 rankings, environmental quality in the united states had gone down by 22% since 1970, while denmark had declined by 11%. in 2000, world leaders, supported by the united nations development programme, defined a set of eight millennium development goals to be attained by 2015, which combine poverty, education, employment, and environmental sustainability. they include human rights and health goals -such as universal primary education, gender equality, and combating aids and other diseases -as well as ensuring environmental sustainability. in the decade since the program began, progress has been made in reducing poverty; expanding educational opportunities for children, especially in africa; slowing deforestation; and providing better water for many people, among others. but progress controlling climate change, halting biodiversity loss, and achieving gender equality has lagged. a later environmental performance index spearheaded by yale and columbia universities ranks 163 countries on 25 performance indicators in 10 well-established policy categories to determine whether and how well countries are meeting established environmental goals. this index incorporates measurements addressing environmental stresses to human health plus ecosystem health and natural resource management, collectively referred to as ecosystem vitality. the categories representing environmental health include the environmental burden of disease and the effects of air and water quality on human health. those representing ecosystem vitality include air and water resources for ecosystems; biodiversity and habitat; and climate change, among others. although hampered by data gaps and the inability to track changes in countries' performance through time, the index provides a sense of countries' relative performance with regard to the environmental challenges faced by all. top performers in the 2010 rankings included iceland, switzerland, costa rica, and sweden. the united states ranked 61st well below japan and nations in the european union. a resource-accounting approach pioneered in the 1990s by geographers rees and wackernagel (1996) translates humans' impact on nature, particularly resource consumption, into a metaphorical ecological footprint. this accounting estimates the area required by a city, town, nation, or other human community to produce consumed resources and absorb generated wastes; it then compares the physical area occupied by a city or country with the area required to supply that city or country's needs. the 29 largest cities of baltic europe, for example, appropriate areas of forest, agricultural, marine, and wetland ecosystems that are at least 565-1130 times larger than the areas of the cities themselves. according to the global footprint network, national ecological footprints in 2010 ranged from a high of 10.7 hectares per person for the united arab emirates to 0.4 hectares per person for timor-leste and 0.6 for afghanistan and bangladesh. the united states' ecological footprint -8.0 hectares per person -tied for fourth among 152 nations with populations of at least 1 million. one-hundred and four of these nations operate under ecological deficits; that is, their 6. cumulative effects (frequency of catastrophic natural disasters; costs of weather-related property damage; human death tolls; government subsidies of environmentally destructive activities such as fishery overcapitalization, below-cost timber sales, water projects, and agricultural supports; replacement costs for ecological services; pricing that reflects environmental costs; ''green'' taxes; rise in polycultural agricultural practices; number of organic farms) a these indicators have been or could be used to monitor status and trends in environmental quality, including dimensions of biotic impoverishment. without a full spectrum of indicators, however, and without coupling them to direct measures of biological condition, only a partial view of the degree of biotic impoverishment will emerge. consumption exceeds the biological capacity of their lands and waters to provide needed resources and absorb their wastes. at their present rates of consumption, these nations are therefore overexploiting either their own resources or those of other nations. by ecological footprint accounting, raising the nearly 7 billion people on earth in the early 21st century to living standards -and thus ecological footprints -equal to those in the united states would require four planets more than the only one we have. clearly, humans are consuming more resources, and discarding more waste, than earth's living systems can produce or absorb in a given time period. this gap is the global sustainability gap the world now faces. most environmental indexes and accounting systems are still human centered; they still do not measure the condition of the biosphere itself. we may know that biodiversity's services are worth huge sums of money and that our hometown's ecological footprint is much bigger than the town's physical footprint, but how do we know whether specific actions damage living systems or that other actions benefit them? how do we know if aggregate human activity is diminishing life on earth? to answer this question, we need measures that directly assess the condition of living systems. biological assessment directly measures the attributes of living systems to determine the condition of a specific landscape. the very presence of thriving living systems -sea otters and kelp forests off the central california coast; salmon, orcas, and herring in pacific northwest waters; monk seals in the mediterranean sea -says that the conditions those organisms need to survive are also present. a biota is thus the most direct and integrative indicator of local, regional, or global biological condition. biological assessments give us a way to evaluate whether monetary valuations, sustainability indexes, and ecological footprints are telling the truth about human impact on the biosphere. biological assessments permit a new level of integration because living systems, including human cultures, register the accumulated effects of all forms of degradation caused by human actions. direct, comprehensive biological monitoring and assessment has been done for many aquatic systems; measures are less developed for terrestrial systems. the index of biological integrity (ibi), for example, was developed in 1981 to assess the health of streams in the us midwest and has since helped scientists, resource managers, and citizen volunteers understand, protect, and restore rivers in at least 67 countries worldwide (karr 2006) . borrowing from the same page as more recent sustainability indexes, ibi takes the concept behind economic indexes like gdp or the consumer price index -that of multiple indicators integrated into a multimetric index -and applies it to animals and plants in bodies of water or other environments. the specific measurements ( table 3 ) are sensitive to a broad range of human effects in waterways, such as sedimentation, nutrient enrichment, toxic chemicals, physical habitat destruction, and altered flows. the resulting index combines the responses of biological parts such as species, as well as processes such as food web dynamics, to human actions. indexes of biological integrity have been developed for a number of aquatic and terrestrial environments; the most widely used indexes for assessing rivers examine fishes and benthic (bottom-dwelling) invertebrates. these groups are abundant and easily sampled, and the species living in virtually any water body represent a diversity of anatomical, ecological, and behavioral adaptations. as humans alter watersheds and water bodies, changes occur in taxonomic richness (biodiversity), species composition (which species are present), individual health, and feeding and reproductive relationships. sampling the inhabitants of a stream can tell us much about that stream and its landscape. biological diversity is higher upstream of wastewater treatment plants than downstream, for example, whereas, at the same location, year-toyear variation is low (figure 2) . biological sampling can also reveal differences between urban and rural streams. for instance, samples of invertebrates from one of the best streams in rural king county, in the us state of washington, contain 27 kinds, or taxa, of invertebrates; similar samples from an urban stream in the city of seattle contain only 7. the rural stream has 18 taxa of mayflies, stoneflies, and caddisflies; the urban stream, only 2 or 3. when these and other metrics are combined in an index based on invertebrates, the resulting benthic ibi (b-ibi) ranks the condition, or health, of a stream numerically ( table 4) . a benthic ibi can also be used to compare sites in different regions. areas in wyoming's grand teton national park where human visitors are rare have near-maximum b-ibis. streams with moderate recreation taking place in their watersheds have b-ibis that are not significantly lower than those with no human presence, but places where recreation is heavy are clearly damaged. urban streams in the nearby town of jackson are even more degraded, yet not as bad as urban streams in seattle. nation-specific biological assessments also can be and are being done. the us environmental protection agency (2006), for example, performed a nationwide survey of stream condition using an ibi-like multimetric index. the survey found that 28% of us stream miles were in good condition in table 3 biological attributes in two indexes of biological integrity comparison with least-disturbed reference sites in their regions, 25% were in fair condition, and 42% were in poor condition (5% were not assessed). the agency has been expanding this effort to include other water resource types, including coastal waters, coral reefs, lakes, large rivers, and wetlands. since 2000, the heinz center (2008) has published two editions of its report on the state of us ecosystems, which seek to capture a view of the large-scale patterns, conditions, and trends across the united states. the center defined and compiled a select set of indicators -specific variables tracking ecosystem extent and pattern, chemical and physical characteristics, biological components, and goods and services derived from the natural world -for six key ecosystems: coasts and oceans, farmlands, forests, fresh waters, grasslands and shrublands, and urban and suburban landscapes. among the many conclusions of the 2008 report were that the acreage burned every year by wildfires was on the increase; nonnative fish had invaded nearly every watershed in the lower 48 states; and chemical contaminants were found in virtually all streams and most groundwater wells, often at levels above those set to protect human health or wildlife. on the plus side, ecosystems were increasing their storage of carbon, soil quality was improving, and crop yields had grown significantly. the massive international un millennium ecosystem assessment (2005) remains the gold standard for synthesizing ecological conditions at a variety of scales. from 2001 through 2005, the project examined the full range of global ecosystems -from those relatively undisturbed, such as natural forests, to landscapes with mixed patterns of human use to ecosystems intensively managed and modified by humans, such as agricultural land and urban areas -and communicated its findings in terms of the consequences of ecosystem change for human well-being. the resulting set of reports drew attention to the many kinds of services people derive from ecosystems, specifically, supporting services, such as photosynthesis, soil formation, and waste absorption; regulating services, such as climate and flood control and maintenance of water quality; provisioning services, such as food, wood, and nature's pharmacopoeia; creek near portland, oregon (united states), taxa richness differed little between years but differed dramatically between sites upstream of a wastewater outfall and sites downstream. (b) taxa richness also differed between two creeks with wastewater outfalls (tickle and north fork deep) and one creek without an outfall (foster). all three streams flowed through watersheds with similar land uses. fig. 2 for graphs of selected b-ibi metrics at these sites. and cultural services from scientific to spiritual. in addition, the reports explicitly tied the status of diverse ecosystems and their capacity to provide these services to human needs as varied as food and health, personal safety and security, and social cohesion. even while recognizing that the human species is buffered against ecological changes by culture and technology, the reports highlighted our fundamental dependence on the flow of ecosystem services and our direct responsibility for the many faces of biotic impoverishment. among other findings, the assessment found that 60% of the services provided by ecosystems are being degraded at levels that derail efforts to stem poverty, hunger, and disease among the poor everywhere. government subsidies that provide incentives to overharvest natural resources are a leading cause of the decline in renewable natural resources. such declines are not limited to coral reefs and tropical forests, which have been on the public's radar for some time; they are pervasive in grasslands, deserts, mountains, and other landscapes as well. moreover, the degradation of ecosystem services could grow worse during the first half of the 21st century, effectively blocking achievement of the united nations' eight millennium development goals. the core message embodied in ecological, especially biological, assessments is that preventing harmful environmental impacts goes beyond narrow protection of clean water or clear skies, even beyond protecting single desired species. certain species may be valuable for commerce or sport, but these species do not exist in isolation. we cannot predict which organisms are vital for the survival of commercial species or species we want for other reasons. failing to protect all organisms -from microbes and fungi to plants, invertebrates, and vertebrates -ignores the key contributions of these groups to healthy biotic communities. no matter how important a particular species is to humans, it cannot persist outside the biological context that sustains it. direct biological assessment objectively measures this context and is fundamental to advancing the kinds of syntheses like those of the millennium ecosystem assessment. every animal is alert to dangers in its environment. a microscopic protozoan gliding through water responds to light, temperature, and chemicals in its path, turning away at the first sign of something noxious. a bird looking for food must decide when to pursue prey and when not, because pursuit might expose the bird to predators. the bird might risk pursuit when it is hungry but not when it has young to protect. animals that assess risks properly and adjust their behavior are more likely to survive; in nature, flawed risk assessment often means death or the end of a genetic line. humans, too, are natural risk assessors. each person chooses whether to smoke or drink, fly or take the train, drive a car or ride a motorcycle and at what speeds. each decision is the result of a partially objective, partially subjective internal calculus that weighs benefits and risks against one another. risk is a combination of two factors: the numerical probability that an adverse event will occur and the consequences of the adverse event. people may not always have the right signals about these two factors, however, and may base their risk calculus on the wrong clues. city dwellers in the united states generally feel that it is safer to drive home on a saturday night than to fly in an airplane, for example. even though the numerical odds of an accident are much higher on the highway than in the air, people fear more the consequences of an airplane falling out of the sky. human society also strives to reduce its collective exposure to risks. governments seldom hesitate to use military power to defend their sovereignty or, albeit more reluctantly, their regulatory power to reduce workplace risks and risks associated with consumer products like automobiles. but people and their governments have been much less successful in defining and reducing a broad range of ecological risks, largely because they have denied that the threats are real. policies and plans generated by economists, technologists, engineers, and even ecologists typically assume that the lost and damaged components of living systems are unimportant or can be repaired or replaced. widespread ecological degradation has resulted directly from the failure of modern society to properly assess the ecological risks it faces. like the fate of old kingdom egypt or easter island, our civilization's future depends on our ability to recognize this deficiency and correct it. risk assessment as formally practiced by various government agencies began as a way to evaluate the effects of toxic substances on human health, usually the effects of single substances, such as pollutants or drugs, from single sources, such as a chemical plant. during the 1990s, the focus widened to encompass mixtures of substances and also ecological risks. ecological risk assessment by the us environmental protection agency (1998) asks five questions: is there a problem? what is the nature of the problem? what are the exposure and ecological effects? (a hazard to which no one or nothing is exposed is not considered to pose any risk.) how can we summarize and explain the problem to affected parties, both at-risk populations and those whose activities would be curtailed? how can we manage the risks? even though these are good questions, ecological risk management has not made any visible headway in stemming biotic impoverishment. its central failing comes from an inability to correctly answer the second question, what is the nature of the problem? our present political, social, and economic systems simply do not give us the right clues about what is at risk. none of society's most familiar indicatorswhether gdp or number of threatened and endangered species -measure the consequences, or risks, of losing living systems. if biotic impoverishment is the problem, then it only makes sense to direct environmental policy toward protecting the integrity of biotic systems. integrity implies a wholeness or unimpaired condition. in present biological usage, integrity refers to the condition at sites with little or no influence from human activity; the organisms there are the products of natural evolutionary and biogeographic processes in the absence of humans. tying the concept of integrity to an evolutionary framework provides a benchmark against which to evaluate sites that humans have altered. directing policy toward protecting biological integrity -as called for in the united states' clean water act, canada's national park act, and the european union's water framework directive, among others -does not, however, mean that humans must cease all activity that interferes with a ''pristine'' earthly biota. the demands of feeding, clothing, and housing billions of people mean that few places on earth will maintain a biota with evolutionary and biogeographic integrity. rather, because humans depend on living systems, it is in our interest to manage our activities so they do not compromise a place's capacity to support those activities in the future; that capacity can be called ecological health. ecological health describes the preferred state of sites heavily used for human purposes: cities, croplands, tree farms, water bodies stocked for fish, and the like. at these places, it is impractical to set a goal of integrity in an evolutionary sense, but we should avoid practices that damage these places to the point where we can no longer derive the intended benefits indefinitely. for example, agricultural practices that leave behind saline soils, depress regional water tables, and erode fertile topsoil faster than it can be renewed destroy the land's biological capacity for agriculture; such practices are unhealthy in both ecological and economic terms. biological integrity as a policy goal redirects our focus away from maximizing goods and services for the human economy toward ways to manage human affairs within the bounds set by the natural economy. it begins to turn our attention away from questions such as, how much stress can landscapes and ecosystems absorb? to ones such as, how can responsible human actions protect and restore ecosystems? in contrast to risk assessment, striving to protect biological integrity is more likely to lead away from mandated corrective actions in the form of technological fixes for environmental problems and toward practices that prevent ecological degradation and encourage ecological restoration. leopold (1949, pp. 224-225) , in a sand county almanac, was the first to invoke the concept of integrity in an ecological sense: ''a thing is right when it tends to preserve the integrity, stability, and beauty of the biotic community. it is wrong when it tends otherwise.'' managing for biological integrity requires the kind of ethical commitment inherent in leopold's words. we are called to curb consumerism and limit population size, to embrace less-selfish attitudes toward land stewardship, and to understand that the biosphere matters. instead of calling on human technical and spiritual wellsprings to manage resources, we have to call on them for managing human affairs. we have to find and use appropriate measurements for all the factors contributing to biotic impoverishment, be they climate change, overharvesting, agriculture, or environmental injustice. measurement of environmental impact founded on the evolutionary idea of integrity allow us to directly assess biotic condition and to compare that condition with what might be expected in a place with little or no human influence. at least then we can make an informed choice: continue with activities that degrade biotic condition or think of an alternative. the ecological world is a complex, variable, and often unpredictable system. when managing for ecological risks, people and their governments need to expect the unexpected and develop formal, yet flexible means of coping with environmental surprises. rather than plunge ahead with projects entailing ecological risks because they can be done, decision makers should follow the precautionary principle, which holds that regulators should act to prevent potential environmental harm even in the absence of certainty. it acknowledges the existence of uncertainty rather than deny it, and it includes mechanisms to safeguard against potentially harmful effects. although inappropriate ecological risk assessment and management are more often the norm today, modern institutions are capable of recognizing ecological threats correctly and responding to them in time, as they did with the montreal protocol. a decade after the agreement's adoption, satellite measurements in the stratosphere indicated that ozone-depleting pollutants were in fact on the decline. given the treaty's success, some policy experts have suggested using it to help curb global warming. specifically, negotiators at the 2010 annual meeting of signatory parties were considering a proposed expansion of the ozone treaty to phase out the production and use of industrial chemicals called hydrofluorocarbons, which have thousands of times the global warming potential of carbon dioxide. even though the montreal protocol was not designed to fight climate change, policymakers in favor of the new proposal say that it could and should be used to achieve broader environmental objectives. early in the 20th century, two sciences of ''home maintenance'' began to flourish: the young science of ecology (from the greek oikos, meaning home) and a maturing neoclassical economics (also from oikos). ecology arose to document and understand the interactions between organisms and their living and nonliving surroundings -in essence, how organisms make a living in the natural economy. in fact, ernst haeckel, who coined the term in the 1860s, defined ecology in an 1870 article as the body of knowledge concerning the economy of nature. neoclassical economics, in contrast, reinforced humans' self-appointed dominion over nature's wealth. it brought unparalleled gains in societal welfare in some places, but it also divorced the human economy from the natural one on which it stands (see figure 1) . by now it is clear to economists and ecologists alike that human actions and their effects have reached scales unprecedented in the history of life. we have altered earth's physical and chemical environment, changed the planet's water and nutrient cycles, and perturbed its climate. we have unleashed the greatest mass extinction in 65 my and distorted the structure and function of nonhuman and human communities worldwide. in trying to make our own living, we have jeopardized the earth's capacity to sustain other species and our own species as well. we are losing the bio in biosphere. now, faced with these unprecedented losses, we need to understand -not deny -the ecological consequences of what we do. we urgently need a new science and art of home maintenance, one that sees the human species' role as ecosystem engineer for what it has become and reconnects human and natural economies. this new science can help us understand and correctly interpret the consequences of human-driven change. by using indicators that measure what matters for sustaining living systems, this new science can make nature visible again and shed new light on the value of the ancient heritage we share with the larger biosphere. it can help us reunite the fragments of our worldview and re-create ethical, social, and ecological bonds that were put aside two centuries ago in the name of progress. and such a science can enable us to reengineer our own social, political, and economic institutions instead of ecosystems. this we must donow -before we impoverish the biosphere and ourselves for all time. the wayfinders: why ancient wisdom matters in the modern world guns, germs, and steel: the fates of human societies evolution, consequences and future of plant and animal domestication collapse: how societies choose to fail or succeed floods, famines, and emperors: el niño and the fate of civilizations the state of the nation's ecosystems: measuring the lands, waters, and living resources of the united states environment, scarcity, and violence intergovernmental panel on climate change (ipcc) (2007) climate change seven foundations of biological monitoring and assessment a sand county almanac: and sketches here and there the challenge for africa. new york: pantheon books. millennium ecosystem assessment (2005) ecosystems and human well-being: synthesis ultimate security: the environmental basis of political stability song of the dodo: island biogeography in an age of extinction the economics of ecosystems and biodiversity: mainstreaming the economics of nature: a synthesis of the approach, conclusions and recommendations of teeb. united nations environment programme guidelines for ecological risk assessment wadeable streams assessment: a collaborative survey of the nation's streams, epa 841-b-06-002 our ecological footprint: reducing human impact on the earth the little things that run the world the earth in transition: patterns and processes of biotic impoverishment a short history of progress key: cord-323703-fsj736dg authors: patterson, grace t.; thomas, lian f.; coyne, lucy a.; rushton, jonathan title: moving health to the heart of agri-food policies; mitigating risk from our food systems date: 2020-08-30 journal: glob food sec doi: 10.1016/j.gfs.2020.100424 sha: doc_id: 323703 cord_uid: fsj736dg our food systems are progressively more industrialized and consolidated with many modern food value chains involving multiple countries and continents, and as such being associated with changes in risk profile and impacts of emerging and re-emerging diseases. disease outbreaks that sweep through a single region can have massive impacts on food supply, while severe outbreaks of human pathogens can disrupt agricultural labor supply or demand for products perceived as ‘unsafe’. market pressures have generally rewarded production of cash crops for fuel and energy dense, low nutrient processed foods over production of fruits and vegetables for local consumption. climbing rates of food-related ncds and pre-existing conditions leave the population increasingly susceptible to infectious diseases that are often driven by or arise from the food system. therefore disease and diet from our food systems cause impacts on human health, and human health issues can impact on the functioning of the food system. the covid-19 outbreak is the most recent example of food system driven disease emergence and of massive supply and demand shocks in the food system, experienced as a direct and indirect result of this disease. the effects of the food system on disease spread (and vice versa) must be addressed in future plans to prevent and mitigate large scale outbreaks. health policies must acknowledge the food system as the base of our health system, as must agri-food policy recognize the pre-eminence of human health (directly and indirectly) in decision making. the health of the global population is underpinned by our food systems, yet these systems are often only nominally included in health policy. in recent times, attention has been given to reformulation and marketing policies in moderate attempts to reduce climbing rates of obesity and non-communicable diseases (ncds) (oecd, 2019) . others focus on reducing foodborne pathogens by regulating processing and enacting trade restrictions (trienekens and beulens, 2001) . many of these policies focus on downstream actors and processes in the food system and neglect the foundations of how our food is produced. while such interventions are easier to enact, they have limited effects on health and are generally reactionary and insufficient to reverse the trajectory of increasing health burdens. in this manuscript we review some of the major human health externalities driven by our current food systems, with an emphasis on livestock-systems and their linkages to the wider food system, the impacts of our response to these externalities and outline key areas in which better integrated health & agri-food policy may mitigate these factors. although the interactions between food systems and our health are many and complex, we outline here four key health externalities arising from these systems of concern across the globe; 1) the dual burden of malnutrition, 2) foodborne disease, 3) antimicrobial resistance and 4) emergence of novel pathogens. despite an increasing ability to produce sufficient calories for the growing human population, distributional inequalities have produced a situation where we face the dual burden of chronic under-nutrition (black et al., 2013) and a global pandemic of overweight and obesity and related ncds, often within the same geographical locality (shrimpton and rokx, 2012) . diet related risks are estimated to be responsible for 255 million (234-274mn) disability adjusted life years (dalys) a year globally, and dietary improvements could potentially prevent one in every five global deaths (afshin et al., 2019) . diet related risks also monopolize large portions of many national health-care budgets (phe, 2017) . from 2020-2050, it is estimated that an average of 8⋅4% of healthcare expenditure in the 36 organisation for economic co-operation and development (oecd) member countries will be spent treating obesity-related diseases, equivalent to us dollars (usd) purchasing power parity 311 billion per year (oecd, 2019) . current food systems exploit demand for affordable, convenient, and tasty food to encourage consumption of nutritionally poor, ultraprocessed foods in both high, middle and low income countries. as economic markets incentivize expanded food value chains, they inevitably produce food with more and more processing to improve taste, texture, and longevity (swinburn et al., 2019) . the processed state of these foods, not necessarily the macronutrient content or energy density, induces increased calorie intake and weight gain associated with exposure to ultra-processed foods (hall et al., 2019) . marginalized groups in high income countries are often reliant on these highly processed, energy-dense, nutrient poor foods to meet their energy needs. ultra-processed foods can be aspirational in low and middle income countries (lmics) and are becoming more affordable and convenient (swinburn et al., 2019) . these foods (and the value chains that produce and market them) are among the most impactful determinants of the growing global burden of food-related ncds. food-related ncds also increase susceptibility to infectious disease. for diseases such as zika, west nile, and dengue death is rare in the absence of any pre-existing conditions (badawi et al., 2018; chan-yeung and xu, 2003) and preliminary analysis suggests that the likelihood of adverse outcomes in sars-cov-2 are significantly higher in patients with hypertension and diabetes (zhou et al., 2018) . this increased risk may be driven by upregulation of the sars-cov-2 receptor ace2 or heightened inflammation in people with these comorbidities (pinto et al., 2020) . these diet-related co-morbidities are visible symptoms of underlying structural inequalities which result in marginalized communities bearing disproportionate disease burdens (garg et al., 2020; icnarc, 2020) . whilst the quantity and nutritional quality of our global diets gains increasing attention, foodborne diseases are also associated with a substantial human health burden, with approximately 33 million dalys lost annually through 31 of the major foodborne diseases, the burden of which are disproportionately borne by lmics (jaffee et al., 2018; li et al., 2019) . thirty-five percent of this burden is attributable to pathogens from animal source foods (asfs) (li et al., 2019) , the increasing demand for which, particularly across lmics, is a major driver in an increasing foodborne disease burden (quested et al., 2010) . specific aspects of livestock value chains associated with an increase foodborne disease risk may differ across 'informal' and 'formal value chains. high prevalence foci of the zoonotic tapeworm taenia solium, etiological agent of neurocysticercosis, are found where free-ranging pig husbandry practises coincide with low levels of sanitation and insufficient implementation of meat inspection . informal value chains, which supply 85-95% of food needs in sub-saharan africa, and the poorly regulated slaughter, processing and retail practises associated with them can result in extensive microbial contamination of products, which may not be sufficiently mitigated by consumer food preparation practises (jaffee et al., 2018) . the formalisation of livestock value chains does not completely mitigate all foodborne disease risks. longer value chains with increasing number of processing or handling steps between 'farm and fork' provide opportunities for cross-contamination, adulteration or spoilage without sufficiently enforced regulation. the progressively intensified systems of livestock production seen increasingly across the globe may also exacerbate the risk posed by microbial hazards. intensive systems bring animals into close proximity under circumstances which induce metabolic and psychological stress (humphrey, 2006; martínez-miró et al., 2016) , increasing the contact opportunities and susceptibility of these populations for disease transmission, including those pathogens which have important implications for food safety. this is particularly evident in monogastric systems, where intensive production contributes to over half of the global pork production and over 70% of global poultry production (steinfeld et al., 2006; thornton, 2010) , and where campylobacter and salmonella spp. have been increasingly prevalent (carrique-mas et al., 2014) . in order to mitigate the effects of high pathogen challenge, which also have production-limiting effects, intensive livestock systems have been heavily reliant on antimicrobials for prophylactic, treatment and growth promotion reasons . resistant bacteria reside in humans, animals, food and the environment and there are no barriers to the transmission of resistance genes between these sites or amongst bacterial species (holmes et al., 2016) . pathways for the spread of resistant organisms, from within animal populations to humans, are well recognized and result from the selective pressures from antimicrobial use in livestock. consequently, antimicrobial use in food-producing animal species represents an important driver of antimicrobial resistance (amr) (marshall and levy, 2011) . the human health burden from amr is forecasted to reach 100 trillion usd by 2050 with a worldwide mortality around 10 million (o'neill, 2016) . in response to the amr crises, there is growing international pressure to reduce the use of antimicrobials within livestock systems and to prohibit the use of antimicrobial growth promoters (agps) in livestock. trade-offs exist, however, between the risks of antimicrobial use and the pressure to ensure global food security. a ban on agps could be associated with a reduction in the value of global meat production of between 13 and 44 billion usd and restricting the use of antimicrobials for prophylactic and therapeutic use whilst retaining current intensive husbandry practices will impact both productivity and animal welfare. lastly, disease emergence and transmission are of growing concern within our intensive, increasingly homogenous and interconnected food systems. lack of genetic diversity has become commonplace in our global food systems, within both livestock and crop production (bennett et al., 2018; khoury et al., 2014) , and is a risk factor for heightened susceptibility to outbreaks of plant and animal disease. this 'monoculture effect' is best illustrated in agronomy, where non-diverse cropping can lead to large scale losses, such as those suffered through rice blast (zhu et al., 2000) and the panama disease epidemic of bananas in the 1960s (ordonez et al., 2015) . similar genetic susceptibilities exist within livestock. porcine reproductive and respiratory syndrome virus, a major disease burden to the global pork sector, has led to exacerbated losses within genetically homogenous herds as compared to herds with a wider genetic pool (lunney et al., 2010; halbur et al., 1998) . as well as the emergence of production limiting pathogens within livestock systems, the emergence of novel, zoonotic pathogens from our food systems is at the forefront of the global consciousness, of which sars-cov-2 is only the latest example in an increasing frequency of such events. novel zoonoses have emerged most commonly from wild mammals such as rodents and bats, which have either adapted to anthropogenic habitat changes, or have increased contact with humans or livestock through agricultural incursions into habitats or establishment of bush-meat value chains (johnson et al., 2020) . examples of direct pathogen spillover from wildlife directly to humans, predominantly associated with formal or informal bushmeat value chains, include hiv and ebola (hahn et al., 2000; kock et al., 2019) . there is also a potential threat to human health from the transmission of disease from wildlife into livestock populations and then further propagation through intensive production (wilcox et al., 2014) . in 1998 cases of the novel febrile encephalitis, nipah virus (niv), emerged amongst pig farmers in malaysia. the intensification of mango and pig production within a localised geographical area are believed to have provided a pathway for the virus, circulating in fruit bats, to infect an intensively managed commercial pig population and subsequently spread to farmers (pulliam et al., 2012) . zoonotic and non-zoonotic disease outbreaks and our responses to the presence or risk of these pathogens can destabilize food systems, leading to increased food insecurity and downstream health and economic effects. fig. 1 illustrates several of the key disease outbreaks of the previous three decades which have had profound impacts on human health and food-security. in europe, outbreaks of bovine spongiform encephalitis (bse) and food and mouth disease (fmd) led to large scale supply and demand shocks in the beef and dairy industries. the use of meat and bone meal in cattle feed was associated with the mid 1980's emergence of the degenerative prion disease bse, which was later found to cause newvariant crutzfeldt-jacob disease (nvcjd) in humans (anderson et al., 1996) . this led to a raft of mitigation measures with short and long-term costs, including large scale culling programmes, new harvesting and processing regulation, a ban on animal by-product use in animal feeds and restrictions on beef and other animal products (including milk, semen and embryos) export from affected countries (kimball and taneda, 2004; thiermann, 2004) . the effects on the food system and health from the fmd outbreak in the uk 2001 also extended beyond production losses (knight-jones and rushton, 2013) . trade restrictions between fmd free and fmd affected countries were sometimes extended to unrelated products. culls produced public outrage against the livestock industry that outlasted the outbreak itself, which together likely contributed to the observed spike of depression and suicide among farmers (thomas et al., 2003) . in areas where a large proportion of the population is dependent on agriculture for both income and food supply, such as ethiopia, endemic fmd has direct effects on the food security of farmers by reducing milk production and oxen availability for cropping (knight-jones and rushton, 2013) . pork is now the most frequently consumed meat per capita worldwide and is responsible for over half of meat consumption in asia (ritchie and roser, 2019) . introduction of african swine fever virus (asfv) into china, home of approximately 50% of the global herd (ritchie and roser, 2019) , resulted in rapid spread throughout the naive pig population. over 10% of pigs have died or been culled (fao, 2019), causing an estimated $140 billion in direct economic losses (moore, 2019) and reverberations through industries including feed mills, veterinary services, restaurants, and tourism. pork prices soared to a peak of 110% year-on-year (moore, 2019), increasing demand for and price of other meat products and leaving poorer households at increased risk of food insecurity and malnutrition (rocha et al., 2016) . similar patterns were seen across the same region less than a decade before in response to outbreaks of highly pathogenic avian influenza where aggressive containment measures drove increases in meat prices and heightened local food insecurity (burgos et al., 2012) . lmics, particularly those in southeast asia, are the site of the majority of industrialized poultry farming. large scale flocks are more likely to contract hpai (h5n1) than small, personal flocks (otte et al., 2008) and experience the greater share of economic effects. yet smallholders, traders, slaughterers, and transporters are at greatest risk of personal negative health outcomes as a result of such diseases, as they often belong to poor households that are unable to weather economic shocks and are sensitive to food insecurity and malnutrition. the west african ebola outbreak of 2014 provides an example of severe, localized effects of an outbreak on the food system due to travel restrictions and market closures (mann et al., 2015) . panic buying caused shortages of some items and dramatic increases in prices. labor shortages resulted in decreased food production and loss of income across communities, particularly in rural regions (de la fuente et al., 2019) . nutrition operations were a low priority among humanitarian response groups and were inadequately supplied (kodish et al., 2019) . they also neglected to consider the needs of communities that did not have ebola outbreaks but had become food insecure from food production losses. after the outbreak, many ebola disease survivors were denied the opportunity to purchase food due to lingering fear of infection. on a global scale, the ongoing covid-19 outbreak has also led to massive perturbations in food systems. lockdowns and retail closures directly cause food insecurity among vulnerable populations, national stockpiling has prompted some countries to halt or reduce exports of staple grains and legumes (reuters, 2020) , and travel restrictions have inhibited farmers across europe from hiring seasonal migrant workers to harvest fruit and vegetable crops (carroll et al., 2020) . workers in food retail and transport that remain employed are experiencing unexpected health risks, severely reducing the ability of some sectors of our food-systems to operate as has been particularly noticeable in the vulnerability of meatpackers and subsequent covid-19-related shutdown of some meat processing plants in usa (apostolidis, 2020) . workers provided with little protection or compensation have in some instances organized strikes to protest their lack of economic and health protection (mcginnis, 2020) and lengthy screening proceedures or social-distancing measures have the potential to dramatically reduce production capacity in some sectors (hailu, 2020) . once social distancing protocols are lifted, medium to large enterprises will likely be best poised to recover and many small businesses integral to local food systems will be unable to bounce back, producing economic knock-on effects on wholesalers, processors, and producers across the supply chain. while food insecurity surges as a result of control measures, food waste is also increasing. it is estimated that over a third of all food produced for human consumption is wasted (fao, 2011), a figure which will grow as shopping patterns change from multiple small shops per week to larger, more infrequent shops (cranfield university, 2020). food systems lack the flexibility needed to adjust to rapidly evolving situations within disease outbreaks, causing still more waste (evans, 2020) . restaurant and coffee shop closures have decreased demand for milk and dairy farmers have been told to pour milk down the drain, yet supermarkets have restricted purchase of milk and other core products (keane, 2020) . closures of large meat processing plants due to worker illness will result in large scale culling of animals across the usa. meanwhile, emergency assistance agencies are running low on supplies to support the deluge of the newly food insecure (power et al., 2020) . despite the undeniable link between our food systems and health, the sustainable development goals (sdgs) do not explicitly link these areas other than within the context of malnutrition (un, 2015) . public health policies must pay greater consideration to the role of food systems as the baseline of population health, while agri-food policies should consider human health as their raison d'être. agri-food policies can promote health in direct and indirect waysglobally 27% percent of employment is in agriculture, this rises to 60% in low income countries (world bank, 2020). sustainable agriculture supports economic prosperity, environmental wellbeing, and social equity (i.e. the triple bottom line), all of which further support stable access to healthy diets and healthy environments. well executed, integrated health-agri-food policy should improve baseline health, mitigate infectious disease risks and increase the resilience of our food systems to protect food security, particularly for the most vulnerable. herein we highlight some key examples of these policies in action, where further strengthening is required, and the dynamics that complicate enactment of health-centered agri-food policy (fig. 2) . evidence-based health-agri-food policies that support access and uptake of healthy diet and exercise, particularly in communities with high inequality, can help reverse the growing trend towards obesity and minimize morbidity and mortality from infectious disease. a multipronged approach is needed to set healthy food preferences at an early age, change the environment to encourage healthy choices (particularly fig. 2 . integration of health and agri-food policy. current and recommended agri-food and health policies to reduce the frequency and impact of disease outbreaks. at the point of purchase), and reduce barriers to expression of healthy preferences (hawkes et al., 2015) . policies that limit consumer exposure to less healthy foods by reducing portion sizes, reformulating foods, and regulating advertising are often blocked or weakened by lobbying groups (swinburn et al., 2019) . there is little immediate financial motivation to adopt more environmentally and health friendly standards, though social demand is increasingly reorienting markets to value transparency and social and environmental aspects of corporate performance. policy interventions that rely on behavioral change, such as consumer awareness campaigns and front of pack labeling, are relatively easier to enact but have limited longevity or effectiveness in the presence of other food stimuli or under stress (hill, 2009; leng et al., 2017) . the dynamics surrounding the adoption, implementation, and effectiveness of regulatory health policy are exemplified by the high fat, salt, and sugar (hfss) taxes that have been adopted across diverse nations with a wide range of outcomes (world health organization regional office for europe, 2015). most slightly reduced consumption of hfss foods but it is unclear what affect, if any, these taxes have on other health indicators in isolation. low income consumers that rely on affordable, energy-dense foods may be unduly burdened by hfss taxes but benefit from alternative price interventions such as subsidization of fruit and vegetables (neff et al., 2009) . while current regulatory interventions in isolation have minimal effect on downstream health indicators, they provide precedent for future regulatory policy and encourage action surrounding the role of large companies in shaping the food environment. significant improvements in global population health will require cooperation of people and companies involved in food systems to fundamentally change how they operate, but some communities have overhauled their local food environments with a ground-up, systemic approach that bypasses many of the barriers encountered on a national level and inspires awareness and innovation. in baltimore, usa, policies have been enacted to re-familiarize consumers with the systems and people that produce their food (department of planning, 2018), incentivize wholesaler produce distribution to small convenience stores, and encourage grocery chains to move into low income communities. city government also supports local ownership of food retail outlets in underserved neighborhoods, cultivating economic growth, a sense of community, and the agency to improve the health issues people see in their neighborhood. baltimore's food policy initiatives demonstrate how to mobilize community members to incorporate agri-food policies across city operations and government, business and economic development, and community organizations to combat the multifactorial roots of poor dietary health. even prior to the covid-19 pandemic the international community had been aware of the increasing threat from emerging zoonotic pathogens, the 'dual burden' imposed by endemic zoonoses on livestock productivity and human health, the biological and chemical hazards present in our food and the looming spectre of a post-antibiotic world. the protection of consumers from zoonoses and foodborne diseases acquired from the consumption of diseased animals or via unhygenic slaughter and processing has been a concern for centuries and formal meat inspection, still broadly recognisable today, was instigated in europe in the 1880's (edwards et al., 1997) . the multi-faceted nature of infectious disease risks within our food-systems and the plethora of public and private actors working within them, with differing roles and responsibilities has driven calls for an integrated 'one health' approach, endorsed at the highest level by the who, oie, fao tripartite and the world bank. despite growing acceptance of this concept internationally and the establishment of several regional and national one health units, there are still major challenges in operationalization. power struggles between ministries for 'ownership' of the movement, poorly integrated policies, lack of interoperability in data systems, and resource constraint for frontline services have all been identified as key barriers, and the multiplication of one health initiatives, specifically those with single issue focus, may risk undermining the strength of the moment (spencer et al., 2019) . this splintering and 'siloisation' of one health can be illustrated by kenya, where four separate one health bodies now exist with separate remits for zoonoses, amr, aflatoxins and pesticides but with no over-arching co-coordinating mechanism across these areas, and with other one health issues such as foodborne disease lacking an inter-ministerial 'home' (kimani et al., 2019) . although challenges exist, the one health approach has been repeatedly employed to mitigate animal and human disease threats, as exemplified by the 2017 response to colistin resistant e. coli (crec) in china (wang et al., 2020) . colistin is used predominantly in the livestock sector but is an important antimicrobial of last resort for human disease. following the discovery of widespread distribution of colistin resistance gene mcr-1 in e. coli, the chinese ministry of agriculture and rural affairs banned use of colistin as a growth promoter in livestock. three years on, significant reduction was observed in the relative abundance of mcr-1 in pigs, crec carriage in pigs and chickens, human carriage of mcr-1 positive e. coli, and human infection with crec. a clearly defined problem and solution, strong political support, and effective inter-agency enforcement contributed to the success of this policy and provides guidance for future approaches to threats to animal and human health. the one health approach will only be as strong as its constituent parts; environmental stewardship is often missed completely, whilst veterinary and human public health systems have often been chronically under-funded in favor of support to primary food production and curative human medicine. strengthening these capacities and meeting countries' commitments under the international health regulations and the oie pathway for veterinary services programmes is integral to the global health security agenda (belay et al., 2017) . the capacity building activities within the ghsa thus far have relied predominately on external donor funding. ultimately, the sustainability of these activities will require resource commitments by national government and a greater acknowledgement of the role of private actors across food systems (kelly et al., 2017) . there an increasing number of private standard setting organizations to enhance global food safety such as the global-good agricultural practises standard (king et al., 2017) . private standards can be excessively high, which improves food safety but may be unattainable for smallholders or farmers in lmics, effectively shutting them out of global markets and further highlighting the need for a collaborative, multi-sectoral approach to food safety. responses to emerging pathogens and outbreaks are often fear driven and reactionary. during the swine flu (h1n1) outbreak, egypt culled their entire pig population, despite pigs not transmitting the virus to humans (atlani-duault and kendall, 2009 ). traceability systems have become more sophisticated and widespread and can support narrower outbreak responses, yet retailers and governments often issue blanket recalls and import restrictions during health crises (van der vorst, 2006) . similar to other components of food safety programs, development of and compliance with better traceability systems largely rest with private companies and may not be harmonized with standards set by public health bodies. to improve response time and accuracy during outbreak events, quality traceability systems across the value chain must be combined with transparency and communication with decision makers. prior to such events, governments should develop and train networks of communication that connect food systems agents, trustworthy spokespeople, and consumers to minimize panic and reactionary measures (who et al., 2019) . national outbreak preparedness plans and risk reduction strategies concerning diseases that originate outside the food system typically do not include considerations for protecting food systems but should be a key factor in planning potential response efforts (ortu et al., 2008) . localities can contribute to outbreak preparedness by conducting food system vulnerability assessments using fault tree analysis across a range of emergencies (biehl et al., 2018) . even when food systems are considered in planning exercises, insufficient follow through, lack of resources, or unwillingness to allocate available resources may render such exercises meaningless. policymakers must prioritize action following planning exercises, as emerging infectious disease and food safety events are increasingly likely as food systems become more globalized. corporate concentration of agricultural sectors both horizontally and vertically has resulted in centralizing control of large swaths of the food system among a few companies, reducing the resilience of food system to internal and external shocks (howard, 2017) . these oligopolies have vast lobbying power, making it difficult to enact structural change. positive change at this level will require market incentives and non-governmental accountability systems similar to what is seen in current efforts to combat climate change (heymann et al., 2015) . reliance on food imports also increases vulnerability to food insecurity. an estimated 1/6th of the global population in 2000 were dependent on food imports for their basic dietary needs (fader et al., 2013) , leading to an unequal power dynamic between net-exporters & net-importers and undermining the development of locally appropriate value-chains. at the production level, agri-food policies which support local farmers and smallholders can strengthen food system resiliency by increasing the diversity of local food systems and protecting against economic shock. one example is the uk government department for the environment food and rural affairs' farm diversification grant scheme, which helped farmers in the uk develop a diverse portfolio of income sources (turner et al., 2006) for this reason. support for agricultural diversification among smallholders in lmics can improve food security, reduce poverty, and strengthen resilience to climate change and market instability, resulting in long term increases in productivity (joshi et al., 2007; world bank, 2018) . however, there are several obstacles to farm-level adoption of diversified farming systems, including start-up costs, potential initial productivity losses, lack of access to technical guidance or support, and market disencentives such as commodity crop subsidies and insurance (harvey et al., 2014) . some farm level barriers can be overcome with an institutionally organized systems or landscape approach that incorporates multiple stakeholders to achieve a collective diversification goal. policymakers should work to increase accessibility of diversification tactics, support research to provide further evidence and guidance for diversification, and ensure follow through on these commitments. while the 55 countries in the african union have pledged to enhance financial investment in agriculture, only nine are on track to reach agricultural research spending goals (vilakazi and hendriks, 2019) . at the consumer level, stable access to food during periods of crisis can be ensured by policies that support emergency food access and flexibility within supply chains. the covid-19 outbreak underscores the need for policies to support and improve access to food assistance agencies, which not only supply emergency food but also help those in need access benefits, job training, and medical resources (environmental audit committee house of commons, 2019). food systems have wide and far-reaching impact on health, economy, and society locally and globally, while health issues and our response to them have major impacts on how food systems operate. a sustainable and healthy food system supports fair wages for individuals across the food value chain, provides affordable, nutritious, diets that are culturally acceptable, operates in an environmentally sustainable manner, and is resilient and adaptable. foodborne disease and emerging infectious disease events should naturally decrease as food systems approach these ideals. this vision is far-removed from the current reality but provides a set of priorities through which agri-food policies should be developed. major challenges exist concerning the reorientation of market incentives and food systems standards, heightened accountability, changes in land use, and cooperation between countries and corporations. yet, consumers are increasingly aware of the broader effects of current food systems and are beginning to widely challenge the status quo. the severity of the current covid-19 outbreak and its unusually far-reaching impacts on food systems will hopefully provide the stimulus and opportunity to for both high level decision makers and the wider consumer base to more seriously focus on restructuring our food systems to better support the health of the global population. lft is supported by the university of liverpool wellcome trust institutional strategic support fund and is a soulsby foundation one health travelling fellow. transmission dynamics and epidemiology of bse in british cattle meatpackers are deeply vulnerable to covid-19. expect a reckoning for us workers | lse covid-19. london sch. econ. polit. scienve covid-19 blog influenza, anthropology, and global uncertainties prevalence of chronic comorbidities in dengue fever and west nile virus: a systematic review and metaanalysis zoonotic disease programs for enhancing global health security the broiler chicken as a signal of a human reconfigured biosphere planning for a resilient urban food system: a case study from baltimore city maternal and child undernutrition and overweight in low-income and middle-income countries poultry, hpai and livelihoods in lao people's democratic republic-a review an epidemiological investigation of campylobacter in pig and poultry farms in the mekong delta of vietnam farmers across europe bank on improvised armies of pickers to save harvest the unintended consequences of stockpiling: food waste impact of the west african ebola epidemic on agricultural production and rural welfare evidence from liberia. department of planning meat inspection: an overview of present practices and future trends sustainable development goals in the uk follow up: hunger, malnutrition and food insecurity in the uk how will covid-19 lockdown impact our eating habits? ahdb spatial decoupling of agricultural production and consumption: quantifying dependences of countries on food imports due to domestic land and water constraints one year on, close to 5 million pigs lost to asia's swine fever outbreak global food losses and food waste: extent, causes and prevention hospitalization rates and characteristics of patients hospitalized with laboratory-confirmed coronavirus disease 2019 -covid-net, 14 states aids as a zoonosis: scientific and public health implications economic thoughts on covid-19 for canadian food processors differences in susceptibility of duroc, hampshire, and meishan pigs to infection with a high virulence strain (vr2385) of porcine reproductive and respiratory syndrome virus (prrsv) ultra-processed diets cause excess calorie intake and weight gain: an inpatient randomized controlled trial of ad libitum food intake climate-smart landscapes: opportunities and challenges for integrating adaptation and mitigation in tropical agriculture smart food policies for obesity prevention global health security: the wider lessons from the west african ebola virus disease epidemic special task force report: can a small-changes approach help address the obesity epidemic ? am understanding the mechanisms and drivers of antimicrobial resistance corporate concentration in global meat processing: the role of government subsidies are happy chickens safer chickens? poultry welfare and disease susceptibility icnarc, 2020. icnarc report on covid-19 in critical care the safe food imperative: accelerating progress in low-and middle-income countries. the safe food imperative: accelerating progress in low-and middle-income countries global shifts in mammalian population trends reveal key predictors of virus spillover risk agricultural diversification and smallholders in south asia -chapter 2 dairy farmers told to cut milk supplies increasing homogeneity in global food supplies and the implications for food security expanding beyond zoonoses: the benefits of a national one health coordination mechanism to address antimicrobial resistance and other shared health threats at the human-animal-environment interface in kenya a new method for assessing the impact of emerging infections on global trade food safety for food security: relationship between global megatrends and developments in food safety the economic impacts of foot and mouth disease -what are they, how big are they and where do they occur? searching for the source of ebola: the elusive factors driving its spillover into humans during the west african outbreak of 2013-2016 a qualitative study to understand how ebola virus disease affected nutrition in sierra leone-a food value-chain framework for improving future response strategies the economic costs of withdrawing antimicrobial growth promoters from the livestock sector. oecd food, agriculture and fisheries papers the determinants of food choice global disease burden of pathogens in animal source foods porcine reproductive and respiratory syndrome virus: an update on an emerging and re-emerging viral disease of swine a review of the role of food and the food system in the transmission and spread of ebolavirus food animals and antimicrobials: impacts on human health causes, consequences and biomarkers of stress in swine: an update brazil's peak soybean export season collides with covid-19. success. farming china's swine fever crisis will impact global trade well into 2020 food systems and public health disparities tackling drug-resistance infections globally: final report and recommendations. the review on antimicrobial resistance the heavy burden of obesity, oecd health policy studies worse comes to worst: bananas and panama disease-when plant and pathogen clones meet pandemic influenza preparedness in africa is a profound challenge for an already distressed region: analysis of national preparedness plans impacts of avian influenza virus on animal production in developing countries ace2 expression is increased in the lungs of patients with comorbidities associated with severe covid-19 health matters: obesity and the food environment how covid-19 has exposed inequalities in the uk food system: the case of uk food and poverty agricultural intensification, priming for persistence and the emergence of nipah virus: a lethal bat-borne zoonosis trends in technology, trade and consumption likely to impact on microbial food safety trade restrictions on food exports due to the coronavirus pandemic meat and dairy production. our world data the 2016 global nutrition report, global nutrition report -from promise to impact: ending malnutrition by 2030 the double burden of malnutrition: a review of global evidence, the double burden of malnutrition zoonotic and vector-borne parasites and epilepsy in low-income and middle-income countries is the current surge in political and financial attention to one health solidifying or splintering the movement? livestock production systems in developing countries: status, drivers, trends the global syndemic of obesity, undernutrition, and climate change: the lancet commission report emerging diseases and implications for global trade mental health of british farmers livestock production: recent trends, future prospects the implications of eu food safety legislation and consumer demands on supply chain information systems the effects of public funding on farmer's attitudes to farm diversification transforming our world: the 2030 agenda for sustainable development global trends in antimicrobial use in food animals product traceability in food-supply chains handbook of quality of life in african societies. international handbooks of quality-of-life changes in colistin resistance and mcr-1 abundance in escherichia coli of animal and human origins following the ban of colistin-positive additives in china: an epidemiological comparative study taking a multisectoral, one health approach: a tripartite guide to addressing zoonotic diseases in countries role of urbanization, land-use diversity, and livestock intensification in zoonotic emerging infectious diseases productive diversification of african agriculture and its effects on resilience and nutrition, productive diversification of african agriculture and its effects on resilience and nutrition world health organization regional office for europe emergence of african swine fever in china genetic diversity and disease control in rice the authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper. key: cord-027885-ua8miwes authors: das, sujata; khanna, charu; singh, shalini; nandi, shilpa; verma, reema title: impact of human microbiome on health date: 2020-03-10 journal: microbial diversity, interventions and scope doi: 10.1007/978-981-15-4099-8_20 sha: doc_id: 27885 cord_uid: ua8miwes the human genome in the recent years, by the advent of technological advancements, has emerged as a major prolocutor for reciprocity between the human body and the food consumed. as known, microbiome comprises all the genetic materials within a microbiota and can thereby be also referred to as metagenome of the microbiota. contemporary researches have revealed the influence of microbiome not only on human mind and health status, but also in wide range of disease switching, ranging from cardio-metabolic diseases, allergies and obesities to life-threatening diseases such as cancer. though the complete mechanism of many diseases is yet unclear, research works have revealed that the metabolites, nutrients and microbes can be regarded as the key players for such physiological state. the major approach of this chapter is to enlighten the interrelationship of the microbiome on the human health either in a synergistic or in an antagonistic manner. in the last few decades, immense initiatives have been taken in understanding the role of microbiome on human health. in the present day, either in the field of therapeutic development or medical treatment, the impact of microbiome is especially apparent in the studies of different microbial communities and the human microbiome. in the recent times, 'omics' has provided a significant impetus on all the facets of biological research which has ushered the field to gain in momentum. the study, which is contemporarily popularised as study of 'human microbiome', is an outcome of the advancement in the field of genomics and other fields of microbiology, which has given the classical microbiology a new outlook and perspective. thereby, the attention was driven and directed towards the genomics, which was the major objective of the human microbiome project (hmp) (turnbough and wilson 2007) . the hmp was used for the characterisation of all the microbial communities living in the human body, eventually switching the interest towards, not only, the type of microbes existing in the human body but also to the role and activity they perform both in the case of a healthy and diseased individual. since 2003, after the publication of the first human genome (chial 2008) , the biomedical research on microbiome has obtained a significant scientific attention. there has been an immense leap from the culture-based surveys of various tissues or organs, for example, of gut and oral cavity, to molecular profiling of the microbial communities and their biochemical products like enzymes, proteins, and amino acids in all the different ecological niches of the human body (eckburg et al. 2005; gill et al. 2006; costello et al. 2009 ) for this subject. this chapter attempts to put an insight into the distribution and diversification of human microbiome, the behaviour of human microbiome on the human health and microbiome as a paradigm for the future nutritional and medicinal strategies for human benefits. over 100 trillion microbes are estimated to be residing both inside and over the surface of humans, possessing genome which is approximately 150 times to that of the entire human being ). this includes microbes of different families such as bacteria, fungi, archaea and viruses, contributing about eight million unique protein-coding genes as compared to the human genome, which comprises only around 22,000 protein-coding genes (tomayko et al. 2013) . this variation and the nature of the organism, specific to the specific anatomic site of the body, is a consequence of their specific growth requirement. the other determinants for such growth include coevolution of microorganism, their extensive interaction amongst each other and with the human host, variation in the composition and function with respect to the population, human life span and inconsistency of the body sites, ecologic condition, difference in the oxygen tension, airway luminal temperature, mechanism of muco-ciliary clearance, sex, genetics and socio-economic status. hence, there has been a development of the concept of interdependence, in variety of physiologic, immunologic and metabolic processes, which ultimately determines the microbiome community in a particular site of the human body system. amongst all the human systemic microbiome, the gut microbiome, composed of the genetic material of the microorganisms in the gut, occupies a very essential and special position. they play an important role in various physiological processes like metabolism, immunity development and nourishment supply. the genotype and the immune system of the host have been shown to contribute towards the development of gut microbiota (thaiss et al. 2016) . in response to environmental factors, such as diet, pathogens and xenobiotic substances, a crosstalk occurs between the human immune system and the microbiome. for instance, the myeloid cells, epithelial layer and the innate lymphoid cells, part of the immune system, crosstalks with the gut microbiota for which the microbiome composition, host physiology and disease susceptibility are the main consequences of such crosstalks and feedback loops between them. along with the bacterial community, like firmicutes and bacteroidetes species (table 20 .1), these interactions are also contributed by the other microbiota like fungi (pothoulakis 2009 ), archaea and viruses (breitbart et al. 2003) . though the understanding of the immunological relationship between the fungi and archaea is limited currently, the trans-kingdom commensalism is expected to be formed from infancy (latuga et al. 2011) . the principal constituents are the bacteriome, virome and mycobiome, whose strong interdependence maintains the functionality of the gut microbiota, if imbalanced may also affect the other systems in various ways. since the time of birth of an individual, when the sterile gut of the neonate gets exposed to the biota of mother's vagina during the vaginal delivery or hospital microbiota in case of caesarean section (which may even include the multidrug-resistant species), the microbes starts their colonisation with an eventual change by the age of 3-5 years, by when an individual starts resembling bacterial community to that of an adult both structurally and functionally (bull and plummer 2014) . in adults, the composition of gut microbiota is uneven throughout the length of the gut. as compared to small intestine, which is rich in the species related to phylum firmicutes, colon on the contrary exhibits the presence of members of phylum bacteroidetes. the microbiome of lumen and that attached to the epithelial lining even show differences. the stool sample exhibited the presence of bacteroides, streptococcus, ruminococcus, lactobacillus, enterococcus, bifidobacterium and clostridium which presented the lumen community while on the mucous layer detected the presence of enterococcus, lactobacillus and clostridium (swidsinski et al. 2005) . during the initiation of hmp, the airways and lungs were exempted from the study, believing these parts to be sterile in nature (moffatt and cookson 2017) . this fact was always acceptable because of the negative results yielded by the various standard microbiological culture tests of the healthy individuals (faner et al. 2017) . its study was also a challenge owing to the difficulty in assessing the lower tract without the invasive techniques such as bronchoscopy. hence, there has been a delay in the systemic microbiome assay until the first study indicating the similarity of bacterial density of this part with the upper small bowel of human body was reported (man et al. 2017 ). this has been made possible due to the advances in molecular techniques independent of culture practices (faner et al. 2017 ). human respiratory system is divided into upper respiratory tract (urt) and lower respiratory tract (lrt) with alveoli, present in the lrt, acquiring the surface area nearly 70 m 2 (man et al. 2017) . this complete tract is occupied by the niche-specific microbiota with higher density dwelling in urt. the development of microbiota has been thought to effect on the morphological genesis of this system (man et al. 2017) . during the first hours of a healthy neonate, non-specific microbes, presumed of maternal origin has been detected. abundance of staphylococcus spp. in the first week, in the urt due to niche specification, has also been detected, which is then occupied by the corynebacterium spp., dominated by the dolosigranulum spp. the moraxella species has its dominance at the age of 4-6 months. the individuals with the possession of such microbiota have been found to possess stable microbiome community along with better airway health (morris et al. 2013; segal et al. 2013 ) this healthy development is prone to disturbance under certain conditions such as usage of antibiotics, oxygen tension, temperature and ph, presence of other siblings, seasonal variations, vaccinations, exposure to smoke and host genetics (man et al. 2017) . attempt to observe the diversity of flora in urt and lrt is well observed (table 20 .1). studies using species specific molecular techniques have declared that the diseasefree arteries and veins are microbe free in nature (jin et al. 2019; sobol 2014 ). very few studies do report the presence of bacterial and viral genome in some vessels of healthy subjects. amongst the microbes, helicobacter pylori and herpes simplex virus are the most prevalent followed by cytomegalovirus, chlamydia pneumoniae, mycoplasma pneumoniae and epstein barr virus and were also detected in healthy aorta, saphenous veins and internal mammary arteries (clifford and hoffman 2015) . studies using species-specific molecular techniques have declared that the diseasefree arteries and veins are microbe free in nature (jin et al. 2019; sobol 2014 ). very few studies do report the presence of bacterial and viral genome in some vessels of healthy subjects. amongst the microbes, helicobacter pylori and herpes simplex virus are the most prevalent followed by cytomegalovirus, chlamydia pneumoniae, mycoplasma pneumoniae and epstein barr virus and were also detected in healthy aorta, saphenous veins and internal mammary arteries (clifford and hoffman 2015) . the human urinary tract along with urine was earlier considered to be 'sterile', until the modern-day research, which has confirmed the presence of microbes in this system, significantly in healthy individuals. the advances in techniques such as 16 s rrna sequencing have considerably helped in revealing the normal microbiota of the human body system. after analysis of the urine, the most common genera reported are lactobacillus (more in women) and streptococcus (more in men), both of which, along with few other groups, deliver a protective role in this system against different pathogens. various bacterial taxa, as illustrated (table 20 .1), had been recognised in healthy adults, but some specific genera such as saccharofermentans, proteiniphilum, parvimonas and jonquetella were found in persons with age more than 70 years. the variation of urinary microbiome related to age and sex may be due to differences in voiding habits, hygiene, urinary metabolites, anatomic structures, hormonal variation and histology. even the vaginal microbiome at premenstrual phase, reproductive age and post-menopausal phase exhibits variation (aragón et al. 2018 ). central nervous system is considered to be one of the most immune privileged systems because of its closed compartmentalisation. it is isolated by physical barriers like blood-brain barriers and blood-cerebrospinal fluid barriers, from the circulatory system (obermeier et al. 2013; ransohoff and engelhardt 2012) . thus, lack of lymphatic drainage, expression of major histocompatibility complex by the parenchymal cells and anti-inflammatory environment of the central nervous system accounts for such privileged status of seclusion from the microbiota (berer and krishnamoorthy 2014) . though, in our body, there exists a bidirectional communication system, involving hormonal, immunological and neural signalling pathways, between the brain and the gut, accessed by the microbial flora of the intestine and certain metabolites, also known as the gut-brain axis. it is estimated that nearly 90% of serotonin (5-ht), a neurotransmitter, is produced in the intestine under the influence of gut microbiota, and the activation of its receptors in the enteric nervous system is responsible for the neuroprotection and adult neurogenesis in the mouse model (de vadder et al. 2018 ). the largest organ forming the external interface of the human body to the environment is the skin. nearly 1.5-2.0 m 2 of the skin covers an average human with 2-3 mm depth. three tissue layers are found in the skin: epidermis, dermis and hypodermis. epidermis is colonised by millions of the microbes such as bacteria, fungi, arthropods and even viruses. it acts as the physical, anatomical and immunological barrier to various pathogens extending protection of the body, unless this barrier is broken or there is an imbalance between commensal and pathogenic organisms resulting to cutaneous or systemic diseases. their acidic ph, continuous shedding of epidermal cells, hydrophobic nature, salinity and association with the antimicrobial compounds make them an efficient barrier (ross et al. 2017) . though microbes do exist on them in spite of the above characteristics, the number of microbes inhabiting the skin ranges from one million to about one billion in each cm 2 . although, human skin is inhabited by quite diverse microflora, but most commonly found bacterial phyla includes proteobacteria, corynebacteria, propionibacteria, bacteroidetes, firmicutes and staphylococcus spp. amongst fungi, malassezia spp., cryptococcus spp., epiciccum spp., aspergillus spp. and rhodotorula spp. are most commonly found. the factors that affect the prevalence and dominance of community on the skin include biological sex, skin depth, skin location (skin thickness, folds, density of hairs), age, health, geographical location, ethnicity, use of lotions, soaps, cosmetics, and antibiotics and hygiene practices. as humans are known to have a constant symbiosis with microorganisms, the human microbial community, inhabiting the various system exhibits their influence on them. there are nearly 100 trillion symbiotic microorganisms that exist on and within the human body and have shown to play very important roles both in human health and disease causation. the influence of this microbiome on various systems is as follows: amongst all the systems, the highest and heterogeneous microbial density resides in colon and is mostly codependent in nature and present along both longitudinal (proximal to distal) and axial (mucosal to lumen) gradients of the gastrointestinal (gi) tract. the microbiome has been reported to aid in food digestion, vitamin biosynthesis, bile acid biotransformation, building of innate immunity, maintenance of intestinal barrier (valdes et al. 2018) , etc. thus, gut is an "essential organ", carrying approximately 150 times more genes than are found in the entire human genome ) (fig. 20.1 ). microbial inhabitants within the host often contribute to metabolism such as: • bile salt metabolism • synthesis of essential and non-essential amino acids • replication of virulence factors in enteric pathogen • pro-drug transformation into active drugs and • metabolism of xenobiotic compounds • antibiotics by chemical transformation (sarkar et al. 2018) gut microbiota and host interaction result in the secretion of a series of metabolites including trimethylamine-n-oxide (tmao); short-chain fatty acid (scfa) such as acetate and butyrate; secondary bile acid; and indoxyl sulphates that activate numerous signalling pathways affecting host physiological processes (jin et al. 2019 ). when a child is born, the immune system at birth is under a relative state of immaturity. the developing immune system is characterised by a skewed t-and b-cell development and a blunted inflammatory cytokine production with respect to the regulatory responses. the consequence of such an underdeveloped immune system is high susceptibility to infections. thereby, the regulatory environment ensures the establishment of the microbiota which ultimately helps in immune regulation and limits the mucosal inflammation following their colonisation (elahi et al. 2013) . many intestinal bacteria also prevent the colonisation of pathogen by producing antimicrobial substances such as bacteriocin (inhibit pathogen growth), by competing for nutrition and attachment sites. this act is termed as barrier/competitive(collado et al. 2010 ). exposure to intestinal bacteria has also been found to prevent certain allergic responses in the hosts. thereby, the commensals, specifically the bacterial species and the products or metabolites derived from them, are considered as an intrinsic regulator of all the immune responses for the upliftment and restoration of human meta-organism's health (belkaid and hand 2014) . gut bacteria are intrinsically linked with the health of our entire body. however, a change in gut microbiota composition, termed as dysbiosis, can result in enhanced susceptibility of the host towards pathology. dysbiosis can cause various diseases such as inflammatory bowel disease (ibd) and irritable bowel syndrome (ibs), chronic kidney disease (ckd), cardiovascular disease (cvd), atherosclerosis, obesity, autism, allergy, asthma, hypertension, coronary artery disease, and heart failure (tang and hazen 2017; backhed, et al., 2012) . (ibd) ibs and ibd are related to bowel disorder with signs and symptoms of abdominal cramping, pain and bloating associated with features of disordered defecation in ibs and ongoing inflammation of all part of gi tract in ibd. although ibs is a multifactorial disease (soares 2014) , intestinal dysbiosis has also been found associated with this disease by facilitating adhesion of enteric pathogens. ibd includes crohn's disease (cd) and ulcerative colitis (uc). gut microbiota has been implicated to have roles in ibd pathogenesis, and the usage of antibiotics may help in reducing inflammation or prevented the same in murine models of disease and in patients. intestinal dysbiosis is most often accompanied by defective intestinal barrier function, which promotes the production of bacterial by-products that are rapidly absorbed and retains in intestinal lumen. when increased absorption is coupled with reduced clearance of these substances by the kidneys, levels of gut derived toxins rise in circulation and may potentiate vascular calcification, atherosclerosis and adverse cardiovascular functioning, which are the clinical conditions in the later stage of ckd. numerous epidemiologic studies have shown a relationship between gut-derived vascular toxins and cardiovascular events in patient with ckd (valdes et al. 2018 ). metabolic origin of traditional cvd risk factors such as obesity, dyslipidaemia and insulin resistance indicates close linkage between the gut microbiome and cvd. recent studies have implicated that atherosclerosis has generated the largest amount of data in association with gut microbiome. according to a hypothesis, gut microbiota metabolites elicit inflammatory cascade by translocating into bloodstream and promote atherosclerosis. oral bacteria causing oral cavities have also been found in atherosclerosis plaque (clifford and hoffman 2015) . in many cvds, heart failure is considered as end stage with a higher rate of morbidity and mortality (jin et al. 2019) . low intestinal perfusion and disruption of intestinal barrier have been reported as reasons for reduced cardiac output and blood redistribution. the translocation of microbiota and endotoxins into blood circulation leads to an aggravated systemic inflammation that further can lead to increased chances of heart failure (peng et al. 2018) . various studies suggested the direct and indirect link between gut microbiota and the development of hypertension (jin et al. 2019) . it is a complex clinical condition and can be influenced by number of factors. it is considered as modifiable risk factor for cvd. in some studies, prevotella, faecalibacterium, klebsiella, clostridium and streptococcus are found in abundance in hypertensive patients, indicating a close association of host microflora to such a clinical condition. other studies demonstrated that reduction in gut microbial diversity, short-chain fatty acid (scfa)-producing bacteria and increase in sympathetic drive to the gut and lactic acid producing bacteria, respectively, have direct role in blood pressure regulation. thus, it can be concluded that blood pressure is closely linked to diversity, richness and evenness of microbiome living in the gut and the improved gut microbiota may be a target for future therapies for hypertension (peng et al. 2018 ). the gut-brain axis plays a major role in central nervous system (cns) and intestinal and immune system functioning, as mentioned earlier. gut-brain axis is the bidirectional biochemical signalling between gi and cns activities, integrating efferent and afferent neural, endocrine, nutrient and immunological signals, providing gut microbiota and its derived metabolites a route to access the brain (bull and plummer 2014; joscelyn and kasper 2014). wang and kasper illustrated that this bidirectional communication system enables brain to command gi functions such as metabolism, peristalsis, mucin production and other immunological functions. gut microbiota is also found to have impact on hypothalamic-pituitary-adrenal (hpa) axis, thus playing a role in body's stress response teitelbaum et al. 2008 ). the gut microbiota has also been shown to synthesise neurotransmitters and neuromodulators. various neurotransmitters produced by bacterial species are presented in table 20 .2. those released neurotransmitter then stimulates epithelial cells to synthesise modulators within the enteric nervous system (ens) or directly acting on primary afferent axons (mcvey neufeld et al. 2013) . moreover, it has been shown that ens plays a major role in fundamental gastrointestinal physiological functions such as motility, fluid secretions and blood flow. furthermore, numerous studies have revealed the correlation between microbiome, microbiota derived products, antibiotics, prebiotics and probiotics and cns (wang and kasper 2014) . anxiety and stress, characteristic mood disorders, associated with nervous, endocrinal and immunological system have also been shown to have an association with the gut microbiota. stressors such as chemical, biological or any environmental stimuli can act as an active component to trigger the anxiety and stress response which ultimately activates the hypothalamic pituitary adrenal (hpa) axis. intestinal dysbiosis and gut pathogens have thereby been shown to cause stress and anxiety. animal models have represented to ameliorate these disorders by using probiotic formulations (messaoudi et al. 2011) . various output and input of role of gut microbiota is depicted in fig. 20 .2. the analysis of microbiome of human body shows both the pathogenic as well as beneficial microbial network (ozturk et al. 2017 ). customizable medicine based on an individual's microbiome is an excellent approach for therapeutic choices based either on exercises or/and medications which considers the genetic makeup of an individual, their health condition and quality of life (hasani-ranjbar and larijani 2017). human genomes are about 99.9% indistinguishable to one another; the generally steady human gene pool does not completely clarify all the phenotypic varieties amongst people. on the other hand, the bacterial biological system, living in each human body, that contributes multiple times larger number of qualities than the human genome, could be significantly unique in relation to an individual. therefore, the human microbiome can be regarded as the true cause of numerous responses that can influence the structure and plenitude of microorganism (hasani-ranjbar and larijani 2017) in the human body. recently various studies have shown that intestinal microbiota is fundamentally associated with various therapeutics as in cardiovascular diseases, cancer, etc. these include narrow spectrum antibiotics along with probiotics, prebiotics and synbiotics, faecal microbiota transplantation, nutritional modulators, immune modulators and phage therapy. in the usa and europe, jointly, in the year 2015, because of the onset of antibiotic resistance amongst pathogens, around 50,000 deaths were witnessed, which was projected to increase to a score of around ten million deaths per year worldwide by 2050 (langdon et al. 2016 ). in addition to the development of resistance, the usage of antibiotics has been reported to disrupt the ecology of the human microbiome. whereby, a dysbiotic microbiome loses the capability to perform vital functions such as nutrient supply, production of vitamin, and defence against the pathogens; thereby it leads to an eventual impairment of the metabolic, immunological and developmental system of the host. for an instance, drug-induced modification of the gut microflora can influence a group of foxp3 + treg cells that control demyelination in exploratory autoimmune encephalomyelitis (eae) (ochoa-repáraz et al. 2010) . another study reflects the antigens produced by bacteroides fragilis (capsule polysaccharide) can protect against eae (cns demyelination) and further in human multiple sclerosis (ochoa-repáraz et al. 2011) . similarly, the effects of common antimicrobial treatments on the gut microbiota have been illustrated in table 20 .3. probiotics are characterised as live microorganisms which are not part of the human host microbiome yet give a medical advantage to the host when administered or directed in sufficient quantity. probiotics have been extensively studied in recent years as it imparts various health benefits by the metabolites it produces in the relief from certain intestinal disorders as well as controlling eae. clinical trials have been able to demonstrate that certain cardio metabolic disorders (cmd), such as type 2 diabetes mellitus (t2d), dyslipidaemia and arterial hypertension, as well as chronic kidney diseases (ckd) can be managed by the ingestion of probiotics (neto et al. 2018) . saccharomyces boulardii has been shown to exert antiinflammatory effect which helps to control inflammation related to the dysbiosis in lumen (rodríguez-nogales et al. 2018) . various metabolites produced by microorganisms present in the host and its associated health benefits are discussed in table 20 .4. prebiotics are those food components that cannot be digested by human body but can be selectively digested by the members of probiotics, and thereby serves as a food fibre for probiotics. recent studies have demonstrated that the use of prebiotics can result in enhancing the ecological performance of the gut microbiota, thus promoting a much more beneficial community (vandeputte et al. 2017) . this conceptualizes that the human microbiota can be enhanced, stabilised and shifted by feeding with certain specific prebiotics such as carbohydrates. however, characterisation of the relationship between the prebiotic and probiotic is still a challenge. the point when the idea of synbiotic was first presented, two setups were proposed: first, where the prebiotic and probiotic segments were independent, each being in charge of a kamal et al. (2019) specific impact or medical advantage, and second, a symbiotic segment, where the probiotic was explicitly structured with a prebiotic substrate that would synergistically support the intensity, survival or metabolic movement of a related probiotic strain in the ecology of gastrointestinal system (krumbeck et al. 2016 ). as of late, two novel methodologies have been proposed for creating such synergistic synbiotics, both being dependent on their ecological function and wellness. first, the in vivo strategy which depends upon the determination and isolation of probiotic strain that would increase in number when a certain population of participants are administered with the specific prebiotic component (krumbeck et al. 2015) . second, called as a multi-taxon insertion, depending on sequencing, based on the identification of genes, would examine the fitness of probiotic strain in relation to the type of prebiotic administered by the usage of libraries of transposon mutants . a list of prebiotics with their probiotic components are given in table 20 .5. sometimes the mere usage of antibiotics is insufficient to treat few diseases and rather requires urgent alternatives to manage the severity of the clinical condition. this has led to the introduction of transplantation of the faecal microbiota. the process involves the separation and delivery of the faecal microbiota from stool of healthy donor to the gastrointestinal tract of the receiver patient, thereby enabling an efficient cure by normalising the microbiota composition. recent investigations have explained the mechanism behind the faecal microbiota transplantation (fmt), which has been used to treat clostridium difficile infection (cdi) for the restoration of the gut microbiome to gain the ability to inhibit clostridium difficile indirectly by competing for nutrients. the faecal microbiota also prevents the colonisation by unwanted microorganism by the activation of immune system as well as by direct release of certain antimicrobial components and other metabolites that helps in the inhibition of vegetative and as well as the sporulated disease-causing organism (khoruts and sadowsky 2016) . table 20 .6 shows the list of diseases and infections which can be treated by using fmt. intake of dietary medications for a long period of time impacts the structure and function of microbiome of the human body. the change in the availability of nutritional content of the diet causes corresponding alteration of the human microbiome. recent findings have suggested that depending upon the intake of dietary, the microbiome richness diversifies. the lower is the genetic richness of microbiome; the lower will be the immune status of the person, relating to abnormal metabolic function and poor anti-inflammatory activity (cotillard et al. 2013; le chatelier et al. 2013 ). the diet patterns may even contribute in managing different diseases (table 20 .7) by way of microbiome. microbiome-based approaches involving antibiotics, probiotics, prebiotics and synbiotics, faecal microbiota transplantation and nutritional modulators correlate directly with the alteration of immune status of an individual focusing on the innate immunity. thereby, the human microbiome is being affected for benefits with the change in immune status. until now, there are inadequate information in this method; however, treatment of inflammatory diseases using steroids is in abundance. intestinal innate and acquired immunity as well as systemic acquired immunity involves various mechanisms for the control of gut microbiome. some of them include change in barrier function, expression of leptin, molecule β, human leukocyte antigen (hla) class i and class ii loci, activation of toll-like receptors, natural killer cells, cd4+ cells and foxp3+ as well as the production of antimicrobial competition for nutrients; direct suppression by antimicrobial peptides; bile-acid-mediated inhibition of spore germination and vegetative growth; activation of immune-mediated colonisation resistance khoruts and sadowsky (2016) ulcerative colitis (uc) increasing the production of short-chain fatty acids, (butyrate); inhibitingth1 differentiation, activity of t cells, leukocyte adhesion; production of inflammatory factors shen et al. (2018) irritable bowel syndrome (ibs) visceral hypersensitivity; altered barrier function, gastrointestinal motility and the gut-brain axis grover et al. (2014) obesity decrease adiposity; alter metabolic phenotype by increased bacterial diversity marotz and zarrinpar (2016) obesity henning et al. (2018) peptides and α-defensins (ticinesi et al. 2019) for the proper functioning of the gut microbiome. phage therapy involves the introduction of explicit bacteriophages that targets a microorganism which in turn has the ability to generate a beneficial microbiome shift. however, a limitation to this strategy is the simultaneous resistance offered by the microorganism in play which is yet to be proven. till date, none of the phage therapy approaches have been established as an fda-approved drug. recently, scientists are working hard to perform phage based killing of microbes, but it is much more complicated. crispr (clustered regularly interspaced short palindromic repeats) is one of the approaches to advance the limitations (lemieux 2019). it is a tool derived from prokaryotic immune system empowered to study and modify organisms with ease and efficiency. the system helps to modify the gut genome of gut microorganisms and bacteriophages. this engineered crispr-cas system ultimately can control gene expression and modulate production of metabolite and protein presenting a new approach for the development of drugs that can target the microbiome (table 20 .8). although these interventions are used as medicinal and nutritional strategies and have been clinically experimented for a successful result, more focus and deliberate attempts are being made for the establishment of such casualty in order to define the functional metabolic change. these therapies are yet not so widely utilised due to the requirement of huge amount of money and time. hence, advancement in this field targeting the relation of human microbiome with health and diseases, identification of the composition and activity of microbiome as well as linking it, and most importantly using technical resources such as bioinformatics to incorporate and fill up the loopholes ascertaining to the models is quite difficult. future research based on these directions is a key to solve the problems and hence enlightening with knowledge about the human microbiome and their influence on the human health. a randomized trial to determine the impact of a digestion resistant starch composition on the gut microbiome in older and mid-age adults the urinary tract microbiome in health and disease defining a healthy human gut microbiome: current concepts, future directions, and clinical applications role of the microbiota in immunity and inflammation microbial view of central nervous system autoimmunity single plasmid systems for inducible dual protein expression and for crispr-cas9/crispri gene regulation in lactic acid bacterium lactococcus lactis impact of antimicrobial therapy on the gut microbiome metagenomic analyses of an uncultured viral community from human feces part 1: the human gut microbiome in health and disease ceftriaxone and cefotaxime have similar effects on the intestinal microbiota in human volunteers treated by standard-dose regimens the human skin microbiome supplementation of diet with galactooligosaccharides increases bifidobacteria, but not insulin sensitivity, in obese prediabetic individuals prebiotic dietary fiber and gut health: comparing the in vitro fermentations of beta-glucan, inulin and xylooligosaccharide colorectal cancer cells increase the production of short chain fatty acids by propionibacterium freudenreichii impacting on cancer cells survival estrogen and microbiota crosstalk: should we pay attention? prebiotic effect of fructooligosaccharides from morinda officinalis on alzheimer's disease in rodent models by targeting the microbiota-gut-brain axis dna sequencing technologies key to the human genome project a diet mimicking fasting promotes regeneration and reduces autoimmunity and multiple sclerosis symptoms evidence for a vascular microbiome and its role in vessel health and disease probiotics in adhesion of pathogens: mechanisms of action bacterial community variation in human body habitats across space and time dietary intervention impact on gut microbial gene richness introducing insoluble wheat bran as a gut microbiota niche in an in vitro dynamic gut model stimulates propionate and butyrate production and induces colon region specific shifts in the luminal and mucosal microbial community gut microbiota regulates maturation of the adult enteric nervous system via enteric serotonin networks healthy dietary pattern and their corresponding gut microbiota profile are linked to a lower risk of type 2 diabetes, independent of the presence of obesity diversity of the human intestinal microbial flora propionate-producing consortium restores antibiotic-induced dysbiosis in a dynamic in vitro model of the human intestinal microbial ecosystem immunosuppressive cd71+ erythroid cells compromise neonatal host defence against infection the microbiome in respiratory medicine: current challenges and future perspectives a diet based on cured acorn-fed ham with oleic acid content promotes anti-inflammatory gut microbiota and prevents ulcerative colitis in an animal mode oral microbiomes: more and more importance in oral cavity and whole body pathophysiological mechanisms of stress-induced intestina damage adaptation of beneficial propionibacteria, lactobacilli, and bifidobacteria improves tolerance toward technological and digestive stresses attenuation of post-myocardial infarction depression in rats by n-3 fatty acids or probiotics starting after the onset of reperfusion metagenomic analysis of the human distal gut microbiome the skin microbiome on the fiftieth anniversary postinfectious irritable bowel syndrome: mechanisms related to pathogens human microbiome as an approach to personalized medicine decaffeinated green and black tea polyphenols decrease weight gain and alter microbiome populations and function in diet-induced obese mice sodium chloride inhibits the suppressive function of foxp3+ regulatory t cells crispr-based engineering of nextgeneration lactic acid bacteria engineering microbes for targeted strikes against human pathogens the effect of synbiotics bifidobacterium infantis and milk oligosaccharides on shaping gut microbiota community structure and nash treatment the role of intestinal microbiota in cardiovascular disease digesting the emerging role for the gut microbiome in central nervous system demyelination impact of early exposure to cefuroxime on the composition of the gut microbiota in infants following cesarean delivery understanding the mechanisms of faecal microbiota transplantation effects of bifidobacterium breve a1 on the cognitive function of older adults with memory complaints: a randomised, double-blind, placebo-controlled trial in vivo selection to identify bacterial strains with enhanced ecological performance in synbiotic applications prebiotics and synbiotics: dietary strategies for improving gut health amelioration of experimental autoimmune encephalomyelitis by probiotic mixture is mediated by a shift in t helper cell immune response the effects of antibiotics on the microbiome throughout development and alternative approaches for therapeutic modulation beyond bacteria: a study of the enteric microbial consortium in extremely low birth weight infants richness of human gut microbiome correlates with metabolic markers phage therapy: turning the tables on bacteria: when engineered to incorporate crispr components, phages may overwhelm bacterial defenses or transform bacterial functions folate-producing lactic acid bacteria reduce inflammation in mice with induced intestinal mucositis metabolic engineering of probiotic saccharomyces boulardii xylooligosaccharide supplementation decreases visceral fat accumulation and modulates cecum microbiome in mice gut microbiome contributes to impairment of immunity in pulmonary tuberculosis patients by alteration of butyrate and propionate producers the microbiota of the respiratory tract: gatekeeper to respiratory health focus: microbiome: treating obesity and metabolic syndrome with fecal microbiota transplantation the microbiome is essential for normal gut intrinsic primary afferent neuron excitability in the mouse gut microbiome diversity and high-fibre intake are related to lower long-term weight gain assessment of psychotropic-like properties of a probiotic formulation (lactobacillus helveticus r0052 and bifidobacterium longum r0175) in rats and human subjects the lung microbiome in health and disease antibiotic-induced shifts in fecal microbiota density and composition during hematopoietic stem cell transplantation comparison of the respiratory microbiome in healthy nonsmokers and smokers average genome size estimation improves comparative metagenomics and sheds light on the functional ecology of the human microbiome gut microbiota and probiotics intervention: a potential therapeutic target for management of cardiometabolic disorders and chronic kidney disease? development, maintenance and disruption of the blood-brain barrier central nervous system demyelinating disease protection by the human commensal bacteroides fragilis depends on polysaccharide a expression gut, bugs, and brain: role of commensal bacteria in the control of central nervous system disease effects of lactobacillus helveticus on murine behavior are dependent on diet and genotype and correlate with alterations in the gut microbiome the potential for emerging microbiomemediated therapeutics in asthma genetic engineering of a temperate phage-based delivery system for crispr/cas9 antimicrobials against staphylococcus aureus interaction between gut microbiome and cardiovascular disease chronic calorie restriction attenuates experimental autoimmune encephalomyelitis anti-inflammatory mechanisms of action of saccharomyces boulardii the anatomical and cellular basis of immune surveillance in the central nervous system intestinal anti-inflammatory effect of the probiotic saccharomyces boulardii in dss-induced colitis in mice: impact on micrornas expression and gut microbiota composition the skin microbiome of cohabiting couples insights into the human gut microbiome and cardiovascular diseases probiotics in gut-bone signaling. in: understanding the gut-bone signaling axis enrichment of lung microbiome with supraglottic taxa is associated with increased pulmonary inflammation relationship between intestinal microbiota and ulcerative colitis: mechanisms and clinical application of probiotics and fecal microbiota transplantation preventive rather than therapeutic treatment with high fiber diet attenuates clinical and inflammatory markers of acute and chronic dss-induced colitis in mice a review on role of microbiome in obesity and antiobesity properties of probiotic supplements irritable bowel syndrome: a clinical review how microbiome impact on the cardiovascular system spatial organization of bacterial flora in normal and inflamed intestine: a fluorescence in situ hybridization study in mice the lactic acid bacterium pediococcus acidilactici suppresses autoimmune encephalomyelitis by inducing il-10-producing regulatory t cells the gut microbiome and its role in cardiovascular diseases in vitro probiotic potential and anti-cancer activity of newly isolated folate-producing streptococcus thermophilus strains chronic peripheral administration of corticotropin-releasing factor causes colonic barrier dysfunction similar to psychological stress the microbiome and innate immunity exercise and immune system as modulators of intestinal microbiome: implications for the gut-muscle axis hypothesis the human microbiome, diet, and health: workshop summary take your medicine": nonadherence issues in patients with ulcerative colitis estimated costs for treatment and prophylaxis of newborn vitamin k deficiency bleeding in tashkent role of the gut microbiota in nutrition and health prebiotic inulin-type fructans induce specific changes in the human gut microbiota the role of microbiome in central nervous system disorders the human microbiota in health and disease genetic determinants of in vivo fitness and diet responsiveness in multiple human gut bacteroides the microbiome in the lower respiratory tract selective and suppressive effects of antibiotics on donor and recipient bacterial strains in gut microbiota determine transmission efficiency of bla ndm-1-bearing plasmids temperate and lytic bacteriophages programmed to sensitize and kill antibiotic-resistant bacteria metabolism of dietary and microbial vitamin b family in the regulation of host immunity park et al. (2017) and yosef et al. (2015) crispri gene regulation berlec et al. (2018) key: cord-355130-a2jc1g0i authors: shrivastava, paul; stafford smith, mark; o’brien, karen; zsolnai, laszlo title: transforming sustainability science to generate positive social and environmental change globally date: 2020-04-24 journal: one earth doi: 10.1016/j.oneear.2020.04.010 sha: doc_id: 355130 cord_uid: a2jc1g0i despite the decades-long efforts of sustainability science and related policy and action programs, humanity has not gotten closer to global sustainability. with its focus on the natural sciences, sustainability science is not able to contribute sufficiently to the global transition to sustainability. this perspective argues for transforming sustainability science into a transdisciplinary enterprise that can generate positive social and environmental change globally. in such transformation, the social sciences, humanities, and the arts can play an important role to address the complex problems of culture, institutions, and human behavior. to realize a truly integrated sustainability science, we need renewed research and public policies that reshape the research ecosystem of universities, funding agencies, science communications, policymaking, and decision making. sustainability science must also engage with society and creatively employ all available sources of knowledge in favor of creating a sustainable earth. global environmental challenges are now manifesting at a planetary scale in the form of climate change, ocean acidification, biodiversity loss, environmental pollution, deforestation, and land-use change, among many others. these characteristics of the anthropocene era have been driven by accelerating socioeconomic trends, including population growth, various measures of consumption and economic growth, urbanization, telecommunications, and other aspects of globalization. the so-called great acceleration, 1 which started in the 1950s, has been compounded by the rapid development and spread of information technologies and telecommunications. 2 decades of international conferences and reports-beginning with the 1972 report the limits to growth by the club of rome and the united nations (un) conference on the human development in that same year and evolving to the creation of the 17 sustainable development goals (sdgs) by the un general assembly in 2015-have warned of the challenges and risks associated with current development pathways and have fostered the evolution of sustainability science at the global scale ( figure 1 ). science has played a pivotal role in helping society to identify and understand the scope and scale of planetary environmental changes. it has also helped to guide policies and practices that address sustainability challenges from local to global scales. in recent years, global-scale challenges have become a growing concern. the 1990s saw the development of a number of global-change research programs and various environmental science initiatives, such as the world climate research program, the international geosphere biosphere program, diversitas, the international human dimensions program, and some attempted unification in the earth systems science program. sustainability science emerged from the recognition of a need to extend global-change research to seek solutions, which in turn requires multidisciplinary and interdisciplinary research. this is slowly moving further from the perceived hegemony of the disciplinary expert toward the transdisciplinary co-design and co-production of research. international science coordination and funding approaches have also evolved over the past decades, as promoted by the international science council, the belmont forum, and others. many of the global-change programs merged into future earth in 2013. 3, 4 in 2018, a unified coordination of the natural and social sciences was advanced by the formation of the international science council through the merger of the international science union and the international social sciences council. this year, the transdisciplinary sustainability research & innovation congress 2020 was planned to unite global researchers, industry practitioners, and world leaders to inspire action and promote sustainability transformation. although the event is now postponed because of the coronavirus disease pandemic, its aim of cultivating a space for sustainability scholarship, innovation, collaboration, and action won't be delayed. global-change research reflects a growing demand for research to become more impactful and solution oriented, as spelled out in the future earth vision, 5 which is aimed at a multiscale (from local to planetary) understanding of environmental changes that transcend national boundaries. over the past decade, global-change research has made significant efforts to be more inclusive of science communities from china, russia, africa, and the global south. global-change research has first and foremost promoted a systems approach to environmental problems. 6 this approach has focused on relationships and interactions among the atmosphere, biosphere, hydrosphere, cryosphere, and soils and explores how they are influenced by (and influence) human activities. an understanding of human impacts on earth system processes gave rise to the notion of planetary boundaries, 1 the idea of tipping points, 7, 8 and the concept of the anthropocene. these scientific metaphors have been useful in articulating the risks of continuing with business as usual and development as usual. it is fair to say that global sustainability science has contributed significantly to a number of global agreements to address the challenges of the anthropocene, most notably the paris agreement and the un agenda 2030, which were signed in 2015. these universal agreements reflect a global consensus to address climate change and strive for sustainable and balanced social and economic development that promotes the well-being of socio-ecological systems. they also provide a powerful statement of intended direction for humanity, as captured in the un's 17 sdgs. they create an imperative for transformative change in response to global challenges. however, such changes have not been forthcoming at the rate and scale required by global agreements. as a consequence, we continue to see increasing concentrations of co 2 in the atmosphere, biodiversity loss, land degradation, and growing inequalities among people and nations. 9, 10 the great acceleration is generated and sustained by powerful economic mechanisms, including globalization, marketization, and financialization, which are based on the mainstream model of doing business and promoting economic growth. 11 to foster growth and maximize profit, companies and other organizations aggressively appropriate value as much as possible from nature and society and externalize the costs to natural ecosystems and to human communities, including future generations. 12, 13 the mainstream business model drives competition and consumption and guarantees that human and social activities continue to drive the overuse of ecosystems beyond their carrying capacity toward breaching several planetary boundaries. 1 the current economic paradigm has led us to the point where the functioning of our global life-support system is no longer assured. 14 the global economy and functioning earth system are on a collision course that will ultimately have dire consequences for humans and the biosphere; we need an urgent course correction, and current research paradigms are not meeting this challenge. in this perspective, we argue that in order to generate positive social and environmental changes globally, sustainability science must transform into a transdisciplinary enterprise. our modest goal here is to lay out the special research challenges posed by the emerging anthropocene era and exhort sustainability science to address these challenges impactfully. we focus our suggestions primarily on research policy measures that shape the conduct of science and agency of scientists and only secondarily on institutional policy measures. this is not intended to be a broad public-policy discussion on sustainability research. sustainability science needs deep integration of the social sciences, humanities, and the arts with its dominant natural sciences. the focus should be on complex problems of nature, culture, institutions, and human behavior and the co-creation of knowledge with stakeholders. to do this, sustainability science and its stakeholders will need to better understand the funding dynamics and higher levels of research funding. to make sustainability science impactful, we need to modify the current research ecosystems. sustainability science must engage with society (economic, social, and cultural spheres) to extend many successful small-scale seeds for change. the goal of sustainability science is to create a sustainable earth by creatively deploying all available knowledge of humanity. the evolution of what is now known as sustainability science has been important, but clearly it is not yet enough to play a pivotal role in social transformations needed for human preservation in the face of accelerating changes of the anthropocene. we argue that we need to transform our approach to research itself. a number of known attributes of global change currently create not only are these changes fast, but in most instances they are also increasing non-linearly, as described by the great acceleration. the human capacity to respond to these changes has not kept pace. some of the historical drivers of social change, such as generational turnover, have slowed as a result of increasing longevity. consequently, cultural and value changes that influence how humans relate to the environment have to occur within a generation rather than over generations. researchers themselves are challenged to think differently and engage in interand transdisciplinary research processes. however, it takes time to develop common understanding between disciplines and engage across different stakeholder knowledge epistemologies; therefore, integrative research risks lagging behind the changes it is addressing (table 1) . spatial connectivity sustainability policies and practices must be local and context specific, yet they are embedded in larger systems that are increasingly and more rapidly connected. although developments in inter-and transdisciplinary research over the past few decades have delivered much success at local levels in agriculture, development, and other domains, replicating this success across scales and globally is genuinely challenging. transformations of social, economic, and institutional systems must also be negotiated across scales. sustainability science research has not reconciled the gap between global-scale problems and locally based solutions. we still do not know how to scale up solutions in an interconnected world (table 1) . social-ecological systems have always been affected by social and cultural norms, vested interests, and power dynamics. however, in today's globalized system, many social norms have been shaped by market-based capitalism and consumption, and the rates and distributed extent of change mean that managing the dynamics of winners and losers is increasingly challenging. for example, oil profits in one part of the world could finance investments that undermine sustainability interventions in another. at a time when there is a need to build engagement across all levels of society, identifying and transforming the nature of power relationships can be difficult. sustainability science has not fully acknowledged or integrated global power dynamics into understandings of change (table 1) . selective embeddedness of science in society science is extremely specialized and operates within universities and corporations, increasingly fulfilling the mandates of government and corporate funders. science does not reach wide swathes of global society, particularly the communities where challenges of the anthropocene are concentrated. many sectors, especially the informal sector of the economy, are not benefiting from scientific knowledge. the informal sector accounts for up to 20% of global output in developed countries and over 33% in developing countries. 15 according to the international labor organization, two billion women and men make their living in the informal economy, which provides over 50% of non-agricultural employment in africa, latin america, and asia. 16 in africa, the informal economy could account for 50%-80% of the gross domestic product (gdp) and 90% of new jobs. 17 even in sectors where science does influence economic and social decisions, it does not always effectively serve broad public interests. science funding for the public good and commons protection is insufficient to address the massive and expanding problems of the anthropocene (table 1) . above all, the challenges involve culture, society, behavior, and institutions. yet, the current approach to sustainability science still pays insufficient attention to the social and human dimensions, and holistic integrated research remains the exception rather than the norm. sustainability science is still dominated by the natural sciences, and there is grossly less investment in the social sciences, the humanities, and arts than in the ''hard sciences,'' as well as a general underinvestment in research overall. the uptake of research insights from the social sciences and the arts and humanities has been limited to the findings that fit within the existing paradigm. 18 particularly debilitating is the failure to open up to new perspectives and different types of knowledge in the name of keeping science ''objective'' and apolitical. even where efforts have been made to integrate social, human, and natural sciences in transdisciplinary research, these successes are challenged by scale. in a recent review, norströ m et al. 19 identified four key principles for good transdisciplinary co-production of knowledge: the engagement by research should be context based, pluralistic, goal oriented, and interactive. these principles, which draw on a great body of understanding from past transdisciplinary successes, are all challenged by the attributes of the anthropocene. engagement must be context specific, yet it seems to be required at all levels, including globally. it must be pluralistic, yet the diversity of stakeholders even at the local level is immense, and more so for global processes. goal-oriented science is challenging with diverse actors across scales, and making engagement interactive, with feedback loops that promote reflection and learning, requires new thinking at the global level. systems thinking emphasizes that the most powerful leverage points for system change come through modifying the design and intent of the system. 20 paradigms-or the shared thought patterns that influence the way that systems are perceived and designed-are also recognized as a powerful driver of systems change. 21 understanding how to achieve paradigm shifts that influence the goals, values, and behaviors of systems is not the normal purview of the natural sciences. in fact, alternative paradigms are usually dismissed rather than encouraged within research systems. although global assessment initiatives such as the intergovernmental science-policy platform on biodiversity and ecosystem services (ipbes) recognize and include cultural beliefs, values, worldviews, and indigenous and local knowledge, 22 many perspectives from the social sciences and humanities have not been taken seriously in sustainability science. for example, research within feminist political ecology, decolonization studies, and ''new materialism'' offers new thinking on ethical and political challenges. 23, 24 to capture the breadth and depth of global challenges, sustainability science needs to support efforts to recognize multiple perspectives and ways of knowing. 25 this includes developing profoundly holistic views on how natural and cultural systems coevolve. 26 interdisciplinary and transdisciplinary research activities in academic institutions are seeking such integration. there have been numerous attempts to encourage transdisciplinary collaboration and team science. 27 in higher-income countries, there is a tradition of collaborative cross-sectoral research, such as the agricultural extension research in the us and that conducted by the cooperative research centres (crcs) in australia. recently, the us national science foundation has encouraged research collaborations across science fields, which it calls ''convergence science,'' by awarding grants for themes such as ''navigating the new arctic.'' global programs such as future earth are developing ''knowledge-action networks'' to promote transdisciplinary research. however, the societal ecosystems in which such initiatives operate do not embrace their logic or provide resources to scale them globally as necessary. current efforts to support integrated and transdisciplinary research do not match the scale of the problem. maintaining the power of conventional, objective science has come at a cost for sustainability science. not only has failure to integrate important insights from the social sciences and environmental humanities limited the perceived ''solution space'' for responding to global challenges, but sustainability science has also failed to engage with the ''how'' of transformative change. it has had limited success in connecting with key stakeholders (policymakers, corporate decision makers, political leaders, and civil society) through a language that they understand. it has also failed to convey not only the sense of urgency underscored by global changes but also the possibilities for responding. currently, research on global environmental change largely speaks to the converted-to those who are influenced by rational arguments and scientific evidence. very little attention has been paid to alternatives that challenge the tenets of conventional science and business as usual. the normative dimension of sustainability science is one area that is often challenging to researchers. the question of which changes are ethical and the ''right ones'' to pursue introduces a normative dimension to science that can also be seen as highly political. however, this is an area where global agreements have provided direction for sustainability in recent years-the sdgs of agenda 2030 clearly articulate normative visions for global sustainability in alignment with providing food, water, and energy to the approximately ten billion people expected to live on earth by 2050, just as the paris agreement clearly states an intent to stay below 2 c warming, and 1.5 c if possible. although they provide a strong statement of goals and intents, both still contain ambiguities around more detailed interpretations relating to different places, levels of governance, and interests, which together can limit their impact for sustainability. to integrate the diversity of interpretations of particular goals, the research system has to open itself up to a wide range of disciplines and epistemologies. many examples of existing research can contribute to a more integrative discourse within sustainability science. however, it is important to consider these different perspectives together rather than as separate boxes or silos. drawing on an integral framework, 28 figure 2 depicts how sustainability science has been largely confined to the domain of ''systems'' science and how less attention has been paid to behavior, culture, and experience. an integral approach to sustainability science includes a more balanced representation of these other three perspectives. below, we highlight some specific examples of research in these other ''quadrants'' to illustrate the potential to broaden the systems-oriented perspective of sustainability science and deepen our understanding of how to respond to environmental change. behavioral change there is still a tendency within sustainability science to draw on the information-deficit model and assume that once individuals ll open access and organizations realize the risks associated with environmental change, they will take action or at least can be manipulated or nudged in that direction. 29 yet, plenty of research in psychology-including research on the relationship between the intrinsic and extrinsic motivation of human actors, i.e., between motivation that is inherent in the task itself and motivation that relies on external rewards or sanctions 30 -shows that human behavior is more complex than this. the latter can result in the crowding-out effect, where external rewards actually reduce the intrinsic motivation of the actors. a crucial result of this mechanism is that people's environmental commitment declines and more environmentally harmful behavior emerges. 31, 32 a key message here is that intrinsic motivation is critical to sustainable behavior within rapidly changing systems. research also describes how mechanisms of moral disengagement enable otherwise considerate people to commit transgressive acts without experiencing personal distress or guilt. in other words, moral control can be selectively disengaged from detrimental conduct through psycho-social mechanisms. 33 selectively activated disengagement strategies (such as moral justification, euphemistic labeling, advantageous comparison, displacement of responsibility, diffusion of responsibility, disregard for or distortion of the consequences, dehumanization, and attribution of blame) are at work in harmful environmental practices of corporations and other economic entities. 34 this points to the need for a more critical engagement with the social structures and systems in which sustainability measures are enacted, as emphasized in social practice theory. 35 cultural change embedding sustainability sciences and practices at a global scale will require a new and evolutionary narrative about hu an integral framework recognizes four perspectives or domains of reality that present different types of knowledge about a subject, in this case sustainability science. global-change research has tended to prioritize the systems domain and put less emphasis on research from the meaning-making, cultural, and behavioral domains. integration does not diminish the systems perspective but rather adds valuable knowledge that can help society meet the sdgs. adapted from wilber. 28 man-environment relationships. research on the power of narratives (language, metaphors, and storytelling) points to the need for new cultural narratives on the journey of humans in the universe across geological time. 36, 37 the current narrative that humans are a special, privileged species destined to dominate over nature is deeply flawed, and it has contributed to a fascination with techno-fixes, such as geoengineering. 38 cultural changes need to embrace research on the power of the arts, aesthetic inquiry, and the humanities to heal the emotional disconnection between humans and nature, which is at the root of our current environmental crisis. the role of artists and art-based practices in achieving sustainability is increasingly being recognized. 39 dutton 40 refers to the art instinct of humans as having survival value across cultures and time. art is ubiquitous in all human communities globally over most of history. communities able to tell better stories and draw better pictures of natural and predatory hazards were able to avoid annihilation more effectively. the rich human legacy of the arts also allows us to connect cognitive understanding to emotional empathy and embodied learning about sustainability. 41 the experiential dimensions of environmental issues are becoming increasingly pronounced through emotions such as hope, grief, anger, and despair. 42 whether and how we identify and respond to environmental problems are also closely tied to the ways that we perceive the world and our place in it. issues such as climate change will be interpreted differently depending on the beliefs, values, and worldviews held by individual or groups. 43 within psychology, meaning making is defined as ''the process of how people construe, understand, or make sense of life events and experiences.'' 44 research in developmental psychology shows that multiple worldviews coexist today and that these can change over generations and within individual lifetimes. acknowledging both differences and developments in perspective-taking capacities and meaning making can provide insights into why people relate to sustainability issues differently and how to connect with a diversity of worldviews. for example, in a study of meaning making among farmers in el salvador as it related to climate-change adaptation, hochachka 44 found differences in the object of awareness, the number of perspectives taken, the complexity of thought, and the scope of time considered in one's understanding of climate change and adaptation. such findings suggest that it could be time for sustainability science to engage with the diversity and dynamics of meaning making rather than to assume that sense making of global challenges is universal and static. in the three research areas described above, one can already see the ways in which researchers are synthesizing knowledge within and across the ''quadrants'' to study the dynamics of sustainability at a deeper level. for example, understanding behavioral changes involves grasping the processes of motivational crowding, understanding socio-cultural changes involves studying the narratives that underpin social values, and understanding experiential responses to environmental change involves indepth research into meaning making. yet, questions remain on how to forge this integrative research more broadly within sustainability science. the integration of different perspectives and domains of knowledge and methodological approaches to research can be enhanced through awareness and education. yet, disciplinary science has privileged cognitive modes of learning, and as disciplines have fragmented, so have the content domains of learning. in highly scientized fields such as medicine, expertise is being isolated and narrowed into increasingly restricted specializations. education for global-change problems needs to be more holistic and contextually anchored through the incorporation of systems and their interconnectedness with culture and experience. education that embraces an integrative discourse recognizes multiple perspectives and the need for critical thinking, reflection, and experience-based learning that leads to action and a sense of agency. 45 one way of integrating different perspectives is through ''action research,'' which focuses on driving change and doing rather than just reaching understanding. 46 otto scharmer and colleagues at the presencing institute created an actionresearch platform at the intersection of science, consciousness, and social change. 47 their ''theory u'' framework and methodology for leading profound innovation proposes that the quality of the results that people create is a function of the quality of awareness, or consciousness, from which the participants in the system operate. the inner shift, from fighting the old to sensing and presencing an emerging future, is at the core of deep leadership work. 48 people are encouraged to expand their thinking from the head to the heart. it is a shift from an ego-system awareness to an eco-system awareness that cares about the well-being of all, including oneself. the presencing institute has reached over 100,000 people in 185 countries to help leaders at any level find new solutions to the disruption that humanity faces. there are a variety of approaches to linking research epistemologies with experiential knowledge systems. the crcs in australia have been a generally successful approach to making public funding contingent on a close working relationship between researchers and stakeholders, the latter of which provide funding input, in a vast array of different topics (https://www. business.gov.au/grants-and-programs/cooperative-research-centres-crc-grants). crc structures increasingly require a formal, even dominant, role for stakeholders in governance; even so, the growing experience suggests that it takes up to 5 years for the diverse partners to really work out a common mission and deliver on the promise of transdisciplinarity to get novel research into action. another promising approach is through greater investment in coordinated, applied, local innovation hubs or sustainable development labs. at community colleges and universities in the us, ''living labs'' are using university operational facilities to create engaged action learning opportunities for students and engaged scholarship opportunities for researchers. 49 examples of integrated and holistic knowledge-creation and action initiatives that embrace more diverse epistemologies are also emerging from social and cultural institutions, as exemplified by the encyclical letter laudato si' by pope francis. 50 the encyclical presents an integral ecology that underscores the human origins of the ecological crisis and proposes fundamental changes in organizing economic life. among the important suggestions put forward by the pope is increased frugality in consumption, and he also acknowledges the intrinsic value of nature. to realize his vision, pope francis initiated the economy of francesco project. 51 this international cooperative platform brings together 5,000 young economists, entrepreneurs, and change makers to co-create and put in place a new economic model in the spirit of integral human development. notwithstanding the examples of integrative approaches described above, sustainability science has not been transformed enough to successfully address the mounting grand challenges faced by society. the challenge of evolving sustainability science cannot be just endogenous and isolated. science is an instrument of society, and it operates within a broader socio-economic, cultural, and technological milieu that sets the operating context for research. at present, the external context is characterized by neoliberal capitalism that sets up competition rather than collaboration; devalues or overlooks social, human, and environmental capitals; fails to see systemic effects; and encourages inequality. 52 science needs to rethink its social relevance and social connections to effectively contribute to the grand challenges of the anthropocene. it must play an active role in the concurrent co-evolution of economies and their underlying cultural assumptions in sustainable directions. within that broader context, scientific institutions and business organizations supporting science would need transformation of their core purpose, administrative processes, and reward systems. sciences would need deep unification across epistemic and disciplinary lines and be willing to play a more active, participatory role in the transformations required for global sustainability in the world at large. at a more synoptic level (see figure 3 ), waddell et al. 53 have argued that research needs to support the establishment of a ''transformation system'' in parallel with the system being transformed (see https://transformationsforum.net/). they outline four strategies that can contribute to transformation: doing change, forcing change, directing change, and co-creating change. according to these authors, most transformations involve a mix of all four types, and research can play a different role in each of these strategies. there is research to be done on actual transformations, as well as on transformation processes, practices, and pathways. although systems thinking emphasizes that addressing design and intent are the strongest leverage points, 20 these ll open access are also the hardest to achieve, particularly when the personal sphere of transformation is considered to be ''external'' or irrelevant to systems change. 43 an integration of experience, culture, behavior, and systems within sustainability science recognizes that the system is not ''out there,'' and it highlights the importance of ''being change,'' i.e., embodying the changes that are considered necessary at all levels, right here and now, and critical to the process of creating an equitable and sustainable future. 54 recognizing that people engage with the future differently is also essential for activating transformations to sustainability. the three horizons framework helps to generate agency and future consciousness by recognizing different ways of addressing uncertainty. this approach distinguishes between incremental and transformative change and emphasizes the need for participatory, reflective, and action-based research. 58 research on how to pre-condition the system for transformation through incremental changes also provides valuable insights on how to create transformative change 56,57 by drawing, for example, on parts of the australian wine industry that were adapting incrementally as opposed to transformationally. 57 when change is seen as a process, incremental changes offer an opportunity to shape the pathways for transformative change, as depicted on the righthand side of figure 3 , and thereby activate easier systems interventions (left side) in order to pre-condition the system for more transformative change in the harder systems characteristics of structure and intent. in practice, this requires a diversity of the intervention strategies (top of cone), as can be seen in examples such as the german energy transition or marriage equality in the us. 55 then arises the question, how can similar insights be applied to sustainability science itself? the cone of increasingly transformative change: systems interventions with increasing leverage (from bottom to top on left side 21, 20 ) as the cone opens out have greater potential to cause transformational change and require a larger application of the four strategies for systems change (top plane 55 ); easier but directed incremental changes can contribute to the transformability of the system (right side). 56, 57 enabling sustainability science action sustainability science is a part of the global system that needs to transform to achieve the sdgs. this means that researchers have to reflect on how science should evolve from the research that is already available on transformations to research that can activate sustainability transformations in the wider world. we note four lessons in this regard, examine incremental changes in research organizations (where a great deal of science is operationalized), and then share some thoughts on how science can drive transformation across society. first, most environmental problems, especially the global ones, are complex, messy, and wicked. too often the problem formulation by researchers is partial and inadequate because they reduce the real-world, multifaceted problems into a single scientific-technical dimension and seek to solve this. instead of providing a precise answer to the wrong question (i.e., committing an ''error of the third kind''), approaches should address the full scope of the problem in question (the scientific-technical, the interpersonal-social, the systemic-ecological, and the existential-spiritual) and then develop some satisficing balance among them. 59 the job of sustainability science is to identify problems and then to produce response options that are substantively adequate and ethically acceptable in broad socio-economic contexts. the same logic can be applied to thinking reflexively about sustainability science itself. second, the growing literature on transitions and transformations in society covers the many dimensions and scales needed for responding to global environmental challenges. [60] [61] [62] much of this research emphasizes small-scale (pilot) experiments as important to demonstrate proof of concepts. it also explores how local success stories can be scaled. 63 we need rapid cross-system learning about the successful ones in a context of changing the institutions and rules (scaling ''up'') and the cultural milieu (scaling ''out'') in which these can be scaled. 64 a growing set of examples and opportunities for scaling can influence how we do sustainability science. 65 we are at a point in time when sustainability science needs to think about how to selectively scale these exemplars massively, including the transformation of science itself. third, as already noted (figure 3) , systems thinking highlights how the strongest leverage points for changing a system are in modifying its design and intent. it is usually much easier to fiddle with parameters and perhaps feedbacks. 20 the resilience literature addresses what makes a system more or less ''transformable.'' 56 it highlights how important well-chosen incremental changes can be to pre-conditioning a system to be more likely to transform in the ''right'' direction when a suitable impetus comes along. therefore, a good strategy for transforming sustainability science (as for transforming the world) should integrate well-directed incremental changes with preparedness to transform at the appropriate opportunity. fourth, enabling sustainability science to be impactful could also involve modifying the identity and role of scientists. historically, the role of scientists has been to be ''objective'' observers of phenomenon, scientific data collectors, unbiased analysts, and accurate recorders and reporters of findings. in a world beset with grand challenges, scientists must also become translators of knowledge, communicators to the public, policymakers, implementors of action, advocates of solutions, and co-designers of the future. they must also listen deeply and maintain reflective, open minds as they engage not just with theory but also with actions that generate a sustainable world. noting these points, we turn to asking how sustainability science could become more pre-adapted to transform (bottom of figure 3 ), as well as actually transform (top of figure 3 ), in order to play a more effective participatory role in global transformations. this must be in the context of the challenges of the anthropocene, i.e., that change is rapid, spatially interconnected, and more than ever influenced by global power dynamics as it is increasingly embedded into all societal systems. acknowledging universities as a key operational venue of sustainability sciences, and knowing how difficult it is to change them, we suggest some modest yet potentially transformative incremental changes. progressing incremental changes in research and public policy scientific research is guided by research policies and public policies, both of which progress through incremental changes. research institutions, especially universities, are being challenged to produce impactful research. it is widely held that good transdisciplinarity (convergence science) is a means to greater and more enduring impact, especially in more complex and value-laden problems, such as many of the sustainability challenges discussed here. such transdisciplinary challenges almost inevitably end up also requiring interdisciplinary approaches. 66, 67 the incremental moves noted above from universities and funders toward promoting more inter-and transdisciplinarity must now be accelerated and empowered in both intent and design. however, there are a number of well-recognized barriers to this-traditional mainstream pressures and incentives (e.g., university tenure based on citation counts and grant dollars or corporate instrumentality of research-delivering products); systemic problems in publication, granting, and training processes (e.g., difficulties in publishing integrated research, funding review processes that favor narrow disciplines, and underresourced student training); and disciplinary research cultures of universities that undermine the commitment to inter-and transdisciplinarity and solving real-world public problems. 68 these barriers need to be systemically addressed. change can be initiated if funders of science articulate more demand for impactful science. funders should also modify the institutional constrains to addressing challenges in the global south, where research funding is woefully inadequate and sustainability problems are also pervasive. universities where researchers are employed should modify tenure and promotion rules and reward systems to actively encourage transdisciplinary, solution-oriented sustainability science. 69 even where inter-and transdisciplinarity are encouraged, we have noted that their co-design and co-production processes can be too slow to meet the rates of change we are experiencing. this could require additional resources to speed up these social processes. many current funding mechanisms and processes do not recognize the need for these additional resources to be effective. however, we must also work harder to recognize where transdisciplinarity is really needed and where it is not. we must obtain better evidence for what works and what doesn't in multistakeholder processes so the resources can be transparently focused where they will have the best effect. a critical impact of science-related public policies occurs via funding priorities. inter-and transdisciplinarity need to be supported by a much greater investment in social science and the humanities if we are to promote an equal partnership across the disciplines. as noted earlier, this investment is miniscule in comparison with that in the natural sciences, even if these also are under-resourced. even if we don't wait for more deep interdisciplinarity, many research areas require greater attention: understanding the deeply social nature of desire and consumption as it expands globally, mapping power dynamics across world consumer demand, exploring how culture change can support global transformations, rethinking the idea of ''progress'' in western cultures, and understanding global social and cultural tipping points and other aspects of mobilizing leverage points for systems change. 8, 70 in addition to this improved investment, there should be a significant effort to improve holistic conceptual models and research practices that draw on multiple traditions. there is potential for increasing resources via government university and private-sector partnering. 71, 72 these could help to resolve epistemological conflicts among the natural sciences, social sciences, humanities, and arts. this is important both interdisciplinarily (e.g., through conceptual models that deeply support the indivisibility and universality of sustainable development as framed by the sdgs) and also transdisciplinarily (e.g., through extended engagement and experimentation with traditional knowledge systems and faith-based systems). another key incremental and ''no-regrets'' step is increased training of researchers. this encompasses building the cohort of early-career researchers comfortable with interdisciplinary and transdisciplinary processes. we should also provide researchers with the skills to engage with stakeholders, convene and facilitate multistakeholder meetings, identify community problems that need to be addressed, and communicate results to the public and to policymakers. effective co-evolution of science and society will also need to address joint training of the next generation of researchers and stakeholders so they will be able work together to meet sustainability challenges. last, we can begin to recognize reflexively that sustainability is itself an open-ended social learning process in which we all need to play a part. the notion of ''being change'' implies the need to ''walk the talk.'' sustainability scientists should be models of sustainable behavior yet also draw attention to the norms and systems that are impediments to sustainability. this means engaging in public debates and contributing to critical and reflective conversations about sustainability. drawing on the experiences described in this paper, we should engage our own organizations to practice sustainability, drive toward net-zero carbon emissions and zero waste, and deliver gender equity and better social outcomes to meet the sdgs in our institutions and communities. 49 driving global transformations to sustainability moving toward a transformational agenda for sustainability science implies not only driving the incremental change in the right direction but also being sufficiently active so that transformational interventions can take place rapidly when the opportunity arises. 73, 74 for this we suggest a number of interlinked approaches. first, building on the incremental efforts already taking place, sustainability science should be pursuing funding and other support for a large network of co-learning innovation hubs 75 in all sorts of contexts. these can be modeled in various ways (e.g., sdgs 10 and cities 76 ). they share the attributes of obtaining close engagement between research and society at a local level in many projects in combination with a strong monitoring, learning, and knowledge-exchange infrastructure that enables success stories to be learned from and scaled rapidly in ways that are sensitive to understanding the contexts in which they will or will not work. 77 this transformation of the ivory-tower model of research not only provides an innovation scaffolding for transformation in society when the opportunity arises but also creates good outcomes at the local level in the meantime. 78, 79 it also engages a much wider portion of society in discussions and learning about sustainability. second, sustainability science needs to engage in an action research mode with the high societal leverage points in such a way that an opportunity for transformation will lead these levers to be pulled in the ''right'' direction. ethically, these should be in areas where the direction is both normatively justified by the framing of goals such as the sdgs and factually justified by research and analysis. examples already noted include dramatic changes to our economic system away from narrow valuation of gdp to broader human well-being, the removal of structural barriers related to the skewed distribution of wealth and consequent inequalities, and a fundamental rejection of simplistic marketbased capitalism, all of which are underpinned by a deep realignment of values to a more collaborative, reflexive, and kinder view of humanity's stewardship of our planetary home. 52 needless to say, such changes would require supporting changes in all areas of institutional and governance design, but these are, of course, past human inventions themselves. the role of sustainability science (and scientists) here would be as activists, as envisaged in waddell et al.'s ''forcing change'' quadrant. 53 third, in addition to playing this activist role, sustainability science needs to have a greater voice in societal processes, such as international un governance discussions or discourse with global religions and traditional societies. the past attitude of scientists has tended to emphasize an autocratic expert identity. the transformed role must be as a partner among equals in seeking the normatively defined outcomes. global-change research has sought this collaborative role, for example, at the un conference on sustainable development (rio+20) and through the intergovernmental panel on climate change and ipbes assessment processes, but in the sdg development process it was still relegated to being one among many lobby groups. the un secretary general's science advisory panel is still seen as a set of experts on the outside of the political process, as are most government scientific advisory boards. this is a failing on both sides of the relationship, and it must be worked on without hubris in policy and other domains. fourth and finally, sustainability science needs to be goal oriented to pursue transformation and accept the challenging roles implied by this. in the absence of clear intentionality in the design of these systems, science is dragged along by the dominant socio-economic forces that themselves should be the subject of change. sustainability science should examine and prepare for its role in all the complementary transformation strategies noted by waddell et al. 53 -understanding when to be an activist and when to partner and being ready to distribute these tasks in a credible way. the community of researchers and their co-design partners also need to accept the goal of transforming themselves to help enable societal transformation in order to meet the sdgs and thereby set the direction for all the above points. maintaining transparency and accepting open debate while seeking sufficient consensus to move forward rapidly to achieve global sustainability and human well-being will be the hardest part of the balancing act. it is difficult to reach final conclusions to the broad critique and transformational vision for sustainability science as explored in this paper. we acknowledge that the realization of this vision could take time, yet the current global situation is challenging assumptions about incremental and transformational change. now is the opportunity for sustainability science to engage and coevolve with the underlying socio-economic roots of environmental changes. can the various branches of science afford to remain at epistemological odds with each other? can research that is driven by funding needs and competition continue to serve private corporate interests rather than the public good? research has not sufficiently emphasized how the most pressing environmental problems are caused and conditioned by social, cultural, economic, and business activities based on individualistic, competitive, profit-seeking models of economics and finance. if there has ever been a time to explore paradigm shifts that support an equitable and sustainable world, this is it. in this article, we have offered some suggestions for moving sustainability science forward on its evolutionary path. profoundly, on this path, the interlinked nature of science and society means that an overall societal commitment to sustainability (as articulated in the un agenda 2030) is a prerequisite for transforming science and society toward sustainability. for this to happen, macroeconomies would need to be designed for human and biosphere well-being rather than for financial economic growth. 80, 81 cultural values to support this economic transition would be based on a more eco-centric vision of life and organizations. [82] [83] [84] [85] [86] [87] transformations like these require a different, more impactful version of sustainability science. the research system of sustainability science itself needs to evolve rather than just seek to change everything else. this means that sustainability scientists and policymakers have to be more reflexive and challenge their own limiting assumptions related to global change. global sustainability research and practices strive to provide adequate food, water, energy, income, education, resilience, voice, jobs, health, and gender and social equity for all in an ecologically safe operating space for humanity. 88 this will require weaving together models of economic growth with models of ecosystem changes and managing winners and losers and the related changes in power dynamics. it will also require addressing the structural economic barriers that have emerged over the past century. the most important of these is wealth and income inequality. 89 currently, 50% of wealth is owned by less than 1% of the world population. two billion people still consume under $2 per day and aspire to increase it. growing inequality makes ''economic growth'' ineffective at addressing the needs of vulnerable sections of society by concentrating wealth with the rich. the co-evolution of sustainability science and sustainable societies implies increasing consumption of some and decreasing consumption of others without degrading ecosystems. the research ecosystem of universities, funding agencies, policymakers, and decision makers needs to be modified in other ways. science operations are different in each country, and there is no one-size-fits-all solution. some general actions for consideration include the following: d universities explicitly prioritize impactful research on environmental grand challenges. d universities adjust their research productivity, measurement methods, and reward systems to beyond academic measures (publications, citations, and grant dollars) to include the impact on social and sustainable development metrics. d funding agencies make more stringent and concrete demands that research should have an impact on sustainability (via the sdgs) in communities. d science communication with policymakers and with the public should be made a priority. many global environmental changes are being addressed outside academia through citizen activism, non-governmental organizations, and social movements. sustainability science could be leading some of these activities instead of merely studying or observing them. however, in order to take such leadership, researchers will need to acknowledge that science is not just a neutral objective pursuit of knowledge when it comes to sustainability. by bringing in reflexivity, values, and ethics, they can justify participation in structural and systemic changes that would produce an equitable and thriving world. scientific pursuit of knowledge involves much more than constructing accurate and analytically powerful representations of the world. knowledge should inspire people to both reflect and act. to change society for the better, we need to creatively activate and employ all available sources of human knowledge in favor of creating an equitable and sustainable earth. sustainability. planetary boundaries: guiding human development on a changing planet social acceleration. a new theory of modernity environment and development. earth system science for global sustainability: grand challenges future earth 2025 vision sets the framework for the programme's contribution to global sustainable development global change and the earth system climate tipping points -too risky to bet against tipping positive change advancing sustainability science for the sdgs the failure of business ethics the corporation: the pathological pursuit of profit and power globalization and self-regulation. the crucial role that corporate codes of conduct play in global business trajectories of the earth system in the anthropocene informal economy and the world bank women and men in the informal economy: a statistical picture raising productivity and reducing risks of household enterprises: diagnostic methodology framework the misallocation of climate research funding principles for knowledge co-production in sustainability research leverage points for sustainability transformation leverage points: places to intervene in a system the ipbes conceptual framework-connecting nature and people new materialisms: ontology, agency, and politics introducing new feminist political ecologies the greening of technology and models of innovation the archipelago of social ecology and the island of the vienna school research collaboration and team science: a state-of-the-art review and agenda a brief history of everything communicating climate change: closing the science-action gap in the oxford handbook of climate change and society the ''what'' and ''why'' of goal pursuits: human needs and the self-determination of behavior not just for the money the moral economy. why good incentives are no substitute for good citizens moral disengagement. how people do harm and live with themselves (macmillan) corporate transgressions through moral disengagement the dynamics of social practice: everyday life and how it changes (sage) rhetorics, literacies, and narratives of sustainability the dream of the earth earthmasters: the dawn of the age of climate engineering restoring our senses, restoring the earth. fostering imaginative capacities through the arts for envisioning climate transformations hand / heart / head aesthetic practice pedagogy for deep sustainability learning hope and grief in the anthropocene: re-conceptualising human-nature relations (routledge) is the 1.5 c target possible? exploring the three spheres of transformation on matryoshkas and meaning-making: understanding the plasticity of climate change climate and society. transforming the future the sage handbook of action research (sage) theory u: leading from the future as it emerges the campus as a living laboratory: using the built environment to revitalize college education (aacc seed center) laudato si': on care for our common home (vatican) the economy of francesco come on! capitalism, short-termism large system change: an emerging field of transformation and transitions radical transformational leadership: strategic action for change agents four strategies for large systems change resilience thinking: integrating resilience, adaptability and transformability informing adaptation responses to climate change through theories of transformation three horizons: a pathways practice for transformation smart thinking for crazy times: the art of solving the right problems the multi-level perspective on sustainability transitions: responses to seven criticisms societal transformation in response to global environmental change: a review of emerging concepts transformation in a changing climate: a research agenda scaling out, scaling up, scaling deep: strategies of non-profits in advancing systemic social innovation emerging new civilization initiative (enci): emergence from emergency bright spots: seeds of a good anthropocene new organizations, old cultures: strategy and implementation of interdisciplinary programs crcs and transdisciplinary research: what are the implications for science? bridging barriers to advance global sustainability positive tipping points in a rapidly warming world universities and the global knowledge economy: a triple helix of university-industry-government relations the new production of knowledge: the dynamics of science and research in contemporary societies (sage) sustainability transitions: an emerging field of research and its prospects innovation studies and sustainability transitions: the allure of the multi-level perspective and its challenges social innovation lab guide sustainable cities and regions (future earth australia and australian academy of sciences) eco-innovation and sustainability management (routledge) managing innovation for sustainability: the challenge of integration and scale managing innovation for sustainability a finer future: creating an economy in service to life prosperity without growth. economics for a finite planet (routledge) towards a steady-state economy (uk sustainable development commission) for the common good: redirecting the economy toward community, the environment, and a sustainable future ecological economics for the anthropocene stewarding sustainability transformations. an emerging theory and practice of sdg implementation ecocentric management for a risk society enlivenment (boll foundation) doughnut economics: seven ways to think like a 21st-century economist capital in the twentieth century key: cord-024329-r8mi42fu authors: tarasiev, andrey; filippova, margarita; aksyonov, konstantin; aksyonova, olga; antonova, anna title: using of open-source technologies for the design and development of a speech processing system based on stemming methods date: 2020-05-05 journal: open source systems doi: 10.1007/978-3-030-47240-5_10 sha: doc_id: 24329 cord_uid: r8mi42fu this article discusses the idea of developing an intelligent and customizable automated system for real-time text and voice dialogs with the user. this system can be used for almost any subject area, for example, to create an automated robot a call center operator or smart chat bots, assistants, and so on. this article presents the developed flexible architecture of the proposed system. the system has many independent submodules. these modules work as interacting microservices and use several speech recognition schemes, including a decision support submodule, third-party speech recognition systems and a post-processing subsystem. in this paper, the post-processing module of the recognized text is presented in detail on the example of russian and english dictionary models. the proposed submodule also uses several processing steps, including the use of various stemming methods, the use of word stop-lists or other lexical structures, the use of stochastic keyword ranking using a weight table, etc. at a present day technologies related to automated control processing systems are rapidly developing. as a part of these modern trends in the development of information technology the creation of effective and technologically advanced solutions for organizing the processing of incoming requests is required. data processing technologies are progressively evolving, with more and more systems replacing human resources every day. automation and the creation of information systems are at the moment the most promising areas of activity of modern society. one of the reasons for the active development of these areas is that automation is the basis for a fundamental change in management processes that play an important role in the activities of companies. thus, there is a need to use a control system, whose operation is aimed at maintaining and improving the operation of the all business processes at all. as an important part of this area an automation of call centers working process require the help of a control device (complex of means for collecting, processing, transmitting information, and generating control signals or commands). the module of speech recognition in this system uses the two most developed at the moment existing solutions yandexspeechkit and googlespeech api. but integration with these third-party services and other related solutions can't provide complete accordance with system requirements including features, adequate recognition quality, scalability, flexibility and so on. as previous part of this work the testing of the speech recognition systems using russian language models including selecting and testing a speech recognition systems and designing architecture of the "twin" automation system was conducted [5] . based on the received information, we can conclude that the yandex system is good at recognizing short expressive phrases, as well as numerals. on contrary, the google api is good at recognizing long phrases and terms. the results of this research are being used in development of the automatic system that calls the customers of "twin" [1, 2, 4] . at the same time, both systems have problems with recognition in terms of noise, uneasy speech tempo and voice defects of the interlocutor. development experience of answer and question automated systems are described in some works: based on frame approach [9] , based on agent approach [10, 11, 15] and conceptual graphs [12] [13] [14] . this is due to the fact that both systems better recognize phonemes and phrases, and individual sounds are worse, especially if there are noises and other factors that distort the quality of the transmitted audio message. this observation is confirmed by the findings obtained by other independent researchers [3] . to eliminate such problems, it is necessary to include pre-and post-processing. these actions can provide requirements of recognition quality. pre-processing uses the dynamic selection of recognition services provided. subsequent processing of the received textual information includes the lemmatization and normalization of individual words in the recognized speech and other methods. for example, in the case of processing numbers, this is the application of tokenization technology. also in previous part of this work the research and selection of methods for evaluating information about part of speech, word form and statistical relationships between words have been done. as the conclusion of this research we can claim that the lemmatization method is most effective for solving the problems of dynamic text processing and recognition. however, to the same extent, this approach is extremely dependent on the exact definition of a part of speech (the lexical category of a word) and the language model. application of this algorithm leads to the fact that the analyzer module receives an array of words in primary form, due to which it is possible to easily recognize part of speech using the rules of the language model. however, this information in practice may not be enough for the further work of the decision-making module and generating a response. in addition, there is the possibility of recognition errors associated with incorrect recognition of the lexical category of a word and its initial form. to obtain a more accurate recognition and processing result, further post-processing mechanisms are used. the implementation of the lemmatization mechanism in the system was performed through some open-source package solutions of morphological processing and lemmatization for the php language. within the framework of this work, the design and development of an automated system named "twin" was carried out and details presents module of recognized text post-processing on the example of the russian and english languages dictionary models. since the approach of reducing the words to normal form and determining the parts of speech is insufficient for the voice robot to understand the interlocutor's speech and for further decision making, the system uses post-processing algorithms for normalized words. this is due to the presence of a number of problems, inaccuracies and recognition errors. in addition, the lemmatization method can occur recognition errors since the lexical category of a word can be incorrectly determined. there is also a big problem with the correct recognition of numerals, which leads to difficulty to identifying numbers, dates, addresses, etc. to solve these problems, several additional processing algorithms are used. many researchers provide several methods to improve quality of natural language processing based on stemming methods. when building the speech recognition module for voice system, we were guided for the most part not by coming up with conceptually new algorithms, but by focusing on the use and adaptation of existing and well-established approaches. the most often usable method based on implementation of a large vocabulary speech recognition system for the inflectional languages such as slavic group languages [8] . based on this approach the following method for additional processing was proposed. conventionally, we can distinguish the following steps of the additional processing of the second stage. • primary processing; • introduction of a keyword system and the use of stochastic keyword ranking; • introduction of a word stop-list system. decisions on the basic additional processing of normalized words are determined on the basis of the given settings and information on the lexical category of the word (part of speech) as a preparatory stage for more serious text analysis methods. the essence of the primary processing of the second stage of text analysis is the use of simple algorithms for automated morphological analysis. to increase the accuracy of recognition of numerals, the information contains numerals goes through the tokenization procedure. also, the system provides for the adjustment of language models, which allows you to customize the perception of numerals, dates, cases, as well as the need to use keywords. in addition, it is possible to configure the methods used for stemming. the introduction of a keyword system allows for significantly more effective recognition of language units. firstly, this is due to the fact that keywords can be tied to specific blocks of the dialogue script, which largely solves the problems associated with the context. this can be useful, for example, when processing paronyms. when creating a script in a special interface, creator of the scenario can set keywords for each person's answer. this dialogue scenario consists of various information blocks, most of which involve the interlocutor's response or expectation of a phrase. moreover, depending on the frequency of mentioning of certain words and phrases, further training takes place. the system, each time making a decision, increases the weight of a certain keyword in the expert table of stochastic data. thus, the likelihood of a particular word appearing during further launches of this dialogue scenario increases depending on the frequency of its appearance and correct recognition earlier. the processed text is transferred to the block of the analyzer of coincidence with keywords. at the same time, both processed normalized words and initially recognized words are transmitted to the system. keywords themselves can be set intentionally in various forms. due to these two conditions, the recognition quality can be significantly improved, and the table of knowledge base weights for the stochastic ranking method will be more trained. a similar approach is widespread in systems using stochastic knowledge bases. in addition, this makes it easy to solve the potential problem of the loss or distortion of the meaning of either a single word or the phrase itself as a whole. that is, if several variants of the same word are specified in the system for a certain block of the script, and both coincide with the original and lemmatized recognized words, then the probability weights for both keywords will significantly increase, and the weight of the script branch will also increase by the weight of both keywords. by default, the conditional weight of each keyword and each branch of the dialogue at the initial moment of execution time of a script is equal to one. when the lemmatized word coincides with the originally recognized word with the keywords, the weight of each keyword increases by one. in this case, the weight of the branch increases by two units. if only the keyword denoting the initial form and the lemmatized language unit coincide, then the weight of the script dialog branch increases by one. based on the dynamically calculated weights of each branch, at every moment a decision is made on the further development of the dialogue scenario taking into account the largest branch weight. this approach allows the author of the script to work out the dialogue features important for the script, which are influenced by the degree and conjugation of the verb, etc. as already mentioned, in addition to setting keywords the system provides for the use of tables of stochastic ranking of words by probability depending on the frequency of their appearance and correct recognition in dialogs. of course, to ensure the correct operation of such a system, it is necessary to implement and maintain a knowledge base. it is worth noting that this storage takes up much less volume than other knowledge base implementations for stochastic stemming methods would occupy. to ensure dynamic interaction, in the system it is implemented as a base tree. the use of the stochastic method is primarily due to the fact that an approach that implements only lemmatization algorithms is not able to provide sufficient reliability for correct speech recognition and decision making, since it places great responsibility on the system operator itself, which may be mistaken due to the human factor or not take into account all the options, which can have a big impact on the course of the dialogue. similar to using the keyword system in a word processing module, stop words are also used. their semantic load is similar to keywords. the essence of this idea is to strictly discard branches when the recognized word coincides with the stop word. this approach allows us to simplify the writing of some dialogue scenarios by providing the user with the opportunity to work with negation and exceptional situations, or to write down fewer keywords and work out less on premediating of potential scenarios. it also allows to speed up the processing of text, due to the fact that some cases can be immediately discarded and not be considered in the future. according to the implementation method, the process of recognizing stop words is practically no different from keywords. a lemmatized word and a primary word are transmitted to the handler in their original form recognized by an external open-source morphy system. these words are compared with stop words. if a match is found, the weight of the stop word in the knowledge base of stochastic rules increases, and the weight of the branch decreases to the minimum possible. thus, during the execution of the further procedure for selecting a scenario vector, this branch cannot be selected in any way. after applying all the rules and treatments, the search for a solution begins in accordance with the rules specified in the script, after which a response is generated. based on everything described earlier, a simplified process of additional processing of a phrase recognized by third-party systems occurs as follows: at the first step, the text recognized by a third-party system is transmitted to the post-processing module. this module analyzes this text in several stages. first of all, the text goes through the procedure of splitting into separate linguistic units -words and statistical grouping by phrases. each word is lemmatized, as a result of which the word in the initial form is returned at the output of the work of the stemming algorithms, and the lexical category of the word is also determined. next, the second stage of processing begins. based on the part of speech defined in the previous step, initial processing is performed. in this case, the rules defined when setting up language models are used, such as processing numerals, tokenization, processing dates, cases for nouns, etc. the processed words and words in their original form, recognized by third-party systems, are transferred to the next data processor, which deals with their comparison with keywords and stop words, which are pre-introduced to the system for the corresponding blocks of the dialogue script. if the words transmitted to the input of the algorithm coincide with the ones specified in the system, the statistical weights of the keywords and corresponding branches of the script increase. the weights of the words specified in the system are recorded in the production knowledge base of stochastic rules. thus, the ranking of words is provided and the model is trained. the more often these or other words and phrases are used when executing a script during dialogs, the more likely it is the appearance of these words in these forms with other passages of this scenario. accordingly, over time, these words will be more correctly recognized. finally, based on which branch, illustrating the possible choice of further development of the dialogue, is gaining the greatest weight, a decision is made and a response is generated. this process can be visualized using the following sequence diagram (see fig. 1 ). we are considering options for highlighting our own developments, such as a system of keywords, into separate freely distributed solutions. the implementation of the lemmatization mechanism in the system was performed through an open-source package solution of morphological processing and lemmatization for the php language -php morphy, which contains the corresponding knowledge bases -dictionaries for various language models. this solution easily integrates with the used development tool stack. this library supports aot and myspell project dictionaries. for the library to work, you need to compile a binary dictionary. when compiling, the dictionary is used in its original form. physically, the dictionary is presented in the form of several files, which are a data store in a key-value format, as well as storage of reference lexical information, grammatical information, end tables, grammes, etc. other word processing packages for processing declension cases, such as petrovitch library also are used. this package also allows to gender determination and develop and apply own linguistic rules. it should be noted that using of open-source systems in not limited by mentioned solutions. flexibility of proposed application and modern technologies itself allows to simple scaling or replacing third-party modules or adding new. moreover, proposed dialogue systems use other open-source packages for different modules that are not limited to post-processing speech recognition module. for example, we use rasa core module for speech-analyze subsystem and chat-bots. mentioned speech-analyze subsystem using as a part of customizable script. open-source solutions provide ability to integrate various implementations of exists methods, such as stemming text recognition methods in proposed case, with each other and own development. due to additional processing, the correct perception of speech and the meaning of what has been said and decision-making occurs in developing system. the development of the system involves the development and implementation of additional functions, such as the sending of statistical data and the creation of hints in the compilation of the script. to do this exists open-source solutions also can be used. analysis of internal statistics will help determine the priority directions for improving the interface and system features. the range of use of the system can be extended, due to the initial flexibility of the structure and wide ability to integrate with third-party solutions, packages and so on. it should be noted that proposed system twin is used in teaching intellectual technologies at the ural federal university on a free basis. we are considering options for highlighting our own developments, such as a system of keywords, into separate freely distributed solutions. testing of the speech recognition systems using russian language models development of decision making software agent for efficiency indicators system of it-specialists the study of the reliability of speech recognition by the system google voice search. cyberleninka.ru development of an intelligent automated system for dialogue and decision-making in real time on the use of acoustic characteristics of speech for the diagnosis of mental states of a person recognition of human emotions by spoken using intelligent data analysis methods large vocabulary continuous speech recognition of an inflected language using stems a framework for representing knowledge in the psychology of computer vision guest editors' introduction: agents and markets computationals-mechanism design: a call to arms knowledge representation: logical, philosophical, and computational foundations conceptual graphs for a database interface conceptual structures: information processing in mind and machine multiagent interaction models for constructing the needs-andmeans networks in open systems the work was supported by act 211 government of the russian federation, contract no. 02.a03.21.0006. key: cord-017841-57rm046y authors: flower, darren r.; perrie, yvonne title: immunomic discovery of adjuvants, delivery systems, and candidate subunit vaccines: a brief introduction date: 2012-09-28 journal: immunomic discovery of adjuvants and candidate subunit vaccines doi: 10.1007/978-1-4614-5070-2_1 sha: doc_id: 17841 cord_uid: 57rm046y mass vaccination, when coupled to profound improvements in general sanitation, has given rise to the most remarkable transformation in public health in human history. yet the development of vaccines remains largely trapped in the past, a hostage to the methodology of pasteur. infectious disease continues to threaten humanity, with new and renascent diseases emerging continually. the last two decades have seen a breath-taking revival in the commercial market for vaccines and the simultaneous emergence of a whole tranche of new technologies that promise to free vaccine development from the muddle of empirical thinking. in this short introduction, we set the scene for this renaissance, and explore how the combination of computational and experimental techniques promise so much for the future development of vaccines and the science of vaccinology. vaccination and sanitation are indisputably the most efficient, and thus costeffective, prophylactic treatments for infectious disease. together they are the firm bedrock upon which the modern world resides. the truth of this is often ignored by those intoxicated by the many distractions of life in the early twenty-first century, from burgeoning social media to the recondite discoveries of the large hadron collider. people say that the internet or the ipad or even facebook have transformed the world. if there is any truth in such assertions, then such transformations are at best shallow and superficial compared to the extraordinary reworking of lives that has transpired over hundreds rather than tens of years. the source of such change can be traced back, in part at least, to the discovery and exploitation of vaccines, vaccination, and vaccinology. for around the first 100 years of the vaccine story, the story was solely that of smallpox. as recently as the late 1960s, some 10-12 million cases of smallpox were recorded in 31 countries, with annual deaths of 2 million. yet today smallpox has, with the exception of a few well-guarded stockpiles, been completely eradicated: there have been no new cases for 30 years. the story of smallpox is thus the high point of the vaccination story; no other disease has been eradicated. polio or poliomyelitis is the next nearest to full eradication, having long been targeted by a systematic, coordinated, worldwide eradication campaign. in 1991, one such programme run by the pan american health organization effected a partial eradication of polio in the western hemisphere. subsequently, the global polio eradication program has radically reduced polio throughout the rest of the world, so that today we can count cases worldwide in the tens or hundreds instead of the hundreds-of-thousands or millions. yet, vaccine-preventable disease still kills millions. infectious and contagious disease cause approximately 25 % of world mortality, particularly in children under five. while in developed countries, mortality for diseases such as diphtheria, polio, or measles is less than 0.1 %, in other parts of the world deaths from such infectious diseases is significant. pertussis, tetanus, influenza, hib, hepatitis b are all responsible for deaths that number in the hundreds-of-thousands. perhaps the most execrable situation is measles, which accounts for 70,000 (over 5) and 540,000 (under 5) deaths. however, the leading global causes of death worldwide remain tuberculosis; diarrhoeal illnesses, especially rotaviruses; hiv/aids; and malaria. in 2010, 8.8 million contracted tb and 1.4 million died. disturbing though these numbers may seem, they nonetheless represent a significant reversal of a once ever-escalating trend. the number with latent tb peaked in 2005 at 9 million, while deaths from tb reached their peak at 1.8 million in 2003. however, these bald numbers are likely to be significant underestimates. let us also look at malaria. murray et al. have recently provided evidence that deaths from malaria over the thirty-year span to 2010 are much higher than previously believed [1] . their epidemiological figures show a peaked distribution over this period, increasing from around a million in 1980, peaking at approximately 1,820,000 in 2004, and then reducing to about 1,240,000 in 2010, with the greatest number dying in africa. these figures are roughly twice the values published by the who. it seems unlikely that the who's estimates for other major diseases are uniformly more accurate. why are these numbers so high? a principal reason is that there are no effective vaccines for either malaria or hiv, two of the who's big three diseases; nor is there expectation that such vaccines will appear in the near future, irrespective of the optimism of those working in the area. and as for tuberculosis-which is carried by around 2 billion people worldwide-the only licensed vaccine has limited efficacy. many viral infections remain recalcitrant threats of the first order. about 350 million people are infected with hepatitis b, 170 million by hepatitis c, and 40 million by human immunodeficiency virus type 1 (hiv-1). this dire situation is further compounded by the threat from the 35 new or previously unknown infectious diseases identified in the past 30 years: hiv, west nile fever, ebola, dengue fever, sars, and the potentially pandemic h5n1 influenza. every year, between 5 and 15 % of the global population becomes infected with a new influenza strain, causing upwards of half-a-million deaths. it is widely thought that there will be a continual emergence of new infectious diseases during the present century: emerging zoonotic infections and antibiotic-resistant bacteria prominent amongst them. in the face of declining birth rates, coupled to decades of ever-enhancing nutrition, advances in treatment regimens and medicines are leading the population of most developed and developing countries figuratively to age: that is for larger and larger proportions of the population to live to maturity and beyond. growth in life expectancy is matched by growth in the diseases of old age. these include neurodegenerative diseases, principally parkinson's or alzheimer's disease; cardiovascular diseases; and stroke. disease has altered significantly in the preceding century. it will continue to alter during the century to come. some alterations we can predict; others will escape the forecaster's eye. disease, particularly infectious disease, has been beaten, or, at least, severely restrained. many factors have conspired to effect this-improved water quality, better precautionary hygiene, improved nutrition, decreased overcrowding-as well as many interventionary measures, principally antibiotic therapy and vaccines. hitherto, vaccines have been an uncompromising success, yet, as we see, so much more needs to be done if the full potential of vaccines is to be achieved. although the licensing and use of vaccines varies between countries, 25-30 commonly licensed vaccines target a range of viral or bacterial infectious diseases, with approximately 14 paediatric diseases targeted during the first few years of life. other than paediatric vaccination, most vaccines are used by travellers to tropical or subtropical regions; a significant minority fight infection in the developing world. vaccination also works to greatly reduce the morbidity of disease, often imbuing lifetime protection; this is particularly important for benign yet economically important infections, such as the so-called common cold. diverse sporadic or epidemic infections of the human respiratory track-as caused by an excess of 200 distinct viruses, such as rsv or, more properly, respiratory syncytial virus, coronaviruses, influenza a and b, rhinoviruses, parainfluenza virus, and cytomegalovirus-remain a principal cause of hospitalisation and community morbidity with an estimated 60 % of gp referrals associated with such infections, and cause the loss of enormous numbers of working days in developed countries. given the recalcitrance, and the immense investment in treatment and prophylaxis, it may be that many of the diseases alluded to above will never be eradicated, as smallpox was, and that vaccines alone will not be enough. we may instead need a complex network of prophylactic and therapeutic measures at least as complex as the diseases themselves in order to effectively reduce the prevalence of the disease and to treat those who become infected. whatever other countermeasures we may have recourse to-artemisinin-based drugs, genetically manipulated vectors, or insecticide-treated bednets-the clinical and cost effectiveness of vaccines means they remain the must-have component in the ongoing search for better means of combating endemic infectious disease. beyond infectious disease lies what is possibly the most underexplored area within the ever burgeoning field of vaccinology and vaccination: vaccines against allergy and allergic disease; vaccines that target so-called lifestyle diseases, such as those deriving from addiction; and vaccines that target chronic diseases, the most important of which is cancer. therapeutic vaccines against cancer are probably the best studied amongst the more novel, innovative, and underexplored areas at the forefront of vaccine discovery. the present overall whole-life risk from cancer, at least in developed countries, runs at or about 40 %. the figure is currently rising. there are approximately 3·2 million new cancer cases in europe each year, resulting in around 1·7 million deaths. in the usa, over one and half million new cases are reported annually. clearly, this is an important disease burden, and thus a key target for the pharmaceutical and biotechnology industries. lifestyle vaccines are another innovation, of which much is expected in certain quarters. they target all kinds of medical problems, ranging from drug addiction through dental caries, all the way to major genetic and multifactorial diseases, including obesity. versatility and flexibility are major hallmarks of the vaccination concept. vaccines take many forms and work in many ways. this facet has been exploited in the development of life-style vaccines. let us look at serious addiction. during 2009, the united nations office on drugs and crime estimated 3.3-6.1 % of the global population abused so-called illicit substances-a serious problem indeed with an enormous implicit health, economic, and behavioural burden, with the worst excesses coming from cannabis abuse and the abuse of amphetamine, cocaine, and opiates. anti-drug vaccines generate antibodies able to bind particular drugs; the drug-antibody complexes thus generated should have too high a molecular weight to penetrate the blood-brain barrier effectively, thus reducing the amount and rate of drug egress into the brain and so inhibiting psychoactive effects at the system level. anti-addiction vaccines have been with us for some time, beginning over 40 years ago, when two proof-of-principle studies [2, 3] demonstrated in rats and in rhesus monkeys that morphine could be used as a hapten in order to create an antibody against morphine addiction. today, addiction vaccines are being developed to target a range of major abused drugs, such as nicotine, cocaine, various amphetamines, and heroin. anti-allergy vaccination also offers great potential for successful commercial exploitation. the prophylaxis and treatment of allergy can now be addressed in many ways, including, notably, recombinant proteins and dna vaccines. vaccines against the common cold or anti-allergy vaccines are similar in mechanism to many lifestyle vaccines. these do not save lives directly but do help to greatly reduce the vast economic burden of disease morbidity. an array of interconnecting factors that have made the pharmaceutical and biotechnology industries re-evaluate the potential of vaccines as a commercially viable product. prior to 1980, there were relatively few vaccines, most targeting major pandemic diseases of the developed or developing worlds. subsequently, partly as a result of enhanced technology as discussed at length in the current book, many vaccines have become available, most recently the cervical papillomavirus vaccine. likewise, there are hundreds upon hundreds of vaccines in trials. the growth rate in the sales of vaccines reflects this feverish and febrile activity: the rate of sales growth for vaccines is something like 16 %, compared to the sluggish drugs market, meandering its desultory way at 4 % per annum. there is profit in vaccines, clearly; what remains problematic for the profit-driven decision-making processes of big pharma is the haphazard and probabilistic nature of vaccine discovery. what the pharmaceutical industry needs is the capacity to apply the same systematic, automated, high-technology approaches used to identify new small-molecule drugs to the discovery and development of vaccines. no right-minded scientist, looking back across the last 200 years, would wish to argue seriously with the contention that the design and development of vaccines is an innately labour-intensive process. the processes deployed to meet the objective of creating new and better vaccines are in desperate need of change. this change must be radical if we hope to simplify such processes. simple processes are hopefully also fast and efficient processes. in the search for subunit vaccine antigens, one technical development-reverse vaccinology-has proved the most profound and hopeful. just over a decade ago, rino rappuoli used the expression "reverse vaccinology" to describe development of vaccines using a genomic-based approach, rather than the ponderous empirical methods favoured then, and still in use today. reverse vaccinology seems about to deliver on its early potential: the european medicines agency is in the process of evaluating novartis's bexsero, the first commercial vaccine developed using the reverse vaccinology approach. the vaccine may become the first vaccine effectively to combat meningococcus b, a disease causing over 50 % of global meningococcal meningitis. a decision on bexsero is expected shortly. during the development of bexsero, new protective protein antigens were identified using genomics: initially over 600 surface-exposed proteins were predicted from the n. meningitidis proteome as molecules liable to host immune surveillance, of which about 350 were then expressed in e. coli. this number was reduced by using these proteins to immunize 350 sets of mice, identifying 91 that could induce antibodies in vivo, 29 of which killed n. meningitidis in vitro. by comparing the genomes of 31 clinical strains, a further subset of proteins offering broad protection could be identified. reverse vaccinology has become perhaps the most famous well-developed approach amongst many advanced approaches now available within the discipline of vaccinology. indeed, a whole range of other, high-technology methods and techniques have been and are being developed to complement and optimise reverse vaccinology. capitalising on its success, these offer new hope in our constant struggle with infection; all we need is for this technology to be fostered, developed, and utilised. this book is intended to fill a gap, if not a void, in current thinking within vaccine design and development by attempting to draw together several disparate strands; and, by doing so, also identify and illuminate some important areas replete with potential. science, in much the same way that all human activities, from the most profound to the most trivial, follows fashion and progresses by tracking trends. whether we think of publically funded science or the pharmaceutical industry, similar phenomena are observed. science follows the money, and money follows consensus. the decision-making process underpinning the strategic direction that policy in both publically funded science and the pharmaceutical industry takes is only in part influenced by science. it is also regrettably in thrall to many, sometimes contradictory, voices: the fickleness of public opinion, vested interests of many hues and flavours, and the myopia of the profit margin, amongst many others. this is because the decision-makers in such organisations are seldom if ever scientists engaged in doing science directly; management and policy, at both the strategic and tactical levels, are often swayed by the prevalence of opinion. in the pharmaceutical industry, for instance, this is manifest as the next big thing: combinatorial libraries, genomics, high-throughput screening, antisense, even molecular modelling; all were hailed as transformative saviours that would remove happenstance and unpredictability from drug discovery-yet as the current parlous state of the pharmaceutical industry readily attests, while all promised much, none really delivered. no single technique can achieve everything, which is why we should always develop a large range of alternatives, both informaticsbased and experimental, all running in parallel. central to computational immunology is the capacity to make accurate predictions. yet, obtaining routes to prediction that are accurate, robust, and dependable continually eludes us. immunoinformatics deals with empirical, datadependent methods. the success and utility of such methods depends very much on the data used to propagate and parameterise them; they cannot escape the severe limitations imposed by the data used to create them. the data from which we build models forms a complex phase space of structural and property variation, which can be extremely multidimensional, with a high degree of interdimensional correlation. when the data we work with is reliable and our knowledge of it is complete, then we can create useful models by applying standard methods from computer science to build accurate and predictive models relating observed biological activity to underlying measurable or predictable properties. usually, such approaches are also much superior when used to interpolate than they are when used to extrapolate. we need complete and thorough data sets effectively and efficiently able to explore the complex relationships between structure and function, necessitating continuous improvement in all aspects of data quality. within the wider context of vaccine design and discovery, we shall in this book describe and explore a range of key alternative strategies and technologies, both informatics-based and experimental, which are, by degrees, both supportive and complementary to reverse vaccinology and more traditional approaches to vaccine discovery. this book looks in turn at reverse vaccinology and the identification of putative candidate antigens, at the discovery of a wide range of different types of adjuvants, and finally at the development of sophisticated new delivery mechanisms, such as liposomes and other applications of nanotechnology. in chap. 2, cafardi et al. review the present state of play with respect to reverse vaccinology, with particular emphasis on how completion of bacterial genomes impinges upon the vaccine discovery. they show how this approach allows the development vaccines that are difficult or near impossible to address with conventional approaches. they also highlight how advances in genome-based techniques and in so-called next-generation sequencing approaches and technologies will help to enhance reverse vaccinology, enabling timely identification of novel candidate antigens for new, emerging, or recrudescent infectious diseases. in chap. 3, flower et al. review the discovery of candidate vaccine antigens in more detail. placing their analysis in the context of emerging ideas about the possible nature of immunogenicity and how it may be propagated by elements of the immune response at the system level, the authors discuss the three main approaches to the identification of novel immunogenic antigens: sequence similarity-based approaches, whereby the antigen nature of a protein is inherited from similar sequences; methods based on identifying the subcellular location of microbial proteins, on the basis that proteins with only certain locations would be accessible to immune surveillance; and the use of empirical alignment-independent approaches to the prediction of antigens. usefully, the chapter also includes discussion of expert systems for antigen discovery. in chap. 4, vordermeier et al. review how genomics and the development of bioinformatics have radically transformed the cattle vaccinology of bovine tuberculosis. within the context of a generalised infrastructure of bioinformatic analytical techniques, the authors describe in detail how the application of comparative in silico transcriptome and genome analysis is able to undertake prospective prioritisation of immunogenic antigens for experimental testing, leading to the identification of candidate subunit vaccines. in chap. 5, he explores the use of epitope-focused immunoinformatic analysis in the prediction of optimal vaccine candidates when undertaking a genome-wide reverse vaccinology exercise. specifically, he describes the web-server vaxign, concentrating on a case study: vaccine design against the virulent bacterium francisella tularensis, where 12 candidates were chosen using a combination of pertinent selection criteria. in chap. 6, dhillon et al. offer us a wide-ranging review of methods and strategies for two important areas of immunoinformatic analysis within the domain of vaccine discovery: predicting the immunogenic subcellular location of microbial proteins and identifying proteins encoded by so-called genomic islands. while in chap. 7, ansari et al. describe a variety of database systems that facilitate immunoinformatics and antigen selection. chapters 8 and 9 look at adjuvants and their discovery. in chap. 8, edwards describes the basis of adjuvant action, and the role played by macromolecular adjuvants. in chap. 9, flower explores and examines different varieties of molecular adjuvant and their discovery, concentrating on small molecule adjuvants, and their systematic identification using virtual screening technology. this topic is put into context by a thorough review of extant adjuvants, molecular mechanisms of adjuvant action, as well as macromolecular adjuvants and how various adjuvants engage pattern recognition receptors of the innate immune system. in addition to the characteristics of the antigen and the adjuvant independently, how the antigen and adjuvant are presented to the immune system has a major impact on the biological output of the vaccine. indeed, the co-delivery and continued association of antigen and adjuvant may be a prerequisite in effective immunisation. this is not a new idea; the ability of alum to promote an antigen depot effect at the site of action has been ascribed as one of its main mechanisms of action for several years. for example, since 1977 the who has recommended that over 80 % of diphtheria toxoid needs to be adsorbed to alum for its effective use [4] . whilst this does not ensure that the antigen remains adsorbed to alum after injection and exposure to interstitial fluid, it is thought to at least initially promote colocation of the antigen with the alum adjuvant. however, alum is not the only adjuvant able to promote the co-delivery of antigens and adjuvants in one system; a range of particulate delivery systems can offer this. examples of such delivery systems include lipid-based systems (e.g., liposomes, niosomes, iscoms) and microparticles. each of these systems offer a suite of advantages and disadvantages and when considering the choice of delivery system attributes including antigenloading capacity, antigen retention and protection both on storage and within the biological milieu, and the ability to incorporate adjuvants within the delivery system all require optimisation and this is without consideration of the ability of the delivery system to act as an adjuvant in its own right. in this book we aim to address these issues by considering several of the most commonly employed particulate vaccine delivery systems. out of these particulate delivery systems liposomes are one of the most established systems; liposomes were first reported as an effective immunological adjuvant for diphtheria toxoids by allison and gregoriadis in 1974 [5] . since then, a large array of understanding on their design has been gathered and strong links between their formulation and function identified. of these parameters, the composition of the liposomes will play a pivotal role. for example, the surface charge of liposomes used for vaccine delivery can influence the interactions between liposomes and protein antigens, and affect how liposomes interact with cells. this manipulation of surface charge can range from the inclusion of anionic lipids such as phosphatidylserine, which may facilitate the targeting of antigen presenting cells through interaction with phosphatidylserine receptors. alternatively, the use of cationic lipids can improve the loading of anionic antigens to the liposomes and upon injection promote a depot effect at the site of injection, promoting the codelivery of antigen and adjuvant to dendritic cells (e.g., [6, 7] ). however, this depot effect is more than electrostatically driven, with the choice of cationic lipids used in the formulation having an impact as explored in chap. 10. in addition to liposomes, there are a range of alternative surfactant-based delivery systems, such as niosomes. these are similar in many ways to liposomes; however, non-ionic surfactants form the main component of their bilayers. the most common composition of niosomes investigated for vaccine delivery is 1-monopalmitoyl glycerol, cholesterol, and dicetyl phosphate. one might argue this is not a niosome formulation due to the inclusion of anionic dicetyl phosphate. however, the addition of charged surfactants has proven to enhance the stability of these vesicles and their inclusion in these vesicles is common practice. generally, niosomes exhibit many similarities to liposomes; however, potential advantages cited include their lack of predisposition to oxidative degradation, as well potential lower cost of components and reduced variability compared to natural phospholipids. whilst with current manufacturing methods, the cost and reproducibility of phospholipids is less of an issue, niosomes still offer a useful alternative to liposome formulations. in particular, niosomes appear an attractive option for oral delivery of vaccines due to their ability to withstand the harsh gastrointestinal environment as outlined in chap. 11. with both liposomes and niosomes, immunostimulatory agents can be easily incorporated within the system and in some cases this can result in restructuring of the particulate delivery system as is the case with iscoms (chap. 12). iscoms are prepared from a mixture of phospholipid, cholesterol, and a saponin (often quil a). whilst a phospholipid/cholesterol mixture would normally form liposomes, the addition of appropriate concentrations of saponin to the mixture can result in restructuring of the system to form spherical, open, cage-like structures around 40 nm in size, as nicely shown in chap. 12. given that their structures are open, iscoms cannot incorporate hydrophilic antigens, and antigens need to display a degree of lipophilicity for inclusion into iscoms. if required, antigens can be modified through a range of methods to incorporate lipophilic regions within their structure, thereby promoting their incorporation into the structure. alternatively, similar to cationic liposomes, cationic iscoms (where cationic components are used to build their structure) can electrostatically bind a range of anionic antigens and enhance their delivery. in chap. 12 the formulation, preparation, and application of iscoms as vaccine adjuvants is considered. however, lipid-based systems are only one group of particulate delivery systems, and polymeric systems have also been extensively studied. in particular the use of biodegradable polymers to formulate nano-and microparticulate delivery systems for vaccines has been widely investigated. much like the lipid-based systems, there is a wide selection of options to consider in the formulation of polymeric nanoparticles and microspheres for vaccine delivery, with polyester polymers offering advantages due to their clinical approved use in a range of medical products. as with the other systems considered, immunostimulatory agents can be incorporated within these polymer constructs and thus these systems can act as delivery systems and adjuvants for antigens. within chap. 13, the design of polymeric microspheres as vaccine adjuvants is considered from the choice of base polymer, through to optimisation of process parameters, and finally to considerations of their stability as a product. whilst much of current research into the development of vaccines has focused on the design of vaccines for administration of a particulate suspension, dry powder vaccines may hold considerable advantages. in chap. 14, the authors consider the design of dry powder vaccines. such vaccines may offer low-cost, temperaturestable products suitable for pulmonary delivery, with the added advantage that the pulmonary route avoids the use of needles (and their associated risks). furthermore, it can allow for the effective delivery of antigens to target cells of the immune system without the harsh conditions faced by orally delivered vaccines. the development of spray-drying methods outlined in chap. 14 supports the ability of such powder vaccines to be delivered using conventional dry powder inhalers already clinically licensed for pulmonary delivery. therefore, by considering these vaccine delivery platforms in conjunction with the appropriate choices for antigen and adjuvant it is hoped that the threefold multicomponent nature of a vaccine can be considered more completely than before. when viewed conceptually, vaccines comprise an important triad. the first part of the vaccine, and in a sense the most important, is the biological component. this is the whole protein, or whole organism, or epitope-based part which confers the ability to be recognised by the immune system. it is this part which differentiates one vaccine from another, an anti-flu vaccine from an anti-tb vaccine. the second part of the vaccine is the adjuvant, which is one of many alternatives, that confers an immunogenicity to many vaccines that they would otherwise not possess. it often does this in a generic fashion, such as via agonising the innate immune system, so that the same adjuvant is quite capable of functioning in many different vaccine formulations. the final and third part of the vaccine is the delivery vehicle, as opposed to the delivery mechanism, such as oral vaccines versus injectable. the vehicle can be things as different as a viral vector or a liposome. an attenuated or heat-treated whole-organism vaccine can be thought of as combining all three parts of this triad in one supra-molecular moiety. of course, this is a gross simplification, and many other things go into deployable vaccine formulations, such as preservatives, contaminants, and other chemical or biological components that so exercise the anti-vaccine lobby. hopefully, this book will encourage us to think of vaccines in these terms, and provides the background necessary to engage with each of the three components of the vaccine triad. with this in mind, the anticipation inherent with this work is indeed simple and straightforward: to foster and foment interest in those areas of vaccinology that this far have not received the level of intense work that they richly deserve. to help achieve this, we have sought to balance optimistic positivity and cold, hard rationality. not all of the approaches described will ultimately bear fruit, but each should, nonetheless, be examined with equal diligence, sedulousness, and assiduity. global malaria mortality between 1980 and 2010: a systematic analysis evidence for active immunity to morphine in mice changes in heroin selfadministration by a rhesus monkey after morphine immunisation world health organization. manual for the production and control of vaccines-tetanus toxoid. blg/undp/77.2 rev liposomes as immunological adjuvants liposomes based on dimethyldioctadecylammonium promote a depot effect and enhance immunogenicity of soluble antigen a liposome-based mycobacterial vaccine induces potent adult and neonatal multifunctional t cells through the exquisite targeting of dendritic cells key: cord-321346-epsp3bbm authors: luppa, peter b.; müller, carolin; schlichtiger, alice; schlebusch, harald title: point-of-care testing (poct): current techniques and future perspectives date: 2011-03-21 journal: trends analyt chem doi: 10.1016/j.trac.2011.01.019 sha: doc_id: 321346 cord_uid: epsp3bbm point-of-care testing (poct) is a laboratory-medicine discipline that is evolving rapidly in analytical scope and clinical application. in this review, we first describe the state of the art of medical-laboratory tests that can be performed near the patient. at present, poct ranges from basic blood-glucose measurement to complex viscoelastic coagulation assays. poct shortens the time to clinical decision-making about additional testing or therapy, as delays are no longer caused by transport and preparation of clinical samples, and biochemical-test results are rapidly available at the point of care. improved medical outcome and lower costs may ensue. recent, evolving technological advances enable the development of novel poct instruments. we review the underlying analytical techniques. if new instruments are not yet in practical use, it is often hard to decide whether the underlying analytical principle has real advantage over former methods. however, future utilization of poct also depends on health-care trends and new areas of application. but, even today, it can be assumed that, for certain applications, near-patient testing is a useful complement to conventional laboratory analyses. a large number of laboratory analyses support correct diagnosis in over 50% of all diseases, in addition to aiding the monitoring of drug therapy in many other cases. laboratory medicine is therefore a vital component in differential diagnosis in the clinic and in local general practice. an affordable, competent system of laboratory diagnostics, in the doctorõs office and hospital, has been made possible through centralization of analysis in purpose-built laboratories or in facilities provided by large hospitals. in contrast to this centralization and increased efficiency in laboratory diagnostics, there has been a recent trend towards a more decentralized diagnostic analysis, so-called point-of-care testing (poct), which occurs directly at patientsõ beds, in operating theatres or outpatient clinics, or at sites of accidents. this modern variety of laboratory medicine is characterized by minimizing instrument size and procedures and the increasing use of current information technology. there are analytical devices available that make it possible to process a whole blood sample in a simple manner, allowing untrained staff to carry out laboratory diagnostics. it is clear that the use of poct shortens the time between sample acquisition and analysis (turnaround time). however, this type of diagnosis is only useful if the results produced lead to immediate therapeutic decisions [1] . however, there is still a paucity of evidence supporting the use of poct and improved patient outcomes [2] . nevertheless, it is not difficult to predict that there will be growth in the development of poct diagnostics for parameters that are at present available only in central laboratories. this growth is supported by new analytical technologies amalgamating issues (e.g., miniaturization, nanoparticle techniques, multiplexing, wireless connectivity, and novel biomarkers). clinical pathology as a discipline needs to be responsible for this field, since adherence to quality-management systems ensures accurate, reliable biochemical-test results for optimal patient care and safety, regardless of whether the individual test is performed in a central laboratory or as poct at the bedside [2] . poct is mainly characterized by proximity to the patient, quantitative or semi-quantitative single measurements, short turnaround time, no sample preparation, no pipetting, use of pre-made reagents, user-friendly dedicated analytical instruments and instant, resultdeduced therapeutic action [3, 4] . all outpatient or ward personnel will be expected to be able to use these analyzers. no previous knowledge in sample analysis should be required. however, there is a continual transition between poct and laboratory-based methods. a generally accepted definition of poct is still under debate. for example, viscoelastic coagulation tests, described below in this article as ''type 4'' poct, are to be performed near the patient in cases of active bleeding. the results have to be available quickly to allow instant therapeutic action. however, the caregiver is mostly overstrained when operating the viscoelastic apparatus, so often these analyses are performed in a central laboratory with online transmission of the sensorgrams to the operating room; so these analyzers can be called ''pseudo-poct''. currently, the annual turnover for poct in vitro diagnostics in europe stands at approximately €3bn, germany contributing €0.9bn of this; the majority comes from blood-sugar-test strips and devices for home testing in diabetes. in the past few years, the market for poct systems grew yearly by more than 10%, but this rapid growth has been slightly dampened since 2007. nevertheless, a marked increase is expected mid-term and long-term [1] . table 1 gives insight into the variety of parameters available at present. 2. current techniques: categorization of the stateof-the-art poct devices poct devices often employ biosensors (fig. 1) . according to the iupac definition, a biosensor is an analytical device for the detection of analytes that combines a biological component with a physicochemical-detector component. this generally occurs through the use of miniaturized analysis systems, where biological components are immobilized on a solid-state surface, which, in turn, interacts with the analyte [5] . these interactions may be detected by using either electrochemical or optical methods. in the latter case, a large role is played by fluorescence or reflection spectroscopy. different parameters, being characteristic for certain diseases, can be detected by using specific biomolecules. poct analyzers currently available can be separated into various groups. we attempt to categorize the instruments according to their practical use in poct and moreover by the following attributes: sensor characteristics, complexity, measuring mode, underlying detection principle and/or sample matrix. here is the simplest form of quantitative poct device, with most of the analysis taking place on the respective test strips. the reader is used only to read the result from the strips where the reaction has already taken place. these test strips are one-use articles. examples include figure 1 . the biosensor as the basis of analysis in many point-of-care testing (poct) instruments. note that the recognition layer may comprise attached recognition elements (antibodies, receptors, aptamers . . .) or immobilized enzymes. in the latter case, the addition of substrates is essential. not illustrated is the fact that the amplified signal is finally processed by microelectronics and displayed. glucometers for home and the hospital poct stations, as well as the i-stat abbott (abbott park, il, usa), a multi-parameter unit-use poct instrument [7] . blood-sugar analysis is, both historically and financially, the most commonly used poct technique. this is valid for in-patient and out-patient care, and for home testing by patients. although generally either capillary or venous blood is used, the results can be presented as blood-glucose or plasma-glucose level, depending on the calibration by the manufacturers. this can make a relevant difference of over 11%, potentially resulting in false therapeutic measures. to minimize the risk of confusion between whole-blood and plasma-glucose results, the international federation of clinical chemistry and laboratory medicine (ifcc) suggested in 2005 that glucose values should be given as plasma levels only, independent of sample type or measuring method. the usa and other countries have already followed this recommendation, and it is also now being implemented in the german-speaking regions [8, 9] . the strong correlation between the international normalized ratio (inr) and the clinical outcome of oral anticoagulation with vitamin k antagonists (warfarin, marcumar) has led to a tight therapy-monitoring regime using inr poct devices. inr testing may be performed at the doctorõs office or by the patient (''home testing''). for the most popular devices see table 3 . all these systems use unit-use test strips and whole blood obtained by finger prick, making them most convenient for the patient [10] . different types and sensitivities of the thromboplastins used and the different signaling types (micromechanical versus electrochemical) cause systematic differences between results obtained with the different inr devices [11] . however, the between-day precision of the devices seems to be satisfactory [12] , so the monitoring should be performed with one single type of device. these instruments are generally more complex than unit-use machines and use different analytical principles [1] : spectrophotometric substrate and enzyme-activity measurement; hematological particle counting; immunoassay; and, sensor-based blood-gas analysis tailored for special poct applications. they use antibody-based immunoassay methodologies. bgas use potentiometric/amperometric or optical sensors for ph, po 2 and pco 2 . additional ion-sensitive electrodes for the measurement of electrolytes and other substrates are available. a selection of systems from the seven marketdominating manufacturers is given in table 4 . optional is the configuration of a bga with a cooximetry unit. this is a most remarkable technical development. the co-oximetry unit [4] is a miniaturized multi-wavelength spectro-photometer, which measures the typical absorption spectra of the various hemoglobin (hb) species in order to distinguish o 2 -hb (oxy-hb) from other hb species and to determine the o 2 -hb saturation [i.e. the percentage of o 2 -hb compared to the total amount of hb, including co-hb, o 2 -hb, deoxygenated hb (hhb with fe 2+ ), and met-hb (hhb with fe 3+ )]. all leading companies on the market have sophisticated oximetry units on board their blood-gas analyzers, which have no counterpart in the central medical laboratory. the only technological diversification is the factor that the hb species are found only inside the erythrocytes. hence, some systems use a cell-lysis step prior to spectrophotometry, whereas others eliminate the erythrocyte-caused light scattering by applying matrixassisted algorithms. these poct compatible machines show a high degree of complexity. although they are valid for use in poct, only qualified personnel should operate them (e.g., a laboratory physician or a trained technical assistant). the combined analysis of plasma clotting, thrombocyte function and fibrinolysis is termed viscoelastic coagulation testing [13] . examples include the rotem (tem international, munich, germany) [14] or the sonoclot from sienco inc (arvada, co, usa). it is also possible to analyze platelet function in terms of in vitro bleeding time or via optical aggregometry [15] . examples here are the pfa 100 from siemens healthcare diagnostics (eschborn, germany) or the verifynow from accumetrics (san diego, ca, usa) [16] and [17] . as described in the introduction, these analyzers have to be called pseudo-poct. the most common example here is continuous glucose monitoring [18] . such analyzing and application systems are already available commercially. they are likely to replace the invasive, intravenous electrode by the minimally invasive location of a microdialysis catheter in subcutaneous tissue (fig. 2) . conversely, other non-invasive methods (e.g., microporation or optical techniques in direct transcutaneous measurement of metabolic parameters) are at least unlikely to prevail. this is mainly due to the broad range of human-skin characteristics concerning thickness, pigmentation and hairiness as well as physiological phenomena (e.g., humidity and salt content). currently available systems for continuous glucose monitoring are the guardian rt from medtronics (minneapolis, mn, usa), the glucoday from a. menarini 2.6. type 6 -molecular biology-based poct devices to detect infectious agents at present on the market, there are many qualitative test strips to detect infectious pathogens. the basic principle in most systems is immunochromatography of a specific microbial antigen (or, more rarely, antibody) in the patient sample (urine, swab, or whole blood). there have also been some attempts to use molecular biological methods [mostly the polymerase chain reaction, (qrt)-pcr] for poct, although these are technically demanding (with a dna/rna-extraction step required), so that they are no rapid tests in the strict sense (yet). due to the complexity of the test procedures and the challenging interpretation of results, future rapid nucleic-acid testing (nat) is more likely to be located in the central laboratory rather than at the bed-side [19] . nevertheless, rapid quantification of dna/rna from various infectious agents will be beneficial for clinical diagnostics. at present, the genexpert system from cepheid (sunnyvale, ca, usa) is the cutting-edge device to automate and to integrate all real-time pcr-based nat steps: sample preparation, dna amplification and detection. the system is a 1-16-site, random-access instrument, integrating real-time cycler, amplification and fluorescence detection. the use of multiple fluorescence dyes permits multiplex assays for detection of several targets within a single sample. the system provides pcr-test results from a raw clinical sample in about 1 h, enabling time-critical nat at the point of need. there is a series of different unit-use microfluidic cartridges to be inserted into the analyzer {e.g., the ''xpert sa nasal complete'' cartridge is designed to detect s. aureus and mrsa colonization from nasal pharyngeal swabs flu a-panel cartridge is already under evaluation [20] }. the currently available novel poct systems are in part forerunners of a new generation of laboratory systems, which will dominate the market in 10-20 yearsõ time (fourth-generation laboratory systems). these methods are characterized through miniaturization, parallel analyses and networking via information technology (it) [1] . 3.1.1. miniaturization. microfluidics, chip technology and electrochemical detection have enabled the construction of miniaturized systems, with which even dna may be directly analyzed in a few microliters of sample. immunoassays and other methods of protein determination may also be carried out by ''micro-total analysis systems'' (ltas). the advantages of miniaturization are obvious: fluid volumes in the nl and pl range reduce not only the amount of reagents required but also the time needed for basic analytical processes (e.g., mixing and equilibrating) [21] . 3.1.2. parallelization. chip technology opens up the possibility of measuring multiple channels or multiple time points in the smallest space; photolithographic techniques allow reaction reservoirs and liquid channels to be etched on a wafer to enable 100 or more measurements to be taken simultaneously. an even higher density of reactions has already been realized in gene analysis with the use of microarrays. appropriate microautomation systems allow multiple laboratory profiles to be established rapidly and cost effectively, thereby opening up the possibility of obtaining an overview of a patientõs situation in just a few minutes. the use of a pattern of laboratory values -in contrast to single parameters -may, at least in principle, open up new diagnostic and prognostic avenues. however, the attendant problems in evaluating and interpreting the massive amounts of data thus obtained remain unsolved. networking. next-generation laboratory systems will generally be networked via it, and an international communication standard (poct1-a) already exists for this purpose. however, current pointto-point communications (e.g., that between decentralized blood-glucose monitors and the laboratory it system) show only a modest beginning. for the future, there have been discussions on individual electronic patient files, in which laboratory values from various sources can be directly input and linked to other results. at present, a wave of novel analytical principles and instruments can be envisioned for the near future, including alternative biological detection elements (scaffolds, aptamers, or anticalins in place of antibodies), sophisticated applications of optical-signal technologies (total internal reflection fluorescence, surface plasmon resonance, reflectometric interference spectroscopy) [22] and new dedicated microarrays for inflammation, malignancies and autoantibody diagnostics. this process will be encouraged through innovation from the communication industry, particularly via wireless networking (wifi) technology. the following details refer to new instruments that are on the market or will shortly to be analyzer. this cartridge-based instrument allows the determination of hba1c, albumin/creatinine ratio (acr), and crp by reflectometry. for testing, whole blood, plasma and urine samples can be used. the advantages of this device are short assay times, all-inone reagent cartridges and its ease of application. tech. this is a hand-held immunoassay machine, which carries out immunological reactions using magnetizable nanoparticles. the particles are labeled with antibodies and exposed to a variable magnetic field. detection is performed with a ''bound-from-free'' separation of reaction components, currently using a frustrated total internal reflection method. nanoparticles can also be used in the so-called giant magnetoresistance effect in signal generation [23] . we expect that the instrument will provide a multi-analyte detection mode, with a spectrum of parameters (e.g., cardiac markers, parathormone and sepsis parameters). this company is developing a new signal-generation and detection technology. the poct instrument uses a piezo film made from polyvinyl fluoride, which acts as a pyroelectric film, thereby enabling an ultrasensitive non-separation assay design for immunoassays (e.g., cardiac markers, thyroid hormones). when led light strikes the piezo film, it causes thermal disturbance with subsequent changes in the charge (signal). however, this is only the case when a carbon particle-labeled immuno complex was previously bound. a labeled antibody, free in the solution and not bound to the surface, does not cause a change in the charge of the piezo film. analyzers. an innovative technology based on an established detection principle is realized in the new multiplate analyzer (dynabyte, munich, germany). the device uses the impedance aggregometry principle [24] for the assessment of platelet function in whole blood. blood thrombocytes are non-thrombogenic in their resting state, but expose receptors on their surface when being activated. this allows them to attach on vascular injuries and artificial surfaces (e.g., the sensor wires). when the platelets aggregate on the multiplate wires, they enhance the electrical resistance between them, which can be continuously recorded. in order to enhance the resistance on the sensor wires, tight attachment of the platelets is required. a number of plateletstimulating test reagents are available for specific, comprehensive platelet diagnostics. 3.2.5. type 5 -continuous measurement with poct systems. for a continuous mode of poct measurements, minimally-invasive microdialysis systems have become accepted as best approach. nevertheless, the most desired clinical access for such probes, the intravenous line, was, for a long time, an unfulfilled clinical desire. only recently, the microeye system of probe scientific ltd. (coventry, uk) enabled continuous, automated monitoring of substances in the circulating blood stream without withdrawing blood from the patient by applying an intravenous microdialysis catheter (http:// www.probescientific.com/microeye/). many companies are currently working on different poct-suitable dna-amplification techniques (e.g., the quantitative detection of the influenza a panel or of enterovirus strains). 3.2.6.1. nanosphere (northbrook, il, usa). the verigene system uses 50-nm gold-nanoparticle-probe technology for the detection of infectious dna or rna. the probe technology uses the change in optical properties of these gold nanoparticles that occurs when two or more nanoparticles are brought into close proximity. the wavelength of light scattered from the surface of the particles is within the visible range and can easily be detected. details of the distancedependent optical properties of gold nanoparticles are portrayed in fig. 3 . 3.2.6.2. enigma (salisbury, wiltshire, uk). this company is working on providing the next generation of integrated and automated real-time pcr-based diagnostics systems. 3.2.6.3. dxna (st. george, ut, usa). a mobile poct technology (genestat) provides rt-pcr-based detection of severe acute respiratory syndrome (sars) or highly pathogenic avian influenza. 3.2.6.4. idaho technology (salt lake city, ut, usa). the filmarray system is a user-friendly automated multiplex pcr. 3.2.6.5. iquum (marlborough, ma, usa). the liat analyzer offers an innovative lab-in-a-tube platform for detection and genotyping of dna/rna viruses and bacteria. a number of novel parameters for poct applications are under discussion. potentially interesting à but still clinically unevaluated à new markers for poct applications are as follows. 3.3.1. ngal. neutrophil gelatinase-associated lipocalin (ngal) tests for acute renal failure [25, 26] . 3.3.2. galectin-3. this b-galactoside-binding protein tests for fibrosis and adverse cardiac remodeling [27, 28] . copeptin. this c-terminal residue of proavp is for early diagnosis of myocardial infarction [29, 30] . the combination of placental growth factor (pigf), soluble fms-like tyrosine kinase 1 (sflt-1) [31] [32] [33] , and endoglin are for early diagnosis of preeclampsia [32, 34] . many of the possibilities of extending the application of poct depend on the wishes, the needs and the practicalities arising from using poct, as well as the advantages and the disadvantages for the patient. many people should be involved in this discussion: clinical and laboratory medical personnel, doctors in practice, political and regulatory authorities, state and private health insurers, industry and, not least, the patient. further-more, applications of poct must be scientifically grounded, evidence based {e.g., via outcome analysis, which has only partly been the case to date [9] } and financially viable. financial viability is strongly associated with the respective national reimbursement system for the various tests, which is, in most countries, mainly a question of healthcare politics. the quality of poct can be assured only through a poct-coordination system, in which the clinical laboratory plays a pivotal role. all poct activities should be discussed and agreed upon between the clinics and the clinical laboratory [35] . there are many examples of fruitful cooperation even today. however, a future necessity will be the more structured search protocol for a new poct method with agreement on the core laboratory method so that the clinical colleagues can assume clinical acceptability of the result variations caused by the poct method. new rules are needed for assessing agreement on a certain analyte result between two different methodologies [36] . in some analytical cases (e.g., step b: the dna probes are hybridized to a dna target in solution. when they are spotted onto the glass slide (step c) these hybridized gold nanoparticles scatter with h · m 2 . the plasmon efffect induces a red light shift with m 2 < m 1 . the scheme presented is in accordance with [48] . blood-glucose or prothrombin-time monitoring), errorgrid analysis can be used to determine cut-offs where differences between central laboratory and poct results may impact clinical decisions [37] . novel statistical approaches are yet to be defined. data management of the poct results is also fundamental to quality [38] . tight surveillance of poct data can show error trends before they affect the results. however, a prerequisite for this is comprehensive connectivity of all poct systems used within a hospital-wide network. newer poct devices have data-storage functions that can collect key information at the time the test is performed and later transmit the result together with the internal quality-control data to a server-based poct data manager software linked to the information system of the hospital. recently developed connectivity standards (e.g., poct1-a) enable different poct devices to share a common interface. poct data-management software programs can be categorized into proprietary and non-proprietary. examples of the first category are cobas it 1000 from roche, radiance from radiometer or rapidcomm from siemens healthcare. non-proprietary software solutions in the eu are pocelerator from conworx (berlin, germany), or, in the usa, quick-linc from telcor (lincoln, ne, usa) [39] . wireless real-time communication of test results and quality data will become a reality in the near future [40] . the number of patients requiring inpatient treatment in a hospital, together with the length of their stay, is likely to decline further in the near future -to the advantage of outpatient treatment and patient observation in their home environment. the importance of poct will increase in these areas. in outpatient care, it is clear what advantage immediate diagnostic and therapeutic decisions offers: the patient may not need to be further investigated. an enlargement of the current parameter spectrum is also important for the doctor in practice; here, in addition to the tests now available [41] , a means of identifying infectious agents would be required. home-care will be extended -apart from glucose and inr monitoring -when new tests for chronic diseases become readily available. the most important trend in this context is termed ''personalized medicine''. in this case, chronic diseases (e.g., diabetes or atherosclerosis) would be diagnosed earlier than by traditional clinical diagnosis, which is focused on the patientõs clinical signs and symptoms. the therapeutic strategies would then be tailored to each patient individually, thus improving the results of treatment (fig. 4) . ''telemedicine'' is increasingly important in this area. instruments used to monitor the patient can, in the right circumstances, send the data obtained directly to the relevant medical service point, which can intervene (e.g., when critical values are exceeded). the effectiveness of home monitoring and thus patient safety are improved. though poct may be used in principle at ''any'' locale, its use today is mainly centered on various areas of the hospital, general practitioners and patient self-monitoring of glucose and inr. above and beyond this, other potential uses have been discussed, or have indeed been, albeit mostly in small numbers, already realized. these include: mobile emergency paramedical care (blood mobiles, mobiles for public events); transport vehicles (e.g., ambulances and helicopters); sport medicine or competitive sport; outbreaks of disease and catastrophes [42, 43] ; remote areas, ''third world'' countries; military use; at the pharmacy; prisons; alternative-medicine practitioners; corporate healthcare operations; nursing homes; veterinary medicine; fitness studios; and, home healthcare in many cases, the equipment currently available, as well as the spectrum of parameters, is only partly suitable; but, future developments will raise the acceptance level and therefore the use of poct in these specific areas. the availability of medical treatment to the population as a whole is particularly important in developing countries. the current western systems (e.g., structured around a large central hospital) are largely unsuitable; rather a decentralized health system offers the most effective means. in this case, a decentralized diagnostic process with poct systems can play a very useful part. there have been a few successful movements in this direction (e.g., in reserves of the australian aborigines) [44] . however, widespread use of the currently available systems is hindered by costs, complexity and unreliability under extreme conditions of heat or humidity. the parameters offered often do not meet those required in these situations. the american national institute of biomedical imaging and bioengineering (nibib) has therefore begun co-operation with india, the aim of which is to develop devices, which are suitable for use at the point of need [45] . the international council for standardization in hematology (icsh) is working on a set of guidelines, which will be applicable worldwide for poct standardization in hematology and which can also be used in developing countries [46] . specialized, novel rapid microbiological tests must be developed to recognize the common infective agents in these countries. the bill and melinda gates foundation has sponsored research in this direction in order to establish a simple poct application for aids patients. this so-called cd4 initiative should make it possible to count the cd4 positive t-lymphocytes in hiv-infected patients, with a view to better control of anti-retroviral therapy. the value of poct in epidemic disease in developing countries can be demonstrated in the following example. tuberculosis treatment is hampered by slow, insensitive diagnostic methods, particularly for the detection of drug-resistant forms. an early diagnosis is essential to reduce mortality and to interrupt transmission, but the insufficient healthcare infrastructure in poor countries limits their accessibility and effect. boehme et al. [47] assessed the performance of the automated molecular identification of mycobacterium tuberculosis (mtb) and the testing of resistance to rifampin (rif) on the genexpert mtb/rif system (mentioned above), applying sputum samples of patients from peru, azerbaijan, south africa, and india. as a result, the authors stated that the mtb/rif test provided sensitive detection of tuberculosis and rifampin resistance directly from untreated sputum in less than 2 h. the test was specific in 604 of 609 patients without tuberculosis (99.2%). as compared with phenotypic drug-susceptibility testing, mtb/rif testing correctly identified 200 of 205 patients (97.6%) with rifampinresistant bacteria and 504 of 514 (98.1%) with rifampin-sensitive bacteria. the device is definitely applicable in rural areas without being compromised by a lack of analytical quality. all future projects should take into account that poct can in principle be brought simply to the patient, but not necessarily the expertise of the laboratory doctor. it is difficult to imagine how complex panels of analyses (e.g., tumor markers or genetic susceptibility tests), supplied via poct, could be interpreted without adequate technical expertise and knowledge. it is therefore vital, in the interest of the patient, that poct is employed by laboratory medical personnel who can suggest appropriate tests and carry them out satisfactorily. a specific problem involves the problem of quality assessment. the wide use of poct in areas outside of the standard hospital environment or by general practitioners can lead to new problems in quality assurance. it is clear today, that use of poct without relevant trained personnel leads to unsatisfactory results and that the end-user has to rely on a competent laboratory medical advisory service, at least when difficulties arise. further development of the relevant equipment is likely to result in inbuilt quality control, so that the current procedures for internal and external controls will become obsolete. however, the problems of end use by untrained or unsupervised personnel will increase, particularly with respect to pre-analysis (patient preparation, sample acquisition, recognition of invalid samples) and post-analysis (documentation, data protection) [1] . this is notably the case where poct is used in areas far from a laboratory (e.g., developing countries). it is an important task for laboratory medicine as a whole to make it clear that poct analysis that meets the medical requirements rests on more than reliable analysis systems. principles and practice of point-of-care testing point-of-care testing key: cord-269821-j4w084u2 authors: gaupp, franziska title: extreme events in a globalized food system date: 2020-06-19 journal: one earth doi: 10.1016/j.oneear.2020.06.001 sha: doc_id: 269821 cord_uid: j4w084u2 our food systems are complex and globally interdependent and are presently struggling to feed the world’s population. as population grows and the world becomes increasingly unstable and subject to shocks, it is imperative that we acknowledge the systemic nature of our food system and enhance its resilience. after the prevalence of undernourishment had declined over the last decades, since 2015, the number of people who suffer from hunger is increasing again. today, more than 820 million people in the world are hungry, and around 2 billion people do not have regular access to nutritious and sufficient food. 1 a growing and more affluent population will further increase the global demand for food and create stresses on land, for example, through deforestation. additionally, food production and agricultural productivity are further affected by an increasingly changing and variable climate, including extreme events, which further undermine the resilience of our systems. we are facing increased frequency, intensity, and duration of climate and extreme weather events, such as heat waves, droughts, and floods. 2 increased temperatures have contributed to land degradation and desertification in many parts of the world. erratic precipitation patterns and unpredictable rainy seasons increase uncertainty about planting times and can lead to crop failure. changing land conditions through human actions on the regional scale, such as deforestation and urban development, can further accentuate these negative impacts at a local level. given that extreme events are expected to further increase through the 21 st century, we need to increase the stability of the global food system. increases in global temperatures will affect processes involved in land degradation (such as vegetation loss or soil erosion) and food security (through crop losses and instabilities in the food supply). a 1.5 c global warming is projected to lead to high risks of food-supply instabilities, including periodic food shocks across regions and spikes in food pricing, and our current trajectory has us on course to significantly exceed this threshold. although climate and climate extremes affect our ability to produce food, food and nutrition security is more than just agricultural productivity. today's food system is globalized and consists of highly interdependent social, technical, financial, economic, and environmental subsystems. it is characterized by increasingly complex trade networks, an increasingly efficient supply chain with market power located in fewer and fewer hands, and increased coordination across interdependent sectors via information technology. between 1992 and 2009, trade connections for wheat and rice doubled and trade flows increased by 42% and 90% respectively. furthermore, market power along the global food supply chains is consolidating. the three largest seed suppliers increased their combined market share from 10% in the 1990s to 55% in 2015. in the uk, the three largest supermarkets hold a market share of 60%. although these emergent network structures of commodity supply chains increase gains in economic efficiency, they lead to systemic instability susceptible to shocks. trade networks are more interconnected and interdependent than ever, and research has shown that they can be intrinsically more fragile than if each network worked independently because they create pathways along which damaging events can spread globally and rapidly. 3 it is true that these trade networks could, for instance, help connect local producers to global markets and increase their incomes, but there is a risk that increasing network densities and complexities might lead to food insecurity in one part of the system as a result of the food system's inherent response to a shock rather than as a result of the initial shock itself. furthermore, local events might trigger consequential failures in other parts of the system through path dependencies and strong correlations and thus lead to amplification of the initial shock through cascading effects. a shock in the integrated global food supply chains can lead to ripple effects in political and social systems. the 2010 droughts in wheat-producing countries such as china, russia, and ukraine, for instance, led to major crop failures, pushing up food prices in the global markets. this in turn contributed to deep civil unrest in egypt, the world's largest wheat importer, as people were facing food shortages and helped the 2011 revolution to spread across the country. although it has been shown that global economic integration continues to strengthen our resilience to smaller shocks through trade adjustments, the current network structure and functional relations create higher vulnerabilities to so-called systemic risks. given how interconnected the food system is globally, taking a systems approach is vital to help us ensure food security. doing so requires an understanding of the systemic characteristics and vulnerabilities of the global interconnected food system. although one extreme event and the consequent crop losses in a single production region can be buffered by highyielding harvests in other places, grain storage, and trade, multiple-breadbasket failure is a growing reason for concern. correlated or compound climate and ll weather events (a combination of correlated climate drivers that surpass the coping capacity of the underlying systems) on both local and global scales could trigger systemic risks in the global food system. locally, non-linear dependencies between heat and drought conditions have been shown to have severe consequences for crop production in specific breadbasket regions, such as the mediterranean. on a global scale, largescale atmospheric circulation patterns, such as el niñ o-southern oscillation or rossby waves, can cause simultaneous extreme heat and rainfall events in different parts of the world. in summer 2018, simultaneous heat extremes in the northern hemisphere were associated with amplified rossby waves, resulting in reduced crop production in central north america, eastern europe, and eastern asia. 4 because those regions include important areas of crop production, such teleconnections have the potential to cause multiple, simultaneous breadbasket failures, posing a risk to global food security. if climate extremes are additionally co-occurring with other hazards, the systemic vulnerability of our global food system becomes apparent. this can currently be observed in east africa and yemen, where the population is fighting against a desert locust upsurge while experiencing conflicts, climate extremes, and coronavirus disease 2019 (covid-19), all of which further constrain surveillance and control operations in the locust crisis. the combination of those multiple hazards has led to warnings that in the second half of 2020, 25 million people will experience acute food insecurity. 5 the climate-agri-food system is characterized by dynamic interactions across spatial and temporal scales, ranging from global to local and from shocks such as heat waves to long-term stressors such as biodiversity loss. nonlinear dynamics in the climate system can lead to rapid and irreversible changes if the system surpasses a certain threshold. one of those so-called climate ''tipping points'' that might have major consequences for europe's climate and lead to significant losses of crop yields is the weakening and potential collapse of the atlantic meridional overturning circulation (amoc). 6 greenland melting and arctic warming are driving an influx of fresh water with lower salinity and density into the north atlantic, which is weakening the overturning circulation, an important part of global heat and salt transport by the ocean. if amoc weakened and even collapsed in the coming decade as a consequence of further warming, europe's seasonality would strongly increase and lead to harsher winters and hotter and drier summers. this is projected to reduce global agricultural productivity and to increase food prices. not all shocks to the globalized food system are directly linked to agricultural productivity or climate. the complexity and inter-connectedness and vulnerability of our globalized food system have become painfully evident in recent weeks after the emergence of a different type of shock to the system: a global pandemic. having started as a health crisis, covid-19 quickly trickled through the political, social, economic, technological, and financial systems. in the food system, lockdown measures and trade and business interruptions all over the world led to cascading effects that are projected to trigger food crises in many parts of the world. although harvests have been successful and food reserves are available, interruptions to global food supply chains have led to food shortages in many places. products cannot be moved from farms to markets, processing plants, or ports. food is rotting in fields because transport disruptions have made it impossible to move food from the farm to the consumer. at the same time, many people have lost their income, and food has become unaffordable to them. the world food programme has warned that by the end of the year, 130 million additional people could face famine. in the fight against covid-19, borders have been closed, and a lack of local production has led to price spikes in some places. in south sudan, for example, wheat and cassava prices have increased by 62% and 41%, respectively, since february. 7 in the coming months, planting, harvesting, and transporting food are likely to further face logistical barriers, which will again exacerbate food shortages and drive up prices. a lack of food access and the related grievance could then lead to further cascading effects, such as food riots and collective violence. besides compound shocks, non-linear dynamics, and cascading events, the complexity of the global food system entails feedback loops that further amplify initial shocks. as a consequence of covid-19, the fear of food shortages and price spikes due to global trade interruptions has led to export restrictions on food in several countries. at the time of writing, 11 countries have active, binding export restrictions on food, as shown by the international food policy research institute's food policy tracker. 8 russia, the world's biggest wheat exporter, announced in april that it will halt exports until july, when farmers will start to bring in grain from the new harvest. other wheat suppliers, such as kazakhstan and ukraine, have also capped sales. importers such as egypt, on the other hand, are hoarding crops in fear of shortages, creating feedback loops that will further drive up prices. especially in lowincome countries, this might make food unaffordable for large parts of the population and increase the threats of food insecurity. the visible and measurable systemic characteristics of the global food system are complemented by hidden links and consequences that become visible only after a shock has hit the system. the current pandemic has revealed structural weaknesses in our food system. little storage and just-in-time supply chains have increased economic efficiency but led to systemic instability. 9 just how vulnerable and easy to disrupt the supply chains in some parts of the world are has become evident in the last several weeks. further food-system interdependences that were previously ignored but are becoming apparent now include the role of schools in providing nutritious food to children. the world food programme estimates that globally, 368 million school children miss out on meals that they would normally receive in schools because classes are shut down. 10 if we want to make the global food system resilient to future shocks, we need to pay attention to both quantifiable and uncertain systemic risks and foodsystem interdependencies. to address the challenges of a globally interconnected, complex food system that is projected to be hit by more frequent and severe extreme events, we need to take a systems approach and one earth 2, june 19, 2020 519 ll acknowledge the systemic characteristics of the global food system (figure 1) . we need better food-system models that can account for systemic risk characteristics and increase our understanding of non-linear dynamics, tipping points, feedbacks, dependence structures, and adaptive behaviors. 11 we need transdisciplinary approaches that bring together different disciplines as well as other forms of expertise, such as knowledge of local practitioners. quantitative and qualitative methods need to be combined, and different model types need to be integrated to link climate hazards with technological and civilizational risks. artificial intelligence and new machine-learning tools can improve our understanding of the food system's complexity but must be complemented by qualitative assessment, such as perceptions, social concerns, or socio-economic impacts. robust data and statistics that are accurate, timely, disaggregated, and accessible are needed for monitoring and early warning systems. systemic risk and systemic opportunities need to be incorporated into the design of food-related policies. 12 effective governance needs to consider the interconnectedness among different parts of the global food system. a network perspective that pays attention to the ll interdependent actors and nodes is required. 13 one solution could be a systemic-risk transaction tax as suggested for the financial markets. 14 individual actors (such as large trading companies or retailers) that increase systemic risks in the network will be taxed proportionally to their marginal contribution to the overall systemic risks. the tax can be seen as insurance for the public against costs arising from cascading failure. additionally, we need global collaboration to work toward better management of trade barriers to ensure that food value chains function even in moments of crises to prevent price spikes and provide ''all people, at all times, [with] physical, social and economic access to sufficient, safe and nutritious food that meets their dietary needs and food preferences for a healthy and active life.'' 15 more shocks are likely to hit our global food system in the future, unleashing the potential for systemic risks to cascade through the system and to lead to major impacts and system breakdowns. these include projected increases in simultaneous climate extremes in breadbasket regions, crop pests, and other, unexpected shocks. if we want to avoid major threats to food security, we need to take a systems perspective in analyzing, managing, and governing the global food system. the state of food security and nutrition in the world: safeguarding against economic slowdowns and downturns special report on climate change and land (intergovernmental panel on climate change globally networked risks and how to respond amplified rossby waves enhance risk of concurrent heatwaves in major breadbasket regions fao makes gains in the fight against desert locusts in east africa and yemen but threat of a food security crisis remains, fao news article running amoc in the farming economy soaring prices, rotting crops: coronavirus triggers global food crisis covid-19 food trade policy tracker coronavirus: rationing based on health, equity and decency now neededfood system expert, the conversation global monitoring of school meals during covid-19 school closures modelling food security: bridging the gap between the micro and the macro scale governance of systemic risks for disaster prevention and mitigation united nations office for disaster risk reduction elimination of systemic risk in financial networks by means of a systemic risk transaction tax global strategic framework for food security and nutrition the author would like to thank the editor and michael obersteiner for valuable suggestions and comments in the development and improvement of the manuscript. the author's work is supported by the receipt project (grant agreement 820712) and by eit climate-kic's ''forging resilient regions deep demonstration.'' key: cord-223669-hs5pfg4b authors: song, jinyue; gu, tianbo; feng, xiaotao; ge, yunjie; mohapatra, prasant title: blockchain meets covid-19: a framework for contact information sharing and risk notification system date: 2020-07-20 journal: nan doi: nan sha: doc_id: 223669 cord_uid: hs5pfg4b covid-19 causes a global epidemic infection, which is the most severe infection disaster in human history. in the absence of particular medication and vaccines, tracing and isolating the source of infection is the best option to slow the spread of the virus and reduce infection and death rates among the population. there are three main obstacles in the process of tracing the infection: 1) patient's electronic health record is stored in a traditional centralized database that could be stolen and tampered with the infection data, 2) the confidential personal identity of the infected user may be revealed to a third party or organization, 3) existing infection tracing systems do not trace infections from multiple dimensions. either the system is location-based or individual-based tracing. in this work, we propose a global covid-19 information sharing system that utilizes the blockchain, smart contract, and bluetooth technologies. the proposed system unifies location-based and bluetooth-based contact tracing services into the blockchain platform, where the automatically executed smart contracts are deployed so that users can get consistent and non-tamperable virus trails. the anonymous functionality provided by the blockchain and bluetooth technology protects the user's identity privacy. with our proposed analysis formula for estimating the probability of infection, users can take measures to protect themselves in advance. we also implement a prototype system to demonstrate the feasibility and effectiveness of our approach. until today, coronavirus has infected more than 11.5 million people and caused nearly 530,000 deaths [3] . in particular, the united states has become the country with the largest number of known infections [4] . every country has its own privacy policy to share information about infected people. it is challenging to share information across the world with reliable privacy protection since different countries have their own privacy policy to share the information of infected people. information sharing in a centralized manner, such as mit, apple, and google who announced their tracking solutions by storing users' personal data in cloud [1] [2] , is highly relying on users' trust. once the personal data has been uploading to the cloud, users generally cannot control the potential abuse. if a comprehensive data security solution is missing, the private user data in the cloud may be hacked for any other harmful purposes. as a global organization, the world health organization (who) collaborates with the governments around the world to share information and enhance the management of epidemic prevention. however, who is losing trust from some countries and cannot obtain sufficient support. some other governments may conceal, falsely report, or hinder from reporting epidemic information. these may create a gaping security hole for global epidemic prevention. to this end, there is no way for individuals to share their information and protect their data privacy simultaneously. government departments and organizations may access the health medical data of all people, and go beyond the scope of their responsibility and duty. for example, some government health departments may locate the personal identity of infected patients, and then enforce them in a centralized isolation shelter, resulting in secondary infections, and restricting personal freedom. actually, apple and google will share the infected individuals' information with health authorities [2] , which means people's personal data privacy and human rights are being violated without their knowledge. currently, there are two separate tracing systems existing: location-based and individual-based contact tracing. locationbased contact tracing always provides a centralized service and records if there are infections in the given locations without the knowledge of infection movement [5] . the individual-based tracing systems only focus on the person-to-person contact via bluetooth [1] [2] , and they do not have a record about where users get infected. who states that the virus could survive on materials surfaces [6] [7] , so the virus has an effect on the people daily activity environment. but the individual-based system cannot trace and estimate the covid-19 effect on the given location. our proposed system combines the locationbased and individual-based systems together for users to access the public area infection status and lookup personal contact tracing history at the same time. our lab already deploys the centralized location-based tracing system [5] and we may merge our two systems together for a future research. blockchain can natively provide the decentralized service to share the information and protect the privacy of people [8] . user information can be packaged in transactions and stored in the blockchain in each computing node. even if the data of one node is manipulated, it will not affect the data's consistency because the manipulated data will not pass the verification by other nodes. a smart contract is a program that runs on the blockchain platform; it can execute instructions in a distributed manner, and maintain output consistency. in our system, the smart contract allows the user to check-in at each location and query the blockchain database for the location infection status. even though mit, apple, and google provide virus contact tracking solutions, the tracking service still brings the following new challenges. the infection transmission factors considered by current existing virus tracking service systems are too simple. the virus is not only carried by the infected person but also remains in the environment, so the user will still be infected by the virus attached to the object surface. therefore, our proposed system not only records the user's contact history but also tracks the user's travel trajectory. protecting user data privacy about his personal health information in a public network is also very challenging. traditional methods of hiding users' personal information, such as removing names, addresses, and social security numbers, cannot fully protect user privacy. so we embedded the function of bluetooth randomization address in the contact tracking service of the system. besides, after users uploading health data and identity information, they lose the ownership and the ability to control the data. apple and google provide anonymous services to users, but the infected patients will be reported to the government health department [2] . we choose the blockchain database as the storage method for the system, combined with the address randomization function of bluetooth, only users can identify and verify their health data and personal information stored in the database. we design and propose a blockchain-based covid-19 contact information sharing and risk notification system that can be used by the world to take preventive measures against epidemics. this system implements the following main functions with smart contracts and bluetooth embedded: (1) users can record their visited location information and personal contact history to the blockchain database. (2) users can update their infection status. (3) the system can update location infection status. (4) the system can notify the users that had been exposed the infected people or infected locations before. (5) the system can estimate the probability of being infected for users based on his location visiting history and personal contacting records. for example, a shopping mall can use our system to detect if this building is infected and the daily infection status. the customers can also query the database to gain the mall infection record before their shopping. the status of an individual is written to the blockchain and cannot be changed. our system is able to protect privacy. we consider a variety of privacy that can be protected by our system. users can regularly change their cellphone bluetooth mac addresses and encode these addresses to virtual ids. then, it is hard to trace individual identity from their public information written in the blockchain. our system will not trace personal identity or report personal health information to authorities. we have the following contributions. • we propose a hierarchical smart contract group in this system, to manage the infection status of each location and transportation. this design reduces the average operation cost and request processing time in the system. • we build and merge location-based and individual person based virus contact tracing and notification system, which tracks the virus infection activity from higher dimensions. • we embed a blockchain database in the system to ensure the safety of users' data and this design avoids the problem that health information may be tampered and stolen in a centralized database. • our system uses a weak random bluetooth address design to generate the user's identity information. this design not only protects the user's privacy but also reduces the congestion of data transmission within the blockchain network. • we propose a mathematical formula for the possibility of user infection, which quantifies the user's contact history and traveling history from multiple dimensions and thus provides a basis for the system's notification service. • we propose an optimal equation for the operating costs of the system, simulate person-to-person contact and user check-in activities in our system, and evaluate the system performance based on the different quantity of users and smart contracts. roadmap: this paper is organized as the following sections: section ii presents the system overview and system components. section iii formulize challenging problems for our system. section iv describes the system design from the perspective of four layers. section v shows how we simulate and evaluate system performance. the last two sections vi and vii present the related work in smart contract tracing and conclusion for this paper. our system can track the locations visited by users and the personal contact history of users with others. when a user updates himself infected, our system will notify other users who have been in direct or indirect contact with him, and provide an assessment of the probability of infection. in a general scenario, the user may visit many public places, such as offices, restaurants, shopping centers, or gyms in one day, so he can use the designed contact tracing service to upload his visiting records including the time and location to our system. then, our system will store the visiting records in decentralized blockchain databases. also, users can check the infection status of a certain location before his visit to ensure safety. when a user reports that his infected status, a smart contract group embedded in our system will update the virus infection status of the locations and transportation that the user visited and took based on his visiting records. our system helps users to track the history of their indirect contact with others because the virus can land on the surfaces of the objects and continue to remain active [9] , and even if the user does not have face-to-face contact with an infected patient, there is still a possibility of being infected after he touches the surface of the virus-contaminated object. our system uses the bluetooth functionality in users' mobile devices to automatically detect the others and then upload their contact records to decentralized blockchain databases. the range of bluetooth detection is relatively small, which is about 5 to 10 meters [10] . so, when the user's mobile phone detects the bluetooth signals, it indicates that there are people nearby. when a user reports himself as infected status, our system will broadcast his infection status and alert other users to check if they have close contact with this infected user. it is important to trace the direct contact recorded by bluetooth because viruses can attach to water vapor and spread through the airborne [11] , and users may be infected by other people in a close range [12] . therefore, uploading users contact history records will help them to track the infection transmission path of the virus and assess users' probability of being infected. our proposed system provides users with this integrated mobile service, which contains the following functionalities: probability of infection: according to the manual for wto medical staff [13] [6] , healthy people could be infected directly and indirectly, where the direct factor is person-toperson contact in a close distance and the indirect factor is that the virus surviving on the surface of the object spread by the patient infects the healthy people after they touch the surface. so the system will evaluate the user's risk from these factors based on the feature data extracted from location tracing and person-to-person contact tracing, like, the length of contact time between users, the spatial distance between users, and the materials of objects in public places. notification of infection: after the estimation of infection probability for this user, the notification function sends a warning alert to him, which reminds him to prepare for virus infection in advance or to seek medical help, before his health condition gets worse. another scenario is when a user reports his infected status, our system will broadcast his virtual identity to other users. then, these users who receive the notification will query the local database to see if he has had any direct or indirect contact with the infected patient and calculate the infection probability. in our system, three main technologies guarantee data security and personal privacy: decentralized database in the blockchain, automatic execution of smart contracts, and randomization of bluetooth mac addresses. (a) data security: our design guarantees that user data will not be manipulated. the blockchain protocol stipulates that the current block will always use the hash value generated from the previous block, so if an attacker manipulates a transaction in a block, he must tamper with all the previous blocks data to ensure his tampering, before the next new block to be mined, verified and accepted by other users. this computing workload is extremely large, so it is almost impossible for an attacker to forcibly manipulate the blockchain data. moreover, users in the network can detect violations of smart contracts, and such attempts to violate the contract are verified to be invalid and those malicious transactions will not be stored in the blockchain. our system contains a smart contract group to handle all user check-in requests for public locations and transportation. since smart contracts have the properties of being unmodifiable, requiring no supervision, and being automatically executed, smart contracts with distributed features ensure that user data cannot be tampered with, or produce malicious results. so, this design ensures the security of user data. decentralized blockchain databases and smart contracts also enhance the usability of our system. since no individual or centralized entity can control all data, there will be no smart contract or system crash due to malicious operations done by some users. (b) identity privacy: we use bluetooth technology to protect user data privacy. the mac addresses uploaded by users are randomly generated by the bluetooth protocol [14] . although these addresses are stored in the blockchain database, each user's mac address is random and is not fixedly bound to the mobile device. it guarantees that users will not be tracked and located by the addresses provided in the network. from another perspective, bluetooth technology frequently replaces the user's mac address [15] , which means a user will gradually use multiple different mac addresses in a unit of time. other people in the surroundings have no way to associate the mac address to this user through investigation. the frequent randomized mac address protects the user's privacy in real life. in order to reduce the operating overhead and internet congestion at the system level, we choose weak randomized generation of bluetooth mac address. users upload their random mac addresses to the system, which is measured by ethereum gas [8] in the system overheads. the larger quantity of random mac addresses, the better privacy protection users could have. but the larger quantity will lead to higher operating costs and network congestion. therefore, we need to find a balance between a sufficiently large quantity of bluetooth random addresses for privacy protection, and a relatively small quantity for lower system operating cost. we highlight four problems: latency, throughput, operating cost, and probability estimation for our proposed covid-19 information sharing and risk notification system and these problems are described by mathematical formulas at a highlevel. in our system, latency is a time difference ∆t about how long the user's latest check-in request can be processed completely by smart contracts and stored in the databases. we consider that latency is affected by the following five factors: 1. the number of users |u| in the system, 2. the frequency freq of users sending requests, 3. the size of one block |b|, 4. the height of the smart contract group scg.height, and 5. the length of the waiting queue |queue| of the smart contract. u includes not only users already exist in the system, but also new users who enter the system within a unit time. the total number of users and the frequency freq of user's check-in determine the total number of user requests per unit time in the system. if the number of user check-in requests exceeds the processing capacity of the smart contract per unit time, the user's requests will enter the waiting queue of the smart contract, which increases the request processing time. we introduce the variable of block size |b|, because it is one of the bottlenecks of the blockchain's development towards high throughput and low latency [16] . the height of the smart contract group scg.height and the length of the queue |queue| in each contract are also important factors that affect latency. in our proposed hierarchical structure, the smart contracts at the same level will not affect the efficiency of processing requests between each other. the requests received by the smart contracts at the current level are all from the higher-level smart contracts, so the hierarchical structure we propose is a tree structure, whose height is described as lg(|scg|). as mentioned before, the smart contract will put the requests that have not been processed in time in the waiting queue. if the number of unprocessed requests is greater than the length of the queue, these requests will be abandoned. then the smart contract needs to wait for other nodes to synchronize the transaction operations and data. this brings a longer latency. we thus establish the latency formula: where we have a series of transition functions δs provided to determine the latency, latency = φ(δ u,freq , δ |b| , δ scg.height,|queue| ). we propose that the three polynomial δs are combined by function φ. in our system, throughput tp refers to the number of user requests that the system can completely handle in a unit time. it can intuitively show the system's ability to process user requests. the greater the throughput, the stronger the processing capacity of the system. throughput is limited by latency, packet loss rate rate pl and bandwidth bw. as described in the previous section, five factors in the system can affect the latency, but the rate pl and bw depends on the network conditions at the user end. therefore, the throughput can be defined as follows: where θ is the transition function with three arguments to determine the tp. operating cost is another important problem we consider. reasonable operating costs determine that our system can serve users stably, and support its operation for a long time. the operating cost in the system is measured by the ethereum gas [8] , which includes the following five influencing factors: location-based and bluetooth-based contact tracing services, health tracing service, the setup and operation of smart contracts. we will explain all these services and components in the next section. since the blockchain decentralized structure, users can query their local blockchain database without consuming gas and we do not include the cost of the database in the operating cost calculation. we consider the number of users and requests have a polynomial relationship with the cost of three tracing services. the setup cost of a smart contract is fixed, but its operating costs will increase with the increase of users. so we have the following cost formula: where loc represents location-based contact tracing service, bt represents bluetooth-based contact tracing service, heal represents health tracing service, and op represents all the operations in the smart contracts like adding a user checkin and getting a location infection status. we define λ to be the transition function to calculate the cost combined by the four factors. then, we measure the average and variance values of the operating cost, which represent the system's stability in an optimal condition. here are the formulas when the system has five factors at minimal cost: args var = arg min var(λ(loc, bt, heal, setup, op)) (4) args avg = arg min avg(λ(loc, bt, heal, setup, op)) (5) therefore, we can provide the optimal arguments with a minimal system cost, which is shown as: args optimal = mincost(args var , args avg , ζ) where we introduce penalty function ζ to adjust five arguments and get the global optimal sets for the cost calculation. in the previous section, we briefly describe the functionality of estimating the infection probability in the health tracing service. since the ground truth data contains only binary values representing infected or not, without a percentage value for the probability of infection, we introduce the statistical logistic regression analysis method with iteratively reweighted least squares (irls) [17] to maximum likelihood estimation and find the optimal arguments. we assume that the data set is relatively clean and there are no outstanding outliers. ground truth dataset contains n user data points (x i , y i ), i = 1, ..., n, where x i is the independent variable tuple with (rssi, ∆t b , ∆t c and ms) and y i is the dependent variable with binary values {0, 1}. we conclude five elements in the tuple affecting the infection probability: • rssi is the received (bluetooth) signal strength index, which represents the distance between users [18] . • ∆t b is bluetooth contact time interval, which indicates how long users detect each other's bluetooth signal from a short distance. • ∆t c is a overlapping time calculated based on two checkin time points t i and t j , which infers the time when two users enter the location, and ∆t c = |t it j |. • ms is a discrete value in set ms representing a virus residual active time period on a material surface [9] . so we have: where β is the set of parameters {β 0 , β 1 , ..., β 4 } for these five arguments including the constant. then, we can use the estimation method irls to get the fitted model and arguments: args optimal [w] = irls(x, y, w, µ) where w is arguments tuple (rssi, ∆t b , ∆t c , ms) and µ is the predicated value from formula (7). in this section, we will define and explain our system components from the perspective of the four layers in the system, and the interactions within the contact tracing mechanisms. our trace and notification system contains four layers for users to trace person-to-person contact via bluetooth, check-in locations, and lookup infection status with other users on the blockchain platform. fig. 1 shows four layers: user interaction layer, mobile service layer, smart contract service layer, and data storage layer. this system provides two primary services for trace and notification: bluetooth-based personal contact trace service and location-based contact trace service. both of the two services are developed on the blockchain platform, and the data generated from these two services is stored in the distributed blockchain databases. the locationbased contact trace is coordinated by the smart contracts in the third layer and the bluetooth-based contact trace is handled in the second layer. (a) user interaction layer at the user interaction layer, we have two entities: user u and location l. users are people who hold bluetooth-enabled mobile phones and have two health statue types: healthy users u normal and infected users u infected . users access to mobile service in the second layer and update their health status in the system based on their medical reports. we assume that users always honestly upload their infection status to our system. a location l is a public place or transportation that users often visit in their daily lives, such as office, restaurant, stadium, bus, and even airplane. location l also has two status types, uninfected location l normal and infected location l infected . if an infected user u infected visited this location, then this location l would be marked as l infected by the system. (b) mobile service layer mobile service layer is the the core handler in our system and it interacts directly with the other three layers. mobile service ms is our proposed mobile phone application in this layer. cooperating with other layers, the mobile service layer is an interface layer for providing users with services, including the following two aspects: contact tracing service based on bluetooth or location, and health tracing service supported by data provided from the blockchain database. contact tracing services include location-based and bluetooth-based, focusing on indirect and direct contact between users. the bluetooth-based service is an integral part of the client's mobile phone service which embeds the bluetooth function. it can sniff other surrounding users' bluetooth devices, and broadcast its own bluetooth random mac address macaddr as a virtual identity. this bluetooth-based service can exchange its user randomized mac address with other users via bluetooth, and then packet received other users' mac addresses macaddrs, time interval timeinterval of the interaction and received signal strength index rssi into the transaction tx, which is broadcast to the blockchain network. in addition, when the user receives other's infection notification from the broadcast transaction tx, our mobile app will automatically query the local blockchain database for the infected user's bluetooth address, check-in information, and check whether the infected user has a direct or indirect contact with current user. the second contact tracing service is the location-based tracing. it accepts the user check-in requests req checkin s from the user interaction layer and it coordinates these requests to different smart contracts in the third layer based on the sender's location. at the same time, the infection status of this location l is affected by the user's check-in request req checkin . if the user u updates the health status as infected u infected and broadcasts it to the smart contract, the location l will be marked as infected l infected by this corresponding smart contract. these two services will be explained step by step in detail and described in mathematical formulas in the next two sections. health tracing service is the third sub-service in mobile service layer. this service has two functions: 1. broadcast user's infected status u infected to alert other users and update the infected status for the locations ls where the infected user visited. 2. estimate the probability of being infected for users prob u (infection). users us at the first layer can update their health status through this service at the second layer, packet their health status {u normal , u infected } in a transaction tx and send it to the smart contract responsible for the infection status at the third layer and broadcast it to other users on the network. once the health status of the infected person has been updated as u infected , people who had contact with the infected person will be prompted with a warning alert provided by this health tracing service. after the normal user u normal receives the warning alert, this health tracking service estimates the probability of the user being infected prob u (infection), based on the data collected from location-based and bluetooth-based tracing services contact regarding the infected person u infected . we consider there is a relationship between received (bluetooth) signal strength index rssi and infection probability p (inf ection) with covid-19. similar to apple and google's design [2] , we use the bluetooth sniff to detect if two users are in a close distance d, which can be measured by the received signal strength indication (rssi) of bluetooth [18] and the author uses low pass filter (lp f ) to reduce the errors in the measurement data. to describe the content of the article without repeating, we only quote the method based on the bluetooth signal strength index. in the following section, we will use the simplified mathematical formulas including lp f by default to reduce errors in measured data as designed. research and experiments made by mit show the covid-19 can spread by airbone [11] , which can reach 26 feet away by a sneeze [12] . so, the closer the user is to the infected person, the easier he is to be exposed to more viruses and become easier to be infected. therefore, we correlate the strength of the bluetooth signal strength with the possibility of being infected by a virus. (c) smart contract service layer the smart contract service layer is the secondary core of our system. the check-in request req checkin generated by the user's visiting location l is processed by the mobile service layer and forwarded to this smart contract service layer, where it is managed by the smart contract group. the smart contract group integrates the contracts according to the administrative hierarchy system. the top level is the smart contract at the state level contract state , followed by the county contract county , then the city contract city , and finally the smallest unit location contract location . location smart contracts contract location belong to the city-level contracts contract city to manage, city-level contracts belong to counties contract county , and county-level contracts belong to states contract state . each contract will only inherit from one superior contract, and will not belong to two different superior contracts at the same time. each location must be in one of these three states: {emptystatus, infectedstatus, cleanstatus}, and the corresponding smart contract contract location dynamically records the infection status of the location l. if an infected user u infected visits this location l, or a user who has visited this location, reports that he is infected, then this location l is considered infected by this user u infected . only after the location is cleaned, or 14 days after being infected, the location l is considered to be in a cleanstatus. in order to save the operating cost of the smart contract contract location and maintain the accuracy of the location l status record, the coming requests reqs trigger the smart contract contract location to check and update the infection status for the location l, otherwise the smart contract contract location will not actively check the infection status of the location l. this design ensures that users can get the latest location status for their requests while avoiding the operations of the smart contract. (d) data storage layer we have deployed a distributed blockchain database db in the data storage layer, and every user and computing node can be synchronized in our network to get a complete database, which is consistent with the data of others. from the perspective of data storage, the traditional centralized database stores all data [19] in one data center; the traditional distributed database stores data in multiple data centers, but each center may not have global complete data [20] . from the data management perspective, traditional databases require a central node to process read and write requests to the database, but a blockchain database does not require a central node to process read and write requests [21] [22] because all users can have the same database locally, to directly query for consistent results. in our system, the blockchain database will store all transactions in the network, including users' bluetooth contact records, check-in information of the visited locations, and the change of the user's public health status. the three tracing services mentioned above, location-based contact tracing, bluetooth-based contact tracing, and health tracing services, except location-based contact tracing requiring smart contracts to update and store infection status to the database, in the other two services, users can query the blockchain database directly for personal contacts and visiting records. current bitcoin and ethereum blockchain database design have problems such as high computational cost, and slow writing and querying data [22] , but there are existing thirdgeneration blockchain databases now whose performance, such as throughput, can be comparable to databases of visa credit card companies [23] [24] . since the third-generation blockchain database has no mature commercial development and lacks a mature platform to support the development of smart contracts, we still deploy our system on the ethereum platform for simulation and evaluation. in this section, we describe the location-based contact tracing service in detail: first, we show entities participating in the service, such as users, smart contracts, and locations; and then, we introduce the interaction activities between entities, including user's check-in a place while his visiting and the user's query of the infection status of a place before visiting. as defined in previous sections, we have location l, which represents a public place or transportation, users u with normal and infected status and smart contracts, who are in a hierarchy structure to process all users' check-in requests. in figure 2 , we illustrate location-based contact tracing service with a simplified state machine. {q, δ, initialstate, cleanstatus} where we have infectedusercheckin, infecteduserupdate, locationiscleaned, wait14days} • initialstate is the null state before the smart contract exists • cleanstatus is the accepting state first, when a user issues a check-in request req checkin through the location-based contact service at the second layer, the system checks whether there is a smart contract contract location corresponding to this location at the third layer. if the contract does not exist, the smart contract of the city contract city where the location is located will create a smart contract contract location for the location. if the contract already exists, the system goes to the next step to process the user's check-in request. in case the coming user is infected, this location enters the next state infectedstatus, otherwise, it enters the cleanstatus. then, when the location is in a state infectedstatus, either after 14 days or if it can be cleaned and disinfected, this location can transform to cleanstatus. finally, in the state cleanstatus, this location is affected by the request of the next user. if the next user is infected u infected , or his past visit is updated to be infected, then this location will return to the previous state infectedstatus. otherwise, it remains in the current cleanstatus. fig. 3 is an example of user u checking in a building in location-based contact tracing service. the check-in information about this user visiting the building, like timestamp t, geographic position geopos and health status {u normal , u infected }, is packaged in a transaction tx with the help of our proposed mobile client app in the second layer. then, the app sends the transaction tx to the smart contract group in the third layer. according to the address of the building geopos, this transaction tx is passed from the state-level contract state to the county-level contract county , to the city-level contract city , and finally transferred to the smart contract contract location corresponding to this building. then, based on the health status u infected for example, provided by the user, the location smart contract changes the infection status to l infected for the building. in this process, the transaction tx that records the user's check-in information will be saved by the blockchain database db. also, the location-based contact tracing service can help the user to get the infection status record of the location l. we need to discuss two scenarios based on whether the user has the contract location network address locally corresponding to the building for example. if the user has checked in this location before or has previously queried the smart contract of this location, the corresponding contract location address should exist in his mobile app. therefore, the user can directly send a request to the contract location network address through the mobile app to get the location infection status. if the user is interacting with this location for the first time, then he needs to get the contract network address first. with the provided the geographic position geopos of this location, the user's mobile app will query the statelevel contract state in the smart contract group, who will transfer this query request from the top level to the city-level contract city based on the provided geopos. if there is a corresponding contract location for this location, the network address of this contract will be returned to the user. otherwise, the city-level smart contract contract city will create a new contract location , and its address will be returned to the user. after receiving the location infection records from smart contract service layer, the request sender can verify the response by querying his local blockchain database for the transaction record. if the infection exists in this location l infected , our proposed mobile app will alert the user. to encourage users to use mobile services more frequently, we have developed a check-in and query incentive mechanism for users. whenever a user checks in a location or queries the corresponding location smart contract, this smart contract will return a slightly more amount of transaction fee to encourage the user to check-in or query more often. the additional fee given to the user is to support the user to use mobile services to broadcast his transaction that contains the bluetooth contact data, check-in information, and update health status. bluetooth-based contact tracing involves all entities except the smart contract group and all layers except smart contract service layer. the mobile app on the second layer packs the bluetooth contact data in the transactions, broadcasts them on the blockchain network, and saves all of them in the blockchain database. similarly, the mobile app processes realtime transactions received about the senders' health status, matches user contact records and reminds users of the danger of contact with infected persons. in the transaction, we will record four elements: a period of users detecting each other ∆t b , the detected mac addresses macaddress, mobile phone model appleinc for example, and the received signal strength index rssi. fig.1 overviews the workflow of bluetooth-based contact tracing. it helps users from the first layer to exchange randomized mac addresses with each other, and then packet the data with a timestamp and received (bluetooth) signal strength index rssi in the transaction, which is sent to the blockchain database in the data storage layer. the signal strength of different bluetooth devices will be different, so we assume that users are using the same type of apple mobile phone, which provides assumption conditions for the next section to calculate the distance between two users through signal strength. except that, this bluetooth-based service will alert users when receiving transactions containing infected health status from another user. fig. 4 shows that the mobile app in layer 2 detects surrounding users through bluetooth, recording the time interval ∆t b of direct contact of the users, the random mac address macaddr of the other user, the type of mobile device devicetype, and the range of rssi. the mobile app will packet these segments into a transaction to broadcast in this blockchain network and store in the blockchain database. and the mobile app will store all the bluetooth mac addresses generated locally for another service functionality health tracing, which will be discussed in the next section. another scenario of bluetooth-based contact tracing is that, if the mobile app receives a transaction containing other user's infected health status and his mac addresses, the bluetoothbased contact tracing will query its local blockchain database based on these mac addresses, and check whether the current user has a contact record with the infected user. if they have a contact history, then the mobile app will alert the user. then, the mobile app will transfer and broadcast this transaction again on the network to alert other users who may have a contact history. we have mentioned in the previous section that there are two types of challenges: 1. the balance between the number of random mac addresses generated by bluetooth and protecting users' privacy, 2. the balance of the number of random mac addresses and network congestion. for the first challenge, we adopt the method of changing the silent period [25] . the silent period refers to the gap time between bluetooth device discarding the old mac address and adopting the new mac address [25] , and during this period, the bluetooth device cannot decide to use the new or old address. the article points out that changing the length of the silent period obviously reduces the duration of the bluetooth device being tracked, and thus the device will not be located easily [25] . this is still an open question, and it is worth our research in the future. for the second challenge, we use weak randomization, which reduces the number of random addresses generated, so fewer transactions are generated for packing these mac addresses. then in the blockchain network, each computing node and the user can achieve data consistency without synchronizing a large number of communication requests. health tracing is the third service in the proposed mobile app and it has two major functionalities: 1). broadcast user's infection status to update contracts infection status and alert other users. 2). estimate the probability of being infected. within the first functionality, users can update their infection status as u infected or u normal . when the user's status becomes infected, the mobile app will automatically two things: 1). update all infection status among contract location s where this user visited in the past 14 days based on his visiting records and 2). broadcast transactions containing his infected status and all the bluetooth randomized mac addresses generated in the past 14 days to alert others who have close contacts with him. within the second functionality, users can estimate the probability of being infected based on the analysis of the four factors rssi, ∆t b , ∆t c , ms. referring to the formula (7) in section 3, we believe these parameters and p(infection) should be formalized in a logit function: where β 4 (ms) is formulized as after accessing real medical data and investigating the public locations, we will calculate and prove this proposed formula. we build a prototype system and evaluate its performance with the following experiments, where we simulate users' daily contact and check-in activities by the poisson distribution equation. in the experiments, we focus on the average cost of processing requests and the total cost to operate our prototype system. first, we introduce the environment for the experiments. then, we evaluate and analyze the system performance on stability and scalability. in the future, we may deploy our system and build benchmarks to evaluate when the real dataset is available. we conduct experiments on the macbook pro with a macos system in version 10.14.5. this machine has an intel i5 cpu with 2.3 ghz and an 8 gb lpddr3 memory. we use a programming language called solidity to develop and implement the smart contract group, which are deployed on a private ethereum blockchain simulated by a software called ganache. then, we use python to code the script for data analysis. our experiments focus on three basic variables affecting performance: 1). the number of |u| increasing from 100 to 600, with even intervals of 100, 2). the user's contact and check-in frequency freq following poisson distribution, and 3). smart contract group size scg.size. based on three quantities of deployed smart contracts and six different numbers of requests increased from 100 to 600, we measure the average gas cost of all requests, and the standard deviation on the average cost of ten rounds experiments. fig. 5 shows that as the quantity of contracts increases from 3 to 18, and the number of requests increases from 100 to 600, the average request gas consumption is reduced by a factor of 5 from 2,000,000 wei to 400,000 wei. fig. 6 shows that with the increase of requests, the deviation of request cost is reduced by 5 times. although the gas amount and deviation in fig. 5 and 6 are very large, the actual overhead is very small. we know that wei is the smallest gas unit in ethereum system and 1 ether is equal to 10 18 wei. then, assuming 1 ether is worth $250, an 400,000 wei request is worth $10 −10 , and the deviation of 500 wei is negligible. also, we find that in the condition of different number of contracts, the average costs trend to be a similar amount around 400,000 wei. system overhead refers to the interaction costs between mobile services and users and operating costs for smart contracts, which are measured by ethereum gas. fig. 7 shows that when the number of requests increases from 50 to 250, and the number of contracts increases from 3 to 18, the overall gas consumption of the system increases linearly, considering the case of the same amount of requests with different amount of contracts, and the case of the amount of contracts with different amount of requests numbers. based on the three measurements, we believe that this prototype system has good stability and scalability. with the increase in the number of requests and contracts, the request cost, which is the major overhead in the system operation, has a stable approaching to a lower boundary. this trend supports the stability of the system. similarly, the system overhead is linear growth, instead of an exponential one, which is an acceptable performance. in the field of contact tracing, mit [1] , apple and google [2] have related products and projects. however, their solutions are either a centralized database that is built in the system or incomplete privacy protection which is provided for users. such designs cannot meet the requirements of user privacy. in terms of data security, the smart contract guarantees the consistency of operating execution and then obtains a consistent output. there is one paper presenting a computing resource trading platform based on a smart contract-based edge computing network [26] . this design uses a similar treestructured smart contract group with ours, but their implementation focuses on how to match users and complete resource trading. and our purpose of smart contracts is to record the infection status for locations. regarding privacy, there are articles that use differential privacy algorithms in iot data processing [27] , but differential privacy methods will return a relatively accurate output with noise added. this conflicts with the property of blockchain because when storing data, the latter block must verify the correctness of the data of the previous block and cannot tolerate deviations. but it could be interesting to see intersection research of these two fields. in this paper, we design this tracing and notification system based on blockchain and smart contract, which provides three types of services: location-based contact tracing, bluetoothbased contact tracing and health tracing services. our system can trace the user's travel and contact history, and can remind the user about his past contacts that may cause infection. in addition, users can also estimate the probability of being infected through the health tracing service. in order to protect users' privacy, they can anonymously send their visiting records and health status on the blockchain platform. at the same time, users can use a large number of random mac addresses provided by bluetooth technology as a temporary identity to further protect privacy. in addition, our smart contract group embedded in the system records the infection status of each location and performs the same sequence of check-in operations to ensure that each user can get the consistent infection results of the location. we also simulate the interaction between users and our prototype system, and then evaluate its performance, including gas consumption, operating stability, and request processing speed. in a simulated environment, our system has a good scalability and good stability. we expect to have real data about user contact records to evaluate our system in the future. pact: private automated contact tracing apple and google partner on covid-19 contact tracing technology covid-19 coronavirus pandemic cases in the u.s we care world covid 19 tracing laboratory testing for coronavirus disease 2019 (covid-19) in suspected human cases: interim guidance protocol for assessment of potential risk factors for coronavirus disease 2019 (covid-19) among health workers in a health care setting ethereum: a secure decentralised generalised transaction ledger byzantium version 7e819ec sustainability of coronavirus on different surfaces covid-19 diagnostic based on mit technology might be tested on patient samples soon a sneeze assessment of risk factors for coronavirus disease 2019 (covid-19) in health workers: protocol for a case-control study bleb: bluetooth low energy botnet for large scale individual tracking handoff all your privacy a review of apples bluetooth low energy continuity protocol on scaling decentralized blockchains machine learning: a probabilistic perspective distance estimation of smart device using bluetooth biomodels database: a free, centralized database of curated, published, quantitative kinetic models of biochemical and cellular systems cassandra: a decentralized structured storage system exploring the attack surface of blockchain: a comprehensive survey how to time-stamp a digital document bigchaindb: a scalable blockchain database operational performance data enhancing wireless location privacy using silent period smart contractbased computing resourcestrading in edge computing a lightweight privacy-preserving data aggregation scheme for fog computing-enhanced iot key: cord-018688-gvk9uazp authors: magid, avi; gesser-edelsburg, anat; green, manfred s. title: the role of informal digital surveillance systems before, during and after infectious disease outbreaks: a critical analysis date: 2018-03-23 journal: defence against bioterrorism doi: 10.1007/978-94-024-1263-5_14 sha: doc_id: 18688 cord_uid: gvk9uazp background one of the main limitations of traditional surveillance systems for disease detection is their inability to detect epidemics in real-time. in addition to syndromic surveillance, a number of informal digital resources have been developed. these systems are based on data collected through media sources such as news reports on the internet, mailing lists, and rss (really simple syndication) feeds. the role of such systems at all stages of the epidemic remains unclear. methods a literature review was carried out on informal digital resources for infectious disease surveillance. we examined the source of information, the manner in which they process and disseminate the information, their role in each phase of disease outbreaks, and whether and to what extent these systems are capable of early detection and management of infectious disease epidemics. results informal digital resources use similar sources of data for surveillance. however, they use different algorithms to create their output, and cover different geographic areas. in this regard, they complement each other with respect to information completeness. there is evidence in the literature on the systems’ usefulness in communicating information to public health professionals, as well as to the general public during and after previous epidemics. retrospective studies of some systems have shown a theoretical decrease in the time of epidemic detection compared to conventional surveillance. however, there is no evidence of the ability for real-time detection. conclusions currently, there is little prospective evidence that existing informal systems are capable of real-time early detection of disease outbreaks. most systems accumulate large amounts of information on a wide variety of diseases, making it difficult to extract critical information. presenting critical information clearly and precisely remains a challenge. not be captured through traditional surveillance, and may be useful to governments and health agencies [4] . these systems are designed to function during all phases of disease outbreak, and are planned to increase sensitivity and timeliness. however, the role of such systems before, during and after infectious disease epidemics and, in particular, whether such systems are currently capable of early detection of epidemics remains unclear. a literature review was carried out to compare informal digital systems with regards to their source of information, the manner in which they process and disseminate the information, their role in each phase of an epidemic, and whether and to what extent these systems are capable of early detection of epidemics. the systems evaluated were promed-mail, global public health intelligence network (gphin), healthmap, medisys, epispider, biocaster, h5n1 google earth mashup, avian influenza daily digest and blog, google flu trends and argus. promed-mail is "an internet based reporting system aimed at rapidly disseminating information on infectious disease outbreaks and acute exposures to toxins that affect human health, including those in animals and in plants grown for food or animal feed" [21] (promed-mail website). promed-mail receives information from a number of sources, such as media reports, official reports, online summaries and local observers. the reports are reviewed and investigated by promed-mail expert team, and then distributed by e-mail to promed subscribers, and published in promed-mail website (promed-mail website). in addition to filtering the received information, promed-mail expert team may also add related information from media, government and other sources [23] . promed-mail was proven as an efficient system during the 2003 outbreak of sars, where information about points of outbreak, including additional information from a british medical journal article, was efficiently disseminated [23] . it should be stressed that promed-mail collects, filters, disseminates and archives it. they do not carry out formal analysis of the information although they provide some evaluation. the global public health intelligence network (gphin) is a biosurveillance system developed by health canada in collaboration with the who. gphin receives as input, information about disease outbreaks arriving from news service items, promed-mail, electronic discussion groups and selected websites, and disseminates information to subscribers using the following decision algorithm. a relevance score is computed for each information item. two thresholds are determined, high and low. if the item relevance score is greater than the high threshold, then it is immediately disseminated to subscribers. if the item relevance score is lower than the low threshold, then it is automatically "trashed". otherwise (if the item relevance score is between the high and the low thresholds), the item goes through human analysis and then disseminated to subscribers [23] . a prominent limitation of gphin efficiency is its reliance on the time in which information about an outbreak or other event if published in one of gphin data sources. nevertheless, gphin is considered efficient in providing earlier warning of events of interest to the international community compared with other systems, as 56% of the 578 outbreaks verified by who between july 1998 and august 2001 were initially picked up by gphin [23] . healthmap is a freely accessible automated electronic information system aimed at facilitating knowledge management and early detection of infectious disease outbreaks by aggregating, extracting, categorizing, filtering and integrating reports on new and ongoing infectious disease outbreaks. data on outbreaks are organized according to geography, time, and infectious disease agent [5] . healthmap receives as input reports received from variety of electronic sources, including online news sources aggregated in websites such as google news, reporting systems such as promed-mail, and validated official reports received from organizations such as the who [5, 11] . an internet search is performed by healthmap every hour, 24 h a day, in order to obtain the required information. search criteria include disease name (scientific and common), symptoms, keywords, and phrases. after collecting the reports, healthmap uses text mining algorithms in order to characterize the reports. characterization includes the following stages: (1) categorization: reports are categorized according to disease and location and relevance is determined. (2) clustering: similar reports are grouped together and exact duplicates are removed. clustering is performed based on similarity of the report's headline, body text, and disease and location categories. (3) filtering: reports are reviewed and corrected by an analyst, and then filtered into five categories -breaking news, warning, old news, context, and not disease related. in order to reduce information overload and to focus on disseminating information regarding outbreaks of high impact, only reports classified as breaking news are overlaid on an interactive geographic map located on healthmap site [5] . among the users of healthmap are the who, the us centers for disease control and prevention, and the european center for disease prevention and control, which use its information for surveillance activities [5, 11] . medical information system (medisys) is an informal automatic public health surveillance system. medisys is designed and operated by the joint research center (jrc) of the european commission, in cooperation with the health threat unit at the european union directorate general for health and consumer affairs and the university of helsinki. medisys collects its information from open-source news media, mainly articles from news pages. medisys categorizes the collected information according to predefined categories and disseminates it to subscribed users by e-mail. the system also provides its user with features and statistics available on its website, including a world map in which event locations are highlighted, aggregated news count per each geographic location presented on graphs, and the most significant event location for the last 25 h. medisys is available in 26 languages (the system collects information in 45 languages, but the website is available in 26 languages). users can filter the information according to language, disease and location, as well as by outbreaks, treatments and legislations. medisys users can also select articles into predefined categories, add comments to these articles, add information, and disseminate them to user-defined groups [12] . argus is an informal biosurveillance system aimed at detecting and monitoring biological events that may be a global health threat to human, plant and animals. the system is hosted at the georgetown university medical center (washington, dc, united states), and funded by the united states government. argus collects information in 40 native languages from media sources, including printed newspapers, electronic media, internet-based newsletters and blogs, as well as from official sources (the world health organization (who) and the world organization for animal health (oie). the system uses bayesian analysis tools for selecting and filtering the collected articles. the process is performed by about 40 regional professional analysts, who monitor several thousand internet sources on a daily basis. by using bayesian analysis tools, the analysts select reports from a dynamic database of media reports. relevance is determined according to a specific set of terms and keywords applicable to infectious diseases surveillance. after filtering the information, events that may indicate the initiation of an outbreak are disseminated to the system users. also disseminated are events that may require investigation [12, 20] . biocaster is an informal surveillance system aimed to collect information on disease outbreaks, filter the information, and disseminate it to users. the system is a part of a research project developed and managed by the national institute of informatics in japan, which involves five institutes in three countries. biocaster focuses mainly on the asia-pacific region. the system collects information by using really system syndication (rss) feeds from more than 1700 sources. information is collected mainly from google news, yahoo! news, and european media monitor, filtered and disseminated in a fully automated manner with no human analysis in any stage. filtered information (about 90 articles per day) is published in three languages (english, japanese and vietnamese). articles are processed and disseminated every hour. in addition, biocaster creates an ontology which covers approximately 117 infectious diseases and six syndromes. the ontology is produced in eight languages (english, japanese, vietnamese, chinese, thai, korean, spanish and french), and is used as an input to global health monitor web portal, which offers its users maps and graphs of health-concerning events [12] . the semantic processing and integration of distributed electronic resources for epidemiology (epispider) is a web-based tool which integrates information gathered from electronic media resources containing health information, as well as from informal surveillance systems, such as promed-mail. the aim is to enhance the surveillance of infectious disease outbreaks.epispider uses promed-mail reports as an input, as well as health news sources that provide rss feeds. by using natural language processing, it extracts location information from the input sources, and geocode them using the yahoo and google geocoding services. after a filtering process, the system generates summaries of promed reports (on a daily basis). these reports are available in the epispider website [13] . google earth combines satellite images, aerial photography and map data to create a 3d interactive template of the world. this template can be used by anyone to add and share information about any subject that involves geographical elements. nature (international weekly journal of science) uses google earth to track the spread of the h5n1 avian flu virus around the globe, and to present a geographic visualization of the spread of h5n1 [19] (nature website). avian influenza daily digest is a digest produced by the united states government. the digest collects raw open source content regarding avian influenza and disseminates it to subscribers. material is disseminated without any processing. users are encouraged to provide with updates and/or clarifications that will be posted in subsequent issues of the digest [2] . google flu trend is designed by google internet company to be a near real-time tool for detection of influenza outbreaks. google flu trend exploits the fact that millions of people worldwide search online for health-related information on a daily basis. the tool was designed based on the assumption that there is an association between the number of people searching for influenza-related topics and the number of people who actually have influenza symptoms, and therefore, an unusual increase in the number of people searching for influenza-related topic on the web may simulate an increase in influenza syndromes. studies performed by google and yahoo have shown that plotting data on searches using influenza-related keywords has led to an epidemic curve that closely matched the epidemic curve generated by traditional surveillance of influenza [4] . google flu trends analyzes a fraction of the total google searches over a period of time, and extrapolates the data to estimate the search volume. the information is displayed in a graph called "search volume index graph". it is claimed by the tool's designers that, according to tool testing, it can detect outbreaks of influenza 7-10 days before it is detected by conventional cdc surveillance [4] . all the studied digital resources use similar sources of data -official reports, as well as media reports, including global media resources, news aggregators, eyewitness reports, internet-based newsletters and blogs. however, they use different algorithms to create their output, and cover different geographic areas. in addition, existing digital resources are different in the manner they filter and analyze the information and may create different output. therefore they complement each other with respect to information completeness. retrospective studies of some systems have shown a theoretical decrease in the time of outbreak detection compared to conventional surveillance. however, evidence of such ability in real time is sparse and unclear. chan et al. [8] have analyzed the average interval between the estimated start of the outbreak to the earliest date of discovery and publication, using who confirmed outbreak reports, as well as promed-mail, gphin and healthmap reports. analysis showed a decrease in intervals over 14 years, which was partially attributed to the emergence of informal digital resources [8] . a retrospective study of argus reports on respiratory disease in mexico showed a significant increase in reporting frequency during the 2008-2009 influenza season relative to that of 2007-2008. the authors suggest that, according to these retrospective results, respiratory disease was prevalent in mexico and reported as unusual much earlier than when the h1n1 pandemic virus was formally identified. however, its connection with the 2009 pandemic is unclear [20] . the google flu trends tool was also retrospectively tested. according to retrospective testing, influenza epidemics can be detected by using google flu trends tool 7-10 days before it is detected by conventional surveillance [6, 7] , however, there are still no prospective evidence to such capability. a retrospective study from china reported that google flu trend search data are correlated with traditional methods of surveillance [14] . another retrospective study tested the real-time detection ability of six informal digital systems, including argus, biocaster, gphin, healthmap, medisys and promed-mail. data from these systems were used to detect epidemics and compared to official data. results suggested that all tested systems have shown retrospective real-time detection ability. moreover, it was found that the combined expertise amongst systems provided a better early detection [3] . unlike retrospective evidence, prospective evidence of informal digital systems capability for early detection of epidemics is sparse. some epidemics have been claimed to be first reported by promed-mail, before they were officially reported by the who [15] . these reports were proved to be reliable, since they were later confirmed by the who. however most of the reports were first published by promed-mail not because the information was not available to the who by this time, but because the who was not authorized to publish them due to lack of conformation [15] . the sars in china (february 10, 2003) is the best known outbreak first reported on promed-mail [17] . a detection in real-time was also demonstrated by gphin during the sars outbreak of 2002. gphin detected sars and issues the first alert to the who more than 2 months before it was first published by the who [16, 18, 24] . however, the time between the gphin alert and the first time it was reported by the who implies that the whole detection process was not shortened due to the gphin alert. retrospective reviews of the polio outbreak of 2013 and 2014 and the ebola outbreak of 2014 showed that informal digital detection preceded official detection by an average of 14.6 days. for example, promed and gphin reported the polio epidemic in cameroon in 2013 23 days after the outbreak began, where the official who report was published 51 days after the outbreak began [1] . however, the digital systems detection did not contribute in real-time to the whole process of outbreak detection and declaration. hence, in real-time it is not an early detection. there is evidence in the literature on the systems' usefulness in communicating the information during previous outbreaks to public health professionals, as well as to the general public. promed-mail and gphin had critical roles in updating public health officials about the sars outbreak in 2002 [4] . such systems are also capable of providing officials, clinicians and the general public with guidance to medical decision making, including the importance of vaccination and other preventive actions [4] . the first report on sars on february 10, 2003 published by promedmail, and the hundreds of subsequent promed-mail reports have helped health professionals worldwide to gather critical details regarding sars, and by this to recognize sars and discover its cause [15] . assessment of correlation between healthmap reports and official government reports reported during the first 100 day of the 2010 haitian cholera outbreak has confirmed that data yielded from informal digital systems were well correlated with data officially reported from the haitian health authorities. moreover, this study has shown that informal digital systems are capable of being used at the early stages of an outbreak not only as an indicator of the outbreak occurrence, but also as a predictive tool by providing a reliable estimation of the reproductive number, a major epidemic parameter [9] . there is no evidence in the literature of the use of informal digital systems after an epiodemic. nevertheless, we believe that data collected during outbreaks through informal digital systems are being used by public health agencies for retrospectively studying the dynamics of epidemic, and for drawing conclusions about the management of the epidemic. there has been impressive progress in the development of informal digital systems for disease surveillance. informal digital systems are widely used by the general public, as well as by health officials. a good example is the goran digital system (the global outbreak and response network) developed by the who, which gather information from number of sources both governmental and informal, including gphin and promed-mail [17] . one of the most prominent suggested advantages of the digital systems is their functioning in early notification of infectious disease outbreaks, before the official notifications, and their contribution to the epidemiological investigation of the disease before official data are available. during epidemics, data gathered and disseminated through official public health authorities are usually not available to public health officials and to policy makers for some time, sometimes due to political and logistic limitations. this period of time is critical for estimating the epidemic dynamics and implementing the response plan [9] . unlike official data, data collected by digital systems are available in near real-time, and may be used for epidemiological assessment. a mandatory requisite for the use of digital systems data for epidemiological investigation of an outbreak is the reliability of the data, as well as their equivalence to official data. in other words, there should be a match between the number of cases derived from the informal data and the number of cases officially reported by public health authorities. indeed, our results have pointed out an example in which a correlation between digital systems data and official data in the first stages of epidemic was confirmed in the data collected from healthmap regarding the 2010 haitian cholera. however, as mentioned by the authors, epidemiological measurements using digital systems data should be also tested in other epidemics, in order to confirm the method's reliability [9] . the fact that the number of subscribers to digital systems is increasing each year [15] makes these systems an efficient tool for globally spreading the information, as well as a tool for epidemiological investigation, complementary to official data. however, despite their theoretical advantage over traditional surveillance, there is no evidence in the literature that information collected through digital system had affected public health policy makers. although we did not find evidence in the literature, we believe that digital systems may also contribute to the public health community after the outbreak ends. the abundance of reports collected and disseminated by these systems during outbreaks creates an epidemiological reservoir, which, due to its availability worldwide, may be used for a post-pandemic investigation and conclusion making. as for early detection of infectious disease outbreaks, we did not find any prospective evidence showing the capability of digital systems of detection infectious disease outbreaks in real-time. our results are consistent with some other studies conclusions, pointing out that currently digital systems are not capable of detecting an outbreak [17, 4] . although there is evidence of informal digital systems publishing reports on outbreaks before official detection (such as in the polio outbreak of 2013 and 2014 pointed out in the results section) [1] , these reports did not actually affect the process of detection. the formal process of detection includes receiving the information, processing the information and using the information. the early digital systems reports were not used in any of the detection phases and did not change the process. it may be viewed as an analogue to screening tests which are effective only if they are capable of changing the natural history of a disease. since there is no evidence of informal digital systems capable of changing the "natural history of outbreak" so far, they cannot be considered useful for early detection. informal digital systems may also have an important role in disease surveillance. incorporating informal digital systems into existing formal systems may improve their performance. a study in the united states showed that combining information gathered from informal digital systems with information received from the texas influenza-like-illness surveillance network (ilinet) improved the ability of predicting hospitalizations due to influenza [22] . another study in the united states showed a good correlation between google flu searches and emergency department influenza-like illness visits [10] . moreover, since digital sources usually contains data not captured through traditional methods, they are used by public health organizations, including the global outbreak alert and response network of the who, which uses digital sources for surveillance on a daily basis [4] . however, the usage of digital systems as a surveillance tool may have some limitations. first, most systems accumulate a huge mass of information on a large variety of diseases, making it difficult to extract critical information. in other words, no integration of the information is performed to yield useful information. the challenge is to present critical information clearly and concisely. second, digital systems are less specific than traditional surveillance systems, mostly due to false alarms, misinformation and information based on rumors [9, 17] . therefore, they may not be solely used but as a complementary tool for traditional surveillance systems [17] . a third limitation is the lack of a response system to early warnings. with the lack of such a system, early warning is not useful, as no practical action is followed by the publication of the information. such a response system may include triggers and decision criteria, which would lead to an appropriate and proportionate response to the threat [17] . to summarize, considerable efforts and resources have been invested in the development of informal digital system for detection of infectious disease outbreaks. as a result, a new generation of informal digital systems has emerged. the most prominent advantage of such systems is their ability to report on an outbreak in near real-time, or, in other words, before the information is officially reported, and by this to be used by public health decision makers for epidemiological assessment and preparation for the pandemic. currently there is no evidence in the literature for their capability to detect an outbreak at its onset. in addition, there are no hard data to prove the benefits of using such systems before and during an outbreak. we do not believe that they can be used to identify early cases, but should be used as a support system for describing the spread of the disease. the challenge is to empirically assess the efficiency of informal digital systems and their use for decision making and interventions during crisis, as well as to test the systems' sensitivity and specificity. a more general informal system, which provides syndromic-based analysis of reports disseminated by all currently existing systems, may be the next step toward disease outbreak detection based on informal systems. digital surveillance for enhanced detection and response to outbreaks factors influencing performance of internet-based biosurveillance systems used in epidemic intelligence for early detection of infectious diseases outbreaks digital disease detection -harnessing the web for public health surveillance surveillance sans frontieres: internetbased emerging infectious disease intelligence and the healthmap project google trends: a web-based tool for real-time surveillance of disease outbreaks google trends: a web-based tool for real-time surveillance of disease outbreaks global capacity for emerging infectious disease detection social and news media anables estimation of epidemiological patterns early in the 2010 haitian cholera outbreak google flu trends: correlation with emergency department influenza rates and crowding metrics healthmap: global infectious disease monitoring through automated classification and visualization of internet media reports landscape of international eventbased biosurveillance scanning the emerging infectious diseases horizon-visualizing promed emails using epispider using google trends for influenza surveillance in south china the internet and the global monitoring of emerging diseases: lessons from the first 10 years of promed-mail internet-based surveillance systems for monitoring emerging infectious diseases public health surveillance and infectious disease detection the global public health intelligence network and early warning outbreak detection: a canadian contribution to global public health event-based biosurveillance of respiratory disease in mexico optimizing provider recruitment for influenza surveillance networks book: handbook of biosurveillance 301 early detection of disease outbreaks using the internet key: cord-010406-uwt95kk8 authors: hu, paul jen-hwa; zeng, daniel; chen, hsinchun; larson, catherine; chang, wei; tseng, chunju; ma, james title: system for infectious disease information sharing and analysis: design and evaluation date: 2007-07-10 journal: ieee trans inf technol biomed doi: 10.1109/titb.2007.893286 sha: doc_id: 10406 cord_uid: uwt95kk8 motivated by the importance of infectious disease informatics (idi) and the challenges to idi system development and data sharing, we design and implement bioportal, a web-based idi system that integrates cross-jurisdictional data to support information sharing, analysis, and visualization in public health. in this paper, we discuss general challenges in idi, describe bioportal's architecture and functionalities, and highlight encouraging evaluation results obtained from a controlled experiment that focused on analysis accuracy, task performance efficiency, user information satisfaction, system usability, usefulness, and ease of use. i ncreasing globalization, combined with accelerating population mobility and more frequent travel, has made the prevention and management of infectious disease outbreaks a growing concern in public health. emerging infectious disease and epidemic outbreaks are particularly important and represent critical challenges facing public health researchers and practitioners [1] , [2] . in addition, potential threats of bioterrorism appear on the horizon [3] . managing infectious disease outbreaks is intrinsically information intensive and requires substantial support for data gathering, integration, analysis, sharing, and visualization [4] . such support requirements are becoming even more challenging because of the diverse, heterogeneous, and complex information available in enormous volumes and different sources that span jurisdictional constituencies both horizontally and vertically. public health professionals such as epidemiologists can be better supported by advanced information systems (is), as vividly manifested by emerging infectious disease informatics (idi)-an interdisciplinary research area that focuses on the design, implementation, and evaluation of advanced systems, techniques, and methods for managing infectious disease and epidemic outbreaks, ranging from prevention to surveillance and detection [5] , [6] . the design and implementation of an effective idi system can be complex and challenging. at the data level, an expanding array of data that pertain to particular diseases, population characteristics, and related health considerations must be collected, organized, and archived, typically by different clinical institutions and health agencies. these data are heterogeneous in their semantics, modeling, granularity, aggregation, availability frequency, and coding/representation. data sharing is critical to the pertinent institutions and agencies, which have to coordinate by explicitly specifying data ownership and access rights, as well as delineating the responsibilities associated with legal and privacy considerations. at the system level, these institutions and agencies often vary in their in-house systems, which adopt proprietary architecture designs and operate on different platforms. as kay et al. [7] point out, most existing systems in public health have been developed in isolation. the challenge and complexity of designing an idi system extends beyond data and system heterogeneity. from the user's perspective, all relevant data must be seamlessly integrated to support his or her surveillance and analysis tasks that are critical to the prevention of and alert about particular disease events or devastating outbreaks. to be effective, an idi system must encompass sophisticated algorithms for the automatic detection of emerging disease patterns and the identification of probable threats or events. an effective idi system also must have advanced computational models that overlay health data for spatial-temporal analysis to support public health professionals' analysis tasks [8] . several additional issues are crucial for system design and implementation, including the integration of multiple heterogeneous source data or systems, data accessibility and security, interfaces with geographic information systems (gis), text document management support, and data or text mining. in particular, idi design requirements include spatial-temporal data analysis and related visualization support. typically, public health professionals approach the surveillance or detection of a probable outbreak as an event for which all related data are dotted and analyzed in spatial and temporal dimensions. furthermore, the value of an idi system is generally determined by the extent to which the system can present data and analysis results through intuitively comprehensible and cognitively efficient visualization. ultimately, an idi system must facilitate and enhance task performance by enabling public health professionals to use heuristics and preferred analysis methods to generate more accurate analysis results within a shorter time window. to support the surveillance and detection of infectious disease outbreaks by public health professionals, we design and implement the bioportal system, a web-based idi system that provides convenient access to distributed, cross-jurisdictional health data pertaining to several major infectious diseases including west nile virus (wnv), foot-and-mouth disease (fmd), and botulism. our system development team is interdisciplinary, consisting of researchers in both is and public health, practitioners, and officials from several state health departments. bioportal supports sophisticated spatial-temporal data analysis methods, and has effective data/information visualization capabilities. the rest of this paper is structured as follows. in section ii, we describe the architecture design of bioportal and highlight its main technical components and functionalities. next, in section iii, we discuss the value of bioportal for infectious disease surveillance and management, derive hypotheses regarding its advantages and effects, and empirically test these hypotheses using a controlled experiment with 33 subjects. to assess bioportal as a whole, we focus our evaluation on users rather than specific algorithms implemented as part of bioportal and examine its effects on their task performances as well as subjective assess-ments of the system. in section iv, we summarize our findings and note that our data support most of the hypotheses tested. our results suggest that bio portal can better support public health professionals' analysis tasks, and is generally considered more usable, useful, and easier to use than the benchmark technology. section v concludes with a summary, discussions of the paper's contributions and limitations, and some future research directions. bioportal is an integrated, cross-jurisdictional idi infrastructure that has been running for testing and research purposes since early 2004 (see www.bioportal.org). although it has not yet been adopted for operational use, it contains more than a dozen realworld data sets contributed by public health partners and other agencies. in this section, we summarize bioportal's architectural design, its main components, data set availability, system functionality, and related outbreak detection research. the information that we present herein establishes the background for the evaluation study reported in the subsequent sections of this paper. fig. 1 illustrates the architecture of the bioportal system, including the data flows between/among the main components of bioportal as well as between bioportal and external data sources. from a system's perspective, bioportal is loosely coupled with state public health information systems in california and new york. it does not change the way these state systems operate. as needed, these systems transmit wnv/botulism information through secure links to the bioportal using mutually agreed protocols. such information is then stored in an internal data store maintained by the bioportal. the system also automatically retrieves data items from sources, such as those from the usgs, and stores them in this internal data store. all the system functions provided by bioportal, including infectious disease data search and query, spatial-temporal visualization, outbreak detection analysis and related modeling, and automatic alert generation based on the results of outbreak detection analysis, are solely based on the data stored in the bioportal internal data store, without further interactions with the contributing data sources. technically speaking, we adopt a data warehousing approach, rather than alternative approaches such as query translation, information linkage, or terminology mapping [9] to address the distributed data integration challenges in idi. this choice of approach is primarily based on the following characteristics of infectious disease data sources and associated analysis needs. first, unlike many other biomedical applications for which it has become increasingly easy to query data sources automatically from remote locations, most infectious disease data sets have been developed primarily for internal use. although accessing the underlying databases through remote queries is technologically feasible, in practice, most idi data providers are unwilling to "open up" their databases. instead, they prefer pushing preprocessed data to (or preprocessing data waiting to be pulled by) a data warehousing system such as bioportal while retaining full control over data fields to be shared (directly at the data level as opposed to at the data access control level). second, the types of queries performed on idi data sources typically are confined to data aggregation requests over particular geographical regions and time periods. therefore, there is no need to strategize complex distributed queries. however, processing speed of the data aggregation is important because such operations must be carried out in large numbers for some outbreak detection analysis approaches (see section ii-c). third, the amount of idi data is relatively small in terms of storage volume because epidemiological information tends to contain a few short data fields, which makes a data warehousing approach feasible. furthermore, overlaps between epidemiological data coverage are rare; therefore, the data warehousing effort becomes relatively manageable. internally, bioportal consists of three main components: a web portal, a data store, and a communication backbone. in section ii-b, we provide the details of each component in more detail; here, we summarize bioportal's implementation environment and the assumptions made on the user's end. bioportal follows a standard three-tier web architecture. the data store component, developed using sql server, provides a data warehouse with information pulled from or pushed by contributing data sources. the communication backbone uses standardcompliant xml formats, and is built as multiple standalone java applications that interface with various data sources using different messaging protocols. most system functions are developed in java using jsp pages to interact with the user. as such, all except one major component of bioportal can be accessed by users through a standard web browser. the exception is the visualization module, which is developed as a standalone java application for improved performance, enhanced interactivity, and greater user interface control and is deployable through the web start technology (assuming that the sun jre environment is installed on the client machine). because the web portal component of bioportal implements the user interface and provides access to all main user functionalities-including: 1) searching and querying available infectious disease-related data sets; 2) visualizing the data sets using spatial-temporal visualization; 3) accessing analysis and outbreak detection functions; and 4) accessing the alerting mechanism-we do not discuss this component as one unit. instead, we briefly summarize our work on 1) and 4) in this section, and then present the bioportal visualization environment. because data analysis and outbreak detection involve innovative spatial-temporal data mining research beyond system implementation, we defer their discussion to section ii-c. 1) portal data store: a main objective of bioportal is to enable users from partnering states and organizations to share data. typically, data from different organizations have different designs and are stored in different formats. to enable data interoperability, we use hl7 standards internally as the main storage format. some data providers (e.g., new york state's hin) have already adopted hl7 and can, thus, send hl7-compliant data to bioportal directly. additional steps are needed to ensure data interoperability for those data providers that do not yet have hl7-compliant data. first, we reach an agreement with them regarding the format (typically a simple home-grown xml format) for their data. second, the data providers modify their data export module to implement this mutually agreed format. third, when data from these providers reach bioportal, a data normalization module maps the customized xml format on to hl7 using predetermined mapping rules implemented by the bioportal team. in effect, the data from the hl7-compliant data providers also are processed by this module, because it removes from them unneeded data attributes, duplications, and common misspellings (based on a customized home-grown dictionary). this normalization module is not intended to resolve structural or semantic incompatibilities in an automated fashion; rather, it converts data to a predetermined format and performs shallow syntactic checking. after being processed by the data normalization module, data are stored directly in bioportal's main data store. this hl7 xml-based design provides a key advantage over an alternative design based on a consolidated database for which the portal data store must consolidate and maintain the data fields for all data sets. when an underlying data set changes its data structure, a portal data store based on the consolidated database must be redesigned and reloaded to reflect the changes, which severely limits system scalability and extensibility. to alleviate potential computational performance problems with our xml-based design, we identify a core set of data fields based on the queries that are likely to be performed frequently. these fields are extracted from all xml messages and stored in a separate database table to enable fast retrieval. 2) communication backbone: the communication backbone component enables data exchanges between bioportal and the underlying data sources. several federal programs have been recently created to promote data sharing and system interoperability in the healthcare domain; the cdc's national electronic disease surveillance system (nedss) initiative is particularly relevant for our research. it builds on a set of recognized national standards such as hl7 for its data format and messaging protocols, and provides basic modeling and ontological support for data models and vocabularies. the nedss and hl7 standards have had major impacts on the development of idi systems. although these standards have not yet been tested in cross-state sharing scenarios, they provide an appropriate foundation for data exchange standards in national and international contexts. bioportal relies heavily on nedss/hl7 standards. the communication backbone component uses a collection of source-specific "connectors" to communicate with contributing sources. we use the connector linking new york's hin system and bioportal to illustrate a typical design. the data from hin to the portal system are transmitted in a "push" manner, i.e., hin send through secure public health information network messaging system (phin ms) messages to the portal at prespecified time intervals. the connector on the portal side runs a data receiver daemon to listen for incoming messages. after a message is received, the connector checks for data integrity syntactically and normalizes the data. the connector then stores the verified message in the portal's internal data store through its data ingest control module. other data sources (e.g., usgs) may have "pull"-type connectors that periodically download information from source web sites, and examine and store those data in the portal's internal data store. in general, the communication backbone component provides data receiving and sending functionalities, source-specific data normalization, and data-encryption capabilities. 3) data confidentiality and access control: data confidentiality, security, and access control are among the key research and development issues for the bioportal project. with regard to system development, programming and rules already developed for new york's hin system constitute the main sources of our design and implementation decisions. because there was no precedent for extending access to a data system across state lines, we needed to develop new access rules for bioportal. we have created new security and user agreement forms for organizations with proposed access as well as individual users within those organizations. in addition, the agencies involved in developing bioportal formally signed a memorandum of understanding prior to sharing any real data. the responsibilities of participating organizations and individuals with access include: 1) the establishment and definition of roles within the agency for access, and the determination of those individuals who fill those roles, including systems for termination of access; 2) security of data physically located on, or transported over the organization's network; 3) protection for the confidentiality of all data accessed, with prohibitions against disclosure of personal or health information to any other agency, person, or public media outlet; and 4) recognition of ownership rights of parties that have provided data. the types of data that must be addressed separately with regard to access are data from humans or owned animals that require the highest levels of confidentiality, data from freeranging wildlife, and data from other systems such as vectors (e.g., mosquitoes for wnv), land use, and so forth. the need for maximum access to track diseases must be balanced against the confidentiality concerns and risks of jeopardizing data reporting to the system. we summarize bioportal's data coverage in table i. 4) data search and alerting: bioportal provides limited data search functions to regular idi users. instead of developing a generic data search interface with a full range of search criteria, after extensive discussions with potential end users (i.e., state and county epidemiologists and public health researchers), we decided to concentrate on search criteria based primarily on location and time. a specialized tabular interface allows users to quickly identify infectious disease cases that occurred at certain locations within a specified period of time. through this interface, the user can also get summary case counts across locations and times with different levels of granularity. an advanced search module is also available to power users. using this module, a power user can build a personalized search interface that includes additional data-set-specific search criteria. because bioportal can perform idi data analysis automatically without user intervention, if potentially interesting events are detected, the concerned individuals (e.g., epidemiologists in charge) should be alerted. we are currently implementing three types of alerting mechanisms. the first one is by e-mail. the second is through the bioportal web interface, so when a user authorizes himself or herself on the bioportal site and an alert message exists, a flashing icon will notify the user of the pending message. the third mechanism is cellular phone notification through an automated web-based short text message interface for urgent alerts. an important role of visualization in the context of large and complex data set exploration is to organize and characterize the data visually to assist users in overcoming information overload problems [10] . bioportal makes available an advanced visualization module, called the spatial temporal visualizer (stv), to facilitate the exploration of infectious disease case data and summarize query results. developed as a generic visualization environment, stv can be used to visualize various spatial-temporal data sets simultaneously. the stv has three integrated and synchronized views: periodic, timeline, and gis. the periodic view provides the user with an intuitive display to identify periodic temporal patterns. the timeline view provides a two-dimensional timeline, along with a hierarchical display of the data elements organized as a tree. the gis view displays cases and sightings on a map. fig. 2 illustrates how these three views can be used to explore infectious disease data sets; the top left panel shows the gis view. the user can select multiple data sets to be shown on the map in a layered manner using the checkboxes (e.g., disease cases, natural land features, land-use elements). the top-right panel corresponds to the timeline view and displays the occurrences of various cases using a gantt chart-like display. the user can also access case details easily using the tree display located to the left of the timeline display. below the timeline view is the periodic view with which the user can identify periodic temporal patterns (e.g., months with an unusually high number of cases). the bottom portion of the interface allows the user to specify subsets of data to be displayed and analyzed. as discussed in section ii-a, to achieve fine-graded interface control and high interactivity, stv has been developed as a standalone java application, which can be deployed transparently across the web. the essential data elements (location, time, and event type) displayed by stv are all captured in the relational tables in the bioportal internal data store. the auxiliary data elements (e.g., case details, needed only when a user wants to learn more about a particular data point) may be re-trieved from the hl7 xml messages stored in the bioportal internal data store. because stv is executed on the client machine, real-time data transmissions between the client machine and bioportal server are necessary. for better performance and shorter response time, stv caches much of the data needed on the client side. in addition to data access, query, and visualization, bioportal provides data analysis capabilities, particularly in the area of spatial-temporal data analysis. in idi applications, measurements of interest such as disease cases are often made at various locations in both space and time. in recent years, interest has increased in answering several central questions, which have great practical importance in outbreak detection and arise from spatial-temporal data analysis and related predictive modeling: how can areas with exceptionally high or low measures be identified? how can observers determine whether unusual measures can be attributed to known random variations or are statistically significant? in the latter case, how should the explanatory factors be assessed? how can statistically significant changes be identified in a timely manner in specific geographic areas? for instance, unusual clustering of dead birds has been proven to be highly indicative of wnv outbreaks. from a modeling and computational perspective, two distinct types of spatial-temporal clustering or hotspot analysis techniques have been developed. the first type is based on various kinds of scan statistics, and has been used with increasing frequency in public health and infectious disease studies [11] . the second type is based on data clustering and its variations, and has found successful application in crime analysis [12] . bio-portal makes both types of methods available through its web interface. in addition, it allows the user to interactively invoke these methods and visually inspect their results through stv. one major computational problem faced by existing methods is that the shapes of potential hotspots are limited to simple, fixed symmetrical shapes for analytical and search efficiency reasons. as a result, when the real underlying clusters do not conform to such shapes, the identified regions are often poorly localized. to overcome this major computational limitation, as part of the bioportal technical research effort, we have developed an alternative and complementary modeling approach called riskadjusted support vector clustering (rsvc). hotspot analysis differs from standard clustering in that clustering must be performed relative to baseline data points (representing a "normal" situation). in rsvc, we apply the "risk adjustment" concept from a crime hotspot analysis approach [12] to incorporate baseline information in the clustering process. the basic intuition behind rsvc is as follows: a robust, svm-based clustering mechanism called support vector clustering allows detection of clusters with arbitrary shapes based on the distances defined over pairs of data points. by adjusting distance measures proportionally to the estimated density of the baseline factor, areas with high baseline density make it more difficult to group data points together as clusters, because the distances between these data points have been adjusted upward. we have also extended our rsvc approach to perform prospective hotspot analysis aimed at monitoring data sources on a continuous basis. for technical details of this bioportal spatial-temporal data analysis work, interested readers are referred to [13] and [14] . we conducted a controlled experiment to evaluate bioportal holistically. our foci were objective user task performance and subjective self-reported system assessments. our evaluation did not involve algorithmic assessments or examine individual components of bioportal (e.g., hotspot analysis and outbreak detection), which have been studied previously [14] . in this section, we discuss the hypotheses tested and detail our evaluation design. we followed the system success evaluation framework by delone and mclean [15] , and focused on evaluating the essential system characteristics of bioportal and its impacts on user task performance measured objectively by analysis accuracy and task completion efficiency. we also examined users' self-reported assessments of bioportal in terms of satisfaction, usability, usefulness, and ease of use, all of which are critical in system evaluations [16] . for benchmark purposes, we included a computer-based spreadsheet program commonly used by public health professionals in their analysis tasks. 1) analysis accuracy: by integrating interrelated data extracted from different sources and presenting them in a visually intuitive and comprehensible way, bioportal can be expected to better support various analysis tasks by public health professionals. therefore, we tested the following hypotheses. h1a : the analysis accuracy that results from the use of bioportal is higher than that associated with the benchmark spreadsheet program. h1b : the accuracy improvement that results from the use of bioportal, as compared with the benchmark spreadsheet program, increases with task complexity. 2) task completion efficiency: by providing convenient access to integrated data extracted from difference sources, together with easy-to-use analytical algorithms and effective visualization, bioportal can be expected to make public health professionals increasingly efficient in their task performance. we, therefore, tested the following hypothesis. h2 : the task completion efficiency associated with bioportal is higher than that observed with the benchmark spreadsheet program. 3) user satisfaction: user satisfaction is a fundamental aspect of system evaluation and embraces user information satisfaction that emphasizes information requirements [17] . because of the critical importance of information support in an idi system, we explicitly focused on user information satisfaction and tested the following hypothesis. h3 : the user information satisfaction that results from the use of bioportal is significantly higher than that observed with the benchmark spreadsheet program. system usability has been shown to affect user adoption, system usage, and satisfaction [18] . several usability instruments have been developed and validated [19] , [20] . of particular importance is the user interaction satisfaction (quis) scale [19] capable of assessing a system in five fundamental usability dimensions-overall reactions to the system, screen layout and sequence, terminology and system information, system learnability, and system capabilities. we tested the following hypothesis. h4 : bioportal is more usable than the benchmark spreadsheet program and shows favorable usability scores in overall reaction to the system, screen layout and sequence, terminology and system information, system learnability, and system capabilities. system usefulness is critical to voluntary use of a new system [21] , [22] , and generally refers to the extent to which an individual considers a system useful in his or her work role. bioportal offers effective data integration support, and has sophisticated built-in functionalities and intuitive visualization designs; as a result, it can be expected to better support the demanding information processing often required in an analysis task. hence, we tested the following hypothesis. h5 : the usefulness of bioportal, as perceived by an individual, is significantly greater than that of the benchmark spreadsheet program. 6) perceived ease of use: perceived ease of use refers to the degree to which an individual considers his or her use of a system to be free of effort [21] . ease of use represents an essential motivation for individuals' voluntary use of a system [23] , and can affect their adoption decisions significantly [22] . hence, we tested the following hypothesis. h6 : the ease of use of bioportal, as perceived by an individual, is significantly greater than that of the benchmark spreadsheet program. we adopted a randomized, between-groups design. our subjects were graduate students attending the management school or the public health school of a major university located in the southwestern united states. all subjects were knowledgeable about computer-based spreadsheets but varied substantially in general public health knowledge. each subject was randomly assigned to use one particular system (bioportal or the spreadsheet program), though we remained mindful of maintaining a balance in the subject-technology assignment. with the assistance of several experienced public health researchers and professionals, we created six analysis scenarios common in public health and then developed a total of 11 experiment tasks accordingly. the assisting experts classified the experiment tasks on the basis of complexity: low, medium, or high. a complete listing of the scenarios and analysis tasks used in the experiment is available upon request. we provide two examples as follows. scenario 1: examine data related to wnv. task 1: in 2002, which county in new york had the highest dead bird count? (complexity = low) task 2: of the three listed bird species, bluejay, crow, and house sparrow, which had the highest number of positive cases of wnv? (complexity = low) scenario 6: determine correlations between the incidence of wnv and dead bird occurrences and mosquito pool counts. task 10: using the bioportal system or the spreadsheets, as assigned, to investigate wnv disease, can you determine whether, during 2002, there is a correlation between the dead bird occurrences and mosquito pool counts? (complexity = high) task 11: (continued with task 10) if so, what correlation do you observe? (complexity = high) to assess an individual subject's accuracy in each task, we consolidated the analyses by the assisting experts to establish a "gold-standard" solution for that task. we measured analysis accuracy using a ten-point scale, with one being completely incorrect and ten being completely correct. we measured task completion efficiency by using the amount of time that a subject needed to complete a task. we evaluated user information satisfaction [17] using a sevenpoint likert scale, with one indicating extreme disagreement and seven indicating extreme agreement. we adapted question items from previous research [21] to measure system usefulness and ease of use, using a seven-point likert scale with one indicating extreme disagreement and seven indicating extreme agreement. we adopted the quis instrument [19] with a nine-point likert scale to evaluate system usability. 1 before the experiment, we used a script to inform the subjects explicitly of our objective and data analysis plan while ensuring them of the necessary information privacy. subjects were asked to provide some demographic information, and self-assessments of their general computer self-efficacy and knowledge about computer-based spreadsheets and public health. we provided each subject with an overview of his or her assigned system and a training session based on sample tasks to illustrate how to use that system. in the experiment, each subject was asked to complete all analysis tasks grouped by analysis scenario and sequenced in increasing complexity, i.e., tasks progressing from low to high complexity. after completing all the tasks, each subject had to complete a questionnaire survey to provide his or her assessment of the system's usability, usefulness, and ease of use, as well as his or her satisfaction with the information support by the system. we imposed a 50-min time limit in the experiment, which was appropriate according to the results of a pilot study [24] . a total of 33 subjects voluntarily participated in the experiment. among them, 17 subjects used bioportal, and the remainder used the spreadsheet program. of those using bioportal, 9 subjects had high domain knowledge and the others were low in domain knowledge. a similar distribution was observed in the spreadsheet group. according to our analysis, the subjects in the bioportal and spreadsheet groups are comparable demographically, and reported similar self-assessments in general computer efficacy and computer-based spreadsheets. we reexamined the reliability of our instrument by assessing its internal consistency [25] . as summarized in table ii , the subjects' evaluative responses showed that almost all constructs exhibited a cronbach's alpha value exceeding the commonly suggested threshold of 0.8 [26] , thus, suggesting adequate reliability of our instrument. we tested the main effect of system (bioportal versus the spreadsheet program) and domain knowledge (low versus high general public health knowledge) as well as their combined effects by performing an analysis of variance (anova) with each dependent variable on the basis of subjects' responses. we also performed a paired t-test to assess the difference in each dependent variable obtained from the subjects using bioportal versus the spreadsheet program. we used the gold-standard result to evaluate the accuracy of each task performed by subjects. for each subject, we aggregated his or her analysis accuracy across all the tasks performed in the experiment and used the overall accuracy to test the hypothesized main effect of system. according to our analysis, the system had a significant effect on analysis accuracy (p-value < 0.01). we further investigated the effect of system on the basis of task complexity, and found that the system's effect on analysis accuracy was insignificant for low-complexity tasks but significant for tasks of medium and high complexity. bioportal's accuracy was greater (mean = 81.94, sd = 21.23) than that of the spreadsheet program (mean = 61.19, sd = 17.92), and the difference was significant at the 0.01 level. thus, our data supported h1a and h1b. based on our analysis, the system showed a significant main effect on task completion efficiency (p-value <0.01). we compared the amount of time required to complete a task using the respective systems and found that, on average, subjects using bioportal could complete an analysis task considerably faster (mean = 36.28 min, sd = 11.33 min) than their counterparts supported by the spreadsheet program (mean = 48.23 min, sd = 5.07 min); the difference was significant at the 0.01 level. thus, our data supported h2. according to our analysis, the main effect of the system on user information satisfaction was significant statistically (pvalue <0.01). overall, subjects using bioportal exhibited higher satisfaction with the information support (mean = 2.34, sd = 1.02) than their counterparts supported by the spreadsheet program (mean = 3.68, sd = 1.23); the difference was significant at the 0.01 level. thus, our data supported h3. according to our analysis, the system had a significant main effect on both overall reactions to the system (p-value <0.01) and system capabilities (p-value <0.05) but not on screen layout and sequence or terminology and system information. the effect on system learnability was somewhat significant statistically. overall, our subjects considered bioportal generally usable and recognized its utilities for supporting their analysis tasks. our evaluation results indicated that the design of bioportal may need to improve in screen layout and sequence, as well as in language (e.g., clarity and user friendliness). our subjects considered their learning to use bioportal not particularly difficult, but its learnability could be enhanced further. according to our comparative analysis of subjects' self-reported assessments of the respective systems, bioportal arguably was more usable than the spreadsheet program in most, but not all, fundamental usability dimensions, though the between-groups differences are not statistically significant. thus, our data partially supported h4. our analysis shows that the system had a significant effect on perceived usefulness (p-value <0.05). overall, our subjects considered bioportal more useful for supporting their analysis tasks (mean = 2.13, sd = 1.06) than the spreadsheet program (mean = 3.47, sd = 1.88). the observed between-groups difference was statistically significant at the 0.05 level. thus, our data supported h5. according to our analysis, the effect of system on perceived ease of use was significant statistically (p-value <0.05). our subjects considered bioportal easier to use (mean = 2.31, sd = 1.06) than the spreadsheet program (mean = 3.24, sd = 0.88). the between-groups difference in perceived ease of use was significant at the 0.01 level. therefore, our data supported h6. the development of advanced idi systems and their routine use by public health professionals are becoming increasingly critical. we report here a significant idi effort, i.e., bioportal that supports cross-jurisdictional data integration with advanced data query, analysis, and visualization capabilities. we conducted a controlled experiment to evaluate bioportal along some fundamental system evaluation dimensions and investigated its effects on user task performance, with particular focus on analysis accuracy, task completion efficiency, and user information satisfaction, system, usability, usefulness, and ease of use. our study generated encouraging findings that suggest desirable effectiveness, usefulness, and ease of use of bioportal. we make several contributions to idi research and practice. first, we designed and implemented an advanced idi system by addressing essential system development challenges pertinent to data/system integration, analysis support, and visualization. second, we conducted a controlled experiment to evaluate bio-portal and its impacts on user task performance. our evaluation had methodological rigor and involved analysis scenarios and tasks common to public health professionals. third, we are contributing to general practices in public health by providing practitioners with a conveniently accessible, easy-to-use system that enables them to generate better analysis results in less time. our future research includes further system enhancements and expanded system evaluations. both system functionalities and usability need further improvement, including hotspot analysis and such usability dimensions as screen layout and sequence and system information. on the evaluation front, the reported evaluation only considers wnv, botulism, and foot-and-mouth disease and emphasizes frequency-and pattern-related analysis tasks. to better mimic real-world challenges in public health, additional and preferably more diverse analysis scenarios and tasks must be considered in future evaluation studies. while our subject choice is appropriate for the intended evaluation purpose and hypothesis testing, future investigations should also involve public health researchers and practitioners, preferably from different institutions and regions. paul jen-hwa hu received the ph.d. degree in management information systems from the university of arizona, tucson. he is an associate professor and david eccles faculty fellow with the david eccles school of business, university of utah, salt lake city. his current research interests include healthcare information systems and management, technology implementation management, electronic commerce, digital government, human-computer interaction, and knowledge management. he has authored more than 30 papers in information systems and ieee journals. he is currently an associate professor and the director of the intelligent systems and decisions laboratory in the department of management information systems, university of arizona, tucson. he is an affiliated professor with the institute of automation, chinese academy of sciences, beijing, china. his research interests include software agents and multiagent systems, complex systems analysis, recommender systems, digital economic institutions, automated negotiation and auction, spatio-temporal data analysis, security informatics, and infectious disease informatics. dr. zeng is the chair of the informs college on artificial intelligence and the vice president for technical activities of the ieee intelligent transportation systems society. hsinchun chen received the bachelor's degree in management science from the national chiao-tung university, taiwan, r.o.c., the m.b.a. degree from the state university of new york, buffalo, and the ph.d. degree in information systems from new york university, new york, ny. he is the mcclelland professor with the department of management information systems, university of arizona, tucson, where he is the founding director of the hoffman e-commerce laboratory as well as the founder and director of the artificial intelligence laboratory. he is the author/coauthor of more than 130 papers published in several journals and conference proceedings. his research interests include intelligence analysis, biomedical informatics, data, text, and web mining, digital library, knowledge management, and web computing. catherine larson received the bachelor's degree in liberal arts (spanish and anthropology) and the master's degree in library and information science from the university of illinois at urbana-champaign, champaign. she is currently the associate director of the artificial intelligence laboratory, department of management information systems, university of arizona, tucson. wei chang received the bachelor's degree from tsinghua university, beijing, china, and the master's degree from the university of arizona, tucson, both in management information systems. he is currently working toward the ph.d. degree in operation research and decision science at the university of pittsburgh, pittsburgh, pa. chunju tseng received the b.s. degree from national taiwan university, taiwan, r.o.c., and the m.s. degree in management information systems from the university of arizona, tucson. he is an assistant research scientist with the department of management information systems, university of arizona. his research interests include web mining, infectious disease surveillance, and humancomputer interaction. he has authored several papers published in conference proceedings. james ma received the b.sc. degree from peking university, beijing, china, and the master's degree in computer science from the university of texas, dallas. he is currently working toward the ph.d. degree in management information systems at the university of arizona, tucson. factors in the mergence of infectious diseases predicting super spreading events during the 2003 severe acute respiratory syndrome epidemics in hong kong and singapore the public health infrastructure and our nation's health disease surveillance and the academic, clinical, and public health communities a novel spatio-temporal data analysis approach based on prospective support vector clustering a national agenda for public health informatics innovative information sharing strategies surveillance and control of infectious diseases at local, national and international levels heterogeneous database integration in biomedicine creating a large-scale content-based airphoto image digital library prospective time periodic geographical disease surveillance using a scan statistic crimestat: a spatial statistics program for the analysis of crime incident locations a comparative study of spatio-temporal data analysis techniques in security informatics spatial-temporal cross-correlation analysis: a new measure and a case study in infectious disease informatics the delone and mclean model of information systems success: a ten-year update evaluating public sector information systems: satisfaction versus impact the measurement of user information satisfaction the usability of everyday technology: emerging and fading opportunities the influence of climate on the epidemiology of bartonellosis in ancash designing the user interface: strategies for effective human-computer interaction perceived usefulness, perceived ease of use, and user acceptance of information technology the technology acceptance model: past, present, and future determinants of perceived ease of use: integrating control, intrinsic motivation, and emotion into the technology acceptance model evaluating an infectious disease information sharing and analysis system validating instruments in mis research applied multiple regression/correlation analysis for the behavioral sciences key: cord-027101-6wq1wqh3 authors: paszyński, maciej; siwik, leszek; podsiadło, krzysztof; minev, peter title: a massively parallel algorithm for the three-dimensional navier-stokes-boussinesq simulations of the atmospheric phenomena date: 2020-05-26 journal: computational science iccs 2020 doi: 10.1007/978-3-030-50371-0_8 sha: doc_id: 27101 cord_uid: 6wq1wqh3 we present a massively parallel solver using the direction splitting technique and stabilized time-integration schemes for the solution of the three-dimensional non-stationary navier-stokes-boussinesq equations. the model can be used for modeling atmospheric phenomena. the time integration scheme utilized enables for efficient direction splitting algorithm with finite difference solver. we show how to incorporate the terrain geometry into the simulation and how to perform the domain decomposition. the computational cost is linear [formula: see text] over each sub-domain, and near to [formula: see text] in parallel over 1024 processors, where n is the number of unknowns and c is the number of cores. this is even if we run the parallel simulator over complex terrain geometry. we analyze the parallel scalability experimentally up to 1024 processors over a prometheus linux cluster with multi-core processors. the weak scalability of the code shows that increasing the number of sub-domains and processors from 4 to 1024, where each processor processes the subdomain of [formula: see text] internal points ([formula: see text] box), results in the increase of the total computational time from 120 s to 178 s for a single time step. thus, we can perform a single time step with over 1,128,000,000 unknowns within 3 min. the number of unknowns results from the fact that we have three components of the velocity vector field, one component of the pressure, and one component of the temperature scalar field over 256,000,000 mesh points. the computation of the one time step takes 3 min on a linux cluster. the direction splitting solver is not an iterative solver; it solves the system accurately since it is equivalent to gaussian elimination. our code is interfaced with the mesh generator reading the nasa database and providing the earth terrain map. the goal of the project is to provide a reliable tool for parallel, fully three-dimensional computations of the atmospheric phenomena. air pollution is receiving a lot of interest nowadays. it is visible, especially in the kraków area in poland (compare fig. 1 ), as this is one of the most polluted cities in europe [1] . people living there are more and more aware of the problem, which causes the raising of various movements that are trying to improve air quality. air pollution grows because of multiple factors, including traffic, climate, heating in the winter, the city's architecture, etc. the ability to model atmospheric phenomena such as thermal inversion over the complicated terrain is crucial for reliable simulations of air pollution. thermal inversion occurs when a layer of warm air stays over a layer of cool air, and the warm air holds down the cool air and it prevents pollutants from rising and scattering. we present a massively parallel solver using the direction splitting technique and stabilized time-integration schemes for the solution of the three-dimensional non-stationary navier-stokes-boussinesq equations. the navier-stokes-boussinesq system is widely applied for modeling the atmospheric phenomena [2] , oceanic flows [3] as well as the geodynamics simulations [4] . the model can be used for modeling atmospheric phenomena, in particular, these resulting in a thermal inversion. it can be used as well for modeling several other important atmospheric phenomena [5, 6] . it may even be possible to run the climate simulation of the entire earth atmosphere using the approach presented here. the time integration scheme utilized results in a kronecker product structure of the matrices, and it enables for efficient direction splitting algorithm with finite difference solver [7] , since the matrix is a kronecker product of three three-diagonal matrices, resulting from discretizations along x, y, and z axes. the direction splitting solver is not an iterative solver; it is equivalent to the gaussian elimination algorithm. we show how to extend the alternating directions solver into non-regular geometries, including the terrain data, still preserving the linear computational cost of the solver. we follow the idea originally used in [8] for sequential computations of particle flow. in this paper, we focus on parallel computations, and we describe how to compute the schur complements in parallel with linear cost, and how to aggregate them further and still have a tri-diagonal matrix that can be factorized with a linear computational cost using the thomas algorithm. we also show how to modify the algorithm to work over the complicated non-regular terrain structure and still preserve the linear computational cost. thus, if well parallelized, the parallel factorization cost is near to o(n/c) in every time step, where n is the number of unknowns and c is the number of cores. we analyze the parallel scalability of the code up to 1024 multi-core processors over a prometheus linux cluster [9] from the cyfronet supercomputing center. each subdomain is processed with 50 × 50 × 100 finite difference mesh. our code is interfaced with the mesh generator [10] reading the nasa database [11] and providing the earth terrain map. the goal of the project is to provide a reliable tool for parallel fully three-dimensional computations of the atmospheric phenomena resulting in the thermal inversion and the pollution propagation. in this paper, we focus on the description and scalability of the parallel solver algorithm, leaving the model formulation and large massive parallel simulations of different atmospheric phenomena for future work. this is a challenging task itself, requiring to acquire reliable data for the initial state, forcing, and boundary conditions. the equations in the strong form are where u is the velocity vector field, p is the pressure, p r = 0.7 is the prandt number, g = (0, 0, −1) is the gravity force, ra = 1000.0 is the rayleigh number, t is the temperature scalar field. we discretize using finite difference method in space and the time integration scheme resulting in a kronecker product structure of the matrices. we use the second-order in time unconditionally stable time integration scheme for the temperature equation and for the navier-stokes equation, with the predictor-corrector scheme for pressure. for example we can use the douglass-gunn scheme [13] , performing an uniform partition of the time inter-valī = [0, t ] as and denoting τ := t n+1 − t n , ∀n = 0, . . . , n − 1. in the douglas-gunn scheme, we integrate the solution from time step t n to t n+1 in three substeps as follows: for the navier-stokes equations, l 1 = ∂ xx , l 2 = ∂ yy , and l 3 = ∂ zz , and the forcing term represents gprrat n+1/2 plus the convective flow and the pressure terms u n+1/2 · ∇u n+1/2 + ∇p n+1/2 treated explicitly as well. the pressure is computed with the predictor/corrector scheme. namely, the predictor step with p − 1 2 = p 0 and φ − 1 2 = 0 computes the pressure to be used in the velocity computations, the penalty steps and the corrector step updates the pressure field based on the velocity results and the penalty step these steps are carefully designed to stabilize the equations as well as to ensure the kronecker product structure of matrix, resulting in the linear computational cost solver. the mathematical proofs of the stability of the formulations, motivating such the predictor/corrector (penalty) steps, can be found in [7, 12] and the references there. for the temperature equation, l 1 = ∂ xx , l 2 = ∂ yy , and l 3 = ∂ zz , and the forcing term represents the advection term treated explicitly u n+1/2 · ∇t n+1/2 . for mathematical details on the problem formulation and its mathematical properties, we refer to [12] . each equation in our scheme contains only derivatives in one direction, so they are of the following form or the update of the pressure scalar field. thus, when employing the finite difference method, we either endup with the kronecker product matrices with submatrices being three-diagonal, or the point-wise updates of the pressure field where α = τ /2 or α = 1, depending on the equation, which is equivalent to these systems have a kronecker product structure m = a x ⊗ b y ⊗ c z where the sub-matrices are aligned along the three axis of the system of coordinates, one of these sub-matrices is three-diagonal, and the other two sub-matrices are scalled identity matrices. from the parallel matrix computations point of view, discussed in our paper, it is important that in every time step, we have to factorize in parallel the system of linear equations having the kronecker product structure. the direction splitting algorithm for the kronecker product matrices implements three steps, which result is equivalent to the gaussian elimination algorithm [14] , each of the three systems is three-diagonal, ⎡ and we can solve it in a linear o(n ) computational cost. first, we solve along x direction, second, we solve along y direction, and third, we solve along z direction. to obtain a reliable three-dimensional simulator of the atmospheric phenomena, we interconnect several components. we interface our code with mesh generator that provides an excellent approximation to the topography of the area [10], based on the nasa database [11] . the resulting mesh generated for the krakow area is presented in fig. 2 . in our system of linear equations, we have several tri-diagonal systems with multiple right-hand-sides, factorized along x, y and z directions. each unknown in the system represents one point of the computational mesh. in the first system, the rows are ordered according to the coordinates of points, sorted along x axis. in the second system, the rows are ordered according to the y coordinates of points, and in the third system, according to z coordinates. when simulating the atmospheric phenomena like the thermal inversion over the prescribed terrain with alternating directions solver and finite difference method, we check if a given point is located in the computational domain. the unknowns representing points that are located inside the terrain (outside the atmospheric domain) are removed from the system of equations. this is done by identifying the indexes of the points along x, y, and z axes, in the three systems of coordinates. then, we modify the systems of equations, so the corresponding three rows in the three systems of equations are reset to 0, the diagonal is set to 1, and the corresponding rows and columns of the three right-hand-sides are set 0. for example, if we want to remove point (r, s, t) from the system, we perform the following modification in the first system. the rows in the first system they follow the numbering of points along x axis. the number of columns corresponds to the number of lines along x axis perpendicular to oy z plane. each column of the right-hand side correspond to yz coordinates of a point over oy z plane. we select the column corresponding to the "st" point. we factorize the system with this column separately, by replacing the row in the matrix by the identity on the diagonal and zero on the right-hand side. the other columns in the first system are factorized in a standard way. ⎡ analogous situation applies for the second system, this time with right-hand side columns representing lines perpendicular to oxz plane. we factorize the "rt" column in the second system separately, by setting the row in the matrix as the identity on the diagonal, and using 0.0 on the right-hand side. the other columns in the second system are factorized in the standard way. similarly, in the third system we factorize the "rs" column separately. the other columns in the third system are factorized in a standard way. ⎡ using this trick for all the points in the terrain, we can factorize the kronecker product system in a linear computational cost over the complex terrain geometry. the computational domain is decomposed into several cube-shape sub-domains. we generate systems of linear equations over each sub-domain separately, and we enumerate the variables in a way that interface unknowns are located at the end of the matrices. we compute the schur complement of the interior variables with respect to the interface variables. we do it in parallel over each of the subdomains. the important observation is that the schur complement matrices will also be three-diagonal matrices. this is because the subdomain matrix is three-diagonal, and the schur complement computation can be implemented as forward eliminations, performed in the three sub-systems, each of them stopped after processing the interior nodes in the particular systems. later, we aggregate the schur complements into one global matrix. we do it by global gather operation. this matrix is also tri-diagonal and can be factorized in a linear cost. later, we scatter the solution, and we use the partial solutions from the global matrix to backward substitute each of the systems in parallel. these operations are illustrated in fig. 3 . we perform this operation three times, for three submatrices of the kronecker product matrix, defined along three axes of the coordinate system. we provide algebraic details below. thus, assuming we have r−1 rows to factorize in the first system (r−1 rows in the interior, and k − r + 1 rows on the interface), we run the forward elimination over the first matrix, along the x direction, and we stop it before processing the r-th row (denoted by red color). this partial forward elimination stopped at the r-th row ensures that below that row we have the schur complement of the first r − 1 rows related with the interior points in the domain with respect to the next k − r + 1 rows related with the interface points (the schur complement is denoted by blue color). this schur complement matrix is indeed tri-diagonal: we perform this operation on every sub-domain, and then we gather on processor one the tri-diagonal schur complements, we aggregate them into one matrix along x direction. the matrix is still a tri-diagonal matrix, and we solve the matrix using linear o(n ) computational cost gaussian elimination procedure with the thomas algorithm. next, we scatter and substitute the partial solutions to sub-system over subdomains. we do it by replacing the last r − k + 1 rows by the identity matrix and placing the solutions into the right-hand side blocks. namely, on the right-hand side we replace rows from r + 1 (denoted by blue color) by the solution obtained in the global phase, to obtain: ⎡ 0 · · · · · · · · · · · · · · · 0ã x 22ã x 23 0 · · · · · · · · · · · · . . . . . . 0 · · · · · · · · · · · · 0 1 0 0 · · · · · · · · · · · · · · · 0 1 and running backward substitutions over each subdomain in parallel. next, we plug the solutions to the right-hand side of the second system along y axis, and we continue with the partial factorization. now, we have s − 1 rows in the interior and l − s + 1 rows on the interface. we compute the schur complements in the same way as for the fist subsystem, thus we skip the algebraic details here. we perform this operation on every sub-domain, then we collect on processor one and aggregate the schur complements into the global matrix along y directions. the global matrix is three-diagonal, and we solve it with thomas algorithm. next, we scatter and substitute the partial solutions to sub-system on each subdomain, and we solve by backward substitutions. finally, we plug the solution to the right-hand side of the third system along z axis, and we continue with the partial factorization. now, we have t − 1 rows in the interior and m − t + 1 rows on the interface. the partial eliminations follow the same lines as for the two other directions, thus, we skip the algebraic details. we repeat the computations for this third direction, computing the schur complements on every sub-domain, collecting them into one global system, which is still three-diagonal, and we can solve it using the linear computational cost thomas algorithm. next, we substitute the partial solution to sub-systems. we replace the last t − m + 1 rows by the identity matrix, and place the solutions into the right-hand side, and run the backward substitutions over each subdomain in parallel. the solver is implemented in fortran95 with openmp (see algorithm 1) and mpi libraries used for parallelization. it does not use any other libraries, and it is a highly optimized code. we report in fig. 4 and table 1 the weak scalability for three different subdomain sizes, 50 × 50 × 100, 25 × 100 × 100, and 50 × 50 × 50. the weak scalability for the subdomains of 49 × 49 × 99 internal points, shows that increasing the number of processors from 4 to 1024, simultaneously increasing the number of subdomains from 4 to 1024, and the problem size from 50 × 50 × 400 to 800 × 800 × 400, results in the increase of the total computational time from 120 s to 178 s for a single time step. thus, we can perform a single time step with over 1,128,000,000 unknowns (three components loop end =nrhs/10; i=1; inverse =1.0/(mat%b(n)-cp(n-1)*mat%a(n)); dmult =mat%a(n) do j = 1,loop end if(i<nrhs-10)then finish = i*(nint+2)-1 dp(n,i) = (d(finish) -dp(n-1,i) * dmult)*inverse dp(n,i+1) = (d(finish+(nint+2)) -dp(n-1,i+1) * dmult)*inverse ... dp(n,i+9) = (d(finish+(nint+2)*9) -dp(n-1,i+9) * dmult)*inverse i=i+10 end if end do of the velocity vector field, and one component of the pressure and the temperature scalar fields over 256,000,000 mesh points) within 3 min on a cluster. for the numerical verification of the code, we refer to [15] . we report in figure 5 and table 2 the strong scalability for six different simulations, each one with box size 50 × 50 × 50, with 8, 16, 32, 64, 128 and 256 subdomains. since the number of nodes is multiplied by the number of unknowns (three components of the velocity vector field, one component of the pressure scalar field and one component of the temperature scalar field), we obtained between 8 × 50 × 50 × 5 = 5 millions, to 256 × 50 × 50 × 5 = 160 millions of unknowns. we can read the superlinear speedup for these plots, which is related to the optimization of cache usage on smaller subdomains, with optimizing the memory transfers to the computational kernel and loop unrolling technique, as illustrated in algorithm 2. in fig. 6 , we show some snapshots from the preliminary simulations. in here, we focused on the description and scalability of the parallel solver algorithm, leaving the model formulation and large massive parallel simulations of different atmospheric phenomena for the future work. this will be a challenging task itself, requiring to acquire reliable data for the initial state, forcing, and boundary conditions. we described a parallel algorithm for the factorization of kronecker product matrices. these matrices result from the finite-difference discretizations of the navier-stokes boussinesq equations. the algorithm allows for simulating over the non-regular terrain topography. we showed that the schur complements are tri-diagonal, and they can be computed, aggregated, and factorized in a linear computational cost. we analyzed the weak scalability over the prometheus linux cluster from the cyfronet supercomputing center. we assigned a subdomain with 50 × 50 × 100 finite difference mesh to each processor, and we increased the number of processors from 4 to 1024. the total execution time for a single time step increased from 120 s to 178 s for a single time step. thus, we could perform computations for a single time step with around 1,128,000,000 unknowns within 3 min on a linux cluster. this corresponds to 5 scalar fields over 256,000,000 mesh points. in future work, we plan to formulate the model parameters, initial state, forcing, and boundary conditions to perform massive parallel simulations of different atmospheric phenomena. european environment agency: air quality in europe -2017 report a review of element-based galerkin methods for numerical weather prediction: finite elements, spectral elements, and discontinuous galerkin parametric vertical coordinate formulation for multiscale, boussinesq, and nonboussinesq ocean modeling turbulent geodynamo simulations: a leap towards earth's core mathematical and physical fundamentals of climate change, chap. 11 -atmospheric dynamics asymptotic modeling of atmospheric flows high-order time stepping for the navier-stokes equations with minimal computational complexity a fast algorithm for direct simulation of particulate flows using conforming grids escience on distributed computing infrastructure. achievements of plgrid plus domain-specific services and tools the shuttle radar topography mission a new class of massively parallel direction splitting for the incompressible navier-stokes equations a general formulation of alternating direction methods matrix computations, 3rd edn direction splitting scheme for navier-stokes-boussinesq system in spherical shell geometries key: cord-340497-8l3gw6sk authors: avgoustaki, dafni despoina; xydis, george title: how energy innovation in indoor vertical farming can improve food security, sustainability, and food safety? date: 2020-09-25 journal: nan doi: 10.1016/bs.af2s.2020.08.002 sha: doc_id: 340497 cord_uid: 8l3gw6sk food safety is an important scientific field, but at the same time a discussion topic of modern society that occupies more and more space of our every day time, dealing with the preparation of food, with its nutritious value, and various transportation and storage ways aiming at preventing food-related sickness. this work compares traditional farming with greenhouses and indoor vertical farming focusing on the challenges and the opportunities for each category. the scope of this work was to stress the role of indoor vertical farming towards this direction. indoor vertical farms can produce high quality and virus-free products that can be locally distributed, inside the urban environment that such investments take place, saving annually millions of tons co2 emissions. beyond that, in this work it was pointed out how energy plays a role in food safety in such systems. it was stressed that indoor vertical farms can act as a demand response aggregator. in large scale units it could play a role to adjust their production according to different electricity prices offered in different time zones throughout the day. this way, the owners under a multi-value business model will create the opportunity to the vertical farm owners not only to improve their production but at the same time absorb inexpensive electricity offered, by creating an additional profit mechanism (multiple revenue streams) under such an approach by entering into contracts with companies in a utility electric region. sustainability of resources and safety in the food production line is a major issue globally. by 2050, it is expected that the global population will reach the 9.8 billion people, 2.4 billion people more that need to be fed (united nations, department of economic and social affairs, 2015) . today, agriculture occupies land equal to the size of south america in order to cover the demand of the global population. based on the assumption that the minimum daily demand of a single person is minimum 2000 kcal, if we maintain the same agricultural practices, we will need additional land equal advances in food security and sustainability # 2020 elsevier inc. issn 2452-2635 all rights reserved. https://doi.org/10.1016/bs.af2s.2020.08.002 1 to the size of brazil (2.1 billion acres) to cover the global food demand (despommier, 2009 ). on the other hand, according to lotze-campen et al. (2008) , the land used for agriculture is projected to be transformed for other purposes such as urbanization, energy production, or infrastructure growth. it is worth to mention, that another crucial challenge that will significantly affect agricultural production in the upcoming years is the rapid increase of the global temperature, as per each degree of temperature rise, 10% of existing agricultural land will be lost (despommier, 2010) . nowadays, climate change is a huge issue since it is expected that the upcoming 50 years will outstandingly affect the agricultural process. the significant increase of the carbon dioxide emission levels from a global perspective-since it constitutes an important impact factor of agricultural productivity-can influence the global economy via the effects on the agriculture's total production rate. in specific, based on mulatu's et al. (2016) research conducted for ethiopia, indicates that the impact of co 2 emissions will decrease 3.5% to 4.5% the real agricultural gdp since it will lead to lower the agricultural productivity and subsequently reduce the amount of traded and non-traded crops. such population increase certainly indicates a significant rise in the required food production, raising concerns on the deficiency, the quantity, and the quality of future food products. we should also take into account the fact that nowadays food travels daily thousands of miles from the production areas to the urban consumers, in order to meet the demand, releasing huge amounts of co 2 . less developed countries such as ethiopia that were mentioned above, apart from global climate change will have to face and other enlarged problems concerning food safety. for example, human excrements that are used as fertilizers (estimation of 50% of the global farming) can cause diseases such as cholera, typhoid fever and numerous parasitic infections (despommier, 2010) . nowadays, even the more developed counties have to face food safety and security problems even if this kind of infectious diseases have been eliminated. it is worth to mention the pandemic of our age, covid-19 caused by virus sars-cov-2 that was initially reported in the province of hubei, wuhan in china. the disease is estimated to have originated from a seafood market in wuhan where wild animals were traded such as marmots, bats, snakes and birds (zhou et al., 2020) . the specific family of viruses, coronaviruses, are known to be transferred from animal to humans. according to zhou et al., 2020 , it is mentioned that 96% of the genetic makeup of covid-19 is matched with the coronavirus found in bats. the uncertainty that is caused globally via covid-19 has caused apart from multiple deaths and lockdowns to most of the european countries, will affect significantly the economy and will cost trillions of dollars in the global economy, during 2020 and beyond (unctad, 2020) . food safety is a major issue of our era, as there are multiple reports of cases worldwide over the last years that have caused food recalls due to bacterial infectious diseases leading to loss of billion dollars. why do we seem to have so many outbreaks concerning food production these days? only in the us, despite the attempts to provide a safe food supply, every year are recorded 48 million foodborne illnesses, 128,000 hospitalizations and 3000 deaths (cdc, 2013) . in 2017-18, e.coli o157: h7 outbreak in the us caused sudden eruption linked to consumption of leafy greens and the romaine lettuce. the pathogen was mainly reported in the regions yuma, az and salinas, california, where greenhouse installations that produce more than 90% of the leafy vegetables and greens in the united states are based. e.coli contamination in the production line almost all of the times originates from the irrigation water used in the fields. additionally, further risk in the contamination process from various bacteria and pathogens comes from the washing of field-grown products after they are harvested, while this step can spread contamination to the whole production. the most regular technique that outdoor farming applies after harvest is to dunk lettuce heads in water tanks from rainfall or irrigation, while most greenhouses apply triple washes with running water from the local network. vertical farms are a novel type of farming in a controlled-environment with a total replacement of solar radiation with artificial lighting that provides the necessary nanometers of the spectrum for the growth and development of plants. in vertical farms, plants grow in soilless cultivation systems such as hydroponic (roots are immersed in multiple substrates, i.e., perlite, rockwool enriched water with nutrient solution), aeroponic (soilless air/ mist solution) or even aquaponic (co-cultivation of fish and hydroponic plants) systems that allow stacking multiple layers or columns of plants horizontally or vertically. vertical farms are located in completely isolated spaces from outdoor environment with thermally insulated installations (especially when at the top floor of the building) and airtight structures that give the opportunity to the farmers to control the environment in terms of temperature, humidity and co 2 (avgoustaki and xydis, 2019) . since vertical farms can theoretically be placed anywhere in the urban network, they allow local, nutritious and fresh consumption for consumers. in specific, a study conducted by jill (2008) , mentioned that food sourced from conventional farming uses 4 to 17 times more fuel compared to locally grown food and emits 5 to 17 times more co 2 . meanwhile, vertical farms may be able to increase the productivity rate in highly urbanized areas that can lead to improvements in the food security of the community. the purpose of the following subchapter is to compare the different farming techniques of outdoor farming, greenhouses and vertical farms in between them in terms of input of resources, the final product in terms of safety and the shelve life of the products in terms of nutrient status and freshness. additionally, we will examine the above criteria for lettuce, which is one of the most important cultivated species in vertical farms and will give us access to multiple data. lettuce belongs to the basic daily diet products; its nature is fragile and can be easily contaminated and spread diseases among the population. in order to make more understandable the concept of resource use efficiency, in fig. 1 the essential resources for growing plants under various farming types are shown. the most vital for plant growth is water, co 2 , light, nutrients, electricity (for ventilation purposes) and heating. as shown in fig. 1 , the definition of resource use efficiency (rue) is given by the ratio of the final plants production to the total input. in order to calculate the total input of a system, we have to summarize the input of resources, the environmental pollutants and the production system. in order to evaluate the sustainability and efficiency of a production system in the food industry, we have to assess three key directions of the system. • rue: the amount of necessary resources to produce. • the cost performance: the ratio of the sales amount to the production cost. • the vulnerability of the system, meaning the deviation of the yield production per year and the quality value per product unit. water is absolutely necessary for all food production such as vegetables, fruits, grains, meat etc. based on nederhoff and stanghellini (2010) , the water use for the global food production reaches at 5400 km 3 and has a rapid increasing rate. the irrigation water-use efficiency can be researched under different scopes and multiple concepts such as storage, delivery distribution of the water to the farm or out of the farm. additional systems that can affect water use efficiency is the ratio of water that is delivered for irrigation and the water that supplies the system. there are various ways we can calculate water use efficiency as one of the major resource inputs in food production that can be accomplished with agronomic ways, engineering or even economic approaches. more analytically, irrigation efficiency estimates the ratio between the diverted water and the consumed by the cultivation, thus it provides water-use measurements that estimate the performance of the irrigation system. on the other hand, water use efficiency is considered an economic concept that in practice evaluates the farm, as it is calculated by the crop yield unit of water diverted (kg/m 3 ). in terms of energy consumption, it is one of the reasons that causes greenhouse gas emissions (ghgs) contributing at the rising global warming. the main gases released by agricultural production are carbon dioxide (co 2 ), methane (ch 4 ) and nitrous oxide (n 2 o). since the global policy makers, organizations, researchers, retailers and producers try to propose and implement novel techniques that identify and reduce ghgs, it is necessary that we will focus and refer to the status of emissions under each farming type and propose mitigation measures in the sector. in order to describe sustainability in agriculture, it is not enough to relate sustainability with the field only from the resources perspective. understanding and evaluating what constitutes a sustainable farming system, it is of vital importance, to furthermore understand the economic and social terms that influence the contemporary issues, values and perspectives of a unique system. economic efficiency reflects to the value that is relative to the cost. in order a resource to reflect an economic value, has to be rare and difficult to obtain, for the market prices to allocate the use of this resource for competitive purposes. for example, even if air and water are essential resources for life giving them high "intrinsic" value, nevertheless under most circumstances they have no economic value due to their sufficiency levels in the environment (ikert, 2001) . they only obtain an economic value in cases of scarcity due to, e.g., high levels of pollution or drought. even if is the most ancient way that people use land, over the last decades with the technological breakthroughs and the numerous innovations introduced, outdoor farming has changed. sensors, satellites and advanced machinery allow farmers to apply more targeted (and precision) agriculture to treat the fields individually according to the needs of the crop and the soil, by dividing it in smaller parts in order to take into consideration the variability level of each unit. to complete the whole picture of climate change issues, an additional evolution process that crucially reduce the growth rate of plants is soil degradation due to excessive floods and droughts. traditional food production systems offer food solutions for people from the beginning of human history. over time, additional innovative techniques were applied in traditional farming in order to rise the productivity rate and reduce the cost and the crops overall footprint. in terms of resources, conventional farming seems to have an increase demand for water use (table 1) as traditional agriculture uses almost 70% of the available fresh water globally. furthermore, a very common problem in terms of sustainability in water use efficiency of conventional farming is the limited soil water-holding capacity that results from the limited mulching of the soil and the consistency in the same fertilizers/soil-preparation practices. scientific results (pimentel et al., 2005) have shown that this maintenance of these practices lead to low soil moisture status and low conservation levels of conventional farming systems. the most used approach for conventional farms is the irrigation efficiency and the water use efficiency. it is worth to mention that the more water applications are applied in a crop, the higher the water delivery losses are. in order to improve the water use efficiency, many farmers apply a combination of hydroponic systems with drip irrigation and smart scheduling of water distribution. hydroponics successfully address the challenge of soil drought and salinity that reduces both yield and crop quality. it should be noted that a decisive factor for the selection of hydroponic systems is the high irrigation water needs that renders the requirement for recirculating water. it becomes apparent that combination of water-saving technologies with limited-water application technologies (such as close-loop hydroponics, drip irrigation, mulching and smart scheduling of water supply) are the most effective solutions for optimizing water use efficiency. regarding land use, growing and producing food to respond to the expanding demand of the world has led agriculture production and food scarcity that can be difficulty bridged. today's farmlands, occupy almost 50% of the global habitable land (ourworldindata.org). we gathered the footprint of the various resources that meet the demand for lettuce production via traditional farming techniques. worth noticing that deforestation is a major problem, since forests are continuously sacrificed against farmland that leads to climate change acceleration and soil inability to maintain water at lower levels. depending on the cultivated variety, the techniques and the season, traditional farmed lettuce has a cultivation cycle between 1.5 and 2.5 months. therefore, farmers have the ability to grow multiple successive crops in the same field throughout a yearly cultivation period in order to increase their yield and income. additional techniques that open-field farmers follow in order to increase their yield and income per hectare (ha) of cultivated land is the density of planting, fertigation (combination of fertilization with irrigation) application and the use of healthy transplants grown in nurseries. assuming that romaine lettuce growing in the mediterranean is planted in distances of 30-50 cm between the rows and 20-35 cm between the plants, then the resulted yield reaches at 75,000-220,000 plants per ha (savvas et al., 2015) . by increasing the planting distance per row by 1 cm, it can lead to a 76% reduction of the total production. harvest period vary depending on the type or the variety of the cultivated crop. for the romaine lettuce grown outdoors, the harvest period is between 55 and 70 days with a typical yield of 25-30 tons/ha. the energy use in outdoor farming is mainly linked to fossil fuels for operations such as soil plowing, sowing, fertilization, harvesting etc. additionally, further electricity is required for pumping (water irrigation), which in developed countries can reach up to 20% of the total fossil fuel usage (despommier, 2010) . conventional farming, unfortunately, is associated to higher emissions in comparison to other types of farming. the majority of the emissions is directly linked to the transportation of the products, also known as food miles. the amount of miles that is required in order for food to travel from the producer to the consumers could release between 11 to 666 kg of co 2 emission depending on the location of the farm (gerecsey, 2018) . since farmlands are often located many kilometers away from the urban centers, where the majority of the end-user is located. food miles emissions represent on average 62% of the total emissions released throughout traditional farming. another important source of co 2 emissions that is linked to traditional farming is the significant amounts of food waste. even if food waste is not only linked to traditional farming, maladministration and mismanagement on-farm losses, and non-marketable crops put traditional farming under the spotlight of high shares of carbon footprint. for the estimation and assessment of the economic efficiency of farming, significant role in the calculation, the resources that bear an "economic" value have played a role. in traditional farming, there is limited motivation to protect and evaluate the quality, use and water maintenance, air, solar radiation and in some cases even soil fertility and productivity. the costs of a farm can vary between two main categories: the variable costs (operational expenses-opex) and the fix costs (capital expenses-capex). in the category of variable costs, all the expenses that cover particular farming actions in a specific period of time such as seeds, fertilizers, chemicals, labor are included. on the other hand, in the fix cost category, all the expenses that will be incurred regardless the process and status of production, building expenses (rent, installations, land) and equipment (irrigation system, machinery) are included. thus, the economic efficiency consists from a combination of technical and other components. based on aurangzeb et al. (2007) and a research that conducted to compare the economic efficiency between traditional farming and mechanized farming systems, it is pointed out that the net income in mechanized farms is significantly higher due to the higher yields/ha than the one of traditional farms. this effect of traditional farms could be explained by the longer time periods in soil preparation, limited tillage practices as well as the high cost requirements of labor expenses (specifically in seasonal workers during harvesting and sowing) in comparison to the high technology and mechanization farming systems. last, another factor that highly affects the final quantity of production is biodiversity. for this reason, the selection and maintenance of mono-cropping techniques that provide a uniformity in the applied practices, can reduce the labor costs and make harvesting easier. however, by cultivating only onespecies crops in the entire field, it can highly influence the biodiversity and make crops more susceptible to pathogen infections. to avoid this effect, traditional farmers apply chemicals and genetically modified organisms to maintain a simple farming system. this practice, though, requires a lot of continuous input of resources and energy (cost). the innovative and high quality mechanization and technological innovation can lead to the increase of production and hence income. multiple practices become more and more vital in traditional farming, as they improve the efficiency of resources use in general and can overall enhance sustainability. concerning the water usage, there are several approaches that new farms bring along in the field and can optimize the existing severe water waste situation. common agronomic measures such as improved crop husbandry and changed crop mix driven by the crop selection, can have a huge impact in improvement of water usage. furthermore, there are various cultivation techniques such as modification of the irrigation infrastructure, which can also influence positively the water use efficiency. last, management actions such as optimal irrigation planning and frequent maintenance irrigation system scheduled maintenance can also influence positively the system's efficiency (wheeler et al., 2015) . due to the growing population, farming has shifted to technologies that enhance significant scale-up of the production via innovative technologies. greenhouses are types of installations, designed to protect and enlarge the cultivation season of various crops. plants growing under greenhouses can grow protected from severe weather conditions such as hail, snow, extreme low temperatures or excessive heat, while at the same time can allow cultivations of out-of-season species. greenhouses first introduced in the 17th century but only on the 19th century were commercially applied in the global market. according to their installed area, greenhouses can be presented with various coverage materials such as plastic, glass, polyethylene and rigid that protect crops from the variability of the outdoor conditions, diffuses solar radiation and traps moisture, which contributes to increased plant growth. the coverage system allows farmers to control the cultivation environment according to each crop preference, as they can apply different techniques that will maintain the heating and the cooling requirements to the desired levels. this way, inside the greenhouses, farmers can develop and maintain the desired microclimate and create a more predictable environment that enhances the final plant yield, achieving higher quality and reduced water consumption compared to open field crops. there are different greenhouse systems that are diversified according to the energy flow inside the greenhouse and the resources flow in the production line. in more details, open greenhouses refer to the structure of the irrigation system, meaning that they do not collect the drained water of the crops for reuse (usually have soil-based crops). these systems seem to have low level of water usage efficiency as they are affected by water losses due to soil depletion and constant water drainage, which drains the excess amount of water with fertilizers. this waste of resources cause significant problems to the environment. usually growers can control the amount of drain as part of the management strategy of resources they follow. the percentage of drain can number between 5% and 50% of the water supply, but can be improved by reusing this drain in the irrigation system. additionally, open greenhouses use window openings as the only mean of dehumidification and cooling technique. there are also the semi-closed systems of greenhouses that have a smaller cooling capacity and window openings, combined with mechanical ventilation air-cooling systems. the combination use of mechanical systems and window openings depending on the cooling demand. concerning the irrigation systems, semi-closed greenhouses reuse the drained nutrient solution by collecting it to a tank that is constantly topped-up with fresh water. in some cases is followed water disinfection in order the collected drain water to be purified for avoiding diseases spread in the crop. to avoid imbalances in the nutrient solution, farmers use various techniques such as bleeding or dumping. in specific, bleeding techniques remove constantly 10% of the drain water, while in the dumping technique the mixing tank gets completely emptied and refilled with fresh water enriched with nutrient solution. finally, closed-systems refer to absolute mechanical support of the cooling and dehumidification system by air treatment units. the air treatment unit consists of a heat exchanger that is connected to a ventilator. the purpose of the ventilator is to withdraw air from the interior of the greenhouse, cool it, dehumidify it, and then distribute it back into the greenhouse. furthermore, in closed-systems water usually follows a close loop that allows the collection, recycle and re-distribution of the irrigation water both for irrigation purposes but also for cooling and heating purposes from inside the distribution pipes between the plant lines (qian, 2017) . concerning the irrigation system in closed-systems of greenhouses, the water does not follow the procedures of bleeding or dumping that are followed in semi-closed systems. on the other hand, the water is constantly recirculated in the mixed tank as it is automatically topped up with the correct and precise amounts of fresh water and each nutrient element. the growers are aware of the status of each nutrient element and are able to adjust it precisely in order not to disrupt the nutrient balance. this process becomes possible because of the high evolvement of automations, sensoring and programming in close greenhouse systems and achieve a 10-50% better water use compared with open greenhouse systems (nederhoff and stanghellini, 2010) . greenhouses have different techniques for irrigation and water collection and highly depend on if greenhouses use soil based techniques or soilless for crop production. another factor that highly influences the final water use and water use efficiency is the type of the system, meaning it is an open system, a semi-open system or a closed-system. however, as can be retrieved from tables 1 and 2 the big difference in water use efficiency can be explained primarily because of the higher production accomplished in greenhouses compared to traditional farming but also because of the lower transpiration in greenhouses. transpiration is the most important factor that influences the water uptake by 90%, thus the control and reduction of transpiration rate can have a huge impact on the water use. transpiration is highly affected by the status of humidity and the irradiation levels inside the greenhouse. the higher the humidity inside the greenhouse the lower the transpiration levels are. if growers manage to control these two factors in the optimal levels for each crop, then there is reduced transpiration level per m 2 , which means lower water usage and therefore better water efficiency. the selection of the applied irrigation system, has also a significant influence. drip irrigation is one of the most popular irrigation techniques in greenhouses. water is located at the foot of each plant with the use of a pipe. drip irrigation has the advantage of saving large water amounts and also can control and maintain the humidity levels of the soil or the hydroponic substrate in constant levels. in that way, water stagnation and puddling of the (2017) food miles 800-1600 km selected substrate mean can easily be avoided. finally, drip irrigation allows the targeted and limited fertilization being dissolved, in the watering system. other irrigation systems are the micro sprinklers that spray water in a range around two meters according to the pressure of the selected nozzle type. this system is mainly used in soil-based greenhouses with sandy soil texture. another very commonly used system is the irrigation with diffusers and is mainly used in narrower areas and the pressure of the diffuser depends on the nozzle that regulates the water supply and flow. finally, other irrigation systems applied in greenhouses are irrigation with hose and underground irrigation mainly found in soil-based greenhouses and present low level of water efficiency. most of the modern greenhouses apply hydroponic solutions that allow plant to grow without soil. in more detail, the word hydroponic comes from the greek words "ύδωρ + πονέω" translated as "water + cultivate," meaning that plants do not grow in soil but in mineral nutrient solutions in water solvent. various substrates in the market replace soil such as perlite, rockwool and zeolite. because of the nature of this technology, plants are permitted to dip directly in their roots into the nutrient-rich solution and subsequently plants can absorb faster the nutrients and in an easier way in comparison with soil-based crops. because of this process, plants grown in hydroponics form smaller root system and can divert more energy for growing their leaves and stems. additionally, smaller root allows more plants in the same area to be grown and harvest higher quantities in comparison to the outdoor farming. the above-described capacity of hydroponic systems, boosts the ability of growing food in limited areas as greenhouses can be. hydroponics consist of a total automated system that pumps water, and pipe-system can be completely auto-controlled. under various handlings and monitoring of every aspect that can be practiced in hydroponic systems, the growers can result into optimal food production results. more specifically, this process gives the opportunity to farmers to control the whole irrigation process of the crops according to the demand of each species and the seasonality. in addition, they can have access to data that can optimize the development rate and the resource footprint of the plants such as (a) the quantity of water that is distributed in each plant, and (b) the amount of nutrient solution that was given to the plants. hydroponics offer a big advantage as they are usually installed in close or semi-close loops that return the excessive water with the enriched nutrient solution back to a collective tank in order to re-distribute it back to the cultivation area. in contrast to the hydroponic solutions, traditional farming experiences huge amounts of resource and water waste as farmlands face the negative effects of soil degradation and the harmful effect of eutrophication (when nutrients from agricultural land create massive increase of phytoplankton populations leading to reduction of oxygen and nutrient reduction of from water and suffocation of multicellular water organisms). unfortunately, in traditional agriculture, excess supply of phosphates and nitrates in the soil can cause nutrient run/off and leaches. furthermore, the close or semi-closed loop of hydroponics categorizes them as more efficient in terms of sustainability process for water efficiency in comparison with traditional farming where most of the water is drained to lower levels of soil that plants cannot access. greenhouses consist of air-sealed cultivation rooms where are installed various automations and technologies that can control and provide the optimal environmental conditions for each crop. according to factors such as location, size of installation, height, outdoor climate conditions, greenhouses use different technologies that can properly adjust the indoor environment to the ideal air conditions. heating is one of the most important processes for space heating inside the growing room, when the outdoor conditions and too hostile for the plants' growth. for heating purposes, the technologies that are usually used vary according to the demand of each case. in general, heating systems use the interior hot air of the greenhouse to transfer heat through a heat exchanger to the stored water that is used as a thermal storage medium. a very common and cheap technique is using water heating systems that consist from plastic bags and ground tubes filled with water placed inside and between the rows of the plants. during daytime, this system absorbs and traps the solar irradiation and during nighttime, the stored heat is transferred in the interior of the greenhouse by releasing heat (sethi and sharma, 2008) . there are electric heaters operated via a thermostat or an automatic timer in order to rise the inside temperature to the desired levels. additional techniques used for heating are rock bed storages, movable insulation and ground air collectors. cooling is a technique of similar importance with heating as it enables to reduce the thermal energy inside a greenhouse and maintain the optimum temperature in each growing stage of the crop. various techniques are used around the world according to the specific climatic conditions, the size and the demand of each case. such techniques can be natural or forced ventilation, fogging and misting, roof cooling and fan-pad systems, as well as shading and reflection systems. the most successful systems are the composite systems since they are giving the opportunity for both heating during the winter period and cooling during the summer period. according to sethi and sharma (2008) , the most promising composite system is the earth-toair-heat exchanger system (eahes) that operates with the underground constant temperature of earth mass and utilize it to transfer or dissipate heat from or to the greenhouse. according to botanists plants are diversified to "long day" plants and "short day" plants based on the photoperiodism needs-meaning on how many hours of light they have to be exposed during the day in order to grow. artificial lighting is a technique that provides greenhouses supplementary lighting in case that the solar radiation does not completely meet the photosynthetic demand of each plant species for optimal growth and development. efficient and proper use of lights in horticulture and with additional boost of reflectors can provide apart from the optimal levels that are required for photosynthesis also can benefit the greenhouses with additional heating (fig. 2 ). heat and energy loss is a common issue in greenhouse and artificial lighting. the latter can become an effective solution that mitigates these losses and add an additional value on the required lighting solutions. the most common types of lamps that are used in greenhouses are high pressure sodium lamps, lighting emitting diodes (led) lamps and ceramic metal halide lamps. energy use into a hydroponic production line is mainly meeting the demand of artificial lighting, heating and cooling loads as well as water pumps. the energy that meets the water pumping needs in a hydroponic system for lettuce is estimated by the average pumping time that is needed to irrigate the plants and the corresponding nominal power of the pump. based on the calculations of kublic et al. (2015) it was estimated that the average irrigation duration for lettuce is four and a half hours of total pumping daily. the energy related to the heating and the cooling loads in a lettuce production greenhouse is estimated by using the following equation the heat transfer coefficient depends on the coverage material of each greenhouse, while the efficiency of cooling and heating systems depends on the height of the greenhouse ceiling. the loss of heat depends on the external climatic conditions and it is a decisive factor of the air technique modification to be used. artificial lighting usage depends on the photoperiod necessary for each species and the active hours of sunlight that plants can absorb for photosynthesis purposes. the active time that lamps have to operate is highly relevant with the location of the greenhouse, meaning that greenhouse areas with limited solar irradiation hours (north part of europe, i.e., netherlands, denmark) have higher demand on artificial lighting in comparison with areas under sunshine (southern part of europe, i.e., spain, greece, italy). furthermore, the duration of the supplementary lighting depends on the nature of the cultivated plants in photoperiodism (if they belong to "long day" or "short day" plants as we mentioned before) . this characteristic can differentiate the need of the plants in total daily radiation and according to the outdoor sunlight, the extra hours that artificial lamps need to operate should be estimated. the ultimate purpose of artificial radiation is to provide to the crop the indispensable photosynthetic active radiation (par) in mol/m 2 /day for optimal yield production. in order to calculate the energy of a mole of photons that reach the canopy the following equation is used: the result value of the above calculation of the energy demand of artificial lighting in the greenhouse is in [kj/kg/year]. food production and consumption is constantly rising, having a significant environmental impact making the implementation of more sustainable practices in food production necessary. in order consumers to satisfy their demand for off-season vegetables and fruits, the necessity of heated greenhouses for production is continually increasing. as it is mentioned in the traditional farming section, food transportation causes huge amounts of ghg emissions. however, this number is lower in comparison to the ghg emissions corresponding to heating hydroponic greenhouses in cold climate areas (ntinas et al., 2016 ) that try to meet high yields in order to meet customers demand. when heating of greenhouses is achieved with the use of natural gas, the consumed energy can reach the 31.6 mj with 2.02 kg of co2 for the production of 1 kg of tomatoes. since the majority of greenhouses use fossil fuels to meet their heating demand such as natural gas, diesel, fossil fuel and liquid petroleum gas, it is of vital importance to strongly limit the greenhouses heat losses, upgrade the heating systems and to shift in utilization of renewable energy sources . heat losses can be minimized with the use of double glazing coverage material or with the use of multiple screens. the upper goal of these measures is to increase the environmental sustainability of greenhouse production lines. as it has already been mentioned, greenhouses combine different energy technologies, automations and digitalization for plants' monitoring, controlling and harvesting. greenhouses is a type of farming that can provide the option to connect with renewable energy resources in order to increase the sustainability of such systems and the energy efficiency of the various treatments that are necessary for mass food production (manos and xydis, 2019) . different types of renewable energy sources such as solar, wind, geothermal, hydroelectric, biofuels, biomass etc., are found all over the world bringing the possibility to greenhouse plants to produce yields under a more sustainable, economical and cost-efficient way (xydis, 2015a) . energy policy strategies in a national and a global level, have as a high priority the support of electricity generation and heating from renewable energy and biofuels (xydis, 2015b) . over the last decades significant improvements in a big variety of significant renewable energy systems, which are ground source-based, solar-based energy systems and wind-based energy systems have been made (koroneos et al., 2009 (koroneos et al., , 2017 . these can be for example electricity-driven heat pumps instead of traditional combustion-based heating systems consumes 25-65% less energy in comparison to a conventional fuel heater (avgoustaki and xydis, 2019) . another advantage that heat pumps present 1.3-2.6 times higher energy efficiency compared to fossil fuel heaters as also 56% -79% reduced co 2 emissions in the cultivation area in comparison with the conventional. there are also examples of greenhouses that use several solar systems that store energy or other photovoltaic systems (pv) that undertake the conversion of solar energy to electricity that meets the heating and cooling needs of greenhouses. based on research conducted by ntinas et al. (2016) , greenhouses that utilize renewable biofuel (wood pellet) present 3-5 times lower global warming potential in comparison with a greenhouse that use fossil fuels for heating purposes (0.4-0.7 kg of co 2 per 1 kg of harvested tomatoes), even when the required energy is the same for both cases. greenhouses in the netherlands use complex technology for production of various cultivars that gather multiple operation during the production such are nurseries, growing bedding plants and transplants. these systems are highly automated and occupy land approximately 10 ha or more (kozai et al., 2016) . even if these machineries occupy a lot of potential cultivated space, they reduce the labor cost and therefore the production cost. without the use of highly automated technology, the average work force required in greenhouses for cultivating purposes is estimated at approximately 8 workers per a 500 m 2 production area. according to penissi et al. (2019) greenhouses produce 112 g of fresh weight of romaine lettuce per m 2 daily while traditional farming produce 10 g of fresh weight of romaine lettuce per m 2 per day. as it can be retrieved from table 2 , the required land use for obtaining 1 kg of fresh romaine lettuce daily is 9m 2 presenting almost 90% of decreased land usage in comparison to traditional farming. in greenhouses there are different variables that based on their priority can offer different benefits to the farmers. these could be the location of the greenhouse, the product type, the access to capital, the required work force and other requirements. high significance in the cost efficiency is also the upfront cost and the ongoing growing cost of the greenhouse that can also lead to higher cost depreciation and development rates of the production unit. based on a comparative study conducted by , a greenhouse farm consisting of a semi-closed system of 675m 2 of growing space in denmark, the opex and capex related with the farm were analyzed. their results showed that by assuming that the wholesale price of greenhouse produced greeneries reached at 7.37€/kg, the annual yield production of harvested products reaches at 16,875 kg/year. it is also presented that the capital expenses for the installation of the greenhouses was calculated at 216,123 € including the hydroponic system and grow unit racks, natural gas, heating and ventilation system, light connection (for supplementary radiation), and electricity distribution. additionally, for the operational expenses the total amount of expenses rises to an annual cost of 152,802 €, including the leasing costs, the electricity demand costs (lighting, ventilation), the natural gas heating cost, the water demand, the labor requirements, the packaging expenses and finally the use of organic material (seeds and nutrients). different greenhouse scenarios were presented and a cash flow analysis in a 20-year projection, indicated that the cumulative gross profit increased in parallel with the increasing wholesale price of greeneries. more specifically, the payback period was calculated much longer than the operational period of the 20 years resulting in negative prices of the net present value (npv), unless the wholesale price of greens increases to 10.37 €/kg or more. indoor vertical farming is an innovative type of closed plant production system that provides the opportunity of a controlled-environment agriculture, which can be controlled according to the crop regardless of the weather conditions. indoor vertical farms use artificial lighting as radiation source in order to cover the demand of plants for growth and development via photosynthesis. vertical farms are based in soilless cultivation techniques such as hydroponics, aeroponics or aquaponics. in addition to the hydroponic systems that recirculate the nutrient solution and benefit greenhouse cultivations, vertical farms use systems that condense and collect the water that is transpired by plants at the cooling panel of the air conditioners and continuously recycle and reuse it for irrigation. some principles concerning the structure elements permeate closedsystems of vertical farms. more specifically, vertical farms are thermally well-insulated and nearly airtight structures that are covered with opaque walls. this characteristic makes the farms capable to totally protect the inside crops from the outdoor climatic conditions and make them able to maintain the indoor conditions to the desired levels without having thermal losses. another characteristic that differentiate vertical farms from greenhouses is the multiple layers of stacked plants in the vertical racks or horizontal columns. this way, the construction provides maximization on the possible yield per unit of land in comparison to both greenhouses and outdoor farming. more specifically, vertical farms, according to the size on the installation, have a multilayer system mostly between 4 and 16 rows or columns with approximately 40 cm of distance between the layers (can slightly vary according to the selected cultivated crop). inside vertical farms airconditioners or heat pumps, which principally are used to reduce the heat generated from the lamps and provide cooling and dehumidification for the crop are installed. furthermore, air-conditioners help to eliminate the water vapor that plants transpire in the cultivation area. fans are installed in order to circulate the air in the culture room; at first to achieve a constant and stable spatial air distribution and secondly to improve the photosynthesis and transpiration status of the plants. key factor in the optimal operation of vertical farms is the co 2 delivery units that stabilize the co 2 levels in the cultivation area at around 1000 ppm during photoperiod (when lamps are on) in order to increase the level that plants photosynthesize. an important characteristic of vertical farms is the nutrient solution unit that distributes the nutrients to the crops, the electrical conductivity control unit (ec) and the ph controller that monitors the level of the nutrient solution. last, it is very important to analyze the radiation systems inside vertical farms as part of the total structure essentials. as mentioned above, vertical farms are equipped with artificial lighting due to absolute lack of solar radiation. lighting is a key factor in plants development and depending on the selected lighting solution, plants can present differentiations in morphology, flowering and biomass production. light is electromagnetic energy that includes visible as also invisible wavelengths. sunlight is a free resource input that provides plants the whole spectrum of several wavelengths, 97% of it is within the range of 280-2800 nm (kozai et al., 2016) . however, according to a number of researchers over the last decades (hogewoning et al., 2010; kim et al., 2004; lin et al., 2013; liu et al., 2011) , it is reported that the most important wavelengths for photosynthesis, morphology of plants and flowering are the wavelengths in the visible (400-700 nm) and the infrared (700-800 nm) spectrum. lighting emitting diodes (leds) offer advantages in comparison with other types of lamps such as fluorescent, incandescent, high-pressure sodium or high-intensity discharge (hid) lamps. these advantages are the robustness, they produce, a stable output that is immediately activated after the electric current flow, have long life (approximately 100,000 h), the opportunity of controlling the light output etc. for this reason, vertical farms focus on applying lighting recipes that combine different nanometers and can promote plants' growth. apart from the spectrum selection of the lamps crucial factors for plants are the dimensions of light, meaning the intensity of light during photoperiod and the duration that lights operate. what has literally been neglected is the potential of indoor vertical farms to act as a demand response provider (aggregators). it may sound weird, but indoor vertical farms could under a multi-value business models create the opportunity to the vertical farm owners to focus on their crop production and at the same time absorb inexpensive electricity offered. usually plants require some hours daylight and fewer darkness. it has been proven that by selecting the hours throughout the day that are not expensive to give the required light, and "give darkness" when electricity price is expensive, has not a significant impact on plants' growth and development. under a mass deployment scenario of such units in major urban environments (xydis, 2012) , the owners and operators of the indoor vertical farms could create an additional profit under such an approach by entering into contracts with companies in a utility electric region. the opportunity to earn (or at least save) significant amounts will or course be related to the size of the indoor farms and create multiple revenue streams. indoor vertical farms have thermally insulated walls and high level of airtightness that allows a better cooling by air-conditioners during the time that lights operate. this process is functioning even during cold winter nights, as the interior temperature can be increased due to the operating lamps that constantly generate heat in the cultivation rooms. the ultimate goal of air-conditioners is to maintain the indoor temperature at the desired levels. however, during the cooling process, a lot of the water portion is lost due to evaporation of plants or evapotranspiration. indoor vertical farms have heat pumps with cooling panels, which can condense and collect this water, recycle it and via the close irrigation loop, reuse it for watering the plants. according to kozai et al. (2016) , only a small part of the irrigated mass water is getting lost to the outside because of the high level of airtightness inside the vertical farm. it is also pointed out in this research, that the airtightness level of vertical farms should not exceed the 0.02 h à1 . this is suggested because this level of airtightness helps to reduce the co 2 losses to the outside environment and at the same time to maintain the sanitize level inside the farm by preventing pathogens, bacteria, dust or insects to enter the area of cultivation. greenhouses compared to indoor vertical farms, do not provide the opportunity of collection, reuse and recycle of the water masses that evapotranspired from plants, because the majority of the water is lost via the ventilation process to the outside area and furthermore most of the water vapor of greenhouses is mainly condensed at the inner walls, making impossible its collection process. another remarkable point that influences the resulted transpiration in indoor vertical farms is the operation of the artificial lighting. more specifically, when lamps do not function, the relative humidity of the room can reach up to 100% (little transpiration in the culture room), and cause physiological and morphological disorders to the plants. in order to solve this issue, farmers operate the lamps in rotation after dividing them in groups (two or three) and each group operates for 12-16 h per day. with this action, a constant heat generation during the day from the lamps that aligns with the 24-h function of the heat pumps that dehumidificate and cool the air in the culture room can be achieved. in order to calculate the water use efficiency in indoor vertical farms the following equation is used: where -wc is the water mass (or weight) that is collected in the cooling panel of the air conditioners for recycling purposes (kg*m à2 *h à1 ), -wp is the alteration in the water mass that is detained by plants and hydroponic substrates (kg*m à2 *h à1 ) and -ws is the irrigated (or supplied) water mass to the indoor vertical farm. in general, co 2 use efficiency in indoor vertical farms is around 0.87-0.89 (when the level of airtightness is between 0.01 and 0.02 h à1 ) and the concertation is around 1000 ppm-unlike greenhouses which achieve approximately a 0.5 cue with closed ventilation system and airtightness level of 0.01 h à1 and co 2 concentration level at 700 ppm (yoshinaga et al., 2000) . based on these data we can estimate that the cue of indoor vertical farms is 0.88/0.5 ¼ 1.8 times higher compared to the greenhouses that do not operate the ventilators and provide co 2 enrichment in the culture room. this phenomenon can be explained because of the amount of co 2 that is released to the outside area from the culture room and keeps increasing with the level of airtightness but also with the difference between the co 2 levels inside and outside. the fact that the co 2 concentration for enrichment in an indoor vertical farm is usually around 1000-2000 ppm in comparison to the greenhouses that have around 700-1000 ppm can be explained based on that. in order to calculate the cue the following equation is used: where -cp is the net photosynthetic rate (μmol m-2 h à1 ), -cs is the enrichment rate of co 2 (μmol m à2 h à1 ) and -cr is the rate of respiration of the workers (if there are) in the culture room (μmol m à2 h à1 ) the light energy of the lamps that is send in the canopy aims to provide the necessary energy that plants need to grow and photosynthesize. however, the salable part of plants can only fix maximum 1-2% of the electrical energy as chemical energy. the remaining 98-99% of the electrical energy that is not absorbed by plants is converted to heat energy into the culture room and the remaining is removed by air-conditioners to the outside area (avgoustaki, 2019). the above-described effect can also explain the negligible heating costs in well thermally insulated indoor vertical farms even in the winter cold nights. nevertheless, indoor vertical farms are based in automations and precision agriculture and all the input resources are measured and validated in order to provide the optimal results in the cultivated crop. for this reason, all farms focus on measurements and optimization of the light energy use efficiency both of the lamps and the plant community. what is important for these measurements in the definition and estimation of the par, which in other words, is the wavelengths of light that are in the visible spectrum of the 400-700 nm and are the ones that drive photosynthesis. par is not a measurement of light; rather it defines the type of light that is necessary for plants to photosynthesize. apart from the type of light, farmers need to know and further metrics of light such as the amount and the spectral quality of par. in order to estimate the light energy use efficiency of lamps (lue l ) we use the following equation: where f is the convention factor from dry mass to chemical energy that is fixed in dry mass (around 20 mj kg à1 ) -d is the increase rate of dry mass of the whole unit of plants or only the salable part of plants in the indoor vertical farm (kg m à2 h à1 ) and -par l is the photosynthetic active radiation emitted by the lamps (mj m à2 h à1 ) respectively, in order to estimate the light energy use efficiency of the plant community (lue p ) is provided by the following equation: where: -par p is the photosynthetic active radiation that is received at the surface area of the cultivation. based on the calculations and experiments conducted by yokoi et al., 2003, it is shown that indoor vertical farms have 1.9 to 2.5 times higher lue p in comparison to the greenhouses. only 1% of the light energy is actually converted into salable portion of plants. nevertheless, there are different techniques which can be applied and can improve the conversion factor to 3% or a little higher. a simple technique that can be followed is the application of interplant lighting, upward lighting, and use of reflectors (fig. 3) . traditional lighting that is located only on top of the crop can cause undesirable shading in dense crops by uneven light distribution and lead to senescence of the leaves that are in lower levels. on the contrary, the application of interplant lighting can provide access of light also in the lower levels of the plants, improve the distribution of light and therefore improve the photosynthetic rate of the crop. according to dueck et al. (2006) , the photosynthetic rate of leaves in low levels is usually negative or nearly zero, but the application of interplant light can increase it in positive values. welldesigned reflectors can significantly enhance the lue l as they can reduce the vertical distance between the canopy and the lamps and increase the distance between the plants or the density, since plants constantly grow. same positive results by interplant lighting have been reported also in greenhouse canopies. the most suitable lamp selection for interplant lighting technique is leds as they have small volume and they perform lower surface temperatures in comparison to fluorescent and other types of light sources. leds have been proven beneficial for reducing the eue l also due to the higher conversion coefficient from electrical energy (0.4) compared to the fluorescent lamps (0.25). although the capital cost of leds is generally higher than the cost of fluorescent lamps, leds have longer operational life and the prices have considerably decreased over the last couple of decades and is expected to continue decreasing. apart from the lighting adjustments, other modifications can improve the lue l such as the control of the environmental conditions. the environment of plants and the ecophysiological status of plants can be enhanced by the optimal selection of air temperature, co 2 concentration, water vapor pressure deficit (vpd), air current speed as well as the combination of ph, electric conductivity (ec) of nutrient solution. these parameters have to be set according to the selected cultivated species. another way to improve the lue l as well as the eue l of the salable part of plants, is to reduce the dry mass of the nonsalable parts of the plants. in indoor vertical farms, the most frequently selected crops for cultivation are leafy vegetables such as lettuce, small fruits and herbs and it is important to limit the percentage of the root mass into less than 10% of the total mass of the plant (kozai et al., 2016) . due to of the cultivation technologies used in indoor vertical farms this is an achievable measure only by minimizing the water stress of plants by controlling the water vapor pressure deficit of the room. if the selected crop is root species, then we can significantly increase the salable portion by harvesting earlier than usual in order to have an edible aerial part. finally, other factors that can also help in increasing the relative annual production capacity (per unit land area) of indoor vertical farms are: • limitation of the culture period between transplanting and harvesting by optimal monitoring and controlling of the environmental conditions • increase of the ratio of cultivation area under each farming type (field, tier, floor, culture bed) • increase of the salable part of plants as also the percentage of salable plants. according to kozai et al. (2016) , it is stated that by applying the abovedescribed techniques, the relative production capacity per land area unit in an indoor vertical farm of 10 layers can rise up to 200-250 times higher compared to outdoor farming, considering that indoor vertical farms already produce 100-150 times more yield than traditional farming (table 3 ). in practice, those techniques could double the efficiency of the whole system. indoor vertical farms use culture beds that are isolated from soil usage and the nutrient solution that enriches the irrigation water is distributed through pumping to the plants. because of the high-automated process of irrigation, the nutrient solution is drained from the culture beds that plants are growing and it follows a close loop by returning to the central nutrient solution tank for recycle and reuse. in order this process to be achieved, nutrient solution is rarely removed to the outside area. this process usually takes place once or twice per year when the level of certain ions such as na + and cl-are not well absorbed by plants and the percentage in the culture beds exceeds the normal levels, requiring discharge. in order this measure to be implemented, the supply of fertilization closes for some days and plants already planted can absorb the nutrient elements existing in the culture beds (kozai et al., 2016) . on the contrary, the fertilizer use efficiency of greenhouses and of fields in traditional farming is relatively low and occasionally can cause on the soil, surface salt accumulation. in order to calculate the fertilizer use efficiency (fue) the following equation is followed: where: -i u is the absorption rate of plants of ion element i that are in the organic fertilizer and -i s is the supply rate of ion element i into the indoor vertical farm. it is worth to be mentioned that the ion element includes the basic elements of fertilization solutions such as nitrogen (no 3 à and no 4 + ), phosphorus (po 4 à ) and potassium (k + ). artificial lighting apart from a key element in the growth of plants indoor, it does increase the energy consumption of vertical farms. shamshiri et al. (2018) , noted that three major operational expenses in a vertical farm are the electricity cost with 25-30% of the total cost, the operational costs (opex) with 27% of the total cost and the capital expenditures (capex) with 18-20% of the total cost. indeed, energy consumption is a significant cost of indoor vertical farms and can be used as an measure for their sustainability levels. many research groups and institutes focus on developing innovative technologies and optimizing the lighting recipes in order to reduce the energy footprint of vertical farms and create a more sustainable and cost efficient type of farming. even if the demand for purchased energy is much higher in indoor vertical farms than in greenhouses, the energy efficiency of the former is significantly higher (graamans et al., 2017) . indoor vertical farms, since are in absolute controlled systems face high efficiency when operating with renewable energy . there are multiple examples of vertical farms that are operating under smart grid systems that generate energy for the demands of the farm via wind turbines or solar panels or even geothermal energy. additional roles in the vertical farm systems towards increasing their efficiency have the connectivity with resourceful batteries that provide the opportunity for smart use of cheap stored electricity from the hours that the electricity prices are lower. an approach gaining constantly more and more attention also under the dynamic pricing concept, where also accurate forecasting plays a crucial role (karabiber and xydis, 2019) . in order to calculate the energy use efficiency for the lamps (eue l ) is followed the below equation where: h is the conversion coefficient of electrical energy to energy of photosynthetic active radiation that is emitted by lamps. for the latest technology of leds this number reaches the 0.3-0.4 (kozai et al., 2016) . apart from the energy that is consumed in order to meet the lighting demands, the energy demand of the heat pumps for the cooling (or heating) processes in the indoor vertical farms should be added to the equation. this type of efficiency is often referred in literature as coefficient of performance of heat pumps for cooling purposes. the coefficient of performance of the heat pumps, in a specific room, increases when the outside temperature decreases. the electrical energy use efficiency for cooling by heat pumps (eue c ) is calculated by the following type: where: -h is the heat energy that the heat pumps remove from the cultivation area (mj*m à2 *h à1 ) and -a is the consumption of electrical energy by the heat pumps (air conditioners) (mj*m à2 *h à1 ). it is worth to mention that the total energy consumption of indoor vertical farms is defined by the sum of the energy consumption of the lamps, the heat pumps/air-conditioners and the electricity demand of other equipment used for the optimal function of the farms such as nutrient solution pumps and air circulation fans. if we focus only in the electricity cost demand of indoor vertical farms, lighting accounts to approx. 80% of the annual electricity energy use (assuming fluorescent lamps of 40 w), while the electricity cost demand for air conditioning is around 16% and 4% the electricity demand of the auxiliary electrical equipment (kozai et al., 2016) . table 3 presents the estimated representative values of resource use efficiencies in an indoor vertical farm that use artificial lighting. it could be concluded (from table 3 ) in comparison to table 1 , that the relative production capacity per land area unit in an indoor vertical farm of 10 layers is 76 to 116 times higher compared to traditional farming and 40 to 80 times higher compared to greenhouse production. indoor vertical farming is a type of farming which by definition is developed to provide enough production in order to meet the local demand in urban areas with continuous increased demand for fresh and nutritious fruits, vegetables and herbs. in general, the most frequently cultivated species are plants that have higher profitability and have a relatively high price. a significant factor on crop selection is the crop to have a short production cycle in order to reduce the required electricity costs for lighting, heating and cooling of the crop and therefore can be harvested as early as possible. additionally, growers prefer plants that have high harvested yield, meaning a high portion of the crop that can be harvested and sold. for example in crops like lettuce and herbs, growers can harvest and sell the whole unit of the plant, while in tomatoes or peppers they can sell only the harvested fruit but at the same time. therefore the electricity used for the rest of the plant, could be considered as a product waste. another key issue in crop selection is the height of the plants, meaning that it is way more preferable the crop to have a compact status in order to be able to reduce the growing distance between multiple plants and grow more at the same available area. plants are also selected according to the perishability level that they present after harvesting and reaching the market. since indoor vertical farms are mainly located in urban or suburban areas, their goal is to produce crops that can increase their self-life (even of perishable crops), by shortening the harvesting and delivery time to the market. another parameter taken into account when selecting crops is the situation in the local market. if, for instance, tomatoes are missing for some reason from the market, then depending on the price they can get, they could be preferred against of another fruit or herb that is in abundance and its price cannot climb up. finally, most suitable crops are those that have year-round productivity in order to be affordable for the farmers to have a year-round market demand that can be profitable despite the continuous operational expenses. the constant production in a yearly basis of the same crop selection, allows also maintenance of the same, specific engineering settings of the crop, avoiding the modifications in the automations' selection that could cause abnormalities from a horticultural perspective. due to the concept of indoor vertical farming and the technology used in the cultivation areas, growing in an urban environment do not advantage the crops due to possible shading of the building, non-fertile soils or dormant soils. this fact can also be considered as one of the major drawbacks as the land price in urban areas is relatively high. concerning this approach, indoor vertical farms are often installed in large warehouses, industrial factories or even abandoned buildings, where the prices are low. according to kozai et al. (2016) , it is stated that indoor vertical farms can produce the same yield of lettuce heads and other leafy greens in only 1% of the land required by traditional farming and 10% compared to a greenhouse construction. based on tables 1-3, it can be retrieved that the land use efficiency of indoor vertical farms (0.3 m 2 ) required for obtaining 1 kg of fresh romaine lettuce per day is almost 97% reduced compared to greenhouses and 100% compared to traditional farming. an indoor vertical farm of 10 layers can produce 3110 g of fresh weight of romaine lettuce per m 2 per day (112 g fw/m 2 /d for greenhouses and 10 g fw/m 2 /day for traditional farming). adenaeuer (2014) mentions that the increase in yield between indoor vertical farms and traditional farming can be increased by 1.5 due to the technology and by 709 due to the technology combined with the stacking ability of the plants. depending on the stacking area and the volume of harvest, cultivation care and crop preparation techniques, the work force can highly vary. , propose that 0.18 workers are necessary per 10,000 kg of yield, resulting in 35% of the annual operational expenses of the farm (depending on the labor cost in each country). the same work force is required for a greenhouse production and approximately half of it for an open field farm. more analytically, according to savvas et al. (2015) , in soil-based crops the labor numbers 34,000 €/ha while a hydroponic greenhouse or indoor vertical farm requires around 64,000 €/ha as production cost. this demand is met by both permanent and by seasonal workers that will be hired for specific labor-intensive operations of the farm (like pruning and harvesting) throughout the year. indoor vertical farms have the advantage that allows them to generate bio-waste as bio-product during the process of edible biomass production. according to the cultivation system that plants grow in (hydroponic, aeroponic or aquaponic), the opportunity to farmers to collect easily all the by-products after the harvest period such as leaves, roots with fibers, stems, or even damaged vegetable and fruits and use it as well waste is offered. based on adenaeuer (2014) , the bio-waste that is collected and used in indoor vertical farms can be 2443 metric tons per year and with daily plant wastes that are collected for the indoor farms of roughly 8.11 tons. since indoor vertical farms use advanced close loop systems, present also the possibility to convert the daily amounts of biowaste and after careful processing to useful resources material for the crop as liquid fertilizer or biofuel (nikas et al., 2018) . the are several cases of installation of indoor vertical farms that have designed specific lines of biowaste management in their production line that only serve this specific purpose. it should be stressed that indoor vertical farms have the option to implement high tech equipment for conversion of food waste into energy production via anaerobic digestion. more specifically, this technology is a biogas recovery system that captures methane from food waste and convert it to heat, steam and electricity to meet the energy demands of the farm. this process requires a close-loop system, which creates biogas from organic material by piping it into the turbine generator. the electricity that is finally produced meets the high-energy demand of indoor vertical farms such as the operation of the lamps. anaerobic digestions is also compatible with aquaponic systems by receiving the organic waste of both fish and plants to produce electricity (agstar, 2020; united states environmental protection agency, 2017). one of the key factors that influences the selection of the farm system is the selling price of the products. according to tasgal (2019) , traditional farming products are 3 to 5 times cheaper in comparison to greenhouse an indoor vertical farming products. more specifically, traditional farming lettuce price usually costs less than 1€/head, while greenhouses lettuce and indoor vertical farm lettuce cost 2-3€/head. additionally, based on the same study, the significant upfront capital requirements of indoor vertical farms can highly limit the pool of market participants. this happens because both the land prices, rents and acquisition of high-technology equipment are significantly higher in comparison with the leasing cost of farmland. on the other hand, , by conducting a comparative analysis between indoor urban farms and greenhouses presented slightly different results. in more detail, they assumed an indoor vertical farm with the same growing space and wholesale price of the greeneries as in the greenhouse facility, of 675 m 2 and 7.37 €/kg respectively. an interesting point is the massively increased production yield that can be achieved in an indoor vertical farm compared to greenhouses, reaching at 33,750 kg of fresh greeneries being annually harvested. the operational expenses of indoor vertical farms according to the examined case reached at 150,800 €/year resulting in almost similar numbers with the greenhouse facility. however, the biggest cost of indoor vertical farms noticed were the capital expenditures reaching at 321,763 € per grow unit, with the most costly equipment the lamps and integral connection of lamps, installation of growing unit racks and the electric distribution of electricity. subsequently, based on their model and the different cash flow analysis, indoor vertical farms present profitable investment opportunities with a high internal rate of return (irr) and a payback period between 3 and 6 years with a wholesale price equal or more than 6.36 €/kg. another research conducted by liaros et al. (2016) , a case scenario of a small iuvf of 100m 2 growing area inside an apartment was presented, showing profitability to smallholders under various scenarios. worth to mention at this point, micro indoor farming in small growing spaces such as containers, garages or even simple rooms can be profitable depending on the demand and the flexibility to rearrange different cultivation parameters aiming for the optimum result. similar findings were also supported by ucal and xydis (2020) . on the other hand, based on a report conducted by agrilyst (2017) indoor micro-farms can be very costly, nevertheless, there are multiple marketing strategies for optimizing the results. according to the united nations (un) projections, the global population will exceed 9.8 billion until 2050, all requiring to meet their food demand. additionally, un estimate that 80% of the global population will be located in urban areas by that time. in order all this increased food demand to be met, it is necessary to produce 70% more nutritious and fresh food. however, at the same time, land experts such as agronomists and ecologists, already warn of the growing shortages in agricultural land, necessary for sufficient food production (al-kodmary, 2018). when it comes to high quality food, the fact that already food prices are climbing high also due to limited agricultural resource inputs such as water and energy is a matter of great concern. over the last decade, the increase demand for more farmland in order to meet global food demand it becomes more and more obvious. as an immediate effect a lot forest areas are substituted by new farmlands in order to supply this demand. at the same time, since cities constantly grow in terms of area they occupy, a lot of farmland is lost due to this expansive urban development. it is important to convert the global production line to a greener form for both human beings but also for the planet. this implies that food production will not sacrifice the attention for the human health against the commercial profit. according to world health organization, more than half of the farms globally, still use for fertilization purposes of their crops raw animal waste that can attract insect as flies or contain weed seeds or even diseases which can contaminate the cultivated crops. subsequently, these techniques can highly affect people's health and can cause diseases. nowadays, the majority of the food is produced in large, industrialized farms and is transported, distributed and sold in supermarkets, grocery stores or multinational food outlets. agronomists, engineers and farmers in order to reduce the production cost and resource footprint of food production and at the same time increase the variety of the available food species for the consumers have developed various techniques. the high centralization level of food supply can allow the possibility of infection from foodborne pathogens and toxins that can poison large numbers of consumers. food usually travels thousands of miles every day leaving huge possibilities for contamination threats as it can be infected in one country and develop pollutant populations in another. because of the high logistic complexity of food supply, it is worth to mention the advantages and drawbacks of each farming type during the whole supply chain. what follows is an exploration of the three subjected farming types, including outdoor farming, greenhouses and indoor vertical farming. we will compare and evaluate products and growing process under the scope of food safety practices. outdoor farming is applied for thousands of years, allowing an unprecedented human development. however, over the past years the continuously increasing demand of the population has led farmers to apply chemical inputs for nourishing of plants, fighting pests, insects and improving soil quality. however, because of its nature, crops growing in the open field are facing all the difficulties from severe weather conditions and the danger of infection from various insects and pathogens. traditional farming is a type of agriculture that allows to multiple plant pathogens, bacteria and insect pests to affect crops, causing scalable losses in global crop production. after heavily tilled farming applications, severe irrigations and monocropped selections, soil has been seriously affected causing depletion of its nutrients, highly requiring additional nutrient solutions that can improve its fertile condition, making it appropriate for cultivation. once crops are harvested, a big after-harvest process and logistic supply has to be followed in order food to be transported from the farmer to customers' table. when we are talking about vegetables and greeneries there is a high level of perishability that needs to be confronted. crops have to keep cool in order to maintain the high fresh and nutritious status. in order farmers to retain a high value for their products, after harvesting, food is transported from the field to processing facilities that are responsible for the cutting, washing of plants in cold water applying centrifugation methods in order to remove the excess water from the products. after removing the roots and fulfilling the described procedure, products begin to decompose. an often procedure that farmers follow is to treat their production with chlorine compounds and/or antioxidants that expand preservation during and after washing. continuously, food is usually packaged and stored in refrigerators and very low temperatures in order to remain in inertia status. however, outdoor farmers are not able to perform refrigeration between harvest and transport of the products for water processing, making it more uncertain in pathogens infection. in order groceries to arrive from the processing facilities to the shelves of the markets, they require on average 2000 to 3500 km, resulting to 4-6 days in transportation. according to kublic et al. (2015) , every three days, products lose 30% of their nutritious value after being harvested and roots' removal, meaning that consumers finally receive severely influenced vegetables in terms of nutritional value. according to the centers for disease control and prevention, each year, "roughly 48 million people (1 in 6)" are food poisoned in the united states. in terms of food safety what products of outdoor farming face is the severe contamination from improper use of manure, either from human fecal that is used as fertilization mainly in developing countries or from contaminated concentrated animal feeding operations (cafos). even if it has been proven by various researchers as a great nutritious source for the crops after proper compostable process, on the other hand the absence of carefulness, targeted application and lack of sanitation can lead to transmission of various types of parasites. a serious parasite that is worth to be mentioned is geohelminths (hookworm, ascaris and whipworm), that can survive their eggs in soil for years when they find the right climate conditions, causing diarrheal diseases as well as permanent learning deficit to children (hotez and pecoul, 2010) . e.coli was a foodborne illness that took high publicity after infecting approximately 265,000 people and causing about 100 deaths, after severe pollution of agricultural water reservoirs in farms of california. to summarize, even if there are multiple technological automations, innovations and outbreaks in outdoor farming over the last decades, the nature of this agricultural type is very open to foodborne illnesses, illnesses extremely difficult to be traced rising the total risks. because of the importance of food safety and in order to avoid further foodborne illnesses, there are several rules that force outdoor farmers to enhance the safety status of their production for the overall benefit of the population. the strictest and most widely recognized organization of food control audits is the global food safety initiatives (gfsi), which was established in 2000 to reduce and control the risks associated with food production as also to streamline and improve the overall food safety while reducing the operating costs. various certifications are provided to farmers including the safe quality food (sqf) and hazard analysis critical control points (haccp) that set the necessary rules and prerequisites of a high-level food safety status. this includes some of the following rules: • it is of significant importance the control and validation of the agricultural water. to be more specific, there are rules that prerequisite the testing of the water quality that is applied via irrigation to the crops, but also the water related to the tangential purposes such as hand-washing of the workers during or after harvest, the ice that refrigerates food and the surfaces that food contacts with. • biological adjustments are often applied in soil for or particular nutritional uses that replace chemical fertilization. it is of vital importance that farmers who follow these techniques to follow specific guidelines for the use of raw manure (such as animal and human feces) as also for the use of stabilization compost in order to maintain a high level of sanitation. • there are rules concerning the compliance of domestic and wild animals either they are working in the farm, invade in the farm or graze. • finally, there are high requirements in workers' health and hygiene that need to be followed in order to prevent the contamination that may source by humans. 3.2.1 food safety status of greenhouses as has already analyzed, there are greenhouses that are soil-based and the more advanced use hydroponic solutions. in hydroponic greenhouses, plants are transported several times according to the growing stage and are monitored throughout the different growing cycles. that give the opportunity to apply the exact resource requirements in every stage, in comparison with soil-based greenhouses and outdoor farms, where the plants remain in the same position until their harvest. another significant advantage of greenhouses in relation with outdoor farming is the high geographical flexibility of installation as it allows a significant reduction of the transit time of the products from the harvesting and processing point to the final consumers. greenhouse plants in an industry that constantly growing, with today's list accounting half of the tomato production and 1/3 of the global pepper production that are distributed in the fresh market (brauther, 2010) . greenhouses are a significant driver of national economies of the agricultural sector because of the high profit margin as also the opportunities for high added-value products. unlike traditional farming products, greenhouse production is highly protected from dangerous elements and various contaminants. however, the technologies that are applied for monitoring and controlling of the environmental conditions do not guarantee crops free of microbes and pathogens. the management practices applied in greenhouses are these that can conduct to growth, survival and spread of foodborne pathogens. a severe contamination thread could be spread by processing equipment since crates and baskets that are used for transportation of products, from propagation tools or even for surfaces that food contacts with. irrigation water is one of the most important food safety risks even in greenhouses as it can be drawn from a wide variety of uncertain sources such as municipality supply, rainwater, underground aquifer, reservoirs or surface water. greenhouses that use untreated surface water as irrigation source face high contamination risks. for example, in 2013 , salmonela saintpaul (cdc, 2013 found to have infected cucumber greenhouses in us that caused the infection of 84 individuals across the country as they consumed imported vegetables with questionable irrigation water status. because of the high risk of infection of consumers, even from more controlled agricultural systems (compared to outdoor farming), regulations for food safety have become stricter by establishing new standards for food production (produce rules). the four areas that these standards focus on are the followings: • health and hygiene this practice targets in maintaining hygienic conditions of the personnel that is occupied in the greenhouse factories, involving criteria for personnel cleanliness, handwashing and use of appropriate gloves. even if handwashing is considered one of the simplest and cost-efficient practices, it has been reported that only 22% of greenhouses practice handwashing before the harvesting process. • irrigation water quality and management since water quality is one of the most crucial and contentious factors, it seems absolutely necessary the mandatory establishment of rules that control the water baseline quality profile. greenhouses withdraw water from a bog variety of sources such as municipality supply, wells, reservoirs and surface ponds. by checking and understanding the quality of the quality of various water sources can provide important information and reduce the risk of contamination. by regulation, greenhouses have to determine frequently microbiological testing on the water sources. furthermore, greenhouses that apply hydroponic solutions in semi-close or closed loops that circulate, recycle and reuse water, have to include filtering treatments that remove possible pathogens before re-applying it. methods that are effective and efficient in water recycling is uv light or disinfectants. • animals and waste significant measure for the protection of crops from foodborne pathogens is also to eliminate the restriction of domestic and wild animals at growing activities inside the greenhouses as well as in the outside area of the buildings. practices that contribute in discouraging animal intrusions can be for example the rapid weeding that will minimize rodents attraction and protection. • sanitation of equipment, tools and greenhouse surfaces foodborne pathogens are usually found all over the greenhouse environment such as harvesting bins and boxes and tarp floor covers of the greenhouse (ilic et al., 2014) . according to produce rule, all the tools and equipment that used in the production line should be inspected, cleaned, sanitized and maintain in this condition throughout the whole production, harvesting and post-harvesting process, in order to prevent contamination. greenhouses in comparison with traditional farming have the advantage of the three-key elements application that can eliminate contamination risk: innovations, automations and control. in specific, innovations provide to greenhouse farms a more secure food safety support such as water filtration systems, integrated pest management and higher quality control systems. automations can reduce the danger of contamination or cross contamination as they minimize or decrease the introduction of foreign specimens. finally, biometric systems provide to growers the ability to detect tracking information concerning the plants. after harvesting, the produce is set up in a traceability system from the greenhouse plant to the customer delivery service. 3.3.1 food safety status of indoor vertical farms leafy greens, vegetables and herbs are considered of high-risk crops since they are usually not cooked but eaten raw. the usual process of consumers is to rinse their purchased greeneries after purchasing them from their grocery store and then consume them. this is not a particularly effective and protective procedure, since harmful pathogens need interference of chemicals to be detached from plants. outdoor farming and most of the greenhouses perform triple-wash on the harvested plants in order to mitigate the contamination risk, as a post-harvest process. this process consists of the pre-washing, a saline wash and the final bathing of greeneries in sanitizing, choline base solutions. unfortunately, this method cause quality reduction to greeneries, as is observed loss of flavor and texture along with the concurrent risk of contamination existing and spreading under the possibility of incorrect application. greens that grow outdoors follow the triple-wash procedure as a postharvest measure for increasing their health status. harvested crops are transported in the processing facility and sorted, rinsed, put in spinners, apply a second rinse, spinner again, third rinse, sorted (again), packed, and then at the end they get delivered at the grocery market. crops that follow the above washing method bear usually on the packaging labels such as "triple-washed" or "pre-washed". even if this method can provide sufficient results in harvested outdoor crops, if the water used for the triplewashing process is polluted with pathogens, then this can spread rapidly to the rest of the harvested crop. for this reason, triple washing cannot categorized as the most effective and guaranteed process. indoor vertical farms apply only nutrient elements in the irrigation system and completely avoid the use any chemicals during the growing period of plants, excluding all the types of pesticides, herbicides and chemical spaying for fertilization. the philosophy of indoor farming depends on monitoring and constant controlling of the crops as also of all the resources that come in and out from the farm and they are isolated from mother nature where many threats and contamination sources may appear. for this reason, indoor farmers suggest that their products do not need to be washed before consumption, as they are already clean by a protected and purified growing process and a quick delivery to local grocery stores. hermetically sealed environments, inside highly controlled spaces that are designed to offer the highest possible level of food safety particularly for the growing period, surround the cultivation rooms of indoor vertical farms. since there are no seasons to be followed as in outdoor farming neither humidity, temperature fluctuations nor long gaps on post-harvesting processes and packaging, indoor farmers can dramatically reduce a potential contamination with precise systems. in addition, the hermetically sealed environment protects crops from being exposed to outside elements such as harmful pests, insects, fungi and bacteria. in one of other type of such systems, aquaponics, co-cultivation of fish with plants is done. this method of cultivation uses very innovative water filtration systems, which extract solids from the fish tanks. continuously, solid break down to beneficial bacteria that transforms them into nitrates. then, the nitrate-rich water circulates to the plant culture area where plants absorb the nutrients and purify the water. since the aquaponic system follows a close-loop, the clean water is circulated and reused into the fish tank. plants that grow in soilless systems can travel along their production process giving the opportunity to be inspected for health status. for example, after sowings, seeds are moved to germination rooms with high humidity that boosts their sprouting. then, seedling is moved to propagation room with controlled climatic conditions that promote their development. next, young plants usually located in the main part of the cultivated room in floating rafts, receiving a nutrient-rich water. after finishing their development and reaching their mature stage, they are daily harvested and shipped. between every translocation of plants, there is intensive quality check to prevent crops' contamination. high precision irrigation systems are used in order to monitor the water that travels throughout the crops. innovative hydroponic or aeroponic methods usually draw water from filtered and drinkable sources and distribute it at each crop often without even touching the salable part of the plants. this is achieved either by the use of water in liquid form, mist or fog that sprays it only into the root section of the plants and not in the parts consumed. extensive sterilization and supplier are also applied methodologies of indoor vertical farms that control and assure the input resources of the farms such as seeds, nutrients that need to be absolutely safe and clean. because of control and monitor mechanisms that are carried out indoor, there is clear advantage of indoor farms. they are aware of the cleaning status of plants and maintain it with further regulations during the cultivation period and finally harvest and deliver a healthy and fresh product. even if indoor vertical farms produce food safer to consume than the open field grown products, bottlenecks and hazards can still be introduced during the growing process of crops. such threats can be dirt and bacteria transferred from the workers and dangerous threats in the nutrient medium that include chemical sources, cleanliness and water safety. further risks can also detected at the post-harvest activities such as trimming, sorting and delivery of the products. thus, it is of vital importance even for indoor farmers to perform high status and certified systems for detection, monitoring, testing and evaluation as in outdoor farming and greenhouses. a study conducted by purdue university (wang et al., 2019) , found that there is also high risk of crops contamination due to pathogen pollution in vegetables grown in hydroponic or aquaponic systems. more specifically, they reported that e.coli o157:h7 was found in fish feces and because of the circulation that close loops systems, it caused water contamination of the plant root surfaces that were in the aquaponic and the hydroponic systems. since fish probably were contaminated by the bacteria, it is important to follow a proper and certified handling, cleaning and sanitizing process in order to reduce the contamination risk in hydroponic and aquaponics. it is a very difficult, time consuming and α costly process to control all the plants even in an indoor vertical farm for having a 100% safe food product. indoor vertical farms use controlled environment of humidity and temperature in order to provide plants the most suitable conditions. however, in the case that unpredictable production errors occur, e.g., technical malfunctions with the engineering equipment, temperature and humidity can get out bounds to unwanted levels and create fertile environment for bacteria growth. this incident could be possibly avoided in the case of traditional farming, as the constant natural air circulation and the sunlight could smooth out some of these errors. bacteria population are not biased, meaning they do not grow or prefer targeted geographic locations, but they are transported to different locations by human activities as they can be brought by clothes, shoes or skin. furthermore, it should be noted that even if indoor farms consist a safer environment compared to other farming types, if a controlled environment develops for some reason bacterial infection, it will be extremely difficult to eliminate the contamination and protect the rest of the growing crop. for this reason, indoor farms follow high sanity level protocols to avoid the possibility of crops' contamination by human contact that involves all the workers involved with various cultivation processes of the plants. that include strict control by imposing the use of facemasks, hair and beard net, footbaths and clean or single-use suits, which can diminish the risk of contamination. another solution for further risk elimination from potential contamination, is the application of innovative technologies that operate extensive integrated pest monitoring. this can be achieved with the use of ultraviolet light outside of the farms that detect possible threats as also air curtains that are installed in every door and can control air that enters the cultivation room protecting it from the danger of contamination. additional solution that can increase the sanitation levels of indoor crops, is the application of certified hvac filters, in order to perform an extensive pest monitoring. indoor vertical farms belong to a novel type of farming cooperating with innovative technologies in order to provide the safest, higher quality and most fresh and nutritious groceries. both advocates and critics of this technology seem to recognize that indoor vertical farms under suitable circumstances (mainly of the high demand on electricity loads), could offer a solution to the safety and sustainability problems faced in traditional farming. however, consumers seem to be more skeptical and critical on this technology. potential explanation of the consumers' skepticism is the uncertainty and lack of trust in other food innovations such as genetically modified crops, food nanotechnology and artificial irradiation that struggled to find acceptance in the market. nevertheless, the subjective knowledge and awareness level of consumers on the indoor vertical farming is still limited, even with the excessive spread of technology and information globally, it is of vital importance to increase the education of people on this new technology by informing them on the actual growing properties and impugn the unjustified myths and dangers. because of the increasing demand of indoor vertical farms and their establishment in the market, many researchers have focused on designing and addressing customer surveys and other research methodologies in order to define the public opinion on this technology and the status of their trust and preference on already existing agricultural production systems. significant angle on these researches is to explore the existing knowledge and perception of customers between the three different farming systems; traditional farming, greenhouses and indoor vertical farms, in respect of the cultivation techniques, safety, resource sustainability, quality and their willingness to buy products from each category. for this reason, primarily it was of high importance to validate that consumers are able to recognize the different agricultural systems between them in order to provide valid, clarify and comprehensive results. different customer studies that investigated customers' opinion on different agricultural methods show a more skeptical belief concerning novel technologies on food production. more specifically, peoples' perception with technological innovations in agriculture are associated with high risks for food production presenting low expectations on the provided benefits of technology used (sparks et al., 1994) . in another research (coyle and ellison, 2017) participants rated higher the greenhouses facilities and the outdoor farms compared to the indoor vertical farms in terms of naturalness in the production process and the final product. concerning the quality status of the final product people also seem to present higher levels of confidence and trust on the greenhouse products and subsequently indoor vertical farms and finally in outdoor farming products. naturalness seems to be a high influencing indicator for consumers' selection globally as also a critical significant factor on the usefulness of the agricultural system. according to j€ urkenbeck et al. (2019) , customers replied that led lighting is not considered a too artificial tool for horticulture and slightly agreed that they do not consider indoor vertical farming too artificial concerning the overall production system. even if consumers in general prefer naturally and traditionally produced food, nevertheless the fact that food of indoor vertical farms grow without chemical additives is highly considered. on the other hand, under a customer research conducted by j€ urkenbeck et al. (2019), it is noticed that consumers seem to present a high acceptance on indoor vertical farming concerning the offering sustainability and the high ecological footprint. people seem to select their purchased food based on their concerns on the naturalness, ethics and environmental status. in more details, 95% of the respondents in that research declare that they put an extra effort to select and buy locally grown food because of its high level in freshness, nutrition and reduced food mile emissions compared to traditional farming methods. on the other hand, a significant share of the consumers evaluates indoor vertical farming as an artificial agricultural process in order to trust their footprint outcome. for this reason, it is pointed out that knowledge, information and nutritional awareness can become a solid solution for the higher acceptance of indoor farming and irradiated food products. respondents of the specific survey showed a strong willing on buying products that were produced in an indoor vertical farm with 46.7% of the total sample, 36.4% partly agreed on that statement, and finally only 16.8% were not willing to purchase these products. however, it should be noted that the perceived behavioral control does not influence the customers' willingness to buy, but it has some influence on the behavioral intension of willingness to purchase the product. overall, the behavioral intention of customers to purchase products from indoor vertical farms is highly dependent on sustainability. under a different analysis, it has become very clear that perceived sustainability of indoor vertical farming is the main reason of acceptance. it has been observed that the more positive the resulted sustainability status of the system is, the higher and the customers' acceptance and willingness to purchase the product is. furthermore, based on the perceived sustainability level of indoor vertical farms it seems that customers increase and their acceptance of this innovative technological food production system. based on these results, we could indicate that the growing involvement and concern of consumers to select products from agricultural systems that present high environmental performance. indoor vertical farming can be very advantageous in terms of resources sustainability since because of the high technology and the soilless cultivation systems it consumes way less on natural resources (e.g., water and nutrients). additionally, indoor vertical farms significantly decrease the co 2 emissions that are correlated to food transportation from the producers and the processing facilities. in specific, indoor vertical farms can provide 100 times higher productivity per year per unit land area compared to traditional farming due to the zero dependence on weather conditions, seasonality and possible infections from insects, pests and bacteria. due to the evolution of technology it is not anymore a prerequisite holding a large area of land for sufficient fresh food production, but the use of multiple layers, optimally controlled (environmental conditions and physiological parameters of the crops and minimum possible loss from crop threats). significant characteristic of indoor vertical farms in terms of sustainability is the minimization food delivered losses. in addition, significant reductions can be observed in the cooling fuel demand, necessary to cool the production in order to be transported in long distances. this can be achieved since indoor vertical farms are usually installed in the urban or suburban areas in shaded and/or abandoned buildings (or even basements) due to the soilless farming techniques and the artificial lighting, providing access to fresh and nutritious greeneries to citizens. finally, one of the significant benefits that indoor vertical farms provide is the ability after proper processing of the use of waste water, crop wastes and excessive co 2 produced in urban areas, as input resources of water, nutrients and co 2 in the culture area. to summarize some of the basic improvements in resource savings provided by indoor vertical farms compared to the immediately following high technology cultivation system, the greenhouses are the following: • indoor vertical farms save 100% of the pesticide use in their interior by maintaining the culture area clean and insect-free. • because of the application of close loop irrigation systems and of the collection, recycle and reuse of the water vapor that plant leaves transpire, indoor vertical farms can reduce up to 95% the water consumption. furthermore, the use of closed loops can decrease up to 50% the fertilizer usage since it is feasible to recirculate and reuse the nutrient solution. • significant land reduction up to 90% can be achieved with the application of indoor vertical farming, due to the important increase (more than 10 times) of the annual productivity of crops per unit land area. • yield variation can also reduced by 90% because of the constant monitoring and control of the crops and the lack of influence from the outdoor environmental conditions. food safety and traceability of products is another important factor highly relevant to indoor vertical farming. even if it does not provide a 100% safety for consumers, despite the fact that crops grow in a controlled environment protected by wildlife, animals, birds and insects, it upgrades the safety and security feeling of the products than those that grow in open field. the majority of the selected cultivated crops of indoor vertical farms are among the species with the higher contamination risk when they grow outdoors or unprotected, because they grow very close to the ground level. furthermore, one of the most crucial factors that greatly affect the possibility of contamination is the water quality that involves during the whole production process, including the irrigation water as also the washing water at the post-harvest processing techniques. farmers of all categories should follow high standards and criteria for the water sources that channel water into the farms as also frequent control and monitor of the crops for potential threats of contamination. it is now clear, that indoor vertical farms are a high necessity for tackling the challenges concerning the conservation of their resources. nevertheless, in order to enhance the environmental sustainability and improve the efficiency and sufficiency of food production supplies for our society, it is necessary to develop more diverse, effective and ecological agricultural systems including both the traditional farms and the greenhouses. further research and experimentation it is absolutely necessary in order both to improve the efficiency of resources in an indoor vertical farm but also to possibly eliminate the possibilities for contamination threats and constantly provide the outmost safe, fresh and nutritious fresh fruits and vegetables to the human population. notwithstanding the promising benefits that are linked with indoor vertical farming, there are also important challenges in the further implementation of this farming system in the future. it is of vital importance further improvements on the efficiency and effectiveness of the equipment that will lead to a significant decrease of the energy demand of the systems. by achieving the reduction of energy demand, it will add extra value in the environmental sustainability of the system but also it would also make it more appealing for the public, the investors and the industry and will increase the viability and profitability. however, it is pointed out by despommier (2011) that there is the opportunity for energy recovery from the non-salable crops' parts and capture of renewable sources of energy that can create zero energy building for hosting indoor vertical farms. at the same time, the whole system of indoor farming can synchronize and manipulate huge amounts of carbon and simultaneously release into the atmosphere oxygen from plants' respiration. significant is also the start-up costs that are associated with indoor vertical farms as it is clear that it is more expensive to develop a vertical greenhouse than a normal greenhouse (fletcher, 2012) . as it has been highlighted by many studies one also key barrier that indoor vertical farmers have to confront is the public resistance to these type of products as social masses face difficulty in accepting indoor vertical farms instead if traditional farming ones because of the natural way that food is produced. additionally, as indoor vertical farms serve the concept of local, fresh food production and they are mainly installed in urban or peri urban areas, they have also to salient the issue of affordability because of the expensive land and space use. for this reason, key factor is the productivity rate of indoor vertical farms that can maintain them profitable and keep them prevailed in the future. more specifically, if indoor vertical farms achieve to produce up to 50 times more yield compared to traditional farming, then they can offset the capital expenditures and the expensive land use. previous research conducted by , presented that indoor urban vertical farms regardless the financing scheme, are much more profitable in comparison to greenhouse constructions. in the specific work, different investment scenarios are presented based on the cash flow analyses and show that iuvf can present high irr (investment return rate) as also a payback period between 2 and 6 years. finally, another drawback that is linked with indoor farming production is the limited variety of crops that can be produced with this technology, such as lettuce, herbs, tomatoes and berries. even if theoretically, all types of crops could be cultivated indoors, that would not be economically feasible due to the highly increased energy demand. thus, low-value agricultural crops such as wheat and barley will continue to grow under economically and environmentally unviable conditions. under these circumstances, the indoor vertical farms have to face a limited production compared to the "limitless" hectares of traditional farming and a reconsideration of scaling up would be particularly costly and complicated. the last years that indoor vertical farming gained more recognition and research interest, a plethora of new studies, prototypes and innovation designs have been presented under the academic and industrial scope. indoor vertical farming presents a high interest and potential to play a critical role in the demanded sustainability in food of urban areas. this becomes even more important by the multiple studies that estimate and analyze the significant increased food demand in urban areas. indoor vertical farming presents important advantages compared to traditional farming, concerning the required sustainability in our times by focusing in three main categories: environmental, economic and social. there is a high demand for further development in automation. this will be scaling up the projects in order to create more feasible scenarios both from economic and commercial perspective. future research is necessary towards a holistic approach via the investigation and the analysis of the full life-cycle of indoor vertical farms and the impact to the environment compared to the traditional farms and greenhouses. up, up and away! the economy of vertical farming state of indoor farming agstar data and trends. united states environmental protection agency the vertical farm: a review of developments and implications for the vertical city benefit cost analysis of the maize crop under mechanized and traditional farming systems in the nwfp optimization of photoperiod and quality assessment of basil plantsgrown in a small-scale indoor cultivation system for reduction of energy demand plant factories in the water-food-energy nexus era: a systematic bibliographic review indoor vertical farming in the urban nexus context: business growth and resource savings basil plants grown under intermittent light stress in a small-scale indoor environment: introducing energy demand reduction intelligent technologies comparison of land, water, and energy requirements of lettuce grown using hydroponic vs. conventional agricultural methods multistate outbreak of salmonella saintpaul infections linked to imported cucumbers (final update) will consumers find vertically farmed produce "out of reach"? the rise of vertical farms the vertical farm: controlled environment agriculture carried out in tall buildings would create greater food safety and security for large urban populations the vertical farm: controlled environment agriculture carried out in tall buildings would create greater food safety and security for large urban populations efficiency of light energy used by leaves situated in different levels of a sweet pepper canopy the future of agriculture may be up sustainable vertical farming outperforms other agricultural methods on co2 outputs plant factories versus greenhouses: comparison of resources use efficiency blue light does-responses of leaf photosynthesis, morphology, and chemical composition of cucumis sativus grown under different combinations of red and blue light manifesto" for advancing the control and elimination of neglected tropical diseases economics of sustainable farming listeria monocytogenes in tomato greenhouses food miles: background and marketing sustainability matters: consumer acceptance of different vertical farming systems electricity price forecasting in danish day-ahead market using tbats green-light supplementation for enhanced lettuce growth under red-and blue-light-emitting diodes solar air conditioning systems impact on the built environment: a thermodynamic approach exergy analysis for a proposed binary geothermal power plant in nisyros island plant factory: an indoor vertical farming system for efficient quality food production comparison of land, water, and energy requirements of lettuce grown using hydroponic vs. conventional agricultural methods technoeconomic evaluation of urban plant factories: the case of basil (ocimum basilicum) the effects of red, blue, and white light-emitting diodes on the growth, development, and edible quality of hydroponically grown lettuce (lactuca sativa l. var. capitata) effects of different spectral lights on oncidium plbs induction, proliferation, and plant regeneration global food demand, productivity growth, and the scarcity of land and water resources: a spatially explicit mathematical programming approach hydroponics: are we moving towards that direction only because of the environment? a discussion on forecasting and a systems review the impact of co2 emissions on agricultural productivity and household welfare in ethiopia. a computable general equilibrium analysis. environment for development water use efficiency of tomatoes in greenhouses and hydroponics spatial planning of biogas processing facilities in greece: the sunflower's capabilities and the waste-to-bioproducts approach carbon footprint and cumulative energy demand of greenhouse and open-field tomato cultivation systems under southern and central european climatic conditions resource use efficiency of indoor lettuce (lactuca sativa l.) cultivation as affected by red: blue ratio provided by led lighting environmental, energetic, and economic comparisons of organic and conventional farming systems crop growth and development in closed and semi-closed greenhouses. thesis recharging greek youth to revitalize the agriculture and food sector of the greek economy survey and evaluation of heating technologies for worldwide agricultural greenhouse applications advances in greenhouse automation and controlled environment agriculture: a transition to plant factories and urban agriculture gene technology, food production, and public opinion: a uk study the economics of local vertical and greenhouse farming are getting competitive multidirectional relationship between energy resources, climate changes and sustainable development: technoeconomic analysis agstar: biogas recovery in the agriculture sector the occurrence of shiga toxin-producing e. coli in aquaponic and hydroponic systems watering the farm: comparing organic and conventional irrigation water use in the murray-darling basin development of an integrated methodology for the energy needs of a major urban city. the case study of athens wind energy integration through district heating. a wind resource based approach a wind energy integration analysis using wind resource assessment as a decision tool for promoting sustainable energy utilization in agriculture small scale plant factories with artificial lighting and wind energy microgeneration: a multiple revenue stream approach effects of leaf area index of tomato seedling population on energy utilization efficiencies in a close transplant production system energy and mass balance of a closed-type transplant production system (part 3): carbon dioxide balance discovery of a novel coronavirus associated with the recent pneumonia outbreak in humans and its potential bat origin key: cord-176677-exej3zwh authors: coveney, peter v.; highfield, roger r. title: when we can trust computers (and when we can't) date: 2020-07-08 journal: nan doi: nan sha: doc_id: 176677 cord_uid: exej3zwh with the relentless rise of computer power, there is a widespread expectation that computers can solve the most pressing problems of science, and even more besides. we explore the limits of computational modelling and conclude that, in the domains of science and engineering that are relatively simple and firmly grounded in theory, these methods are indeed powerful. even so, the availability of code, data and documentation, along with a range of techniques for validation, verification and uncertainty quantification, are essential for building trust in computer generated findings. when it comes to complex systems in domains of science that are less firmly grounded in theory, notably biology and medicine, to say nothing of the social sciences and humanities, computers can create the illusion of objectivity, not least because the rise of big data and machine learning pose new challenges to reproducibility, while lacking true explanatory power. we also discuss important aspects of the natural world which cannot be solved by digital means. in the long-term, renewed emphasis on analogue methods will be necessary to temper the excessive faith currently placed in digital computation. the extent to which reproducibility is an issue for computer modelling is more profound and convoluted however, depending on the domain of interest, the complexity of the system, the power of available theory, the customs and practices of different scientific communities, and many practical considerations, such as when commercial considerations are challenged by scientific findings. 5 for research on microscopic and relatively simple systems, such as those found in physics and chemistry, for example, theory -both classical and quantum mechanical -offers a powerful way to curate the design of experiments and weigh up the validity of results. in these and other domains of science that are grounded firmly on theory, computational methods more easily help to confer apparent objectivity, with the obvious exceptions of pathological science 6 and fraud 7 . for the very reason that the underlying theory is established and trusted in these fields, there is perhaps less emphasis than there should be on verification and validation ("solving the equations right" and "solving the right equations", respectively 8 ) along with uncertainty quantification-collectively known by the acronym vvuq. by comparison, in macroscopic systems of interest to engineers, applied mathematicians, computational scientists and technologists and others who have to design devices and systems that actually work, and which must not put people's lives in jeopardy, vvuq, is a way of life -in every sense -to ensure that simulations are credible. this vvuq philosophy underpins advances in computer hardware and algorithms that improve our ability to model complex processes using techniques such as finite element analysis, and computational fluid dynamics for end-toend simulations in virtual prototyping and to create digital twins. 9 there is a virtuous circle in vvuq, where experimental data hone simulations, while simulations hone experiments and data interpretation. in this way, the ability to simulate an experiment influences validation by experiment. in other domains, however, notably biology and biomedical sciences, theories have rarely attained the power and generality of physics. the state space of biological systems tends to be so vast that detailed predictions are often elusive, and vvuq is less well established, though that is now changing rapidly as, for example, models and simulations begin to find clinical use 10 . despite the often stated importance of reproducibility, researchers still find various ways to unwittingly fool themselves and their peers 11 . data dredging-also known as blind big data, data fishing, data snooping, and p-hackingseeks results that can be presented as statistically significant, without any knowledge of the structural characteristics of the problem, let alone first devising a hypothesis about the underlying mechanistic relationships. while corroboration or indirect supporting evidence may be reassuring, when taken too far it can lead to the interpretation of random patterns as evidence of correlations, and to conflation of these correlations with causative effects. spurred on by the current reward and recognition systems of academia, it is easier and very tempting to quickly publish one-off findings which appear transformative, rather than invest additional money, energy and time to ensure that these one-off findings are reproducible. as a consequence, a significant number of 'discoveries' turn out to be unreliable because they are more likely to depend on small populations, weak statistics and flawed analysis [12] [13] [14] [15] [16] . there is also a temptation to carry out post hoc rationalisation or harking, 'hypothesizing after the results are known' and to invest more effort into explaining away unexpected findings than validating expected results. most contemporary research depends heavily on computers which generate numbers with great facility. ultimately, though, computers are themselves tools that are designed and used by people. because human beings have a capacity for self-deception 17 , the datasets and algorithms that they create can be subject to unconscious biases of various kinds, for example in the way data are collected and curated in data dredging activities, a lack of standardized data analysis workflows 18 , or the selection of tools that generate promising results, even if their use is not appropriate in the circumstances. no field of science is immune to these issues, but they are particularly challenging in domains where systems are complex and many dimensional, weakly underpinned by theoretical understanding, and exhibit non-linearity, chaos and long-range correlations. with the rise of digital computing power, approaches predicated on big data, machine learning (ml) and artificial intelligence (ai) are frequently deemed to be indispensable. ml and ai are increasingly used to sift experimental and simulation data for otherwise hidden patterns that such methods may suggest are significant. reproducibility is particularly important here because these forms of data analysis play a disproportionate role in producing results and supporting conclusions. some even maintain that big data analyses can do away with the scientific method. 19 however, as data sets increase in size, the ratio of false to true correlations increases very rapidly, so one must be able to reliably distinguish false from true if one is able to find robust correlations. that is difficult to do without a reliable theory underpinning the data being analysed. we, like others 20 , argue that the faith placed in big data analyses is profoundly misguided: to be successful, big data methods must be more firmly grounded on the scientific method. 21 far from being a threat to the scientific method, the weaknesses of blind big data methods serve as a timely reminder that the scientific method remains the most powerful means we have to understand our world. in science, unlike politics, it does not matter how many people say or agree about something: if science is to be objective, it has to be reproducible ("within the error bars"). observations and "scientific facts and results" cannot depend on who is reporting them but must be universal. consensus is the business of politics and the scientific equivalent only comes after the slow accumulation of unambiguous pieces of empirical evidence (albeit most research and programmes are still funded on the basis of what the majority of people on a review panel thinks is right, so that scientists who have previously been successful are more likely to be awarded grants 22,23 .) there is some debate about the definition of reproducibility 24 . some argue that replicability is more important than reproducibility. others maintain that the gold standard of research should be 're-testability', where the result is replicated rather than the experiment itself, though the degree to which the 'same result' can emerge from different setups, software and implementations is open to question. 25 by reproducibility we mean the repetition of the findings of an experiment or calculation, generally by others, providing independent confirmation and confidence that we understand what was done and how, thus ensuring that reliable ideas are able to propagate through the scientific community and become widely adopted. when it comes to computer modelling, reproducibility means that the original data and code can be analysed by any independent, sceptical investigator to reach the same conclusions. the status of all investigators is supposedly equal and the same results should be obtained regardless of who is performing the study, within well-defined error bars -that is, reproducibility must be framed as a statistically robust criterion because so many factors can change between one set of observations and another, no matter who performs the experiment. the uncertainties come in two forms: (i) "epistemic", or systematic errors, which might be due to differences in measuring apparatus; and (ii) "aleatoric", caused by random effects. the latter typically arise in chaotic dynamical systems which manifest extreme sensitivity to initial conditions, and/or because of variations in conditions outside of the control of an experimentalist. by seeking to control uncertainty in terms of a margin of error, reproducibility means that an experiment or observation is robust enough to survive all manner of scientific analysis. note, of course, that reproducibility is a necessary but not a sufficient condition for an observation to be deemed scientific. in the scientific enterprise, a single result or measurement can never provide definitive resolution for or against a theory. unlike mathematics, which advances when a proof is published, it takes much more than a single finding to establish a novel scientific insight or idea. indeed, in the popperian view of science, there can be no final vindication of the validity of a scientific theory: they are all provisional, and may eventually be falsified. the extreme form of the modern machine-learners' pre-baconian view stands in stark opposition to this: there is no theory at all, only data, and success is measured by how well one's learning algorithm performs at discerning correlations within these data, even though many of these correlations will turn out to be false, random or meaningless. moreover, in recent years, the integrity of the scientific endeavour has been open to question because of issues around reproducibility, notably in the biological sciences. confidence in the reliability of clinical research has, for example, been under increasing scrutiny. 5 in 2005, john p. a. ioannidis wrote an influential article about biomedical research, entitled "why most published research findings are false", in which he assessed the positive predictive value of the truth of a research finding from values such as threshold of significance and power of the statistical test applied. 26 he found that the more teams were involved in studying a given topic, the less likely the research findings from individual studies turn out to be true. this seemingly paradoxical corollary follows because of the scramble to replicate the most impressive "positive" results and the attraction of refuting claims made in a prestigious journal, so that early replications tend to be biased against the initial findings. this 'proteus phenomenon' has been observed as an early sequence of extreme, opposite results in retrospective hypothesis-generating molecular genetic research 27 , although there is often a fine line to be drawn between contrarianism, wilful misrepresentation and the scepticism ('nullius in verba') that is the hallmark of good science. 28 such lack of reproducibility can be troubling. an investigation of 49 medical studies undertaken between 1990-2003 -with more than 1000 citations in total -found that 16% were contradicted by subsequent studies, 16% found stronger effects than subsequent studies, 44% were replicated, and 24% remained largely unchallenged. 29 in psychological science, a large portion of independent experimental replications did not reproduce evidence supporting the original results despite using high-powered designs and original materials when available. 30 even worse performance is found in cognitive neuroscience. 13 scientists more widely are routinely confronted with issues of reproducibility: a may 2016 survey in nature of more than 1576 scientists reported that more than 70% had tried and failed to reproduce another scientist's experiments, and more than half had failed to reproduce their own experiments. 31 this lack of reproducibility can be devastating for the credibility of a field. computers are critical in all fields of data analysis and computer simulations need to be reliable -validated, verified, and their uncertainty quantified -so that they can feed into real world applications and decisions be they governmental policies dealing with pandemics, for the global climate emergency, the provision of food and shelter for refugee populations fleeing conflicts, creation of new materials, the design of the first commercial fusion reactor, or to assist doctors to test medication on a virtual patient before a real one. reproducibility in computer simulations would seem trivial to the uninitiated: enter the same data into the same program on the same architecture and you should get the same results. in practice, however, there are many barriers to overcome to ensure the fidelity of a model in a computational environment. 32 overall, it can be challenging if not impossible to test the claims and arguments made by authors in published work without access to the original code and data, and even in some instances the machines the software ran on. one study of what the authors dubbed 'weak repeatability' examined 402 papers with results backed by code and found that, for one third, they were able to obtain the code and build it within half an hour, while for just under half they succeeded with significant extra effort. for the remainder, it was not possible to verify the published findings. the authors reported that some researchers are reluctant to share their source code, for instance for commercial and licensing reasons, or because of dependencies on other software, whether due to external libraries or compilers, or because the version they used in their paper had been superseded, or had been lost due to lack of backup. many detailed choices in the design and implementation of a simulation never make it into published papers. frequently, the principal code developer has moved on, the code turns out to depend on exotic hardware, there is inadequate documentation, and/or the code developers say that they are too busy to help. 33 there are some high-profiles examples of these issues, from disclosure of climate codes and data, 34 to delays in sharing codes for covid-19 pandemic modelling. 35 if the public are to have confidence in computing models that could directly affect them, transparency, openness and the timely release of code and data are critical. in response to this challenge, there have been various proposals to allow scientists to openly share code and data that underlie their research publications: runmycode [runmycode.org] and, perhaps better known, github [github.com]; share, a web portal to create, share, and access remote virtual machines that can be cited from research papers to make an article fully reproducible and interactive; 36 papermâché, another means to view and interact with a paper using virtual machines; 37 various means to create 'executable papers' 38, 39 ; and a verifiable result identifier (vri), which consists of trusted and automatically generated strings that point to publicly available results originally created by the computational process. 40 in addition to external verification, there are many initiatives to incorporate verification and validation into computer model development, along with uncertainty quantification techniques to verify and validate the models. 41 in the united states, for example, the american society of mechanical engineers has a standards committee for the development of verification and validation v&v procedures for computational solid mechanics models, 42 guidelines and recommended practices have been developed by the national aeronautics and space administration (nasa); 43 the us defense nuclear facilities safety board backs model v&v for all safety-related nuclear facility design, analyses, and operations, while various groups within the doe laboratories (including sandia, los alamos, and lawrence livermore) are conducting research in this area. 44 in europe, the vecma (verified exascale computing for multiscale applications) project 45 is developing software tools that can be applied to many research domains, from the laptop to the emerging generation of exascale supercomputers, in order to validate, verify, and quantify the uncertainty within highly diverse applications. the major challenge faced by the state of the art is that many scientific models are multiphysics in nature, combining two or more kinds of physics, for instance to simulate the behaviour of plasmas in tokamak nuclear fusion reactors 46 , electromechanical systems 47 or in food processing 48 . even more common, and more challenging, many models are also multiscale, which require the successful convergence of various theories that operate at different temporal and/or spatial scales. they are widespread at the interface between various fields, notably physics, chemistry and biology. the ability to integrate macroscopic universality and molecular individualism is perhaps the greatest challenge of multiscale modelling 49 . as one example, we certainly need multiscale models if we are to predict the biology, medicine that underpin the behaviour of an individual person. digital medicine is increasingly important and, as a corollary of this, there have been calls for steps to avoid a reproducibility "crisis" of the kind that has engulfed other areas of biomedicine. 50 although there are many kinds of multiscale modelling, there now exist protocols to enable the verification, validation, and uncertainty quantification of multiscale models. 51 the vecma toolkit 52 , which is not only open source but whose development is also performed openly, has many components: fabsim3, to organise and perform complex remote tasks; easyvvuq, a python library designed to facilitate verification, validation and uncertainty quantification for a variety of simulations 53, 54 ; qcg pilot job, to provide the efficient and reliable execution of large number of computational jobs; qcg-now, to prepare and run computational jobs on high performance computing machines; qcg-client, to provide support for a variety of computing jobs, from simple ones to complex distributed workflows; easyvvuq-qcgpilotjob, for efficient, parallel execution of demanding easyvvuq scenarios on high performance machines; and muscle 3, to make creating coupled multiscale simulations easier, and to then enable efficient uncertainty quantification of such models. the vecma toolkit is already being applied in several circumstances: climate modelling, where multiscale simulations of the atmosphere and oceans are required; forecasting refugee movements away from conflicts, or as a result of climate change, to help prioritise resources and investigate the effects of border closures and other policy decisions 55 ; for exploring the mechanical properties of a simulated material at several length and time scales with verified multiscale simulations; and multiscale simulations to understand the mechanisms of heat and particle transport in fusion devices, which is important because the transport plays a key role in determining the size, shape and more detailed design and operating conditions of a future fusion power reactor, and hence the possibility of extracting almost limitless energy; and verified simulations to aid in the decision-making of drug prescriptions, simulating how drugs interact with a virtual version of a patient's proteins, 56 or how stents will behave when placed in virtual versions of arteries. 57 recent years have seen an explosive growth in digital data accompanied by the rising public awareness that their lives depend on "algorithms", though it is plain to all that any computer code is based on an algorithm, without which it will not run. under the banner of artificial intelligence and machine learning, many of these algorithms seek patterns in those data. some -emphatically not the authors of this paper -even claim that this approach will be faster and more revealing than modelling the underlying behaviour notably by the use of conventional theory, modelling and simulation. 58 this approach is particularly attractive in disciplines traditionally not deemed suitable for mathematical treatment because they are so complex, notably life and social sciences, along with the humanities. however, to build a machine-learning system, you have to decide what data you are going to choose to populate it. that choice is frequently made without any attempt to first try to understand the structural characteristics that underlie the system of interest, with the result that the "ai system" produced strongly reflects the limitations or biases (be they implicit or explicit) of its creators. moreover, there are four fundamental issues with big data that are frequently not recognised by practitioners 58 : complex systems are strongly correlated, so they do not generally obey gaussian statistics; no datasets are large enough for systems with strong sensitivity to rounding or inaccuracies; correlation does not imply causality; and too much data can be as bad as no data: although computers can be trained on larger datasets than the human brain can absorb, there are fundamental limitations to the power of such datasets (as one very real example, mapping genotype to phenotype is far from straightforward), not least due to their digital character. all machine-learning algorithms are initialised using (pseudo) random number generators and have to be run vast numbers of times to ensure that their statistical predictions are robust. however, they typically make plenty of other assumptions, such as smoothness (i.e. continuity) between data points. the problem is that nonlinear systems are often anything but smooth, and there can be jumps, discontinuities and singularities. not only the smoothness of behaviour but also the forms of distribution of data regularly assumed by machine learners are frequently unknown or untrue in complex systems. indeed, many such approaches are distribution free, in the sense that there is no knowledge provided about the way the data being used is distributed in a statistical sense. 58 often, a gaussian ("normal") distribution is assumed by default; while this distribution plays an undeniable role across all walks of science it is far from universal. indeed, it fails to describe most phenomena where complexity holds sway because, rather than resting on randomness, these typically have feedback loops, interactions and correlations. machine learning is often used to seek correlations in data. but in a real-world system, for instance in a living cell that is a cauldron of activity of 42 million protein molecules 59 , can we be confident that we have captured the right data? random data dredging for complex problems is doomed to fail where one has no idea which variables are important. in these cases, data dredging will always be defeated by the curse of dimensionality -there will simply be far too much data needed to fill in the hyperdimensional space for blind machine learning to produce correlations to any degree of confidence. on top of that, as mentioned earlier, the ratio of false to true correlations soars with the size of the dataset, so that too much data can be worse than no data at all. there are practical considerations too. machine-learning systems can never be better than the data they are trained on, which can contain biases 'whether morally neutral as toward insects or flowers, problematic as toward race or gender, or even simply veridical, reflecting the status quo distribution of gender with respect to careers or first names'. 60 in healthcare systems, for example, where commercial prediction algorithms are used to identify and help patients with complex health needs, significant racial bias has been found. 61 machine learning systems are black boxes, even to the researchers that build them, making it hard for their creators, let alone others, to assess the results produced by these glorified curve-fitting systems. precise replication would be nearly impossible given the natural randomness in neural networks and variations in hardware and code. that is one reason why blind machine learning is unlikely to ever be accepted by regulatory authorities in medical practice as a basis for offering 8 drugs to patients. to comply with the regulatory authorities such as the us food and drug administration and the european medicines agency, the predictions of a ml algorithm are not enough and it is essential that an underlying mechanistic explanation is also provided, one which can explain not only when a drug works but also when it fails, and/or produces side effects. there are even deeper problems of principle in seeking to produce reliable predictions about the behaviour of complex systems of the sort one encounters frequently in the most pressing problems of twenty-first century science. we are thinking particularly, in life sciences, medicine, healthcare and environmental sciences, where systems typically involve large numbers of variables and many parameters. the question is how to select these variables and parameters to best fit the data. despite the constant refrain that we live in the age of "big data", the data we have available is never enough to model problems of this degree of complexity. unlike more traditional reductionist models, where one may reasonably assume one has sufficient data to estimate a small number of parameters, such as a drug interacting with a nerve cell receptor, this ceases to be the case in complex and emergent systems, such as modelling a nerve cell itself. the favourite approach of the moment is of course to select machine learning, which involves adjustments of large numbers of parameters inside the neural network "models" used; these can be tuned to fit the data available but have little to no predictability beyond the range of the data used because they do not take into account the structural characteristics of the phenomenon under study. this is a form of overfitting. 63 as a result of the uncertainty in all these parameters, the model itself becomes uncertain as testing it involves an assessment of probability distributions over the parameters and, with nowhere near adequate data available, it is not clear if it can be validated in a meaningful manner. 64 for some related issues of a more speculative and philosophical nature in the study of complexity, see succi (2019) . compounding all this, there is a fundamental problem that undermines our faith in simulations which arises from the digital nature of modern computers, whether classical or quantum. digital computers make use of four billion rational numbers that range from plus to minus infinity, the so-called 'single-precision ieee floating-point numbers', which refers to a technical standard for floating-point arithmetic established by the institute of electrical and electronics engineers in the 1950s; they also frequently use double precision floating-point numbers, while half-precision has become commonplace of late in the running of machine learning algorithms. however, digital computers only use a very small subset of the rational numbers -so-called dyadic numbers, whose denominators are powers of 2 because of the binary system underlying all digital computers -and the way these numbers are distributed is highly nonuniform. moreover, there are infinitely more irrational than rational numbers, which are ignored by all digital computers because to store any one of them, typically, one would require an infinite memory. leaving aside the mistaken belief held by some that a very few repeats of, say, a molecular dynamics simulation is any replacement for (quasi) monte carlo methods based on ensembles of replicas, these findings strongly suggest that the digital simulation of all chaotic systems, found in models used to predict weather, climate, molecular dynamics, chemical reactions, fusion energy and much more, contain sizeable errors of a nature that hitherto have been unknown to 9 most scientists. by the same token, the use of data from these chaotic simulations to train machine learning algorithms will in turn produce artefacts, making them unreliable. this shortcoming produced generic errors of up to 20 per cent in the case of the bernoulli map along with pure nonsense on rare occasions. one might ask why, if the consequences can be so substantial, these errors have not been noticed. the difficulty is that for real world simulations in turbulence and molecular dynamics, for example, there are no exact, closed form mathematical solutions for comparison so the numerical solutions that roll off the computer are simply assumed to be correct. given the approximations involved in such models, not to speak of the various sources of measurement errors, it is never possible to obtain exact agreement with experimental results. in short, the use of floating point numbers instead of real numbers contributes additional systematic errors in numerical schemes that have not so far been assessed at all 66 . for modelling, we need to tackle both epistemic and aleatoric sources of error. to deal with these challenges, a number of association for the advance of artificial intelligence, aaai, conferences 76 ) found that only 6% of the presenters shared the algorithm's code. 77 the most commonly-used machine learning platforms provided by big tech companies have poor support for reproducibility. 78 studies have shown that even if the results of a deep learning model could be reproduced, a slightly different experiment would not support the findings-yet another example of overfitting-which is common in machine learning research. in other words, unreproducible findings can be built upon supposedly reproducible methods. 79 rather than continuing to simply fund, pursue and promote 'blind' big data projects, more resources should be allocated to the elucidation of the multiphysics, multiscale and stochastic processes controlling the behaviour of complex systems, such as those in biology, medicine, healthcare and environmental science. 21 finding robust predictive mechanistic models that provide explanatory insights will be of particular value for machine learning when dealing with sparse and incomplete sets of data, ill-posed problems, exploring vast design spaces to seek correlations and then, most importantly, for identifying correlations. where machine learning provides a correlation, multiscale modelling can test if this correlation is causal. there are also demands in some fields for a reproducibility checklist, 80 to make ai reproducibility more practical, reliable and effective. another suggestion is the use of so-called "model cards" -documentation that accompanies trained machine learning models which outline the application domains, the context in which they are being used and their carefully benchmarked evaluation in a variety of conditions, such as across different cultural, demographic, and phenotypic groups; 81 and proposals for best practice in reporting experimental results which permit for robust comparison. 82 despite the caveat that computers are made and used by people, there is also considerable interest in their use to design and run experiments, for instance using bayesian optimization methods, such as in the field of cognitive neuroscience 83 and to model infectious diseases and immunology quantitatively. 84 when it comes to the limitations of digital computing, research is under way by boghosian and pvc to find alternative approaches that might render such problems computable on digital computers. among possible solutions, one that seems guaranteed to succeed is analogue computing, an older idea, able to handle the numerical continuum of reality in a way that digital computers can only approximate. 85 in the short term, notably in the biosciences, better data collection, curation, validation, verification and uncertainty quantification procedures of the kind described here, will make computer simulations more reproducible, while machine learning will benefit from a more rigorous and transparent approach. the field of big data and machine learning has become extremely influential but without big theory it remains dogged by a lack of firm theoretical underpinning ensuring its results are reliable. 21 indeed, we have argued that in the modern era in which we aspire to describe really complex systems, involving many variables and vast numbers of parameters, there is not sufficient data to apply these methods reliably. our models are likely to remain uncertain in many respects, as it is so difficult to validate them. in the medium term, ai methods may, if carefully produced, improve the design, objectivity and analysis of experiments. however, this will always require the participation of people to devise the underlying hypotheses and, as a result, it is important to ensure that they fully grasp the assumptions on which these algorithms are based and are also open about these assumptions. it is already becoming increasingly clear that 'artificial intelligence' is a digital approximation to reality. moreover, in the long term, when we are firmly in the era of routine exascale and perhaps eventually also quantum computation, we will have to grapple with a more fundamental issue. even though there are those who believe the complexity of the universe can be understood in terms of simple programs rather than by means of concise mathematical equations, 86,87 digital computers are limited in the extent to which they can capture the richness of the real world. 85,88 freeman dyson, for example, speculated that for this reason the downloading of a human consciousness into a digital computer would involve 'a certain loss of our finer feelings and qualities' 89 . in the quantum and exascale computing eras, we will need renewed emphasis on the analogue world and analogue computational methods if we are to trust our computers. 85 the turing way" -a handbook for reproducible data science the need for open source software in machine learning as concerns about non-reproducible data mount, some solutions take shape addressing scientific fraud. science (80-) bad pharma: how drug companies mislead doctors and harm patients betrayers of the truth: fraud and deceit in the halls of science quantification of uncertainty in computational fluid dynamics assessing the reliability of complex models: mathematical and statistical foundations of verification, validation, and uncertainty quantification editorial: special issue on verification, validation, and uncertainty quantification of cardiovascular models: towards effective vvuq for translating cardiovascular modelling to clinical utility how scientists fool themselves -and how they can stop low statistical power in biomedical science: a review of three human research domains empirical assessment of published effect sizes and power in the recent cognitive neuroscience and psychology literature a survey of the statistical power of research in behavioral ecology and animal behavior evaluating the quality of empirical journals with respect to sample size and statistical power. ouzounis ca power failure: why small sample size undermines the reliability of neuroscience the evolution and psychology of self-deception variability in the analysis of a single neuroimaging dataset by many teams the end of theory: the data deluge makes the scientific method obsolete the scientific method in the science of machine learning big data need big theory too the matthew effect in science: the reward and communication systems of science are considered. science (80-) the matthew effect in science funding muddled meanings hamper efforts to fix reproducibility crisis reproducible research: a minority opinion why most published research findings are false early extreme contradictory estimates may appear in published research: the proteus phenomenon in molecular genetics research and randomized trials why selective publication of statistically significant results can be effective contradicted and initially stronger effects in highly cited clinical research estimating the reproducibility of psychological science. science (80-) 1,500 scientists lift the lid on reproducibility reproducibility in scientific computing published online 2014:1-37. 34. the disclosure of climate data from the climatic github -imperial college london -code for modelling estimated deaths and cases for covid19 share: a web portal for creating and sharing executable research papers paper mâché: creating dynamic reproducible science a provenance-based infrastructure to support the life cycle of executable papers a universal identifier for computational results a comprehensive framework for verification, validation, and uncertainty quantification in scientific computing verification, validation and uncertainty quantification (vvuq) concepts of model verification and validation vecma verified exascale computing for multiscale applications multidimensional multiphysics simulation of nuclear fuel behavior multiphysics simulation innovative food processing technologies: advances in bridging the gaps at the physics-chemistry-biology interface the reproducibility crisis in the age of digital medicine multiscale modelling: approaches and challenges building confidence in simulation: applications of easyvvuq a library for verification, validation and uncertainty quantification in high performance computing a generalized simulation development approach for predicting refugee destinations ensemble-based steered molecular dynamics predicts relative residence time of a 2a receptor binders semi-intrusive multiscale metamodelling uncertainty quantification with application to a model of in-stent restenosis big data: the end of the scientific method unification of protein abundance datasets yields a quantitative saccharomyces cerevisiae proteome semantics derived automatically from language corpora contain human-like biases dissecting racial bias in an algorithm used to manage the health of populations. science (80-) weapons of math destruction. crown random house the problem of overfitting the evolution of scientific knowledge: from certainty to uncertainty a new pathology in the simulation of chaotic dynamical systems on digital computers of naturalness and complexity metascience could rescue the 'replication crisis the unity of knowledge psychologists strike a blow for reproducibility increasing transparency through a multiverse analysis the science of team science brazilian biomedical science faces reproducibility test policy: nih plans to enhance reproducibility the academy of medical sciences. reproducibility and reliability of biomedical research: improving research practice state of the art: reproducibility in artificial intelligence missing data hinder replication of artificial intelligence studies. science (80-) out-of-the-box reproducibility: a survey of machine learning platforms unreproducible research is reproducible the machine learning reproducibility checklist (version 1.0) model cards for model reporting show your work: improved reporting of experimental results neuroadaptive bayesian optimization and hypothesis testing host genotype and time dependent antigen presentation of viral peptides: predictions from theory. sci rep from digital hype to analogue reality: universal simulation beyond the quantum and exascale eras the wolfram physics project: a project to find the fundamental theory of physics a new kind of science. wolfram media a class of models with the potential to represent fundamental physics the authors are grateful for many stimulating conversations with bruce boghosian, daan crommelin, ed dougherty, derek groen, alfons hoekstra, robin richardson & david wright. authors' contributions all authors contributed to the concept and writing of the article. the authors have no competing interests.funding statement p.v.c. is grateful for funding from the uk epsrc for the ukcomes uk high-end computing consortium (ep/r029598/1), from mrc for a medical bioinformatics grant (mr/l016311/1), the european commission for the compbiomed, compbiomed2 and vecma grants (numbers 675451, 823712 and 800925 respectively) and special funding from the ucl provost. key: cord-209166-1drg5gzp authors: tah, rajdeep; mazumdar, sarbajit; parida, krishna kant title: study of the curvature of liquid surface surrounding a rotating spherical object in gravity free space date: 2020-08-26 journal: nan doi: nan sha: doc_id: 209166 cord_uid: 1drg5gzp concept of curvature of liquid surrounding a spherical surface seems obvious in daily life, but based on earthly conditions everywhere. however, our understanding about the concept seems more transparent when we keep the system out of the usual earthly condition i.e. without gravity. although existence of forces like adhesion and cohesion along liquid surface come to the fore even in the presence of other force like gravitational ones, but without gravity these forces are solely responsible for kind of observable phenomenon. also, we introduced a form of force responsible for providing a form of potential dominating over the gravitational one. the discussion was provided an ingenious approach, by conserving surface energy, it still explains a lot about what can be done more to explore other properties of rotating liquids in free space. whenever we think of curvature of a surface associated to a system, the first thing which comes to our mind is that it is related to the profile of the surface i.e. the physical state of the system's surface. in mathematical sense, the curvature is defined as the amount by which a curve deviates from being a straight line or the amount by which a surface deviates from being a plane. the curvature of curves drawn on a surface is the main tool for the defining and studying of the curvature of the surface. now, in our case we tend to study the curvature of liquid surrounding a spherical surface which is in-turn rotating at an angular velocity ω but in the absence of a major factor i.e. gravity. the absence of gravity in our case is a key factor which helps us to study our system in a more transparent and effective way i.e. it can be used to study the curvature properties of fluids in gravity-free space. although we are neglecting gravity in our system, we will still consider the existence of other factors (like cohesion forces; adhesion forces; etc) which contribute for the change in the curvature of the rotating system's surface. in the absence of gravity, this factors will be solely responsible for the change in the properties of the system. further, we proceed to calculate the restriction on the angular velocity beyond which the centrifugal force will tend to spill the fluid out from the system and following it, we will try to study the various other properties of the system by conserving the surface energy through a much more ingenious approach. in section 2 we will describe our system by calculating the equatorial bulge (n) and also by deriving the equation of the spheroid formed by the fluid due to the rotation of the system. then in section 3 we will derive the expression for the central fluid pressure (p 0 ) and following it we will derive the expression for the containing force (f(x, y, z)) of the system. after deriving both the previously mentioned expressions, we will derive the expression for the angular velocity of the system after finding out the moment of inertia (i net ) and surface area (s ) of the system in section 4. then in section 5 we will find the expressions for the containing force (f(x)) along x-axis, limiting ω and the containing potential (ψ(x)) along x-axis respectively which we are accounting under restricted conditions. after that in section 6 we will conclude our results which we inferred from the previous sections using various concepts followed by acknowledgement in section 7 and references respectively. we are considering a metal sphere is present in free space, with no net force on the system, and a liquid layer had completely engulfed the metal ball. the system is an isolated one, with no presence or effect of gravity and standard atmospheric pressure of 1 atm. the physical conditions of the system can be compared to that present inside the international space station where habitable conditions are present with the exception of gravity.. now the complete system is rotated with a constant angular velocity ω and was allowed to rotate with the same angular velocity. we can say that many forces are acting within the system and primary one will be the gravitational force between the metal ball and the liquid layer. the system seems more or like a planet with a solid metallic core and a liquid atmosphere. but we have taken effect to to gravitational forces to be very negligible, since the mass of the metal ball as well as that of the liquid layer is very small. also the gravitational constant g is of the value 6.674 × 10 −11 m 3 kg −1 s −2 , which is too small to make the gravitational force effective for calculations. hence we have to take adhesion forces between the liquid particle and metal, and cohesion forces within the liquid particles to be main cause for keeping the complete system intact. let the radius of the metal ball be r and the initial height of the liquid layer is taken to be m. now the system is made up of material of different densities. let the density of the liquid layer surrounding it be ρ and that of the metal ball be ρ m respectively. now from the initial state, since the complete system is being rotated, then the shape of the metal ball won't change, but the shape of the liquid layer will change due to non-rigid properties of it. due to presence of centrifugal force, the liquid will try to flatten out in the space along equatorial plane, resulting in the formation of a spheroid shaped drop of liquid surrounding the spherical metal ball. according to the section 2, as the value of angular velocity increases the height of fluid layer (m) along z− axis will decrease resulting an equatorial bulge along xy plane. let the decrease of fluid layer along z−axis be h and let the value of the bulge be n. since the volume of the fluid is constant throughout the process, to calculate the value of the bulge we are going to conserve the volume of initial state and final state. let the volume of liquid at initial state be v 1 and that at final state be v 2 , so we have: since v 1 = v 2 , we can write: so finally we have the lengths of all semi major axes of the spheroid so equation of the spheroid will be: 3 pressure potential and containing force consider the equilibrium of an incompressible fluid with a constant angular velocity ω in some inertial frame of reference. according to standard newtonian dynamics the force balance equation takes form where ∇p is the static fluid pressure, ρ is the mass density and in our case ψ is the potential for containing force. so the equation can be modified into where ψ is the centrifugal potential, ψ = − ω(x 2 + y 2 ) 2 so, the equation becomes where p 0 is central fluid pressure and the pressure at the outer boundary of the fluid surface must be zero, otherwise there would be a force imbalance across the boundary. hence p = 0 which implies now as we know, the effect of gravitational potential is much minimal than another potential taken under consideration in the equation for central fluid pressure. the cause of this potential can be explained by presence of another form of force different from the gravitational one. this force in some form, is related to the surface formation of the liquid, which also includes surface tension (γ) of the fluid in the potential equation. now surface tension of the fluid leads to the laplace pressure, which is the pressure difference between the inside and the outside of a curved surface that forms the boundary between a gas region and a liquid region, and is given by: where r 1 and r 2 are the principle radii curvature of the fluid layer. and hence, pressure due to the potential for containing force can be represented by: so the final equation for containing force potential is: for general ellipsoid, the principle radii of curvature depend on the mean curvature(h) and the gaussian curvature(k) [1] . hence, that means equation 7 can be modified as: now, as we know that h = so we get the general form of potential; so, the general form of containing force will be hence; now to analyse the potential and the containing force we only consider it's contribution along x axis. so the simplified equations for potential and force will be: we are calculating for which angular velocity (ω) the fluid layer along z− axis just touches the surface of the metal sphere. to find the required angular velocity we have to use conservation of energy. now since the rotation of the complete system is resulting in the change in the shape of the surface, hence total rotational energy can be considered to be transferred to the overall change in the surface energy due to changed shape of the fluid layer. let γ wa and γ ws be the interfacial energy between water layer and the gas or air present outside and between water layer and solid metal surface respectively.let ∆e 1 and ∆e 2 be the initial and final surface energies of the system respectively. also, let the initial and final surface areas of the fluid layer be s and s respectively. then we can write: for conserving total energy of the system, we can say that total energy available initially will be equal to the total energy available finally with the system. hence we can write: change in rotational kinetic energy = change in surface energy now, moment of inertia (i net ) of the overall system can be divided into three parts, i.e., moment of inertia of the overall spheroid(i 1 ), moment of inertia of the cavity (i 2 )(which is to be subtracted from that of the spheroid) and moment of inertia of the metal ball (i 3 ). hence we can write: i net = i 1 − i 2 + i 3 now, therefore we have, using equation 1, we have now the surface area of the spheroid with equation now for our case we have a = (r + m) 1 + m r and c = r, hence ; so using the value of s and i net we can find the value of ω as: now for m << r; we can approximate the value of ω by using taylor's series [5] up to o(1) and from that we get the expression of ω as: the simplified expression for force in equation 9 can be plotted with respect to distance measured along x-axis as shown in figure 4 . now as we can see that we had restricted the plot of f(x) from the radius of the metal sphere r, up to the semi-major axis along x axis or y axis, a = (r + m) 1 + m r , which corresponds to the force values f max and f min respectively. the restriction comes with the reason that the considered containing force is limited to a particular value of x beyond which, the magnitude of force becomes inconsistent with the physical system as fluid is present from the surface of the metal sphere up to the liquid and air interface. the force at x = a is given by f min = f(a) = 2a 2 γ ρ . this agrees with the fact that for the system being static or without any rotation, this force is responsible for bringing the surface of the liquid to the equilibrium position, i.e. into the spherical shape. now during rotation, this force is balanced by the centrifugal force acting per unit mass along the x direction. using this concept we can find a bound on the angular velocity (ω) for which the water layer remains in shape of the spheroid without spilling out from the system. now if we give ω > 2aγ ρ , the water will start spilling out from the outer surface as the force at the boundary is minimum. similarly, with same conditions, we can define the containing potential of the system along x direction from radius r to the value a as shown in figure 5 . the potential trend also have physical significance, since potential at the surface of the spheroid surface of the liquid is zero. now with regards to the f max , this force is balanced by the surface of the metal sphere, since the metal sphere is rigid and is not deformable. we are able to highlight certain points which may not seem possible to replicate on earthly conditions, by removing effects on gravity but keeping other conditions around it as such. under theoretical considerations, we were able to predict specific aspects like how much angular velocity is required in order to get the desired shape of the liquid drop with condition of having very little trace of liquid at region along z axis, also called polar region. we have to introduce the concept of containing force, which is other than gravitational force present between the particles of the liquid and the metal sphere. the reason behind introduction of such concept within our system is also explained as we are not considering very massive system like that of a planet and its atmospheric layer. we also explained how the containing force is helpful for explaining the presence of a potential, which is different from gravitational potential as explained in the maclaurin spheroid [2] . if we consider the gravitational force and corresponding gravitational potential, the expression for angular velocity (ω) is already derived as: (3 − 2e 2 ) sin −1 e − 6 e 2 (1 − e 2 ), e = eccentricity of the spheroid further we proceed to calculate the restriction on the angular velocity beyond which, the centrifugal force will tend to spill the fluid from the system, leaving all the expressions explaining the system to be inconsistent. the system we tried to explain in the paper seems to have least amount of applications in current physics and engineering fields, but it has the potential to explain about the considerations related to outer space machineries with special focus on lubrication systems. curvature of the ellipsoid with cartesian coordinates rt, sm and kp would like to thank school of physical sciences (sps) and academic section of niser, bhubaneswar where they got the opportunity to interact with wonderful members and professors who helped them a lot with the basics of fluid dynamics. they also acknowledge the support of their parents who constantly kept them motivated throughout the project during the covid-19 pandemic and didn't let their morale down. key: cord-033137-xezwbs4f authors: fan, jingfang; meng, jun; ludescher, josef; chen, xiaosong; ashkenazy, yosef; kurths, jürgen; havlin, shlomo; schellnhuber, hans joachim title: statistical physics approaches to the complex earth system date: 2020-10-03 journal: phys rep doi: 10.1016/j.physrep.2020.09.005 sha: doc_id: 33137 cord_uid: xezwbs4f global warming, extreme climate events, earthquakes and their accompanying socioeconomic disasters pose significant risks to humanity. yet due to the nonlinear feedbacks, multiple interactions and complex structures of the earth system, the understanding and, in particular, the prediction of such disruptive events represent formidable challenges to both scientific and policy communities. during the past years, the emergence and evolution of earth system science has attracted much attention and produced new concepts and frameworks. especially, novel statistical physics and complex networks-based techniques have been developed and implemented to substantially advance our knowledge of the earth system, including climate extreme events, earthquakes and geological relief features, leading to substantially improved predictive performances. we present here a comprehensive review on the recent scientific progress in the development and application of how combined statistical physics and complex systems science approaches such as critical phenomena, network theory, percolation, tipping points analysis, and entropy can be applied to complex earth systems. notably, these integrating tools and approaches provide new insights and perspectives for understanding the dynamics of the earth systems. the overall aim of this review is to offer readers the knowledge on how statistical physics concepts and theories can be useful in the field of earth system science. the earth behaves as an integrated system comprised of geosphere, atmosphere, hydrosphere, cryosphere as well as biosphere components, with nonlinear interactions and feedback loops between and within them [1] . these components can be also regarded as self-regulating systems in their own right, and further broken down into more specialized subsystems. nevertheless, the growing understanding of the multi-component interactions between physical, chemical, biological and human processes suggests that one should bring different disciplines together and take into account the earth system as a whole. such studies and results initiate the emergence of a new 'science of the earth'-earth system science (ess) [2] . the ess framework has already demonstrated its potential as a powerful tool for exploring the dynamical and structural properties of how the earth operates as a complex system. the ess has emerged in the early to mid-20th century, and has developed rapidly during the last decades. its historical evolution can be briefly outlined into four phases [2] : (i) precursors and beginnings (pre-1970s) . the systemic nature of earth was mainly described and emphasized by some conceptualizations, such as vernadsky's biosphere concept that life has a strong influence on the physical and chemical properties of earth [3] , and lovelock's gaia hypothesis that earth is a synergistic and self-regulating, complex system [4] . these conceptualizations play vital roles in the contemporary understanding of the earth system. in particular, the international geophysical year (igy) 1957-1958 promoted the development of international science and led to the emergence of two contemporary paradigms, modern climatology and plate tectonics [5, 6] . (ii) founding a new science (1980s). in the 1980s, the newly emerging recognition of earth as an integrated entity: the earth system, was called for by a series of workshops and conference reports. in particular, the bretherton diagram developed by the national aeronautics and space administration (nasa) [7] was the first systems-dynamics representation of the earth system to couple the physical climate system and biogeochemical cycle. the brundtland report published by the world commission on environment and development in 1987 [8] developed guiding principles for a sustainable development and recognized the importance of the environmental problems for the earth system. some international organizations were also established in this stage, for example, the world climate research programme (wcrp) aims to determine the predictability of the climate and the effects of human activities on the climate. the international geosphere-biosphere programme (igbp) was launched in 1987 in order to coordinate international research on global-scale and regional-scale interactions between earth's biological, chemical and physical processes and their interactions with human systems. the intergovernmental panel on climate change (ipcc) was created in 1988 to provide policymakers with scientific assessments on climate change, its implications and potential future risks, as well as to put forward adaptation and mitigation options. (iii) going global (1990s-2015) . due to great research efforts of international programmes such as the igbp, and the widespread use of the bretherton diagram, ess then developed rapidly, from the 'new science of the earth' movement to a global one. in particular, the international human dimensions programme (ihdp) on global environmental change was founded with the aim to frame, develop and integrate social science research on global change. (iv) contemporary ess (beyond 2015). in the 21st century, the ess framework was well established, and initiated a new programme, future earth, which was integrated by several international organizations or programmes, such as the igbp and ihdp. future earth's mission is to accelerate transformations to global sustainability through research and innovation. following ref. [1] , we highlight the key organizations, publications, campaigns and events that characterize well the evolution of ess in fig. 1 . among the pioneering publications for the evolution of ess, hans joachim schellnhuber introduced and developed a fundamental concept for ess: the dynamic, co-evolutionary relationship between nature and human factors at the planetary scale [9] . he proposed that the earth system e can be conceptually represented by the following mathematical form: where n = (a, b, c, . . .); h = (a, s). this model suggests that the overall earth system contains two main components: n stands the ecosphere and h is interpreted as the human drivers. n is composed of an alphabet of intricately interacting planetary sub-spheres a (atmosphere), b (biosphere), c (cryosphere), etc. the human factor h is much more subtle: a means the 'physical' sub-component and the 'metaphysical' sub-component s reflects the emergence of a 'global subject'. this work provided the conceptual framework for fully integrating human dynamics into an earth system and built a unified understanding of the earth. there exist numerous tools and approaches that support the evolutionary development of the ess. however, it is worth noting that they can be integrated into three interrelated foci: observations, modeling and computer simulations, assessments and syntheses [2] . the earth observations are usually referring to the instrumental data, for example, that are collected by the meteorological stations or polar orbiting and geostationary satellites. however, such data sets only extend, at best, for about one-to-two centuries into the past. in order to extend our reach beyond thousands or even millions of years ago, climate proxies fill this gap. there are multiple proxy records, including coral records [10] , marine-sediment [11] , stalagmite time series [12] , tree rings [13] as well as the vostok [14] and epica [15] ice core record that help us to better understand the past earth system. mathematical models are currently key methods for the understanding and projecting of climate and earth systems. these models include different variants from conceptual climate models-energy balance models (ebms) [16] to more complex earth models, the general circulation models (gcms) [17] . in addition, the integrated assessment models (iams) were designed to take human dynamics as an integral component into account and aim to understand how human development and societal choices affect each other and the natural world, including climate change [18, 19] . based on the gcms, the earth systems models of intermediate complexity (emics) were developed to investigate the earth's systems on long timescales or at reduced computational cost [20] . these models provide knowledge-integration to explore the dynamic properties and basic mechanisms of the earth system across multiple space and time scales. the assessments and syntheses also played crucial roles in building new scientific knowledge, linking the scientific and political communities, and facilitating new research fields based on the feedback from the political sector. some new concepts and theory have indeed arisen from the evolution of ess, including the emerging concept of sustainability [21] , anthropocene [22] , tipping elements [23] , planetary boundaries framework [24] , such as planetary thresholds and state shifts [25] , etc. taken together, they create powerful ways to better understand and project the future trajectory of the earth system. yet, despite the rapid development of the ess, the nature and statistical properties of extreme climate events and earthquakes remain elusive and debated, and furthermore, the existence of early warning signals of these phenomena is still a major open question. as a consequence, in this review, we will present several novel approaches based on or stemmed from statistical physics, that could enhance our understanding of how the earth system could evolve. in particular, the interdisciplinary perspective on statistical physics and the earth system yields to an improvement of the prediction skill of high-impact disruptive events within the climate and earthquake systems. statistical physics is a branch of physics that draws heavily on the laws of probability and the statistics of many interacting components. it can describe a wide variety of systems with an inherently stochastic nature, aiming to predict and explain measurable properties and behaviours of macroscopic systems. it has been applied to many problems including fields of physics, biology, chemistry, engineering and also social sciences. note that statistical physics does not focus on the dynamic of every individual particle but on the macroscopic behaviour of a large number of particles. the basic theory and ideas of statistical physics are depicted in many textbooks, such as [26] [27] [28] . sethna motivated the relationship between statistical physics and complex systems in his book [29] as follows: "many systems in nature are far too complex to analyze directly. solving for the motion of all the atoms in a block of ice -or the boulders in an earthquake fault, or the nodes on the internetis simply infeasible. despite this, such systems often show simple, striking behavior. we use statistical mechanics to explain the simple behavior of complex systems." a complex system is usually defined as a system composed of many components which interact with each other. regarding the large variety of components and interrelations, the earth system thus can be interpreted as an evolving complex system. the concepts and methods of statistical physics can infiltrate into ess, in particular, (i) critical phenomena used to construct a network. the climate networks approach allows to study the interrelationship between the different locations on the globe and thus represent the global behavior of the climate system, e.g., how energy and matter are transferred from on location to another. these networks have been used successfully to analyze, model, understand, and even predict various climate phenomena [44] [45] [46] [55] [56] [57] [58] [59] [60] [61] [62] [63] [64] [65] [66] [67] [68] [69] [70] . a visualization of a climate network is shown in fig. 3 as an example. we first provide an introduction of definitions, notations and basic quantities used to describe the topology of a network, and then present an overview on how to construct a climate network. in this section we review basic characteristics and metrics used to describe and analyze networks, most of them come from graph theory [72] . graph theory is a large field containing many branches but we present only a small fraction of those results here, focusing on the ones most relevant to the study of the complex earth system. a network, also called a graph in the mathematical literature, is a collection of vertices connected by edges. vertices and edges are also called nodes and links in computer science, sites and bonds in physics, and actors and ties in sociology [40] . throughout this review we will denote the number of vertices by n and the number of edges by m , which is also a common notation in the mathematical and physics literature. most of the networks we will study in this review have at most a single edge between any pair of vertices, i.e., self-edges or self-loops not allowed. a simple representation of a network for many purposes is the adjacency matrix a, with elements a ij such that a ij = 1 if there is an edge between vertices i and j, 0 otherwise. we should notice that the diagonal matrix elements are all zero in the adjacency matrix. in some situations it is useful to represent edges as having a strength, weight, or value to them, usually a real number. such weighted or valued networks can be represented by giving the elements of the adjacency matrix values equal to the weights of the corresponding connections. a directed network or directed graph, also called a digraph for short, is a network in which each edge has a direction, pointing from one vertex to another. such edges are themselves called directed edges. for this case, the adjacency matrix is usually not symmetric, and becomes a ij = 1 if there is an edge from j to i, 0 otherwise. degree the degree of a vertex in a network is the number of edges connected to it. we denote the degree of vertex i by k i . for an undirected network of n vertices the degree can be written in terms of the adjacency matrix (eq. 3) as the number of edges m is equal to the sum of the degrees of all the vertices divided by 2, so the mean degree k of the node in an undirected graph is k = 1 n n i=1 k i = 2m/n . the concept of degree is more complicated in directed networks. in a directed network each vertex or node has two degrees. the in-degree k in i is the number of in-going edges connected to a node and the out-degree k out j is the number of out-going edges. from the adjacency matrix of a directed network eq. 3, they can be written bearing in mind that the number of edges in a directed network is equal to the total number of in-going ends of edges at all vertices, or equivalently to the total number of out-going ends of edges. one of the most fundamental properties of a network that can be measured directly is the degree distribution, or the fraction p (k) of nodes having k connections (degree k). a well-known result for the erdős-rényi [73] network is that its degree distribution follows a poissonian, p (k) = e −z z k /k!, where z = k is the average degree. as shown in fig. 2e , despite the fact that the position of the edges is random, a typical random graph is rather homogeneous, the maximum number of the nodes having the same number of edges. however, direct measurements of the degree distribution for real networks, such as the internet [74] , www [38] , email network [75] , metabolic networks [76] , airline networks [77] , neuronal networks [78] , and many more, show that the poisson law does not apply. but they exhibit an approximate power-law degree distribution p (k) = ck −λ , where c is a normalization factor. the constant λ is known as the exponent of the power law. values in the range 2 ≤ λ ≤ 3 are typical, although values slightly outside this range are possible and are observed occasionally [79] . networks with power-law degree distributions are called scale-free networks. the simplest strategy to determine the scale-free properties is to look at a histogram of the degree distribution on a log-log plot, as we did in fig. 2f , to see if we have a straight line. several models have been proposed for the evolution of scale-free networks, each of which may lead to a different ensemble. the first proposal was the preferential attachment model of barabási and albert, which is known as the barabási-albert model [38] . several variants of this model have been suggested, see, e.g., in ref. [80] . the extent to which nodes cluster together on very short scales in a network is measured by the clustering coefficient. the definition of clustering is related to the number of triangles in the network. the clustering is high if two nodes sharing a neighbor have a high probability of being connected to each other. the most common way of defining the clustering coefficient is: ( number of triangles ) × 3 ( number of connected triples) . here a "connected triple" means three nodes uvw with edges (u, v) and (v, w). the factor of three in the numerator arises because each triangle is counted three times when the connected triples in the network are counted. we can also get a local clustering coefficient c i for a single vertex by defining define c i = the number of triangles connected to vertex i the number of triples centered on vertex i . that is, to calculate c i , we go through all distinct pairs of vertices that are neighbors of i, count the number of such pairs that are connected to each other, and divide by the total number of pairs. c i represents the average probability that a pair of i's friends are friends of one another. for vertices with degree 0 or 1, for which both numerator and denominator are zero, we assume c i = 0. then the clustering coefficient for the whole network [37] is the average c = 1 n i c i . in both cases, the clustering is in the range 0 ≤ c ≤ 1. a graph g 1 consisting of a set p 1 of nodes and a set e 1 of edges is a subgraph of a graph g = {p, e} if all nodes in p 1 are also nodes of p and all edges in e 1 are also edges of e. the simplest examples of subgraphs are cycles, trees, and complete subgraphs [81] . a cycle of order k is a closed loop of k edges such that every two consecutive edges and only those have a common node. that is, graphically a triangle is a cycle of order 3, while a rectangle is a cycle of order 4. the average degree of a cycle is equal to 2, since every node has two edges. a tree, as shown in fig. 4a , is a connected, undirected network that contains no closed loops. here, by "connected" we mean that every node in the network is reachable from every other via some path through the network. a river network is an example of a naturally occurring tree with directed links [82] . the most important property of trees is that, since they have no closed loops, there is exactly one path between any pair of nodes. thus the number of edges in a tree is always exactly k − 1 edges. complete subgraphs of order k, also called k-clique, contain k nodes and all the possible k(k − 1)/2 edges-in other words, they are completely connected [see fig. 4b ]. let us consider the er model, in which we start from n isolated nodes, then connect every pair of nodes with a probability p. most generally, we can ask whether there is a critical probability that marks the appearance of arbitrary subgraphs consisting of k nodes and l edges. a few important special cases that directly give an answer to this question [81] are: (i) the threshold probability of having a tree of order k is p c (n ) = cn −k/(k−1) ; (ii) the threshold probability of having a cycle of order k is p c (n ) = cn −1 ; (iii) the critical probability of having a complete subgraph of order k is p c (n ) = cn −2/(k−1) . generally, a network is always composed of many separate subgraphs or components, i.e., groups of nodes connected internally, but disconnected from others. in each such component there exists a path between any two nodes, but there is no path between nodes in different components. a network component with size proportional to that of the entire network, n , is called a giant component. similar to component, the concept of community is also one of the most fundamental properties in complex networks [83] . the nodes of the network might be joined together into tightly connected groups, whereas between them there are still links but less connections. a single node of the network may belong to more than one community, and most of the actual networks are made of highly overlapping communities of nodes [84] . since community structures are quite common in real networks, community finding and detection are thus of great importance for better understanding the function of a network. an example of overlapping k-clique communities is shown in fig. 4c . a good review on the community detection in graphs can be found in ref. [85] . network models erdős-rényi model : it can be interpreted by the following two well-studied graph ensembles, g n,m the ensemble of all graphs having n vertices and m edges, and g n,p the ensemble consisting of graphs with n vertices, where each possible edge is realized/connected with probability p. these two families, initially studied by erdős and rényi [73] , are known to be similar if m = n 2 p. they are referred to as erdős-rényi (er) model. these descriptions are quite similar to the microcanonical and canonical ensembles studied in statistical physics [86] . the er model has traditionally been the dominant subject of study in the field of random graphs [81] , with poissonian degree distributions, see eq. (7). barabási-albert model : it is based on two simple assumptions regarding network evolution [38] . (i) growth: starting with a small number (m 0 ) of nodes, at every time step, a new node with m ( m 0 ) edges that link the new node to m different nodes already present in the system is added. (ii) preferential attachment: this is the heart of the model. when choosing the nodes to which the new node will be connected, the probability π that the new node will be connected to node i depends on the degree k i of node i, such that π (k i ) = ki j kj . in this process, after t time steps this results in a network with n = t + m 0 nodes and mt edges. theoretical and numerical results show that this network model evolves into a power-law degree distribution, see eq. (8) . watts-strogatz model : in 1998, watts and strogatz [37] proposed a one-parameter model that interpolates between a regular lattice and a random graph. the details behind the model are the following: (i) start with order : start with a ring lattice with n nodes in which every node is connected to its closest k neighbors. in order to have a sparse but connected network at all times, consider n k ln(n ) 1. (ii) randomize: randomly rewire each edge of the lattice with probability p such that self-connections and duplicate edges are excluded. this process introduces pn k/2 long-range edges. the resulting network properties of this model are smallworld and high clustering coefficient. specifically, a small-world network is referring to a network where the characteristic path length l grows proportionally to the logarithm of the number of nodes, l ∝ log n [37]. our climate system is made up of an enormous number of nonlinear sub-systems having mutual nonlinear interactions and feedback loops active on a wide-range of temporal and spatial scales. therefore, modeling the earth climate system from the point of view of complex networks can clearly provide critical insights into the underlying dynamics of the evolving climate system. as discussed above, in cns, geographical regions of earth are regarded as nodes, and the bivariate statistical analysis of similarities between pairs of climatological variables time series represent the links. in general, there are five steps for the cns construction and analysis [46] , the procedure is displayed in fig. 5 , adapted from [87] . step (i): nodes, the nodes in cns are usually defined as locations in a longitude-latitude spatial grid at various resolutions. (ii): climatological variable, we select the suitable climatological time series to be analyzed, e.g., surface air temperature, sea surface temperature, precipitation, wind, etc. some pre-processing is also often needed. for example, to avoid the strong effect of seasonality, we usually subtract the mean seasonal cycle and divide by the seasonal standard deviations for each grid point time series. (iii): edges, in this step we compute the statistical similarity that quantifies the interdependencies between pairs of time series. the strength of each edge is correlation based. there are many measures for quantifying the interdependencies of time series, here we mention the types of cns by correlation measures. for example, a pearson correlation climate network, where we use pearson correlation to quantify the cross-correlations between time series; while an event synchronization climate network uses the event synchronization method; and a mutual information climate network is based on mutual information. (iv): construction, in this step we construct the cn which typically involves some thresholding criterion to select only statistically significant edges. considering, for example, that the significant links have w ij values above a given threshold, w c , then the adjacency matrix is determined as where h is the heaviside function. we then investigate the obtained network by using various network characteristics. finally, in step (v): climatological interpretation, the results of this analysis are interpreted in terms of dynamical processes of the climate system (e.g., atmospheric circulation, ocean currents, plenty of waves, etc.). in the following, we will demonstrate how to construct a pearson correlation climate network. suppose that the climatic variable is the daily surface air temperature, either from observations, proxy reconstructions, reanalyses, or simulations, gathered at static measurement stations, or provided at grid cells. at each node i of the network, we calculate the daily atmospheric temperature anomalies t i (t) (actual temperature value minus the climatological average which is then divided by the climatological standard deviation) for each calendar day. for obtaining the time evolution of the weight of the link between a pair of nodes i and j, we follow [66] and compute, for each time windowing (such as month or year) t over the whole time span, the time-delayed cross-correlation function defined as and where the brackets denote an average over the past t days, according to here, τ is the time lag spanning from [0, τ max ] days. the reliable estimate of the background noise level, i.e., the values of the τ max was discussed in [88, 89] . based on the correlation functions, eqs. (12)and (13), a weighted and directed link between nodes i and j was defined in refs. [59, 61, 64] . this is done by identifying the value of the highest peak (or lowest valley) of the cross-correlation function and denote the corresponding time lag of this peak (valley) as θ i,j . the sign of θ i,j indicates the direction of each link; i.e., when the time lag is positive (θ i,j > 0), the direction of the link is from j to i [64] . the positive and negative links and their weights are determined via c i,j (τ ), and are defined as and where "max" and "min" are the maximum and minimum values of the cross-correlation function, "mean" and "std" are the mean and standard deviation. typical time series and their cross-correlation functions are shown in fig. 6 . for demonstration, two nodes are selected, a node i is located in the southwest atlantic and a node j is in the south american continent (fig. 6a) . the near surface daily air temperature anomalies are shown in fig. 6b for the time period between 2014 to 2018. the cross-correlation function between the time series is shown in fig. 6c , where the absolute value of a maximum cross-correlation function is much larger than the level of noise and the minimum value. since θ i,j = 2 > 0, then the direction of the link is from j to i. according to eq. (15), we calculate its weight to be w + i,j = 5.71. this value represents 5.71 standard deviations above the noise level, i.e., highly significant. finally, networks can be constructed by establishing links between pairs of nodes with weights larger than some significant threshold. the advantage of this method is that it overcomes the problem of strong auto-correlation values in the data [88] . . the direction of this link is from j to i with weight w + i,j = 5.71. the event synchronization method provides an alternative way to construct a network from climate observations: event synchronization cn. it was originally proposed by quian quiroga et al. [90] , where they measured synchronization and inferred time delays between signals in neuroscience. event synchronization is based on the relative timings of events, such as rainfall, in a time series and is defined, e.g., by the crossing of a threshold or by local maxima or minima, etc. for instance, an extreme rainfall event is defined as the day where its precipitation is above the 99th percentiles of all days. event synchronization is especially appropriate for studying extreme events. the degree of synchronization is obtained from the number of quasi-simultaneous events and the delay is calculated from the precedence of events in one time series (signal) with respect to the other. event synchronization starts by constructing two event series from two discrete univariate time series, x and y . an event l that occurs at x at time t x l is considered to be synchronized with an event m that occurs at y at time t y m within a time lag ±τ xy here, l = 1, 2, . . . , e x , and m = 1, 2, . . . , e y . e x and e y are the number of events in the x and y respectively. the number of times an event appears in x shortly after it occurs in y is counted: and analogously to eq. (18) we can define c(x|y). finally, the symmetrical and anti-symmetrical combinations are used to measure the synchronization of the events and their delay behavior, respectively. they are normalized to 0 q xy 1 and −1 q xy 1. it should be noted that if several extreme events are very close in one record, then only the first one is considered. particularly, q xy = 1 if and only if the events of the signals are fully synchronized. in addition, if the events in x always precede those in y , then we get q xy = 1. the event synchronization method has been found useful for the study of electroencephalogram signals [90] , neurophysiological signals [91] , and the patterns of extreme rainfall events [92] [93] [94] [95] [96] . the mutual information method is also one of the common tools for quantifying the interdependencies between time series and for constructing climate networks. the mutual information of two random variables is a measure of the mutual dependence between the two variables. the concept of mutual information is intricately linked to that of entropy of a random variable [97] . let x and y be a pair of random variables with values over the space x × y. if their joint distribution is p(x, y), and their associated marginal probability distribution functions are p(x) and p(y), then the mutual information is defined as states with zero probability of occurrence are ignored. intuitively, m i is a measure of the inherent dependence expressed in the joint distribution of x and y relative to their corresponding joint distribution under the assumption of independence. mutual information, therefore, measures dependence and nonlinearity, in fact: m i = 0 if and only if x and y are independent random variables, i.e., p(x, y) = p(x)p(y). moreover, mutual information is non-negative, symmetric and can also be computed with a time lag [98] . after we get the mutual information coefficient, eq. (21), for each pair of nodes, we can construct a mutual information climate network. it has been shown that the mutual information cn can well capture the westward propagation of sea surface temperature (sst) anomalies that occur in the atlantic multidecadal oscillation [99] . to summarize, in this chapter we have described different network characteristics and presented various linear and nonlinear tools of time series analysis, which can be used to construct, define and characterize cns. linear and nonlinear methods include pearson correlation, event synchronization, and informationtheory measures such as entropy and mutual information. there are also some other powerful tools, such as, spectral analysis, empirical orthogonal function analysis and symbolic ordinal analysis that can be used to reconstruct cns [46] . percolation originally has been used to describe the movement and filtering of fluids through porous materials [100] . the idea of percolation was also considered by flory and stockmayer on polymerization and the sol-gel transition [101] . in recent years, it has been a cornerstone in the theory of spatial stochastic processes with broad applications to diverse problems in fields such as statistical physics [102] [103] [104] [105] , phase transitions [106, 107] , materials [108, 109] , epidemiology [110] [111] [112] [113] , networks [30, 38, 40, [114] [115] [116] [117] [118] [119] [120] [121] , colloids [122, 123] , semiconductors [124] , traffic [125] , turbulence [126] , as well as earth systems [33, 65, 67, [127] [128] [129] [130] . percolation theory also plays a pivotal role in studying the structure, robustness and functions of complex systems [131] . it is well known that in lattices and other ordered networks [106, 107, 132] , for dimensions greater than one, a percolation phase transition occurs. the percolation process is a simple model in which the nodes (sites) or edges (bonds) are occupied with some probability p and unoccupied with probability q = 1 − p. take a regular lattice as an example (see fig. 2a) . a system is regraded as percolating if there is a path from one side to the other parallel one, passing only through occupied bonds and sites. when such a path exists, the component or cluster of sites that spans the lattice is called the spanning cluster or the infinite percolation cluster. at low concentration p, the sites are either isolated or form small clusters of nearestneighbor sites. two sites belong to the same cluster if they are connected by a path of nearest-neighbor sites. at large p values, on the other hand, at least one path between opposite sides exist (see fig. 7 ). the percolation phase transition occurs at some critical density p c that depends on the type and dimensionality of the lattice. for many complex networks, the notion of side does not exist. however, the ideas and tools of percolation theory can still be applied [51] . the main difference is that the condition for percolation is no longer the existence of spanning cluster, but rather having a cluster containing o(n ) nodes. if such a component exists, we call it giant component, that was discussed in section 2.1. indeed, the condition of the existence of a giant component applies also to lattice networks, and therefore it can be used as a more general condition than the spanning property. there exist two types of percolation, site percolation, where all sites are with probability p occupied and 1 − p empty. in bond percolation, however, it is the bonds which are occupied with probability p and above p c they form a giant component of connected sites. in this review, we will focus on the critical phenomena of percolation near the percolation threshold p c , where for the first time a giant component is formed or the system collapses [133] . these aspects are called critical phenomena, and the fundamental theory to describe them is the scaling theory from phase transitions. the concept of phase transition is usually used to describe transitions between solid, liquid, and gaseous states of matter for thermodynamic physical systems, where an ordered phase (e.g., solid) changes into a disordered phase (e.g., liquid) at some critical temperature t c [26] . another classical example of a phase transition is the magnetic phase transition, explained by the ising model, where a spontaneous magnetization m > 0 appears, at low temperature without any external field. while increasing temperature, the spontaneous magnetization decreases and vanishes at t c . percolation is indeed a simple geometrical phase transition, where the percolation threshold p c distinguishes a phase of finite clusters (disordered phase) from a phase of an infinite cluster (ordered phase). the occupied probability p of sites or bonds plays the same role as the temperature in the thermal phase transition. they are usually called control parameters in statistical physics. the percolation order parameter p ∞ in the relative size of the infinite cluster which is defined as the fraction of the sites belonging to the infinite cluster. for p < p c , there exist only finite clusters, thus, p ∞ = 0; for the case p > p c , however, p ∞ is analogous to the magnetization below t c and behaves near p c as a power law it describes the order in the percolation system and is therefore called the order parameter. we show in fig. 8 , how p ∞ behaves as a function of p, for both bond and site percolation in the 2d square lattice model, as well as, for site percolation in the er network, respectively. it is worth noting that we adopt p as the control parameter in fig. 8 , which results p ∞ being a monotonic increasing function. when p is gradually decreased (from 1), it can be regarded as removal (or attack) of nodes or links and the system collapses after removing a fraction of 1 − p c . thus, 1 − p c can be a measure of the resilience of the system, i.e., how close it is to collapse. the finite components distribution near criticality follows the scaling form [106, 107] n s ∼ s −τ e −s/s ξ . where s is the component size, and n s represents the number of components of size s. at the percolation threshold s ξ ∼ |p − p c | −σ diverges and the tail of the distribution behaves as a power law with the critical exponent σ. for any given site, the probability that it belongs to a cluster of size s is sn s . let us define ρ(p) as the probability that any given site is part of a finite cluster, which is the first moment of the cluster size distribution. hence, for any given site of any given finite cluster, the probability w s (p) that the cluster is of size s is with ∞ s=1 w s (p) = 1. for any given site of any given finite cluster, the average size s(p) of the cluster is note that, the average size s(p) , excluding the infinite cluster, diverges near the critical point as, the correlation function g(r) is the probability that a site at position r from an occupied site in a finite cluster belongs to the same cluster. typically, for large r ≡ |r|, there is an exponential cutoff, i.e., g(r) ∼ e −r/ξ , at the correlation length ξ. the correlation length ξ is defined as . (28) ξ measures the mean distance between two sites on the same finite cluster. when p approaches p c , ξ increases as the quantities p ∞ and s are analogous to the magnetization m and the susceptibility χ in magnetic systems. β, γ and ν are called the critical exponents and describe the critical behavior of the percolation phase transition. next, we will briefly introduce some fundamental measurements that are used to characterize the structural properties of a percolation cluster. for more details, the readers can refer to ref. [106] . the fractal concept was introduced into the physical world by mandelbrot [134] and applied to percolation by stanley [135] to describe the cluster shapes at the percolation threshold p c . the infinite cluster is selfsimilar on all length scales, and can be regarded as a fractal. the fractal dimension d f is defined as how, on average, the mass m of the cluster within a sphere of radius r from a site of the cluster changes with r, for length scales smaller than the correlation length ξ, a fractal structure exists. for length scales larger than ξ, the cluster becomes homogeneous. this can be summarized as the relative size of the giant component, p ∞ can also be expressed as the fractal dimension d f of percolation clusters can be related to the critical exponents β and ν. substituting eqs. (22) and (29) into eq. (32) , yields d f = d − β ν . fractal analysis was also applied in the study of complex networks [136] . generally, there are two basic methods to calculate the fractal dimensions of a given system, i.e., using either the box counting method [137] or the cluster growing method [138] . (i) the box counting method: let n b be the number of boxes of linear size l b that are needed to cover the object, the fractal dimension d f is then given by it means that the average number of nodes m b (l b ) within a box of size l b is, the fractal dimension d f can be obtained by a power law fit. (ii) the cluster growing method: similar to eq. (30) , the dimension d f can be calculated by where m c is the average mass of the cluster, defined as the average number of nodes within linear size in the cluster. note that, the above methods are difficult to apply directly to networks, since networks are generally not embedded in space. in order to measure the fractal dimension of networks one usually combines the concept of renormalization [136] , i.e., for each chemical size l b , boxes are placed until the network is covered. then each box is replaced by a node (renormalization), and the renormalized nodes are connected if there is at least one link between the no-renormalized boxes. this procedure is repeated and the number of boxes the fractal dimension d min has been used to describe the structural properties of the shortest path between two arbitrary sites in the same cluster. let be the path length, which is often called the "chemical distance" [139] , it scales with r as ∼ r dmin , and the number of sites within is where d is often called the "graph dimension" or "chemical" dimension. combining eqs. (30) , (35) and (36), we obtain the relation between d min , d and d f , d = d f dmin . besides the fractal dimension exponents d min , d and d f discussed above, there are also some other exponents such as the backbone exponent and the red bonds exponents to describe the fractal dimensions of the substructures composing percolation clusters [26, 107] . the scaling hypotheses of phase transitions was developed by kenneth g. wilson during the last century and honoured by the 1982 nobel prize. the scaling theory of percolation clusters relates the critical exponents of the percolation transition to the cluster size distribution, n s (p). according to the scaling hypotheses, it is possible to state the following relation for n s (p), where τ is the fisher exponent [102] , and f (x) is scaling function following f (x) = exp(−x), which rapidly decays to zero. the correlation length ξ is defined as the root mean square distance between occupied sites on the same finite cluster for all clusters, see eq. (28) . for clusters with s sites, the root mean square distance between all pairs of sites on each cluster, is and thus, close to the percolation threshold p c , r s ∼ s 1/d f , and we obtain from the above equation to calculate the sums in eq. (40), we transform them into integrals, and obtain which yields the scaling relations between ν, σ and τ where d is the system's dimension. consider the k-th moment of the cluster size distribution which scales in the critical region as, next we consider the percolation order parameter p ∞ , which behaves as [106] , combining with eq. (22), we have, similarly, we obtain for k = 2, the relation thus, the critical exponents are not independent of each other but satisfy two sets of scaling and hyperscaling relations. the scaling relations can be easily expressed as by the eqs. (42) , (46) and (47 j o u r n a l p r e -p r o o f an interesting property of percolation is universality, which is a fundamental principle of behavior at or near a phase transition critical point. as a result, the critical exponents depend only on the dimensionality of the system, and are independent of the microscopic interaction details of the system. the behavior of a system is characterized by a set of critical exponents, as discussed in the last section. if two systems have the same values of critical exponents, they belong to the same universality class. the universality property is a general feature of phase transitions. a phase transition is also characterized by scaling functions that govern the finite-size behaviours [140] . the concept of finite-size scaling provides a versatile tool to study the percolation transition. usually, a lattice or network is expected to undergo a continuous percolation phase transition during a random occupation or failure process [81] . explosive, hybrid and genuinely discontinuous percolation for processes that are not random but competitive link-addition processes has attracted much attention in recent years [116, [141] [142] [143] [144] [145] [146] [147] [148] [149] [150] [151] [152] . the description of the explosive percolation model is shown in fig. 9 . instead of performing a finite-size scaling analysis at the critical phase transition point, the finite-size scaling analysis on the percolation properties and critical scaling of the size of the largest gap in the order parameter is considered. for instance, fan et al. [153] developed a new universal gap scaling theory and propose six gap critical exponents to describe the universality of percolation phase transitions. this theoretical framework can be applied for both continuous and discontinuous percolation in various lattice and network models by using finite-size scaling functions. starting with an empty lattice, or network system with n isolated nodes, bonds or links are added randomly or by competitive link-addition processes one by one. the control parameter is denoted as r, which represents the link density r = t /m , with m being the maximal number of edges. the order parameter is the size of the largest cluster, given by the largest connected component in the entire system. during the evolution of the system, we record the size of the largest cluster s(t ) at time step t , and calculate its largest one-step gap ∆ the step with the largest jump defines t c and its relative transition point as r c . moreover, the percolation strength is defined as the size of the largest connected component at t c , i.e., s c = s(t c ). critical phenomena such as percolation exhibit scale-free behaviors that are quantified by scaling relations, see discussions in previous sections. similarity, the averages∆,r c ands c are anticipated to exhibit the following power-law relations [153] , as a function of l 1 , where β 1 , ν 1 and d f 1 are three critical exponents describing the universal class of the percolation, and r c (∞) is the percolation threshold in the thermodynamic infinity limit, l → ∞. their corresponding fluctuations , are also investigated, where d is the number of independent realizations. we expect that χ ∆ , χ rc and χ sc decay algebraically with l with the following scaling relations where β 2 , ν 2 and d f 2 constitute another set of critical exponents. the universality class of the percolation is characterized by the new six gap critical exponents. note that they are highly related to the standard percolation exponents, and not fully independent from each other. the relationships between the gap exponents and the standard percolation critical exponents are derived as [153] , in particular, the values of β 1 can imply the order of the percolation. that is, β 1 = 0 indicates the percolation is a first order transition; whereas models with 0 < β 1 < 1 are continuous [120, 147] . we present the numerical results for bond percolation on a 2d square lattice in fig. 10 , where we obtain β 1 = β 2 ≈ 0.104, 1/ν 1 = 1/ν 2 ≈ 1/ν, and r c (∞) ≈ 0.5, and d f 1 = d f 2 ≈ 1.895. all results are in agreement with the known theoretical values [103] . 1 the size of the system is n = l d . the notion of tipping point: "little things can make a big difference", was first published by malcolm gladwell in his book [154] . it means, at a particular moment in time, a small change can result a large and long-term consequences in a complex system. tipping points are usually associated with bifurcations [155] . a tipping point is defined as "the moment of critical mass, the threshold, the boiling point". many complex systems experience sudden shifts in behavior, often referred to as tipping points or critical transitions, ranging from climate [23, [156] [157] [158] [159] [160] [161] , ecosystems [158, [162] [163] [164] [165] [166] [167] , to social science [168, 169] , financial markets [170, 171] , medicine [172] [173] [174] and event macroeconomic agent-based models [175] . complex systems can shift abruptly from one state to another at tipping points, which may imply growing a threat and risk of abrupt and irreversible changes [161] . it is thus of great practical importance to understand the theoretical mechanisms and predict the tipping phenomena. although predicting such critical points before they occur is notoriously difficult. theory proposes the existence of generic early-warning signals (ews) that may indicate for a wide class of systems if a critical threshold is approaching [176] [177] [178] . ews is currently one of the most powerful tools for predicting critical transitions. the tipping point analysis technique provides a vital tool to anticipate, detect and predict tipping points in complex dynamical systems. the methodology usually combines monitoring memory in time series, includes dynamically derived lag-1 autocorrelation [179] , power-law scaling exponent of detrended fluctuation analysis (dfa) [180, 181] , and power-spectrum-based analysis [182] . defining tipping points: following ref. [177] , a tipping point is the corresponding critical point at which the future state of the system is qualitatively changed. a single control parameter is identified as (ρ), for which there exists a threshold (ρ c ), from which a small perturbation (δρ > 0) leads to a qualitative change (f ) in a crucial feature of the system (f ), after some time (t > 0). the actual change (∆f ) is defined as: in this definition, the critical threshold (ρ c ) is considered as the tipping point, beyond which a qualitative change occurs. note that such changes may occur immediately or even much later after the cause. types of tipping points: the theoretical mechanisms behind tipping phenomena in a complex system can be effectively divided into three distinct categories: bifurcation-induced, noise-induced and ratej o u r n a l p r e -p r o o f dependent tipping [183] . bifurcation, means that a small change in forcing (ρ) past a critical threshold causes a large, nonlinear change in the system state, thus meeting the tipping point definition in eq. (59) . given a conceptualized open system [184] , where ρ(t) is in general a time-varying input. in the case that ρ is constant, we refer eq. (60) as the parameterized system with parameter λ and to its stable solution as the quasi-static attractor. if ρ(t) passes through a bifurcation point of the system where a quasi-static attractor loses stability, it is intuitively clear that a system may 'tip' directly as a result of varying that parameter. we present two bifurcation-induced tipping point examples in fig. 11 . where the system's states are described by and for fig. 11 a and b, respectively. in general, as a system approaches a bifurcation tipping point, where its current state becomes unstable, it leads to a shift to an alternative attractor. noise-induced, noise-induced transitions between existing stable states of a complex system (fig. 11c ), can also be regarded as tipping points [185] , however, they do not meet the definition of forced changes, as in eq. (59) . noise-induced tipping points mean that noisy fluctuations result in the system departing from a neighbourhood of a quasi-static attractor. for example, pikovsky and kurths studied the coherence resonance in a noise-driven excitable fitz hugh-nagumo system and uncovered that the effect of coherence resonance is explained by different noise dependencies of the activation and the excursion times [186] . the abrupt warming events during the last ice age, known as dansgaard-oeschger events, provide a noise-induced tipping real-world example [185] . in addition, sutera [187] studied noise-induced tipping points in a simple global zero-dimensional energy balance model with ice-albedo and greenhouse feedback [188] . their results indicate a characteristic time of 100, 000 years for random transitions between 'warm' and 'cold' climate states, which match very well with the observed data. the noise-induced tipping points approach has also successfully been used for modelling changes in other climate models and phenomena [189] . in contrast to approaching bifurcations, it was found that noise-induced transitions are fundamentally unpredictable and show none of the ews [177] . rate-dependent, in which the system fails to track a continuously changing quasi-static attractor. to better understand the phenomenon of rate-dependent tipping, ashwin et al. introduced a linear model with a tipping radius and discussed three examples where rate-dependent tipping appears [183] . given a system for f ∈ r n and the parameter ρ has a quasi-static equilibriumf (ρ) with a tipping radius r > 0, then for some initial f 0 with f 0 −f (ρ) < r, the evolution of f with time is given by where m is a fixed stable linear operator (i.e. e m t → 0 as t → ∞ ). ρ(t) is a time-varying parameter, that represents the input to the subsystem. the tipping radius may be related to the basin of attraction boundary for the nonlinear problem. this model shows only rate-dependent tipping-because m is fixed and there is no bifurcation in the system and no noise is present. in particular, the model can be generalized to include m and r that vary with ρ(t). eq. (63) can be solved with the initial condition f (0) = f 0 to give note that the rate-dependent tipping was also observed in the zero-dimensional global energy balance model [188] . besides the above three tipping types, there is potentially another type, a reversible tipping point. in fig. 11d , we show an example of a reversible tipping point, in which a mono-stable system exhibits non-linear but reversible change [159] . in ref. [23] lenton et al. introduced the term tipping elements to describe large-scale components of the earth system that may pass a tipping point, and assessed where their tipping points lie. they emphasized that the human activities may play a vital role to push the components of the earth system past their tipping points, resulting in profound impacts on our social and natural systems. they also defined the subset of policy-relevant tipping elements having the following conditions: (i) the first condition is described in eq. (59); (ii) the second one is related to the "political time horizon" t p ; (iii) the third one is called "ethical time horizon" t e ; (iv) the last one is that a qualitative change should correspondingly be defined in terms of impacts. according to these four aforementioned conditions, we review here some policy-relevant potential tipping elements in the climate system, as shown in fig. 12 . for each tipping element, we consider its (1) feature of the system f (see eq. (59)) or direction of change, (2) control parameter(s) ρ, (3) critical value(s) ρ c , (4) transition timescale t as well as (5) key impacts. arctic sea-ice. for both summer and winter arctic sea-ice, the area coverage is declining and the ice has thinned significantly over a large area [190] . in the ipcc projections with ocean-atmosphere gcms, half of them will become ice-free in september during this century [191] , at a polar temperature −9 • c [192] . the decline in the areal extent can be regarded as the feature of the system, denoted by f ; the local air temperature ∆t air and ocean heat transport can be regarded as control parameters; a numerical value of the 27 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 61 62 63 64 65 j o u r n a l p r e -p r o o f critical threshold is still lacking in literature; the transition timescale is about t ∼ 10 years; the declining of the arctic sea-ice can amplify warming and cause ecosystem changes. greenland ice sheet. it has been reported that the greenland ice sheet is melting at an accelerating rate [193] , which could add additional 7 meters to sea level over thousands of years if it passes a particular threshold. the decrease of the ice volume can be regarded as f , a feature of system; the local air temperature ∆t air is a control parameter; the critical value ρ c ∼ 3 • ; the transition timescale t can reach about 300 years. west antarctic ice sheet. according to the ipcc report [193] , the amundsen sea embayment of west antarctica might have already passed a tipping point, i.e., the 'grounding line' (where ice, ocean and bedrock meet) is retreating irreversibly. this could also destabilize the rest of the west antarctic ice sheet [194] . it has been found, using paleoclimatology data, that such widespread collapse of the west antarctic ice sheet occurred repeatedly in the past. similar to the greenland ice sheet, the decrease of the ice volume can be regarded as f ; the local air temperature ∆t air is the control parameter; the critical value ρ c ∼ 5 − 8 • ; the transition timescale t can reach 300 years. the collapse of the west antarctic ice sheet may lead to about 5 meters of sea-level rise on a timescale of centuries to millennia [23] . atlantic circulation. the atlantic meridional overturning circulation (amoc) is one of earth's major ocean circulation systems, redistributing heat and affecting the climate. research has provided evidence for a weakening of the amoc by about 3 ± 1 sverdrups (around 15 per cent) 2 since the mid-twentieth century [160] . the amoc is also considered as one of the main tipping elements of the earth system [23, 156] . the overturning of the atlantic circulation can be regarded as f ; the additional north atlantic freshwater input is the control parameter; the critical value ρ c ∼ 0.1 − 0.5 sv; the transition timescale t can reach 100 years. a slowdown of the amoc is associated with a southward shift of the tropical rainfall belt by influencing the intertropical convergence zone, and a warming of the southern ocean and antarctica [17] . el niño-southern oscillation (enso). enso, the interannual fluctuation between anomalous warm and cold conditions in the tropical pacific, is one of the most influential coupled ocean-atmosphere climate phenomena on earth [195] [196] [197] . it has been reported that extreme el niño events are projected to likely increase in frequency in the 21st century [198] . the tipping point behavior of enso in a warming world was discussed in ref. [199] . the amplitude of enso is regarded as f ; the zonal mean thermocline depth, thermocline sharpness in the east equatorial pacific, and the strength of the annual cycle are the control parameters; the transition timescale t can reach 100 years. a stronger el niño usually causes more extreme events (e.g., floods, droughts, or severe storms), which have serious consequences for economies, societies, agriculture and ecosystems. indian summer monsoon. the indian summer monsoon rainfall (ismr) has a decisive influence on india's agricultural output and economy. the monsoon season (from june to september) can bring drought and food shortages or severe flooding, depending on how much rain falls. the land-to-ocean pressure gradient drives the monsoon circulation, related to the moisture-advection feedback [200] . the ismr shows a declining trend since 1953 [201] . it has been reported that under some plausible decadal-scale scenarios of land use, greenhouse gas and aerosol forcing, the indian summer monsoon switches between two metastable regimes corresponding to the "active" and "weak" monsoon phases [23] . thus, the indian summer monsoon can be also regarded as a tipping element. in particular, the ismr is regarded as f ; the planetary albedo over india is the control parameter; the critical value ρ c = 0.5 sv; and the transition timescale t is 1 years. amazon rainforest. tropical forests play a vital role in the global carbon cycle [202] and are the home of more than half of the known species worldwide [203] . deforestation and climate change are destabilizing the tropical forests with annual deforestation rates of around 0.5% since the 1990s, with a strong increase in recent years [204] . an empirical finding suggests that the observed tropical forest fragmentation is near to the critical point in three continents, including the americas, africa and asia-australia [205] . in particular, we focus on the amazon rainforest, since it is the world's largest rainforest and is home to one in ten known species [161] . if forests are close to tipping points, the amazon dieback could release 90 gigatonnes carbon dioxide. finding the tipping point of the amazon rainforest is thus essential for us to stay within the emissions budget. here the rainforest in the amazon is regarded as f ; the precipitation and dry season length are the control parameters; the critical value ρ c = 1, 100 mm/year; its transition timescale t is about 50 years. besides the seven above mentioned tipping elements, there are also some potential policy-relevant tipping elements in the climate system, such as, sahara/sahel and west african monsoon, boreal forest, antarctic bottom water, tundra, permafrost, marine methane hydrates, ocean anoxia and arctic ozone. the readers who are interested can find them in ref. [23] . strong evidence indicates that "tipping points are under way has mounted in the past decade. domino effects have also been proposed" [161] . as discussed above, many complex dynamical systems, in particular climate systems, can have tipping points and imply risks of unwanted collapse. although predicting such tipping points before they are reached is a big challenge, the existence of generic ews provide useful indicators for anticipating such critical transitions [176, 178] . hence, if an early warning of a tipping point can be identified, then it could help broader society, scientists and policymakers to perform early actions to reduce system collapsed related damages. thus, numerous studies have been dedicated to detecting and predicting these critical transitions, often making use of the ews. in this review, we will highlight the tipping point analysis techniques that are used to anticipate, detect and forecast critical transitions in a dynamical system. critical slowing down near tipping points the "critical slowing down" phenomenon has been suggested as indicators of whether a dynamical system is getting close to a critical threshold [206] . this happens, for instance, at the fold bifurcation ( fig. 11 ), often associated with tipping points. this indicates that the rate at which a system recovers from small perturbations becomes slow, and the slowness can be inferred from rising "memory" in small fluctuations in 29 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 61 62 63 64 65 j o u r n a l p r e -p r o o f the state of a system. for example, fig. 13 demonstrates that the critical slowing down has an indicator that the system has lost resilience and may be tipped more easily into an alternative state (as reflected by lag-1 autocorrelation) [178] . in order to illustrate the relation between the critical slowing down phenomenon, increased autocorrelation and increased variance, here we show a simple example that reveals the underlying mechanism. we consider a simple autoregressive model of first order, wherex is the stable equilibrium of the model, ∆t is the period and λ is the recovery speed. here y n is the deviation of the state variable x from the equilibrium, ε n is a random number obtained from a standard normal distribution and σ is the standard deviation. if λ and ∆t are independent of y n , eq. (65) can be written as: the lag-1 autocorrelation α ≡ e λ∆t is zero for white noise and close to one for red noise. the expectation value of a first-order autoregressive process y n+1 = c + αy n + σε n is let's consider c = 0, then the expectation value equals zero and the variance to be the return speed to equilibrium decreases, when the system is close to the critical point. this implies that λ tends to zero and α approaches to one. according to eq. (68), the variance tends to infinity. in summary, the critical slowing down leads to an increase in lag-1 autocorrelation (α) and in the resulting pattern of fluctuations (variance). slowing down causes the intrinsic rates of change to decrease, and thus the state of the system becomes more like its past state, i.e., the autocorrelation increases. the resulting increase in "memory" can be measured in a variety of ways from the frequency spectrum of the system. autocorrelation function. the lag-1 autocorrelation function (acf(1)) indicator is a simple way to provide an ews for an impending tipping event. for instance, held and kleinen have shown that the autocorrelation increases in the vicinity of a bifurcation in a model of the thermohaline circulation [179] ; dakos et al. found that the autocorrelation increases before eight well-known climate transitions in the past data [157] . the coefficient of the correlation between two values in a time series is called the acf. for example the acf for a time series y n , see eq. (66) in the autoregressive model, is given by: corr(y n , y n−s ), s = 1, 2, ..., where s is the time gap and is called the lag. in particular, a lag-1 autocorrelation is the correlation between values that are one time step apart. more generally, a lag s autocorrelation is the correlation between values that are s time steps apart. the acf scaling exponent, γ, is the power-law decay of the autocorrelation function with increasing lag s [207] . let's denote c(s) as the autocorrelation with lag s of time series, then the scaling is defined as for long-range correlations. notably, for short-range correlated records, c(s) decays exponentially and only acf(1) is indicative of a data variability close to a tipping point. detrended fluctuation analysis. slowing down causes an increase in memory, which can also be measured using detrended fluctuation analysis (dfa) [180] . see [180] for the detailed method. dfa is often used to detect long-range correlations or the persistence of diverse time series including dna sequences [208] , heart 30 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 61 62 63 64 65 j o u r n a l p r e -p r o o f rate [209] [210] [211] , earthquakes [212] , and also climate records [213] . if the time series is long-range correlated, the fluctuation function, f (n), increases according to a power-law relation: where n is the window size and α the dfa scaling exponent. here, is the cumulative sum or the "profile" of a time series x i , and y z t is the fitting polynomial, z stands for the (polynomial) order of the dfa. the dfa exponent α is calculated as the slope of a linear fit to the log-log graph of f (n) vs. n. it has been found that the dfa exponent in the temporal range 10 ≤ n ≤ 100 is sensitive to changes in a dynamical system, which is similar to acf(1) [181] . power spectrum. recently, prettyman et al. introduced a novel scaling indicator based on the decay rate of the power spectrum (ps) [214] . ps analysis partitions the amount of variation in a time series into different frequencies. when a system is close to a critical transition, it tends to show spectral reddening, i.e., higher variation at low frequencies [215] . the ps scaling exponent β is calculated by estimating the slope of the power spectrum s(f ) of the data, from the scaling relationship analytically, the three scaling exponents have the linear relationship: it has been reported that the ps-indicator is a useful technique which behaves similarly to the related acf(1)-and dfa-indicators. in addition, it also shows signs of providing an ews for a real geophysical system, tropical cyclones, whereas the acf(1)-indicator fails [214] . besides the slower recovery from perturbations, increased autocorrelation and memory [see eq. (67)], increased variance [measured as standard deviation, see eq. (68)] is another possible indicator of a critical slowing down as a critical transition is approached. for example, carpenter and brock have shown that the variance increases in the vicinity of a bifurcation in a lake model [216] . flickering before transitions. another notable ews is a system's back and forth oscillation between two stable states in the vicinity of a critical transition. this oscillation has been called flickering and was observed on the model of lake eutrophication [217] and trophic cascades [218] . skewness and kurtosis. in addition to the aforementioned ews before a catastrophic bifurcation, two further precursors, observed when approaching a critical transition and thus suggested as ews, are changes in the skewness and kurtosis of the distribution of states [219] [220] [221] . while skewness indicates asymmetry in the distribution -with a negative skew indicating a right-sided concentration and a positive skew indicating the opposite. skewness is the standardized third moment around the mean of a distribution, similarly, kurtosis is a measure of the "peakedness" of the distribution -with positive kurtosis indicating a peak higher than the one of a normal distribution and negative kurtosis indicating a lower peak. kurtosis is the standardized fourth moment around the mean of a distribution estimated as, in the last section, we first highlighted the theoretical background of tipping points that may occur in non-equilibrium dynamics before critical transitions. nonetheless, it poses much of a challenge to detect ews in real complex systems such as, climate, social and ecological systems. developing reliable predictive systems based on these generic properties can strengthen our capacity to navigate systemic failure and guide us for designing more resilient systems. 31 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 61 62 63 64 65 j o u r n a l p r e -p r o o f we will briefly review emerging precursors of transitions in different real systems, including climate, ecosystems, medicine and finance. an abrupt climate change occurs when the earth system is forced to cross a threshold to a new climate state [222] . large, abrupt, and widespread climate changes include the carboniferous rainforest collapse [223] , younger dryas [224] , greenhouse-icehouse transition [225] , dansgaard-oeschger events, heinrich events and paleocene-eocene thermal maximum [226] . all these rapid climate changes could be explained as critical transitions [176] . for instance, a significant increase in autocorrelation is regarded as a precursor of eight well-known climate transitions [157] . a flickering phenomenon was found to precede the abrupt end of the younger dryas cold period, which can be also regarded as a precursor [227] . in ecology, tipping point analysis has also become a major focus of research. for example, an ews was found in the destabilization of exploited fish stocks, where harvesting leads to increased fluctuations in fish populations [228] . the emerging of ews has been demonstrated in lake and marine systems [229, 230] . in physiology, abrupt transitions were found in epileptic seizures and asthma attacks [174] . for example, flickering may occur before epileptic seizures. in finance, market dynamics may contain information indicating an abrupt change. for example, increased trade volatility may occur before a main shock [231] . for other precursors of transitions in real systems, the reader is referred to ref. [176] . entropy is an important concept arising from statistical mechanics. it is a characteristic that describes the state of a system composed of smaller components, and it has been used to be a general measure of complexity, with widespread applications. in classical thermodynamics, entropy is related to the loss of energy during an irreversible process, developed by rudolf clausius in the early 1850s. the thermodynamic entropy s is derived from the heat flow δq at a fixed temperature t , note that the entropy of a system is defined only if it is in a thermodynamic equilibrium. the statistical mechanics definition of entropy was developed by ludwig boltzmann in 1870s via analyzing the statistical behavior of the microscopic components of a system as here s is the entropy of the macrostate, k b is the boltzmann's constant, and ω standing the total number of possible microstates that yields the macrostate. when viewed in terms of information theory, claude shannon developed the very general concept of information entropy, a fundamental cornerstone of information theory, to describe an analogous loss of information [232] . it is a measure of the amount of information that is missing before reception. the definition of the information entropy is, where k s = 1/ log(2) (in bits), p i is the probability of each state. in quantum statistical mechanics, the concept of entropy has been developed by john von neumann and is generally referred to as "von neumann entropy". for a quantum-mechanical system described by a density matrix ρ, the von neumann entropy is, where tr denotes the trace operator. there exist also many other types of entropy, such as gibbs, residual, approximate, sinai-kolmogorov, sample, multiscale. entropy has been proven useful in many real-world systems, including analysis of dna sequences [233] , cosmology and astrophysics [234] [235] [236] , economics [237, 238] , and climate systems [239] [240] [241] . each definition of entropy could give better results for some systems but fails for others. we will discuss in the following that entropy has three related interpretations [29] as a measure (i) irreversible changes, (ii) disorder and (iii) uncertainty. 32 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 61 62 63 the idea of irreversibility is central to the understanding of entropy. a process that is not reversible is usually called irreversible. this concept arises in thermodynamics. it has been reported that all complex dynamic natural processes are irreversible [242] . for an isolated system with an irreversible process, the entropy never decreases. this is known as the second law of thermodynamics. in order to better understand the behavior of entropy in an irreversible process, we consider the carnot heat engine and the corresponding carnot cycle. as shown in fig. 14a , given by a piston with pressure p (external), and two heat baths, one at a hot temperature t h and the other at a cold temperature t c with some material (a monatomic ideal gas) inside the piston. during one carnot cycle, q h heat flows out of the hot bath, heat q c flows into the cold bath, resulting in a net work w = q h − q c . carnot imagined a fully reversible heat engine, and the carnot cycle moves the piston in and out with the following four steps (see, fig. 14 the gas is connected to the hot bath, and the piston moves outward at a varying pressure and a fixed temperature t h . in this step, heat q h flows into the piston. (2) (ii → iii): the piston expands at varying pressure p . (3) (iii → iv): the expanded gas is then connected to the cold bath and compressed at a fixed temperature at t c . in this step, the heat q c flows out. (4) (iv → i): the piston is compressed and returns to the original state. in this step, there is no heat transfer. the net work w done by the piston is the area inside p-v loop (integration) in fig. 14b , according to the ideal gas law, where n is the number of particles, the total energy of the ideal gas inside the piston is given by the equipartition theorem where e stands for the kinetic energy. during the first step, we get similarly, for the third step, we have 33 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 61 62 63 the above equation substituting eq. (84) into the heat flow, eqs. (81) and (81), we yield carnot's fundamental result, let us define the heat flow ∆e = q at a fixed temperature t . then the entropy change ∆s is note that the above is equivalent to eq. 74. this means that for a reversible carnot engine, the entropy flows from the hot bath q h /t h equals the entropy flows from the piston q c /t c , i.e., no entropy is created or destroyed. however, for any irreversible real engines, entropy will increase. traditionally, another interpretation of entropy is described as a measurement of the disorder or randomness of a system. in thermodynamics, the entropy of mixing is the increase of entropy when two or more different types of particles are mixed without chemical reactions. consider that n molecules of an ideal gas are separated in a vessel with two equal volumes v . the total unmixed entropy s un is [29] s un = 2k b log v n/2 /(n/2)! . here we assume that the two separated ideal gases have the same masses and the same total energy. let the partition between the gases be removed and they are allowed to mix. then the mixed entropy s m becomes according to the eqs. (87) and (88), we obtain the increased entropy, the above equation means that there will be a gain k b log 2 in entropy per molecule during the mixing process. this is since the temperatures and pressures are equal, and removing the partition of the vessel does not involve any heat transfer, thus mixing of gases (e.g., by diffusion), always results in an increasing entropy. the mixing is spontaneous! the diffusion of initially separated gases results in an increase in entropy. the process has increased the random distribution of molecules. therefore, it is appropriate to suggest a relationship between entropy and complexity (disorder). the third interpretation of entropy is as a measure of the uncertainty or ignorance of a complex system. this interpretation is more general and strongly related to the information and memory. in this interpretation, the entropy is not an intrinsic property but representing our knowledge about the system. next, we will focus on the non-equilibrium entropy and the information entropy to insulate how entropy can be interpreted as uncertainty. the second law of thermodynamics states that the entropy of an isolated system never decreases, and is unchanged if and only if all processes are reversible. what we are interested in 34 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 61 62 63 64 65 j o u r n a l p r e -p r o o f is how to define the entropy for non-equilibrium systems. generally, for both non-equilibrium and equilibrium systems, we use a probability distribution ρ, to define an ensemble of states. given a probability distribution of a discrete set of states, the entropy is where p i is the probability of i state. in the case of continuum distributions, the entropy becomes we consider a microcanonical ensemble, in which ρ equil = 1/ (ω(e)δe), the non-equilibrium of the entropy is shifted from the equilibrium (eq. (75)), and the entropy is for quantum systems, the non-equilibrium entropy can be described by the density matrix ρ, as in eq. (77). for non-thermodynamic systems, the temperature variable does not exist. therefore we do not need to use boltzmann's constant k b , but the constant k s = 1/ log(2). then eq. (76) becomes here the entropy is measured in bits. this is called information entropy, which was introduced by shannon [232] . information entropy represents an ensemble of possible messages or images, and has important implications in communication technologies (message passing) and computer science (data compression). note that the non-equilibrium shannon entropy satisfies the following properties: (1) it is maximum for equal probabilities; (2) it is unaffected by extra states of zero probability; and (3) it changes for conditional probabilities. for more details, we refer the reader to ref. [29] . in the following, we will highlight several types of entropy, including approximate entropy (apen), sample entropy (sampen) and system sample entropy (syssampen), which have been developed to quantify the complexity in non-linear time-series. the apen was introduced by pincus in 1991 [243] motivated by the exact regularity statistics, kolmogorov-sinai (ks) entropy [244] and the k 2 entropy [245] . it is used as a quantification of regularity in time-series data, in particular for relatively short and noisy data sets. apen has a wide range of applications in areas from medical data, such as heart rate [243] , to finance system [246] , psychology [247] and human factors engineering [248] . definition of apen: given a time series of data u(1), u(2), . . . , u(n ). there are n raw data equidistant in time. it forms a sequence of vectors here, m is an integer representing the length of the data. define the embedding distance d[x(i), x(j)] [249] between vectors x(i) and x(j) as the maximum difference in their respective scalar components. then use where r is a positive real number which specifies a filtering level. based on the c m i (r), the apen is defined as 35 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 61 62 63 64 65 j o u r n a l p r e -p r o o f for fixed m and r, eq. (95) becomes in particular, given n data points, the statistic formula is implemented one should note that a similar entropy, called eckmann-ruelle (e-r) entropy [244] , has been defined as, despite their algorithms being very similar, apen(m, r) is not intended to be an approximate value of the e − r entropy. compared to the k − s, e − r and k 2 entropy, apen(m, r) has the following advantages [250] : (i) less affected by noise; (ii) it is robust to outliers; (iii) lower computational demand, with good confidence; (iv) apen(m, r) is finite for stochastic, noisy deterministic and composite processes; (v) increasing apen(m, r) corresponds to intuitively increasing process complexity. apen(m, r) has been applied to classify the electroencephalogram (eeg) in psychiatric diseases, such as schizophrenia [251] , epilepsy [252] , and addiction [253] . motivated by the concept of apen, richman and moorman developed the sample entropy (sampen) to assess the complexity of physiological time-series signals, diagnosing diseased states [254] . sampen is a modification of apen, but has three advantages: (i) sampen agrees much better than apen statistics with the theory for random numbers having known probabilistic character over a broad range of operating conditions; (ii) maintains relative consistency and (iii) has a residual bias for very short record lengths. definition of sampen: similar to the definition of apen: given a time series of n points, the maximum difference of their corresponding scalar components. let b i be the number of vectors x m (j) within r of x m (i) and let a i be the number of vectors x m+1 (j) within r of x m+1 (i). here r represents the tolerance for accepting matches. it is convenient to set the tolerance as r × sd, the standard deviation of the data set. then the sample entropy is defined as: it is clear that a will always be smaller or equal to b. therefore, sampen will be always either zero or positive. a smaller value of sampen indicates more self-similarity in the data set or less noise [254] . note that the parameters n , m, and r must be fixed for each calculation. one limitation of the aforementioned entropy is that it can only be applied to univariate or bivariate time series (cross-apen) [254] . for a complex system with multivariate time series and spatial-temporal structures, meng et al. proposed the so called syssampen and applied it to study the climate system. based on the syssampen, they could measure the complexity (disorder) of a system composed of temperature anomaly time series and forecast the magnitude of an el niño event with a prediction horizon of 1 year and high accuracy [241] . definition of syssampen: given n interdependent time series x α (t) (α = 1, 2, ..., n ; t = 1, 2, ..., l) of length l composing the system: 1. we select sub-records k of length m < l, starting at each q-th data point, i.e., starting at t = k × q + 1 = 0 × q + 1, 1 × q + 1, 2 × q + 1, ..., as long as k × q + m ≤ l. thus a specific sub-sequence 36 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 61 62 63 64 65 j o u r n a l p r e -p r o o f then we select n subsequences from each time series and construct a set of n × n template vectors from the system, i.e., where σ α and σ β are the standard deviations of the time series x α (t) and x β (t) respectively. γ defines the similarity criterion and is a nonzero constant. 2. to examine the probability that two time series, which are close at m data points, still will be close at the next p data points, we construct analogously another set θ(m + p, q, n) by selecting sub-records of length m + p. to make the number of template vectors of length m equal to that of length m + p, we choose n ≤ l−m−p q + 1. in order to reduce the parameter degrees of freedom and save calculation time, we take p = q, then n ≤ l−m p . we assume that two template vectors from the set θ(m + p, q, n) 3. the syssampen of the system is defined as where a is the number of close vector pairs from the set θ(m + p, q, n), b is the number of close vector pairs from the set θ(m, q, n), and l ef f (n) = n * p + m, is the number of days that is used in the calculation of the syssampen. 37 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 61 62 63 64 65 j o u r n a l p r e -p r o o f note that, when n = 1, p = 1, and l ef f = l, the syssampen is equivalent to the classical sampen [254] . appropriate parameter values have to be identified since only certain value combinations can be used to estimate a system's complexity with considerable accuracy. in order to achieve this, meng et al. introduced two tests [241] : spatial asynchrony test and temporal disorder test. the syssampen was calculated for the climate system composed of the near surface air or sea surface temperature anomaly time series in the niño 3.4 region. it was found that a strong positive correlation between the el niño magnitudes and the values syssampen of its previous calendar year exists (fig. 15a,b) . fig. 15c demonstrates the calculation of the accuracy level, which allows to choose effective parameter combinations. in addition, to reveal the mathematical meaning of the syssampen, fig. 16 shows the logistic map as an example of applying the syssampen to estimate the system complexity and compare it with the lyapunov exponents. it is found that higher (lower) values of the syssampen are strongly associated with higher (lower) lyapunov exponents, which indicates that the syssampen can well capture the complexity of the system. the earth's climate system is a highly complex and interactive system as defined by the global atmospheric research programme (garp) of the world meteorological organization (wmo) in 1975, as being composed of five major components: the atmosphere, the hydrosphere, the cryosphere, the land surface and the biosphere. and in 1992, the united nations' framework convention on climate change (fccc) defined the climate system as 'the totality of the atmosphere, hydrosphere, biosphere and geosphere and their interactions'. fig. 17 shows a schematic representation of the most important components of the climate system and their potential changes. the atmosphere is the gaseous envelope surrounding the solid planet and provides oxygen to most animal life at the earth's surface. it is the most unstable and rapidly changing 38 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 61 62 63 64 65 j o u r n a l p r e -p r o o f component. currently, the atmosphere is made up primarily of nitrogen (78.1%) and oxygen (20.9%). there are also a number of trace gases that cause of great concern for the future of the planet, including carbon dioxide (co 2 ), methane (ch 4 ) and ozone (o 3 ). they are referred to as greenhouse gases (ghgs) and, along with water vapor, provide the earth with the greenhouse effect. their effect is to keep our earth warm, but not too warm. the hydrosphere is the water on a planet, including oceans, seas, rivers, lakes and underground water. the cryosphere is the portion of the earth's surface where water is in solid form, consisting of land ice (including ice shelves and glaciers), snow and sea ice. it impacts the climate system greatly through its high albedo. variations in the volume of the ice sheets are regarded as one of the major factors for sea level rise. land surface, i.e., the "solid" earth, creates the distribution of continents, ocean basins, mountain ranges, etc. it influences the transformation of short-wave to long-wave radiation, reflectivity of the earth's surface, reservoir of dust, transfer of momentum and energy of the land surface. the biosphere is the total sum of all living things on the planet, including the organic cover of the land masses (vegetation, soil) and marine organisms. it plays a vital role in exchanging of carbon between the different reservoirs, such as the concentration of co 2 in the atmosphere, as well as the balances of other gases [255] . there are numerous interactions between the components, by exchanging mass, heat and momentum. for instance, the ocean-atmosphere interaction is a strongly-coupled system exchanging water vapour and heat through evaporation, among others. each climate system component operates on a rather broad of characteristic temporal and spatial scales. note that the climate system itself is often considered as part of the broader earth system [2] . a fundamental factor in climate science analyses is the climate data, which provides information on changes in the earth's ecosystem and supplies decision makers with a reliable knowledge base on the climate crisis and its impact. in general, there are two data collection methods to gather quantitative and qualitative climate data (also briefly discussed in section 1.1 ): observations (instrumental, proxies and reanalysis) and climate models simulations. observational data of the climate system are based on direct measurements and remote sensing from satellites, radar and other platforms. the first meteorological stations were established in europe and north america. global-scale observations in the instrumental era began in the mid-19th century for temperature and other climatic variables. the increase and quality of the network of observations and the technology supporting the collection and storage of data have rapidly evolved form 1950 onwards. paleoclimate reconstructions extend some proxy records back hundreds to millions of years, including coral records [10, 256] , tree rings records [13] for the last few millennia, as well as stalagmite δ 18 o time series [12] , central-mediterranean sediment [11] and ice-core records [257] . climate studies require data of consistent spatial resolution and accuracy over long time intervals. to satisfy this goal, some numerical weather prediction centers have started to produce so-called reanalyses that are obtained by subtle data assimilation techniques which efficiently combine observational and numerical data. for example, the european centre for medium-range weather forecasts (ecmwf) [258] provides the following reanalysis datasets: era5, era-interim, cera-sat, cera-20c, era-20cm, era-40, era-20c and so on 3 . the ncep-ncar reanalysis i/ii, 20th century reanalysis and north american regional reanalysis (narr) 4 are produced in collaboration by the u.s. national centers for environmental prediction (ncep) and the national center for atmospheric research (ncar) [259] . the jra-25 and jra-55 reanalysis 5 are produced by the japan meteorological agency (jma) [260, 261] . note that most climatic variables or fields in these reanalysis data agree very well with observed data, such as the geopotential fields over the continents of the northern hemisphere. however, substantial differences persist in some fields over the southern hemisphere or those that are poorly observed [262] [263] [264] [265] [266] . more details are summarized in ref. [267] . climate models are considered as the primary tools available for investigating the response of the climate system to various forcings, for making climate predictions on seasonal to decadal time scales and for making projections of future climate over the coming century and beyond. climate models could be defined as a mathematical representation of the climate system based on physical, chemical and biological principles. 3 see https://www.ecmwf.int/en/forecasts/datasets/browse-reanalysis-datasets 4 see https://psl.noaa.gov/data/gridded/reanalysis/ 5 see https://climatedataguide.ucar.edu/climate-data/jra-55/ 39 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 61 62 63 64 65 j o u r n a l p r e -p r o o f the models used in climate research range from simple energy balance models to complex earth system models (esms), which can be slow and costly to use, even on high-performance computers, and the results can only be approximations [17] . here, we provide a brief overview of the basic types of climate models: (i) energy balance models propose a highly simplified version of the dynamic of the climate system. they are zero-or one-dimensional models describing the surface temperature as a function of the energy balance of the earth, where the number of dimensions, from zero to three, refers to the number of independent space variables used in the model domain. for instance, the global mean surface temperaturet s can be expressed as: here r i and r o are the absorbed shortwave radiation and outgoing longwave radiation. c is the heat capacity of earth system, in units of j · m −2 · k −1 , α is the planetary albedo, m is the transmissivity of the atmosphere, a number less than 1 that represents the greenhouse effect of the earth's atmosphere, σ ≈ 5.67 × 10 −8 w · m −2 · k −4 is the stefan-boltzmann constant, µ is an insolation parameter equal to unity for present-day conditions. (ii) atmosphere-ocean general circulation models (aogcms) are the most comprehensive standard climate models that are assessed in the ipcc fourth assessment report (ar). they are used to understand the dynamics of the physical components of the climate system (such as, atmosphere, ocean, land and sea ice), and for making projections based on future ghgs and aerosol forcing. (iii) earth system models (esms) are the current state-of-the-art models, and they expand on aogcms by including representation of various biogeochemical cycles such as those involved in the carbon cycle, the sulphur cycle, or ozone. these models provide the most comprehensive tools available for simulating the past and future response of the climate system to external forcing, in which biogeochemical feedbacks play an important role. (iv) regional climate models rcms are limited-area models with representations of climate processes comparable to those in the atmospheric and land surface components of aogcms, though typically run without interactive ocean and sea ice. rcms are often used to dynamically 'downscale' global model simulations for some particular geographical region to provide more detailed information. in addition, based on the gcms, the intermediate complexity models were developed to investigate the earth systems on long timescales or at a reduced computational cost [20] . a special application is to achieve reliable predictions of extreme events on a sub-seasonal scale. taken together, climate models provide a comprehensive view of the variability and long-term changes in the climate systems. however, assessing and forecasting climate extreme events, such as el niño events and extreme rainfall, still poses challenges. the difficulties are mainly due to the intrinsically rare and disruptive natural conditions. they are strongly influenced by weather patterns, modes of variability, thermodynamic processes and various feedbacks. in the following, we will provide an in-depth and detailed description of how the statistical physics methodologies introduced in section. 2 can be applied to investigate and (or) predict various climate phenomena, such as el niño-southern oscillation (enso), indian summer monsoon (ism), extreme rainfall, atmospheric circulation and atlantic meridional overturning circulation (amoc). one of the most important climate phenomena on year-to-year time scales is the el niño southern oscillation (enso). as mentioned in section 2.3, enso can be regarded as a tipping element of the earth system. el niño is the warm phase of the enso and is associated with a band of warm ocean water that develops in the central and east-central equatorial pacific, sometimes called el niño basin [44] . the cool phase of enso is called la niña, with ssts in the eastern pacific below average. enso significantly impacts earth's ecosystems and human societies, by influencing temperatures and precipitation around the globe, including the americas, india and surrounding tropical continents [268] [269] [270] . these remote effects are known as teleconnections [271] . definition of el niño: in this review, we use the oceanic niño index (oni) to define and distinguish the el niño and la niña events. the oni is one of the primary indices used to monitor the oni. it is 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 61 62 63 fig. 18 . if the oni > +0.5 • c for at least five consecutive months, then the event is defined as an el niño; whereas, when oni is < −0.5 • c for at least five consecutive months, the corresponding event is regarded as a la niña. the oni is use for the operational definition of the enso phase by noaa 6 . there are also some other indices to measure the el niño events, such as, niño 1+2, 3, 3.4, 4 and trans-niño indexes 7 . besides the sst, enso also influences other climate variables like atmosperic pressure gradients, upper ocean currents and the thermocline depth [272] . enso transition mechanism in brief: in the following, we will describe the physical mechanism of enso. bjerknes first postulated that the ocean-atmosphere interaction is essential for enso and is now referred to as the bjerknes feedback [273] . this positive feedback mechanism is understood to be a prominent mechanism necessary for the development of enso. consider an initial sst anomaly in the central/eastern tropical pacific. this anomaly reduces the east-west sst gradient and hence weakens the walker circulation [274] , resulting in a westerly wind anomaly. the westerly wind anomaly, in turn, drives the ocean circulation change (easterly winds shoal the equatorial thermocline and induce upwelling in the eastern pacific, keeping the east cool) that further reinforces the sst anomaly. as a result of the positive feedback, the tropical pacific reaches a warm state, i.e., el niño. tziperman and yu pointed out that the westerly wind bursts, while partially stochastic, seem an inherent part of the large-scale deterministic enso dynamics [275] . besides the positive bjerknes feedback, the oscillatory nature of enso requires a negative feedback to turn the coupled ocean-atmosphere system from a warm (el niño) phase to a cold (la niña) phase. different negative feedbacks have been proposed since the 1980s associated with the delayed oscillator, the western pacific oscillator, the recharge-discharge oscillator, and the advective-reflective oscillator. the delayed oscillator is a mechanism for the oscillatory nature of enso, which was proposed by mccreary based on the rossby wave reflection at the ocean western boundary [276] . suarez and schopf introduced the delayed oscillator explaining the oscillatory feature of enso to emphasize the delayed effects of oceanic wave reflection at the ocean western boundary [277] . cane and zebiak developed the first coupled ocean-atmosphere model, and used it to predict enso [278, 279] . the conceptual delayed oscillator model is represented as follows: where t represents the sst anomaly in the equatorial eastern pacific, a, b, η and ε are constants. the first term on the right hand side of eq. (102) stands for the bjerknes positive feedback between the ocean and the atmosphere. the second term represents the delayed negative feedback of wave reflection at the ocean western boundary. the last term is a cubic damping term. it has been found that the delayed oscillator overlooks the role of the ocean-atmosphere interaction in the western pacific and also assumes that wave reflection at the ocean eastern boundary is not important [280] . since the delayed oscillator does not consider the western-pacific anomaly pattern, weisberg and wang [281] developed a western-pacific oscillator model for enso. this model stresses that the equatorial wind anomalies in the far western pacific play an important role in the evolution of enso. the western-pacific oscillator model is represented as: where t is the sst anomaly in the equatorial eastern pacific, h is the thermocline-depth anomaly in the off-equatorial western pacific, and τ 1 and τ 2 are the equatorial zonal wind-stress anomalies in the central pacific and the western pacific, respectively. all model parameters are constants. it has been found that the growth and decrease in sea level over the western pacific ocean are related to enso [282] . based on these ideas jin [283, 284] developed a recharge-discharge oscillator model for enso that is represented by: where t is the sst anomaly in the equatorial eastern pacific and h is the thermocline-depth anomaly in the equatorial western pacific. the model parameters c, d, ε, e and r h are constants. in particular, eqs. (108) and (109) represent the discharge and recharge of tropical pacific ocean heat content. picaut et al. [285] proposed a conceptual model of the advective-reflective oscillator for enso. they found that the positive feedback results from ocean zonal currents that advect the western-pacific warm pool (wpwp) toward the east. there are three negative feedbacks that tend to push the wpwp back to the western pacific, include: (i) anomalous zonal current associated with wave reflection at the ocean western boundary, (ii) anomalous zonal current associated with wave reflection at the ocean eastern boundary and (iii) mean zonal current converging at the wpwp's eastern edge. unlike the other three oscillator models, the advective-reflective oscillator model does not have a set of simple and heuristic equations. instead, they used a linear wind-forced ocean numerical model forced by wind anomalies, which were associated with the zonal current of the first baroclinic kelvin and first meridional rossby waves [195] . moreover, based on the dynamics and thermodynamics of zebiak and cane's coupled model, as well as all previous enso oscillator models, wang [286] developed a unified enso oscillator ocean-atmosphere model with the hypothesis that enso may be a multi-mechanism phenomenon and their relative importance may depend on time. the unified enso oscillator model is formulated as: where t, h, τ 1 and τ 2 are variables that stand for the sst anomalies in the equatorial eastern pacific, the thermocline-depth anomalies in the off-equatorial western pacific, the zonal wind-stress anomalies in the equatorial central pacific and the zonal wind-stress anomalies in the equatorial western pacific, respectively. for a given set of parameters, eqs. (110)(113) can oscillate on interannual time scales. el niño forecasting: since the 1990s, both dynamical and statistical models have been used to model and forecast el niño events, providing the society the opportunity to prepare for associated hazards such as heavy rains, floods and droughts [287, 288] . although there are about 17 dynamic and 8 statistical models in cpc/iri currently providing enso forecasts routinely, high prediction skill is generally limited to about six months ahead, see the description in fig. 19 . the reason is the presence of the so-called "spring predictability barrier" (spb), where errors are greatly amplified due to the coupled feedbacks in the equatorial ocean-atmosphere system [289, 290] . after the spring, the ability of the models to predict successfully becomes increasingly better. in general, a poor skill in forecasts can be divided into two categories: imperfections in the forecast system (including, error in model parameterization of sub-grid-scale motions, the relative scarcity of points 44 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 61 62 63 with data, errors in the measurement of the data), and fundamental limits of predictability [272] . the fundamental limits are usually coming from the complex and chaotic nature of a system. to overcome the limits of the spb in el niño forecasting, several cn-based approached were developed. for example, by analyzing the cn, ludescher et al. found that a large-scale cooperative mode exists one calendar year before the warming event, linking the el niño region and the rest of the ocean [291] . this mode is probably due to the emergence of el niño acting as an autonomous component in the cn [61] . all nodes used in the cn are shown in fig. 20a , where the 14 grid points in the el niño basin [61] are in red and outside this domain there are 193 grid point in blue. the data is the daily surface air temperature (sat) from ncep/ncar reanalysis i. as described in section 2, the first step is to remove the seasonal trend by minus climatological average for each calendar day. and then, the links between the el niño basin and outside (see fig. 20a ) are considered. the strength w i,j (t) of each link is computed according to eq. (15). the mean strength s(t) of the dynamical links in the cn is obtained by simply averaging over all individual link strengths. fig. 20b , shows the time evolution of s(t) for the learning (top) and forecasting (bottom) phases. they found that before an el niño event, s(t) tends to increase. they marked an alarm when s(t) crosses a threshold θ = 2.82 (see fig. 20b ). the prediction accuracy, through receiver operating characteristic (roc)-type analysis (explained later), is much better than the 12-months forecasts based on climate models. based on these findings regarding the temporal evolution of the cn, ludescher et al. successfully predicted the onset of the 2014-2016 el niño event [292] . motivated by this work, meng et al. developed a multidisciplinary approach combining climate, network, and percolation theory to predict the onset of el niño [65] . their method can forecast el niño events 1 year-ahead, with a high prediction accuracy of 70%, and a low false alarm of only 4%. different from [291] , they consider the global cn with initially 726 isolated nodes. links are added one by one according to the link strength, i.e., first adding the link with the highest weight, and continue selecting links ordered by decreasing weight. during the evolution of the cn, they found that the structure of the network changes violently approximately one year ahead of el niño events, and thus suggest to use the largest size change of the largest cluster (percolation cluster) ∆ [see eq. (48)] as a percolation-based precursor, to forecast el niño events. in addition, based on finite size scaling analysis, they found that the percolation process is discontinuous. to forecast both the onset and magnitude of el niño events, another sophisticated network-based ap-proach was developed by meng et al. [66] . they proposed a new forecasting index based on cn links representing the similarity of low frequency temporal temperature anomaly variations between different sites in the niño 3.4 region. they found that significant upward trends in the index forecast the onset of el niño approximately 1 year ahead, and the highest peak since the end of last el niño forecasts the magnitude of the following event. their index was also tested on several datasets, including era-interim, ncep reanalysis i, pcmdi-amip 1.1.3 and ersst.v5. they defined the degree of coherence/disorder of the niño 3.4 region as, where n is the number of nodes in the niño 3.4 region and c (t) i,j (θ) stands for the max value of the timedelayed cross-correlation function, see eqs. (12) and (13) . the forecasting index (fi) was proposed as a function of time (months), where a = 0 indicates that the average of ln(c) includes the current month, while m is the total number of months preceding t since jan. 1981. we show the time evolution of the fi and oni in fig. 21 by using the era-interim dataset. based on the temporal evolution of the fi, they successfully predicted the onset of the 2018-2019 el niño event, see the purple star above the purple arrow in fig. 21b . note that one can use the peak of the fi to predict the magnitude of the following el niño event. this peak value is determined from the end of the last event to the start of a new event, which means that we should wait until the new event begins to occur. to further improve the forecasting skill, in particular, the magnitude forecasting with a long lead time. meng et al. [241] developed the syssampen method, see more details in sec. (2.4.5) . it is defined in eq. (100) and shown in fig. 15a . the correlation between the magnitude of the el niño events and the syssampen can reach to r = 0.99 [see fig. 15b ]. they further used this correlation to forecast the magnitude of an el niño with a prediction horizon of 1 year and high accuracy (i.e., root mean square error = 0.23 • c for the average of the individual datasets forecasts). in particular, for the 2018-2019 el niño event, they forecasted a weak el niño with a magnitude of 1.11±0.23 • c, compared to the observed value 0.9 • c. recently, feng et al. combined machine learning techniques and climate networks to apply to el niño predictions, especially for the occurrence of el niño events and the development of the nino 3.4 index over time [293] . in ref. [294] , petersik and dijkstra applied gaussian density neural network and quantile regression neural network ensembles to predict enso. classical machine learning models for prediction usually only predict a point value, e.g., the oni. in contrast, their models forecast directly a probability distribution for the oni. in particular, they found that their machine learning models have a high-correlation skill (r = 0.5) for long lead times (12 months ahead) for an evaluation period between 1963 and 2017. in ref. [295] , ham, kim and luo used transfer learning to train a convolutional neural network on historical simulations and reanalysis, and developed the heat map analyses. they found that a statistical forecast model employing a deep-learning approach produces skillful enso forecasts for lead times of up to one and a half years. a review of enso prediction based on modern machine learning and artificial neural networks was reported in ref. [296] . enso remote impacts: teleconnections as introduced above, the enso phenomenon mainly lies in the pacific ocean. however, its effects are felt over a large part of the earth. these remote effects are usually called teleconnections, emphasizing that changing climatic conditions in one place can affect areas far from the source [271, 297] . a teleconnection is usually indicated by the correlation between the values observed at two separate locations or regions. it is related to a pattern of variability, associated with atmospheric wave propagation, the presence of ocean currents, etc. 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 61 62 63 indonesia and northern australia, much drier and warmer conditions in mozambique, while the western usa tends to be wetter. fig. 22 shows weather patterns statistically associated with el niño conditions during the boreal winter months december-january-february. the regions that have been shown with some degree of reliability to be affected by warm enso phases are highlighted, for example, warm-vs.-cold and wet-vs.-dry anomalies. the effects for cold phases of enso, la niña, are in approximately the same regions, but with the opposite sign. the basic mechanism of enso remote influences are produced in the atmosphere via large-scale atmospheric waves, such as rossby waves. rossby waves are fundamental for the understanding of weather systems in the atmosphere and the large-scale circulation in the ocean. they depend fundamentally upon the variation of the coriolis parameter with latitude. in the so called β-plain approximation, the coriolis parameter varies linearly with latitude where where φ is the latitude, ω is the angular speed of the earth's rotation, and r is the mean radius of the earth. the wavelength of atmospheric rossby wave is about 6000 km. the atmospheric wave dynamics are akin to those observed in the ocean for enso in that they involve a transfer of atmospheric mass by anomalous winds, which in turn affect pressure and winds. in order to assess if a region has an enso connection, fan et al. applied the pearson correlation cns approach to investigate the global remote impacts of el niño and la niña [64] . they refer to the niño 3.4 region as the el niño basin (enb). the cns were constructed by using only directed links from the enb to regions outside the enb ("in"-links) based on the global daily sat fields of the ncep/ncar reanalysis and ear-interim datasets. the links' strengths are computed according to eqs. (12) , (13) and (15) . they 48 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 61 62 63 64 65 j o u r n a l p r e -p r o o f identified the value of the highest peak of the absolute value of the cross-correlation function c y i,j (τ ) and denote the corresponding time lag as θ y i,j . the adjacency matrix of a cn became where h(x) is the heaviside step function for which h(x ≥ 0) = 1 and h(x < 0) = 0. the "in" and "out" degrees of each node are defined as i y i = j a y j,i , o y i = j a y i,j respectively, quantifying the number of links into a node or out from a node. the total "in" weights for each node outside the enb using are defined as the values of in(c y i ) and in(w y i ) reflect the impacts of the enb. specifically, if there are no "in" links for a node, both the "in" degree and "in" weights are zero, indicating no impact of enb. based on the oni, the time is divided into el niño , la niña and normal years. the "in"-weighted degree fields for el niño and la niña are calculated by taking the average of the same type of events where s = y∈ey(ly) i y i , and "ey" and "ly" refer to the years in which el niño and la niña start. the number of nodes with zero in-degree is defined as n y and the average in-weights per node is fig. 23 (a-d) shows that, the affected regions by el niño and la niña, are characterized by relatively high in-weights (using c and w ). for comparison, mean winter (dec-feb) temperature anomalies during el niño and la niña years are shown in fig. 23 (e-f). a quantitative analysis of the area that is affected/unaffected during el niño and la niña years is shown in fig. 24 , where el niño and la niña years are respectively emphasized by the red and blue shading. the network analysis reveals strongly (high c y ) localized (low n y ) impacts of enso, i.e., these stronger in-weighted activities are found to be concentrated in very localized areas, whereas a large fraction of the globe is not influenced by the events (low n y means that fewer nodes are influenced). it has been reported that there are different types of el niño events, such as the canonical eastern pacific (ep) and the modoki central pacific (cp) types [298, 299] . they are classified based on spatial patterns of the sst anomaly. for instance, the strongest sst anomalies associated with the ep types are located off the coast of south america. however, for cp types, the strongest sst anomalies are located near the international date line. in ref. [300] , wiedermann et al. proposed an index based on evolving cns to objectively discriminate different types of el niño and la niña episodes. in particular, they find that their network index displays a sharp peak during ep events, whereas during cp events it remains close to the observed values during normal years. based on the cns analysis, lu et al. developed a new approach to estimate the impacts of el niño events in advance, as well as predicted the types of el niño events [301] . the indian summer monsoon (ism) is the most prominent among the world's monsoon systems and has a decisive influence on india's agricultural output and economy. the ism delivers more than 70% of the country's annual rainfall. the prediction of the ism timing (onset date and withdrawal date) as well as the ism rainfall is a vital issue for the indian subcontinent and strongly affects the gross domestic product of the 49 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 61 62 63 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 61 62 63 64 65 j o u r n a l p r e -p r o o f country, up to 22% of which is determined by agriculture [302] . a slight deviation of the timing manifested as delay (or early arrival) may lead to drastic droughts (floods). also, swings in the amount of rainfall, even with variations of only 10%, can cause severe flooding or drought, causing damages to infrastructure and loss of crops and livelihoods of the population [303] . the onset of the ism takes place abruptly and the long lead-time operational forecasts for the onset, withdrawal and rainfall amount have shown little skill in the recent decades. ism onset and withdrawal forecasting several statistical climate models have been developed for ism onset and withdrawal dates forecasting with different timescales from short-range (up to 4 days), medium-range (4-10 days) [304, 305] to extended-range (10-30 days) and long-range (more than 30 days) forecasts [306] . in particular, the indian meteorological department (imd) provides a forecast of the monsoon onset 21 days in advance, with an accuracy of ± 4 days. note that most of the ism forecasting models are based on the input values, including sst, mean sea level pressure, tropospheric moisture, moist static energy, outgoing longwave radiation, wind fields, etc [307] [308] [309] [310] [311] . however, there are still impending challenges in the existing forecasting methods: (i) the false monsoon onsets mostly related to non-monsoonal atmospheric circulation systems [312] ; (ii) the limitations in predicting a withdrawal data earlier than 1st september. to overcome these challenges, stolbova et al. proposed a novel tipping elements approach to accurately forecasts the ism onset and withdrawal dates [95] . this approach relies on the combination of two methodologies: the climate network analysis and the nonlinear dynamics. first, the analysis of the network of extreme rainfall events allows to identify and reveal the teleconnection between the most active hubs, the eastern ghats (eg) and north pakistan (np). second, the application of the nonlinear dynamics through the analysis of fluctuations allows to detect the critical transitions, which are used to define the tipping elements of the ism. later, the eg and np were chosen as optimal observation locations or reference points (rps) to predict the ism monsoon onset and withdrawal. it was discovered that there is a tipping point between these rps for the sat and relative humidity, which yields a very successful prediction scheme. the ism cn is focused on the monsoon region (62.5-97.5 • e, 5.0-40.0 • n) with a spatial resolution of 2.5 degrees, which results in 15 × 15 = 225 grid points, see fig. 25 . the near sat (t ) at 1000 hpa, relative humidity (rh) at 1000 hpa, and wind fields at 700 hpa were used. the variance σ 2 of (t ) and (rh) for each grid point are firstly calculated as [95] where x(t) stands for a time series, w is the length of the time window, d is the number of days before the onset of the ism, y is a given year, and t * (y) is the onset date for the given year y. the time average value of σ 2 is simply expressed asσ where y stands for the total number of years. the results are shown in fig. 25 . it has been confirmed that the eg and np regions experience the highest growth ofσ 2 for both t and rh, when approaching the monsoon onset date. it has been further discovered that there exist two intersections in the rps for the average time series during the period from 1951 to 2014. at both intersections, it coincides with the mean values of the onset and withdraw as determined by the imd with a standard deviation of ±5 days for the eg region, see fig. 26 . thus, this allows to derive a prediction scheme for forecasting the onset and withdrawal of the ism in the eg, based on the trends of t and rh in the rps. next, a linear trend estimation for the two rps is performed and compared with the trends of the mean time series of the training period (1951) (1952) (1953) (1954) (1955) (1956) (1957) (1958) (1959) (1960) (1961) (1962) (1963) (1964) (1965) . the slopes of the trends for the rps provided an estimation of an early, normal, or late monsoon arrival: a greater than average slope of t will lead to an earlier than 52 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 61 62 63 64 65 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 61 62 63 usual onset, and vice versa. trends of rh in the rps in comparison with the average trends for the training period add up to the predictability of the onset: higher than average values of the relative humidity lead to a late onset, and vice versa. it has also been found that the ism onset coincides with the date when t in the eg and in np become equal (see fig. 26a ). therefore, for the forecasting of the onset, one should predict when t for the eg will abruptly decrease and intersect t for np. finally, the prediction scheme of the withdrawal is based on the symmetry of t changes in np during the year. the withdrawal is estimated as the intersection of the projected t decrease in np and the t in the eg during the monsoon season (see fig. 26c ). the prediction is regarded as successful if the time difference between the predicted onset and the real one is ≤ 7 days for the onset and ≤ 10 days for the withdrawal [95] . the proposed scheme using t results in 74% successful predictions of the onset when made on day 125 of the year (april 10 ). the prediction scheme for the withdrawal succeeds in 84% of the years when made on day 205 (july 25). based on this prediction scheme, they successfully predicted both the onset and withdrawal since 2016 in the eg region 8 . the ism is one of the most prominent monsoon systems. it affects the indian subcontinent, where it is one of the oldest and most anticipated weather phenomena. several theories have been proposed to explain 8 for more detailed and updated information have a look at pik's information page on the indian summer monsoon forecast: https://www.pik-potsdam.de/services/infodesk/forecasting-indian-monsoon. 54 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 61 62 63 64 65 j o u r n a l p r e -p r o o f the underlying mechanism of the monsoon. for example, the sea breeze theory: because of the differences in the specific heat capacity of land and water, continents heat up faster than seas. consequently, the air above coastal lands heats up faster than the air above seas. these create areas of low air pressure above coastal lands compared with pressure over the seas, resulting moisture-laden winds to flow from the seas onto the lands. on reaching land, these winds rise because of the geographical relief, cooling adiabatically and leading to orographic rains. however, the understanding and predictability of ism are still evolving. the all india rainfall index (airi) is the total amount of ism june-to-september rainfall averaged over the entire indian subcontinent. it is the imd's primary indicator for monitoring the ism rainfall [313] . an accurate long-term prediction of the ismr is crucial for taking timely actions and mitigation activities. over the last few decades, great efforts have been undertaken to understand the basic physics of the monsoon, and to improve the prediction skill for the airi [314] [315] [316] [317] [318] [319] [320] . however, both methods have the challenge of unstable relationships with the predictors over time, as observed during the recent years due to a weakened coupling between the boundary forcing and the indian monsoon [321, 322] . in particular, the forecasting skill (cross correlation) of the official operational forecasts made by imd statistical models is -0.12 for 1989-2012, for the five ensemble models' multi-model ensemble is 0.09 and the four apcc/clipas models' mme skill is 0.24 after 1989. it was discovered that the recent failure is largely due to the models' inability to capture new predictability sources emerging during the recent global warming [303] . traditionally, the april-may sst anomaly in the niño 3 or niño 3.4 region was used as a predictor for the ismr prediction. this is since the ismr variations are primarily driven by the ep-enso through tropical teleconnections [314, 315] . this inverse relationship between the airi and ep-enso, however, has weakened in recent decades [270] . it has been found that the cp-enso has also distinct teleconnections and affects on the ismr [323] . based on this physical mechanism, wang et al. in order to achieve the goal of a long-term and reliable prediction of the ism rainfall, fan et al. constructed a series of dynamical and physical cns based on the global near sat field [324] . it was uncovered that some characteristics of the directed and weighted cns can serve as efficient long-term predictors for ism rainfall forecasting. the developed prediction method produces a forecast skill of 0.5 with a 5-month lead-time in advance by using the previous calendar year's data. the skill of the forecasting, is comparable to the current state-of-the-art models, however, with quite a short (i.e., within one month) lead-time. the new network-based prediction approach is described as follows. cn construction: based on the ncep/ncar reanalysis daily sat anomalies data, a cn is constructed for each calendar year from 1948 to the present. in the work, 726 nodes are selected, such that the globe is covered approximately homogeneously [61] . the link strength and direction are determined based on a similarity measure between the temperature anomaly time series of the nodes [see eqs. (12)(16) ]. a pair of nodes is defined to be connected only if their link is within the top 5% positive strength, which is also corresponding to a statistical significance of above the 95% confidence level. network predictors mining: to mine the dynamic network predictors for the airi, the cross correlation between the observed airi and the time series of network in-degree k y i [see eq. (6)] for each node i on the globe is calculated. because the forecasting skill of the airi became very poor in the imd operational forecasts and other models since the year 1989 [303] , the record is divided into two consecutive periods, i.e., (i) 1950-1988, as the training period and (ii) 1989-2018, as the retrospective forecast period. for the training period, the correlation coefficients are shown in fig. 27a . it was found that the in-degree index for 55 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 61 62 63 64 65 j o u r n a l p r e -p r o o f some nodes captures well the behavior of the airi. particularly, the node (in the region marked by a yellow box in fig. 27a ) having the maximal correlation r is located in the south atlantic ocean [(30 • s, 30 • w)]. fig. 27b shows that the value of this maximal correlation is r = 0.49, with a student t-test significance of p < 0.01. therefore, the in-degree index of this key node is regarded as the optimized network predictor for the airi. airi forecasting: next, the forecasting capability of the network predictor during the period 1989-2018 is tested. to obtain the forecasted value of the airi for one specific year, the forecasted year's predictor value (i.e., the previous year's network in-degree of the key node) is substituted into the least square linear regression equation. this equation used for prediction is derived using only the past information. the independent forecast skill for the period 1989-2018 becomes then ∼ 0.50, which is significantly higher than the imd operational forecast skill [see fig. 27c ]. the result indicates the good accuracy of the network-based predictor with the long lead-time of 5 months. finally, based on 2018's data, the airi for 2019 is forecasted as 949.87 mm, a 7% departure percentage compared to 9 the long-term average, which is 887.5 mm. the actual value of the airi for 2019 was 968.3 mm. in the following, a potential physical mechanism is discussed. from a meteorological perspective, the teleconnection between the key node's location in the mid-latitude of the south west atlantic ocean and the indian subcontinent is no coincidence. the orography of the eastern coast of the south american continent is prone to the formation of an anticyclone at mid-latitude due to the interaction with mid-latitude westerlies (winds that blow from the west at the surface level between 30 • and 60 • s). moreover, there is the influence of the south polar jet stream, which appears within the upper air westerlies. the acceleration/deceleration of the south polar jet stream induces areas of low/high pressure, respectively, which links to the formation of cyclones and anticyclones. because the south polar jet stream strengthens and weakens seasonally, and from year to year, it synchronizes with the variability in the strength of the circulation centered around the location of the key node. one of the possible data-based teleconnection paths is shown in fig. 27d . hence, the key node location is sort of the nexus of the south atlantic circulation, the south polar jet stream and the main atmospheric circulations patterns over the indian ocean. it explains why we observe a significant correlation between the network characteristics of the key node and the airi. further analyses are performed and reveal that the physical mechanisms are related to (i) the network delayed enso teleconnection, i.e., the in-degree index is locally minimized in the previous calendar year of the el niño onset. (ii) the enso-monsoon teleconnection [270, 325, 326] , i.e., the airi anomaly and the niño 3.4 sst anomaly index are negatively correlated. the spatial correlation maps between the niño 3.4 sst anomaly index with both the sst as well as the rainfall index for all nodes are presented in fig. 28 . these results indicate that the niño 3.4 sst anomaly index is significantly anti-correlated with the sst in the equatorial indian ocean and also heavily influences the rainfall pattern over the indian land region. moreover, the network-based approach can be also successfully applied to forecast the indian homogeneous region rainfall as a prediction scheme, including two specific homogeneous regions [324] : (1) the central india rainfall index; (2) the east & northeast india rainfall index. in addition, there is solid evidence which reveals that climate change affects the forecast skills. the considerable warming trend in the last 70 years -the 850 hpa temperature in the key node area has risen 2.5 • c -substantially impacted the key node region. an increase of the in-degree index and a change in the structure of the network itself are observed. concurrently with these changes, the prediction skill of this forecasting method for the ism rainfall amount is improving substantially [324] . extreme precipitation events have produced more rain and have become more common since the 1950s in many regions of the world. heavy precipitation may pose threats to our society. the most immediate impact is the threat of flooding. this risk can be heightened in urban and agricultural areas. in addition to flooding, extreme precipitation also increases the risk of landslides. excessive precipitation can also degrade water quality, harming human health and ecosystems. thus is is crucial to predict extreme rainfall events 9 the observed airi data can be found in https://mausam.imd.gov.in/imd_latest/contents/rainfall_time_series.php. 56 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 61 62 63 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 61 62 63 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 61 62 63 and to establish an early warning system for them. however, the analysis of spatial patterns of co-variability of such events is challenging because traditional techniques based on principal component analysis of the covariance matrix only capture the first two statistical moments of the data distribution and are not suitable to analyze the behavior in the tails of the respective distributions [327] . to overcome these challenges, malik et al. applied the cns approach to analyze the spatial and temporal patterns of extreme rainfall during the ism [92, 328] . this approach is based on the event synchronization method (see section. 2.1.3). in ref. [328] , the dataset is a daily precipitation gridded set for 1961-2004 developed as part of the project -asian precipitation highly resolved observational data integration towards the evaluation of water resources 10 . then they extracted the data for the south asian region at a 0.5 degree resolution. for calculating the event synchronization, two different thresholds of α = 94th percentile and α = 90th percentile were chosen to obtain the extreme event time series, referring to very heavy and heavy rainfall events, respectively. next, they computed the strength of synchronization q ij and the delay behaviour q ij between each pair of the grid sites i and j [see eq. (20) ]. after normalizing it holds, that 0 ≤ q ij ≤ 1 and −1 ≤ q ij ≤ 1, where q ij = 1 means complete synchronization, and q ij = 0 stands for the absence of synchronization; and q ij = 1 means that events at i precede events in j. the cn is then constructed by selecting only pairs of sites that show strong correlations, and the time lags between the events are used to define the direction of the links. the spatial structures, organization, and scales of the extreme rainfall over india during the ism period were investigated. fig. 29 exhibits that the degree centrality [see eq. (4)] of the cns reveals well-defined spatial structures in the monsoonal extreme precipitation [92] . based on the median of the geographical length of the links, calculated using the formula for the spherical earth projected onto a plane, it has been uncovered that extreme rainfall events are synchronized up to 250 km for most of the region. they further found that such a spatial organization opens the possibility for predictions of a probable spatial extent of monsoonal rainfall activity without delay, e.g., for most of the subcontinent, the accuracy of the prediction is above 70% and even reaches up to 100% in certain places [92] . this event synchronization cn approach was also applied to predict extreme rainfall events in the central andes of south america [94] . based on the real-time satellite-derived rainfall data, trmm 3b42v7 [329] , boers et al. were able to predict more than 60% of the rainfall events above the 99th percentile in the 10 it is freely downloadable from: https://www.chikyu.ac.jp/precip/. 59 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 61 62 63 64 65 j o u r n a l p r e -p r o o f central andes, and even 90% during el niño conditions. the interplay of northward migrating frontal systems and a low-level wind channel from the western amazon to the subtropics are considered as the responsible mechanism. extreme rainfall events are defined as times with threshold α = 99% for all dec-jan-feb seasons in the spatial domain (85 • w, 30 • w) and (40 • s, 15 • n) , at a resolution of 0.25 • , and 3-hourly temporal resolution for the time period from 1998 to 2012. after the construction of the event synchronization cn (see section 2.1.3), the network divergence ∆s is defined as where s in and s out stand the in-strength and out-strength respectively at each node. positive values of ∆s indicate sinks of the network; negative values indicate sources. the strength out of and into a region r is where |r| denotes the number of grid points contained in r. fig. 30a shows the network divergence ∆s (defined in eq. (123)), which estimates the dynamics and temporal order of extreme rainfall in south america. the boxes labelled 1 to 7 are used for the tracking of extreme events. it was found that the most pronounced source region of the rainfall network is south eastern south america (sesa), defined as the box ranging from (60 • w, 53 • w) to (35 • s, 30 • s). fig. 30b depicts where synchronized extreme events occur within 2 days after extreme events occurred in sesa, measured by s out i (sesa). for comparison, s out i (sesa) is shown in fig. 30c . this analysis reveals that extreme events in sesa are followed by extreme events along a narrow band following the eastern andean slopes up to western bolivia, while they are only preceded by extreme events to the southwest. based on the propagation of extreme rainfall from sesa to the eastern central andes (eca), see fig. 30d , a prediction for the extreme rainfall for the eca within 2 days is made after they occurred in sesa. it should be noted that these extreme rainfall events could not be forecasted by using other methods. extreme rainfall over the globe the event synchronization cn method was also applied to investigate the global pattern of extremerainfall by boers et al. [96] . the gauge-calibrated, satellite-derived rainfall dataset trmm 3b42 v731, with daily temporal resolution, from 50 • n to 50 • s for the period 1998-2016 is used. the extreme rainfall event thresholds are 80th, 81th, . . . , 99th of the wet days (>1 mm) for the jun-jul-aug season. note that consecutive days with rainfall above the threshold are considered as single events and placed on the first day of the occurrence. to study the robustness of the method, global precipitation climatology project32 and ncep/ncar reanalysis 1 data were also tested. first, the synchronization of extreme events for each pair of nodes is computed (see section 2.1.3). network links are added between two nodes if the corresponding synchronization values are significant, p < 0.005. the global distribution of the spatial distances for which significant synchronizations occur was studied. this distribution decays as a power law p(d) ∼ d −α , with an exponent α very close to 1 for distances d < 2, 500 km, but exhibits a super-power-law behaviour for longer distances. the scale-break indicates that the significant links can be divided into two distinct classes: (i) links associated with regional weather systems with distances up to 2, 500 km, which include mesoscale convective systems and tropical cyclones; and (ii) links associated with global-scale teleconnections. such teleconnections are generally understood to be caused either by direct signal transport due to large-scale atmospheric circulations or by propagating waves triggered by disturbances of these circulations [96] . in fig. 31 , the teleconnection pattern for southcentral asia (sca) for events above the 95th percentile is presented. pronounced link bundles connecting sca with eastern asia, the african tropics, large parts of europe and the eastern coast of north america, 60 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 61 62 63 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 61 62 63 64 65 j o u r n a l p r e -p r o o f 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 61 62 63 64 65 j o u r n a l p r e -p r o o f as well as the southern hemisphere extratropics are apparent. further analysis reveals that the rossby waves can be regarded as the physical mechanism underlying these global teleconnection patterns. earth's atmosphere, made up essentially of the gases that surround our planet, consists of circulation patterns that move air from one location to another and from the surface to top elevations. the atmospheric circulation is controlled by earth's rotation, barometric pressure, topography, ocean currents, and differences in temperature, salinity, etc. latitude structure of the circulation since latitudinal gradients in solar energy input are dominant drivers of the atmospheric circulation, its primary features depend on the latitude. we show an idealised depiction of the large-scale atmospheric circulation on earth in fig. 32 , which schematizes the main features of the circulation, both in a latitude-height plane and in the horizontal, without yet considering longitudinal variations due to continents, oceans, etc. this represents the circulation in an average over all longitudes, known as a zonal average. as we see, there are three large-scale convection cells in both hemisphere. (i) the hadley cell is a thermally driven, overturning circulation that tends to rise in the tropics and sink at slightly higher latitudes. warming from the surface near the equator is transferred upward through a deep layer by convection clouds. the rising air spreads poleward and cools slowly, returning to the surface and then moves toward the equator. roughly speaking, the hadley circulation transports heat to about 30 • latitude, and the average effect of the transient weather disturbances transports the heat further poleward. (ii) the ferrel cell is also called mid-latitude cell. in the ferrel cell, air flows poleward and eastward near the surface and equatorward and westward at higher altitudes; this movement is the reverse of the airflow in the hadley cell. it was the first to account for the westerly winds between latitudes 35 • and 60 • in both hemispheres. (iii) the polar cell is the smallest and weakest cell, which extends from between 60 • and 70 • north and south to the poles. air in these cells sinks over the highest latitudes and flows out towards the lower latitudes at the surface. note that the idealized picture of the atmospheric circulation based on latitude is a reasonable first approximation, but land-ocean contrasts and other variations in longitude are also important. climate change strongly affects the mode of atmospheric circulations, especially, the hadley cell, which plays a pivotal role in the earth's climate by transporting energy and heat poleward [330] [331] [332] . an analysis of satellite observations indicates a poleward expansion of the hadley cell by 2 • latitude from 1979 to 2005 [330] . a possible mechanism for the changes in the hadley cell and its relation to global warming has been reported [331] . also, a possible mechanism for the changes in the hadley cell's strength and its relation to global warming was developed in [332] . both aforementioned observations and theories suggest a weakening and poleward expansion of the hadley cell under global warming. as is well known, the locations of the subtropical dry zones and the major tropical/subtropical deserts are associated with the subsiding branches of the hadley cell [333] . therefore, the poleward expansion of the hadley cell may result in a drier future in some tropical/subtropical regions [331] . in addition, the poleward migration of the location of the tropical cyclone maximum intensity is also considered as a consequence of the expansion of the hadley cell [334] . the strength of the hadley cell is calculated using the observed zonal-mean meridional wind in the stream function ψ [335] , where v is the meridional velocity in pressure coordinates, r is the mean radius of the earth, and p is the pressure. the operators¯and [ ] stand for temporal and zonal averaging, respectively. when computing the ψ field, we usually assume ψ = 0 at the top of the atmosphere and integrating eq. (126) downward to the surface. the intensity of the hadley cell is much stronger in winter than in summer. to quantify the edges 63 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 61 62 63 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 61 62 63 64 65 j o u r n a l p r e -p r o o f of the hadley cell, the maximum of the absolute value of ψ at 500 hpa is first determined (ψ 500 ), and then the edges are identified as the first latitude poleward of the maximum at which ψ 500 becomes zero [331] . theories of the hadley cell suggest that the meridional extent should scale with the height of the tropopause h [336] , where h was computed from temperature data as the lowest pressure level at which the lapse rate decreases to 2 • c/km [337] , ∆ h is the tropospheric mean meridional potential temperature gradient, and ω is the angular velocity of the earth. from the thermodynamic equation and the mass continuity equation [335] , the meridional overturning stream function ψ of the circulation scales as, where ∆ v is the dry static stability of the tropical troposphere and τ is the characteristic overturning time scale of the circulation. although the conventional analysis of satellite observations and the climate change simulations suggests a poleward expansion of the hadley cell, there are still two challenges. (i) the latitude-longitude structure of this expansion is not fully resolved. (ii) in contrast to models and theoretical considerations, which predict a decreasing intensity, an increasing trend was found in the intensity of the hadley cell in reanalysis datasets [338] . to overcome these unsolved issues, fan et al. developed an approach based on network and percolation frameworks to study the impacts of climate changes on the atmospheric circulations [67] . they found an abrupt transition during the evolution of the climate network, indicating a consistent poleward expansion of the largest cluster that corresponds to the tropical area, as well as a weakening of the strength of link in the tropics. this was found in both the reanalysis data and the cmip5 simulations. the underlying mechanism for the observed expansion of the tropical cluster was linked to the weakening and expansion of the hadley cell. in ref. [67] , the cns were embedded into a two-dimensional lattice, where only nearest neighbor links is considered. the strength of each link are calculated based on eq. (15), with the month-to-month temperature difference. the links are sorted in decreasing order of strength and then added one by one according to the decreasing strength. more specifically, the nodes that are more similar are connected first. existing clusters grow when a new link connects one cluster to another one. then the cn undergoes an abrupt and statistically significant phase transition, i.e., exhibiting a significant discontinuity in the order parameter g 1 , the relative size of the largest cluster. due to the earth's spherical shape, the largest component in the climate networks is redefined as where φ i is the latitude of node i. the percolation threshold is determined according to eq. (48), i.e., the step with the largest jump is regarded as the phase transition point. fig. 33(a) shows the cn component structure in the global map at the percolation threshold. just before this jump, the cn is characterized by three major communities; the largest one is located in the tropical region; the second and third largest are located in the high latitudes of the southern and northern hemispheres. fig. 33(b) depicts the relative size of the largest cluster, g 1 , as a function of the link occupation probability r in the evolution of the cn. it has been found that g 1 exhibits an abrupt jump at the percolation threshold r c ≈ 0.53. the probability density function (pdf) of the weight w i,j of links is shown in fig. 33(d) . to determine the temporal evolution of the largest component g c , and its intensity w c (the weight of the critical link that leads to the largest transition), a sequence of networks based on successive and nonoverlapping temporal windows with lengths of 5 years each was constructed. the results suggested that the 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 61 62 63 tropical cluster is expanding poleward, meanwhile, its intensity decreases significantly with time. the robust weakening and poleward expansion of the tropical component have been observed both in the reanalysis data and in 31 cmip5 models. the network-percolation approach was also used to identify the climate change response, by comparing the topology of the tropical component for the first and last twenty years of the 21st century, i.e., 2080-2100 vs. 2006-2026. it was found that some regions, for example, northern india, southern africa, and western australia have a higher probability to be influenced by the tropical component, whereas the impacts in other regions, e.g., the northeast pacific, will become weaker in the future. the atlantic meridional overturning circulation (amoc) is a crucial part of the climate system in the north atlantic. it has a major impact on climate because of its redistributing meridional heat transport [339, 340] . the amoc has been identified as one of the important tipping elements of the earth system [23] . changes in the amoc have not only affected the north atlantic and surrounding landmasses, but have also had global impacts [156] . for example, negative change leads to increasing storminess in southern europe [341] , connects to above average sea-level rise at the us east coast [342] , and associates with increasing drought in the sahel (for both frequency and intensity) [343] . recently, caesar et al. provided solid evidence for a weakening of the amoc by about 3 ± 1 (around 15%) sverdrups since the mid-twentieth century [160] . this weakening is revealed by the consisting of a pattern of cooling in the subpolar atlantic ocean and warming in the gulf stream region (see fig. 34 ). the slowdown of the amoc was found in both in a high-resolution climate model (cm2.6) in response to increasing atmospheric carbon dioxide concentrations, and in the observed hadisst data since the late 19th century. an improved sst-based amoc index (the subpolar cold patch) was developed, which is optimized in its regional and seasonal coverage to reconstruct amoc changes. in particular, it was found that the amoc decline since the 1950s is very likely to be largely anthropogenic. this slowdown of the amoc is mainly caused by the gradually varying freshwater forcing in the northern north atlantic. the amoc is the zonally averaged volume transport and its strength at 26 • n in the atlantic and is now routinely monitored by the rapid-mocha array [344] . mean patterns of the meridional overturning circulation in the atlantic are determined from the community earth system model (cesm) simulation 67 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 61 62 63 64 65 j o u r n a l p r e -p r o o f [345, 346] . the amoc has its maximum at about 1000 m depth around the separation latitude of the gulf stream with a strength of about 20 sv. since a future collapse of the amoc has been identified as a tipping element, it is therefore crucial to develop early warning indicators for such a potential collapse. as we discussed in section 2.3, various techniques have been developed and successfully applied to detect early warning indicators in single time series used based on the concepts of critical slowing down [177] . in particular, early warning indicators for the tipping point of the amoc have been discussed in [179, 181] . recently, several cn-based warning-signals have been proposed by analyzing the correlations for the amoc collapse that efficiently monitor spatial changes in deep ocean circulation. specifically, the values of the mean degree, its kurtosis, assortativity, and clustering increase when approaching a tipping point [69, 70] . the cn methodology is applied to temperature time series from an idealized ocean model of the amoc, as well as to amoc strength time series from a coupled atmosphere-ocean gcm. in addition, the optimal locations of measurement of the amoc are formulated to provide early warning signals of a collapse through the analysis of the performance of this indicator. next, we will provide an overview of these cn-based early warning indicators for the collapse of the amoc. mheen et al. [69] used a dimensional (meridional-depth) idealized ocean model of the amoc, where tipping points can be explicitly computed. in this model, there are two active tracers: temperature t and salinity s, which are related to the density ρ by a linear equation of state [347] where α t and α s are the thermal expansion and saline contraction coefficients, respectively, and ρ 0 , t 0 and s 0 are reference quantities. the surface freshwater forcing f s , which can be applied as a virtual salt flux, is where φ indicates latitude, φ n is the northern boundary of the equatorially symmetric domain, γ is the strength of the background freshwater forcing, and η is a white noise term. β is the strength of an anomalous freshwater flux which is only applied over the area [40 • n, 60 • n], and q is a constant used to normalize the surface-integrated salt flux from 0 to 1 for all parameter values. as shown in fig. 35 , the bifurcation diagram of the amoc in the idealized ocean model indicates the existence of tipping points at l 1 and l 2 . the model was discretized on a 32 × 16 spatial grid, with four values of β close to l 1 , labeled a, b, c and d. the output of this model is the temperature field time series. next, an undirected and unweighted cn is constructed based on the pearson correlation method (see sec. 2.1) with no time lags and a threshold c c = 0.7 [69] , i.e, if the correlation c i,j > c c , the corresponding two nodes i and j are connected. four network-based indicators e d , e c , k d and k c were developed to study the dynamic evolution of the amoc. here e d is defined as the expectation value of the normalized degree distribution d/d max ; e c is defined as the expectation value of the normalized clustering coefficients c/c max ; k d is the kurtosis of the degree distribution; and k c is the kurtosis of the clustering coefficients distribution. these four network indicators are presented in fig. 36 . it was found that e d increases steeply and smoothly when the tipping point l 1 is approached. the explanation for this increase is based on the increased dominance of the first eof in the variance of the solution in a large part of the domain. the same holds for the indicator e c (fig. 36g) . the kurtosis distributions k d and k c (fig. 36h) show even more clearly a strong increase when the tipping point l 1 is approached before the collapse. note that the cns were constructed from the full spatial temperature field using a sliding window of 5000 years with a shift of 1000 years. the results were not sensitive to the sliding window length. for comparison, previously suggested early warning indicators of the transition in the same amoc time series, such as, lag-1 autocorrelation, dfa, standard deviation (see our discussion in section 2.3) are also plotted in fig. 36 and tend to exhibit a rapid increase/decrease when approaching the tipping point. in particular, the coefficient of the lag-1 autocorrelation (fig. 36c ) reaches its maximum near the tipping point indicating a critical slowdown of the amoc. however, it has been found that the indicator is not monotonic and also increases when the amoc is still far from the transition. the dfa analysis shown in fig. 36d does not indicate early warnings of a transition. fig. 36e depicts the standard deviation of the amoc record. it shows a steady increase toward 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 61 62 63 the transition but it is difficult to set a threshold for an alarm. in fig. 36f , the fraction of the time spent below the 0.5 percentile (estimated based on the first 5000 years) of the amoc strength is shown. this indicator displays a much sharper increase, but it is a rather ad-hoc measure which depends on a calibration in the far past [69] . the same cns-based analysis was also performed in a more realistic fast met office/uk universities simulator (famous) model by feng et al. [70] . the famous model is a reduction for hadley centre coupled model with a lower resolution ocean and atmosphere component. the annual mean of amoc strength data were obtained from a control simulation and from a freshwater-perturbed (referred to as "hosing") simulation of the famous model. in the hosing simulation, the freshwater flux over the extratropical north atlantic was increased linearly from zero to 1.0 sv over 2000 years [70] . the complete amoc streamfunction field for each of the six 100-year equilibrium simulations has been considered. it was found that when the freshwater forcing is increased, a high degree in the network -indicating high spatial amoc correlations -first appears at nodes in the south atlantic at about 1000 m depth. the kurtosis k d of the cn was introduced as an effective indicator to capture the changes in the topology of the degree field. for the hosing simulation, there is a strong increase of k d to values far extending those for the control simulation significantly before the collapse time. however, the critical slowdown indicators variance and lag-1 autocorrelation do not show any early warning signal. moreover, the kurtosis k d can also be used to determine the optimal observation locations of the amoc, which provides a strong anomalous signal at least 100 years before the transition. the topography or bathymetry of the earth shows complex multifractal structures and scaling properties [34, [348] [349] [350] , which can be regarded as a consequence of plate tectonic processes. revealing the relations between geometrical features of terrestrial surfaces and their internal geological processes has long been a fundamental challenge in the earth sciences. the plate tectonic theory is usually used to study most of the major surface topographic features of the earth [351] . it has been reported that there exist strong connections between the ocean bottom topography and the earth's climate [352] . moreover, the surface topography of the earth plays a remarkable role in the dynamical evolution of oceans, especially, in regard to global climate change and sea level rising. sea level rise is one of the major consequences of global climate warming [353] and the effects in combination with storm surges and other extreme events have been observed [354] . decreasing global co2 emissions is crucial for limiting the risks of sea-level rise. it is estimated that the median sea-level rise will be between 0.7 and 1.2 m until the year 2300 even within the constraints of the paris agreement [355] . the world's large ice sheets in greenland and antarctica are considered to be the largest possible contributors to sea level rise. if these ice sheets melt completely, the sea level would rise about 7 m, 5 m and 53 m from the greenland ice sheet, the west antarctic ice sheet, and the east antarctic ice sheet, respectively. there is thus a great interest in clarifying/predicting the influenced areas in response to sea level rise. recently, the etopo1 global relief model was released and provided new opportunities for a better understanding of earth's surface processes based on geomorphic signatures [356] . etopo1 is a 1 arc-minute global relief model of the earth's surface that integrates land topography and ocean bathymetry and was developed by noaa. it was built from global and regional data sets and used to calculate the volumes of the world's oceans and to derive a hypsographic curve of earth's surface (see fig. 37 ). in this section, we will review statistical properties of the earth geometric surface relief using percolation theory. their fractal structure and scaling will also be discussed. in particular, the present mean sea level on earth will be shown to coincide with the critical threshold in a percolation description of the global topography; strong evidence reveals abrupt transitions that occurred during the evolution of the earth's relief network, indicative of a continental/cluster aggregation. this could help us to identify the critical nodes or locations that will be more exposed to global climate change. self-similarity and long-range correlations are remarkable features of the earth's surface topography [34] . starting about 30 years ago, new ideas in nonlinear dynamics, particularly fractals and scaling, provoked an explosive growth of research both in modeling and in experimentally characterizing the solid earth geophysics including the topography, for a review see ref. [357] . the power spectrum s of linear transects of earth's topography follows the scaling relation s(k) ∼ k −βc , where k stands for the wave number [34] . such a scaling relation was used to measure the self-similarity of the earth's surface topography. moreover, similar scaling relations have been identified in earth's sea-floor topography [358] , the topology of river networks [359] , as well as natural rock surfaces [360] . the long-range correlation features of the earth's surface topography can be described in the following way. the exponent β c in the power-law spectrum is related to the hurst exponent h in fractional brownian motion (fbm) via β c = 2h + 1, which results in h 0.5 for the earth's topography. whereas further universal multifractal parameters estimated for specific parts have resulted in a much more complex structure, i.e., h = 0.46, 0.66, 0.77 for bathymetry, continents and continental margins, respectively [348] . the third characteristic of the earth's surface topography is the well known bimodal distribution, as a consequence of plate tectonic processes [361] . it indicates the topographic dichotomy of continents and ocean basins. although the classical pate tectonic theory provided a framework that might explain most of the major surface topographic features of the earth, a percolation-based description as discussed in sec.(2.2) was developed to study the geometrical features of the earth from a statistical physics perspective [127, 130] . the analysis is based on high-resolution etopo1 global relief records. the resolution is 1 arc-minute, i.e., n = 10800 × 21600 grid points. the present mean sea level (zero height) is assumed as a vertical datum of the height relief h(φ i , θ i ), where φ i , θ i are the corresponding latitude and longitude of grid point i. in ref. [127] , the hypothetical water level was considered as the percolation control parameter. when it is decreased from the highest to lowest available heights on earth, there occurs a geometrical percolation phase transition at a critical level h c around which most parts of landmass join together. fig. 38 illustrates the landmass cluster aggregation by decreasing the sea level from h = 100 m (top) to h = −3520 m (bottom). to determine the percolation threshold, the probability of any nodes to be part of the largest island was defined as the order parameter; the mean island size χ (analogous to the susceptibility of the system) was defined as, χ = s s 2 n s (h)/ s sn s (h), where n s (h) denotes the average number of islands of size s at level h and the prime on the sums stands for the exclusion of the largest island; the correlation length ξ is defined as the average distance of nodes belonging to the same island cluster, ξ 2 = s 2r 2 s s 2 n s (h)/ s s 2 n s (h), where r s is the radius of gyration of a given s cluster. it has been found that the order parameter for islands has an abrupt jump around the zero height level h = 0, i.e., right at the present mean sea level (fig. 39a) . it was furthermore found, that both quantities χ and ξ become divergent at the present mean sea level, see fig. 39b . from these results, the most remarkable observation is that the critical level h c coincides with the present mean sea level h = 0 on earth. this may suggest the important role of water on earth and shed new light on the tectonic plate motion [127] . the same percolation analysis was also performed for the oceanic clusters, i.e., the hypothetical water level was increased from the lowest to highest. fig. 39b gives rise to a discontinuous jump in the oceanic order parameter at around −3640 m. further investigation in the lunar topography reveals various characteristic features of the moon. it was found that the critical level for the moon has the same amount of land and oceans at the threshold, indicating a purely geometrical phase transition [127] . it has been reported that a random network or lattice system always undergoes a continuous percolation phase transition and shows standard scaling features [81] . yet, the order of the percolation transition for the earth's topography is still an open question. to answer this, fan et al. developed a more sophisticated percolation-based method [130] . the percolation model was defined as follows: starting from an unoccupied 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 61 62 63 lattice, the sites are occupied one by one according to their ranking, i.e., first choosing the site with the largest height, then the second, etc. at each step, the fraction of occupied sites p increases by the inverse of the total number of sites n in the earth's relief landscape. by this procedure, a configuration of occupied sites is continually obtained at every p. here all the grid points in the etopo1 global relief model were ranked according to their height h(φ i , θ i ), from the largest to the smallest value. in the following, the size of the percolation landmass (oceanic) clusters s were calculated based on eq. (129) due to the earth's spherical nature. the size of the i-th gap g i at each fraction p is defined as follows: specifically, here g 1 denotes the largest gap, g 2 indicates the second largest gap, etc. the larger the gap g i is, the larger are the two clusters before merging. the dynamical evolution of the largest landmass cluster s as a function of the fraction of occupied nodes p is shown in fig. 40a . it was found that earth's relief network undergoes several abrupt and statistically significant phase transitions, i.e., exhibiting a discontinuity in the order parameter s. fig. 40b shows the network clusters landmass structure at the percolation threshold (just before the largest gap g 1 ). the results reveal that the network, just before this jump, is characterized by four major communities: the largest one being the afro-eurasia continental landmass, the second largest cluster is the americas, the third is located in antarctica, and the fourth is oceania. notably, there exists a critical node (64.458333 • n, 171.141667 • w) that connects the largest and second largest cluster at the percolation threshold p c ≈ 0.321, with altitude level h = −43 m, under the current sea level. the same analysis has also been applied for the oceanic clusters, i.e., the nodes are occupied one by one according to their hight level in increasing order. it also has given rise to a discontinuous jump in the oceanic order parameter at the percolation threshold p c ≈ 0.379 with an altitude level h = −3621 m. this is very close to the result h = −3640 m reported in ref. [127] . note that the critical node, (59.908333 • s, 161.308333 • e), connects the atlantic+indian ocean plate to the pacific plate. it is worth noting that the role of the largest cluster on the landmass structures is different from that on the oceanic one. these differences reveal the complex and different structure of the earth's topography (continents) and bathymetry (oceans). this dichotomy is manifested in the well-known bimodal distribution of the earth's topography [361] . to demonstrate the order of the percolation phase transition in earth's relief network, a finite-size effects analysis was performed by altering the resolution of the nodes. the largest gap g 1 (l) was calculated for a given system size l. here, l is defined as the number of nodes in the zonal direction. according to the eq. (49), if g 1 (l) → 0 as l → ∞, the corresponding giant cluster is assumed to undergo a continuous 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 61 62 63 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 61 62 63 64 65 j o u r n a l p r e -p r o o f percolation; otherwise, the corresponding percolation is assumed to be discontinuous [147, 153] . in addition, the size of the order parameter at the percolation threshold [just before the largest jump g 1 ], s c (l), was also studied [see eq. (51)]. the results are shown in fig. 41a and b , which indicate a discontinuous percolation since (1) g 1 (l) tends to be a non-zero constant; (2) d f − d = 0, also indicates a discontinuous percolation. for comparison, a randomized topography obtained from the shuffling (spatial randomizing the height profile) of the original data was also considered (see fig. 41a and b) . the numerical results for the shuffled case, suggest a continuous percolation transition with known critical exponents β/ν = 5/48 ≈ 0.104 and d f = 91/48 [106, 107] , as expected when the long-range correlations were destroyed. in order to reveal the origin of the discontinuity, fan et al. [130] further studied the percolation on 2d fbm surfaces with hurst exponent h [362, 363] . the parameter h is usually between 0 and 1, where 0 is very noisy, and 1 is smoother. the earth's rough surfaces can be modeled via a fbm [364] , and the estimation for the earth continents topography is h = 0.66 [348] . next, the percolation analysis on fbm surfaces with h = 0.66 was performed. similar to the real network, the largest cluster s also exhibited abrupt transitions around p ≈ 0.3. the largest gap g 1 (h, l) (average) as a function of system size l is shown in fig. 41c . it was found that the percolation on fbm surfaces with h = 0.66 is discontinuous, since g 1 (h, l) tends to be a non-zero constant when l → ∞. the percolation threshold p c (h, l) corresponding to the largest gap during the evolution of site percolation is shown in fig. 41d . the values are robust and agree well with the real data where p c ≈ 0.321 [see fig. 38 ]. this indicates that the combined percolation and fbm methods can be used as an efficient tool to investigate the earth's surface topography. earthquakes are one of the most devastating natural disasters that plague society. thus, reliable and skillful earthquake forecasts over different time scales (from days to decades) are essential for establishing rational seismic risk reduction strategies and to enhance preparedness and resilience. the earthquake process is a complex spatio-temporal phenomenon, and has been thought to be an example of self-organised criticality systems, in which events occur as cascades on a wide range of sizes, each determined by fine details of the rupture process. despite rapid progress in the latter part of the 20th century, the study of earthquakes, like the science of many other complex natural systems, is still in its juvenile stages of exploration and discovery. the scientific challenge is how to understand the earthquake phenomena that are both profound and practical, in particular, the deterministic prediction of specific event sizes, their locations, and times. recently, probabilistic forecasting, based on statistical patterns of earthquake occurrence, became a much more realistic goal, and has been actively explored and tested in global initiatives [36] . in this section, we will first present a description of the phenomenological laws of earthquake occurrence, and scale invariance and the intertime distribution is also briefly discussed. then we will review how the statistical mechanics approach (memory analysis) can be applied to improve the forecasting skill for real earthquake catalogs. first, we introduce some fundamental quantities that characterize earthquake occurrence. this information, such as occurrence time, hypocentral location, earthquake magnitude and depth, is usually reported in seismic catalogs available online for different geographic areas, for instance, the united states geological survey (usgs) earthquake catalog 11 . we present here the main phenomenological laws for the statistical distribution of these quantities. the most common method of describing the size of an earthquake is the richter magnitude scale m, which takes the logarithm of the ground displacement as measured by a seismograph, and applies a correction which varies with the distance from the earthquake to the seismograph [365] . the characteristic size of the fractured area l, as well as the faulting duration τ , can be both expressed in terms of the magnitude as l ∝ τ ∝ 10 0.5m . next, we will give some well established empirical basic laws of earthquakes. in seismology, the gutenberg-richter (gr) law expresses the relationship between the magnitude m and cumulative number of earthquakes n in any given region and time period with magnitude larger or equal to m, which exhibits an exponential decay [366] log n = a − bm, where a and b are fitting parameters. we show the gutenberg-richter law for the israeli earthquake catalog as an example, in fig. 42 . the b-value of the gutenberg and richter law has been calculated using the approach described by marzocchi and sandri [367] . the value was found to be equal to 0.97 ± 0.02. the intercept of the fit line defines the a-value that explains the earthquake rate for the region selected. in our case a = 5.36. as expected, different catalogs exhibit different seismicity levels quantified by the magnitude range and by the total number of events, corresponding to different values of the constant a. conversely, the parameter b appears to be quite independent of the geographical region and the temporal interval. typical experimental results provide b 1 for tectonic earthquakes. the second empirical law, the omori law, was first described by the seismologist omori in 1894 [368] , where n(t) is the aftershock rate, t is the time elapsed from the mainshock occurrence; k and c are empirical constants controlling, respectively, the total number of aftershocks n(t) and the onset of the power law decay. p is a third constant, which modifies the decay rate and typically falls in the range 0.7-1.5. the magnitude of an event understandably influences the number of aftershocks triggered by it, i.e., the larger the mainshock magnitude, the larger is the total number of triggered aftershocks. this property is known as the productivity law, stating that the number of aftershocks n a [integral of eq. (134)] belonging to a sequence increases exponentially with the mainshock magnitude m m , the parameter α m is typically small for swarm-type activity, and large for clear primary aftershock sequences [369] . the distribution of waiting times between seismic events has generated much attention and discussion over the last decades, since it was considered to have a universal scaling form [370] . bak et al. proposed, for the first time, a unified scaling law for the waiting times between earthquakes, expressing a hierarchical organization in time, space, and magnitude. they considered the california earthquake catalog and covered the region with a grid of cells of size l × l. then they analyzed the inter-event times for each cell and calculated a histogram of the inter-event times p s,l whose magnitudes are greater than m = log(s); this was repeated for different values of l and minimum magnitudes (see fig. 43a ). it was found that if they rescaled the inter-event times t by s −b l d f (where b is the gutenberg-richter b -value and d f is the spatial fractal dimension), and rescaled p s,l by t α , where α is some exponent, the distribution of waiting times all appeared to collapse onto a single curve (fig. 43b) . the scaling hypothesis was thus expressed as where α ≈ 1 can be identified as the omori-law exponent for aftershocks, b ≈ 1 is the b value in the gutenberg-richter law, and d f ≈ 1.2 describes the 2d fractal dimension of the location of epicenters projected onto the surface of the earth [370] . subsequently, corral [371] has discovered that the probability density function d xy (τ ) of the interoccurrence time τ can be simplified to the scaling form where r xy stands for the mean seismic rate that refers to the (x, y) region. the scaling function f can be expressed by a generalized gamma distribution, 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 61 62 63 [372] carried out an extensive analysis to determine the distribution of the inter-event time based on the epidemic-type aftershock sequences (etas) model (see more details in the following section), and they found that it is only approximately universal and of a gamma form, assuming that p in eq. (134) has a value close to 1, and the branching ratio of seismicity, the average number of events each event triggers, is around 0.7-1. then molchan [373] published a rigorous mathematical study on the earthquake inter-event time, and proved that if the scaling distribution would be universal, the functional form must follow an exponential function. touati et al. analyzed the variation of the distribution for both real data (scec catalog) and etas simulation data, and suggested that it is not universal [374] . they have shown that the distribution is a bimodal mixture distribution dominated by correlated aftershocks at short waiting times and independent events at longer times. there are various statistical earthquake models for describing the occurrences process of earthquakes that can be used for real-time earthquake forecasts [375] . the principle of these models is to evaluate the probabilities of earthquake occurrence by using a point process approach. among the different models, the etas model, which describes the features of earthquake clustering of mainshocks, foreshocks and aftershocks, has become a standard model for testing hypotheses and a starting point for short-term earthquake forecasts [376] [377] [378] [379] . here we will focus on the etas model, which is used to generate synthetic earthquake catalogs. the etas model was developed by ogata [369, 380, 381] who observed that seismic activity can be well described by the gutenberg-richter law eq. (133) , and the omori law eq. (134) . the conditional rate in the etas model is given by, where t i are the times of the past events and m i are their magnitudes; h t = {(t i , m i ); t i < t} is the history of occurrence. λ(t|h t ) provides the probability to have an earthquake above a threshold magnitude m z at time t, given the earthquake history. here, a = k/c p is the occurrence rate of earthquakes in the omori law at zero lag [374] , and α m is called the productivity parameter defined in eq. (135) . the etas model can also be written in terms of a stochastic integral [382] , where n (ds, dm) = 1 if an infinitesimal element (ds, dm) would include an event (t i , m i ) for some i, otherwise n (ds, dm) = 0. the five parameters (µ, a, c, α m , p) represent some characteristics of seismic activity of the region. therefore, they vary spatially, and also temporally in some cases. then the etas model was extended to a space-time phase with the conditional intensity function [383] , where µ(x, y) still is the background intensity, which is a function of space, but not of time; κ(m) is the expected number of events triggered from an event of a magnitude m in the form g(t) is the probability density function of the occurrence times of the triggered events, f (x, y|m) is the spatial distribution of the triggered events, which can be expressed in the following two ways: a short-range gaussian decay, or a long-range inverse power decay, the etas model has been successfully used for operational earthquake forecasting, e.g., the complex amatrice-norcia seismic sequence [384] , next-day earthquake forecasts for the japan region [385] and california region [386] . more general discussions on modeling seismic time series by the point process approach are given in ref. [387] . in this subsection, we will review the memory analysis that has been applied to seismic records. the first method is the so-called detrended fluctuation analysis (dfa), which is used to detect long-range correlations or long-term memory of diverse time series, such as dna sequences and climate records [180] . details of the dfa are presented in sec. 2.3.3. according to eq. (70), if the time series has a long-term memory, the fluctuation function f (n) increases according to a power-law relation with a exponent α. the dfa has been applied to earthquake interoccurrence times for the first time by lennartz et al. [388] . they tested the real and the synthetic records for long-term correlations by employing the first two orders of the detrended fluctuation analysis dfa0 and dfa1 on the scec catalog (1995) (1996) (1997) (1998) and the ncsn catalog (1995-1998). they found that there exists a long-term memory between seismic events in the californian catalogs, which show up in characteristic fluctuations in both magnitudes and interocurrence times. both dfa0 and dfa1 have resulted in a dfa exponent α ≈ 0.75, independently of the threshold m z and the catalog, see fig. 45 . a similar analysis, dfa2, has been applied to the real israeli and italian catalogs by fan et al. [212] . they found that the exponent α was also very close to 0.75 for the inter-occurrence time series between earthquake events with different magnitude threshold m z . figs. 46a-d show the dfa2 results. they also performed dfa on other seismic variables, such as the number of earthquake events and the released energy within a coarse time window dt. the energy of earthquakes was defined as s(t) = where e(t) denotes the number of events that occurred between t and t + dt, and m l (t) denotes the magnitude of the event. to deal with more homogeneous time series, fan et al. switched to s(t) = log(s(t)) for s(t) > 1 and zero for s(t) ≤ 1 and e(t) = log(e(t)) for e(t) > 1 and zero for e(t) ≤ 1 [212] . next, applying the dfa analysis for the time series s(t) and e(t) in the israeli and italian catalogs, with different time windows dt, were tested. it was found that for both regions and for all studied magnitudes, the value of the scaling exponent α is quite robust ∼ 0.75, i.e., the size of dt does not affect the memory exponent α. thus, the return intervals, the number of events and the released energy are significantly long-term correlated and have a very similar scaling exponent. the memory analysis has also been applied on the etas model. the dfa2 of the inter-occurrence times, with the parameters a = 6.26, µ = 0.2, p = 1.1, α m = 1.5 and c = 0.007, see eq. (139) , yields α ≈ 0.75 and is shown in fig. 46e . another common method for studying memory in the occurrence of earthquakes is called conditional probability (cp), which was introduced by livina et al. [389] . considering a time series of recurrence intervals, they sorted it in ascending order and divided it into four 25% quantiles; i.e., the first quantile, q 1 , represents the shortest 25% of waiting times, etc. the distribution of recurrence times τ , that follow a prior recurrence time τ 0 , p (τ |τ 0 ), was studied, where τ 0 belongs to either one of the quantiles at the extremes, q 1 or q 4 . note that for records without memory, p (τ |τ 0 ) should be independent of τ 0 and should be identical to p (τ ). for the real data, livina et al. found that p (τ |τ 0 ) depends strongly on the previous recurrence time τ 0 , such that short recurrence times lead to short ones, and long recurrence times follow long ones (see figs. 47a, c for the israeli and italian catalogs). to quantify and measure the level of memory, fan et al. analyzed the cumulative distribution function (cdf) of the recurrence times and denoted the cdf for q, q 1 and q 4 as cq(τ ), cq 1 (τ ) and cq 4 (τ ), respectively. the strength of the memory for q 1 is defined as and similarly, the level of memory for q 4 is thus, 0 ≤ ρ 1 ≤ 1 and −1 ≤ ρ 4 ≤ 0, and higher |ρ 1 | (or |ρ 4 |), imply stronger memory and ρ 1 = 0 (or ρ 4 = 0), implies no memory [212] . the results are shown in fig. 47b and d for the israeli and italian earthquake catalogs. they found ρ 1 = 0.248, ρ 4 = −0.193 for q 1 and q 4 of the israeli catalog, whereas ρ 1 = 0.260, ρ 4 = −0.153 for q 1 and q 4 of the italian catalog [212] . further study suggests that the values of ρ are robust and do not depend on m z . to study the dependence of the correlations (α, ρ 1 , and ρ 4 ) on the geographical location (tectonic setting), they performed the dfa and cp analysis for other earthquake catalogs, including new zealand, southern california earthquake center, japan unified high-resolution relocated catalog for earthquakes and preliminary determination of epicenters global earthquake catalogs. it has been found that for all catalogs and for all studied magnitudes the values of α, ρ 1 , and ρ 4 are quite robust. finally, the memory analyses dfa2 and cp, were performed in etas models, by measuring the coefficients α, ρ 1 and ρ 4 , as shown in figs. 46 and 47. the results indicate that the empirically observed earthquake memory (for israeli catalog) can be reproduced only for a narrow range of the model's parameters. moreover, the origin of memory in the etas model is influenced by: (i) the model's background (noise) rate parameter µ which, affects the memory through the interference of temporally overlapping aftershock subsequences, i.e., smaller µ leads to stronger memory, and (ii) the branching ratio n = ac p−1 β β−αm : the exponent relating the production of aftershocks as a function of magnitude, α m , and the power p of omori's law can also affect the memory through the branching ratio of the etas model, i.e., smaller p and larger α m result in a stronger memory (see figures. 11 and 12 in ref. [212] ). more recently, a lagged cp method was introduced by zhang et al. to study a possible long-term memory in both the interevent times and distances of earthquakes [390] . the cp of times and distances were defined as ρ (τ k |τ 0 ) and ρ (r k |r 0 ), respectively, where τ 0 (r 0 ) belongs to the q 1 or q 3 group. τ k (r k ) is the lagged k-th inter-event time that follows τ 0 (r 0 ). in order to quantify the level of memory, s (τ k |τ 0 ) ≡ 1 − s 13 was introduced, where s 13 is the common area of the pdf between q 1 and q 3 . here s (τ k |τ 0 ) ∈ [0, 1]. similarly, s (r k |r 0 ) represents the level of memory for the inter-event distances. interestingly, it has been shown that s(k) can be expressed by the following scaling form, where d u and a are constants, b = 1 as for the gutenberg-richter law. the distribution of s (τ k |τ 0 ), s (r k |r 0 ) and their corresponding scaling functions for the italian catalog are presented in fig. 48 , where d u = 0.14 and a = 0.09 for the inter-event times τ ; d u = −0.08 and a = 0.24 for the inter-event distances r. the scaling functions shown in fig. 48c and d suggest a crossover between two distinct power-law relations with a = 0 . 5 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 61 62 63 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 61 62 63 f (x) ∼ x −γ . both scaling functions exhibit a significant crossover at k · 10 bmz 10 5 . these results indicate that the memory measure of different grid sizes and different magnitude thresholds can be rescaled into a single function. however, it was found that the memory function in the etas model is very different from that of real earthquake catalogs, i.e., the model's memory is weaker (stronger) on short (long) timescale compared to the real catalogs. moreover, the model does not exhibit a clear crossover observed in the real catalogs. earthquakes are one of the most destructive natural disasters in the world. skilled and reliable earthquake forecasting remains an ultimate goal. the term earthquake prediction is usually referring to the specification of the occurrence time, location or magnitude of a future earthquake. however, "the term earthquake forecasting usually refers to the evaluation of the occurrence probability p σ of an earthquake inside a hypercell of volume σ centered in the point ω = (t, x, y, z, m). therefore, the fundamental quantity is the occurrence probability p σ ( ω| ) conditioned on the whole set of information available at time t" [36] . the probability of having an earthquake at time t, in the k-th interval conditioned to the previous history is where λ is the local conditional rate as defined in eqs. (139) or (141) , ω k denotes the center of the k-th cell. the forecasting probability, p σ the integral over σ of the occurrence rate λ ( ω k |h t k ), depends on 86 the volume of the hypercell σ, i.e., the smaller σ, the more accurate is the evaluation of λ. secondly, the temporal extension t of σ also influences the forecasting accuracy. according to the time scale of t , one can separate arbitrarily long-term from short-term forecasting [391] : earthquake long-term forecasting, for t in the interval of decades to centuries; intermediate-term forecasting, for t of the order of months and short-term forecasting, for t in the interval from few seconds up to several weeks. short-term forecasting is usually subdivided into post-seismic and pre-seismic forecasting, referring to the aftershock and foreshock of a mainshock. it is notable that the post seismic forecasting is still very important from the point of view of risk management, since aftershocks can have sizes comparable or even larger than their triggering mainshock. long-term forecasting, however, is probably the most relevant from the engineering point of view, such as urban planning risk assessment. based on the early warning foreshock observations, one could successfully predict some big earthquakes, such as the 1975 haicheng, china earthquake [392] and the 1995 kozani-grevena, greece earthquake [393] . nevertheless, most earthquakes do not present significant precursory patterns, e.g., the 2004 parkfield (california) earthquake [394] . thus the evaluation of foreshocks or precursory patterns is still an open question. since most earthquake models are based on the wellestablished empirical laws, aftershock spatio-temporal occurrence clustering, as the etas model, they are able, in some degree, to provide reliable risk assessment only for the long-term and the post-seismic short-term forecasting but not for the foreshocks. nevertheless, how to validate a forecasting model is still of great importance. in the following, we will review various methods for evaluating earthquake predictions and earthquake forecasts. the receiver operating characteristic (roc) diagram is a graphical plot that illustrates the prediction quality of a binary classifier system as its discrimination threshold is varied. for evaluating earthquake predictions, if an earthquake is expected ("yes") with a probability p σ > p t , where p t is a threshold, we call it a hit, if an event has occurred; if earthquakes are not expected ("no") in the cell, we call it a miss, if an event has occurred. each "yes" prediction in which no corresponding earthquake is observed is a false alarm, and each "no" prediction in which no earthquake occurred is a correct rejections. in this representation, there are four possible combinations of alarm declaration and event observation, see table. 1 for the number of each of these contingencies. here we define a as the number of hits, b as the number of false alarms, c as the number of misses and d as the number of correct rejections, respectively. we define a true positive rate (t p r) as and false positive rate (f p r) as when t p r and f p r are plotted together on the square [0,1] × [0,1], the resulting metric is called the roc, whereas the perfect prediction corresponds to the point with coordinates t p r = 1 and f p r = 0, the diagonal line represents the long-term behavior of random guessing. one can obtain the full roc curve by changing p t continuously. here we show an aftershock forecasting roc curve by using a deep learning method in fig. 49 as an example. closely related to the roc is the molchan diagram, which is an error diagram that allows to compare the prediction of the test model and another generic reference model [396] . it is a plot of the miss rate, m = 1−t p r, and the fraction of experiment space-time volume, f , occupied by alarms or yes predictions. the best prediction in the molchan diagram is the point (f = 0, m = 0). in particular, the probability of obtaining h or more hits by chance, given that there have been n observed target earthquakes, is described by the binomial distribution where n i stands the binomial coefficient. one can obtain a confidence level curve in the molchan a b figure the n-test is used to access how well the total number of forecasted earthquakes n f orecast matches the number of events observed n obs [397] . the target earthquakes are assumed to follow a generalized poisson distribution, and the mass function of the negative binomial distribution (nbd) can be expressed as [384] f (n; r, p) = γ(r + n) n!γ(r) p n (1 − p) r , where γ(r) is the gamma function, n is the number of target earthquakes, and r, p are the two parameters of the distribution. then nbd can be derived as f (n; r, p) = ∞ 0 f 1 (n; λ)f 2 λ; r, 1 − p p dλ, where f 1 (n; λ) is the poisson distribution and λ is assumed to follow a gamma distribution where k, θ are the parameters of the gamma distribution with r = k and p = θ 1+θ . next, we calculate the parameters k and θ of the gamma distribution. to interpret the n-test results, one can use a one-sided test with an effective significance value α ef f , which is half of the intended significance value α = 0.05. fig. 50c shows an example of the n-test. the space test (s-test) the s-test is a likelihood test where only the spatial distributions of the forecast and the observation are considered [398] . the s-test can be summarized by a where s i is the i-th simulated spatial likelihood, s s is the set of simulated spatial likelihoods, s is the likelihood of the spatial forecast relative to the observed, n{a} is the number of elements in a set {a}. if ζ is less than critical significance value α, the observed spatial distribution is inconsistent with the forecast. however, values close to 1 indicate optimal spatial forecasts. we show an example of the s-test in fig. 50a . besides the aforementioned methods, there are still various other methods that have been developed to evaluate the accuracy of a forecasting model, such as, the likelihood test (l-test), the likelihood ratio test (r-test) and the magnitude test (m-test). an instructive review on the the evaluation of earthquake predictions and earthquake forecasts can be found in ref. [399] . as discussed in the last section, we studied the possible origin of memory in earthquakes for both real catalogs and etas models. here, we will show that the memory analysis can significantly improve the short-term forecasting rate for the real earthquake catalog. in estimating the etas model parameters for a given earthquake catalog, the etas parameters are commonly inverted from the data based on the pointprocess maximum likelihood (ml) method, by the davidon-fletcher-powell algorithm [380] or by simulated annealing [400] . for example, by using the ml method, one can obtain µ = 0.2, a = 6.26, α m = 1.4, 139), for the italian catalog [400] . when considering the memory, however, the etas model can reproduce the same memory as in real catalogs, only for a small range of parameter values, i.e., µ = 0.2, a = 6.26, α m = 1.5, p = 1.1 and c = 0.007 [212] . as a test case the cumulative number of events, nc, after the capitignano 5.7 main shock that happened on 18 january 2017 [401] , was forecasted by using the etas model. the cumulative number at time t is calculated by integrating eq. (139) from 0 to t. this way the forecasting accuracy within 14 days after the main shock can be improved by more than 20% by considering the memory. moreover, a revised and generalized etas model was proposed recently by zhang et al., in which the short-and long-term/distance memory was reproduced accurately [402] . the new etas model is also found to significantly improve earthquake forecasting for the italian and south california earthquake catalogs. in this article, we have reviewed statistical physics and complex networks-based techniques that advance our knowledge on the complex earth system, a relatively novel branch of geophysics. these techniques can help to address the understanding as well as the prediction of climate variability, earth geometric relief features and earthquakes. in this section we try to draw a few concluding remarks and address the perspectives that remain still open for future research. we believe that these novel approaches will attract the attention of scientists in the related fields. in section 2 we started by describing the overall methodology based on statistical physics for analyzing important aspects of complex earth subsystems. it includes complex networks, percolation theory, tipping points analysis, entropy theory and complexity. although it was shown that the aforementioned approaches can be successfully applied to earth systems individually or in a combination, there is still a lot of work ahead in order to develop a proper and comprehensive framework. among the different open problems to be solved, we highlight the following four: i) the need of gathering a better knowledge and understanding on the underlying physical mechanisms; ii) the need of extending concepts of cns to a more comprehensive description, such as multilayer climate networks [403] . this is since the climate is a coupled and multilayered 90 system; iii) the need of developing a universal theory of critical phenomena (percolation) in climate systems, in particular for out-of-equilibrium systems; iv) the need of proposing a principle on how to anticipate the tipping points and entropy of the earth system by means of time series of multiple spatio-temporal variables. in section 3, we have continued by reviewing the applications of statistical physics approaches to different earth subsystems. specifically, five weather and climate dynamical scenarios were highlighted in section 3.1, including, el niño-southern oscillation, indian summer monsoon, extreme rainfall, atmospheric circulation and atlantic meridional overturning circulation. we suggested that the evolving cns (and other combined approaches) interactions and patterns can serve as novel predictors which significantly improve the predictions. beside the above mentioned scenarios, the cns method was also widely applied to other high-impact phenomena, such as the westward propagation of the atlantic multidecadal oscillation [99] , the detection of teleconnection paths [62] , the identifying of causal gateways and mediators [404] , the early prediction of extreme stratospheric polar vortex states [405] , the impacts of carbon dioxide (co2) on global sat [406] , etc. in spite of the great achievements, there are still many unexplored problems related to climate systems that merit further attention. therefore, further topics are needed to be investigated, for example, i) how human activities have changed and continue to change the earth's oceanic and atmospheric composition, some of these changes have a direct or indirect impact on the energy balance of the earth and are thus drivers of climate change; ii) the statistical mechanics and cns behaviours in earth system models, ranging from simple energy balance models to general circulation models; iii) the projected short-term and long-term abrupt phase transitions in paleoclimate, in particular, the role of sea ice and snowball-earth initiations [407] [408] [409] ; iv) improving the predictability of severe convective weather processes including thunderstorms, hail and tornadoes by using the cns approach. in section 3.2, we have reviewed the current literature on the statistical features and the percolation framework of the earth's surface topography. we have pointed out the evidence for abrupt transitions that occurred during the evolution of the earth's relief network, indicative of a continental/cluster aggregation. a further analysis via fractional brownian motion models suggests that long-range correlations may play a key role in the observed discontinuity on earth. this research may facilitate the understanding of the geometrical phase transition on earth, but also can be used to identify the critical nodes for future global change in earth's relief network. in particular, i) the future movement of tectonic plates, as well as ii) the dynamic evolution of these critical nodes are of key importance and thus need to be further addressed. in section 3.3, we first presented a description of the well established empirical basic laws of earthquakes, including the gutenberg-richter, omori and productivity laws. we then discussed the scaling hypothesis of the inter-event distributions. an epidemic-type aftershock sequence model (etas) and various methods for evaluating earthquake predictions and earthquake forecasts have been also reviewed. at last, we have shown that the two proposed memory analysis approaches, dfa and cp, can significantly improve the short-term forecasting rate for real earthquake catalogs. nevertheless, earthquake prediction research has been plagued by controversy, and it remains an outstanding challenge. besides the improvement of shortterm aftershock forecasting, we also emphasize the potential of new directions. i) the wide applications of tipping points and memory analysis in theoretical earthquake models which account for accelerated moment release and foreshock occurrence together with other precursory patterns, where mainshocks were treated as characteristic earthquakes within a seismic cycle [410] . ii) the development of an earthquake forecasting model which is implemented for describing foreshock organization and memory behaviors. iii) motivated by the successful applications of network theory to the prediction of climate phenomena, the proposing of an earthquake network framework based on the occurrence time series [411] , or in particular the seismic waves (elastic body and shear waves) and elastogravity signals preceding direct seismic waves [412] . one of the key features of statistical physics-based approaches reviewed here is the ability to better and reliably forecast the complex earth phenomena, such as el niño events, extreme rainfall in the eastern central andes, indian summer monsoon, the collapse of the atlantic multidecadal oscillation and the occurrence of earthquakes. moreover, we observed that artificial intelligence and deep learning techniques [413] have achieved great success during recent years in many fields, such as phase transitions in statistical physics [414] [415] [416] , data-driven earth system science [417] , enso forecasts [294, 295] , indian monsoon rainfall [418] , as well as the forecasting aftershock patterns [395] and detecting earthquakes [419] in seismic systems. in fact, we are confident that the combination and complement of network-based and artificial intelligence-based skills will boost each other. for instance, an application of machine learning to network attribute vectors (products similarity) can predict successful and failing firms with much better accuracy than current state of the art techniques for market forecasting [420] . in addition, deep learning systems can be regarded as complex networks, which thus gain some insights into the structural and functional properties of the computational graph resulting from the learning process [421] . as a final remark, there is still room for many approaches considering the analysis of climate resilience of societies, ecosystems and economies by using the complex network theory. we currently lack appropriate models to better understand or predict the effects of cascading failures [30] triggered by the increasing adverse effect of extreme climate/weather events on interdependent critical infrastructures. closing this knowledge gap is crucial for developing means to achieve both climate and infrastructure resilience. another very relevant subject of investigation that will certainly attract huge attention is the assessing and quantifying of the complex climate-health relationships. there is an overwhelming consensus that climate conditions, including temperatures and spatial-temporal distribution of precipitation, has key implications for human health [422] . moreover, the morbidity and outbreak of some disease, such as the vector-borne (malaria and dengue fever) disease, the ongoing covid-19 pandemic [423] , and influenza are strongly affected by the climate change or the environment [424] [425] [426] [427] [428] . however, the climatic influences are often excluded from consideration in the traditional epidemiologic models [113] , e.g., the sir, sirs and seir models. what we need to emphasize is that traditional epidemiologic models with taking into account the climate factor could be a very promising road towards a deeper understanding of epidemic processes and assess the health consequences of climate change both regionally and globally. 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 61 62 63 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 61 62 63 64 65 j o u r n a l p r e -p r o o f 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 61 62 63 64 65 j o u r n a l p r e -p r o o f 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 61 62 63 64 65 j o u r n a l p r e -p r o o f 106 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 61 62 63 64 65 j o u r n a l p r e -p r o o f 107 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 61 62 63 64 65 j o u r n a l p r e -p r o o f journal pre-proof global change and the earth system: a planet under pressure, global change -the igbp series the emergence and evolution of earth system science atmospheric homeostasis by and for the biosphere: the gaia hypothesis a vast machine: computer models, climate data, and the politics of global warming the rejection of continental drift: theory and method in american earth science earth system science overview: a program for global change, national aeronautics and space administration our common future: report of the world commission on environment and development earth system' analysis and the second copernican revolution climatic trends and interdecadal variability from south-central pacific coral records two millennia of climate variability in the central mediterranean the asian monsoon over the past 640,000 years and ice age terminations low-frequency signals in long tree-ring chronologies for reconstructing past temperature variability climate and atmospheric history of the past 420,000 years from the vostok ice core orbital and millennial antarctic climate variability over the past 800,000 years energy balance climate models climate change 2013: the physical science basis, contribution of working group i to the fifth assessment report of the intergovernmental panel on climate change integrated assessment models of global climate change how well do integrated assessment models simulate climate change? earth system models: an overview sustainable development of the biosphere geology of mankind tipping elements in the earth's climate system a safe operating space for humanity trajectories of the earth system in the anthropocene phase transitions and critical phenomena introduction to modern statistical mechanics introduction to statistical physics statistical mechanics: entropy, order parameters, and complexity catastrophic cascade of failures in interdependent networks critical transitions in nature and society critical phenomena in atmospheric precipitation the percolation phase transition in sea ice stochastic models for the earth's relief, the shape and the fractal dimension of the coastlines, and the number-area rule for islands self-organized criticality and earthquakes statistical physics approach to earthquake occurrence and forecasting collective dynamics of 'small-world' networks emergence of scaling in random networks complex networks: structure and dynamics networks: an introduction the statistical physics of real-world networks the architecture of the climate network climate networks around the globe are significantly affected by el niño complex networks in climate dynamics networks in climate modelling disease outbreaks in realistic urban social networks scale-free networks: complex webs in nature and technology dynamical processes on complex networks identification of influential spreaders in complex networks complex networks: structure, robustness and function network science statistical mechanics of complex networks what do networks have to do with climate? topology and predictability of el niño and la nina networks pattern of climate network blinking links follows el niño events the backbone of the climate network an exploration of climate data using complex networks dominant imprint of rossby waves in the climate network global climate network evolves with north atlantic oscillation phases: coupling to southern pacific ocean emergence of el niño as an autonomous component in the climate network teleconnection paths via climate network direct link detection network approaches to climate science network analysis reveals strongly localized impacts of el niño percolation framework to describe el niño conditions forecasting the magnitude and onset of el niño based on climate network climate network percolation reveals the expansion and weakening of the tropical component under global warming improved el niño forecasting by cooperativity detection interaction network based early warning indicators for the atlantic moc collapse deep ocean early warning signals of an atlantic moc collapse review: visual analytics of climate networks introduction to graph theory on the evolution of random graphs on power-law relationships of the internet topology scale-free topology of e-mail networks lethality and centrality in protein networks the architecture of complex weighted networks scale-free brain functional networks scale-free networks are rare connectivity of growing random networks random graphs connectivity, fragmentation, and extinction risk in dendritic metapopulations community structure in social and biological networks uncovering the overlapping community structure of complex networks in nature and society community detection in graphs fast monte carlo algorithm for site or bond percolation unified functional network and nonlinear time series analysis for complex systems science: the pyunicorn package influence of autocorrelation on the topology of the climate network interpretation of link fluctuations in climate networks during el niño periods event synchronization: a simple and fast method to measure synchronicity and time delay patterns nonlinear multivariate analysis of neurophysiological signals analysis of spatial and temporal extreme monsoonal rainfall over south asia using complex networks complex networks identify spatial patterns of extreme rainfall events of the south american monsoon system prediction of extreme floods in the eastern central andes based on a complex networks approach tipping elements of the indian monsoon: prediction of onset and withdrawal complex networks reveal global pattern of extreme-rainfall teleconnections elements of information theory assessing the direction of climate interactions by means of complex networks and information theoretic tools are north atlantic multidecadal sst anomalies westward propagating? percolation and conduction molecular size distribution in three dimensional polymers. i. gelation1 statistical mechanics of dimers on a plane lattice percolation theory at the critical dimension percolation, statistical topography, and transport in random media flow phenomena in rocks: from continuum models to fractals, percolation, cellular automata, and simulated annealing introduction to percolation theory fractals and disordered systems an experimental approach to the percolation of sticky nanotubes tunneling and nonuniversality in continuum percolation systems random graphs with arbitrary degree distributions and their applications percolation on heterogeneous networks as a model for epidemics contact network epidemiology: bond percolation applied to infectious disease prediction and control epidemic processes in complex networks network robustness and fragility: percolation on random graphs clique percolation in random networks explosive percolation in random networks bootstrap percolation on complex networks core percolation on complex networks networks formed from interdependent networks general clique percolation in random networks influence maximization in complex networks through optimal percolation percolation in interacting colloids casimir-like forces at the percolation transition electronic properties of doped semiconductors percolation transition in dynamical traffic network with evolving critical bottlenecks conformal invariance in two-dimensional turbulence percolation description of the global topography of earth and the moon percolation-based precursors of transitions in extended systems percolation phase transition of surface air temperature networks under attacks of el ni ñ o/la ni ñ a percolation framework of the earth's topography recent advances in percolation theory and its applications percolation theory resilience of the internet to random breakdowns the fractal geometry of nature cluster shapes at the percolation threshold: and effective cluster dimensionality and its connection with critical-point exponents self-similarity of complex networks origins of fractality in the growth of complex networks dimension of spatially embedded networks the chemical distance distribution in percolation clusters universal critical amplitudes in finite-size scaling construction and analysis of random networks with explosive percolation explosive percolation in scale-free networks explosive growth in biased dynamic percolation on two-dimensional regular lattice networks cluster aggregation model for discontinuous percolation transitions explosive percolation transition is actually continuous explosive percolation is continuous impact of single links in competitive percolation explosive percolation is continuous, but with unusual finite size behavior continuous percolation phase transitions of random networks under a generalized achlioptas process avoiding a spanning cluster in percolation models anomalous critical and supercritical phenomena in explosive percolation explosive phenomena in complex networks universal gap scaling in percolation the tipping point: how little things can make a big difference nonlinear dynamics and chaos: with applications to physics ocean circulation and climate during the past 120,000 years slowing down as an early warning signal for abrupt climate change global resilience of tropical forest and savanna to critical transitions arctic climate tipping points observed fingerprint of a weakening atlantic ocean overturning circulation climate tipping points -too risky to bet against catastrophic shifts in ecosystems spatial vegetation patterns and imminent desertification in mediterranean arid ecosystems early warning signals of extinction in deteriorating environments generic indicators for loss of resilience before a tipping point leading to population collapse the sudden collapse of pollinator communities predicting tipping points in mutualistic networks through dimension reduction experimental evidence for tipping points in social convention social tipping dynamics for stabilizing earth's climate by 2050 systemic risk in banking ecosystems multiple tipping points and optimal repairing in interacting networks epileptic seizures may begin hours in advance of clinical onset: a report of five patients prediction of epileptic seizures: are nonlinear methods relevant? self-organized patchiness in asthma as a prelude to catastrophic shifts tipping points in macroeconomic agent-based models early-warning signals for critical transitions early warning of climate tipping points anticipating critical transitions detection of climate system bifurcations by degenerate fingerprinting mosaic organization of dna nucleotides a modified method for detecting incipient bifurcations in a dynamical system generalized early warning signals in multivariate and gridded data with an application to tropical cyclones tipping points in open systems: bifurcation, noise-induced and ratedependent examples in the climate system early warning of climate tipping points from critical slowing down: comparing methods to improve robustness tipping points: early warning and wishful thinking coherence resonance in a noise-driven excitable system on stochastic perturbation and long-term climate behaviour catastrophes and resilience of a zero-dimensional climate system with ice-albedo and greenhouse feedback noise-induced transitions in a barotropic β-plane channel arctic sea ice decline: faster than forecast future abrupt reductions in the summer arctic sea ice does the arctic sea ice have a tipping point? ipcc special report on the ocean and cryosphere in a changing climate collapse of the west antarctic ice sheet after local destabilization of the amundsen basin nonlinear physical oceanography: a dynamical systems approach to the large scale ocean circulation and el niño an introduction to the dynamics of el niño and the southern oscillation the el niño-southern oscillation phenomenon increasing frequency of extreme el niño events due to greenhouse warming el niño/southern oscillation response to global warming is the indian summer monsoon stable against global change? recent unprecedented weakening of indian summer monsoon in warming environment global carbon budget tropical forests in a changing environment determination of tropical deforestation rates and related carbon losses from global patterns of tropical forest fragmentation a universal law of the characteristic return time near thresholds detecting long-range correlations with detrended fluctuation analysis long-range correlation properties of coding and noncoding dna sequences: genbank analysis long-range anticorrelations and non-gaussian behavior of the heartbeat discrimination of the healthy and sick cardiac autonomic nervous system by a new wavelet analysis of heartbeat intervals correlated and uncorrelated regions in heart-rate fluctuations during sleep possible origin of memory in earthquakes: real catalogs and an epidemic-type aftershock sequence model indication of a universal persistence law governing atmospheric variability a novel scaling indicator of early warning signals helps anticipate tropical cyclones the potential role of spectral properties in detecting thresholds in the earth system: application to the thermohaline circulation rising variance: a leading indicator of ecological transition flickering gives early warning signals of a critical transition to a eutrophic lake state leading indicators of trophic cascades changing skewness: an early warning signal of regime shifts in ecosystems turning back from the brink: detecting an impending regime shift in time to avert it methods for detecting early warnings of critical transitions in time series illustrated using simulated ecological data abrupt climate change rainforest collapse triggered carboniferous tetrapod diversification in euramerica was the younger dryas triggered by a flood? global cooling during the eocene-oligocene climate transition abrupt climate change: inevitable surprises rapid oceanic and atmospheric changes during the younger dryas cold period fishing elevates variability in the abundance of exploited species catastrophic regime shifts in ecosystems: linking theory to observation shallow lakes theory revisited: various alternative regimes driven by climate, nutrients, depth and lake size the crash of '87: was it expected? the evidence from options markets a mathematical theory of communication entropic fluctuations in dna sequences black holes and entropy particle creation by black holes entropy and area the entropy law and the economic problem, valuing the earth: economics, ecology on the practical limits to substitution entropy and climate. i: erbe observations of the entropy production of the earth entropy and climate. ii: simple models complexity-based approach for el niño magnitude forecasting before the spring predictability barrier approximate entropy as a measure of system complexity ergodic theory of chaos and strange attractors estimation of the kolmogorov entropy from a chaotic signal irregularity, volatility, risk, and financial market time series physiological time-series analysis: what does regularity quantify? evaluation of eye metrics as a detector of fatigue:, human factors detecting strange attractors in turbulence approximate entropy (apen) as a complexity measure entropy and complexity measures for eeg signal classification of schizophrenic and control participants epileptic eeg classification based on extreme learning machine and nonlinear features decreased cortical complexity in methamphetamine abusers physiological time-series analysis using approximate entropy and sample entropy climate change 2007: the physical science basis: contribution of working group i to the fourth assessment report of the intergovernmental panel on climate change the response of enso flavors to mid-holocene climate: implications for proxy interpretation high-resolution record of northern hemisphere climate extending into the last interglacial period the era-interim reanalysis: configuration and performance of the data assimilation system the ncep/ncar 40-year reanalysis project the jra-25 reanalysis the jra-55 reanalysis: general specifications and basic characteristics hayashi spectra of the northern hemisphere mid-latitude atmospheric variability in the ncep-ncar and ecmwf reanalyses intercomparison of near-surface temperature and precipitation extremes in amip-2 simulations, reanalyses, and observations southern hemisphere midlatitude atmospheric variability of the ncep-ncar and ecmwf reanalyses comparative energetics of era-40, jra-25 and ncep-r2 reanalysis, in the wave number domain examination of the global lorenz energy cycle using merra and ncep-reanalysis 2 the physics of climate variability and climate change north american precipitation and temperature patterns associated with the el niño/southern oscillation (enso) inter-decadal modulation of the impact of enso on australia on the weakening relationship between the indian monsoon and enso interdecadal modulation of enso teleconnections climate change and climate modeling atmospheric teleconnections from the equatorial pacific some simple solutions for heat-induced tropical circulation quantifying the dependence of westerly wind bursts on the large-scale tropical pacific sst a model of tropical ocean-atmosphere interaction a delayed action oscillator for enso a model el niño-southern oscillation experimental forecasts of el niño a review of enso theories a western pacific oscillator paradigm for the el niño-southern oscillation the dynamic response of the equatorial pacific oceanto atmospheric forcing an equatorial ocean recharge paradigm for enso. part i: conceptual model an equatorial ocean recharge paradigm for enso. part ii: a stripped-down coupled model an advective-reflective conceptual model for the oscillatory nature of the enso unified oscillator model for the el niño-southern oscillation predictability of el niño over the past 148 years el niño in a changing climate current approaches to seasonal to interannual climate predictions the 'spring predictability barrier' for enso predictions and its possible mechanism: results from a fully coupled model improved el niño forecasting by cooperativity detection very early warning of next el niño climatelearn: a machine-learning approach for climate prediction using network measures probabilistic forecasting of el niño using neural network models deep learning for multi-year enso forecasts the application of machine learning techniques to improve el niño prediction skill the atmospheric bridge: the influence of enso teleconnections on air-sea interaction over the global oceans el niño modoki and its possible teleconnection contrasting eastern-pacific and central-pacific types of enso a climate network-based index to discriminate different types of el niño and la niña on the impacts of el niño events: a new monitoring approach using complex network analysis statistical analysis of indian rainfall and rice productivity anomalies over the last decades rethinking indian monsoon rainfall prediction in the context of recent global warming skill of medium-range forecasts over the indian monsoon region using different parameterizations of deep convection prediction of indian summer monsoon in short to medium range time scale with high resolution global forecast system (gfs) t574 and t382 prediction of indian summer monsoon onset using dynamical subseasonal forecasts: effects of realistic initialization of the atmosphere onset and withdrawal of indian summer monsoon objective definition of the indian summer monsoon onset comparison of definitions of indian summer monsoon onset: better representation of rapid transitions of atmospheric conditions combining regional moist static energy and enso for forecasting of early and late season indian monsoon rainfall and its extremes an index for predicting the onset of monsoon over kerala the dynamics of double monsoon onsets new statistical models for long-range forecasting of southwest monsoon rainfall over india empirical prediction of the summer monsoon rainfall over india monsoons: processes, predictability, and the prospects for prediction long-lead statistical forecasts of the indian summer monsoon rainfall based on causal precursors seasonal prediction of the indian monsoon evaluation of the ensembles multi-model seasonal forecasts of indian summer monsoon variability indian summer monsoon rainfall simulation and prediction skill in the cfsv2 coupled model: impact of atmospheric horizontal resolution climate models produce skillful predictions of indian summer monsoon rainfall prediction of indian summer monsoon: status, problems and prospects monsoon prediction -why yet another failure? unraveling the mystery of indian monsoon failure during el niño network-based approach and climate change benefits for forecasting the amount of indian monsoon rainfall monsoon and enso: selectively interactive systems on the el niño-indian monsoon predictive relationships machine learning and data mining approaches to climate science: proceedings of the 4th international workshop on climate informatics spatial structures and directionalities in monsoonal precipitation over south asia the trmm multisatellite precipitation analysis (tmpa): quasi-global, multiyear, combined-sensor precipitation estimates at fine scales enhanced mid-latitude tropospheric warming in satellite measurements expansion of the hadley cell under global warming a mechanism for future changes in hadley circulation strength in cmip5 climate change simulations robust responses of the hydrological cycle to global warming the poleward migration of the location of tropical cyclone maximum intensity atmospheric and oceanic fluid dynamics nonlinear axially symmetric circulations in a nearly inviscid atmosphere determining the tropopause height from gridded data has the hadley cell been strengthening in recent decades? improved estimates of global ocean circulation, heat transport and mixing from hydrographic data continuous, array-based estimates of atlantic ocean heat transport at 26.5 • n global and european climate impacts of a slowdown of the amoc in a high resolution gcm hotspot of accelerated sea-level rise on the atlantic coast of north america consequences of rapid ice sheet melting on the sahelian population vulnerability temporal variability of the atlantic meridional overturning circulation at 26.5 • n the community earth system model: a framework for collaborative research the community earth system model version 2 (cesm2) spurious multiple equilibria introduced by convective adjustment multifractal earth topography self-stabilized fractality of seacoasts through damped erosion universality classes of optimal channel networks orogenic andesites and plate tectonics connections between ocean bottom topography and earth's climate anthropogenic forcing dominates global mean sea-level rise since 1970 from the extreme to the mean: acceleration and tipping points of coastal inundation from sea level rise committed sea-level rise under the paris agreement and the legacy of delayed mitigation action noaa technical memorandum nesdis ngdc scaling and multifractal fields in the solid earth and topography statistical features of sea-floor topography testing statistical self-similarity in the topology of river networks broad bandwidth study of the topography of natural rock surfaces the origin of continents and oceans, courier corporation percolation in a fractional brownian motion lattice fractal growth phenomena dynamics of fractal surfaces new empirical relationships among magnitude, rupture length, rupture width, rupture area, and surface displacement frequency of earthquakes in california a review and new insights on the estimation of the b-valueand its uncertainty on the after-shocks of earthquakes detection of precursory relative quiescence before great earthquakes through a statistical model unified scaling law for earthquakes long-term clustering, scaling, and universality in the temporal occurrence of earthquakes universal" distribution of interearthquake times explained interevent time distribution in seismicity: a theoretical approach, pure and applied geophysics origin and nonuniversality of the earthquake interevent time distribution stochastic models for earthquake occurrence foreshocks explained by cascades of triggered seismicity analyzing earthquake clustering features by using stochastic reconstruction detecting fluid signals in seismicity data through statistical earthquake modeling on the increase of background seismicity rate during the sequence: apparent variation or fluid-driven triggering?on the increase of background seismicity rate during the 1997-1998 umbria-marche sequence statistical models for earthquake occurrences and residual analysis for point processes statistical model for standard seismicity and detection of anomalies by residual analysis an introduction to the theory of point processes: volume i: elementary theory and methods, 2nd edition, probability and its applications, an introduction to the theory of point processes space-time point-process models for earthquake occurrences, annals of the institute of statistical mathematics earthquake forecasting during the complex amatrice-norcia seismic sequence next-day earthquake forecasts for the japan region generated by the etas model a spatiotemporal clustering model for the third uniform california earthquake rupture forecast (ucerf3-etas): toward an operational earthquake forecasta spatiotemporal clustering model for the ucerf3-etas seismicity analysis through point-process modeling: a review, pure and applied geophysics long-term memory in earthquakes and the distribution of interoccurrence times memory in the occurrence of earthquakes scaling laws in earthquake memory for interevent times and distances statistical short-term earthquake prediction predicting the 1975 haicheng earthquake from precursors to prediction: a few recent cases from greece implications for prediction and hazard assessment from the 2004 parkfield earthquake deep learning of aftershock patterns following large earthquakes strategies in strong earthquake prediction earthquake likelihood model testing likelihood-based tests for evaluating space-rate-magnitude earthquake forecastslikelihood-based tests for evaluating space-rate-magnitude earthquake forecasts evaluating earthquake predictions and earthquake forecasts: a guide for students and new researchers estimation of the parameters of etas models by simulated annealing the campotosto seismic gap in between the 2009 and 2016-2017 seismic sequences of central italy and the role of inherited lithospheric faults in regional seismotectonic settings improved earthquake forecasting model based on long-term memory in earthquake the structure and dynamics of multilayer networks identifying causal gateways and mediators in complex spatio-temporal systems early prediction of extreme stratospheric polar vortex states based on causal precursors rossby waves detection in the co2 and temperature multilayer climate network dynamics of a snowball earth ocean transitions across melancholia states in a climate model: reconciling the deterministic and stochastic points of view functional shape of the earthquake frequency-magnitude distribution and completeness magnitude earthquake networks as a tool for seismicity investigation: a review, pure and applied geophysics observations and modeling of the elastogravity signals preceding direct seismic waves deep learning machine learning phases of matter, nature physics advance online publication learning phase transitions by confusion unveiling the predictive power of static structure in glassy systems deep learning and process understanding for data-driven earth system science deep learning for predicting the monsoon over the homogeneous regions of india convolutional neural network for earthquake detection and location topology of products similarity network for market forecasting deep learning systems as complex networks social and economic impacts of climate covid-19: consider cytokine storm syndromes and immunosuppression climate-driven variation in mosquito density predicts the spatiotemporal dynamics of dengue climate change and influenza: the likelihood of early and severe influenza seasons following warmer than average winters global environmental drivers of influenza forecasting the spatial transmission of influenza in the united states exposure to air pollution and covid-19 mortality in the united states: a nationwide cross-sectional study, medrxiv appendix a acronyms 92 we would like to acknowledge gratefully all colleagues with whom we maintained interactions and discussions on the topic in our report.in particular, we would like to thank a. agarwala, n. this work was partly supported by the 'east africa peru india climate capacities -epicc' project, which is part of the international climate initiative (iki). the federal ministry for the environment, nature conservation and nuclear safety (bmu) supports this initiative on the basis of a decision adopted by the german bundestag. the potsdam institute for climate impact research (pik) is leading the execution of the project together with its project partners the energy and resources institute (teri) and the deutscher wetterdienst (dwd). s.h. thanks the italian ministry of foreign affairs and international cooperation jointly with the israel ministry of science, technology, and space (most); the israel science foundation, and the eu h2020 project rise for financial support. this appendix contains two tables of acronyms used throughout the paper: table a.2 contains the scientific acronyms and table a. 3 the institutional ones. key: cord-130507-baheh8i5 authors: benreguia, badreddine; moumen, hamouma; merzoug, mohammed amine title: tracking covid-19 by tracking infectious trajectories date: 2020-05-12 journal: nan doi: nan sha: doc_id: 130507 cord_uid: baheh8i5 nowadays, the coronavirus pandemic has and is still causing large numbers of deaths and infected people. although governments all over the world have taken severe measurements to slow down the virus spreading (e.g., travel restrictions, suspending all sportive, social, and economic activities, quarantines, social distancing, etc.), a lot of persons have died and a lot more are still in danger. indeed, a recently conducted study~cite{ref2} has reported that 79% of the confirmed infections in china were caused by undocumented patients who had no symptoms. in the same context, in numerous other countries, since coronavirus takes several days before the emergence of symptoms, it has also been reported that the known number of infections is not representative of the real number of infected people (the actual number is expected to be much higher). that is to say, asymptomatic patients are the main factor behind the large quick spreading of coronavirus and are also the major reason that caused governments to lose control over this critical situation. to contribute to remedying this global pandemic, in this paper, we propose an iot (internet of things) investigation system that was specifically designed to spot both undocumented patients and infectious places. the goal is to help the authorities to disinfect high-contamination sites and confine persons even if they have no apparent symptoms. the proposed system also allows determining all persons who had close contact with infected or suspected patients. consequently, rapid isolation of suspicious cases and more efficient control over any pandemic propagation can be achieved. in december 2019, a novel virus has emerged in wuhan city. this disease, named coronavirus or covid-19, has quickly spread throughout china and then to the entire world. as of may 2020, more than 200 countries and territories have been affected and over 4 million people have been diagnosed with the virus. since no cure or vaccine has been found and since tests cannot be applied on a large scale to millions of persons, governments had and still have no choice but to take severe actions such as border closing, travel canceling, curfews, quarantines, and contact precautions (facemasks, social distancing, and self-isolation). the authorities have also implemented strategies that aim to rapidly detect infections using different cutting-edge medical tools (such as thermal cameras, blood tests, nasal swabs, etc.). on the one hand, these measurements, which for the time being constitute the only possible solution, have succeeded to slow down the contagion spreading, but on the other hand, they have also caused considerable economic damages, especially to countries with brittle economies (suspension of all sorts of activities: social, economic, educational, etc.). the rapid spreading of coronavirus is due to the continuous person-to-person transmission [2, 3, 4] . in addition to this, a recent study has also suggested that a second factor is playing a major causal role in this high virus spreading; namely the stealth transmission [1] . the coronavirus can take 14 days before the appearance of symptoms. during this incubation period, asymptomatic patients, called undocumented patients, can infect large communities of people. in turn, these newly infected persons, who will remain unaware of their illness (until they eventually develop the symptoms), can also infect larger communities, thus, leading to an uncontrollable domino effect [1, 5, 6] . accordingly, to confine and effectively eliminate the coronavirus, it is crucial and mandatory to possess an efficient investigation system that allows determining (1) highly infectious places and (2) all the persons who were in contact with patients who have recently tested positive. indeed, persons who are known to be in direct relationship with a patient (such as family members, friends, and coworkers) can be easily determined and tested. however, numerous other persons could have also been in contact with this infected individual. all these persons, who cannot be easily determined, can contribute to the widespread of the virus. to remedy this issue, in this paper, we propose an iot investigation system that can determine the exact trajectories of infected persons (exact coordinates labeled with time). hence, as shown in figure 1 , the proposed solution allows determining areas of high contamination and also provides a quick detection mechanism of undocumented patients who are known to be the main and most important factor in the rapid widespread of coronavirus. these persons (resp. places) can be tested/confined even if they had no blatant symptoms (resp. closed and properly disinfected). public building 1 public building 2 public building 3 t ra je ct o ry 1 t r a je c t o r y 2 in te rs e ct io n 2 high risk of infection during the presence of the patient and hours after he leaves. uninfected person x intersection 1 t ra je ct o ry 3 uninfected person y figure 1 : infection tracking: example of historical trajectories taken by a covid-19 patient and all persons (resp. places) who he might have encountered/infected (resp. visited). the investigation system presented in this paper is a proposition to governments and lawmakers. so, it needs to be approved and adopted only in the case of highly dangerous pandemics and public health emergencies. in summary, to properly operate, the proposed system must be continuously fed with the coordinates of persons who are in public crowded places (mainly using iot devices that can identify persons and report their locations). although this proposed solution can open large debates from the standpoint of privacy and human rights, during extreme critical situations in which the entire human race might be at stake, the urgency of saving lives becomes of higher priority. in such circumstances, the proposed technique can be quickly applied as a last resort by higher authorities (governments, who 2 , etc.) while giving guarantees about (1) protecting the privacy of people, and (2) disabling this system once the outbreak is contained. the remainder of this paper is organized into five sections as follows. section 2 describes and details the proposed system. section 3 presents a small-scale implementation example of this proposal. section 4 addresses the benefits that the proposed investigation system can bring to the state-of-the-art mathematical disease spreading/prediction models. section 5 discusses the main advantages and shortcomings of the proposed solution. finally, section 6 concludes the paper and provides some recommendations. in the proposed system, a big-data architecture is considered to archive the continuously collected trajectories of persons. this archiving structure, which is inspired by the big data model proposed in [12] , must (as previously mentioned) be fed by iot devices that can (1) determine coordinates of persons during their outdoor activities and (2) send the collected data to the system. we point out that persons staying at home or in their vehicles are assumed to be isolated (i.e., they cannot infect other persons nor they can be infected). consequently, they do not need to activate the coordinates collection process. on the contrary, to ensure their safety by ensuring the proper operation of the system, it is necessary for any person leaving his/her house to activate coordinates collection and save his/her tracks. as figure 2 depicts, the proposed architecture has three main basic layers; namely, data collection, data storage, and data leveraging. in the remainder of this section, we describe each of these layers and provide their specific complementary tasks. as shown in figure 2 , the data gathering process is divided into two parallel independent tasks. whereas the first one is related to collecting the necessary health data, the second is responsible for collecting the geolocalization coordinates of persons. reporting the coronavirus deaths and all the newly confirmed and recovered cases is a basic stepping stone in the overall investigation process. for instance, once a new case has been discovered, the latter must be immediately reported to the automatic investigation system to allow it to find all the individuals who could have been infected by this person. in the same context, given the fact that patients are the main factor in the system, once some of them have recovered from the virus, their statuses must be immediately changed from active to recovered, and so forth. on the contrary to the second task (i.e., geolocalization data gathering) in which data is automatically captured and reported by the employed iot devices, in this first task, data must be manually entered by health authorities. for example, hospitals can be responsible for providing the newly infected cases, and all the other indispensable updates. different approaches can be considered to collect the coordinates of persons who left their houses. for example, (1) relying on telecom and it infrastructures, (2) equipping public buildings with appropriate devices, or (3) utilizing dedicated tracking apparatuses. • telecom/high-tech companies: using telecommunication technologies, smartphones can be easily instructed (programmed) to periodically report their actual location to a dedicated storage system (phone-number, gps-coordinates, time). • public buildings: by installing face recognition cameras on the entries/exits of public buildings, the identity of visitors can be easily determined and then sent to the designated infrastructure (person-id, building-id, entry-time, exit-time). this way, based on the information provided by the different public buildings and facilities, the tracks of infected people can be easily determined. the following lines provide an example of places that were visited by an infected person p1: (p1, building, (entry, t1), (exit, t2)), (p1, shopping mall, (entry, t3), (exit, t4)), (.., .., (.., ..), (.., ..)), (p1, airport a, (entry, tm), (exit to plane, tn)), (p1, airport b, (entry from plane, tx), (exit, ty)), (.., .., (.., ..), (.., ..). the main drawback of this second data collection approach is that it cannot be easily implemented in most countries due to the lack of appropriate underlying identification/recognition systems. however, this solution can be utilized in the case where authorities fail to convince people of using their cellphones as tracking devices. • specific solutions and electronic devices: the two aforementioned approaches can be adopted independently or in combination. actually, during dangerous outbreaks where rapid tracking of infected people becomes an urgency, in addition to personal smartphones and public indoor (entry/exit) cameras, governments can consider using other tracking techniques like for instance electronic bracelets, public outdoor security cameras, drones, or even satellites. furthermore, to consolidate the previous two approaches, solutions such as bluetooth or nfc 3 can also be considered [7, 8, 9, 10, 11] . in the same context, in the event of technical issues related to gps, governments can opt for alternative efficient geo-localization techniques [8, 10] . as their name implies, the iot devices (responsible for collecting person tracks) report the required data using the internet (via wired or mobile wireless networks: 4/5g, ...). so, as shown in figure 2 , to avoid losing important information in the case of internet disconnection, each device must store locally the collected data. once reconnected, these iot devices can then send the gathered data to the system. finally, it is worth mentioning that to ensure the success of the whole virus tracking process, it companies, public institutions, and third-parties must contribute to collecting and reporting the required data. big data, which refers to enormous datasets, has five essential characteristics known as the 5 vs: volume (data size), velocity (data generation frequency), variety (data diversity), veracity (data trustworthiness and quality), and finally value (information or knowledge extracted from data). to address the issues related to storing and processing huge data volumes, extensive research has been done and numerous efficient solutions have been proposed (data ingestion, online stream processing, batch offline processing, distributed file systems, clustering, etc.). these techniques provide both efficient distributed storage and fast processing. currently, research is more focused on big data leveraging (i.e., the last v which consists of turning data into something valuable using data analytics, machine learning, and other techniques) [12, 13] . this point, which represents the main contribution of our work, will be detailed in the next subsection. as stated in [12] , an ideal big data storage/processing infrastructure must adhere to the four following major requirements: (1) time and space efficiency, (2) scalability, (3) robustness against failures and damages, and (4) data security/privacy. for instance, regarding this last point, since the collected data in our context is personal and highly sensitive, strict laws and regulations must be imposed to protect it. the established privacy model must determine who can access data (governments, higher health authorities, who organization, etc.), under what constraints, for how long data can be kept, and to whom this data can be distributed, etc. moreover, in addition to data privacy, all conventional data security mechanisms must also be considered (confidentiality, integrity, and availability). to ensure the proper operation of the system, data that is continuously coming from the various considered iot sources (such as sensors, smartphones, cameras, bracelets, etc.) must provide three main information: person-id, coordinates, and time ( table 1 ). the received trajectories of persons are recorded as an immutable discrete set of points (coordinates) labeled with time. for example, according to table 1 , the recorded traces of person p1 along with their corresponding time instants are: (c1, t1), (c4, t3), (c5, t4), (c2, t5), and (c3, t6). note that in table 1 , for any given person, time is a monotone function that must keep growing (it cannot go back). note also that more than one person can exist in the same coordinates at the same time. for example, p1 with p3 at (c2, t5) and p1 with p5 at (c3, t6). in the considered context, the frequency according to which data must be collected (i.e., the second v) has a direct significant impact on both the first and last vs (i.e., data size and data value). high-quality trajectories can be achieved by increasing the frequency of data collection. this, however, can considerably increase the volume of data. in contrast, low collection frequencies reduce the data size but yield low trajectory quality. so, a tradeoff between the quality of trajectories and the size of data must be defined. the main goal of the proposed system is to track the main sources of infection, whether they would be humans or places. but, in reality, the quality of the collected data can have a deep impact on the system's performance; it can contribute to its efficiency or deficiency. to prevent useless noisy data from harming or affecting the decisions of the investigation process, a filtering mechanism must be added ( figure 3) . if it appears that the filtered data is useless, it can then be safely deleted. for example, data collected from highways and main roads is not of value. in this scenario, users are inside their respective cars and the risk of infection is nil (similarly to houses, people inside the same vehicle are considered as isolated). concretely speaking, the responsibility of data filtering can be left to authorities, which can decide which geographic zones or areas are of interest. in the proposed system, only two operations can be performed on the collected geolocalization data: storage and leveraging (figure 2 ). this section presents our three proposed algorithms, which exploit the gathered data to (1) find and classify suspected cases (i.e., persons who met with confirmed patients or other suspected cases), (2) determine black areas (zones with high contamination probability), and (3) find all persons who visited black areas (also considered as probably infected persons). for the convenience of the presentation, table 2 compiles all the variables utilized by the different proposed algorithms presented respectively in the following three subsections. based on the stored data (both recorded trajectories of people and health information), the main goal is to deduce all possible infections. as previously mentioned, the proposed investigation system does not only find the suspected cases but also categorizes them into several disjoint subsets denoted suspected t,d ( figure 4 and table 2 ). the variable d, which stands for distance or degree, indicates how close/far is a person to a confirmed patient. the initial class of persons suspected t,0 represents the set of all patients (confirmed cases). if set suspected t,1 is defined (suspected t,1 = φ) then this means that it contains all individuals who had direct contact with at least a confirmed patient p c from suspected t,0 (∀ p c ∈ suspected t,0 ). as regards the elements (persons) of suspected t,2 , they had no direct intersection with a positive case from suspected t,0 definition common (pi, ci, ti) a recorded tuple: person identity, coordinates, and the corresponding collection time. set of all trajectories. set of all confirmed patients. a patient is defined as (pi, ti). aux a boolean that indicates whether an end-user is a suspected case. s server side. c client side. algorithm of figure an index to explore all black areas. to find and categorize suspected cases into disjoint subsets, the investigation system s performs the following steps: input: t rajectory; p atient; if (∃(p ersoni, ci, ti) ∈ t rajectory ) and (∃(p ersonj, ci, ti) ∈ t rajectory ) with ((p ersoni, ti) / ∈ suspected t,d−1 ) and ((cd − ti) ≤ ip )) then suspected t,d ← suspected t,d ∪ {(p ersonj, ti)}; (set of confirmed cases), but they have certainly met at least an element from suspected t,1 . in general, for i >= 2, each element of suspected t,i has certainly met at least an element from suspected t,(i−1) , but none from suspected t,(i−2) . to properly operate, the proposed approach must be given the initial set of patients suspected t,0 (suspects at distance 0). as shown by the algorithm of figure 5 , at each iteration, based on the precedently entered/calculated set suspected t,(i−1) , the new set of suspected persons suspected t,i is determined. note that a person is considered to be suspected if he has met a patient or another suspected person during the incubation period (line 4: (cd −t i ) ≤ ip ). the algorithm stops when the previously entered/calculated set to find black zones, the investigation system s performs the following steps: input: t rajectory; a; p atient; if ((∃(p ersoni, * ) ∈ p atient and (∃(p ersonj, c k , ti) ∈ t rajectory ) ) then count k ← count k + 1 suspected t,(i−1) is empty (which means that the next set of suspected persons suspected t,i cannot be calculated). thus, after its execution, the proposed algorithm gives a classification (partition) of all individuals stored in the system: suspected t,0 , suspected t,1 , ..., suspected t,d (suspected t set). the remaining unclassified persons are considered uninfected. they have not met with confirmed patients from suspected t,0 nor with suspected persons from suspected t,i (with i > 0). when a user (client) sends a query to the investigation system (algorithm of figure 6 ), he will receive to which class he belongs (i.e., suspected t,0 , suspected t,1 , suspected t,2 , ... or negative category). using the distance d (rather than the identity of persons) allows informing (reassuring or alerting) users about their current statuses without exposing the privacy of other users of the system. the distance d can be seen as a warning, the more a user's distance is close to zero, the higher the risk of infection will be, and vice versa ( figure 4) . finally, we point out that in the proposed system, geolocalization data is periodically collected every ∆t period. to avoid any synchronization issue that might occur during this process, time differences that are less or equal to ∆t are ignored. for example, if a person (p i , c i , t i ) has the same coordinates as another person (p j , c j , t j ) and the difference between the collection instants (t i and t j ) of these coordinates (c i and c j ) is less or equal to ∆t then ti and tj will be seen as equal (t i = t j ). another major problem that must be tackled is the possibility of virus transmission without direct human contact. this can happen, for instance, when an undocumented patient visits a public place and leaves the virus there (he touches objects found in that area, he sneezes, coughs, etc.). in such a scenario, as long as the virus stays alive, the probability of contamination remains very high. the algorithm of to find all persons who visited black areas, the investigation system s performs the following steps: input: t rajectory; ba; if ∃(p ersoni, * ) ∈ suspectt,sthen aux ← true; k ← s else aux ← f alse endif strates the process followed to find all black areas. the key idea consists of determining if numerous patients have visited the same location. if so, then this zone is likely an infectious area of high viral transmission. more concretely, for each public location a k ∈ a (where a is the predefined set of all public areas), the number of confirmed patients who have visited this area is determined using the count k variable. if this number overtakes the predefined threshold α, the corresponding location a k is then considered to be a black area. as depicted in figure 8 and algorithm of figure 9 , once black areas have been determined, all persons who have been in these infectious zones can be straightforwardly found. more specifically, based on the set of captured trajectories, the proposed algorithm finds each person p i who has visited at least one black area ba k . each of these found suspected people will be added to the corresponding suspect t,k set (list of all suspected persons who have frequented black area k at time t). in other terms, after its execution, the algorithm depicted in figure 9 gives a different classification (partition) of all individuals stored in the system: suspect t,0 , suspect t,1 , ..., suspect t,k (suspect t set). the unclassified persons are considered uninfected (they have not visited any infectious areas ba k ∈ ba). accordingly, the investigation system can be queried about black areas and the people who frequented them. this section describes the small-scale version of our proposed system that we have implemented. the main purpose of this demonstration is to show the applicability of the ideas discussed earlier in the paper. in brief, the implemented system has three essential parts ( figure 10 ): (1) mobile phone application for end-users, (2) automatic investigation mechanism (described in sections 2.2 and 2.3), and (3) an interface dedicated to the government and health authorities. the developed mobile application collects the required geo-localization information by sending the gps longitude and latitude coordinates along with the corresponding time to the investigation system. this operation is repeated periodically every x unit of time. to avoid redundancies (useless data), during this defined period (i.e., x time unit), when a user remains in the same location, his coordinates will not be reported to the system nor stored in the phone local database. similarly, if a user moves for a certain ... distance of y meters or less, he will be considered as stationary and his coordinates will not be collected. also, as previously mentioned, in the event of internet disconnection, each smartphone will store locally the gathered data and wait for reconnection. the application can offer numerous services to the end-users. for instance, users can review their historical trajectories during any possible correct period that they can define ( figure 11) . a second most important service that can also be offered is allowing end-users to know if they have met infected people. the investigation system is continuously fed with both the locations of users and health data (e.g., confirmed patients, closed cases, etc.). figure 12 provides an example of historical trajectories taken by a single user of the system. as previously stated in the paper, the collected locations of persons are stored as immutable append-only data (since there is no need to change the gathered tracks, no updates are allowed). due to the continuous periodic data gathering, an important storing space is needed. however, for demonstration, in this small-scale evaluation version, a single pc was considered to store both health and localization data. numerous functionalities can be offered to the government, health authorities, and other entities that are responsible for monitoring, evaluating, and controlling the outbreak. the most important functionality is the ability to search for novel suspected cases. to do so, the authorities must first provide the investigation system with the necessary new coronavirus cases, deaths, and recovered patients. upon discovering and entering a new infected case, the proposed system can find all people who had contact with this patient ( figure 13 ). as previously explained, the proposed system classifies the newfound suspected people into disjoint subsets. for example, if we assume that the newly discovered patient p i belongs to subset s 0 , then, all persons who had direct contact with him will belong to s 1 . and, all persons who had direct contact with persons from s 1 will belong to s 2 , and so forth. this way, categories of high and low risk can be easily determined. once determined, all suspected cases will be (1) stored in the system and (2) informed to immediately start self-isolation at home. the authorities can also apply several techniques to determine if suspected persons are violating the imposed quarantines. the investigation system must be designed so that (1) it helps the authorities in their endeavor of fighting coronavirus, and (2) it protects the privacy of users. the latter goal can be achieved through the designed gui and offered functionalities, which must be defined based on a well-established privacy model. mathematical modeling of infectious diseases is a powerful tool that allows analyzing, understanding, and predicting the behavior of pandemics [14, 16, 18] . for instance, these models can be utilized to help authorities set up the best strategies for successful pandemics control. in the following three subsections, we will briefly show how different mathematical prediction models can benefit from the proposed investigation system. more exactly, how can the two generated lists/sets (suspected t and suspect t ) of (1) suspected/infected persons and (2) foci of infection (black areas) help attain a more accurate realistic estimate of disease spreading rate. more recently, a new mathematical model, called θ-seihrd 4 , has been specifically proposed for the coronavirus disease [15] . this model assumes that the pandemic spatial distribution inside a territory is omitted. θ-seihrd also assumes that people in a territory are characterized to be in one of the following nine compartments: s (susceptible), e (exposed), i ( infectious), iu (infectious but undetected), hr (hospitalized or in quarantine at home), hd (hospitalized that will die), rd (recovered after being previously detected as infectious), ru (recovered after being previously infectious but undetected) or finally d (dead by . the most important parameters of this model that determine the pandemic spreading are: β hd . these parameters represent the disease contact rates (day −1 ) of a person in both the corresponding compartments (e, i, iu, ...) and territory (i) (without taking into account the control measurements). indeed, it is very hard to determine these contacts rate without having an idea about who contacted whom. also, the contacts between persons are not sufficient to estimate the coronavirus contact rates. this disease can be transmitted without direct contact with patients (e.g., touching an object previously used by an infected person). the determination of both sets suspected t and suspect t helps to accurately estimate the contacts rate and to determine the set of infectious but undetected (iu) persons. in addition to this, our proposed investigation system can also help to determine the pandemic spatial distribution inside any monitored territory. as it is well-known, it is very important to understand diseases spreading, determine the safe and risky areas, and take control measurements with insight. sir 5 is one of the most studied mathematical models for the spreading of infectious diseases [16, 17, 18] . in this model, the most important parameters that determine the pandemic spreading are α, γ, and β. where α is the probability of becoming infected, γ is the number of infected people (new infections are the result of contact between infectives and susceptibles), and β is the average number of transmissions from an infected person (determined by the chance of contact and the probability of disease transmission). the major difficulty or drawback of this model is estimating these parameters. the answer to this question is our proposed investigation system. seir 6 is also one of the most important mathematical models for the spreading of infectious diseases [19] . in this model, the main parameter that determines the pandemic spreading is the contact rate β(t), which is the average number of susceptibles in a given population contacted per infective per unit time. the main difference between seir and sir lies in the addition of a latency period. more details can be found in [19, 20, 21, 22] . endowing the seir model with accurate contact information can help it obtain better results about the spreading (prediction) of infectious diseases. as previously highlighted, this is exactly where our proposed solution comes in handy. it can help to understand and estimate important parameters for numerous mathematical models proposed for infectious diseases. in this section, we provide the main advantages and disadvantages of the proposed investigation system. first, among the numerous interesting features of this iot system we mainly cite: • this system allows tracking the trajectories of infected persons, and this, days before the appearance of their symptoms. the system also allows determining all persons who were in close contact with infected patients. thus, the former can be immediately confined and the proper measurements can be rapidly taken by health authorities. since all persons who have met infected patients can be identified earlier (i.e., parents, friends, colleagues, and most importantly, those who cannot be easily identified using conventional techniques), the proposed tracking system allows an early outbreak control. • the proposed system can be utilized to identify black zones, which can be the main source of virusspreading. as previously explained, a given location is said to be a source of contamination if numerous infected persons have visited it (the intersection zones of high infections can be identified based on the trajectories of infected persons). • according to [2] , the coronavirus can live for several hours without a host. to remedy this, the proposed system can be configured to find all uninfected persons who have visited locations that were previously visited by other infected persons (and this, hours after the patients have left this location). • the proposed system can help reduce the economic damages generated by the suspension of all activities. instead of shutting down all sports, social and economic activities, the authorities can impose confinement on only a few people. • in addition to coronavirus, the proposed solution can be utilized for any potential outbreak which might be more powerful and have a higher spreading pace. despite its advantages, the proposed solution suffers from some issues that must be adequately tackled: • for instance, if certain persons do not respect the safety instructions (e.g., they did not intentionally or unintentionally save their trajectories when leaving their houses or cars), it will be impossible to determine whether they have met infected persons. the absence of one or several trajectories does not mean the total failure of the proposed investigation system. indeed, the efficiency of this system is proportional to its users. if x% of the population has been involved, then approximately x% of the persons who had close contact with patients will be determined. • the proposed system can be empowered with extra functionalities such as machine (deep) learning abilities. moreover, the system can be utilized to determine whether citizens are respecting social distancing and also to check whether the persons who are suspected to be infected are respecting the imposed confinement. in this work, we proposed a system that can quickly determine all persons who are suspected to be infected by the coronavirus. while being specific to this virus, the proposed solution can also be applied to control other pandemics. the objective (as specified in the paper) is not tracking people but tracking extremely dangerous viruses. to ensure a practical and more efficient application of this proposal, the following points must be taken into account: • system utilization: to keep the public health situation under control, it is recommended to launch such an automatic investigation process as early as dangerous rapid infections start taking place. in the case where the outbreak achieves large scales, the operation of detecting, tracking, and surveilling a huge number of infected people might become useless. • system efficiency: for a more efficient system, we recommend exploiting all possible existing resources that can enhance the quality of the collected data (cellphones, security camera footage, credit card records, etc.). • data privacy: finally, to ensure the privacy and security of the collected data, which is personal and highly sensitive, an authority of trust must manage this whole process. substantial undocumented infection facilitates the rapid dissemination of novel coronavirus (sars-cov2) clinical characteristics and imaging manifestations of the 2019 novel coronavirus disease (covid-19):a multi-center study in wenzhou city first known person-to-person transmission of severe acute respiratory syndrome coronavirus 2 (sars-cov-2) in the usa patients with respiratory symptoms are at greater risk of covid-19 transmission transmission of covid-19 in the terminal stage of incubation period: a familial cluster stealth transmission' fuels fast spread of coronavirus outbreak decentralized human trajectories tracking using hodge decomposition in sensor networks cts: a cellular-based trajectory tracking system with gps-level accuracy smar-tits: smartphone-based identification and tracking using seamless indoor-outdoor localization hermes: pedestrian real-time offline positioning and moving trajectory tracking system based on mems sensors urska demsar & a. stewart fotheringham analysis of human mobility patterns from gps trajectories and contextual information a big data architecture for automotive applications: psa group deployment experience big automotive data: leveraging large volumes of data for knowledge-driven product development the mathematics of infectious diseases mathematical modeling of the spread of the coronavirus disease 2019 (covid-19) taking into account the undetected infections. the case of china. communications in nonlinear science and numerical simulation a contribution to the mathematical theory of epidemics mathematical modeling in infectious disease, clinical microbiology and infection ł2013 european society of clinical microbiology and infectious diseases the sir model of infectious diseases seasonality and period-doubling bifurcations in an epidemic model a seir model for control of infectious diseases with constraints optimal control of a seir model with mixed constraints and l1 cost mathematical modeling of biological processes key: cord-324944-ixh3ykrc authors: mitsakakis, konstantinos; d'acremont, valérie; hin, sebastian; von stetten, felix; zengerle, roland title: diagnostic tools for tackling febrile illness and enhancing patient management date: 2018-12-05 journal: microelectron eng doi: 10.1016/j.mee.2018.10.001 sha: doc_id: 324944 cord_uid: ixh3ykrc most patients with acute infectious diseases develop fever, which is frequently a reason to visit health facilities in resource-limited settings. the symptomatic overlap between febrile diseases impedes their diagnosis on clinical grounds. therefore, the world health organization promotes an integrated management of febrile illness. along this line, we present an overview of endemic and epidemic etiologies of fever and state-of-the-art diagnostic tools used in the field. it becomes evident that there is an urgent need for the development of novel technologies to fulfill end-users' requirements. this need can be met with point-of-care and near-patient diagnostic platforms, as well as e-health clinical algorithms, which co-assess test results with key clinical elements and biosensors, assisting clinicians in patient triage and management, thus enhancing disease surveillance and outbreak alerts. this review gives an overview of diagnostic technologies featuring a platform based approach: (i) assay (nucleic acid amplification technologies are examined); (ii) cartridge (microfluidic technologies are presented); (iii) instrument (various detection technologies are discussed); and at the end proposes a way that such technologies can be interfaced with electronic clinical decision-making algorithms towards a broad and complete diagnostic ecosystem. people in low-and middle-income countries (lmics), primarily in sub-saharan africa, south-east asia, and latin america, suffer dramatically from infectious diseases, some of which are endemic, while others occur in the form of epidemics. a combination of factors leads to the perpetuation of this situation, including the tropical climate, the simultaneous presentation of multiple diseases, innate or acquired host factors (micronutrient deficiency, immunodeficiency, co-morbidities), lack of resources (low income) to procure medicines and vaccines or other preventive tools, lack/misuse (due to lack of training or information) of (suitable) diagnostic tools, increase of pathogen resistance to antimicrobial medicines, as well as social and community factors. as a result, in 2010, 64% of deaths worldwide were still due to infectious diseases; pneumonia, malaria, and sepsis were the leading causes of death, particularly in africa [1] . moreover, while the borders between tropical diseases -thought to be confined around the equator (such as ebola) -and infectious diseases classically related to northern countries (such as influenza) were discrete, climate change and the increase in population migration contributed to a blurring of these borders, thus making the spread of febrile illnesses a global reality and concern. many people with acute infectious diseases develop fever, which is a frequent complaint, observed in approximately 80% of pediatric and adult patients presenting with an acute medical condition to health facilities. the estimated incidence of febrile episodes in malaria endemic countries varies according to studies but is generally between 2 and 7 episodes per person per year in children < 5 years [2] . despite the fact that all these infectious diseases cause fever, they can result from very different types of pathogens -parasites, viruses, bacteriaeach of which requires a different type of treatment and management. malaria, which was a major cause of fever a few decades ago, has now https://doi.org/10.1016/j.mee.2018. 10 .001 given the still high (and often increasing) numbers of febrile (and related) illness cases and accompanied mortality globally, the (re-) emergence of global threats (e.g. ebola in 2014) and the increasing resistance to antimicrobial medicines, there have been several conventions at the international level with the aim to define a roadmap to tackle these challenges. the most significant conclusions and decisions deriving from summit meetings are summarized below. the world health organization (who) has changed its policy regarding the case management of malaria, to shift from presumptive treatment to laboratory-based confirmation of malaria before treatment [8] . thus, wide access to malaria diagnosis is advocated to protect available antimalarial medicines against the development of resistance. according to the who 2014 global report on surveillance and antimicrobial resistance, the emergence of drug resistance due to misuse of antimicrobials is one of the major current public health threats worldwide. new tools that could mitigate this threat are urgently required [9] . in a summary of the lessons learned from the 2014 ebola epidemic, bill gates mentioned that: "we need to invest in better disease-surveillance and laboratory-testing capacity, for normal situations and for epidemics. routine surveillance systems should…detect early signs of an outbreak beyond their sentinel sites and be quickly scaled up during epidemics. they should be linked with national public health laboratories to enable robust monitoring and response." [10] . in an ebola-focusing summit that was convened by the paul allen foundation in april 2015, one of the conclusions stated that "the greatest unmet need…is a field-test for use in community or village settings… to help determine whether isolation and/or referral is indicated." [11] . during an expert meeting at who in june 2015 focusing on interoperability standards, it was concluded that there is a clear need for interface systems between diagnostic tools and e-health technologies, aiming at both patient management and epidemics control [12] . these conclusions and directives coming from multiple sites strongly demonstrate the need and the motivation that exists to support the development of new, innovative diagnostic tools that can efficiently resolve the aforementioned challenges related to febrile illnesses. the first step to improve the management of patients with febrile illness is to estimate the distribution of the various syndromes and pathogens that cause fever. even though the distribution of fevercausing diseases varies by geography, season, age and immunity of patients and the level of care, some findings were similar among studies [2] . these were related to cosmopolitan diseases found in most places worldwide, such as pneumonia or urinary tract infection. for other diseases, with various levels of endemicity, such as typhoid or rickettsiosis, or that occur rather in an epidemic way, such as arbovirus infections, the prevalence varied significantly according to the geographic location and, even more, according to the season or the calendar year. in studies that have recorded the clinical presentation of patients (and not only their laboratory results), the causes of fever in outpatients could be classified into four main syndromes: 1) acute respiratory infections (ari, of any type); 2) diarrhea (gastroenteritis); 3) fever with another clear focus (e.g. meningitis or skin infection); and 4) non-specific fevers [13] (each diagnostic platform described in section 5 focuses on at least one of the aforementioned cases). even though important diagnostic challenges remain regarding the first three syndromes, clear clinical guidelines have been developed by who, such as the integrated management of childhood illness -imci [14] , to help clinicians to assess their patients and to decide on their treatment. because they correspond to localized infections, these syndromes are generally diagnosed using the best available clinical predictors, while diagnostic tools are indeed not included. in contrast, regarding the 4th syndrome (non-specific fevers), very few guidelines are available. the main challenge faced by clinicians is the management of patients with fever without specific symptoms or signs to guide them to the source of infection, combined, in endemic areas, with a negative malaria test result (the "negative syndrome" as labeled by health workers in tanzania). these non-specific fevers, also called "fever without focus", are caused by a wide range of pathogens ( fig. 1 and table 1 ) that cannot be identified without accurate laboratory tests. recent studies that aimed at determining the etiologies of fever [13, [15] [16] [17] [18] have identified the following important diseases as causes of non-specific fever: (i) malaria continues to be a major global health problem. according to the 2016 world malaria report, 91 countries were still malaria endemic [19] . there were an estimated 212 million cases of malaria worldwide in 2016 and an estimated 429,000 deaths, while 90% of all malaria deaths occurred in africa. malaria prevalence varies widely, from 0 to 32% of all fevers. indeed, the decrease in malaria transmission in many places in africa [20, 21] has led to significant heterogeneity in transmission. the prevalence of malaria among patients with febrile illness has, therefore, become unpredictable and has triggered the switch of the who malaria case management policy from presumptive treatment, to treatment based on laboratory confirmation [8] . (ii) urinary tract infection was an important cause of fever in young children (generally younger than two years old) that cannot be suspected on the basis of a history of urinary symptoms at that age (and therefore, it is included in the category of non-specific fevers). the prevalence among all children under five with febrile illness was 1-6%. (iii) enteric fever was a significant cause of fever in children older than two years old and in adults, accounting for 2-10% of all fevers, with salmonella typhi being the most frequent pathogen identified, followed by salmonella non-typhi. (iv) other bloodstream infections, mainly due to streptococcus pneumoniae and escherichia coli, accounted for about 2% of all fevers. other systemic bacterial diseases, such as leptospirosis (0.2-13%), rickettsiosis (1-25%), q fever (1-8%), and brucellosis (5%) were also important causes of fever, especially in adults who were more exposed to zoonoses and other environmental-related diseases than children. (v) dengue was also an important cause and seemed to emerge in several african countries. its prevalence is extremely variable, not so much geographically but temporally (seasonal and year to year variation). (vi) cosmopolitan viruses, such as human herpesvirus 6, parvovirus b19, epstein-barr virus, and cytomegalovirus, which are transmitted mainly through close contact within family members, were mostly prevalent in children, accounting for 9% of all fevers and 22% of non-specific fevers in children [13] , while adults were more susceptible to vector-borne diseases such as chikungunya or west nile virus. acute hiv was found in a few patients. (vii) co-infection with two or even more pathogens was frequent (in one study, 23% of pediatric patients had more than one disease simultaneously, fig. 2 ), especially when an outbreak overlapped with an endemic situation, as it happened in a fever study in tanzania in 2013. suddenly, in january 2014, a significant outbreak of dengue started, and at the peak of the epidemic, almost 80% of patients presenting with fever were infected with dengue virus of serotype 2 [6] . because of the absence of diagnostic tools outside the study sites, most of these patients were considered as malaria cases and treated with antimalarial medicines or, in case that malaria tests where available, they were considered as possibly infected by bacteria and treated with antibiotics. (viii) regarding hiv, chronic hiv is an important co-morbidity, especially for adults (40% were positive in two tanzanian studies, while hiv prevalence in the corresponding community was 5 to 10%), and should, thus, be systematically screened for, which is unfortunately not the case presently in africa. whatever the etiology, the first step in assessing febrile patients is to triage them as having a severe rather than a mild illness [22] . objective point-of-care (poc) biosensors (such as oximeters), in addition to the subjective assessment of clinical danger signs (such as lethargy or severe dehydration), are helpful to correctly triage patients and should be integrated in the evaluation process [23] . when patients present with a mild disease, ambulatory management with appropriate home-based treatment is sufficient [24, 25] . the main challenge is then to identify the few patients who will benefit from antimicrobial treatment among all those with self-limited infection. presumptive treatment with antimicrobials should be avoided as most of these infections are of viral origin (fig. 2) , especially in children (except for malaria that can be ruled out by a rapid diagnostic test). follow-up of patients to detect persistent or new symptoms and signs will then allow for the detection of rare clinical failures. at the primary care level, the decision to administer an antimicrobial should be based on an initial classification of the condition in the four categories mentioned in section 2.2, combined with an accurate test specific for each syndrome that can distinguish between bacterial and viral etiology. as the existing host biomarkers lack accuracy (for example, c-reactive protein to identify the few bacterial pneumonia cases among the many viral respiratory infections), they should be combined with clinical predictors (e.g. fast breathing [26] ) to improve the overall specificity [23] . patients with a rather severe febrile illness (most of them satisfy the criteria for sepsis) should be admitted and assessed for the different etiologies of fever, irrespective of the syndrome they present with. in sepsis, the symptoms and signs are indeed often unspecific to allow for initial classification into the four syndromes mentioned in section 2.2 and all possibilities should be considered [15] . under these conditions, accurate laboratory diagnosis is important, and the infections listed in section 2.2 (taking into account specific exposures mentioned by the patients and the local epidemiology) should be tested for, whenever possible. the results of the laboratory tests should then still be interpreted in the clinical context, i.e. the pre-test probability of the disease and the likelihood ratios of clinical predictors. indeed, for laboratory tests that have imperfect accuracy, these elements should be considered to calculate the post-test probability and be able to safely include or exclude certain diseases [27] . if this probability is above the "treatment threshold" (above which one treats the patients instead of continuing investigating them using tests), the appropriate antimicrobial should be administered [28] . if the probability is below the diagnostic threshold, no specific treatment is required, besides adjunctive treatment (e.g. i.e. acute respiratory infections, gastroenteritis, skin infections, meningitis and naso-pharyngeal infections (infections due to enterovirus, adenovirus or influenza, however presenting without any respiratory symptom). source: figure provided by v. d'acremont, adapted from [13] . classification of fever-causing pathogens in africa (parasitic, bacterial, viral, fungal systemic infections). "na": not applicable; "?": unknown; "naats": nucleic acid amplification technologies; "lft": lateral flow test. diseases not included because generally considered after more than one week of fever: sinusitis, visceral leishmaniasis, amoebic liver abscess. diseases not included because, although they are fairly prevalent, they rarely present with fever: pertussis, cryptosporidium. diseases not included because clinical diagnosis is generally sensitive enough and specific enough: measles. a medium sensitivity in hiv patients with low cd4 cell count (urinary test). k. mitsakakis et al. microelectronic engineering 201 (2018) intravenous rehydration in patients with severe diarrhea) and treatment for chronic conditions. if the probability falls between these two thresholds, further testing (when available) may be required and a second-line test should then be used. in practice, antimicrobials are often administered presumptively while waiting for the test results (except for malaria that can be excluded in < 15 min based on the results of a rapid test). in summary, evidence-based integrated management of fever is a complex process, both at hospital and primary care levels, that would clearly benefit from electronic clinical decision algorithms (ecda) based on mobile technology [23, 29, 30] to guide clinicians through the necessary steps, reach an accurate evaluation of the disease probability, and decide on the most appropriate treatment and management. electronic clinical decision algorithms are e-health tools pertaining to the broader category of "computerized decision support systems" (cdss). contrary to differential diagnosis generators (ddx) that provide a long list of possible diagnoses and have shown poor accuracy [31] , ecda provide structured decision pathways that align with the clinician's workflow and guide the clinician throughout the consultation, including on final treatments and advice (section 6). the diagnostic tools target in-and outpatients that visit urban and rural hospitals, peripheral health facilities and laboratories, and community level health posts. in particular, the following broad categories of target groups are identified: (i) patients living in resource-limited countries are clearly in need of new diagnostic tests for infectious diseases. these patients are very diverse in their immune status, the chronic conditions they may suffer from, and their ability and willingness to pay for a diagnostic test. (ii) people traveling from endemic to non-endemic areas. although it is difficult to determine the number of returning travelers and migrants to northern countries that are infected with an endemic disease, because of variation in reporting methods for different pathogens, an estimated 20,000-30,000 cases of travelers with malaria are, for example, reported in europe each year [32] . the number of people crossing borders between the southern countries is much higher and represents also an important target group. they can be tested at airports, harbors, cross-border controls, immigration control checkpoints, where reliability and short time-to-result tests are required. (iii) pre-and post-vaccination, elimination, and screening programs run by local governments, international organizations, and pharmaceutical industries. disease prevention and elimination programs should be supported by comprehensive evidence to demonstrate the underlying prevalence of the pathogen in question and the efficacy of the strategy proposed. using a multiplexingcapable diagnostic platform allows for effective modeling of the: (a) true prevalence and variation of multiple species, enabling the monitoring of impact of the intervention on disease dynamics; (b) local and regional differences in pathogen distribution. (iv) epidemics surveillance, particularly of interest in: (a) sentinel sites, where the diagnostic tools can be installed at strategic geographic locations, in order to cover several regional, cultural, environmental, and epidemiological settings; (b) ministries of health and regional alarm centers that should regularly receive reports from the sentinel sites for assessment of the frequency of patients and the diversity of diseases. the diagnostic systems should operate independently of patients' age, although there are some inherent difficulties to acquire proper quality sample matrix from an infant (e.g. sputum for alleged respiratory infection). furthermore, for some tests, such as blood culture or polymerase chain reaction (pcr) for bloodstream bacterial infections, the same diagnostic tool may have different performance in different age groups, due to different pathogen load [33] . not all diagnostic tools are, thus, suitable for all cases. in addition, the training level of the users, the facilities available at each level of care, and the target patient population are parameters that developers of novel diagnostic tools must take into consideration. there is an extensive debate regarding whether diagnostic tests with short time-to-result, but applicable only in laboratories, are true poc technologies or if the term "poc" should be used for tests that can be performed "under the tree". considering that diagnosis should be closely associated with treatment and that, at the peripheral level, there is often shortage of health staff and medicines, the "point-of-care" becomes by default the "point" where "proper care" can be provided, and this may well be a clinic, a peripheral lab, or a hospital, depending on how well-equipped these settings are, especially in terms of human resources [34] . the diversity of available diagnostic systems, but more importantly of target groups and local requirements, has posed the need to define generic requirements. to this scope, the "assured" criteria were developed [35] to summarize the characteristics of the ideal diagnostic test for the developing world, which should be affordable by the target groups; sensitive and specific; user-friendly by limited trained personnel; rapid and robust; equipment-free; and delivered to those in need. these criteria were initially developed for sexually transmitted infections but are generic for developing countries-targeted diagnostics. however, it has been evident that the users' requirements call for elaboration (or "fine-tuning") of these criteria. for example, the "equipment-free" criterion may be risky, because saving the test results in the device decreases the risk of mis-interpretation by inexperienced personnel. therefore, there is an increasing trend to use mobile technology to read results provided by lateral flow tests (lfts). moreover, there is need for guidance on how to select patients who should be tested, interpret test results in the clinical context, and triage patients who should be referred and admitted due to severe illness. in case of epidemics, where several patients arrive simultaneously at the checkpoint, the capacity of the tool to process several samples at the same time is an advantage. the assured criteria, together with additional specific requirements, are summarized in target product profiles (tpps), which technology developers should meet during their development stages. examples of tpps exist for tuberculosis, ebola, and differentiation between viral and bacterial infections, and are driven by the who, the médecins sans frontières (msf), the foundation for innovative new diagnostics (find), and other organizations [36, 37] . microscopy for screening of malaria parasites by examining a drop of blood smeared on a slide after proper staining remains a widely used diagnostic tool. giemsa-stained thick and thin blood films are used to detect the presence of plasmodium parasites, to quantify the parasite density, and to identify the species [38] . considering the progress in light technology and the use of light emitting diodes (leds), microscopy has become more easy to use. however, it still requires well-trained personnel and a degree of experience to prepare the samples and to interpret the results. poor slide/ blood film preparation may lead to artifacts which are mistaken for parasites, while in reality they are bacteria, fungi, dirt, or cell debris. as a consequence, poor specificity leads to poor diagnostic quality [39] . two studies in tanzania have shown that the low quality of malaria microscopy and the lack of trust by clinicians in microscopy outcomes resulted in higher mortality among patients with non-malaria febrile illnesses than patients with malaria [40, 41] . an additional major disadvantage of this method is the fact that microscopy cannot identify sources of fever other than malaria (except for borreliosis, which is a relatively rare cause of acute fever). thus, in the case of malaria-negative diagnosis, or in the case of co-infection of malaria with some viral/bacterial agent, the microscopy-only diagnosis is not sufficient for clinicians to decide on the suitable treatment. for bloodstream bacterial infections, including typhoid fever, the main test used in equipped laboratories is conventional bacterial culture. for pneumonia, culture is also based on respiratory secretions, including sputum (but it is then impossible to distinguish between chronic carriage and infection), bronchoalveolar lavage, or pleural fluid. detection of pneumococcal bacteremia can orient the diagnosis; however, this test results in low yields, due to the relative fragility of these bacteria. indeed, the main general drawback of blood culture is the inability, except for a few cases, to diagnose localized infections such as pneumonia or urinary tract infection. for typhoid, in addition to blood culture, stool culture and/or bone marrow culture are used but are rarely available. other limitations are that cultures take several days to provide results and that collection of high quality samples from pediatric patients is rather difficult and often leads to false results [42, 43] . in adults, sensitivity of blood culture is limited by the low bacterial load. specificity of blood culture is also hampered by contamination by cutaneous bacteria during blood withdrawal, because sample acquisition from two different sites to overcome this problem is rather challenging in practice, especially in children. lateral flow tests [44] consist of a plastic device containing a composite assay strip flanked by a reagent/sample pad at one end and an absorption pad at the other. the strip contains two or more lines striped into the nitrocellulose membrane: one or more test line(s) consist of a disease-specific antigen or antibody and one line is used as a control. thus, both antigen-or antibody-specific tests can be performed. the reagent pad holds the dried conjugate consisting of colloidal goldlabeled antibodies or antigens specific for each test. the reaction is performed by adding the sample (typically finger-prick whole blood, or urine) to the sample pad, followed by the addition of a few drops of buffer. the sample is mixed with the conjugate, forming antibody-gold complexes that flow through the nitrocellulose membrane. when specific antibodies/antigens are present in the sample, they bind the respective conjugate at the test zone, indicating a positive result through the captured red-colored gold nanoparticles carrying the antibody/antigen of interest (fig. 3) . multiplexing is possible with the lfts in two ways: either via multiple analytes on the same strip (one sample inlet for all), or via several strips on the same cassette (as many inlets as the number of strips). the former option is more challenging and less generic because it depends on the assays and the (compatibility of) reagents with all target proteins to be detected. the results are read visually after 10-20 min, depending on the test. a valid test result is obtained if staining of the control line is observed. the equipment-free readout, however, is not sufficiently subjective, while it becomes even more complicated when (semi)quantitative readout is desired, e.g. to assess the intensity of the strip line. in addition to the mis-interpretation and ambiguity of the test results, there is also the risk of data loss, which is not the case when a readout equipment (with data storage and transmission capacity) is used. especially in the case of multi-strip cassettes, the use of a readout unit may help to diminish the risk of confusion between results from different lines. thus, a current trend in lft diagnostics is towards an automated readout, such as the deki-reader that has been well validated in the field for malaria testing [46] . several technologies have been developed based on colorimetric (e.g. colloidal gold or colored latex) or fluorescence detection, by commercial suppliers: esequant™ lft strip reader by qiagen lake constance (germany); an imaging system by axxin inc. (australia); the handheld image capture lft reader by detekt biomedical, lcc (usa); the cpoc™ reader from lre medical (germany); the handheld dcn fluorescent test visualizer (usa) [47] . although these systems are well suited for developed countries, they are often not affordable for lmics. a more promising approach is the use of smartphones, which has several advantages over other systems to: (i) become increasingly accessible in africa; (ii) be used as an image processing device with the built-in camera and suitable software; (iii) be able to support an application (app) for processing the acquired data; and (iv) enable global positioning system (gps) tracking of patients during an epidemic (following the appropriate ethics rules and after related permissions). furthermore, data storage and transmission to external databases offers a high added value to lfts. the camera can also typically scan 1-or 2-d barcodes on the lft cassette in order to identify the type of test. to this direction, the non-profit global health organization "global solutions for infectious diseases (gsid)" [48] is developing a mobile tool to read, digitize, and transmit poc diagnostic results for important infectious diseases. in cooperation with the university of washington (seattle, usa) and dimagi (boston, usa), gsid developed an open-source software to capture the results of many different poc tests and report them immediately via wireless technology. a proof-of-concept study was conducted in zimbabwe in 2014 [49] . other examples of this approach are the mreader of mobile assay (usa), the assayquest™ from clearbridge bioloc (singapore), and the skansmart and skaneasy products from skannex (norway). extensive work on this field has been performed regarding software development, for example by novarum dx™ (uk), or the commcare mobile platform by dimagi (usa). due to the relatively low complexity in manufacturing lfts, there are > 100 manufacturers worldwide for clinical applications (which is why we are not able to provide a list of these manufacturers). the average price of an lft is approximately us$1. several studies assessed the performance of lfts [50] [51] [52] [53] and in a non-exhaustive list of infections, lfts currently exist for malaria, dengue, typhoid, hiv, hepatitis b virus (hbv), hepatitis c virus (hcv), and recently ebola and lassa [47] . however, the average quality of these tests is inversely proportional to the multitude of suppliers. it is often the case that their accuracy is inadequate [54] . the main drawback of lfts that detect antibodies is that, at the acute phase, antibodies are often not yet present and the tests lack sensitivity. also, the dynamics of igm/igg can be complex and hard to interpret, especially when re-infection is possible (e.g. dengue). another problem is that antibodies -even igm -persist for several weeks after acute infection, which decreases the specificity of the test (e.g. chikungunya). for this reason, the who has a standard operating procedure for pre-qualification, and publishes and regularly reviews recommendations for lfts for particular diseases, especially malaria and hiv [54, 55] . nucleic acid amplification technologies (naats) are based on sequence-specific recognition and amplification of unique target regions in the genome of pathogens to be detected. thus, naats are highly specific and sensitive, as single microorganisms can be detected. compared to microscopy (the reference in malaria diagnosis), naats are much more sensitive, while in contrast to culture methods (the reference in bacterial disease diagnosis), naat process times can be reduced from days to hours. the following sections present the most frequently used naats. polymerase chain reaction (pcr) represents the most commonly used naat-based diagnostic technology [56] . in this reaction, a thermostable polymerase (taq, from thermus aquaticus) is employed. two primers are used that flank the target region of the template dna to be amplified. a three-step process is then repeated~30-45 times: (i) a denaturation step at~95°c, where the double-stranded dna (dsdna) template is denatured resulting in two single-stranded dna (ssdna) molecules; (ii) a primer annealing step at~68°c, where the primers are hybridized to the ssdna target region to be amplified; and (iii) an elongation step at~72°c, where the polymerase generates a copy (amplicon) of the target region starting from the primer in 3′-to 5′-end direction. in an ideal reaction, the targeted region of the template dna is doubled after each cycle. the (endpoint) amplicons are detected via gel-electrophoresis (non-specific method) or by using different kinds of specific molecular probes that generate an amplicon-specific signal. here, the signal also depends on the template concentration. the need for cyclic heating and cooling to perform the previously described steps for pcr renders this method relatively time-consuming (several hours depending on the configuration and the thermocycler) and energy-consuming (a power supply is required). recently advanced methods in dna amplification use isothermal naats (inaats) [57, 58] , which overcome the need for thermal cycling. these methods are less energy consuming than pcr and can be potentially executed in battery or solar panel operated devices, which is an important advantage for their application at the point of care. furthermore, as a consequence of continuous amplification rather than cycling, the reaction will often generate detectable products faster than pcr. a disadvantage of inaats is in general (not referring to specific methods) that quantitation is difficult as they do not follow a strictly controllable reaction scheme. in all methods mentioned above and in the next sections, rna can also be amplified by the addition of a reverse transcriptase (rt), which transcribes rna into complementary dna (cdna) for further amplification. in the following sections some of the main isothermal amplification technologies are briefly described. nucleic acid sequence based amplification (nasba) [59] is used to detect rna and is performed at 41°c. due to the fact that it targets rna, nasba was first used for the rapid diagnosis and quantification of hiv-1 [60] . nasba reaction is facilitated by three enzymes (avian myeloblastosis virus reverse transcriptase, rnase h, and t7 rna polymerase) leading to the amplification product. in brief, during the amplification process, cdna is initially produced from rna templates through primer (primer 1) annealing to the template rna and reverse transcription forming a cdna from the latter. during cdna production, rnase h hydrolyzes the rna template. the resulting cdna with the promoter sequence is annealed to a second primer (primer 2) and double-stranded dna (dsdna) is produced with reverse transcription. following this step, t7 rna polymerase transcribes an rna molecule from the previously generated dsdna. the above procedure is cyclic and in each round the number of produced rna is increased. a unique advantage of this method is that it can selectively amplify rna in the presence of high dna background, while the process can also start with a dna template. in this case, the rnase h step in the non-cyclic phase is not necessary. with nasba, amplification of a nucleic acid sequence to > 10 9 copies can be accomplished in~90 min. loop-mediated isothermal amplification (lamp) was developed by eiken chemical [61] and is based on a bst polymerase with strand displacement activity. thermal denaturation of dsdna is not required, as the polymerase itself can denature dsdna prior to elongation. due to the reaction temperatures employed (60-65°c), dsdna is in a metastable condition and the structure may hybridize/dehybridize spontaneously. this enables primers to bind to the template dna in melted regions. six primers are used resulting in high specificity. the forward (f3) and backward (b3) primers, together with the forward inner primer (fip) and backward inner primer (bip), generate an artificial starting structure with single-stranded loop regions in each end. based on this "dumb-bell" structure, a cauliflower-like structure is generated with the fip and bip primers binding at the loop region. loop primers (lf, lb) may help to decrease the reaction time [62] . this process results in very fast generation of dsdna products that can be detected either with intercalating dyes or with specific probes [63] in potentially < 10 min. in short, lamp is advantageous due to: (i) high specificity, through the use of a minimum of four primers that define six binding sites on the target; (ii) sensitivity equal to, or higher than pcr; (iii) robustness against inhibiting compounds. recombinase polymerase amplification (rpa) [64] is typically performed at 37-42°c. recombinase/primer filaments scan the template dna for homologous sequences. if a match is found, recombinase mediates the hybridization of the primer to the complementary sequence. the denatured part of the template dna is stabilized by singlestrand binding (ssb) proteins and the polymerase can extend the primer complementary to the template sequence. due to the low reaction temperature, the reaction may start at will, with the addition of mg 2+ . this may be necessary, especially in applications where ambient temperature is the same as the reaction temperature. a recent international quality assessment of molecular detection of rift valley fever virus via rpa performed equally well as the best rt-pcr tests [65] . helicase-dependent amplification (hda) is typically performed at 63°c. two primers are used to initiate dna replication by a polymerase. in a first step, the dsdna unwinding activity of a helicase is exploited to achieve denaturation [66] . subsequently, the primers anneal to the single-stranded dna and then are extended by the dna polymerase. each dna duplex is doubled in one cycle. the produced dsdna molecules are separated by helicase and the chain reaction is repeated. similar to rpa, single-strand binding proteins are used to stabilize the denatured ssdna templates. the simplicity of the method, the uncomplicated cycle reaction, and its high speed (especially for circular hda (chda), speed can reach 100 bp/s) are the major advantages of hda [67] . hda has been demonstrated to be efficient with several different types of sample matrices, including urine, stool, blood, and plasma [57] . further promising dna isothermal amplification techniques, such as strand displacement amplification (sda) [68] , nicking enzyme amplification reaction (near) [69] , strand invasion based amplification (siba) [70] , or rolling circle amplification (rca) [71] are available and advantageous in various applications. despite the advantages of naats regarding sensitivity, a drawback is the relatively high cost of the reagents, compared to the cost of lfts. the main cost driver in naats is typically the amplification enzyme (the polymerase). cost further increases when viral rna should be amplified and a reverse transcriptase should be incorporated in the reaction mix. in the case of some inaats, the assay design can be very complicated; for example, primer design in lamp or the simultaneous use of three enzymes, as required in nasba. costs increase further, when enzymes are not pre-stored in liquid, but in dry format (e.g. lyophilized). such approaches may be required to help enzyme stability at the challenging environmental conditions of tropical regions. however, enzyme activity loss may be observed in lyophilized compared to liquid reagents. further problems may arise as naats inherently measure both living and dead pathogens (as both can provide template dna). this may lead to false positive diagnosis, for example during monitoring the progress of a patient over a treatment period. from the infrastructure point of view, specialized laboratory facilities are needed. in this case, well-trained personnel must also be employed to achieve proper assay set-up. in case of multiplexed assays, the risk of human error (e.g. pipetting error) increases, even with experienced personnel. to reach satisfying sensitivity, it is generally still necessary to purify nucleic acids from raw sample materials, such as blood, sputum, or urine. such purification processes may represent > 50% of the manual workload. the labor costs for manual workflows add substantially to the overall costs. this section summarizes poc and near-patient platforms for nucleic acid extraction and amplification, representing a big category of emerging and promising technologies that address the end-users' needs expressed in sections 2.3 and 2.4, and overcome some challenges outlined in section 4.2 related to lack of automation. in this context, naats mentioned in section 4 should be integrated into automated systems in order to overcome the need for specialized personnel as well as laboratory infrastructure. this includes reagent pre-storage in the system for full hands-off operation. the automated platforms must withstand the challenging environmental conditions (temperature, humidity) of the tropical regions. there are dozens of technologies for diagnostics, in various stages of development, used in various settings and detecting various targets. the list is long, and it would be impossible to include all in the present review, which does not aim to provide a list of all existing technologies, but to present those that apply in the context defined in the previous sections. within this framework, we applied a specific methodology and followed certain (inclusion/exclusion) criteria to select which technologies to present. in particular, we included those that: (i) focus on febrile illness and other syndromic infectious diseases with the potential to be expanded to febrile illness (thus, we excluded technologies for blood chemistry/clinical chemistry analyzers); (ii) include at least nucleic acid amplification testing, which can also be combined with immunoassays (thus, we excluded technologies for cd4 cell counting); (iii) are at least at late stage development, pre-commercial, or commercial stage (thus, we excluded academic, research, or laboratorylevel solutions). the description of each platform is divided in two sections: the performance as well as the technology and operating principle behind the platform. the former refers to issues such as portability, modularity, time-to-result, etc. the latter describes the core technical component(s) and main innovative features, e.g. the detection technology, the fluidics handling, and the manufacturing technology. in short: (i) in terms of detection technology, most platforms use optical methods (fluorescence in real-time signal monitoring or imaging mode; or light scattering as in verigene®), thus, it appears that progress on the label-free technologies is still required to reach commercial level. exceptions are the q-poc™ that uses silicon nanowires for detection and easynat™ that uses an integrated lft. (ii) regarding fluidics, it is noteworthy that not all described technologies implement microfluidics but also "macro"fluidics. in particular, enigma® ml and cobas® liat use cartridges that incorporate macrofluidic handling (e.g. syringes, plunges). fluid handling is mostly pressure-driven, although centrifugal microfluidics are increasingly used (e.g. labdisk, genepoc™, liaison® mdx). (iii) manufacturing-wise, the majority of the cartridges are plastic disposables, with some exceptions that are based on cmos (complementary metal-oxide-semiconductor) technologies such as the vereplex™ chip and the q-poc™ silicon nanowire array. (iv) in terms of amplification, all platforms allow for both dna and rna analysis. most of them use rt-pcr although isothermal amplification is also increasingly used (e.g. in alere™ i, easynat™, labdisk). notably, the assembly "assay-cartridge-instrument" is common in all cases, constituting a platform-based approach. regarding performance, some of the platforms aim at single or few pathogens (e.g. genexpert®, alere™ i, easynat™, cobas® liat, q-poc™, genepoc™, liaison® mdx), while others follow a syndromic approach, offering panels of several pathogens being simultaneously tested (e.g. filmarray®, vereplex™ biosystem, verigene®, diagcore®, labdisk). furthermore, it is important to emphasize that many of the technologies have a sample-to-answer time > 1 h (except alere™ i, cobas® liat, and q-poc™), while the cost is in the range of tens of us$ for the cartridge and several thousands of us$ for the instrument. therefore, naats seem to be outcompeted by lfts. however, the added value that multiplex naat platforms offer in comparison to the lfts should be taken into account. moreover, the cost should be considered in a broad perspective, taking into account "hidden" materials and labor costs when comparing with the standards of care. a comprehensive overview of the technologies and some of their technical features are shown in table 2 . performance features (e.g. time-to-result, portability) are mentioned in the platform description but not in the table, as the latter intends to focus mostly on technical aspects. furthermore, the information provided regarding the performance characteristics is based on the most up-to-date search on publicly available resources. the authors cannot exclude the possibility that these data may be updated in the future by the developers/manufacturers during the progress of the platform, or that manufacturers terminate or delay the development/manufacturing/market entry due to corporate or financial reasons. furthermore, table 3 summarizes some "customer" related information such as prices of instrument and test, pathogens/syndromes detected, and regulatory status. this information is provided for those platforms that public information is available, and it may vary between the european and us market. the genexpert® system ( fig. 4(a) ) from cepheid [72] is a widely used system in developing countries implementing molecular diagnostics to identify tuberculosis (tb) [73] [74] [75] . despite the complexity of its cartridge (assembly of more than five parts), the system was adopted by resource-limited countries due to the endorsement of the xpert® mtb/rif test for tb by the who in 2010 and the financial support by the global fund for aids, malaria, and tuberculosis. the system contains several additional versions to detect drug-resistant tb, hiv, mrsa, chlamydia trachomatis, neisseria gonorrhea [76] , while it included ebola testing during the 2014 outbreak. the time-to-result is estimated to be 60 min. the system of the xpert® series is developed in single-cartridge or multi-cartridge configurations for the end-users to decide which option is more suitable to their needs: low/medium throughput (1-, 2-, 4-, 16-cartridge system) for the point-of-care, or high throughput (80-cartridge station) for centralized laboratories. the most portable system of the xpert® series aims to truly decentralize tb diagnosis. the genexpert® omni* ( fig. 4(b) ) is a single-cartridge version, battery operated and wirelessly connected to the cloud (operable also via smartphone), which allows highly decentralized utility and real-time data transmission. the price of a test is us$40-60 in the us market, however there are special arrangements with ngos that subsidize this test for tb in developing countries, thus eventually costing slightly below us$10 (excluding shipping and distribution; this is the sales price ex works). the genexpert® iv device cost is approximately us$17,000, while the genexpert® omni* costs less than us$3,000. the genexpert® is a sample-to-answer system, where sample preparation and extraction of nucleic acids take place in the cartridge in an automated way as the necessary reagents are pre-stored. only a few minutes of hands-on steps are required for liquefaction of crude samples such as sputum. regarding fluidics, the cartridge contains a total of 11 chambers and reservoirs ( fig. 5(a) ). at its core, there is a cylindrical rotary valve, which is handled by a plunger rod that moves vertically, as soon as the cartridge is inserted in the processing device. the interface between the valve and the plunger activates the liquids (sample and pre-stored liquid reagents) movement between chambers, by positive or negative pressure through small openings at the base of the reservoirs. an important component of the cartridge is an integrated filter, which is located at the base of the valve body and captures the microorganisms. subsequently, a sonic horn approaches the filter area and mediates the mechanical lysis of the microorganisms and the release of nucleic acids. the latter are pumped through a chamber where beads with freezedried amplification reagents are stored. these are rehydrated upon sufficient filling of the corresponding chamber (the filling level acts also as internal fluidic quality control). the entire mixture is then directed to the pcr reaction tube, located at the back of the cartridge [79] . regarding detection, the pcr tube is surrounded by heating/cooling plates that enable fast thermal response, and by optical blocks that enable amplification and real-time fluorescence-based pcr product detection. in contrast to traditional thermal cycling systems, in which all samples are subjected to the same time/temperature/optical protocol, each sample in a genexpert® system can be subjected to a different protocol. one more innovative feature of the processing module is the intelligent cooling/heating optical reaction (i-core) module [80] , a sophisticated, solid-state, miniaturized microelectronic and micro-optical system capable of performing pcr reactions with multichannel fluorescence detection in < 30 min. each i-core includes a four-channel optics system capable of exciting and detecting multiple fluorescent dyes in the same reaction tube, thereby being able to accurately measure four dna targets simultaneously. the filmarray® (fig. 6 ) from biomérieux [83] is a benchtop diagnostic instrument that has three fda-cleared panels: the respiratory panel [84] , the blood culture identification panel [85] , and the gastrointestinal panel [86, 87] . filmarray® responded to the 2014 ebola outbreak and received emergency use authorization (eua) from the us fda, based on the filmarray® biothreat panel that specifically detects ebola zaire. a field study was conducted in sierra leone in patients and healthcare workers [88] . only a few minutes hands-on preparation time is required before the cartridge is inserted in the processing device, including the loading of the pouch into the loading block, the insertion of the hydration solution into the pouch, and the loading of the sample. the simplicity of use, the lack of refrigeration storage requirements, and the multiplexing capacity are positive features for use by limitedtrained personnel. up to 27 different targets can be analyzed per test, yet the throughput is still low, as only one patient can be screened per test run [89] . in addition, the manufacturer's list price for the reader (~us$40,000) and reagents (~us$130/cartridge) may not be affordable in resource-limited settings. the filmarray® is a sample-to-answer system, whose core technology is based on the freeze-dried vacuum reagent pouch, and on the nested multiplexed pcr that allows for the detection of multiple organisms. the microfluidic pouch ( fig. 6(b) ) consists of two areas, namely the fitment, where the reagents are pre-loaded and freeze-dried, and the main body. these two are bonded via heat welding. the fitment is fabricated with injection molding of polypropylene. the main body is fabricated with two sheets of a polyester/polypropylene film that contain a copolymer adhesive layer. these layers are welded together using heated plates. the fitment contains 12 reservoirs (a in fig. 6(b) ) that contain the biochemical reagents. during the entire manufacturing process, vacuum is maintained in each well through a set of plunges (b). it is very important to maintain the vacuum in the fitment and the blisters because: (i) it helps to maintain the freeze-dried nature of the components and, thus, their long-term storage; (ii) it allows for the injection of the sample and the hydration solutions in the pouch in the correct volume; (iii) it minimizes the formation of bubbles in the microfluidic system. the sample matrix is a naso-pharyngeal swab for the respiratory panel (different matrices are used for other panels). a total of 300 μl liquefied sample (100 μl sample and 200 μl lysis buffer) are used for the test. the user injects the hydration buffer (used for rehydrating the freeze-dried pre-stored reagents) and the sample in two differently designated inlets. the vacuum in the pouch automatically draws the correct volume, thus eliminating the need for precise measuring and pipetting by the user. liquid handling is applied through pneumatic pressure and "bladder" assembly. this contains a bladder plate on which several inflatable bladders are located, each corresponding to one chamber of the pouch. the assembly is subjected to different gas pressures and as it (and the bladders) is externally in contact with the pouch chambers, any change in the volume of the bladders causes liquid to move between the chambers of the pouch. the bladders are pressed through pneumatically-driven pistons (figs. 2, 3 in [90] ). the sample moves to the lysis chamber where the filmarray® performs mechanical lysis (bead beating) using ceramic (zirconium silicate) beads. the procedure follows the "boom chemistry" [91] . the released nucleic acids are bound on silica-coated magnetic beads, which are transported from the lysis chamber into the purification chamber, using a magnet integrated in the device. in this chamber, any remaining cellular or viral debris are removed through washing. an elution step releases the nucleic acids from the magnetic beads. the 1st stage pcr follows (15-20 cycles, including a reverse transcription step to convert rna into cdna), where dozens of primer pairs of all target molecules are mixed for multiplex pcr. a dilution step follows with the addition of a mixture of a fresh mastermix containing an intercalating fluorescent dna dye. subsequently, the mixture is aliquoted in a high-density array of 102 microfluidic wells where the 2nd stage (nested) pcr [92] takes place. the 2-step pcr is used to eliminate limitations associated with single-step multiplex pcr. at the end of the 2nd stage pcr, the temperature is slowly increased and the fluorescence in each well is measured to create a melting curve, the shape and position of which can be used for differentiation of amplification products with differences of < 2°c in melting temperature [93] . for the two stages of pcr, the instrument has two peltier elements. a blue led is used to illuminate the array at the 2nd stage pcr and the fluorescence emitted by the dyes is imaged by a ccd camera. alere inc. markets the molecular diagnostics point-of-care system alere™ i (fig. 7(a), [94] ) for the detection of influenza a and b [95] , human respiratory syncytial virus (rsv), as well as group a streptococcus (clia-waived, different cartridges for virus and bacterium detection). recent studies have shown that the alere™ i system exhibited at least equal or superior sensitivity over conventional rapid tests [96, 97] . the rapid isothermal amplification technology implemented by alere™ i (near, [69] ) enables the sample-to-answer analysis within 15 min. the instrument itself is portable and stand-alone (no need for a laptop), has a user-friendly interface, data storage capacity, and interface ability. the test price, as of 2014, is approximately us$70-85 while the device costs approximately us$8,500. the sample matrix is a naso-pharyngeal swab eluted in transport medium. the analysis procedure is from sample to answer with prestored reagents. the single use disposable test cartridge consists of three parts (fig. 7) . the test base is inserted into the first available device slot. the test base has two reaction tubes, each of which contains a lyophilized pellet with a pair of templates (similar to primers) for the specific amplification of the target nucleic acid, and a fluorescentlylabeled molecular beacon designed to specifically identify the amplified targets. the sample receiver is inserted in the second available device slot. this cartridge contains the elution buffer to elute the pathogens from the swab (10 s stirring of the swab in the cartridge). the transfer cartridge is pressed against the sample receiver to collect the necessary amount of material and then transferred and locked over the test base in order to initiate amplification. the amplified products are detected via dual-channel fluorescence, deriving from the fluorescent probes. indepth technical design or information regarding the cartridge are not publicly available in detail for this system. another system from the same manufacturer is the alere™ q hiv-1/2 detect (fig. 8, [98] ). it is a fully automated platform for hiv-1 and hiv-2 detection within 50 min based on real-time pcr with multiplexing capability [99, 100] . it is suitable for point-of-care use due to its portable nature, battery operation, no laptop requirement, easy to use touch screen, while the cartridge can be stored at room temperature without need for cold chain. the alere™ q platform has been launched in multiple developing countries, initially for qualitative hiv, and allows for finger-prick detection of hiv virus in whole blood collected directly into the cartridge (25 μl are sufficient) that is then inserted into an instrument for automated processing, extraction, amplification, and detection. technology and operating principle of the alere™ q alere™ q hiv-1/2 detect is a sample-to-answer system. lysis is based on chaotropic salts acting upon the sample and releasing the nucleic acids. biotinylated oligonucleotides that are specific to hiv-1 and hiv-2 genome hybridize with the post-lysis released viral rna, and are subsequently bound on streptavidin-sepharose particles. after washing, reverse transcription and pcr follow. detection of the pcr product is the core technological innovation of the system. it is based on competitive reporter monitored amplification (cma) technology utilizing an array of immobilized oligonucleotide probes and complementary fluorescently labeled reporter oligonucleotides in solution [101] . these reporters compete with the amplicons for hybridization on the surface probes. as in the beginning there are only few amplicons, labeled reporters hybridize with the surface-probes, thus, the initial signal is high. as the pcr reaction progresses, the labeled reporters preferably bind to the amplicons, therefore, less reporters are bound on the microarray. consequently, the progress of pcr is monitored via the decrease of fluorescence from the microarray spots until a plateau in the amplification reaction is reached. fluorescence imaging follows the progress of the reaction, as one image is collected during each annealing cycle. regarding fluidics (fig. 8(b) ), the sample is inserted in a capillary (position 1), the inlet being specially designed to receive finger/heel prick. a control window (5) helps the user to assess the filling level. the cap (6) ensures hermetic sealing and minimizes the risk of sample spilling or aerosols exiting the cartridge, thereby avoiding any contamination risk. dry reagents are pre-stored in internal compartments, along with a buffer reservoir (4) . a fluidic network of valves in the cartridge regulates the liquid/air movement. a septum, which is pricked by a needle connected to the pneumatic module of the cartridge, is used to apply air pressure and move the liquids into the different compartments. the compartment (3) is the reaction chamber where pcr takes place and the microarray is located. the enigma® ml (minilab™) (fig. 9) is a point-of-care platform commercialized by enigma® diagnostics, which, since mid-2017, is in the process of selling their intellectual property underlying its point-of-care molecular diagnostics platform [104] (nevertheless the platform is described here as one of the most representative ones). although the main application of the system has been on respiratory tract infections [105] , its functional and performance characteristics would enable potential use in febrile illness diagnosis. indicatively, the pipeline of infectious diseases includes influenza a and b, as well as rsv. the enigma® ml requires no laptop due to its internal controller and user-friendly touch screen. the instrument is available in a modular configuration, i.e. in a single-module up to a six-module option (for one to six cartridges, respectively). each module can operate independently via the central controller, therefore, different assays (requiring different protocols) from the same or different specimens can be performed at the same time. the overall analysis time for each module is 95 min. the analytical and diagnostic verification process of the fluab-rsv assay on the enigma® ml that led to the subsequent ce marking was completed in july 2013, at guy's and st. thomas' nhs foundation trust, london, uk [105] . the molecular analysis is based on real-time pcr. all required reagents are pre-stored in the cartridge allowing for fully automated analysis with a very short hands-on time of 2 min, while there is no need for cold chain as the reagents can be stored at ambient temperature. the biochemical analysis is initiated with a combination of thermal and chemical lysis to release nucleic acids, which are then captured by magnetic beads. magnetic beads are robotically transferred in situ to two washing buffers for purification and removal of any inhibitory agents. the last step before amplification is the elution of the nucleic acids from the beads, followed by mixing with the amplification reagents (freeze-dried). each test contains bacteriophage ms2 as an internal process control. the cartridge consists of three main blocks (fig. 10) : (i) the sample inlet block, where the swab is introduced in the collection tube that is then clamped onto the cartridge; (ii) the block with the pre-stored (liquid) reagents (indicated with yellow in fig. 10) ; and (iii) the block with specific tools (movable components such as pipettors, magnetic wand, and foil cutter). the core of the cartridge operation is based on robotic actuation of fluids between the vessels in order to facilitate the biochemical reactions, handling of beads, etc. in particular, a robotic arm picks any movable items and inserts them in the corresponding liquid tubes [107] . a highly innovative feature of the system is the pcr reaction vessel [108] . it consists of an electrically conducting polymer (ecp), which acts as a resistive heating element. at the same time it is arranged in such a way that the electrically conducting profile differs in different areas of the vessel (along its axis), thereby controlling thermal gradients that are necessary for thermocycling. the different electrical conducting profile is regulated during the manufacturing of the vessel by varying its radial thickness (key parameter), thus varying unevenly its resistance profile. the vessel consists of a combination of a highly conductive material (aluminum) and an ecp, the two being separated by an insulating layer. the aforementioned induced temperature gradients also assist in convection-based liquid mixing in the reaction vessel. the ecp fabrication technology is typically injection molding. detection is achieved via hybridization probes and fluorescent resonant energy transfer (fret) [109] . taking advantage of this effect, the enigma® ml system uses two probes to hybridize with the amplified dna/rna product, acting as energy donors and acceptors. when amplification occurs, the two probes hybridize and are located in close proximity, thus, the energy of the donor (induced by the excitation source) is transferred to the acceptor, which, in turn, emits this energy as fluorescence. the latter is then monitored in real time during the amplification process, as the amount of the acceptor fluorescence is proportional to the amount of the generated pcr product. roche diagnostics offers the cobas® liat (lab-in-a-tube) system [110] as a compact solution for point-of-care molecular diagnostics (fig. 11) . the system is currently used for detecting: (i) influenza a and b and rsv viral rna in naso-pharyngeal swab specimens from patients with signs and symptoms of respiratory infection in conjunction with clinical and epidemiological risk factors; (ii) streptococcus pyogenes (group a β-hemolytic streptococcus, strep a) in throat swab specimens from patients with signs and symptoms of pharyngitis. however, it is not a multiplexed system, as the aforementioned infections are detected in different cartridges. the instrument is compact and stand-alone (laptop-free) with small footprint operating in such way that minimizes possible interference by, and contamination of the user. with minimum training needed, the system can provide results in as fast as 20 min. the cost of the device and cartridge is approximately €25,000 and €55, respectively. although the system has not yet been used for febrile illness diagnosis in developing countries, its performance features make it a good candidate. technology and operating principle cobas® liat is a sample-to-answer platform thanks to the full integration of all necessary reagents in its cartridge. the in situ sample preparation starts with mixing the sample (200 μl) with chaotropic agents inducing pathogen lysis, followed by the "bind-wash-elute" protocol (boom-chemistry [91] ), which involves binding of nucleic acids in silica-shelled magnetic beads, subsequent washes and a final elution step. nucleic acids are then ready for real-time pcr with internal controls. the core technology of the system is the cartridge, which consists of a rigid frame and a transparent flexible tubule. the latter consists of several pouch segments, which are separated via breakable seals and are flexible enough to be compressed. the system uses peristaltic fluidic actuation for liquid handling. after the cartridge is loaded, the analyzer, which contains clamps, plunges, and actuators, controls the selective release of reagents from tube segments, leading to mixing and propulsion of liquids by precisely applying pressure to the pouches. the pressure needed to compress the tubule to unload the pouch is approximately 1 atm. the analyzer also provides control of reaction conditions at different temperatures, and alternating movement of the reaction mix between two different temperature zones for rapid pcr (fig. 12) . the outcome of the reaction appears as a real-time fluorescence signal, detected by the six-channel photometer module in the analyzer [112] . from the fabrication perspective, the tubule is based on polyolefins and is fabricated via injection molding or blow molding. in order to serve specific processing needs, some tubules are surface-processed via plasma treatment or introduction of additives. the wall material is multi-layered, with the inner layer being biocompatible, while the outer is resistant to gas permeability. the flexible tubule is supported by a rigid frame, which is made of polypropylene with lower melting temperature than the tubule to ensure uniform melting during sealing. according to the relevant patent [112] (although the patent does not ensure that these exact methods are used in the final production) sealing between these components is achieved via welding of the flexible tubule on the outer frame using thermal and/or ultrasonic sources. an alternative approach is to seal the two components with a hot-melt adhesive joint with ethylene vinyl acetate, or with uv cure epoxy. the dimensions of the frame, surrounding the tubule cartridge, are 150 mm (height) × 25 mm (width). the agency for science, technology and research (a*star), singapore, stmicroelectronics, italy, and veredus laboratories, singapore [114] have partnered to develop a lab-on-a-chip for diagnosis of infectious diseases (fig. 13) . the system is already in the market and was used by the brazilian authorities as the tool of choice during the 2014 world cup. a number of application-specific cartridges have been developed, some of which are: vereflu™ (for respiratory infections); veremtb™ (for tuberculosis); verethreat™ (for biothreat agents such as anthrax, small pox, plague and tularemia); verevet™ (for multiple avian pathogens); veremers™ (for the middle east respiratory syndrome coronavirus). in 2013 the partnership announced the launch of veretrop™ for multiple tropical diseases, providing the necessary multiplexity that febrile illness diagnosis requires. the cost per veretrop™ test is approximately us$100. sample preparation is performed ex situ, either via spin columns or an automated extraction kit, and the overall time-to-result (including extraction, amplification, optical readout) adds up to approximately 3.5 h. vereflu™ [115] and veretrop™ [116] assay chips and multi drug resistant tuberculosis [117] have been clinically validated. the core of the system is based on a lab-on-a-chip cartridge (the "in-check" platform, fig. 14) in the size of a standard microscope slide. it contains a silicon chip covered with a glass coverslip and bonded to a plastic support. an on-chip printed circuit board (pcb) provides the necessary electric contacts as an interface to the processing instrument (e.g. for temperature control). the silicon chip has distinct processing areas (fig. 14) : (i) the sample inlet microfluidic ports with buried microchannels to facilitate easy chip filling and fluid displacement. (ii) the pcr area (12 × 8.5 mm 2 ), which contains four pcr chambers, with 1 μl liquid volume capacity, each. four pairs of independently controlled aluminum resistors are used to heat the silicon substrates and fluidic reactors, thus providing the thermocycling environment. the temperature is regulated by the temperature control system (tcs) in the chip processing device. (iii) the outlet holes, which provide microfluidic contact to (iv) the post-pcr hybridization and detection area, which consists of a 3.5 × 9 mm 2 microarray with 126 spots, where oligonucleotide probes are spotted using a piezo-array system [119] . fabrication is performed with a semiconductor-based mems process technology, named simich at 8″ wafer diameter, which allows for the fabrication of micro-mechanical, sensor, and microfluidic components. more details on the process steps are available in [120] . fig. 14 shows some key microfabricated components of the chip and a cross-sectional view of the entire lab-on-a-chip device. post-fabrication processing includes surface chemical treatment in order to immobilize dna probes in the microarray area, and to achieve biocompatibility of the pcr area for dna amplification [121] . post-amplification detection is performed in a separate, dedicated instrument, through fluorescent microarray detection of chip-hybridized amplification products using an image acquisition camera. quantumdx [123] is a uk-based nanomedtech company that develops and commercializes affordable handheld sample-to-result diagnostic and dna sequencing technologies using nanowire biosensors and innovative microfluidic technologies. their collaboration with the institute of microelectronics (ime) of the agency for science, technology and research (a*star), singapore, is strategic for the fabrication of the key microelectronic components. the q-poc™ platform (fig. 15 ) of quantumdx is a handheld, battery-powered molecular diagnostic device that promises a sample-to-answer dna-based result in as few as 15 min. it contains integrated reagents with current stability targets of up to 40°c for up to 18 months. the system can test one patient per run and is a pioneering approach to the handheld sequencing market. in 2014, quantumdx partnered with find in order to develop a solution for the combined detection of tb and drug susceptibility determination for the causative agent of tb. the q-poc™ system seems to be especially promising for antimicrobial resistance profiling given that many different genes can be screened through a nanowire array. there are several other assays in the pipeline, for diagnosis of e.g. hiv, sexually transmitted infections, neglected diseases, as well as for tumor profiling [124] . the core of q-poc™ lies in its biosensor chip, which is equipped with a silicon nanowire (sinw) array based field-effect biosensor. sinws have large surface-to-volume ratio, tunable electric properties, and exhibit high potential as biosensors, thus enabling next-generation point-of-care diagnostics. the binding events are detected in a labelfree way through changes in the nanowires' resistance, effectively converting the genetic code into a binary code and providing the output in a digitized format. in particular, upon hybridization of the target dna on the sinw, the charge density (and subsequently the electric field) changes. this is reflected as a change in the resistance of the nanowire, which produces the biosensing signal (fig. 16) . the more negative charges present during hybridization, the higher the resistance of the n-type sinw sensor. this principle was shown by zhang et al., by varying the hybridization sites (thus varying the distance of the charge layer) but keeping the total number of charges unchanged [126] . functionalization of sinws is performed with peptide nucleic acid (pna), instead of dna oligonucleotides. the neutral nature of pna minimizes the formation of dense charge layer near the nanowire surface, subsequently minimizing the signal to noise ratio. the n-type sinws feature typical dimensions of (w × h × l) 50 nm × 60 nm × 100 μm and are fabricated using standard cmoscompatible top-down approaches, through the oxidation of polysilicon beams patterning using conventional photolithography on p-type test wafers (more details available in gao et al. [127] ). this method is compatible with mass-manufacturing, is reproducible and convenient, and allows for scaling the sensing area and multiplexing as required by the application. with a capacity of 1000 nanowires in each silicon biosensor chip, the multiplexing potential becomes huge [128] . the sample-to-answer operation is achieved by housing the biosensor chip in a microfluidic cartridge (fig. 17) . ustar biotechnologies (hangzhou) ltd. [130] commercializes its proprietary isothermal amplification technology into an equipment-free diagnostic system, the easynat™ (fig. 18) . therefore, the system is especially attractive for resource-limited settings and is intended for batch testing in microscopy centers. however, the system lacks multiplexity (1 target/cartridge) and requires hands-on extraction steps (centrifuge, vortex, syringe). the extraction is performed ex situ using an equipment-free extraction kit. after boiling lysis, a syringe is used to extract and elute the dna using appropriate buffers. initial tests with easynat™ kit have been performed in clinical studies in tanzania [131] and china [132] for tuberculosis. the manufacturers note that the reagents require refrigeration ("storage in −20°c"). the system is based on an isothermal amplification method, called cross priming amplification (cpa), which uses multiple (5-8) crosslinked primers to amplify dna or rna at a constant temperature of reprinted with permission from [126] . copyright © 2008 american chemical society. 63°c [134] . due to its isothermal nature, amplification does not require any complex temperature control system: a water bath or a heating block are used in order to maintain constant temperature and incubate the cpa assays. following the 60 min amplification step, the reaction tube is transferred to the cartridge, where a lateral flow immunochromatographic strip provides the visual readout after 15-30 min. the patented cross-contamination-proof (xcp) nucleic acid detection device (fig. 19 ) offers the advantage of minimizing the risk of amplicon cross-contamination [135] , through: (i) maintaining closed reaction tubes after amplification (thus avoiding release of amplicon aerosols); (ii) using an enclosed unit (the amplicon tube) to insert the reaction tube; (iii) placing the amplicon tube into an additional enclosed unit (the detection device). running buffer and reaction tubes are inserted in the amplicon cartridge. the amplicon cartridge is subsequently inserted into the detection chamber. as the sealing of the detection chamber is closed, it activates a blade that punctures simultaneously the two tubes. the running buffer drives the amplicon to the adjacent lateral flow strip and amplification results are visually readable (fig. 19(c) ). the detection principle in the lateral flow nucleic acid testing strip is based on a combination of the nucleic acid hybridization and immunoassay principles. a probe is used, which carries a colored particle, and can hybridize with the amplicon. the complex is then captured by the antibody on the nitrocellulose membrane of the test strip. in the absence of an amplification product, there is no interaction/hybridization with the probe, which then flows uncaptured through the membrane to the absorbent pad ( fig. 19(b) ). (i) meander-shaped microfluidic channel filled with reagents that are released at will to area (g)/(h). in particular, reagents may include various nucleotide mixtures and wash solutions, all separated by air bubble(s), in order to flow sequentially and in a well-controlled way. when nucleotides flow sequentially over a nanowire-immobilized single stranded oligonucleotide, then according to the hybridization sequence, the local charge changes, causing a resistance change in the sinw. image source: used from [129] . luminex corp. commercializes the verigene® rp flex system, (fig. 20) (developed and previously commercialized by nanosphere), which received an fda approval in 2015 [138] . the target application is respiratory tract infections and the system covers a broad panel including 13 viruses and 3 bacteria [139] . analysis is performed from naso-pharyngeal swabs. verigene® also offers other panels such as gastrointestinal, while it also identifies eight gram-negative organisms and resistance markers from blood culture [140] . the full-automation mode of the system (pre-stored reagents) requires minimum hands-on time (< 5 min), while the overall time-to-result is 2 h. the system is benchtop and consists of two components, the reader (as a controller) and the processor sp, performing the automated test processing steps. it is a modular system since one reader, which acts as the user interface and central control unit, can handle several processors sp. each verigene® test uses four single-use consumables: an extraction tray, a tip holder assembly, an amplification tray, and a test cartridge. these are all loaded in the processor sp for all automated processing the glass support of the test cartridge, which carries the microarray, is inserted in the reader of (a) for detection. image sources: (a) used from [138] ; (b,c) reprinted from [141] , copyright © 2009, with permission from elsevier. steps, specimen extraction, target amplification, and hybridization. fluidic handling is performed via an in-system servo actuator and positive displacement pump, e.g. syringe pump, in combination with pneumatic valves. a comprehensive sequence of steps is provided in one of the company's patents [142] and the system's technical bulletin [138] . after sample processing in the extraction and amplification trays, nucleic acids are transported to the test cartridge. at the end of the procedure, the substrate (which supports a glass slide with the microarray for capturing the target nucleic acids) must be removed from the cartridge and placed in the reader (readout takes a few seconds). all necessary reagents are contained in the cartridge. the amplification technology is based on multiplex rt-pcr between heating and cooling zones followed by hybridization to target specific capture oligonucleotides in a microarray format. end-point detection is achieved with gold nanoparticles (aunps), which constitute the core technology of the system (proprietary nanogrid technology, fig. 21 ). with dimensions of 13-15 nm, the advantage of aunps over conventional fluorophores is the increased sensitivity by several orders of magnitude (e.g. the light scattered from one nanoparticle is equivalent to the light emitted from 500,000 fluorophores [143] ). after amplification is completed, the workflow for detection is as follows: inside the test cartridge, the amplified targets are hybridized to capture oligonucleotides that are pre-immobilized on the glass slide microarray. after removal of the unbound dna, the hybridization products are labelled with aunps. subsequently, intermediate oligonucleotides are flushed into the chamber and bind specifically to the immobilized complex, leaving a poly-a tail sticking out and being available for subsequent attachment of poly-t-bound aunps, which act as probes. after removal of unbound aunps, captured aunps are covered by silver nps (during the analysis procedure) to enhance the scattered light. thus, the overall size of the nps becomes larger and the subsequent optical signal is enhanced. illumination begins with an led (λ max 630 nm) shining through the edge of the glass slide (acting as a waveguide to the led illumination). the evanescent wave that is generated through the glass slide and towards the np coated surface causes light scattering from the illuminated au/silvernps towards the camera, which captures images of the array multiple times and at various exposure times [144] . notably, the aunp-based detection technology may be adapted for protein analysis as well. the spanish molecular diagnostics company stat-dx [146], acquired by qiagen in january 2018, has been developing the diagcore® (fig. 22 ) platform operating as a near-patient testing solution for low test volumes or as a platform to cover mid-throughput requirements. the company announced in early 2018 the approval of ce-ivd clearance for the system and the respiratory panel. stat-dx expects full commercial launch of the diagcore® in the 2nd quarter of 2018. the the cartridges of diagcore® are pre-filled with dry and liquid reagents with a shelf-life stability of 12 months at room temperature. the cartridges can be inoculated either using swab directly (with the appropriate cartridge housing for sample elution [147] ), or using liquid samples of volumes from 100 μl to 1.5 ml. automated cell lysis is used for releasing nucleic acids from pathogens. nucleic acid purification follows using high quality extraction columns. the sample is then subjected to rapid thermocycling for amplification; eight real-time pcr reaction chambers with six-plex capability allow for the simultaneous detection of a total of 48 targets. regarding fluidics, the cartridge includes a transfer module that moves laterally along a guide that is located within the cartridge in order to align various fluid ports and to control the movement of fluids through the cartridge channels and chambers [148] . fig. 23(a) shows a typical cartridge from the back side, where the areas designated with the numbers #110, #114, and #116 represent the testing unit, the dilution/dosing unit, and the fluid guiding unit between various chambers, respectively. the testing unit is shown in more detail in fig. 23(b) . the fluidic testing arrangement is aligned with the gravity field in order to avoid formation of bubbles in the various chambers. the inlet port of the structure is indicated as #204, while the top chamber (#216) is unvented and acts as an air reservoir for the air trapped within the microchannels. by avoiding venting, the elimination of aerosol leakage out of the cartridge is achieved. channel openings (#206a, #206b) are used as fluidic controls, where optic sensing defines whether there is liquid or not. a mixing chamber is included (#208). its large crosssection causes turbulent liquid flow, which enhances mixing (passive mixing). the chamber #210 is the actual reaction chamber (with a volume of up to 50 μl) serving also for detection. chamber #212 contains dry stored reagents. by applying variable pressure in the inlet port #204, the liquid may be pushed to chamber #212, rehydrate the reagents, which are then pulled back to chamber #210 in order to participate in the reaction. the structure indicated in fig. 23 (b) may be repeated several times, with a common inlet port and different stored reagents, in order to serve in geometrically multiplexed reactions. the concept of centrifugal microfluidics (in contrast to pressuredriven microfluidics that all the aforementioned platforms use) has evolved to a mature and powerful technology for automation of diagnostic workflows in the fields of clinical chemistry, immunodiagnostics, nucleic acid analysis and others. this microfluidic approach benefits in particular from the straightforward mechanism for liquid actuation based on centrifugal forces that are a result of simple rotation of the cartridge with defined spinning frequencies [150] . no external pressure sources or complicated (micro)pumping systems are required, which reduces the risk of cross-contamination (due to openings in the cartridge) and allows for the building of simple, portable processing devices with small footprint and weight. these facts qualify centrifugal microfluidics as a technology for automation of diagnostics at the point of care. a main advantage of centrifugal microfluidics is the employment of microfluidic modules ("unit operations"), each one serving a particular fluidic functionality for: liquid handling (e.g. metering and aliquoting [151, 152] , valving and switching [153] [154] [155] , pumping [156, 157] or mixing [158, 159] ); assay integration (reagent pre-storage and supply [160, 161] ); separation [162, 163] ; detection [162, 164] ; blood plasma separation; nucleic acid purification and amplification, or washing and blocking. a comprehensive overview on unit operations and process chains is provided by strohmeier et al. [165] . combination of these unit operations and process chains allows for the integration of complex laboratory processes into one system for fully automated sample-to-answer analysis. challenges are mainly in the field of cartridge-integrated sample preparation required for completely integrated sample-to-answer solutions [165] [166] [167] . apart from the world-to-disk interface that defines how the sample can be added by the user to the cartridge, the preparation of complex samples such as sputum, a classical specimen in diagnostics e.g. for detection of tuberculosis [81] , is an unsolved issue yet, as samples can be diverse in their physical constitution, and harsh chemicals or strong forces are required for pretreatment. further challenges are the handling of increased sample volumes (> 1 ml) of blood or urine that are required when the pathogen load is low or when the required clinical sensitivity for diagnosis is high. the maximum sample volume is typically limited by the available footprint of the disk and it has to be considered that e.g. for nucleic acid extraction by classical bind-wash-elute chemistries, equal volumes of lysis and binding buffer are required for sample treatment, hence posing further challenges to cartridge integrated reagent pre-storage. from the research perspective, increased application of disk-based systems has been recently observed in resource-limited settings, especially in the field of immunodiagnostics. a disk-based test for detection of a four-parameter liver assay panel for defining liver function has been demonstrated to be completed within 20 min by nwankire et al. [168] using a portable, battery powered processing device and including field testing in a laboratory in nigeria. furthermore, hosseini et al. [169] have implemented a novel mixing method using microspheres on disk in order to detect dengue virus antigen ns1. the following sections provide a short overview of some centrifugal microfluidic platforms that are at late development or market stage. the inclusion criteria in this review were that the technologies focus on infectious diseases and have at least a few publications in the field. one representative platform of centrifugal microfluidics at late stage development is the labdisk (fig. 24) from hahn-schickard [170] and imtek, university of freiburg, germany. the technology has been applied in a variety of applications ranging from clinical chemistry [164, 171] , to immunoassay [172] and nucleic acid analysis, indicating universal use. in the latter field, two significant advances in fully automated sample-to-answer (nucleic acid extraction and amplification) analysis were announced, particularly for neonatal sepsis, detecting s. warneri, h. influenza, e. coli, and s. agalactiae [173] , as well as for h3n2 influenza virus [174] . the amplification was based on pcr and the time-to-result was 3-3.5 h. yet, when the labdisk was used to integrate rpa isothermal amplification for the detection of biological warfare pathogens (b. anthracis, f. tularensis, and y. pestis) the total time-toresult was < 45 min [175] . in addition, the processing device for isothermal amplification operates with a power consumption of < 75 w, which facilitates its application in remote environments [176] and its potential use with battery or solar panels. the application of the labdisk at the point of care in resourcelimited settings has been demonstrated with the detection of yellow fever virus in laboratory and field settings in senegal using rpa and 20 min amplification time [177] . recently, a labdisk was developed for the detection of malaria, dengue, chikungunya, pneumonia, and typhoid fever that used lamp providing the necessary means for nucleic acid based multiplex amplification with a time-to-result between 70 and 120 min [178] . currently, one disadvantage of labdisk is that, while several parameters can be analyzed per disk, only one disk, corresponding to one patient, can be processed per test run. the concept behind the labdisk is to translate laboratory processes and steps into microfluidic unit operations and interface them towards fully functional sample-to-answer solutions. sample volumes can range from (sub)μl to several hundreds µl. one of the labdisk innovations is the pre-storage of liquid reagents in stick-packs [160] . these pouches tightly seal the buffers and release them on demand upon centrifugation. the manufacturing technology is microthermoforming of polymer foils [179] , a technology that is routinely used in the macroscale for blister packages, but was adapted to the microscale for micro/nanostructures in the labdisk (fig. 25) . typical foil thicknesses are between 150 and 300 μm. a mold and a thin polymer foil placed above the mold are heated at a temperature near the glass transition temperature of the polymer foil. when the conditions are favorable, air is blown from the top so that the foil takes the shape of the mold. main process parameters are the applied air pressure and the temperatures of mold and foil. various materials can be used, with most preferable candidates being the cyclic olefin polymer/copolymer (cop, coc) and polycarbonate (pc), depending on the application requirements. sealing is performed either via a pressure sensitive adhesive foil (containing nanocapsules, which break and release adhesive upon application of pressure) or via thermal sealing using one more foil material (e.g. coc on coc). the elastomeric molds are suitable for prototyping, where fast iterations are required. however, each elastomeric mold may be worn out after repeated use for some tens of cartridges. thus, for large manufacturing series it is more efficient to use a metal mold. it may be more challenging to deform the foil (and methods such as coating on metal are used), however it is economically more sustainable and the durability of one metal mold can be sufficient for tens of thousands of disks. from the commercial point of view, the genepoc™ system (fig. 26 ) from genepoc inc. [181] is a simple and integrated portable instrument for the prevention and early detection of infectious diseases. the system received in 2017 fda clearance for its clostridium difficile assay and a ce mark for its group b streptococcus assay. in the future, genepoc inc. plans to develop cartridges for detection of respiratory, gastrointestinal, neonatal, and hospital-acquired infections, as well as sexually transmitted diseases. the system operates in a sample-to-answer mode, with minimum hands-on steps (a few minutes). the manufacturers declare a time-to-result of up to 1 h. importantly, the system operates in a stand-alone mode, without the need for laptop, which is replaced with a touch screen instead. its universal sample-to-cartridge interface (accepts liquefied matrix) allows compatibility with several types of swabs, urine, etc. [182] . the cartridge consists of only three pieces of plastic and has a wedge-shaped form (unlike an entire disk), which allows the device to process up to eight cartridges simultaneously, thus offering an increased throughput that is often necessary in cases of "extreme" poc testing [182] . the genepoc™ disposable is composed of three distinct operational sectors: (i) world-to-chip interface: 100 to 200 μl of clinical sample conditioned in genepoc™ sample buffer is inserted in the disposable with a transfer pipette. the developers mention that the use of blood is not feasible yet, as it would require off-chip microbial preconcentration. subsequently, the inlet chamber is sealed with a lid that prevents aerosols from leakage. a series of siphons and passive valves prime the liquid to the next chambers. (ii) universal sample preparation, starting with pathogen mechanical lysis: a metallic disk is magnetically actuated in the chamber where glass beads are present leading to efficient lysis and release of nucleic acids. the lysate is centrifugally driven to a dilution chamber, where pcr inhibitors are inactivated by heating up to 95°c and with proper ionic condition adjustment of the extracted material in order to optimize subsequent amplification. (iii) the amplification and detection sector, which is the only assay-specific area of the cartridge where rt-pcr is performed (reagents are drystored). this sector has three chambers available for amplification (thermocycling is achieved via air heating of the overall process chamber). the device includes four non-overlapping excitation and emission wavelengths for fluorescence detection of chemical dyes, therefore, up to 12 targets (4 in each chamber) can be detected simultaneously, and under rotation. further details regarding cartridge material and fabrication methods are not available by the manufacturer. the liaison® mdx (fig. 27 , formerly 3m cycler with simplexa™ integrated assays) is a centrifugal-based automated platform commercialized by diasorin inc. (formerly by focus diagnostics, which is currently owned by, and is part of diasorin group [184]). the system has acquired fda/ce-ivd approval and its target applications currently aim at respiratory tract infections for influenza a and b as well as rsv viruses, hsv 1 and 2, group a streptococcus, and c. difficile [185] . the system is classified as benchtop and is operated via a laptop. the cost per cartridge is approximately €30, while the instrument costs approximately €40,000. the device is compatible with two types of disks, targeting different types of end-user specifications (both are made of polypropylene with a nucleic acid compatible adhesive): (i) the direct amplification disk has eight individual slots/cartridges (all are connected into one disk, but one or more can be used simultaneously). the sample volume is 50 μl, which is mixed with buffer (50 μl) and the sample-to-answer time is 60 min based on high-speed thermocycling and rapid rt-pcr using high heating/cooling rates (> 5°c/s and > 4°c/s, respectively). samples require no pre-processing (dna/rna extraction) and direct amplification is applicable. (ii) the universal disk is a 96-well singleuse cartridge able to perform multiple assays per disk. the reaction volume is 10 μl. the device has four excitation and emission wavelengths for optical multiplexing in addition to the geometric (multichamber) multiplexing. when the universal disk is used, extraction should be performed ex situ and the eluate is inserted into the disk. a rather different approach from the typical benchtop-based or fully-automated systems has been suggested by the "diagnostics-in-a-suitcase (dias)", initially developed for the emerging avian influenza a (h7n9) virus [186] . in contrast to the aforementioned systems, where the core was a (micro)fluidic cartridge handled in a fully automated way, the dias system does not proclaim miniaturization or fully-integrated components and requires a few hands-on steps. yet, it smartly includes all the necessary biochemical components (extraction and amplification reagents), tools (gloves, pipettes, tube tracks, pipette boxes, waste container), and equipment (a tube scanner, vortex, microcentrifuge, even a mask in case of a highly infectious disease/epidemic), within a suitcase for a complete on-site analysis. it is an entire portable lab, which functions with solar panels or power packs (fig. 28) . the workflow includes a typical sample preparation starting from the lysis of the pathogens, extraction of nucleic acids by binding to magnetic beads, two washing steps, and elution. this process requires 30 min. the subsequent amplification step is conducted via rt-rpa and the time-to-positive is 15 min. in total, nine pipetting steps are required, which can be reduced by replacing some liquid components with lyophilized reagents. during the 2014 ebola outbreak, the system was applied in field trial in guinea in collaboration with the institut pasteur in dakar, the public health institute of guinea, the university of stirling, the robert koch institute, and twistdx ltd. [187] . this innovative approach tackled several problems including lack of (constant) electricity; lack of cold chain for material storage; risk and time loss from transferring infectious material from the field to central laboratories for analysis; and the need for fast time-to-result for healthcare providers who screened patients for ebola virus. a natural drawback of the dias system is the lack of connectivity with all the accompanied consequences (manual registration of data, risk of loss, ambiguous storage of data, etc.). furthermore, it requires a certain degree of user training for the handling of manual steps. along a similar concept, msf is developing a small-scale, autonomous diagnostic bacteriology laboratory based on appropriate and feasible, existing or adapted techniques, at affordable cost, with high accessibility and ease of use, which may respond to clinical needs in rt-pcr/isothermal . the feasibility of using robust blood culture bottles with visual readout, with a new type of media that bypasses the need for blood and, thus making it easy to use by non-experts, is being currently evaluated. a simple pathogen identification system, restricted to genus, as well as an antibiotic sensitivity testing system, restricted to the six antibiotics used frequently at the peripheral level, have also been developed. the target patient populations are children presenting with severe illness, those with risk factors for bloodstream infections, such as neonates, malnourished or hiv positive children, those who fail to respond to first-line antibiotic treatment, hospitalized war patients, and patients with trauma or prior surgery by providing organism-directed antibiotic therapy. with the developed autonomous diagnostic laboratory, it will be possible to acquire information regarding more general questions such as proportions and antibiotic resistance profiles of key bacterial pathogens, thereby providing evidence-based information for antibiotic policy (appropriate prescribing) and allowing for the improvement of epidemiological investigations as well as supporting healthcare infection control in emergencies and in neglected populations in resource-limited settings. novel diagnostic platforms, such as those mentioned in the previous sections, require validation in the target patient population, as well as in users. this should be a requirement for all new diagnostic tests, but it is even more important for point-of-care tests that are often used in nonselected patients (for example, any patient with fever attending any level of the health system) and by users with no technical background. before large-scale implementation in resource-limited countries is decided, the test should also preferably be endorsed by who and by international donors and governments that provide funding. unfortunately, many point-of-care tests have been launched in the market with only limited laboratory validation using convenient patient samples (for example that have a high pathogen load), or even cultured pathogens in relatively high concentration. field evaluation of the performance of the test in real patients, who often harbor low pathogen loads, is indeed often undertaken only after registration of the product. this is due to the lack of national and international regulatory mechanisms applicable to diagnostic tests, which, in contrast, exist for medicines [189] . the net result is that health workers, especially those working in private clinics, and even patients going to pharmacies, have access to poc tests that have in fact very low sensitivity and/or specificity. for example, lft for chikungunya is commercialized, although it has a sensitivity of maximum 50% in the field [190] [191] [192] . the gap between the performance measured in the laboratory and that in the field is due to several factors, depending on the type of pathogen and technology used. the sensitivity of naats to detect bacteria in the blood compared to blood cultures is often disappointing [33] . even though naats are able to detect additional cases compared to blood-culture, they are not positive in all blood culture-positive patients [193] because bacterial load can be very low, especially in adult patients with robust immunity. this explains the failure of numerous molecular-based multiplex platforms licensed in northern countries, aiming at diagnosing rapidly the cause of sepsis in admitted patients. fig. 29 . the whole diagnostic test development process, from innovation, to validation and application. this development process is in reality a reiterative process that is more circular than linear. source: v. d'acremont. these tests work well for blood cultured for a few days but not for native blood, which implies that the potential gain of time, essential in these patients, is lost [194] . tests based on the detection of antibodies, even of igm type, often lack sensitivity during the very first days of an acute febrile episode, but also specificity because of antibody persistence for several weeks. these challenges show how important it is to undertake clinical trials in real conditions to assess first the real performance of the test in the field (accuracy against the best available standard), and then the impact of using such tests on the management of patients (efficacy, measured through the impact on the health outcome of patients, cure rate after a certain number of days, secondary hospitalizations, or deaths and sequelae), done by routine health staff in their real working environment (effectiveness, measured generally through the level of compliance to test results and guidelines, and the appropriateness of antimicrobial prescriptions) (fig. 29 ). outcomes measured in these trials should be related to the health outcome of patients and the public health benefits, and not only to the diagnosis based on laboratory reference standards (which for some diseases are anyway suboptimal or even non-existent). "we have to treat the patient, not a test result". to follow this adage, it is even more important nowadays that a continually increasing number of diagnostic tools is becoming available at the point of care. that should, however, not be interpreted as it has been for years with malaria microscopy, as an invitation to disregard test results and treat diseases presumptively based on clinical elements with low sensitivity and/or specificity [195] . this adage rather suggests that, because no diagnostic test is 100% perfect, their results do not provide a black-orwhite answer, but rather an indication on the disease probability. this probability depends on two factors: (i) the prevalence of the disease in the corresponding patient population (pre-test probability), and (ii) the performance (positive and negative likelihood ratios) of the test used. when a disease is rare in the population (low pre-test probability) even if the test has a reasonable accuracy, the post-test probability after a positive result remains low. for example, if a typhoid rapid test with 80% sensitivity and 80% specificity (and thus, a likelihood ratio (lr) of 0.8/(1-0.8) = 4) is used in an area of asia where prevalence of typhoid fever among patients with febrile illness is 20%, the post-test probability will be 50% (it can be calculated using fagan's nomogram [196] ), which suggests that it is reasonable to implement an antibiotic treatment to that patient. if the same test is used in an area of africa where the prevalence is 2%, the post-test probability will only be 7.5%, which hardly justifies treating the patient for typhoid but rather to consider alternative diagnoses. based on these principles, decision charts can be built to precisely guide clinicians on how to use diagnostic tests and prescribe medicines in the most rational way. paper versions of such guidelines have been developed by who and unicef, and details on their strengths and weaknesses can be found in the literature [24, 25, 197] . these guidelines, however, were deemed difficult to be adopted and properly implemented, due to several reasons including the long duration of the training required and the conflicting recommendations provided in different guidelines due to the time required for harmonization and then update of all paper material at large scale. mobile electronic technologies are promising tools to overcome these challenges. an electronic algorithm is, indeed, able to guide clinicians through the specific branches that apply to their patients, to accelerate training by teaching health workers directly on the mobile support (and even integrating training modules into the device), and to help to disseminate quickly to all health workers updated versions of e-algorithms through 3g connection. fig. 30 provides a schematic representation of an integrated tool that aims at managing children with febrile disease at primary care level in resource-limited countries (derived from the existing e-poct tool [23] ). the tool integrates a few key clinical data, biosensors measuring respiratory rate, oxygen saturation, cardiac rate, glycemia, and hemoglobinemia, as well as rapid diagnostic tests detecting malaria antigen (other pathogens can be added according to local endemicities) and host biomarkers (to differentiate bacterial from viral infections). it then provides guidance to health workers on when to refer a sick child, and whether to prescribe or not an antimicrobial (and other type of medicines), while it also calculates drug dosage based on weight or age. it is beyond the scope of this review to elaborate on the content and performance of existing e-algorithms and platforms for the integrated management of children with febrile disease, however, the reader can refer to a recent review published by keitel et al. [30] . the consultation process data collected through the computerized decision-support tools used by clinicians and sent with 3g remote connection to a userfriendly data management system can be used for surveillance and outbreak detection. to document the cause of patient's fever, and at the same time the cause of the potential epidemic, a decision-support system should integrate the results of a pathogen poc test, through a reader directly connected to the tablet supporting the clinical e-algorithm, and if possible also remotely to the central server. the source of information would be patients seen at routine health facilities tested by poc tools that have a direct impact on case management (e.g. malaria or typhoid), but also from a restricted number of sentinel sites where additional poc test, aimed at detecting pathogens of particular interest (that cause large epidemics, even if non-treatable such as dengue, or rare such as ebola virus disease), would be used. the data accumulated on a daily or weekly basis (depending on the level of 3g connection availability) at the central level would allow health authorities to conduct real-time disease surveillance, through weekly/monthly reports, and would also enable immediate outbreak alarms when the preset incidence threshold for the surveyed disease has been reached. from the central server, automated feedback reports can also be created, sent to clinicians' tablets and discussed during supervision visits at the health facility. training e-tools, aimed at improving management of patients in general or in the particular situation of an outbreak, could also be sent to the tablet to guide clinicians. finally, a patient file supported by an electronic card owned by the patient, could be synchronized at each new consultation with the data collected by clinicians in the tablet (fig. 31) . such a fully integrated system seems to be overoptimistic; however, each of its components has already proven to be feasible and the challenge now is to find appropriate and sustainable diagnostic and information and communication technology (ict) platforms that can be deployed at large scale and in the long term. to address the clinically complex differential diagnosis of an episode of fever, that is a frequent presenting syndrome, this review outlined some point-of-care and near-patient diagnostic technologies that can assist to this scope. in the long, but non-exhaustive, list of described technologies, none seems to satisfy simultaneously all criteria of end-users' needs, target product profiles, and the assured criteria. regarding performance, some technologies may be fully automated and multiplexed, but they are prohibitively expensive; some are cost-effective, but compromise sensitivity or multiplexity. in contrast, some solutions offer multiplexity, yet they are benchtop, require laptop, electricity, and are barely portable (due to size and weight). regarding technology, most of the described platforms implement nucleic acid amplification (pcr-or isothermal-based) and almost none of them includes a parallel immunoassay analysis to measure the host response to an infection. a few of the suggested approaches are equipment-free but therefore lack connectivity and interfacing ability. however, one can observe a common rationale in the structure of the described technologies. the vast majority are based on the combination of three components: the assay, the cartridge, and the readout device. several variations may exist in the format of the assay, the cartridge, or the detection principle; however, this three-element model seems to be the backbone of all described diagnostic platforms. this modular nature should motivate the developers and manufacturers to consider more (semi)open and adaptable platforms to address end-users' needs in a dynamic way, being able to quickly modify their panels and adapt to endemic or epidemic needs. the review concludes with the need of electronic decision trees to support diagnostic tools, as interactive assistance to clinicians. such algorithms are derived from paper-based guidelines to computerized versions (which enforce the provision of output for any possible combination of clinical elements, which is not the case for traditional guidelines), operating on 3g mobile tools such as tablets. they receive, as input, data from several (poc and non-poc) diagnostic tests, which are processed via clinical algorithms, to provide clinicians with guidance regarding the handling of the patient (admission, treatment, etc.). such interoperability between ict components and interfaced physical systems (diagnostic tests) for decision-support can provide solutions of clinical and public health utility, therefore, this trend is expected to increase rapidly, offering high impact diagnosis in developing countries, and transforming the global healthcare landscape. this process, being rather complex and requiring know-how from a variety of disciplines and partners, requires multiple interactions between universities, industries, governmental and non-governmental organizations, as well as philanthropic institutions, that are not only based in wealthy countries but also in resource-limited countries. to increase the chances to make progress, most successful technologies should be supported and developers be motivated to partner with each other. it is only with such a spirit that the development pathway can be coherent and harmonized, and the new tools eventually may be implemented at large scale. looking into future perspectives, it seems that a combination of technological and non-technological factors will be critical to decide whether the diagnostic poc technologies can be used in the management of patients with febrile illness. from the technological perspective, it would be interesting to observe the landscape in the next 5-8 years and whether label-free detection technologies can be transferred from the laboratory to the clinics, and then to the market. magnetic field, electrochemical, gravimetric (e.g. surface acoustic waves), and micromechanical (e.g. cantilever based) detection technologies are some laboratory-mature fig. 31 . schematic representation of the potential integration of a mobile patient management system (efever) and a centralized electronic disease surveillance system (emergence). source: valérie d'acremont. candidates that could potentially substitute fluorescence detection and offer the advantage of cheaper read-out devices. regarding label-free technologies, it is important to consider how they can potentially be integrated into sample-to-answer systems, in view of the requirements outlined in sections 2.3 and 2.4. in addition, it is important to keep track of the progress on the lft-based nucleic acid analysis and detection, a concept that is rapidly catching up and combines the easiness of lfts with the molecular analysis. the transition from the laboratory to the market is crucial and depends on multiple parameters. a fundamental one is the sustainable and scalable manufacturability of the disposable cartridges (in terms of materials and processes). ideally, developers should have access to fabrication facilities that could perform the scale-up manufacturing in two phases: first, from prototyping to medium scale manufacturing (a few thousands or tens of thousands cartridges/chips/components per year) in order to serve clinical validation studies; and in a next level to aim for large scale manufacturing (tens of thousands to hundreds of thousands, that are fairly realistic quantities for infectious diseases of global impact). special attention should be given to the heterogeneous integration of components, i.e. when a system consists of not only plastic/silicon/glass core components, but integrates biological and biochemical agents, which are necessary for the sample-to-answer analysis. issues of stability and compatibility of (bio)chemical reagents with cartridge materials and manufacturing processes, adsorption (and loss) of reagents may prove critical for reaching the desired limit of detection. a future perspective is also that the diagnostic tools are no longer operating in a stand-alone mode, but in a broader "ecosystem". to this direction, integration of ict is of primary importance, as it is the means to connect a variety of different tools and generate networks (existing "traditional" approaches, reference methods, and new innovative tools), create databases, and process data (since we are in the era of big data). along these lines, the combination of nucleic acid with protein biomarker testing seems to be a path for more reliable diagnosis, especially in case of syndromes caused by several different pathogens of bacterial, viral, parasitic, or fungal nature. further elaborating on the ecosystem approach, and especially for diagnostics for resource-limited countries, special business model developments are required that would render marketization in the target areas attractive. large industries or small and medium enterprises (smes) may choose their own way to market, but a new path may be to try marketing in partnership with the pharmaceutical industry. it is obvious that diagnostics and therapeutics are the two sides of the same coin, and only through their combination and interplay can healthcare truly progress. especially in an era that antimicrobial resistance rises dramatically, this exact issue could and should become the bridge to unite the worlds of diagnostics and therapeutics. one parameter that should also be considered during the transition of the poc technologies from the laboratory to the market is the healthimpact modeling, including health economics and cost-impact analysis. one technology may be (comparatively) cheap but may not offer added clinical value; another may be (seemingly) expensive but it may prove overall cheaper than the existing test because the clinical impact is higher. the developers/manufacturers should compare the manufacturing cost of their technology to the real cost of the same test within their healthcare systems. a substitution of an existing standard of care by an innovative technology would be welcome if: it can lead to the same clinical impact at an overall lower price; or it can offer higher clinical impact at the same price. when assessing novel technologies, one should consider what they actually aim to substitute (material savings, time and labor savings, etc.) in order to have a complete view of the cost savings. last but not least is the perspective of adaptability and usability by the potential end-users. whether in resource-limited countries or in the more wealthy part of the world, a technology may be blocked from being used, even if it performs in the best way, due to reasons related to constraints and reluctances that end-users have. a behavioral change of the end-users (through several means, e.g. electronic decision algorithms, e-learning material, supervision tools) is the key factor to increase acceptability and trust towards new technologies. in addition, end-users should be reassured that these emerging tools do not aim to substitute them but to assist them in decision making, for their benefit, and for the benefit of the patients and the whole community. global, regional, and national causes of child mortality: an updated systematic analysis for 2010 with time trends since informal consultation on fever management in peripheral health care settings: a global review of evidence and practice, world health organization invasive salmonella infections in areas of high and low malaria transmission intensity in tanzania reduction of anti-malarial consumption after rapid diagnostic tests implementation in dar es salaam: a before-after and cluster randomized controlled study typhoid fever and the challenge of nonmalaria febrile illness in sub-saharan africa detection of a dengue outbreak through systematic screening of febrile patients in dar es salaam global health experts accuse who of "egregious failure" on ebola world health organization the next epidemic-lessons from ebola report of the ebola innovation summit beyond malaria -causes of fever in outpatient tanzanian children integrated management of childhood illness etiology of severe non-malaria febrile illness in northern tanzania: a prospective cohort study causes of non-malarial fever in laos: a prospective study aetiology of acute febrile episodes in children attending korogwe district hospital in north-eastern tanzania acute uncomplicated febrile illness in children aged 2-59 months in zanzibaraetiologies, antibiotic treatment and outcome world malaria report, world health organization reduction in the proportion of fevers associated with plasmodium falciparum parasitaemia in africa: a systematic review the effect of malaria control on plasmodium falciparum in africa between emergency triage assessment and treatment (etat) course, world health organization a novel electronic algorithm using host biomarker point-of-care-tests for the management of febrile illnesses in tanzanian children (e-poct): a randomized, controlled noninferiority trial integrated management of childhood illness by outpatient health workers: technical basis and overview. the who working group on guidelines for integrated management of the sick child integrated management of adolescent and adult illness: interim guidelines for first-level facility health workers at health centre and district outpatient clinic: acute care, world health organization clinical features for diagnosis of pneumonia in children younger than 5 years: a systematic review and meta-analysis ruling a diagnosis in or out with "sppin" and "snnout": a note of caution refining clinical diagnosis with likelihood ratios new algorithm for managing childhood illness using mobile technology (almanach): a controlled non-inferiority study on clinical outcome and antibiotic use in tanzania electronic clinical decision algorithms for the integrated primary care management of febrile children in low resource settings -review of existing tools evaluation of symptom checkers for self diagnosis and triage: audit study fever in the returned traveler pcr using blood for diagnosis of invasive pneumococcal disease: systematic review and meta-analysis point-of-care testing for infectious diseases: diversity, complexity, and barriers in low-and middle-income countries tropical infectious diseases: diagnostics for the developing world target product profile for a diagnostic assay to differentiate between bacterial and nonbacterial infections and reduce antimicrobial overuse in resource-limited settings: an expert consensus malaria diagnosis: a brief review learner's guide admission diagnosis of cerebral malaria in adults in an endemic area of tanzania: implications and clinical description overdiagnosis of malaria in patients with severe febrile illness in tanzania: a prospective study detection of salmonella typhi by nested polymerase chain reaction in blood, urine, and stool samples mix-infection of s. typhi and paratyphi a in typhoid fever and chronic typhoid carriers: a nested pcr based study in north india lateral flow assays from lateral flow devices to a novel nano-color microfluidic assay clinical performance of an automated reader in interpreting malaria rapid diagnostic tests in tanzania lateral flow immunoassay systems: evolution from the current state of the art to the next generation of highly sensitive, quantitative rapid assays field evaluation of a camera-based mobile health system in low-resource settings the comparative accuracy of 8 commercial rapid immunochromatographic assays for the diagnosis of acute dengue virus infection comparative field performance and adherence to test results of four malaria rapid diagnostic tests among febrile patients more than five years of age in evaluation of four rapid diagnostic tests for the diagnosis of plasmodium vivax in korea comparative performance of aldolase and lactate dehydrogenase rapid diagnostic tests in plasmodium vivax detection diagnostic test performance: summary results of who product testing of malaria rdts: rounds 1-6 the unusual origin of the polymerase chain reaction isothermal nucleic acid amplification technologies for point-of-care diagnostics: a critical review nucleic acid amplification: alternative methods of polymerase chain reaction nucleic acid sequence-based amplification nasba isothermal enzymatic in vitro nucleic acid amplification optimized for the diagnosis of hiv-1 infection loop-mediated isothermal amplification of dna accelerated reaction by loop-mediated isothermal amplification using loop primers simultaneous multiple target detection in realtime loop-mediated isothermal amplification dna detection using recombination proteins international external quality assessment of molecular detection of rift valley fever virus helicase-dependent isothermal dna amplification isothermal dna amplification in vitro: the helicase isothermal in-vitro amplification of dna by a restriction enzyme-dna polymerase system isothermal reactions for the amplification of oligonucleotides multiplex strand invasion based amplification (msiba) assay for detection of chlamydia trachomatis and neisseria gonorrhoeae rolling circle dna synthesis: small circular oligonucleotides as efficient templates for dna polymerases clinical validation of xpert mtb/rif for the diagnosis of extrapulmonary tuberculosis genexpert tb 8: a pointof-care diagnostic pilot genexpert mtb/rif on bronchoalveolar lavage samples in children with suspected complicated intrathoracic tuberculosis: a pilot study review of use of a new rapid real-time pcr, the cepheid genexpert ((r)) (xpert) ct/ng assay, for chlamydia trachomatis and neisseria gonorrhoeae: results for patients while in a clinical setting technology for automated, rapid, and quantitative pcr or reverse transcription-pcr clinical testing future of dna diagnostic testing point-of-care nucleic acid testing for infectious diseases filmarray, an automated nested multiplex pcr system for multi-pathogen detection: development and application to respiratory tract infection rapid identification of pathogens from pediatric blood cultures by use of the filmarray blood culture identification panel multicenter evaluation of the biofire filmarray gastrointestinal panel for etiologic diagnosis of infectious gastroenteritis spectrum of enteropathogens detected by the filmarray gi panel in a multicentre study of community-acquired gastroenteritis use of the filmarray system for detection of zaire ebolavirus in a small hospital in comparison of the filmarray respiratory panel and prodesse real-time pcr assays for detection of respiratory pathogens sample preparation for difficult sample types, us patent rapid and simple method for purification of nucleic acids specific synthesis of dna in vitro via a polymerasecatalyzed chain reaction product differentiation by analysis of dna melting curves during the polymerase chain reaction evaluation of alere i influenza a&b for rapid detection of influenza viruses a and b multicenter clinical evaluation of the novel alere i strep a isothermal nucleic acid amplification test detection of influenza a and b with the aleretm i influenza a & b: a novel isothermal nucleic acid amplification assay, influenza other resp detection and differentiation of hiv-2 using the point-ofcare alere q hiv-1/2 detect nucleic acid test accurate early infant hiv diagnosis in primary health clinics using a point-of-care nucleic acid test competitive reporter monitored amplification (cma)-quantification of molecular targets by real time monitoring of competitive reporter hybridization the enigma® ml fluab-rsv assay: a fully automated molecular test for the rapid detection of influenza a, b and respiratory syncytial viruses in respiratory specimens performance of a novel point-of-care molecular assay for detection of influenza a and b viruses and respiratory syncytial virus (enigma minilab) in children with acute respiratory infection reaction vessel comprising electrically conducting polymer as heating element fluorescence-based detection methods and apparatus, world patent sample processing a rapid and automated sample-to-result hiv load test for near-patient application vereflu™: an integrated multiplex rt-pcr and microarray assay for rapid detection and identification of human influenza a and b viruses using lab-on-chip technology an integrated lab-on-chip for rapid identification and simultaneous differentiation of tropical pathogens a lab-on-chip based platform for fast molecular diagnosis of multi-drug resistant tuberculosis novel biochip platform for nucleic acid analysis develop the "in-check" platform for diagnostic applications integrated pcr amplification and detection processes on a lab-on-chip platform: a new advanced solution for molecular diagnostics silicon nanowire biosensor for highly sensitive and rapid detection of dengue virus dna sensing by silicon nanowire: charge layer distance dependence silicon nanowire arrays for label-free detection of dna company profile: quantumdx group limited methods and kits for nucleic acid sequencing, us patent detection of mycobacterium tuberculosis by easynat diagnostic kit in sputum samples from tanzania a multicenter study of cross-priming amplification for tuberculosis diagnosis at peripheral level in china cross-priming amplification for rapid detection of mycobacterium tuberculosis in sputum specimens enclosed unit for rapid detection of a target nucleic acid amplification product, us patent rapid detection of haemophilus parasuis using cross-priming amplification and vertical flow visualization nucleic acid assay system for tier ii labs and moderately complex clinics to detect hiv in lowresource settings accessed date comparison of xtag respiratory virus panel and verigene respiratory virus plus for detecting influenza virus and respiratory syncytial virus rapid testing using the verigene gram-negative blood culture nucleic acid test in combination with antimicrobial stewardship intervention against gram-negative bacteremia evaluation of the nanosphere verigene® system and the verigene® f5/f2/mthfr nucleic acid tests method of preparing nucleic acids for detection, us patent the use of gold nanoparticles in diagnostics and detection multiplexed, rapid detection of h5n1 using a pcr-free nanoparticle-based genomic microarray assay emerging technologies for the clinical microbiology laboratory swab elution chamber in a test cartridge, us patent test cartridge with integrated transfer module, us patent fluidic system for performing assays, us patent microfluidic lab-on-achip platforms: requirements, characteristics and applications centrifugation and capillarity integrated into a multiple analyte whole-blood analyser aliquoting on the centrifugal microfluidic platform based on centrifugo-pneumatic valves multifunctional microvalves control by optical illumination on nanoheaters and its application in centrifugal microfluidic devices centrifugopneumatic valve for metering of highly wetting liquids on centrifugal microfluidic platforms event-triggered logical flow control for comprehensive process integration of multi-step assays on centrifugal microfluidic platforms pneumatically pumping fluids radially inward on centrifugal microfluidic platforms in motion centrifugo-dynamic inward pumping of liquids on a centrifugal microfluidic platform batch-mode mixing on centrifugal microfluidic platforms centrifugal micromixery miniature stickpackaging -an industrial technology for pre-storage and release of reagents in labon-a-chip systems real-time stability testing of air-dried primers and fluorogenic hydrolysis probes stabilized by trehalose and xanthan optical sensing system based on wireless paired emitter detector diode device and ionogels for lab-on-a-disc water quality analysis all-in-one centrifugal microfluidic device for size-selective circulating tumor cell isolation with high purity sensitivity enhancement for colorimetric glucose assays on whole blood by onchip beam-guidance centrifugal microfluidic platforms: advanced unit operations and applications one-step pathogen specific dna extraction from whole blood on a centrifugal microfluidic device point-of-care diagnostics for global health a portable centrifugal analyser for liver function screening microsphere integrated microfluidic disk: synergy of two techniques for rapid and ultrasensitive dengue detection fully integrated whole blood testing by real-time absorption measurement on a centrifugal platform magnetic chemiluminescent immunoassay for human c-reactive protein on the centrifugal microfluidics platform rapid and fully automated bacterial pathogen detection on a centrifugal-microfluidic labdisk using highly sensitive nested pcr with integrated sample preparation labdisk with complete reagent prestorage for sample-toanswer nucleic acid based detection of respiratory pathogens verified with influenza a h3n2 virus dna based sample-toanswer detection of bacterial pathogens on a centrifugal microfluidic foil cartridge portable, centrifugal microfluidic platform for automatic molecular diagnostics in a low gravity environment rapid molecular assays for the detection of yellow fever virus in low-resource settings the feverdisk: multiplex detection of fever-causing pathogens for rapid diagnosis of tropical diseases lab-on-a-foil: microfluidics on thin and flexible films microstructuring of polymer films for sensitive genotyping by real-time pcr on a centrifugal microfluidic platform the genepoc platform, a rational solution for extreme point-of-care testing influenza and respiratory syncytial virus detection in clinical specimens without nucleic acid extraction using focus direct disc assay is substantially equivalent to the traditional methods and the focus nucleic acid extraction-dependent rt-pcr assay diagnostics-in-a-suitcase: development of a portable and rapid assay for the detection of the emerging avian influenza a (h7n9) virus development and deployment of a rapid recombinase polymerase amplification ebola virus detection assay in guinea in 2015 point-of-care tests for diagnosing infections in the developing world evaluation of two igm rapid immunochromatographic tests during circulation of asian lineage chikungunya virus detection of chikungunya virus antigen by a novel rapid immunochromatographic test evaluation of a rapid assay for detection of igm antibodies to chikungunya evaluation of serological diagnostic tests for typhoid fever in papua new guinea using a composite reference standard the era of molecular and other non-culture-based methods in diagnosis of sepsis use of clinical algorithms for diagnosing malaria diagnostic tests 4: likelihood ratios integrated management of childhood illness: caring for newborns and children in the community the authors k.m., s.h., f.v.s., and r.z. acknowledge the european commission for funding under the fp7 project "discognosis" (ga-318408), the h2020 project "diagoras" (ga-633780), and the h2020 project "dmc-malvec" (ga-688207).the author v.d.a. thanks blaise genton for critical reading of the manuscript. conflicts of interest: none. key: cord-164916-v5w6senz authors: elhenawy, mohammed; komol, md mostafizur rahman; ashqar, huthaifa i.; almannaa, mohammed hamad; masoud, mahmoud; rakha, hesham a.; rakotonirainy, andry title: developing a novel crowdsourcing business model for micro-mobility ride-sharing systems: methodology and preliminary results date: 2020-07-30 journal: nan doi: nan sha: doc_id: 164916 cord_uid: v5w6senz micro-mobility ride-sharing is an emerging technology that provides access to the transit system with minimum environmental impacts. significant research is required to ensure that micro-mobility ride-sharing provides a better fulfilment of user needs. in this study, we propose a novel business model for the micro-mobility ride-sharing system where light vehicles such as electric scooters and electric bikes are crowdsourced. this new model consists of three entities, the suppliers, the customers, and a management party, which is responsible for receiving, renting, booking, and demand matching with offered resources. the proposed model has the potential to allow the suppliers to define the location of their private e-scooter/e-bike and the period of time they are available for rent, match it with a particular demand, and then offer suppliers the opportunity to get their e-scooters/e-bikes rented and returned at the end of the renting period to the same (nearby) location. the management party will need to match the e-scooter/e-bike to a series of renting demands with the last demand as a destination very close to the initial location of the e-scooter/e-bike at the start of the renting period. one potential advantage of the proposed model is that it shifts the charging and maintenance efforts to a crowd of suppliers. at present days, micro-mobility is a promising urban mobility solution [1] . the term micromobility deals with the incorporation of a short-trip by small vehicle operation. when transportation mobility is restrained to a very limited range of trip for light vehicles only, it is called micro-mobility [2] . vehicles of light categories such as bicycles, motorbike, electric bikes (e-bikes), electric scooters (e-scooters), shared bicycles, and some riding devices like skateboards are considered as micro-mobility rides [3] . micro-mobility ride sharing is a marvelling technique in providing sustainability at this current hazards of over-growing population, traffic congestion and greenhouse impact [4] . environment pollution and carbon dioxide emission can be minimised to an enormous amount of utilising micro-mobility rather than regular motor vehicles [5, 6] . recently, the public interest on micro-mobility like bikesharing systems (bss) or scooters-sharing systems (sss) is proliferating with the rapid transformation of the transportation system. especially for traffic congested routes, this strategy gives an affordable and rapid passage opportunity, saving civilians from intolerable waiting and the squander of time [7] [8] [9] . micro-mobility ride-sharing system is categorised as four generations [10] where dock-based systems are considered as latest updated versions of the first two generations [11, 12] . the emergence of fourth-generation dockless ride-sharing has brought a revolution in the micromobility ride-sharing market [13] due to mass public deliberation to the sharing economy, sustainability and health transports [14] . the early invention of micro-mobility ride-sharing strategy was initiated as dockless bike-sharing, but this system was not prioritised enough due to the lack of technologies to fulfil the system drawbacks. people rather accepted dock-based ride-sharing as more flexible and reliable in comparison to the dockless ride-sharing. however, in modern days, the new available technologies have assured the dockless ride-sharing far more flexible and reliable to users expectation [15] . moreover, this system is handy on installation, less intricate for smartcard integration system on transit and facilitated with advanced power assisting features [10] . rather the dock-based system causes an extra infrastructure cost to be paid for the agglomeration and locking security of micro-mobility rides [15] . nonetheless, the dockless system is considered as more flexible than the dock-based system, their advantages and superiority of implementation varies based on the feature of locations [16] and also on the purpose of the trip, how fast user walks, destination, budgets for the rides, reliability value and any other operators attributes [17] . even the dockless micro-mobility system is bound to a lot of limitations. many of such businesses got collapsed like a short flash of fireworks due to the hardship in maintaining sustainability, oversized fleets and vandalism [15, 18, 19] . moreover, in dockless system, range of the distance to be travelled by scooters is needed to be restricted due to the limitation of battery power charge of scooters. otherwise, the commuters will requisite to abandon the shared scooter in the halfway to their destination if the charge drops below usability condition [16] . in some european countries, private e-scooter is overgrowing rapidly [20] and shared scooter policy is being observed in withdrawal eyes in those places. for extensive use of micro-mobility rides in public roads, it often causes annoyance and conflict with the safety of regular pedestrians [21] . that is why there is also a profusion of complaining over the exaggeration of light vehicles which impact negatively over the business idea of micro-mobility ride sharing. so, the clarification of ambiguity of previous inventions as well as evaluating novel ideas on the micro-mobility ride-sharing system is a pivotal research focus of the modern era. considering all the challenges and hurdling experiences with conventional micro-mobility ride sharing systems, a massive improvement and modifications are desired to cover the drawbacks pertaining to this field. still, a lot of further research is required to give the micro-mobility ridesharing system a better fulfilment of users' expectation. the innovation of business models like micro-mobility ride-sharing is also getting popular concern and being welcomed in public appearance if it can fulfil challenges confronted by the dock-based and dockless ride-sharing systems. in this research paper, a novel business model is demonstrated over the micro-mobility ride-sharing system focusing on recovering flaws of conventional strategies. the proposed model offers numerous advantages features for all classes included in this business strategy. another application to this model is during the periods of pandemics and after the lockdown is ended and restrictions are eased gradually. this model has the potential to serve as a solution to the issues arise after people can return to normal life routines for economic recovery. however, precautions are required to minimize the chance of exposing areas to a second wave of the pandemic. social distancing is one of the most important counter-measures against the spread of a pandemic, which needs to be in effect for months after the peak. social distancing is a challenging issue in cities where public transportation is the main commuting means. consequently, this model could be deployed in some areas of the city to meet some of the demand due to abandoning public transportation. sharing the micro-mobility rides for public necessity and the business expansion over this strategy is not an unprecedented scheme. this idea first evolved around 1965, but for the lack of technologies to track down customers, the users used to remain anonymous and miserable incidents like stealing and destroying of bikes frequently occurred which led the strategy to collapse for practical implementation [22] [23] [24] . as the technology arises with smartphone and gps system, customers can easily track and locate the nearest available sharable rides through gps, and they can even unlock them using their smartphones [16, 25] . it is spreading in greater extend from the last decades with the advancement of technology and management policy [26] . so, researchers are driven to put immense effort to improve micro-mobility ride-sharing management and technologies as it is a popular concern and being used ubiquitously. as time flies, the micro-mobility ride-sharing system is developed and modified with both technology and policy improvements. an innovative simulation study of models without redistribution and with simple redistribution are proposed for more effectual bike redistribution system [27] . when customers intend to rent a bike, they need to know the rental information exploitation mechanisms about the pick-up and the return of bikes. this helps in fulfil their demand properly and prevent probable queue making during renting. in another research, machine learning-based univariate and multivariate regression algorithms have been implemented to model available bikes in rental stations [28] [29] [30] . a greedy randomised adaptive search procedure (grasp) algorithm has also been utilised and improved for bicycle rebalancing problem to solve unavailability of bicycles to rent [31] . random forrest (rf) and multi-layer perception (mlp) have been used to predict the bike availability in different rental stations using the real-world data of some swiss cities. here, the pick-up and the return of the bike is optimised to maintain balance for bike availability based on the prediction [32] . nowadays, mobile phone apps are also being initiated and developed to locate nearby station to rent and ride availability. also, route planning and possible parking for commuters are being presented through these apps [33] . for solving ride unavailability in rental stations, attempts are also taken using policymaking. the characteristics of customers in renting rides from different stations have been evaluated. the tendency of choosing an alternative station with less available bikes is found among customers than the preferred ones [34] . dockless ridesharing is getting superior with economic advantages in the bike-sharing market by implementing remote mobile payment system and the emergence of big-data computation [8, 15, [35] [36] [37] . but for the dockless ride-sharing system, the redistribution due to the imbalance of pick up and the return of rides is more challenging. it is found that maintaining a win-win situation to customers by providing monetary incentives in renting rides is highly effective for balancing the redistribution in the dockless ride-sharing system [38] . e-scooter share trip trajectories were provided at the street link level with precise construction of trajectory trip inventories [39] . contribution of characters individuals to the reduction of heat alert through the bike-sharing system was analysed in research where the factor of age and gender was also showed different behaviour. also, different weather and environmental condition and the ability to expand the system [40] are found to be responsible for the variance of micro-mobility ride hiring by users [41, 42] . apart from age and gender, trip purpose, time of the day, day of the week, population density, median household income and some other demographic and external factors were found as a reagent in other researches on washington dc and austin, tx for the variability of ride-sharing [43, 44] . to understand people's perception and opinion over micro-mobility ride-sharing, researchers accumulated data from social media like twitter, facebook and utilised machine learning approach to evaluate the current toleration and support of ride-sharing business at the present time [45] . complexity and sophistication of mobile applications that operate in micro-mobility ride-sharing assistance are found as an influential factor behind the use of ride-sharing. the young generation is highly acquaintance with complex apps, and they have easily been driven to use micro-mobility as they properly understand the feature and facilities of the system through the apps. however, people of old age highly decline these apps, and they are really sophisticated for them to understand the facility and benefit of this system. for them, an easier version of apps sounds recommended [46] . considering previous literature as mentioned above, it is found that micro-mobility ridesharing system is being developed gradually and this system needs a lot of future improvement and modification to solve myriads of challenges that people face every day in operating this business practice. in this research, a new business model for the micro-mobility ride-sharing system is proposed where light vehicles such as electric scooters, electric bikes are crowdsourced. an advantage of this new model is that it shifts the charging and maintenance efforts to the crowd of the suppliers. in conventional ride-sharing system, the maintenance cost and charging cost due to high repositioning rates of shared-scooters is much higher [16] , which often gets challenging to afford by customers or business party. but in our proposed strategy, this cost will be covered by suppliers, and both customers and maintenance party will be relieved from the burden of sharing this cost. however, the incentive payments by the customers to suppliers will cover some of its expense as shared policy. the proposed model consists of three entities. the suppliers, who are the people who are offering their private e-scooters/e-bikes 1 for hire. the customers, who are the people that create hiring demand. finally, the management party, which is responsible for receiving hiring booking demand and matching it with offered resources. the proposed model allows the suppliers to define the location of their private e-scooter/e-bike and the period of time they are available for rent. the model will allow suppliers to get their e-scooters/e-bikes hired and returned at the end of the renting period to the same location or another near location. this means that the management party needs to match the e-scooter/e-bike to a series of hiring demands with the last demand as a destination very close to the initial location of the escooter/e-bike at the start of the hiring period. an advantage of this new model is that it shifts the charging and maintenance efforts to the crowd of the suppliers. in conventional ride-sharing system, the maintenance cost and charging cost due to high repositioning rates of shared-scooters is much higher [16] , which often gets challenging to afford by customers or business party. but in our proposed strategy, this cost will be covered by suppliers, and both customers and maintenance party will be relieved from the burden of sharing this cost. however, the incentive payments by the customers to suppliers will cover some of its expense as shared policy. there are many approaches that could be used to match the supply and demand in a ride-sharing system. depending on the booking scheme allowed by the model, we can choose the matching approach. the first possible approach is a data-driven one, which is suitable for real time booking. the naivest variant of the data-driven approach is what can be referred to the two-leg round-trip approach. the current e-scooter service is assumed to be meeting only 50% of the total demand volume, thus; we used the available data about the current trips to estimate the origin-destination matrix of non-observed/hidden demand. consequently, we plan routes such that we assign e-scooters that are located at route endpoints to service this route. the e-scooters will be assigned trips only over this route such that it travels back and forth between the two ends of the route and the destination of the last trip is the e-scooters location at the starting of the day. this will require the management application to ask the renters to enter the origin and destination of the trip to find them the e-scooter that assigned to this route. moreover, the management should not allow the e-scooters to get out of their assigned routes. in this approach, the planned routes may vary based on the day-of-the-weak and hour-of-the-day to meet the expected demand. this route variations makes the model flexible and capable of meeting the demand in a more efficient way. allowing more than two-leg round-trip is more complicated than a data-driven approach. we use the estimated o-d matrix to establish routes between more than two points such that an e-scooter can move freely between this subset of points during its renting period. however, at the end of the rental period, the final destination of the e-scooter should be as closer as possible to its initial location. in this paper, we will use an agent-based modelling (abm) approach to establish simple management rules which are suitable for the two-leg round-trip approach. in this subsection we discuss the abm framework of the proposed new model. we established a set of simple rules that will yield a suitable behaviour of the proposed e-scooter model. our model has three types of agents namely; 1. e-scooter agent, 2. central agent, and 3. demand generator agent. the e-scooter agent has four state variables namely, the home location, the current location, availability, and current battery range. the other two agents do not have state variables. however, the demand generator has one behaviour rule. every clock tick, the demand generator looks at the trips data and informs all e-scooter agents of new demand requests including trip information. in other terms, if there are concurrent demands, the demand generator agent chooses one of them to broadcast and wait until the demand is processed then broadcasts another one to the concurrent demand requests. the e-scooter agent rule checks if its current location is the same as the origin of the trip, its battery level, and the availability of e-scooters. if all of the above conditions are satisfied, then the e-scooter agent sends an expression of interest (eoi) to the central agent. the central agent receives eios to serve a particular demand from all e-scooter agents and chooses the winner based on the system's own rules. in this experimental work, we proposed five rules to choose the winner as follows; the first rule chooses an e-scooter agent randomly from a subset of e-scooter agents, which is submitted to the eoi. the second rule chooses the e-scooter agent that submitted the eoi, and it has the largest remaining charge in its battery. the third rule chooses an e-scooter agent randomly from a subset of e-scooter agents, which submitted eoi, and the destination of the demand (i.e. trip) is the home location of the e-scooter agent. the fourth rule chooses the e-scooter agent, which submitted eoi, the destination of the demand (i.e. trip) is the home location of the e-scooter agent, and it has the largest remaining charge in its battery. the fifth rule drops the demand if there is no any eoi. this study used a dataset that was collected in the city of austin, texas and is publicly available by the city of austin [47] . the dataset contains about 9.2 million trips taken by users for either e-bikes or e-scooters from december 2018 to january 2020. each trip is represented in a row and each of which has 18 features: trip id, device id, vehicle type (e-scooter or e-bike), trip duration (in seconds), trip distance (in meter), start time of the trip, end time of the trip, month, hour, day of week, year, council district (both start and end), and census tract (both start and end). table 1 shows the total number of e-scooter and e-bikes that operate in the city. we should highlight that the data is reidentified by replacing the location of origin and destination of each trip by the census tracts of these locations. we removed all the trips that are missing the start census tract and/or end census tract and consequently constructed the origindestination (o-d) matrix. the number of trips after removing the above trips reached to 9,174,541 trips (originally it was 9,231,107 trips). based on the o-d matrix we have two observations. first, most of the trips are concentrated between particular census tracts and secondly, a significant percentage of trips started and ended in the same census tract as shown in figure 1 . both of those observations are expected because the majority of the trips are escooter trips and these trips are usually short trips and e-scooters are deployed in a limited area at the austin downtown area. to this end, in the following analysis we used a reduced o-d by selecting fourteen census tracts shown in figure 1 (c) and (d), which include almost 90% of the total trips in austin, tx. it is apparent that the large numbers on the diagonal of the matrix in figure 1 (d) means that a high percentage of the trips start and end in the same census tract. in this section, we used the trips data of the reduced o-d matrix to investigate the concept of the new model. in other terms, we used a real dataset of the reduced o-d matrix to run our proposed abm for different combinations of the central agent rules and chose the combination that satisfies our target performance. we used two criteria to evaluate the aggregated behaviour of the abm when changing the central agent rules. the first criterion is called home index (hi). we defined hi as the percentage of e-scooter agents that ended up at the users' home location at the end of the operation hours, as follows; where is an e-scooter that ended up at a user's home at the end of hours of operation, and is number of the e-scooters that satisfied one demand at least during the hours of operation. the second criterion is the percentage of satisfied demand (psd), which is the ratio of the demand/trip met at least once during the hours of operation over the total number of trips. we ran the proposed abm simulation using the 389-day worth of data. we assumed that all escooters have the same maximum battery range in the same run. moreover, we assumed that the operation hours started with the same number of e-scooters at each of the fourteen origins for each run. we simulated the proposed abm six times using a maximum battery range of (35, 45 , and 60 km) and different number of e-scooters at each origin of (50, and 100). hi and psd were calculated for every day at the different combination of the proposed centre agent rules. to compare the result, we used mixed effect gamma regression model to explain the variability in hi and psd in terms of the combination of the proposed rules. table 2 shows the indicators variables used to encode the tested combination. table 2 the indicator variables used to encode the tested combination. the e-scooter trips of the 389 days were used in the abm environment to simulate the proposed model and estimate the hi for the 389 days. figure 2 shows the box plots of the hi results versus the different scenarios. the figures show that scenarios 3 and 4 yield higher hi than the other two scenarios, as it includes the rule of which destination of the demand (i.e. trip) is the home location of the e-scooter agent. moreover, we used the gamma mixed effect model to test whether the differences in hi results between the scenarios are statistically significant. as shown in table 3 , the indicator variables corresponding to scenarios 3 ( 2 ) and 4 ( 3 ) are significant. however, only when the system contains 100 e-scooters, the indicator variable for scenario 2 is significant. results of hi show that when the system contains 50 e-scooters, hi of scenarios 3 and 4 are significantly larger than the hi of the scenario 1. it also shows that at the same condition of 50 e-scooters, hi of scenario 2 is not statistically significant. finally, it shows that at 100 e-scooters condition, hi of scenarios 2, 3, and 4 are significantly higher than hi of scenario 1. table 3 the p-values of the scenario's indicator variables. we next compare between the four scenarios in terms of the percentage of satisfied demand (psd) at different conditions of the e-scooters number and battery ranges. figure 3 shows the box plots of the pds versus the scenarios. the figures show that almost all scenarios have the same psd values at the same conditions and psd increases as the used number of e-scooters in the model increases at each census tract. we also modelled psd results using mixed effect gamma regression model as a function in the scenarios. the p-values of the indicator variables corresponding to the scenarios are shown in table 4 . these p-values dictate that scenario 3 is different from the scenario 1 at all conditions. recently, the public interest on micro-mobility modes has been proliferating with rapid pace transforming the transportation systems in many cities. micro-mobility offers affordable and rapid commute opportunity, saving users from intolerable waiting and the squander of time in congested areas. during the covid-19 pandemic, surveys showed that more people have shifted to micro-mobility ride-sharing modes making it less affected by covid-19 compared to other public transportation sectors like buses and trains. in this research, we propose a new business model for the micro-mobility ride-sharing systems, where e-scooters and e-bikes are crowd-sourced. this novel model consists of three entities including the suppliers, the customers, and the management party. the proposed model is believed to offer several advantages features for all classes included in this business strategy. we hypothesis that this type of business-based models is getting popular and being welcomed in public appearance and has the potential to change the micro-mobility sharing system market, if it succeeds. an agent-based modelling (abm) framework was built to control the proposed model using five different rules. we tested the model using a dataset that contains nine million e-scooter trips in austin, tx. we used two criteria to evaluate the aggregated behaviour of the abm model when changing the central agent rules over four different scenarios. we used two criterions, the home index (hi) and the percentage of satisfied demand (psd). to compare the results, we used mixed effect gamma regression model to explain the variability in the four scenarios in terms of the combination of the proposed rules. the gamma mixed effect model was used to test whether the differences are statistically significant. we ran the agent-based simulation six times using three maximum battery ranges (35, 45 , and 60 km) and different number of e-scooters at each origin (50, and 100). our results show the two criterions were met for the four agent levels, meaning that the business-based e-scooter model achieved the two operation criterions, namely, (1) the e-scooter end up at their home location at the end of day (2) the demand is partially satisfied. both criterions will improve as we increase the available number of e-scooters and the battery ranges. a promising application of this novel model could be in a crowded city centre, where employees arrive at their offices early in the morning using e-scooters/e-bikes and they stay until 5:00 pm. these e-scooter/e-bike could be offered for rent during the entire period they are at offices under the constrains that at the end of the office hours the e-scooter/e-bike is returned to the same point or nearby point they were at the beginning of the day and have a residual charge enough for the owner to go back home. e-scooters on the ground: lessons for redesigning urban micro-mobility adoption intentions for micro-mobility -insights from electric scooter sharing in taiwan notes on the practices and appearances of e-scooter users in public space optimal investment and management of shared bikes in a competitive market car free cities: pathway to healthy urban living urban and transport planning, environmental exposures and health-new concepts, methods and tools to improve health in cities spatiotemporal comparative analysis of scooter-share and bike-share usage patterns in integrating shared autonomous vehicle in public transportation system: a supply-side simulation of the first-mile service in singapore transit-oriented autonomous vehicle operation with integrated demand-supply interaction understanding the diffusion of public bikesharing systems: evidence from europe and north america public bikesharing in north america: early operator understanding and emerging trends strategic design of smart bike-sharing systems for smart cities dockless bike-sharing systems: what are the implications? a spatial framework for planning station-based bike sharing systems understanding the usage of dockless bike sharing in singapore understanding spatio-temporal heterogeneity of bike-sharing and scooter-sharing mobility unreliability of delay caused by bike unavailability in bike share systems challenges of collaborative governance in the sharing economy: the case of free-floating bike sharing in shanghai unravel the landscape and pulses of cycling activities from a dockless bike-sharing system e-scooter companies find success despite business model difficulties smart bikes: public transportation for the 21st century will smart bikes succeed as public transportation in the united states? bike-sharing: history, impacts, models of provision, and future smartphone transportation mode recognition using a hierarchical machine learning classifier and pooled features from time and frequency domains bikeshare: a review of recent literature analyzing bike repositioning strategies based on simulations for public bike sharing systems: simulating bike repositioning strategies for bike sharing systems modeling bike availability in a bike-sharing system using machine learning network-wide bike availability clustering using the college admission algorithm: a case study of san francisco bay area bike share travel time modeling: san francisco bay area case study an improved grasp for the bike-sharing rebalancing problem bike usage forecasting for optimal rebalancing operations in bike-sharing systems using mobile phones to crowd-source user flow data for assessing bike sharing site suitability bike commuters contribution to balance shared bike systems during peak load traffic state prediction: a traveler equity and multi-modal perspective smart bike-sharing systems for smart cities quantifying the effect of various features on the modeling of bike counts in a bike-sharing system how does dockless bike-sharing system behave by incentivizing users to participate in rebalancing? exploratory analysis of real-time e-scooter trip data in predicting station locations in bike-sharing systems using a proposed quality-of-service measurement: methodology and case study heat warnings and avoidance behavior: evidence from a bike-sharing system modeling bike counts in a bike-sharing system considering the effect of weather conditions bike lanes and other determinants of capital bikeshare trips a comparative analysis of e-scooter and e-bike usage patterns: findings from the city of public opinion on dockless bike sharing: a machine learning approach exploring travelers' characteristics affecting their intention to shift to bike-sharing systems due to a sophisticated mobile app dockless vehicle trips -open data the authors confirm contribution to the paper as follows: study conception and design: all authors; analysis and interpretation of results: all authors; draft manuscript preparation: all authors. all authors reviewed the results and approved the final version of the manuscript. key: cord-103490-cy6db8bf authors: gerritse, emma j.; hasibi, faegheh; vries, arjen p. de title: bias in conversational search: the double-edged sword of the personalized knowledge graph date: 2020-10-20 journal: nan doi: 10.1145/3409256.3409834 sha: doc_id: 103490 cord_uid: cy6db8bf conversational ai systems are being used in personal devices, providing users with highly personalized content. personalized knowledge graphs (pkgs) are one of the recently proposed methods to store users' information in a structured form and tailor answers to their liking. personalization, however, is prone to amplifying bias and contributing to the echo-chamber phenomenon. in this paper, we discuss different types of biases in conversational search systems, with the emphasis on the biases that are related to pkgs. we review existing definitions of bias in the literature: people bias, algorithm bias, and a combination of the two, and further propose different strategies for tackling these biases for conversational search systems. finally, we discuss methods for measuring bias and evaluating user satisfaction. which is a structured form of information about "entities personally related to a user, their attributes and the relations between them" [2] . pkgs provide the system with (locally stored) rich information about the user, to tailor the answers as much to their liking as possible [2] . however, introducing such a data resource and personalizing search results is not without risks: personalization can lead to echo-chamber effect and reinforce bias [4] . bias in search and recommendation systems can influence human decision making, contribute to societal and political biases, and impact the health of our society [14] . examples of bias in current search and social media websites include google's anti-brexit bias 1 , facebook's filter bubbles during us presidential election 2 , and linkedin's gender bias 3 . bias can get even more severe in a conversational setting because: (i) answers in conversational systems are concise and there is less chance of showing diversified results compared to the traditional ten blue links interface, and (ii) users tend to access information that are in line with their prior views [12] , and conversational systems provide personalized results to optimize for high user satisfaction and engagement, thereby intensifying bias. this raises several questions about bias in conversational search. can forms of bias be introduced in this personalized search setting, which would not happen so easily in other search settings? how will conversational systems influence the behavior of their users? and, can users influence the behavior of their conversational agents? what are the sources of bias and how can we measure bias? how to mitigate bias while keeping users engaged and satisfied? in this position paper, we discuss these questions from multiple angles. we consider the risk of different types of biases, and the places in the system architecture where these might be introduced; a major challenge being the difficulty to distinguish between bias and preference. we discuss how bias can be introduced by search algorithms, specifically when constructing pkgs from users' personal data and using them to provide personalized responses. we further explain how pkgs play the role of a double-edged sword to on one hand amplify bias but on the other hand detect and mitigate bias (section 2). next, we argue that the community has to consider conversational search systems in regard to questions of bias, and find solutions to address it (section 3). the main usage of a conversational system is a convenient form of tackling search; not an assistant to educate the user. if we would correct system results for bias too strongly, the debiasing process can only degrade user satisfaction. conversational search therefore faces quite a challenge with respect to design ethics: not only do we need to think through the biases that we encounter and find solutions to counter those biases, we also have to do so in a manner that maintains user satisfaction. we outline possible approaches to help avoid the reinforcement of biases, while keeping the attraction of personalization to improve user satisfaction. finally, we discuss methods to measure bias and user satisfaction (with respect to bias measures) in conversational systems (section 4). baeza-yates [1] enumerates different types of bias on the web and groups them into three categories: (i) bias that involves only algorithms, (ii) bias that originates from people, and (iii) bias that involves both algorithm and people. we discuss the different types of biases we expect to encounter in conversational search along the same categories. knowledge graph construction bias. knowledge graphs (kgs) are structured repositories of data and powerful means to provide a machine understandable form of knowledge. it is envisaged that public, domain-specific, and personalized knowledge graphs (pkgs) can be used to empower conversational search systems [2] . while generation of general purpose knowledge graphs (e.g., yago and wikidata) and domain-specific knowledge graphs (e.g., geonames and musicbrainz) can involve bias, construction of personalized knowledge graphs can be even a greater source of bias. pkgs can be built from social media feeds, search history [11] , conversation history, and other sources of information for which users give the system access permission (e.g., online shops and contacts). the choice of data source by itself can already introduce bias in pkgs. if the system relies on publicly available social media feeds (e.g., twitter [18] ), pkgs may be biased towards only one aspect of users' preferences: people often use different social media websites for different purposes; e.g., twitter for work and instagram for casual usage. even if the system gets access to the content of a user's private social media feeds, the question remains whether the pkg is diversified enough or not: the user might avoid sharing some aspects of her life in social media. therefore, conversational systems need to account for the incompleteness of pkgs and incorporate it into their search and recommendation algorithms. otherwise, this can negatively affect users' satisfaction, as the user encounter undesired bias in search and recommendation results (e.g., observing only work-related events instead of diversified ones). another source of bias in constructing pkgs is time [2] . timebased events can greatly influence search behavior. for example, during election times people are more likely to share their opinion about politics, even when normally politics is not an important aspect of their lives. another example is that during the covid-19 outbreak in 2020, many people used social media to discuss the epidemiology, even though epidemiology is normally not in their interests. when using social media for knowledge graph construction, this can lead to some kind of time-based bias or time-based clutter, which will "somehow" have to be cleaned up after the event. search algorithms bias. bias can also occur in search and recommendation algorithms. while not much research covers the biases in conversational search, some research has been carried out to measure bias in neural ranking models and embedding algorithms. in a recent study, rekabsaz and schedl [16] found that gender bias in document ranking is intensified when using neural models and in particular the ones that are based on contextual embeddings like bert [8] . bias has been also observed in the word and graph embeddings themselves; e.g., social bias in wikidata graph embeddings [9] and gender bias in word embeddings (like word2vec) [3] . while debiasing embeddings may appear as an immediate solution to mitigate bias, incorporating debiased embeddings in information retrieval algorithms may not immediately affect the ranking outcome, as confirmed by some initial results in [10] . people with their activities and preferences themselves form another source of bias in conversational systems. a study on age demographic of conversational systems' users in the united states showed that the most active users are 30-44 years old 4 . indeed, people's preferences vary by their age group, and therefore a large population of a certain age group can introduce certain biases in the system. for example, a study on recommender systems [19] shows that that preference of phone color differs per age group, and recommendations can be tailored to the age group of users. not only age group, but also gender, location, and language of people can affect the outcome of conversational systems, often causing results biased towards the majority groups. conversational systems are highly interactive and are often designed to be personalized. these two features make conversational systems prone to a vicious cycle of bias that is generated by a biased user interacting with a biased algorithm, thereby creating a filter-bubble effect. personalized knowledge bases are double-edged swords in such a setting: they can be used to accelerate personalization and therefore providing even more biased results to the user, or they can be used to detect a biased user and help to diversify results. drawing a line between personalization and diversification is a challenge and varies for different scenarios. here, we enumerate three different scenarios as examples: • a user wants to order the same guitar strings (s)he previously ordered. however, these might not be the highest quality strings. should the system recommend other strings or guide the user to buy the previously bought strings? • a user has a certain political preference and asks a political question about her favorite party. should the system takes users' preferences into account, or provide a neutral answer? • a user searches for a known conspiracy, e.g., fake news about covid-19 outbreak. knowing that the user trusts the source of information based on available information in pkg, how the system should respond to the query? in the following section, we propose some solutions that a system can take to handle bias. conversational search systems can take a wide range of strategies against bias, from being completely ignorant to chastising users. in this section, we discuss three main strategies that these systems can take and argue against being ignorant. in this setting, the system does not detect bias and takes no action against it. depending on the search algorithm, the system may provide nonsensical responses or get along with users and provide them with what they ask. such systems can fall into the trap of creating a vicious cycle of second-order bias (generated from people and algorithmic bias) [1] and then become their own enemy. an example of such a scenario is microsoft's ai chatbot that had to be taken down 16 hours after release, because of learning harmful intents from some twitter users 5 . we argue that being ignorant and not accounting for bias is not an option for conversational search systems, especially because they are meant to work in a highly personalized setting. in this strategy, systems detect bias and offer the user control through its ui/ux, from being viciously biased or offering diversified results; see figure 1 for an example. there is a wide spectrum of actions that systems take in this strategy, ranging from complete neutralization of results to playing along with users until reaching a certain threshold (e.g., inappropriate user requests). choosing a spot in this wide spectrum of approaches is a challenge and requires carrying out user studies. in addition, users may have different tolerance for interacting with diversified answers: some may appreciate it and others may abandon the system altogether. most of the current commercial systems choose to "play along" with users or take a middle approach of not answering or providing "do not know" answers to gender and sexual questions [5] . this, however, may not be an ideal setting for the well being of all users. providing users with an option to choose a degree of diversification (and therefore mitigate bias), e.g. with a ui control like a slider, may alleviate these issues. this does however raise additional implementation challenges, especially for data-driven approaches (like neural networks). incorporating knowledge graphs and in particular pkgs can help detecting bias in the first place and further control it by providing factual and diversified responses (e.g., alternating between nodes of pkgs) to reduce bias. another approach is being reflective about bias: both user and system detect bias and explicitly reflect it to each other. so here debiasing is the responsibility of both user and system. in the case of user bias, the system notifies the user in an appropriate language and suggests alternatives (see figure 2 ). educating users with an explicit response may be considered as an over-reaction, however, it is desired for some cases; e.g., healthrelated fake news in a pandemic like covid-19, sexual harassment, this topic seems to be controversial. according to the who, adding pepper to your soup does not help prevent covid-19. adding pepper does not help against covid-19, it seems to be a myth. here is a link to the website of the world health organisation, discussing why this does not work, and other myths surrounding covid-19. figure 2 : example of the reflective strategy, where the system detects that the user is biased towards fake news, and the system supplies them with an alternative. or cases when the user is gathering confrontational viewpoints for an argument. users may also correct an unnecessary bias of system and help to create a balance between bias and preference. examples of such scenarios include: users changing their interests and opinions over time, or searching for something either on the spur of the moment or on someone else's request. in such cases, systems should provide the user with the source of bias and the option to mediate it. pkgs, while being a source of bias, can help the explainability of the system and enable users to influence the behavior of systems by editing their own pkg. in this section, we propose methods to measure bias in conversational search and further evaluate user satisfaction when applying debiasing methods. here, we do not aim to provide formal evaluation measures for bias, focusing instead on methods that can identify bias. researchers have raised challenges of measuring bias in online information [15] and search engines [13] . measuring bias in conversational search raises even more challenges, due to the difficulties of evaluating the systems themselves. while test collections like trec cast [7] and quac [6] exist, they are often restricted to the question-answering part of conversational systems. evaluating multiple aspects of a conversational system is often performed with user experiments, and is mainly performed by considering general user satisfaction. we argue that a system's ability to handle bias should be measured separately and be an integral element of user evaluations in personalized conversational search systems. another option to measure bias in conversational systems is to have an evaluation method that does not require relevance judgments. in [16] , one such method is discussed for computing gender bias in search results. this is performed by computing a term frequency based score with predefined gendered words. similar approaches can be employed to measure the bias of an answer to a query, not only with respect to gender but other aspects like political bias. it is important to note that bias is a relative concept and depending on the context, a biased answer may not be always undesired. one way of measuring bias is computing the results with and without pkgs; e.g., by using the general purpose knowledge graphs. if the difference is rather large, then it indicates that personalization has occurred in a large degree (which is not always an undesired behavior). this is indeed under the assumption that global knowledge graphs are fair representation of information. one can also generate the results with an opposite knowledge graph, where nodes and relations of user's pkg are replaced with opposite information. we acknowledge that this concept does not generalize, as opposite information may not be available for all nodes. however, this solution may provide interesting insights for certain biases like gender and politics. consider for example swapping "female", "right-wing", and "conservative" with "male", "left-wing", and "progressive", and compare the bias scores as described in [16] . this indicates the influence of these nodes on the results. if there is a large difference between the two polarities, it means that personalization has occurred to a large extent. changing system behavior to handle bias, either by diversifying results or offering a reflective option, may change user satisfaction. it is expected that users do not appreciate the feeling of being judged or educated by their conversational assistant. it is therefore wise to first study the best strategy of handling bias, before investigating technical aspects of offering these solutions. to this end, users satisfaction can be measured by wizard experiments, similar to [17] . here, a wizard is a human who pretends to be a conversational search system and interacts with a human. these studies can be performed by instructing a wizard to offer perspectives to questions of human test subjects and measure user satisfaction. in this paper, we touched upon some aspects of bias in conversational search. we raised questions about bias in the personalized search setting, especially using personalized knowledge graphs. we discussed the challenge of distinguishing between bias and preference, caused by the inherent bias of user preferences and also limitations of offering diversified results in conversational settings. we enumerated different types of bias present in conversational search, being algorithmic bias, people bias and bias introduced by both algorithm and people. we also suggested possible solutions for handling bias, based on diversification and mutual reflection of bias between user and system. lastly, we suggested methods of measuring bias and evaluating user satisfaction. bias in conversational search is an aspect that requires extra attention and we urge the community to keep it in mind while developing conversational search systems. bias on the web personal knowledge graphs: a research agenda man is to computer programmer as woman is to homemaker? debiasing word embeddings bias in algorithmic filtering and personalization #metoo alexa: how conversational systems respond to sexual harassment quac: question answering in context trec cast 2019: the conversational assistance track overview bert: pre-training of deep bidirectional transformers for language understanding measuring social bias in knowledge graph embeddings effect of debiasing on information retrieval measuring demonstrated potential domain knowledge with knowledge graphs events and controversies: influences of a shocking news event on information seeking measuring search engine bias facts-ir: fairness, accountability, confidentiality, transparency, and safety in information retrieval on measuring bias in online information do neural ranking models intensify gender bias exploring conversational search with humans, assistants, and wizards personal knowledge base construction from text-based lifelogs we know what you want to buy: a demographic-based system for product recommendation on microblogs key: cord-353241-ityhcak7 authors: zhu, hanliang; podesva, pavel; liu, xiaocheng; zhang, haoqing; teply, tomas; xu, ying; chang, honglong; qian, airong; lei, yingfeng; li, yu; niculescu, andreea; iliescu, ciprian; neuzil, pavel title: iot pcr for pandemic disease detection and its spread monitoring date: 2020-01-15 journal: sens actuators b chem doi: 10.1016/j.snb.2019.127098 sha: doc_id: 353241 cord_uid: ityhcak7 during infectious disease outbreaks, the centers for disease control need to monitor particular areas. considerable effort has been invested in the development of portable, user-friendly, and cost-effective systems for point-of-care (poc) diagnostics, which could also create an internet of things (iot) for healthcare via a global network. however, at present iot based on a functional poc instrument is not available. here we show a fast, user-friendly, and affordable iot system based on a miniaturized polymerase chain reaction device. we demonstrated the system’s capability by amplification of complementary deoxyribonucleic acid (cdna) of the dengue fever virus. the resulting data were then automatically uploaded via a bluetooth interface to an android-based smartphone and then wirelessly sent to a global network, instantly making the test results available anywhere in the world. the iot system presented here could become an essential tool for healthcare centers to tackle infectious disease outbreaks identified either by dna or ribonucleic acid. dengue fever, ranked as the most important mosquito-borne viral disease with pandemic potential, emerged as a serious public health concern. the world health organization (who) reported that in the past fifty years, the number of cases has increased 30 times, causing huge human and economic cost which has yet to be tackled [1] . according to studies [2, 3] , there were an estimated 390 million annual cases of dengue fever and an estimated 3.9 billion people in 128 countries are at risk of being infected by dengue fever virus (denv) with different serotypes. the denv is affecting numerous areas in eastern and southeast asia, such as china [4] ; the lowlands of nepal [5] ; the slums in delhi, india [6] ; hanoi, vietnam [7] ; and iran [8] as well as other parts of the world, such as the middle east, north africa [9] , and south america [10] . due to the lack of effective treatment of severe dengue fever [11] , accurate and early detection methods to identify the denv serotype [5, 12, 13] are required in order to provide careful patient monitoring and to prevent the disease's progression to a more severe stage. mobile laboratories suitable for tackling highly contagious diseases such as ebola, have been developed [14] , but they are too bulky and expensive for non-contagious denv, malaria or similar diseases. an easy-to-use, affordable, truthful and reliable point of care (poc) device to perform rapid and economical denv diagnostic tests in high-risk areas is badly needed. moreover, it is of the utmost importance to immediately report the results of the poc, including its time and location, to authorities in a centralized location to take required measures. these days, a vast amount of the human population has possession of a cell phone with an embedded global positioning system (gps) connected to mobile networks via base stations using different generations of cellular mobile communications, such as second ( telecommunications system) or fourth (long term evolution (lte)/ worldwide interoperability for microwave access). connecting the easy to use and cost-effective poc devices providing the denv diagnoses via a mobile network would create an internet of things (iot) [15] for healthcare [16, 17] , an essential tool to tackle any infectious disease outbreak. the iot would speed up the information transfer from poc devices to a centralized location and then based on models using big data analysis [12, 18, 19] , suggest targeting specific sites. the iot would be able to communicate with the poc systems, inform its operator where to go next to perform testing, remotely change the test protocols or provide other important information to the poc device user. this year, the fifth generation of cellular mobile communications will be released with a massively increased data rate compared to those of previous generations. nevertheless, the iot for poc applications such as denv testing can rely on the oldest generation of 2g, as the poc communication with the centralized location requires a low data rate, an important feature for cash strapped countries of the developing world. denv is often detected by a paper-based immunochromatographic test either alone [20] or in combination with malaria [21] , enzymelinked immunosorbent assay (elisa) [22] , and/or reverse transcription polymerase chain reaction (rt-pcr) [23] (allowing identification of the denv serotype) [12] . the paper-based lateral flow immunechromatographic test is fast and easy to usetherefore is often used in remote areas. however, due to its lower sensitivity as well as specificity, a confirmation using a second detection method (elisa and/or rt-pcr) is often required. the elisa method relies on antibody production by the human immune system, the method being more suitable for advanced stages of the disease. the rt-pcr is the preferred method as, in principle, it can detect a single copy of specific ribonucleic acid (rna) [24, 25] having both sensitivity and specificity required for early disease detection (lowering the cost of treatment). unfortunately, rt-pcr tests are typically only carried out in hospitals or certified diagnostic laboratories after the onset of denv symptoms. the rt-pcr method is highly specific and reliable; the problem is that not every infected patient makes it to the hospital to be positively diagnosed. as an outcome, the diagnostic results are not available as quickly and comprehensively as required for disease outbreak control. recently, a lot of effort was invested in the development of a portable system for poc diagnostics [26] [27] [28] [29] . numerous methods were developed using a smartphone for data communications as well as fully integrated systems [30] such as a smartphone-based optical system for high throughput [19] . we previously reported a hand-held polymerase chain reaction (pcr) system capable of four simultaneous reactions, demonstrating its performance by amplification of complementary deoxyribonucleic acid (cdna) of avian influenza a (h7n9) rna, proving its principle of operation [31] . later on, a reverse transcription step was added and rna of an ebola virus was detected with rt-pcr while using human transcript glyceraldehyde-3-phosphate dehydrogenase rna as a positive control [31] . here, we report an iot pcr device that can be used for pandemic disease detection and its spread monitoring. it is a portable system with a weight of ≈ 170 g and physical dimensions of (≈ 60 × ≈ 100 × ≈ 34) mm 3 equipped with bluetooth (bt) communication between the pcr and the android wireless tool, such as a smartphone or a tablet. we utilized it to test cdna of a denv, which could also be used for rna detection. once the denv was detected, the unit sent this information, including the gps coordinates to a centralized location via the lte network, thus helping the authorities to map the disease spread (as suggested in fig. 1 ). the core of the system consists of four virtual reaction chambers (vrc) on a hydrophobically coated glass (fig. 2c) which were successfully tested earlier for ultrafast real-time pcr with external optics [32] , and a fully integrated real-time pcr [33] . the glass was placed on a silicon chip made by micro-electro-mechanical system (mems) technology developed earlier [32] . we improved the layout of the mems chip containing heaters and sensors using a nanolithography toolbox [34] by adding an electrically grounded guard ring between the thin film sensor and the heater. we also increased the chip size to (≈ 18 × ≈ 18 mm) 2 to assure easier handling. the chip was placed on its own printed circuit board (pcb) and was connected to the motherboard of the pcr unit by a connector for simple replacement and calibration ( fig. 2b and d) . the electronics as well as the optics ( fig. 2a) was also improved but it is not a subject of this contribution. the pcr systems were then assembled and calibration were ready for deployment. we tested the pcr device performance using cdna of denv prepared by synthesis. real field testing of the device would have to start with sample collection and preparation followed by reverse transcription before the pcr can be conducted [25, 35] . also, reagents for single step qrt-pcr are needed. more details are in the discussion section. we selected an amplicon with a total length of 177 base pairs (bp) from 9937 bp to 10113 bp, as follows: acaagtagaacaacctggtccatacacgccaaacatgaatggatg acaacggaagacatgctgacagtctggaacagggtgtggattcaaga aaacccatggatggaagacaaaactccagtggaatcatgggaggaaa tcccatacttgggaaaaagagaagaccaatggtgcggc using forward and reverse primers with sequences acaagtagaacaacctggtccat and gccgcaccattggtcttctc, respectively. we prepared a solution to conduct the pcr by mixing ≈ 0.3 μl of a commercial polymerase with contents of 5 units μl −1 , ≈ 1 μl of a pcr buffer consisting of 100 mm tris (hydroxymethyl) aminomethane-hcl (ph ≈ 8.3), 500 mm kcl, ≈ 15 mm mgcl 2 , ≈ 0.8 μl of a deoxyribonucleotide triphosphate mixture consisting of adenine, thymine, cytosine, and guanine each with a concentration of ≈ 2.5 mm, ≈ 0.4 μl of forward as well as reverse primer, ≈ 0.5 μl of eva green intercalating dye with original concentration 20× (diluted as per manufacturer instruction), ≈ 1 μl of bovine serum albumin with contents of ≈ 5 mg·ml −1 , and ≈ 1 μl of cdna template with varied contents depending on the experiment. furthermore, the total volume was adjusted by adding ≈ 4.6 μl of sterilized water to a total volume of ≈ 10 μl. prior to testing on an iot pcr device, we verified the master mix performance and its values of critical threshold (c t ) and the melting temperature (t m ) using a commercial real-time pcr system (supplementary section a) beginning with a hot start at 95°c for 30 s followed by 40 cycles of pcr amplification consisting of dna denaturation at 95°c for 8 s, primer annealing at 60°c for 30 s, and dna sequence elongation at 72°c for 10 s, then followed by melting curve analysis (mca) from 72°c to 95°c. once the master mix performance was verified, we ran the same protocol on the iot pcr system. wireless communication between mobile platforms, such as a mobile phone or tablet, and the pcr system was provided via a bt module, which comprised a commercial unit supporting bt version 2.1. this unit has its own dedicated 5 v power supply with a universal serial bus (usb) connector, including ac-dc converter and voltage stabilizer, making it compatible with a standard usb power supply or power bank. the independent power supply for the bt module eliminates its influence on pcr system stability. data transfer between the bt unit and pcr system is conducted bi-directionally via a universal asynchronous receiver-transmitter interface. based on a request from a mobile system, such as a smartphone or tablet, the last pcr measurement data are sent to the mobile system. we developed an application (app) for android devices that allows them to receive data via the bt communication module from the pcr system, save them into a text file, and represent them in graphical format. the smartphone display is split into two parts: the top is used to show the pcr amplification curve and the bottom for mca. the system has the option of sending the data to a dedicated place with gps coordinates to inform authorities about the presence of the infectious disease. after pairing the android device with the bt unit of the pcr system, data from the pcr system are downloaded into the device and plotted on its display. they can also be automatically sent via lte network to a dedicated place monitoring the disease outbreak. for convenience, we also created a personal computer (pc) app for pcr system programming (fig. 3a) either via usb or bt interface. we performed two basic real-time pcr experiments. the first test was conducted with four samples having a master mix with identical contents of the cdna of denv template to verify real-time pcr system uniformity and repeatability as well as temperature settings by performing mca. the second test mimicked the actual field test having three samples and a no template control (ntc) (supplementary section b) . the captured analog-to-digital converter output values of pcr fig. 1 . the schematic diagram demonstrates the iot system that could be applied for denv spread monitoring. once the sample potentially containing the denv is processed at point of interest location, the results of diagnoses as well as gps coordinates of the location are automatically transferred via the user's mobile phone interface through a global network to a control center. all results can be collected as cloud data by a network to create a disease outbreak map showing the disease outbreak area and carry out continuous monitoring. amplification data, as well as mcas, were displayed on the integrated thin film transistor (tft) screen of the pcr unit. then they were transferred to the smartphone via bt to be displayed by the mobile system and also further processed by subtracting the background noise and normalization. the data were then averaged from four consecutive runs and plotted in a linear (fig. 3b) and logarithmic (fig. 3c) scale. we then calculated the c t as the value of the cycle number with 10% of relative fluorescence amplitude as (20.07 ± 0.17) cycle (mean ± standard deviation from four measurements), showing an excellent point to spot result uniformity. we also extracted the mca (fig. 3d ) and performed the curve fitting using a modified boltzmann curve obtaining a t m value of (81.98 ± 0.16)°c (mean ± standard deviation from four measurements). we subsequently performed a numerical differentiation of the fitted curves for better visualization (fig. 3e) by showing the values of t m as peaks of the -df/dt curve. concurrently, we performed identical testing using a commercial real-time pcr system getting comparable results for both, c t and t m (supplementary section a) . then, the data containing the time of experiment and gps location could be sent via lte to the centralized location for further analysis of the disease spreading. we developed an iot pcr system equipped with bt communication and a supporting android app for smartphones as well as a pc app for convenient pcr programming with a projected manufacturing cost between 100-200 usd. this real-time pcr is a robust device with no moving parts and uses four standard light emitting diodes (leds) with a diameter of 5 mm as a light source with a lifetime of more than 50,000 h. the most fragile part is the micromachined silicon chip, here mounted on its own replaceable pcb. we performed a set of experiments using a cdna of denv with 40 cycles of a three-step pcr protocol followed by an mca taking ≈ 34 min in total. the testing time can be shortened either by system redesign [36] or using probe-based pcr and performing a single pcr cycle as fast as 10 s [32] , thus having a total required time for the 40 cycle pcr protocol as short as ≈ 6 min. the system can be further expanded using multiplexing just by reprogramming and effectively doubling the throughput [36] , reprogramming the pcr readout, or using multicolor leds with a dual bandpass filter. once the pcr was completed, the data were transferred via bt to a portable android platform and then they could be sent via lte network together with the pcr unit number, time of test completion and gps coordinates of its location to a centralized location. the positive result could indicate the spread of the infectious disease on a map in a similar form as the hypothetical results from xi'an, shaanxi province, p. r. china (fig. 4) . we performed the first test in our laboratory capturing pcr data as well as the gps location, and then we emulated the tests performed through the city by capturing the locations only (supplementary section c). this pcr with bt and android-based wireless communication could be a key part of an iot system for healthcare ready for deployment to tackle infectious disease outbreaks. an actual approach to tackle infectious disease spreading would require obtaining samples from patients, processing them depending on the body fluid type, then mixing them with the reagents and pre-concentrating them into volume compatible with the miniaturized pcr, which is 0.5 μl or less. samples collected from cheek swabs or from saliva have very low contents of dna, thus hindering the pcr analysis. there are readily available single-step kits for real-time pcr systems capable of processing samples as they are collected [37, 38] . the only requirement is mixing the sample with these reagents. after the mixing step, the cells are enzymatically lysed, so that dna/rna from cells is released, followed by heating to stop the lysis enzymes [39] and activation of the polymerase, which is then followed by pcr thermal cycling. the last two steps would be performed on the hand-held pcr. samples that originate from blood contain albumin and other pcr inhibitors; these have to be first removed by a method compatible with hand-held pcr as shown before [25, 35] using functionalized paramagnetic beads. this sample preparation also includes sample preconcentration to process small sample volumes by hand-held pcr. any part of the system getting into contact with the actual sample has to be disposable due to extremely high contagion of sample to be tested. both systems, potential sample preparation as well as the pcr reaction should be conducted on a disposable microscope cover slip glass coated with suitable fluorosilanes or teflon making their surface hydrophobic [40] . all other parts of the system can be non-disposable since they do not come into contact with either the reagents or the sample. once the test is completed, the used glass objects are disposed of and are then replaced with fresh ones. the device is now ready making the entire process of diagnoses economical. here we tested a denv as a typical representative of an infectious disease spread by mosquitos. the same system can also be applied to monitor the spreading of other diseases, such as malaria. in principle, its deployment could also be possible for highly contagious diseases such as ebola, severe acute respiratory syndrome (sars), and other viruses, but in such circumstances, the system would have to be handled by highly trained professionals due to the highly infectious nature of the viruses. also, for practical reasons, the hand-held pcr system would have to be redesigned for autoclaving to prevent any contamination of the operator. we tested an iot real-time pcr system and demonstrated that the device sensitivity and reproducibility were improved. the pcr device performance is comparable with that of a commercial system. the protocol consisting of 40 cycles to detect the cdna of a denv required ≈ 34 min. the system was connected via bt communication with an android-based smartphone and then with the whole world via lte. this could become a basic block of the iot, thus helping to tackle infectious disease outbreaks. the device can be used for other diseases such as any type of influenza, malaria, and human immunodeficiency virus. it can be used as a "first-defender" tool to perform rapid early diagnosis and, more importantly, the data collected can be used to prevent pandemic outbreaks. pavel neuzil, ciprian iliescu and hanliang zhu wrote the manuscript, hanliang zhu performed the experiments and data analyses, and pavel podesva improved and assembled the pcr systems. xiaocheng liu and haoqing zhang fabricated the pcr chip, tomas teply programmed the internal pcr software as well as the android app; honglong chang participated in the organizing and writing of the manuscript; ying xu conceived the pcr as an iot device and participated in writing the manuscript; airong qian, yingfeng lei, and yu li chose the cdna sequence and designed the primers; andreea niculescu wrote the iot part of the manuscript; and pavel neuzil designed the portable pcr concept as well as the micromachined chip. authors declare no competing interests. global brief on vector-borne diseases, who the global distribution and burden of dengue refining the global spatial limits of dengue virus transmission by evidence-based consensus characterizing a large outbreak of dengue fever in guangdong province cost-effective real-time reverse transcriptase pcr (rt-pcr) to screen for dengue virus followed by rapid single-tube multiplex rt-pcr for serotyping of the virus widespread fear of dengue transmission but poor practices of dengue prevention: a study in the slums of delhi cost-of-illness and the health-related quality of life of patients in the dengue fever outbreak in hanoi in 2017 emerging and re-emerging infectious diseases in iran, iran abu-raddad, dengue in the middle east and north africa: a systematic review the burden of dengue fever and chikungunya in southern coastal ecuador: epidemiology, clinical presentation, and phylogenetics from the first two years of a prospective study dengue fever: causes, complications, and vaccine strategies internet-of-things and big data for smarter healthcare: from device to architecture, applications and analytics development of a fluorogenic rt-pcr system for quantitative identification of dengue virus serotypes 1-4 using conserved and serotype-specific 3' noncoding sequences mobile laboratories for ebola and other pathogens a communitybased iot personalized wireless healthcare solution trial a comprehensive review on usage of internet of things (iot) in healthcare system the internet of things in healthcare potential applications and challenges, it prof innovative healthcare systems for the 21st century a multichannel smartphone optical biosensor for high-throughput point-of-care diagnostics evaluation of the mrl diagnostics dengue fever virus igm capture elisa and the panbio rapid immunochromatographic test for diagnosis of dengue fever in jamaica multiplexed, patternedpaper immunoassay for detection of malaria and dengue fever comparison of capture immunoglobulin m (igm) and igg enzyme-linked immunosorbent assay (elisa) and nonstructural protein ns1 serotype-specific igg elisa for differentiation of primary and secondary dengue virus infections rapid detection, serotyping and quantitation of dengue viruses by taqman real-time one-step rt-pcr an integrated fluorescence detection system for lab-on-a-chip applications clockwork pcr including sample preparation point-of-care diagnostics: recent developments in a connected age portable biosensing devices for point-of-care diagnostics: recent developments and applications from chipin-a-lab to lab-on-a-chip: towards a single handheld electronic system for multiple application-specific lab-on-a-chip (asloc) advances in addressing technical challenges of point-of-care diagnostics in resource-limited settings smart biosensors for multiplexed and fully integrated point-of-care diagnostics palm-sized device for point-of-care ebola detection ultra fast miniaturized real-time pcr: 40 cycles in less than six minutes rapid detection of viral rna by a pocket-size real-time pcr system the nanolithography toolbox catching bird flu in a droplet doubling throughput of a real-time pcr single-cell rt-pcr in microfluidic droplets with integrated chemical lysis comparison of reverse transcription-quantitative polymerase chain reaction methods and platforms for single cell gene expression analysis ultrahigh-throughput mammalian single-cell reverse-transcriptase polymerase chain reaction in microfluidic drops disposable real-time micropcr device: lab-on-achip at a low cost he is currently a ph.d. student of school of mechanical engineering in npu. his research interests include microfluidics and miniaturization and application of pcr systems he was a postdoctoral research fellow at the npu for over two years and he is currently a researcher in but. his research interests include analytical chemistry, material chemistry and mems degrees from npu in 2013 and 2016, respectively. he is currently a ph.d. student of school of mechanical engineering in npu. his research interests include adhesion force of gecko-inspired nanostructure and its application haoqing zhang received the b.s. and m.s. degrees from the xi'an university of technology in 2011 and 2015, respectively. he is currently a ph.d. student of school of mechanical engineering in npu. his research interests include droplet pcr and digital pcr systems currently he is a lecturer and member of microsystems group in the department of microelectronics, fee ctu in prague. his interests include programming of microcontrollers, sensors application and electronic circuits design she had worked in r&d in medical device industry. she also obtained emba from wharton school of business, university of pennsylvania. she worked as a scientist at fraunhofer-ibmt in germany prior to joining npu as a research associate in the microfluidic systems lab respectively. currently, he is a faculty member as associate professor and vice director of department of microbiology in fourth military medical university. he has published over 40 peer-reviewed journal and conference papers, and about 5 chinese patents as principle investigator from fourth military medical university in 1995, 2002, and 2005, respectively. currently, he is currently an associate professor with faculty of life sciences, npu. he has published over 31 sci papers, and one international pct patent and six national invention patents. he is a member of the china cancer society's cancer mark professional committee, the shaanxi provincial cell biology society, and the shaanxi provincial cancer mark professional committee. his interests include in antibody drugs and tumor cell biology her research interests include user experience and interaction design. currently, she is working on several projects focusing on user interfaces for ai and iot applications he is member of the academy of romanian scientists. and now work in national institute for research and development in microtechnologies he is currently a professor with the ministry of education key laboratory of micro and nano systems for aerospace supplementary material related to this article can be found, in the online version, at doi: https://doi.org/10.1016/j.snb.2019.127098. key: cord-260985-ria9v2p6 authors: mcdarby, geraldine; reynolds, lindy; zibwowa, zandile; syed, shams; kelley, ed; saikat, sohel title: the global pool of simulation exercise materials in health emergency preparedness and response: a scoping review with a health system perspective date: 2019-07-29 journal: bmj glob health doi: 10.1136/bmjgh-2019-001687 sha: doc_id: 260985 cord_uid: ria9v2p6 simulation exercises (simex) are an established tool in defence and allied security sectors, applied extensively in health security initiatives under national or international legislative requirements, particularly the international health regulations (2005). there is, however, a paucity of information on simex application to test the functionality of health systems alongside emergency preparedness, response and recovery. given the important implications health services resilience has for the protection and improvement of human life, this scoping review was undertaken to determine how the publicly available body of existing global simex materials considers health systems, together with health security functions in the event of disruptive emergencies. the global review identified 668 articles from literature and 73 products from institutional sources. relevant screening identified 51 materials suitable to examine from a health system lens using the six health system building blocks as per the who health system framework. eight materials were identified for further examination of their ability to test health system functionality from a resilience perspective. simex are an effective approach used extensively within health security and emergency response sectors but is not yet adequately used to test health system resilience. currently available simex materials lack an integrated health system perspective and have a limited focus on the quality of services delivered within the context of response to a public health emergency. the materials do not focus on the ability of systems to effectively maintain core services during response. without adjustment of the scope and focus, currently available simex materials do not have the capacity to test health systems to support the development of resilient health systems. dedicated simex materials are urgently needed to fill this gap and harness their potential as an operational tool to contribute to improvements in health systems. they can act as effective global goods to allow testing of different functional aspects of health systems and service delivery alongside emergency preparedness and response. the work was conducted within the scope of the tackling deadly diseases in africa programme, funded by the uk department for international development, which seeks to strengthen collaboration between the health system and health security clusters to promote health security and build resilient health systems. ► simulation exercises (simex) are established, practised in defence and security sectors which are later recognised in national and international regulations (e.g international health regulations, 2005). ► the importance of simex as an improvement tool has yet to be identified to test capacities for health system resilience alongside emergency preparedness. what are the new findings? ► currently available global pool of simex materials lack an integrated health system perspective with a limited focus on the maintenance of routine quality health services delivered during response to a public health emergency. ► without adjustment of the scope and focus, currently available global pool of publicly available simex materials do not have the capacity to test health system resilience. what do the new findings imply? ► participation of health system authorities or stakeholders in the development, conduct of simex and accountability of findings for improvement in health system strengthening is needed. ► dedicated simex materials are urgently needed to fill gaps identified in global resources to harness the potential of simex as an operational tool to contribute to improvements in health system strengthening. simulation exercises (simex) are an established tool in defence and allied security sectors, applied extensively in health security initiatives under national or international legislative requirements, particularly the international health regulations (2005) . there is, however, a paucity of information on simex application to test the functionality of health systems alongside emergency preparedness, response and recovery. given the important implications health services resilience has for the protection and improvement of human life, this scoping review was undertaken to determine how the publicly available body of existing global simex materials considers health systems, together with health security functions in the event of disruptive emergencies. the global review identified 668 articles from literature and 73 products from institutional sources. relevant screening identified 51 materials suitable to examine from a health system lens using the six health system building blocks as per the who health system framework. eight materials were identified for further examination of their ability to test health system functionality from a resilience perspective. simex are an effective approach used extensively within health security and emergency response sectors but is not yet adequately used to test health system resilience. currently available simex materials lack an integrated health system perspective and have a limited focus on the quality of services delivered within the context of response to a public health emergency. to disease outbreaks. 1 health workers were significantly more likely to be infected than the general population, with this increased risk largely attributable to the poor quality of infection prevention and control (ipc) practices and emergency preparedness. [1] [2] [3] guinea, liberia and sierra leone lost between 1.5% and 8.1% of their country's doctors, nurses and midwives to evd, translating into significant reductions in the healthcare provision. 2 ineffective surveillance systems enabled evd to spread locally as well as across borders. 4 5 during an outbreak, as with any public health emergency (phe), the resilience of a country's health system is tested in real time. the capacity to respond to an outbreak and maintain essential services creates a surge in demand for critical resources. health system resilience has been defined as 'the capacity of health actors, institutions, and populations to prepare for and effectively respond to crises; maintain core functions when a crisis hits; and, informed by lessons learned during the crisis, reorganize if conditions require it.' 4 resilient health systems protect human life and are linked with positive health outcomes during a phe as well as in its aftermath. 4 a lack of health system resilience is associated with excess morbidity and mortality due to the phe as well as from other causes, which can be at least partially attributed to a reduction in access to quality health services. [6] [7] [8] it has been estimated that a 50% reduction in access to healthcare services during the west african evd resulted in increased child and maternal mortality as well as increased mortality from other infectious diseases. 6 8 these indirect deaths-10 623-were not insignificant. 6 thus, it is critical that a mechanism exists to test and build resilient health systems in order to reduce excess morbidity and mortality from future phes. national and international preparedness for phes has long been discussed on a global scale, especially in health security forums that seek to address challenges and gaps in meeting core international health regulations (ihr) (2005) capacities. one component of the revised ihr (2005) monitoring and evaluation framework includes simulation exercises (simex), which are defined by the who as 'forms of practice, training, monitoring or evaluation of capabilities involving the description or simulation of an emergency, to which a described or simulated response is made.' 9 simex have historically been an established tool in defence and allied security sectors, with recent adaptation to health security efforts in the context of disruptive emergencies, natural and man-made. they are now being used as a tool to assess compliance with national-for example, the uk civil contingencies act 2004-and international legislative requirements, notably ihr 2005. 10 there is, however, a paucity of information on the application of these exercises to test the functionality of health systems alongside emergency preparedness, response and recovery. simex are also an ideal opportunity to test preparedness of the various functions of the health system, particularly health service delivery, in response to phes. this would contribute to the development of strong linkages between health systems and health security sectors supporting an integrated approach towards building resilient health systems. given the important implications health services resilience has for the protection and improvement of human life, this scoping review was undertaken to determine how the body of existing global simex materials considers the testing of health system resilience, together with health security preparedness, and response functions in the event of acute and/or protracted phes. recognising the dispersion of simex materials, a scoping review of academic literature as well as an institutional search was undertaken. 11 a wide search strategy was employed to identify all relevant materials. searches of different hazard types, exercise types and subject areas were combined (online supplementary appendix 1). the initial search returned 668 articles for screening with an additional 17 identified through bibliography review. titles and abstracts were reviewed against inclusion and exclusion criteria (table 1) , reducing the number for fulltext review to 32. for the institutional search, institutions involved in the development, implementation or evaluation of simex were identified by experts in the field, as well as through relevant articles. their websites were searched for applicable materials. ultimately, 31 institutions, ranging from academic to national response agencies, were reviewed (online supplementary appendix 2), identifying 73 materials which were reduced to 19 following the screening of aims and objectives (figure 1). the final materials identified (n=51; online supplementary appendix 3) (32 emerging from the literature and 19 emerging from institutional review) were analysed by two independent reviewers for their scope to test aspects of health systems using the who health system framework, consisting of six independent but inter-related building blocks (table 2) . resilience was considered to be addressed if materials demonstrated evidence of an integrated emergency response, maintenance of essential functions or reorganisation of services within the context of a phe. a smaller number of materials (n=8) were bmj global health ► does the material look at aspects of health service delivery in the event of a simulated event? ► does the material test a function of specific building blocks of a health system in consideration of other related building blocks? ► does the material test preparedness and response of healthcare facilities alongside national incident management system? ► does the material look at impact of emergencies on provision of routine essential health services? ► does the material look at how phes impact health service delivery at subnational and district-level facilities? ► does the material examine the standard of care during phe? identified for examination of their scope to test resilience from a functional perspective (figure 1 and box 1). materials were excluded from this portion of the analysis if they did not report either an exercise or objectives in sufficient detail to support this analysis. this portion of the analysis (n=8) involved five journal articles and three guidance materials (online supplementary appendix 4). a library of publicly available materials to support the development of simex was compiled in parallel to the review process. there was no involvement of patients or the public in this study, in either the design of the methods or in the conduct of the study. results simex consideration of health system building blocks the majority (63%) of the materials (n=51) identified exclusively tested preparedness and response to a phe, without considering them as typical functions of a resilient health system (figure 2). a limited number were identified that tested aspects of health system resilience or recovery (figure 2). the categorisation of the scope of material was based on the research bmj global health team's interpretation, that is, if the exercises dealt with preparing and responding to a phe or if they dealt with aspects of recovery or response. a few materials self-identified as testing health security or testing for contingency, and so warranted extra categories. the materials identified looked at aspects of health security across different geographical levels (international, national, regional and facility); they more frequently examined the regional/ district (43%) and facility levels (31.4%) ( figure 3) . the most frequent hazard type (37%) simulated was biological which included pandemic influenza and other emerging infections ( figure 4) . though often not the explicit focus, all reviewed materials (n=51) tested at least one building block or an aspect of a health system building block (figure 5). a functional approach was apparent, though functions were generally tested in a narrow way both within and across relevant building blocks, rather than integrated across all relevant health system building blocks. ipc was the most commonly tested service delivery function with adherence to case management guidelines and standard operating procedures (sop) being a frequent focus of testing. 8 the quality of health services provided in the context of the response or measures of the maintenance of essential health services were lacking. surge capacity was the most frequently tested function of health workforce, though a narrow approach was often apparent. exercises tested aspects of surge capacity such as staffing, however, this was not addressed within other building blocks in relation to the invariable increased demands on services and supplies. a narrow approach to health information systems was also apparent, with three materials (out of 51) focusing on surveillance systems and only limited aspects of risk communication tested. only one identified material tested the triggering of a response plan using a surveillance system. 12 leadership and governance (from a security perspective) was the most frequently addressed building block, with many materials testing aspects of this building block such as response plans and roles and responsibilities of responders. however, alignment with national structures and guidelines was not apparent, nor was consideration given to decision in risk identification. while access to medicines and supplies was frequently alluded to, testing of access to mechanisms or supply chain resilience was infrequent. financing was alluded to in only 17% of materials (n=51), but testing of mechanisms to access funding in the event of phes was not identified. tabletop exercise/discussion was the most frequent approach identified (figure 6). they are less expensive and faster to execute, particularly when considering large groups of stakeholders. the limitation with this approach bmj global health is limitation in scope to adequately simulate phes and test the individual and integrative aspects of health systems. simex consideration of health system functionality and system underpinning eight materials were identified for further analyses on the functionality/system aspect of the six building blocks, using a set of questions (box 1). most materials tested activation of appropriate emergency response mechanisms and structures within the respective administrative levels tested, for example, a facility-level exercise testing activation of all appropriate response mechanisms within the health facility. some materials tested activation across different levels within the system, for example, a health facility responding to an outbreak activating regional or national response systems. emergency response systems were generally assumed to have been activated, without testing system triggering. as was apparent with the building block analysis, healthcare functions were generally not tested in an integrated way. for example, a mass dispensing drill had no regard for the parallel response structures with which they would have to integrate during a response. 13 similarly, a facility response to a sarin attack failed to integrate with national response agencies. 13 14 reporting of alignment with national command and control structures or response plans was limited as was evidence of consideration of the impact of the phe on other health system-level facilities or management structures. materials to test the impact of emergencies on primary healthcare (phc) or its response were extremely limited and community resilience materials, where identified, failed to link with health systems. [15] [16] [17] the materials were limited in their focus on testing the quality of services delivered in the context of response to a phe. where present, measures tended to focus on clinical aspects of care rather than system and process measures. measures to test the maintenance of essential services from a quality perspective were not identified in any of the eight selected materials. limitations of the review the application of the health system framework to review simex materials introduced a degree of subjectivity. in reality, it is the same health system that provides routine healthcare, emergency-specific healthcare and response to a shock impacting public health. this was addressed through discussion as well as with input from health system and security experts in who. similarly, the approach to the analyses conducted required sufficient detail in the identified materials in relation to exercises or objectives, which led to the exclusion of a number of relevant materials as they were not present either with sufficient details or as a package (written narrative, scenario, injects and postexercise report). both the academic literature and institutional materials were analysed using the same approach. this likely led to an underestimation of effect in relation to academic literature as not all that was tested may have been reported, and an overestimation in relation to institutional materials as objectives may not have been applied effectively within exercises. a publication bias also likely exists in relation to health security exercise reports and materials given the sensitive nature of findings as well as in relation to materials developed and delivered by private companies. while multiple institutions and organisations (n=31) known to be involved in simex were contacted to identify unpublished materials, it is likely that there remains a pool of materials not made available as they were considered proprietary and/or sensitive. however, the objectives of the exercise are based on publicly available materials that are accessible and can be used as global resources, as such access-restricted materials fall beyond the scope of this review. in general, materials identified were from countries with developed capacities for emergency preparedness and response-materials from other settings may not have percolated the literature and may not have an institutional home where materials are placed. however, the inclusion of institutions known to support simex within low income settings is likely to reduce the impact of the bias towards high-income countries within the findings of the work. the exclusion of non-english materials has the potential to introduce a cultural bias, although materials in the review included materials from most continents (north america, europe, africa, asia and australia). the extent of any cultural bias is therefore limited, and unlikely to affect the findings of the work. despite these limitations, there is no reason to suspect that materials not included in this review differ systematically from those included in such a way as to negate the findings. discussion while all materials identified tested aspects of health systems, there was limited evidence of an integrated health system approach. health system building blocks were touched on from a preparedness perspective, and often tested in a fragmented and isolated way without addressing interlinkages. ipc was frequently tested in the context of response to a pandemic or other emerging infection, with a focus on governance structures and adherence to case management and sops. ipc is central to the response to any emerging infection as was highlighted within the evd outbreak in west africa where poor ipc practices contributed to significant health worker transmission, leading to reductions in response as well as essential healthcare delivery. 2 in a similar instance, a lack of strict adherence to ipc guidelines was associated with ongoing healthcare facility transmission during the severe acute respiratory syndrome outbreak in both toronto and taiwan in 2003. 18 an integrated approach to ipc as a critical function of quality health service delivery in these simex materials is thus required. the availability and timeliness of emergency financing is critical in determining the timeliness and effectiveness of coordinated efforts in any emergency response. despite this, limited consideration was given to the supply of resources required to meet the surge in demand, including staff, diagnostics, medicines or personal protective equipment, and to accessing the financing necessary to meet these demands. 19 it is therefore of utmost importance that the rapid mobilisation of financing should be regularly tested in simex, taking into account the response required as well as consideration of maintaining quality essential health services. health information systems, though often alluded to, were rarely tested in a robust and integrated way, with a lack of focus on surveillance systems in particular. the only material identified that tested activation of an emergency plan using syndromic surveillance failed to trigger activation of the response plan. 12 the pivotal role played by data and information systems in routine healthcare delivery is even greater in the context of emergencies. a strong and reliable health information system ensures understanding of the epidemiology of disease, and is critical in coordination, communication and management of response efforts. testing this functionality should be an integral part of simex conducted at any level. consideration of the integration of preparedness and response across health system levels was lacking, with services and facilities tending to test their own response capabilities in isolation. phc, despite being the likely first point of care in many phes, was rarely considered in the simex materials reviewed either in terms of supporting preparedness capacity or in terms of the effect of the phe on phc. 15 phc plays a central role in surveillance which was highlighted within the evd outbreak in west africa, where failure to identify evd when it first presented within the community in guinea led to a significant delay in response with a concomitant lack of containment. despite evidence from evd west africa that community linkages can support health system response in the face of phes, the current global pool of simex materials were unlikely to link with community aspects and where community preparedness materials existed they rarely linked with health systems. 16 17 the importance of integrating private healthcare facilities into emergency preparedness and response capabilities was highlighted by the recent dengue outbreak in khyber pakhtunkhwa in 2017. a private health facility, unfamiliar with emergency response protocols, failed to activate emergency response mechanisms in a timely manner. this, alongside a lack of case management guidelines, contributed to prolonged community transmission. 20 no evidence of integration of private healthcare facilities into health system preparedness and response was identified within the current pool of global simex materials. while there is international consensus of the need to focus on quality in healthcare, the focus on the quality of services delivered in the context of a phe was lacking. 21 22 where materials did focus on service quality, they tended to focus on clinical aspects of care. system and process measures were only identified in drills that tested mass dispensing capabilities in the context of a pandemic or biological attack. these capabilities represented parallel bmj global health response structures and their linkages with appropriate health system structures were not tested, nor was the communication required to divert individuals from normal health services tested. 23 24 no materials identified included measures to test the maintenance of or quality of essential health services during the response to a phe. evidence from west africa shows that the indirect mortality and morbidity associated with discontinuity of health services as well as poor quality health services was significant across the three countries, with the biggest impact on maternal and child health. 6 the current global pool of simex materials are limited in its ability to test health service resilience alongside preparedness and response. this in turn limits the opportunity to practically bridge health security and health systems at different administrative levels. special attention is required in using simex approaches to drive sustainability of investment in health security preparedness or disease-specific programmes to proactively position available scarce resources into sector-wide development of health systems for all public health hazards per ihr (2005). simexercises are a well-practised method of testing and promoting emergency preparedness and response for local, national and global health threats. while much experience exists globally in this area, the proprietary nature of some of the materials creates a missed opportunity for the sharing of knowledge across global health security and preparedness communities. it would be in the interest of the global community to develop a mechanism to support sharing of lessons learnt that respects the integrity of private organisations involved in simex development and delivery. the effectiveness of simex has been demonstrated in identifying gaps in emergency response plans, skills and associated resources. 11 25 the lack of an integrated health system perspective in the current global pool of simex materials limits their ability to support health system functionality and strengthening in the context of phes. this, along with their lack of focus on the quality of response and the maintenance of quality essential health service functions, means that they do not have the capacity to support health system resilience. the incorporation of a health system perspective into simex materials has the potential to enhance health system strengthening and the development of resilience alongside emergency response and health security capabilities. an integrated approach to simex including health security, emergency preparedness and health systems is required to address the gap identified by this review. as a result of these findings, an off-the-shelf simex package that addresses health system aspects within the context of response to a phe is being developed, which will be freely available for all countries, particularly low-income countries. this could be further supported through the cross involvement in ongoing exercises and after-action reviews to enhance connectivity and support the development of shared ownership of improvement recommendations. these new-generation simex materials could be collated in a global repository that could be accessed by national authorities. such a collaborative approach would allow the leveraging of the considerable expertise in simex present within health security and emergency preparedness sectors. lessons learnt from this integrated approach will allow health systems to be built better, function better, which will ultimately lead to the protection and improvement of human life. world health organisation. health worker ebola infections in guinea, liberia and sierra leone: a preliminary report health-care worker mortality and the legacy of the ebola epidemic ebola virus disease in health care workers--sierra leone what is a resilient health system? lessons from ebola building resilient health systems: a proposal for a resilience index effects of response to 2014-2015 ebola outbreak on deaths from malaria, hiv/aids, and tuberculosis, west africa the ebola outbreak and staffing in public health facilities in rural sierra leone: who is left to do the job? counting indirect crisis-related deaths in the context of a low-resilience health system: the case of maternal and neonatal health during the ebola epidemic in sierra leone world health organisation. development, monitoring and evaluation of functional core capacity for implementing the international health regulations (2005): concept note world health organisation. after action reviews and simulation exercises: under the international health regulations monitoring & evaluation framework (ihr mef) what is the value of health emergency preparedness exercises? a scoping review study a test of syndromic surveillance using a severe acute respiratory syndrome model the planning, execution, and evaluation of a mass prophylaxis full-scale exercise in cook county sarin exposure: a simulation case scenario emergency preparedness toolkit for primary care providers academic-community partnership to develop a novel disaster training tool for school nurses: emergency triage drill kit whole community: planning for the unthinkable tabletop exercise sars in healthcare facilities the financial logistics of disaster: the case of hurricane katrina who paints grim picture of anti-dengue preparedness. reliefweb high-quality health systems in the sustainable development goals era: time for a revolution delivering quality health services: a global imperative for universal health coverage ready or not: analysis of a no-notice mass vaccination field response in philadelphia hospital emergency preparedness: push-pod operation and pharmacists as immunizers a scoping review of evaluation methods for health emergency preparedness exercises contributors ssa conceptualised the study. ssa, gm and zz designed the methods. gm, lr and ssa conducted the literature search. gm and lr collected the data. gm, lr and zz developed the figures. gm, lr, ssa, zz, ek and ssy all contributed to data analysis, data interpretation, manuscript writing and reviewing.funding this study was funded by world health organization.competing interests none declared. provenance and peer review not commissioned; externally peer reviewed. open access this is an open access article distributed in accordance with the creative commons attribution non commercial (cc by-nc 4.0) license, which permits others to distribute, remix, adapt, build upon this work non-commercially, and license their derivative works on different terms, provided the original work is properly cited, appropriate credit is given, any changes made indicated, and the use is non-commercial. see: http:// creativecommons. org/ licenses/ by-nc/ 4. 0/. key: cord-323973-wszo9s3d authors: zhu, hanliang; zhang, haoqing; ni, sheng; korabečná, marie; yobas, levent; neuzil, pavel title: the vision of point-of-care pcr tests for the covid-19 pandemic and beyond date: 2020-07-20 journal: trends analyt chem doi: 10.1016/j.trac.2020.115984 sha: doc_id: 323973 cord_uid: wszo9s3d infectious diseases, such as the most recent case of covid-19, have brought the prospect of point-of-care (poc) diagnostic tests into the spotlight. a rapid, accurate, low-cost, and easy-to-use test in the field could stop epidemics before they develop into full-blown pandemics. unfortunately, despite all the advances, it still does not exist. here, we critically review the limited number of prototypes demonstrated to date that is based on a polymerase chain reaction (pcr) and has come close to fulfilling this vision. we summarize the requirements for the poc-pcr tests and then go on to discuss the pcr product-detection methods, the integration of their functional components, the potential applications, and other practical issues related to the implementation of lab-on-a-chip technologies. we conclude our review with a discussion of the latest findings on nucleic acid-based diagnosis. st century has witnessed exponential growth in science and technology; however, sadly and ironically, it has succumbed to devastating coronavirus infections: severe acute respiratory syndrome (sars), middle east respiratory syndrome (mers), and now coronavirus disease 2019 . [1, 2] as of july 7, 2020, a total of 11,500,302 covid-19 cases had been reported across the world, together with 535,759 deaths. [3] the sars and mers coronaviruses were transmitted indirectly from bats to humans through civets and camels, respectively, as intermediate hosts, [1] but the one responsible for covid -19 , has yet to be identified. [4] one of the main reasons that outbreaks such as these occur is the ability of viruses to evolve rapidly and change their animal hosts; this was seen with spanish influenza, the single-largest pandemic of the last century, which was probably caused by a virus that was transmitted from ducks to domestic pigs and then to humans, in the densely populated region of southern china. [5] the virus was eventually transmitted to the usa and europe after world war i. recombination among viruses as they change their hosts is a natural process. there is, therefore, always a strong probability of new epidemics emerging from novel types of viruses, especially in densely inhabited regions ( figure 1 ). the emergence of a coronavirus epidemic in the southern provinces of china has been predicted for some time, given the presence of multiple bat species that are infected with many different coronaviruses, in these highly populated areas. [6] coronaviruses and influenza viruses tend to be diagnosed through the detection of their specific genomes, in particular their ribonucleic acid (rna) sequences. rapid and precise screening of the public is essential to identify and quarantine infected people, thereby limiting or potentially eliminating the spread of the virus. the viral load of coronaviruses is high, making it easily detectable in symptomatic patients. unfortunately, there is also a high percentage of asymptomatic individuals, making an accurate disease diagnosis of the population a challenging job unless the entire population is tested. [7] a low level of limit-of-detection (lod) is crucial to shift the diagnostic window of opportunity toward the start of the infection process, to detect newly infected individuals. the test can be based on immunoassays, using antibodies to detect a specific antigen produced by the body's immune system or polymerase chain reactions (pcr) to detect a viral genome sequence. the pcr technically, once a disease emerges, researchers identify the infectious agent by sequencing its genome, after which the newly obtained sequence is compared against sequences in the databases to evaluate its similarity to other known viruses. determination of a sequence that is truly unique to the newly characterized virus represents a crucial step in the development of an effective diagnostic procedure. furthermore, such a sequence should be selected from a region of the viral genome that is not variable between its different isolates and is not expected to evolve and mutate quickly. when a suitable sequence is determined, the design and synthesis of the relevant pcr primers can be performed routinely. in the case of rna viruses, the reverse transcription that converts viral rna to cdna represents a crucial step, allowing the subsequent application of all methodologies based on pcr ( figure 2 ). the raw sample and the reagents used in the sample preparation often contain a significant level of pcrinhibiting factors; thus, proper sample preparation-whether manual or automated-is an essential step for successful detection. [8] the success of virus detection in real-world biological samples is highly dependent on the volume of the analyzed sample. during the initial phases of infection, the viral loads in patients may be near or below the test lod. [9] the analysis of larger volumes of biological fluids with a low viral load is likely to reduce the risk of false-negative results, but microfluidic point-of-care (poc) devices are typically unable to handle ml-scale volumes in a short time due to the requirement of slow flow rates. multiple approaches have been developed to address this problem. flow-through capture membranes [10] have been used to capture nucleic acids in a short period of time, allowing the direct amplification of the captured material without any elution step, detecting as few as ≈10 copies of dna with flow rates as high as ≈1 ml·min -1 . magnetic beads [11, 12] were used to isolate the rna of avian influenza h5n1 and sars from a ≈40 µl sample of blood; this is an insufficient volume on which to perform reliable coronavirus diagnostics. carbon nanotubes [13] or aptamers [14] have been used to capture rna from influenza a and h5n1 from avian flu viruses, respectively, and to promote their enrichment in samples. here, we look back and identify those technologies that appeared since the emergence of sars in late 2002. it is our aim to put landmark developments under the spotlight, critically evaluate them, and raise questions to identify the gaps that are currently hindering a commercial poc-pcr test. there have also been other recent outbreaks of infectious diseases that have threatened global human health, such as ebola virus disease in the democratic republic of the congo in 2018, measles in burundi, and yellow fever. additionally, the lower respiratory infections, tuberculosis, and human immunodeficiency virus (hiv) disease were listed among the top ten global causes of death in 2016. [15] the pcr test on samples prepared from blood and urine specimens is capable of detecting the virus in the early stages of the disease, resulting in early diagnosis and subsequent isolation of infected patients to block transmission. for a comprehensive snapshot of the field, we refer the reader to in-depth reviews on the topic that have appeared in recent years. [16] [17] [18] figure 2. a conceptual demonstration of the detection of viruses in the field, based on real-time rt-pcr at poc. the process starts with a nasal swap to obtain a sample that might contain the virus. the sample is purified using paramagnetic beads and viruses are captured, followed by virus lysis to release the rna, and purification then, the rna is reverse transcribed to cdna; the number of cdna molecules is multiplied by the pcr, and the results are displayed and possibly transferred to a centralized laboratory via a mobile device. [19] 2. pcr and its technology a. the principles and brief history of pcr single-step rt-pcr is a technique derived from the original pcr [20] to perform a reverse transcription followed by the pcr temperature cycling. it requires a mixture of reverse transcriptase, dna polymerase, short oligonucleotides complementary to the cdna target, called primers, free nucleotides, and bivalent salts, such as mgcl 2 . the rt-pcr starts with reverse transcription, typically conducted at ≈60°c, followed by a hot start performed at ≈93˚c, destroying the reverse transcriptase and activating the polymerase. typically, there are 35-45 cycles consisting of a double stranded dna (dsdna) denaturation at 93°c, primer annealing at a temperature ranging between 55°c and 65°c, and extension at 72°c to finish the dsdna. the three temperature protocols can be simplified by a two-step temperature protocol, whereby the annealing and elongation are conducted at the same temperature. [21] the classical endpoint detection system, which required an external pcr product detection by hybridization or electrophoresis, was modified by adding a fluorescent agent, [22, 23] either intercalating dye or a fluorescent probe that converts the endpoint pcr into a real-time pcr, also known as a qpcr. pcr-based systems are, in principle, capable of detecting a single copy of rna or dna, with the assumption that the single dna/rna copy is presented in the sample and not lost during the sample preparation. the pcr/rt-pcr is a far superior technique in comparison with antibody-based assays due to the much smaller diagnostic window, i.e., it stops providing false-negative results earlier than the antibody-based assays. an alternative to pcr is a ligase chain reaction with short templates, whereby the products are only one base pair longer than the combined length of both the primers. [24] b. other techniques based on the amplification of nucleic acids a group of new techniques, called isothermal amplification of nucleic acids, has been introduced with loopmediated isothermal amplification (lamp) [21] and nucleic acid sequence-based amplification (nasba) [25] as its main representative, with an important aim of bypassing pcr-related patents. papers that have described these techniques start with the claim that lamp is far superior to pcr because complicated temperature cycling is not required for isothermal amplification. however, temperature cycling is a very simple engineering method that helps to improve the pcr specificity. proportional-integral-derivative controllers that include temperature sensors and solid-state relays are cheap and readily available. in addition to that, lamp still needs a closed-feedback loop system heater to maintain the system's temperature in a range of 50°c to 60°c. both real-time pcr and real-time lamp require a fluorescence detection system, and due to the rather weak fluorescent output signal the fluorescent signal related electronic processing circuits is far more demanding than those for temperature control. although there is no requirement for temperature cycling, lamp is a complicated process. given that nothing comes for free, a simple lamp requires six primers, but a pcr needs only two; thus, it does not seem to be correct to assume the simplicity of lamp. nevertheless, the lamp system has its merits, which include the fact that thermal cycling is not required, resulting in better thermal isolation and, therefore, less power consumption compared with pcr. also, lamp can be designed to simplify the readout by colorimetry or nephelometry, instead of conventional fluorescence detection. [26] there are other isothermal nucleic acid amplification techniques using different principles and temperatures, such as strand displacement amplification, [27] rolling circle amplification, [28] nicking enzyme amplification reaction, [29] recombinase polymerase amplification, [30] and catalytic hairpin amplification. [31] c. microfluidics and pcr miniaturization following in the footsteps of microelectromechanical systems (mems), the field of microfluidics-or lab-on-achip-emerged in the early 1990s, with the grand vision of producing a micro-total-analysis system (μtas). [32] two analytical techniques pioneered the field: capillary electrophoresis (ce) [33] and pcr. [34] the first demonstrations of both techniques independently on the microchip format led to the pcr-ce integration [35] later on by implementation of a real-time pcr to a portable unit for a poc-pcr. [36] since then, important innovations have taken place in terms of materials, [37] as well as the method of implementation, [38] extending the boundaries of this field. however, nearly three decades on, these efforts have yet to bear fruit in daily life outside the laboratory. one cannot help but wonder: why has the development and deployment of practical poc-pcr testing taken so much time? this is a pressing question, especially at a dire time like the present, when the world is battling another once-in-a-century pandemic, covid-19. [39] this is the raison d'être of μtas. this background should not be taken as a criticism of the slow development because the task of making portable systems for viral diagnostics with minimal false-positive and -negative results is complex, and great progress has indeed been made in recent years. pcr runs on a platform which cycles the temperature of the reaction between two or three set points for over 30 to 40 cycles. the reaction time, is often limited by heating and cooling rate within each cycle determined by power dissipation (p) and system thermal time constant (τ). it is reduced with a reduced thermal mass (inclusive of the sample and the chamber) and with decreased isolation of this thermal mass from surrounding. both features can be fulfilled by miniaturization, through mems technology and microfluidics, which has lowered the reaction time from >30 min to several min in the past decade [40] or even less [41] as: where δt is the time taken by the system to change the temperature from one pcr step to another (δt), and c is its thermal capacitance. the total reaction time depends on a series of parameters, such as the chip size, the pcr master mix volume determining the value of c, the thermal conductivity of the substrate (g), and the temperature cycling rate. here, the miniaturization of the pcr results in a smaller value of c, which proportionally lowers the ∆t. recently, an extremely fast pcr with a 0.4 s·cycle -1 has been demonstrated and resulted in an incredible total reaction time of less than 15 s. [41] this fast pcr could be combined with a fast melting curve analysis to achieve a total detection time of less than 1 min. [42] the pcr can also utilize passive cooling, with a cooling rate determined by its τ value: = . (2) increasing the value of g leads, ultimately, to a faster system, unfortunately, the cooling-rate increase comes with a cost, making the system power demanding as: and power demanding systems are not suitable for battery-operated poc devices. nevertheless, the system heating/cooling rate can still be increased without compromising the power increase by minimizing the sample volume. isothermal amplification systems benefit from the absence of a cooling requirement, making the τ value irrelevant. then, there is an option to thermally isolate the heated part with a g value as low as possible to consume very little energy-a valuable feature of portable, battery-operated systems. poc-pcr requires fast thermal cycling and precise temperature control. there are demanding requirements for the design of temperature modules for miniaturized pcr devices. heating methods determine the temperature ramping rate and are currently divided into contact and noncontact heating. the contact heating method utilizes embedded/external heat sources, such as deposited thin films or external peltier elements; [43] this method increases thermal mass, inevitably hindering fast thermal transitions during reactions. the non-contact heating method was developed using hot air [44] or infrared radiation as a heat source and directly heats the sample in small volumes. [45] these contact and non-contact heating methods employed for microchips depend on the material of the substrate, the reaction volume, and the structure of the chip. thermal isolation must also be considered in a miniaturized pcr system because thermal cross-talk deteriorates the chip's performance, especially in multifunctional reaction chambers on a single chip. [46] furthermore, poor thermal isolation results in heat loss from the temperature zones to the surroundings, while the suspended pcr chamber enables good thermal isolation. a hollow reaction chamber has been made out of micromachined silicon (si) that is integrated with both the heater and the sensor and is connected by a narrow beam to the substrate to achieve thermal isolation. [47] in addition, isolated layers with high thermal resistance have been applied to provide excellent thermal isolation; [48] however, they result in more complex fabrication and additional stress in different layers. materials with low thermal conductivity, such as glass or plastics, could provide excellent thermal isolation for pcr chips, thereby avoiding the temperature cycling of the entire device. [49] 4. materials the implementation of microfluidics has led to the development of miniaturized pcr systems that offer portability and save time, thereby facilitating applications in the realm of poc diagnostics. researchers have developed a family of miniaturized devices based on different types of pcr. materials such as si, [50] glass, [51] polydimethylsiloxane (pdms), [52] polycarbonate, [53] and polymethylmethacrylate (pmma), [54] used for fabricating microfluidic chips, are also available for the substrate of pcr chips. each material has a number of merits and demerits, based on its individual properties. glass is used to make pcr chips due to its transparency and low auto-fluorescence. a multifunctional platform suitable for the on-chip detection of biomolecules consists of different thin films integrated on a single glass substrate that can also be optically and thermally coupled with another glass. [51] however, the low thermal conductivity of glass leads to an uneven temperature distribution and slow heating/cooling rates. pdms is currently one of the most widely used polymers for microfluidic devices due to its biocompatibility, low cost, and simple processing. [55] a disposable pdms-glass bonding chamber has been utilized to perform pcr with an intercalating reusable electrode part for thermal cycling. [52] however, the evaporation of the pcr solution, located in the pdms chamber, at a denaturation step may lead to cross-talk because pdms is porous. pmma is another popular candidate. [56] inexpensive and versatile pmma pcr chips can be fabricated by laser ablation technology, followed by bonding methods such as low-temperature bonding that uses optically clear adhesive film and liquid optically clear adhesive. [54] additionally, the nonspecific adsorption between pmma and dna is minimal. the surface properties of microfluidic channels/chambers must be considered because the interaction between biomolecules in the pcr solution and the channels/chambers affects pcr efficiency. a high surfaceto-volume ratio of microchannels/chambers leads to the non-specific adsorption of the enzymes, limiting or even prohibiting the reaction. [57] two methods have been employed to solve this problem. one involves the use of silanization to modify the surface permanently. silanization is accomplished by filling the channel with a silanizing agent and heating the filled chip for a period of time, then removing the agent and washing the chip. alternatively, solutions such as bovine serum albumin (bsa), [58] polyvinylpyrrolidone, [59] glycerol, [60] polyethylene glycol 8000, [59] and tween 20 [61] are commonly added into the pcr solution to reduce the nonspecific adsorption, thereby improving the pcr efficiency. chips made of si and glass can be cleaned using solutions such as piranha (h 2 so 4 /h 2 o 2 ), since the solution performs the mineralization of organic materials, and no rna/dna can be left behind. nevertheless, general practitioners prefer disposable systems because they are safer in terms of cross-contamination. in the future, pcr chips could well be able to be cleaned and reused, once the cleaning process has shown its consistency. the first conventional thermal cycler, called mr. cycle, was a time-domain system, as are most current pcr devices. samples are placed at fixed locations on a heater whose temperature is modulated according to the required pcr protocol. the heaters can be based on dissipating joule heat with active heating and passive cooling, or by utilizing thermoelectric coolers with active heating, as well as cooling. the pcr master mix is dispensed into a stationary location, such as droplets or microwells, and microheaters are then used to conduct the thermal cycling beneath the droplets or wells. there are several techniques that can be employed to split samples to form droplets. a few µl of pcr master mix is pipetted onto a hydrophobic/oleophobic glass coverslip and covered by mineral oil, forming a virtual reaction chamber (vrc). [47] alternatively, the droplets, including cells separated by mineral oil, form an emulsion and are loaded into the microfluidic channel and are then transferred to different positions within the chip, after which they are subjected to a series of procedures such as cell lysis, dna extraction, and purification. [62] then, the thermal cycling is conducted by heating this emulsion or vrc, and cameras capture the subsequent fluorescence. a variety of chip structures are fabricated by micromachining with different wells or microfluidic channels in materials such as si, glass, and polymers, typically pdms or pmma. samples are loaded into either wells or channels for subsequent dna amplification by pcr. [63] ii. space domain the pcr master mix is pumped into a channel or chamber in the chip, typically with two/three zones maintained at constant temperatures. [34] the reaction is performed when the solution is passing through these temperature areas by denaturation, annealing, and extension. this is an old technique that was used in the early days of pcr systems, with three baths with different temperatures and a laboratory technician or robot to move a basket with samples from bath to bath. currently, this technique is still utilized in miniaturized systems, such as flow-through and rotational types. the flow-through method is a typical space-domain pcr method. the solution is pumped into a microfluidic channel made of polymers with different temperature regions, and a number of microheaters are applied to provide different temperature zones for denaturation, annealing, and elongation when performing the pcr. these zones maintain constant temperatures, and only the temperature of the samples is changed while it is moved through the zones with different temperatures. [49] iii. integration with sample preparation the nucleic acids to be detected are first isolated from the bacteria or viruses using technologies such as lysis, pre-concentration, and purification. these procedures can be fulfilled on-chip or off-chip using bench-top instruments. a number of fully integrated systems have so far been developed, starting with the non-portable genexpert from cepheid, which was first utilized for anthrax detection by the united states postal service, [64] and then with different primers to perform hiv and tuberculosis diagnostics in south africa. [65] despite being nonportable, it is a true sample-to-answer system that uses injection-molded plastic preloaded cartridges with lyophilized compounds, including a pcr master mix, as well as infectious agent-specific primers. their prohibitive capital cost has unfortunately restricted the spread of these systems, given that those employed in south africa were heavily subsidized. we demonstrated the first monolithic si-based chip integrating a micromixer for diluting blood sample, a microfilter for isolating and chemical lysing of leukocytes, a binder for capturing and purifying nucleic acids, and microvalves for fluidic control. [66, 67] the captured dna was amplified in a pcr chip followed by the product detection using si nanowires. [68] a large sample volume to be processed needed to be stored in a plastic cartridge next to the si chip, and the si chip experienced processing problems due to its small channel cross section. the first step is to filter out red blood cells, while effectively capturing white blood cells for subsequent lysis. the dna-capturing system was based on a sio 2 surface inside the chip, was orders of magnitude smaller than the one based on microfiber membranes or silica beads. finally, the use of si nanowires as dna sensors offers somewhat questionable repeatability. the replacement of a conventional pcr with a qpcr would make life easier because there would be no sample manipulation after pcr, and qpcr is well established technique. nevertheless, the system had limited portability. standard pcr (end-point) typically utilizes electrophoresis of either a gel or capillary type to detect pcr products after thermal cycling. a pcr master mix is pumped into a microchannel fabricated in a microfluidic chip, and pcr is performed using microheaters. after the reaction, the solution is taken out, and gel electrophoresis (ge) is conducted to check the presence and specificity of the amplicons. [69] alternatively, the solution is transferred to a ce column on a chip with electrodes on both sides, and the products are detected by ce. [70] detection by ge/ce made the chip or system design complicated, hindering the system miniaturization for poc diagnostics. inspired by commercial real-time pcr instruments, optical detection modules, such as optical fibers [71] and lock-in amplifiers, [72] were then integrated into miniaturized qpcr systems while intercalating dyes such as sybr green or probe were added into the pcr master mix, and the reaction was monitored in real time. furthermore, additional types of qpcr systems combined with other technologies have been developed to improve performance. a pico-liter droplet array generated by double-inkjet printing was demonstrated to be capable of performing parallel qpcr with an enhanced throughput. [73] a complementary metal oxide semiconductor was integrated with the pcr chip used for multiplexing detection, which enhanced detection sensitivity. [74] electrowetting on dielectric technology is well adapted for single-cell isolation, mrna purification, and subsequent multiplex qpcr at the single-cell level. [75] 7. the vision and the reality the miniaturized pcr was first envisioned in the early 1990s, and since then numerous pcr microchips have emerged. most of these pcr microchips have required off-chip sample preparation and even off-chip detection. these undesired features hinder complete freedom from laboratory settings and give rise to the tongue-in-cheek expression for these devices: chip-in-a-lab. [76] the sample preparation, which involves the extraction and purification of nucleic acids from bodily fluids, plays a determining role in the accuracy of the test, which heavily depends on the effective removal of pcr inhibitors such as hemoglobin. however, the diversity and complexity of bodily fluid samples, together with the sample volume requirement, make it difficult to combine sample preparation with pcr on a microchip or in a cartridge. few studies have undertaken the challenge of demonstrating a fully integrated pcr system, where sample preparation, amplification, and detection occur in one platform. in this section, we describe these integrated solutions and the issues preventing their poc use. the first of these was arguably a unit reported ≈15 years ago based on the esensor microarray chip ( figure 3a ). [77] the esensor was a printed circuit board featuring a thin-film metal electrode array (4 × 4 array), with dna capture probes to hybridize the pcr products for multiplexed electrochemical detection. the overall unit was a fully integrated system made up of pumps, valves, and fluidic micromixers, all made free from any fabricated moving parts and based on thermopneumatic expansion and electrolysis for pumping, cavitation microstreaming for mixing, and phase transition for fluidic valving. eliminating such parts simplified not only the design but also the fabrication, thus lowering the overall cost. the cost was further reduced by patterning the electrodes through the standard printed circuit board (pcb) process and forming the polycarbonate channels and chambers through computerized numerical control-machining. cavitation mixing was induced by a pair of piezoelectric disks mounted on the sample chamber and the microarray detection chamber, while electrolytic pumping was activated by platinum wires inserted into the pumping chambers. a magnet was mounted on the pcr chamber to trap and enrich the immunomagnetic capture beads that carried target pathogens or cells from the sample chamber for subsequent thermal lysis and asymmetric pcr. the unit was shown to work for pathogenic bacteria detection from whole rabbit blood by spiking it with escherichia coli, after which a single nucleotide polymorphism analysis was performed. despite the device's simple fabrication process, it entailed post-fabrication steps, such as introducing phase-changing material wax into the valves and conditioning the channel's surface. despite the claim that it is completely self-contained in terms of reagents and waste storage, the reagents were loaded at the time of sample introduction, which added to the in-use handling. other technical issues included the low efficiency of the pcr due to the immunomagnetic beads (≈50%) and their low cell-capture efficiency of ≈40%, as well as the low sensitivity of electrochemical detection. furthermore, the overall assay time was relatively long as sample preparation, amplification, and detection took ≈1 h each. a biochip featuring an integrated pcr with solid-phase extraction (spe) and ce detection has been proposed and tested. [78] bacillus anthraces (anthrax) and bordetella pertussis have been detected within ≈30 min,. while this is impressive, the biochip was not a self-contained unit with reagents and waste storage. reagents had to be externally supplied during each test and the sample had to be lysed off-chip. both these steps have to be integrated when providing a biochip or a cartridge for a portable diagnostic system. the possible inhibition of pcr by reagent traces of spe was avoided through the integration of membrane valves that decoupled the two process sites. the valves, however, involved multiple structural layers, adding further complexity to the fabrication. moreover, the valve activation required compressed air from an external line, which increased the hardware requirement, in addition to the infrared mediated thermal cycling, high-voltage product separation, and laser-induced fluorescence detection. the spe site had to be packed with silica beads, which added to the post-fabrication handling. despite all these technical issues, however, the study demonstrates the feasibility of a highly rapid sample-in answer-out capability. a portable and affordable real-time pcr system has been developed. the core of the system is a micromachined si chip integrated with a thin-film metal heater and a resistive temperature detector type of sensor. [47] the basic philosophy the researchers had in mind was to develop a cheap system with disposable parts in contact with the sample to avoid sample-to-sample cross-contamination, and, therefore, the mems chip was separated from the sample by a disposable microscope coverslip. the sample was placed together with a volume of a few μl, covered with mineral oil to prevent evaporation, forming a vrc. the first device was rather slow with sample and mineral oil volumes of 5 µl and 10 µl, respectively. the temperature was controlled externally via a personal computer, and the fluorescence was captured by an external microscope. separate to the pcr system, the same group also developed a miniaturized fluorescent system with the vision of integrating it later on with the pcr system. [79] then, the mems system was redesigned, and the sample volume was lowered to increase the speed to perform 40 pcr cycles in less than 6 min, [40] after which everything was integrated into a single system, demonstrating real-time rt-pcr detection in a sample containing the rna of the h5n1 avian flu virus in a single vrc-which is clearly insufficient for actual testing. [80] the system was evolved into a world-smallest real-time pcr capable of detecting four samples at a time and demonstrated the ability to perform real-time pcr of cdna from the h7n9 avian influenza virus, [81] rt-pcr from the ebola virus rna, [72] and finally, it was integrated with a bluetooth communication system that detected cdna from the dengue fever virus. [19] the system was even combined with the sample preparation step to detect rna from the h5n1 avian flu [11] or sars, [12] although they were far from perfect and required further improvement. furthermore, the vrc looked like an open system, but the system should be converted into a closed system to increase user comfort and avoid the possibility of accidental contamination. a sample-to-answer system that runs an automated sample preparation and real-time rt-pcr in an all-in-one cartridge has been developed and tested for influenza (h1n1), achieving an lod of 100 copies·ml -1 . [83] the cartridge was a three-dimensional acrylic block with chambers that extended between the top and bottom surfaces and featured millimeter-sized interconnecting channels ( figure 3b) . one chamber contained a silica membrane for nucleic acid adsorption, while another was designated for waste storage. all the remaining chambers were preloaded with liquid reagents, and the cartridge was completely sealed to reduce the risk of viral exposure or sample-to-sample cross-contamination. a nasopharyngeal swab sample, diluted in viral transport media, was injected, together with a lysis buffer, into the silica membrane chamber from a syringe equipped with a needle by punching the seal. the sample and reagent liquids were pneumatically manipulated within the cartridge under pressure, and a vacuum was applied from syringe pumps through the seal-piercing connectors. further fluidic control was provided by built-in surface tension valves. the amplification took place in three separate vials, each containing a frozen pcr master mix, which were inserted into the cartridge prior to the test. all the hardware was built in a desktop system that completed a fully automated test within 2.5 h. the test duration could be further shortened through miniaturization, which could also minimize the large dead volume introduced by the commercial silica membrane, as well as avoid diluting the viral rna due to a large eluent volume for elution consistency. moreover, the absence of mixing strategies implemented within the pcr vials led to a slightly lower pcr efficiency. otherwise, the cartridge appears to be simple and amenable to injection molding for mass dissemination. the fully automated sample-to-answer detection of influenza type h3n2 was based on a centrifugal microfluidic disk [84] named the labdisk ( figure 3c ). the disk was fully enclosed with preloaded reagents comprising liquid stick packs for nucleic acid extraction, air-dried specific pcr primers with fluorescence probes, and a lyophilized rt-pcr master mix. after loading the sample, the disk started to rotate creating a centrifugal force, triggering a series of events. first, the content of each stick pack was released into a designated chamber at a well-defined number of revolutions per min, breaking the fragile seals of the stick packs. then, the silica-coated magnetic beads captured and transported the nucleic acids from the lysis chamber into subsequent chambers for washing and release, with the aid of an external stationary magnet. the subsequent eluent was aliquoted into eight reaction cavities, dissolving the lyophilized rt-pcr master mix before thermal cycling through convective heating. the entire test took less than ≈3.5 h, with an lod of ≈2.4 × 10 4 of viral rna copies per ml of sample. all the hardware was packed into a lightweight shoebox-sized unit suitable for poc use, with the only manual step being the sample loading. the centrifugal pumping, together with the surface tension valving, simplified the overall design, allowing the disk to be fabricated by microthermoforming a polymer foil via hot embossing. the thermoformed foil was then sealed by a pressuresensitive adhesive tape. although the lod shown is fourfold lower than the clinically relevant level, the disk needs to be further developed to process viscous clinical specimens, such as sputum, through sample liquefaction and homogenization. a further reduction in analysis time and a demonstration of the ability to conduct multiplexing for sub-typing viral strains would be likely to bring the disk closer to practical poc use. the detection of the hepatitis c virus (hcv) in serum was demonstrated using a system based on a thermoformed plastic cartridge [85] where the reagents were confined into discrete wells isolated from each other by a layer of oil. the first well contained an acidic binding buffer that induced a positive surface charge on the ph-responsive magnetic particles mixed into a serum sample before being introduced. accordingly, the nucleic acids were captured by the charged particles and transported from well to well as the cartridge went through a series of translations and rotations between a pair of opposing stationary magnets ( figure 3d ). the surface charge was neutralized by an alkaline pcr solution, and the nucleic acid release was triggered. following the removal of the particles from the well, rapid thermal cycling took place, administered by the surrounding heat block through the thermoformed thin layer of plastic. a fluorescent signal was captured via a confocal detector placed above the well. more than a dozen clinical serum samples were tested, each analyzed within an hour and generating lods as low as 45 iu per 10 µl. while these results are encouraging, the unit gave rise to some of the same concerns as the others. first, the rt-pcr enzymes required refrigeration; this issue might be difficult to address by keeping the enzymes lyophilized because the pcr solution itself served as the rna eluate necessary for rehydration. second, the unit analyzed blood serum, which must be extracted beforehand, while the unit must be evaluated for other bodily samples relevant to respiratory infections such as sputum or a nasopharyngeal swab. third, the dynamic range should match the entire range of viral loads encountered clinically. fourth, the unit faced the issue of equally allocating particles among the wells, each of which has a distinct set of primers specific to the target serotype. fifth, the greater spread of the data was an issue relevant to the retrieval of viral rna but could be addressed by increasing the eluate volume by an order of magnitude, at the expense of test sensitivity. the processes of dna purification, pcr, post-pcr clean up, and inline injection, as well as ce separation, were all combined into a single microchip for forensic human identification ( figure 3e ). [86] the microchip included a miniaturized pdms pump and valves, a fluidized bed of immunomagnetic beads in bifurcating channels, a 250 nl pcr chamber, and a capillary electrophoresis channel with a double-t junction. the dna purification was realized through a sequence-specific capture of dna by a fluidized bed of streptavidin-coated magnetic beads. this, however, required off-chip handling, fragmenting the genomic dna, and then modifying the fragments with biotin-labeled capture probes. moreover, the dna capture efficiency was rather low (5.4%). an external magnet was applied to arrest the beads in the bifurcating channels and then manually displaced to transport the beads into the pcr chamber. the pcr master mix, including biotin-labeled primers, was introduced, and the pcr products were then concentrated into a narrow band within a streptavidin-modified gel plug inside the double-t junction. the thermal release of the band led the ce separation and detection. the microchip was accompanied by a small-footprint, battery-powered instrument capable of a full nineplex short tandem repeat (str) typing from 2.5 ng input dna, as well as forensic str analysis from oral swabs within 3 h. nevertheless, the high level of integration creates complex fabrication and post-fabrication handling, including the alignment and bonding of multiple layers of glass and a pdms membrane. all reagents had to be externally supplied and the sample lysed outside the microchip. table 1 lists the prototypes discussed above and compares their important features. the labdisk stands out as the sole cartridge that is fully enclosed with reagents and a waste storage. it can be stored at room temperature owing to the lyophilized pcr master mix. reagent evaporation and cross-mixing are addressed by the individually sealed stick packs that keep the reagents isolated. this isolation, along with the plastic housing, renders the cartridge robust against handling and transportation. the use of centrifugal microfluidics and silica-coated magnetic beads facilitates on-chip pumping, valving, and fluidic mixing that are realized in a simple design that can be produced through thermoforming. moreover, the cartridge runs on an instrument very similar to the cd or dvd players of the past. however, several challenges remain. the cartridge must be further developed to handle clinical specimens. the overall test duration should be reduced to below 1 h. finally, all these prototypes, albeit underdeveloped and very limited in number, are encouraging. however, it is imperative that many more studies are geared towards a poc-pcr test, instead of stand-alone pcr microchips, so that the next outbreak can be contained promptly. the time is running out for the vision before it fades away into a dream and a legacy for future generations. table 1 a comparison between the different systems currently available or under development. "cfu" stands for "colony forming unit," "c" for "copy," "hcv" for "hepatitis c virus," and "str" for "short tandem repeat," "na" for "not applicable," "ns" for "not stated," "lod" for "limit of detection", "ma" for "microarray", "ce" for "capillary electrophoresis," and "iu" for "infection unit" waste one of the most important practical challenges to be resolved for any poc molecular diagnostic device is sample preparation. ideally, it should be a sample-to-answer system with no human interaction; this is especially important for poc diagnostics, such as for sars in the past and currently for covid-19. during the sars outbreak in singapore, which was one of the worst-hit countries, the tan tock seng hospital was almost the only clinic capable of carrying out routine sars diagnostics. prospective patients' only option for testing was to get there, leading to a high chance of cross-infection. it was seen to be greatly beneficial to have a system for poc to ensure that prospective patients did not have to go anywhere, but rather stay at home and call health care providers to come to their location to conduct infectious disease diagnostics, and not simply collect a sample. this would demand a small system that is easily portable, with samples sealed to avoid sample-to-sample cross-contamination, thus suppressing the number of false-negative results. sample preparation is definitely a problem, and many attempts have been made to simplify these crucial steps. there are too many pcr inhibitors in human body fluid samples, such as blood and saliva. over the years, there has been continuous research into the development of pcr reagents, and especially into methods aimed at simplifying the sample preparation steps and fully integrating the sample preparation with pcr. single-step direct pcr has also been developed, greatly simplifying the sample preparation process; only hemoglobin has to be removed before performing pcr to diagnose malaria infection. [87] from the standpoint of practicality, the extraction and isolation of the dna/rna of pathogens from saliva are most desirable for poc applications. several studies have explored this area, including standard labor-intensive techniques. [88] figure 4 the systems currently available for covid-19 poc testing (poct). (a) a system performing 25 reactions at a time using magnetic beads for sample preparation, followed by a lamp. [89] (b) cobas® liat® system by roche originally designed as a universal molecular biology platform for diagnoses based on a real-time pcr. [90] (c) id now, made by abbott laboratories, originally sought to diagnose seasonal flu; it has currently been converted into a covid-19 system, based on an isothermal dna amplification originally developed for the seasonal flu test. [91] (d) bosch's new microfluidic system is capable of virus diagnostics; the sample preparation is integrated with a multiplexed end-point pcr. [92] pcr systems that offer multiplexing capability are preferred because they can reduce the test time for multiple diseases. this can be particularly beneficial during the flu season because different virus strains often present during one season. the differential diagnostics of community-acquired pneumonias can also benefit from this methodological approach, allowing not only the detection of multiple viruses and bacteria in one pcr reaction but also their relative quantification in the examined sample. such a quantification may lead to the correct determination of the disease-causing agent by selecting the most suitable therapy, [93] thus tackling the spread of a pandemic disease. the application of the broad-range pcr in multiplex arrangements can be used to detect a suspect group of viruses, giving rise to more detailed analyses in the initial phases of outbreaks. [94, 95] the pcr for poc and other applications should fulfill the essential requirements of portability, operability by untrained personnel, fast and adequate accuracy for screening reliability, and giving the minimum information for the publication of quantitative real-time pcr experimental guidelines. [96] miniaturization enjoys a big advantage in poc applications due to its portability and excellent accuracy and specificity; however, the complications arising from system operation outside the specialized laboratory environment as well as by untrained people pose the biggest challenge for the development of such devices. developments in the past have led to the commercialization of sample-to-answer systems that are more or less portable. one of the early birds in this regard was genexpert, [97] a cartridge-based system that performed a fully automated nucleic acid amplification technique. it was designed for the detection of healthcare-associated infections, critical infectious diseases, sexual health, viruses, and cancers. its development was accelerated by concerns over anthrax in the usa [64] and via the call for a proposal by the bill and melinda gates foundation to tackle hiv and tuberculosis in south africa. [65] the system simplifies the testing procedure as it integrates sample preparation with qpcr/rt-pcr. the system is designed to be employed in areas lacking sophisticated facilities to conduct conventional testing. it is not easily portable but it does offer the option of multicolor multiplexing. there was another push to release pcr systems for poc applications and this has contributed to disease diagnosis during pandemics such as the 2014 ebola outbreak. starting in early 2014, an unprecedented ebola outbreak occurred in west africa. on august 8, 2014, the world health organization (who) declared it a public health emergency of international concern[98] and initiated an emergency use assessment and listing (eual) mechanism to encourage research and development (r&d) of early access to unregistered products of in-vitro diagnostics [99] . cepheid received a $3.3 million grant, co-financed by the paul g. allen family foundation and the bill & melinda gates foundation, to develop the genexpert ebola assay based on the genexpert system. it took only ≈5 months from development for it to get listed in eual by who. the genexpert ebola assay is fully automated and only requires the placing of the patient sample into the cartridge and inserting the cartridge into a compact desktop-level instrument. it takes ≈2 h for an entire assay, from sample to answer, and features high detection accuracy. [100] filmarray biothreat-e is another product that was given emergency use authorization from both the food and drug administration (fda) and who. the assay is also a cartridge-based automatic rt-pcr system with a turnaround time of only 1 h. [101] the covid-19 global pandemic has greatly boosted the determination and motivation of researchers worldwide to speed up the development of poct systems, as well as the number of companies prepared to fund such work. some have adapted existing devices, while others have made new ones. the systems that were available in march 2020 mostly contained immunoassays [102] ; one was pcr-based, involving clusters of regularly interspaced short palindromic repeats, known as clustered regularly interspersed short palindromic repeats (crispr), with a crispr-associated protein, known as cas, performing the detection of the isothermal amplification products. other companies that have developed systems to diagnose seasonal flu have made use of different primers to convert these systems into poct for covid-19. the eiken chemical co. in japan has developed a system for multiple testing that combines sample preparation with lamp, and the optical detection of the results with rna purification is based on magnetic force ( figure 4a ). [89] it employed a microfluidic cartridge with 25 wells, allowing for multiplexed detection. roche created a poct system called cobas® liat®, a fast and fully automated sample-to-answer system based on a pcr capable of testing samples in 20 min or less ( figure 4b ). it utilized a sealed-tube design that eliminated the operators' contact with the reagents. its closed system with multiple controls also reduces the potential for human error. [90] id now, from abbott laboratories, is a fully integrated sample-to-answer system that was originally developed to diagnose a seasonal flu ( figure 4c ), [91] and is currently available with modified primers to diagnose the covid-19 virus. [103] it uses the isothermal amplification of nucleic acid, and it is capable of processing two samples at a time, with a claimed 5 min wait required for positive and 15 min for negative results. recently, bosch began r&d of a poct system; the result of their efforts is the vivalytic system ( figure 4d ), a molecular diagnostic device based on a microwell array that is capable of detecting the coronavirus in less than 2.5 h, using a fully sealed cartridge. the vivalytic system performs sample preparation, after which it performs a multiplex pcr, followed by microarray-detection, to allow the identification of sars-cov-2. [92] currently, the large-scale practical deployment of the above-mentioned devices to combat the pandemic spread of covid-19 will allow their advantages and disadvantages to be quickly determined, after which further development can be encouraged. more systems will emerge in the near future in the context of the covid-19 pandemic, after which companies are likely to switch their interest to systems that are more viable from an economic perspective. as of july 7, 2020, there were 104 approved test kits and systems approved by the u.s. fda for emergency use authorization, with the vast majority being based on a real-time rt-pcr technique with or without sample preparation. [104] given the development of communication technology, more and more devices are envisioned to be connected to form a network called the internet of things (iot), a burgeoning industry that is projected to change human society tremendously by bringing about convenience, ubiquitous information, and intelligence to improve our lives in a fundamental way; the health care industry can greatly benefit from this technological advancement. there have also been developments in personal healthcare monitoring devices linked with wearable healthcare monitoring, such as electrocardiogram and blood o 2 measurement devices. the data collected can be used for remote and/or real-time disease monitoring and predictive model building. furthermore, it is easy to predict that a diagnostic device such as pcr, with high connectivity and data transmission capabilities, can become highly impactful in pandemic management. we have demonstrated such a pcr in a pseudo dengue fever test; [19] however, in order to make it a true iot device we have to improve the compatibility of its communication module and its back-end data analysis capability. origin and evolution of pathogenic coronaviruses genomic characterisation and epidemiology of 2019 novel coronavirus: implications for virus origins and receptor binding coronavirus disease (covid-19) situation report-169 rna based mngs approach identifies a novel human coronavirus from two individual pneumonia cases in 2019 wuhan outbreak evolution and ecology of influenza a viruses, microbiol bat coronaviruses in china asymptomatic coronavirus infection: mers-cov and sars-cov-2 (covid-19) an emerging coronavirus causing pneumonia outbreak in wuhan, china: calling for developing therapeutic and prophylactic strategies viral load kinetics of mers coronavirus infection flow-through capture and in situ amplification can enable rapid detection of a few single molecules of nucleic acids from several milliliters of solution catching bird flu in a droplet clockwork pcr including sample preparation a rapid and label-free platform for virus capture and identification from clinical samples aptasensors for detection of avian influenza virus h5n1 the top 10 causes of death a review on viral biosensors to detect human pathogens multiplex pcr system for the rapid diagnosis of respiratory virus infection: systematic review and meta-analysis recent advances in lab-on-a-chip technologies for viral diagnosis iot pcr for pandemic disease detection and its spread monitoring specific enzymatic amplification of dna invitro -the polymerase chain-reaction revealing the secrets of pcr simultaneous amplification and detection of specific dna sequences kinetic pcr analysis: real-time monitoring of dna amplification reactions polymerase chain reaction/ligase detection reaction/hybridization assays using flow-through microfluidic devices for the detection of low-abundant dna point mutations characteristics and applications of nucleic acid sequence-based amplification (nasba) colorimetric detection of loop-mediated isothermal amplification reaction by using hydroxy naphthol blue strand displacement amplification-an isothermal, in vitro dna amplification technique rolling circle amplification: a versatile tool for chemical biology, materials science and medicine specific versus nonspecific isothermal dna amplification through thermophilic polymerase and nicking enzyme activities microfluidic lab-on-a-foil for nucleic acid analysis based on isothermal recombinase polymerase amplification (rpa) real-time detection of isothermal amplification reactions with thermostable catalytic hairpin assembly miniaturized total chemical analysis systems: a novel concept for chemical sensing micromachining a miniaturized capillary electrophoresis-based chemical analysis system on a chip chemical amplification: continuous-flow pcr on a chip functional integration of pcr amplification and capillary electrophoresis in a microfabricated dna analysis device a battery-powered notebook thermal cycler for rapid multiplex real-time pcr analysis effect of materials for micro-electro-mechanical systems on pcr yield microfluidic platforms for lab responding to covid-19-a once-in-a-century pandemic? ultra fast miniaturized real-time pcr: 40 cycles in less than six minutes extreme pcr: efficient and specific dna amplification in 15-60 seconds integrated extreme real-time pcr and high-speed melting analysis in 52 to 87 seconds multiplex polymerase chain reaction (pcr) on a su-8 chip automated polymerase chain reaction in capillary tubes with hot air a point-of-need infrared mediated pcr platform with compatible lateral flow strip for hpv detection a novel miniaturized pcr multi-reactor array fabricated using flip-chip bonding techniques disposable real-time micropcr device: lab-on-a-chip at a low cost precise temperature control and rapid thermal cycling in a micromachined dna polymerase chain reaction chip all-plastic, lowpower, disposable, continuous-flow pcr chip with integrated microheaters for rapid dna amplification versatile digital polymerase chain reaction chip design, fabrication, and image processing multifunctional system-on-glass for lab-on-chip applications design and experiment of a pdms-based pcr chip with reusable heater of optimized electrode high sensitivity pcr assay in plastic micro reactors pmma microfluidic chip fabrication using laser ablation and low temperature bonding with oca film and loca characterization of polydimethylsiloxane (pdms) properties for biomedical micro/nanosystems comparison of biocompatibility and adsorption properties of different plastics for advanced microfluidic cell and tissue culture models a bsa coated noa81 pcr chip for gene amplification inhibition of on-chip pcr using pdms-glass hybrid microfluidic chips multi-chamber pcr chip with simple liquid introduction utilizing the gas permeability of polydimethylsiloxane on-chip quantitative detection of pathogen genes by autonomous microfluidic pcr platform single-cell rt-pcr in microfluidic droplets with integrated chemical lysis chipbased analysis of exosomal mrna mediating drug resistance in glioblastoma evaluation of the cepheid genexpert® system for detecting bacillus anthracis integrating community-based interventions to reverse the convergent tb/hiv epidemics in rural south africa microfluidic systems for extracting nucleic acids for dna and rna analysis silicon-based microfilters for whole blood cell separation nucleic acid extraction, amplification, and detection on si-based microfluidic platforms miniaturized polymerase chain reaction device for rapid identification of genetically modified organisms integrated capture, concentration, polymerase chain reaction, and capillary electrophoretic analysis of pathogens on a chip a miniaturized quantitative polymerase chain reaction system for dna amplification and detection palm-sized device for point-of-care ebola detection a novel picoliter droplet array for parallel real-time polymerase chain reaction based on double-inkjet printing a fully integrated cmos fluorescence biochip for multiplex polymerase chain-reaction (pcr) processes an ewod-based microfluidic chip for single-cell isolation, mrna purification and subsequent multiplex qpcr from chip-in-a-lab to lab-on-a-chip: towards a single handheld electronic system for multiple application-specific lab-on-a-chip (asloc) self-contained, fully integrated biochip for sample preparation, polymerase chain reaction amplification, and dna microarray detection a fully integrated microfluidic genetic analysis system with sample-in-answer-out capability an integrated fluorescence detection system for lab-on-a-chip applications rapid detection of viral rna by a pocket-size real-time pcr system handheld real-time pcr device esensor® a microarray technology based on electrochemical detection of nucleic acids and its application to cystic fibrosis carrier screening a self-contained all-in-one cartridge for sample preparation and real-time pcr in rapid influenza diagnosis labdisk with complete reagent prestorage for sample-to-answer nucleic acid based detection of respiratory pathogens verified with influenza a h3n2 virus sample-to-answer droplet magnetofluidic platform for point-of-care hepatitis c viral load quantitation integrated dna purification, pcr, sample cleanup, and capillary electrophoresis microchip for forensic human identification human malaria diagnosis using a single-step direct-pcr based on the plasmodium cytochrome oxidase iii gene consistent detection of 2019 novel coronavirus in saliva fully automated molecular diagnostic system "simprova" for simultaneous testing of multiple items lab-in-a-tube: real-time molecular point-ofcare diagnostics for influenza a and b using the cobas® liat® system comparison of the id now influenza a & b 2, cobas influenza a/b, and xpert xpress flu point-of-care nucleic acid amplification tests for influenza a/b virus detection in children a sample-in-digital-answerout system for rapid detection and quantitation of infectious pathogens in bodily fluids comprehensive molecular testing for respiratory pathogens in community-acquired pneumonia identification of a novel coronavirus in patients with severe acute respiratory syndrome coronavirus as a possible cause of severe acute respiratory syndrome minimum information for publication of quantitative real-time pcr experiments use of the genexpert tuberculosis system for hiv viral load testing in india ebola virus disease norms and standards: assessing new medical products in health emergencies: the eual procedures performance of the genexpert ebola assay for diagnosis of ebola virus disease in sierra leone: a field evaluation study clinical evaluation of the biofire filmarray((r)) biothreat-e test for the diagnosis of ebola virus disease in guinea fast, portable tests come online to curb coronavirus pandemic laboratory diagnosis of emerging human coronavirus infections-the state of the art emergency use authorizations. 2020, pp. approved kits and tests for sars recently there is a boom in development of portable microfluidic based system for sars-cov-2 molecular diagnostics.most of those techniques approved by us fda is based on a reverse transcription polymerase chain reaction (rt-pcr) drawback of the rt-pcr methods such as sample preparation to remove pcr inhibitors and perform sample preconcentration ☒ the authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.☐the authors declare the following financial interests/personal relationships which may be considered as potential competing interests: key: cord-024088-020rgz5t authors: radandt, siegfried; rantanen, jorma; renn, ortwin title: governance of occupational safety and health and environmental risks date: 2008 journal: risks in modern society doi: 10.1007/978-1-4020-8289-4_4 sha: doc_id: 24088 cord_uid: 020rgz5t occupational safety and health (osh) activities were started in the industrialized countries already 150 years ago. separated and specific actions were directed at accident prevention, and the diagnosis, treatment and prevention of occupational diseases. as industrialization has advanced, the complexity of safety and health problems and challenges has substantially grown, calling for more comprehensive approaches. such development has expanded the scope, as well as blurred the borders between specific activities. in the modern world of work, occupational safety and health are part of a complex system that involves innumerable interdependencies and interactions. these are, for instance, safety, health, well-being, aspects of the occupational and general environment, corporate policies and social responsibility, community policies and services, community social environment, workers’ families, their civil life, lifestyles and social networks, cultural and religious environments, and political and media environments. a well-functioning and economically stable company generates resources to the workers and to the community, which consequently is able to maintain a positive cycle in development. a high standard of safety and health brings benefits for everyone: the company, the workers and the whole community. these few above-mentioned interactions elucidate the need for an integrated approach, and the modelling of the complex entity. if we picture osh as a house, this integrated approach could be the roof, but in order to build a stable house, it is also necessary to construct a solid basement as a foundation to the house. these basement "stones" are connected to each other, and are described in more detail in sections 4.1-4.3. section 4.1 focuses on the existing hazards, while section 4.2 mainly considers the exposure of workers to health hazards. health, due to its complexity, however, is not only influenced and impaired by work-related hazards, but also by hazards arising from the environment. these two sub-chapters are thus linked to section 4.3. in addition, the safety levels of companies may affect the environment. the strategies and measures needed for effective risk management, as described in section 4.1, therefore also contribute to reducing the risks to the environment. in the case of work that is done outdoors, the hazards arising from the environment understandably have to be given special attention. here, the methods applied to tackle the usual hazards at workplaces are less effective. it is necessary to develop protective measures to avoid or minimize hazards present in the environment. namely, agriculture, forestry and construction involve these types of hazards, and affect high numbers of workers on a global scale. finally, hazards in the environment or in leisure-time activities can lead to strain and injuries which -combined with hazards at work -may result in more severe health consequences. as an example one can mention the hazardous substances in the air causing allergies or other illnesses. another example is the strain on the musculoskeletal system from sports and leisuretime activities causing low back pain and other musculoskeletal disorders. depending on the type of hazard, the three topics, namely, safety, health and the environment, may share the common trait that the proper handling of risks, i.e., how to reduce probabilities and/or consequences of unwanted events is not always possible within a risk management system. this is true when one moves into the realm of uncertainty, i.e., when there is uncertain, insufficient or no knowledge of the consequences and/or probabilities (see chapter 5). 1. integrated multi-sectorial bodies for policy design and planning (national safety and health committee). 2. comprehensive approach in osh activities. 3. multi-disciplinary expert resources in inspection and services. 4. multi-professional participation of employers' and workers' representatives. 5. joint training in integrated activities. 6. information support facilitating multi-professional collaboration. international labour office (ilo) (2003) international labour conference, 91st session, report vi, ilo standards-related activities in the area of occupational safety and health: an in-depth study for discussion with a view to the elaboration of a plan of action for such activities. sixth item on the agenda. international labour office, geneva. what are the main challenges arising from the major societal changes for business/companies and workers/employees? how can these challenges be met in order to succeed in the growing international competition? what is the role of occupational safety and health (osh) in this context? the above-mentioned changes create new possibilities, new tasks and new risks to businesses in particular, and to the workers as well. in order to optimize the relation between the possibilities and the risks (maximize possibilities -minimize risks) there is a growing need for risk management. risk management includes all measures required for the target-oriented structuring of the risk situation and safety situation of a company. it is the systematic application of management strategies for the detection, assessment, evaluation, mastering and monitoring of risks. risk management was first considered exclusively from the point of view of providing insurance coverage for entrepreneurial risks. gradually the demands of jurisdiction grew, and the expectations of users and consumers increased with regard to the quality and safety of products. furthermore, the ever more complex problems of modern technology and ultimately the socioeconomic conditions have led to the development of risk management into an independent interdisciplinary field of work. risks can be regarded as potential failures, which may decrease trust in realizing a company's goals. the aim of risk management is to identify these potential failures qualitatively and quantitatively, and to reduce them to the level of a non-hazardous and acceptable residual risk potential. the development and formulation of a company's risk policy is regarded as the basis of effective risk management. this includes, first and foremost, the management's target concept with respect to the organization of work, distribution of labour, and competence of the departments and persons in charge of risk management. risk issues are important as far as acceptance of technology is concerned. it is not enough to reduce the problem to the question of which risks are tolerable or acceptable. it appears more and more that, although the risks themselves are not accepted, the measures or technologies causing them are. value aspects have an important role in this consideration. a positive or negative view of measures and technologies is thus influenced strongly by value expectations that are new, contradictory and even disputed. comparing risks and benefits has become a current topic of discussion. the relation between risks and benefits remains an unanswered question. the general public has a far broader understanding of the risks and benefits of a given technology than the normal understanding professed by engineering sciences which is limited to probability x harm. the damage or catastrophe potential, qualitative attributes such as voluntary nature and controllability also play an important role in the risk assessment of a technology. a normative setting for a certain, universally accepted risk definition according to engineering science is therefore hardly capable of consensus at the moment. the balanced management of risks and possibilities (benefits) is capable of increasing the value of a company. it may by far surpass the extent of legal obligations: for example, in germany, there is a law on the control and transparency for companies (kontrag) . the respective parameters may be defined accurately as follows: • strategic decisions aim to offer opportunities for acquiring benefit, taking into consideration risks. • risks that can have negative consequences to the technological capacities, the profitability potential, the asset values and the reputation of a company, as well as the confidence of shareholders are identified and measured. • the management focuses on important possibilities and risks, and addresses them adequately or reduces them to a tolerable level. the aim is not to avoid risks altogether, but to create opportunities for promoting proactive treatment of all important risks. the traditional occupational health hazards, such as physical, chemical and biological risks, as well as accidents, will not totally disappear as a consequence of change, nor will heavy physical work. about 30-50% of workers are still exposed to such hazards. there is thus need to still develop risk assessment, prevention and control methods and programmes for these often well-known hazards. in many industrialized countries, prevention and control programmes have had a positive impact by reducing the trends of occupational diseases and accidents, particularly in big industries. some developing countries, however, show an increase in traditional hazards. international comparisons, however, are difficult to make because of poor coverage, underreporting, and poor harmonization of concepts, definitions and registration criteria. statistics on occupational accidents are difficult to compare, and therefore data on their total numbers in europe should be viewed with caution. the majority of countries, however, have shown declining trends in accident rates irrespective of the absolute numbers of accidents. some exceptions to this general trend have nevertheless been seen. the accident risk also seems to shift somewhat as regards location, so that instead of risks related to machines and tools, particularly the risks in internal transportation and traffic within the workplace grow in relative importance. this trend may increase in future, particularly as the work place, as well as the speed and volume of material flows are increasing. a threat is caused by lengthened working hours, which tend to affect the vigilance of workers and increase the risk of errors. small-scale enterprises and micro-enterprises are known to have a lower capacity for occupational health and safety than larger ones. in fact, a higher accident risk has been noted in medium-sized companies, and a lower risk in very small and very large enterprises. we can conclude that this is due to the higher mechanization level and energy use in small and medium-sized enterprises (sme) compared with micro-enterprises, which usually produce services. on the other hand, the better capacity of very large enterprises in safety management is demonstrated by their low accident rates. the production of chemicals in the world is growing steadily. the average growth has been between 2-5% a year during the past 2-3 decades. the total value of european chemical production in 1998 was about usd 500 billion, i.e. 32% of the world's total chemical production, and it has increased 35% in the 10-year period of 1985-1995. the european union (eu) is the largest chemical producer in the world, the usa the second, and japan the third. there are some 100,000 different chemical entities in industrial use, but only about 2,500 are so-called high-production volume (hpv) chemicals produced (and consumed) in amounts exceeding 1,000 tons a year. the number of chemicals produced in amounts of 10-1,000 tons a year is about 25,000. but volume is not necessarily the most important aspect in chemical safety at work. reactivity, toxicological properties, and how the chemicals are used, are more important. the european chemical companies number some 39,000, and in addition there are 36,000 plants producing plastics and rubber. surprisingly, as many as 96% of these are smes employing fewer than 50 workers, and 70% are micro-enterprises employing fewer than 10 workers. thus, the common belief that the chemical industry constitutes only large firms is not true. small enterprises and self-employed people have much less competence to deal with chemical risk assessment and management than the large companies. guidance and support in chemical safety is therefore crucial for them. the number of workers dealing with chemicals in the european work life is difficult to estimate. the chemical industry alone employs some 1.7 million workers in europe, i.e. about 7% of the workforce of manufacturing industries. about 30%, i.e. over 500,000 work in chemical smes. but a much higher number of workers are exposed in other sectors of the economy. there is a distinct trend showing that the use of chemicals is spreading to all sectors, and thus exposures are found in all types of activities: agriculture, forestry, manufacturing, services and even in high-tech production. the national and european surveys on chemical exposures in the work environment give very similar results. while about 25% of the eu work-ers were exposed to hazardous chemicals, the corresponding figure in central and eastern european countries may be much higher. the workers are exposed simultaneously to traditional industrial chemicals, such as heavy metals, solvents and pyrolytic products, and to "new exposures", such as plastics monomers and oligomers, highly reactive additives, cross-linkers and hardeners, as well as to, for example, fungal spores or volatile compounds in contaminated buildings. this implies that some 40 million people in the eu15 are exposed at work, and usually the level of exposure is one to three orders of magnitude higher than in any other environment. about the same proportion (25% of the workforce, i.e. 500,000) of the finnish workers in the national survey reported exposure. the chemicals to which the largest numbers of workers are exposed occur typically in smes; they are e.g. detergents and other cleaning chemicals, carbon monoxide, solvents, environmental tobacco smoke, and vegetable dusts. european directives on occupational health and safety require a high level of protection in terms of chemical safety in all workplaces and for all workers. risk assessment and risk management are key elements in achieving these requirements. the risk assessment of chemicals takes place at two levels: a) systems-level risk assessment, providing a dose-response relationship for a particular chemical, and serving as a basis for standard setting. risk assessment at the systems level is carried out in the pre-marketing stage through testing. this consequently leads to actions stipulated in the regulations concerning standards and exposure limits, labelling and marking of hazardous chemicals, limitations in marketing, trade and use. in this respect, the level of safety aimed at remains to be decided. is it the reasonably achievable level or, for example, the level achieved by the best available technology? the impact is expected to be system-wide, covering all enterprises and all workers in the country. this type of risk assessment is an interactive practice between the scientific community and the politically controlled decision making. a high level of competence in toxicology, occupational hygiene and epidemiology is needed in the scientific community. and the decision makers must have the ability to put the risk in concern into perspective. in most countries the social partners also take part in the political decision making regarding occupational risks. b) workplace risk assessment directed at identifying the hazards at an individual workplace and utilizing standards as a guide for making decisions on risk management. risk assessment at workplace level leads to practical actions in the company and usually ensures compliance with regulations and standards. risk assessment is done by looking at the local exposure levels and comparing these with standards produced in the type a) risk assessment. risk management is done through preventive and control actions by selecting the safest chemicals, by controlling emissions at their source, by general and local ventilation, and by introducing safe working practices. if none of the above is effective, personal protective devices must be taken into use. noise is a nearly universal problem. the products of technology, which have freed us from the day-to-day survival struggle, have been accompanied by noise as the price of progress. however, noise can no longer be regarded as an inevitable by-product of today's society. not only is noise an undesirable contaminant of our living environment, but high levels of noise are frequently present in a variety of work situations. many problems arise from noise: annoyance, interference with conversation, leisure or sleep, effects on work efficiency, and potentially harmful effects, particularly on hearing. in short, noise may affect health, productivity, and well-being. the selection of appropriate noise criteria for the industry depends on knowledge of the effects of noise on people, as well as on the activities in which they are engaged. many of the effects are dependent on the level, and the magnitude of the effects varies with this level. hearing damage is not the only criterion for assessing excessive noise. it is also important to consider the ability and ease of people to communicate with each other. criteria have therefore been developed to relate the existing noise environment to the ability of the typical individual to communicate in areas that are likely to be noisy. the effects of noise on job performance are difficult to evaluate. in general, one can say that sudden, intermittent, high-intensity noise impedes efficient work more than low-intensity and steady-state noise. the complexity of the task with which noise interferes plays a major role in determining how much noise actually degrades performance. two common ways in which noise can interfere with sleep are: delaying the onset of sleep, and shifting sleep stages. one effect of noise that does not seem to depend strongly on its level is annoyance. under some circumstances, a dripping water faucet can be as annoying as a jackhammer. there are no generally accepted criteria for noise levels associated with annoyance. if the noise consists of pure tones, or if it is impulsive in nature, serious complaints may arise. new information and communication technologies (ict) are being rapidly implemented in modern work life. about 65% of workers have computers at work, and about 35% are e-mail and internet users. there are three main problem areas in the use of new ict at work. these are: 1) the visual sensory system, 2) the cognitive processes, and 3) the psychomotoric responses needed for employing hand-arm systems. all three have been found to present special occupational health and even safety problems, which are not yet fully solved. the design of new more user-friendly technology is highly desirable, and the criteria for such technology need to be generated by experts in neurophysiology, cognitive psychology and ergonomics. it is important to note that the productivity and quality of information-intensive work requiring the use of ict depends crucially on the user-friendliness of the new technology interface, both the hardware and software. communication and information technologies will change job contents, organization of work, working methods and competence demands substantially in all sectors of the economy in all countries. a number of new occupational health and safety hazards have already arisen or are foreseen, including problems with the ergonomics of video display units, and musculoskeletal disorders in shoulder-neck and arm-hand systems, information overload, psychological stress, and pressure to learn new skills. the challenge to occupational health and safety people is to provide health-based criteria for new technologies and new types of work organization. it is also important to contribute to the establishment of healthy and safe work environments for people. in the approved and draft standards of the international standardization organization, iso, there are altogether about 50 different targets dealing with the standardization of eyesight-related aspects. vision is the most important channel of information in information-intensive work. from the point of view of seeing and eye fatigue, the commonly used visual display units (vdu) are not the most optimal solutions. stability of the image, poor lighting conditions, reflections and glare, as well as invisible flicker, are frequent problems affecting vision. the displays have, however, developed enormously in the 1990s, and there is evidence that the so-called flat displays have gradually gained ground. information-intensive work may increasingly load the vision and sense of hearing, particularly of older workers. even relatively minor limitations in vision or hearing associated with ageing have a negative effect on receiving and comprehending messages. this affects the working capacity in information-intensive work. the growing haste in information-intensive work causes concern among workers and occupational health professionals. particularly older workers experience stress, learning difficulties and threat of exclusion. corrective measures are needed to adjust the technology to the worker. the most important extension of the man-technology interface has taken place in the interaction of two information-processing elements: the central nervous system and the microprocessor. the contact is transmitted visually and by the hands, but also by the software which has been developed during the 1990s even more than the technology itself. many problems are still associated with the immaturity of the software, even though its user-friendliness has recently greatly improved. the logic and structure of the software and the user systems, visual ergonomics, information ergonomics, the speed needed, and the forgiving characteristics of programs, as well as the possibility to correct the commands at any stage of processing are the most important features of such improvements. also the user's skills and knowledge of information technology and the software have a direct effect on how the work is managed and how it causes workload. the user-friendliness and ergonomics of the technology, the disturbing factors in the environment, haste and time pressure, the work climate, and the age and professional skills of the individual user, even his or her physical fitness, all have an impact on the cognitive capacity of a person. this capacity can to a certain extent be improved by training, exercise and regulating the working conditions, as well as with expert support provided for the users when difficulties do occur. the use of new technologies has been found to be associated with high information overload and psychological stress. the problem is not only typical for older workers or those with less training, but also for the super-experts in ict who have shown an elevated risk of psychological exhaustion. there are four main types of ergonomic work loads: heavy dynamic work that may overload both the musculoskeletal and cardiovascular system; re-petitive tasks which may cause strain injuries; static work that may result in muscular pain; and lifting and moving heavy loads, which may result in overexertion, low back injury, or accidental injuries. visual ergonomics is gaining in importance in modern work life. the overload of the visual sensory system and unsatisfactory working conditions may strain the eye musculature, but can also cause muscle tension in the upper part of the body. this effect is aggravated if the worker is subjected to psychological stress or time pressure. in addition to being a biological threat, the risk of infections causes psychological stress to workers. the improved communication between health services and international organizations provides help in the early detection and control of previously unknown hazards. nevertheless, for example, the danger related to drug abusers continues to grow and present a serious risk to workers in, for example, health services and the police force. some new viral or re-emerging bacterial infections also affect health care staff in their work. the increase in the cases of drug-resistant tuberculosis is an example of such a hazard. the goal of preventive approaches is to exert control on the cause of unwelcome events, the course of such negative events or their outcome. in this context, one has to decide whether the harmful process is acute (an accident) or dependent on impact duration and stimulus (short-, medium-, and long-term). naturally, the prevention approaches depend on the phases of the harmful process, i.e. whether the harm is reversible, or whether it is possible only to maintain its status, or to slow down the process. it is assumed here that a stressful factor generates an inter-individual or intra-individual strain. thus the effects and consequences of stress are dependent on the situation, individual characteristics, capabilities, skills and the regulation of actions, and other factors. the overall consideration is related to work systems characterized by work contents, working conditions, activities, and actions. system performance is expected of this work system, and this system performance is characterized by a performance structure and its conditions and requirements (figure 4 .2). the performance of the biopsychosocial unit, i.e. the human being, plays an important role within the human performance structure (see figures 4.2 and 4.3). the human being is characterized by external and internal features, which are closely related to stress compatibility, and thus to strain. in this respect, preventive measures serve to optimize and ensure performance, on the one hand, and to control stress and strain, on the other. preventive measures aim to prevent bionegative effects and to facilitate and promote biopositive responses. • the internal factors affecting performance are described by performance capacity and performance readiness. • performance capacity is determined by the individual's physiological and psychological capacity. • performance readiness is characterized by physiological fitness and psychological willingness. • the external factors affecting performance are described by organizational preconditions/requirements and technical preconditions/requirements. • regarding the organizational requirements, the organizational structure and organizational dynamics are of significance. • in the case of technical requirements, the difficulties of the task, characterized by machines, the entire plant and its constructions, task content, task design, technical and situation-related factors, such as work layout, anthropometrics, and quality of the environment, are decisive (table 4 .1). mental stress plays an increasing role in the routine activities of enterprises. through interactive models of mental stress and strain, it is possible to represent the development of mental strain and its impairing effects (e.g. tension, fatigue, monotony, lack of mental satisfaction). it is important to distinguish the above-mentioned impairing effects from each other, since they can arise from different origins and can be prevented or eliminated by different means. activities that strain optimally enhance health and promote safe execution of work tasks. stress essentially results from the design parameters of the work system or workplace. these design parameters are characterized by, e.g.: • technology, such as work processes, work equipment, work materials, work objects; • anthropometric design; • work techniques, working hours, sharing of work, cycle dependence, job rotation; • physiological design that causes strain, fatigue; • psychological design that either motivates or frustrates; • information technology, e.g. information processing, cognitive ergonomic design; • sociological conditions; and • environmental conditions, e.g. noise, dust, heat, cold. the stress structure is very complex, and we therefore need to look at the individual parameters carefully, taking into account the interactions and links between the parameters at the conceptual level. the design parameters impact people as stress factors. as a result, they also turn into conditions affecting performance. such conditions can basically be classified into two types: a person's internal conditions, characterized in particular by predisposition and personality traits, and a person's external conditions, determined mainly by the design parameters. when we look at performance as resulting from regulated or reactive action, we find three essential approaches for prevention: • the first approach identifies strain. it is related to anatomical, biochemical, histological, physiological characteristic values, typical curves of organ systems, the degree of utilization of skills through stress, and thus the degree of utilization of the dynamics of physiological variables in the course of stress. • the second approach is related to the control of strain. the aim is to identify performance limits, the limits of training and practice, and to put them into positive use. adaptation and fatigue are the central elements here. • the third approach for prevention is related to reducing strain. the aim is to avoid harm, using known limits as guidelines (e.g. maximum workplace concentration limit values for harmful substances, maximum organspecific concentration values, biological tolerance values for substances, limit values for noise and physical loads). however, the use of guideline values can only be an auxiliary approach, because the stress-strain concept is characterized by highly complex connections between the exogenous stress and the resulting strain. an objectively identical stress will not always cause the same level of strain in an individual. due to action regulation and individual characteristic values and curves of the organ systems (properties and capabilities), differences in strain may occur. seemingly identical stress can cause differing strain due to the superposition of partial stress. combinations of partial stress can lead to compensatory differences (e.g. physiological stress can compensate for psychological stress) or accumulation effects. partial stress is determined by the intensity and duration of the stress, and can therefore appear in differing dimensions and have varying effects. in assessing overall stress, the composition of the partial stress according to type, intensity, course and time is decisive. partial stress can occur simultaneously and successively. in our considerations, the principle of homeostasis plays an important role. however, optimizing performance is only a means to an end in a prevention programme. the actual purpose is to avoid harm, and thus to control strain. harm is a bionegative effect of stress. the causative stress is part of complex conditions in a causal connection. causal relationships can act as dose-effect relationships or without any relation to the dose. in this respect, the causative stress condition can form a chain with a fixed or variable sequence; it can add up, multiply, intensify or have an effect in only specific combinations, and generate different effects (e.g. diseases). we are thus dealing with a multicausal model or a multi-factor genesis. low back pain is an example of a complex phenomenon. the incidence of musculoskeletal disorders, especially low back pain, is rapidly increasing. several occupational factors have been found to increase the risk for low back pain. some studies indicate that psychosocial and work-related conditions are far more accurate in the prognosis of disability than are physical conditions. chronic low back pain is perceived as a phenomenon which encompasses biological, social and psychological variables. according to the model of adaptation, the goal of reducing risks is to increase a person's physical abilities (i.e. flexibility, strength, endurance), the use of body mechanics, techniques to protect the back (following the rules of biomechanics), to improve positive coping skills and emotional control. the following unfavourable factors leading to back pain have been identified at workplaces: • the lifting of too heavy loads. • working in a twisted or bent-down position. • work causing whole-body vibration. • working predominantly in a sitting position. • carrying heavy loads on the shoulders. the prevention of acute back pain and the prevention of work disability must entail several features. one important element is work safety, which can be maximized by screening a worker's physical and intellectual capacities, by ensuring ergonomic performance of the work procedures, and by increasing awareness of proper working techniques that do not strain the back. the use of adaptation programmes makes it possible to attain a higher performance level and to be able to withstand more strain (figure 4.4) . research-based methods of training optimize and improve performance. they are a means for controlling stress and strain with the aim of preventing bionegative effects and facilitating and promoting biopositive responses. the stress (load) and strain model and human performance can be described as follows: • causative stress generates an inter-individual or intra-individual strain. • the effects and consequences depend on a person's properties, capabilities, skills and the regulation of actions, individual characteristics of the organ systems, and similar factors. • within the performance structure, the performance of the biopsychosocial unit, i.e. the human being, plays an important role. the human being is characterized by external and internal factors, which in turn are closely related to stress compatibility and thus to strain. the connection between stress and harm plays a significant role in the research on occupational health hazards. how should this connection be explored? different hypotheses exist in replying to this question, but none of them have been definitively proven. the three most common hypotheses today are: stress occurring in connection with a person's life events. the number and extent of such events is decisive. problem-coping behaviour and/or social conditions are variables explaining the connection between stress and harm. 3. the additive stress hypothesis. the ability to cope with problems and the social conditions has an effect on harm which is independent of the stress resulting from life events. when we refer to the complexity of risks in this context of occupational safety our focus shall be on the enterprise. there are different kinds of risks to be found in enterprises. many of them are of general importance, i.e. they are in principle rather independent of an enterprise's size or its type of activity. how to deal with such risks shall be outlined to some extent here. in order to treat those risks at work successfully resources are needed whose availability often depends on the enterprise's situation. the situation in enterprises usually is a determining indicator for available resources to control and develop safety and health and thus performance of the enterprise and its workers and employees through appropriate preventive measures. this situation has been described to some extent in chapter 2. big companies usually have well-developed safety and health resources, and they often transfer appropriate policies and practices to the less developed areas where they operate. even in big enterprises, however, there is fragmentation of local workplaces into ever smaller units. many of the formerly in-built activities of enterprises are outsourced. new types of work organizations are introduced, such as flat and lean organizations, increase of telework and call centres, many kinds of mobile jobs and network organizations. former in-company occupational health services are frequently transferred to service providers. this leads to the establishment of high numbers of micro-enterprises, small scale enterprises (sses), small and medium-sized enterprises (smes) and self-employed people. sses and smes are thus becoming the most important employers in the future. from a number of studies there is evidence that at least among a part of sses and smes awareness of osh risks is low. both managers and workers often do not see the need to improve occupational safety and health or ergonomic issues and their possibilities and benefits by reducing or eliminating risks at work. as these types of enterprises, even more the self-employed, do not have sufficient resources or expertise for implementing preventive measures, the need for external advisory support, services and incentives is evident and growing. interpersonal relations in sses and smes being generally very good provides a strong chance for effectively supporting them. other special features in the structure of small and medium-sized enterprises to be considered are: • direct participation of the management in the daily activities; • the management structure is designed to meet the requirements of the manager; • less formal and standardized work processes, organizational structures and decision processes; • no clear-cut division of work: -wide range of tasks; and -less specialization of the employees; • unsystematic ways of obtaining and processing information; • great importance of direct personal communication; • less interest in external cooperation and advice; • small range of internal, especially long-term and strategic planning; and • stronger inclination of individual staff members to represent their own interests. the role of occupational health services (ohs) in smes is an interdisciplinary task, consisting of: • risk assessment: -investigation of occupational health problems according to type of technology, organization, work environment, working conditions, social relationships. • surveillance of employees' health: -medical examinations to assess employees' state of health; and -offering advice, information, training. • advice, information, training: -measures to optimize safety and health protection; and -safe behaviour, safe working procedures, first aid preparedness. different kinds of risks are found in enterprises (see table 4 .2). these different types of risks need to be handled by an interlinked system to control the risks and to find compromises between the solutions. figure 4 .7 illustrates these linkages. the promotion of safety and health is linked to several areas and activities. all of these areas influence the risk management process. the results of risk treatment not only solve occupational health and safety problems, but they also give added value to the linked fields. specific risk management methods are needed to reach the set goal. one needs to know what a risk is. the definition of risk is essential: a risk is a combination of a probability -not frequency -of occurrence, and the associated unwelcome outcome or impact of a risk element (consequence). risk management is recognized as an integral part of good management practice. it is a recurring process consisting of steps which, when carried out in a sequence, allow decision making to be improved continuously. risk management is a logical and systematic method of identifying, analyzing, evaluating, treating, monitoring and communicating risks arising during any activity, function, or process in a manner enabling the organization to minimize losses and maximize productive opportunities. different methods are available for analyzing problems. each method is especially suited to respond to certain questions and less suited for others. a complex "thinking scheme" is necessary for arranging the different analyses correctly within the system review. such a scheme includes the following steps: 1. defining the unit under review: the actual tasks and boundaries of the system (a fictitious or a real system) must be specified: time, space and state. 2. problem analysis: all problems existing in the defined system, including problems which do not originate from the system itself, are detected and described. 3. causes of problems: all possible or probable causes of the problems are identified and listed. 4. identifying interaction: the dependencies of the effect mechanisms are described, and the links between the causes are determined. 5. establishing priorities and formulating targets: to carry out this step, it is necessary to evaluate the effects of the causes. 6. solutions to the problems: all measures needed for solving the individual problems are listed. the known lists usually include technical as well as non-technical measures. since several measures are often appropriate for solving one problem, a pre-selection of measures has to be done already at this stage. however, this can only be an approach to the solution; the actual selection of measures has to be completed in steps 7 and 8. 7. clarifying inconsistencies and setting priorities: as the measures required for solving individual problems may be inconsistent in part, or may even have to be excluded as a whole, any inconsist-encies need to be clarified. a decision should then be made in favour or against a measure, or a compromise may be sought. 8. determining measures for the unit under review: the measures applicable to the defined overall system are now selected from the measures for the individual problems. 9. list of questions regarding solutions selected for the overall system: checking whether the selected measures are implementable and applicable for solving the problems of the overall system. 10. controlling for possible new problems: this step consists of checking whether new problems are created by the selected solution. the close link between cause and effect demands that the processes and sub-processes must be evaluated uniformly, and risks must be dealt with according to a coordinated procedure. the analysis is started by orientation to the problem. this is done in the following steps: 1. recognizing and analyzing the problem according to its causes and extent, by means of a diagnosis and prediction, and comparison with the goals aimed at. 2. description and division of the overall problem into individual problem areas, and specifying their dependencies. 3. defining the problem and structuring it according to the objectives, time relation, degree of difficulty, and relevance to the goal. 4. detailed analysis of the causes, and classification in accordance to the possible solution. the analysis of the problem should be integrated into the overall analytical process in accordance with the thinking schemes described earlier. the relevance and priorities related to the process determine the starting point for the remaining steps of the analysis. analyses are divided into quantitative and qualitative ones. quantitative analyses include risk analyses, that is, theoretical safety-related analysis methods and safety analyses, e.g. classical accident analyses. qualitative analyses include failure mode and effect analyses, hazard analyses, failure hazard analyses, operating hazard analyses, human error code and effect analyses, information error and effect analyses. the theoretical safety-related analysis methods include inductive and deductive analyses based on boolean models. inductive analyses are, e.g. fault process analyses. deductive analyses are fault tree analyses, analytical processes and simulation methods. theoretical safety-related analysis methods which are not based on boolean models are stochastic processes, such as markow's model, risk analyses and accident analyses which, as a rule, are statistical or probability-related analyses. a possible scheme to begin with is shown in figure 4 .9. since absolute safety, entailing freedom from all risks, does not exist in any sphere of life, the task of those dealing with safety issues is to avert hazards and to achieve a sustainable reduction of the residual risk, so that it does not exceed a tolerable limit. the extent of this rationally acceptable risk is also influenced by the level of risk which society intuitively considers as being acceptable. those who propose definitions of safety are neither authorized nor capable of evaluating the general benefit of technical products, processes and services. risk assessment is therefore focused at the potential harm caused by the use or non-use of the technology. the guidelines given in "a new approach to technical harmonization and standards" by the council resolution of 7 may 1985 are valid in the european union. the legal system of a state describes the protective goals, such as protection of life, health, etc., in its constitution, as well as in individual laws and regulations. as a rule, these do not provide an exact limit as to what is still a tolerable risk. this limit can only be established indirectly and unclearly on the basis of the goals and conceptions set down by the authorities and laws of a state. in the european union, the limits are expressed primarily in the "basic safety and health requirements". these requirements are then put into more concrete terms in the safety-related definitions issued by the bodies responsible for preparing industrial standards. compliance with the standards is voluntary, but it is presumed that the basic requirements of the directives are met. the term which is opposite to "safety" is "hazard". both safe and hazardous situations are founded on the intended use of the technical products, processes and services. unintended use is taken into account only to the extent that it can be reasonably foreseen. the risks present in certain events are, in a more narrow sense, unwelcome and unwanted outcomes with negative effects (which exceed the range of acceptance). unwelcome events are • source conditions of processes and states; • processes and states themselves; and • effects of processes and states which can result in harm to persons or property. an unwelcome event can be defined as a single event or an event within a sequence of events. possible unwelcome events are identified for a unit under review. the causes may be inherent in the unit itself, or outside of it. in order to determine the risks involved in unwelcome events, it is necessary to identify probabilities and consequences. the question arises: are the extent and probability of the risk known? information is needed to answer this question. defining risk requires information concerning the probability of occurrence and the extent of the harm of the consequences. uncertainty is given if the effects are known but the probability is unknown. ignorance is given if both the effects and the probability are unknown. figure 4 .10 shows the risk analysis procedure according to the type of information available. since risk analyses are not possible without practical, usable information, it is necessary to consider the nature of the information. the information is characterized by its content, truth and objectivity, degree of confirmation, the possibility of being tested, and the age of the information. the factors determining the content of the information are generality, precision and conditionality. the higher the conditionality, the smaller is the generality, and thus the smaller the information content of the statement. truth is understood as conformity of the statement with the real state of affairs. the closer that the information is to reality, the higher is its information content, and the smaller its logical margin. the degree of controllability is directly dependent on the information content: the bigger the logical margin, the smaller the information content, and thus the higher the probability that the information content will prove its worth. in this respect, probability plays a role in the information content: the greatest significance is attributed to the logical hypothetical probability and statistical probability of an event. objectivity and age are additional criteria for any information. the age and time relation of information play a particularly important role, because consideration of the time period is an important feature of analysis. as a rule, information and thus the data input in the risk analysis consist of figures and facts based on experience, materials, technical design, the organization and the environment. in this regard, most figures are based on statistics on incidents and their occurrences. factual information reveals something about the actual state of affairs. it consists of statements related to past conditions, incidents, etc. forecast-type predictions are related to real future conditions, foretelling that certain events will occur in the future. explanatory information replies to questions about the causes of phenomena, and provides explanations and reasons. it establishes links between different states based on presumed cause-effect relationships. subjunctive information expresses possibilities, implying that certain situations might occur at present or in the future, thus giving information about conceivable conditions, events and relationships. normative information expresses goals, standards, evaluations and similar matters; it formulates what is desirable or necessary. the main problem with risk analyses is incomplete information, in particular regarding the area of "uncertainty". in the eu commission's view, recourse to the so-called precautionary principle presupposes that potentially dangerous effects deriving from a phenomenon, product or process have been identified via objective scientific evaluation, and that scientific evaluation does not allow the risk to be determined with sufficient certainty. recourse to the precautionary principle thus takes place in the framework of general risk management that is concretely connected to the decision-making process. if application of the precautionary principle results in the decision that action is the appropriate response to a risk, and that further scientific information is not needed, it is still necessary to decide how to proceed. apart from adopting legal provisions which are subject to judicial control, a whole range of actions is available to the decision-makers (e.g. funding research, or deciding to inform the public about the possible adverse effects of a product or procedure). however, the measures may not be selected arbitrarily. in conclusion, the assessment of various risks and risk types which may be related to different types of hazards requires a variety of specific risk assessment methods. if one has dependable information about the probability and consequences of a serious risk or risky event, one should use the risk assessment procedure shown in figure 4 .11. • major industrial accidents; • damage caused by dangerous substances; • nuclear accidents; • major accidents at sea; • disasters due to forces of nature; and • acts of terrorism. • dangerous substances discharged (fire, explosion); • injury to people and damage to property; • immediate damage to the environment; • permanent or long-term damage to terrestrial habitats, to fresh water, to marine habitats, to aquifers or underground water supplies; and • cross-border damage. • technical failure: devices, mountings, containers, flanges, mechanical damage, corrosion of pipes, etc.; • human failure: operating error, organizational failure, during repair work; • chemical reaction; • physical reaction; and • environmental cause. system analysis is the basis of all hazard analyses, and thus needs to be done with special care. system analysis includes the examination of the system functions, particularly the performance goals and admissible deviations in the ambient conditions not influenced by the system, the auxiliary sources of the system (e.g. energy supply), the components of the system, and the organization and behaviour of the system. geographical arrangements, block diagrams, material flow charts, information flow charts, energy flow charts, etc. are used to depict technical systems. the objective is to ensure the safe behaviour of the technical systems by design methods, at least during the required service life and during intended use. qualitative analyses are particularly important in practice. as a rule, they are form sheet analyses and include failure mode and effect analyses, which examine and determine failure modes and their effects on systems. the preliminary hazard analysis looks for the hazard potentials of a system. the failure hazard analysis examines the causes of failures and their effects. the operating hazard analysis determines the hazards which may occur during operation, maintenance, repair, etc. the human error mode and effect analysis examines error modes and their effects which occur because of wrong behaviour of humans. the information error mode and effect analysis examines operating, maintenance and repair errors, fault elimination errors and the effects caused by errors in instructions and faulty information. theoretical analysis methods include the fault tree analysis, which is a deductive analysis. an unwelcome incident is provided to the system under review. then all logical links and/or failure combinations of components or partial system failures which might lead to this unwelcome incident are assembled, forming the fault tree. the fault tree analysis is suited for simple as well as for complex systems. the objective is to identify failures which might lead to an unwelcome incident. the prerequisite is exact knowledge about the functioning of the system under review. the process, the functioning of the components and partial systems therefore need to be present. it is possible to focus on the flow of force, of energy, of materials and of signals. the fault process analysis has a structure similar to that of the fault tree analysis. in this case, however, we are looking for all unwelcome incidents as well as their combinations which have the same fault trigger. analysis of the functioning of the system under review is also necessary for this. analyses can also be used to identify possible, probable and actual risk components. the phases of the analysis are the phases of design, including the preparation of a concept, project and construction, and the phases of use which are production, operation and maintenance. in order to identify the fault potential as completely as possible, different types of analyses are usually combined. documentation of the sufficient safety of a system can be achieved at a reasonable cost only for a small system. in the case of complex systems, it is therefore recommended to document individual unwelcome incidents. if solutions are sought and found for individual unwelcome incidents, care should be taken to ensure that no target conflicts arise with regard to other detail solutions. with the help of the fault tree, it is possible to analyse the causes of an unwelcome incident and the probability of its occurrence. decisions on whether and which redundancies are necessary can in most cases be reached by simple estimates. four results can be expected by using a fault tree: 1. the failure combination of inputs leading to the unwelcome event; 2. the probability of their occurrence; 3. the probability of occurrence of the unwelcome event; and 4. the critical path that this incident took from the failure combination through the fault tree. a systematic evaluation of the fault tree model can be done by an analytical evaluation (calculation) or by simulation of the model (monte-carlo method). a graphic analysis of the failure process is especially suited to prove the safety risk of previously defined failure combinations in the system. the failure mode and effect analysis and the preliminary hazard analysis as mentioned previously, no method can disclose all potential faults in a system with any degree of certainty. however, if one starts with the preliminary hazard analysis, then at least the essential components with hazard potential will be defined. the essential components are always similar, namely, kinetic energy, potential energy, source of thermal energy, radioactive material, biological material, chemically reactive substance. with the fault tree method, any possible failure combinations (causes), leading to an unwelcome outcome, can then be identified additionally. re-160 are especially suited for identifying failures in a system which pose a risk. liability parameters can be determined in the process, e.g. the frequency of occurrence of failure combinations, the frequency of occurrence of unwelcome events, non-availability of the system upon requests, etc. the failure effect analysis is a supplementary method. it is able to depict the effects of mistakes made by the operating personnel, e.g. when a task is not performed, or is performed according to inappropriate instructions, or performed too early, too late, unintentionally or with errors. it can pinpoint also effects resulting from operating conditions and errors in the functional process or its elements. an important aspect of all hazard analyses is that they are only valid for the respective case under review. every change in a parameter basically requires new analyses. this applies to changes in the personnel structure and the qualification of persons, as well as to technical specifications. for this reason, it is necessary to document the parameters on which each analysis is based. the results of the hazard analyses form the basis for the selection of protective measures and measures to combat the hazards. if the system is modified, the hazards inherent in the system may change, and the measures to combat the hazards may have to be changed as well. this may also mean that the protective measures or equipment which existed at time x for the system or partial system in certain operating conditions (e.g. normal operation, set-up operation, and maintenance phase) may no longer be compatible. different protective measures, equipment or strategies may then be needed. however, hazard analyses do not merely serve to detect and solve potential failures. they form the basis for the selection of protective measures and protective equipment, and they can also test the success of the safety strategies specified. a selection of methods used for hazard analysis is given in annex 2 to section 4.1. risk assessment is a series of logical steps enabling the systematic examination of the hazards associated with machinery. risk assessment is followed, whenever necessary, by actions to reduce the existing risks and by implementing safety measures. when this process is repeated, it eliminates hazards as far as possible. risk assessment includes: • risk analysis: -determining the limits of machinery; -identifying hazards; and -estimating risks. • risk evaluation. risk analysis provides the information required for evaluating risks, and this in turn allows judgements to be made on the safety of e.g. the machinery or plant under review. risk assessment relies on decisions based on judgement. these decisions are to be supported by qualitative methods, complemented, as far as possible, by quantitative methods. quantitative methods are particularly appropriate when the foreseeable harm is very severe or extensive. quantitative methods are useful for assessing alternative safety measures and for determining which measure gives best protection. the application of quantitative methods is restricted to the amount of useful data which is available, and in many cases only qualitative risk assessment will be possible. risk assessment should be conducted so that it is possible to document the used procedure and the results that have been achieved. risk assessment shall take into account: • the life cycle of machinery or the life span of the plant. • the limitations of the machinery or plant, including the intended use (correct use and operation of the machinery or plant, as well as the consequences of reasonably foreseeable misuse or malfunction). • the full range of foreseeable uses of the machinery (e.g. industrial, nonindustrial and domestic) by persons identified by sex, age, dominant hand usage, or limiting physical abilities (e.g. visual or hearing impairment, stature, strength). • the anticipated level of training, experience or ability of the anticipated users, such as: -operators including maintenance personnel or technicians; -trainees and juniors; and -general public. • exposure of other persons to the machine hazards, whenever they can be reasonably foreseen. having identified the various hazards that can originate from the machine (permanent hazards and ones that can appear unexpectedly), the machine designer shall estimate the risk for each hazard, as far as possible, on the basis of quantifiable factors. he must finally decide, based on the risk evaluation, whether risk reduction is required. for this purpose, the designer has to take into account the different operating modes and intervention procedures, as well as human interaction during the entire life cycle of the machine. the following aspects in particular must be considered: • construction; transport; • assembly, installation, commissioning; • adjusting settings, programming or process changeover; • instructions for users; • operating, cleaning, maintenance, servicing; and • checking for faults, de-commissioning, dismantling and safe disposal. malfunctioning of the machine due to, e.g. • variation in a characteristic or dimension of the processed material or workpiece; • failure of a part or function; • external disturbance (e.g. shock, vibration, electromagnetic interference); • design error or deficiency (e.g. software errors); • disturbance in power supply; and • flaw in surrounding conditions (e.g. damaged floor surface). unintentional behaviour of the operator or foreseeable misuse of the machine, e.g.: • loss of control of the machine by the operator (especially in the case of hand-held devices or moving parts); • automatic (reflexive) behaviour of a person in case of a machine malfunction or failure during operation; • the operator's carelessness or lack of concentration; • the operator taking the "line of least resistance" in carrying out a task; • behaviour resulting from pressure to keep the machine running in all circumstances; and • unexpected behaviour of certain persons (e.g. children, disabled persons). when carrying out a risk assessment, the risk of the most severe harm that is likely to occur from each identified hazard must be considered, but the greatest foreseeable severity must also be taken into account, even if the probability of such an occurrence is not high. this objective may be met by eliminating the hazards, or by reducing, separately or simultaneously, each of the two elements which determine the risk, i.e. the severity of the harm from the hazard in question, and the probability of occurrence of that harm. all protective measures intended to reach this goal shall be applied according to the following steps: this stage is the only one at which hazards can be eliminated, thus avoiding the need for additional protective measures, such as safeguarding machines or implementing complementary protective measures. 3. information about the residual risk. information for use on the residual risk is not to be a substitute for inherently safe design, or for safeguarding or complementary protective measures. risk estimation and evaluation must be carried out after each of the above three steps of risk reduction. adequate protective measures associated with each of the operating modes and intervention procedures prevent operators from being prone to use hazardous intervention techniques in case of technical difficulties. the aim is to achieve the lowest possible level of risk. the design process is an iterative cycle, and several successive applications may be necessary to reduce the risk, making the best use of available technology. four aspects should be considered, preferably in the following order: 1. the safety of the machine during all the phases of its life cycle; 2. the ability of the machine to perform its function; 3. the usability of the machine; and 4. the costs of manufacturing, operating and dismantling the machine. the following principles apply to technical design: service life, safe machine life, fail-safe and tamper-proof design. a design which ensures the safety of service life has to be chosen when neither the technical system nor any of its safety-relevant partial functions can be allowed to fail during the service life envisaged. this means that the components of the partial functions need to be exchanged at previously defined time intervals (preventive maintenance). in the case of a fail-safe design, the technical system or its partial functions allow for faults, but none of these faults, alone or in combination, may lead to a hazardous state. it is necessary to specify just which faults in one or several partial systems can be allowed to occur simultaneously without the overall system being transferred into a hazardous state (maximum admissible number of simultaneous faults). a failure or a reduction in the performance of the technical system is accepted in this case. tamper-proof means that it is impossible to intentionally induce a hazardous state of the system. this is often required of technical systems with a high hazard potential. strategies involving secrecy play a special role in this regard. in the safety principles described here, redundant design should also be mentioned. the probability of occurrence and the consequences of damage are reduced by multiple arrangements, allowing both for subsystems or elements to be arranged in a row or in parallel. it is possible to reduce the fault potential of a technical system by the diversification of principles: several different principles are used in redundant arrangements. the spatial distribution of the function carriers allows the possibilities to influence faults to be reduced to one function. in the redundant arrangements, important functions, e.g. information transmission, are therefore designed in a redundant manner at different locations. the measures to eliminate or avoid hazards have to meet the following basic requirements: their effect must be reliable and compulsory, and they cannot be circumvented. reliable effect means that the effect principle and construction design of the planned measure guarantee an unambiguous effect, that the components have been designed according to regulations, that production and assembly are performed in a controlled manner, and that the measure has been tested. compulsory effect includes the demand for a protective effect which is active at the start of a hazardous state and during it, and which is deactivated only when the hazardous state is no longer present, or stops when the protective effect is not active. technical systems are planned as determined systems. only predictable and intended system behaviour is taken into account when the system is designed. experience has shown, however, that technical systems also display stochastic behaviour. that is, external influences and/or internal modifications not taken into consideration in the design result in unintended changes in the system's behaviour and properties. the period of time until the unintended changes in behaviour and/or in properties occur, cannot be accurately determined; it is a random variable. we have to presume that there will be a fault in every technical system. we simply do not know in advance when it will take place. the same is true for repairs. we know that it is generally possible in systems requiring re-pair to complete a repair operation successfully, but we cannot determine the exact time in advance. using statistical evaluations, we can establish a timedependent probability at which a "fault event" or "completion of a repair operation" occurs. the frequency of these events determines the availability of the system requiring repair. technical systems are intended to perform numerous functions and, at the same time, to be safe. the influence of human action on safety has to be taken into account in safety considerations as well (i.e. human factor). a system is safe when there are no functions or action sequences resulting in hazardous effects for people and/or property. risks of unwelcome events (in the following called "risk of an event") are determined on the basis of the experience (e.g. catalogue of measures) with technical systems. in addition to this, safety analyses are used (e.g. failure mode and effect analysis, hazard analysis, failure hazard analysis, operating hazard analysis, information error analysis), as well as mathematical models (e.g. worst-case analysis, monte-carlo procedure, markow's models). unwelcome events are examined for their effects. this is followed by considerations about which design modifications or additional protective measures might provide sufficient safety against these unwelcome events. the explanations below present the basic procedure for developing safety-relevant arrangements and solutions, i.e. the thinking and decision-making processes, as well as selecting criteria that are significant for the identification of unwelcome events, the risk of an event, the acceptance limits and the adoption of measures. before preparing the final documentation, it is essential to verify that the limit risk has not been exceeded, and that no new unwelcome events have occurred. the sequence scheme describes the procedure for developing safety arrangements and for finding solutions aiming to avoid the occurrence of unwelcome events which exceed the acceptance limits, by selecting suitable measures. in this context, it is assumed that: • an unwelcome event is initially identified as a single event within a comprehensive event sequence (e.g. start-up of a plant), and the risk of an event and limit risk are determined. • the selection of technical and/or non-technical measures is subject to a review of the content and the system, and the decision regarding a solution is then made. • the number of applicable measures is limited, and therefore it may not be possible to immediately find a measure with an acceptable risk for a preliminary determination of the unwelcome event. • implementation of the selected solution can result in the occurrence of a new unwelcome event. • in the above cases, a more concrete, new determination of the unwelcome event and/or the unit under review, or the state of the unit under review, and another attempt at deciding upon measures may lead to the desired result, although this may have to be repeated several times before it is successful. in the case of complex event sequences, several unwelcome events may become apparent which have to be tackled by the respective set of measures. in accordance with the sequence scheme, the unit under review and its state have to be determined first. this determination includes information on, e.g., • product type, dimension, product parts/elements distinguished according to functional or construction aspects, if applicable; • intended use; • work system or field of application; • target group; • supply energy, transformed in the product, transmitted, distributed, output; • other parameters essential to safety assessment according to type and size of the product; • known or assumed effects on the product or its parts (e.g. due to transport, assembly, conditions at the assembly site, operation, maintenance); • weight, centre of gravity; • materials, consumables; • operating states (e.g. start-up, standstill, test run, normal operation); • condition (new, condition after a period in storage/shutdown, after repair, in case of modified operating conditions and/or other significant changes); and • known or suspected effects on humans. the next step is the identification of unwelcome events. they are source conditions of processes and states, or processes and states themselves. they can be the effects of processes and states which can cause harm to people or property. an unwelcome event can be a single event or part of a sequence of events. one should look for unwelcome events in sequences of processes and functions, in work activities and organizational procedures, or in the work environment. care has to be taken that the respective interfaces are included in the considerations. deviations and time-dependent changes in regard to the planned sequences and conditions have to be taken into account as well. the risk of an unwanted event results from the probability statement which takes into account both • the expected frequency of occurrence of the event; and • the anticipated extent of harm of the event. the expected frequency of occurrence of an event leading to harm is determined by, e.g., • the probability of the occurrence itself; • the duration and frequency of exposure of people (or of objects) in the danger zone, e.g. -extremely seldom (e.g. during repair), -seldom (e.g. during installation, maintenance and inspection), -frequently, and -very frequently (e.g. constant intervention during every work cycle); • the influence of users or third parties on the risk of an event. the extent of harm is determined by, e.g., • the type of harm (harm to people and/or property); • the severity of the harm (slight/severe/fatal injury of persons, or corresponding damage to property); and • number of people or objects affected. in principle, the safety requirements depend on the ratio of the risk of an event to the limit risk. criteria for determining the limit risk are, e.g., • personal and social acceptance of hazards; • people possibly affected (e.g. layman, trained person, specialized worker); • participation of those affected in the process; and • possibilities of averting hazards. the safety of various technical equipment with comparable risk can, for instance, be achieved • primarily by technical measures, in some cases; and • mainly by non-technical measures, in other cases. this means that several acceptable solutions with varying proportions of technical and non-technical measures may be found for a specific risk. in this context, the responsibility of those involved should be taken into consideration. technical measures are developed on the basis of e.g. the following principles: • avoiding hazardous interfaces (e.g. risk of crushing, shearing); hazard sources (e.g. radiation sources, flying parts, hazardous states and actions as well as inappropriate processes); • limiting hazardous energy (e.g. by rupture disks, temperature controllers, safety valves, rated break points); • using suitable construction and other materials (e.g. solid, sufficiently resistant against corrosion and ageing, glare-free, break-proof, non-toxic, non-inflammable, non-combustible, non-sliding); • designing equipment in accordance with its function, material, load, and ergonomics principles; • using fail-safe control devices employing technical means; • employing technical means of informing (e.g. danger signal); • protective equipment for separating, attaching, rejecting, catching, etc.; • suction equipment, exhaust hoods, when needed; • protection and emergency rooms; and • couplings or locks. technical measures refer to, e.g., • physical, chemical or biological processes; • energy, material and information flow in connection with the applied processes; • properties of materials and changes in the properties; and • function and design of technical products, parts and connections. the iterative (repeated) risk reduction process can be concluded after achieving adequate risk reduction and, if applicable, a favourable outcome of risk comparison. adequate risk reduction can be considered to have been achieved when one is able to answer each of the following questions positively: • have all operating conditions and all intervention procedures been taken into account? • have hazards been eliminated or their risks been reduced to the lowest practicable level? • is it certain that the measures undertaken do not generate new hazards? • are the users sufficiently informed and warned about the residual risks? • is it certain that the operator's working conditions are not jeopardized by the protective measures taken? • are the protective measures compatible with each other? • has sufficient consideration been given to the consequences that can arise from the use of a machine designed for professional/industrial use when it is used in a non-professional/non-industrial context? • is it certain that the measures undertaken do not excessively reduce the ability of the machine to perform its intended function? there are still many potential risks connected with hazardous substances about which more information is needed. because the knowledge about the relation between their dose and mode of action is not sufficient for controlling such risks, more research is needed. the following list highlights the themes of the numerous questions related to such risks: • potentially harmful organisms; • toxicants, carcinogens; • pesticides, pollutants, poisonous substances; • genetically engineered substances; • relation between chemical and structural properties and toxicity; • chemical structure and chemical properties and the relation to reactivity and reaction possibilities of organic compounds to metabolic reaction and living systems; • modes of action, genotoxicity, carcinogenicity, effects on humans/animals; • potentially harmful organisms in feedstuffs and animal faeces; • viruses and pathogens; • bacteria in feedstuffs and faeces; • parasites in feedstuffs and animal faeces; • pests in stored feedstuffs; • probiotics as feed additives; and • preservatives in feedstuffs. violent actions damaging society, property or people have increased, and they seem to spread both internationally as well as within countries. these new risks are difficult to predict and manage, as the very strategy of the actors is to create unexpected chaotic events. certain possibilities to predict the potential types of hazards do exist, and comprehensive predictive analyses have been done (meyerson, reaser 2002) . new methodologies are needed to predict the risk of terrorist actions, and also the strategies for risk management need to be developed. due to the numerous background factors, the preparedness of societies against these risks needs to be strengthened. table 4 .3 lists important societal systems which are vulnerable to acts of terrorism. the situation in the developing countries needs to be tackled with specific methods. one has to answer the following questions: • what specific examples of prevention instruments can be offered? • what are the prerequisites for success? • how can industrialized countries assist the developing countries in carrying out preventive actions? • how should priorities be set according to the available resources? one possibility is to start a first-step programme, the goal of which is higher productivity and better workplaces. it can be carried out by improving • storage and handling of materials: -provide storage racks for tools, materials, etc.; -put stores, racks etc. on wheels, whenever possible; -use carts, conveyers or other aids when moving heavy loads; -use jigs, clamps or other fixtures to hold items in place. • work sites: -keep the working area clear of everything that is not in frequent use. • machine safety: -install proper guards to dangerous tools/machines; -use safety devices; -maintain machines properly. • control of hazardous substances: -substitute hazardous chemicals with less hazardous substances; -make sure that all organic solvents, paints, glues, etc., are kept in covered containers; -install or improve local exhaust ventilation; -provide adequate protective goggles, face shields, earplugs, safety footwear, gloves, etc.; -instruct and train workers; -make sure that workers wash their hands before eating and drinking, and change their clothes before going home. • lighting: -make sure that lighting is adequate. • social and sanitary facilities: -provide a supply of cool, safe drinking water; -have the sanitary facilities cleaned regularly; -provide a hygienic place for meals; -provide storage for clothing or other belongings; -provide first aid equipment and train a qualified first-aider. • premises: -increase natural ventilation by having more roof and wall openings, windows or open doorways; -move sources of heat, noise, fumes, arc welding, etc., out of the workshop, or install exhaust ventilation, noise barriers, or other solutions; -provide fire extinguishers and train the workers to use them; -clear passageways, provide signs and markings. • work organization: -keep the workers alert and reduce fatigue through frequent changes in tasks, opportunities to change work postures, short breaks, etc.; -have buffer stocks of materials to keep work flow constant; -use quality circles to improve productivity and quality. risk combination of the probability of an event and its consequences. the term "risk" is generally used only when there is at least a possibility of negative consequences. in some situation, risk arises from the possibility of deviation from the expected outcome or event. outcome of an event or a situation, expressed in quality and in quantity. it may result in a loss or in an injury or may be linked to it. the result can be a disadvantage or a gain. in this case the event or the situation is the source. in connection with every analysis it has to be checked whether the cause is given empirically, or follows a set pattern, and whether there is scientific agreement regarding these circumstances. note 1 there can be more than one consequence from one event. note 2 consequences can range from positive to negative. the consequences are always negative from the viewpoint of safety. extent to which an event is likely to occur. note 1 iso 3534-1:1993 gives the mathematical definition of probability as "a real number in the interval 0 to 1 attached to a random event. it can be related to a long-run relative frequency of occurrence or to a degree of belief that an event will occur. for a high degree of belief the probability is near 1". note 2 frequency rather than probability may be used in describing risk. degrees of belief about probability can be chosen as classes or ranks such as: rare/unlikely/moderate/likely/almost certain, or incredible/improbable/ remote/occasional/probable/frequent. remark: informal language often confuses frequency and probability. this can lead to wrong conclusions in safety technology. probability is the degree of coincidence of the time frequency of coincidental realization of a fact from a certain possibility. coincidence is an event which basically can happen, may be cause-related, but does not occur necessarily or following a set pattern. it may also not occur (yes-or-no-alternative). data for probability of occurring with specific kinds of occurrence and weight of consequences can be: in a statistical sense: empirical, retrospective, real in a prognostic sense: speculative, prospective, probabilistic occurrence of a particular set of circumstances regarding place and time. an event can be the source of certain consequences (empirically to be expected with certain regularity). the event can be certain or uncertain. the event can be a single occurrence or a series of occurrences. the probability associated with the event can be estimated for a given period of time. task range by which the significance of risk is assessed. note 1 risk criteria can include associated costs and benefits, legal and statutory requirements, socio-economic and environmental aspects, the concerns of stakeholders, priorities and other inputs to the assessment. the way in which a stakeholder views a risk based on a set of values or concerns. note 1 risk perception depends on the stakeholder's needs, issues and knowledge. note 2 risk perception can differ from objective data. exchange or sharing of information about risk between the decision-makers and other stakeholders. overall process of risk analysis and risk evaluation. systematic use of information to identify sources and to estimate the risk. note 1 risk analysis provides a basis for risk evaluation, risk treatment, and risk acceptance. note 2 information can include historical data, theoretical analyses, informal opinions, and the concerns of stakeholders. process used to assign figures, values to the probability and consequences of a risk. note 1 risk estimation can consider cost, benefits, the concerns of stakeholders, and other variables, as appropriate for risk evaluation. process of comparing the estimated risk against given risk criteria to determine the significance of a risk. process of selection and implementation of measures to modify risk. note 1 risk treatment measures can include avoiding, optimizing, transferring or retaining risk. actions implementing risk management decisions. note 1 risk control may involve monitoring, re-evaluation, and compliance with decisions. process, related to a risk, to minimize the negative and to maximize the positive consequences (and their respective probabilities). actions taken to lessen the probability, negative consequences or both, associated with a risk. limitation of any negative consequences of a particular event. decision not to become involved in, or action to withdraw from, a risk situation. sharing with another party the burden of loss or benefit of gain, for a risk. note 1 legal or statutory requirements can limit, prohibit or mandate the transfer of a certain risk. note 2 risk transfer can be carried out through insurance or other agreements. note 3 risk transfer can create new risks or modify existing ones. note 4 relocation of the source is not risk transfer. acceptance of the burden of loss, or benefit of gain, from a particular risk. note 1 risk retention includes the acceptance of risks that have not been identified. note 2 risk retention does not include means involving insurance, or transfer in other ways. this includes risk assessment, risk treatment, risk acceptance and risk communication. risk assessment is risk analysis, with identification of sources and risk estimation, and risk evaluation. risk treatment includes avoiding, optimizing, transferring and retaining risk. → risk acceptance → risk communication harm physical injury or damage to the health of people or damage to property or the environment [iso/iec guide 51]. note 1 harm includes any disadvantage which is causally related to the infringement of the object of legal protection brought about by the harmful event. note 2 in the individual safety-relevant definitions, harm to people, property and the environment may be included separately, in combination, or it may be excluded. this has to be stated in the respective scope. potential source of harm [iso/iec guide 51]. the term "hazard" can be supplemented to define its origin or the nature of the possible harm, e.g., hazard of electric shock, crushing, cutting, dangerous substances, fire, drowning. in every-day informal language, there is insufficient differentiation between source of harm, hazardous situation, hazardous event and risk. circumstance in which people, property or the environment are exposed to one or more hazards [iso/iec guide 51]. note 1 circumstance can last for a shorter or longer period of time. event that can cause harm [din en 1050] . 1 the hazardous event can be preceded by a latent hazardous situation or by a critical event. combination of the probability of occurrence of harm and the severity of that harm [iso/iec guide 51]. note 1 in many cases, only a uniform extent of harm (e.g. leading to death) is taken into account, or the occurrence of harm may be independent of the extent of harm, as in a lottery game. in these cases, it is easier to make a probability statement; risk assessment by risk comparison [din en 1050] thus becomes much simpler. note 2 risks can be grouped in relation to different variables, e.g. to all people or only those affected by the incident, to different periods of time, or to performance. the probabilistic expectation value of the extent of harm is suitable for combining the two probability variables. note 4 risks which arise as a consequence of continuous emission, e.g. noise, vibration, pollutants, are affected by the duration and level of exposure of those affected. risk which is accepted in a given context based on the current values of society [iso/iec guide 51]. the acceptable risk has to be taken into account in this context, too. note 2 safety-relevant definitions are oriented to the maximum tolerable risk. this is also referred to as limit risk. note 3 tolerability is also based on the assumption that the intended use in addition to a reasonably predictable misuse of the products, processes and services, is complied with. freedom from unacceptable risk [iso/iec guide 51]. note 1 safety is indivisible. it cannot be split into classes or levels. note 2 safety is achieved by risk reduction, so that the residual risk in no case exceeds the maximum tolerable risk. existence of an unacceptable risk. note 1 safety and danger exclude one another -a technical product, process or service cannot be safe and dangerous at the same time. means used to reduce risk [iso/iec guide 51]. note 1 protective measures at the product level have priority over protective measures at the workplace level. preventive measure means assumed, but not proven, to reduce risk. risk remaining after safety measures have been taken [din en 1050] . note 1 residual risk may be related to the use of technical products, processes and services. systematic use of available information to identify hazards and to estimate their risks [iso/iec guide 51]. determination of connected risk elements of all hazards as a basis for risk assessment. decision based on the analysis of whether the tolerable risk has been exceeded [iso/iec guide 51]. overall process of risk analysis and risk evaluation [iso/iec guide 51]. use of a product, process or service in accordance with information provided by the supplier [iso/iec guide 51]. note 1 information provided by the supplier also includes descriptions issued for advertising purposes. use of a product, process or service in a way not intended by the supplier, but which may result from readily predictable human behaviour [iso/iec guide 51]. safety-related formulation of contents of a normative document in the form of a declaration, instructions, recommendations or requirements [compare en 45020, safety related]. the information set down in technical rules is normally restricted to certain technical relations and situations; in this context, it is presumed that the general safety-relevant principles are followed. a procedure with the aim to reduce risk of a (technical) product, process or service according to the following steps serves to reach the safety goals in the design stage: • safety-related layout; • protective measures; • safety-related information for users. function inevitable to maintain safety. a function which, in case of failure, allows the tolerable risk to be immediately exceeded. depending on the situation, it is possible to use one method or a combination of several methods. intuitive hazard detection spontaneous, uncritical listing of possible hazards as a result of brainstorming by experts. group work which is as creative as possible. writing ideas down (on a flip chart) first, then evaluating them. technical documentation (instructions, requirements) is available for many industrial plant and work processes, describing the hazards and safety measures. this documentation has to be obtained before continuing with the risk analysis. the deviations between the set point and the actual situation of individual components are examined. information on the probability of failure of these elements may be found in technical literature. examining the safety aspects in unusual situations (emergency, repair, starting and stopping) when plans are made to modify the plant or processes. a systematic check of the processes and plant parts for effects in normal operation and in case of set point deviations, using selected question words (and -or -not -too much -too little?). this is used in particular for measurement, control units, programming of computer controls, robots. all possible causes and combinations of causes are identified for an unwanted operating state or an event, and represented in the graphic format of a tree. the probability of occurrence of an event can be estimated from the context. the fault tree analysis can also be used retrospectively to clarify the causes of events. additional methods may be: human reliability analysis a frequency analysis technique which deals with the behaviour of human beings affecting the performance of the system, and estimates the influence of human error on reliability. a hazard identification and frequency analysis technique which can be used at an early stage in the design phase to identify and critically evaluate hazards. operating safety block program a frequency analysis technique which utilizes a model of the system and its redundancies to evaluate the operating safety of the entire system. classifying risks into categories, to establish the main risk groups. all typical hazardous substances and/or possible accident sources which have to be taken into account are listed. the checklist may be used to evaluate the conformity with codes and standards. this method is used to estimate whether coincidental failures of an entire series of different parts or modules within a system are possible and what the probable effects would be. estimate the influence of an event on humans, property or the environment. simplified analytical approaches, as well as complex computer models can be used. a large circle of experts is questioned in several steps; the result of the previous step together with additional information is communicated to all participants. during the third or fourth step the anonymous questioning concentrates on aspects on which no agreement is reached so far. basically this technique is used for making predictions, but is also used for the development of new ideas. this method is particularly efficient due to its limitation to experts. a hazard identification and evaluation technique used to establish a ranking of the different system options and to identify the less hazardous options. a frequency analysis technique in which a model of the system is used to evaluate variations of the input conditions and assumptions. a means to estimate and list risk groups; reviews risk pairs and evaluates only one risk pair at a time. overview of data from the past a technique used to identify possible problem areas; can also be used for frequency analysis, based on accident and operation safety data, etc. a method to identify latent risks which can cause unforeseeable incidents. cssr differs from the other methods in that it is not conducted by a team, and can be conducted by a single person. the overview points out essential safety and health requirements related to a machine and simultaneously to all relevant (national, european, international) standards. this information ensures that the design of the machine complies with the issued "state of the art" for that particular type of machine. the "what-if" method is an inductive procedure. the design and operation of the machine in question are examined for fairly simple applications. at every step "what-if" questions are asked and answered to evaluate the effect of a failure of the machine elements or of process faults in view of the hazards caused by the machine. for more complex applications, the "what-if" method is most useful with the aid of a "checklist" and the corresponding work division to allocate specific features of the process to persons who have the greatest experience and practice in evaluating the respective feature. the operator's behaviour and professional knowledge are assessed. the suitability of the equipment and design of the machine, its control unit and protective devices are evaluated. the influence of the materials processed is examined, and the operating and maintenance records are checked. the checklist evaluation of the machine generally precedes the more detailed methods described below. fmea is an inductive method for evaluating the frequency and consequences of component failure. when operating procedures or operator errors are investigated, then other methods may be more suitable. fmea can be more time-consuming than the fault tree analysis, because every mode of failure is considered for every component. some failures have a very low probability of occurrence. if these failures are not analyzed in depth this decision should be recorded in the documentation. the method is specified in iec 812 "analysis techniques for system reliability -procedure for failure mode and effects analysis (fmea)". in this inductive method, the test procedures are based on two criteria: technology and complexity of the control system. mainly, the following methods are applicable: • practical tests of the actual circuit and fault simulation on certain components, particularly in suspected areas of performance identified during the theoretical check and analysis. • simulation of control behaviour (e.g. by means of hardware and/or software models). whenever complex safety-related parts of control systems are tested, it may be necessary to divide the system into several functional sub-systems, and to exclusively submit the interface to fault simulation tests. this technique can also be applied to other parts of machinery. mosar is a complete approach in 10 steps. the system to be analyzed (machinery, process, installation, etc.) is examined as a number of sub-systems which interact. a table is used to identify hazards and hazardous situations and events. the adequacy of the safety measures is studied with a second table, and a third table is used to look at their interdependency. a study, using known tools (e.g. fmea) underlines the possible dangerous failures. this leads to the elaboration of accident scenarios. by consensus, the scenarios are sorted in a severity table. a further table, again by consensus, links the severity with the targets of the safety measures, and specifies the performance levels of the technical and organizational measures. the safety measures are then incorporated into the logic trees and the residual risks are analyzed via an acceptability table defined by consensus. ilo (1987) 1994, 1995, 1996, 1997, 1998, 1999 the risks to health at work are numerous and originate from several sources. their origins vary greatly and they cause vast numbers of diseases, injuries and other adverse conditions, such as symptoms of overexertion or overload. traditional occupational health risk factors and their approximate numbers are given in table 4 .4. the exposure of workers to hazards or other adverse conditions of work may lead to health problems, manifested in the workers' physical health, psysical workload, psychological disturbances or social aspects of life. workers may be exposed to various factors alone or in different types of combinations, which may or may not show interaction. the assessment of interacting risk factors is complex and may lead to substantial differences in the final risk estimates when compared with estimates of solitary factors. examples of interaction between different risk factors in the work environment are given in table 4 .5. the who estimate of the total number of occupational diseases among the 3 billion workers of the world is 160 million a year. this is likely to be an under-estimate due to the lack of diagnostic services, limited legislative coverage of both workers and diseases, and variation in diagnostic criteria between different parts of the world. the mortality from occupational diseases is substantial, comparable with other major diseases of the world population such as malaria or tuberculosis. the recent ilo estimate discloses 2.4 million deaths a year from work-related causes in the world including deaths from accidents, dangerous substances, and occupational diseases. eightyfive percent (85%) of these deaths take place in developing countries, where the diagnostic services, social security to families and compensation to workers are less developed. although the risk is decreasing in the industrialized world, the trend is increasing in the rapidly industrializing and transitory countries. a single hazard alone, such as asbestos exposure, is calculated to cause 100,000 cancers a year with a fatal outcome in less than two years after diagnosis (takala 2005). the incidence rates of occupational diseases in well registered industrialized countries are at the level of 3-5 cases/10,000 active employees/year, i.e., the incidence levels are comparable with major public health problems, such as cardiovascular diseases, respiratory disorders, etc. in the industrialized countries, the rate of morbidity from traditional occupational diseases, such as chemical poisonings, is declining, while musculoskeletal and allergic diseases are on the increase. about 200 biological factors that are hazardous to workers' health have been identified in various work environments. some of the new diseases recognized are blood-borne infections, such as hepatitis c and hiv, and exotic bacterial or viral infections transmitted by increasing mobility, international travelling and migration of working people. also some hospital infections and, e.g., drug-resistant tuberculosis, are being contracted increasingly by health care personnel. in the developing countries the morbidity picture of occupational diseases is much less clear for several reasons: low recognition rates, rotation and turnover of workers, shorter life expectancy which hides morbidity with a long latency period, and the work-relatedness of several common epidemic diseases, such as malaria and hiv/aids (rantanen 2003). the estimation of so-called work-related diseases is even more difficult than that of occupational diseases. they may be about 10-fold more prevalent than the definite occupational diseases. several studies suggest that siegfried radandt, jorma rantanen and ortwin renn work-related allergies, musculoskeletal disorders and stress disorders are showing a growing trend at the moment. the prevention of work-related diseases is important in view of maintaining work ability and reducing economic loss from absenteeism and premature retirement. the proportion of work-relatedness out of the total morbidity figures has been estimated and found surprisingly high (nurminen and karjalainen 2001 , who 2002 ) (see table 4 .6). the public health impact of work-related diseases is great, due to their high prevalence in the population. musculoskeletal disorders are among the three most common chronic diseases in every country, which implies that the attribution of work is very high. similarly, cardiovascular diseases in most industrialized countries contribute to 50% of the total mortality. even a small attributable fraction of work-relatedness implies high rates of morbidity and mortality related to work. the concept of disease is in general not a simple one. when discussing morbidity one has to recognize three different concepts: 1. illness = an individual's perception of a health problem resulting from either external or internal causes. 2. disease = an adverse health condition diagnosed by a doctor or other health professional. 3. sickness = a socially recognized disease which is related to, for example, social security actions or prescription of sick leave, etc. when dealing with occupational and work-related morbidity, one may need to consider any of the above three aspects of morbidity. a recognized occupational disease, however, belongs to group 3, i.e. it is a sickness defined by legal criteria. medical evidence is required to show that the condition meets the criteria of an occupational disease before recognition can be made. there are dozens of definitions for occupational disease. the content of the concept varies, depending on the context: a) the medical concept of occupational disease is based on a biomedical or other health-related etiological relationship between work and health, and is used in occupational health practice and clinical occupational medicine. b) the legal concept of occupational disease defines the disease or conditions which are legally recognized as conditions caused by work, and which lead to liabilities for recognition, compensation and often also prevention. the legal concept of occupational disease has a different background in different countries, often declared in the form of an official list of occupational diseases. there is universal discrepancy between the legal and medical concept, so that in nearly all countries the official list of recognized occupational diseases is shorter than the medically established list. this automatically implies that a substantial proportion of medically established occupational diseases remain unrecognized, unregistered, and consequently also uncompensated. the definition of occupational disease, as used in this chapter, summarizes various statements generated during the history of occupational medicine: an occupational disease is any disease contracted as a result of exposures at work or other conditions of work. the general criteria for the diagnosis and recognition of an occupational disease are derived from the core statements of various definitions: 1. evidence on exposure(s) or condition(s) in work or the work environment, which on the basis of scientific knowledge is (are) able to generate disease or some other adverse health condition. 2. evidence of symptoms and clinical findings which on the basis of scientific knowledge can be associated with the exposure(s) or condition(s) in concern. 3. exclusion of non-occupational factors or conditions as a main cause of the disease or adverse health condition. point 3 often creates problems, as several occupationally generated clinical conditions can be caused also by non-occupational factors. on the other hand, several factors from different sources and environments are involved in virtually every disease. therefore the wordings "main cause" or "principal cause" are used. the practical solution in many countries is that the attribution of work needs to be more than 50%. usually the necessary generalizeable scientific evidence is obtained from epidemiological studies, but also other types of evidence, e.g. well documented clinical experience combined with information on working conditions may be acceptable. in some countries, like finland, any disease of the worker which meets the above criteria can be recognized as an occupational disease. in most other countries, however, there are official lists of occupational diseases which determine the conditions and criteria on which the disease is considered to be of occupational origin. in 1985 who launched a new concept: work-related disease (who 1985) . the concept is wider than that of an occupational disease. it includes: a) diseases in which the work or working conditions constitute the principal causal factor. b) diseases for which the occupational factor may be one of several causal agents, or the occupational factor may trigger, aggravate or worsen the disease. c) diseases for which the risk may be increased by work or work-determined lifestyles. the diseases in category (a) are typically recognized as legally determined occupational diseases. categories (b) and (c) are important regarding the morbidity of working populations, and they are often considered as important targets for prevention. in general, categories (b) and (c) cover greater numbers of people, as the diseases in question are often common noncommunicable diseases of the population, such as cardiovascular diseases, musculoskeletal disorders, and allergies and, to a growing extent, stressrelated disorders (see table 4 .6). the concept of work-related disease is very important from the viewpoint of occupational health risk assessment and the use of its results for preventive purposes and for promoting health and safety at work. this is because preventive actions in occupational health practice cannot be limited only to legally recognized morbidity. the lists of occupational diseases contain great numbers of agents that show evidence on occupational morbidity. according to the ilo recommendation r194 (2002): list of occupational diseases, the occupational diseases are divided into four main categories: 1. diseases resulting from single causes following the categories listed in table 4 .4. the most common categories are physical factors, chemical agents, biological factors and physical work, including repetitive tasks, poor ergonomic conditions, and static and dynamic work. 2. diseases of the various organs: respiratory system, nervous system, sensory organs, internal organs, particularly liver and kidneys, musculoskeletal system, and the skin. 3. occupational cancers. 4. diseases caused by other conditions of work. research on risk perception shows differences in how different types of risks are viewed. instant, visible, dramatic risk events, particularly ones that cause numerous fatalities or severe visible injuries in a single event generally arouse much attention, and are given high priority. on the other hand, even great numbers of smaller events, such as fatal accidents of single workers, arouse less attention in both the media and among regulators, even though the total number of single fatal accidents in a year may exceed the number of fatalities in major events by several orders of magnitude. occupational diseases, with the exception of a few acute cases, are silent, develop slowly, and concern only one or a few individuals at a time. furthermore, the diseases take months or years to develop, in extreme cases even decades, after the exposure or as a consequence of accumulation of exposure during several years. as occupational health problems are difficult to detect and seriously under-diagnosed and under-reported, they tend to be given less priority than accidents. the perception of occupational disease risk remains low in spite of their severity and relatively high incidence. particularly in industrialized countries, the extent of occupational health problems is substantially greater than that of occupational accidents. on a global scale, the estimated number of fatalities due to occupational accidents is 375,000 and the respective estimate for fatalities due to work-related diseases is 1.8 million a year, giving a fatal accident/fatal disease ratio of 1 to 5. the corresponding ratio in the eu-15 is 1 to 20 (takala 2005). the risk distribution of ods is principally determined by the nature of the work in question and the characteristics of the work environment. there is great variation in the risk of ods between the lowest and highest risk occupations. in the finnish workforce, the risk between the highest risk and the lowest risk occupations varies by a factor of 30. the highest risk occupations carry a risk which is 4-10 times higher than the average for all occupations. the risk of an occupational disease can be estimated on the basis of epidemiological studies, if they do exist in the case of the condition in question. on the other hand, various types of economic activity, work and occupations carry different types of risks, and each activity may have its own risk profile. by examining the available epidemiological evidence, we can recognize high-risk occupations and characterize the typical risks connected with them ( figure 4 .12, table 4.7). as an example, the risk of occupational asthma, dermatosis or musculoskeletal disorders is common in several occupations, but not in all. there may be huge differences in risks between different occupations. the 10 occupations carrying the highest risk for occupational asthma, occupational skin diseases and work-related tenosynovitis, in 1986-1991, are shown in table 4 .8. assessment of the risk of occupational diseases has an impact on research priorities. table 4 .9 shows the priorities for research in four countries. the similarity of the priorities is striking, revealing that the problems related to the risks of occupational diseases are universal. the diagnosis of occupational diseases is important for the treatment of the disease, and for prevention, registration and compensation. the diagnosis is based on information obtained from: a) data on the work and the work environment usually provided by the employer, occupational health services, occupational safety committee, or expert bodies carrying out hygienic and other services for the workplace. b) information on the health examination of individual workers. the authorities in many countries have stipulated legal obligations for high-risk sectors to follow up the workers' health and promote early detection of changes in their health. occupational health services keep records on examinations. c) workers with special symptoms (for example, asthmatic reactions) are taken into the diagnostic process as early as possible. epidemiological evidence is a critical prerequisite for recognizing causal relationship between work and disease. epidemiology is dependent on three basic sources of information on work and the work environment: (a) exposure assessment that helps to define the "dose" of risk factor at work, (b) the outcome assumed to occur as a biological (or psychological) response to the exposures involved, and (c) time, which has a complex role in various aspects of epidemiology. all these sources are affected by the current dynamics of work life which has major impact on epidemiological research and its results. exposure assessment is the critical initial step in risk assessment. as discussed in this chapter, accurate exposure assessment will become more difficult and cumbersome than before in spite of remarkable achievements in measurement, analysis and monitoring methods in occupational hygiene, toxicology and ergonomics. great variations in working hours and individu-alization of exposures, growing fragmentation and mobility increase the uncertainties, which are multiplied. structural uncertainty, measurement uncertainty, modelling uncertainty, input data uncertainty and natural uncertainty amplify each other. as a rule, variation in any direction in exposure assessment tends to lead to underestimation of risk, and this has severe consequences to health. personal monitoring of exposures, considering variations in individual doses, and monitoring internal doses using biological monitoring methods help in the control of such variation. a monofactorial exposure situation in the past was ideal in the assessment because of its manageability. it also occurs usually as a constant determinant for long periods of time and can be regularly and continuously measured and monitored. this is very seldom the case today, and exposure assessment in modern work life is affected by discontinuities of the enterprise, of technologies and production methods, and turnover of the workforce, as well as the growing mobility and internationalization of both work and workers. company files that were earlier an important source of exposure and health data no longer necessarily fulfil that function. in addition, the standard 8-h time-weighted average for exposure assessment can no longer be taken as a standard, as working hours are becoming extremely heterogeneous. assessment of accurate exposure is thus more and more complex and cumbersome, and new strategies and methods for the quantification of exposure are needed. three challenges in particular can be recognized: a) the challenge arising from numerous discontinuities, fragmentation and changes in the company, employment and technology. although in the past company data were collected from all sources that were available, collective workroom measurements were the most valuable source of data. due to the high mobility of workers and variation in the work tasks, personal exposure monitoring is needed that follows the worker wherever he or she works. special smart cards for recording all personal exposures over years have been proposed, but so far no system-wide action has been possible. in radiation protection, however, such a personal monitoring system has long been a routine procedure. b) the complex nature of exposures where dozens of different factors may be involved (such as those in indoor air problems) and acting in combinations. table 4 .4 gives a list of exposing factors in modern work life, many of which are difficult to monitor. c) new, rapidly spreading and often unexpected exposures that are not well characterized. often their mechanisms of action are not known, or the fast spread of problems calls for urgent action, as in the case of bovine spongiform encephalopathy (bse) in the 1990s, sars outbreak in 2003, and in the new epidemics of psychological stress or musculoskeletal disorders in modern manufacturing. the causes of occupational diseases are grouped into several categories by the type of factor (see table 4 .4). a typical grouping is the one used in ilo recommendation no. 194 . the lists of occupational diseases contain diseases caused by one single factor only, but also diseases which may have been caused by multifactorial exposures. exposure assessment is a crucial step in the overall risk assessment. the growing complexity of exposure situations has led to the development of new methods for assessing such complex exposure situations. these methods are based on construction of model matrices for jobs which have been studied thoroughly for their typical exposures. the exposure profiles are illustrated in job exposure matrices (jem) which are available for dozens of occupations (heikkilä et al. 2005 , guo 2005 . several factors can cause occupational diseases. the jem is a tool used to convert information on job titles into information on occupational risk factors. jem-based analysis is economical, systematic, and often the only reasonable choice in large retrospective studies in which exposure assessment at the individual level is not feasible. but the matrices can also be used in the practical work for getting information on typical exposure profiles of various jobs. the finnish national job-exposure matrix (finjem) is the first and so far the only general jem that is able to give a quantitative estimation of cumulative exposure. for example, finjem estimates were used for exposure profiling on 43 chemical exposures and several other cancer-risk factors for 393 occupational categories. the jem analysis has been further developed into task specific exposure matrices charting the exposure panorama of various tasks (benke et al. 2000 (benke et al. , 2001 . as the previous mono-causal, mono-mechanism, mono-outcome setting has shifted in the direction of multicausality, multiple mechanisms and multioutcomes, the assessment of risks has become more complex. some outcomes, as mentioned above, are difficult to define and measure with objective methods and some of them may be difficult to recognize by exposed groups themselves, or even by experts and researchers. for example, the objective measurement of stress reactions is still imprecise in spite of improvements in the analysis of some indicator hormones, such as adrenalin, noradrenalin, cortisol, prolactin, or in physical measurements, such as galvanic skin resistance and heart rate variability. questionnaires monitoring perceived stress symptoms are still the most common method for measuring stress outcomes. thanks to well organized registries, particularly in germany and the nordic countries, data on many of the relevant outcomes of exposure, such as cancer, pneumoconiosis, reproductive health disturbances and cardiovascular diseases can be accumulated, and long-term follow-up of outcomes at the group level is therefore possible. on the other hand, several common diseases, such as cardiovascular diseases, may have a work-related aetiology, but it may be difficult to show at individual level. the long-term data show that due to changes in the structure of economies, types of employment, occupational structures and conditions of work, many of the traditional occupational diseases, such as pneumoconiosis and acute intoxications have almost disappeared. several new outcomes have appeared, however, such as symptoms of physical or psychological overload, psychological stress, problems of adapting to a high pace of work, and uncertainty related to rapid organizational changes and risk of unemployment. in addition, age-related and work-related diseases among the ageing workforce are on the increase (kivimäki et al. 2006 , ilmarinen 2006 . these new outcomes may have a somatic, psychosomatic or psychosocial phenotype, and they often appear in the form of symptoms or groups of symptoms instead of well-defined diagnoses. practising physicians or clinics are not able to set an icd (international statistical classification of diseases and health-related conditions)-coded diagnosis for them. in spite of their diffuse nature, they are still problems for both the worker and the enterprise, and their consequences may be seen as sickness absenteeism, premature retirement, loss of job satisfaction, or lowered productivity. thus, they may have even a greater impact on the quality of work life and the economy than on clinical health. many such outcomes have been investigated by using questionnaire surveys among either representative samples of the whole workforce or by focusing the survey on a specific sector or occupational group. the combination of data from the surveys of "exposing factors", such as organizational changes, with questionnaire surveys of "outcomes", such as sickness absenteeism, provides epidemiological information on the association between the new exposures and the new outcomes. there are, however, major problems in both the accurate measurement of the exposures and outcomes, and also, the information available on the mechanisms of action is very scarce. epidemiology has expanded the focus of our observations from crosssectional descriptions to longitudinal perspectives, by focussing attention on the occurrence of diseases and finding associations between exposure and morbidity. such an extension of vision is both horizontal and vertical, looking at the causes of diseases. time is not only a temporal parameter in epidemiology, but has also been used for the quantification of exposure, measurement of latencies, and the detection of acceleration or slowing of the course of biological processes. as the time dimension in epidemiology is very important, the changes in temporal parameters of the new work life also affect the methods of epidemiological research. the time dimension is affected in several ways. first, the fragmentation and discontinuities of employment contracts, as described above, break the accumulation of exposure time into smaller fragments, and continuities are thus difficult to maintain. collecting data on cumulative exposures over time becomes more difficult. the time needed for exposure factors to cause an effect becomes more complex, as the discontinuities typical to modern work life allow time for biological repair and elimination processes, thus diluting the risk which would get manifested from continuous exposure. the dosage patterns become more pulse-type, rather than being continuous, stable level exposures. this may affect the multi-staged mechanisms of action in several biological processes. the breaking up of time also increases the likelihood of memory bias of respondents in questionnaire studies among exposed workers, and thus affects the estimation of total exposures. probably the most intensive effect, however, will be seen as a consequence of the variation in working hours. for example, instead of regular work of 8 hours per day, 40 hours per week and 11 months per year, new time schedules and total time budgets are introduced for the majority of workers in the industrial society. the present distribution of weekly working hours in finland is less than 30 hours per week for one third of workers, regular 35-40 hours per week for one third, and 45-80 hours per week for the remaining third. thus the real exposure times may vary substantially even among workers in the same jobs and same occupations, depending on the working hours and the employment contract (temporary, seasonal, part-time, full-time) (härmä 2000 , piirainen et al. 2003 . such variation in time distribution in "new work life" has numerous consequences for epidemiological studies, which in the past "industrial society" effectively utilized the constant time patterns at work for the assessment of exposures and outcomes and their interdependencies. the time dimension also has new structural aspects. as biological processes are highly deterministic in terms of time, the rapid changes in work life cannot wait for the maturation of results in longitudinal follow-up studies. the data are needed rapidly in order to be useful in the management of working conditions. this calls for the development of rapid epidemiological methods which enable rapid collection of the data and the making of analyses in a very short time, in order to provide information on the effects of potential causal factors before the emergence of a new change. often these methods imply the compromising of accuracy and reliability for the benefit of timeliness and actuality. as occupational epidemiology is not only interested in acute and short-term events, but looks at the health of workers over a 30-40-year perspective, the introduction of such new quick methods should not jeopardize the interest and efforts to carry out long-term studies. epidemiology has traditionally been a key tool in making a reliable risk assessment of the likelihood of the adverse outcomes from certain levels of exposure. the new developments in work life bring numerous new challenges to risk assessment. as discussed above, the new developments in work life have eliminated a number of possibilities for risk assessment which prevailed in the stable industrial society. on the other hand, several new methods and new information technologies provide new opportunities for collection and analysis of data. traditionally, the relationship between exposure and outcome has been judged on the basis of the classical criteria set by hill (1965) . höfler (2005) crystallizes the criteria with their explanations as the following: 1. strength of association: a strong association is more likely to have a causal component than is a modest association. 2. consistency: a relationship is observed repeatedly. 3. specificity: a factor influences specifically a particular outcome or population. 4. temporality: the factor must precede the outcome it is assumed to affect. ing dose of exposure or according to a function predicted by a substantive theory. 6. plausibility: the observed association can be plausibly explained by substantive matter (e.g. biological) explanations. 7. coherence: a causal conclusion should not fundamentally contradict present substantive knowledge. 8. experiment: causation is more likely if evidence is based on randomized experiments. 9. analogy: for analogous exposures and outcomes an effect has already been shown. the hill criteria have been subjected to scrutiny, and sven hernberg has analyzed them in detail from the viewpoint of occupational health epidemiology. virtually all the hill criteria are affected by the changes in the new work life, and therefore methodological development is now needed. a few comments on causal inference are made here in view of the critiques by rothman (1988), hernberg (1992) and höfler (2005) : the strength of association will be more difficult to demonstrate due to the growing fragmentation that tends to diminish the sample sizes. the structural change that removes workers from high-level exposures to lower and shorterterm exposures may dilute the strength of effect, which may still prevail, but at a lower level. consistency of evidence may also be affected by the higher variation in conditions of work, study groups, multicultural and multiethnic composition of the workforce, etc. similarly, in the multifactorial, multi-mechanism, multi-outcome setting, the specificity criterion is not always relevant. the temporal dimension has already been discussed. in rapidly changing work life the follow-up times before the next change and before turnover in the workforce may be too short. the outcomes may also be defined by the exposures that have taken place long ago but have not been considered in the study design because historical data are not available. the biological gradient may be possible to demonstrate in a relatively simple exposure-outcome relationship. however, the more complex and multifactorial the setting becomes, the more difficult it may be to show the doseresponse relationship. the dose-response relationship may also be difficult to demonstrate in the cases of relatively ill-defined outcomes which are difficult to measure, but which can be detected as qualitative changes. biological plausibility is an important criterion which in a multimechanism setting may at least in part be difficult to demonstrate. on the other hand, the mechanisms of numerous psychological and psychosocial outcomes lack explanations, even though they undoubtedly are work-related. the missing knowledge of the mechanism of action did not prevent the establishment of causality between asbestos and cancer in a pleural sack or a lung. as many of the new outcomes may be context-dependent, the coherence criterion may be irrelevant. similarly, many of the psychosocial outcomes are difficult to put into an experimental setting, and it can be difficult to make inferences based on analogy. all of the foregoing implies that the new dynamic trends in work life challenge epidemiology in a new way, particularly in the establishment of causality. knowledge of causality is required for the prevention and management of problems. the hill criteria nevertheless need to be supplemented with new ones to meet the conditions of the new work life. similarly, more definitive and specific criteria and indicators need to be developed for the new exposures and outcomes. many of the challenges faced in the struggle to improve health and safety in modern work life can only be solved with the help of research. research on occupational health in the rapidly changing work life is needed more than ever. epidemiology is, and will remain, a key producer of information needed for prevention policies and for ensuring healthy and safe working conditions. the role of epidemiology is, however, expanding from the analysis of the occurrence of well-defined clinical diseases to studies on the occurrence of several other types of exposure and outcome, and their increasingly complex associations. as the baseline in modern work life is shifting in a more dynamic direction, and many parameters in work and the workers' situation are becoming more fragmented, incontinuous and complex, new approaches are needed to tackle the uncertainties in exposure assessment. the rapid pace of change in work life calls for the development of assessment methods to provide up-todate data quickly, so that they can be used to manage these changes and their consequences. many new outcomes which are not possible to register as clinical icd diagnoses constitute problems for today's work life. this is particularly true in the case of psychological, psychosocial and many musculoskeletal outcomes which need to be managed by occupational health physicians. methods for the identification and measurement of such outcomes need to be improved. the traditional hill criteria for causal inference are not always met even in cases where true association does exist. new criteria suitable for a new situation should be established without jeopardizing the original objective of ascertaining the true association. developing the bayesian inference further through utilization of a priori knowledge and a holistic approach may provide responses to new challenges. new neural network softwares may help in the management of the growing complexity. the glory of science does not lie in the perfection of a scientific method but rather in the recognition of its limitations. we must keep in mind the old saying: "absence of evidence is not evidence of absence". instead, it is merely a consequence of our ignorance that should be reduced through further efforts in systematic research, and particularly through epidemiology. and secondly, the ultimate value of occupational health research will be determined on the basis of its impact on practice in the improvement of the working conditions, safety and health of working people. changing conditions of work, new technologies, new substances, new work organizations and working practices are associated with new morbidity patterns and even with new occupational and work-related diseases. the new risk factors, such as rapidly transforming microbials and certain social and behavioural "exposures" may follow totally new dynamics when compared with the traditional industrial exposures (self-replicating nature of microbials and spreading of certain behaviours, such as terrorism) (smolinski et al. 2003 , loza 2007 . several social conditions, such as massive rural-urban migration, increased international mobility of working people, new work organizations and mobile work may cause totally new types of morbidity. examples of such development are, among others, the following: • mobile transboundary transportation work leading to the spread of hiv/aids. • increased risk of metabolic syndrome, diabetes and cardiovascular diseases aggravated by unconventional working hours. • increased risk of psychological burnout in jobs with a high level of longterm stress. • virtually a global epidemic of musculoskeletal disorders among vdu workers with high work load, psychological stress and poor ergonomics. the incidences of occupational diseases may not decline in the future, but the type of morbidity may change. the direction of trend in industrialized countries is the prominence of work-related morbidity and new diseases, while the traditional occupational diseases such as noise injury, pneumoconiosis, repetitive strain and chemical intoxications may continue to be prevalent in developing countries for long periods in the future. the new ergonomics problems are related to light physical work with a considerable proportion of static and repetitive workload. recent research points to an interesting interaction between unergonomic working conditions and psychological stress, leading to a combined risk of musculoskeletal disorders of the neck, shoulders and upper arms, including carpal tunnel syndrome in the wrist. the muscle tension in static work is amplified by the uncontrolled muscular tension caused by psychological stress. furthermore, there seems to be wide inter-individual variation in the tendency to respond with spasm, particularly in the trapezius muscle of neck, under psychological stress. about 40% of the health complaints of working-aged people are related to musculoskeletal disorders, of which a substantial part is work-related. the epidemics have been resistant against preventive measures. new regulatory and management strategies may be needed for effective prevention and control measures (westgaard et al. 2006, paoli and merllié 2001) . the 21st century will be the era of the brain at work and consequently of psychological stress. between 35% and 50% of eu workers in certain occupations report psychological stress due to high time pressure at work (parent-thirion et al. 2007 ). the occurrence of work-related stress is most prevalent in occupations with tight deadlines, pressure from clients, or the high level of responsibility for productivity and quality given to the workers. undoubtedly, the threat of unemployment increases the perception of stress as well. as a consequence, for example, in finland some 40% of workers report symptoms of psychological overload and about 7% show clinical signs of burn out. these are not the problems of low-paid manual workers only, but also, for example, highly educated and well-paid computer super-experts have an elevated risk of burnout as a consequence of often self-committed workload (kalimo and toppinen 1997) . unconventional and ever longer working hours are causing similar problems. for example, one third of finns work over 45 hours a week, and of these 25% work over 50 hours, and 8% often work 65-80 hours per week. it is important to have flexibility in the work time schedules, but it is counterproductive if the biologically determined physiological time rhythms of the worker are seriously offended. over 40% have a sleep deficit of at least one hour each day, and 22% are tired and somnolent at work (härmä et al. 2000) . the toughening global competition, growing productivity demands and continuous changes of work, together with job insecurity, are associated with increased stress. up to 30-50% of workers in different countries and different sectors of the economy report high time pressure and tight deadlines. this prevents them from doing their job as well as they would like to, and causes psychological stress. psychological stress is particularly likely to occur if the high demands are associated with a low degree of self-regulation by the workers (houtman 2005) . stress, if continuous, has been found to be detrimental to physical health (cardiovascular diseases), mental health (psychological burnout), safety (accident risks), and musculoskeletal disorders (particularly hand-arm and shoulder-neck disorders). it also has a negative impact on productivity, sickness absenteeism, and the quality of products and services. the resulting economic losses due to sickness absenteeism, work disability and lower quality of products and services are substantial. the prevention of stress consists not only of actions targeted at the individual worker. there is also a need for measures directed at the work organization, moderation of the total workload, competence building and collaboration within the workplace (theorell 1997). the support from foremen and supervisors is of crucial importance in stress management programmes. another type of psychological burden is the stress arising from the threat of physical violence or aggressive behaviour from the part of clients. in finland some 4% of workers have been subjected to insults or the threat of physical violence, 13% have experienced sexual harassment, and 10% mental violence or bullying at work. the risk is substantially higher for female workers than for men. stress has been found to be associated with somatic health, cardiovascular diseases, mental disorders and depression. one of the new and partly re-emerging challenges of occupational health services is associated with the new trends in microbial hazards. there are several reasons for these developments, for instance, the generation of new microbial strains, structural changes in human habitations with high population densities, growing international travel, and changes possibly in our microbiological environment as a consequence of global warming. of the 10 to 100 million species in the world, about 3 million are microbes. the vast majority of them are not pathogenic to man, and we live in harmony and symbiosis with many of them. we also use bacteria in numerous ways to produce food, medicines, proteins, etc. the pathogenic bacteria have been well controlled in the 20th century; this control had an enormous positive impact on human health, including occupational health. but now the microbial world is challenging us in many ways. new or re-emerging biological hazards are possible due to the transformation of viruses, the increased resistance of some microbial strains (e.g. tuberculosis and some other bacterial agents) and the rapid spread of contaminants through extensive overseas travelling (smolinski et al. 2003) . the scenarios of health hazards from the use of genetically manipulated organisms have not been realized, but biotechnological products have brought along new risks of allergies. a major indoor air problem is caused by fungi, moulds and chemical emissions from contaminated construction materials. new allergies are encountered as a consequence of the increasingly allergic constitution of the population and of the introduction of new allergens into the work environment. health care personnel are increasingly exposed to new microbial hazards due to the growing mobility of people. evidence of high rates of hepatitis b antigen positivity has been shown among health care workers who are in contact with migrants from endemic areas. along with the growing international interactions and mobility, a number of viral and re-emerging bacterial infections also affect the health of people engaged in health care and the care of the elderly, as well as personnel in migrant and refugee services, in social services and other public services. this section applies the general framework for risk governance (chapter 3) to the area of environmental risks. why should we include this topic in a book that is dominantly dealing with occupational health risks and safety issues? there are two major reasons for this decision: 1. most risks that impact health and safety of human beings are also affecting the natural environment. it is therefore necessary for risk managers to reflect the consequences of risk-taking activities with respect to workers, the public and the environment. these risk consequences are all interconnected. our approach to foster an integral approach to risk and risk management requires the integration of all risk consequences. 2. environmental risks are characterized by many features and properties that highlight exemplary issues for many generic risk assessment and management questions and challenges. for example, the question of how to balance benefits and risks becomes more accentuated, if not human life, but damage to environmental quality is at stake. while most people agree that saving human lives takes priority over economic benefits, it remains an open question of how much environmental change and potential damage one is willing to trade off against certain economic benefits. this section is divided into two major parts. part 1 will introduce the essentials of environmental ethics and the application of ethical principles to judging the acceptability of human interventions into the environment. part 2 addresses the procedures for an analytic-deliberative process of decision making when using the risk governance framework developed in chapter 3. it should be noted that this section draws from material that the author has compiled for the german scientific council for global environmental change and that has been partially published in german in a special report of the council (wbgu 1999). the last section on decision making has borrowed material from an unpublished background document on decision making and risk management that dr. warner north and the author had prepared for the us national academy of sciences. should people be allowed to do everything that they are capable of doing? this question is posed in connection with new technologies, such as nanotubes, or with human interventions in nature, such as the clearance of primaeval forests so that the land can be used for agriculture. intuitively everyone answers this question with a definitive "no": no way should people be allowed to everything that they are capable of doing. this also applies to everyday actions. many options in daily life, from lying to minor deception, from breaking a promise up to going behind a friend's back, are obviously actions that are seen by all well-intentioned observers as unacceptable. however, it is much more difficult to assess those actions where the valuation is not so obvious. is it justified to break a promise when keeping the promise could harm many other people? actions where there are conflicts between positive and negative consequences or where a judgement could be made one way or the other with equally good justification are especially common in risk management. there is hardly anyone who wilfully and without reason pollutes the environment, releases toxic pollutants or damages the health of individuals. people who pursue their own selfish goals on the cost and risk of others are obviously acting wrongly and every legislator will sanction this behaviour with the threat of punishment or a penalty. but there is a need for clarification where people bring about a benefit to society with the best intentions and for plausible reasons and, in the process, risk negative impacts on others. in ethics we talk about "conflicting values" here. most decisions involving risks to oneself or others are made for some reason: the actors who make such interventions want to secure goods or services to consumers, for example, to ensure long-term jobs and adequate incomes, to use natural resources for products and services or to use nature for recycling waste materials from production and consumption that are no longer needed. none of this is done for reasons of brotherly love, but to maintain social interests. even improving one's own financial resource is not immoral mere for this reason. the list of human activities that pose risks onto others perpetrated for existential or economic reasons could be carried on into infinity. human existence is bound to taking opportunities and risks. here are just a few figures: around 12,000 years ago about 5 million people lived on the earth. under the production conditions those days (hunter-gatherer culture) this population level was the limit for the human species within the framework of an economic form that only interfered slightly with man's natural environment. the neolithic revolution brought a dramatic change: the carrying capacity of the world for human beings increased by a factor of 10 and more. this agrarian pre-industrial cultural form was characterized by tightly limited carrying capacity, in around 1750 the earth was capable of feeding approx. 750 million people. today the world supports 6 billion people -and this figure is rising. the carrying capacity in comparison to the neolithic age has thus increased thousand-fold and continues to grow in parallel to new changes in production conditions (fritsch 1993; kesselring 1994; mohr 1995) . the five "promethean innovations" are behind this tremendous achievement of human culture: mastering fire, using the natural environment for agriculture, transforming fossil fuels into thermal and mechanical energy, industrial production and substituting material with information (renn 1996) . with today's settlement densities and the predominantly industrial way of life, the human race is therefore dependent on the technical remodelling of nature. without doubt, it needs this for survival, especially for the well-being of the innumerable people, goods and services that reduce the stock of natural resources. with regard to the question of the responsibility of human interventions in nature, the question cannot be about "whether" but -even better -about "how much", because it is an anthropological necessity to adapt and shape existing nature to human needs. for example, the philosopher klaus michael meyer-abich sees the situation as follows: ". . . we humans are not there to leave the world as though we had never been there. as with all other life forms, it is also part of our nature and our lives to bring about changes in the world. of course, this does not legitimise the destructive ways of life that we have fallen into. but only when we basically approve of the changes in the world can we turn to the decisive question of which changes are appropriate for human existence and which are not" (meyer-abich 1997). therefore, to be able to make a sensible judgement of the balance between necessary interventions into the environment and the risks posed by these interventions to human health and environmental quality, the range of products and services created by the consumption of nature has to be considered in relation to the losses that are inflicted on the environment and nature. with this comparison, it can be seen that even serious interventions in nature and the environment did not occur without reflection, but to provide the growing number of people with goods and services; these people need them to survive or as a prerequisite for a "good" life. however, at the same time it must be kept in mind that these interventions often inflict irreversible damage on the environment and destroy possible future usage potentials for future generations. above and beyond this, for the human race, nature is a cradle of social, cultural, aesthetic and religious values, the infringement of which, in turn, has a major influence on people's well-being. on both sides of the equation, there are therefore important goods that have to be appreciated when interventions in nature occur. but what form should such an appreciation take? if the pros and cons of the intervention in nature have to be weighed against each other, criteria are needed that can be used as yardsticks. who can and may draw up such criteria, according to which standards should the interventions be assessed and how can the various evaluative options for action be compared with each other for each criterion? taking risks always involves two major components: an assessment of what we can expect from an intervention into the environment (be it the use of resources or the use of environments as a sink for our waste). this is the risk and benefit assessment side of the risk analysis. secondly, we need to decide whether the assessed consequences are desirable. whereas the estimate of consequences broadly falls in the domain of scientific research and expertise, with uncertainties and ambiguities in particular having to be taken into account (irgc 2005, klinke and renn 2002) , the question about the foundations for evaluating various options for action and about drawing up standards guiding action is a central function of ethics (taylor 1986) . ethics can provide an answer to the question posed in the beginning ("should people be allowed to do everything that they are capable of doing?") in a consistent and transparent manner. in section 4.3.2, environmental ethics will be briefly introduced. this review is inspired by the need for a pragmatic and policy-oriented approach. it is not a replacement for a comprehensive and theoretically driven compendium of environmental ethics. environmental ethics will then be applied to evaluate environmental assets. in this process, a simple distinction is made between categorical principles -that must under no circumstances be exceeded or violated -and compensatory principles, where compensation with other competing principles is allowed. this distinction consequently leads to a classification of environmental values, which, in turn, can be broken down into criteria to appreciate options for designing environmental policies. in section 4.3.3, these ideas of valuation will be taken up and used to translate the value categories into risk handling guidelines. at the heart of the considerations here is the issue of how the aims of ethically founded considerations can be used to support and implement risk-based balancing of costs and benefits. for this purpose, we will develop an integrative risk governance framework. the concept of risk governance comprises a broad picture of risk: not only does it include what has been termed "risk management" or "risk analysis", it also looks at how risk-related decision making unfolds when a range of actors is involved, requiring co-ordination and possibly reconciliation between a profusion of roles, perspectives, goals and activities. indeed, the problem-solving capacities of individual actors, be they government, the scientific community, business players, ngos or civil society as a whole, are limited and often unequal to the major challenges facing society today. then the ideas of the operational implementation of normative and factual valuations are continued and a procedure is described that is capable of integrating ethical, risk-based and work-related criteria into a proposed procedural orientation. this procedure is heavily inspired by decision analysis. answering the question about the right action is the field of practical philosophy, ethics. following the usual view in philosophy, ethics describes the theory of the justification of normative statements, i.e. those that guide action (gethmann 1991 , mittelstraß 1992 , nida-rümelin 1996a , revermann 1998 . a system of normative statements is called "morals". ethical judgements therefore refer to the justifiability of moral instructions for action that may vary from individual to individual and from culture to culture (ott 1999) . basically, humans are purpose-oriented and self-determined beings who act not only instinctively, but also with foresight, and are subject to the moral standards to carry out only those actions that they can classify as good and justifiable (honnefelder 1993) . obviously, not all people act according to the standards that they themselves see as necessary, but they are capable of doing so. in this context, it is possible for people to act morally because, on the one hand, they are capable of distinguishing between moral and immoral action and, on the other, are largely free to choose between different options for action. whether pursuing a particular instruction for action should be considered as moral or immoral is based on whether the action concerned can be felt and justified to be "reasonable" in a particular situation. standards that cross over situations and that demand universal applicability are referred to as principles here. conflicts may arise between competing standards (in a specific situation), as well as between competing principles, the solution of which, in turn, needs justification (szejnwald-brown et al. 1993) . providing yardsticks for such justification or examining moral systems with respect to their justifiability is one of the key tasks of practical ethics (gethmann 1998) . in ethics a distinction is made between descriptive (experienced morality) and prescriptive approaches, i.e. justifiable principles of individual and collective behaviour (frankena 1963 , hansen 1995 . all descriptive approaches are, generally speaking, a "stock-taking" of actually experienced standards. initially, it is irrelevant whether these standards are justified or not. they gain their normative force solely from the fact that they exist and instigate human action (normative force of actual action). most ethicists agree that no conclusions about general validity can be drawn from the actual existence of standards. this would be a naturalistic fallacy (akademie der wissenschaften 1992, ott 1999) . nevertheless, experienced morality can be an important indicator of different, equally justifiable moral systems, especially where guidance for cross-cultural behaviour is concerned. this means that the actual behaviour of many people with regard to their natural environment reveals which elements of this environment they value in particular and which they do not. however, in this case, too, the validity of the standards is not derived from their factuality, but merely used as a heurism in order to find an adequate (possibly culture-immanent) justification. but given the variety of cultures and beliefs, how can standards be justified inter-subjectively, i.e. in a way that is equally valid to all? is it not the case that science can only prove or disprove factual statements (and this only to a certain extent), but not normative statements? a brief discourse on the various approaches in ethics is needed to answer this question. first of all, ethics is concerned with two different target aspects: on the one hand, it is concerned with the question of the "success" of one's own "good life", i.e. with the standards and principles that enable a person to have a happy and fulfilled life. this is called eudemonistic ethics. on the other hand, it is concerned with the standards and principles of living together, i.e. with binding regulations that create the conditions for a happy life: the common good. this is called normative ethics (galert 1998 , ott 1999 . within normative ethics a distinction is made between deontological and teleological approaches when justifying normative statements (höffe 1987) . deontological approaches are principles and standards of behaviour that apply to the behaviour itself on the basis of an external valuation criterion. it is not the consequences of an action that are the yardstick of the valuation; rather, it is adhering to inherent yardsticks that can be used against the action itself. such external yardsticks of valuation are derived from religion, nature, intuition or common sense, depending on the basic philosophical direction. thus, protection of the biosphere can be seen as a divine order to protect creation (rock 1980 , schmitz 1985 , as an innate tendency for the emotional attachment of people to an environment with biodiversity (wilson 1984) , as a directly understandable source of inspiration and joy (ehrenfeld 1993) or as an educational means of practising responsibility and maintaining social stability (gowdy 1997) . by contrast, teleological approaches refer to the consequences of action. here, too, external standards of valuation are needed since the ethical quality of the consequences of action also have to be evaluated against a yardstick of some kind. with the most utilitarian approaches (a subset of the teleological approaches) this yardstick is defined as an increase in individual or social benefit. in other schools of ethics, intuition (can the consequence still be desirable?) or the aspect of reciprocity (the so-called "golden rule": "do as you would be done by") play a key role. in the approaches based on logical reasoning (especially in kant), the yardstick is derived from the logic of the ability to generalize or universalize. kant himself is in the tradition of deontological approaches ("good will is not good as a result of what it does or achieves, but just as a result of the intention"). according to kant, every principle that, if followed generally, makes it impossible for a happy life to be conducted is ethically impermissible. in this connection, it is not the desirability of the consequences that captures kant's mind, but the logical inconsistency that results from the fact that the conditions of the actions of individuals would be undermined if everyone were to act according to the same maxims (höffe 1992) . a number of contemporary ethicists have taken up kant's generalization formula, but do not judge the maxims according to their internal contradictions; rather, they judge them according to the desirability of the consequences to be feared from the generalization (jonas 1979 or zimmerli 1993 should be mentioned here). these approaches can be defined as a middle course between deontological and teleological forms of justification. in addition to deontological and teleological approaches, there is also the simple solution of consensual ethics, which, however, comprises more than just actually experienced morality. consensual ethics presupposes the explicit agreement of the people involved in an action. everything is allowed provided that all affected (for whatever reason) voluntarily agree. in sexual ethics at the moment a change from deontological ethics to a consensual moral code can be seen. the three forms of normative ethics are shown in figure 4 .13. the comparison of the basic justification paths for normative moral systems already clearly shows that professional ethicists cannot create any standards or designate any as clearly right, even if they play a role in people's actual lives. much rather it is the prime task of ethics to ensure on the basis of generally recognized principles (for example, human rights) that all associated standards and behaviour regulations do not contradict each other or a higher order principle. above and beyond this, ethics can identify possible solutions that may occur with a conflict between standards and principles of equal standing. ethics may also reveal interconnections of justification that have proved themselves as examination criteria for moral action in the course of their disciplinary history. finally, many ethicists see their task as providing methods and procedures primarily of an intellectual nature by means of which the compatibility or incompatibility of standards within the framework of one or more moral systems can be completed. unlike the law, the wealth of standards of ethics is not bound to codified rules that can be used as a basis for such compatibility examinations. every normative discussion therefore starts with the general issues that are needed in order to allow individuals a "good life" and, at the same time, to give validity to the principles required to regulate the community life built on common good. but how can generally binding and inter-subjectively valid criteria be made for the valuation of "the common good"? in modern pluralistic societies, it is increasingly difficult for individuals and groups of society to draw up or recognize collectively binding principles that are perceived by all equally as justifiable and as self-obliging (hartwich and wewer 1991, zilleßen 1993) . the variety of lifestyle options and subjectiernization. with increasing technical and organizational means of shaping the future, the range of behaviour options available to people also expands. with the increasing plurality of lifestyles, group-specific rationalities emerge that create their own worldviews and moral standards, which demand a binding nature and validity only within a social group or subculture. the fewer cross-society guiding principles or behaviour orientations are available, the more difficult is the process of agreement on collectively binding orientations for action. however, these are vital for the maintenance of economic cooperation, for the protection of the natural foundations of life and for the maintenance of cohesion in a society. no society can exist without the binding specification of minimum canons of principles and standards. but how can agreement be reached on such collectively binding principles and standards? what criteria can be used to judge standards? the answers to this question depend on whether the primary principles, in other words, the starting point of all moral systems, or secondary principles or standards, i.e. follow-on standards that can be derived from the primary principles, are subjected to an ethical examination. primary principles can be categorical or compensatory (capable of being compensated). categorical principles are those that must not be infringed under any circumstances, even if other prin-219 fication of meaning (individualization) are accompanying features of mod-ciples would be infringed as a result. the human right to the integrity of life could be named here as an example. compensatory principles are those where temporary or partial infringement is acceptable, provided that as a result the infringement of a principle of equal or higher ranking is avoided or can be avoided. in this way certain freedom rights can be restricted in times of emergency. in the literature on ethical rules, one can find more complex and sophisticated classifications of normative rules. for our purpose to provide a simple and pragmatic framework, the distinction in four categories (principles and standards; categorical and compensatory) may suffice. this distinction has been developed from a decision-analytical perspective. but how can primary principles be justified as equally valid for all people? although many philosophers have made proposals here, there is a broad consensus today that neither philosophy nor any other human facility is capable of stating binding metacriteria without any doubt and for all people, according to which such primary principles should be derived or examined (mittelstraß 1984) . a final justification of normative judgements cannot be achieved by logical means either, since all attempts of this kind automatically end either in a logical circle, in an unending regression (vicious cycle) or in a termination of the procedure and none of these alternatives is a satisfactory solution for final justification (albert 1991). the problem of not being able to derive finally valid principles definitively, however, seems to be less serious than would appear at first glance. because, regardless of whether the basic axioms of moral rules are taken from intuition, observations of nature, religion, tradition reasoning or common sense, they have broadly similar contents. thus, there is broad consensus that each human individual has a right to life, that human freedom is a high-value good and that social justice should be aimed at. but there are obviously many different opinions about what these principles mean in detail and how they should be implemented. in spite of this plurality, however, discerning and well-intentioned observers can usually quickly agree, whether one of the basic principles has clearly been infringed. it is more difficult to decide whether they have clearly been fulfilled or whether the behaviour to be judged should clearly be assigned to one or several principles. since there is no finally binding body in a secular society that can specify primary principles or standards ex cathedra, in this case consensus among equally defendable standards or principles can be used (or pragmatically under certain conditions also majority decisions). ethical considerations are still useful in this case as they allow the test of generalization and the enhancement of awareness raising capabilities. in particular, they help to reveal the implications of such primary principles and standards. provided that primary principles are not concerned (such as human rights), the ethical discussion largely consists of examining the compatibility of each of the available standards and options for action with the primary principles. in this connection, the main concerns are a lack of contradictions (consistency), logical consistency (deductive validity), coherence (agreement with other principles that have been recognized as correct) and other, broadly logical criteria (gethmann 1998) . as the result of such an examination it is entirely possible to reach completely different conclusions that all correspond to the laws of logic and thus justify new plurality. in order to reach binding statements or valuations here the evaluator can either conduct a discussion in his "mind" and let the arguments for various standards compete with each other (rather like a platonic dialogue) or conduct a real discussion with the people affected by the action. in both cases the main concern is to use the consensually agreed primary principles to derive secondary principles of general action and standards of specific action that should be preferred over alternatives that can be equally justified. a plurality of solutions should be expected especially because most of the concrete options for action comprise only a gradual fulfilment and infringement of primary principles and therefore also include conflicting values. for value conflicts at the same level of abstraction there are, by definition, no clear rules for solution. there are therefore frequently conflicts between conserving life through economic development and destroying life through environmental damage. since the principle of conserving life can be used for both options a conflict is unavoidable in this case. to solve the conflicts, ethical considerations, such as the avoidance of extremes, staggering priorities over time or the search for third solutions can help without, however, being able to convincingly solve this conflict in principle to the same degree for all (szejnwald-brown et al. 1993) . these considerations lead to some important conclusions for the matter of the application of ethical principles to the issue of human action with regard to the natural environment. first of all, it contradicts the way ethics sees itself to develop ethics of its own for different action contexts. just as there can be no different rules for the logic of deduction and induction in nomological science, depending on which object is concerned, it does not make any sense to postulate an independent set of ethics for the environment (galert 1998) . justifications for principles and moral systems have to satisfy universal validity (nida-rümelin 1996b). furthermore, it is not very helpful to call for a special moral system for the environment since this -like every other moral system -has to be traceable to primary principles. instead, it makes sense to specify the generally valid principles that are also relevant with regard to the issue of how to deal with the natural environment. at the same time standards should be specified that are appropriate to environmental goods and that reflect those principles that are valid beyond their application to the environment. as implied above, it does not make much sense to talk about an independent set of environmental ethics. much rather, general ethics should be transferred to issues relating to the use of the environment (hargrove 1989) . three areas are usually dealt with within the context of environmental ethics (galert 1998 ): • environmental protection, i.e. the avoidance or alleviation of direct or indirect, current or future damage and pollution resulting from anthropogenic emissions, waste or changes to the landscape, including land use, as well as the long-term securing of the natural foundations of life for people and other living creatures (birnbacher 1991a ). • animal protection, i.e. the search for reasonable and enforceable standards to avoid or reduce pain and suffering in sentient beings (krebs 1997 , vischer 1999 ). • nature conservation, i.e. the protection of nature against the transforming intervention of human use, especially all measures to conserve, care for, promote and recreate components of nature deemed to be valuable, including species of flora and fauna, biotic communities, landscapes and the foundations of life required there (birnbacher 1991a) . regardless which of these three areas are addressed we need to explore which primary principles be applied to them. when dealing with the environment, the traditional basic and human rights, as well as the civil rights that have been derived from them, should be just as much a foundation of the consideration as other areas of application in ethics. however, with regard to the primary principles there is a special transfer problem when addressing human interventions into nature and the environment: does the basic postulate of conservation of life apply only to human beings, to all other creatures or to all elements of nature, too? this question does not lead to a new primary principle, as one may suspect at first glance. much rather, it is concerned with the delineation of the universally recognized principle of the conservation of life that has already been specified in the basic rights canon. are only people included in this principle (this is the codified version valid in most legal constitutions today) or other living creatures, too? and if yes, which ones? should non-living elements be included as well? when answering this question, two at first sight contradictory positions can be derived: anthropocentrism and physiocentrism (taylor 1986 , ott 1993 , galert 1998 . the anthropocentric view places humans and their needs at the fore. nature's own original demands are alien to this view. interventions in nature are allowed if they are useful to human society. a duty to make provisions for the future and to conserve nature exists in the anthropocentric world only to the extent that natural systems are classed as valuable to people today and subsequent generations and that nature can be classed as a means and guarantor of human life and survival (norton 1987 , birnbacher 1991b . in the physiocentric concept, which forms an opposite pole to the anthropocentric view, the needs of human beings are not placed above those of nature. here, every living creature, whether humans, animals or plants, have intrinsic rights with regard to the chance to develop their own lives within the framework of a natural order. merit for protection is justified in the physiocentric view by an inner value that is unique to each living creature or the environment in general. nature has a value of its own that does not depend on the functions that it fulfils today or may fulfil later from a human society's point of view (devall and sessions 1984 , callicott 1989 , rolston 1994 , meyer-abich 1996 . each of these prevailing understandings of the human-nature relationship has implications that are decisive for the form and extent of nature use by humans (elliot 1995 , krebs 1997 . strictly speaking, it could be concluded from the physiocentric idea that all human interventions in nature have to be stopped so that the rights of other creatures are not endangered. yet, not even extreme representatives of a physiocentric view would go so far as to reject all human interventions in nature because animals, too, change the environment by their ways of life (e.g. the elephant prevents the greening of the savannah). the central postulate of a physiocentric view is the gradual minimization of the depth of interventions in human use of nature. the only interventions that are permitted are those that contribute to directly securing human existence and do not change the fundamental composition of the surrounding natural environment. if these two criteria were taken to the extreme, neither population development beyond the boundaries of biological carrying capacity nor a transformation of natural land into pure agricultural land would be allowed. such a strict interpretation of physiocentrism would lead to a radical reversal of human history so far and is not compatible with the values and expectations of most people. the same is true for the unlimited transfer of anthropocentrism to dealings with nature. in this view, the use of natural services is subjected solely to the individual cost-benefit calculation. this can lead to unscrupulous exploitation of nature by humans with the aim of expanding human civilization. both extremes quickly lead to counter-intuitive implications. when the issue of environmental design and policy is concerned, anthropocentric and physiocentric approaches in their pure form are found only rarely, much rather they occur in different mixtures and slants. the transitions between the concepts are fluid. moderate approaches certainly take on elements from the opposite position. it can thus be in line with a fundamentally physiocentric perspective if the priority of human interests is not questioned in the use of natural resources. it is also true that the conclusions of a moderate form of anthropocentrism can approach the implications of the physiocentric view. table 4 .10 provides an overview of various types of anthropocentric and physiocentric perspectives. if we look at the behaviour patterns of people in different cultures, physiocentric or anthropocentric basic positions are rarely maintained consistently (bargatzky and kuschel 1994 ; on the convergence theory: birnbacher 1996) . in the strongly anthropocentric countries in the west, people spend more money on the welfare and health of their own pets than on saving human lives in other countries. in the countries of the far east that are characterized by physiocentrism, nature is frequently exploited even more radically than in the industrialized countries of the west. this inconsistent action is not a justification for one view or the other, it is just a warning for caution when laying down further rules for use so that no extreme -and thus untenable -demands be made. also from an ethical point of view, radical anthropocentrism should be rejected just as much as radical physiocentrism. if, to take up just one argument, the right to human integrity is largely justified by the fact that causing pain by others should be seen as something to avoid, this consideration without a doubt has to be applied to other creatures that are also capable of feeling pain (referred to as: pathocentrism). here, therefore, pure anthropocentrism cannot convince. in turn, with a purely physiocentric approach the primary principles of freedom, equality and human dignity could not be maintained at all if every part of living nature were equally entitled to use the natural environment. under these circumstances people would have to do without agriculture, the conversion of natural land into agricultural land and breeding farm animals and pets in line with human needs. as soon table 4 .10 different perspectives on nature. adapted from renn and goble (1996: 4). as physiocentrism is related to species and not to individuals as is done in some biocentric perspectives human priority is automatically implied; because where human beings are concerned, nearly all schools of ethics share the fundamental moral principle of an individual right to life from birth. if this right is not granted to individual animals or plants, a superiority of the human race is implicitly assumed. moderate versions of physiocentrism acknowledge a gradual de-escalation with respect to the claim of individual table 4 .10 different perspectives on nature (continued). adapted from renn and goble (1996: 4). life protection. the extreme forms of both physiocentrism and anthropocentrism are therefore not very convincing and are hardly capable of achieving a global consensus. this means that only moderate anthropocentrism or moderate biocentrism should be considered. the image of nature that is used as a basis for the considerations in this section emphasizes the uniqueness of human beings vis-à-vis physiocentric views, but does not imply carte blanche for wasteful and careless dealings with nature. this moderate concept derives society's duty to conserve nature -also for future generations -from the life-preserving and life-enhancing meaning of nature for society. this is not just concerned with the instrumental value of nature as a "store of resources", it is also a matter of the function of nature as a provider of inspiration, spiritual experience, beauty and peace (birnbacher and schicha 1996) . in this context it is important that human beings -as the addressees of the moral standard -do not regard nature merely as material and as a way towards their own self-realization, but can also assume responsibility for conservation of their cultural and so-cial function, as well as their existential value above and beyond the objective and technically available benefits (honnefelder 1993) . one of the first people to express this responsibility of human stewardship of nature in an almost poetic way was the american ecologist aldo leopold, who pointed out people's special responsibility for the existence of nature and land as early as the 1930s with the essay "the conservation ethics". his most well-known work "a sand county almanac" is sustained by the attempt to observe and assess human activities from the viewpoint of the land (a mountain or an animal). this perspective was clearly physiocentric and revealed fundamental insights about the relationship between humans and nature on the basis of empathy and shifting perspectives. his point of view had a strong influence on american environmental ethics and the stance of conservationists. although this physiocentric perspective raises many concerns, the idea of stewardship has been one of the guiding ideas for the arguments used in this section (pickett et al. 1997) . we are morally required to exercise a sort of stewardship over living nature, because nature cannot claim any rights for itself, but nevertheless has exceptional value that is important to man above and beyond its economic utility value (hösle 1991) . since contemporary society and the generations to come certainly use, or will use, more natural resources than would be compatible with a lifestyle in harmony with the given natural conditions, the conversion of natural land into anthropogenically determined agricultural land cannot be avoided (mohr 1995) . many people criticized human interventions into natural cycles as infringements of the applicable moral standards of nature conservation (for example, fastened onto the postulate of sustainability). but we should avoid premature conclusions here, as can be seen with the example of species protection. for example, where natural objects or phenomena are concerned that turn out to be a risk to human or non-human living creatures, the general call for nature conservation is already thrown into doubt (gale and cordray 1994) . not many people would call the eradication of cholera bacteria, hiv viruses and other pathogens morally bad (mittelstraß 1995) if remaining samples were kept under lock and key in laboratories. also, combating highly evolved creatures, such as cockroaches or rats meets with broad support if we ignore the call for the complete eradication of these species for the time being. an environmental initiative to save cockroaches would not be likely to gain supporters. if we look at the situation carefully, the valuation of human behaviour in these examples results from a conflict. because the conservation of the species competes with the objective of maintaining human health or the objective of a hygienic place to live, two principles, possibly of equal ranking, come face to face. in this case the options for action, which may all involve a gradual infringement of one or more principles, would have to be weighed up against each other. a general ban on eradicating a species can thus not be justified ethically, in the sense of a categorical principle, unless the maintenance of human health were to be given lower priority than the conservation of a species. with regard to the issue of species conservation, therefore, different goods have to be weighed up against each other. nature itself cannot show society what it is essential to conserve and how much nature can be traded for valuable commodities. humans alone are responsible for a decision and the resulting conflicts between competing objectives. appreciation and negotiation processes are therefore the core of the considerations about the ethical justification of rules for interventions. but this does not mean that there is no room for categorical judgements along the lines of "this or that absolutely must be prohibited" in the matter of human interventions into the natural environment. it follows on from the basic principle of conserving human life that all human interventions that threaten the ability of the human race as a whole, or a significant number of individuals alive today or in the future, to exist should be categorically prohibited. this refers to intervention threats to the systemic functions of the biosphere. such threats are one of the guiding principles that must not be exceeded under any circumstances, even if this excess were to be associated with high benefits. in the language of ethics this is a categorical principle, in the language of economics a good that is not capable of being traded. the "club" of categorical prohibitions should, however, be used very sparingly because plausible trade-offs can be thought up for most principles, the partial exceeding of which appears intuitively. in the case of threats to existence, however, the categorical rejection of the behaviour that leads to this is obvious. but what does the adoption of categorical principles specifically mean for the political moulding of environmental protection? in the past, a number of authors have tried to specify the minimum requirements for an ethically responsible moral system with respect to biosphere use. these so-called "safe minimum standards" specify thresholds for the open-ended measurement scale of the consequences of human interventions that may not be ex-ceeded even if there is a prospect of great benefits (randall 1988, randall and farmer 1995) . in order to be able to specify these thresholds in more detail the breakdown into three levels proposed by the german scientific council for global environmental change is helpful (wbgu 2000) . these levels are: • the global bio-geochemical cycles in which the biosphere is involved as one of the causes, modulator or "beneficiary"; • the diversity of ecosystems and landscapes that have key functions as bearers of diversity in the biosphere; and • the genetic diversity and the species diversity that are both "the modelling clay of evolution" and basic elements of ecosystem functions and dynamics. where the first level is concerned, in which the functioning of the global ecosystem is at stake, categorical principles are obviously necessary and sensible, provided that no one wants to shake the primary principle of the permanent preservation of the human race. accordingly, all interventions in which important substance or energy cycles are significantly influenced at a global level and where globally effective negative impacts are to be expected are categorically prohibited. usually no stringently causal evidence of the harmful nature of globally relevant information is needed; justified suspicion of such harmfulness should suffice. later in this chapter we will make a proposal for risk valuation and management how the problem of uncertainty in the event of possible catastrophic damage potential should be dealt with (risk type cassandra). on the second level, the protection of ecosystems and landscapes, it is much more difficult to draw up categorical rules. initially, it is obvious that all interventions in landscapes in which the global functions mentioned on the first level are endangered must be avoided. above and beyond this, it is wise from a precautionary point of view to maintain as much ecosystem diversity as possible in order to keep the degree of vulnerability to the unforeseen or even unforeseeable consequences of anthropogenic and nonanthropogenic interventions as low as possible. even though it is difficult to derive findings for human behaviour from observations of evolution, the empirically proven statement "he who places everything on one card, always loses in the long run" seems to demonstrate a universally valid insight into the functioning of systemically organized interactions. for this reason, the conservation of the natural diversity of ecosystems and landscape forms is a categorical principle, whereas the depth of intervention allowed should be specified on the basis of principles and standards capable of compensation. the same can be said for the third level, genetic and species protection. here too, initially the causal chain should be laid down: species conservation, landscape conservation, maintaining global functions. wherever this chain is unbroken, a categorical order of conservation should apply. these species could be termed primary key species. this includes such species that are not only essential for the specific landscape type in which they occur, but also for the global cycles above and beyond this specific landscape type thanks to their special position in the ecosystem. probably, it will not be possible to organize all species under this functional contribution to the surrounding ecosystem, but we could also think of groups of species, for example, humus-forming bacteria. in second place there are the species that characterize certain ecosystems or landscapes. here they are referred to as secondary key species. they, too, are under special protection that is not necessarily under categorical reservations. their function value, however, is worthy of special attention. below these two types of species there are the remaining species that perform ecosystem functions to a greater or lesser extent. what this means for the worthiness for protection of these species and the point at which the precise limit for permitted intervention should be drawn, is a question that can no longer be solved with categorical principles and standards, but with the help of compensatory principles and standards. generally, here, too, as with the issue of ecosystem and landscape protection, the conservation of diversity as a strategy of "reinsurance" against ignorance, global risks and unforeseeable surprises is recommended. it remains to be said that from a systemic point of view, a categorical ban has to apply to all human interventions where global closed loops are demonstrably at risk. above and beyond this, it makes sense to recognize the conservation of landscape variety (also of ecosystem diversity within landscapes) and of genetic variety and species diversity as basic principles, without being able to make categorical judgements about individual landscape or species types as a result. in order to evaluate partial infringements of compensatory principles or standards, which are referred to in the issue of environmental protection, we need rules for decision making that facilitate the balancing process necessary to resolve compensatory conflicts. in the current debate about rules for using the environment and nature, it is mainly teleological valuation methods that are proposed (hubig 1993 , ott 1993 . these methods are aimed at: • estimating the possible consequences of various options for action at all dimensions relevant to potentially affected people; • recording the infringements or fulfilments of these expected consequences in the light of the guiding standards and principles; and • then weighing them according to an internal key so that they can be weighed up in a balanced way. on the positive side of the equation, there are the economic benefits of an intervention and the cultural values created by use, for example, in the form of income, subsistence (self-sufficiency) or an aesthetically attractive landscape (parks, ornamental gardens, etc.); on the negative side, there are the destruction of current or future usage potentials, the loss of unknown natural resources that may be needed in the future and the violation of aesthetic, cultural or religious attributes associated with the environment and nature. there are therefore related categories on both sides of the equation: current uses vs. possible uses in the future, development potentials of current uses vs. option values for future use, shaping the environment by use vs. impairments to the environment as a result of alternative use, etc. with the same or similar categories on the credit and debit side of the balance sheet the decision is easy when there is one option that performs better or worse than all the other options for all categories. although such a dominant (the best for all categories) or sub-dominant option (the worst for all categories) is rare in reality, there are examples of dominant or sub-dominant solutions. thus, for example, the overfelling of the forests of kalimantan on the island of borneo in indonesia can be classed as a sub-dominant option since the short-term benefit, even with extremely high discount rates, is in no proportion to the long-term losses of benefits associated with a barren area covered in imperata grass. the recultivation of a barren area of this kind requires sums many times the income from the sale of the wood, including interest. apparently there are no cultural, aesthetic or religious reasons for conversion of primary or secondary woodland into grassland. this means that the option of deforestation should be classed as of less value than alternative options for all criteria, including economic and social criteria. at best, we can talk about a habit of leaving rainforests, as a "biotope not worthy of conservation", to short-term use. but habit is not a sound reason for the choice of any sub-optimum option. as mentioned at the start of this chapter, habit as experienced morality, does not have any normative force, especially when this is based on the illusion of the marginality of one's own behaviour or ignorance about sustainable usage forms. but if we disregard the dominant or sub-dominant solutions, an appreciation between options that violate or fulfil compensatory standards and principles depends on two preconditions: best possible knowledge of the consequences (what happens if i choose option a instead of option b?) and a transparent, consistent rationale for weighing up these consequences as part of a legitimate political decision process (are the foreseeable consequences of option a more desirable or bearable than the consequences of option b?) (akademie der wissenschaften 1992). adequate knowledge of the consequences is needed in order to reveal the systemic connections between resource use, ecosystem reactions to human interventions and socio-cultural condition factors (wolters 1995) . this requires interdisciplinary research and cooperation. the task of applied ecological research, for example, is to show the consequences of human intervention in the natural environment and how ecosystems are burdened by different interventions and practices. the economic approach provides a benefit-oriented valuation of natural and artificial resources within the context of production and consumption, as well as a valuation of transformation processes according to the criterion of efficiency. cultural and social sciences examine the feedback effects between use, social development and cultural self-perception. they illustrate the dynamic interactions between usage forms, socio-cultural lifestyles and control forms. interdisciplinary, problem-oriented and system-related research contribute to forming a basic stock of findings and insights about functional links in the relationship between human interventions and the environment and also in developing constructive proposals as to how the basic question of an ethically justified use of the natural environment can be answered in agreement with the actors concerned (wbgu 2000) . accordingly, in order to ensure sufficient environmental protection, scientific research, but especially transdisciplinary system research at the interface between natural sciences and social sciences is essential. bringing together the results of interdisciplinary research, the policy-relevant choice of knowledge banks and balanced interpretation in an environment of uncertainty and ambivalence are difficult tasks that primarily have to be performed by the science system itself. how this can happen in a way that is methodslogically sound, receptive to all reasonable aspects of interpretation and yet subjectively valid will be the subject of section 4.3.3. but knowledge alone does not suffice. in order to be able to act effectively and efficiently while observing ethical principles, it is necessary to shape the appreciation process between the various options for action according to rational criteria (gethmann 1998) . to do this it is, first of all, necessary to identify the dimensions that should be used for a valuation. the discussion about the value dimensions to be used as a basis for valuation is one of the most popular subjects within environmental ethics. to apply these criteria in risk evaluation and to combine the knowledge aspects about expected consequences of different behavioural options with the ethical principles is the task of what we have called risk governance. what contribution do ethics make towards clarifying the prospects and limits of human interventions into the natural environment? the use of environmental resources is an anthropological necessity. human consciousness works reflexively and humans have developed a causal recognition capacity that enables them to record cause and effect anticipatively and to productively incorporate assessed consequences in their own action. this knowledge is the motivating force behind the cultural evolution and the development of technologies, agriculture and urbanization. with power over an ever-increasing potential of design and intervention in nature and social affairs over the course of human history, the potential for abuse and exploitation has also grown. whereas this potential was reflected in philosophical considerations and legal standards at a very early stage with regard to moral standards between people, the issue of human responsibility towards nature and the environment has only become the subject of intensive considerations in recent times. ethical considerations are paramount in this respect. on the one hand, they offer concrete standards for human conduct on the bases of criteria that can be generalized, and, on the other hand, they provide procedural advice about a rational and decision-and policy-making process. a simple breakdown into categorical rules and prohibitions that are capable of being compensated can assist decision makers for the justification of principles and standards on environmental protection. as soon as human activities exceed the guidelines of the categorical principles, there is an urgent need for action. how can we detect whether such an excess has happened and how it can be prevented from the very outset that these inviolable standards and principles be exceeded? here are three strategies of environmental protection to be helpful for the implementation of categor-ical guidelines. the first strategy is that of complete protection with severe restrictions of all use by humans (protection priority). the second strategy provides for a balanced relationship between protection and use, where extensive resource use should go hand in hand with the conservation of the ecosystems concerned (equal weight). the third strategy is based on optimum use involving assurance of continuous reproduction. the guiding principle here would be an intensive and, at the same time, sustainable, i.e. with a view to the long term, use of natural resources (use priority). the following section will present a framework for applying these principles into environmental decision making under risk. the main line of argument is that risk management requires an analytic-deliberative approach for dealing effectively and prudently with environmental risks. assessing potential consequences of human interventions and evaluating their desirability on the basis of subsequent knowledge and transparent valuation criteria are two of the central tasks of a risk governance process. however, the plural values of a heterogeneous public and people's preferences have to be incorporated in this process. but how can this be done given the wealth of competing values and preferences? should we simply accept the results of opinion polls as the basis for making political decisions? can we rely on risk perception results to judge the seriousness of pending risks? or should we place all our faith in professional risk management? if we turn to professional help to deal with plural value input, economic theory might provide us an answer to this problem. if environmental goods are made individual and suitable for the market by means of property rights, the price that forms on the market ensures an appropriate valuation of the environmental good. every user of this good can then weigh up whether he is willing to pay the price or would rather not use the good. with many environmental goods, however, this valuation has to be made by collective action, because the environmental good concerned is a collective or open access good. in this case a process is needed that safeguards the valuation and justifies it to the collective. however, this valuation cannot be determined with the help of survey results. although surveys are needed to be able to estimate 234 the breadth of preferences and people's willingness to pay, they are insufficient for a derivation of concrete decision-making criteria and yardsticks for evaluating the tolerability of risks to human health and the environment. • firstly, the individual values are so widely scattered that there is little sense in finding an average value here. • secondly, the preferences expressed in surveys change much within a short time, whereas ethical valuations have to be valid for a long time. • thirdly, as outlined in the subsection on risk perception, preferences are frequently based on flawed knowledge or ad hoc assumptions both of which should not be decisive according to rational considerations. what is needed, therefore, is a gradual process of assigning trade-offs in which existing empirical values are put into a coherent and logically consistent form. in political science and sociological literature reference is mostly made to three strategies of incorporating social values and preferences in rational decision-making processes (renn 1997) . firstly, a reference to social preferences is viewed solely as a question of legitimate procedure (luhmann 1983 , vollmer 1996 . the decision is made on the basis of formal decision-making process (such as majority voting). if all the rules have been kept, a decision is binding, regardless of whether the subject matter of the decision can be justified or whether the people affected by the decision can understand the justification. in this version, social consensus has to be found only about the structure of the procedures; the only people who are then involved in the decisions are those who are explicitly legitimated to do so within the framework of the procedure decided upon. the second strategy is to rely on the minimum consensuses that have developed in the political opinion-forming process (muddling through) (lindbloom 1959 (lindbloom , 1965 . in this process, only those decisions that cause the least resistance in society are considered to be legitimate. in this version of social pluralism groups in society have an influence on the process of the formation of will and decision making to the extent that they provide proposals capable of being absorbed, i.e. adapted to the processing style of the political system, and that they mobilize public pressure. the proposal that then establishes itself in politics is the one that stands up best in the competition of proposals, i.e. the one that entails the fewest losses of support for political decision makers by interest groups. the third strategy is based on the discussion between the groups involved (habermas 1971 , renn 2004 ). in the communicative exchange among the people involved in the discussion a form of communicative rationality that everyone can understand evolves that can serve as a justification for collectively binding decisions. at the same time, discursive methods claim to more appropriately reflect the holistic nature of human beings and also to provide fair access to designing and selecting solutions to problems. in principle, the justification of standards relevant to decisions is linked to two conditions: the agreement of all involved and substantial justification of the statements made in the discussion (habermas 1981) . all three strategies of political control are represented in modern societies to a different extent. legitimation conflicts mostly arise when the three versions are realized in their pure form. merely formally adhering to decisionmaking procedures without a justification of content encounters a lack of understanding and rejection among the groups affected especially when they have to endure negative side effects or risks. then acceptance is refused. if, however, we pursue the opposite path of least resistance and base ourselves on the route of muddling through we may be certain of the support of the influential groups, but, as in the first case, the disadvantaged groups will gradually withdraw their acceptance because of insufficient justification of the decision. at the same time, antipathy to politics without a line or guidance is growing, even the affected population. the consequence is political apathy. the third strategy of discursive control faces problems, too. although in an ideal situation it is suitable for providing transparent justifications for the decision-making methods and the decision itself, in real cases the conditions of ideal discourse can rarely be adhered to (wellmer 1992) . frequently, discussions among strategically operating players lead to a paralysis of practical politics by forcing endless marathon meetings with vast quantities of points of order and peripheral contributions to the discussion. the "dictatorship of patience" (weinrich 1972) ultimately determines which justifications are accepted by the participants. the public becomes uncertain and disappointed by such discussions that begin with major claims and end with trivial findings. in brief: none of the three ways out of the control dilemma can convince on its own; as so often in politics, everything depends on the right mixture. what should a mixture of the three elements (due process, pluralistic muddling through and discourse) look like so that a maximum degree of rationality can come about on the basis of social value priorities? a report by the american academy of sciences on the subject of "understanding environmental risks" (national research council 1996) comes to the conclusion that scientifically valid and ethically justified procedure for the collective valuation of options for risk handling can only be realized within the context of -what the authors coin -an analytic-deliberative process. analytic means that the best scientific findings about the possible consequences and conditions of collective action are incorporated in the negotiations; deliberative means that rationally and ethically transparent criteria for making trade-offs are used and documented externally. moreover, the authors consider that fair participation by all groups concerned is necessary to ensure that the different moral systems that can legitimately exist alongside each other should also be incorporated in the process. to illustrate the concept of analytic-deliberative decision making consider a set of alternative options or choices, from which follow consequences (see basic overview in dodgson et al. 2000) . the relationship between the choice made, and the consequences that follow from this choice, may be straightforward or complex. the science supporting environmental policy is often complicated, across many disciplines of science and engineering, and also involving human institutions and economic interactions. because of limitations in scientific understanding and predictive capabilities, the consequences following a choice are normally uncertain. finally, different individuals and groups within society may not agree on how to evaluate the consequences -which may involve a detailed characterization of what happens in ecological, economic, and human health terms. we shall describe consequences as ambiguous when there is this difficulty in getting agreement on how to interpret and evaluate them. this distinction has been further explained in chapter 3 (see also klinke and renn 2002) . environmental assessment and environmental decision making inherently involve these difficulties of complexity, uncertainty, and ambiguity (klinke and renn 2002) . in some situations where there is lots of experience, these difficulties may be minimal. but in other situations these difficulties may constitute major impediments to the decision-making process. to understand how analysis and deliberation interact in an iterative process following the national research council (nrc) 1996 report, one must consider how these three areas of potential difficulty can be addressed. it is useful to separate questions of evidence with respect to the likelihood, magnitude of consequences and related characteristics (which can involve complexity and uncertainty) from valuation of the consequences (i.e. ambiguity). for each of the three areas there are analytical tools that can be helpful in identifying, characterizing and quantifying cause-effect relationships. some of these tools have been described in chapter 3. the integration of these tools of risk governance into a consistent procedure will be discussed in the next subsections. the possibility to reach closure on evaluating risks to human health or the environment rests on two conditions: first, all participants need to achieve closure on the underlying goal (often legally prescribed, such as prevention of health detriments or guarantee of an undisturbed environmental quality, for example, purity laws for drinking water); secondly, they need to agree with the implications derived from the present state of knowledge (whether and to what degree the identified hazard impacts the desired goal). dissent can result from conflicting values as well as conflicting evidence. it is crucial in environmental risk management to investigate both sides of the coin: the values that govern the selection of the goal and the evidence that governs the selection of cause-effect claims. strong differences in both areas can be expected in most environmental decision-making contexts but also in occupational health and safety and public health risks. so for all risk areas it is necessary to explore why people disagree about what to do -that is, which decision alternative should be selected. as pointed out before, differences of opinion may be focused on the evidence of what is at stake or which option has what kind of consequences. for example: what is the evidence that an environmental management initiative will lead to an improvement, such as reducing losses of agricultural crops to insect pests -and what is the evidence that the management initiative could lead to ecological damage -loss of insects we value, such as bees or butterflies, damage to birds and other predators that feed on insects -and health impacts from the level of pesticides and important nutrients in the food crops we eat? other differences of opinion may be about values -value of food crops that contain less pesticide residue compared to those that contain more, value of having more bees or butterflies, value of maintaining indigenous species of bees or butterflies compared to other varieties not native to the local ecosystem, value ascribed to good health and nutrition, and maybe, value ascribed to having food in what is perceived to be a "natural" state as opposed to containing manufactured chemical pesticides or altered genetic material. separating the science issues of what will happen from the value issues of how to make appropriate trade-offs between ecological, economic, and human health goals can become very difficult. the separation of facts and values in decision making is difficult to accomplish in practical decision situations, since what is regarded as facts includes a preference dependent process of cognitive framing (tversky and kahneman 1981) and what is regarded as value includes a prior knowledge about the factual implica-tions of different value preferences (fischhoff 1975) . furthermore, there are serious objections against a clear-cut division from a sociological view on science and knowledge generation (jasanoff 1996) . particularly when calculating risk estimates, value-based conventions may enter the assessment process. for example, conservative assumptions may be built into the assessment process, so that some adverse effects (such as human cancer from pesticide exposure) are much less likely to be underestimated than overestimated (national research council 1983) . at the same time, ignoring major sources of uncertainty can evoke a sense of security and overconfidence that is not justified from the quality or extent of the data base (einhorn and hogarth 1978) . perceptions and world views may be very important, and difficult to sort out from matters of science, especially with large uncertainties about the causes of environmental damage. a combination of analytic and deliberative processes can help explore these differences of opinions relating to complexity, uncertainty, and ambiguity in order to examine the appropriate basis for a decision before the decision is made. most environmental agencies go through an environmental assessment process and provide opportunities for public review and comment. many controversial environmental decisions become the focus of large analytical efforts, in which mathematical models are used to predict the environmental, economic, and health consequences of environmental management alternatives. analysis should be seen as an indispensable complement to deliberative processes, regardless whether this analysis is sophisticated or not. even simple questions need analytic input for making prudent decisions, especially in situations where there is controversy arising from complexity, uncertainty, and ambiguity. in many policy arenas in which problems of structuring human decisions are relevant, the tools of normative decision analysis (da) have been applied. especially in economics, sociology, philosophical ethics, and also many branches of engineering and science, these methods have been extended and refined during the past several decades. (edwards 1954 , howard 1966 , north 1968 , howard et al. 1972 , north and merkhofer 1976 , behn and vaupel 1982 , pinkau and renn 1998 , van asselt 2000 , jaeger et al. 2001 . da is a process for decomposing a decision problem into pieces, starting with the simple structure of alternatives, information, and prefer-ences. it provides a formal framework for quantitative evaluation of alternative choices in terms of what is known about the consequences and how the consequences are valued (hammond et al. 1999 , skinner 1999 . the procedures and analytical tools of da provide a number of possibilities to improve the precision and transparency of the decision procedure. however, they are subject to a number of limitations. the opportunities refer to: • different action alternatives can be quantitatively evaluated to allow selection of a best choice. such evaluation relies both on a description of uncertain consequences for each action alternative, with uncertainty in the consequences described using probabilities, and a description of the values and preferences assigned to consequences. (explicit characterization of uncertainty and values of consequences) • the opportunity to assure transparency, in that (1) models and data summarizing complexity (e.g., applicable and available scientific evidence) (2) probabilities characterizing judgement about uncertainty, and (3) values (utilities) on the consequences are made explicit and available. so the evaluation of risk handling alternatives can be viewed and checked for accuracy by outside observers. (outside audit enabled of basis for decision) • a complex decision situation can be decomposed into smaller pieces in a formal analytical framework. the level of such composition can range from a decision tree of action alternatives and ensuing consequences that fits on a single piece of paper, to extremely large and complex computerimplemented models used in calculating environmental consequences and ascribing probabilities and values of the consequences. a more complex analysis is more expensive and is less transparent to observers. in principle, with sufficient effort any formal analytical framework can be checked to assure that calculations are made in the way that is intended. (decomposition possible to include extensive detail) on the other hand, there are important limitations: • placing value judgements (utilities) on consequences may be difficult, especially in a political context where loss of life, impairment of health, ecological damage, or similar social consequences are involved. utility theory is essentially an extension of cost-benefit methods from economics to include attitude toward risk. the basic trade-off judgements needed for cost-benefit analysis remain difficult and controversial, and often, inherently subjective. (difficulties in valuing consequences) • assessing uncertainty in the form of a numerical probability also poses difficulties, especially in situations when there is not a statistical data base on an agreed-on model as the basis for the assessment. (difficulty in quantifying uncertainty, assigning probabilities) • the analytical framework may not be complete. holistic or overarching considerations or important details may have been omitted. (analytical framework incomplete) • da is built upon an axiomatic structure, both for dealing with uncertainty (i.e., the axiomatic foundation of probability theory), and for valuing consequences (i.e., the axiomatic basis for von neumann-morgenstern utility theory). especially when the decision is to be made by a group rather than an individual decision maker, rational preferences for the group consistent with the axioms may not exist (the "impossibility" theorem of arrow, 1951) . so in cases of strong disagreements on objectives or unwillingness to use a rational process, decision analysis methods may not be helpful. decision analytical methods should not be regarded as inherently "mechanical" or "algorithmic", in which analysts obtain a set of "inputs" about uncertainty and valuing consequences, then feed these into a mathematical procedure (possibly implemented in a computer) that produces an "output" of the "best" decision. da can only offer coherent conclusions from the information which the decision maker provides by his/her preferences among consequences and his/her state of information on the occurrence of these consequences. where there is disagreement about the preferences or about the information, da may be used to implore the implications of such disagreement. so in application, there is often a great deal of iteration (sensitivity analysis) to explore how differences in judgement should affect the selection of the best action alternative. da thus merely offers a formal framework that can be effective in helping participants in a decision process to better understand the implications of differing information and judgement about complex and uncertain consequences from the choice among the available action alternatives. insight about which factors are most important in selecting among the alternatives is often the most important output of the process, and it is obtained through extensive and iterative exchange between analysts and the decision makers and stakeholders. the main advantage of the framework is that it is based on logic that is both explicit and checkable -usually facilitated by the use of mathematical models and probability calculations. research on human judgement supports the superiority of such procedures for decomposing complex decision problems and using logic to integrate the pieces, rather than relying on holistic judgement on which of the alternatives is best (this is not only true for individual decisions, see heap et al. 1992 : 36ff., jungermann 1986 ; but also for collective decisions, see heap et al. 1992 : 197ff., pettit 1991 . one should keep in mind, however, that "superior" is measured in accordance with indicator of instrumental rationality, i.e. measuring means-ends effectiveness. if this rationality is appropriate, the sequence suggested by da is intrinsically plausible and obvious. even at the level of qualitative discussion and debate, groups often explore the rationale for different action alternatives. decision analysis simply uses formal quantitative methods for this traditional and common-sense process of exploring the rationale -using models to describe complexity, probability to describe uncertainty, and to deal with ambiguity, explicit valuation of consequences via utility theory and other balancing procedures, such as cost-benefit or cost-effectiveness analyses. by decomposing the problem in logical steps, the analysis permits better understanding of differences in the participants' perspective on evidence and values. da offers methods to overcome these differences, such as resolving questions about underlying science through data collection and research, and encouraging tradeoffs, compromise, and rethinking of values. based on this review of opportunities and shortcomings we conclude that decision analysis provides a suitable structure for guiding discussion and problem formulation, and offers a set of quantitative analytical tools that can be useful for environmental decisions, especially in conjunction with deliberative processes. da can assist decision makers and others involved in, and potentially affected by, the decision (i.e., participants, stakeholders) to deal with complexity and many components of uncertainty, and to address issues of remaining uncertainties and ambiguities. using these methods promises consistency from one decision situation to another, assurance of an appropriate use of evidence from scientific studies related to the environment, and explicit accountability and transparency with respect to those institutionally responsible for the value judgements that drive the evaluation of the alternative choices. collectively the analytical tools provide a framework for a systematic process of exploring and evaluating the decision alternatives -assembling and validating the applicable scientific evidence relevant to what will happen as the result of each possible choice, and valuing how bad or how good these consequences are based on an agreement of common objectives. yet, it does not replace the need for additional methods and processes for including other objectives, such as finding common goals, defining preferences, revisiting assumptions, sharing visions and exploring common grounds for values and normative positions. the value judgements motivating decisions are made explicit and can then be criticized by those who were not involved in the process. to the extent that uncertainty becomes important, it will be helpful to deal with uncertainty in an orderly and consistent way (morgan and henrion 1990). those aspects of uncertainty that can be modelled by using probability theory (inter-target variation, systematic and random errors in applying inferential statistics, model and data uncertainties) will be spelled out and those that remain in forms of indeterminacies, system boundaries or plain ignorance will become visible and can then be fed into the deliberation process (van asselt 2000, klinke and renn 2002) . the term deliberation refers to the style and procedure of decision making without specifying which participants are invited to deliberate (national research council (nrc) 1996, rossi 1997) . for a discussion to be called deliberative it is essential that it relies on mutual exchange of arguments and reflections rather than decision making based on the status of the participants, sublime strategies of persuasion, or social-political pressure. deliberative processes should include a debate about the relative weight of each argument and a transparent procedure for balancing pros and cons (tuler and webler 1999) . in addition, deliberative processes should be governed by the established rules of a rational discourse. in the theory of communicative action developed by the german philosopher jürgen habermas, the term discourse denotes a special form of a dialogue, in which all affected parties have equal rights and duties to present claims and test their validity in a context free of social or political domination (habermas 1970 (habermas , 1987b . a discourse is called rational if it meets the following specific requirements (see mccarthy 1975 , habermas 1987a , kemp 1985 , webler 1995 . all participants are obliged: • to seek a consensus on the procedure that they want to employ in order to derive the final decision or compromise, such as voting, sorting of positions, consensual decision making or the involvement of a mediator or arbitrator; • to articulate and criticize factual claims on the basis of the "state of the art" of scientific knowledge and other forms of problem-adequate knowledge; (in the case of dissent all relevant camps have the right to be represented); • to interpret factual evidence in accordance with the laws of formal logic and analytical reasoning; • to disclose their relevant values and preferences, thus avoiding hidden agendas and strategic game playing; and • to process data, arguments and evaluations in a structured format (for example a decision-analytic procedure) so that norms of procedural rationality are met and transparency can be created. the rules of deliberation do not necessarily include the demand for stakeholder or public involvement. deliberation can be organized in closed circles (such as conferences of catholic bishops, where the term has indeed been used since the council of nicosia), as well as in public forums. it may be wise to use the term "deliberative democracy" when one refers to the combination of deliberation and public or stakeholder involvement (see also cohen 1997 , rossi 1997 . what needs to be deliberated? firstly, deliberative processes are needed to define the role and relevance of systematic and anecdotal knowledge for making far-reaching choices. secondly, deliberation is needed to find the most appropriate way to deal with uncertainty in environmental decision making and to set efficient and fair trade-offs between potential over-and under-protection. thirdly, deliberation needs to address the wider concerns of the affected groups and the public at large. why can one expect that deliberative processes are better suited to deal with environmental challenges than using expert judgement, political majority votes or relying on public survey data? • deliberation can produce common understanding of the issues or the problems based on the joint learning experience of the participants with respect to systematic and anecdotal knowledge (webler and renn 1995 , pidgeon 1997 ). • deliberation can produce a common understanding of each party's position and argumentation and thus assist in a mental reconstruction of each actor's argumentation (warren 1993 , tuler 1996 . the main driver for gaining mutual understanding is empathy. the theory of communicative action provides further insights in how to mobilize empathy and how to use the mechanisms of empathy and normative reasoning to explore and generate common moral grounds (webler 1995). • deliberation can produce new options and novel solutions to a problem. this creative process can either be mobilized by finding win-win solutions or by discovering identical moral grounds on which new options can grow (renn 1999) . • deliberation has the potential to show and document the full scope of ambiguity associated with environmental problems. deliberation helps to make a society aware of the options, interpretations, and potential actions that are connected with the issue under investigation (wynne 1992, de marchi and ravetz 1999) . each position within a deliberative discourse can only survive the crossfire of arguments and counter-arguments if it demonstrates internal consistency, compatibility with the legitimate range of knowledge claims and correspondence with the widely accepted norms and values of society. deliberation clarifies the problem, makes people aware of framing effects, and determines the limits of what could be called reasonable within the plurality of interpretations (skillington 1997) . • deliberations can also produce agreements. the minimal agreement may be a consensus about dissent (raiffa 1994) . if all arguments are exchanged, participants know why they disagree. they may not be convinced that the arguments of the other side are true or morally strong enough to change their own position; but they understand the reasons why the opponents came to their conclusion. in the end, the deliberative process produces several consistent and -in their own domain -optimized positions that can be offered as package options to legal decision makers or the public. once these options have been subjected to public discourse and debate, political bodies, such as agencies or parliaments can make the final selection in accordance with the legitimate rules and institutional arrangements such as majority vote or executive order. final selections could also be performed by popular vote or referendum. • deliberation may result in consensus. often deliberative processes are used synonymously with consensus-seeking activities (coglianese 1997 ). this is a major misunderstanding. consensus is a possible outcome of deliberation, but not a mandatory requirement. if all participants find a new option that they all value more than the one option that they preferred when entering the deliberation, a "true" consensus is reached (renn 1999) . it is clear that finding such a consensus is the exception rather than the rule. consensus is either based on a win-win solution or a solution that serves the "common good" and each participant's interests and values better than any other solution. less stringent is the requirement of a tolerated consensus. such a consensus rests on the recognition that the selected decision option might serve the "common good" best, but on the expense of some interest violations or additional costs. in a tolerated consensus some participants voluntarily accept personal or group-specific losses in exchange for providing benefits to all of society. case studies have provided sufficient evidence that deliberation has produced a tolerated consensus solution, particularly in siting conflicts (one example in schneider et al. 1998) . consensus and tolerated consensus should be distinguished from compromise. a compromise is a product of bargaining where each side gradually reduces its claim to the opposing party until they reach an agreement (raiffa 1994) . all parties involved would rather choose the option that they preferred before starting deliberations, but since they cannot find a win-win situation or a morally superior alternative they look for a solution that they can "live with" knowing that it is the second or third best solution for them. compromising on an issue relies on full representation of all vested interests. in summary, many desirable products and accomplishments are associated with deliberation (chess et al. 1998) . depending on the structure of the discourse and the underlying rationale deliberative processes can: • enhance understanding; • generate new options; • decrease hostility and aggressive attitudes among the participants; • explore new problem framing; • enlighten legal policy-makers; • produce competent, fair and optimized solution packages; and • facilitate consensus, tolerated consensus and compromise. in a deliberative setting, participants exchange arguments, provide evidence for their claims and develop common criteria for balancing pros and cons. this task can be facilitated and often guided by using decision analytic tools (overview in merkhofer 1984) . decision theory provides a logical framework distinguishing action alternatives or options, consequences, likelihood of consequences, and value of consequences, where the valuation can be over multiple attributes that are weighted based on tradeoffs in multi-attribute utility analysis (edwards 1977) . a sequence of decisions and consequences may be considered, and use of mathematical models for predicting the environmental consequences of options may or may not be part of the process (humphreys 1977 , bardach 1996 , arvai et al. 2001 ): a) the structuring potential of decision analysis has been used in many participatory processes. it helps the facilitator of such processes to focus on one element during the deliberation, to sort out the central from the peripheral elements, provide a consistent reference structure for ordering arguments and observations and to synthesize multiple impressions, observations and arguments into a coherent framework. the structuring power of decision analysis has often been used without expanding the analysis into quantitative modelling. b) the second potential, agenda setting and sequencing, is also frequently applied in participatory settings. it often makes sense to start with problem definition, then develop the criteria for evaluation, generate options, assess consequences of options, and so on. c) the third potential, quantifying consequences, probabilities and relative weights and calculating expected utilities, is more controversial than the other two. whether the deliberative process should include a numerical analysis of utilities or engage the participants in a quantitative elicitation process is contested among participation practitioners . one side claims that quantifying helps participants to be more precise about their judgements and to be aware of the often painful trade-offs they are forced to make. in addition, quantification can make judgements more transparent to outside observers. the other side claims that quantification restricts the participants to the logic of numbers and reduces the complexity of argumentation into a mere trade-off game. many philosophers argue that quantification supports the illusion that all values can be traded off against other values and that complex problems can be reduced to simple linear combinations of utilities. one possible compromise between the two camps may be to have participants go through the quantification exercise as a means to help them clarify their thoughts and preferences, but make the final decisions on the basis of holistic judgements (renn 1986) . in this application of decision analytic procedures, the numerical results (i.e. for each option the sum over the utilities of each dimension multiplied by the weight of each dimension) of the decision process are not used as expression of the final judgement of the participant, but as a structuring aid to improve the participant's holistic, intuitive judgement. by pointing out potential discrepancies between the numerical model and the holistic judgements, the participants are forced to reflect upon their opinions and search for potential hidden motives or values that might explain the discrepancy. in a situation of major value conflicts, the deliberation process may involve soliciting a diverse set of viewpoints, and judgements need to be made on what sources of information are viewed as responsible and reliable. publication in scientific journals and peer review from scientists outside the government agency are the two most popular methods by which managers or organizers of deliberative processes try to limit what will be considered as acceptable evidence. other methods are to reach a consensus among the participants up front which expertise should be included in the deliberation or to appoint representatives of opposing science camps to explain their differences in public. in many cases, participants have strong reasons for questioning scientific orthodoxy and would like to have different science camps represented. many stakeholders in environmental decisions have access to expert scientists, and often such scientists will take leading roles in criticizing agency science. such discussions need to be managed so that disagreements among the scientific experts can be evaluated in terms of the validity of the evidence presented and the importance to the decision. it is essential in these situations to have a process in place that distinguishes between those evidence claims that all parties agree on, those where the factual base is shared but not its meaning for some quality criterion (such as "healthy" environment), and those where even the factual base is contested (foster 2002) . in the course of practical risk management different conflicts arise in deliberative settings that have to be dealt with in different ways. the main conflicts occur at the process level (how should the negotiations be conducted?), on the cognitive level (what is factually correct?), the interest level (what benefits me?), the value level (what is needed for a "good" life?) and the normative level (what can i expect of all involved?). these different conflict levels are addressed in this subsection. first of all, negotiations begin by specifying the method that structures the dialogue and the rights and duties of all participants. it is the task of the chairman or organizer to present and justify the implicit rules of the talks and negotiations. above and beyond this, the participants have to specify joint rules for decisions, the agenda, the role of the chairman, the order of hearings, etc. this should always be done according to the consensus principle. all partners in the negotiations have to be able to agree to the method. if no agreement is reached here the negotiations have to be interrupted or reorganized. once the negotiation method has been determined and, in a first stage, the values, standards and objectives needed for judgement have been agreed jointly, then follows the exchange of arguments and counter arguments. in accordance with decision theory, four stages of validation occur: • in a first stage, the values and standards accepted by the participants are translated into criteria and then into indicators (measurement instructions). this translation needs the consensual agreement of all participants. experts are asked to assess the available options with regard to each indicator according to the best of their knowledge (factual correctness). in this context it makes more sense to specify a joint methodological procedure or a consensus about the experts to be questioned than to give each group the freedom to have the indicators answered by their own experts. often many potential consequences remain disputed as a result of this process, especially if they are uncertain. however, the bandwidth of possible opinions is more or less restricted depending on the level of certainty and clarity associated with the issue in question. consensus on dissent is also of help here in separating contentious factual claims from undisputed ones and thus promotes further discussion. • in a second stage, all participating parties are required to interpret bandwidths of impacts to be expected for each criterion. interpretation means linking factual statements with values and interests to form a balanced overall judgement (conflicts of interests and values). this judgement can and should be made separately for each indicator. in this way, each of the chains of causes for judgements can be understood better and criticized in the course of the negotiations. for example, the question of trustworthiness of the respective risk management agencies may play an important role in the interpretation of an expected risk value. then it is the duty of the participating parties to scrutinize the previous performance of the authority concerned and propose institutional changes where appropriate. • third stage: even if there were a joint assessment and interpretation for every indicator, this would by no means signify that agreement is at hand. much rather, the participants' different judgements about decisionmaking options may be a result of different value weightings for the indicators that are used as a basis for the values and standards. for example, a committed environmentalist may give much more weight to the indicator for conservation than to the indicator of efficiency. in the literature on game theory, this conflict is considered to be insoluble unless one of the participants can persuade the other to change his preference by means of compensation payments (for example, in the form of special benefits), transfer services (for example, in the form of a special service) or swap transactions (do, ut des). in reality, however, it can be seen that participants in negotiations are definitely open to the arguments of the other participants (i.e. they may renounce their first preference) if the loss of benefit is still tolerable for them and, at the same time, the proposed solution is considered to be "conducive to the common good", i.e. is seen as socially desirable in public perception. if no consensus is reached, a compromise solution can and should be reached, in which a 'fair' distribution of burdens and profits is accomplished. • fourth stage: when weighing up options for action formal methods of balancing assessment can be used. of these methods, the cost-benefit analysis and the multi-attribute or multi-criteria decision have proved their worth. the first method is largely based on the approach of revealed "preferences", i.e. on people's preferences shown in the past expressed in relative prices, the second on the approach of "expressed preferences", i.e. the explicit indication of relative weightings between the various cost and benefit dimensions (fischhoff et al. 1982) . but both methods are only aids in weighing up and cannot replace an ethical reflection of the advantages and disadvantages. normative conflicts pose special problems because different evaluative criteria can always be classified as equally justifiable or unjustifiable as explained earlier. for this reason, most ethicists assume that different types and schools of ethical justification can claim parallel validity, it therefore remains up to the groups involved to choose the type of ethically legitimate justification that they want to use (ropohl 1991 , renn 1997 . nevertheless, the limits of particular justifications are trespassed wherever primary principles accepted by all are infringed (such as human rights). otherwise, standards should be classed as legitimate if they can be defended within the framework of ethical reasoning and if they do not contradict universal standards that are seen as binding for all. in this process conflicts can and will arise, e.g. that legitimate derivations of standards from the perspective of group a contradict the equally legitimate derivations of group b (shrader-frechette 1988). in order to reach a jointly supported selection of standards, either a portfolio of standards that can claim parallel validity should be drawn up or compensation solutions will have to be created in which one party compensates the other for giving up its legitimate options for action in favour of a common option. when choosing possible options for action or standards, options that infringe categorical principles, for example, to endangering the systematic ability of the natural environment to function for human use in the future and thus exceeding the limits of tolerability are not tolerable even if they imply major benefits to society. at the same time, all sub-dominant options have to be excluded. frequently sub-dominant solutions, i.e. those that perform worse than all other options with regard to all criteria at least in the long term, are so attractive because they promise benefits in the short term although they entail losses in the long term, even if high interest rates are assumed. often people or groups have no choice other than to choose the sub-dominant solution because all other options are closed to them due to a lack of resources. if large numbers of groups or many individuals act in this way, global risks become unmanageable (beck 1996) . to avoid these risks intermediate financing or compensation by third parties should be considered. the objective of this last section of chapter 4 was to address and discuss the use of decision analytic tools and structuring aids for participatory processes in environmental management. organizing and structuring discourses goes beyond the good intention to have all relevant stakeholders involved in decision making. the mere desire to initiate a two-way communication process and the willingness to listen to stakeholder concerns are not sufficient. discursive processes need a structure that assures the integration of technical expertise, regulatory requirements, and public values. these different inputs should be combined in such a fashion that they contribute to the deliberation process the type of expertise and knowledge that can claim legitimacy within a rational decision-making procedure (von schomberg 1995). it does not make sense to replace technical expertise with vague public perceptions, nor is it justified to have the experts insert their own value judgements into what ought to be a democratic process. decision analytic tools can be of great value for structuring participatory processes. they can provide assistance in problem structuring, in dealing with complex scientific issues and uncertainty, and in helping a diverse group to understand disagreements and ambiguity with respect to values and preferences. decision analysis tools should be used with care. they do not provide an algorithm to reach an answer as to what is the best decision. rather, decision analysis is a formal framework that can be used for environmental assessment and risk handling to explore difficult issues, to focus debate and further analysis on the factors most important to the decision, and to provide for increased transparency and more effective exchange of information and opinions among the process participants. the basic concepts are relatively simple and can be implemented with a minimum of mathematics (hammond et al. 1999) . many participation organizers have restricted the use of decision analytic tools to assist participants in structuring problems and ordering concerns and evaluations, and have refrained from going further into quantitative trade-off analysis. others have advocated quantitative modelling as a clarification tool for making value conflicts more transparent to the participants. the full power of decision analysis for complex environmental problem may require mathematical models and probability assessment. experienced analysts may be needed to guide the implementation of these analytical tools for aiding decisions. skilled communicators and facilitators may be needed to achieve effective interaction between analysts and participants in the deliberative process whose exposure to advanced analytical decision aids is much less, so that understanding of both process and substance, and therefore transparency and trust, can be achieved. many risk management agencies are already making use of decision analysis tools. we urge them to use these tools in the context of an iterative, deliberative process with broad participation by the interested and affected parties to the decision in the context of the risk governance framework. the analytical methods, the data and judgement, and the assumptions, as well as the analytical results should be readily available and understood by the participants. we believe that both the risk management agencies and the interested groups within the public that government agencies interact with on environmental decisions should all gain experience with these methods. improper or premature use of sophisticated analytical methods may be more destructive to trust and understanding than helpful in resolving the difficulties of complexity, uncertainty, and ambiguity. bioterrorism, preparedness, attack and response. elsevier beyond the job exposure matrix (jem): the task exposure matrix (tem) chapter 4 occupational safety and health and environmental risks a task exposure database for use in the alumina and primary aluminium industry work-related neck and upper limb musculoskeletal disorders. european agency for safety and health at work finnish national job-exposure matrix (finjem) in register-based cancer research. people and work toimivat ja terveelliset työajat (well-functioning and healthy working times). finnish institute of occupational health, ministry of social affairs and health and ministry of labour finnish database of occupational exposure measurements (fdoem). finnish institute of occupational health, finland. ioha, pilanesberg introduction to occupational epidemiology the environment and disease: association or causation? the bradford hill considerations on causality: a counterfactual perspective work-related stress. european foundation for the improvement of living and working conditions the ageing workforce -challenges for occupational health recommendation concerning the list of occupational diseases and the recording and notification of occupational accidents and diseases: r 194. international labour conference. international labour office työuupumus suomen työikäisellä väestöllä (burn-out among the working-aged population in finland). finnish institute of occupational health, helsinki work-related disease risk in different occupations. työterveiset 3, special issue. information newsletter of the finnish institute of occupational health ammattitaudit 2000 (occupational diseases 2000). finnish institute of occupational health work is related to a substantial portion of adult-onset asthma incidence in the finnish population work stress in the etiology of coronary heart disease -a meta-analysis occupational injury and illness in the united states. estimates of costs, morbidity, and mortality the psychology of extremism and terrorism: a middle-eastern perspective musculoskeletal disorders and the workplace: low back and upper extremities testing a structured decision approach: value focused thinking for deliberative risk communication the eight-step path of policy analysis the invention of nature ökologische fragen im bezugsrahmen fabrizierter unsicherheiten quick analysis for busy decision makers grundzüge der ökologischen ethik natur" als maßstab menschlichen handelns. zeitschrift für philosophische forschung landschaftsschutz und artenschutz. wie weit tragen utilitaristische begründungen? in: nutzinger hg vorsorge statt nachhaltigkeit -ethische grundlagen der zukunftsverantwortung on the intrinsic value of nonhuman species who should deliberate when? assessing consensus; the promise and performance of negotiated rule making procedure and substance in deliberative democracy risk management and governance: a post-normal science approach deep ecology: living as if nature mattered multi-criteria analysis: a manual. department of the environment, transport and the regions the theory of decision making how to use multi-attribute utility measurement for social decision making beginning again: people and nature in the new millennium confidence in judgment: persistence of the illusion of validity environmental ethics hindsight versus foresight: the effect of outcome knowledge on judgment under uncertainty the experienced utility of expected utility approaches the role of experts in risk-based decision making. hse risk assessment policy unit. web manuscript. www.trustnetgovernance.com/library/pdf/doc2 ökologie und konsensfindung: neue chancen und risiken making sense of sustainability: nine answers to "what should be sustained? literaturreview und bibliographie. graue reihe nr. 12. europäische akademie zur erforschung von folgen wissenschaftlich-technischer entwicklungen ethische aspekte des handelns unter risiko rationale technikfolgenbeurteilung the value of biodiversity decision aiding, not dispute resolution: a new perspective for environmental negotiation towards a theory of communicative competence vorbereitende bemerkungen zu einer theorie der kommunikativen kompetenz theorie des kommunikativen handelns. suhrkamp, frankfurt/m the philosophical discourse of modernity smart choices: a practical guide to making better decisions marketing und soziale verantwortung foundations of environmental ethics systemsteuerung und "staatskunst": theoretische konzepte und empirische befunde. leske und budrich the theory of choice: a practical guide politische gerechtigkeit. grundlegung einer kritischen philosophie von recht und staat. suhrkamp, frankfurt/m welche natur sollen wir schützen? gaia philosophie der ökologischen krise decision analysis: applied decision theory the foundations of decision analysis the decision to seed hurricanes application of multi-attribute utility theory risk governance: towards an integrative appraoch. irgc beyond epistemology: relativism and engagement in the politics of science risk, uncertainty and rational action das prinzip verantwortung. versuch einer ethik für die technologische zivilisation judgment and decision making: an interdisciplinary reader planning, political hearings, and the politics of discourse ökologie global: die auswirkungen von wirtschaftswachstum, bevölkerungswachstum und zunehmendem nord-süd-gefälle auf die umwelt a new approach to risk evaluation and management: risk-based, precaution-based and discourse-based management naturethik im überblick the science of muddling through the intelligence of democracy. decision making through mutual adjustment legitimation durch verfahren. suhrkamp, frankfurt/m translator's introduction comparative analysis of formal decision-making approaches sustainable development? wie nicht nur die menschen eine "dauerhafte" entwicklung überdauern können ist biologisches produzieren natürlich? leitbilder einer naturgemäßen technik methodische philosophie umwelt. bemerkungen eines philosophen zum umweltverträglichen wirtschaften committee on institutional means for assessing risk to public health (1983) risk assessment in the federal government: managing the process ethik des risikos angewandte ethik. die bereichsethiken und ihre theoretische fundierung a tutorial introduction to decision theory a methodology for analyzing emission control strategies why preserve natural variety? ökologie und ethik. ein versuch praktischer philosophie. ethik in den wissenschaften zur ethischen bewertung von biodiversität. externes gutachten für den wbgu. unveröffentlichtes manuskript decision theory and folk psychology towards a comprehensive conservation theory the limits to safety? culture, politics, learning and manmade disasters environmental standards. scientific foundations and rational procedures of regulation with emphasis on radiological risk management the art and science of negotiation what mainstream economists have to say about the value of biodiversity benefits, costs, and the safe minimum standard of conservation decision analytic tools for resolving uncertainty in the energy debate ökologisch denken -sozial handeln: die realisierbarkeit einer nachhaltigen entwicklung und die rolle der sozial-und kulturwissenschaften ein diskursives verfahren zur bildung und begründung kollektiv verbindlicher bewertungskriterien a model for an analytic-deliberative process in risk management the challenge of integrating deliberation and expertise: participation and discourse in risk management a regional concept of qualitative growth and sustainability -support for a case study in the german state of baden-württemberg was heißt hier bioethik? tab-brief theologie der natur und ihre anthriopologisch-ethischen konsequenzen values in nature and the nature of value ob man die ambivalenzen des technischen fortschritts mit einer neuen ethik meistern kann? participation run amok: the costs of mass participation for deliberative agency decisionmaking ist die schöpfung noch zu retten? umweltkrise und christliche verantwortung experiences from germany: application of a structured model of public participation in waste management planning environmental ethics politics and the struggle to define: a discourse analysis of the framing strategies of competing actors in a "new introduction to decision analysis corporate environmentalism in a global economy respect for nature. a theory of environmental ethics meanings, understandings, and interpersonal relationships in environmental policy discourse. doctoral dissertation designing an analytic deliberative process for environmental health policy making in the u.s. nuclear weapons complex the framing of decisions and the psychology of choice perspectives on uncertainty and risk akzeptanzbeschaffung: verfahren und verhandlungen the erosion of the valuespheres. the ways in which society copes with scientific, moral and ethical uncertainty can participatory democracy produce better selves? psychological dimensions of habermas discursive model of democracy welt im wandel: erhaltung und nachhaltige nutzung der biosphäre. jahresgutachten 1999 discourse in citizen participation. an evaluative yardstick the craft and theory of public participation: a dialectical process a brief primer on participation: philosophy and practice system, diskurs, didaktik und die diktatur des sitzfleisches konsens als telos der sprachlichen kommunikation? in: giegel biophilia: the human bond with other species rio" oder die moralische verpflichtung zum erhalt der natürlichen vielfalt risk and social learning: reification to engagement die modernisierung der demokratie im zeichen der umweltproblematik wandelt sich die verantwortung mit technischem wandel? key: cord-286438-jz7gktaz authors: scherer, h.; quast, a. title: olfactory system date: 2002-11-02 journal: international encyclopedia of the social & behavioral sciences doi: 10.1016/b0-08-043076-7/03478-1 sha: doc_id: 286438 cord_uid: jz7gktaz there are two different olfactory systems in man: one for self-preservation (classical olfactory system) and one for the propagation of the species (vomeronasal system). both systems have a considerable impact on subcortical centers and particularly on emotional reactions, but only stimulation of the classical olfactory system is consciously perceived. the stimulus threshold of both systems is extremely low, but that of the olfactory system varies strongly with the nutritional status. the anatomy, physiology, and pathophysiology are described. the olfactory system belongs to the chemical senses (smell, taste). we are dealing here with a sensory system whose receptive field is situated in the upper part of the nose and whose information is taken up and transmitted via a cns protrusion, the olfactory nerves, or olfactory bulb. this sensory function essentially serves the purpose of self-preservation by enabling individuals to recognize food, assess its quality, and receive warning signals (burnt smell, poisons). the stimulus threshold is extremely low in humans, and yet humans are poor smellers compared with animals. the stimulus threshold varies considerably according to the nutritional status. the nerve fibers of the olfactory system are spread much wider in the cns than is the case with any other sensory system. the olfactory system exerts an essential part of its influence on subcortical centers like the limbic system. thus, smell has a high emotional component, which accounts for its great social importance. this explains why olfactory impressions are used to express behavior patterns in all languages. in german, for example, the expression ich kann dich nicht riechen (i cannot smell you) means 'i don't like you. ' humans have special inherited and acquired preferences (hedonics). thus, for example, the smell of a forest elicits a positive emotion in many people, whereas negative feelings are associated with the smell of isobutyric acid (sweat). because of the broad spread of the secondary and tertiary olfactory fields in the cns, an odorant can evoke its strong conscious reaction only initially; the sense of smell must then adapt rapidly to be ready for new odorants, and we lose the perception. apart from the olfactory sense for self-preservation, referred to henceforth as the 'classical olfactory system,' the nose has a second chemical sense that long received little attention in humans: the vomeronasal organ or system. its purpose lies in the perception of pheromones within the sexual sphere and thus in the propagation of the species as well as in signaling and controlling aggressions. while hormones distribute messages within the body, pheromones take over communication to the outside (jellinek). little is known about the neuronal pathways and connections of the vomeronasal system. an intensive and exclusively subcortical process is probably involved, which means that we are not consciously aware of the stimulation of this organ. as with the classical olfactory sense, marked hedonics also exist here that are controlled by the immunological system. the zone of smell of the classical olfactory system in the nose lies in the upper part of the nasal cavity. it covers an area of about 5 cm on either side of the nose, specifically in the region of the superior nasal conchae, the septum and the ethmoid bone ( fig. 1) . this region harbors about 100 million bipolar olfactory cells in humans (220 million in dogs). the sensory epithelium also contains stem cells with the lifelong capacity for replacing olfactory cells that have died. the peripheral processes of the bipolar sensory cells rise to the mucosal surface (fig. 2) . they bear up to 1,000 hairs (cilia) on which the primary contact takes place between aromatic molecules and receptors. the hairs differ in length among the various organisms. the longer the hairs, the more points of contact for the aromatic molecules. organisms with long sensory hairs are therefore described as macrosmatic, those with short ones as microsmatic. the hairs must be moistened constantly so as not to be destroyed by desiccation. the olfactory zone thus has special mucous glands which, in contrast to other glands in the nose, do not form proteolytic enzymes. odorants would otherwise be broken down into substances with a different smell prior to contact with the sensory cells. anatomy of the nose with the olfactory zone the aromatic molecules dock at special receptor proteins situated on the hairs. these proteins activate signal proteins (g proteins) which in turn trigger a cascade that sends an electric signal (action potential) along the central process of the cell to the bulb of the olfactory nerve (reed 1992) . this bulb contains cell accumulations (granules), each granule corresponding to a specific receptor molecule (reed 1992) . the expression of the specific olfactory receptor proteins is genetically determined (buck and axel 1991), one gene encoding each receptor molecule. altogether, we possess about 1,000 different olfactory receptors. approximately 1 percent of our genes are used here, which corresponds to the largest family of genes found in humans. this underscores the profound importance of the sense of smell in man (axel 1995) . there are about 30,000 odorants. humans can recognize about 3,000 of these but only differentiate 300. we have a poor memory for smells, a much poorer olfactory than visual memory. thus, we can only remember about 20 odorants. odorants must be volatile and lipid-soluble, otherwise they cannot be perceived. however, perception does not take place solely in the area of the olfactory detail of the olfactory mucosa with ciliated bipolar sensory cells. the cilia differ in length. (a) cilia in microsmatic organisms (for example, humans); (b) cilia in macrosmatic organisms (for example, dogs, eels, salmons) sensory cells. thus, it is possible to distinguish between odorants that excite the olfactory system almost exclusively, for example, coffee, cinnamon, tar, or the scent of roses, and odorants that stimulate both the olfactory system and fibers of the sensory trigeminal nerve in the nose, for example, menthol or camphor (sensation: cool) or formalin and vinegar (sensation: pungent). in addition, there are odorants that stimulate both the olfactory system and taste fibers on the base of the tongue, for example, pyridine (sensation: bitter) and chloroform (sensation: sweet). the vomeronasal organ is situated in the anterior and lower mucosal areas of the nasal septum (fig. 3) . it comprises a 2-8 mm pouch with a diameter of 0.2-2 mm. the numerous mucous glands opening into this pouch ensure constant irrigation. at the end of the pouch there are elongated sensory cells with small mobile saccules resembling handles. numerous blood vessels and nerve fibers are situated basal to this cell layer. the precise spread of these fibers and of the information thus transmitted to the cns is not known. however, stimulation of the vomeronasal organ is not perceived consciously. the vomeronasal organ develops between the 37th and 43rd prenatal day in humans (smith and bhatnagar 2000) . at this time, it is morphologically identical to its counterpart in animals, for which chemosensory activity has been demonstrated. its structure changes between the 12th and 14th week. the mucosal cells develop cilia and lose contact to nerves. thus, there are discussions as to whether the organ loses its function in humans at a very early stage, that is, in infancy. however, some findings (monti-bloch et al. 1998 , grosser et al. 2000 have demonstrated that the vomeronasal organ can be stimulated directly and specifically by a pheromone in adults (see below). odorants of graded concentration (e.g., 'sniffin' sticks, butanol test (gudziol) are inhaled to test perception within the scope of the classical olfactory system (hummel et al. 1997) . the results are based largely on subjective data. since this leaves open the possibility anatomy of the nasal septum of simulation and aggravation, expert opinions always require clarification by taste tests with substances that can only be smelled (e.g., peppermint solution) or by objective examination procedures. in the objective smell test, an odorant is introduced into the nose by pulsed application, and the brain waves are measured via the vertex synchronous to inhalation. the evoked test curve obtained by summation indicates the activation of the olfactory system-but not necessarily the perception of the odorant-in the cerebral cortex. moreover, no qualitative statements can be made on the question of whether an odorant is perceived as such. very complex apparatuses were used to investigate the threshold of the classical olfactory sensation. it is 1-2i10 − "% mol\l in humans and 2i10 − "' mol\l in eels. from the fact that only 10 molecules excite a receptor cell and 40 trigger a smell perception, it may be calculated that the olfactory system of the eel will be stimulated by even a thimbleful of rosewater poured into 16 times the amount of water in lake constance and well distributed (jung 1994) . this good olfactory sense of fish surely also explains why a salmon usually finds his home river. it probably smells its specific odorants, and this should give us at least some idea of the impact our water pollution has on the olfactory system of fish. measurements have shown that the olfactory threshold changes significantly in relation to the nutritional status. thus, the threshold is 1-2i10 − "% mol\l in a fasting state and rises to 3.5-5.5i10 − "% mol\l after a meal. the biochemical basis for this threshold variation is the sugar content in the blood. sugar is the nutritive substance of the cns. it makes sense for the olfactory threshold to differ in the fasting state: hungry humans and animals require a good olfactory sense for self-preservation, since they must hunt for 'prey' or use the olfactory organ for orientation (salmon fast when returning to their home river; their olfactory sense is thus optimal). one of the secrets of the famous art of modern french cooking (nou elle cuisine) is to maintain the fasting state and thus the good olfactory sense of the guest by serving very small portions in the individual courses. it is especially important to go easy on sweet foods and to serve dry sherry as an aperitif, for example. sweet foods should only be consumed as a dessert. conversely, serving a sweet aperitif, for example, port wine or a rich appetizer can mask the taste of a poorly seasoned dish. there are individuals with a particularly keen olfactory sense. they are usually highly paid for testing wines and other luxury items like coffee or tea or may also work as perfumers. on the other hand, there are people who cannot perceive certain odorants. there are, for example, such specific, genetically determined anosmias for isobutyric acid (sweat), hydrogen cyanide (bitter almond), and the scent of freesias. more than 80 specific anosmias have been identified thus far. contrary to subjective reports by women, no change has been detected in the classical olfactory sense during the menstrual cycle (hummel et al. 1997) . in 1986 national geographic magazine published an article on the sense of smell in humans. for illustration, they included six encapsulated odorants to be identified after being released by rubbing. this evolved into a gigantic global smell test with 1.5 million participants. some of the results are physiologically significant. the test clearly demonstrated, for example, that men have a poorer sense of smell than women (something women have always said). this difference is enhanced in old age. the olfactory sense is best at the age of 20 and declines continuously with age (about 40-50 percent by the age of 80). on the other hand, the ability to smell the scent of a rose hardly decreases at all. roses are, therefore, a suitable present for older people. smokers consider themselves poor smellers and they are quite right. different odorants trigger completely different physical reactions in humans. the scent of jasmine and lemon increases alertness, while that of lavender and roses have a calming effect. a japanese company introduces a lemon scent into their air conditioning system in the morning to increase motivation, the scent of roses at noon to calm tempers, and different wood fragrances in the afternoon to mobilize energy reserves (pm 1\1990). buying behavior can also be stimulated. adding aromatics to the air lengthens customers' shopping time by 16 percent, makes them 15 percent more willing to buy, and increases sales by 6 percent (sto$ hr 1995). there are many secretory glands in the olfactory zone of the nasal mucosa. their primary function is to moisten and thus protect the cilia of the olfactory cells, but they also rinse out odorants. thus, the organ of smell very quickly regains its readiness for a new stimulation. the olfactory sense adapts very quickly though to odorants that are present constantly. in other words, an odorant is no longer perceived as such after a few minutes. a dog that has taken up a trail must, therefore, run in zigzags, since he would otherwise lose the scent by adaptation of his olfactory sense. this also applies to perfume users. a person smelling a perfume will no longer perceive it after a short time if the wearer remains at a constant distance. hedonics are understood to be the assessment of smell sensations. they can be positive or negative but differ from organism to organism and also among members of the same species. thus, the smell of decay is negative for humans but positive for vultures. europeans value the smell of pizza spices more highly than that of soybean oil. the opposite is true for asians. hedonics can be inherited (smell of decay) and acquired and are already passed on during gestation. fetuses are known to react to olfactory stimuli in the mother after the 28th week of pregnancy. thus, newborn rabbits already show a selective preference for the food their mothers preferred. the literature on the anatomy and physiology of the vomeronasal organ in humans is extensive and in part extremely controversial. thus, shoyi et al. found that the vomeronasal system has very high sensitivity to various stimulating substances, though the sensory cells in the nasal pouch do not possess distinct cilia like the olfactory cells of the classic olfactory system. in contrast, smith and bhatnagor (2000) already detected a transformation into ciliated cells in infancy. on the one hand, a physiological receptor function has been negated in man (trotier et al. 2000) . on the other hand, women randomized in a double-blind manner reacted specifically to the male pheromone androstadienone when applied to their vomeronasal organ in picogram quantities. direct application of the female pheromone pregna-4,20-diene-3,6-dione (vomeropherin pdd) to the vomeronasal organ of men elicited a significant and dose-dependent parasympathomimetic reaction and also decreased serum lh and testosterone (monti-bloch et al. 1998 ). the threshold for pheromone detection is extremely low in animals. hence, a male silk moth will find a female even at a distance of 10 km, though not if it is sitting under a glass bell. as mentioned above, the stimulating substances of the vomeronasal system are pheromones, androstenone, and androstenol being among those found in humans. they are formed in the nasolabial groove, the axilla, the mammillae and the sexual organs. we do not perceive consciously their stimulation of the vomeronasal organ. if we think we are smelling pheromones, these are bacterial breakdown products of the respective substances, for which we also develop marked hedonics. famous in this connection is a letter napoleon wrote to his wife, josephine, requesting that she no longer wash herself because he was coming home from the field. pheromones control not only sexual activity but also aggressive behaviors among conspecifics (dulac 2000) . dogs mark their territory, for example. smelling pheromones is particularly important for newborn creatures, especially while still helpless, for example, blind animals. they smell their mother's mammary glands via pheromones. kangaroo babies are very small creatures at birth. guided by pheromones, they find their way unaided to the mother's pouch, where the mammary glands are situated. hedonics for pheromones are controlled genetically (wedekind and penn 2000, wedekind and furi 1997) . they are influenced by the immune system. thus, hedonics for the pheromones of a partner are known to be all the more positive, the greater the difference between the immunocompetence of a couple, as measured by the major histocompatibility complex. this is nature's attempt to prevent marriages between brothers and sisters. hedonics has been found to undergo a reversal in the course of pregnancy, during which time a previously preferred partner is not very attractive. this effect is biologically understandable. the reversal only lasts as long as the pregnancy. however, women taking oral contraceptives also show a reversal of hedonics, and this circumstance harbors an implicit threat to their partnership. normosmia: normal sense of smell anosmia: total absence of the sense of smell dysosmia: partial impairment of the sense of smell (hyperosmia, hyposmia) anosmia and dysosmia: specific, genetic: lack of receptor proteins respiratory: -marked deviations of the nasal septum -turbinate hyperplasia -mucosal swelling (for example, in colds) -nasal polyps -tracheotomized patients epithelial: -drying of the nose (ozena) -exogenous noxae (cadmium, chromic acid, butylene glycol, benzoic acid, hydrogen selenide (sometimes even one breath of a noxa will permanently destroy the sense of smell) -overgrowth of the olfactory epithelium by normal nasal mucosa neural: -virus infections, especially those associated with the common cold (rhinoviruses, coronaviruses, adenoviruses) -traumas in an anteroposterior (for example, boxing) or posteroanterior (falling on the back of the head) direction that shift the brain tangentially to the skull base, rupturing the olfactory nerve fibers at the point where they pass through it (fig. 1) endocrine: -cushing's syndrome, hypothyroidism, diabetes, b12 deficiency central: -traumas, tumors, influenza infection, meningitis parosmia: smelling a substance that is not there; the affected individual knows that the substance smelled is not there cause: often nasal (partial destruction of olfactory epithelium) but also michels' classic (1959) can be read as an extended argument that continually returns in various forms to the following syllogism: (a) strong organization is necessary to achieve collective democratic goals, (b) strong organization requires delegation of popular sovereignty to an autonomous and self-perpetuating leadership cadre, and, (c) an autonomous and self-perpetuating leadership cadre is incompatible with true democracy. the paradox in the syllogism is evident: organizations whose professed goals are democratic cannot themselves be democratic. michels' argument was strongly influenced by his friend and mentor max weber's perspective on bureaucracy (weber 1978). following weber, michels argued that the differentiation of an organizational elite from the rank-and-file was a product of the technical division of labor necessary in complex mass organizations. weber's pessimistic analysis of the 'iron cage' of modern rationality was reflected in michels' own pessimism about the dilemma faced by the party rank-and-file: the benefits of bureaucratic organization were manifold, but could only be realized by forsaking popular sovereignty. weber also argued that the 'routinization of charisma' that led to modern bureaucratic organization was driven by the concerns of the administrative staff to secure and stabilize their position. in michels' argument, the conservative tendency of organizational elites stems from their concern to maintain their status and livelihood. as in weber, michels' argument about mass bureaucratic organization has two elements which coexist in some degree of tension. on the one hand, the rise of bureaucracy is driven by the demand for services provided by a stunningly efficient organizational machine; on the other hand, bureaucracy is a mech-anism of elite domination (kitschelt 1989, pp. 69-70 distinguishes between a 'weak' theory of oligarchy based on organizational efficiency and a 'strong' theory based on elite domination). thus, michels moves back and forth from arguing that oligarchy is explained by the masses' adoration of and gratitude towards their leaders to arguing that it is explained by elites' ability to co-opt dissent through control over patronage and the media. the tension can be highlighted as follows: despite the leaders' subversion of the revolutionary goals and democratic aspirations of the masses, the masses remain grateful and obedient. to explain this tension, michels turned to another source of intellectual influence: a conservative view of the masses associated with the italian elite theorists vilfredo pareto and gaetano mosca and the french sociologist gustave le bon. pareto and mosca drew a sharp distinction between the elites and the masses and argued that the competence and energy of the elites made it possible for them to rule the unenterprising masses. influenced by le bon's study of the irrationality of crowds, michels argued that the masses are weakly organized and hence politically immature and even incompetent. unable to organize themselves, the masses have a strong need for leadership. the relatively strong emphasis on psychological factors in political parties-such as the argument that the masses have 'a need to pay adoring worship' to their leaders -seems to derive from le bon (though we cannot forget weber's analysis of charisma). the psychological distance between elites and masses leads the rank-and-file to behave in a deferential way towards their leaders. while elite theory is often seen as a rejection of the marxist analysis of class struggle-and michels certainly used his analysis to deflate the optimism of marxism-elements of a marxian analysis can also be seen in political parties. at numerous points, michels suggests that the organizational asymmetry between elites and masses is akin to a class struggle. with their monopoly over the means of production (organization), the elite is essentially able to extract surplus rents from the social movement. he analyzes the organizational differentiation of elites and masses as a process of embourgeoisement. even (or especially) leaders of proletarian origin become culturally, educationally, and financially differentiated from their original social class and consequently lose all fraternal feeling towards the working class. michels' articulation of the iron law of oligarchy has had a broad impact on subsequent studies of political parties, social movements, and organizations. nevertheless, the basic thrust of this subsequent literature has been to qualify and nuance michels' portrait of the distribution of power in organizations. one of the first important studies to follow up on the molecular logic of smell r 1991 a novel multigene family may encode odorant receptors: a molecular basis for odor recognition sensory coding of pheromone signals in mammals behavioral and electrophysiological effects of androstadienone, a human pheromone sniffin' sticks': olfactory performance assessed by the combined testing of odor identification, odor discrimination and olfactory threshold ein durchbruch in der parfuemerie? dragoco report 1\99 modulation of serum testosterone and autonomic function through stimulation of the male human vomeronasal organ (vno) with pregna-4,20-diene-3,6-dione signaling pathways in odorant detection key: cord-356062-7q5n4t97 authors: nan title: cumulative pharmacological activity index volumes 1-30 date: 2005-12-31 journal: studies in natural products chemistry doi: 10.1016/s1572-5995(05)80101-2 sha: doc_id: 356062 cord_uid: 7q5n4t97 publisher summary this chapter lists the important subjects on pharmacological activity that are discussed in the publication studies in natural products chemistry, volumes 1–30, such as abdominal constriction test, acanthoic acid, acetaminophen, parkinson's disease, photodynamic activity, prostaglandins, and oleanolic acid. the terms are mentioned along with the page numbers in which they are discussed in the publication. use of psycotria colorata in 30:205 19(4--+ 3)-abeo-8 c~, 13(s)-epoxylabda-4(18), 14-diene 29:102 activity in ebv bioassay system 29:102 19-nor-abieta-4( 18),8,11,13-tetraen-7one 29:100 activity in ebv assay system 29:100 abieta-8,11,13-trien-7-one 29:99 activity in ebv assay system 29:99 18-nor11, 7a, 15triol 29:99 activity in ebv assay system 29:99 11, 15, activity in ebv assay system 29:99 abieta-8,11,13-triene-7o~,l 5,18-triol 7acetate 29:99 activity in ebv assay system 29:99 abietic acid 29:99 activity in tpa assay system 29:99 activity in crg (+)-5-epi-acetomycin 10:447 antitumor activity of 10:447 2(x-acetoxy-14-hydroxy-15-iso-valeroyloxy-9-oxo-costunolide 29:89 activity in nfkb assay system 29:89 activity in tnf(x assay system 29:89 9cx-acetoxy-4 ~, 15-epoxymiller-1 ( 10)zenolide 29:89 activity in nfkb assay system 29:89 3~-acetoxy-813-isobutyryloxy-reynosin 29:87 activity in mam-2 assay system 29:87 activity in dif assay system 29:87 15-acetoxy-9 ~-hydroxy-8 [3-methacryloyloxy-14-oxo-acanthospermolide 29:89 activity in nfkb assay system 29:89 15-acetoxy-9cx-methacryloyloxy-8 ~hydroxy-14-oxo-acantho-spermolide 29:89 activity in nfkb assay system 29:89 3-acetoxycostunolide 29:89 activity in nfkb assay system 29:89 15-acetoxy-eremantholide b 29:89 activity in nfkb assay system 29:89 activity in tnf~ assay system 29:89 9~-acetoxy-miller-1 ( 10) 30:191, 192, 203 development of 30:192, 203 use of 30:191 analgesic effects 26:401, 403, 404, 406, 416, 418; 30:205, 208 5:512,747;10:78, 117;11:134;12:398;14:145;20:245, 712 ;21:21,108,109,190,207,213,214, 230,237,262,266,267,275,284,601, 615 ;23:41,126,164,235,258,355,468, 375,472,473,531,534,800;24:573, 856,857;26:226,1002;28:140,688; 30:328 11:130;12:192; 17:283 ;21:188,202,223,239,364,437; 23:649;24:953-954 21:186,192,197, 203,206,213,214,218,221,226, 227,233-235,223,238,240,389, 391,399,404,405,435,599-662; 22:626;23:3,4,271,341,344,348, 468,473,798,801354,368 ;26:227, 228,553,1098;28:4;66,473,475,686; 30:327,738,629 against candida albicans 30:327,629 from hypericum roeperanum 30:629 of (e)-8(17), 12-1abdadiene-15,16-dial 23:798 of (e)-8b, 17-epoxylabd-12-ene-15,16-deol 23:798 of (e)-8 [3,17-epoxylabol-12-ene-15,1619:511,748;21:132, 133,149,252,269,664,665 ;23:316, 533,534,687,704 ;24:742,743 ;30:226, 396,397 13:647;17:137,376;18:775;21:130, 134,153,152,312,591,594,690,616, 656,657;22:93,119,307,345,361; 23:158,297,512,602,832;25:43; 26:340,341,343,391,398,400,401, 403,404,406,415,418,482,556,558, 741,759,1035 ;27:660,858 ;28:200, 257,293 ;671,844;30:206,207,224, 225,292,320,689 21:674,744; 26:229,790,813,887,789,790,791, 800,805,807,810,811,821,828,830 -microbial activity 5:752;10:443; 15:327-339;18:776-778;21:153, 235,257,401,571,573,591,596,597, 599,606;22:626;23:94,153,180,233, 237,249,262,268,267,346,353,522; 24:64,69,206,330,331-334,402,403, 404,406,417,418,443,470,485,859, 1091,1092,1189;26:64,330,401,402, 1189;27:807;28:62,224,257,293; 28:698 22:308,321,345 ;23:395,534,535,742, 772,773,845 ;25:59;30:173,224,225, 289,320,321,524,561,743,745; 26:250,255,509,556,672,763,768, 887,1005,1006;28:4,16,17,63,179, 257,293 1:275,316-320-408; 17:17 ;21:137,405,420,435,436,638, 704,733;22:676;23:74,96,126,132, 139,395,445,522,696,721; 24:272-275 ;24:288,845,847,849; 25:43,430,431,438,450-453;26:56, 482,706;27:803;28:211,517,519,559, 560,566;30:3,28,29,55,56,58,59,64, 65,224,314,395,589,690,777,840 :461,563;10:608, 609,619,620; 17:146 ;21:132,148, 150,393,435,600,615,662,690,718; 22:307,361 ;23:233,268,233,346,395, 473,512,531,703;25:114;26:169,222, 224,226,344,452,741,759;27:482; 28:257,293 ;30:224,324-326,393,394, 395,398,401-405,407,410-412 132,148,162, 223,236,238,263,265,269,284,297, 366,388,390,410,419,421,431,571, 578,591,598,601,603,620,694 ;23:63, 93,95,108,111,129,126,107-152,153, 158,179,267,271,285,395-453, 455-485,491,497,642-643 ;739;24:25, 799,845 ;27:178,377,431,421,443, 693,778,801,924 ;24:353-376; 27:185-225,332,659;28:109,212,228, 229,340;29:743 ;30:4,26,172,175, 200,224,303,310,395,404,483,484, 485,495,496,497,499,562,588,603, 637,831,840,843, acaricidal enzymatic activity 24:995-998 tx-cembra-2,7,11-triene-4,6-diol 29:100 activity in ebv assay system 29:100 activity in pkc assay system 29:100 activity in skin-1 assay system 29:100 activity in odc assay system 29:100 ~-cembra-2,7,11-triene-4,6-diol 29:100 activity in ebv assay system 29:100 activity in skin-1 assay system 29:100 activity in odc assay system 29:100 of (-)-deoxypodophyllotoxin 30:589 of (-)-hemone 30:590 of (-)-nymphone 30:590 of (-)-yatein 30:590 of (+)-corytuberine 30:589 of (+)-epiaschantin 30:590 of (+)-epimagnolin 30:590 of (+)-epiyangambin 30:590 of (+)-hernovine 30:589 of (+)-laurotetanine 30:589 of (+)-magnoflorine 30:589 of (+)-malekulatine 30:590 of (+)-n-formyldehydroovigerine 30:589 of (+)-n-formylnornantenine 30:589 of (+)-n-formylovigerine 30:589 of (+)-n-hydroxyhemangerine 30:589 of (+)-n-methylhemangerine 30:590 of (+)-ovigerine 30:590 of (+)-ovihernangerine 30:590 of (+)-vateamine-2' [3-n134,136,150, 174,257,259,260,262,263,265,267, 269,270,271,273,274,284,311,364, 410,634,643,656,661,690,704,744; 23:96,97,187,218,238,249,250,253, 255,272,274,277,290,292,299,308, 317,701,274 ;24:294,352,740,867; 26:212-213,1185 as anti-fungal agents 24:406 2 [3,5-epoxy-5,10-dihydroxy-6c~ange lo y-lo xy-9 [3-is obutylo xygermacran-8ot, 12-olide 29:89 activity in inos assay system 29:89 activity in nfkb assay system 29:89 7 a, 8 c~-ep oxy-6 or-hydro xyabieta-9(11), 13-dien-12-one 29:99 activity in ebv assay system 29:99 9,10or-epoxy-9,10-seco-abieta4-o-phenyl-2-propionyl-neu5ac-cx2me 27:123 4-t-butyl-dimethyl-silyl ether 27:127 5-azido-5-deamino-neu5ac-c~2me 27:119 5-n-benzyloxycarbonyl-neu-cx2me 27:119 5-n-propionyl-neu-c~2me 27:119 7-deoxy-neu5 ac-c~2me 27:123,127 8-amino-8-deoxy-neu5 ac 27:121,122 8-azido-8-deoxy-neu5ac 27:121,122 8-tosyl-neu5acme ester 27:121,122 9-amino-9-deoxy-neu5 ac 27:121,122 9-amino-9-deoxy-neu5ac-c~2me 27:119 9-azido-9-deoxy-neu5 ac 27:121,22 9-azido-9-deoxy-neu5ac-c~2me 27:119 9-o-acetyl-neu5 ac-( ,590 activity of (-)-deoxypodophyllotoxin in 30:589 activity of (-)-hemone in 30:590 activity of (-)-nymphone in 30:590 activity of (-)-yatein in 30:590 activity of (+)-corytuberine in 30:589 activity of (+)-epiaschantin in 30:590 activity of (+)-epimagnolin in 30:590 activity of (+)-epiyangambin in 30:590 activity of (+)-hernovine in 30:589 activity of (+)-laurotetanine in 30:589 activity of (+)-magnoflorine in 30:589 activity of (+)-malekulatine in 30:590 activity of (+)-n-activity of hydroxyhernangerine in 30:589 activity of (+)-n-formylnornantenine in 30:589 activity of (+)-n-formyldehydroovigerine in 30:589 activity of (+)-n-formylovigerine in 30:589 activity of (+)-ovigerine in 30:590 activity of (+)-ovihernangerine in 30:590 activity of (+)-vateamine-2'13-noxide in 30:590 activity of 7-formyldehydrohernangerine in 30:589 activity of 7-formyldehydronornantenine in 30:589 activity of 7-formyldehydroovigerine in 30:589 activity of 7-hydroxy-6-methoxy12-hydroxy -6,7-seco-abieta-8,11,13trien-6,7-dial 29:99 activity in ebv assay system 29:99 7-hydroxy-6-methoxy-1-methyliso22:510,512,513,516,518,519, 521,522,525-529,531,534 achyranthes aspera l 9,459,503,753,754,311; 17:313,441 ;20:108-113,273,613; 24:215,228-232,503-509,739-798; 26:229,790,813,887;30:560,562,567, 570,590 chemo-differentiation therapy 30:498 of acute monocytic leukemia chemopreventive activity 30:591,597, 766 in mouse mammary organ culture model 30:591 in vitro 30:597 in vitro bioassays for 30:591 of chlorella vulgaris 30:766 ofisoquinoline alkaloids 30:597 of lignans 30:597 chemopreventive agent 26:219 genkwanin as chemopreventive efficacy 27:386 of limonene carcinogenesis 27:386 chemoprophylaxis 26:674 chemosensitizers 30:595 antimalarial drugs as 30:595 chemotaxonomic markers 26:1128 chemotherapeutic agent 30:5,330,742 atovaquone 30:330 for colon cancer 30:5 for multi-drug resistant cancers 30:5 for ovarian cancer 5 for taxol-resistant breast cancer chemotherapeutic intervention 30:397 targets for chemotherapy drug 27:436 antitumor activity of 27:436 chest pain 30:203 use of ginseng root in 30:203 childhood disintegrative disorder (cdd) gram-negative bacteria 30:693 maytenus species against gram-positive bacteria 23:68;30:629, 693 actamycin against 23:68 antimicrobial activity of 30:629 inhibitor maytenus species against grandiflorolic acid 29:100 activity in ebv bioassay system grandiflorolic acid angelate 29:100 activity in ebv bioassay system grandisin 26:230 trypanosomicidal activity of grandmal seizures granulocyte phagocytosis 26:47,55 ofjenisseensosides c 26:47,55 ofjenisseensosides d green tea 27:417,420 biological activity of 757 effect of general analogues on pgf2 na-induced contraction griseofulvin 24:934 as anti-fungal agents grob-type fragmentation 24:728,731 growth 30:61 of tumor cells 30:61 growth inhibition 17:142 growth inhibitor activity 7:407 guaiazulene 5:363,369 as an anti-inflammatory agent guidimacrin 27:831 antitumor mechanism 584 neurodegeneration 30:224 reperfusion injuries 30:224 respiratory disorders 30:224 role of free radicals in 30:224 human growth hormone human hek 293 cell line 30:781 biological response of human hl-60 leukemia cells 30:496 differentiation of 30:496 inhibition of 30:496 proliferation of human leukemia cells 18:269 human leukocyte elastase 16:727 inhibitor human leukocyte elastase (hle) 30:830,831,843 homologous sequences of 30:831 plasminogen activator (u-pa) 30:830 human lung carcinoma 23:290;30:5 discodermolide against human lung carcinoma) 30:691 phenolic triterpenes against 30:691 human medulloblastoma 23:290 human melanoma 23:255 human mesangial cell proliferation 30:308 in vitro human monoblastic leukemia cells 30:498 effect of hydroxyurea (hu) 30:498 growth inhibition of 30:498 human monoblastic leukemia u937 cells 30:498 growth inhibition of human neuroblastoma nbla-n-5 cells tamoxifen activity against 12:390 human neuroblastoma sh-sy-5y cells 12:390 human neutrophils 12:390 human neutropil protein kinase c 12:389 human onchocerciasis 12:9 ivermectin for human papilloma virus 30:394 pathogenesis of 30:394 cause of hepatocellular carcinoma human papilloma virus (hpv) 23:97 human papilloma virus type 11 30:406 microbicidal compound against human papilloma virus type 40 30:406 microbicidal compound against 30:406 human platelets 12:390 study of protein kinase c in 12:390 by staurosporine 12:390 human rotavirus (hrv) 30:406,412 cause of dehydrating gastroenteritis 30:406 member of 30:406 reoviridae type of human serotonin transporter gene (slc6a4) 30:376 regulation of neuronal activity human spermatozoa motility 21:675 human synovial pla2 25:697 inhibitor anti-glycemic effects 24:902-904 anti-hyperglycemic effects of -o-methyl-20-hydroxyecdysone activity in 29:28 pinnatasterone activity in 29:28 polypodine b 2-o-cinnamate activity in muscarinic achr antagonists 21:56 muscarinic antagonist 22:19 muscarinic receptor 21:95 muscarinic receptor antagonists 22:734 muscarinic-l,2-receptor 21:95 muscle pain 24:898 aconitine for relieving activation effects of 24:906 medicinal uses of 24:906 protein kinase c activation effects 24:906 muscular p-388 (murine lymphocytic leukemia) 30:588 activity of (-)-deoxypodophyllotoxin against 30:588 activity of (-)-yatein against p-388 lymphocytic leukemia cell 25:764 p388 mouse leukemia 12:390 staurosperine activity against p-388 murine leukemia cells 21:411 pachyman 5:288,317 antitumor activity of 5:317 pachyrrhizus erosus 24:779,822-826 anti-feeding activity of 24:825 pachyrrhizus palmatilobis 24:822 biological activity of 24:823 paclitaxel 240 (+)-acetoxypinoresinol dimethyl ether as 26:241 arctigenin as 26:241 arctigenin methyl ether as 26:241 chamigrenal as 26:244 cinnamophilin as 26:240 dehydroschisandrol a as 26:245 denudatin as 458 fptase inhibition by 24:458 paw oederma 30:207 inhibition of 30:207 2-pectenotoxin 19:580 cytoxic activity of 19:580 pectinasic pectolinaringenin 30:725,748 antispasmodic activity of 30:748 pedunculariside 29:84 activity in cox assay system 29:84 activity in epp assay system pelargonidin (anthocyanidin) 29:585 effects on lela 29:585 effects on penates sp. 28:713 as ~-glucosidase inhibitor 28:713 penicillium turbatum 11:194 antibiotic a 26771b by 11:194 penstemide 7:441 activity against p-388 lymphocytic leukemia penstemon rosseus 24:838 anti-fungal activity pentacyclic triterpenes 30:207 protein kinases inhibition by 564 in lipid autoxidation 3,4,6-p enta-o-gallo yl-~ -d-g luc o s e (galloyl ester) 29:589 effects on 229 biological activity of 28:229 8-prenyleriodictyol 28:229 biological activity of 28:229 prenylflavones 28:225 anti-human immunodeficiency virus (hiv) activity of 28:226 prenylfiavonoid 30:207 antinocicieptive effect of antitumor promoting activities of 29:720 as cytomegalovirus protease inhibitors 29:720 gastro-intestinal problems 29:720 use in hodgkins disease 29:720 use in infections 29:720 use in lupus 29:720 use in osteomyelitis 29:720 use in parkinson's disease 29:720 use in psoriasis 29:720 use in respiratory problems 29:720 use in skin ulcerations 29:720 use in syphilis 29:720 6-prenylnaringenin prevention by chitosan 27:436 of myelotoxicity 27:436 primary biliary cirrhosis 25:463 primary granulosa cells 25:269 apoptosis in 25:269 primary solid-tumor growth 30:64 effect of triterpenoids on 30:64 pristimarin 30:692 cytotoxic activity of prl1 & prl2 proproteins 29:592 effects on cysteine protease 851 probes 27:332 activity of 27:346 procurcumenol 29:90 activity in tnfct assay system procyanidin b 1 (dimeric flavan-3-ol) 29:580 effects on ace procyanidin b2 (dimeric flavan-3-ol) 29:580 effects on ace procyanidin b-5 3,3'-di-o-gallate 29:580 effects on ace procyanidin c2 (trimeric flavan-3-ol) 29:580 effects on ace 29:580 procyanidin polymer (flavan-3-ol polymer) 29:580 effects on ace prodrug monotherapy 21:157 natural anthracyclines for 21:157 prodrugs 21:157 of natural anthracyclines pro-inflammatory cytokines 25:461 pro-inflammatory substances 30:206 in chronic painful inflammatory diseases 30:206 proliferation 30:368,378 effect of serotonergic neurotransmission on 30:368 serotonin action on 30:378 prolysine 25:388 prophylactic effects pro-nociceptive prostaglandins 30:192 in inflammation pro-nociceptive transmitter 30:194 release inhibition of 30:194 prooxidant activities 30:745 in thiobarbituric acid assays 30:745 of chemiluminescence 30:745 of tert-butyl hydroperoxide 30:745 prooxidant property 30:524 of carotenoids 30:524 prophylactic drugs prostaglandin e2-dependent flavonoids cytoprotection effect pgh2) 25:595 prostaglandin inhibitors 30:192 clinical use of 30:192 use in rheumatism 30:192 use in osteoarthritis 30:192 use in headache 30:192 use in dental surgery anticancer clavulones 16:366 as antiviral compound 24:541-544 as pain mediator 30:192 in mast cells 30:192 nociceptive effect of prostate cancer prevention 30:525 role of lycopene protease inhibitor proteins from plants 29:596 effects on metallo protease inhibitors 24:487,488 567 cereal bifunctionals as 29:567 chymotrypsin as 29:568 destructive potential of 29:568 in alzheimer's disease 29:567 in angiogenesis 29:567 in cancer inflammatory disease 29:567 in protozoal infection 29:567 in viral infection 29:567 kunitz as 29:567 mustard family of 29:567 of metallocarboxypeptidase 29:567 ofserine protease 29:567 pepsin as 29:568 phytocystatins as 29:567 potato type 29:567 proteins as 29:567 serpin as 29:567 trypsin as 29:568 proteases 24:1005;29:567 enzymatic activity 24:1005 in apoptosis 29:567 in blood clotting 29:567 in cell division 29:567 in digestion 29:567 in extracellular matrix digestion 29:567 in inflammatory responses 29:567 protein kinase a (pka) 192 in regulation of ionotropic receptors protein kinase activity 28:677 ofxestoquinolide protein kinase c (pkc) activator 30:70 protein kinase c inhibition 24:573; 25:46,488 by xestocylamine 24:573 protein kinase inhibition 30:207 by pentacyclic triterpenes protein phosphatase inhibitors 27:874 protein phosphatases 25:707 inhibitor of 25:707 protein synthesis 30:407 inhibitor of 30:407 protein transduction domain (ptd) proteinaceous receptor 25:371 proteinase activity 21:150 protein-coupled receptor 27:821 g-proteine kinase c activity 21:638 proteins protein-tyrosine kinase activity 27:842 offlavonoid aglycones 27:842 of glycosides 27:842 ofkoelreuteria henryi protein-tyrosine kinase inhibitory activities proteolytic activity 30:841 proteolytic systemin inactivation protium kleinii 30:206 anti-inflammatory effect of 30:206 antinociceptive effect of 30:207 protocatechuic acid (3,4-dihydroxybenzoic acid) 29:589 effects on proton pump inhibitors (ppis) 25:612 proton-translocating nadh:q oxidoreductase 28:435 high-affinity inhibitors protozoocidal activity 30:329,741,742 against leishmania 30:329 against plasmodium 30:329 against tryponosoma 30:329 genus baccharis 30:741 in vitro 30:329 of neo-clerodane diterpenoids 30:742 ofquinone derivative 30:329 protozoocidal compounds 30:740 use of proxyelocytic leukaemia (hl-60) prunella vulgaris 30:401 antiviral activity of psammaplin a (bisprasin) 28:693 effects on bacillus subtilis psammaplysilla purpurea 28:693 antimicrobial activity of 28:693 as tyrosine kinase inhibitor pseudobersana mossambicensis 20:478 bioactive steroids from pseudoguaiane 11 a,13-dihydrohelenalin acetate 29:90 activity in cro assay system 627 against cytomegalo virus 30:627 against human immunodeficiency virus-1 30:627 against influenza virus 30:627 as virucidal agents 30:627 retroviral activity of 7:421 pseudolaric acid b 13:653 pseudomonas aeruginosa 30:739 antibacterial activity against 30:739 antifungal activity against pseudorabies virus 30:404 in vitro replication of 30:404 role ofheparin 30:404 psii inhibitors 26:368,371 psk in mitosis 25:377 psk-receptor 25:378 psk-a activity 25:378 psoriasis 30:484 use of lc~,25(oh)2d3 30:484 psychiatric disorders 30:369 anorexia 30:369 bipolar disorder 30:369 bulimia 30:369 effect of circadian activity on 30:369 obsessive compulsive disorder 30:369 panic disorder 30:369 schizophrenia 30:369 seasonal effective disorder 30:369 unipolar depression 30:369 psychodelic drugs 26:820 cocaine as 26:820 morphine as 26:820 semisynthetic lsd as 26:820 psychovegetative disorders 22:643,684 psycotria colorata 30:205 to relieve abdominal pain puberulin a 26:243,244 as paf-induced inhibitor pulegone 29:83 activity in am assay system 29:83 pulmericin 16:299 antifungal activity of 16:299 antileukemic activity of 16:299 cytotoxic activity 16:299 pulmonary vascular injury 30:70 role of serine protease in 30:70 role of thrombin pumiliotoxin alkaloids 27:238,239 alio-ptx 323 b alkaloid 27:249,250 biological activity of punta toro virus 30:411 treatment of 30:411 purifying blood 26:50 herniaria hirsuta in 26:50 purine receptors putative receptor proteins 25:398 putative satiety factor 24:910 effect of cholecystokinin 24:910 pyranocoumarin 27:429 biological activity pyranoid carbasugars 29:466 as cellular messengers 29:467 as insulin release mediators 29:467 biological activities of 29:466 effects on glycosidase enzyme 29:467 effects on glycosyltransferase enzyme 29:467 galactosyltrans ferase inhibition by pyridoacridine alkaloids 28:639 cytotoxic effects on kb cells 524 glycosidase inhibitors of 10:524 pyrrolomycin antibiotics 25:794 pytressin-induced coronary spasm antileukaemic activity of 4'-quercetagetin (6-hydroxy quercetin hexahydroxyflavone) (flavonol) 29:573 effects on 4'-pentahydroxyflavone) 29:578;30:208,749 analgesic effect of quercetin 3-o-c~-l-arabinopyranoside 28:64 anti-oxidant activity quercetin 3-o-c~-l-rhamnopyranoside 28:64,65 anti-oxidant activity of quercetin 3-o-[3-d-galactopyranoside 28:64 anti-oxidant activity o-galloyl)-glucoside 29:580 effects on ace quercetin-3-o-ot-l-rhamnopyranoside 28:64 anti-oxidant activity quercitrin (quercetin-3-rhamnoside) 29:581 effects on ace quillaia 26:55 of adjuvant activity 26:55 quinidine 25:347 quinine 25:344 290 antitumor activity of 10:117 inhibitor of dna quinoline drugs 25:348 quinoline drug family quinoline-based antimalarials 25:344 quinoline-ring antimalarial drugs 25:327 quinolines 25:327 quinolizidine derivatives 27:284,285 testing against mycobacterium tuberculosis 541-544 as antiviral quinone methides 30:694 antiplasmodial activity of 30:694 quinones rabidaea platyphylla 22:513 for epilepsy 22:513 rabies virus 30:409 inhibitor of 30:409 rabies virus infection 30:409 in chicken-embryo-related cells 30:409 rabies virus replication 30:409 in nerve fibers rachitic bone healing potencies 484 of vitamin d2 30:484 of vitamin d3 30:484 of vitamin d4 30:484 of vitamin d5 30:484 of vitamin d6 30:484 of vitamin d7 radical scavenging activity 23:362 of coumarin 23:335 radioligands 30:814 from brain membrane synaptosomes ranitidine 25:612,617 ranp-triggered signal transduction raphe nuclei 30:368 role in cognitive functions ras oncogenes 22:621 rat brain synaptosomes 30:807 p-affinity rat mammary carcinogenesis model 30:592 cancer chemopreventive activity rat serum vitamin d-binding protein (dbp) 30:494 binding assay for rebaudioside a 29:101 activity in tpa bioassay system tpa bioassay system 8-receptor 22:296 ~-receptor 22:296 8-receptor affinity 30:814 binding properties of 30:814 ~t-receptor affinity 30:814 binding properties of 30:814 of glycopeptide 30:814 8-receptor agonists k-receptor agonists 30:797,806 pharmacological activity of -receptor agonists 30:797 pharmacological activity of 30:797 8-receptor antagonists 30:797 pharmacological activity of k-receptor antagonists 30:797 pharmacological activity of 30:797 ~t-receptor antagonists 30:797 pharmacological activity of 30:797 receptor binding 27:377 receptor binding assay 30:810 in vitro 30:810 mouse vas deferens (mvd) activity in 30:810 of guinea-pig ileum (gpi) 30:810 receptor binding properties 30:815 of tyr-d-ala-phe-[[3-d-glc(oac)4]tyr-pro-ser-nh2 30:815 of tyr-d-ala-phe-asp-val-val-gly-nh2 (deltorphin c) 30:815 of oac)4]-gly-nh2: 30:815 of tyr-d-ala-phe-gly-tyr-pro-ser-nh2 (deltorphin c) 30:815 of tyr-d-ala-phe-gly-tyr-pro-thr([3-d-glc)-gly-nh2 30:815 of tyr-d-ala-phe-gly-tyr-pro-thr c )-val-vai-gly-nh2 receptor tyrosine kinase (rtk) 25:512 receptors 27:363;30:421 and cell-cell adhesion 30:421 and cell-cell communication 30:421 antigenic determinants of 30:421 for bacteria 30:421 ofchiral proteins 27:363 with enzymatic activity 27:824 without enzymatic recombinant prourokinase (pro-uk) 30:839 as fibrinolytic enzymes recombinant tissue-type plasminogen activator 30:839 as fibrinolytic enzymes 30:839 red wine 27:430 biological activity of 27:430 redox enzymes redox inhibitors 30:225 of 5-1ipoxygenase repandusic acid (hydrolysable tannin) 29:573 effects on hiv-1 protease 29:573 repellent properties 28:395 of amblyomma variegatum 28:395 reperfusion injuries 30:224 role of free radicals rescorcinolic lipids 30:165-167 effects on enzymatic activity 30:165-167 interaction with proteins reserpine 25:538 as immunosuppressive drug resistance-modifiers 30:595 antimalarial drugs as resistant plasmodium species 30:595 in chloroquine sensitivity resorcinolic lipids 30:119,158,160,162-164 as antifungal fluids 30:160 as growth reulators 30:162-163 as hair restoration-lotion 30:160 used in gingival infections respiratory ailments 24:847 respiratory depression 30:799 due to analgesic alkaloid 30:799 respiratory disorder 30:224 role of free radicals in 30:224 respiratory infection 30:408 respiratory syncytial virus 30:395 respiratory toxins 24:1002,1063 respiratory tract infections 26:50 herniaria hirsuta in 26:50 response decay mechanism 25:491 response modifiers resveratrol 27:430 biological activity of 27:430 resveratrol 28:582 effects in llc-bearing mice 28:582 effects on tumor 597 treatment of 30:597 use of hernandia moerenhoutiana in 30:597 use of hernandia nymphaeifolia 596 antimalarial activity of 30:595 antiplasmodial activity of 30:596 as platelet aggregation inhibitor 30:594 effect on antiplasmodial activity 30:596 retinal 29:111;30:520,521 activity in am bioassay system 29:111 in visual signal transduction 30:520 retinoic acid 29:111 activity in am, ebv, skin-1, odc bioassay systems 29:111 retinoic acid 30:493,498,521 in cell differentiation 30:521 in development 30:521 in growth regulation 30:521 retinoid retinol 29:111 activity in am retinol acetate 29:111 activity in am bioassay system retinol palmitate 29:111 activity in am bioassay system 29:111 retroviral enzymes 30:397 inhibitor of 408 causative agent of immunodeficiency syndrome 30:394 inhibitor of rett's disorder (rtt) 30:372,373,384 by mutations in mecp2 gene 30:373 genetic basis of 30:373 reutericyclin 28:127 as antibiotic reverse transcriptase 30:226,395,397 role in hiv replication cycle 30:226 role in viral replication rhazinilams 29:362 as microtubule poisons rheedia gardneriana 30:207 antinocicieptive effect of 30:207 rheumatism 26:50 herniaria hirsuta in 26:50 rheumatism 30:192,691 use of non-steroidal antiinflammatory drugs (nsaids) 30:192 use of prostaglandin inhibitors 30:192 rheumatoid arteriosclerosis 30:70 rheumatoid arthritis 21:152;22:310; 26:170,486;30:70,204,225 in treatment of cph82 26:170 in rats 30:204 reagent-induced 30:204 treatment of 30:225 use of kalopanax pictus rhoifolin (5, 4'-dihydroxy -7-rhamnosyl-glucosyl-flavone) 29:573 effects on hiv-1 protease 29:573 rhombenone 24:456 fptase inhibition by ricinoleic acid 30:193 analgesic action of 30:193 rickettsial pathogens 28:394 cowdria ruminantium as rifamycin-type macrolides 24:545,546 antiviral microbial-derived compound 24 rift valley fever infection 30:411 treatment of rimantidine 27:108 for treatment of influenza in infections 814 activity of deltorphin b in 30:807 of glycopeptide 30:814 rna virus 30:394 arenavirus type of 30:394 bunyavirus type of 30:394 coronavirus 30:394 influenza virus 30:394 measles virus 30:394 parainfluenza virus 30:394 picornavirus type of 30:394 rabies virus 30:394 reovirus type of 30:394 rotavirus type of rna-dependent rna polymerase activity 30:407 virion-associated rna-directed dna-polymerases 29:127 inhibition by sf-9 cell line 29:33 activity in mam-3assay system 29:100 antileukemic activity 8:18 sarcoptes mites 28:410 cause of human scabies 28:410 symptoms sarcoptes scabiei 28:409 cause of mange mites 28:409 cause of scabies sargasm horneri 30:400,405 anti-cmv activity of 30:405 polysaccharide (ps) from 30:400 sarkomycin 8:150 as antitumor agent 8:150 satratoxins 30:743 antiviral activity of 204 scavenging effects 23:443 of tannic acid 23:443 sch 207278 24:451 fptase inhibition by 24:451 schisandra 26:183 display platelet activating factor antagonist activity 26:183 effect on cardiovascular system 26:183 effect on heart rate 26:183 for treatment of hepatitis schisandrol a 26:245 as paf-induced inhibitor schistosomiasis (bilharzia) 7:405 schistosomicidal activity 1:545 schizonticidal drug 26:837 schizophrenia 101 activity in crg bioassay system 29:101 from scoparia dulcis scropolioside a 29:84 activity in tpa assay system 29:84 scrovalentinoside 29:84 activity in tpa assay system scutellaria baicalensis 30:55,56,69, 254,289 in allergric inflammatory disease 431 sdb 21:695 inhibitory effect of 21:695 sdc 21:695 inhibitory effect of 21:695 sdz-249-665 30:201 antinociceptive activity of 30:201 anti-hyperalgesic activity of 30:201 seasonal affective disorder 30:369 effect of circadian activity on 30:369 seaweeds 30:404 anti-cmv activity of activity in ebv assay system 29:100 1,2-secoemetine derivatives 6:485 amoebicidal activity of 6:485 secoiridoids 26:330 antimicrobial activity of 26:330 second messenger 25:516 secondary metabolites 28:3,432,617 acaricidal activity of 28:432 biological activity of 28:617 secondary metastatic tumor growth 30:64 effect of triterpenoids on 599 arnica as 22:559 artocarpus heterophyllus as basilicum polystachyon for 22:514 sedative activity 21:673 sedative in convulsions 22:518 crocus sativus l. for 22:518 sedative in epilepsy 22:535 withania somnifera l. as 22:535 g-selective agonist 30:807 dermorphin 30:807 8-selective antagonist tyr-tic-phe-nh2 selective antitumor activity 15:355 selective cytotoxicity 13:648 8-selective opioid agonist 30:817 deltorphin b 30:817 g-selective opioid peptide 30:802 8-selective opioid peptide of ici 19944 30:811 ofk opioids 30:811 8,g-selectivity 30:819 in vivo 30:819 of glyco analogues semilicoisoflavone b 28:229 biological activity of 28:229 semiochemicals semisynthetic lsd 26:820 as psychodelic drug 10-dihydro xy-7-methoxy-naphtho sendai virus infection sensory neurons 30:194 hyperpolarisation of 30:194 8er264 inhibitors serine 27:850 activitiy of serine 30:836 c~2-macroglobulin inhibition by serine protease inhibitor proteins 29:617 arrowhead pis api-a & api-b as cp-thionin as 29:617 effects on barley malt cysteine endoproteinases 29:617 effects on chymotrypsin 29:617 effects on subtilisin 29:618 effects on subtilisin eleusine double-headed try-~x-amylase inhibitor i-2 as 29:617 from brassica nigra 29:618 from brassica napus 29:618 from cassia fistula 29:617 from eleusine coracana 29:617 from hordeum vulgare 29:617 from phaseolus angularis from sagittaria sagittifolia 29:617 from sinapis arvensis 29:618 from vigna unguiculata 29:617 hordeum lipid transfer proteins as 840 blood clotting factors as 29:570 cathepsin g as 29:570 chymase as 29:570 chymotrypsin as 29:570 granzymes as 29:570 in angiogenesis 29:570 in blood clotting 29:570 in cytosolic proteolysis 29:570 in digestion 29:570 in inflammation 29:570 in proprotein processing 29:570 in tissue remodelling 29:570 kallikrein as 29:570 plasmin as 29:570 prolyl endopeptidases as 29:570 role in blood coagulation 30:70 role in platelet activation 30:70 role in pulmonary vascular injury serine proteases (subtilases) 25:388 cysteine proteinase inhibitor 25:370 poly phenol oxidase 25:370 serine proteinase inhibitor serine proteinase inhibitor i 25:370 as defense proteins serine-threonine-specific receptor protein kinases serotonergic innervations 30:377 autoradiographic imaging 30:377 in cerebral cortex 30:377 in neonatal rats by immunohistochemical techniques 30:377 physiological role of 377 serotonergic neurons 30:368,376 in raphe nuclei 30:376 role in 30:regulation of neurogenesis 627 as hyperforin inhibitor 30:627 as neurotransmitter 30:367 effect on synaptogenesis 30:378 in apoptosis 30:367 in cell proliferation 30:367 in presynaptic neuron 30:381 morphogenic properties of 30:367 neurochemical connection of 30:368 receptor activity of -ht) receptors 30:376,378, 382,383 in brain 30:383 in limbic brain regions 30:383 pharmacological aspects of serotonin (5-ht) re-uptake inhibitors 30:368 mechanism of serotonin (5-ht) synthesis 30:370,376, 377 in autistic children serotonin (5-ht) 1ar receptor antagonists 30:378 in posmatal treatment -ht)sa receptor 30:375 in brain development 30:375 in phenotypic behavioral irregularities 30:375 in purkinje cells 30:375 mrna expression of 30:375 serotonin action 30:378 on apoptosis 30:378 on maturation 30:378 on neuronal functioning 30:378 on proliferation serotonin agonists 25:531 anticomplementary activity of 25:46 role of cyclic amp-dependent protein kinase 25:46 delayed-type allergy suppressant activity of 25 serotonin synthesis inhibitor 30:378 parachlorophenylalanine as serum tc 27:420 biological activity of 27:420 serum transaminase 27:415,417,420 biological activity of 27:415 serum transaminase activity 25:469 of curcumia longa l serum triglyceride 27:404 biological activity sesquiterpene lactone 7:426 sesquiterpene quinones 5:429 antimicrobial activity of 5:429 cytotoxic activity of sesquiterpenoids drimane-type synthesis 29:127 avian myeloblastosis virus (amv) inhibition by 127 by jauch 29:127 sex pheromone sexual potency 28:4 ofbroussoflavonol g 28:4 sgot 30:292 activities of 30:292 sgpt 30:292 activities of 21:654 shikimic acid derivatives 29:478 antitumoral activity of 29:479 shinpterocarpin 28:229 biological activity of 28:229 sialic acids 27:103 biological function of 27:103 model for sialyltransferase activity 16:81 side effect 30:194,799 of analgesic alkaloid 30:799 of opioids sigmoidin a 28:229 biological activity sigmoidin b 28:229 biological activity of 28:229 signal reception 30:377 in autistic children 30:377 signal transduction mechanisms 25:518 signal transduction pathways signal transductors/activators of transcription (stats) 25:519 silandrin 26:255 as antihepatotoxic agent 26:255 silchristin 26:255 as antihepatotoxic agent 26:255 sildenafil 30:155 used for erectile dysfunction silymonin 26:255 as antihepatotoxic agent 26:255 simocyclinon 29:319 antibiotic activity of 29:319 cytostatic effects of 29:319 sindbis virus 17:135 sinomenine 25:472,476 hepatoprotective effect glycoside) 29:590 effects on pep 29:590 skin cancer 5:747 skin diseases 30:732 skin inflammation 30:323 anthrones used for 30:323 skk moth 1:704-706 juvenile hormone from slaframine 27:252 biological activity slaframine alkaloids 27:250,255 as muscarinic agonist 27:255 sleeping sickness smenoquinone 5:434,425 antimicrobial activity of smooth muscle relaxation 24:875-925 as biological action 24:875 of kampo medicines snake bite 30:691 treatment of 30:691 use of crossopetalum gaumeri 30:691 sobrerol 29:83 activity in mam-2 assay system 29:83 activity in gst assay system 29:83 activity in ras assay system 29:83 social phobia sodium salt of caffeic acid tetramer 24:742,743 anti-hiv activity of 24:742 soilborne fungi 21:181 bioactive metabolites from soilborne phytopathogens 21:182 biological control of 21:182 solandelactones 24:455 fptase inhibition by solanidane-induced teratogenicity 23:573 solid tumors neovasularization 25:593 somatostain 25:265 as a~ adenosine agonists somatostatin receptor 21:72;22:26; 25:530 type of g-protein-linked receptor 25:530 sonodione 30:566 from hernandia sonora 30:566 sores 30:616 tricyclic acylphloroglucinols sorivudine 24:474,486 as anti-viral agent 24 sorocein f 28:230 biological activity of 28:230 sos chromotest 22:623 soyabean saponins 25:222,223 hypocholesterdemic effects soyasaponins 27:398 anti-obesity action of 27:398 biological activity of 27:399 soybean trypsin inhibitor (sbti) spasmolytic activity 23:358 ;28:257,293; 30:264,561,748 of hernandia moerenhoutiana 30:561 ofhernandia voyronii 30:561 of imperatorin 23:350,358 of plant polyphenols 28:257,293 of thymus satureioides 30:264 on guinea pig ileum 30:748 on smooth muscles specionin 10:425 antifeedant activity of 10:425 spermicidal activity 26:53 of gypsophila paniculata sphingolipid receptor ca 2+ channels 25:535 sphingolipid receptors 25:534 sphinxolide 10:153;17:17 antitumor activity of 17:17 spider mite 1:702 hatching inhibitor spiroalkyl analogs 28:357 biological activity spirulina platensis 30:399,404,408 anti-influenza activity of 30:408 biological activity of 30:404 calcium spirulan from 30:399 polysaccharide (ps) from spleen/adipose tissue weight 30:56,57, 66 1203 effect of fucoidan on 30:56,57 effect of oleic acid on 30:65 spontaneous apoptosis 26:926 spontaneous metastasis squarroside a 26:31,50 immuno-modulatory effect of 26:31 from vaccaria segetalis squash family serine protease inhibitors 29:614 bd-ti-ii as 29:614 cmti-i as 29:614 cmti-iii as 29:614 cmti-iv as 29:615 cpgti-i as 29:615 cpti-ii as factor xiia, kallikrein, trypsin 29:615 effects on lysyl endopeptidase 29:614 effects on trypsin 29:614 effects on trypsin 29:615 effects on trypsin 29:616 effects on xa, xiia, kallikrein, plasmin, trypsin 29:614 elti-i as 29:615 from bryonia dioica 29:614 from citrullus vulgaris 29:614 from cucumis melo 29:614 from cucumis melo 29:614 from cucumis sativus 29:614 from cucurbita maxima 29:614 from cucurbita maxima 29:614 from cucurbita pepo 29:615 from cucurbita pepo 29:615 from ecballium elaterium 29:615 from echinocystis lobata 29:615 from lagenaria leucantha 29:615 from luffa acutangula 29:615 from luffa cylindrica cucurbitaceae) 29:616 from tricosanthes 29:616 hmti-i as 29:614 lati as 29:615 lati-ii as 29:615 lldti-i as 29:615 lldti-ii as 29:615 mcei-i as stachenone 29:101 activity in ebv bioassay system 29:101 standishinal 29:100 activity in ebv assay system 29:100 standishinal diacetate 29:100 activity in ebv assay system staphyllococcus aureus 30:693 minimal inhibitory concentration (mic) of staphyllococcus epidermidis 30:693 minimal inhibitory concentration (mic) of staphyllococcus saprophyticus 30:693 minimal inhibitory concentration (mic) of staphyllococcus warnieri 30:693 minimal inhibitory concentration (mic) of staphylococcus aureus 30:628,739 antibacterial activity against 30:739 antibacterial properties of 30:628 antifungal activity against starfish fertilisation inhibitor 28:712 callyspongin b as 28:712 stavudin 24:474,486,488 as anti-viral agent 24:474 steganes 29:360,399 cytotoxicity of 29:360,399 tubulin assembly inhibition by 29:360 antitubulin activity of 29:399,400 microtubules assembly inhibition by sterculia urens roxb. 30:405 antiviral activity of 30:405 steroid hormones 27:823 steroid saponins 7:426 teratogenic metabolites of 7:21-24 toxic cardiac active steroids sterol biosynthesis 25:303 inhibitor of 25:303 6a-sterol sulfate 28:702 cytotoxicity against sterol sulfates haplosamate a 28:703 as hiv-1 integrase inhibitor 28:703 sterols 25:43 antitumor activity of 25:43 antiinflammatory activity of 25:43 stevia stevia extract 27:307,310,314 for diabetics stevia leaves 27:315 immunological activity stevia plant 27:315 allergenic activity steviol 27:304,305,307,312 acute toxicity tests of 27:307 effect on fertility 27:312 mutagenicity tests with tpa bioassaysystem 29:101 activity in skin-1 bioassay system 29:101 acute toxicity tests of 27:307 allergenicity problems of 27:315 chronic toxicity studies of 27:306,307,308 effect on fertility 27:309,310 for phenylketonuria 27:315 mutagenicity tests for stigmasterol 30:208,505,570,719 analgesic effect of stilbene derivatives 27:405,409;28:579 biological activity of 27:405,409 in llc-bearing mice 28:579 tumor growth inhibition by 28:579 stimulatory 24:845 biological activity 24:845 stimulant agent 23:642 ofteucrium sp stimulus-response mechanisms 25:515 streptococci 29:535 pathogenicity of 29:535 serological groups streptokinase/streptodomase 25:271 streptolygidin 28:130 antibiotic activity of 28:130 as bacterial rna polymerase 28:inhibitor 130 as terminal dna transferase striga asiatica 30:162 infection with ofbioactive metabolites 21:251 of scopadulan-type diterpenoids structure activity relationship studies (sar) 30:195,494,800 of anandamide 30:196 of aspirin 30:195 ofopioid analogues 30:800 of vitamin d analogues 30:493 structure dereplication 29:658 bioactive natural product database in 29:670 of melanin inhibition strychnine 25:530 as immunosuppressive drug 25:530 strychnopentamine 26:1062 anticancer activity strychnos bisindole 26:1062 anticancer activity strychnos monoindole 26:1061 anticancer/protozoal subacute toxicity 27:307 from animals 27:307 of stevioside 588 effects on furin 29:588 sudhl-4-1ymphoma 21:138 sugar cane cystatins 29:593 effects on cysteine proteases 29:593 suicide inhibition 27:348 of 8'-hydroxylase 27:348 suicide inhibitors 9 sulfatobastadin 13 28:695 as endothelin a receptor inhibitor sulfide-containing pyrroloiminoquinones 28:685 as antileukaemic agents sulfur-containing cyclic peptides 28:678 biological activities of 28:678 cytotoxicity of sulphated polysaccharide (ps) 30:398, 400,405 anti-cmv activity of 30:405 anti-hiv activity of 30:398 antiviral activity against hiv 30:400 effect on viral replication 30:400 from marine sources 30:400 inhibitory effect of 30:398 mechanisms of action sulphated polysaccharide (ps) extracts 30:399 inhibitory activity of sulphonylurea drugs 25:534 sunflower cystatin phytocystatin 29:593 effects on cathepsin h ficin 29:593 effects on papain 29:593 sunflower multicystatin 29:593 effects on papain supercritical fluid extraction 21:576 superoxide dismutase superoxide dismutase activity 21:669 suppressing activity 30:308 on cells proliferation zizyphus saponin i as 27:41 zizyphus saponin ii sweroside 25:471,476 serum alt activity of 25 symbiotic marine microorganisms 23:185 bioactive metabolites 23:185 sympathetic ganglia 30:782 blockade of 30:782 sympathetic nerve activity 30:787 sympathomimetic amines 12:411 synapic potential mediation 28:318 by non-nmda receptors 28:318 synaptic actions 30:381 of serotonin synaptic dopamine transporters 25:539 synaptic glycine transporters 25:539 synaptic serotonin transporters 25:539 synaptogenesis 30:368,377 effect of serotonergic neurotransmission on 30:368 role in rodents 30:377 role of serotonin in 30:377 syndrome type-la 26:1130 adipose tissue distribution as 26:1130 cerebellar dysfunction as 26:1130 liver insufficiency as 26:1130 peripheral neuropathy as 26:1130 psychomotor retardation as synergistic activity 21:673 synergistic effect 21:608,611; 30:498,596 ofhervelines b 30:596 ofhervelines c 30:596 of retinoids 30:498 of vitamin d derivatives 30:498 synergistic interaction 21:698 of acyclovir(acv) 21:698 ofganciclovir (gcv) 21:698 with ganciclovir synergistic purgative actions 30:306 of rheinanthrone 30:306 of aloe-emodin-anthrone 30:306 synthetic pharmacological agents 30:224 antioxidant activity of 30:224 synthetic podolactones 28:484 allelopathic activity synthetic psychotropic agents 24:1093 syringaresinol 24:741 anti-platelet aggregation activity of systemic wound response protein 25:402 systemin activity 25:373 assay for ]-val-val-gly-nh2 30:815 receptor binding properties of receptor binding properties tyr-pro-ser-nh2 (deltorphin c) 30:815 receptor binding properties of -d-glc)-gly-nh2 30:815 receptor binding properties of oac)4]-gly-nh2 30:815 receptor binding properties of -d-glc)-tyr-pro-ser-nh2 30:815 receptor binding properties of -d-g lc )-v a 1-v al-gly-nh2 30:815 receptor binding properties of tyrosinase inhibitory activity 21:587 tyrosine kinase tyrosine-specific kinase 19:178 inhibitor of tyr-tic-nh2 30:809 activity in functional bioassay tyr-tic-phe-nh2 30:806 as 5 selective antagonist 30:806 u2 66 myeloma cell 25:273 ucn-01-ucn-02 12:386 antitumor activity of 12:395 protein kinase inhibitor ulapualides a 19:609 antifungal activity unsaturated carbapyranoses 29:475 as enzyme inhibitors 29:475 as herbicidal 29:475 biological properties of 29:475 unsaturated ketonucleosides 4:253 tumor inhibition by u-plasminogen activator (u-pa) 30:844 urdamycin a 11:134 antifungal activity of 11:134 antitumor activity ureterolithiasis-drug-therapy 27:382 urinary tract infections 26:50 herniaria hirsuta in 26:50 urine retention 30:194 as opioid's side effect 30:194 uroterpenol 29:83 activity in mam-2 assay system 29:83 activity in ras assay system ursane triterpene 29:585 effect on chy 29:585 ursolic acid 28:18,40;29:588 effects on hiv-1 protease 29:575 effects on lela 29:588 inhibition of hiv-1 protease dimerization by ursolic acid methyl ester (ursene triterpene) 29:575 effects on hiv-1 protease 29:575 usambarensine 26:1062 anticancer activity of 26:1060 uterine tumor 30:27 effect of pironetin derivatives in 30:27 uusambarine 26:1062 anticancer activity uvaol (triterpene) 29:588 effects on urs-12-ene-3,28-diol) (ursene triterpene) 29:575 effects on vaccaria segetalis 26:50 imunomodulatory effect of 50 vaccination 30:408 for respiratory infection as neuraminidase inhibitors 29:487 as c~-amylase inhibitor 29:486 c~-glucosidase inhibitors of 7:46;10:518 yeast a-glucosidase inhibitor of 10:518 valinuoctins 24:459 fptase inhibition by valiolamine 13:189,195 yeast c~-glucosidase inhibitor valiolamines 29:484 as glucosidase inhibitor 29:484 biological potential of 29:484 in clinical trials 29:484 in treatment of diabetes 29:484 valproic acid 22:507 vancomycin 25:791 vancoresmycin 28:150 activity against gram-positive bacteria vanilloid receptor agonist 30:202 olvanil as 30:202 capsaicin as 30:202 structure of vanilloid receptor antagonist 30:201, 202 capsazepine as 30 varicella-zoster virus 30:394 cause of varroajacobsoni 28:387 toxicity of varroa mites 28:383 vascular physiological action 30:55,69 of natural products vasodilating activity 21:379 vasodilator 23:358 scoparone as vasodilator effect 25:595 of acetylcholine vasodilatory activity 30:275 ofsatujera obovata vasoprotective agent 22:443 vasoprotective effects 28:302 of green tea vateamine-2' [3-n-oxide 30:567, 590,595 activity in human cancer cell line 30:589 activity in murine cancer cell line 30:589 as platelet aggregation inhibitor 30:594 cytotoxic effect of 30:589 veinotonic activities veraguensin 26:230 trypanosomicidal activity verapamil 25:534,673 as caz+-channel blocker 25:487 vermisporin 28:125 antimicrobial activity vernonia amygdalina 28:400 tick toxicity of 28:400 verrucarins 30:743 arenavirus junin 30:743 herpes simplex virus type ii (hsv-2) verrucosin 26:228 ofantiviral activity 26:228 vertebrate toxicity 21:98 vesicant activity vesicular glutamate transporters 25:538 vesicular monoamine transporter type 2 (vmat2) inhibitor vesicular stomatitis virus (vsv) 30:743,752 replication of 30:743 vesititol 28:224 anti-microbial activity of 28:224 vestitol 28:243 anti-helicobacter pylori activity of 28:243 veterinary medicine 28:383 for ectoparasites control 28:383 vetiver grass 28:399 for controlling ticks viagra (sildenafil) 25:541 vicolides 29:89 activity in ctn bioassay system 458 anticancer activity of 14:805 viral diseases 30:732 viral dna 30:226 integration of 12:332;21:98,193, 263,270,312,398,430,585,589,591, 594,643,659,670,673,675,690;23:59, 216,808;26:217;28:481 ;30 :629 against bone resorption 21:720, 721 against h+,k+-atpase 21:719 against staphyllococcus aureus 30:629 mechanism action of 30:225 of (-)-(8,8a-di-epi-swainsonine 12:325 of (-)-swainsonine 12:327 o f (+) cyasterone activity in 29:32 2-deoxy-20-hyroxyecdysone activity in 29:32 14-deoxymuristerone a activity in 29:32 2 [3,3 [3-dihydro xy-513-chole st-7en-6-one activity in 29:32 3 [3,14a-dihydroxy-5 [3-cholest-7en-6-one activity in 29:32 ecdysone activity in 29:32 313-hydro xy-5 [3-cho le st-6-one activity in 29:32 20-hydroxyecdysone activity in 29:32 inokosterone activity in 29:32 kc cell bioassay as 29:30 makisterone a activity in 29:32 muristerone a activity in 29:32 podecdysone a activity in 29:32 ponasterones activity in 29:32 213,313,513,14 a-tetrahydro x ycholest-7-en-6-one activity in 29:32 2[3,3 ~ ,25-tridihydroxy-513cholest-6-one activity in 29:32 viticosterone e activity in 29:32 insect control 21:611 insect juvenile hormone analogues 14:391-397 from thujone 14:391-397 insect pheromones 11: [415] [416] [417] via diisopropylethanediol esters 11:415-417 insect repellant 5:757 insecticidal activity 18:229,704;21:252, 254,262,266,267,269,270,271,278, 285,379,595,611;23:666;667,670, 677,678;24:25,799,845,866;26:231, 232,426,471,479,485;28:477; 30:153-154 activity of (-)-deoxypodo phyllotoxin in 30:589 activity of (-)-hernone in 30:590 activity of (-)-nymphone in 30:590 activity of (-)-yatein in 30:590 activity of (+)-corytuberine in 30:589 activity of (+)-epiaschantin in 30:590 activity of (+)-epimagnolin in 30:590 activity of (+)-epiyangambin in 30:590 activity of (+)-hernovine in 30:589 activity of (+)-laurotetanine in 30:589 activity of (+)-magnoflorine in 30:589 activity of (+)-malekulatine in 30:590 activity of (+)-n-hydroxyhemangerine in 30:589 activity of (+)-n-formylnornantenine in 30:589 activity of (+)-n-formyldehydroovigerine in 30:589 activity of (+)-n-formylovigerine in 30:589 activity of (+)-ovigerine in 30:590 activity of (+)-ovihernangerine in 30:590 activity of (+)-vateamine-2' [3-noxide in 30:590 activity of 7-formyldehydrohernangerine in 30:589 activity of 7-formyldehydronornantenine in 30:589 activity of 7-formyldehydroovigerine in 30:589 activity of 7-hydroxy-6-methoxy1:467,468;2:278,279,286, 289;6:108;7:103,110,121-123; 17:235,244 ;29:87 ;30:193,206 activity in ebv assay system 29:87 activity in skin 1 assay system 29:87 activity in crg assay system 29:87 activity in cro assay system 29:87 activity in aa assay system 29:87 activity in brk assay system 29:87 activity in cps assay system 29:87 activity in htm assay system 29:87 activity in paf assay system 26:3,4,10,13,15, 18,27,30,42,54,57;30:407 anti-inflammatory activity of 26:27 antiviral activity of 30:407 saponins 1:305; 402; 7:155, 156, 190, [426] [427] [428] [429] [430] [431] [432] 434, 435; 18:649 ajugasterone c activity in 29:28 amarasterone a activity in 29:28 amarasterone b activity in 29:28 cyasterone activity in 29:28 ponasterone a 2p-glucoside activity in 29:28 pterosterone activity in 29:28 rubrosterone activity in 29:28 sengosterone activity in 29:28 stachysterone c activity in 29:28 capitasterone activity in 29:28 sarcophytol a 29:100 activity in ebv assay system 29:100 activity in colon-2 assay system 29:100 activity in liver-2 assay system 29:100 activity in lung-1 assay system 29:100 activity in mam-3 assay system 29:100 activity in pancr assay system 29:100 activity in skin-2 assay system 29:100 sarcophytol b 8:18;29:100 activity in skin-2 assay system 29:100 use of platycodon grandiflorum key: cord-265410-khwzdi79 authors: bartlett, stuart; wong, michael l. title: defining lyfe in the universe: from three privileged functions to four pillars date: 2020-04-16 journal: life (basel) doi: 10.3390/life10040042 sha: doc_id: 265410 cord_uid: khwzdi79 motivated by the need to paint a more general picture of what life is—and could be—with respect to the rest of the phenomena of the universe, we propose a new vocabulary for astrobiological research. lyfe is defined as any system that fulfills all four processes of the living state, namely: dissipation, autocatalysis, homeostasis, and learning. life is defined as the instance of lyfe that we are familiar with on earth, one that uses a specific organometallic molecular toolbox to record information about its environment and achieve dynamical order by dissipating certain planetary disequilibria. this new classification system allows the astrobiological community to more clearly define the questions that propel their research—e.g., whether they are developing a historical narrative to explain the origin of life (on earth), or a universal narrative for the emergence of lyfe, or whether they are seeking signs of life specifically, or lyfe at large across the universe. while the concept of “life as we don’t know it” is not new, the four pillars of lyfe offer a novel perspective on the living state that is indifferent to the particular components that might produce it. we contend that most standard definitions of life are restrictive and may blind future astrobiological research from life that is hiding in plain sight. nasa's current working definition of life is "a self-sustaining chemical system capable of darwinian evolution." while this is a fair description of the life that is present at this moment on planet earth, searching the universe for phenomena that fit this definition is similar to playing darts by focusing solely on the bullseye. for the uninitiated darts player, there are three problems with aiming only for the bullseye in a game of darts: (1) it is hard to hit because it occupies a tiny area of the dart board; (2) it is not the highest scoring region on the board (triple 60 for 180 is); (3) there are other high-scoring regions that should also be considered as targets. hence, in the search for extraterrestrial life, we must consider that: (1) life exactly as we know it may be rare in the universe, but a more general class of phenomena with life-like characteristics may be far more common; (2) there may be systems, yet to be discovered or even imagined, that more successfully satisfy the living criteria than even earthly life does; (3) by loosening our constraints on the definition of life, we open ourselves up to exploring the full parameter space of physical and chemical interactions that may create life. we are also motivated by the often heated nature of the origins-of-life field, which has branched into various camps, each seeking to describe their own "one true" scenario for the origin of life. we posit that most of the contentious behavior within the field stems from differences in assumptions about what constitutes life and therefore what life's onset must have been. as [1] wrote, "without a definition for life, the problem of how life began is not well posed." by redefining the vocabulary of the living state, we hope to engender greater mutual understanding and communication between the various communities engaged in origins-of-life research. the remainder of section 1 reviews some of the major issues leading to discord in the origins-of-life field that compelled us to seek a more general definition of life. in section 2, we introduce our new expansive definition of life-which we call lyfe (pronounced "loif")-and discuss how such a definition might engender new perspectives on origins-of-life research. finally, in section 3, we take the first steps toward imagining phenomena that could be classified as lyfe. many origins-of-life theories focus on explaining the emergence of a "privileged function"-a specific aspect of modern-day earthly biology that is assumed to have been present at its emergence. often, these privileged functions are also implicitly assumed to be more fundamental to life in that once the privileged function is established, the rest of life's functions should naturally emerge [2] . another way of viewing privileged functions is the idea of conserved quantities, essential in physics and engineering. origins researchers often try to pin down functional or material aspects of life that they believe must have been present and conserved from the outset. so important are such characteristics that without them, life no longer deserves to be called life (according to such an approach). one example is the so-called "chemistry conservation principle" as invoked by [3] . their argument is summarized thus: "the chemical traits of organisms are more conservative than the changing environment and hence retain information about ancient environmental conditions" [4] . under this principle, a search for the context of life's origins is essentially a search for prebiotic environments that match cellular conditions as closely as possible. examples of privileged functions include template-driven replication of rna, sets of reactions forming early metabolisms, and compartmentalization by lipid membranes (see figure 1 ). this leads to the various "x-first" theories for the emergence of life. for instance, in the so-called "strong rna-first" theory, "darwinian evolution is thought to be necessary for nonintelligent matter to self-organize to produce behaviors that we ascribe to biology" [5] , and the onset of darwinism would occur with the abiotic formation of long-chained rna polymers. conversely, in many "metabolism-first" theories, life is viewed "like other self-organizing systems in the universe, as an inevitable outcome of particular disequilibria" [6] . in this view, life, first and foremost, serves to dissipate these specific disequilibria, using disequilibria conversion engines to maintain its own low-entropy state [7, 8] . furthermore, in "compartment-first" theories, "compartmentalization of primitive biochemical reactions within membrane-bound water micro-droplets is considered an essential step in the origin of life" [9] . this spatial organization and molecular crowding is often mediated by self-assembling lipid vesicles, conferring heightened functionalilty to other biomolecules [10, 11] . the focus on privileged functions leads these theories to different favored environments for life's emergence-a source of great tension within the community. laboratory experiments indicate that surficial environments-energized by uv radiation, exposed to (perhaps transient) reducing atmospheric conditions, and/or supplied with the relevant molecules from exogenous sources-are ideal for rna synthesis, e.g., [12] . on the other hand, deep-sea alkaline hydrothermal vents appear to be the focusing centers for the redox and ph disequilibria that could drive the onset of proto-metabolic pathways, e.g., [6] . however, geothermal pools are favored for the spontaneous membrane assembly of lipid vesicles due, in part, to their low concentrations of divalent cations [13] . every theory seeking to explain a privileged function of earthly biology contains the implicit assumption that the privileged function was present at the emergence of life and that such a function is fundamental to life. however, given the inherent lack of fossil or geological records, there is little to no evidence that any of these functions actually were present at life's beginnings. based on phylogenetic studies, our knowledge of earth's earliest lifeforms ceases at the last universal common ancestor (luca). such studies have shed light on luca's nature-most likely a cellular being with a chemiosmotic metabolism whose genetics were written in dna/rna. however, luca was almost certainly not the original lifeform on earth (see, e.g., [14] ). furthermore, phylogenetic reconstructions of luca are inherently problematic and potentially unreliable due to the limits of bioinformatic techniques. there exists an "event horizon," to appropriate a term from astrophysics, in origins-of-life studies ( figure 2 ). "top-down" approaches, such as molecular phylogenetics, use clues in extant life to trace the history of life back towards its origin. these can take us back only as far as luca. "bottom-up" approaches, which aim to simulate the synthesis of prebiotic molecules and/or the onset of proto-living structures and functions, may one day succeed in producing an abiogenesis. however, this "test-tube origin" will not be luca. furthermore, top-down approaches are limited in their insight into the origin of life because the tree of life that we see today can be satisfied by numerous theoretical histories ( figure 3 ). these histories can involve multiple geneses that never converge, resulting in completely different lineages that either go extinct or are still present as a "shadow biosphere" [15] . plausible origins-of-life narratives can also involve horizontal gene transfer between different lineages, perhaps rampant in a protobiological ribofilm [16] , such that luca is actually a plurality of lifeforms: last universal common ancestral set (lucas) [17] . if so, then the "tree" of life may be a more fitting metaphor than initially imagined: while it branches upward into the diversity of beings that have inhabited our planet post-luca(s), so too does it radiate downward in a tangled root web of early living experiments to which we will never be privy. there is a deeper underlying issue: different origins-of-life researchers may not be seeking analogous explanations. ref. [18] defined three distinct categories of origin narratives: historical, synthetic, and universal ( figure 4 ). in purple are trajectories that describe historical narratives: how an abiotic earth developed into life as we know it. the many trajectories with the same beginning and end points represent the different proposed hypotheses for the origin of life on earth. in dotted orange are synthetic explanations. these may seek: (1) to recreate natural life on earth, though likely through a different trajectory than the natural origin; (2) alter natural life to create new forms of life. in blue and green are hypothetical alien origins of life (lyfe); these and the trajectories that resulted in earth life converge at a point in parameter space that universal narratives seek to describe. in red is a system that did not pass through those universal requirements, thus evolving into an end product that fulfills some but not all of life's pillars. based on [18, 19] . historical origin narratives deal with the origin of life on earth, constrained by our knowledge of early earth environments and informed by what we observe in the products of that process: luca and the modern tree of life. all privileged function theories (section 1.1) are historical narratives. synthetic origin narratives describe experiments where researchers direct the creation of new life in the laboratory. such narratives may deal with the transition between nonlife and life, or they could create new forms of life from pre-existing forms of life, via directed evolution [20] or an artificially expanded basis set for encoding information [21] . as previously discussed, artificial abiogenesis experiments may be tuned to approximate historical narratives, but caution is warranted in conflating the two, due to the "clean lab" nature of artificial experiments and the vast uncertainty about prebiotic environments. universal narratives describe steps that are necessary for abiogenesis anywhere. they are relatively unconstrained by the details of the early earth, the biosphere's particular trajectory on our planet, or the chemical nature of life as we know it-all of these are but a single expression of the phenomena that result from a universal narrative. it is not yet certain that universal narratives exist, but some attempts have been made to explain life's functions from an abstract, fundamental physics perspective, e.g., [22] [23] [24] [25] . hence, the different narratives that have been developed in origins-of-life research do not address a single scientific question but many. one commonly addressed question is the origin of life on earth, which is naturally extended to the genesis of earthly life on other worlds. however, there is a completely separate question of the origin of life-like systems, whether earthly or not, in alien environments. yet another question asks how we might create, in a completely synthetic fashion, new structures that we would deem living. all origins-of-life questions-be it to explain the origin of chemiosmotic coupling in earthly life, the circumvention of the "error catastrophe" in any template-driven replicating system, or the creation of dynamical order in general-are valid, but they are not necessarily asking the same thing. these disparate aims and goals can cause the origins-of-life community to suffer from confusion (where there should be mutual understanding), friction (where there should be an amicable exchange of ideas), or apathy (where there should be genuine interest in one another's progress). this paper introduces a new vocabulary and a new definition of life in response to these concerns. we seek to reframe the definition of life in a more expansive way while recognizing the need to signify the specific kind of life that earthly forms represent. thus, we have come up with a new term-lyfe. henceforth, we will refer to life (as we know it) and lyfe (as it could be, in the most general sense). the two designations are distinguished as follows: • life represents life as we know it; it uses the specific disequilibria and classes of components of earthly life. life is an autocatalytic network of organometallic chemicals in aqueous solution that records and processes information about its environment in molecular form and achieves dynamical order by dissipating any subset of the following disequilibria: redox gradients, chemiosmotic gradients, visible/thermal photons, etc. lyfe represents any hypothetical phenomenon in the universe that fulfills the fundamental processes of the living state, regardless of the disequilibria or components that it harnesses or uses. lyfe is any hypothetical phenomenon that maintains a low-entropy state via dissipation and disequilibria conversions, utilizes autocatalytic networks to achieve nonlinear growth and proliferation, employs homeostatic regulatory mechanisms to maintain stability and mitigate external perturbations, and acquires and processes functional information about its environment. the concept of "life as we don't know it" is not new. however, traditional definitions of life struggle to clearly delineate boundaries between "life as we know it" and "life as we don't know it." furthermore, many traditional definitions are equally fuzzy with regard to when "life as we don't know it" becomes sufficiently far from "life as we know it" that it should no longer be considered life. for instance, according to nasa's definition of life (section 1), should a self-sustaining chemical system that is found evolving in a non-darwinian fashion be considered "life as we don't know it" or not alive at all? to remedy this, we have developed our criteria for lyfe based on four fundamental processes. we agree with the general sentiment of [26] that "life is a verb, not a noun," a remark based on the view that life operates by dissipating planetary redox gradients, shuttling electrons and transducing that disequilibrium into other dynamic configurations [27] . while the dissipation of free energy is certainly the first necessary aspect of life, we contend that it must be accompanied by three other processes-autocatalysis, homeostasis, and learning-to form a sufficient description of the living state. in other words, we define lyfe as any physical system that exhibits all four of the processes described below. (furthermore, life fulfills each of these via the specific mechanisms described in italics.) dissipation-lyfe cannot exist at equilibrium. the second law of thermodynamics, in the presence of free energy transduction mechanisms, permits the coupling of exergonic processes to the endergonic, organized configurations of lyfe. using an array of nanoscale molecular machines, life dissipates external chemical disequilibria and/or converts low-entropy photons into high-entropy waste heat, transducing these disequilibria into other disequilibria (e.g., endergonically building up proton gradients and high [atp]/[adp]). to perform useful work, life converts atp → adp + p i , which dissipates the [atp]/[adp] disequilibrium [28, 29] . autocatalysis-the ability of a system to exhibit exponential growth of representative measures of size or population in ideal conditions. the property of autocatalysis can appear in different forms-including self-catalysis, cross-catalysis, and network autocatalysis-as long as the effect leads to exponential growth of a suitable metric under ideal conditions. a cultured system of microorganisms exhibits autocatalytic population growth due to cellular replication in resource-abundant conditions. homeostasis-the ability of a system to maintain key internal variables within ranges of ideal set points. in a dynamic world of perturbations, coupled with the exponential growth described above, a lyving system must have means to limit the variation of its internal systems when external conditions change. life performs homeostasis with networks of sensors, receptors, and effectors. the substance under homeostatic regulation (e.g., calcium ions) typically binds with receptors and promotes the release of further substances (e.g., hormones). these indicator compounds then stimulate an appropriate response mechanism to return the substance level to within the desired window. learning-the ability of a system to record information about its external and internal environment, process that information, and carry out actions that feed back positively on its probability of surviving/proliferating. darwinian evolution is one commonly cited biological learning process (e.g., [30] [31] [32] ) among a much larger set of learning processes that living systems perform. for example, there are widely studied examples of biological learning within the realm of neuroscience, permitted by a range of neuronal and synaptic interactions (e.g., [33] [34] [35] ). in addition, there is a growing list of non-neural learning systems, including gene regulatory networks [36] [37] [38] , protein interaction networks [39, 40] , and other epigenetic mechanisms (e.g., [41, 42] ). many examples fall under the general framework of associative learning, which has been exhibited by non-neural organisms such as slime moulds [43, 44] . darwinism mingles with these other learning processes (and perhaps other hitherto undiscovered forms) to create the incredible diversity and complexity of the biosphere. hence, "learning" is an umbrella term for this large and incompletely understood set of processes. while these four pillars of lyfe are derived from observations of life as we know it (after all, life must be a subset of lyfe), this new definition is far more expansive. the four pillars constitute necessary and sufficient requirements of the lyving state while remaining divorced from the specific components that make up the system. this is illustrated by the fact that there are numerous systems that perform the same pillars but are quite distinct in form (as will be discussed in section 2.1). hence, the universality of the term "lyfe" is derived from the reasonable expectation that it can be applied to as yet undiscovered (or uninvented) systems that exist at myriad scales across the universe. there may even be a class of systems, still undiscovered and undescribed, that performs all four pillars of lyfe and some fifth pillar as well. such systems might be deemed super-lyfe. while the discovery of super-lyfe would certainly be paradigm-shifting, we remain agnostic about its existence for now. we take this moment to emphasize that our definition of lyfe applies at the system level. the pillars that can be ascribed to a certain system depend specifically on the boundaries with which we use to refer to that system. for example, which pillars do viruses perform? a single virus in isolation cannot perform any of the pillars. viruses in a system consisting of viruses, bacteria, and nutrients can perform autocatalysis and, through the coercion of their bacterial hosts, dissipation. viruses in a biosphere that is coevolving with its environment will cause not only autocatalysis and dissipation but learning (through evolution) as well. in certain ecosystems, viruses may even impart homeostatic attributes to the system by introducing auxiliary metabolic genes to their hosts and recycling organic matter via lysis [45] . thus, the argument about whether a virus (or a mule, to cite another classic paradox) is alive becomes irrelevant under the assumption that "lyfeness" does not emerge at the molecular, cellular, or organismal level. like others have suggested [46, 47] , we contend that the living state may best be assessed at an ecosystem or planetary scale. as an example of a pillar being manifested at the system level, consider learning. one might suggest that learning rate naturally applies to individual species, but in our opinion it is more effectively interpreted at the system level. this is because every species evolves in concert with other species and its abiotic environment. hence, when one species learns, other species in the system that do not become extinct must also learn. even though humans are doing a great deal of learning, other species that we share the planet with are learning how to cope with the consequences and changes due to our learning (e.g., fungal species learning to degrade plastics [48] [49] [50] or humans learning to counter pathogens with antiobiotics, which then learn resistance strategies [51] ). we note also that the importance and relevance of the pillars are separate from the ease with which we can measure their presence. for example, the measurement of the autocatalytic property is likely to be nontrivial, unless the organisms/system in question can be cultured. in particular, biosignatures from distant exoplanets will likely not be sufficient to reveal the autocatalytic property, unless those signatures are collected over very long periods. hence, for this pillar there is a difference between significance (this property is fundamental to the definition of lyfe) and ease of measurement (it may not be easy to assess). however, sometimes the autocatalytic property reveals itself in dramatic fashion. as we write, the world as we know it is being turned upside down by an entity that is both considered not to be alive and comprises only a small rna genome and a small set of proteins. every chart that has tracked covid-19 has shown exponential growth (autocatalysis) in the first phase, demonstrating how tiny biological entities can show extreme nonlinear dynamical changes in a short time. beyond granting a simple "checklist" of criteria for determining whether or not a dynamical system is alyve, the four pillars also help us place lyfe in the context of other phenomena in the universe. we define lyfe as any system that performs all four pillars and sublyfe as any system that performs some but not all of those functions (see figure 5 ). below, we list examples of phenomena that correspond to the numbered regions in figure 5 . dissipation only: thermal diffusion, or any thermodynamically irreversible process. homeostasis only: an ideal gas at equilibrium. an isolated system such as this always relaxes back to equilibrium after an internal or external fluctuation. dissipation and autocatalysis: fire is a frequently discussed example of dissipation and autocatalysis. it exhibits homeostasis of certain variables (e.g., burn temperature naturally stays within certain bounds), but its inability to fully regulate its behavior or learn from experience keeps it relegated to the nonliving world. another relevant example would by the exponential growth of products in nonlinear chemical reactions (e.g., the formose reaction). dissipation and homeostasis: a damped harmonic oscillator converts kinetic energy to thermal energy and always returns to its equilibrium position. dissipation and learning: an artificial neural network is an example system that learns and is dissipative but does not necessarily exhibit autocatalytic growth or homeostasis (e.g., it does not by itself maintain the temperature of its own hardware). one could argue that their usefulness compels us to produce them at an exponential rate, but that is another discussion. dissipation, autocatalysis, and learning: a living system that wipes itself out by tragedy of the commons. examples might include invasive species introduced to an island that destroy their food sources so fast that the food sources are damaged beyond recovery. one might also suggest anthropic climate change as another example. note that these cases depend critically on where one draws the boundary of the system (e.g., to include humans or not). indeed, this form of sublyfe or sublife is less likely to occur because if the system is capable of learning, then in principle it could learn how to regulate itself homeostatically (unless it cannot learn fast enough). 7. dissipation, homeostasis, and learning: a "smart" house thermostat that monitors occupant behavior over time. this system cannot replicate but consumes free energy, is capable of primitive learning, and can regulate its local temperature. 8. dissipation, autocatalysis, and homeostasis: thermal gray-scott reaction-diffusion spots. certain nonequilibrium chemical patterns have been shown to grow exponentially and also regulate their local temperature [52] [53] [54] [55] . 9. all four: lyfe (which includes life). regarding homeostasis in equilibrium systems (region 2 in figure 5 ), there are some delicate subtleties. our contention that homeostasis occurs in such isolated systems is simply the fact that they are the archetype of stability (by definition). however, the point is arguable from both sides. one might argue that at the instant a fluctuation occurs, there is a momentary creation of free energy. however, harnessing such a fluctuation would require measurement and information-processing, and, as [56] showed, to accomplish this in a finite memory system requires erasure, which cannot be done for free and hence there is in fact no free energy created by fluctuations of an equilibrium system. in general, any perturbation of such a system will disappear in finite time as the system re-relaxes, hence we allow for homeostasis in equilibrium systems (in contrast, biological homeostatic processes generally occur through the consumption of free energy). there are several notable macromolecular complexes that are both dissipative and autocatalytic but not necessarily homeostatic (regions 3 and 6 of figure 5 ). we already discussed viruses above, which are protein-gene complexes. amyloids (prions) are essentially peptide conformational viruses, since they propagate via a pathological spread of their own conformation on existing peptides (of different conformation) [57] [58] [59] . there are also autocatalytic genes, known as transposons: "transposable or mobile elements capable of parasite-like proliferation in the host genome" [60] [61] [62] [63] . if we also consider the biochemical examples of self-replicating micelles and droplets [64, 65] , we see that the subset of entities that are dissipative and autocatalytic potentially deserve a dedicated category of their own. we note that such a category would also include phenomena at higher levels in the hierarchy of life. for instance, internet memes are clearly autocatalytic and dissipative (given the computational energetic costs involved in their proliferation and communication). furthermore, the social-media-membership-plus-meme system learns collectively, placing it in region 6 of figure 5 . furthermore, in analogy to biological viruses, misinformation stories that are only weakly correlated with reality can be amplified by various effects to the point of causing disruptive socio-political impacts. in this subsection, we have presented a list of increasingly lyfe-like phenomena, which may give the illusion that the origins of lyfe always proceed in a simple, stepwise manner-i.e., that a prebiotic dissipative structure must first exhibit exponential growth (perhaps replication), acquire homeostatic regulatory abilities, then finally learning. in our view, it is also plausible that relatively simple systems capable of rudimentary information processing can arise de novo and that the ability of these systems to optimize over time their dissipative, autocatalytic, and homeostatic traits will determine their ultimate fate (see also [66] ). for instance, the first life on earth almost certainly did not use dna for information storage or any recognizable enzymes in its metabolic network. after innumerable tugs of war between chance and necessity, evolution produced the familiar macromolecules that we observe today. in section 2.2, we turn to how the concept of lyfe can change our approaches to investigations of the origins of life. with regard to the origins-of-life narratives discussed in section 1.1, lyfe encompasses any system that fulfills the four pillars described above but may fulfill the three classic privileged functions-replication, metabolism, and compartmentalization-using components not used by earthly life. the idea that these three privileged functions define the necessary and sufficient conditions for life was explored in great theoretical detail within the artificial life field and the concepts of autopoiesis [67] and the "chemoton" [68] . figure 6 shows a "cube" whose vertices represent living systems with different combinations of components fulfilling the three privileged functions. at one vertex, life fulfills these privileged functions using the mechanisms of rna/dna, chemiosmosis, and lipid membranes. directly connected to life are instances of lyfe that share two of the three mechanisms with life. two steps removed from life are instances of lyfe that share only one of the three mechanisms with life. furthermore, at the opposite vertex is a lyfe form completely unlike earthly life. in figure 6 , life is positioned at the apex, because this is the end goal of most origins-of-life hypotheses. in origins-of-life narratives that involve stepwise emergence, an abiotic system might follow a trajectory that moves from one vertex to another, finally arriving at life. an example of this is illustrated by the dashed purple line in figure 6 , which denotes a narrative in which the abiotic synthesis of rna was the first step in life's onset, followed by compartmentalization inside of lipid membranes, followed by the emergence of chemiosmotic metabolism (e.g., an rna world hypothesis). the way this diagram is drawn, origins-of-life narratives would be described by trajectories that move "upward" towards a convergence upon the general biochemical composition of life as we know it. however, the "cube" shape of figure 6 allows one to easily rotate the figure in the mind's eye so that any combination of fulfilling mechanisms to the three privileged functions is at the apex-perhaps as a lyfeform would see it. performing this mental gymnastics sparks two realizations: (1) the earliest stages of life's evolution may have passed through phases that fully meet the living criteria but that we would identify as lyfe; (2) origins-of-life research should be open to imagining and seeking lyfe-like solutions to the three privileged functions because they might represent attractors that our particular biological history did not find or settle in but alien lyfe did. the more detailed reasons for why a system might traverse these alternative pathways or not are a vast topic of philosophical debate, beyond the scope of the present work. as discussed in section 1.2, it is difficult to discern the components that the first life form used based on the highly evolved and complex machinery that present-day life uses. in the spirit of metaphor, we would like to highlight two analogies that are helpful when considering these issues. the first is the striking differences in composition and structure between scaffolding and finished products. the second is an analogy between biological evolution and the history of locomotives (figure 7 ). consider a state-of-the-art japanese shinkansen (with a "metabolism" run by superconducting magnets cooled with liquid helium, "compartmentalization" made of composite materials such as carbon fiber, polymers and alloys, and "information processing" by a computer guidance system). by examining the shinkansen and knowing nothing else, could one deduce the origin of the locomotive (a "metabolism" run by burning fossil fuels to boil water into steam, "compartmentalization" made of cast iron and wood, and "information processing" in the form of a human conductor)? given that train components can be swapped wholesale for one another by human hands, this analogy may appear inappropriate with regard to the commonly held view of biological evolution as a gradual process that is driven by the natural selection of minute tweaks to pre-existing elements. for example, in so-called "onion hypotheses" regarding the evolution of life [69] [70] [71] , it is assumed that life began with a metabolic core of reactions, around which all further layers of complexity were laid. however, as [72] argued, the evolution of life has progressed through several "major transitions," many of which reshaped biology's information processing capabilities. it is quite possible that at the earliest stages of life, such transitions were rampant, as primitive living systems battled for supremacy in a relatively flat fitness landscape, trading components with ease via horizontal transfers of information and material. upon the invention of a mechanism that conferred greater selective advantage than the rest, that machinery-be it the ribosome or atp synthase-would become a stable attractor in earthy biology's evolutionary trajectory. in his book seven clues to the origin of life, ref. [73] analogizes the lineage of life to a long rope made of overlapping fibers, in which no one fiber stretches from beginning to end. he writes: "there is then a simple way in which the central control machinery of organisms could have been updated: through a gradual takeover. a rope of hemp fibers at one end could gradually transform into a rope with only sisal fibers in it, by hemp fibers fading out and sisal fibers fading in." in our opinion, the truth is probably closest to a hybrid of these rope and onion concepts. life's emergence and early stages are probably well represented by the rope concept, with different fibers joining, contributing, and leaving the rope. building from the "long rope" analogy, we suggest that cairn-smith's "takeovers" could have been sudden rather than gradual. however, once evolution stumbled upon a fiber with superior efficiency or stability, that feature would rapidly become integrated and grow into a central thread of the rope of life, forever ingrained in the biosphere's molecular core. layers of function accumulating around the rope core would prevent core components from being lost from the rope. this results in an "onion-like" history that can be peeled apart via modern techniques like molecular phylogeny and metabolomics. note that the 'locking-in' of functionality occurs in many complex systems including economic, technological, and political systems. core systems that interface with many higher subsystems become so functionally entwined with the various modules that any changes to the core systems would be detrimental or catastrophic to the coherent whole. this can be true even if there are superior candidate systems for the core. for example, many european cities grew organically over long periods and now have a somewhat fractal organization. compare this to the much more planned grid structures of american cities. changing the fundamental structure of european cities is now too costly so they are stuck with their undesigned layouts. it is also quite plausible that the modern components of core biochemical systems performed different functions originally. the biosphere is replete with examples of such exaptation: the shift in function or co-optation of a component for another use. feathers originally granted warmth and signaling abilities before they became tools of flight; wings were likely used to augment running speed before being used for flight [74] ; the water oxidizing complex that ushered in the age of oxygenic photosynthesis may have originally been used for manganese oxidation [75] . for further examples of exaptation, see the seminal work by [76] and citations therein. thus, we should be careful not to unequivocally equate a certain component's universality in the modern biosphere with its necessity at the emergence of life. perhaps long strands of nucleotides were not the first information processing system; perhaps polyphosphate chains were not the first "energy currency" of life; perhaps the first membranes were not composed of organic hydrocarbons. origins-of-life narratives that seek abiotic pathways to synthesize the specific "building blocks" of life operate under the assumptions that those molecules: (1) were involved in the origins of life; (2) performed the same functions at the origins of life that they do today; (3) exhibited functionality that follows form, i.e., once all of the materials of life were synthesized and colocated, the complex information processing abilities of life would simply emerge (some have even described this third assumption as a form of modern-day "vitalism" [77] ). this narrow approach is necessarily blind to scenarios where life-likeness could begin using alternative components, as might have occurred not only on earth but elsewhere in the universe. so, when investigating the emergence of life, what are we looking for if not specific biomolecules? the definition of lyfe offers an answer: we seek a system that exhibits dissipation, autocatalysis, homeostasis, and learning. by basing the criteria for lyfe on generic processes-rather than specific components that perform specific tasks-we open our minds to the exploration of all systems that display these emergent properties, freeing ourselves from the restrictions of precise chemical recipes whose prescriptions contain assumptions that may limit our explorations of the emergence of life-like behavior in the universe. the origins-of-life community has suggested many hypotheses that involve stepwise emergence through lyfe-like phases. using our classification, these emergent systems could be classified as lyfe or sublyfe because they utilize radically different components than present-day life to achieve one or more of the four pillars of lyfe. this section describes a few salient examples of origins hypotheses that utilize alternative components at the origin of life. some authors have hypothesized that the earliest metabolic systems may have been thioester-driven [70] (also note the concerns raised by [78] ), rather than phosphate-driven, due to the inaccessibility of inorganic phosphate on the early earth [79] (also note contradictory opinions [80] ). phosphorous is a key constituent of modern biology; it is found in atp, the universal energy currency of life, metabolic cofactors like nadh, and information storage molecules like dna and rna. however, ref. [81] identified a plausible phosphate-free metabolism using a systems biology approach. within the biosphere, a phosphate-independent metabolism exists that heavily depends on iron-sulfur and transition metal enzymes-which themselves have been linked to geochemical scenarios for the emergence of biochemistry [82] [83] [84] . network-based algorithms suggest that various environmental parameters could result in a thioester-based proto-metabolic network similar to the reductive tricarboxylic acid cycle [85] . this would result in a "thioester world", which, once phosphate became bioavailable, transitioned to the modern "phosphate world" that features atp and nucleotides [70] . thus, the thioester world, if it ever existed, would have been a primitive but thriving biosphere of lyfeforms. self-replicating clay minerals have been suggested as the first informational structures of life [86, 87] . in this hypothesis, information is contained in the defects, irregularities, and aperiodic features in crystals' chemical and spatial structures. because crystals grow through the addition of planar, complementary layers, this information can be "replicated" when growing crystals break. these crystal genotypes can manifest as phenotypes that affect the environment in which the crystal is growing as well as the catalytic power of the crystal [73] . of particular interest is the autocatalytic potential of clay-organic systems. for example, it has been suggested that the origin of life could have involved iron-rich clays that could perform light-induced charge transfer to reduce co 2 to functional organic molecules [69] . clay minerals are also known to promote polynucleotide synthesis [88, 89] . in this hypothesis for the emergence of life, an "organic takeover" would eventually ensue, in which organic-organic autocatalysis superseded clay-organic systems. thus, this scientific narrative has clay minerals providing the scaffold for organic biochemistry: a clay-organic form of lyfe. recently, green rust (formally known as fougerite), an fe 2+ /fe 3+ oxyhydroxide, has been suggested as the fundamental seed of life [90] . this metastable mineral is hypothesized to have been a constituent of hadean submarine alkaline hydrothermal vents. due to its reduction-oxidation capabilities, green rust is thought to have been an important driver of organosynthesis in these contexts [90] . within its flexible, anhydrous interlayers, nitrate can be converted to ammonium [91, 92] , and it is hypothesized that inorganic carbon can be converted to pyruvate, which can then be aminated to alanine. at the same time, the theory suggests that green rust could act as a primitive inorganic pyrophosphatase-making it one of the first nanoengines of life [93] . some have even hypothesized that the variable fe 2+ /fe 3+ cations might have served as a primitive information-storage system [86, 93, 94] . if green rust (or a similar mineral) played the role of a molecular machine at the emergence of life, then that protometabolic system could be classified as lyfe. in emergence hypotheses that occur in hydrothermal systems, compartmentalization is not achieved through spontaneously assembling lipid membranes but by a maze of inorganic mineral pore spaces. only after the invention of lipid biosynthesis and the subsequent invention of cell wall biochemistry did life-like cells emerge and escape their hydrothermal confines [95] . however, before they became free-living prokaryotes, the complex chemical networks lodged inside their mineral enclosures could still be considered alyve (or subalyve before achieving all four pillars of lyfe). other researchers have considered amyloids-polypeptides with a unique β-sheet fold-as the first self-replicating biological entity. it is a longstanding problem that rna synthesis and polymerization is a challenge in most abiotic environments (e.g., [96, 97] ), although several advances have been made in this area recently (e.g., [98] ). amyloid-based replicators offer an alternative to rna: their monomers are more readily synthesized in prebiotic scenarios; they are stable under early earth conditions; they are self-assembling, replicative, catalytic, and may be capable of adapting to changes in their environment [57, 58] . although amyloidosis is most famously known for being associated with human diseases [99] , modern life uses functional amyloidal proteins to its benefit, from biofilm formation to dehydration resistance to long-term memory [58] . should the earliest living entities have comprised an "amyloid world," rather than an "rna world," this would represent a unique type of lyfe. the biochemistry of life-from its water-based nature to its heavy reliance on chnops-is a reflection of earth's physical and chemical conditions. other worlds that feature potentially similar habitats-for instance, hydrothermal systems on europa or enceladus-may be inhabited by living creatures that are biochemically similar to life. however, on worlds that occupy radically different physical and chemical spaces to those of earth, any exobiology that exists would surely be lyfe, not life. one intriguing possibility is titan, the only other world in the solar system known to possess stable bodies of liquid on its surface. despite titan's broad-brush similarities to earth (atmospheric pressure, geomorphology, an active methalogical cycle akin to earth's hydrological cycle, etc.), if exobiology exists on titan, it would have emerged and evolved in an environment incredibly dissimilar to earth. major environmental differences include: extremely low surface temperatures of ∼94 k (resulting in exotic van der waals-driven organic chemistry [100, 101] ); a lack of available oxygen for biochemistry (resulting in hypotheses for n-substituted biochemistries [102] ); and a nonpolar ch 4 -c 2 h 6 solvent (resulting in speculations for alternative membrane structures). addressing the final difference in more detail, ref. [103] theorized that acrylonitrile (c 2 h 3 cn) could form stable membrane-like structures called azotosomes in titan's lakes and seas. although acrylonitrile has recently been detected in titan's atmosphere [104] , recent quantum mechanical calculations have shown that azotosome membranes do not self-assemble in titan-like conditions [105] . however, this may not rule out the possibility that lyfe-like entities can construct them. alternatively, membranes may not be required for lyfe on titan because the extraordinarily low temperatures already prevent the dissolution of macromolecules, and membrane-like structures would inhibit the diffusion of metabolites in and out of a cold, stationary lyving system [105] . should such a membrane-less exobiological entity exist, it would certainly be a provocative example of lyfe. in the spirit of imagining lyfe as we do not yet know it, we now present a hypothetical form of life that uses an alternative dissipative/metabolic system: the transduction of mechanical work into chemical disequilibria. earthly life utilizes various external free energy sources, from redox couples to solar radiation to gradients of proton density and even electron density (e.g., electrotrophs [106] ). macroscopic organisms are known to exploit abiotic sources of mechanical work and use them to their advantage; examples of this include salmon hitching rides on turbulent eddies to swim upstream [107, 108] , birds catching thermals to higher altitudes, and humans building wind turbines and hydroelectric power plants. however, there is no cataloged example of an organism that transduces mechanical work directly into its metabolism. we find this rather surprising, since the reverse process is such a fundamental component of all life, i.e., the conversion of chemiosmotic gradients to rotational motion using the atp synthase family of molecular motors [109] . this rotational motion is most well known for synthesizing atp from adp and p i , but it is also used to produce cellular movement in fluid environments. this occurs through the flagellar motor proteins, close relatives of the atp synthases [110, 111] . flagellar motors dissipate the free energy of atp hydrolysis in eukaryotes, or ion motive forces in prokaryotes, for the production of rotational motion. rotation of the central rotor causes the flagellar filament to spin, and the structure of the filament allows the rotational motion to produce translational movement of the organism in water (this motion has been compared to the mechanism by which corks are thrust from a bottle by the rotation of a corkscrew [112] ). why could this process not be reversed to power a mechanotrophic organism? imagine a single-celled organism in flowing water that is anchored to a rock via a pilus-like tether filament (like a person parasailing behind a boat). assume that such an organism is equipped with the type of flagellar motor proteins that have been studied in swimming bacteria or single-cell eukaryotes. the flow of water causes rotational motion of flagellar filaments, and this rotational motion is used for atp synthesis (or perhaps some other endergonic chemical step in more exotic lyfe), as shown in figure 8 . whether this is feasible depends on the thermodynamic and fluid dynamic conditions. note that such a mechanotrophic organism might have a different design to the archetypal swimming bacterium. for example, it may use a torsionally stiff tether attached to a molecular generator molecule (the flagellum being driven), and the whole cell might rotate around the tether-generator complex. in this case the flagellum would be fixed relative to the cell body (at the opposite end to the tether-generator complex) and would rotate with the cell. the hydrodynamic details, efficiencies, and energetics of such an organism will be explored in detail in a future work. here we simply wish to highlight the possibility of such an organism. this would be a prime example of a lyfeform, though it is also possible that such an organism exists on earth but has simply gone unnoticed to date. we have presented a new conceptual definition of life that encompasses four pillars: dissipation, autocatalysis, homeostasis, and learning. any physical system exhibiting all of these necessary and sufficient features could be classified as lyfe, our designation for the set of all systems fulfilling the criteria, including the more specific case of life on earth. this is in contrast to the idea of privileged functions, the pursuit of which has guided much of the research in the origins-of-life field. systems that show differing levels of similarity to life (e.g., a different metabolic system or information-processing apparatus), could be considered lyfe or sublyfe. this would include phases of life's emergence before it arrived upon the familiar metabolic cycles and pathways, dna/rna information storage and processing, and lipid membranes. in the context of astrobiology, our conceptual framework can help clarify the objectives and discussions surrounding origins and biosignature research. when seeking to explain the emergence of biology on earth or signatures of exobiology that are directly analogous to life on earth, it would be appropriate to refer to life. on the other hand, when addressing questions related to more general systems that share the properties of life but differ in chemical or structural specifics, it would be appropriate to refer to lyfe. for example, finding unicellular chemotrophs resembling archaea on europa would constitute a discovery of life. on the other hand, finding methane-based biology on titan would constitute a discovery of lyfe. whether exobiology can emerge from a multitude of abiotic states or just one is a profound scientific question. indeed, our ignorance in this matter is what motivates a conceptual definition of life that is agnostic to specific components and highlights processes instead. only by seeking other instances of lyfe can we know if life is a proper subset of lyfe or if life and lyfe are identical sets. by framing life as a subset of a larger class of hypothetical phenomena called lyfe, we encourage the search for lyfe as we do not know it. this search can lead us to a deeper scientific understanding of what, how, and why we are: if we find that lyfe exists only in the form of life, that would suggest that the specific molecular components of life dictate the functionality of the lyving state (in line with a reductionist point of view). however, if we find that lyfe exists in myriad forms, that would suggest that "lyfeness" is emergent. finally, we also suggest a hypothetical lyfeform that transduces mechanical energy from a flowing fluid into its metabolism, a so-called mechanotroph. such an organism has not been cataloged, and hence remains in the realm of lyfe. however, since no one is looking for it, it may be hiding in plain sight on earth, which would render it a member of the life set. in closing, we facetiously submit that this journal should henceforth be rebranded as lyfe because of its expansive scope and open-mindedness towards publishing ideas such as ours. the algorithmic origins of life the origin of life: models and data origin of first cells at terrestrial, anoxic geothermal fields physico-chemical and evolutionary constraints for the formation and selection of first biopolymers: towards the consensus paradigm of the abiogenic origin of life when did life likely emerge on earth in an rna-first process? chemsystemschem the drive to life on wet and icy worlds turnstiles and bifurcators: the disequilibrium converting engines that put metabolism on the road escapement mechanisms and the conversion of disequilibria; the engines of creation designs for life: protocell models in the laboratory molecular crowding and early evolution lipid vesicles chaperone an encapsulated rna aptamer opinion: studies on the origin of life-the end of the beginning coupled phases and combinatorial selection in fluctuating hydrothermal pools: a scenario to guide experimental approaches to the origin of cellular life signatures of a shadow biosphere the ribofilm as a concept for life's origins ancient horizontal gene transfer and the last common ancestors hidden concepts in the history and philosophy of origins-of-life studies: a workshop report a strategy for origins of life research directed evolution: bringing new chemistry to life hachimoji dna and rna: a genetic system with eight building blocks statistical physics of self-replication dissipative adaptation in driven self-assembly origins of life: a problem for physics, a key issues review universal scaling across biochemical networks on life is a verb what is life?: the physical aspect of the living cell frankenstein or a submarine alkaline vent: who is responsible for abiogenesis? frankenstein or a submarine alkaline vent: who is responsible for abiogenesis? probably approximately correct: nature's algorithms for learning and prospering in a complex world evolutionary connectionism: algorithmic principles underlying the evolution of biological organisation in evo-devo, evo-eco and evolutionary transitions how can evolution learn? integrated information theory: from consciousness to its physical substrate molecular circuits for associative learning in single-celled organisms associative learning in biochemical networks predictive behavior within microbial genetic networks protein molecules as computational elements in living cells wetware: a computer in every living cell computational functions in biochemical reaction networks advances in physarum machines: sensing and computing with slime mould habituation in non-neural organisms: evidence from slime moulds phage puppet masters of the marine microbial realm the origin and nature of life on earth: the emergence of the fourth geosphere life as a planetary phenomenon recent trends and advances in the biodegradation of conventional plastics microbial degradation and other environmental aspects of microplastics/plastics biodegradation of polyethylene microplastic particles by the fungus aspergillus flavus from the guts of wax moth galleria mellonella antibacterial resistance worldwide: causes, challenges and responses review why is life? an assessment of the thermodynamic properties of dissipative, pattern-forming systems emergence of competition between different dissipative structures for the same free energy source a precarious existence: thermal homeostasis of simple dissipative structures delving deeper into homeostatic dynamics of reaction diffusion systems with a general fluid dynamics and artificial chemistry model irreversibility and heat generation in the computing process on the possible amyloid origin of protein folds self-propagating β-sheet polypeptide structures as prebiotic informational molecular entities: the amyloid world a prebiotic template-directed peptide synthesis based on amyloids dna transposons and the evolution of eukaryotic genomes a virophage at the origin of large dna transposons inevitability of genetic parasites self-synthesizing dna transposons in eukaryotes autocatalytic self-replicating micelles as models for prebiotic structures replicating vesicles as models of primitive cell growth and division probing complexity: thermodynamics and computational mechanics approaches to origins studies autopoiesis and cognition: the realization of the living organization of chemical reactions into dividing and metabolizing units: the chemotons speculations on the origin and evolution of metabolism origin of the genetic code is found at the transition between a thioester world of peptides and the phosphoester world of polynucleotides peeling the onion: ribosomes are ancient molecular fossils the major transitions in evolution seven clues to the origin of life: a scientific detective story identification of avian flapping motion from non-volant winged dinosaurs based on modal effective mass analysis how manganese empowered life with dioxygen (and vice versa). free. radic exaptation-a missing term in the science of form fougerite: the not so simple progenitor of the first cells the abiotic chemistry of thiolated acetate derivatives and the origin of life are polyphosphates or phosphate esters prebiotic reagents? phosphorus in prebiotic chemistry remnants of an ancient metabolism without phosphate mineral surfaces, geochemical complexities, and the origins of life on the origin of biochemistry at an alkaline hydrothermal vent evolution of the first metabolic cycles environmental boundary conditions for the origin of life converge to an organo-sulfur metabolism layered double hydroxide minerals as possible prebiotic information storage and transfer compounds clay minerals and the origin of life clay minerals mediate folding and regioselective interactions of rna: a large-scale atomistic simulation study catalytic activity of hammerhead ribozymes in a clay mineral environment: implications for the rna world the simple organizing seed of all life? life kinetics of nitrate reduction by green rusts-effects of interlayer anion and fe(ii):fe(iii) ratio fougerite a natural layered double hydroxide in gley soil: habitus, structure, and some properties. in clay minerals in nature-their characterization, modification and application figuring out how life first took off is (much like) rocket science! crystals and life on the origins of cells: a hypothesis for the evolutionary transitions from abiotic geochemistry to chemoautotrophic prokaryotes, and from prokaryotes to nucleated cells the rna dreamtime: modern cells feature proteins that might have supported a prebiotic polypeptide world but nothing indicates that rna world ever was a simpler origin for life unified prebiotically plausible synthesis of pyrimidine and purine rna ribonucleotides protein misfolding, amyloid formation, and human disease: a summary of progress over the last decade a co-crystal between benzene and ethane: a potential evaporite material for saturn's moon titan prospects for mineralogy on titan oxygen-free biochemistry: the putative chn foundation for exotic life in a hydrocarbon world membrane alternatives in worlds without oxygen: creation of an azotosome alma detection and astrobiological potential of vinyl cyanide on titan can polarity-inverted membranes self-assemble on titan? sci. adv. 2020, 6 electromicrobiology: realities, grand challenges, goals and predictions fish exploiting vortices decrease muscle activity extraction of flow energy by fish and birds in a wavy stream atp synthase-a marvellous rotary engine of the cell the turn of the screw: the bacterial flagellar motor bacterial flagellar motor this article is an open access article distributed under the terms and conditions of the creative commons attribution we extend our heartfelt thanks to the members of the caltech astrobiology reading group, with whom many of the ideas in this paper were passionately discussed. the authors declare no conflict of interest. the following abbreviations are used in this manuscript:luca last universal common ancestor key: cord-263450-v6vdg8os authors: shegogue, daniel; zheng, w. jim title: object-oriented biological system integration: a sars coronavirus example date: 2005-05-15 journal: bioinformatics doi: 10.1093/bioinformatics/bti344 sha: doc_id: 263450 cord_uid: v6vdg8os motivation: the importance of studying biology at the system level has been well recognized, yet there is no well-defined process or consistent methodology to integrate and represent biological information at this level. to overcome this hurdle, a blending of disciplines such as computer science and biology is necessary. results: by applying an adapted, sequential software engineering process, a complex biological system (severe acquired respiratory syndrome-coronavirus viral infection) has been reverse-engineered and represented as an object-oriented software system. the scalability of this object-oriented software engineering approach indicates that we can apply this technology for the integration of large complex biological systems. availability: a navigable web-based version of the system is freely available at http://people.musc.edu/~zhengw/sars/software-process.htm contact: zhengw@musc.edu supplementary information: supplemental data: table 1 and figures 1–16. a paradigm shift is occurring with the study of individual cellular components progressing toward the study of the cell at the system level (ideker et al., 2001) . however, no well-defined process or effective integration methodology, in combination with a widely accepted system representation has been developed to provide a comprehensive view of a biological system. biological systems are currently presented in a variety of forms, such as relational databases (galperin, 2004) , diagrams (kohn, 1999; peleg et al., 2002) , ontology (ashburner et al., 2000) and markup language (hucka et al., 2003) , but they are integrated through an ill-defined, informal process. this assortment of information integration methods can hamper biological studies, especially of large complex systems. a well-defined process and integration technology can orchestrate system-level integration efforts by biologists, computer scientists and engineers, and provide consistency and manageability during integration. current advanced engineering systems and biological systems have followed a convergent evolution (csete and doyle, 2002) . * to whom correspondence should be addressed. this resemblance is particularly evident in a comparison between object-oriented software and typical biological systems (table 1 and supplemental table 1 ). both systems are based on components and protocols, and their layered control mechanisms allow them to efficiently handle complexity. this complexity is further managed by subsystems, both in biological and object-oriented software systems. furthermore, these systems contain static time-independent and dynamic time-dependent relationships. this resemblance has led to a very similar architecture and system-level organization, making it possible to represent a biological system as an object-oriented software system. to effectively construct a high-quality object-oriented software system, three components are necessary. first, a well-defined software engineering process, such as the waterfall model, rational unified process, agile process, etc. (graham, 2001) , is needed to specify stepwise activities and define what should be accomplished during software development. second, object-oriented technology, a paradigm of software development, including object-oriented programming, analysis, design and database centered on the object concept, is vital for object-oriented software development. this technology is used to dissect real-world problems and create an object-oriented software system to solve these problems. third, a widely accepted object-oriented modeling language, such as the unified modeling language (uml) (rumbaugh et al., 1999) , should be used to represent this object-oriented system. this visual representation can help to organize and communicate a complex software system as small, manageable pieces without losing system integrity. while these components were specifically designed for successful software development, they can be adapted for biological system information integration. the similarity between biological and object-oriented software systems at the system organization level indicates that we can represent one by another. therefore, the integration of biological information at the system level can be approached by reverse engineering the biological system into an object-oriented software architecture. following a well-defined software development process to reverse engineer a biological system offers significant advantages. although simple systems can be easily modeled by an indiscriminate approach, a formal approach is necessary for the creation of complex system models, such as those found in biology. moreover, when this methodology is applied to biological system modeling, the distinct phases of software development allow tasks best suited for call-back functions, observer/visitor patterns (gamma et al., 1997) feedback controls one part of the system originates a signal that affects the behavior of the other components of the system and this interaction in return affects the behavior of the subsystem where the signal originated component dynamics life cycle of individual objects life cycle of individual proteins the birth and death of individual components a see also discussion section. the concepts of biological system and object-oriented software systems are compared and the commonalities among these concepts are listed. we only used the cell system to represent a typical biological system in this table. this comparison focuses on the system organization and functionality. we provide correlations between the main object-oriented concepts (i.e. object, composition, inheritance, polymorphism and encapsulation). however, biological and object-oriented system concepts are not necessarily a one-to-one mapping. modeling of some of the biological system components and a further explanation of object-oriented concepts can be found in the main text. biologists or computer scientists to be divided and coordinated. in addition, applying a well-defined software engineering process and object-oriented methodology provide an effective means to capture specifications from experimental data and integrate the biological system information. finally, this process provides a guideline for the development of an integrated biological system, represented as an object-oriented software architecture, in a widely accepted objectoriented modeling language (such as uml), which can facilitate communication about complex systems among software engineers, biologists and other users. recently, biologists have also begun to develop and specify processes for biological data integration, such as the one used for the transpath database (schacherer et al., 2001) , but this activity has been fairly limited and has not gained the community-wide acceptance comparable with popular software engineering processes. to demonstrate the efficacy of a well-defined software engineering process in the translation of a biological system to a model grounded in object-oriented principles, we used uml in the development of a severe acquired respiratory syndrome-coronavirus (sars-cov) model. sars-cov, which killed nearly 800 people and infected 8000 worldwide, consists of 28 structural and non-structural proteins. although the viral infection and replication process is not entirely resolved, some details have emerged. briefly, the virus binds to the cellular ace2 receptor via its spike (s) protein, then fuses with the host cell membrane (li et al., 2003) . genomic rna is released, and gene one, encoding the non-structural replicase proteins is translated. the replicase proteins are cleaved by the papain-like proteinase (rota et al., 2003) and 3c-like protease (fan et al., 2004) also contained within gene one. the replicase produces the structural protein mrnas from the remaining genome, which are subsequently translated. the structural proteins (ying et al., 2004) consist of the membrane (m) (rottier et al., 1986) and envelope (e) (yu et al., 1994) proteins, which form the viral envelope, the s protein that binds the cellular receptor (xiao et al., 2003) and nucleocapsid (n) protein, which associates with the viral rna to form the ribonucleoprotein (ying et al., 2004) . the recently identified transmembrane proteins u274 and u122 may also serve as structural proteins (fielding et al., 2004; tan et al., 2004) . the virus assembles in an intermediate compartment between the golgi and endoplasmic reticulum (lai and cavanagh, 1997) , and is released via the constitutive exocytic pathway (lai and cavanagh, 1997) . in this paper, we demonstrate that a complex biological system can be reverse-engineered, and the resulting software representation can capture the static and dynamic relationships contained with the system. information collection to define the requirements and collect the information necessary for model generation, two approaches were necessary. first, annotations of the sars viral gene products were obtained through an automated pipeline, geneatlas, implemented by accelrys, inc. (kitson et al., 2002) . this pipeline can efficiently identify the function of a gene or a protein through homology searches and structural analysis (yan et al., 2003) . second, an extensive literature review was manually conducted. detailed information about the sars-cov and viral infection were collected. because of the relatively recent appearance of sars-cov, when information was not available, functions and relationships were extrapolated from homologous genes in other viruses. the attributes and the interactions of the viral proteins were captured using crc cards as described by shegogue and zheng (2004) (for an example of a crc card, see supplemental figure 1 ). use case development based on the information gathered, a use case was developed to reflect the viral infection process (see supplemental figure 2 ). the use case also serves to define the boundary and scope of the sars-cov model. therefore, although the infection process may involve other elements such as the immune system, this study was limited, as defined in the use case, to those events that directly involve the interaction between the virus and the cell. use case diagram generation to define the high-level interaction between the sars-cov and the cell, a uml use case diagram was generated (see supplemental figure 3 ). this diagram was based on the boundaries defined in the use case. this and other uml diagrams in this study are generated using microsoft visio pro. for a review on uml models see http:// bdn.borland.com/article/0,1410,31863,00.html conceptual model generation to provide an overview of the system and its interrelationships a conceptual model, or simplified class diagram, was generated based on the information defined in the requirement-gathering phase. this conceptual model integrated biological information, and represented the viral and cellular components involved in viral infection and their relationships in uml notation (see supplemental figure 4 ). by applying object-oriented analysis, the sars virus and the cellular components involved were decomposed into objects and the component relationships were realized. however, information regarding component properties is hidden. this intermediate artifact defines the organization of the biological system and provides an overview of the components and their relationships. system sequence diagram generation to capture the sequence of events occurring between the sars-cov and cell at the system-level, a system sequence diagram was created (see supplemental figure 5 ). state diagram generation state diagrams were created to capture the transitions and different states that a cellular component can exist. in addition, multiple concurrent states can be illustrated using this uml notation. an example of a state diagram for the m protein is shown in supplemental figure 6 . additional state diagrams for the s, e and n proteins can be found at http://people.musc.edu/∼zhengw/sars/state%20diagrams/ web%20page/statechart_index.htm sequence, activity and class diagram generation sequence, activity and class diagrams have been used as an example to demonstrate the feasibility of generating an object-oriented representation of the sars-cov and cell system. to generate these diagrams, objects representing corresponding bioentities are created and their essential attributes are captured. interactions among objects are also identified. for each interaction, a corresponding method is generated. the nature of the interaction determines the method parameters. to generate sequence diagrams the sequence of events is captured. scenarios are also generated for object interactions and used to generate activity diagrams. the information captured in the sequence diagram and activity diagrams are used, along with the bioentities attributes, to generate class diagrams. model validation models are validated by referring back to the crc cards and use case to determine if the requirements set forth in the requirementgathering phase have been met. web-based implementation these diagrams, along with crc cards for objects in the system, were incorporated into a web-based system to deliver our object-oriented representation to the users (http://people. musc.edu/∼zhengw/sars/software-process.htm). to capture the static and dynamic relationships that occur between sars-cov and a cell, models were constructed by following an adapted, linear, sequential software process containing distinct phases of requirement gathering, analysis and design (fig. 1) . this approach is suitable because of the relative simplicity of the viral infection process. since the major activities (such as requirement gathering, analysis and design) of this sequential software process serve as the foundation for other advanced software engineering processes, these advanced processes can be applied to modeling more complex biological systems. the requirement-gathering phase entails collecting all available information about the system, generating crc cards (see supplemental figure 1 ) to capture biological entity (bioentity) functions and collaborators, and defining a use case (see supplemental figure 2 ). the analysis phase is used to dissect the interrelationships among components involved in viral infection and serves as a basis for the detailed models generated in the design phase. here, the gathered information is used to create a use case diagram (see supplemental figure 3 ), conceptual model (see supplemental figure 4 ) and system sequence diagram (see supplemental figure 5 ). in the design phase, information from the analysis and requirement-gathering stages is incorporated to develop a detailed model, which represents the static (class diagram, see supplemental the major events of sars-cov infection include virus binding, membrane fusion, viral rna replication, structural proteins translation, viral assembly and release. sequence diagrams were created to capture the dynamic nature of these processes as an object-oriented software system. a high-level representation of the sars viral infection is reflected chronologically in figure 2 . simple (such as proteins) or complex (such as viral replicase or virus) bioentities, identified by literature searches, are modeled as objects, which are represented by rectangles with vertical lifelines. object functions, which are implemented by the methods contained within the objects, are used to represent the functions of these bioentities. for example, a viral replicase can take a viral rna as a template and generates a new viral rna through replication. similarly, the viral_replicase object function, replicate(), takes a viral rna as a template and generates new viral_rna. to capture interactions between objects, one object can call a method of another object by connecting object lifelines in the sequence diagram (fig. 2) . this invocation of an object function by another is described as one object sending a message to another object. in this way, real-world processes may be captured using an object-oriented approach. for instance, the cell object can call the translation machinery to translate the viral replicase by sending a message translate(in: positive_genomic_rna), where the value after the 'in' is an object, positive_genomic_rna, that must first be passed into the method. to complete the process, the translation machinery will use this positive_genomic_rna as a template to translate and create a 'viral replicase' object, which is sent back to the cell. the cell now contains a copy of the viral replicase for later use. together, this message passing captures the cellular process of translation of a viral rna into a viral replicase. alternatively, a message need not be passed between objects. a self-call may include self-checks or autoactivation signals. in the case of the assemblevirus() function call, the cell requires two components, structural protein and viral rna, to generate the virus. however, a self-call might only indicate whether an event has occurred and not accept any parameters. for example, the bindvirus() method only indicates whether a virus has bound the cell. together, the events leading to the creation of a virus can be chronologically ordered and can mimic the viral infection process. additional details that reflect the rna replication process, translation of the structural proteins and viral assembly are available in supplemental figures 7-9 . collectively, these diagrams demonstrate that the activities of a biological system can be represented as part of the dynamic processes of an object-oriented software system. to capture additional features of the dynamic architecture, activity diagrams were created to reflect the sars-cov and viral infection process. unlike the sequence diagram, which captures best-case scenario events, the action sequence or flow of the activity diagram can portray alternative outcomes. for instance, as shown in figure 3 , a virus may bind a receptor if one is available, or else this action ends and the flow is directed to the final state. once bound, if the s protein undergoes a conformational change that exposes the fusion peptide, then the virus can fuse with the cellular membrane. alternatively, the virus may detach from the cell, or if a neighboring cell is available cell-cell fusion may be initiated or the flow may be directed to the final state, indicating that infection failed. if the virus is able to fuse with the membrane, release of the n protein will decoat the positive genomic rna, or else this action will also end. once decoated, the viral rna is replicated after which the virus is assembled and released. these subactivities are presented in greater detail in supplemental figures 11-13 . in addition, supplemental figure 10 illustrates a high-level view reflecting only the major stages of the viral infection process. overall, these activity diagrams define the main success and alternative scenarios that may occur during viral infection. complex bioentities and their relationships are contained within the biological system encompassing the cell and sars-cov. these relationships include sars-cov binding to the cell receptor, viral protein interacting with the translation machinery and rna interacting with the viral replicase. to capture this static architecture, class diagrams were generated that model the components, operations and interrelationships that occur between the sars-cov and cell. specifically, figure 4 , a high-level class diagram, captures the major components of the sars virus and viral infection as objects and their associations using an object-oriented representation. these objects, where applicable, were given attributes that describe important characteristics, or if changed, might alter the function of a fig. 2 . a high-level sequence diagram for major sars viral infection events. bioentities (cell, virus, translation machinery and viral replicase) are modeled as objects. these objects are shown at the top of the diagrams with vertical lifelines ( ), representing extensions of the objects below. functions and events associated with these bioentities are modeled as messages sent to the corresponding objects. these message calls, indicated by solid arrows, are made between objects via connection of their lifelines. messages contain a message name followed by parameters, which must be passed into the object and/or outputted from the object, e.g. translation machinery executes its function of translating viral mrna by receiving a translate message 'translate (in: positive_genomic_rna)' from the cell. this message accepts a positive_genomic_rna as a template, indicated by 'in: positive_genomic_rna', then a 'viral_replicase' is created and returned. these return values are displayed below the message call as a named dotted arrow extending in the opposite direction as the original message call. where return values are not explicitly indicated, as in an object calling itself ( ), an 'out' followed by a return value is substituted. events in gray boxes are shown in more detail in supplemental figures 7-9. component. the object functions, which parallel bioentity functions, were generated from the sequence diagrams. these functions or operations are a declaration of the methods that an object may use. in addition, protein complexes can be represented, as in the case of the viral replicase, as complex objects, which are composed of many other objects (see supplemental figure 15 ). similarly, utilizing composition allows the virus object to mimic a real-world virus, which contains structural proteins, positive genomic rna and a membrane (fig. 4) . larger systems such as organelles and pathways can also be contained within the object-oriented software system in the form of modules, subsystems or packages. in this way, class diagrams allow a biological system to be captured as a layered system, e.g. the cellular translation machinery may be represented as a single object, even though it has a very complex mechanism and regulation. this complexity could also be captured in a subsystem that specifically focuses on the cellular translation machinery. this layered structure indicates that the object-oriented technology can be used to represent the layered structure of complex biological systems. in addition, the uml notation provides a mechanism to specify inheritance, which may be used to indicate an object that is the foundation for other objects. for instance, structural protein and gene1 viral protein precursor inherit the properties of the viral protein object (fig. 4) . binary associations are also used to capture associations between objects. these associations may contain cardinalities, which indicate how many of one object interacts with another. for instance, a virus can infect one cell and a cell can be infected by one to many viruses (fig. 4) . additional class diagrams showing a higher level of detail and focusing on the interactions and components of the ) are captured during different time periods. this diagram can represent sequential as well as concurrent events. concurrent events are represented by forks ( ) and merges ( ). this architecture signifies that multiple events are possible simultaneously and the occurrence of at least one of these events is enough to continue the flow of control. in contrast, the flow of control may also be conditional, e.g. decision diamonds ( ) represent a point at which alternative events can occur. at these decision diamonds, guard conditions must be satisfied for the flow of control to continue, or else the flow of control follows an alternative route. structural proteins, replicase complex and viral replication process are available in supplemental figures 14-16 . these class diagrams demonstrate that the static structure of a biological system can be represented as an object-oriented model. together, the models generated using the described object-oriented methodology yield a software system representation of a biological system, capturing both static and dynamic relationships. here, we have demonstrated that a complex biological system can be modeled following a systematic, well-defined software engineering process. we have leveraged the power of the uml to construct our object-oriented software representation of the sars-cov and its infection process. even though uml has been proposed as a tool to model biological systems (bornberg-bauer and paton, 2002; roux-rouquie et al., 2004) , and an overview of the modeling process used in the development of biological simulation software is available (johnson et al., 2004) , without a well-defined methodology and software-engineering process, such as object-oriented analysis and design, transforming biological information into uml becomes an ad hoc activity. however, by applying object-oriented analysis and design, biological entities and related information can be systematically transformed into an object-oriented system by this well-defined methodology. furthermore, uml is created to represent object-oriented software systems. if a biological system itself is directly represented by uml, but not as an object-oriented software system, this representation may be confusing and the full capabilities of uml may not be achieved. reverse engineering a biological system into an object-oriented system ensures that the object-oriented system can be accurately and completely represented by uml. additional tools such as class-responsibility-collaboration cards (wilkinson, 1998) , which can be used to capture the attributes, collaborators and functions of the annotated object, may also be employed to capture data not fully represented by the uml notation. one major problem for modeling and integrating biological information is the lack of information: experimental observations without known underlining mechanisms. this challenge can be met by applying object encapsulation: defining the behavior of an object through its operations that are accessible to other objects, but hiding the underlining mechanism of the operations and object attributes from other objects (graham, 2001) . for example, to communicate that the translation machinery translates a viral protein does not require that every component of the translation machinery be detailed. specifically, to capture this information we gave the translation machinery a function called 'translate'. this function will create a viral protein from the viral mrna without defining the real process of how the viral protein is created. this approach can encapsulate the unknown cellular process inside a well-known function or behavior, and model the biological system without understanding every detail of the system. object-oriented models have additional applications in the construction of biological systems. the layered structure of the objectoriented model allows one to capture various levels of detail such as population, organism, organ, pathway or cell system. this layering can be used to create compartments, which define spatial relationships between cellular components. webb and white (2003) have recently modeled these compartments as classes. however, the compartment issue may be addressed using packages or subsystems. the flexibility of the object-oriented model and uml tool also allow an assortment of biological information to be captured and subsequently translated into mathematical models. it has been shown by priami et al. (2001) and webb and white (2003) that rate equations can be generated using uml sequence and state diagrams. together, an object-oriented approach is capable of modeling enzymatic reactions and metabolic pathways (freier et al., 2003) . however, challenges still exist for fully representing a biological system as an object-oriented system. significantly, because the breadth and depth of biological systems make a complete description of biological systems intractable, a collaborative effort is essential. to integrate new information into existing models, a system, such as a web portal, will have to be implemented that also allows experts to contribute to model maturation. the alliance for cellular signaling (http://www.signaling-gateway.org/) has already set a precedence for such a collaboration. this approach is especially suitable for object-oriented system development, since the modular nature of an object-oriented system allows the modification of individual components without affecting other parts of the system. together, these approaches will distribute the burden of creating these models. because portions of a model might be derived from information that contains varying degrees of confidence, in the future, uml models might benefit from the addition of a confidence indicator to indicate whether portions of a model are derived from experimental data or are based on hypothesis and speculation. while this study has focused on the construction of a web-based system for navigation of the layered structure of a biological system, future work will also strive to integrate information into a searchable database, and implement software systems to simulate biological processes. finally, to extend this application to a more complex biological system, it will be necessary to adopt an advanced iterative process, such as the rational unified process (kruchten, 2003) , or apply advanced techniques, such as façade pattern (gamma et al., 1997) , in object-oriented design to reduce the complexity of the system. this will allow the development of more sophisticated methods to reverse engineer biological systems and significantly enhance the study of biological complexity at a system level. we demonstrate that by applying an adapted, sequential software engineering process, a complex biological system (sars viral infection) can be reverse-engineered and represented as an object-oriented software system. similar to software engineering projects, the well-defined software process makes the biological system information integration repeatable, controllable, scalable and manageable. the resulting object-oriented system not only captures information about individual components but also displays the system-level architecture and interrelationships among system components. furthermore, this object-oriented model captures system dynamics, in that the key events and the process of viral infection are also described. our research demonstrates that object-oriented analysis and design can be employed as an effective methodology to integrate biological information, and the uml representation is a comprehensive approach to capturing the complexity of biological systems, integrating biological information and facilitating communication between biologists and computer scientists. supported by a grant (de-fg02-01er121) from the department of energy. gene ontology: tool for the unification of biology. the gene ontology consortium conceptual data modelling for bioinformatics reverse engineering of biological complexity biosynthesis, purification, and substrate specificity of severe acute respiratory syndrome coronavirus 3c-like proteinase characterization of a unique group-specific protein (u122) of the severe acute respiratory syndrome coronavirus iudb: an object-oriented system for modelling, integration and analysis of gene controlled metabolic networks the molecular biology database collection: 2004 update design patterns basic concepts the systems biology markup language (sbml): a medium for representation and exchange of biochemical network models a new approach to decoding life: systems biology simulating complex intracellular processes using objectoriented computational modeling functional annotation of proteomic sequences based on consensus of sequence and structural analysis molecular interaction map of the mammalian cell cycle control and dna repair systems the rational unified process: an introduction, 3rd edn the molecular biology of coronaviruses angiotensin-converting enzyme 2 is a functional receptor for the sars coronavirus modelling biological processes using workflow and petri net models application of a stochastic name-passing calculus to representation and simulation of molecular processes characterization of a novel coronavirus associated with severe acute respiratory syndrome predicted membrane topology of the coronavirus protein e1 using the unified modelling language (uml) to guide the systemic description of biological processes and systems the unified modeling language reference manual the transpath signal transduction database: a knowledge base on signal transduction networks capturing biological information with class-responsibility-collaboration cards a novel severe acute respiratory syndrome coronavirus protein, u274, is transported to the cell surface and undergoes endocytosis uml as a cell and biochemistry modeling language using crc cards: an informal approach to object-oriented development the sars-cov s glycoprotein: expression and functional characterization assessment of putative protein targets derived from the sars genome proteomic analysis on structural proteins of severe acute respiratory syndrome coronavirus mouse hepatitis virus gene 5b protein is a new virion envelope protein we thank e. voit for critical reading of this manuscript. d.s. is supported by nlm training grant 5-t15-lm007438-02. w.j.z is partly key: cord-017062-dkw2sugl authors: singh, indu; swami, rajan; khan, wahid; sistla, ramakrishna title: delivery systems for lymphatic targeting date: 2013-10-08 journal: focal controlled drug delivery doi: 10.1007/978-1-4614-9434-8_20 sha: doc_id: 17062 cord_uid: dkw2sugl the lymphatic system has a critical role in the immune system’s recognition and response to disease, and it is an additional circulatory system throughout the entire body. most solid cancers primarily spread from the main site via the tumour’s surrounding lymphatics before haematological dissemination. targeting drugs to lymphatic system is quite complicated because of its intricate physiology. therefore, it tends to be an important target for developing novel therapeutics. currently, nanocarriers have encouraged the lymphatic targeting, but still there are challenges of locating drugs and bioactives to specific sites, maintaining desired action and crossing all the physiological barriers. lymphatic therapy using drug-encapsulated colloidal carriers especially liposomes and solid lipid nanoparticles emerges as a new technology to provide better penetration into the lymphatics where residual disease exists. optimising the proper procedure, selecting the proper delivery route and target area and making use of surface engineering tool, better carrier for lymphotropic system can be achieved. thus, new methods of delivering drugs and other carriers to lymph nodes are currently under investigation. the lymphatic system was fi rst recognised by gaspare aselli in 1627, and the anatomy of the lymphatic system was almost completely characterised by the early nineteenth century. however, knowledge of the blood circulation continued to grow rapidly in the last century [ 1 ] . two different theories are proposed which are in favour of origin of the lymphatic vessels. firstly, centrifugal theory of embryologic origin of the lymphatics was described in the early twentieth century by sabin and later by lewis, postulating that lymphatic endothelial cells (lecs) are derived from the venous endothelium. later the centripetal theory of lymphatic development was proposed by huntington and mcclure in 1910 which describes the development of the lymphatic system beginning with lymphangioblasts, mesenchymal progenitor cells, arising independently of veins. the venous connection to the lymphatic system then happens later in development [ 2 ] . the lymphatic vessels in the embryo are originated at mid-gestation and are developed after the cardiovascular system is fully established and functional [ 3 ] . a dual origin of lymphatic vessels from embryonic veins and mesenchymal lymphangioblasts is also proposed [ 4 ] . recent studies provide strong support of the venous origin of lymphatic vessels [ 5 -8 ] . the recent discovery of various molecular markers has allowed for more in-depth research of the lymphatic system and its role in health and disease. the lymphatic system has recently been elucidated as playing an active role in cancer metastasis. the knowledge of the active processes involved in lymphatic metastasis provides novel treatment targets for various malignancies. the lymphatic system consists of the lymphatic vessels, lymph nodes, spleen, thymus, peyer's patches and tonsils, which play important roles in immune surveillance and response. the lymphatic system serves as the body's second vascular system in vertebrates and functions co-dependently with the cardiovascular system [ 9 , 10 ] . the lymphatic system comprises a single irreversible, open-ended transit network without a principal driving force [ 9 ] . it consists of fi ve main types of conduits including the capillaries, collecting vessels, lymph nodes, trunks and ducts. the lymphatic system originates in the dermis with initial lymphatic vessels and blind-ended lymphatic capillaries that are nearly equivalent in size to but less abundant than regular capillaries [ 9 , 11 ] . lymphatic capillaries consist of a single layer of thin-walled, non-fenestrated lymphatic endothelial cells (lecs), alike to blood capillaries. the lecs, on the contrary to blood vessels, have poorly developed basement membrane and lack tight junctions and adherent junctions too. these very porous capillaries act as gateway for large particles, cells and interstitial fl uid. particles as large as 100 nm in diameter can extravasate into the interstitial space, get phagocytosed by macrophages and are ultimately passed on to lymph nodes [ 11 -14 ] . lymphatic capillary endothelial cells are affi xed to the extracellular matrix by elastic anchoring fi laments, which check vessel collapse under high interstitial pressure. these initial lymphatics, under a positive pressure gradient, distend and create an opening between loosely anchored endothelial cells letting for the entry of lymph, a protein-rich exudate from the blood capillaries [ 12 , 15 , 16 ] . in initial lymphatic vessels, overlying endothelial cell-cell contacts prevent fl uid refl ux back into the interstitial space [ 17 , 18 ] . after the collection of lymph by the lymphatic capillaries, it is transported through a system of converging lymphatic vessels of progressively larger size, is fi ltered through lymph nodes where bacteria and particulate matter are removed and fi nally goes back to the blood circulation. lymph is received from the initial capillary lymphatic by deeper collecting vessels that contain valves to maintain unidirectional fl ow of lymph. these collecting vessels have basement membranes and are surrounded by smooth muscle cells with intrinsic contractile activity that in combination with contraction of surrounding skeletal muscles and arterial pulsations propels the lymph to lymph nodes [ 19 -21 ] . the collecting lymphatic vessels unite into lymphatic trunks, and the lymph is fi nally returned to the venous circulation via the thoracic duct into the left subclavian vein [ 22 , 23 ] . the fl ow of lymph toward the circulatory system is supported by increases in interstitial pressure as well as contractions of the lymphatic vessels themselves. roughly 25 l of lymphatic fl uid enters the cardiovascular system each day [ 11 ] . the key functions of the lymphatic system are maintenance of normal tissue fl uid balance, absorption of lipids and fat-soluble vitamins from the intestine and magnetism and transport of immune cells. lymphatics transport the antigen-presenting cells as well as antigens from the interstitium of peripheral tissues to the draining lymph nodes where they initiate immune responses via b-and t-cells in the lymph nodes [ 9 , 12 , 24 , 25 ] . tissue fl uid balance is maintained by restoring interstitial fl uid to the cardiovascular system [ 9 ] . although capillaries have very low permeability to proteins, these molecules as well as other macromolecules and bacteria accumulate in the interstitium. due to the accumulation of these large molecules in the interstitium, signifi cant tissue oedema would result. the lymphatic system offers the mechanism by which these large molecules re-enter the blood circulation [ 26 ]. the lymphatic system is the site of many diseases such as metastitial tuberculosis (tb), cancer and fi lariasis [ 27 ] . due to the peculiar nature and anatomy of the lymphatic system, localisation of drugs in the lymphatics has been particularly diffi cult to achieve. the lymphatic system has an active role in cancer metastasis. although many cancers may be treated with surgical resection, microscopic disease may remain and lead to locoregional recurrence. conventional systemic chemotherapy cannot prove effective for delivering drugs to the lymphatic system without dose-limiting toxicities [ 28 ]. lymphatic system functions in the clearance of particulate matter from the interstitium following presentation to lymph nodes have created interest in developing microparticulate systems to target regional lymph nodes. molecule's composition is important in determining uptake into the lymphatics and retention within the lymph nodes. colloidal materials, for example, liposomes, activated carbon particles, emulsions, lipids and polymeric particulates, are highly taken up by the lymphatics; that's why nowadays these substances are emerging as potential carriers for lymphatic drug targeting [ 29 ] . the vast majority of drugs following oral administration are absorbed directly into portal blood, but a number of lipophilic molecules may get access to the systemic circulation via the lymphatic pathway [ 30 , 31 ] . intestinal lymphatic transport of lipophilic molecules is signifi cant and presents benefi ts in a number of situations: the lymphatic system also acts as the primary systemic transport pathway for b-and t-lymphocytes as well as the main route of metastatic spread of a number of solid tumours [ 36 , 37 ] . therefore, lymphatic absorption of the immunomodulatory and anticancer compounds may be more effective [ 38 , 39 ] . the presence of wide amounts of hiv-susceptible immune cells in the lymphoid organs makes antiretroviral drug targeting to these sites of tremendous interest in hiv therapy. this strategy comprises once again targeting nanosystems to immune cell populations, particularly macrophages. also evidence further suggests that lymph and lymphoid tissue, and in particular gut-associated lymphoid tissue, play a major role in the development of hiv and antivirals which target acquired immunodefi ciency syndrome (aids) may therefore be more effective when absorbed via the intestinal lymphatics [ 40 , 41 ] targeting drugs to lymphatic system is a tough and challenging task, and it totally depends upon the intricate physiology of the lymphatic system. targeting facilitates direct contact of drug with the specifi c site, decreasing the dose of the drugs and minimising the side effects caused by them. currently, nanocarriers have encouraged the lymphatic targeting, but still there are challenges of locating drugs and bioactives to specifi c sites, maintaining desired action and crossing all the physiological barriers. these hurdles could be overcome by the use of modifi ed nanosystems achieved by the surface engineering phenomena. from the growing awareness of the importance of lymph nodes in cancer prognosis, their signifi cance for vaccine immune stimulation and the comprehension that the lymph nodes harbour hiv as well as other infectious diseases stems the development of new methods of lymph node drug delivery [ 47 -50 ] . new methods of delivering drugs and other carriers to lymph nodes are currently under investigation. lymph node dissemination is the primary cause of the spread of majority of solid cancers [ 51 ] . in regard to cancer metastasis, the status of the lymph node is a major determinant of the patient's diagnosis. the most important factor that determines the appropriate care of the patient is correct lymph node staging [ 52 ] . but patient survivals have been shown to improve by the therapeutic interventions that treat metastatic cancer in lymph nodes with either surgery or local radiation therapy [ 53 ] . viraemia is an early indication of primary infection with hiv followed by a specifi c hiv immune response and a dramatic decline of virus in the plasma [ 54 ] . long after the hiv virus can be found in the blood, hiv can be found in high levels in mononuclear cells located in lymph nodes. viral replication in these lymph nodes has been reported to be about 10-to 100-fold higher than in the peripheral blood mononuclear cells [ 55 ] . standard oral or intravenous drug delivery to these lymph node mononuclear cells is diffi cult [ 56 ] . even if highly active antiretroviral therapy (haart) can reduce plasma viral loads in hiv-infected patients by 90 %, active virus can still be isolated from lymph nodes even after 30 months of haart therapy. lymph nodes are the key element of the life cycle of several parasite organisms, including fi laria. lymphatic vessels and lymph nodes of infected patients can carry adult worms. this adult fi laria obstructs the lymphatic drainage that results into swelling of extremities that are distal to the infected lymph node. these very symptoms of swollen limbs in patients with fi larial disease have been termed elephantiasis. the eradication of adult worms in lymph nodes is not frequently possible, and commonly a much extended course of medical therapy is required for it to be successful [ 57 ] . new methods of curing anthrax have become a burning interest following the recent outburst of anthrax infections and deaths in the usa as a result of terrorism. in anthrax infection, endospores from bacillus anthracis that gain access into the body are phagocytosed by macrophages and carried to regional lymph nodes where the endospores germinate inside the macrophages and become vegetative bacteria [ 58 ] . according to one literature, computed tomography of the chest was performed on eight patients infected with inhalational anthrax. mediastinal lymphadenopathy was found in seven of the eight patients [ 59 ] . in another case report of a patient, the anthrax bacillus was shown to be rapidly sterilised within the blood stream after initiation of antibiotic therapy. however, viable anthrax bacteria were still present in postmortem mediastinal lymph node specimens [ 60 ] . treatment and control of these diseases are hard to accomplish because of the limited access of drugs to mediastinal nodes using common pathways of drug delivery. also, the anatomical location of mediastinal nodes represents a diffi cult target for external beam irradiation. newer methods to target antituberculosis drugs to these lymph nodes could possibly decrease the amount of time of drug therapy. tb requires lengthy treatment minimum of approximately 6 months probably because of its diffi culty in delivering drugs into the tubercular lesions. the tb infection is caused by mycobacteria that invade and grow chiefl y in phagocytic cells. lymph node tb is the most common form of extrapulmonary tb rating approximately as 38.3 %. this is frequently found to spread from the lungs to lymph nodes. in one study, total tb lymph node involvement was found as 71 % of the intrathoracic lymph nodes, 26 % of the cervical lymph nodes and 3 % of the axillary lymph nodes [ 61 ] . targeted delivery of drugs can be achieved utilising carriers with a specifi ed affi nity to the target tissue. there are two approaches for the targeting, i.e. passive and active. in passive targeting, most of the carriers accumulate to the target site during continuous systemic circulation to deliver the drug substance, the behaviour of which depends highly upon the physicochemical characteristics of the carriers. whereas much effort has been concentrated on active targeting, this involves delivering drugs more actively to the target site. passive targeting involves the transport of carriers through leaky tumour vasculature into the tumour interstitium and cells by convection or passive diffusion. further, nanocarriers and drug then accumulate at the target site by the enhanced permeation and retention (epr) effect [ 62 ] . the epr effect is most prominent mainly in cancer targeting. moreover, the epr effect is pertinent for about all fast-growing solid tumours [ 63 ] . the epr effect will be most positive if nanocarriers can escape immune surveillance and circulate for a long period. very high local concentrations of drug-loaded nanocarriers can be attained at the target site, for example, about 10-to 50-fold higher than in normal tissue within 1-2 days [ 64 ] . however, there exist some limitations for passively targeting the tumour; fi rst is the degree of tumour vascularisation and angiogenesis which is important for passive targeting of nanocarriers [ 65 ] . and, second, due to the poor lymphatic drainage in tumours, the interstitial fl uid pressure increases which correlates nanocarrier size relationship with the epr effect: larger and long-circulating nanocarriers (100 nm) are more retained in the tumour, whereas smaller molecules easily diffuse [ 66 ] . active targeting is based upon the attachment of targeting ligands on the surface of the nanocarrier for appropriate receptor binding that are expressed at the target site. the ligand particularly binds to a receptor overexpressed in particular diseased cells or tumour vasculature and not expressed by normal cells. in addition, targeted receptors should be present uniformly on all targeted cells. targeting ligands are either monoclonal antibodies (mabs) and antibody fragments or non-antibody ligands (peptidic or not). these can also be termed as ligand-targeted therapeutics [ 67 , 68 ] . targeting approaches for lymphatic targeting are shown in fig. 20 .1 . current research is focussed on two types of carriers, namely, colloidal carriers and polymeric carriers. targeting strategies for lymphatics are shown in fig. 20 much effort has been concentrated to achieve lymphatic targeting of drugs using colloidal carriers. the physicochemical nature of the colloid itself has been shown to be of particular relevance, with the main considerations being size of colloid and hydrophobicity. the major purpose of lymphatic targeting is to provide an effective anticancer chemotherapy to prevent the metastasis of cancer cells by accumulating the drug in the regional lymph node. emulsions are probably well-known particulate carriers with comparative histories of research and have been widely used as a carrier for lymph targeting. hashida et al. demonstrated that injection of water-in-oil (w/o) or oil-in-water (o/w) emulsions favoured lymphatic transport of mitomycin c via the intraperitoneal and intramuscular routes and uptake into the regional lymphatics was reported in the order of o/w > w/o > aqueous solution. the nanoparticle-in-oil emulsion system, containing anti-fi larial drug in gelatin nanoparticles, was studied for enhancing lymphatic targeting [ 69 ] . pirarubicin and lipiodol emulsion formulation was developed for treating gastric cancer and metastatic lymph nodes [ 70 , 71 ] . after endoscopic injection of the pirarubicin-lipiodol emulsion, the drug retained over 7 days at the injection site and in the regional lymph node. hauss et al. in their study have explored the lymphotropic potential of emulsions and self-emulsifying drug delivery systems (sedds). they investigated the effects of a range of lipid-based formulations on the bioavailability and lymphatic transport of ontazolast following oral administration to conscious rats and found that all the lipid formulations increased the bioavailability of ontazolast comparative to the control suspension, the sedds promoted more rapid absorption and maximum lymphatic transport is found with the emulsion [ 72 , 73 ] . lymphatic delivery of drug-encapsulated liposomal formulations has been investigated extensively in the past decade. liposomes possess ideal features for delivering therapeutic agents to the lymph nodes which are based on their size, which prevents their direct absorption into the blood; the large amount of drugs and other therapeutic agents that liposomes can carry; and their biocompatibility. the utility of liposomes as a carrier for lymphatic delivery was fi rst investigated by segal et al. in 1975 [ 74 ] . orally administered drug-incorporated liposomes enter the systemic circulation via the portal vein and intestinal lymphatics. drugs entering the intestinal lymphatic through the intestinal lumen avoid liver and fi rst-pass metabolism as they fi rst migrate to lymphatic vessels and draining lymph nodes before entering systemic circulation. lymphatic uptake of carriers via the intestinal route increases bioavailability of a number of drugs. for oral delivery of drug-encapsulated liposomal formulations, intestinal absorbability and stability are the primary formulation concerns. ling et al. evaluated oral delivery of a poorly bioavailable hydrophilic drug, cefotaxime, in three different forms: liposomal formulation, aqueous-free drug and a physical mixture of the drug and empty liposomes [ 75 ] . the liposomal formulation of the drug turned out to exhibit a 2.7-fold increase in its oral bioavailability compared to the aqueous dosage and a 2.3-fold increase for the physical mixture. they also accounted that the liposomal formulation leads to a signifi cant enhancement of the lymphatic localisation of the drug relative to the other two formulations. as a result, liposome systems emerged as useful carriers for poorly bioavailable hydrophilic drugs, promoting their lymphatic transport in the intestinal lymph as well as their systemic bioavailability. conventional liposomal formulations contain anticancer drugs incorporated in them for intravenous infusion in treating various types of cancers. doxil, a chemotherapeutic formulation of pegylated liposomes of doxorubicin, is widely used as fi rst-line therapy of aids-related kaposi's sarcoma, breast cancer, ovarian cancer and other solid tumours [ 76 -80 ] . liposomal delivery of anticancer drug actinomycin d via intratesticular injection has shown greater concentration of the drug in the local lymph nodes. furthermore, a study by hirnle et al. found liposomes as a better carrier for intralymphatically delivered drugs contrasted with bleomycin emulsions [ 81 ] . systemic liposomal chemotherapy is preferred mainly because of its reduced side effects compared to the standard therapy and improved inhibition of the anticancer drugs from enzymatic digestion in the systemic circulation. effective chemotherapy by pulmonary route could overcome various lacunas associated with systemic chemotherapy like serious non-targeted toxicities, poor drug penetration into the lymphatic vessels and surrounding lymph node and fi rst-pass clearance concentrating drugs in the lungs and draining lymphatics in the case of oral delivery. latimer et al. developed liposomes of paclitaxel and a vitamin e analogue α-tocopheryloxy acetic acid (α-tea) in an aerosol formulation for treating murine mammary tumours and metastases [ 82 ] . similarly, lawson et al. performed a comparative study for the anti-proliferative effi cacy of a 9-nitro-camptothecin (9-nc)-encapsulated dilauroylphosphatidylcholine liposomal delivery, α-tea and a combination therapy of 9-nc and α-tea, in a metastatic murine mammary tumour model. liposome-encapsulated individual as well as combination treatment was delivered via an aerosol for curing metastases of lungs and of the surrounding lymph node. the animals treated with the combination therapy were found to have less proliferative cells compared to the animals treated with 9-nc alone when immunostained with ki-67. the in vivo anticancer effi cacy studies demonstrated that the combination treatment greatly hindered the tumour progression compared to each treatment alone, leading to the prolonged survival rate [ 83 ] . high levels of drugs could be targeted to lymph nodes containing tb using liposomal antituberculosis drug therapy [ 84 ] . deep lung lymphatic drainage could also be visualised using 99mtc radioactive marker-incorporated liposomes. in addition, botelho et al. delivered aerosolised nanoradioliposomal formulation to wild boars and observed their deep lung lymphatic network and surrounding lymph nodes [ 85 ] . also, this technique has offered new information of the complicated structure of lymphatic network and has emerged as a new and non-invasive molecular imaging technique for the diagnosis of early dissemination of lung cancers as compared to the conventional computed tomography. solid lipid nanoparticles (sln) could be a good formulation strategy for incorporating drugs with poor oral bioavailability due to low solubility in gi tract or pre-systemic hepatic metabolism (fi rst-pass effect) permitting transportation into the systemic circulation through the intestinal lymphatics. bargoni et al. have performed various studies on absorption and distribution of sln after duodenal administration [ 86 -89 ] . in one study, 131 i-17-iodoheptadecanoic acid-labelled drug-free sln were delivered into the duodenal lumen of fed rats, and transmission electron microscopy and photon correlation spectroscopy results of the lymph and blood samples verifi ed the transmucosal transport of sln [ 86 ] . in a later study of tobramycin-loaded sln after duodenal administration, the improvement of drug absorption and bioavailability was ascribed mostly to the favoured transmucosal transport of sln to the lymph compared to the blood [ 88 ] . the same group conducted a study using idarubicin-loaded sln, administered via the duodenal route rather than intravenous route, and observed enhancement in drug bioavailability [ 89 ] . reddy et al. prepared etoposide-loaded tripalmitin (etpl) sln radiolabelled with 99mtc and administered the etpl nanoparticles subcutaneously, intraperitoneally and intravenously, to mice bearing dalton's lymphoma tumours, and 24 h after subcutaneous administration, gamma scintigraphy and the radioactivity measurements showed that the etpl sln revealed a clearly higher degree of tumour uptake given via subcutaneous route (8-and 59-fold higher than that of the intraperitoneal and intravenous routes, respectively) and reduced accumulation in reticuloendothelial system organs [ 90 ] . targeting therapies are of great potential in small cell lung cancer considering intrathoracic lymph node metastasis occurring in approximately 70 % of the limited stage patients and to nearly 80 % of the extensive stage patients [ 91 ] . considering the case of non-small cell lung cancer, extensive rate of metastasis of lymphatics is seen in greater than 80 % of stage iv patients [ 92 ] . videira et al. compared the biodistribution of inhaled 99mtc-d,l -hexamethylpropyleneamine oxime (hmpao)radiolabelled sln with that of the free tracer administered through the same route, and gamma scintigraphic results specifi ed that the radiolabelled sln were primarily cleared from lungs via the lymphatics [ 93 , 94 ] . nanocapsules tend to be the most promising approach for lymphatic targeting because of their possibility of attaining distinct qualities with an easy manufacturing process. nanocapsules coated with hydrophobic polymers could be easily captured by lymphatic cells in the body, when administered, because the hydrophobic particle is generally recognised as a foreign substance. the lymphatic targeting ability of poly(isobutylcyanoacrylate) nanocapsules encapsulating 12-(9-anthroxy) stearic acid upon intramuscular administration was evaluated and compared with three conventional colloidal carriers [ 69 ] . in vivo study in rats proved that poly(isobutylcyanoacrylate) nanocapsules retained in the right iliac regional lymph nodes in comparison with other colloidal carriers following intramuscular administration. for effective targeted and sustained delivery of drugs to lymph, several polymeric particles have been designed and studied. the polymers are categorised in two types based on their origin either natural polymers like dextran, alginate, chitosan, gelatin, pullulan and hyaluronan or synthetic polymers like plga, pla and pmma. dextran a natural polysaccharide has been used as a carrier for a range of drug molecules due to its outstanding biocompatibility. bhatnagar et al. synthesised cyclosporine a-loaded dextran acetate particles labelled with 99mtc. these particles gradually distributed cyclosporine a all through the lymph nodes following subcutaneous injection into the footpad of rats [ 95 ] . dextran (average molecular weights of 10, 70 and 500 kda)-conjugated lymphotropic delivery system of mitomycin c has been studied and it was reported that after intramuscular injection in mice, this mitomycin c-dextran conjugates retained for a longer period in regional lymph nodes for nearly 48 h while the free mitomycin was quickly cleared. hyaluronan, also called as hyaluronic acid, is a natural biocompatible polymer that follows lymphatic drainage from the interstitial spaces. cai et al. demonstrated a novel intralymphatic drug delivery method synthesising a cisplatin-hyaluronic acid conjugate for breast cancer treatment. following subcutaneous injection into the upper mammary fat pad of female rats, most of the carrier localised in the regional nodal tissue compared to the standard cisplatin formulation [ 96 ] . poly(lactide-co-glycolide) as synthetic polymer that is used to prepare biodegradable nanospheres has been accounted to deliver drugs and diagnostic agents to the lymphatic system. similarly, nanospheres coated with block copolymers of poloxamers and poloxamines with radiolabelled 111 in-oxine are used to trace the nanoparticles in vivo. upon s.c. injection, the regional lymph node showed a maximum uptake of 17 % of the administered dose [ 97 ] . dunne et al. synthesised a conjugate of block copolymer cis-diamminedichloroplatinum(ii) (cddp) and poly(ethylene oxide)-block-poly(lysine) (peo-b-plys) for treating lymph node metastasis. one animal treatment with 10 wt.% cddp-polymer resulted into limited tumour growth in the draining lymph nodes and prevention of systemic metastasis [ 98 ] . johnston and coworkers designed a biodegradable intrapleural (ipl) implant of paclitaxel consisting gelatin sponge impregnated with poly(lactide-co-glycolide) (plga-ptx) for targeting thoracic lymphatics. in rat model, this system exhibited lymphatic targeting capability and showed sustained drug release properties [ 99 ] . kumanohoso et al. designed a new drug delivery system for bleomycin by loading it into a small cylinder of biodegradable polylactic acid to target lesions. this system showed signifi cantly higher antitumour effect compared to bleomycin solution and no treatment [ 100 ] . to treat lesions, a new biodegradable colloidal particulatebased nanocarrier system was designed to target thoracic lymphatics and lymph nodes. various nano-and microparticles of charcoal, polystyrene and poly(lactideco-glycolide) were studied for the lymphatic distribution after intrapleural implantation in rats, and after 3 h of intrapleural injection, the lymphatic uptake was observed [ 101 ] . kobayashi et al. utilised dendrimer-based contrast agents for dynamic magnetic resonance lymphangiography [ 102 ] . gadolinium (gd)-containing dendrimers of different sizes and molecular structures (pamam-g8, pamam-g4 and dab-g5) (pamam, polyamidoamine; dab, diaminobutyl) are used as contrast agents. size and molecular structure play a great role in distribution and pharmacokinetics of dendrimers. for example, pamam-g8 when injected intravenously had a comparatively long life in the circulatory system with minimum leakage out of the vessels, whereas pamam-g4 cleared rapidly from the systemic circulation due to rapid renal clearance but had immediate survival in lymphatic circulation. the smaller-sized dab-g5 showed greater accumulation and retention in lymph nodes useful for lymph node imaging using mr-lg. gadomer-17 and gd-dtpadimeglumine (magnevist) were evaluated as controls. imaging experiments revealed that all of the reagents are able to visualise the deep lymphatic system except gd-dtpa-dimeglumine. to visualise the lymphatic vessels and lymph nodes, pamam-g8 and dab-g5 were used, respectively. while pamam-g4 provided good contrast of both the nodes and connecting vessels, gadomer-17 was able to visualise lymph nodes, but not as clear as gd-based dendrimers. kobayashi also delivered various gd-pamam (pamam-g2, pamam-g4, pamam-g6, pamam-g8) and dab-g5 dendrimers to the sentinel lymph nodes and evaluated its visualisation with other nodes. the g6 dendrimer provided excellent opacifi cation of sentinel lymph nodes and was able to be absorbed and retained in the lymphatic system [ 103 ] . using a combination of mri and fl uorescence with pamam-g6-gd-cy, the sentinel nodes were more clearly observed signifying the potential of the dendrimers as platform for dual imaging. kobayashi et al. further overcame the sensitivity limitation and depth limitations of each individual method by the simultaneous use of two modalities (radionuclide and optical imaging). making use of pamam-g6 dendrimers conjugated with near-infrared (nir) dyes and an 111 in radionuclide probe, multimodal nanoprobes were developed for radionuclide and multicolour optical lymphatic imaging [ 104 , 105 ] . later kobayashi also proposed the use of quantum dots for labelling cancer cells and dendrimer-based optical agents for visualising lymphatic drainage and identifying sentinel lymph nodes [ 106 ] . polylysine dendrimers have been best used for targeting the lymphatic system and lymph nodes. carbon nanotubes (cnt) possess various mechanochemical properties like high surface area, mechanical strength and thermal and chemical stability which cause them to be versatile carriers for drugs, proteins, radiologicals and peptides to target tumour tissues. hydrophilic multiwalled carbon nanotubes (mwnts) coated with magnetic nanoparticles (mn-mwnts) have emerged as an effective delivery system for lymphatic targeting following subcutaneous injection of these particles into the left footpad of sprague dawley rats; the left popliteal lymph nodes were dyed black. mn-mwnts were favourably absorbed by lymphatic vessels following their transfer into lymph nodes and no uptake was seen in chief internal organs such as the liver, spleen, kidney, heart and lungs. gemcitabine loaded in these particles was evaluated for its lymphatic delivery effi ciency and mn-mwnts-gemcitabine displayed the maximum concentration of gemcitabine in the lymph nodes [ 107 ] . mcdevitt et al. synthesised tumour-targeting water-soluble cnt constructs by covalent attachment of monoclonal antibodies like rituximab and lintuzumab using 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid (dota) as a metal ion chelator while the fl uorescent probe was fl uorescein. cnt-([ 111 in] dota) (rituximab) explicitly targeted a disseminated human lymphoma in vivo trials compared to the controls cnt-([ 111 in] dota) (lintuzumab) and [ 111 in]rituximab [ 108 ] . tsuchida and coworkers evaluated the drug delivery effi ciency of water-dispersed carbon nanohorns in a non-small cell lung cancer model. polyethylene glycol (peg)-doxorubicin conjugate bound oxidised single-wall carbon nanohorns (oxswnhs) injected intratumourally into mice bearing human non-small cell lung cancer (nci-h460) caused a signifi cant retardation of tumour growth. histological analyses showed (probably by means of interstitial lymphatic fl uid transport), migration of oxswnhs to the axillary lymph node occurred which is a major site of breast cancer metastasis near the tumour [ 109 ] . shimada et al. described a silica particle-based lymphatic drug delivery system of bleomycin and compared its therapeutic effi cacy to that of free bleomycin solution in a transplanted tumour model in animals. silica particle-adsorbed bleomycin showed considerable inhibitory effect on tumour growth and lymph node metastasis compared to free bleomycin solution [ 110 ] . activated carbon particles of aclarubicin are used for adsorption and sustained release into lymph nodes. upon subcutaneous administration into the fore foot-pads of rats these particles showed signifi cantly elevated distribution of aclarubicin to the auxiliary lymph nodes compared to aqueous solution of the drug [ 111 ] . activated carbon particles of aclacinomycin a, adriamycin, mitomycin c and pepleomycin have also been used by another group for adsorption. higher level of drug concentration was maintained in the new dosage form than in the solution form [ 112 ] . antibody-drug conjugates enhance the cytotoxic activity of anticancer drugs by conjugating them with antibodies. antibodies conjugated with cytostatic drugs such as calicheamicin have been used for the treatment of various lymphomas, including non-hodgkin b-cell lymphoma (nhl), follicular lymphoma (fl) and diffuse large b-cell lymphoma (dlbcl) [ 113 -116 ] . cd20 b-cell marker is expressed on the surface membrane of pre-b-lymphocytes and mature b-lymphocytes. the anti-cd20 mab rituximab (rituxan) is now the most potential antibody for the treatment of non-hodgkin b-cell lymphomas (b-nhl) [ 117 ] . rituximab-conjugated calicheamicin elevated the antitumour activity of rituximab against human b-cell lymphoma (bcl) xenografts in preclinical models [ 118 ] . cd22 is a b-lymphoid lineage-specifi c differentiation antigen expressed on the surface of both normal and malignant b-cells. hence, the cd22-specifi c antibody could be effective in delivering chemotherapeutic drugs to malignant b-cells. also, cd22 (siglec-2) antibodies targeting to cd22 are suited for a trojan horse strategy. thus, antibody-conjugated therapeutic agents bind to the siglec and are carried effi ciently into the cell [ 119 ] . a lot of interest has been seen in clinical progress of the conjugated anti-cd22 antibodies, especially inotuzumab ozogamicin (cmc-544) [ 120 ] . cd30 is expressed in the malignant hodgkin and reed-sternberg cells of classical hodgkin lymphoma (hl) and anaplastic large-cell lymphoma. younes and bartlett reported an ongoing phase i dose-escalation trial in relapsed and refractory hl patients with seattle genetics (sgn-35) , a novel anti-cd30-antibody-monomethylauristatin e conjugate. sgn-35 was stable in the blood and released the conjugate only upon internalisation into cd30-expressing tumour cells [ 121 ] . huang et al. constructed (anti-her2/ neu -igg3-ifn α ), another antibody-drug conjugate, and examined its effect on a murine b-cell lymphoma, 38c13, expressing human her2/ neu , and this signifi cantly inhibited 38c13/her2 tumour growth in vivo [ 122 ] . hybrid systems use combination of two or more delivery forms for effective targeting. khatri et al. prepared and investigated the in vivo effi cacy of plasmid dna-loaded chitosan nanoparticles for nasal mucosal immunisation against hepatitis b. chitosan-dna nanoparticles prepared by the coacervation process adhered to the nasal or gastrointestinal epithelia and are easily transported to the nasal-associated lymphoid tissue (nalt) and peyer's patches of the gut-associated lymphoid tissue (galt) both as iga inductive site [ 123 ] , in which chitosan-dna might be taken in by m cell, and transported across the mucosal boundary and thereby transfect immune cells within nalt or galt [ 124 ] . a work demonstrates targeting of three peptides containing sequences that bind to cell markers expressed in the tumour vasculature (p24-nrp-1 and p39-flt-1) [ 125 , 126 ] and tumour lymphatics (p47-lyp-1) [ 127 ] and were tested for their ability to target 3(nitrilotriacetic acid)-ditetradecylamine (nta3-dtda) containing liposomes to subcutaneous b16-f1 tumours. signifi cantly, a potential antitumour effect was seen after administration of doxorubicin-loaded peg750 liposomes engrafted with p24-nrp-1. hybrid liposomes composed of l -α-dimyristoylphosphatidylcholine and polyoxyethylene (25) dodecyl ether prepared by sonication showed remarkable reduction of tumour volume in model mice of acute lymphatic leukaemia (all) treated intravenously with hl-25 without drugs after the subcutaneous inoculation of human all (molt-4) cells was verifi ed in vivo. prolonged survival (>400 %) was noted in model mice of all after the treatment with hl-25 without drugs [ 128 ] . in a report, lyp-1 peptide-conjugated pegylated liposomes loaded with fl uorescein or doxorubicin were prepared for targeting and treating lymphatic metastatic tumours. the in vitro cellular uptake and in vivo near-infrared fl uorescence imaging results confi rmed that lyp-1-modifi ed liposome increased uptake by tumour cells and metastatic lymph nodes. in another study, in vitro cellular uptake of peg-plga nanoparticle (lyp-1-nps) was about four times that of peg-plga nanoparticles without lyp-1 (nps). in vivo study, about eight times lymph node uptake of lyp-1-nps was seen in metastasis than that of nps, indicated lyp-1-np as a promising carrier for targetspecifi c drug delivery to lymphatic metastatic tumours [ 129 ] . currently, surgery, radiation therapy and chemotherapy are the principal methods for cancer treatment. gene therapies may act synergistically or additively with them. for example, another case demonstrated that replacement of the p53 (protein 53) gene in p53-defi cient cancer cell lines enhanced the sensitivity of these cells to ad-p53 (adenovirus-expressed protein 53) and cisplatin (cddp) and resulted into greater tumour cell death [ 130 ] . later, son and huang [ 131 ] stated that treatment of cddp-resistant tumour cells with cddp increased the sensitivity of these cells to transduction by dna-carrying liposomes. also, chen et al. [ 132 ] described that to improve tumour killing, herpes simplex virus thymidine kinase (hsv-tk) and interleukin (il) expression can be combined. on the whole, greater therapeutic effect can be achieved by effectively combining conventional cancer treatments and gene therapy together. mainly colloidal carriers have emerged as potential targeting agents to lymphatic system. physicochemical properties affect the effi ciency of colloid uptake into the lymphatic system [ 28 ]. these properties include size, number of particles, surface charge, molecular weight and colloid lipophilicity. physicochemical properties are altered by adsorption of group of hydrophilic polymers like poloxamers and poloxamines to the particle surface. these properties modifi ed the biodistribution of particles in vivo, particularly the avoidance of the reticuloendothelial system (res) upon intravenous administration [ 133 , 134 ] . in one study, it was opined that opsonisation may cause alteration of the particle surface in vivo [ 135 ] . size could be important factor in defi ning the behaviour of particulates after subcutaneous injection. small particles with diameter less than a few nanometres generally exchanged through the blood capillaries, whereas larger particles of diameters up to a few tens of nanometres absorbed into the lymph capillaries. but particles over a size of few hundred nanometres remain trapped in the interstitial space for a long time [ 136 ] . christy et al. have shown a relationship between colloid size and ease of injection site drainage using model polystyrene nanospheres after subcutaneous administration to the rat [ 137 ] . results showed distribution of polystyrene nanospheres in the size range 30-260 nm 24 h after administration and 74-99 % of the recovered dose retained at the administration site, and as particle diameter increased, drainage became slower. it has been proposed earlier that the optimum colloid size range for lymphoscintigraphic agents is l0-50 nm [ 138 ] . size has less importance when colloids are administered intraperitoneally (i.p.) within the nanometre size range, as drainage is only from a cavity into the initial lymphatics; hence, no diffusion is required through the interstitial space [ 28 ]. the size limit of the open junctions of the initial lymphatic wall is the only barrier to uptake from the peritoneal cavity into the lymphatics [ 139 ] . more number of particles at the injection site decreases their rate of drainage, owing to increased obstruction of their diffusion through the interstitial space [ 139 , 140 ] . scientists at nottingham university investigated this effect using polystyrene nanospheres of 60 nm. following administration to the rat, the concentration range of nanospheres was approximately 0.05-3.0 mg/ml. lower lymphatic uptake was seen on increasing the concentration of nanospheres in the injection volume due to slower drainage from the injection site. injecting oily vehicles intramuscularly to the rat, the effect of injection volume has been studied. increasing volume of sesame oil accelerated oil transport into the lymphatic system. upon s.c. administration, volumes of aqueous polystyrene particle suspensions have been investigated in the range 50-150 μl [ 39 ]. surface charge studies have been done utilising liposome as colloidal carrier. the surface charge of liposomes affected their lymphatic uptake from s.c. and i.p. injection sites. negatively charged liposomes showed faster drainage than that for positive liposomes after i.p. administration [ 141 ] . patel et al. also indicated that liposome localisation in the lymph nodes followed a particular order negative > positive > neutral [ 142 ] . macromolecule having high molecular weight has a decreased ability for exchange across blood capillaries and lymphatic drainage becomes the route of drainage from the injection site which shows a linear relationship between the molecular weight of macromolecules and the proportion of the dose absorbed by the lymphatics. for a compound to be absorbed by the lymphatics, the molecular weight should range between 1,000 and 16,000 [ 141 , 143 ] . the effect of molecular weight becomes negligible when targeting carriers to the lymphatic system as the molecular weight of a colloidal carrier is generally less than 1,000 da. the most important determinant of the phagocytic response and so lymphatic uptake is the lipophilicity of a colloid [ 144 ] . opsonins generally unite with lipophilic rather than hydrophilic surfaces; hence, the hydrophilic particles show reduced phagocytosis [ 145 ] . hydrophobic polystyrene nanospheres adsorbed with hydrophilic block copolymers showed drastic reduction in phagocytosis prior to i.v. administration [ 146 ] . in the case of polystyrene nanospheres of 60-nm diameter, peo chains of the poloxamers and poloxamines adsorbed onto the surface of the particle described the relationship between interstitial injection site drainage and lymph node uptake in rat [ 144 ] . uncoated nanospheres of this diameter showed reduced drainage from the injection site with 70 % of the administered dose remaining after 24 h. the adsorption of block copolymers can enhance the drainage from the injection site such that levels remaining at the injection site may be as little as 16 % after 24 h, with very hydrophilic polymers such as poloxamine 908. uptake of nanospheres into the regional lymph nodes may also be improved by the adsorption of block copolymers with intermediate lengths of polyoxyethylene, such as poloxamine 904. this polymer may sequester up to 40 % of the given dose by the lymph nodes after 24 h [ 147 ] . surface modifi cation could prove as an effective strategy for potential targeting to lymphatic system. the infl uence can be quoted in following ways. coating of a carrier with hydrophilic and sterically stabilised peg layer can successfully enhance lymphatic absorption, reducing specifi c interaction of particle with the interstitial surrounding, and inhibit the formation of too large particle structure [ 49 ] . surface modifi cation of liposomes with peg also does not have a significant effect on lymph node uptake. small liposomes coated with peg showed greatest clearance from the s.c. injection site with small 86-nm peg-coated liposomes having <40 % remaining at the injection site at 24 h. larger neutral and negatively charged liposomes had a clearance >60 % remaining at the initial s.c. injection site. however, this smaller amount of large liposomes that were cleared from the injection site was compensated by better retention in the lymph node [ 148 ] . oussoren et al. reported that the amount of liposomes cleared from the injection site was somewhat greater with the peg-coated liposomes [ 149 ] . this improved clearance did not result in improved lymph node retention because the fraction of peg liposomes retained by the lymph node is decreased. phillips et al. also studied the slightly improved clearance of peg-coated liposomes from the s.c. injection site [ 148 ] . porter and coworkers demonstrated that pegylation of poly-l -lysine dendrimers resulted into better absorption from s.c. injection sites and stated that the extent of lymphatic transport may be improved by increasing the size of the pegylated dendrimer complex. they estimated the lymphatic uptake and lymph node retention properties of several generation four dendrimers coated with peg or 4-benzene sulphonate after subcutaneous administration in rats. for this surface modifi cation study, three types of pegs with molecular weights of 200, 570 or 2,000 da were taken. peg200-derived dendrimers showed rapid and complete absorption into the blood when injected subcutaneously, and only 3 % of the total given dose was found in the pooled thoracic lymph over 30 h, whereas peg570-and peg2000derived dendrimers showed lesser absorption, and a higher amount was recovered in lymphatics (29 %) over 30 h. however, the benzene sulphonate-capped dendrimer was not well absorbed either in the blood or in lymph following subcutaneous injection [ 150 ] . carriers capped with nonspecifi c human antibodies as ligands showed greater lymphatic uptake and lymph node retention compared to uncoated one at the s.c. site. liposomes coated with the antibody, igg, have been shown to increase lymph node localisation of liposomes to 4.5 % of the injected dose at 1 h, but this level decreased to 3 % by 24 h [ 151 ] . in a study, the liposomes containing positively charged lipids had approximately 2-3 times the lymph node localisation (up to 3.6 % of the injected dose) than liposomes containing neutral or negatively charged lipids (1.2 % of the injected dose) [ 149 ] . attachment of mannose to the surface of a liposome increased lymph node uptake by threefold compared to control liposomes [ 152 ] . another study demonstrated hbsag entrapped dried liposomes with their surfaces modifi ed with galactose. pharmacokinetic study in rats showed that galactosylated liposomes delivered higher amounts of hbsag to the regional lymph nodes than other ungalactosylated formulations [ 153 ] . lectin is another ligand that can be attached to the carriers for improved targeting to intestinal lymphatics. bovine serum albumin containing acid phosphatase model protein and polystyrene microspheres conjugated with mouse m-cell-specifi c ulex europaeus lectin. ex vivo results showed that there was favoured binding of the lectin-conjugated microspheres to the follicle-associated epithelium. final results indicated that coupling of ligands such as lectin specifi c to cells of the follicleassociated epithelium can improve the targeting of encapsulated candidate antigens for delivery to the peyer's patches of the intestine for better oral delivery [ 154 ] . to improve carrier retention in lymph nodes, a new method of increasing lymphatic uptake of subcutaneously injected liposome utilises the high-affi nity ligands biotin and avidin. biotin is a naturally occurring cofactor and avidin is a protein derived from eggs. avidin and biotin are having extremely high affi nity for each other. for instance, upon injection, the avidin and the biotin liposomes move into the lymphatic vessels. biotin liposomes that migrate through the lymphatic vessels meet the avidin resulting in an aggregate that becomes trapped in the lymph nodes [ 155 , 156 ] . the biotin liposome/avidin system has promising potential as therapeutic agent for delivery to lymph nodes. it can be applied not only to s.c. targeting of lymph nodes but also to intracavitary lymph node targeting [ 50 ] . different ligands with their application in lymphatic targeting are represented in table 20 .1 . the lymphatics have the potential to play a major role in anticancer treatment as lymphatic spread is recognised to precede haematological spread in many cancers including melanoma, breast, colon, lung and prostate cancers. currently, the focus is on the development of drug carriers that can localise chemotherapy to the lymphatic system, thus improving the treatment of localised disease while minimising the exposure of healthy organs to cytotoxic drugs. the delivery of novel carriers to lymph nodes for therapeutic purposes has much promise. giving importance to the lymphatic route in metastasis, this delivery system may have great potential for targeted delivery of various therapeutic agents to tumours and their metastatic lymph nodes. various delivery systems have been discussed here but colloidal carriers, especially, liposomes have been the carrier of choice to date. the purpose of this review is to provide an improved and effective lymphotropic system with a satisfactory quality for clinical use and to establish a preparation method applicable for industrial production. surface-engineered lymphotropic systems may prove as an effective carrier for anti-hiv, anticancer and oral vaccine delivery in near future. 3. delivery of antigens to gut-associated lymphoid tissue (galt) intestinal delivery [ 154 ] 4. microparticles active targeting of peripheral lymph nodes doppler ultrasonography contrast agent [ 159 ] 5. lymph vaccine delivery [ 148 -150 ] 6. 13. block copolymer of poloxamine and poloxamer nanospheres regional lymph nodes [ 144 ] 14. lyp-1 nanoparticles, liposomes targeted to lymphatic vessels and also in tumour cells within hypoxic area antitumour [ 129 ] 15. liposomes targeting to lymph node mediastinal lymph node targeting [ 155 ] 16. liposome targeting to lymph node increased lymph node retention [ 145 , 151 ] 25 the physiology of the lymphatic system the anatomy and development of the jugular lymph sacs in the domestic cat (felis domestica) on the origin of the lymphatic system from the veins and the development of the lymph hearts and thoracic duct in the pig dual origin of avian lymphatics lineage tracing demonstrates the venous origin of the mammalian lymphatic vasculature an essential role for prox1 in the induction of the lymphatic endothelial cell phenotype prox1 function is required for the development of the murine lymphatic system live imaging of lymphatic development in the zebrafi sh developmental and pathological lymphangiogenesis: from models to human disease tumor lymphangiogenesis and melanoma metastasis cardiovascular physiology new insights into the molecular control of the lymphatic vascular system and its role in disease advanced colloid-based systems for effi cient delivery of drugs and diagnostic agents to the lymphatic tissues the structure of lymphatic capillaries in lymph formation specifi c adhesion molecules bind anchoring fi laments and endothelial cells in human skin initial lymphatics focal adhesion molecules expression and fi brillin deposition by lymphatic and blood vessel endothelial cells in culture the second valve system in lymphatics evidence for a second valve system in lymphatics: endothelial microvalves ultrastructural studies on the lymphatic anchoring fi laments new horizons for imaging lymphatic function lymphatic smooth muscle: the motor unit of lymph drainage the fi ne structure and functioning of tissue channels and lymphatics clinically oriented anatomy lymphangiogenesis in development and human disease acyclic nucleoside phosphonate analogs delivered in ph-sensitive liposomes liposomes for drug targeting in the lymphatic system liposomes to target the lymphatics by subcutaneous administration novel method of greatly enhanced delivery of liposomes to lymph nodes current concepts in lymph node imaging old friends, new ways: revisiting extended lymphadenectomy and neoadjuvant chemotherapy to improve outcomes targeted delivery of indinavir to hiv-1 primary reservoirs with immunoliposomes studies on lymphoid tissue from hivinfected individuals: implications for the design of therapeutic strategies lymphoid tissue targeting of anti-hiv drugs using liposomes a randomized clinical trial comparing single-and multi-dose combination therapy with diethylcarbamazine and albendazole for treatment of bancroftian fi lariasis infección bacteriana por ántrax bioterrorism-related inhalational anthrax: the fi rst 10 cases reported in the united states fatal inhalational anthrax in a 94-year-old connecticut woman extrapulmonary tuberculosis: clinical and epidemiologic spectrum of 636 cases nanoparticles for drug delivery in cancer treatment polymeric drugs for effi cient tumor-targeted drug delivery based on epr-effect exploiting the enhanced permeability and retention effect for tumor targeting drug targeting and tumor heterogeneity does a targeting ligand infl uence nanoparticle tumor localization or uptake? high affi nity restricts the localization and tumor penetration of single-chain fv antibody molecules vcam-1 directed immunoliposomes selectively target tumor vasculature in vivo lymphatic targeting with nanoparticulate system a lymphotropic colloidal carrier system for diethylcarbamazine: preparation and performance evaluation evaluation of endoscopic pirarubicin-lipiodol emulsion injection therapy for gastric cancer targeted lymphatic transport and modifi ed systemic distribution of ci-976, a lipophilic lipid-regulator drug, via a formulation approach self-emulsifying drug delivery systems (sedds) of coenzyme q 10: formulation development and bioavailability assessment liposomes as vehicles for the local release of drugs enhanced oral bioavailability and intestinal lymphatic transport of a hydrophilic drug using liposomes delivery of liposomal doxorubicin (doxil) in a breast cancer tumor model: investigation of potential enhancement by pulsed-high intensity focused ultrasound exposure reduced cardiotoxicity and comparable effi cacy in a phase iii trial of pegylated liposomal doxorubicin hcl (caelyx™/doxil®) versus conventional doxorubicin for fi rst-line treatment of metastatic breast cancer doxil offers hope to ks sufferers liposomal doxorubicin (doxil): in vitro stability, pharmacokinetics, imaging and biodistribution in a head and neck squamous cell carcinoma xenograft model caelyx/doxil for the treatment of metastatic ovarian and breast cancer patent blue v encapsulation in liposomes: potential applicability to endolympatic therapy and preoperative chromolymphography aerosol delivery of liposomal formulated paclitaxel and vitamin e analog reduces murine mammary tumor burden and metastases novel vitamin e analogue and 9-nitro-camptothecin administered as liposome aerosols decrease syngeneic mouse mammary tumor burden and inhibit metastasis use of liposome preparation to treat mycobacterial infections nanoradioliposomes molecularly modulated to study the lung deep lymphatic drainage solid lipid nanoparticles in lymph and plasma after duodenal administration to rats duodenal administration of solid lipid nanoparticles loaded with different percentages of tobramycin transmucosal transport of tobramycin incorporated in sln after duodenal administration to rats. part i-a pharmacokinetic study pharmacokinetics and tissue distribution of idarubicin-loaded solid lipid nanoparticles after duodenal administration to rats infl uence of administration route on tumor uptake and biodistribution of etoposide loaded solid lipid nanoparticles in dalton's lymphoma tumor bearing mice metastatic patterns in small-cell lung cancer: correlation of autopsy fi ndings with clinical parameters in 537 patients metastatic pattern in non-resectable non-small cell lung cancer solid lipid nanoparticles (sln) for controlled drug delivery-a review of the state of the art lymphatic uptake of pulmonary delivered radiolabelled solid lipid nanoparticles infl ammation imaging using tc-99m dextran intralymphatic chemotherapy using a hyaluronan-cisplatin conjugate lymph node localisation of biodegradable nanospheres surface modifi ed with poloxamer and poloxamine block co-polymers block copolymer carrier systems for translymphatic chemotherapy of lymph node metastases translymphatic chemotherapy by intrapleural placement of gelatin sponge containing biodegradable paclitaxel colloids controls lymphatic metastasis in lung cancer enhancement of therapeutic effi cacy of bleomycin by incorporation into biodegradable poly-d, l-lactic acid targeting colloidal particulates to thoracic lymph nodes comparison of dendrimer-based macromolecular contrast agents for dynamic micro-magnetic resonance lymphangiography delivery of gadolinium-labeled nanoparticles to the sentinel lymph node: comparison of the sentinel node visualization and estimations of intra-nodal gadolinium concentration by the magnetic resonance imaging multimodal nanoprobes for radionuclide and fi ve-color near-infrared optical lymphatic imaging a dendrimer-based nanosized contrast agent dual-labeled for magnetic resonance and optical fl uorescence imaging to localize the sentinel lymph node in mice multicolor imaging of lymphatic function with two nanomaterials: quantum dot-labeled cancer cells and dendrimerbased optical agents hydrophilic multi-walled carbon nanotubes decorated with magnetite nanoparticles as lymphatic targeted drug delivery vehicles tumor targeting with antibody-functionalized, radiolabeled carbon nanotubes waterdispersed single-wall carbon nanohorns as drug carriers for local cancer chemotherapy enhanced effi cacy of bleomycin adsorbed on silica particles against lymph node metastasis derived from a transplanted tumor selective distribution of aclarubicin to regional lymph nodes with a new dosage form: aclarubicin adsorbed on activated carbon particles carbon dye as an adjunct to isosulfan blue dye for sentinel lymph node dissection safety, pharmacokinetics, and preliminary clinical activity of inotuzumab ozogamicin, a novel immunoconjugate for the treatment of b-cell non-hodgkin's lymphoma: results of a phase i study therapeutic potential of cd22-specifi c antibody-targeted chemotherapy using inotuzumab ozogamicin (cmc-544) for the treatment of acute lymphoblastic leukemia antibody-targeted chemotherapy with cmc-544: a cd22-targeted immunoconjugate of calicheamicin for the treatment of b-lymphoid malignancies preclinical anti-tumor activity of antibody-targeted chemotherapy with cmc-544 (inotuzumab ozogamicin), a cd22-specifi c immunoconjugate of calicheamicin, compared with non-targeted combination chemotherapy with cvp or chop rituximab (rituxan®/mabthera®): the fi rst decade cd20-specifi c antibody-targeted chemotherapy of non-hodgkin's b-cell lymphoma using calicheamicin-conjugated rituximab siglecs as targets for therapy in immune-cell-mediated disease clinical activity of the immunoconjugate cmc-544 in b-cell malignancies: preliminary report of the expanded maximum tolerated dose (mtd) cohort of a phase 1 study objective responses in a phase i dose-escalation study of sgn-35, a novel antibody-drug conjugate (adc) targeting cd30, in patients with relapsed or refractory hodgkin lymphoma targeted delivery of interferonalpha via fusion to anti-cd20 results in potent antitumor activity against b-cell lymphoma chitosan for mucosal vaccination polysaccharide colloidal particles as delivery systems for macromolecules suppression of tumor growth and metastasis by a vegfr-1 antagonizing peptide identifi ed from a phage display library antiangiogenic and antitumor activities of peptide inhibiting the vascular endothelial growth factor binding to neuropilin-1 a tumor-homing peptide with a targeting specifi city related to lymphatic vessels chemotherapy with hybrid liposomes for acute lymphatic leukemia leading to apoptosis in vivo lyp-1-conjugated nanoparticles for targeting drug delivery to lymphatic metastatic tumors successful treatment of primary and disseminated human lung cancers by systemic delivery of tumor suppressor genes using an improved liposome vector exposure of human ovarian carcinoma to cisplatin transiently sensitizes the tumor cells for liposome-mediated gene transfer combination gene therapy for liver metastasis of colon carcinoma in vivo polymeric microspheres as drug carriers the organ distribution and circulation time of intravenously injected colloidal carriers sterically stabilized with a blockcopolymerpoloxamine 908 fate of liposomes in vivo: a brief introductory review the characterisation of radio colloids used for administration to the lymphatic system effect of size on the lymphatic uptake of a model colloid system radiolabeled colloids and macromolecules in the lymphatic system electron microscopic studies on the peritoneal resorption of intraperitoneally injected latex particles via the diaphragmatic lymphatics lymphatic transport of liposomeencapsulated drugs following intraperitoneal administration-effect of lipid composition assessment of the potential uses of liposomes for lymphoscintigraphy and lymphatic drug delivery failure of 99-technetium marker to represent intact liposomes in lymph nodes effect of molecular weight on the lymphatic absorption of water-soluble compounds following subcutaneous administration surface engineered nanospheres with enhanced drainage into lymphatics and uptake by macrophages of the regional lymph nodes serum opsonins and liposomes: their interaction and opsonophagocytosis physicochemical principles of pharmacy targeting of colloids to lymph nodes: infl uence of lymphatic physiology and colloidal characteristics evaluation of [(99m) tc] liposomes as lymphoscintigraphic agents: comparison with [(99m) tc] sulfur colloid and [(99m) tc] human serum albumin lymphatic uptake and biodistribution of liposomes after subcutaneous injection: iii. infl uence of surface modifi cation with poly(ethyleneglycol) pegylation of polylysine dendrimers improves absorption and lymphatic targeting following sc administration in rats lymph node localization of non-specifi c antibody-coated liposomes modifi ed in vivo behavior of liposomes containing synthetic glycolipids enhanced lymph node delivery and immunogenicity of hepatitis b surface antigen entrapped in galactosylated liposomes targeted delivery of antigens to the gut-associated lymphoid tissues: 2. ex vivo evaluation of lectinlabelled albumin microspheres for targeted delivery of antigens to the m-cells of the peyer's patches avidin/biotin-liposome system injected in the pleural space for drug delivery to mediastinal lymph nodes pharmacokinetics and biodistribution of 111 in avidin and 99 tc biotin-liposomes injected in the pleural space for the targeting of mediastinal nodes folate-peg-ckk 2-dtpa, a potential carrier for lymph-metastasized tumor targeting nanotechnology in cancer therapeutics: bioconjugated nanoparticles for drug delivery molecular targeting of lymph nodes with l-selectin ligand-specifi c us contrast agent: a feasibility study in mice and dogs hyaluronan in drug delivery lymphatic targeting of zidovudine using surfaceengineered liposomes alginate/chitosan microparticles for tamoxifen delivery to the lymphatic system homing of negatively charged albumins to the lymphatic system: general implications for drug targeting to peripheral tissues and viral reservoirs key: cord-212813-yrca1hij authors: winkelmann, ricarda; donges, jonathan f.; smith, e. keith; milkoreit, manjana; eder, christina; heitzig, jobst; katsanidou, alexia; wiedermann, marc; wunderling, nico; lenton, timothy m. title: social tipping processes for sustainability: an analytical framework date: 2020-10-09 journal: nan doi: nan sha: doc_id: 212813 cord_uid: yrca1hij societal transformations are necessary to address critical global challenges, such as mitigation of anthropogenic climate change and reaching un sustainable development goals. recently, social tipping processes have received increased attention, as they present a form of social change whereby a small change can shift a sensitive social system into a qualitatively different state due to strongly self-amplifying (mathematically positive) feedback mechanisms. social tipping processes have been suggested as key drivers of sustainability transitions emerging in the fields of technological and energy systems, political mobilization, financial markets and sociocultural norms and behaviors. drawing from expert elicitation and comprehensive literature review, we develop a framework to identify and characterize social tipping processes critical to facilitating rapid social transformations. we find that social tipping processes are distinguishable from those of already more widely studied climate and ecological tipping dynamics. in particular, we identify human agency, social-institutional network structures, different spatial and temporal scales and increased complexity as key distinctive features underlying social tipping processes. building on these characteristics, we propose a formal definition for social tipping processes and filtering criteria for those processes that could be decisive for future trajectories to global sustainability in the anthropocene. we illustrate this definition with the european political system as an example of potential social tipping processes, highlighting the potential role of the fridaysforfuture movement. accordingly, this analytical framework for social tipping processes can be utilized to illuminate mechanisms for necessary transformative climate change mitigation policies and actions. there is growing concern that global climate change is reaching a point where parts of the earth system are starting to pass damaging climate tipping points (1): in particular, part of the west antarctic ice sheet (wais) appears to already be collapsing because of irreversible retreat of grounding lines (2, 3) which in turn is expected to trigger loss of the rest of the wais (4) . other tipping points may be close: a recent systematic scan of earth system model projections has detected a cluster of abrupt shifts between 1.5 and 2.0°c of global warming (5) , including a collapse of labrador sea convection with far-reaching impacts on human societies. the abrupt degradation of tropical coral reefs is projected to be almost complete if warming reaches 2.0°c (6, 7) . the possibility of the global climate tipping to a 'hothouse earth' state has even been posited (8) . against this backdrop, there is a growing consensus that avoiding crossing undesired climate tipping points requires rapid transformational social change, which may be propelled (intentionally or unintentionally) by triggering social tipping processes (9, 10) or "sensitive intervention points" (11, 12) . examples for such proposed social tipping dynamics include divestment from fossil fuels in financial markets, political mobilization and social norm change, socio-technical innovation (911, 13, 14) . equally, if human societies do not act collectively and decisively, climate change could conceivably trigger undesirable social tipping processes, such as international migration bursts, food system collapse or political revolutions (15) . social tipping processes have received recent attention, as they encompass this sort of rapid, transformational system change (9, 10, 13, 15) . here we develop an analytical framework for social tipping processes. drawing upon expert elicitation and a comprehensive literature review, we find that the mechanisms underlying social tipping processes are categorically different from other forms of tipping, as they uniquely have the capacity for agency, they operate on networked social structures, have different spatial and temporal scales, and a higher degree of complexity. following these distinctions, we present a definitional framework for identifying social tipping processes for sustainability, where under critical conditions, a small perturbation can induce non-linear systemic change, driven by positive feedback mechanisms and cascading network effects. we adopt this framework to understand potential social tipping dynamics in the european political system, where the fridaysforfuture movement (16) pushes the system towards criticality, generating the conditions for shifting climate policy regimes into a qualitatively different state. the proposed framework aims to establish a common terminology to avoid misconceptions, including the notions of agency, criticality as well as the manifestation and intervention time horizons in the context of social tipping. in this way, the framework can serve to connect literatures and science communities working on social tipping, social change, complex contagion dynamics and evidence from behavioral experiments (e.g. 14, 17). we start by reviewing the characterization of tipping points across the natural and social sciences. over the last 150 years, a suite of concepts and theories describing small changes with large systemic effects has been developed at the intersection of natural and social sciences. more recently, the concepts of tipping points and tipping elements have been broadly adopted by both natural and social scientists working within the field of climate change. while the concept of 'tipping' originated in the natural sciences (18, 19) , social scientists made extensive use of the idea in the 20th century, often without using the terminology of tipping. famously, schelling (20) , following grodzins (21) , developed a theory of tipping processes to explain racial segregation in us neighbourhoods. granovetter (22) modeled collective behavior as a tipping process that depends on passing individual thresholds for participation in riots or strikes. kuran (23) described political revolution in terms of tipping dynamics, while gould and eldridge (24) distinguish phases of policy change and stability in terms of 'punctuated equilibrium'. gladwell (25) popularised the concept of 'tipping points', exploring contagion effects ("fads and fashions"), sometimes triggered by specific events. several recent studies have examined tipping processes within contemporary social systems. homer-dixon (26) and battison (27) explored the 2008 financial crisis as a tipping phenomenon. nyborg (14, 28) discussed shifts in norms and attitudes, for example regarding smoking behaviors. centola (17) associated tipping points with the "critical mass phenomenon", wherein 20-30% of a population becoming engaged in an activity can be sufficient to tip the whole society. similarly, rockström et al. (29) highlighted this so-called pareto effect in the context of decarbonization transitions. kopp et al. (15) distinguished different social tipping elements within the realm of policy, new technologies, migration and civil conflict that are sensitive to "climate-economic shocks". here, a tipping element is a system or subsystem that may undergo a tipping process. since the mid 1990s, ecologists and social-ecological systems (ses) researchers have also developed an extensive body of research on tipping processes using the terminology of 'regime shifts' and 'critical transitions' (e.g. [30] [31] [32] . recognizing the impacts of human development on various ecosystems, this body of work often models ecological regime shifts as a consequence of social drivers. less attention, however, has been paid to sudden changes in social systems triggered by ecosystem changes. there is a rich literature on the collapse of past civilizations (e.g. 33, 34) and the potential role of tipping points in that (35) . recently, cumming and peterson (36) brought this together with work on ecological regime shifts, proposing a "unifying social-ecological framework" for understanding resilience and collapse. further, rocha et al. (37) noted that tipping dynamics can be produced by the interactions between climatic, ecological and social regime shifts. the concept of climate tipping elements introduced by lenton et al. (1) and schellnhuber (38) , has been increasingly adopted within earth and climate sciences. climate tipping elements are defined as at least sub-continental-scale components of the climate system that can undergo a qualitative change once a critical threshold in a control variable, e.g., global mean temperature, is crossed. positive feedback mechanisms at the critical threshold drive the system's transition from a previously stable to a qualitatively different state (1). other scholars, e.g., levermann et al. (39) , suggest a somewhat narrower definition of climate tipping elements by introducing additional characteristics, such as (limited) reversibility or abruptness. the tipping elements identified so far include biosphere components such as the amazon rainforest (40) (41) (42) and coral reefs (6, 7) , cryosphere components such as the ice-sheets on greenland and antarctica (43) , and large-scale atmospheric or oceanic circulation systems including the atlantic meridional overturning circulation (44, 45) . their tipping would have far-reaching impacts on the global climate, ecosystems and human societies (e.g. 8, 46). in response to the concept of climate tipping points, social scientists are re-engaging with this concept yet again, creating an additional layer of tipping scholarship with an emphasis on the need for and possibility of deliberate tipping of social systems onto novel development pathways towards sustainability (e.g. 11, 47) . scholars argue in particular that the rapid, non-linear change of social tipping dynamics might be necessary to speed up societies' responses to climate change, and to achieve the goals of the paris agreement. it is this element of acceleration, propelled by positive feedbacks, that makes the concept of tipping particularly interesting. for example, otto and donges et al. (9) reported expert elicitations identifying social tipping elements relevant for driving rapid decarbonization by 2050. rapid-paced changes are a distinctive feature potentially differentiating tipping dynamics from many other forms of social change, including incremental (policy or institutional) changes, or more radical (socio-technical) transitions or societal transformations. over the last decade, the literature on deliberate transitions and transformations towards sustainability has expanded significantly, exploring the dynamics that lead to the reorganization of social, economic or political systems (e.g. 48, 49) . in many ways, this literature and the emerging work on social tipping are interested in very similar phenomena: fundamental shifts in the organization of social or social-ecological systems -a movement from one stable state to anotherincluding a change in power relations, resource flows, as well as actor identities, norms and other meanings (48) . transformations can be fast, but speed is generally not one of their defining characteristics. this temporal feature of social tipping points -rapidity of change compared to the system's normal background rate of change -combined with the fact that tipping processes can be triggered by a relatively small disturbance of the system is motivating scholarship on leverage or 'sensitive intervention points', e.g. farmer et al. (12) , who identified such potentially high-impact intervention opportunities, e.g., financial disclosure, choosing investments in technology and political mobilization that may be key for triggering decarbonization transitions. based on a bibliometric and qualitative review of these various bodies of literature across the natural and social sciences, milkoreit et al. (10) proposed the following general definition of (social) tipping: "the point or threshold at which small quantitative changes in the system trigger a nonlinear change process that is driven by system-internal feedback mechanisms and inevitably leads to a qualitatively different state of the system, which is often irreversible." milkoreit et al. (10) further noted there is a need to recognize and identify potential differences between climatic (or ecological) and social tipping processes to gain a deeper understanding of these phenomena. given this diverse and nascent field, there is a clear need for consensus as to what defines social tipping processes, as well as an understanding of how these processes are similar and diverge from dynamics in other non-social systems. further, there are currently limited examples of social tipping elements in the context of sustainability transitions presented within the broader literature (9, 12, 13, 15) . here we explore the characterization of tipping processes within the natural and social sciences, examining how social and climate tipping processes are differently conceptualized. we draw upon a mixed qualitative methodological approach to illuminate these differences and key distinctions. initially, core differences were identified and discussed via expert elicitation (50) . a selected group of 25 experts from across the climate and social sciences were invited to take part in an expert elicitation workshop, that focused on identifying a common definition for social tipping processes, as well as the characterization of their dynamics. this workshop was convened in june 2018 in cologne, germany. the workshop participants were split into cross-disciplinary breakout groups, to independently identify the dynamics of social tipping processes. then, each of these groups reported their findings to the broader plenary, for discussion, consolidation, reconciliation and clarification. the process was then repeated for further clarification within the breakout groups. through this iterative inductive and deductive process, several unique themes and characteristics were identified from the broader set of codes, resulting in the key differences in and definition of social tipping processes presented below. drawing upon the differences identified in the expert elicitation workshop, we then review and synthesize the emerging field of social tipping processes, particularly in comparison to the related climate and ecological tipping dynamics. we then draw upon these unique characteristics to develop a common definition for social tipping processes, which we explore using the example of the fridaysforfuture student movement. social and climate systems' tipping processes exhibit several broad, fundamental differences in their structure and underlying mechanisms: (i) agency is a main causal driver of social tipping processes, (ii) the quality of social networks and associated information exchange provides for specific social change mechanisms not available in non-human systems, (iii) climate and social tipping processes occur at different spatial and temporal scales, and (iv) social tipping dynamics exhibit significantly more complexity than climatic ones. the most important characteristic differentiating social from climate tipping processes is the presence of agency. while a significant body of work (e.g. 51), including latour's actor-network theory (52) , addresses different forms and effects of non-human or more-than-human agency, here, we focus on a more narrow understanding of agency that is based on consciousness and cognitive processes such as foresight, planning, normative-principled and strategic thinking, that allow human beings to purposefully affect their environment on multiple temporal and spatial scales. while humans have a generally poor track record of utilizing their agentic capacities especially with regard to shaping the future (e.g. [53] [54] [55] , they appear unique in their capacity to transcend current realities with their decisions. agency in this more narrow sense can be understood as the human capacity to exercise free will, to make decisions and consciously chart a path of action (individually or collectively) that shapes future life events and the environment (56) . the notion of intentionality inherent in the idea of agency implies that human actors are not only able to adapt to changes in their environment, but also deliberately create such changes. non-human life forms can also be engaged in deliberate changes of their environment (e.g., beavers building dams), but the cognitive quality of these actions differs from those of humans, which can be based on different forms of knowledge and meaning about the world, moral norms and principles, or ideas about desirable futures. agency allows individuals and societies to be proactive rather than merely responsive in their relationships with other humans or the environment through planning, goal setting and strategic decision-making, which links decisions and behaviors in the present with consequences and realities in the (distant) future (57) . governance scholars address this social-cognitive capacity for forethought and goal-pursuit in terms of anticipation (58) and imagination (10) , which can be tied to a set of futuring methods (59, 60) . the ability to anticipate and imagine futures enables humans and their societies (53, 54) -as opposed to animal communities or ecosystems -to transcend the present and shape the future according to our values and goals (61), possibly increasing the prospects for human survival in times of fast and significant environmental change (56, 62) . although this ability has been underutilized in the past, especially in the context of responding to climate change (63), it is a crucial dimension of the human repertoire of tools to create change and to ensure its long-term well-being. agency interacts with many of the additional differentiating characteristics we identify below in important ways. for example, agency plays a role in the creation of social networks, institutions and meaning, i.e., the production of the structures of social systems. these network structures in turn enable and constrain agency (e.g. 64, 65) . physical climate tipping elements, such as ice sheets or ocean circulations, lack that ability to intentionally act and adapt. however, the adaptive capacity of ecosystems can be interpreted as a form of non-human agency and learning mechanism (66), see also supplementary information s2. while scholarship on non-human agency, including that of animals, inanimate objects, landscape features or ecosystems (e.g. 67, 68) might expand our understanding of agency, the cognitive abilities that characterize human agency, especially long-term and strategic thinking, do not exist in the non-human or inanimate worlds. understanding the nature of social networks is crucial for studying social tipping. while both natural (including physical and ecological) and social systems can be structurally characterized as networks and studied using a network science approach (69), social systems differ from natural systems in the quality of the networks' nodes and interconnections and the processes and dynamics facilitated and impacted by these particular network characteristics. social systems feature additional network levels of information transmission (cultural and symbolic) that are largely restricted to human societies compared to natural systems (70) . network qualities unique to social systems: networks in social and natural systems share various commonalities such as the existence of fundamental nodes and links (69) . in contrast to most natural systems, however, social networks have the capacity to intentionally generate new nodes, which include socially constructed entities such as organizations and movements (71) . new nodes can be created through cultural, political or legal means, as can the rules for their interactions with other existing nodes. social system nodes are unique in that they have richer cognitive realities, particularly agency and forethought. these nodes often have conflicting vested interests, which may be more short-sighted than future oriented. relationships in social networks can consist of shared meanings -especially norms, identities and other ideas -and a vast variety of cultural, economic and political relationships (e.g., employment, citizenship), all of which are not as pronounced or non-existent in less complex human societies and nature. hence, social network links are more diverse than links in natural systems and enable different kinds of network processes. for example, links between nodes in social networks are not necessarily dependent on physical co-presence, due to technologically enabled connections or the presence of more abstract interrelations such as shared norms, values or interpersonal relationships. social network dynamics can be of a purely ideational nature (e.g., the subject of the study of opinion and belief dynamics), but also involve material changes (e.g., resource extraction, movement and transformation for economic purpose). markets are unique social networks, involving both ideational and material network processes. in the anthropocene, the intensity and speed of socially networked interaction has increased dramatically, largely due to new media, digitalization, more efficient means of transportation, lower travel costs, and overall increased mobility, which is likely to increase spreading rates, while at the same time affecting the stability of the network itself (72) (73) (74) . generally, social tipping can either occur on a given network (e.g., through spreading dynamics changing the state of nodes (75) or change the network structure itself (see figure 1 ). the structural network changes generated by social tipping processes include transitions from centralistic or hierarchical to more polycentric (neuromorphic) structures in urban systems, energy distribution and generation networks (76, 77) . structural changes can manifest on large and small-scale spatial networks across multiple social structure levels. in order to capture these network tipping processes, quantifiers from complex network theory such as modularity, degree distribution, centrality or clustering can be used (69) . 3, 1975 to january 3, 1977) . node colors indicate different party membership and links between nodes are drawn if the corresponding members agree on 66% of all votes in the considered two-year period. the lower network shows the same for the 110th united states congress (january 3, 2007, to january 3, 2009 ). the transition from a closely entangled to an almost fragmented topology indicates a polarisation between democratic and republican party members over time (16) . temporal and spatial scales: scales can differ greatly between social tipping and climate tipping processes and are more ephemeral for social tipping than for climate tipping. temporally, tipping in social systems manifests more commonly on the scale of months to decades, while for the climate tipping elements range from years to millennia. human actors tend to focus on more short-term consequences or outcomes, as complex issues (such as climate change) with longer timeframes are often harder to assess (79) . within social systems, fund manager performance is evaluated quarterly, politicians often think in electoral cycles, business operates with annual or five-year forecasts, while individual practices and dispositions are constantly evaluated and reevaluated (80) (81) (82) . in natural systems, however, it might take decades, centuries or even millennia for outcomes of change processes to become detectable (see figure 2 ). both social and climate tipping elements can be ordered spatially (1, 39, 83) , although social tipping elements cannot always be precisely located geographically. social scientists and economists have long grouped systems and processes as existing on the macro-, meso-and micro-levels (or some variation thereof), whereby some social systems (e.g., financial markets, political systems, technologies) consist of interdependent subsystems existing on multiple spatial levels. social tipping processes can also display spatial-temporal ephemerality. while climate tipping elements have a known spatial extent and dimensionality (with often a comparable extent in latitude and longitude and a generally much smaller extent in altitude) and have persisted in their current stable state for thousands (if not millions) of years, social tipping processes do not have a spatial extent or effective dimensionality that is known ex-ante and they can emerge (move into a critical state) and disappear (move out of a critical state) over time. (84) , tabara (11) and lenton (13) . complexity: social tipping processes occur in complex adaptive systems (85-87) as opposed to the complex but non-adaptive physical climate system. as such they can exhibit comparatively greater complexity in the (i) drivers, (ii) mechanisms and (iii) resulting pathways of social tipping processes, as well as the aforementioned ephemerality in their spatial-temporal manifestations, including a potentially fractal and varying dimensionality and a more complex interaction topology (88, 89) . social tipping processes can rarely be linked to a single common control parameter, such as is the case with global mean temperature in climate tipping dynamics. for most of the climate tipping elements like the ice sheets or the atlantic meridional overturning circulation, the control variables such as local air temperature, precipitation or ocean heat transport, can often be translated or downscaled into changes in global mean temperature as one common driver (1, 38). however, for social tipping processes, multiple, interrelated factors are often identified as forcing the critical transition. for example, shifts in social norms regarding smoking (14) can be linked to several, entwined factors, such as policies, taxation, advertising and communication, social feedbacks (e.g., via normative conformity), or individual preference changes. centola et al. (17) show that tipping in social convention is possibly explained by a single parameter: the size of the committed minority). at larger scales, the collapse of complex civilizations has been linked to multiple interacting causes, and whilst disagreement abounds over the balance of causes in particular cases, there is general agreement that multiple factors were at play (33) . this kind of causality -multiple interacting, distributed causes across varying scales -are a key characteristic of complex systems (90) , contrasting starkly with conventional notions of causality involving bivariate relationships (one cause and one effect). further, due to their potential for agency and adaptive plasticity, social systems are open to a larger number of mechanisms that could cause a tipping process and various pathways of change that a tipping process could follow towards a greater number of potentially stable post-tipping states (91) . climate tipping processes are often modeled as bi-or multistable, where the directional outcomes of forcing are to some extent known or knowable, e.g., based on paleoclimatic data and processbased earth system modelling. given a specific forcing change, one can predict in what state the element will restabilize as well as the "net" effects of the tipping process on larger earth systems. based on this understanding, the tipping of climate system elements is generally perceived as undesirable and often as part of pushing the earth system out of the "safe operating space for humanity" (92, 93) . in contrast, for social systems, it is often unclear what a final stable state of the system will look like, or even whether the changes resulting from a tipping process will be normatively considered "positive" or "negative". as clark and harley (94) point out, the characteristics of complexadaptive social systems, including the diversity of actors and elements and the different outcomes generated by local and global interactions, imply that the development pathways of these systems are less predictable. further, a social tipping process can generate new and destroy existing actor types (e.g., identities, institutions) and their behaviors. cross-scale dynamics and local differences are important to understand the emergent system structure and change dynamics, but predictive capacities, e.g., regarding the timing of a social tipping point or the boundaries between different stable states, do not yet exist (94) . hence, the term 'managing transitions' is less useful than the idea of navigating a transformation pathway. the political nature of social change processes (95) -different actors within a social community pursuing different, sometimes opposing, interests and visions for a reorganization of a social system while bringing to bear different resources and strategies -further exacerbate this situation. actors can deliberately generate new feedback dynamics that support or slow change, even after a tipping point has been passed, and they can actively work to adjust the direction of change. from the discussion above, it follows that a definition of social tipping process should take a microperspective and incorporate network effects and agency in addition to common tipping characteristics already explored in the review by milkoreit if a tipping element is already in a critical condition, where the stability of its current state is low, there may be a time window during which an agential intervention might prevent an unwanted tipping process by moving the system into an uncritical condition (see also si text s1). alternatively, if a tipping element is not already in a critical condition, there may be a time window during which some intervention might move it into a critical condition in order to bring about a desired tipping process. the small change triggering the tipping process could be either (i) a localized modification of the network structure (e.g., a change on the level of single nodes, small groups of nodes or links) or of the state of agents or subsystems, (ii) small changes of macroscopic parameters or properties, or (iii) small external perturbations or shocks. we deliberately do not require the trigger to be a single driving parameter. this is because we expect that a social tipping process could be triggered by a combination of causes rather than a single cause. furthermore, a social tipping element may be tipped by several different combinations of causes. consequently, for social tipping elements we cannot always expect at this point to identify a common aggregate indicator (such as global mean temperature in the case of climatic tipping elements) and a well-defined 'threshold' for this indicator at which the system will tip (see also the discussion on complexity above). note that social tipping as defined here is a unique form of social change, e.g., distinct from climate economic shocks (15) and more specific than socio-technical transitions (96, 97) . further, social tipping also denotes a shift to a qualitatively different state, and such, is different from standard business cycles or causes of seasonality. as such, social tipping presents a particular process of social change, where a system undergoes a transformation from one qualitatively different state to another, after being in a more critical state and affected by a potentially small triggering event. we propose several filtering criteria to focus on social tipping processes (i) that have the potential to be relevant to global sustainability in future earth system tractories and (ii) where human interventions can occur within a pertinent intervention time horizon on the order of decades and will have consequences within a political/ethical time horizon on the order of hundreds of years. (i) relevance of social tipping for global sustainability the social tipping process can impact a wide array of social systems, such as technological or energy systems, political mobilization, financial markets and sociocultural norms. we consider social tipping processes to be relevant here that have an impact on the biophysical earth system or on macro-scale social systems. the qualitative change in a 'relevant' social tipping process significantly affects the future state of the earth system in the anthropocene directly or indirectly through interactions with other social tipping processes. relevance can hence be defined in terms of impacts on biophysical earth system properties such as global mean temperature, biosphere integrity or other planetary boundary dimensions. for example, tipping dynamics to a political system could result in policy regime changes, affecting substantial reductions in greenhouse gas emissions (9, 12). furthermore, we consider social tipping processes that have relevant impacts on macro-social systems and can be triggered by changes in the same biophysical earth systems, for example, mass migration due to climate impacts (84, 98) . we are interested in potential social tipping processes in which humans have the agency to substantively intervene. for example, such interventions could be via technological or physical capacities of agential or structural actors. this therefore places emphasis on human intervention, such as decreasing the likelihood of extreme weather events via mitigation efforts, or triggering socio-technological changes towards decarbonization. we define intervention and ethical time horizons as follows: intervention time horizon human agency interferes with a social tipping element, such that decisions and actions taken between now and an 'intervention time horizon' could influence whether (or not) the system tips. we suggest to consider only social tipping processes with an intervention time on the order of 10 years (9), which arguably presents a practical limit of human forethought (99) and of future-oriented political agency. for example, international governance efforts for global sustainability challenges, such as the ozone regime or the sustainable development goals, tend to work with similar time horizons. similarly, social tipping processes for rapid decarbonization to meet the paris climate agreement would have to be triggered within the next few years (9), with ambitious emissions reduction roadmaps aiming for peak greenhouse gas emissions in 2020 (29, 100) . the intervention time horizon is analogous to the 'political time horizon' defined for climate tipping elements in lenton et al. (1). the time to observe these relevant consequences should lie within an 'ethical time horizon'. this recognizes that consequences manifesting too far in the future are not relevant to the current discourse on how contemporary societies impact earth systems. such an ethical time horizon could consider only social tipping processes which can have relevant consequences within the next centuries at most, corresponding to an upper life expectancy of the next generations of children born. currently, international climate policies, including those of the european union (eu) are insufficient to meet the +1.5°c or +2°c goals of the paris agreement (101) . while european policy makers presume to lead global mitigation efforts and characterize their actions as ambitious (102, 103) , actual policy measures and proposals have been lagging behind this aspiration (104) . eu countries emit about a tenth of the world's emissions, and a policy change towards more rapid decarbonization would not only have significant direct impacts on the climate system, but likely have indirect effects on the policies of other major emitters. but what kinds of sociopolitical processes can lead to these necessary changes? could such changes result from social tipping dynamics? public opinion is a crucial factor in policy formation, where the public can be understood as a "thermostat" signalling what is politically feasible (105, 106) . shifts in public opinion can punctuate previously stable and 'sticky' institutions, leading to policy change (107) . increased activism and public concern regarding climate change can generate new coalitions, or shift the priorities of existing ones (108, 109) . here we examine the european political system as an example of and how social tipping processes could be triggered as a result of large-scale public activism and social movements. the european political system is composed of networks of agents (i.e., activists, decision-makers and organizations) with a range of social and political ties and is structured in nested and overlapping subsystems (i.e., national group, transnational political coalitions). viewed through the lens of social tipping, european political dynamics present a 'social system', embedded within the broader international political and climate change governance community 'environment'. driven by the fridaysforfuture movement (16) (among other things), a groundswell of bottom-up support for more proactive climate policies has recently developed among european citizens, resulting in routine mass demonstrations and historical wins for green parties in the 2019 european parliamentary elections, as well as in federal elections in austria, belgium and switzerland. the european political system could be moving towards a critical 'state', creating the conditions for a tipping process towards radical policy change, bringing european climate policy in line with the paris agreement. accordingly, the european political system could constitute a potential 'social tipping element', where as it nears critical conditions, a small change to the system or its broader environment could lead to large-scale macroscopic changes, affected by cascading network dynamics and positive feedback mechanisms. such transformations could involve establishing more aggressive mitigation strategies that connect goals (such as remaining below +2°c, 50% emissions reductions by 2030, zero carbon emissions by 2050) with measures and pathways that have a reasonable chance to achieve them (i.e., investment in negative emission technologies, increased carbon taxation policies etc.). the fridaysforfuture movement has been pushing the european political system towards criticality, where it becomes more likely that the system will be propelled into a qualitatively different state. the movement was set off and inspired by a single swedish high school student choosing to protest on the steps of the riksdag for meaningful climate action. greta thunberg's protest quickly spread through the european social-political networks until more than a million students have been participating in weekly protests. this growing bottom-up pressure on the european climate policy-makers (16, 110) has created an opening for significant policy change. the european political system consists of embedded subsystems at multiple scales. at the national scale, for example, the german socio-political system responded strongly to the activities of the similarly, several national western european green parties received historically strong electoral support in the may 2019 european parliamentary elections (such as in belgium, germany, finland, france and luxembourg). this increased support is also reflected in polling data in germany, where the green party has been effectively equal with the conservative party as the preferred political party of german voters in the latter half of 2019 ( figure 3 , panels c and d). subsequently, germany introduced its first ever federal climate change laws, mandating that the country meet its 2030 goals (a ~55% reduction in ghg emissions) and establishing pathways to carbon neutrality by 2050. currently, only a limited set of countries have enacted national climate change laws, and germany is one of the largest and most diverse economies to propose such actions. this presents the possibility for policy diffusion and transfer to other states (111) , particularly considering the influential role germany plays within the european union. climate policy entrepreneurs could build upon momentum to further capitalize on windows of opportunity, pushing climate change proposals prominently into national and supra-national governmental agendas before the ephemeral moment passes (112) . the 2020 covid-19 pandemic has placed new priorities on the policy agenda, also reflected in issue salience of climate change (see also fig. s1 in supplementary materials). as political and behavioral responses to covid-19 have led already to a significant temporary reduction in greenhouse gas emissions (113) , this shock could be further leveraged to reinforce climate action -future economic recovery packages should set european economies on a pathway towards carbonneutrality, rather than return to the old normal (114, 115) . drawing from this social tipping framework, the european political system may remain near a critical state. it remains unclear whether the covid-19 shock has supplanted climate change, or whether both remain on the political agenda. for example, discussions of a "green new deal" remain at the core of covid-19 economic recovery plans within the european union. the sociopolitical dynamics have likely moved the germany political subsystem further towards criticality, but it remains largely unknown whether this will result in tipping towards a qualitatively different state, in germany or in the broader european political system. rather, these judgements can likely only be made in hindsight, observing whether the system remained stable, moved towards criticality or experienced tipping dynamics. such an analysis in line with the proposed framework requires specific process tracing, identifying the key moments, actors, networks, mechanisms affecting criticality, the triggering event (threshold), and the positive feedback dynamics propelling the system towards qualitative changes. much attention is often paid to the specific triggering event, but it is rarely one single actor or action which accounts for the entirety of the tipping process. rather a full account needs to be made of all of the previous and related processes that have further placed the system towards criticality, allowing for such changes to become more likely. accordingly, for a tipping process to occur at the scale of the entire european political system, moving it into a state of decarbonization that is aligned with the paris agreement, a series of additional social movements and protests, or other shifts within the system or the environment, may be required. while we identify the role of fridaysforfuture in creating critical conditions, or potentially triggering the social transformations required for global sustainability, recent literature has identified further tipping candidates which could have generally "positive" effects on global sustainability. for example, divestment and reinvestment present candidates for rapid decarbonization and processes to achieve climate targets (9, 12). in this case, intervention times range from years to decades, depending on the social structure level (9). previous studies note that the adoption of technologies and behaviors such as rapid uptake of autonomously driven electric vehicles (if socially licensed), rapid change in dietary preferences reducing meat consumption and associated land-use and climate impacts can follow an epidemic-type model of diffusing across social networks (13, 15) . alternatively, social tipping processes can lead to states of criticality with less desirable outcomes: recently it has been shown that climate change has contributed to the emergence of infections carried by mosquitoes, like dengue fever or zika, which could be accelerated further by increased mobility, e.g., through denser air traffic networks (75) . the thermal minimum for transmission of the zika virus could in fact give rise to a threshold behaviour (116) . changes to the local environment may enact "push" factors, resulting in large scale migrations (117, 118) . further, increased global mean temperature has been suggested to increase the likelihood of civil conflicts (84) . these social tipping processes are of great interest to policy makers, as it is desirable to potentially trigger or facilitate "positive" tipping (11, 13) , while at the same time, mitigating the effects of potential "negative" outcomes. social tipping processes have been recently recognized as potentially key pathways for generating the necessary shifts for sustainability. drawing upon this emerging field, this paper develops a framework for characterizing social tipping processes. we find that mechanisms underlying social tipping processes are more likely to exhibit the unique characteristics of agency, social-institutional and cultural network structures, they occur across different spatial and temporal scales to climate tipping, and the nature of tipping can be more complex. social tipping processes thus present qualitatively different characteristics to those shared by climate tipping processes. accordingly, this paper develops a common framework for the unique characteristics of social tipping processes. we identify social tipping as a process, resultant of a complex system of drivers, resulting in shifting a system into a more (or less) critical state. it can thus serve to structure and inform future data analysis and process-based modelling exercises (118, 119) . even so, while there is an emerging focus on social tipping dynamics (9-13), there remains great difficulty in pinpointing tipping events and generalizing the emerging dynamics. drawing from natural tipping dynamics, previous work on social tipping has often focused on identifying specific trigger events or critical thresholds in macroscopic system variables in analogy to identifying for instance critical temperature thresholds in the context of climate tipping (10) . in natural systems the underlying dynamics are more deterministic and often can be directly observed, allowing for the identification of specific thresholds and events. while social systems comprise a much more open and complex system, one that is constantly adapting and where dynamics are often incredibly complex, interrelated and cannot be directly observed. accordingly, one could observe the same event across ten similar social systems, and could potentially observe ten unique outcomes. as such, anticipating a specific trigger, making causal inferences, or having generalizability in expected effects are all greatly limited within social systems. further, social tipping points are sometimes also understood as a point in time, rather than a point in a complex parameter space. such an approach makes it difficult to identify social tipping processes, as they often do not contain easily observable macroscopic thresholds nor temporal markers for change. rather, a complex adaptive systems viewpoint is required, understanding the multitude of interrelated processes and social structures driving change, and not focusing on a single trigger or threshold. accordingly, our framework proposed here focuses on identifying the processes and mechanisms of such change, and not a single triggering event, where the interplay of micro-level changes embedded within adaptive structural conditions can affect systemic changes. the notion of a critical state is central within our framework. changing conditions to the system's environment can cause it to enter more (or less) critical states, such that a single, or multiplicative action, can effect a systemic change. it is these changing conditions, and specifically the processes and dynamics underlying them, that are of analytical importance. drawing upon the analogy of a tipping coal wagon (15), it is not the single, specific piece of coal that caused the wagon to tip, but rather the processes by which the wagon was filled with enough coal that any single piece (placed at a number of different locales) could cause such tipping. accordingly, the specific triggering event of a social tipping process could be somewhat random or arbitrary, as the conditions are critical enough such that any event with enough magnitude could have triggered these dynamics. it is therefore key to focus on the processes and mechanisms underlying the nature of such critical states which allow some trigger event to cause contagion dynamics. from social network models, we can deduce which kind of structural features make a system less resilient and thus more prone to social tipping (119). one example is polarization, where social network models and social mediabased data analyses have shown that in polarized states with nearly disconnected network communities which in themselves are highly connected, contagion processes are more likely to occur (120) (121) (122) . behavioral experiments and corresponding conceptual modelling approaches suggest that minority groups can initiate social change dynamics in the emergence of new social conventions (17, 119) . furthermore, a rich social science literature has noted an array of factors (i.e. political institutions, technological or behavioral adaptation, environmental, normative and attitudinal) effective in shifting the social conditions surrounding climate change (14) . a better understanding of critical states as demanded by our framework may help to identify early warning signals that could possibly indicate that a social-ecological system is close to a critical state in specific situations (30, 123) . social tipping processes present a specific type of social change -characteristized by non-linear shifting states driving by positive feedbacks -which is similar to, but conceptually distinct from, other forms of social change. similar to how we explore the differences between natural and social tipping processes, further research should engage with social tipping in comparison to other forms of social change (such as historical institutionalist perspectives, social movements, policy feedbacks, complex systems). one of the greatest challenges lies in dealing with multiple, entangled drivers of tipping processes on different scales -temporal, spatial or social structural levels -and different levels of agency and heterogeneous agents and subsystems. in order to further understand the dynamics arising from these various levels of agency, it is crucial to identify examples from different subfields (economics, political science, demographics). a key current limitation in applying our framework is finding and operationalizing empirical data describing actual spreading processes on networks across these different levels, particularly compared to macro-economic data and public opinion polls (124) , even though first steps in this direction are being made (125, 126) . particularly data on the social structures and networks is notoriously difficult to access. while there have been advances in developing modeling frameworks (119, 127) to simulate social tipping dynamics, linking these theoretical modelling to empirical data and behavioral experiments requires more attention. even if predictive modeling (i.e., the kind of deterministic, time-forward modeling we know from earth system models for instance) of such social dynamics in the sense of inferring time trajectories is very difficult or even conceptually unfeasible, such process-based modelling of social tipping dynamics can be very crucial to understand the nature of critical states also in realworld social situations. lastly, we focus here specifically on social tipping processes relevant for mitigating climate change, or sustainability more broadly, fitting within the previous literature. but, such a framework for social tipping dynamics is generalizable to other areas of study and social phenomena (such as the 2020 rapid social movements and public opinion dynamics surrounding racial inequality in the united states). while we explore one example of social tipping in detail, further inquiry is required to test the distinctiveness of social tipping processes, as well as the utility of the proposed definition to other social tipping processes. systematizing the types of social tipping processes, and exemplary case studies, would help to further illustrate these forms of change. research is also warranted into establishing typical timescales of social tipping; understanding how network structures affect social tipping dynamics; identifying typical network structures of systems entering critical states; discerning the temporal aspects of how effects travel through different social network structures; and gaining a better understanding of the origin of spreading processes. data acquisition, analysis and process-based modelling could all play a role in this research agenda. a wealth of social media data is available to study potential social tipping processes. however, this kind of data has mostly yet to be adopted within the context of earth system analysis and tipping dynamics. social tipping processes could be decisive for the future of the earth system in the anthropocene: some rapid shifts in social systems are, in fact, necessary to meet the targets of the paris agreement and the sustainable development goals (8) . while we focus here on processes relevant for future trajectories of the earth system, we suggest that further analysis could use or adapt our definition to characterize other types of general social tipping processes (i.e. revolutions or rapid transformations). we also recognize that tipping processes within ecosystems present an interesting intermediary case between social and physical climate tipping as they typically incorporate characteristics from both realms. they are also crucial in determining future trajectories of the earth system (see preliminary discussion in the si). understanding, identifying and potentially instigating some social tipping processes is highly relevant for the future of the anthropocene, particularly with regard to the potential role in triggering rapid transformative change needed for effective earth system stewardship (9, [11] [12] [13] . resulting trajectory would still move at some time ″ > ′ into some state ″ (which will usually depend on the influence exerted) that is qualitatively different from * . similarly, an uncritical condition, ( ∘ , ∘ ), is tippable by a decision maker if there is a possible trajectory ( ( ), ( )) %2%3 , starting in ( ∘ , ∘ ) at some time ′, that the decision maker can force and to follow, and this trajectory would move into some state ″ at some time ″ > ′ that is qualitatively different from ∘ (a tippable uncritical state roughly corresponds to what others call a 'sensitive intervention point' ). at any time at which the system is not in an unmanageable critical state, the prevention time is the time interval it takes before some quasi-inertial trajectory has moved it into an unmanageable critical state. in other words, at time zero it is the largest time interval so that, when no intervention takes place until time , for all > 0 with < , the system would not be in an unmanageable critical state at time . similarly, at any time at which the system is in a tippable uncritical state, the triggering time is the time interval it takes before some quasi-inertial trajectory has moved it into an uncritical state that is no longer tippable. in other words, at time zero it is the largest time interval so that, when no intervention takes place until time , for all > 0 with < , the system would not be in a tippable uncritical state at time . we only consider social tipping processes for which the prevention or triggering time is smaller than some intervention time horizon. ecosystem tipping processes share properties of physical climate tipping dynamics in atmosphere, ocean and cryosphere in that they can often be described by a common driver, as well as that of deliberative social tipping elements in that they have adaptive capacity, and can therefore be regarded as intermediate. but, as previously noted, human agential capacity is far greater than those of other species. similarly to human social systems, ecosystems are comprised of interacting living organisms, they can be viewed as networks with components that can adapt (e.g., food webs). this is different from physical tipping elements such as the cryosphere elements (e.g., melting of permafrost) which do not typically exhibit the same networked structures. within the nominally 'climate' tipping elements are some major biomes -notably boreal forests, the amazon rainforest, and coral reefs -that are composed of living organisms and exhibit ecological network structures. indeed changing interactions between the living elements of these systems may be key to tipping dynamics -for example epidemic bark beetle infestation of boreal forests triggered by climate warming allowing the beetles to complete two life cycles rather than one within a season (128) . thus these biotic tipping elements lie towards smaller scale ecosystems in the continuum, and tend to be more closely related to social systems in spatial and temporal scales compared to the typically much larger and more slowly changing physical climate tipping elements. these differences give rise to a proposed ordering of tipping elements, ranging from (1) the physical climate tipping elements via (2) ecosystem tipping elements to (3) social tipping elements (table s1 ). tipping elements in the earth's climate system retreat of pine island glacier controlled by marine ice-sheet instability marine ice sheet collapse potentially under way for the thwaites glacier basin, west antarctica stability of the west antarctic ice sheet in a warming world catalogue of abrupt shifts in intergovernmental panel on climate change climate models limiting global warming to 2 °c is unlikely to save most coral reefs others, coral reefs in the anthropocene trajectories of the earth system in the anthropocene defining tipping points for social-ecological systems scholarship-an interdisciplinary literature review positive tipping points in a rapidly warming world sensitive intervention points in the post-carbon transition tipping positive change others, social norms as solutions tipping elements and climate--economic shocks: pathways toward integrated assessment. earths future concerns of young protesters are justified experimental evidence for tipping points in social convention a tilting water meter for purposes of experiment sur l'équilibre d'une masse fluide animée d'un mouvement de rotation dynamic models of segregation metropolitan segregation threshold models of collective behavior sparks and prairie fires: a theory of unanticipated political revolution punctuated equilibrium comes of age the tipping point: how little things can make a big difference (little, brown and company synchronous failure: the emerging causal architecture of global crisis complexity theory and financial regulation on social norms: the evolution of considerate smoking behavior a roadmap for rapid decarbonization critical transitions in nature and society regime shifts, resilience, and biodiversity in ecosystem management thresholds in ecological and social-ecological systems: a developing database the collapse of complex societies collapse, environment, and society sunk-cost effects and vulnerability to collapse in ancient societies unifying research on social--ecological resilience and collapse cascading regime shifts within and across scales tipping elements in the earth system others, potential climatic transitions with profound impact on europe tipping points' for the amazon forest land-use and climate change risks in the amazon and the need of a novel sustainable development paradigm global resilience of tropical forest and savanna to critical transitions multistability and critical thresholds of the greenland ice sheet ocean circulation and climate during the past 120,000 years observed fingerprint of a weakening atlantic ocean overturning circulation risk of multiple interacting tipping points should encourage rapid co 2 emission reduction sendzimir, others, tipping toward sustainability: emerging pathways of transformation studying the complexity of change: toward an analytical framework for understanding deliberate socialecological transformations societal transformation in response to global environmental change: a review of emerging concepts elicitation of expert opinions for uncertainty and risks the agency of nature or the nature of agency? reassembling the social: an introduction to actor-network-theory late lessons from early warnings: science, precaution, innovation toward a psychology of human agency human agency in social cognitive theory gaia 2.0 anticipatory governance for social-ecological resilience imagining transformative futures: participatory foresight for food systems change selfefficacy beliefs of adolescents (iap mindmade politics: the cognitive roots of international climate governance the consequences of modernity an invitation to reflexive sociology how can evolution learn? material agency: towards a non-anthropocentric approach prologue: archaeology, animism and non-human agents inheritance systems and the extended synthesis statistical physics of social dynamics the network society: from knowledge to policy runaway world: how globalization is reshaping our lives the condition of postmodernity the hidden geometry of complex, network-driven contagion phenomena polycentric systems for coping with collective action and global environmental change humanity on the move: unlocking the transformative power of cities a multilayer network dataset of interaction and influence spreading in a virtual world ordinary science intelligence': a science-comprehension measure for study of risk and science communication, with notes on evolution and climate change the political business cycle political business cycles 40 years after nordhaus electoral business cycle in industrial democracies why the right climate target was agreed in paris quantifying the influence of climate on human conflict socialecological systems as complex adaptive systems: modeling and policy implications understanding the complexity of economic, ecological, and social systems adaptive systems: an introduction to computational models of social life self-similarity of complex networks origins of fractality in the growth of complex networks introduction to the theory of complex system exploring non-linear transition pathways in social-ecological systems schellnhuber, others, a safe operating space for humanity planetary boundaries: guiding human development on a changing planet sustainability science: towards a synthesis exploring the governance and politics of transformations towards sustainability ontologies, socio-technical transitions (to sustainability), and the multi-level perspective the multi-level perspective on sustainability transitions: responses to seven criticisms climate and conflict cognitive representations of the future: survey results three years to safeguard our climate paris agreement climate proposals need a boost to keep warming well below 2 °c climate change policy in the european union: confronting the dilemmas of mitigation and adaptation? assessing the european union's global climate change leadership: from copenhagen to the paris agreement the paris agreement and the inherent inconsistency of climate policymaking the public as thermostat: dynamics of preferences for spending degrees of democracy: politics, public opinion and policy agendas and instability in american politics an advocacy coalition framework of policy change and the role of policy-oriented learning therein theories of the policy process the rhetorical limitations of the #fridaysforfuture movement the mechanisms of policy diffusion agendas, alternatives, and public policies temporary reduction in daily global co 2 emissions during the covid-19 forced confinement after covid-19, green investment must deliver jobs to get political traction a covid-19 recovery for climate temperature drives zika virus transmission: evidence from empirical and mathematical models migration as an adaptation to climate change causality chains in the international migration systems approach a network-based microfoundation of granovetter's threshold model for social tipping echo chambers and viral misinformation: modeling fake news as complex contagion consensus and polarization in competing complex contagion processes early warning signals of regime shifts in coupled humanenvironment systems futurict: participatory computing to understand and manage our complex world in a more sustainable and resilient way fundamental structures of dynamic social networks interaction data from the copenhagen networks study earth system modeling with endogenous and dynamic human societies: the copan:core open world-earth modeling framework a first assessment of the impact of the extreme 2018 summer drought on central european forests general: we are very grateful to william c. clark, anne-sophie crépin, niklas harring, matthew ives, j. doyne farmer, wolfgang lucht, and john schellnhuber, for providing helpful insights and comments. we thank the participants of two dominoes workshops on social tipping dynamics held at gesis leibniz institute for social science, cologne, in summer 2018 and 2019 for foundational and framing discussions. the authors declare no competing interests. in this section, we give a more formal version of the definition of 'social tipping process' given in the main text, as a reference for mathematically inclined readers.after defining what we mean by a social system and its environment, we first classify their possible states into critical, unmanageable, uncritical, and tippable conditions, and then finally define the notions of prevention time and triggering time. by a social system, , we mean a set of agents together with a network-like social structure, that interacts in some form with the rest of the world, called the environment, , of the system, such that, if no "perturbation" or deliberate "influence" by some decision-maker occurs, and together can only follow certain "quasi-inertial" (or "default") trajectories restricted by the agency of the system's agents. let (%) and (%) denote the states that and are actually in at time .a critical condition for the system is a pair of possible system and environment states, ( * , * ), such that there exists another possible pair of states, ( ′, ′), with the following properties:1. the state pair ( ′, ′) is no further away in state space from ( * , * ) than a certain "small" distance, , that represents the possible magnitude of "local" perturbations in (affecting only few agents or network links directly) or small changes in that are considered sufficiently "likely" to care about, with respect to some suitable distance function . in other words, (( ′, ′), ( * , * )) < . 2. if and were in state ( ′, ′) at any time ′, there is a quasi-inertial trajectory that would move at some later time ″ > ′ into some state ″ that is "qualitatively" different from * . this move represents a "global" (i.e., affecting a very large fraction of the agents) and "significant" change in the system (but not necessarily in its environment).if such a change actually happens, the time point ′ (not the state!) at which it starts may be called the tipping point or less ambiguously the triggering time point, and the system behavior within the time interval from ' to ″ is called the corresponding tipping process. an uncritical condition for and then is any pair of states that is not critical.a critical condition is unmanageable for an actor that may influence or in some way if there exists a possible pair of states, ( ′, ′), with (( ′, ′), ( * , * )) < and the following property:• assume that and were in state ( ′, ′) at any time ' and afterwards the state of and would follow any trajectory ( ( ), ( )) %2%3 that the actor can force it to follow. then the key: cord-276218-dcg9oq6y authors: kim, jihoon; koo, bon-kyoung; knoblich, juergen a. title: human organoids: model systems for human biology and medicine date: 2020-07-07 journal: nat rev mol cell biol doi: 10.1038/s41580-020-0259-3 sha: doc_id: 276218 cord_uid: dcg9oq6y the historical reliance of biological research on the use of animal models has sometimes made it challenging to address questions that are specific to the understanding of human biology and disease. but with the advent of human organoids — which are stem cell-derived 3d culture systems — it is now possible to re-create the architecture and physiology of human organs in remarkable detail. human organoids provide unique opportunities for the study of human disease and complement animal models. human organoids have been used to study infectious diseases, genetic disorders and cancers through the genetic engineering of human stem cells, as well as directly when organoids are generated from patient biopsy samples. this review discusses the applications, advantages and disadvantages of human organoids as models of development and disease and outlines the challenges that have to be overcome for organoids to be able to substantially reduce the need for animal experiments. the use of classical cell line and animal model systems in biomedical research during the late twentieth and early twenty-first centuries has been successful in many areas, such as improving our understanding of cellular signalling pathways, identifying potential drug targets and guiding the design of candidate drugs for pathologies including cancer and infectious disease. the value of the achievements these model systems have made possible is proved by the fact that their use has become near universal in biomedical research today. historically, the investigation of disease mechanisms in animal models has progressed along a common discovery pipeline, whereby biological processes were initially investigated by genetic screens in invertebrates, followed by an analysis of evolutionary conservation in mammalian model systems, eventually leading to clinical translation to humans. the common principles of animal development and organ physiology derived from this approach have led to a detailed mechanistic understanding of many human diseases. nevertheless, extrapolating results from model systems to humans has become a major bottleneck in the drug discovery process. furthermore, recent studies have identified biological processes that are specific to the human body and cannot be modelled in other animals. these include, for example, brain development, metabolism and the testing of drug efficacy. the emergence of human in vitro 3d cell culture approaches using stem cells from different organs has therefore received widespread attention as having the potential to overcome these limitations. attempts to model the biology of human organsincluding the differentiation of human stem cells in 2d, in either the presence or absence of a 3d matrix; bio-printing of human cells; and the culture of cells in a microfluidic device ('organ-on-a-chip') 1 -were made prior to the emergence of organoids and have shown some potential for drug screening or human disease research. however, organoids are unique, in that they are self-organizing, 3d culture systems that are highly similar to -and in some cases, histologically indistinguishable from -actual human organs [2] [3] [4] [5] [6] [7] [8] . one feature that is common to all organoids is that they are generated from pluripotent stem cells (pscs) or adult stem cells (adscs; also known as tissue stem cells) by mimicking human development or organ regeneration in vitro 9 . the analysis of organoid formation can thus provide valuable information about the mechanisms underlying human development and organ regeneration, highlighting their value for basic biological research in addition to their potential application in pharmaceutical drug testing and molecular medicine. the potential of organoids to complement existing model systems and extend basic biological research, medical research and drug discovery into a more physiologically relevant human setting is becoming ever more widely appreciated. however, the development of organoid technology is still in its infancy in comparison to established cell line and animal models, with challenges still to overcome. in this review, we describe examples of psc-derived and adsc-derived organoid models that have shown great potential and promise in modelling human disease for a broad spectrum of life stages, from early development through to adulthood. many excellent reviews have already described the various organoid human organoids: model systems for human biology and medicine model systems [9] [10] [11] [12] [13] [14] , highlighted their relevance for disease modelling 15, 16 and outlined organoid-associated protocols 17 . this review will instead discuss organoids as a novel model system for understanding human development and genetics that complements, and in the future might reduce, the use of animal models. we emphasize the differences between the mouse and human systems, evaluating the advantages and disadvantages of animal models and human organoid models. we also highlight the tools and methodologies that are available for human organoid studies, in the hope that this information will lead basic biologists to consider using this emerging platform for the study of human pathophysiology. the diversity of biological model systems building on the fundamental idea that biological mechanisms are conserved throughout evolution, biomedical research has traditionally focused on only a handful of model organisms. these models are typically robust, fast-growing species that generate a large number of offspring in a short time and can be propagated at low cost in the laboratory. researchers benefit greatly from the use of an established model organism, as there are already detailed descriptions of its development and physiology, a large body of experimental techniques and many community resources, including reagents and species-specific databases. while some model organisms, such as yeast (saccharomyces cerevisiae), fruit flies (drosophila melanogaster) and mice (mus musculus), have a long tradition of use in research, other model systems have been established more recently but nevertheless have helped broaden our knowledge by presenting us with a new set of experimental tools. these include the worm, caenorhabditis elegans, whose reproducible cell lineage allows unparalleled analysis of cell fate decisions 18 , and the zebrafish, danio rerio, the first vertebrate system in which saturation loss-of-function mutagenesis screening was performed 19, 20 . c. elegans became a widely used experimental model from the mid 1970s, whereas the first large-scale genetic screens in d. rerio were conducted in the 1990s 21, 22 . however, although it is extensive, the information obtained from animal models does not completely reflect human physiology. in this respect, human organoids (which emerged in the early 2010s) 3, 5, 23, 24 can be seen as a novel experimental model that bridges the gap between animal models and human beings. the most commonly used model organisms currently are s. cerevisiae, c. elegans, d. melanogaster, zebrafish and the common house mouse. in addition, for cancer studies, patient-derived xenografts (pdx) and cancer cell lines are also used. each model has its own unique advantages and limitations (fig. 1 ). for example, 60-80% of genes in c. elegans have human orthologues, and about 42% of human disease genes have orthologues in c. elegans [25] [26] [27] . moreover, many signalling pathways have been evolutionarily conserved from this species. the largely deterministic nature of c. elegans development makes it an ideal model for cell lineage analysis and has led to the discovery of key cell death pathways, yet pattern formation in c. elegans is different from that in other organisms, and mechanistic principles of its development are often not fully conserved. in addition, other aspects of the physiology of c. elegans, such as its lifespan, metabolism and immune system, are very different from human physiology. among mammalian model systems, the availability of highly advanced genetic tools and germline-competent www.nature.com/nrm mouse embryonic stem cells (escs) that enable the generation of genetically engineered mouse models makes the mouse the most preferred system. nevertheless, limitations remain with regard to the physical inaccessibility of early development, as compared with other, egg-laying models with visible development, the requirement for expensive animal facilities in which to maintain the mice and some important differences between mouse and human physiology (box 1). the zebrafish was developed as a vertebrate model for large-scale genetic studies in the 1990s, to overcome some of the limitations of the mouse model: the transparent zebrafish embryo and this model's potential to reproduce in huge numbers with a manageable price tag allowed zebrafish researchers to uncover fundamental principles of early embryonic development. however, the zebrafish model suffers from a hugely complex genome, a lack of comprehensive in vitro culture systems and the fact that it shares only a limited degree of biological similarity with humans. over the past 30 years, the use of these various, equally valuable animal model systems, as wells as 2d human cancer cell lines, has led to an explosion of knowledge about human development and mechanisms of disease, but it has also revealed the limitations of these same model systems in emulating human pathophysiology. a number of biological phenomena that are specific to humans are not amenable to being reproduced in animal models. the human brain, for example, is far more complex than its mouse counterpart, owing partly to human-specific developmental events and mechanisms 28 . neurons in the human cortex, for example, arise from a cell type, outer radial glia, that is not present -or is present only in minute numbers -in rodents 28 . human physiology is also profoundly different from the mouse model system: it is perhaps unsurprising that there are huge differences in metabolism between humans and laboratory models, given that humans develop far more slowly than the models 29 . common drugs such as ibuprofen 30 and warfarin 31 are metabolized in the liver in such different manners in humans and rodents that, in humans, ibuprofen is prescribed for pain, fever and inflammation, and warfarin is prescribed as an anticoagulation drug, whereas both drugs are toxic to rats. finally, and in contrast to all animal models, humans are not inbred. understanding human genetic diversity and its influence on disease onset and progression and drug responses is a prerequisite for developing personalized medical treatments and requires the establishment of human-specific model systems. the advent of human induced pluripotent stem cell (ipsc) technology and diverse human adsc culture methods has made it possible, for the first time, to generate laboratory models specific to an individual 32 . reprogramming other cells into ipscs has become a routine laboratory procedure, but generating disease models from those cell lines remains challenging. early approaches focused on the differentiation of ipscs into one type of cell (for example, neurons and cardiomyocytes) and their culture in 2d 33, 34 . more recently, culture methods have been developed to mimic in vivo organ development in 3d, allowing more complex tissue structures and diverse cell types to be modelled simultaneously 1, 9, [35] [36] [37] [38] [39] . in this methodology, human ipscs are sequentially exposed to a course of differentiation cues in order to simulate the stages of a human developmental process. during this process, differentiated ipscs aggregate to form first an organ bud, and later organoids, that faithfully mimic the mature organ structure, including multiple cell types and the interactions between them. human adsc-derived organoids have also emerged as an alternative organoid system that consists of a simpler structure composed mainly of cell types found in the epithelium 3 . in contrast to the complicated process of ipsc reprogramming followed by differentiation to the required organ type, these organoids can be generated from biopsies isolated directly from the organ of interest or from diseased patient tissue. however, the establishment of human adsc-derived organoids is limited by accessibility to the tissue and prior knowledge of the culture conditions for that tissue, while an ipsc line, once established from a patient, can be used to repeatedly generate different tissue models without any time limit (that is, beyond the patient's lifespan) 40 . in summary, organoids constitute an improvement in the generation of model systems, with cell types that more closely resemble, and are in similar conditions to, those found in the human body 7, 41 . diverse organoid systems provide a useful variety in their complexity and modelling capability, ranging from the simple epithelial structures derived from adscs 3 to more complex cultures box 1 | differences between human and mouse stem cells the difference between mice and humans is evident in the culturing of their stem cells. pluripotent mouse embryonic stem cells (escs) were established in the early 1980s 45, 46 . leukaemia inhibitory factor (lif) was the first pluripotent stem cell (psc) factor identified, with the later discovery that 2i (the kinase inhibitors pd0325901 and chir99021) and lif (together termed 2ilif) are necessary as well as sufficient for the culture of escs from all mouse strains [176] [177] [178] [179] . the first human pscs to be successfully cultured were human escs, equivalent to mouse epi-stem cells that have lost germ-cell potential 44, 180, 181 . initial cultures were propagated in media that were based on basic fibroblast growth factor, but for decades researchers continued to search for appropriate culture conditions in which to maintain human pscs in the naive, pluripotent state [182] [183] [184] [185] [186] [187] [188] . universally agreed-upon culture conditions for the maintenance of naive human psc cultures remain elusive, with various reports suggesting different medium compositions including the use of a pkc inhibitor (gö6983), rock inhibitor (y-27632), mek inhibitor (pd98059), gsk inhibitor (im12), braf inhibitor (sb590885), lck/src inhibitor (wh-4-023) or wnt5a 182, 184, 189, 190 . a similar fundamental discrepancy between mouse and human cultures, in their requirements for additional media components, has also been observed in a number of adult stem cell (adsc)-derived organoid culture systems. however, one commonality appears to be that inhibitors of tgfβ and p38 signalling must be added to the growth factors used in murine adsc cultures in order to enable the long-term culture of human adscs 9 . for example, mouse colonic organoids require epidermal growth factor, noggin, r-spondin and wnt as growth factors, while human colonic organoids additionally require two inhibitors, a83-01 and sb202190 (which block tgfβ and p38 signalling, respectively) 3 , in order to attain a similar level of longevity and robustness. the differences between mouse and human systems in both psc-derived and adsc-derived organoid cultures suggest rather surprising fundamental differences between these species in the intrinsic signalling pathways that are activated in their stem cells and that are required for stem cell identity, and not just differences during development or during an immune response. this confirms the need for human-based laboratory model systems in order to fully understand human development and pathophysiology. part of neocortex where neuronal progenitors reside. a breed with genetically closely related individuals. induced pluripotent stem cell (ipsc). pluripotent stem cells reprogrammed from somatic cells. nature reviews | molecular cell biology generated from multiple germ layers 6 obtained via multistep psc differentiation protocols 42, 43 . it is expected that, in the near future, the variety of organoid models available, coupled with advances in the technology, will provide a series of powerful and efficient platforms for studying human development, physiology and disease. the past years have seen substantial progress in the development and use of human-cell-based model systems for the study of human biology, including both pluripotent and adsc-based systems. the introduction of human ipsc technology. the first human pscs, known as human escs, were reported in 1998 (ref. 44 ), nearly two decades after the initial discovery of murine pluripotent cell lines in 1981 (refs 45, 46 ). the human stem cell lines were predicted to be useful for the study of human developmental biology, drug discovery and cell therapy -in spite of the technical limitations imposed by the lack of knowledge of appropriate differentiation protocols at that time. as the generation of human psc lines required the sacrifice of human embryos at the blastocyst stage, strong ethical concerns were raised 47 . in 2007 the debate around human pscs was largely circumvented by the introduction of human ipsc technology, which converts an ordinary differentiated fibroblast from an adult human into a pluripotent cell 33 . reprogramming to a pluripotent state is usually achieved by forced expression of a specific set of transcription factors 33 , and the pluripotent cells can then be differentiated into specific cell types. ipsc technology not only allowed the generation of patient-specific stem cells but also enabled researchers to work with an unlimited supply of human stem cells and stem cell-derived tissues. furthermore, ipsc technology allows the banking of patient-derived stem cells 48 . there are still technical limitations when using ipscs -for example, the use of oncogenes for reprogramming, the genetically unstable nature of the reprogramming and the low efficiency of reprogramming. all these technical hurdles made it difficult to obtain error-free ipsc lines from patients 49 . but these limitations have been at least partly overcome by the use of non-integrating vectors and standardized quality control protocols to avoid or screen out unwanted genetic alterations 49 . line-to-line variability caused by the genetic heterogeneity of humans has also been resolved by the use of isogenic controls generated by genetic engineering using crispr-cas9 technology 50 . these improvements have enabled researchers to use ipsc-derived specialized cell types -for example, neurons, cardiomyocytes, haematopoietic progenitor cells and pancreatic β-cells -in disease modelling and drug screening 51 . however, just over a decade after their introduction, 3d organ culture methods have greatly increased the utility of human ipscs. ipsc-derived organoid models. human psc-derived organoids are generated by guided differentiation protocols that mimic developmental processes identified through previous work, both in vitro and in vivo (fig. 2) . as our knowledge of human development is very limited, most early studies that aimed at producing organoids with properties similar to those of human tissues were based on parallels drawn from mouse development. in principle, to generate an organoid, the entire process of organ development from pscs should be faithfully mimicked. in reality, it is nearly impossible in vitro to provide all biochemical cues that drive cell differentiation and 3d tissue assembly and organization at precisely the right times, places and concentrations at which they would occur during embryonic development. fortunately, cells in vitro tend to follow a semi-autonomous differentiation trajectory, as they do in vivo, and three main types of protocols are utilized to generate functional organoids. in the case of brain organoids, human pscs are initially guided to differentiate into embryoid bodies before further differentiation towards the neuroectodermal lineage. once the cell aggregates contain the developmental precursors for brain tissue patterning, the rest of the developmental steps occur spontaneously in a spinning bioreactor 5, 52 . for the development of liver primordia, guided differentiation to hepatoblasts (hepatocyte precursors) was not sufficient to form a complete set of organ precursors, which requires cells from different lineages. it was known from studies of mouse hepatogenesis that cell-to-cell communication between endothelial, mesenchymal and hepatic endoderm cells was important, so based on this knowledge the first human liver primordia were created by mixing human psc-derived hepatoblasts, mesenchymal cells and endothelial cells. through self-condensation and organization, these three cell types assemble in vitro to make an aggregate that mimics the architecture of developing human liver primordia 23, 53 . other organoids require more precise, lengthy protocols in order to acquire the appropriate progenitor cell types for the target epithelium. many organoids that model endoderm-derived organs (such as oesophagus, stomach, colon, intestine and lung) undergo stepwise differentiation protocols, in which the timing, concentration and combination of specific growth factors and chemical inhibitors for modulating key developmental signalling pathways are crucial to developing the desired epithelial tissue in a manner analogous to fetal development 24, 43, [54] [55] [56] [57] . in all cases, the process of organoid formation involves three crucial steps. first, key signalling pathways regulating developmental patterning are activated or inhibited (using commercially available morphogens and signalling inhibitors) in order to establish a correct regional identity during stem cell differentiation. usually this is achieved through induction of the signalling events that have been identified in the mouse as establishing cell fates in vivo. second, media formulations that allow proper terminal differentiation of the desired cell types within the organoid are developed, generally following established methods of 2d culture or inspired by the murine developmental process. finally, cultures are grown in a way that allows their expansion in three dimensions, which is achieved either by aggregating cells into 3d structures or by embedding the cultures into a 3d matrix. control cell lines with the same genetic background as experimental cells. three-dimensional pluripotent stem cell aggregates. culture system in which nutrients are supplied while cells are agitated. adscs can be cultured in the presence of niche factors that function to maintain the stem cells in an undifferentiated state while allowing stem cell differentiation. as a result, the cultured stem cells can generate adsc-derived organoids, which are composed of an epithelial monolayer that mimics the 3d architecture and contains the cell types of the desired organ to be modelled 58 . the first steps in the development of adsc-derived organoids, however, are to identify and isolate the appropriate population of adult stem cells and to understand their niche requirements. obtaining the first mouse intestinal organoid cultures required not only the identification of the intestinal stem cell population expressing the selective marker for these cells, the leucine-rich repeat-containing g protein-coupled receptor 5 (lgr5), but also the essential niche factors to support stem cell activity 59 . the re-creation of the intestinal stem cell niche in vitro was inspired by mouse genetic studies that had shown that epithelial proliferation and stem cell self-renewal are dependent on epidermal growth factor (egf) and wnt activity, while differentiation is controlled by bone morphogenetic protein (bmp) kidney organoids after it was discovered that sb202190 prevents secretory lineage differentiation, this inhibitor was replaced by the addition of insulin-like growth factor 1 (igf1) and fibroblast growth factor 2 (fgf2) 4 ; this modification enables concurrent multilineage differentiation and self-renewal in human gut organoids. all these changes and refinements to culture conditions, which are the results of another decade of research, improved the quality of human gut organoid cultures, enabling researchers to produce organoids that more closely resemble tissue in vivo. nevertheless, mouse organoids remain more similar to the in vivo tissue of origin than do human organoids. generation of other adsc-derived organoids. using the procedure described above, multiple types of organoids from various tissues have been generated by modifying the basic medium that was initially developed for intestinal organoid culture ( fig. 2; fig. 3 ). in most cases, a mouse organoid culture system was first established and then adapted to human cells. mouse colonic organoids can be grown through the addition of wnt3a 3 . mouse stomach pyloric organoids and corpus organoids were grown with only slight modifications to the original protocol for mouse intestinal organoids 60, 61 . mouse liver and pancreas ductal organoids were also obtained similarly from injury-activated lgr5-positive progenitors 62, 63 . the number of organoids generated from diverse mouse tissues and organs is growing, and for each, previous knowledge of the signalling processes that comprise the organ-specific or tissue-specific stem cell niche environment has been key to developing the appropriate protocols. adsc-derived human organoids have also become widely available (fig. 3) . they have been generated from almost all endoderm-derived tissues (intestine, colon, stomach, liver, pancreas, lung, bladder and so forth) 3,7,41,64-70 and from gender-specific tissues (prostate, endometrium, fallopian tube and mammary gland) 8,71-75 . additional components frequently required for the growth of human cultures, in comparison to the mouse cultures, are a tgfβ pathway inhibitor (such as a83-01) and a p38 mapk inhibitor (such as sb202190) 9 . as for mouse organoids, human organoids can be derived from minimal amounts of tissue biopsies and can be cultured indefinitely, thus forming the basis for the building of living biobanks, which are an important resource in biomedical research. following the establishment of human stem cell-based organoids, various human diseases have been modelled using these systems. for example, human organoids have been used to study infectious diseases, inheritable genetic disorders and cancer. moreover, with the advent of various genetic-engineering tools, pathogenic genes and mutations can be directly tested in organoids derived from cells isolated from healthy donors, which enables us to perform human genetic studies in controlled genetic backgrounds. the human brain is the most highly developed brain in the animal kingdom and the most complex organ in our body. because of its distinct complexity, it has been very difficult to model human brain development using animal systems. human brain organoids, however, have now been successfully used to model human brain development and disease. the first disease studied in this model was microcephaly; unlike rodent models, human brain organoids with a mutation in cdk5rap2 display a significantly reduced size, with a smaller number of progenitors that undergo premature neurogenesis 5 . cdk5rap2 is localized at the centrosome and is required for correct orientation of the mitotic spindle, so that a mis-orientation of the cleavage plane prevents the expansion of the progenitor pool by symmetric division in the patient-derived organoids. this human experimental system was also crucial in determining the relationship between the zika virus (zikv) and microcephaly. during the recent zikv outbreak, a link was initially suggested between middle part of the stomach, connected with cardia, fundus and pylorus. collection of biomaterials, such as cancer organoids, used for research. www.nature.com/nrm neurological disorders, including microcephaly, and zikv [76] [77] [78] [79] . nevertheless, even though zikv could be detected in microcephalic fetal tissues, the causal relationship and the mechanism by which zikv might compromise brain development were not clear. a number of studies have used 3d human stem cell-derived systems, including neurosphere culture and brain organoid models, to reveal the effect of zikv infection on human brain development 80, 81 . thus, the human organoid model provides a physiologically relevant platform for studying zikv pathogenesis. further studies with human brain organoids have determined that brazilian zikv strains possess increased virulence as compared to an african strain 82 , and that the ns2a protein of zikv causes decreased neural stem cell proliferation 83 . various chemical compounds that alleviate the hypomorphic effect of zikv have also been identified using the human brain organoid system 84 . these studies highlight the importance of human brain organoids as a model system in understanding the pathology and mechanistic basis of genetic and infectious diseases, as well as in discovering potential therapeutic reagents. as exemplified by the zikv studies above, a human model system is preferable to animal models when studying infectious disease pathogenesis, because pathogens often have a narrow species or tissue tropism; that is, they infect only certain species, and sometimes only specific cell types. for decades there were not any methods to culture human noroviruses, which cause acute gastroenteritis and can be fatal in young children or in old or immunocompromised individuals 85 . it recently became possible to successfully grow norovirus in culture by using a human intestinal organoid-derived epithelial monolayer, which led to the finding that certain viral strains specifically require both certain bile components and the activity of the host galactoside 2-alphal-fucosyltransferase 2 (fut2) enzyme for infection 86 , which mirrors the patterns of epidemiological studies. rotaviruses, which also cause diarrhoeal disease 87 , are another example of human intestinal organoids being used to cultivate patient-derived viral strains. the rapid replication of the virus was detected within one day of inoculation. an antiviral cytokine, a vp7-neutralizing antibody and ribavirin were demonstrated to suppress viral replication in the organoid system 88, 89 . human airway organoids are suitable models for the study of respiratory viruses. respiratory syncytial virus (rsv) is a single-stranded rna virus that is a significant threat to young infants and the elderly 41 . it was shown to successfully infect human airway organoids and to cause dramatic changes to epithelial morphology and function: the ns2 protein of rsv was shown to cause increased motility of airway epithelial cells 41 . the human airway organoid system has also been used to assess the infectivity of animal-borne influenza viruses [90] [91] [92] . the diversity of two viral surface molecules -haemagglutinin and neuraminidase -has previously made it difficult to predict the infectivity of these viruses in humans 93 . the human airway organoids faithfully simulate human airway epithelium, and so provide a platform for rapidly testing the infectivity of newly emerging influenza viruses. organoids have also been used to co-cultivate human epithelia with bacteria (for example, helicobacter pylori) and with protozoan parasites (for example, cryptosporidium) 64, [94] [95] [96] . the list of pathogens that can be grown in the surrogate human organ system is growing rapidly. we expect that, in the near future, numerous microbiome strains from the human gut or glands will be analysed by using organoids to study infectious diseases in a human cell context, and that organoid infection models will be used as screening platforms to identify novel drug candidates (box 2). human organoids are important resources for precision medicine. for example, they can be useful for selecting an appropriate drug for patients with genetic diseases or cancer. cystic fibrosis (cf) is a relatively common genetic disease with approximately 90,000 patients worldwide. although loss of function of the cftr gene, encoding a chloride channel regulating the mucosal environment, is the main cause, nearly 2,000 mutations have been reported that affect the function or expression of cftr, making this a genetically heterogeneous disease 97 . while two drugs -vx-770 (a cftr potentiator that improves chloride-channel activity) and vx-809 (a cftr corrector that helps mutant protein folding) -are already available for patients with cf who carry specific cftr mutations in combination with the common mutation f508del, evaluating the efficacy of these drugs or discovering new drugs for patients with different mutations remains challenging. rectal organoids isolated from small endoscopic biopsies have nonstructural protein 2a, encoded in a virus genome. an effect that causes small size of a body part. the ability of a virus to infect various species and cells. an approach to treating individual patients with the best possible drug, based on the individual's genetic background and disease characteristics. numerous epidemics and pandemics have threatened human health in history (of which a few examples are plague (yersinia pestis), spanish flu, smallpox, aids/hiv, ebola virus, severe acute respiratory syndrome (sars)-coronavirus (cov) and zika virus (zikv); who, disease outbreaks), heavily disrupting human society. the emergence of the novel human coronavirus sars-cov-2 was first reported in wuhan, china, at the end of 2019 (ref. 191 ). sars-cov-2, reported to have crossed the species barrier, causes coronavirus disease-19 in humans 192 . owing to the lack of targeted antiviral medicines and a vaccine against covid-19, the virus has spread globally and the who has declared covid-19 a pandemic (who, coronavirus disease (covid-19) pandemic). developing a vaccine and therapeutics against covid-19 has become a priority, and human organoids can play a crucial role in advancing research, by providing human cells from different organs that are susceptible to sars-cov-2 infection. several research groups have successfully shown that sars-cov-2 can infect and propagate in primary human organoids derived from liver and gut, as well as in pluripotent stem cell-derived organoids modelling blood vessels and the kidney [193] [194] [195] . sars-cov-2 uses the same protein, viral receptor angiotensin-converting enzyme 2 (ace2), as sars-cov to enter the host cells. ace2 is expressed in multiple tissues and cell types, enabling the virus to infect other tissues beyond the lung [196] [197] [198] [199] . the ability of sars-cov-2 to infect different organs was modelled with human organoids [193] [194] [195] . moreover, it has been shown that the viral infection of human blood vessel and kidney organoids can be inhibited by human recombinant soluble ace2, a drug candidate initially developed for sars-cov 194 . structural studies of the spike proteins of sars-cov-2 and sars-cov bound to ace2 predicted a similar result [200] [201] [202] . these studies have provided additional evidence that human organoids can serve as an effective research platform for the study of human diseases, including the outbreak of new viruses such as sars-cov-2 and zikv. nature reviews | molecular cell biology been used in forskolin-induced swelling (fis) assays [98] [99] [100] to assess forskolin-camp signalling-induced cftr channel activity, and it was found that the assay can faithfully predict patient responses to individual drugs and to combined treatments 99, 100 . it was also found that with this relatively simple in vitro assay it is possible to identify which patients with rare mutations would respond to, and could therefore be treated with, existing therapies 99,100 , which could be life-changing for those individuals. the rectal-organoid-based fis assay screen is currently being performed by hubrecht organoid technology for patients in the netherlands with cf. human organoids also play a prominent role in enhancing our understanding of human cancers. traditionally, human cancer cell lines, mouse cancer models or pdx have been the main experimental platforms for studies of human cancer. however, with the advent of adsc-derived organoid technology, transformed cancer tissues have been grown in vitro as cancer organoids (also known as tumouroids or canceroids), demonstrating that this technology is applicable to diseased tissue as well as to normal epithelia. patient samples from colon 101-105 , brain 106, 107 , prostate 108 , pancreas [109] [110] [111] , liver 112 , breast 113 , bladder 68 , stomach [114] [115] [116] , oesophageal 117 , endometrial 118 and lung 41,119 cancers have readily been cultured as cancer organoid models. recently, an analysis of drug responses in patients and in their matched cancer organoids led to the conclusion that responses to the drugs are highly similar in the two settings: a drug with no antitumour activity in the organoids did not demonstrate efficacy in the matched patient, and drugs that showed an effect in the organoid cultures were matched by a patient response in close to 90% of cases 120 . this initial study has been corroborated by several studies with larger cohorts 105, 121, 122 . the sample size and cancer types analysed remain limited, and more rigorous investigation will be needed before cancer organoids can be routinely adopted as in vitro patient 'avatars' . nevertheless, the rapidly increasing number of patient-derived cancer organoids and their use in xenograft formation and molecular profiling have already accelerated cancer research, and patient-derived organoid models in the future may provide an in vitro screening platform to predict the best therapeutic options for individual patients. capecchi reported highly frequent homologous recombination events in mouse escs 123 . by contrast, homologous recombination was found to be an extremely rare event in human pscs 124 . later studies reported that double-strand breaks (dsbs) facilitate homologous recombination events as part of the dna repair mechanism in human cell lines 125 . much effort has been directed at establishing an efficient method for generating dsbs at a specific target locus in order to harness this repair machinery in introducing desired genetic changes such as repair sequence or pathogenic mutation into the target locus. this was initially accomplished by the use of meganucleases (endonucleases with a long recognition site (12-40 bps)), zinc finger nucleases 126 and transcription activator-like effector nucleases 127 , but with varying levels of target specificity and activity. the development of the crispr−cas9 endonuclease technology has made diverse methods of genetic engineering readily available to all researchers [128] [129] [130] [131] . unlike the previous technologies, the cas9 endonuclease is guided to the genomic sequence of interest as a means to generate a dsb by a guide rna sequence (grna), making the system highly versatile and easy to apply 132 . there are numerous examples of genetic engineering of human pscs using this system, including the generation of isogenic cell lines with specific mutations. such isogenic cell lines serve as an important control for genetic analysis in human pscs, due to the strong phenotypical variation among different cell lines. furthermore, large-scale loss-of-function analyses have been performed in many human cancer cell lines and human pscs through the multiplexing of grnas in retroviral or lentiviral vectors, to provide genome-wide targeting coverage. combining organoid systems with crispr-cas9 genome editing broadens the applications of the organoid system in many ways. for example, this system has been used to model monogenic disorders such as cf. human gut organoids with f508del, causing misfolded cftr channel protein that leads to rapid degradation, were precisely corrected into the normal sequence by crispr-cas9; the engineered organoids with the corrected cftr amino acid sequence showed restored channel activity of cftr in vitro 133 , showing the causal relationship between the mutation and disease phenotype, as well as the possibility of using a similar strategy to generate autologous organoids for transplantation. crispr-cas9 technology has also been employed to identify and introduce oncogenic driver mutations to normal epithelial organoids. two groups have independently reported the minimum set of mutations that can closely model a metastatic human colon cancer 134, 135 . simultaneous knockout of multiple genes or conditional gene knockout strategies have also been developed for adsc-derived organoids and for pscs that can be used for psc-derived organoids [136] [137] [138] . crispr grna library screening analysis has been performed in multiple labs to maximize the utility of human organoids in genetic studies 139, 140 . these developments in genetic engineering, combined with human psc and human organoid technologies, have opened new opportunities for studies of human genetics, as it is possible to perform genetic experiments in small human organ models that closely reflect human physiology. organoids should largely be considered as a model system currently under development: much of the current excitement around the technology is based on their enormous potential rather than on what has already been achieved. nevertheless, organoids could potentially revolutionize disease research in a profound manner (fig. 4) . generating animal models for a specific disease requires pre-existing insight into either the causative conditions or the genes involved. animal models are typically created by applying harmful conditions to animals or by www.nature.com/nrm manipulating the genes responsible for the disorder, while organoid models can be directly generated from affected patients without prior knowledge of the specific genes responsible. this is particularly relevant for multigenic disorders such as inflammatory bowel disease, provided that the pathology is caused by the affected epithelium, and for cancers, where cancer organoids can be directly isolated from the patient [141] [142] [143] [144] [145] [146] . human organoid cultures have a number of potential benefits over animal models (box 3): organoids provide faster and more robust outcomes, are more readily accessible and provide both a more accurate representation of human tissue and a larger quantity of material to work with than animal models do. the mouse is one of the animal model systems most frequently used to explore human biology and disease, owing to its similarity to humans, in comparison with other animal models, and to the ability to generate transgenic and knockout mouse strains. however, the generation of a conventional transgenic mouse model to address questions regarding human disease generally takes more than a year, even with the technological advance of crispr-cas9-mediated precision genome editing [147] [148] [149] . furthermore, differences in microbiota and pathogen composition between animal models and humans, as well as the failure of certain phenomena observed in mice to translate directly to humans, limit the utility of animal models in human disease research 150 . (2) biobanking, whereby samples obtained from patients can be used to generate patient-derived organoids and stored as a resource for future research; (3) disease modelling, to understand the mechanisms of human diseases such as infectious diseases, inheritable genetic disorders and cancer using various laboratory techniques, including omics and drug-screening analyses; and (4) precision medicine, in which patient-derived organoids can be used to predict response to drugs and as resources for regenerative medicine coupled with genetic engineering. ecm, extracellular matrix. diseases caused by more than a single genetic factor. human stem cell-derived organoid culture is widely expected to bridge the remaining gaps between animal models and humans, primarily because the source material for organoid culture is a stem cell derived from a human 35, 40 . the length of time required to establish the research platform is also faster for the organoid model than for animal models: a human organoid culture can be established within a few weeks or months with a high success rate, thus supporting the use of patient-derived organoids in personalized medicine to provide robust personalized data, including individual mutation profiles and drug responses. organoid handling is also relatively easy, with researchers being able to handle a large number of organoid lines at the same time, although the initial determination of culture conditions for a new tissue type is rather complicated. variability and single-cell profiling. in spite of the many advantages detailed above, human organoid culture remains under development, and numerous efforts to advance the technology are still in progress. one of the most pressing issues in organoid technology is the variability of the system: individual reports have described contrasting methods to generate organoids from stem cells, but a widely accepted, standardized protocol is still missing. for instance, human gut organoids derived in different laboratories, using human adscs and human pscs, are different, even though both are termed 'gut organoids' 2,3,24 . the possibility for further improvement of an already well-defined method was recently reported by showing that more refined culture conditions provide better cellular diversity and culture efficiency 4 . the lack of widely accepted, standardized protocols and guidance is an important issue to overcome in order to reduce the variability of the system from research group to group. a collective effort should be made to set clear guidelines and ways to assess the quality and validity of each system. single-cell profiling technologies for transcriptome and epigenome analysis may be key in terms of highly accurate assays suitable for this purpose. with these assays, it would be possible to compare every cell type present in the organoids with their in vivo counterpart at the level of transcriptome and epigenome. in addition, individual differences such as age and genetic background might introduce further variability to the human organoid system, which, while potentially challenging, could also present an opportunity to assess the role of person-to-person variability in human biology. additional considerations to be addressed include the modelling of cell-to-cell communication with stromal cell populations and the development of vasculature in organoid systems, although vascularization has been observed in some cases upon transplantation 23, 151 . significant progress has recently been achieved with blood vessel organoids 152 , which have a great potential to impact research into vascular diseases such as diabetes. however, despite steady progress, vascularization still remains a difficult obstacle to overcome. several reports already exist of organoid co-culture systems containing pathogens such as bacteria, viruses and parasites 153, 154 , and moreover, organoid co-cultures with mesenchymal and/or immune cell populations have shown exciting progress in recent years and are of great interest as means to better model human disease 23, [155] [156] [157] [158] [159] [160] [161] [162] . nevertheless, organoid systems already contain a large degree of complexity, and it is important to acknowledge the challenge of adding additional components to an already complex system. both simple and complex organoid systems have their pros and cons, and therefore it is most important to use the most appropriate level of complexity for a given study. for example, cancer organoids containing only the epithelial component can be sufficient to test the efficacy of most cancer drugs. however, for immuno-oncology therapies or for an assessment of drug metabolism and availability to the target organ, more complex systems containing immune cells, mesenchymal cells and/or endothelial cells together with the organoids will be necessary. unfortunately, a standardized protocol or guidelines concerning these matters are still lacking, so individual researchers are left to determine the most appropriate system for themselves. we foresee that full standardization of many of these protocols will be achieved in the near future, in a manner similar to increases in both the quality and reproducibility of human ipsc technology. assessing interactions with other organs and the environment. one clear drawback of organoid systems is the lack of interorgan communication. human organoid systems fundamentally mimic a part of the human body, not the entire body. therefore, human organoids are limited to the reproduction of organ-specific or tissue-specific microphysiology, a limitation to bear in mind prior to entering this exciting new field. however, efforts are already in progress to overcome • human-derived: human organoids represent human physiology, rather than being a 'human-like' or 'similar' system. • rapid: adult stem cell-derived and pluripotent stem cell-derived organoids can be established rapidly and easily. • robustness: once established, scale-up is usually possible for large-scale genomic screening and drug screening. • genetic manipulation: most modern genetic engineering tools can be applied to induced pluripotent stem cells or directly to organoid systems. • personalization: induced pluripotent stem cells and organoids can be obtained from individuals. • cellular components: the microenvironment is sometimes lacking, particularly in adult stem cell-derived organoids. co-culture systems with other cell types are not firmly established. • standardization: protocols for organoid establishment and quality control are not globally standardized. • relatively costly: organoids cost less than mouse or fish models, but they are relatively expensive compared to traditional cell lines, fly, yeast or worm models. • scale: studies at the level of whole organs are difficult. • heterogeneity: owing to diversity between individuals and protocols, outcomes may vary from group to group. www.nature.com/nrm this limitation. for example, multiple organoids have been connected in order to study communication between the liver, pancreas and gastrointestinal tract 163 ; cell migration between the developing forebrain and hindbrain [164] [165] [166] ; and the interaction between the brain and hormone-producing organs 167 . the development of tools to help us model organ-level communication will progress, although this capacity is likely to lag in comparison to progress in other areas of the field, owing to the complex nature of the studies undertaken. of note, efforts to bring together the fields of organoid research and organ-on-a-chip research are particularly exciting, potentially resulting in an 'organoid-on-a-chip' technology 168, 169 . we foresee, for example, the potential generation of a chamber device that enables the separate culture of distinct organoid types, thus preventing the uncontrolled fusion of organoids while permitting organoid-organoid communication. finally, the effect of extracellular matrix composition on organoid culture is yet to be defined; uncertainty in the composition of the extracellular matrix can heavily influence the outcomes in chemical screening or genetic screening of human organoids. diverse efforts have been made, with some impressive successes in recent years [170] [171] [172] [173] [174] [175] . this obstacle should be overcome not only as a means to produce more robust human model systems, but also to allow the translation of human organoid technology to regenerative medicine, where 'good manufacturing practice' requires all raw materials, including matrix materials, to be fully defined. furthermore, work is ongoing towards the development of organoid culture platforms for large-scale production, organoid-based high-content screening platforms and micro-organoids-on-a-chip as miniature, finely controlled systems. for all these systems, it will be extremely important to know how to manufacture a synthetic, versatile extracellular matrix. despite the remaining challenges, human organoids hold great potential in clinical translational research, owing to the advantages outlined above and to rapid, ongoing technology development. from the initial full 'laboratory life cycle' , which started with the isolation of patient samples, to the establishment of organoids and their cryopreservation, organoid technology has expanded to embrace genetic manipulation, various omics and drug-screening analyses and diverse co-culture system with viruses, bacteria and parasites (fig. 4) . thus, technologies and experimental procedures that were developed in other model systems can now be applied to human organoid systems, which will accelerate our understanding of human biology and enable us to validate hypotheses and models generated from studies in animal model systems. given the rapid technical advances in the field, we believe that human organoid systems will provide unprecedented opportunities to improve human health. published online xx xx xxxx progress and potential in organoid research single lgr5 stem cells build crypt-villus structures in vitro without a mesenchymal niche provide the first example of organoids derived from adscs isolated from mouse gut long-term expansion of epithelial organoids from human colon, adenoma, adenocarcinoma, and barrett's epithelium human intestinal organoids maintain self-renewal capacity and cellular diversity in niche-inspired culture condition cerebral organoids model human brain development and microcephaly report that the complexity of human brain development can be modelled by human psc-derived organoids kidney organoids from human ips cells contain multiple lineages and model human nephrogenesis long-term expansion of functional mouse and human hepatocytes as 3d organoids long-term, hormone-responsive organoid cultures of human endometrium in a chemically defined medium in this review, huch and koo summarize the development of various organoid culture systems and compare mouse and human systems organoids: a historical perspective of thinking in three dimensions organogenesis in a dish: modeling development and disease using organoid technologies dishing out mini-brains: current progress and future prospects in brain organoid research organoids: modeling development and the stem cell niche in a dish modeling development and disease with organoids organoid models for translational pancreatic cancer research organoids as an in vitro model of human development and disease stem cell models of human brain development the embryonic cell lineage of the nematode caenorhabditis elegans large-scale mutagenesis in the zebrafish: in search of genes controlling development in a vertebrate large scale genetics in a small vertebrate, the zebrafish the zebrafish issue of development the genetics of caenorhabditis elegans this is the first report of organ bud formation through self-condensation of cells from different lineages directed differentiation of human pluripotent stem cells into intestinal tissue in vitro identify a step-by-step procedure to generate human intestinal organoids derived from pscs wormbase: a multi-species resource for nematode biology and genomics the worm in us -caenorhabditis elegans as a model of human disease genome-wide rnai screens in caenorhabditis elegans: impact on cancer research development and evolution of the human neocortex metabolic costs and evolutionary implications of human brain development predictability of metabolism of ibuprofen and naproxen using chimeric mice with human hepatocytes cyp2c9-catalyzed metabolism of s-warfarin to 7-hydroxywarfarin in vivo and in vitro in chimeric mice with humanized liver pluripotent stem cell-derived organoids: using principles of developmental biology to grow human tissues in a dish induction of pluripotent stem cells from adult human fibroblasts by defined factors induced pluripotent stem cell lines derived from human somatic cells a critical look: challenges in differentiating human pluripotent stem cells into desired cell types and organoids disease modeling in stem cell-derived 3d organoid systems organoid and assembloid technologies for investigating cellular crosstalk in human brain development and disease human kidney organoids: progress and remaining challenges liver organoids: from basic research to therapeutic applications disease modelling in human organoids long-term expanding human airway organoids for disease modeling stepwise differentiation of pluripotent stem cells into retinal cells modelling human development and disease in pluripotent stem-cell-derived gastric organoids embryonic stem cell lines derived from human blastocysts establishment in culture of pluripotential cells from mouse embryos isolation of a pluripotent cell line from early mouse embryos cultured in medium conditioned by teratocarcinoma stem cells human embryonic stem cells: research, ethics and policy human ipsc banking: barriers and opportunities advances in pluripotent stem cells: history, mechanisms, technologies, and applications stem cells, genome editing, and the path to translational medicine pluripotent stem cells in disease modelling and drug discovery guided self-organization and cortical plate formation in human brain organoids generation of a vascularized and functional human liver from an ipsc-derived organ bud transplant 3d modeling of esophageal development using human psc-derived basal progenitors reveals a critical role for notch signaling esophageal organoids from human pluripotent stem cells delineate sox2 functions during esophageal specification wnt/beta-catenin promotes gastric fundus specification in mice and humans in vitro generation of human pluripotent stem cell derived lung organoids the intestinal stem cell identification of stem cells in small intestine and colon by marker gene lgr5 differentiated troy+chief cells act as reserve stem cells to generate all lineages of the stomach epithelium lgr5 +ve stem cells drive self-renewal in the stomach and build long-lived gastric units in vitro in vitro expansion of single lgr5 + liver stem cells induced by wnt-driven regeneration unlimited in vitro expansion of adult bi-potent pancreas progenitors through the lgr5/r-spondin axis in vitro expansion of human gastric epithelial stem cells and their responses to bacterial infection a novel human gastric primary cell culture system for modelling helicobacter pylori infection in vitro long-term culture of genome-stable bipotent stem cells from adult human liver expansion of adult human pancreatic tissue yields organoids harboring progenitor cells with endocrine differentiation potential tumor evolution and drug response in patient-derived organoid models of bladder cancer basal cells as stem cells of the mouse trachea and human airway epithelium reconstruction of the mouse extrahepatic biliary tree using primary human extrahepatic cholangiocyte organoids development of organoids from mouse and human endometrium showing endometrial epithelium physiology and long-term expandability quantification of regenerative potential in primary human mammary epithelial cells identification of multipotent luminal progenitor cells in human prostate organoid cultures single luminal epithelial progenitors can generate prostate organoids in culture the notch and wnt pathways regulate stemness and differentiation in human fallopian tube organoids a developmental and genetic classification for malformations of cortical development: update 2012 zika virus and microcephaly: why is this situation a pheic? detection and sequencing of zika virus from amniotic fluid of fetuses with microcephaly in brazil: a case study zika virus associated with microcephaly zika virus depletes neural progenitors in human cerebral organoids through activation of the innate immune receptor tlr3 zika virus impairs growth in human neurospheres and brain organoids show the utility of complex brain organoids for translational zika virus research the brazilian zika virus strain causes birth defects in experimental models zika-virus-encoded ns2a disrupts mammalian cortical neurogenesis by degrading adherens junction proteins identification of small-molecule inhibitors of zika virus infection and induced neural cell death via a drug repurposing screen epidemiology of human noroviruses and updates on vaccine development replication of human noroviruses in stem cell-derived human enteroids demonstrate that organoid culture systems can support research on difficult pathogens that previously could not be cultivated rotavirus vaccines human intestinal enteroids: a new model to study human rotavirus infection, host restriction, and pathophysiology modeling rotavirus infection and antiviral therapy using primary intestinal organoids the emergence of influenza a h7n9 in human beings 16 years after influenza a h5n1: a tale of two cities differentiated human airway organoids to assess infectivity of emerging influenza virus influenza viruses en route from birds to man influenza virus neuraminidase structure and functions modeling infectious diseases and host-microbe interactions in gastrointestinal organoids persistence and toxin production by clostridium difficile within human intestinal organoids result in disruption of epithelial paracellular barrier function modelling cryptosporidium infection in human small intestinal and lung organoids recent strategic advances in cftr drug discovery: an overview a functional cftr assay using primary cystic fibrosis intestinal organoids report the use of organoids in precision medicine for patients with cystic fibrosis characterizing responses to cftr-modulating drugs using rectal organoids derived from subjects with cystic fibrosis rectal organoids enable personalized treatment of cystic fibrosis prospective derivation of a living organoid biobank of colorectal cancer patients a colorectal tumor organoid library demonstrates progressive loss of niche factor requirements during tumorigenesis preserved genetic diversity in organoids cultured from biopsies of human colorectal cancer metastases patient-derived colorectal cancer organoids upregulate revival stem cell marker genes following chemotherapeutic treatment patient-derived organoids can predict response to chemotherapy in metastatic colorectal cancer patients a patient-derived glioblastoma organoid model and biobank recapitulates interand intra-tumoral heterogeneity patient-derived organoids (pdos) as a novel in vitro model for neuroblastoma tumours organoid cultures derived from patients with advanced prostate cancer organoid models of human and mouse ductal pancreatic cancer pancreatic cancer organoids recapitulate disease and allow personalized drug screening human pancreatic tumor organoids reveal loss of stem cell niche factor dependence during disease progression human primary liver cancer-derived organoid cultures for disease modeling and drug screening a living biobank of breast cancer organoids captures disease heterogeneity human gastric cancer modelling using organoids a comprehensive human gastric cancer organoid biobank captures tumor subtype heterogeneity and enables therapeutic screening divergent routes toward wnt and r-spondin niche independency during human gastric carcinogenesis organoid cultures recapitulate esophageal adenocarcinoma heterogeneity providing a model for clonality studies and precision therapeutics patient-derived organoids from endometrial disease capture clinical heterogeneity and are amenable to drug screening patient-derived lung cancer organoids as in vitro cancer models for therapeutic screening patient-derived organoids model treatment response of metastatic gastrointestinal cancers a rectal cancer organoid platform to study individual responses to chemoradiation patient-derived organoids predict chemoradiation responses of locally advanced rectal cancer site-directed mutagenesis by gene targeting in mouse embryoderived stem cells gene targeting in human pluripotent cells chimeric nucleases stimulate gene targeting in human cells enhancing gene targeting with designed zinc finger nucleases a tale nuclease architecture for efficient genome editing rna-guided genetic silencing systems in bacteria and archaea multiplex genome engineering using crispr/cas systems rna-guided human genome engineering via cas9 targeted genome engineering in human cells with the cas9 rna-guided endonuclease the next generation of crispr-cas technologies and applications functional repair of cftr by crispr/cas9 in intestinal stem cell organoids of cystic fibrosis patients report the first study to apply crispr-cas9-based gene correction in an organoid system sequential cancer mutations in cultured human intestinal stem cells modeling colorectal cancer using crispr-cas9-mediated engineering of human intestinal organoids one-step generation of conditional and reversible gene knockouts a protocol for multiple gene knockout in mouse small intestinal organoids using a crispr-concatemer simultaneous paralogue knockout using a crispr-concatemer in mouse small intestinal organoids pooled in vitro and in vivo crispr-cas9 screening identifies tumor suppressors in human colon organoids genome-scale crispr screening in human intestinal organoids identifies drivers of tgf-beta resistance alterations in the epithelial stem cell compartment could contribute to permanent changes in the mucosa of patients with ulcerative colitis dna methylation defines regional identity of human intestinal epithelial organoids and undergoes dynamic changes during development single cell analysis of crohn's disease patient-derived small intestinal organoids reveals disease activity-dependent modification of stem cell properties dna methylation and transcription patterns in intestinal epithelial cells from pediatric patients with inflammatory bowel diseases differentiate disease subtypes and associate with outcome organoids in cancer research somatic inflammatory gene mutations in human ulcerative colitis epithelium one-step generation of mice carrying mutations in multiple genes by crispr/cas-mediated genome engineering generating genetically modified mice using crispr/cas-mediated genome engineering one-step generation of mice carrying reporter and conditional alleles by crispr/ cas-mediated genome engineering exploring host-microbiota interactions in animal models and humans vascularized and complex organ buds from diverse tissues via mesenchymal cell-driven condensation human blood vessel organoids as a model of diabetic vasculopathy organoids in immunological research modeling host-virus interactions in viral infectious diseases using stem-cellderived systems and crispr/cas9 technology human fetal tnf-alpha-cytokineproducing cd4 + effector memory t cells promote intestinal development and mediate inflammation early in life organoid modeling of the tumor immune microenvironment generation of tumor-reactive t cells by co-culture of peripheral blood lymphocytes and tumor organoids 3d model for car-mediated cytotoxicity using patient-derived colorectal cancer organoids co-culture with intestinal epithelial organoids allows efficient expansion and motility analysis of intraepithelial lymphocytes a primary human macrophage-enteroid co-culture model to investigate mucosal gut physiology and host-pathogen interactions mesenchymal stem cells increase alveolar differentiation in lung progenitor organoid cultures anatomically and functionally distinct lung mesenchymal populations marked by lgr5 and lgr6 modelling human hepato-biliarypancreatic organogenesis from the foregut-midgut boundary fused cerebral organoids model interactions between brain regions fusion of regionally specified hpsc-derived organoids models human brain development and interneuron migration assembly of functionally integrated human forebrain spheroids understanding the role of steroids in typical and atypical brain development: advantages of using a "brain in a dish" approach towards a human-on-chip: culturing multiple cell types on a chip with compartmentalized microenvironments multisensor-integrated organs-onchips platform for automated and continual in situ monitoring of organoid behaviors extracellular matrix hydrogel derived from decellularized tissues enables endodermal organoid culture development of collagen-based 3d matrix for gastrointestinal tract-derived organoid culture peg-4mal hydrogels for human organoid generation, culture, and in vivo delivery mechanically and chemically defined hydrogel matrices for patient-derived colorectal tumor organoid culture designer matrices for intestinal stem cell and organoid culture growth of epithelial organoids in a defined hydrogel inhibition of pluripotential embryonic stem cell differentiation by purified polypeptides myeloid leukaemia inhibitory factor maintains the developmental potential of embryonic stem cells the ground state of embryonic stem cell self-renewal validated germline-competent embryonic stem cell lines from nonobese diabetic mice new cell lines from mouse epiblast share defining features with human embryonic stem cells derivation of pluripotent epiblast stem cells from mammalian embryos derivation of novel human ground state naive pluripotent stem cells induction of a human pluripotent state with distinct regulatory circuitry that resembles preimplantation epiblast derivation of naive human embryonic stem cells systematic identification of culture conditions for induction and maintenance of naive human pluripotency epigenetic resetting of human pluripotency naive pluripotent stem cells derived directly from isolated cells of the human inner cell mass resetting transcription factor control circuitry toward ground-state pluripotency in human systematic identification of culture conditions for induction and maintenance of naive human pluripotency application of small molecules favoring naive pluripotency during human embryonic stem cell derivation clinical features of patients infected with 2019 novel coronavirus in wuhan coronaviridae study group of the international committee on taxonomy of viruses. the species severe acute respiratory syndrome-related coronavirus: classifying 2019-ncov and naming it sars-cov-2 recapitulation of sars-cov-2 infection and cholangiocyte damage with human liver ductal organoids inhibition of sars-cov-2 infections in engineered human tissues using clinical-grade soluble human ace2 sars-cov-2 productively infects human gut enterocytes a pneumonia outbreak associated with a new coronavirus of probable bat origin sars-cov-2 entry factors are highly expressed in nasal epithelial cells together with innate immune genes angiotensin-converting enzyme 2 is a functional receptor for the sars coronavirus sars-cov-2 cell entry depends on ace2 and tmprss2 and is blocked by a clinically proven protease inhibitor structure, function, and antigenicity of the sars-cov-2 spike glycoprotein structural basis for the recognition of sars-cov-2 by full-length human ace2 structural basis of receptor recognition by sars-cov-2 the authors thank a. kavirayani and c. hindley for proofreading, and s. bae for initial design of the artwork. work in j.a.k.'s laboratory is supported by the austrian academy of sciences, the austrian science fund (grant z_153_b09) and an advanced grant from the european research council. b.-k.k.'s laboratory is supported by the austrian academy of sciences, the european research council, the human frontier science program and the interpark bio-convergence center grant program. the authors contributed equally to the writing and revisions of the article. the authors declare no competing interests. nature reviews molecular cell biology thanks h. clevers, p. liberali and the other, anonymous, reviewer(s) for their contribution to the peer review of this work. springer nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations. who, disease outbreaks: www.who.int/emergencies/ diseases/en/ who, coronavirus disease 19 (covid-19) pandemic: https:// www.who.int/emergencies/diseases/novel-coronavirus-2019 key: cord-017543-60q9iecq authors: tian, wei-chang; finehout, erin title: microfluidic applications in biodefense date: 2008-08-23 journal: microfluidics for biological applications doi: 10.1007/978-0-387-09480-9_10 sha: doc_id: 17543 cord_uid: 60q9iecq nan the growing threat of bioterrorism attacks, combined with repeated outbreaks of emerging infectious diseases, underlines the importance of infrastructure improvement for the detection and diagnosis of biowarfare agents and emerging pathogens. the growing threats posed by terrorists and rogue nations⎯as evidenced by iraq's acknowledgement following the first gulf war that it had loaded biological weapons, and multiple biological "incidents" worldwide⎯has raised serious concerns about bioterrorism attacks directed against the united states and other nations. following the october 2001 anthrax attacks in the united states, biodefense has become an area of utmost national and international urgency [1] [2] . this incident sparked the testing of tens of thousands of samples for the presence of anthrax, straining the laboratory response network (lrn) system. the new york city experience after the anthrax attacks is telling [3] . the increase in incoming samples went from one every several months to about 6,000 in two weeks, requiring a coordinate growth of analytical staff and laboratories by over twenty-fold. the operational expectation had been that any surge would primarily be composed of human clinical samples; instead most of the samples were environmental. a recent study of acute care facilities in mississippi found that the diagnostic capacity of hospitals would be overwhelmed by a weapon of mass destruction (wmd) attack [4] ; similar conclusions regarding the lack of diagnostic surge capacity in alternate locales were reached in other studies [5] [6] . the destructive potential of genetically engineered bioagents is huge. toxic genes can be hidden in innocuous organisms and expressed at high levels. expression timing and genotypic specificity could be controlled to maximize impact and potentially limit spread to a defined racial pool. the purported accomplishments of the soviet bioweapon program [7] accentu-ate how real the problem is: sophisticated state-sponsored bioweapons programs have already genetically engineered bacteria and viruses to increase their devastating impact on human populations. "capitalizing" on post-1972 advances in biotechnology such as genetic engineering, the soviet union program researched and produced a range of weapons employing smallpox, anthrax, plague, and other dangerous pathogens [8] . fortunately, terrorists and rogue states have not yet fully incorporated biological weapons into their arsenals, to our knowledge [9] . the detection of bioengineered organisms presents even greater challenges than detecting conventional pathogens, and may require multi-tiered screening, including high resolution detection of target genes and dna sequencing. similar to the needs for biodefense of engineered organisms, naturally emerging infectious diseases present another major threat to human health, through the natural spread of these organisms, their rapid evolution to human hosts, and the potential for bioterrorism using these agents. the recent outbreaks of severe acute respiratory syndrome (sars) and bovine spongiform encephalopathy (bse), commonly known as mad-cow disease, have raised global concerns on the need for rapid identification of causative agents and infected individuals before the virus spreads beyond control. hantavirus pulmonary syndrome and west nile virus are examples of additional infectious diseases now emerging in the us. rapid molecular diagnostic methods and monitoring platforms that can adaptively be configured for newly emerging infectious diseases or newly engineered bioagents will be essential for combating these diseases and for biodefense applications. past incidents and the dangers of future bioterrorism attacks highlight the critical need for improved field-and laboratory-based systems to detect, identify, and subtype bioagents [10] [11] . as will be seen, the state-of-art of biodefense systems today is operational but rudimentary: all us mail is screened at sorting centers. there is a strong demand from the u.s. government for next-generation systems for civilian and military applications. biodefense monitoring equipment has even more stringent requirements: the size of the current equipment, and the acquisition and operating costs place severe constraints on widespread implementation and deployment. new analytical systems are needed that are scalable, more automated, and capable of rapid deployment in response to surging needs or field operations. the equipment needs to be smaller, use less reagents, be simpler to use, more integrated, and automated-all attibutes of microfluidic systems, making microfluidics an ideal platform to fulfill biodefense needs. this review summarizes approaches to detecting and characterizing biological threat agents for both civilian and military biodefense; describes biodefense programs in place and under development; and delineates some of the approaches where microfluidics is presently being applied to monitor, detect, and characterize biothreat agents. some of the primary needs are outlined, and the challenges to design and build fully integrated microfluidic systems are described. the majority of the review surveys microfluidic technologies that might or could be used in future biodefense systems. sections cover microscale sample preparation methods; immunomagnetic separations and immunoassays; proteomics; polymerase chain reaction (pcr), quantitative pcr (qpcr) and other nucleic acid amplification methods; dna microarrays, microelectrophoresis, and finally integrated lab-on-a-chip systems. there are two basic biodefense detection approaches: detect-to-warn and detect-to-treat. detect-to-warn systems aim to identify biothreats rapidly enough to provide sufficient warning to prevent exposure by the threat. detect-to-treat systems aim to identify the causative agent for diagnostic purposes and thereby to direct healthcare workers to the most effective treatment as quickly as possible. for all systems, low false alarm rates (far) and affordable acquisition and operating costs are essential for widespread adoption. civilian biodefense is based upon surveillance to detect biothreat agents, response networks to warn and direct the treatment of the affected population [12] , and the development of countermeasures [13] . bioterrorism incidents, releases of a bioagent in a form that can harm individuals or larger populations, can range from mailing of toxin or bacteria, to release of aerosols in high profile events, to attacks on the food supply. bioshield, a countermeasures program [14] , is a 10-year, $5.6 billion u.s. program for the advanced development and purchase of medical countermeasures. acquisition programs have been announced to counter bacillus anthracis (anthrax), variola virus (smallpox), botulinum toxins, and radiological/nuclear agents. the primary programs that have been implemented for civilian biodefense detection include screening of all postal mail at sorting centers, the bio-watch program, selected localized screening in subways and other undisclosed locations, and the lrn. the currently deployed systems principally use full volume or meso-scale fluidics. despite the need for more advanced detect-to-warn biodefense detection for the general public, these systems have largely remained undeveloped in large part due to the complexity of integrating the complete process. the largest monitoring program is biowatch [15-16], a joint effort by the department of homeland security (dhs), the centers for disease control and prevention (cdc), and the environmental protection agency (epa). biowatch is a 'detect-to-treat' program and monitors the air in at least 31 cities (and as many as 120 cities) [17] for significant release of bioagents. dhs does not report cities monitored by the biowatch program or the assays used. in 2004 and 2005, dhs funding for the biowatch was $26.8 million for the epa and $28.5 million for the cdc [18]. the epa is responsible for continual air sampling by aerosol collectors that trap airborne particles onto filters. the filters are collected for analysis every 24 hours. the cdc is responsible for the analysis of the filters at state and local public health laboratories, and developing new protocols in coordination with both the department of energy (doe) national laboratories and the epa. the assays are generally acknowledged to be pcr amplified detection of specific targets using classified primer sequences. the list of target agents is similarly classified but thought to include at least anthrax, smallpox, plague, and tularemia. the biowatch program has now processed over 2 million samples without a false positive [19]-an impressive accomplishment. however, the daily sampling frequency and the amount of coverage of the biowatch program still leaves the civilian populace vulnerable. dhs has been funding the biological autonomous networked detectors (band) program as a next-generation system to alleviate some of the deficiencies of the biowatch program. the original goals of the band program were continuous air monitoring with sample analysis every three hours. the detection limits were 100 organisms or 10 ng of toxin per 17,000 liters of air processed, with a very low far. the instrument was planned to have an acquisition cost of $25,000, an annual operating cost of $15,000, and run autonomously for 30 days. additional requirements were for dimensions of 2 ft 3 and the ability to detect up to 20 organisms and tox-ins. the band participants have almost uniformly taken a microfluidics approach. their efforts are described in more technical detail in a later section of this chapter. in addition to detecting bioagents in the primary environment, a major effort has gone into upgrading the response of the medical community to detect unannounced attacks [20] , since the expectation is that in many scenarios the first alert will be in the form of patients presenting at doctors' offices or hospitals [21] . biodefense systems are also required to monitor food and water sources [22-23], suspect powders, the exposure of first responders, and test for decontamination after treatment of personnel, equipment, and key environments. the u.s. military biodefense programs aim to detect and identify biological warfare agents that an enemy might use to degrade forces, contaminate bases, and spread confusion throughout command and control systems. various defense programs are delivering technologies that are beginning to counter these vulnerabilities. for example, the portal shield program is designed for facilities protection; the joint biological agent identifiation and diagnostic system (jbaids) is designed for both detection and diagnostics of environmental and clinical samples [24] , and the joint biological point detection system (jbpds) is designed for detect-to-warn capabilities in the field. portal shield is an array-able sensor system developed to provide early warning of biological attacks for high-value, fixed-site assets, such as air bases and port facilities. portal shield is designed to detect and identify threats simultaneously within 25 min. it is programable to survey continuously as well as perform random or directed sampling. portal shield was deployed in the persian gulf region in february 1998 during operation desert thunder, and the current instrumentation is about two thirds the size of a typical office desk. it's fully modularized, selfcontained, and can detect eight different agents. as many as 18 sensor units may be arrayed in a given area and are able to communicate with each other, so there is no reliance on just one of them sounding an alarm. using an array system, the false positive rate diminishes towards zero. jbaids is the dod's first common platform for identification and diagnostic confirmation of biological agent exposure or infection. jbaids block i is currently operational as a real-time pcr instrument with fda approved assays. the current jbaids block i system (and jbtds systems) utilizes manual sample preparation (disrupting the cell or spore and extracting the nucleic acid or protein of interest) which results in: 1) complex operator procedures that may result in human error, 2) increased operational costs, and 3) support and logistical requirements that preclude remote operations. the intent of jbaids block iii, next generation diagnostics (ngd), is to establish a new system incorporating the capabilities of block i and block ii capabilities (table 10 .1) and adding immunoassay capabilities and the ability to identify up to 50 agents including toxins in 15 minutes using automated, miniaturized sample preparation integrated with analysis for nucleic acids and proteins, in a hand held or smaller format. jbpds is planned to detect, identify, and warn against the presence of up to 24 biowarfare agents at discrete points within a given field environment. jbpds is being designed with a sampler, trigger detection, and identification technologies that allow it to rapidly and automatically detect and identify biological threat agents. jbpds was committed to initial limited production and procurement during fy2006, with planned full production in 2007. future jbpds improvements will reduce size, weight, power consumption, and reagent use while increasing the number of agents recognized, sensitivity, and system reliability. a number of the requirements of the above programs may be met through the implementation of microfluidics. for example, reduced consummable use, weight and size are directly tied to the drastic reduction of reaction volume employed in microfluidic systems and the use of miniaturized microfluidic components. increasing the number of recognized agents can also be accomodated without high order reaction multiplexing by using multi-channel microfluidic devices with single-plex reactions carried out in parallel. in theory, with microfabricated microchips adding more channels is simple a 'cut and paste' exercise once the issues of connections and integation are solved. there are considerable issues in integrating all processing and analysis steps in a 'hands-free' device. however, microfluidics requires a 'hands-free' implementation once samples are loaded since typically there is nothing the user can do to intervene. the detection of biothreat agents today can be segmented into laboratory detection and field detection methods, and by the type of sample matrix processed (reviewed in [11, [25] [26] [27] [28] ). the detection must be sensitive, specific for the test organism(s), and may require substantial up-front sample preparation before the read-out assay can be performed. there are a number of commercial tests available today to detect biothreats using nucleic acid, immunological, and biochemical methods. the appropriate test may be determined by the level of information required (i.e., phenotypic or genotypic), timeframe, and the consequences. in general, nucleic acid tests are more sensitive, but require a higher order of skilled operators and more sophisticated equipment than immunological tests, and cannot detect toxins. confounding the problem is the need to assess samples for the presence of many possible biothreat agents; the detection of genetically engineered organisms potentially designed to evade standard detection methods; and the vast numbers of people crossing international borders (600 million international travelers per year [27]). sample matrix and sample preparation methods are key variables in biodefense detection. targets may be contained in air, food, water, bodily fluids, powders, swabs, swipes, cloth, or filters, among many other possibilities. significant reductions in sample volume are required and sample preparation methods must remove contaminants such as metal ions, heme, humic acids, and other compounds, which inhibit pcr or other assays. to enable input of samples into microfluidic systems, the target analyte needs to be highly concentrated without concentrating inhibitors. sample preparation issues are further detailed in section 10.5. traditional identification of pathogens is often tedious and prolonged, involving batteries of tests that often take days or even longer to confirm. standard clinical laboratory identification of bacillus anthracis serves as an example [12] . bacillus anthracis testing by a lrn level a laboratory begins with growth of the organism, a gram stain, capsule observation and routine culturing on sheep blood agar. this is followed by observation of colony morphology, motility, sporulation, and hemolysis. the presumptive identification can take up to 96 hours with additional days for confirmation. confirmatory testing at level b labs (state and federal laboratories) consists of phage lysis and immuno-fluorescence assays of a cell wall and capsule. level c laboratories determine antimicrobial susceptibility and apply more advanced technology including pcr, qpcr, and time-resolved fluorescence measurements. finally, at level d, labs (cdc), in-depth molecular characterization is performed using multiple-locus vntr (variable-number tandem repeat) analysis (mlva), 16s rdna ribotyping and other methods, including sequencing to provide subtyping information for identification [29] . full characterization can include complete genomic sequencing to identify the exact strain variant for epidemiological and forensic analysis. similarly the armed forces institute of pathology uses dna extraction, dna quantification, qpcr of unique genetic targets, 16s rrna gene sequencing, amplified fragment length polymorphism polymerase chain reaction (aflp-pcr), and repetitive element polymerase chain reaction dna fingerprinting to characterize strains [30]. the results are compared to extensive databases that have been assembled [31-32]. for more rapid detection in the field, current detection methods use immunoassays, pcr, and other molecular typing methods to provide information on suspected biothreat samples. direct fluorescence assay with monoclonal antibodies to cell wall and capsule components have also worked well for b. anthracis [33] . real-time pcr or qpcr methods are frequently performed [29] and can yield rapid identification in the field, using the lightcycler (roche), genexpert (cepheid), jbaids (idaho technology) or other systems. dna sequencing of 16s ribosomal sequences, plasmids, or variable regions can yield the highest resolution identification, but is relatively slow and has a low throughput compared to other methods; sequencing has not yet been adapted to field applications. one of the most advanced field detection concepts is qpcr packaged in portable devices. researchers at lawrence livermore national laboratories (llnl) demonstrated real-time detection of pcr products in a miniaturized silicon reactor with thin film heaters and integrated fluorescence detection [34] . this work was extended at llnl to a 10-channel advanced nucleic acid analyzer and a portable version was devised [35] . cepheid developed commercial versions of this device with integrated (multimicroliter) sample processing for qpcr analysis, now incorporated into the northrop grumman biohazard detection system (bds), used by the postal system for monitoring mail facilities. the bds incorporates upstream air sampling with the genexpert (cepheid), which prepares dna from a fluidized aerosol sample and then performs qpcr. the system is fully integrated, automated, and reports data to a central point. the biowatch program grew in part from the biological aerosol sentry and information system (basis) project developed at the llnl and los alamos national laboratory. basis used filters to collect aerosolized particulate samples at large events such as the olympics. filters were analyzed by pcr in separate laboratory facilities using largely manual protocols [36] . llnl also developed the autonomous pathogen detection system (apds) [37] as a stand-alone, autonomous aerosol detection device. apds, a podium-sized system, monitors air for all three biological threat agent types (bacteria, viruses, and toxins) by continuously performing aerosol collection, sample preparation, and multiplexed biological tests. the apds first employs fluorescent bead-based immunoassays to detect more than ten agents. if a positive signal is detected, a second tier confirmation using qpcr is enabled. apds systems have been field tested at major transportation centers and at special events [32] . a number of companies (e.g., tetracore, alexeter technologies) have developed and market lateral flow immunoassays for field detection of a number of relevant biothreat agents. these are strips of porous materials to which sample is added to one end, and during migration to the other end, encounters a region containing antibodies specific to the target along with a chromophore. while most immunoassays are not particularly sensitive, they are the normally used field screening method and are also used in situations where large numbers of negative samples are anticipated. rapid detection, identification, and subtyping analysis of pathogenic organisms and toxins are critical needs for biodefense and for the management of emerging infectious diseases. autonomous systems that can detect and provide initial identification of bioagents are required for field monitoring and to provide any reasonable degree of protection to civilians. laboratories require automated systems that can rapidly genotype microorganisms from human samples, environmental samples, or food and differentiate at the strain level and better direct treatment. all of these newly developed systems must detect nucleic acids and/or toxins in varying amounts, formats, and in many different matrices. they will need to be completely automated or simple to use; incorporate advanced technologies including sample preparation starting from primary samples (aerosols, blood, etc.), molecular detection, automation, microfluidics, and bioinformatics; reduce reagent consumption and space requirements; and provide cost and performance advantages compared to present systems. analytical techniques such as pcr, vntr, mlva, aflp, and single molecule detection are well suited to analysis on microfluidic systems. in addition, the systems should be capable of accommodating new assays as they become available. the burgeoning field of microfluidics can offer remedies that fulfill many of these needs and is thus becoming an ever increasingly desired component of next-generation systems. microfluidics can provide the fundamental platform technology that reduces the footprint, minimizes reagent consumption, and fully automates monitoring and analytical equipment for operation in the field, monitoring of cities, and detection in the clinic. however, microfluidics faces many challenges before it is ready for wide-spread deployment. the first challenge is interfacing microfluidics with the full scale samples input at the front end. 'real world' samples may be measured in milliliter volumes while microfluidics typically manipulates nanoliter volumes: therefore the sample must be concentrated or many orders-of-magnitude of detection sensitivity will be sacrificed at the front end of the system. in addition, for biodefense and many other applications, a potentially dilute target must be detected from what can be very large volumes. the band program specification for civilian protection is 100 organisms in 17,000 liters of air. to work in a microfluidic system, the 'real world' input sample must therefore be reduced in volume by orders-of-magnitude while still achieving the necessary signal-to-noise to maintain sensitivity. this means taking 100s of microliters of liquids or thousands of liters of air and concentrating the input sample into nanoliters before further microfluidic processing and reactions can take place. this can be achieved by chemically, biochemically, or physically concentrating the sample. paramagnetic beads provide one elegant solution to both the 'macro-tomicro' interface and specificity. the beads, typically about several microns, can specifically or non-specifically capture nucleic acids, cells, viruses, or toxins, from large volumes of solution and move samples from the full volume world of milliliters into a hundreds of nanoliters. when a capture chemistry such as immunomagnetic separations or hydridization is performed with the beads, they can extract the desired target from high backgrounds and clutter. paramagnetic beads simplify sample handling in the microfluidic world by minimizing the positioning demands on the fluidic system since magnetics can be used to recapture beads at any location and potentially eliminate diffusional losses. once the sample has been introduced into the submicroliter realm, the next challenge is integrating the workflow steps in a microfluidic device. as we will see, microfluidics has been applied to the individual processes and proof-of-concept publications on almost any conceivable individual step are a proof of the potential of microfluidic approaches. however, while numerous microfluidic components are well developed in academic or research settings, a key challenge for microfluidics is to either (1) fully integrate all processes to build complete 'sample-to-answer' microfluidic systems, or (2) seamlessly interface microfluidic components with each other and with 'full scale' components into a complete system. the later requires interfacing components (or modules)-such as upstream samples from aerosol collectors, swabs, and blood-with sample preparation components and downstream analytical devices, such as real-time pcr or mass spectroscopy. ideally best of breed modules could be interfaced once microfluidic interfaces and connections are standardized. in any case, full process integration within a microfluidic system or module must be accomplished. the integration requires the coupling of different reactions, which may have multiple steps of sample purification, reagent addition, mixing, separation, and detection. microvalves and micropumps are invaluable to isolate processes or reactions as individual steps and to move fluids to integrate different steps into a workflow. the fourth challenge is designing manufacturable biodefense microfluidic systems. the system should be lower in costs to build and operate, preferably by an order-of-magnitue. the 'valley of death' from proof of concept to product must be crossed and scalable manufacturing capabilities must be implemented with only low volumes as early adopters provide the initial orders. the commercial challenge of crossing the valley of death without prior governmental commitment is substantial. the focus of the remainder of this review is on the microfluidic technologies that can provide advanced rapid detection and identification of bioagents. we review microfluidic technologies that may be used with an emphasis on critical battlefield needs and civilian biodefense. in each section, the technology is briefly introduced, how microfluidics is being applied to advance the state of the art is reviewed, and how this is being applied to biodefense is described. in the final sections, fully integrated microfluidics systems are assessed and some of the devices under development for biodefense are presented. other chapters in this book review the general state of microfluidics and many of the different formats having components that might be applicable to biodefense. starting from the sample, microfluidics can be applied to lyse organisms, concentrate, pre-separate, and purify components for further processing. state-of-the-art rapid, automated, miniaturized, modular universal sample preparation systems are required to prepare nucleic acids and proteins from biological samples in order to detect and identify high priority bioagents. this section describes some of the technologies that can be drawn upon to create a front-end that interacts with 'real world, full volume' samples. analysis of intracellular nucleic acids or proteins requires that cells be disrupted by physical (sonication, heating, or bead beating) or chemical means. the most challenging biothreat organisms to disrupt are the gram positive bacterial spores, bacillus anthracis and clostridium botulinum. sonication and bead beating are the most common ways to disrupt spores today. microfluidic-based cell disruption using sonication has been reported. belgrader and coworkers from llnl reported in 1999 the development of a mini-sonication device that disrupted spores in 30 s; when coupled with a mini-chip pcr instrument, the complete analysis took 30 min [38] . this work was further extended at cepheid where spores were lysed by a sonication horn (in conjunction with glass beads) through a flexible interface using a pressurized microfluidics cartridge [39] then the sonicated lysate was pcr amplified after reagent addition in a disposable cartridge [40] . marentis et al. developed a piezoelectric microfluidic mini-sonicator and determined that it could lyse eukaryotic cells and spores with an efficiency of 50% lysis of b. subtilis spores in 30 s [41] . laser induced disruption in a polydimethylsiloxane (pdms) microchip has been reported for b. atrophaeus spores using a laser absorbing matrix at fluencies below 20 mj/cm 2 and without matrix above that level [42] . small laser diodes and carboxyl-terminated magnetic beads have been shown to disrupt e. coli, gram-positive vegetative bacteria, and hepatitis b virus mixed with human serum in a microchip with real time detection [43] . following lysis, the cellular material often requires separation into component fractions. beads, gels, and membranes can also be incorporated to perform pre-separations to remove inhibitors or concentrate samples. they can provide high mass transfer rates and be made from polymer, silica, or other substrates for microchip liquid chromatography and electrochromatography applications. agilent has developed a commercial polymer microfluidic chip for hplc separation using ablation of polyimide to form channels [44] . a microfluidic technique which is increasingly applied is the use of monoliths as a stationary phase (reviewed in [45] [46] [47] ). the monoliths can be made with a variety of surface chemistries, pore sizes, and functionalized coatings. dielectrophoretic separations can also be performed on microdevices [48] ; in this regard, the nanogen digital array will be discussed in the microarray section. biodefense samples are derived from a wide variety of substrates and matrices. the matrix may contain complex mixtures including inhibitory compounds (e.g., mold, hemes, indigo, humic and fulvic acids, chelating agents, dnases, rnases, and proteases) that interfere with dna amplification, the gold standard for bioagent identification. the dod critical reagents program now provides standardized test kits composed of commonly encountered inhibitory compounds to aid biodefense development and testing. a number of approaches have been taken to purify nucleic acids before analysis at a full volume. early work showed that dilution of the sample can relieve inhibition contained in soil extracts for pcr amplification reactions [55]. however, dilution may not be an option if target concentrations are low. low-melting-temperature agarose has been used to extract dna from soil samples [56] . solid phase extraction that adsorb analytes onto columns, beads, and surfaces and spun separation gels in column format are commonly used to purify dna before analysis. multistep purifications such as organic extracts combined with sephadex columns have also been developed. while these methods are effective, they were best suited for research laboratory environments due to their reliance on supplementary equipment, trained personnel, and time-consuming procedures [57]. most are not amenable to a microfluidic format. microfluidics and microchips are now being applied to miniaturize dna extractions and concentrate nucleic acids. . silicon microfluidic channels have been modified with amino silanes and dna selectively eluted with alkaline rinses [63] . immobilized beads in microchannels increased the extraction efficiency from serum 88-fold compared with free beads [64] . an integrated microfluidic device was developed to pre-treat whole blood samples using a micro-filter, micro-mixer, micro-pillar array, micro-weir and porous matrix [65] . sol gels in capillary chromatography have been reviewed [66] , as have monoliths for preconcentration and sample extraction [67] . the landers group reported silica bead purification of dna with chaotrophic agents and sol gel immobilization [68] on microchips, and demonstrated purification of b. anthracis dna [69] . they have demonstrated a silica-based monolithic column in a fused-silica capillary [70] and on a glass microchip [71] with extraction of model dna from complex samples with efficiencies of 70%. for whole blood and other mixtures they combined a c18 reverse phase column, to remove proteins and other compounds, with a monolithic column to create a dual phase sample preparation microchip [72] [73] . the authors of this chapter have been developing a device, beadstorm™, for automated magnetic bead purification technology on microfluidic handling on microchips ( fig. 10 .1) at microchip biotechnologies inc. the plastic sample processing cube, about 1 in³, with a 800 ul processing chamber is integrated with pneumatically actuated microvalves on a glass microchip to direct pressure-driven flows consisting of fluids, beads, and samples among reagent and reaction reservoirs. microvalves replace both conventional valves and tubing between reservoirs providing a leak-free, self-contained fluid transport. the beadstorm module manipulates input liquid and swabs of biological samples by bead purifying the samples, fully preparing them for downstream analysis such as pcr and immunoassays. immunomagnetic separations have also been performed in this format. the beadstorm device has been successfully used to automate dna extraction from buccal swabs for str amplification. liquid blood samples have also been successfully prepared by the beadstorm module with automated preparation of dna from liquid blood sample in less than 5 minutes. immunological techniques are widely used for rapid purification and detection of cells, viruses, and proteins and are the most widely used diagnostic method in clinical medicine. the primary antibody is typically attached to a solid surface such a microtiter plate or a bead. the secondary antibody is added to generate a 'sandwich' assay that can provide a highly specific and rapid readout. the antibodies can be polyclonal, with wide variation of specificity from batch-to-batch, or monoclonal, which can be produced repeatedly with identical avidity to the corresponding antigen. sandwich assays are well developed for use in laboratories, clinics, and field applications, and are the most common type of assays for toxin testing. there are many immunoassays that are being applied to biodefense and microfluidic immunoassays will be increasingly important in the future. immunomagnetic separation (ims) is a powerful technology that allows targets to be captured and concentrated in a single step using a mechanically simplified format that employs paramagnetic beads and a magnetic field (reviewed in [74] [75] [76] ). ims is used to capture, concentrate, and then purify specific target antigens, proteins, toxins, nucleic acids, cells, and spores in a single step. ims works by binding a specific affinity reagent, typically an antibody, to paramagnetic beads, which are only magnetic in the presence of an external magnetic field. the beads can be added to complex samples such as aerosols, liquids, bodily fluids, or food. after binding of the target to the affinity reagent (which itself is bound to the paramagnetic bead) the bead is captured by application of a magnetic field. unbound or loosely bound material is removed by washing purifing the target from other, unwanted materials in the original sample. a similar approach can purify nucleic acids using a complementary nucleic acid strand attached to a bead. because beads are small and bind high levels of target, when the beads are concentrated by magnetic force, they form bead beds measured in the hundreds of nanoliters (or as low as a single bead), thus concentrating the target at the same time it is purified. the purified and concentrated targets can be conveniently transported, denatured, lysed or analyzed on-bead, or eluted off-bead for further sample preparation and analysis. immunomagnetic separations are commonly used as an upstream purification step before qpcr, electrochemiluminescence, and magnetic force discrimination. as mentioned, paramagnetic beads provide an excellent solution to the macroscale-to-microscale interface: beads are an almost ideal vehicle to purify samples at the macroscale from large volumes and concentrate the specific biomolecules or targets to the nanoscale for introduction into microfluidics devices. immunomagnetic separations are used widely for the detection of microorganisms in food and agriculture. typically, immunomagnetic beads coated with the appropriate antibody are added to material that had been homogenized in a stomacher. the pathogenic strain escherichia coli o157:h7 can be detected directly in ground beef with greater sensitivity than traditional plate enumeration methods by using ims before plating [77] . ims has also been coupled with qpcr [78] , fluorescence microscopy, and solid-phase laser scanning cytometry [79] . key parameters affecting detection sensitivity were shown to include the amount of nanoparticles per assay, immunoreaction incubation time, concentration of the target organism, matrix background, and interferents. generally, interference by non-target microorganisms is less than 1% [80] . ims can also be combined with pcr to effectively increase the specificity of the overall process by combining the specificity of antibody-antigen recognition and the sensitivity of pcr. immuno-quantitative pcr for s. aureus enterotoxin b (seb) detection was found to be 1,000 times more sensitive than enzyme linked immunosorbent assays (elisa), with little cross-reactivity [81] . immunomagnetic separations have been adapted to microfluidic devices. in one application, electromagnetic gradients were generated using cu micro-coil arrays embedded in a silicon substrate with magnetic pillars composed of nicop alloy with an integrated sensing coil to produce tunable, localized magnetic forces that were able to trap up to 80% of applied particles [82] . a miniature, integrated microfluidic device to separate magnetic particles from laminar flows was developed by the whitesides group and demonstrated to be effective in separating live e. coli on magnetic beads [83] . clinical applications development includes capture of targeted t cells from blood in bead beds contained in microfluidic channels. these studies resulted in 20-37% t cell capture, but only allowed flow rates of 3 ul/min [84] . rates of hundreds of microliters per minute are required for most applications that can be achieved by increasing flow rates or by upstream deployment of ims or other technologies that specifically capture and concentrate targets. in addition to performing ims purifications, extensive work has been done on developing and integrating complete immunoassays in microfluidic devices. immunoassays include immunochromatographic lateral flow devices, elisa, ims-electrochemiluminescence, time-resolved fluorescence, and magnetic force assays. in addition to widespread clinical applications, immunoassays are routinely used to detect biosecurity threats [85] [86] [87] . future immunoassays will continue to exploit advances in antibody production and screening, miniaturization, integration, and multiplexing [88] . lateral flow assays are very simple and compatible with portable applications. a drop of test solution in buffer is added to a pad containing antibodies coupled to colloidal gold or other labels. the antibody and antigen, if present, bind and wick down the pad laterally and intercept detection lines that contain capture antibody-gold complexes. the gold aggregates due to the bivalency of the antibodies and produces a line that is visible by eye. home pregnancy tests are probably the best-known lateral flow devices. lateral flow devices are simple to use, require little training or equipment, and have been devised to detect biothreat agents. simple dipstick immunoassays for e. coli o157:h7 can detect 1 cell per g in ground beef, after outgrowth of the sample [89] . a comparison of lateral flow assays in a handheld device with elisas and pcr found the sensitivity of the lateral flow assays were approximately one-hundredth of elisas, which were in turn one-tenth the sensitivity of pcr assays [90] . lateral flow devices are being improved with microfluidic technologies. monolithic beds have been combined with electrophoretic separations to produce a fast (<10 min), sensitive assay for saliva analysis [91] . an elisa detected staphylococcal enterotoxin b (seb) in a handheld assay for food at about 50 pg/g of matrix using lateral flow [92] . an integrated microfluidic device with sample preparation (filtration and mixing) has been described to detect botulinum neurotoxin directly from whole blood [93] . elisa is the most commonly used form of immunoassay. elisas use an antibody bound to a solid phase support, such as a microtiter plate, to capture analytes from liquids. after washing away unbound material, a secondary antibody with a label or coupled to an enzyme (e.g., horseradish peroxidase) is used to produce a visual or fluorescent readout. elisas are relatively inexpensive, scalable to 96-and 384-well technologies, and can be sensitive and specific, depending on the antibody pairs. early work with polyclonal antibodies demonstrated detection of the enterohemorrhagic e. coli o157:h7 in food at about 1 cell/g sensitivity [94] . anti-s. aureus enterotoxin b (seb) antibody has been immobilized on carboxylated polystyrene microparticles and a competitive assay between fitc-labeled seb was developed to detect 0.125 ng/ml of seb in drinking water and 0.5 ng/ml in whole milk [95] . seb has also been assayed by direct labeling of secondary antibody with detection limits of 100 pg/well [96] [97] . immunoassays are being adapted to microfluidic devices. microfluidic molded silicone integrated devices have successfully detected botulinum neurotoxin serotype a with results equivalent to full volume assays [98] . microfluidic immunoassays in plastic devices offer the affordability of plastics, the availability of diverse microfabrication methods, and many well-developed polymer surface modifications [99] . a heterogeneous immunoassay with antigens immobilized on pdms-coated glass microchips with electrokinetic-control for multiple analyte detection had detection limits for e. coli o157:h7 of 3 ug/ml in an automated prototype [100] . a poly(methyl methacrylate) (pmma) microfluidic immunoassay device was modified with poly(ethyleneimine) (pei), an amine-bearing polymer, to increase antibody binding ten-fold [101] ; the authors believe this is due to the spacer effect as well as the addition of amine groups. due to the smaller dimensions, the microchip reactions were ten-fold faster than 96 well plates and had a dynamic range of 5 to 1000 ng/ml. an early study using glass capillary tubes as a solid support to assay e. coli o157:h7 employed a competitive-based immunoassay and achieved a detectable limit of 1 cfu per 10 g of ground beef [102] . capillaries have also been employed to separate complex matrices and detect a model antigen at 10 pm with immunoaffinity chromatography using dual syringe pumps, a silica bead packed bed, and laser-induced fluorescence [103] . beads have been used on microchips to separate binding steps from the secondary detection to reduce background [104] and to mix immunoassays on microchips [105] . microfluidic immunoassays are being adapted for biodefense needs. liu and coworkers [106] developed multi-stage integrated microfluidics for immunoassays utilizing electrochemical detection. micropumps and circuits were integrated to perform parallel immunoassays for model organisms with enzyme-generated signal detected by active cmos circuitry with resulting sensitivity in the fm range [106] . a multi-analyte array biosensor (maab) was developed at the naval research laboratory (nrl) to detect multiple target agents in complex samples using a novel fluidics cube module to control the flow of solutions over six different immunoarray sensors in a small portable device with an evanescent wave detector [107] . the maab could rapidly detect three toxins: ricin, staphylococcal enterotoxin b, and cholera toxin [108] , and s. typhimurium [109] . liquid array-based immunoassays with multiplexed detection have also been developed and tested for model organisms [110] . yang and coworkers at nanogen exploited the unique characteristics of their addressable arrays to develop a device that performed automated electric-fielddriven immunocapture and dna hybridization [111] . immunomagnetic separations have been combined with electrochemiluminescence (ecl) detection [85] . ecl-based assays contain ruthenium labels, which emit light when electrochemically reduced. in ecl detection, tripropylamine can be oxided at the surface of an array of electrodes, and in turn it reduces the ruthenium, which then emits light. background noise is reduced since the reaction activation is localized and controlled by the electrode. several commercial systems are available, and over 50 immunoassays are available on one clinically aimed system. ecl has been further developed with microelectrodes manufactured using screen-printing of carbon inks onto microtiter plates. for biodefense, work in 1995 showed that the ecl detection of biotoxoids and 100 bacillus anthracis spores in less than one hour [112] . an ecl assay was compared with fluorogenic chemiluminescence (fcl) for the detection of biological threat agents [113] . seb at a concentration of 1 pg/ml has been detected in a range of matrices using ecl in a 30 min immunoassay and found to be significantly better than elisa reactions [114] . clostridium botulinum toxins a, b, e, and f were detected at about 100 pg/ml in clinical samples and food using ims ecl detection, about the same sensitivity as elisa, but with much more rapid time to results [115] . ecl immunoassays have also been used to detect ricin at 0.1 ng/ml [116] . in the future, ecl detection may well be integrated with microfluidics to produce fully integrated and sensitive laboratory and portable detection systems for biothreat agents. time resolved fluorescence uses lanthanide chelate labels that have very long fluorescent decay times and large stokes shifts [85] . the secondary antibody can use a lanthanide such as europium that fluoresces in an enhancer solution. the long decay time can lead to a very low background and sensitivity that are an order of magnitude better than traditional eli-sas, but with greater variability [117] . commercial full scale systems are in use to detect biothreat agents including franciscella tularensis, clostridium botulinum toxin, and seb with detection at a range of low pg/ml [118] . immunoassays have been integrated with magnetic detectors to produce microfluidic systems that are being applied to biodefense and other fields. using this approach, the strength of intermolecular interactions can be measured by the force required to disrupt a bond when the target is attached to a magnetic bead [119] [120] . the magnetic bead serves as the label which can be detected by microfabricated magnetoresistive transducers on microchips. multiple analytes can be measured in less than 15 min in an array format with sensitivity close to elisa [121] . model spores and viruses can be detected at about 10 5 cfu/ml and 10 7 pfu/ml, respectively, while seb was detected at 10 ng/ml. multiple samples can be measured simultaneously and magnetic force is tolerant of different types of analytes. multiplexed femtomolar detection of proteins from complex mixtures has been shown in a format that may be adapted for a handheld platform for both nucleic acid hybridization assays and immunoassays [122] . proteomic approaches for biodefense rely on identification of proteins and peptides to evaluate and characterize potential biothreat agents. primary proteomic approaches for biodefense include separation of proteins and peptides by mass spectrometry platforms [123] , two-dimensional gel analysis [124] , and protein arrays. proteomic approaches are being used to build a cyberinfrastructure of niaid-funded centers that are applying these tools for biodefense to develop vaccines and proteomic targets [125] . data from mass spectrometry, yeast two-hybrid (y2h), gene expression profiles, x-ray and nmr for bacillus, brucella, cryptosporidium, salmonella, sars, toxoplasma, vibrio and yersinia, human tissue libraries, and mouse macrophages have been developed [126]. microfluidics is being widely adapted to proteomic systems as upstream devices and nozzle systems for mass spectroscopy, as reviewed in [127] [128] . in essence, microfluidics is compatible with the low flow rates, small sample volumes, and microseparations that are required for mass spectrometry. both microfabricated nozzles and hplc devices are presently commercially available. the various developments are beyond the scope of this review, but are described in chapter 3 of this book. for biodefense applications, protein profiling has been developed at sandia national laboratory into an autonomous microfluidics system combining microfluidics sample preparation modules with microchip gel electrophoresis [129] . this system is fully automated, with a total 10 min sample preparation and detection time. sensitivity for b. subtilis spores was 16 agent-containing particles per liter of air. protein arrays [130] containing either antibodies to different epitopes or to different proteins arrayed on a solid surface, have been applied to characterize and type biothreats. a protein chip for the arraytube platform was developed that uses a microtube-integrated protein chip that accomplishes detection using the classical sandwich assay and horseradish peroxidase colorimetric substrate. [131] . invitrogen has been developing its highdensity protein microarrays (protoarrays™) for detection of plague, smallpox, anthrax, and a number of hemorrhagic diseases, such as ebola and dengue fever. phage display, where the probes on the array are selected from billions of clones, is a potentially powerful application of protein arrays and may be adopted for biodefense [132] , as are peptide arrays. for nucleic acids following sample preparation, amplification technologies can be applied that greatly increase the signal. the best known and most used dna amplification method is pcr [133] . pcr uses thermal cycling to exponentially amplify dna using a thermally stable dna polymerase. each cycle of amplification doubles the amount of template, thereby exponentially increasing the amount of target, and can amplify from as little as a single copy of dna. the specificity of the amplification is determined by the pair of primers that initiate the amplification. pcr has become a standard clinical and research technique for nucleic acid testing (reviewed in [134] [135] [136] ) and for biodefense [137] . many different variations of the basic pcr reaction have been developed, including qpcr, nested pcr, multiplexed pcr, and single nucleotide polymorphism pcr. qpcr has revolutionized the detection of specific dna sequences in the laboratory, clinic, and field (reviewed in [138] [139] ). qpcr quantifies the amount of original target sequence using a fluorescently-labeled probe that acts as a reporter and detection system. in one version, qpcr employs a fluorescently-labeled probe that contains a quencher that suppresses the fluorescent signal. during the replication process, the signal becomes un-quenched, emitting light. as the amplification progresses, the fluorescent signal increases. the presence and amount of target dna in the sample can be determined from the cycle number when the signal increases over a threshold value, commonly called the cycle threshold (c t ). multiplexed reactions make it possible to detect multiple agents in each reaction vessel. the amount of dna produced can also be measured non-specifically using dna intercalating dyes and other (specific) probes such as molecular beacons. qpcr has specificity and sensitivity equivalent to pcr, while simultaneously amplifying, detecting, and quantifying the original dna target in a single, contained reaction. pcr and qpcr are the most widely used methods to detect and identify biowarfare agents by identifying target dna sequences in the laboratory and field [140] . multiplexed pcr assays for virulence factors on two plasmids, pxo1 and pxo2, in b. anthracis were able to distinguish it from closely related strains [141] . highly specific assays can identify b. anthracis using pxo1, pxo2, protective antigen (paga), and capsular protein b (capb) [142] are commercially available for clinical samples [143] [144] . b. anthracis has been detected from soil samples at 10 spores per ml [145] , in aerosols [146] , and in food [147] . qpcr detection assays have been multiplexed to detect four biothreat agents, y. pestis, f. tularensis, b. anthracis and b. mallei, simultaneously using molecular beacons complementary to conserved 16s rrna targets [148] , and with minor groove binding probes with a sensitivity of 1 fg of target with no cross reactivity [149] . melting point curves combined with the multiplex amplification helped distinguish b. anthracis from y. pestis and leishmania [150] , and between members of the b. cereus group [151] . the performance of three commonly used qpcr instruments was compared and generally comparable limits of detection, sensitivity, and specificity were found [152] . the genexpert system (cepheid) is a standard instrument for semi-automated sample preparation and qpcr (using the smartcycler as the qpcr platform). its performance has been evaluated and the incorporation of the nucleic acid purification component of the sample preparation has been credited with its 1,000-fold improved detection over the smartcycler alone for b. anthracis sterne spores due to removal of inhibitors and concentration of the sample [153] . miniaturized pcr has been an area of intense development with devices developed that use microchips and capillaries to perform thermal cycling in stationary formats and continuous flow regimes. the advantages of miniaturized pcr devices are more rapid assay times, low reagent consumption, and potential integration with upstream sample preparation modules. miniaturization of pcr, including discussion on the types of common designs, issues with surface chemistries that can inhibit pcr, coating procedures, and heating strategies has been reviewed [154] [155] . miniaturized pcr was initially performed in glass capillary tubing. capillaries are off-the-shelf items that are suitable for sub-microliter reactions. wittver first demonstrated (in 1989) pcr amplification of dna in sealed capillary tubes using a hot air cycler [156] . this basic design became the rapid cycler (idaho technologies), the forerunner to razor, in use today for biodefense applications. sample volumes were reduced to 1-10 ul with cycling times of less than 15 min [157] . a medium-throughput automated capillary sample preparation system that processed 1,000 one ul samples per day was developed [158] ; the acapella-1k (u. washington, seattle) utilized a mechanical capillary handling system, air cycling, and a piezoelectric reagent dispenser [159] . jovanovich and co-workers developed a 500 nl sample preparation system also using an air cycler with a 384-channel capillary cassette (fig. 10.2) . this system used 150 um id capillaries without a coating and standard pcr conditions (except for elevated mg +2 concentrations). dna samples were forced onto the surface of the glass capillary using chaotrophic agents, the dna-coated capillary was then evacuated, and 500 nl of pcr reagents re-filled the tube by capillary action. pcr reaction efficiencies were equivalent to full volume reactions. capillaries have many applications in microfluidics but are currently more difficult to integrate with other functions than microchips. the integration requires robust microfluidic connectors. the first miniaturized pcr reactions in a microchip were performed in an etched silicon wafer by northrup and coworkers, reported in 1993 [160] . wilding et al. reported successful cycling in 5 and 10 ul silicon reactors cycled by an external peltier device in 1994 [161] . pcr reactions have been performed in 87 nl in silicon devices [162] , which was expanded on by belgrader et al. by addition of capillary electrophoresis on microchip [163] . belgrader and coworkers further reported pcr amplification in seven minutes [164] . the first miniaturized thermal cycler with real time detection was from northrup and colleagues at llnl and used silicon based reaction chambers with diode detectors and integrated heaters [165] , this same group devised a portable battery-powered unit [166] . many other reports of qpcr devices have followed [167] [168] [169] . some of the challenges of microfluidic qpcr are control of liquid positioning, bubble formation, sealing chambers using microfluidic valves, and surface interactions. liquids and targets can be manipulated by precise positioning by micropumps, by capture onto a surface, or by exploiting beads and solid phase chemistries. microvalves can restrict the liquid solutions to proper chambers. one group has simultaneously sealed the reaction chamber with valves that serve as the macro-to-micro interface [170] . for poly(cyclic olefin) plastic fabrication, gel valves were used to confine amplicons before integrated on-chip gel electrophoresis [171] . bubble formation has been found to be primarily due to surface wetting properties of the chamber [172] . surface interactions can be minimized by improving the surface chemistry or by increasing channel dimensions to over 125 microns. a challenge for microfluidics is improving heating and cooling rates while maintaining good uniformity. one approach has been to microfabricate resistive heaters and sensors directly onto microchips. this has been used to achieve 20 o c/s heating and 10 o c/s cooling rates to detect upper respiratory tract infection microorganisms in 15 min [173] . a second approach, pioneered by the landers group, used infrared heating to rapidly heat water in fluidic channels [174] [175] ; cycle times as low as 17s were achieved with successful pcr amplification and cycle sequencing. ir driven pcr was integrated on a microchip with upstream solid-phase extraction of dna using silica beads in sol gel to isolate dna from an anthrax spore-spiked nasal swab; amplified target dna sequences were detected on-chip with a total processing time of 25 min [176] . a third approach is flowthrough devices with fixed-temperature zones. control of temperature and uniformity is simplified, and power consumption is low since the zones stay at a constant temperature. an added advantage is that cycling times can be reduced due to the elimination of temperature ramping. the challenge to this approach is to reduce surface interactions which can degrade pcr reaction performance. this can be accomplished using surface coatings, by controlling surface to volume ratios, or by using emulsions. soper's group has built flowthrough devices combining flowthrough pcr in a polycarbonate chip and a pmma chip using detection with a ligase assay [177] . reverse transcription and pcr have been integrated in a flow through glass microchip with 55 um channels integrating different temperature zones; 30 cycles of pcr amplification were achieved in 6 min [178] . a ferrofluidic actuator has also been used to move a pcr sample plug rapidly between temperature zones [179] . a hybrid chip with silicon and pmma has performed high speed pcr with three heated zones [180] . pcr amplification in small volumes has also been accomplished in microarray formats. the target dna is spotted onto a glass slide that has covalently attached primers, enabling amplification of bacterial target dna [181] . this approach has been applied to identify bacteria using amplification of rdna sequences [182] . the solexa dna sequencer uses a similar process to amplify clusters of pcr products from a single template molecule on a surface. one of the first miniaturized real-time pcr instruments was developed for biodefense applications. the advanced nucleic acid analyzer (anaa), devised at llnl, used an array of 10 silicon reaction chambers with thinfilm resistive heaters and solid-state optics to rapidly test samples for simulants of biothreat agents with detection limits of 100 to 1,000/ml [183] [184] . a compact version of a qpcr instrument with a notebook computer, two reaction modules with integrated four-color fluorescence detection was developed and shown to detect bacillus spores [166] . a handheld device, the handheld advanced nucleic acid analyzer (hanaa), also developed at llnl, used plastic reaction tubes with silicon and platinum-based thermal cycler units and two light emitting diodes to detect bacteria including b. anthracis ames [185] . the llnl group, working with sandia, went on to develop the apds, which performs continuous monitoring with a multiplexed immunoassay trigger and confirmatory qpcr assays. this has been demonstrated for aerosolized b. anthracis, yersinia pestis, bacillus globigii, and botulinum toxin [186] . llnl and sandia also developed a "biobriefcase" device with a smaller footprint that has multiplexed, autonomous detection with immunoassays, toxin assays, and qpcr. the biobriefcase incorporated inhibitor removal and concentrated the sample by mixing aerosol-collected liquid with a chaotrophic agent prior to silica bead bed purification [187] . the llnl group has also developed a 10-plex pcr amplification with hybridization to beads upstream of a flow cytometry readout; 1000 samples were processed in 8 hours [188] . the lightcycler (idaho technology) has been adapted into a "ruggedized" advanced pathogen identification device, rapid, which has been used for field analysis of bioagents and pathogens [189] . y. pestis was detected at about 20 genome equivalents in 75 min using this system. the latest innovation from llnl is amplification in emulsion of single copy dna in a lab-on-a-chip format; the system produces 10 pl droplets that can perform qpcr with thresholds exceeded significantly earlier than conventional instruments [190] . in addition to detecting dna targets, biodefense needs require that rna viruses be detected. detection of two rna-based viruses-dengue virus and enterovirus 71 has been demonstrated with a pdms microchip that integrates reverse transcriptase and pcr amplification [191] . parallel reverse transcriptase-pcr assays have been performed in 450 pl and shown to detect as little as 34 copies [192] . a fully integrated handheld device using isothermal nucleic acid sequence-based amplification (nasba) [193] , which specifically amplifies rna from primers, was built and shown to have comparable results to laboratory instruments. in addition to pcr there are a number of other nucleic acid amplification technologies that are being adapted for biodefense: strand displacement amplification (sda), loop-mediated isothermal amplification (lamp), exponential amplification reaction (expar), and rolling circle amplification (rca). sda uses the primer-directed nicking activity of a restriction enzyme and the strand displacement activity of an exonuclease-deficient polymerase to amplify dna [194] . sda can achieve 10 8 to 10 10 amplification in about 15 min [195] . fluorescence resonance energy transfer (fret) probes can be incorporated and real time instruments for the clinical laboratory are in commercial use [196] [197] . expar is a dna amplification reaction that produces short (8-22 nucleotides) in a linear or exponential amplification in isothermal homogeneous assays [198] . expar can be extremely rapid with amplification of greater than 10 6 . both real time [199] and end point formats have been developed. expar has been applied to biodefense applications as part of the band project and in other projects as a quick, specific amplification technique. lamp is an isothermal dna amplification method that relies on autocycling strand-displacement dna synthesis. four sets of primers are used with the thermostable bacillus stearothermophilus (bst) dna polymerase that has high strand displacement activity [200] . the reaction produces a white precipitate, magnesium pyrophosphate, which can be easily detected as an indicator of a positive amplification reaction [201] . the specificity and sensitivity are competitive with pcr, and in some cases more sensitive than nested pcr [202] . rca is an isothermal method that uses phi29 dna polymerase to amplify circular dna [203] . phi29 dna polymerase is a single subunit with excellent processivity and can amplify >10 7 fold [204] . rca is widely used for whole genome amplification, for scarce material, and can nonspecifically amplify trace amounts of dna. rca is routinely used at genome centers preparing template for sample preparation for dna sequencing [205] . rca has also been used for cell free cloning of genomic dna that might be lethal to cells [206] . rca is a powerful tool for forensic and biodefense applications. dna microarrays have many potential applications in biodefense. dna microarray technology is a widely used powerful technique that uses large arrays of microspots of dna on a solid support or beads to detect complementary dna or rna products from a sample. (protein arrays were briefly discussed in the proteomics section). for rna samples, amplification of rna is commonly done by first performing reverse transcriptase to create dna from rna, and then the resulting dna can be amplified using standard dna amplification methods such as pcr, or whole genome amplification, or by in vitro transcription followed by another cycle of re-verse transcriptase. fluorescent labeling can be introduced into the sample at various steps in the process. typically, tens of thousands of spots, each containing a unique sequence, are interrogated in a single experiment with fluorescent detection. the data can represent a fingerprint of the transcriptional state of an organism (e.g. biothreat agent, or of the response of a human potentially infected with the agent), identify dna sequences present in an organism, or resequence organisms [207] [208] . the strength of the microarray platform is the depth of characterization. the 10,000s of analytes measured on a single microarray slide can generate massive amounts of data. in the future, dna microarrays may be displaced or challenged by digital gene expression methods using next-generation dna sequencing to produce 100,000's or more sequences from a sample or single mrna detection and enumeration strategies (e.g., nanostring). microarrays have been combined with pcr amplification to identify and genetically discriminate b. anthracis from closely related bacterial species from the b. cereus group and determine if the strains harbor plasmids [209] . dna microarrays can potentially detect multiple pathogens in a single sample. the fda has developed a microarray, fda-1, to screen for several food pathogens and virulence factors, including seb [210] . saic developed its phase i band project based upon a fully automated system that would collect air samples and then analyze them for pathogens using microarrays. after collection and filtration, reverse transcriptase and pcr amplification were used before hybridization to a dna microarray in a cartridge for ten min. the data was then analyzed for fingerprints that indicated the presence of threat agents. microarray sample preparation is a complicated, multistep process that is dependent on the variability of individual operators. microfluidics, with its potential to automate and integrate processes, has been applied to simplify and standardize sample preparation (reviewed in [211] ). this seminal work was performed by anderson and colleagues at affymetrix, where a miniaturized integrated microdevice was developed to prepare samples by accessing 10 reagents and performing 60 automated operations before performing hybridizations to a microarray. anderson and coworkers elegantly demonstrated a plastic miniaturized sample preparation system for microarray sample preparation and analysis by hybridization, and showed the detection of mutations in the hiv genome from serum samples [212] . the advantages of microfluidics for microarray biodefense applications are in-tegration of sample preparation steps, potential reductions in reagent cost, ease of use, and decreased hybridization times. the soper group has integrated microarrays with microfluidic technology in plastics. a microarray was fabricated in pmma using uv exposure of the polymer surface, coupling of amine-terminated oligonucleotide probes to the surface, and washing the surface [213] . the hybridization and allelespecific ligase detection reactions were performed in a polycarbonate flowthrough biochip. the system could screen for mutations in 20 min. work by liu and coworkers at motorola and then combimatrix has developed biochips that integrate sample preparation, pcr, and microarray detection [214] . electrochemical pumps were used with paraffin-based single-use microvalves to regulate flow. detection of pathogens from whole blood [214] , identification of influenza virus [215] , and gene expression from a cell line [216] were shown. a microelectronic array system was developed by nanogen for microarray testing [217] . this system employs dielectrophoresis as a sample preparation method and the hybridization of nucleic acid to probes attached to electrodes is accelerated by application of an electrical potential. the electrodes are covered with a hydrogel. detection is performed with fluorescence probes [218] . electrophoresis is a powerful separation technology with many biodefense applications. the advent of capillary electrophoresis (ce) and the multichannel capillary array electrophoresis (cae) propelled the applications of electrophoresis by increasing separation speeds, automating the analysis, and improving data reliability. most notably, cae technology was partially responsible for the early completion of the human genome project and has become the separation method of choice for most nucleic acid applications. several pcr-based methods for detailed laboratory characterization of microorganisms have been developed that rely on electrophoretic separation. in this section, we first review several of the separation based typing methods for bacteria identification and discrimination, describe the application of microfluidics on microchips to microelectrophoresis, and discuss applications of dna sequencing in a microfluidic platform to biodefense [219] . vntr loci analysis [220] is a powerful laboratory tool for identifying bacteria, humans, and other organisms. analogous to microsatellite genotyping, vntr pcr amplification detects regions where genetic drift has created variable numbers of tandem repeats inserted into the genome, plasmids, or other extra-chromosomal elements. vntr has been used to identify y. pestis [221] , mycobacterium tuberculosis [222] , and numerous other organisms. mlva [223] extend vntr to assay multiple alleles and provide a fingerprint, analogous to forensics identification by microsatellite dna analysis. keim and co-workers have identified a set of eight vntr regions that are diagnostic for b. anthracis and characterized 426 b. anthracis isolates into 89 distinct genotypes [223] . mlva was used to subtype the anthrax strains from the bioterrorist attack in 2001 within eight hours of receiving isolates [224] . mlva has been applied to type closely related bacillus strains but at times required additional information for confirmatory determination of b. anthracis [225] . mlva types of bacillus anthracis could be further differentiated by single-nucleotide repeats [226] . aflp is a pcr-based method that can fingerprint microbes [227] , type organisms, and identify phyllogenetic relationships. aflp is rapid-it employs a relatively simple multi-step workflow with standard reagents regardless of the organism typed. aflp has been applied to differentiate a wide variety of microorganisms at the subspecies level [228] , including b. anthracis [229] and is widely used to classify plants, yeasts, and other organisms. the aflp process begins with a two-enzyme restriction digest followed by ligation of fluorescently labeled restriction half-site adapters to reconstruct the restriction site at the end of the fragments and serve as pcr primers. two or three degenerate nucleotides on the end of the adapter reduce the complexity of the amplified products during the high stringency pcr amplification. by selection of proper restriction enzyme pairs, fluorescently labeled fragments in the range of 100 to 1,000 bases can be produced, and then are separated and detected by capillary electrophoresis. the resultant pattern is a fingerprint of the test organisms' genomic and extra-chromosomal restriction patterns. a post-labeling fluorescent method using dye-terminator chemistry can visualize both rflp and aflp products [230] . cdna-aflp can also be performed to visualize the gene expression pattern of an organism [231] . subtle strain-to-strain variations can be characterized by single nucleotide polymorphisms (snps). snp typing of intragenic spacers in the 16s-23s region has been shown to differentiate closely related bacillus strains including b. cereus from b. anthracis [232] . the identification of the specific strain of a biothreat organism can help microbial forensics trace the origin and determine whether multiple incidents were caused by release of identical organisms and therefore share a common origin. to determine strain variations, the genomic sequence can be determined by dna sequencing. following the september 11th attacks, the cdc sequenced the 16s rrna gene to definitely identify b. anthracis from culture-negative clinical specimens of patients with confirmed anthrax [233] . in other studies, 183 16s rrna and 74 23s rrna sequences for all species in the b. cereus group showed disagreement with phenotyping clustering, but by utilizing rrna together with gyrb sequences these workers could discriminate between groups [234] . ruppitsch and coworkers showed for a wider variety of strains that sequencing of the 16s rrna gene is not always sufficient, but that additional sequencing of intragenic sequences can increase resolution and thus differentiate bioagents [235] . cae is based upon separation in a capillary, itself a type of microdevice. in this review, we use microelectrophoresis to connote separations on microchips. the interested reader is referred to reviews [236] [237] [238] for details of chip construction, coatings, operation, and equipment. most commonly, glass microchips are employed but plastic devices have also been developed [239] . microchips have the potential to simultaneously separate hundreds of samples in minutes. microchips typically consume only picoliters of samples and thus can be well matched to microscale sample preparation volumes. fluorescently labeled amino acids [240] , dna restriction fragments [241] [242] [243] , pcr products, short oligonucleotides [244] , and sequencing ladders [245] have been separated by microchip capillary electrophoresis [246] . the analyses are extremely rapid, from less than a minute for oligonucleotides to less than 20 minutes for dna sequencing [247] . we note that next generation microfluidic sequencing-by-synthesis [248] , microfluidic pyrosequencing [249] , sequencing by ligation, and nanopores may, in the future, have biodefense applications for detecting genetically modified organisms and digital gene expression. interfacing upstream microfluidic sample preparation with microscale separations is challenging. the classic twin-t injector defines roughly a 100 to 500 picoliter volume in a short sample plug (100 to 500 um) in the separation channel with electrokinetic loading [250] . this is only a small fraction of the sample, even if the prepared sample is only 100 nl: for biodefense applications this means losing orders of magnitude of potential sensitivity during the analysis step. this can be ameliorated with isotachaphoresis, field amplified stacking, dna binding, and other methods that locally increase the sample concentration. isotachaphoresis concentrates samples between leading and trailing electrolytes and can increase detection limits by 50-fold or more for dna [251] [252] , and stack up to a million-fold [253] . field amplified sample stacking, routinely used in capillary electrophoresis, has been adapted to microchip electrophoresis using pressure driven flows to move low osmotic strength samples into position for stacking on the column. concentration effects range from 65-fold increase in signals [254] to 180-fold for model compounds [255] . separations on microchips are most developed for dna separations. in 1998, 96 hemochromatosis samples were genotyped in less than 8 min by microelectrophoresis on a microchip [256] . locus-specific, multiplex pcr products specific for deletions causing duchene/becker muscular dystrophy have been separated on a silicon-glass microchip, and t-cell receptorgamma genes and immunoglobulin heavy chain gene on glass microchips [257] . fluorescently-labeled cttv pcr samples and short tandem repeats have been analyzed on single-channel ce microchips [258] . the mathies laboratory genotyped 384 hemochromatosis samples in less than 6 min, with detection by a four-color rotary confocal fluorescence scanner [259] . dna sequencing on microchips was first performed in 1995 in the mathies' lab [260] ; fluorescently labeled dna sequencing fragment ladders were separated on glass microchips with a denaturing polyacrylamide sieving matrix and readlengths of about 200 bases obtained in 15 min. mathies' lab later reported readlengths of about 500 bases in about 30 min in single channel [261] . jovanovich's group reported the first multichannel dna sequencing in an array of 16 microchannels with readlengths of 450 bases in 15 min in an automated instrument [247] . high throughput dna sequencing in 96 channels obtained readlengths of 430 high quality bases [262] . to achieve longer readlengths, microchips with 40 to 50 cm separation channels were constructed and readlengths of 600 to 800 bases reported with plates containing up to 384 channels [263] . separations on plastic microchips have also been achieved. one of the first microelectrophoresis devices, in 1993, used a twin t injector on a pdms substrate to separate proteins and dna [289] . plastic substrates offer the advantage of potential low cost fabrication once high production volumes are achieved, but have been plagued by higher background fluorescence signals than glass or quartz devices and require specialized surface coatings [264] . while a detailed review is outside the scope of this article, injection molded microelectrophoresis devices have achieved separation of dna fragments [265] [266] and dna sequencing [267] . hot embossed pmma with ir detection was shown to sequence dna out to 450 bases in linear polyacrylamide [268] . for biodefense, the breakdown products of g-type and v-type nerve agents can be assayed by ce and microelectrophoresis [269] . wang et al. [270] combined microchip electrophoresis with derivatization and electrochemical detection of thiol-containing degradation of v-type nerve agents at micromolar concentrations in less than four min. a microchemlab portable device, developed at sandia national laboratory, with a fused silica microfluidic separation chip, a miniature lif detector, and high voltage power supplies [271] can assay proteins and toxins at nanomolar concentrations [272] . the achievement of working devices for both microfluidic based sample preparation and analysis has created the possibility for complete system integration to meet biodefense needs. for robust biodefense applications, sample preparation and analysis must be integrated either directly in a monolithic format or by microfluidic connections. as described above, for dna microarrays sample preparation has been integrated with analysis, as first shown by anderson, and brought to commercial product by combimatrix. today, one of the most advanced areas for complete system integration has been the integration of upstream sample preparation reaction steps with microelectrophoresis on microchips. microchip and capillary-based analysis systems have the advantages of high-resolution separations with extremely fast separation times, automation, and nanoliter-scale consumption of reagents. integration of pcr reactions in microfabricated devices with microelectrophoresis was first demonstrated in 1996 in collaboration be-tween northrup's llnl group and the mathies laboratory; pcr amplification was performed in a silicon device mounted on a glass separation device and complete analysis was achieved in 20 min for a cloned human gene and 45 min for a bacterial genomic target [273] . early work exploited the small volumes in capillaries to prepare samples and analyze them by capillary electrophoresis. swerdlow and colleagues developed an automated prototype with a sample loop in an air cycler coupled to a separation capillary [274] . a fully integrated miniaturized integrated microsystem using capillaries was shown to perform all steps including pcr amplification in 125 nl and separations starting from buccal cells [275] . integration of pcr reactions in capillaries with separations in capillaries has been shown to be effective for dna typing from blood [276] and other materials for human and viral targets [277] . pcr reactions and dna cycle sequencing in capillaries and capillary separations have been fully integrated to create a microvolume system with readlengths of 257 bases in 4 hrs from human dna [278] . pcr reaction chambers have now been monolithically integrated with microelectrophoresis to create fully integrated microsystems that perform pcr on the same device as the separation. burns et al. integrated sample preparation, gel electrophoresis, and on-chip detection for low resolution separation of dna fragments [279] . the mathies lab showed pcr amplification and microelectrophoresis separation to determine sex of humans from dna in 15 min [280] . a portable system with 200 nl pcr reactors, solid state lasers, pneumatically actuated on-chip micropumps, and microelectrophoresis was able to detect 2-3 e. coli or s. aureus cells in less than 10 min including determining drug resistance [281] . the mathies lab has extended their dna sequencing on microchips upstream to include integrated nanovolume sample preparation and purification. first, nanoscale pcr reactions were combined with capillary electrophoresis analysis [282] . then, sample preparation, cleanup, and dna separations were combined to integrate dna sequencing, as shown in fig. 10 .3, [283] . a 250 nl cycle sequencing reaction was performed on microchip, and then moved by micropumps onto an acrylamide gel capture matrix with an oligonucleotide hybridization probe to capture the target dna in a 60 nl capture chamber. the affinity capture removes template dna, desalts, and pre-concentrates the sample for microchip electrophoresis for higher injection efficiencies. the sample is then electrophoresed into an injector and separated on microchip. readlengths up to 556 bases were obtained from 1 fmol of template. the key to the integration was pneumatically actuated microvalves and micropumps [288] . the landers laboratory has completely integrated pcr, sample cleanup, and capillary electrophoresis on microchips, as shown in fig. 10 .4. a device with pcr reactions, ir mediated heating, pneumatically actuated onchip micropumps, and microelectrophoresis has achieved amplification and good separations in less than 12 min [284] . they have detected of b. anthracis from whole blood of asymptomatic mice and bordetella pertussis from nasal aspirate of a patient using on-chip nucleic acid purification with a 550 nl pcr reactor coupled to microelectrophoresis analysis with control by pneumatic microvalves [285] . fully integrated 'industrial strength' microfluidics is being applied to homeland security and biodefense in the band program by several groups including microfluidic systems inc, iquum, and us genomics. iquum (www.iquum.com) is developing a liat™ "detect-to-treat" system. the system has a disposable cassette containing reagents and equipment that contains air sampling, sample preparation, and real time detection using pcr in a "lab-in-a-tube" [286] . the sample moves through segmented tube sections containing the reagents using peristaltic pumps and moves back and forth between temperature zones to amplify the dna. us genomics (www.usgenomics.com) has taken a very different approach. single stranded dna is prepared from aerosol samples and a set of fluorescently labeled probes added. the probes bind to their homologous targets, if present. the labeled dna is then moved through a narrow channel one molecule at a time where it is interrogated by laser-induced fluorescence. the resulting pattern of binding sites is a fingerprint that can identify known and unknown agents from a database [287] . one advantage of the approach is the analysis of individual molecules is done without apriori knowledge of the sequence. this potentially enables detection of genetically engineered or previously unknown pathogens. in the future, next generation dna sequencers, mass spectroscopy, and dna microarrays will provide additional solutions to begin to address genetically engineered organisms. microfluidics will become an increasing important element of the future biodefense portfolio. as shown in this and other chapters in this volume, microfluidic components are available in many different shapes and formats: proof-of-concept of just about any imaginable type of sample extraction, lysis, pre-separations, sample preparation, assay, separation, and detection component has been demonstrated in research settings, at universities, research facilities, national laboratories, and in industry. the missing component is the integration of the many parts into complete working systems that are robust, manufacturable, and maintainable. complete integration of complex workflows has proved elusive in full volume devices and in microfluidics. to fully integrate a microfluidic system for biodefense requires taking a sample of several hundred microliters or larger and processing it completely to yield an answer. the chemical and biochemical workflow usually has many steps that all have to be developed, tested, and integrated. in addition, of course, surface chemistries, temperature, and other variables must be controlled. the work described in this review has shown the promise for microfluidics for many types of genomic and proteomic devices. fully microfluidic integrated devices that can input samples and perform sample preparation and analysis are just beginning to appear, as are field portable microfluidic genetic analyzers. while there are some examples of success at the research and prototype levels, there are only a few fully integrated solutions and fewer commercial successes. two main microfluidic elements are needed for full integration. the first element is an appropriate level of control of the microfluidic volumes. this typically requires microfluidic valves, a pumping mechanism, and routers to move, mix, and split, and aliquot liquids. single use valves enable many applications in a disposable cartridge, while programmable valves that enable multiple uses are required for environmental monitoring and reusable applictions. pneumatically driven programmable microvalves are only now becoming more mature and are in use in academia and industry to control fluid flows and mixing. these microvalves [288] have proved invaluable in the integration by the mathies and landers groups, and in industry to integrate processes onto monolithic microdevices. the second enabling element will be to connect different microfluidic devices together using standard connections. in microelectronics, usb, firewire, and other standard connectors allow devices to interconnect by defining the interface and the protocols. microfluidics now needs to establish standards for connections that will enable 'best in class' microfluidic devices to work together in a 'plug and play' manner. a symposium of industry and academia with government representation, including dod, dhs, and nist, to discuss achievable initial standards would be invaluable and may serve to begin the path towards interconnectivity for microfluidics for biodefense and other applications. in this regard, the physical dimension of spacing for connectors needs to be standardized, and standard protocols for transfer of materials developed. the future for microfluidics will be bright as individual steps are optimized and integrated. future microfluidic systems will connect directly to 'real world' samples and fully integrate upstream sample concentration and analysis in a single autonomous device. the full integration of micro-fluidic processes will enable man-portable and then hand-held biodefense devices. eventually, if biothreats become pervasive, microfluidic home and business security devices akin to smoke detectors may provide the massive sampling capability needed to detect to warn the public. the public and the biodefense community await the transformation that complete microfluidic integration of biodefense detection can bring to increase the biosecurity of the world. fear of terrorism in new york after the september 11 terrorist attacks: implications for emergency mental health and preparedness bioterrorism-a new challenge for public health laboratory response to anthrax bioterrorism chemical or biological terrorist attacks: an analysis of the preparedness of hospitals for managing victims affected by chemical or biological weapons of mass destruction hospital infectious disease emergency preparedness: a survey of infection control professionals physicians' preparedness for bioterrorism and other public health priorities the knowledge report to congressional requesters, biological weapons: effort to reduce former soviet threat offers benefits, poses new risks the looming threat of bioterrorism biological threat characterization research: a critical component of national biodefense multiplex diagnostic platforms for detection of biothreat agents bioterrorism: implications for the clinical microbiologist medical countermeasures to wmds: defense research for civilian and military use project bioshield: what it is, why it is needed, and its accomplishments so far comparative genomics tools applied to bioterrorism defense a two-component direct fluorescent-antibody assay for rapid identification of bacillus anthracis report to congressional requesters: bioterrorism information technology strategy could strengthen federal agencies' abilities to respond to public health emergencies apds: the autonomous pathogen detection system a minisonicator to rapidly disrupt bacterial spores for dna analysis lysing bacterial spores by sonication through a flexible interface in a microfluidic system a microfluidic cartridge to prepare spores for pcr analysis microfluidic sonicator for q-disruption of eukaryotic cells and bacterial spores for dna analysis laser induced disruption of bacterial spores on a microchip microchip-based one step dna extraction and qpcr in one chamber for rapid pathogen identification the fundamental aspects and applications of agilent hplc-chip monolithic continuous beds as a new generation of stationary phase for chromatographic and electro-driven separations recent development of monolithic stationary phases with emphasis on microscale chromatographic separation a plastic microchip for nucleic acid purification fabrication of amino silane-coated microchip for dna extraction from whole blood microfluidic chip for high efficiency dna extraction continuous flow microfluidic device for cell separation, cell lysis and dna purification advances in sol-gel based columns for capillary electrochromatography: sol-gel open-tubular columns less common applications of monoliths: preconcentration and solid-phase extraction microchip-based purification of dna from biological samples evaluation of silica resins for direct and efficient extraction of dna from complex biological matrices in a miniaturized format dna extraction using a tetramethyl orthosilicate-grafted photopolymerized monolithic solid phase microchip-based macroporous silica sol-gel monolith for efficient isolation of dna from clinical samples microfluidic-based dna purification in a two-stage, dual-phase microchip containing a reversed-phase and a photopolymerized monolith microfluidic chip-based protein capture from human whole blood using octadecyl (c18) silica beads for nucleic acid analysis from large volume samples microfluidic system integration in sample preparation chip-sets -a summary microfluidic immunoaffinity separations for bioanalysis bacterial separation and concentration from complex sample matrices: a review detection and recovery rates achieved using direct plate and enrichment/immunomagnetic separation methods for escherichia coli o157:h7 in minced beef and on bovine hide rapid detection of escherichia coli o157:h7 by immunomagnetic separation and qpcr sensitive detection of escherichia coli o157:h7 in food and water by immunomagnetic separation and solid-phase laser cytometry magnetic nanoparticle-antibody conjugates for the separation of escherichia coli o157:h7 in ground beef immunoquantitative qpcr for detection and quantification of staphylococcus aureus enterotoxin b in foods magnetic-based microfluidic platform for biomolecular separation combined microfluidic-micromagnetic separation of living cells in continuous flow immunomagnetic t cell capture from blood for pcr analysis using microfluidic systems immunological methods for detection and identification of infectious disease and biological warfare agents immunoassay of infectious agents heightened sense for sensing: recent advances in pathogen immunoassay sensing platforms microfluidic immunosensor systems dipstick immunoassay to detect enterohemorrhagic escherichia coli o157:h7 in retail ground beef detection of francisella tularensis in biological specimens using a capture enzyme-linked immunosorbent assay, an immunochromatographic handheld assay, and a pcr microfluidic immunoassays as rapid saliva-based clinical diagnostics detection of staphylococcal enterotoxin b (seb) by enzyme-linked immunosorbent assay and by a rapid hand-held assay microfluidic tectonics platform: a colorimetric, disposable botulinum toxin enzyme-linked immunosorbent assay system rapid procedure for detecting enterohemorrhagic escherichia coli o157:h7 in food development of a fluorescent latex microparticle immunoassay for the detection of staphylococcal enterotoxin b (seb) a simple and rapid fluorescence-based immunoassay for the detection of staphylococcal enterotoxin b a rapid and sensitive magnetic bead-based immunoassay for the detection of staphylococcal enterotoxin b for high-through put screening integrated bioassays in microfluidic devices: botulinum toxin assays bead-based microfluidic immunoassays: the next generation an electrokineticallycontrolled immunoassay for simultaneous detection of multiple microbial antigens surface modification for enhancing antibody binding on polymer-based microfluidic device for enzyme-linked immunosorbent assay a solid phase fluorescent capillary immunoassay for the detection of escherichia coli o157:h7 in ground beef and apple cider a capillary-based microfluidic instrument suitable for immunoaffinity chromatography microfluidic elisa on non-passivated pdms chip using magnetic bead transfer inside dual networks of channels enzymatically-generated fluorescent detection in micro-channels with internal magnetic mixing for the development of parallel microfluidic elisa integrated microfluidic biochips for immunoassay and dna bioassays a portable array biosensor for detecting multiple analytes in complex samples array biosensor for detection of toxins detection of salmonella enterica serovar typhimurium by using a rapid, array-based immunosensor multiplexed liquid arrays for simultaneous detection of simulants of biological warfare agents an integrated, stacked microlaboratory for biological agent detection with dna and immunoassays sensitive detection of biotoxoids and bacterial spores using an immunomagnetic electrochemiluminescence sensor detection of biological threat agents by immunomagnetic microsphere-based solid phase fluorogenic-and electro-chemiluminescence rapid and sensitive immunomagnetic-electrochemiluminescent detection of staphyloccocal enterotoxin b rapid detection of clostridium botulinum toxins a, b, e, and f in clinical samples, selected food matrices, and buffer using paramagnetic bead-based electrochemiluminescence detection an activity-dependent assay for ricin and related rna n-glycosidases based on electrochemiluminescence comparison of dissociation-enhanced lanthanide fluorescent immunoassays to enzyme-linked immunosorbent assays for detection of staphylococcal enterotoxin b, yersinia pestis-specific f1 antigen, and venezuelan equine encephalitis virus rapid and sensitive detection of biological warfare agents using time-resolved fluorescence assays a biosensor based on magnetoresistance technology giant magnetoresistive sensors and superparamagnetic nanoparticles: a chip-scale detection strategy for immunosorbent assays array biosensor for simultaneous identification of bacterial, viral, and protein analytes rapid, femtomolar bioassays in complex matrices combining microfluidics and magnetoelectronics proteomics for biodefense applications: progress and opportunities a prototype two-dimensional capillary electrophoresis system fabricated in poly(dimethylsiloxane) an emerging cyberinfrastructure for biodefense pathogen and pathogen host data microfluidic systems in proteomics microfabricated devices: a new sample introduction approach to mass spectrometry autonomous microfluidic sample preparation system for protein profile-based detection of aerosolized bacterial cells and spores biomarker discovery using protein microarray technology platforms: antibody-antigen complex profiling a simple and rapid protein array based method for the simultaneous detection of biowarfare agents phage display for detection of biological threat agents enzymatic amplification of beta-globin genomic sequences and restriction site analysis for diagnosis of sickle-cell anemia molecular diagnosis of medical viruses nucleic acid-based methods for the detection of bacterial pathogens: present and future considerations for the clinical laboratory advances in nucleic acid-based diagnostics of bacterial infections nucleic acid approaches for detection and identification of biological warfare and infectious disease agents qpcr in the microbiology laboratory cockerill fr 3rd, smith tf (2006) qpcr in clinical microbiology: applications for routine laboratory testing multiplexing for the detection of multiple biowarfare agents shows promise in the field identification and characterization of bacillus anthracis by multiplex pcr analysis of sequences on plasmids pxo1 and pxo2 and chromosomal dna fluorescent detection techniques for q-multiplex strand specific detection of bacillus anthracis using rapid pcr detection of bacillus anthracis dna by lightcycler pcr application of rapid-cycle q-polymerase chain reaction for the detection of microbial pathogens: the mayo-roche rapid anthrax test sensitive and rapid quantitative detection of anthrax spores isolated from soil samples by qpcr application of the qpcr for the detection of airborne microbial pathogens in reference to the anthrax spores real-time nucleic acid-based detection methods for pathogenic bacteria in food molecular beacons for multiplex detection of four bacterial bioterrorism agents simultaneous qpcr detection of bacillus anthracis, francisella tularensis and yersinia pestis a novel semiquantitative fluorescencebased multiplex polymerase chain reaction assay for rapid simultaneous detection of bacterial and parasitic pathogens from blood rapid genotypic detection of bacillus anthracis and the bacillus cereus group by multiplex qpcr melting curve analysis detection of biological threat agents by qpcr: comparison of assay performance on the r.a.p.i.d., the lightcycler, and the smart cycler platforms evaluation of the cepheid genexpert system for detecting bacillus anthracis miniaturized pcr chips for nucleic acid amplification and analysis: latest advances and future trends microchip pcr automated polymerase chain reaction in capillary tubes with hot air minimizing the time required for dna amplification by efficient heat transfer to small samples capillary tube resistive thermal cycling acapella-1k, a capillary-based submicroliter automated fluid handling system for genome analysis dna amplification with a microfabricated reaction chamber pcr in a silicon microstructure development of a microchamber array for picoliter pcr functional integration of pcr amplification and capillary electrophoresis in a microfabricated dna analysis device pcr detection of bacteria in seven minutes a miniature analytical instrument for nucleic acids based on micromachined silicon reaction chambers a battery-powered notebook thermal cycler for rapid multiplex real-time pcr analysis disposable q-micropcr device: labon-a-chip at a low cost ultra fast miniaturized qpcr: 40 cycles in less than six minutes towards a portable microchip system with integrated thermal control and polymer waveguides for qpcr world-to-chip microfluidic interface with built-in valves for multichamber chip-based pcr assays integrating polymerase chain reaction, valving, and electrophoresis in a plastic device for bacterial detection microfluidic handling of pcr solution and dna amplification on a reaction chamber array biochip micromachined polymerase chain reaction system for multiple dna amplification of upper respiratory tract infectious diseases infrared-mediated thermocycling for ultrafast polymerase chain reaction amplification of dna infrared temperature control system for a completely noncontact polymerase chain reaction in microfluidic chips a simple, valveless microfluidic sample preparation device for extraction and amplification of dna from nanoliter-volume samples polymerase chain reaction/ligase detection reaction/hybridization assays using flow-through microfluidic devices for the detection of low-abundant dna point mutations microfabricated device for dna and rna amplification by continuous-flow polymerase chain reaction and reverse transcription-polymerase chain reaction with cycle number selection automated chip-based device for simple and fast nucleic acid amplification high-throughput pcr in silicon based microchamber array microarray-based detection of bacteria by on-chip pcr microarray-based identification of bacteria in clinical samples by solid-phase pcr amplification of 23s ribosomal dna sequences rapid pathogen detection using a microchip pcr array instrument a miniature analytical instrument for nucleic acids based on micromachined silicon reaction chambers a handheld real time thermal cycler for bacterial pathogen detection autonomous detection of aerosolized bacillus anthracis and yersinia pestis flow through pcr module of biobriefcase. smart medical and biomedical sensor technology iii a multiplexed pcr-coupled liquid bead array for the simultaneous detection of four biothreat agents a q-fluorescence polymerase chain reaction assay for the identification of yersinia pestis using a field-deployable thermocycler on-chip, q-, single-copy polymerase chain reaction in picoliter droplets miniature qpcr system for diagnosis of rna-based viruses parallel picoliter qpcr assays using microfluidics characteristics and applications of nucleic acid sequence-based amplification (nasba) detection of m. tuberculosis dna using thermophilic strand displacement amplification strand displacement amplification: a versatile tool for molecular diagnostics homogeneous q-detection of single-nucleotide polymorphisms by strand displacement amplification on the bd probetec et system isothermal reactions for the amplification of oligonucleotides isothermal dna amplification coupled with dna nanosphere-based colorimetric detection loop-mediated isothermal amplification of dna sensitive and inexpensive molecular test for falciparum malaria: detecting plasmodium falciparum dna directly from heat-treated blood by loop-mediated isothermal amplification use of loop-mediated isothermal amplification of the is900 sequence for rapid detection of cultured mycobacterium avium subsp. paratuberculosis rapid amplification of plasmid and phage dna using phi 29 dna polymerase and multiply-primed rolling circle amplification tem-pliphi, phi29 dna polymerase based rolling circle amplification of templates for dna sequencing templiphi: a sequencing template preparation procedure that eliminates overnight cultures and dna purification cell-free cloning using phi29 dna polymerase potential of dna microarrays for developing parallel detection tools (pdts) for microorganisms relevant to biodefense and related research needs potential applications of dna microarrays in biodefense-related diagnostics identification of bacillus anthracis by multiprobe microarray hybridization multipathogen oligonucleotide microarray for environmental and biodefense applications merging microfluidics with microarray-based bioassays a miniature integrated device for automated multistep genetic assays fabrication of dna microarrays onto polymer substrates using uv modification protocols with integration into microfluidic platforms for the sensing of low-abundant dna point mutations self-contained, fully integrated biochip for sample preparation, polymerase chain reaction amplification, and dna microarray detection validation of a fully integrated microfluidic array device for influenza a subtype identification and sequencing fully integrated miniature device for automated gene expression dna microarray processing microelectronic array system for molecular diagnostic genotyping: nanogen nanochip 400 and molecular biology workstation active microelectronic array system for dna hybridization, genotyping and pharmacogenomic applications chemical and biological threat-agent detection using electrophoresis-based lab-on-a-chip devices variable number of tandem repeat (vntr) markers for human gene mapping diversity in a variable-number tandem repeat from yersinia pestis discrimination of m. tuberculosis complex bacteria using novel vntr-pcr targets multiple-locus variable-number tandem repeat analysis reveals genetic relationships within bacillus anthracis molecular subtyping of bacillus anthracis and the 2001 bioterrorism-associated anthrax outbreak, united states comparison of minisatellite polymorphisms in the bacillus cereus complex: a simple assay for large-scale screening and identification of strains most closely related to bacillus anthracis singlenucleotide repeat analysis for subtyping bacillus anthracis isolates aflp: a new technique for dna fingerprinting evaluation of the dna fingerprinting method aflp as an new tool in bacterial taxonomy genetic comparison of bacillus anthracis and its close relatives using amplified fragment length polymorphism and polymerase chain reaction analysis frflp and faflp: medium-throughput genotyping by fluorescently post-labeling restriction digestion visualization of differential gene expression using a novel method of rna fingerprinting based on aflp: analysis of gene expression during potato tuber development strategy for identification of bacillus cereus and bacillus thuringiensis strains closely related to bacillus anthracis sequencing of 16s rrna gene: a rapid tool for identification of bacillus anthracis use of 16s rrna, 23s rrna, and gyrb gene sequence analysis to determine phylogenetic relationships of bacillus cereus group microorganisms suitability of partial 16s ribosomal rna gene sequence analysis for the identification of dangerous bacterial pathogens capillary electrophoresis on microchips (review) microfabricated electrophoresis systems for dna sequencing and genotyping applications: current technology and future directions high resolution dna separations using microchip electrophoresis plastic microchip electrophoresis for analysis of pcr products of hepatitis c virus micromachining a miniaturized capillary electrophoresis-based chemical analysis system on a chip ultra-high-speed dna fragment separations using microfabricated capillary array electrophoresis chips high-speed separations on a microchip high-speed dna genotyping using microfabricated capillary array electrophoresis chips high-speed separation of antisense oligonucleotides on a micromachined capillary electrophoresis device optimization of high-speed dna sequencing on microfabricated capillary electrophoresis channels automated parallel dna sequencing on multiple channel chips microfluidic device reads up to four consecutive base pairs in dna sequencing-by-synthesis pyrosequencing in a microfluidic flow-through device micromachining a miniaturized capillary electrophoresis-based chemical analysis system on a chip tandem isotachophoresis-zone electrophoresis via base-mediated destacking for increased detection sensitivity in microfluidic systems preconcentration and separation of double-stranded dna fragments by electrophoresis in plastic microfluidic devices on-chip millionfold sample stacking using transient isotachophoresis sample preconcentration by field amplification stacking for microchip-based capillary electrophoresis online sample preconcentration using field-amplified stacking injection in microchip capillary electrophoresis high-throughput genetic analysis using microfabricated 96-sample capillary array electrophoresis microplates degenerate oligonucleotide primed-polymerase chain reaction and capillary electrophoretic analysis of human dna on microchipbased devices dna typing in thirty seconds with a microfabricated device microfabricated 384-lane capillary array electrophoresis bioanalyzer for ultrahigh-throughput genetic analysis ultra-high-speed dna fragment separations using microfabricated capillary array electrophoresis chips microfabricated capillary array electrophoresis dna analysis systems high throughput dna sequencing with a microfabricated 96-lane capillary array electrophoresis bioprocessor optimization of high-performance dna sequencing on short microfabricated electrophoretic devices modification of poly(methyl methacrylate) microchannels for highly efficient and reproducible electrophoretic separations of double-stranded dna high-performance genetic analysis on microfabricated capillary array electrophoresis plastic chips fabricated by injection molding modification of a poly(methyl methacrylate) injection-molded microchip and its use for high performance analysis of dna application of disposable plastic microfluidic device arrays with customized chemistries to multiplexed biochemical assays near-infrared time-resolved fluorescence lifetime determinations in poly(methylmethacrylate) microchip electrophoresis devices analysis of nerve agents using capillary electrophoresis and laboratory-on-a-chip technology microchip capillary electrophoresis with electrochemical detection of thiol-containing degradation products of v-type nerve agents microchip separations of protein biotoxins using an integrated hand-held device hand-held microanalytical instrument for chip-based electrophoretic separations of proteins functional integration of pcr amplification and capillary electrophoresis in a microfabricated dna analysis device fully automated dna reaction and analysis in a fluidic capillary instrument capillary-based fully integrated and automated system for nanoliter polymerase chain reaction analysis directly from cheek cells high-throughput polymerase chain reaction analysis of clinical samples by capillary electrophoresis with uv detection on-line coupling of polymerase chain reaction and capillary electrophoresis for automatic dna typing and hiv-1 diagnosis on-line integration of pcr and cycle sequencing in capillaries: from human genomic dna directly to called bases an integrated nanoliter dna analysis device fully integrated pcr-capillary electrophoresis microsystem for dna analysis integrated portable genetic analysis microsystem for pathogen/infectious disease detection single-molecule dna amplification and analysis in an integrated microfluidic device microfabricated bioprocessor for integrated nanoliter-scale sanger dna sequencing on-chip pressure injection for integration of infrared-mediated dna amplification with electrophoretic separation a fully integrated microfluidic genetic analysis system with sample-in-answerout capability nucleic acid-based technologies ramp up alternative methods for amplifying and detecting specific dna sequences dna mapping using microfluidic stretching and single-molecule detection of fluorescent site-specific tags an integrated microfluidic processor for single nucleotide polymorphism-based dna computing miniaturized electrophoresis in planar foramtas produced by semiconductor fabrication techniques key: cord-269200-9h2mmp0j authors: al-azri, nasser hammad title: antifragility amid the covid-19 crisis: making healthcare systems thrive through generic organisational skills date: 2020-10-05 journal: sultan qaboos univ med j doi: 10.18295/squmj.2020.20.03.001 sha: doc_id: 269200 cord_uid: 9h2mmp0j nan d isasters and crises are disruptive to the structures and functions of communities. however, the challenges they present also bring opportunities for learning and changing at both individual and organisational levels. healthcare systems owe much of their developments and advancements to crisis situations. hence, healthcare organisations need to pay attention to these opportunities for thrivingand not just surviving-amid the chaos of adversities. thriving through and within crises requires systems to be antifragile. taleb coined the term "antifragility" to describe systems that "benefit from shocks" and "thrive and grow when exposed to volatility, randomness, disorder, and stressors and love adventure, risk, and uncertainty". 1 taleb advances the notion that "by grasping the mechanisms of antifragility we can build a systematic and broad guide to non-predictive decision making under uncertainty in business, politics, medicine, and life in general". 1 in other words, antifragile systems need to be built and designed so they not only survive during a crisis but thrive through it. the current global outbreak of covid-19 presents exceptional challenges that can serve as an opportunity for healthcare systems to thrive and boost their antifragility as the pandemic continues to spread and evolve over time. this makes the current time a valuable chance for healthcare systems to develop their organisational skills proactively in tandem with the development of the crisis itself. this opportunity to thrive, however, should not be taken for granted. instead, healthcare systems and organisations need to be prepared, guided and directed towards this thriving learning mode during the crisis. unfortunately, many healthcare systems are ill-prepared for pandemic crisis management and are not prepared at all for the opportunity to thrive under such circumstances. in 2019, both the global preparedness monitoring board and the global health security index reported a global lack of preparedness for health emergencies worldwide. 2,3 during a pandemic, health systems are typically absorbed in managing the chaos that ensues and these systems find themselves exhausted afterwards. hence, they may lose a great opportunity to thrive amid the chaos of a crisis. it is the responsibility of healthcare leaders to guide their systems through the transition of surviving the crisis to thriving in it. much like individuals, organisations have mechanisms and skills that allow them to survive and thrive. living organisms might survive with the provision of minimal requirements for living such as breathing, eating and drinking. however, these organisms need more proactive efforts for thriving and growing. each living organism has its own mechanisms for surviving and thriving. similarly, organisations and systems can survive with the provision of their basic requirements but thriving requires planned proactive efforts that best suit the organisation. there are two types of organisational skills used during crises: specific and generic. 4 on the one hand, specific skills are needed to manage a particular crisis, such as the development of management protocols and measures to manage the outbreak of covid-19. hence, specific skills can be viewed as the 'survival skills' necessary to allow the system to continue functioning despite the crisis. on the other hand, generic skills are necessary for the organisation to carry out its functions and duties to achieve its goals and are usually applied to routine duties. these generic skills are also the platform for applying specific skills during a crisis. generic skills include aspects such as leadership, teamwork and communication, and they can enhance the antifragility of the system. although generic organisational skills are not specific to a single type of crisis, they are critical for initiating, building and maintaining specific organisational capabilities before as well as during a crisis. moreover, because they are applicable to routine duties, generic skills remain relevant after the crisis. if these skills are well developed, they can help an organisation thrive well beyond the crisis, adding to the system's reservoir of antifragility mechanisms. much of the current literature concerning covid-19 management is focused on the specific organisational skills needed to control the outbreak. however, little is being said about generic organisational skills as the basis of specific skills and how they should be realised. hence, the focus of this article is on crisis management through the lens of five critical generic organisational skills that give healthcare systems an opportunity to grow and thrive amid the covid-19 pandemic. vuca is a military term used to describe situations characterised by volatility, uncertainty, complexity and ambiguity. 5 disasters and crises are deemed to have a vuca nature. in the disruptive crisis environment of vuca, some systems become fragile and breakdown under pressure, while others remain robust and, relatively, indifferent to change. yet, there are systems that take the opportunity of the disaster to grow and develop their antifragility amid the crisis. many systems choose to adapt and survive, but antifragile ones evolve, grow and thrive, becoming more resilient in the future. 6 the following discussion briefly presents an example of five generic organisational skills that give healthcare systems a chance to boost their antifragility in a vuca environment like the current covid-19 crisis. these skills include leadership, teamwork, communication, system design and learning and feedback. leadership is a cornerstone for any system or organisation. the whole activity of a system revolves around the leadership style manifested in that system. leadership is not an individual task but rather a mindset that diffuses throughout the organisation. almost all other organisational skills, whether specific or generic, are dependent in their execution on organisational leadership, and crisis management relies heavily on leadership. 7 leadership is essential to build capacity and mitigate risks during pandemics. 2 the covid-19 outbreak has shown that leaders need to act now. 8 in particular, vuca environments, like the covid-19 pandemic, require unique leadership characteristics. lawrence suggests a set of leadership characteristics known as vuca prime to be used in vuca environments; 5 these characteristics include vision, understanding, clarity and agility. to be properly prepared, leaders need to have developed these traits well ahead of a vuca situation. this is achieved through practice and applying the correct type of leadership styles to daily/routine situations prior to a crisis. how systems and organisations perform is defined by the integrative activities of their actors. teams are the backbone of healthcare systems and teamwork is a rate-limiting factor for organisational outcomes. the execution of healthcare activities cannot be achieved without coordinated teamwork. however, a crisis is a hard test for teams as it stretches the systems' needs beyond the normal capacity of the system. teams in healthcare systems are the source of power in the system. in fact, there is sufficient evidence that teamwork is more important than technical prowess for preventing and mitigating a major crisis or disaster in high-risk fields such as healthcare. 9 unfortunately, it is not uncommon during a crisis for some managers to strip teams of their professional autonomy and ability to cooperate with others through the piling up of structures such as procedures and formal rules. 10 as systems are stretched beyond their means during a crisis, more gaps appear at critical times, and the organisational capacity for patching and stitching that prevails during routine work is no longer sufficient. undoubtedly, teamwork has been proved essential to address concerns related to the covid-19 pandemic, 11 and systems cannot succeed in managing the crisis without good teamwork. a crisis is a time for teamwork; leaders must equip their organisations with this invaluable skill. a single, reliable tool during a crisis has always been communication. over several decades, communication has been recognised as a key element for successful crisis management. 12 moreover, poor communication has been repeatedly associated with crisis management failure. 13 the joint commission in the united states asserts that "the success or failure of an eop [emergency nasser hammad al-azri operations plan] is often determined by timely access to communication that ensures the flow of critical information". 14 the covid-19 crisis has shown how effective communication is serving as a key factor in managing the pandemic through proper flow of information, handling uncertainty and fear and promoting behavioural changes in the community. 15 however, leaders must realise that this critical, key element cannot be developed overnight, especially during a crisis. instead, communication requires time and proactive efforts well in advance of the crisis in order to establish itself in the system. a crisis provides a live test for the quality of established communication strategies within an organisation. nevertheless, it is also a lesson for future planning. it is a fundamental concept in biology and physiology that the structure of a system determines its functions. 16 using a medical analogy, one might think of the system's structure as the anatomy and the system's processes and functions as the physiology. for any system to serve specific outcomes, it is not enough to adjust the physiological functions through enforcing new or modified processes, policies, protocols and standards of the system unless the anatomical structure of the system is fit for the intended purpose. as has been shown during the covid-19 crisis, many healthcare systems failed as their anatomy was not made to meet the physiological adaptations needed during a crisis. a system that is not structurally designed to meet the challenges of crisis management will not be able to cope with the stressors resulting from required functional changes during a disaster. although it might be late to review and modify a system's design during an emergency status, how the system responds to the challenges offers invaluable insight into how the anatomical structure of the system might present a constraint to its processes and functionality. this insight should inform a better, future system designone that takes into account the needs that may arise during crisis management, and beyond. social systems, such as healthcare, are informationbonded. they change their behaviour in response to information flow within the system. 17 hence, understanding the centrality of learning and feedback in such systems is massive as it provides the means for the system to adapt to a changing external, as well as internal, environment. the quality of embedded learning and feedback functionality within a system determines, to a large extent, the quality of change in response to a dynamic environment. the vuca nature of reality during any crisis precludes the ability to forecast much of the changes and prepare for them in advance. in such a dynamic and constantly unfolding environment, meadows once said: "we can't control systems or figure them out. but we can dance with them!". 18 responding within such a dynamic environment requires consciously planned and continuously monitored learning and feedback loops that allow the system to self-organise its behaviour as it emerges within the crisis. disasters and crises disrupt the 'normal' activities of systems and organisations. however, amid this disruption, crises also offer organisations the potential to grow and thrive. the current covid-19 pandemic is a good example of such a potential. as much as healthcare systems focus on their specific organisational skills to mitigate the effects of the crisis, one must also pay attention to the generic organisational skills through which they can develop and improve. by focusing on both specific and generic organisational skills, systems will not only survive through the crisis but will also grow and thrive towards antifragility. antifragility and thriving are, therefore, a reflection of how much we have invested in our generic organisational skills through leadership, teamwork, communication, system's design, and learning and feedback. systems will, almost always, output during a crisis whatever inputted in them at routine times. antifragile: how to live in a world we don't understand global preparedness monitoring board. a world at risk global health security index. global health security index everyday crisis management developing leaders in a vuca environment making sense of disaster medicine: a hands-on guide for medics thinking through crisis: improving teamwork and leadership in high-risk fields responding to covid-19 -a once-in-a-century pandemic? six simple rules: how to manage complexity without getting complicated covid-19: a critical care perspective informed by lessons learnt from other viral epidemics communicating throughout katrina: competing and complementary conceptual lenses on crisis communication crisis communication failure: a case study of typhoon morakot emergency management in health care: an all-hazards approach effective health communication -a key factor in fighting the covid-19 pandemic structure determines function systems thinking: managing chaos and complexity: a platform for designing business architecture thinking in systems: a primer key: cord-296253-bxyzhsfs authors: elham, elzat; wumaier, reziya; wang, chengji; luo, xiangying; chen, tao; zhong, nanshan title: anatomic evidence shows that lymphatic drainage exists in the pituitary to loop the cerebral lymphatic circulation date: 2020-05-30 journal: med hypotheses doi: 10.1016/j.mehy.2020.109898 sha: doc_id: 296253 cord_uid: bxyzhsfs respiratory infections can result in intracranial infections and unknown neurological symptoms. the central nervous system lacks classical meningeal lymphatic (circulation) drainage, and the exact underlying mechanisms of how immune cells from the peripheral lymphatic system enter the central nervous system (cns) remain unknown. to determine whether the perinasal lymphatic system or lymphatic vessels are involved in cerebral immune defence and play a role in causing cns infections (especially respiratory tract-related infections), we performed an anatomic study to investigate the drainage differences between the perinasal and intracerebral lymphatic systems by using injection of evans blue and anatomic surgery, together with immunohistochemistry and immunofluorescence assays. surprisingly, we found that (1) the pituitary (adenohypophysis) is involved and is rich in lymphatic vessels and (2) perinasal tissue could communicate with central pituitary lymphatic vessels in a specific and unidirectional manner. taken together, our study may be the first to anatomically demonstrate the existence of novel lymphatic vessel structures in the pituitary, as well as their communication with the perinasal (lymphatic) tissue. our findings suggest the existence of an ultimate loop for “classical” meningeal lymphatic drainage and are relevant to cerebral infection and immune defence. respiratory infections can result in intracranial infections and unknown neurological symptoms. the central nervous system lacks classical meningeal lymphatic (circulation) drainage, and the exact underlying mechanisms of how immune cells from the peripheral lymphatic system enter the central nervous system (cns) remain unknown. to determine whether the perinasal lymphatic system or lymphatic vessels are involved in cerebral immune defence and play a role in causing cns infections (especially respiratory tract-related infections), we performed an anatomic study to investigate the drainage differences between the perinasal and intracerebral lymphatic systems by using injection of evans blue and anatomic surgery, together with immunohistochemistry and immunofluorescence assays. surprisingly, we found that (1) the pituitary (adenohypophysis) is involved and is rich in lymphatic vessels and (2) perinasal tissue could communicate with central pituitary lymphatic vessels in a specific and unidirectional manner. respiratory infections (e.g., fungi, bacteria, and coronavirus) can result in unknown intracranial infections and consequent neurological symptoms (1) (2) (3) . for example, in the current covid-19 epidemic in china, 78 (36.4%) of 214 patients with covid-19 were admitted with neurological symptoms to wuhan union hospital (2) , and we observed 2 cases of diabetes insipidus (di) related to pituitary disorder in patients with severe covid-19 (in the first affiliated hospital of guangzhou medical university). it is generally believed that pathogens cause intracranial infection by entering the subarachnoid space via nasopharyngeal or middle ear passages, blood flow, blood-brain, and cerebrospinal fluid (csf) barriers, although we still cannot explain the existence of pathogens in the csf, as the blood-brain barrier (bbb) can prevent the transmission of pathogens to the meninges(1). csf originates from the choroid plexus of the intracranial lateral ventricle (4) . the reflux of csf to the lymphatic system plays an important role in cerebral immunity (4, 5) . csf is drained through meningeal lymphatic vessels, which allow immune cells to enter draining lymph nodes (dlns) and play an important role in cerebral immune defence. however, the exact underlying mechanisms of how immune cells from the peripheral lymphatic system enter the central nervous system (cns) remain unknown (4, 6) . the perinasal lymphatic system is the first-line barrier of respiratory immunity against pathogen invasion of the respiratory tract and body (7) . respiratory infections can lead to cns infections, but it is unclear whether the perinasal lymphatic system and lymphatic vessels are involved in cerebral immune defence and play a role in cns infections caused by respiratory pathogens (1, 7) . to elucidate the roles of the perinasal lymphatic system during cerebral infection (especially respiratory-related infections) and cerebral immune defence, we carried out an anatomic study to investigate the drainage differences between the perinasal and intracerebral lymphatic systems. under an anatomic (20× magnification) microscope, we dissected the mouse intracranial nervous system after injection of evans blue (perinasal lymphatic reflux assay) and found that lymphatic vessels that exist in the pituitary and loop the cerebral lymphatic circulation are responsible for the perinasal-pituitary lymphatic drainage. the lyve1-alexa 488 antibody was purchased from ebioscience (catalogue # 53-0443-80) and used at a 1:250 dilution. anti-cd31 was purchased from abcam (catalogue # ab222783) at a 1:100 dilution. anti-rabbit igg (h+l) highly cross-adsorbed secondary antibody was purchased from invitrogen co., ltd., (catalogue # a32740) and used at a 1:1000 dilution. evans blue and other reagents were purchased from sangon biotech (shanghai) and were of high analytical grade. mice (balb/c, 7 weeks old) were divided into different groups (5 for each group). for the treated groups, mice were anaesthetized with pentobarbital sodium (70 mg/kg) by intraperitoneal injections and then subcutaneously injected with 0.1 ml of evans blue (5%) by microsyringe via either or both limbs, the tail and the perinasal area (e.g., bilateral the hindlimbs, the second toe of the dorsal feet, both flanks, the dorsal sides of the bilateral forelimbs, the bilateral retroauricular regions, the parietal midpoint between both ears, the tip of the nose, and the bilateral ventral mucosae of the tongue). the control groups received saline instead. after injection, the mice were placed on a heating pad at a stable (25°c) temperature for 4 hours and then euthanized (350 mg/kg) for anatomic analysis. in brief, with the abdomen facing down, the dorsal fur of the mouse was moistened with saline, after which the dorsal skin was cut transversely and then longitudinally to the mouse nose to fully expose the skull with scissors. then, the cervical muscles were cut off the skull from the foramen magnum to expose the brain. the brain was then removed with tweezers; nerves connecting the brain were also cut off to expose the pituitary for observation under a zeiss operating microscope (zeiss opmi primo ceiling-mount microscope, 20× magnification). photos were taken with a canon 5dsr camera (micro lens: canon ef 100 mm f/2.8l is usm; micro flash: canon mr-14ex ii). the pituitary was removed from the mouse, immersed in 4% pfa for fixation, and embedded in paraffin. the pituitary was transversely sectioned (leica cm 1950) and adhered to a glass slide. the sections were then deparaffinized, after which they underwent antigen retrieval, 3% hydrogen peroxide solution blocking for endogenous peroxidase and 3% bsa blocking. the blocking buffer was discarded, and primary antibody in pbs was added to the section and incubated in a wet box at 4°c overnight. the glass slides were washed with pbs three times (5 min each) after incubation. then, the slides were incubated with secondary antibody (hrp-labelled) for 50 min at room temperature. the sections then underwent dab and harris haematoxylin staining after three washes in pbs. then, the section was mounted using neutral balsam, and the blue staining was reversed in ammonia water. photos were taken under a fluorescence microscope (leica dmi4000b). the sections and slide preparation, antigen retrieval, bsa blocking, and primary and secondary antibody incubation processes were the same as the aforementioned conditions used for immunohistochemistry. after staining with secondary antibodies, the glass slide was washed in pbs three times (5 min each). after the section dried slightly, dapi was added to the sample, followed by 10 min of room temperature incubation in the dark. the glass slide was then washed again three times and mounted using an antifluorescence quenching mounting agent. photos were taken under a confocal microscope (lsm710 laser confocal microscope, zeiss). adobe photoshop, fiji image analysis, and graphpad prism software were chosen for image processing. two to four hours after the subcutaneous injection of evans blue into the mouse limbs, tail, and perinasal area, we unexpectedly observed a light-blue, well-discriminated, regular "birds eye" region in the central pituitary (8) (fig. 1a; we then dissected other untreated mice and found that the "birds eye" region exists in the central pituitary, which was identifiable but can be carefully discriminated from peripheral white matter ( fig. 1c ; see green arrow and circle). we further performed pathological staining and confirmed that the "birds eye" region was actually the posterior pituitary or adenohypophysis(8) (fig. 1d) . moreover, lymphatic endothelial growth factor was found to be highly expressed in the "birds eye" region ( fig. 1e and we acknowledge that the brain lacks classic lymph circulation according to our current understanding. however, it remains unclear how respiratory pathogens cause intracranial infection, how immune cells enter the brain for immune defence and whether the peripheral lymphatic system communicates with the central nervous system in addition to the meningeal lymphatic draining system. a, colocalization analysis of lyve-1 and cd31 under the scope in figure 2d (upper right, the most colocalized area in figure 2d was selected and marked with a yellow line; scale bar: 100 μm). b, immunofluorescence analysis showed no colocalization of lyve-1 and cd31 (fiji image analysis). funding: the study was financially supported by grants from the national natural science we declare that none of the authors has any potential conflicts of interest with regard to this manuscript. a, colocalization analysis of lyve-1 and cd31 under the scope in figure 2d (upper right, the most colocalized area in figure 2d was selected and marked with a yellow line; scale bar: 100 μm). b, immunofluorescence analysis showed no colocalization of lyve-1 and cd31 (fiji image analysis). nasal lymphatics as a novel invasion and dissemination route of bacterial meningitis neurological manifestations of hospitalized patients with covid-19 in wuhan, china: a retrospective case series study pituitary aspergillus infection structural and functional features of central nervous system lymphatic vessels implications of the discovery of brain lymphatic pathways current understanding of lymphatic vessels in the central nervous system rapid transepithelial transport of prions following inhalation neuronal m<sub>3</sub> muscarinic acetylcholine receptors are essential for somatotroph proliferation and normal somatic growth none of the authors has any potential conflicts of interest. key: cord-256408-bf79lj4f authors: jayasinghe, saroj title: social determinants of health inequalities: towards a theoretical perspective using systems science date: 2015-08-25 journal: int j equity health doi: 10.1186/s12939-015-0205-8 sha: doc_id: 256408 cord_uid: bf79lj4f a systems approach offers a novel conceptualization to natural and social systems. in recent years, this has led to perceiving population health outcomes as an emergent property of a dynamic and open, complex adaptive system. the current paper explores these themes further and applies the principles of systems approach and complexity science (i.e. systems science) to conceptualize social determinants of health inequalities. the conceptualization can be done in two steps: viewing health inequalities from a systems approach and extending it to include complexity science. systems approach views health inequalities as patterns within the larger rubric of other facets of the human condition, such as educational outcomes and economic development. this anlysis requires more sophisticated models such as systems dynamic models. an extension of the approach is to view systems as complex adaptive systems, i.e. systems that are 'open' and adapt to the environment. they consist of dynamic adapting subsystems that exhibit non-linear interactions, while being 'open' to a similarly dynamic environment of interconnected systems. they exhibit emergent properties that cannot be estimated with precision by using the known interactions among its components (such as economic development, political freedom, health system, culture etc.). different combinations of the same bundle of factors or determinants give rise to similar patterns or outcomes (i.e. property of convergence), and minor variations in the initial condition could give rise to widely divergent outcomes. novel approaches using computer simulation models (e.g. agent-based models) would shed light on possible mechanisms as to how factors or determinants interact and lead to emergent patterns of health inequalities of populations. health outcomes are increasingly perceived from a systems approach that is more holistic and non-reductionist [1] . the author in a recent paper extended the systems approach to incorporate principles of complexity science and to conceptualize population health outcomes as an emergent property of a dynamic and open, complex adaptive system [2] . the current paper explores these themes further and applies the principles of systems approach and complexity science (i.e. systems science) to conceptualize social determinants of health inequalities (sdhi). the paper begins with a brief overview of the existing models of sdhi, and then proposes a two step approach to remodel our perspectives and outlines possible implications. interest in studying social influences of population health outcomes dates back to at least the 19th century. pioneering researchers in this area include rudolf virchow who reported on the role of political economy and poverty in causing an epidemic of plague in upper silesia of prussia, and friedrich engels on the link between high mortality and poor living conditions of the working class in england [3] . subsequently salvador allende's work in chile attempted to show the role of social and political factors in generating health inequalities in populations [3] . more recent expansion of a population-based approach to inequalities includes the work of geoffrey rose in the 1980's [4] . he distinguished between causes of incidence of a population group and causes of disease in individuals. the extreme example he proposes was to assume that every member of society smoked 20 cigarettes a day, which if investigates through cohort studies and case-control studies, will lead to the conclusion that incidence of lung cancer is determined by genetic predispositions. these smoking patterns are a reflection of social norms, values, traditions and their customs, in a historical context. thus, societies with lower per-capita rates of smoking have a significantly lower incidence of lung cancer. the balances or imbalances of norms and social structures within the population lead to distinct patterns of individual behaviours in smoking rates or varying rates of disease prevalence or incidence among social groups, i.e. inequalities. sick individuals represent the extremes of the population mean. recent literature uses the term social determinants of health inequalities (sdhi) to denote contexts, social norms, social structures, and their determinants. three main pathways have been advanced to explain the generation of health inequalities from the social environment [5] . (a) "social selection", or social mobility which implies that health determines socioeconomic position, rather than the reverse. thus, healthier persons will move towards better socioeconomic positions, compared to less healthier, leading to inequalities. (b)"social causation" proposes that a range of unequally spread material, psycho-social and behavioural factors, give rise to inequalities in health outcomes [6, 7] . material factors include varying income levels and investments across infrastructure beneficial to the community. psychosocial factors are the chronic stresses that arise from perceptions and experiences of personal status in an unequal society. behavioral factors are, for example, the higher rates of smoking observed in poorer groups that lead to differential rates of diseases and mortality [8, 9] . (c) a "life course perspective" suggests that a multitude of factors across the life span (e.g. maternal malnutrition during fetal period, poor educational facilities in childhood, occupations with physical hazards etc.) determine and manifest disease trends observed over time. the ecosocial approach attempts to integrate these ecological, social and biological factors in disease causation through a dynamic process of their 'embodiment' i.e. "we literally incorporate biological influences from the material and social world" [10, 11] . there are several biological pathways that explain how social environment gets 'embodied' on health. one is the 'stress biology approach' whereby stress leads to chronic stimulation of the hypothalamo-pituitaryadrenal axis and the resultant hormonal changes increase risks of hypertension, insulin resistance, and hyperglycaemia [6] . the other relates to the advancing field of epigenetics, i.e. non-genetic modifiers of gene expression that are transmitted to future generation. they provide an explanation as to how environmental effects convert to lasting physiological changes and behavioral changes. an example is how psychological stress alters dna methylation and histones in stress-sensitive brain regions such as the hippocampus, amygdala, and prefrontal cortex. these changes modulate subsequent gene expression [12] . another pathway is through modifications of telomeres in dividing somatic cells. telomeres are dna repeat sequences, which together with accompanying telomere-binding proteins, cap and protect chromosome ends. their length shortens with each cycle of replication, which is an expression of accelerated ageing as this process ultimately leads to a loss of capacity to replicate. it has been shown that lower socio-economic groups demonstrate shortening of the length of telomeres, thus suggesting that these groups acquire features of accelerated ageing with its consequences [13, 14] . the commission of sdh (csdh) re-conceptualizes health inequalities by integrating these theories and concepts further [5, 15] . they describe sdhi to have a context, structural mechanisms and socioeconomic positions of individuals. it assumes a crucial role for the "context" which includes social systems (e.g. education system, labour market), culture (e.g. racism and caste) and political systems (e.g. structure of the state, redistributive policies). the structure of the state in relation to welfare and redistribution of wealth is recognized as a dominant institution. the 'context' should be viewed as a dynamic concept, having a historical past, a present and future trajectory. structural mechanisms that are rooted in institutions and processes within the context generate stratifications in society according to socio-economic position, income or wealth, educational achievements and access, occupation, gender, race/ethnicity and other dimensions. these are inter-related dimensions and could act as proxies for each other. for example, in a heavily market-driven individualized society, incomes or wealth are good proxy indicators for socio-economic position. the socioeconomic position in turn is a key stratifier in most contemporary societies and reflects a hierachical system consisting of power, prestige and access to resources. the sdhi operate through a group of intermediary determinants to mould health outcomes. the main groups of intermediary determinants of health are: material circumstances (e.g. quality of housing, exposure to pollution, financial means to purchase quality food, and work environment); psychosocial circumstances (e.g. levels of stress and social support); behavioural factors (e.g. rates of tobacco and alcohol consumption, nutrition and physical activity) biological factors (e.g. genetic predisposition to diseases in different population groups) factors) and the health system (e.g. access to quality care in populations). increasingly, research evidence reports a widening range of influencing material circumstance (such as availability of safe water and sanitation, agricultural policies and food security, access to health and social care services, unemployment, under-employment and working conditions, access to housing, the living environment, access to education, and availability of transport) [15, 16] . those holding higher positions in the hierarchies of social stratification (e.g. higher socio-economic position or most affluent) would hold an advantageous position in accessing resources, information and environments that are more favourable to better health outcomes. an implicit and explicit recognition of an inter-related web of factors functioning as a system runs through the above discourse. rose's concept of causes of incidence in a population group, implies that the population functions as a cohesive 'whole' or system, rather than being a mere collection of independent individuals. similarly, the concept of sdhi proposed by the csdh describes a system that consists of elements such as, a context, structural mechanisms, and intermediary determinants. these are related both as influencers as well as through feedback mechanisms. however, as with most concepts related to health outcome, sdhi implicitly and explicitly accepts certain elements of a newtonian view of reality (i.e. reductionism, linearity and hierarchy) [2, 17] . an example of this reductionist approach is the descriptions of a single factor that influences health outcomes (e.g. socio-economic stratification of mortality due to asthma) and selecting interventions that focus on a single determinant (e.g. improving thermal comfort in houses that have inadequate warmth) [18] . another assumption prevalent in this discourse is linearity, which assumes that determinants of inequalities can be applied across a wide range of contexts. for example, differential access to healthcare or education is explicitly or implicitly assumed to lead to variations in outcomes, almost in a linear fashion [6, 7, 17] . this view does not give adequate credit to unintended consequences commonly seen in reality. for example, mobile phones have improved connectivity, but their use while driving have become an important cause of road traffic accidents, a feature that was never predicted at the outset. another key concept is the role of hierarchies or power, position and access to resources (e.g. in the understanding of socio-economic position). the concept of hierarchies is implicitly used to explain the process of sdhi as exemplified by terms such as proximate or distal determinants of health inequalities. this indicates a clear path of influences that arise 'distal' to the population group (e.g. labour laws that determine wage structure) and affect it through more 'proximal' factors that are closer to the population (e.g. income) [6, 7] . the statistical methods of estimating the effects of determinants also imply other features of the mechanistic reductionist paradigm. earlier generation of studies used relatively simple statistical models such as odds ratios [19] . these methods assume that explanatory variables have a cause and-effect-pathway (i.e. unidirectional path of influences) and do not encompass positive or negative feed-back loops between the outcome variable and explanatory variables. for example, poor education leads to higher probability of ill-health, which combined together lowers the level of employment and ability to generate income, which in turn influences the ability to live in a more affluent neighbourhood and therefore reduces opportunities for further education, and higher grades of occupations, as well has increases exposure to more polluting environments. they also ignore interrelations among individuals. for example health education could have a positive health impact on an individual, that could indirectly improve the health of the individual's friends. more sophisticated statistical methods, e.g. generalized linear models such as multiple regression, logistic regression and poisson regression, account for multiple explanatory variables and to those that are not normally distributed. however, these analyses do not include feedback loops and interrelations among individuals. the latter require multilevel or hierarchical regressions models [20] . they 'implicitly assume that these effects can be isolated from each other and do not allow for feedback loops or reciprocal interactions between groups and individuals, or between outcomes and predictors' [21, 22] . systems science combines systems theory and complexity science. systems theory states that properties of a complete system cannot be predicted by disaggregating, analyzing and exploring its individual constituent parts alone [23] . complexity applies systems theory to open and adaptive systems (i.e. complex adaptive systems) and views health outcomes as an emergent property of such systems [1, 2] . the next sections propose a two-step process to visualize sdhi from a systems science perspective, firstly, by shifting away from a reductionist paradigm towards a systems approach and secondly, by enriching this with principles of complexity science. in applying the systems approach, a point of departure from the conventional model of thinking is to consider population health outcomes as one of many components of the human condition or the 'standard of living' of a population. this human condition has several other facets or components such as educational attainment, economic well-being, and social status. these components too exhibit patterns of inequalities, in a manner similar to health outcomes. health inequalities should be viewed as patterns in health outcomes that arise in association with other patterns of human condition, and lie within this milieu. extricating health outcomes from these other human conditions and exploring it individually is therefore arbitrary, though justified on grounds of interest, existing disciplinarity and convenience of tackling one component at a time sdhi can be visualized from a systems approach using a matrix that captures at least part of the elements of the human condition system. each group of columns will represent a facet of the human condition (e.g. health outcomes, educational attainment). for comparison between countries, the parameters should be uniform and preferably continuous variables. the bundle of parameters in health outcomes could include life expectancy at birth (leb), vaccination rate of children at 5 years, inverse of infant mortality rate (imr), inverse of maternal mortality rates (mmr), and inverse of childhood malnutrition rates. similarly, the parameter bundle on educational attainment could be percentage enrolment by 6 years, percentage in secondary level education, and per-capita investment in education, and for socio-economic status, the per-capita income, and employment rates. each row could be for a particular year or for specific intervals (e.g. 2005 to 2010). an appropriate colour coding for the parameters will give rise to a pattern in the matrix that will be a fingerprint of the human condition of a particular country. the whole matrix represents the totality of the human condition as an output. a similar method of visualization is used to illustrate worldwide trends in life expectancy at birth from 1970 to 2010 published in the human development report [24] . this process will also demonstrate the disadvantages of narrowing our perspective to a particular health outcome (e.g. obesity rates) or its determinants (e.g. physical activity). when such single parameters are selected from the matrix, we are in reality focusing on a single facet of health outcomes extracted from a column of the matrix. instead, the preferred option is to express data on several other health related outputs such as imr, mmr, leb, and morbidity rates. this will immediately highlight unintended consequences, a feature of complex adaptive systems. once a predetermined bundle of health outcomes are used to describe health, e.g. leb, imr, and mmr, comparisons between countries or regions become easier. the second step is to incorporate an inequalities dimension to this form of visualization. this could be done on standardized measures of inequalities or stratifiers (e.g. across income or wealth or educational attainment, or geographic region) over a period or 10-20 years (see fig. 1 ). this will allow tracking of trends within a country. for example, a stratifier such as income could impact across several categories of the human condition. this impact could be visualized using the following summary measure of inequalities: a) health inequalities: odds ratios between highest and lowest income quintile in parameters such as imr, mmr, under-5 year vaccination rates, childhood nutrition, leb b) educational attainment: odds ratios between highest and lowest income quintiles in education such as primary school enrollment, secondary school enrollment, and tertiary education. c) inequalities in social capital: odds ratios between highest and lowest income quintiles in measures of social capital, contact with social networks, one option is for individual countries to have several of these charts, one for each stratifier, and track selected measures of inequalities (e.g. ratios of imr, leb, mmr, primary school enrollment) over a period (e.g. every 5 years). each cell will be filled by the result of the odds ratio. in order to give an immediate visual impression, a simple set of rules could be observed to colour the cells in the matrix. the colour coding could be such that the cells showing extreme values of a ratio (e.g. above an odds ration of 2) and having the widest the inequalities will receive red colour and the least receive green, with other intermediates receiving light green, yellow, and orange. the precise values at which the colour transits would be arbitrary (e.g. one may choose a ratio of 2 and above to be red and below 0.5 to be green) see fig. 1 . however, it is necessary to be consistent in order to allow for comparisons along the time axis and between countries or regions. a series of matrices developed for each stratifer in a country, could be analysed subsequently using hierarchical clustering algorithms (e.g. hierarchical clustering and k-means clustering) and computational methods (e.g. organizing maps and artificial neural networks) [25] . hierarchical clustering will produce a 'tree' of those cells that are closest to each other (e.g. those with closest values as percentages). heat maps can also be used to illustrate the clustering and it increasingly used to describe multimorbidity [26] . such analyses from individual countries and the emerging patterns of health outcomes will give invaluable insight on the processes that are involved in giving rise to these particular patterns. it is possible that this method will demonstrate clusters of inequality measures that would not be expected from usual epidemiological parameters. an analogous situation was seen with data from acutely ill patients in intensive care units, where by clusters of physiological parameters were observed that could not be defined by conventional knowledge [27] . the third step is to view the system as a dynamic entity and to include dynamic dimension to the approach. describing outcomes at a given time is a mere snapshot of a dynamic process and analogous to prognostication of diabetes and its risks by using a single random blood glucose level. extending this further, the predictions become more robust if a series of observations are made and this dynamism is incorporated into the model by using rates of change of parameters (e.g. rate of decline of imr). parallels are seen in systems medicine where serial analyses of the pulsatile secretion of insulin (rather than fasting insulin) was found to be an early feature of diabetes rather than a single value [28] . data can now be gathered relatively inexpensively using crowd-sourcing techniques (i.e. gathering information by engaging with a large number of people usually via the internet) and real-time surveillance of information in twitter and other social media [29, 30] . a systems approach views the human condition and their determinants as causes as well as outcomes. they are interlinked in a causal web, rather than a linear path of social determinants leading to a range of health outcome. the feedback loops lead to outcomes influencing their causations. for example, low income and deprivation lead to poorer health outcomes, which in turn makes the group even poorer and worsening health outcomes. in order to operationalize the above, more sophisticated models such as systems dynamic models are required [31] [32] [33] . analogous models are now being developed for specific disorders such as obesity [34] . a systems approach to sdhi emphasizes the role of the context in determining health inequalities. the relationships between the variations in health outcomes and other parameters would be more or less unique to that particular system. the interpretation of the outputs is heavily dependent on the context and less generalizable across different nations, social groups, or environments. this is yet another reason to explore natural experiments to identify causal pathways and interventions for a given context [21] . the next advance is to incorporate complexity science to the systems perspective. this will require perceiving the patterns of human condition in a given population group to be an emergent property of a complex adaptive system (cas). the latter is a special type of system that is 'open' to its environment, with non-linear interactions among a dynamic set of other interconnected systems and subsystems. other systems include the political system (e.g. the predominant political governance system), the physical environment (e.g. pollution levels or transport), and biological systems (e.g. genetic predispositions), within the context of a larger social system [2] . one could position this within the context of global ecosystems (e.g. the biosphere, the atmosphere, the hydrosphere, geosphere and cryosphere). a feature of a cas is that it has emergent properties, i.e. it is almost impossible to predict precisely what it the larger system will evolve to, by using knowledge relating to the interactions among its components and sub-components [35] . rather than mathematical modeling where equations are the basis of the model, some complexity scientists use simulations using computer-based programmes (e.g. python) [36] . the building blocks of these simulation models are considered to be as fundamental and irreducible as mathematic equations, and are being hailed as a 'new kind of science' [37] . if the analogy of the matrix of sdhi used in the previous section is applied, the picture will be one of continuously changing colours and patterns that are not predictable. complexity scientists would mimic these patterns through computer simulations using simple rules that describe the inter-relationships among parameters. of the commonly used computer simulations, those most likely to mimic patterns of health outcomes seen in the matrix model are agent-based models, cellular automata, and networks. initial work on agent-based models by schelling showed that segregation in a city (often attributed to racial discrimination) can be simulated by simple rules applied to a grid where each cell represents a house [38] . consider an example where 90 % percent of houses are occupied randomly, either by blue or red, in approximately equal proportions. a programme will simulate future scenarios if they follow two simple rules: place each house where at least 2 of 8 encircling them are of similar colour as the house, and avoid the cell if they have one or none of same colour. within a few iterations, the emerging model begins to mimic cities that are segregated by race, poverty, or social status. these simulations provide evidence (but no proof ) that several alternate pathways could potentially explain urban clusters of race or poverty, rather than discrimination. extrapolating to health outcomes, they challenge area-wise aggregation of health outcomes are through pathway such as the neighbourhood effects or the effects of urbanization on health outcomes [38] . cellular automata is another simulation where the programme places lines of square cells next to each other (e.g. you fill the cell with a black cell, if two of the eight cells around the cell of interest are black) as in a checker board [37, 39] . the interesting observations include the following: minor changes in the rules and initial conditions lead to emergence of widely divergent patterns, some lead to recurring patterns, and similar patterns arise with different starting points and different set of rules. if this metaphor is applied to sdhi, it illustrates the uniqueness of the context to a particular pattern of health outcomes in a population, and the theoretical possibility that widely divergent determinants lead to similar patterns of outcomes in different situations. networks perceive systems as interlinked webs. the strengths and numbers of links among nodes can be varied to generate emergent properties mimicking reality. milgram pioneered the concept of small world networks, in which the degrees of separation between two individual members is considerably small compared to the given population [40] . this was illustrated by an experiment that showed an average six intermediate acquaintances separate any two people on the planet. thus a highly infectious disease could theoretically spread to all 7 billion on the globe in about six incubation periods! another improvement of this simulation is to incorporate the highly connected super-spreaders who are disproportionately relevant to rapid spread of the disease, called scale-free networks [41] . in this model, new members of the network connect preferentially to those who already have a large number of contacts (i.e. analogous to individuals befriending the most popular members of a network). the resulting number of contacts per individual takes a power-law distribution. the recent epidemic of severe acute respiratory syndrome (sars) was such an example where a significant proportion of the infections were due to 'super-spreaders' [42] . these networks resemble patterns observed in the spread of information along social networks and obesity in geographic areas giving rise to amazing patterns of spread [43] . characterizing outcomes of the human condition (including health inequalities) as patterns that emerge in a complex adaptive system has several implications. firstly, isolating health outcomes from the other human conditions are problematic because they are features or dimensions of a larger picture and therefore more sophisticated forms of systems analyses will be required to shed light on sdhi. secondly, emerging patterns of a complex adaptive system cannot be estimated with precision by using the known interactions among its components (such as economic development, political freedom, health system, culture etc.). different combinations of the same bundle of factors or determinants can give rise to similar patterns or outcomes (i.e. property of convergence), and minor variations in the initial condition could give rise to widely divergent outcomes. this questions the ability to generalize with precision, the determinants of health outcomes from one contextual environment to another. thus the favoured models would be those developed for the particular context using longitudinal data and using systems dynamics of feedback and interrelationships. finally, computer simulation models (e.g. agent-based models) would shed light on possible mechanisms as to how factors or determinants interact and lead to emergent patterns of health outcomes of populations. the metaphor needs to shift from the current visualizations of health outcomes as rigid bar diagrams demonstrating the step-wise gradient to a more vibrant multi-coloured canvass with changing hues. complex systems analysis: towards holistic approaches to health systems planning and policy conceptualising population health: from mechanistic thinking to complexity science who, and what, causes health inequities? reflections on emerging debates from in exploratory latin american/north american workshop sick individuals and sick populations a conceptual framework for action on the social determinants of health. social determinants of health discussion paper 2 policy and practice. geneva: world health organization social organization, stress, and health biology and health inequality commentary: understanding it all-health, meta-theories, and mortality trends social determinants of health: present status, unanswered questions, and future directions theories for social epidemiology in the 21st century: an ecosocial perspective epi + demos + cracy: linking political systems and priorities to the magnitude of health inequalities evidence, gaps and a research agenda stress and anxiety across the lifespan: structural plasticity and epigenetic regulation socioeconomic status and telomere length: the west of scotland coronary prevention study is socioeconomic status associated with biological aging as measured by telomere length? commission on social determinants of health. closing the gap in a generation: health equity through action on the social determinants of health. geneva: world health organization tackling the wider social determinants of health and health inequalities: evidence from systematic reviews housing improvements for health and associated socio-economic outcomes health inequality: an introduction to theories, concepts and methods multilevel analysis in public health research next steps in understanding the multilevel determinants of health simulation models for socioeconomic inequalities in health: a systematic review general system theory the real wealth of nations: pathways to human development analyzing microarray data using cluster analysis multimorbidity in heart failure: a community perspective identification of complex metabolic states in critically injured patients using bioinformatics cluster analysis impaired pulsatile secretion of insulin in relatives of patients with non-insulin-dependent diabetes crowd-sourcing: novel childhood predictors of adult obesity twitter: a good place to detect health conditions system dynamics modeling for public health: background and opportunities integrating social and biological factors in health research: a systems view methods in public health: dynamics, networks, and agents a systems-oriented multilevel framework for addressing obesity in the 21st century emergence: from chaos to order. massachusetts: persus books think complexity a new kind of science dynamic models of segregation theory of self reproducing automata the small world problem epidemic spreading in scale-free networks transmission dynamics of the etiological agent of sars in hong kong: impact of public health interventions submit your next manuscript to biomed central and take full advantage of: â�¢ convenient online submission â�¢ thorough peer review â�¢ no space constraints or color figure charges â�¢ immediate publication on acceptance â�¢ inclusion in pubmed, cas, scopus and google scholar â�¢ research which is freely available for redistribution the author has no financial or non-financial competing interests (political, personal, religious, ideological, academic, intellectual, commercial or any other) to declare in relation to this manuscript. the author is responsible for conceptualising the issues in the article and writing the manuscript. the author qualified with a mbbs (hons) from university of colombo, and was awarded md (colombo), frcp (london) and md (bristol). he is an academic in the university of colombo, sri lanka, and a practicing physician at the national hospital of sri lanka, colombo. he has a special interest in social determinants of health, poverty, health equity, and the application of complexity science to health. key: cord-029674-qcu7vm05 authors: ioannidou, alexandra; desjardins, richard title: the political economy of adult learning systems date: 2020-07-24 journal: zfw doi: 10.1007/s40955-020-00166-z sha: doc_id: 29674 cord_uid: qcu7vm05 nan spheres of political economies (pp. 25-26) . the exploration of the linkages between skills and nation-specific institutions such as labour market, industrial relations as well as welfare state formed consequently one of the most interesting research strands in the comparative study of education and training in contemporary period: the political economy of skill formations regimes. the political economy of adult learning systems has been developed as an offshoot of the debates on comparative welfare state research and considers contributions from different disciplinary perspectives (political science, sociology, education economics) to understanding the causes and consequences of cross-national diversity in adult learning systems across developed democracies. the core argument in this research strand is that adult learning systems are embedded in specific economic and social arrangements, "they lie at the intersection of a variety of other systems including a nation's education and training system, labour market and employment system and other welfare state and social policy measures" (desjardins 2017, p. 232) . investigating the role of "institutional complementarities" or "institutional packages" in shaping adult learning systems contributes to the large body of literature triggered by the voc approach in comparative political economy of skill formation regimes (mayer and solga 2008; busemeyer and trampusch 2012) . yet, analyses of the impact of institutional settings on adult learning systems build at present a relatively small research strand, which mainly focuses at explaining differences in participation in adult learning (cf. blossfeld et al. 2014; roosma and saar 2016) . very little scholarship deals with exploring variation in adult learning systems on the grounds of existing institutional complementarities and how the latter affect patterns of coordination and outcomes of adult learning systems (desjardins and rubenson 2013; desjardins 2017) . this issue of the zeitschrift für weiterbildungsforschung focusing on the political economy of adult learning systems aims at furthering this debate and addressing questions dealing with the role of diverse institutions in framing adult learning systems. this includes questions on the impact of institutional structures on participation, of governance modes and policy interventions on outcomes of adult learning systems or issues related to the political economy of educational research. the topic met high interest and attracted sound scholarly work from all over the world. richard desjardins and alexandra ioannidou introduce in their fokus-article "the political economy of adult learning systems: some institutional features that promote adult learning participation" into the interdisciplinary research strand of the political economy of adult learning systems. they review recent multi-disciplinary work and different typologies that have emerged out of the field of comparative economics and comparative politics and show how this work has influenced adult education research. drawing on quantitative analysis of piaac, ials and other oecd data, they provide empirical evidence on cross-national patterns of organized adult learning. this evidence reveals shortcomings in the explanatory framework of existing typologies from comparative welfare state and comparative politics research, despite their virtues. desjardins & ioannidou suggest that there are some specific institutional features that seem to be more proximally related to adult learning systems k and appear to play a leading role in explaining the cross-national patterns of variation in the take up, provision and distribution of organised adult learning. among them: open and flexible formal education structures, public support for education, active labour market policies and programmes that target socially disadvantaged adults. explaining variation in participation in adult learning across countries as well as within countries is a challenge for researchers. improvements with regard to availability and accessibility of international comparable data and advancements in empirical methods enable new insights in this respect. the next two contributions by miroslav stefanik and veronika philipps deal with explaining cross national participation patterns in adult learning in europe focusing specifically on the participation of disadvantaged groups. miroslav stefanik in his contribution "multi-layered perspective on the barriers to learning participation of disadvantaged adults" takes a closer look at disadvantaged adults and proposes a multilevel explanatory model that aims to explain the variability behind participation in adult learning. departing from available theoretical and empirical research on constraints with regard to participation in adult learning, he explores the barriers to participation among disadvantaged adults across europe. he focuses on employed adults, specifically on three vulnerable sub-groups: low-skilled; young and low-skilled, and immigrants. he uses microdata from the european union labour force survey (eu-lfs) for 28 european countries and considers also regional variability by applying a multilevel modelling technique. his model groups the explanatory variables into individual-level determinants, household-level, jobrelated, employer-level related as well as system-level characteristics. comparing the results across the vulnerable groups and types of determinants yields interesting insights in understanding the variability in adult learning participation across europe. veronika philipps in her contribution "job-related further education of the elderly in europe: do institutions matter?" investigates country differences in the participation in further education for older people from an institutional-theoretical perspective. departing from the empirical evidence that people in their late employment age are rarely involved in job-related further education in europe she poses the question whether institutions matter. in line with the literature she argues that institutions function in conjunction with one another and that institutional configurations of the labour market and education system account for the relative disadvantages of older people in participation in further education. she identifies four combinations of factors and examines her hypotheses with qualitative comparative analysis (qca) drawing on data from the european union adult education survey (aes) in 26 countries. in contrast to other quantitative methods, the qca allows for examining not only the influence of isolated factors but also the effect of combinations of institutions. the results of her analysis illustrate how important institutional configurations are for the explanation of disparities in further education. in particular, the existence of far-reaching state and well-developed company structures concerning the provision of continuing education as well as a dual vocational training system are crucial for the appearance of relatively lower disadvantages for older people in a given country. the next two contributions deal with policy changes and educational reforms in adult learning systems in three different countries: switzerland, canada and aotearoa new zealand. michael geiss reconstructs in his contribution "in steady search for optimization: the role of public and private actors in switzerland's political economy of adult education" the changes in the political economy of adult education in switzerland since the middle of the 20th century. he argues that, contrary to the literature, the swiss adult education system is neither exclusively a market-led nor a stakeholder-led regime and deals with the contradictory accounts of the swiss adult education system. he shows that alongside with the ongoing power of the employers, industry and professional associations, and the liberal-minded national authorities in switzerland, there are several other corporate actors that played an important role in recent developments in the swiss adult education system. the article underlines that "history matters" and that the current situation cannot be understood without considering the several government initiatives since the 1970s as well as the efforts of the private national continuing education association sveb. it concludes with reflections on the expected impact of the first national law on continuing education, which was enacted in 2017. methodologically, the analysis is based on historical institutionalism and concentrates on path dependencies and critical junctures. judith walker examines in her contribution "comparing adult education 'systems': canada and aotearoa new zealand" recent policy initiatives in adult education and training in the two countries in relation to previous political and educational reforms, taking an explicit comparative perspective. both canada and aotearoa new zealand are liberal market economies (lme) with high standards of living, high levels of education, and long histories in the evolution of non-formal adult education. the reforms in both countries share many similarities with their emphasis in skill development, increasing immigration and cultural diversity, and a need to address the legacy of colonisation and marginalisation of indigenous populations. yet, walker assigns a dearth of adult education infrastructure in canada as well as insufficient coordination across the institutions due to a fragmented fiscal federalism. in contrast, new zealand's centralised policy making, extreme neoliberal reforms of the 1980s and 1990s and subsequent reaction to them during the following decades, resulted in the creation of structures and institutions that allowed for a highly coordinated, regulated, professionalised, and centralised adult education system. walker's contribution is in the tradition of comparative education research, which takes the nation state as unit of analysis and shows how two lmes prioritise and approach adult education and training in relation to their unique socio-cultural-historical-political contexts. this reveals also in their reaction to the covid-19 pandemic. michael schemmann, dörthe herbrechter and martina engels choose a different approach in their contribution about "researching the political economy of adult learning systems: theoretical amendments and empirical findings". their focus lays with the level of adult education organisations, which is hardly taken into account when analysing the political economy of adult learning systems. they introduce neo-institutional ideas to specify aspects of the institutional environment of adult education organizations and outline how these theoretical amendments complement the political economy approach and the governance perspective. by re-analysing interview data from public adult education organizations in germany they aim at exploring the potential of their theoretical amendments. their findings provide first empirical indications that the institutional conditions that shape adult learning systems can be further specified with regard to the adult education organizations and their institutionally embedded interdependency relationships with other (organized) actors in their environment. finally, shalini singh looks at the political economy of adult educational research focusing on the "impact of international large-scale assessments on adult education systems". she takes as a starting point the fact that international largescale assessments (ilsas) have become a predominant tool for the measurement of learning outcomes and the sustainability of education systems all over the globe, often despite resistance from several stakeholders. she claims that especially after the adoption of the sustainable development goals (sdgs) in 2015 ilsas influence state policies directly or indirectly, irrespectively of whether states participate in them or not. she discusses the role of ilsas in shaping education systems in general and adult education systems in particular. her methodology includes document analysis, comparative analysis of four ilsas and the indicators developed by the global alliance to monitor learning (gaml) as well as mapping of policy linkages between ilsas and employability policies. the selected ilsas include the programme for the international assessment of adult competencies (piaac) by the oecd, the skills towards employability and productivity (step) survey by the world bank group (wbg), school-to work transition survey (stws) by the ilo and literacy assessment and monitoring program (lamp) by the unesco institute of statistics (uis). the paper views the changes in adult education systems as a part of global policy shift led by the oecd with ilsas being some of the effective catalysts and acting as windows of opportunity for introducing changes in the education systems. together, these contributions on the main topic help to highlight that theory building and comparative empirical evidence are indispensable for furthering the development of this research strand. we hope that we were able to contribute to these efforts with this issue and would like to thank the editors and the editorial team of the zfw for their support. in the rubric forum, this issue presents two articles. in their contribution "assessing media pedagogical competence of adult education teachers", bernhard schmidt-hertha and matthias rohs address the (digital) media competence of adult educators, a topic that acquired an increasing importance due to the corona pandemic. it is surprising that despite the intensive academic discussion on the topic, it remains largely unclear what can be understood by 'media-pedagogical competencies'. this includes the question whether the concept describes a specific competence or rather a cross-sectional competence. to develop a structural model of digital media competence, the authors proceed in two steps: first, they engage with the international theoretical discussion; then, they include the specific practical knowledge of experts in adult education. the structure of the model comprises the following facets: field competence, subject-related media competence, subject-didactic competence, pedagogical competence as well as attitudes and self-monitoring. this model (except for media-related competence in different subjects) is tested by using a sam-ple of more than 600 adult education teachers. the empirical results largely confirm the assumptions of the developed model. the article "reversing the matthew principle-adult education in the context of low numeracy" by luise krejcik and anke grotlüschen addresses another topic that has been intensely discussed for many years in educational research generally as well as in adult education research: the so-called matthew effect that points to the relation between prior educational experience and participation in adult education. this effect has been confirmed by research multiple times. using a re-analysis of piaac-data, the authors examine adults with low numerical skills in order to find out whether this effect may be reverse if one does not take into account only participation, but the volume of participation in adult education. the re-analysis shows that both on an oecd-average and in the majority of the nordic countries, adults with low numerical skills tend to participate less in further education than adults with high numerical skills. but if they participate, the duration of participation is longer than average, also if sociodemographic and occupational factors are controlled. the authors discuss distinct country-specific differences in the context of different welfare state policies. adult learning in modern societies. an international comparison from a life-course perspective the political economy of collective skill formation political economy of adult learning systems. comparative study of strategies, policies and constraints participation patterns in adult education: the role of institutions and public policy frameworks in resolving coordination problems varieties of capitalism. the institutional foundations of comparative advantage skill formation: interdisciplinary and cross-national perspectives comparing adult learning systems. an emerging political economy adults who do not want to participate in learning. a cross-national european analysis of their perceived barriers open access this article is licensed under a creative commons attribution 4.0 international license, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the creative commons licence, and indicate if changes were made. the images or other third party material in this article are included in the article's creative commons licence, unless indicated otherwise in a credit line to the material. if material is not included in the article's creative commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. to view a copy of this licence, visit http://creativecommons.org/licenses/by/4. 0/. key: cord-027304-a0vva8kb authors: achermann, guillem; de luca, gabriele; simoni, michele title: an information-theoretic and dissipative systems approach to the study of knowledge diffusion and emerging complexity in innovation systems date: 2020-05-23 journal: computational science iccs 2020 doi: 10.1007/978-3-030-50423-6_19 sha: doc_id: 27304 cord_uid: a0vva8kb the paper applies information theory and the theory of dissipative systems to discuss the emergence of complexity in an innovation system, as a result of its adaptation to an uneven distribution of the cognitive distance between its members. by modelling, on one hand, cognitive distance as noise, and, on the other hand, the inefficiencies linked to a bad flow of information as costs, we propose a model of the dynamics by which a horizontal network evolves into a hierarchical network, with some members emerging as intermediaries in the transfer of knowledge between seekers and problem-solvers. our theoretical model contributes to the understanding of the evolution of an innovation system by explaining how the increased complexity of the system can be thermodynamically justified by purely internal factors. complementing previous studies, we demonstrate mathematically that the complexity of an innovation system can increase not only to address the complexity of the problems that the system has to solve, but also to improve the performance of the system in transferring the knowledge needed to find a solution. formalization and organization of a network becomes strategic to accelerate the flow of information and knowledge and the emergence of innovation [2] . several forms of reticular organization (hierarchical, heterarchical, according to the centrality of elements, according to the transitivity of element, etc.) can be conceptualized within that context. evolutionary economics and technology studies highlight (neo-schumpeterian) models to understand the plurality of evolution cases, depending on the initial forms of organization, but also on the ability of a system to adapt to systemic crises. in this work we study, from an information-theoretical perspective, the relationship between the structure of an innovation network, the noise in its communication channels and the energy costs associated with the network's maintenance. an innovation network is here considered to encompass a variety of organisations who, through their interactions and the resulting relationships, build a system conducive to the emergence of innovation. this system is identified by the literature [3] with different terms, such as innovation ecosystem, [4] problem-solving network, [5] or innovation environment [6] . in this system, the information channels transfer a multitude of information and knowledge which, depending on the structural holes, [7, 8] but also on the absence of predetermined receivers, are subject to information "noise" [9] . the more the information is distorted in the network, the more energy is needed to transfer accurate information, in order to keep performance of the innovation network high. the idea we propose is that the structure of an innovation system evolves to address the heterogeneity in the quality of communication that takes place between its members. in particular, we argue that the noise in a network increases the complexity of the network structure required for the accurate transfer of information and knowledge, and thus leads to the emergence of hierarchical structures. these structures, thanks to their fractal configuration, make it possible to combine high levels of efficiency in the transmission of information, with low network maintenance costs. this idea complements previous studies that have analysed the relationship between the structure of an innovation network, on one hand, and the complexity of the problem to be solved and the resulting innovation process, on the other, [10] by focusing on communication noise and cost of network structure maintenance. to the existing understanding of this phenomenon we contribute by identifying a thermodynamically efficient process which the network follows as it decreases in entropy while simultaneously cutting down its costs. this model is based on the analysis of a network composed of two classes or categories of organisations, which operate within the same innovation system [11] . these classes are represented by a central organisation called seeker, which poses a research question to a group of other organisations, called problem-solvers, and from which in turn receives a solution. it has been suggested [12] that one of the problems that the innovation system has to solve, and for which it self-organises, is the problem of effective diffusion of knowledge between problem-solvers and solution-seekers, as this can be considered as a problem sui generis. the theory on the diffusion of knowledge in an innovation system suggests that this problem is solved through the evolution of modular structures in the innovation network, which implies the emergence of organisations that act as intermediary conduits of knowledge between hyperspecialised organisations in the same innovation environment [13] . a modular structure is, in network theory, connected to the idea of a hierarchical or fractal structure of the network, [14] and is also characterised by scale-invariance; [15] the latter is a particularly important property, because if innovation systems have it as an emergent property of their behaviour, this allows them to be considered as complex adaptive systems [16] . it has been suggested that scale-invariance property of an innovation system might emerge as the result of horizontal cooperation between its elements, [17] which try to reach the level of complexity required to solve a complex problem; but it is not yet clear how does a complex structure emerge when the complexity of the problem does not vary, which is a phenomenon observed empirically [18, 19] . in this paper we show how complexity can also vary as a result of a non-uniform distribution of the cognitive distance between organisations of the network, and of the adaptation required to solve the problem of knowledge diffusion among them. our contribution to the theoretical understanding on the self-organising properties of innovation systems is that, by framing the problem of heterogeneous cognitive distance between organisations under the theory of dissipative systems, we can explain in thermodynamically efficient terms the reduction in entropy of an innovation system, as an emergent adaptation aimed at reducing costs of maintenance of the system's structure. the theoretical framework which we use for this paper is comprised by four parts. first, we will frame the innovation system as a thermodynamically-open system, which is a property that derives from the fact that social systems also are [20] . second, we will see under what conditions a system can undertake self-organisation and evolution. this will allow us to consider an innovation system as a complex adaptive system, should it be found that there are emergent properties of its behaviour which lead to an increase in complexity. third, we will frame the innovation system as a dissipative system, which is a property also shared by social systems [21] . dissipative systems are characterised by the fact that a variation in the level of their entropy tends to happen as a consequence of their changed ability to process inputs, and we will see how this applies for innovation systems. lastly, we will study cognitive distance as it applies to a network of innovation, in order to show how a spontaneous reduction in it leads to an increase in complexity of the network. systems. an open thermodynamic system is defined as a system which exchanges matter and energy with its surrounding environment, [22] and among them are found all social systems, which are open systems due to their exchanging of energy with the surrounding environment [23] . social systems are also dynamical systems, because their structure changes over time through a process of dynamical evolution [24] . innovation systems are some special classes of social systems, [25] which can thus also be considered as open systems [26] . in addition to this, like all social systems, innovation systems are also capable of selforganisation, [27] which is a property that they inherit from social systems [28] . there is however a property which distinguishes innovation systems from the generic social system: that is, the capacity of the former to act as problem-solving environments [11] . an innovation system possesses the peculiar function of developing knowledge, [29] which is not necessarily possessed by the general social system [30] . it has been theorised that developing and distributing knowledge [31] is the method by which the innovation system implements the function of solving problems, [32, 33] and we will be working within this theoretical assumption. the innovation system, for this paper, is therefore framed as a thermodynamically-open social system which solves problems through the development and diffusion of knowledge. evolution and self-organisation. like all other social systems, [34] an innovation system undertakes evolution [35] and changes in complexity over time [36] . the change in complexity of a system, in absence of any central planning or authority, is called in the literature self-organisation [37] . self-organisation in a system implies that the system's components do not have access to global information, but only to information which is available in their immediate neighbourhood, and that upon that information they then act [28] . innovation systems evolve, with a process that may concern either their members, [38] their relationships and interactions, [39] the technological channels of communication, [40] the policies pursued in them, [41] or all of these factors simultaneously [42] . for the purpose of this work we will limit ourselves to consider as evolution of an innovation system the modification of the existing relationships between its members, and the functions which they perform in their system. this process of evolution of the innovation system is characterised by self-organisation, [43] and it occurs along the lines of both information [44] and knowledge flows within the system [45] . the selforganisation of an innovation system is also the result of evolutionary pressures, [46] and we will here argue that one form of such pressures is cognitive distance between organisations within a network of innovation, whose attempt at reduction may lead to modifications in the relationships within the system and to the emergence of complex structures. while it has also been suggested that variations in the complexity of an innovation system might be the consequence of intrinsic complexity of the problems to be solved, [47] it has also been suggested that problems related to the transfer of knowledge within the elements of the system can, by themselves, generate the emergence of complex network structures, through a process which is thermodynamically advantageous. dissipative innovation systems. as the innovation system acquires a more complex structure, its entropy decreases. if one assumes that the decrease in entropy follows the expenditure of some kind of energy by the system, without which its evolution towards a lower-entropy state is not possible, then it follows that the innovation system can be framed as a dissipative system. this is a consequence of the theory which, in more general terms, suggests that all social systems can be considered as dissipative systems; [48] and, among them, innovation systems can thus also be considered as dissipative systems [49] . the application of the theory of dissipative structures [50] to the study of social systems has already been done in the past, [51, 52] and it has also been applied to the study of innovation systems specifically, [53, 54] to understand the process by which new structures evolve in old organisational networks [55] . by framing the problem in this manner the emergence of a hierarchical structure in a dissipative innovation system can be considered as a process through which the innovation system reaches a different level of entropy in its structure, [56] by means of a series of steps which imply sequential minimal variations in the level of entropy of the system, and lead to the emergence of complexity [57] . cognitive distance as noise. the process of transferring knowledge between organisations presumes the existence of knowledge assets that are transferred [58] . companies embedded in an innovation system are therefore characterised by an intellectual or knowledge capital, [59] which is the sum of the knowledge assets which they possess, [60] and which in turn are the result of the individual organisation's path of development, [61] and of the knowledge possessed by the human and technological components of the organisation [62] . any two organisations do not generally share the same intellectual capital, and therefore there are differences in the knowledge assets which they possess, and in the understanding and representation which they create about the world. this difference is called "cognitive distance" in the literature on knowledge management, and it refers to the difficulty in transferring knowledge between any two organisations [63] . the theory suggests that an innovation network has to perform a trade-off between increasing cognitive distance between organisations, which means higher novelty value, and increasing mutual understanding between them, which gives higher transfer of knowledge at the expenses of novelty [64] . it has been argued that if alliances (that is, network connections) are successfully formed between organisations with high cognitive distance between their members, this leads to a higher production of innovation by that alliance, [65] as a consequence of the relationship between cognitive distance and novelty, as described above. it has also been argued that the measure of centrality of a organisation in an innovation network is a consequence of the organisation's impact on the whole knowledge governance process, with organisations contributing more to it located more centrally in the network [66] . we propose that this known mechanism might play a role in the dynamic evolution of an innovation system, in a manner analogous to that of noise in an information system. the idea is that an organisation generally possessing a lower cognitive distance between multiple components of a network might spontaneously become a preferential intermediary for the transfer of knowledge within the innovation system, and as a consequence of this a hierarchical network structure emerges out of a lower-ordered structure. 2 the structure of the network and its evolution the modeling of the process of evolution of a network of innovation is conducted as follows. first, we imagine that there are two different structures of the ego-network of an innovation seeker company that are the subject of our analysis. the first is a horizontal network, in which a seeker organisation is positioned in a network of solvers, which are all directly connected with the seeker organisation in question. the second is a hierarchical or fractal network, in which a structure exists that represents the presence of intermediaries in the transfer of knowledge between the seeker organisation and the solving organisations in the same network. all nodes besides the seeker organisation being studied in the first scenario, and all nodes at the periphery of the hierarchical structure of the second scenario, are from here on called solvers. there are n nodes in the ego-network of an innovation seeker company. the n nodes in the horizontal network are all solver nodes, while the n nodes in the hierarchical network are divided into two classes of nodes: the intermediaries comprised of m nodes, and the solvers, comprised of m 2 nodes (fig. 1) . in order to make the two network structures comparable we impose the additional condition that the total number of nodes in the two networks is the same, which is satisfied for n ¼ m 2 þ m. we also impose the additional condition that each of the n solver nodes in the periphery of the horizontal network has at least m link neighbours belonging to n, as this allows us to describe a dynamical process which leads from the horizontal network to the hierarchical network without the creation of new links. the hierarchical network always possesses a lower entropy than the horizontal network comprised of the same number of nodes. this can be demonstrated by using as a measure of entropy shannon's definition, [67] which calculates it as the amount of information required to describe the current status of a system, accordingly to the formula below: this measure of entropy can be applied to a social network by assigning the random variable x to the flattened adjacency matrix of the edges of the network, as done by others [68] . the adjacency matrices of the two classes of networks in relation to the size n + 1 of the same network are indicated in the tables below, for the specific values m ¼ 2 ! n ¼ 6 ( table 1) . in general, for any value m ! 2, if a horizontal network has n solver nodes, one seeker node is connected to all other n nodes, and all solver nodes are additionally connected to m solver nodes each, where m þ m 2 ¼ n. in a hierarchical network with m intermediary nodes and m 2 solver nodes, the seeker node is connected to m intermediary nodes, and each of the intermediary nodes is connected to m solver nodes. the general formulation of the adjacency matrix is indicated below, in relation to the value of m ( table 2 ). the adjacency matrices can be flattened by either chaining all rows or all columns together, in order to obtain a vector x which univocally corresponds to a given matrix. this vector has a dimensionality of n þ 1 ð þ 2 , having been derived from an n + 1 by n + 1 matrix. the vector x which derives from flattening can then be treated as the probability distribution over a random binary variable, and shannon's measure of entropy can be computed on it. for the horizontal network, the vector x horizontal has value 1 two times for each of the peripheral nodes because of their connection to the centre, and then again twice for each of the peripheral nodes. this means that the vector x horizontal corresponds to the probability distribution (2). for the hierarchical network, the vector x hierarchical has value 1 two times for each of the m intermediary nodes, and then 2 times for each of the m 2 nodes. the probability distribution associated with the vector x hierarchical is therefore (3) the hierarchical network systematically possesses a lower level of entropy than a horizontal network with the same number of nodes, as shown in the graph below (fig. 2) . since we consider the network as a dissipative system, the lower level of entropy implies an expected higher energetic cost of maintenance for the lower-entropy structure. it follows from this theoretical premise that the hierarchical network should either allow the system to receive a higher input, or emit a lower output, or both simultaneously, lest its structure would decay to a higher entropy form, the horizontal one. an innovation system which starts evolving from a horizontal structure would tend to develop a hierarchical structure as a solution to the problem of transfer of knowledge in a network where cognitive distance is not uniformly distributed, as we will see in this paragraph. this can be shown by considering the hierarchical network as an attractor for the dynamical evolution of a horizontal network, under condition that the cognitive distance between pairs of nodes is distributed non-uniformly. stationary states. for the context of this paper, as we model a finite-state network which operates on discrete time, which models the dynamics of a dissipation systems which evolves over time [69] . these functions have the form depicted below, with x k ð þ being the state of the system at time k, u k ð þ being the input to the system at k, and y k ð þ being the output of the system. if the system does not undertake change in its internal structure, having already reached a stationary state, then x k þ 1 ð þ¼x k ð þ. as we want to study whether the system spontaneously evolves from a horizontal to a hierarchical structure, we can assume that x k þ 1 ð þ¼f hiearchical x k ð þ; u k ð þ ð þ¼x k ð þ which can only be true if either the input u k ð þ is 0, which is not the case if the system is active, or if u k þ 1 ð þ¼u k ð þ. for the innovation system this condition is valid if minor variations in the structure of the network associated with it do not lead to a significant variation of the input to the system, which means that no advantages in the receipt by the seeker of solutions found by the solver should be found. if this is true, and if the hierarchical structure is an attractor for the corresponding horizontal network, then we expect the input of the horizontal network to increase as it acquires a modular structure and develops into a hierarchical network. input of the system. the input function of the system depends on the receipt by the seeker organisation of a solution to a problem found by one of the peripheral solver organisations, as described above. let us imagine that at each timestep the solver organisations do indeed find a solution, and that thus the input u k ð þ depends on the number of solver nodes, and for each of them on the probability of correct transmission of knowledge from them to the seeker organisation, which increases as the cognitive distance between two communicating nodes decreases. if this is true, then the input to the horizontal network is a function of the form u horizontal n k ; p k ð þ, where n is the number of solver nodes, and p is the cognitive distance in the knowledge transmission channel. similarly, the input to the hierarchical network is a function of the form u hierarchical m 2 k ; q k à á which depends on the m 2 solver nodes in the hierarchical network, and on the parameter q which describes the cognitive distance. n and m are such that as they increase so do, respectively, u horizontal and u hierarchical ; while p and q are such that, as they decrease, so do respectively u horizontal and u hierarchical increase. it can be also noted that it can then be argued that if p q then u horizontal [ u hiearchical , which means that the system would not evolve into a hierarchical network. it can also be noted that, if n and m 2 are sufficiently large, then lim n;m! þ 1 n m 2 à á ¼ 1 and therefore any difference between the number of solvers in the two network structures would not play a role in the input to the innovation system. from this follows that u hiearchical [ u horizontal ! q \ p; that is, that the input to the innovation system with a hierarchical structure is higher than the input to the innovation system with a horizontal structure, if the cognitive distance between the members of the former is lower than the cognitive distance between the members of the latter. output of the system. as per the output of the system, we can imagine that there is a cost to be paid for the maintenance of the communication channels from which the seeker receives solutions from the solvers. if the system is in a stationary state, the condition y k þ 1 ð þ¼y k ð þ must be valid, as it follows from the considerations that u k þ 1 ð þ¼u k ð þ. if the system is not in a stationary state, as the input to the system increases, so should the output, under the hypothesis of dissipative system described above. a graphical representation of the evolution of the system from higher to lower entropy state is thus presented below (fig. 3) . the seeker organisation would at each step receive a solution transferred by one of its link neighbours, with the indication of the full path through which the communication has reached it. the seeker would then pay a certain cost, an output with the terminology of dissipative systems, for the maintenance of the channel through which the solution has been transferred to it successfully. such channels increase in intensity or weight, and are more likely to be used in subsequent iterations. on the contrary, channels through which a solution has not been received in a given iteration are decreased in intensity or weight, and are less likely to be used in the future. a process such as the one described would eventually, if enough iterations are performed, lead to the withering links between nodes with higher cognitive distance, and to the preservation of links between nodes with a lower cognitive distance. new connections are not formed, because cognitive distance is considered to be an exogenous parameter in this model, which does not vary once the innovation system starts evolving. real-world phenomena are not characterised by this restriction, which should be considered when analysing real-world systems under this model. the originality of this paper consists in the framing of an innovation system under different theoretical approaches, such as that of thermodynamically-open systems, selforganisation and evolution, dissipative systems, and cognitive distance, which, when combined, highlight another way of understanding the overall operation and the evolution of innovation systems. from this perspective, the process which we here describe evolu on hierarchical network fig. 3 . evolution of a branch of the innovation network from a higher to a lower entropy structure, from left to right. the letters p and q define respectively a high and a low cognitive distance between peers. accounts for an emergent complexity of the innovation system, which can occur without central planning and on the basis of information locally available by its members. this seems to confirm the theory according to which innovation systems can self-organise to solve, among others, the problem of transfer of knowledge among their members. this seems also to suggest that, if the only form of proximity which matters is cognitive, and not geographical, organisational, or other, it might be possible to infer cognitive distance between the members of an innovation system on the basis of the way in which their relationships change over time. the theoretical prediction which this model allows to make is that, should a connection between members of an innovation system be preserved while others are dropped, this means that the cognitive distance between pairs of nodes with surviving connections is lower than that of other nodes in their ego-networks. the modelling of the evolution of an innovation system that we propose also shows that, if an innovation system starts its evolution with a centrally, highly-connected organisation in a largely horizontal network of solver, where the cognitive distance between each pair of nodes is not uniformly distributed, then the system would evolve towards a lower-entropy hierarchical structure, in order to solve the problem of transfer of knowledge from the organisations at the periphery of the innovation system to the central organisation. our finding is consistent with the theory on modularity as an emergent property of complex adaptive innovation systems. subsequent research might apply the mathematical model described in this paper to a longitudinal study of the evolution of real-world innovation networks, in order to test whether the theory related to the spontaneous emergence of a hierarchical structure of innovation networks can be empirically supported. on the theoretical plane, further research could expand the understanding of the evolution of an innovation network by adding considerations related to the role which geographical and organisational proximity have in the development of the network, and add these factors to the model proposed. issues related to perturbation of the network, limit cycle of its evolution, and self-organised criticality in connection to our model may also be explored in subsequent works. sociologie et épistémologie evolution and structure of technological systems -an innovation output network leveraging complexity for ecosystemic innovation what is an innovation ecosystem networks for innovation and problem solving and their use for improving education: a comparative overview value creation from the innovation environment: partnership strategies in university spin-outs structural holes: the social structure of competition knowledge management, intellectual capital, structural holes, economic complexity and national prosperity innovation as a nonlinear process, the scientometric perspective, and the specification of an 'innovation opportunities explorer the role of the organization structure in the diffusion of innovations innovation contests, open innovation, and multiagent problem solving network structure and the diffusion of knowledge the limits to specialization: problem solving and coordination in "modular networks hierarchical organization in complex networks scale-free and hierarchical structures in complex networks what is a complex innovation system? cooperation, scale-invariance and complex innovation systems: a generalization growing silicon valley on a landscape: an agent-based approach to high-tech industrial clusters developing the art and science of innovation systems enquiry: alternative tools and methods, and applications to sub-saharan african agriculture prigogine's model for self-organization in nonequilibrium systems the evolution of dissipative social systems the meaning of open systems social systems knowledge, complexity and innovation systems institutional complementarity and diversity of social systems of innovation and production thermodynamic properties in the evolution of firms and innovation systems networks, national innovation systems and self-organisation self-organisation and evolution of biological and social systems functions of innovation systems: a new approach for analysing technological change on the sociology of intellectual stagnation: the late twentieth century in perspective disciplinary knowledge production and diffusion in science a knowledge-based theory of the organisation-the problemsolving perspective thinking: a guide to systems engineering problem-solving social complexity: patterns, processes, and evolution the evolution of economic and innovation systems a complexity-theoretic perspective on innovation policy emergence versus self-organisation: different concepts but promising when combined the evolution of innovation systems understanding evolving universityindustry relationships innovation as co-evolution of scientific and technological networks: exploring tissue engineering from technopoles to regional innovation systems: the evolution of localised technology development policy perspectives on cluster evolution: critical review and future research issues innovation, diversity and diffusion: a selforganisation model social information and self-organisation self-organization, knowledge and responsibility the dynamics of innovation: from national systems and "mode 2" to a triple helix of university-industry-government relations the value and costs of modularity: a problem-solving perspective a dissipative network model with neighboring activation the analysis of dissipative structure in the technological innovation system of enterprises modern thermodynamics: from heat engines to dissipative structures lessons from the nonlinear paradigm: applications of the theory of dissipative structures in the social sciences self-organization and dissipative structures: applications in the physical and social sciences understanding organizational transformation using a dissipative structure model technological paradigms, innovative behavior and the formation of dissipative enterprises a dissipative structure model of organization transformation entropy model of dissipative structure on corporate social responsibility revisiting complexity theory to achieve strategic intelligence defining knowledge management: toward an applied compendium enterprise knowledge capital intellectual capital-defining key performance indicators for organizational knowledge assets the dynamics of knowledge assets and their link with firm performance management mechanisms, technological knowledge assets and firm market performance problems and solutions in knowledge transfer empirical tests of optimal cognitive distance industry cognitive distance in alliances and firm innovation performance the impact of focal firm's centrality and knowledge governance on innovation performance the mathematical theory of communication the physics of spreading processes in multilayer networks dissipative control for linear discrete-time systems key: cord-290856-6de0mwg0 authors: abbo, aharon (ronnie); miller, asaf; gazit, talya; savir, yonatan; caspi, oren title: technological developments and strategic management for overcoming the covid-19 challenge within the hospital setting in israel date: 2020-07-31 journal: rambam maimonides med j doi: 10.5041/rmmj.10417 sha: doc_id: 290856 cord_uid: 6de0mwg0 the coronavirus disease 2019 (covid-19) pandemic has remarkably challenged health care organizations and societies. a key strategy for confronting the disease implications on individuals and communities was based on harnessing multidisciplinary efforts to develop technologies for mitigating the disease spread and its deleterious clinical implications. one of the main challenging characteristics of covid-19 is the provision of medical care to patients with a highly infective disease mandating the use of isolation measures. such care is complicated by the need for complex critical care, dynamic treatment guidelines, and a vague knowledge regarding the disease’s pathophysiology. a second key component of this challenge was the overwhelming surge in patient burden and the relative lack of trained staff and medical equipment which required rapid re-organization of large systems and augmenting health care efficiencies to unprecedented levels. in contrast to the risk management strategies employed to mitigate other serious threats and the billions of dollars that are invested in reducing these risks annually by governments around the world, no such preparation has been shown to be of effect during the current covid-19 pandemic. unmet needs were identified within the newly opened covid-19 departments together with the urgent need for reliable information for effective decision-making at the state level. this review article describes the early research and development response in israel under the scope of in-hospital patient care, such as non-contact sensing of patients’ vital signs, and how it could potentially be weaved into a practical big picture at the hospital or national level using a strategic management system. at this stage, some of the described technologies are still in developmental or clinical evidence generation phases with respect to covid-19 settings. while waiting for future publications describing the results of the ongoing evidence generation efforts, one should be aware of this trend as these emerging tools have the potential to further benefit patients as well as caregivers and health care systems beyond the scope of the current pandemic as well as confronting future surges in the number of cases. the coronavirus disease 2019 (covid-19) pandemic, caused by the severe acute respiratory syndrome coronavirus 2 (sars-cov-2), has spread around the globe during the first half of 2020 and become a leading cause for mortality worldwide. an unprecedented multinational and multidisciplinary effort is being conducted to confront and minimize its implications on individuals and communities. health care systems have faced a rapidly spreading infection which required extreme isolation measures on one hand-yet providing mass, extensive, and highly professional critical care treatment amidst a new disease with unknown pathophysiology. while our understanding of the disease is advancing, and treatment guidelines are continuously being rewritten, the mortality rates of admitted patients are still high. the clinical manifestation includes a respiratory distress syndrome sometimes requiring long periods of mechanical ventilation, 1 accompanied by a cytokine storm. supportive care is challenging due to the long-term need for mechanical ventilation and the requirement for prone position ventilation and the associated clinical presentation: blood coagulation disorders, 1 such as venous thrombosis and pulmonary thromboembolic events, 2 renal failure, 2 and cardiac manifestations, such as myocarditis with myocardial injury, 2-4 right ventricular dysfunction, 1 and arrhythmias. 3 the complex clini-cal presentation, the extremely large number of patients (around 12.6 million infected worldwide and >562,000 deaths as of july 12, 2020), combined with a prolonged recovery period, 1 overwhelmed health care systems worldwide. all these mandated flexibility, agility, and adoption of new protocols and technologies across the board, from the field responders (health care personnel and health care system executives) to governmental agencies and policy makers. due to the high infection rate, multidisciplinary teams had to adapt quickly and make crucial (and sometimes unpopular) decisions in a short time while basing their decision-making on often unreliable, limited information. effective response had to be decisive and predicative to the chain of events within a narrow time window, facing an exponential disease spread, while the health of citizens and the economy were at stake. in contrast to the risk management strategies employed to mitigate military threats, or cyber or terrorist attacks, and the billions of dollars which are invested in reducing these risks annually by governments around the world, no such preparation has been shown to be of effect during the covid-19 pandemic in most countries. unmet needs were identified both within the newly opened covid-19 departments at the hospitals and within the branches of academia. the threat magnitude, combined with the globally forced economy shut-down, quickly and uniquely brought in strong players with extensive research and development capabilities: civil companies together with army units, large organizations such as army/aviation industries, and small startups-all trying to find solutions to the covid-19 crisis and its implications. this review article describes the early research and development response in israel under the scope of in-hospital patient care, and how it could potentially be weaved into a practical strategic big picture that could help confront the next wave or any upcoming health crisis. left out of this discussion are the technologies for disease diagnostics, personal protective gear solutions, and telemedicine aimed at caring for patients outside the hospitals. these are major topics affecting hospital congestion and deserve a separate discussion. the risk associated with disease spread to and through health care workers raised the need for telemetry of patients' status across rooms, often without direct line of sight to some or all patients. the traditional non-invasive measurements of vital signs-blood pressure, heart rate, temperature, respiration rate, and oxygen saturation-had to be transferred to a remote control room. the lack of a sufficient amount of telemetry monitors that would allow a wired or wireless transfer of data to the control room is a key challenge, especially when considering the relatively sudden and unexpected deterioration patterns associated with the disease. this challenge is valid for both critical patients and patients with moderate-to-severe disease. the covid-19 outbreak facilitated efforts to monitor vital signs without the need for physical contact as well as investigating old and new physiological measurements to allow appropriate patient monitoring. as body temperature distribution and respiratory dynamics are among the primary indicators for covid-19 infection, 1,5-7 monitoring both these indicators without contact of the subject with a sensor from a distance of a few meters is a key challenge. [8] [9] [10] inferring core body temperature at a medical resolution (an error of about 0.3°c) from afar (more than 2 meters) is not a trivial task as it requires an accurate estimation of the core body temperature based on the detection of the absolute tissue temperature. similarly, inferring remote and contactless respiratory dynamics is of paramount importance and a major challenge based on current technologies. thermal imaging is a method in which a camera's sensor translates infrared signals into images, which represent the heat emitted from the captured objects. 11 uncooled bolometric sensors can sense radiation from objects at a temperature of -40°c to 140°c and can map body and tissue surface temperature remotely. much research has been conducted in recent years in order to apply thermal imaging techniques for medical uses. 11 the advantages of thermal imaging as a diagnostic tool arise from the fact that it is a low-cost, non-invasive, safe, and fast method that (most importantly in the covid-19 case) does not require direct contact with the patient. besides fever detection, 10,12 thermal imaging videos were used to infer physiological parameters related to respiratory patterns, such as breathing rate and tidal volume, by monitoring temperature changes around the nostrils. [13] [14] [15] an additional use of thermal imaging that is currently under investigation is the assessment of peripheral perfusion. changes in peripheral perfusion could be used for the detection of pathologies associated with damaged blood flow and for early diagnosis of shock states. 11 hand temperature distribution 16 and foot thermal distribution were used to identify peripheral arterial disease. 17, 18 differences between peripheral organs (ears) and core body temperature 19 and thermal gradient along the arm 20 were used for shock diagnosis. one of the promising approaches to detect respiratory dynamics from a distance involves the usage of doppler radar. 21 these radars are not affected by factors such as temperature, light, and humidity but are extremely sensitive to motion and vibration in the vicinity of the subject. to overcome these limitations, a system that includes both doppler radar and thermal imaging was developed. 22 signal integration from both systems was conducted by harnessing machine learning methods. the usage of machine learning occurs on two levels, technical and semantic. on the technical level, it is being used to improve the measurement of the physiological parameters. for example, deep convolutional networks are used for inferring the core temperature based on videos alone without any biophysical model inputs. on the semantic level, clinical status is inferred by aggregating the dynamical data (e.g. the entire body temperature distribution and the respiratory dynamics rather than just common parameters such as core temperature and respiratory rate). the system could be used at the hospital/emergency room entrance in order to test visitors or new patients at a distance without the need for personnel contact, serving as a first contact triage point. the application can allow to similarly trace vital signs in indoor environments (e.g. in emergency rooms). this system was codeveloped during the covid-19 crisis by the israel aerospace industry (iai) and the technion and was applied at rabin medical center emergency room, where it was used for remote patient monitoring. another approach for remote vital sign monitoring was recently suggested by binah.ai (ramat-gan, israel), an approach relying solely on a smartphone camera either by taking a self-picture or by placing a finger over the camera for a few seconds. 23 another system utilizes a piezoelectric sensor integrated into a plate placed under the patient's bed mattress (earlysense, ramat-gan, israel). 24 the device senses the mechanical vibrations and movements created by the patient. those are analyzed in order to provide contact-free, continuous monitoring of bed-ridden patients and may help in early detection of patient deteriorations. it is prudent to wait for clinical validation of noncontact vital sign sensing of the above-mentioned systems. radar-based sensors (utilizing low-power radio frequency wavelength) are embedded in a system which is used for heart failure management by measuring lung fluid content (reds heart failure monitor™, sensible medical, netanya, israel). [25] [26] [27] the system measures the dielectric properties of tissues. low-power electromagnetic signals are emitted through the right mid-thorax, and the signals received after passing through the tissue reflect their combined dielectric properties. these, in turn, are mostly affected by the fluid content of the tissue in the path of the signal. the system is currently being investigated in covid-19 patients as a tool that could potentially assist in following up the respiratory status of the covid-19 patients and assist in managing those who require mechanical ventilation. the high covid-19 contamination risk, in addition to the shortage of protection gear and its encumbrance, led to a policy of limited entries into contaminated wards (usually one to two times during a 12-hour shift), in order to reduce staff-patient interactions unless an urgent need rises. this new condition in patient care required a new approach. when physicians and nurses contact the covid-19 patients the heavy protection gear limits visibility and hearing. the use of stethoscope or direct visualization of the nasopharynx is discouraged, thus restricting physical exam to observation alone. the use of existing ultrasound systems (point-of-care ultrasound [pocus], handheld) to assess patient pulmonary status and diagnose lung pathologies has been reported in covid-19 patients. 28, 29 audio and/ or visually enhanced scopes (tyto care, netanya, israel) help clinicians evaluate patients' cardiovascular, pulmonary, and upper respiratory tract status 30 and enable a complete direct bedside physical exam. this represents an example of how a tool invented and marketed for remote patient home monitoring became relevant as a bedside tool. the expected overwhelming surge of patients during this crisis, the lack of staff trained in the medical intensive care unit (micu), the requirement for operation within large halls (e.g. underground parking domains) using personal protective equipment (ppe), and the inherent uncertainties associated with a new pandemic may all coalesce to an austere environment with chaotic patterns of communication that may severely hamper the quality of care. consequently, all entries to a quarantined domain have to be planned and executed flawlessly in order to avoid unnecessary re-entering, much like a spacewalk or entering a battle-zone. similarly to these scenarios, there is a need for continuous and organized communication between the field teams at the ward or micu and the control and command center (located in the non-infected environment) that need to integrate and operate the logistics and supportive para-clinical arms. in israel, combat management strategies, communications, and other technologies were rapidly applied for the covid-19 environments. application of such technologies from the battle field to the medical field required mutual adaptations (of both the technology and medical teams) to maximize the operational capabilities of the team. the c 4 i™ system is a command and control tactical system integrating computing, communication, and intelligence information (for health care applications this applies for patient sensors), developed for military use by elbit systems. a collaborative effort between elbit and rambam health care campus modified the c 4 i system for medical purposes and developed ex-teams ™. ex-teams is a dual wifi and cellular and cloud-based communication and command platform. it was installed as a software app into push-to-talk mobile phones of all medical and para-clinical teams that operate in the hospital setting caring for covid-19 patients. a key component of the system is the transformation of medical teams to predefined communication groups organized according to the clinical setting with strict hierarchical communication rules, thereby reducing the levels of the aforementioned chaos (figure 1 ). the cornerstone of the sys-tem is that each clinical team automatically communicate with the relevant team members using pushto-talk group communication. in addition, the system allows location tracking and automated locationbased group switching, thereby allowing each member of the hospital clinical team arriving to a clinical domain to automatically join the relevant clinical team "micro-environment." the location-based system allows hospital clinical command to better conthe figure delineates the organized and hierarchal group communication within a covid-19 department comprising one unit using ex-teams™. the same set-up can be applied to more than one patient care unit within the department. the ex-teams communication network enables communication between the medical staff within the unit (black arrows). the intensive care unit's md (icu md) and rn (icu rn) move between the control center and the units, according to clinical needs or get a real time consultation (blue arrows) from within the infected environment (dash-dots blue line) from their colleagues in the control and command center located in a sterile and safe environment (dashed purple line). each person in the unit, working within the infected environment can also communicate with a hospital management representative (purple box) in the department level control and command center. for every employee in each unit, a location-based system allows calling into action the emergency team and helps them navigate to the exact location where they are needed (orange arrows). in addition, the system allows in-person communication with the paramedical services outside of exteams (not shown). icu, intensive care unit; md, medical doctor; mngmnt., management; rep., representative; rn, registered nurse; lpn, licensed practical nurse. trol key personnel (e.g. icu physicians) and to allocate them according to their location and the clinical need. the addition of a location-based communication may also help to monitor the staff efforts in each clinical section and to allocate resources accordingly and help cut contamination trails if contamination occurs. the system prioritizes response efforts so as to provide efficient mission-focused allocation of workforce and assets, for example rapid assignment and deployment of emergency teams. additionally, the system allows to receive real-time push-to-video, pictures, peer-to-peer calls, and chats across personnel and between the medical teams and the command and control units. finally, a key challenge in the setting of covid-19 is the limited ability to record clinical notes for the electronic medical record (emr) under ppe. to overcome this challenge, the platform can potentially allow a voice medical record (vmr) to be recorded: a short, "tweet-like," 1.5minute voice clip, describing the clinical status of the patient, available to all personnel working in the facility. all the transmitted information is encrypted, maintaining medical confidentiality. the implementation of the system in covid-19 departments initially in rambam and later on in 15 additional hospitals across israel allowed clinical teams to easily communicate using ppe and to manage, control, and execute the complex tasks performed by a large and diverse workforce operating in a wide range of locations. 31 once each ward was equipped with a control room, then (as in usual times) all information available on the patients (directly and indirectly) can be entered into e-records. connecting all control rooms to a central one at the hospitals and from each hospital to the national level was the next logical step in order to provide a reliable central real-time dashboard. during the early phases of the covid-19 crisis, a real-time hospital command and control tool was evidently needed in order to manage the medical and logistical aspect of the crisis. to maximize hospitals' efficiency a system integrating patients' clinical data, medical staff status, critical clinical resources, and asset allocation into a single dashboard was required. the coview™ system (a collaboration between the department of government medical centers within the ministry of health, the directorate of defense research and development within the ministry of defense, israel defense forces, and specialists from the hi-tech industry) was developed for this goal, integrating defense concepts, big data analytics, and health care protocols. the system reflects the status of all covid-19 patients at all hospitals and admission facilities, in order to enable decision makers to make an efficient, evidence-based, optimized response. using artificial intelligence algorithms, the system can analyze aggregated data from patient monitors and electronic charts, alerting medical staff to potential deterioration of specific patients, or analyze treatment protocols in specific hospitals (figure 2 ). one could centrally control and optimize urgently needed (sometimes scarce or costly) resources such as ppe, drugs, mechanical ventilators, or extracorporeal membrane oxygenation (ecmo) systems. moreover, professional advisors such as high-level experts can monitor the situation at each hospital, the treatment protocol used, and its efficacy and can help direct hospital staff across the country as needed. 32, 33 while witnessing the current global surge of cases, health care systems must be prepared also for a potential winter surge with the common flu, a different pandemic, or another health crisis-hopefully with a better readiness than what has been seen so far in most countries during the covid-19 pandemic. there are many lessons to be learned from the events in our small nation during the past half year. several conclusions relevant to this discussion should be mentioned:  caring for massive numbers of critically ill patients who require strict isolation while maintaining health care personnel safety is challenging and requires "out of the box" thinking.  a central, real-time overview of hospitals' status (occupancy, patient condition, logistics, etc.) is fundamental for effective decision-making and resource allocation. this can be helpful to any organization operating during crisis, more so to public health care systems which usually operate on tight budgets and should be prepared for a sudden and extensive stretch in the capacity they need to provide.  an effective, safe, and widely spread remote patient monitoring capacity is needed for two reasons: to minimize contact between caregivers and patients and to keep hospital beds for the sickest patients who need intensive care. this can be done by keeping the moderately sick patients at home (equipped with sensitive, remote, artificial intelligence-based sensors), while being able to alert whenever and wherever a referral to the hospital is required. in addition, the experience acquired during the covid-19 pandemic is expected to have a tremendous potential on the continuum of care for chronic disease management like diabetes, heart failure, chronic kidney disease, chronic inflammatory conditions, and high-risk pregnancy. the number of existing technologies and tools, and those in development, is expected to grow tremendously. as covid-19's natural progress proceeds, experience of using such tools and evidence generation needs to be published in order to facilitate a wider adoption of appropriate tools that may benefit patients in the future. for this to succeed, the health care systems and insurers should be prepared with an appropriate infrastructure, including effective reimbursement strategy. the ideal outcome would be for these technologies to provide effective and progressive health care, from the community and regional hospital level to the national level. different patient data generated by the various sensors/systems (left) and data regarding medical staff competence and critical clinical equipment can be transferred to each local hospital coview™ system, for each patient record (electronic medical record [emr]). patient data from the tactic system (ex-teams™, right) can be uploaded to the relevant patient emr, while logistics information is uploaded to the relevant administrative databases. the aggregated data (center) can be analyzed at both the hospital and the national levels and presented using a realtime dashboard at each level using the coview™ platform. the ex-teams system (right) enables tactical organization of the hospital personnel and is used as the operation platform in the organization. the suggested data flow scheme may further facilitate organizational feedback loops to further assist hospitals to perform better and reflect the overall status of the situation to the national decision makers (national mission control dashboard, above). hd/ir, high-definition/infrared; vmr, voice medical record. clinical course and risk factors for mortality of adult inpatients with covid-19 in wuhan, china: a retrospective cohort study autopsy findings and venous thromboembolism in patients with covid-19: a prospective cohort study clinical characteristics of 138 hospitalized patients with 2019 novel coronavirusinfected pneumonia in wuhan, china covid-19 and the cardiovascular system who scientific and technical advisory group for infectious hazards. covid-19: what is next for public health? review of the clinical characteristics of coronavirus disease 2019 (covid-19) the novel coronavirus pneumonia emergency response epidemiology team. vital surveillances: the epidemiological characteristics of an outbreak of 2019 novel coronavirus diseases (covid-19)-china contact-based methods for measuring respiratory rate respiration rate monitoring methods: a review analysis of ir thermal imager for mass blind fever screening comparison of 3 infrared thermal detection systems and selfreport for mass fever screening ir thermography-based monitoring of respiration phase without image segmentation noncontact tidal volume measurement through thin medium thermal imaging a novel method for extracting respiration rate and relative tidal volume from infrared thermography altered hand temperatures following transradial cardiac catheterization: a thermography study the identification of higher forefoot temperatures associated with peripheral arterial disease in type 2 diabetes mellitus as detected by thermography use of infrared thermography to detect early alterations of peripheral perfusion -evaluation in a porcine model early sepsis recognition based on ear localization using infrared thermography a pilot study of the leicester ed medical infrared imaging protocol in fever and sepsis monitoring and analysis of respiratory patterns using microwave doppler radar institute of technology. radar & optical sensors to remotely monitor covid-19 combat the spread of covid-19 with realtime, remote video-based vital signs monitoring the earlysense solution: use of continuous monitoring for isolation patients validation of remote dielectric sensing (redstm) technology for quantification of lung fluid status: comparison to high resolution chest computed tomography in patients with and without acute heart failure relationship between noninvasive assessment of lung fluid volume and invasively measured cardiac hemodynamics remote dielectric sensing (redstm)-guided patient management of ambulatory heart failure patients reduces rehospitalization rates is there a role for lung ultrasound during the covid-19 pandemic? a preliminary study on the ultrasonic manifestations of peripulmonary lesions of non-critical novel coronavirus pneumonia (covid-19). ssrn electron j tyto care helps the fight against covid-19 rambam healthcare campus in israel joins forces with elbit systems to deploy a management & control technology israeli army's idea lab aims at a new target: saving lives fda greenlights icu ai for predicting fatal covid-19 complications. fierce biotech key: cord-294784-r84td2i0 authors: meessen, bruno title: health system governance: welcoming the reboot date: 2020-08-10 journal: bmj glob health doi: 10.1136/bmjgh-2020-002404 sha: doc_id: 294784 cord_uid: r84td2i0 nan though a rather recent concept, governance is as old as humanity. over the last two decades, governance has received a lot of attention from the global health community. the governance lens has been applied to, among others, health, 1 health systems, 2 health system strengthening, 3 health system resilience, 4 primary healthcare 5 and hospitals. 6 substantial knowledge and understanding have been accumulated in the process. yet, recent reviews of frameworks have also reported a certain conceptual confusion and lack of progress with the empirical agenda. 2 no framework has managed to impose itself so far. is the health system governance research programme experiencing a stalemate? we don't think so. recent contributions indicate that a conceptual reboot is on its way. a number of researchers are moving away from the government-centred perspective to an understanding of governance as the organisation by human beings of their collective action. the main goal of this paper is to make the case for this extended approach to governance and to explore its implications, both for research and action. the first section consists in a quick summary on the emergence and development of the concept of health system governance. in the second section, a formalised expression of the new approach to governance is sketched; at its centre is the choice set of actions available to groups of individuals. we use the covid-19 pandemic, one of the biggest collective action problems faced in the history of humanity, to illustrate our point. in the third section, we explore some key benefits attached to the collective agency approach. the paper ends with some suggestions of ways to move forward. in the field of international development, the mainstream view that governance is a determinant of development outcomes follows decades of work developed by the world bank. 7 8 two who reports have been pivotal in establishing a similar view for health systems: the world health report 2000 9 and, 7 years later, the report entitled 'everybody's business: strengthening health systems to improve health outcomes'. 10 the core contribution of the world health report 2000 was to put forward a proposition on how performance of health systems should be conceptualised and measured. its definition of health system performance focused on outcomes which can be attributed to health interventions. the report also looked at determinants of performance. a whole chapter was dedicated to the concept of stewardship, understood as the central responsibility of the government for the overall performance of a country's health system. ► the literature on health system governance is growing. alternative frameworks have been proposed, but none has really imposed itself so far. the empirical agenda is progressing slowly. ► there is a turning point among recent publications: a move away from a government-centred perspective of governance to a broader understanding of governance as the people's organisation of their collective action. ► in this paper, we argue that what matters is the choice set of actions that groups of individuals can undertake, that is, their collective agency. ► the focus on collective agency broadens the perspective for action: the governance of the health system is not only about the ministry of health doing well certain things, it is about groups of individuals being able to organise their collective action, through the state, but also through other mechanisms. ► the collective agency approach opens avenues for research. for example, governance is both an explanatory and an outcome variable. a governance intervention (explanatory variable) may be effective to improve some health outcomes, but also disempower collective action for some groups of the population (outcome variable). by the publication of the 2007 report, the concept of stewardship had evolved to 'governance and leadership'. the term 'governance' better captures the fact that health systems are increasingly complex and that in its steering of the health system, a ministry of health has to coordinate with a large set of actors and also to be accountable. the very title of the report acknowledges that health systems are fundamentally collective action problems. however, in subsequent who documents, the distinction between governance and leadership was dropped and many contributors to the field fell back on a ministry of health-centred understanding of governance (see reference 11) . the biggest challenge with governance as a concept is probably that it seems to elude measurement. the dominant approach to solve this problem has been to mimic the world bank's approach of state governance and define dimensions. this has led to a proliferation of frameworks. 2 12 13 in a report for the european health system observatory in which they compared existing 'frameworks', greer et al concluded that such frameworks were long, normative and arbitrary lists of dimensions or items, 14 and noted that the power of these frameworks to help improve policies still had to be demonstrated. indeed, most have limited empirical validation. 15 this highlights the main limitation with the 'good governance' approach: normative choices (defining how things should be) take a central role in determining both the empirical and the policy agenda. normative orientations are probably inevitable in this field, the problem is that they are rarely cast with sufficient exposure of the values and interests underlying them. this exposes formalisation and subsequent empirical research to arbitrariness or even bias, as authors may be promoting their view of the world or the one that legitimates the theory of change implemented by their agency. because of their constituencies, agencies may be tempted to promote specific governance mechanisms or downplay dimensions that may arouse hostility from some policy actors. recently, several authors have tried to lay down firmer foundations to the governance agenda. for siddiqi et al, 'governance comprises the complex mechanisms, processes and institutions through which citizens and groups articulate their interests, mediate their differences and exercise their legal rights and obligations'. 13 in 2014, abimbola et al explored the lessons from the common-pool resources literature to enlighten collective action for primary healthcare. 5 to our knowledge, this was the first time that the health system governance literature was connecting with the pioneering work of elinor ostrom. if anyone has studied collective action and reflected on how to move from a positivist programme to more prescriptive messages, it is ostrom. 16 in 2017, in a review of the literature, abimbola et al pointed to the shortcomings of the government-centred approach and made the point for a more comprehensive approach to governance and its underlying institutional arrangements. 17 the same year, fryatt et al also came with a more comprehensive approach of governance 18 -it is also marked by their adoption of a non-normative definition of governance-'how societies make and implement collective decisions'. the same year, pyone et al took a similar approach: 'governance is defined as the rules (both formal and informal) for collective action and decision making in a system with diverse players and organisations while no formal control mechanism can dictate the relationship among those players and organisations. 2 adopting such broad and less normative definitions reduces the risk of excluding certain variables from the scope of analysis. with this new view, governance can be summarised as the organisation by human beings of their collective action. we characterise it as a 'reboot' because the focus of the health system governance agenda shifts from the government to the people. governance of the health system is not just about the ministry of health doing certain things well, it is not even about the ministry of health collaborating with other actors, it is about groups of individuals being able to organise their collective action, also through the state, but not exclusively. in the next section, we propose a formalisation of this new perspective. by adopting the extended formulation of governance, we de facto lose our analytical 'anchor': the organisation. as far as health systems are concerned, no longer can our thinking and analysis be organised around the coordination functions played by the ministry of health. what would then be the new variables of interest? our proposition is to organise the analysis around four main sets of variables: (1) the set of collective action problems to solve (let us call it p) (2) the group of individuals facing this p (g),(3) the set of possible actions (a) that members of g can take at a time t in order to handle p and (4) the conditions (c) determining the choice set a. a collective action problem can be defined as any problem whose solution requires some coordination between potentially benefiting individuals. it can be of various natures: a pandemic to contain, child mortality and the need to reduce it, the performance of a specific hospital. g can be any grouping of persons of relevance: investors, local community, the medical profession, a nation, the world population. for sure, it is not limited to civil servants working for the ministry of health. p and g are closely linked. the staff of a hospital (g) will be busy with solving a large set of problems: availability of services, organisation of work, quality of care, management of interpersonal conflicts. many collective action problems require coordination at the level of different gs. as a pandemic, covid-19 requires action at the global level (eg, under the leadership of who, the international monetary fund, etc), bmj global health but also at the national and community levels. obviously, a multitude of gs creates coordination issues: different groups have diverse interests and sometimes conflicting interests (cf. siddiqi et al's definition). governance is a lot about overcoming such tensions, including, but not exclusively, through mechanisms such as governments. we propose to put the collective agency held by the group, the choice set a, at the centre of the analysis. it can include actions of very different natures. some actions are generic (eg, stating the problem, agreeing on common goals, adopting rules), others are specific to the problem. covid-19 can be addressed by closing borders, testing, restricting movement, forbidding social gatherings, treatment. an action belongs to a if it is really feasible by g. the set of possible actions a is itself determined by a set of conditions (c): the size and composition of g, the nature, quantity and distribution of resources (including information and trust) endowed by its members, their preferences, organisations (eg, the ministry of health) and other institutional arrangements in place, as well as external factors such as available technology or security. we do not doubt that future work will generate a more granular view of these conditions and their inter-relationships. adopting a collective agency approach to governance has benefits on at least three levels. it creates space for theoretical and empirical research independent from normative preferences. it allows (1) description of the different sets of interest (p, g, c, a) at different periods of time; (2) the study of how sets and variables related to each other; (3) the study of how sets and variables are determined across time (historical studies, path dependency); (4) the linking of all these variables to other variables of interest. all these aspects can be investigated, in a neutral manner, without some prejudices on some standards of 'good governance'. this opens new territories for health system governance researchers. for example, some researchers may want to study how the actual collective agency of a group is also a result of history. indeed, the capacity of a group to develop health interventions may be partly determined by earlier collective events. good examples of such phenomena are provided by the recent stream of work establishing a link between slave trade or colonial history with trust and capacity to implement collective action in some regions of africa. 19 20 covid-19 reveals that this can also play the other way round. in south korea, the painful experience with the middle east respiratory syndromerelated coronavirus outbreak in 2015 generated a lot of learning which expanded the set of actions available for the national response to covid-19. 21 other analysts may want to reorganise the 'order' of the variables. for the last 20 years, we have looked at governance as a 'building block' contributing to health system performance, the latter being measured in terms of health, responsiveness, financial protection outcomes. by equating governance with collective agency, we can, at last, conceptually handle the fact that our collective agency may also be impacted by health policies-that is, be an outcome variable. with covid-19, we have seen how health policies may affect our individual and collective rights. some watchdogs are even worried about long-lasting regressions in terms of civic rights. this new perspective could lead to a better recognition of the contribution of health systems to broader political goals (eg, consolidation of the social contract). by moving the centre of gravity of the analysis (from the ministry of health to our collective agency), the research programme undergoes a double shift which will generate new insights. the first shift is that we now take a neutral approach toward coordination mechanisms. ministries of health, rightly, receive a lot of attention. but let us keep in mind that they are quite modern institutions. our proposition is compatible with the study of institutions organising the practice of medicine in ancient greece or during the islamic golden age, for instance. this is also a reminder that even in our societies, a ministry of health is just one coordination mechanism among others. as stressed by pyone et al's definition, it is the whole nexus of institutions that matters. again, covid-19 has shown the need to broaden the scope of attention. we have seen how some resources critical for a performing health system (eg, personal protective equipment, test reagents or medicines) are nowadays more governed by global markets than by ministries of health. we have witnessed the spread of conspiracy theories on social media and the subsequent erosion of trust in health authorities. understanding better other coordination mechanisms (eg, social norms, judicial system, the market, social media) seems a prerequisite before calling to an authority for implementing any corrective measure. the second shift is a repositioning of institutional arrangements in the analysis. we do not deny that there is great convenience in anchoring governance analyses on organisations. institutions are key for collective actionthey assign rights and thus reduce uncertainty and coordination costs. 22 as an organisation, a ministry of health constitutes a stable platform. it can issue policies, which are themselves malleable institutions. still, organisations and institutions are just instruments. 23 what ultimately matters to people is the set of actions at their disposal to solve their problems. as analysts we should not forget that this set is determined by more conditions than just institutions. a crucial condition is power. integrating it into the analysis requires going beyond the mere observation that institutions are in place. the right to strike has bmj global health its intrinsic value, but the impact of a strike will depend on how it disrupts the economy and thus empowers the unions in the negotiation. another key factor is trust. low trust in organisations limits options. the covid-19 crisis has provided examples of nations whose response has been constrained by the growing distrust in the leadership. the collective agency proposition also allows a more opened discussion about what 'good governance' might be. it provides a ranking approach which does not bring straight on the preferences of the authors for, for example, a too specific governance modality. indeed, it gives us probably the least normative ranking criterion possible: for given g and p, it is correct to say that conditions c 1 are superior to conditions c 2 , if a 2 is larger than a 1 . this is not trivial. for instance, from the perspective of a local community (g), a legal system (c 1 ) allowing to set up a community health association is superior to a system (c 2 ) which does not permits that. from the perspective of investors, entrepreneurs or consumers, a social system guaranteeing the rule of law and respect of contracts is also superior to one which does not guarantee such conditions. of course, a choice set a will rarely dominate all the others and more elaborate criteria will be needed to decide on the inescapable trade-offs. this approach will meet its own limitations, but at least, it will lay bare the normative issues encompassed by the health system governance agenda. such an approach valuing 'real rights for collective action' is not without firm moral foundations. it is aligned with the concept of primary goods put forward by rawls 24 or the concept of capabilities developed by nussbaum nd sen. 25 obviously, operationalisation will require to list collective capabilities of importance and establish rules for fair treatment of different groups. we believe this could be done in generic terms (eg, capability for members to appoint a representative to the governing body of the group), but also be tailored to the g, a and p of interest. over these last years, a new view on health system governance has been emerging. a growing number of authors proposed to take collective action as the central issue. we think it is a healthy development, as it will allow to better disentangle the empirical, normative and prescriptive agendas. frameworks and concepts are themselves a source of power and influence; the conceptualisation of governance is, by essence, an area where contributors should be vigilant about their positionality. our message is not that past research and policy guidance should be wiped out. conceptual and empirical efforts dedicated to identifying dimensions of interest (transparency, accountability, etc) and supportive institutional mechanisms remain very valuable. 26 27 in the end, governments formally take on much of the responsibility for governance; reminding them their duties towards their citizens, especially for 'common goods for health', 28 should remain a central task of multilateral agencies. our point is that this must be embedded in a broader perspective. today, we are far from being conceptually and methodologically equipped to capture the actual rights of the groups of individuals having a stake in health systems. collective agency should be our new conceptual, empirical and prescriptive horizon. we hope that this paper is a useful step in this direction. the collective agency approach to health system governance surely raises its own challenges. its value will depend on how useful it proves when employed in empirical research, reflection and action. it encompasses a risk of misuse, for instance, to legitimate more privatisation, ill-conceived decentralisation, societal fragmentation or the unchecked growth of digital giants. time will tell whether it leads to real progress for people, especially the most vulnerable. at short term, we must be ready to address heads-on some possible tensions, for instance, when a policy is effective to improve some health outcomes but also disempower groups of the population. governance is both an explanatory and an outcome variable for health systems. some of the collective capabilities to protect or to expand may include some sensitive issues (eg, capability to associate or to access reliable information, including through whistle blowers), but there is no escape: excluding them is analytically wrong. we hope that with the collective agency approach, the global health community will manage to get the issue of governance taking off, both as a field of study and an area of intervention. the covid-19 crisis indicates that it should happen now. governance for health in the 21st century frameworks to assess health systems governance: a systematic review health governance: principal-agent linkages and health system strengthening governance and capacity to manage resilience of health systems: towards a new conceptual framework towards people-centred health systems: a multi-level framework for analysing primary health care governance in low-and middle-income countries a framework for assessing hospital governance world development report 1997: the state in a changing world health systems: improving performance everybody's business: strengthening health systems to improve health outcomes: who's framework for action monitoring the building blocks of health systems: a handbook of indicators and their measurement strategies a review of health governance: definitions, dimensions and tools to govern framework for assessing governance of the health system in developing countries: gateway to good governance governance: a framework an approach to addressing governance from a health system framework perspective beyond markets and states: polycentric governance of complex economic systems institutional analysis of health system governance health sector governance: should we be investing more? the slave trade and the origins of mistrust in africa violence and indirect rule: evidence from the congo free state national response to covid-19 in the republic of korea and lessons learned for other countries institutions, institutional change and economic performance economics, organization and management a theory of justice the quality of life measuring governance: accountability, management and research strategies for policy success: achieving 'good' governance financing common goods for health: fundamental for health, the foundation for uhc acknowledgements the paper benefited a lot from the critical comments key: cord-237721-rhcvsqtk authors: welch, charles; lahnala, allison; p'erez-rosas, ver'onica; shen, siqi; seraj, sarah; an, larry; resnicow, kenneth; pennebaker, james; mihalcea, rada title: expressive interviewing: a conversational system for coping with covid-19 date: 2020-07-07 journal: nan doi: nan sha: doc_id: 237721 cord_uid: rhcvsqtk the ongoing covid-19 pandemic has raised concerns for many regarding personal and public health implications, financial security and economic stability. alongside many other unprecedented challenges, there are increasing concerns over social isolation and mental health. we introduce textit{expressive interviewing}--an interview-style conversational system that draws on ideas from motivational interviewing and expressive writing. expressive interviewing seeks to encourage users to express their thoughts and feelings through writing by asking them questions about how covid-19 has impacted their lives. we present relevant aspects of the system's design and implementation as well as quantitative and qualitative analyses of user interactions with the system. in addition, we conduct a comparative evaluation with a general purpose dialogue system for mental health that shows our system potential in helping users to cope with covid-19 issues. the covid-19 pandemic has changed our world in unimaginable ways, dramatically challenging our health system and drastically changing our daily lives. as we learned from recent large-scale analyses that we performed on social media datasets and extensive surveys, many people are currently experiencing increased anxiety, loneliness, depression, concerns for the health of family and themselves, unexpected unemployment, increased child care or homeschooling, and general concern with what the future might look like. 1 research in expressive writing (pennebaker, 1997b) and motivational interviewing (miller and rollnick, 2012) has shown that even simple interactions where people talk about one particular experience can have significant psychological value. numerous studies have demonstrated their effectiveness in improving peoples mental and physical health (vine et al., 2020; pennebaker and chung, 2011; resnicow et al., 2017) . both expressive writing and motivational interviewing rely on the fundamental idea that by putting emotional upheavals into words, one can start to understand them better and therefore gain a sense of agency and coherence of the thoughts and emotions surrounding their experience. in this paper, we introduce a new interview-style dialogue paradigm called expressive interviewing that unites strategies from expressive writing and motivational interviewing through a system that guides an individual to reflect on, express, and better understand their own thoughts and feelings during the pandemic. by encouraging introspection and selfexpression, the dialogue aims to reduce stress and anxiety. our system is currently online at https://expressiveinterviewing.org and available for anyone to try anonymously. expressive writing. expressive writing is a writing paradigm where people are asked to disclose their emotions and thoughts about significant life upheavals. originally studied in the scope of traumatic experiences (pennebaker and beall, 1986) , study participants are usually asked to write about an assigned topic for about 15 minutes for one to five consecutive days. later studies expanded to specific experiences such as losing a job (spera et al., 1994) . expressive writing has been shown to be effective on both physical and mental health measures by multiple meta-analyses (frattaroli, 2006) , finding its association with drops in physician visits, positive behavioral changes, and longterm mood improvements. no single theory at present explains the cause of its benefits, but it is believed that the process of expressing emotions and constructing a story may play a role for participants in forming a new perspective on their lives (pennebaker and chung, 2011) . motivational interviewing. motivational interviewing (mi) is a counseling technique designed to help people change a desired behavior by leveraging their own values and interests. the approach accepts that many people looking for a change are ambivalent about doing so as they have reasons to both change and sustain the behavior. therefore, the goal of an mi counselor is to elicit their client's own motivation for changing by asking open questions and reflecting back on the client's statements. mi has been shown to correlate with positive behavior changes in a large variety of client goals, such as weight management (small et al., 2009) , chronic care intervention (brodie et al., 2008) , and substance abuse prevention (d'amico et al., 2008) . dialogue systems. with the development of deep learning techniques, dialogue systems have been applied to a large variety of tasks to meet increasing demands. in recent work, afzal et al. (2019) built a dialogue-based tutoring system to guide learners through varying levels of content granularity to facilitate a better understanding of content. henderson et al. (2019) applied a response retrieval approach in restaurant search and booking to provide and enable the users to ask various questions about a restaurant. ortega et al. (2019) built an open-source dialogue system framework that navigates students through course selection. there are also dialogue system building tools such as google's dialogflow 2 and ibm's watson assistant, 3 which enable numerous dialogue systems for customer service or conversational user interfaces. chatbots for automated counseling. two dialogue systems for automated counseling services available on mobile platforms are wysa 4 and woebot. 5 these chatbots provide cognitive behavioral therapy with the goal of easing anxiety and depression by allowing users to express their thoughts. a study of wysa users over three months showed that more active users had significantly improved symp-toms of depression (inkster et al., 2018) . another study shows that young students using woebot significantly reduced anxiety levels after two weeks of using the conversational agent (fitzpatrick et al., 2017) . these findings suggest a promising benefit of automated counseling for the nonclinical population. our system is distinct from wysa and woebot in that it is designed specifically for coping with covid-19 and allows users to write more topic related free-form responses. it asks open-ended questions and encourages users to introspect, and then provides visualized feedback afterward, whereas the others have a conversational logic mainly based on precoded multiple choice options. our system conducts an interview-style interaction with the users about how the covid-19 pandemic has been affecting them. the interview consists of several writing prompts in the form of questions about specific issues related to the pandemic. during the interview, the system provides reflective feedback based on the user's answers. after the interaction is concluded, the system presents users with detailed graphical and textual feedback. the system's goal is to encourage users to write as much as possible about themselves, building upon previous findings regarding the psychological value of writing about personal upheavals and the use of reflective listening for behavioral change (pennebaker, 1997b; miller and rollnick, 2012) . to achieve this, the system guides the interaction by asking four main open-ended questions. then, based on users responses, the system provides feedback and asks additional questions whenever appropriate. in order to provide reflective feedback, the system automatically detects the topics being discussed (e.g., work, family) or emotions being felt (e.g., anger, anxiety), and responds with a reflective prompt that asks the user to elaborate or to answer a related question to explore that concept more deeply. for instance, if the system detects work as a topic of interest, it responds with "how has work changed under covid? what might you be able to do to keep your career moving during these difficult times?" during the formulation of the guiding questions used by our system, we worked closely with our psychology and public health collaborators to identify a set of questions on covid-19 topics that would motivate individuals to talk about their personal experience with the pandemic. we formulated the following question as the system's conversation starting point: [major issues] what are the major issues in your life right now, especially in the light of the covid outbreak? we also formulated three follow-up questions, which were generated after several refining iterations. 6 the order of these questions is randomized across users of the system. [looking forward] what do you most look forward to doing once the pandemic is over? [advice to others] what advice would you give other people about how to cope with any of the issues you are facing? [grateful] the outbreak has been affecting everyone's life, but people have the amazing ability to find good things even in the most challenging situations. what is something that you have done or experienced recently that you are grateful for? our system's capability for language understanding relies on identifying words belonging to various lexicons. this simple strategy allowed us to quickly develop a platform upon which we intend to implement a more sophisticated language understanding ability in future work. when a user responds to one of the main prompts, the system looks for words belonging to specific topics and word categories. the system examines the user responses to identify dominant word categories or topics and triggers a reflection from a set of appropriate reflections. 7 if none of these types are matched, it responds with a generic reflection. the word categories are derived from the liwc, wordnet-affect and mpqa lexicons (pennebaker et al., 2001; strapparava et al., 2004; wiebe et al., 6 we removed an additional question about how people's lives have changed since the outbreak, as well as a question about what people missed the most about their previous lives. 7 a dominant word category is defined as a word type, where the frequency of occurrence is at least 50% higher than the second highest frequency category for that group. 2005) and include pronouns (i, we, others), negative emotion (anger, anxiety, and sadness), positive emotion (joy) and positive and negative words. the covid-19 related topics include finances, health, home, work, family, friends, and politics. most of the topics are covered by the liwc lexicon, with the exception of politics. for this category, we use the politics category from the roget's thesaurus (roget, 1911) and add a small number of proper nouns covered in recent news (e.g. trump, biden, fauci, sanders). we formulate a set of specific reflections for each word category and topic, which were refined by our psychology and public health collaborators. for instance, if the dominant emotion category is anxiety, the system responds "you mention feelings such as fear and anxiety. what do you think is the best way for people to cope with these feelings?" initially, we also considered reflections for different types of pronouns, but found that they did not steer the dialogue in a meaningful direction. instead, we flag responses with dominant use of impersonal pronouns and lack of references to the self and reflect that fact back to the user and further ask them how they are specifically being affected. we also crafted generic reflections to be applicable to a large number of situations though the system does not understand the content of what the user has said (e.g. "i see. tell me about a time when things were different", and "i hear you. what have you tried in the past that has worked well"). after the interview, the system provides visual and textual feedback based on the user's responses and provides links to resources (i.e., mental health resources) appropriate given their main concerns. the visual feedback consists of four pie charts showing the relative usage of different word categories, including: discussed topics (work, finance, home, health, family, friends and politics), affect (positive, negative), emotions (anger, sadness, fear, anxiety, joy), and pronouns (i, we, other). the textual feedback includes a comparison with others (to normalize the user's reactions) and interpretations of where the user falls within normalized scales. the system also presents a summary of the most and least discussed topics and how they compare to the average user, along with normalized values for meaningfulness, self-reflection, and emotional tone (using a 0-10 scale) along with textual descriptors for the shown scale values. 8 these metrics are inspired by previous work on expressive writing and represent the self-reported meaningfulness, usage of self-referring pronouns, and the difference in positive and negative word usage (pennebaker, 1997a) . finally, the system provides relevant resources for further exploration (e.g. for the work topic it lists external links to covid related job resources and safety practices). the system is implemented as a web interface so it is accessible and easy to use. the interface is built with the django platform and jquery and uses python on the backend (django software foundation, 2019). before the interaction users are asked to report on a 1-7 scale: (1) [life satisfaction] how satisfied they are with their life in general, and (2) [stress bef ore ] what is their level of stress. the user then proceeds to the conversational interaction with our system. after the interaction, the user is asked again about (3) (5) [meaningful] how meaningful their interaction was. once this is submitted, the user can proceed to the feedback page and view details about what they wrote and how their interaction compares to a sample of recent users. the user is finally presented with a list of resources triggered by the topics discussed. we made an effort to make our system appear human-like to make users more comfortable while interacting with it, although this can vary for different individuals. in future work, we hope to explore individual personas and more sophisticated rapport building techniques. we named our dialogue agent 'c.p.', which stands for computer program. this name acknowledges that the user is interacting with a computer, while at the same time it makes the system more human by assigning it a name. when responding to the user, c.p. pauses for a few seconds as if it is thinking and then proceeds to type a response one letter at a time with a low probability of making typos -similarly to how human users would type. after the system was launched (and up to when we conducted this analysis), we had 174 users interact with the system. we analyze these interactions to evaluate system usefulness, user engagement, and reflection effectiveness. system usefulness. we examine the system's ability to help users cope with covid-19 related issues by analyzing the different ratings provided by users before and after their interaction with c.p. throughout this discussion, we use ∆stress to indicate how the users stress rating differs before and after the interaction: ∆stress = stress af ter -stress bef ore . negative values for ∆stress are therefore an indicator of stress reduction, whereas positive values for ∆stress reflect an increase in stress. we start by measuring the spearman correlation between the different ratings for the 174 interactions with c.p. results are shown in table 1 . the strongest correlation we observe is between the personal and meaningful ratings, suggesting that interactions that are more meaningful appear to feel more personal, or vice versa. we also observe a strong negative correlation between ∆ stress and the meaningfulness of the interaction, suggesting that the interactions that the users found to be meaningful are associated with a reduction in stress. user engagement. we examine user engagement by analyzing the time users spend in the interaction and the number of words they write throughout the session. figure 1 shows histograms of the session lengths in the number of words used by the user and of the session duration in seconds. the rightmost column of table 2 shows spearman correlation coefficients between user ratings and the length and duration of the sessions. we find a significant negative correlation between stress bef ore and stress af ter with session duration and number of words, suggesting an association between user engagement and lower stress. there is also a weak negative correlation between duration of session and reduction in stress (∆stress). we also investigate if there is a relationship between the pre-and post-session ratings and how engaged a user was with each prompt in terms of length of and duration in writing their response. table 2 shows spearman correlation coefficients for these relationships. it appears that life satisfaction has no correlation with the length of any prompt response except a potentially weak negative correlation with length on the major issues prompt (p = 0.052). a lower rating may relate with having more personal challenges to write about. stress bef ore has a weak negative correlation between the number of words used and the duration spent in the response to looking forward. higher stress may relate to present concerns, which may make one less inclined to spend time thinking and writing about positive aspects of their future than someone with less stress. we presume this could be the case for the grateful prompt, which likewise correlates weakly and negatively with stress bef ore . stress af ter has a negative correlation between duration spent on every prompt response except for the time spent on major issues. this could be a reflection of the fact that those who have a lot to write about major issues in their life also incur high levels of stress. the personal rating shows no correlations with the duration spent on any of responses, except potentially advice to others (p = 0.074). we do observe weak negative correlations between personal ratings and response lengths on major issues and looking forward, and potentially on grateful (p = 0.054) and advice to others (p = 0.08). perhaps if a user writes more, there is a greater expectation for more personal reflections. we discuss engagement related to reflections more deeply in the next section. the meaningful rating shows weak negative correlations with length on major issues, advice to others, and possibly on grateful (p = 0.052) and looking forward (p = 0.062). we do not observe a significant correlation with duration on major issues or grateful, but we do observe positive correlations between duration and looking forward and advice to others. users who spend more time thinking about advice they would give others facing their issues may find the interaction more meaningful, and may experience benefits having reflected on their agency in managing their challenges. reflection effectiveness. to investigate the effectiveness of expressive interviewing reflections, we compare the reflections that were triggered for users whose stressed decreased to the reflections that triggered for the users whose stress increased. for each of these user groups, we compute the dominance of each reflection as its proportion of times it was triggered out of all reflections triggered. in figure 2 , we compare the dominance of each reflection across these user groups by dividing the reflection dominance in the decreased-stress group by that of the increased-stress group. importantly, we observe that all emotion reflections and more topic reflections were triggered at a higher rate for users whose stress decreased, whereas more generic reflections were triggered at a higher rate for users whose stress increased. while we do not presume that increased stress was due to generic reflections, the correspondence between emotion and topic reflections with stress reduction aligns with expectations of effective reflections from motivational interviewing-generic reflections and specific reflections resemble simple reflections and complex reflections respectively, as referred to in motivation interviewing. while both types of reflections serve a purpose, complex reflections both communicate an understanding of what the client has said and also contribute an additional layer of understanding or a new interpretation for the user, whereas simple reflections focus on the former (rollnick and allison, 2004) . in qualitatively analyzing the instances where generic reflections were triggered, we observe that contextual appropriateness seems to be the best indicator of their success (in terms of ability to elicit a deeper thought, feeling, or interpretation) given that the user was invested in the experience. as these generic reflections are selected at random, their contextual appropriateness was inconsistent, illuminating the scenarios in which they are more or less appropriate. for instance, out of the seven times the reflection "interesting to hear that. how does what you say relate to your values?" was triggered for the increased-stress users, one user expanded on their previous message, one expressed confusion about the question, and another copied and pasted the definition of core values 9 as their response. two other instances of this reflection were triggered when a user had expressed negative feelings such as worry and feeling lazy which appeared misplaced, and the last case was triggered by a message that was not readable. out of the thirteen times the same reflection was triggered for the decreased-stress group, one user expressed not havfigure 2 : the dominance of each reflection triggered for users whose stress decreased divided by each reflection's dominance for users whose stress increased. scores above 1 (red line) correspond to a decrease in stress; score below 1 correspond to an increase in stress. see table 3 for sample reflections, including the generic reflections. ing much to say, another gave one word responses before and after, and all others expanded on their previous message in relation to their values or gave a simple response to indicate a degree that it relates. this reflection appeared more "successful" (based on if the user expanded on their previous message or values) when it was triggered by a message with more neutral to positive sentiment, such as when the user was expressing what they were looking forward to, or when they had several pieces of advice to offer for a friend in their situation, as opposed to one with more negative sentiment like the messages expressing worry or laziness. in instances of other generic reflections, we observed that another issue for appropriateness was whether the reflection matched the user's frame of thought in terms of past, present, or future. for instance, the reflection "i see. tell me about a time when things were different," best matched scenarios when users described thoughts about changes to their daily lives, but not when users described future topics such as what they were looking forward to, nor when they were already describing the past. based on our observations of the reflections in action, we have three main takeaways. first, topic and emotion specific reflections are more associated with the group of users whose stress decreased. these reflections are only triggered if the system determines a dominant topic or emotion, which depends on the effectiveness of its heuristics, as well as the amount of detail and context that a user expresses. this leads to the next takeaway, that the system appears to be more effective when users approach the experience with an intention for expression, or conversely it seems less effective when the intent to not engage and express is explicit. third, the generic reflections were developed with the intent to function in generic contexts, but we learned in practice that some clashed with emotional and situational content or were confusing given the context. as we did observe many, if not more, successful instances of generic reflections, we are able to contrast these contexts to the unsuccessful contexts, and can develop a heuristic for selecting the generic reflections rather than selecting at random, as well as adapt the language of our current generic reflections to be more appropriate for the expressive interviewing setting. to assess the extent to which our expressive interviewing system delivers an engaging user experience, we conduct a comparative study between our system and the conversational mental health app woebot (fitzpatrick et al., 2017) . we recruited 12 participants and asked them to interact independently with each system to discuss their covid-19 related concerns. more specifically, we asked them to use each system for 10-15 minutes and provide evaluative feedback pre-and post-interaction. to avoid cognitive bias, we randomized the order in which each participant evaluated the systems. in addition, we randomized the order in which the evaluation questions are shown. before interacting with either system, participants rated their life satisfaction and their stress level. after the interaction, participants reported again their stress level and rated several aspects of their interaction with the system, including ease of use, usefulness (in terms of discussing covid-19 related issues and motivation to write about it), overall experience, and satisfaction using mainly binary scales. for example, the questions "did <system> motivate you to write at length about your thoughts and feelings? yes/no" and "how useful was c.p. to discuss your concerns about covid? useful/not useful" assess whether the system encouraged the user to write about their thoughts and feelings about covid and whether the system provided guidance for it. tables 4 and 5 show the percentage of users that provided positive or high scores (> 3 on a 7-point scale) for each of these aspects after interacting with both systems. stress bef ore 91% 91% stress af ter 73% 64% as observed, there are fewer participants reporting high levels of stress after using either system. however, we see a smaller fraction of participants reporting high levels of stress after interacting with expressive interviewing, thus suggesting that our system was more effective in helping participants to reduce their stress levels. overall, participants reported that expressive interviewing was easier to use, more useful to discuss their covid concerns and motivated them to write more than woebot. similarly, users reported a more meaningful interaction and a better overall experience. however, it is important to mention that woebot was not specifically designed for discussing covid-19 concerns and it is of more general purpose than our system. nonetheless, we believe that this comparison provides evidence that a dialogue system such as expressive interviewing is more effective in helping users cope with covid-19 issues as compared to a general purpose dialogue system for mental health. we followed the suggestions of previous research on automated mental health counseling and adopted the goals of being respectful of user privacy, following evidence based methods, ensuring user safety, and being transparent in system capabilities (kretzschmar et al., 2019) . the practices of motivational interviewing and expressive writing have numerous studies supporting their efficacy (miller and rollnick, 2012; pennebaker and chung, 2007) . the combination of these methods in an interviewing format has not previously been studied and we intend to continue publishing our findings as the user population expands and becomes more diverse. we will also continue to improve our system and assessment. we have taken efforts to secure user data. we do not ask for identifiers and data is stored anonymously by session id. the website is secured with ssl. data is only accessible to researchers directly involved with our study. our study has been approved by the university of michigan irb. in this paper, we introduced an interview-style dialogue system called expressive interviewing to help people cope with the effects of the covid-19 pandemic. we provided a detailed description on how the system is designed and implemented. we analyzed a sample of 174 user interactions with our system and conducted qualitative and quantitative analyses on aspects such as system usefulness, user engagement and reflection effectiveness. we also conducted a comparative evaluation study between our system and woebot, a general purpose dialogue system for mental health. our main findings suggest that users benefited from the reflective strategies used by our system and experienced meaningful interactions leading to reduced stress levels. furthermore, our system was judged to be easier to use and more useful than woebot when discussing covid-19 related concerns. in future work we intend to explore the applicability of the developed system to other healthrelated domains. table 6 : average ratings grouped by order that the prompts appeared. all sessions begin with "major issues." figure 6 : histograms of the number of words of each user message preceding the generic reflections, grouping users whose stressed increased and decreased. figure 7 : histograms of the number of words of each user message after the generic reflections, grouping users whose stressed increased and decreased. figure 8: top: before and after stress ratings by users whose stress increased after interaction with c.p. middle: before and after stress ratings by users whose stress remained the same after interaction with c.p. bottom: before and after stress ratings by users whose stress decreased after interaction with c.p. the bars are ordered by the magnitude of change (top and bottom), or by the static stress rating (middle). development and deployment of a large-scale dialog-based intelligent tutoring system motivational interviewing to change quality of life for people with chronic heart failure: a randomised controlled trial brief motivational interviewing for teens at risk of substance use consequences: a randomized pilot study in a primary care clinic django software foundation delivering cognitive behavior therapy to young adults with symptoms of depression and anxiety using a fully automated conversational agent (woebot): a randomized controlled trial experimental disclosure and its moderators: a meta-analysis. psychological bulletin polyresponse: a rank-based approach to task-oriented dialogue with application in restaurant search and booking an empathy-driven, conversational artificial intelligence agent (wysa) for digital mental well-being: real-world data evaluation mixed-methods study can your phone be your therapist? young peoples ethical perspectives on the use of fully automated conversational agents (chatbots) in mental health support motivational interviewing: helping people change ad-viser: a dialog system framework for education & research writing about emotional experiences as a therapeutic process confronting a traumatic event: toward an understanding of inhibition and disease expressive writing, emotional upheavals, and health. foundations of health psychology expressive writing: connections to physical and mental health linguistic inquiry and word count: liwc writing about emotional experiences as a therapeutic process efficient allocation of public health and behavior change resources: the "difficulty by motivation" matrix roget's thesaurus of english words and phrases motivational interviewing. the essential handbook of treatment and prevention of alcohol problems pediatric nurse practitioners' assessment and management of childhood overweight/obesity: results from 1999 and 2005 cohort surveys expressive writing and coping with job loss wordnet affect: an affective extension of wordnet feelings in many words: natural emotion vocabularies as windows on distress and well-being annotating expressions of opinions and emotions in language. language resources and evaluation this material is based in part upon work supported by the precision health initiative at the university of michigan, by the national science foundation (grant #1815291), and by the john templeton foundation (grant #61156). any opinions, findings, and conclusions or recommendations expressed in this material are those of the authors and do not necessarily reflect the views of the precision health initiative, the national science foundation, or john templeton foundation. key: cord-267978-05hxrpi1 authors: nuzzo, jennifer b.; meyer, diane; snyder, michael; ravi, sanjana j.; lapascu, ana; souleles, jon; andrada, carolina i.; bishai, david title: what makes health systems resilient against infectious disease outbreaks and natural hazards? results from a scoping review date: 2019-10-17 journal: bmc public health doi: 10.1186/s12889-019-7707-z sha: doc_id: 267978 cord_uid: 05hxrpi1 background: the 2014–2016 ebola outbreak was a wake-up call regarding the critical importance of resilient health systems. fragile health systems can become overwhelmed during public health crises, further exacerbating the human, economic, and political toll. important work has been done to describe the general attributes of a health system resilient to these crises, and the next step will be to identify the specific capacities that health systems need to develop and maintain to achieve resiliency. methods: we conducted a scoping review of the literature to identify recurring themes and capacities needed for health system resiliency to infectious disease outbreaks and natural hazards and any existing implementation frameworks that highlight these capacities. we also sought to identify the overlap of the identified themes and capacities with those highlighted in the world health organization’s joint external evaluation. sources of evidence included pubmed, web of science, oaister, and the websites of relevant major public health organizations. results: we identified 16 themes of health system resilience, including: the need to develop plans for altered standards of care during emergencies, the need to develop plans for post-event recovery, and a commitment to quality improvement. most of the literature described the general attributes of a resilient health system; no implementation frameworks were identified that could translate these elements into specific capacities that health system actors can employ to improve resilience to outbreaks and natural hazards in a variety of settings. conclusions: an implementation-oriented health system resilience framework could help translate the important components of a health system identified in this review into specific capacities that actors in the health system could work to develop to improve resilience to public health crises. however, there remains a need to further refine the concept of resilience so that health systems can simultaneously achieve sustainable transformations in healthcare practice and health service delivery as well as improve their preparedness for emergencies. health system resilience has been previously defined as "the capacity of health actors, institutions, and populations to prepare for and effectively respond to crises; maintain core functions when a crisis hits; and, informed by lessons learnt during the crisis, reorganize if conditions require it" [1, 2] . for many countries, the 2014-2016 ebola outbreak in west africa was a wake-up call regarding the critical importance of having resilient health systems. in each of the three countries most affected by ebola, a fragile health system was quickly overwhelmed by the complexity of tracking cases, the need to create and disseminate communication strategies, and the challenges of safely caring for a surge of critically ill patients. health workers were 21-32 times more likely to have been infected with the virus than members of the general public [3] . sickened health workers could no longer care for ebola patients, and poor infection control in healthcare facilities contributed to nosocomial ebola transmission. in turn, heightened risks of nosocomial ebola infection increased public fear around hospitalization [4] . rather than helping to contain ebola, health systems became an amplifier of disease, exacerbating the human, economic, and political toll of the outbreak. similarly, unprepared health systems across the world inadvertently contributed to disease transmission during recent epidemics of severe acute respiratory syndrome (sars) and middle east respiratory syndrome (mers) [5] . health systems that were unprepared for disasters were also unable to provide essential services, even in highly developed countries (e.g., canada during sars [6] , korea with mers [7] , and the us following hurricane sandy [8] ). many countries have committed resources and efforts toward health system strengthening based on these recent disasters, but actionable plans and approaches to build resilient health systems have not yet achieved consensus. independent reviews of the global response to the 2014-2016 ebola outbreak have stressed the importance of establishing metrics to assess and monitor progress towards improving countries' capacity to respond to public health emergencies [9] [10] [11] . in 2016, the world health organization (who) created the international health regulations (ihr) joint external evaluation (jee) tool-a framework and process designed to measure countries' capacities to implement the requirements of the ihr, which include the ability to prevent, detect, and respond to public health emergencies of international concern [12] . since its introduction, the jee has become an important tool used by countries to assess their capacities for infectious disease outbreaks and other public health emergencies. to-date, more than 100 countries have conducted jee assessments [13] . some countries that have undergone jee assessments have also begun to develop action plans to address gaps found in their jees. despite this progress, health facilities continue to be vulnerable to public health emergencies [14] . important work has been done to describe the general attributes of a resilient health system [1, 2, [15] [16] [17] . for example, kruk et al. describe a resilient health system as one that is "integrated with existing efforts to strengthen health systems," able to "detect and interpret local warning signs and quickly call for support," able to provide care for a diverse population, able to "isolate threats and maintain core functions," and is able to "adapt to health shocks" [2] . however, as highlighted by turenne et al., there continues to be a lack of clarity around the conceptualization of health systems resilience [18] . the aim of this scoping review was to draw from existing literature to characterize specific capacities required to build resilient health systems in the face of infectious disease emergencies and natural hazards, with an emphasis on highlighting potential efforts that health system actors (e.g. health facilities and health service delivery organizations that are not always well-integrated in government-led preparedness initiatives) could pursue to achieve desired health outcomes during health crises. we also sought to examine the extent to which capacities that are associated with resilient health systems are addressed by existing frameworks for measuring and motivating countries' health security, such as the jee. we searched the scholarly and grey-literature databases to identify which capacities should be included in a framework for assessing and improving health system resilience to infectious disease outbreaks and natural hazards. we also sought to determine whether there were existing frameworks that highlighted these capacities that could be used in low-, middle-, and high-income settings. for the purposes of our research, we used the who definition of health systems, defined as "all the activities whose primary purpose is to promote, restore, or maintain health" [19] . specifically, we integrated literature in the following three areas: health security, health systems strengthening, and quality improvement. the aims of this research were to characterize the impacts that infectious disease outbreaks and natural hazards have on health systems; to identify challenges in maintaining health service delivery during outbreaks and natural hazards; and to identify strategies for effecting sustainable change in health systems-strengthening efforts. literature databases included pubmed, web of science, and oaister. key search terms were informed by, but not inclusive of, kruk et al.'s definition of a resilient health system, and included "health system," "health system strengthening," "resilience," "recover," "quality improvement," "infectious disease," "outbreak," "natural disaster," "global health security," "pandemic," "outbreak response," and "essential functions," as well as a variety of different pathogens responsible for recent infectious disease outbreaks (e.g., sars, ebola) and natural hazard types (e.g., cyclone, earthquake). 1 see additional file 1: table s1 for the full electronic search strategy. additionally, we examined the websites of major relevant public health organizations (who, the rockefeller foundation, cdc stacks) to identify articles and frameworks not indexed in the aforementioned databases. all but one of the search results were filtered to include only those articles published during or after 2002, to capture literature emanating in the wake of the sars epidemic, up until february 2018, the end of the study period. however, one search term did included articles published during or after 1990, to capture more broadly those resources that focused on essential health functions. only english-language articles were considered. we included documents if they described health system capacities that could potentially strengthen health system resilience to either infectious disease threats or natural hazards. documents were excluded if they described health capacities that were outside the aims of this research, as defined previously (i.e. articles that were purely about public health capacities that did not mention the relationship of these capacities to the healthcare system). for example, articles that described the importance of a trained epidemiologic workforce (a public health capacity) in outbreak identification and mitigation would be excluded. articles about the importance of engagement between ministries of health and the public would be excluded; however, articles about the importance of engagement between healthcare facilities and ministries of health would be included. documents were also excluded if the article described resilience in contexts outside of natural hazards and infectious disease outbreaks (i.e. armed conflict situations). each of the research team members (7 in total) was assigned a set of articles to review. each article title was reviewed by the assigned researcher for relevancy using the previously mentioned inclusion and exclusion criteria, followed by a review of the abstract for those titles deemed relevant. all articles deemed relevant after title and abstract review were then read in their entirety by the assigned researcher, providing a final set of articles for analysis. article references were also reviewed to identify important literature not located in the primary search. articles were then thematically coded by the assigned researcher using qrs international's nvivo 11 coding software [20] and a qualitative coding instrument developed from a priori themes previously identified in other resilience checklists [1, 2, 21, 22] . additional topics of interest that did not fit into the previously identified thematic rubric were coded as "other" for further review during data analysis. after completion of coding, through a process of inductive and deductive reasoning, the researchers identified a final list of themes and associated key literature that described the critical capacities necessary for health system resilience to infectious disease outbreaks and natural hazards. we then sought to identify areas of overlap between the health system resilience themes and capacities identified in our literature search, and the specific health security capacities that are the focus of the jee. the search yielded a total of 1108 articles after the removal of duplicates (additional file 3: figure s1 ). one hundred and fifty-eight articles were read in their entirety, of which 132 were deemed to be relevant and underwent thematic coding. after the completion of coding, we identified 77 key documents that described 16 high-level themes of health system resilience, which are summarized in additional file 1: table s2 (see additional file 2: appendix a and appendix b for a comprehensive breakdown of sources organized by theme and author). thirty-nine papers focused primarily on infectious diseases, while another 12 addressed natural hazards. the remaining 26 papers were not threat-specific, but rather articulated general principles for strengthening health systems and described baseline capacities required for health system functioning. while the themes found in our search were consistent with the five elements of a resilient health system previously outlined by kruk et al. [1, 2] , we also identified three additional themes not included in previous reviews, including the need to: develop policies for determining what level of care will be delivered when the level of demand exceeds existing resources; plan for post-event recovery; and commitment to quality improvement that ensures integration of lessons learned. for example, mehta et al. described the need to develop "altered standards of care" for responses to mass casualty events, which might include shifting resources to save as many lives as possible (i.e., triaging patients differently during emergencies as compared to normal operating conditions) and allowing for group isolation of patients that would normally be boarded in single rooms [23] . the literature identified a number of issues that must be addressed during the recovery phase of a public health emergency, including the need for grief and psychological counseling [24] , after-action assessment and revision of emergency response plans [25] , and rebuilding of social cohesion and trust [26] . a commitment to continuous quality improvement was also identified as an important component of resilient health systems, including making hospital performance ratings mandatory and publicly available to encourage peer competition with the primary goal being the overall improvement of hospital performance [27] . in integrating literature across subject areas, we were able to identify multiple references to the capacities necessary to achieve the 16 health system resilience attributes identified in our scoping review, which are summarized below (also see additional file 1: table s2 ). core health service capabilities: a resilient health system sustains baseline levels of routine healthcare delivery during a public health emergency [28] [29] [30] [31] [32] [33] [34] [35] . barriers to healthcare access: a resilient health system dismantles barriers to healthcare access so that the public accesses care during emergencies [36, 37] . maintaining critical infrastructure and transportation: a resilient health system develops plans to weather interruptions in critical infrastructure and transportation [24, 25, [38] [39] [40] [41] [42] [43] [44] [45] [46] [47] [48] [49] [50] . timely and flexible access to emergency/crisis financing: a resilient health system has timely, flexible access to financing so that it can better prepare for and respond to public health emergencies [24, 29, 36, 37, [39] [40] [41] [51] [52] [53] [54] [55] [56] [57] [58] [59] . leadership and command structure: a resilient health system has a clear and flexible command structure that has been established prior to an event and is exercised frequently [24, 29, 57, [60] [61] [62] [63] [64] [65] . collaboration, coordination, and partnerships: a resilient health system collaborates and coordinates with partners within and outside of the health system [2, 29, 39, 45, 57, 66, 67] . communication: a resilient health system has clear channels of communication between health system actors and other sectors, risk communication protocols, and robust engagement with patient populations [60, 68] . flexible plans and management structures: a resilient health system has flexible plans and management structures to cope with rapidly evolving circumstances [1, [69] [70] [71] . legal preparations: a resilient health system has made legal preparations to address challenges that may emerge during a crisis [1, 45, 49, 59, 62, 72, 73] . surge capacity: a resilient health system is able to call on human and capital resources to "surge" the level of care during public health emergencies [36, 74] . altered standards of care: a resilient health system has adaptable response plans to guide them in allocating scarce resources and healthcare services [23, 75] . health workforce: a resilient health system has an adequate, trained, and willing work force [4, 22, 36, 40, 45, 53, 54, 76, 77] . medical supplies and equipment: a resilient health system has access to medical supplies and equipment, including personal protective equipment, antivirals, and ventilators, during a crisis [40, 53, [78] [79] [80] . infection prevention and control (ipc): a resilient health system has implemented strong ipc measures, including staff training, standardized protocols, a dedicated ipc focal point, and dedicated treatment units [4, 22, 39, 47, 51, 54, [81] [82] [83] [84] [85] [86] . commitment to quality improvement: a resilient health system requires a commitment to continuous quality improvement that promotes excellence and garners the trust of the community [15, 27, 39, 54, 69, 87, 88] . plans for post-event recovery: resilient health systems have plans for post-event recovery that address a broad range of issues [24-26, 34, 40, 59, 73, 89-93] . the capacities that we identified are associated with different actors in health systems. some of the capacities identified in our review could potentially be developed by individual health facilities. for example, kim et al. discussed one health system's plan to develop alternate care centers that could be deployed during an influenza pandemic, including the infrastructure that needs to be in place to ensure adequate functioning, such as transport of patients to the center [42] . other capacities identified in the scoping review concerned the health system more broadly and would likely be addressed by national governmental authorities. for example, hanefeld et al. noted "the nature of the funding and financing mechanism as a core aspect enabling or hindering health systems' ability to respond to a shock" [29] . no frameworks were identified in the search that translated these high-level themes into specific and actionable steps that health system actors can employ to improve and support health system resilience to both infectious diseases and natural hazards, and that can be undertaken in low-, middle-, and high-income settings alike. frameworks that did articulate specific capacities were either 1) only applicable in the us context [22] or 2) did not cohesively address both infectious diseases and natural disasters [50] . for example, meyer et al. created a checklist for health sector resilience to highconsequence infectious diseases [22] , but the data for this checklist was informed by the us domestic response to the 2014-2016 west africa ebola outbreak. while some of the identified capacities may be generalizable to other countries, some are only pertinent in the us context. similarly, the hospital safety index, a tool developed by who, does identify capacities that are relevant to some health facilities, but the tool is largely aimed at evaluating the vulnerability of hospital infrastructure to natural hazards (an updated version includes limited consideration of the potential impact of infectious disease threats to hospitals) [50] . only two of the health system resilience themes and capacities identified in our literature search directly overlapped with the specific health security capacities that are the focus of the jee-namely ipc and communication (see additional file 1: table s3 ). specifically, the jee indicator on antimicrobial resistance does address ipc, but only within the context of healthcareassociated infections and associated ipc programs [12] . additionally, the literature we collected emphasized the importance of communication between health system actors, other sectors, and the community during outbreaks [60, 68] . the jee contains a very detailed section on communication that specifically calls out for the need for communication and coordination between stakeholders, including healthcare workers; for systems for rumor management through healthcare workers; and for formal communication mechanisms with the hospital and healthcare sector [12] . otherwise, health facilities are not directly addressed in the jee framework. there are some indicators in the jee that do address the themes identified in the literature review, but only within the context of public health. for example, the literature suggests that health facilities need access to financing during emergencies to cover the added costs of preparing for and responding to emergencies [24, 29, 36, 37, [39] [40] [41] [51] [52] [53] [54] [55] [56] [57] [58] [59] . the jee indicator on national, legislation, policy and financing does address whether countries have the financing to fulfill their obligations under the ihr, which includes "regulations or administrative requirements, or other governmental instruments governing public health surveillance and response" [12] . however, it does not specifically address financing within the context of health facilities, although countries could choose to include them in efforts to develop capacities in those areas. finally, some of the themes identified in our review could be leveraged through the development of other capacities that are the focus of the jee. for example, the jee does not explicitly assess how healthcare facilities should address barriers to healthcare access, such as long travel distances, the high cost of medical care, and public distrust. however, it does address the importance of risk communication and community engagement during an emergency. these relationships could potentially be leveraged by the healthcare system during an emergency to improve the public's trust in and subsequent use of the healthcare system. to date, much of the literature that specifically references health system resilience has focused on high-level attributes, rather than identifying specific capacities that health systems need to be resilient to infectious disease outbreaks and natural hazards. for example, kruk et al.'s five attributes of a resilient health system include a system that is "self-regulating," with the ability to "quickly identify and isolate a threat and target resources to it" [1] . by integrating literature from across different disciplines, we were able translate these high-level themes into actionable corresponding capacities that health systems need to respond to infectious disease outbreaks and natural hazards. for example, the literature highlighted numerous ipc protocols and practices that are important for the control of infectious disease threats, including the need for front-line healthcare workers to conduct travel histories [22] and the need to establish dedicated and multidisciplinary ipc committees to coordinate and guide healthcare staff on how to safely manage patients with infectious diseases [54, 84] . an article by palagyi et al., published after this review was conducted, also highlights the importance of these capacities that we identified [94] . additionally, the literature highlighted three themes not previously identified as attributes of a resilient health system, which warrant consideration in future efforts to define health system resilience. we present the capacities that we identified across the literature as merely the beginning of an effort to define capacities that health system actors need to be prepared for infectious disease outbreaks and natural hazards. further scholarship in these areas could support efforts to translate research findings into best practices in public health and healthcare practice and improve health outcomes following public health emergencies of all kinds. notably, the jee does identify the capacities necessary to implement the ihrs to protect against public health emergencies of international concern, but lacks guidance for health facilities at the patient-provider interface [12] . moreover, many of the capacities assessed in the jee presume the existence and functioning of core health system capacities, yet these capacities themselves are not explicitly addressed in jee assessments. for example, while the presence of a national laboratory system-a jee indicator-is a critical capacity to have during an outbreak, it requires that healthcare providers and the proper supplies be available to collect patient samples (e.g., blood, sputum, etc.). ideally, efforts to improve health system resilience would complement and build upon those foundational capacities presumed by the jee process. the results of this literature review point to a need for increased integration of efforts to advance health security and health systems strengthening across the globe. several high-priority elements for health systems resilience likely exist at the nexus of health systems strengthening, health system resilience, and health security; further work is needed to determine the most effective co-investments in global health security and health system strengthening that enable more robust health system responses at the local, national, regional, and global levels during emergent crises [95] . identifying those areas of overlap can help to actualize the jee's priority areas in health security, and also strengthen key components of national health systems such that their overall resilience is enhanced. while we strived to capture all relevant health systems literature, a potential limitation to our review is the lack of consistency and definitional clarity with which terms like "health system" and "resilience" are used throughout the medical and public health literature. it is possible that we may have missed relevant articles that describe these concepts using different terms. we also found in the literature an overrepresentation of papers detailing health system impacts of certain events and, thus, our findings may not include considerations from other events not represented in the literature. despite these limitations, we think our review serves to deepen the understanding of the specific capacities that health systems need to prepare for infectious diseases and natural hazards. the themes and capacities identified in our literature review provide an initial step in refining the concept of health system resilience to enable actors across the various sectors of the health system to take action to be able to respond and recover from infectious disease outbreaks and natural hazards. there remains a need to further define the concept of resilience so that health systems can simultaneously achieve sustainable transformations in public health practice and health service delivery as well as improve their preparedness for emergencies. in the same way that the jee tool has helped motivate countries to assess and improve their core public health capacities, an implementationoriented health system resilience framework could help translate the important components of a health system identified in this review into specific capacities that actors in the health system could work to develop to improve resilience to public health crises. moreover, such an effort may help to integrate foundational health system capacities into national efforts to improve core public health capacities. supplementary information accompanies this paper at https://doi.org/10. 1186/s12889-019-7707-z. additional file 1: table s1 . electronic search strategy. table s2 . summary of key themes and associated key evidence. table s3 . overlap of scoping review themes with joint external evaluation indicators. additional file 2: appendix a: coding framework--the full coding framework is provided, along with definitions of each code, and the number and first author/publication year of all sources that were coded into each theme. appendix b: citations are provided for all sources referenced in appendix a. additional file 3: figure s1 . selection of sources of evidence. what is a resilient health system? lessons from ebola building resilient health systems: a proposal for a resilience index who | ebola health worker infections a retrospective and prospective analysis of the west african ebola virus disease epidemic: robust national health systems at the foundation and an empowered who at the apex transmission characteristics of mers and sars in the healthcare setting: a comparative study critical role of nosocomial transmission in the toronto sars outbreak mers outbreak in korea: hospital-to-hospital transmission lessons from sandy -preparing health systems for future disasters will ebola change the game? ten essential reforms before the next pandemic. the report of the harvard-lshtm independent panel on the global response to ebola monitoring of disease outbreak preparedness: a shared framework assessments | global health security agenda strategic partnership for ihr and health security (sph) | building stronger health systems for sustainable health security governance and capacity to manage resilience of health systems: towards a new conceptual framework everybody's business --strengthening health systems to improve health outcomes: who's framework for action monitoring the building blocks of health systems: a handbook of indicators and their measurement strategies conceptual analysis of health systems resilience: a scoping review health systems: improving performance [internet]. world health organization nvivo qualitative data analysis software. qrs international pty ltd a community checklist for health sector resilience informed by hurricane sandy lessons from the domestic ebola response: improving health care system resilience to high consequence infectious diseases disaster and mass casualty management in a hospital: how well are we prepared? the health impact of the 2014-15 ebola outbreak hospital preparedness for chemical and radiological disasters the role of fear-related behaviors in the 2013-2016 west africa ebola virus disease outbreak future of japan's system of good health at low cost with equity: beyond universal coverage mortuary provision in emergencies causing mass fatalities towards an understanding of resilience: responding to health systems shocks ebola impact on african health systems entails a quest for more international and local resilience: the case of african portuguese speaking countries district health officer perceptions of pepfar's influence on the health system in uganda likely effect of the 2014 ebola epidemic on hiv care in liberia beyond vertical and horizontal programs: a diagonal approach to building national immunization programs through measles elimination disability and health-related rehabilitation in international disaster relief community-based health care is an essential component of a resilient health system: evidence from ebola outbreak in liberia health system resilience: lebanon and the syrian refugee crisis china's public health-care system: facing the challenges the initial health-system response to the earthquake in strengthening health systems while responding to a health crisis: lessons learned by a nongovernmental organization during the ebola virus disease epidemic in sierra leone even when you are afraid, you stay": provision of maternity care during the ebola virus epidemic: a qualitative study disasters; the 2010 haitian earthquake and the evacuation of burn victims to us burn centers role of hospitalists in an offsite alternate care center (acc) for pandemic flu quantifying surgical capacity in sierra leone: a guide for improving surgical care challenges of hospital response to the twin earthquakes of hurricanes katrina and rita: evacuee healthcare efforts remote from hurricane affected areas survey of the bam earthquake survivors' opinions on medical and health system services the link between the west african ebola outbreak and health systems in guinea, liberia and sierra leone: a systematic review. glob health estimation of surgery capacity in haiti: nationwide survey of hospitals the ebola outbreak in western africa: ethical obligations for care paho/who health emergencies -the hospital safety index | paho/who [internet]. pan american health organization / world health organization preparedness, evaluation, and care of pediatric patients under investigation for ebola virus disease: experience from a pediatric designated care facility ebola virus epidemic in west africa: global health economic challenges, lessons learned, and policy recommendations. value health reg issues developing health system surge capacity: community efforts in jeopardy keeping health facilities safe: one way of strengthening the interaction between disease-specific programmes and health systems strengthening the health system while investing in haiti effects of the global financial crisis on health in high-income oecd countries: a narrative review primary healthcare system capacities for responding to storm and flood-related health problems: a case study from a rural district in central vietnam retention of health workers in rural sierra leone: findings from life histories progress and challenges of disaster health management in china: a scoping review pandemic (h1n1) 2009: setting up a multi-agency regional response centre--a toolkit for other public health emergencies response to a serious flood: the st joseph's healthcare experience we and the nurses are now working with one voice": how community leaders and health committee members describe their role in sierra leone's ebola response how can health systems be strengthened to control and prevent an ebola outbreak? a narrative review preparing the health system to respond to ebola virus disease assessment of hospital emergency management in the beijing area partnerships for community resilience: perspectives from the los angeles county community disaster resilience project public health preparedness in california: lessons learned from seven health jurisdictions [internet]: rand corporation sars: a health system's perspective from bouncing back, to nurturing emergence: reframing the concept of resilience in health systems strengthening maternal health care in the time of ebola: a mixed-method exploration of the impact of the epidemic on delivery services in monrovia people-centred health systems: building more resilient health systems in the wake of the ebola crisis the sars commission interim report: sars and public health in ontario a hospital system's response to a hurricane offers lessons, including the need for mandatory interfacility drills. health aff proj hope bridging complexity theory and resilience to develop surge capacity in health systems barriers to integrating crisis standards of care principles into international disaster response plans: workshop summary the national academies collection: reports funded by national institutes of health) preparedness planning and care of patients under investigation for or with ebola virus disease: a survey of physicians in north america what adaptation to research is needed following crises: a comparative, qualitative study of the health workforce in sierra leone and nepal best practices for healthcare facility and regional stockpile maintenance and sustainment: a literature review the uk's multidisciplinary response to an ebola epidemic distribution of selected healthcare resources for influenza pandemic response in cambodia rapid assessment of ebola-related implications for reproductive, maternal, newborn and child health service delivery and utilization in guinea infection prevention and control of the ebola outbreak in liberia impact of the ebola outbreak on health systems and population health in sierra leone rapid assessment of ebola infection prevention and control needs--six districts mapping infectious disease hospital surge threats to lessons learnt in singapore: a systems analysis and development of a framework to inform how to decide on planning and response strategies estimation of needed isolation capacity for an airborne influenza pandemic strengthening resilience: a priority shared by health 2020 and the sustainable development goals [internet]. world health organization defining pharmaceutical systems strengthening: concepts to enable measurement recovery toolkit: supporting countries to achieve health service resilience [internet]. world health organization applying a new framework for public health systems recovery following emergencies and disasters: the example of haiti following a major earthquake and cholera outbreak ebola in the context of conflict affected states and health systems: case studies of northern uganda and sierra leone vertical interventions and system effects; have we learned anything from past experiences? rebuilding transformation strategies in post-ebola epidemics in africa health system preparedness for emerging infectious diseases: a synthesis of the literature who | health security and health systems strengthening -an integrated approach publisher's note springer nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations not applicable. this research was supported by the rockefeller foundation. the rockefeller foundation had no role in the study design, data collection, data analysis and interpretation, in the writing of the report, or the decision to submit it for publication. key: cord-027387-5ga212w8 authors: ma, yibing; feng, xuetao; jiao, jile; peng, zhongdong; qian, shenger; xue, hui; li, hua title: smart fire alarm system with person detection and thermal camera date: 2020-05-25 journal: computational science iccs 2020 doi: 10.1007/978-3-030-50436-6_26 sha: doc_id: 27387 cord_uid: 5ga212w8 fire alarm is crucial for safety of life and property in many scenes. a good fire alarm system should be small-sized, low-cost and effective to prevent fire accidents from happening. in this paper we introduce a smart fire alarm system used in kitchen as a representative scenario. the system captures both thermal and optical videos for temperature monitoring and person detection, which are further used to predict potential fire accident and avoid false alarm. thermal videos are used to record the temperature change in region-of-interests, for example, cookware. yolov3-tiny algorithm is modified for person detection and can be iteratively improved with the hard examples gathered by the system. to implement the system on an edge device instead of a server, we propose a high-efficiency neural network inference computing framework called turingnn. comprehensive rules enable the system to appropriately respond to different situations. the proposed system has been proved effective in both experiments and numerous cases in complex practical applications. fire prevention is crucial for the safety of life and property in residential and public scenes such as restaurants, hotels and factories. according to statistics [1, 2] , cooking is the leading cause of hotel fires (46%) and one out of every 12 hotels reports a structural fire per year in us. a fire accident usually happens when the cookware is kept heated on the stove without supervision. although fire regulations have been made to prevent fire accidents, they are sometimes ignored or disobeyed. the earlier the accidents can be detected, the easier they can be dealt with and the fewer damages can be caused. therefore, it is necessary to build a smart alarm system for fire prevention. the system needs two essential modules, potential fire sensing and proper disposal strategy. there are various methods for fire sensing such as by detecting smoke, carbon monoxide or flame. however, these methods work only when the fire has broken out. in contrast, monitoring the temperature of combustibles makes it possible to prevent fire before it occurs. there are three necessary ingredients for most fires, i.e. fuel, oxygen and heat. it is feasible to predict potential fire accidents when the temperature rises too quickly or near the ignition point. temperature measurement sites in application scenario, e.g. stoves in kitchen, are usually numerous and irregularly distributed. consequently, it is more practical to deploy remote and wide-ranged thermal cameras than single-point thermometers to monitor temperature. nevertheless, merely monitoring temperature may lead to the problem of false alarm when someone is working in the scene, for example a chef in the kitchen. as a correction, it is necessary to judge the presence of persons. the task can be fulfilled by person detection. with the development of computer vision and deep learning, person detection has been widely researched and various algorithms have been proposed. surveillance cameras have become common in public places, providing clear and realtime videos. but there are still challenges for person detection in different sites. for instance, chefs in kitchen usually wear white suits with masks and tall hats, which are rare in public datasets and difficult to detect for existing models. moreover, the system should be reliable, low-cost and easy to deploy. transmitting all the videos to one computing server is a waste of network bandwidth. instead, person detection should be implemented on distributed mobile processors on an edge device and only detection results are sent to communication server. to ensure the speed of person detection, a device-specific deep learning acceleration framework is necessary. in this paper, we propose a smart fire alarm system with bi-spectrum camera and embedded industry personal computer (ipc). the system is applied in kitchens and the diagram is shown in fig. 1 . the camera has an optical lens and a thermal lens to capture visible videos and monitor the temperature of cookware respectively. the processor performs person detection with yolov3-tiny [3] model. the algorithm is modified to deal with special appearance and occlusions of chefs in kitchen, and the inconsistency of aspect ratio between different training datasets. to implement the system on a mobile processor, we propose a high-performance neural network inference computing framework called turingnn. person detection and temperature analysis are completed in real time on the ipc. when the temperature reaches preset thresholds and no person is detected, staff in charge are alarmed with buzzers in the kitchen, phone calls or instant messages from mobile applications. meanwhile, the ipc reports an alarm along with current temperature, video frames and detection results to the server. in severe cases when temperature gets too high or rises too fast, the ipc sends a signal to controller to shut off the stove. as the system runs, incorrect person detection cases are collected to improve the model, and disposal rules can be updated according to feedback. the system has been applied for kitchen fire alarm in freshhema [4] , a combination of fresh food supermarket and restaurant with over 130 chain stores all over china. by adjusting detection model and disposal rules, we can apply the system to various application situations. the remainder of the paper is organized as follows. section 2 reviews the related work on fire alarm systems and person detection. section 3 presents the details of the proposed system. section 4 introduces the experiments and shows the respective results. section 5 concludes the whole paper and gives some directions for future work. fire safety solutions have been widely researched for years. a fire alarm system has a number of devices working together to detect fire and warn people through visual and audio appliances when smoke, flame, carbon monoxide or other emergencies occur. these alarms may be activated automatically from smoke detectors and heat detectors or be activated via manual fire alarm activation devices like manual call points. siemens [5] proposed a solution of fire detection in kitchens with fire detector which consists of smoke, carbon monoxide and flame detectors. however, this solution only works after fire has happened. moreover, although siemens claims that selectable parameter settings make the detectors immune to deceptive phenomena, they are not well adaptive to environment changes. cheng et al. [6] proposed a fire safety device for stove-top burner. it senses the temperature of the cooking utensil and automatically shuts off the flow of electricity or gas to the burner when the temperature of the cooking utensil exceeds threshold. however, the device does not consider the presence of persons, which may cause false alarms. there are some other fire alarm solutions such as [19] [20] [21] , which are similar to [5] and [6] . the alarms are made after the fire happens, which lead to delayed handling. thermal camera (also called infrared camera) is a device that creates an image using infrared radiation. all objects with temperature higher than absolute zero (−273.15°c) radiate infrared constantly. infrared thermal imager is a device that receives infrared radiation from objects and converts it into visible-light image. this thermal image corresponds to the temperature distribution field on the surface of the object, which is essentially the infrared radiation distribution map of all parts of the object. different from the 400-700 nm range of the visible light camera, thermal cameras are sensitive to wavelengths from about 1 lm to about 14 lm. when sensitive enough, infrared camera can capture accurate temperature of every position in an image, and further calculate the temperature change rate in a video. in kitchens, cooking is similar to fire accident when only monitoring heat, flame or smoke. therefore, person detection is an essential task in an intelligent fire alarm system to prevent false alarms. compared with general object detection, there are more challenges in person detection, such as various clothing, appearance, poses and different viewpoints of the camera. with the development of computer vision and deep learning, various object detection algorithms are proposed such as faster r-cnn [7] , ssd [8] and yolo [9] . although new algorithms are constantly proposed and better performances have been achieved, they usually require lots of computing resources and long inference time. in application scenarios, deep learning models should be economical and efficient on mobile platforms with limited computing power. numerous techniques have been proposed to accelerate deep learning models on low-power devices. for example, model quantization refers to the process of reducing the number of bits that represent a number [22] . in the context of deep learning, the predominant numerical format used for research and for deployment has so far been 32bit floating point. however, the pursuit for reduced bandwidth and computing requirements of deep learning models has driven research into using lower-precision numerical formats. it has been proved that weights and activations can be represented using 8-bit integers without incurring significant loss in accuracy. by combining various acceleration techniques, researchers have built deep learning inference frameworks for mobile devices, such as tensorflow lite [11] and ncnn [12] . the frameworks make deep learning models faster and smaller while maintain or even improve the performance by combining multiple optimization methods. despite the convenience of public frameworks, there are still adaption problems for specific devices and network structures, and there is still space for further optimization. the workflow of our system is demonstrated in fig. 2 . it consists of four modules, (a) temperature measurement, (b) person detection, (c) turingnn and (d) disposal rules. the temperature is measured with thermal videos. first of all, the locations of stoves are manually annotated in thermal videos, as shown in fig. 3 right. the temperature of target locations is sampled with an adjustable time interval. the heating curve is fit with least square method according to the discrete temperature data points in observation period. the slope of the curve, namely current temperature ascent rate r, is calculated as where the last n temperature data points are used for calculation and n is set to 20 in our system, t denotes the temperature check time interval and is set according to current temperature as shown in table 1 , y i represents the values of the n temperature data points. if the difference between the current temperature and the previous temperature is more than 20°c, the current temperature is ignored as a noise. person detection is deployed on optical video because the color and texture of persons are more obvious in optical video compared with thermal video, as shown in fig. 3 . in order to achieve accurate and efficient person detection, the small model of yolov3tiny is utilized in our system. yolov3-tiny is pruned from yolov3 [3] with fewer layers and scales. compared to the large model of yolov3, yolov3-tiny is 5 times faster with small accuracy loss. the network structure of yolov3-tiny is shown in fig. 4 . the model extracts feature map (fm) of input image with a 13-layer backbone network. yolov3-tiny predicts bounding boxes at two different scales (with stride 8 and 16) with the idea of feature pyramid network for object detection (fpn) [10] . two detection headers, which are separately built on the top of two feature maps with different scales, are responsible for detecting objects with different sizes. as shown in fig. 4(b) , each grid in the detection header is assigned with k different anchors, and thus predicts k detections that consist of 4 bounding box offsets, 1 objectiveness and c class predictions. in our case, k is set to 3 and c is 1 to only detect person. the final result tensor of detection header has a shape of n â n â (k(4 + 1 + c)) where n â n denotes the spatial size of the last convolutional feature map. three modifications are proposed to make it more suitable for our system. wide augmentation, which means data augmentation while keeping aspect ratio. to deal with the difference of person appearance between public datasets and kitchen and to guarantee the generalization ability of the model, we train it with both public datasets and application dataset of surveillance videos. as shown in fig. 5(a) and fig. 5(b) , public datasets have various aspect ratios while surveillance videos have fixed aspect ratio of 16:9 . when training the model, if we directly resize all images into the same aspect ratio, some ground truths will suffer from severe distortion, as illustrated in fig. 5(c) . therefore, we keep the aspect ratio of images unchanged by adding borders to the image in data augmentation, as demonstrated fig. 5(d) . ignore label. the original yolo distinguishes positive and negative objects with an intersection-over-union (iou) threshold. however, the ground truth is ambiguous in some cases such as crowd, heavy occlusion and blur. to deal with this issue, we introduce "ignore" label in annotation. the object is annotated with ignore label if it is a crowd of persons, a part of a person or when we are not sure if the object is a person or not. in training, if the iou between a box proposal and any ground truth box with ignore label is larger than threshold, the proposal is ignored in loss calculation. in this way the noise of unclear targets is avoided. iterative improvement. with the operation of the system, the incorrect person detection instances are accumulated along with the false fire alarms. the instances can be used as hard examples (images that cause the model make errors) to improve the detection model. the new model is finetuned from current model with new instances as well as original training data, which effectively avoid overfitting. each time the model is updated, the accumulation of hard examples and the next iteration becomes slower. finally, the model turns stable and accurate. turingnn is a novel deep learning inference framework optimized for mobile heterogeneous computing on android, ios, linux and windows devices. it has optimization solutions for most neural network models, operating systems and mobile processors. when developing turingnn, we set goals in various aspects, such as customization, resource-saving, high performance and easy-using. to achieve all these goals, comprehensive technical solutions and tricks are considered. data rearrangement. rearrange data for the underlying hardware to reduce the bottleneck of memory reading. for example, the technique of im2col is demonstrated in fig. 6 . a straightforward implementation of convolution is not well suited for fast execution on cpu. when dealing with convolutional layers in a neural network, turingnn rearranges the 2 â 2 kernel matrix into a 4 â 1 array. meanwhile the n â m input feature map (image) is reshaped into a p â 4 matrix by putting the elements of 2 â 2 neighborhood into a row, where p = (n -2 + 1)(m -2 + 1). in this way, the operation of convolution is translated to matrix multiplication. the cpu can then take advantage of special parallel hardware (sse or mmx) to speed up convolution substantially. quantitative calculation. 8-bit data is calculated for the underlying hardware to increase data multiplication and throughput. the quantitative calculation chart is shown in fig. 7 . firstly, turingnn runs a model of 32-bit floating point (fp32) with a calibration dataset and records the maximum and minimum of the feature map in each layer. during inference, the weights and input in each layer are converted into 8-bit integers (int8) with the recorded maximum and minimum, and then the output is turned back to fp32. the quantization and dequantization formula are noted in eq. (2), where f and q are the fp32 and int8 values, while f max and f min are the maximum and minimum fp32 values of each layers. as shown in fig. 7 left, the dequantization of the previous operation and the quantization of the next operation will cancel each other out. therefore, turingnn combines the dequantization and quantization before each operation when there are multiple quantified operations in succession, as illustrated in fig. 7 right. multithreading. turingnn makes full use of the ability of multithreading. when the compiler supports openmp, we set the concurrent number according to the hardware device, and the concurrent time-consuming cycle will use multi-core to complete the operation. on apple's platform, we use grand central dispatch (gcd) to dispatch the time-consuming cycle, and the system dynamically allocates the concurrent number, so as to obtain better performance than openmp. on android and linux platform, using thread pool for multithreading management and control can make more efficient use of multi-core and large-small-core hardware resources. memory layout. different data arrangement formats are designed for different operators. for example, operators such as convolution and pool run on the memory layout of batch number/channel/height/width (nchw), while operators such as post and split use the memory layout of nhwc. when the memory layouts of adjacent operators are inconsistent, conversion operators are inserted. fusing. when transforming models, we merge layers that do not need to exist independently to reduce the amount of calculation. for example, by merging batch normalization into convolution, the cost of batch normalization is saved while the operation result is not affected. the disposal rules of industrial personal computer (ipc) in our system are demonstrated in fig. 2 . the optical lens and thermal lens focus on the same view of the kitchen. person detection is continuously run in optical videos. if no person is detected, current temperature of cookware is measured from thermal videos. when food is fried, the oil temperature rises fast and then slows down. according to this rule, different temperature thresholds are set for temperature analysis, as listed in table 1 . as the temperature rises, the check interval becomes shorter, the accent rate threshold gets smaller and the disposal action becomes more urgent. when the temperature or the ascent rate reaches a threshold, the corresponding disposal actions are triggered. each time the system sends an alarm, the frames of the optical and infrared videos are saved along with person detection results and temperature data. incorrect person detection results will be fed back to improve person detection algorithm. the bi-spectrum camera in our system (fig. 8) is dh-tpc-bf2221-t manufactured by dahua technology [13] . it has an optical lens and a thermal lens to capture optical videos and monitor temperature. the thermal lens has many advantages such as fast response (latency less than 40 ms), wide temperature measurement range (−20°c to 550°c) and small error (less than 5°c). the type of ipc ( fig. 9) is uno-2372g produced by advantech [14] . it has intel ® atom baytrial e3845 quad core processor up to 1.91 ghz and 4 gb ddr3l memory. to guarantee the generalization of the person detection model, we build the training and testing dataset with our application dataset and multiple public datasets including pascal voc (only person) [15] , coco2017 (only person) [16] , kitti [17] and citypersons [18] . there are 60000 and 80000 images in application dataset and public datasets respectively, and 20% of images in each dataset are randomly chosen as test set and the remainder for training. the results of the following experiments are based on the test set. the results of different person detection algorithms are listed in table 2 . compared with original yolov3-tiny, the model with ignore label improves the precisions of both public and application datasets, showing its general effectiveness. as a contrast, wide augmentation significantly improves the performance on application dataset while not affects the performance on public datasets. the reason is that wide augmentation adapts the model to the highly concentrated aspect ratio of the application dataset (16:9) with input size of 384 â 224 instead of the original 288 â 288. by combining ignore label and wide augmentation (il â wa), the model achieves better scores on application dataset, showing the coordination of the two modifications. the last line indicates the model finetuned with 500 hard examples. it further improves the precision on application dataset, proving the effectiveness of iterative model improvement. we speed up the person detection model above with different model acceleration frameworks, and the inference time is listed in table 3 . darknet [3] is the original framework, and all the models have the same precision. the inference time is calculated with the same processor of our system. it is clear that all the acceleration frameworks have large speedup over original darknet. moreover, turingnn shows the advantage in efficiency over the other open-source frameworks. it is 17.4% faster than ncnn [12], 48.5% faster than tensorflow lite [11] and 370% faster than darknet, indicating the effectiveness of the optimizations in turingnn. in summary, the features of existing fire alarm systems and the proposed system are compared in table 4 . it is obvious that our system has advantage over existing ones in accuracy, speed, cost and so on. the proposed fire alarm system has been applied for kitchen safety in freshhema for over 6 months. an alarm is defined as effective if the oil temperature has risen into the ranges in table 1 and nobody is near the stove, i.e. both the temperature measurement and person detection are correct. for each store, the system makes over 100 effective alarms per month. the alarmed cases do not necessarily lead to fire accident, but the warnings do reduce potential fire risks and improve the safety awareness of the staff. in this paper we propose a smart kitchen fire alarm system. the proposed system prevents fire accidents by monitoring the temperature of cookware with thermal cameras. moreover, the system achieves quick response and avoids false alarm via person detection. by using yolov3-tiny algorithm and model quantification framework turingnn, we are able to implement the system on small-scale low-cost edge devices for practical situations. experiments demonstrate the effectiveness of our system. future research will focus on person detection on thermal videos and improving the precision and recall of the system with the detections of both optical and thermal videos. national fire protection association: structure fires in hotel and motels fire administration: hotel and motel fires yolov3: an incremental improvement fire detection in kitchens fire safety device for stove-top burner. patent, us5945017a, united sates faster r-cnn: towards real-time object detection with region proposal networks ssd: single shot multibox detector you only look once: unified, real-time object detection feature pyramid networks for object detection dahua technology advantech the pascal visual object classes challenge 2012 (voc2012) results microsoft coco: common objects in context. in: fleet are we ready for autonomous driving? the kitti vision benchmark suite citypersons: a diverse dataset for pedestrian detection installation, service, and inspection for a range of systems nearby engineers inc.: fire suppression systems for commercial kitchens encore fire protection inc.: protecting your kitchen: restaurant fire safety systems quantizing deep convolutional networks for efficient inference: a whitepaper microsoft coco: common objects in context key: cord-183016-ajwnihk6 authors: carrillo, dick; nguyen, lam duc; nardelli, pedro h. j.; pournaras, evangelos; morita, plinio; rodr'iguez, dem'ostenes z.; dzaferagic, merim; siljak, harun; jung, alexander; h'ebert-dufresne, laurent; macaluso, irene; ullah, mehar; fraidenraich, gustavo; popovski, petar title: containing future epidemics with trustworthy federated systems for ubiquitous warning and response date: 2020-10-26 journal: nan doi: nan sha: doc_id: 183016 cord_uid: ajwnihk6 in this paper, we propose a global digital platform to avoid and combat epidemics by providing relevant real-time information to support selective lockdowns. it leverages the pervasiveness of wireless connectivity while being trustworthy and secure. the proposed system is conceptualized to be decentralized yet federated, based on ubiquitous public systems and active citizen participation. its foundations lie on the principle of informational self-determination. we argue that only in this way it can become a trustworthy and legitimate public good infrastructure for citizens by balancing the asymmetry of the different hierarchical levels within the federated organization while providing highly effective detection and guiding mitigation measures towards graceful lockdown of the society. to exemplify the proposed system, we choose the remote patient monitoring as use case. in which, the integration of distributed ledger technologies with narrowband iot technology is evaluated considering different number of endorsed peers. an experimental proof of concept setup is used to evaluate the performance of this integration, in which the end-to-end latency is slightly increased when a new endorsed element is added. however, the system reliability, privacy, and interoperability are guaranteed. in this sense, we expect active participation of empowered citizens to supplement the more usual top-down management of epidemics. the covid-19 pandemic has clearly shown that, in many senses, the world as a whole was not prepared for dealing with a disease of such magnitude. although a final assessment is infeasible at this point (october, 2020), in which a second wave is creeping back in europe and is poised to rage across the continent by fall 2020 [1] the current statistics available about covid-19 indicate that the most successful policies for monitoring and controlling the virus propagation are employing various digital technologies and connectivity [2] , [3] . these technologies can be used in two principal ways: (1) to provide status and predictions of the epidemiological spread and facilitate actions, such as administration of diagnostic tests or preparation of medical equipment; (2) to implement active policies that facilitate societal processes and safe citizen movement, such as shop schedules, organized delivery of goods, and similar. the success of such informed policies is also strongly related to the timing of their implementation for the respective place, as virus contagion is a spatiotemporal phenomenon that usually leads to exponential growth in the number of infected individuals [4] . unfortunately, even the most successful timely interventions are in some way overreaching, shutting down abruptly most of the economic activities and creating social distancing between citizens. while acknowledging that this is our current societal need to contain the propagation of this pandemic, our main hypothesis is that, in the near future, digital technologies and wireless connectivity can enable a graceful lockdown by having the following main roles. • facilitate selective lockdowns by allowing flexible transfer of the work, learn and play activities online to the desired extent. • assist in maintaining physical distancing and tracking the spreading of the disease, while offering digital tools to control the level of lockdown/reopening. • provide real-time information for targeted and efficient testing. note that these are also important today (october, 2020), when a second wave is forcing several countries to rethink a stepby-step reopening based on the aforementioned points. this will be achieved based on an integrated solution for the ubiquitous and privacy-preserving data acquisition, explainable predictive methods for contagion risk assessment, and digitized policies for selective lockdown and scheduling of societal activities. data will be locally acquired via online social networks, personal smart phone devices and internet-of-things (iot) sensors in general. it is foreseen that an effective global solution shall be decentralized, yet hierarchical (systemof-systems), to provide fine-tuned coordination among the federated entities to orchestrate actions and then avoid both the human rights cost of overly stringent solutions [5] and the human life cost of "hands-off" approaches. beyond more traditional centralized policy-making, such a decentralized platform, which will be built using trustworthy distributed ledger technology (dlt), should also provide citizens with arxiv:2010.13392v1 [cs.dc] 26 oct 2020 tools for more direct participation in mitigation measures based on tailored individual incentives. in this sense, we expect to bring managerial decisions closer to the citizens. with such a federated governance, we attempt to convert the usually passive data acquisition process to an active participatory one. hence, our aim is to move away from a purely top-down management approach. in addition, the proposed platform aims at achieving the minimal use of personal data, processed with the maximum possible privacy-preservation to eliminate global health risks, while protecting the economy [6] . the covid-19 pandemic has demonstrated that nations and governments need to be better prepared and coordinated to detect and react to global threats focusing on early detection, early response, moving beyond traditionally passive disease monitoring based on voluntary reporting systems. hence, a rapid, participatory responsive detection system must be in place to support public health officials. a synergistic relationship with public health officials, policymakers, and citizens' active participation will be critical to align the mandate for public health with the protection of privacy, freedom and democracy [7] , [8] . in this context, one main factor is to design a special set of incentives that would allow the citizens to provide secured anonymized access to their data while actively participating in the crowd platform to support early disease detection, a public information system, and possible mitigation measures. building up such an incentive structure that maps those tradeoffs is a key aspect (beyond purely technical ones) for the success of the proposed federated architecture. furthermore, this system should be constructed and evaluated rigorously following ethical guidelines as in [3] , [9] . our contribution in this paper is threefold: 1) a comprehensive analysis of epidemiological models, data collection, and wireless connectivity is done based on key relevant scientific references on pandemics. 2) a federated global epidemiological warning system is proposed based on dlts. 3) a proof of concept of the integration between dlt and nb-iot is used to evaluate the wireless network performance on the iot infrastructure supporting a remote patient monitoring use case. the rest of the paper is organized as follows: section ii presents a discussion of epidemiological models and their limitations. section iii describes the relevance of wireless connectivity on pandemic scenarios. in section iv, the federated global epidemiological warning system is proposed, here is also detailed the proof of concept of the integration of dlt and narrowband iot (nb-iot). section v is reserved for some discussion and future perspectives. biomedical data alone do not contain the information required for preventing and mitigating pandemics. in a highly interconnected globalized society, social interactions, environmental data, spatiotemporal events, and collective nonlinear phenomena observed on complex infrastructures such as transport systems, require the mining of heterogeneous pandemic big data that are a result of a complex system of systems processes. in fact, modern disease monitoring systems, as the global public health intelligence network (gphin) [10] and the global outbreak alert and response network (goarn) [11] , and event-based approaches that use a combination of web crawlers, artificial intelligence, and public health expertise in the detection of indicators [12] , [13] provide goodalthough limited-examples of successful platforms to identify latent indicators of an outbreak. to go beyond those existing solutions, data scale, contextualization, and granularity are key requirements for data quality, which is usually orthogonal to privacy preservation, timely processing and analysis as well as storage and processing cost. in the following, more details will be provided about existing epidemiological models and epidemic-related data collection. models for emerging epidemics come in various forms, all relying on differently coarse-grained individuals' data. state-of-the-art population-level models for covid-19 rely on metapopulation models with an underlying susceptible-exposed-infectious-recovered disease dynamics [14] . the metapopulations represent regions surrounding travel hubs and are interconnected through mobility data, for example, data from airlines, public transportation systems, and traffic control systems. the disease dynamics itself accounts for the fact that exposed individuals can be presymptomatic and unaware that they infect others, as suggested by early case data [15] . unfortunately, while this model is effective at providing large-scale forecasts and predicting importations at aggregated levels, it is less informative at the individual level. however, this more granular information is necessary to accurately estimate the probability of such epidemics given a single importation event, which is rare given the population size. this particular event has the potential to become the initial point of a widespread contagion in a region not reached by epidemics until that point. since this event basically depends on one individual, aggregated level statistics provide poor description of the heterogeneity of individual level behavior and mobility. given this, most governments around the world introduced stay-at-home policies to reduce the mobility of their citizens, which should result in the now famous flattening of the curve. this expression refers to the process of slowing down the virus spread to keep the need for hospital care below the health care capacity. different variations of the stay-at-home policy have been introduced, e.g. allowing citizens to move in a predefined radius to walk with their pets and exercise, restricting the number of times per day or the number of people per household that can go out. as previously argued, this is, understandably, a necessary abrupt solution for now, but it can become more graceful by properly utilizing crowdsourced data and active citizens' participation. b. data collection: current activities and potential existing sources at higher levels, mining existing data sets collected by different types of service providers is an obvious and powerful way to quantify to which extent citizens follow stay-at-home policies, thereby providing an essential means of assessing their effectiveness. for example, smart grid systems can provide information about the changes in the energy consumption in different areas suggesting that people, on average, spend more time at home. the network usage information from broadband operators can also be used as an indicator of change in human behavior related to their mobility. another source of information are mobility data sets from cellular network operators that are already present on the network. smart appliances, like smart tvs, smart fridges, and smart light bulbs can be used to provide information about the overall time spent at home versus the time spent outside. finally, information collected by several smart city applications, such as public transport usage and traffic, could also be leveraged to estimate the change in mobility patterns at the level of places, buildings and vehicles. at a lower granularity, proximity-tracing platforms have been proposed to collect data at the individual level, for instance the pan-european privacy-preserving proximity tracing (pepp-pt) [16] , often in partnership with mobile service providers and operators. by aggregating proximity data over time, these approaches can follow data protection regulations. among others, two of the largest big tech companies apple and google proposed a joint solution that appears to focus on the protection of user privacy, for example by keeping people anonymous in the central servers and making data submission voluntary. different technologies are being proposed to automate and extend contact tracing (e.g. shared databases, gps traces of confirmed cases, contact tracing through bluetooth). the use of bluetooth-based solutions, either in the form of the third-party dedicated apps or as a feature built into the mobile devices' operating systems, seems to be the most promising solution to date. although these solutions are a new source of granular data, they cannot cover all relevant forms of infection, namely asymptomatic infections, and infections occurring out of proximity through shared surfaces (e.g. doorknobs). in this sense, our hope is to combine the metapopulation and individual tracing models in a citizen science framework to consider more possible routes of infection as well as both proximity (e.g. close contact) and mobility data (e.g. location visited). in addition to this, machine learning methods are expected to combine expert knowledge (e.g. from virologists and sociologists) with global data collected from different sources for fitting powerful predictive models to high-dimensional data. in doing so, the target is to leverage the different straights of different data sources at different granularity for privacy protection and allow for more thorough probabilistic forecasts. ultimately this will allow for graceful lockdowns, tailored incentives for citizens' participation, and fine-tuned legitimate interventions. fine-grained data collection approaches raise two major concerns as highlighted in [17] : (1) user privacy; and (2) trustworthiness of the shared information. clearly, sharing location and health information with third-party entities can lead to the misuse of the data set for instance through unwanted advertising and health insurance implications. the impact of fake health information on the contact tracing system should be analyzed to assess its implications in terms of access to testing. all these applications rely on the premise that users will trust and adopt these applications. this brings us to an additional problem that could hamper these approaches. the adoption of new technologies is hard to predict, and it might introduce bias for example related to age, wealth, level of education, and state of country development. citizens' participation in warning and response of epidemic outbreaks requires new incentives that reward the responsible use of citizens' personal data for protecting public health, while penalize and prevent citizens' profiling actions, manipulative nudging and power misuse. designing new data fusion schemes tailored to fuel data analytics processes for prevention and mitigation of pandemics is an open research question: which smart phone sensor data can model compliance of stay-at-home policies? how such models can be enhanced with social media, smart grid, or transport data? pandemics require that we revisit and potentially reinvent how data should be managed and how systems should be designed to manage data in a more responsible way [9] . more specifically, discovering new ways to turn private sensitive data of citizens into public good, while preventing massive surveillance, profiling, and discriminatory analytics, becomes imperative. to pioneer this capability, new transparent and accountable data commons [18] , for instance personal data stores [19] [21] , to which citizens have incentives to contribute data, are required. the citizens retain the right for their data to be "forgotten" [22] . ultimately, they have control and give their consent to how these data are used. these data commons are designed to maximize the use of techniques for privacy preservation [6] , [23] , for example homomorphic encryption, differential privacy, anonymity, and obfuscation, while citizens are made aware of the privacy risks they experience. the previous section covered different aspects related to the data required to support a fine-grained epidemiological model. it is argued that a ubiquitous system can be employed to manage such data in a privacy-preserving manner. here, we identify wireless connectivity and internet of things (iot) devices as a way to collect data following those principles. wireless connectivity and iot devices come in many flavors, but can, in general, be classified into (1) personal devices, such as mobile phones and earbuds; and (2) unattended connected machines and things, such as surveillance cameras and motion sensors. there are three principal sources of epidemic-relevant data acquired through wireless connectivity: (1) online social networks; (2) personal smart phone and mobile data; and (3) sensory and internet of things (iot) devices. in critical events, people tend to use online social networks to post comments about the emergency and learn from other users' comments. as a result, online social networks become a rich source of diverse information that could help to understand the main characteristics of the crises and their potential magnitude in early stages. for instance, in [24] , authors collected comments posted in the chinese social media channel weibo during the first month of the covid-19 epidemic in wuhan (china) to understand the evolution trend of the public opinion. a similar approach was used on twitter in [25] , [26] . government entities can use this approach to give more attention to the needs of the public during the beginning of the epidemic and adjust their responses accordingly. another form of data content, normally provided by explicit consent from the citizens, is the gps location data from personal devices. at the application level, the twitter platform offers various user information, geographical location being one of the most important for a wide range of applications. however, only a few users enable their geolocation as public. to discover the user's geolocation (geographical region or exact geocoordinates), there are mainly two approaches called contentbased and network-based algorithms. the first one uses textual contents from tweets, and the latter uses the connections and interactions between users. in [27] , a neural network model based on multiview learning by combining knowledge from both user-generated content and network interaction is proposed to infer users' locations. this source of information comes in many different forms, ranging from the use of metadata in cellular mobile networks for user localization up to the metadata associated with different applications, such as twitter. the use of data 1 collected in mobile networks during the covid-19 pandemic has received significant attention [28] . agglomerated mobile network data can be used to verify if interventions, such as school closures, are effective, and help to understand the geographical spread of an epidemic. different types of mobile network metadata are collected at different levels of the communication system, and they offer varying levels of information. at the lowest level, localization of mobile network users is possible by evaluating the strength of the wireless signal received at base stations. depending on how many base stations are connected to the device, the location can be determined at the level of entire cells or down to a few meters using triangulation. the resolution offered by cellular mobile localization maybe too coarse for detecting citizens' proximity and potential infections by face-to-face interactions. nevertheless, even lowresolution location data can give insight into behavioral patterns (how much time is spent at home, office, and events) of individuals. proximity detection can be enhanced by using the metadata from bluetooth devices, such as beacons and discovery messages (e.g., [29] ). wi-fi is also a technology that is used ubiquitously, and its metadata can provide information on user proximity. for example, proximity can be inferred by comparing the lists of access points that each device can see within a given time interval [30] . fusing the metadata from these different sources, along with the context information (how many family members are at home, which events) is very relevant for monitoring the epidemic. many applications and devices become an important tool to provide epidemic-relevant data. these can be related to surveillance/thermal cameras, drones and even wearable devices. for example, surveillance cameras can be employed to count the number of people entering and exiting a specific area. in other scenarios, thermal cameras are used with specialized settings to focus on human skin temperature range, and infrared spot sensors for individual temperature scanning. a similar application to identify individual or group activities in a given place is based on motion sensors as infrared lasers, or ultrasonic sensors. in this approach, the motion sensors are installed and deployed in a specific environment to recognize different human activities. other important source of iot data are the smart healthcare devices, for instance, remote monitoring systems to check body temperature, which is a key sign in the support of homeostasis. other popular applications are oxygen saturation monitoring based on beat oximetry, electrocardiogram monitoring with a specialized framework to estimate the heart rate, elderly monitoring using doppler radar to identify risk movements of elderly people, sugar level monitoring, and blood pressure monitoring. these iot devices provide information that is analyzed by the iot data to support specific applications. we envision a federated and decentralized coordination system for epidimiological warning and response. this system is federated by citizen communities that crowd-source data, personal smart phone devices, community-level iot devices (e.g. lorawan networks) and other computational resources on the edge. it can scale organically and bottom-up to citylevel, national-level and ultimately at global-level to coordinate in a socially responsible way the international actions of public health organizations and governments. this scaling requires tailored incentives that align public health policy-making with citizens' privacy and autonomy. distributed ledgers with secure crypto-economic incentive models running on edge serves and scaling-up on-demand at a global level using wireless connectivity and public cloud infrastructure are the means to support the federated nature of this proposed system. figure 1 illustrates the our vision. the proposed federated global epidemiological warning and response system is built upon epidemic-relevant information obtained through wireless connectivity introduced in section iii, and the internet infrastructure including fiber-optic and satellite links. thus, in addition to the devices that are the data sources (e.g., smart phones, wearables, smart appliances, and cameras) with their applications and existing communication networks, there is a need for a computer infrastructure dedicated to store and process the epidemic-relevant data in a secure and privacy-preserving manner. it is argued that this infrastructure also needs to follow a similar federated organization based on the geographic locations. each municipality, city, county, or larger neighborhood will rely on edge servers to process their respective data contents. the premise is to keep the computations as local as possible. however, larger-scale computations related to the interrelations between locations (e.g. mobility, commuting, and traveling) requires data from these different places. once again, the federated organization supports this collaborative sharing (up and downstream between the federated entities), but it will probably require computationally more demanding algorithms. it is possible that those computational tasks require more powerful cloud servers, or collaborative parallel computing at the edges. for security reasons, dlts shall be employed to store data from the edge servers at the global level. at the technical level, the following aspects deserve special attention. in this paper, a privacy-by-design approach is proposed with which several critical operations of the federated global epidemiological system can be performed; for instance, decentralized data analytics [31] , [32] , social interactions analysis [33] , [34] , decentralized planning and resource allocation [35] and federated learning [36] , [37] . such operations integrate in a smart way state-of-the-art techniques, for instance, informational self-determination, homomorphic encryption, differential privacy, obfuscation, and anonymity. often, privacy may limit the quality of service known as the system utility as the accuracy and quality of data are deteriorated to hide information content. for instance, predicting the risk of infections for individuals based on an epidemiological network model is a graph-based semi-supervised learning problem. privacy-preserving semisupervised learning over graphs has been considered in [38] . however, the precise trade-off between (lowering of) privacy protection and learning accuracy is largely open. pareto optimal trade-offs can be configured and regulated by tuning the parameters of the privacy techniques as previously shown in [6] . monetary and other incentives can be used to coordinate data sharing choices in a crowd. however, new types of social incentives are required for such an epidemiological system; for instance, incentives related to well-being, receiving solidarity, and longterm payoffs. 2) interoperability: the big data required by the proposed solution have to be interoperable, i.e. the several applications that are providing data to be used to accomplish a specific task have to operate together and share a common "understanding" [39] . this can be achieved by employing standards for health informatics such as iso ts22220:2011, ts21090:2011, or ts13606. for instance, the data collected from different sources can be used to predict refinements to patient care or new drug contraindication [40] . this key issue, though, has deserved little attention in large-scale epidemiological studies [41] , [42] ; it is usually assumed that heterogeneous data sources are compatible with each other. in practice, though, the highly heterogeneous data sources lead to poor interoperability, which creates barriers to effectively combat pandemics like covid-19, as indicated by [43] . 3) user interfaces: a successful platform also involves a suitable end-user interface [44] . in this sense, data consumption by public health officials and global health agencies will require user-friendly web-based interfaces, using common dashboarding techniques. however, beyond this, the proposed federated platform has to consider citizen participation and the heterogeneity of end-users. therefore, the following characteristics should be taken into consideration in the design of the platform: it should be (a) explainable/accountable to improve for instance awareness and engagement [45] ; (b) gamified to engage and incentivize participation [46] , [47] ; and (c) customizable for different user groups at an international level. the proposed architecture has to articulate the key elements from data acquisition to analytics, following the best practices related to privacy and cyber-security. at the acquisition level, in addition to the existing data retrieval from the web, wirelessconnected devices will send data to edge nodes that will be associated with specific regions. data will be anonymously preprocessed at the edge, including some intelligent detection of anomalies or event detection, and then sent to regional (cloud) servers to run a more complete model that will fuse the geolocated timestamped data and run detection models based on explainable ai approaches combined with mathematical computational methods. in this case, crowd-sourcing models are applied based on hardware as a public good in an approach similar to the diaspora social network, also extending this approach to software and data. regional models will be associated in federations resembling the governance structure of the actual regions under interest, which is customizable to local policies/governance models. following this federated and collaborative organization, it will be possible to build organically a fine-tuned early detection system at the global level in a decentralized yet hierarchical manner to support graceful lockdowns. this would turn the proposed federated architecture into a holarchy, where each level is independent and self-sustained, but it can also be encapsulated at a level above to capture new goals [48] . this system will rely on dlts to guarantee a trustful system without a responsible third party and minimize the risk of data manipulation [49] . distributed ledgers could also be used to facilitate different (crypto-economic) incentive mechanisms, such as token curated registries. its main objective is to build a crowd-sensing trustworthy platform based on privacypreserving methods to detect potential harmful symptoms in a specific region in almost real-time and flag them to relevant authorities. this alarm needs to be accurate, reliable, and explainable. it will also require a user-tailored interface that could empower citizens by providing detailed explanations and easy-to-access information, while being a tool for policymakers (from the city level to global organizations) designing the correct interventions (a variety of incentives, sanctions, and other persuasive measures) given the specific context of the epidemic in some specific region in a given period of time. to obtain insights from a practical implementation, we define a specific use case that matches the premises of the proposed architecture. this key industry use case focuses on remote patient monitoring. here, many issues in treating chronic patients could be reduced or resolved through more efficient patient care. in the case of pandemics, such as covid-19, the remote monitoring of diagnoses patient can represent a key difference to avoid the virus acceleration. besides that, the application should guarantee privacy and security on the gathered data. to guarantee people participation, the system can provide incentives, such as bonus or tax compensations. in the context of the proposed federated global epidemiological warning system, the remote patient monitoring is a representative use case, in which the integration between dlts and iot devices plays a key role. we analyze this integration through an experimental setup based on nb-iot, which is one of the most representative low-power wide-area network (lpwan) technologies in cellular networks. considering that the proposed architecture requires massive end devices deployment, low power consumption, superior coverage, and worldwide deployment, nb-iot becomes a key solution to support the federated global epidemiological warning system. some details of the integration setup are described in the next sub-section, emphasizing data workflow, and end-to-end (e2e) latency. we consider latency because the experimental results indicate that it is the most sensible network parameter when dlts are built in the top of the connectivity system. in this section, we provide brief explanation of the integration between the nb-iot network and the dlt-based system, which is illustrated in figure 2 . in a conventional nb-iot system, the uplink data generated by the user equipments (ues) is transmitted though specialized packet messages and routed toward an edge data center to be stored and processed (yellow background in fig. 2 representing a standard-nb-iot). at this point, the monitoring system has no control on the collected data, so modification, corruption, and losses may occur. conversely, in our dltenabled nb-iot setup, the uplink data generated by the ues is transmitted to a randomly chosen group of endorsing peers of hyperledger fabric, a type of dlt used in our study, as transaction proposals (check the purple background in fig. 2) . then, each of the peers signs the transaction using the elliptic curve digital signature algorithm (ecdsa) and adds the signature before returning the signed message back to the iot devices (ues). the peers that provide an endorsement of the ledger send an update to the application, but do not immediately apply the proposed update to their copy of the ledger. instead, a response is sent back to the iot device to confirm that the transaction proposal is correct, has not been previously submitted to the ledger, and has a valid signature. therefore, the security increases with the number of endorsing peers. in addition, smart contracts can be executed to update or query the ledger. then the iot device broadcast the confirmed transaction proposals along with the confirmation to the ordering service. the received transactions are ordered chronologically to create blocks. these transaction blocks are delivered to all peers for validation. then, the peers append the block to the ledger, and the valid transactions are committed to the current state database. finally, a confirmation message is transmitted back to the iot devices to report that the submitted transaction has been immutably published to the ledger. the important of confirmation is analyzed in [50] . 2) results: we evaluate the performance of an integrated dlt and iot system with permissioned hyperledger fabric and nb-iot. our experimental setup is based on hyperledger fabric v1.4, nb-iot development kits sara evk n211 and one nb-iot amarisoft enb station. we compare the e2e latency of conventional nb-iot with dlt-based nb-iot in various scenarios. the number of endorsing peers is varied from 1 to 4 peers. the iot device transmits packet every minute, and the total number of messages transmitted is around 1000. the figure 3 shows that the end-to-end (e2e) latency is computed based on the total uplink and downlink transmission latency and validation latency in the ledger. we observe that when adding an new endorsing node to the same channel, this will increase the endorsing latency. in comparison with conventional nb-iot system, the dlt-based nb-iot perform a higher latency due to the validation process from ledger to verify transaction as well as building block. this is a trade-off between dlt-based system and conventional iot system. dlt-based iot system guarantees the trust; however, the latency is slightly degraded. a federated global epidemiological warning system was proposed. based on it, an specialized use case focused on remote patient monitoring was considered. as one critical piece of this use case is the integration of dlts and iot devices, we implemented an experimental integration setup between dlts and nb-iot. in this study, we conclude that the latency increased when the number of endorsing peers is increased. results indicate that in average the latency is increased 0.25 seconds per endorsed peer that is added. however, this increases the guarantees in terms of secrecy, privacy, and interoperability. this experimental setup also reveals additional technology challenges, such as the performance of dlt-based system with massive number of connections, as well as the trade-off between privacy, interoperability and system performance. although the proposed architecture is designed to be fair among all citizens, few important practical points still deserve attention. good quality data usually follow the wealth distribution over all scales of the proposed federation. in other words, good quality data is very likely to be less available in poorer regions of a city. the same is valid for poor regions within a country and poor countries. this existing gap needs to be considered when building incentives for participation, otherwise the proposed solution has the potential of reinforcing inequality [51] . another current issue we cannot neglect is the public perception of wireless technologies. in this sense, part of the general public perceives iot as insecure [52] and fifth generation (5g) as a health hazard [53] to the point of claiming an astonishing causal relation between 5g and covid-19, which has caused destruction of base stations across uk. beyond these conspiracy theories that are hard to combat, there exist legitimate concerns of anonymized data not being anonymous [54] , and of novel surveillance techniques introduced in times of crisis that are maintained for monitoring (legally or illegally) populations, e.g. post-9/11 surveillance in the usa [55] . all these have to be carefully addressed from the early stages of the system development. furthermore, it has to be clear that neither technology nor data can prevent another outbreak on their own, but can only provide the extremely valuable tools to enable the holy grail of controlling epidemics: early detection, early response along all relevant actors within the federated organization. in other words, technology and data detect and identify potential harms and suggest actions and reactions, but the final diagnostic and further interventions are due to the responsible institutions within the federated structure and citizens' active participation. our proposal answers those challenges based on informational self-determination as the way to build trustworthy and secure public infrastructure that shall enable graceful lockdowns as advocated here. in this sense, the proposed solution introduces a more balanced management strategy, moving away from purely top-down approaches toward a participatory system where the citizens are active. we further expect that, even without being implemented, the high-level architecture introduced here could offer important technological suggestions to decisionmakers of how to start smoothly resuming activities after lockdowns. second wave covid-19 pandemics in europe: a temporal playbook digital technology and covid-19 on the responsible use of digital data to tackle the covid-19 pandemic special report: the simulations driving the world's response to covid-19 can china's covid-19 strategy work elsewhere optimization of privacy-utility trade-offs under informational self-determination will democracy survive big data and artificial intelligence? pandemics meet democracy. experimental evidence from the covid-19 crisis in spain the ethics of ai ethics: an evaluation of guidelines the global public health intelligence network and early warning outbreak detection gphin, goarn, gone" the role of the world health organization in global disease surveillance and response revitalizing the global public health intelligence network (gphin) intelligence and global health: assessing the role of open source and social media intelligence analysis in infectious disease outbreaks the effect of travel restrictions on the spread of the 2019 novel coronavirus presymptomatic transmission of sars-cov-2-singapore pan-european privacy-preserving proximity tracing contact tracing mobile apps for covid-19: privacy considerations and related trade-offs privacy in iot blockchains: with big data comes big responsibility make users own their data: a decentralized personal data store prototype based on ethereum and ipfs blockchain as a notarization service for data sharing with personal data store blockchain-based personal data management: from fiction to solution the future of privacy lies in forgetting the past privacypreserving mechanisms for crowdsensing: survey and research challenges characterizing the propagation of situational information in social media during covid-19 epidemic: a case study on weibo how the world's collective attention is being paid to a pandemic: covid-19 related 1-gram time series for 24 languages on twitter event detection system based on user behavior changes in online social networks: case of the covid-19 pandemic twitter user geolocation using deep multiview learning mobile phone data and covid-19: missing an opportunity?" arxiv e-prints quantifying sars-cov-2 transmission suggests epidemic control with digital contact tracing inferring person-to-person proximity using wifi signals practical secure aggregation for privacy-preserving machine learning engineering democratization in internet of things data analytics mining social interactions in privacy-preserving temporal networks ieee/acm international conference on advances in social networks analysis and mining (asonam) decentralized privacy preserving services for online social networks decentralized collective learning for self-managed sharing economies federated machine learning: concept and applications federated multi-task learning privacy preserving semi-supervised learning for labeled graphs semantic interoperability as key to iot platform federation fundamentals of clinical data science preparing data at the source to foster interoperability across rare disease resources improving semantic interoperability of big data for epidemiological surveillance are high-performing health systems resilient against the covid-19 epidemic? a pandemic influenza modeling and visualization tool participatory disease surveillance systems: ethical framework participatory surveillance based on crowdsourcing during the rio 2016 olympic games using the guardians of health platform: descriptive study applications of crowdsourcing in health: an overview hierarchical self-awareness and authority for scalable self-integrating systems trusted wireless monitoring based on distributed ledgers over nb-iot connectivity communication aspects of the integration of wireless iot devices with distributed ledger technology potential biases in machine learning algorithms using electronic health record data a longitudinal analysis of the public perception of the opportunities and challenges of the internet of things the 5g debate in new zealand-government actions and public perception anonymization and risk making sense from snowden: what's significant in the nsa surveillance revelations key: cord-287765-nsdequl9 authors: taiwo, olutosin; ezugwu, absalom e. title: smart healthcare support for remote patient monitoring during covid-19 quarantine date: 2020-09-15 journal: inform med unlocked doi: 10.1016/j.imu.2020.100428 sha: doc_id: 287765 cord_uid: nsdequl9 social distancing and quarantining are now standard practices which are implemented worldwide since the outbreak of the novel coronavirus (covid-19) disease pandemic in 2019. due to the full acceptance of the above control practices, frequent hospital contact visits are being discouraged. however, there are people whose physiological vital needs still require routine monitoring for improved healthy living. interestingly, with the recent technological advancements in the areas of internet of things (iot) technology, smart home automation, and healthcare systems, contact-based hospital visits are now regarded as non-obligatory. to this end, a remote smart home healthcare support system (shhes) is proposed for monitoring patients’ health status and receiving doctors’ prescriptions while staying at home. besides this, doctors can also carry out the diagnosis of ailments using the data collected remotely from the patient. an android based mobile application that interfaces with a web-based application is implemented for efficient patients-doctors dual real-time communication. sensors are incorporated in the system for automatic capturing of physiological health parameters of patients. also, a hyperspace analogue to context (hac) was incorporated into the current monitoring framework for service discovery and context change in the home environment towards accurate readings of the physiological parameters and improved system performance. with the proposed system, patients can be remotely monitored from their homes, and can also live a more comfortable life through the use of some features of smart home automation devices on their phones. therefore, one main significant contribution of this study is that patients in self-isolation or self-quarantine can use the new platform to send daily health symptoms and challenges to doctors via their mobile phones. thus, improved healthy living and a comfortable lifestyle can still be achieved even during such a problematic period of the 2019 covid-19 pandemic that has already recorded 20,026,186 million cases so far with 734,020 thousand deaths globally. independent and convenient, healthy living is the aim of any human being no matter their age, gender, location or health status. however, there are limitations due to age, illness, medication, hospitalization, epidemic, pandemic and other circumstances. health monitoring systems have evolved to assist convenient healthy living, more accessible communication between healthcare givers and patients for close monitoring, measurement of vital health parameters, routine consultation and overall healthy living. moreover, with the recent advances in information and communication technologies (ict) through the adoption of internet of things (iot) technology, smart health monitoring and support systems now have a higher edge of development and acceptability for enhanced healthy living. the study conducted by zikali [1] , revealed that with the rapid increase in the population of older or senior citizens, patients who require health monitoring have also increased exponentially. the same study predicts that by the year 2045 the number of senior citizens who are considered the most vulnerable in society will exceed the number of children and young adults as a recent population census shows an increase in older people. however, a shortage of home health helpers, nursing assistants and home healthcare givers is looming worldwide, which makes care for the elderly expensive. therefore, a health monitoring system can play a vital role in lessening physical contact, hospitalization, consultation time, queuing list and overall health cost for a patient while also reducing workload, burden and stress on medical staff [2] . advancements in information and communication technologies for connectivity anywhere and anytime make a valuable contribution to the development of the modern healthcare system utilized in telemedicine solutions and other portable medical platforms. the advent of smart home technologies proposes healthy living and improved quality of healthcare support services for the elderly and handicapped for independent and comfortable lifestyles while at home, instead of nursing homes, hospitals or other confinement facilities. the healthcare module, as a part of the smart home automation system, will improve healthcare facilities for patients while at home or in remote locations outside the hospitals. thus, there is a reduction in depression that arises from loneliness in the hospital wards for patients. the doctors can monitor patients from their office, prescribe medication and view measured vital health parameters for a remote diagnosis. also, the rapid improvement of software and hardware technologies in the smart home healthcare system, makes it possible for patients, especially the elderly or disabled, to control certain home appliances with ease from devices such as smartphones, tablets, laptops, internet, etc. a smart home healthcare environment comprises of numerous computing devices acting proactively on behalf of users, thereby making such an environment pervasive. therefore, for decision making in a smart home healthcare environment, user context and preferences are some of the vital features to be considered for the user to make any choice of interest among available resources and services in any given situation [3] . also, the context-aware paradigm gives an insight into some factors within the home that influence the output of physiological readings. these factors are explained in this paper. the user context refers to any information that could be used to describe the situation of an entity (person) concerning their physiological medical status or needs. in this paper, we propose a mobile application-based prototype smart home healthcare system for efficient and effective health monitoring for the elderly and disabled for their convenient and independent living while at home. a section of the proposed system allows the patient to remotely upload or capture essential health symptoms information during an era of a pandemic such as the ongoing covid-19 disease for their doctor's assisted diagnosis. the new healthcare support system measures and records specific health parameters such as weight, pulse, blood pressure, glucose level and body temperature. also, it assists with the control of a few home appliances for the conducive home living of the user. this system is also designed to send a reminder to patients on the use of certain medications with input supplied by the user. an algorithm was developed based on hyperspace analogue context (hac) for a pervasive environment, to address the need for choice for the user among numerous devices placed in the smart home healthcare system. the technical contributions of this paper are as follows: • a proposal for a smart health support system for remote patients monitoring during quarantines. • implementation of iot based smart home healthcare support framework capable of reducing unnecessary burdens on the hospitals due to disease outbreak. mainly, the new system which is also able to provide essential comforts using the iots enabled home appliances only, encourages patients with severe and critical conditions to utilize hospital facilities. • extraction and interpretation of patient's health data from wearable, built-in, and mobile sensors for medical diagnoses and prescriptions. the remainder of this paper is organized as follows: section 2 provides the review of related works and overview of health monitoring support systems, section 3 discusses the proposed system motivation, conceptualization, and design methodology, while in section 4, the details of the new smart healthcare support system implementation are discussed. finally, section 5 provides concluding remarks, recommendations, and future directions. advances in the development of smart home automation technologies and e-healthcare systems allow people to enjoy in-home medical services without staying in hospitals. remote health monitoring of patients through home health care technologies assists healthcare givers, medical personnel and physicians to reach out to patients without physical contact or presence of patients at clinics or hospitals. health care technologies also save cost, expenditure and stress for patients as they do not need to travel before seeing their healthcare givers or medical personnel. there have been many published articles in the area of smart health care system, e-health and remote healthcare. authors in [4] proposed an e-health care system for monitoring patients' vital physiological parameters by doctors from any location. the proposed system is capable of collecting the required data from the patient and making it available and visible to the doctor for action. the web application is a feature of the system that allows the doctor to record the patient's information, input notes for advice, prescription and dosage of drugs while also allowing the patient to key in measured psychological parameter values and display of information received from the doctor. smart tv application was used for reminding patients daily about their activities, medications and other events. lastly, the system has the feature of a mobile application with the same functionality as the web application but with an added advantage of access from anywhere and at any time. sparsh and agarwal [5] described a remote health monitoring system for the collection of blood pressure values from patients through mobile phones. values recorded on mobile phones are supplied and displayed to doctors or caregivers through the web interface in the system. doctors can monitor and manage the patient's condition through the system and provide feedback to the patient remotely. a secure iot based modern healthcare system using body sensor network (bsn) referred to as bsn-care was proposed by authors in [6] . the proposed system measures and monitors physiological parameters such as blood pressure, electrocardiogram (ecg), and electroencephalography conditions in the body through wearable sensors. the values of measured parameters are collected and sent to the local processing unit, which is also the coordinator of the system. data received by the bsn-care server from the body of the patient are fed into the database for analysis. based on the analysis and degree of abnormality in the values, the system alerts either the family member, local physician or emergency unit contact of the patient. the system is secured using a lightweight anonymous authentication protocol, which confirms the identity of anyone using the bsn-care server. for privacy, data integrity and data freshness, the offset codebook (ocb) authentication encryption scheme was used. minh pham et al. [7] presented a cloud-based smart home environment named coshe for a home healthcare wearable unit, a private cloud and robot assistant. the coshe system collects physiological, motion and audio signals from residents thorough non-invasive wearable sensors and thus provides information about the daily activities and location of residents in the home. comprehensive health data are provided to caregivers and caretakers through a web application built on the cloud server of the system. the system also has a hydration monitoring application for continuous monitoring of water consumption levels and daily fluid requirements of the patient. hydration monitoring is achieved by the use of acoustic data collected from microphones and body activity context derived from a smartwatch accelerometer in the system. a smart home integrated system that runs on an android operating system, for ambient assisted living for people living with dementia, was presented by eren demir [8] . the design of the system allows the collection, recording and transmission of data through cloud application. the system involves seven types of sensors to detect a person's position, whether standing or sitting, flame detection and use of specified appliances in the home. also, the sensors remind or alert the patient if he or she forgets to carry out specific tasks in time. a switch is also installed in the system to detect if the light is on or off. the system is also designed to identify the activities of patients and send information to the doctor or caregiver. data are retrieved from different sensors placed in specific locations in the home for processing. bilal and khaled [9] proposed a wireless remote-control home automation system for the elderly, handicapped, decrepit and disabled persons. the system was designed for easy movement, control and monitoring of essential home appliances for people with locomotion difficulty. users of the system can control or monitor appliances through a remote control device that sends orders wirelessly using xbee trans-receivers. features of the remote control are control buttons for different appliances and lcd for message notifications. the proposed system was implemented using a wooden prototype version. min chen et al. [10] proposed an e-healthcare management system based on a secondgeneration rfid system. the system for monitoring the medical condition of the patient, communication between the patient and the doctor or health caregiver as the case may be is through video conferencing calls via the internet. the system is also capable of information collection and transformation for medical emergencies handling. physiological signals such as temperature, blood rate and heart rate are obtained from the patient through a body sensor attached to a particular part of the patient's body. the system also maintains a healthcare database of the user's profile and medical history. andrea et al. [11] proposed a smart sensing technique based on an integrated sensor network for monitoring the user's home and environment in order to derive information about the user's health status and behaviour. the authors' proposed platform includes sensors that are biomedical, wearable and unobtrusive for monitoring physiological parameters such as ecg, heart rate, breathing waveform, breathing rate, blood pressure and so on. an application on a smart device such as a tablet was proposed for the user's interaction with the sensors. data collected through the application are further sent to the cloud for storage and data analytics towards services for the elderly. moreover, abdelsalam et al. [12] designed a smart home-based software architecture for assisted monitoring of diabetic patients. the system is intended to monitor activity, diet and exercise compliance of patients and evaluates the effect of alternative medicine and regimen through personal connected devices and smart home technologies. wearable devices are used as connectivity to technology in the architecture. an android smartphone application for elderly, assisted independent living at home was developed by muhammad et al. [13] . the application tracks and monitors the daily activities of the user and serves as a reminder for the scheduled activities of the patient. the system also gives alerts in case of incomplete, critical and overlooked scheduled activities. other functions and actions performed by the system include tracking of environmental conditions (humidity, temperature, location and gas leakage), giving details of the patient's activity list and reminders to family members and healthcare givers. hossain [14] proposed a smart home healthcare system to fulfil the needs of older adults for continued care. the patient's health condition is monitored through the use of multimodal inputs (speech and video) in the proposed system. videos and speeches of the patient are captured continuously through sensors from video cameras, and microphones installed in the home, transmitted to a dedicated cloud for processing, and classification scores are produced. doctors prescribe and render services through audio or video messages depending on the outcome of the classification score, whether normal, tensed or in pain. saiteja et al. [15] proposed a smart home health monitoring system for remote monitoring of diabetes and blood pressure in patients. the system assists in analysing the patient's blood pressure and glucose readings from home, sending a notification to the caregiver or healthcare provider if an abnormality is detected and also predicting the status of hypertension and diabetes in patients by training results obtained from the readings. for the model training, support vector machine classification was employed to provide effective and efficient training task. also, the system is capable of sending alerts and real-time notifications from home about the patient's health to a registered physician or clinic. kashif et al. [16] developed a smart home system based on information and communication technologies (ict) for the elderly, using an android platform. the system was developed to improve the quality of life for elderly persons, prevent electricity wastage and preserve human energy simultaneously through remote access. the system also controls environmental parameters according to the health status and living needs of the elderly and triggers alerts in the case of intrusion and home invasion. the provision of services through assisted means enables a comfortable and convenient lifestyle. the primary role of any smart home automation system is to assist users in remotely controlling and monitoring appliances. with this in mind, we are motivated to develop a system that not only controls and monitors the home but also supports an improved healthy lifestyle of users. smart home automation as an emerging area of iot has been applied in various areas such as: easy and assisted daily living especially for the provision of support to humans [17] , remote control of home appliances [18, 19] , detection of movement in the house [20] , energy management in the home [21] and security [22] , and provision of healthcare services to out-patients, disabled and elderly persons [10, 23, 24] . however, the design of a system for both health monitoring and home control is yet to be fully explored. considering this, we look at a scenario whereby john has just been discharged from the hospital but still needs his physiological vitals monitored closely by his doctor, and he is advised to bed-rest at home. for john to enjoy being at home, he needs some convenience, such as putting on the television while lying on the bed, and controlling a fan or light while still in bed. we decided to aid people in john's condition by proposing a smart homehealthcare automation system. the proposed system monitors and records physiological parameters in a module, sends recorded parameters to the doctor and controls the home as well. the second motivation comes from the pandemic currently ravaging the globe. social distancing, less physical contact and staying at home orders are issued by the government to control the spread of the virus. people who have been in contact with positively tested individuals, but who are not showing symptoms, are also counselled to self-isolate or selfquarantine for some days. positive patients with mild symptoms are advised to observe quarantine. the self-isolation or quarantine can be observed from home while the affected person sends signs or symptoms of any ailment observed to the doctor at regular intervals. to this end, we are motivated to broaden the scope of the smart home healthcare system to accommodate the upload of symptoms affected by covid-19 from the comfort of their respective homes. there may be some instances whereby the user might need to change his position in the house, increase or decrease the home temperature, and other environmental factors that might affect the physiological parameters recorded. to address the external factors that influence the values of the physiological readings, we build on the discovery approach used by authors in [25] . the approach is based on hyperspace analogue to context (hac) in a pervasive environment. the hac concept implemented for the current study is discussed below. in smart home-healthcare, hac is a formal model to define the multi-dimensional context in space [25] healthcare in a smart home comprises different elements such as the user, location of the user, his activities, environmental conditions and other related parameters. to adequately understand the correlation between the user and his activities, context is required. according to [26] , context is any information that can be used to describe the situation of an entity. dimensions for hac are formed based on specific context types. due to the complexity of a pervasive environment, the number of dimensions may be large [3] . for our work, the significant fractions of applicable dimensions are used, as indicated in most context descriptions. the major context used to describe the situation of the user are location, time, physiological condition, activity and atmospheric and environmental conditions. to get an accurate value of a specific physiological parameter such as blood pressure, the position of the patient is of significant consideration. for instance, it is not recommended to take the readings of blood pressure while standing. position influences the values obtained either on the systolic or diastolic value. therefore, the recommended position is sitting straight up or lying down comfortably [27] . location can also be added to the position to effectively record the range of the value. time influences the results and conclusions of doctors for prescription of medications. time describes activity happening and surrounding. some medications or prescriptions are recommended to be taken with breakfast and others at night. if a patient misses the right time for taking a specific dosage, it may trigger some health conditions which the doctor will need to consider before concluding the patient's health condition. weather and indoor environmental conditions, for example, absolute humidity and temperature influence physiological conditions of the patient in so many ways. a patient might have a high fever yet his body temperature has a reading that falls within the normal range during the winter season. at the same time, a patient without a high fever might have a high trigger in his body temperature when readings are taken in a hot environment or a closed room with a heater on. therefore, atmospheric conditions must be taken into consideration for a better summary of the health of a patient. subsequently, we now present some significant definition of terminologies used in this study concerning service discovery and context change relative to the hac. definition 1 (n-dimensional hac): a n-dimensional hac is a space =< , ,⋅⋅⋅ , > where represents each dimension as a type of context. dimension in hac refers to an informative label used to describe other data in the space alongside their type and value set for a specific context. examples are time, physiological condition, location and position. the values of a dimension can be continuous or discrete, depending on the data type [25] . this work will take into consideration blood pressure with a normal value range between 110/70mhg and 140/90mhg, and normal sugar level values between 70 and 100mg/dl. > where ∈ . the context of a device or appliance in the home is described as a point in hac. in our work, the context of john may be< = , = !" >, where #$ denotes blood pressure; when the bp value is not within an expected range, it can be put as < = %&" , = "' !" >. a context scope ( is a subspace in , ( =< ) , ) , ⋯ ) >, where ) ⊆ . a context scope limits the value sets for the dimensions. it is mostly used to describe a condition. for example, < ) ℬ,-= .110, 111, ⋯ ,1402, ) ℬ,) = .70,71, ⋯ ,902 >, where ) ℬ,-describes the systolic value for normal blood pressure and ) ℬ,) describes the normal diastolic value of blood pressure readings. blood pressure has both upper and lower bound values measured in units of millimetres of mercury (mmhg). the readings are always given in pairs with the upper bound (systolic) value first, followed by the lower bound (diastolic) value. movement of a patient must be taken into consideration in addition to monitoring and measurement of physiological parameters. important information about a patient's health condition can also be derived by monitoring the walking pattern [28] . to denote the movement operation of a patient in the home concerning health influencing factors, context change is required. the context change is an operation to change a context point. it is defined as × ∆7 =< ∆ , ∆ , ⋯ , >, ∆ denotes the new value for a dimension, 8 = × ∆ . if does not change, ∆ = ∅. the above definitions are applied to our shhes environment for detecting the factors that can influence the output of measured physiological parameters. for instance, if the indoor temperature of the home is high, it might affect the body temperature of the patient. likewise, if a patient is in a standing position while blood pressure is being measured, the corresponding value of the measured blood pressure might be high either on the systolic or diastolic readings. to further apply the hac contexts in our work, an algorithm (algorithm 1) was developed on steps to consider these factors in order to get actual readings and for diagnosis by the doctor. with the advent of the iot, remote health monitoring, consultations and prescription have been made a reality while patients are at home. iot technologies have made medical equipment smart; examples are sensors, actuators, microcontrollers and boards that have made it easier for doctors to have the patient's data and oversee their health without a visit to the hospital. on the patients' side, it has greatly assisted in stress reduction as patients do not need to waste time in queues in hospitals, and again, they can send and receive information from their doctors through iot enabled systems for health monitoring. furthermore, the physiological parameters of patients can be measured and transmitted to the database for a doctor's perusal towards clinical diagnosis and advice on treatment. in this covid-19 era experienced globally, infected patients and people in contact with the infected patients are counselled to self-isolate or quarantine depending on the level of infection. we propose a smart home health monitoring system referred to as shhes in this paper. the proposed system described in our work is intended to perform a dual function of controlling home appliances as well as monitoring and recording the patient's physiological data such as blood pressure, body temperature, pulse rate, body weight and sugar level and other symptoms related to a specific virus. the values of measured physiological parameters are keyed into the system wirelessly through sensors or manually by the caregiver for transmission to the doctor. the main aim of the system is to assist out-patients and patients in prescribed isolation to live a comfortable life at home and reduce the stress of visiting hospitals often before consultation with their doctors. disabled and older adults are also factored into consideration of our system as the system is designed for comfortable daily living in the home. in our proposed system, the home is remotely controlled through a developed mobile application installed on a smartphone, and the user can also communicate with his doctor through another module in the same application. medical conditions, such as chronic hypertension and diabetes, were used as the focus of our design. the system takes into consideration the role of family member or caregiver who will be responsible for helping the patient while at home. hence, the proposed system has three major users: patient, doctor and home caregiver. in the proposed system, patients can upload values of physiological health parameters, chat with doctors, leave a message and book an appointment. figure 1 shows the architecture of the proposed system design. the architecture includes the home appliances, smartphone, sensors, actuators, wi-fi module, and an arduino board, which serves as the microcontroller. in the system, sensors are installed for measurement of the indoor temperature or humidity levels, and detection of motion and smoke. in our work, communication between the user and the home is wireless. the arduino microcontroller collects data from various sensors, home appliances and physiological devices through the esp8266 wireless module and transmits it over the internet to the user. the user receives and sends commands to the home through the developed android mobile application to the microcontroller for necessary actions. the selected controlled area for the work is a room in the house. features to be controlled by the application are the temperature, cooling system and other basic home appliances like the light switch and television. for temperature control of the home, the desired temperature is entered by the user through the developed mobile application and saved in the memory unit of the arduino microcontroller. the temperature is measured using the dht11 humidity and temperature sensor. the arduino continuously reads the temperature and compares it with the value inserted by the user; if the value measured by the system is lower than the desired value given as input by the user, a notification is sent to the user, and the system automatically puts on the heater to make the area warm. if the measured value is higher than the user's desired value, the fan is automatically turned on, and a notification is also sent to the user via the mobile application. for control of other appliances in the home, the user selects the desired appliance he wants to control on the mobile application and a signal is sent to the arduino microcontroller for necessary action on the appliance. the communication between the user, the devices and appliances is wireless using the esp8266 wi-fi module. the user's physiological parameters are measured manually using conventional equipment such as a sphygmomanometer for blood pressure, a thermometer for body temperature and a glucometer for sugar level, with the assistance of the healthcare or family member caregiver at home. the values of the measured parameters and symptoms of the virus are sent to the doctor via the internet. the values are stored in the created database for the developed system and the doctor views the parameters and other information from the patient such as message, chat and complaints through the developed web application. on the other hand, the physiological body parameters can be obtained through the use of body sensors and the values are received wirelessly by the microcontroller. if the measured parameters received are not within the normal specified range as discussed earlier, our algorithm 1 is used to perform basic checks using the hac context types. the microcontroller sends the received values to the database and the values are then transmitted over the internet to the doctor for further diagnosis. the doctor communicates with the patient via the mobile application by placing a call, leaving a message or using the chat box option on the platform. several devices are involved in iot systems, thus the need for a gateway that serves as a means of integration between the devices in the system and communication modules. home appliances are connected to the arduino board for control through relay pins on the board. also, data generated from the smart sensors used for measuring physiological parameters of the patient are sent to the database and from there transmitted over the internet to the doctor for action. the doctor also communicates with the patient by sending information through the internet. steps involved in the operations of the smart home healthcare system are stated in algorithm 1. the algorithm works based on the hac definitions to perform necessary actions for accurate readings. if the readings do not fall within the expected range, context change, position and other factors in the hac are put into consideration for subsequent readings. the following terminologies are used: • ha stands for home appliances. • : ; stands for network connectivity • hc stands for home control • pd stands for physiological devices • pd1 stands for blood pressure devices with the range as < ) ℬ,-= .110, 111, ⋯ 1402, ) ℬ,) = .70,71, ⋯ 902 >. • pd2 stands for sugar level device with the range between 70 to 100mg/dl • pd3 stands for temperature device with the range between 36.1℃ and 37.2℃ as presented in the architecture, the proposed smart home healthcare system has both user's and doctor's sides. at the user side, two main parties are involved: registered user and the family member responsible for overseeing and taking care of the patient at home. the user (patient) can perform the following basic functions on the system after successful registration: • control home devices • check for medication and prescription • send symptoms of the virus to the doctor while under isolation • chat with the doctor or leave a message • enter physiological values of measured parameters manually • book appointment with the doctor • view medical data. the role of the family member in the system is to assist the patient with uploading and retrieval of information when necessary, communication with the doctor on behalf of the patient, and also use the application for home control. the doctor, after successful registration and login, can communicate with the patient either by chats or message, view messages from patients, prescribe medications, diagnose any ailment from the received physiological vitals and provide other medical consultations. hardware components of the system include the following: sensors: body temperature sensor, blood pressure sensor, pulse rate sensor, etc. are used in the system for collection of data from the patient's body and transmitted wirelessly to the microcontroller via the home network gateway for storage in the database and forwarding to the doctor over the internet. the body sensors are used to ease the stress of manual input. arduino board: microcontroller oversees the functionality of the system. iot appliances and devices are connected to the arduino board for collection and transmission of data within the system and also to the mobile application for home control. with the digital pins present in boards, home appliances settings are relayed and hence controlled. the functionality of home appliances is highly dependent on boards. gateway: several iot devices are involved in this system. to this end, we decided to use a gateway to unify the communication of devices. the gateway serves as an intermediary between the home appliances, the microcontroller and the internet. the major role of the gateway is the unification of network connection from various devices. the proposed system is to be used through the mobile application and the web-based application. patients connect with home appliance and doctor through the mobile application to carry out the functions previously highlighted. the patient's physiological parameters are sent to the doctor automatically with the aid of body sensors deployed for capturing the values or are sent via manual input of values on the mobile application platform. with the help of service discovery explained through hac earlier, the performance of services in the home are displayed, changes can be discovered and results reflected in the system. the android studio integrated development environment was used for designing and developing the android mobile application for the control of home appliances, health devices and communication with the doctor from home. figure 2 shows the home page of the designed mobile application. the user can gain access to the mobile application by registering on the application; after successful registration, access can be gained to login ( figure 3 ) and then directed to the desired page either as patient or family member depending on the information submitted. the user registers home appliances to be controlled, and there is an option to add more appliances for future purposes. after the registration of devices, home appliances that can be controlled are displayed (figure 4) . the application is designed to allow manual input data in case the sensors irritate the patient's body, and automatic measurement of physiological conditions is not possible. for automatic capturing of health data, the user will click on the parameter to send while on the health parameter page. the health interface of the mobile application allows the user to perform functions like chatting, leave a message or check the prescription of previous medication from the doctor ( figure 5) . also, the mobile application can be used to communicate with the doctor concerning covid-19 diagnoses, especially when the patient is observing self-isolation at home or government obligatory quarantine. specific symptoms are monitored and checked at intervals and sent to the doctor with the range of the readings (figure 6) . a response is received after the patient has successfully keyed-in the values and responses for the covid-19 symptoms. the application is designed to analyse the input received and give a response for the next action to be taken by the patient. colour code red means the patient should report to the nearest hospital or covid-19 treatment facility with immediate effect, an orange colour indicates that the patient is exhibiting above 80% of covid-19 symptoms and should be screened and further tested as soon as possible, a yellow colour indicates that the patient is exhibiting very mild symptoms of the virus and hence should continue to self-isolate and avoid contact with people while the green colour indicates that the patient is not showing any symptoms at all. based on the input received in figure 6 , figure 7 shows a mild indication and the necessary action to be taken by the patient. the doctor will further advise and prescribe necessary medications based on the parameters received at the doctor's end. from the doctor's side, a web-based application was designed which can be managed by an administrator. users and family members are to register with the doctor to have their data in the database similar to the hospital filing system. the doctor and administrator are the major users of the web application. it is designed for monitoring patients' health, delivery of services such as consultations, prescriptions, evaluation of physiological values for diagnosis, treatment or further directive on medical tests required. the system is designed for the doctor to manage as many patients that are registered on the platform. for the creation of a portal for the user, user supply information such as id number, sex, blood group, genotype and other vital details are recorded on the system. the interfaces for the web application are shown in the figures below. figure 8 shows the interface of the web application giving an overview of what the application is all about. after successful registration, the login page of the doctor is displayed in figure 9 , while figure 10 shows the doctor's page in the application. a family member taking care of the patient from home also needs to register with the doctor and have their records in the database since he or she has the assigned role of communicating with the doctor on the patient's behalf ( figure 11 ). in summary, the main goal of the developed hybrid smart healthcare based android mobile web application framework discussed in this paper serves the vast purpose of enabling remote monitoring of patients concerning those diseases that might require constant visits to the hospitals even though such diseases can be diagnosed and treated remotely by doctors. the proposed mobile application similarly offers features that can monitor the patient's condition in real-time, which can help reduce hospital costs and most importantly protect the hospital against any further exposure of patients to highly contagious diseases, especially during a pandemic like the covid-19 where most hospitals are grappling with high numbers of patients. the application of internet of things in smart home automation has led to a great deal of improvement in convenient living, remote access to home appliances, mobile health care and improved social lifestyle primarily for senior citizens. combining home automation with the healthcare system helps alleviate stress, reduces the cost of living and gives room for remote communication between doctors and patients. in this work, we have proposed a smart home health care system for the sick, elderly and handicapped. the current work was focused mainly on making life more convenient for those with health challenges who need to visit the hospital regularly. the new system has been developed in order to reduce the number of hospital visits, queues in the hospital and reduction in the cost of taking care of the sick. the system performs a dual role of both health monitoring and control of essential home appliances; with this, users can enjoy social life and still have their health managed and monitored especially during an era of the pandemic. the proposed method will have a great impact on the quality of life by reducing the transmission rate of communicable diseases. patients diagnosed and under treatment for a disease such as covid-19 will not have any cause to move about frequently and thus, quality of life is ensured and transmission rate is reduced. the on-going phase of the current system is its physical deployment with iot devices, a testing phase of the mobile application using the real-world scenario and documentation of feedbacks for improvement. it is recommended that after rigorous testing and evaluation, the proposed system can be deployed in hospitals for use in various units. the designed mobile and web application, once fully developed, can be plugged into existing web domains of hospitals as a portal and can be launched as a fresh application for hospitals without existing domains. it is also recommended that new features such as a physiological data capturing device be incorporated into the current system. as a future direction, we plan to extend our application beyond android platform to other ios platforms for wide adaptability. with the efficient technique presented in this paper, it is believed that this research can be extended to other areas of iot such as agriculture for monitoring of livestock and consultation of farmers with veterinary doctors towards diagnosis, prescription and treatment of diseases in livestock in farms. also, the new system can be extended for use in the pharmaceutical sector. the doctors can send prescriptions to the pharmacist for recommendation of dosage and possible dispensing of medications to patients. finally, it will be of interest to carry out an evaluation of the overall performance of the proposed system using different mathematical and statistical evaluation tools. no suitable care for sa's elderly smart health monitoring systems: an overview of design and modeling context-driven personalized service discovery in pervasive environments e-health monitoring system remote health monitoring using mobile phones and web services bsn-care: a secure iot-based modern healthcare system using body sensor network delivering home healthcare thorugh a cloud-based smart home envrionment (coshe) smart home assistant for ambient assited living of elderly people with dementia smart home automation system for elderly and handicapped people using xbee a 2g-rfid-based e-healthcare system a smart sensing architecture for domestic monitoring: methodological approach and experimental validation smart home-based health platform for behavioral monitoring and alteration of diabetes patients daily life activity tracking application for smart homes using android smartphone patient status monitoring for smart home healthcare smart home health monitoring system for predicting type 2 diabetes and hypertension smart home for elderly living using wireless sensor networks and an android application robot-enabled support of daily activities in smart home environments smart gsm based home automation system design & implementation of smart house control using labview iot based smart home automation system using sensor node real-time energy control approach for smart home energy management system design and implementation of an iot-based smart home security system a home mobile healthcare system for wheelchair users an iot-aware architecture for smart healthcare systems context-driven personalized service discovery in pervasive environments understanding and using context tips to measure your blood pressure correctly gait disorders in adults and the elderly: a clinical the authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper. key: cord-277918-a2iisve0 authors: malik, asif iqbal; sarkar, biswajit title: disruption management in a constrained multi-product imperfect production system date: 2020-06-26 journal: j manuf syst doi: 10.1016/j.jmsy.2020.05.015 sha: doc_id: 277918 cord_uid: a2iisve0 over several decades, production and inventory systems have been widely studied in different aspects, but only a few studies have considered the production disruption problem. in production systems, the production may be disrupted by priorly unknown disturbance and the entire manufacturing plan can be distorted. this research introduces a production-disruption model for a multi-product single-stage production-inventory system. first, a mathematical model for the multi-item production-inventory system is developed to maximize the total profit for a single-disruption recovery-time window. the main objective of the proposed model is to obtain the optimal manufacturing batch size for multi-item in the recovery time window so that the total profit is maximized. to maintain the matter of multi-product, budget and space constraints are used. a genetic algorithm and pattern search techniques are employed to solve this model and all randomly generated test results are compared. some numerical examples and sensitivity analysis are given to explain the effectiveness and advantages of the proposed model. this proposed model offers a recovery plan for managers and decision-makers to make accurate and effective decisions in real time during the production disruption problems. a supply chain comprises of facilities and entities that are involved in transforming raw materials into completed products and later delivered to end customers through the supplier. mostly, conventional supply chains are designed for problem-free and smoothly-operating environments. however, in reality, some unexpected events may occur during the production process such as labor strikes, natural disasters, machine breakdowns, raw materials shortage, and transportation problems. these events are almost unavoidable and cause disruptions at different stages of a supply chain system. in real-life supply chain environments, production disruption is one of very familiar disruptions/ interruptions. in literature, production disruption is defined as any form of disturbance during a production process, including power cut, tool failure, machine breakdown, material shortage, or any type of manmade or accidental interruption [39, 40] . in 1995, a disastrous earthquake hit kobe and destroyed all available transportation links to the site. this earthquake affected the toyota production setup negatively and a large amount of revenue was lost due to this unavoidable event [50] . recovering from production interruptions due to these kinds of events without having a proper response results in an additional cost for the organization, which is considerably high [31] . the most recent example of this type of production disruptions is the covid-19 epidemic and it caused manufacturers like hyundai and fiat chrysler automobiles nv to halt their productions [16] . batch production systems are a well-known and very popular technique in advanced manufacturing setups [5, 46] . in these manufacturing setups, both single and multiple products depending on the type of products are produced and delivered in batches. batch production helps to reduce cost and increase profitability, the production lot size is determined to minimize the cost of the manufacturing system. there are numerous industries that produce single or multiple products using the batch production technique [27, 48, 53] . however, in real-life cases, there are lots of risk factors involved and these should be taken into consideration when a system is analyzed [37] . the system may face production interruptions due to the above mentioned inevitable events or any other type of production system failure. because of the imperfect production, the process reliability (which is usually less than 100%) is also an important factor in real-life production systems. it is very difficult to obtain a system which produces all perfect products. production disruption may affect the organization financially and the reputation of the organization in the market may suffer due to the shortage of goods and customer demand that is not fulfilled during this period. to avoid financial and reputation losses, the organization should have a recovery plan for these types of inevitable events [38] . the organization can use such a plan in a time of need and quickly alleviate the disruption effect. another aspect of production systems is the impact they have on the environment and human lives. this is why production disruption has recently gathered the interest of researchers. moreover, reliability of the system is highly related to production disruption, and if the system is more reliable, the chances of moving from in-control to out-of-control are rare, resulting in a less imperfect production [19] . usually, rejected imperfect products are either reworked to obtain perfect quality or disposed. disposing of rejected products is more dangerous for the environment and should be avoided. one of the most widely studied research topics is the analysis of production-inventory systems. in early studies, researchers developed models under ideal conditions with simple assumptions. later, a singleitem imperfect production inventory control model with reworking was considered by sana [43] . pal et al. [30] incorporated price dependent production rate in an inventory model under imperfect conditions. sarkar et al. [45] studied a single-stage production-inventory model for rework at the same production setup with random defective rates and backorders. cárdenas-barrón and sana [8] investigated a two-echelon supply chain model for multiple items under promotion dependent demand and delayed payments. moreover, cárdenas-barrón et al. [6] proposed a multi-period supplier selection problem for multiple items in an inventory model. an improved multi-stage imperfect production model was analyzed by kim and sarkar [20] under controllable lead time. further, cheikhrouhou et al. [9] developed an inventory inspection model for order size and sample size with the return of imperfect products. recently, malik and sarkar [26] studied an inventory control model for a coordination supply chain management to reduce the leadtime by considering two different modes of transportation. malik and kim [24] introduced a flexible production model with production rate and economic lot size optimization considering the relation between production rates and carbon emissions. multi-product production inventory models have been considered by several researchers for previous two decades. a multi-product optimal production quantity model was developed by pasandideh et al. [32] with imperfect production, permissible rework, and a space constraint. taleizadeh et al. [51] analyzed a multi-item production model with limited production capacity and service level constraint. further, taleizadeh et al. [52] updated the imperfect production model with a random defective rate under limited production capacity. they minimized total cost on optimal values of back ordered quantity, cycle length, and production quantity. pirayesh and poormoaied [41] solved a multi-item imperfect production model under limited production capacity by using the genetic algorithm and swarm optimization. pasandideh et al. [33] developed a multi-item lot size inventory model considering budget and space constraints. recently, malik and sarkar [25] analyzed a multi-product model with uncertain demand, service level and storage space constraints to optimize the order quantity and process quality. recently, researchers focused on developing different types of disruption recovery models for supply chain and production-inventory systems. they investigated supply chain systems for several types of disruptions such as supply disruption [21, 29] , demand disruption [14, 1, 17] , transportation disruption [57, 13, 34] , machine breakdowns [22, 2, 59] and production disruption [39] . one can find a recent review on rescheduling of production systems in uhlmann and frazzon [55] . studies for optimal recovery strategies in production disruptions are scarce and limited. xia et al. [58] introduced a disruption management general approach for the two-stage production-inventory system. they incorporated a penalty cost of deviations from the original plan to the new plan and a recovery time window for production disruption. the work of xia et al. [58] was extended for real-time recovery strategy in a single-stage perfect production-inventory system, by hishamuddin et al. [12] . the model considered constrained non-linear optimization problem for a known disruption period. two different solution methodologies, heuristic and evolutionary algorithms were used and compared the results. paul et al. [37] suggested a real-time production disruption management plan for a two-stage batch production system under the reliability considerations. initially, they developed a single (independent) production disruption recovery model and then extended it to multiple (dependent) disruptions scenarios. further, paul et al. [39] extended model of hishamuddin et al. [12] to imperfect production system with random production disruptions and a uniform random distribution was followed by disruption occurrences. they introduced a mathematical model that deals with both dependent and independent (single and multiple) disruptions on a realtime basis. moreover, a three-stage production-inventory model with single and multiple disruptions was proposed by paul et al. [38] . they considered perfect production and presented a heuristic solution and a standard search algorithm to solve the model. paul et al. [40] proposed a three-stage supply chain model for mitigation of production disruptions. they developed three different models for different scenarios, an ideal plan with infinite planning horizon, a predictive disruption mitigation plan, and a reactive disruption mitigation plan. the model was solved by a heuristic approach and optimization with lingo software and compared the results of both. another important factor that should be considered when developing a production-inventory model is the process reliability, which has a significant impact on system cost and profit. lulu and black [23] studied the multi-component manufacturing and assembly environment and analyzed the impact of process unreliability on the performance of the manufacturing production system. they proved that process unreliability results in lower system utilization. cheng [11] defined the production process reliability as the percentage of non-defective products produced in a system. he considered process reliability for a single-period inventory system and formulated it as a geometric problem. a production model with production process reliability was developed by bag et al. [3] . they considered the set-up cost, production period, and process reliability as the decision variables for that model. sarkar [44] analyzed an economic manufacturing quantity model for an imperfect production process under the effects of inflation while considering reliability as the decision variable. paul et al. [36] analyzed a production-inventory system using process reliability and uncertain demand. furthermore, paul et al. [35] introduced a two-stage batch production-inventory system for real-time disruption management under consideration of the process reliability. they formulated the model for both kinds of disruptions, single and multiple (dependent and independent), and revised the solution with changed parameters. see table 1 for the contributions from different authors, only the most relevant papers are included. the research contributions regarding the production type, disruption type, reliability, and constraints are given. in previous studies, no one has considered the production-disruption problem for multi-item production-inventory systems. in this study, the production disruption model for a single-stage single-product production-inventory system introduced by hishamuddin et al. [12] is considered. they considered a perfect production-inventory system facing production disruptions during the production cycle. in their model, they assumed all the produced products are perfect. however, in real life situations production systems also produce some defective products due to system limitations. production disruptions due to inevitable events such as natural disasters or machine breakdowns cannot be predicted in advance. however, when the production system experiences disruption, the original plan is revised and updated quickly to come to alleviate the effects of the disruption. till now in literature, no study is available regarding multi-product imperfect production systems under disruption. this research considered a single-stage multi-product imperfect production-inventory system that faces random production disruptions at different stages of the production cycle. in this paper, the authors first developed a multi-constrained non-linear mathematical model to deal with the production disruption in an imperfect production system where multiple products are produced. the developed mathematical model is solved by two standard search algorithms, genetic algorithm (ga) and pattern search (ps). authors have generated number of test problems for the production disruption by using a uniform distribution. numerical examples are presented to demonstrate the effectiveness of the proposed research and the obtained results from both search techniques, ga and ps, are compared. the production system is faced with eight types of costs: setup cost, production cost, holding cost, inspection cost, imperfect products rejection cost, interest and depreciation cost, backorder cost, and lost sales cost. the research contributions regarding the production type, disruption type, reliability, and constraints are given in table 1 . based on the research gap from the literature review and authors' contribution table, the overall contribution of this paper is twofold 1 first, a multi-item single-stage production system is studied under the consideration of unavoidable production disruption. to the best of authors' knowledge, in existing literature, only a very few studies exist with production disruption scenario while none of them has considered multi-item production system. in practical environments, multi-item production systems are getting more famous among manufacturers. therefore, it is need of time for managers and manufacturers to have multi-item production recovery models with disruptions during production cycles. 2 second, most of the existing models do not consider budget and space limitations for economic and production decisions while these two factors are the most important for multi-item production-inventory systems. this model considers limitations over both, budget and space, to make the model more practical and real-life problembased. the remainder of the paper is formulated as follows. in section 2, the problem definition, notation, and assumptions are given. a mathematical model is given in section 3. section 4, presents the solution approach, and numerical examples are given in section 5. section 6 consists of a sensitivity analysis. finally, conclusions are given in section 7. in this section, first the problem definition is given, which indicates the major motivation for this research. after that, notation and assumptions are given for the mathematical model. in manufacturing systems, it is no surprise that disruption problem can emerge at any time within the manufacturing uptime. in this particular section, the disruption problem in a multi-item production system is described. almost every manufacturing system may face disruption during the production run-time and recover from disruption; it is a difficult and formidable task for the manufacturer. usually, it is very difficult to devise a recovery plan after the occurrence of the disruption. therefore, manufacturers should have a recovery plan prior to disruption. when disruption happens, it takes time to get to a point, where the manufacturer can start production again. thus, the system loses time due to this recovery process. occasionally the system may even face another disruption. however, if the system faces another disruption after recovering from the previous one, researchers will consider it as a single-disruption case. this is because the second disruption will not be affected by the previous disruption and recovery plan, and the system recovers from the second disruption on the basis of the original recovery plan. paul et al. [39] developed a single item production disruption model, but this model does not consider any constraint for the budget or space. most of the studies have been for the single-item production system because of the complexity of the multiitem production system. this paper presents the first attempt at a multiitem production system recovery plan for a disrupted system. this model considers a multi-item production system for the manufacturer, with limits on the available budget and storage space for the inventory. fig. 1 (a) shows an ideal plan for the single-stage batch productioninventory system where multiple products are produced. these products are produced in batches. after the completion of each batch, there is a production down time which is the summation of idle time and setup. in an ideal production-inventory system, the production quantity for each cycle i is x in,0 (i = 1, 2, .. ., m), and summation of these quantities is equal to q n . the recovery plan is a updated schedule that includes the revised production quantities in each cycle, and ensuring the maximization of the total profit in the recovery time window. the number of future cycles allocated to return to the original production schedule from the disrupted cycle, is known as the recovery time table 1 summary of different authors' contributions. banerjee [4] single-item, perfect production − − − widyadana and wee [56] single-stage, single-item, − − − perfect production sarkar [44] single-stage, single-item, − product production imperfect production reliability capacity paul et al. [36] single-stage, single-item, single and multiple system production perfect production production reliability capacity disruptions cárdenas-barrón and sana [7] single-stage, single-item, − − − perfect production paul et al. [38] three-stage, single-item, production system production perfect production disruption reliability capacity kim and sarkar [20] multi-stage, multi-item, − − budget imperfect production constraint saha et al. [42] single-stage, single-item, − − − perfect production this model single-stage, multi-item, production system production imperfect production disruption reliability capacity, budget, and space constraint "−" this key word is not available in paper. journal of manufacturing systems 56 (2020) 227-240 window [39] , and is decided by the management of the organization. fig. 1 (b) presents a disrupted production-inventory system and a recovery plan after the occurrence of production disruption. the production system becomes inoperable for a certain period (t dn ) due to a disruption and after then it operates normally. the recovery plan starts just after the ending point of the disruption period and continues during the recovery time window. it takes m number of production cycles during the recovery time window to returns the production process to its original or normal schedule. decision variablesx in production quantity of product n for cycle i of the recovery time window, i = 1, 2, .. ., mparameterss tn setup time of machine n for each cycle (year)δ in idle time of machine n for cycle i (year)d n demand of product n per year (units/year)h n holding cost for product n per unit per year ($/unit/year)rreliability of the production process, which is known from historical data of the production systemq n economic lot size for product n per ideal production cycle with process reliability r (units/cycle)a n setup cost of machine n per setup ($/setup)p n production rate for product n (units/year) in a 100% reliable systemmnumber of cycles in the recovery time window to get to the original production plan (given from the management)t dn disruption period (years)q n pre-disruption production quantity of product n (units)t 0n production time (years) for = q ( ) n of each acceptable unit of product n, this must be greater than 1s n space occupied per unit of product n (meter sq./ unit)wtotal budget ($)stotal space(meter sq./ unit)nmaximum number of machines and products, a positive integer this paper is based on the following assumptions: (1) this model is developed for a multi-product single-machine imperfect production system. in this system, multiple products are produced on the same machine; for example, different types of gears or bearings manufacturing. (2) to fulfill the customer's demand, the production rate is always higher than the demand for each product. therefore, shortages are not allowed in the model. (3) during the production cycle, the system may go under the disruption due to unexpected factors (i.e. labors strikes, material shortages, or power cut off) and production stops. (4) the recovery cycle starts immediately after a production disruption occurs. the recovery cycle is the time in which management update the current production plan. the management would like to get to the original production plan within the reasonable number of production cycles after a production disruption happens. in this time window, the managers utilize the ideal time to produce products to over come the delay in production process caused by the production disruption during the disruption period [12] . (6) this model is for an imperfect production system. however, all the produced products are well inspected before dispatch. in this system, all defective products are separated and rejected [39] . hence, no rework is done in the system. (7) the production system has a restriction on the available space for inventory storage. the total available storage space is s and the system should not violate this limit (see [28] ). (8) there is an investment limit for the manufacturer for the production cost. the total available budget for production cost is b and the production cost should not exceed this budget limit. in this section, the disruption problem for a multi-item productioninventory system is described and formulated. in real life, for multiitem production systems, different types of disruptions may occur, and to manage the system, it is necessary to devise a recovery plan. if a system faces another disruption after the recovery time window of the first disruption, it can still be managed as a single-disruption problem because the second disruption is not affected by the previous disruption and recovery plan. this model considers the multiple products singledisruption problem. using the lot-for-lot (lfl) policy of banerjee [4] and the single-item batch production of sarker and khan [49] , the proposed model obtains the economic lot size (q n ). the the total cost for the inventory system (by adding the above-given equations) is given by the mathematical expressions for the setup cost, production cost, holding cost, inspection cost, rejection cost, and interest and depreciation cost are needed to calculate for total cost calculation. these costs are considered in this model for the disruption recovery time window. all the costs involved in the total cost of the production system tc per item are derived as follows: setup cost (sc) the first step for starting production is devising the production/ manufacturing setup. all the fixed costs, which are experienced every time a single item is produced, are consolidated as the setup cost, and all these fixed costs are mostly associated with the physical activities required within the setup to produce the quantity or lot. these physical activities include setting up the machine, work center, or assembly line. (2) when a manufacturer produces a product or a lot of a given quantity, this requires a certain amount of resources per product. the production cost is the unit production cost for each product multiplied by the total number of units produced for each product. here, the production costs are calculated as the product of production cost per unit cp n , production rate p n , and the total production time. thus, it can be found as follows: all produced products are stored in the warehouse at a cost. to calculate the holding cost, first, an average inventory is calculated. the manufacturing system begins producing products and faces production disruption after some production time t 0n , and from the start to the time when disruption occurs, the produced quantity is q n . the system faces disruption for the time period t dn , the setup time is s tn , the production run time for the first cycle is t 1n , and the system holds the predisruption quantity q n for the entire time of the production cycle (see fig. 1 ). thus, the total produced quantity is calculated (see appendix a) as the unit holding cost multiplied by the total inventory in the recovery time window, which is the area under the curve in the recovery time window, is the total inventory holding cost: all produced products are inspected, and a fixed cost is incurred to inspect all products. the product inspection cost is all produced products are inspected and the defective products are separated from the perfect products. further, all imperfect products are rejected and thrown out of the manufacturing system at a fixed cost. the process reliability is r and the rate of rejection is (1 − r) . the total rejection cost is the rejection cost per unit rejected multiplied by the number of units rejected after inspection: the total cost of interest and depreciation equation is considered to be a general power function (for instance, see [11] ). it is inversely proportional to the setup cost a n and directly proportional to the process reliability r. hence, the cost of interest and depreciation is the backorder is the quantity that is not fulfilled at the time of order but is delivered at a later date when the required quantity is available. when the demand quantity is not available at the time of order and the customer will not wait for the stock to be replenished, the lost sales cost is incurred. the lost sales cost is the unit lost sales cost multiplied by the lost sales units. therefore, the lost sales cost (see appendix c) is the total cost per item (tc) is calculated by combining setup cost, production cost, inventory holding cost, inspection cost, interest and depreciation cost, backorder cost, and lost sales cost, and is given by the revenue generated by the acceptable items, during the recovery time window, is calculated as selling price per unit multiplied by the demand quantity. the above profit is maximized subject to the following budget, space, demand, capacity, delivery and transportation constraints: in real life production systems, the manufacturer has a limit for the available budget. thus, the manufacturer never exceeds this budget limit in terms of the costs, i.e., the total production cost is less than or equal to the budget limit, i.e.; ) . every production setup has a limited storage capacity for the inventory, which usually considered as space constraint. the space constraint ensures that the space occupied by all products is less than or equal to the total space available at the production setup as follows: n n n n 1 in (15) production capacity constraint when the system functions according to the original production plan without facing any disruption, the production quantity is equal to the desired number. the following constraint ensures the cycle production quantity in an ideal system for all the products:: the following constraints set the production in each cycle of the recovery time window as less than or equal to the production quantity for the ideal system. these two constraints ensures that the production quantity in each cycle of the recovery time window is less than the production quantity in each cycle before the occurrence of the production disruption. this is because of the transportation and delivery requirements. the model is developed considering the recovery window production capacity. the production capacity is limited in that the system can never produce more than the capacity during the disruption recovery period. it gives the recovery time window production capacity constraint as the process reliability is always less than or equal to one and is only equal to 1 when the production system is considered fully reliable. however, in real-life systems, process reliability is always less than 1. thus, the system has reliability r which affects the overall rate of production of the item and must be higher than the total demand. as the reliability of the system is less than 1 (r < 1), the production rate can be such that the combined rp n is higher than the demand: the following equations represent the ideal time non-negativity conditions: the following two equations ensure the delay time non-negativity: finally, all the decision variables are non-negative: 1, 2, ..., ; 1, 2. .., . in (27) in literature, various analytical techniques have been used to solve small-and medium-sized problems. however, finding the optimal solution in a complex non-linear constrained problems by using the analytical approaches presented in the literature is very difficult or nearly impossible because of heavy computing overheads. consequently, there is also a need to reduce the time, and enhance the precision and quality of the solutions for the optimization of complex real-life problems. the proposed multiitem recovery model is a complex constrained mathematical model and it cannot be solved with the analytical procedure because of its complexity. however, this mathematical model is solved by using the two global search metaheurisitc techniques, (1) genetic algorithm (ga) and (2) pattern search (ps). these metaheurisitics are not local search-based and usually these are population-based. these techniques were used because of their ability to effectively deal with a large number of parameters in complex optimization problems. to obtain the solution, we coded the model in matlab r2015b and solved using the optimization toolbox. we executed this on an intel core i5 with a 3.20 ghz cpu and 8gb ram. a single disruption problem given in this model can be solved via the help of search algorithms. to validate the model results, two search techniques a genetic algorithm (ga) and pattern search (ps), were implemented in matlab r2015b. ga is a natural genetic-based general purpose optimization technique that mostly explores the given search space [15] . pattern search is also a search technique for use within a given search space and is easy to apply to nonlinear optimization problems with constraints. we compare the results obtained by both search techniques at the end of the next section. major steps of the proposed solution algorithm for real-time based single-disruption model can be found as follows: solution algorithm 1 input all data for the ideal system and get q n by using the eq. (1). 2 assign the values x in0 = q n 3 put i = 1 for disruption for all products i = 1, 2, 3 4 input pre-disruption quantity q n and the disruption period (t dn ). 5 generate initial population for the recovery cycle from the start of the disruption. 6 solve the proposed model with two, genetic algorithm and pattern search, search 7 update the x in value as a revised lot size from (6) and note down the revised production plan. 8 stop. journal of manufacturing systems 56 (2020) 227-240 in this section, we inspect results for the single-disruption case. in this study, we define a disruption situation as the coalition of the predisruption situation and the duration of the disruption. we consider these parameters as uniform random variables in this study. to test the recommended model within the intervals, over 50 disruption test problems are generated by varying the parameters. we solve the proposed model by both the ga and ps search techniques and analyze and compare the obtained results. the ranges of the parameters are given in table 2 and the input data for single disruption problem is given in table 3 . the parameters used for the ga and ps to execute the model and obtain the solution are as follows parameters for ga • all other parameters in the optimization tool box were set as the default. to examine the results, most of the input data from [39] were used: from the above data, x in = q n can be easily calculated: below the results for each example are given where only the best results out of thirty runs are provided. example 1. in the first numerical example, a disruption problem with the upper and lower bounds is considered. the results for example 1 are given in table 4 for ga and for ps. in the second numerical example, a disruption problem is considered with budget and space constraints, as well as all the upper and lower bounds mentioned in the above mathematical model. the results for example 2 are given in table 5 for ga and for ps. example 3. in the third and last numerical example, a disruption problem is considered with all the constraints as well as the upper and lower bounds already mentioned in the above model and the data given above. the results for example 3 are given in table 6 for ga and for ps. to judge the consistency, best results of 24 independent runs have been compared for two different search techniques (ga and ps). all the table 2 parameter ranges. test problems have been generated randomly within the ranges given in table 2 . the obtained average percentage of error or deviation for the two search techniques was 0.00118% and it can be considered as negligible. to calculate the average value of percentage deviation, following equation was used the comparison of results for 24 test runs, obtained from ga and ps, is presented in table 7 . from the comparison of results, one can see that the results are very much consistent for both, ga and ps, search techniques. in this part, sensitivity analysis for test instance 1 of example 3 are given. a sensitivity analysis is performed for each of the five key parameters t dn , q n , b n , and l n . the pattern search technique was used for the sensitivity analysis. the total effect of the parameter changes −50%, −25%, +25%, and +50% are calculated, and details are given in table 8 . for characterizing the impact, the sensitivity analysis is performed different parameters. from the sensitivity analysis table, one can see that the total profit decreases with the increase of the disruption time t dn and unit lost sales cost l n . the total profit increases in the reverse case for disruption time and unit lost sales cost. however, profit is increasing with the increase of the pre-disruption quantity q n and unit backorder cost b n and vice versa. overall profit is more sensitive for the two parameters, the unit lost sales cost l n and the reliability of the system r, and it changes values very quickly with a smaller change in the parametric value. figs. 2, 3, and 4 show the changes in the total profit with respect to disruption period, pre-disruption quantity, backorder cost, and lost sales cost. for each analysis, only one parameter is changed and the remaining parameters are kept constant. authors make these analyses for both the search techniques, ga and ps. fig. 2 shows the changes in the total profit with the disruption period. the total profit decreases with the increase in the disruption period. the profit decreases linearly when the disruption period is higher than 0.0035 and the authors depict it in fig. 2. fig. 3 presents the impact of pre-disruption quantity on the profitability of the system. the total profit increases with the increase in pre-disruption quantity and increment are more when the predisruption quantity is closer to the production quantity x in0 for the ideal cycle. fig. 4shows the increment in lost sales cost has a strong impact on the profit and causes a linear decrease in overall profit for the production system. the trend is the same for both ga and ps. this production disruption model can be applied to the production systems where the production environment is imperfect and producing multiple items at the same setup. more importantly, in multiple item production systems, the manufacturer must consider the proposed or suitable type of storage space limit and budget limitations over different costs and investments. the managers of any production industry are always anxious about breakdowns (especially random breakdown) due to defective production. any disruption may occur within a production setup and it can cause defective production or production with low quality. therefore, the management aims to control the breakdown anyhow. to do this, they generally consider corrective maintenance or preventive maintenance or both, but all these maintenance policies are too costly and it is difficult to maintain and based on literature, only disruptions are available in a single-stage production system. nowadays, many complex systems are made, where disruptions may occur anytime. the outcomes of this model will benefit industries to maximize profit. managers can predict the amount of disruption affected products; they can predict how much time the production process will be suspended due to production such that they can commit with other players for selling their products. finally, by the outcomes, the industry can make proper scheduling of throughput of finished products and the arrival rate of raw material for any multi-product production system. authors assumed the lot size is fixed throughout the planning horizon. however, the lot size may be split to meet the transportation capacities i.e. truck capacity. we also assumed the demand is known and fixed. while in practical situations the demand may fluctuate in different conditions. therefore, management should take care of these parameters while applying the proposed approach. the numerical problems are used to develop the methods of making recovery plans after the production disruption occurs which can present managers with some examples to solve the disruption problems in real environment. in a real operation of production systems, there are a number of variables and constants. these numerical are surely not to analyze all possible situations, but they can • demonstrate the effectiveness of the methods, • indicate how to make the recovery plans after the production disruption occurs, • present the advantages of the solution methodology developed for the production system, and • provides a comparison of results and time taken to solve the problem by ga and ps search techniques. in production systems, disruption can occur at any time within the production run-time. without a recovery plan for such disruptions, the organization may face huge financial and reputation losses. furthermore, imperfect production lines are very common in real-life and greatly impact a company's profit and loss. the aim of this study was to develop a recovery plan for a disrupted single-stage multi-product imperfect production system. a mathematical model with constraints was developed for single-stage multi-product imperfect production systems. space and budget constraints were considered, based on real life experiences. for implementation in the real-time recovery planning of a disrupted production system, a few examples were provided. these examples were solved using the pattern search (ps) and genetic algorithm (ga) solution approaches. similar results were obtained by both solution approaches. in addition to this, sensitivity analyses were performed to show the impact on total profit; these were performed while changing one parameter and keeping all others constant. this model only considers single production disruption or multidisruption without the effect of the previous disruption, which is a major limitation of this model. immediate extension of this model is possible by considering multiple dependent production disruptions. further, this model can be extended to different directions related to production or supply chain. the first extension of this model can be for the fuzzy or uncertain environment, parameter like demand can be considered as a triangular or trapezoidal fuzzy number. several researchers considered different parameters as fuzzy for inventory models [47] , and it would be a new direction if fuzzy parameters are combined with production disruption problem. the fuzzy nature of parameters will have a noticeable impact on the system and profit. secondly, this model can be extended with the probabilistic proportion for defective products and all imperfect products reworked in the same setup or with separate setup, see for reference [18] . another possible extension can be, multi-stage production with stochastic lead time demand [20] . few other future research directions include considering the case of multiple shipments in a supply chain [10] with multi-stage imperfect production and random defective rate [54] . the authors declare no conflicts of interest. the backorder cost is calculated as coordinating a supply chain system for production, pricing and service strategies with disruptions rescheduling of parallel machines with stochastic processing and setup times a production inventory model with fuzzy random demand and with flexibility and reliability considerations a joint economic-lot-size model for purchaser and vendor optimal manufacturing batch size with rework in a singlestage production system-a simple derivation a new approach to solve the multi-product multi-period inventory lot sizing with supplier selection problem a production-inventory model for a two-echelon supply chain when demand is dependent on sales teams? initiatives multi-item eoq inventory model in a two-layer supply chain while demand varies with promotional effort optimization of sample size and order size in an inventory model with quality inspection and return of defective items optimisation of multi-stage jit production-pricing decision: centralised and decentralised models and algorithms an economic production quantity model with flexibility and reliability considerations a disruption recovery model for a single stage production-inventory system a recovery model for a two-echelon serial supply chain with consideration of transportation disruption a recovery mechanism for a two echelon supply chain system under supply disruption constrained optimization via genetic algorithms predicting the impacts of epidemic outbreaks on global supply chains: a simulation-based analysis on the coronavirus outbreak (covid-19/sars-cov-2) case turn bad into good: using transshipment-before-buyback for disruptions of stochastic demand impact of random defective rate on lot size focusing work-in-process inventory in manufacturing system an improved way to calculate imperfect items during long-run production in an integrated inventory model with backorders multi-stage cleaner production process with quality improvement and lead time dependent ordering cost competition and cooperation in a single-retailer twosupplier supply chain with supply disruption solving single machine scheduling under disruption with discounted costs by quantum-inspired hybrid heuristics effect of process unreliability on integrated manufacturing/ production systems a constrained production system involving production exibility and carbon emissions optimizing a multi-product continuous-review inventory model with uncertain demand, quality improvement, setup cost reduction, and variation control in lead time coordinating supply-chain management under stochastic fuzzy environment and lead-time reduction a sustainable production-inventory, model for a controllable carbon emissions rate under shortages a hybrid genetic and imperialist competitive algorithm for green vendor managed inventory of multi-item multi-constraint eoq model under shortage a multi-echelon supply chain model for reworkable items in multiple-markets with supply disruption joint pricing and ordering policy for two echelon imperfect production inventory model with two cycles a multi-echelon production-inventory system with supply disruption a parameter-tuned genetic algorithm to solve multi-product economic production quantity model with defective items, rework, and constrained space a multiproduct single machine economic production quantity model for an imperfect production system under warehouse construction cost managing sudden transportation disruptions in supply chains under delivery delay and quantity loss development of a production inventory model with uncertainty and reliability considerations a disruption recovery model in a production-inventory system with demand uncertainty and process reliability real time disruption management for a two-stage batch production-inventory system with reliability considerations a disruption recovery plan in a three-stage productioninventory system managing disruption in an imperfect production-inventory system a quantitative model for disruption mitigation in a supply chain gpso-ls algorithm for a multi-item epq model with production capacity restriction optimal retailer investments in green operations and preservation technology for deteriorating items a production-inventory model in an imperfect production process an inventory model with reliability in an imperfect production process an economic production quantity model with random defective rate, rework process and backorders for a single stage production system manufacturing setup cost reduction and quality improvement for the distribution free continuous-review inventory model with a service level constraint periodic review fuzzy inventory model with variable lead time and fuzzy demand how does an industry reduce waste and consumed energy within a multi-stage smart sustainable biofuel production system? an optimal batch size for a production system operating under periodic delivery policy the resilient enterprise: overcoming vulnerability for competitive advantage multiproduct single-machine production system with stochastic scrapped production rate, partial backordering and service level constraint economic production quantity model with repair failure and limited capacity a sustainable development framework for a cleaner multi-item multi-stage textile production system with a process improvement initiative optimal batch quantity in a cleaner multi-stage lean production system with random defective rate production rescheduling review: opportunities for industrial integration and practical applications optimal deteriorating items production inventory models with random machine breakdown and stochastic repair time the impact of transportation disruptions on supply chain performance real-time disruption management in a two-stage production and inventory system resilient adaptive control based on renewal particle swarm optimization to improve production system energy efficiency the work is supported by the national research foundation of korea (nrf) grant funded by the korea government (msit) (nrf-2020r1f1a1064460). range of data ) 1 5414 6164 6164 6164 6164 1 2 6042 6803 6892 6892 6892 3148441.09 3 6599 7549 7549 7472 7549 1 5139 6164 6164 6164 6164 2 2 5667 6892 6892 6892 6892 3148065.77 3 6174 7549 7549 7549 7549 1 5664 6164 6164 6164 6164 3 2 6217 6892 6892 6892 6892 3147491.57 3 6749 7549 7549 7549 7549 test product type 1 5414 6164 6164 6164 6164 1 2 6042 6892 6892 6892 6892 3148460.78 3 6599 7549 7549 7549 7549 1 5139 6164 6164 6164 6164 2 2 5667 6892 6892 6892 6892 3148065.76 3 6174 7549 7549 7549 7549 1 5664 6164 6164 6164 6164 3 2 6217 6892 6892 6892 6892 3147491.55 3 6749 7549 7549 7549 key: cord-255036-vpw40g40 authors: zhang, l.z.; zhu, d.s.; deng, x.h.; hua, b. title: thermodynamic modeling of a novel air dehumidification system date: 2004-08-14 journal: energy build doi: 10.1016/j.enbuild.2004.06.019 sha: doc_id: 255036 cord_uid: vpw40g40 a novel air dehumidification system is proposed. the proposed system incorporates a membrane-based total heat exchanger into a mechanical air dehumidification system, where the fresh air flows through the enthalpy exchanger, the evaporator and the condenser subsequently. thermodynamic model for the performance estimation of the combined system is investigated. processes of the fresh air and the refrigerant are studied. two additional specific programs are devised to calculate the psychrometrics and the thermodynamic properties of the refrigerant r134a. annual energy requirement is 4.15 × 10(6) kj per person, or 33% saving from a system without energy saving measures. it is often necessary to control and modify the water vapor content in the air: the operation is quite usual in air conditioning. normally the water vapor content of atmospheric air is small, some tens of grams per kilo of air; nonetheless, due to the very high heat of vaporization, the latent heat content in air conditioning is of the same order of the sensible one. the relative importance of latent load increases with the ventilation rates to buildings. higher ventilation rates are dictated both by better comfort requirements and by the most recent standards such as the ashrae 62/99 [1] . more recently, people's concern on indoor air quality has been greatly deepened since the outbreak of the severe acute respiratory syndrome (sars) epidemic that molested south china and some other parts of the world in this spring and caused a loss of 17.9 billion us$ (or 1.3% gdp) in china alone and 59 billion us$ globally [2] . the mechanisms of sars virus spreading is complex and remains a mystery till now. however, it is believed that enough fresh air ventilation is helpful in decreasing the possibility of being infected by the disease. ventilation air is the major source of moisture load in air conditioning. as shown in fig. 1 for a moisture load estimation of a medium size retail store [3] , ventilation air constitutes about 68% of the total moisture load in most commercial buildings. as a consequence, treatment of the latent load from the ventilation air is a difficult and imminent task for hvac engineers, especially in hot and humid climates like south china. traditionally, latent load and sensible load are treated in a coupled way. because air is not only for ventilation, but also a heat transfer medium, and large quantities of air is needed to extract the sensible load, energy requirements are very high. another problem with this technique is that in transit seasons, humidity will lose control [4] . there is an increasing trend to separate the treatment of sensible and latent load by using an independent humidity control system. according to this concept, the latent load of a room is treated by an independent humidity control system, while the sensible load is treated with some other alternative cooling techniques such as chilled-ceiling panels. since the circulating air is dramatically reduced, energy consumption can be reduced substantially. another benefit is that chilled water or suction temperatures can be raised, resulting in increased equipment efficiency and decreased operating costs. it is estimated that 30% of the energy used www.elsevier.com/locate/enbuild energy and buildings 37 (2005) 279-286 could be saved with chilled-ceiling panels combined with independent humidity control system, in comparison to a traditional coupled system [5, 6] . nevertheless, due to the hot and humid climates in south china, energy for moisture control with an independent humidity control system still accounts for 25% of the total energy for air conditioning. thus, energy recovery measures must be combined to an independent humidity control system. in this study, a novel air dehumidification system, namely, mechanical dehumidification with membrane-based total heat exchanger, is proposed. the thermodynamic modeling of the system is performed. through hour-by-hour analysis, the annual primary energy consumptions for the systems proposed are discussed. a schematic of the new air dehumidification system modeled in this work is shown in fig. 2 (a) and its psychrometrics are shown in fig. 2 (b). in this system, a membrane based total heat exchanger is used to pre-cool the fresh air before it is pumped to a refrigeration system for air dehumidification. the total heat exchanger has a membrane core where the incoming fresh air exchanges moisture and temperature simultaneously with the exhaust air. in this manner, the total heat or enthalpy from the exhaust is recovered, and both the temperature and the humidity of the fresh air are decreased, which results in energy saving. then the cooled and dehumidified fresh air is flowed through the condenser of the refrigeration system, to satisfy the requirements of supply air: 7 g/kg humidity and 20 8c in temperature. this system is relatively simple, since the membrane system has no moving parts, and is compact. desiccant wheels may be another choice, however, in some circumstances, gas or other thermal heat may be difficult to find. the control of the new system is easier than a conventional compression dehumidification system, because no other components except a membrane system is added to the system. the differences are the operating parameters are changed, and much fan power is needed. the membrane system operated in near atmospheric pressure condition, the maintenance is easy. this study is to estimate the energy performance of a novel air dehumidification system by thermodynamic analysis. the modeling methodology of the refrigeration cycle is a steady-state one. they are sufficient to reflect the coefficient of performance or the energy performance of the refrigeration cycle. even though more detailed models are proposed by some authors, see [7, 8] , they are compli-list of symbols a tot total exchange area (m 2 ) c 0 constants in sorption curves cop coefficient of performance c p specific heat (kj kg à1 k à1 ) d wm water diffusivity in membrane (kg m à1 s à1 ) h specific enthalpy (kj/kg) k s convective mass transfer coefficient in supply side (kg m à2 s à1 ) m_ mass flow rate (kg/s) dp total pressure rise (pa) q heat (kw) cated and case-sensitive. in other words, the distributed parameter models in such studies are strongly based on design parameters of a specific refrigeration system. those detailed models just gave very generalized equations for the calculation of heat and mass transport in each component and rely heavily on manufacturers' product empirical data. further, this study gives a general view of energy savings in sub-tropical regions, which do not rely on a specific system. furthermoreover, the more detailed partial difference models in other studies are difficult and slow in finding a solution. it is difficult to use such models in a wholeyear-hour-by-hour simulation. those more complicated models are usually necessary to study transient responses and start-up characters of a refrigeration cycle, which is beyond the scope of this study. the effects of the refrigeration cycle on the whole system are demonstrable. a thermodynamic model of the refrigeration system is formulated based on the processes of refrigerant r134a shown in fig. 3 . saturated r134a liquid at point 4 flows through an expansion valve and becomes wet vapor at point 5. refrigerant at this state flows to the evaporator (also the dehumidifier) where it chills and dehumidifies the fresh air and evaporates to point 6 and further superheats to point 1. then the refrigerant vapor is pumped by a compressor to point 2 where the vapor is displaced to the condenser and condensates from state 2 to 4 through 3. the superheat is set to 5 8c. it should be noted that the exact degree of superheating may be affected by many factors, such as evaporator, expansion valve, and compressor, and are strongly related to operating conditions, control strategies. experiments found superheating are in the range of 2-16 8c; and the superheating increases with air temperature in the evaporator, from 3 to 14 8c when the expansion valve is fully open. from viewpoint of energy use, too large superheating is not good. generally, 3-14 8c superheating are possible. refrigerant superheating refers to the superheating in the evaporator. superheating from tubes can be neglected with well tube-insulating, since superheating from such sources is harmful to energy performance and should be prevented. the effects of heating in the compressor are included in the compressor's isentropic efficiency. the specific enthalpy of the refrigerant at the compressor exit is calculated by where h s is the isentropic efficiency; h 2 , is the specific enthalpy at the condensing pressure by isentropic compression from the evaporating pressure. experimental results have shown that the isentropic efficiency is a weak function of the displacement volume, and varies linearly with the compressor speed. in this analysis, a constant isentropic efficiency of 0.75 is assumed, neglecting the rotational speed of the compressor. the specific enthalpy after the expansion valve is calculated by the degree of dryness at the inlet of the evaporator where h 5 , is the specific enthalpy of the saturated liquid refrigerant at the evaporating pressure. the electricity consumed by the compressor where m_ f is the mass flow rate of refrigerant (kg/s); h motor is the motor efficiency, which is considered as 0.75. the energy load of the evaporator heat rejected in the condenser refrigeration efficiency cooling and dehumidification of the incoming fresh air are performed in the evaporator. a detailed modeling of the evaporator and the condenser are rather complicated [7, 8] . usually, they are divided into regions associated to the phase of the refrigerant. each region constitutes a separate heat exchanger. in the case of the condenser, the superheated vapor, the condensation and subcooled liquid regions are considered, whereas for the evaporator it is divided into the evaporating and superheated vapor regions. for each region, the refrigerant side and air side convective heat transfer coefficients need be calculated from the established correlations for single-phase and two-phase flow. for the evaporator, when the average fin surface temperature is calculated to be less than the local water dew point of the air stream, moisture condensation will occur. under these conditions, the air heat transfer coefficients can no longer be calculated as in dry conditions, and a water mass balance must be carried out. in this case, the enthalpic method, proposed by threlkeld [9] and introduced in ashrae handbook [10] was most adequate for use. according to this procedure, the driving force for heat transfer is assumed to be the difference between the saturated enthalpy of the air flowing over the fins and a fictitious saturated air enthalpy evaluated at the refrigerant temperature. the analysis of air cooling and dehumidifying coils requires coupled, non-linear heat mass transfer relationships. while the complex heat mass transfer theory that is presented in many textbooks is often required for cooling coil design, simpler models based on effectiveness concepts are usually more appropriate for energy estimation. these techniques are resulted from basic heat and moisture transfer equations for simultaneous heat and moisture transport. therefore, in this study, to ease the analysis, thermal performance of the heat exchangers regions is evaluated by the (e, ntu) method. according to this procedure, the heat exchanger effectiveness is defined as where t hi and t ci are the inlet temperatures of the hot and cold fluids, respectively (k). because the operations are set to fluctuate around the design points, constant evaporator and condenser effectiveness are assumed in the simulations. then the air states at the outlets of evaporator and condenser could be obtained. the heat extracted in the evaporator is calculated by where m_ a is the mass flow rate of fresh air stream (kg/s); h ab and h ac are the specific enthalpies of air at point b and c, respectively. similarly, heat rejected at this portion of the condenser is governed by it should be noted that an energy balance requires that eq. (9) equals to eq. (5); while q c1 calculated from eq. (10) is only a portion of that calculated by eq. (6). in the study, the evaporating temperature is fixed to 5 8c, while the refrigerant flow rate and the condensing temperature varies according to the cooling load of the evaporator and the outside weather conditions. usually, a 10 8c log mean temperature difference between the condensing refrigerant and the air flowing through it is required. energy saving for conditioning fresh air can be achieved by the application of heat recovery ventilators, in which a fraction of energy is recovered from the exhaust air to the supply air. there are presently two kinds of techniques available for heat recovery ventilators: sensible heat exchangers and energy wheels. the sensible heat exchangers are simple and reliable, but they cannot recover latent energy. energy wheels, which could save latent energy, are problematic due to rotating moving parts. hydrophilic polymer membranes provide a new alternative. some membranes such as polyethersulphone, cellulose triacetate, and polyvinylchloride, are useful in separating moisture from the vapor/air mixture, due to their strong affinity to the water molecule. the strong affinity also leads to the high permeation difference between water and air. the permeability ratio of water to air ranges from 460 to 30,000. in other words, gases other than vapor can hardly permeate through the membrane. these features have led to the development of a novel ventilator, the so-called membrane based enthalpy exchanger in which both the sensible heat and the moisture are transferred simultaneously through the membrane. since the membranes are very thin, both the heat and the moisture transfer rates can be very high. furthermore, no water condensation happens in the membrane system, which prevents hygienic problems of microorganism growth that is common for cooling coils. in this study, a membrane-based enthalpy recovery ventilator developed in the laboratory is used [11] . two effectiveness: sensible effectiveness (e s ) and latent effectiveness (e l ) are defined. air state at point b in fig. 2 is calculated by: the membrane-based energy recovery ventilator is just like a traditional plate-type heat recuperator. the only difference is that hydrophilic membranes are used in place of metal plates. a total number of transfer units is used to reflect the sensible heat transfer in an exchanger. for the membrane exchanger that has equal area on both sides of the air streams, the total number of transfer units for sensible heat is [12] : where u is the total heat transfer coefficient (kw m à2 k à1 ), a tot is the total exchange area (m 2 ), m_ a is the mass flow rate (kg/s), and c pa is the specific heat of air (kj kg à1 k à1 ). the sensible effectiveness is a function of two dimensionless parameters, ntu and c 0 min =c 0 max , the ratio of minimum to maximum heat capacity rate of the two air streams. for un-mixed cross flows, it can be expressed as [11] [12] moisture transfer is more complicated due to the couplings between moisture transfer and operating conditions, as well as membrane materials. by clarification of moisture resistance in membrane, a form similar to eq. (14) for latent effectiveness has been proposed and validated by the authors as e l ¼ 1 à exp expðàntu 0:78 l m_ min =m_ max þ à 1 ntu à0:22 and and where k s is convective mass transfer coefficient in supply side (kg m à2 s à1 ); d the membrane thickness, and d wm is the water diffusivity in membrane (kg m à1 s à1 ); c 0 and w max are constants in sorption curves, which determine the equilibrium between the membrane and moisture at its surface by where u is the moisture uptake in membrane at two surfaces (kg kg à1 ). the convective mass transfer coefficient k can be estimated from convective heat transfer coefficient by a sensible effectiveness of 0.8 is easily obtained with a membrane system, and the latent effectiveness is about 0.9 of the sensible effectiveness according to a case study in [11] [12] . fan energy is an important factor in the annual energy consumption of an hvac system. fan (pump) performance can be characterized by its efficiency, which itself is dependent on operational air flow rate. mostly, rated volumetric flow rate, pressure rise and efficiency are available from the manufacturer. then rated power can be calculated as where v a is air (water) volumetric flow rate (m 3 /h), dp is total pressure rise (pa), h fan is fan (pump) efficiency. the simulations are conducted on an hour-by-hour basis. the air flow rates are dictated as 45kg/h per person. the operating hours are from 9:00 to 18:00. fan efficiency is selected as 0.6. for the convenience of comparison, energy consumed in the form of electricity is converted to primary energy by a factor of 3.3. in addition, two specific programs was written to calculate the psychrometrics and the thermodynamic properties of r134a. it is based on a cubic equation of state [13] . the calculations are on a per person basis. it is recommended that each person has 45 kg/h of fresh air. the cop varies with both the evaporating temperature and the condensing temperature. the influence of the condensing temperatures on the cop is shown in fig. 4 . as can be seen, the cop decreases with increasing condensing temperatures. when the condensing temperature increases from 20 to 50 8c, the system cop decreases from around 8.0-2.5. following correlation could be formulated for the relation for the refrigeration evaporating at 5 8c: the effects of the evaporating temperature on the cop are shown in fig. 5 . the system cop rises with increasing evaporating temperatures. in fact, when the evaporating temperature increases from à10 to 25 8c, cop is improved from 2.0 to 6.5. a correlation has been formulated for the analysis of the system performance: cop ¼ 0:0001t 3 e þ 0:0016t 2 e þ 0:0746t e þ 2:6377 (25) where the condensing temperature is fixed as 45 8c. fig. 6 shows the distribution of the cop of the refrigeration system during a year. as indicated, in winter, the system has higher performance, in contrast, when it's hot in summer, the system cop decreases, which will in return deteriorate the energy requirements. in the analysis, the system is used to treat the latent load solely, and the sensible load of the room is around 50 w/m 2 , which will be extracted by chilled-ceiling panels. results indicate that the annual total energy requirement is 4.15 â 10 6 kj per person. in comparison, an air dehumidification with no energy recovery measures would consume more than 6.2 â 10 6 kj per person of primary energy. in other words, energy saving is 33%. considering local primary energy price of 0.007usd/mj, the annual economic return for each person would be 15usd, which is not a small amount, considering china is still a developing country and the energy price is low. with oil prices growing very fast now, we believe the savings potential could be higher in future. fig. 7 plots the primary energy requirements by the dehumidification system for each person in each month in a year. in hot and humid regions like guangzhou, air dehumidification is required most of the year. the energy values vary from 1.5 â 10 5 to 4.3 â 10 5 kj per month per person. in january and february, when it is the dry season, energy requirements are the least. in august, it is the most humid season, required energy is the largest. in office buildings, the moisture load from ventilation air comprises almost all the moisture load. the percentages of the energy required in each month to the annual total energy are shown in fig. 8 . five months from may to september account for 60.5% of the yearly energy load. other 7 months account for the rest 39.5%. south china has a long hot and humid summer. the energy for air dehumidification is substantially large. to save energy while ensuring a healthy built environment with enough fresh air ventilation, an independent air dehumidification system with energy recovery is necessary. concerns on indoor air quality have prompted the researches of novel air dehumidification techniques. this study proposed a new air dehumidification system in which a mechanical dehumidification is combined with a membranebased enthalpy exchanger. an hour-by-hour simulation reveals that the novel independent air dehumidification system could save 33% of primary energy. with this system, the annual total primary energy used for independent air dehumidification is around 4.2â10 6 kj per person. standard 62-1999, ventilation for acceptable indoor air quality economic toll of sars, china youth daily dehumidification equipment advances energy savings potential of chilledceiling combined with desiccant cooling in hot and humid climates indoor humidity behaviors associated with decoupled cooling in hot and humid climates thermal comfort and energy consumption of the radiant ceiling panel system, comparison with the conventional all-air system cyrano: a computational model for the detailed design of plate-fin-and-tube heat exchangers using pure and mixed refrigerants numerical simulation of a variable speed refrigeration system thermal environmental engineering energy estimating and modeling methods membrane-based enthalpy exchanger: material considerations and clarification of moisture resistance effectiveness correlations for heat and moisture transfer processes in an enthalpy exchanger with membrane cores thermophysical properties of 1,1,1,2-tetrafluoroethane (r134a) this project 50306005 is supported by national natural science foundation of china. this project is also supported by the natural science foundation of south china university of technology. key: cord-032466-1nfp1hcs authors: gong, liang; söderlund, henrik; bogojevic, leonard; chen, xiaoxia; berce, anton; fast-berglund, åsa; johansson, björn title: interaction design for multi-user virtual reality systems: an automotive case study date: 2020-09-22 journal: procedia cirp doi: 10.1016/j.procir.2020.04.036 sha: doc_id: 32466 cord_uid: 1nfp1hcs virtual reality (vr) technology have become ever matured today. various research and practice have demonstrated the potential benefits of using vr in different application area of manufacturing, such as in factory layout planning, product design, training, etc. however, along with the new possibilities brought by vr, comes with the new ways for users to communicate with the computer system. the human computer interaction design for these vr systems becomes pivotal to the smooth integration. in this paper, it reports the study that investigates interaction design strategies for the multi-user vr system used in manufacturing context though an automotive case study. due to the fast development in the domain of communication and an ongoing trend of digitization and digitalization, manufacturing enterprises are facing important challenges in today's market environments: a continuing tendency towards reduction of product development times and shortened product lifecycles. in addition, there is an increasing demand of customization, being at the same time in a global competition with competitors all over the world. this trend, which is inducing the development from macro to micro markets, results in diminished lot sizes due to augmenting product varieties (high-volume to low-volume production) [1] . to cope with this augmenting variety as well as to be able to identify possible optimization potentials in the existing production system, it is important to have a precise knowledge of the product range and characteristics manufactured and/or assembled in this system. in this context, the main challenge in modelling and analysis is now not only to cope with single products, a limited product range or existing product families, but also to be able to analyze and to compare products to define new product families. it can be observed that classical existing product families are regrouped in function of clients or features. however, assembly oriented product families are hardly to find. on the product family level, products differ mainly in two main characteristics: (i) the number of components and (ii) the type of components (e.g. mechanical, electrical, electronical). classical methodologies considering mainly single products or solitary, already existing product families analyze the product structure on a physical level (components level) which causes difficulties regarding an efficient definition and comparison of different product families. addressing this virtual reality (vr) technology is attracting increasing attention in various industries during the past decade. it is the result of the ever-matured vr technology as well as the great potential to improve existing practices in different industries. the manufacturing industry is among those that actively pushing to find out the applications of using vr. there are growing studies shows that much effort are spending on the vr applications in the manufacturing industry [1][4] . it has shown that vr technologies have great advantages to improve areas like factory layout planning, product design, training, etc., especially for globally distributed manufacturing companies that have different functional team located in different parts of the world [3] , [5] [8] . however, while the studies have showed the promising future of vr usages in manufacturing, they have also pointed out the new challenges that are hinder the vr integration process [7] , [9] , [10] . the usability related issues of vr systems is one of the most important to be addressed, as it would affect end users acceptance, which would ultimately affect the process of vr integration in manufacturing. these issues are resulted from the new interaction mediums that comes with the vr systems. different from the "window, icon, menu, and pointing device" (wimp) or direct manipulation interaction style, vr systems use handheld controllers, haptic devices or even voice and gesture recognition for users to interact with the systems. jacob et al. pointed out that there is a lack of established interaction design principles and frameworks for post-wimp systems [11] . in this study, we have chosen a globally distributed manufacturing company as the case to study the different possibilities of interaction design approaches for the multi-user vr system used in the design review process. two iterations of vr system development and evaluation have been conducted. virtual reality (vr) technology is attracting increasing attention in various industries during the past decade. it is the result of the ever-matured vr technology as well as the great potential to improve existing practices in different industries. the manufacturing industry is among those that actively pushing to find out the applications of using vr. there are growing studies shows that much effort are spending on the vr applications in the manufacturing industry [1][4] . it has shown that vr technologies have great advantages to improve areas like factory layout planning, product design, training, etc., especially for globally distributed manufacturing companies that have different functional team located in different parts of the world [3] , [5] [8] . however, while the studies have showed the promising future of vr usages in manufacturing, they have also pointed out the new challenges that are hinder the vr integration process [7] , [9] , [10] . the usability related issues of vr systems is one of the most important to be addressed, as it would affect end users acceptance, which would ultimately affect the process of vr integration in manufacturing. these issues are resulted from the new interaction mediums that comes with the vr systems. different from the "window, icon, menu, and pointing device" (wimp) or direct manipulation interaction style, vr systems use handheld controllers, haptic devices or even voice and gesture recognition for users to interact with the systems. jacob et al. pointed out that there is a lack of established interaction design principles and frameworks for post-wimp systems [11] . in this study, we have chosen a globally distributed manufacturing company as the case to study the different possibilities of interaction design approaches for the multi-user vr system used in the design review process. two iterations of vr system development and evaluation have been conducted. available online at www.sciencedirect.com virtual reality (vr) technology is attracting increasing attention in various industries during the past decade. it is the result of the ever-matured vr technology as well as the great potential to improve existing practices in different industries. the manufacturing industry is among those that actively pushing to find out the applications of using vr. there are growing studies shows that much effort are spending on the vr applications in the manufacturing industry [1][4] . it has shown that vr technologies have great advantages to improve areas like factory layout planning, product design, training, etc., especially for globally distributed manufacturing companies that have different functional team located in different parts of the world [3] , [5] [8] . however, while the studies have showed the promising future of vr usages in manufacturing, they have also pointed out the new challenges that are hinder the vr integration process [7] , [9] , [10] . the usability related issues of vr systems is one of the most important to be addressed, as it would affect end users acceptance, which would ultimately affect the process of vr integration in manufacturing. these issues are resulted from the new interaction mediums that comes with the vr systems. different from the "window, icon, menu, and pointing device" (wimp) or direct manipulation interaction style, vr systems use handheld controllers, haptic devices or even voice and gesture recognition for users to interact with the systems. jacob et al. pointed out that there is a lack of established interaction design principles and frameworks for post-wimp systems [11] . in this study, we have chosen a globally distributed manufacturing company as the case to study the different possibilities of interaction design approaches for the multi-user vr system used in the design review process. two iterations of vr system development and evaluation have been conducted. the research process as well as the findings are presented and discussed in the rest of the paper. vr technology is not new; the first vr system was successfully implemented with a hmd that presents a user with stereoscopic 3d view slaved to a sensing device, which tracks the user's head movement [12] . ever since then, research effort are continuing spending on this area. korves and loftus described vr systems based on the different setups and thus categorized them as:  desktop system.  wide-screen projection system.  immersive cave system.  immersive vr system using hmds [13] . thanks to the latest advancement in hardware and software of vr devices, immersive vr system with hmds is becoming more and more viable in many industries, thus it again attracted much attention in academia and industry during the last 10 years. in this emerging domain of vr, there are many studies on the different issues of integrating it to the manufacturing process. most of them have shown that vr can improve existing practice in the manufacturing industry. for example, wiendahl and harmschristian have shown that immersive vr is an important tool for collaborative factory planning, especially when multiple viewpoints of users are visualized [14] . menck stated that the vr based collaborative planning tool can extend the communication and facilitate cooperation beyond existing organizational boundaries, which would reduce the complexity of work and increase the work efficiency [15] . at the same time, some studies also pointed out that the vr systems is also bringing new challenges for both developers and end users [10] , [16] , [17] . those challenges can be categorized into two major groups. one is associated with creating the realistic virtual environment. the challenge lies on the complexity of generating realistic enough models that can represent the real world so that enough context is provided to perform the intended tasks in vr. this involves the data source integration and data compatibility issues when reuse data already existed for other purposes in vr [18] [20] . another is the usability related issues of vr systems. it is reported that some early users of vr systems have difficulty understanding the interaction logic and some even feel motion sick [21] , [22] . instead of the already established style of human computer interaction for desktop or mobile application, the vr interaction design is new and need further studies to develop supporting guideline and framework. another worth noting point is that, most reported vr systems are designed for single user. in the real world, multiuser engagement is the norm for most activities. therefore, one argument is that if it is not multi-user, then it is not virtual reality. based on the above description, this study has chosen the immersive vr system with hmds as the basic setup to study the interaction design of multi-user vr systems for manufacturing. an automotive company was selected as the case to study how interaction design would affect manufacturing companies from adopting the multi-user vr system. the company's research and development department is located in gothenburg of sweden, while the manufacturing plants are situated across different regions of china. it makes the selected company a perfect case for the purpose of this study. the team that was actively involved in this study was the manufacturing engineering (me) team. they are mainly responsible for developing all body-in-white (biw) process. therefore, the industrial base of the study was set as fixture design and review process using multi-user vr system. the first step to develop the multi-user vr system is to gather the data needed for the intended task. for the purpose of this study, we used two types of data sources: cad models of the new fixture design and point cloud data of the factory environment. the cad models are created by me team using catia tm [23] in the format of jt. however, as the jt files contains many irrelevant information for the vr scenario such as the internal structure of the fixture, the provided jt files were further optimized using pixyz [24] by removing unnecessary surfaces and converting the files to the fbx format which can be directly imported to the vr development platform. the point cloud data was captured onsite of their chinese manufacturing plants using 3d laser scanning technology. it is a realistic version of virtual representation of the real factory environment. studies have shown the point cloud environment provides more accurate contextual information compare to the simplified version of the completely computer generated cad models. the disadvantage of point cloud data is that there is no surfaces or triangle meshes, but millions of points, which requires excessive computing power to rendering them in real time for vr system. it is also difficult to interact with the point cloud data in vr due to this limitation. therefore, the point cloud data was processed with adjusted level of density based on the area of interest. simple surfaces such as floor and walls were also meshed using mathematic algorithm. unity3d [25] was chosen as the development platform to integrate different data sources and program the needed functionality. the unity plugin pun2 [26] was used to handle the network and synchronization for different users. htc vive headsets were used as the vr hardware throughout the study. in this study, two versions of the multi-user vr system were developed in sequence. the pilot version aimed at testing the feasibility of the multi user concepts as well as getting some feedback of the desired functionalities for the second version. therefore, minimal effort were spent on user interface design, but focus on making the connection reliable and synchronization with low latency. the vr view is shown in fig. 1 . a second version was later developed based on the feedback gathered through the pilot workshops with additional features such as customized user avatars. moreover, two sets of interaction design approaches were implemented to further understand user's preference in multi-user vr system. in addition to the common practice that uses 3d trackable controller to trigger different functionalities in vr (illustrated in fig. 2 ), 2d graphic user interfaces (gui) were added on top of the projected screen so that conventional mouse and keyboard can also be used to activate the same functionalities (illustrated in fig. 3 ). the pilot version were tested in two occasions. it was first tested for the cross continent connection from gothenburg in sweden with fellow colleagues in national institute of standards and technology (nist) in united states. this test was to verify the stability of the connection and the quality of real time synchronization of both objects and audio. after the verification, it was later tested in a workshop hosted at university site in sweden. five engineers from the case company as well as two senior researchers from fraunhofer-chalmers centre (fcc) participated in the workshop. all participants tested the multi-user vr system in pairs and performed the intended design review task in vr. after the hands-on experience with the system, a following-up discussion was held to get feedback and potential improvement for the second version. the second version was developed based on the feedback collected from the pilot workshops. then it was tested in case company's facility in gothenburg. there were 14 participants from different groups of the case company joined the test. the participants' role ranging from process developer to vice president of the case company. a tutorial session was held to introduce the study and the basic functions of the multi-user vr system. then all participants tested it in pairs to perform the design review task (illustrated in fig. 4) . after the test, each participants filled in a scale-rating questionnaire regarding their general experience of the interaction design and a semistructured interview was held to collect qualitative data for better analysis of the rating result. the two tests have shown that the multi-user vr system can provide enough context for relevant engineers from different sites to collaborative perform the fixture review process in vr. the connection is stable while objects of interest and audio synchronization are reliable. the scale-rating questionnaire are a series of statements for the participants to rate based on their experience. the results are analyzed with the qualitative feedback from the semistructure interview. a portion of the questionnaire results are illustrated in fig. 5 . with the statements listed in the vertical axis. it shows that all participants believe this type of multi-user vr system would be great benefit for their daily work. the customized user avatars allow users to easily identify their colleagues in vr, therefore increased their feeling of being presence in the virtual environment. most participants still prefer controller interaction than the 2d gui. but when there is an admin role that supervise the vr review session from the projected screen, then it would be preferable to have the 2d gui for the admin to control the review process. when it comes to the question whether all users should have the same functions, the view are diverse. while 10 out of 16 believe the point cloud data make the virtual model more realistic, some questioned the necessity, especially for design review tasks that are independent from the factory environment. in this study, a multi-user vr system was developed to support the design review process for globally distributed manufacturing companies. we focused on the interaction design of such system through two iterations of development and evaluation. it has affirmed interaction design of vr systems is of great importance for manufacturing companies to widely adopt and benefit from the latest advancements of vr technologies. it is generally believe that the quality of the vr environment is pivotal to the user experience, which would affect whether vr systems can be widely adopted and used in manufacturing companies [27] [29] . to put it another way, previous findings emphasis the importance of creating realistic virtual environment to attract users to switch from conventional work procedure to vr. the feedback we collected partly affirm it. as features such as customized user avatars and point cloud factory environment, which would improve the realistic level of the vr environment, received positive ratings from the participants. however, the test result also hinted that not all the design review work would need those features. another aspect that need to consider is that those features would take longer time and much more effort to implement. therefore, it should be judged by the individual cases whether to add those qualityimprovement features in the vr systems. the new interaction medium brought by the vr system is a challenge for both developer and end users. today, most people are used to the wimme interaction from the personal computer as well as touch screen in smart phone and tablets. due to the specific setup with hmd in vr systems, it is impossible to transfer the matured interaction design approaches from those platforms directly to the vr world. instead, handheld controllers with multiple buttons and various haptic devices are created to support the interaction in vr. however, these devices and interaction logic are new to most of the users. previous studies have shown that this might be one of the hinder for vr usage [11] , [30] , [31] . the result of this study shows that the new interaction devices did make it difficult for new users to engage in the tasks at the beginning, but it also shows that going backward to find ways of using those matured medium in vr is not a favorable option either. most users expressed the idea that they can learn the new ways rather fast and the tests also proved that most of them managed to use the different functions smoothly at the end. however, there is no established knowledge or standards that they can be referred to when they were first presented with a vr system. this should be improved by joint effort from academia and vr industry to further study and establish uniform framework for interaction design in vr. additionally, technologies such as voice and gesture recognition are becoming increasingly viable with the latest development in deep learning and artificial intelligence (ai) [32] . many practitioners and researchers are starting to integrate those new technologies to explore new ways of user interaction in the vr [33] , [34] . due to the complex nature of manufacturing environment, there might be some negative factors such as the noise level of factory are too high for voice recognition. it is still worth the effort to have future studies that can explore voice and gesture interaction for vr systems used in the manufacturing industry. this study has set out to investigate appropriate interaction design approaches for multi-user vr system used in the me process. a multi-user vr system was developed and evaluated to in two iterations with academia and industrial partners. the results indicates that multi-user vr system can complement and improve the existing me processes. new interaction medium comes with vr system is a challenge for the wider usage in manufacturing, but also an opportunity to further develop principles and standards of interaction design so that the manufacturing companies can also enjoy then benefit brought by the latest advancement of vr technologies. an immersive and collaborative visualization system for digital manufacturing introducing quantitative analysis methods into virtual environments for real-time and continuous ergonomic evaluations a novel facility layout planning and optimization methodology adaptation of high-variant automotive production system using a collaborative approach virtual manufacturing as a way for the factory of the future the use of virtual reality techniques during the design process: from the functional definition of the product to the design of its structure improvement of manufacturing processes with virtual reality-based cip workshops virtual and augmented reality technologies for product realization virtual factory: an integrated framework for manufacturing systems design and analysis virtual reality applications in manufacturing industries : past research , present findings , and future directions reality-based interaction: a framework for post-wimp interfaces a head-mounted three dimensional display the application of immersive virtual reality for layout planning of manufacturing cells virtual factory design -a new tool for a co-operative planning approach collaborative factory planning in virtual reality a survey on human-computer interaction in mixed reality state of the art of the virtual reality applied to design and manufacturing processes rationalizing virtual reality based on manufacturing paradigms virtual reality-based approach to manufacturing process planning virtual reality approaches for immersive design virtual reality and 3d imaging to support collaborative decision making for adaptation of long-life assets real walking in virtual environments for factory planning and evaluation catia 3d experience pixyz software what is unity? photon unity networking framework for realtime multiplayer games and applications a novel vr tool for collaborative planning of manufacturing process change using point cloud data a fast parametric deformation mechanism for virtual reality applications virtual plastic injection molding based on virtual reality technique immersive authoring of tangible augmented reality content : a user study tangible user interfaces: past, present, and future directions deep learning-based human motion recognition for predictive context-aware human-robot collaboration mr : an interdisciplinary framework for mixed reality experience design and criticism cognitive load by context-sensitive information provision using binocular smart glasses in an industrial setting this work is funded by vinnova (swedish agency for innovation systems) through the summit project. this work is also within the sustainable production initiative and the production area of advance at chalmers university of technology. the support is gratefully acknowledged.i would also like to express my sincere thanks to all the medical staff that are fighting with the covid-19. keep safe and victory is ours! key: cord-353787-24c98ug8 authors: jackson, j. a. title: immunology in wild nonmodel rodents: an ecological context for studies of health and disease date: 2015-04-27 journal: parasite immunol doi: 10.1111/pim.12180 sha: doc_id: 353787 cord_uid: 24c98ug8 transcriptomic methods are set to revolutionize the study of the immune system in naturally occurring nonmodel organisms. with this in mind, the present article focuses on ways in which the use of ‘nonmodel’ rodents (not the familiar laboratory species) can advance studies into the classical, but ever relevant, epidemiologic triad of immune defence, infectious disease and environment. for example, naturally occurring rodents are an interesting system in which to study the environmental stimuli that drive the development and homeostasis of the immune system and, by extension, to identify where these stimuli are altered in anthropogenic environments leading to the formation of immunopathological phenotypes. measurement of immune expression may help define individual heterogeneity in infectious disease susceptibility and transmission and facilitate our understanding of infection dynamics and risk in the natural environment; furthermore, it may provide a means of surveillance that can filter individuals carrying previously unknown acute infections of potential ecological or zoonotic importance. finally, the study of immunology in wild animals may reveal interactions within the immune system and between immunity and other organismal traits that are not observable under restricted laboratory conditions. potentiating much of this is the possibility of combining gene expression profiles with analytical tools derived from ecology and systems biology to reverse engineer interaction networks between immune responses, other organismal traits and the environment (including symbiont exposures), revealing regulatory architecture. such holistic studies promise to link ecology, epidemiology and immunology in natural systems in a unified approach that can illuminate important problems relevant to human health and animal welfare and production. recent technological advances in the de novo sequencing and analysis of nucleic acids are revolutionizing the measurement of gene expression in nonmodel organisms, with promising applications in the study of the immune system (1) . although other phenotypic measurements of immunity remain relevant and useful, albeit limited in scope or technically difficult to apply in nonmodel organisms (2) , these advances mean that studying the immunology of such organisms in the natural environment has become easier and can take on a genomewide perspective embodied, for example in techniques such as rnaseq. this can, in turn, be accompanied by powerful analytical approaches derived from systems biology (3) and statistical methodologies applied in ecology. when these elements are combined with the monitoring of natural fluctuation or experimental perturbation, it opens up the possibility of 'reverse engineering' the regulatory architecture of the immune system and its interaction with other organismal traits and with natural environmental pressures (3) . such approaches, using natural systems, complement the strengths and weaknesses of modern immunology (4) . here, the great strengths are derived from the very refined use of inbred and genetically manipulated mice under controlled conditions that negate environmental variation. this is very successful for unpicking the structure of molecular pathways and workings of cellular populations, but relevance for natural environmental variation disappears where genetically unrepresentative individuals are studied under homogenous laboratory conditions and in the absence of a natural flora and fauna of symbionts (4, 5) . (here symbiont is defined as any organism involved in an intimate association with the host, including parasitic, commensal and mutualistic associations.) the present review will be concerned with how this 'blind spot' in modern immunology can be addressed by a focus on natural populations. it will scan the horizon for unique ways in which studies of nonmodel rodents can contribute to our wider understanding of the biology of the immune system and the way it interacts with the environment to determine health. additionally, it will consider how immunological measurement, interpreted in the light of paradigms from laboratory mouse immunology, can define individual variation relevant to ecological and epidemiological studies of infectious disease in the natural environment (1, 6) . rather than produce an exhaustive list of possible interests, though, this review will concentrate on three broad reasons to study immunology in naturally occurring vertebrate hosts, reasons that seem particularly exciting because they could have major practical implications for human health and the welfare and productivity of domesticated animals. each of these themes will be considered in turn and then the reasons why nonmodel rodents (species excluding mus musculus, m. domesticus and rattus norvegicus) may be particularly useful models. finally, selected case studies will be discussed that have begun to approach some of the issues raised. whilst the focus in these case studies is on the measurement of expression in selected candidate genes, they also illustrate the potential for future work using broader transcriptomic approaches, such as rnaseq. much of the ill-health experienced in modern human populations is not caused directly by infectious agents but linked to aberrant inflammation (7) . thus, conditions including cancer (8) , diabetes (9) , asthma and allergies (10) and various neurological (11, 12) and psychiatric disorders (13) have, in part, immunological aetiologies. trends in these immune-based conditions have been, broadly, upward in 'westernized' human populations, the short time scale indicating the involvement of an environmental variable or variables (14) associated with 'modern' anthropogenic environments. in addition, the effects of environmental factors on individuals are likely to be modulated by genetic variation inherited from wild ancestral populations (15, 16) amplifying individual variability in propensity to disease. a major challenge facing biomedical science, then, is to pinpoint the environmental causes for this variance in health. in particular, given the observed epidemiological trends in immunologically based disease, the questions might be asked: what are the immunological changes that occur in the transition from natural to more anthropogenic environments, and what triggers these changes? several causal mechanisms have been considered, but to parasite immunologists one particularly influential and intuitive (but unproven) line of thought is that increased dysregulation of the immune system is ultimately caused by the host's co-adaptation to stimuli from co-evolved symbionts. these symbionts might include macroparasites and other agents of chronic infection that tend to be lost in anthropogenic environments (17) . this idea is embodied in what has been termed the 'hygiene' (5) or 'old friends' (13) hypothesis. surprisingly, modern immunology is not well placed to take up the challenge of identifying real-world environmental drivers of the immune phenotype. this is because the remarkable laboratory models that have been established for revealing the molecular details of immunological pathways are unsuited to studying how these pathways interact with complex environments under natural conditions. it has often been noted that the genetics of laboratory mice does not reflect the natural situation (4). typically, laboratory lineages are partly or completely inbred and often generated in a haphazard way that makes the range of allelic variation fixed in their genomes unrepresentative of natural variation (18) . in the case of the fully inbred (isogenic) lines, their genomewide homozygosity is itself highly unnatural. whilst these disadvantages would perhaps be overcome (19) by a range of carefully generated wild-derived inbred and outbred lines (20) , a much less tractable limitation lies in the inability to recreate natural environmental influences in the laboratory (5) . thus, wild animals experience a range of complex symbiont exposures and environmental stressors that cannot be sufficiently replicated in captivity (5) . as such, wild mammals (and especially wild nonmodel rodents, due to some of the advantages discussed below) would seem a natural starting point to approach the problem of identifying environmental stimuli that drive immunological development and homeostasis in the wild. as previously considered in more detail by friberg et al. (5) , progress could be made either through in situ studies in natural populations tracking the effects of environmental variables using manipulative experimental or observational approaches (see for example, the wood mouse case study below), or through transplantation of naturally occurring lineages to (and monitoring of the changes occurring in) experimentally manipulated anthropogenic environments. not all humans in modern environments develop immunologically based diseases (even though increasing numbers do), and those that succumb often have identifiable genetic predispositions. as noted above, causative environmental factors likely exert their effects upon a background of significant immunogenetic variability inherited from wild ancestral populations. the subject area of wild rodents as models for this immunogenetic variability was reviewed in detail by turner and paterson (15) and will only be considered here sufficiently to provide a general overview relevant to the present article. briefly, a parallel challenge to the one of identifying environmental factors driving immunopathological phenotypes in anthropogenic environments described above, then, is the one of revealing genetic variation that places individuals at risk (15) . in other words, finding genetic variation that natural selection has shaped in a way that, although adaptive in some natural settings, has the potential to be maladaptive in anthropogenic environments. such variation results from balancing selection, or from directional selection or genetic drift that has not proceeded to fixation, in ancestral populations. here, selective agents (for example, naturally occurring pathogens) that were present in the past, or neutral processes, may have driven into wild populations alleles that are broadly deleterious in novel anthropogenic environments. where potentially deleterious genes have been fixed, though, only variation due to environmental factors (see section above) or the wider genetic background is important. immunogenetic polymorphism driven by balancing or directional selection is thought to cause at least some of the variation underlying immunopathological conditions in modern humans (21) (22) (23) (24) (25) (26) and could, equally, be responsible for a great deal of natural variation in wild animals. although it is increasingly recognized that adaptive evolution has structured the polymorphism in genes controlling the vertebrate immune system (26) (27) (28) , our knowledge of the dynamics of this selection is still rudimentary. crucially, the types of genes and pathways that tend to undergo balancing selection, or frequent intermittent directional selection, in the natural environment are poorly known. also, the nature of the selection involved and the phenotypic manifestations of the polymorphisms are not well understood. studies of wild populations (29) (30) (31) (32) (33) are pivotal in this regard, as the wild is the only place in which natural selection, and the phenotypes upon which it operates, can be measured. furthermore, given the conservation of the mammalian immune system, it seems likely that similar genes and pathways may be the target of pathogen-mediated balancing selection across taxa. future studies into the causes of immunogenetic variation (and the characterization of the associated phenotypes) in natural populations are thus likely to feed insights into the biomedical and veterinary fields through focussing attention on the types of gene predisposed to drive immunopathology due to ancestral adaptive evolution. another goal of immunological studies in wildlife is to provide an improved understanding of the dynamics of infection in natural populations (6, 34) . and rodents are of particular interest in this regard, given their ecological importance and role as reservoirs for many zoonotic infectious agents (35, 36) . in general, the dynamics of zoonotic and other ecologically important infectious diseases are likely to be influenced by heterogeneities amongst individual hosts (37) , which will affect disease severity and transmission. as the immune system is the host's defence against infectious agents, variation within it is likely to generate much of this heterogeneity (32, 38) . the measurement of immunological variation in real ecological contexts may, then, allow us to better define individual heterogeneity, and moreover, to address its environmental causes and epidemiological consequences (1, 39) . this could help anticipate infectious disease risks in the environment. for example, such information may help to identify variations in immune defence that occur in time [e.g. seasonal (40) (41) (42) ] or space [e.g. habitat-specific or along invasion fronts (43, 44) ] and that alter susceptibility to infectious agents; or it may help identify species (45, 46) , or subsets within populations (47) , whose immunological profiles indicate an increased likelihood to serve as permissive reservoir hosts for certain pathogens. for example, studies on variability in the expression of tnfa and mx2 genes in bank voles (myodes glareolus) have suggested the possibility of environmentally driven landscape-level patterns that may affect the epidemiology of zoonotic puumala hantavirus (47). as the most numerous group of mammals (~1500 species) the rodents represent, through sheer weight of numbers and their wide distributions, very significant potential reservoirs for emerging zoonotic infections. much emphasis has recently been placed on emerging and re-emerging infectious diseases from wildlife reservoirs [for example, the one health initiative (48) ] and the importance of undertaking pro-active monitoring programmes. in addition to infection risk for humans and domesticated animals, it is likely that epidemic and endemic infectious disease may also contribute significantly to the dynamics of wildlife themselves and indirectly to higher level ecosystem functioning. discovery of infectious agents of ecological importance, or that present a risk of zoonotic emergence is, however, limited by the fact that diagnostic techniques are specific to individual pathogens, or groups of pathogens. whilst next-generation sequencing (ngs) approaches are becoming available that allow very broad nonspecific surveys for pathogen sequences, these are costly and many individuals would have to be processed were a population to be sampled randomly. given this, the detection of potential emergent infectious agents in wildlife could, in some cases, be facilitated by the monitoring of immunological expression (49) . this would narrowly focus attention on individuals with aberrant immunological profiles that might be indicative of infection states. for example, these individuals could then be selected for further ngs studies (50, 51) in order to detect pathogen-specific sequences correlating with the aberrant immune expression profiles. whilst endemic reservoirs may be highly co-adapted with their pathogens and be relatively asymptomatic (52) (perhaps without anomalous immune expression profiles), it is sometimes the case that infectious agents emerging from wildlife reservoirs may do so via an intermediate 'amplifier' host that does succumb to significant disease. such hosts may shed more infective particles into the environment than the endemic reservoir and also show signs of acute immune responses. for example, putative amplifier hosts may have been involved in the emergence of the sars coronavirus. here, the ultimate natural reservoir appears to include horseshoe bats (rhinolophus spp.), but the infection was likely transmitted to human hosts indirectly via other wild mammals, including the palm civet (paguma larvata) (53), which is highly susceptible to the virus and sheds high titres of infective particles (54) . in scenarios where the aim is to identify emergent disease risks before any infectious agent is specifically identified, immune expression studies may provide a way to filter potential diseased amplifier individuals from natural populations and focus attention on these for further study. it is also likely that the pattern of immune expression may be indicative of the type of pathogen involved [as is increasingly being exploited in medical diagnostics (55) ] and that this may help target subsequent efforts at identification. studies in wild nonmodel rodents may also yield unexpected general insights into the fundamental biology of the mammalian immune system. thus, the laboratory model of mouse immunity cannot properly address many aspects of environmental variation seen in nature, and studies in humans are also limited in this way and by what tissues may be sampled or experimental approaches undertaken. on the other hand, immune responses in wild animals, once they can be interpreted using post-genomic and systems biology approaches, may reveal functional pathways and interaction networks that were not previously understood, precisely because they relate to stressful environmental conditions that cannot ethically be replicated in laboratory or domestic animals, or in humans. an example where a focus on natural populations may feed-back insights into general immunology is given in the section below dealing with immunodynamics in field vole populations (56) . included in the fundamental biology of the immune system might be the costs of immunity embodied in classical ecological immunology (16, 57) . this revolves around the concept of trade-offs: that immune responses impose a penalty in the form of competition for energy allocation to, or functional interference with, other life-history traits. understanding these costs, and the interaction of immunity with other organismal traits, is an active and exciting area of research (16) that may generate insights relevant to human health and biomedical science. there is also relevance to agriculture, where production traits reflecting reproductive or growth parameters might interact with immunity in ways that are not yet fully appreciated. whilst many advances have been made in the field relating to costs of immunity (16) , further studies in natural populations using the type of holistic, genomewide approach possible with transcriptomic methods hold the promise of further advances. why not just well-studied laboratory or farmed species? some studies may necessarily focus on particular species or local faunas because of specific concerns about infectious disease risks. where more general questions are to be answered, though, an obvious starting point for studies of immune function in natural systems would be to use the wild counterparts of standard laboratory models or other well-studied domesticated species. in particular, house mice (covered elsewhere in this special issue) would seem an obvious choice given their status as a central model in immunology. this would allow the use of robust measurements based on pre-existing antibody reagents (see next section) and would also allow interpretation of immunological patterns in the context of a very detailed organism-specific knowledge base. looking beyond the small number of laboratory and domesticated species, though, several considerations make it advisable to additionally consider other naturally occurring species. firstly, as they occur in the 'wild' today, rats and mice may have patchy population structures that are narrowly focussed on unnatural anthropogenic environments and are the result of a long history of anthropogenically linked dispersal. in the case of the mouse, for example, dispersal is believed to have occurred from the fertile crescent in the near east (58) across most of the globe following the expansion of human civilizations (59) . this history of co-habitation and dispersal means that house mice may harbour symbiont assemblages restricted by lineage sorting (extinction) during dispersal/colonization events and, as invasive organisms, they may have acquired new infections in their relatively recent evolutionary history. in response to selective forces acting during dispersal and gain/loss of infectious threats, such invasive species may also have undergone rapid genetically based changes in immune function (43, 60) . these considerations (lack of a natural symbiont flora/fauna, an unusual evolutionary history of dispersal and frequent occupation of anthropogenic habitats) make rats and mice weaker natural models to assess 'hygiene hypothesis' or 'old friends' ideas. here, where the hypothesis is that immunopathological phenotypes in anthropogenic environments arise from a lack of stimuli from co-evolved symbionts, models are needed where the host co-exists in its natural setting with a complete assemblage of co-evolved symbionts. in contrast to the house mouse and norway rat, common naturally occurring murine and microtine rodent species (e.g. apodemus, myodes and microtus spp. in europe), whose ecology has been well studied, often occur in relatively extensive, evenly distributed and persistent populations. these are likelier to have been stably linked in the long term with natural or quasi-natural habitats and with a diverse, co-adapted symbiont assemblage. transcriptomic studies are facilitated by a previously annotated genome, and this may affect the choice of study species. increasing genomic information is becoming available for several well-studied taxa, including the european species apodemus sylvaticus, microtus agrestis and m. glareolus, and annotated genomes have been assembled for peromyscus maniculatus and microtus ochrogaster in north america. however, transcriptomic studies can be carried out through de novo assembly without a pre-existing species-specific genome (61) and allow the exploitation of almost any species for ecological immunology studies. naturally occurring rodents recently used for ecological immunological studies in other parts of the world include, for example, organisms as diverse as the capybara (hydrochoerus hydrochaeris) (62, 63) or the pallid atlantic forest rat (delomys sublineatus) (64) in south america. in general, naturally occurring rodents represent unparalleled models for work in ecological immunology and immunogenetics. whilst their close relation to the laboratory mouse allows interpretation in terms of modern mechanistic immunology, their high population densities, amenability to longitudinal sampling and experimental manipulation in the field, short generation times, relatively spatially static populations and, ultimately, their adaptability to the laboratory environment, make these organisms uniquely tractable study systems. given the diversity and abundance of rodents, it is likely that most researchers will have, at close hand, naturally occurring rodent systems that may serve as useful ecological models or that are practically relevant as reservoirs of transmissible infectious disease. finally, it should not be forgotten that there is very likely to be value, for its own sake, in carrying out studies across a wide diversity of host systems (16) . this will be useful in revealing the generalities in immune function across species and will also provide insights from the specialized adaptations that individual species use to meet specific sets of circumstances. there is a strong emphasis in modern immunology on the use of antibody reagents against immunological biomolecules for robust molecular phenotypic measurements. gene expression measurements at the mrna level, in comparison, are generally considered a more problematical approach that is 'resorted to' if necessary, particularly in the case of analyses of individual genes by real-time pcr (qpcr). this is based primarily on the fact that the expression of bioactive protein may not always track upstream mrna concentrations (65) . complex kinetics in the pathway between mrna and protein, and the stability of the proteins themselves, can prevent a simple relationship. often there is poor concordance of matched transcriptomic and proteomic data sets across a range of organisms (66) . such a lack of gene-by-gene correlation, though, is much less relevant than the information content that transcriptomic profiles carry in relation to the biological status of the individual. this information content is attested to by the increasing use of transcriptomic approaches in modern biology. more specifically, early indications in wild rodents (1, 47, 56, 64) suggest that gene expression measurements, especially if interpreted with the complexity of post-transcriptional dynamics in mind, do contain much useful information. practical issues restrict the usefulness of antibody-based methods in nonmodel rodents. due to structural variability in many immune molecules (especially canonical cytokines and cell surface markers), the transferability of commercially available off-the-shelf reagents amongst rodents, even within the subfamily murinae, is limited. whilst some commercial assays and reagents may indeed be found to cross-react amongst rodent species (for example, many commercial antibodies targeting immunoglobulins), this approach may involve trialling dozens of others unsuccessfully. this has been the experience of researchers targeting cytokines in the cricetid, peromyscus maniculatus, in north america (67), or even the murine, apodemus sylvaticus, in western europe (49, 68) . thus, developing a broad panel of assays may require the de novo production of monoclonal and/or polyclonal antibodies, which is technically feasible but is costly and time-consuming (4-6), especially given that two separate antibodies may be required per target (if setting up a microplate elisa, for example). a promising and more economical strategy will be to explore further the potential of established and emerging nucleic acids measurement (4, 6, 69) . this approach can focus on panels of candidate genes, selected on the basis of the existing knowledge base for mouse immunological pathways. even more powerfully, rnaseq (70, 71) , because it encompasses the whole transcriptome (10s of thousands of genes, depending on sequencing coverage), largely removes concerns associated with measuring single genes as it allows a focus on whole pathways whose concerted variation is much more likely to reflect the phenotype. this is a technique that, although very expensive per sample, can be used in an unbiased way to identify informative and reliable marker genes that can then be measured by cheaper technologies in larger sample sizes. as an approach directed at the entire transcriptome, it is likely to supersede oligonucleotide microarray methods, due to general technical superiority (72) and, particularly in the case of studies in nonmodel species, the requirement of microarrays for prior sequence information. targeting of single genes (or small panels of genes) may be carried out by qpcr (quantitative real-time pcr, often abbreviated as rt-pcr) or other emerging technologies that measure nucleic acids more directly, for example digital pcr (73) or new developments of microarray-like systems (74) . the latter technologies are likely to be technically more robust than qpcr, which although still invaluable, suffers from sensitivity to variable reaction kinetics amongst sam-ples; however, they are currently more expensive, and in the case of digital pcr less applicable to high-throughput applications. finally, the point should be made that, whatever molecular measurement approach taken (protein or rnabased), this should ultimately be cross-referenced to functional measures of immunity (such as susceptibility to pathogens). in natural populations, this cross-referencing can be achieved either by observational epidemiological studies, or, more powerfully (and with greater difficulty), by manipulative experimental infectious challenges. the advent of genomewide transcriptomic (and other high throughput) measurements arguably allows rapid progress in the study of immunity in natural systems through exploratory, data-led approaches that might loosely be covered by the term 'reverse engineering' (75) . biologists often attempt to explain natural systems using a forwardengineering-like philosophy, where a high level model or concept is used to direct the interpretation of data. although this approach (in some form or other) is likely to remain indispensible and also corresponds to many biologists idea of the basic scientific method, it is vulnerable to arbitrary choice of starting model and is not necessarily the exclusive, or most direct, route to understand complex systems (especially when starting from a low knowledge base). in contrast, in reverse engineering, large sets of responses (as generated by, for example, transcriptomic or multiplexed protein measurements) can be correlated with environmental and organismal variables of interest across perturbations (either natural or experimental), allowing interaction networks to be inferred (3). this can help establish how molecular pathways interact with each other and the environment to generate the observed phenotype. a reverse-engineering-like philosophy has, in part, featured in the examples dealt with below, and although these work with limited panels of measurements, they illustrate the potential for future studies using broader transcriptomic approaches. the immune system has co-evolved with commensal microbes to the extent that it requires cues from these organisms to programme its normal development (76, 77) . moreover, as a result of this, immunopathological phenotypes are to be expected where microbial exposures occur that are outside the parameters within which natural selection has operated during evolutionary history (77, 78) . this developmental interaction between microbiota and immune system is, in part, channelled by toll-like receptors (tlrs) of the innate immune system (76, 79, 80) . the specificities of these receptors (81) and the inflammatory programmes they recruit are essentially directed against microbes. however, recent studies in wild rodents, which will briefly be reviewed here, suggest that natural exposures to macroparasites modify systemic tlr-mediated responses to bacterial molecular patterns. macroparasites, given their widespread occurrence in natural vertebrate populations, may thus be part of an extended co-evolved interaction network, involving commensal microbes and innate antimicrobial responses, which can drive the development and homeostasis of the immune system. cotgrave forest, nottinghamshire a wood mouse population at cotgrave forest, nottinghamshire, was monitored (49, 68, 82) over time, with a series of cross-sectional samples between 2006 and 2008. a range of ex vivo tlr-mediated responses (to defined tlr agonists) were measured in cultured splenocytes from subsets of animals. due to a pattern of positive covariation amongst tlr-mediated tumour necrosis factor alpha (tnf-a) protein responses (tlrs 2, 3-5, 7, 9) measured in the early part of the sample series, and the especially strong associations of tlr2-mediated tnf-a production with measures of macroparasite infection, this last response in particular was chosen to be measured in all samples. focussing on tlr2-mediated response across the whole study period, this was found to be associated with certain macroparasites, especially the gastrointestinal heligmosomatid nematode, heligmosomoides polygyrus, and the blood-sucking ectoparasitic louse, polyplax serrata. the magnitude and direction of the associations, though, changed across the study period. the changing associations corresponded to different environmental conditions following a perturbation (population crash) in the mouse population during the middle part of the study. early in the study, the abundances of mice and macroparasites were high, but following the mouse population crash, the later part of the study was characterized by lower macroparasite abundance. corresponding changes occurred in the association between macroparasites (p. serrata and h. polygyrus) and tlr2-mediated tnf-a responses, with a strong negative coefficient before the perturbation (at high infection levels) and a positive one subsequently (at lower infection levels). complementary laboratory experiments using the mouse-heligmosomoides bakeri model supported a causal effect of nematode infection upon tlr-mediated responses. this model is relevant because h. bakeri is a very close relative (59, 83) of the h. polygyrus occurring in wood mice. previously na€ ıve inbred mice exposed to single (cba, balb/c, c57bl/6, swr) and trickle h. bakeri infections (balb/c, c57bl/6), typically up-regulated tlr responses signalling through myeloid differentiation primary response gene 88 (myd88) at some point during the infection course, usually coinciding with peak standing worm burdens. this is consistent with a permissive effect of myd88 signalling on gastrointestinal nematode infection demonstrated through experiments with myd88 à/à and interleukin one receptor one (il1r1) à/à mice (84, 85) . it is interesting, though, that in the trickle infection experiments, resistance developed in both balb/c and c57bl/6. this contrasts with more permissive infection phenotypes previously achieved under similar experimental regimens, where the susceptible strain c57bl/6 supports chronic infection and can continue accumulating worms to the point of lethality (86) . furthermore, in wild wood mice there is a linear accumulation of h. polygyrus with age and no indication of acquired immunity in the form of abundance as a decelerating function of age indicators (82) . it may also be significant that the laboratory experiments, in initially na€ ıve animals, produced results consistent with the positive abundance -tlr response association seen in the wood mouse population during times of low parasite abundance, but were not consistent with the negative association seen at times of high parasite abundance. the latter negative association (and perhaps the permissive infection phenotypes noted above) could be accounted for by the well-known immunosuppressive effects of chronic heligmosomatid infections. these effects would likely involve adaptive regulatory t-cell responses that could feedback negatively onto innate immune responses (87) . taken together, all of this information indicates that macroparasite infection exposures may have significant and context-dependent effects on the innate responses that mediate interactions of the immune system with microbes. the mechanism for this remains to be determined, but as discussed by friberg et al. (5) , the possibilities include effects on tlr signalling by worms that are direct, via secreted immunomodulators, or indirect, via feedback from adaptive immune responses. other indirect effects could be mediated by altered exposure to microbes or innate damage signals resulting from the activities of macroparasites at their site of infection. there is some reason to believe that one or both of these last mechanisms might be important. thus, heligmosomatid excretory-secretory products (88) , and the regulatory (87) and th2 (89) immune responses that these parasites typically trigger, generally reduce tlr-mediated signalling. in the laboratory experiments, though, heligmosomatid infections actually increased tlr responses (perhaps consistent with activation by tlr ligands, which are primarily microbialor damage-associated patterns). this re-emphasizes the possibility that co-infections with macroparasites contribute to the network of interactions between commensal microbes and the immune system. moreover, due to the epidemiological association of ectoparasitic lice with systemic responses in the cotgrave forest study, it seems that antiparasite responses at peripheral sites beyond the gut (78) may also be involved. the existence of such an extended interaction network is highly relevant to our understanding of how microbiotal exposures programme immunity. because macroparasites are often absent in anthropogenic environments (5) , this may contribute to the disruption of co-evolved interactions (cf. the hygiene hypothesis). thus, studies in a wild system have pointed towards the need for further work to establish the role of natural macroparasite communities in the formation of microbiotal assemblages and the contribution of this to health. background evolutionary fitness in the natural environment is not measureable in the laboratory, and so studies of hostpathogen community dynamics in the wild are essential to fully understand immune responses in their wider context as components of antipathogen strategies that maximize fitness. studies in wild field voles, briefly reviewed below, have aimed to identify distributional infection patterns associated with different antipathogen strategies in natural populations and to link these to expression signatures in immune-relevant genes. such gene expression markers can then be used to track the life-history correlates of different putative strategies and may also give insights into the immunological mechanisms involved. when considering adaptations to infection exposures, two strategies are available to a host and these may often be deployed together, although there may be some emphasis on one relative to the other. these strategies are resistance, where the host prevents infection (denies access to the pathogen), and tolerance, where the host allows access to the pathogen whilst actively mitigating the negative effects of infection. identifying patterns of tolerance and resistance in natural populations is problematical but can be approached using a general framework like that summarized in jackson et al. (56) . in cross-sectional samples ('snapshot' destructive samples of individuals), tolerance in identifiable groups of animals may be measured as the regression slopes (reaction norms) of fitness measures (for example, body condition) against infection load. here there is the problem that, in individuals from natural populations, the infection dose and the time course of infection are not standardized. reaction norms, though, can also be supplemented by consideration of the phase curves (temporal trajectories of fitness in relation to infection load) of infection courses in longitudinally sampled individual animals. these allow a known infection load to be related to fitness measurements at a later time point. in the study briefly reviewed below, both approaches were used, with initial identification of a tolerance-like pattern in cross-sectional samples, which was then corroborated and extended by focussed analyses in longitudinal samples. immune gene expression profiles in field voles at kielder forest, northumberland gene expression measurements are increasingly being used in studies of infectious disease in nonmodel rodent systems (1, 6, 49, 64, (90) (91) (92) . in work carried out in the well-studied kielder voles (m. agrestis) system (39), measurements of a panel of candidate genes (representing different immunological pathways) were taken in sets of cross-sectional and longitudinal samples from individual voles at two sites in each of two seasons (2008) (2009) (2010) . this design aimed to capitalize on the respective strengths of the two sampling modes: the greater range and precision of measurement in destructively sampled 'snapshot' cross-sectional samples (where more tissues can be interrogated and manipulated in the laboratory), also the stronger inference of causality in time series data from repeat-sampled individually marked animals (longitudinal samples). in cross-sectional samples, expression profiles were measured in ex vivo stimulatory assays of cultured splenocytes (with stimulants including tlr2 and tlr7 agonists and mitogen), whilst in longitudinal samples, constitutive expression was recorded in peripheral blood. in addition, a range of infection and condition measures were recorded in the sample animals. early analyses in a partial cross-sectional data set for immune gene expression (2008) (2009) , and without considering pathogen data, suggested the value of the measurement approach through the existence of significant variation of expression in relation to season, life-history stage and individual condition (1) . further analyses, on the full data set, searched for patterns of resistance or tolerance to pathogens. initially focussing on the more detailed cross-sectional data, and using body and organ condition (weight adjusted for standard length) as a fitness measure, it was possible to recognize a predominant pattern indicative of tolerance to macroparasite infection in mature males (where macroparasites accumulated with age indicators and were associated with increasing body condition) and a pattern indicative of resistance in immature males (where macroparasite abundances were decelerating functions of age indicators and not associated with body condition) (56) . although unexpected, the positive association of body condition with macroparasite infection in mature males was in the context of negative changes in other life-history components and not inconsistent with a negative overall impact of infection exposure on fitness. high expression of the transcription factor gata-binding factor 3 (gata3) in mitogen-stimulated splenocytes was found to mark both tolerant animals (amongst mature males) and resistant animals (amongst immature males) in the cross-sectional set. furthermore, analyses of timelagged associations in mature males in the longitudinal data suggested that macroparasite infections triggered gata3 responses [as might be expected in laboratory models (93) (94) (95) ], which in turn gave rise to increases in body condition. this corroborated the cross-sectional analyses and supported a hypothesis that gata3 activity stimulated by macroparasites is part of a complex of (tolerance) responses leading to the readjustment of body condition in mature males. constitutive gata3 expression in peripheral blood also correlated with survival in longitudinally monitored animals, with high relative expression of gata3 predicting poorer survival in younger animals but having a progressively more positive effect on survivorship with increasing age. in this observational field study, the existence of confounding processes that might produce the cross-sectional patterns attributed to tolerance should be considered. indeed, the multifaceted nature of the kielder study, with cross-sectional and longitudinal components providing a range of measurements in different population strata, increases the opportunities for comparing predictions to data. two main confounding processes might be relevant in terms of their potential to generate tolerance-like patterns. one of these is differential mortality (dm): where, amongst animals heavily infected with macroparasites, those in poor condition die more quickly, biasing the average condition of the survivors upwards. another possibility is correlated risk (cr): where parasite load may be linked to good condition because hosts in good condition forage more or range more and encounter more parasite infective stages as a result. the dm and cr scenarios were poorer explanations for the observed patterns from a number of perspectives. under cr, increased condition would precede increased acquisition of parasites and the gata3 responses they trigger; but in temporal series for individual mature males, these two sets of events, in reality, occurred in the reversed sequence. this observed sequence and the direction of association also contradict the prediction of dm of a negative effect of macroparasite infection (and the gata3 responses triggered) on subsequent condition (which follows if infection is a major cause of mortality)in reality there was a positive association. perhaps most importantly, the dm and cr scenarios do not explain the age-specific changes in the relationship between gata3 expression and survival in longitudinal data, or in the relationship between gata3, condition and macroparasite infection in the cross-sectional data. thus, if dm were true, gata3 expression in peripheral blood would be expected to show a consistent negative association with individual survival, especially in classes of animal with an apparent gata3associated tolerance pattern. however, in reality gata3 expression decreases survival in smaller males (where a tolerance pattern is not seen) but tends to increase survival in larger animals (which do show the gata3-associated tolerance pattern). furthermore, if cr were true, better conditioned animals would generally have higher parasite exposure and higher gata3 expression; but in reality, this is only seen in mature males and not in immature males. it might also be argued that a special case of cr, which could explain the latter age-specific pattern, is that better conditioned mature males range more, or have more social contacts, due to increased breeding activity. even in this case, though, the wide-ranging males would be expected to have better testis condition (if undergoing increased behaviours associated with breeding); but in our study, gata3 expression was negatively correlated with testis condition. thus, the details of the study were consistent with a hypothesis of elevated gata3 expression mediating resistance in immature males and tolerance in mature males; there were major inconsistencies with alternative dm and cr interpretations. gata3 is a master transcription factor involved in the differentiation and development of th2 (t-helper type 2 cell) cells (96) and would be expected to mark th2 activity in the splenocyte cultures studied. the seemingly dual aspect of gata3 (involved in tolerance and resistance) is not biologically implausible, given that th2 responses have long been associated with resistance to macroparasite infections in laboratory models but are also linked to wound-healing mechanisms that might be involved in tolerance (97) . it would seem possible, however, that gata3expressing th2 cells might drive different downstream effector responses in resistance and tolerance, and this is one aspect that is worthy of further study. whilst regulatory components of the immune system have previously been considered as possible mediators of tolerance (98) , through their ability to dampen effector responses, this was not supported in the kielder study. thus, the antiinflammatory cytokines interleukin (il) 10 and transforming growth factor beta one (tgf-b1) and the transcription factor forkhead box p3 (foxp3), whose expression characterizes many regulatory t-cell subsets, were measured but were not associated with tolerance patterns. gata3 expression in tolerant males was associated with a complex of life-history readjustments likely to impact fitness (represented by schematic phase curves in figure 1 ). apart from the increase in body condition, there was a decrease in a fecundity indicator (testis condition) and increasing survival as animals aged. possibly, then, the tolerance strategy increases fitness via improvements in residual reproductive value (potential for future reproduction) during macroparasite infection. this is epidemiologically significant, as the transmission of infectious agents may become focussed through tolerant subsets of the population with consequences for the population dynamics, life histories and co-evolutionary dynamics of the interacting organisms. but there are also insights that can be fed back into laboratory immunology. the results raise the possibility that th2 cell activity may show ontogenetic changes, sometimes mediating resistance and at other times mediating tolerance. this dichotomy could be relevant to the lab-oratory as, typically, laboratory experiments are carried out in restricted life-history stages under restricted environmental conditions, and this may have skewed our view of what responses are likely to occur in systems outside of the laboratory. there is also a relevance to vaccination strategies in real-world situations, which may need to encompass the possibility that, under some conditions and in some subsets of a population, similar immunogenic stimuli may result in resistance or tolerance responses (that might not be protective in terms of preventing infection, but could have some benefit in terms of ameliorating disease). this may be especially significant, given that most vaccine adjuvants used in practical contexts are th2-inducing aluminium salts (alums). although much interesting work has been done in nonmodel rodents using narrow focusses on individual immune responses, a renewed effort addressing the immune system (and its interaction with wider organismal traits and the environment) in a more holistic way seems likely to pay dividends. the pivotal technological means to do this are now accessible, in the form of nextgeneration sequencing analyses of the transcriptome. early indications from studies using qpcr panels of candidate genes suggest that gene expression measurements in natural populations do convey interpretable information about individual status, especially where combined with defined stimulatory treatments of cultured cell populations. associations occur with season, life-history stage and body condition. furthermore, there are strong indications that infection pressures are key drivers of many aspects of expression in the immune system in nature and that, as a result of these pressures, wild mammals can be confidently predicted to adopt phenotypes very different to those seen in laboratory rodents. all of this supports the utility of immune gene expression measurements for ecologically and epidemiologically motivated studies; it also confirms the interest for our basic understanding of the immune system: where the diverse combinations of environmental conditions seen in the wild may reveal interaction networks that remain hidden under controlled laboratory conditions. finally, studying the diversity of immune function in natural and anthropogenic environments (and, ultimately, further dissection of this variation under experimental conditions) will help us resolve the environmental stimuli (and genotype 9 environment interactions) that affect the formation of immunopathological phenotypes such as those responsible for so much variation in human health and animal welfare. δ body condition δ survival probability δ testis condition large mature male immature male gata3 blood gata3 mit-s m gata3 mit-s m figure 1 life-history responses in male field voles (microtus agrestis) associated with gata3 expression triggered by macroparasite exposures, represented schematically as phase curves (temporal tracks through the plotted parameter space). gata3 mit-stim , gata3 expression in cultured mitogen-stimulated splenocytes; gata3 blood , constitutive gata3 expression in peripheral blood. the analysis of immunological profiles in wild animals: a case study on immunodynamics in the field vole, microtus agrestis beyond phytohaemagglutinin: assessing vertebrate immune function across ecological contexts towards a system level understanding of non-model organisms sampled from the environment: a network biology approach wild immunology macroparasites, innate immunity and immunoregulation: developing natural models measuring immune system variation to help understand host-pathogen community dynamics regulation of the immune system by biodiversity from the natural environment: an ecosystem service essential to health inflammation and cancer diagnosis and classification of autoimmune diabetes mellitus microbiome diversity and asthma and allergy risk the role of innate and adaptive immunity in parkinson's disease molecular mechanisms linking neuroinflammation and neurodegeneration in ms microbiota, immunoregulatory old friends and psychiatric disorders hygiene hypothesis and autoimmune diseases wild rodents as a model to discover genes and pathways underlying natural variation in infectious disease susceptibility the history of ecoimmunology and its integration with disease ecology review series on helminths, immune modulation and the hygiene hypothesis: immunity against helminths and immunological phenomena in modern human populations: coevolutionary legacies? the origins and uses of mouse outbred stocks inbred strains derived from feral mice reveal new pathogenic mechanisms of experimental leishmaniasis due to leishmania major wild-derived mouse stocks: an underappreciated tool for aging research cystic fibrosis heterozygote resistance to cholera toxin in the cystic fibrosis mouse model cellular mechanism for the protective effect of haemoglobin s against p. falciparum malaria a defunctioning polymorphism in fcgr2b is associated with protection against malaria but susceptibility to systemic lupus erythematosus the heterozygote advantage in phenylketonuria human snp links differential outcomes in inflammatory and infectious disease to a foxo3-regulated pathway signatures of environmental genetic adaptation pinpoint pathogens as the main selective pressure through human evolution an evolutionary analysis of antigen processing and presentation across different timescales reveals pervasive selection multiple instances of ancient balancing selection shared between humans and chimpanzees naturally occurring toll-like receptor 11 (tlr11) and toll-like receptor 12 (tlr12) polymorphisms are not associated with toxoplasma gondii infection in wild wood mice polymorphisms at the innate immune receptor tlr2 are associated with borrelia infection in a wild rodent population signatures of selection acting on the innate immunity gene toll-like receptor 2 (tlr2) during the evolutionary history of rodents genetic diversity in cytokines associated with immune variation and resistance to multiple pathogens in a natural rodent population evidence for selection at cytokine loci in a natural population of field voles (microtus agrestis) fitness consequences of immune responses: strengthening the empirical framework for ecoimmunology rodent-borne diseases and their risks for public health fluctuating rodent populations and risk to humans from rodent-borne zoonoses. vector borne zoonotic superspreading and the effect of individual variation on disease emergence transmission consequences of coinfection: cytokines writ large? host-parasite biology in the real world: the field voles of kielder photoperiod, ambient temperature, and food availability interact to affect reproductive and immune function in adult male deer mice (peromyscus maniculatus) seasonal changes in vertebrate immune activity: mediation by physiological trade-offs seasonal changes in immune function surveillance for microbes and range expansion in house sparrows adaptive evolution during an ongoing range expansion: the invasive bank vole (myodes glareolus) in ireland mass extinctions, biodiversity and mitochondrial function: are bats 'special' as reservoirs for emerging viruses? immune defense and reproductive pace of life in peromyscus mice landscape features and helminth co-infection shape bank vole immunoheterogeneity, with consequences for puumala virus epidemiology zoonoses and one health: a review of the literature temporal anomalies in immunological gene expression in a time series of wild mice: signature of an epidemic? elm: enhanced lowest common ancestor based method for detecting a pathogenic virus from a large sequence dataset next generation sequencing technologies for insect virus discovery type iii ifns in pteropid bats: differential expression patterns provide evidence for distinct roles in antiviral immunity a review of studies on animal reservoirs of the sars coronavirus molecular evolution analysis and geographic investigation of severe acute respiratory syndrome coronavirus-like virus in palm civets at an animal market and on farms pathogen-specific local immune fingerprints diagnose bacterial infection in peritoneal dialysis patients an immunological marker of tolerance to infection in wild rodents ecological immunology: costly parasite defences and tradeoffs in evolutionary ecology phylogeography and postglacial expansion of mus musculus domesticus inferred from mitochondrial dna coalescent, from iran to europe heligmosomoides bakeri: a new name for an old worm? the ecoimmunology of invasive species full-length transcriptome assembly from rna-seq data without a reference genome parasitism and physiological trade-offs in stressed capybaras differences in natural antibody titres comparing free-ranging guanacos (lama guanicoe) and capybaras (hydrochoerus hydrochaeris) are associations of immune gene expression, body condition and parasite burden detectable in nature? a case study in an endemic rodent from the brazilian atlantic forest translation control: bridging the gap between genomics and proteomics? the utility of protein and mrna correlation profiling helper t cell subset gene expression in deer mice immunomodulatory parasites and toll-like receptor-mediated tumour necrosis factor alpha responsiveness in wild mammals hantavirus immunology of rodent reservoirs: current status and future directions immune-and wound-dependent differential gene expression in an ancient insect exploring local immunological adaptation of two stickleback ecotypes by experimental infection and transcriptomewide digital gene expression analysis rna-seq transcriptional profiling of peripheral blood leukocytes from cattle infected with mycobacterium bovis digital pcr strategies in the development and analysis of molecular biomarkers for personalized medicine evaluation of high throughput gene expression platforms using a genomic biomarker signature for prediction of skin sensitization reverse engineering and identification in systems biology: strategies, perspectives and challenges intestinal microbiota: shaping local and systemic immune responses interactions between the microbiota and the immune system environmental biodiversity, human microbiota, and allergy are interrelated tlr-2-activated b cells suppress helicobacterinduced preneoplastic gastric immunopathology by inducing t regulatory-1 cells metabolic syndrome and altered gut microbiota in mice lacking toll-like receptor 5 tlr-mediated innate immune recognition macroparasites at peripheral sites of infection are major and dynamic modifiers of systemic antimicrobial pattern recognition responses heligmosomoides polygyrus: one species still myd88 signaling inhibits protective immunity to the gastrointestinal helminth parasite heligmosomoides polygyrus essential role for tlr4 and myd88 in the development of chronic intestinal nematode infection genetic variation in resistance to repeated infections with heligmosomoides polygyrus bakeri, in inbred mouse strains selected for the mouse genome project cd4 + cd25 + regulatory t cells control innate immune reactivity after injury impairment of dendritic cell function by excretory-secretory products: a potential mechanism for nematode-induced immunosuppression th2 cytokines down-regulate tlr expression and function in human intestinal epithelial cells tnf-alpha expression and promoter sequences reflect the balance of tolerance/ resistance to puumala hantavirus infection in european bank vole populations heligmosomoides polygyrus infection is associated with lower mhc class ii gene expression in apodemus flavicollis: indication for immune suppression? expression profiling of lymph node cells from deer mice infected with andes virus saag-4 is a novel mosquito salivary protein that programmes host cd4(+) t cells to express il-4 blood feeding by the rocky mountain spotted fever vector, dermacentor andersoni, induces interleukin-4 expression by cognate antigen responding cd4(+) t cells the immune response to parasitic helminths: insights from murine models gata3 and the t-cell lineage: essential functions before and after t-helper-2-cell differentiation evolution of th2 immunity: a rapid repair response to tissue destructive pathogens decomposing health: tolerance and resistance to parasites in animals acknowledgements i gratefully acknowledge the colleagues with whom i have worked on nonmodel rodent systems and whose ideas have influenced my own thinking, also acknowledged is funding from the leverhulme trust (rpg-301) and nerc (ne/l013517/1). key: cord-032561-x3qbqy69 authors: liu, gengqi; lovell, jonathan f.; zhang, lei; zhang, yumiao title: stimulus-responsive nanomedicines for disease diagnosis and treatment date: 2020-09-02 journal: int j mol sci doi: 10.3390/ijms21176380 sha: doc_id: 32561 cord_uid: x3qbqy69 stimulus-responsive drug delivery systems generally aim to release the active pharmaceutical ingredient (api) in response to specific conditions and have recently been explored for disease treatments. these approaches can also be extended to molecular imaging to report on disease diagnosis and management. the stimuli used for activation are based on differences between the environment of the diseased or targeted sites, and normal tissues. endogenous stimuli include ph, redox reactions, enzymatic activity, temperature and others. exogenous site-specific stimuli include the use of magnetic fields, light, ultrasound and others. these endogenous or exogenous stimuli lead to structural changes or cleavage of the cargo carrier, leading to release of the api. a wide variety of stimulus-responsive systems have been developed—responsive to both a single stimulus or multiple stimuli—and represent a theranostic tool for disease treatment. in this review, stimuli commonly used in the development of theranostic nanoplatforms are enumerated. an emphasis on chemical structure and property relationships is provided, aiming to focus on insights for the design of stimulus-responsive delivery systems. several examples of theranostic applications of these stimulus-responsive nanomedicines are discussed. one of the challenges of traditional drug delivery systems is the lack of specific targeting capability, which leads to dose-limiting side effects [1, 2] . some drugs can be rapidly metabolized, and low bioavailability necessitates large injection doses but unsatisfactory therapeutic effects. for example, it is generally necessary to increase the dosage of anticancer drugs for the desired therapeutic effect, to an extent that also causes undesirable side effects [3, 4] . in addition, orally administered medicines need to evade the risk of degradation in the acidic/enzymatic environment in the gastrointestinal tract [5, 6] . all of these problems can potentially be addressed with stimulus-responsive nanotechnology. utilizing specific microenvironmental conditions at diseased sites or alternatively by using external stimuli, nanotherapeutics can release active therapeutic ingredients specifically at desired spots in a controlled and targeted manner. as early as 1978, tanaka described the phase transition of polyacrylamide polymers [7] , and in the same year blumenthal reported thermosensitive liposomes [8] . findings such as these laid the groundwork for stimulus-responsive drug delivery systems, which have been developed for more than 40 years. stimulus-responsive nanomaterials have been designed for a differences between the microenvironment of diseased tissues (e.g., arthritic tissues, tumor cells, and other diseases) and normal tissues can be used as endogenous stimuli to trigger drug release. endogenous stimuli include lower ph values in tumor cells, high concentrations of glutathione (gsh) and reactive oxygen species (ros) in inflammatory tissues and cancer tissues, and specific enzymatic activity at the target sites [9, 10] . in addition to these endogenous stimuli above, exogenous stimuli such as magnet, light, and ultrasound can realize real-time tracking of drug distribution, disease diagnosis and disease management. stimulus-responsive systems responsive to one single stimulus or multiple stimuli have been used to treat or diagnose a variety of diseases, such as cancers (colon cancer [11, 12] , breast cancer [13, 14] , lymphoma [15, 16] ), inflammatory diseases (inflammatory bowel disease [17, 18] , neurodegenerative diseases [19] ), diabetes [20, 21] and others. some nanomedicines that could be considered stimulus-responsive have successfully gone into clinical trials. thermodox (thermosensitive doxorubicin liposomes) used heat to release the cargo from liposome [22] ; other successful examples include opaxio [23] (involving on enzyme-degradable polymer), nanotherm [24] (based on magnetic iron oxide nanoparticles), and auroshell [25] (based on thermosensitive gold nanoshells). stimulus-responsive systems also have potential for bioimaging. they can enhance the contrast in specific diseased area in response to internal or external stimuli for real-time monitoring. imine [34] hydrazone [35, 36] ester [37] acetal [38, 39] cis-aconityl [40] orthoester [41, 42] silyl ether [43] (c) twenty minutes after mice were injected with deionized water, mg micromotors and inert polystyrene (ps) particles, superimposed fluorescent images of the whole stomach of mice were collected (both mg micromotors and ps microparticles were loaded with did dye and encapsulated in a ph-sensitive polymer shell). reproduced with permission from the publisher of corresponding reference [45] . [34] hydrazone int. j. mol. sci. 2020, 21, x for peer review 4 of 44 imine [34] hydrazone [35, 36] ester [37] acetal [38, 39] cis-aconityl [40] orthoester [41, 42] silyl ether [43] (c) twenty minutes after mice were injected with deionized water, mg micromotors and inert polystyrene (ps) particles, superimposed fluorescent images of the whole stomach of mice were collected (both mg micromotors and ps microparticles were loaded with did dye and encapsulated in a ph-sensitive polymer shell). reproduced with permission from the publisher of corresponding reference [45] . [35, 36] ester int. j. mol. sci. 2020, 21, x for peer review 4 of 44 imine [34] hydrazone [35, 36] ester [37] acetal [38, 39] cis-aconityl [40] orthoester [41, 42] silyl ether [43] (c) twenty minutes after mice were injected with deionized water, mg micromotors and inert polystyrene (ps) particles, superimposed fluorescent images of the whole stomach of mice were collected (both mg micromotors and ps microparticles were loaded with did dye and encapsulated in a ph-sensitive polymer shell). reproduced with permission from the publisher of corresponding reference [45] . [37] acetal imine [34] hydrazone [35, 36] ester [37] acetal [38, 39] cis-aconityl [40] orthoester [41, 42] silyl ether [43] (c) twenty minutes after mice were injected with deionized water, mg micromotors and inert polystyrene (ps) particles, superimposed fluorescent images of the whole stomach of mice were collected (both mg micromotors and ps microparticles were loaded with did dye and encapsulated in a ph-sensitive polymer shell). reproduced with permission from the publisher of corresponding reference [45] . [38, 39] cis-aconityl imine [34] hydrazone [35, 36] ester [37] acetal [38, 39] cis-aconityl [40] orthoester [41, 42] silyl ether [43] (c) twenty minutes after mice were injected with deionized water, mg micromotors and inert polystyrene (ps) particles, superimposed fluorescent images of the whole stomach of mice were collected (both mg micromotors and ps microparticles were loaded with did dye and encapsulated in a ph-sensitive polymer shell). reproduced with permission from the publisher of corresponding reference [45] . [40] orthoester imine [34] hydrazone [35, 36] ester [37] acetal [38, 39] cis-aconityl [40] orthoester [41, 42] silyl ether [43] (c) twenty minutes after mice were injected with deionized water, mg micromotors and inert polystyrene (ps) particles, superimposed fluorescent images of the whole stomach of mice were collected (both mg micromotors and ps microparticles were loaded with did dye and encapsulated in a ph-sensitive polymer shell). reproduced with permission from the publisher of corresponding reference [45] . imine [34] hydrazone [35, 36] ester [37] acetal [38, 39] cis-aconityl [40] orthoester [41, 42] silyl ether [43] (c) twenty minutes after mice were injected with deionized water, mg micromotors and inert polystyrene (ps) particles, superimposed fluorescent images of the whole stomach of mice were collected (both mg micromotors and ps microparticles were loaded with did dye and encapsulated in a ph-sensitive polymer shell). reproduced with permission from the publisher of corresponding reference [45] . [43] int. j. mol. sci. 2020, 21, x for peer review 4 of 44 imine [34] hydrazone [35, 36] ester [37] acetal [38, 39] cis-aconityl [40] orthoester [41, 42] silyl ether [43] (c) twenty minutes after mice were injected with deionized water, mg micromotors and inert polystyrene (ps) particles, superimposed fluorescent images of the whole stomach of mice were collected (both mg micromotors and ps microparticles were loaded with did dye and encapsulated in a ph-sensitive polymer shell). reproduced with permission from the publisher of corresponding reference [45] . besides the development of drug delivery systems, ph-responsive systems can also be used for tumor detection and image-guided surgery [46] . by taking advantage of the difference of ph values in different target tissues in vivo, ph-responsive systems represent an intriguing strategy in drug delivery system. however, the difference of ph values between target and healthy tissues may not always differ significantly, so responsive systems that rely only on ph may be limited by low specificity and sensitivity. therefore, ph responsive system can be combined with other stimulus conditions such as light, redox, enzymes and others with the aim of improved selectivity for drug release in diseased tissues [47, 48] . redox conditions can be used for another type of stimulus-responsive system. these include gsh-sensitive systems, which have attracted much recent attention. gsh-sensitive systems can have stability in normal tissues but undergo release of cargo in diseased tissues in response to higher concentrations of gsh. for example, the concentration of gsh, as a tumor marker in cancer cells is 4-7 fold higher than in normal cells [49, 50] . the intracellular concentration of gsh (1-10 mm) is about three orders of magnitude higher than that in the blood (2-10 µm) [51, 52] . disulfide bonds and diselenium bonds have been widely used in the design of redox stimulus responsive systems (table 2 ). disulfide bonds have also been used in chemical sensors. shi et al. designed and synthesized a nanodrug carrier based on disulfide bond-doped organosilicon-micelle hybrid nanoparticles, the surface of which was modified with disulfide-bonded peg and amide-bonded polyethylenimine (pei). this nano-drug carrier exhibited excellent blood circulation ability and accumulation performance in tumor tissues, and the side effects of anticancer drugs on not-carcinogenic tissues were also reduced because of such rational design [53] . besides the development of drug delivery systems, ph-responsive systems can also be used for tumor detection and image-guided surgery [46] . by taking advantage of the difference of ph values in different target tissues in vivo, ph-responsive systems represent an intriguing strategy in drug delivery system. however, the difference of ph values between target and healthy tissues may not always differ significantly, so responsive systems that rely only on ph may be limited by low specificity and sensitivity. therefore, ph responsive system can be combined with other stimulus conditions such as light, redox, enzymes and others with the aim of improved selectivity for drug release in diseased tissues [47, 48] . redox conditions can be used for another type of stimulus-responsive system. these include gsh-sensitive systems, which have attracted much recent attention. gsh-sensitive systems can have stability in normal tissues but undergo release of cargo in diseased tissues in response to higher concentrations of gsh. for example, the concentration of gsh, as a tumor marker in cancer cells is 4-7 fold higher than in normal cells [49, 50] . the intracellular concentration of gsh (1-10 mm) is about three orders of magnitude higher than that in the blood (2-10 µm) [51, 52] . disulfide bonds and diselenium bonds have been widely used in the design of redox stimulus responsive systems (table 2) . disulfide bonds have also been used in chemical sensors. shi et al. designed and synthesized a nanodrug carrier based on disulfide bond-doped organosilicon-micelle hybrid nanoparticles, the surface of which was modified with disulfide-bonded peg and amidebonded polyethylenimine (pei). this nano-drug carrier exhibited excellent blood circulation ability and accumulation performance in tumor tissues, and the side effects of anticancer drugs on notcarcinogenic tissues were also reduced because of such rational design [53] . disulfide [53] diselenium [54, 55] thioether selenium tellurium [56] [57] [58] [59] [60] [61] oxalate ester [62, 63] vinyldithioether [64] thioketal [65] pba/pbe [66] compared with sulfur, the electronegativity of selenium is relatively low, and the atomic radius of selenium is larger, so selenium has some unique properties such as high reactivity and sensitivity ( table 2 ). the bond energy of s-s is 240 kj·mol −1 , whereas the bond energy of se-se bond is only 172 kj·mol −1 , suggesting that se-se bond may have higher sensitivity than disulfide bond; that is, se-se bond has the potential to release drug faster [54] . wei et al. demonstrated that nanodrug carriers with diselenides could release more drugs using poly (ester carbamate) triblock copolymers (paur-se-se) with se-se bonds compared with poly (ester carbamate) triblock copolymers (paur-s-s) [53] diselenium int. j. mol. sci. 2020, 21, x for peer review 5 of 44 besides the development of drug delivery systems, ph-responsive systems can also be used for tumor detection and image-guided surgery [46] . by taking advantage of the difference of ph values in different target tissues in vivo, ph-responsive systems represent an intriguing strategy in drug delivery system. however, the difference of ph values between target and healthy tissues may not always differ significantly, so responsive systems that rely only on ph may be limited by low specificity and sensitivity. therefore, ph responsive system can be combined with other stimulus conditions such as light, redox, enzymes and others with the aim of improved selectivity for drug release in diseased tissues [47, 48] . redox conditions can be used for another type of stimulus-responsive system. these include gsh-sensitive systems, which have attracted much recent attention. gsh-sensitive systems can have stability in normal tissues but undergo release of cargo in diseased tissues in response to higher concentrations of gsh. for example, the concentration of gsh, as a tumor marker in cancer cells is 4-7 fold higher than in normal cells [49, 50] . the intracellular concentration of gsh (1-10 mm) is about three orders of magnitude higher than that in the blood (2-10 µm) [51, 52] . disulfide bonds and diselenium bonds have been widely used in the design of redox stimulus responsive systems (table 2) . disulfide bonds have also been used in chemical sensors. shi et al. designed and synthesized a nanodrug carrier based on disulfide bond-doped organosilicon-micelle hybrid nanoparticles, the surface of which was modified with disulfide-bonded peg and amidebonded polyethylenimine (pei). this nano-drug carrier exhibited excellent blood circulation ability and accumulation performance in tumor tissues, and the side effects of anticancer drugs on notcarcinogenic tissues were also reduced because of such rational design [53] . disulfide [53] diselenium [54, 55] thioether selenium tellurium [56] [57] [58] [59] [60] [61] oxalate ester [62, 63] vinyldithioether [64] thioketal [65] pba/pbe [66] compared with sulfur, the electronegativity of selenium is relatively low, and the atomic radius of selenium is larger, so selenium has some unique properties such as high reactivity and sensitivity ( table 2 ). the bond energy of s-s is 240 kj·mol −1 , whereas the bond energy of se-se bond is only 172 kj·mol −1 , suggesting that se-se bond may have higher sensitivity than disulfide bond; that is, se-se bond has the potential to release drug faster [54] . wei et al. demonstrated that nanodrug carriers with diselenides could release more drugs using poly (ester carbamate) triblock copolymers (paur-se-se) with se-se bonds compared with poly (ester carbamate) triblock copolymers (paur-s-s) [54, 55] thioether selenium tellurium besides the development of drug delivery systems, ph-responsive systems can also be used for tumor detection and image-guided surgery [46] . by taking advantage of the difference of ph values in different target tissues in vivo, ph-responsive systems represent an intriguing strategy in drug delivery system. however, the difference of ph values between target and healthy tissues may not always differ significantly, so responsive systems that rely only on ph may be limited by low specificity and sensitivity. therefore, ph responsive system can be combined with other stimulus conditions such as light, redox, enzymes and others with the aim of improved selectivity for drug release in diseased tissues [47, 48] . redox conditions can be used for another type of stimulus-responsive system. these include gsh-sensitive systems, which have attracted much recent attention. gsh-sensitive systems can have stability in normal tissues but undergo release of cargo in diseased tissues in response to higher concentrations of gsh. for example, the concentration of gsh, as a tumor marker in cancer cells is 4-7 fold higher than in normal cells [49, 50] . the intracellular concentration of gsh (1-10 mm) is about three orders of magnitude higher than that in the blood (2-10 µm) [51, 52] . disulfide bonds and diselenium bonds have been widely used in the design of redox stimulus responsive systems (table 2) . disulfide bonds have also been used in chemical sensors. shi et al. designed and synthesized a nanodrug carrier based on disulfide bond-doped organosilicon-micelle hybrid nanoparticles, the surface of which was modified with disulfide-bonded peg and amidebonded polyethylenimine (pei). this nano-drug carrier exhibited excellent blood circulation ability and accumulation performance in tumor tissues, and the side effects of anticancer drugs on notcarcinogenic tissues were also reduced because of such rational design [53] . disulfide [53] diselenium [54, 55] thioether selenium tellurium [56] [57] [58] [59] [60] [61] oxalate ester [62, 63] vinyldithioether [64] thioketal [65] pba/pbe [66] compared with sulfur, the electronegativity of selenium is relatively low, and the atomic radius of selenium is larger, so selenium has some unique properties such as high reactivity and sensitivity ( table 2 ). the bond energy of s-s is 240 kj·mol −1 , whereas the bond energy of se-se bond is only 172 kj·mol −1 , suggesting that se-se bond may have higher sensitivity than disulfide bond; that is, se-se bond has the potential to release drug faster [54] . wei et al. demonstrated that nanodrug carriers with diselenides could release more drugs using poly (ester carbamate) triblock copolymers (paur-se-se) with se-se bonds compared with poly (ester carbamate) triblock copolymers (paur-s-s) [56] [57] [58] [59] [60] [61] oxalate ester besides the development of drug delivery systems, ph-responsive systems can also be used for tumor detection and image-guided surgery [46] . by taking advantage of the difference of ph values in different target tissues in vivo, ph-responsive systems represent an intriguing strategy in drug delivery system. however, the difference of ph values between target and healthy tissues may not always differ significantly, so responsive systems that rely only on ph may be limited by low specificity and sensitivity. therefore, ph responsive system can be combined with other stimulus conditions such as light, redox, enzymes and others with the aim of improved selectivity for drug release in diseased tissues [47, 48] . redox conditions can be used for another type of stimulus-responsive system. these include gsh-sensitive systems, which have attracted much recent attention. gsh-sensitive systems can have stability in normal tissues but undergo release of cargo in diseased tissues in response to higher concentrations of gsh. for example, the concentration of gsh, as a tumor marker in cancer cells is 4-7 fold higher than in normal cells [49, 50] . the intracellular concentration of gsh (1-10 mm) is about three orders of magnitude higher than that in the blood (2-10 µm) [51, 52] . disulfide bonds and diselenium bonds have been widely used in the design of redox stimulus responsive systems (table 2) . disulfide bonds have also been used in chemical sensors. shi et al. designed and synthesized a nanodrug carrier based on disulfide bond-doped organosilicon-micelle hybrid nanoparticles, the surface of which was modified with disulfide-bonded peg and amidebonded polyethylenimine (pei). this nano-drug carrier exhibited excellent blood circulation ability and accumulation performance in tumor tissues, and the side effects of anticancer drugs on notcarcinogenic tissues were also reduced because of such rational design [53] . disulfide [53] diselenium [54, 55] thioether selenium tellurium [56] [57] [58] [59] [60] [61] oxalate ester [62, 63] vinyldithioether [64] thioketal [65] pba/pbe [66] compared with sulfur, the electronegativity of selenium is relatively low, and the atomic radius of selenium is larger, so selenium has some unique properties such as high reactivity and sensitivity ( table 2 ). the bond energy of s-s is 240 kj·mol −1 , whereas the bond energy of se-se bond is only 172 kj·mol −1 , suggesting that se-se bond may have higher sensitivity than disulfide bond; that is, se-se bond has the potential to release drug faster [54] . wei et al. demonstrated that nanodrug carriers with diselenides could release more drugs using poly (ester carbamate) triblock copolymers (paur-se-se) with se-se bonds compared with poly (ester carbamate) triblock copolymers (paur-s-s) [62, 63] vinyldithioether int. j. mol. sci. 2020, 21, x for peer review 5 of 44 besides the development of drug delivery systems, ph-responsive systems can also be used for tumor detection and image-guided surgery [46] . by taking advantage of the difference of ph values in different target tissues in vivo, ph-responsive systems represent an intriguing strategy in drug delivery system. however, the difference of ph values between target and healthy tissues may not always differ significantly, so responsive systems that rely only on ph may be limited by low specificity and sensitivity. therefore, ph responsive system can be combined with other stimulus conditions such as light, redox, enzymes and others with the aim of improved selectivity for drug release in diseased tissues [47, 48] . redox conditions can be used for another type of stimulus-responsive system. these include gsh-sensitive systems, which have attracted much recent attention. gsh-sensitive systems can have stability in normal tissues but undergo release of cargo in diseased tissues in response to higher concentrations of gsh. for example, the concentration of gsh, as a tumor marker in cancer cells is 4-7 fold higher than in normal cells [49, 50] . the intracellular concentration of gsh (1-10 mm) is about three orders of magnitude higher than that in the blood (2-10 µm) [51, 52] . disulfide bonds and diselenium bonds have been widely used in the design of redox stimulus responsive systems (table 2) . disulfide bonds have also been used in chemical sensors. shi et al. designed and synthesized a nanodrug carrier based on disulfide bond-doped organosilicon-micelle hybrid nanoparticles, the surface of which was modified with disulfide-bonded peg and amidebonded polyethylenimine (pei). this nano-drug carrier exhibited excellent blood circulation ability and accumulation performance in tumor tissues, and the side effects of anticancer drugs on notcarcinogenic tissues were also reduced because of such rational design [53] . disulfide [53] diselenium [54, 55] thioether selenium tellurium [56] [57] [58] [59] [60] [61] oxalate ester [62, 63] vinyldithioether [64] thioketal [65] pba/pbe [66] compared with sulfur, the electronegativity of selenium is relatively low, and the atomic radius of selenium is larger, so selenium has some unique properties such as high reactivity and sensitivity ( table 2 ). the bond energy of s-s is 240 kj·mol −1 , whereas the bond energy of se-se bond is only 172 kj·mol −1 , suggesting that se-se bond may have higher sensitivity than disulfide bond; that is, se-se bond has the potential to release drug faster [54] . wei et al. demonstrated that nanodrug carriers with diselenides could release more drugs using poly (ester carbamate) triblock copolymers (paur-se-se) with se-se bonds compared with poly (ester carbamate) triblock copolymers (paur-s-s) [ besides the development of drug delivery systems, ph-responsive systems can also be used for tumor detection and image-guided surgery [46] . by taking advantage of the difference of ph values in different target tissues in vivo, ph-responsive systems represent an intriguing strategy in drug delivery system. however, the difference of ph values between target and healthy tissues may not always differ significantly, so responsive systems that rely only on ph may be limited by low specificity and sensitivity. therefore, ph responsive system can be combined with other stimulus conditions such as light, redox, enzymes and others with the aim of improved selectivity for drug release in diseased tissues [47, 48] . redox conditions can be used for another type of stimulus-responsive system. these include gsh-sensitive systems, which have attracted much recent attention. gsh-sensitive systems can have stability in normal tissues but undergo release of cargo in diseased tissues in response to higher concentrations of gsh. for example, the concentration of gsh, as a tumor marker in cancer cells is 4-7 fold higher than in normal cells [49, 50] . the intracellular concentration of gsh (1-10 mm) is about three orders of magnitude higher than that in the blood (2-10 µm) [51, 52] . disulfide bonds and diselenium bonds have been widely used in the design of redox stimulus responsive systems ( table 2 ). disulfide bonds have also been used in chemical sensors. shi et al. designed and synthesized a nanodrug carrier based on disulfide bond-doped organosilicon-micelle hybrid nanoparticles, the surface of which was modified with disulfide-bonded peg and amidebonded polyethylenimine (pei). this nano-drug carrier exhibited excellent blood circulation ability and accumulation performance in tumor tissues, and the side effects of anticancer drugs on notcarcinogenic tissues were also reduced because of such rational design [53] . disulfide [53] diselenium [54, 55] thioether selenium tellurium [56] [57] [58] [59] [60] [61] oxalate ester [62, 63] vinyldithioether [64] thioketal [65] pba/pbe [66] compared with sulfur, the electronegativity of selenium is relatively low, and the atomic radius of selenium is larger, so selenium has some unique properties such as high reactivity and sensitivity ( table 2 ). the bond energy of s-s is 240 kj·mol −1 , whereas the bond energy of se-se bond is only 172 kj·mol −1 , suggesting that se-se bond may have higher sensitivity than disulfide bond; that is, se-se bond has the potential to release drug faster [54] . wei et al. demonstrated that nanodrug carriers with diselenides could release more drugs using poly (ester carbamate) triblock copolymers (paur-se-se) with se-se bonds compared with poly (ester carbamate) triblock copolymers (paur-s-s) [65] pba/pbe besides the development of drug delivery systems, ph-responsive systems can also be used for tumor detection and image-guided surgery [46] . by taking advantage of the difference of ph values in different target tissues in vivo, ph-responsive systems represent an intriguing strategy in drug delivery system. however, the difference of ph values between target and healthy tissues may not always differ significantly, so responsive systems that rely only on ph may be limited by low specificity and sensitivity. therefore, ph responsive system can be combined with other stimulus conditions such as light, redox, enzymes and others with the aim of improved selectivity for drug release in diseased tissues [47, 48] . redox conditions can be used for another type of stimulus-responsive system. these include gsh-sensitive systems, which have attracted much recent attention. gsh-sensitive systems can have stability in normal tissues but undergo release of cargo in diseased tissues in response to higher concentrations of gsh. for example, the concentration of gsh, as a tumor marker in cancer cells is 4-7 fold higher than in normal cells [49, 50] . the intracellular concentration of gsh (1-10 mm) is about three orders of magnitude higher than that in the blood (2-10 µm) [51, 52] . disulfide bonds and diselenium bonds have been widely used in the design of redox stimulus responsive systems ( table 2 ). disulfide bonds have also been used in chemical sensors. shi et al. designed and synthesized a nanodrug carrier based on disulfide bond-doped organosilicon-micelle hybrid nanoparticles, the surface of which was modified with disulfide-bonded peg and amidebonded polyethylenimine (pei). this nano-drug carrier exhibited excellent blood circulation ability and accumulation performance in tumor tissues, and the side effects of anticancer drugs on notcarcinogenic tissues were also reduced because of such rational design [53] . disulfide [53] diselenium [54, 55] thioether selenium tellurium [56] [57] [58] [59] [60] [61] oxalate ester [62, 63] vinyldithioether [64] thioketal [65] pba/pbe [66] compared with sulfur, the electronegativity of selenium is relatively low, and the atomic radius of selenium is larger, so selenium has some unique properties such as high reactivity and sensitivity ( table 2 ). the bond energy of s-s is 240 kj·mol −1 , whereas the bond energy of se-se bond is only 172 kj·mol −1 , suggesting that se-se bond may have higher sensitivity than disulfide bond; that is, se-se bond has the potential to release drug faster [54] . wei et al. demonstrated that nanodrug carriers with diselenides could release more drugs using poly (ester carbamate) triblock copolymers (paur-se-se) with se-se bonds compared with poly (ester carbamate) triblock copolymers (paur-s-s) [66] compared with sulfur, the electronegativity of selenium is relatively low, and the atomic radius of selenium is larger, so selenium has some unique properties such as high reactivity and sensitivity ( table 2 ). the bond energy of s-s is 240 kj·mol −1 , whereas the bond energy of se-se bond is only 172 kj·mol −1 , suggesting that se-se bond may have higher sensitivity than disulfide bond; that is, se-se bond has the potential to release drug faster [54] . wei et al. demonstrated that nanodrug carriers with diselenides could release more drugs using poly (ester carbamate) triblock copolymers (paur-se-se) with se-se bonds compared with poly (ester carbamate) triblock copolymers (paur-s-s) containing s-s bonds. at similar gsh concentrations (30 mm), 85% of encapsulated dox was released by paur-se-se micelles within 48 h, while 67% of the drug was released by paur-s-s micelles. in addition, the antitumor effect of dox-loaded paur-se-se was six fold higher than that of s-s analogues due to faster cleavage of the diselenium bond and enhanced drug release [55] . in a recent study, a new type of redox-responsive drug delivery system was developed based on the nano-metal-organic framework. its core structure was formed by zirconium ions (zr 4+ , metal nodes), 2,5-disulfanylterephthalic acid (bdc-(sh) 2 , organic ligand) and benzoic acid (ba, modulator), the sulfhydryl group on the anticancer drug, 6-mercaptopurine, formed a disulfide bond with bdc-(sh) 2 . this disulfide-based nanomaterial exhibited excellent intracellular drug release ability, and its cytotoxicity to cancer cells was shown to be three times higher than normal cells [67] . ros-responsive systems are also an effective strategy to control drug release by utilizing the ros accumulated in inflammation sites or other diseased tissues [68] . it has been shown that the concentration of ros in inflammatory tissue is 10-100 times higher than that in normal tissue [69] . there are two chemical mechanisms commonly used in ros-responsive systems. one is ros-induced non-breaking hydrophobic-hydrophilic transition and the other is ros-induced structural dissociation ( table 2 ). ros can oxidize chalcogens (e.g., s, se, te) [56, 57] , causing oxygen atoms to form covalent bonds with chalcogens, and polarizing groups to form hydrogen bonds with environmental water molecules. thus, hydrophobic-hydrophilic transition of the carrier backbone was induced without damaging the chemical structure of the drug carrier. among the chalcogen elements, tellurium-containing compounds have more potential than other chalcogen elements due to their lower electronegativity and lower toxicity [58] . additionally, it has been shown that tellurium-containing compounds have higher oxidation responsiveness than selenium-containing compounds and sulfur-containing compounds [59, 60] . xu et al. reported a synergistic therapeutic nanoplatform using near-infrared light-responsive cisplatin for cancer therapy. cisplatin (cddp) and indocyanine green (icg) were simultaneously loaded in nanocarriers made of amphiphilic tellurium-containing block copolymer peg-pute-peg. tellurium atoms in nanocarriers can be easily oxidized by ros (stimulated by near-infrared laser) produced by indocyanine green. tellurium oxidation weakens the coordination with cisplatin, thereby releasing cisplatin and achieving better anti-tumor effect [61] . in addition, ros can react with chemical structures such as thioketal (tk), phenylboronic acid/ester (pba/pbe), oxalate ester [62, 63] , vinyldithioether [64] and proline oligomers, leading to the cleavage of these structures. in general, tk groups are easily oxidized to generate acetone and two other thiol-containing fragments in the presence of ros. a novel amphiphilic peptide-drug conjugate for cancer targeted therapy has recently been reported. hydrophilic cyclic peptides and hydrophobic cytotoxin epothilone b (epo b) were linked by ros responsive tk groups. when nanotherapeutics entered tumor cells, tk groups could be cleaved due to high levels of intracellular ros, thereby releasing epo b, which effectively inhibited cell proliferation [65] . h 2 o 2 can specifically oxidize pbe/pba and subsequently induce the formation of borate ester and hydroxybenzyl alcohol [66] . in addition, amino acids such as proline, histidine and arginine are also found to be susceptible to ros-mediated and metal-catalyzed oxidation [70, 71] . for example, murthy et al. developed thioketal-based nanoparticles that could degrade selectively in the presence of ros in inflamed intestinal tissue. in responsive to abnormally high levels of ros in the sites of intestinal inflammation, thioketal nanoparticles could effectively deliver and release sirna to diseased sites, protecting mice from dextran sodium sulphate (dss)-induced colitis [72] . in addition to treating inflammation, ros-responsive systems can also be used for cancer treatment. ge et al. designed ros-responsive nanoparticles made up by a thioketal linker (tl), poly (ε-caprolactone) and poly (n,n-dimethylacrylamide) (pcl-tl-pdma). amphiphilic di-block copolymers with pcl-b-poly (2-guanidinoethyl methacrylate) (pcl-pgema) were used to encapsulate the anticancer drug paclitaxel and a photosensitizer, achieving efficient cell uptake and enhanced anticancer efficacy [73] (figure 3 ). complexity and diversity of the microenvironment in vivo, delivery systems based on specific redox molecular mechanism that can release cargo in a controllable manner are preferred but not well established yet. further investigations are expected to address these potential issues. enzymes have attracted attention in various nanobiotechnology applications owing to their specific biological targeting and catalytic properties. in the microenvironment of the diseased sites, the levels of some enzymes can be abnormal; therefore, enzyme-responsive systems represent an appealing strategy for the development of responsive drug carriers. enzymes can specifically target the affected sites, thereby regulating drug release. hydrolases, including proteases, lipases and glycosidases, have been widely explored in enzyme-responsive systems. oxidoreductases, such as nad(p)h, quinone oxidoreductase isoenzyme i (nqo1) and glucose oxidase (gox) have also been studied [74] . proteases play important role in biological processes, such as differentiation, angiogenesis, hormone synthesis, digestion, wound repair, hemostasis, inflammation, coagulation, immune response, necrosis and apoptosis [75] . some proteases are usually overexpressed in diseased tissues, and their activation can be used to release drugs for the development of protease-targeted therapies. cell uptake under 660 nm light irradiation was improved. a synergistic effect of photodynamic therapy (pdt) and chemotherapy was achieved through efficient cell uptake and drug release. reproduced with permission from the publisher of corresponding reference [73] . redox stimulus-responsive systems need high sensitivity and accuracy for fast triggered responses in diseased tissues, but stability under normal conditions. however, considering the complexity and diversity of the microenvironment in vivo, delivery systems based on specific redox molecular mechanism that can release cargo in a controllable manner are preferred but not well established yet. further investigations are expected to address these potential issues. enzymes have attracted attention in various nanobiotechnology applications owing to their specific biological targeting and catalytic properties. in the microenvironment of the diseased sites, the levels of some enzymes can be abnormal; therefore, enzyme-responsive systems represent an appealing strategy for the development of responsive drug carriers. enzymes can specifically target the affected sites, thereby regulating drug release. hydrolases, including proteases, lipases and glycosidases, have been widely explored in enzyme-responsive systems. oxidoreductases, such as nad(p)h, quinone oxidoreductase isoenzyme i (nqo1) and glucose oxidase (gox) have also been studied [74] . proteases play important role in biological processes, such as differentiation, angiogenesis, hormone synthesis, digestion, wound repair, hemostasis, inflammation, coagulation, immune response, necrosis and apoptosis [75] . some proteases are usually overexpressed in diseased tissues, and their activation can be used to release drugs for the development of protease-targeted therapies. for example, a new type of polymer poly (ethylene glycol) diacrylate (pegda) have been designed that could form spherical polymeric nanoparticles when mixed with peptide. the nanoparticles can be hydrolyzed by the overexpressed matrix metalloproteinase in lung lesions and then release the drugs [76] . in addition, it has been shown that the use of protease triggered drug release can enhance the therapeutic effect of drugs, and also reduce the toxicity and other side effects of drugs [77] . cathepsin-b is normally expressed in the lysosomes of human cells, but is also overexpressed in many invasive and metastatic cancers. therefore, cathepsin-b has been used as one of the targets of the enzyme-responsive systems. the amino acid sequence of gly-phe-leu-gly (gflg) can be degraded in the presence of cathepsin-b, so it can be applied for the development of nanodrug carriers. for example, peg, anticancer drug cisplatin and gflg peptide were used to be assembled for cisplatin prodrug units. icg was used to co-assemble with cathepsin-responsive cisplatin multidrug nanoplatforms. after cell uptake, the gflg peptide structure was degraded by cathepsin b in lysosomes, which in turn released icg and cisplatin prodrugs for cancer treatment [78] . among many kinds of matrix metalloproteinases, mmp2 has been one of the most widely explored for stimulus-responsive systems because it is overexpressed in cancer tissues. mmp2-acting substrates have been used for drug delivery and molecular imaging. for example, torchilin et al. synthesized an octapeptide (gly-pro-leu-gly-ile-ala-gly-gln) as a mmp2-sensitive linker for the conjugation of long-chain peg to liposomes and encapsulation of the cell-penetrating peptide tatp. when octapeptide (gplgiagq) was cleaved by mmp2, tatp was exposed, giving rise to the increased uptake of liposome particles by tumor cells [79] . in 2016, similar structures were also used in drug-loaded liposomes for pancreatic cancer treatment [80] . to improve permeability, antitumor efficacy and biodegradability, a size-shrinkable drug delivery system has been designed based on polysaccharide-modified dendrimers, which contained poly (amidoamine), hyaluronic acid and mmp2-sensitive peptide linker (plglag). the system retained good stability in blood circulation, and underwent degradation upon the action of mmp2, leading to enhanced uptake in tumor cells [81] . in addition, polypeptide gplgvrg [82, 83] , polypeptide gplgvrgdg [84] and triglyceride monostearate (tgms) [85] have also been synthesized for mmp2 sensitive drug delivery systems. phospholipase is another therapeutic target that can be overexpressed in infectious diseases, inflammatory diseases and tumors. in several inflammatory diseases or cancer cell types, the expression of phospholipase a2 (spla 2 ) has been shown to be higher [86] [87] [88] [89] . pla 2 is often used as a trigger condition in liposomal drug delivery systems, which specifically hydrolyzes sn-2 ester bonds in phospholipids, resulting in direct release of active drugs or hydrolysis of liposomes [90] . for example, colchicine-containing phosphatidylcholinase-responsive liposomes have been developed that are stable in human blood circulation. at the same time, liposomes could release colchicine-containing fatty acids upon the action of high levels of phospholipase a2. the latter further hydrolyzed and released colchicine analogues by non-specific enzymes [91] . besides, in a recent report, an enzyme reactive liposome containing 1,2-dipalmitoyl-sn-glycerin-3-phosphocholine (dppc) and 1-o-stearyl-2-retinoic acid receptor (rar)-c6-sn-glycerin-3-phosphoglycerol with c6-rar as a prodrug was designed. in the absence of spla 2 , the ic50 of c6-rar prodrug in mt-3 breast cancer cell line was 110 µm, while the ic50 of c6-rar prodrug decreased to 10 µm after adding spla2, indicating that the prodrug was hydrolyzed into c6-rar and lyso-o-spg by spla 2 , in response to phospholipase [92] . oxidoreductase has also been used as a potential target of stimulus-responsive systems due to its important role in intracellular oxidative environment. gox is a kind of oxidoreductase tested for stimulus-response systems. it catalyzes the oxidation of β-d-glucose to d-glucose-δ-lactone and hydrogen peroxide with oxygen as electron acceptor [93, 94] . glucose responsive systems have been assessed for the treatment of diabetes [95, 96] . for example, willner et al. recently used zeolite imidazolium frameworks (zif-8 nmofs) as a glucose-responsive carrier to release insulin and vascular endothelial growth factor (vegf) aptamers, which inhibited angiogenesis by binding to vegf proteins. zif-8 was stable under neutral physiological conditions but degraded under acidic conditions. insulin or vegf aptamers and gox were integrated into zif-8 nmofs as biocatalysts. gox catalyzed the aerobic oxidation of glucose to produce gluconic acid. local acidification of nmofs degraded zif-8, thereby releasing insulin and vegf aptamers [97] . besides, nqo1 has been widely used in stimulus-responsive therapy systems due to its overexpression in tumors and other diseases [98, 99] . in a recent report, a nqo1-responsive multifunctional polymeric vesicle covalently bound to a photosensitizer (coumarin and nile blue) was prepared. without being triggered by nqo1, both fluorescence emission and photodynamic therapy (pdt) capability were turned off due to the "double quenching" effect. upon cellular uptake, highly expressed nqo1 triggered the self-immolative cleavage of the quinone trimethyl lock, which then led to the release of photosensitizers, near infrared (nir) emission and pdt activation, thereby realizing real-time monitoring and treatment of cancers [100] . glucosidase is an enzyme that can hydrolyze glycosidic bonds. its concentration in diseased tissues can be higher than that in normal tissues. therefore, glucosidase can be used to design enzyme-responsive system. it was reported that the concentration of α-amylase could increase by 85-fold in the tumor environment [101] , so it is an effective anticancer therapeutic approach to design glycosylated drug carriers to release anticancer drugs upon catalysis of glycosidase. for example, scanlan et al. used glycosylated 1,8-naphthalimide as a fluorescent probe for tumor treatment and diagnosis. the glycosylated 4-amino-1,8-naphthalimide derivatives with chemical structures including the glycosidic bond can be selectively hydrolyzed by glycosidase to release naphthalimide. it was shown that naphthalimide was uptaken by cells only after the glycan unit was hydrolyzed, indicating high targeting capability of the delivery system [102] (figure 4) . nmofs degraded zif-8, thereby releasing insulin and vegf aptamers [97] . besides, nqo1 has been widely used in stimulus-responsive therapy systems due to its overexpression in tumors and other diseases [98, 99] . in a recent report, a nqo1-responsive multifunctional polymeric vesicle covalently bound to a photosensitizer (coumarin and nile blue) was prepared. without being triggered by nqo1, both fluorescence emission and photodynamic therapy (pdt) capability were turned off due to the "double quenching" effect. upon cellular uptake, highly expressed nqo1 triggered the selfimmolative cleavage of the quinone trimethyl lock, which then led to the release of photosensitizers, near infrared (nir) emission and pdt activation, thereby realizing real-time monitoring and treatment of cancers [100] . glucosidase is an enzyme that can hydrolyze glycosidic bonds. its concentration in diseased tissues can be higher than that in normal tissues. therefore, glucosidase can be used to design enzyme-responsive system. it was reported that the concentration of α-amylase could increase by 85fold in the tumor environment [101] , so it is an effective anticancer therapeutic approach to design glycosylated drug carriers to release anticancer drugs upon catalysis of glycosidase. for example, scanlan et al. used glycosylated 1,8-naphthalimide as a fluorescent probe for tumor treatment and diagnosis. the glycosylated 4-amino-1,8-naphthalimide derivatives with chemical structures including the glycosidic bond can be selectively hydrolyzed by glycosidase to release naphthalimide. it was shown that naphthalimide was uptaken by cells only after the glycan unit was hydrolyzed, indicating high targeting capability of the delivery system [102] (figure 4 ). in spite of extensive development of enzyme-responsive systems, there are many drawbacks for them. the level of enzyme expression might be different in different patients, so whether they are sufficiently expressed in a target population is questionable. in addition, the specificity might be another issue. for example, various types of mmps have cross-reactivity. another consideration is whether the cleavage of the enzyme-responsive substrates and polymers is feasible in complex biological environment. the therapeutic effect of one single enzyme stimulus-responsive system might not be specific enough. in spite of extensive development of enzyme-responsive systems, there are many drawbacks for them. the level of enzyme expression might be different in different patients, so whether they are sufficiently expressed in a target population is questionable. in addition, the specificity might be another issue. for example, various types of mmps have cross-reactivity. another consideration is whether the cleavage of the enzyme-responsive substrates and polymers is feasible in complex biological environment. the therapeutic effect of one single enzyme stimulus-responsive system might not be specific enough. solid tumors have a variety of physiological barriers (such as high interstitial pressure and dense extracellular matrix) which affect the uptake of nanoparticles [103] . photothermal therapy is a minimally-invasive treatment for cancer, which relies on the thermal stress caused by light irradiation at a specific wavelength [104, 105] . pdt is another commonly used light-triggered strategy for disease treatments. some examples of photosensitizers include photofrin, visudyne, chlorin e6 and oligo (p-phenylene vinylene) derivative (opv). they can be activated by light to produce ros, which can not only directly kill cancer cells, but also induce vascular damage, cause membrane oxidation and affect the permeability of cell membrane, which facilitates the penetration of anticancer drugs [32] . in a recent report, researchers designed a nano drug carrier that combines photosensitizer protoporphyrin, chemical drug doxorubicin (dox) and apatite (apa). with light stimulation, dox and apa were released and ros was generated owing to porphyrins. apa competed with p-glycoprotein (p-gp) transporter to reduce its enzyme catalytic activity and dox was used to treat tumor tissues; thus, the synergistic treatment of chemotherapy and phototherapy was achieved [106] (figure 5 ). solid tumors have a variety of physiological barriers (such as high interstitial pressure and dense extracellular matrix) which affect the uptake of nanoparticles [103] . photothermal therapy is a minimally-invasive treatment for cancer, which relies on the thermal stress caused by light irradiation at a specific wavelength [104, 105] . pdt is another commonly used light-triggered strategy for disease treatments. some examples of photosensitizers include photofrin, visudyne, chlorin e6 and oligo (pphenylene vinylene) derivative (opv). they can be activated by light to produce ros, which can not only directly kill cancer cells, but also induce vascular damage, cause membrane oxidation and affect the permeability of cell membrane, which facilitates the penetration of anticancer drugs [32] . in a recent report, researchers designed a nano drug carrier that combines photosensitizer protoporphyrin, chemical drug doxorubicin (dox) and apatite (apa). with light stimulation, dox and apa were released and ros was generated owing to porphyrins. apa competed with pglycoprotein (p-gp) transporter to reduce its enzyme catalytic activity and dox was used to treat tumor tissues; thus, the synergistic treatment of chemotherapy and phototherapy was achieved [106] ( figure 5 ). the mechanism of smart acp-dox + apa micelles against multidrug resistance (mdr), for promoting the synergistic antitumor efficacy. reproduced with permission from the publisher of corresponding reference [106] . the mechanism of smart acp-dox + apa micelles against multidrug resistance (mdr), for promoting the synergistic antitumor efficacy. reproduced with permission from the publisher of corresponding reference [106] . in addition to photothermal therapy and pdt, light-responsive strategies have also been applied in the design of prodrug systems and drug delivery carriers. light-responsive structure is generally a robust strategy because it is easy to control with good accuracy and minimal invasiveness. light-responsive systems for drug release are often based on the cleavage of prodrugs with light-sensitive structures or the changes of photosensitive molecules upon light stimulation, so as to release the conjugated or encapsulated drugs [107, 108] . lovell et al. developed near-infrared light-responsive liposomes doped with hexyloxyethy-pyropheophorbide (hpph) entrapped into the bilayers. the anticancer drug doxorubicin (dox) was encapsulated inside the light-sensitive liposomes. when nir light was irradiated at the target site, hpph-liposomes opened the bilayer structure and then released dox, but when nir light was off, the stable bilayer structure of hpph-liposomes was reformed [109] . common photolytic functional groups include o-nitrobenzyl derivatives (onb), anthracene [110] , coumarin esters [111] , arylmethyl [112] and pyrenylmethyl ester [113] (table 3) . among these, o-nitrobenzyl derivatives have been widely used because of their fast photolysis rate and simple synthesis process. for example, willner et al. designed a light-responsive and ph-responsive dna microcapsule. light-responsive capsules are assembled by o-nitrobenzyl phosphate groups with dna layers used to stabilize the microcapsule shell (table 3) . when stimulated by light, o-nitrobenzyl groups were cleaved, causing the degradation of microcapsules and the release of the cargo [114] . another report showed that the o-nitrobenzyl linker containing carbamate bond had a better photolysis rate and a slower hydrolysis rate than other o-nitrobenzyl derivatives (containing an ester bond or amide bond), so that it could maintain higher stability in vivo and can degrade faster after light stimulation [115] . besides, baker et al. chose onb group as the core of photosensitive protective group and conjugated with poly (amidoamine) (pamam) dendrimer to develop a drug delivery platform for methotrexate (mtx) loading. it was demonstrated that this photochemical approach can be used for in vivo delivery of anticancer drugs [116] . ultraviolet-visible (uv-vis) light could be used for the trigger of o-nitrobenzyl derivatives and aryl methyl photolysis functional groups; however, it is difficult to achieve tissue penetration for better therapeutic performance. compared with uv and visible light, near-infrared light with wavelengths in the 700-1000 nm range can penetrate into deeper tissues. nir could be used to cleave, isomerize or rearrange molecules through the two-photon absorption (tpa) process and nir-uv upconversion (uc) process. the theoretical analysis on the principle of tpa process was first proposed in 1930s [117] . however, the generation of tpa requires high excitation energy, and there are still some problems in practical applications. in the uc process, the luminescence center can absorb the energy of two or more photons, and then generate one emitted photon whose energy is higher than that of a single excited photon. additionally, unlike the tpa process, each process of photon absorption is like a second-order element reaction due to the existence of a real intermediate state in the uc process, which has a higher probability than the simultaneous absorption of two molecules [117, 118] . upconversion nanoparticles (ucnps) based on rare earth ions provide a new method for the nir light-responsive system. for example, zou and his colleagues designed a near-infrared light-triggered nanocomposite peg-nmab-pla-ucnps (pnp-uc) to achieve photo-controllable release of dox for cancer treatment. under the irradiation of 980 nm nir light, 2-nitrobenzyl group was triggered for its cleavage to release dox. compared with other ucnps-based nir responsive systems, this new nanocomposite has many advantages, such as high drug encapsulation efficiency, fast light responsiveness at low power density and less heating effect, showing potential in pharmaceutical and biomedical applications [119] . common photoisomerization materials include azobenzene-based materials, spiropyran (sp)-based materials, and diarylethene-based materials (table 3) . azobenzene is an azo group containing an aryl group, with cis and trans isomers. it shows a significant π-π transition in the uv region and a faint π-π transition in the visible region. therefore, upon irradiation of uv light, the trans structure of azobenzene changes into cis structure. additionally with heat or visible light, azobenzene can change from cis structure to trans structure [120] . liu and colleagues assembled azobenzene-functionalized dna strands using ucnp to construct nano-pumps and load dox into nano-pumps. with 980 nm nir irradiation, the continuous rotational inversion motion of azo molecules caused dna hybridization and de-hybridization, thereby leading to the release of dox. this novel light-responsive drug delivery system has shown good biocompatibility and excellent performance in both in vitro and in vivo therapies [121] . sps are an important photoisomeric compounds. under the uv light, the spiro-c-o bond in the spiropyran structure breaks open, leading to an increase in the degree of pi-electron conjugation of the whole molecular system, forming a long conjugate chain and the colored ring-opened merocyanine (mc). mc can be reversibly transformed into a colorless ring-closed sp under specific wavelength or heating conditions. taking the advantage of this special property of sp, a novel n,n-bis(acryloyl) cystamine crosslinked poly (acrylic acid-co-spiropyran methacrylate) nanogel containing disulfide bonds was recently reported as a multi-stimulus responsive nanocarrier [122] . upon the stimulation of low ph value or uv light, the hydrophobic sp isomerizes into hydrophilic mc, making the nanogel swollen. with the addition of reductant agent, the structure of nanogel was destroyed after disulfide bond was oxidized and cleaved, which caused the release of the loaded drug. cytotoxicity experiments showed that dox-loaded nanogels could effectively kill cancer cells, while their cytotoxicity could be enhanced by uv light irradiation. in addition, isomerized mcs in nanogels could emit strong green light after illumination, so they could also be used for fluorescent cell imaging. dna hybridization and de-hybridization, thereby leading to the release of dox. this novel lightresponsive drug delivery system has shown good biocompatibility and excellent performance in both in vitro and in vivo therapies [121] . sps are an important photoisomeric compounds. under the uv light, the spiro-c-o bond in the spiropyran structure breaks open, leading to an increase in the degree of pi-electron conjugation of the whole molecular system, forming a long conjugate chain and the colored ring-opened merocyanine (mc). mc can be reversibly transformed into a colorless ringclosed sp under specific wavelength or heating conditions. taking the advantage of this special property of sp, a novel n,n-bis(acryloyl) cystamine crosslinked poly (acrylic acid-co-spiropyran methacrylate) nanogel containing disulfide bonds was recently reported as a multi-stimulus responsive nanocarrier [122] . upon the stimulation of low ph value or uv light, the hydrophobic sp isomerizes into hydrophilic mc, making the nanogel swollen. with the addition of reductant agent, the structure of nanogel was destroyed after disulfide bond was oxidized and cleaved, which caused the release of the loaded drug. cytotoxicity experiments showed that dox-loaded nanogels could effectively kill cancer cells, while their cytotoxicity could be enhanced by uv light irradiation. in addition, isomerized mcs in nanogels could emit strong green light after illumination, so they could also be used for fluorescent cell imaging. anthracene [110] coumarinyl ester [111] arylmethyl [112] pyrenylmethyl ester [113] o-nitrobenzyl [114] [115] [116] azobenzene [120, 121] spiropyan [122] [110] coumarinyl ester dna hybridization and de-hybridization, thereby leading to the release of dox. this novel lightresponsive drug delivery system has shown good biocompatibility and excellent performance in both in vitro and in vivo therapies [121] . sps are an important photoisomeric compounds. under the uv light, the spiro-c-o bond in the spiropyran structure breaks open, leading to an increase in the degree of pi-electron conjugation of the whole molecular system, forming a long conjugate chain and the colored ring-opened merocyanine (mc). mc can be reversibly transformed into a colorless ringclosed sp under specific wavelength or heating conditions. taking the advantage of this special property of sp, a novel n,n-bis(acryloyl) cystamine crosslinked poly (acrylic acid-co-spiropyran methacrylate) nanogel containing disulfide bonds was recently reported as a multi-stimulus responsive nanocarrier [122] . upon the stimulation of low ph value or uv light, the hydrophobic sp isomerizes into hydrophilic mc, making the nanogel swollen. with the addition of reductant agent, the structure of nanogel was destroyed after disulfide bond was oxidized and cleaved, which caused the release of the loaded drug. cytotoxicity experiments showed that dox-loaded nanogels could effectively kill cancer cells, while their cytotoxicity could be enhanced by uv light irradiation. in addition, isomerized mcs in nanogels could emit strong green light after illumination, so they could also be used for fluorescent cell imaging. anthracene [110] coumarinyl ester [111] arylmethyl [112] pyrenylmethyl ester [113] o-nitrobenzyl [114] [115] [116] azobenzene [120, 121] spiropyan [122] [111] arylmethyl dna hybridization and de-hybridization, thereby leading to the release of dox. this novel lightresponsive drug delivery system has shown good biocompatibility and excellent performance in both in vitro and in vivo therapies [121] . sps are an important photoisomeric compounds. under the uv light, the spiro-c-o bond in the spiropyran structure breaks open, leading to an increase in the degree of pi-electron conjugation of the whole molecular system, forming a long conjugate chain and the colored ring-opened merocyanine (mc). mc can be reversibly transformed into a colorless ringclosed sp under specific wavelength or heating conditions. taking the advantage of this special property of sp, a novel n,n-bis(acryloyl) cystamine crosslinked poly (acrylic acid-co-spiropyran methacrylate) nanogel containing disulfide bonds was recently reported as a multi-stimulus responsive nanocarrier [122] . upon the stimulation of low ph value or uv light, the hydrophobic sp isomerizes into hydrophilic mc, making the nanogel swollen. with the addition of reductant agent, the structure of nanogel was destroyed after disulfide bond was oxidized and cleaved, which caused the release of the loaded drug. cytotoxicity experiments showed that dox-loaded nanogels could effectively kill cancer cells, while their cytotoxicity could be enhanced by uv light irradiation. in addition, isomerized mcs in nanogels could emit strong green light after illumination, so they could also be used for fluorescent cell imaging. anthracene [110] coumarinyl ester [111] arylmethyl [112] pyrenylmethyl ester [113] o-nitrobenzyl [114] [115] [116] azobenzene [120, 121] spiropyan [122] [112] pyrenylmethyl ester dna hybridization and de-hybridization, thereby leading to the release of dox. this novel lightresponsive drug delivery system has shown good biocompatibility and excellent performance in both in vitro and in vivo therapies [121] . sps are an important photoisomeric compounds. under the uv light, the spiro-c-o bond in the spiropyran structure breaks open, leading to an increase in the degree of pi-electron conjugation of the whole molecular system, forming a long conjugate chain and the colored ring-opened merocyanine (mc). mc can be reversibly transformed into a colorless ringclosed sp under specific wavelength or heating conditions. taking the advantage of this special property of sp, a novel n,n-bis(acryloyl) cystamine crosslinked poly (acrylic acid-co-spiropyran methacrylate) nanogel containing disulfide bonds was recently reported as a multi-stimulus responsive nanocarrier [122] . upon the stimulation of low ph value or uv light, the hydrophobic sp isomerizes into hydrophilic mc, making the nanogel swollen. with the addition of reductant agent, the structure of nanogel was destroyed after disulfide bond was oxidized and cleaved, which caused the release of the loaded drug. cytotoxicity experiments showed that dox-loaded nanogels could effectively kill cancer cells, while their cytotoxicity could be enhanced by uv light irradiation. in addition, isomerized mcs in nanogels could emit strong green light after illumination, so they could also be used for fluorescent cell imaging. anthracene [110] coumarinyl ester [111] arylmethyl [112] pyrenylmethyl ester [113] o-nitrobenzyl [114] [115] [116] azobenzene [120, 121] spiropyan [122] [113] o-nitrobenzyl dna hybridization and de-hybridization, thereby leading to the release of dox. this novel lightresponsive drug delivery system has shown good biocompatibility and excellent performance in both in vitro and in vivo therapies [121] . sps are an important photoisomeric compounds. under the uv light, the spiro-c-o bond in the spiropyran structure breaks open, leading to an increase in the degree of pi-electron conjugation of the whole molecular system, forming a long conjugate chain and the colored ring-opened merocyanine (mc). mc can be reversibly transformed into a colorless ringclosed sp under specific wavelength or heating conditions. taking the advantage of this special property of sp, a novel n,n-bis(acryloyl) cystamine crosslinked poly (acrylic acid-co-spiropyran methacrylate) nanogel containing disulfide bonds was recently reported as a multi-stimulus responsive nanocarrier [122] . upon the stimulation of low ph value or uv light, the hydrophobic sp isomerizes into hydrophilic mc, making the nanogel swollen. with the addition of reductant agent, the structure of nanogel was destroyed after disulfide bond was oxidized and cleaved, which caused the release of the loaded drug. cytotoxicity experiments showed that dox-loaded nanogels could effectively kill cancer cells, while their cytotoxicity could be enhanced by uv light irradiation. in addition, isomerized mcs in nanogels could emit strong green light after illumination, so they could also be used for fluorescent cell imaging. anthracene [110] coumarinyl ester [111] arylmethyl [112] pyrenylmethyl ester [113] o-nitrobenzyl [114] [115] [116] azobenzene [120, 121] spiropyan [122] [ dna hybridization and de-hybridization, thereby leading to the release of dox. this novel lightresponsive drug delivery system has shown good biocompatibility and excellent performance in both in vitro and in vivo therapies [121] . sps are an important photoisomeric compounds. under the uv light, the spiro-c-o bond in the spiropyran structure breaks open, leading to an increase in the degree of pi-electron conjugation of the whole molecular system, forming a long conjugate chain and the colored ring-opened merocyanine (mc). mc can be reversibly transformed into a colorless ringclosed sp under specific wavelength or heating conditions. taking the advantage of this special property of sp, a novel n,n-bis(acryloyl) cystamine crosslinked poly (acrylic acid-co-spiropyran methacrylate) nanogel containing disulfide bonds was recently reported as a multi-stimulus responsive nanocarrier [122] . upon the stimulation of low ph value or uv light, the hydrophobic sp isomerizes into hydrophilic mc, making the nanogel swollen. with the addition of reductant agent, the structure of nanogel was destroyed after disulfide bond was oxidized and cleaved, which caused the release of the loaded drug. cytotoxicity experiments showed that dox-loaded nanogels could effectively kill cancer cells, while their cytotoxicity could be enhanced by uv light irradiation. in addition, isomerized mcs in nanogels could emit strong green light after illumination, so they could also be used for fluorescent cell imaging. anthracene [110] coumarinyl ester [111] arylmethyl [112] pyrenylmethyl ester [113] o-nitrobenzyl [114] [115] [116] azobenzene [120, 121] spiropyan [122] [120 dna hybridization and de-hybridization, thereby leading to the release of dox. this novel lightresponsive drug delivery system has shown good biocompatibility and excellent performance in both in vitro and in vivo therapies [121] . sps are an important photoisomeric compounds. under the uv light, the spiro-c-o bond in the spiropyran structure breaks open, leading to an increase in the degree of pi-electron conjugation of the whole molecular system, forming a long conjugate chain and the colored ring-opened merocyanine (mc). mc can be reversibly transformed into a colorless ringclosed sp under specific wavelength or heating conditions. taking the advantage of this special property of sp, a novel n,n-bis(acryloyl) cystamine crosslinked poly (acrylic acid-co-spiropyran methacrylate) nanogel containing disulfide bonds was recently reported as a multi-stimulus responsive nanocarrier [122] . upon the stimulation of low ph value or uv light, the hydrophobic sp isomerizes into hydrophilic mc, making the nanogel swollen. with the addition of reductant agent, the structure of nanogel was destroyed after disulfide bond was oxidized and cleaved, which caused the release of the loaded drug. cytotoxicity experiments showed that dox-loaded nanogels could effectively kill cancer cells, while their cytotoxicity could be enhanced by uv light irradiation. in addition, isomerized mcs in nanogels could emit strong green light after illumination, so they could also be used for fluorescent cell imaging. anthracene [110] coumarinyl ester [111] arylmethyl [112] pyrenylmethyl ester [113] o-nitrobenzyl [114] [115] [116] azobenzene [120, 121] diarylethene [123] [124] [125] [126] [127] [128] [129] [130] [131] diarylethenes (daes) are also popular photoisomeric molecules with many useful properties, such as easy modification, fatigue resistance, high thermal stability and significant changes in optical and electronic properties after photoisomerization [123] ( table 3 ). the performance and application of diaryl ethylene mainly depend on the modification of its parental structure, that is, the design and selection process of alkene bridges and aryl units. one common way is the modification of aryl units such as substituting the benzene ring of diaryl ethylene with thiophene ring [124] (which could improve the stability of the closed loop) and modifying the alkene bridge using five-membered rings [123] [124] [125] [126] [127] [128] [129] [130] [131] diarylethenes (daes) are also popular photoisomeric molecules with many useful properties, such as easy modification, fatigue resistance, high thermal stability and significant changes in optical and electronic properties after photoisomerization [123] (table 3) . the performance and application of diaryl ethylene mainly depend on the modification of its parental structure, that is, the design and selection process of alkene bridges and aryl units. one common way is the modification of aryl units such as substituting the benzene ring of diaryl ethylene with thiophene ring [124] (which could improve the stability of the closed loop) and modifying the alkene bridge using five-membered rings such as alkene bridges (such as cyclopentene [125] , thiazole [126] , imidazole [127] , furan [128] ) or using six-membered rings (such as benzoquinone [129] , coumarin fluorophores [130] , benzothiazole [131] ). in addition, some light-sensitive molecules-vitamin b12 derivatives [132, 133] , ruthenium complexes [134] , etc.-have also been applied to light-sensitive systems. still, further development of light-stimulated systems is hindered by some factors. for example, for the treatment of solid tumors, penetration depth, effective area, power intensity and irradiation time are some important factors to be considered for the light-responsive therapy. in addition, many photosensitizers and photothermal agents have inherent toxicity or photo-toxicity. ongoing research is still making efforts for the application of light stimulation systems for clinical advances. temperature is a frequently studied stimuli for responsive delivery systems. inflammatory pathological sites and the hyperthermic nature of tumors can be used as internal stimuli [135, 136] . another strategy is to increase the temperature by applying an external heat source. ideally, the thermal response nanocarrier should maintain its stability at body temperature and release the drug rapidly when the diseased site is heated. thermal stimulus-responsive polymers usually have lower critical solubility temperature (lcst) or upper critical solubility temperature (ucst), and the former is more widely used. for example, poly (n-isopropylacrylamide) (pnipam) and its derivatives have been used in the design of thermal stimulation response systems because their corresponding lcst is about 32 • c, which is close to the physiological temperature of human body [137] [138] [139] . a core-shell drug delivery system was designed to improve the solubility of drug by encapsulating hydrophobic drugs in nanoparticles with thermo-responsive materials as the shell. luo et al. developed a core-shell micelle that used hydrophilic thermo-responsive material pnipam as a shell for the encapsulation of the drug of paclitaxel (ptx). both drug and shell were conjugated by diselenide bond (pnipam-sese-ptx). the nanoparticles exhibited dual characteristics of temperature-responsiveness and redox-responsiveness, with high loading capacity (54.1%) and encapsulation efficiency (72.3%) [140] . compared with pnipam that cannot be degraded in vivo, temperature-sensitive oligomers containing oligo (ethylene glycol) (oeg) potentially has better biocompatibility. jayakannan et al. developed a dual stimulus-responsive amphiphilic copolymer formed by copolymerization of hydrophobic polymerizable monomers with methacrylamide based on hydrophobic 3-pentadecylphenol (pdp) and oligopolyethylene glycol (peg) methacrylate, which thus was temperature sensitive and enzyme sensitive. the nanoparticles formed from the amphiphilic copolymer, which remained stable in normal tissues in vivo with less than 20% drug release, while above lcst, the nanoparticles had an up to 90% drug release rate in two hours due to the thermal response property of acrylamide copolymers. in the presence of esterase, the drug release rate could exceed 95% within 12 h. such type of dox-polymer showed better anticancer efficacy [141] . in addition to thermosensitive hydrogels and thermosensitive polymers, temperature-sensitive liposomes have also been investigated. since yatvin first proposed the concept of temperature-sensitive liposomes (tsl) in the late 1970s [8] , tsl has been attracting much attention from molecular design to clinical applications. thermodox is an example of temperature-sensitive liposomes, which was tested in phase iii clinical trials for hepatocellular carcinoma and phase ii clinical trials for breast cancer. its drug release rate is 5-50 fold higher than other liposomal drugs at elevated temperatures. porter et al. developed a novel polymer-modified temperature-sensitive liposome (ptsl) for the delivery of dox for the treatment of cancers. by reversible addition-break chain transfer (raft) polymerization of n-isopropylacrylamide (nipaam) and ph-responsive propylacrylic acid (paa) copolymers with temperature responsiveness, copolymers with dual ph/temperature phase transition properties were obtained. when attached to liposomes, these copolymers can cause membrane destruction with a ph/temperature-dependent manner. compared with traditional thermosensitive formulations, ptsl exhibited enhanced release profiles, significantly reduced thermal dose thresholds and better stability in serum with minimal drug leakage over time. therefore, these liposomes have the potential of significantly reducing damage to healthy tissue commonly associated with liposomal cancer therapies [142] (figure 6 ). was tested in phase iii clinical trials for hepatocellular carcinoma and phase ii clinical trials for breast cancer. its drug release rate is 5-50 fold higher than other liposomal drugs at elevated temperatures. porter et al. developed a novel polymer-modified temperature-sensitive liposome (ptsl) for the delivery of dox for the treatment of cancers. by reversible addition-break chain transfer (raft) polymerization of n-isopropylacrylamide (nipaam) and ph-responsive propylacrylic acid (paa) copolymers with temperature responsiveness, copolymers with dual ph/temperature phase transition properties were obtained. when attached to liposomes, these copolymers can cause membrane destruction with a ph/temperature-dependent manner. compared with traditional thermosensitive formulations, ptsl exhibited enhanced release profiles, significantly reduced thermal dose thresholds and better stability in serum with minimal drug leakage over time. therefore, these liposomes have the potential of significantly reducing damage to healthy tissue commonly associated with liposomal cancer therapies [142] (figure 6 ). for temperature-sensitive systems, thermo-specificity is one of the biggest issues because it is usually hard for chemistry to be exquisitely specific. for the future research directions, temperatureresponsive drug carriers with higher sensitivity to the minimum temperature change, better stability and enhanced safety profile in normal tissues are greatly desired. compared with other stimuli such as light radiation and ultrasonic, magnetic force is an intriguing condition for external stimuli-responsiveness because it has almost no physical interaction with the human body. additionally, owing to spatial focusing, magnetic stimulus-responsive systems can overcome some limitations of traditional delivery systems, such as difficulty of passing through physiological barrier in vivo and lack of specificity to diseased tissues. magnetic fields was first proposed as external triggers for drug release in 1960 [143] . magnetic hyperthermia caused by the rotation of magnetic nanoparticles when exposed to alternating magnetic field, and the heat generated by magnetic loss will dissipate to the surrounding tissues. it is generally believed that the heat generated by magnetic nanoparticles is based on mechanisms of internal rotation axis and external movement, namely, thermal rotation and diffusion relaxation of magnetic moment. in terms of local drug release, the thermodynamic phase and conformation transition of polymeric for temperature-sensitive systems, thermo-specificity is one of the biggest issues because it is usually hard for chemistry to be exquisitely specific. for the future research directions, temperature-responsive drug carriers with higher sensitivity to the minimum temperature change, better stability and enhanced safety profile in normal tissues are greatly desired. compared with other stimuli such as light radiation and ultrasonic, magnetic force is an intriguing condition for external stimuli-responsiveness because it has almost no physical interaction with the human body. additionally, owing to spatial focusing, magnetic stimulus-responsive systems can overcome some limitations of traditional delivery systems, such as difficulty of passing through physiological barrier in vivo and lack of specificity to diseased tissues. magnetic fields was first proposed as external triggers for drug release in 1960 [143] . magnetic hyperthermia caused by the rotation of magnetic nanoparticles when exposed to alternating magnetic field, and the heat generated by magnetic loss will dissipate to the surrounding tissues. it is generally believed that the heat generated by magnetic nanoparticles is based on mechanisms of internal rotation axis and external movement, namely, thermal rotation and diffusion relaxation of magnetic moment. in terms of local drug release, the thermodynamic phase and conformation transition of polymeric nanoparticles depends on their lcst/ucst and then expands/contracts, possibly leading to the drug release [144, 145] . zink et al. developed a magnetic nanoparticle with superparamagnetic particles as the core and thermo-responsive peptide phe-phe-glycine-glycine (n-porcine linolenic acid a) as the nano-valve. this novel class of mesoporous silica nanoparticles (msns) could maintain stability and safety under normal environment in human body. once the magnetic core is triggered by magnetic field, the heat generated can lead to the change of its structure, giving rise to the release of drugs [146] (figure 7) . nanoparticles depends on their lcst/ucst and then expands/contracts, possibly leading to the drug release [144, 145] . zink et al. developed a magnetic nanoparticle with superparamagnetic particles as the core and thermo-responsive peptide phe-phe-glycine-glycine (n-porcine linolenic acid a) as the nano-valve. this novel class of mesoporous silica nanoparticles (msns) could maintain stability and safety under normal environment in human body. once the magnetic core is triggered by magnetic field, the heat generated can lead to the change of its structure, giving rise to the release of drugs [146] ( figure 7 ). magnetic transfection or magnetically-aided transfection is a method to control gene delivery and improve transfection efficiency for gene therapy. the magnetic transfection method is based on the principle of magnetic targeted drug delivery proposed by widder et al. in 1978 [147] . in 2002, mah et al. first linked the magnetic microspheres to recombinant adeno-associated virus 2 (aav2) gene vector through heparin for c12s cells and mice in vivo [148] . in the same year, plank et al. associated gene carriers with superparamagnetic iron oxide nanoparticles coated with polycation polyethyleneimine. it was shown that the magnetic-assisted transfection method improved the transfection efficiency of vectors by at least three orders of magnitude by either viral or non-viral vectors [149] . ultrasound is also commonly used in stimulus responsive system. compared with other external stimuli, ultrasound is non-invasive and can improve drug release profile and drug permeability. the mechanism is based on acoustic cavitation, which means the formation and activity of gas-filled bubbles in the medium under the action of ultrasound [150] . these gas-filled bubbles can be produced either naturally or manually (microbubbles, mbs). with microbubbles, ultrasound cavitation affects the permeability of the plasma membrane, enhances cell uptake of drugs, or allows internalization of other cell-impermeable substances. even in the absence of microbubbles, highintensity ultrasound can still induce drug uptake by cells. these characteristics enable ultrasoundmediated drug delivery systems to improve the absorption efficiency of weak or non-permeable drugs by cells [151] . porous lipid polymer hybrid microbubbles (lipid/plga mbs) have also been developed using water/oil/water (w/o/w) double emulsification process, which addressed the limitation of low drug encapsulation efficiency of lipid-based mbs and poor ultrasound imaging ability of polymer-based mbs. compared with pure plga mbs, lipid/plga mbs had hollow microcapsule structure, which reduced the cavitation threshold intensity and enhanced the ultrasonic imaging ability of mbs. in addition, the increased surface area and porous structure of lipid/plga mbs make them have good drug encapsulation properties. using this method, controllable drug release and real-time monitoring of drug release could be achieved [152] . another example is that zheng et al. developed ultrasound stimulus-responsive microbubbles for ultrasound imaging, which magnetic transfection or magnetically-aided transfection is a method to control gene delivery and improve transfection efficiency for gene therapy. the magnetic transfection method is based on the principle of magnetic targeted drug delivery proposed by widder et al. in 1978 [147] . in 2002, mah et al. first linked the magnetic microspheres to recombinant adeno-associated virus 2 (aav2) gene vector through heparin for c12s cells and mice in vivo [148] . in the same year, plank et al. associated gene carriers with superparamagnetic iron oxide nanoparticles coated with polycation polyethyleneimine. it was shown that the magnetic-assisted transfection method improved the transfection efficiency of vectors by at least three orders of magnitude by either viral or non-viral vectors [149] . ultrasound is also commonly used in stimulus responsive system. compared with other external stimuli, ultrasound is non-invasive and can improve drug release profile and drug permeability. the mechanism is based on acoustic cavitation, which means the formation and activity of gas-filled bubbles in the medium under the action of ultrasound [150] . these gas-filled bubbles can be produced either naturally or manually (microbubbles, mbs). with microbubbles, ultrasound cavitation affects the permeability of the plasma membrane, enhances cell uptake of drugs, or allows internalization of other cell-impermeable substances. even in the absence of microbubbles, high-intensity ultrasound can still induce drug uptake by cells. these characteristics enable ultrasound-mediated drug delivery systems to improve the absorption efficiency of weak or non-permeable drugs by cells [151] . porous lipid polymer hybrid microbubbles (lipid/plga mbs) have also been developed using water/oil/water (w/o/w) double emulsification process, which addressed the limitation of low drug encapsulation efficiency of lipid-based mbs and poor ultrasound imaging ability of polymer-based mbs. compared with pure plga mbs, lipid/plga mbs had hollow microcapsule structure, which reduced the cavitation threshold intensity and enhanced the ultrasonic imaging ability of mbs. in addition, the increased surface area and porous structure of lipid/plga mbs make them have good drug encapsulation properties. using this method, controllable drug release and real-time monitoring of drug release could be achieved [152] . another example is that zheng et al. developed ultrasound stimulus-responsive microbubbles for ultrasound imaging, which can be converted into porphyrin nanomaterials with fluorescent and photoacoustic activity upon low-frequency ultrasound pulses. larger microcarriers could reach the tumor vasculature (independent of epr effect), and its principle is that the energy of ultrasound could rupture microbubbles, pushing nanomaterials into the tumor interstitium. these nanomaterials (porphyrins) could further utilize photoacoustic imaging (pai) and fluorescence for multistep multimodal imaging in tumor-bearing mice [153] . liu et al. recently designed an ultrasound responsive photoacoustic (pa) probe based on microbubbles containing gold nanoparticles. in this design, gold nanoparticles were encapsulated in the lipid shell of mbs and filled with sulfur hexafluoride gas, thereby forming mbs with strong cavitation characteristics and low toxicity. au@lip mbs exhibited lower nir pa signal. when triggered by ultrasound, mbs was ruptured and au@lip aggregates were formed, showing enhanced nir pa signals. background-free pa imaging was achieved by subtracting the pa image before us stimulation from the pa image after us stimulation [154] (figure 8 ). although ultrasound-responsive nanotechnology shows some advantages, its side effects (such as skin irritations, transient pain and nerve injury) should be also considered [155, 156] . the intensity, frequency and duty cycle should be carefully selected for the optimized therapeutic effects and reduced side effects. can be converted into porphyrin nanomaterials with fluorescent and photoacoustic activity upon low-frequency ultrasound pulses. larger microcarriers could reach the tumor vasculature (independent of epr effect), and its principle is that the energy of ultrasound could rupture microbubbles, pushing nanomaterials into the tumor interstitium. these nanomaterials (porphyrins) could further utilize photoacoustic imaging (pai) and fluorescence for multistep multimodal imaging in tumor-bearing mice [153] . liu et al. recently designed an ultrasound responsive photoacoustic (pa) probe based on microbubbles containing gold nanoparticles. in this design, gold nanoparticles were encapsulated in the lipid shell of mbs and filled with sulfur hexafluoride gas, thereby forming mbs with strong cavitation characteristics and low toxicity. au@lip mbs exhibited lower nir pa signal. when triggered by ultrasound, mbs was ruptured and au@lip aggregates were formed, showing enhanced nir pa signals. background-free pa imaging was achieved by subtracting the pa image before us stimulation from the pa image after us stimulation [154] (figure 8 ). although ultrasound-responsive nanotechnology shows some advantages, its side effects (such as skin irritations, transient pain and nerve injury) should be also considered [155, 156] . the intensity, frequency and duty cycle should be carefully selected for the optimized therapeutic effects and reduced side effects. the mechanism of self-immolation is that two chemical bonds in the inactive precursor are associated by the self-immolation spacer. the precursor usually contains a protective group (pg), self-immolation spacer (sis) and a target compound (tc). after appropriate stimulus, the protective group is removed, and then a step-by-step decomposition reaction similar to "domino" is produced, resulting in the release of target compounds (figure 9 ). philip et al. proposed the concept of selfimmolative structures in 1981 that a pristine drug can be released without chemical modification after the bond between the carrier component and the drug component is cleaved [157] . self-immolative linkers have subsequently been widely used in prodrug design [158, 159] , sensors [160, 161] , drug delivery [162, 163] and other applications. the mechanism of self-immolation is that two chemical bonds in the inactive precursor are associated by the self-immolation spacer. the precursor usually contains a protective group (pg), self-immolation spacer (sis) and a target compound (tc). after appropriate stimulus, the protective group is removed, and then a step-by-step decomposition reaction similar to "domino" is produced, resulting in the release of target compounds (figure 9 ). philip et al. proposed the concept of self-immolative structures in 1981 that a pristine drug can be released without chemical modification after the bond between the carrier component and the drug component is cleaved [157] . self-immolative linkers have subsequently been widely used in prodrug design [158, 159] , sensors [160, 161] , drug delivery [162, 163] and other applications. can be converted into porphyrin nanomaterials with fluorescent and photoacoustic activity upon low-frequency ultrasound pulses. larger microcarriers could reach the tumor vasculature (independent of epr effect), and its principle is that the energy of ultrasound could rupture microbubbles, pushing nanomaterials into the tumor interstitium. these nanomaterials (porphyrins) could further utilize photoacoustic imaging (pai) and fluorescence for multistep multimodal imaging in tumor-bearing mice [153] . liu et al. recently designed an ultrasound responsive photoacoustic (pa) probe based on microbubbles containing gold nanoparticles. in this design, gold nanoparticles were encapsulated in the lipid shell of mbs and filled with sulfur hexafluoride gas, thereby forming mbs with strong cavitation characteristics and low toxicity. au@lip mbs exhibited lower nir pa signal. when triggered by ultrasound, mbs was ruptured and au@lip aggregates were formed, showing enhanced nir pa signals. background-free pa imaging was achieved by subtracting the pa image before us stimulation from the pa image after us stimulation [154] (figure 8 ). although ultrasound-responsive nanotechnology shows some advantages, its side effects (such as skin irritations, transient pain and nerve injury) should be also considered [155, 156] . the intensity, frequency and duty cycle should be carefully selected for the optimized therapeutic effects and reduced side effects. the mechanism of self-immolation is that two chemical bonds in the inactive precursor are associated by the self-immolation spacer. the precursor usually contains a protective group (pg), self-immolation spacer (sis) and a target compound (tc). after appropriate stimulus, the protective group is removed, and then a step-by-step decomposition reaction similar to "domino" is produced, resulting in the release of target compounds (figure 9 ). philip et al. proposed the concept of selfimmolative structures in 1981 that a pristine drug can be released without chemical modification after the bond between the carrier component and the drug component is cleaved [157] . self-immolative linkers have subsequently been widely used in prodrug design [158, 159] , sensors [160, 161] , drug delivery [162, 163] and other applications. the self-immolation mechanism can be categorized into electron rearrangement and intramolecular cyclization. electron rearrangement is mainly based on the structure of quinone or its derivatives (such as thioquinone methide or azaquinone methide). the mechanism of electron rearrangement includes 1,4-elimination, 1,6-elimination, 1,8-elimination and β-elimination. the electron rearrangement reaction usually contain aromatic ring structure with hydroxyl [164, 165] , amino [166, 167] or mercaptan groups [168, 169] . when they are masked by protective groups, the electron rearrangement process of these functional groups is inhibited. when the external stimulus triggers the cleavage of the protective groups, these functional groups undergo irreversible self-immolation process driven by positive entropy or the generation of stable products. common protection groups that can be triggered by various stimuli are summarized in table 4 . self-immolative spacers based on electron rearrangement are mostly based on 1,4-elimination or 1,6-elimination [170, 171] . additionally, 1,8-cleavage may occur in para amino (or hydroxy) cinnamyl alcohol or coumarin alcohol. in contrast, self-immolative spacer groups based on continuous combinations (such as 1,8-elimination of naphthalene or 1,10-elimination of biphenyls) usually do not result in the release of drug groups because the high energy barrier destroys the aromaticity, and the repulsion of the hydrogen atom in adjacent biphenyl prevents the formation of the planar structure needed for electron rearrangement [172] . the cyclization reaction is based on alkyl chain or aromatic spacer groups [173] [174] [175] . once the cleavage is triggered, the nucleophilic attack of carbonyl or electrophilic aliphatic carbon atoms could result in the cyclization of spacer groups. as of the electron rearrangement, the self-immolation cyclization is driven by the formation of positive reaction entropy and thermodynamic stable products (such as 5-membered and 6-membered rings). additionally, the triggers of self-immolation include chemical reagents, enzymes, light and others. self-immolative linkers that can be triggered by chemical reagents; boc(tert-butoxycarbonyl), fmoc(9-fluorenylmethyl) and 3-oxobutyl carbamate are typically ph-responsive for organic synthesis [176] . boc and fmoc are protecting groups for protecting amino groups, and the fmoc is the only widely used amino acid protecting group of carbamates which can be dissociated in weak base condition. these functional groups undergo self-immolation under some specific triggering conditions (trifluoroacetic acid, piperidine), which in turn releases the attached cargo [177, 178] . redox-triggered self-immolative linkers could be broadly divided into three categories by triggers: transition metal (e.g., zn, pd) based reagents [179] [180] [181] , reducing reagents (dtt, gsh or tcep) [182] , and oxidant (e.g., h 2 o 2 ) [183] [184] [185] [186] . disulfide bonds are the most widely used reductant-responsive linkers. wu et al. employed α, α-dimethyl groups with disulfide bonds, so that p-dithiobenzyl (dtb) intermediates could maintain faster self-immolation rate and improve the stability. this novel self-immolative linker is expected to promote the design of targeted drug delivery systems and achieve traceless drug release [187] . similarly, self-immolative linkers with h 2 o 2 as trigger condition have also been widely used in stimulus-responsive systems in various fields. recently, clausen et al. reported the synthesis of arylboronic acid-based hydrogen peroxide-responsive methotrexate and aminopterin prodrugs. this new prodrug can deliver drugs to the lesion of chronic rheumatoid arthritis, so that the side effects of the drug on normal cells were significantly reduced [186] . enzyme sensitive self-immolative linkers are also commonly used in enzyme responsive systems. plasmin can induce the hydrolysis of tripeptides, which in turn causes the cleavage of self-immolative linkers and release of drugs [172, 188] . penicillin g amidase (pga) and bovine serum albumin (bsa) can be used to trigger the cleavage of phenylacetamide or carbamate bonds to release the linked cargo [189, 190] . β-galactoside and β-glucuronide are functional groups that can be cleaved by β-galactosidase and β-glucuronidase, respectively, and these two functional groups can also be combined with other self-immolative linkers [191] [192] [193] [194] . in addition, dibenzyl phosphate derivatives are also used for enzymatic (alkaline phosphatase) self-immolative design for the delivery and release of fluorescent probes and prodrugs [195, 196] . enzymatically activated self-immolation is usually slow and requires synergy with other self-immolative linkers. light-activated self-immolation does not require an additional step for synergy. a variety of similar structures based on nitrobenzyl groups can undergo self-immolation under uv light [197, 198] . examples of self-immolative structure triggered by nir light are polymers with o-nitrobenzyl group as the capping group [199, 200] and polymers based on coumarin derivatives [201, 202] . are polymers with o-nitrobenzyl group as the capping group [199, 200] and polymers based on coumarin derivatives [201, 202] . [176, 177] piperidine/ morpholine ph [178] zn/acoh redox [180] pd (pph3)4 redox [179, 181] dithiothreitol redox [182, 187] thiols redox [182] h2o2 redox [183] [184] [185] [186] plasmin enzyme [172, 188] penicillin-gamidase (pga) enzyme [190] bsa enzyme [189] β-glucuronidase enzyme [191] [192] [193] β-galactosidase enzyme [194] alkaline phosphatase enzyme [195, 196] uv light/ nir light light [197] [198] [199] [200] light [201, 202] h + ph [176, 177] are polymers with o-nitrobenzyl group as the capping group [199, 200] and polymers based on coumarin derivatives [201, 202] . [176, 177] piperidine/ morpholine ph [178] zn/acoh redox [180] pd (pph3)4 redox [179, 181] dithiothreitol redox [182, 187] thiols redox [182] h2o2 redox [183] [184] [185] [186] plasmin enzyme [172, 188] penicillin-gamidase (pga) enzyme [190] bsa enzyme [189] β-glucuronidase enzyme [191] [192] [193] β-galactosidase enzyme [194] alkaline phosphatase enzyme [195, 196] uv light/ nir light light [197] [198] [199] [200] light [201, 202] piperidine/ morpholine ph [178] int. j. mol. sci. 2020, 21, x for peer review 18 of 44 are polymers with o-nitrobenzyl group as the capping group [199, 200] and polymers based on coumarin derivatives [201, 202] . [176, 177] piperidine/ morpholine ph [178] zn/acoh redox [180] pd (pph3)4 redox [179, 181] dithiothreitol redox [182, 187] thiols redox [182] h2o2 redox [183] [184] [185] [186] plasmin enzyme [172, 188] penicillin-gamidase (pga) enzyme [190] bsa enzyme [189] β-glucuronidase enzyme [191] [192] [193] β-galactosidase enzyme [194] alkaline phosphatase enzyme [195, 196] uv light/ nir light light [197] [198] [199] [200] light [201, 202] zn/acoh redox [180] int. j. mol. sci. 2020, 21, x for peer review 18 of 44 are polymers with o-nitrobenzyl group as the capping group [199, 200] and polymers based on coumarin derivatives [201, 202] . [176, 177] piperidine/ morpholine ph [178] zn/acoh redox [180] pd (pph3)4 redox [179, 181] dithiothreitol redox [182, 187] thiols redox [182] h2o2 redox [183] [184] [185] [186] plasmin enzyme [172, 188] penicillin-gamidase (pga) enzyme [190] bsa enzyme [189] β-glucuronidase enzyme [191] [192] [193] β-galactosidase enzyme [194] alkaline phosphatase enzyme [195, 196] uv light/ nir light light [197] [198] [199] [200] light [201, 202] are polymers with o-nitrobenzyl group as the capping group [199, 200] and polymers based on coumarin derivatives [201, 202] . [176, 177] piperidine/ morpholine ph [178] zn/acoh redox [180] pd (pph3)4 redox [179, 181] dithiothreitol redox [182, 187] thiols redox [182] h2o2 redox [183] [184] [185] [186] plasmin enzyme [172, 188] penicillin-gamidase (pga) enzyme [190] bsa enzyme [189] β-glucuronidase enzyme [191] [192] [193] β-galactosidase enzyme [194] alkaline phosphatase enzyme [195, 196] uv light/ nir light light [197] [198] [199] [200] light [201, 202] dithiothreitol redox [182, 187] are polymers with o-nitrobenzyl group as the capping group [199, 200] and polymers based on coumarin derivatives [201, 202] . [176, 177] piperidine/ morpholine ph [178] zn/acoh redox [180] pd (pph3)4 redox [179, 181] dithiothreitol redox [182, 187] thiols redox [182] h2o2 redox [183] [184] [185] [186] plasmin enzyme [172, 188] penicillin-gamidase (pga) enzyme [190] bsa enzyme [189] β-glucuronidase enzyme [191] [192] [193] β-galactosidase enzyme [194] alkaline phosphatase enzyme [195, 196] uv light/ nir light light [197] [198] [199] [200] light [201, 202] thiols redox [182] int. j. mol. sci. 2020, 21, x for peer review 18 of 44 are polymers with o-nitrobenzyl group as the capping group [199, 200] and polymers based on coumarin derivatives [201, 202] . [176, 177] piperidine/ morpholine ph [178] zn/acoh redox [180] pd (pph3)4 redox [179, 181] dithiothreitol redox [182, 187] thiols redox [182] h2o2 redox [183] [184] [185] [186] plasmin enzyme [172, 188] penicillin-gamidase (pga) enzyme [190] bsa enzyme [189] β-glucuronidase enzyme [191] [192] [193] β-galactosidase enzyme [194] alkaline phosphatase enzyme [195, 196] uv light/ nir light light [197] [198] [199] [200] are polymers with o-nitrobenzyl group as the capping group [199, 200] and polymers based on coumarin derivatives [201, 202] . [176, 177] piperidine/ morpholine ph [178] zn/acoh redox [180] pd (pph3)4 redox [179, 181] dithiothreitol redox [182, 187] thiols redox [182] h2o2 redox [183] [184] [185] [186] plasmin enzyme [172, 188] penicillin-gamidase (pga) enzyme [190] bsa enzyme [189] β-glucuronidase enzyme [191] [192] [193] β-galactosidase enzyme [194] alkaline phosphatase enzyme [195, 196] uv light/ nir light light [197] [198] [199] [200] light [201, 202] plasmin enzyme [172, 188] are polymers with o-nitrobenzyl group as the capping group [199, 200] and polymers based on coumarin derivatives [201, 202] . [176, 177] piperidine/ morpholine ph [178] zn/acoh redox [180] pd (pph3)4 redox [179, 181] dithiothreitol redox [182, 187] thiols redox [182] h2o2 redox [183] [184] [185] [186] plasmin enzyme [172, 188] penicillin-gamidase (pga) enzyme [190] bsa enzyme [189] β-glucuronidase enzyme [191] [192] [193] β-galactosidase enzyme [194] alkaline phosphatase enzyme [195, 196] uv light/ nir light light [197] [198] [199] [200] light [201, 202] penicillin-g-amidase (pga) enzyme [190] int. j. mol. sci. 2020, 21, x for peer review 18 of 44 are polymers with o-nitrobenzyl group as the capping group [199, 200] and polymers based on coumarin derivatives [201, 202] . [176, 177] piperidine/ morpholine ph [178] zn/acoh redox [180] pd (pph3)4 redox [179, 181] dithiothreitol redox [182, 187] thiols redox [182] h2o2 redox [183] [184] [185] [186] plasmin enzyme [172, 188] penicillin-gamidase (pga) enzyme [190] bsa enzyme [189] β-glucuronidase enzyme [191] [192] [193] β-galactosidase enzyme [194] alkaline phosphatase enzyme [195, 196] uv light/ nir light light [197] [198] [199] [200] light [201, 202] bsa enzyme [189] int. j. mol. sci. 2020, 21, x for peer review 18 of 44 are polymers with o-nitrobenzyl group as the capping group [199, 200] and polymers based on coumarin derivatives [201, 202] . [176, 177] piperidine/ morpholine ph [178] zn/acoh redox [180] pd (pph3)4 redox [179, 181] dithiothreitol redox [182, 187] thiols redox [182] h2o2 redox [183] [184] [185] [186] plasmin enzyme [172, 188] penicillin-gamidase (pga) enzyme [190] bsa enzyme [189] β-glucuronidase enzyme [191] [192] [193] β-galactosidase enzyme [194] alkaline phosphatase enzyme [195, 196] uv light/ nir light light [197] [198] [199] [200] light [201, 202] β-glucuronidase enzyme [191] [192] [193] are polymers with o-nitrobenzyl group as the capping group [199, 200] and polymers based on coumarin derivatives [201, 202] . [176, 177] piperidine/ morpholine ph [178] zn/acoh redox [180] pd (pph3)4 redox [179, 181] dithiothreitol redox [182, 187] thiols redox [182] h2o2 redox [183] [184] [185] [186] plasmin enzyme [172, 188] penicillin-gamidase (pga) enzyme [190] bsa enzyme [189] β-glucuronidase enzyme [191] [192] [193] β-galactosidase enzyme [194] alkaline phosphatase enzyme [195, 196] uv light/ nir light light [197] [198] [199] [200] light [201, 202] β-galactosidase enzyme [194] int. j. mol. sci. 2020, 21, x for peer review 18 of 44 are polymers with o-nitrobenzyl group as the capping group [199, 200] and polymers based on coumarin derivatives [201, 202] . [176, 177] piperidine/ morpholine ph [178] zn/acoh redox [180] pd (pph3)4 redox [179, 181] dithiothreitol redox [182, 187] thiols redox [182] h2o2 redox [183] [184] [185] [186] plasmin enzyme [172, 188] penicillin-gamidase (pga) enzyme [190] bsa enzyme [189] β-glucuronidase enzyme [191] [192] [193] β-galactosidase enzyme [194] alkaline phosphatase enzyme [195, 196] uv light/ nir light light [197] [198] [199] [200] light [201, 202] alkaline phosphatase enzyme [195, 196] are polymers with o-nitrobenzyl group as the capping group [199, 200] and polymers based on coumarin derivatives [201, 202] . [176, 177] piperidine/ morpholine ph [178] zn/acoh redox [180] pd (pph3)4 redox [179, 181] dithiothreitol redox [182, 187] thiols redox [182] h2o2 redox [183] [184] [185] [186] plasmin enzyme [172, 188] penicillin-gamidase (pga) enzyme [190] bsa enzyme [189] β-glucuronidase enzyme [191] [192] [193] β-galactosidase enzyme [194] alkaline phosphatase enzyme [195, 196] uv light/ nir light light [197] [198] [199] [200] light [201, 202] uv light/ nir light light [197] [198] [199] [200] are polymers with o-nitrobenzyl group as the capping group [199, 200] and polymers based on coumarin derivatives [201, 202] . [176, 177] piperidine/ morpholine ph [178] zn/acoh redox [180] pd (pph3)4 redox [179, 181] dithiothreitol redox [182, 187] thiols redox [182] h2o2 redox [183] [184] [185] [186] plasmin enzyme [172, 188] penicillin-gamidase (pga) enzyme [190] bsa enzyme [189] β-glucuronidase enzyme [191] [192] [193] β-galactosidase enzyme [194] alkaline phosphatase enzyme [195, 196] uv light/ nir light light [197] [198] [199] [200] light [201, 202] nir light light [201, 202] conventional drug delivery systems lack controlled drug release schemes whereas stimulus-responsive drug delivery systems or drug conjugates with rational design can release loaded drugs at specific sites triggered by various endogenous or exogenous stimuli. the human body is a complex collection of various microenvironments, so occasionally it may not suffice if only one stimulation condition is used for the design of stimulus-responsive systems. in this section, we mainly describe the combination of dual or multiple stimuli-responsive conditions for the design of nanoplatforms to improve the specificity and accuracy of nanotherapeutic systems. although single stimulus-responsive systems can solve the problems of specificity and side effects to some extent, the microenvironment within tumor tissues is complex. by contrast, dual or multiple stimuli-responsive nanocarriers can better detect subtle changes in diseased tissues. the traditional combination of ph and temperature has certain defects, such as possible premature drug release in blood or normal tissue species [203, 204] , and the ph responsive system might be affected by various factors. to solve the problem of premature leakage, cui et al. made n-isopropylacrylamide-methacrylic acid-octadecyl acrylate (nipam-maa-oda) copolymer liposomes as nanodrug carriers that can be responsive to dual stimulates of ph and temperature [205] . in recent years, the combination of light and ph has also become a popular choice for dual response systems. for example, researchers have conjugated ph-sensitive i-motif dna (converted from single-stranded structure to c-quadruplex structure in acidic ph environment) to gold nanostars (gns) with as 1411 used as a targeting structure. it has been discovered that a-gns/dna/dox nanocomposites have strong photothermal conversion ability, and the combination of ph and nir irradiation can effectively trigger drug release. this nanocomposite showed good stability in not-carcinogenic tissues, and therapeutic effect and biocompatibility were achieved using the construct for combined chemoand photo-therapy [206] (figure 10 ). in addition to the combination with photothermal therapy, ph responsive systems can also be combined with the uv light-responsive systems. host-guest interaction of β-cyclodextrin with azobenzene has also been studied, but most supramolecular polymer drug carriers are single stimulus-responsive with low delivery accuracy [207] [208] [209] . chen et al. recently constructed supramolecular polymers harnessing host-guest interaction between β-cyclodextrin and azobenzene. β-cyclodextrin was combined with ph-sensitive hydrophilic poly(2-(dimethylamino) ethyl methacrylate, and azobenzene was modified with hydrophobic poly(ε-caprolactone), enabling nanodrug micelles to be responsive to both ph and uv light. additionally, higher anticancer activity and stronger cancer cell inhibition than free dox could be achieved using this method [210] . dual stimuli-responsive system using ph and redox is also a common combination. with low ph and high gsh levels in tumor cells, a strong controlled release effect can be achieved. recently, ph/redox-responsive mixed polymeric micelles formed by self-assembly of two amphiphilic diblock copolymers (poly(ethylene glycol) methyl ether-b-poly(β-amino ester)) (mpeg-b-pae) and poly(ethylene glycol) methyl ether-grafted disulfide bond-poly(β-amino ester) (pae-ss-mpeg) have been developed, which released drugs at low ph and high gsh concentration in tumor cells [211] . in addition, there are also many combinations, such as ph/ros responsive system [212, 213] , ph/enzyme responsive system [214, 215] and others. as a significant characteristic stimulus condition in vivo, the combination of ph with other stimulus has shown promise for cancer treatments. redox and ros responsive systems are often used in combination with exogenous stimuli to achieve better stability and controlled release schemes. these two stimuli conditions can be combined to form a dual stimuli-responsive systems [216] . for example, xu et al. copolymerized two camptothecin (cpt) prodrug monomers with disulfide bond and oxalate bond on β-cyclodextrin and hydrophilic poly (ethylene glycol) methyl ether methacrylate (oegma) to prepare a novel dual redox stimulus-responsive prodrug. this prodrug has small particle size, high stability, excellent biocompatibility, good permeability, improved safety and high anti-tumor efficiency. this prodrug strategy is expected to provide a feasible method for advanced chemotherapy [217] . in addition, redox/light responsive system is also an effective strategy for cancer treatments due to the scheme of dual triggers [218, 219] . in a recent report, a thioacetal-based ros-sensitive amphiphilic copolymer (ptk) was developed to load nir cyanine dye ir 780 and dox to obtain stable nanoparticles (ir780/dox@ptk). ir 780 was used as photosensitizer, photothermal agent and imaging contrast agent simultaneously [220, 221] . upon the irradiation of 808 nm nir light, ir 780 could not only initiate pdt and ptt, but also used for photothermal imaging. the generated singlet oxygen ( 1 o2) led to the cleavage of thioacetal linker in ptk and the release of dox, enabling chemotherapy and phototherapy [222] . pu et al. synthesized an organic semiconducting pro-nanoenzyme (ospe) recently, which could be activated by nir light irradiation. ospe is made by binding semiconductor polymer nanoparticles (spn) to proenzyme (based on cytotoxic ribonuclease a) via singlet oxygen ( 1 o2)-sensitive linkers. when spn released singlet oxygen, it could not only carry out photodynamic therapy, but also released proenzyme to degrade cancer-specific rna, the synergistic treatment by chemo/phototherapy for cancers [223] (figure 11 ). other dual/multiple stimuli-responsive systems for cancer treatments include ph/magnetic responsive systems [224, 225] and other ph-based multiple stimuli-responsive systems [226] [227] [228] . redox and ros responsive systems are often used in combination with exogenous stimuli to achieve better stability and controlled release schemes. these two stimuli conditions can be combined to form a dual stimuli-responsive systems [216] . for example, xu et al. copolymerized two camptothecin (cpt) prodrug monomers with disulfide bond and oxalate bond on β-cyclodextrin and hydrophilic poly (ethylene glycol) methyl ether methacrylate (oegma) to prepare a novel dual redox stimulus-responsive prodrug. this prodrug has small particle size, high stability, excellent biocompatibility, good permeability, improved safety and high anti-tumor efficiency. this prodrug strategy is expected to provide a feasible method for advanced chemotherapy [217] . in addition, redox/light responsive system is also an effective strategy for cancer treatments due to the scheme of dual triggers [218, 219] . in a recent report, a thioacetal-based ros-sensitive amphiphilic copolymer (ptk) was developed to load nir cyanine dye ir 780 and dox to obtain stable nanoparticles (ir780/dox@ptk). ir 780 was used as photosensitizer, photothermal agent and imaging contrast agent simultaneously [220, 221] . upon the irradiation of 808 nm nir light, ir 780 could not only initiate pdt and ptt, but also used for photothermal imaging. the generated singlet oxygen ( 1 o 2 ) led to the cleavage of thioacetal linker in ptk and the release of dox, enabling chemotherapy and phototherapy [222] . pu et al. synthesized an organic semiconducting pro-nanoenzyme (ospe) recently, which could be activated by nir light irradiation. ospe is made by binding semiconductor polymer nanoparticles (spn) to proenzyme (based on cytotoxic ribonuclease a) via singlet oxygen ( 1 o 2 )-sensitive linkers. when spn released singlet oxygen, it could not only carry out photodynamic therapy, but also released proenzyme to degrade cancer-specific rna, the synergistic treatment by chemo/phototherapy for cancers [223] (figure 11 ). other dual/multiple stimuli-responsive systems for cancer treatments include ph/magnetic responsive systems [224, 225] and other ph-based multiple stimuli-responsive systems [226] [227] [228] . inflammation is an immune response to infection and tissue damage so that the body can be protected from injury. however, it can also cause many diseases such as asthma, cardiovascular diseases, neurodegenerative diseases and autoimmune diseases (including rheumatoid arthritis, systemic lupus erythematosus and other diseases) [229, 230] . these inflammatory chronic diseases can seriously affect health, and there are many obstacles to the treatment of the inflammatory chronic diseases. inflammation is an immune response to infection and tissue damage so that the body can be protected from injury. however, it can also cause many diseases such as asthma, cardiovascular diseases, neurodegenerative diseases and autoimmune diseases (including rheumatoid arthritis, systemic lupus erythematosus and other diseases) [229, 230] . these inflammatory chronic diseases can seriously affect health, and there are many obstacles to the treatment of the inflammatory chronic diseases. the characteristic conditions within the microenvironment of inflammatory tissues include lower ph value and higher ros concentration [231, 232] . therefore, ph/ros dual stimulus-responsive system is the most widely used treatment strategy for inflammation therapy [233, 234] . for example, almutairi et al. designed a ros-reactive dextran-drug conjugate (nap-dex) and blended nap-dex with an acid-sensitive acetal-dextran polymer (ac-dex) to obtain ph/ros dual stimulus-responsive nanoparticles. the ros-responsive pba-modified anti-inflammatory drug naproxen was used. when the nanodrug was stimulated by h 2 o 2 and acidic environment, the ac-dex and pba structure of the nanodrug micelle was cleaved, thereby releasing naproxen (nap) for the treatment of inflammatory tissues. dual stimuli-responsive nanodrug micelles are more effective in scavenging ros. compared with free naproxen, dual stimuli-responsive nanoparticles reduced the levels of proinflammatory cytokines il-6 and tnfα by 120 times and 6 times respectively [235] . in addition to lower ph and higher ros concentrations, inflammation of tissues tends to have slightly increased local temperature (1-2 • c above ambient temperature) [236] . therefore, the temperature/ph dual stimulus-responsive system has also been applied in the treatment of inflammation [237, 238] . a microbead system was designed with a porous poly (d, l-lactic-co-glycolic acid) (plga) shell coupled with a gelatin plug (thermal-responsive switch). additionally, n-palmitoyl chitosan (npcs) (ph-responsive switch) was employed as a core. vancomycin was loaded in the nanoparticles as the inflammatory therapeutic drug. this dual stimuli-responsive drug mbs could release the drug only in the presence of both stimuli (temperature and ph), preventing unexpected drug release due to accidental stimulus [239] . inflammatory bowel disease (ibd) is a chronic condition of idiopathic inflammation. ibd can affect the entire gastrointestinal tract (gi) and increases the risk of colorectal cancer [240] . the incidence of ibd worldwide has increased and effective treatment approaches are needed. therefore, drug delivery systems utilizing ibd microenvironment characteristic conditions (low ph, colonic enzymes, and high ros concentration) as responsive factors have attracted extensive attention [241, 242] . li et al. reported a nano-platform of oxidatively sensitive dextran (oxidex) with its exterior modified by chitosan (cs) and then further encapsulated by ph-sensitive hydroxypropyl methylcellulose acetate succinate (hpmcas). the intestinal-specific antibiotic rifampicin was used as a model drug to form ph/ros dual stimulus-responsive nanodrug composites. this novel nanodrug composite remained stable in the upper gastrointestinal tract, but hpmcas were cleaved in the intestine, thereby releasing nanodrug particles. triggered by higher ros levels, rifampicin is released into inflamed tissues. compared with traditional enteric drug formulations, this nanodrug composite effectively reduced the permeability of drugs at the intestinal epithelium, preventing non-specific absorption and side effects [243] (figure 12 ). in addition, some oral drug formulations responsive to ph have been used for clinical treatments. examples include 5-aminosalicylic acid encapsulated in capsules made from copolymers of acrylic acid derivatives and methyl methacrylate (eudragit ® ), such as salofalk ® , calitoflak ® , claversal ® , pentasa ® and other brands [244] [245] [246] . other inflammation examples include neuroinflammation, which represents an abnormal condition of central nervous system in many neurodegenerative diseases [247] . some serious neurodegenerative diseases include alzheimer's disease, epilepsy, huntington's disease and parkinson's disease [248] [249] [250] . strategies for the treatment of these diseases are desired and some ongoing efforts have been made on stimulus-responsive systems that can target excessive ros concentrations in neuroinflammatory tissues [251, 252] . oral medication is the preferred route of administration because of better patient compliance. however, many drugs themselves (protein drugs/polypeptide drugs) are sensitive to the acidic and proteolytic environment of the gastrointestinal tract, so oral administration remains a challenge. factors for oral administration that should be considered include (1) the activity of biopharmaceutical macromolecules in the gastrointestinal (git) environment, (2) the permeability in the intestine, and (3) drug molecules can be absorbed into the systemic circulation through the intestine [30, 253] . diabetes mellitus is an endocrine disease characterized by hyperglycemia, which is difficult to cure and can lead to various serious complications. the conventional way to treat diabetes is to inject insulin using subcutaneous administration, but the development of an effective orally administered insulin formulation would be preferred. the first attempt to treat diabetes with oral insulin can date back to 1922 but the failure made researchers realize that the main obstacle to oral delivery of biomacromolecules was mainly from the human body itself [254] . chitosan/insulin/heparin sodium (cs/ins/hs) nanoparticles were synthesized by ionic gel method, in which heparin sodium with three acidic functional groups can enhance the stability of the nanoparticle system in the stomach. mucosal affinity for cs/ins/hs in the small intestine were also improved due to the interaction of ins/hs with the positive charge on chitosan. in addition, acrylate-grafted-carboxymethyl starch (cms-g-aa) and oral medication is the preferred route of administration because of better patient compliance. however, many drugs themselves (protein drugs/polypeptide drugs) are sensitive to the acidic and proteolytic environment of the gastrointestinal tract, so oral administration remains a challenge. factors for oral administration that should be considered include (1) the activity of biopharmaceutical macromolecules in the gastrointestinal (git) environment, (2) the permeability in the intestine, and (3) drug molecules can be absorbed into the systemic circulation through the intestine [30, 253] . diabetes mellitus is an endocrine disease characterized by hyperglycemia, which is difficult to cure and can lead to various serious complications. the conventional way to treat diabetes is to inject insulin using subcutaneous administration, but the development of an effective orally administered insulin formulation would be preferred. the first attempt to treat diabetes with oral insulin can date back to 1922 but the failure made researchers realize that the main obstacle to oral delivery of biomacromolecules was mainly from the human body itself [254] . chitosan/insulin/heparin sodium (cs/ins/hs) nanoparticles were synthesized by ionic gel method, in which heparin sodium with three acidic functional groups can enhance the stability of the nanoparticle system in the stomach. mucosal affinity for cs/ins/hs in the small intestine were also improved due to the interaction of ins/hs with the positive charge on chitosan. in addition, acrylate-grafted-carboxymethyl starch (cms-g-aa) and methacrylic acid (maa) were used to synthesize ph/amylase dual stimuli-responsive hydrogels (cms-g-aa/pmaa). drug-loaded hydrogels can be contracted in the stomach to ensure the stability of nanoparticles. however, in the small intestine the hydrogel can be swollen so that insulin could be released, resulting from the carrier degradation by intestinal amylase. additionally, because of chitosan and heparin sodium, the reversible opening of the pathway between adjacent intestinal epithelial cells allowed insulin molecules to pass through the intestinal epithelial cells. this approach can also be used for the design of orally administered insulin formulations [255] . in addition to oral insulin, many stimulus-responsive systems have been designed for the treatment of other diseases such as helicobacter pylori [256] , various cancers [257] [258] [259] . for example, oupciky et al. reported a nanostructured lipid carrier (nlc)-based ptt formulation that can be orally administered. nlc showed its excellent biocompatibility, degradability and stability in the gastrointestinal tract. under nir light, nlc loaded with ir780 can be used for photothermal treatment on cancer cells [260] (figure 13 ). zhang et al. developed a supramolecular elastomer gel based on poly (acryloyl 6-aminocaproicacid) (pa6aca) and poly (methacrylic acid-ethyl acrylate) (eudragit l 100-55), which remained stable in acidic gastric environment, but dissolved in the small intestine with neutral ph, allowing safe passage through the stomach and into the intestine afterwards. under acidic conditions, carboxyl groups were protonated, and the inter-chain hydrogen bonds between carboxyl groups and amide units on pa6aca and l 100-55 formed a loosely cross-linked supramolecular network with water trapped inside, providing good elasticity and stability. under neutral ph conditions, the carboxyl groups in the molecule underwent deprotonation, and the disappearance of hydrogen bonds made the gel dissolve quickly. this type of gel material could be used as a good carrier for oral drugs with the advantages of strong elasticity, easy compression and folding. the use of this material for oral delivery drug was also evaluated in pigs [261] . stimulus-responsive oral drug delivery systems are still under development and much efforts are being endeavored in order to improve delivery efficiency and targeting capability and to reduce the side effects (e.g., potential pathogenic risk of agents used for increased intestinal permeability). methacrylic acid (maa) were used to synthesize ph/amylase dual stimuli-responsive hydrogels (cms-g-aa/pmaa). drug-loaded hydrogels can be contracted in the stomach to ensure the stability of nanoparticles. however, in the small intestine the hydrogel can be swollen so that insulin could be released, resulting from the carrier degradation by intestinal amylase. additionally, because of chitosan and heparin sodium, the reversible opening of the pathway between adjacent intestinal epithelial cells allowed insulin molecules to pass through the intestinal epithelial cells. this approach can also be used for the design of orally administered insulin formulations [255] . in addition to oral insulin, many stimulus-responsive systems have been designed for the treatment of other diseases such as helicobacter pylori [256] , various cancers [257] [258] [259] . for example, oupciky et al. reported a nanostructured lipid carrier (nlc)-based ptt formulation that can be orally administered. nlc showed its excellent biocompatibility, degradability and stability in the gastrointestinal tract. under nir light, nlc loaded with ir780 can be used for photothermal treatment on cancer cells [260] (figure 13 ). zhang et al. developed a supramolecular elastomer gel based on poly (acryloyl 6-aminocaproicacid) (pa6aca) and poly (methacrylic acid-ethyl acrylate) (eudragit l 100-55), which remained stable in acidic gastric environment, but dissolved in the small intestine with neutral ph, allowing safe passage through the stomach and into the intestine afterwards. under acidic conditions, carboxyl groups were protonated, and the inter-chain hydrogen bonds between carboxyl groups and amide units on pa6aca and l 100-55 formed a loosely crosslinked supramolecular network with water trapped inside, providing good elasticity and stability. under neutral ph conditions, the carboxyl groups in the molecule underwent deprotonation, and the disappearance of hydrogen bonds made the gel dissolve quickly. this type of gel material could be used as a good carrier for oral drugs with the advantages of strong elasticity, easy compression and folding. the use of this material for oral delivery drug was also evaluated in pigs [261] . stimulusresponsive oral drug delivery systems are still under development and much efforts are being endeavored in order to improve delivery efficiency and targeting capability and to reduce the side effects (e.g., potential pathogenic risk of agents used for increased intestinal permeability). non-invasive bioimaging approaches have been used for observation of biological activities and disease diagnosis. commonly used bioimaging techniques include optical imaging, magnetic resonance imaging (mri), ultrasound (us), computed tomography (ct), positron emission tomography (pet). photoacoustic imaging is another emerging imaging modality that shows potential for future clinical use. in this section, we will mainly introduce the latest research results of stimulus responsive nano-systems used for bio-imaging. among the optical imaging methods, fluorescence imaging plays an important role because of its high resolution and low cost. for higher accuracy and specificity, optical imaging is also synergistically used with chemo-pharmacotherapy [262, 263] . huang et al. recently reported a ph responsive nanocarrier using nir-ii dye-based multifunctional telechelic glycopolymer (ttq-tc-pfru). the nir-ii dyes in pfru-btz-pbob nanoparticles could be used for optical imaging and non-invasive bioimaging approaches have been used for observation of biological activities and disease diagnosis. commonly used bioimaging techniques include optical imaging, magnetic resonance imaging (mri), ultrasound (us), computed tomography (ct), positron emission tomography (pet). photoacoustic imaging is another emerging imaging modality that shows potential for future clinical use. in this section, we will mainly introduce the latest research results of stimulus responsive nano-systems used for bio-imaging. among the optical imaging methods, fluorescence imaging plays an important role because of its high resolution and low cost. for higher accuracy and specificity, optical imaging is also synergistically used with chemo-pharmacotherapy [262, 263] . huang et al. recently reported a ph responsive nanocarrier using nir-ii dye-based multifunctional telechelic glycopolymer (ttq-tc-pfru). the nir-ii dyes in pfru-btz-pbob nanoparticles could be used for optical imaging and photothermal therapy. additionally, anticancer drug bortezomib (btz) was conjugated for chemotherapy. this novel nir-ii dye-based drug-loaded nanoparticles showed potential because of their precise targeting capability and the real time imaging modality to achieve better therapeutic effects [264] . in addition, a site-selective in situ growth-induced self-assembly method was reported recently. ph-responsive micelles were synthesized based on site-specific human serum albumin-poly(2-(diisopropylamino) ethyl methacrylate) (hsa-pdpa) conjugates. icg could be loaded into the core of micelles. when present in the weak acid microenvironment in tumors, the nanoparticles rapidly decomposed into protonated moieties, allowing the fluorescence enhancement by 3-6 fold compared to non-stimulus-responsive nanoprobe and icg [265] (figure 14a ). photothermal therapy. additionally, anticancer drug bortezomib (btz) was conjugated for chemotherapy. this novel nir-ii dye-based drug-loaded nanoparticles showed potential because of their precise targeting capability and the real time imaging modality to achieve better therapeutic effects [264] . in addition, a site-selective in situ growth-induced self-assembly method was reported recently. ph-responsive micelles were synthesized based on site-specific human serum albuminpoly(2-(diisopropylamino) ethyl methacrylate) (hsa-pdpa) conjugates. icg could be loaded into the core of micelles. when present in the weak acid microenvironment in tumors, the nanoparticles rapidly decomposed into protonated moieties, allowing the fluorescence enhancement by 3-6 fold compared to non-stimulus-responsive nanoprobe and icg [265] (figure 14a ). [265] . (b) ros significantly enhanced the ultrasound signal [266] . (c) mr contrast could be enhanced in the tumor microenvironment by atp/ph stimuli [267] . (d) pa signal could be significantly enhanced in tumor microenvironment by no/ph stimulation [268] . (e) pet imaging enabled realtime monitoring of enzyme/nir/ph stimulus-responsive drug delivery systems [269] . (f) ph and nir responsive system resulted in significant enhancement of ct contrast [270] . reproduced with permission from the publishers of corresponding references. us has become one of the most commonly used medical imaging techniques due to its excellent portability, noninvasiveness and low cost. however, ultrasound contrast imaging typically requires the use of diffusible microbubbles to diffuse into the surrounding media to enhance contrast. with the diffusion of gas, these microbubbles will be quickly cleared in vivo, for this reason the ultrasonic imaging has a shorter imaging time [266, 271, 272] (figure 14b ). in recent reports, a ph responsive solid ultrasound nanosensor (sun) was developed that did not require the generation of bubbles. the sun consists of three parts, a multi-empty silicone shell, a solid silica core and a ph-sensitive coating on its surface. this new type of sun could expand and shrink responsive to the change of [265] . (b) ros significantly enhanced the ultrasound signal [266] . (c) mr contrast could be enhanced in the tumor microenvironment by atp/ph stimuli [267] . (d) pa signal could be significantly enhanced in tumor microenvironment by no/ph stimulation [268] . (e) pet imaging enabled real-time monitoring of enzyme/nir/ph stimulus-responsive drug delivery systems [269] . (f) ph and nir responsive system resulted in significant enhancement of ct contrast [270] . reproduced with permission from the publishers of corresponding references. us has become one of the most commonly used medical imaging techniques due to its excellent portability, noninvasiveness and low cost. however, ultrasound contrast imaging typically requires the use of diffusible microbubbles to diffuse into the surrounding media to enhance contrast. with the diffusion of gas, these microbubbles will be quickly cleared in vivo, for this reason the ultrasonic imaging has a shorter imaging time [266, 271, 272] (figure 14b ). in recent reports, a ph responsive solid ultrasound nanosensor (sun) was developed that did not require the generation of bubbles. the sun consists of three parts, a multi-empty silicone shell, a solid silica core and a ph-sensitive coating on its surface. this new type of sun could expand and shrink responsive to the change of ph value owing to the ph-sensitive coating. when the concentration of hydrogen ions decreased, the ultrasound imaging was significantly enhanced [273] . magnetic resonance imaging (mri) is a powerful technology for diagnosis and bioimaging of diseases because of its non-invasiveness, reasonable imaging time, deep tissue penetration and high resolution [274, 275] . zhang et al. recently developed a triple stimulus (gsh/ph/nir)-responsive nanocarrier based on magnetic hollow porous carbon nanoparticles (mhpcn). the outer shell of mhpcn consists of two layers: the inner layer with iron oxide (fe 3 o 4 ) as contrast agent for mri and the outer layer with fluorescent carbon nanodots as the outer layer. dox was selected as a model drug and folic acid (fa) was crosslinked for targeting purpose. this new triple stimulus-responsive nanodrug carrier can achieve synergistic photothermal/chemical therapy of tumor guided by mri under the synergistic stimulation of low ph, high concentration of gsh and external nir light in tumor cells [276] . in addition, a strategy using magnetic resonance/near-infrared fluorescence (mr/nirfl) was developed to determine the location of tumors. using ph and nir as stimuli, chemo/photothermal therapy could be achieved for efficient cancer nanotheranostics [277] . yang et al. used polyphenols as phase transfer agents to increase the solubility of hydrophobic magnetic nanoparticles. meanwhile, polyphenols also promoted the self-assembly of nanoparticles. the core of this assembly is atp/ph responsive for the dual imaging of mr/fl and photothermal therapy. when the nano-assembly entered the tumor microenvironment with lower ph value and higher concentration of atp, they showed enhanced mr contrast (due to the strong binding affinity of atp to fe 3 o 4 ) and significantly altered fluorescence signal (the protonation of hydroxyl groups in tannic acid (ta). thus, functional activities in tumors could be tracked using this mr responsive nanoplatform [267] (figure 14c ). photoacoustic (pa) imaging is an emerging biomedical imaging modality with deep penetration depth, high spatial resolution, and high selectivity that combines the advantages of optical imaging and ultrasound imaging [278] . when laser irradiation is applied to biological tissues, pa signals are generated in the tissues, and the detailed information of human internal tissues could be collected and reconstructed in computer. tan et al. designed a nitric oxide (no)/ph activatable theranostic nanoprobe (datn), taking advantage of high concentration of nitric oxide and low ph in tumor microenvironment. benzo [c] [1, 2, 5] thiadiazole-5,6-diamine was used for the stimulus responsive π-receptor-π-donor (d-π-a-π-d) molecules. in the tumor microenvironment, datn could turn on pa signals for tumor specific pa imaging in vivo. using these dual stimuli, the pa signal intensity of datn was 9.8 times higher than that no responsive system and 132 times higher that of acid responsive system [268] (figure 14d ). pu recently reported the synthesis of a fluoro-photoacoustic polymeric renal reporter (fprr) for noninvasive near-infrared fluorescence (nirf) and pa dual imaging of drug-induced acute kidney injury (aki) in mice. fprr consists of dextran (used to promote renal clarity), hemicyanine cyoh (used for imaging), and γ-glutamate (enzyme-responsive moiety). fprr exhibited high renal clearance efficiency in living mice, it also enhanced nirf and pa signals for real-time molecular imaging of aki after the enzyme responsive moiety is cleaved. this nanoplatform showed potential for early detection of aki in clinics [279] . positron emission tomography (pet) can provide precise biological information on metabolic processes with whole-body penetration and excellent sensitivity at the molecular level using radiopharmaceuticals. it has been widely used in clinical disease treatment and diagnosis [280] . when used in combination with computed tomography (ct), the clinical effect of pet can be improved for disease diagnosis such as tumors and heart diseases [281] . for example, hyaluronic acid (ha)-functionalized mos 2 was designed with the surface modified by pei and 64 cu (positron emitter with a half-life of 12.7 h). the resulting mos 2 -pei-ha multifunctional nanoplatform was endowed with the property of triple stimuli (ph/haase/nir) responsiveness. this new nanocarrier can be used as contrast agent for pet imaging, providing an effective way to monitor the treatment process and optimize disease management plan [269] (figure 14e ). chen et al. developed a multimodal imaging synergistic therapy based on amphiphilic iron oxide-gold janus nanoparticles (fe 3 o 4 -au-jnps) in response to ph stimulus. it was shown that under the guidance of pet, mr and pa multimodal imaging, fe 3 o 4 -au-jnps could effectively carry out ros-mediated cancer treatment. after intravenous injection of [ 64 cu] radiolabeled jnp vesicles, pet imaging showed that the drug vesicles remained stable in the blood, but gradually dissociated in the liver, showing reduced hepatotoxicity. pet imaging modality provided effective real-time monitoring of the whole treatment process [282] . computed tomography (ct) collects information of anatomical structures via x-ray scans and cross-sectional images are reconstructed by computer operation. due to its penetration capability and fast scanning speed, ct is one of the most widely used clinical imaging methods. it is also usually used in combination with mri, pa and other imaging modalities to achieve better imaging results. for example, as coronavirus disease 2019 (covid-19) is spreading worldwide, ct has served as an important imaging tool for the assessment of covid-19. ct scans can spot abnormalities of lungs such as multiple opacities (denser, more profuse and confluent) [283] . in other applications such as cancer diagnosis and treatment, ct also plays a significant role and is mostly used in combination with other imaging approaches. for example, yu et al. recently developed bismuth nano-raspberries (bi-bsa nrs) modified by bsa. bi-bsa nrs have high drug loading efficiency (~69%) and can release the cargo in response to ph and nir stimuli. the efficiency of ct contrast agent is improved by infrared thermal and pa. 100% tumor treatment rate was achieved by chemo-photothermal therapy. in conclusion, this bi-bsa nrs have potential for the application of multimodality imaging and combination therapy [270] (figure 14f ). in summary, nanotechnology-based therapeutics have developed rapidly in recent years, and a wide variety of novel strategies have been explored. particularly, stimulus-responsive nano-systems warrant attention as they show promise in targeting and controlled release in response to endogenous or exogenous stimuli. owing to their unique advantages, stimulus-responsive systems have been widely investigated as a strategy well-suited for nanotherapeutics. furthermore, dual/multiple stimulus-responsive systems have been investigated for more accurate disease diagnosis and management. self-immolative linkers with different cleavage and self-immolation rates hold potential as a versatile technology to address controlled drug release. stimulus-responsive systems have shown promise for the treatment of various cancers, ibd, neuroinflammation, gastrointestinal diseases, bio-imaging and many others. however, the translational potential, especially for the highly complex chemically modified systems, is a concern. drug-device combinations for external stimuli are more complicated than drug-alone approaches from regulatory perspectives. for example, for cancer treatment, stimulus-responsive systems treating only local tumors would require one to find advantages over the well-established modalities of surgery, radiotherapy and other ablation approaches, which do not have the complications of drugs. in addition, toxicity of not only the api, but also the carrier system, is another factor for consideration. nevertheless, with smart and rational design, stimulus-responsive nanotechnology adds a new weapon to drug delivery and molecular imaging arsenal, and warrants further research and development. funding: this work is supported by the start-up grant at tianjin university (y.z.) and one-thousand young talent program (y.z.). the authors declare no conflict of interest. stimuli-responsive nanomaterials for biomedical applications remotely triggerable drug delivery systems external triggering and triggered targeting strategies for drug delivery overcoming limitations in nanoparticle drug delivery: triggered, intravascular release to improve drug penetration into tumors bacteria-mediated tumor therapy utilizing photothermally-controlled tnf-α expression via oral administration emerging frontiers in drug delivery collapse of gels and critical endpoint design of liposomes for enhanced local release of drugs by hyperthermia ph-responsive nanoparticles for drug delivery integrated nanoparticles to synergistically elevate tumor oxidative stress and suppress antioxidative capability for amplified oxidation therapy targeted mmp-2 responsive chimeric polymersomes for therapy against colorectal cancer development of a theranostic prodrug for colon cancer therapy by combining ligand-targeted delivery and enzyme-stimulated activation stimuli-responsive polymeric prodrug-based nanomedicine delivering nifuroxazide and doxorubicin against primary breast cancer and pulmonary metastasis stimuli-responsive mesoporous silica nps as non-viral dual sirna/chemotherapy carriers for triple negative breast cancer telomerase responsive delivery of doxorubicin from mesoporous silica nanoparticles in multiple malignancies: therapeutic efficacies against experimental aggressive murine lymphoma doxorubicin loaded ph responsive biodegradable aba-type amphiphilic peg-b-aliphatic polyketal-b-peg block copolymer for therapy against aggressive murine lymphoma bifunctional succinylated ε-polylysine-coated mesoporous silica nanoparticles for ph-responsive and intracellular drug delivery targeting the colon rational design of polyphenol-poloxamer nanovesicles for targeting inflammatory bowel disease therapy rational design of a stimuli-responsive polymer electrode interface coupled with in vivo microdialysis for measurement of sialic acid in live mouse brain in alzheimer's disease glucose-and h2o2-responsive polymeric vesicles integrated with microneedle patches for glucose-sensitive transcutaneous delivery of insulin in diabetic rats functionalized gold nanoparticle-polypyrrole nanobiocomposite with high effective surface area for electrochemical/ph dual stimuli-responsive smart release of insulin lyso-thermosensitive liposomal doxorubicin: an adjuvant to increase the cure rate of radiofrequency ablation in liver cancer in vitro and in vivo metabolism of paclitaxel poliglumex: identification of metabolites and active proteases efficacy and safety of intratumoral thermotherapy using magnetic iron-oxide nanoparticles combined with external beam radiotherapy on patients with recurrent glioblastoma multiforme feasibility study of particle-assisted laser ablation of brain tumors in orthotopic canine model ph-sensitive nano-systems for drug delivery in cancer therapy the relevance of tumour ph to the treatment of malignant disease extracellular ph distribution in human tumours causes and consequences of tumour acidity and implications for treatment biointerfacial self-assembly generates lipid membrane coated bacteria for enhanced oral delivery and treatment targeted delivery of small and macromolecular drugs stimuli-responsive nanomedicines for overcoming cancer multidrug resistance a. ph-dependent equilibrium swelling properties of hydrophobic polyelectrolyte copolymer gels ph-sensitive dissociable nanoscale coordination polymers with drug loading for synergistically enhanced chemoradiotherapy synthesis of a triple-responsive double hydrophilic block copolymer prodrug using a reducible raft-atrp double-head agent probe-inspired nano-prodrug with dual-color fluorogenic property reveals spatiotemporal drug release in living cells hollow chitosan-silica nanospheres as ph-sensitive targeted delivery carriers in breast cancer therapy micelles based on acid degradable poly(acetal urethane): preparation, ph-sensitivity, and triggered intracellular drug release acetal-functionalized pillar (5)arene: a ph-responsive and versatile nanomaterial for the delivery of chemotherapeutic agents doxorubicin-conjugated biodegradable polymeric micelles having acid-cleavable linkages synthesis of highly ph-responsive glucose poly(orthoester) facile fabrication of double-walled polymeric hollow spheres with independent temperature and ph dual-responsiveness for synergetic drug delivery incorporation and controlled release of silyl ether prodrugs from print nanoparticles acetal-linked pegylated paclitaxel prodrugs forming free-paclitaxel-loaded ph-responsive micelles with high drug loading capacity and improved drug delivery micromotors spontaneously neutralize gastric acid for ph-responsive payload release a transistor-like ph nanoprobe for tumour detection and image-guided surgery multistimuli-responsive pegylated polymeric bioconjugate-based nano-aggregate for cancer therapy intracellular release of doxorubicin from core-crosslinked polypeptide micelles triggered by both ph and reduction conditions smart drug delivery systems: from fundamentals to the clinic noninvasive imaging of tumor redox status and its modification by tissue glutathione levels intracellular microenvironment responsive pegylated polypeptide nanogels with ionizable cores for efficient doxorubicin loading and triggered release glutathione metabolism and its implications for health gradient redox-responsive and two-stage rocket-mimetic drug delivery system for improved tumor accumulation and safe chemotherapy bond energy data summarized enhanced bioreduction-responsive biodegradable diselenide-containing poly(ester urethane) nanocarriers thioether phosphatidylcholine liposomes: a novel ros-responsive platform for drug delivery self-assembled redox dual-responsive prodrug-nanosystem formed by single thioether-bridged paclitaxel-fatty acid conjugate for cancer chemotherapy synthetic applications of organotellurium chemistry γ-ray-responsive supramolecular hydrogel based on a diselenide-containing polymer and a peptide ultra-sensitive ros-responsive tellurium-containing polymers near-infrared light stimuli-responsive synergistic therapy nanoplatforms based on the coordination of tellurium-containing block polymer and cisplatin for cancer treatment hydrogen peroxide-responsive micelles self-assembled from a peroxalate ester-containing triblock copolymer in vivo imaging of hydrogen peroxide with chemiluminescent nanoparticles visible light-induced singlet oxygen-mediated intracellular disassembly of polymeric micelles co-loaded with a photosensitizer and an anticancer drug for enhanced photodynamic therapy nanoparticles formed from rgd-epothilone b conjugate for targeted cancer therapy synthesis of a phenylboronic ester-linked peg-lipid conjugate for ros-responsive drug delivery glutathione-responsive nanoscale mofs for effective intracellular delivery of the anticancer drug 6-mercaptopurine stimuli-responsive nanocarriers for drug delivery the smart drug delivery system and its clinical potential proline isomerization-regulated tumor microenvironment-adaptable self-assembly of peptides for enhanced therapeutic efficacy ros-cleavable proline oligomer crosslinking of polycaprolactone for pro-angiogenic host response orally delivered thioketal nanoparticles loaded with tnf-α-sirna target inflammation and inhibit gene expression in the intestines ros-responsive polymeric nanocarriers with photoinduced exposure of cell-penetrating moieties for specific intracellular drug delivery enzyme-responsive nanoparticle systems proteases: multifunctional enzymes in life and disease enzyme-responsive hydrogel microparticles for pulmonary drug delivery polymer-directed enzyme prodrug therapy. i. hpma copolymer-cathepsin b and pk1 as a model combination cascade-promoted photo-chemotherapy against resistant cancers by enzyme-responsive polyprodrug nanoplatforms matrix metalloprotease 2-responsive multifunctional liposomal nanocarrier for enhanced tumor targeting an mmp-2 responsive liposome integrating antifibrosis and chemotherapeutic drugs for enhanced drug perfusion and efficacy in pancreatic cancer mmp-2-sensitive ha end-conjugated poly(amidoamine) dendrimers via click reaction to enhance drug penetration into solid tumor smart asymmetric vesicles with triggered availability of inner cell-penetrating shells for specific intracellular drug delivery peg-sheddable polyplex micelles as smart gene carriers based on mmp-cleavable peptide-linked block copolymers modular design and facile synthesis of enzyme-responsive peptide-linked block copolymers for efficient delivery of doxorubicin matrix metalloproteinase-responsive pegylated lipid nanoparticles for controlled drug delivery in the treatment of rheumatoid arthritis phospholipase a2 enzymes: physical structure, biological function, disease implication, chemical inhibition, and therapeutic intervention expression of group iia secretory phospholipase a2 increases with prostate tumor grade overexpression of group ii phospholipase a2 in human breast cancer tissues is closely associated with their malignant potency presence of secretory group iia and v phospholipase a2 and cytosolic group ivα phospholipase a2 in chondrocytes from patients with rheumatoid arthritis phospholipase a2s in cell injury and death phospholipidic colchicinoids as promising prodrugs incorporated into enzyme-responsive liposomes: chemical, biophysical, and enzymological aspects phospholipase a2-susceptible liposomes of anticancer double lipid-prodrugs a new glucose oxidase fromaspergillus niger: characterization and regulation studies of enzyme and gene kinetics of glucose oxidase excretion by recombinant aspergillus niger glucose-responsive nanoparticles for rapid and extended self-regulated insulin delivery erythrocyte-membrane-camouflaged nanoplatform for intravenous glucose-responsive insulin delivery glucose-responsive metal-organic-framework nanoparticles act as "smart" sense-and-treat carriers nad(p)h:quinone oxidoreductase1 (dt-diaphorase) expression in normal and tumor tissues efficacy of beta-lapachone in pancreatic cancer treatment: exploiting the novel, therapeutic target nqo1 cytosolic nqo1 enzyme-activated near-infrared fluorescence imaging and photodynamic therapy with polymeric vesicles a unique case of breast carcinoma producing pancreatic-type isoamylase glycosidase activated release of fluorescent 1,8-naphthalimide probes for tumor cell imaging from glycosylated 'pro-probes' factors controlling the pharmacokinetics, biodistribution and intratumoral penetration of nanoparticles functional nanomaterials for phototherapies of cancer light-activated ros-responsive nanoplatform codelivering apatinib and doxorubicin for enhanced chemo-photodynamic therapy of multidrug-resistant tumors applications of light-responsive systems for cancer theranostics biologically active molecules with a "light switch porphyrin-phospholipid liposomes permeabilized by near-infrared light tunable luminescent lanthanide supramolecular assembly based on photoreaction of anthracene a self-assembled coumarin-anchored dendrimer for efficient gene delivery and light-responsive drug delivery photocontrolled nuclear-targeted drug delivery by single component photoresponsive fluorescent organic nanoparticles of acridin-9-methanol photoinduced reversible worm-to-vesicle transformation of azo-containing block copolymer assemblies prepared by polymerization-induced self-assembly light-responsive and ph-responsive dna microcapsules for controlled release of loads photolabile linkers: exploiting labile bond chemistry to control mode and rate of hydrogel degradation and protein release a photochemical approach for controlled drug release in targeted drug delivery anti-stokes shift luminescent materials for bio-applications upconversion and anti-stokes processes with f and d ions in solids copolymer upconversion nanocomposites for triggered drug release in vitro and in vivo light-controlled tools a dna-azobenzene nanopump fueled by upconversion luminescence for controllable intracellular drug release photo, ph and redox multi-responsive nanogels for drug delivery and fluorescence cell imaging diarylethenes for memories and switches reversible photoswitching of triplet-triplet annihilation upconversion using dithienylethene photochromic switches synthesis and comparative photoswitching studies of unsymmetrical 2,3-diarylcyclopent-2-en-1-ones photochromic properties of terarylene derivatives having a π-conjugation unit on central aromatic ring photoswitchable organocatalysis: using light to modulate the catalytic activities of n-heterocyclic carbenes reporting the release of caged species by a combination of two sequential photoreactions, a molecular switch, and one color of light a contribution to the design of molecular switches: novel acid-mediated ring-closing−photochemical ring-opening of 2,3-bis(heteroaryl)quinones (heteroaryl = thienyl, furanyl, pyrrolyl) dual-mode fluorescence switching of photochromic bisthiazolylcoumarin synthesis and photochromism of a novel diarylethene compound containing terminal benzothiazole unit light-responsive platform for launching therapeutic agents on command drug delivery via cell-conveyed phototherapeutics photoactivation of anticancer ru complexes in deep tissue: how deep can we go? chem. a eur temperature-responsive smart nanocarriers for delivery of therapeutic agents: applications and recent advances multifunctional up-converting nanocomposites with smart polymer brushes gated mesopores for cell imaging and thermo/ph dual-responsive drug controlled release release kinetics of benzoic acid and its sodium salt from a series of poly(n-isopropylacrylamide) matrices with various percentage crosslinking the targeted behavior of thermally responsive nanohydrogel evaluated by nir system in mouse model thermoresponsive poly(n-vinyl caprolactam)-coated gold nanoparticles: sharp reversible response and easy tunability dual-stimuli-responsive paclitaxel delivery nanosystems from chemically conjugate self-assemblies for carcinoma treatment enzyme and thermal dual responsive amphiphilic polymer core-shell nanoparticle for doxorubicin delivery to cancer cells thermosensitive liposomes modified with poly(n-isopropylacrylamide-co-propylacrylic acid) copolymers for triggered release of doxorubicin magnetism in medicine thermoresponsive polymer brush-functionalized magnetic manganite nanoparticles for remotely triggered drug release controlled release of doxorubicin loaded within magnetic thermo-responsive nanocarriers under magnetic and thermal actuation in a microfluidic channel magnetically stimulated drug release using nanoparticles capped by self-assembling peptides magnetic microspheres: a model system for site specific drug delivery in vivo improved method of recombinant aav2 delivery for systemic targeted gene therapy magnetofection: enhancing and targeting gene delivery by magnetic force in vitro and in vivo acoustic cavitation: a possible consequence of biomedical uses of ultrasound microbubble contrast agents: targeted ultrasound imaging and ultrasound-assisted drug-delivery applications lipid/plga hybrid microbubbles as a versatile platform for noninvasive image-guided targeted drug delivery in situ conversion of porphyrin microbubbles to nanoparticles for multimodality imaging ultrasound-responsive conversion of microbubbles to nanoparticles to enable background-free in vivo photoacoustic imaging short and long term efficacy of high intensity focused ultrasound therapy for advanced hepatocellular carcinoma extracorporeal high intensity focused ultrasound ablation in the treatment of 1038 patients with solid carcinomas in china: an overview a novel connector linkage applicable in prodrug design polymeric persulfide prodrugs: mitigating oxidative stress through controlled delivery of reactive sulfur species alkylamine-substituted perthiocarbamates: dual precursors to hydropersulfide and carbonyl sulfide with cardioprotective actions development of a cysteine-conjugatable disulfide fret probe: influence of charge on linker cleavage and payload trafficking for an anti-her2 antibody conjugate small molecule as fluorescent probes for monitoring intracellular enzymatic transformations engineered ph-responsive mesoporous carbon nanoparticles for drug delivery reactive oxygen species (ros)-responsive polymersomes with site-specific chemotherapeutic delivery into tumors via spacer design chemistry rational design of latent fluorophores from water-soluble hydroxyphenyltriazine dyes suitable for lipase sensing disassembly kinetics of quinone-methide-based self-immolative spacers that contain aromatic nitrogen heterocycles supramolecular hydrogels for enzymatically triggered self-immolative drug delivery quinone-methide species, a gateway to functional molecular systems: from self-immolative dendrimers to long-wavelength fluorescent dyes development of a drug-release strategy based on the reductive fragmentation of benzyl carbamate disulfides synthesis and evaluation of paclitaxel-loaded gold nanoparticles for tumor-targeted drug delivery real-time monitoring of drug release single-triggered ab6 self-immolative dendritic amplifiers elongated multiple electronic cascade and cyclization spacer systems in activatible anticancer prodrugs for enhanced drug release prodrug strategies based on intramolecular cyclization reactions oxidation degradable aliphatic polycarbonates with pendent phenylboronic ester syntheses and kinetic studies of cyclisation-based self-immolative spacers programmable microcapsules from self-immolative polymers self-immolative linkers as caps for the design of gated silica mesoporous supports intramolecular cyclization for stimuli-controlled depolymerization of polycaprolactone particles leading to disassembly and payload release use of dithiasuccinoyl-caged amines enables cos/h2s release lacking electrophilic byproducts 4-bis(hydroxymethyl)aniline as a building block for oligomers with self-eliminating and multiple release properties convergent synthesis of geometrically disassembling dendrimers using cu(i)-catalyzed c−o bond formation disulfide-based self-immolative linkers and functional bioconjugates for biological applications dual-responsive nanoparticles release cargo upon exposure to matrix metalloproteinase and reactive oxygen species exponential molecular amplification by h2o2-mediated autocatalytic deprotection of boronic ester probes to redox cyclers effects of electronics, aromaticity, and solvent polarity on the rate of azaquinone-methide-mediated depolymerization of aromatic carbamate oligomers synthesis and evaluation of hydrogen peroxide sensitive prodrugs of methotrexate and aminopterin for the treatment of rheumatoid arthritis stabilizing p-dithiobenzyl urethane linkers without rate-limiting self-immolation for traceless drug release design and synthesis of new protease-triggered co-releasing peptide-metal-complex conjugates self-immolative polymers activity-based optical sensing enabled by self-immolative scaffolds: monitoring of release events by fluorescence or chemiluminescence output a gadolinium chelate for detection of β-glucuronidase: a self-immolative approach synthesis and antitumor properties of bqc-glucuronide, a camptothecin prodrug for selective tumor activation benzyl ether-linked glucuronide derivative of 10-hydroxycamptothecin designed for selective camptothecin-based anticancer therapy aryl sulfate is a useful motif for conjugating and releasing phenolic molecules: sulfur fluorine exchange click chemistry enables discovery of ortho-hydroxy-protected aryl sulfate linker an improved fluorogenic substrate for the detection of alkaline phosphatase activity synthesis, and pharmacokinetic evaluation of phosphate and amino acid ester prodrugs for improving the oral bioavailability of the hiv-1 protease inhibitor atazanavir light activation for the versatile and accurate kinetic analysis of disassembly of self-immolative spacers sterically-controlled self-immolation in phosphoramidate linkers triggered by light single uv or near ir triggering event leads to polymer degradation into small molecules multi-stimuli-responsive self-immolative polymer assemblies red and near-infrared light-cleavable polymers increasing materials' response to two-photon nir light via self-immolative dendritic scaffolds ph-sensitive, serum-stable and long-circulating liposomes as a new drug delivery system tumor-selective targeted delivery of genes and antisense oligodeoxyribonucleotides via the folate receptor ph and thermo dual-stimuli-responsive drug carrier based on mesoporous silica nanoparticles encapsulated in a copolymer-lipid bilayer facile construction of i-motif dna-conjugated gold nanostars as near-infrared and ph dual-responsive targeted drug delivery systems for combined cancer therapy light-triggered reversible assemblies of azobenzene-containing amphiphilic copolymer with β-cyclodextrin-modified hollow mesoporous silica nanoparticles for controlled drug release light-triggered specific cancer cell release from cyclodextrin/azobenzene and aptamer-modified substrate controlled drug release from cyclodextrin-gated mesoporous silica nanoparticles based on switchable host-guest interactions dual stimuli-responsive supramolecular self-assemblies based on the host-guest interaction between β-cyclodextrin and azobenzene for cellular drug release dual ph/redox-responsive mixed polymeric micelles for anticancer drug delivery and controlled release reactive oxygen species synergistic ph/h2o2-responsive poly(l-lactic acid)-block-poly(sodium 4-styrenesulfonate)/citrate-fe(iii)@zif-8 hybrid nanocomposites for controlled drug release intracellular enzyme-triggered assembly of amino acid-modified gold nanoparticles for accurate cancer therapy with multimode a smart drug delivery system responsive to ph/enzyme stimuli based on hydrophobic modified sodium alginate enzyme/ph dual-responsive polymer prodrug nanoparticles based on 10-hydroxycamptothecin-carboxymethylchitosan for enhanced drug stability and anticancer efficacy glutathione and reactive oxygen species dual-responsive block copolymer prodrugs for boosting tumor site-specific drug release and enhanced antitumor efficacy smart unimolecular micelle-based polyprodrug with dual-redox stimuli response for tumor microenvironment: enhanced in vivo delivery efficiency and tumor penetration gsh and light dual stimuli-responsive supramolecular polymer drug carriers for cancer therapy dual-stimuli-responsive microparticles ir-780 dye loaded tumor targeting theranostic nanoparticles for nir imaging and photothermal therapy sentinel lymph node mapping by a near-infrared fluorescent heptamethine dye nir-triggered multifunctional and degradable nanoplatform based on an ros-sensitive block copolymer for imaging-guided chemo-phototherapy photoactivatable organic semiconducting pro-nanoenzymes dual stimuli-guided lipid-based delivery system of cancer combination therapy non-magnetic injectable implant for magnetic field-driven thermochemotherapy and dual stimuli-responsive drug delivery: transformable liquid metal hybrid platform for cancer theranostics multi-stimuli responsive mesoporous carbon nano-platform gated by human serum albumin for cancer thermo-chemotherapy reduction/temperature/ph multi-stimuli responsive core cross-linked polypeptide hybrid micelles for triggered and intracellular drug release synthesis of temperature, ph, light and dual-redox quintuple-stimuli-responsive shell-crosslinked polymeric nanoparticles for controlled release reactive oxygen species in inflammation and tissue injury inflammation responsive logic gate nanoparticles for the delivery of proteins a dual ph/redox responsive copper-ligand nanoliposome bioactive complex for the treatment of chronic inflammation a ph/ros dual-responsive and targeting nanotherapy for vascular inflammatory diseases inflammation-responsive drug-conjugated dextran nanoparticles enhance anti-inflammatory drug efficacy responsive polyelectrolyte complexes for triggered release of nucleic acid therapeutics dual thermo-and ph-sensitive network-grafted hydrogels formed by macrocrosslinker as drug delivery system thermo and ph dual actuating smart porous anodic aluminum for controllable drug release injectable microbeads with a thermo-responsive shell and a ph-responsive core as a dual-switch-controlled release system inflammatory bowel disease bioresponsive drug delivery systems in intestinal inflammation: state-of-the-art and future perspectives reactive oxygen species responsive nanoplatforms as smart drug delivery systems for gastrointestinal tract targeting ph and reactive oxygen species-sequential responsive nano-in-micro composite for targeted therapy of inflammatory bowel disease nanoparticulate drug delivery systems targeting inflammation for treatment of inflammatory bowel disease drug delivery strategies in the therapy of inflammatory bowel disease short-term adverse effects of 5-aminosalicylic acid agents in the treatment of ulcerative colitis inflammation in neurological disorders: a help or a hindrance? neuroscience van der valk, p. inflammation in neurodegenerative diseases role of immunity and inflammation in the pathophysiology of neurodegenerative diseases dysregulation of mitochondrial calcium signaling and superoxide flashes cause mitochondrial genomic dna damage in huntington disease positively charged polyprodrug amphiphiles with enhanced drug loading and reactive oxygen species-responsive release ability for traceable synergistic therapy core-cross-linked nanoparticles reduce neuroinflammation and improve outcome in a mouse model of traumatic brain injury a review of advanced oral drug delivery technologies facilitating the protection and absorption of protein and peptide molecules insulin in hospital and home dual stimuli-responsive nanoparticle-incorporated hydrogels as an oral insulin carrier for intestine-targeted delivery and enhanced paracellular permeation development of ph-responsive chitosan/heparin nanoparticles for stomach-specific anti-helicobacter pylori therapy oral delivery of ph-responsive alginate microbeads incorporating folic acid-grafted solid lipid nanoparticles exhibits enhanced targeting effect against colorectal cancer: a dual-targeted approach in vitro evaluation of ph-responsive nanoscale hydrogels for the oral delivery of hydrophobic therapeutics ph-responsive lignin-based nanomicelles for oral drug delivery oral nanostructured lipid carriers loaded with near-infrared dye for image-guided photothermal therapy a ph-responsive supramolecular polymer gel as an enteric elastomer for use in gastric devices amphiphilic semiconducting polymer as multifunctional nanocarrier for fluorescence/photoacoustic imaging guided chemo-photothermal therapy dual-stimuli responsive and reversibly activatable theranostic nanoprobe for precision tumor-targeting and fluorescence-guided photothermal therapy nir-ii dye-based multifunctional telechelic glycopolymers for nir-iia fluorescence imaging-guided stimuli-responsive chemo-photothermal combination therapy site-selective in situ growth-induced self-assembly of protein-polymer conjugates into ph-responsive micelles for tumor microenvironment triggered fluorescence imaging dual-enzyme-loaded multifunctional hybrid nanogel system for pathological responsive ultrasound imaging and t2-weighted magnetic resonance imaging polyphenol-inspired facile construction of smart assemblies for atp-and ph-responsive tumor mr/optical imaging and photothermal therapy nitric oxide-activated "dual-key-one-lock" nanoprobe for in vivo molecular imaging and high-specificity cancer therapy intelligent mos2 nanotheranostic for targeted and enzyme-/ph-/nir-responsive drug delivery to overcome cancer chemotherapy resistance guided by pet imaging dual-stimuli responsive bismuth nanoraspberries for multimodal imaging and combined cancer therapy microbubbles in medical imaging: current applications and future directions injectable microbubbles as contrast agents for diagnostic ultrasound imaging: the key role of perfluorochemicals dynamic solid-state ultrasound contrast agent for monitoring ph fluctuations in vivo molecular imaging by mri advances in high-field magnetic resonance imaging triple stimuli-responsive magnetic hollow porous carbon-based nanodrug delivery system for magnetic resonance imaging-guided synergistic photothermal/chemotherapy of cancer hp-β-cd functionalized fe3o4/cnps-based theranostic nanoplatform for ph/nir responsive drug release and mr/nirfl imaging-guided synergetic chemo/photothermal therapy of tumor noninvasive laser-induced photoacoustic tomography for structural and functional in vivo imaging of the brain fluoro-photoacoustic polymeric renal reporter for real-time dual imaging of acute kidney injury the merging of biology and imaging into molecular imaging positron emission tomography basic science of pet and pet/ct self-assembled responsive bilayered vesicles with adjustable oxidative stress for enhanced cancer imaging and therapy correlation of chest ct and rt-pcr testing in coronavirus disease 2019 (covid-19) in china: a report of 1014 cases this article is an open access article distributed under the terms and conditions of the creative commons attribution (cc by) license key: cord-284934-mkxfa2nr authors: davis, mark m title: systems immunology date: 2020-07-30 journal: curr opin immunol doi: 10.1016/j.coi.2020.06.006 sha: doc_id: 284934 cord_uid: mkxfa2nr systems biological approaches to immunology have grown exponentially in the past decade, especially as broad approaches to data collection have become more accessible. it is still in its infancy; however, largely descriptive, and looking for the main drivers of particular phenomena, such as vaccination effects or pregnancy. but this lays the ground work for an increasingly sophisticated appreciation of subsystems and interactions and will lead to predictive modeling and a deeper understanding of human diseases and interactions with pathogens. "imagine that the gods are playing some great game like chess . . . you don't know the rules of the game, but you're allowed to look at the board at least from time to time and . . . from these observations, you try to figure out what the rules are . . . " richard feynman while this quote is meant to apply to science generally, i find it especially relevant to where we are now in applying systems approaches to immunology. it also is an important reminder of how all science begins with observation. this contradicts the conventional wisdom that one must start with a hypothesis and quickly get to 'mechanistic' data by the end of the paper. hypotheses and mechanisms are surely where we want to go, but careful observation and analysis must come first. only as more data accumulates are hypotheses worth having. i am biased, but i believe the modern form of systems immunology began in 2008-2009, with a relatively concurrent publication that i wrote entitled 'a prescription for human immunology' [1] together with the first data papers by sekaly [2] and pulendran [3] , where both groups analyzed yellow fever vaccine responses using gene array data and other data. the theme of all of these was that we needed new approaches to human immunology, since so much of what we do in mouse work cannot be done in humans. furthermore we needed data that was not dependent on what we know about the mouse immunology, because many failed translational efforts suggest that the mouse is not a reliable predictor of human results (and not just in immunology). and also that at least part of the way forward was to look closely at vaccine responses, since these stimulate the immune system broadly and are relatively easy to get underway. but first some definitions and the overarching logic. i think that the success of a systems approach to an area depends on how well the data to be obtained captures the most important aspects of system. early systems work focused on signaling pathways in yeast for example [4] which makes sense for a single cell organism, where the cells can be thought of as uniform entities (although they were not!). but the immune system is much more complex, with many very different cell types and subsets and activation state differences. furthermore these cells 'talk' to each other with many different cytokines. and develop different antigen receptor repertoires that are shaped by experience. luckily, we now have very sophisticated methods to phenotype the different cell types and quantitate most of the different cytokines. and new methods to characterize the tcr and bcr repertoires. importantly, this can be done at the single cell level, which is important in distinguishing what different subpopulations may be doing versus the aggregate. much less accessible in humans are the different tissues and organs where immune cells mature and mount responses in, but there has been considerable progress recently here as well [5 ] . but why study the immune system as a system? because that's what it is-the different cell types and molecules work together to mount and modulate responses. while it has been necessary to take a piecemeal approach to something as complicated as the immune system for most of the history of our field, it shouldn't stop there, and the next order of business (and much ongoing work) is to understand the interplay of its various components such that we can actually predict the contribution of each part to a given response. here blood is the most valuable resource in that it is one of the mostly easily obtainable clinical material and contains both most of the key cell types and many of the cytokines. and these cells are circulating, especially after any immunological stimulations, such as a vaccination. another reason why this approach is particularly valuable for human work is that the key interactivities of a system are revealed by perturbations-thus the more and the more varied perturbations employed, the more can be learned. here humans have an embarrassment of riches, with thousands of drug treatments, dozens of vaccines, thousands of infectious diseases and so on. and there is broad genetic variation and environmental exposures in different parts of the world. plus humans typically live a long time and so longitudinal studies can be undertaken to see how the immune system varies with age. but inbred mice and monkeys can be very useful in a systems approach as well, there are many manipulations, both immunological, infectious and genetic that are well known in mice and would reveal immune circuitry quickly and efficiently. and it would be much easier to go into mechanistic depth than with humans, of course. but another reason for parallel systems studies in mice is to discover differences in mouse versus human responses that might explain why mouse models of disease fail to predict human responses or their heterogeneity so often. monkeys are attractive because they are much more likely to produce similar responses to humans (although this is not a given) but they are a more limited option because of their expense. i think of systems immunology as divisible into four phases, as depicted in table 1 . the first being a 'discovery' phase where you use the many available technology platforms to 'cast a big net' over an area or phenomenon of interest and see what you catch. this is mostly where we are now, finding expected or unexpected connections between parts of the immune system and a disease for example. a second phase would be where attention is paid to the interactions of different parts of the system in order to discern which parts correlate or anti-correlate. the third and fourth phases are where we have enough understanding of the major drivers of activity to predict what will happen, first in a subsystem and ultimately in the whole thing. a spoiler alert at this point would be that three and four are still quite distant! an excellent early example of phase 1 was the observation of pulendran et al. that the stimulation of tlr5 on murine b cells was critical for them to progress to antibody secretion and the plasma cell stage. and that this stimulation was dependent on flagellin produce by the microbiome [6] . interestingly, while the mouse experiment was suggested by gene expression data from human vaccination, thus far this same relationship does not seem to be operative in human vaccination, although the microbiome does have interesting effects in human vaccines [7 ] . an example of the second phase has been in the immunology of pregnancy, where brodin et al. on the differences from birth to early life of children born naturally versus cesaerian births is very interesting, in that they start out having very distinct but reproducible phenotypes but then converge soon afterwards [8 ] . other groups have also developed very deep 'omics approaches to pregnancy, identifying the major immunological and other shifts that occur and relating the immunological data with microbiome and metabolomic data [9 ,10 ] reviewed in brodin [11] . these analyses clearly set the stage for further work on what can go wrong in pregnancy, which if one could know in advance would be important clinically. another area of longstanding interest centers around vaccination and infectious diseases. the recent sars-cov-2 pandemic where different individuals have very different outcomes is a dramatic example of individual immune variation, but not enough data is available at this point to say much, or to cite papers. but we do have very interesting systems studies of other diseases to mention, particular those of chien and khatri [12 ] on latent infection with mycobacterium tuberculosis. here an extensive mass cytometry panel analysis comparing latently infected with unifected individuals to show distinct differences in peripheral b cells and t cell types. most importantly they used a program to convert gene expression data in other studies to cell subsets [13 ] to find (and confirm) that nk cell levels drop dramatically in subjects that develop active tb disease, and that they return to baseline after antibiotic treatment. in the context f vaccination, there are many reports, but perhaps the most important f the most recent crop is that of tsang and co-workers who followed p their earlier correlation of successful influenza vaccine responses with higher baseline levels of memory b cells in the periphery [14] with a very recent study expanding on that theme and extending it to an autoimmune disease [15 ] . that higher levels of the raw material of an antibody response at the outset would be this predictive seems simplistic, but there is a definite link here and this may reflect an underlying dynamic within the system. whether it applies to other parts of an immune response is less clear. another important area to be tackled is the general influence of the immune system on aging and health problems in general. the absence of almost any medically validated biomarkers has fostered a whole industry of 'immune boosters' that while popular with the public, have no basis in fact. here the earlier work of furman and colleagues [16] established an important link between the inflammasome pathway and cardiovascular disease. also noteworthy is a time course of stroke patients that uses machine learning of large immunological datasets (as do many of those already cited) to show a very dynamic of immune activity. these early studies lay the groundwork for potential interventions, as well as identifying useful biomarkers that have predictive potential. here it is worth noting how valuable, but rare, longitudinal studies are. there is no better control for an individual than themselves, and following them over an extended time can be very revealing. the framingham study is one of the longest running and has been very valuable for general medicine over time. but there are few studies that use modern immunological assays over time. here the stanford-ellison study is small (135 individuals) but has been going since 2007 and has been a rich source of insight into how peoples immune systems age and the relationships to common diseases. a number of papers have emerged from this cohort, including the furman paper cited above, most recently alpert et al. [17 ] have seen that older individuals have a specific 'trajectory' in their cellular phenotype where different individuals have different immune starting points, but generally change with the same slope. they also derived an inflammatory cell type score that was more predictive of cardiovascular disease than one derived from methylation patterns (considered the best indicator prior). combining both the s-e longitudinal data and much larger cross sectional cohorts to cover over one thousand individuals, sayed et al. [18 ] , used machine learning and other computational methods to subset individuals and linked these subsets to specific disease risks and inflammation associated with aging. also critically important in the development of systems immunology is the development of specialized computational and statistical methods to extract meaning from the large amounts of data that are generated and solve some of the many problems that come up. here the widespread use of gene arrays (and now single cell rnaseq) expression data has resulted in masses of data. being able to simultaneously assay all the genes in the genome was a revolutionary advance, and it is still the richest data source obtainable from blood or tissues, but gene expression data is inherently noisy and there is also no guarantee that a given transcript is translated. but here khatri and colleagues have developed innovative ways to combine study results such that common themes and gene signatures emerge clearly. this they have done in multiple studies, most importantly in tb, where they have been able to re-analyze multiple conflicting studies to derive a three-gene signature for predicting who will develop tb disease versus the vast majority of people who will not [19 ,20] . other important tools for systems analysis are programs that combine different data sets to identify important relationships between the immune system and metabolism for example, or the microbiome [9 ,10 ] . another area of immunology where computational tools have been sorely needed has been in the area of tcr and bcr repertoire analysis. while it is easy now to generate massive data sets of millions of these sequences, parsing them into useful insights has been a struggle. particularly in human t cell responses there can be hundreds or thousands of tcr sequences for each ab tcr specificity peptide-mhc. here several new programs have come to the fore [21, 22, 23 ] that allow one to reduce even very large tcr sequence sets into a much smaller set of shared specificities. in closing i see a bright future for systems immunology, which will only become more useful with time. i am reminded of the famous words of theodosius dobszhansky, who said that "nothing in biology makes sense except in the light of evolution." to which i would say that the equivalent here would be that "nothing in immunology makes sense except in the context of infectious diseases and self-nonself discrimination." in the case of the former influence, the review by prof. quintana-merci is very timely [24 ] . nothing declared. papers of particular interest, published within the period of review, have been highlighted as: of special interest of outstanding interest a prescription for human immunology yellow fever vaccine induces integrated multilineage and polyfunctional immune responses tlr5-mediated sensing of gut microbiota is necessary for antibody responses to seasonal influenza vaccination antibiotics-driven gut microbiome perturbation alters immunity to vaccines in humans the effects on the flu antibody response after antibiotic administration to human volunteers. a template for loss risk interventional studies stereotypic immune system development in newborn children a cell phenotype analysis using impossibly small blood samples to show differences in the immune system of newborns born by ceasarian versus natural birth multimodal immune phenotyping of maternal peripheral blood in normal human pregnancy multiomics modeling of the immunome, transcriptome, microbiome, proteome and metabolome adaptations during human pregnancy intensely broad analyses of maternal blood across the gestational age spectrum, show active immunological and other changes, and highlighting connections between different components new approaches to the study of immune responses in humans a multi-cohort study of the immune factors associated with m. tuberculosis infection outcomes a mass cytometry comparison of latent mtb infected individuals versus healthy controls, showing that latency is not an immunologically quiescent state. they were also able to extend the reach of their informatically to characterize a dramatic drop in peripheral nk cells just before disease onset leveraging heterogeneity across multiple datasets increases cell-mixture deconvolution accuracy and reduces biological and technical biases global analyses of human immune variation reveal baseline predictors of postvaccination responses broad immune activation underlies shared set point signatures for vaccine responsiveness in healthy individuals and disease activity in patients with lupus further validation of baseline values being predictive of immune responses, and extension in the prediction of sle flares expression of specific inflammasome gene modules stratifies older individuals into two extreme clinical and immunological states a clinically meaningful metric of immune age derived from highdimensional longitudinal monitoring longitudinal data on aging shows a clear trajectory of aging but with different set points for different individuals. also a cell phenotype score predictive of cardiovascular disease an inflammatory clock predicts multi-morbidity, immunosenescence and cardiovascular aging in humans an analysis of over 1000 individuals with intensive immune monitoring with different proposed subsets having different predispositions for disease risk. also a cytokine-based signature of inflammation having predictive value host-response-based gene signatures for tuberculosis diagnosis: a systematic comparison of 16 signatures genome-wide expression for diagnosis of pulmonary tuberculosis: a multicohort analysis identifying specificity groups in the t cell receptor repertoire quantifiable predictive features define epitope-specific t cell receptor repertoires analyzing the mycobacterium tuberculosis immune response by t-cell receptor clustering with gliph2 and genome-wide antigen screening a much improved algorithm for parsing even millions of tcr sequences into specificity groups to complement other omics data human immunology through the lens of evolutionary genetics an important reminder of the importance of infectious diseases and their likely role in the evolution of the immune system of human beings key: cord-292024-ae7rauc6 authors: fulop, t.; larbi, a.; hirokawa, k.; cohen, a. a.; witkowski, j. m. title: immunosenescence is both functional/adaptive and dysfunctional/maladaptive date: 2020-09-15 journal: semin immunopathol doi: 10.1007/s00281-020-00818-9 sha: doc_id: 292024 cord_uid: ae7rauc6 alterations in the immune system with aging are considered to underlie many age-related diseases. however, many elderly individuals remain healthy until even a very advanced age. there is also an increase in numbers of centenarians and their apparent fitness. we should therefore change our unilaterally detrimental consideration of age-related immune changes. recent data taking into consideration the immunobiography concept may allow for meaningful distinctions among various aging trajectories. this implies that the aging immune system has a homeodynamic characteristic balanced between adaptive and maladaptive aspects. the survival and health of an individual depends from the equilibrium of this balance. in this article, we highlight which parts of the aging of the immune system may be considered adaptive in contrast to those that may be maladaptive. due to the current covid-19 pandemic, there is much interest in the immune system of older individuals, who are at highest risk of severe disease and death [1] [2] [3] [4] [5] . it seems to be common knowledge that infections and diseases are increased in the elderly and may be considered associated with the aging process itself [6] [7] [8] [9] . in the meantime, there is an unprecedented increase both in life expectancy and in the number of centenarians and semi-supercentenarians (several "blue zones" where these oldest old individuals are "enriched" compared with the general population exist on earth) [10, 11] . many changes have been described in the innate and adaptive immune systems with aging [12] [13] [14] [15] [16] [17] [18] [19] [20] . these contentions seem to represent completely contradictory trends which may be difficult to reconcile. interestingly, two decades ago a common denominator of aging and age-related diseases called inflammaging was described by franceschi et al. [9, [21] [22] [23] , a concept that was largely based on the previously described age-related immune changes called as immunosenescence [24, 25] . as a corollary, this became for geroscience and the nine hallmarks of aging one of the basic underlying mechanisms of aging designated as "intercellular communication" [26] [27] [28] . however, a new appreciation of the data found in older adults suggests that not all changes in the immune system generally considered detrimental are actually harmful, and concomitantly the immune system of the elderly is able to perform better than it was previously suspected when adequately challenged [29, 30] . as mentioned, the common paradigm is that age-related diseases (ards) and the decreased vaccine response are somehow linked to immune alterations with aging, commonly called immunosenescence [16, 20] . however, several recent observations challenge this idea. first, the elderly are in fact able to sustain an adequate vaccine response compared with young subjects [30] . second, for semi-supercentenarians the most important survival factor was proposed to be inflammation [31] [32] [33] . third, the immune checkpoint inhibitors are as efficient in elderly as in young [34, 35] . lastly, most infections are not fatal in the elderly, including covid-19, and not all older adults suffer from numerous age-related diseases [36, 37] . therefore, it could be conceptualized that the age-related immune changes may be a mix of adaptation/resilience and maladaptation/allostatic load, which are closely related to what was called the immunobiography or immunoadaptation, in line with the well-known physiological concepts hormesis and homeodynamics [25, [38] [39] [40] . in this article, we propose a framework to understand adaptation and maladaptation of the immune system and their consequences for elderly. thus, we will describe a holistic conceptualization of the immune system changes during aging, called the "adaptage theory." the main role of the immune response is the protection of the organism from all external and internal challenges in the form of pathogenic microorganisms as well as transformed (neoplastic) or damaged (injured) cells [7, 23] . the immune system is fully equipped to perform this complex task. however, many reports state that with aging there is not a single part of this response which would not undergo changes, and these changes were uniformly conceptualized as being only "bad," deteriorative changes, and their consequences could be only detrimental for elderly [41] [42] [43] . this notion generated the concept of removing the problem by the rejuvenation of the immune system of the aged individuals to the level of young subjects [44, 45] , however in the physiological environment of an otherwise old organism. usually, the aging-associated changes in the immune system are described separately for its innate and adaptive compartments. here we will try to describe the unified concept of these age-related changes. when we consider aging, we consider time. so, the immune system today is not the same as yesterday, neither in young nor in elderly persons. this means that at every moment challenges are arising in our external and internal environment which should be dealt with by the immune system [38] and that these challenges build on the previous state. this implies that every event occurring during life will consequently change or sculpt the immune response [25] . so, during life, we have many aggressions which can be halted by the innate immune system alone, or if they are more serious, they will need the involvement of the adaptive immune system [46, 47] . thus, in any case, the first encounter of an external or internal pathogen is with the cells of innate immune system [48] . this system is fully equipped to deal with all aggressors in a very fast time frame starting from minutes to days. this system is composed of neutrophils, monocytes/macrophages, dendritic cells (dc) and nk cells, and numerous soluble mediators secreted by these cells, including cytokines and chemokines [49] . by itself, this system is powerful enough to clear an aggression without harm [50, 51] but also in special circumstances to turn against its own by sustaining inflammation, over time leading to chronic inflammatory diseases [52] [53] [54] . among these are, for example, rheumatoid diseases, atherosclerosis, cancer, or asthma, the precursory stages of which may be arising very early in life [55, 56] . acute inflammation is meant to eliminate everything which is harmful for the organism and ends with a (near) complete return to the original state. however, if the aggression is chronic, or if the innate immune system is not set back because the regulatory mechanisms are overwhelmed, the inflammatory process can become chronic and potentially harmful. when we think about a chronic process, we suppose that time plays a role; thus, aging (occurring with time) is a relevant factor [57] . this means that all these pathologies are considered ards. nevertheless, we should mention that the chronic nature of such stimulation has already somehow elucidated the occurrence (explained the beginnings) of these diseases early in life [57] . the concept of trained innate immune memory sheds some light how the innate immune system may orchestrate the eventual alertness or chronicity of the innate immune response. at each aggression, innate immune cells such as monocytes are activated [58] [59] [60] [61] [62] . "trained" at each occasion by intracellular immunometabolic changes/reprogramming as well as by subsequent epigenetic imprint, these cells will increase their inflammatory response [63] [64] [65] . however, this process may take two different paths. one path is the controlled, wellorchestrated innate immune memory, which helps to resolve aggressions rapidly and stop the inflammatory process, but nonetheless remains at a higher activation/readiness state [58] [59] [60] [61] [62] . the other pathway is less controlled, which can lead to what has been called "immune paralysis" [66] . not only will the response not be adequate, but these cells are already overstimulated, so a new stimulus may not trigger an efficient response and the functionality of the innate immune cells will decrease when it should increase. this has been shown also for some specific phagocytic cells' functions with aging [67] . a basal state of activation of the innate cells was demonstrated with aging, which may lead to a preparedness and readiness of the system [68] [69] [70] [71] . this may help the innate immune system to be reactive more quickly and at a higher level. interestingly, many tissue-resident and blood-born innate immune cells, including neutrophils and microglia, display hyperactivation phenotypes with aging [72, 73] . however, it was also shown that this may lead to a relative immune paralysis state resulting in the blunting of the innate cells functionality when specifically challenged. it should be mentioned that this does not mean that these phagocytic cells may not enhance their functions, but not in all elderly and not to the level as would be expected in case of young. this is corroborated by the findings that most elderly subjects may efficiently clear an infection, such as influenza or even sars-cov-2 [14, 16, 37, 74] . certainly, this happens in fewer cases than in young subjects, but it is not impossible. thus, this is a perfect example of the adaptation/maladaptation concept of the aged immune system. moreover, the innate immune response can also determine how the adaptive immune system will react [75, 76] . cells have evolved which seem to be intermediate between the innate and the adaptive immune system and may react faster than standard adaptive immune system but keep its characteristics [76] [77] [78] . these innate lymphoid cells (ilc) are broadly classified into three main subsets: ilc1 (ifn-γ producing), ilc2 (il-4, il-5, and il-13 producing), and ilc3 (il-17 or il-22 producing or both) [79, 80] . ilcs are either present in the tissues or in the circulation. they can rapidly react to microbial and cytokine signals. they also rapidly induce the cxcl13 chemokine, which is the ligand of cxcr5 playing a role in the tissue accumulation of these ilcs [81, 82] . by their rapid response, these cells also contribute to the eradication of the microbes constantly invading the human organism and permanently threatening its overall health. the innate-like t cells comprise the cells displaying a γδtcr, mucosa-associated invariant t (mait), inkt (invariant natural killer t), gemt (germline-encoded mycolyl lipid-reactive), and innate-like b cells [83] . these cells recognize foreign/self-lipid presented by nonclassical mhc molecules, such as cd1d, mr1, and cd1a [84] . they are activated during the early stages of bacterial infection and act as a bridge between the innate and adaptive immune systems. unlike their conventional counterparts, innate t cells rapidly recognize foreign pathogen signals and manifest immediate effector functions after activation [85] . these innate t cells are implicated in immune responses to viral infections such as cmv, are able to recognize a broad range of cancer cells, and react to stress-induced molecules, such as mhc class i-related chains a and b (mica and micb) that are expressed on virus-infected cells and also participate in the general antimicrobial defense [86] [87] [88] . the nature of the antigens recognized by innate t cells is also diverse and broadly non-overlapping, involving metabolites, bacterial products, and lipids. inkt cells have been principally shown to respond to glycolipids, γδ t cells are potently activated by (e)-4-hydroxy-3-methyl-but-2-enylpyrophosphate (hmbpp) [89] , and mait cells can be activated by riboflavin metabolitesreduced 6-hydroxy methyl-8-δ-ribitylumazine (rrl-6-c h 2 o h ) , a s w e l l a s f o l i c a c i d m e t a b o l i t e , 6formylpterin(6fp) [90] . so, they have some characteristics of the innate immunity such as rapid effector functions but also adaptive immunity because of the rearrangement of their tcr and thymic selection. this allows innate t cells to perform effector immune responses much earlier than conventional t cells and act as an additional "bridge" between innate and adaptive immune responses. studies demonstrate that unconventional t cells do indeed contribute to the ability of host organisms to clear and control certain bacterial infections. these cells are able to efficiently travel to the sites of inflammation and initiate rapid responses by means of cytokine production and cytotoxic activities. in the description of immune changes with aging, there are a few places for these essential cells. larbi and his group devoted a lot of work to understand how these cells work during aging [91, 92] . they have shown that vδ2+ t cells composition and functionality are not altered in older adults. they have further shown that peripheral vδ2+ phenotype, functional capacity (cytokines, cytotoxicity, proliferation), and gene expression profile are specific to this subset when compared against all other αβ and γδ t cells in aging. also, hallmarks of senescence including telomere length, epigenetic profile, and dna damage response of vδ2+ differ from all other αβ and γδ t cells. finally, they have shown that vδ2+ are resistant to cellular aging due to their unique epigenetic and transcriptomic signatures. these findings constitute another type of adaptation in view to compensate/complement the changes observed in adaptative t cells during aging. future work should unravel whether this potential of being resilient to stressors in vδ2+ could be promoted in other cell type and consequently exploited to lead to better response to infections and in the field of cancer immunotherapy or designing a vaccine utilizing vδ2+ properties for the elderly. group 2 ilc (ilc2) respond to the alarmin cytokine il-33 and are potent producers of il-5 and il-13 as well as a variety of other effector molecules in vitro and in vivo [93] . tissueresident ilc2 are implicated in tissue repair, tissue remodeling, and metabolic homeostasis [94] . recently, it was shown in the choroid plexus of the brain that ilc2 in the aged brain are long-lived and capable of reversibly switching between cell cycle dormancy and proliferation [95] . they are relatively resistant to cellular senescence and exhaustion under replication stress, leading to enhanced self-renewal capability. when activated in vitro and transferred intracerebroventricularly, they revitalized the aged brains and enhanced cognitive function of aged mice. these results suggest that aging may expand a unique population of brain-resident ilc2 with enhanced cellular fitness and potent neuroprotective capability, by decreasing neuroinflammation, cellular senescence, and exhaustion and moreover capable of self-renewing. it is still unclear whether similar ilc2 cells may be found elsewhere in aging organism, but this is another cell type which may resist the "detrimental" effect of aging and show adaptative traits even if is occurring locally. the subsets of memory or memory-like t and b cells with innate-like properties have been observed to accumulate with aging [42, [96] [97] [98] [99] [100] . the increased numbers and activity of certain innate or innate-like immune cell subsets with aging might be considered host responses to compensate for the drastic decline in adaptive immune cell development and function [95] . these cells mirror the findings in vδ2+ t cells as described above and show the plasticity and adaptability of the innate and innate-like cells with aging, probably maintaining some physiologically important functions when the other part of the system is fainting [91] . thus, we can say that the effects of aging on the immune system are much more complicated than a commonly preached generalized decline in immune cell development, phenotype, and function. once an aggression occurs and the innate response is inefficient or insufficient to clear it, the innate system should drive the adaptive immune system to react [75, 76] . this is carried out by coordinated and efficient actions by the cells and the soluble mediators of the innate immune system. the antigens should be efficiently presented, and the mediators should prime the adaptive immunity. it has often been stated that antigen presentation is altered with aging; however, this is still controversial and probably depends on the strength of the innate stimulation [101] [102] [103] [104] [105] [106] [107] . dendritic cells (dcs) are one type of innate immune cell known for antigen presentation, being key components linking innate and adaptive immunity through priming of naïve t cells and shaping adaptive t cell responses [108, 109] . there are two major dc subsets in human peripheral blood: conventional dcs (cdcs) and plasmacytoid dcs (pdcs). these dc subsets recognize different pathogen-associated molecular patterns by expressing distinct repertoires of toll-like receptors (tlrs) and other receptors [110, 111] . engagement of specific dc subset tlrs in turn triggers distinct immune response pathways. for example, pdcs produce a large amount of interferon alpha (ifn-a) in response to virus infection [112] . as mentioned, age effect studies of human dcs have been controversial [107] . while some studies demonstrated agedependent declines, others have shown no difference in the number of circulating dcs [113, 114] . several studies have also indicated age-related functional changes in dcs, such as impaired expression of tlrs [115] ; decreased production of cytokines, chemokines, and ifn-a after tlr stimulation [112] [113] [114] [115] [116] ; and increased responses to self-antigen [117] . an earlier study has shown that the b cell and monocyte antigen presentation in the frame of mhc did not change with age. however, what was interesting in the data reported in one study is that among the 11 elderly even if the monocyte antigen presentation was homogenously maintained, there were 4 elderly with less bcr antigen presentation [106] . even if most of the results suggest a change in antigen presentation, the various steps leading to it are differentially affected by age from the number of dcs through the immunoproteasome processing and the final mhc dependent presentation like in b cells. interestingly, almost a quarter century ago, one of us (jmw) had demonstrated that the level of expression of mhc class i (h-2) molecules on spleen t cells of old mice is higher than on the surface of young splenic t cells; this effect was concomitant with the significant (homeostatic?) decrease of the level of transporter of antigenic peptides (tap)-1 in the same cells [118] . these observations were later supported by assounga et al. [119] . contrarily, expression of mhc class ii on macrophages derived from old mice was reported to be decreased compared with young animals [120] . these data even if contradictory may also illustrate that in the immune system, the changes are not uniform, and the system is self-adapted to compensate eventual failure of some part of the whole system. however, other non-antigen processing and presentation functions of apc, such as ability to become activated, costimulation, or cytokine/chemokine production, may play a role in the difficulties of the immune response to progress to the adaptive immune response [121] . more human studies are needed to clearly elucidate which processes are conserved and which are not in the antigen presentation process. however, once the innate immune response is able to present the antigens, the adaptive immune response takes over with b and t lymphocytes' activation [75] . these main cell types executing the adaptive immune response have many different subpopulations [122] . this part of the immune system has been extensively studied in human aging [123] [124] [125] [126] . there is a very broad consensus considering the adaptive immune system as the "devil" which is responsible for all the misfortunes of the immune system with age [25, 28] . the most important changes are the decrease in the naïve immune cell compartment of the cd4 + and cd8 + t lymphocytes, however being more important in the latter [12, 16, 96, 97] . concomitantly, the tcr repertoire variability is also drastically decreased [127, 128] . in contrast, there is a concomitant increase in memory t and b cells which represent the subpopulation which already encountered antigens [129] [130] [131] . this quickens the adaptive response to cognate antigens, which is evolutionarily beneficial for the survival of an (older) individual, provided he/she stays in the general area where they spent their childhood and where meeting new, previously unknown pathogens is normally infrequent. the contrary would be quite strange if we consider from an evolutionary perspective that the immune system should react to all internal and external stimulation. this state of the adaptive immune system only reflects what we called earlier as immunobiography or immune history [38, 132] . so, in this case, it should be considered neither bad nor good; it reflects only the reality of the role of the immune system. this is simply a life-long adaptation to life. certainly, the decrease of naïve cells in the elderly may not be advantageous for the survival of the individual when an encounter occurs with a completely new antigen such as the sars-cov-2 causing current (2020) pandemics of covid-19 [15] . as we already know, these individuals may die in proportions significantly higher than young covid-19 patients [1, 2] . the cause of this decrease of naïve t cells is the involution of the thymus which starts early in life [13] . this also should have some adaptative reasons in the life of an individual. the maintenance of such an organ (with one of the highest proportions of concurrently dividing cells) is very costly in terms of metabolic energy; so, as most of the naïve t cells have been generated early in life, there is no need for its full function later in life. also, if it is maintained, some errors in the regulatory mechanisms may occur with time, and some diseases such as autoimmune disease may increase. on the other hand, it should be stated that once more nature has foreseen a compensatory mechanism for lack of naïve t cells of thymic origin, which is their homeostatic proliferation in the periphery [133] . there is an unsettled debate among researchers as to whether this phenomenon fully replaces the lost tcr repertoire expressed by the thymus-generated naïve t cells. however, long survival of centenarians demonstrates that it should be an efficient compensatory mechanism. it is of note that many nonagenarians and centenarians recovered form covid-19 despite the apparent lack of naïve t cells [134] . the increase of the memory (mainly cd8 + ) t cells is also considered quite damaging for old individuals [135, 136] . the reason is that their numerous proliferation cycles have led to what is called a senescent state or senescence [137] [138] [139] . this senescent state precludes their capacity to proliferate, mainly due to decreased cd28 expression resulting in slower and weaker activation during a known antigenic encounter, leading to decreased protection [18, 140] . in the meantime, senescent t cells, analogously to other senescent cells arising with age in the body, produce large amounts of proinflammatory cytokines (a phenomenon called senescence-associated secretory phenotype, sasp) as stated by the inflammaging characteristics of the human immune system [141, 142] . this could substantially impede the response to recall antigens in specific cases, but in the meantime, the clinical and experimental evidence both point to the fact that even in the elderly, this part of the adaptive immune system may function efficiently. in the meantime, these cells are also able to contribute to the maintenance of innate immune preparedness. the sasp phenotype elicits an autocrine role on senescent cells, but it is also involved in the recruitment of immune cells, such as macrophages, neutrophils, and natural killer (nk) cells in order to eliminate the senescent cells themselves [143] . concomitantly, upon accumulation of senescent cells, the production of cytokines is enhanced, along with the recruitment of immune cells, jointly paving the way towards the installation of inflammaging [14, 57] . the cytomegalovirus (cmv) infection was considered one of the most important triggers of accumulation of senescent t cells [144, 145] . this consideration generated many studies to evaluate whether senescence of the immune cells could be considered detrimental or beneficial. certainly, the accumulation of senescent cells has numerous maladaptive aspects [146, 147] ; but, as many recent studies have shown, it also has adaptive aspects [148] . finally, the exact percentage of the senescent/exhausted t cells at different ages and what role they may play physiologically is not clearly settled in this context. it may be that they (mainly exhausted t cells) are like the reserve in the army which can awaken when the well-trained t cells are not able to combat pathologies like cancer [149] [150] [151] . in view of the continuous production of immune cells, it seems likely that part of accumulated memory immune cells is not truly irreversibly senescent but rather exhausted, exhibiting reduced functional capabilities which can possibly be reversed when the circumstances may necessitate it [152] . with aging, not only phenotypic changes but also functional changes occur in the adaptive system, which may or may not be always related to the t cell subpopulations but may be due directly to the immunobiography [38] . there are documented changes in the main signaling pathways in t cells, including the tcr, costimulatory receptors, and cytokine receptors, which lead to the decrease of cytokine production, proliferation, cytotoxicity, and differentiation [153] [154] [155] [156] . moreover, the metabolism of these cells is also affected because of the changes in the mtor pathway either favoring the autophagy or the anabolic effects of its stimulation [157] . the changes from oxphos in the resting state to aerobic glycolysis characterizing activated t cells are delayed and less intense in aging t cells [158, 159] . these changes in the functioning may arise from the membrane changes with aging altering the immune synapse formation and consequently the cognate signaling pathways [160, 161] . the changes either in the feedforward or feedback pathways are very complex-as the experimental results suggest-but together, we can say that they may decrease the efficiency of the t and b cell responses with age in certain circumstances which can really have detrimental consequences for the individual [162] [163] [164] . there are also new data that some pathways (precisely the jak-stat pathway [165] ) in the resting t cells are activated with age. to summarize the immune changes in the continuity from the innate to the adaptive immune response, it can be concluded that the immune response in old individuals is complex and heterogenous and probably is more dependent on the type of challenge than from the age of the subjects [166, 167] . furthermore, many compensatory and redundant mechanisms are inbuilt to assure an adequate immune response even in the very elderly. thus, there is an equilibrium between adaptation and maladaptation, and this balance may be disrupted depending on the type, the degree, and the acuteness of aggression (fig. 1 ). the performance of the immune system cannot be completely evaluated without discussing the role of the inbuilt immune suppressor mechanisms which may also be affected by age [168] [169] [170] . first, the role of tregs is double; on the one hand, they should control the extent of the inflammation, and on the other hand, they should avoid the autoimmune processes arising continuously in the body and assure the attrition of the immune response once the danger has been eradicated [168, 171] . given the central role of treg cells in immune homeostasis, age-related loss of treg function would be predicted to render the host susceptible to excessive immunity, encountered in elderly humans as a syndrome of chronic low-grade inflammation [172] . conversely, age-dependent gain of treg activity would expose the host to greater risk of immune failure, such as the rising risk of malignancies and infections in the aging population [171] . emerging data suggest that some treg populations, specifically naturally occurring tregs (ntreg), seem to accumulate with advancing age, whereas inducible tregs (itreg) appear to be less available in the older host, though this is controversial [118] . human studies assessing peripheral blood of aged versus young individuals seem to concur that the percentage of cd4 + tregs is increased in older individuals [173, 174] . all these data indicate that the cd4 + treg compartment expands with age, relative to the total cd4 + t cells. human cd4 + foxp3 + tregs from older individuals show enhanced foxp3 expression compared with tregs from young individuals, while the remainder of their fig. 1 . the balance between the adaptive/maladaptive patterns of an individual's aging immune system. adaptive and maladaptive immune changes can be found simultaneously in the same individual and in the same arms of the immune response (innate or adaptive). concurrent (also balanced or imbalanced) changes in other body systems will affect the immune system. complex integrated immunobiography over time will determine which state (balanced/adapted or imbalanced/maladapted) will predominate and towards which destiny (resilience and longevity, or aging-related diseases, frailty and earlier death) the organism will be pushed phenotypic makeup (gitr, ctla-4, cd127 low ) is unchanged [175] . the implication of this finding is that although tregs retain most of their functional properties, which would be responsible for suppressing immune activation against infection and tumors, while in the meantime, they may fail to control autoimmune inflammation. however, autoimmune diseases are relatively rare in elderly [176] . such an abnormality suggests that an apparent paradox is observed during aging as an aberrant inflammation coexists with immune hyporesponsiveness to infection, vaccination, and tumors [9] . like cd4 + tregs, the percentage of cd8 + foxp3 + tregs has been shown to be significantly increased in the blood of older individuals [177] . given the critical role of tregs in immune homeostasis, any decline in treg competence would inevitably lead to a disbalance of protective and pathogenic immunity and would favor chronic, relentless, and possibly tissuedamaging inflammation [171] . in reality, as already discussed above, aging is indeed associated with a low-grade but clinically not manifest inflammation (inflammaging) which is not out of control. this indicates that the increased treg number sufficiently control inflammaging, in contrast to what it is commonly stated. in contrast, increased numbers of tregs could be considered detrimental in the suppression for the specific adaptive immune responses, mainly in infections and eventually in cancer [168] . it is of note that in cancer tregs were demonstrated to be immunosuppressive directly in the tumor microenvironment [178] . it is also questionable whether their role in the specific immune response is detrimental or regulatory, as the age-related inflammatory diseases start at middle age and not in old age. the well-regulated treg activity may also prevent a hyperreactivity of the immune system, as is seen in the present covid-19 infection in a form of cytokine storm. there is another major immunoregulatory/suppressor cell type, namely, myeloid derived suppressor cells (mdscs) [170, 179] . the mdscs are specialized immunosuppressors which can control the functions of all other immune cells, thus preventing excessive inflammatory responses [180] . there is convincing evidence that the aging process increases the frequencies of circulating mdscs in humans [181] . verschoor et al. [182] revealed that the levels of the cd11b + cd15 + and granulocytic mdscs were increased in the blood of community-dwelling seniors (61-76 years) and especially in frail elderly people (67-99 years). furthermore, it was shown that the mdscs induce the increase of tregs [170, 183] . in this way, there is an immunosuppressive network which is created as individuals are aging as coined by salminen et al. [184] . it seems that tgf-β, il-10, and no, secreted by mdscs, are the major soluble mediators maintaining the functions of this age-related immunosuppressive network [185] . there is an abundant literature indicating that tgf-β signaling suppresses the functions of cd4 + [186] and cd8 + [187] t cells as well as dcs [188] and nk cells [189] . in particular, tgf-β inhibits the signaling pathways of cd28 and mtor kinase [190] . il-10 also inhibits the cd28mediated signaling in t cells by activating shp-1 tyrosine phosphatase-1 [191] . together, the immunosuppressive network increase with aging may have several pathological consequences [184, 185] , but however, in the spirit of immunobiography, this can also be considered an adaptation to decrease the lifelong activation process of the innate immune system (innate immune memory) but unfortunately in the meantime downregulate adaptive immune activation. among the elderly, the number of centenarians and semisupercentenarians is steadily increasing, especially in the blue zones [192] . there is a debate as to whether centenarians and semi-supercentenarians are the result of selection or whether they represent the way that humans should physiologically age, with others simply having risk factors that preclude them from attaining this age even if we consider their genetic specificities. centenarians are a category of exceptionally aged individuals that succeed in preventing or delaying the onset of age-related disorders such as cv disease, type 2 diabetes mellitus (t2dm), alzheimer's disease, or cancer [193, 194] . it is also well-known that these centenarians do not avoid major diseases but are able to counteract their deleterious effects, which most of the other elderly are not capable of. probably the advantage that they have is what we could call the "adaptage," which during their immunobiography resulted in the development of resilience which could overcome the many dysfunctions and lead to a maintained functionality [192] . the centenarians have adapted their immune responses which maintain an adequate functionality and, in the meantime, control inflammaging [31, 32, 195] . this means that they have an efficient anti-inflammaging machinery which may be constituted from immunosuppressive cells and soluble mediators like circulating gp130 to compensate for their increased il-6 level [32] . in this context, pawelec's group has shown a positive correlation between increased frequency of tregs and survival in the elderly, which suggests the increasing importance of maintaining a balance between the adaptation and the maladaptation of immune responses and inflammation as we grew old [196] . one example of this adaptation/maladaptation is the microbiome largely influencing/shaping the functioning of all physiological systems, particularly that of the immune system in centenarians [193, [197] [198] [199] . extreme longevity seems also to be associated with a unique shift of the gut microbiome c h a r a c t e r i z e d b y e n r i c h m e n t i n a k k e r m a n s i a , bifidobacterium, and christensenellaceae [200] . on one hand, microbiota in centenarians show more diversity than in young old, resembling more that found in young subjects [201] . here, certain aspects of the changes in centenarians and semi-supercentenarians are proinflammatory, such as the decline in the abundance of the putative butyrate producers like faecalibacterium (phylum firmicutes), while on the other hand within the bacteroidetes phylum, rikenellaceae (alistipes) and porphyromonaceae (parabacteroides, odoribacter, porphyromonas), also butyrate producers, were found to be increased in all centenarians [201] with the concomitant decrease of prevotella (phylum bacteroidetes) richness, sustaining the changes in microbiota holding strong antiinflammatory activity [202, 203] . thus, the dysbiosis, proinflammatory change in microbiota composition in the oldest old, is compensated by the increase of anti-inflammatory bacteria which are metabolically more active than the former [204] . thus, the microbiota of centenarians support both the increase in the inflammatory load found in the sera but also a compensatory mechanism which is maintaining a fine balance, nevertheless favoring longevity. we should also mention that not all centenarians are made equal. a recent study by tedone et al. [192] studied telomere length and telomerase activity in t cells from low-and highperforming centenarians. they identified several parameters that are different between high-and low-performing centenarians: (a) the amount of proliferation following in vitro stimulation is dramatically greater in high-performing centenarians compared with 67-to 83-year-old controls and lowperforming centenarians; (b) telomere length is greater in the high-performing centenarians; and (c) telomerase activity following stimulation is greater in the high-performing centenarians. in addition, this study has validated a number of genes whose expression was directly related to telomere length which may influence the risk and progression of multiple aging-associated conditions. together, centenarians have better resilience and biological reserves to effectively cope with multiple issues originated from their immunobiography. they can also better cope with inflammaging as they are able to mount a powerful antiinflammaging response neutralizing the overall presence of inflammatory processes [32, 205] . it was clearly demonstrated in case of the semi-supercentenarians in whom the most powerful determinant of longevity was the presence of controlled inflammation [33] . they are the perfect example of hormesis and homeodynamic balance, as they have all the described supposed detrimental effect of aging on the immune system, but they have a powerful compensatory mechanism, a perfect demonstration of the "adaptage" concept of the immune changes. the understanding of adaptation/maladaptation of the immune system with aging was never more relevant than during the covid-19 pandemic. it is widely stated that the old individuals are more susceptible [6, 9, 206, 207] . this should help us to better understand the immune changes with aging which may be detrimental and which may be beneficial. as mentioned, not all elderly who are infected will progress to the severe stage and will not die either [208] [209] [210] [211] . the sars-cov-2 infection is a two-step inducer of the immune response. at the beginning, a functional immune system could contain the infection even in elderly [212, 213] . an effective innate and adaptive immunity is required to try to advance the response until efficient antibody production begins. most of the elderly can fight the infection but perhaps will not establish protective immunity with ab. in those where the immune system is less efficient, the virus will propagate, and a massive destruction of the tissues, especially those possessing abundant ace2 receptors, will ensue [214, 215] . damaged cells will induce massive inflammation via the stimulation of macrophages producing the cytokine storm. as the lung is the main acute target for sars-cov-2, it could be that the lung epithelial cells may also participate in the inflammatory mediators' production [216] . this is not far from resembling the pathomechanism of sepsis [217, 218] . it is debatable whether good health is an advantage or not for the course of sars-cov-2 infection, as the huge immune response during the second phase may be a strong disadvantage [219, 220] . it is also of note that among the elderly who died in the hospitals, only 1% had comorbidities [221] . in the nursing homes, the situation is different as the old persons living in these institutions are not only comorbid but very ill; otherwise they would not be in these institutions [222] [223] [224] [225] . in their cases, already the first step is deficient, so they die as in the case of influenza from the immunosuppressive effect of the virus and the following increased infection by bacteria or fungi [226, 227] . these differences in the elderly reflect their heterogeneity and also the differences of the immune status in each individual. what would it take to move from the "damn-age" concept to the "adaptage" concept? first of all, biology of aging research should dissociate itself from what can be called ageism. the idea that young individuals are by definition in a better state is a cultural belief, not a biological fact [227] . thus, perhaps not all data should be interpreted as detrimental, but considered from an evolutionary and immunobiographic perspective, including the concepts of balanced adaptation/maladaptation as the norm. it should also move from the "good versus bad" concept that biology does not recognize, as these are moral concepts. furthermore, researchers should not derive unequivocal conclusions from mouse data applied to humans, as they do not always follow the same path. in fact, the majority of the immune system-related data refer to mouse data without sufficient warning about the profound difference between murine biology and human biology. these data are highly misleading and lead to many failures when applied to humans. one recent example is the failure of vaccination and use of monoclonal antibodies for treating alzheimer's disease [228] . one of the most important criticisms of human studies is the heterogeneity of humans, which increases with age. this is absolutely true, but this is not a disadvantage but a wealth. this is the only way to unravel how the immune and other related physiological systems age. the immune system and physiology more generally are canonical examples of complex adaptive systems [229, 230] , and it is well-known that linear, cause-and-effect models perform poorly for understanding such systems [231] ; nonetheless, much of the research in these fields has applied methodology based on controlled experiments, which assume that results in a tightly controlled environment (e.g., lab mice) are easily extrapolatable, rather than methodologies that embrace heterogeneity and complexity. undoubtedly, the latter are challenging, but they are also essential if we want to arrive at robust conclusions. this is more and more emphasized in research papers when they deal with aging. the one mentioned for centenarians is a bright example of this necessity if we want to understand what is occurring during aging [192] . another example is a study published a few years ago, which has shown how to identify the drivers of the interindividual diversity of the human immune system, which is crucial to understand their consequences for immune-mediated diseases. by examining the transcriptional responses of 1000 individuals to various microbial challenges, they have shown that age and sex influence the expression of many immunerelated genes, but their effects were overall moderate, whereas genetic factors affect a smaller gene set but with a stronger effect. these results enable us to understand the regulatory role of interindividual variants in the pathogenesis of immune-related diseases and improve our understanding of the respective effects of age, sex, and genetics on immune response variation [166] . furthermore, better understanding not only of the changes in the adaptive t cell compartment but also in the innate compartment occurring with aging will help to better understand how the inflammaging concept may also have some adaptive characteristics and not only be considered the driver of agerelated diseases [14] . it is of paramount importance to assess the role of the trained immune memory in aging humans [38, 67] . furthermore, this would help also to design better vaccines. this is demonstrated by the success of shingrix a vaccine designed for elderly to prevent herpes zoster reactivation, which uses a special adjuvant to help boost the innate immune stimulation toward an effective adaptive immune response in 80 to 90% of old individuals [29] . this would perhaps end the misleading concept that elderly do not respond to vaccination. as we have already stated, this is not the fault of the immune changes with aging but the use of inadequate vaccines, such as in the case of the influenza vaccines, and in particular vaccines that were designed for the immune systems of younger individuals. the increased success of the quadrivalent vaccine is the proof that elderly can built efficient and protective responses [232] [233] [234] . finally, the understanding of the holistic nature as well as the step by step progression of the immune system should lead not to the "single pill" intervention model but to the multimodal/multistep intervention model. aging is a natural process which ultimately results in death. however, the aging process should not be defined only by the perspective of death, which is inevitable. concomitantly, it is often conceptualized that aging is the major underlying cause of all alterations occurring with aging and leading to agerelated diseases, forgetting that these disease processes started long before the beginning of old age. however, the aging process is very different for each individual, which means that aging is not occurring uniformly in every human being. this underlies the plasticity of the physiological functions' changes accompanying aging, including those of the immune system. the immune system has evolved to protect us from aggressions and challenges. it is well established that its dysfunction can lead to pathologies in the elderly. however, not all elderly will suffer from these diseases: more and more are reaching a very old age, with centenarians and semi-supercentenarians having a relatively well-functioning immune system. so, the immune system during life is depending on its individual, unique immunobiography, consequently developing concomitantly adaptative and maladaptive aspects of its functioning. the balance between these functions will determine how the person will age (fig. 1) . it is possible that the effect of age may only be detrimental, as the most common paradigm is stating. however, a more positive and balanced consideration of the immune response with aging will permit us to intervene in a judicious manner. still, many studies are needed to learn how to not intervene when it is not necessary and how to intervene in a multimodal/multistep way where it is needed. better lifestyle, vaccines, and microbiome modulation will help to increase the adaptaging part of the immune system for a healthier life of old individuals. epidemiology working group for ncip epidemic response, chinese center for disease control and prevention (2020) the epidemiological characteristics of an outbreak of 2019 novel coronavirus diseases (covid-19) in china severe outcomes among patients with coronavirus disease 2019 (covid-19) -united states characteristics of and important lessons from the coronavirus disease 2019 (covid-19) outbreak in china: summary of a report of 72 314 cases from the chinese center for disease control and prevention casefatality risk estimates for covid-19 calculated by using a lag time for fatality care for critically ill patients with covid-19 the role of immunosenescence in the development of age-related diseases the immune system and its dysregulation with aging aging of the immune system. mechanisms and therapeutic targets inflamm-aging. an evolutionary perspective on immunosenescence blue zones: lessons from the world's longest lived identification of a blue zone in a typical chinese longevity region the twilight of immunity: emerging concepts in aging of the immune system contributions of age-related thymic involution to immunosenescence and inflammaging human inflammaging immunosenescence in aging: between immune cells depletion and cytokines up-regulation immunosenescence and its hallmarks: how to oppose aging strategically? a review of potential options for therapeutic intervention macrophages in age-related chronic inflammatory diseases markers of t cell senescence in humans does the human immune system ever really become "senescent inflammaging and 'garb-aging inflammaging: a new immune-metabolic viewpoint for agerelated diseases inflammaging and human longevity in the omics era immunity and inflammation: from jekyll to hyde immunosenescence and inflamm-aging as two sides of the same coin: friends or foes? front immunol geroscience: linking aging to chronic disease the emergence of geroscience as an interdisciplinary approach to the enhancement of health span and life span the hallmarks of aging efficacy of an adjuvanted herpes zoster subunit vaccine in older adults persistence of immune response to an adjuvanted varicella-zoster virus subunit vaccine for up to year nine in older adults inflammation, not cholesterol, is a cause of chronic disease inflammaging and anti-inflammaging: the role of cytokines in extreme longevity inflammation, but not telomere length, predicts successful ageing at extr eme old age: a longitudinal study of semisupercentenarians immune checkpoint inhibitors and elderly people: a review does patient age influence anti-cancer immunity? the first 75 days of novel coronavirus (sars-cov-2) outbreak: recent advances, prevention, and treatment benign covid-19 in an immunocompromised cancer patient -the case of a married couple immunobiography and the heterogeneity of immune responses in the elderly: a focus on inflammaging and trained immunity vaccination in the elderly: the challenge of immune changes with aging inflammation, genetic background and longevity immunosenescence: implications for response to infection and vaccination in older people the impact of senescence-associated t cells on immunosenescence and agerelated disorders immune system dysfunction in the elderly immunosenescence and vaccine failure in the elderly: strategies for improving response a sestrindependent erk-jnk-p38 mapk activation complex inhibits immunity during aging innate immunity: impact on the adaptive immune response a brief review of the basics of immunology: the innate and adaptive response a brief journey through the immune system a brief outline of the immune system introduction to the immune system macrophage polarization in chronic inflammatory diseases: killers or builders? how the innate immune system senses trouble and causes trouble the immune system in atherosclerosis inflammation and cancer role of the peripheral innate immune system in the development of alzheimer's disease the integration of inflammaging in age-related diseases bacille calmette-guerin induces nod2-dependent nonspecific protection from reinfection via epigenetic reprogramming of monocytes innate immune memory: an evolutionary perspective trained immunity: an ancient way of remembering innate immune memory: the latest frontier of adjuvanticity bcg vaccination protects against experimental viral infection in humans through the induction of cytokines associated with trained tmmunity epigenetic programming of monocyte-to-macrophage differentiation and trained innate immunity ) mtor-and hif-1α-mediated aerobic glycolysis as metabolic basis for trained immunity epigenetics and trained immunity the itaconate pathway is a central regulatory node linking innate immune tolerance and trained immunity from inflamm-aging to immune-paralysis: a slippery slope during aging for immune-adaptation phosphoinositide 3-kinase inhibition restores neutrophil accuracy in the elderly: toward targeted treatments for immunosenescence host resistance and immune aging signal transduction and functional changes in neutrophils with aging impairment of shp-1 down-regulation in the lipid rafts of human neutrophils under gm-csf stimulation contributes to their agerelated, altered functions aging, microglial cell priming, and the discordant central inflammatory response to signals from the peripheral immune system aging microglia-phenotypes, functions and implications for age-related neurodegenerative diseases immunosenescence: a systems-level overview of immune cell biology and strategies for improving vaccine responses adaptive immunity the biology of innate lymphoid cells the roles for innate lymphoid cells in the human immune system spatial and temporal mapping of human innate lymphoid cells reveals elements of tissue specificity innate lymphoid cells (ilcs): cytokine hubs regulating immunity and tissue homeostasis innate lymphoid cells: major players in inflammatory diseases group 3 innate lymphoid cells mediate early protective immunity against tuberculosis human nkp44+ group 3 innate lymphoid cells associate with tumorassociated tertiary lymphoid structures in colorectal cancer why innate lymphoid cells? cd1d restriction and th1/th2/th17 cytokine secretion by human vδ3 t cells cd1d-lipid antigen recognition by the γδ tcr ) γ/δ t cell subsets in human aging using the classical α/β t cell model histone deacetylase inhibitor modulates nkg2d receptor expression and memory phenotype of human gamma/delta t cells upon interaction with tumor cells nkg2d-and t-cell receptor-dependent lysis of malignant glioma cell lines by human γδ t cells: modulation by temozolomide and a disintegrin and metalloproteases 10 and 17 inhibitors the vγ9vδ2 t cell antigen receptor and butyrophilin-3 a1: models of interaction, the possibility of co-evolution, and the case of dendritic epidermal t cells an overview on the identification of mait cell antigens mapping of γ/δ t cells reveals vδ2+ t cells resistance to senescence vδ2+ and α/ß t cells show divergent trajectories during human aging the development of adult innate lymphoid cells innate lymphoid cells: 10 years on activation of group 2 innate lymphoid cells alleviates aging-associated cognitive decline n g immunosenescence to improve responses to vaccines a b-cell subset uniquely responsive to innate stimuli accumulates in aged mice riley rl in senescence, age-associated b cells secrete tnfα and inhibit survival of b-cell precursors toll-like receptor 7 (tlr7)-driven accumulation of a novel cd11c + b-cell population is important for the development of autoimmunity ageassociated b cells: a t-bet-dependent effector with roles in protective and pathogenic immunity impact of aging on antigen presentation cell function of dendritic cells systems-level analysis of innate immunity innate immunosenescence: effect of aging on cells and receptors of the innate immune system in humans aging of the innate immune system inflammation and neutrophil immunosenescence in health and disease: targeted treatments to improve clinical outcomes in the elderly characterization of mhc-ii antigen presentation by b cells and monocytes from older individuals radiation-and age-associated changes in peripheral blood dendritic cell populations among aging atomic bomb survivors in japan dendritic cells and the control of immunity monocyte-derived dendritic cells as antigenpresenting cells in t-cell proliferation and cytokine production toll-like receptor signaling and its inducible proteins tlr signalling and the function of dendritic cells circulating, interferon-producing plasmacytoid dendritic cells decline during human ageing peripheral blood dendritic cells and monocytes are differently regulated in the elderly aging is associated with a numerical and functional decline in plasmacytoid dendritic cells, whereas myeloid dendritic cells are relatively unaltered in human peripheral blood age-associated decrease in tlr function in primary human dendritic cells predicts influenza vaccine response dendritic cells in human aging increased reactivity of dendritic cells from aged subjects to self-antigen, the human dna reciprocal expression of p-glycoprotein and tap1 accompanied by higher expression of mhc class i antigens in t cells of old mice winter) memory lymphocytes of young and old c57bl/6 mice express high levels of class i major histocompatibility complex (h-2 kb) protein immunosenescence of macrophages: reduced mhc class ii gene expression global analyses revealed age-related alterations in innate immune responses after stimulation of pathogen recognition receptors from "truly naïve" to "exhausted senescent" t cells: when markers predict functionality immunosenescence: participation of t lymphocytes and myeloid-derived suppressor cells in agingrelated immune response changes human t cell immunosenescence and inflammation in aging t cells and immune functions of plasma extracellular vesicles are differentially modulated from adults to centenarians diversity and clonal selection in the human t-cell repertoire age-related decrease in tcr repertoire diversity measured with deep and normalized sequence profiling similar but different: virtual memory cd8 t cells as a memory-like cell population aging of antiviral cd8(+) memory t cells fosters increased survival, metabolic adaptations, and lymphoid tissue homing activation of mir-21-regulated pathways in immune aging selects against signatures characteristic of memory t cells cutting edge: synapse propensity of human memory cd8 t cells confers competitive advantage over naive counterparts successful and maladaptive t cell aging covid-19 and immunity in aging populations -a new research agenda the impact of aging on memory t cell phenotype and function in the human bone marrow aging and cytomegalovirus infection differentially and jointly affect distinct circulating t cell subsets in humans memory t cell homeostasis and senescence during aging memory t-cell homeostasis and senescence during aging t cell replicative senescence in human aging senescence of t lymphocytes: implications for enhancing human immunity divergent mechanisms of metabolic dysfunction drive fibroblast and t-cell senescence human cd8(+) emra t cells display a senescence-associated secretory phenotype regulated by p38 mapk immunosurveillance of senescent cells: the bright side of the senescence program impact of cmv upon immune aging: facts and fiction cmv seropositivity and t-cell senescence predict increased cardiovascular mortality in octogenarians: results from the newcastle 85+ study the reserve-capacity hypothesis: evolutionary origins and modern implications of the trade-off between tumor-suppression and tissue-repair age and age-related diseases: role of inflammation triggers and cytokines. front immunol dissecting aging and senescence-current concepts and open lessons defining 't cell exhaustion overcoming t cell exhaustion in infection and cancer molecular and cellular insights into t cell exhaustion is there a positive side to t cell exhaustion? front immunol signal transduction changes in cd4(+) and cd8(+) t cell subpopulations with aging signaling pathways in aged t cells -a reflection of t cell differentiation, cell senescence and host environment membrane signal transduction in t cells in aging humans cellular signaling in the aging immune system longitudinal study of leukocyte dna methylation and biomarkers for cancer risk in older adults does metabolic reprogramming underpin age-associated changes in t cell phenotype and function? free radic biol med metabolic reprogramming in memory cd4 t cell responses of old adults age-associated decline in effective immune synapse formation of cd4(+) t cells is reversed by vitamin e supplementation aging, immunosenescence and membrane rafts: the lipid connection signal inhibition by the dual-specific phosphatase 4 impairs t cell-dependent b-cell responses with age decline in mir-181a expression with age impairs t cell receptor sensitivity by increasing dusp6 activity downregulation of inhibitory src homology 2 domain-containing phosphatase-1 (shp-1) leads to recovery of t cell responses in elderly defective signaling in the jak-stat pathway tracks with chronic inflammation and cardiovascular risk in aging humans distinctive roles of age, sex, and genetics in shaping transcriptional variation of human immune responses to microbial challenges personal aging markers and ageotypes revealed by deep longitudinal profiling regulatory t cells and the immune aging process: a mini-review an association between immunosenescence and cd4(+)cd25(+) regulatory t cells: a systematic review immunosenescence: the potential role of myeloid-derived suppressor cells (mdsc) in age-related immune deficiency homeostasis and function of regulatory t cells in aging immune aging and autoimmunity functional regulatory t cells accumulate in aged hosts and promote chronic infectious disease reactivation cd4 cd25high regulatory t cells are not impaired in patients with primary sjögren's syndrome the number of human peripheral blood cd4+ cd25high regulatory t cells increases with age aging of the immune system: a risk factor for autoimmunity? the frequency of regulatory cd3+cd8+cd28-cd25+ t lymphocytes in human peripheral blood increases with age tumorinfiltrating regulatory t cells: phenotype, role, mechanism of expansion in situ and clinical significance myeloid-derived suppressor cells as regulators of the immune system the immunobiology of myeloidderived suppressor cells in cancer parameters of the immune system and vitamin d levels in old individuals. front immunol blood cd33(+)hla-dr(-) myeloid-derived suppressor cells are increased with age and a history of cancer correlation between mdsc and immune tolerance in transplantation: cytokines, pathways and cell-cell interaction er stress activates immunosuppressive network: implications for aging and alzheimer's disease activation of immunosuppressive network in the aging process the tgf-β-smad3 pathway inhibits cd28-dependent cell growth and proliferation of cd4 t cells cellintrinsic transforming growth factor-β signaling mediates virusspecific cd8+ t cell deletion and viral persistence in vivo the role of tgf-β signaling in dendritic cell tolerance canonical tgf-β signaling pathway represses human nk cell metabolism tgf-β inhibits the activation and functions of nk cells by repressing the mtor pathway il-10 inhibits cd28 and icos costimulations of t cells via src homology 2 domain-containing protein tyrosine phosphatase 1 telomere length and telomerase activity in t cells are biomarkers of highperforming centenarians the gut microbiota of centenarians: signatures of longevity in the gut microbiota profile gut microbiota changes in the extreme decades of human life: a focus on centenarians cellular immune activation in sardinian middle-aged, older adults and centenarians ccr4+ regulatory t cells accumulate in the very elderly and correlate with superior 8-year survival the interaction of intestinal microbiota and innate lymphoid cells in health and disease throughout life gut microbiota and aging through ageing, and beyond: gut microbiota and inflammatory status in seniors and centenarians comparison of the gut microbiota of centenarians in longevity villages of south korea with those of other age groups comparative analysis of the gut microbiota in centenarians and young adults shows a common signature across genotypically non-related populations recognition of commensal microflora by toll-like receptors is required for intestinal homeostasis gut microbiota signatures of longevity pérez-martínez p (1866) gut microbiota and aging-a focus on centenarians inflammaging and antiinflammaging: a systemic perspective on aging and longevity emerged from studies in humans inflamm-aging: why older men are the most susceptible to sars-cov-2 complicated outcomes. reprints (www.preprints. org) sars-cov-2 and covid-19 in older adults: what we may expect regarding pathogenesis, immune responses, and outcomes china medical treatment expert group for covid-19. clinical characteristics of coronavirus disease 2019 in china clinical course and risk factors for mortality of adult inpatients with covid-19 in wuhan, china: a retrospective cohort study clinical characteristics of patients who died of coronavirus disease 2019 in china risk factors for severity and mortality in adult covid-19 inpatients in wuhan 1) the many faces of the anti-covid immune response imbalanced host response to sars-cov-2 drives development of covid-19 organ-protective effect of angiotensin-converting enzyme 2 and its effect on the prognosis of covid-19 a hypothesis for pathobiology and treatment of covid-19: the centrality of ace1/ace2 imbalance severe acute respiratory syndrome coronavirus 2 (sars-cov-2): an overview of viral structure and host response the immune system regulation in sepsis: from innate to adaptive sepsis and immunosenescence in the elderly patient: a review immune response to sars-cov-2 and mechanisms of immunopathological changes in covid-19 the first, holistic immunological model of covid-19: implications for prevention, diagnosis, and public health measures immune-epidemiological parameters of the novel coronavirus -a perspective covid-19 pandemic: palliative care for elderly and frail patients at home and in residential and nursing homes editorial: covid-19 and older adults the coronavirus and the risks to the elderly in long-term care covid-19 and older adults: what we know invasive pulmonary aspergillosis complicating severe influenza: epidemiology, diagnosis and treatment is aging biology ageist? anti-amyloid-β monoclonal antibodies for alzheimer's disease: pitfalls and promise systems immunology: just getting started complex adaptive systems complex systems dynamics in aging: new evidence, continuing questions vaccines to prevent infectious diseases in the older population: immunological challenges and future perspectives control of adaptive immunity by the innate immune system influenza and bacterial superinfection: illuminating the immunologic mechanisms of disease publisher's note springer nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations key: cord-296129-rkadl46r authors: macfall, janet; lelekacs, joanna massey; levasseur, todd; moore, steve; walker, jennifer title: toward resilient food systems through increased agricultural diversity and local sourcing in the carolinas date: 2015-09-18 journal: j environ stud sci doi: 10.1007/s13412-015-0321-1 sha: doc_id: 296129 cord_uid: rkadl46r biological and agricultural diversity are connected to food security through strengthened resilience to both anthropogenic and natural perturbations. increased resilience to stress via increased biodiversity has been described in a number of natural systems. diversity in food production can be considered on the following three levels: (a) genetic diversity as reflected in the range of cultivars which can be selected for production; (b) species diversity, captured through production of a wide range of crops on each farm; and (c) broad ecosystem diversity, described by the diversity of production between farms and within the broader food system. a network of locally based food producers and entrepreneurs provides opportunity for high diversity at each network stage, with increased adaptive capacity and the ability for rapid response to disturbance. we argue that production techniques that use carefully planned diverse plantings, such as biointensive cultivation, increase resilience by increased water use efficiency, yield and nutrient retention while reducing pressure from pests and pathogens. we present a model for a diverse, distributed food system in the north carolina piedmont and analyze an existing distributed network by a food hub in south carolina. through these models, we argue that a shift in the food network has the potential to increase local food security by having food more reliably available where it is needed and by contributing to local resilience through community economic development. the shift in food production and distribution systems serves multiple goals: when crop loss occurs, other crops still contribute to overall harvest, reducing net loss. diverse on-farm production can support a more distributed network of food aggregators, processors, and markets than the current approach of large-scale consolidation. finally, a distributed food supply network supported with diverse agricultural products can increase resilience by providing access to diversified markets for producers and improved food access to consumers with more food choices, while expanding the need for skilled jobs supporting the regionally based food industry. the term bresilience^has been used in many social, economic, and ecological contexts with varied meanings and interpretations. in its broadest form, the term resilience refers to the ability to recover from or to adjust to change (gunderson 2000) . uncertainties in an era of rapid change in agriculture have led to uncertainty in the capacity to meet growing food needs. concerns about chronic malnutrition, environmental degradation, livelihood security, food safety and hygiene, and equitable access raise important questions about how and where food is grown and eaten. in this paper, resilience is defined as the capacity for a system to absorb disturbance and to reorganize, while retaining system function and self-organizing processes (gunderson 2000; folke et al. 2004; folke et al. 2010) . resilience can be measured by the magnitude of disturbance that can be absorbed before the system is redefined into a different state (gunderson 2000) . in the case of agriculture and food security, resilience can be considered from the perspective of food availability with a return to a predictable and adequate supply following a disturbance. in this case, disturbances may occur in production (as with the loss of a crop following a plant disease or pest outbreak, severe weather, or climate change), transportation, processing, distribution, and access/supply. locally based and controlled food production systems with high diversity can provide opportunities for adaptive capacity through the ability to rapidly respond to disturbance and to changing conditions in production and market conditions (hendrickson and heffernen 2002) . the prevailing perception is that food production is predictable with a constancy of relationships. as anyone who has been involved with food production, processing, and distribution has experienced, uncertainty in food production and post-harvest handling is common-and risks are high. adaptive capacity and adaptive management acknowledge that the system being managed will always change, so humans can respond by adjusting the system quickly (gunderson 2000) . a growing body of evidence highlights the importance of biodiversity for ecosystem functionality (peterson et al. 1998; fischer et al. 2006; kerkhoff and enquist 2007) . although food and agricultural systems are highly managed, they are still guided by ecological principles through provision of essential natural resources, ecological function, and ecosystem services. just as with natural systems, biodiversity within food systems can be considered across a range of spatial scales from single farms to larger landscapes. food system diversity also includes social dimensions such as food distribution and access. sustaining ecosystem function in agricultural activities is essential for sustaining farm livelihoods and the food supply (tomimatsu et al. 2013) . functional redundancy within an ecosystem may increase resilience to environmental fluctuations, facilitating successful reorganization of ecological systems. high response diversity may ensure ecosystem functionality, providing a range of ways to respond to environmental change and uncertainty (folke et al. 2004) . biodiversity in agricultural systems can improve crop protection and soil fertility through these expanded functionalities (altieri 1999) . below, we discuss two examples of risks from biodiversity loss, the southern corn leaf blight epidemic of 1970 and porcine epidemic diarrhea, which demonstrate the negative possible outcomes of genetic uniformity in food systems. building on this, the insurance hypothesis for ecosystem resilience through conservation of functional diversity and responsive scale may be transferable to food systems. in the case of food systems, diversity in farm scale and number, diversity of crops, type of market opportunities, and higher numbers of farms equate to high numbers of species that respond differently to external pressures in ecosystems where diversity provides binsurance^if one component of the system declines or is lost. to overcome such risks, species with similar traits may be functionally redundant; if a crop is lost because of high disturbance sensitivity (drought or disease, for example), other crops will still provide agricultural product (mori et al. 2013) . some techniques such as biointensive agriculture which emphasize high biological diversity are most easily adaptable to small acreage. a concern can be about yield loss under such production techniques. however, intensively managed biointensive farms can produce yields which are comparable and often greater than conventional methods, while enhancing ecosystem structure and functions such as biogeochemical cycles and pest population controls. the contemporary food system is built on a complex network of related activities, ranging from on-farm production to harvest and sale to distribution, processing, and marketing, ending with consumer access, purchase, consumption and resource, and waste recovery. many related factors including environmental, social, and economic disruptions have the potential to contribute both chronic and acute disturbance to all points in this complex system. it is important to consider resilience at all points in the food system, minimizing vulnerability in each individual sector. therefore, it is crucial to link the shift in food production systems with a shift in the food distribution mechanisms. indeed, greater crop diversity offers opportunities for more diverse markets, and a locally embedded network of markets for food sale and access may provide greater resilience by insuring multiple points of entry for sale and access if the system is disrupted. as the two models below, north carolina central piedmont network and the south carolina food hub demonstrate, decentralized models that link producers to consumers provide opportunities for farmers that utilize high-yield, low input techniques such as biointensive and other agroecological techniques a convenient and reasonable access to markets. since the techniques can be developed both in urban and rural areas on smaller acreage farms, they can reduce the upfront capitalization costs for a start-up farm and also provide access to food related entrepreneurial opportunities and to food for low-income, ethnically and racially diverse consumers. this paper explores the link between diversity and resilience in our food networks-from production to distribution. we first illustrate risks from loss of biological diversity in food production systems and discuss how biointensive techniques can help overcome such risks. we then discuss two models of food value chains in the carolinas and how a shift in food distribution mechanisms built on principles of biological diversity can help build food security and community resilience. we also discuss the challenges of establishing such models on the ground and thus offer guidelines for practitioners on how to establish decentralized/local/ networked food production, distribution, and access systems. conditions of extreme genetic uniformity for production efficiency. the outcome was the 1970 epidemic of southern corn leaf blight, causing a crop loss of over $1 billion (priced at 1970 dollars) (american phytopathological society 2015). corn leaf blight is caused by a fungus, an ascomycete, cochliobolus heterostrophus (also known as bipolaris maydis, or its name at the time of the epidemic, helminthosporium maydis). the fungus was common across the southern corn production areas, with annual losses of around 2.3 % under normal production. cultural practices to reduce leaf wetness and maintain general resistance throughout the corn population were the most affordable and consistently effective means of control. stubble left following harvest was plowed under to enhance decay, with corn production moved to other fields the following year (levings iii 1990; schumann 1991) . early in the twentieth century, corn breeders found that greatest yields were produced by progeny from the crossing of two genetically different inbred parental lines (buckner 2014). production of seed from defined crosses, however, required carefully controlled pollination and removal of the corn tassels. the detasseling process is very labor intensive and must be done by hand before pollen shedding. a genetic factor was discovered that caused plants to become male sterile, the texas male-sterile (tms) cytoplasm (levings iii 1990) . this meant that plants used for corn production no longer had to be de-tasseled prior to pollination-a huge economic benefit to the corn industry. by 1970, nearly 85 % of the corn produced in the usa contained tms cytoplasm. genetically, two changes occurred simultaneously in the host (the corn) and the pathogen populations. the corn crop now had the new tms gene throughout the population, and a genetic change occurred in the population of the fungal pathogen. a new race, named race t, evolved which had genes to produce a toxin that only affected the tms plants. the fungus could now complete its life cycle more quickly and was able to infect not only the leaves and husks but also the developing ears. for disease to occur, three conditions must be met-a susceptible host, a virulent pathogen contacting the host, and an appropriate environment. the genetic uniformity of the corn with the tms genes introduced the susceptible host. the genetic change in the pathogen, evolved in response to changes in the host genetics, greatly increased the virulence. the weather during the summer of 1970 was warm and wet, ideal conditions for disease to develop. the infection started in early summer in the southeastern united states. by early fall, corn across the entire east coast and westward past the mississippi river had become infected, with 80-100 % crop loss in many areas. the result was near complete loss of the corn crop that year, with a huge economic impact. fortunately, producers returned to still available non-tms lines of corn, again requiring manual detasseling. with the return to production of corn varieties lacking the tms genes, epidemics of this disease have not returned. however, as current corn production becomes less diverse, similar risks may again become important. most pork producers raise hogs for specific markets, resulting in limited genetic variability. breeds and hybrid lines are often selected for uniformity and product predictability, such as size, feed conversion, time to market, and/or meeting the requirements of the integrator or marketing company (martinez and zering 2004) . the disease porcine epidemic diarrhea has had widespread and damaging impacts throughout the pork industry. the disease was first confirmed in the usa in april 2013. it is caused by a highly transmissible coronavirus genetically related to strains found in china (stevenson et al. 2013 ). this disease can be contracted by pigs of all ages; 100 % mortality often occurs with suckling pigs. the incubation period is only 12-24 h. symptoms include vomiting and severe diarrhea, frequently followed by dehydration and death. there are no effective vaccines or pharmaceutical therapies for the disease at this time. the disease is highly contagious and environmentally stable. a tiny amount of material taken from intestines of an infected animal can be highly diluted (diluted 10 -8 ) and still remain infective. air-borne transmission is under investigation. trucks and trailers used for dead haul, transport of animals to processing plants, feed deliveries, trash removal and other activities on infected farms have likely been associated with disease spread (national pork board 2014). the toll on the swine industry has been high. of the 63 million hogs in the usa, about 7 million have been lost to this disease (us department of agriculture 2014). some individual swine farms have lost as many as 30,000 pigs. in response to the rapid disease spread, quarantine measures were put into place in fall 2013. farm visits were not allowed, and movement of people and materials between farms and processors was restricted and monitored. biosecurity measures, isolating one farm from another and preventing movement of infectious material between farms, have been the only effective method for control (national pork board 2014). separation of farms across north carolina and across the country is comparable to high landscape level diversity with species separation in natural systems, similar to the space between farms and sanitation providing a physical barrier to pathogen movement. most hogs are grown by independent farmers, who are producing on contract for sale of the animals to a larger integrator (animal processor). for example, one such integrator has contracts with over 2000 independent farmers for swine production across the usa. the integrators are requiring quarantine between farms as part of the contractual agreement. many of these independent farmers are small to mid-sized concentrated animal feeding operations (us environmental protection agency 2015) . when compared to production on only a few very large swine farms (>100,000 animals), this distributed national network of independent, smaller farms provides opportunities for implementation of the needed biosecurity measures and physical separation. farms can be widely spaced across the landscape, reducing potential for aerial transmission, and strict sanitation measures have been effectively implemented with farm isolation. in addition, risk is distributed across the 2000 farmer network, rather than being concentrated in only a few centralized production farms. this suggests that resilience in the hog industry may be dependent on small farms producing animals independently of each other, rather than depending on large, centralized concentrated animal feeding operations. it also argues that genetic diversity between growers and by each independent grower should be increased to add potential disease resistance and resilience within the industry. in agricultural systems focused on local market channels, resilience can be enhanced by a diversification of on-farm products as well as by distribution of more smaller farms across the landscape, as suggested by the above two examples (thrupp 2000) . on farms, production risk is distributed across the products being grown for harvest and sale. each product represents an ecologically functional group, with varied ranges of tolerance for conditions within the production environment. for example, some crop plants may have a wider or shifted range of tolerance for temperatures or available moisture. based on observations from natural systems, biological and agricultural diversity can be considered from several perspectives: & diversity of markets based on diverse crops and production some crop production techniques mimic and enhance ecological processes and function within agricultural settings. biointensive production is an example of one approach which is focused on application of principles of agroecology (grow biointensive 2014). biointensive production emphasizes high crop spatial and temporal diversity via crop selection and multi-cropping practices. soil is highly managed for fertility and biological activity through an initial deep tillage and frequent carbon and nitrogen additions through green manures and compost. these techniques are often most suitable for small-scale farming and are able to provide product to the local food supply network for sale and distribution. there is also efficient land use and minimal reliance on mechanized equipment. these options are severely limited in conventional agriculture which is dependent on high acreage, uniform mechanized production (jeavons 2006) . a number of studies in natural systems have shown a correlation between high biodiversity and pressures from pests and pathogens (janzen 1970; connell 1971; wright 2002; peterman et al. 2008; terborgh 2012; bagchi et al. 2014 ). in some ecosystems, there is a feedback between pest and pathogen populations and biological diversity, with pests/ pathogen pressures reduced as individuals of each species become more widely separated with increasing biological diversity, reducing the potential for disease and insect spread. production methods which can enhance agricultural diversity such as organic production have been compared to conventional production in a number of studies. two large-scale meta-analysis studies have suggested that high acreage crop yield with organic production is generally lower when conventional methods are used. however, the yield differences between the two methods varied widely and were highly contextual, differing between crop groups, regions, site characteristics, and cropping techniques (di ponti et al. 2012; seufert et al. 2012 ). in addition, organic production can be done in large acreage monoculture, minimizing biodiversity (mission 2014). in contrast, some techniques that are most easily adaptable to small acreage, intensively managed farms can produce yields which are comparable and often greater than conventional methods, while enhancing ecosystem structure and functions such as biogeochemical cycles and pest population controls. biointensive techniques with high density diverse plantings have shown a 25-400 % increase in production with improved water use efficiency, as for tomato, basil, and brussel sprouts (jeavons 2001; jeavons 2006; bomford 2009; grow biointensive 2014) . in another study evaluating yield of nine onion cultivars, 45 kg/10 m 2 were produced with conventional practice compared to 160 kg/10 m 2 with biointensive techniques (moore 2010 ). in addition, energy efficiency was improved from an energy efficiency ratio of 0.9:1 with conventional tillage compared to 51:1 with biointensive production-meaning 51.5 cal of food were produced for 1 cal used for production. with biointensive production, most of the energy input was from direct human input (renewable) rather than from fossil fuels (moore 2010) . high biological diversity is a characteristic of biointensive production, with at least 13 different types of plants grown together, including food for local consumption, food for sale, and compost crops. one farm in california, woodleaf farm, grows over 200 different crops on 3 ha of land with 7 ha in woodland and meadow, using similar cultivation techniques. this farm notes anecdotal evidence that populations of beneficial insects have increased while pests and pathogens are minimized (woodleaf 2014). enhanced productivity, water use efficiency, creation of unique microclimates, and pest/pathogen management are also attributed to high crop diversity (woodleaf 2014). soil management in biointensive systems is directed toward enhancing ecosystem processes (e.g., nitrogen fixation, mineral immobilization) which are associated with soil fertility (perrings et al. 2006; d'haene 2012) . common practices include double-digging soils (loosening two layers of soil) to a depth of 0.7 m with annual amendments of cured compost and green manures. communities of bacteria acting synergistically can suppress plant pathogens, promoting plant growth and crop production (kinkel et al. 2011; mendes 2011; hadar and papadopoulou 2012; gaiero 2013 ) and regulating carbon flux (fitter et al. 2005) . biologically active soil amended with organic materials will also provide ecosystem services by retaining water in soil, regulating biogeochemical processes, increasing cation exchange capacity to enhance nutrient retention and plant availability and reducing material loss from the production beds (cooperband 2002) . environmental benefits with biointensive and similar types of agricultural production include (jeavons 2001 from 2007 to 2012, average farm size in the usa increased from 418 to 434 acres, while the number of farms declined by about 100,000. significantly, the number of small farms, those most suitable for biointensive and similar types of production (1-9 acres) declined nationally from 232,849 to 223,634. the number of large farms (â�¥2000 acres) increased from 80,393 to 82,207 during the same period of time (us department of agriculture 2014). these trends suggest that agricultural production in the usa has the potential for decreased resilience, as farming becomes more consolidated into large acreage operations growing a small number of crops on each farm. biointensive and similar practices have the potential to greatly enhance the adaptive capacity of the food production network, increasing crop/product diversity, while shortening the supply chain from production to consumption. they also have the potential to increase productivity per unit area, while protecting and enhancing ecosystem health. through more diversified agricultural production, as described above, greater resilience can also be attained within locally based food systems and associated economies. a distributed and regional network of small-scale aggregators, processors, distributors, and markets has the capacity to make venues for sale of farm products more accessible to small farmers, especially as they diversify their crops. greater crop diversity offers opportunities for more diverse markets, especially in specialty and ethnic foods. a distributed network can foster regional job growth within the food system and enhance food access to underserved communities. a distributed network of producers, buyers, processors, and consumers that share adjacency also have more opportunity for sharing information and knowledge such as technical advice, information about buyers and markets, new regulations, market opportunities, and new innovative practices. the distributed network can also support current models of centralized markets by consolidating products from small growers into larger quantities for sale/purchase. currently, large aggregators leave little room for small producers who cannot meet minimum quantities required for sale. they also usually require a substantial supply of single, uniform crops, creating a disincentive for on-farm crop diversification. in contrast, small producers have found much of their success by selling directly to consumers through farmers markets and community supported agricultural (csa) models (low and vogel 2011) . among us farmers, in 2012, nearly 8 % of farms sold primarily through direct-to-consumer (dtc) markets, including farmer markets and csas while another 30 % used dtc marketing in addition to other venues. the number of american farms with dtc sales increased by 17 % and sales increased by 32 % between 2002 and 2007; however, between 2007 and 2012, the number of farms with dtc sales increased 5.5 %, with no change in total dtc sales. that dtc sales did not increase may be a reflection that consumer demand has been met (low and vogel 2011) . effectiveness of these direct market outlets for small farmers can be place-based and the cost of marketing can be high. additionally, most farmers benefit by having a combination of sale approaches and types of crops to market. as in other industries, market diversification in the produce business is a means to minimize risks-a diversity of market connections supports a financially sustainable farm enterprise (izumi et al. 2010; vogt and kaiser 2008 ; us department of agriculture 2010). additionally, the food system benefits by having a diversity of local and global channels for the distribution, storage, and value-added processing of food through an increased base of skilled labor. in the event of a disruption (natural disaster, economic collapse, contamination of a supply chain), there are multiple alternative channels that can supply a population's food needs, including supplies closer to communities needing food. disaster notwithstanding, the resilience of the local economy may be increased because of the geographically-linked, skilled jobs that are created: farming, logistics, marketing, storage and distribution, value-added processing, equipment maintenance, input supplier, and many other jobs that cannot be outsourced to non-local entities. in the current national food system, where food is grown and harvested several states away and aggregated by large, centralized corporations, these jobs and skills are not available locally. an increase in non-transferable, geographically dependent, skilled jobs increases the resilience of the local economy immediately. according to the the north carolina commission on workforce development, state of the north carolina workforce report (2007), rural areas of north carolina continue to lose employment opportunities, and bmiddle jobs^that supplied a family-sustaining wage for workers with little formal education are disappearing rapidly. the median family income in north carolina is $46,450, substantially below the national median of $53,004, with 17 % of the population living in poverty. median incomes in rural counties are often 10 % or more below the state median (u.s. census bureau 2014). central north carolina is also challenged by many communities having limited access to food. north carolina is ranked 10th in the nation for degree of food hardships, and greensboro, north carolina in the north carolina piedmont, has been identified as the most food-limited metropolitan area in the nation (food research and action center 2015) . for many small farmers in the piedmont region of north carolina, farm income is a supplemental, yet crucial, part of their household economy. in 2012, about half of the people identified as farmers (50,218 individuals) in north carolina by the us department of agriculture did not farm full time. of those, 19,563 worked more than 200 days off farm (us department of agriculture 2014). for many of these families, small farm operations provide much needed household income to supplement off-farm, low-wage job earnings (us department of agriculture 2014). small farming operations also present economic opportunities for partially-or fullyretired growers, as well as opportunities for beginning producers interested in scaling up to full-time farm businesses. when farmers utilize high-yield, low input techniques such as biointensive and other agroecological methods, farming businesses can be developed in both urban and rural areas, as smaller acreage farms (typically 0.5-5 acres) are proving their viability. reduced land and equipment requirements also reduce the upfront capitalization costs for a start-up farm. one of the barriers that small farmers experience when developing a farm enterprise is identifying convenient and reasonable access to markets for the sale of their products. generally, individual small growers do not have the physical infrastructure (e.g., cold storage), product volume or market access to sell to institutions or buyers that can pay a fair wholesale price and provide a consistent market for their products. there is room for growth and diversification in the local food sector. according to the us department of agriculture, in 2004 north carolinians spend $35 billion per year on food (us department of agriculture 2004, center for environmental farming systems 2013, 2014a, b). although spending on local foods is difficult to track, cefs is encouraging north carolina consumers to spend at least 10 % of their food dollars on locally produced food. the north carolina 10 % campaign is in the third year of its promotion to bring local foods spending closer to this benchmark. at 10 %, $3.5 billion would be generated for the local economy, increasing opportunities for entrepreneurial jobs, skilled employment, and healthy foods. at the same time, there is a renewed interest in local foods to foster healthier north carolinians and protect and preserve the rural landscape (curtis et al. 2010 ). there is a tremendous untapped opportunity for selling locally grown produce to indirect markets such as stores, restaurants, institutions, and distributors, particularly for small growers that cannot market their produce directly to consumers. developing a marketing network geared toward these small and micro-farmers simultaneously builds the resilience of local economies through job creation, the resilience of the food system through a diversified, decentralized network of small suppliers, and resilience in production through cultivation of diversified crops. many of the jobs that have been created since the end of the recession have been low-wage service jobs. rural areas of north carolina continue to lose jobs, while metropolitan areas slowly begin adding new jobs (forter et al. 2013) . this slow economic recovery is a reflection of a lack of resilience in the local economy-largely in response to changes in the manufacturing foundation of the region. fostering locally embedded sustainable, high-yield/low-input agriculture and markets for product sale has the potential to replace the former manufacturing economic foundation. potential for distribution, sale and access to locally produced foods in the north carolina piedmont region to assist local governments in fostering sustainable neighborhoods through food, a model was designed to describe a decentralized, produce aggregation network serving smalland micro-producers within the north carolina piedmont (piedmont together 2013; walker 2014). questions of economic and food system resilience were addressed by asking: what opportunities exist to build the supply and demand for local produce while engaging and involving rural and urban, low-resource, diverse communities in ways that generate individual and community wealth and security? more specifically, what sectors are not currently engaged in local food system efforts that hold potential for growing their businesses while contributing to a more robust local food system? (walker 2014) . the economy of the piedmont region of north carolina during the latter half of the 20th century-dependent largely on low-skilled manufacturing jobs in textiles, furniture, and tobacco in urban areas and tobacco production in rural areasis a prime example of low resilience, as attested by the lingering effects of the recent economic downturn. similarly, while the prevalence and growth of farmers markets across the piedmont is certainly a welcomed addition, a food system dependent on a small number of relatively high-priced, geographically distant markets with limited hours lacks the resilience possible in a food system that includes markets characterized by a high diversity of product, price, location, and methods of sale. the goal of the model designed to address the participation gaps in the local food system described above was to enhance resilient economic development through creative and sustainable diversification of employment opportunities and market types within the local foodshed through opportunities for aggregation, storage, and sale of local food products, exploiting the growing diversity of locally grown agricultural products. figure 1 shows the model developed. dashed boxes, focusing on micro-, small-, and medium-scale aggregators, are assets that could provide opportunities for sale and distribution of local foods from small-and micro-scale producers that currently lack many opportunities for indirect sales of their products. these small-scale aggregators would fill a niche that links a diverse set of producers (part-and full-time, rural and urban) and diverse agricultural products to retail outlets that cater to populations that currently lack access to local food, such as small rural grocers, urban convenience stores, locally owned mid-priced restaurants, and city-and county-managed institutions. diversifying crops also provides opportunities for consumer access to more diverse produce and other foods, such as culturally important or desired foods. this model demonstrates one method by which resilience is increased through supplementing and integrating with the existing, larger-scale food system, not creating a parallel, stand-alone blocal^system. resilience, defined earlier as the capacity for a system to absorb disturbance and to reorganize, while retaining system function, structure, and self-organizing processes, can best be achieved within the food system by more food produced, distributed, and consumed within local geographies, while keeping the larger-even national-structures that fill gaps which cannot be filled locally or where those structures inform and support local efforts. another important concept model is illustrated in fig. 2 , where the inner ring of the diagram shows a direct-toconsumer relationship within a typical local food system. while this smaller model is more highly favored by local food system activists who place a priority on bknowing one's farmer,^it can exclude opportunities for generating community wealth, resilience, and new jobs through the multiplier effect: increasing the number of times a dollar cycles through a community increases the economic impact of that dollar on the local economy (morgan 2006) . a study by the iowa state university concluded that buying local food has a multiplier effect of 1.4-2.6 throughout the wider local economy, depending on the rural or urban context and commodities and scale of the community economy (swenson 2007) . additionally, current research suggests that adding one skilled job in the tradable sector generates 2.5 jobs in local goods and services, with high potential for the non-tradable sector (moretti 2010) . the larger circle in fig. 2 illustrates the inclusion of these bvalue-added^industries, including aggregators, institutions and wide variety of retail outlets. the larger circle also illustrates multiple points of entry for community members into a food production/aggregation/processing/distribution network. programs such as farm incubators can provide training and technical assistance in sustainable production techniques (e.g., biointensive, agroecology), access to on-farm resources and infrastructure, initial entry into post-harvest processing/marketing, and land access opportunities to facilitate the start of new businesses. additionally, underutilized facilities such as convenience stores, restaurants, grocery stores, and local institutions can provide the capacity for storage, post-harvest processing, and/or distribution that can serve as points of entry for people interested in starting non-farm food system enterprises. retail outlets such as restaurants, small grocers, institutions, and innovative outlets, for example mobile or pop-up markets, can widen the distribution of the local food network increasing (walker 2014) access for community members who would not normally be a part of a regional foodshed based only around direct farm-toconsumer marketing. currently, there are a number of projects, programs, and initiatives geared toward assisting mid-scale farmers who wish to enter mainstream markets. for example, started in 1997, the north carolina farm to school program now facilitates sale of locally grown food to 87 of the 100 counties in north carolina. during the 2013-2014 school year, $1,350, 263 was spent purchasing fruits and vegetables grown in north carolina, which were then served in schools (north carolina department of agriculture and consumer services 2014). both approaches are necessary to build a resilient local food system that can absorb disturbances such as natural disasters and disruptions to the national economy and transportation network. the models illustrated in figs. 1 and 2 highlight the opportunities for new, small-scale, locally-based markets that can be the portal for sale of products by locallyconnected producers, thus increasing the resilience of the food system through dynamic, adaptive, and responsive enterprises. to achieve this increase in locally sourced food through decentralized, networked distribution channels, three main issues must be addressed. 1) many of the resources for the promotion of local foods development over the past decade have gone to supporting mid-and larger-scale farms within north carolina. large foundations and universities have focused on helping farmers who were affected by the tobacco buyout program transition to other viable crops, as well as the renewed public health focus on obesity, diabetes, and stroke prevention. this led to much public support for getting fresh, local foods into a variety of markets and food access programs. working with larger farmers and distributors has made a difference in the prevalence of local foods available to consumers. however, smallscale farmers are often not able to sell to these markets. for example, during the 2012-2013 school year, the north carolina farm to school program purchased strawberries from only 7 farms and sweet potatoes from 11 farms. for comparison, there are 4155 north carolina farms of 1-9 acres identified in the us department of agriculture agricultural census, who are also potential suppliers for this program (north carolina department of agriculture and consumer services 2014; us department of agriculture 2014). in order to engender greater resilience of the local food system, more attention needs to be paid to the contribution potential for smalland micro-scale farms. 2) nationally, the local food movement has often been hegemonic in its lack of inclusion of minority participantsbe they underrepresented racial, ethnic, class, or genders (hinrichs 2002; jarosz 2008) . greater attention paid to the barriers for participation by individuals, businesses, and communities that are not currently part of the local food movement will help in creating greater equity of food access, economic opportunity, and environmental health (holley 2012) . it will also expand the range of products available for production, processing, and purchase, increasing both social and economic resilience within the local network. in the same way that a diversity of scale and market type will strengthen the ecosystem of the local food system in the piedmont region, greater involvement from a diversity of stakeholders throughout the system will engender more dynamism and resilience (page 2007; holley 2012) . expanding and diversifying the community of growers and non-farm entrepreneurs will also expand markets and market access to parts of the community now underserved by a centralized food marketing network. 3) for many small farmers, resources and equipment that would facilitate sale to a network are not available. for example, the cost of an 8 ã� 10â�² cold storage unit is typically $4000-$10,000. for many farmers who have substantial capital, off-farm income, or access to credit, this is attainable. however, need for capital is a barrier for most low-resource farmers. the local food models outlined above for central north carolina's small and mid-sized farmers and aggregators can be described in a set of five bdesign guidelines^that increase economic, social, and agricultural resilience. these guidelines are intended as a beginning resource for people interested in starting or adding to some aspect of the decentralized aggregation and cold storage local food network. they are suggestions that can be utilized by decision makers, designers, and planners that may be working on other community-wide issues such as transportation, housing, and economic development. having design guidelines that summarize a model of diversification and resilience based on food system localization allows for the inclusion of ideas generated through academic research into practical, applied community practice. guideline #1: promote networks and nodes intent: develop a regional food system that utilizes complexity (a system of networks and nodes) as a mechanism for achieving greater economic and food system resiliency. a. look for opportunities to situate aggregation and storage facilities throughout the region and close to production farms, while encouraging strong interconnectedness throughout. b. cluster small aggregation and storage facilities near one another, forming bnodes^of the local food supply chain. fifty miles has been defined as a maximum distance between aggregation facilities in rural north carolina (bruno and hossfeld 2009) . facilities may be closer in the more urban piedmont. guideline #2: engender equity and inclusivity intent: build on the strengths of the piedmont triad region by integrating cross-barrier collaborations into the aggregation or cold storage business initiative. a. understand the political geography of your community. while doing business in a new area may present challenges, new business opportunities, collaborators, and markets also await. b. build new lines of community rapport across racial, economic, class, and language boundaries. c. work to promote access to financing and entrepreneurial assistance for diverse communities that are specific to current trends in food system development. guideline #3: plan for appropriate transportation options intent: ensure that the cold storage chain of all produce is maintained from farm to fork and that handling is appropriately documented. each segment of the supply chain should work in tandem with other segments. a. ensure that there is access to a refrigerated truck or trailer to maintain the cold chain. b. know the acceptable upper and lower temperature and humidity limits of the produce being aggregated. c. manage traceability of products. keep all appropriate records regarding the place of origin, place of sale, and storage notes for all produce that moves through the aggregation facility. guideline #4: design an effective management plan intent: design a management structure and plan that addresses the goals and limitations of your aggregation or cold storage business, while adhering to all regulatory requirements. a. choose a management structure that fits with your business. a simple cold storage unit leased to several farmers who are marketing their own produce to aggregators will still require facility management, rent collection, and marketing to new farmers. b. ensure that your facility is compliant with all regulatory requirements, including appropriate recordkeeping. track and document temperature and humidity through the duration of the entire supply chain. c. develop procedures that allow access to storage space by clients when needed and ensure that everyone who enters keeps appropriate logs for food safety requirements. guideline #5: build appropriately-sized cold storage facilities intent: invest in infrastructure wisely. cold storage units can be re-configured, and their temperature and humidity changed with relative ease if planned for up front. however, there is an economy of scale, even when working exclusively with small-scale farmers and buyers. develop a business plan that helps to determine what and when your enterprise should build. a. estimate how much cold storage space you need based on the amount of produce your suppliers will need to store. b. approximate energy costs ahead of time and investigate alternative energy as a way to lower the utility bill. c. know what the optimal temperature and humidity settings are for each produce type you will store and aggregate. d. determine what storage system you will use within the cold storage units based on the farmers and buyers you are working with. some smaller growers who are storing their own produce to sell at a local farmers' market may prefer industrial shelves where they can store produce boxes, while larger growers may prefer palettes and use palette jacks to move their product around. also, there is a need to consider the storage of certified organic and gap-certified produce, which need to remain segregated from non-certified foods in order to maintain their certification. the distributed network of aggregation and distribution centers proposed for the piedmont of north carolina provides an integrated, regional model, reducing transport needs for farmers and expanding distribution, sale, and access. a south carolina food hub has been successful in making local foods more available, providing an aggregation/distribution/service network to benefit farmers and consumers. growfood carolina is presented below as a case study of a food hub that is helping the charleston food system move toward a fledgling level of resilience. growfood carolina provides a model for one of the nodes in fig. 2 -an aggregation/distribution hub that connects small-scale growers to consumers in the same region. it also has the potential to serve as a central hub networked to smaller aggregation and value-added processing centers throughout the region, like the spokes of a wheel. charleston, south carolina is the center of one of the southeast's real estate development booms, aided by an influx of retirees moving to the area due to its climate and the availability of still-relatively affordable land. charleston county has grown from 350,000 in 2010 to almost 373,000 in 2013, a 6.5 % increase (us census 2014). a corollary to the above growth and international recognition for charleston's reputation is the recent development of a vibrant, local food culture. green grocer farms on wadmalaw island, located outside of charleston is representative of this growing local food culture. in the 1990s, the farm grew a variety of row crops under regional organic certification, which were sold in savannah and atlanta. now, 15 years later, pastured eggs and pastured raw milk are produced, and the farm is unable to keep up with local demand for their products. the growth in demand for local foods is also seen at the charleston farmers market. ten years ago there were approximately five farmers; now there are over twenty-six farms selling a variety of local and regional products, including meat, eggs, milk and cheese, vegetables, flowers, shrimp, rice and fruit. one other area of growth is in the local restaurant industry, where almost every local restaurant advertises that they support local farmers and purchase local food, with one nationally recognized restaurant, husk, advertising a strictly regional menu. growfood carolina-a regional food hub growfood carolina is a working example of how a food hub contributes to resilience in the lowcountry of south carolina. the us department of agriculture defines a food hub as ba centrally located facility with a business management structure facilitating the aggregation, storage, processing, distribution, and/or marketing of locally/regionally produced food products^(us department of agriculture 2010). food hubs have operational services which can range from aggregation to distribution and sale, including branding, promotion, packaging, and product storage. they can also assist with producer services, ranging from transportation, training, business guidance, value-added product development, and providing liability insurance, to an often key service, linking buyers with producers. food hubs can also help with the promotion of community/social missions, such as distribution of products to food deserts, raising awareness about benefits of supporting local agricultural businesses, providing opportunities to community youth, offering composting and other forms of regionally-appropriate farming/agricultural workshops. growfood carolina is a subsidiary of south carolina's leading environmental advocacy group, coastal league (ccl), started in 1989 with the mission to bprotect the natural environment of the south carolina coastal plain and to enhance the quality of life in our communities by working with individuals, businesses, and government to ensure balanced solutions( coastal conservation league 2014). to realize this mission, in 2009, ccl started two bin-house^policy and advocacy groups to help protect the coastal corridor of south carolina. one group focuses on climate change and energy, while the other group focuses on sustainable agriculture. the ccl sustainable agriculture committee helps ccl's overall strategy of land conservation: if farming is not economically viable, then small and mid-sized farmers will be squeezed out of the market with subsequent changes in land use. thus, ccl actively supports local, sustainable agriculture. to support their mission, growfood was created to provide infrastructural help to regional small-and mid-sized farmers. in 2010, a 6500-ft 2 warehouse in downtown charleston was donated to ccl. after renovating it and hiring a general manager, growfood officially opened for business in 2011. the warehouse contains 1400ft 2 of refrigerated space. although not climate controlled, the building meets leadership in energy and environmental design (leed) standards. growfood also owns a 15-ft refrigerated truck purchased with funds from a us department of agriculture grant and has an urban garden demonstration plot on site where they host various workshops and school groups. the warehouse is located directly off interstate i-26 leading inland to columbia, sc, and is close to hwy 17, a north/south artery. this location is strategic, allowing growfood to meet its mission as a food hub, consolidating production and distribution within a 120-mile radius. this radius allows growfood to work with farmers growing produce from georgia to north carolina, and ccl has plans to open a second hub in greenville, south carolina that will service the upstate, mountain region of south carolina. farms within the 120-mile radius contact the growfood general manager, who visits the farm for bfull transparency.ã� restaurants and retail stores, and growfood either delivers the produce, or retail customers come to purchase it from the warehouse. after a sale, 80 % of the purchase price returns to the grower, and 20 % remains with growfood to help cover operating costs. growfood does all sales, marketing, and distribution via a two-person marketing team, and they work with over 300 local businesses, distributing 5 days per week. this requires five fulltime staff and two part-time drivers. in 2011, they had five growers and 15 customers, and as of 2013, they have 35 growers with 85 regular restaurant and 10 retail customers. currently, demand far outstrips supply, demonstrating demand for additional local foods and food hub services in the south carolina foodshed. with 2135 farms from 1 to 9 acres in size in south carolina, clearly, there is additional production and distribution capacity (us department of agriculure 2014). the goal of the ccl staff is to expand both growfood as well as the local farming community, helping them to diversify, increase production and transition to organic production. therefore, besides retail distribution, growfood is an advocate for small-scale, sustainable farming. south carolina currently has 15 certified organic growers, compared to over 700 in north carolina. to expand the number of sc organic growers, ccl does outreach and education about the benefits of sustainable agriculture, focusing especially on the next generation of farmers. the average age of farmers in south carolina is 63. there are currently not enough young farmers to supply current demand, let alone create new farms that can help meet growing regional food needs. but this also presents an entrepreneurial opportunity for increasing the resilience of the regional food system through creation of new, small, sustainable farms if land, training, and resources for start-up business are made available. dirt works incubator farm on johns island outside of charleston is working to support development of new farmers starting their new farm businesses in the area. growfood as a food hub is helping the community in three important areas. & bthe environment.^use of sustainable production techniques that improves the soil with each cropping cycle and use of the food near the production farms, reducing transportation impacts & bdiversify local agriculture.^smaller farms have the adaptive capacity for increased diversity of crops in production both on-farm and between farms, also increasing nutritional and preference opportunities for consumers. & brural economic development.^as the manager of growfood stated, bcash flow is the most important thing for a grower.^this cash flow allows farmers to be economically solvent, allowing them to continue farming. median income in south carolina is $44,623, well below the national average, with 18 % living in poverty. despite the ready support of local restaurants, it is recognized the client base needs to expand, so further goals are to sell to local schools, hospitals, and retail supermarkets, and to advocate for a year-round farmers' market. lastly, their larger goal is to have 80 % of charleston's food come from within the 120-mile radius of the food hub by 2030. if successful, charleston and the lowcountry will demonstrate a powerful move toward food resilience as a disturbance response. thus, ccl's goal is to develop an aggregated food hub network in the state, as is proposed for the north carolina piedmont in the previous section, while working on education and training, advocating for policy shifts at the state level, and creating food network market resilience. food hubs can potentially add redundancy and resilience at regional scales and across industries (production, distribution, consumption) and thus act as catalysts fostering reorganization of regional food networks. as anyone who has farmed knows, if a farm cannot make money, then there will be no farm. food hubs can become key nodes in food networks, and because they interact with farmers and retailers on a daily basis, they can serve as a platform to quickly disseminate information, creating synergies between and among the diverse members of a food system. rapid information transfer throughout the food network strengthens adaptive capacity for growers, processors, and distributors, allowing rapid response to changes in market and/or environmental conditions. however, a note of caution: food hubs may not be appropriate in all places, and local production may not fill all food needs. even growfood is years away, at best, from helping create a critical mass of local farms that can provide even a small percentage of the lowcountry's daily food needs. the food hub model in south carolina and the proposed model for north carolina may also be appropriate for other regions, extending the capacity of the regional food networks with strengthened resilience. it is an open question as to whether our global food system contains the capacity to respond to a variety of disturbances, which may threaten the integrity of system function and structure and the ability to provide needed food to all people. by definition, if such structure and function were to collapse and be unable to reorganize, such a food system lacks resilience, with potentially severe human consequences. however, adaptive capacity and adaptive management acknowledge that a system being managed will always change, so humans can respond by adjusting the system in response to disturbance and changing conditions (gunderson 2000 (gunderson , 2001 (gunderson , 2002 . in the case of agriculture and food security, resilience can be considered from the perspective of food availability with a return to a predictable, dependable, safe supply following disturbance. this issue of the journal of environmental studies and science grapples with diverse perspectives of this challenge (marten and atalan-helicke 2015) . while appearing to make agricultural production more consistent and predictable, genetic uniformity also has the potential to increase risk from pests, pathogens, and other disturbances, as highlighted earlier in this paper. in contrast, high biological diversity has the capacity to decrease risk with genetic diversity within a single crop and through distribution of risk between a variety of products being grown for harvest and sale. these insights should help us think more clearly about what are long-term goals for a truly resilient food system. small-scale, locally-managed farm operations using agroecological techniques such as biointensive agriculture have the adaptive capacity for increasing biological and ecological diversity both on and between farms, increasing resilience through the ability to rapidly respond to changing environmental and market conditions. highly managed, low acreage production systems such as the biointensive technique have shown greater yields per unit area than conventional production systems reliant on larger acreage. these highly managed systems also result in more efficient water use, reduced pest/pathogen pressures, and protection of adjacent environments. interspersing small farms with large farms and returning some lands to a non-farmed condition to provide localized ecosystem services has the potential to engender social, economic, and environmental resilience through regionally-enhanced, responsive, and more highly-integrated food networks. development of small, diverse farming operations can help communities respond to chronic economic stress by providing supplemental income for some growers and opportunities to build larger, full-time farm businesses for others. high-yield, low acreage techniques can also make farming accessible to a diverse community of people who had not been involved with agriculture, such as urban residents, minorities, and women. however, one barrier to small farmers is often inconvenient access to markets for sale of their products. to have a resilient food system, you have to make it economically viable to farm. therefore, it is crucial to link the shift in food production systems with the shift in food distribution networks. there is a growing emphasis for the need for new, locally based markets that can be the portal for sale of products by small-scale, locally connected producers. indeed, several regional food hubs and networks have emerged in the usa that aim to close the gap between producers and consumers. regional food network and hub models can increase opportunities for direct consumer purchase, or they can serve as aggregation centers for food to be passed into a centralized distribution network, connecting with large volume sellers such as grocery stores, processing facilities, and wholesale distributors. a food system that is predicated upon supporting and cultivating farmers of all scales, who utilize various sustainable production methods for a diversity of crops, and have a variety of market outlets will inevitably be stronger, more resilient, and more adaptable to change than a system that only responds to one scale or type of farmer or one type of market. using biological and agricultural diversity to expand locally based, sustainable farming systems, foster new farmers and food entrepreneurs, and build distributed aggregation, processing and marketing networks that focus on triple bottom line benefits-environmental, social, and economic-have the potential to strengthen our food security and our communities, providing resilience to both acute and long-term stress. the role of ecological biodiversity in agroecosystems historical perspectives of plant diseases pathogens and insect herbivores drive rainforest plant diversity and composition do tomatoes love basil but hate brussels sprouts? competition and land-use efficiency of popularly recommended and discouraged crop mixtures in biointensive agriculture systems southeastern north carolina food system project: an assessment of market opportunities and limited resource farmers in s.e. north carolina. final report statewide-infrastructure-map.html center for environmental farming systems (2014a) nc growing together: connecting local foods to mainstream markets on the role of natural enemies in preventing competitive exclusion in some marine animals and in rain forest trees. dynamics of populations building soil organic matter with organic amendments from farm to fork: a guide to building north carolina's sustainable local food economy, a center for environmental farming systems report the crop yield gap between organic and conventional agriculture biodiversity, ecosystem function, and resilience: ten guiding principles for commodity production landscapes biodiversity and ecosystem function in soil regime shifts, resilience, and biodiversity in ecosystem management resilience thinking: integrating resilience, adaptability and transformability the state of working north carolina inside the root microbiome: bacterial root endophytes and plant growth promotion ecology action, one earth, many gardens ecological resilience-in theory and application suppressive composts: microbial ecology links between abiotic environments and healthy plants opening spaces through relocalization: locating potential resistance in the weaknesses of the global food system the practice and politics of food system localization market diversification and social benefits: motivations of farmers participating in farm to school programs herbivores and the number of tree species in tropical forests the city in the country: growing alternative food networks in metropolitan areas biointensive sustainable mini-farming i: the challenge the implications of scaling approaches for understanding resilience and reorganization in ecosystems a coevolutionary framework for managing disease-suppressive soils the texas cytoplasm of maize: cytoplasmic male sterility and disease susceptibility direct and intermediate marketing of local foods in the united states introduction to the symposium on american food resilience pork quality and the role of market organization deciphering the rhizosphere microbiome for diseasesuppressive bacteria mission 2014: feeding the world. organic industrial agriculture energy efficiency in small-scale biointensive organic onion production in pennsylvania, usa. renewable agric and food syst local multipliers response diversity determines the resilience of ecosystems to environmental change north carolina farm to school program the difference: how the power of diversity creates better groups, firms, schools and societies janzen-connell effects are widespread and strong enough to maintain diversity in grasslands ecological resilience, biodiversity and scale community choices, regional solutions plant diseases: their biology and social impact comparing the yields of organic and conventional agriculture emergence of porcine epidemic diarrhea virus in the united states: clinical signs, lesions and viral genomic se determining the methods for measuring the economic and fiscal impacts associated with organic crop conversion in iowa enemies maintain hyperdiverse tropical forests state of the north carolina workforce: an assessment of the state's labor force demand and supply linking agricultural biodiversity and food security: the valuable role of agrobiodiversity for sustainable agriculture sustaining ecosystem functions in a changing world: a call for an integrated approach department of agriculture, agricultural marketing service, north carolina: population profile, transportation and marketing getting to scale with regional food hubs regulatory definitions of large cafos, medium cafos and small cafos. access may still a time to act: a review of institutional marketing of regionally-grown food planning for a networked produce storage and aggregation system for the piedmont region plant diversity in tropical forests: a review of mechanisms of species coexistence key: cord-010681-tmpxs9og authors: dondapati, srujan kumar; stech, marlitt; zemella, anne; kubick, stefan title: cell-free protein synthesis: a promising option for future drug development date: 2020-03-20 journal: biodrugs doi: 10.1007/s40259-020-00417-y sha: doc_id: 10681 cord_uid: tmpxs9og proteins are the main source of drug targets and some of them possess therapeutic potential themselves. among them, membrane proteins constitute approximately 50% of the major drug targets. in the drug discovery pipeline, rapid methods for producing different classes of proteins in a simple manner with high quality are important for structural and functional analysis. cell-free systems are emerging as an attractive alternative for the production of proteins due to their flexible nature without any cell membrane constraints. in a bioproduction context, open systems based on cell lysates derived from different sources, and with batch-to-batch consistency, have acted as a catalyst for cell-free synthesis of target proteins. most importantly, proteins can be processed for downstream applications like purification and functional analysis without the necessity of transfection, selection, and expansion of clones. in the last 5 years, there has been an increased availability of new cell-free lysates derived from multiple organisms, and their use for the synthesis of a diverse range of proteins. despite this progress, major challenges still exist in terms of scalability, cost effectiveness, protein folding, and functionality. in this review, we present an overview of different cell-free systems derived from diverse sources and their application in the production of a wide spectrum of proteins. further, this article discusses some recent progress in cell-free systems derived from chinese hamster ovary and sf21 lysates containing endogenous translocationally active microsomes for the synthesis of membrane proteins. we particularly highlight the usage of internal ribosomal entry site sequences for more efficient protein production, and also the significance of site-specific incorporation of non-canonical amino acids for labeling applications and creation of antibody drug conjugates using cell-free systems. we also discuss strategies to overcome the major challenges involved in commercializing cell-free platforms from a laboratory level for future drug development. proteins whose functionality is not well characterized form a large percentage of entries in many of the currently available biological databases, including the protein data bank (pdb), and there is a constantly growing demand for reliable and fast synthesis and characterization methods. when it comes to drug discovery, proteins are key components as they can have therapeutic potential themselves (e.g., antibodies, coagulation factors, hormones, growth factors, enzymes, and antimicrobial peptides), but also because they could serve as drug targets for diverse diseases (as ion channels, receptors, enzymes, and transporters, for example) [1] [2] [3] [4] [5] [6] [7] . a large proportion of approved pharmaceutical drugs target human proteins. beyond that, protein-based therapeutics, such as antibody-drug conjugates, represent a significant percentage of total drug molecules currently approved. they are poised to grow further with increased gene expression technology, improved protein engineering, and refined bioinformatics tools. some proteins are very difficult to express in traditional cell-based systems and this can hamper our ability to define the mechanism of action and structure-function relationship of the individual protein, knowledge which aids the development of drugs targeting these proteins [1] [2] [3] [4] . generally, to exploit and fine-tune the structural and functional characteristics of a protein, it needs to be expressed and purified with high quality by using recombinant expression technology. traditionally, escherichia coli-based systems were widely used for the production of recombinant proteins due to simplicity in preparation and operation, and cost effectiveness. as a result, broad research and standardization from several years was performed using e. coli-based expression systems, resulting in their often-cited utilization as a state-of-the-art protein expression system [8] . for complex therapeutic proteins, membrane proteins (mps) originating from humans, and virus-like proteins (vlps), mammalian expression systems fulfill all the requirements like post-translational modifications (ptms), cofactors, and chaperones for correct folding and efficient production. however, batch-tobatch variation in cell culture may be a source of process variation. additionally, overexpression of mps might be toxic for the cultivated cells, resulting in cell death or truncated and misfolded proteins [9] . ideally, synthesized proteins are functionally folded and exhibit appropriate ptms. due to the lack of extensive research and low yields in recombinant protein expression, many mps are not yet crystallized, thus limiting the computer-aided drug discovery efforts. due to the growing demand for the production of protein biologics and drug discovery targeting proteins, alternative strategies for protein synthesis should be developed. new expression technologies where proteins can be expressed in a simple way and which allow high throughput screening of different reaction conditions, different genes, and different supplements in a cost-effective manner are extremely important for future drug development. in this review, we give an overview of recent advances in cell-free (cf) synthesis platforms and their diverse applications. additionally, we focus on human and therapeutic proteins produced by different types of cf systems and how these cf protein synthesis (cfps) methods can further play a prominent role in future drug development. cfps systems use crude cell extracts prepared from cells of choice by lysis followed by many steps of washing to remove the cell debris and genomic dna [10, 11] . these cell extracts can be stored at − 80 °c for years and can be used by thawing just before the reaction. such extracts contain all the principal components necessary for transcription and translation, such as aminoacyl-trna synthetase (aas), ribosomes, and factors necessary for elongation, initiation, and transcription. protein synthesis can be realized by combining cell extracts with necessary substrates like amino acids, energy substrates, dna, cofactors, salts, and nucleotides. depending on the biochemical properties of the protein and its end application, the appropriate cf system can be selected. cfps is a fast protein production system since it does not require transfection or cell culture and lacks cell viability constraints. due to its openness, cfps platforms offer additional advantages when compared with cell-based expression methods. a comparative analysis of cf and cellbased approaches is shown in table 1 . for complex proteins, eukaryotic cf systems are ideal as they contain the endogenous microsomes derived from the endoplasmic reticulum (er), enabling co-translational translocation of proteins and er-based ptms [10, 11, 18, 19] . there has been a constant improvement in the quality of lysate preparation, system optimization, linear templatebased protein synthesis, and reduction of process costs, which has led to the preparation of cost-effective systems suitable for commercial purposes. a general scheme of cf protein production is depicted in fig. 1 . cfps platforms are based on either prokaryotic or eukaryotic origin. among the prokaryotic cf systems, extracts based on e. coli are regularly used and are available commercially for cfps of a diverse range of proteins. very recently, cf systems based on bacillus subtilis [32] , pseudomonas putida [33] , streptomyces [34] , and vibrio [12] have been optimized well at the laboratory level due to the ease of preparation of cf lysates. a wide range of detailed protocols is currently available for the preparation of e. coli-based lysates. among the eukaryotic cf systems, extracts based on rabbit reticulocyte lysate (rrl), wheat germ, insect spodoptera frugiperda 21 (sf21), chinese hamster ovary (cho), and cultured human cells are regularly used. an increasing number of eukaryotic cf systems have so far reached technical maturity and become commercially available. requires dna template to be cloned in a plasmid [15] toxic proteins ideal choice for the synthesis of most of the toxic proteins due to high toxic tolerance of cf systems toxic proteins may be difficult to synthesize [16] membrane proteins suitable for a wide range of mps of different sizes mp overexpression can lead to cell toxicity and death [17] [18] [19] membrane protein solubilization to solubilize mps, supplements can be added directly to the reaction mixture in the form of nanodiscs, detergents or liposomes (prokaryotic systems and wheat germ systems) or by using endogenous microsomes or proteoliposomes (eukaryotic systems) not possible to add supplements externally during translation mps have to be purified and reconstituted into liposomes or detergents for functional analysis [9, 20] reliability most of the reports related to cfps are currently limited to the research and laboratory level, but progress towards the drug discovery pipeline has been made recently most reliable and state of the art for protein production and drug discovery purposes, and approved by drug authorities [21] functional characterization compared with cell-based systems, standardized biophysical, biochemical assays are limited, but progress has been made recently a wide range of standardized biophysical and biochemical techniques are available for proteins synthesized by cell-based systems [22] protein yields and downstream applications yields range from µg/ml (complex proteins) to several mg/ml (cytosolic proteins and few mps) with more complex proteins downstream applications are simple and protein can be purified and reconstituted immediately after synthesis yields can be very high, in the range of mg/ml downstream applications are possible but need to additionally lyse the cells for mps [11, 21, 22] post-translational modifications (ptms) ptms possible (mostly in eukaryotic cf systems with translationally active microsomes) limited ptms in prokaryotic and eukaryotic lysates lacking endogenous microsomes. o-glycosylation not possible all ptms are possible including o-glycosylation [13] incorporation of non-standard amino acids ideal choice for the incorporation of single and multiple noncanonical amino acids difficult to incorporate non-canonical amino acids due to cell membrane barrier and cytotoxic effects [23, 24] scale of reaction volume ranging from few µl (chip-based and batch-based in an eppendorf tube) to 100 l reaction (in a fermenter) typical reactions require a minimum of 5 ml. there are exceptional cases where it is performed in 60 µl spots [25] [26] [27] [28] flexibility completely open system and easy to manipulate the reaction conditions with lack of cell membrane constraints completely closed system and difficult to manipulate [22] automation cf systems can be automated with high throughput screening of multiple templates, starting in an elisa plate format generally difficult to automate due to the requirement of larger volumes and aseptic techniques [29] point of care production of biologics lyophilized cf lysates are suitable for the production of therapeutic proteins next to the emergency settings very difficult due to its time-consuming process and requirements of large infrastructure including manufacturing facilities, transport, and cold storage facilities [30, 31] recently, several eukaryotic cf extracts based on tobacco [35] , leishmania [36] , neurospora [37] , yeast cells [38] , and human blood cells [39] were characterized and optimized for a limited number of proteins at the laboratory level. there is a growing trend in the development of novel cf platforms for taking advantage of the genetic tools available in the literature and the abundant literature available on the in vivo expression of proteins. prokaryotic cf systems based on e. coli are most commonly used for protein production towards drug development due to their simplicity and a vast literature available on the utilization of these cells. protein synthesis starts with crude cell extracts prepared from e. coli cells that contain the translation machinery along with all the essential components required for translation. a modified and reconstituted cf synthesis system known as the pure system (protein synthesis using recombinant elements), where all the components of the translation machinery are purified and added individually along with the dna template to produce the protein, has been reported [40] . this is a highly controlled system compared with crude extract methods. a major advantage of the pure system is that protein factors participating in the initiation, elongation, and termination of the protein synthesis process are identified and can be adapted individually to the cf system's requirements. although the naturally occurring ptm machinery is not available in the e. coli lysates, recently proteins with n-glycosylation were synthesized by using e. coli extracts enriched with glycosylation components, including oligosaccharyltransferases (osts) and lipid-linked oligosaccharides (llos) [41] . using release factor (rf1) deficient e. coli lysates, proteins were phosphorylated by incorporation of non-canonical amino acids, which will be addressed in a later part of this review [42] . due to a constantly growing demand for more complex proteins of pharmaceutical value, cf systems based on eukaryotic lysates have been developed to produce highquality proteins. cf systems based on wheat germ lysates (wgl) are among the most popular eukaryotic platforms due to their capacity to produce eukaryotic proteins with high yields [43] . cfps based on wgl have been used frequently for the discovery of novel vaccine candidates as well as for producing several proteins of high quality for structural analysis. despite the high yields and quality of the lysate, this system does not offer all the ptms like glycosylation and does not support the solubilization of complex mps [13] . in the case of wheat germ and rrl, there are no translationally active endogenous microsomes present in the system. in the case of rrl, exogenous microsomes are typically supplied from the canine pancreas for protein translation [13, 44] . it is quite laborious and difficult to enrich rrls with heterologous microsomes. cf systems derived from cultured insect (sf21) cells represent the most popular eukaryotic-based approach for synthesizing a wide variety of proteins. sf21 lysates contain translationally active endogenous er membranes, thereby supporting the signal peptide-mediated translocation of proteins across the membrane, and further provides functions such as signal peptide cleavage, post-translational modifications like n-glycosylation, and lipid modification [13, 14, 45] . cho cell-based expression is well established and is approved for the large-scale synthesis of several biologics by the fda because it undergoes human-compatible ptms. nearly 70% of the approved mammalian therapeutic proteins are currently expressed in cho cells. however, these cells have limitations when it comes to difficult-to-express proteins like overexpression of complex mps, toxic proteins, and multi-subunit proteins as discussed above. cf systems based on cho lysates are evolving as an alternative strategy for the expression of difficult-to-express proteins [13, [17] [18] [19] 46] . apart from many general advantages of cf systems, cho-based cf systems retain most of the features of cho cells while being more flexible due to the lack of cell membrane boundaries. cho-based lysates harbor endogenous microsomal vesicles enabling translocation of transmembrane proteins and secretory proteins. furthermore, ptms of de novo synthesized mps, such as glycosylation, are possible using cho lysate. thus, using cho cell lysate for cfps has a potential value and enables new opportunities, in particular, the high-yield production of pharmaceutically relevant mps [13, [17] [18] [19] . there is a significant increase in the number of publications based on cho lysates for cfps. table 2 compares different cf systems and their advantages and limitations with some selected examples. fig. 1 general scheme depicting the overall process of cell-free protein production. aatrna aminoacyl-trna, aas aminoacyl-trna synthetase, atp adenosine triphosphate, ef elongation factor, gsh glutathione, gssg glutathione-disulfide, gtp guanosine-5'-triphosphate, if initiation factor, ires internal ribosome entry site, mp membrane protein, ncaa non-canonical amino acid, pdi protein disulfide isomerase, peg polyethylene glycol, ptm post-translational modification, r ribosomes, t-rna transfer rna, tf transcription factor, utr untranslated region, vlp virus like particle chinese hamster ovary (cho) mimic the cho cell-based production ptms (n-glycosylation, disulfide bridging, and lipidation) suitable for a wide range of eukaryotic and complex proteins presence of translational active endogenous microsomes [45] high yields in cecf mode endotoxin free lysates used for point-of-care testing [30] low yields especially in the batch mode [58] cost ineffective and difficult to establish unlike e. colibased system streptokinase (cecf): 500 µg/ml [59] human tlr9 receptor [18] (3-h batch): 21 µg/ml (48-h cecf): 900 µg/ml hegfr [46] (batch): 40 µg/ml (cecf): 800 µg/ml cfps can be performed in different formats. the batchbased format is the most commonly used method both in the prokaryotic and eukaryotic systems. this method is relatively fast and cheap, and synthesis can be performed within 1.5-3 hours depending on the system. e. coli-based systems can provide protein yields ranging from 100 µg/ ml to 2-3 mg/ml. although the yields from batch-based eukaryotic systems are comparatively low, mps are automatically incorporated into microsomal membranes and the functionality can be addressed immediately after the synthesis [62] . for researchers who would like to further scale up the protein yields via batch-based eukaryotic systems, a repetitive batch-based synthesis format has been proposed where the microsomes incorporating the mp of interest generated in an initial synthesis reaction can be added to a fresh cf synthesis reaction that has been depleted of its microsomes [19, 45] . another popular cf synthesis format that has been used for a rapid increase in the protein yields is the so-called continuous exchange cell-free synthesis platform (cecf). in this format, a semi-permeable dialysis membrane separates the reaction chamber and a feed chamber and thereby a feed chamber provides the fresh reaction components and enriches the reaction chamber. in exchange, the inhibitory components accumulated during the reaction are removed [14, 17, 18, 46] . typically, the cecf format prolongs the reaction time and increases the protein yields. until now, the cecf format has been used to increase the protein yield by multiple fold, and is widely used as cf platforms (table 2 ). this section highlights some of the key parameters that might influence the protein production using cf lysates. designing synthetic dna and sequence manipulation for cf synthesis by adding regulatory elements plays a significant role in high-yield protein production. in eukaryotic cf systems, initiation factors (ifs) in particular limit the initiation of protein synthesis, thereby leading to low protein yields. one alternative is to use internal ribosome entry site (ires) elements found in the 5′-untranslated region (5′utr) of the different viral genomes upstream of the start codon for cap-independent translation initiation [14, 18, 62] . ires elements from three different viral sources were compared for their translational efficiency in sf21, cho, and human leukemia k562 cf lysates. the ires from the cricket paralysis virus (crpv) typically increased protein yields by a factor of 3-5 [62] . inserting the crpv-ires into the corresponding vector upstream of the epidermal growth factor receptor (egfr) gene, and using the cecf reaction format, egfr yields were significantly increased to more than 100-fold compared with batch reaction format without crpv-ires [14] . additionally, replacement of the initiator codon (atg) to a gcu-codon in combination with the crpv-ires resulted in a further improvement of protein expression levels in cho and k562 cf systems [62] . the vector backbone also plays an important role in cfps. a detailed study comparing commercially available vectors harboring the luciferase gene in combination with crpv-ires showed that there is a significant 5-fold increase in protein yield with a change in the vector backbone [19] . species-independent translational sequences (sits) are another group of synthetic 5′utrs capable of initiating cap-independent translation in multiple prokaryotic and eukaryotic cf systems [66] . typically, polymerase chain reaction (pcr) products are generated with sits downstream of the t7 promoter and upstream of the start codon atg [66] . the 3′ hairpin region of the sits increases the residence time of the preinitiation complex in the vicinity of the start codon [66] . using l. tarentolae cfps in the presence of genes encoding 58 rab encoding variable fragments in combination with a universal sits, nearly a full complement of human rab gtpases were produced with a yield of around 30 µg/ml [36] . similarly, egfp with a yield of around 300 µg/ml [66] , and an active multisubunit enzyme heterodimeric farnesyl transferase (ftase) [36] were synthesized using the l. tarentolae cfps [36] . codon optimization is another important parameter that plays a crucial role in increasing the expression yields of proteins. codon optimization has been shown to influence the translation efficiency of several proteins [71] . by taking advantage of the cf lysates derived from n. crassa and s. cerevisiae, transcription and translation reactions were uncoupled for ribosome profiling, which provided strong biochemical evidence that codon optimization enhances the rate of translational elongation, thereby affecting the ribosome traffic on the mrna [72] . on the one hand, codon optimization usually improves protein yields, but on the other hand, it was shown that faster translation rates might negatively affect the protein folding and function of the individual protein [72, 73] . this problem often cannot be solved even by altering the trna population in the case of cfps. the addition of anti-spliced leader oligonucleotide to l. tarentolae cell extracts suppressed the translation of endogenous l. tarentolae mrnas, thus increasing the translation efficiency of exogenously supplied mrna [65] . using the er-specific signal sequence of honeybee melittin (melittin signal peptide) instead of the native signal peptide increased the translocation of synthesized proteins such as wnt proteins, single-chain antibody variable fragments, and the htlr9-ectodomain into microsomes in the case of sf21 and cho-based cf systems [18, 59, 74, 75] . iterative optimization processes are required to develop highyield cfps. factors that influence both protein quality and quantity include reaction temperature, reaction time, plasmid concentration, salt concentration, t7 polymerase, and other supplements. the influence of these factors on the synthesis rates is also protein specific. very recently, cfps of human toll-like receptor protein (htlr9) in cho-based lysates has been reported by using a cecf method with high yields of around 0.9 mg/ml. by increasing the temperature from 27 to 30 °c, the protein yields were increased by almost 50%. stable monitoring and maintenance of ph throughout the entire cf reaction along with sufficient adenosine triphosphate (atp) supply are essential for efficient and maximum yield protein production. by using amino acid decarboxylase, the ph is controlled throughout the cf reaction [76] . supplementation of chaperones influences the functional folding of many proteins. supplementation of chaperones such as groes/el and dnak/dnaj/grpe in prokaryotic cf systems was used to increase the yield and solubility of colicin m from 16 to 100%, resulting in enhanced cellkilling activity [77] . li et al. demonstrated that by using cfps based on wheat germ extracts, expression of j-domain containing chaperone proteins (dnajb12 and dnajb14) along with potassium channels plays a critical role in the folding, stabilization, and tetramerization of k+ channels [78] . ion concentrations (potassium and magnesium) in the cf reaction have a significant effect on protein production. in the case of cho-based cecf reactions, an increase in the magnesium ion (mg 2+ ) concentration from 3.9 to 22.5 mm led to a 3.9-fold increase in egfr yield [46] . for efficient regeneration of atp, several methods have been developed in cf systems. in prokaryotic systems, compounds like phosphoenolpyruvate (pep), glucose + glutamate decarboxylase, glucose-6-phosphate, fructose-1,6-biphosphate, acetyl phosphate, maltodextrin, and creatine phosphate are widely used as energy sources [79] . in eukaryotic cf systems, a combination of creatine phosphate and creatine kinase is typically used for energy regeneration. apart from these, phosphoglycerate (b. subtilis), and polyphosphate are used in cf systems [80, 81] . cf systems have evolved over the last decade from their use as a prototype method in research laboratories to commercial and large-scale applications. in this section, the utility of cf systems in mp synthesis, antibody production, vaccine development, protein labeling, and antimicrobial peptide synthesis are addressed. mps are structurally and functionally diverse, and constitute 30% of the proteins encoded in the human genome. drugs targeting mps such as ion channels, transporters, and g-protein coupled receptors (gpcrs) represent 12 out of the top 20 global revenues in the pharmaceutical industry [3] . due to the presence of transmembrane domains, ranging from 1 to 24, these proteins are highly hydrophobic and are very challenging to express by traditional cell-based systems. expression of human proteins in heterologous cellular hosts is very much limited due to the difference in their lipid composition, which can prevent the mps from attaining maximum functionality [9] . synthesis of mps by cell-based methods often leads to cytotoxicity, aggregation, and improper folding [9] . to analyze mp functionality, the protein needs to be folded properly and in the appropriate hydrophobic environment. cf systems derived from prokaryotic, as well as eukaryotic lysates lacking endogenous microsomes, require specific supplements for the solubilization of mps in the form of detergents, nanodiscs, or liposomes. non-ionic and zwitterionic detergents are commonly used as supplements in the majority of cfps reactions for the solubilization of mps during their production. detergent-solubilized mps can be either used directly for functional analysis or may be reconstituted into liposomes by mixing with artificial lipids followed by detergent removal [9, 82, 83] . alternatively, nanodiscs (nds) and liposome-based reconstitution are detergent-free strategies where nds and liposomes, prepared and characterized externally, could be supplemented directly into the reaction mixture for the reconstitution [9, 84] . a detailed review of the cf synthesis of mps and the usage of solubilization supplements for isolation and functional analysis is presented in the literature [9] . some of the advantages of nds are easy purification and flexibility in using different lipids and membrane scaffold proteins for creating different sizes, and their availability as monodisperse and homogenous nds. nonetheless, nds have their limitations, particularly when working with a protein whose functionality depends on its orientation and also working with transporter proteins. liposome-based reconstitution covers the limitations of the nds, but the separation of liposomes after the cf reaction is quite challenging and often suffers from disruption due to osmotic instability. further, such passive reconstitution strategies do not offer the advantages of post-translational modifications within native membranes and are limited for mps whose function does not depend on active translocon-based translation. cf systems derived from eukaryotic lysates equipped with endogenous microsomes (e.g., sf21, cho, cultured human cells, and tobacco-by2) satisfy all the necessary requirements for proper folding of mps. the microsomes offer a native environment and intact translocon machinery for a proper embedment and folding of mps [13, 18, 19, 45, 46, 51, 59] . there are continuing efforts in analyzing the functionality of microsomal reconstituted mps, indicated by a good number of publications reporting on this reconstitution strategy, which should help the pharmaceutical industry to develop more dynamic drug screening assays involving mps [9, 46, 83] . here we present recent works on ion channels, transporters, and gpcrs, which constitute more than 40% of the major drug targets in the pharma industry [85] . ion channels constitute approximately 19% of all currently existing human drug targets and play a crucial role in diverse physiological processes involving cell excitability, neuronal transmission, metabolism, sensory transduction, cognition, and electrolyte homeostasis. transporters mediate the translocation of a variety of substrates across biological membranes [86] . the solute carrier (slc) family is the largest class of transporters and is implicated in metabolic conditions and diseases, and in the transport of drugs. these proteins typically have 9-12 transmembrane domains and are difficult to express by traditional methods [2, 9, 50, 87] . slc transporters are an emerging drug target class and the molecular target of several approved inhibitor drugs [2] . despite this, these classes of proteins remain largely unexplored in recent years due to the high costs involved and lack of proper expression methods [9, 53] . table 3 highlights some of the selected publications using cf methods for synthesis, reconstitution, and functional analysis of ion channels and transporters. the most widely used method of reconstitution for functional analysis is detergent-based reconstitution into liposomes or passive integration of mps into liposomes and nds. the majority of the functional assays were performed with plbe in the case of ion channels and substrate uptake assays in the case of transporters. gpcrs transduce extracellular stimuli to the inside of cells, after activation by a variety of different molecules such as neurotransmitters, hormones, odorants, and peptides, thereby triggering several signal transduction cascades. the involvement of gpcrs in almost all processes in living cells has resulted in significant pharmaceutical interest in this protein class, and the development of robust and high-throughput-suitable assays for the discovery of novel ligands and drugs targeting these proteins. in principle, the screening of ligands can be performed in whole-cell assays by measuring a downstream signaling event, or in cf assays, which are decoupled from the living organism. usually, these decoupled methods are preferable for high-throughput screenings, as they are easy to handle and therefore amenable for automation and downsizing. these parameters can be well combined with cfps. an automated cf synthesis procedure for the production of different mps is already reported [97] . this procedure might be further expanded for the parallel analysis and identification of molecules that target different gpcrs. to date, only a few studies have analyzed in detail the activity of receptors produced by cf systems. the main reason for this is that there are limited well established activity assays. this section addresses possible activity assays that might be transferred to cf systems in the future. radioligand binding assays, the gold standard for identifying binding molecules, are already adapted for gpcrs that have been synthesized in eukaryotic and prokaryotic cf systems, and demonstrate similar binding affinities in comparison with in vivo produced gpcrs [20, [98] [99] [100] . alternatively, fluorescently labeled ligands can be analyzed by an optical read-out system using eukaryotic cf systems harboring endogenous membrane structures [101] . nevertheless, for these systems, radiolabeled or fluorescently labeled ligands are required, thereby limiting the analysis mainly to gpcrs with already known ligands. in addition, simple ligand binding assays usually do not differentiate between an agonistic and an antagonistic effect of the bound substance. in this context, measuring downstream signaling to distinguish between an activating and inhibitory ligand is preferable. one possible method of choice is the receptor-mediated coupling of g proteins [102] . this early event immediately follows after gpcr activation and is detected by the binding of [ 35 s]gtpγs to gα subunits. this method is not yet established in cf systems but might be transferable assuming the presence of gα proteins in the eukaryotic lysate. alternatively, the gα proteins can be additionally co-synthesized to the target gpcr or directly applied to the reaction based on the open nature of cf systems. after gpcr activation, gtp binding and hydrolysis should be detectable. in addition to ligand binding and g protein coupling, intra-and intermolecular interactions can be visualized by förster and bioluminescence resonance energy transfer techniques. different sensor models are known in living cells [103] . the monitoring of intermolecular interactions can be performed as well in cf systems using the already established in vivo models. one model includes the tagging of the c-terminus of a gpcr of interest to a fluorophore (gfp/yfp) and fusing a binding partner such as β-arrestin to luciferase or a second fluorophore [104] . upon activation of the gpcr, β-arrestin binds to the receptor and both tags are in close proximity, resulting in a measurable energy transfer. this model requires active g protein-coupled receptor kinases for the phosphorylation of the c terminus of the synthesized gpcr to get recognized by β-arrestins. this requirement has to be analyzed in detail in the individual cf systems. the second known in vivo model visualized intramolecular changes after agonist and antagonist binding by introducing fluorophores into the third extracellular loop and the c terminus of different gpcrs. upon activation, the distance between both fluorophores changes and an alteration in the energy transfer can be measured [105, 106] . initial experiments to transfer these energy transfer-based sensors were recently performed in cf systems [107] . both models can be applied to high-throughput analyses. in summary, the successful cf synthesis of a variety of gpcrs has been demonstrated in recent years and a transfer of these gpcr production systems to a drug discovery format in a high-throughput manner has recently started. in the near future, we might see novel technologies for ligand screenings, thereby utilizing the advantage of the automatization and downsizing capacity of cf systems. the gold standard for synthesis, development, and production of antibody-based drugs (based on full-length antibodies) is mammalian cell culture-based expression systems. although cultivation of mammalian cells is well established and widely used, the development of monoclonal antibodies (mabs) and antibody-drug conjugates (adcs) remains time-consuming and challenging. thus, methods for highthroughput screenings, especially in the early-stage evaluation of antibody candidates, are valuable. in view of this, the use of the cf technology constitutes a promising strategy to shorten the time from antibody discovery to production. using cf technology, antibodies can be produced in a flexible scale within a couple of hours. besides the synthesis of individual antibodies, cf technology can be used to display libraries of antibodies. in contrast to phage and yeast display, in vitro cf systems such as ribosome and mrna display are open, and thus result in higher library sizes. in theory, the size of the library is only limited by the quantity of supplemented mrna/dna, the volume of the cf reaction, and the number of ribosomes within the system, resulting in library sizes of ~ 10 12−15 /ml cf reaction [108] . in comparison, phage and yeast display exhibit library diversities of ~ 10 6 -10 10 . selection technologies such as ribosome display [109] , mrna display [110] , and cis display [111] have been developed based on reticulocyte lysate [110] and e. coli cf systems [109] . these systems focused on smaller antibody fragments because their functionality does not rely on the assembly of multiple polypeptide chains. nonetheless, recently two groups have succeeded in developing completely cf display technologies that allow the selection of fab fragments [112] . the challenge to assemble the heavy and light polypeptide chain (hc/lc) of the fab fragment was approached in different ways. while sumida et al. succeeded by combining mrna display based on two mrna sub-libraries, one encoding hc, the other one encoding lc, with in vitro compartmentalization pcr to link and then amplify hc and lc gene pairs [112] , stafford et al. developed a ribosome display method where they displayed only one of the two fab chains, while the other one was not presented in display format [113] . successful synthesis of different antibody formats, including single-chain variable fragments (scfvs), fab fragments, as well as complete iggs, has already been shown in e. coli [114, 115] , sf21 [10, 116] , reticulocyte [110] , wheat germ [117] , and cho cf systems [46, 75, 118] . furthermore, the upscaling of cf reactions to the liter-scale [25, 115] as well as downscaling [119] and high-throughput applications [120] have been demonstrated. in addition, advances in bioorthogonal reaction chemistries have paved the way to expand the possibilities for adc development. the site-specific introduction of non-canonical amino acids into a genetically engineered sequence can be used to create site-specifically labeled adcs [121] . currently, seven adcs are approved for therapy. to date, all of these adcs have been generated by coupling of mabs to the cytotoxic linker-payload via surface-exposed lysines, or partial disulfide reduction and conjugation to free cysteines, which typically results in a controlled but heterogeneous adc population with varying numbers and positions of drug molecules attached to the mab [122] . homogeneous adc populations can be achieved by introducing the payload at one or more defined positions. by developing a bioorthogonal trna/synthetase pair, zimmerman et al. have shown that the optimized non-canonical amino acid para-azidomethyll-phenylalanine (pamf) can be site-specifically incorporated into the tumor-specific, her2-binding igg trastuzumab [123] . subsequently, the cytotoxic linker payload dbco-peg-monomethyl auristatin (dbco-peg-mmaf) was conjugated to pamf via strain-promoted azide-alkyne cycloaddition (spaac) copper-free click chemistry. in the context of dual-functioning molecules, bispecific antibodies have also emerged as promising anti-cancer agents. one of the advantages of these proteins is their capability to target two different epitopes simultaneously, thereby increasing target engagement, where mono-specific antibodies might fail [124] . due to their open design, cfps reactions can easily be manipulated, for example by varying the template ratios and concentrations of hc and lc. for example, xu et al. showed the successful assembly of bispecific 'knobs-into-holes' antibodies in multiple scaffolds by using an e. coli-based cf expression platform [125] . taken together, antibody evolution, selection, and engineering can dramatically benefit from the technological advances in the field of cfps. (1) novel display technologies based on cf methods enable the in vitro evolution of multimeric proteins and allow for more sophisticated protein engineering. (2) due to the very short time frame from synthesis to functional testing, cf systems can accelerate antibody construct evaluation by a repetitive (one after one) and/or parallel screening. (3) the introduction of non-canonical amino acids expands the chemical repertoire and thus the possibilities to modify and improve antibody-based therapeutics. advanced labeling technologies allow for a very fast qualitative analysis of drug-to-antibody ratio (dar), linker, linker/position, drug, drug/position (research application), and allow full control of the adc design (commercial application). cf systems are becoming a potential option for synthesizing vaccine antigens. most of the vaccine antigens produced by cf systems to date have used e. coli and wgl. recent progress on eukaryotic cf systems may offer additional advantages. in this context, eukaryotic cf systems are endotoxin free and lack the complex plasma membrane that makes the protein purification simple. some of the antigens synthesized by using cf systems are highlighted in table 4 . they are able to induce a strong immune response in experimental animals and could serve as a proof of concept for future vaccine development. using recent advances in cfps technology, a freeze-dried, cell-free (fd-cf) expression system was created based on e. coli cf lysates [31] . using this fd-cf technique, diphtheria toxoid antigen variants (dt5 and dt6) were produced following rehydration with water and functional characterization of the synthesized proteins was verified following administration in mice and measuring the immune response [31] . the fd-cf method could enable the production of on-demand, point-of-care biologics requiring just the simple addition of water for activation and synthesis. recently, cf-based expression has proven successful in producing difficult-to-express proteins like major outer membrane protein (mmomp) of chlamydia spp., a major vaccine antigen. using e. coli-based cfps, mmomp was synthesized in a native trimeric form in the presence of nanolipoproteins (nlps) with a yield of around 1.5 mg/ml. when injected into mice in the presence of an adjuvant, the protein elicited an enhanced humoral immune response [126] . this method of synthesizing and simultaneous incorporation of antigens into nlps using a cf approach is a promising method for future vaccine development. conjugate vaccines are one of the safest and most effective biologics [127] . bioconjugate vaccines are produced using protein glycan coupling technology (pgct). however, pgct has its own limitations such as time-consuming in vivo processes. additionally, fda-approved carrier proteins, such as toxins derived from clostridium tetani and corynebacterium diphtheria, have not yet been demonstrated to be compatible with an e. coli-based production process. relevant ptms are often difficult to synthesize in e. coli cf systems. meanwhile, there have been further advances in using bacterial glycoengineering combined with cfps for producing bioconjugate vaccines. this cf glycoprotein synthesis (cfgps) used glycooptimized e. coli extracts integrating both n-linked glycosylation and protein synthesis. using cfgps, two bioconjugate vaccines were synthesized against f. tularensis and e. coli o78 [146, 147] . besides posttranslational modification, the assembly of macromolecular structures in cf systems is highly ambitious. virus-like particles (vlps), for example, are nanoscale structures that are formed from the self-assembly of viral proteins without the viral genome responsible for the infection. usually, vlps mimic the capsid structure of the real virus. vlp antigens are vaccine candidates for several diseases [148] . one of the vaccine candidates, which is currently in clinical trials, contains vlp antigens addressing noroviruses responsible for gastroenteritis in humans [149] . cf synthesized vlps were structurally confirmed by electron microscopy [150] . using e. coli cf systems, antimicrobial colicins (colicin m, la, e1, and e2) have been synthesized with high yields (around 300 µg/ml) and solubility. the synthesized colicins are able to effectively kill the target cells without any purification [151] . antimicrobial peptides (amps) are another class of defense molecules that have a wide spectrum of targets; for example, bacteria, viruses, fungi, parasites, and cancer. amps are evolving as alternatives to antibiotics [152] . using lyophilized e. coli cf lysates, ten different amps have been synthesized successfully and the functionality of plasmodium falciparum wheat germ interaction of pfmsa180 with cd47 was confirmed by erythrocyte binding assay [133] . antigen-specific igg responses to lsa3-c were profiled by an alphascreen assay [134] . western blotting and elisa confirmed the interaction of purified recombinant msp11 with human sera [135] . immunization of purified pfron12 with freund's complete adjuvant into japanese white rabbits generated pfron12 antisera [136] . rabbits immunized with expressed pfripr produced specific antibodies [137] . anti-exp1 antibodies were generated by immunization of recombinant exp1 [138] . wheat germ proteoliposomal engineered cldn-5 antigens induced anti-cldn5-5-ecr antibodies in mice [145] αcd19-id small diabody (db) molecule containing both a b-cell-targeting moiety (anti-cd19) and a lymphoma id, 38c13-scfv idiotype-specific single-chain variable fragment of the immunoglobulin from the 38c13 mouse b-cell lymphoma, cldn-5 claudin-5, when non-canonical amino acids (ncaas) are incorporated into proteins, novel functional, structural, and imaging properties can be generated. this synthetic biology application is fast emerging and has wide applications such as incorporating precise ptms and adding novel functions to proteins [23, 24, 42, 45] . by taking advantage of the openness of the cfps, one can add the machinery responsible for the co-translational incorporation of ncaas directly to the standard reaction components. one possible method to incorporate ncaa is to use precharged trnas harboring the ncaa. one of the most commonly used methods is the amber suppression technology using an orthogonal pair of aminoacyl-trna synthetase/trna (o-trna/aars pairs from distinct organisms), which functions independent of endogenous aarss and trnas in the host and is used to direct the incorporation of ncaas to specific positions such as the amber stop codon (uag). after incorporation of an ncaa with a reactive group, bioorthogonal click reactions can be performed to conjugate a molecule of interest. the most general biorthogonal click reactions for conjugating molecular probes or polymers are the copper-catalyzed azide-alkyne cycloaddition (cuaac), staudinger-ligation, photo click cycloaddition, strain-promoted azide-alkyne cycloaddition (spaac) and inverse electron-demand diels-alder cycloadditions (iedda + spiedac). using e. colibased cf systems, cui et al. showed the incorporation of two fluorescent labels, bodipy fluorophore and tamra-dibo, by using a precharged trna + orthogonal system for fret measurements [153] . using sf21-based cf systems, quast et al. demonstrated the incorporation of p-azido-l-phenylalanine at defined amber positions in parallel in the two subunits of the human egfr protein dimer. later, the azido group of the incorporated azfs was coupled by photoaffinity cross-linking using a bis-combo linker to create a stable synthetic dimer of egfr [14] . the dimerized protein shows autophosphorylation in the presence of tyrosine kinase. in general, release factor 1 (rf1) competes with orthogonal ncaa-trna for the amber codon, which results in truncated products along with successfully suppressed products. so, cf lysates derived from genetically modified e. coli lacking release factor 1 (rf1) can be used to enhance the incorporation efficiency of ncaas. using the orthogonal system and e. coli-based cfps, human mek1 kinase with ptms was synthesized up to milligram quantities by site-specific, co-translational incorporation of phosphoserine at specific positions [154] . various polyethylene glycol (peg) moieties have been widely used to decorate therapeutic proteins. the peg moiety usually offers high stability and extends the half-life of proteins while in circulation inside the body. the food and drug administration (fda) has recognized peg moieties as safe due to their structural flexibility, hydrophilicity, and minimal toxicity, and several pegylated drugs have been approved by the fda. using sf21-based cf systems, a site-specific pegylated human epo was produced and characterized by autoradiography [45] . apart from the amber suppression strategy, there are other strategies like frameshift suppression, sense codon reassignment, and unnatural base pairing. a detailed review of prominent methods for the incorporation of ncaas into proteins using cfps has been recently published [23] . a wide range of commercial cf systems is available in the market based on lysates derived from diverse sources. as well, a few companies provide services for cf synthesis of proteins. some of the products derived from the cf systems based on e. coli lysates are already in clinical trials, such as adcs targeting cd74 and folate receptor alpha highly expressed in myeloma and cancer cells (sutro biopharma, inc, usa). table 5 lists commercial systems currently available for the cf production of proteins. evolving cf systems from a laboratory level to a robust production platform is necessary to fulfill their potential. prior to full realization of cf systems as emerging tools for drug discovery and evaluation, several factors need to be addressed, like synthesis of the high-quality functional protein with proper folding and ptm, cost of production, scalability, and safety issues. a more detailed understanding of the components in the cf lysates will substantially improve the quality and stability of the extract preparation. the quantity of the protein depends on the translation efficiency of the cf system. the most important factors that influence the protein yields are quality of the celllysate, reaction conditions, and template optimization as addressed in section 3.2. to increase the translation efficiency, further efforts are required to increase the quality of lysate production. this can be achieved by using genetic engineering tools to remove the factors responsible for nucleic acid degradation, ribosome inactivation, and protein degradation. brodiazhenko et al. showed that genomic disruption of genes encoding ribosome-inactivating factors (hpf in b. subtilis and stm1 in s. cerevisiae) has improved the activities of bacterial and yeast translation systems [54] . in this context, advanced engineering tools like crispr cas could help to improve the translation efficiency of the cf systems [155] . activation and enrichment of translation-relevant factors could also increase translation efficiency [63] . when it comes to eukaryotic cfps platforms, translocation through microsomes currently remains a black box. optimizing the efficiency of coupling translation and translocation needs to be addressed. the most important issue with cf systems, especially when working with cecf systems, is to maintain the balance between the amount of protein synthesized and the stability and quality of the protein. although cecf has been capable of producing 0.6-1 mg protein per ml, especially with the mammalian expression systems, only a small fraction of the produced protein was subject to detailed functional analysis [15, 155] . this is one of the reasons why the functional assays are limited to binding assays (gpcr, tlr, antibody), plbe (ion channels), and colorimetric assays (enzymes). by optimizing the redox conditions, the problem of ab translocation into the lumen of microsomes is addressed already [75, 155] . however, when it comes to the synthesis of complex transmembrane proteins in mammalian systems, the insertion efficiency might be already saturated at the low synthesis rates due to restrictions on the level of the translocon's functionality. a more detailed analysis of lipid composition and proteins constituting the microsomes present in the insect, cho, and human-derived lysates will help to improve the quality of synthesized membrane proteins. one could use alternative supplements like nanodiscs or liposomes reconstituted from microsomal membranes to support mp integration [156] . intense efforts on designing novel and improved mammalian cf systems should be maintained as the majority of the drug targets are related to complex eukaryotic proteins. optimizing cf reactions in order to decrease protein aggregation during the purification processes and increasing the quality of the protein purification, especially when using the cecf method, is strongly required. another point to address in the field of cfps is to decrease the costs of production, especially in the preparation of cf lysates and the individual reaction components. substantial costs arise from the usage of phosphorylated energy systems, cofactors, nucleotides, amino acids, and dna. alternative energy regeneration systems are available in the place of phosphorylated substrates (e.g., glucose, maltodextrin, etc.) for sustainable atp regeneration throughout the synthesis reaction [157] [158] [159] . use of nucleoside monophosphates instead of nucleoside triphosphates as the nucleotide source in the cf systems could be another cost-effective parameter [159] . avoiding the use of exogenous trnas and cyclic amps and reducing the concentration of amino acids and nucleotides are some of the cost-effective parameters one could optimize during protein synthesis. additionally, new high-cell-density cultivation strategies and improvement in the quality of cell lines by genetic engineering could help to produce cost-effective high-quality cf systems. costs can also be decreased by engineering and optimization of eukaryotic lysates to extend the lifetime of these systems, thereby increasing the yield of the produced protein. there has been considerable progress in the point-ofcare production devices for on-demand biologic synthesis of small quantities of therapeutic proteins using cho lysates and e. coli lysates through on-site good manufacturing practice (gmp) [30] . this type of miniaturized device could be useful for quick testing of proteins and thus help in treating common and rare diseases, and cfps could help solve the challenges associated with in vivo expression. due to the open nature of the cf systems, proteins can be modified with chemically synthesized glycans by bioconjugate chemistries. this will help to increase the quality and therapeutic efficiency of the synthesized proteins. there is an exponential increase in the number of publications from the last 5 years using cf lysates for producing a wide range of proteins [160] . due to the increased awareness of the biosynthetic potential of the cf systems, protocols becoming simpler, improvement in the lysate quality, and its applicability in the preparation of a diverse range of proteins, there will be unexpected outcomes in the field of protein production towards future drug development. funding this research is supported by the european regional development fund (efre) and the german ministry of education and research (bmbf, no. 031b0078a). open access this article is licensed under a creative commons attribution-noncommercial 4.0 international license, which permits any non-commercial use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the creative commons licence, and indicate if changes were made. the images or other third party material in this article are included in the article's creative commons licence, unless indicated otherwise in a credit line to the material. if material is not included in the article's creative commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder.to view a copy of this licence, visit http://creat iveco mmons .org/licen ses/by-nc/4.0/. ion channels in drug discovery and safety pharmacology slc transporters as therapeutic targets: emerging opportunities unexplored therapeutic opportunities in the human genome properties of protein drug target classes antibodies and venom peptides: new modalities for ion channels prospects for pharmacological targeting of pseudokinases advances in development of antimicrobial peptidomimetics as potential drugs. molecules new tools for recombinant protein production in escherichia coli: a 5-year update membrane protein synthesis in cell-free systems: from bio-mimetic systems to bio-membranes cell-free protein synthesis for producing difficult-to-express proteins a user's guide to cell-free protein synthesis establishing a high-yielding cell-free protein synthesis platform derived from vibrio natriegens cell-free protein synthesis: pros and cons of prokaryotic and eukaryotic systems high-yield cell-free synthesis of human egfr by iresmediated protein translation in a continuous exchange cellfree reaction format cellfree synthesis of functional and endotoxin-free antibody fab fragments by translocation into microsomes cell-free synthesis and characterization of a novel cytotoxic pierisin-like protein from the cabbage butterfly pieris rapae improving the recombinant human erythropoietin glycosylation using microsome supplementation in cho cell-free system cell-free synthesis of human toll-like receptor 9 (tlr9): optimization of synthesis conditions and functional analysis cell-free systems based on cho cell lysates: optimization strategies, synthesis of "difficultto-express" proteins and future perspectives systematic optimization of cell-free synthesized human endothelin b receptor folding cell-free protein synthesis: applications come of age cell-free synthetic biology: engineering in an open world cotranslational incorporation of non-standard amino acids using cell-free protein synthesis cell-free protein synthesis from genomically recoded bacteria enables multisite incorporation of noncanonical amino acids microscale to manufacturing scale-up of cell-free cytokine production-a new approach for shortening protein production development timelines on-chip automation of cell-free protein synthesis: new opportunities due to a novel reaction mode highthroughput transfection of interfering rna into a 3d cell-culture chip microarray platform affords improved product analysis in mammalian cell growth studies automated production of functional membrane proteins using eukaryotic cell-free translation systems point-of-care production of therapeutic proteins of good-manufacturing-practice quality on-demand biomolecular manufacturing development of a bacillus subtilis cell-free transcription-translation system for prototyping regulatory elements development of a pseudomonas putida cell-free protein synthesis platform for rapid screening of gene regulatory elements establishing a high yielding streptomyces-based cell-free protein synthesis system tobacco by-2 cellfree lysate: an alternative and highly-productive plant-based in vitro translation system leishmania cell-free protein expression system the cell free protein synthesis system from the model filamentous fungus neurospora crassa a combined cell-free transcription-translation system from saccharomyces cerevisiae for rapid and robust protein synthe rapid recombinant protein expression in cell-free extracts from human blood the pure system for the cell-free synthesis of membrane proteins single-pot glycoprotein biosynthesis using a cell-free transcription-translation system enriched with glycosylation machinery the application of cell-free protein synthesis in genetic code expansion for post-translational modifications wheat germ systems for cell-free protein expression cell-free expression and functional reconstitution of homo-oligomeric alpha7 nicotinic acetylcholine receptors into planar lipid bilayers cell-free protein synthesis as a novel tool for directed glycoengineering of active erythropoietin high-yield production of "difficult-to-express" proteins in a continuous exchange cell-free system based on performance benchmarking of four cell-free protein expression systems production and stabilization of the trimeric influenza hemagglutinin stem domain for potentially broadly protective influenza vaccines cell-free expression of a functional pore-only sodium channel high-level cell-free production of the malarial lactate transporter pffnt as a basis for crystallization trials and directional transport studies cell-free production of pore forming toxins: functional analysis of thermostable direct hemolysin from vibrio parahaemolyticus functional characterization of cell-free expressed kv1.3 channel using a voltage-sensitive fluorescent dye crystallization of the membrane protein hvdac1 produced in cell-free system elimination of ribosome inactivating factors improves the efficiency of bacillus subtilis and saccharomyces cerevisiae cell-free translation systems cell-free protein synthesis from fast-growing vibrio natriegens membrane assembly of the functional kcsa potassium channel in a vesicle-based eukaryotic cellfree translation system cell-free synthesis of functional human epidermal growth factor receptor: investigation of ligand-independent dimerization in sf21 microsomal membranes using non-canonical amino acids accelerating the production of druggable targets: eukaryotic cell-free systems come into focus cell-free production of a therapeutic protein: expression, purification, and characterization of recombinant streptokinase using a cho lysate functional g-protein-coupled receptor (gpcr) synthesis: the pharmacological analysis of human histamine h1 receptor (hrh1) synthesized by a wheat germ cell-free protein synthesis system combined with asolectin glycerosomes accelerated pharmaceutical protein development with integrated cell free expression, purification, and bioconjugation ires-mediated translation of membrane proteins and glycoproteins in eukaryotic cell-free systems an efficient mammalian cell-free translation system supplemented with translation factors a technique to increase protein yield in a rabbit reticulocyte lysate translation system host-regulated hepatitis b virus capsid assembly in a mammalian cell-free system species-independent translational leaders facilitate cell-free expression cell-free expression, purification and immunoreactivity assessment of recombinant fasciola hepatica saposin-like protein-2 exploiting leishmania tarentolae cell-free extracts for the synthesis of human solute carriers an optimized yeast cell-free system: sufficient for translation of human papillomavirus 58 l1 mrna and assembly of virus-like particles a cell-free expression and purification process for rapid production of protein biologics synthesis of membrane proteins in eukaryotic cell-free systems codon usage influences the local rate of translation elongation to regulate co-translational protein folding nonoptimal codon usage influences protein structure in intrinsically disordered regions cell-free eukaryotic systems for the production, engineering, and modification of scfv antibody fragments cell-free synthesis of functional antibodies using a coupled in vitro transcription-translation system based on method for cell-free protein synthesis involved with ph control with amino acid decarboxylase optimizing cell-free protein synthesis for increased yield and activity of colicins tetrameric assembly of k(+) channels requires er-located chaperone proteins energy systems for atp regeneration in cell-free protein synthesis reactions recent advances in development of cell-free protein synthesis systems for fast and efficient production of recombinant proteins effects of atp regeneration systems on the yields and solubilities of cell-free synthesized proteins combining in vitro folding with cell free protein synthesis for membrane protein expression membrane protein production in escherichia coli cell-free lysates reconstitution and functional characterization of ion channels from nanodiscs in lipid bilayers challenges in the development of functional assays of membrane proteins a comprehensive map of molecular drug targets a cell-free translocation system using extracts of cultured insect cells to yield functional membrane proteins in vitro synthesis of a major facilitator transporter for specific active transport across droplet interface bilayers functional reconstitution of cell-free synthesized purified kv channels the sensorless pore module of voltage-gated k + channel family 7 embodies the target site for the anticonvulsant retigabine cell free expression and functional reconstitution of eukaryotic drug transporters coupled cell-free synthesis and lipid vesicle insertion of a functional oligomeric channel mscl mscl does not need the insertase yidc for insertion in vitro enhanced functional expression of aquaporin z via fusion of in situ cleavable leader peptides in escherichia coli cell-free system green fluorescent protein changes the conductance of connexin 43 (cx43) hemichannels reconstituted in planar lipid bilayers a small viral potassium ion channel with an inherent inward rectification incorporation of adenine nucleotide transporter, ant1p, into proteoliposomes facilitates atp translocation and activation of encapsulated luciferase synthesis and site-directed fluorescence labeling of azido proteins using eukaryotic cell-free orthogonal translation systems modulation of g-protein coupled receptor sample quality by modified cell-free expression protocols: a case study of the human endothelin a receptor production of g protein-coupled receptors in an insect-based cell-free system cellfree synthesis of a functional g protein-coupled receptor complexed with nanometer scale bilayer discs qualifying a eukaryotic cell-free system for fluorescence based gpcr analyses principles: extending the utility of [35s]gtp gamma s binding assays optical probes based on g proteincoupled receptors -added work or added value? the role of beta-arrestins in the termination and transduction of g-protein-coupled receptor signals a flash-based fret approach to determine g protein-coupled receptor activation in living cells intramolecular and intermolecular fret sensors for gpcrs-monitoring conformational changes and beyond a combined cell-free protein synthesis and fluorescence-based approach to investigate gpcr binding properties in vitro selection and evolution of functional proteins by using ribosome display antibody-ribosome-mrna (arm) complexes as efficient selection particles for in vitro display and evolution of antibody combining sites cis display: in vitro selection of peptides from libraries of proteinâ€"dna complexes antibodies to watch in 2019 in vitro selection of fab fragments by mrna display and gene-linking emulsion pcr in vitro fab display: a cell-free system for igg discovery engineering toward a bacterial "endoplasmic reticulum" for the rapid expression of immunoglobulin proteins aglycosylated antibodies and antibody fragments produced in a scalable in vitro transcription-translation system production of functional antibody fragments in a vesicle-based eukaryotic cell-free translation system efficient synthesis of a disulfide-containing protein through a batch cellfree system from wheat germ development of a cho-based cellfree platform for synthesis of active monoclonal antibodies sealable femtoliter chamber arrays for cell-free biology high-throughput screening of biomolecules using cell-free gene expression systems synthesis of site-specific antibody-drug conjugates using unnatural amino acids location matters: site of conjugation modulates stability and pharmacokinetics of antibody drug conjugates production of site-specific antibody-drug conjugates using optimized non-natural amino acids in a cell-free expression system dual targeting strategies with bispecific antibodies production of bispecific antibodies in "knobs-into-holes" using a cell-free expression system cell-free production of a functional oligomeric form of a chlamydia major outer-membrane protein (momp) for vaccine development protein carriers of conjugate vaccines: characteristics, development, and clinical trials efficacy of a potential trivalent vaccine based on hc fragments of botulinum toxins a, b, and e produced in a cellfree expression system hela based cell free expression systems for expression of plasmodium rhoptry proteins ralp1 is a rhoptry neck erythrocytebinding protein of plasmodium falciparum merozoites and a potential blood-stage vaccine candidate antigen a vaccine directed to b cells and produced by cell-free protein synthesis generates potent antilymphoma immunity cellfree production of scfv fusion proteins: an efficient approach for personalized lymphoma vaccines pfmsa180 is a novel plasmodium falciparum vaccine antigen that interacts with human erythrocyte integrin associated protein (cd47) pv1, a novel plasmodium falciparum merozoite dense granule protein, interacts with exported protein in infected erythrocytes anti-msp11 igg inhibits plasmodium falciparum merozoite invasion into erythrocytes in vitro antibodies against a plasmodium falciparum ron12 inhibit merozoite invasion into erythrocytes identification of plasmodium falciparum reticulocyte binding protein homologue 5-interacting protein, pfripr, as a highly conserved blood-stage malaria vaccine candidate. vaccine plasmodium falciparum exported protein 1 is localized to dense granules in merozoites wheat germ cell-free system-based production of malaria proteins for discovery of novel vaccine candidates immunological characterization of plasmodium vivax pv32, a novel predicted gpi-anchored merozoite surface protein assessing sequence plasticity of a virus-like nanoparticle by evolution toward a versatile scaffold for vaccines and drug delivery malaria derived glycosylphosphatidylinositol anchor enhances anti-pfs25 functional antibodies that block malaria transmission immunogenicity of glycosylphosphatidylinositolanchored micronemal antigen in natural plasmodium vivax exposure wheat germ cell-free system-based production of hemagglutinin-neuraminidase glycoprotein of human parainfluenza virus type 3 for generation and characterization of monoclonal antibody engineered membrane protein antigens successfully induce antibodies against extracellular regions of claudin-5 toy building set method for rapid in vitro synthesis of bioconjugate vaccines via recombinant production of n-glycosylated proteins in prokaryotic cell lysates immunodrugs: therapeutic vlpbased vaccines for chronic diseases immunogenicity of takedas bivalent viruslike particle (vlp) norovirus vaccine (nov) candidate in children from 6 months up to 4 years of age cell-free protein synthesis of norovirus virus-like particles rapid production and characterization of antimicrobial colicins using escherichia coli-based cell-free protein synthesis application of antimicrobial peptides of the innate immune system in combination with conventional antibiotics-a novel way to combat antibiotic resistance? front cell infect microbiol combining sense and nonsense codon reassignment for site-selective protein modification with unnatural amino acids robust production of recombinant phosphoproteins using cell-free protein synthesis biosynthesis of membrane dependent proteins in insect cell lysates: identification of limiting parameters for folding and processing cell-free synthesis of membrane proteins: tailored cell models out of microsomes methods for activating natural energy metabolism for improving yeast cellfree protein synthesis: 15/639 cell-free gene expression: an expanded repertoire of applications an economical method for cell-free protein synthesis using glucose and nucleoside monophosphates cell-free biosensors for biomedical applications key: cord-291786-66c8xta0 authors: he, shaobo; peng, yuexi; sun, kehui title: seir modeling of the covid-19 and its dynamics date: 2020-06-18 journal: nonlinear dyn doi: 10.1007/s11071-020-05743-y sha: doc_id: 291786 cord_uid: 66c8xta0 in this paper, a seir epidemic model for the covid-19 is built according to some general control strategies, such as hospital, quarantine and external input. based on the data of hubei province, the particle swarm optimization (pso) algorithm is applied to estimate the parameters of the system. we found that the parameters of the proposed seir model are different for different scenarios. then, the model is employed to show the evolution of the epidemic in hubei province, which shows that it can be used to forecast covid-19 epidemic situation. moreover, by introducing the seasonality and stochastic infection the parameters, nonlinear dynamics including chaos are found in the system. finally, we discussed the control strategies of the covid-19 based on the structure and parameters of the proposed model. at the end of 2019, a novel coronavirus disease (covid-19) was declared as a major health hazard by world health organization (who). at present, this disease is rapidly growing in many countries, and the global number of covid-19 cases is increasing at a rapid rate. this coronavirus is a kind of enveloped, single stranded and positive sense virus which belongs to the rna coronaviridae family [1, 2] . in early december of 2019, this infectious disease has begun to outbreak in wuhan, the capital city of hubei province, china. until now, the epidemic in china is basically under control, but there are still many infections around the world. to defeat the epidemic, scientists in different fields investigated the covid-19 from different points of view. those aspects include pathology, sociology perspective, the infection mechanism and prediction [3] [4] [5] [6] [7] [8] . in the history of mankind, there are many other outbreak and transmission of diseases such as dengue fever, malaria, influenza, pestilence and hiv/aids. how to built a proper epidemiological model for these epidemics is a challenging task. some scientists treat the disease spread as a complex network for forecast and modeling [9, 10] . for the covid-19, bastian prasse et al. [10] designed a network-based model which is built by the cities and traffic flow to describe the epidemic in the hubei province. at present, the sis [11, 12] , sir [13] and seir [14, 15] another way for the simulation of epidemics. lots of research works have been reported. it shows that those sis, sir and seir models can reflect the dynamics of different epidemics well. meanwhile, these models have been used to model the covid-19 [16, 17] . for instance, tang et al. [17] investigated a general seirtype epidemiological model where quarantine, isolation and treatment are considered. moreover, there are also other methods for modeling of the covid-19 [18, 19] . wang et al. [19] applied the phase-adjusted estimation for the number of coronavirus disease 2019 cases in wuhan. thus in this paper, we try to propose a seir model to simulate the process of covid19. chaos widely exits in nature and man-made systems including those biological systems [20] [21] [22] [23] [24] . according to the famous logistic map, it shows that the natural evolution of the population size could be chaotic. however, it is not a good thing to find chaos in the seir model. unfortunately, chaos in the sir, sis and seir models has been investigated by many researchers. generally, the seasonality and stochastic infection are introduced to the system for the nonlinear dynamics. for instance, kuznetsov and piccardi [25] investigated the bifurcations of the periodic solutions of seir and sir epidemic models with sinusoidally varying contact rate. meanwhile, the fractional-order seir epidemic model has aroused research interests of scientists. he et al. [26] investigated the epidemic outbreaks using the sir model, and a hard limited controller is designed for the control of the system. however, on the one hand, those sir and seir models cannot always show the nature of the covid-19, and we need to modify the system. on the other hand, the nonlinear dynamics of the system should be investigated. thus, we need to get more information on the dynamics of the epidemic system. the rest of this paper is organized as follows. in sect. 2, the modified seir model is designed and the descriptions of the system are presented. in sect. 3, the seir model is applied to the covid-19 data of hubei province where the pso algorithm is introduced to estimate the parameters. in sect. 4, the seasonality and stochastic infection are introduced to the model and the dynamics of the system is investigated. in sect. 5, the structure, parameters on the dynamics of the system and how to control the epidemic of the system are discussed. section 6 is the summary of the analysis. the classical seir model has four elements which are s (susceptible), e (exposed), i (infectious) and r (recovered). thus, n ¼ s þ e þ i þ r means the total number of people. the basic hypothesis of the seir model is that all the individuals in the model will have the four roles as time goes on. the seir model has some limitations for the real situations, but it provides a basic model for the research of different kinds of epidemic. based on the basic seir model, we proposed a new model which is denoted by where s, e, i 1 , i 2 , r, h and q are the system variables. the descriptions of those variables are presented in table 1 . the description of the system parameters is illustrated in table 2 , and the relationship between different variables is shown in fig. 1 . in this model, the infectious class is divided into two parts, i 1 and i 2 . meanwhile, we consider the quarantined class and hospitalized class in the model according to the real situation. for example, if one got coronavirus-like obviously, as shown in fig. 1 , we consider two main channels in the proposed model. the first one goes to s ! e ! i 1 ! r, and the second channel goes to s ! q ! i 2 ! h ! r. the first case shows the natural process of the epidemic, and it is a typical seir model. the second channel considers the control from the government including the quarantine and hospital. as a result, the designed model is an improved version of the seir model. if there is no quarantine (q 2 ¼ 0), hospital treatment / ¼ 0 and the recovered is immune to the virus (a ¼ 0), the model becomes to the classical seir model. however, there are always quarantine and hospital treatment. meanwhile, there is no evidence that the recovered is immune to the covid-19. thus, we need to considered these factors in the model. in this paper, we have n 6 ¼ s þ e þ i 1 þ i 2 þ r þ q þ h according to eq. (1) since there is an external input k. obviously, n is not a constant and it varies over time when k 6 ¼ 0. since there are a large population under voluntary home quarantine. thus, for a chosen place, n is not the total population of that place, but it can be estimated by adding the final number of recoveries and deaths. 3 estimation of the model parameters particle swarm optimization (pso) algorithm is a famous population-based stochastic optimization algorithm motivated by intelligent collective behavior, such as the foraging process of bird group [27] . in pso algorithm, each particle represents a bird, and the algorithm starts with a random initialization of the particle locations. for one iteration, each particle keeps track of its own best position and the population's best position to update its position and velocity. considering a one-dimensional optimization problem, the velocity and the position of the particle i is defined by and where v i ðtþ and x i ðtþ are velocity and position of the particle i at the t-th iteration, respectively. c 1 and c 2 are the contact and infection rate of transmission per contact from infected class v probability of transmission per contact from exposed individuals h 1 , h 2 transition rate of exposed individuals to the infected class c 1 , c 2 recovery rate of symptomatic infected individuals to recovered u rate of infectious with symptoms to hospitalized / recovered rate of quarantined infected individuals k rate of the quarantined class to the recovered class q 1 , q 2 transition rate of quarantined exposed between the quarantined infected class and the wider community k external input from the foreign countries the learning factors. r 1 and r 2 are random numbers between 0 and 1. p b;i ðtþ represents the best position of the particle i at the t-th iteration, and p g ðtþ represents the population's best position at the t-th iteration. x is called inertia weight, which is very important for the search process of pso algorithm. here, it is defined by [28] xðtþ where t and t are the current and maximum iterations of the pso algorithm, respectively, and x min and x max are the minimal and maximum value of the inertia weight, respectively. suppose that we meet a minimum optimization problem, the implementation steps of the pso algorithm are summarized as follows: step 1: the position of each particle is randomly initialized. step 2: calculate the fitness value fðx i ðtþþ of the particle i, and find the p b;i ðtþ and the p g ðtþ. step 3: if fðx i ðtþþ\fðp b;i ðtþþ, then replace the p b;i ðtþ by the x i ðtþ. step 4: if fðx i ðtþþ\fðp g ðtþþ, then replace the p g ðtþ by the x i ðtþ. step 5: calculate the inertia weight by eq. (4). step 6: update velocity and position of the particle i according to eqs. (2) and (3), respectively. step 7: repeat the steps 3-6 until the termination criterion is satisfied. in this section, through the actual covid-19 data from hubei province, the pso algorithm is utilized to estimate the parameters of the proposed seir model to fit the real situation. the covid-19 data come from the official website of the wuhan municipal health commission (http://wjw.wh.gov.cn/), and some actual data are listed in table 3 . in the face of the pressure of epidemic prevention and control, wuhan government announced to seal off the city from all outside contact on january 23rd, 2020. then, other cities in hubei province also took the ''closure city'' measure. the covid-19 epidemic situation of hubei is relatively stable after january 23rd, 2020, so we chose to study the data between january 24th and april 12th. the initial values setting of seir model is presented in table 4 , where n is the total population of hubei affected by the covid-19 epidemic in january 24th, 2020, and e is calculated based on the number of confirmed patients. i 1 is an estimated value based on i 2 , and the other initial values are originated from the actual data. the system parameters of seir model are calculated by the actual data, as shown in table 5 . however, there is no accurate statistics of the rate of infectious to hospitalized u and the recovered rate of quarantined infected individuals /. here, the two parameters are estimated by the pso algorithm with the actual data of r and h. the settings of pso algorithm are set as: population of particle swarm np ¼ 40, learning factors c 1 ¼ c 2 ¼ 2, maximal iteration t ¼ 100 and the search spaces u; / 2 ð0; 0:1. the covid-19 epidemic situation in hubei is divided into two stages: the outbreak stage (the first 19 days) and the inhibition stage (the 20th day to the end). in the outbreak stage, according to the actual data of r and h, the u and the / are estimated to u ¼ 0:2910, / ¼ 0:0107, respectively. the error convergence curve of pso is shown in fig. 2 . after the outbreak stage, due to the continuous assistance from other provinces and other countries, the epidemic in hubei began to enter the inhibition stage. in this stage, the error convergence curve of pso is given in fig. 3 , and the estimated u and / changed to u ¼ 0:0973; / ¼ 0:0416, respectively. the estimated and actual trajectories in the two stages are shown in fig. 4 . in the first stage, although there are some errors between the estimated number and the actual number, it shows that the estimated values match well with the real situation. however, the accuracy is not satisfying in the second stage which shows that the real data are smaller than the estimated values, but the trend is basically the same. there are two reasons for the deviation. one is that only two parameters, namely the rate of infectious to hospitalized u and the recovered rate of quarantined infected individuals /, are estimated, while the rest of the parameters are set as a matter of experience. moreover, the control measures for containing the error ×10 4 fig. 3 the error convergence curve of pso algorithm in the inhibition stage outbreak are more and more powerful; thus, the system parameter should be time varying variables. for instance, compared with the outbreak stage, the hospitalization rate decreased a lot, and the cure rate increased nearly four times, which means that the number of confirmed infected cases is declining a lot and the number of patients recovering is increasing rapidly. 4 nonlinear dynamics of the model the 0-1 test algorithm is employed to verify the existence of chaos in the model. if a set of discrete time series xðnþðn ¼ 1; 2; 3; . . .þ represents a one-dimensional observable data set obtained from the modified seir system, then the following two real-valued sequences are defined as [30] p n ð þ ¼ x i ð þ, and g 2 p 5 ; 4p 5 â ã . by plotting the trajectories in the (p, s)-plane, the state of the system can be identified. usually, the bounded trajectories in the (p, s)-plane imply the dynamics of the time series is regular, while brownian-like (unbounded) trajectories imply chaos. the seasonality is widely found in the epidemic models [26, 29] , and it can make the system more complex. indeed, there is no report showing that the effect of seasonality for covid-19 spread since this epidemic outbreaks only about half year until now. but we try to introduce the seasonality to the system and analyze chaos in the system from an academic point of view. meanwhile, there are individual differences and many unpredictable factors in the epidemic infection. thus, the noise is an important factor considered in our analysis. here, three cases are analyzed to show how the system parameter a, seasonality and stochastic infection affect the dynamics of the system. case 1: the parameter b 1 contains seasonality and stochastic infection, and the three contact and infection rate parameters are defined as where b 0 ¼ 2 â b 1 ¼ 60, e 1 and e 2 are degree of the seasonality and stochastic infection, respectively. n t ð þ h i is the white gaussian independent noises, and it has the properties of n t ð þ h i ¼ 0 and n t ð þ; n s ð þ h i¼ d t à s ð þ. the analysis results of the system with different parameters are shown in fig. 5 . in fig. 5a , b, we set e 1 ¼ 0, e 2 ¼ 0 and a ¼ 0:08. it shows that the system is convergent to a limited region. thus, the system is not chaotic without the seasonality and stochastic infection. when e 1 ¼ 0:8 and e 2 ¼ 0, the system has seasonality but no stochastic infection. it shows that fig. 4 actual and estimated trajectories for the epidemic situation in hubei province the system now is chaotic according to the p à s plot. as shown in fig. 5c that the attractor looks like a limited circle. however, there are many lines in the ''circle'' as it is illustrated in fig. 5d . if e 1 ¼ 0:8 and e 2 ¼ 0:2, there are both seasonality and stochastic infection in the system. at this time, the complex dynamics is observed in the system, as shown in fig. 5f -h. case 2: the parameter b 2 contains seasonality and stochastic infection, while the other two infection rate parameters are constants. thus, the parameters are defined as where b 0 ¼ 2 â b 2 ¼ 60, e 1 and e 2 are the degree of the seasonality and stochastic infection, respectively. firstly, we let e 1 ¼ 0:8 and e 2 ¼ 0. if a ¼ 0:02, a ¼ 0:03, a ¼ 0:04747 and a ¼ 0:08, different kinds of chaotic attractors are shown in fig. 6a -d, respectively. meanwhile, we let e 1 ¼ 0:8 and e 2 ¼ 0:2, the chaotic attractors with different values of a are shown in fig. 6e -h. the p à s plots of attractors of fig. 6b -d are shown in fig. 6i -k, respectively. it verifies the existence of chaos in the model. where b 0 ¼ 2v ¼ 60:8, e 1 and e 2 are the degree of the seasonality and stochastic infection, respectively. let e 1 ¼ 0:8, e 2 ¼ 0 and a ¼ 0:0133, the phase diagram is shown in fig. 7a . it shows in fig. 7b that the attractor is chaotic. if we set e 2 ¼ 0:2, the phase diagram is illustrated in fig. 7c , where a much wider range in the phase space when the system has stochastic infection. when e 1 ¼ 0:8, e 2 ¼ 0 and a ¼ 0:08, the phase diagram is shown in fig. 7d , while the time series are shown in fig. 7e . the proposed system is an improved seir system with quarantined class and hospitalized class. as with other sir and seir models, this model can also generate chaos with given parameters. and the existence chaos is verified by the 0-1 test method. in this section, we consider the dynamics of the proposed seir model. in refs. [26, 29] , the parameters of the seir model are set where the infection rate b is set as quite large values. for instance, in ref. [29] , b ¼ 108 for the proposed seir dengue fever model. to further analyze dynamics of the system, we choose case 2 as an example to show the bifurcations of the proposed system. the parameters are set as b firstly, let e 1 ¼ 0:8, e 2 ¼ 0, and the parameter a varies from 0.02 to 0.08 with step size of 0.00024. the bifurcation diagram is shown in fig. 8a . meanwhile, if e 2 ¼ 0:2, the corresponding bifurcation diagram is fig. 8b . obviously, the bifurcation diagram of fig. 8a has no noise, while that in fig. 8b does. it shows that the stochastic infection makes the system fluctuate more volatile. secondly, let a ¼ 0:08, e 2 ¼ 0, and the parameter e 1 ¼ 0:8 varies from 0.02 to 0.08 with step size of 0.004. the bifurcation diagrams with e 1 are shown in fig. 9 . it shows that when there exists stochastic infection, the bifurcation diagram shows more complex behaviors of the system. as shown in figs. 8 and 9, it shows that the system has rich dynamics with both parameters a and e 1 . when the system has stochastic infection, the system will become more complex. we hold the opinion that chaos is also the nature of the system, and the seasonality and the temporary immunity rate can change the dynamics of the system. in this section, the spectral entropy (se) algorithm [31] is employed to analyze complexity of the proposed seir system, and steps are presented as follows. for a given time series {x(n), n=0, 1, 2, á á á, l-1} with a length of l, let xðnþ ¼ xðnþ à x, where x is the mean value of time series. its corresponding discrete fourier transform (dft) is defined by where k ¼ 0; 1; á á á ; l à 1 and j is the imaginary unit. if the power of a discrete power spectrum with the k th frequency is jxðkþj 2 , then the ''probability'' of this frequency is defined as when the dft is employed, the summation runs from k ¼ 0 to k ¼ n=2 à 1. the normalization entropy is denoted by [31] where lnðn=2þ is the entropy of a completely random signal. obviously, the more balanced the probability distribution is, the higher complexity (the larger entropy) the time series is. the larger measuring value means higher complexity, and vice versa. based on the above complexity algorithms, multiscale complexity algorithm is designed. for a onedimensional discrete time series fxðnþ : n ¼ 0; 1; . . .; n à 1g, the consecutive coarsegrained time series are constructed by [32] where 1 j ½n=s, s is the scale factor which represents the length of the non-overlapping window, and ½á denotes the floor function. obviously, when s ¼ 1, the sequence y s is the original sequence fxðnþ; n ¼ 0; 2; 3; á á á ; l à 1g. thus, the complexity of y 1 is the complexity of the original sequence. in this paper, the multiscale complexity is defined as [31] in this paper, we set s max ¼ 20. fix e 1 ¼ 0:8 and vary the parameter a from 0.02 to 0.08 with step size of 0.00024. mse complexity analysis results are shown in fig. 10a, b, where fig. 10a for e 2 ¼ 0 and fig. 10b for e 2 ¼ 0:2. fix a ¼ 0:08 and the parameter e 1 varies from 0.1 to 1 with step size of 0.0036. the complexity analysis results with e 1 are shown in fig. 10c, d. here, e 2 ¼ 0 is employed in fig. 10c , while e 2 ¼ 0:2 is used in fig. 10d . the complexity analysis results with parameters show that the stochastic infection does not affect the complexity of the system. the system has higher complexity when a takes values between 0.03 and 0.06, and e 1 takes those values which are larger than 0.6. fix e 2 ¼ 0, vary a from 0.02 to 0.08 with step size of 0.0006 and e 1 varies from 0.1 to 1 with step size of 0.009. the complexity analysis result in the a à e 1 plane is shown in fig. 11 . obviously, the higher complexity region is located in the right side of the parameter plane, where a 2 ½0:4; 1 and e 1 2 ½0:025; 0:055. since the complexity measure results are obtained based on the generated time series, mse provides an effective way for the dynamics analysis of the system. when there is higher complexity, the behavior of the model is more complex, vice versa. for a epidemic system, high complexity means outbreak. thus, we can use the complexity measure algorithm to monitor the dynamics of the proposed seir system. in this paper, the parameters of the system are mainly chosen by two means including the references and the pso algorithm. usually, the parameters can be set according to the existing work. for instance, the contact and infection rate parameters are defined according to refs. [26, 29] . also, there are some other references which show some parameters of the system. moreover, since there is actual covid-19 data from hubei province, we use the pso algorithm to estimate the parameters of the system. as shown above, the system has rich dynamics with the given parameters, especially when the parameters b 1 , b 2 and v have seasonality and stochastic infection. the existence of chaos is verified by the 0-1 test, and complexity of the generated time series is measured. here, different sets of parameters are summarized in table 6 . it should noted that, when the parameters are set as the set a, they seem ''large.'' however, those parameters should be multiplied by s/n. thus, they are reasonable for the proposed model. according to our analysis, it shows that chaos are found in the system with those ''large parameters.'' in fact, we want to explore the nonlinear dynamics of the proposed system and to study how does chaos occur in the system. figure 12 shows the evolution of the system with different parameters. the parameters used the set d, and different colors lines in the figure including magenta color lines (m), blue color lines (b), red color lines (r) and green color lines (g) are obtained using the following parameters: it shows that the number of infected class (i 1 , i 2 ) and hospitalized class (h) is different with different parameters. when u ¼ 0:0009, there is a peak value for i 2 . it means that if the hospital reception capacity is limited, the infected class (i 2 ) will increase dramatically. however, when u ¼ 0:8, the infected class (i 2 ) keeps a relative low level; thus, the infection can be controlled well. as shown in fig. 12 , when a ¼ 0:5, it is quite difficulty for the system to become convergent. the reason is obvious because those recovered can be infected again, and a closed loop system is observed. because no reports show that the recovered class is immune to the covid-19, we need to be aware of those recovered to be infected again. here, the evolution of the system with parameters of set e is shown in fig. 13 , which shows how all the classes of the system affect the dynamics. generally, all the classes except the recovered class r will converge to zero. however, fig. 13 is simulated with external input k ¼ 100 and a ¼ 0:5. since there is no evidence that the recovered class is immune to the virus, this makes the system hard to converge. thus, it shows in fig. 13 that these variables converge to zero slowly. in the early stage of the covid-19 epidemic, the epidemic situation in hubei province presents an uncontrollable trend. however, due to the low population contact rate, high hospitalization rate and high cure rate, the epidemic was quickly controlled after 20 days. therefore, the government's attention, people's self-awareness and sufficient medical resources are the key to eliminate the threat of covid-19. to get better estimation results, we need to built a proper model and also need to set proper parameters for the systems. to the knowledge of authors, the parameters of the system change as time since the control from the government is different along with time. thus, we can also treat the parameters as functions of time. if the values of b 1 , b 2 and v are large, the system can even become chaotic. when q 2 takes larger values, it means that there are more people which have like covid-19-symptoms. in fact, the government should take stronger and harsher measures to increase isolation, especially there are many potential infections. meanwhile, if the quarantine is done well, the values of b 1 , b 2 and v will be also much smaller; thus, it is helpful to control the spread of the epidemic disease. in this paper, a seir model is proposed for the covid-19. parameters of the system are estimated by the pso algorithm, and dynamics of the system is investigated. finally, how the parameters affect the dynamics of the system is discussed and the control strategies are presented. the conclusions of this paper are given as follows. (1) the proposed model has considered the quarantine and treatment, so it is more suitable for the dynamics of the epidemic of covid-19. (2) the pso algorithm provides a good way for parameter estimation of the seir model. and according to the application to the data of hubei province, the accuracy is acceptable. the main trends of the epidemic evolution are illustrated. (3) nonlinear dynamics of the system is investigated by means of bifurcation diagram, mse algorithm and 0-1 test algorithm. it shows that, for the given parameters, if there exists seasonality and stochastic infection, the system can generate chaos. (4) some control suggestions are suggested based on the proposed model. meanwhile, we found that the dynamics of the system is different with different sets of parameters. emerging coronaviruses: genome structure, replication, and pathogenesis coronaviruses: structure and genome expression a novel coronavirus from patients with pneumonia in china 2019-ncov, fake news, and racism 2019-ncov in context: lessons learned? genomic variance of the 2019-ncov coronavirus cross-species transmission of the newly identified coronavirus 2019-ncov analysis and forecast of covid-19 spreading in china, italy and france networks and epidemic models network-based prediction of the 2019-ncov epidemic outbreak in the chinese province hubei a simple sis epidemic model with a backward bifurcation a networked sis disease dynamics model with a waterborne pathogen a stochastic sirs epidemic model with nonlinear incidence rate a simple seir mathematical model of malaria transmission analysis of a fractional seir model with treatment a time-dependent sir model for covid-19 estimation of the transmission risk of the 2019-ncov and its implication for public health interventions nowcasting and forecasting the potential domestic and international spread of the 2019-ncov outbreak originating in wuhan, china: a modelling study phase-adjusted estimation of the number of coronavirus disease 2019 cases in wuhan, china chaos analysis in attitude dynamics of a flexible satellite chaotic vibrations of flexible shallow axially symmetric shells theory of hybrid dynamical systems and its applications to biological and medical systems complexity, analysis and control of singular biological systems complexity in the muscular blood vessel model with variable fractional derivative and external disturbances bifurcation analysis of periodic seir and sir epidemic models epidemic outbreaks and its control using a fractional order model with seasonality and stochastic infection particle swarm optimization algorithm: an overview parameter identification of fractional-order discrete chaotic systems epidemiology of dengue fever: a model with temporary cross-immunity and possible secondary infection shows bifurcations and chaotic behaviour in wide parameter regions the 0-1 test algorithm for chaos and its applications complexity analysis and dsp implementation of the fractional-order lorenz hyperchaotic system multiscale entropy analysis of complex physiologic time series publisher's note springer nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations conflict of interest the authors declare that they have no conflict of interest. key: cord-292623-mxdlii77 authors: arji, goli; ahmadi, hossein; nilashi, mehrbakhsh; a. rashid, tarik; hassan ahmed, omed; aljojo, nahla; zainol, azida title: fuzzy logic approach for infectious disease diagnosis: a methodical evaluation, literature and classification date: 2019-09-26 journal: biocybern biomed eng doi: 10.1016/j.bbe.2019.09.004 sha: doc_id: 292623 cord_uid: mxdlii77 this paper presents a systematic review of the literature and the classification of fuzzy logic application in an infectious disease. although the emergence of infectious diseases and their subsequent spread have a significant impact on global health and economics, a comprehensive literature evaluation of this topic has yet to be carried out. thus, the current study encompasses the first systematic, identifiable and comprehensive academic literature evaluation and classification of the fuzzy logic methods in infectious diseases. 40 papers on this topic, which have been published from 2005 to 2019 and related to the human infectious diseases were evaluated and analyzed. the findings of this evaluation clearly show that the fuzzy logic methods are vastly used for diagnosis of diseases such as dengue fever, hepatitis and tuberculosis. the key fuzzy logic methods used for the infectious disease are the fuzzy inference system; the rule-based fuzzy logic, adaptive neuro-fuzzy inference system (anfis) and fuzzy cognitive map. furthermore, the accuracy, sensitivity, specificity and the receiver operating characteristic (roc) curve were universally applied for a performance evaluation of the fuzzy logic techniques. this thesis will also address the various needs between the different industries, practitioners and researchers to encourage more research regarding the more overlooked areas, and it will conclude with several suggestions for the future infectious disease researches. fuzzy logic approach for infectious disease diagnosis: a methodical evaluation, literature and classification show that the fuzzy logic methods are vastly used for diagnosis of diseases such as dengue fever, hepatitis and tuberculosis. the key fuzzy logic methods used for the infectious disease are the fuzzy inference system; the rule-based fuzzy logic, adaptive neuro-fuzzy inference system (anfis) and fuzzy cognitive map. furthermore, the accuracy, sensitivity, specificity and the receiver operating characteristic (roc) curve were universally applied for a performance evaluation of the fuzzy logic techniques. this thesis will also address the various needs between the different industries, practitioners and researchers to encourage 1. an infectious disease can be defined as a situation that is created by the incursion of a human body by harmful agents which will in turn hurt the body and may spread to other people as well [1] . infectious diseases are the major cause of illness and death for any given population [2, 3] . human immunodeficiency virus infection and acquired immune deficiency syndrome (hiv/aids), severe acute respiratory syndrome (sars), h1n1 influenza and poliomyelitis are examples of an infectious disease [3, 4] . while globally the incidence of the infectious disease varies greatly between countries, in the 21st century, the main incidences are hand-foot-mouth disease, hepatitis b, and tuberculosis [3] . these diseases have a significant impact on the global health and economies [5] . despite numerous valuable achievements regarding the procedures for preventing and controlling of various diseases, infectious diseases remain a massive threat to a population's well-being [6] . aspects such as the increasing in antimicrobial resistance, increase in population and environmental changes are important in an infectious disease transmission [7] . of 57 million mortality reported per year throughout the world, 14.9 million were related to infectious diseases; which represent more than 25% of the overall deaths [3] . additionally, clinical diagnosis and detection of the contaminated population are critical elements in controlling and supervising an infectious disease [8] . for the diagnosis of different diseases, there are different essential criteria that should be interpreted by the caregivers [9] . in a clinical situation, an analysis physician combines a patient's medical history, clinical symptoms, physical examination and laboratory findings [10] . due to the elusive and complex nature of clinical decisionmaking, they may be accomplished with unwanted errors [11] . in other word, medical diagnosis is an error-prone process, which occurs as the result of logical thinking [10] . mathematical models are essential means for demonstrating the cause and effect relationship and evaluating the evidence for decisiveness regarding infectious diseases [12] . computer-aided methods have the potential for examining the complexity of an infectious disease dynamics [11, 12] . computational tools are essential for understanding epidemiological patterns in a disease outbreak [13] . for handling the uncertainty of decision-making, studies have tried to explain the decision-making process in a medical setting by using the boolean or binary structure [11] . fuzzy logic is considered as a vigorous technique for modelling ambiguity in medical practice [14] . in medical field, most medical concepts are fuzzy [15, 16] . these concepts usually are difficult to formalize and measure [17] . fuzzy logic is making a decision in an inaccuracy, uncertainty and incompleteness environment [14] . fuzzy methods deal with the classes whose boundaries are unclear and elusive [18] . fuzzy logic concepts were introduced in 1965 [10] . the medical field was one of the first fields in which fuzzy theory was implemented [19] . fuzzy classes are given a degree of membership that is intermediary between 0 and 1 [18] . some of the cases for a medical use of the fuzzy set theory are the myci, internist and doctormoon applications [10, 20] . therefore, for these application methods, different types of research have been conducted in the disease diagnosis field. so, the major objective of the current study is to examine the researches in which fuzzy logic techniques have been applied in infectious diseases so to determining its trends and methods, through the processes of conducting a systematic literature review (slr). the current thesis is structured in the fallowing order. section 2 exemplifies the procedures or the methodology of the current slr, section 3 will present a complete report of the current systematic review. section 4 will be dedicated to the discussion of this research. section 5 will be dedicated to the implications of this study, its future research prospects and to the limitations of this evaluation and the last section will be the conclusion. we carried out an slr to define the influence of using the fuzzy logic methods in infectious diseases. the three main questions of this slr are as follow: (rq1) which domain of an infectious disease was more interesting in past researches? (rq2) which fuzzy methods were more dominant in infectious disease data analysis? (rq3) which performance evaluation methods were more frequently applied in previous reviews? for the gathering of the relevant information from all the eligible articles, a data extraction method was used for getting the detailed answer of the research questions. (rq1) which domain of an infectious disease was more interesting in past researches? (rq2) which fuzzy techniques were more dominant in an infectious disease data analysis? (rq3) which performance evaluation procedures were more frequently applied in the previous reviews? to answer the research questions and to produce the extracted data, numerous methods were used. generally, a narrative combining approach to answering different research questions was applied. furthermore, various visualization techniques such as tables and charts were used according to the research questions. current part is dedicated to the outcome of the current slr. first, we will demonstrate an overall description of the outcome of selecting the appropriate studies; and then, all the obtained outcomes will be categorized for each study questions independently. by searching for the four aforementioned databases, the 372candidate paper was removed as shown in fig. 1 . then, based on using the exclusion conditions, 162 studies were excluded. the other 210 articles were investigated meticulously to choose relevant studies. by reviewing the title, abstract and the keywords, merely papers that have at least one of the inclusion measures were used. eventually, 40 papers were used to get an answer to the research questions and data extraction for this methodical evaluation. fig. 2 is showing the distribution of eligible papers per year. according to the diagram below, the frequency of papers relevant to the use of fuzzy logic in an infectious disease was roughly stable during the first 5 years. however, as shown in the next chart, there is a persistent trend in the number of finally, the spread of the relevant articles based on a journal or conference, has been explained in table 1 . based on the obtained results expert systems with applications journal was used by researchers for publishing their studies in the 9.6 percent of instances. rq1: which domain of an infectious disease was more interesting in past researches? articles related to the precise domain of fuzzy methods application in an infectious disease, which have been considered from the past section, are summarized in the following section. fig. 3 illustrates the distribution of numerous fuzzy techniques that have been used in an infectious disease data analysis. based on this figure, the results revealed that the critical application domain of the fuzzy methods in an infectious disease were relevant to dengue fever (15% of papers), hepatitis and tuberculosis (10% of papers). in addition, the other papers were related to diseases such as pulmonary infection (7.5% of papers), urinary tract infections (utis), human immune deficiency viruses (hiv) and meningitis (5% of papers). for all the other diseases, there is one paper per each disease (2.5% of papers). appendix a, table a1 lists eligible articles according to the specific type of disease, their objective, their inward and outward variables and findings. as shown in fig. 3 , the distribution of the different fuzzy methods in various disease data analysis was as fallows. in articles published for dengue fever, 33.33% of the papers applied fuzzy set theory and the adaptive neuro-fuzzy inference system (anfis) method in data analysis while 16.6% of the papers used association rule mining. 50% of selected papers that related to hepatitis, employed neuro-fuzzy classifier as a fuzzy technique, while the other 50% were devoted to fuzzy inference system and fuzzy decision support system (fdss). furthermore, in tuberculosis studies, 50% of the papers used rule-based fuzzy logic and 50% of the papers applied the gaussian-fuzzy neural network method. rq2: which fuzzy techniques were more dominant in an infectious disease data analysis? fuzzy inference systems are increasingly becoming more predominant in the area of fuzzy logic methods where 15% of the selected papers in the current slr are related to this particular method. fuzzy inference systems are used for the processes of mapping the inward variables to appropriate outward [21, 22] . fuzzy inference process incorporates three key concepts: the membership functions, the fuzzy set operations, and the inference procedures [23] . a fuzzy inference system can be divided mainly to four portions as follows: fuzzification, weighting, assessment of inference procedures, and the de-fuzzification [24] . dragovit et al. utilized the fuzzy inference system to decide the probability of having peritonitis in patients. by using this system, the fuzzy rules enable the automation of the clinical decision making process in an imprecise and complicated conditions [25] . urinary tract infections (utis) are considered amongst one of the utmost predominant bacterial infections. ibrisimovic et al. has suggested a fis fuzzy model, to provide the necessary support that the caregivers need for explaining the aftermath of a microscopic urine analysis. to create the model's various variables such as the colony forming units (cfu), white blood cells (wbc) and the red blood cells (rbc) as well as the turbidity of urine specimen used for inward variables, and the risk of a uti as an outward variable. the end result has revealed that the use of the fuzzy methods simplifies and secures the clarification of urine analysis [26] . putra and munir proposed a method for diagnosis of measles, german measles and varicella. the data for those diseases were used because of the similarity of their infection mechanism and symptoms. a built fuzzy inference system b i o c y b e r n e t i c s a n d b i o m e d i c a l e n g i n e e r i n g 3 9 ( 2 0 1 9 ) 9 3 7 -9 5 5 takes in the inward variables that represent the possible symptoms that may appear in each disease. cough, runny nose, sore throat, conjunctivitis, koplik's spot, diarrhea, headache, swollen neck or ear, loss of appetite, malaise, pimples/crust skin, joint pain used as inward variables. the application effectively identified 19 out of 25 accurate diseases throughout its testing stage [27] . the anfis method was proposed by jang and its notions then were used in other fields [22] . this method works by setting a list of features by using an amalgam of learning rules which will incorporate the back-propagation incline in error digestion and a tiniest squares method. this method can be implemented as the basis for creating a set of if-then guidelines with suitable association of functions to brand the inward and outward variables [22] . anfis method has been notably successful in disease diagnosis in the past few years. as an example, the major groups of hepatitis in human beings are hepatitis a, hepatitis b and hepatitis c with the main symptoms being malaise (a common sick feeling), fever, and muscle pain, loss of appetite, nausea, vomiting, diarrhea, and jaundice. viral hepatitis disease can be diagnosed by blood test analysis and interpretations. dogantekin et al. developed an anfis system to be used for hepatitis diagnosis. the precision achieved in this automated system of diagnosis was at about 94.16% [28] . faisal et al. conducted a research using an adaptive neurofuzzy inference system to diagnose a dengue fever. the general precision of the developed method is 86.13% with its sensitivity being at 87.5% and its specificity being at 86.7% [29] . this method was used in the campisi research as well to determine the amount of the risk factors of an infection disease in relationship with oral candidiasis (oc) [30] . additionally, shariati has recommended a method of diagnosis for both hepatitis and thyroid diseases. the researchers in this study compared the outcome of the anfis technique with the support vector machine (svm) and the artificial neural networks techniques. then, they demonstrate that this technique had an improved outcome in being a precise diagnosis in comparison with the previous techniques [31] . the information in a fuzzy rule-based system is typically represented by using an if-then statement. rule-based fuzzy logic includes two portions: the precursor portion are the relevant conditions which are known as the inward variable(s), and the subsequent portion which expresses the outward variable(s). mamdani and sugeno are two different types of fuzzy rulebased systems. in the mamdani technique, the precursor, as well as the subsequent section, comprises fuzzy statements that reveal the value of the variables, while in sugeno method, the subsequent portion displays a nonlinear affiliation among both the inward and outward variables [32, 33] . because of the complex nature of the numerous diseases, this technique can improve the infectious disease diagnosis and the treatment of such diseases as hiv and tuberculosis. based on this, sloot et al. efficiently used rule-based fuzzy logic to uncover the drug-resistance in hiv patients [34] . furthermore, semogan et al. has created a clinical decision supporting system based on the fuzzy logic and the rule-based model that determine different classes of tuberculosis to assess respiratory diseases. in this system variables such as cough, cough duration, body temperature, fever duration, sputum discoloration, nose sputum, afternoon chills, night sweats, weight loss, and loss of appetite have been used in the diagnosis [35] . b i o c y b e r n e t i c s a n d b i o m e d i c a l e n g i n e e r i n g 3 9 ( 2 0 1 9 ) 9 3 7 -9 5 5 the fuzzy cognitive map (fcm) is a modelling technique that defines the connections between concepts such as variables, the inwards and the outwards by the methods of previous knowledge and experience. kosko has introduced fcm in 1986. fcm is usually used to demonstrate the cause and effect relationship between the different concepts in a given system. fcms are used in numerous fields such as in engineering, error detection and medicine [36, 37] . in this regard, mei et al. has applied fcm to describe how factors such as people's emotions and cognition functions influence each other to create epidemic infection [38] . fcm was also used by the mago et al. research to develop the required knowledge-based structure used for recognizing the specific causes and the symptoms of meningitis disease in children. this system can be implemented as a dependable instrument in supporting the physician's to better their decision making processes [39] . rq3: which performance evaluation techniques were more frequently applied in the previous reviews? performance evaluation procedures are among the furthermost significant indicators for determining the quality of the artificial intelligence techniques [40] . overall, performance evaluation procedures are classified into two main types; the first being the single scaler techniques and second being the graphical techniques. the sensitivity, specificity and the accuracy indicators are grouped into the single scaler techniques. receiver operating characteristic (roc) curve, cost-line, and lift graph are clustered together in graphical methods. anyhow, graphical methods cannot simply be clarified and analyzed as single scalar method [41] [42] [43] . fig. 5 indicates the most noticeable performance evaluation indicators used in eligible papers. for the current slr, most qualified studies did not use any kind of indicators for analyzing the performance of the fuzzy techniques. among the fuzzy techniques, anfis and fis techniques were evaluated by single scaler techniques and graphical evaluation techniques. the major objective of this slr was to select and examine the various studies relevant to the employment of the fuzzy logic techniques in an infectious disease. in this regard, 40 studies were selected and analyzed from an original number of 372 candidate studies. this section is dedicated to the discussion of the major findings of this study. rq1: which domain of an infectious disease was more interesting in past researches? in last few years, it has been obvious from the results of analytic studies conducted in our research that there is a growing interest in studying the relevance of the fuzzy logic techniques for an infectious diseases diagnosis applications. the studies have shown that there is an increase in the number of published articles from 5% in 2005 up to 17.5% of the evaluated papers in 2017. this growing trend may prove the popularity of the fuzzy techniques between different academic papers and its valuable effects in identifying the infectious disease trends. additionally, 13 different fuzzy logic techniques have been used in the evaluated papers. from a medical point of view, in fig. 3 we found that the main application domain of fuzzy logic in infectious disease was related to dengue fever, hepatitis and tuberculosis respectively. the other noticeable outcome that we have found was the b i o c y b e r n e t i c s a n d b i o m e d i c a l e n g i n e e r i n g 3 9 ( 2 0 1 9 ) 9 3 7 -9 5 5 fact that the evaluated papers were very diverse in terms of disease types. because of the big impact on global health by infectious diseases, determining the different features of these diseases are a valuable source for improving our knowledge and ability to predict how a disease will spread in a population [2, 3] . the ability to comprehend and control an infectious disease can be obtained by the usage of mathematical models [44] . the newly arising infectious agents such as hiv, the severe acute respiratory syndrome (sars), the mid-eastern respiratory syndrome (mers) coronaviruses; the west nile virus; the nipah virus; the drug-resistant pathogens; novel influenza a strains and the ebola virus outbreak were considered as big challenge to health in the recent century [6] . using computeraided diagnosis techniques like the fuzzy logic technique can be useful in determining the main factors associated with the infectious diseases occurrence and epidemics. regarding this, in another field of research in relationship to the vaccination strategies for infectious diseases it has been shown that infectious diseases such as influenza have a seasonal pattern in which case mathematical techniques such as the fuzzy logic techniques is considered a vital instrument in the process of forecasting the viral development from one year to another. this technique can also deliver the required scientific confirmation which will help us decide the amount of vaccine treatment, its efficiency, its financial costs and its pattern of contact in any given population [6, 45, 46] . it has been shown that the fuzzy based techniques have an essential part in the determination of infectious disease outbreaks. as an example for that, we have the hiv outbreaks, these techniques can identify at what time a viral transmission has occurred, its outbreak stage and the sexual behavior pattern of the specific population in which a disease is spreading. moreover, the infectious diseases annihilation and elimination techniques dictate that the transmission process itself has to be the target not the disease. regard this, the data sources are typically scattered and the collective effect of the numerous control techniques are complex and are dependent on the rate of transmission [47] . rq2: which fuzzy techniques were more dominant in an infectious disease data analysis? as shown in fig. 4 , there is a various number of fuzzy techniques employed in the chosen papers. based on this studied section we can mostly classify the fuzzy techniques into four main classes; the fuzzy inference system, the rulebased fuzzy logic, anfis and the fuzzy cognitive map. the fuzzy inference system and the anfis technique were used as shown in 17.5% and 12.5% of candidate papers, respectively. fis is based on the 'if-then' rules and it can be used to predict the behavior of a various uncertain situations [48] . as seen in the distribution of the studied articles, we can say that fuzzy techniques are efficient means for modelling unclear disease conditions such as in the case of transmissible disease diagnosis. additionally, in the last few years, there has been substantial interest amongst the researchers to apply the anfis technique in the case of an infectious disease. anfis combines the positive effects of both ann and fis in an influential tool for disease diagnosis. this technique does not require excessive knowledge in the modelling and training system. these techniques are usually valuable for the situation, which are in many cases complicated, with a nonlinear behavior pattern. this work approach has created a relationship between the inward and the outward features by the means of the neurons [49] . rq3: which performance evaluation procedures were more frequently applied in the previous reviews? performance evaluation procedures are usually employed as a valued method in determining the quality of the numerous fuzzy techniques [40] . as shown in fig. 5 in the current slr, amongst the single scaler techniques, the sensitivity, specificity and the accuracy are frequently utilized in the evaluation of a developed technique. additionally, in graphical evaluation techniques, the roc curve was the other measure of performance evaluation that was used in the adequate papers. these indicators are to be considered significant when reporting and assessing a diagnostic technique. scientists have to provide suitable information about the sensitivity, specificity, and the projected values when describing a computer based diagnostic technique end results and this information must contain how those metrics are concluded and also what are its appropriate interpretations [43, 49] . although those indicators are promising in qualified studies, nonetheless, they typically generate an inadequate image of the indicators performance and thus it is possible to lose a certain amount of valuable information. moreover, it is possible that the employment of a single-scaler technique will not identify the full scope of a performance assessment. therefore, a comprehensive and dependable assessment must reflect all the numerous parts of performance distinctive quality. in this methodical review, the studies related to the employment of the fuzzy logic techniques in an infectious disease were assessed, and depending on the acquired outcomes, we can notice an interest amongst the researchers regarding this specific field of research. in last few years, a large number of the transmissible diseases that were supposed to be eliminated have made a comeback. certain factors such as manufacturing, agricultural practices, wars, changes in lifestyles, development and urbanization, and environmental change are all effective in the appearance and reappearance of an infectious disease [50] . this slr's result demonstrates that even though these are the most of the infectious diseases that were investigated, but there is still more area that needs to be covered. nevertheless, more work should be carried out on the appearance and reappearance diseases domains such as h1n1, sars, zoonosis and the rift valley fever. internationally, there is a lack of an integrated framework for reporting infectious disease [51] . additionally, an infectious diseases information system has an inadequate support for data analysis and generating predictive techniques centered on artificial intelligence. an integrated analytical framework that offers functions as in a progressive data analysis capability and a visualization support is of a critical importance [51, 52] . there is serious need for creating an atmosphere for collecting, distributing, reporting, assessing, and picturing the infectious disease data and to provide support for decisionmaking tools regarding disease prevention, recognition, and controlling [53, 54] . infectious disease observation and controlling has demanded an interdisciplinary work. to have the ability to achieve those objectives, the employment of geographic information systems (gis), three-dimensional information analysis, machine learning and visualization applications and techniques became a must. because of the significance of the infectious diseases at the global level, it is essential to simultaneously develop an incorporated infectious disease dataset and to make the specialized analytical and diagnostic methods all at the same time. additionally, there was slight conversation in the incorporated literature about three-dimensional information analysis for an infectious disease occurrence. three-dimensional information evaluation techniques may be useful in determining the concentration pattern of a disease occurrence and to make the required association from the determined patterns to the measureable procedures [55, 56] . furthermore, the social networks information study has facilitated the evaluation of the association amongst the population in a particular social setting. methods that correlate spatial and social network data analysis are unusual but have the capacity to promote the determination of a spreading progress of an infectious disease [57] . or we can say, the deployment of a unified spatial and social network structure to define the spreading of an infectious pathogens in a given populace will allow insights into both the understanding to the disease method of distribution and the possible process associated with the observed patterns. since there are various studies regarding social media information analysis, we recommend the use of this dataset for the prediction of an infectious disease epidemic. this research has its limitations. even with the use of a broad search approach, some of the publications regarding the fuzzy logic deployment in an infectious disease could not be recovered, as in the case of grey literature and reports that were not published in surveyed digital databases, which we have reviewed. thus, it is recommended that additional slr papers should be carried out to go through the other noticeable databases. additionally, some studies did not report clearly on the performance assessment technique. in conclusion, only the english language publications were included, therefore future studies could be expanded to incorporate relevant papers which are published in other languages. in this study we have identified, classified, and defined the use of the fuzzy logic techniques in infectious diseases. 40 studies were scrutinized and the main conclusions can be briefed as follows: (1) the key application field of the fuzzy logic in an infectious disease was related to dengue fever, hepatitis and tuberculosis, (2) amongst the fuzzy logic techniques fuzzy inference system, rule-based fuzzy logic, anfis and fuzzy cognitive map are commonly used in many studies, and (3) the major performance evaluation indicators such as the sensitivity, specificity, and the accuracy the roc curve is employed. in addition, this study highlights the absence of an integration of infectious disease information systems in order to provide a valuable datasets in this domain. additionally, using machine learning and visualization applications for information analysis is essential. even though in the current slr there is a diversity of infectious diseases that are investigated, there is only one article per each disease. there is additional need to use the fuzzy logic methods for infectious disease detection and prediction. it appears that one of the causes for a limited number of relevant articles to infectious diseases is in the difficulty in obtaining adequate research data. finally, we expect that a mounting number of infectious diseases datasets will be mostly obtainable in the forthcoming years because of raising collaboration amongst medical practitioners and researchers and that this would lead to additional studies of the machine learning techniques that can be useful in this regard. the generated results are more reliable for the prediction of tb in patients. 35 langarizadeh [82] 2014 meningitis rule-based fuzzy logic this system is used to distinguish between bacterial and aseptic meningitis, by using fuzzy logic. gram stain, white blood cell (wbc) count in cerebrospinal fluid (csf), percentage of polymorphonucleocytes in csf, csf protein, csf/ serum glucose ratio, wbc count in blood, percentage of blood neutrophils, blood c-reactive protein (crp), and platelet (plt) count. bacterial and aseptic meningitis this suggested system has shown great efficiency in regards to its ability to differentiating between the bacterial and aseptic meningitis. 36 omisore [83] 2017 tuberculosis genetic-neuro-fuzzy proposing a genetic-neuro-fuzzy for the diagnosis of tuberculosis. the study has proposed fcms for determining the symptoms and causes for meningitis patients. b i o c y b e r n e t i c s a n d b i o m e d i c a l e n g i n e e r i n g 3 9 ( 2 0 1 9 ) 9 3 7 -9 5 5 biological sciences curriculum study nih curriculum supplement series. bethesda, md: national institutes of health how urbanization affects the epidemiology of emerging infectious diseases epidemiological features of and changes in incidence of infectious diseases in china in the first decade after the sars outbreak: an observational trend study clinical aspects of influenza a (h1n1) in hivinfected individuals in sã£o paulo during the pandemic of global trends in emerging infectious diseases modeling infectious disease dynamics in the complex landscape of global health ecology of increasing diseases: population growth and environmental degradation the challenge of emerging and re-emerging infectious diseases the concept of disease-vague, complex, or just indefinable? from vagueness in medical thought to the foundations of fuzzy reasoning in medical diagnosis diseases diagnosis using fuzzy logic methods: a systematic and meta-analysis review modeling uncertainty in clinical diagnosis using fuzzy logic a computational model to monitor and predict trends in bacterial resistance is there a need for fuzzy logic? fuzzy logic and measles vaccination: designing a control strategy face recognition with triangular fuzzy set-based local cross patterns in wavelet domain fuzzy logic and hospital admission due to respiratory diseases using estimated values by mathematical model profiles of multiple causes of death related to hiv/aids in the cities of sao paulo and santos, brazil fuzzy-trace theory, risk communication, and product labeling in sexually transmitted diseases fuzzy logic and exponential smoothing for mapping implementation of dengue haemorrhagic fever in surabaya mu'arifin assessing water quality in rivers with fuzzy inference systems: a case study adaptive network based fuzzy inference system (anfis) training approaches: a comprehensive survey adaptive neuro-fuzzy inference system for evaluating dysarthric automatic speech recognition (asr) systems: a case study on mvml-based mamdani-type fuzzy inference system for evaluation of tax potential. recent developments and the new direction in soft-computing foundations and applications a boolean consistent fuzzy inference system for diagnosing diseases and its application for determining peritonitis likelihood a fuzzy model to predict risk of urinary tract infection implementation of fuzzy inference system in children skin disease diagnosis application automatic hepatitis diagnosis system based on linear discriminant analysis and adaptive network based on fuzzy inference system adaptive neuro-fuzzy inference system for diagnosis risk in dengue patients risk factors of oral candidosis: a twofold approach of study by fuzzy logic and traditional statistic comparison of anfis neural network with several other anns and support vector machine for diagnosing hepatitis and thyroid diseases fuzzy rule-based models for decision support in ecosystem management adaptive weighted fuzzy rule-based system for the risk level assessment of heart disease a grid-based hiv expert system a rule-based fuzzy diagnostics decision support system for tuberculosis unsupervised learning techniques for fine-tuning fuzzy cognitive map causal links a fuzzy cognitive map approach to support urban design individual decision making can drive epidemics: a fuzzy cognitive map study supporting meningitis diagnosis amongst infants and children through the use of fuzzy cognitive mapping a systematic literature review and classification of knowledge discovery in traditional medicine predictive model evaluation for phm cost curves: an improved method for visualizing classifier performance sensitivity, specificity, accuracy, associated confidence interval and roc analysis with practical sas implementations. nesug proceedings: health care and life sciences evolution and emergence of infectious diseases in theoretical and real-world networks seasonality of viral infections: mechanisms and unknowns introducing vaccination against serogroup b meningococcal disease: an economic and mathematical modelling study of potential impact targeting asymptomatic malaria infections: active surveillance in control and elimination adaptive neuro-fuzzy approach for wind turbine power coefficient estimation sensitivity, specificity, and predictive values: foundations, pliabilities, and pitfalls in research and practice public health threat of new, reemerging, and neglected zoonoses in the industrialized world towards a national infectious disease information infrastructure a case study in west nile virus and botulism annual national conference on digital government research digital government society of north america editorial overview-the role of information systems in healthcare: current research and future trends participatory epidemiology: use of mobile phones for community-based health reporting measuring vaccine confidence: analysis of data obtained by a media surveillance system used to analyse public concerns about vaccines visualization and analytics tools for infectious disease epidemiology: a systematic review spatial analysis of crimean congo hemorrhagic fever in iran evolution of a location-based online social network: analysis and models a data-driven epidemiological prediction method for dengue outbreaks using local and remote sensing data a fuzzy inference map approach to cope with uncertainty in modeling medical knowledge and making decisions a fuzzy reedfrost model for epidemic spreading a mathematical model with control to analyse the dynamics of dengue disease transmission in urban colombo a medical decision support system for disease diagnosis under uncertainty a predictive method for hepatitis disease diagnosis using ensembles of neuro-fuzzy technique accurate segmentation of leukocyte in blood cell images using atanassov's intuitionistic fuzzy and interval type ii fuzzy set theory assessing airborne transmission of foot and mouth disease using fuzzy logic automating the clinical stools exam using image processing integrated in an expert system comparison of adaptive neuro-fuzzy inference system and artificial neutral networks model to categorize patients in the emergency department designing and implementation of fuzzy expert system for diagnosis of herpes zoster detecting borderline infection in an automated monitoring system for healthcare-associated infection using fuzzy logic fuzzy expert system for tropical infectious disease by certainty factor fuzzy risk assessment tool for microbial hazards in food systems intelligent curation system for blood infections using fuzzy inference system in android mathematical modelling and a systems science approach to describe the role of cytokines in the evolution of severe dengue mathematical modelling of immune parameters in the evolution of severe dengue optimal neuro-fuzzy control of hepatitis c virus integrated by genetic algorithm evaluation of pulmonary function tests by using fuzzy logic theory ebinformatics: ebola fuzzy informatics systems on the diagnosis, prediction and recommendation of appropriate treatments for ebola virus disease (evd) an experimental comparison of fuzzy logic and analytic hierarchy process for medical decision support systems a grid-based hiv expert system gfnn: gaussian-fuzzy-neural network for diagnosis of tuberculosis using sputum smear microscopic images a belief rule based expert system to assess tuberculosis under uncertainty a fuzzy expert system for distinguishing between bacterial and aseptic meningitis a genetic-neuro-fuzzy inferential model for diagnosis of tuberculosis application of evolutionary fuzzy cognitive maps for prediction of pulmonary infections supporting meningitis diagnosis amongst infants and children through the use of fuzzy cognitive mapping key: cord-336599-r8xgnz87 authors: yaacoub, jean-paul a.; salman, ola; noura, hassan n.; kaaniche, nesrine; chehab, ali; malli, mohamad title: cyber-physical systems security: limitations, issues and future trends date: 2020-07-08 journal: microprocess microsyst doi: 10.1016/j.micpro.2020.103201 sha: doc_id: 336599 cord_uid: r8xgnz87 typically, cyber-physical systems (cps) involve various interconnected systems, which can monitor and manipulate real objects and processes. they are closely related to internet of things (iot) systems, except that cps focuses on the interaction between physical, networking and computation processes. their integration with iot led to a new cps aspect, the internet of cyber-physical things (iocpt). the fast and significant evolution of cps affects various aspects in people’s way of life and enables a wider range of services and applications including e-health, smart homes, e-commerce, etc. however, interconnecting the cyber and physical worlds gives rise to new dangerous security challenges. consequently, cps security has attracted the attention of both researchers and industries. this paper surveys the main aspects of cps and the corresponding applications, technologies, and standards. moreover, cps security vulnerabilities, threats and attacks are reviewed, while the key issues and challenges are identified. additionally, the existing security measures are presented and analyzed while identifying their main limitations. finally, several suggestions and recommendations are proposed benefiting from the lessons learned throughout this comprehensive review. cyber physical systems (cps) are designated as essential components of the industrial internet of things (iiot), and they are supposed to play a key role in industry v4.0. cps enables smart applications and services to operate accurately and in real-time. they are based on the integration of cyber and physical systems, which exchange various types of data and sensitive information in a real-time manner [1] . the development of cps is being carried out by researchers and manufacturers alike [2] . given that cps and industry v4.0 offer a significant economic potential [3] , the german gross value will be boosted by a cumulative of 267 billion euros by 2025 upon the introduction of cps into industry v4.0 [4] . a cps is identified as a network of embedded systems that interact with physical input and output. in other words, cps consists of the combination of various interconnected systems with the ability to monitor and manipulate real iotrelated objects and processes. cps includes three main central components: sensors, aggregators and actuators. moreover, cps systems can sense the surrounding environment, with the ability to adapt and control the physical world [5] . this is mainly attributed to their flexibility and capability to change the run-time of system(s) process(es) through the use of realtime computing [6] . in fact, cps systems are being used in multiple domains (see fig. 1 ), and embedded in different systems such as power transmission systems, communication systems, agricultural/ecological systems, military systems [7] , [8] , and autonomous systems (drones, robotics, autonomous cars, etc.) [9] , [10] . that, in addition to medical care domains to enhance the medical services [11] . moreover, cps can be used in supply chain management to enable echo-friendly, transient, cost efficient, and safe manufacturing process. despite their numerous advantages, cps systems are prone to various cyber and/or physical security threats, attacks and challenges. this is due to their heterogeneous nature, their reliance on private and sensitive data, and their large scale deployment. as such, intentional or accidental exposures of these systems can result into catastrophic effects, which makes it critical to put in place robust security measures. however, this could lead to unacceptable network overhead, especially in terms of latency. also, zero-day vulnerabilities should be minimized with constant software, applications and operating system updates. recently, several research works addressed the different security aspects of cps: the different cps security goals were listed and discussed in [12] , [13] , [14] , [15] ; maintaining cps security was presented in [16] ; cps security challenges and issues were presented in [17] , [18] ; some of the security issues were reviewed, including big data security [19] , [20] , iot storage issues [21] , and operating system vulnerabilities [22] ; several security and privacy solutions using cryptographic algorithms and protocols were discussed in [23] , [24] . however, none of the existing works presented a comprehensive view of cps security in terms of threats, vulnerabilities, and attacks based on the targeted domain (cyber, physical, or hybrid). hence, this paper presents a detailed overview of the existing cyber, physical and hybrid attacks, and their security solutions including cryptographic and non-cryptographic ones. • cyber-physical attacks are presented in relation to the targeted cyber and/or physical system/device, and the corresponding vulnerabilities of each such domain. • risk assessment: a qualitative risk assessment method is presented to evaluate the risk and exposure levels for each cps system, while proposing suitable security countermeasures. • security measures and their limitations are discussed and analyzed, including recent cryptographic and noncryptographic solutions. • forensics solutions are also presented and discussed about securely extracting evidence and thus, to improve forensics investigations. • lessons: various lessons are learnt throughout this survey including how to protect real-time data/information communication among resource-constrained cps devices, and how to achieve protection of cps security goals such as confidentiality, integrity, availability and authentication. • suggestions & recommendations are presented about how to mitigate and overcome various cyber, physical and hybrid threats, vulnerabilities, attacks, challenges and issues for a safe cps environment. aside from the introduction, this paper is divided into six main sections as follows. section ii presents some background about cps including their layers, components, and models. section iii discusses and details the key cps threats, attacks and vulnerabilities in addition to listing and describing several real-case cps attacks, and the main persistent challenges and issues. section v assesses and evaluates the risks associated with cps security attacks, especially in a qualitative risk assessment manner. section v presents and analyzes the main cps security solutions including cryptographic, noncryptographic, and forensics ones. section vi highlights the lessons learnt throughout this study. section vii provides key suggestions and recommendations for a safe and secure cps environment. section viii concludes the presented work. in this section, we present the cps architecture, its main layers and components, as well as the main cps models. the architecture of cps systems consists of different layers and components, which rely on different communication protocols and technologies to communicate among each other across the different layers. 1) cps layers: the cps architecture consists of three main layers, the perception layer, transmission layer, and application layer, which are presented and described in fig. 2 . the analysis of the security issues at the various cps layers is based on the work in [25] . • perception layer: it is also known as either the recognition or the sensing layer [26] . it includes equipment such as sensors, actuators, aggregators, radio-frequency identification (rfid) tags, global positioning systems (gps) along with various other devices. these devices collect real-time data in order to monitor, track and interpret the physical world [27] . examples of such collected data include electrical consumption, heat, location, chemistry, and biology, in addition to sound and light signals [28] , depending on the sensors' type [29] . these sensors generate real-time data within wide and local network domains, before being aggregated and analyzed by the application layer. moreover, securing actuators depends on authorized sources to ensure that both feedback and control commands are error-free and protected [30] . generally, increasing the security level requires an endto-end encryption scheme at each layer [31] . therefore, heavyweight computations and large memory requirements would be introduced [32] . in this context, there is a need for the design of efficient and lightweight security protocols, which take into consideration the devices capabilities and the security requirements. • transmission layer: it is also known as the transport layer or network layer, and it is the second cps layer [29] . this layer interchanges and processes data between the perception and application layers. data transmission and interaction is achieved through the internet for this purpose, various protocols are used to address the increase in the number of internet-connected devices, such as the internet protocol version 6 (ipv6) [33] . this layer also ensures data routing and transmission using cloud computing platforms, routing devices, switching and internet gateways, firewalls and intrusion detection/prevention systems (ids/ips) [34] , [35] . before outsourcing data contents, it is essential to secure their transmission to prevent intrusions and malicious attacks including malware, malicious code injection [36] , denial of service/distributed denial of service (dos/ddos), eavesdropping, and unauthorised access attacks [37] . this introduces a challenge, especially for resource-constrained devices due to the imposed overhead in terms of the required processing and power resources [38] . • application layer: it is the third and most interactive layer. it processes the received information from the data transmission layer and issues commands, which are executed by the physical units including sensors and actuators [39] . this is done by implementing complex decision-making algorithms based on the aggregated data [40] . moreover, this layer receives and processes information from the perception layer before determining the rightly invoked automated actions [29] . in fact, cloud computing, middleware, and data mining algorithms are used to manage the data at this layer [41] . protecting and preserving privacy requires protecting private data from being leaked. the most known protective approaches include anonymization, data masking (camouflage) [42] , [43] , privacy-preserving, and secret sharing [31] . moreover, this layer also requires a strong multi-factor authentication process to prevent unauthorised access and escalation of privilege [44] . due to the increase in the number of internet-connected devices, the size of the generated data has become a significant issue [21] . therefore, securing big data calls for efficient protection techniques to process huge amounts of data in a timely and efficient manner [45] . 2) cps components: cps components are used for sensing information [5] , or for controlling signals (fig. 3) . in this regard, cps components are classified into two main categories: sensing components (sc) that collect and sense information, and controlling components (cc) that monitor and control signals. • sensing components: are primarily located at the perception layer and consist of sensors that collect data/information and forward them to aggregators. then, this data/information is sent to the actuators for further analysis to ensure accurate decision making. in the following, we list the main cps sensing components. -sensors: collect and record real-world data following a correlation process named "calibration", to assess the correctness of the collected data [46] . sensing data is essential since the decisions that will be made are based on the analysis of this data. -aggregators: are primarily located at the transmission layer (i.e routers, switches and gateways) to process the received data/information from sensors, before issuing the corresponding decision(s). in fact, data aggregation is based on the collected information about a specific target, where this information is gathered and summarized following a statistical analysis. online analytical processing (olap) is a prime data aggregation type used as an online reporting mechanism for processing information [46] . -actuators: are located at the application layer to make the information visible to the surrounding environment based on the decisions made by the aggregators. since actuators highly depend on other network nodes, then each action performed by the cps relies on an earlier data aggregation sequence [5] . also in terms of operations, actuators process electrical signals as input and generate physical actions as output [46] . • controlling components: are used to control signals and they play a key role in signal control, monitoring and management to achieve higher levels of accuracy and protection against malicious attacks or accidents, mainly signal jamming, noise and interference. as a result, the reliance on programmable logic controllers (plcs) and distributed control system (dcss) along with their components (i.e programmable automation controller (pac) [47] , operational technology/information tech-nology (ot/it) [48] , control loop/server [49] , and human-machine interface (hmi)/graphical user interface (gui) [50] ) has become highly essential. next, we list the different types of control systems that are used in cps systems: • programmable logic controllers (plc): were initially developed to replace hard-wired relays, and are considered as industrial digital computers that control the manufacturing processes such as robotic devices performance and/or fault diagnosis processing; hence achieving better flexibility and resiliency. • distributed control systems (dcs): are computerized control systems that allow the autonomous controllers' distribution throughout the system using a central operator supervisory control. as a result of the remote monitoring and supervision process, the dcs's reliability is increased, whilst its installation cost is reduced. in some cases, dcs can be similar to supervisory control and data acquisition (scada) systems. • remote terminal units (rtu): or "remote telemetry unit" [51] , are electronic devices controlled by a microprocessor such as the master terminal unit (mtu) [52] . unlike the plc, they do not support any control loop nor control algorithm(s). thus, making them more suitable for wireless communications over wider geographical telemetry areas. rtu's main task is to interface scada to the physical object(s) using a supervisory messaging system that controls these objects through the system's transmission of telemetry data. in fact, both rtus and plcs use a small computerized "artificial brain" (central processing unit (cpu)) to process inputs and outputs from sensing devices and pumping equip-ment [53] ; hence using ieds (intelligent electronic devices) to transmit data flow or trigger an alarm in case of any intrusion. table i presents a comparison of the common points and differences between plcs and rtus. concerning the relation between components and layers, it can be seen that sensing components are mainly deployed at the perception and transmission layers, while the controlling components are deployed at the application layer. cps models can be divided into three main types: • timed actor cps: this model focuses on the functional aspects based on behaviour and correctness, along with the non-functional aspects that are based on performance and timing. a theory was introduced in [54] with a functional and classical refinement that restricts certain behaviour set, improving efficiency while reducing complexity. the main focus is on the refinement based on the "earlier-the-better" principle since it offers the ability to identify deterministic abstractions of non-deterministic systems [55] . in fact, these time-deterministic models are less prone to state explosion problems, with the ability to derive analytical bounds easier [56] . • event-based cps: in such models, an event must be sensed and detected by the proper cps components, before the actuation decisions are made. however, individual component timing constraints vary depending on the non-deterministic system delay, which is caused by the different cps actions including sensing, actuating, communication and computing [57] . in [58] , hu et al. stated that time constraints can be handled through the use of an event-based approach, which uses cps events to ensure the system's communication, computation, and control processes. this allows the cps to be more suitable and more useful for spatio-temporal information. • lattice-based event model in [59] , the cps events are represented according to the event type, along with the internal and external event attributes. if these events are combined, they can be used to define a spatio-temporal property of any given event, while also identifying all the components that were observing the event. • hybrid-based cps model hybrid cps systems are heterogeneous systems that are made up of two distinct interactive system types, continuous state (physical dynamic systems) and discrete-state (discrete computing systems) [60] , [61] . both development and evolution depend on the response of discrete transient events represented by finite state machines, and the the dynamic behaviour represented by differential/difference equation(s) [62] . unlike other cps models, hybrid cps is interconnected via a network, which makes it prone to delays. moreover, hybrid cps systems do not support any hierarchical modeling, and are not suitable for modeling concurrent systems. hence, hybrid systems modeling challenges caused by cps were discussed by benveniste et al. in [63] . in fact, cps system network latency issues were addressed and solved by kumar et al. using a real-time hybrid authentication method [64] , while a configurable real-time hybrid structural testing for cps was presented by tidwell et al. in [65] . finally, an event driven monitoring of cps based on hybrid automata was presented by jianhui in [66] . in a similar manner to most networking systems, security services were not incorporated into cps systems by design, leaving the door open for various vulnerabilities and threats to be leveraged by attackers to launch security attacks. this is also due to the heterogeneous nature of cps devices since they operate in different iot domains and communicate using different technologies and protocols. cps security threats can be classified as cyber or physical threats, as explained below, and if combined, these can result into cyber-physical threats. 1) cyber threats: the main attention on industrial iot security was highly focused on cyber threats rather than physical threats for many reasons, as cited in [18] . this includes the electrical grid evolution into an advanced metering infrastructure (ami), which resulted into the rise of newly unknown cyber threats aside from scada vulnerabilities [67] , [68] , [69] . electronic attacks are now easier to launch from any device, unlike physical attacks that require physical presence and physical tools. moreover, the smart meter interfacing and interconnection with other meters in the near-me area network (nan) and home area network (han) increase its exposure to various remote threats. finally, electronic attacks are difficult to mitigate and overcome in the absence of the right prevention and defensive countermeasures. for further details on cyber threat intelligence, a brief survey of cps security approaches was presented in [14] . for further information about cyber security threats, more details can be found in [70] , [71] . since cyber security is not limited to a single aspect, it can be considered from different perspectives, such as: • centring information: which requires protecting the data flow during the storage phase, transmission phase, and even the processing phase. • oriented function: which requires integrating the cyberphysical components in the overall cps. • oriented threat: which impacts data confidentiality, integrity, availability, and accountability [70] . the above issues make cps systems prone to: • wireless exploitation: it requires knowledge of the system's structure and thus, exploiting its wireless capabilities to gain remote access or control over a system or possibly disrupt the system's operations. this causes collision and/or loss of control [72] . • jamming: in this case, attackers usually aim at changing the device's state and the expected operations to cause sold with plc-like features digital computers designed for output arrangements and multiple inputs electronic device controlled by a microprocessor automates electro-mechanical processes interfaces scada physical objects physical media with process, relays, motion control and networking uses supervisory system messages to control objects does support control loops and algorithms does not support control loops and algorithms immune to electrical noise, resistant to vibration low to null immunity against electrical noise and vibration suitable for local geographical areas suitable for wider geographical telemetry areas mainly iec standards wired/wireless communications damage by launching waves of de-authentication or wireless jamming signals, which would result into denial of device and system services [73] . • reconnaissance: an example of such a threat is where intelligence agencies continuously perform operations targeting a nation's computational intelligence (ci) and industrial control system (ics) mainly through a malware spread [74] . this results in violating data confidentiality due to the limitation of traditional defenses [75] , [76] . • remote access: this is mainly done by trying to gain remote access to the cps infrastructure, for example, causing disturbances, financial losses, blackouts, as well as industrial data theft and industrial espionage [77] . moreover, havex trojans are among the most dangerous malware against icss, as they can be weaponized and used as part of cyber-warfare campaign management against a nation's cps [78] . • disclosure of information: hackers can disclose any private/personal information through the interception of communication traffic using wireless hacking tools [16] , violating both privacy and confidentiality [79] . • unauthorised access: attackers try to gain an unauthorized access through either a logical or physical network breach and to retrieve important data, leading to a privacy breach [80] . • interception: hackers can intercept private conversations through the exploitation of already existing or new vulnerabilities leading to another type of privacy and confidentiality breach [72] . • gps exploitation: hackers can track a device or even a car by exploiting (gps) navigation systems, resulting in a location privacy violation [81] , [72] . • information gathering: software manufacturers covertly gather files and audit logs stored on any given device in order to sell this huge amount of personal information for marketing and commercial purposes in an illegal manner. 2) physical threats: cps systems are recently evolving into the industrial domain by introducing an advanced metering infrastructure (ami), and neighbourhood area networks (nans), along with data meter management systems to maintain the robustness of cps in industrial domains [82] . in fact, physical threats might be classified according to the following three factors: are well protected. this is due to the fact that these stations are well-manned and well-guarded based on the implementation of access controls, authorisation and authentication mechanisms such as usernames and passwords, access cards, biometrics and video surveillance. however, the main concern is related to the less protected power-generating sub-stations since transmission lines are vulnerable to sabotage attacks and disruption. in fact, smart meters are also vulnerable to a number of threats as explained in [83] . to address this problem, smart meters must be tamper-resistant by relying on outage detection or even host-based intrusion detection. however, it is almost impossible to prevent physical tampering or theft by adversaries (such as advanced persistent threats (apts)), except that it is possible to mitigate the risk and reduce its impact. • loss: the most worrying scenario is having more than a single substation failure caused by a malicious attacker. in case of a severe damage in the smart grid, a total blackout of major metropolitan areas may occur for several hours [84] . a real-case scenario includes the cascading blackout that managed to hit the u.s. on august 14th, 2003 [85] , caused by the people liberation army (pla), which is a chinese politically-motivated group [86] . • repair: it can be based on a self-healing process [87] , which is based on the ability to either sense faults or disruptions, whilst isolating the problem and sending alerts to the corresponding control system to automatically reconfigure the back-up resources in order to continuously provide the necessary service. the aim is to ensure a fast recovery in as short of a time as possible. however, critical components do suffer from either a lack or a limited backup capability. therefore, self-healing can respond faster to a severe damage. some of the threats associated with cps systems include: • spoofing: it consists of masquerading the identity of a trusted entity by a malicious unknown source. in this case, attackers are capable of spoofing sensors, for example, by sending misleading and/or false measurements to the control center. • sabotage: sabotage consists of intercepting the legal communication traffic and redirecting it to malicious third party or disrupting the communication process. for example, attackers can sabotage physically exposed cps components across the power grid, to cause a service disruption or even denial of service that leads to either total or partial blackout. • service disruption or denial: attackers are capable of physically tampering with any device to disrupt a service or to change the configuration. this has serious effects, especially in the case of medical applications. • tracking: since devices are physically exposed, an attacker can gain access to a given device, and/or even attach a malicious device or track the legal ones. in the following, we present the main cps vulnerabilities that can be targeted by the above-mentioned threats. a vulnerability is identified as a security gap that can be exploited for industrial espionage purposes (reconnaissance or active attacks). hence, a vulnerability assessment includes the identification and analysis of the available cps weaknesses, while also identifying appropriate corrective and preventive actions to reduce, mitigate or even eliminate any vulnerability [88] . in fact, cps vulnerabilities are divided into three main categories: • network vulnerabilities: include weaknesses of the protective security measures, in addition to compromising open wired/wireless communication and connections, including man-in-the-middle, eavesdropping, replay, sniffing, spoofing and communication-stack (network/transport/application layer) [89] , back-doors [90] , dos/ddos and packet manipulation attacks [91] . • platform vulnerabilities: include hardware, software, configuration, and database vulnerabilities [36] . • management vulnerabilities: include lack of security guidelines, procedures and policies. vulnerabilities occur due to many reasons. however, there are three main causes of vulnerabilities: • assumption and isolation: it is based on the "security by obscurity" trend in most cps designs. therefore, the focus here is to design a reliable and safe system, taking into consideration the implementation of necessary security services, without assuming that systems are isolated from the outside world. • heterogeneity: cps systems include heterogeneous third party components which are integrated to build cps applications. this has resulted in cps becoming a multivendor system, where each product is prone to different security problems [93] . • usb usage: this is a main cause of cps vulnerabilities, such as the case of the stuxnet attack that targeted iranian power plants, since the malware is inside the usb. upon plugging it, the malware spread across several devices through exploitation and replication. • bad practice: is primarily related to a bad coding/weak skills that lead to the code to execute infinite loops, or to become too easy to be modified by a given attacker. • spying: cps systems are also prone to spying/surveillance attacks, mainly by using spyware (malware) types that gain a stealthy access and remain undetected for years with the main task to eavesdrop, steal and gather sensitive/confidential data and information. • homogeneity: similar cyber-physical system types suffer from the same vulnerabilities, which once exploited, can affect all the devices within their vicinity, a prime example is the stuxnet worm attack on iranian nuclear power plants [94] . • suspicious employees: can intentionally or inadvertently damage or harm cps devices, by sabotaging and modifying the coding language, or granting remote access to hackers through the opening of closed ports or plugging in an infected usb/device. thus, cps vulnerabilities can be of three types, including cyber, physical, and when combined, they result into a cyberphysical threat. 1) cyber vulnerabilities: since ics heavily relies on open standard protocols including inter-control center communications protocol (iccp) [95] and transmission control protocol/internet protocol (tcp/ip) [96] , ics applications are prone to security attacks. in fact, iccp suffers from a critical buffer overflow vulnerability [89] and also lacks the basic security measures [97] . in fact, the remote procedure call (rpc) protocol [98] and icss are prone to various vulnerabilities including the stuxnet (1 & 2) [99] , [100] , [101] and duqu malware (1.0, 1.5 & 2.0) attack types [102] , [103] , [104] , gauss malware [105] , [106] , [102] , and red october malware [107] , [108] , as well as shamoon malware (1, 2 & 3) [109] , [110] , [111] , mahdi malware [112] , [113] , [114] , and slammer worm [115] . open/non-secure wired/wireless communications such as ethernet are vulnerable to interception, sniffing, eavesdropping, wiretapping and wardialing and wardriving attacks [116] , [117] , [118] and meet-in-the-middle attacks [119] . short-range wireless communications are also vulnerable, since they can be captured, analysed, damaged, deleted or even manipulated by insiders [120] . moreover, employees' connected devices to ics wireless network, if not secure, are prone to botnet, remote access trojan and rootkit attacks, where their devices will be remotely controlled by an attacker [121] . long-range wireless communications are vulnerable to eavesdropping, replay attacks, and unauthorized access attacks. yet, sql injection remains the most web-related vulnerability since attackers can access any server database without authorization through the injection of a malicious code that keeps on running endlessly once executed without the user's knowledge [122] . since many medical devices heavily rely on wireless communications, they are prone to a large number of wireless attacks including jamming, modification and replay attacks due to the lack of encryption. moreover, gps and the device's microphone are now becoming a tracking tool, allowing the identification of the target's location, or intercepting the in-car conversations through eavesdropping [13] . by default, ics relies on modbus and dnp3 protocols to monitor and send control commands to sensors and actuators. in [16] , humayed et al. stated that the modbus protocol lacks basic security measures such as encryption, authentication and authorization. this has made it prone to eavesdropping, wiretapping, and port-scan [123] , with the risk of the controller being spoofed through false data injection [124] . the dnp3 protocol is also prone to the same vulnerabilities and attacks, with one main difference which is the integration of cyclic redundancy check (crc) as an integrity measure [125] . moreover, windows server services were vulnerable to remote code execution [99] , with more attacks being achieved through the exploitation of buffer overflow vulnerabilities in any running operating system (os). moreover, power system infrastructure of smart grids is prone to the same vulnerabilities as ics, modbus and dnp3, since they are based on the same protocols. as a result, iec 61850 protocol was introduced in substations' communications, which lack security properties and are prone to eavesdropping attacks. therefore, leading to interference attacks [126] , or false information injection attacks [127] . in [128] , santamarta et al. analysed the available documentation of smart meters, and located a "factory login" account used to perform basic configurations. this gives the user full control over a smart meter and leads to power disruption, wrong decision making and targeting neighbouring smart meters within the same network. in addition, many devices are prone to battery exhausting attacks [73] . gollakota et al. [129] and halperin et al. [130] exploited the implantable cardioverter defibrillator (icd) wireless vulnerabilities through injection attacks. the authors also showed that smart cars are vulnerable to various attack types. in [131] , radcliffe, revealed another vulnerability with continuous glucose monitoring (cgm) devices being vulnerable to replay attacks. the cgm device was spoofed with the injection of incorrect values. this is due to the fact that security considerations were not made when the smart cars were designed [132] . in fact, the controller area network (can) protocol suffers from many vulnerabilities, which if exploited could result in attacks against smart cars. this will increase the likelihood of a dos attack [133] . a tire-pressure monitoring system (tpms) is also vulnerable to eavesdropping and spoofing due to the lack of encryption [134] . in addition, adaptive cruise control (acc), which forms a part of the can network can be directly exploited [13] . in fact, a well-equipped attacker is able to interrupt acc sensors' operations by adding noise or spoofing. thus, controlling the car by either reducing, increasing its speed or even causing collisions. 2) physical vulnerabilities: physical tampering may result into misleading data in cyber-physical components. in fact, physical attacks with cyber impact were studied in [135] . the physical exposure of ics components is classified as a vulnerability due to the insufficient physical security provided to these components. thus, making them prone to physical tampering, alteration, modification or even sabotage. cps field devices (i.e smart grids, power grids, supply chains etc.) are prone to the same ics vulnerabilities since a large number of physical components is exposed without physical security, making them prone to physical destruction. therefore, in [136] , mo et al. stressed on detection and prevention solutions. in [16] , humayed et al. stated that medical devices are vulnerable to physical access along with the possibility of installing malware into them, or even modifying the device's configurations, risking the patient's health. moreover, a physical access to any medical device is also a vulnerability since an attacker can retrieve the device's serial number to launch targeted attacks [131] . as listed above, cps systems suffer from various vulnerabilities making them prone to different types of attacks, which are discussed next. in this section, we present the different types of attacks that target the different aspects of cps systems, including cyber and physical ones: 1) physical attacks: physical attacks were more active in past years, especially against industrial cps systems [137] , [138] . many of these attacks were already presented in [139] . nonetheless, this paper presents a broader range of physical attack types: • infected items: this includes infected cds, usbs, devices and drives such as the case of the stuxnet worm [140] , which upon their insertion into a cyberphysical device, a covert malware is installed containing a malicious software. • abuse of privilege: this attack occurs when rogue or unsatisfied employees access the server rooms and installation areas within the cps domain. this allows them to insert a rogue usb for infection through the installation of malicious malware/code or as keystroke, or to capture confidential data. • wire cuts/taps/dialing: since communication lines including telephony and wi-fi of many cyber-physical headquarters (hqs) are still physically visible, attackers can cut the wires or wiretap into them to intercept the communicated data [117] . • fake identity: this attack occurs when attackers masquerade themselves as legitimate employees, with enough experience to fool the others. they mainly act as cleaners to gain an easier access and better interaction with other employees. a prime example of that is australia's maroochy water breach in 2000 [141] . • stalkers: these are usually legal employees who act curious (with malicious intents) by being on the shoulder of cps administrators and engineers to acquire their credentials to blackmail or sell them to other competing cps organisations. • cctv camera interception: this includes intercepting the footage of closed-circuit television cameras that are securing entry and key points within cps areas. this can be done by distorting the signals of cameras, cutting off the communication wires, deleting the footage, gaining access to the remote control and monitoring area, etc., before performing a physical attack in an undetected manner. • key-card hijacking: this includes cloning legitimate cards that are stolen from employees, or creating lookalike genuine copies to gain full/partial access and to compromise the cps domain. • physical breach: this attack requires gaining an illegal physical access to the system, mainly through a physical breach such as the case of the springfield pumping station in 2011 [142] , a backdoor such as the case of us georgia water treatment plant in 2013 [143] , or an exploited security gap such as the case of the canadian telvent company in 2012 [144] . this allows an attacker to damage and shut-down network-connected manufacturing systems and cps devices, resulting into loss of availability and productivity. • malicious third party software provider: the main purpose of this attack is to target the company's cps by compromising the legitimate "industrial control systems" software, such as the case of the georgia nuclear power plant shutdown in 2008 [145] . this includes replacing legitimate files in their repositories with a malware that will be installed to offer remote access functionalities to control or compromise a given system. • abuse of privilege: is mainly led by insiders or "whistleblowers" to perform or help perform a (cyber)-attack from within. such high privilege grants them the ability to conduct these attacks by exposing valuable knowledge on cps systems' vulnerabilities and weaknesses. this abuse of privilege can take many forms. -physical tampering: including gaining unauthorised or masqueraded authorised access to restricted areas to damage cps systems, devices, modify their operational mode, inject malicious data/information or steal confidential documents. -unauthorised activities: are based on performing suspicious tasks, such as opening/closing pumping stations, increasing/decreasing power voltage, opening closed ports, communicating with an external entity, network traffic redirection or information leakage. • social engineering: can take many deceptive forms [91] such as reverse engineering (impersonating a techysavvy), baiting (selling malicious usbs or software), tailgating (following authorised personnel) or quid pro quo (impersonating technical support teams), and is based on the art of manipulating people (either mentally or emotionally) to reveal confidential information by manipulating their emotions to gain their trust to reveal sensitive information related to a cps, plc or ics system. recently, cps systems became the new target of hackers for espionage, sabotage, warfare, terrorism, and service theft [146] , mainly as part of cyber-warfare [147] , cybercrimes [148] , [149] , (cyber)-terrorism [150] , [151] , [152] , (cyber)-sabotage [153] (such as cyber-attacks against estonia in 2007 [154] , and georgia in 2008 [155] ), or (cyber)espionage [156] , [157] . the lack of (cyber)-security revealed a serious issue with possibly drastic effects [12] , especially in countries like lebanon [158] , [159] . 2) cyber attacks: in recent years, there was a rise in the rate of cyber-attacks targeting cps and iocpt with very devastating consequences. according to current studies carried out by [160] , [161] , cps is highly prone to malicious code injection attacks [162] and code-reuse attacks [163] , along with fake data injection attacks [164] , zero-control data attacks [165] , and finally control-flow attestation (c-flat) attacks [160] . such attacks can result into a total blackout targeting cps industrial devices and systems as presented in table ii. • eavesdropping: eavesdropping includes the interception of non-secure cps network traffic to obtain sensitive information (passwords, usernames, or any other cps information). eavesdropping can take two main forms:passive by listening to cps network message transmission, and active by probing, scanning or tampering the message by claiming to be a legitimate source. • cross-site scripting: or xss occurs when third-party web resources are used to run malicious scripts in the targeted victim's web browser (mainly a targeted cps engineer, contractor, workers, etc. ) by injecting malicious coding script into a website's database. xss can achieve session hijacking, and in some cases, can log key strokes along and remotely accesses a victim's machine. • sql injection: or sqli targets cps database-driven websites to read and/or modify sensitive data, along possibly executing administrative operations such as database shutdown, especially when cps systems are still relying on sql for data management [166] . • password cracking: aim to target the authenticity of cps users [167] , [168] (mainly engineers and managers) by trying to crack their passwords using bruteforce [169] , dictionary [170] (mitigated by using key exchange [171] ), rainbow table [172] , birthday (mitigated by hashing) [173] or online/offline password guessing attacks [174] to gain access to the password database, or to the incoming/outgoing network traffic. therefore, it is important to prevent such escalation from taking place [175] , [176] . • phishing: has many types such as e-mail phishing, vishing, spear phishing or whaling that target some or all cps users (such as engineers, specialists, businessmen, chief executive officers (ceos), chief operations officers (coo), or/and chief financial officers (cfo)), through impersonation of business colleagues or service providers. • replay: includes intercepting transmitted/received packets between icss, rtus, and plcs through impersonation to cause delays that affect cps's real-time operations and affect their availability. in some cases, these intercepted packets can be modified, which would seriously hinder normal operations. • dos/ddos: dos attacks target the cyber-physical system resources and are launched from a large number of locally infected devices. ddos attacks are usually exploited by botnets, whereby a large number of infected devices simultaneously launch a ddos attack from different geo-graphical locations. dos attacks can take many forms (i.e blackhole [177] , teardrop [178] ), while ddos can take the following forms (i.e ping-of-death [179] , smurf [180] and black energy series (be-1, be-2 and be-3 [181] , [182] , [183] ), all targeting cps systems. -tcp syn flood: exploits the tcp handshake process by constantly sending requests without responding back to the server, causing the server to constantly allocate space awaiting a reply [184] . this leads to a buffer overflow and causes the cyberphysical system to crash. • malicious third party: includes software that covertly exploit data aggregation network and compromises them, mainly using botnets, trojans or worms to infiltrate information through a cps encrypted channel from an internal system (i.e plc, ics or rtu) through the reliance on trusted third party in disguise, to a botnet commandand-control server. thus, targeting cpss [185] and amis [186] . • watering-hole attack: the attacker scans for any cyberphysical security weakness. once a weakness is identified, the chosen cps website will be manipulated by a "watering hole", where a malware will delivered by exploiting the targeted cps system mainly through backdoor, rootkits or zero-day exploit [187] . • malware: is used to compromise cps devices in order to steal/leak data, harm devices or bypass access control systems. the malware can take many forms, however, the main forms that target cps are briefly listed and presented in the following. -botnets: this includes exploiting cps devices vulnerabilities to turn them into bots or zombies, mainly to conduct hardly-traceable ddos attacks (i.e ramnit (2015) [188] , mirai (2016) [189] , smominru botnet (2017) [188] , mootbot (2020) [190] , wild-pressure and victorygate (2020).) -trojan: is a disguised malware that seems legitimate and tricks users to download it. upon download, the trojan infects the device and offers a remote access to steal data credentials and monitor users activities. this also includes remote access trojans which in turn, can be used to turn a device into a bot (i.e turla (2008) [191] , minipanzer/megapanzer (2009) [192] , gh0st rat (2009) [193] , shylock (2011) [194] , coreflood (2011) [195] , darkcornet (2012) [196] , memz (2016) [197] , tinybanker (2016) [198] and banking.br android botnet (2020)). -virus: it can replicate and spread to other devices through human/non-human intervention. viruses spread by attaching themselves to other executable codes and programs to harm cps devices and steal information. -worms: spread by exploiting operating system vulnerabilities to harm host networks by carrying payloads to steal, modify and delete data, or overload to web-servers (aside stuxnet, flame and duqu, i.e acode red/code red ii (2001) [199] , nimda (2001) [200] , triton (2017 [201] )). -rootkit: is designed to remotely and covertly access or control a computer to execute files, access/steal information or modify system configurations (i.e moonlight maze (1999) [202] , and blackhole exploit kit (2012) [203] ). -polymorphic malware: constantly and frequently changes its identifiable to evade being detected to become unrecognizable against any pattern-matching detection technique. -spyware: is a malicious software covertly installed on a device without the user or authorization knowledge, for spying purposes (e.g surveillance, reconnaissance, or scanning). in fact, they can be used for future cyber-attack purposes (i.e projectsauron (2011) [204] , dark caracal (2012) [205] , red october (2013) [107] , warriorpride (2014) [206] , fin-fisher (2014) [207] , and covid-19 spyware.) -ransomware: is a malicious software that holds and encrypts cps data as a ransom by exploiting cps vulnerabilities, targeting oil refineries, power grids [208] , manufacturing facilities, medical centers and encrypting all data-backups until a ransom has been paid. a prime example of that is the siskey (2016) (2019) and ekans (2020) ransomware [211] , [212] , [213] , [214] . • side-channel: is based on the information gained from the implemented cps system such as timing information, power consumption and electromagnetic leaks that can be exploited. for this reason, some of the most infamous cyber-attacks deserve being mentioned (table ii) . moreover, for further details, you can refer to [139] . in fact, do et al. presented a much more detailed attack description as early as 1980s in [142] . however, this paper aims to classify the occurrence of these attacks as early as 2000 and based on, but not limited to, political, religious, and criminal motives. after reviewing the main cps attacks, it is essential to assess their associated risks to design the convenient countermeasures. in the next section, the risks associated with the different cps security attacks are evaluated. given the different threats, attacks and vulnerabilities that the cps domain suffers from, it is important to highlight the main failures than cps systems suffer from. these failures can either be minor (limited damage) or major (severe damage). in fact, further details can be found in [222] , where avizienis et al. presented a well-defined and detailed explanation in this regards. • content failure: means that the content of the delivered information is inaccurate, which would result into some functional system failure. content failure can be either numerical or non-numerical (i.e alphabets, graphics, sounds or colours). • timing failure: means that the timing of information delivery (transmission/receiving) is delayed or interrupted (received/transmitted too early or too late). this would affect the decision making process and may cause data management issues. • sensors failure: means that the sensors are no longer functioning properly, and would seriously hinder the decision making process due to misinformation, or bringing a cps system to a sudden halt. a similar case occurred in 2005, at taum sauk hydroelectric power station [216] . • silent failure: occurs when there is no message sent or received in a distributed system. • babbling failure: occurs when the information is delivered, causing the system to malfunction and to operate in a babbling manner. • budget failure: occurs when the cost of implementing a cyber-physical system outweighs the budget set, before ever reaching the testing level. this is mainly caused by poor planning. • schedule failure: occurs when the schedule set for planning, testing and evaluating a given cps is not achieved due to further upgrades, additional testing, or inadequacy for users needs. • service failure: occurs when having an error propagates through the service interface and affects its decision making or/and normal performance ability. this failure can either cause a partial or full cps system failure either temporarily or permanently. • consistent/inconsistent failures: a consistent failure occurs when a given service is identically perceived by all cps users. an inconsistent failure takes place when all cps users differently perceive an incorrect service (i.e bohrbugs, mandelbugs, heisenbugs and byzantine failures) [223] . evaluating risks is essential to assess the risk's economic impact of an attack on any cps system, before managing it. such management is based on assessing and analysing the risk before mitigating it, then deploying the right security measures according to the level of severity and risk impact (see fig. 4 ). risk management is implemented in order to identify, analyse, rank, evaluate, plan and monitor any possible risk through risk assessment. • identifying risks: identification is based on uncovering and recognising risks that can negatively affect a project/project outcome and describing it [224] . • analysing risks: risks likelihood and consequence must be determined once they are identified, to understand the nature of a risk. • ranking risks: risks rank is evaluated according to the risk magnitude, based on the combination of both risk likelihood and consequence in case it occurred. • evaluating risks: based on their ranks, risks are either deemed as acceptable or require serious treatment and urgent attention. • planning risks response: highest ranked risks are assessed to treat, modify and mitigate them to once again achieve an acceptable risk level. therefore, risk mitigation strategies are created, along with the deployment of preventive and contingency plans. • monitoring and reviewing risks: risks are constantly monitored, tracked and reviewed. in case of any suspicious activity, these risks are mitigated before any serious threat occurs. risk assessment is implemented to minimize the impact of a given attack [225] . in fact, risks are evaluated based on calculating the average loss in each occurring event [226] . additionally, several risk assessment methods, as well as various techniques to secure cps were revealed in [25] . in fact, since most studies are focused on securing enterprise systems in order to assess risks, security became an emerging issue that imposes a serious risk on cps [227] . as a result in [228] , [229] , lu et al. presented an adequate risk assessment method. the main security focus was based on transferring it from risk assessment, to computer risk assessment (cra), to network risk assessment (nra) with a heavy reliance on the internet [230] . asset identification: is also important, since it is a resource value that can either be tangible, or intangible that impacts daily transactions and services [231] . in fact, cps assets can be divided between cyber assets, physical assets, and cyber-physical assets. finally, since asset quantization is estimated from both direct and indirect economic losses [232] , it is important to determine the asset value (av). risk is assessed based on its possible impact on cps systems. it is divided into three main types: • high impact: in case the risk has occurred, this can result in devastating and damaging effects on cps systems. it is used to evaluate and mitigate persistent advanced threats [233] . • medium impact: in case of its occurrence, the impact is less severe. however, it also imposes a serious threat against cps. it is used to evaluate and mitigate advanced threats [234] . • low impact: in case this risk has occurred, its impact is not severe, nor has damaging effects. as a result, its impact is very limited and can be easily mitigated. it is used to evaluate and mitigate basic threats [235] . risk mitigation requires the adaptation and implementation of a well-built management strategy in addition to cyber and physical security in order to counter-espionage, theft, or/and terrorist attacks. such a mitigation model also requires, data security and protection, as well as anti-counterfeit and supply chain risk management [236] . these models should also be supported by both forensic and recovery plans. this can help in analyzing cyber-attacks whilst coordinating and cooperating with the responsible agencies to identify external cyber-attack vectors [237] . therefore, preventive, detective, repressive and corrective logical security measures can be adopted. as a result, a qualitative risk assessment table is presented (see table iii the cost of security attacks can take many forms, and the main ones are highlighted as follows: • delays: cps systems may be prone to service delays, which may affect their performance and render them inactive (blackout, burnout) until the issue is sorted either through maintenance or back up. • affected performance: system delays due to a malicious (cyber-attack)/non-malicious (accident) event can gradually affect the cps performance and cause it to operate in an abnormal manner which can seriously affect the decision making process. • additional spending: may be required to tackle the advanced persistent threat attempts and zero-day attacks, which require additional spending in terms of security protection in a defense-in-depth manner. • loss of life: can be the result of flooding, radioactivity, fire or electric shock due to hazardous or intentional acts. • disclosure of information: can affect cps businesses and business trades and put the privacy of users at risk of having their personal information being exposed. before proceeding any further, it is important to classify cps components as critical, moderate and non-critical, to identify the risk of an event occurrence (malicious/hazard) along its impact to define the proper security measures (basic, standard or advanced), as seen in fig. 5 . while adopting all possible security measures might be costly in all terms (i.e. complexity, financial cost, delay, etc.), risk management is key for selecting the convenient security solutions. in the next section, the different security solutions proposed to defend the security issues are reviewed. while these security solutions aim at preventing, detecting or correcting system damage, the cps forensics aim at knowing the system issues causes, which help in reducing and preventing future attacks. thus, the main cps forensics solutions are also reviewed. securing cps is not a straightforward task. for this reason, various existing solutions are mentioned and discussed in this section. already existing testing tools are also introduced. all of these schemes are presented to protect cps domains against attacks that target the confidentiality, integrity, availability, authentication and privacy of both data and systems as seen in fig. 6 . according to national institute of standards and technology (nist) guidelines [243] , [244] , ensuring trust between iot and cps, should consist of various multi-factors. this is due to both iot and cps systems relying on safety, security, privacy, consistency, dependability, resiliency, reliability, interaction and coordination, all of which are combined to form a well-designed and trustworthy system. if this condition is satisfied, a perfect cps mechanism is achieved. as a result, several cps testing tools were used to evaluate the security of industrial control devices upon their development (see table iv) . for further details, these tools are explained in [245] . moreover, several security certifications are also discussed, reviewed, analysed and compared according to their different aspects [245] (see table v) . in the following, the main cps security requirements are defined and discussed. • privacy: in cps, a huge data collection process is constantly taking place, and this is what most people are not aware of [256] , [257] . therefore, a person has the right to access his own data, along with being given the right to know what type of data is being collected about them by data collectors, and to whom these data is being given or sold to. however, this also requires preventing the illegal/unauthorised access to the user's personal data and their information disclosure [258] , [259] . • dependability: intelligent physical world (ipw) ensures that the cps adaptive behaviour is achieved to bring a higher dependability and ensure the right quality of service (qos) through the adoption of fault-tolerance mechanisms in a timely manner. dependability includes two other qualities, safety and reliability. safety is often an objective defined in terms of the organisation's [243] . this is due to the negative impact of cybersecurity risks, where vulnerabilities can be compromised and exploited by a hacker, or due to cps failure. hence, safety is of a high concern for iot, cps and (internet of cyber-physical things) iocpt users alike. while reliability is based on the ability to adapt to changing conditions to overcome and recover from any possible disruption either based on cyber or/and physical attacks led by adversaries, in addition to natural disasters [243] . physical systems rely on timing and proper functionality. however, in case of any possible mismatch, unreliability and uncertainty can cause problems and disruptions for cps services. therefore, maintaining a high reliability requires reducing the uncertainty levels. in fact, it is also recommended to implement error-correction algorithms to sort electronic components imperfect reliability [260] . as a result, rajamaki et al. in [260] stated that cps behaviour can be predictable through the implementation and use of artificial intelligence or/and even machine learning (ml) schemes. this allows the prediction of the so called "next-time system state". • resiliency: cps must be resilient to overcome accidents and malicious attacks. therefore, cps logical and physical systems are prone to cyber security vulnerabilities from a security aspect. this included the demonstration of carshark software tools that control a car in [133] , along with the successful design of a virus in 2010 which attacked siemens plant-control systems [261] , along with how hackers broke into the united states federal aviation administration (us faa) air traffic control system in 2009 [262] . resiliency is achieved by each cps component in a base architecture (ba) presented in [263] , where each communication and physical connection path between elements is granted access by the ba's connectors. this requires the ba system to know and identify every possible path, while overcoming any connection disruption. moreover, in case the elements were inconsistent, a multi-view editor will be deployed to make corrections. • interaction and coordination: are essential to maintain an all-time operational cps security. in [58] , hu et al. stated that cps interaction and coordination between cyber and physical system elements are a key aspect. in fact, the main physical world characteristics are based on the constant system change over time. however, the cyber world characteristics are based on sequence series with no temporal semantics. moreover, two basic approaches are presented to study and analyse this problem. these approaches are based on the "cyberizing" the physical (ctp) aspect through the introduction of cyber-properties and interfaces into physical systems, and "physicalizing" the cyber (ptc) where cyber-software components are to be represented in real-time [264] . • operational security (opsec): operational security (opsec) was introduced in 1988 to ensure physical security, information security, and personnel security [265] through careful planning, risk assessment and risk management [266] . its primary task is to ensure operational effectiveness by denying any adversary access to public/private information; hence controlling information and observable actions about a given cyber-physical system, especially in hostile environments/areas [265] . one of its key benefits is providing means to develop cost-effective security measures to overcome a given threat. to achieve this task, opsec involves five main steps: -critical information identification: includes identifying which information, if targeted, can effectively degrade a cps's operational effectiveness or place its potential organizational success at risk, and develop an initial plan to protect it. -threat analysis: includes determining an adversary's potential and capabilities to gather, process, analyze, and use the needed information. -vulnerability analysis: includes studying the weaknesses of a given cyber-physical system and the strengths of an adversary. thus, building a possible view over how a potential adversary might exploit this security gap to perform a security breach. -risk assessment: risks are assessed based on the threat and vulnerability levels combined, depending on how high or how low these levels are. risk assessment levels include evaluating the cost of implementing the right security measures by ensuring a trade-off between the effective cost and benefit balance. -appropriate application countermeasures: once the trade-off is achieved in the earlier phase, the appropriate countermeasures are then developed to offer the best protection of cps against these ongoing threats in terms of feasibility, cost, and effectiveness. • system hardening: system hardening can be used to defend a wider range of threats. therefore, it is highly recommended to isolate critical applications that lack the proper security measures, from any os that is not trusted in order to boost the iocpt and cpt security. in [267] , shepherd et al. analysed different trustcomputing technologies along with their applications in the cps domain. according to [268] , such analysis included a trusted platform module (tpm), trusted execution environments (tee), secure elements (se), and encrypted execution environment (e3), to increase the os's integrity. moreover, the authors' work in [269] has successfully achieved a higher security level in the presence of untrustworthy components. this allowed the improvement of cps by enhancing system's integrity. however, if the graph-based optimization was combined with parameters, it can provide a reasoning basis to ensure an overall system integrity [270] . therefore, it is essential to set the right privileges (task-based, role-based, rulebased, etc..) and strong password complexity policies in order to enhance the security level. moreover, this also includes getting rid of old unused accounts and open yet unused ports to reduce the exposure to remote wireless attacks. as a result, cps nature must be considered before achieving any design. in [136] , mo et al. presented a cyber-physical security by combining systems-theoretic with cyber-physical security controls. the adoption of security measures has many benefits when it comes to protecting cps components, layers and domains. however, despite these advantages, cps systems are impacted by the application of these security measures, which can be summarized as follows: • reduced performance: security measures can partially or fully affect the performance of a given cps, in the absence of careful consideration for a balanced securityperformance trade-off. this can affect normal operations and requires more human interventions to manually assign services and domains. • higher power consumption: is a serious issue, especially for resource-constrained and battery-limited cps end devices. a higher power consumption means a shorter lifespan and a higher cost to maintain their availability. • transmission delays: transmitted/received data is prone to delays due to the additional encryption process that is being added to thwart passive/active eavesdropping and sniffing attacks. despite the protective advantage that is offers, this is unacceptable in a real-time cps systems. • higher cost: higher security levels are associated with higher computational costs, which are not limited to the initial capital spending phase, but also include training, update, and operational phases. • compatibility issues: some cps systems are not compatible with the employed security measures and vice versa. this can be due to the software in-use, firmware, operating system, etc. • operational security delays: upon the deployment of any security service, there is a training phase that precedes the full operational security mode, and during which the service is temporarily ineffective or basic and thus, prone to attacks. maintaining a secure cps environment is not an easy task due to the constant increase of challenges, integration issues and limitation of the existing solutions including the lack of security, privacy and accuracy. nonetheless, this can be mitigated through different means including cryptographic and non-cryptographic solutions as seen in fig. 7 . • safety critical: in such a cps type, an attack can lead to loss of life or to chronic deadly diseases, with significant damage to the environment such as fire, floods, radioactivity (e.g. chernobyl in 1986 and fukushima in 2011) incidents [271] , [272] . • mission critical: for this type of cps, an attack can result into a fatal/non-fatal, total/partial failure of a cps to achieve its objectives [273] . • business critical: in such a cps type, an attack can result into huge financial and economic losses, damaged reputation and loss of cps contractors and clients. • security critical: for this type of cps, an attack can result into a security breach of the cyber-physical system (security gap, exploitable vulnerability, rootkits, backdoors, etc.). cryptographic measures are mainly employed to secure the communication channel from active/passive attacks, along any unauthorized access and interception, especially in scada systems [274] . in fact, traditional cryptography approaches based on utilizing ciphers and hash function are not easily applied to cps including iocpt due to power and size constraints. as a result, the main focus should be limited to data security alone, instead it should maintain and ensure the efficiency of the overall system process along. therefore, various solutions were presented. in [23] kocabas et al. conducted their own survey which was dedicated to conventional and emerging encryption schemes which could be employed to offer secure data storage and sharing. in [24] , lai et al. reviewed and discussed prominent cryptographic authentication and encryption methods [275] to secure distributed energy resources (der) systems, while providing recommendations on applying cryptography to der systems. in [276] , ding et al. presented an overview of recent advances on security control and attack detection of industrial cps, especially against denial-of-service, replay, and deception attacks. in [15] , sklavos et al. presented a tutorial that discusses the implementation efficiency of communications confidentiality, user authentication, data integrity and services availability, along attacks and modern threats with their countermeasures. many solutions were presented to maintain a secure cps environment by fulfilling its main security goals. in [277] , adam et. al. presented a novel framework to understand cyber-attacks and cps risks. their framework offers a novel approach to ensure a comprehensive study of cps attack elements, including the attacker and his objectives, cyber exploitation, control-theoretic and physical system properties. in [232] , stouffer et al. provided a comprehensive ics security guideline that is related to technical controls including intrusion detection systems (ids), access controls (ac), firewalls, and operational controls including training, awareness and personnel security. in [97] , security experts were able to gain the employees' credentials due to their lack of awareness and training, using phishing and social engineering techniques through a simulated attack. in [34] , sommestad et al. conducted a keyword mining comparison, and concluded that the main focus was either on operational controls, or technical controls only. in [278] , sharma et al. presented a novel multi-level network security evaluation scheme (nses) that represent five different levels of security. therefore, providing a holistic view over whether nses is suitable for wireless sensor networks (wsn) security for iot/cps/iocpt applications. nses offers recommendation for network administrators on early design phases to achieve the right security needs. as a result, this paper classifies these solutions in terms of them fulfilling one of the following security goals: • confidentiality: securing cps communication lines is essential. as a result, various cryptographic solutions were presented. in [279] , the authors presented a solution based on the use of compression techniques before being encrypted. their solution reduces the overhead and mitigates the problem. since, lightweight cryptography became the centre of attention with various lightweight block ciphers being presented by different authors, including an ultra-lightweight block cipher by bogdanov et al. [280] and a low-latency block cipher for pervasive computing applications [281] . this was due to their lowcost and low-latency with the ability to provide cryptographic blocks for any resource constrained, normal, industrial, or even medical devices. in [282] , shahzad, et al. suggested the installation of encryption-decryption modules at both ends of non-secure modbus communication to protect its connection from confidentiality attacks. thus, requiring an additional overhead to convert plaintexts into ciphertexts and vice versa. in [283] , the american gas association (aga) presented its aga-12 standard to provide "bump-in-the-wire" encryption services for cps, but at the expense of large latency overheard [284] . in [ [296] . ssu is complementary to the existing siem architectures, and it can transparently intercept its communication control channels along with its physical process input/output lines to constantly assess both security and operational status of plc or rtu. another approach was also presented in [297] , by asem et. al to overcome mitm, replay and command modification attacks by providing an encryption level for the transferred packets, along with the use of hardware cipher models. in [298] , cao et al. presented a layered approach with the aim of protecting sensitive data. their techniques relied on hash chains that provide a layered protection for both high and low security levels zones along with a lightweight key management mechanism. thus, preventing attackers from intercepting data from a higher security level zone. therefore, ics applications vendors should work on releasing compatible versions of their applications to ensure that the ics operators will not resort to older versions of vulnerable os [22] . their presented approach revealed the ineffectiveness of interception, injection and denial of service attacks, along with the ability of their openplc project to overcome man-in-the-middle attacks through data encryption, without interfering with its own real-time characteristics. • authentication: authentication is the first line of defense that should be well-built, designed and maintained [302] , [303] , [259] , [304] . as a result, in [130] , halperin et al. presented a public key-exchange authentication mechanism to prevent unauthorized parties from gaining access. their mechanism relies on external radio frequency rather than batteries as an energy source. in fact, out-ofband authentication were deployed in certain wearable devices, where the authentication mechanism uses additional channels including audio and visual channels [73] . on the other hand, medical cps (mcps) biometrics, including mainly heart rates and blood pressure [305] , can possibly be used to generate a key to encrypt and secure the body sensor network communication [73] . in [ • privacy preserving preserving the privacy of users' big data is not an easy task. as a result, various privacy preserving techniques were presented to solve this issue including differential privacy and homomorphic encryption. -differential privacy: limits the disclosure of private real-time big-data and information during its transmission. in [311] , keshk et al. studied the feature reduction role along privacy protection levels using independent component analysis (ica) as a technique on big power cps data. results revealed that ica is more secure without breaching confidential data and offers a better privacy preservation and data utility. in [312] , j. feng et al. presented a lightweight privacy-preserving high-order bi-lanczos scheme in integrated edge-fog-cloud architectural paradigm for big data processing. user's privacy is achieved using an homomorphic cryptosystem, while computation overheads are offloaded using privacy-preserving tensor protocols. in [313] , ye et al. presented a secure and efficient outsourcing differential privacy (dp) scheme to solve data providers issues related to being vulnerable to privacy attacks. in [314] , zhang et al. presented a practical lightweight identity-based proxy-oriented outsourcing with public auditing scheme in cloudbased mcps, by using elliptic curve cryptography to achieve storage correctness guarantee and proxyoriented privacy-preserving property. -homomorphic encryption: for a better data confidentiality and privacy protection, homomorphic encryption techniques were adopted. in [315] , zhang et al. presented a secure estimation based on kalman filtering (sekf) using a multiplicative homomorphic encryption scheme with a modified decryption algorithm to reduce network overhead and enhance the confidentiality of the communicated data. in [316] , kim et al. a fully homomorphic encryption (fhe) as an advanced cryptographic scheme to directly enable arithmetic operations on the encrypted variables without decryption. moreover, a tree-based computation of sequential matrix multiplication is introduced to slow down the decrease of the lifespan. in [317] , min et al. presented a parallel fully homomorphic encryption algorithm that supports floatingpoint numbers to achieve an efficient ciphertext operation without decryption. results revealed that the ability to limited application problems while meeting the efficient homomorphic encryption requirements in cloud computing environment. many noncryptographic solutions were also presented to mitigate and eliminate any possible cyber-attack or malicious event. this was done by implementing intrusion detection systems (ids), firewalls and honeypots. as a result, various solutions presented by various authors are mentioned and discussed. various ids methodology types are available due to the availability of different network configurations [318] . each ids methodology is characterised by its own advantages and drawbacks when it comes to detection, configuration, cost, and their placement in the network. in [268] , almohri et al. stated that various research activities were implemented to detect attacks against the cps. these attacks are split into two main models. physics-based model, which defines normal cps operations in cps through anomaly detection. cyber-based model which is used in order to recognize potential attacks as listed in [319] , [320] . in fact, existing approaches were mainly designed to detect specific attacks against specific applications, including unmanned aerial vehicles (uav) [321] , industrial control processes [322] , and smart grids [323] . in [324] , zimmer et al. exploited the possibility of a worst case execution time, through obtaining information using a static application analysis in order to detect malicious code injection attacks in cps. in [325] , mitchell et al. analysed a behaviour-rule specification-based technique to employ ids mainly in medical cps. the authors also presented the transformation of behaviour rules in a state machine, which can detect any suspicious deviation initiated from any medical device behaviour specification. -intrusion detection system placement: ids can be placed at the border router of any given iot network, in one or many given hosts, or in every physical object to ensure the required detection of attacks. simultaneously, ids may be able to generate a communication overhead between the lln (low power lossy networks) nodes and the border router due to the ids ability to frequently query the network state. in fact in [326] , zarpel at al. described three main ids placement strategies (see fig. 8 ): fig. 8 : ids structure * distributed ids: d-idss are being employed in every physical lln object, whilst being optimized in each resource-constrained node. therefore, a lightweight distributed ids was presented. in [327] , oh et al. identified a lightweight algorithm matching the attack signatures, and the packet payloads, while suggesting other techniques that require less matching numbers to detect any possible attack. in [328] , lee et al. suggested their own lightweight method that allows them to monitor a node's energy consumption by assigning nodes to monitor their neighbours in the distributed placement. these nodes are defined as "watchdogs". in [329] , cervantes et al., presented a solution called "intrusion detection of sinkhole attacks on ipv6 over low -power wireless personal area networks (6lowpan) for iot" (inti), which combines their concepts of trust and reputation with the watchdogs nodes to mainly detect and mitigate sinkhole attacks. this included the node's role possibly changing every time a network is reconfigured or an attack event has occurred. * centralized ids: c-ids is mainly deployed in centralized components. this allows all data to be gathered and transmitted by the lln to the internet across the border. therefore, centralised ids can analyse all of the exchanged traffic between the lln and the internet. in fact, it is not enough to only detect attacks involving nodes within the lln, since it is difficult to monitor each node during an occurring attack [330] . in [331] , cho et al. presented their solution which is based on analysing all the packets that pass through the border router between physical and network domains. however, the main task is based on how to overcome a botnet attack. in [332] , [333] , kasinathan et al. deployed a centralized placement that allows them to take into consideration the possibility of overcoming dos attacks, where in case of a dos attack, the ids data transmission would not be affected. in [334] , wallgren et al. employed their centralized approach which is placed in the border router to detect the attacks that target the physical domain. * hybrid ids: h-ids utilizes both concepts of centralized and distributed placements, by combining their advantages and overcoming their drawbacks. the initial approach allows the network to be organised into clusters with the main node of each cluster being able to host an ids instance before taking the responsibility for monitoring other neighbouring nodes. therefore, hybrid ids placements can be designed in order to consume more resources than a distributed ids placement. in [335] , le et al. followed the same approach, through the use of a hybrid placement using a relatively small number of "watchdogs" nodes covering the network. this offered them the ability to sniff the communication of its surrounding neighbours in order to indicate whether a node was compromised or not. therefore, reducing the communication overhead. in [336] , le et al. also managed to organize the network into smaller clusters with a cluster head for each, using the same number of nodes. this allowed an ids instance to be placed in each cluster head, with each cluster member reporting its own related information and other neighbours related information to the cluster head. in the second approach, ids modules were placed in, both the border router and other network nodes with the presence of a central component. in [337] , raza et al. presented their own ids named as svelte, where the border router hosts are given the task of processing intensive ids modules that are responsible for detecting any intrusion attempt by analysing the routing protocol low-power and lossy device's (rpl) network data. based on pongle et al.'s work [338] , network nodes were responsible for any detectable changes in their neighbourhood. moreover, network nodes were also responsible for sending information about their surrounding neighbours to their centralized module which is deployed in the border router having the main assigned responsibility of storing and analysing data. thus, making it easier to detect and intrusion while identifying attacks in their early stages. in [339] , thanigaivelan et al. presented an ids, which allocates different responsibilities to the network nodes and also to the router's border. thus, ensuring a cooperative combined work amongst them, with the ids module monitoring neighbouring nodes, detecting any intrusion attempt, and sending notifications to the ids modules. -intrusion detection methods: the four main ids methods are signature-based, anomaly-based, behaviour-based and hybrid based. in fact in [326] , these methods were presented, while testing methods and techniques were classified into five main categories, depending on their detection mechanism . * signature based: such a detection technique is very fast and easy to configure. however, it is only effective for detecting known threats. thus, showing a high weakness against unknown threats mainly polymorphic malwares and crypting services. despite its limited capability, signature based ids is very accurate, and also very effective at detecting known threats, with an easy way to understand mechanism. however, this approach is ineffective against the detection of both new and variants of known attacks, due to their matching signature remaining unknown, and constantly updating its signature patches [340] , [341] . in [327] , oh et al.'s aimed to reduce the computational cost by comparing attack signatures and packet payloads. in [342] , liu et al. presented a signaturebased ids that employs an "artificial immune system" (ais) mechanism with detectors being modelled as immune cells with an ability to classify any datagram as malicious or non-malicious according to the matching signature. such approach can evolve into the adaptation ability new conditions in new environments that are being monitored. in [332] , kasinathan et al. integrated a signature-based ids into the network framework, with the objective of being able to detect dos attacks against 6lowpan-based networks. this ids was implemented through the adaptation of "suricata4" used for 6lowpan networks, with the main objective of reducing the false alarm rate. in [333] , kasinathan et al. presented a signaturebased approach as an extension of their presented approach in [332] . * behaviour based: behaviour based can be classified as a set of rules and thresholds implemented to define the expected behaviour of the network's components including both nodes and protocols. this approach is capable of detecting any intrusion as soon as the network behaviour deviates from its original behaviour. behaviour-based acts in the same way as the anomaly-based detection with a slight difference from specification-based systems where a human expert is needed to manually define each specification rule. thus, providing a lower false-positive rate than the anomaly based detection [343] , [344] . therefore, there will be no need for any training phase, since they are implemented to operate instantly. however, such an approach is not fit for all scenarios, and may become time consuming and error prone. in [345] , misra et al. presented their new approach to protect the iot middleware from ddos attacks, by triggering an alert whenever the request number exceeds the threshold line. in [335] , le et al. presented a different specification-based approach, aimed at detecting rpl attacks [346] , by specifying the rpl behaviour through network monitoring operation and malicious action detection. in [336] , le et al.'s work was extended. their experimentation resulted in a high true-positive rate, where false positive rates were low throughout their experimentation, whilst also causing an energy overhead compared to a typical rpl network as stated in [326] . in [347] , amaral et al. presented a specification-based ids that grants the network administrator the ability to create and maintain rules in order to detect any potential attack. whenever the rule is violated, the ids would right away send an alert to the event management system (ems) that correlates these alerts for different available nodes in a given network. the success of misra et al. [345] and amaral et al. [347] approaches highly relied on the expertise of the network administrator, as well as his experience and skills combined. therefore, in case of any wrong specifications, it will cause an excessively high false-positive rate and/or a high false-negative rate, leading to a possibly serious risk that threatens the network's security. * anomaly based: this type compares system's activities instantly with the ability to generate an alert whenever a deviation from normal behaviour is detected. however, such a detection method suffers from a high false positive rate [343] , [348] , [349] . in [331] , cho et al. presented a botnet detection scheme using the anomaly-based method, by computing an average for each three metrics composing the normal behaviour profile. this was achieved before the system monitors the network's traffic and raises the alert whenever a metric violates the already defined computed averages. in [350] , gupta et al. presented their own architecture for a wireless ids, by applying the necessary computational intelligence algorithms which are used in order to a construct normal profile behaviour. moreover, a distinct normal behaviour profile will be implemented for each different ip address being assigned. in [328] , lee et al. suggested that energy consumption should be classified as parameter in order to be used in analyzing each node's behaviour. thus, defining a regular energy consumption model for each mesh-under routing scheme and route-over routing scheme, where each node will monitor its own energy consumption. in case the node deviates, the ids classifies the node as malicious and removes it. in [351] , summerville et al. successfully managed to develop a deep-packet anomaly detection approach aimed at reducing the run on resource constrained iot devices, by using a bit-pattern matching technique which performs a feature selection. in their experimental evaluation, they used internet enabled devices against four main attack types (including sqli, worms, etc..), and results have shown low false-positive rates. in [339] , thanigaivelan et al. successfully introduced an iot distributed internal anomaly detection system, that monitors the node's data rate and packet size. moreover , in [338] pongle and chavan presented an ids that is designed specifically in order to detect wormhole attacks in iot devices, in addition to presenting three main algorithms to detect network anomalies. as a result, their experiment revealed that the system has achieved a true positive rate of 94% when tested against wormhole detection, whilst scoring an 87% when it came to detecting both, the attack, and the attacker launching it. in [352] , k. demertzis et al. presented an advanced spiking one-class anomaly detection framework (soccadf) based on the evolving spiking neural network algorithm. this algorithm implements a one-class classification methodology in an innovative applicable way, due to it being exclusively trained with data to characterise normal ics operations. moreover, this algorithm can detect any divergence in behaviours and abnormalities that are associated with apt attacks. the authors stated that soccadf is highly suitable for difficult problems, and applications with a huge amount of data. according to their results, the authors stated that soccadf has a better performance at a very fast learning speed, with higher accuracy, reliability, and efficiency, and it outperforms the other approaches. * radio-frequency based: in [353] , stone et al. presented a radio-frequency based anomaly detection method for programmable logic controllers in the critical infrastructure [354] . their experimental results have demonstrated that the use of a single collected waveform response provides sufficient separability to enable the differentiation between anomalous and normal operational conditions. however, in case of using multi-time domain waveform response, their performance significantly degrades. to solve this problem, the authors presented anomaly detection method based on rf fingerprint feature retrieved from the waveform amplitude, phase, and frequency response to ensure a qualitative differentiation between an anomalous and normal operating conditions. in [355] , stone et al. also presented an rf-based methodology to detect anomalous programmable logic controller behaviours with a superior timedomain rf emissions performance. the cincinnati bell any distance (cbad) approach reached a threat agent detection and response (tadr) detection rate higher than 90% benchmark realised at an signal power ratio (snr) higher or equal to 0 db. despite these results, this approach is prone to rf noise, signal degradation and coding loops. in [356] , stephen et al. presented a timing-based side channel analysis technique to help control system operators in detecting any firmware and ladder logic programs modification to the programmable logic controllers. this approach allows a field device to be fingerprinted upon deployment to create an supplicate baseline fingerprint. various fingerprints of the device are taken and compared to the baseline in order to detect and alert operators of both intentional and unintentional modifications in programmable logic controllers. * hybrid based: it is based on using a specification-based techniques of signature-based, and anomaly-based detection in order to maximize their advantage whilst minimizing their drawbacks. in [337] , raza et al. presented a hybrid ids known as svelte which offers the right trade-off between storage cost of signature-based methods, and computational cost of anomalybased methods. in [357] , krimmling et al. tested their anomaly and signature-based ids using the ids evaluation framework that they presented. their results revealed the failure of each approach in detecting certain attacks alone. as a result, the authors combined these approaches to cover and detect a wider attack range. in [329] it is not enough to encrypt, detect and protect against passive and active attacks. in fact, aside from identifying the source of the attack, it is also important to know how the attack was performed despite of the challenges [372] . hence, there an urgent need for the forensics domain to enhance the forensics tools and techniques to retrieve and analyze logs of events that took place before, during and after the incident. in fact, cps forensic analysis is still in its early stages of development, due to the ics specialized nature along with its proprietary and poorly documented protocols [373] . in [374] , awad et al. surveyed the digital forensics applied to scada systems and covered the challenges that surround them. therefore, presenting the current state-of-the-art device and networkspecific tools. in [375] , grispos et al. presented a forensicby-design framework that ensure the integration of forensics principles and concepts in mcps. in [376] , h. al-khateeb et al. shed a light on a new approach where a blockchainbased chain-of-custody may be simultaneously established to the generated preidentified data (data of interest) by an iot device. in [377] , chan et al. described a novel security block method for detecting memory variable changes that may affect the integrity of programmable logic controllers and efficiently and effectively enhancing security and forensics. this is done by by adding monitoring and logging mechanisms to plcs. therefore, ensuring faster anomaly detection with higher accuracy, less overhead and adjustable impact. in [378] , ahmadi et al. presented a federated blockchain (bc) model that achieves forensic-readiness by establishing a digital chain-of-custody (coc) and a cps collaborative environment to qualify as digital witnesses (dw) to support post-incident investigations. in [379] , parry et al. presented a high speed hardware-software network forensics tool that was specifically designed for capturing and replaying data traffic in scada systems. experimental results guaranteed preserving the original packet ordering with improvement in data capture and replay capabilities. in [380] , cebe et al. presented a blockchain infrastructure by integrating a vehicular public key infrastructure (vpki) to achieve membership establishment and privacy along a fragmented ledger related to detailed vehicular data. moreover, identities pseudonyms were used to preserve users' privacy. in [381] , p. taveras presented a high level software application that detects critical situations like abnormal changes of sensor reads and traffic over the communication channel, mainly. therefore, helping by improving critical infrastructure protection and providing appropriate scada forensics tools for incident response and forensics analysis. in [382] , ahmed et. al. presented a testbed of three ipps (industrial physical processes) using real-world industrial equipment including plc. the authors stated that their presented testbed is useful in cyber-security, education (scada systems) and forensics research including plc analysis and programming. moreover, their testbed includes fully functional physical processes which are deemed very essential for both research and pedagogical efforts. in [383] , yau and chow presented a novel methodology which logs relevant memory address values, that are being used by programmable logic controller programs, in addition to their timestamps. this methodology can be extremely valuable in a forensic investigation in case of an ics incident. this is realized by applying machine learning techniques to the logged data in order to identify any anomalous programmable logic controller operation. in [384] saman et. al. combined symbolic execution with model checking to analyse any malicious plc code bound injection. their combined approach can also be used for forensic purposes including the identification of the areas where the code injection took place, along with which part of the code caused its execution. in [385] , mcminn et al. presented a firmware verification tool used for the forensics analysis of trials of the altered firmware codes to gain unauthorised access over ics networks. such verification is achieved either though the analysis of the plc's captured data to check whether the plc's firmware is modified or not. in [386] , kleinmann et al. presented an accurate ids that utilizes a deterministic finite automaton that models the network traffic with a 99.26% accuracy, after analysing and observing the highly periodic network traffic of siemens s7 plc. in [387] , saranyan et al. provided a comprehensive forensic analysis of network traffic generated by the pccc (programmable controller communication commands) protocol, and also presented prototype tool that extracts updates of the programmable logic and crucial configuration information. authors also stated that their proof-of-concept tool, "cutter", which is capable of parsing the content of pccc messages, extracts and presents digital artifacts in a human-readable form such as simple mail transfer protocol (smtp) configuration. moreover, the smtp configuration can be retrieved from the network log and can be parsed, too. in [377] , chan et. al. presented a novel security block method that enhances ics security and forensics by adding monitoring and logging mechanisms to plcs, and ics's key components. their results demonstrated that their approach increased the anomaly detection range, speed and accuracy with a slight performance impact and a reduced network overhead. thus, ensuring a more enhanced, efficient and effective forensic investigation procedure. in [388] , yua et al. described the design and implementation of a novel plc logging system. to overcome the inadequacy of information in forensics investigations, their logging system is used to extract data from siemens s7 communications protocol traffic. this logging system also helps in recording the evidence based on the exchanged data between the plc and other network devices. thus, providing key information about the attack source, actions and timelines. the choice of simatic s7 plc is due to their widespread use [389] and successful exploitation by insidious stuxnet malware. in [390] , chan et al. focused on the logging mechanism of a siemens plc, including the siemens total integrated automation portal v13 program (siemens tia portal, known as siemens step-7). the author's methodology performs an effective and practical forensics analysis of the plc. moreover, it focuses on siemens plc along with an installed computer workstation with the siemens tia portal (previously targeted by stuxnet). during the evaluation and analysis of the existing presented security solutions, several limitations can be deduced, presented and discussed as follows: • asymmetric cryptography: introduces overhead in terms of latency and resources. the asymmetric nature of certain cryptographic work [285] , [292] leaves cps's real-time communication prone to network latency and overhead due to delays in the encryption/decryption process. • weak device/user authentication scheme: many of the presented authentication techniques [130] , [73] , [306] , [308] are not very suitable for a secure appliance, due to the lack of multi-factor authentication schemes to protect cps systems from unauthorised users and access. • cps forensics field: are still prone to many challenges including the lack of tools, skills and responses against any potential anti-forensics activity [372] , [373] . • inefficient honeypot & deception system: despite of the recently proposed techniques in [366] , [368] , [370] , [371] , there are no appropriate honeypot techniques that can be specifically adopted to protect cps systems, especially in the wake of industry v4.0. • lack of firewall protection: firewall solutions including [358] , [359] are not very applicable and suitable for employment into the cps domain, nor they offer an effective protection. the best solution requires dynamic firewalls, as well as application and next generation firewall types. • inefficient intrusion detection systems: despite the availability of various ids types such as anomalybased [352] , behaviour-based [345] and signaturebased [333] , these are generally applied within iot-based domains and not specifically designed to protect cps systems. to secure cps, many lessons were learnt as how to maintain and achieve their required security goals. among such lessons: 1) maintaining security services: new lightweight cryptographic solutions are required to secure cps and iocpt in real-time operations but with minimum computational complexity. these cryptographic solutions can help ensure the following security services: • confidentiality: there is a need for a new class of lightweight block or stream cipher algorithms to secure cps resource-constrained real-time communications. recently, a new approach was presented, and it is based on the dynamic key-dependent cipher structure and it requires two or one iteration with few operations [391] , [392] , [393] , [394] . a set of these solutions can be applied at the physical layer [395] , [393] , [394] . • message/device integrity: this includes the protection of cps data and devices' integrity from any physical/logical alteration(s). this can be done by ensuring that the operating system, applications, and software are securely designed and without any flaws to prevent tampering, with strong cryptographic hash functions (sha256, sha384 and sha512). in this end, a new lightweight hash function was presented in [396] and it requires a single round compared to the existing ones. • device/data availability: requires the need for computational resources along with verified backups, and a self-healing ability of cps in such a way to recover immediately from availability attack types. also, maintaining data availability is as necessary [397] , and this can be done by defining a multi-secure connection [398] [403] . 2) strong device/user authentication: an efficient device/user mutual multi-factor authentication scheme is necessary,along with enhancing verification and identification phases based on attribute access-control privileges (least-privilege) to ensure non-repudiation and stronger accountability. 3) protecting digital evidences: this is highly important since most of the advanced attacks focus on eliminating any source of evidence that traces back to the attack source, such as the case of shamoon, duqu, flame and stuxnet malware types [404] , [109] , [75] . furthermore, modern digital forensics solutions should define new countermeasures to preserve digital forensics logs. 4) enhancing security policy: in many cases, cps attacks occurred by insiders (by accident or on purpose). accordingly, all employees must undergo a screening process before recruitment, and have their privileges suspended outside working hours and monitored their actions in the case of advanced tasks. this means that cps security policy should be contain new rules to limit access and to reduce the potential damage. 5) smart cooperation with non-cryptographic solutions: intrusion detection systems should be hybrid in all terms and should be coordinated in an efficient manner with firewalls and dynamic honeypot systems. 6) enforcing compliance: by respecting users' privacy through ensuring data access regulatory compliance that processes cps's big data via clouds, especially when stored by utility providers (trusted third party (ttp)) to prevent any data leakage and users privacy violations. therefore, maintaining a suitable trade-off between users privacy and systems' security and performance, while also ensuring firmer accountability measures [405] , [406] . 7) achieving trade-off: is essential for maintaining systems' availability, safety and security [407] , [408] . therefore, such a trade-off must be achieved based on the combination of these three key requirements while taking into consideration available budget and cost requirements in terms of risk assessment: • availability & safety: both features are linked together since issues related to the safety of a cps system also affect its operational availability. to ensure this trade-off, verified back-ups of computational devices must always be considered in the planning phase, as a second line of defense to handle any sudden service/system disruption (power cuts, blackouts, pumping stoppage), or maintenance (updates, renovation, installation, etc. different security measures could be adopted and enhanced to enhance the protection against various threats and attacks. these include: • prioritization & classification: of critical cps components and assets before assessing, managing and analysing risks to ensure the proper budget spending on the right choice of security measures (basic, standard or advanced) in accordance to their costs compared to the likelihood of the occurrence of a given incident and its impact. • careful financial planning & management: must be conducted in terms of available budget and needed costs/resources to protect critical/non-critical cps assets and components. • lightweight dynamic key dependent cryptographic algorithms: these solutions can be used to to ensure several security services such as message confidentiality, integrity and authentication, which are mandatory during any secure cps communications. this can be done by using new generation of cryptographic algorithms, which were presented in [392] , [409] , [410] . the advantage of these solutions that it can reach a good balance between security and performance level. the robustness against attacks were proved since a dynamic key is used per message (or a set of messages; depend of application constraints and requirements). moreover, this dynamic key is used to produce a set of cryptographic primitives and update cryptographic primitives. this means different ciphertext can be obtained for the same plaintext since different cryptographic primitives are used. while, the effectiveness is validates since these algorithms require only one round iteration and uses simple operations in addition to avoid diffusion operation. the new generation of these cryptographic algorithms reduce the required latency, resources and computation overhead, which help cps devices to preserve better their main functionalities. • defining privileges: this should be considered as the most suitable access control policy, which assigns permissions and rights depending on the users' roles/tasks/attributes when it comes to accessing cps, and removing these access rights upon completing the task or upon the employee's leave. this also includes the use of the least privilege policy. therefore, the definition of privilege should be done based on attribute based access control (abac), where policies combined with attributes specify access authorizations. note that abac makes access control decisions based on boolean conditions of attribute values. it provides a high level of granularity, which is necessary to make cps control access scheme more secure. • strong entity multi-factor authentication: unfortunately, entity authentication schemes that are based on a single factor of authentication (you have, you know, you do or you are) are not resistant enough against authentication attacks, which are increasingly becoming more dangerous. the first line of defense in any system is the entity authentication scheme since any entity authentication attack can lead to confidentiality, integrity and/or availability attack. recently, the concept of multi-factor authentication was applied by combining two or more factors: 1) "you are" which includes device fingerprint, user fingerprint, hand geometry, iris scan, retina scan, etc., and 2) "you have" which includes cryptographic keys to increase its robustness against authentication attacks such as the ones described in [411] , [412] . this mechanism should be an essential requirement in cps systems, in addition to the use of the geographical location. the advantage of these solutions is their ability to reduce false positives, and to complicate the authentication attacks since several factors should be broken instead of one. consequently, this limits the access only to authorised entities and personnel (devices/users). • strong password & dynamic hashing process: passwords are considered as the "you know" authentication factor. however, several attacks such as rainbow and hash table attacks can be applied. in order to prevent them from occurring, after a periodic interval, passwords must be re-hashed with a new dynamic nonce for each user. moreover, a secure cryptographic hash function should be used such as sha-3 and sha-2 (variant 512). this avoids birthday attacks and reduces rainbow/hash table attacks. • secure and protected audit: can be done by using an audit manager system that collects and stores logs in a distributed system. a possible solution that can be applied in this context was presented recently in [413] . this limits any insider attempt against a cyber-physical system and it preserves the digital evidence of internal and external attacks to trace them back. • enhanced non-cryptographic solutions: require the need for hybrid ids/ips systems or ai-based ids/ips (using machine learning algorithms), along with advanced firewalls (i.e application and next generation firewalls) [414] , and dynamic honeypots [415] to prevent any future security breach based on a vulnerability exploit. this can be done by employing lightweight ids/ips and especially the anomaly-based ones. in fact, one should select the anomaly detection algorithm according to the cps device constraints, which can be statistical for limited ones or based on machine algorithm, such as random forest, for powerful cps devices. on the other hand, signature-based techniques can be applied at the gateway (gw) where all network traffic can be analyzed. • secure & verified backups: this is essential to maintain the cps data availability and to avoid data destruction or alteration by ensuring robustness against dos/ddos and ransowmare attacks, especially that such attacks may result in total blackouts as in the case of the us. this can be done by using lightweight data protection solutions such as the ones presented in [399] . • forensic efforts: are essential to retrieve the traces of any occurring attack. also, new solutions against antiforensic techniques should be introduced to preserve any digital evidence [413] . this is realized by recovering logs and monitoring network and system behaviour, which can successfully limit various reconnaissance attempts. however, the newly introduced forensics tools must be compatible with different cps devices' software/hardware, especially resource constrained devices, and must also be resistant against anti-forensics attempts. • enhanced incident response: includes the ability to identify, alert and respond to a given incident. moreover, incident recovery and incident investigation plans should be put in place to mitigate attacks. this provides protection against non-intentional technical and operational failures (power shortage, blackout) through backup plans, and from intentional failures (cyber-attacks), through cert (computer emergency response) [416] , csirt (computer security incident response) [417] , and ircf (incident response and computer forensics) teams [418] , [419] . as such, cps scientists and engineers must undergo further education and training to ensure an enhanced and efficient cyber, physical and computational environment with secure computing and communications. • real time monitoring: running real-time systems using specialised forensics or non-forensics tools and methods is essential to prevent any cyber-physical system accidental or non-accidental failure. this enables constant checking and monitoring of cps devices' behaviour and hence, the detection of any cyber-attack attempt in its early stages. • security check: and employee screening must be done for each employee before and during the job to eliminate and contain any possible insider/whistle-blower attempt. therefore, signing agreements [420] such as non-disclosure agreement (nda), confidentiality agreement (ca), confidential disclosure agreement (cda), proprietary information agreement (pia) or secrecy agreement (sa) is highly recommended. such security checks are essential especially in critical areas such as nuclear power plants [421] . • periodic employee training: includes periodic awareness training of ics and plc employees on the best cyber-security practices based on their level and knowledge, with the ability to detect any suspicious behaviour or activity. moreover, employees must be trained over various security threats and wrong practices such as avoiding the installation of any software update, how to counter social-engineering and phishing attempts, while also maintaining accountability in case of wrong doings. • periodic pen testing & vulnerability assessment: must be maintained in a periodic manner to enforce system auditing, detecting threats, and mitigating them in a real-time manner before they are discovered and exploited by an attacker under the zero-day exploit conditions. • periodic risk assessment: must also be enforced to study the likelihood and impact of a given risk against a critical/non-critical cyber-physical system based on a qualitative or/and quantitative risk assessment and a cost-benefit analysis (cba), to classify the risk based on acceptable/non-acceptable level and to mitigate it as early as possible. • up-to-date systems: cyber-physical systems must be kept up-to-date in terms of software, firmware and hardware through constant verified patches and updates [422] . moreover, such systems must be secured at different levels of their implementations (layered protection), with the ability to mitigate and tackle a given attack to reduce its impact and prevent further escalation and damage. furthermore, usb ports must be physically and logically removed to prevent any payload injection, and plc systems behaviour and activities must be constantly monitored for any suspicious/abnormal behaviour [422] . • ai security solutions: artificial intelligence is used in ids/ips anomaly detection schemes or in "you are" or "you do" entity authentication schemes. in fact, ai is now being considered as a game-changing solution against a variety of cyber-physical attacks targeting cps systems, devices and communication points. despite the time consuming process of training an ai system, the accuracy of detection and prevention are much higher than any human intervention. recent advancements in machine learning, and especially in deep learning, can make cps systems more secure, robust and resistant against cyber-physical attacks. • defense in-depth: most of the existing solutions offer protection against a single attack aspect or a security requirement. instead, there is need for a multi-purpose security solution that ensures the best protection at each operational layer (perception, transmission and application) of cps. for example, the two most known international standards for functional safety in the automotive industry, the iso 26262 [423] and iec 61508/edition2 [424] , [425] should be respected and applied. this ensures a safe cps implementation based on the functional safety, which includes the safety integrity level (sil) basics [426] which in turn, rely on the probability of failure on demand (pofod) and the risk reduction factor (rrf) to ensure a much more accurate and efficient hazard and risk analysis (hra) [426] , [424] , mainly in the electronic control units (ecu) [427] , [428] ). • cps security & privacy life-cycle: finally, to sum up this work, our paper presents a combined operational and functional safety/security (ofss) life-cycle that ensures a successful and safe cps employment as seen in fig. 9 ). this framework is derived from iso 26262 and iec 61508/edition2 protocols and their approach towards ensuring the cps functional safety/security. the framework consists of six main phases: -phase 1: devising a plan to design a cps system by following a well-defined time-table and schedule in accordance to the needed budget and corresponding costs. this also requires the assistance of humans (businessmen, engineers, workers, etc.) and nonhuman assets (vehicles, machines, etc.). -phase 2: requires a careful risk and hazard analysis, which consists of a proper risk management and asset classification, as well as the mutual connection between the two to ensure an accurate decision-making over the adoption of the right security measures/counter-measures. -phase 3: defines the right functional safety, security and dependability requirements along their key components/mechanisms that are essential to mitigate a risk/hazard and to reduce their likelihood and impact in case of their occurrence. -phase 4: consists of evaluating the performance of cps in terms of the recently introduced functional safety, security and dependability measures in an operational manner where a performance management and analysis will be conducted to ensure a proper/mutual security-performance, safetyperformance and dependability-performance tradeoffs. -phase 5: once the performance is evaluated, the cyber-physical system is tested and validated to detect any remaining software/hardware bug, security gap, or performance issue to apply the required modifications before being commissioned. if the testing is unsuccessful, the process restarts again to find where the issue took place. if successful, the cps will head towards further commissioning before being officially deployed. -phase 6: upon successful testing, the deployed cps system will undergo a trial phase to evaluate its operational status, while monitoring its behaviour and performance before becoming fully operational. cps systems are key components of industry v4.0, and they are already transforming how humans interact with the physical environment by integrating it with the cyber world. the aim of implementing cps systems, either within or outside iot (iocpt), is to enhance the products' quality and systems' availability and reliability. however, cps systems suffer from various security and privacy issues that can degrade their reliability, safety, efficiency, and possibly hindering their wide deployment. in this paper, we first overview all components within cps systems and their interconnections including iot systems, and we focus on the main cps security threats, vulnerabilities and attacks, as related to the components and communication protocols being used. then, we discuss and analyze the recently available cps security solutions, which can be categorized as cryptographic and non-cryptographic solutions. next, we highlight the important lessons learnt throughout, and accordingly, we present suggestions and recommendations with respect to the various security aspects, services, and best practices that must be put in place to ensure resilient and secure cps systems, while maintaining the required performance and quality of service. acknowledgement this paper is supported with funds from the maroun semaan faculty of engineering and architecture at the american university of beirut. a cyber-physical systems architecture for industry 4.0-based manufacturing systems industry 4.0: a survey on technologies, applications and open research issues predictive manufacturing system-trends of next-generation production systems industry 4.0: huge potential for value creation waiting to be tapped. deutsche bank research cascading data corruption: about dependencies in cyber-physical systems: poster ninth ieee international symposium on object and component-oriented real-time distributed computing autonomic computing technologies for cyber-physical systems smart monitoring of potato crop: a cyber-physical system architecture model in the field of precision agriculture industrial and medical cyber-physical systems: tackling user requirements and challenges in robotics role of cyber security in drone technology securing internet of medical things systems: limitations, issues and recommendations situation management, and impact assessment ii; and visual analytics for homeland defense and security ii a survey of remote automotive attack surfaces. black hat usa a brief survey of security approaches for cyberphysical systems cryptography and security in internet of things (iots): models, schemes, and implementations cyberphysical systems security-a survey challenges and research directions for heterogeneous cyber-physical system based on iec 61850: vulnerabilities, security requirements, and security architecture. future generation computer systems cyber-physical systems and their security issues a survey of security and privacy in big data a survey of security and privacy in big data a survey on internet of things: security and privacy issues survey of scada security challenges and potential attack vectors emerging security mechanisms for medical cyber physical systems cryptography considerations for distributed energy resource systems cyber physical systems security: analysis, challenges and solutions internet of things (iot) security: current status, challenges and prospective measures securing physical processes against cyber attacks in cyber-physical systems a survey on the internet of things security future internet: the internet of things architecture, possible applications and key challenges identity-based key agreement and encryption for wireless sensor networks security of the internet of things: perspectives and challenges security of distributed, ubiquitous, and embedded computing platforms. wiley handbook of science and technology for homeland security research on the architecture of internet of things scada system cyber security-a comparison of standards scada-specific intrusion detection/prevention systems: a survey and taxonomy cyber security in power systems protecting industrial control systems from electronic threats expanding gate level information flow tracking for multilevel security the design of ics testbed based on emulation, physical, and simulation (eps-ics testbed) cyber 32 security for cyber physcial systems: a trust-based approach security architecture on the trusting internet of things camouflage: automated anonymization of field data data masking system and method cyber-physical systems: a security perspective lightweight security solutions for the internet of things internet of things (iot): a vision, architectural elements, and future directions. future generation computer systems time synchronization of automation controllers for power applications an open and flexible cybersecurity training laboratory in it/ot infrastructures method and apparatus providing remote reprogramming of programmable logic devices using embedded jtag physical layer and protocol sustainable and flexible industrial human machine interfaces to support adaptable applications in the industry 4.0 paradigm automated remote telemetry paging system guide to supervisory control and data acquisition (scada) and industrial control systems security. national institute of standards and technology remote terminal industrial control communication system the earlier the better: a theory of timed actor interfaces bundle: a group-based programming abstraction for cyber-physical systems context-sensitive synthesis of executable functional models of cyber-physical systems co-simulation framework for design of time-triggered cyber physical systems robust cyber-physical systems: concept, models, and implementation. future generation computer systems a concept lattice-based event model for cyber-physical systems the algorithmic analysis of hybrid systems hybrid system modeling and autonomous control systems cyber-physical systems modeling based on extended hybrid automata hybrid systems modeling challenges caused by cyberphysical systems anuradha annaswamy, kai lampka, and lothar thiele. a hybrid approach to cyberphysical systems verification towards configurable real-time hybrid structural testing: a cyber-physical system approach event driven monitoring of cyber-physical systems based on hybrid automata vulnerability assessment of cybersecurity for scada systems scada systems and their vulnerabilities within the smart grid: can they be defended from a cyber attack vulnerability analysis of network scanning on scada systems in power and energy society general meeting-conversion and delivery of electrical energy in the 21st century smart grid security technology comprehensive experimental analyses of automotive attack surfaces sok: security and privacy in implantable medical devices and body area networks leveraging information security and computational trust for cybersecurity deconstructing flame: the limitations of traditional defences a survey scada of and critical infrastructure incidents security and privacy challenges in the smart grid an evaluation of cyber threats to industrial control systems security and privacy for implantable medical devices challenges and research directions in medical cyber-physical systems automotive system security: challenges and state-of-the-art intelligent substation automation systems for robust operation of smart grids ieee innovative smart grid technologies-asia (isgt asia) petri net modeling of cyber-physical attacks on smart grid securing the electricity grid. the bridge blackout in the united states and canada: causes and recommendations, us-canada power system outage task force cyberwar: taking stock of security and warfare in the digital age realizing the smart grid imperative risk management and critical infrastructure protection: assessing, integrating, and managing threats, vulnerabilities and consequences a taxonomy of cyber attacks on scada systems backdoors and holes in network perimeters cyber security of water scada systems-part i: analysis and experimentation of stealthy deception attacks the myths and facts behind cyber security risks for industrial control systems in quest of benchmarking security risks to cyber-physical systems cyber attack: a dull tool to shape foreign policy smart grid technologies: communication technologies and standards transmission control protocol/internet protocol (tcp/ip) packet-centric wireless point to multi-point (ptmp) transmission system architecture scada security in the light of cyber-warfare rpc: remote procedure call protocol specification version 2 reliability of exploits and consequences for decision support stuxnet worm impact on industrial cyberphysical system security lessons from stuxnet the cousins of stuxnet: duqu, flame, and gauss duqu 2.0: a comparison to duqu duqu: a stuxnet-like malware found in the wild gauss malware: nation-state cyber-espionage banking trojan related to flame, stuxnet stuxnet-risk & uncertainty in the first salvo of global cyber warfare. the sais red october and its reincarnation analysis and triage of advanced hacking groups targeting western countries critical national infrastructure: apt28, red october, and regin the middle east under malware attack dissecting cyber weapons saudi arabia's response to cyber conflict: a case study of the shamoon malware incident cybercrime and analysis of laws in kingdom of saudi arabia world war c: understanding nation-state motives behind today's advanced cyber attacks the role of malware in reported cyber espionage: a review of the impact and mechanism the trajectory of iran's nuclear program inside the slammer worm an ontology for network security attacks cyberattacks on scada systems war, peace, or stalemate: wargames, wardialing, wardriving, and the emerging market for hacker ethics a meet-in-the-middle attack on 8-round aes integrating physical and cyber security resources to detect wireless threats to critical infrastructure wireless vulnerability of scada systems scada security: a new concerning issue of an inhouse egat-scada an experimental investigation of malware attacks on scada systems rose tsang. cyberthreats, vulnerabilities and attacks on scada networks attack taxonomies for the modbus protocols evaluating electricity theft detectors in smart grid networks cyber security in the smart grid: survey and challenges here be backdoors: a journey into the secrets of industrial firmware they can hear your heartbeats: non-invasive security for implantable medical devices pacemakers and implantable cardiac defibrillators: software radio attacks and zero-power defenses hacking medical devices for fun and insulin: breaking the human scada system securing vehicles against cyber attacks experimental security analysis of a modern automobile security and privacy vulnerabilities of in-car wireless networks: a tire pressure monitoring system case study cyber/physical security vulnerability assessment integration cyber-physical security of a smart grid infrastructure cyber-physical attacks and defences in the smart grid: a survey secure estimation and control for cyber-physical systems under adversarial attacks cyber-security incidents: a review cases in cyber-physical systems stuxnet malware and natanz: update of isis december 22 lessons learned from the maroochy water breach security of scada systems against cyber-physical attacks. ieee aerospace and electronic systems magazine fbi probes georgia water plant break-in on terror concern telvent hit by sophisticated cyber-attack, scada admin tool compromised cyber incident blamed for nuclear power plant shutdown securing the smart grid: next generation power grid security cyber-physical systems: an overview of design process, applications, and security cyber threats impacting critical infrastructures weapons against cyber-physical targets risk of terrorism to cyber-physical and organizational-societal infrastructures future of all technologies-the cloud and cyber physical systems cybercrime and risks for cyber physical systems: a review an exceptional war that ended in victory for estonia or an ordinary e-disturbance? estonian narratives of the cyber-attacks in 2007. the institute ecole supérieure en informatique electronique et automatique cyber attacks on estonia: short synopsis. double shot security russian operational art in the russo-georgian war of malware classification for cyber physical system (cps) based on phylogenetics cybercrime and risks for cyber physical systems 2019 does lebanon possess the capabilities to defend itself from cyber-theats? learning from estonia's experience.(c2019) cyber warfare awareness in lebanon: exploratory research c-flat: control-flow attestation for embedded systems software towards automated dynamic analysis for linux-based embedded firmware code injection attacks on harvard-architecture devices return-oriented programming: systems, languages, and applications targeted attacks on teleoperated surgical robots: dynamic model-based detection and mitigation data-oriented programming: on the expressiveness of non-control data attacks data management issues in cyber-physical systems cyber-physical attacks: a growing invisible threat privilege escalation attacks on android a study of passwords and methods used in brute-force ssh attacks fast dictionary attacks on passwords using time-space tradeoff extended password key exchange protocols immune to dictionary attack ioannis papaefstathiou, and charalampos manifavas. fast, fpga-based rainbow table creation for attacking encrypted mobile communications hash function balance and its impact on birthday attacks guess again (and again and again): measuring password strength by simulating password-cracking algorithms preventing privilege escalation xmandroid: a new android evolution to mitigate privilege escalation attacks black hole attack in mobile ad hoc networks denial of service attack and classification techniques for attack detection analysis of ping of death dos and ddos attacks smurf-based distributed denial of service (ddos) attack amplification in internet threat analysis of blackenergy malware for synchrophasor based real-time control and monitoring in smart grid blackenergy-what we really know about the notorious cyber attacks blackenergy malware used in ukraine power grid attacks resisting syn flood dos attacks with a syn cache taking control: design and implementation of botnets for cyber-physical attacks with cpsbot short-term risk assessment of botnet attacks on advanced metering infrastructure smart factories in industry 4.0: a review of the concept and of energy management approached in production based on the internet of things paradigm botnet protocol inference in the presence of encrypted traffic ddos in the iot: mirai and other botnets the social roles of bots: evaluating impact of bots on discussions in online communities regin, an old but sophisticated cyber espionage toolkit platform reconsidering human dignity in the new era building a gh0st malware experimentation environment anonymity vs. trust in cybersecurity collaboration a coreflood investigation of nanofluid enhanced oil recovery to catch a ratter: monitoring the behavior of amateur darkcomet rat operators in the wild how cybercriminals can abuse chat platform apis as c&c infrastructures it threat evolution in q1 2016 global routing instabilities triggered by code red ii and nimda worm attacks nimda worm analysis triton: the first ics cyber attack on safety instrument systems cyber security: the lifeline of information and communication technology blackhole exploit kit: rise & evolution. malware research team technical paper how port security has to evolve to address the cyber-physical security threat: lessons from the sauron project cyber threat report big game hunting: the peculiarities in nation-state malware research the smartphone who loved me: finfisher goes mobile how cyber-attacks in ukraine show the vulnerability of the us power grid jigsaw: an investigation and countermeasure for ransomware attacks current trend, challenges, and research directions ransomware in industrial control systems. what comes after wannacry and petya global attacks? comprehensive survey on petya ransomware attack protection model of pcs of subway from attacks type «wanna cry»,«petya» and «bad rabbit» iot ransomware attacks: detection, prevention and cure slammer worm crashed ohio nuke plant network overview of the taum sauk pumped storage power plant upper reservoir failure electricity grid in us penetrated by spies. the wall street journal infiltrating critical infrastructures with nextgeneration attacks. fraunhofer institute for secure information technology (sit) ukraine claims hackers caused christmas power outage challenges and threats of ukraine's national cyber security in hybrid war the ransomware "petya" as a challenge to the cybersecurity of ukraine, main factors of spreading this virus in the focus of ukraine, the steps taken by the authorities to combat this phenomenon and suggest ways to improve such activities using experience of other countries. phd thesis, victor zhoghov the ransomware "petya" as a challenge to the cybersecurity of basic concepts and taxonomy of dependable and secure computing fault-tolerant distributed cyber-physical systems: two case studies challenges for securing cyber physical systems risk assessment of information and communication systems-analysis of some practices and methods in the electric power industry adaptive fault-tolerance faulttolerance for cyber-physical systems threat modeling for security assessment in cyberphysical systems a new multilevel framework for cyber-physical system security an analysis of cyber physical system security theories cyber-physical system risk assessment an impact review on internet of things attacks guide to industrial control systems (ics) security. nist special publication the big four-what we did wrong in advanced persistent threat detection classification of security threats in information systems type of security threats and it's prevention stress test scenario: sybil logic bomb cyber catastrophe. cambridge risk framework series cyberphysical systems: the next computing revolution a study on secure shell (ssh) protocol hardware designs for security in ultra-low-power iot systems: an overview and survey new security perspectives around byod comments of the sierra club historical analysis of us onshore hazardous liquid pipeline accidents triggered by natural hazards cyber-physical systems in manufacturing analysis of cyber security for industrial control systems security testing methods and techniques of industrial control devices achilles -the world's first man-in-the-middle web security tool dark reading. breakingpoint unveils firestorm cyber tomography implementation of the canfd protocol in the fuzzing tool bestorm codenomicon defensics finds risks that lurk in your protocols mu studio performance suite peach fuzzing platform. peach fuzzer unraveling scada protocols: using sulley fuzzer an introduction to spike, the fuzzer creation kit. presentation slides) exida certification -iec 61508, iec 61511, iec 62443, iso 26262 isasecure -iec 62443-4-2 -edsa certification pu-abe: lightweight attribute-based encryption supporting access policy update for cloud assisted iot mohamed mohamed, and giovanni russello. c-absc: cooperative attribute based signcryption scheme for internet of things applications information security and innovation; guide to secure technology innovation initiatives data security and privacy preservation in cloud storage environments based on cryptographic mechanisms resilience of cyber-physical system: a case study of safe school environment virus attacks siemens plant-control systems hackers broke into faa air traffic control system an architectural approach to the design and analysis of cyber-physical systems state estimation in spatially distributed cyber-physical systems: bounds on critical measurement drop rates operational security management in violent environments risk assessment and risk management: review of recent advances on their foundation secure and trusted execution: past, present, and future-a critical review in the context of the internet of things and cyber-physical systems on threat modeling and mitigation of medical cyber-physical systems process authentication for high system assurance security optimization of dynamic networks with probabilistic graph modeling and linear programming environmental consequences of the chernobyl accident and their remediation: twenty years of experience. report of the chernobyl forum expert group 'environment' sti/pub/1239, 2006, international atomic energy agency radioactivity release from the fukushima accident and its consequences: a review ensuring safety, security, and sustainability of mission-critical cyber-physical systems cryptographic protection of scada communications part 1: background, policies and test plan restricted authentication and encryption for cyber-physical systems a survey on security control and attack detection for industrial cyber-physical systems a multi-layered and kill-chain based security analysis framework for cyber-physical systems haytham elmiligi, and musfiq rahman. network security evaluation scheme for wsn in cyber-physical systems trustworthiness of medical devices and body area networks present: an ultra-lightweight block cipher prince-a low-latency block cipher for pervasive computing applications real time modbus transmissions and cryptography security designs and enhancements of protocol sensitive information aga-12, part 2 performance test results. pacific northwest national laboratories revisiting a watermark-based detection scheme to handle cyber-physical attacks secure and efficient communication in cyber-physical systems through cryptography and complex event processing continuous analytics on geospatial data streams with wso2 complex event processor solving the grand challenge using an opensource cep engine logarithmic encryption scheme for cyber-physical systems employing fibonacci q-matrix vehicular ad hoc networks (vanets): status, results, and challenges a comprehensive survey on vehicular ad hoc network lightweight attribute based encryption scheme for mobile cloud assisted cyberphysical systems secure pub-sub: blockchain-based fair payment with reputation for reliable cyber physical systems postquantum enabled cyber physical systems design and implementation of a security framework for industrial control systems improving network security monitoring for industrial control systems sans scada and process control security survey on plc network security a layered encryption mechanism for networked critical infrastructures on the interdependence of reliability and security in networked control systems attacks against process control systems: risk assessment, detection, and response embedding encryption and machine learning intrusion prevention systems on programmable logic controllers proud: verifiable privacy-preserving outsourced attribute based signcryption supporting access policy update for cloud assisted iot applications. future generation computer systems pcs, a privacy-preserving certification scheme cloud data storage security based on cryptographic mechanisms secure key-exchange protocol for implants using heartbeats physical layer security for wireless implantable medical devices multi-factor authentication in cyber physical system: a state of art survey an authentication scheme with identity-based cryptography for m2m security in cyber-physical systems plc access control: a security analysis advanced key-management architecture for secure scada communications elena sitnikova, and benjamin turnbull. privacy-preserving big data analytics for cyber-physical systems practical privacypreserving high-order bi-lanczos in integrated edge-fog-cloud architecture for cyber-physical-social systems secure and efficient outsourcing differential privacy data release scheme in cyber-physical system identity-based proxy-oriented outsourcing with public auditing in cloud-based medical cyber-physical systems secure kalman filter state estimation by partially homomorphic encryption encrypting controller using fully homomorphic encryption for security of cyber-physical systems a privacy protection-oriented parallel fully homomorphic encryption algorithm in cyber physical systems intrusion detection system and intrusion prevention system: a comparative study unearthing stealthy program attacks buried in extremely long execution paths a sharper sense of self: probabilistic reasoning of program behaviors for anomaly detection with context sensitivity adaptive intrusion detection of malicious unmanned air vehicles using behavior rule specifications limiting the impact of stealthy attacks on industrial control systems cyberphysical system security for the electric power grid time-based intrusion detection in cyber-physical systems behavior rule specification-based intrusion detection for safety critical medical cyber physical systems a survey of intrusion detection in internet of things a malicious pattern detection engine for embedded security systems in the internet of things a lightweight intrusion detection scheme based on energy consumption analysis in 6lowpan detection of sinkhole attacks for supporting secure routing on 6lowpan for internet of things intrusion detection systems for wireless sensor networks: a survey management enabling the future internet for changing business and new computing services: 12th asia-pacific network operations and management symposium denial-of-service detection in 6lowpan based internet of things an ids framework for internet of things empowered by 6lowpan routing attacks and countermeasures in the rpl-based internet of things specification-based ids for securing rpl from topology attacks a specification-based ids for detecting attacks on rpl-based network topology svelte: realtime intrusion detection in the internet of things real time intrusion and wormhole attack detection in internet of things distributed internal anomaly detection system for internet-of-things intrusion detection system: a comprehensive review computer and information security handbook research on immunity-based intrusion detection technology for the internet of things a survey of intrusion detection techniques for cyber-physical systems a survey of intrusion detection systems in wireless sensor networks a learning automata based solution for preventing distributed denial of service in internet of things internet of things: a survey on enabling technologies, protocols, and applications policy and network-based intrusion detection system for ipv6-enabled wireless sensor networks an introduction to intrusion-detection systems guide to intrusion detection and prevention systems (idps). nist special publication computational intelligence based intrusion detection systems for wireless communication and pervasive computing networks ultralightweight deep packet anomaly detection for internet of things devices a spiking one-class anomaly detection framework for cyber-security on industrial control systems radio-frequency-based anomaly detection for programmable logic controllers in the critical infrastructure security for critical infrastructure scada systems. sans reading room detecting anomalous programmable logic controller behavior using rf-based hilbert transform features and a correlation-based verification process using timing-based side channels for anomaly detection in industrial control systems integration and evaluation of intrusion detection for coap in smart city applications research of paired industrial firewalls in defense-in-depth architecture of integrated manufacturing or production system on the use of open-source firewalls in ics/scada systems argus: an orthogonal defense framework to protect public infrastructure against cyber-physical attacks real time failure prediction of load balancers and firewalls a partition-driven integrated security architecture for cyber-physical systems the use of deception techniques: honeypots and decoys. handbook of information security towards high-interaction virtual ics honeypots-in-a-box rethinking the honeypot for cyber-physical systems honeybot: a honeypot for robotic systems investigation of cyber crime conducted by abusing weak or default passwords with a medium interaction honeypot defense strategies against network attacks in cyber-physical systems with analysis cost constraint based on honeypot game model conceal: a strategy composition for resilient cyber deception-framework, metrics and deployment a novel architecture for cyber-physical security in industrial control networks deception-as-defense framework for cyber-physical systems scada systems: challenges for forensic investigators programmable logic controller forensics tools, techniques, and methodologies: a survey of digital forensics for scada systems medical cyber-physical systems development: a forensicsdriven approach blockchain for modern digital forensics: the chain-of-custody as a distributed ledger enhancing the security and forensic capabilities of programmable logic controllers federated blockchain-based tracking and liability attribution framework for employees and cyber-physical objects in a smart workplace a network forensics tool for precise data packet capture and replay in cyberphysical systems block4forensic: an integrated lightweight blockchain framework for forensics applications of connected vehicles scada live forensics: real time data acquisition process to detect, prevent or evaluate critical situations a scada system testbed for cybersecurity and forensic research and pedagogy detecting anomalous programmable logic controller events using machine learning detecting industrial control malware using automated plc code analytics a firmware verification tool for programmable logic controllers accurate modeling of the siemens s7 scada protocol for intrusion detection and digital forensics scada network forensics of the pccc protocol a forensic logging system for siemens programmable logic controllers exploiting siemens simatic s7 plcs forensic analysis of a siemens programmable logic controller lightweight, dynamic and efficient image encryption scheme one round cipher algorithm for multimedia iot devices. multimedia tools and applications a physical encryption scheme for low-power wireless m2m devices: a dynamic key approach. mobile networks and applications an efficient ofdm-based encryption scheme using a dynamic key approach a survey on ofdm physical layer security erdia: an efficient and robust data integrity algorithm for mobile and wireless networks an efficient secure storage scheme based on information fragmentation erss-rlnc: efficient and robust secure scheme for random linear network coding preserving data security in distributed fog computing secure and resilient scheme for data protection in unattended wireless sensor networks additively homomorphic encryption and fragmentation scheme for data aggregation inside unattended wireless sensor networks a simple approach for securing iot data transmitted over multi-rats design and realization of efficient & secure multi-homed systems based on random linear network coding on the feasibility of online malware detection with performance counters idbased user-centric data usage auditing scheme for distributed environments security sla based monitoring in clouds aligning cyber-physical system safety and security security analysis on cyber-physical system using attack tree efficient & secure cipher scheme with dynamic key-dependent mode of operation lightweight stream cipher scheme for resource-constrained iot devices lightweight multifactor mutual authentication protocol for iot devices secure and lightweight mutual multi-factor authentication for iot communication systems distlog: a distributed logging scheme for iot forensics improving network security: next generation firewalls and advanced packet inspection devices a dynamic honeypot design for intrusion detection the human and computer as a team in emergency management information systems computer security incident response team development and evolution guide to integrating forensic techniques into incident response incident response & computer forensics the importance of having a non-disclosure agreement how to measure employee reliability plc code-level vulnerabilities assessing automotive functional safety microprocessor with iso 26262 hardware requirements introduction and revision of iec 61508 introduction to iec 61508 assessing and computing the safety integrity level (sil) for turbo machinery protection electronic control unit us patent 9,036,026. declare the following financial interests/personal relationships which may be considered as potential competing interests: jean-paul yaacoub is a master student in the department of electrical and lebanon. hassan noura is a research associate in the department of electrical and computer engineering at the american university of beirut (aub), lebanon. nesrine kaaniche is lecturer in cybersecurity and expert in cryptographic solutions at the university of salford, school of computing ali chehab is a professor in the department of electrical and key: cord-290448-oeeg754b authors: shah, darshil; sahu, dipit; kini, abhishek; bagaria, vaibhav title: improvising the surgical helmet system for aerosol-generating procedures in the or: surgeon designed 3d printed mould for augmented filtration system date: 2020-09-24 journal: j clin orthop trauma doi: 10.1016/j.jcot.2020.09.030 sha: doc_id: 290448 cord_uid: oeeg754b 1. background: the aim of this paper is to describe the process of designing and developing a mould for filter placement via 3d printing on top of the surgical helmet. this mould was designed to affix a filter material on top of the helmet system for use during the covid 19 pandemic. 2. method: the authors performed 3d scanning of the stryker surgical helmet (stryker t5, ref 400–610, us patents 6,973,677:7,753,682) and created a negative template of the top of the helmet. a mould for filter placement was printed and fitted onto the top of the surgical helmet. this construct was tested to evaluate the surgeon's comfort, aerosol filtration efficiency etc. 3. result: the helmet provided adequate comfort, showed no evidence of staining on spill test and the filter passed the industry filtration efficiency standards. 4. conclusion: the 3d printed mould is an inexpensive, efficient, and comfortable design to augment personal protection ability of the stryker helmet system. this process can be extrapolated to 3d print templates for other surgical helmets. orthopaedic procedures are traditionally considered aerosol-generating procedures (agp) [1, 2] . reaming, lavage, and use of saw ( figure 1 ) have all shown to produce aerosols that vary in size from 0.7 -5 microns [1, 3] surgical helmet systems are routinely used by arthroplasty surgeons to reduce the peri-operative infection rates and were also used as personal protective equipment [2, 3, 4] . however, their use during covid 19 pandemic [5] is not recommended by the manufacturers and the orthopaedic community [6, 7] . while anesthesia patient safety foundation recommended that healthcare workers wear an n 95 mask with a face shield while performing agp [8] , there are no clear guidelines in orthopaedics on how to augment the protection while performing agp. many orthopaedic surgeons tend to rely on the use of powered air-purifying respirators (papr) to augment the standard personal protection attire [9] . these papr draw air through a hepa (high efficiency particulate air) or n95 grade equivalent filter and create a positive pressure environment inside the suit so that the air inside it is filtered and devoid of any virus-containing aerosol [10] . these papr systems are expensive and cumbersome to use [11] . surgical helmets, on the other hand, are readily available in the operating room and familiar to use for most orthopaedic surgeons. if improved and tested for efficacy, these may serve as a viable, easy to use, and inexpensive alternative to papr. the process of 3d (three dimensional) printing is pivotal for creating prototypes and newer designs for industrial use from aircraft to healthcare equipment in a short period. these 3d printed prototype designs can then be used for testing and subsequently improvised for enduse. orthopaedic surgeons too have been routinely using rapid prototyping also known as 3d printing in a varied clinical setting [12] . these include creating bio-model for surgical j o u r n a l p r e -p r o o f planning and simulation, designing customised jigs and cutting blocks, and for designing implants. over the years, engineers have collaborated with orthopaedic surgeons to enhance the patient outcome. this paper describes the process of designing and applying 3d printing technology to enhance the functionality of the surgical helmet system for use during the pandemic period. i. design & deliberation process: when the reports and warnings on the use of the surgical helmet system were issued [13] , the authors embarked on a design process that would facilitate the optimal use of the helmet system. as users of these helmet systems for almost 2 decades, it was ascertained that modification of the system will be a valuable addition to the personal protection armamentarium. the designers thoroughly researched the mechanism of action of n95 masks and hepa filters. this enabled better understanding about the process of aerosol generation, the filtration mechanism of the filters and how the same can be used for augmenting the helmet. the filters could act by blocking the aerosol thereby preventing their concentration under the surgical hood and toga. equipped with the information, cut sheets of n 95 were affixed on top of the helmet system with adhesive tape and their comfort evaluated while operating in a simulated environment. the comfort of the construct was ensured and a template for the mould was designed. j o u r n a l p r e -p r o o f the initial design was a plain rectangle sheet of acrylonitrile butadiene styrene (abs) plastic frame, 3d printed, to match the size of the fan grill located at the top of the helmet. however, the fit of this design was not adequate and demonstrated gaps and leakage at the periphery. to have a press-fit seal, this design was subsequently modified as follows. (figure 2) ii. scanning the helmet system and getting a negative template: a 3d scanner, einscan pro 2x by shining 3d (hanghzou, china) was used to scan the helmet and obtain a standard tessellation language (stl) file of the helmet. a negative template of this file was created using 3d slicer software v 4.10.2 [14] . an appropriately fitting and locking mould template was cartographically created on blender 3d v 2.9 (blender institute, general public license) iii. generation of cad model: the process of creating this cad model was very similar to those used to create patient-specific jigs for knee arthroplasty. the surgeons and engineers worked on the blender 3d, which is also used for creating the jigs for trauma cases. the only difference being that the input files were not generated from ct (computed tomography) scan or dicom (digital imaging & communications in medicine) images but through the data obtained from scanning of the helmet. the process is described in figure 3 . iv. printing process: once the cad file was virtually inspected and tested for fit, and locking mechanism -it was then made printer ready. the cad model was converted into stl file format which was exported for printing. two types of printers i.e. fused deposition j o u r n a l p r e -p r o o f 5 modelling (fdm) and selective laser sintering (sls) were tried. the quality and finish of the sls printer was superior and subsequent prints were taken using this technology ( figure 4) highlight of the design: the highlights of design include 1. customized 3d printed design that accurately fits the stryker helmet system (michigan, usa). 2. the cad process is standardized and can be used for any helmet systems. 3. unique snap locking mechanism created by generating negative grooves that lock on the grid of the fan system eliminating the need for any additional locking mechanism. 4. a unique dual frame mechanism that allows change of the inner filters periodically. the key to the filtration system is the type of filter used. niosh certified n95 material was procured from magnum health & safety pvt. ltd. (thane, india) and cut to the appropriate size. the unique dual frame mechanism ensures a complete fit. these filters are disposable and as per manufacturer's instruction was to be replaced after 40 hours. as a matter of abundant precaution, it was recommended that they are changed after each positive or suspected positive covid case. tests: to verify the appropriateness and efficacy of the system, the following tests were conducted: i. surgeon comfort: 10 surgeons were asked to wear the helmet for 2 surgeries each and ask to rate their comfort after wearing this modified helmet system. their response to the use was registered as a) felt no difference b) felt that airflow is decreased c) felt suffocated ii. spill test: while not completely relevant here as the system would be mounted on the top of the helmet system where the blood products or spill was unlikely to reach, this test was conducted to complete the standard testing protocol for filtration devices. a coloured liquid was sprayed from distance 2, 3, and 5 feet and any spill as assessed by visualizing for any colour or wetting of the inner surface of the filter was assessed at 30 secs and 3 min. iii. filtration efficacy for the filter: standard tests were used for measuring the efficacy of the filter for 0.3-micron particle size. these particles were generated using the aerosol generator with sodium chloride aerosol. the lowest filter efficiency or highest penetration data point were recorded. the condensation particle counters (cpc) measured the particle concentration. ii. surgeon comfort: 20 responses were recorded from 10 surgeons who used the system in the operating room. 19 responses were 'a 'and one response was 'b'. 9 of 10 surgeons reported that they felt no difference, one surgeon felt that in one of the two surgeries the flow had decreased, however, he did not experience suffocation. iii. spill test: 2ml of blue ink was sprayed using a 20 cc syringe from a distance of 2 feet. the wettability of the inner surface was visually inspected and manually felt. there was no staining at 30 secs and 3 min. the filtration efficiency of the filter was tested on tsi 8130 filter tester using sodium chloride as an aerosol (0.3 micron particle size), passed at the rate of 85 litres per minute ( figure 5 ). the test was performed on three samples, all of which had a filtration efficiency consistently above the permissible limit of 95% with less than 24mm of water breathing resistance (table 1) . there are several risks in performing surgeries that lead to the production of aerosols [1, 15] . in orthopedics nearly, all procedures can be classified as aerosol-generating as most involve the use of the drill, saw, and use of pulse lavage system. although unproven, it was speculated that the use of positive pressure theatres, space suits, and diathermy during these procedures increased the risks of the spread of infection to healthcare professionals [13] . while most emergency surgeries were performed, the elective surgeries were deferred. with pandemic showing signs of plateauing and abating, the surgeons have started to resume elective procedures [16, 17] . balancing patient care and ensuring healthcare personnel safety is of prime importance in the current scenario. the proposed solution of augmenting the surgical helmet system will be an added layer of safety for healthcare professionals including orthopedic surgeons, scrub nurses, and anesthesia teams. the use of rapid prototyping technique by orthopedic surgeons for pelvi-acetabular fractures, spinal deformities, and complex peri-articular fixation is well known [12] . as familiarity with the technology grew, surgeons collaborated with the engineers to design patient-specific jigs for percutaneous fixation, osteotomies, and joint replacement [18] . the unique design of this design is the anatomical fit over the grid of the existing helmet system and the ease with which filter can be changed. modifications of helmet system exist [21] , however, the simplicity of our design and cost -effectiveness will enable mass production of the augmented system which can be easily modified to fit helmet systems of other companies. moreover, since only the n 95 filter needs to be changed periodically, the modification is financially sustainable. we were able to 3d print this augmented system for [21] is required in our system. there is no need to sterilise our system, whereas the modification done by erickson et. al. may require sterilisation of the tubes as they can easily contaminate surgical gowns of fellow assistant/nurse. as per our knowledge, no similar fixture has been described in the literature. using a negative mould to generate an exact mirror print is a standard prototyping technique and has been harnessed for producing this design. a two-frame snapfit design makes the changing of filter an easy task for the operator. this may be especially relevant when operating on suspected or proven covid positive patients. in such a scenario, the filter can be discarded and the rest of the unit may be disinfected along with the mould template in a standard fashion. while this particular design process was for the stryker j o u r n a l p r e -p r o o f extended to all kinds of commercially available helmet systems. evaluating the efficiency and safety of any new product design is paramount to ensure its widespread intended usage. there has been a lot of debate about the particle filtration efficiency standards for these devices. several studies have shown that this is not the same as bacterial filtration efficiency (bfe) or virus filtration efficiency (vfe). a virus is submicron in size averaging 0.025 microns and thus much smaller than bacteria which are around 3 microns. while intuitively it may seem that to be effective the filter must prevent sub microns from entering the hood system but this is not the case. the virus cannot survive on their own and for them to be transmitted they need to be suspended in the aerosolized droplet [19, 20] . studies have shown that most aerosols are less than 5 microns in size [1] . the particle efficiency studies done on this construct have revealed a 95.24 % reduction in the particle count. the tests conducted on the filter were as per norms laid down by the cdc (center of disease control) [23] . most surgeons were initially apprehensive to use this system fearing it would compromise their comfort. most healthcare workers have reported discomfort and excessive sweating with the use of the recommended ppe and the surgeons felt the same apprehension. however, the study conducted to evaluate surgeon comfort found that in 19 of 20 instances, the operator did not find any discomfort or difference from their usual practice. only in one instance did the surgeon experience mild discomfort not amounting to the need for its removal while performing the procedure. moreover, after submission of this manuscript, the augmented filtration system has been distributed amongst five tertiary care hospitals in the city. over 200 surgeries have been performed by approximately 50 surgeons, using this system. no surgeon had a negative feedback with j o u r n a l p r e -p r o o f respect to the breathing discomfort/ excessive sweating etc, thereby adding strength to our test result findings. this modified helmet system has the potential of being used not only by the orthopedic surgeons while performing agp but also by any frontline healthcare professionals. these include anesthetists, scrub nurses, and intensivists. as a part of the comfort test, anesthetists and scrub nurses used it and reported in favour. while not explicitly tested in other subgroups, it will likely be adapted for its comfort and enhanced safety. despite its unique design, interdisciplinary collaboration, and successful efficiency and safety testing, the modified system does have its limitations. the efficiency of the system is dependent on the type of filter used and the end-user must ensure that this is sourced from a certified agency. furthermore, the parameters used to measure 'surgeon comfort' were subjective in nature and was difficult to quantify. given the nature of the epidemic and the urgent need for adaption of this safety measure, a lengthier multi-centric study with multiple operators would have been difficult. however, for long term use, a larger study with a variety of healthcare professionals is recommended. the 3d printed mould for filter placement is an inexpensive, efficient, and comfortable design to augment the personal protection of the stryker helmet system. interdisciplinary collaboration for innovation seems to be the way forward in solving some common problems during these uncommon pandemic times. particle sizes of infectious aerosols: implications for infection control. the lancet respiratory medicine covid-19 coronavirus: recommended personal protective equipment for the orthopaedic and trauma surgeon risk of conjunctival contamination in total joint arthroplasty utilization of an orthopedic hood as personal protective equipment for intubation of coronavirus patients: a brief 3d printing of face shields during covid-19 pandemic: a technical note does a surgical helmet provide protection against aerosol transmitted disease? acta orthopaedica surgical helmet systems" or "body exhaust suits" affect contamination and deep infection rates in arthroplasty? a systematic review helmet modification to ppe with 3d printing during the covid-19 pandemic at duke university medical center: a novel technique field-testing method for loose-fitting powered air-purifying respirators equipped with hepa filters surgical helmets and sars infection intelligent operating room (inor) for orthopaedic surgery use of rapid prototyping and three-dimensional reconstruction modeling in the management of complex fractures perioperative considerations in urgent surgical care of suspected and confirmed covid-19 orthopaedic patients: operating room protocols and recommendations in the current covid-19 pandemic 3d slicer as an image computing platform for the quantitative imaging network eye protection in orthopaedic surgery: an in vitro study of various forms of eye protection and their effectiveness orthopaedic surgical selection and inpatient paradigms during the coronavirus (covid-19) pandemic resuming arthroplasty: a well aligned and a balanced approach in the covid-19 era a paradigm shift in surgical planning and simulation using 3dgraphy: experience of first 50 surgeries done using 3d-printed biomodels transmission of covid-19 virus by droplets and aerosols: a critical review on the unresolved dichotomy virus transmission during orthopedic surgery on patients with covid-19 -a brief narrative review helmet modification to ppe with 3d printing during the covid-19 pandemic at duke university medical center: a novel technique laboratory performance evaluation of n95 filtering facepiece respirators, 1996. mmwr weekly 3d printing from diagnostic images: a radiologist's primer with an emphasis on musculoskeletal imaging-putting the 3d printing of pathology into the hands of every physician the authors confirm they have no conflicts of interest. key: cord-004995-5jmjejbp authors: hunt, hamish c.; wilkinson, james s. title: optofluidic integration for microanalysis date: 2007-09-11 journal: microfluid nanofluidics doi: 10.1007/s10404-007-0223-y sha: doc_id: 4995 cord_uid: 5jmjejbp this review describes recent research in the application of optical techniques to microfluidic systems for chemical and biochemical analysis. the “lab-on-a-chip” presents great benefits in terms of reagent and sample consumption, speed, precision, and automation of analysis, and thus cost and ease of use, resulting in rapidly escalating adoption of microfluidic approaches. the use of light for detection of particles and chemical species within these systems is widespread because of the sensitivity and specificity which can be achieved, and optical trapping, manipulation and sorting of particles show significant benefits in terms of discrimination and reconfigurability. nonetheless, the full integration of optical functions within microfluidic chips is in its infancy, and this review aims to highlight approaches, which may contribute to further miniaturisation and integration. the convergence of microfabrication technologies, novel materials systems, and techniques for chemical and biochemical analysis is enabling the realisation of the lab-ona-chip (loc) or micro-total analysis system (ltas) (manz et al. 1990 ). this research is driven by the demand for fast, low-cost, automated chemical analysis using minimal sample and reagent volumes, in a multiplicity of applications (demello 2006) , including interrogation of individual molecules (craighead 2006) . the scale of integration, low cost and robustness of microfabrication approaches enabled the ubiquitous presence of the mobile phone, hand-held computer and ccd camera, to name a few, in the last century. it is often suggested that products incorporating loc technology will find similarly widespread use early in this century, for example in personal medicine, food safety, water management and security. cost and reliability for use by non-specialist personnel will be key issues in achieving this goal, which could advance personalised preventative medicine and public safety, and improve efficiency in food and water use. as its name suggests, the loc requires integration of many of the tools found in the (bio)chemistry laboratory. these may include means for sample and reagent presentation such as channels, pumps and valves, chambers for mixing and reaction, heaters for reaction control, devices for trapping, separation and selection, devices for cell lysis, and for chemical analysis (el-ali et al. 2006 ). the last decade has seen rapid growth in demonstrations of these functions in chips fabricated using either ''hard lithography'' in semiconductors or glass or ''soft lithography'' (xia and whitesides 1998) in polymers such as pdms. the former builds upon the functionality of silicon mems and the enormous infrastructure of the microelectronics industry and points to its example of cost reduction in complex electronic systems. the latter exploits the benefits of improved compatibility with biological systems and the simplicity of rapid prototyping in polymers. the emergence of preferred materials for the loc will depend upon the degree to which components can be standardised and selected for integration to satisfy large-volume applications. optical approaches are the most widely used for chemical analysis in microsystems, as they have an excellent track-record in chemical analysis, usually show the lowest limits of detection (lechuga 2007 ) and provide the greatest chemical or morphological information on the species being analysed. more recently there has been increasing interest in using optical forces as a non-contact means for trapping and separation in microsystems, and for driving pumps. the use of light for ''optoporation'' of cells in microsystems has also been demonstrated (soughayer et al. 2000) . users of microfluidic systems for (bio)chemical analysis are familiar with conventional optical systems for chemical analysis and increasingly with the optical manipulation approaches first demonstrated by ashkin (1970) . however, the majority of optical functions demonstrated in microfluidic systems use external microoptical systems, or hybrid embedding of optical fibres, which, while providing much flexibility, do not offer the robustness, stability, operator-independence, and potential for mass manufacture of fully integrated approaches. integrated optics, first proposed by miller (1969) , allows the construction of optical circuits in or on planar substrates, guiding and processing light in dielectric cores of elevated refractive index defined using photolithography. the original motivation for this innovation was in optical telecommunications, but its application to chemical sensing was soon demonstrated in 1974 (mitchell 1977) , where bilirubin in neonatal whole blood was measured in a waveguide evanescent field. integrated optics is widely used in biosensing and chemical sensing research, but there have been rather few demonstrations in true microfluidic systems. until recently, most integrated optical circuits consisted of only one or two devices, but the field appears to be maturing with a drive for standardisation of materials, devices and interfaces, allowing much denser integration (smit et al. 2007) , and proposals for photonic circuit foundries which will allow ''fab-less'' researchers with design and measurement tools to access high-quality device fabrication. the confluence of integrated optics with integrated fluidics in the future loc shows great potential. many excellent reviews of microfluidics (erickson and li 2004) for cell manipulation and analysis (yi et al. 2006a) , ltas (dittrich et al. 2006) , optical manipulation (dholakia and reece 2006) , and optofluidic devices for non-chemical applications (psaltis et al. 2006) , have recently been published, and this paper does not set out to revisit these topics comprehensively. instead, the present review focuses on recent advances in the field of full optofluidic integration for (bio)chemical analysis in microsystems, where optical functions have been incorporated in a microfluidic system, and especially where the demonstration of an optical function appears to lend itself to an integrated approach. a key loc function is the isolation and trapping of individual entities such as biological cells to allow detailed analysis, and separation of cell types among large populations according to size, for example, before analysis. the most common approaches employ hydrodynamic trapping (khademhosseini et al. 2005) , switched electro-osmotic flow (fu et al. 1999) or dielectrophoresis ), but configurations, which employ light have advantages in terms of increased flexibility of liquid medium composition (wang et al. 2005 ) and excellent discrimination (hart et al. 2006) . the principles and stateof-the art of optical manipulation are extensively reviewed in (dholakia and reece 2006) . in short, the forces at work may be broadly described as the ''radiation pressure'' (rp) forces driving particles in the direction of light propagation and the gradient forces, similar to those exploited in dielectrophoresis (dep), driving particles of higher refractive index than the surrounding medium to the region of maximum optical intensity. thus in a weakly focussed (or weakly diffracting) beam, a particle is drawn onto the beam axis and propelled along it, while in a tightly focussed beam or optical tweezer (ashkin et al. 1986 ), the forwarddirected rp forces may be balanced by the backwarddirected gradient forces to trap the particle near the focus, as rapid diffraction leads to a large intensity gradient towards the focus. while optical forces have been used to trap and cool atoms in vacuum, particles of diameters from a few tens of nanometres to ten microns or so can be stably trapped in liquid samples using typical optical components. in this section the use of freely propagating light, evanescent fields and photonic landscapes in microfluidic devices are reviewed, with emphasis on the potential for integration of the optical functions. optical manipulation using externally focussed beams in microfluidic systems sheds light on the potential applications, advantages and disadvantages of integrated approaches. the use of longitudinal optical propulsion for chinese hamster ovary cell sorting using radiation pressure was demonstrated by buican et al (1987) as the ''basis of a miniaturized and automated cell biology laboratory''. terray et al. (2005) exploited optical propulsion in a weakly focussed beam combined with opposing fluid flow in a microfluidic system to achieve optical separation or chromatography (imasaka 1998) of bacillus anthracis spores and mulberry pollen, dependent upon particle size and refractive index. this was followed, using the configuration shown in fig. 1 , by separation of b. anthracis and b. thuringiensis (hart et al. 2006 ) and sample concentration in which every cell of the selected type flowing in the channel was sorted (hart et al. 2007) . another approach to microfluidic optophoresis was demonstrated by zhang et al. (2004) , in which reduction of cell flow velocity due to a single-beam trap crossing the channel was used to identify cancerous and non-cancerous cells by time-of-flight, with detection through scattering from a probe laser beam. tightly-focussed single beam traps are particularly suited to subwavelength particles (svoboda and block 1994) and have been exploited for the study of the scattering spectra of individual trapped silver nanoparticles of order 100 nm diameter, yielding information on their morphology-dependent resonances (prikulis et al. 2004 ). an array of single beam traps generated using a vcsel array was used to optically capture and manipulate in three dimensions a 2 · 2 array of red blood cells, and independently release them (flynn et al. 2002) . further biological applications of this system are described by ozkan et al. (2003) , where two implementations of optical diversion of polystyrene microspheres between two outputs of a microfluidic junction under electro-osmotic flow are demonstrated, using the scattering force or the gradient force, respectively. wang et al. (2005) then built upon the latter and demonstrated optical switching in a microfluidic fluorescence-activated cell sorter (facs) citing improved buffer medium compatibility when compared with electrokinetic and dielectrophoretic methods. throughput of gfpexpressing hela cells was up to 100 cells/s and recovery rates were [85%. no evidence of cell stress was observed in the recovered populations. stable trapping is particularly important when acquiring weak signals such as raman spectra, as it allows improved noise reduction. ramser et al. (2005) employed a single beam trap in an electro-osmotically controlled microfluidic system to observe raman spectra of individual erythrocytes during the oxygenation cycle. the trapping beam entered from one side of the flow channel, while the raman excitation and collection was configured on the opposite side of the channel, as shown in fig. 2 , allowing independent optimisation. the oxygenation cycle was clearly observed, but the high-intensity trapping illumination at a wavelength of 1,064 nm was observed to accelerate transfer of hb to methb, and lack of this deleterious effect at a trapping wavelength of 830 nm was noted. neuman et al. (1999) conducted detailed multiwavelength measurements and showed that wavelengths of 830 and 970 nm are preferred for minimising cell damage. buican et al. (1987) noted that a counterpropagating dual-beam trap may be preferred over the single-beam trap in practical cell manipulation, and this was emphasised by jess et al. (2006) who realised a dual beam fibre trap [first demonstrated by constable et al. (1993) ] for raman spectroscopy of cells in a simple microfluidic system, yielding localised raman spectra from within primary trapped human keratinocites. the divergent fields result in a large area trap and allow large objects such as cells to be held and manipulated with greater positional control and reduced risk of photodamage when compared with conventional optical tweezers. an elegant application of a dual beam fibre trap to study the deformability of cells as a new biological marker for disease was demonstrated by guck et al. (2005) , and is illustrated in fig. 3 . the trap captures a cell in a microflow channel, centres it in the channel, and stretches the cell towards the opposing fibres to an extent dependent upon the incident power. the power dependent deformation, observed on a ccd camera, depends upon the cell rigidity, and may be used to distinguish between normal and cancerous human breast epithelial cells, for example. fig. 1 optical chromatography glass micro-flowcell; a construction of the microfluidic device showing the pathway for fluid and the laser beam focused on the channel, b illustration of a separation, in which sample particles are constrained to the focal point of the beam by the size of the separation channel that is filled by the laser beam (hart et al. 2007) . reproduced with permission from the optical society of america fig. 2 schematic of the experimental apparatus where a raman spectrometer is combined with the optical tweezers in a doublemicroscope configuration (ramser et al. 2005) . reproduced with permission from the royal society of chemistry microfluid nanofluid (2008) 4:53-79 55 2.2 two-dimensional separation a 100-lm long line-trap, rather than a point trap, has been generated by focussing a diode laser bar in a microflow channel (applegate et al. 2004) . bovine red blood cells were captured by the line trap, which was placed at an angle to the flow direction, and the cells were conveyed along it before being released at its end, thereby deflecting cells of a certain size; time-dependent interruption of the beam may be used to sort cells into different fluidic output channels. subsequently, optical waveguides written into a block of fused silica placed under the microfluidic system were used to excite fluorescence in line-trapped dye-doped polystyrene microspheres, allowing fluorescent-based activation of sorting (applegate et al. 2006) . the use of a potential landscape or optical lattice (korda et al. 2002; macdonald et al. 2003) , where interference produces an array of traps has been employed to selectively deflect monodisperse 2 lm protein microcapsules from a externally pumped stream of polydisperse particles, and to sort erythrocytes from lymphocytes, in a simple microflow system (macdonald et al. 2004) . this approach has the advantage of flexibility in tailoring the potential barriers and thus the interconnection of traps for particle sorting. paterson et al. (2005) observed that the requirement for external pumping of the fluid could be removed by breaking the symmetry of the lattice, for example by tilting the potential, and milne et al. (2007) demonstrated simultaneous separation of four sizes of silica sphere between 2 and 7 lm into parallel output flows using an acoustooptically generated potential landscape, demonstrating simple programmable reconfigurability of the landscape. further approaches to deflecting particles dependent upon their optical properties include the use of a composite microlens array to form a linear array of ''optical rods'' across a 2-d flow plane (sun et al. 2006b ). integration of waveguides from which light emerges into a microfluidic channel is an attractive advance upon the use of external lenses or the hybrid integration of individual optical fibres to realise dual-beam traps, in terms of robustness, alignment and potential for mass production. cran-mcgreehin et al. (2006a, b) demonstrated integration of semiconductor lasers emitting directly into a microfluidic channel, as shown in fig. 4 , to realise two dual-beam traps and demonstrate trapping of polymer spheres and shunting of these spheres between traps. the device was realised in a gaas/algaas heterostructure with inas quantum dots emitting at 1290 nm, with microflow channel realised using chemically assisted ion-beam etching. this approach provides the potential for monolithic integration of all the optics and fluidics in highly functional devices, and potentially removes the need for any optical connection to the chip, which is often the most difficult aspect particularly for users not specialised in optics. propulsion of particles in an evanescent field was demonstrated by kawata and sugiura (1992) , where light incident on the base of a prism beyond the critical angle resulting in an evanescent field in water was used to propel polystyrene and glass spheres of diameter between 1 and 27 lm at velocities up to 20 lm/s along the surface of the prism. the evanescent approach allows convenient manipulation at a surface, improved stability as the radiation does not propagate through the liquid medium and, with the use of appropriate apertures, greater spatial localisation through the near-field, but the fields are normally restricted to within a few hundred nanometres of the surface. forces in an evanescent field were exploited by garcés-chávez et al. (2005) to guide and trap red blood cells over an area of order 1 mm 2 . a grating of period 12 lm was imaged on the base of a prism and the cells assembled along the lines of high intensity and moved along them under radiation pressure. a counter-propagating beam with the same grating pattern was used to produce the same gradient force pattern but equal and opposite radiation pressure forces parallel to the surface, thereby producing stationary traps. control of the relative beam powers was used to control the velocity of cells from stationary to about 1 lm/s. mellor and bain (2006) used a similar two-beam geometry without fig. 3 optically induced surface forces leading to trapping and stretching of cells (guck et al. 2005) . reproduced with permission from biophysical society the grating image, and both with and without resulting interference fringes, to study the interplay of trapping and optical binding forces on collections of spheres of order 500 nm diameter to produce ordered arrays with controlled sphere spacings. optical waveguides provide evanescent fields and have the advantages of greater stability and potential for integration. kawata and tani (1996) demonstrated the trapping and propulsion of polystyrene latex and metallic spheres along a channel optical waveguide at velocities up to 14 lm/s, and suggested the use of a standing wave for positional control, and application to cell sorting according to ''size, chemical composition and shape''. grujic et al. (2005) demonstrated optical sorting of 6-lm diameter polystyrene latex particles into the two output branches of a y-junction glass waveguide, following external identification of the particles to be selected. gaugiran et al. (2005) demonstrated increased propulsion velocity, using a highindex silicon nitride waveguide with a larger proportion of power carried in the evanescent field, and also demonstrated the trapping and propulsion of red blood cells and yeast cells at velocities up to 1 lm/s on these waveguides. gold nanoparticles have a high scattering cross-section compared with latex particles and biological cells and have potential as tags in sorting of biological species. hole et al. (2005) trapped and propelled 250 nm diameter gold spheres; individual particle trajectories exhibited velocities up to 500 lm/s, showing promise for high throughput sorting. recently schroll et al. (2007) have demonstrated bulk transport of liquid with mesoscopic spatial variations in refractive index under radiation pressure. in addition to directly manipulating particles in microfluidic systems, there is a need for pumps and mixers to manipulate fluids themselves. while there are many demonstrations of pumps in microsystems, optically actuated pumps and mixers may have the advantages of simple reconfigurability, integration and small size, compared with external and piezo-electric pumps, and fewer constraints on the properties of the liquid medium compared to electrokinetic pumps. colloidal particles, appropriately assembled into a microflow channel or chamber and manipulated using optical traps to provide pump action, are the most widely reported, with the two-lobe ''gear pump'' shown in fig. 5 formed from four 3 lm particles, and a peristaltic pump formed from six such particles, being demonstrated by terray et al. (2002) . the particles comprising the pump were individually assembled and manipulated using scanning laser optical trapping, using a piezo-electric mirror under computer control to realise a time-averaged trapping pattern, with flow rates of 3 lm/s being achieved. ladavac and grier (2004) employed an array of ring traps, or optical vortices, formed by using a hologram to split laser light into two rows of three circular traps of few micron diameter; 800 nm silica spheres in the fluid were drawn into the rings and propelled around them by transfer of angular momentum. the helical phase functions for the optical modes driving the rings in the upper and lower rows were chosen such that their rotations were in opposite senses, so that the circulating rings of colloidal spheres caused the liquid between the rows to flow with velocities up to 5 lm/s. leach et al. (2006) constructed a micropump by optically trapping two birefringent vaterite spheres of 6 lm diameter in a microflow channel and causing them to rotate in opposite directions, achieving a pump speed of up to 8 lm/s (200 fl/s). a single input beam was split using a wollaston prism and waveplates to provide two traps with circular polarisation in opposite sense, imparting angular momentum to the spheres and causing them to rotate in opposite senses. rotation of a microgear with geometrical birefringence, using either circular polarisation to impart angular momentum or rotation of linear polarisation, in a conventional optical trap was demonstrated by neale et al. (2005) . the microgears, a few microns in diameter, were fabricated in su-8 by e-beam lithography, released from their substrate, placed in the microflow system and positioned and rotated by the optical trap. micropumps in which the rotors are fabricated around fixed shafts and rotated using optical forces have been demonstrated by kelemen et al. (2006) and maruo and inoue (2006) . kelemen et al. incorporated an eccentrically positioned optical waveguide illuminating one side of the propeller to rotate it using scattering forces. all components were realised on a glass substrate by direct two-photon writing in photopolymers, and a rotation rate of 2 hz was achieved with waveguide power of about 25 mw. although reconfigurability is limited in this approach, as the propeller is ''trapped'' by an axle rather than by an optical trap, no complex external optics is required, the pump does not have to be assembled optically, and rotation is simply driven by waveguide power. maruo and inoue demonstrated a micropump shown in fig. 6 , with two counter-rotating meshing rotors in a protrusion in a 5 lm · 7 lm channel, rotating around fixed shafts, which achieved a pump rate up to 0.7 lm/s. all the structures were written by direct two-photon writing in photopolymer, and the rotors were driven by ''time-divided laser scanning'', dividing the beam and scanning the two resultant beams in opposite circular trajectories using galvano scanners. detection and analysis of chemical and biochemical species in microfluidic systems is challenging due to short optical path-lengths, small sample volumes, and the need to analyse individual particles or molecules. for the loc to fulfil its potential in terms of parallelism and throughput, high-speed, multiparameter and multiplexed detection is required, creating heightened demand for low noise, high specificity, miniaturization and ruggedness. conventional optical techniques for macro analysis such as fluorescence, absorption, and chemiluminescence have been applied to microfluidic systems, and recently good reviews have been published on optical detection methods for microfluidic devices (götz and karst 2007a; yi et al. 2006b ). less common methods for optical detection in microfluidic devices, such as raman spectroscopy, are reviewed by (viskari and landers 2006) . the majority of optical detection systems in microfluidics address flow cytometry or capillary electrophoresis (ce). in common with demonstrations of optical trapping and propulsion in microsystems, most detection systems rely upon rather complex bulk-optical systems external to the microchannel, such as confocal microscopes, or hybrid integration by embedding optical fibres, rather than the full on-chip integration of optical functions likely to be required for highly functional integrated systems (craighead 2006) . while the majority of demonstrations use optical transmission directly through the fluidic channel, a few exploit evanescent field configurations where heterogeneous detection at a surface is carried out, exploiting the rapid diffusion to surfaces, which occurs in thin laminar flow channels. there are undoubtedly some challenges in applying evanescent sensing techniques, and integrated optical waveguides (lambeck 2006) , to microfluidic systems, such as the robustness and reversibility of chemically specific films and surface fouling, but biosensor research and microfluidics research appear artificially distinct at (maruo and inoue 2006) . reproduced with permission from the american institute of physics present. this section reviews optical detection schemes for chemical analysis in microfluidic systems, divided according to the principal optical phenomena employed: scattering, absorption, refractive index, fluorescence, raman spectroscopy, and thermal lensing. this review is limited to microfluidic systems, but there are many sophisticated optical systems which have been applied to biosensing and chemical analysis which use less advanced fluidics, but which have potential for future incorporation in the loc. light scattering techniques applied to collections of particles, such as turbidimetry, are well established, but microfluidics enables scattering measurements to be made on individual particles of size comparable with the wavelength, with high throughput. scattering is probably the simplest method for cell counting in a microchannel, but can be extended to yield information on cell size, shape and internal and external structure according to the angular scattering spectrum, and is commonly combined with fluorescence measurements to yield chemically specific information. this is a natural extension of traditional bulk flow cytometry systems where scattering may be employed to increase detection reliability and the resolvability of fluorescent signals. laser light is focussed on a detection volume and the scattering caused by a refractive index contrast between the particle entering that volume and the surrounding fluid allows detection of particles. large angle scattering can give information a particle's surface roughness and internal structures; whilst small angle or forward scattering can yield information of a particle's size. given the information which can be gained, scattering is surprisingly underutilised in microfluidic devices, perhaps due to the fact that most implementations are dependant on alignment of bulk external optics at the necessary angles. nevertheless, miniaturisations of flow cytometers using microfluidics have been developed, taking advantage of light scattering from particles. schrum et al. (1999) used scattering from fluorescently labelled 2 lm diameter and unlabelled 1 lm diameter polystyrene latex particles to demonstrate flow cytometry in a microfluidic device, distinguishing them through fluorescence and size, and counting them at a rate of 13.5 hz, using radiation from an ar ion laser. subsequently, mcclain et al. (2001) extended this work to perform flow cytometry on fluorescentlylabelled e. coli at a rate of up to 85 hz, observing marginally better particle resolution using scattering. witek et al. (2004) studied electromigration of e. coli and red blood cells in polymer microflow channels, using backscatter from focussed light from a 680 nm laser diode to monitor cell transport. pamme et al. (2003) analysed polystyrene microspheres of diameters between 1 and 9 lm in a microflow channel using scattering alone from incident light at a wavelength of 633 nm. optical fibres to collect scattering were fixed externally on top of the microfluidic device at 15°and 45°t o the incident light beam, as shown in fig. 7 . while particles between 3 and 9 lm diameter were readily distinguished at throughputs of order 150 hz, the lower scattering intensities from smaller particles reduced the signal-to-noise ratio (snr) to the point where 1 and 2 lm particles could not be reliably distinguished. the same group then analysed magnetic particles of diameter 2.8 and 4.5 lm under magnetophoretic separation. similar apparatus was used as before but with scatter collection angles of 25°and 35°to the incident light (pamme et al. 2006) . wang et al. (2004) incorporated optical waveguides aligned photolithographically across the flow channel to perform scattering measurements on polystyrene microspheres of diameters 2.8, 4.6, 5.8 and 9.1 lm, avoiding the need for optical alignment and improving mechanical stability of the device. an integrated microlens was used to control the beam shape of incident light being delivered to the microchannel, and optical fibres were connected at the input and outputs. the channel, waveguides and lens were realised in a single su-8 photoresist-processing step. measurements of forward scattering were made by fig. 7 photograph of the microfluidic particle scattering apparatus; two optical fibres were mounted above the microchip for scattering detection. (pamme et al. 2003) . reproduced with permission from the royal society of chemistry microfluid nanofluid (2008) 4:53-79 59 collection in an output waveguide at an angle of 5°, and the particles of different size were clearly distinguished. the design allowed for optical extinction and large-angle scattering to be measured, but this was not discussed in detail. incorporating this approach in a dielectrophoresis structure the authors were subsequently able to separate and count viable and non-viable yeast cells ). integration of further components close to the scattering particles may be expected to yield more detailed information on cells. detection through optical absorption is common in chemical analysis, employing either direct absorption in the uv/ visible wavelength range or a colorimetric assay, for example. its simplicity, and the lack of need for chemical derivitisations for fluorescent tagging, has led to it being the most widely used detection technique in conventional microseparations. however, the short optical path length inherent in miniaturization of fluidic channels leads to weak optical absorption competing with a high background transmission, and consequent difficulty in achieving low detection limits. this has been addressed in conventional capillary systems using z-shaped and u-shaped channels (e.g. moring et al. 1993 ) with longitudinal transmission. the problem becomes more severe in on-chip systems, and several approaches to increasing the absorption have been demonstrated, such as liquid-core waveguiding, multipass configurations, hollow prism cells, integrated waveguides and ''slow light''. detection limit depends upon good stability and reduced noise as much as increased sensitivity, rendering waveguiding approaches attractive. to enhance absorption in microfluidic devices, liang et al. (1996) microfabricated a planar u-shaped absorption cell, with the base of the u placed between excitation and collection fibres in photolithographically defined channels, and achieved a 10-fold increase in absorbance, using a path length of 140 lm compared with transverse measurement through the 20 lm depth of the channel. mogensen et al. (2001) integrated planar channel waveguides into a uchannel design for the coupling of light into the detection region, to overcome alignment issues observed with optical fibres, and used a larger optical path length of 750 lm, increasing this to 1,000 lm in a subsequent polymer device (mogensen et al. 2003) . they further increased this to an effective optical path length of 1.2 mm using novel pure silica integrated waveguides shown in fig. 8 ; using absorbance at a wavelength of 254 nm a detection limit of 3 lg ml -1 was achieved for paracetamol, and electrophoretic separation allowed caffeine, paracetamol, and ketoprofone to be distinguished. launching and collecting light across long path-length channels has also been achieved using total internal reflection from micromachined v-grooves placed either side of the fluidic channel ) and used for a glucose assay on whole blood, where a limit of detection (lod) of 200 lm was achieved. salimi-moosavi et al. (2000) reported a multi-reflection cell, achieving five to tenfold enhancements of optical path lengths. mortensen and xiao (2007) have recently modeled the use of ''slow light'' structures for optical path-length enhancement. periodic structures such as liquid-infiltrated photonic crystals could be incorporated within a flow channel and potentially yield large enhancements particularly near the band edges. duggan et al. (2003) achieved an optical path length of 5 mm by using a teflon fluoropolymer capillary as both the flow channel and a liquid-core waveguide, and teflon af coated waveguiding microflow channels in silicon have also been realised (datta et al. stripe of sio 2 that acts as a waveguide . reproduced with permission from wiley-vch verlag gmbh & co. kgaa 2003). however, extending the physical path-length along the direction of flow also degrades the spatial resolution of species in capillary separation systems, resulting in a tradeoff between enhancement and resolution. performance of absorbance based measurements can also be improved by incorporating additional on-chip optical components, such as microlenses and micro-apertures, to reduce stray light and hence increase the effective optical path length (ro et al. 2005) . zhu et al. (2006) improved collection efficiency by incorporating bare silicon detectors within the pdms fluidic chip and reported a lod for bovine serum albumin of 1 lg ml -1 with channels of 60-400 lm depth. llobera and co-workers developed monolithically integrated hollow prism microchannels combined with optical components in a single-step pdms microfabrication process (llobera et al. 2004 (llobera et al. , 2005 . the device, shown schematically in fig. 9 , consisted of channels to embed input and output fibres and cylindrical biconvex microlenses to collimate the light, pass it through the hollow prism filled with analyte, reflecting from a prism wall to enhance path-length, and refocus it on the output fibre. lods of 1.83 and 0.683 lm were achieved for fluorescein and methyl orange, respectively, and ph measurements performed using methyl orange. an alternative approach to overcoming the low uv absorptions obtained in microchannel separations due to the short optical path-length is to preconcentrate the sample. isotachophoretic (itp) preconcentration has been combined with zone electrophoresis (ze) to lower the lod on detection of two flavonoids down to 1.2 and 0.2 lg ml -1 ), a 32-fold enhancement compared with zone electrophoresis alone. jiang and pau (2007) fabricated an su 8 rib waveguide in a pdms microchannel with a spiral geometry resulting in a length of 110 mm within a chip area of 4 cm 2 . the fluid to be sensed acts as a cladding and its spectroscopic properties are probed using the evanescent field. hu et al. (2007) realised a chalcogenide glass waveguide in a pdms microchannel, in principle allowing transmission and evanescent absorption spectroscopy at mid-ir wavelengths. special waveguide designs can achieve higher sensitivity for evanescent detection than ''free space'' designs of the same physical path length (veldhuis et al. 2000) , rendering them potentially attractive in on-chip labs. fluorescence techniques are by far the most commonly used for optical detection in microfluidic devices, having the prime advantage that the signal is usually readily separable from the scattered incident light. compared to other detection techniques, fluorescence delivers high sensitivity, allowing detection of single molecules as reviewed by dittrich and manz (2005) , and has gained wide acceptance in conventional bulk instruments. in some cases, the autofluorescence of species (molecules, viruses, cells, etc) may be detected; otherwise a fluorophore or quantum dot must be attached to act as a tag to be detected. tagging has the disadvantage that additional sample preparation must be carried out, but usually yields lower fluorescent background and improved confidence in the measurement. due to the vast literature on this topic, in this section only work published within the last 2 years will be discussed to highlight the most recent advances in applications of fluorescence in microfluidic systems. a wide variety of light sources are used for excitation, including lasers, leds and lamps. lasers and ''laserinduced fluorescence'' (lif) are most commonly employed in microfluidics, due to diffraction-limited focussing and the potential for 2-photon excitation. the increasing range of wavelengths, continually reducing cost due to use in consumer electronics, good stability, ease of switching, and low power requirements when using semiconductor lasers reinforce this trend. longer wavelength fluorophores (600 nm \ k abs \ 800 nm) tend to be gaining ground due to the reduced background fluorescence and wider availability of high-quality compact optical components at these wavelengths. recent research has emphasised electrokinetic methods for concentrating species to be detected, thereby improving lod, with most using conventional external optical apparatus such as confocal microscopes. it has been shown that field-amplified stacking injection (fasi) in microfluidic capillary electrophoresis devices can increase sensitivity of detection of fluorescent dyes (gong et al. 2006) . using a microfluidic chip with several channel networks, detection of immunoassays of insulin secretions from multiple independent pancreatic islets can be continuously monitored with an lod of 10 nm (dishinger and kennedy 2007) . sun and yin (2006a) reported a multi-depth microfluidic device for single cell analysis. human carcinoma fig. 9 schematic of the system for detection with a hollow abbe prism (llobera et al. 2004) . reproduced with permission from the royal society of chemistry cells which tend to aggregate and settle were separated using this device and the cell constituents separated by ce and analysed using lif. flow cytometry is an important function to benefit from microfluidics technology, with many continuous-flow microfluidic devices demonstrating fluorescent-activated cell sorting (lfacs). morgan and co-workers holmes et al. 2006) reported devices that used dielectrophoresis (dep) to focus particles in a microchannel stream. figure 10 shows a micro flow cytometer chip, which focused particles in both the vertical and horizontal dimensions and detected 6 lm diameter fluorescent latex particles. using careful optimisation of the excitation intensity to limit fluorophore saturation, jung et al. (2007a) obtained an extremely low lod of 100 attom, and single molecule detection, of alexa fluor 488 by lif in a ce chip flow with isotachophoretic separation. hydrodynamic focussing has also been applied in microfluidic systems with fluorescence detection. yang et al. (2006) realised a portable system incorporating a microfluidic device with hydrodynamic focussing and onchip pumping and applied it to detection and sorting of fluorescent dye-labelled human lung cancer cells, with a count rate of 120 min -1 and an error rate better than 2%. simonnet and groisman (2006) created high-throughput flow cytometers (17,000 s -1 ) in molded pdms with ''3d'' hydrodynamic focussing, and applied them to tagged yeast cells. kamei and wada (2006) built upon earlier work (kamei et al 2005) demonstrating microfluidic separation of biomolecules, and realised a detection platform shown in fig. 11 , which included a 2 mm diameter half-ball lens for fluorescence collection, a microstructured interference filter deposited directly on a pin photodiode, and an aperture through the centre of the detector and filter via which excitation light from a 488 nm frequency-doubled vcsel was introduced. a planar microfluidic ce device was placed in contact with the half-ball lens, exciting fluorescein in the microchannel and allowing fluorescence to be collimated, filtered and collected. the device achieved an lod of 7 nm fluorescein and was used to demonstrate the separation and detection of haeiii-digested øx174 bacteriophage dna. breakthrough of scattered excitation light is often the limiting factor in performance of fluorescencebased systems. fu et al. (2006) performed a detailed study of the directionality of scattered excitation light and fluorescence, which enabled maximization of the fluorescent to scattered power ratio by selecting the fluorescence collection angle. excitation light was incident orthogonally to the microfluidic substrate and collected in the plane of the substrate from the chip sidewall, as shown in fig. 12 , with angles of 45°and 135°to the flow-channel exhibiting the best performance. an lod of 1.1 pm fluorescein was achieved, and the device demonstrated ce of tagged arginine and phenylalanine. waveguiding structures have been exploited for fluorescence-based detection in microfluidic systems. mazurczyk et al. (2006) realised ion-exchanged waveguides in sodalime glass integrated with etched microfluidic channels and used fibre-coupled laser light at 532 nm for excitation of the cy3 dye. collection was carried out orthogonally from the top of the chip using a lens or bonded fibre, achieving an lod as low as 0.5 nm cy3, and collinearly in a waveguide on the opposite side of the channel, achieving an lod of 1 lm cy3. the orthogonal configuration was then used to demonstrate ce of cy3 and cy3-tagged streptavidin. bernini et al. (2006) used antiresonant reflecting optical waveguide (arrow) structures to realise microfluidic channels that guided excitation light in the liquid, an alternative approach to the teflon-clad waveguides described above. although the liquid core has a lower refractive index than the surrounding media, the dual-layer cladding forms a high reflectivity mirror, which traps the light without invoking total internal reflection. laser light was coupled into the flow channel via a fibre inserted into the hydrodynamically focused sample stream and two detector fibres were inserted in microchannels perpendicular to the main channel to capture fluorescent light from passing particles. . reproduced with permission from elsevier the system was tested with human t leukaemia cells with diameters of about 20 lm. the rapid, low-cost, detection of bacteria in food and drink is an important application for micro flow cytometry. small numbers of pseudomonas cells in milk (yamaguchi et al. 2006 ) and e. coli in buffer (inatomi et al. 2006) were detected using fluorescence microscopy following attachment of fluorescent dyes. a more portable pdms immunoassay system that detects the e. coli o157:h7 bacterial antigen with an lod of 0.3 ng ll -1 has been reported (xiang et al. 2006) . a laser diode and optical fibre-based fluorescence excitation and collection module was employed, with the distal end of the multimode fibre placed against the bottom side of the thin glass fluidic substrate. li et al. (2006) employed an optical fibre embedded in an etched channel in the substrate and a blue (470 nm) laser diode for detection of fluorescene-5-isothiocyanate (fitc)-tagged neurotransmitters dopamine and epinephrine in a micro ce chip. fluorescence correlation spectroscopy (fcs) is a powerful technique for the determination of the physical properties of small numbers of fluorescent or dye-tagged molecules. to ensure that only small numbers of molecules are studied at one time, the excitation volume is minimised by focussing to a small volume and using confocal detection. a correlation analysis of the temporal fluorescence fluctuations of particles entering the optical detection volume yields information on the diffusion characteristics and other physical properties of the molecules under study. yeh et al. (2006) assembled an fcs system with a complex pdms microfluidic network, consisting of on-chip serpentine channels for mixing, interrogation chambers, reservoirs and valves, as shown in fig. 13 . in a single flowchip, the drug doxorubicin was mixed to produce eight parallel streams of different concentration; each stream was then mixed with a complex of dna with transcription factor sp1, and incubated in eight parallel interrogation chambers. sp1-dna dissociation was then determined by fcs, exploiting the different diffusion kinetics of differently sized entities to determine the fractions of bound and unbound dna. the resultant dose-response titration curve yielded the sp1-dna binding inhibition versus drug concentration. rapid detection of low numbers of viruses for medical diagnostics is an important field of application of microfluidics. zhang et al. (2006) detected dengue virus with fluorescence cross-correlation spectroscopy (fccs), using dengue-specific antibody in a pdms microfluidic array structure. the virus and antibody were fluorescently labelled with two different fluorophores, excited at 488 and 633 nm, and the emission separated by a dichroic filter onto two apds. viruses bound to antibodies entering the detection volume exhibited positively cross-correlated fig. 11 cross-sectional view of the fluorescence detection platform on which a capillary electrophoresis chip is mounted. laser light is introduced from below through an aperture (kamei and wada 2006) . reproduced with permission from the american institute of physics fig. 12 schematic diagram of angle-resolved fluorescence detection apparatus . reproduced with permission from the american chemical society fluorescence signals due to their motion being coupled. this study suggests that detection of a single virus is achievable with this system. hollars et al. (2006) also performed a cross-correlation of two fluorescence labelled probes excited and emitting at different wavelengths, in a flowing system. in this case total internal reflection was used to excite the dye-tagged analyte in an evanescent field in the microchannel. short non-amplified dna sequences were measured to sub 100 pm levels in less than 1 min. advances have also been made in systems employing conventional lamps, which allow great flexibility in excitation wavelength by using wavelength filters, but generally show poorer limits of detection. yan et al. (2006) used an hg-lamp with excitation wavelengths from 300 to 600 nm and a carefully engineered detection system with a microfluidic ce chip to achieve an lod for fitc of 7 · 10 -10 m and for fitc-tagged cystine of 9.6 nm. götz and karst (2007b) used a xenon arc lamp as an excitation source combined with a spectrograph and ccd, providing more detailed information through the complete emission spectrum in the visible wavelength range (350-800 nm). three rhodamine dyes were used to characterise the system, applied to a standard microfluidic chip performing ce for separation of analytes. peaks in the elution spectra could be assigned to different pure analytes and unexpected impurities were detected both by ce separation and within an elution peak using spectral information. using similar apparatus the group were then able to detect thiols found in cosmetics with an lod of 2 lm (revermann et al. 2007) and taurine found in drinks with concentrations as low as 60 am (götz et al. 2007c ). mitra et al. (2006) fabricated a microdischarge device stacked on top of a microfluidic device as an alternative excitation light source producing emission in the uv for intrinsic fluorescence excitation. the microdischarge is created in an air gap between a metal anode and saturated salt solution in a reservoir acting as a cathode. light-emitting diodes (leds) are attractive sources for microfluidic measurements as they are cheap, compact, and efficient and may have higher brightness than conventional lamps. destandau et al. (2007) have reported a simple yshaped microfluidic channel and a simple led and pmt detecting potassium with a lod of 0.5 mm using calyxbodipy dye, which has fluorescence intensity sensitive to potassium concentration. kim et al. (2006) realised a highly integrated fluorescent microfluidic detection chip comprising the microfluidic channels, silicon detector with interference filter, and an integrated organic led (oled) deposited directly on to glass as a thin-film structure. the microchannels were etched in glass with dimensions 70 lm width and 20 lm depth, and the oled, emitting at 530 nm, was deposited on the reverse of the substrate. a p-i-n photodiode with a deposited interference filter was buried in pdms, which was then bonded to the glass microchannel. the device was characterised using tetramethylrhodamine dye (tamra) and achieved an lod of 10 lm. hofmann et al. (2006) realised microchannels in a pdms slab, which had been doped with lysochrome dye and acted as a high-quality filter itself. 1 mm thick samples doped with 1,200 lg ml -1 sudan ii, for example, showed .01% transmission at 500 nm and [80% transmission above 570 nm. irawan et al. (2006) realised microchannels for use as fluorescence detection cells in pmma optical fibre and plastic clad silica optical fibre by laser ablation. microchannels of dimension 100 lm width by 210 lm depth were etched into the fibre cores and fluorescein, excited by a blue led (centre wavelength at 470 nm), measured with an lod of 5 ng l -1 . protein and dna detection and manipulation are major fields of application in microfluidic systems due to the fig. 13 the pdms-based microfluidic chip used for investigation of the dissociation of sp1-dna complex by dox. a the layout of microchannels (red) and the mechanical on-off valves (blue). b a zoom-in of the mixing portion on the microfluidic chip (yeh et al. 2006) . reproduced with permission from oxford university press benefits of rapidity, flow control and small sample and reagent volumes at these scales. shen et al. (2006) developed an array of ce microchannels connected to reservoirs in soda-lime glass to perform parallel detection of fluorescent-tagged biomolecules. light from a diode-pumped solid-state laser at 473 nm was line-focussed across the parallel measurement and reference channels and a ccd camera with filter was used to detect fluorescence simultaneously from all channels. parallel ce separation allowed simultaneous detection of many fluorescently labelled molecules such as amino acids, proteins and nucleic acids. a similar system was realised in pmma by liu et al. (2006) , working in the same group, and successfully applied to rapid detection of multiplex pcr products for sars and hepatitis b. huang et al. (2007) fabricated a sophisticated microfluidic network in pdms to analyse low-copy number proteins in a single cell by single molecule detection. individual cells were trapped, lysed, and the proteins in the lysate bound with fluorescently labelled antibodies and then separated using ce. a line-focused laser beam ensured that all molecules across the width and depth of the detection microchannel (40 lm · 1.8 lm) were detected, while in the direction of flow it was still tightly focussed to reduce background fluorescence. edgar et al. (2006) performed detection of fluorescein-labelled amino acids contained in 10 fl droplets. the device consisted of a t-channel 3 lm wide by 3 lm deep that separates a ce channel (50 lm wide by 50 lm deep) from the droplet generation input by using an immiscible liquid. lif is then performed in the ce channel. oh et al. (2006) genetically engineered e. coli cells to cause them to express capture proteins and present them at the membrane surface. dep cell traps were used to immobilise the cells in 200 lm wide microchannels, which then captured fluorescently labelled target molecules. dual wavelength fluorescence measurements were performed on the cells to detect the bound molecules and reference to the number of capture ligands. this technique provides a convenient method to produce surfaces with immobilised capture molecules. direct uv-excited fluorescence offers the potential for label-free protein analysis in individual cells. hellmich et al. (2006) performed direct uv fluorescence measurements on amino acids and proteins, using a frequency-quadrupled nd:yag laser and photomultiplier. individual cells were optically trapped and electrically lysed on an pdms/quartz chip, and the lysate separated by ce. optimisation of the confocal optics and addition of carbon black into the pdms led to a reduction in background florescence and an lod of 25 nm for tryptophan. dna hybridisation to a free dna probe sequence in solution is much faster than to immobilised dna sequences, due to the diffusion-limited kinetics of the latter. thus detection of hybridisation of target and probe in a microflow system, with suitable mixing, presents considerable advantages in terms of speed. lee and co-workers (yea et al. 2006; kim et al. 2007 ) implemented a fluorescence resonance energy transfer (fret) system in a pdms microfluidic device with an ''alligator teeth'' structure for efficient mixing in otherwise laminar flow, shown in fig. 14b . as the probe and hybridised target is not immobilised, washing cannot be used to remove the unbound target, so an alternative labelling scheme is required. yea et al. (2006) employed a system where both the target dna and probe strands were labelled, and relative intensities yielded the fraction of hybridised dna. to avoid the need to label the target dna, kim et al (2007) then employed a molecular beacon, shown in fig. 14a , where one end of a single-stranded oligonucleotide has a fluorophore attached and the other end has a quencher attached. in the absence of the target sequence, emission is quenched as the probe sequence forms a loop and the fluorophore and quencher are in close proximity. hybridisation of the complementary target causes the loop to open, the fluorophore and quencher to separate and fluorescence to be observed. liu et al. (2007) report a portable genetic analysis system for forensic applications and have validated it using samples extracted from oral swabs and human bone extracts. the glass microfluidic chip performed the polymerase chain reaction (pcr) and capillary electrophoresis followed by parallel four-wavelength fluorescence detection using a 488 nm frequency-doubled diode laser and confocal optics, with optical fibre as confocal aperture. multiplexed analysis with multiple fluorescent labels is limited by the spectral overlap of fluorophore emission. pregibon et al. (2007) created single and multi probe particles encoded with a spatial fluorescent pattern for multiplexed detection of dna oligomers at 500 attomoles. the encoded particles were created in a continuous microflow system, shown in fig. 15a , where laminar fluorescently labelled monomer and probe-labelled monomer flows are introduced adjacently. elongated polymer particles (*90 lm wide by 30 lm deep by up to 270 lm long) were formed in the channel by exposing the monomers to 30 ms bursts of uv to photopolymerise them. the shape of the particles was defined by a mask in the uv projection system, which included the 20-dot pattern at one end, yielding more than one million possible codes. the resultant particles, which have a fluorescent graphically encoded region and covalently coupled probe-carrying regions, as shown in fig. 15b , are collected in a reservoir. after incubation with the fluorescent-labelled target the particles are read by passing them through a microfluidic channel a little wider than the particle width. the particles are hydrodynamically focused and aligned along the channel and when they pass through the detection region the fluorescent intensities are detected using a ccd camera and analysed in five ''lanes'' along the particles, corresponding to the rows of dots in the code, so that the particle's code is read and the presence of the target is detected. the external optics employed in microfluidic lif systems is often complex. huang et al. (2006a) performed on-chip pcr and ce in pdms/pmma/glass multilayer device with an embedded optical fibre for fluorescence collection to reduce the complexity of collection. they detected s. pneumoniae bacteria and the dengue-2 virus by separating and detecting amplified dna products. suitably modified microbeads may be used as probes for biomolecular detection in microfluidic systems. for example, the beads may be functionalised with antibodies and binding of the fluorescent-tagged antigen enables fluorescent detection in a microfluidic device, with multiplexing being achieved using beads containing different fluorophores. detection on beads means that the antibody does not have to be immobilised in the microfluidic device, and the beads can be readily manipulated within a microflow system. yun et al. (2006) realised physical pdms gate valves within a microfluidic channel to isolate individual beads, as shown in fig. 16 . using 10 lm diameter polystyrene beads modified with human igg specifically bound to protein a on the bead surface, an lod of 0.1 lg ml -1 for anti-human igg conjugated with cdse/zns quantum dots (qds) was achieved, using a fluorescence microscope to excite and detect. riegger et al. (2006) performed sandwich immunoassays for hepatitis a and tetanus in a centrifugally propelled disk-based microfluidic system with an led light source. cdse qds were used to identify the beads and fluorescent polystyrene microspheres were used to identify presence of the bound antigen. haes et al. (2006) achieved an lod of 1 fm for staphylococcal enterotoxin b (seb) detection using a microfluidic device with a system of microchannels and micro-weirs. displacement assays were carried out on a glass chip using lif and electrokinetic fluid pumping. silica beads modified with antibodies specific to seb were trapped in the weirs, and fluorescent-tagged seb bound to these in situ. the displacement assay was carried out by introducing the sample containing seb and observing fluorescence from displaced tagged seb downstream from the beads. the beads could be replaced, for repeated assay, or used again a limited number of times. refractive index based techniques are more prone to influence by non-specific effects than fluorescence techniques but are extremely attractive for chemical and biochemical sensing due to the lack of need for a label. however, rather few papers describe integration of refractive index based detection techniques with true microfluidic systems, and key examples are reviewed in this section. costin et al. (2003) demonstrated measurement of diffusion coefficients and molecular masses in a microfluidic chip, which could play a useful role in on-chip ce and hplc systems. the sample stream and mobile phase stream were merged in a detection channel and interdiffusion between the streams was monitored using the angular deflection of laser light by the resultant refractive index gradient. two detection points, one just as the streams merge and one downstream, were used to monitor the extent of diffusion over the length of the channel, and the relative deflections yielded information on analyte diffusion coefficient and molecular weight. leung et al. (2004) analysed bubbles in microchannels 40 lm wide and 30 lm deep, for application in on-line reaction monitoring and safety applications. as a bubble passes through the microchannel the deflection of laser light due to the fig. 14 a schematic representation of the molecular beacon and its operating principle. target hybridization leads to the separation of the fluorophore (fam) and quencher (dabcyl) and a consequent fluorescent signal. b schematic representation of an alligator-teeth-shaped pdms microfluidic channel (kim et al. 2007) . reproduced with permission from the japanese society for analytical chemistry refractive index perturbation is recorded using a positionsensitive detector. performing statistical analysis on the data the system is able to determine bubble size and formation frequency. using a hollow abbe prism as a microfluidic chamber, as described above for absorption monitoring (fig. 9) , llobera et al. (2004) were able to detect changes in refractive index through dispersion of the prism, as well as the absorption of a liquid. the device consisted of channels to embed input and output fibres, biconvex microlenses to focus the light, reservoirs, and the abbe prism monolithically integrated in pdms. the spectral transmission of a fabry-perot cavity is highly sensitive to intracavity refractive index and may enable imaging of refractive index distributions. shao et al. (2006) fabricated a fabry-perot (fp) microfluidic cavity in which microchannels of up to 30 lm deep and 200 lm wide were etched in pyrex and the top and bottom coated with 35 nm thick gold layers to serve as reflectors. using an led as a broadband source and measuring the transmission spectrum of the device, they performed intracavity measurements on single particles such as yeast cells; human red and white blood cells could be distinguished, although improved quantification is needed. song et al. (2006) realised a microfluidic network incorporating a dual-fibre fabry-perot, with channels for insertion of cells and two buffer solutions, two single mode fibres with high reflectivity gold-coated ends crossing the fluid channel to fig. 15 a schematic diagram of dot-coded particle synthesis showing polymerization across two adjacent laminar streams to make single-probe, halffluorescent particles (shown in b). c diagrammatic representation of particle features for encoding and analyte detection. d differential interference contrast (dic) image of particles generated by using the scheme shown in a. e to g overlap of fluorescence and dic images of single-probe (e), multiprobe (f, bottom), and probe-gradient (g, left) encoded particles. scale bars indicate 100 mm in (d), (f), and (g) and 50 mm in (e) (pregibon et al. 2007) . reproduced with permission from aaas fig. 16 schematic view of each step in capturing of a single microbead: a bead introduction, b inlet gate opens by applying a vacuum to the gate control channel and c gate closed by stopping the vacuum on the gate control channel resulting in a single bead captured for sensing (yun et al. 2006) . reproduced with permission from the institute of physics act as the fp cavity and as optical input and output, and a pressure-based cell-holder to trap the cell between the two fibres, as shown in fig. 17 . the cavity was 35.5 lm long and two buffer solutions having different refractive indices were alternated while the cell was trapped in position by the cell holder. the differences in the spectra with the two buffer media were reported to yield the effective refractive index of the cell with a standard deviation of ±0.2%, and cell size with an sd of ±4% for repeated measurements of the same cell, being released and retrapped. there is a vast literature on evanescent wave refractive index based biosensing, based on dielectric waveguides or on surface plasmon resonance, for instance, and these are subject of excellent recent reviews by lambeck (2006) and phillips and cheng (2007) . integration of these sensors with microfluidic circuits is essential for repeatable performance, integration of reference sensors, enhanced interaction of analyte and surface, and low-cost manufacture, and the loc may benefit greatly from these welldeveloped techniques. mach-zehnder interferometers (mzis) are one of the most sensitive waveguide devices for evanescent refractive index measurement (quigley et al. 1999) , particularly if active electro-optic control is employed (heideman and lambeck 1999) . lechuga and co-workers have reported the development of an mzi using cmos compatible technology integrated with an su-8 microfluidic channel sepúlveda et al. 2006) , achieving an lod for bulk refractive index of 3.8 · 10 -6 . ymeti et al. (2005) realised a waveguiding young interferometer (yi) on silicon with a glass microfluidic circuit. light is inserted into a waveguide and is split into four channels. the outputs of the four waveguides are focused by a cylindrical lens to a ccd camera to record the interference pattern. each of the four-waveguide channels is exposed in a sensing window, and microfluidic channels are realised on a separate wafer, which is then bonded to the yi over the sensing windows. the device was used to analyse an immunoreaction between human serum albumin (hsa) and a-hsa and achieved a refractive index resolution of 6 · 10 -8 . the authors later reported that a similar yi device, shown in fig. 18 , could detect herpes simplex virus type 1 (hsv-1) at concentrations of 850 particles ml -1 and that the sensor may be able to achieve sufficient sensitivity to detect single virus binding (ymeti et al. 2007) . in this case, a macrofluidic flow system was used, but the use of microfluidics was predicted to reduce the sample volume to a few microlitres and reduce time response to seconds. gratings are attractive components for interrogation of refractive index in microfluidic systems, as they allow relaxed optical alignment compared with direct end-coupling into waveguides (duveneck et al. 2002) . yuen et al. (2005) realised a polymer h-shaped microfluidic device integrated with a grating for sensing, in which a reference fluid and analyte fluid could be passed over the grating under the same conditions and measured simultaneously. laser light is incident through a polariser and focused via a lens to provide many angles to couple into the grating. light is coupled into the waveguide at the angle where the grating provides phase matching, and this angle changes depending on the refractive index of the material at the grating surface; the light then couples out of the waveguide and is recorded by a ccd camera. tests with biotin/ streptavidin assays were conducted to demonstrate detection of small molecule binding. sarov et al. (2006) employed a grating realised with a pmma microprism on a glass microchannel lid, requiring multi-step fabrication. laser light enters the prism under total internal reflection, so that the grating is irradiated and an evanescent field penetrates the microchannel. reflected and diffracted light exits the prism and the energy redistribution into the diffracted orders allows the determination of the refractive index and the absorption coefficient of the fluid. the device had an index sensitivity of 5 · 10 -4 and an absorption coefficient sensitivity of 3.5 · 10 6 m -1 . choi and cunningham (2006) fabricated grating structures with a period of 550 nm as the sensors on the floor of a microchannel, by molding followed by deposition of a thin titania film. using a white light source the grating is irradiated perpendicular to the grating with polarised light, and the reflected light, whose wavelength depends strongly on the refractive index of the medium in contact with the grating, is directed back to an imaging spectrometer. the key advantage of this approach is that an array of small sensor patches (*10 · 10 lm) can be imaged as independent sensors, allowing the potential for dense integration. the device detects changes of refractive index in the microchannel as a shift in the peak wavelength of reflection. discrimination surface plasmon resonance is an alternative means of detecting refractive index changes in an evanescent field, which has achieved great success through the biacore tm system (malmqvist 1993) . detection depends upon angle-or wavelength-dependent coupling to a lossy surface plasma wave at a metal surface, whose velocity changes with refractive index resulting in a change in reflected power. the potential for low reagent and sample volumes and fast reactions is driving further miniaturization and integration of spr devices in microfluidic systems. furuki et al. (2001) realised photopatterned microchannels with floors coated with a gold film. the device was excited using a prism in the kretschmann configuration where light is reflected from the 60 nm thick gold layer, tunneling through it to excite a surface plasmon on the liquid side of the film in each channel. a sequence of cysteamine, photobiotin, and avidin was bound to the surface and measured by spr. the results were consistent with those measured by atomic force microscopy, and the channel sample volume was reduced to about 8 nl. wheeler et al. (2004) fabricated micro-flow cells designed to be attached to the commercial spreeta tm spr sensor by soft lithography in pdms using conventional photoresist processing. refractive index changes due to biotinylated bovine serum albumin, streptavidin, biotinylated protein a, and human immunoglobulin each bound to the preceding layer were monitored. using volumes as low as 73 nl, the data were consistent those obtained with conventional flow cells of volume 8 ll. recently more sophisticated microfluidic spr devices have been reported. huang et al. (2006b) realised a complex integrated flow circuit with many microfluidic components, and detection regions for three analytes in one channel. the optical excitation and detection was again achieved using an external prism in the kretschmann configuration. temperature control was incorporated into the device, as all refractive index based sensors are sensitive to temperature variations. molecular imprinted polymer (mip) films were used as the recognition element for specific biomolecules. mips specific to testosterone, cholesterol, and progesterone were pattered on the gold and detection of these three analytes successfully demonstrated at physiologically normal concentrations. this microfluidic spr system demonstrated direct detection of low molecular weight analytes, higher association rates between analyte and mip compared with a conventional spr system due to more efficient interaction between biomolecules and mip films, and reduced sample consumption. lei et al. (2007) integrated a microfluidic vortex pump into a hot embossed pmma micro flow cell, with integrated spr sensor. the gold layer was not deposited directly in the microchannel, but on the surface of a prism attached to an open microchannel, thereby sealing it. the sensitivity of the spr technique was enhanced by exploiting the phase change on reflection as well as the amplitude change, in a bulk interferometer. an estimated sensitivity of *10 ng ml -1 was achieved for the binding reaction of bsa antibodies to bsa immobilised molecules on the gold surface. making use of a simple t-shaped pdms microchannel on a glass wafer with gold patches, kurita et al. (2006) realised a portable spr system with a concentration range of 5 pg ml -1 to 100 ng ml -1 for b-type natriuretic peptide, a marker of cardiovascular risk. this indirect measurement involved incubation with enzyme labelled antibody, attachment of the unbound antibody at a position upstream of the spr sensor, production of thiocholine by interaction of enzyme and acetylthiocholine in solution, and accumulation of the thiocholine on the gold spr pad. fig. 18 four-waveguide integrated optical young's interferometer sensor. waveguides 1, 2, and 3 are sensing arms, and waveguide 4 is the reference arm (ymeti et al. 2007) . reproduced with permission from the american chemical society microfluid nanofluid (2008) 4:53-79 69 3.5 raman spectroscopy light incident on a material may exhibit raman scattering at shifted wavelengths (raman 1928 ) due to energy being lost to molecular vibrations (stokes shift) or imparted by them (anti-stokes shift). raman spectroscopy thus provides molecular fingerprint information similar to that provided by ir spectroscopy, and is a powerful technique for highly specific label-free detection of species. raman spectroscopy has the advantage that it may be carried out at wavelengths where the absorption of water is weak and where optical instrumentation is straightforward, but has the disadvantage that the scattered signals are very weak. several approaches have been adopted to obtain measurable signals in microfluidic systems, including surfaceenhanced raman scattering (sers), resonance raman scattering, and long-term integration for a stationary particle. in this section two important types of raman spectroscopy applied to microfluidic devices are discussed. surface-enhanced raman spectroscopy (sers) exploits resonant excitation of surface plasmons on metallic surfaces to enhance raman scattering by many orders of magnitude (kneipp et al. 2002) . metals with a large and negative real part and a small imaginary part of dielectric permittivity, usually gold and silver, are required for efficient enhancement, and suitable surfaces for plasmon excitation include colloids, roughened and patterned films, and self-assembled structures. sers is emerging as a competitor to fluorescence with the benefit that no tagging is required (although surface-enhanced resonance raman (serrs) may employ a tag) and raman spectra show narrow lines characteristic of the material, allowing greater multiplexing. however, its application to microfluidic devices is complicated as, unlike fluorescence, the species under detection must be in close proximity to the metal surface for enhancement to take place. reliable quantitative measurements are difficult to achieve as sers enhancement is strongly dependent on the shape, size, and proximity of metallic features. for example, colloids are attractive enhancers in microfluidic systems as they may be analysed while flowing through the system and do not use a fixed surface within the flow channel which may become fouled. the highest enhancements using colloids occur in ''hot-spots'' where two particles are within a few nanometres of each other. adequate control of measurement conditions such as the degree of colloid aggregation, particle sizes of the colloids, and the inhomogeneous distribution of molecules on a metal surface is challenging. using a fixed patterned surface within the microflow channel has the advantage of providing a constant surface, with the disadvantage of its potential fouling and degradation. connatser et al. (2004) vapour deposited *20 nm silver onto pdms microfluidic channels to create partially embedded clusters. raman spectroscopy was performed in a conventional raman microscope with 633 nm laser illumination for detection of riboflavin and resorufin following ce separation in the channel. the raman spectra for riboflavin and resorufin gave readily distinguishable peaks at wavenumber shifts of 1200 and 600 cm -1 , respectively. novel structures such as nanowells patterned by soft lithography in pdms allow more controlled production of ''hot spots'' for sers, yielding enhancement factors of 10 7 in a pdms/glass microflow channel, configured as shown in fig. 19 , compared to a device with a smooth pdms surface (liu and lee 2005) . collection of sers spectra from flowing colloids can provide more reproducible results because of temporal averaging of the geometry and improved heat dissipation. nirode et al. (2000) demonstrated direct on-column sers in 100 lm diameter ce microcapillaries using silver colloids with nanomolar sensitivities for rhodamine 6g. flowing colloidal particles and analyte molecules show a tendency to adhere to microflow channel walls, contaminating subsequent measurements. strehle et al. (2007) performed analysis on aqueous droplets contained within flowing lipophilic tetradecane and moved along the channel by external pumps, thereby avoiding contact between colloid and the channel walls. crystal violet detections within the 60-180 nl droplets were performed in a concentration range of 10 -5 -10 -6 m, and quantification achieved by recording the amplitude of the 1177 cm -1 raman peak. fig. 19 schematic diagram of integrated microfluidic chip with nanowell structures for sers, and the raman detection apparatus (liu and lee 2005) . reproduced with permission from the american institute of physics lee and co-workers realised pdms microfluidic chips where the colloidal silver solution is mixed with the analyte using the ''alligator-teeth'' structure ( fig. 14b) in the microchannel, and the raman spectrum is acquired in a downstream detection region using a conventional raman microscope and excitation at 514.5 nm. one device was used to detect cyanide anions for pollution monitoring using a raman peak at 2100 cm -1 , achieving an lod of 1.0 ppb or below (yea et al. 2005) . using similar devices this group also reported (1) detection of two different dyelabelled dna nucleotides without the need for pcr or microspotting; tamra and cy3 dyes were used for labelling with an lod estimated at 10 -11 m (park et al. 2005) , and (2) the detection of methyl parathion insecticide with an lod of 0.1 ppm . recently, again using a similar device, molecular beacons were used to hybridise with the target dna and detected using fluorescence and sers. in the case of the sers measurements, silver colloids were mixed with the beacon solution and the dna to be hybridised was added through another inlet. the raman signal was strongly reduced if the analyte was complementary to the beacon as the change in conformation of the beacon caused the sers-active labels to be separated from the colloid (jung et al. 2007b) . cooper and co-workers employed surface-enhanced resonance raman spectroscopy (serrs) in a microfluidic system (keir et al. 2002; docherty et al. 2004) . the wavelength of the excitation source is selected to be at an analyte absorption, resulting in an increase in sensitivity and selectivity of the system, enabling improved discrimination of analytes from contaminants. a microfluidic device in glass was realised, in which silver colloid was formed on chip by insertion of an aqueous solution of agno 3 and of nabh 4 in naoh solution into two branches of a t-junction, producing a stream of aggregated colloids flowing down the centre of the third branch (keir et al. 2002) . the analyte was introduced at a downstream t-junction and 10 fmol of an azo dye derivative of trinitrotoluene (tnt) was detected using a conventional sers system with 514 nm laser excitation. a similar microfluidic device was realised by the same group in pdms, and yielded identification of up to three oligonucleotides of e. coli sequences labelled with different dyes (docherty et al. 2004) advances in optical trapping in microfluidic systems were described in sect. 2. laser tweezer raman spectroscopy (ltrs) exploits a single-beam optical gradient trap to hold and isolate a particle while performing raman spectroscopy upon it. such trapping applies to larger objects such as cells, where detection of raman scattering is less problematic, and has the advantages that the object can be held away from surroundings, to limit background interferences, can be held stationary for extended periods and then released, to allow temporal integration of spectra and improved signal to noise ratio, and can be moved in and out of the raman probe volume, to allow background subtraction and noise reduction. the optical gradient trap has been employed for some time to study raman spectra of levitated objects (thurn and kiefer 1984) . however, it is only recently that ltrs has been used in conjunction with microfluidics. xie et al. (2005) fabricated a simple microfluidic system with two microfluidic chambers joined by a microchannel. individual yeast cells and bacteria were trapped and identified from their raman spectra in one chamber and then moved to separate regions in the collection chamber for subsequent processing using the tweezer. methods to improve the sensitivity of ltrs systems by reducing or cancelling background and environmental interferences are being continually refined and incorporated in microfluidic environments. the use of separate wavelengths for trapping and raman scattering adds an extra dimension to ltrs systems. creely et al. (2005) realised a macrofluidic dual-wavelength ltrs system with excitation of the raman spectra at 785 nm, and trapping at 1,064 nm. individual living yeast cells were trapped, a raman spectrum obtained for between 0.2 and 180 s, moved out of the raman excitation beam so that a background measurement could be acquired under the same experimental conditions, and then the spectrum had the background subtracted. the approach employed a single microscope system using an excitation power and trapping power both\10 mw, allowing live cells to be observed for over 2 h, and detailed information on temporal changes in the cells to be obtained. recently rusciano et al. (2006) have demonstrated a dual-beam microfluidic system, where the raman probe beam is kept in a fixed position whilst the trapping beam position oscillates so that the trapped object is moved through the raman probe volume twice in each period of oscillation, thereby modulating the raman signal by a frequency 2f. this can then be demodulated with low noise using a lock-in amplifier, removing any stray light or unmodulated background fluorescence. the resultant signal includes a spurious component also modulated by 2f due to the photon scattering of the solvent volume. however, this second component is easily distinguished as it is shifted by p/2 radians with respect to the desired signal. dual-wavelength systems have also been realised, employing two separate microscopes, allowing greater independence in optimisation of trapping and raman detection, and applied to red blood cells, yeast cells and resonance raman spectroscopy of red blood cells (geßner et al. 2004; ramser et al. 2004) . ramser et al (2005) have developed their approach for application to a microfluidic chip for resonance raman spectroscopy of red blood cells (fig. 2) , allowing the oxygenation cycle of an individual erythrocyte to be studied. in the case of detection of small numbers of biomolecules, laser tweezer raman spectroscopy may be combined with sers to enhance raman signals by trapping micronsized metal colloid clusters or metal-coated glass spheres (xie and li 2002; geßner et al. 2004) , and taking the raman spectra of molecules absorbed on them. light incident upon a liquid sample may be absorbed by the sample, which then undergoes non-radiative relaxation and heats the surrounding volume. this heat changes the refractive index of the medium, usually lowering it. if a laser excitation beam is focussed on a small volume, the refractive index change is increased and a thermal lens (tl) is created by the thermal gradient. a laser probe beam can then be used to detect these changes. a thermal lens microscope (tlm) exploits this phenomenon by focussing a coaxial excitation beam and probe beam on the fluid through an objective lens, and detecting the power in the probe beam passing through a pinhole after filtering out the excitation wavelength, as shown for nanoparticle detection in fig. 20 . a modulated excitation laser and synchronous detection is used to reduce noise and improve detection limit. sensitivity to absorption is extremely high, as is required in microfluidic systems with small volumes, but tl is generally not able to distinguish between absorbing species, although thermal lens spectroscopy yields some compositional information. kitamori et al. (2004) , writing on the principles of tlm and its application to imaging and microfluidics, observe that the chemical specificity obtainable through separation of analyte species in microfluidics is complemented by the great sensitivity of thermal lens detection. further miniaturisation of the tlm is required for practical use in microfluidic systems as the optics used tends to be quite bulky. kitamori and co-workers have made a large contribution to the development of continuous-flow chemical processing incorporating tlm in microfluidic devices, and demonstrated its potential applications, with 0.1 nm detection limits in 7 fl detection volumes, for example (tokeshi et al. 2001) . a three-dimensional microchannel network on chip with pressure-driven flow that produced multi-phase laminar flows was able to perform 20 micro operations such as mixing and liquid-liquid extraction. the device was used to extract and analyse fe(ii) and co(ii) ions using changes in absorption spectrum (kikutani et al. 2004) . a simpler device which also makes use of multi-phased flows demonstrated an lod of 7 · 10 -8 m for carbaryl pesticide, two orders of magnitude lower than conventional spectrophotometric methods for (smirnova et al. 2006 ). these systems employed single wavelength excitation at 532 nm and detection at 633 nm. a tunable thermal lens spectrometer system using a xe lamp and monochromator as excitation source (tamaki et al. 2005 ) enabled determination of the thermal lens spectrum of non-scattering and turbid solutions, demonstrating a clear advantage over absorption detection by measuring the spectrum largely regardless of the strong light scattering background. the thermal gradient required for thermal lensing was obtained by exploiting rapid thermal conduction between the glass substrate and the liquid, rather than by tight focussing of the excitation beam. application to cultured cells was demonstrated (goto et al. 2005 ) by monitoring nitric oxide release from macrophage-like cells stimulated by lipopolysaccharide using a colorimetric reaction in a microfluidic device with localized temperature control. a successful assay was achieved within 4 h using 3,000 cells compared to 24 h and 10 5 -10 6 cells for conventional batch methods. uv excitation tlm at 266 nm was employed for detection of adenine aqueous solutions without labelling in a fused-silica microchip. the sensitivity obtained was 350 times higher than a conventional spectrophotometric method with an lod of 1.4 · 10 -8 m (hiki et al. 2006 ). detection of single and multiple 50 nm gold nanoparticles was demonstrated using tlm, as shown in fig. 20 and their optically driven fixation to the wall of the glass microchannel using the same beam was also demonstrated, having the potential for sensitive nanoparticles detection and production of microchip sers substrates . a glass microchip consisting of a microchamber for cell culture connected to a microchannel for tlm detection was used to monitor intercellular messengers . rat hippocampus nerve cells were fig. 20 principle of detection of individual nanoparticles by thermal lens microscopy . reproduced with permission from the japanese society for analytical chemistry cultured in the microchamber to form a neural network, and the retrograde messenger was then detected in the microchannel. chirality of low-volume liquid samples was measured in a microfluidic device by using tlm in which the handedness of circularly polarised excitation light was modulated (yamauchi et al. 2006a) . left-and right-circularly polarized light may be absorbed differentially in a chiral medium, and this is detected as a modulation of the detected tlm signal. miniaturised thermal lens systems have been realised utilizing, for example, optical fibres, fibre wavelength multiplexer for the two diode laser sources (658 nm excitation and 785 nm probe) and selfoc microlenses. lods of 3.7 · 10 -8 m for sunset yellow and 7.7 · 10 -9 m for nickel (ii) phthalocyanine-tetrasulfonic acid, tetrasodium salt (nip) were achieved, showing a rather small reduction in performance compared with the conventional bulk-optical detection system . employing both tlm detection and fluorescence in a microfluidic device with similar miniaturised optics yielded an lod 6.3 nm for (nip) and 3 nm for cy5 (yamauchi et al. 2006b ). kakuta et al. (2006) realised a semi-automated heterogeneous immunoassay system for brain natriuretic peptide (bnp), using tlm to detect bnp within a concentration range of 0.1-100 pg ml -1 through reaction resulting in a blue-coloured product. kitagawa et al. (2006) used ce separation in fused silica capillaries connected to a microchannel on a chip. the system achieved an lod of 3.6 · 10 -7 m for the azo dye, sudan red, more than two orders of magnitude lower than that of conventional absorbance detection. micellar electrokinetic chromatography with sample concentration by ''sweeping'' (quirino and terabe 1998) reduced the lod to 1.2 · 10 -12 m. mawatari et al. (2005) used the sensitivity of the tlm signal to focusing position to optimise precise focusing in a portable tl apparatus, in a manner similar to the optical disk drive, using a micro-optical pick-up unit rather than a microscope. adjustments of the depth were made by determination of the astigmatism of the reflected excitation beam, and the width by monitoring transmitted scattered light of the probe beam. an lod of 30 nm was achieved for xylene cyanol solution, with a sensitivity between 20 and 100 times higher than that obtained by spectrophotometry. this laboratory then presented a similar miniaturised tlm system, but with reflective rather than transmissive optics, shown in fig. 21 , reducing alignment complexity, and only requiring optical access from one side of the microfluidic chip. this was achieved by forming an aluminium mirror on the reverse of the microchip. the reflected probe beam was detected with 40 times higher sensitivity when compared to a spectrophotometer, with an lod of 60 nm for xylene cyanol solution (mawatari and shimoide 2006) . ghaleb and georges have reviewed and analysed the use of crossed-beam systems, in which the excitation beam and probe beam are perpendicular to each other, in comparison with the collinear approach normally used in tlm. in the former case, the heated sample acts as a cylindrical lens for the probe beam, and excitation light exhibits less interference with detection (ghaleb and georges 2004) . they subsequently performed a detailed optimisation of crossedbeam systems for chopping frequency, sample size and flow rate in fused silica capillaries (ghaleb and georges 2005) . the miniaturised and automated laboratory, micro-totalanalysis system, or loc, presents great benefits in terms of reagent and sample consumption, speed, precision, and automation of analysis, and thus cost and ease of use, resulting in growing adoption of microfluidic approaches for practical measurements, and the potential for their widespread use in society. in this review we have discussed a representative cross-section of recent applications of optical techniques in microfluidic systems for chemical and biological analysis, with an emphasis on further miniaturisation of the optical functions. optical techniques are the established front-runners for high sensitivity detection in microfluidic systems. while optical telecommunications benefited from needing large numbers of devices providing a few very well defined functions, optical sensing must satisfy many very diverse applications so that the optical phenomenon to be probed and the required sensitivity, specificity, speed and cost may require a different technological solution for each fig. 21 illustration of the optical arrangement of a reflective thermal lens detection device (mawatari and shimoide 2006) . reproduced with permission from the royal society of chemistry application (culshaw 2004) . the challenge is greater in microfluidic systems where the sample volumes and optical path-lengths are small, though this also provides advantages for single molecule or single cell analysis. most present systems rely upon bulk-optical systems external to the fluidic chip, such as confocal microscopes, which render portable miniaturised systems difficult to realise, although this approach does have the advantage of easy reconfigurability of the optical system. scattering has been used to yield size and shape information on cells in flow cytometry and it is expected that further advances in interrogation of scattering distributions, perhaps combined with fluorimetry, will generate powerful new automated cell recognition and sorting systems. detection through optical absorption is common in macrofluidic chemical analysis, often not requiring labelling; however, the short path length leads to poor detectability and a variety of approaches are being adopted to enhance absorption through using a variety of ''multipass'' systems and sample concentration steps. thermal lensing is advancing fast as an extremely sensitive method of performing absorption-dependent assays not requiring long path lengths. fluorescent detection, while usually requiring labelling, is so sensitive and has achieved such wide acceptance that it is by far the most dominant technique. the trends here are in moving to more stable tags such as quantum dots, and longer wavelength semiconductor sources for compactness, low cost and reduced background fluorescence. improvements are also being made in geometrical configuration by incorporating multiple parallel channels, optimising fluorescence collection angle and realising compact multiwavelength chip interrogation units. refractive index measurement is being exploited in fabry-perot etalon cavities for cell analysis and in evanescent approaches such as integrating dielectric waveguides or metal films supporting surface plasma waves within microchannels. these techniques do not require the use of labels and therefore need careful referencing or integration in separation systems to reduce the potential for non-specific interferences. nonetheless the ease of mass-production of the evanescent devices using conventional microelectronics processing renders them particularly attractive for fully integrated systems. raman spectroscopy is gaining ground in direct label-free detection and identification of species. normally, surface enhanced raman spectroscopy must be used to obtain sufficient signal, and this either uses nanostructured metal films on the walls of the fluidic channels, which can degrade or become blocked, or metallic nanoparticles added to the fluid to be analysed, which then flow out of the detection region with the sample after analysis. laser-tweezer raman spectroscopy may be used to trap and analyse larger objects such as cells without enhancement, or for species attached to enhancing beads, with the advantages that signal integration times can be long and an object can be held away from any interfering surface. optical trapping, manipulation and sorting, which extend the well-known optical tweezer technique, are growing in appeal, often as complements to hydrodynamic or electrokinetic approaches. optical sorting of particles such as cells has been carried out in several different implementations by using forces dependent on the optical characteristics of the particles to modify their velocity in a flowing liquid in a novel form of chromatography. alternatively, arrays of optical traps have been used to capture particles and release them at will; concurrent optical detection may activate the release in miniaturised versions of fluorescence-activated cell-sorters, for example. in addition to long-term trapping of cells or beads for lownoise raman detection, optical traps have been used for other measurements on individual cells, including cell rigidity. rather than tightly focussed single-beam traps, opposing dual-beam traps show advantages for these applications as they are more suited to large objects and, using lower intensities, are less likely to cause cell damage. not requiring lenses, these traps are particularly simple to implement monolithically with photolithographically positioned integrated lasers or waveguides. for twodimensional sorting in a fluidic plane, the optical potential landscape produced by interference or other patterning of a two-dimensional array of optical traps provides a particularly elegant and flexible means of separation. evanescent techniques, where a prism or waveguide is used to generate propulsion and trapping optical forces close to a surface, have been employed for sorting, guiding and separation at a surface. this approach has the advantage that the optical path does not pass through the bulk of the sample and that the intensity distribution at the surface may be very precisely controlled. however, the optical fields only reach a few hundred nanometres into the sample, so objects to be manipulated must be very close to the surface. a wide variety of optically driven micropumps have been realised, having fewer constraints on liquid medium than electrokinetic pumps, and flexible external control of the impellors without direct electrical connection to the microfluidic chip. in some cases the micropumps are completely optically configured within the channel by collecting and controlling the motion of beads, yielding great flexibility in configuration. this review set out to study progress towards the miniaturisation of optical functions in the loc, where significant advances are being made in both miniaturisation of the instrumentation and on the chips themselves. the vast majority of optical functions are still carried out with bulky external optics, but it is envisaged that with closer collaboration between the optics, biosensor and microsystems communities, low-cost mass-produced integrated systems will emerge. in the sort term, further integration of optical functions will render the loc more costly and less flexible, but the example of the microelectronics industry shows that mass-production will ultimately bring these costs down and allow widespread use of optically integrated microsystems, if further standardisation of protocols and commonality of technological platforms can be achieved. optical trapping, manipulation, and sorting of cells and colloids in microfluidic systems with diode laser bars microfluidic sorting system based on optical waveguide integration and diode laser bar trapping acceleration and trapping of particles by radiation pressure observation of a single-beam gradient force optical trap for dielectric particles development and characterization of an integrated silicon micro flow cytometer microfluidic-optical integrated cmos compatible devices for label-free biochemical sensing automated single-cell manipulation and sorting by light trapping single-step fabrication and characterization of photonic crystal biosensors with polymer microfluidic channels metal-polymer nanocomposites for integrated microfluidic separations and surface enhanced raman spectroscopic detection demonstration of a fibre-optical light-force trap diffusion coefficient measurement in a microfluidic analyzer using dual-beam microscale-refractive index gradient detection application to on-chip molecular size determination future lab-on-a-chip technologies for interrogating individual molecules monolithic integration of microfluidic channels and semiconductor lasers integrated monolithic optical manipulation dual wavelength optical tweezers for confocal raman spectroscopy optical fiber sensor technologies: opportunities and-perhaps-pitfalls microfabrication and characterisation of teflon afcoated liquid core waveguide channels in silicon control and detection of chemical reactions in microfluidic systems a novel microfluidic flow-injection analysis device with fluorescence detection for cation sensing. application to potassium serial immunoassay in parallel on a microfluidic chip for monitoring hormone secretion from living cells single-molecule fluorescence detection in microfluidic channels-the holy grail in ltas micro total analysis systems. latest advancements and trends the first serrs multiplexing from labelled oligonucleotides in a microfluidics lab-on-a-chip a non-invasive analysis method for on-chip spectrophotometric detection using liquidcore waveguiding within a 3d architecture planar waveguides for ultra-high sensitivity of the analysis of nucleic acids capillary electrophoresis separation in the presence of an immiscible boundary for droplet analysis cells on chips integrated microfluidic devices parallel transport of biological cells using individually addressable vcsel arrays as optical tweezers laser-induced fluorescence detection system for microfluidic chips based on an orthogonal optical arrangement surface plasmon resonance sensors utilizing microfabricated channels extended-area optically induced organization of microparticles on a surface optical manipulation of microparticles and cells on silicon nitride waveguides identification of biotic and abiotic particles by using a combination of optical tweezers and in situ raman spectroscopy photothermal spectrometry for detection in miniaturized systems: relevant features, strategies and recent applications signal optimisation in cw-laser crossed-beam photothermal spectrometry: influence of the chopping frequency, sample size and flow rate on-line sample preconcentration using field-amplified stacking injection in microchip capillary electrophoresis development of a microchip-based bioassay system using cultured cells recent developments in optical detection methods for microchip separations wavelength-resolved fluorescence detector for microchip capillary electrophoresis separations quantitative on-chip determination of taurine in energy and sports drinks sorting of polystyrene microspheres using a y-branched optical waveguide sensitivity enhancement for colorimetric glucose assays on whole blood by on-chip beamguidance optical deformability as an inherent cell marker for testing malignant transformation and metastatic competence bead-assisted displacement immunoassay for staphylococcal enterotoxin b on a microchip discovery of a significant optical chromatographic difference between spores of bacillus anthracis and its close relative, bacillus thuringiensis sample concentration using optical chromatography remote opto-chemical sensing with extreme sensitivity: design, fabrication and performance of a pigtailed integrated optical phase-modulated mach-zehnder interferometer system improved native uv laser induced fluorescence detection for single cell analysis in poly(dimethylsiloxane) microfluidic devices uv excitation thermal lens microscope for sensitive and nonlabeled detection of nonfluorescent molecules monolithically integrated dyedoped pdms long-pass filters for disposable on-chip fluorescence detection velocity distribution of gold nanoparticles trapped on an optical waveguide bio-assay based on single molecule fluorescence detection in microfluidic channels high throughput particle analysis: combining dielectrophoresis article focussing with confocal optical detection fabrication and testing of planar chalcogenide waveguide integrated microfluidic sensor an integrated microfluidic chip for dna/rna amplification, electrophoresis separation and on-line optical detection a microfluidic system with integrated molecular imprinting polymer films for surface plasmon resonance detection counting low-copy number proteins in a single cell optical chromatography. a new tool for separation of particles application of a microfluidic device for counting of bacteria compact fluorescence detection using in-fiber microchannels-its potential for lab-on-achip applications dual beam fibre trap for raman microspectroscopy of single cells integrated waveguide with a microfluidic channel in spiral geometry for spectroscopic applications detection of 100 am fluorophores using a high-sensitivity on-chip ce system and transient isotachophoresis fast and sensitive dna analysis using changes in the fret signals of molecular beacons in a pdms microfluidic channel development of the microchip-based repeatable immunoassay system for clinical diagnosis contact-lens type of micromachined hydrogenated amorphous si fluorescence detector coupled with microfluidic electrophoresis devices microfluidic genetic analysis with an integrated a-si:h detector movement of micrometer-sized particles in the evanescent field of a laser beam optically-driven mie particles in an evanescent field along a channeled waveguide serrs. in situ substrate formation and improved detection using microfluidics integrated optical motor cell docking inside microwells within reversibly sealed microfluidic channels for fabricating multiphenotype cell arrays poly(dimethylsiloxane)-based packaging technique for microchip fluorescence detection system applications rapid dna hybridisation analysis using a pdms microfluidic sensor and a molecular beacon micro wet analysis system using multi-phase laminar flows in threedimensional microchannel network thermal lens microscopy and microchip chemistry toward million-fold sensitivity enhancement by sweeping in capillary electrophoresis combined with thermal lens microscopic detection using an interface chip surfaceenhanced raman scattering and biophysics evolution of a colloidal critical state in an optical pinning potential landscape on-chip enzyme immunoassay of a cardiac marker using a microfluidic device combined with a portable surface plasmon resonance system microoptomechanical pumps assembled and driven by holographic optical vortex arrays integrated optical sensors for the optical domain an optically driven pump for microfluidics new frontiers in optical biosensing quantitative analysis of methyl parathion pesticides in a polydimethylsiloxane microfluidic channel using confocal surfaceenhanced raman spectroscopy a vortex pump-based optically-transparent microfluidic platform for biotech and medical applications continuous real-time bubble monitoring in microchannels using refractive index detection separation of catecholamines by microchip electrophoresis with a simple integrated laser-induced fluorescence detector microfabrication of a planar absorbance and fluorescence cell for integrated capillary electrophoresis devices nanowell surface enhanced raman scattering arrays fabricated in soft-lithography for label-free biomolecular detections in integrated microfluidics analysis of multiplex pcr fragments with pmma microchip integrated portable polymerase chain reaction-capillary electrophoresis microsystem for rapid forensic short tandem repeat typing poly(dimethylsiloxane) hollow abbe prism with microlenses for detection based on absorption and refractive index shift optimization of poly(dimethylsiloxane) hollow prisms for optical sensing integrated isotachophoretic preconcentration with zone electrophoresis separation on a quartz microchip for uv detection of flavonoids microfluidic sorting in an optical lattice cell cytometry with a light touch: sorting microscopic matter with an optical lattice biospecific interaction analysis using biosensor technology miniaturized total chemicalanalysis systems-a novel concept for chemical sensing optically driven micropump produced by three-dimensional two-photon microfabrication reflective thermal lens detection device portable thermal lens spectrometer with focusing system quantitative detection and fixation of single and multiple gold nanoparticles on a microfluidic chip by thermal lens microscope a novel concept of the integrated fluorescence detection system and its application in a lab-on-a-chip microdevice flow cytometry of escherichia coli on microfluidic devices array formation in evanescent waves integrated optics: an introduction fractionation of polydisperse colloid with acousto-optically generated potential energy landscapes absorption spectroscopy in scattering samples using integrated-optics microfluidic discharge-based optical sources for detection of biochemicals monolithic integration of optical waveguides for absorbance detection in microfabricated electrophoresis devices integration of polymer waveguides for optical detection in microfabricated chemical analysis systems pure-silica optical waveguides, fiber couplers, and highaspect ratio submicrometer channels for electrokinetic separation devices high speed simultaneous single particle impedance and fluorescence analysis on a chip optical improvements of a z-shaped cell for high-sensitivity uv absorbance detection in capillary electrophoresis slow-light enhancement of beer-lambert-bouguer absorption all-optical control of microfluidic components using form birefringence characterization of photodamage to escherichia coli in optical traps on-column surface-enhanced raman spectroscopy detection in capillary electrophoresis using running buffers containing silver colloidal solutions microfluidic protein detection through genetically engineered bacterial cells optical manipulation of objects and biological cells in microfluidic devices counting and sizing of particles and particle agglomerates in a microfluidic device using laser light scattering: application to a particle-enhanced immunoassay on-chip free-flow magnetophoresis: separation and detection of mixtures of magnetic particles in continuous flow highly sensitive signal detection of duplex dye-labelled dna oligonucleotides in a pdms microfluidic chip: confocal surface-enhanced raman spectroscopic study light-induced cell separation in a tailored optical landscape recent advances in surface plasmon resonance based techniques for bioanalysis multifunctional encoded particles for high-throughput biomolecule analysis optical spectroscopy of single trapped metal nanoparticles in solution developing optofluidic technology through the fusion of microfluidics and optics sensitivity enhancement of integrated optical sensors by use of thin high-index films exceeding 5000-fold concentration of dilute analytes in micellar electrokinetic chromatography a new type of secondary radiation resonance raman spectroscopy of optically trapped functional erythrocytes a microfluidic system enabling raman measurements of the oxygenation cycle in single optically trapped red blood cells quantitative analysis of thiols in consumer products on a microfluidic ce chip with fluorescence detection read-out concepts for multiplexed bead-based fluorescence immunoassays on centrifugal microfluidic platforms integrated light collimating system for extended optical-path-length absorbance detection in microchip-based capillary electrophoresis phase sensitive detection in raman tweezers a multireflection cell for enhanced absorbance detection in microchip-based capillary electrophoresis devices diffraction under total internal reflection for micro-fluidic analysis monitoring of intercellular messengers released from neuron networks cultured in a microchip liquid transport due to light scattering microchip flow cytometry using electrokinetic focusing optical biosensor microsystems based on the integration of highly sensitive mach-zehnder interferometer devices single-cell detection using optofluidic intracavity spectroscopy parallel analysis of biomolecules on a microfabricated capillary array chip high-throughput and high-resolution flow cytometry in molded microfluidic devices micro-multiphase laminar flows for the extraction and detection of carbaryl derivative refractive index measurement of single living cells using on-chip fabry-perot cavity characterisation of cellular optoporation with distance a reproducible surface-enhanced raman spectroscopy approach. online sers measurements in a segmented microfluidic system novel multi-depth microfluidic chip for single cell analysis composite-microlens-arrayenabled microfluidic sorting optical trapping of metallic rayleigh particles tunable thermal lens spectrometry utilizing microchannel-assisted thermal lens spectrometry microfluidic control using colloidal devices enhanced optical chromatography in a pdms microfluidic system determination of subyoctomole amounts of nonfluorescent molecules using a thermal lens microscope: subsingle-molecule determination thermal lens micro optical systems raman-microsampling technique applying optical levitation by radiation pressure sensitivity enhancement in evanescent optical waveguide sensors unconventional detection methods for microfluidic devices measurements of scattered light on a microchip flow cytometer with integrated polymer based optical elements microfluidic sorting of mammalian cells by optical force switching dielectrophoresis microsystem with integrated flow cytometers for on-line monitoring of sorting efficiency poly(dimethylsiloxane) microfluidic flow cells for surface plasmon resonance spectroscopy cell transportation via electromigration in polymer-based microfluidic devices soft lithography miniaturized immunoassay microfluidic system with electrokinetic control raman spectra and optical trapping of highly refractive and non-transparent particles raman sorting and identification of single living micro-organisms with optical tweezers simple detection of small amounts of pseudomonas cells in milk by using a microfluidic device circular dichroism thermal lens microscope for sensitive chiral analysis on microchip miniaturized thermal lens and fluorescence detection system for microchemical chips highly sensitive fluorescence detection with hg-lamp and photon counter in microchip capillary electrophoresis a cell counting/sorting system incorporated with a microfabricated flow cytometer chip ultra-sensitive trace analysis of cyanide water pollutant in a pdms microfluidic channel using surface-enhanced raman spectroscopy fast and sensitive analysis of dna hybridization in a pdms micro-fluidic channel using fluorescence resonance energy transfer a microfluidic-fcs platform for investigation on the dissociation of sp1-dna complex by doxorubicin microfluidics technology for manipulation and analysis of biological cells optical and electrochemical detection techniques for cell-based microfluidic systems integration of microfluidics with a fourchannel integrated optical young interferometer immunoassay fast, ultrasensitive virus detection using a young interferometer sensor self-referencing a single waveguide-grating sensor in a micron-sized deep flow chamber for label-free biomolecular binding assays a microfluidic chip for measurement of biomolecules using microbead-based quantum dot fluorescence assay time-of-flight optophoresis analysis of live whole cells in microfluidic channels toward the detection of a single virus particle in serum integrated microfluidic uv absorbance detector with attomol-level sensitivity for bsa acknowledgments the authors are grateful to dr olav gaute hellesø, university of tromsø, norway, for useful comments on the manuscript. key: cord-030026-4jew57ce authors: vasala, antti; hytönen, vesa p.; laitinen, olli h. title: modern tools for rapid diagnostics of antimicrobial resistance date: 2020-07-15 journal: front cell infect microbiol doi: 10.3389/fcimb.2020.00308 sha: doc_id: 30026 cord_uid: 4jew57ce fast, robust, and affordable antimicrobial susceptibility testing (ast) is required, as roughly 50% of antibiotic treatments are started with wrong antibiotics and without a proper diagnosis of the pathogen. validated growth-based ast according to eucast or clsi (european committee on antimicrobial susceptibility testing, clinical laboratory standards institute) recommendations is currently suggested to guide the antimicrobial therapy. any new ast should be validated against these standard methods. many rapid diagnostic techniques can already provide pathogen identification. some of them can additionally detect the presence of resistance genes or resistance proteins, but usually isolated pure cultures are needed for ast. we discuss the value of the technologies applying nucleic acid amplification, whole genome sequencing, and hybridization as well as immunodiagnostic and mass spectrometry-based methods and biosensor-based ast. additionally, we evaluate the potential of integrated systems applying microfluidics to integrate cultivation, lysis, purification, and signal reading steps. we discuss technologies and commercial products with potential for point-of-care testing (poct) and their capability to analyze polymicrobial samples without pre-purification steps. the purpose of this critical review is to present the needs and drivers for ast development, to show the benefits and limitations of ast methods, to introduce promising new poct-compatible technologies, and to discuss ast technologies that are likely to thrive in the future. evidence-based treatments of infectious diseases in outpatient clinics. rapid diagnostics are needed for both pathogen identification and resistance testing. the prevalence of amr may be very high for some species in certain geographic locations. according to the current recommendations on ast (antimicrobial susceptibility testing), pure culture isolates are used to test the effect of antimicrobial drugs. this is required as the sample matrix (blood, urine, mucosal) as well as the number and proportions of different microbial species may vary a lot in polymicrobial samples. it may be unclear whether the detected microbe is pathogenic or merely a commensal species. despite significant progress in diagnostic technologies in recent years, most patients with infectious diseases are still treated empirically and thus antibiotics are heavily overused (li et al., 2016; mashalla et al., 2017) . even in western countries, 30% of antibiotic prescriptions are considered to be either unnecessary or suboptimal (centers for disease control prevention, 2018). current diagnostic tests serve hospitalized patients rather well, but they are often not available in outpatient clinics. for typical growth-based ast, several cultivation rounds are required: enrichment cultivations (e.g., blood cultures) to increase the number of bacteria, plate cultivations to obtain pure cultures, and finally ast for liquid or plate cultures using various antimicrobial loads. microbiology laboratories apply the eucast-accepted breakpoint values to define whether the microbe is susceptible or resistant to the tested antibiotic. they use the disk diffusion method or other systems calibrated to eucast standards. altogether, ast may require several days. rapid molecular diagnostics has been discussed in many excellent reviews (pulido et al., 2013; plüddemann et al., 2015; li et al., 2017b; maurer et al., 2017; syal et al., 2017; maugeri et al., 2019) . they present the progress in nucleic acid amplification technology (naat), electrochemical methods, microarrays, micro-and nanoparticles, as well as mass spectrometry applications, but also emphasize that very few of the molecular methods have acquired fda approval. the review of david boyle, "tuberculosis diagnostics technology landscape" is worth reading, since, although not focusing on ast, it presents comprehensively new affordable molecular diagnostic technologies available in standard microscopy stations, particularly in developing countries (boyle, 2017) . the most up-to-date and concise progress compendia in the field of ast can be found in congress presentations, lectures and webinars. prof. mark fisher's webinar "rapid antimicrobial susceptibility testing" (arup, 2020; scientific resource for research and education: educational resources-rapid antimicrobial susceptibility testing | university of utah) is particularly useful. the innovations in electronics, biosensor techniques, optics, microfluidics, hybridization technologies and dna amplification technologies have yielded new approaches in ast. unfortunately, the scientific papers on these technologies do not sufficiently relate these findings to the practical needs in poct. the requirement of a microbiology laboratory and the time and resources needed for the enrichment of cultures, preparation of pure cultures. and sample treatments is often not sufficiently considered. these requirements also easily blur the total costs of ast. for these reasons, the deployment of new molecular methods for ast has been very slow (doern, 2018) . the standards of care for antibiotic prescription are quite consistent in most european countries and the usa and apply evidence-based id and ast when available. in total, urinary and respiratory tract infections form a significant part of acute infections. quick and accurate diagnosis for these diseases already at outpatient settings could efficiently restrict the spread of amr bacteria and allow an early isolation of the carrier and correct treatment. rapid diagnosis would also allow the prompt dismantling of unnecessary patient isolation, saving money and resources. however, technical improvements translate into benefits only if the structured communication and interpretation of the results are applied by the clinicians (maurer et al., 2017) and the cost of these technologies are reasonable. vital emergency diagnostics for septicaemia has received a lot of resources (marco, 2017; hughes, 2018) . new sensitive methods such as t2mr (t2biosystems, usa) can quickly detect molecular targets directly from clinical samples, enabling rapid pathogen identification and detection of resistance factors. however, growth-based ast for blood requires a fairly high bacterial count for enrichment cultures and a well-equipped microbiology laboratory. the achievements in blood testing do not necessarily relate well to the antimicrobial stewardship in the front-line: healthcare settings. optimal antimicrobial therapy policy would require (1) fast point of care analysis, (2) identification of the etiological agent, (3) finding of an efficient antibiotic, and (4) determination of the functional dosage. according to prof. gunnar kahlmeter (chairman, eucast general committee) (kahlmeter, 2016) , the key questions for any new ast technology are: • is it generally applicable or suitable only for one infection (for example sepsis or one resistance type)? • what is the capacity? how many organisms/agents per hour can be processed? • has the technology been validated against reference methods? • are there any reference installations? • is scientific literature available? • when will it be on the market? complete answers are hard to dig up, but this review tries to address these questions. we present the needs and drivers for ast development, increase the understanding about the role of rapid ast in diagnostics of infectious diseases, show the benefits and limitations of ast methods, introduce the key poct-compatible technologies, and contemplate on which ast technologies are likely to thrive in the future. bacteria can acquire resistance to antibiotics by several mechanisms. the antibiotic can be degraded or chemically modified (by acetylation, phosphorylation, nucleotidylation, adp-ribosylation, mono-oxygenation, glycosylation). the drug intake can be prevented, or efflux can be enhanced. some resistance mechanisms are based on the reprogramming of cell wall synthesis. even slight changes in the target molecule, e.g., a point mutation in the ribosomal protein, can render the antibiotic inefficient. the overwhelming variety of antimicrobials and resistance mechanisms complicates ast. genotypic (nucleic acid-based) methods can only find resistances that are searched for, and the potentially found resistance genes are not necessarily from the actual pathogenic organism. according to eucast and clsi guidelines, reliable antibiotic resistance diagnostics requires phenotypic testing, i.e., an experimental test whether the microorganism grows in the presence of the antibiotic. these methods work regardless of the resistance mechanism and give answers to the practical questions: which antibiotic is efficient and which dose should be applied in the therapy. classical ast techniques such as broth microdilution, disk diffusion, gradient tests, agar dilution and breakpoint tests are based on continuous exposure of a bacterial isolate to a set of antimicrobials, followed by a visual detection of growth. the use of advanced optoelectronic systems, fiber optics, microfluidics and indicator dyes sensitive to redox-state or ph can further enhance the sensitivity and performance of optical systems. several commercial systems have streamlined and partly automatized the follow-up of ast cultures. systems like vitek and microscan perform automated turbidity measurement for multiwell liquid cultures. bd phoenix system tm applies a redox indicator to enhance the detection of organism growth. these systems have turnaround times as short as 4 h for id and 6-8 h for susceptibility testing (she and bender, 2019) . the ce-marked alfed 60 ast tm system (alifax, italy) uses sensitive laser-light scattering technology to detect bacterial growth in a liquid culture broth and provides antimicrobial susceptibility results directly from positive blood culture bottles within 4-6 h. such broth dilution-based systems use ready-made ast cassettes or cards containing positive controls and wells with increasing concentrations of antibiotics. they provide continuous growth monitoring and can analyze mic patterns for a large group of organisms through their extensive databases. pathogen identification (id) is usually a preliminary step of ast. for blood samples, microscopy and gram-staining are nearly always performed, as gram-positive bacteria in general have a more limited variety of antibiotic resistances and less problems with multidrug resistance. in ast, the following sequence is typically applied: first clinical samples are cultured to obtain pure isolates. then identification (with maldi-tof mass spectrometer, if available) is performed. thereafter, ast and mic determination is performed according to eucast or clsi standards. this sequence, in total, requires several days. standard healthcare settings do not have advanced microbiology laboratories with mass spectrometry instruments. their arsenal for the diagnosis of infectious diseases may be limited to immuno-chromatographic strip tests (aka lateral flow tests = lf or "dip-sticks") applied to the detection of viruses (e.g., influenza) and bacterial pathogens causing sexually transmitted diseases. quick identification can efficiently restrict the search palette for certain antibiotics. hence mass spectrometry has become a versatile workhorse in clinical laboratories. it is routinely applied for bacterial id as soon as isolated colonies are available. through the simultaneous measurement of several metabolites a biochemical signature of microbes can be obtained. matrixassisted laser desorption/ionization time of flight (maldi-tof) applies laser energy to evaporate the matrix-bound sample, that is then immediately analyzed. when frequent sampling is applied, maldi-tof can even provide semi-quantitative growth rate data (maxson et al., 2017) . bruker corp. (germany) has launched test kits such as bt star-carba assay for ast based on antibiotic degradation monitoring. ast for blood cultures has been applied after a short-term cultivation on agar plates followed by susceptibility testing using vitek ast cards selected on the basis of maldi-tof analysis (idelevich et al., 2014; mauri et al., 2017) . by applying the mbt-astra tm test with maldi biotyper for id and ast, identification of mycobacterial strains resistant to rifampicin, isoniazid, linezolid, ethambutol, clarithromycin and rifabutin can be obtained 1 week faster than through routine cultivation-based ast (ceyssens et al., 2017) . the ms approaches combined with naat or microfluidics will be presented in "future technologies" section. many novel methods claim to perform ast in minutes or in few hours. such statements usually ignore the need of timeconsuming steps such as enrichment cultures and isolation of pure cultures (figure 1) . methods based on naat, nucleic acid hybridization or immunodiagnostics in principle allow the use of non-purified polymicrobial clinical samples. a short cultivation with a pre-determined antibiotic load followed by naat (e.g., isothermal amplification) can reveal ar, and even provide a rough estimate of the minimal inhibitory concentration (mic) for the tested antibiotics. most rapid growth-based ast methods perform end-point analysis only, whilst others rely on frequent sampling from the cultivation chamber. some sensitive immunodiagnostic systems however provide real on-line growth monitoring (figure 2) . biosensor technologies detecting changes in microbial metabolism, movement or heat production have not yet provided convincing clinical demonstrations. fast, reliable, easy-to-use and inexpensive systems applicable to ast in outpatient clinics are still elusive (van belkum et al., 2019a) . figure 2 | usability landscape of rapid ast technologies. naat, nucleic acid amplification technology; tpx, immunodetection based on two-photon excitation fluorometry; multipath, immunodiagnostic method applying magnetic spheres for cell separation, fluorescent nanoparticles for labeling and non-microscopic imaging. frontiers in cellular and infection microbiology | www.frontiersin.org counting of bacteria on agar plates by microscopy techniques is possible long before they have reached the number providing visible colonies. e. coli colonies visible by eye contain roughly 5 × 10 6 bacteria, but by microscopy microcolonies formed by 120 cells can already be detected (london et al., 2010) . drug susceptibility (mods) for mycobacterium tuberculosis can be assessed by observing cell aggregates (cords) microscopically in sealed microtiter plates (den hertog et al., 2014) . the growth direct system by rapid micro biosystems inc. detects microcolonies with digital imaging by illuminating them with blue light and directing the cellular autofluorescence directly onto a ccd chip without magnification. the average time for e. coli detection by this autofluorescence was 3.1 h compared to an average of 8.5 h for the visual plate counting method. although the idea of applying this to ast has been declared in the patent application, clinical studies for ast have not yet been presented. automated microscopy systems can provide real time growth curves, and quantitative bacterial counts have been presented. multiplexed automated digital microscopy (madm) applying fluorescent in situ hybridization (fish) has been commercialized by accelerated diagnostics (usa) for rapid online ast (metzger et al., 2014; chantell, 2015) . the accelerate pheno r system can separate impurities from clinical samples (e.g., blood or urine) by brief electrophoresis, which runs impurities into a gel. after this a change of the electric field polarity repels the microbes back to the liquid. a fluorescence signal is detected every 10 min from samples taken from the bacterial culture multiplying in mueller-hinton media . this system seems to be currently the only fda-approved growth-based rapid diagnostic ast system (doern, 2018) . the performance of accelerate pheno r has been demonstrated with many clinical studies, e.g., with urinary tract infections and bloodstream infections marschal et al., 2017; descours et al., 2018) . with 232 positive blood cultures tested, the overall essential agreement with routine methods was 95.1%, and the time needed for ast was decreased by 42 h in comparison to standard growth-based analysis. id could be obtained in 1.5 h and ast in 7 h . fish is a highly specific method to visualize the presence of the target organism in a quantitative manner. the pna-fish technology applies peptide nucleic acid probes which allow more rapid and specific binding than dna or rna probes (perry-o'keefe et al., 2001; almeida et al., 2009; cerqueira et al., 2011) . it is applied in the commercial quickfish technology (opgen, usa) which performs id by targeting 16s rrna (enroth et al., 2019) . xpressfish specifically detects the meca gene in staphylococcus, allowing, when used subsequent to quickfishbased identification, diagnosis of methicillin resistance already 2 h after the blood culture turns positive (salimnia et al., 2014) . since a temperature of 55 • c is needed for target cell permeabilization, fixing and hybridization, pna-fish systems are not applicable to on-line growth monitoring. fda-approved systems are available also from biomérieux (durham, nc). clinical laboratories applying mass spectrometry are unlikely to need pna-fish technology; the bruker maldi septityper r kit pbp2a, e.g., can detect the meca-encoded pbp2a-protein in 1 h with fairly low costs. in actively growing cells, rna is more abundant than dna and thus a good target for probing. especially precursor rrna (pre-rrna), the intermediate stage in the formation of mature rrna, is a good indicator of bacterial metabolism, viability, and growth rate (halford et al., 2013) . the biosensorbased ast (b-ast) system from genefluidics inc. (ca, usa) measures bacterial growth by quantifying 16s rrna molecules with an electrochemical biosensor. this system uses speciesspecific probes and integrates nanotechnology, plastic micro electromechanical system and microfluidics . this system achieved a detection limit of 10 4 cfu ml −1 in rapid ast of clinical urine and blood samples (liu et al., 2014) . mohan et al. demonstrated the simultaneous detection of uropathogens and the host biomarker lactoferrin in urinary tract infection, but reached only 89% sensitivity in the pathogen identification . in 2018 genefluidics announced ce-ivd marking for the utimax tm kit, which provides id in 30 min and ast in 2 h from urine with an overall sensitivity of 100% and specificity of 98.2% (genefludics inc., n.d.). the first generation "molecular tests" such as restriction fragment length polymorphism, pulsed-field electrophoresis, multiple locus tandem repeat analysis, multi-locus sequence typing and virulence genotyping were suitable rather for typing and outbreak investigation than for ast. since these technologies require a high amount of purified nucleic acid, they do not allow rapid diagnostics. however, hybridization-based approaches and molecular beacon systems have persisted and are creatively combined with naat technologies. naat is a very powerful tool for pathogen identification, especially when combined with a syndromic approach. many diagnostic panels provided e.g., by biomérieux, elitech, bosch, eplex, qiagen, or becton dickinson, include detection of specific ar-genes. they can provide clinically relevant results, especially in cases where a detailed antibiogram is not needed. for example, pathogen such as bordetella, legionella, mycoplasma, chlamydia trachomatis, or neisseria gonorrhea exhibit quite few antibiotic resistances. the detection of specific ar genes, however, cannot give an undisputed proof of antibiotic resistance. the identified ar genes do not necessarily relate to the pathogen causing the disease, or the found resistance gene may not be functional. naat neither defines the mics nor directly indicates which antibiotics should be used. an advantage of naat is that the tests can be relatively quickly updated for newly emerging pathogens and resistance factors. quantitative pcr (qpcr) allows a rough quantification of microbes. quantitative reverse transcription pcr (qrt-pcr) can additionally assess the expression level of resistance genes after exposure to different antibiotic loads and thus provide rough mic values. the cost of devices and reagents for qrt-pcr are unfortunately currently far beyond the level acceptable for routine ast. naat is a powerful tool for the identification of both bacterial and viral pathogens. in principle it allows the use of patient samples without enrichment cultivations. however, due to the risk of losing the template during nucleic acid extraction and the sensitivity of dna polymerases to impurities in the sample matrix, enrichment cultivations and nucleic acid purification are often necessary. due to the vast choice of different fluorescent labels, several target genes can be conveniently tested in parallel from the same sample. the systems can reach further sensitivity and specificity by applying hybridization to dna arrays. a good number of fdaapproved multiplexed diagnostic panels are already available. such commercial systems include xpert r (cepheid inc.), eplex (genmark diagnostics), unyvero (curetis ag), bd max (becton-dickinson), septifast (roche), magicplex (seegene), novodiag (mobidiag), and genomera (abacus diagnostics). the xpert r technology combines sample preparation, real-time pcr and nucleic acid analysis with molecular beacons. the eplex system provides electrochemical detection of the amplified sequences, wherein the detection is achieved by hybridizing ferrocene-labeled probes with the sample dna (nijhuis et al., 2017) . hybridization techniques and microfluidics have been implemented also to novodiag's genomera microarray platform. it applies time-resolved fluorescence detection of the amplified product on a sealable plastic chip packed with dry chemistry. also curetis, becton-dickinson, roche and seegene products apply cartridge-based integrated designs (hughes, 2018) . fast analytics of pcr products enhance the throughput of naat systems. t2biosystems has recently launched a test panel able to detect 13 resistance genes from both gram-positive and gram-negative pathogens directly from blood. the amplification products are detected by magnetic resonance after hybridization with dna probes conjugated with superparamagnetic particles (hong nguyen et al., 2019) . pcr/electrospray ionization-mass spectrometry (iridica pcr/esi-ms by abbott laboratories inc., usa) allows the detection of >750 different bacterial species in a single test (strålin et al., 2016) . the high resolution melting system (hrm, by thermofisher) identifies variations in nucleic acid sequences by detecting small differences in pcr melting curves. a melting curve analysis for real-time quantitative pcr or digital pcr (wherein the sample is partitioned into a large number of individual wells each containing either 1 or 0 targets) performed for growing bacterial cultures, revealed both id and antimicrobial susceptibility profiles for e. coli, e. faecalis, p. mirabilis and s. aureus in ∼6.5 h, when analyzed by machine learning algorithms (athamanolap et al., 2017) . for routine analytics, such systems are still too expensive, and they require separate kits for dna extraction and pcr. however, new enzymes and technologies such as ligase chain reaction (lcr) (barany, 1991) , nucleic acid sequence-based amplification (nasba) (compton, 1991) , strand displacement amplification (sda) (walker et al., 1992) and loop-mediated isothermal amplification (lamp) (notomi et al., 2000) have simplified nucleic acid amplification, made it more robust (often allowing the use of samples without na extraction), enabled miniaturization and cut down the costs of instrumentation by allowing naat at a constant (isothermal) temperature. nuclisens r easyq r (biomerieux) was the first automated system to combine nasba and real-time detection using molecular beacon probes. it enabled the fast detection of klebsiella carbapenemase genes (spanu et al., 2012) . lcr has been successfully used for the detection of ciprofloxacin and doxycycline resistance genes in bacillus anthracis, francisella tularensis, and yersinia pestis (oblath et al., 2013) . lcr can be easily integrated into detection systems such as electrochemical and magnetic biosensors, quantum dots, quartz crystal and leaky surface acoustic surface biosensors, surface enhanced raman scattering (sers), chemiluminescence and fluorescence resonance energy transfer (oblath et al., 2013) . high-throughput multiplex genotyping can be achieved also by dnazyme (dna oligonucleotides) technology, rolling circle amplification (rca) and strand displacement amplification (sda) techniques. lamp is especially robust, since it is less sensitive to inhibitors than standard pcr. this allows analysis after a minimal processing of blood (curtis et al., 2008) , urine or stool (francois et al., 2011) . lamp is applicable to low-resource field settings where dna or rna extraction is not possible. however multiplexing approaches are less developed for lamp than for pcr (sahoo et al., 2016) . immunodetection is a specific and sensitive method for the identification of bacterial pathogens (verma et al., 2013) , toxin proteins (zhu et al., 2014) , and viruses. since immunodetection does not necessarily require disruption of the target microbes, it can potentially provide pathogen identification and growth monitoring in a single step. it is applicable as simple lateral flow (lf) tests, but can also be integrated to biosensor technology, microfluidics and even to dna/rna-based analysis. companies like mizuho medy, alere, and beckton dickinson have launched several easy-to-use stick tests for clinical diagnostics of (influenza) viruses and bacteria causing sexually transmitted diseases. the binders are typically antibodies that are immobilized onto strips, micro/nanoparticle beads or biosensor surfaces providing an efficient and specific target binding. the detection antibody can be labeled with fluorescent dyes or redox enzymes to provide a quantitative signal. only few products are available for the direct detection of antibiotic resistance proteins. the lf-test developed by kitao et al. detects chloramphenicol resistance in p. aeruginosa samples (kitao et al., 2010) . alere inc. has launched an immunochromatography test for the detection of mrsa, based on a pbp2a-specific chicken igy antibody (yamada et al., 2013) . the pbp2a sa culture colony test can identify mrsa in 6 min (trienski et al., 2013; delport et al., 2016) . coris bioconcept (belgium) has launched tests for the detection of carbapenemases (oxa-48-like, kpc, and ndm type) from enterobacterial isolates (bogaerts et al., 2013; glupczynski et al., 2017) . boutal et al. have presented lf tests for the carbapenemases tx-m-15, ndm, oxa-48-like, kpc, imp, and vim (boutal et al., 2017) . lf tests work well with isolated clinical isolates. they are, however, generally not applicable for direct analysis of clinical samples. the commercial multipath tm platform (first light diagnostics inc., usa) applies non-magnified digital imaging for the detection of biomolecules tagged with antibody-coated fluorescent nanoparticles. the maripoc r system by arcdia ltd, based on two-photon excitation fluoroscopy technology (tpx), is already in diagnostic use for immunogenic detection of pathogens. these two technologies will be discussed closer in the following sections. mass spectrometry is likely to become tightly integrated into other ast technologies, especially in the diagnostics of septicaemia. in the maldi-tof direct-on-target microdroplet growth assay (dot-mga), sample droplets (culture plus antibiotics in 6 µl volume) are spotted directly onto disposable ms-target plates, incubated for 3-4 h and then analyzed with ms (idelevich et al., 2018) . screening panels for esbl and ampc β-lactamases of enterobacteria are already available (correa-martínez et al., 2019) . the fast progress in microfluidics, biosensor technologies, isothermal amplification-based naat, and immunodetection has recently provided several potent systems which may eventually change the paradigms of ast. gradientech's quickmic system combines microfluidics with automated time-lapse photomicrography to follow growth inhibition along a linear drug gradient. it measures the greyscale intensity changes in the images caused by the formation of microcolonies and provides ast in 2-5 h (malmberg et al., 2016) . the system is currently seeking ce-ivd marking and fda approval for ast in blood samples. q-linea astar r applies time-lapse microscopy to fully automated monitoring of blood cultures as well as preparation and monitoring of bacterial isolates. it processes 12 samples at a time and 50 samples a day, delivering true mic values for up to 48 antibiotics within 6 h. the system does not perform id, but it can be connected to any id system. clinical trials will start in 2020. the bacterioscan 216dx system (st. louis, mo, usa) measures both a sample's optical density (od) and the scattered intensity by forward laser light scattering, allowing 10-100-fold higher sensitivity compared to normal od measurements. this system can process 16 samples simultaneously and perform realtime continuous growth measurement. it can detect bacterial growth in 3 h from clinical urine samples containing >10 4 cfu/ml of e. coli, staphylococcus aureus, pseudomonas aeruginosa, bacillus antracis, yersinia pestis, or burkholderia pseudomallei (bugrysheva et al., 2016; hayden et al., 2016; montgomery et al., 2017) . the bacterioscan 216r rapid ast system is currently under testing. the ocelloscope tm system (biosense solutions, denmark) applies digital time-lapse angled field microscopy and image analysis for standard 96-well plates. the time-to-result for positive blood cultures ranged from 1 to 4.2 h (fredborg et al., 2015) . however, the overall performance for ast has not yet been sufficiently tested. changes in individual cell morphology or size indicate growth long before bacteria have multiplied. single-cell morphological analysis (scma) uses bright-field microscopy to determine antibiotic-induced changes in cells immobilized on an agarose channel chip (choi et al., 2014) . the commercial multipath tm platform applies non-magnified digital imaging for the detection of biomolecules tagged with antibody-coated fluorescent nanoparticles. antibody-coated magnetic particles are used to bind to target cells and to pull them down to the camera surface, thus eliminating background signal and enabling a wash-free assay for clinical samples. the system counts individual targets in large areas and performs growth monitoring for multiple targets cells and enables the determination of mic values. the multipath tm system is currently seeking fda clearance. the two-photon excitation fluoroscopy technology (tpx), commercialized by arcdia ltd. (finland), allows separation-free detection of biological molecules in small reaction volumes by immunodetection (vakkila et al., 2015) . the maripoc r test system was developed for rapid pathogen identification. it applies polystyrene microparticles as solid carriers for immunocomplex formation, that leads to three-component immunocomplexes (monoclonal antibody-antigen-labeled monoclonal antibody) on the microspheres in proportion to the analyte concentration. the detection of the immunoassay fluorescence signal is achieved by two-photon excitation from the surface of individual microspheres. this mechanism allows the use of unpurified clinical samples and on-line monitoring of viable cells in ast cultures. at present this system is under clinical testing for ast. nanostring technologies inc. combine genotypic and phenotypic ast through rna detection. the gophast-r platform detects mrna expression signatures in bacteria after antibiotic exposure. the system can be used directly for positive blood culture bottles. it couples machine learning analysis of transcriptional changes with the detection of resistance genes (bhattacharyya et al., 2019) . colorimetric sensor arrays provide an inexpensive method to detect volatile organic compounds (vocs) associated with microbial metabolism (lonsdale et al., 2013; lim et al., 2014) . specific technologies inc (usa) has commercialized the small molecule sensor (sms) array technology which reacts with the metabolic products of bacteria produced during their growth. their reveal-ast printed sensor array system responds to the volatiles emitted during growth producing a colorimetric pattern. it reveals also species id with 94% accuracy (sharp, 2020) . the slipchip/dlamp technology by caltech integrates seamlessly cultivation and semiquantitative smartphone-based visual analysis of na products (schoepp et al., 2017) . this system, currently being commercialized by talis inc. (usa), will be described closer in the chapter "poct-compatible technologies." progress in whole genome sequencing (wgs) technologies has made them a feasible system for pathogen id and ast (van belkum and rochas, 2018) . 3rd generation systems such as illumina miniseq, pacific biosciences pacbio sequel system, or oxford nanopore minion and promethion can provide fairly long reads at high speed. in principle, wgs can simultaneously provide fast pathogen id, epidemiological typing, and detection of drug susceptibility genes. since wgs provides a massive amount of data in fragmented form, sophisticated software is needed to interpret the results (quainoo et al., 2017) . the european committee on antimicrobial susceptibility testing reviewed in 2017 the development status of wgs for ast (ellington et al., 2017) . they concluded that, for most bacteria, the available evidence for wgs as an ast tool is still either poor or non-existent and thus inadequate for clinical decision making. they pointed out the urgent need for a single database of all known resistance genes/mutations to facilitate comparison between different systems and bioinformatics tools. kadlec et al. combined smartphone technology to a microphotometric system applying microwell plates coated with antibiotics and the yellow redox indicator dye tetrazolium salt wst-8, which turns orange by the metabolic activity of growing cells. the system could correctly monitor the growth of several pathogens associated with urine tract infection. samples with concentrations of 10 1 to 10 6 cfu/ml could be tested directly without preliminary enrichment cultivations (kadlec et al., 2014) . feng et al. presented an automated smartphone-based device with a 3d-printed attachment holding a microwell plate. a light-emitting diode array and fiber-based optics enabled detection of turbidity changes in wells already after 1 min (feng et al., 2016) . this system, tested for 17 antibiotics targeting gram-negative bacteria on clinical isolates of k. pneumoniae, provided drug susceptibility interpretation with accuracy of 99.23%. cui et al. have presented a smartphone-based system for the monitoring of viable bacteria in droplet-based single-cell microdroplet cultures (cui et al., 2018) . in this system, single bacteria were encapsulated in monodisperse microdroplets. this dspc (digital standard plate count) platform could quantify e. coli and b. subtilis in 6 h, compared to 24 h needed for traditional plate counting. these smartphone demonstrations discussed above were performed using pure culture isolates. smartphone-based systems applying immunodetection have been demonstrated for id, but so far not for growthbased ast. wang et al. introduced a microwell plate-based microphotometric system, which applied a field-of-view adapter and a microprism array between the mobile phone camera and the 96-well plates. in a serological analysis (771 patient samples in 12 serology assays for bacterial/viral infections) the system exhibited 97.59∼99.90% analytical accuracy in pathogen identification with costs of ∼50 usd per 96-well plate and analytical quality sufficient for poct . an inexpensive (under 100 usd) smartphone-based monitoring system for nucleic acid amplification, smart-lamp, was introduced by barnes et al. the system contains a hot plate for isothermal amplification, two flexible cables and 96 led lights fitted into a cardboard box. the bacticount software detects the emitted green light as a result of a successful amplification and automatically determines the genome copy number in real time. smart-lamp was shown to work well with diverse gramnegative and gram-positive pathogens in biological specimens, giving in ∼1 h results with matched standard cultivation-based tests. reliable pathogen id was obtained for spiked urea and blood samples as well as for urea samples of sepsis patients (10 5 −10 8 cfus). the small sample size (2 µl per reaction), however, limits its use for very diluted samples (barnes et al., 2018) . priya et al. have successfully coupled loop-mediated isothermal amplification (rt-lamp) and sensitive quenching of unincorporated amplification signal reporters (quasr) detection technologies to visual detection with a smartphone. the portable "lamp box" was successfully used for the sensitive and specific detection of zika, chikungunya, and dengue viruses (priye et al., 2017) . this system has not yet been applied to growth-based ast. choi et al. have developed a rapid antimicrobial susceptibility testing system, drast. it can determine the ar from a positive blood culture bottle in 6 h (choi et al., 2017) . the sample is mixed with agarose and inoculated into a well of a plastic microchip. addition of cultivation medium forms a liquid bridge between the growth chamber and the satellite well, which contains the antibiotic agent. using microscopic detection of bacterial colony formation in agarose, the total time-to-result was only 6 h with a wide range of bacterial concentrations. the tested clinical isolates (n=206) included 16 gram-negative species and seven grampositive species, and the drast system agreed with a standard microdilution test with an accuracy rate of 91.11% (choi et al., 2017) . matsumoto et al. described a microfluidic channel method for rapid (3 h) ast for p. aeruginosa by automated microscopic detection of cell number and cell morphology (matsumoto et al., 2016) . their drug susceptibility testing microfluidic device (dstm) consisted of five sets of four microfluidic channels and allowed simultaneous microscopic observation. susceptibilities to the antibiotics (pre-dried into each channel) were evaluated by the differences in cell number and shape between drug-treated and control cells. hundred and one clinically isolated strains of p. aeruginosa tested with dstm correlated strongly with the results obtained using the conventional microbroth dilution method (matsumoto et al., 2016) . this system waits for applicability testing with other organisms. in nanowell ast, morphotyping with a phase contrast microscopy and optical signal analysis is performed for 0.5 µl cultures. antibiotic susceptibility data can be obtained for uropathogens in <4 h. the system showed a total categorical agreement of 97.9% with standard disk diffusion assays, but a careful standardization of cell densities prior to cultivation was found necessary (veses-garcia et al., 2018). mezger et al. presented a generic method for rapid species identification and ast after 0.5-2 h cultivation (mezger et al., 2015) . cultured bacteria from urine samples were lysed by sodium hydroxide and heat, and dna was captured onto magnetic beads. padlock probes targeting the 16s rrna gene were hybridized, ligated and amplified by the circle-to-circle amplification method. optical imaging system then performed digital quantification. antibiotic susceptibility profiles of e. coli for ciprofloxacin and trimethoprim could be determined with 100% accuracy in 3.5 h (mezger et al., 2015) . devices detecting volatile compounds, so-called electronic noses (enose), can recognize a smell-print characteristic for bacterial species and their metabolic profile. since 1982, electronic noses have been applied in diagnostics (persaud and dodd, 1982) , but mainly for pathogen id. the cyranose system (smiths detection) was able to distinguish between controls and samples positive for s. aureus, s. pneumoniae, haemophilus influenzae, and p. aeruginosa in upper respiratory tract infections (lai et al., 2002) . gas chromatography connected to ion mobility spectrometry (gc-ims e-nose) could reliably distinguish bacterial infections from viral respiratory tract infections (lewis et al., 2017) . saviauk et al. performed an applicability test for the chempro 100i ion mobility spectrometry sensor (environics inc.). they could discriminate mrsa from mssa with 83% sensitivity and 100% specificity (saviauk et al., 2018) and were able to identify also other pathogens (p. aeruginosa, enterococcus, e. coli, and clostridium perfringens) from culture plates with 78% accuracy. in order to access the ast market, the enose systems should outperform the simple and inexpensive colorimetric sensor array system "reveal-ast" (specific technologies, usa) which is applicable to growth-based ast. real-time qpcr can detect quantitative differences between cultures exposed to various antibiotics and different concentrations. already a 15 min cultivation can provide a detectable increase of nucleic acids (schoepp et al., 2017) . although qpcr devices are expensive and require experienced personnel for their operation, low-cost devices based on isothermal amplification might change the game thoroughly. applying chip electronics and microfluidics, caltech (the technical university of california) has developed a device applicable to ast. this system is currently under commercialization by slipchip corp and will be described closer later in this review in the section "poc-compatible technology." biosensors are devices that measure biological or chemical reactions by generating signals proportional to the concentration of an analyte in the reaction. exposure to antibiotics causes detectable changes in bacterial membranes, morphology, metabolism, movements, mass, heat production and nucleic acid content. in microcalorimetry approaches, heat production correlates with the number of cells arising over time (von ah et al., 2009 ). this approach is applicable to both solid and liquid cultures (howell et al., 2012) . dynamic heat flow patterns have served species identification from urine samples (bonkat et al., 2012) . isothermal microcalorimetry revealed vancomycin-resistant staphylococcus aureus in <8 h (entenza et al., 2014) . butini et al. applied isothermal microcalorimetry to real-time monitoring of microbial viability in biofilms in the presence or absence of antimicrobial compounds (butini et al., 2018) . microcalorimetric methods, although fast and sensitive, require pure cultures and a fairly high number of bacterial cells. in 2017, the swedish company symcel announced an extensive 28-months clinical testing of their microcalorimeter calscreener tm for ast. however, currently no clinically validated microcalorimetry systems are available. for some antibiotics (beta-lactams, chloramphenicol) amr can be assayed by the follow-up of antibiotic degradation. the byg carba test detects conductivity changes caused by the enzymatic hydrolysis reaction of imipenem antibiotics on an electrode coated with polyaniline, which is highly sensitive to changes in ph or redox potential. with a loop-full of bacteria (10 µl) from a fresh plate as a sample, this home-made inexpensive instrument could detect carbapenem resistance in <35 min displaying 95% sensitivity and 100% specificity in comparison to pcr-based analysis (bogaerts et al., 2016) . mecklenburg et al. developed an assay that directly detects the thermal signal generated from the enzymatic breakdown of antibiotics. the system was able to distinguish between penicillinase and metalloβ-lactamase (mecklenburg et al., 2017) . its value for clinical work needs to be evaluated, as it requires pure cultures and does not provide pathogen id. a variety of electrochemical reporters for cell viability have been applied to viability analysis and drug susceptibility measurements. the system by besant et al. uses resazurin dye (an oxidation-reduction indicator) to monitor cells trapped in nanoliter wells (besant et al., 2015) . within 1 h the microfabricated device could detect the response of e. coli and k. pneumoniae exposed to ampicillin and ciprofloxacin in urine samples spiked with bacteria in concentrations as low as 1 cfu/µl. the level of commercialization of this technology is not known. in microelectromechanical systems (mems) the deflections associated with the micromotions of bacteria attached to a microcantilever provide a signature of bacterial metabolism. such changes can indicate growth long before the bacteria replicate. with bacteria captured in bi-material microchannel cantilevers, quantitative antibiograms have been obtained for e. coli and s. aureus within 2 h (etayash et al., 2016) . the bacteria absorb infrared photons and release heat to the support matrix by a process of vibrational energy relaxation, inducing bending of the bimetallic cantilever proportional to the quantity of the released energy. high sensitivity, corresponding to a single cell per µl, was obtained with listeria-containing samples. the researchers plan to further integrate sample separation techniques into this biomaterial cantilever platform. lifescale analytics (nc, usa) has already launched a commercial product which correlates cantilever vibration to biomass for mic determination (burg et al., 2007) . this system performs automated cell counting, mass measurement, and visual observation of liquid samples for ast in <3 h, but requires cell concentrations above 10 4 cells/ml. the company has not yet presented peer-reviewed clinical ast studies for this instrument. micromotions are affected by flowing liquids, and inefficient transfer of antibiotics to immobilized bacteria can distort the results. therefore pre-enrichment and pre-purification of bacteria may be necessary (li et al., 2017b; syal et al., 2017) . a low-cost and rapid biosensor has been developed employing photoluminescence emission of photo-corroded gaas/algaas biochips. growing bacteria protect the biosensor surface against photo-corrosion, while non-growing or dead cells give a higher signal. these biochips exposed to a e. coli and legionella pneumophila cultures (w/o antibiotics) were capable of quantifying electrically charged bacteria in 4.5 h (nazemi et al., 2017) . the field effect enzymatic detection (feed) biosensor platform can detect extremely low bacterial concentrations (below 10 c.f.u./ml). an electrical field between the working electrode and the immune complex multiplies the biocatalytic output current, enabling a direct detection of bacteria without sample processing (shi et al., 2018) . these biosensors apply horse radish peroxidase (hrp) as a redox source in the sandwich hybridization complex. commercial devices applying this technology for ast may potentially emerge in coming years. flow cytometry (fc) can provide excitation/emission spectra of cells and give information about cell-counts, morphology and viability, enabling ast in 2-3 h. due to the large amount of raw data, mathematical methods such as adaptive multidimensional statistics must be applied for analysis (huang et al., 2015) . successful demonstrations with clinical polymicrobial samples are currently missing. flow cytometric assays struggle with complex patient samples, inefficient staining, the presence of autofluorescence, the inability to differentiate cellular damage for the influence of antibiotics, and lack of clinical databases for validation. flow cytometry instruments are very costly, making this technology an unlikely candidate for poct. atomic force microscope (afm) allows real time monitoring of bacterial activity. this principle is applicable to both cultivable and non-cultivable cells (longo et al., 2013) , but is an expensive and tedious system for clinical work. plasmonic imaging and tracking (pit) can be used for the monitoring of nanometerscale motions of single bacterial cells before and after antibiotic addition. the observed image contrast fluctuations were found to indicate changes in bacterial metabolism long before cell replication . asynchronous magnetic bead rotation (ambr) is a non-microscopy-based approach capable of monitoring individual cells for elongation, generation time, lag time, division, as well as sensitivity to antibiotics. it has been successfully tested with e. coli attached to anti-e. coli functionalized beads (kinnunen et al., 2011) . this system is still at the research stage and a complex technology regarding handling, setup and the need for expertise (schumacher et al., 2018) . lab-on-a-chip systems combine one or several laboratory functions into a single integrated circuit. the microfluidic ast platforms typically apply channels containing pre-loaded dried antibiotics. following on-chip cultivation, the channels can be monitored with a detection device such as a phase contrast microscope. some systems use agar to immobilize the bacteria and to form growth chambers. also gradient-forming microfluidic platforms have been applied (luka et al., 2015; campbell et al., 2016) . he et al. introduced a chip applying antibody-coated glass beads to capture e. coli o157 and to provide fluorescence detection for id and ast. their device had an integrated antibiotic release system, which could identify this strain at a range of 10 4 -10 8 cfu/ml within 30 min (he et al., 2014) . li et al. integrated antibody-coated carbon nanotubes with isothermal amplification. the carbon nanotube multilayer could perform selective capture, cultivation and release of the bacteria. after cultivation the antibody-entrapped bacteria were lysed, and their dna was accurately quantified by lamp. this system could detect e. coli o157:h7 and its toxin at concentrations as low as 1 cfu/ml without any complicated instrumentation (li et al., 2017a) . its use for growth-based ast has not yet been presented. researchers at hong kong baptist university have developed a multidimensional ast system for growth-based ast, providing detection through automated microscopy in 4 h. they established a hydrogel microfluidic chip which simulates drug diffusion and pathogen killing processes inside the human body. due to the chip's multidimensionality, several antibiotics, nutrients or immunologic substances could be tested simultaneously. this system is currently under commercialization liu et al., 2017a,b) . weibull et al. have presented a nanowell ast device capable of real-time optical reading and growth data analysis. this "stationary nanoliter droplet array" (snda) system can provide id and ast for urine samples within one working shift. a filtering process first isolates bacteria from the clinical urine samples into a growth medium, supplemented with 10% resazurin (oxidation-reduction indicator). applying 12 bacteria-antibiotic combinations precise mic determinations could be obtained in 3 h, which is 6-fold faster than traditional broth microdilution assays in 96-well plates. however, follow-up studies with clinical isolates of both gram-negative and grampositive bacteria are needed (weibull et al., 2014) . veses-garcia et al. applied 0.5 µl cultures for uropathogens on a 672-nanowell slide equipped with optical signal analysis. they were able to define a precise minimum inhibitory concentration for 70 clinical e. coli isolates. algorithm-assisted optical analysis determined antibiotic susceptibility in 3 h 40 min, showing a total categorical agreement of 97.9% (veses-garcia et al., 2018) . the "integrated comprehensive droplet digital detection" (ic 3d) system is capable of detecting bacteria directly from diluted blood within 1.5-4 h. it consists of bacteria-specific dnazyme-based sensors, a droplet microencapsulation system, lysozyme and a 3d particle counter system. the ongoing work aims to develop an automated, portable device for multiplexed and rapid detection of antibiotic-resistant strains (kang et al., 2014) . the ultrafast parallelized microfluidic platform consists of four arrays (each holding 110 pl droplets with 1-4 bacteria) which are screened by dynamic imaging over 2 h. this imaging-based ast was successfully tested with four types of pathogens causing urinary tract infection (uti) (kang et al., 2019) . systems with a low price and high speed are needed in all healthcare sectors, but especially in outpatient clinics and in developing countries. point-of-care tests (poct) could bring the diagnostics into outpatient clinics and thus facilitate evidence-based medication in places where extensive prescription of antibiotics happens. very few rapid ast systems are currently available, but the ongoing development is promising. david boyle has made an excellent review of affordable technologies and devices applicable to "standard microscopy stations" (boyle, 2017) . the devices presented below can provide a fast pathogen identification. some of these are already now applicable to ast, and some devices can be even applied to diagnostics of non-infectious diseases as well. scanogen inc. (usa) develops dna-based (nonamplification-based) "single-molecule biosensors" bound to microparticles, which can convert the hybridization signal to an optical signal. the device contains an inexpensive and low-power light-emitting diode ring and uses disposable sample cartridges, abolishing the need for manual sample preparation. the company is currently developing diagnostic assays for infectious diseases and drug resistance. the microbedx technology (microbedx inc., ca, usa) applies a transformational ribosomal rna-based assay on a microfluidic disc platform. the disc holds 150 nucleic acid capture probes spotted onto a glass slide. this system can discriminate four clinically relevant staphylococcus species that differ by a single nucleotide polymorphism (snp) in diagnostic probe sequences. the protocol includes hybridization, washing, rinsing, and drying steps and does not require purification of the target nucleic acids (peytavi, 2005) . in a clinical testing phase financed by nih during the years 2018-2019 (so far unpublished), microbedx has tested urologic, a highly automated instrument and cartridge system that performs rapid pathogen id and ast in 30 and 150 min, respectively. the alveo platform (alveo technologies inc, ca) applies hybridization-based electrochemical nucleic acid detection in single-use cartridges. the cloud-connected device is capable of analyzing 100 infectious diseases. sample preparation, nucleic acid amplification, and real-time hybridization-based detection are performed in a single isothermal microfluidic channel. this concept has not yet been applied to growth-based ast. the loopamp tm technology by eiken chemical corp. (china) has already received who endorsement for tuberculosis diagnostics (noncommercial culture drug-susceptibility testing methods for screening patients at risk for multidrug-resistant tuberculosis: policy statement, 2011). the loop-mediated isothermal amplification (strand displacement reaction) employs four different primers designed for six distinct regions on the target gene. it has been used successfully for the detection of neisseria meningitidis in clinical cerebrospinal fluid samples (lee et al., 2015) and for the detection of the oxa-23 carbapenemase gene of acinetobacter baumannii. schoepp et al. (2017) demonstrated with 51 clinical samples that the antibiotic exposure time in phenotypic ast can be shortened to 15 min when dlamp (digital real-time loop-mediated isothermal amplification) or dpcr (droplet-pcr) is applied. the changes in dna concentrations (control vs. antibiotic-treated samples) were determined by a "digital singlemolecule counting" system after incubation. in dlamp, the target molecules or lysed cells are partitioned into thousands of nanodroplets so that each compartment contains approximately a single molecule (schoepp et al., 2017) . the 1 nl droplets with dna concentrations relevant to clinical urine tract infection samples showed 1.23-fold difference in amplification products between resistant and susceptible strains, and 98.1% of the tested samples matched the standard ast results. the lamp chemistry was optimized and applied to a slipchip microfluidic device equipped with an electrophoretic system concentrating the bacteria in the sample. this concept was tested for ast of positive blood cultures. the results were consistent with standard microdilution tests or the bd phoenix system when several broad-spectrum antibiotics and clinical e. coli samples as well as the s. aureus atcc 6538 strain were tested (yi et al., 2019) . the talis biomedical corporation (ca, usa) is currently commercializing dast/slipchip technology to a product which combines a single-use cartridge integrating fluid partitioning for parallel treatment with different antimicrobials, reagent additions, target lysis, extraction and amplification. the naat-based system presented by priye et al. (2017) applies a reverse-transcription loop-mediated isothermal amplification (rt-lamp) coupled with the quasr technique (quenching of unincorporated amplification signal reporters). the device was found to be five times more sensitive than traditional poct for the detection of the dengue, chikungunya, or zika viruses. hassibi et al. have presented a fully integrated, miniaturized semiconductor biochip with closed-tube detection chemistry for multiplex na amplification and sequence analysis, which they claimed to have a high dynamic quantification range for the microbial load, while at the same time performing comprehensive mutation analysis on up to 1,000 sequences or strands simultaneously in <2 h. this chip was able to correctly detect and quantify multiple dna and rna respiratory viruses in clinical samples, while at the same time detecting 54 drugresistance-associated mutations in six genes of mycobacterium tuberculosis (hassibi et al., 2018) . quantumdx ltd (uk) aims to launch a miniaturized portable diagnostic platform that runs on battery power. this q-poc tm device would be a portable molecular diagnostic instrument with built-in na amplification for multiplexed diagnostics and drug susceptibility testing within 20 min. it applies nanowires coated with specific probes for the detection of genetic variants and has cloudbased connectivity to share and utilize epidemiologic data. this technology is currently under clinical evaluation for diagnostics of malaria (protozoan plasmodium falciparum) and tuberculosis (mycobacterium tuberculosis). the device, although not designed for growth-based ast, might have a very wide applicability range including non-infectious diseases such as cancer or genetic disorders. the outcome of the clinical tests has not yet been published. the multipath instrument (first light diagnostics inc., usa) applies fluorescence-labeled nanoparticles attaching to the target cells by immunobinding. the nanoparticle/cell complex can then be pulled toward the detection system by magnetic beads conjugated with another microbe-specific antibody. thereby the system is capable of monitoring viable cells using non-magnified digital imaging. a poct-compatible device is currently under testing. immunodiagnostic tpx-technology (two-photon excitation fluorometry), commercialized by arcdia ltd (finland) to maripoc and mariast platforms, can analyze 40-100 samples a day. it has a cloud-based connectivity allowing efficient sharing of epidemiologic data. tpx allows the use of untreated polymicrobial clinical samples on-line, providing bacterial id in 20 min and antibiotic resistance in a few hours. unlike most other ast systems, it can also provide sensitive immunodetection of viruses sanbonmatsu-gámez et al., 2015; bruning et al., 2018. poct devices using smartphone technology have long been used for personalized medication applications such as the colorimetric analysis of urine strips (barnes et al., 2018) . the use of smartphones in growth-based ast for miniaturized cultures has been presented in several studies (kadlec et al., 2014; schoepp et al., 2017; cui et al., 2018; hernández-neuta et al., 2019) , although instrument manufacturers seem to be reluctant to incorporate standard commercial smartphones to their systems. photolithography, plotting, plasma etching, inkjet printing, cutting, and wax printing can be used to pattern paper for inexpensive diagnostic tests (lópez-marzo and merkoçi, 2016). paper-based systems have been applied for the immunodetection of helicobacter pylori, chlamydia infections and zika virus infections, but platforms integrating lf-testing into growthbased ast have not been presented. due to the small sample size (at lowest a single bacterial cell), rapid ast may not give results representing the whole bacterial population in the diagnostic sample. this problem can be mitigated by analyzing a large number of individual cells. an example of such a strategy is the stochastic confinement of bacteria in nanoliter droplets, so that a high number of individual droplets can be analyzed quickly and efficiently (boedicker et al., 2008) . the direct analysis of clinical samples also demands the use of valid internal standards. additionally any growth-based analysis may utterly fail if the growth rate is very low or the microbes cease to grow due to the lack of specific growth factors, a non-optimal atmosphere, or the accumulation of growth-inhibiting substances. for intracellular pathogens the use of naat may be necessary. compared to standard microbial cultivations, favorable growth conditions may be difficult to establish for miniaturized set-ups. these problems can be mitigated by analyzing cell viability, morphology or movements. miniaturization can improve the performance and capacity of cultivation-based ast only if the need for manual steps is minimized. as reagent dilutions are often done before loading onto the chip device, set-up complexity may stay a similar level as in broth dilution methods. due to the relatively low number of pathogens in most clinical samples and the impurities of the sample matrices, miniaturized cultivations benefit from methods concentrating the cells and removing the impurities. the fish-based accelerate pheno system applies electrokinetic/electrophoretic system for these purposes. coarse cell sorting for blood samples can be achieved through simple inertial microfluidic systems, where centrifugal (inertial) force drives bacteria to the outer side of a spiral-formed microcapillary tube, while blood cells stay on the inner side (bhattacharyya et al., 2017) . some systems bypass the problem of impurities by bringing the target close to the biosensor surface with magnetic and antibody-coupled beads or nanoparticles. miniaturization sets high demands to the standardization of the conditions, as the samples should represent similar growth states and culture densities. robots may be needed for accurate pipetting. serious doubts concerning the ability of accelerated cultures to fully substitute standard growth-based tests have been presented. accelerated cultivations may struggle to differentiate between wild type and resistant strains (leclercq et al., 2013; kahlmeter, 2014; maurer et al., 2017) . this is especially a concern in the case of induced expression of the resistance factors, e.g., in the detection of ampc in enterobacteria or macrolide-resistance in streptococci (leclercq and courvalin, 2002; jacoby, 2009; harris and ferguson, 2012) . substantial overall savings can be obtained with rapid ast through the reduction of hospital days, disability days and the saving of lives (cassini et al., 2019) . despite the potential total cost savings, the high price of molecular testing forms an efficient barrier for the installation of new technologies. a price tag ranging from $100 to $250 per test is common for molecular tests (li et al., 2017b) . for a typical 500-bed community hospital, detailed multiplex testing of positive blood cultures by naat could cost more than $500,000/year in reagents alone (she and bender, 2019) . also the instrument prices may be significant. the total costs of testing are quite difficult to estimate. the prices of test kits and diagnostic instruments are based on the distributors' offers. testing costs include clinical sampling and usually also cultivations in microbiology labs (enrichment, preparation of bacterial isolates) before ast can be performed. as an example, patel et al. estimated the total costs for mass spectrometry-based ast to be roughly 79 e per patient, when the cost of the maldi-tof device, reagents, pharmacist time and the antimicrobial stewardship program are pooled together (patel et al., 2017 ). yet the reagent costs for one ms-sample can be pressed close to 1 e. a typical maldi-tof ms system with all the accessories, software databases and maintenance is costly (up to e200,000 per year) (wieser et al., 2012) . this implies that the use of the instrument must be high for an acceptable cost efficiency. this unfortunately rules out their use in outpatient clinics. ms-instruments are, however, very versatile and can be applied also to other routine diagnostics in central laboratories (vrioni et al., 2018) . direct inoculation from plates to automated identification systems such as vitek, microscan and others has been validated and used in many clinical laboratories. these systems apply multiwell liquid cultures and have a turnaround time as short as 4 h for id and 6-8 h for ast (she and bender, 2019) , but the prices of these instruments are high. the cost of any rapid ast should not significantly exceed the cost level currently considered acceptable for routine testing: this might be roughly 30 to 50 e, including sampling, culturing and ast (eu, 2013) . five phenotypic tests for a targeted detection of enterobacterial carbapenemases have been recently evaluated for their performance and costs. all these tests required the use of culture isolates. per sample, the costs of multiplexed pcrbased analysis was 30 e, immunochromatographic methods ∼15 e, a colorimetric assay 5 e and carbapenem hydrolysis test 1 e per sample (baeza et al., 2019) . apart from turnover time, also the capacity of the test system is important. among automated microscopy systems, accelerate pheno handles one sample per unit module (max. four modules per device, cost per sample ∼250 e), whilst the q-linea astar system can handle 50 samples per day. the capacities of naat-based systems can be even higher. automated mic-determination systems such as vitek-2 can manage tens of samples per time. for poct usage, however, a throughput capacity of a few tens of samples per time would be sufficient. several identification systems based on naat, na hybridization and immunodiagnostics are already available for rapid diagnostics, but only few are applicable to poct. this is due to high costs, lack of appropriate facilities or expert labor, or insufficient performance with clinical samples. multiplexing and high-throughput capacities are important for central laboratories, but most healthcare settings process only few samples per time. poct use rules out expensive devices, systems requiring demanding sample preparation, and systems requiring standard microbiology laboratory facilities. naat serves well for the identification of viral and bacterial pathogens, and in some cases provides also the detection of ar genes. however, the commercial multiplexed diagnostic naat panels are far too expensive for routine diagnostics. immunochromatographic tests, especially dip-sticks, are a low-cost and handy option for virus diagnostics and detection of inflammatory factors. unfortunately systems capable of detection of antibiotic resistance proteins directly from clinical samples without enrichment cultures, culture isolation or sample purification do not yet exist. the routine diagnostic tools for infectious diseases should serve a wide field of requirements: both the identification of the pathogen (viral and bacterial), the detection of antibiotic resistance and the determination of the correct antibiotic dosing. systems based on microscopy are not compatible with the detection of viruses or inflammatory factors. diagnostic systems differ very much in their potential to provide all relevant data for medication ( table 1) . multiplexing naat-based systems can seamlessly incorporate the detection of emerging resistance-related genes or mutations. however, every update for a diagnostic panel requires new validations. while central laboratories have resources to validate new tests against the standardized tests, smaller healthcare units are bound to use tests validated by authorities such as the fda. the few systems currently capable of providing simultaneous id and growth-based ast directly from clinical samples are based either on fish (accelerate pheno), immunodiagnostics (e.g., mariast, multipath, immunobiosensors), or digital ast (dast) ( table 1) . as their properties and application areas differ, all these systems may have a good future. rapid growth-based diagnostic systems rely on accelerated cultivations. for this reason, it is necessary to validate every new technology carefully against the standard tests accepted by eucast and clsi. the mic breakpoints (sensitive, resistant, or in-between) must be re-defined for each system and each tested organism. this requirement definitely retards the deployment of new technologies. while most novel ast systems only perform an endpoint analysis, the systems based on microscopy, heat production, movements, immunodetection or detection of mass changes in principle facilitate on-line monitoring. biosensor-based ast systems, however, yet miss convincing clinical demonstrations. in practice clinical diagnosis should be obtained during office hours, meaning <8 h from sampling to results. this does not leave time for enrichment cultivations or preparation of culture isolates. the strategy "take a sample, store and analyze later" works fine for diagnosis of slowly advancing infections, but also for naat-based systems which do not require viable microbes. growth-based ast, on the other hand, need fresh samples to ensure pathogen survival until the point of analysis. regarding the speed and the need to handle clinical polybacterial samples, the immunodiagnostic tpx-technology (arcdia ltd), growth-based fish (accelerate pheno), the multipath digital imaging technology based on nanoparticles for labeling and magnetic beads for capturing (first light diagnostics inc.) , and the naat-based dast (talis inc.) seem promising options for rapid point-of-care testing of antimicrobial susceptibility. the talis system is, however, not yet on the market. immunobiosensors are still in their infancy, but may in the future become important in testing non-culturable microbes. lateral flow (immuno-chromatographic "dip-sticks") systems would be an ideal product format for clinical work, but their applicability has been so far proven only with isolated cultures (i.e., colonies on plate). due to the plethora of different resistance mechanisms, naat struggles with the detection of antibiotic resistances in gramnegative bacteria (maurer et al., 2017) and with the analysis of samples containing commensal flora. naat may fail to identify several esbl genes and genes providing fluoroquinolone or aminoglycoside resistance. naat is, however, a powerful and necessary technology for the detection of fastidious, slowgrowing or intracellular pathogens, toxin-producing bacteria such as e. coli o157:h7, and viruses (miller et al., 2018) . still, the increased use of naat has raised a concern about the fate of bacterial samples required for further studies (marder et al., 2017; mcadam, 2017) . since naat does not require viable samples, subsequent epidemiologic studies or cultivation-based confirmatory ast may be impossible with the stored samples. whole genome sequencing (wgs) is still in its infancy regarding (kahlmeter, 2016) . generally applicable or restricted to certain infections? in principle all growth-based rapid ast systems are generic and work with culture isolates. however, for polymicrobial clinical samples they must be coupled with specific probes or antibodies which provide id. therefore, specific test panels have been developed e.g., for respiratory, urinary, and blood samples. the pathogen load may not be high enough for direct analysis, and especially blood samples may require culturing prior to analysis. ast for fastidious, non-culturable, or intracellular pathogens call for naat. the complexity of the sample matrix affects the choice of the diagnostic system and the methods for sample preparations. capacity: how many organisms/agents per hour can be processed dna-arrays and pcr systems (including multiplexed cassette designs) have a high throughput capacity. mass-spectrometry performed on pcr products can handle hundreds of samples per hour in central laboratories. in outpatient clinics speed is more essential than the capacity. high multiplexing (parameters per sample) and high-throughput capacity (number of samples) may be challenging to combine. progress in naat, immunodiagnostics, biosensor technologies and microfluidics has yielded several systems capable of analyzing tens of samples per day or even during a single work shift. has the technology been validated against reference methods? so far quite few quick technologies have received fda-approval. currently they include pcr-tests, cartridge-based naat-systems and accelerate pheno (automated microscopy). several clinical trials are in progress to achieve ce-marking or fda-clearance. are there any reference installations? commercial analysis systems in general do have, and manufacturers tend to publish successful clinical trials. however, finding a lab which lines up to the specific needs may be challenging. is scientific literature available? for mature commercial systems scientific references can be fairly easily found. for near-market products this is much more challenging. companies often only declare on-going tests, but provide only limited info about the progress. scientific articles typically present proof-of-concept level data obtained with isolated cultures spiked into sample matrices. when on market? many systems are already available, but they may have a limited scope for id/ast. due to lack of clinical data, some systems have a "research use only" status. some have been accepted only for veterinary use. commercially mature products include several naat systems, fish-systems and immunodiagnostic system. its use for rapid ast. the required bioinformatics is challenging, and universal open databases are needed to interpret the results. in the near future, the progress in chip, microfluidics and biosensor technologies may provide new inexpensive ast systems. integration of many sophisticated technologies will be needed to resolve problems with a low initial pathogen number and the presence of contaminating sample matrices. several scientific publications have already demonstrated the successful use of smartphone optics and telecommunication capacity for monitoring of microwell or microcapillary cultivations, ph and redox changes and for delivering the read-outs of biosensor data (berg et al., 2015; feng et al., 2016; cui et al., 2018; hernández-neuta et al., 2019) . the obvious lack of ipr protection for smartphone-based analytic devices and the requirement to validate analytic devices as an entity unfortunately discourages commercialization of these technologies. standard growth-based technologies based on disc diffusion and broth dilution still dominate in ast. they are slow and require pure cultures, but in other aspects serve the purpose well. only few rapid growth-based ast methods work directly with polymicrobial clinical samples, which is required in poct. sensitive growth monitoring can be achieved either by frequent sampling (applicable to disruptive methods like fish or naat) or by on-line immunodiagnostic methods. dast with chipbased microfluidics devices and isothermal amplification can potentially revolutionize phenotypic ast. with this approach, thousands of individual single bacterium droplet samples can be categorized according whether the amount of amplified dna reaches the limit defined for growing cells. the fish-based accelerate pheno system has already reached fda approval. the maripoc device based on immunodetection with twophoton excitation fluoroscopy allows non-disruptive microbial identification. its clinical validation for ast should be followed with interest, as this technology enables the use of nonpurified clinical samples and also allows follow-up studies to confirm the results. additionally it already provides a rapid and sensitive identification of both bacterial and viral pathogens. the multipath technology (first light diagnostics inc.) may provide a functional platform for automated on-line detection by applying fluorescent nanoparticles for signaling, magnetic beads for binding, and non-microscopic imaging for detection. multiplexing cartridge-based naat solutions are likely to reach a significant customer base in central laboratories, since they offer high speed, work well with non-culturable bacteria and viruses and possess an excellent high-throughput power for pathogen id. however, all new emerging technologies struggle to meet all of the criteria prof. kahlmeter set for ast technologies ( table 2) . as none of the presented technologies is optimal in all aspects, it is probable that many of them will reach a large customer base. therefore the consensus statement of the piamr amr-rdt working group on antimicrobial resistance and rapid diagnostic testing is still valid: "there is no single major, or broadly accepted, technological breakthrough that leads the field of rapid ast platform development" (van belkum et al., 2019b) . the described technologies have been taken into consideration without any pre-selection and have been judged only by their applicability to clinical diagnostics. av was the main responsible for the acquisition and structuring of the scientific literature. the text and conclusions were processed by all the authors. vh and ol mediated valuable contacts to the expert biotech scientists and healthcare experts consulted for this review. all authors contributed to the writing and accepted the final version. this work was supported by the business finland project dare (dnro 2769/31/2018), which evaluated the current and emerging technologies for ast and the possibilities to develop new innovative products for ast testing. the authors declare that this study received funding from fimlab laboratories. the funder was not involved in the study design, collection, analysis, interpretation of data, the writing of this article or the decision to submit it for publication. development and application of a novel peptide nucleic acid probe for the specific detection of cronobacter genomospecies (enterobacter sakazakii) in powdered infant formula scientific resource for research, and education: educational resources -rapid antimicrobial susceptibility testing | university of utah integrated bacterial identification and antimicrobial susceptibility testing using pcr and high-resolution melt rapid phenotypic antimicrobial susceptibility testing using nanoliter arrays comparison of five methods for detection of carbapenemases in enterobacterales with proposal of a new algorithm genetic disease detection and dna amplification using cloned thermostable ligase rna signatures allow rapid identification of pathogens and antibiotic susceptibilities smartphone-based pathogen diagnosis in urinary sepsis patients cellphone-based hand-held microplate reader for point-of-care testing of enzyme-linked immunosorbent assays rapid electrochemical phenotypic profiling of antibiotic-resistant bacteria rapid phenotypic antibiotic susceptibility testing through rna detection simultaneous detection of genotype and phenotype enables rapid and accurate antibiotic susceptibility determination detecting bacteria and determining their susceptibility to antibiotics by stochastic confinement in nanoliter droplets using plug-based microfluidics validation of carbapenemase and extendedspectrum β-lactamase multiplex endpoint pcr assays according to iso 15189 evaluation of the byg carba test, a new electrochemical assay for rapid laboratory detection of carbapenemase-producing enterobacteriaceae rapid detection of urinary tract pathogens using microcalorimetry: principle, technique and first results development and validation of a lateral flow immunoassay for rapid detection of ndm-producing enterobacteriaceae unitaid tb diagnostics -naat for microscopy stations rapid detection and monitoring of human coronavirus infections rapid antimicrobial susceptibility testing of bacillus anthracis, yersinia pestis, and burkholderia pseudomallei by use of laser light scattering technology weighing of biomolecules, single cells and single nanoparticles in fluid real-time antimicrobial susceptibility assay of planktonic and biofilm bacteria by isothermal microcalorimetry microfluidic advances in phenotypic antibiotic susceptibility testing attributable deaths and disability-adjusted life-years caused by infections with antibiotic-resistant bacteria in the eu and the european economic area in 2015: a population-level modelling analysis be antibiotics aware: smart use, best care | features | cdc pna-fish as a new diagnostic method for the determination of clarithromycin resistance of helicobacter pylori matrix-assisted laser desorption ionizationtime of flight mass spectrometry for combined species identification and drug sensitivity testing in mycobacteria a surface acoustic wave sensor modified from a wireless transmitter for the monitoring of the growth of bacteria multiplexed automated digital microscopy for rapid identification and antimicrobial susceptibility testing of bacteria and yeast directly from clinical samples use of the accelerate pheno system for identification and antimicrobial susceptibility testing of pathogens in positive blood cultures and impact on time to results and workflow surface plasmon resonance biotechnology for antimicrobial susceptibility test direct, rapid antimicrobial susceptibility test from positive blood cultures based on microscopic imaging analysis a rapid antimicrobial susceptibility test based on single-cell morphological analysis nucleic acid sequence-based amplification rapid detection of extended-spectrum β-lactamases (esbl) and ampc β-lactamases in enterobacterales: development of a screening panel using the maldi-tof ms-based direct-on-target microdroplet growth assay potential impact of flow cytometry antimicrobial susceptibility testing on the clinical management of gramnegative bacteremia using the smartphone-based rapid quantification of viable bacteria by single-cell microdroplet turbidity imaging rapid detection of hiv-1 by reverse-transcription, loop-mediated isothermal amplification (rt-lamp) rapid detection of meticillin-resistant staphylococcus aureus bacteraemia using combined three-hour short-incubation matrixassisted laser desorption/ionization time-of-flight ms identification and alere culture colony pbp2a detection test evaluation of a microcolony growth monitoring method for the rapid determination of ethambutol resistance in mycobacterium tuberculosis evaluation of the accelerate pheno tm system for rapid identification and antimicrobial susceptibility testing of gram-negative bacteria in bloodstream infections the slow march toward rapid phenotypic antimicrobial susceptibility testing: are we there yet? the role of whole genome sequencing in antimicrobial susceptibility testing of bacteria: report from the eucast subcommittee infectious diseases evaluation of quickfish and maldi sepsityper for identification of bacteria in bloodstream infection rapid detection of staphylococcus aureus strains with reduced susceptibility to vancomycin by isothermal microcalorimetry microfluidic cantilever detects bacteria and measures their susceptibility to antibiotics in small confined volumes as regards a union financial aid towards a coordinated control plan for antimicrobial resistance monitoring in zoonotic agents high-throughput and automated diagnosis of antimicrobial resistance using a cost-effective cellphone-based micro-plate reader robustness of a loop-mediated isothermal amplification reaction for diagnostic applications rapid antimicrobial susceptibility testing of clinical isolates by digital time-lapse microscopy genefluidics announces ce-ivd marking of utimax tm uropathogen identification (id) and antimicrobial susceptibility testing (ast): genefluidicslifescience. available online at prospective evaluation of the okn k-set assay, a new multiplex immunochromatographic test for the rapid detection of oxa-48-like, kpc and ndm carbapenemases rapid antimicrobial susceptibility testing by sensitive detection of precursor rrna using a novel electrochemical biosensing platform antibiotic therapy for inducible ampc β-lactamase-producing gram-negative bacilli: what are the alternatives to carbapenems, quinolones and aminoglycosides? multiplexed identification, quantification and genotyping of infectious agents using a semiconductor biochip rapid antimicrobial susceptibility testing using forward laser light scatter technology a novel microbead-based microfluidic device for rapid bacterial identification and antibiotic susceptibility testing use of the accelerate pheno system for identification and antimicrobial susceptibility testing of pathogens in positive blood cultures and impact on time to results and workflow performance of the t2bacteria panel for diagnosing bloodstream infections mass-sensitive biosensor systems to determine the membrane interaction of analytes application of a microcalorimetric method for determining drug susceptibility in mycobacterium species rapid cytometric antibiotic susceptibility testing utilizing adaptive multidimensional statistical metrics advances and trends in sepsis diagnostics rapid identification of microorganisms from positive blood cultures by maldi-tof mass spectrometry subsequent to very shortterm incubation on solid medium rapid direct susceptibility testing from positive blood cultures by the matrix-assisted laser desorption ionization-time of flight mass spectrometrybased direct-on-target microdroplet growth assay ampc -lactamases a cell phone-based microphotometric system for rapid antimicrobial susceptibility testing defining antibiotic resistance-towards international harmonization rapid phenotypic susceptibility testing article rapid detection of single bacteria in unprocessed blood using integrated comprehensive droplet digital detection ultrafast parallelized microfluidic platform for antimicrobial susceptibility testing of gram positive and negative bacteria new technologies for rapid bacterial identification and antibiotic resistance profiling monitoring the growth and drug susceptibility of individual bacteria using asynchronous magnetic bead rotation sensors development of an immunochromatographic assay for the rapid detection of aac(6 ′ )-iae-producing multidrug-resistant pseudomonas aeruginosa a highy multiplexed broad pathogen detection assay for infectious disease diagnostics two-photon excitation fluorometry in detection identification of upper respiratory bacterial pathogens with the electronic nose eucast expert rules in antimicrobial susceptibility testing resistance to macrolides and related antibiotics in streptococcus pneumoniae clinical evaluation of a loop-mediated isothermal amplification (lamp) assay for rapid detection of neisseria meningitidis in cerebrospinal fluid identifying volatile metabolite signatures for the diagnosis of bacterial respiratory tract infection using electronic nose technology: a pilot study a systematic review of antibiotic prescription associated with upper respiratory tract infections in china selective capture and rapid identification of e. coli o157:h7 by carbon nanotube multilayer biosensors and microfluidic chip-based lamp emerging microtechnologies and automated systems for rapid bacterial identification and antibiotic susceptibility testing colorimetric sensor array allows fast detection and simultaneous identification of sepsis-causing bacteria in spiked blood culture rapid bacterial antibiotic susceptibility test based on simple surfaceenhanced raman spectroscopic biomarkers rapid antimicrobial susceptibility testing with electrokinetics enhanced biosensors for diagnosis of acute bacterial infections microfluidics for combating antimicrobial resistance a multiplexed, gradient-based, full-hydrogel microfluidic platform for rapid, high-throughput antimicrobial susceptibility testing an automated system for rapid non-destructive enumeration of growing microbes rapid detection of bacterial resistance to antibiotics using afm cantilevers as nanomechanical sensors the use of colorimetric sensor arrays to discriminate between pathogenic bacteria paper-based sensors and assays: a success of the engineering design and the convergence of knowledge areas microfluidics integrated biosensors: a leading technology towards lab-on-a-chip and sensing applications a biosensor platform for rapid antimicrobial susceptibility testing directly from clinical samples a novel microfluidic assay for rapid phenotypic antibiotic susceptibility testing of bacteria detected in clinical blood cultures molecular methods for septicemia diagnosis incidence and trends of infections with pathogens transmitted commonly through food and the effect of increasing use of cultureindependent diagnostic tests on surveillance -foodborne diseases active surveillance network evaluation of the accelerate pheno system for fast identification and antimicrobial susceptibility testing from positive blood cultures in bloodstream infections caused by gram-negative pathogens assessment of prescribing practices at the primary healthcare facilities in botswana with an emphasis on antibiotics: findings and implications a microfluidic channel method for rapid drugsusceptibility testing of pseudomonas aeruginosa identification and antibiotic-susceptibility profiling of infectious bacterial agents: a review of current and future trends advances in rapid identification and susceptibility testing of bacteria in the clinical microbiology laboratory: implications for patient care and antimicrobial stewardship programs identification by mass spectrometry and automated susceptibility testing from positive bottles: a simple, rapid, and standardized approach to reduce the turnaround time in the management of blood cultures semi-quantitative maldi-tof for antimicrobial susceptibility testing in staphylococcus aureus unforeseen consequences: culture-independent diagnostic tests and epidemiologic tracking of foodborne pathogens a biosensing strategy for fast profiling of antibiotic resistance rapid simultaneous identification and quantitation of staphylococcus aureus and pseudomonas aeruginosa directly from bronchoalveolar lavage specimens using automated microscopy a general method for rapid determination of antibiotic susceptibility and species in bacterial infections a guide to utilization of the microbiology laboratory for diagnosis of infectious diseases: 2018 update by the infectious diseases society of america and the american society for microbiologya clinical validation of integrated nucleic acid and protein detection on an electrochemical biosensor array for urinary tract infection diagnosis prospective evaluation of light scatter technology paired with matrix-assisted laser desorption ionization-time of flight mass spectrometry for rapid diagnosis of urinary tract infections downloaded from monitoring growth and antibiotic susceptibility of escherichia coli with photoluminescence of gaas/algaas quantum well microstructures comparison of eplex respiratory pathogen panel with laboratory-developed real-time pcr assays for detection of respiratory pathogens noncommercial culture and drug-susceptibility testing methods for screening patients at risk for multidrug-resistant tuberculosis: policy statement loop-mediated isothermal amplification of dna a microfluidic chip integrating dna extraction and real-time pcr for the detection of bacteria in saliva cost analysis of implementing matrix-assisted laser desorption ionization-time of flight mass spectrometry plus real-time antimicrobial stewardship intervention for bloodstream infections filter-based pna in situ hybridization for rapid detection, identification and enumeration of specific micro-organisms analysis of discrimination mechanisms in the mammalian olfactory system using a model nose microfluidic device for rapid (andlt;15 min) automated microarray hybridization position paper on anti-microbial resistance diagnostics a smartphone-based diagnostic platform for rapid detection of zika, chikungunya and dengue viruses progress on the development of rapid methods for antimicrobial susceptibility testing whole-genome sequencing of bacterial pathogens: the future of nosocomial outbreak analysis loop mediated isothermal amplification: an innovative gene amplification technique for animal diseases an international, prospective, multicenter evaluation of the combination of advandx staphylococcus quickfish bc with meca xpressfish for detection of methicillinresistant staphylococcus aureus isolates from positive blood cultures analytical performance of the automated multianalyte point-ofcare maripoc r for the detection of respiratory viruses electronic nose in the detection of wound infection bacteria from bacterial cultures: a proof-of-principle study differential dna accessibility to polymerase enables 30-minute phenotypic-lactam antibiotic susceptibility testing of carbapenem-resistant enterobacteriaceae rapid pathogen-specific phenotypic antibiotic susceptibility testing using digital lamp quantification in clinical samples in vitro antimicrobial susceptibility testing methods: agar dilution to 3d tissue-engineered models specific reveal tm -specific technologies. specif. reveal. ast hours not days advances in rapid molecular blood culture diagnostics: healthcare impact, laboratory implications, and multiplex technologies culture-free bacterial detection and identification from blood with rapid, phenotypic, antibiotic susceptibility testing evaluation of the new nuclisens easyq kpc test for rapid detection of klebsiella pneumoniae carbapenemase genes (blakpc) mbt-astra: a suitable tool for fast antibiotic susceptibility testing? the iridica pcr/electrospray ionization-mass spectrometry assay on bronchoalveolar lavage for bacterial etiology in mechanically ventilated patients with suspected pneumonia cell-on-hydrogel platform made of agar and alginate for rapid, low-cost, multidimensional test of antimicrobial susceptibility antimicrobial susceptibility test with plasmonic imaging and tracking of single bacterial motions on nanometer scale current and emerging techniques for antibiotic susceptibility tests antibiotic susceptibility testing via plasmonic imaging and tracking evaluation and use of a rapid staphylococcus aureus assay by an antimicrobial stewardship program united nations meeting on antimicrobial resistance detection of group a streptococcus from pharyngeal swab samples by bacterial culture is challenged by a novel maripoc point-of-care test developmental roadmap for antimicrobial susceptibility testing systems developmental roadmap for antimicrobial susceptibility testing systems laboratory-based and point-of-care testing for mssa/mrsa detection in the age of whole genome sequencing elisa-based identification and detection of microbes rapid phenotypic antibiotic susceptibility testing of uropathogens using optical signal analysis on the nanowell slide isothermal micro calorimetry-a new method for mic determinations: results for 12 antibiotics and reference strains of e. coli and s. aureus maldi-tof mass spectrometry technology for detecting biomarkers of antimicrobial resistance: current achievements and future perspectives strand displacement amplification-an isothermal, in vitro dna amplification technique analytical validation of an ultra low-cost mobile phone microplate reader for infectious disease testing bacterial nanoscale cultures for phenotypic multiplexed antibiotic susceptibility testing maldi-tof ms in microbiological diagnostics-identification of microorganisms and beyond (mini review) detection of methicillin-resistant staphylococcus aureus using a specific anti-pbp2a chicken igy antibody a multiplex loopmediated isothermal amplification assay for rapid screening of acinetobacter baumannii and d carbapenemase oxa-23 gene direct antimicrobial susceptibility testing of bloodstream infection on slipchip recent developments in antibody-based assays for the detection of bacterial toxins we thank to dr. pierre bogaerts, who pointed out which practical issues are important in clinical diagnostics of microbial diseases. conflict of interest: vh was employed by fimlab laboratories.the remaining authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.copyright © 2020 vasala, hytönen and laitinen. this is an open-access article distributed under the terms of the creative commons attribution license (cc by). the use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner(s) are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. no use, distribution or reproduction is permitted which does not comply with these terms. key: cord-264749-m1awr1rm authors: saad, julian m.; prochaska, james o. title: a philosophy of health: life as reality, health as a universal value date: 2020-03-18 journal: palgrave commun doi: 10.1057/s41599-020-0420-9 sha: doc_id: 264749 cord_uid: m1awr1rm emphases on biomarkers (e.g. when making diagnoses) and pharmaceutical/drug methods (e.g. when researching/disseminating population level interventions) in primary care evidence philosophies of health (and healthcare) that reduce health to the biological level. however, with chronic diseases being responsible for the majority of all cause deaths and being strongly linked to health behavior and lifestyle; predominantly biological views are becoming increasingly insufficient when discussing this health crisis. a philosophy that integrates biological, behavioral, and social determinants of health could benefit multidisciplinary discussions of healthy publics. this manuscript introduces a philosophy of health by presenting its first five principles of health. the philosophy creates parallels among biological immunity, health behavior change, social change by proposing that two general functions—precision and variation—impact population health at biological, behavioral, and social levels. this higher-level of abstraction is used to conclude that integrating functions, rather than separated (biological) structures drive healthy publics. a philosophy of health provides a framework that can integrate existing theories, models, concepts, and constructs. a philosophy of health w hat is health? is it a state of the body or the mind? is health primarily a natural, biological state or a holistic, value-laden state? naturalistic and holistic philosophies of health have provided very important, but very different, perspectives of population health. naturalistic views (e.g. as seen in boorse, 1997) provide insight into physical, natural, biological, or physiological processes that are tangible (in the material sense), observable, and measurable with modern technology. complementarily, holistic views contend that value-laden phenomena (e.g. vital goals, meaning, and purpose) play a central role in population health (nordenfeldt, 2007) . a dialog, or as we see it, an important dialectic among naturalistic and holistic perspectives plays out between the biostatistical theory of health (bst) and the holistic theory of health (hth). the bst posits that a person is healthy if and only if, all natural organs function normally given a statistically normal environment (boorse, 1997) . the hth posits that a person is healthy if and only if (given standard circumstances) he/she has the ability to attain their vital goals (nordenfeldt, 2007) . in addition to defining health, each philosophy defines disease. the bst poses that disease is the internal state of impairment to the normal functioning of organs (boorse, 1997) . in the hth, an organ dysfunction is a disease if and only if the organ's process reduces the person's ability to pursue vital goals or life-purpose (nordenfelt, 2007) . in bst health is the absence of disease; and in hth, health is not the absence of biological disease, but is the whole person's ability to function in relation to vital goals. both naturalistic and holistic perspectives guide important observations of health and disease. when one considers health through the bst one pays close attention to the functions of the internal, biological functioning of the human being. when one considers health through the hth, one pays close attention to the functioning of an individual, in relation to their external, societal/ cultural functions. is there a hybrid model that accounts for both internal and external functioning? wakefield's (2014) harmful dysfunction analysis (hda) creates a hybrid model that integrates natural-and value-laden phenomena when conceptualizing disease. hda asserts that a person suffers from a disorder/disease if (1) the condition causes harm (as judged by the standards of the person's culture); or if (2) the person's internal, natural processes cannot perform normal functioning (as judged by the standards set by evolution). hda creates a hybrid model that can integrate perspectives of the bst (i.e. by considering internal organ functioning); and the hth (i.e. by considering external societal/goal functioning). however, while hda may define health processes in relation to disease, it serves primarily as an integrative model of disease. is there an integrative model of health that can account for natural and value-laden functions? schroeder (2012) identifies a significant, common thread among these competing (or perhaps complementing) philosophies: functionalism. the researcher suggests that each philosophy is concerned with the functioning of organisms. although the bst, hth, and the hda might not agree on which functions inform the first principles of health, schroder (2012) uses higherlevel abstraction to identify one common first principle: the state of functioning in an organism impacts its state of health. when paralleling the three philosophies based upon functioning one might observe that (1) bst declares an individual healthy if its organs function normally; (2) hth declares an individual healthy if he/she can function in relation to vital goals; and (3) hda declares an individual unhealthy if internal mechanisms cannot perform natural, evolutionary functions, and/or when a condition prevents a person from functioning in relation to goals/norms/values. through this higher-level abstraction, an integration of seemingly separate philosophies of health is made possible. learning from leaders in the field. as we attend to these philosophies of health, we too observe how discussions about functions and functioning produce integrative perspectives. although a definition of "function" is not explicitly stated in the above research, it appears that nordenfeldt (2007) , boorse (1997) , wakefield (2014) , and schroeder (2012) are each discussing functions as pre-existent (i.e. either from evolution, personal goalsetting, cultural tradition) processes-with-purposes. whether one is describing a value-laden function (e.g. decision-making in pursuit of a valuable career) or an evolutionary-biological function (e.g. the heart beating for circulation), each process (i.e. decision-making processes or cardiac processes) serves identifiable purposes (e.g. maintained financial stability or maintained blood flow). whether an organ is functioning normally in relation to the body or a human being is functioning in relation to vital goals, it appears that both perspectives consider if an active "process" (i.e. an organ's activity, an individual's activity) can express its "purpose" (i.e. evolutionary-purpose, life-purpose). in the present manuscript we will propose that naturalistic and holistic perspectives can be integrated within a single philosophy of health. we will propose two universal functions-termed precision and variation-that can account for both natural functions and value-laden functions of the existing philosophies. this functional language will support a higher level of abstraction that integrates, rather than separates, biological functions, behavioral functions, and social functions under a philosophy of health. the need for new perspectives in population health. the chronic disease crisis beckons the need for an updated philosophy of health that can account for biological, behavioral, and social functioning. why? chronic diseases, which account for 60% of all-cause deaths worldwide (chartier and cawthorpe, 2016) , do not emerge from naturalistic, biological, or physical contact with an illness. rather, chronic diseases do emerge in biological functions (e.g. tumor proliferation in an organ) after prolonged contact with health risk behaviors and lifestyle factors that active the conditions (mokdad et al., 2018; edington, 2009; li et al., 2018) . chronic diseases are not curable by purely naturalistic or biological means (e.g. pharmaceuticals). rather, some diseases may be effectively prevented or intervened on through healthy behavior (dansinger et al., 2005; daubenmier et al., 2007) . population health risk behaviors are unique determinants of population health because researchers can actively observe how they simultaneously alter biological functioning (e.g. chronic smoking alters cells in lung tissue), behavioral functioning (e.g. chronic smoking alters decision-making and daily habits) and social functioning (e.g. chronic smoking creates an economic, social, and healthcare burden) of the population. these behaviors not only have biological, behavioral, and social implications for the individual doing the behavior, but also have intergenerational and interpersonal effects. the individual who binges on refined sugar not only puts themselves at risk of diabetes, but can put their future offspring at risk. the individual who smokes two packs of cigarettes per day not only puts themselves at risk of lung cancer, but can put their housemates at risk of lung cancer from second-hand smoke. therefore, the chronic disease crisis is neither purely naturalistic, nor purely value-laden; rather it reflects an integration of natural and value-laden phenomena. there remains a real need for principles of health that can integrate existing naturalistic and holistic perspectives of population health. the principles since april 7, 1948, the constitution of the world health organization (2010) has utilized an intuitive definition of health by suggesting that health is "a state of complete physical, mental, and social well-being." while this definition might be intuitive and even accessible to a wide audience; the defininition is not necessarily researchable across health disciplines. integrating principles of health might begin with a common-sense definition of health that can also be upheld across existing naturalistic and holistic perspectives. without operationally defining functions that drive physical, mental, and social well-being, it is a challenge for multidisciplinary collaborators to unite under the who mission. further, without a common definition of health, important communications from patients to doctors, from subjects to researchers, from researchers to collaborators, and from peer-reviewers to peer-reviewees, can become fragmented or lost in translation. in the proceeding sections, a common-sense definition of health is used to present the first principles of a philosophy of health. principle 1: "health" is the state of maintainable-ease of functioning. a "disease" is a state of prolonged-dysfunction that prevents ease. chronic diseases emerge from prolonged exposure to dysfunctional behaviors like smoking, alcohol abuse, unhealthy diet, and inactivity (mokdad et al., 2018) that also create dysfunctional expressions of life functions. smoking creates dysfunctional breathing; alcohol abuse creates dysfunctional drinking; sugar binging creates dysfunctional eating; and sedentary behavior creates dysfunctional moving. when these health risk behaviors lead to chronic disease, they have already prolonged dysfunctional breathing, drinking, eating, and/or moving. the chronic smoker breathes in smoke so frequently that he no longer experiences an ease-of-breathing. rather, his breathing becomes short and shallow. prior to the emergence of lung tumors, the chronic smoker prolongs dysfunctional patterns of breathing. the "couch potato" sits so frequently that he no longer experiences an ease-of-movement. rather his movement becomes rigid and limited. prior to the emergence of cardiovascular dysfunction or obesity, the sedentary person prolongs dysfunctional patterns of movement. if chronic smoking facilitates prolonged-dysfunction in breathing, and sedentary behavior facilitates prolonged-dysfunction in movement, what do functional breathing and moving look like? healthy breathing and moving (as well as eating and drinking) are characteristic of an ease of one's functioning that can be maintained in normal conditions. for example, the chronic smoker and the "couch potato" might report momentary-ease in breathing and posture when engaging in their health risk behaviors; but they do not maintain that ease outside of smoking or sitting. conversely, the yogi might report that their yoga practices expose them to momentary dis-ease in breathing and moving that lead to maintainable-ease in breathing and movement in everyday life. in contrast to disease as a prolongeddysfunction, healthy functioning can be commonly sensed as a maintainable-ease of functioning. when observing a disease, perhaps we are observing a prolonged-dysfunction that prevents ease. rather than define health as the absence of disease (as seen in bst), notice here how we instead define disease in relation to health; and we define health in relation to maintainability, ease, and functioning. consideration of "maintainable-ease of functioning" will allow us to consider how not all "dis-ease" is bad (i.e. exposure to acute dis-ease/stress maintains healthy functioning in the long-term); and not all "ease" is good (i.e. avoidance of stress and prolonged "comfort" creates fragility seen in sedentary behavior). we propose that: 1. dysfunction parallels a state of "dis-ease"; and prolongeddysfunction parallels the state of disease. 2. function parallels a state of "ease"; and maintainable-ease of functioning parallels the state of health. this definition of health will be applied in the proceeding principles to integrate naturalistic and holistic perspectives of population health. principle 2: health emerges from maintainable-ease of functioning at multiple levels. maintainable-ease of functioning in the general population can be observed at the level of the cell, the self, and the society simultaneously. cooperation across multiple levels of functioning is required for the organization and adaptation of living systems (nowak and sigmund, 2005; antonucci and webster, 2014) . when developing an integrative model of health, it is important to consider how biological cells, individuals, and the larger society simultaneously play a role in population health (xavier da silveira dos santos and liberali, 2019; antonucci and webster, 2014) . in this philosophy, we define health from three levels: cells, selves, and societies. what happens when these levels do not function in cooperation? when the functioning of cells disrupts the functioning of the self, a state dis-ease in the self can follow. for example, prolonged dysfunction in autoimmune conditions can lead to prolonged dysfunction for the (individual's sense of) self by triggering depression, decreased motivation, or anxiety (lougee et al., 2000; garud et al., 2009) . the reverse can also be true. when the functioning of the self (i.e. one individual) disrupts the functioning of their cells, a state dis-ease in the cells can also follow. for example, prolonged sugar binging and addictive eating can lead to prolonged high blood sugar and pancreatic dysfunction seen in diabetes (de koning et al., 2011; imamura et al., 2015) . cells and selves are not separate. when the functioning of the self disrupts the functioning of the society we observe a state dis-ease in the society. for example, one person's unprotected sex with multiple partners can also lead to epidemics and social conflicts. the reverse can also be true. when the functioning of the society disrupts the functioning of the individual, a state dis-ease in the self can follow. for example, dysfunctional social conditions (as seen in rutter, 1998) , can lead to prolonged psychological and behavioral dysfunctions of individuals. selves and societies are not separate. when the functioning of society disrupts the functioning of cells, a state of dis-ease in the cells can also follow. for example, prolonged dysfunction in society in the form of misguided values about cleanliness, can lead to over-sanitization practices that create superbugs and antibiotic-resistant bacteria (zaccheo et al., 2017; finkelstein et al., 2014; bower and daeschel, 1999) . the reverse can also be true. when the functioning of cells disrupts the functioning of the society, a state of dis-ease in the society can follow. prolonged dysfunction in cells from naturally occurring parasites (e.g. yersinia pestis [cui et al., 2013] ) can lead to prolonged dysfunctions like the economic collapse following 14th century black death (haensch et al., 2010) . cells and societies are not separate. what does health look like when these levels work together? recent reports on the blue zones (i.e. the areas of the world where populations live significantly longer and healthier than the average) demonstrate that healthy functioning at these levels enhances physical longevity and mental wellbeing in populations (buettner, 2012; poulain et al., 2013) . buettner (2012) reports on how blue-zone populations intentionally and habitually enrich their physical bodies with healthy eating and physical activity. in addition to integrating physical and behavioral practices, these communities also integrate behavioral and social practices, such as, goal-setting, meditations/prayer, social engagement, pursuit of purpose, and community gathering. humor is used by individuals and groups as a means to practice ease when challenges present themselves (buettner, 2012) . blue zone communities place value upon physical/natural, behavioral and social processes, generating them intentionally and habitually. both states of ease and dis-ease can teach us about the contributions of cells, selves, and societies to population health. although it is important to be able to observe the levels separately to describe their contributions, it is also important to consider how the levels integrate to impact healthy publics. we acknowledge that meaningful changes can be observed above and below these levels (e.g. at the level of the biosphere and genome). however, this initial paper will introduce levels that are most proximal and accessible to the experience of a general readership (fig. 1) . principle 3: health emerges from systems whose primary purpose is to generate maintainable-ease of functioning at a respective level. we propose that systems exist at each level with the purpose of generating maintainable-ease of functioning at that level. the biological immune system, an individual's system of health behaviors, and the social system will be observed as systems that generate maintainable-ease of functioning in cells, selves, and societies respectively (fig. 2 ). principle 3a: the biological immune system is directly responsible for maintainable-ease of functioning at the level of the cell. throughout the course of human evolution, the complexity and biodiversity of the human body continued to increase (rodríguez et al., 2012) . what keeps the trillions of cells and microorganisms in cooperation in a human body? the biological immune system maintains functional cells (rodríguez et al., 2012) . although it is documented that the functioning of the biological immune system has implications for behavioral functioning (ader, 1974 (ader, , 2000 johnston et al., 1992; cdc, 2016) and social functioning (cdc, 2016; reidel, 2005; cutler and miller, 2005) the system's primary purpose is supporting functioning in the cellular/biological system. principle 3b: health behavior is directly responsible for maintainable-ease of functioning at the level of the self. throughout the course of time, the complexity of human behavior, has continued to increase (boulding and khalil, 2002) . what keeps an individual in a state of balance during times of rapid change? one's system of health behaviors (e.g. one's practices of breathing, drinking, eating, and moving) maintain a functional self. although it is well documented that the behavior of the individual impacts biological functioning (fadel, 2013 (fadel, , 2015 and social functioning (omer et al., 2009 ), one's system of health behaviors directly impacts one's experience of (or one's 'sense of') their "self". principle 3c: the social system is directly responsible for maintainable-ease of functioning at the level of the society. throughout history, the social diversity of human societies continued to increase. during periods of rapid increases in social diversity and cultural integration, what supported cooperation in the society? social systems (e.g. public governments, private social organizations, religious/spiritual organizations) emerge to maintain a functional society. although it is well documented that a social system can impact biological functioning (cdc, 2016; riedel, 2005; cutler and miller, 2005) and behavioral functioning (buettner, 2012) , the social system's primary role is to maintain functions at the level of the society. principle 3d: by considering health as maintainable-ease of functioning generated by systems, we have the ability generalize health across levels. to observe health at the level of the cell, the self, and the society simultaneously, we consider systems that support maintainable-ease of biological, behavioral, and social functioning. the biological immune system, an individual's system of health behaviors, and the social system make meaningful contributions to the functioning of cells, selves, and societies, respectively. while these systems are not the only systems that impact each level (e.g. one's cardiovascular system impacts cells, one's "personality" impacts the self, the environment impacts society), the biological immune system, health behavior, and the social system have great implications for population health from their respective levels; and they can be operationalized at these levels based upon their functions. by considering health as maintainable-ease of functioning (rather than maintained biological structures) at multiple levels, we set a point of reference from which to integrate important determinants of population health. when taking the structuralist's perspective, the biological immune system, health behavior, and social systems appear as distinctly separated. when taking a functionalist's perspective, the biological immune system (i.e. the integration of host defense functions and microbiota functions), one's (system of) health behaviors (i.e. the integration of decisionmaking/executive functions and habits/habitual life functions), and the social system (i.e. the integration of population values and population behaviors) appear together in a philosophy of health. fig. 1 the levels of functioning. this philosophy of health investigates three levels of health: cell, self, and society. the level of the cell accounts for biological functioning within human beings. the level of the self accounts for first-person functioning of each human being. the level of the society accounts for group functioning of human beings. fig. 2 the systems at each level. each system is responsible for generating maintainable-ease of functioning at a level. the biological immune system is responsible at the level of the cell. a human's system of health behaviors is responsible at the level of the self. the social system is responsible at the level of the society. article palgrave communications | https://doi.org/10.1057/s41599-020-0420-9 principle 4: each system employs two general functions-variation and precision-to generate maintainable-ease of functioning at a level. the functionalist perspective allows us to observe systems based upon their functions. the biological immune system will be observed as an integration of host defense functions and microbiota functions (hooper and littman macpherson, 2012) ; (2) an individual's system of health behaviors will be observed as an integration of decisions/executive functions and habits/habitual life functions (de bruin et al., 2016; verplankern, 2005; norman et al., 1998; prochaska et al., 1994; prochaska et al., 1991) ; and the social system will be observed as an integration of actively functioning values and population-wide behaviors that function in relation to those values (dowling and pfeffer, 1975; cotgrove and duff, 1981) . by researching the role of these functions at each level, we distilled two general functions of each system: variation and precision. variation appears in the functions of each system that generate a range of abilities, the "varied-abilities", that sustain health in presently changing conditions. the microbiota, habits/ habitual life functions and population behaviors will be observed (in principle 4a) as the variation-functions of the biological immune system, health behavior, and the social system, respectively. precision appears in those functions that prioritize and organize the patterns of variation that can sustain health at a level in future, changing conditions. the host-defense functions, decision-making/executive functions, and values systems will be observed (in principle 4b) as the precision-functions in the biological immune system, health behavior, and the social system, respectively. consideration of a complementary relationship among precision and variation is not novel. precision and variation have been discussed as central to the development of neural and biological systems (hiesinger and bassem, 2018) . discussions of precision and variation have also provided important insight into research on the biological immune system (albert-vega et al., 2018; brodin et al., 2015) . through this philosophy, one can go beyond biological systems to observe how precision (in the form of hostdefense functions, decision-making/executive functions, and values) and variation (in the form of microbiota functions, habits/habitual life functions, and population-wide behaviors) integrate to generate to maintainable-ease of functioning in cells, selves, and societies simultaneously (fig. 3) . principle 4a: variation is responsible for generating the range of abilities, the "varied-abilities", that can express ease-of-functioning in presently changing conditions. without functional variation, life is fragile because the present environment is always changing (taleb and blyth, 2011) . fragile systems' inability to experience changing conditions (in part) relates to limited variability. conversely, adaptive system's ability to experience changing conditions (in part) relates to functional variability (taleb, 2012) . when one microorganism in the microbiome takes over, biological fragility reflects a state of infection. when one habit takes over, behavioral fragility reflects a state of an addiction/dependence. when one population behavior takes over (e.g. when economic participation or access to food is restricted to a small percentage of the population) social fragility reflects a state of social/civil unrest. the human microbiota is comprised of trillions of microorganisms, such as bacteria, fungi, and viruses. when variability in the human microbiota exists, an ease of functioning, or "homeostasis" in cells can be expressed in the present biological/ ecological environment bogaert et al., 2011; claesson et al., 2011) . research demonstrates that variation in the microbiota impacts the health of human cells by metabolizing complex carbohydrates, converting proteins to neural signals, and modulating diurnal rhythms that maintain biological homeostasis (clemente et al., 2012; rothe and blaut, 2012; blaut and clavel, 2007; de vadder et al., 2014) . when variation in the microbiota is dramatically limited or changed (e.g. following antibiotic overuse), cellular tissue in the human body is fragile and vulnerable to infections, allergies, and inflammatory outbreaks (francino, 2016) . when one's habitual life functions (e.g. breathing, drinking, eating, and moving) and one's healthy habits (e.g. one's weekly exercise schedule, or weekly meal preparation) can be expressed freely, an ease of functioning is felt by one-self in the present environment. when life functions are no longer expressed with ease (e.g. breathing and movement are compromised due to prolonged sedentary lifestyle), or when a single habit takes over one's lifestyle (e.g. smokes breaks "must" occur every 30 min), an individual is vulnerable to stressful outbreaks and chronic states (al'absi, 2011; conrad et al., 2007; suess et al., 1980; león and sheen, 2003; parrott, 1999; koob, 2008) . when the basic human rights in a society are preserved in the present (e.g. right to life, freedom of speech; right to property), human populations have the ability to freely engage in the population-wide behaviors (e.g. health behaviors, social behaviors, economic behaviors) that support a functioning society. health behaviors drive health and longevity. social behaviors drive communication and cooperation. economic behaviors drive goods and resources. when these population-wide behaviors are chronically restricted in a population (e.g. poor access to health care, oppression of free-speech, economic crash), societies become vulnerable to social/civil unrest [as commented historically by victor frankl (1985) , alexander solzhenitsyn (2003) , franklin d. roosevelt (1941) , and dr. martin luther king (1985) ]. variation is essential so that a system has varied-abilities that can express ease-of-functioning in present environmental conditions. dramatic and prolonged restrictions to variation in the microbiota, habits/habitual life functions, and population-wide behaviors characterize fragile and vulnerable states in cells, selves, and societies. conversely, functional-variation supports resilience, robustness, and antifragility (taleb, 2012) . this does not mean that infinite variation is desirable; however, in this philosophy, precision is responsible for organizing expressions of variation so that the system does not degrade into unpredictably random variation or chaos (see principle 4b). fig. 3 precision and variation in each system. maintainable-ease of functioning is generated by two functions in each system: precision and variation. the human microbiota, habits, and population-wide behaviors evidence variation in cells, selves and societies respectively. the host defense system, decisions, and values evidence precision in cells, selves and societies respectively. palgrave communications | https://doi.org/10.1057/s41599-020-0420-9 article palgrave communications | (2020) 6:45 | https://doi.org/10.1057/s41599-020-0420-9 | www.nature.com/palcomms principle 4b: precision is responsible for prioritizing and organizing the patterns of variation that maintain ease-of-functioning in future, changing conditions. some environmental changes are too challenging for ease to be expressed in the present. however, following an exposure to challenging conditions, some systems adapt and become more functional (taleb, 2012) . without the ability to functionally organize after stressors, a system degrades into disorder or chaos over time. host-defense functions, decision-making/executive functions and values systems prioritize and organize variation in the microbiota, habits/habitual life functions, and population behaviors respectively. when a pathogen invades the biological system, precise responses must occur to organize this potentially chaotic situation. at the level of the cell, a functional host-defense system (comprised of the innate, adaptive and complement immune system branches) organizes the biological system so that functional invaders (i.e. symbionts) and healthy cells are maintained and dysfunctional invaders (i.e. pathogens) and damaged cells are removed (hoeb et al., 2004; janeway, 1992; janeway and medzhitov, 2002; janeway et al., 2014) . when precision is dysfunctional, the host-defense system may (1) fail to prioritize responses to a costly invasion, leading to a state of infection; or (2) the host-defense system might prioritize dysfunctional responses to the cells of body that prolong a state of autoimmunity (naor and tarcic, 1982) . when a bad habit emerges, precise responses must occur to organize this potentially chaotic situation. at the level of the self, functional decision-making (or at smaller scales executive functioning) prioritizes and organizes behavior so that functional expressions of habit (or at smaller scales, habitual life functions) are prioritized regularly, and dysfunctional ones are replaced or minimized (de bruin et al., 2016; prochaska et al., 1994; prochaska and prochaska, 2016; prochaska et al., 1988; redding et al., 2011; weissenborn and duka, 2003; bickel et al., 2012) . when dysfunctional, decisions may (1) fail to prioritize responses that remove a costly expression of habit (e.g. a teen started smoking cigarettes to "be cool" and now has to smoke in the bathroom before each class to get through the day; by not deciding to move at work, one's breathing becomes shallow and movement becomes rigid); or decisions may (2) prioritize habits that prolong dysfunction despite knowing the dangerous consequences (e.g. an adult continues smoking cigarettes despite knowing the family's history of lung cancer; an adolescent continues binge on sugar despite a diabetes diagnosis). when dangerous population-wide behaviors threaten life in a society, precise responses must occur to organize this potentially chaotic situation. at the level of society, the agreed upon values organize the social system so that functional population behaviors are prioritized and dysfunctional population behaviors are minimized. functional values prioritize behaviors that support the society (e.g. as seen when societies mandate that students get certain vaccines before attending university), while also setting standards that remove/replace behaviors that threaten the society (e.g. new laws create legal repercussions for risk behaviors in society). without values that functionally prioritize populationwide behavior, society may (1) fail to prioritize responses to a dysfunctional population behavior (e.g. as seen during aids epidemic of the 1980s due to insufficient public health values around safe sex); or society may (2) prioritize dangerous behaviors that can prolong societal dysfunction (e.g. the antibiotic resistance crisis (ventola, 2015; michael et al., 2014) has been attributed in part to the over-valuing or over-use of antibiotic medications in healthcare practices). precision is essential so that a system can maintain ease-offunctioning in future, changing conditions. when precision does not adequately detect the presence of costly conditions, a response may not be prioritized (e.g. as seen during acute infection, addiction/dependence following a surgery, the aids outbreak in the 1980s). when precision prioritizes responses that prevent ease longitudinally, dysfunction is prolonged (e.g. autoimmunity, continued smoking despite family history of cancer, misguided values that create an antibiotic-resistant bacteria). through dysfunctional-precision, the conditions for life in cells, selves, and societies becomes disordered over time. through functional-precision, a system prioritizes responses that maintain ease-of-functioning in future conditions. prioritizing functional microorganisms (i.e. symbionts) supports the developing life of cells; prioritizing functional habits (e.g. weekly exercise) and habitual life functions (e.g. diaphragmatic breathing and relaxed movement) supports the developing life of the self; and prioritizing functional population behaviors (e.g. access to functional health care, economic resources; access to social support) supports the developing life of the society. principle 5: health is valued by a system when precision-andvariation generate maintainable-ease of functioning. health is de-valued by a system when precision or variation prevent maintainable-ease of functioning. by defining precision-andvariation, we can better understand maintainable-ease of functioning in population health: • functional-variation generates ease-of-functioning in the present (e.g. fluid and variable motion reflects an ease and variability of one's movement); while functional-precision prioritizes expressions that can maintain ease-of-functioning in the future (e.g. prioritizing challenging exercise for 20 min each day may lead to an ease in bodily movement long term). • dysfunctional-variation prevents ease-of-functioning in the present (e.g. prolonged sitting might lead to rigid movement and shallow breathing); while dysfunctional-precision might prioritize expressions that prevent ease in the future (e.g. rather than focus on relaxing breathing and movement on work breaks, one decides to drink alcohol to relax). without functional-variation, life is fragile and vulnerable to changing conditions of the present. without functional-precision, life becomes disorganized from the system's exposure to changing conditions across time. when functional-and-integrated, precision-and-variation value maintainable-ease of functioning in cells, selves, and societies. when dysfunctional or fragmented, precision or variation can de-value maintainable-ease of functioning in cells, selves, or societies. if maintainable-ease of functioning can be valued in cells, selves, and societies, we will likely observe healthy publics. five principles of health are presented: (1) health is the maintainable-ease of functioning; (2) maintainable-ease of functioning emerges from multiple levels; (3) at each level, maintainable-ease of functioning is generated by systems; (4) each system employs two functions, precision-and-variation, that generate maintainable-ease of functioning; and (5) health is valued by a system if precision-and-variation generate maintainable-ease of functioning. through these five principles, both naturalistic and holistic perspectives can be considered simultaneously because maintainable-ease of functioning is relevant to biological functioning (e.g. as described in bst) and personal/social, goal-oriented functioning (e.g. as described in hth). this philosophy can also be used to investigate how naturalistic and holistic phenomena have informed past healthcare interventions. what do vaccine interventions, behavior change interventions, and social change interventions have in common? when successful, these interventions enhance both precision and variation. vaccine interventions can enhance both the precision of the host-defense functions and variation in the microbiome. during a vaccine intervention, the microbiome is exposed to a new variation in the form of a new virus (reidel, 2005) . through this exposure, the precision of host defense functions can adapt to prioritize maintainable-ease of functioning in the microbiome in the future. how? the host-defense system produces antibodies that allow the immune system to respond effectively and efficiently to this virus when exposed to it again in the future (janeyway, 2014) . although the precision of the immune system has been enhanced to handle historical threats through vaccines (e.g. for small pox, chickenpox, measles), new viruses like the coronavirus can still emerge. with this philosophy, vaccine developers and public health officials might not only ask the question, "how do we combat the coronavirus?" researchers, vaccine developers and public health officials may also ask the functional question: "how do we enhance the precision of the host-defense system and the variation of the human microbiome to adapt following an exposure to the coronavirus?" behavior change interventions can enhance both the precision in one's decisions and the variation in one's habits. during a behavior change intervention, a person's existing habits are exposed to a new variation in habit. for example, the beginning of a new exercise intervention exposes the individual's current habits/habitual functioning to changes in movement and breathing (i.e. exercise) that may also change their patterns of eating and hydration. through this exposure, a person's decisionmaking might adapt to prioritize maintainable-ease of functioning in the individual's lifestyle. how? some behavior change interventions train one's decision-making to remove or "countercondition" unhealthy habits, by replacing them with healthy habits (prochaska et al., 1988) . although modern behavior change interventions have shaped the precision of decisionmaking during health behavior change (e.g. of smoking, diet, alcohol use, inactivity), new problems for health behavior still emerge when the individual is exposed to a new, potentially addictive technology. with this philosophy, behavior change interventionists and health officials might not only ask the question, "how do we support good decision-making of individuals?" researchers, behavior change technology developers, and public health officials may also ask the functional question: "how do we enhance the precision of one's decisions and the variation of one's habits following the exposure to a new, potentially addictive technology?" public health campaigns disseminated by social organizations can enhance the precision of the population's health values and variation in population-wide health behaviors. leading up to first surgeon general's advisory committee on smoking and health (1964) , the u.s. department of health had become increasingly aware of (i.e. exposed to) variations in a population health behavior. if populations smoked, then populations were more likely to develop lung cancer, laryngeal cancer, or chronic bronchitis (cdc, 2018) . following this exposure to (the consequences of) population smoking behavior, society's values shifted to prioritize health. how? the federal cigarette labeling and advertising act of 1965 was adopted, and the public health cigarette smoking act of 1969 was adopted to create new health values. this shift in values prioritized new variations in population health behavior by: (1) requiring a health warning on cigarette packages; (2) banning cigarette advertising in the broadcasting media; and (3) calling for an annual report on the health consequences of smoking (cdc, 2018) . since these first initiatives adult smoking rates have fallen from about 43% (in 1965) to about 18% today; and mortality rates from lung cancer, the leading cause of cancer death, are declining (department of health and human services, 2014). although the precision of the population's values has been enhanced to impact population behaviors (e.g. the tobacco laws described above supported healthy change), new chronic states can still emerge following exposure to social changes (e.g. the invention of the juul impacted high school and college aged populations). with this philosophy, public policy officials and public health researchers might not only ask the question, "how do we create new laws to protect population health from nicotine addiction?" they may also ask the functional question: "how do we enhance the precision of the population's values and the variation of the population's behavior following the invention of a new nicotine delivery system technology (e.g. flavored juuls)?" previously we described that without functional variation, life is fragile when exposed to present changing conditions; and without functional precision, life becomes disorganized from exposure to changing conditions across time. when successful, the above interventions upon biological, behavioral, and social functioning have a common theme: each facilitates exposures to biological, behavioral or social conditions that support (1) increasingly complex/diverse variation; and (2) increasingly organizable precision. exposure, not avoidance, has facilitated population health in these interventions. while healthcare systematically prioritizes biological exposures in the form of vaccine interventions, they do not systematically prioritize behavioral or social exposures. however, it is documented that exposure to healthy behaviors in youth prevents risk behaviors in adolescence (velicer et al., 2000) ; and exposure to community-based health initiatives can support population health (dulin et al., 2018; cdc, 2018) . given that systematic biological exposures in the form of vaccination have led to a global control of some acute infectious diseases (tangermann et al., 2007) ; might systematic behavioral and social exposures (especially in youth) be needed to enhance global campaigns toward the control of chronic disease? a functional language of health is central to the success of a philosophy of health. why? the levels are not separate, but rather are continuously connecting with one another. a good philosophy of health should have the ability to discuss assessment, diagnosis, intervention, and prevention across levels, across systems, across cultural populations, and across time. using the common language of precision and variation creates discussions that connect the levels and integrate research disciplines. a case (to) study: mental health as between-level functioning in this philosophy. historically, and still too often, health professionals have an expertise at one level, that limits their prescription of interventions to that level. this can actually create barriers to a complete solution when a health problem is multileveled. while a person's mental health is typically assessed based upon their firstperson experience of thoughts, feelings, and behaviors; symptoms can be triggered by biological, physiological, behavioral, psychological, and/or social dysfunction. most clinicians typically do not have the ability to assess and address all forms functioning. so if one person, john, is meeting with a clinician who specializes in primary care medicine, he may only be prescribed a biological intervention like medication. if john is meeting with a clinician who specializes in behavioral medicine, he may only be prescribed a health behavior change intervention. if john is meeting with a clinician who specializes in a certain theory of psychotherapy, he may only be prescribed a psychotherapy intervention based on the clinician's training. if john is meeting with a clinician who specializes in social work, he may only be prescribed a group, community or social intervention. while the above specializations have been helpful in establishing an empirical bases for mental health interventions, overspecialization can be problematic when a multi-leveled solution is needed. in addition, it can also be problematic when a levelspecific solution is needed that the clinician cannot provide (e.g. when psychotherapy is needed but a clinician only has the ability to prescribe psychiatric medication). technology poses a multileveled issue for population mental health in 2020. selves have more social connection then ever in history, yet societies are characterized by increasing rates of depression and loneliness (sum et al., 2008; hammond, 2020; srivastava and tiwari, 2013; twenge, 2017) . researchers might use this philosophy of health to facilitate between-level conversations that address seemingly paradoxical outcomes that emerge during this new age of rapid technological growth. to do this, a researcher might first begin by asking questions about functioning at each level; second, ask questions about processes between the levels; and third, concurrently ask questions at and between levels. first: begin by asking questions at each level. novel challenges face the igeneration (and their parents) due to technology's novel impacts on the development of individual and social functioning (twenge, 2017) . for example, if john's decisions (self-precision) and habits (self-variation) remain consistent during school hours because his parents do not let him have a phone; but his class' social behaviors around him (society-variation) change dramatically because everyone else at school uses the newest smartphone application to talk during class; will john's mental health suffer? although his parents' intentions are to protect john, the contrast between his behavior (self's precision-and-variation) and the population social behavior (society-variation) can impact john's health. notice here how we have not yet considered functions that connect the self to the society (e.g. john's thoughts and feelings). rather we first consider (or contrast) functioning at the level of the self (i.e. john's decisions-and-habits) and the society (i.e. population social behavior) in accordance with principles 1-5 (see figs 1-3). second: look for functional processes that connect the levels. one person's thoughts and emotions/feelings are processes that help to integrate the functioning of one-self within the functioning of a society. how might john's thoughts and feelings connect his (sense of) self to his society? perhaps john's parents teach him that it is important to feel separate from his classmates during class so he can think clearly in class; and that he can feel connected to his friends by inviting them over to communicate together after school. this parenting may impact john's thoughts and feelings during school. if john's parents do not talk with him about this topic, john may experience different thoughts and feelings during school hours. when kept to one-self, thoughts and emotions are foundational to an internal sense of self as one functions in the larger society; and, when acted upon, thoughts and feelings can become verbal communication (e.g. speech) and non-verbal communication (e.g. body language, facial expressions) that form an external sense of self that is visible to the society. the (internal) experience of and (external) communication of thoughts, feelings and actions form the foundation of all systems of psychotherapy (prochaska and norcross, 2018) . this view can be particularly helpful as researchers begin to investigate how smart technology impacts developmental changes to the self within the society beginning in youth. third: concurrently ask questions at and between levels. perhaps, a clinical researcher is interested in investigating protective mental health factors in the igeneration; and they hypothesize that lower rates of loneliness, anxiety, and depression will be seen in subjects that do not respond to text messages immediately. the researcher might investigate further by using the philosophy to develop questions for the research subjects: "(1) do you use conscious decision-making (self-precision) to prevent yourself from habitually responding to your phone when a text appears (self-variation)? (2) how fast do other's in your social group typically respond to texts (society-variation)? (3) what changes in thoughts and feelings are experienced (internal self-society connection) after you communicate via text (external self-society connection)?" perhaps this researcher also wants to investigate how those who are addicted to the technology perceive nonresponders. the clinical researcher might again apply the philosophy: "(1) how fast do other's in your social group typically respond to your texts (society-variation)? (2) do you experience changes in thought and feeling (internal self-society connection) when others do not respond to you within an hour (societyvariation)? (3) how do you communicate those thoughts and feelings (external self-society connection) with others when they do not respond for a prolonged period of time (society-variation)?" future research might use this method to gather and organize levels of information on mental health factors across different self-and societal-conditions. the processes that form our mental health form a functional connection between self and society. if mental health is a reflection of the self-society connection, what might be a reflection of the self-cell connection? physiological health evidences a functional connection between our sense of self and our cells. for example, aerobic exercise is a health behavior that stimulates changes to variations in breathing and movement. by engaging in this behavior, the biological cells of the body are also stimulated via various physiological processes. breathing will stimulate cellular functioning via the cardiovascular and respiratory systems; and movement will stimulate cellular functioning via the cardiovascular, musculoskeletal, and central nervous systems. while all physiological systems are working in collaboration in the body, certain changes to behavioral and biological functioning will stimulate certain physiological systems. by viewing health through this lens, between-level observations join the philosophy: biological functions emerge at the level of the cell; physiological functioning emerges as the cell-self connection; behavioral functions emerge at the level of the self; psychological/ mental functioning emerges as the self-society connection; and social functions emerge at the level of the society. future papers will explore maintainable-ease of functioning at and between levels. future directions: new images of healthcare integration and new perspectives of healthcare innovation. by considering this integrative philosophy, one can define health based upon a tangible connectedness, rather than separateness, of cells, selves, and societies. we provide image 4 as a way to visualize the common paths to the health of healthy publics. when researchers observe that a host defense system is changing cellular functions following an infection, they may also expect these changes to have an impact [along path 1] on expressions of habitual or physiological functions (e.g. immune function can stimulate the sensation of "achiness" or "pain" altering one's physical movement, breath rate, hydration, and hunger) (kelley, 2003; johnson et al., 1992; danzer, 2009) . when researchers observe an individual deciding to engage in health behavior change following an addiction, they may also expect these changes to have an impact [along path 2] on the group-behavior of their family system or social systems. when researchers observe changes to society's values following a newly detected problem (e.g. laws ban cigarette advertising in broadcasting media; public health standards mandate certain vaccines before attending school), they may also expect that these changes can have an impact on behavioral functions of individuals [along path 2] and biological functions of cells/organs [along path 3]. these levels are continually integrating along these common paths to the health of healthy publics (fig. 4) . when attending to this connectedness new, important questions can have new answers. what function does modern technology serve in population health and healthcare? if technology algorithms prioritize variations in population behaviors, then they fulfill a role as society-level precision. when modern technologies like machine learning (ml) technology and computer tailored interventions (cti) prioritize patterns of population behavior, we can see profound impacts on social change in a society. although one might argue that technologies can be used by individual-level functions, the algorithms that are currently deployed and updated on devices interface with bigdata gathered on population behaviors (manogaran and lopez, 2017; dinov, 2016; mullainathan and spiess, 2017; cheng et al., 2017) . in this paper, we identified that precision can be functional or dysfunctional. similarly, technologies can support or prevent healthy population behavior. some technologies prioritize health behavior in populations by tracking physical activity and providing feedback on activity progress; while others prevent healthy behavior by prioritizing sedentary behavior through video-gaming. some social media technologies facilitate social communication with distant friends and relatives that supports wellbeing; while others facilitate conflictual communication that diminishes wellbeing. given that modern technology can support or hinder health, we believe it is important that healthcare can prioritize technological innovations that value health in cells, selves, and societies. to do this, technology innovators might seek to value a higher order construct (e.g. maintainable-ease of functioning) in their algorithms. medical technology is currently used to titrate the doses of vaccines so that maintainable-ease of biological functioning (i.e. inoculation) is made available to the general population. when biological exposures are not properly titrated, infections can become active in the population and health is no longer valued at the level of the cell. similarly, when behavioral and social exposures are not tailored to the needs of individuals and groups, populations can become resistant to healthy change, and health is no longer valued at the level of the self and the society. behavior change researchers prochaska and prochaska (2016) report that when individuals and populations are not ready for a change, interventions that force individuals or populations to take action can increase resistance and prolong dysfunction. by tailoring (or what they term "staging") behavioral and social level interventions, computer tailored interventions upon behavioral and social functioning are made possible (prochaska et al., 2001; velicer et al., 2000, prochaska and prochaska, 2016) . despite these advances, there remains a need for technological advances that can make maintainable-ease of behavioral and social functioning available to the general population. future healthcare interventions could benefit from ml algorithms that tailor behavioral and social exposures to enhance precision-and-variation. research already demonstrates that tailoring interventions for biological precision (albert-vega et al., 2018) and biological variation (brodin et al., 2015) can impact long-term biological functioning. future innovations might seek to use technology to tailor behavioral and social interventions to generate maintainable-ease of functioning. through the functional language used in this paper we hope readers are inspired to present new questions, new comments, and new perspectives about needed healthcare innovations. behaviorially conditioned immunosuppression on the development of psychoneuroimmunology immune functional assays, from custom to standardized tests for precision medicine rethinking cells to society are executive function and impulsivity antipodes? a conceptual reconstruction with special reference to addiction metabolic diversity of the intestinal microbiota: implications for health and disease variability and diversity of nasopharyngeal microbiota in children: a metagenomic analysis a rebuttal on health evolution, order and complexity. routledge bower ck, daeschel ma (1999) resistance responses of microorganisms in food environments variation in the human immune system is largely driven by non-heritable influences the blue zones: 9 lessons for living longer from the people who've lived the longest from 'big 4'to 'big 5': a review and epidemiological study on the relationship between psychiatric disorders and world health organization preventable diseases assessing suicide risk and emotional distress in chinese social media: a text mining and machine learning study composition, variability, and temporal stability of the intestinal microbiota of the elderly the impact of the gut microbiota on human health: an integrative view psychophysiological effects of breathing instructions for stress management environmentalism, values, and social change historical variations in mutation rate in an epidemic pathogen, yersinia pestis the role of public health improvements in health advances: the twentieth-century united states comparison of the atkins, ornish, weight watchers, and zone diets for weight loss and heart disease risk reduction cytokine, sickness behavior, and depression the contribution of changes in diet, exercise, and stress management to changes in coronary risk in women and men in the multisite cardiac lifestyle intervention program a rct comparing daily mindfulness meditations, biofeedback exercises, and daily physical exercise on attention control, executive functioning, mindful awareness, self-compassion, and worrying in stressed young adults sugar-sweetened and artificially sweetened beverage consumption and risk of type 2 diabetes in men microbiota-generated metabolites promote metabolic benefits via gut-brain neural circuits volume and value of big healthcare data organizational legitimacy: social values and organizational behavior examining neighborhood and interpersonal norms and social support on fruit and vegetable intake in low-income communities zero trends: health as a serious economic strategy neural control of the circulation during exercise in health and disease reflex control of the circulation during exercise childhood infections, antibiotics, and resistance: what are parents saying now? antibiotics and the human gut microbiome: dysbioses and accumulation of resistances interaction between psychiatric and autoimmune disorders in coeliac disease patients in the northeastern united states distinct clones of yersinia pestis caused the black death the evolution of variability and robustness in neural development interactions between the microbiota and the immune system consumption of sugar sweetened beverages, artificially sweetened beverages, and fruit juice and incidence of type 2 diabetes: systematic review, metaanalysis, and estimation of population attributable fraction the immune system evolved to discriminate infectious nonself from noninfectious self innate immune recognition immunobiology: the immune system in health and disease mechanisms of stress: a dynamic overview of hormonal and behavioral homeostasis cytokine-induced behavior a role for brain stress systems in addiction sugar and hormone connections impact of healthy lifestyle factors on life expectancies in the us population psychiatric disorders in first-degree relatives of children with pediatric autoimmune neuropsychiatric disorders associated with streptococcal infections (pan-das) a survey of big data architectures and machine learning algorithms in healthcare the antimicrobial resistance crisis: causes, consequences, and management the state of us health, 1990-2016: burden of diseases, injuries, and risk factors among us states machine learning: an applied econometric approach control of autoimmune responses induced with modified self antigens the concepts of health and illness revisited dynamic typology clustering within the stages of change for smoking cessation evolution of indirect reciprocity vaccine refusal, mandatory immunization, and the risks of vaccine-preventable diseases spatial and temporal variability of the human microbiota does cigarette smoking cause stress? the blue zones: areas of exceptional longevity around the world measuring processes of change: applications to the cessation of smoking patterns of change: dynamic typology applied to smoking cessation evaluating a population-based recruitment approach and a stage-based expert system intervention for smoking cessation baseline stage, severity, and effort effects differentiate stable smokers from maintainers and relapsers jenner and the history of smallpox and vaccination immune systems evolution. sensing in nature message to congress evolution of the gut microbiota and the influence of diet developmental catch-up, and deficit, following adoption after severe global early privation rethinking health: healthy or healthier than? the gulag archipelago, 1918-56: an experiment in literary investigation. random house srivastava a, tiwari rp (2013) effect of excess use of cell phone on adolescent's mental health and quality of life the effects of psychological stress on respiration: a preliminary study of anxiety and hyperventilation internet use and loneliness in older adults the black swan of cairo: how suppressing volatility makes the world less predictable and more dangerous random house incorporated tangermann rh, nohynek h, eggers r (2007) global control of infectious diseases by vaccination programs igen: why today's super-connected kids are growing up less rebellious, more tolerant, less happy-and completely unprepared for adulthood-and what that means for the rest of us. simon and schuster. us department of health and human services (2014) the health consequences of smoking-50 years of progress. a report of the surgeon general velicer wf the antibiotic resistance crisis: part 1: causes and threats habits and implementation intentions. the abc of behavioural change the biostatistical theory versus the harmful dysfunction analysis, part 1: is part-dysfunction a sufficient condition for medical disorder acute alcohol effects on cognitive function in social drinkers: their relationship to drinking habits basic documents. world health organization xavier da silveira dos santos a biocides and "superbugs this research was supported by funding from the national institutes of health's (nih) national cancer institute (nci). the authors declared the following potential competing interests: jop, ph.d. is a founder of the transtheoretical model (ttm) of behavior change, and is the director of the cancer prevention research center at the university of rhode island. jms, m.ed. is currently a ph.d. student conducting dissertation research on population health and behavior change under jop's supervision at the university of rhode island. correspondence and requests for materials should be addressed to j.m.s.reprints and permission information is available at http://www.nature.com/reprintspublisher's note springer nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.open access this article is licensed under a creative commons attribution 4.0 international license, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the creative commons license, and indicate if changes were made. the images or other third party material in this article are included in the article's creative commons license, unless indicated otherwise in a credit line to the material. if material is not included in the article's creative commons license and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. to view a copy of this license, visit http://creativecommons.org/ licenses/by/4.0/. key: cord-263261-xhem8l39 authors: tulchinsky, theodore h. title: bismarck and the long road to universal health coverage date: 2018-03-30 journal: case studies in public health doi: 10.1016/b978-0-12-804571-8.00031-7 sha: doc_id: 263261 cord_uid: xhem8l39 the 2015 sustainable development goals (sdgs) state that all united nations member states have agreed to try to achieve universal health coverage by 2030. this includes financial risk protection, access to quality essential health care services and access to safe, effective, quality and affordable essential medicines and vaccines for all. universal health coverage (uhc) means inclusion and empowerment for all people to access medical care, including treatment and prevention services. uhc exists in all the industrial nations except the us, which has a mixed public-private system and struggles with closing the gap between the insured and the uninsured population. middleand low-income countries face many challenges for uhc achievement, including low levels of funding, lack of personnel, weak health management, and issues of availability of services favoring middleand upper-class communities. community health services for preventive and curative health services for needs in populations at risk for poor health in low-income countries must be addressed with proactive health promotion initiatives for the double burden of infectious and noncommunicable diseases. each nation will develop its own unique approach to national health systems, but there are models used by a number of countries based on principles of national responsibility for health, social solidarity for providing funding, and for effective ways of providing care with comprehensiveness, efficiency, quality, and cost containment. universal access does not eliminate social inequalities in health by itself, including a wide context of reducing social inequities. understanding national health systems requires examining representative models of different systems. health reform is necessarily a continuing process as all countries must adapt to face challenges of cost constraints, inequalities in access to care, aging populations, emergence of new disease conditions and advancing technology including the growing capacity of medicine, public health and health promotion. the growing stress of increasing obesity, diabetes, and other chronic diseases, requires nations to modify their health care systems. learning from the systems developed in different countries helps to learn from the processes of change in other countries. the world health organization (who) defines a health system as: "the people, institutions and resources, arranged together in accordance with established policies, to improve the health of the population they serve, while responding to people's legitimate expectations and protecting them against the cost of ill-health through a variety of activities whose primary intent is to improve health. it is a set of elements and their relationship in a complex whole, designed to serve the health needs of the population. health systems fulfill three main functions: health care delivery, fair treatment to all, and meeting health expectations of the population." who's world health reports (2000, 2006, 2013) focused on health systems financing and management in the search for universal health coverage. under the globally endorsed sdgs, universal health coverage (uhc) is designated goal 3 (health and wellbeing), target 3.8: "achieve universal health coverage (uhc), including financial risk protection, access to quality essential health care services, and access to safe, effective, quality, and affordable essential medicines and vaccines for all". box 8.1 outlines who building blocks for uhc. universal access is a means of assuring that the economic barrier to health care is mostly if not completely removed for the total population and may lead to increased access to medical and hospital services for those previously excluded. while uhc increases access to medical care and health indices, it does not, of itself, guarantee achievement of many important health targets. allocation of resources is an even more fundamental problem to address the needs of those with the highest risk of early disability or avoidable premature death. a system of national health must be able to allocate resources to meet those needs and must not simply be a payment system for doctors and hospitals. changing demographics, medical advances and epidemiological challenges including social and health inequalities also be addressed with high priority. this case study provides the background and experience of the development of uhc over the past century and a half, with lessons learned for consideration in how-and what-is done to achieve this goal. most industrialized countries have implemented national health programs such as health insurance systems or national health services. each system developed in the political, social, and historical context of the country-and continues to evolve. medium-and lowincome countries are also struggling to achieve universal access to care and health for all by expanding primary health care and social security plans which provide benefits to workers and for certain vulnerable populations-primarily mothers and children. as they move up the scale of economic development, developing countries must also address the problem of how to decrease morbidity and mortality, achieve equity in access to health care, and expand the funding basis for health care through national health insurance. some countries experience rapid economic development, but lag behind in directing increased national wealth towards improving health status. this is often due to a lack of focused political commitment, trained policy analysts, and cultural adaptation to the crucial importance of public health. each national health system has its own characteristics and challenges. systems management requires continuous evaluation based on welldeveloped information systems, trained health management personnel, societal involvement through all levels of government, as well as the private sector, professional organizations and advocacy groups. there is no defined "gold standard" plan for providing universal access to health care that is suitable for all countries. each country develops and modifies a program of national health appropriate to its own political and cultural needs and available resources. however, there are evolving patterns in health care organization, so that networking within and between countries ensures that they can-and do-learn from one another (box 8.2). barriers to necessary health care can be geographic, ethnic, cultural, social, lack of information and awareness, psychological, financial, and poverty. removing financial barriers to care is necessary and constructive, but not sufficient to address the health problems of individuals and of a society. equity in financial access with universal coverage is vital to population and individual health since anyone can have serious illness at any time. but equally important, long-term preventive care and health promotion are essential to good population and individual health standards. inequities exist in all societies, but many countries have successfully reduced these by poverty alleviation, job creation, education, and other programs that reduce interregional, socioeconomic, and demographic differences in health. special attention to high-risk groups in a population is essential. groups at-risk may be based on age, gender, ethnicity, genetic legacy, occupation, risky lifestyle, location of residence, religion, sexual orientation, economic status, or other factors that increase susceptibility to disease, premature death, or disability. services must be based on need and not only demand, which can escalate costs by over-servicing. health systems planning needs to promote access to patient care, but also those services that reach the entire population, especially people at high risk who are often least able to seek and access appropriate care. a program that provides equal access for all may not achieve the objective of better health for the population unless accompanied by other box 8. 2 key elements of national health systems 1. a tradition of government and nongovernmental initiatives to improve health of the population. 2. public administration and regulation; public-private partnerships. 3. intersectoral cooperation with education, social services and the private sector. 4. demographic, economic, and epidemiologic monitoring. 5. health targets monitored with accessible data systems. 6 . public health programs, including strong elements of health promotion. 7. universal coverage by public insurance or service system. 8. access to a broad range of health services. 9. strategic planning for health and social policies. 10 . monitoring health status indicators. 11. recognition of special needs of high-risk groups and related issues. 12. portability and accessibility of benefits when changing employer or residence. 13. efforts to reduce inequity in regional and socio-demographic accessibility and quality of care. 14. adequacy of financing. 15. cost containment. 16 . efficient use of resources for a well-balanced health system. 17 . consumer satisfaction and choice of primary care provider. 18 . provider satisfaction and choice of referral services. 19 . promotion of high-quality service. 20. promote patient and staff safety. 21. comprehensive public health and health promotion programs. 22 . comprehensive primary, secondary, and tertiary levels of medical care. 23. well-developed information and monitoring systems. 24 . continual policy and management review. 25 . promotion of standards and accreditation of services, professional education, training, research. 26. governmental and private provision of services. 27 . decentralized management and community participation. 28 . assurance of ethical standards of care for all. 29 . conduct epidemiological, basic sciences and health systems research. 30 . preparation for mass casualties from disasters and terrorism. important governmental, community and personal self-care activities. these include enactment and enforcement of environmental and occupational health laws, food safety, nutrition standards, clean water, improved rural care, higher educational levels, and provision of health information to the public. additional national programs are needed to promote health generally and to reduce specific risk factors for morbidity and mortality. responsibility for health lies not only with medical and other health professionals, but also with governmental and voluntary organizations, the community, the family, and the individual. individual access to an essential "basket of services" as a prepaid insured benefit is fundamental to a successful national health program. each country addresses this issue according to its means and traditions, but cost-effective evidence-based methods of meeting a countrys epidemiologic and demographic needs should be prioritized. coverage and payments for heart transplantation, for example, may be beyond the means of a health system, but early and aggressive management of hypertension, smoking, poor diet, physical inactivity, and rapid care for acute myocardial infarction are effective in saving lives at modest cost and containing the need for more intrusive health care interventions. prevention is cost-effective and should be integral to the development of service priorities within the insured benefits with incentives included in the "basket of services". globalization affects health systems around the world not only in the ease of spread of infectious diseases, but in increased access to modern preventive, diagnostic, treatment modalities. access to antiretroviral drugs has dramatically changed the face of hiv/aids globally, including in low-income countries with support of international and bilateral donors. the same is true for vaccines, including the mmr (measles, mumps, rubella, 2 doses), hib (hemophilus influenza b), rotavirus, pneumococcal pneumonia and hpv (human papillomavirus) vaccines, which will save millions of children's lives and foster well being in the coming decade. information technology, migration of medical professionals, and internalization of educational standards are all global health issues affecting national health systems. health systems in all countries are facing common problems in population health, with rising population age, hypertension, obesity and diabetes prevalence, and rising health care costs. health systems research capacity is important in each country as it attempts to cope with rapid changes in population health and individual health needs with limited resources. development of research capacity enables improved capacity of decision-makers for informed, cost-effective decisions. in developing countries, low levels of funding for health in general-including research-impede evidence-based health system development and training of the new health workforce. strengthening reporting systems of data aggregation, as well as economic and epidemiologic analysis, are vital for health policy and management. national health systems from germany, uk, canada, us and russia are presented here as representing major models of organization. these organizational models influence health care system formulation in both developing and developed countries, as well as for countries restructuring their health services. health care systems and financing are under pressure everywhere, not only to assure access to health for all citizens, but also to keep up with advancing medical technology, and contain the cost increase at sustainable levels. because a health system is judged by more than its cost and measure of medical services, indicators of health status of the population, as well as morbidity and mortality are vital and should be available for the public through community organizations and the media. this topic has developed a complex terminology of its own. the world health organization (who) helps development of national health systems as shown in box 8.3. universal health coverage is defined as ensuring that all people have access to needed health promotion, preventive, curative and rehabilitative health services, of sufficient quality to be effective, while also ensuring that people do not suffer financial hardship when paying for these services. g good health is essential to sustained economic and social development and poverty reduction. g access to needed health services is crucial for maintaining and improving health. g at the same time, people need to be protected from being forced into poverty because of the cost of health care. g a well-functioning health system working in harmony is built on having: trained and motivated health workers; a well-maintained infrastructure; a reliable supply of medicine and technologies; backed by adequate funding; strong health plans; evidence-based policies. who assists in creating resilient health systems by supporting countries to: g "develop, implement, and monitor solid national health policies, strategies and plans. g assure the availability of equitable integrated people-centered health services at an affordable price. g facilitate access to affordable, safe, and effective medicine and health technologies. g strengthen their health information systems and evidence-based policy-making, and to provide information and evidence on health-related matters." source: world health organization. health systems. available at: http://www.who.int/ healthsystems/about/en/ (accessed 2 may 2017). health systems are meant to improve health and quality of life, as measured by quantitative and qualitative methods. the human development index (hdi) provides a standard method of comparison which combines many health and social indices into a summary figure for social development of countries. these include life expectancy at birth, gross domestic product (gdp) per capita, child mortality, education and others. table 8 .1 shows life expectancy, still a valued health status indicator, for some industrialized, mid-level, and developing countries. comparisons between countries health indicators are useful to portray relative international health status among nations. the foundations of public responsibility for health care systems go back to ancient greece and rome where city states employed municipal doctors to service the poor and slaves. in the medieval and renaissance periods, monasteries and nunneries provided charitable care to the poor while professional guilds provided prepaid medical care and other social benefits to members and their families. these later evolved into the friendly (benevolent) societies, as mutual benefit programs that provided for burials, pensions, and payment for health services for members. in the twentieth century, these developed through collective bargaining into health insurance plans through private or professionally sponsored insurers, and labor unionã�sponsored health plans. governmental responsibility for health systems evolved in public health and health protection systems in the nineteenth and twentieth centuries and continues to evolve to face new challenges as well as preventive and treatment capacities. the health systems described highlight the unique and common features of national health systems in the search for "health for all", and policies for making health a priority in resource allocation, policy priority for human rights, and for socioeconomic development. figure 8 .1 indicates the 1995ã�2014 trends in total health expenditures as percent of gross domestic products of selected countries in the european region of who. german and swedish expenditures rose to between 11% and 12%, in the united kingdom to over 9% while israel is relatively stable under 8% and the russian federation expenditures rose to 7% of gdp. germany's health care system today is characterized by participation as well as sharing of decision-making powers between the states (laâ¨nder), the federal government and civil society organizations. since 2009, statutory health insurance (shi) has been mandatory for all citizens and permanent residents pay a uniform contribution of 15.5 percent of their income (gesetzliche krankenversicherung) with 118 sickness funds (krankenkassen, january 2016). shi covers 85 percent of the population, who have the right to choose their preferred sickness fund for a comprehensive range of services. the sickness funds are linked to associations of physicians accredited to treat patients covered by shi. private health insurance (phi) covers 11 percent of the population for designated groups such as civil servants. others (4%) such as the military are included in other specific governmental programs. since the 1990s financial incentives are being introduced to improve quality and efficiency of care along with beneficiaries right to choose between sickness funds increasing competition and a market orientation. hospitals are paid by diagnosis related groups (drgs)-i.e., payment by diagnostic category rather than hospital length of stay, adopted from us experience. physicians are paid by a capitation system-i.e., a fixed payment for each person registered for care with a doctor for a fixed period of time (as opposed to fee-for-service) in the doctor's medical associations. longterm care is covered by a federal mandatory program. germany expends 11.3 percent of gdp (2015) on health, one of the highest levels among eu members, with 73 percent from public sources and 27 percent privately sourced. in 2014, germany had 6.2 acute care beds per 1,000 beds per 1000 population, nearly 40 percent above the rate for the original eu countries (3.8 per 1,000). of these, 48 percent of beds were in publicly owned, 34 percent in private nonprofit, and 18 percent in private for-profit hospitals. busse et al. (2017) describes reforms since its founding in 1883 gradually achieving universal coverage. the system is also seeking greater cost effectiveness as compared to neighboring countries. in europe, many countries developed taxation or social security models based on the bismarckian approach, with compulsory contributions by workers and their employers to a national social security system. this then financed approved services usually paid through private medical practice with fee-for-service payment. many european countries and japan gradually developed similar forms of compulsory health insurance for workers and their families following world war i, or later after world war ii, expanding to universal coverage health insurance systems. this model is used in france, belgium, the netherlands, japan, switzerland, and latin america as well as post-soviet health reforms and countries of eastern europe (cee). the israeli system, adopted in 1995, based on the bismarckian model is mandatory national health insurance in which everyone must choose one of four long-standing sick funds now called health organizations. they compete for members, and are paid a per capita sum for which they are obliged to provide comprehensive services including hospital, primary care, and preventive services. the services improved vastly under national health insurance, with services kept up to date with annual additions to the statutory "basket of services." health statistics show israel as among the top countries for life expectancy, with rapidly falling mortality from strokes, coronary heart diseases, and cancers. consumer satisfaction is high, maternal and child health are stressed, a low hospital bed to population ratio, while health expenditures are relatively modest and a stable per capita health expenditure just under eight percent of gdp (lancet 2017). william beveridge was born in 1879 in bengal, india, where his father was a judge in the indian civil service. he trained as a lawyer coming to prominence in the british liberal government of 1906ã�14 when he advised david lloyd george (chancellor of the exchequer from 1908 to 1915, prime minister from 1916 to 1922) on old age pensions and national insurance. in 1911, initiated by lloyd-george, influenced by the german compulsory health insurance scheme, the liberal government of great britain introduced the national health insurance act. it was compulsory for all wage earners between 16 and 70 years of age. this was a two-part plan based on a worker and employer contributory system for both unemployment insurance and for medical care for workers and their families. administration was through approved mutual benefit societies (the friendly societies), some based on insurance companies, and others by professional associations and trade unions. general practitioner services were paid on a capitation basis rather than a salary, preserving their status as self-employed professionals. initially this plan covered one-third of the population increasing to half by 1940, however there was disruption due to mass unemployment during the great depression starting in 1929 and continuing to the late 1930s. in the early days of world war ii, the british government established a national emergency medical service for hospitals in preparation for the anticipated large-scale civilian casualties that were expected during the blitz bombing by nazi germany. this established national health planning and rescued many hospitals from near bankruptcy due to the effects of the great depression in the united kingdom (uk). during world war ii, at the behest of prime minister winston churchill, beveridge developed a postwar social reconstruction program. the beveridge report of 1942, social insurance and health services, outlined the concept of a future welfare state including a national health service, placing medical care in the context of general social policy for the total population. the wartime government coalition approved the principle of a national health service, which had wide public support, despite opposition from the medical association. in 1945, the newly elected labour government of clement attlee took up the recommendations of beveridge to introduce the national insurance act (1946) as a comprehensive system of unemployment, sickness, maternity, and pension benefits funded by employers, employees and the government. the national health service (nhs) act was instituted in 1948 under the leadership of aneurin bevan, against continued opposition from medical organizations, as a universal state health service in britain. the nhs provides a nationally tax-based financed, universal coverage system providing free care by general practitioners, specialists, hospitals, and public health services. this includes diagnosis and treatment of illnesses at home or in hospital, including dental and optometric care. the original nhs structure was divided into three separate services: hospital, general practitioner, and community health services. the hospital and specialist services were under the authority of 14 regional boards. general practitioners worked under national contracts, and community health services, such as public health, home nursing and health visitors, midwives, maternal-and child care, came under the control of the county and city local authorities. all units reported to the minister of health and his staff. the hospital bed supply in the uk in 2014 was just under half the rate in france and one third of the rate of beds in germany per 1,000 population. hospital based specialists are salaried but highly independent; general practitioners ran their own practices and provided the foundation of the nhs system. over time, this tripartite structure evolved to some degree of integration of gp and community health services, along with hospitals under hospital trusts reporting to regional health authorities. the nhs, with periodic reforms, is still in place in the uk and well accepted by the population and-over time-even by conservative governments and by the medical profession. there are differences between the nhs systems of the uk: england, scotland, wales and northern ireland each operate their own nhs, albeit with funding and structure of the central nhs. regional disparities in health indicators still exist despite changes in funding giving greater resources within regions (north-south divide) of england; each of the four has their own, policy directions. social class and geographic inequities in health within the nhs have been recognized since the 1970s with a series of reports and analyses showing large gaps in life expectancy, avoidable (i.e., preventable) mortality between the south and north of england and even more so with scotland and significantly poorer health indicators. the marmot report on inequalities from 2010 indicated the scope of the problem: "people living in the most deprived neighborhoods will on average die seven years earlier than people living in the richest neighborhoods. even more disturbing, people living in poorer areas not only die sooner, but spend more of their lives with disability-an average total difference of 17 years. the review has estimated the cost of health inequalities in england: productivity losses of d31ã�33 billion every year; lost taxes and higher welfare payments in the range of d20ã�32 billion per year; and additional nhs healthcare costs well in excess of d5.5 billion per year." the "beveridge model" is a term used for the national health service model, which has since been adopted by many european countries and should be regarded as a strong model for countries reforming their universal health care systems, such as spain and italy. the scottish nhs diverges from the central english nhs in addressing inequalities by a focus on the health sector as the sole responsibility for reduction of inequalities. the english nhs and other government agencies see the problem more broadly and adopted poverty-fighting measures with some success in improving mortality and morbidity social and health disparities since 2000. the nhs system remains generally popular in providing health security for all, and reaching good outcome measures despite regional inequities. no change of governing political party has led to dismantling the nhs for a privatized health system over the seven decades since its inception. canada: national health insurance tc (tommy) douglas was born in falkirk, scotland and immigrated at the age of 10 with his working class family to winnipeg, manitoba, canada. he developed osteoarthritis and the doctors were going to amputate his leg as the family lacked funds for long-term medical care. his leg was saved by a senior surgeon who refused the amputation. this made tommy a lifelong advocate and fighter for publicly administered, universal health care for all. he became a baptist minister and entered politics winning the saskatchewan general election of 1944 for the ccf party in a massive victory. it was the first democratic socialist government elected in north america. he held the office for 17 years, during which time he pioneered many major social and economic reforms. canada (population 35.5 million) is a federal state and a constitutional monarchy with parliamentary systems at national and provincial/territorial levels. health is primarily a provincial responsibility, but federal funding and standards play an important role in the canadian health system. local authorities also carry out many primary public health services including sanitation, water safety, and supervision of food safety, among other responsibilities. the provinces/territories are responsible for the funding of hospital, community, home and long-term care, as well as mental and public health services. starting in the 1930s, federal grants-in-aid were given to the provinces/ territories for categorical health programs, such as cancer and public health services programs. since the sars (severe acute respiratory syndrome) epidemic in 2003, the canadian federal government has increased its capacity in public health with a new federal department of public health, regional laboratories and encouragement of many schools of public health across the country. canada's national health program evolved as a system of provincial health insurance with federal government financial support and standards. initiatives for national health insurance in canada go back to the 1920s, but definitive action occurred only after world war ii. the federal government regulates drug and medical device safety, funds research and provides services to the native indigenous population groups, the military, rcmp (royal canadian mounted police) and federal prison inmates. services for veterans were later transferred to provincial medicare programs. the development of national health insurance was largely due to the bitter experience of the great depression of the 1930s, a strong agrarian cooperative movement, and the collective wish for a better society following world war ii. in 1946, the social democratic cooperative commonwealth federation (ccf) party under the leadership of tommy douglas formed the government of saskatchewan, a large wheat-growing province of one million people on the western prairies. the national universal health insurance program evolved from the provincial initiatives led by tommy douglas, now considered "the father of canada's universal medicare plan." douglas established the saskatchewan hospital insurance and diagnostic services act in 1946 under provincial public administration. in 1956 a federal cost-sharing formula began providing approximately 50 percent cost-sharing with greater levels of funding going to the poorer provinces. by 1961, all 10 provinces and two territories had implemented hospital insurance plans, in a twotiered national health insurance plan-i.e., universal provincial/territorial health plans with federal standards and cost-sharing. in 1962, again in saskatchewan, the medical care insurance plan (medicare) was implemented after a bitter doctors' strike. in 1961, the federal government appointed a royal commission on health services (the hall commission) which in 1964 recommended adoption of the saskatchewan model across the country with federal support and standards. the saskatchewan plan was rapidly followed by similar plans in other provinces encouraged by generous federal costsharing. the federal government cost-shares provincial and territorial programs. provinces/territories must adhere to the standards of the canada health act (1984), which defines services to be covered for hospital, diagnostic, and physician services. there is federal funding support for provincial/territorial public health, long-term care, home care and community mental health services. this federal legislation was expanded to provide co-funding for provincial/territorial medicare plans, which over a short period brought all canadians into provincially administered systems of publicly financed health care, while retaining the private practice model of medical care. hospital care is provided mostly through non-profit, non-governmental hospitals. developed over the period 1946ã�71, the provincial/territorial health insurance plans were promoted by federal governmental cost-sharing, political support, and national standards. the plans were initially financed by taxation and premiums, but later solely by general tax revenues with federal support under the canada health act of 1984. federal standards required the provincial plans to be: publicly administered; comprehensive in coverage of health services; universal; portable across provinces; and, accessible without user fees. federal reimbursement to the provinces/territories initially covered 25 percent of national average medical care expenditures per capita and 25 percent of the actual expenditures by each individual province. this provided higher-than-national-average rates of support to poorer provinces as well as portability between provinces/territories. by 1971, all provinces had implemented such plans, and a high degree of health services equity was achieved across the country. care is provided by private medical practitioners on a fee-for-service basis under negotiated medical fee schedules with no extra billing allowed. hospitals are operated by nonprofit voluntary, religious organizations or municipal authorities, with payment by block budgets. per capita spending on health in canada is relatively modest in comparison with that of the us, but above oecd averages. public spending as a percent of total health expenditures is close to the oecd average (see box 8.4 ). this medicaretype plan was later adopted in a number of other countries including australia. medicare is still popular in canada, with support from all political parties and by most medical professionals. medicare and federal cost-sharing weighed in favor of the poorer provinces, allowing these to catch up in health care services and standards with the richer provinces. the canadian health program differs substantively from those of the united kingdom and the united states. health systems are important in the political and cultural life of a country. each within its own tradition is attempting to ensure population health through public or private means, to constrain the rate of cost increases. comparisons using various health indicators can be controversial, but the canadian universal health service or insurance coverage seems to have improved the health status of the population more rapidly than similar indicators for the total us population, but not necessarily for all segments of the population. after decades of focus on developing national health insurance, canada became a leading innovator in health promotion prevention (see chapter 21) . the canadian health program established universal coverage for a comprehensive set of health benefits without changing the basic practice of medicine from individual medical practice on a fee-for-service basis. poorer provinces were able to use the federal cost-sharing mechanism to raise standards of health services, and a high degree of health services equity was achieved across the country. rapid increases in health care costs led to a review of health policies in 1969 (the federalã�provincial committee on the costs of health services). the resulting report stressed the need to reduce hospital beds and develop lower-cost alternatives to hospital care, such as home-based care and long-term care. federally-led initiatives during this period extended coverage to include home-based care and long-term nursing home care, while restricting federal participation in cost-sharing to the rate of increases in the gross national product (gnp). since then, many provincial and federal reports have examined the issues in health care and recommended changes in financing, cost-sharing, hospital services, development of primary care, and other community services. in 1974, a new approach to health was outlined by the federal minister of health, marc lalonde, in a landmark public policy document, a new perspective on the health of canadians. this report described the health field theory in which health was seen as a result of genetic, lifestyle, and environmental issues, as well as medical care itself. as a result, health promotion became a feature of canadian public policy, with the objective of changing personal lifestyle habits to decrease cross-cutting risky behaviors such as smoking, obesity, and physical inactivity. the pioneering work in nutrition from the national nutrition survey published in 1971 led to the adoption of federal mandatory enrichment regulations for basic foods with essential vitamins and minerals. this and other initiatives in the 1980s led to the ottawa charter on health promotion (see chapter 21) , which has had a global impact with the foundation of health promotion as a crucial new aspect of public health and health system policy. the canadian health system being primarily the responsibility of the provinces/territories had a down side. during the sars pandemic of 2003, the provinces dealt with it and were found to be lacking strong public health institutions adequate to the task. following high level reviews of the sars episode the federal government established a cdc-like institution, regional laboratories capable of infectious disease challenges and eight schools of public health across the country to ensure continuing development of a competent public health workforce. universal health care needed to be supplemented by introduction of lalonde-initiated health promotion and equally so a strong microbiologic public health component to ensure rapid and competent responses to new emerging health challenges. how does the canadian public view the universal public single payer medicare run by the provinces with federal guidelines and cost-sharing program? despite complaints, mostly from us sources, the canadian public appreciates their health protection very much. in 2004, the canadian broadcasting corporation (cbc) television conducted a program over many months called "the greatest canadian," with 10 candidates and advocates. this included a call to all people in canada to nominate their greatest canadian. canadians from coast to coast were asked to vote and chose tommy douglas, known as the "father of medicare" and selected by national polling as "the greatest canadian of all time." the canadian public is proud of their medicare plan, and appreciates the security and social protection as a great achievement for everyone in the country. australia, taiwan, and south korea have adopted national health insurance systems similar to the canadian model. the us (population 322 million, gdp per capita usd $56,066 in 2015) has a system of government based on the federal constitution, with 50 states each having its own elected government. the constitution gives primary responsibility for health and welfare to the states, while direct federal services are provided to armed forces, veterans, and indigenous (native) americans. the federal government has established a major leadership role in national health by the development of national standards, national regulatory powers, funding, and information systems. the federal level has many governmental structures for regulation of food, drugs, and environment, as well as for research, public health services, training programs and health insurance systems for the elderly and the poor. the us has the world's costliest health care system with over 86 percent health insurance coverage, but universal access remains elusive, and population health indicators are well below many less-wealthy countries. however, the us has through trial and error experimentation made major contributions to the content and organization of public health systems, which are important for strengthening health systems in medium-and low-income countries as well as influencing countries with universal health systems (see chapter 15) . clearly, the us can learn from other countries as well (see box 8.5) . in 1798, the federal government established the us marine hospital service to provide hospitals for sick and disabled merchant seamen. this later became the uniformed us public health service commissioned corps (usphs) headed by the surgeon general (1873). services were added for native americans, military personnel and their families (through the veterans affairs department), the food and drug administration (fda), the national institutes of health (nih), the centers for disease control (cdc) and many other world class federal programs of research, service and teaching. other departments and legislation were added to promote nutrition and hygiene, establish state, municipal, and county health departments, and regulate drugs and health hazards. in 1921, the sheppard-towner act established the federal children's bureau that administered grants to assist states to operate maternal and child health programs. from the 1920s, labor unions won health insurance benefits through collective bargaining, which became the main basis for prepayment for health care in the united states until today. in 1927, the committee on the costs of medical care recommended a universal national health program. this initiative was set aside during the great depression of 1929ã�39. the us social security act (ssa) of 1935 was introduced by president franklin d. roosevelt as part of the "new deal" to alleviate the mass suffering of the people during this very traumatic period in the us (and europe). the ssa was intended to include national health insurance, but this part of the ssa was set aside largely due to strong opposition of the insurance industry and the organized medical profession. the ssa provides financial benefits for widows, orphans, and the disabled, as well as pensions for the elderly, and provided a base for future reform including health insurance. with the outbreak of world war ii, a significant percentage of eligible military recruits were found unfit for compulsory service due to preventable health conditions. this, and the wish to maintain population health, led president roosevelt to initiate regulations in 1941 for fortification of "enriched" foods reaching a majority of the population including salt with iodine, flour with iron and vitamin b complex, and milk with vitamin d. during world war ii (1941ã�45), governmental health insurance was provided to many millions of americans serving in the armed forces, along with their families. at the same time, health benefits through voluntary insurance for workers were vastly expanded in place of wage increases and this became the major method of prepayment for health care for a majority of the population. at the end of the war, millions of veterans were eligible for health care through the veterans administration (va), which established a national network of federal hospitals and primary care services. in 1946, president truman attempted to bring in national health insurance, but the legislation (the wagner-murray-dingell bill) failed in the us congress. one section of the bill was approved, enabling the federal government to initiate a program to upgrade country-wide hospital facilities, while limiting the beds to population ratio, under the hill-burton act (see chapter 15) . legislation also provided massive federal funding for the newly established national institutes of health (nih) to fund and promote research to strengthen public and private medical schools, teaching hospitals, and research facilities. in 1946, president truman established the federally-assisted school lunch program through the department of agriculture bringing nutritious meals to many (millions increasing from 7 million in 1946 to 30 million in 2016) of school children throughout the us. in the 1950s, the federal government also established the centers for disease control and prevention (cdc) and increased assistance for state and local public health activities and encouraged expansion of schools of public health across the country. in the us during the 1960s through to the 1990s, rapid health cost increases were attributed to many factors including the lack of a national health insurance mechanism. the plethora of health insurance systems fostered high costs and restrictions on access due to pre-existing conditions. other factors for rapid cost increases included an increasing elderly population, high levels of morbidity in the poor population, the spread of aids, rapid innovation and costly medical technology, specialization, high laboratory and diagnostic imaging costs, and large-scale public investment in medical education, research and health facility construction. the us system includes a mix of public health insurance and service programs (medicare, medicaid, veterans administration, indian health services, and military health coverage) which provide for a significant part-36.5 percent in 2014-of the us population. however, the majority (66%) is covered by the private insurance industry through employer-employee contracts which developed rapidly as the dominant health insurance sector with minimal government regulation. the cost of private health insurance to employers included in labor contracts of their employees and pensioners has become very high. in 2008, general motors reported to a senate hearing that the cost of health insurance per car produced was double the direct cost of labor and more than the cost of steel per car. this impinged on competitiveness in price with for example with japan which has a successful universal governmental health insurance plan with public-private mix of services. the affordable care act (aca) introduced by president barack obama in 2010 brought some 16 million previously uninsured persons into public and private insurance, increased governmental regulation to ensure fair pricing and payment and, especially, to abolish the past abuses of the "pre-existing condition" exclusions from insurance. other equally important factors were high levels of preventable hospitalization, institutional orientation of the health system, high administrative costs due to multiple private billing agencies in the private insurance industry, high incomes especially for specialist physicians, and high medical malpractice insurance costs. the pressure for cost constraint came from government, industry, and the private insurance industry. (see chapter 15). private medical practice, with payment by fee-for-service, was the major form of medical care in the us until the 1990s. most hospitals were operated through a mix of nonprofit agencies, including federal, state, and local governments, and voluntary and religious organizations, but a growing percentage are privately owned, for-profit (from 7.8% of beds in 1975 to 20.6% in 2013). in an effort to contain costs, the diversity of insurance systems promoted experimentation with organizational systems. health maintenance organizations (hmos) and other forms of managed care systems grew rapidly to become the predominant method of organizing health care in the united states. prepaid group practice (pgp) originated from private companies contracting to provide medical care, especially in remote mining camps and construction sites. in the 1940s, new york city sponsored the health insurance plan of greater new york to provide prepaid medical care for residents of urban renewal and low-income housing areas. this was later extended to include organized union groups such as municipal employees and garment industry workers. pgp became best known in the kaiser permanente network developed for workers of henry j. kaiser industries, at the boulder dam and grand coulee dam construction sites in the 1930s. kaiser permanente health plans now provide care for millions of americans in many other states. initially opposed by the organized medical profession and the private insurance industry, pgp gained acceptance by providing high-quality, less-costly health care. this became attractive to employers and unions alike, and later to governments seeking ways to constrain increases in health costs. since the 1970s, the generic term health maintenance organization (hmo) was promoted by the federal government in the hmo act by president richard nixon in 1973. hmos, which operate their own clinics and staff (i.e., the staff model), or through contracts with medical groups as preferred provider organizations (ppos), have become an accepted, if criticized, part of medical care in the united states and an important alternative to fee-for-service, private practice medicine. in 2011, 70.2 million americans were registered in hmo plans or 22.5 percent of the total us population. in recent years, the terms accountable care organizations (aco), patient-centered medical home (pcmh) and population health management system (phms) have come into wide use to denote organizations that take responsibility for comprehensive care for enrolled patients, with payment based on a form of capitation rather than fee-for-service. acos are present in all 50 states, washington, dc, and puerto rico, with the population covered increasing from 2.6 million in 2011 to 23.5 million in 2015. the aco comes in different models, but many include a hospital base and may be linked to independent practice associations (ipas), and specialty groups, or hospital medical staff organizations, or in a network of hospitals linked with other providers as an organized delivery system. these are not-for-profit group practices led by doctors who are salaried and subject to rigorous annual professional review. this model may be adaptable on a wider scale to improve quality and cost effective care to improve health of americans. in 1983, a prospective payment system, called diagnosis-related groups (drgs), was adopted for medicare, to encourage more efficient use of hospital care, with payment by categories of diagnosis. the drg is a classification system, for inpatient stays, categorizing possible diagnoses into more than 20 major body systems and subdivides them into almost 500 groups for the purpose of medicare reimbursement. this replaced the previous system of paying by the number of hospital days, or per diem or by itemized billing which encouraged longer hospital stays. drgs provided incentives for hospitals to diagnose and treat patients expeditiously and effectively. payment for medicare and medicaid patients shifted to this method placed the public insurance plans in a stronger position for payments to hospitals. in many states this has also become standard for patients with private health insurance as well. during the late 1980s, the term managed care was introduced, expanding from hmos of the kaiser permanente type to include both non-profit and for-profit systems. these include independent practice associations (ipas), which operate with physicians in private practice, and preferred provider organizations (ppos), which provide insured care by doctors and other providers associated with the plan to the enrolled members or beneficiaries at negotiated prices. the drg payment system and hmos or managed care systems reduced hospital utilization. while total costs of health care increased in this period, without reduction of hospital utilization the increase would have been considerably higher. in 1994, president clinton tried to introduce a health plan based on federally administered compulsory universal health insurance through the place of employment. a state could opt to form its own health insurance program including through its own department of health. physicians could contract with health insurance plans to provide care on a fixed-fee schedule, or in hmos, whether based on group or individual practice. the clinton health plan failed in congress mainly due to well financed opposition by the insurance industry and the organized medical community. in addition, opposition was also widespread among the majority of the population who already had good insurance benefits under their employment-based health insurance plans or medicare. their interest was in keeping the status quo so that the bill was defeated. following the failure of the clinton national health insurance proposal, managed care experienced tremendous growth. managed care systems have been able to cut costs in health care in ways that the us government could not. in the us as a whole, in addition to the nearly 58 million persons enrolled in hmos, another 91 million persons are enrolled in ppos, with 25 percent of medicaid and 10 percent of medicare beneficiaries in various "managed care plans". the search for cost containment led to the development of a series of important innovations in health care delivery, payment, and information systems. hmos demonstrated that good care provision can be operated efficiently with lower hospital admission rates than care provided on a fee-for-service basis. the managed care systems brought about profound changes in health care organization in the united states. in 2010, president barack obama established the patient protection and affordable care act/health care and education reconciliation act of 2010, widely known as the affordable care act (aca or obamacare) bringing health insurance to millions of previously uninsured americans when it went into effect in 2014 (see box 8.6). the aca requires most companies to cover their workers, and mandates that everyone has coverage or pay a fine. aca also requires insurance companies to accept all newcomers, regardless of any preexisting conditions, and assists people unable to afford insurance. this legislation covers young people under their parents' health insurance plans until the age of 26, covering 2.5 million young americans. it eliminated other limits on coverage, allowing those who had already reached a lifetime limit to be eligible for coverage. the affordable care act introduced discounts as large as 50 percent for pharmaceuticals for seniors. health care reform is currently a contentious issue with the donald trump government planning to repeal the obama health care reforms to be replaced with a plan still under development. us health care spending increased from 13.1 percent of gdp in 1995 to 16.6 percent in 2014, threatening the ultimate insolvency of medicare and cutbacks in medicaid in the near future. lack of universal access and the empowerment it potentially brings encourages an alienation or non-engagement with early health care for the socially disadvantaged sector of the population. this promotes inappropriate reliance on emergency department care and hospitalization in response to under-treated health needs. with large numbers of uninsured persons and many others lacking adequate health insurance, access and utilization of preventive care are below the levels needed to achieve social equity in health in the us. this is especially true for maternal-and child-health and for chronic diseases such as diabetes, hypertension, cancer, and heart disease. infant mortality rates in the united states vary greatly by race and ethnicity. as measured by the infant mortality rate, the rate among non-hispanic black mothers was 2.4 times higher than the rate for white non-hispanic mothers. a significantly higher rate of infant mortality exists among puerto rican and american indian populations compared with the national average. cdc reports that maternal mortality rates have increased in the united states between 2000 and 2013 from 14.5 to 17.3 per 100,000 live births possibly due to changes in reporting and increase in chronic illnesses and influenza during pregnancy particularly in the african american population. in 2000, the department of health and human services (dhhs) released healthy people 2010 with two main goals: "increase the quality and years of healthy life" and "eliminate health disparities." these goals focus on 28 specific areas developed by over 350 national membership organizations and 250 state health, mental health, substance abuse, and environmental agencies. many states have adopted use of these targets as their own measures of health status and performance. the us public health service, in cooperation with the national center for health statistics, regularly make available a wide set of data for updating health status and process measures relating to these national health goals. various preventive health initiatives are in place to try to alleviate health disparities, which successfully improved immunization coverage of us infants to meet national health targets, as well as for lead and other efforts directed toward poor population groups. in 2002, a program called racial and ethnic adult disparities in immunization initiative was introduced in order to improve influenza and pneumococcal vaccinations among minorities aged 65 and over. the us department of agriculture's women, infants and children (wic) program enables millions of poor americans to have good nutritional security. the wic program covers pregnant women, breastfeeding women (up to infant's first birthday), non-breastfeeding postpartum women (up to 6 months after the birth of an infant or after pregnancy ends) and infants and children (up to their fifth birthday). wic serves 53 percent of all infants born in the united states. the benefits include: supplemental nutritious foods, nutrition education and counseling at wic clinics, screening, and referrals to other health, welfare and social services such as completion of immunization and special needs counseling. school lunch programs are widespread under a federally assisted meal program operating in over 100,000 public and non-profit private schools and residential child care institutions, providing nutritionally balanced, low-cost or free lunches to more than 31 million children each school day in 2012. nutrition support for pregnant women and children in need, alleviates some of the ill effects of poverty in the united states, but lack of health insurance affects these groups severely especially in chronic disease, trauma, and other diseases of poverty. health disparities are a complex problem that goes beyond the issue of uninsured americans. low-income and illegal immigrants face challenges to access medical insurance. new immigrants must wait five years before they are eligible for medicaid. the structure of the medical system plays an important role in an individual's ability to obtain medical care. this includes convenience of making an appointment, office hours, waiting times, and transportation. a lack of health literacy also plays a role in an individual's ability to seek medical attention. individuals not fluent in english experience communication gaps. in 2003, it was estimated that an excess of usd $58 billion a year is spent on health care in the united states as a result of low health literacy. in certain areas of the country, medical facilities are scarce. minorities are under-represented in medical professions. black, latino, and native american populations make up approximately six percent of the physician workforce, although these populations represent over 26 percent of the population in the united states. health disparities remain an important social and political issue in the united states. the office of minority health (omh) of the department of health and human services was established in 1986 to address issues of health disparities among racial and ethnic minorities. important health disparities exist in america in relation to region of residence, with the southern states having high rates of obesity, stroke, and coronary heart disease mortality, which are thought to be due to customary diets rich in fatty and salty foods. state health departments will need to address these issues in order to reduce gaps in life expectancy due to lifestyle factors which are grounded in tradition and poverty as well as lack of health insurance. one of the main goals of healthy people 2020 is to eliminate health disparities. the us has developed extensive information systems of domestic and international importance. the cdc publishes the mmwr (morbidity and mortality weekly report), which sets high standards in disease reporting and policy analysis. the us national center for health statistics (nchs), the health care financing administration (hcfa), the us public health service (usphs), the food and drug administration (usfda), the national institutes of health (nih), and many nongovernmental organizations (ngos) carry out data collection, publication, and health services research activities important for health status monitoring. national nutrition surveillance and other systems of health status monitoring are reported in the professional literature and in publications of the cdc. national monitoring of hospital discharge information facilitates the understanding of patterns of utilization and morbidity. these information systems are vital for epidemiologic surveillance and managing the health care system. us surgeon general reports have an important influence on health systems not only in the united states, but also internationally. the cdc created the national center for public health informatics (ncphi) in 2005 to provide leadership and coordination of shared systems and services, to build and support a national network of integrated, standards-based, and interoperable public health information systems. this is meant to strengthen capabilities to monitor, detect, register, confirm, report, and analyze data, as well as provide feedback and alerts on important health events. this will enable partners to communicate evidence that supports decisions that impact health. electronic medical and personal health records are now widely used. these protect patient privacy and confidentiality, and serve legitimate clinical and public health needs. media coverage of health-related topics is extensive, and is important to promote health consciousness in the public. however, the sheer volume of information may make it difficult to discern which information is most relevant, and due to misinformation on internet sites, can also create opposition to public health initiatives such as the refusal to vaccinate children. public levels of health knowledge grow steadily, but vary widely by social class and educational levels. in 1979, the us surgeon general's report healthy people set a series of national health targets for a wide variety of public health issues. the program defined 226 objectives in 15 program areas within the three categories of prevention, protection, and promotion. these goals and objectives were formulated based on research and consultation by experts in different fields who participated in a conference by the us public health service. consensus is based on position papers, studies, and conferences involving the national governmental health agencies, the national academy of science institute of medicine, and professional organizations such as the american academy of pediatrics (aap), the us preventive health services task force, and the american college of obstetrics and gynecology (acog). many private individuals and organizations contribute to this effort, including state and local health agencies, representatives of consumer and provider groups, academic centers, and voluntary health associations. these targets are periodically assessed as performance indicators of the us health system and then updated. progress made during the 1980s included major reductions in death rates for three of the leading causes of death: heart disease, stroke, and unintentional injuries. infant mortality decreased, as did the incidence of vaccine-preventable infectious diseases. the latest iteration, healthy people 2020, identifies national health priorities. it strives to increase public awareness and understanding of the determinants of health, disease, disability, and opportunities for progress. it defines measurable objectives and goals for federal, state, and local authorities in the areas of health promotion, health protection, preventive services, surveillance and data systems, and age-related and special population groups. the final reviews of healthy people 2000 showed significant decreases in mortality from coronary heart disease and cancer. healthy people 2020 renews this effort to establish national targets which are adopted by state level governments and strongly influence policy in health insurance systems. the us has managed to achieve many of the targets set by the 1979 surgeon general's healthy people report. at the same time, the average annual increases in health care expenditures in the united states slowed markedly from the 1986ã�90 period with average annual increases of 10.7 percent, falling to under 7 percent annually between 1995 and 2005. this is partly due to lower general inflation rates (,3%), but also cost-containment measures being adopted by government insurance (medicare and medicaid) programs, the health insurance industry, the growth of managed care, and rationalizing the hospital sector by downsizing and promoting lower-cost alternative forms of care. national health insurance was delayed by congressional rejection of the clinton health plan. president barack obama's 2010 affordable care act (aca) provided millions of previously uninsured americans health insurance within better regulated private insurance or in state-run medicaid plans, but in 2017 is facing "repeal and replace" efforts by the president trump administration and republican congress. a number of possibilities exist to extend health insurance coverage: state health insurance initiatives with federal waivers and cost-sharing; a federal single payer universal coverage plan based on the federal medicare model or a federal-state medicaid model. the us health system is often called a costly and inefficient nonsystem. there are many stakeholders and providers, high costs, and poorer population health results than those achieved in other industrialized countries such as britain, germany, and canada. the health system is diffused with high levels of coverage for diverse insurance plans through employment-based insurance along with publicly financed and administered health insurance (e.g., medicare, medicaid, aca). inequalities are a significant health challenge in the us along with the uninsured, poverty, aging of the population, rising levels of obesity and diabetes. the principle of universal access through public insurance for all is still a highly politicized issue in the united states, although public acceptance seems to be gradually growing. the us has a reputation for good to outstanding quality of medical care, but for those without insurance, services are limited to hospital emergency care only. important ethnic, social, and regional inequities in health status are still present, but not necessarily greater than in countries with universal access health care plans such as the uk nhs. further, there are many parallel programs in the united states that have important positive public health content, such as universal school lunch programs, nutrition support for poor women, infants, and children (the wic program); food stamps for the working poor; fortification of basic foods, free care for the uninsured in emergency departments, medicare for the elderly, medicaid for the poor, and aca coverage for the near-poor. box 8.7 shows the challenges of the us health system. despite rapid increases in health care expenditures during the 1970s and 1980s, despite improved health promotion activities and rapidly developing medical technology, the health status of the american population g preventive programs strong tradition; screening for cancer; smoking reduction; food fortification, school lunch programs; nutrition support for poor pregnant women and children (wic); g hospitals obliged to provide emergency care to all regardless of insurance status, citizenship, legal status or ability to pay has improved less rapidly than that in other western countries and universal coverage has not been achieved. us performance measures are lower than many middle-and high-income countries with much lower per capita health expenditures, including measures such as infant mortality rates and life expectancy. infant mortality in the us remains high in comparison to oecd countries and ranks 34 th among all countries in 2012 (estimated). even the rate of infant mortality of the white population of the united states was higher than that of 16 countries that spent much less per person and a lesser percentage of gnp per capita on health care. life expectancy at birth in the united states in 2014 was below that of 24 countries, just behind costa rica, portugal and slovenia. in 2014, the us life expectancy at birth was 78.8 years, well below the oecd average of 81.6 years. social inequities in these health status indicators are further evidence of failures of the united states health system to reach its full potential, despite its being the costliest system in the world and its high quality for those with access (commonwealth fund, 2008) . the advent of the aca (obamacare) introduced in 2010 brought health insurance to millions of americans, but is challenged as unaffordable. the us still lacks a universal single payer health plan of canadian or european tradition, but the aca is a huge step forward in america where the working poor are in large measure excluded from access to health care except for emergencies. the struggle for universal access and cost containment are still formidable political and societal challenges for the united states. in 1918, following the russian revolution, the soviet union (ussr) introduced its national health plan for universal coverage within a state-run system of health protection. the soviet model, designed and implemented by nikolai semashko, provided free health care for all as a governmentfinanced and -organized service. it brought free health services to the population, with a system of primary-and secondary-care based on the principles of universal and equitable access to care through district organization of services. it achieved control of epidemic and endemic infectious diseases and expanded services into the most remote areas of the vast under-developed country. this model was also applicable in countries included in the ussr following world war ii until the collapse of the ussr. the model developed in the former soviet union in 1917 by semashko brought free health care with governmental management by republic and regional authorities according to national norms set out by the ministry of finance. since the 1930s health care became available for all with mostly underdeveloped basic infrastructure for health care including human resources. the semashko plan provided universal access to preventive and curative care, and control of infectious disease in a uniform plan, with many republics previously having only primitive care available, achieving national standards of services and improved health indicators. since the 1960s, an "epidemiologic transition" was occurring characterized by declining mortality from infectious diseases and rising death rates from non-infectious diseases. life expectancy increased since 1995, still remains far below levels in many medium-income developed countries. the transition in health systems following the collapse of the soviet union in 1991 took different paths for the socialist central and eastern european countries (cee) as compared to the core countries of the soviet union, called the commonwealth of independent states (cis). the cee countries moved rapidly to dismantle their soviet, centrally managed sanitary-epidemiological system (sanepid) system with decentralization while retaining universal coverage with central funding, but with local authority participation in some cases. most cee and cis countries have introduced health insurance systems, with more out-of-pocket payments (both formal and informal), and efforts to strengthen primary health care, with family medicine delivered by general practitioners. in most cases central authorities also maintained responsibility for epidemiological surveillance and environmental monitoring with some transferring responsibilities for environmental health in other ministries. the cee and cis countries maintained similar levels of health expenditures as percent of gdp between six and seven percent over the past decade, while the original european union (eu) countries reached an average of 10 percent of gdp. the cis acute care hospital bed capacity ratio declined to six per 1000 population in 2014 far higher than cee countries (declined to 4.6 per 1000), which were higher than the western countries, although all country groups were declining (see chapter 15) . the importance of these differences lies in the fact that total resources allocated for health in the soviet system was relatively low while the allocation allowed hospital care to consume some 70 percent of total expenditures compared with less than 50 percent in western countries. the outcome of this allocation of resources was weakness in development of primary care, prevention and community care in favor of an over-developed hospital bed supply. the russian federation adopted a mandatory health insurance (mhi) plan in 1993 to open up additional funding for health care in the face of severe governmental funding constraints. it remains a highly centralized system and is struggling to provide universal access to basic care. despite this, death rates from avoidable causes such as stroke and coronary heart disease have declined in the past decade and life expectancy has risen modestly, but remaining far below western as well as former socialist countries of central and eastern europe. developing national health systems with universal access has been a long process in high-income countries and is an important goal for all countries including medium-and low-income countries to promote improving access to health for the total population. the commonwealth fund published an outstanding international profile of selected health care systems in highincome countries (2015) including: australia, canada, china, denmark, england, france, germany, india, israel, italy, japan, the netherlands, new zealand, norway, singapore, sweden, switzerland, and the united states. global spending on health is expected to increase from us$7.83 trillion in 2013 to $18.28 (uncertainty interval 14.42ã�22.24) trillion in 2040 (in 2010 purchasing power parity-adjusted dollars). we expect per-capita health spending to increase annually by 2.7% (1.9ã�3.4) in high-income countries, 3.4% (2.4ã�4.2) in upper middle-income countries, 3.0% (2.3ã�3.6) in lower middle-income countries, and 2.4% (1.6ã�3.1) in low-income countries. low-and medium-income countries face major difficulties in developing universal health coverage, especially in terms of financial and professional resources. a study of global health care financing (dielman et al lancet 2016) reported on health expenditures from 184 countries, including public, donor, and private ("out of pocket") payments between 1995 and 2014. high-income countries spent more, and mostly from public sources, increasing expenditures by an estimated three percent per year. medium income countries increased their health spending more than three-four percent per year and low-income countries by two percent. economic development was positively associated with total health spending and a gradual shift away from a reliance on development assistance and out-of-pocket spending towards government spending. in 2014, 59.2 percent of all health spending was financed by the government, although in low-income and lower-middleincome countries, 29 percent and 58 percent of spending was out-of-pocket, 35.7 percent and three percent respectively was with development assistance. recent growth in development assistance for health has been tepid. between 2010 and 2016, it grew annually at 1.8 percent, and reached usd $37 ã� 6 billion in 2016. nonetheless, there is a great deal of variation revolving around these averages. in 2016 countries spending less than five percent of gdp on health, included many in asia, the middle east and sub-saharan africa (institute of health metrics and evaluation, 2016). while there is wide variation in health spending in low-and lowermiddle-income countries and there is overall increased spending in absolute terms, there is still a heavy reliance on out-of-pocket spending and development assistance, which itself is growing very slowly. this indicates that medium-and low-income countries are not providing the financial means to develop universal health access insurance plans. economic growth also does not translate into adequate funding for universal health care without dramatic changes in policy and decreased dependency on donor aid. international agencies-such as who-are promoting the search for ways to provide universal and equitable care, while controlling costs and improving efficiency in low-and middle-income countries. the 1948 universal declaration of human rights, article 25 states: "(1) everyone has the right to a standard of living adequate for the health and well-being of himself and of his family, including food, clothing, housing and medical care and necessary social services, and the right to security in the event of unemployment, sickness, disability, widowhood, old age or other lack of livelihood in circumstances beyond his control. (2) motherhood and childhood are entitled to special care and assistance. all children, whether born in or out of wedlock, shall enjoy the same social protection." the universal declaration of human rights specific inclusion of access to medical care for all should be seen as a priority in planning universal health insurance (uhi) for promotion of access to health needs for remote rural populations as well as urban poor, and displaced persons. this also applies to conditions of warfare, civil strife, natural disasters as well as incitement to and actual genocide. as said previously, the global consensus of the mdgs (2000ã�15) and the sdgs (2015ã�30) have undertaken to implement key elements of this important declaration. it is easier to be pessimistic than optimistic in the potential for success, but the significant achievements of the mdgs in poverty reduction, educational equity between the genders and in reduction of child and maternal mortality as well as in control of hiv, malaria and tuberculosis are signs of important progress and future possibilities. national governments must take up the financial burdens and management of expanding health systems as well as contributory advances in education, environment and other government sectors toward achieving these goals. bilateral aid and international donors are vital, but they cannot achieve or sustain all this without national commitments and resources. national health systems are essential to provide universal access to health care, but must be developed recognizing that restraint in increasing costs, equity in access and quality, as well as efficiency and effectiveness in use of resources are vital to achieve health targets and equity in population health. in the united states, a study of ethnic differences in utilization of services among medicare beneficiaries who have the same entitlements show significant differences indicating lesser use of preventive services such as mammography and higher rates of lower limb amputation for diabetes indicating poorer management of diabetes. studies in the united kingdom also show sharp differences in mortality rates by region of residence that correlate with socioeconomic gradations. universal access alone does not guarantee equality so that the design of service systems needs to take into account differing needs of groups or regions at higher risk and greater need. universal access by itself is important, but not sufficient to reduce inequalities, which have more complex needs than medical care alone. universal coverage health insurance must be developed with great care to avoid mistakes made in many countries in previous decades of promoting rapid increase in health expenditures to the benefit of the middle class while rural and poor urban populations linger in relatively poor health. a universal health insurance plan without strong incentives for prevention and community health will find itself in a trap of punishing the poor for the benefit of the rich. population health experience of the past century has shown the power of public health, in all its aspects, to raise life expectancy and quality, yet inequalities still plague all health systems. this provides an ethical challenge in planning, resource allocation and political support. beyond financing and resource allocation, there are many "nontariff" barriers to health. even in highly developed national health systems, social class, place of residence, education level, and ethnicity play significant roles in morbidity and mortality rates. addressing important health risk factors other than medical or hospital care is vital. the disease-risk factors of diet, smoking, physical fitness, nutrition status including obesity, and untreated hypertension. such conditions are not necessarily managed even where all residents of a country are insured for health care. social class, ethnic and regional differences in morbidity and mortality exist due to povertyassociated factors, such as insecurity, lack of control over one's life, lack of financial means or knowledge to purchase healthy foods, as well as fear, loneliness and depression. these are issues that are important and must be addressed in public health policy to reduce inequalities in health and the achievement of national health goals and equity. models of financing of universal health insurance include a variety of methods: general taxation; social security by employee-employer payments through payroll deductions; private insurance under contracts between employee and employer; and private out-of-pocket payments. taxation financing can be mainly through progressive income tax, resource taxes, surcharges or "sin taxes" (e.g., on cigarettes, alcohol, gasoline) and excise taxes along with local property and business licensing taxation where local authorities have a management role. funding by general tax revenues at national or state levels or shared between the two levels provides for more local administration while sharing in costs may be the most equitable way of raising funds. many countries use social security systems based on employerã� employee contributions to pay for health services. the who, the world bank and oecd promote universal health insurance (uhi) for middle-income countries. the advantage will be to reduce the heavy burden of out-of-pocket payments, which are 60 percent of health expenditures in many emerging countries. universal health insurance provides security for individuals and families against catastrophic health events, for regular medical and hospital care, and for ageing populations with increasing health needs. oecd recommends increasing health expenditures, which improves life expectancy, and to allow uhi implementation. even a 10 percent increase in national health spending has been shown to reduce child mortality across many countries. universal health insurance must include promotion of greater efficiency in health care, such as shifting of services from hospital care to outpatient and primary care along with community and home-based care (see chapter 15) . the process requires developing new health care provider roles with emphasis on outreach to groups with greater than average need, promoting public health and preventive care such as for underserved rural or urban communities or groups at special risk for disease such as cardiovascular disease (cvd) and diabetes, making use of epidemiologic and sociologic health data and information systems. universal health insurance undoubtedly contributes to improving health indicators such as life expectancy by coverage of the total population, systematizing financing of the health system and providing access to the population. however, without good management of resource allocation, universal health insurance cannot guarantee achievement of important health targets. allocation of resources is a fundamental problematic aspect of universal health insurance. national health policy governing universal health insurance must invest adequately in health promotion and disease prevention in order to reduce excessive allocation and utilization of hospital care. continuous monitoring and evaluation are essential to a health system, but not only for traditional outcome indicators, such as infant, child and maternal mortality rates, and disease-specific mortality rates. these are all valuable indicators of population health, but not sufficient. input, process and outcome indicators are important and necessary to include, such as supply and distribution of resources e.g., primary care, maternity centers, hospital beds; process measures e.g., immunization rates, incidence of vaccine-preventable diseases, growth patterns and anemia rates in infancy and childhood, food fortification, micronutrient supplements to risk group, prenatal delivery and neonatal care. outcome measures include prevalence of disabling conditions morbidity and mortality rates. disability adjusted life years (dalys) and quality adjusted life years (qalys) help change the emphasis from mortality to quality of life measures as part of the evaluation. national health systems require data systems that generate information needed for this continuous process of monitoring. monitoring of hospitalizations, length of stay, health-care facility acquired (nosocomial) infection, readmission rate by diagnosis and many more indicators, compliance with standards of care such as in infection control, surgical and maternal mortality, including infection and error rates, and other qualitative measures are now part of monitoring and payment systems. high-quality academic centers are needed for training epidemiologic, sociologic, and economic analyses professionals as well as health system managers and to carry out the studies and research vital for health progress. health systems are large-scale employers and among the largest economic sectors in their respective countries, with 7ã�16 percent of gdp in middle-and high-income countries and, therefore, a major factor in the total national economy. but the gap between countries is very high. many countries have per capita spending of less than usd $100 per year, so that inadequate resources prevent people from receiving quality health care, without unaffordable out of pocket expenditures. in contrast, in many high-income countries annual health expenditures are above usd $5,000 per capita. donor aid to low-income countries from bilateral or international agencies or other donors rose rapidly from 2000 with an estimated $10 billion usd to a peak of usd $37 billion in 2012, with only a modest change up to 2016. low-income nations, many of which are undergoing important economic development, are under-spending in national allocations to the health sector and remain highly reliant on international aid. a goal of five to six percent of gdp spent on health is widely regarded as a minimum to provide the health care needed in any country. a 2016 study published in lancet by the institute for health metrics and evaluation, indicates that only one of 37 low-income countries, and 36 out of 98 of middle-income countries, are expected to meet the target of five percent. low rates of national health expenditures in countries will be a serious limiting factor in improved health and universal access, especially if preventive care is unable to compete for resources as compared to clinical and hospital services. all countries face problems of financing, cost constraint, overcoming structural inefficiencies, and funding incentives for high quality and efficiency in health services. national health systems are necessarily complex, but go well beyond medical and hospital care. the quality of the community infrastructure-sewage, water, roads, communication, urban planning-social support such as pensions and welfare for the disabled, widows, orphans and others in need are essential for population health. attention to the quantity and quality of food (i.e., food and nutritional security), levels of education, and professional organization are all parts of this continuum. national health systems are not only a matter of adequacy and methods of financing and assuring access to services; they must also address health promotion, national health targets, and adapt to the changing needs of the population, the environment, and with a broad intersectoral approach to health of the population and the individual. the structure, content, and quality of a health system plays a vital role in the social and economic development of a society and its quality of life. universal access is increasingly widely accepted as essential to reduce the social inequalities in health. even when income gaps are high. however, vulnerable populations with higher levels of risk than those of the general population are still relatively deprived even under classical universal insurance systems. the key common factors of elevated vulnerability are poverty, isolation by geographic location, physical access by reasons of residency location, ethnicity, education and institutional barriers which reduce access. these inequality factors are the achilles heel of classical universal health insurance and service systems most of which have sought health promotion measures. there can be little doubt that universal access to health insurance or service systems reduces inequalities, but they require imaginative and outreach-oriented approaches to reach those urban and rural poor, people of aboriginal descent, those with an income lower than the poverty threshold, the unemployed, the homeless, and those who have not completed secondary education. societal programs to increase family disposable income for the poor are effective in reducing the health inequities. the two are complementary and equally important in social policy. in the united states more than ten percentages of the population are without any, or have inadequate, health insurance. loss of health coverage with change of place of employment and the rapidly increasing cost of private health insurance generated widespread pressure for a national health program. the business community, too, loses confidence in voluntary health insurance as costs of health insurance mounted rapidly and as a cost of employment in an increasingly harms the competitive international business climate. narrow planning for health systems ignores this message at the risk of missing their targets of improved health indicators, such as those adopted by the united nations-i.e., the millennium development goals and sustainable development goals. the mdgs and sdgs represent a growing movement of globalization of health with economic and political dimensions and greater stress on human rights to health policy. they are particularly relevant to lmics (low-and middle-income countries), but high-income countries have health inequalities that require new approaches based on outreach poverty abatement, and health promotion concepts. mdgs and sdgs presented a challenge to establish common data systems for performance measures to monitor effectiveness of policies and programs. this helps to build capacity for target-oriented health planning in low-and middle-income countries (lmics). a holistic view of health for all must take into account the many reasons for health disparities and disadvantage to the poor in health status. insurance to pay for doctors, hospitals, laboratories and imaging centers is necessary, but not sufficient, to raise population health standards for all. the "nontariff barriers"-i.e., issues beyond payment for services which may be addressed with incentives in payment systems, not only to reduce hospital length-of-stay, but to reduce health-care acquired infections, reaching out to chronically ill people with health promotion measures such as nutritional support, pneumonia and influenza immunization, hypertension control, cancer screening, and many other features of public health promotion. since the 1880s, when bismarck introduced national health insurance in germany as part of social security with funding though sick funds, many countries have grappled in unique ways with developing health care systems. national health insurance systems developed through social security and social welfare systems, by national health insurance, or options to provide access to health services. in canada national health insurance provides universal coverage through national support for provincial health plans, paid for by general taxation, with national criteria. in the united states, president lyndon johnson established social security-based health insurance for the elderly and the poor through amendments to the social security act of 1935, and president barack obama extended health insurance through the affordable care act of 2010. the uk national health service-with the northern ireland, scottish and welsh nhs run semi-independently-was established in 1948, providing a state-run system of medical, hospital, preventive, and community health care. though not discussed here, nordic and other european health systems provide universal coverage with involvement from all three levels of government, but over 80 percent of expenditures are funded through public sources. in denmark, norway and sweden county councils are central to funding and management; in finland, the municipalities provide most of the health care. the former socialist countries have gone through painful periods of transition. many of these countries have developed free-market systems with dynamic growth in national economies along with health system reform. health systems in transition have adapted with great gains in longevity and reduced mortality from preventable diseases in many former socialist countries in central and eastern europe. others have had difficulties addressing the "missed epidemiologic transition" from infectious disease to control of noncommunicable disease but have begun to make progress in the 21 st century. globally, public and private donor partnerships have emerged to help the poorest countries cope with overwhelming health problems of raising immunization coverage levels, reducing child and maternal mortality, managing hiv, tuberculosis, malaria, diarrheal and respiratory diseases and vaccinepreventable diseases in keeping with the mdgs based on a consensus of all member nations of the un. the objectives and specific targets included: reducing poverty, improving equal access of boys and girls to primary education, reducing child and maternal mortality, managing significant diseases such as hiv, tuberculosis, and malaria, along with improving the environment. reaching the targets for achieving these goals depends on developing infrastructures of health systems that provide access for all and distribution to meet geographic and social inequities in health. each country needs to develop its own system, but can learn from the experience of others. the purpose of this case study is to highlight the unique and common features, including positive and negative lessons learned from national health systems. observing and learning can help in defining needs for countries lacking but aspiring to achieve universal health systems, including positive and negative challenges. universal access is an important means of assuring that the economic barrier is removed for the total population, leading to increased access to medical and hospital services for those previously lacking the means to reach these services. universal access systems have been achieved in most industrialized countries. however, the us has not achieved this goal even with, by far, the highest health expenditures of oecd countries. this is due mainly to political gridlock despite success with its single payer system for medicare for the elderly. for low-income countries, the rates of health expenditures at present and forecast for the coming decades will be insufficient to achieve universal access systems. there must be a fundamental political change in national policies with health as a higher priority for funding and leadership. universal healthcare access is still a work in progress. the goal of universal access is a worthy one: to make health care accessible to all. the advent of universal access, however, is not assured given low levels of funding in many countries most in need of improved access but strengthening health systems: the role and promise of policy and systems research. geneva: global forum for health research alliance for health systems policy and research. world health organization. what is health policy and systems research (hpsr)? 2017. geneva: world health organization achieving a high-performance health care system with universal access: what the united states can learn from other countries health spending in the united states and the rest of the industrialized world the publicã�private pendulum-patient choice and equity in sweden uk health dividesã�where you live can kill you disease and disadvantage in the united states and in england lessons from the east-china's rapidly evolving health care system the affordable care act at five years gatekeeping in health care the organization of personal health services. milbank quart noncommunicable diseases: stepping up the fight: how the russian federation is collaborating with other commonwealth of independent states' countries comparing health systems in four countries: lessons for the united states germany: health system review germany and health 1: statutory health insurance in germany: a health system shaped by 135 years of solidarity, self-governance, and competition how canada compares: results from the commonwealth fund 2015 international health policy survey of primary care physicians healthy people 2020: topic areas at a glance. national center for health statistics, last reviewed 6 national health expenditure fact sheet, last modified 21 health and health care in israel: an introduction. lancet. 2017. special series international health care system profiles international profiles of health care systems united kingdom: health system review reforming the russian health care system the fragmentary federation: experiences with the decentralized health system in russia health care reform: lessons from canada ata international conference on primary health care the development of social security in america. social security administration national spending on health by source for 184 countries between regional health inequalities in england. uk office for national statistics, department of health health care systems in the eu: a comparative study. directorate general for research systems science for universal health coverage health systems special edition. health systems: more evidence, more debate evolution and patterns of global health financing 1995ã�2014: development assistance for health, and government, prepaid private, and out-of-pocket health spending in 184 countries reforming sanitaryepidemiological service in central and eastern europe and the former soviet union: an exploratory study effects of race and income on mortality and use of services among medicare beneficiaries global health: a pivotal moment of opportunity and peril a systematic review of studies comparing health outcomes in canada and the united states how does the quality of care compare in five countries? financing global health 2016: development assistance, public and private health spending for the pursuit of universal health coverage disease control priorities in developing countries kaiser family foundation. us global health policy strengthening health systems to provide rehabilitation services a new perspective on the health of canadians. ottawa, on: department of national health and welfare, 1974a new perspective on the health of canadians. ottawa, on: department of national health and welfare access to care, health status, and health disparities in the united states and canada: results of a cross-national population-based survey social medicine vs professional dominance: the german experience universal health care: lessons from the british experience a system in name only-access, variation, and reform in canada's provinces the unequal health of europeans: successes and failures of policies a comparative analysis of health policy performance in 43 european countries nordic health systems: recent reforms and health policy challenges. copenhagen: who regional office for europe on behalf of the european observatory on health systems and policies the history of health care in canada the marmot review final report: fair society, healthy lives a precious jewel-the role of general practice in the english nhs canada health system review reinventing public health: a new perspective on the health of canadians and its international impact international health care systems international profiles of health care systems the global campaign for the health mdgs: challenges, opportunities, and the imperative of shared learning red medicine: socialized health in soviet russia measuring the health of nations: updating an earlier analysis organization for economic cooperation and development (oecd) 2016. oecd reviews of health care systems organization for economic cooperation and development (oecd) organization of economic cooperation and development (oecd) organization of economic cooperation and development (oecd) improving health services delivery in developing countries: from evidence to action russian federation. health system review health reform in central and eastern europe and the former soviet union national health systems of the world national health systems throughout the world building on values: the future of health care in canada comparing health and health care use in canada and the united states a survey of primary care physicians in 11 countries ):w1171ã�w1183. available at rocky road from the semashko to a new health model universalism, responsiveness, sustainability-regulating the french health care system integrated ambulatory specialist care-germany's new health care sector ethics of resource allocation and rationing medical care in a time of fiscal restraint-us and europe addressing the epidemiologic transition in the former soviet union: strategies for health system and public health reform in russia the new public health brave new world: the welfare state united nations human development report transforming our world., agenda items 15 and 116 sustainable development goals. 17 goals to transform our world healthy development. the world bank strategy for health, nutrition, and population results spotlight on nutrition: unlocking human potential and economic growth world bank life expectancy at birth, total (years) world health organization. global health observatory. world health statistics 2016: monitoring health for the sdgs everybody's business: strengthening health systems to improve outcomes: who's framework for action. geneva: world health organization world health report 2006: working together for health. geneva: world health organization health systems: improving performance european health report 2012: charting the way to well-being. copenhagen: world health organization research for universal health coverage world health organization. european health for all database (hfadb) world health organization. health systems: health system financing universal health coverage: sustainable development goal 3, health relying heavily on donors and out-of-pocket payments. the devil is in the details. 1. universal health insurance (uhi) or national health service systems are essential for advancing population health and should be give high priority in policy and funding by national governments and international aid agencies in middle-and low-income countries in the coming decades. 2. universal health insurance or service systems cannot be expected to succeed without continuing development of public health and health promotion as equal needs for population health and to achieve sdgs. 3. all countries seeking health development will need to raise public support for financing health systems by raising health expenditures to more than five -six percent of gdp. 4. all countries addressing these issues should endeavor to expand training to include bachelor and master degree training in public health and health systems management in order to raise the professional leadership and management levels to lead in the complexities of health systems in the challenges ahead. health promotion to hospice care on par with acute and rehabilitation care hospitals as essential, but managed so as to avoid unnecessary economic domination of the health system and potentially damaging health-care infections and trauma. 6. reaching out to populations-at-risk and in need of preventive care and health promotion by multi-professional and paraprofessional teamse.g., community health workers, is vital to address chronic care needs and prevent their complications, for remote villages or urban poverty areas, or to groups of people with chronic disease conditions. 7. health information systems including development and implementation of epidemiology and information technology for monitoring of disease and quality of care require emphasis. 8. immunization and nutritional support for prevention of infectious diseases, chronic diseases and micronutrient deficiency conditions are crucial for population health and should be given high priority in health system development. 9. health policy management is vital to achieving universal health coverage to advance population health, but it must be seen as part of health in all strategies and the sdgs to be effective within financial limitations and cost restraint. 10. health promotion must be developed in all its aspects to raise population and professional awareness with educational and legal means to reduce risk factors in population health. health systems to promote efficient use of resources and achievement of specified health targets? 4. what methods may be incorporated into national health systems to promote quality of care? 5. how can developing countries achieve universal health care, and at the same time work toward national health targets such as upgrading maternal and child heath, control of infectious diseases and preventing chronic diseases? 6. how can low-income countries address the low public expenditure on health to reduce dependence on global financial aid for sustainable development goals (sdgs)? key: cord-133273-kvyzuayp authors: christ, andreas; quint, franz title: artificial intelligence: research impact on key industries; the upper-rhine artificial intelligence symposium (ur-ai 2020) date: 2020-10-05 journal: nan doi: nan sha: doc_id: 133273 cord_uid: kvyzuayp the trirhenatech alliance presents a collection of accepted papers of the cancelled tri-national 'upper-rhine artificial inteeligence symposium' planned for 13th may 2020 in karlsruhe. the trirhenatech alliance is a network of universities in the upper-rhine trinational metropolitan region comprising of the german universities of applied sciences in furtwangen, kaiserslautern, karlsruhe, and offenburg, the baden-wuerttemberg cooperative state university loerrach, the french university network alsace tech (comprised of 14 'grandes 'ecoles' in the fields of engineering, architecture and management) and the university of applied sciences and arts northwestern switzerland. the alliance's common goal is to reinforce the transfer of knowledge, research, and technology, as well as the cross-border mobility of students. in the area of privacy-preserving machine learning, many organisations could potentially benefit from sharing data with other, similar organisations to train good models. health insurers could, for instance, work together on solving the automated processing of unstructured paperwork such as insurers' claim receipts. the issue here is that organisations cannot share their data with each other for confidentiality and privacy reasons, which is why secure collaborative machine learning where a common model is trained on distributed data to prevent information from the participants from being reconstructedis gaining traction. this shows that the biggest problem in the area of privacy-preserving machine learning is not technical implementation, but how much the entities involved (decision makers, legal departments, etc.) trust the technologies. as a result, the degree to which ai can be explained, and the amount of trust people have in it, will be an issue requiring attention in the years to come. the representation of language has undergone enormous development of late: new models and variants, which can be used for a range of natural language processing (nlp) tasks, seem to pop up almost monthly. such tasks include machine translation, extracting information from documents, text summarisation and generation, document classification, bots, and so forth. the new generation of language models, for instance, is advanced enough to be used to generate completely realistic texts. these examples reveal the rapid development currently taking place in the ai landscape, so much so that the coming year may well witness major advances or even a breakthrough in the following areas: • healthcare sector (reinforced by the covid-19 pandemic): ai facilitates the analysis of huge amounts of personal information, diagnoses, treatments and medical data, as well as the identification of patterns and the early identification and/or cure of disorders. • privacy concerns: how civil society should respond to the fast increasing use of ai remains a major challenge in terms of safeguarding privacy. the sector will need to explain ai to civil society in ways that can be understood, so that people can have confidence in these technologies. • ai in retail: increasing reliance on online shopping (especially in the current situation) will change the way traditional (food) shops function. we are already seeing signs of new approaches with self-scanning checkouts, but this is only the beginning. going forward, food retailers will (have to) increasingly rely on a combination of staff and automated technologies to ensure cost-effective, frictionless shopping. • process automation: an ever greater proportion of production is being automated or performed by robotic methods. • bots: progress in the field of language (especially in natural language processing, outlined above) is expected to lead to major advances in the take-up of bots, such as in customer service, marketing, help desk services, healthcare/diagnosis, consultancy and many other areas. the rapid pace of development means it is almost impossible to predict either the challenges we will face in the future or the solutions destined to simplify our lives. one thing we can say is that there is enormous potential here. the universities in the trirhenatech alliance are actively contributing interdisciplinary solutions to the development of ai and its associated technical, societal and psychological research questions. utilizing toes of a humanoid robot is difficult for various reasons, one of which is that inverse kinematics is overdetermined with the introduction of toe joints. nevertheless, a number of robots with either passive toe joints like the monroe or hrp-2 robots [1, 2] or active toe joints like lola, the toyota robot or toni [3, 4, 5] have been developed. recent work shows considerable progress on learning model-free behaviors using genetic learning [6] for kicking with toes and deep reinforcement learning [7, 8, 9] for walking without toe joints. in this work, we show that toe joints can significantly improve the walking behavior of a simulated nao robot and can be learned model-free. the remainder of this paper is organized as follows: section 2 gives an overview of the domain in which learning took place. section 3 explains the approach for model free learning with toes. section 4 contains empirical results for various behaviors trained before we conclude in section 5. the robots used in this work are robots of the robocup 3d soccer simulation which is based on simspark 1 and initially initiated by [10] . it uses the ode physics engine 2 and runs at an update speed of 50hz. the simulator provides variations of aldebaran nao robots with 22 dof for the robot types without toes and 24 dof for the type with toes, naotoe henceforth. more specifically, the robot has 6 (7) dof in each leg, 4 in each arm and 2 in its neck. there are several simplifications in the simulation compared to the real nao: all motors of the simulated nao are of equal strength whereas the real nao has weaker motors in the arms and different gears in the leg pitch motors. joints do not experience extensive backlash rotation axes of the hip yaw part of the hip are identical in both robots, but the simulated robot can move hip yaw for each leg independently, whereas for the real nao, left and right hip yaw are coupled the simulated naos do not have hands the touch model of the ground is softer and therefore more forgiving to stronger ground touches in the simulation energy consumption and heat is not simulated masses are assumed to be point masses in the center of each body part the feet of naotoe are modeled as rectangular body parts of size 8cm x 12cm x 2cm for the foot and 8cm x 4cm x 1cm for the toes (see figure 1 ). the two body parts are connected with a hinge joint that can move from -1 degrees (downward) to 70 degrees. all joints can move at an angular speed of at most 7.02 degrees per 20ms. the simulation server expects to get the desired speed at 50 hz for each joint. if no speeds are sent to the server it will continue movement of the joint with the last speed received. joint angles are noiselessly perceived at 50hz, but with a delay of 40ms compared to sent actions. so only after two cycles, the robot knows the result of a triggered action. a controller provided for each joint inside the server tries to achieve the requested speed, but is subject to maximum torque, maximum angular speed and maximum joint angles. the simulator is able to run 22 simulated naos in real-time on reasonable cpus. it is used as competition platform for the robocup 3d soccer simulation league 3 . in this context, only a single agent was running in the simulator. the following subsections describe how we approached the learning problem. this includes a description of the design of the behavior parameters used, what the fitness functions for the genetic algorithm look like, which hyperparameters were used and how the fitness calculation in the simspark simulation environment works exactly. the guiding goal behind our approach is to learn a model-free walk behavior. with model-free we depict an approach that does not make any assumptions about a robot's architecture nor the task to be performed. thus, from the viewpoint of learning, our model consists of a set of flat parameters. these parameters are later grounded inside the domain. the server requires 50 values per second for each joint. to reduce the search space, we make use of the fact that output values of a joint over time are not independent. therefore, we learn keyframes, i.e. all joint angles for discrete phases of movement together with the duration of the phase from keyframe to keyframe. the experiments described in this paper used four to eight of such phases. the number of phases is variable between learning runs, but not subject to learning for now, except for skipping phases by learning a zero duration for it. the robocup server requires robots to send the actual angular speed of each joint as a command. when only leg joints are included, this would require to learn 15 parameters per phase (14 joints + 1 for the duration of the phase), resulting in 60, 90 and 120 parameters for the 4, 6, 8 phases worked with. the disadvantage of this approach is that the speed during a particular phase is constant, thus making it unable to adapt to discrepancies between the desired and the actual motor movement. therefore, a combination of angular value and the maximum amount of angular speed each joint should have is used. the direction and final value of movement is entirely encoded in the angular values, but the speed can be controlled separately. it follows that: -if the amount of angular speed does not allow reaching the angular value, the joint behaves like in version 1. -if the amount of angular speed is bigger, the joint stops to move even if the phase is not over. this almost doubles the amount of parameters to learn, but the co-domain of values for the speed values is half the size, since here we only require an absolute amount of angular speed. with these parameters, the robot learns a single step and mirrors the movement to get a double step. feedback from the domain is provided by a fitness function that defines the utility of a robot. the fitness function subtracts a penalty for falling from the walked distance in x-direction in meters. there is also a penalty for the maximum deviation in y-direction reached during an episode, weighted by a constant factor. in practice, the values chosen for f allenp enalty and a factor f were usually 3 and 2 respectively. this same fitness function can be used without modification for forward, backward and sideward walk learning, simply by adjusting the initial orientation of the agent. the also trained turn behavior requires a different fitness function. f itness turn = (g * totalt urn) − distance (2) where totalt urn refers to the cumulative rotation performed in degrees, weighted by a constant factor g (typically 1/100). we penalize any deviation from the initial starting x / y position (distance) as incentive to turn in-place. it is noteworthy that other than swapping out the fitness function and a few more minor adjustments mentioned in 3. 3 , everything else about the learning setup remained the same thanks to the model-free approach. naturally, the fitness calculation for an individual requires connecting an agent to the simspark simulation server and having it execute the behavior defined by the learned parameters. in detail, this works as follows: at the start of each "episode", the agent starts walking with the old model-based walk engine at full speed. once 80 simulation cycles (roughly 1.5 seconds) have elapsed, the robot starts checking the foot force sensors. as soon as the left foot touches the ground, it switches to the learned behavior. this ensures that the learned walk has comparable starting conditions each time. if this does not occur within 70 cycles (which sometimes happens due to non-determinism in the domain and noise in the foot force perception), the robot switches anyway. from that point on, the robot keeps performing the learned behavior that represents a single step, alternating between the original learned parameters and a mirrored version (right step and left step). an episode ends once the agents has fallen or 8 seconds have elapsed. to train different walk directions (forward, backward, sideward), the initial orientation of the player is simply changed accordingly. in addition, the robot uses a different walk direction of the model-based walk engine for the initial steps that are not subject to learning. in case of training a morphing behavior (see 4.5) , the episode duration is extended to 12 seconds. when a morphing behavior should be trained, the step behavior from another learning run is used. this also means that a morphing behavior is always trained for a specific set of walk parameters. after 6 seconds, the morphing behavior is triggered once the foot force sensors detect that the left foot has just touched the ground. unlike the step / walk behavior, this behavior is just executed once and not mirrored or repeated. then the robot switches back to walking at full speed with the model-based walk engine. to maximize the reward, the agent has to learn a morphing behavior that enables the transition between learned model-free and old model-based walk to work as reliably as possible. finally, for the turn behavior, the robot keeps repeating the learned behavior without alternating with a mirrored version. in any case, if the robot falls, a training run is over. the overall runtime of each such learning run is 2.5 days on our hardware. learning is done using plain genetic algorithms. the following hyperparameters were used: more details on the approach can be found in [11] . this section presents the results for each kind of behavior trained. this includes three different walk directions, a turn behavior and a behavior for morphing. the main focus of this work has been on training a forward walk movement. figure 2 shows a sequence of images for a learned step. the best result reaches a speed of 1.3 m/s compared to the 1.0 m/s of our model-based walk and 0.96 m/s for a walk behavior learned on the nao robot without toes. the learned walk with toes is less stable, however, and shows a fall rate of 30% compared to 2% of the model-based walk. regarding the characteristics of this walk, it utilizes remarkably long steps 4 . table 1 shows an in-depth comparison of various properties, including step duration, length and height, which are all considerably bigger compared to our previous model-based walk. the forward leaning of the agent has increased by 80.4%, while 28.1% more time is spent with both legs off the ground. however, the maximum deviation from the intended path (maxy ) has also increased by 137.8%. table 1 : comparison of the previously fastest and the fastest learned forward walk once a working forward walk was achieved, it was natural to try to train a backward walk behavior as well, since this only requires a minor modification in the learning environment (changing the initial rotation of the agent and model-based walk direction to start with). the best backward walk learned reaches a speed of 1.03 m/s, which is significantly faster than the 0.67 m/s of its model-based counterpart. unfortunately, the agent also falls 15% more frequently. it is interesting just how backward-leaning the agent is during this walk behavior. it could almost be described as "controlled falling" 5 (see figure 3 ). sideward walk learning was the least successful out of the three walk directions. like with all directions, the agent starts out using the old walk engine and then switches to the learned behavior after a short time. in this case however, instead of continuing to walk sideward, the agent has learned to turn around and walk forward instead, see figure 4 . the resulting forward walk is not very fast and usually causes the agent to fall within a few meters 6 , but it is still remarkable that the learned behavior manages to both turn the agent around and make it walk forward with the same repeating step movement. it is also remarkable that the robot learned that it is quicker with the given legs at least for long distances to turn and run forward than to keep making sidesteps. with the alternate fitness function presented in section 3, the agent managed to learn a turn behavior that is comparable in speed to that of the existing walk engine. despite this, the approach is actually different: while the old walk engine uses small, angled steps 7 , the learned behavior uses the left leg as a "pivot", creating angular momentum with the right leg 8 . figure 5 shows the movement sequence in detail. unfortunately, despite the comparable speed, the learned turn behavior suffers from much worse stability. with the old turn behavior, the agent only falls in roughly 3% of cases, with the learned behavior it falls in roughly 55% of the attempts. one of the major hurdles for using the learned walk behaviors in a robocup competition is the smooth transition between them and other existing behaviors such as kicks. the initial transition to the learned walk is already built into the learning setup described in 3 by switching mid-walk, so it does not have to be given special consideration. more problematic is switching to another behavior afterwards without falling. to handle this, the robot simply attempted to train a "morphing" behavior using the same model-free learning setup. the result is something that could be described as a "lunge" (see figure 6 ) that reduces the forward momentum sufficiently to allow it to transition to the slower model-based walk when successful. 9 however, the morphing is not successful in about 50% of cases, resulting in a fall. we were able to successfully train forward and backward walk behaviors, as well as a morphing and turn behavior using plain genetic algorithms and a very flexible model-free approach. the usage of the toe joint in particular makes the walks look more natural and human-like than that of the model-based walk engine. however, while the learned behaviors outperform or at least match our old modelbased walk engine in terms of speed, they are not stable enough to be used during actual robocup 3d simulation league competitions. we think this is an inherent limitation of the approach: we train a static behavior that is unable to adapt to changing circumstances in the environment, which is common in simspark's non-deterministic simulation with perception noise. deep reinforcement learning seems more promising in this regard, as the neural network can dynamically react to the environment since sensor data serves as input. it is also arguably even less restrictive than the keyframe-based behavior parameterization we presented in this paper, as a neural network can output raw joint actions each simulation cycle. at least two other robocup 3d simulation league teams, fc portugal [8] and itandroids [9] , have had great success with this approach, everything points towards this becoming the state-of-the-art approach in robocup 3d soccer simulation in the near future, so we want to concentrate our future efforts here as well. retail companies dealing in alcoholic beverages are faced with a constant flux of products. apart from general product changes like modified bottle designs and sizes or new packaging units two factors are responsible for this development. the first is the natural wine cycle with new vintages arriving at the market and old ones cycling out each year. the second is the impact of the rapidly growing craft beer trend which has also motivated established breweries to add to their range. the management of the corresponding product data is a challenge for most retail companies. the reason lies in the large amount of data and its complexity. data entry and maintenance processes are linked with considerable manual effort resulting in high data management costs. product data attributes like dimensions, weights and supplier information are often entered manually into the data base and are often afflicted with errors. another widely used source of product data is the import from commercial data pools. a means of checking the data thus acquired for plausibility is necessary. sometimes product data is incomplete due to different reasons and a method to fill the missing values is required. all these possible product data errors lead to complications in the downstream automated purchase and logistics processes. we propose a machine learning model which involves domain specific knowledge and compare it a heuristic approach by applying both to real world data of a retail company. in this paper we address the problem of predicting the gross weight of product items in the merchandise category alcoholic beverages. to this end we introduce two levels of additional features. the first level consists of engineered features which can be determined by the basic features alone or by domain specific expert knowledge like which type of bottle is usually used for which grape variety. in the next step an advanced second level feature is computed from these first level features. adding these two levels of engineered features increases the prediction quality of the suggestion values we are looking for. the results emphasize the importance of careful feature engineering using expert knowledge about the data domain. feature engineering is the process of extracting features from the data in order to train a prediction model. it is a crucial step in the machine learning pipeline, because the quality of the prediction is based on the choice of features used to training. the majority of time and effort in building a machine learning pipeline is spent on data cleaning and feature engineering [domingos 2012] . a first overview of basic feature engineering principles can be found in [zheng 2018 ]. the main problem is the dependency of the feature choice on the data set and the prediction algorithm. what works best for one combination does not necessarily work for another. a systematic approach to feature engineering without expert knowledge about the data is given in [heaton 2016 ]. the authors present a study whether different machine learning algorithms are able to synthesize engineered features on their own. as engineered features logarithms, ratios, powers and other simple mathematical functions of the original features are used. in [anderson 2017 ] a framework for automated feature engineering is described. the data set is provided by a major german retail company and consists of 3659 beers and 10212 wines. each product is characterized by the seven features shown in table 1. the product name obeys only a generalized format. depending on the user generating the product entry in the company data base, abbreviating style and other editing may vary. the product group is a company specific number which encodes the product category -dairy products, vegetables or soft drinks for example. in our case it allows a differentiation of the product into beer and wine. additionally wines are grouped by country of origin and for germany also into wine-growing regions. note that the product group is no inherent feature like length, width, height and volume, but depends on the product classification system a company uses. the dimensions length, width, height and the volume derived by multiplicating them are given as float values. the feature (gross) weight, also given as a float value, is what we want to predict. as is often the case with real world data, a pre-processing step has to be performed prior to the actual machine learning in order to reduce data errors and inconsistencies. for our data we first removed all articles missing one or more of the required attributes of table 1. then all articles with dummy values were identified and discarded. dummy values are often introduced due to internal process requirements but do not add any relevant information to the data. if for example the attribute weight has to be filled for an article during article generation in order to proceed to the next step but the actual value is not know, often a dummy value of 1 or 999 is entered. these values distort the prediction model when used as training data in the machine learning step. the product name is subjected to lower casing and substitution of special german characters like umlauts. special symbolic characters like #,! or separators are also deleted. with this preprocessing done the data is ready to be used for feature engineering. following this formal data cleaning we perform an additional content-focused pre-processing. the feature weight is discretized by binning it with bin width 10g. volume is likewise treated with bin size 10ml. this simplifies the value distribution without rendering it too coarse. all articles where length is not equal to width are removed, because in these cases there are no single items but packages of items. often the data at hand is not sufficient to train a meaningful prediction model. in these cases feature engineering is a promising option. identifying and engineering new features depends heavily on expert knowledge of the application domain. the first level consists of engineered features which can be determined by the original features alone. in the next step advanced second level features are computed from these first level and the original features. for our data set the original features are product name and group as well as the dimensions length, width, height and volume. we see that the volume is computed in the most general way by multiplication of the dimensions. geometrically this corresponds to all products being modelled as cuboids. since angular beer or wine bottles are very much the exception in the real world, a sensible new feature would be a more appropriate modelling of the bottle shape. since weight is closely correlated to volume, the better the volume estimate the better the weight estimate. to this end we propose four first level engineered features: capacity, wine bottle type, beer packaging type and beer bottle type which are in turn used to compute a second level engineered feature namely the packaging specific volume. figure 1 shows all discussed features and their interdependencies. let us have a closer look at the first level engineered features. the capacity of a beverage states the amount of liquid contained and is usually limited to a few discrete values. 0.33l and 0.5l are typical values for beer cans and bottles while wines are almost exclusively sold in 0.75l bottles and sometimes in 0.375l bottles. the capacity can be estimated from the given volume with sufficient certainty using appropriate threshold values. outliers were removed from the data set. there are three main beer packaging types in retail: cans, bottles and kegs. while kegs are mainly of interest to pubs and restaurants and are not considered in this paper, cans and bottles target the typical super market shopper and come in a greater variety. in our data set, the product name in case of beers is preceded by a prefix denoting whether the product is packaged in a can or a bottle. extracting the relevant information is done using regular expressions. not, though, that the prefix is not always correct and needs to be checked against the dimensions. the shapes of cans are the same for all practical purposes, no matter the capacity. the only difference is in their wall thickness, which depends on the material, aluminium and tin foil being the two common ones. the difference is weight is small and the actual material used is impossible to extract from the data. a further distinction for cans in different types like for beer and wine is therefore unnecessary. regarding the german beer market, the five bottle types shown in figure 2 the engineered feature beer packaging type assigns each article identified as beer by its product group to one of the classes bottle or can. the feature beer bottle type contains the most probably member of the five main beer bottle types. packages containing more than one bottle or can like crates or six packs are not considered in this paper and were removed from the data set. compared to beer the variety of commercially sold wine packagings is limited to bottles only. a corresponding packaging type attribute to distinguish between cans and bottles is not necessary. again there are a few bottle types which are used for the majority of wines, namely schlegel, bordeaux and burgunder ( figure 3 ). deciding what product is filled in which bottle type is a question of domain knowledge. the original data set does not contain a corresponding feature. from the product group the country of origin and in the case of german wines the region can be determined via a mapping table. this depends on the type of product classification system the respective company uses and has not to be valid for all companies. our data set uses a customer specific classification with focus on germany. a more general one would be the global product classification (gpc) standard for example. to determine wine growing regions in non-german countries like france the product name has to be analyzed using regular expressions. the type of grape is likewise to be deduced from the product name if possible. using the country and specifically the region of origin and type of grape of the wine in question is the only way to assign a bottle type with acceptable certainty. there are countries and region in which a certain bottle type is used predominantly, sometimes also depending on the color of the wine. the schlegel bottle, for example, is almost exclusively used for german and alsatian white wines and almost nowhere else. bordeaux and burgunder bottles on the other hand are used throughout the world. some countries like california or chile use a mix of bottle types for their wines, which poses an additional challenge. with expert knowledge one can assign regions and grape types to the different bottle types. as with beer bottles this categorization is by no means comprehensive or free of exceptions but serves as a first step. the standard volume computation by multiplying the product dimensions length, width and height is a rather coarse cuboid approximation to the real shape of alcoholic beverage packagings. since the volume is intrinsically linked to the weight which we want to predict a packaging type specific volume computation is required for cans and especially bottles. the modelling of a can is straightforward using a cylinder with the given height ℎ and a diameter of the given width and length . thus the packaging type specific volume is: a bottle on the other hand needs to be modelled piecewise. its height can be divided into three parts: base, shoulders and neck as shown in figure 4. base and neck can be modeled by a cylinder. the shoulders are approximated by a truncated cone. with the help of the corresponding partial heights ℎ , ℎ ℎ and ℎ we can compute coefficients , ℎ and as fractions of the overall height ℎ of the bottle. the diameters of the bottle base and the neck opening are given by and and are likewise used to compute the ratio . since bottles have circular bases, the values for width and length in the original data have to be the same and either one may be used for . these four coefficients are characteristic for each bottle type, be it beer or wine (table 3) . with their help, a bottle type specific volume from the original data length, width and height can be computed which is a much better approximation to the true volume than the former cuboid model. the bottle base can be modelled as a cylinder as follows: the bottle shoulders have the form of a truncated cone and are described by formula 3: the bottle neck again is a simple cylinder: summing up all three sections yields the packaging type specific volume for bottles: ur-ai 2020 // 18 the experiments follow the multi-level feature engineering scheme as shown in figure 1 . first, we use only the original features product group and dimensions. then we add the first level engineered features capacity and bottle type to the basic features. next the second level engineered feature packaging type specific volume is used along with the basic features. finally all features from every level are used for the prediction. after pre-processing and feature engineering the data set size is reduced from 3659 to 3380 beers and from 10212 to 8946 wines. for prediction of the continuous valued attribute gross weight, we use and compare several regression algorithms. both the decision-tree based random forests algorithm (breimann, 2001) and support vector machines (svm) (cortes, 1995) are available in regression mode (smola, 1997) . linear regression (lai, 1979) and stochastic gradient descent (sgd) (taddy, 2019) are also employed as examples of more traditional statics-based methods. our baseline is a heuristic approach taking the median of the attribute gross weight for each product group and use this value as a prediction for all products of the same product group. practical experience has shown this to be a surprisingly good strategy. the implementation was done in python 3.6 using the standard libraries sk-learn and pandas. all numeric features were logarithmized prior to training the models. the non-numeric feature bottle type was converted to numbers. the final results were obtained using tenfold cross validation (kohavi, 1995) . for model training 80% of the data was used while the remaining 20% constituted the test data. we used the root mean square error (rsme) (6) as well as the mean and variance of the absolute percentage error (7) as metrics for the evaluation of the performance of the algorithms. all machine learning algorithms deliver significant improvements regarding the observed metrics compared to the heuristic median approach. the best results for each feature combination are highlighted in bold script. the results for the beer data set in table 4 show that the rsme can be more than halved, the mean almost be reduced to a third and the variance of quartered compared to the baseline approach. the random forest regressor achieves the best results in terms of rsme and for almost all feature combinations except basic features and basic features combined with the packaging type specific volume, in which cases support vector machines prove superior. linear regression and sgd are are still better than the baseline approach but not on par with the other algorithms. linear regression shows the tendency to improved results when successively adding features. sgd on the other hand exhibits no clear relation between number and level of features and corresponding prediction quality. a possible cause could be the choice of hyper parameters. sgd is very sensitive in this regard and depends more heavily upon a higher number of correctly adjusted hyper parameters than the other algorithms we used. random forests is a method which is very well suited to problems, where there is no easily discernable relation between the features. it is prone to overfitting, though, which we tried to avoid by using 20% of all data as test data. adding more engineered features leads to increasingly better results using random forest with an outlier for the packaging type specific volume feature. svm are not affected by only first level engineered features but profit from using the bottle type specific volume. regarding the wine data set the results depicted in table 5 are not as good as for the beer data set though still much better than the baseline approach. a reduction of the rsme by over 29% and of the mean by almost 50% compared to the baseline were achieved. the variance of could even be limited to under 10% of the baseline value. again random forests is the algorithm with the best metrics. linear regression and svm are comparable in terms of while sgd is worse but shows good rsme values. in conclusion the general results of the wine data set show not much improvement when applying additional engineered features. 6 discussion and conclusion the experiments show a much better predicting quality for beer than for wine. a possible cause could be the higher weight variance in bottle types compared to beer bottles and cans. it is also more difficult to correctly determine the bottle type for wine, since the higher overlap in dimensions does not allow to compute the bottle type with the help of idealized bottle dimensions. using expert knowledge to assign the bottle type by region and grape variety seems not to be as reliable, though. especially with regard to the lack of a predominant bottle type in the region with the most bottles (red wine from baden for example), this approach should be improved. especially bordeaux bottles often sport an indentation in the bottom, called a 'culot de bouteille'. the size and thickness of this indentation cannot be inferred from the bottle's dimensions. this means that the relation between bottle volume and weight is skewed compared to other bottles without these indentations, which in turn decreases prediction quality. predicting gross weights with machine learning and domain-specifically engineered features leads to smaller discrepancies than using simple heuristic approaches. this is important for retail companies since big deviations are much worse for logistical reasons than small ones which may well be within natural production tolerances for bottle weights. our method allows to check manually generated as well as data pool imported product data for implausible gross weight entries and proposes suggestion values in case of missing entries. the method we presented can easily be adapted to non-alcoholic beverages using the same engineered features. in this segment, plastics bottles are much more common than glass ones and hence the impact of the bottle weight compared to the liquid weight is significantly smaller. we assume that this will cause a smaller importance of the bottle type feature in the prediction. a more problematic kind of beverage is liquor. although there are only a few different standard capacities, the bottle types vary so greatly, that identifying a common type is almost impossible. one of the main challenges of our approach is determining the correct bottle types. using expert knowledge is a solid approach but cannot capture all exemptions. especially if a wine growing region has no predominant bottle type and is using mixed bottle types instead. additionally many wine growers use bottle types which haven't been typical for their wine types because they want to differ from other suppliers in order to get the customer's attention. assuming that all rieslings are sold in schlegel bottles, for example, is therefore not exactly true. one option could be to model hybrid bottles using a weighted average of the coefficients for each bottle type in use. if a region uses both burgunder and bordeaux bottles with about equal frequency, all products from this region could be assigned a hybrid bottle with coefficients computed by the mean value of each coefficient. if an initially bottle type labeled data set is available, preliminary simulations have shown that most bottle types can be predicted robustly using classification algorithms. the most promising strategy, in our opinion, is to learn the bottle types directly from product images using deep neural nets for example. with regard to the ever increasing online retail sector, web stores need to have pictures of their products on display, so the data is there to be used. quality assurance is one of the key issues for modern production technologies. especially new production methods like additive manufacturing and composite materials require high resolution 3d quality assurance methods. computed tomography (ct) is one of the most promising technologies to acquire material and geometry data non-destructively at the same time. with ct it is possible to digitalize subjects in 3d, also allowing to visualize their inner structure. a 3d-ct scanner produces voxel data, comprising of volumetric pixels that correlate with material properties. the voxel value (grey value) is approximately proportional to the material density. nowadays it is still common to analyse the data by manually inspecting the voxel data set, searching for and manually annotating defects. the drawback is that for high-resolution ct data, this process it very time consuming and the result is operator-dependent. therefore, there is a high motivation to establish automatic defect detection methods. there are established methods for automatic defect detection using algorithmic approaches. however, these methods show a low reliability in several practical applications. at this point artificial neural networks come into play that have been already implemented successfully in medical applications [1] . the most common networks, developed for medical data segmentation, are by ronneberger et al., the u-net [2] and by milletari et al., the v-net [3] and their derivates. these networks are widely used for segmentation tasks. fuchs et al. describes three different ways of analysing industrial ct data [4] . one of these contains a 3d-cnn. this cnn is based on the u-net architecture and is shown in their previous paper [5] . the authors enhance and combine the u-net and v-net architecture to build a new network for examination of 3d volumes. in contrast, we investigate in our work how the networks introduced by ronneberger et al. and milletari et al. perform in industrial environments. furthermore, we investigate if derivates of these architectures are able to identify small features in industrial ct data. in industrial ct systems, not only in the hardware design but also in the resulting 3d imaging data differs from medical ct systems. voxel data from industrial parts differ from medical data in the contrast level and the resolution. state-of-the-art industrial ct scanner produce one to two order of magnitude larger data sets compared to medical ct systems. the corresponding resolution is necessary to resolve small defects. medical ct scanners are optimised for a low xray dose for the patient, the energy of x-ray photons are typically up to 150 kev, industrial scanner typically use energies up to 450 kev. in combination with the difference of the scan "object", the datasets differ significantly in size and image content. to store volume data there are a lot of different file formats. some of them are mainly used in medical applications like dicom [6] , nifti 1 or raw. in industrial applications vgl 3 , raw and tiff 4 are commonly used. also depending on the format, it is possible to store the data slice wise or as a complete volume stack. industrial ct data, as mentioned in previous section, has some differences to medical ct data. one aspect is the size of the features to be detected or learned by the neural network. our target is to find defects in industrial parts. as an example, we analyse pores in casting parts. these features may be very small, down to 1 to 7 voxels in each dimension. compared to the size of the complete data volume (typically larger than 512 x 512 x 512 voxel), the feature size is very small. the density difference between material and pores may be as low as 2% of the maximum grey value. thus, it is difficult to annotate the data even for human experts. the availability of real industrial data of good quality, annotated by experts, is very low. most companies don't reveal their quality analysis data. training a neural network with a small quantity of data is not possible. for medical applications, especially ai applications, there are several public datasets available. yet these datasets are not always sufficient and researchers are creating synthetic medical data [7] . therefore, we decided to create synthetic industrial ct data. another important reason for synthetic data is the quality of annotations done by human experts. the consistency of results is not given for different experts. fuchs et al. have shown that training on synthetic data and predicting on real data lead to good results [4] . however, synthetic data may not reflect all properties of real data. some of the properties are not obvious, which may lead to ignoring some varieties in the data. in order to achieve a high associability, we use a large numbers of synthetic data mixed with a small number of real data. to achieve this, we developed an algorithm which generates large amounts of data, containing a large variation of aspects, needed to generalize a neural network. the variation includes material density, pore density, pore size, pore amount, pore shape and size of the part. there are some samples that could be learned easily, because the pores are clearly visible inside the material. however, some samples are more difficult to be learned, because the pores are nearly invisible. this allows us to generate data with a wide variety and hence the network can predict on different data. to train the neural networks, we can mix the real and synthetic data or use them separately. the real data was annotated manually by two operators. to create a dataset of this volume we sliced it into 64x64x64 blocks. only the blocks with a mean density greater than 50% of the grayscale range are used, to avoid too much empty volumes in the training data. another advantage of synthetic data is the class balance. we have two classes, where 0 corresponds to material and surrounding air and 1 for the defects. because of the size of the defects there is a high imbalance between the classes. by generating data with more features than in the real data, we could reduce the imbalance. reducing the size of the volume to 64x64x64 also leads to better balance between the size of defects compared to full volume. in table 1details of our dataset for training, evaluation and testing are shown. the synthetic data will not be recombined to a larger volume as they represent separate small components or full material units. the following two slices of real data ( figure 1 ) and synthetic data (figure 2 ) with annotated defects show the conformity between the data. ur-ai 2020 // 26 hardware and software setup deep learning (dl) consist of two phases: the training and its application. while dl models can be executed very fast, the training of the neural network can be very time-consuming, depending on several factors. one major factor is the hardware. the time consumed can be reduced by the factor of around ten when graphics cards (gpus) are used. [8] to cache the training data, before it is given into the model, calculated on the gpu, a lot of random-access memory (ram) is used [9] [10] [11] . our system is built on a dual cpu hardware with 10 cores each running at 2.1 ghz and a nvidia gpu titan rtx 5 with 24gb of vram and 64gb of regular ram. all measurements in this work concerning training and execution time are related to this hardware setup. the operating system is ubuntu 18.4lts. anaconda is used for python package management and deployment. the dl-framework is tensorflow 6 2.1 and keras as a submodule in python 7 . based on the 3du-net [12] and 3dv-net [3] architecture compared from paichao et al. [13] we created modified versions which differ in number of layers and their hyperparameters. due to the small size of our data, no patch division is necessary. instead the training is performed on the full volumes. we actually do not use the z-net enhancement proposed in their paper. the input size, depending on our data, is defined to 64x64x64x1 with 1 dimension for channel. the incoming data will be normalized. as we have a binary segmentation task, our output activation is the sigmoid [14] function. based on paichao et al. [13] the convolutional layer of our 3du-nets have a kernel size of (3, 3, 3) and the 3dv-nets have a kernel size of (5, 5, 5). as convolution activation function we are using elu [14] [15] and he_normal [16] as kernel initialization [17] . the adam optimisation method [18] [19] is used with a starting learning rate of 0.0001, a decay factor of 0.1 and the loss function is the binary cross-entropy [20] . figure 3 shows a sample 3du-net architecture where downwards max pooling and upwards transposed convolution are used. compared to figure 4 , the 3dv-net, where we have a fully convolutional neural network, the descend is done with a (2, 2, 2) convolution and a stride of 2 and ascent with transposed convolution. it also has a layer level addition of the input of this level added to the last convolution output of the same level, as marked by the blue arrows. to adapt the shapes of the tensors for adding them, the down-convolution and the last convolution of the same level, have to have the same number of kernel filters. our modified neural network differ in the levels of de-/ascending, the convolution filter kernel size and their hyperparameters, shown in table 2 . the convolutions on one level have the same number of filter kernel. after every down convolution the number of filters is multiplied by 2 and on the way up divided by 2. training and evaluation of the neural networks the conditions of a training and a careful parameters selection is important. in table 3 the training conditions fitted to our system and networks are shown. we are also taking into account that different network architectures and number of layers are better performing on different learning rates, batch size, etc. to evaluate our trained models, we are mainly focusing on the iou metric, also called jackard index, which is the intersection over union. this metric is widely used for segmentation tasks and compares the intersection over union between the prediction and ground truth for each voxel. the value of iou range between 0 and 1, whereas the loss values range between 0 and infinite. therefore, the iou is a much clearer indicator. an iou close to 1 indicates a high intersectionprecision between the prediction and the groundtruth. our networks where trained between 30 and 90 epochs until no more improvement could be achieved. both datasets consist of a similar number of samples, which means the epoch time is equivalent. one epoch took around 4 minutes. figure 5 shows the loss determined based on the evaluation data. as described in fehler! verweisquelle konnte nicht gefunden werden., all models are trained on and evaluated against the synthetic dataset gdata and on the mixed dataset mdata. in general, the loss achieved by all models is higher on mdata because the real data is harder to learn. a direct comparison between the models is only possible between models with the same architecture. the iou metric shown in figure 6 . here the evaluation is sorted based on the iou metric. if we compare the loss of unet-mdata with unet-gdata, which are nearly the same for mdata, with their corresponding iou (unet-mdata (~0.8) and unet-gdata (~0.93)), we can see that a lower loss does not necessarily lead to higher iou score. if only the loss and iou are considered, the unets tend to be better than the vnets. as a conclusion, considering the iou metric for model selection, the unet-gdata is the best performing model and vnet-gdata the least performing. the evaluation loss determined based on the evaluation data sorted from lowest to highest. the evaluation iou determined based on the evaluation data sorted from lowest to highest. after comparing the automatic evaluation, we show prediction samples of different models on real and synthetic data ( table 4) . rows 1 and 2 show the comparison between unet-gdata and vnet-gdata, predicting on a synthetic test sample. the result of unet-gdata exactly hits the groundtruth, whereas the vnet-gdata prediction has a 100% overlap to the groundtruth but with surrounding false positive segmentations. in row 3 and 4 both models predict the groundtruth plus some false positive segmentations in the close neighbourhood. in row 5 and 6 the prediction results of the same two models on real data is shown, taking into account that both models are not trained on real data. unet-gdata delivers a good precision with some false positive segmentations in thegroundtruth area and one additional segmented defect. this shows that the model was able to find a defect which was missed by the expert. vnet-gdata shows a very high number of false positive segmentations. in this paper, we have proposed a neural network to find defects in real and synthetic industrial ct volumes. we have shown that neural networks, developed for medical applications can be adapted to industrial applications. to achieve high accuracy, we used a large variety of features in our data. based on the evaluation and manually reviewing random samples we have chosen the unet architecture for further research. this model achieved great performance on our real and synthetic dataset. in summery this paper shows that the artificial intelligence and their neural networks will take an import enrichment in industrial issues. stress can affect all aspects of our lives, including our emotions, behaviors, thinking ability, and physical health, making our society sick -both mentally and physically. among the effects that the stress and anxiety can cause are heart diseases, such as coronary heart disease and heart failure [5] . due this information, this research will present a proposal to help people handling stress using the benefit of technology development and to set patters of stress status as way to propose some intervention, once the first step to controlling stress is to know the symptoms of stress. the stress symptoms are very board and can be confused with others diseases according the american institute of stress [15] , for example the frequent headache, irritability, insomnia, nightmares, disturbing dreams, dry mouth, problems swallowing, increased or decreased appetite, or even cause other diseases such as frequent colds and infections. in view of the wide variety of symptoms caused by stress, this research intends to define, through physiological signals, the patterns generated by the body and obtained by wearable sensors and develop a standardized database to apply the machine learning. hand, advances in sensor technology, wearable devices and mobile growth would help to online stress identification based on physiological signals and delivery of psychological interventions. currently with the advancement of technology and improvements in the wearable sensors area, made it possible to use these devices as a source of data to monitor the user's physiological state. the majority of the wearable devices consist of low-cost board that can be used to the acquisition of physiological signals [1, 10] . after the data are obtained it is necessary apply some filters to clear signal, without noise or distortions aiming to use some machine learning approaches to model and predict these stress states [2, 11] . the wide-spread use of mobile devices and microcomputers, as raspberry pi, and its capabilities presents a great possibility to collect, and process those signs with an elaborated application. these devices can collect the physiological signals and detect specific stress states to generate interventions following the predetermined diagnosis based on the standards already evaluated in the system [9, 6] . during the literature review it was evident the presence of few works dedicated to evaluating comprehensively the complete cycle of biofeedback, which comprises using the wearable devices, applying machine learning patterns detection algorithms, generate the psychologic intervention, besides monitoring its effects and recording the history of events [9, 3] . stress is identified by professionals using human physiology, so wearables sensors could help on data acquisition and processing, through machine learning algorithms on biosignal data, suggesting psychological interventions. some works [6, 14] are dedicated to define patterns as experiment for data acquisition simulation real situations. jebelli, khalili and lee [6] showed a deep learning approach that was used to compare with a baseline feedforward artificial neural network. schmidt et al. [12] describes wearable stress and affect detection (wesad), one public dataset used to set classifiers and identify stress patterns integrating several sensors signals with the emotion aspect with a precision of 93% in the experiments. the work of gaglioli et al. [4] describe the main features and preliminary evaluation of a free mobile platform for the selfmanagement of psychological stress. in terms of the wearables, some studies [13, 14] evaluate the usability of devices to monitory the signals and the patient's well-being. pavic et al. [13] showed a research performed to monitor cancer patients remotely and as the majority of the patients have a lot of symptoms but cannot stay at hospital during all treatment. the authors emphasize that was obtained good results and that this system is viable, as long as the patient is not a critical case, as it does not replace medical equipment or the emergency care present in the hospital. henriques et al. [5] focus was to evaluated the effects of biofeedback in a group of students to reduce anxiety, in this paper was monitored the heart rate variability with two experiments with duration of four weeks each. the work of wijman [8] describes the use of emg signals to identify stress, this experiment was conducted with 22 participants, evaluating both the wearables signals and questionnaires. in this section will be described the uniqueness of this research and the devices that was used. this solution is being proposed by several literature study about stress patterns and physiological aspects but with few results, for this reason, our project will address topics like experimental study protocol on signals acquisition from patients/participants with wearables to data acquisition and processing, in sequence will be applied machine learning modeling and prediction on biosignal data regarding stress (fig. 1) . the protocol followed to the acquisition of signals during all different status is the trier social stress test (tsst) [7] , recognized as the gold standard protocol for stress experiments. the estimated total protocol time, involving pre-tests and post-tests, is 116 minutes with a total of thirteen steps, but applied experiment was adapted and it was established with ten stages: initial evaluation: the participant arrives, with the scheduled time, and answer the questionnaires; habituation: it will take a rest time of twenty minutes before the pre-test to avoid the influence of events and to establish a safe baseline of that organism; pre-test: the sensors will be allocated ( fig. 2 ), collected saliva sample and applied the psychological instruments. the next step is explanation of procedure and preparation: the participant reads the instructions and the researcher ensures that he understands the job specifications, in sequence, he is sent to the room with the jurors (fig. 3) , composed of two collaborators of the research, were trained to remain neutral during the experiment, not giving positive verbal or non-verbal feedback; free speech: after three minutes of preparation, the participant is requested to start his speech, being informed that he cannot use the notes. this will follow the arithmetic task: the jurors request an arithmetic task in which the participant must subtract mentally, sometimes, the jurors interrupt and warn that the participant has made a mistake; post-test evaluation: the experimenter receives the subject outside the room for the post-test evaluations; feedback and clarification: the investigator and jurors talk to the subject and clarify what the task was about; relaxation technique: a recording will be used with the guidelines on how to perform a relaxation technique, using only the breathing; final post-test: some of the psychological instruments will be reapplied, saliva samples will be collected, and the sensors will still be picking up the physiological signals. based on literature [14] and wearable devices available the signals that was selected to analysis is the ecg, eda and emg for an initial experiment. this experimental study protocol on data acquisition started with 71 participants, where data annotation each step was done manually, from protocol experiment, preprocessing data based on features selection. in the machine learning step, it was evaluated the metrics of different algorithms as decision tree, random forest, adaboost, knn, k-means, svm. the experiment was made using the bitalino kit -plux wireless biosignals s.a. (fig. 4 ) composed by ecg sensor, which will provide data on heart rate and heart rate variability; eda sensor that will allow measure the electrical dermal activity of the sweat glands; emg sensor that allows the data collect the activity of the muscle signals. this section will describe the results in the pre-processing step and how it was made, listing all parts regarded to categorization and filtering data, evaluating the signal to know if it has plausibility and create a standardized database. the developed code is written in python due to the wide variety of libraries available, in this step was used the libraries numpy and pandas, both used to data manipulation and analysis. in the first step it is necessary read the files with the raw data and the timestamp, during this process the used channels are renamed to the name of the signal, because the bitalino store the data with the channel number as name of each signals. in sequence, the data timestamp is converted to a useful format, with goal to compare with the annotations, after time changed to the right format all channels unused are discarded to avoid unnecessary processing. the next step is to read the annotations taken manually in the experiment, as said before, to compare the time and classify each part of the experiment with its respective signal. after all signals are classified with its respective process of the tsst, each part of the experiment is grouped in six categories, which will be analyzed later. the first category is the "baseline", with just two parts of the experiment, representing the beginning of the experiment, when the participants had just arrived. the second is called of "tsst" comprises the period in which the participant spoke, the third category is the "arithmetic" with the data in acquired in the arithmetic test. the others two relevant categories are the "post_test_sensors_1" and "post_test_sensors_2", with its respective signals in the parts called with the same name. every other part of the experiment was categorized as "no_category", in sequence, this category is discarded in function of it will not be necessary in the machine learning stage. after the dataframe is right with all signals properly classified, the columns with the participants number and the timestamp are removed of the dataframe. the next step is evaluated the signal, to verify if the signal is really useful in the process of machine learning. for this, it is analyzed the signals using the biosppy library, which performs the data filtering process and makes it possible to view the data. finally, the script checks the volume of data present in each classification and returns the value of the smallest category. this is done because it was found that the categories have different volumes of data, which would become a problem in the machine learning stage, by offering more data from a determinate category than from the others. due this fact, the code analyzes the others categories and reduce its size until all categories stay with the same number of rows in each category (); after this the dataframe is exported in a csv file, to be read in the machine learning stage. the purpose of this article is to describe some stages of the development of a system for the acquisition and analysis of physiological signals to determine patterns in these signals that would detect stress states. during the development of the project was verified that there are data gaps in the dataframe in the middle of the experiment in some participants; a hypothesis about the motivation of this had happened is the sampling of the acquisition of bitalino regarding communication issues in some specifics sampling rates. it evaluate the results obtained when reducing this acquisition rate, however, it is necessary to carefully evaluate the extent to which the reduction in the sampling rate will interfere with the results. during the evaluation of the plausibility of the signals, it was verified that there are evident differences between the signals patterns in the different stages of the process, thus validating the protocol followed in the acquisition of the standards. the next step in this project is implement the machine learning stage, applying different algorithms as svm, decision tree, random forest, adaboost, knn and k-means; besides to evaluate the results using metrics like accuracy, precision, recall and f1. the next steps of this research will support the confirmation of the hypothesis raised about being able to define patterns of physiological signals to detect stress states. from the definition of the patterns, a system can be applied that identifies the acquisition of the signals and, in real time, performs the analysis of these data based on the machine learning results. therefore we can detect the state of the person and that the psychologist can indicate a proposal intervention and monitor whether the decrease is occurring. technological developments have been influencing all kinds of disciplines by transferring more competences from human beings to technical devices. the steps inculde [1]: 1. tools: transfer of mechanics (material) from the human being to the device 2. machines: transfer of energy from the human being to the device 3. automatic machines 1 : transfer of information from the human being to the device 4. assistants: transfer of decisions from the human being to the device with the introduction of artificial intelligence (ai), in particular its latest developments in deep learning, we let the system (in step 4) take over our decisions and creation processes. thus, tasks and disciplines that were exclusively reserved for humans in the past can now co-exist or even take the human out of the loop. it is no wonder that this transformation is not stopped at disciplines such as engineering, business, agriculture but also affects humanities, art and design. each new technology has been adopted for artistic expression-just see the many wonderful examples in media art. therefore, it is not surprising, that ai is going to be established as a novel tool to produce creative content of any form. however, in contrast to other disruptive technologies, ai seems particular challenging to be accepted in the area of art because it offers capabilities we thought once only humans are able to perform-the art is no longer done by artists using new technology to perform their art, but the art is done by the machine itself without the need for a human to intervene. the question of "what is art" has always been an emotionally debated topic in which everyone has a slightly different definition depending on his or her own experiences, knowledge base and personal aesthetics. however, there seems to be a broad consensus that art requires human creativity and imagination as, for instance, stated by the oxford dictionary "the expression or application of human creative skill and imagination, typically in a visual form such as painting or sculpture, producing works to be appreciated primarily for their beauty or emotional power." every art movement challenges old ways and uses artistic creative abilities to spark new ideas and styles. with each art movement diverse intentions and reasons for creating the artwork came along with critics who did not want to accept the new style as an artform. with the introduction of ai into the creation process another art movement is trying to be established which is fundamentally changing the way we see art. for the first time, ai has the potential to take the artist out of the loop, to leave humans only in the positions of curators, observers and judges to decide if the artwork is beautiful and emotionally powerful. while there is a strong debate going on in the arts if creativity is profoundly human, we investigate how ai can foster inspiration, creativity and produce unexpected results. it has been shown by many publications that ai can generate images, music and the like which can resemble different styles and produce artistic content. for instance, elgammal et al. [2] have used generative adversarial networks (gan) to generate images by learning about styles and deviating from style norms. the promise of ai-assisted creation is "a world where creativity is highly accessible, through systems that empower us to create from new perspectives and raise the collective human potential" as roelof pieters and samim winiger pointed out [3] . to get a better understanding of the process on how ai is capable to propose images, music, etc. we have to open the black box to investigate where and how the magic is happening. random variations in the image space (sometimes also referred to as pixel space) are usually not leading to any interesting result. this is because semantic knowledge cannot be applied. therefore, methods need to be applied which constrain the possible variations of the given dataset in a meaningful way. this can be realized by generative design or procedural generation. it is applied to generate geometric patterns, textures, shapes, meshes, terrain or plants. the generation processes may include, but are not limited, to self-organization, swarm systems, ant colonies, evolutionary systems, fractal geometry, and generative grammars. mccormack et al. [4] review some generative design approaches and discuss how art and design can benefit from those applications. these generative algorithms which are usually realized by writing program code are very limited. ai can change this process into data-driven procedures. ai, or more specifically artificial neural networks, can learn patterns from (labeled) examples or by reinforcement. before an artificial neural network can be applied to a task (classification, regression, image reconstruction), the general architecture is to extract features through many hidden layers. these layers represent different levels of abstractions. data that have a similar structure or meaning should be represented as data points that are close together while divergent structures or meanings should be further apart from each other. to convert the image back (with some conversion/compression loss) from the low dimensional vector, which is the result of the first component, to the original input an additional component is needed. together they form the autoencoder which consists of the encoder and the decoder . the encoder compresses the data from a high dimensional input space to a low dimensional space, often called the bottleneck layer. then, the decoder takes this encoded input and converts it back to the original input as closely as possible. the latent space is the space in which the data lies in the bottleneck layer. if you look at figure 1 you might be wondering why a model is needed that converts the input data into a "close as possible" output data. it seems rather useless if all it outputs is itself. as discussed, the latent space contains a highly compressed representation of the input data, which is the only information the decoder can use to reconstruct the input as faithfully as possible. the magic happens by interpolating between points and performing vector arithmetic between points in latent space. these transformations result in meaningful effects on the generated images. as dimensionality is reduced, information which is distinct to each image is discarded from the latent space representation, since only the most important information of each image can be stored in this low-dimensional space. the latent space captures the structure in your data and usually offers some semantic meaningful interpretation. this semantic meaning is, however, not given a priori but has to be discovered. as already discussed autoencoders, after learning a particular non-linear mapping, are capable of producing photo-realistic images from randomly sampled points in the latent space. the latent space concept is definitely intriguing but at the same time non-trivial to comprehend. although latent space means hidden, understanding what is happening in latent space is not only helpful but necessary for various applications. exploring the structure of the latent space is both interesting for the problem domain and helps to develop an intuition for what has been learned and can be regenerated. it is obvious that the latent space has to contain some structure that can be queried and navigated. however, it is non-obvious how semantics are represented within this space and how different semantic attributes are entangled with each other. to investigate the latent space one should favor a dataset that offers a limited and distinctive feature set. therefore, faces are a good example in this regard because they share features common to most faces but offer enough variance. if aligned correctly also other meaningful representations of faces are possible, see for instance the widely used approach of eigenfaces [5] to describe the specific characteristic of faces in a low dimensional space. in the latent space we can do vector arithmetic. this can correspond to particular features. for example, the vector a smiling woman representing the face of a smiling woman minus the vector a neutral woman representing a neutral looking woman plus the vector a neutral man representing a neutral looking man resulted in the vector a smiling man representing a smiling man. this can also be done with all kinds of images; see e.g. the publication by radford et al. [6] who first observed the vector arithmetic property in latent space. a visual example is given in figure 2 . please note that all images shown in this publication are produced using biggan [7] . the photo of the author on which most of the variations are based on is taken by tobias schwerdt. in latent space, vector algebra can be carried out. semantic editing requires to move within the latent space along a certain 'direction'. identifying the 'direction' of only one particular characteristic is non-trivial since editing one attribute may affect others because they are correlated. this correlation can be attributed to some extent to pre-existing correlations in 'the real world' (e.g. old persons are more likely to wear eyeglasses) or bias in the training dataset (e.g. more women are smiling on photos than men). to identify the semantics encoded in the latent space shen et al. proposed a framework for interpreting faces in latent space [8] . beyond the vector arithmetic property, their framework allows decoupling some entangled attributes (remember the aforementioned correlation between old people and eyeglasses) through linear subspace projection. shen et al. found that in their dataset pose and smile are almost orthogonal to other attributes while gender, age, and eyeglasses are highly correlated with each other. disentangled semantics enable precise control of facial attributes without retraining of any given model. in our examples, in figures 3 and 4 , faces are varied according to gender or age. it has been widely observed that when linearly interpolate between two points in latent space the appearance of the corresponding synthesized images 'morphs' continuously from one face to another; see figure 5 . this implies that also the semantic meaning contained in the two images changes gradually. this is in stark contrast to having a simple fading between two images in image space. it can be observed that the shape and style slowly transform from one image into the other. this demonstrates how well the latent space understands the structure and semantics of the images. other examples are given in section 3. even though our analysis has focused on face editing for the reasons discussed earlier it holds true also for other domains. for instance, bau et al. [9] generated living rooms using similar approaches. they showed that some units from intermediate layers of the generator are specialized to synthesize certain visual concepts such as sofas or tvs. so far we have discussed how autoencoders can connect the latent space and the image semantic space, as well as how the latent code can be used for image editing without influencing the image style. next, we want to discuss how this can be used for artistic expression. while in the former section we have seen how to use manipulation in the latent space to generate mathematical sound operations not much artistic content has been generatedjust variations of photography like faces. imprecision in ai systems can lead to unacceptable errors in the system and even result in deadly decisions; e.g. at autonomous driving or at cancer treatment. in the case of artistic applications, errors or glitches might lead to interesting, non-intended, artifacts. if those errors or glitches are treated as a bug or a feature lies in the eye of the artist. to create higher variations in the generated output some artists randomly introduce glitches within the autoencoder. due to the complex structure of the autoencoder these glitches (assuming that they are introduced at an early layer in the network) occur on a semantic level as already discussed and might cause the models to misinterpret the input data in interesting ways. some could even be interpreted as glimpses of autonomous creativity; see for instance the artistic work 'mistaken identity' by mario klingemann [10] . so far the latent space is explored by humans either by random walk or intuitive steering into a particular direction. it is up to human decisions if the synthesized image of a particular location in latent space is producing a visually appealing or otherwise interesting result. the question arises where to find those places and if those places can be spotted by an automatized process. the latent space is usually defined by a space of ddimensions for which it is assumed the data to be represented as multivariate gaussian distributions n (0, i d ) [11] . therefore, the mean representation of all images lies in the center of the latent space. but what does that mean for the generated results? it is said that "beauty lies in the eyes of the beholder", however, research shows that there is a common understanding of beauty. for instance, averaged faces are perceived as more beautiful [12] . adopting these findings to latent space let us assume that the most beautiful images (in our case faces) can be found in the center of the space. particular deviations from the center stand for local sweet spots (e.g. female and male, ethnic groups). these types of sweet spots can be found by common means of data analysis (e.g. clustering). but where are interesting local sweet spots if it comes to artistic expression? figure 6 demonstrates some variation in style within the latent space. of course, one can search for locations in the latent space where particular artworks from a given artist or art styles are located; see e.g. figure 7 where the styles of different artists, as well as white noise 2 , have been used for adoption. but isn't lingering around these sweet spots not only producing "more of the same"? how to find the local sweet spots which can define a new art style and can be deemed truly creative? or do those discoveries of new art style lie outside of the latent space, because the latent space is trained within a particular set of defined art styles and can, therefore, produce only interpolations of those styles but nothing conceptually new? so far we have discussed how ai can help to generate different variations of faces and where to find visually interesting sweet spots. in this section, we want to show how ai is supporting the creation process by applying the discussed techniques to other areas of image and object processing. 3 probably, different variations of image-to-image translation are the most popular approach at least if looking at the mass media. the most prominent example is style transfer -the capability to transfer the style of one image to draw the content of another (examples are shown in figure 7 ). but mapping an input image to an output image is also possible for a variety of other applications such as object transfiguration (e.g. horse-to-zebra, apple-to-orange, season transfer (e.g. summer-to-winter) or photo enhancement [13] . while some of the just mentioned systems are not yet in a state to be widely applicable, ai tools are taking over and gradually automating design processes which used to be time-consuming manual processes. indeed, the most potential for ai in art and design is seen in its application to tedious, uncreative tasks such as coloring black-and-white images [14] . marco kempf and simon zimmerman used ai in their work dubbed 'deepworld' to generate a compilation of 'artificial countries' using data of all existing countries (around 195) to generate new anthems, flags and other descriptors [15] . roman lipski uses an ai muse (developed by florian dohmann et al.) to foster his/her inspiration [16] . because the ai muse is trained only on the artist's previous drawings and fed with the current work in progress it suggests image variations in line with roman's taste. cluzel et al. have proposed an interactive genetic algorithm to progressively sketch the desired side-view of a car profile [17] . for this, the user has taken on the role of a fitness function 4 through interaction with the system. the chair project [18] is a series of four chairs co-designed by ai and human designers. the project explores a collaborative creative process between humans and computers. it used a gan to propose new chairs which then have been 'interpreted' by trained designers to resemble a chair. deep-wear [19] is a method using deep convolutional gans for clothes design. the gan is trained on features of brand clothes and can generate images that are similar to actual clothes. a human interprets the generated images and tries to manually draw the corresponding pattern which is needed to make the finished product. li et al. [20] introduced an artificial neural network for encoding and synthesizing the structure of 3d shapes which-according to their findings-are effectively characterized by their hierarchical organization. german et al. [21] have applied different ai techniques trained by a small sample set of shapes of bottles, to propose novel bottle-like shapes. the evaluation of their proposed methods revealed that it can be used by trained designers as well as nondesigners to support the design process in different phases and that it could lead to novel designs not intended/foreseen by the designers. for decades, ai has fostered (often false) future visions ranging from transhumanist utopia to "world run by machines" dystopia. artists and designers explore solutions concerning the semiotic, the aesthetic and the dynamic realm, as well as confronting corporate, industrial, cultural and political aspects. the relationship between the artist and the artwork is directly connected through their intentions, although currently mediated by third-parties and media tools. understanding the ethical and social implications of ai-assisted creation is becoming a pressing need. the implications, where each has to be investigated in more detail in the future, include: -bias: al systems are sensitive to bias. as a consequence, the ai is not being a neutral tool, but has pre-decoded preferences. bias relevant in creative ai systems are: • algorithmic bias occurs when a computer system reflects the implicit values of the humans who created it; e.g. the system is optimized on dataset a and later retrained on dataset b without reconfiguring the neural network (this is not uncommon, as many people do not fully understand what is going on in the network, but are able to use the given code to run training on other data). • data bias occurs when your samples are not representative of your population of interest. • prejudice bias results from cultural influences or stereotypes which are reflected in the data. -art crisis: until 200 years ago painting served as the primary method for visual communication and was a widely and highly respected art form. with the invention of photography, painting began to suffer an identity crisis because painting-in its current form then-was not able to reproduce the world as accurate and with as low effort as photography. as a consequence visual artists had to change to different forms of representations not possible by photography inventing different art styles such as impressionism, expressionism, cubism, pointillism, constructivism, surrealism, up to abstract expressionism. at the time ai can perfectly simulate those styles what will happen with the artists? will artists still be needed, be replaced by ai, or will they have to turn to other artistic work which yet cannot be simulated by ai? -inflation: similar to the image flood which has reached us the same can happen with ai art. because of the glut, nobody is valuing and watching the images anymore. -wrong expectations: only esthetic appealing or otherwise interesting or surprising results are published which can be contributed to similar effects as the well-known publication bias [22] in other areas. eventually, this is leading to wrong expectations of what is already possible with ai. in addition, this misunderstanding is fueled by content claimed to be created by ai but has indeed been produced-or at least reworked-either by human labor or by methods not containing ai. -unequal judgment: even though the raised emotions in viewing artworks emerge from its underlying structure in the works, people also include the creation process in their judgment (in the cases where they know about it). frequently, becoming to know that a computer or an ai has created the artwork, in the opinion of the people it turns boring, has no guts, no emotion, no soul while before it was inspiring, creative and beautiful. -authorship: the authorship of ai-generated content has not been clarified. for instance, is the authorship of a novel song composed by an ai trained exclusively on songs by johann sebastian bach belonging to the ai, the developer/artist, or bach? see e.g. [23] for a more detailed discussion. -trustworthiness: new ai-driven tools make it easy for non-experts to manipulate audio and/or visual media. thus, image, audio as well as video evidence is not trustworthy anymore. manipulated image, audio, and video are leading to fake information, truth skepticism, and claims that real audio/video footage is fake (known as the liar's dividend ) [24] . the potential of ai in creativity has just been started to be explored. we have investigated on the creative power of ai which is represented-not exclusively-in the semantic meaningful representation of data in a dimensionally reduced space, dubbed latent space, from which images, but also audio, video, and 3d models can be synthesized. ai is able to imagine visualizations that lie between everything the ai has learned from us and far beyond and might even develop its own art styles (see e.g. deep dream [25] ). however, ai still lacks intention and is just processing data. those novel ai tools are shifting the creativity process from crafting to generating and selecting-a process which yet can not be transferred to machine judgment only. however, ai can already be employed to find possible sweet spots or make suggestions based on the learned taste of the artist [21] . ai is without any doubt changing the way we experience art and the way we do art. doing art is shifting from handcrafting to exploring and discovering. this leaves humans more in the role of a curator instead of an artist, but it can also foster creativity (as discussed before in the case of roman lipski) or reduce the time between intention and realization. it has the potential, just as many other technical developments, to democratize creativity because the handcrafting skills are not so much in need to express his/her own ideas anymore. widespread misuse (e.g. image manipulation to produce fake pornography) can limit the social acceptance and require ai literacy. as human beings, we have to ask ourselves if feelings are wrong just because the ai never felt alike in its creation process as we do? or should we not worry too much and simply enjoy the new artworks created no matter if they are done by humans, by ai or as a co-creation between the two ones? [1] aims to design and implement a machine learning system for the sake of generating prediction models with respect to quality checks and reducing faulty products in manufacturing processes. it is based on an industrial case study in cooperation with sick ag. we will present first results of the project concerning a new process model for cooperating data scientists and quality engineers, a product testing model as knowledge base for machine learning computing and visual support of quality engineers in order to explain prediction results. a typical production line consists of various test stations that conduct several measurements. those measurements are processed by the system on the fly, to point out problematic products. among the many challenges, one focus of the project is on support for quality engineers. preparation of prediction models is usually done by data scientists. but the demand for data scientists is increasing too fast, when a big number of products, production lines and changing circumstances have to be considered. hence, a software is needed which quality engineers can operate directly and leverage the results from prediction models. based on quality management and data science standard processes [2] [3] we created a reference process model for production error detection and correction which includes needed actors and associated tasks. with ml system and data scientist assistance we bolster the quality engineer in his work. to support the ml system, we developed a product testing model which includes crucial information about a specific product. in this model we describe the relation to product specific features, test systems, production lines sequences etc. the idea behind this, is to provide metadata information which in turn is used by the ml system instead of individual script solutions for each product. a ml model with good predictions has often a lack of information about the internal decisions. therefore, it is beneficial to support the quality engineer with useful feature visualizations. by default, we support the quality engineer with 2d -3d feature plots and histograms, in which the error distribution is visualized. on top, we developed further feature importance measures based on shap values [4] . these can be used to get deeper insight for particular ml decisions to significant features which get lower ranked by standard feature importance measures. medicine is a highly empirical discipline, where important aspects have to be demonstrated using adequate data and sound evaluations. this is one of the core requirements, which were emphasized during the development of the medical device regulation (mdr) of the european union (eu) [1] . this applies to all medical devices, including mechanical and electrical devices as well as software systems. also, the us food & drug administration (fda) recently set a focus on the discussions about using data for demonstrating the safety and efficacy of medical devices [2] . beside pure approval steps, they foster the use of data for optimization of the products, as nowadays data can be acquired more and more, using modern it technology. in particular, they pursue the use of real world evidence, i.e. data that is collected through the lifetime of a device, for demonstrating improved outcomes. [2] such approaches require the use of sophisticated data analysis techniques. beside classical statistics, artificial intelligence (ai) and machine learning (ml) are considered to be powerful techniques for this purpose. currently, they gain more and more attention. these techniques allow to detect dependencies in complex situations, where inputs and/or outputs of a problem have high-dimensional parameter spaces. this can e.g. be the case when extensive data is collected from diverse clinical studies or also treatment protocols from local sites. furthermore, ai/ml based techniques may be used in the devices themselves. for example, devices may be developed which are considered to improve complex diagnostic tasks or find individualized treatment options for specific medical conditions (see e.g. [3, 4] for an overview). for some applications, it already has been demonstrated that ml algorithms are able to outperform human experts with respect to specific success rates (e.g. [5, 6] ). in this paper, it will be discussed how ml based techniques can be brought onto the market including an analysis of appropriate regulatory requirements. for this purpose, the main focus lies on ml based devices applied in the intensive care unit (icu) as e.g. proposed in [7, 8] . the need for specific regulatory requirements comes from the observation, that ai/ml based techniques pose specific risks which need to be considered and handled appropriately. for example, ai/ml based methods are more challenging w.r.t. bias effects, reduced transparency, vulnerability to cybersecurity attacks, or general ethical issues (see e.g. [9, 10] ). in particular cases, ml based techniques may lead to noticeably critical results, as it has been shown for the ibm watson for oncology device. in [11] , it was reported that the direct use of the system in particular clinical environments resulted in critical treatment suggestions. the characteristics of ml based systems led to various discussions about their reliability in the clinical context. it requires to find appropriate ways to guarantee their safety and performance. (cf. [12] ) this applies to the field of medicine / medical devices as well as ai/ml based techniques in general. the latter was e.g. approached by the eu in their ethics guidelines for trustworthy ai [9] . driven by this overall development, the fda started a discussion regarding an extended use of ml algorithms in samd (software as a medical device) with a focus in quicker release cycles. in [13] , it pursued the development of a specific process which makes it easier to bring ml based devices onto the market and also to update them during their lifecycle. current regulations for medical devices, e.g. in us or eu, do not provide specific guidelines for ml based devices. in particular, this applies to systems which continuously collect data in order to improve the performance of the device. current regulations focus on a fixed status of the device, which may only be adapted in a minor extent after the release. usually, a new release or clearance by the authority is required, when the clinical performance of a device is modified. but continuously learning systems exactly want to do such improvement steps using additional real-world data from daily applications without extra approvals (see fig. 1 ). basic approaches for ai/ml based medical devices. left side: classical approach, where the status of the software has to be fixed after the release / approval stage. right side: continuously learning system where data is collected during the lifetime of the device without a separated release / approval step. in this case, an automatic validation step has to guarantee proper safety and efficacy. in [13] , the fda made suggestions how this could be addressed. it proposed the definition of so called samd pre-specifications (sps) and an algorithm change protocol (acp), which are considered to represent major tools for dealing with modifications of the ml based system during its lifetime. within the sps, the manufacturer has to define the anticipated changes which are considered to be allowed during the automatic update process. in addition, the acp defines the particular steps which have to be implemented to realize the sps specifications. see [13] for more information about sps and acp. but the details are not yet well elaborated by the fda at the moment. the fda requested for suggestions with respect to this. in particular, these tools serve as a basis for performing an automated validation of the updates. the applicability of this approach depends on the risk of the samd. in [13] , the fda uses the risk categories from the international medical device regulators forum (imdrf) [14] . this includes the categories state of healthcare situation or condition (critical vs. serious vs. noncritical) and significance of information provided by samd to healthcare decision (treat or diagnose vs. drive clinical management vs. inform clinical management) as the basic attributes. according to [13] , the regulatory requirements for the management of ml based systems are considered to depend on this classification as well as the particular changes which may take place during the lifetime of the device. the fda categorizes them as changes in performance, inputs, and intended use. such anticipated changes have to be defined in the sps in advance. the main purpose of the present paper is to discuss the validity of the described fda approach for enabling continuously learning systems. therefore, it uses a scenario based technique to analyze whether validation in terms of sps and acp can be considered adequate tools. the scenarios represent applications of ml based devices in the icu. it checks its consistency with other important regulatory requirements and analyzes pitfalls which may jeopardize the safety of the devices. additionally, it discusses whether more general requirements can be sufficiently addressed in the scenarios, as e.g. proposed in ethical guidelines for ai based systems like [9, 10] . this is not considered as a comprehensive analysis of the topics, but as an addition to current discussions about risks and ethical issues, as they are e.g. discussed in [10, 12] . finally, the paper proposes own suggestions to address the regulation of continuously learning ml based systems. again, this is not considered to be a full regulatory strategy, but a proposal of particular requirements, which may overcome some of the current limitations of the approach discussed in [13] . the overall aim of this paper is to contribute to a better understanding of the options and challenges of ai/ml based devices on the one hand and to enable the development of best practices and appropriate regulatory strategies, in the future. within this paper, the analysis of the fda approach proposed in [13] is performed using specific reference scenarios from icu applications, which are particularly taken from [13] itself. the focus lies on ml based devices which allow continuous updates of the model according to data collected during the lifetime of the device. in this context, sps and acp are considered as crucial steps which allow an automated validation of the device based on specified measures. in particular, the requirements and limitations of such an automated validation are analyzed and discussed, including the following topics / questions.  is automated validation reasonable for these cases? what are limitations / potential pitfalls of such an approach when applied in the particular clinical context?  which additional risks could apply to ai/ml based samd, in general, which go beyond the existing discussions in the literature as e.g. presented in [9, 10, 12] ?  how should such issues be taken into account in the future? what could be appropriate measures / best practices to achieve reliability? the following exemplary scenarios are used for this purpose. ur-ai 2020 // 56  base scenario icu: ml based intensive care unit (icu) monitoring system where the detection of critical situations (e.g. regarding physiological instability, potential myocardial infarcts or sepsis) is addressed by using ml. using auditory alarms, the icu staff is informed to initiate appropriate measures to treat the patients in these situations. this scenario addresses a 'critical healthcare situation or condition' and is considered to 'drive clinical management' (according to the risk classification used in [13] ).  modification "locked": icu scenario as presented above, where the release of the monitoring system is done according to a locked state of the algorithm.  modification "cont-learn": icu scenario as presented above, where the detection of alarm situations is continuously improved according to data acquired during daily routine, including adaptation of performance to sub-populations and/or characteristics of the local environment. in this case, scs and acp have to define standard measures like success rates of alarms/detection and requirements for the management of data, update of the algorithm, and labeling. more details of such requirements are discussed later. this scenario was presented as scenario 1a in [13] with minor modifications. this section provides the basic analysis of the scenarios according to the particular aspects addressed in this paper. it addresses the topics automated validation, man-machine interaction, explainability, bias effects, and confounding, fairness and non-discrimination as well as corrective actions to systematic deficiencies. according to standard regulatory requirements [1, 15, 16] , validation is a core step in the development and for the release of medical devices. according to [17] , a change in performance of a device (including an algorithm in a samd) as well as a change in particular risks (e.g. new risks, but also new risk assessment or new measures) usually triggers a new premarket notification (510(k)) for most of the devices which get onto the market in the us. thus, such situations require an fda review for clearance of the device. for samd, this requires to include an analytical evaluation, i.e. correct processing of input data to generate accurate, reliable, and precise output data. additionally, a clinical validation as well as the demonstration of a valid clinical association need to be provided. [18] this is intended to show that the outputs of the device appropriately work in the clinical environment, i.e. have a valid association regarding the targeted clinical condition and achieve the intended purpose in the context of clinical care. [18] thus, based on the current standards, a device with continuously changing performance usually requires a thorough analysis regarding its validity. this is one of the main points, where [13] proposes to establish a new approach for the "cont-learn" cases. as already mentioned, sps and acp basically have to be considered as tools for automated validation in this context. within this new approach, the manual validation step is replaced by an automated process with only reduced or even no additional control by a human observer. thus, it may work as an automated of fully automatic, closed loop validation approach. the question is whether this change can be considered as an appropriate alternative. in the following, this question is addressed using the icu scenario with a main focus on the "cont-learn" case. some of the aspects also apply to the "locked" cases. but the impact is considered to be higher in the "cont-learn" situation, since the validation step has to be performed in an automated fashion. human oversight, which is usually considered important, is not included here during the particular updates. within the icu scenario, the validation step has to ensure that the alarm rates stay on a sufficiently high level, regarding standard factors like specificity, sensitivity, area under curve (auc), etc. basically, these are technical parameters which can be analyzed according to an analytical evaluation as discussed above. (see also [18] ) this could also be applied to situations, where continuous updates are made during the lifecycle of the device, i.e. in the "cont-learn". however, there are some limitations of the approach. on the one hand, it has to be ensured, that this analysis is sound and reliable, i.e. it is not compromised according to statistical effects like bias or other deficiencies in the data. on the other hand, it has to be ensured that the success rates really have a valid clinical association and can be used as a sole criterion for measuring the clinical impact. thus, the relationship between pure success rates and clinical effects has to be evaluated thoroughly and there may be some major limitations. one major question in the icu scenario is, whether better success rates really guarantee a higher or at least sufficient level of clinical benefit. this is not innately given. for example, a higher success rate of the alarms may still have a negative effect when the icu staff relies more and more on the alarms and subsequently reduces attention. thus, it may be the case that the initiation of appropriate treatment steps may be compromised even though the actually occurring alarms seem to be more reliable. in particular, this may apply in situations where the algorithms are adapted to local settings, like in the "cont-learn" scenario. here, the ml based system is intended to be optimized to subpopulations in the local environment or to specific treatment preferences at the local site. according to habituation effects, the staff's expectations get aligned to the algorithm's behavior to a certain degree after a period of time. but when the algorithm changes or an employee from another hospital or department takes over duties in the local unit, the reliability of the alarms may be affected. in these cases, it is not clear whether the expectations are well aligned with the current status of the algorithmeither in the positive or negative direction. since the data updates of the device are intended to improve its performance w.r.t. detection rates, it is clear that significant effects on user interaction may happen. under some circumstances, the overall outcome in terms of the clinical effect may be impaired. evaluation of such risks have to be addressed during validation. it is questionable whether this can be performed by using an automatic validation approach which focuses on alarm rates but does not include an assessment of the associated risks. at least a clear relationship between these two aspects has to be demonstrated in advance. it is also unclear, whether this could be achieved by assessment of pure technical parameters which are defined in advance as required by the sps and acp. usually, ml based systems are trained to a specific scenario. they provide a specific solution for this particular problem. but they do not have a more general intelligence and reasoning about potential risks, which were not under consideration at that point of time. such a more general intelligence can only be provided when using human oversight. in general, it is not clear whether technical aspects like alarms lead to valid reactions by the users. in technical terms, alarm rates are basically related to the probability of occurrence of specific hazardous situations. but they do not address a full assessment of occurrence of harm. however, this is pivotal for risk assessment in medical devices, in particular for risks related to potential use errors. this is considered to be one of the main reasons why a change in risk parameters triggers a new premarket approval in the us according to [17] . also, the mdr [1] sets high requirements to address the final clinical impact and not only technical parameters. basically, the example emphasizes the importance to consider the interaction between man and machine, or in this case, the algorithm and its clinical environment. this is addressed in the usability standards for medical devices, e.g. iso 62366 [19] . for this reason, the iso 62366 requires that the final (summative) usability evaluation is performed using the final version of the device (in this case, the algorithm) or an equivalent version. this is in conflict with the fda proposal which allows to perform this assessment based on previous versions. at most, a predetermined relationship between technical parameters (alarm rates) and clinical effects (in particular, use related risks) can be obtained. for usage of ml based devices, it remains crucial to consider the interaction between the device and the clinical environment as there usually are important interrelationships. the outcome of an ml based algorithm always depends on the data it gets provided. whenever an input parameter is omitted, which is clinically relevant, the resulting outcome of the ml based system is limited. in the presented scenarios, the pure alarm rates may not be the only clinically relevant outcomes. even though, such parameters are usually the main focus regarding the quality of algorithms, e.g. in publications about ml based techniques. this is due to the fact, that such quality measures are commonly considered the best available objective parameters, which allow a comparison of different techniques. this even more applies to other ml based techniques which are also very popular in the scientific community, like segmentation tasks in medical image analysis. here the standard quality measures are general distance metrics, i.e. differences between segmented areas. [20] they usually do not include specific clinical aspects like the accuracy in specific risk areas, e.g. important blood vessels or nerves. but such aspects are key factors to ensure the safety of a clinical procedure in many applications. again, only technical parameters are typically in focus. the association to the clinical effects is not assessed accordingly. this situation is depicted in fig. 2 for the icu as well as image segmentation cases. additionally, the validity of an outcome in medical treatments depends on many factors. regarding input data, multiple parameters from a patient's individual history may be important for deciding about a particular diagnosis or treatment. a surgeon usually has access to a multitude of data and also side conditions (like socio-economic aspects) which should be included in an individual diagnosis or treatment decision. his general intelligence and background knowledge allow him to include a variety of individual aspects, which have to be considered for a specific case-based decision. in contrary, ml based algorithms rely on a more standardized structure of input data and are only trained for a specific purpose. they lack a more general intelligence, which allows them to react in very specific situations. even more, ml based algorithms need to generalize and thus to mask out very specific conditions, which could by fatal in some cases. in [13] , the fda presents some examples where changes of the inputs in an ml based samd are included. it is surprising, that the fda considers some of them as candidates for a continuous learning system, which does not need an additional review, when a tailored sps/acp is available. such discrepancies between technical outcomes and clinical effects also apply to situations like the icu scenario, which only informs or drives clinical management. often users rely on automatically provided decisions, even when they are informed that this only is a proposal. again, this is a matter of man-machine interaction. this gets even worse due to the lack of explainability which ml based algorithms typically have. [9, 21] when surgeons or more general users (e.g, icu staff) detect situations which require a diverging treatment because of very specific individual conditions, they should overrule the algorithm. but users will often be confused by the outcome of the algorithm and do not have a clear idea how they should treat conflicting results between the algorithm's suggestions and their own belief. as long as the ml based decision is not transparent to the user, they will not be able to merge these two directions. the ibm watson example, referenced in the introduction shows, that this actually is an issue [11] . this may be even more serious, when the users (i.e. healthcare professionals) fear litigation because they did not trust the algorithm. in a situation, where the algorithm's outcome finally turns out to be true, they may be sued because of this documented deviation. because of such issues, the eu general data protection regulation (gfpr) [22] requires that the users get autonomy regarding their decisions and transparency about the mechanisms underlying the algorithm's outcome. [23] this may be less relevant for the patients, who usually have only limited medical knowledge. they will probably also not understand the medical decisions in conventional cases. but it is highly relevant for responsible healthcare professionals. they require to get basic insights how the decision emerged, as they finally are in charge of the treatment. this demonstrates that methods regarding the explainability of ml based techniques are important. fortunately, this currently gets a very active field. [21, 24] this need for explainability applies to locked algorithms as well as situations where continuous learning is applied. based on their own data-driven nature, ml based techniques highly depend on a very high quality of data which are provided for learning and validation. in particular, this is important for the analytical evaluation of the ml algorithms. one of the major aspects are bias effects due to unbalanced input data. for example, in [25] a substantially different detection rate between white and colored people was recognized due to unbalanced data. beside ethical considerations, this demonstrates dependencies of the outcome quality on sub-populations, which may be critical in some cases. even though, the fda proposal [13] currently does not consequently include specific requirements for assessing bias factors or imbalance of data. however, high quality requirements for data management are crucial for ml based devices. in particular, this applies to the icu "cont-learn" cases. there have to be very specific protocols that guarantee that new data and updates of the algorithms are highly reliable w.r.t. bias effects. most of the currently used ml based algorithms fall under the category of supervised learning. thus, they require accurate and clinically sound labeling of the data. during the data collection, it has to be ensured how this labeling is performed and how the data can be fed back into the system in a "cont-learn" scenario. additionally, the data needs to stay balancedwhatever this means in a situation where adaptions to sub-populations and/or local environments are intended for optimization. it is unclear, whether and how this could be achieved by staff who is only operating with the system but possibly does not know potential algorithmic pitfalls. in the icu scenario, many data points probably need to be recorded by the system itself. thus, a precise and reliable recording scheme has to be established which automatically avoids imbalance of data on the one hand and fusion with manual labelings on the other hand. basically, the sps and acp (proposed in [13] ) are tools to achieve this. the question is whether this is possible in a reliable fashion using automated processes. a complete closed loop validation approach seems to be questionable, especially when the assessment of clinical impact has to be included. thus, the integration of humans including adequate healthcare professionals as well as ml/ai experts with sufficient statistical knowledge seems reasonable. at least, bias assessment steps should be included. as already mentioned, this is not addressed in [13] in a dedicated way. further on, the outcomes may be compromised by side effects in the data. it may be the case, that the main reason for a specific outcome of the algorithm is not a relevant clinical parameter but a specific data artifact, i.e. some confounding factor. in the icu case, it could be the case, that the icu staff reacts early to a potentially critical situation and e.g. gives specific medication in advance to prevent upcoming problems. the physiological reaction of the patient can then be visible in the data as some kind of artifact. during its learning phase, the algorithm may recognize the critical situation not based on a deeper clinical reason, but on detecting the physiological reaction pattern. this may cause serious problems as shown subsequently. in the presented scenario, the definition of clinical situation and the pattern can be deeply coupled by design, since the labeling of the data by the icu staff and the administration of the medication will probably be done in combination at the particular site. this may increase the probability of such effects. usually, confounding factors are hard to determine. even when they can be detected, they are hard to be communicated and managed in an appropriate way. how should healthcare professionals react, when they get such potentially misleading information (see discussion about liability). this further limits the explanatory power of ml based systems. when confounders are not detected, they may have unpredictable outcomes w.r.t. the clinical effects. for example, consider the following case. in the icu scenario, an ml based algorithm gets trained in a way that it basically detects the medication artifact as described above during the learning phase. in the next step, this algorithm is used in clinical practice and the icu staff relies on the outcome of the algorithm. then, on the one hand, the medication artifact is not visible unless the icu staff administers the medication. on the other hand, the algorithm does not recognize the pattern and thus does not provide an alarm. subsequently, the icu staff does no act appropriately to manage the critical situation. in particular, such confounders may be more likely in situations where a strong dependence between the outcome of the algorithm and the clinical treatment exists. further examples of such effects were discussed in [7] for icu scenarios. the occurrence of confounders may be a bit less probable in pure diagnostic cases without influence of the diagnostic task onto the generation of data. but even here, such confounding factors may occur. the discussion in [10] provided examples where confounders may occur in diagnostic cases e.g. because of rulers placed for measurements on radiographs. in most of the publications about ml based techniques, such side effects are not discussed (or only in a limited fashion). in many papers, the main focus is the technical evaluation and not the clinical environment and the interrelation between technical parameters and clinical effects. additional important aspects which are amply discussed in the context of ai/ml based systems are discrimination and fairness (see e.g. [10] ). in particular, the eu puts a high priority of their future ai/ml strategy on fairness requirements [9] . fairness is often closely related to bias effects. but it goes beyond to more general ethical questions, e.g. regarding the natural tendency of ml based systems to favor specific subgroups. for example, the icu scenario "cont-learn" is intended to optimize w.r.t. to specifics of sub-populations and local characteristics, i.e. it tries to make the outcome better for specific groups. based on such optimization, other groups (e.g. minorities, underrepresented groups) which are not well represented may be discriminated in some sense. this is not a statistical but a systematic effect. superiority of a medical device for a specific subgroup (e.g. gender, social environment, etc.) is not uncommon. for example, some diagnosis steps, implants, or treatments achieve deviating success rates when applied to women in comparison to men. this also applies to differences between adults and children. when assessing bias in clinical outcome in ml based devices, it will probably often be unclear whether this is due to imbalance of data or a true clinical difference between the groups. does an ml based algorithm has to adjust the treatment of a subgroup to a higher level, e.g. a better medication, to achieve comparable results, when the analysis recognized worse results for this subgroup? another example could be a situation where the particular group does not have the financial capabilities to afford the high-level treatment. this could e.g. be the case in a developing country or in subgroups with a lower insurance level. in these cases, the inclusion of socio-economical parameters into the analysis seems to be unavoidable. subsequently, this compromises the notion of fairness as basic principle in some way. this is nothing genuine to ml based devices. but in the case of ml based systems with a high degree of automation, the responsibility for the individual treatment decision more and more shifts from the health care professional to the device. it is implicitly defined in the ml algorithm. in comparison to human reasoning, which allows some weaknesses in terms of individual adjustments of general rules, ml based algorithms are rather deterministic / unique in their outcome. for a fixed input, they have one dedicated outcome (when we neglect statistical algorithms which may allow minor deviations). differences of opinions and room for individual decisions are main aspects of ethics. thus, it remains unclear how fairness can be defined and implemented at all when considering ml based systems. this is even more challenging as socioeconomical aspects (even more than clinical aspects) are usually not included in the data and analysis of ml based techniques in medicine. additionally, they are hard to assess and implement in a fair way, especially when using automated validation processes. another disadvantage of ml based devices is the limited opportunities to fix systematic deficiencies in the outcome of the algorithm. let us assume that during the lifetime of the icu monitoring system a systematic deviation of the intended outcome was detected, e.g. in the context of post-market surveillance or due to an increased number of serious adverse events. according to standard rules, a proper preventive respectively corrective action has to be taken by the manufacturer. in conventional software devices, the error simple should be eliminated, i.e. some sort of bug fixing has to be performed. for ml based devices it is less clear, how bug fixing should work especially when the systematic deficiency is deeply hidden in the data and/or ml model. in these cases, there usually is no clear reason for the deficiency. subsequently, the deficiency cannot be resolved in a straightforward way using standard bug fixing. there is no dedicated route to find the deeper reasons and to perform changes which could cure the deficiencies, e.g. by providing additional data or changing the ml model. even more, other side effects may easily occur, when data and model are changed manually by intent to fix the issue. 4 discussion and outlook in summary, there are many open questions, which are not yet clarified. there still is little experience how ml based systems work in clinical practice and which concrete risks may occur. thus, the fda's commitment to foster the discussion about ml based samd is necessary and appreciated by many stakeholders as the feedback docket [26] for [13] shows. however, it is a bit surprising that the fda proposes to substantially reduce its very high standards in [13] at this point of time. in particular, it is questionable whether an adequate validation can be achieved by using a fully automatic approach as proposed in [13] . ml based devices are usually optimized according to very specific goals. they can only account for the specific conditions that are reflected in the data and the used optimization / quality criteria. they do not include side conditions and a more general reasoning about potential risks in a complex environment. but this is important for medical devices. for this reason, a more deliberate path would be suited, from the author's perspective. in a first step, more experience should be gained w.r.t. to the use of ml based devices in clinical practice. thus, continuous learning should not be a first hand option. first, it should be demonstrated that a device works in clinical practice before a continuous learning approach should be possible. this could also be justified from a regulatory point-of-view. the automated validation process itself should be considered as a feature of the device. it should be considered as part of the design transfer which enables safe use of the device during its lifecycle. as part of the design transfer, it should be validated itself. thus, it has to be demonstrated that this automated validation process, e.g. in terms of the sps and acp, works in a real clinical environment. ideally, this would have been demonstrated during the application of the device in clinical practice. thus, one reasonable approach for a regulatory strategy could be to reduce or prohibit the options for enabling automatic validation in a first release / clearance of the device. during the lifetime, direct clinical data could be acquired to demonstrate a better insight into the reliability and limitations of the automatic validation / continuous learning approach. in particular, the relation between technical parameters and clinical effects could be assessed on a broader and more stable basis. based on this evidence in real clinical environments, the automated validation feature could then be cleared in a second round. otherwise, the validity of the automated validation approach would have to be demonstrated in a comprehensive setting during the development phase. in principle, this is possible when enough data is available which truly reflects a comprehensive set of situations. as discussed in this paper, there are many aspects which do not render this approach impossible but very challenging. in particular, this applies to the clinical effects and the interdependency between the users and clinical environment on the one hand and the device, including the ml algorithm, data management, etc., on the other hand. this also includes not only variation in the status and needs of the individual patient but also the local clinical environment and potentially also the socioeconomic setting. following a consequent process validation approach, it would have to be demonstrated that the algorithm reacts in a valid and predictable way no matter which training data have been provided, which environment have to be addressed, and which local adjustments have been applied. this also needs to include deficient data and inputs in some way. in [20] , it has been shown that the variation of outcomes can be substantial, even w.r.t. rather simple technical parameters. in [20] , this was analyzed for scientific contests ("challenges") where renowned scientific groups supervised the quality of the submitted ml algorithms. this demonstrates the challenges validation steps for ml based systems still include, even w.r.t. technical evaluation. for these reasons, it seems adequate to pursue the regulatory strategy in a more deliberate way. this includes the restriction of the "cont-learn" cases as proposed. this also includes a better classification scheme, where automated or fully automatic validation is possible. currently, the proposal in [13] does not provide clear rules when continuous learning is allowed. it does not really address a dedicated risk-based approach that defines which options and limitations are applicable. for some options, like the change of the inputs, it should be reviewed, whether automatic validation is a natural option. additionally, the dependency between technical parameters and clinical effects as well as risks should get more attention. in particular, the grade of interrelationship between the clinical actions and the learning task should be considered. in general, the discussions about ml based medical devices are very important. these techniques provide valuable opportunities for improvements in fields like medical technologies, where evidence based on high quality data is crucial. this applies to the overall development of medicine as well as to the development of sophisticated ml based medical devices. this also includes the assessment of treatment options and success of particular devices during their lifetime. data-driven strategies will be important for ensuring high-level standards in the future. they may also strengthen regulatory oversight in the long term by amplifying the necessity of post-market activities. this seems to be one of the promises the fda envisions according to their concepts of "total product lifecycle quality (tplc)" and "organizational excellence" [13] . also, the mdr strengthens the requirements for data-driven strategies in the pre-as well as postmarket phase. but it should not shift the priorities for a basically proven-quality-in-advance (exante) to a primarily ex-post regulation, which boils down to a trial-and-error oriented approach in the extreme. thus, we should aim at a good compromise between pushing these valuable and innovative options on the one hand and potential challenges and deficiencies on the other hand. computer-assisted technologies in medical interventions are intended to support the surgeon during treatment and improve the outcome for the patient. one possibility is to augment reality with additional information that would otherwise not be perceptible to the surgeon. in medical applications, it is particularly important that demanding spatial and temporal conditions are adhered to. challenges in augmenting the operating room are the correct placement of holograms in the real world, and thus, the precise registration of multiple coordinate frames to each other, the exact scaling of holograms, and the performance capacity of processing and rendering systems. in general, two different scenarios can be distinguished. first, applications exist, in which a placement of holograms with an accuracy of 1 cm and above are sufficient. these are mainly applications where a person needs a three-dimensional view of data. an example in the medical field may be the visualization of patient data, e.g. to understand and analyse the anatomy of a patient, for diagnosis or surgical planning. the correct visualization of these data can be of great benefit to the surgeon. often only 2d patient data is available, such as ct or mri scans. the availability of 3d representations depend strongly on the field of application. in neurosurgery 3d views are available but often not extensively utilized due to their limited informative value. additionally computer monitors are a big limitation, because the data can not be visualized in real world scale. further application areas are the translation of known user interfaces into augmented ur-ai 2020 // 67 reality (ar) space. the benefit here is that a surgeon refrains from touching anything, but can interact with the interface in space using hand or voice gestures. applications visualizing patient data, such as ct scans, only require a rough positioning of the image or holograms in the operation room (or). thus, the surgeon can conveniently place the application freely in space. the main requirement is then to keep the holograms in a constant position. therefore, the internal tracking of the ar device is sufficient to hold the holograms at a fixed position in space. the second scenario covers all applications, in which an exact registration of holograms to the real world is required, in particular with a precision below 1 cm. these scenarios are more demanding, especially when holograms must be placed precisely over real patient anatomy. to achieve this, patient tracking is essential to determine position and to follow patient movements. the system therefore needs to track the patient and adjust the visualization to the current situation. furthermore, it is necessary to track and augment surgical instruments and other objects in the operating room. the augmentation needs to be visualized at the correct spatial position and time constraints need to be fulfilled. therefore, the ar system needs to be embedded into the surgical workflow and react to it. to achieve these goals modern state of the art machine learning algorithms are required. however, the computing power on available ar devices is often not yet sufficient for sophisticated machine learning algorithms. one way to overcome this shortcoming is the integration of the ar system into a distributed system with higher capabilities, such as the digital operating theatre op:sense (see fig. 2 ). in this work an augmented reality system holomed [4] (see fig. 1 ) is integrated into the surgical research platform for robot assisted surgery op:sense [5] . the objective is to enable high-quality and patient-safe neurosurgical procedures in order to increase the surgical outcome by providing surgeons with an assistance system that supports them in cognitively demanding operations. the physician's perception limits are extended by the ar system, which bases on supporting intelligent machine learning algorithms. ar glasses allow the neurosurgeon to perceive the internal structures of the patient's brain. the complete system is demonstrated by applying this methodology to the ventricular puncture of the human brain, one of the most frequently performed procedures in neurosurgery. the ventricle system has an elongated shape with a width of 1-2 cm and is located in a depth of 4 cm inside the human head. patient models are generated fast (< 2s) from ct-data [3] , which are superimposed over the patient during operation and serve as a navigation aid for the surgeon. in this work the expanded system architecture is presented to overcome some limitations of the original system where all information were processed on the microsoft hololens, which lead to performance deficits. to overcome these shortcomings the holomed project was integrated into op:sense for additional sensing and computing power. to achieve integration of ar into the operation room and the surgical workflows, the patient, the instruments and the medical staff need to be tracked. to track the patient, a marker system is fixated on the patient head and registration from the marker system to the patient is determined. a two-stage process was implemented for this purpose. first the rough position of the patient's head is determined on the or table by applying a yolo v3 net to reduce the search space. then a robot with a mounted rgb-d sensor is used to scan the acquired area and build a point cloud of the same. to determine the patient's head in space as precisely as possible a two-step surface matching approach is utilized. during recording, the markers are also tracked. with known position of the patient and the markers, the registration matrix can be calculated. for the ventricular puncture a solution is proposed to track the puncture catheter to determine the depth of insertion into the human brain. by tracking the medical staff the system is able to react to the current situation, e.g. if an instrument is passed. in the following the solutions are described in detail. our digital operating room op:sense (illustrated in fig. 2a) to detect the patient's head, the coarse position is first determined with the yolo v3 cnn [6] , performed on the kinect rgb image streams. the position in 3d is determined through the depth stream of the sensors. the or table and the robots are tracked with retroreflective markers by the arttrack system. this step reduces the spatial search area for fine adjustment. the franka panda has an attached intel realsense rgb-d camera as depicted in fig. 3 . the precise determination of the position is performed on the depth data with surface matching. the robot scans the area of the coarsely determined position of the patient's head. a combined surface matching approach with feature-based and icp matching was implemented. the process to perform the surface matching is depicted in fig. 4 . in clinical reality, a ct scan of the patient head is always performed prior to a ventricular puncture for diagnosis, such that we can safely assume the availability of ct data. a process to segment the patient models from ct data was proposed by kunz et al. in [3] . the algorithm processes the ct data extremely fast in under two seconds. the data format is '.nrrd', a volume model format, which can easily be converted into surface models or point clouds. the point cloud of the patient's head ct scan is the reference model that needs to be found in or space. the second point cloud is recorded from the realsense depth stream mounted on the panda robot by scanning the previously determined rough position of the patient head. all points are recorded in world coordinate space. the search space is further restricted with a segmentation step by filtering out points that are located on the or table. additionally, manual changes can be made by the surgeon. in a performance optimization, the resolution of the point clouds is reduced to decrease processing time without loosing too much accuracy. the normals of both point clouds generated from ct data and from the recorded realsense depth stream are subsequently calculated and harmonised. during this step, the harmonisation is especially important as the normals are often misaligned. this misalignment occurs because the ct data is a combination of several individual scans. for alignment of all normals, a point inside the patient's head is chosen manually as a reference point, followed by orienting all normals in the direction of this point and subsequently inverting all normals to the outside of the head (see fig. 5 ). after the preprocessing steps, the first surface fitting step is executed. it is based on the initial alignment algorithm proposed by rusu et al. [8] . an implementation within the point cloud library (pcl) is used. therefore fast point feature histograms need to be calculated as a preprocessing step. in the last step an iterative closest point (icp) algorithm is used to refine the surface matching result. after the two point clouds have been aligned to each other the inverse transformation matrix can be calculated to get the correct transformation from marker system to patient model coordinate space. as outlined in fig. 6 , catheter tracking was implemented based on semantic segmentation using a full-resolution residual network (frrn) [7] . after the semantic segmentation of the rgb stream of the kinect cameras, the image is fused with the depth stream ur-ai 2020 // 71 to determine the voxels in the point cloud belonging to the catheter. as a further step a density based clustering approach [2] is performed on the chosen voxels. this is due to noise especially on the edges of the instrument voxels in the point cloud. based on the found clusters an estimation of the three dimensional structure of the catheter is performed. for this purpose, a narrow cylinder with variable length is constructed. the length is changed accordingly to the semantic segmentation and the clustered voxels of the point cloud. the approach is applicable to identify a variety of instruments. the openpose [1] library is used to track key points on the bodies of the medical staff. available ros nodes have been modified to integrate openpose in the op:sense ros environment. the architecture is outlined in fig. 7 . in this chapter the results of the patient, catheter and medical staff tracking are described. the approach to find the coarse position of a patient's head was performed on a phantom head placed on the or table within op:sense. multiple scenarios with changing illumination and occlusion conditions were recorded. the results are depicted in fig. 8 and the evaluation results are depicted in table 1 . precision detection of the patient was performed with a two-stage surface matching approach. different point cloud resolutions were tested with regard to runtime behaviour. voxel grid edge sizes of 6, 4 and 3 mm have been tested, with a higher edge size corresponding to a smaller point cloud. the matching results of the two point clouds were analyzed manually. an average accuracy of 4.7 mm was found with an accuracy range between 3.0 and 7.0 mm. in the first stage of the surface matching, the two point clouds are coarsely aligned as depicted in fig. 9 . in the second step icp is used for fine adjustment. a two-stage process was implemented as icp requires a good initial alignment of the two point clouds. ur-ai 2020 // 73 for catheter tracking a precision of the semantic segmentation between 47% and 84% is reached (see table 3 ). tracking of instruments, especially neurosurgical catheters, are challenging due to their thin structure and non-rigid shape. detailed results on catheter tracking have been presented in [7] . the 3d estimation of the catheter is shown in fig. 10 . the catheter was moved in front of the camera and the 3d reconstruction was recorded simultaneously. over a long period of the recording over 90% of the catheter are tracked correctly. in some situations this drops to under 50% or lower. the tracking of medical personnel is shown in fig. 11 . the different body parts and joint positions are determined, e.g. the head, eyes, shoulders, elbows, etc. the library yielded very good results as described in [1] . we reached a performance of 21 frames per second on a workstation (intel i7-9700k, geforce 1080 ti) processing 1 stream. fig. 11 . results of the medical staff tracking. ur-ai 2020 // 75 4 discussion as shown in the evaluation, our approach succeeds in detecting the patient in an automated two-stage process with an accuracy between 3 and 7 mm. the coarse position is determined by using a yolo v3 net. the results under normal or conditions are very satisfying. the solution performance drops strongly under bright illumination conditions. this is due to large flares that occur on the phantom as it is made of plastic or silicone. however, these effects do not occur on human skin. the advantage of our system is that the detection is performed on all four kinect rgb streams enable different views on the operation area. unfavourable illumination conditions normally don't occur on all of these streams. therefore a robust detection is still possible. in the future the datasets will be expanded with samples with strong illumination conditions. the following surface matching of the head yields good results and a robust and precise detection of the patient. most important is a good preprocessing of the ct data and the recorded point cloud of the search area, as described in the methods. the algorithm does not manage to find a result if there are larger holes in the point clouds or if the normals are not calculated correctly. additionally, challenges that have to be considered include skin deformities and noisy ct data. the silicone skin is not fixed to the skull (as human skin is), which leads to changes in position, some of which are greater than 1 cm. also the processing time of 7 minutes is quite long and must be optimized in the future. the processing time may be shortened by reducing the size of the point clouds. however, in this case the matching results may also become worse. catheter tracking [7] yielded good results, despite the challenging task of segmenting a very thin ( 2.5 mm) and deformable object. additionally, a 3d estimation of the catheter was implemented. the results showed that in many cases over 90% of the catheter can be estimated correctly. however, these results strongly depend on the orientation and the quality of the depth stream. using higher quality sensors could improve the detection results. for tracking of the medical staff openpose as a ready-to-use people detection algorithm was used and integrated into ros. the library produces very good results, despite medical staff wearing surgical clothing. in this work the integration of augmented reality into the digital operating room op:sense is demonstrated. this makes it possible to expand the capabilities of current ar glasses. the system can determine the precise patient's position by implementing a two-stage process. first a yolo v3 net is used to coarsly detect the patient to reduce the search area. in a second subsequent step a two-stage surface matching process is implemented for refined detection. this approach allows for precise location of the patient's head for later tracking. further, a frnn-based solution to track the surgical instruments in the or was implemented and demonstrated on a thin neurosurgical catheter for ventricular punctures. additionally, openpose was integrated into the digital or to track the surgical personnel. the presented solution will enable the system to react to the current situation in the operating room and is the base for an integration into the surgical workflow. due to the emergence of commodity depth sensors many classical computer vision tasks are employed on networks of multiple depth sensors e.g. people detection [1] or full-body motion tracking [2] . existing methods approach these applications using a sequential processing pipeline where the depth estimation and inference are performed on each sensor separately and the information is fused in a post-processing step. in previous work [3] we introduce a scene-adaptive optimization schema, which aims to leverage the accumulated scene context to improve perception as well as post-processing vision algorithms (see fig. 1 ). in this work we present a proof-of-concept implementation of the scene-adaptive optimization methods proposed in [3] for the specific task of stereomatching in a depth sensor network. we propose to improve the 3d data acquisition step with the help of an articulated shape model, which is fitted to the acquired depth data. in particular, we use the known camera calibration and the estimated 3d shape model to resolve disparity ambiguities that arise from repeating patterns in a stereo image pair. the applicability of our approach can be shown by preliminary qualitative results. in previous work [3] we introduce a general framework for scene-adaptive optimization of depth sensor networks. it is suggested to exploit inferred scene context by the sensor network to improve the perception and post-processing algorithms themselves. in this work we apply the proposed ideas in [3] to the process of stereo disparity estimation, also referred to as stereo matching. while stereo matching has been studied for decades in the computer vision literature [4, 5] it is still a challenging problem and an active area of research. stereo matching approaches can be categorized into two main categories, local and global methods. while local methods, such as block matching [6] , obtain a disparity estimation by finding the best matching point on the corresponding scan line by comparing local image regions, global methods formulate the problem of disparity estimation as a global energy minimization problem [7] . local methods lead to highly efficient real-time capable algorithms, however, they suffer from local disparity ambiguities. in contrast, global approaches are able to resolve local ambiguities and therefore provide high-quality disparity estimations. but they are in general very time consuming and without further simplifications not suitable for real-time applications. the semi-global matching (sgm) introduced by hirschmuller [8] aggregates many feasible local 1d smoothness constraints to approximate global disparity smoothness regularization. sgm and its modifications are still offering a remarkable trade-off between the quality of the disparity estimation and the run-time performance. more recent work from poggi et al. [9] focuses on improving the stereo matching by taking additional high-quality sources (e.g. lidar) into account. they propose to leverage sparse reliable depth measurements to improve dense stereo matching. the sparse reliable depth measurements act as a prior to the dense disparity estimation. the proposed approach can be used to improve more recent end-to-end deep learning architectures [10, 11] , as well as classical stereo approaches like sgm. this work is inspired by [9] , however, our approach does not rely on an additional lidar sensor but leverages a priori scene knowledge in terms of an articulated shape model instead to improve the stereo matching process. we set up four stereo depth sensors with overlapping fields of view. the sensors are extrinsically calibrated in advance, thus their pose with respect to a world coordinates system is known. the stereo sensors are pointed at a mannequin and capture eight greyscale images (one image pair for each stereo sensor, the left image of each pair is depicted in fig. 3a) . for our experiments we use a high-quality laser scan of the mannequin as ground truth. we assume that the proposed algorithm has access to an existing shape model that can express the observed geometry of the scene in some capacity. in our experimental setup, we assume a shape model of a mannequin with two articulated shoulders and a slightly different shape in the belly area of the mannequin (see fig. 2 ). in the remaining section we use the provided shape model to improve the depth data generation of the sensor network. first, we estimate the disparity values of each of the four stereo sensors with sgm without using the human shape model. let p denote a pixel and q denote an adjacent pixel. let d denote a disparity map and d p ,d q denote the disparity at pixel location p and q. let p denote the set of all pixels and n the set of all adjacent pixels. then the sgm cost function can be defined as where d(p, d p ) denotes the matching term (here the sum of absolute differences in a 7 × 7 neighborhood) which assigns a matching cost to the assignment of disparity d p to pixel p and r(p, d p , q, d q ) penalizes disparity discontinuities between adjacent pixels p and q. in sgm the objective given in (1) is minimized with dynamic programming, leading to the resulting disparity mapd = arg min d e(d). as input for the shape model fitting we apply sgm on all four stereo pairs leading to four disparity maps as depicted in fig. 4a . to be able to exploit the articulated shape model for stereo matching we initial need to fit the model to the 3d data obtained by classical sgm as described in 3.2. to be more robust to outliers we do only use disparity values from pixels with high contrast and transform them into 3d point clouds. since we assume that the relative camera poses are known, it is straight forward to merge the resulting point clouds in one world coordinate system. finally the shape model is fitted to the merged point cloud by optimizing over the shape model parameters, namely the pose of the model and the rotation of the shoulder joints. we use an articulated mannequin shape model in this work as a proxy for an articulated human shape model (e.g. [2] ) as proof-of-concept and plan to transfer the proposed approach on real humans in future work. once the model parameters of the shape model are obtained we can reproject the model fit to each sensor view by making use of the known projection matrices. fig. 3b shows the rendered wireframe mesh of the fitted model as an overlay on the camera images. for our guided stereo matching approach we then need the synthetic disparity map which can be computed from the synthetic depth maps (a byproduct of 3d rendering). we denote the synthetic disparity image by d synth . one synthetic disparity image is created for each stereo sensor, see fig. 4b . in the final step we exploit the existing shape model fit, in particular the synthetic disparity image d synth of each stereo sensor and combine it with sgm (inspired by guided stereo matching [9] ). our augmented objective is defined as with the introduced objective is very similar to sgm and can be minimized in a similar fashion leading to the final disparity estimation in our scene-adaptive depth sensor network to summarize our approach, we exploit an articulated shape model fit to enhance sgm with minor adjustments. to show the applicability of our approach we present preliminary qualitative results. the results are depicted in fig. 4 . using sgm without exploiting the provided articulated shape model leads to reasonable results, but the disparity map is very noisy and no clean silhouette of the mannequin is extracted (see fig. 4a ). fitting our articulated shape model to the data leads to clean synthetic disparity maps as shown in fig. 4c , with a clean silhouette. in the belly area the synthetic model disparity map (fig. 4b) does not agree with the ground truth (fig. 4d) . the articulated shape model is not general enough to explain the recorded scene faithfully. using the guided stereo matching approach, we construct a much cleaner disparity map than sgm. in addition, the approach takes the current sensor data into account and exploits an existing articulated shape model. in this work we have proposed a method for scene-adaptive disparity estimation in depth sensor networks. our main contribution is the exploitation of a fitted human shape model to make the estimation of disparities more robust to local ambiguities. our early results indicate that our method can lead to more robust and accurate results compared to classical sgm. future work will focus on a quantitative evaluation as well as incorporating sophisticated statistical human shape models into our approach. inverse process-structure-property mapping abstract. workpieces for dedicated purposes must be composed of materials which have certain properties. the latter are determined by the compositional structure of the material. in this paper, we present the scientific approach of our current dfg funded project tailored material properties through microstructural optimization: machine learning methods for the modeling and inversion of structure-property relationships and their application to sheet metals. the project proposes a methodology to automatically find an optimal sequence of processing steps which produce a material structure that bears the desired properties. the overall task is split in two steps: first find a mapping which delivers a set of structures with given properties and second, find an optimal process path to reach one of these structures with least effort. the first step is achieved by machine learning the generalized mapping of structures to properties in a supervised fashion, and then inverting this relation with methods delivering a set of goal structure solutions. the second step is performed via reinforcement learning of optimal paths by finding the processing sequence which leads to the best reachable goal structure. the paper considers steel processing as an example, where the microstructure is represented by orientation density functions and elastic and plastic material target properties are considered. the paper shows the inversion of the learned structure-property mapping by means of genetic algorithms. the search for structures is thereby regularized by a loss term representing the deviation from process-feasible structures. it is shown how reinforcement learning is used to find deformation action sequences in order to reach the given goal structures, which finally lead to the required properties. keywords: computational materials science, property-structure-mapping, texture evolution optimization, machine learning, reinforcement learning the derivation of processing control actions to produce materials with certain, desired properties is the "inverse problem" of the causal chain "process control" -"microstructure instantiation" -"material properties". the main goal of our current project is the creation of a new basis for the solution of this problem by using modern approaches from machine learning and optimization. the inversion will be composed of two explicitly separated parts: "inverse structure-property-mapping" (spm) and "microstructure evolution optimization". the focus of the project lies on the investigation and development of methods which allow an inversion of the structure-property-relations of materials relevant in the industry. this inversion is the basis for the design of microstructures and for the optimal control of the related production processes. another goal is the development of optimal control methods yielding exactly those structures which have the desired properties. the developed methods will be applied to sheet metals within the frame of the project as a proof of concept. the goals include the development of methods for inverting technologically relevant "structure-property-mappings" and methods for efficient microstructure representation by supervised and unsupervised machine learning. adaptive processing path-optimization methods, based on reinforcement learning, will be developed for adaptive optimal control of manufacturing processes. we expect that the results of the project will lead to an increasing insight into technologically relevant process-structure-property-relationships of materials. the instruments resulting from the project will also promote the economically efficient development of new materials and process controls. in general, approaches to microstructure design make high demands on the mathematical description of microstructures, on the selection and presentation of suitable features, and on the determination of structure-property relationships. for example, the increasingly advanced methods in these areas enable microstructure sensitive design (msd), which is introduced in [1] and [2] and described in detail in [3] . the relationship between structures and properties descriptors can be abstracted from the concrete data by regression in the form of a structure-property-mapping. the idea of modeling a structure-property-mapping by means of regression and in particular using artificial neural networks was intensively pursued in the 1990s [4] and is still used today. the approach and related methods presented in [5] always consist of a structure-property-mapping and an optimizer (in [5] genetic algorithms) whose objective function represents the desired properties. the inversion of the spm can be alternatively reached via generative models. in contrast to discriminative models (e.g. spm), which are used to map conditional dependencies between data (e.g. classification or regression), generative models map the composite probabilities of the variables and can thus be used to generate new data from the assumed population. established, generative methods are for example mixture models [6] , hidden markov models [7] and in the field of artificial neural networks restricted boltzmann machines [8] . in the field of deep learning, generative models, in particular generative adversarial networks [9] , are currently being researched and successfully applied in the context of image processing. conditional generative models can generalize the probability of occurrence of structural features under given material properties. in this way, if desired, any number of microstructures could be generated. based on the work on the spm, the process path optimization in the context of the msd is treated depending on the material properties. for this purpose, the process is regarded as a sequence of structure-changing process operations which correspond to elementary processing steps. shaffer et al. [10] construct a so called texture evolution network based on process simulation samples, to represent the process. the texture evolution network can be considered as a graph with structures as vertices, connected by elementary processing steps as edges. the structure vertices are points in the structure-space and are mapped to the property-space by using the spm for property driven process path optimization. in [11] one-step deformation processes are optimized to reach the most reachable element of a texture-set from the inverse spm. processes are represented by so called process planes, principal component analysis (pca) projections of microstructures reachable by the process. the optimization then is conducted by searching for the process plane which best represents one of the texture-set elements. in [12] , a generic ontology based semantic system for processing path hypothesis generation (matcalo) is proposed and showcased. the required mapping of the structures to the properties is modeled based on data from simulations. the simulations are based on taylor models. the structures are represented using textures in the form of orientation density functions (odf), from which the properties are calculated. in the investigations, elastic and plastic properties are considered in particular. structural features are extracted from the odf for a more compact description. the project uses spectral methods such as generalized spherical harmonics (gsh) to approximate the odf. as an alternative representation we investigate the discretization in the orientation-space, where the orientation density is represented by a histogram. the solution of the inverse problem consists of a structure-property-mapping and an optimizer: as [4] described, the spm is modeled by regression using artificial neural networks. in this investigation, we use a multilayer perceptron. differential evolution (de) is used for the optimization problem. de is an evolutionary algorithm developed by rainer storn and kenneth price [13] . it is a optimization method, which repeatedly improves a candidate solution set under consideration of a given quality measure over a continuous domain. the de algorithm optimizes a problem by taking a population of candidate solutions and generating new candidate solutions (structures) by mutation and recombination existing ones. the candidate solution with the best fitness is considered for further processing. so, for the generated structures the reached properties are determined using the spm. the fitness f is composed of two terms: the property loss l p , which expresses, how close the property of a candidate is to the target property, and the structure loss l s , which represents the degree of feasibility of the candidate structure in the process the property loss is the mean squared error (mse) between the reached properties p r ∈ p r and the desired properties p d ∈ p d : considering the goal that the genetic algorithm generates reachable structures, a neural network is formed which functions as an anomaly detector. the data basis of this neural network are structures that can be reached by a process. the goal of anomaly detection is to exclude unreachable structures. the anomaly detection is implemented using an autoencoder [14] . this is a neural network (see fig. 1 ) which consists of the following two parts: the encoder and the decoder. the encoder converts the input data to an embedding space. the decoder converts the embedding space as close as possible to the original data. due to the reduction to an embedding space, the autoencoder uses data compression and extracts relevant features. the cost function for the structures is a distance function in the odf-space, which penalizes the network if it produces outputs that differ from the input. the cost function is also known as the reconstruction loss: with s i ∈ s as the original structures,ŝ i ∈ˆ s as the reconstructed structures and λ = 0.001 to avoid division by zero. when using the anomaly detection, the autoencoder determines a high reconstruction loss if the input data are structures that are very different from the reachable structures. the overall approach is shown in fig. 2 and consists of the following steps: 1. the genetic algorithm generates structures. 2. the spm determines the reached properties of the generated structures. 3. the structure loss l s is determined by the reconstruction loss of the anomaly detector for the generated structures with respect to the reachable structures. 4. the property loss l p is determined by the mse of the reached properties and the desired properties. 5. the fitness is calculated as the sum of the structure loss l s and the property loss l p . the structures, resulting from the described approach form the basis for optimal process control. due to the forward mapping, the process evolution optimization based on texture evolution networks ( [10] ) is restricted to a-priori sampled process paths. [11] relies on linearization assumptions and is applicable to short process sequences only. [12] relies on a-priori learned process models in the form of regression trees and is also applicable to relatively short process sequences only. ur-ai 2020 // 88 as an adaptive alternative for texture evolution optimization, that can be trained to find process-paths of arbitrary length, we propose methods from reinforcement learning. for desired material properties p d . the inverted spm determines a set of goal microstructures s d ∈ g, which are very likely reachable by the considered deformation process. the texture evolution optimization objective is then to find the shortest process path p * starting from a given structure s 0 , and leading close to one of the structures from g. where p = (a k ) k=0,...,k ; k t is a path of process actions a, t is the maximum allowed process length. the mapping e(s, p) = s k delivers the resulting structure, when applying p to the structure s. here, for the sake of simplicity, we assume the process to be deterministic, although the reinforcement learning methods we use are not restricted to deterministic processes. g τ is a neighbourhood of g, the union of all open balls with radius τ and center points from g. to solve the optimization problem by reinforcement learning approaches, it must be reformulated as markov decision process (mdp), which is defined by the tuple (s, a, p, r). in our case s is the space of structures s, a is the parameter-space of the deformation process, containing process actions a, p : s × a → s is the transition function of the deformation process, which we assume to be deterministic. r g : s × a → r is a goalspecific reward function. the objective of the reinforcement learning agent is then to find the optimal goal-specific policy π * g (s t ) = a t that maximizes the discounted future goal-specific reward where γ ∈ [0, 1] discounts early attained rewards, the policy π g (s k ) determines a k and the transition function p (s k , a k ) determines s k+1 . for a distance function d in the structure space, the binary reward function r g (s, a) = 1, if d(p (s, a), g) < τ 0, otherwise (6) if maximized, leads to an optimal policy π * g that yields the shortest path to g from every s for γ < 1. moreover, if v g is given for every microstructure from g, p from eq. 4 is identical with the application of the policy π * ζ , where ζ = arg max g [v g ]. π * g can be approached by methods from reinforcement learning. value-based reinforcement learning is doing so by learning expected discounted future reward functions [15] . one of these functions is the so called value-function v . in the case of a deterministic mdp and for a given g, this expectation value function reduces to v g from eq. 4 and ζ can be extracted if v is learned for every g from g. for doing so, a generalized form of expectation value functions can be learned as it is done e.g. in [16] . this exemplary mdp formulation shows how reinforcement learning can be used for texture evolution optimization tasks. the optimization thereby is operating in the space of microstructures and does not rely on a-priori microstructure samples. when using off-policy reinforcement learning algorithms and due to the generalization over goal-microstructures, the functions learned while solving a specific optimization task can be easily transferred to new optimization tasks (i.e. different desired properties or even a different property space). industrial robots are mainly deployed in large-scale production, especially in the automotive industry. today, there are already 26.1 industrial robots deployed per 1,000 employees on average in these industry branches. in contrast, small and medium-sized enterprises (smes) only use 0.6 robots per 1,000 employees [1] . reasons for this low usage of industrial robots in smes include the lack of flexibility with great variance of products and the high investment expenses due to additional peripherals required, such as gripping or sensor technology. the robot as an incomplete machine accounts for a fourth of the total investment costs [2] . due to the constantly growing demand of individualized products, robot systems have to be adapted to new production processes and flows [3] . this development requires the flexibilization of robot systems and the associated frequent programming of new processes and applications as well as the adaption of existing ones. robot programming usually requires specialists who can adapt flexibly to different types of programming for the most diverse robots and can follow the latest innovations. in contrast to many large companies, smes often have no in-house expertise and a lack of prior knowledge with regard to robotics. this often has to be obtained externally via system integrators, which, due to high costs, is one of the reasons for the inhibited use of robot systems. during the initial generation or extensive adaption of process flows with industrial robots, there is a constant risk of injuring persons and damaging the expensive hardware components. therefore, the programs have to be tested under strict safety precautions and usually in a very slow test mode. this makes the programming of new processes very complex and therefore time-and cost-intensive. the concept presented in this paper combines intuitive, gesture-based programming with simulation of robot movements. using a mixed reality solution, it is possible to create a simulation-based visualization of the robot and project, to program and to test it in the working environment without disturbing the workflow. a virtual control panel enables the user to adjust, save and generate a sequence of specific robot poses and gripper actions and to simulate the developed program. an interface to transfer the developed program to the robot controller and execute it by the real robot is provided. the paper is structured as follows. first, a research on related work is conducted in section 2, followed by a description of the system of the gesture-based control concept in section 3. the function of robot positioning and program creation is described in section 4. last follow the evaluation in section 5 and conclusion in section 6. various interfaces exist to program robots, such as lead-trough, offline or walk-trough programming, programming by demonstration, vision based programming or vocal commanding. in the survey of villani et al. [4] a clear overview on existing interfaces for robot programming and current research is provided. besides the named interfaces, the programming of robots using a virtual or mixed reality solution aims to provide intuitiveness, simplicity and accessibility of robot programming for non-experts. designed for this purpose, guhl et al. [5] developed a generic architecture for human-robot interaction based on virtual and mixed reality. in the marker tracking based approach presented by [6] and [7] , the user defines a collision-free-volume and generates and selects control points while the system creates and visualizes a path through the defined points. others [8] , [9] , [10] and [11] use handheld devices in combination with gesture control and motion tracking. herein, the robot can be controlled through gestures, pointing or via the device, while the path, workpieces or the robot itself are visualized on several displays. other gesture and virtual or mixed reality based concepts are developed by cousins et al. [12] or tran et al. [13] . here, the robots perspective or the robot in the working environment is presented to the user on a display (head-mounted or stationary) and the user controls the robot via gestures. further concepts using a mixed reality method enable an image of the workpiece to be imported into cad and the system automatically generates a path for robot movements [14] or visualizing the intended motion of the robot on the microsoft hololens, that the user knows where the robot will move to next [15] . other methods combine pointing at objects on an screen with speech instructions to control the robot [16] . sha et al. [17] also use a virtual control panel in their programming method, but for adjusting parameters and not for controlling robots. another approach pursues programming based on cognition, spatial augmented reality and multimodal input and output [18] , where the user interacts with a touchable table. krupke et al. [19] developed a concept in which humans can control the robot by head orientation or by pointing, both combined with speech. the user is equipped with a head-mounted display presenting a virtual robot superimposed over the real robot. the user can determine pick and place position by specifying objects to be picked by head orientation or by pointing. the virtual robot then executes the potential pick movement and after the user confirms by voice command, the real robot performs the same movement. a similar concept based on gesture and speech is persued by quintero et al. [20] , whose method offers two different types of programming. on the one hand, the user can determine a pick and place position by head orientation and speech commands. the system automatically generates a path which is displayed to the user, can be manipulated by the user and is simulated by a virtual robot. on the other hand, it is possible to create a path on a surface by the user generating waypoints. ostanin and klimchik [21] introduced a concept to generate collision-free paths. the user is provided with virtual goal points that can be placed in the mixed reality environment and between which a path is automatically generated. by means of a virtual menu, the user can set process parameters such as speed, velocity etc.. additionally, it is possible to draw paths with a virtual device and the movement along the path is simulated by a virtual robot. differently to the concept described in this paper, only a pick and place task can be realized with the concepts of [19] and [20] . a differentiation between movements to positions and gripper commands as well as the movement to several positions in succession and the generation of a program structure are not supported by these concepts. another distinction is that the user only has the possibility to show certain objects to the robot, but not to move the robot to specific positions. in [19] a preview of the movement to be executed is provided, but the entire program (pick and place movements) is not simulated. in contrast to [21] , with the concept presented in this paper it is possible to integrate certain gripper commands into the program. with [21] programming method, the user can determine positions but exact axis angles or robot poses cannot be set. overall, the approach presented in this paper offers an intuitive, virtual user interface without the use of handheld devices (cf. [6] , [7] , [8] , [9] , [10] and [11] ) which allows the exact positions of the robot to be specified. compared to other methods, such as [12] , [13] , [14] , [15] or [16] , it is possible to create more complex program structures, which include the specification of robot poses and gripper positions, and to simulate the program in a mixed reality environment with a virtual robot. in this section the components of the mixed reality robot programming system are introduced and described. the system consists of multiple real and virtual interactive elements, whereby the virtual components are projected directly into the field of view using a mixed reality (mr) approach. compared to the real environment, which consists entirely of real objects and virtual reality (vr), which consists entirely of virtual objects and which overlays the real reality, in mr the real scene here is preserved and only supplemented by the virtual representations [22] . in order to interact in the different realities, head-mounted devices similar to glasses, screens or mobile devices are often used. figure 1 provides an overview of the systems components and their interaction. the system presented in this paper includes kukas collaborative, lightweight robot lbr iiwa 14 r820 combined with an equally collaborative gripper from zimmer as real components and a virtual robot model and a user interface as virtual components. the virtual components are presented on the microsoft hololens. for calculation and rendering the robot model and visualization of the user interface, the 3d-and physics-engine of the unity3d development framework is used. furthermore, for supplementary functions, components and for building additional mr interactable elements, the microsoft mixed reality toolkit (mrtk) is utilized. for spatial positioning of the virtual robot, marker tracking is used, a technique supported by the vuforia framework. in this use case, the image target is attached to the real robot's base, such that in mr the virtual robot superimposes the real robot. the program code is written in c . the robot is controlled and programmed via an intuitive and virtual user interface that can be manipulated using the so-called airtap gesture, a gesture provided by microsoft hololens. ur-ai 2020 // 95 to ensure that the virtual robot mirrors the motion sequences and poses of the real robot, the most exact representation of the real robot is employed. the virtual robot consists of a total of eight links, matching the base and the seven joints of iiwa 14 r820: the base frame, five joint modules, the central hand and the media flange. the eight links are connected together as a kinematic chain. the model is provided as open source files from [23] and [24] and is integrated into the unity3d project. the individual links are created as gameobjects in a hierarchy, with the base frame defining the top level and are limited similar to those of the real robot. the cad data of the deployed gripping system is also imported into unity3d and linked to the robot model. the canvas of the head-up displayer of the microsoft hololens is divided into two parts and rendered at a fixed distance in front of the user and on top of the scene. at the top left side of the screen the current joint angles (a1 to a7) are displayed and on the left side the current program is shown. this setting simplifies the interaction with the robot as the informations do not behave like other objects in the mr scene, but are attached to the head up display (hud) and move with the user's field of view. the user interface, which consists of multiple interactable components, is placed into the scene and is shown at the right side of the head-up display. at the beginning of the application the user interface is in "clear screen" mode, i.e. only the buttons "drag", "cartesian", "joints", "play" and "clear screen" and the joint angles at the top left of the screen are visible. for interaction with the robot, the user has to switch into a particular control mode by tapping the corresponding button. the user interface provides three different control modes for positioning the virtual robot: -drag mode, for rough positioning, -cartesian mode, for cartesian positioning and -joint mode, for the exact adjustment of each joint angle. figure 2 shows the interactable components that are visible and therefore controllable in the respective control modes. depending on the selected mode, different interactable components become visible in the user interface, with whom the virtual robot can be controlled. in addition to the control modes, the user interface offers further groups of interactable elements: -motion buttons, with which e.g. the speed of the robot movement can be adjusted or the robot movement can be started or stopped, -application buttons, to save or delete specific robot poses, for example, -gripper buttons, to adjust the gripper and -interface buttons, that enable communication with the real robot. this section focuses on the description of the usage of the presented approach. in addition to the description of the individual control modes, the procedure for creating a program is also described. as outlined in section 3.2, the user interface consists of three different control modes and four groups of further interactable components. through this concept, the virtual robot can be moved efficiently to certain positions with different movement modes, the gripper can be adjusted, the motion can be controlled and a sequence of positions can be chained. drag by gripping the tool of the virtual robot with the airtap gesture, the user can "drag" the robot to the required position. additionally, it is possible to rotate the position of the robot using both hands. this mode is particularly suitable for moving the robot very quickly to a certain position. cartesian this mode is used for the subsequent positioning of the robot tool with millimeter precision. the tool can be translated to the required positions using the cartesian coordinates x, y, z and the euler angles a, b, c. the user interface provides a separate slider for each of the six translation options.the tool of the robot moves analogously to the respective slider button, which the user can set to the required value. joints this mode is an alternative to the cartesian method for exact positioning. the joints of the virtual robot can be adjusted precisely to the required angle, which is particularly suitable for e.g. bypassing an obstacle. there is a separate slider for each joint of the virtual robot. in order to set the individual joint angles, the respective slider button is dragged to the required value, which is also displayed above the slider button for better orientation. to program the robot, the user interface provides various application buttons, such as saving and removing robot poses from the chain and a display of the poses in the chain. the user directs the virtual robot to the desired position and confirms using the corresponding button. the pose of the robot is then saved as joint angles from a1 to a7 and one gripper position in a list and is displayed on the left side of the screen. when running the programmed application, the robot moves to the saved robot poses and gripper positions according to the defined sequence. for a better orientation, the robots current target position changes its color from white to red. after testing the application, the list of robot poses can be sent to the controller of the real robot via a webservice. the real robot then moves analogously to the virtual robot to the corresponding robot poses and gripper positions. the purpose of the evaluation is how the gesture-based control concept compares to other concepts regarding intuitiveness, comfort and complexity. for the evaluation, a study was conducted with seven test persons, who had to solve a pick and place task with five different operating concepts and subsequently evaluate them. the developed concept based on gestures and mr was evaluated against a lead through procedure, programming with java, programming with a simplified programming concept and approaching and saving points with kuka smartpad. the test persons had no experience with microsoft hololens and mr, no to moderate experience with robots and no to moderate programming skills. the questionnaire for the evaluation of physical assistive devices (quead) developed by schmidtler et al [25] was used to evaluate and compare the five control concepts. the questionnaire is classified into five categories (perceived usefulness, perceived ease of use, emotions, attitude and comfort) and contains a total of 26 questions, rated on an ordinal scale from 1 (entirely disagree) to 7 (entirely agree). firstly, each test person received a short introduction to the respective control concept, conducted the pick and place task and immediately afterwards evaluated the respective control concept using quead. all test persons agreed that they would reuse the concept in future tasks (3 mostly agree, 4 entirely agree). in addition, the test persons considered the gesture-based concept to be intuitive (1 mostly agree, 4 entirely agree), easy to use (5 mostly agree, 2 entirely agree) and easy to learn (1 mostly agree, 6 entirely agree). two test persons mostly agree and four entirely agree that the gesture-based concept enabled them to solve the task efficiently and four test persons mostly agree and two entirely agree that the concept enhances their work performance. all seven subjects were comfortable using the gesturebased concept (4 mostly agree, 2 entirely agree). overall, the concept presented in this paper was evaluated as more comfortable, more intuitive and easier to learn than the other control concepts evaluated. in comparison to them, the new operating concept was perceived as the most useful and easiest to use. the test persons felt physically and psychologically most comfortable when using the concept and were most positive in total. in this paper, a new concept for programming robots based on gestures and mr and for simulating the created applications was presented. this concept forms the basis for a new, gesture-based programming method, with which it is possible to project a virtual robot model of the real robot into the real working environment by means of a mr solution, to program it and to simulate the workflow. using an intuitive virtual user interface, the robot can be controlled by three control modes and further groups of interactable elements and via certain functions, several robot positions can be chained as a program. by using this concept, test and simulation times can be reduced, since on the one hand the program can be tested directly in the mr environment without disturbing the workflow. on the other hand, the robot model is rendered into the real working environment via the mr approach, thus eliminating the need for time-consuming and costly modeling of the environment. the results of the user study indicate that the control concept is easy to learn, intuitive and easy to use. this facilitates the introduction of robots and especially in smes, since no expert knowledge is required for programming, programs can be created rapidly and intuitively and processes can be adapted flexibly. in addition, the user study showed that tasks can be solved efficiently and the concept is perceived as performance-enhancing. potential directions of improvement are: implement various movement types, such as point-to-point, linear and circular movements in the concept. this makes the robot motion more flexible and efficient, since positions can be approached in different ways depending on the situation. another improvement is to extend the concept with collaborative functions of the robot, such as force sensitivity or the ability to conduct search movements. in this way, the functions that make collaborative robots special can be integrated into the program structure. a further approach for improvement is to engage in a larger scale study. in 2019 the world's commercial fleet consists of 95,402 ships with a total capacity of 1,976,491 thousand dwt. (a plus of 2.6 % in carrying capacity compared to last year) [1] . according to the international chamber of shipping, the shipping industry is responsible for about 90 % of all trade [2] . in order to ensure the safe voyage of all participant in the international travel at sea, the need for monitoring is steadily increasing. while more and more data regarding the sea traffic is collected by using cheaper and more powerful sensors, the data still needs to be processed and understood by human operators. in order to support the operators, reliable anomaly detection and situation recognition systems are needed. one cornerstone for this development is a reliable automatic classification of vessels at sea. for example by classifying the behaviour of non cooperative vessels in ecological protected areas, the identification of illegal, unreported and unregulated (iuu) fishing activities is possible. iuu fishing is in some areas of the world a major problem, e. g., »in the wider-caribbean, western central atlantic region, iuu fishing compares to 20-30 percent of the legitimate landings of fish« [3] resulting in an estimated value between usd 700 and 930 million per year. one approach for gathering information on the sea traffic is based on the automatic identification system (ais) 3 . it was introduced as a collision avoidance system. as each vessel is broadcasting its information on an open channel, the data is often used for other purposes, like training and validating of machine learning models. ais provides dynamic data like position, speed and course over ground, static data like mmsi 4 , shiptype and length, and voyage related data like draught, type of cargo, and destination about a vessel. the system is self-reporting, it has no strong verification of transmission, and many of the fields in each message are set by hand. therefore, the data can not be fully trusted. as harati-mokhtari et al. [4] stated, half of all ais messages contain some erroneous data. as for this work, the dataset is collected by using the ais stream provided by aishub 5 , the dataset is likely to have some amount of false data. while most of the errors will have no further consequences, minor coordinate inaccuracies or wrong vessel dimensions are irrelevant, some false information in vessel information can have an impact on the model performance. classification of maritime trajectories and the detection of anomalies is a challenging problem, e.g., since classifications should be based on short observation periods, only limited information is available for vessel identification. riveiro et al. [5] give a survey on anomaly detection at sea, where shiptype classification is a subtype. jiang et al. [6] present a novel trajectorynet capable of point-based classification. their approach is based on the usage of embedding gps coordinates into a new feature space. the classification itself is accomplished using an long short-term memory (lstm) network. further, jiang et al. [7] propose a partition-wise lstm (plstm) for point-based binary classification of ais trajectories into fishing or non-fishing activity. they evaluated their model against other recurrent neural networks and achieve a significantly better result than common recurrent network architectures based on lstm or gated recurrent units. a recurrent neural network is used by nguyen et al. in [8] to reconstruct incomplete trajectories, detect anomalies in the traffic data and identify the real type of a vessel. they are embedding the position data to generate a new representation as input for the neural network. besides these neural network based approaches, other methods are also used for situation recognition tasks in the maritime domain. especially expert-knowledge based systems are used frequently, as illegal or at least suspicious behaviour is not recorded as often as desirable for deep learning approaches. conditional random fields are used by hu et al. [9] for the identification of fishing activities from ais data. the data has been labelled by an expert and contains only longliner fisher boats. saini et al. [10] propose an hidden markov model (hmm) based approach to the classification of trajectories. they combine global-hmm and segmental-hmm using a genetic algorithm. in addition, they tested the robustness of the framework by adding gaussian noise. in [11] fischer et al. introduce a holistic approach for situation analysis based on situation-specific dynamic bayesian networks (ssdbn). this includes the modelling of the ssdbn as well as the presentation to end-users. for a bayesian network, the parametrisation of the conditional probability tables is crucial. fischer introduces an algorithm for choosing these parameters in a more transparent way. important for the functionality is the ability of the network to model the domain knowledge and the handling of noisy input data. for the evaluation, simulated and real data is used to assess the detection quality of the ssdbn. based on dbns, anneken et al. [12] implemented an algorithm for detecting illegal diving activities in the north sea. as explained by de rosa et al. [13] an additional layer for modelling the reliability of different sensor sources is added to the dbn. in order to use the ais data, preprocessing is necessary. this includes cleaning wrong data, filtering data, segmentation, and calculation of additional features. the whole workflow is depicted in figure 1 . the input in form of ais data and different maps is shown as blue boxes. all relevant mmsis are extracted from the ais data. for each mmsi, the position data is used for further processing. segmentation into separate trajectories is the next step (yellow). the resulting trajectories are filtered (orange). based on the remaining trajectories, geographic (green) and trajectory (purple) based features are derived. for each of the resulting sequences, the data is normalized (red), which results in the final dataset. only the 6 major shiptypes in the dataset are used for the evaluation. these are "cargo", "tanker", "fishing", "passenger", "pleasure craft" and "tug". due to their similar behaviour, "cargo" and "tanker" will combined to a single class "cargo-tanker". figure 1 : visualization of all preprocessing steps. input in blue, segmentation in yellow, filtering in orange, geographic features in green, trajectory feature in purple and normalization in red. four different trajectory features are used: ur-ai 2020 // 105 -time difference -speed over ground -course over ground -trajectory transformation as the incoming data from ais is not necessarily uniformly distributed in time, there is a need to create a feature representing the time dimension. therefore, the time difference between two samples is introduced. as the speed and course over ground is directly accessible through the ais data, the network will be directly fed with these features. the vessel's speed is a numeric value in 0.1-knot resolution in the interval [0; 1022] and the course is the negative angle in degrees relative to true north and therefore in the interval [0; 359]. the position will be transformed in two ways. the first transformation, further called "relative-to-first", will shift the trajectory to start at the origin. the second transformation, henceforth called "rotate-to-zero", will rotate the trajectory, in such a way, that the end point is on the x-axis. additional to the trajectory based features, two geographic features are derived by using coastline maps 6 and a map of large harbours. the coastline map consists of a list of line strips. in order to reduce complexity, the edge points are used to calculate the "distance-to-coast". further, only a lower resolution of the shapefile itself is used. in figure 2 , the resolution "high" and "low" for some fjords in norway are shown. due to the geoindex' cell size set to 40 km, a radius of 20 km can be queried. the world's 140 major harbours based on the world port index 7 are used to calculate the "distance-to-closest-harbor". as fishing vessels are expected to stay near to a certain harbour, this feature should support the network to identify some shiptypes. the geoindex' cell size is set for this feature to 5,000 km, resulting in a maximum radius of 2,500 km. the data is split into separate trajectories by using gaps in either time or space, or the sequence length. as real ais data is used, package loss during the transmission is common. this problem is tackled by splitting the data if the time between two successive samples is larger than 2 hours, or if the distance between two successive samples is large. regarding the distance, even though the great circle distance is more accurate, the euclidean distance is used. for simplification the distance value is squared and as a threshold 10 −4 is used. depending on latitude this corresponds to a value of about 1 km at the equator and only about 600 m at 60 • n. since the calculation includes approximation a relatively high threshold is chosen. as the neural network depends on a fixed input size, the data is split into fitting chunks by cutting and padding with these rules: -longer sequences are split into chunks according to the desired sequence length. -any left over sequence shorter than 80 % of the desired length is discarded. -the others will be padded with zeroes. this results in segmented trajectories of similar but not necessarily same duration. as this work is about the vessel behaviour at sea, stationary vessels (anchored and moored vessels) and vessels traversing rivers are removed from the segmented trajectories. the stationary vessels are identified by using a measure of movement in a trajectory: where n as the sequence length and p i its data points. a trajectory will be removed if α stationary is below a certain threshold. a shapefile 8 containing the major and most minor rivers (compare ??) is used in order to remove the vessels not on the high seas. a sequence with more than 50 % of its points on a river is removed from the dataset. in order to speed up the training process, the data is normalized in the interval [0; 1] by applying here, for the positional features a differentiation between "global normalization" and "local normalization" is taken into account. the "global normalization" will scale the input data for the maximum x max and minimum x min calculated over the entire data set, while "local normalization" will estimate the maximum x max and minimum x min only over the trajectory itself. as the data is processed parallel, the parameters for the "global normalization" will be calculated only for each chunk of data. this will result in slight deviations in the minimum and maximum, but for large batches this should be neglectable. all other additional features are normalized as well. for the geographic features "distance-to-coast" and "distance-to-closest-harbor" the maximum distance, that can be queried depending on grid size, is used as x max and 0 is used as the lower bound x min . the time difference feature is scaled using a minimum x min of 0 and the threshold for the temporal gap since this is the maximum value for this feature. speed and course are normalized using 0 and their respective maximum values. for the dataset, a period between 2018-07-24 and 2018-11-15 is used. altogether 209,536 unique vessels with 2,144,317,101 raw data points are included. using this foundation and the previously described methods, six datasets are derived. all datasets use the same spatial and temporal thresholds. in addition, filter thresholds are identical as well. the datasets differentiate in their sequence length and by applying only the "relativeto-first" transformation or additionally the "rotate-to-zero" transformation. either 360, 1,080, or 1,800 points per sequence are used resulting in approximate 1 h, 3 h, or 5 h long sequences. in figure 3 , the distribution of shiptypes in the datasets after applying the different filters is shown. for the shiptype classification, neural networks are chosen. the different networks are implemented using keras [14] with tensorflow as backend [15] . fawaz et al. [16] have shown, that, despite their initial design for image data, a residual neural network (resnet) can perform quite well on time-series classification. thus, as foundation for the evaluated architectures the resnet is used. the main difference to other neural network architectures is the inclusion of "skip connections". this allows for deeper networks by circumventing the vanishing gradient problem during the training phase. based on the main idea of a resnet, several architectures are designed and evaluated for this work. some information regarding the structure are given in table 1 . further, the single architectures are depicted in figures 4a to 4f . the main idea behind these architectures is to analyse the impact of the depth of the networks. furthermore, as the features itself are not necessarily logically linked with each other, the hope is to be able to capture the behaviour better by splitting up the network path for each feature. to verify the necessity of cnns two multilayer perceptron (mlp) based networks are tested: one with two hidden layers and one with four hidden layers, all with 64 neurons and fully connected with their adjacent layers. the majority of the parameters for the two networks are bound in the first layer. they are necessary to map the large number of input neurons, e. g., for the 360 samples dataset 360 * 9 = 3,240 input neurons, to the first hidden layer. each of the datasets is split into three parts: 64 % for the training set, 16 % for the validation set, and 20 % for the test set. for solving or at least mitigating the problem of overfitting, regularization techniques (input noise, batch normalization, and early stopping) are used. small noise on the input in the training phase is used to support the generalization of the network. for each feature a normal distribution with a standard deviation of 0.01 and a mean of 0 is used as noise. furthermore, batch normalization is implemented. this means, before each relulayer a batch normalization layer is added, allowing higher learning rates. therefore, the initial learning rate is doubled. additionally, the learning rate is halved if the validation error does not improve after ten training epochs, improving the training behaviour during oscillation on a plateau. in order to prevent overfitting, an early stopping criteria is introduced. the training will be interrupted if the validation error is not decreasing after 15 training epochs. to counter the dataset imbalance, class weights were considered but ultimately did not lead to better classification results and were discarded. the different neural network architectures are evaluated on a amd ryzen threadripper batch normalization and the input noise is tested. the initial learning rate is set to 0.001 without batch normalization and 0.002 with batch normalization activated. the maximum number of epochs is set to 600. the batch sizes are set to 64, 128, and 256 for 360, 1,080, and 1,800 samples per sequence respectively. in total 144 different setups are evaluated. furthermore, 4 additional networks are trained on the 360 samples dataset with "relative-to-first" transformation. two mlps to verify the need of deep neural networks, and the shallow and deep resnet trained without geographic features to measure the impact of these features. (f) "rtz" with 1,800 samples shown. the first row shows the results for the "relative-to-first" (rtf) transformation, the second for the "rotate-to-zero" (rtz) transformation. the results for the six different architectures are depicted in figure 5 . for 360 samples the shallow resnet and the deep resnet outperformed the other networks. in case of the "relative-to-first" transformation (see figure 5a ), the shallow resnet achieved an f 1 -score of 0.920, while the deep resnet achieved 0.919. for the "rotate-to-zero" transformation (see figure 5d ), the deep resnet achieved 0.918 and the shallow resnet 0.913. in all these cases the regularization methods lead to no improvements. the "relative-to-first" transformation performs slightly better overall. for the datasets with 360 samples per sequence, the standard resnet variants achieve higher f 1 -scores compared to the split resnet versions. but this difference is relatively small. as expected, the tiny resnet is not large and deep enough to classify the data on a similar level. for the "relative-first" transformation and trajectories based on 1080 samples (see figure 5b ), the split resnet and the total split resnet achieve the best results. the first performed well with an f 1 -score of 0.913, while the latter is slightly worse with 0.912. in both cases again the regularization did not improve the result. for the "rotateto-zero" transformation (see figure 5e ), the shallow resnet achieved an f 1 -score of 0.907 without any regularization and 0.905 with only the the noise added to the input. for the largest sequence length of 1,800 samples, the split based networks slightly outperform the standard resnets. for the "relative-to-first" transformation (see figure 5c ), the split resnet achieved an f 1 -score of 0.911, while for the "rotate-to-zero" transformation (see figure 5f ) the total split resnet reached an f 1 -score of 0.898. again without noise and batch normalization. to verify, that the implementation of cnns is actually necessary, additional tests with mlps were carried out. two different mlps are trained on the 360 samples dataset with "relative-to-first" transformation since this dataset leads to best results for the resnet architectures. both networks lead to no results as their output always is the "cargo-tanker" class regardless of the actual input. the only thing the models are able to learn is, that the "cargo-tanker" class is the most probable class based on the uneven distribution of classes. an mlp is not the right model for this kind of data and performs badly. the large dimensionality of even the small sequence length makes the use of the fully connected networks impracticable. probably, further hand-crafted feature extraction is needed to achieve better results. to measure the impact the feature "distance to coast" and "distance to closest harbor" have on the overall performance, a shallow resnet and a deep resnet are trained on the 360 sample length data set with the "relative-to-first" transformation excluding these features. the trained networks have f 1 -scores of 0.888 and 0.871 respectively. this means, by including this features, we are able to increase the performance by 3.5 %. the "relative-to-first" transformation compared to the "rotate-to-zero" transformation yields the better results. especially, this is easily visible for the longest sequence length. a possible explanation can be seen in the "stationary" filter. this filter removes more trajectories for the "relative-to-first" transformation than for the additional "rotate-to-zero" transformation. a problem might be, that the end point is used for rotating the trajectory. this adds a certain randomness to the data, especially for round trip sequences. in some cases, the stretched deep resnet is not able to learn the classes. it is possible, that there is a problem with the structure of the network or the large number of parameters. further, there seems to be a problem with the batch normalization, as seen in figures 5c and 5e . the overall worse performance of the "rotate-to-zero" transformation could be because of the difference in the "stationary" filter. in the "rotate-to-zero" dataset, fewer sequences are filtered out. the filter leads to more "fishing" and "pleasure craft" sequences in relation to each other as described in section 3.6. this could also explain the difference in class prediction distribution since the network is punished more for mistakes in these classes because more classes are overall from this type. for the evaluation, the expectation based on previous work by other authors was, that the shorter sequence length should perform worse compared to the longer ones. instead the shorter sequences outperform the longer ones. the main advantages of the shorter sequences are essentially the larger number of sequences in the dataset. for example the 360 samples dataset with "relative-to-first" transformation contains about 2.2 million sequences, while the corresponding 1,800 sample dataset contains only approximately 250,000 sequences. in addition, the more frequent segmentation can yield more easily classifiable sequences: the behaviour of a fishing vessel in general contains different characteristics, like travelling from the harbour to the fishing ground, the fishing itself, and the way back. the travelling parts are similar to other vessels and only the fishing part is unique. a more aggressive segmentation will yield more fishing sequences, that will be easier to classify regardless of observation length. the shallow resnet has the overall best results by using the 360 samples dataset and the "relative-to-first" transformation. the results for this setup are shown in the confusion matrix in figure 6 . as expected, the tiny resnet is not able to compete with the others. the other standard resnet architectures performed well, especially on shorter sequences. the split architectures are able to perform better on datasets with longer sequences, with the shallow resnet achieving similar performance. comparing the number of parameters, all three architectures have about 400,000 the shallow resnet about 50,000 more, the total split resnet about 40,000 less. only on the dataset with more sequences, the deep resnet performs well. this correlates with the need of more information due to the larger parameter count. due to the reduced flexibility, the split architecture can be interpreted as a "head start". this means, that the network has already information regarding the structure of the data, which in turn does not need to be extracted from the data. this can result in a better performance for smaller datasets. all in all, the best results are always achieved by omitting the suggested regularization methods. nevertheless, the batch normalization had an effect on the learning rate and needed training epochs: the learning rate is higher and less epochs are needed before convergence. based on the resnet, several architectures are evaluated for the task of shiptype classification. from the initial dataset based on ais data with over 2.2 billion datapoints six datasets with different trajectory length and preprocessing steps are derived. further to the kinematic information included in the dataset, geographical features are generated. each network architecture is evaluated with each of the datasets with and without batch normalization and input noise. overall the best result is an f 1 -score of 0.920 with the shallow resnet on the 360 samples per sequence dataset and a shift of the trajectories to the origin. additionally, we are able to show, that the inclusion of geographic features yield an improvement in classification quality. the achieved results are quite promising, but there is still some room for improvement. first of all, the the sequence length used for this work might still be too long for real world use cases. therefore, shorter sequences should be tried. additionally, interpolation for creating data with the same time delta between two samples or some kind of embedding or alignment layer might yield better results. as there are many sources for additional domain related information, further research in the integration of these sources is necessary. comparison of cnn for the detection of small ojects based on the example of components on an assembly many tasks which only a few years ago had to be performed by humans can now be performed by robots or will be performed by robots in the near future. nevertheless, there are some tasks in assembly processes which cannot be automated in the next few years. this applies especially to workpieces that are only produced in very small series or tasks that require a lot of tact and sensitivity, such as inserting small screws into a thread or assembling small components. in conversations with companies we have found out that a big problem for the workers is learning new production processes. this is currently done with instructions and by supervisors. but this requires a lot of time. this effort can be significantly reduced by modern systems, which accompany workers in the learning process. such intelligent systems require not only instructions that describe the target status and the individual work steps that lead to it, but also information on the current status at the assembly workstation. one way to obtain this information is to install cameras above the assembly workstation and use image recognition to calculate where an object is located at any given moment. the individual parts, often very small compared to the work surface, must be reliably detected. we have trained and tested several deep neural networks for this purpose. we have developed an assembly workstation where work instructions can be projected directly onto the work surface using a projector. at a distance, 21 containers for components are arranged in three rows, slightly offset to the rear, one above the other. these containers can also be illuminated by the projector. thus a very flexible pick-by-light system can be implemented. in order for the system behind it to automatically switch to the next work step and, in the event of errors, to point them out and provide support in correcting them, it is helpful to be able to identify the individual components on the work surface. we use a realsense depth camera for this purpose, from which, however, we are currently only using the colour image. the camera is mounted in a central position at a height of about two meters above the work surface. thus the camera image includes the complete working surface as well as the 21 containers and a small area next to the working surface. the objects to be detected are components of a kit for the construction of various toy cars. the kit contains 25 components in total. some of the components vary considerably from each other, but some others are very similar to each other. since it is the same with real components of a production, the choice of the kit seemed appropriate for the purposes of this project. object detection, one of the most fundamental and challenging problems in computer vision, seeks to local object instances from a large number of predefined categories in natural images. until the beginning of 2000, a similar approach was mostly used in object detection. keypoints in one or more images of a category were searched for automatically. at these points a feature vector was generated. during the recognition process, keypoints in the image were again searched, the corresponding feature vectors were generated and compared with the stored feature vectors. after a certain threshold an object was assigned to the category. one of the first approaches based on machine learning was published by viola and jones in 2001 [1] . they still selected features, in their case they were selected by using a haar basis function [2] and then using a variant of adaboost [3] . starting in 2012 with the publication of alexnet by krizhevsky et al. [4] , deep neural networks became more and more the standard in object detection tasks. they used a convolutional neural network which has 60 million parameters in five convolutional layers, some of them are followed by max-pooling layers, three fully-connected layers and a final softmax layer. they won the imagenet lsvrc-2012 competition with a error rate almost half as high as the second best. inception-v2 is mostly identical to inception-v3 by szegedy et al. [5] . it is based on inception-v1 [6] . all inception architectures are composed of dense modules. instead of stacking convolutional layers, they stack modules or blocks, within which are convolutional layers. for inception-v2 they redesigned the architecture of inception-v1 to avoid representational bottlenecks and have more efficient computations by using factorisation methods. they are the first using batch normalisation in object detection tasks. in previous architectures the most significant difference has been the increasing number of layers. but with the network depth increasing, accuracy gets saturated and then degrades rapidly. kaiming et al. [7] addressed this problem with resnet using skip connections, while building deeper models. in 2017 howard et al. presented mobilenet architecture [8] . mobilenet was developed for efficient work on mobile devices with less computational power and is very fast. they used depthwise convolutional layers for a extremely efficient network architecture. one year later sandler et al. [9] published a second version of mobilenet. besides some minor adjustments, a bottleneck was added in the convolutional layers, which further reduced the dimensions of the convolutional layers. thus a further increase in speed could be achieved. in addition to the neural network architectures presented so far, there are also different methods to detect in which area of the image the object is located. the two most frequently used are described briefly below. to bypass the problem of selecting a huge number of regions, girshick et al. [10] proposed a method where they use selective search by the features of the base cnn to extract just 2000 regions proposals from the image. liu et al. [11] introduced the single shot multibox detector (ssd). they added some extra feature layers behind the base model for detection of default boxes in different scales and aspect ratios. at prediction time, the network generates scores for the presence of each object in each default box. then it produces adjustments to the box to better match the object shape. there is just one publication over the past few years which gives an survey of generic object detection methods. liu et al. [12] compared 18 common object detection architectures for generic object detection. there are many other comparisons of specific object detection tasks. for example pedestrian detection [13] , face detection [14] and text detection [15] . the project is based on the methodology of supervised learning. thereby the models are trained using a training dataset consisting of many samples. each sample within the training dataset is tagged with a so called label (also called annotation). the label provides the model with information about the desired output for this sample. during training, the output generated by the model is then compared to the desired output (labels) and the error is determined. this error on the one hand gives information about the current performance of the model and, on the other hand it is used for further mathematical computations to adjust the model's parameters, so that the model's performance improves. for the training of neural networks in the field of computer vision the following rule of thumb applies: the larger and more diverse the training dataset, the higher the accuracy that can be achieved by the trained model. if you have too little data and/or run it through the model too often, this can lead to so-called overfitting. overfitting means that instead of learning an abstract concept that can be applied to a variety of data, the model basically memorizes the individual samples [16, 17] . if you train neural networks for the purpose of this project from scratch, it is quite possible that you will need more than 100,000 different images -depending on the accuracy that the model should finally be able to achieve. however, the methodology of the so-called transfer learning offers the possibility to transfer results of neural networks, which have already been trained for a specific task, completely or partially to a new task and thus to save time and resources [18] . for this reason, we also applied transfer learning methods within the project. the training dataset was created manually: a tripod, a mobile phone camera (10 megapixel format 3104 x 3104) and an apeman action cam (20 megapixel format 5120x3840) were used to take 97 images for each of the 25 classes. this corresponds to 2,425 images in total (actually 100 images were taken per class, but only 97 were suitable for use as training data). all images were documented and sorted into close-ups (distance between camera and object less than or equal to 30 cm) and standards (distance between camera and object more than 30 cm). this procedure should ensure the traceability and controllability of the data set. in total, the training data set contains approx. 25% close-ups and approx. 75% standards, each taken on different backgrounds and under different lighting conditions (see fig. 2 ). the labelimg tool was used for the labelling of the data. with the help of this tool, bounding boxes, whose coordinates are stored in either yolo or pascval voc format, can be marked in the images [19] . for the training of the neural networks the created dataset was finally divided into: ur-ai 2020 // 118 -training data (90% of all labelled images): images that are used for the training of the models and that pass through the models multiple times during the training. -test data (10% of all labelled images): images that are used for later testing or validation of the training results. in contrast to the images used as training data, the model is presented these images for the first time after training. the goal of this approach, which is common in deep learning, is to see how well the neural network recognizes objects in images, that it has never seen before, after the training. thus it is possible to make a statement about the accuracy and to be able to meet any further training needs that may arise. the training of deep neural networks is very demanding on resources due to the large number of computations. therefore, it is essential to use hardware with adequate performance. since the computations that run for each node in the graph can be highly parallelized, the use of a powerful graphical processing unit (gpu) is particularly suitable. a gpu with its several hundred computing cores has a clear advantage over a current cpu with four to eight cores when processing parallel computing tasks [20] . these are the outline parameters of the project computer in use: -operating system (os): ubuntu 18.04.2 lts -gpu: geforce r gtx 1080 ti (11 gb gddr5x-memory, data transfer speed 11 gbit/s) for the intended comparison the tensorflow object detection api was used. tensorflow object detection api is an open source framework based on tensorflow, which among other things provides implementations of pre-trained object detection models for transfer learning [21, 22] . the api was chosen because of its good and easy to understand documentation and its variety of pre-trained object detection models. for the comparison the following models were selected: -ssd mobilenet v1 coco: [11, 23, 24] -ssd mobilenet v2 coco: [11, 25, 26] -faster rcnn inception v2 coco: [27] [28] [29] -rfcn resnet101 coco: [30] [31] [32] to ensure comparability of the networks, all of the selected pre-trained models were trained on the coco dataset [33] . fundamentally, the algorithms based on cnn models can be grouped into two main categories: region-based algorithms and one-stage algorithms [34] . while both ssd models can be categorized as one-stage algorithms, faster r-cnn and r-fcn fall into the category of region-based algorithms. one-stage algorithms predict both -the fields (or the bounding boxes) and the class of the contained objects -simultaneously. they are generally considered extremely fast, but are known for their trade-off between accuracy and real-time processing speed. region-based algorithms consist of two parts: a special region proposal method and a classifier. instead of splitting the image into many small areas and then working with a large number of areas like conventional cnn would proceed, the region-based algorithm first proposes a set of regions of interest (roi) in the image and checks whether one of these fields contains an object. if an object is contained, the classifier classifies it [34] . region-based algorithms are generally considered as accurate, but also as slow. since, according to our requirements, both accuracy and speed are important, it seemed reasonable to compare models of both categories. besides the collection of pre-trained models for object detection, the tensorflow object detection api also offers corresponding configuration files for the training of each model. since these configurations have already shown to be successful, these files were used as a basis for own configurations. the configuration files contain information about the training parameters, such as the number of steps to be performed during training, the image resizer to be used, the number of samples processed as a batch before the model parameters are updated (batch size) and the number of classes which can be detected. to make the study of the different networks as comparable as possible, the training of all networks was configured in such a way that the number of images fed into the network simultaneously (batch size) was kept as small as possible. since the configurations of some models did not allow batch sizes larger than one, but other models did not allow batch sizes smaller than two, no general value for all models could be defined for this parameter. during training, each of the training images should be passed through the net 200 times (corresponds to 200 epochs). the number of steps was therefore adjusted accordingly, depending on the batch size. if a fixed shape resizer was used in the base configurations, two different dimensions of resizing (default: 300x300 pixels and custom: 512x512 pixels) were selected for the training. table 1 gives an overview of the training configurations used for the training of the different models. in this section we will first look at the training, before we then focus on evaluating the quality of the results and the speed of the selected convolutional neural networks. when evaluating the training results, we first considered the duration that the neural networks require for 200 epochs (see fig. 3 ). it becomes clear that especially the two region based object detectors (faster r-cnn inception v2 and rfcn resnet101) took significantly longer than the single shot object detectors (ssd mobilenet v1 and ssd mobilenet v2). in addition, the single shot object detectors clearly show that the size of the input data also has a decisive effect on the training duration: while ssd mobilenet v2 with an input data size of 300x300 pixels took the shortest time for the training with 9 hours 41 minutes and 47 seconds, the same neural network with an input data size of 512x512 pixels took almost three hours more for the training, but is still far below the time required by rfcn resnet101 for 200 epochs of training. the next point in which we compared the different networks was accuracy (see fig. 4 ). we focused on seeing which of the nets were correct in their detections and how often (absolute values), and we also wanted to see what proportion of the total detections were correct (relative values). the latter seemed to us to make sense especially because some of the nets showed more than three detections for a single object. the probability that the correct classification will be found for the same object with more than one detection is of course higher in this case than if only one detection per object is made. with regard to the later use at the assembly table, however, it does not help us if the neural net provides several possible interpretations for the classification of a component. figure 4 shows that, in this comparison, the two region based object detectors generally perform significantly better than the single shot object detectors -both in terms of the correct detections and their share of the total detections. it is also noticeable that for the single shot object detectors, the size of the input data also appears to have an effect on the comparison point on the result. however, there is a clear difference to the previous comparison of the required training durations: while the training duration increased uniformly with increasing size of the images with the single shot object detectors, such a uniform observation cannot be made with the accuracy, concerning the relation to the input data sizes. while ssd mobilenet v2 achieves good results with an input data size of 512x512 pixels, ssd mobilenet v1 delivers the worst result of this comparison for the same input data size (regarding the number of correct detections as well as their share of the total detections). with an input data size of 300x300 pixels, however, the result improves with ssd mobilenet v1, while the change to a smaller input data size has a deteriorating effect on the result with ssd mobilenet v2. the best result of this comparison -judging by the absolute values -was achieved by faster r-cnn inception v2. however, in terms of the proportion of correct detections in the total detections, the region based object detector is two percentage points behind rfcn resnet 101, also a region based object detector. we were particularly interested in how neural networks would react to particularly similar, small objects. therefore, we decided to investigate the behavior of neural networks within the comparison using an example to illustrate the behavior of the three very similar objects. figure 5 shows the selected components for the experiment. for each of these three components we examined how often it was correctly detected and classified by the compared neural networks and how often the network misclassified it with which of the similar components. the first and the second component was detected in nearly all cases by both region based approaches. the classification by inception-v2 and resnet-101 failed in about one third of images. the ssd networks detected the object in just one of twenty cases but mobilenet classified this correct. it has been surprising, that the results for the third component looks very different to the others (see fig. 6 ). ssd mobilenet v1 correctly identified the component in seven of ten images and did not produce any detections that could be interpreted as misclassifications with one of the similar components. ssd mobilenet v2 did not detect any of the three components, as in the two previous investigations. the results of the two region based object detectors are rather moderate. faster r-cnn inception v2 has detected the correct component in four of ten images, but still five misclassifications with the other two components. rfcn resnet101 has caused many misclassifications with the other two components. only two of ten images were correctly detected but it had six misclassifications with the similar components. an other important aspect of the study is the speed, or rather the speed at which the neural networks can detect objects, especially with regard to later use at the assembly table. for the comparison of the speeds on the one hand the data of the github repository of the tensorflow object detection api for the individual neural nets were used, on the other hand the actual speeds of the neural nets within this project were measured. it becomes clear that the speeds measured in the project are clearly below the achievable speeds that are mentioned in the github repository of the tensorflow object-detection api. on the other hand, the differences between the speeds of the region based object detectors and the single shot object detectors in the project are far less drastic than expected. we have created a training dataset with small, partly very similar components. with this we have trained four common deep neural networks. in addition to the training times, we examined the accuracy and the recognition time with general evaluation data. in addition, we examined the results for ten images each of three very similar and small components. none of the networks we trained produced suitable results for our scenario. nevertheless, we were able to gain some important insights from the results. at the moment, the runtime is not yet suitable for our scenario, but it is also not far from the minimum requirements, so that these can easily be achieved with smaller optimizations and better hardware. it was also important to realize that there are no serious runtime differences between the different network architectures. the two region based approaches delivered significantly better results than the ssd approaches. however, the results of the detection of the third small component suggest that mobilenet in combination with a faster r-cnn could possibly deliver even better results. longer training and training data better adapted to the intended use could also significantly improve the results of the object detectors. team schluckspecht from offenburg university of applied sciences is a very successful participant of the shell eco marathon [1] . in this contest, student groups are to design and build their own vehicles with the aim of low energy consumption. since 2018 the event features the additional autonomous driving contest. in this area, the vehicle has to fulfill several tasks, like driving a parcour, stopping within a defined parking space or circumvent obstacles, autonomously. for the upcoming season, the schluckspecht v car of the so called urban concept class has to be augmented with the according hardware and software to reliably recognize (i. e. detect and classify) possible obstacles and incorporate them into the software framework for further planning. in this contribution we describe the additional components in hard-and software that are necessary to allow an opitcal 3d object detection. main criteria are accuracy, cost effectiveness, computational complexity for relative real time performance and ease of use with regard to incorporation in the existing software framework and possible extensibility. this paper consists of the following sections. at first, the schluckspecht v system is described in terms of hard-and software components for autonomous driving and the additional parts for the visual object recognition. the second part scrutinizes the object recognition pipeline. therefore, software frameworks, neural network architecture and final data fusion in a global map is depicted in detail. the contribution closes with an evaluation of the object recognition results and conclusions. the schluckspecht v is a self designed and self build vehicle according to the requirements of the eco marathon rules. the vehicle is depicted in figure 1 . the main features are the relatively large size, including driver cabin, motor area and a large trunk, a fully equipped lighting system and two doors that can be opened separately. for the autonomous driving challenges, the vehicle is additionally equipped with several essential parts, that are divided into hardware, consisting of actuators, sensors, computational hardware and communication controllers. the software is based on a middle ware, can-open communication layers, localization, mapping and path planning algorithms that are embedded into a high level state machine. actuators the car is equipped with two actors, one for steering and one for braking. each actor is paired with sensors for measuring steering angle and braking pressure. environmental sensors several sensors are needed for localization and mapping. backbone is a multilayer 3d laser scanning system (lidar), which is combined with an inertial navigation system that consists of accelerometers, gyroscopes and magnetic field sensors all realized as triads. odometry information is provided from a global navigation satellite system (gnss) and two wheel encoders. the communication is based on two separate can-bus-systems, one for basic operations and an additional one for the autonomous functions. the hardware can nodes are designed and build from the team coupling usb-, i2c-, spi-and can-open-interfaces. messages are send from the central processing unit or the driver depending on drive mode. the trunk of the car is equipped with an industrial grade high performance cpu and an additional graphics processing unit (gpu). can communication is ensured with an internal card, remote access is possible via generic wireless components. software structure the schluckspecht uses a modular software system consisting of several basic modules that are activated and combined within a high level state ma-chine as needed. an overview of the main modules and possible sensors and actuators is depicted in figure 2 localization and mapping the schluckspecht v is running a simultaneous localization and mapping (slam) framework for navigation, mission planning and environment representation. in its current version we use a graph based slam approach based upon the cartographer framework developed by google [2] . we calculate a dynamic occupancy grid map that can be used for further planning. sensor data is provided by the lidar, inertial navigation and odometry systems. an example of a drivable map is shown in figure 3 . this kind of map is also used as base for the localization and placement of the later detected obstacles. the maps are accurate to roughly 20 centimeters, providing relative localization towards obstacles or homing regions. path planning to make use of the slam created maps, an additional module calculates the motion commands from start to target pose of the car. the schluckspecht is a classical car like mobile system which means that the path planning must take into account the non holonomic kind of permitted movement. parking maneuvers, close by driving on obstacles or planning a trajectory between given points is realized as a combination of local control commands based upon modeled vehicle dynamics, the so called local planner, and optimization algorithms that find the globally most cost efficient path given a cost function, the so called global planner. we employ a kinodynamic strategy, the elastic band method presented in [3] , for the local planning. global planning is realized with a variant of the a* algorithm as described in [4] . middleware and communication all submodules, namely, localization, mapping, path planning and high-level state machines for each competition are implemented within ur-ai 2020 // 129 the robot operating system (ros) middleware [5] . ros provides a messaging system based upon the subscriber/publisher principle. the single modules are capsuled in a process, called node, capable to asynchronously exchange messages as needed. due to its open source character and an abundance on drivers and helper functions, ros provides additional features like hardware abstraction, device drivers, visualization and data storage. data structures for mobile robotic systems, e. g. static and dynamic maps or velocity control messages, allow for rapid development. the lidar sensor system has four rays, enabling only the incorporation of walls and track delimiters within a map. therefore, a stereo camera system is additionally implemented to allow for object detection of persons, other cars, traffic signs or visual parking space delimiters and simultaneously measure the distance of any environmental objects. camera hardware a zed-stereo-camera system is installed upon the car and incorporated into the ros framework. the system provides a color image streams for each camera and a depth map from stereo vision. the camera images are calibrated to each other and towards the depth information. the algorithms for disparity estimation are running around 50 frames per second making use of the provided gpu. the object recognition relies on deep neural networks. to seamlessly work with the other software parts and for easy integration, the networks are evaluated with tensorflow [6] and pytorch [7] frameworks. both are connected to ros via the opencv image formats providing ros-nodes and -topics for visualization and further processing. the object recognition pipeline relies on a combination of mono camera images and calibrated depth information to determine object and position. core algorithm is a deep learning approach with convolutional neural networks. ur-ai 2020 // 130 main contribution of this paper is the incorporation of a deep neural network object detector into our framework. object detection with deep neural networks can be subdivided into two approaches, one being a two step approach, where regions of interest are identified in a first step and classified in a second one. the second are so called single shot detectors (like [8] ), that extract and classify the objects in one network run. therefore, two network architectures are evaluated, namely yolov3 [9] as a single shot approach and faster r-cnn [10] as two step model. all are trained on public data sets and fine tuned to our setting by incorporating training images from the schluckspecht v in the zed image format. the models are pre-selected due to their real time capability in combination with the expected classification performance. this excludes the current best instance segmentation network mask r-cnn [11] due to computational burdens and fast but inaccurate networks based on the mobilenet backbone [12] . the class count is adapted for the contest, in the given case eight classes, including the relevant pedestrian, car, van, tram and cyclist. for this paper, the two chosen network architectures were trained in their respective framework, i. e. darknet for the yolov3 detector and tensorflow for the faster r-cnn detector. yolov3 is used in its standard form with the darknet 53 backbone, faster r-cnn is designed with the resnet 101 [13] backbone. the models were trained on local hardware with the kitti [14] data set. alternatively, an open source data set from the teaching company udacity, with only three classes (truck, car, pedestrian) was tested. to deal with the problem of domain adaptation, the training images for yolov3 were pre-processed to fit the aspect ratio of the zed camera. the faster r-cnn net can cope with ratio variations as it uses a two stage approach for detection based on regions of interest pooling. both networks were trained and stored. afterward, their are incorporated into the system via a ros node making use of standard python libraries. the detector output is represented by several labeled bounding boxes within the 2d image. three dimensional information is extracted from the associated depth map by calculating the center of gravity of each box to get a x and y coordinate within the image. interpolating the depth map pixels accordingly one gets the distance coordinate z from the depth map to determine the object position p(x, y, z) in the stereo camera coordinate system. the ease of projection between dieeferent coordinate systems is one reason to use the ros middleware. the complete vehicle is modeled in a so calle tranform tree (tf-tree), that allows the direct interpolation between different coordinate systems in all six spatial degrees of freedom. the dynamic map, created in the slam subsystem, is now augmented with the current obstacles in the car coordinate system. the local path planner can take these into account and plan a trajectory including kinodynamic constraints to prevent collision or initiate a breaking maneuver. both newly trained networks were first evaluated upon the training data. exemplary results for the kitti data set are shown in figure 4 . the results clearly indicate an advantage for the yolov3 system, both in speed and accuracy. the figure depicts good results for occlusions (e. g. the car on the upper right) or high object count (see the black car on the lower left as example). the evaluation on a desktop system showed 50 fps for yolov3 and approximately 10 fps for faster r-cnn. after validating the performance upon the training data, both networks were started as a ros node and tested upon real data of the schluckspecht vehicle. as the training data differs from the zed-camera images in format and resolution, several adaptions were necessary for the yolov3 detector. the images are cropped in real time before presented to the neural net to emulate the format of the training images. the r-cnn like two stage networks are directly connected to the zed node. the test data is not labeled as ground truth. it is therefore not possible to give quantitative results for the recognition task. table 1 gives a quantitative overview of the object detection and classification, the subsequent figures give some expression of exemplary results. the evaluation on the schluckspecht videos showed an advantage for the yolov3 network. main reason is the faster computation, which results in a frame rate nearly twice as high compared to two stage detectors. in addition, the recognition of objects in the distance, i. e. smaller objects is a strong point of yolo. the closer the camera gets, the bigger is the balance shift towards faster r-cnn, that outperforms yolo on all categories for larger objects. what becomes apparent is a maximum detection distance of approximately 30 meters, from which on cars become to small in size. figure 6 shows an additional result demonstrating the detection power for partially obstructed objects. another interesting finding was the capability of the networks to generalize. faster r-cnn copes much better with new object instances than yolov3. persons with so far unknown cloth color or darker areas with vehicles remain a problem for yolo, but ur-ai 2020 // 133 commonly not for the r-cnn. the domain transfer from training data in berkeley and kitti to real zed vehicle images proved problematic. this contribution describes an optical object recognition system in hard-and software for the application in autonomous driving under restricted conditions, within the shell eco marathon competition. an overall overview of the system and the incorporation of the detector within the framework is given. main focus was the evaluation and implementation of several neural network detectors, namely yolov3 as one shot detector and faster r-cnn as a two step detector, and their combination with distance information to gain a three dimensional information for detected objects. for the given application, the advantage clearly lies with yolov3. especially the achievable frame rate of minimum 10 hz allows a seamless integration into the localization and mapping framework. given the velocities and map update rate, the object recognition and integration via sensor fusion for path planning and navigation works in quasi real-time. for future applications we plan to further increase the detection quality by incorporating new classes and modern object detector frameworks like m2det [15] . this will additionally increase frame rate and bounding box quality. for more complex tasks, the data of the 3d-lidar system shall be directly incorporated into the fusion framework to enhance the perception of object boundaries and object velocities. a few useful things to know about machine learning feature engineering for machine learning an empirical analysis of feature engineering for predictive modeling input selection for fast feature engineering random forests support vector regression machines strong consistency of least squares estimates in multiple regression ii business data science: combining machine learning and economics to optimize, automate, and accelerate business decisions global product classification (gpc) a study of cross-validation and bootstrap for accuracy estimation and model selection automatic liver and tumor segmentation of ct and mri volumes using cascaded fully convolutional neural networks convolutional networks for biomedical image segmentation v-net: fully convolutional neural networks for volumetric medical image segmentation self-supervised learning for pore detection in ct-scans of cast aluminum parts generating meaningful synthetic ground truth for pore detection in cast aluminum parts nema ps3 / iso 12052, digital imaging and communications in medicine (dicom) standard, national electrical manufacturers association ct-realistic lung nodule simulation from 3d conditional generative adversarial networks for robust lung segmentation deep learning hardware: past, present, and future a survey on specialised hardware for machine learning a survey on distributed machine learning hardware for machine learning: challenges and opportunities 3d u-net: learning dense volumetric segmentation from sparse annotation z-net: an anisotropic 3d dcnn for medical ct volume segmentation activation functions: comparison of trends in practice and research for deep learning fast and accurate deep network learning by exponential linear units (elus) delving deep into rectifiers: surpassing human-level performance on imagenet classification toward deeper understanding of neural networks: the power of initialization and a dual view on expressivity adam: a method for stochastic optimization diffgrad: an optimization method for convolutional neural networks tversky loss function for image segmentation using 3d fully convolutional deep networks a low-power multi physiological monitoring". processor for stress detection. ieee sensors using heart rate monitors to detect mental stress positive technology: a free mobile platform for the self-management of psychological stress exploring the effectiveness of a computer-based heart rate variability biofeedback program in reducing anxiety in college students psychological stress and incidence of atrial fibrillation continuously updated, computationally efficient stress recognition framework using electroencephalogram (eeg) by applying online multitask learning algorithms (omtl) ten years of research with the trier social stress test trapezius muscle emg as predictor of mental stress poptherapy: coping with stress through pop-culture du-md: an open-source human action dataset for ubiquitous wearable sensors stress recognition using wearable sensors and mobile phones introducing wesad, a multimodal dataset for wearable stress and affect detection feasibility and usability aspects of continuous remote monitoring of health status in palliative cancer patients using wearables detection of diseases based on electrocardiography and electroencephalography signals embedded in different devices: an exploratory study stress effects". the american institute of stress der smarte assistent can: creative adversarial networks, generating" art" by learning about styles and deviating from style norms creative ai: on the democratisation & escalation of creativity generative design: a paradigm for design research eigenfaces for recognition unsupervised representation learning with deep convolutional generative adversarial networks large scale gan training for high fidelity natural image synthesis interpreting the latent space of gans for semantic face editing visualizing and understanding generative adversarial networks mistaken identity spectral normalization for generative adversarial networks beauty is in the ease of the beholding: a neurophysiological test of the averageness theory of facial attractiveness unpaired image-to-image translation using cycleconsistent adversarial networks colorization for anime sketches with cycle-consistent adversarial network artificial muse using evolutionary design to interactively sketch car silhouettes and stimulate designer's creativity the chair project-four classics deepwear: a case study of collaborative design between human and artificial intelligence grass: generative recursive autoencoders for shape structures co-designing object shapes with artificial intelligence systematic review of the empirical evidence of study publication bias and outcome reporting bias ki-kunst und urheberrecht -die maschine als schöpferin? public law research paper no. 692; u of maryland legal studies research paper no inceptionism: going deeper into neural networks proactive error prevention in manufacturing based on an adaptable machine learning environment. artificial intelligence: from research to application: the upper-rhine artificial intelligence symposium ur-ai the benefits fo pdca crisp-dm 1.0: step-by-step data mining guide interpretable machine learning for quality engineering in manufacturing-importance measures that reveal insights on errors regulation (eu) 2017/745 of the european parliament and of the council of 5 april 2017 on medical devices -medical device regulation (mdr) use of real-world evidence to support regulatory decision-making for medical devices. guidance for industry and food and drug administration staff high-performance medicine: the convergence of human and artificial intelligence artificial intelligence powers digital medicine dermatologist-level classification of skin cancer with deep neural networks chestx-ray8: hospital-scale chest x-ray database and benchmarks on weaklysupervised classification and localization of common thorax diseases an attention based deep learning model of clinical events in the intensive care unit the artificial intelligence clinician learns optimal treatment strategies for sepsis in intensive care the european commission's high-level expert group on artificial intelligence: ethics guidelines for trustworthy ai key challenges for delivering clinical impact with artificial intelligence ibm's watson supercomputer recommended 'unsafe and incorrect' cancer treatments, internal documents show towards international standards for the evaluation of artificial intelligence for health proposed regulatory framework for modifications to artificial intelligence/machine learning (ai/ml)-based software as a medical device (samd) artificial-intelligence-and-machine-learning-discussion-paper.pdf 14. international medical device regulators forum (imdrf) -samd working group medical device software -software life-cycle processes general principles of software validation. final guidance for industry and fda staff deciding when to submit a 510(k) for a change to an existing device. guidance for industry and food and drug administration staff software as a medical device (samd): clinical evaluation. guidance for industry and food and drug administration staff international electrotechnical commission. iec 62366-1:2015 -part 1: application of usability engineering to medical devices why rankings of biomedical image analysis competitions should be interpreted with care what do we need to build explainable ai systems for the medical domain /679 of the european parliament and of the council of 27 april 2016 on the protection of natural persons with regard to the processing of personal data and on the free movement of such data (general data protection regulation -gdpr) artificial intelligence in healthcare: a critical analysis of the legal and ethical implications explainable artificial intelligence: understanding, visualizing and interpreting deep learning models association between race/ethnicity and survival of melanoma patients in the united states over 3 decades docket for feedback -proposed regulatory framework for modifications to artificial intelligence/machine learning (ai/ml)-based software as a medical device (samd) openpose: realtime multi-person 2d pose estimation using part affinity fields a density-based algorithm for discovering clusters in large spatial databases with noise fast volumetric auto-segmentation of head ct images in emergency situations for ventricular punctures a system for augmented reality guided ventricular puncture using a hololens: design, implementation and initial evaluation op sense-a robotic research platform for telemanipulated and automatic computer assisted surgery yolov3: an incremental improvement. arxiv deep learning based 3d pose estimation of surgical tools using a rgb-d camera at the example of a catheter for ventricular puncture fast point feature histograms (fpfh) for 3d registration joint probabilistic people detection in overlapping depth images towards end-to-end 3d human avatar shape reconstruction from 4d data scene-adaptive optimization scheme for depth sensor networks a taxonomy and evaluation of dense two-frame stereo correspondence algorithms advances in computational stereo a comparative analysis of cross-correlation matching algorithms using a pyramidal resolution approach fast approximate energy minimization via graph cuts stereo processing by semiglobal matching and mutual information guided stereo matching a large dataset to train convolutional networks for disparity, optical flow, and scene flow estimation pyramid stereo matching network microstructure-sensitive design of a compliant beam microstructure sensitive design of an orthotropic plate subjected to tensile load microstructure sensitive design for performance optimization on the design, analysis, and characterization of materials using computational neural networks texture optimization of rolled aluminum alloy sheets using a genetic algorithm finite mixture models a tutorial on hidden markov models and selected applications in speech recognition information processing in dynamical systems: foundations of harmony theory generative adversarial nets building texture evolution networks for deformation processing of polycrystalline fcc metals using spectral approaches: applications to process design for targeted performance linear solution scheme for microstructure design with process constraints matcalo: knowledge-enabled machine learning in materials science differential evolution -a simple and efficient adaptive scheme for global optimization over continuous spaces reinforcement learning: an introduction hindsight experience replay industrieroboter für kmu. flexible und intuitive prozessbeschreibung toward efficient robot teach-in and semantic process descriptions for small lot sizes survey on human-robot collaboration in industrial settings: safety, intuitive interfaces and applications concept and architecture for programming industrial robots using augmented reality with mobile devices like microsoft hololens robot programming using augmented reality robot path and end-effector orientation planning using augmented reality spatial programming for industrial robots based on gestures and augmented reality spatial programming for industrial robots through task demonstration augmented reality based teaching pendant for industrial robot intuitive robot tasks with augmented reality and virtual obstacles development of a mixed reality based interface for human roboter interaction a hands-free virtual-reality teleoperation interface for wizard-of-oz control mixed reality as a tool supporting programming of the robot communicating robot arm motion intent through mixed reality head-mounted displays intuitive industrial robot programming through incremental multimodal language and augmented reality development of mixed reality robot control system based on hololens interactive spatial augmented reality in collaborative robot programming: user experience evaluation comparison of multimodal heading and pointing gestures for co-located mixed reality human-robot interaction robot programming through augmented trajectories in augmented reality interactive robot programming using mixed reality a taxonomy of mixed reality visual displays experimental packages for kuka manipulators within ros-indus-trial 24. siemens: ros-sharp a questionnaire for the evaluation of physical assistive devices (quead) unctad: review of maritime transport 2019 (2019) last accessed 2019-11-19. 2. international chamber of shipping report of the second meeting of the regional working group on illegal, unreported and unregulated (iuu) fishing automatic identification system (ais): data reliability and human error implications maritime anomaly detection: a review trajectorynet: an embedded gps trajectory representation for point-based classification using recurrent neural networks partition-wise recurrent neural networks for point-based ais trajectory classification a multi-task deep learning architecture for maritime surveillance using ais data streams identifying fishing activities from ais data with conditional random fields a segmental hmm based trajectory classification using genetic algorithm wissensbasierte probabilistische modellierung für die situationsanalyse am beispiel der maritimen überwachung detecting illegal diving and other suspicious activities in the north sea: tale of a successful trial source quality handling in fusion systems: a bayesian perspective tensorflow: large-scale machine learning on heterogeneous systems deep learning for time series classification: a review rapid object detection using a boosted cascade of simple features general framework for object detection a decision-theoretic generalization of on-line learning and an application to boosting imagenet classification with deep convolutional neural networks rethinking the inception architecture for computer vision going deeper with convolutions deep residual learning for image recognition mobilenets: efficient convolutional neural networks for mobile vision applications mobilenetv2: inverted residuals and linear bottlenecks rich feature hierarchies for accurate object detection and semantic segmentation ssd: single shot multibox detector deep learning for generic object detection: a survey pedestrian detection: an evaluation of the state of the art a survey on face detection in the wild: past, present and future text detection and recognition in imagery: a survey information visualizations used to avoid the problem of overfitting in supervised machine learning data science for business: what you need to know about data mining and data-analytic thinking automatic object detection from digital images by deep learning with transfer learning gpu asynchronous stochastic gradient descent to speed up neural network training tensorflow: tensorflow object detection api: ssd mobilenet v2 coco faster r-cnn: towards real-time object detection with region proposal networks tensorflow object detection api: faster rcnn inception v2 coco. online 29. tensorflow: tensorflow object detection api: faster rcnn inception v2 coco r-fcn: object detection via region-based fully convolutional networks 31. tensorflow: tensorflow object detection api: rfcn resnet101 coco multi-scale feature fusion single shot object detector based on densenet references 1. shell: the shell eco marathon real-time loop closure in 2d lidar slam kinodynamic trajectory optimization and control for car-like robots experiments with the graph traverser program robot operating system automatic differentiation in pytorch ssd: single shot multibox detector yolov3: an incremental improvement rich feature hierarchies for accurate object detection and semantic segmentation mobilenets: efficient convolutional neural networks for mobile vision applications deep residual learning for image recognition are we ready for autonomous driving? the kitti vision benchmark suite m2det: a single-shot object detector based on multi-level feature pyramid network the upper-rhine artificial intelligence symposium ur-ai 2020we thank our sponsor! main sponsor: esentri ag, ettlingen this research and development project is funded by the german federal ministry of education and research (bmbf) and the european social fund (esf) within the program "future of work" (02l17c550) and implemented by the project management agency karlsruhe (ptka). the author is responsible for the content of this publication. underlying projects to this article are funded by the wtd 81 of the german federal ministry of defense. the authors are responsible for the content of this article.this work was developed in the fraunhofer cluster of excellence "cognitive internet technologies". the upper-rhine artificial intelligence symposium key: cord-189096-85v2jgx0 authors: muhlenbach, fabrice title: a methodology for ethics-by-design ai systems: dealing with human value conflicts date: 2020-10-15 journal: nan doi: nan sha: doc_id: 189096 cord_uid: 85v2jgx0 the introduction of artificial intelligence into activities traditionally carried out by human beings produces brutal changes. this is not without consequences for human values. this paper is about designing and implementing models of ethical behaviors in ai-based systems, and more specifically it presents a methodology for designing systems that take ethical aspects into account at an early stage while finding an innovative solution to prevent human values from being affected. two case studies where ai-based innovations complement economic and social proposals with this methodology are presented: one in the field of culture and operated by a private company, the other in the field of scientific research and supported by a state organization. artificial intelligence (or "ai") is often defined as the simulation on a machine of so-called "intelligent" processes. being both an applied and theoretical field, this discipline of computer science is covering the range from weak ai (can machines act intelligently?) to strong ai (can machines really think?) [1] . for the past decade, the first approach has brought ai back to the forefront, especially with the development of new machine learning techniques, such as deep learning models [2] . this technique has created extremely effective ai applications in the area of pattern recognition or information selection problems for decision making, with programs that extract information from raw data and learn to improve their skills from existing examples. with this learning process, ai systems can perform complex tasks in place of humans. however, the arrival of this new technique has brought a number of ethical issues. firstly, artificial intelligence programs reason in a simplistic way, but the real world is complex and full of unexpected events, which the machine has great difficulty in dealing with. secondly, when an ai program learns about data collected on past situations, it performs statistical deductions and transforms correlations between variables into implication relationships. this can lead to problems with dramatic consequences such as gender bias in resume analysis support system by denying women access to managerial positions [3] , or racial bias in legal decision support system to predict future criminals [4] . the concern for the developers is to optimize some specific criteria, e.g., efficiency and usability. for instance, online e-commerce marketplaces seek to optimize the relevance of the connection between supply and demand. they collect a maximum of information from consumers and suppliers, and use a whole set of ai-derived strategies to maximize the matching between the different parties. attention-economybased companies seek to keep their users on their websites and apps for as long as possible to offer them advertising. the collection of personal data leads to personalization which may have certain advantages, such as recommending more relevant content, but also important problems relating to the preservation of privacy, attacks on democracy [5] , or the limitation of access to diversity [6] . intelligent system algorithms are black boxes that are impossible to understand, they are unregulated and difficult to question in the case of the presence of bias, and in some cases they amplify inequalities [7] . the highlighting of these problems has led to reactions from states and civil society. some initiatives, for example in the usa (asilomar ai principles, 1 "ai now" institute...), in canada (montréal declaration for a responsible development of artificial intelligence 2 ), or in france (villani report "for a meaningful artificial intelligence" 3 ), with the production of reports, charters, lists of principles or laws, are moving in the direction of taking ethical concerns into account early in the development of ai systems. however, there is not much information provided on how to make this possible. for designing and implementing models of ethical behaviors, this paper proposes in the following a methodology for building ethics-by-design ai systems. this methodology is explained and illustrated by means of two innovative applications developed during phd works supervised by the author. we will explain how these ai applications are able to respond to the problem of human value conflicts. the main idea of the three-step methodology proposed here is to take advantage of collective intelligence. the introduction of technologies derived from artificial intelligence leads to risks of undermining values for which the different stakeholders may have contrasting views. step: innovations in an economic and social world the first step of the methodology is to set up an economic and social model respectful of human values where the contribution of ai technologies can be integrated as a complement to the skills of human beings. the quote "they who can give up essential liberty to obtain a little temporary safety, deserve neither liberty nor safety" attributed to benjamin franklin makes us think that certain values are necessarily contradictory: liberty/privacy vs. security/safety. when resources are limited, choices need to be made. these choices will necessarily follow certain values and favor certain behaviors supported by some groups of individuals over others. however, in the case of adding a new technology such as ai, we must find a way not to optimize a single value at the expense of others. it is important to try always to find an alternative and innovative solution that guarantees respect for all human values. for example, in our study made in the field of predictive judicial analytics [8] , we came to the conclusion that the values of knowledge and trust are essential to give credit to decisions produced by intelligent systems. if the deep learning models cannot guarantee understanding by specialists (i.e., lawyers), they must be replaced by other more comprehensible models (rule-based approaches, bayesian methods), otherwise these ai models will no longer be able to contribute to their role as a tool to serve justice and help defend litigants. the methodology continues with the clarification of the main values involved in the introduction of a new ai system for all stakeholders by setting up a decision-making tool called the "ethical matrix." this tool was initially proposed by ben mepham to facilitate judgements on bioethic questions [9] , in order to identify the values that are threatened. by focusing more specifically on a small number of ethical principles (e.g., respect for wellbeing, for autonomy and for justice) on a given subject (e.g., the impact of new technologies in food and agriculture), it is possible to elicit the problems and concerns of different stakeholders or interest groups. the selected ethical principles constitute values that form the columns of the matrix. the rows of the matrix consist of each stakeholder caught up with the issue in question. each cell of the matrix specifies the main criterion to be satisfied for a stakeholder for a given principle. one of the most important difficulties of the ethical matrix approach concerns the choice of the values to use. a value is a way of being and acting for a person or a group of people, and people consider that certain behaviors resulting from a value are more desirable than others. from antiquity (e.g., with plato) to the present day, authors have proposed lists of fundamental values, such as the axiology -defined as the philosophical science of values-proposed by the philosopher max scheler [10] , or the "theory of basic human values" proposed by the social psychologist shalom h. schwartz [11] . the arrival of artificial intelligence highlights a specific value: the efficiency. by taking into account a greater number of variables on a greater number of examples that no human being is capable of accumulating in a lifetime, intelligent programs are able to accomplish very complex tasks brilliantly and effortlessly. however, this efficiency, introduced in a disruptive manner in fields which until now felt protected, such as medicine or justice, seems to be predatory for other values. faced with this difficult task, it seems important to us to select a list of basic values, for example the 10-item list of values selected by the montréal declaration for a responsible development of artificial intelligence [12] : well-being, respect for autonomy, protection of privacy and intimacy, solidarity, democratic participation, equity, diversity inclusion, prudence, responsibility, and sustainable development. however this list is neither necessary nor exhaustive. depending on the application context, values more likely than others to be affected by an ai technology must replace certain values among the 10 or be added to this list. the values of individuals can differ greatly from one individual to another, or from one social group to another. it is then recommended to take into account all possible stakeholders, even those who cannot express themselves (e.g., animals, nature). in the field of artificial intelligence, the stakeholders refers especially to researchers, developers, manufacturers, providers, policymakers, and users [13] . in the generic ethical matrix manual [14] , a protocol has been proposed for these stakeholders to meet in workshops in order to take into account the diversity of opinions. in the last step, the plural visions and their consequences on some human values provided by the matrix allow to draw the guidelines for integrating human values throughout the design process and this development by following the principles of the "value sensitive design" (vsd) introduced by batya friedman and her colleagues in the late 1980s [15] . the vsd theory has influenced the dutch approach to responsible innovation [16] . in this approach, human values are integrated throughout the design process and this development is done using a threephase survey: conceptual, empirical and technological [17] . there are many different methods of applying this theoretical approach in practice to real-world problems [18] . among the advice given by the authors, they recommend starting with a value, a technology or a context of use. then they propose to identify the direct and indirect stakeholders, as well as the benefits and harms for each stakeholder group. the benefits and harms are then mapped onto the corresponding values, allowing the identification of potential value conflicts. with the advent of the internet, it was predicted that this would be an opportunity for musical creation aimed at a niche audience. unfortunately, it did not happen. apart from a few notable exceptions, access to music via the internet has only strengthened trends: popular music, artists or genres have become even more popular, and lesser-known artists have found it even more difficult to find their audience, producing an impoverishment of musical diversity [19] . one of the reasons for the lack of diversity is linked to what eli pariser calls the "filter bubble" [6] . personalized recommendations are based on the processing of big data: the algorithms recommend to individuals products that are similar, or products appreciated by individuals sharing common traits, or popular products. with machine learning, recommendation algorithms will identify people's profiles, extract the most prototypical traits, and make only recommendations corresponding to a caricature of everyone's interests. the modus operandi of the recommender systems may be suitable for mainstream consumers who want to go directly to the greatest hits. however, this "superstar effect" -where the winner takes all [19] -impoverishes musical diversity and does not allow musical niches to emerge, ending up stifling a musical creativity that can no longer reconnect with its audience. if the digital music market is held only by major record labels supporting a musical offer limited to a few superstars, are the music and musical artists produced by independent record labels destined to disappear? in addition, we have become accustomed to paying the container (e.g., a subscription to an internet service provider, a nice smartphone) but not the cultural content as such (e.g., music and videos on online video-sharing platform, mobile applications on app stores). as the new digital use is mainly free, how can we make people accept to pay to access to unknown musical content produced by indie labels? according to the methodology proposed here, the first step consists of a response from the economic and social world. such a response exists in the form of a company offering the general public innovative solutions to discover independent creations. the french social economy company 1dlab 4 has decided to take up this challenge. to be able to give an answer to the question of how to support the varied musical creation produced by independent record labels, this company has found a two-level solution. the first level solution is economic. the company proposes a fairer remuneration model to the creators (artists and content producers). the business plan works according to a businessto-many model (b2m). the company provides a music streaming platform (1dtouch, dimusic) but also works in the physical world to build a network of places and actors for favoring new ways of sharing, meeting, discovering and creating a cultural diversity. the streaming platform access is possible by a subscription fee paid by the project located partners (libraries, showrooms, work's councils, administrative divisions...) to their members. the second level solution was proposed in the phd work of pierre-rené lhérisson, supervised by pierre maret and the author of this paper: it is a technical innovation allowing the general public to discover pleasant independent creations with bold recommendation tools that has been designed following an ethics-by-design approach [20] . the views on values such as music (or more generally to culture as a whole), equity, diversity, trust and usefulness are very different depending on whether the stakeholders 4 1dlab -innovation, culture & territories: http://en.1d-lab.eu/ are the consumers (who listen to music), the creators of cultural contents (artists), the media services providers or the independent record labels. following the second step of our methodology, we build an ethical matrix (table i) allowing to see how to develop an innovative solution capable of meeting all the criteria. in the third step of the methodology, we follow an approach inspired by the vsd theory to integrate early in the design the values defined in the previous step for the development of a fair tool for recommending indie content. first of all, we developed a distance measure between musical items (music, songs, artists, music genres) based on different criteria (text information of the music, properties extracted from the sound signal, musical classification performed by experts) as well as on the perceptual distance sensation obtained through an experiment with listeners. then we proposed an "optimal diversity function" using this multicriteria distance measure in order to find a compromise between the values diversity and efficiency (or usefulness). to get this optimal diversity function, we projected the distances between items on the (negative) mexican hat wavelet function to establish clear distinction between similar, dissimilar, and sufficiently dissimilar items ( figure 1 ). 5 fig. 1 . mexican hat and optimal diversity function. the optimal diversity is obtained when the items are not perceived too similar nor too dissimilar. for example, a listener enjoys trip hop music, a musical genre resulting from the fusion of hip hop and electronic music. based on this information, it is unreasonable to recommend music located at other ends of the spectrum of musical genres (e.g., classical music or hard rock). on the contrary, it is more relevant for the listener to recommend slightly different items, such as music of the acid house, indie soul or dub type, depending on whether the musical orientation is respectively rather in the direction of jazz, funk music or reggae (figure 2 ). each item (music, song, artist or musical genre) is located in a multidimensional space where it is always possible to find other items at more or less close distances, therefore of a more or less optimal diversity value. in order to guarantee respect for the equity value, it is thus possible to orient the sense fig. 2 . musical genres and optimal diversity. compared to a given item (here, trip hop genre), the diversity will be optimal when it is far enough from the item in question, but not too much (e.g., acid house, electro jazz, dub or indie soul can be recommended genres because the diversity is optimal). of diversity towards basins of attraction where artists are less recommended than others in order to make them discovered. to maintain the trust of the user to the recommendations made by the streaming platform, the system indicates to the user that the proposed recommendations are bold, i.e., they are not targeting to the music that the listener is used to listening to, but that there is a chance that the user may still like it. if, after having tried these recommended musics in a daring way, the listeners express their dissatisfaction, the personalization can adapt by decreasing the distance of optimum diversity and the system can thus propose musics closer to what they already appreciate. if, on the other hand, the listeners have eclectic tastes and want bolder discoveries, the distance of optimum diversity can increase for these people. finally, by taking into account the opinions of the various players in a music platform system at a very early stage, it is possible to propose an innovative solution guaranteeing respect for a priori contradictory ethical values. iv. case study 2: research-paper recommender systems in scientific digital libraries access to knowledge is a major problem for human beings. this issue affects populations of all ages and at all levels: education for all the children of the planet, fight against (digital) illiteracy, and access to expert knowledge for all scientists, etc. in the latter case, there are notable initiatives that are part of an open science perspective, but articles published in scientific journals or renowned conference proceedings still remain mainly the property of scientific publishers and their access is most often made by purchasing articles or by subscribing to the publisher's platform. yet this access to knowledge is necessary for the production of knowledge because researchers are science prosumers: researchers "consume" and "produce" knowledge, and this activity takes one of the two following paths: exploration or exploitation. in the exploitation phase, researchers use their existing knowledge to produce new knowledge by following a discovery process specific to their discipline. in the exploratory phase, researchers update their knowledge by discussing with colleagues in their laboratory, reading books or scientific articles, or attending seminars and conferences. exploratory research is an essential phase of researcher's activity [21] , and exploitation and exploration are two complementary phases that feed on each other. in addition to the cost of accessing these sources of knowledge, even if these scientific digital platforms are multidisciplinary, the way they are designed and the means they offer to access interesting information will not promote the knowledge transfer between disciplines. each scientific discipline has its own vocabulary, its own jargon. without knowing the good keywords to enter in the search engine of the digital library platform, scientists thus find themselves isolated in a "filter bubble" [6] preventing them from accessing the content of articles from other disciplines. however, results can be obtained when the research transcends the limits of a discipline. it is well established that exchanges (knowledge transfers) between disciplines are very fruitful [22] . faced with these problems, the french state research organization (cnrs) has proposed a solution that is part of the social and economic world -as the first step in our methodology-by launching an ambitious project in two parts. the first part concerns the creation of a scientific digital library accessible to all french research institutions called istex 6 [23] , resulting from a massive resource acquisition policy, obtained by entering into operating contracts with a lot of international and french-speaking publishers. the second part concerns the exploitation of these istex resources with text and data mining techniques [24] . together with the phd student hussein al-natsheh and with djamel a. zighed (cosupervisor), we had the opportunity to lead a 4-people team working on this goal: provide assistance to users of istex resource by offering recommender systems that promote disciplinary diversity. for researchers (whether they produce or seek knowledge), scientific publishers, research organizations and public authorities, interests diverge greatly, and their respective views on values such as knowledge, equity, diversity, trust or usefulness do not involve the same issues, which can lead to conflicts, as shown on the ethical matrix (second step: table ii ). in the third step of the methodology, we integrate the values of the ethical matrix into the design of an innovative solution, following the vsd approach. here, we focus on the value of diversity which is threatened by the value of a precisionbased usefulness. as part of the projects funded by the cnrs and intended to benefit from the istex platform, we have proposed to develop a research-paper recommender system whose purpose is to favor diversity. instead of being focused on the sole criterion of accuracy of results, as most other systems do [25] , we propose a system able to recommend scientific papers through a semantic similarity model [26] based on computational linguistics methods and word embedding techniques [27] , as shown on figure 3 . in this system, the user does not enter a set of keywords but a list of articles dealing with a given subject of interest. the content (abstract, plain text) of these articles is projected in a multidimensional space constructed by the text representation of the scientific papers of the multidisciplinary digital library (blue arrow in figure 3 ). as the representation space is constructed by semantic similarities resulting from a proximity of the presence of terms in the texts, the more or less distant neighborhood will bring articles to recommend (arrows in green) comprising more or less diversity with the input articles. in the experiments we launched, we collaborated with sports science researchers who were working on a test to detect motor skills. thanks to our system, we have been able to recommend research papers to the sports science researchers -deemed relevant by them-from different disciplines such as psychology, linguistics and defectology (e.g., studies on sign language used by deaf people, another form of complex movements with meaning). this kind of transfer of knowledge fig. 3 . semantic-similarity based recommendation of scientific papers. first, a vector representation of the scientific terms is created with word embedding techniques from the contents of the research articles of the library (represented by white dots in a multidimensional space). second, the interesting papers provided by the user are projected in the vector representation space (represented by red dots) to define a target area of interest. third, the neighboring articles of the target (black dots) in the vector representation space are activated to recommend diverse items, sometimes coming from other disciplines, but connected through semantic links to the input papers. between disciplines has hitherto never been established. by examining in detail the different values involved using the ethical matrix, it was possible to explain them better and to realize that the usefulness of the system does not necessarily imply the quest for precision: diversity also matters. the early integration of the diversity value in the design of the research-paper recommender system allows researchers in the exploratory phase to have access to knowledge from various sources. with this recommender system, istex platform promotes scientific diversity and allows all scientific disciplines to be represented with equity and all their articles to be recommended. from an epistemological point of view, this diversity is essential because otherwise a researcher using a scientific digital library will only have access to sources of information from his/her own discipline. the result will be that the documents found will only confirm the initial point of view of the researcher using this digital library, thus calling into question one of the golden rules of science which is the property of "falsifiability" [28] . ai is often associated with robotization, and robotization can be understood as "automation" but also as "the process of turning a human being into a robot." that is why the arrival of ai can scare some individuals, giving rise to feelings of technophobia with the fear of mass unemployment due to jobs performed by robots or intelligent computer systems. moreover, there is a rejection of ai because this technology may be seen as a kind of automated and not human -if not "inhuman"-thinking. the species of the human being is homo sapiens, which means "wise man" in latin. the human being is not simply characterized as being intelligent, that is to say artificial intelligence -a modern approach the deep learning revolution amazon scraps secret ai recruiting tool that showed bias against women machine bias mindf*ck: cambridge analytica and the plot to break america the filter bubble: what the internet is hiding from you weapons of math destruction: how big data increases inequality and threatens democracy artificial intelligence and law: what do people really want? example of a french multidisciplinary working group a framework for the ethical analysis of novel foods: the ethical matrix formalism in ethics and non-formal ethics of values universals in the content and structure of values: theoretical advances and empirical tests in 20 countries montréal declaration for a responsible development of artificial intelligence responsible artificial intelligence: how to develop and use ai in a responsible way ethical matrix manual value sensitive design: shaping technology with moral imagination handbook of ethics, values, and technological design: sources, theory, values and application domains value sensitive design and information systems a survey of value sensitive design methods digital music and the "death of the long tail fair recommendations through diversity promotion exploratory search: from finding to understanding scientific discovery: computational explorations of the creative process scientific and technical information department -cnrs, white paper -open science in a digital republic white paper -open science in a digital republic -strategic guide research-paper recommender systems: a literature survey semantic search-by-examples for scientific topic corpus expansion in digital libraries distributed representations of words and phrases and their compositionality the logic of scientific discovery. london and new york: routledge classics having an ability to find solutions to complex problems, but also has a nature of a discerning, wise, and sensible being.the events that we are currently experiencing -covid-19 crisis, climate change-produce abrupt changes in all areas, bringing new challenges in the fields of health, economy, ecology, security, justice, science, culture. . . with the collective intelligence of human beings, let ai and ict be the means pushing us to be innovative in order to respond to these problems in an appropriate manner, and may this response be done wisely in order to guarantee respect for human values. key: cord-023913-pnjhi8cu authors: foreman, stephen; kilsdonk, joseph; boggs, kelly; mouradian, wendy e.; boulter, suzanne; casamassimo, paul; powell, valerie j. h.; piraino, beth; shoemaker, wells; kovarik, jessica; waxman, evan(jake); cheriyan, biju; hood, henry; farman, allan g.; holder, matthew; torres-urquidy, miguel humberto; walji, muhammad f.; acharya, amit; mahnke, andrea; chyou, po-huang; din, franklin m.; schrodi, steven j. title: broader considerations of medical and dental data integration date: 2011-10-08 journal: integration of medical and dental care and patient data doi: 10.1007/978-1-4471-2185-5_4 sha: doc_id: 23913 cord_uid: pnjhi8cu dental health insurance coverage in the united states is either nonexistent (medicare and the uninsured), spotty (medicaid) and limited (most employer-based private benefit plans). perhaps as a result, dental health in the united states is not good. what public policy makers may not appreciate is that this may well be impacting medical care costs in a way that improved dental benefits would produce a substantial return to investment in expanded dental insurance coverage. have been rising at double digit rates. most employers have been dropping health care coverage rather than expanding it ( kaiser family foundation 2010 ) . medicare trust funds are bankrupt (social security and medicare boards of trustees 2011 ). adding coverage would exacerbate an already alarming problem. medicaid funding is a major source of state government defi cits. many states are slashing medicaid coverage during this time of crisis (wolf 2010). improving medicaid dental coverage during times of budget crisis would meet substantial political resistance. strikingly, strong and increasing evidence suggests relationships between oral health and a range of chronic illnesses. for example, recent fi ndings show relationships between periodontal infl ammatory conditions and diabetes, myocardial infarction, coronary artery disease, stroke, preeclampsia and rheumatoid arthritis. this suggests that improved oral health may well have the potential to reduce the incidence of chronic diseases as well as their complications. if chronic disease incidence is reduced it may be possible to avoid medical care costs related to treating them. it would be important to know more about the extent to which improved oral health could reduce health care costs and improve lives. there are few, if any, studies of the costs of providing medicare dental benefi ts, the costs of improving the medicaid dental benefi t or the cost of providing dental insurance to the uninsured. there are a few studies that indicate that periodontitis increases medical care costs, perhaps by as much as 20% (ide et al. 2007; albert et al. 2006) . 1 ideally there should be a controlled study to assess the benefi t of providing dental coverage through a government payer system. for a preliminary inquiry we can consider work already done and using some cost and benefi t estimates, determine whether it is possible that benefi ts of extending dental coverage may outweigh costs. the failure of medicare to cover dental care has engendered some (albeit not much) public debate. in 2003, congress enacted the medicare prescription drug, improvement, and modernization act (medicare part d). by 2009 medicare provided $56.6 billion in benefi t payments for outpatient prescription drugs and medicaid paid 15.7 billion for outpatient prescription drugs (center for medicare and medicaid services 2010 ) . benefi ciaries provided billions more in the form of monthly part d premiums. the expense of the medicare prescription drug program and the controversy surrounding its enactment may well have eroded public support for increased medicare coverage. so while there has been no shortage of effort paid to improving medicare, the one common theme in all of the recent initiatives is that dental care has been conspicuously 1 a new study by hedlund, jeffcoat, genco and tanna funded by cigna of patients with type ii diabetes and periodontal disease found that medical costs of patients who received maintenance therapy were $2483.51 per year lower than patients who did not. cigna, research from cigna supports potential association between treated gum disease and reduced medical costs for people with diabetes, http://newsroom.cigna.com/newsreleases/research-from-cigna-supports-potential-association-between-treated-gum-disease-and-reduced-medical-costs-for-people-with-diabetes. omitted. as a result, 43 million medicare recipients in 2009 (us census bureau 2011 ) continue to have no dental insurance coverage through medicare. 2 medicaid dental coverage is an optional benefi t that states may or may not elect to provide. in medicaid, both the state and the federal government provide funds to cover healthcare services to eligible patients. the bulk of the money comes from the federal government. because the medicaid dollars are limited and coverage for systemic diseases has precedence, medicaid coverage of dental care has been spotty. even where it has been provided, payments to dental providers have been so low as to make it diffi cult or impossible for medicaid benefi ciaries to obtain adequate dental care (broadwater 2009 ) . the 2008 recession increased the number of medicaid eligible individuals nationwide. further, the federal budget defi cits of the past few years have reduced the federal contribution to state medicaid programs. the combination of increases in the number of benefi ciaries and diminished revenues has caused a number of states to eliminate or curtail medicaid dental coverage (ehow 2011 ; mullins et al. 2004 ) . the result, 49 million medicaid benefi ciaries in the us (us census bureau 2011 ) in 2009 either had no dental insurance coverage or inadequate coverage. approximately 52 million people in the united states do not have health insurance (kaiser family foundation 2010 ) . presumably, they have no dental insurance either. further, not every employer provides dental insurance. a 1995 cdc survey found that 44.3% of adults do not have dental insurance coverage (centers for disease control 1997 ) . a 2006 montana survey found that 53% of employers who offer health insurance do not offer dental insurance coverage (montana business journal 2006 ) . in 2009 there were approximately 202 million people enrolled in health insurance plans (us census bureau 2011 ) . if half (a rough combination of the cdc and montana percentages) of them do not have dental insurance it is likely that an additional 101 million (nonelderly, non-poor) people in the us do not have dental insurance coverage. finally, the term "dental insurance" is actually a misnomer. 3 dental policies cover routine treatments, offer discounts for more complex treatment and impose a low yearly on total payments. in fact, it has been called "part insurance, part prepayment and part large volume discount" (manski 2001 ) . effectively, many (if not most) people who have dental insurance fi nd it coverage to be quite restrictive. for example, many impose a small yearly cap ($1,500 is common) or large coinsurance amounts (50% for orthodontia, for example) (rubenstein 2005 ) . even with discounts it is easy for many people to exceed the annual limit. given the lack of dental insurance coverage it is not surprising that the status of oral health in the us is not particularly good. in 2002 approximately 26.5% of adults between the ages of 35 and 44 had untreated caries, 42% had decayed, missing and fi lled tooth surfaces and more than one-half of adults had gingival bleeding (dental, oral and craniofacial data resource center of the national institute of dental and craniofacial research 2002 ) . three fourths of adults in the us have gingivitis and 35% have periodontitis (mealey and rose 2008 ) . if these levels of untreated disease were applied to most systemic diseases, there would be public outcry. over the past decade evidence has been building that there is a relationship between dental disease, particularly periodontal disease, and chronic illnesses. mealey and rose note that there is strong evidence that "diabetes is a risk factor for gingivitis and periodontitis and that the level of glycemic control appears to be an important determinant in this relationship" (mealey and rose 2008 ) . moreover, diabetics have a six times greater risk for worsening of glycemic control over time compared to those without periodontitis and, periodontitis is associated with an increased risk for diabetic complications. for example, in one study more than 80% of diabetics with periodontitis experienced one or more major cardiovascular, cerebrovascular or peripheral vascular events compared to 21% of the diabetic subjects without periodontitis (thorstensson et al. 1996 ) . also, a longitudinal study of 600 type 2 diabetics found that the death rate from ischemic heart disease was 2.3 times higher in subjects with severe periodontitis and the death rate from diabetic nephropathy was 8.5 times higher (saremi et al. 2005 ) . clinical trials have demonstrated that treatment of periodontal disease improved glycemic control in diabetics (miller et al. 1992 ) . moreover, investigations have found an association between periodontal disease and the development of glucose intolerance in non-diabetics (saito et al. 2004 ) . while it is diffi cult to establish causality and it is possible that other factors infl uence periodontal disease and medical complications, these studies suggest that treatment of periodontitis substantially improves health and greatly reduces medical complications related to diabetes. similarly, periodontitis is associated with cardiovascular disease and its complications including ischemia, atherosclerosis, myocardial infarction and stroke. a study by slade and colleagues found both a relationship between periodontitis and elevated serum c-reactive protein levels (systemic marker of infl ammation and documented risk factor for cardiovascular disease) as well as a relationship among body mass index, periodontitis and crp concentrations (slade et al. 2003 ) . hung and colleagues evaluated the association between baseline number of teeth and incident tooth loss and peripheral arterial disease. they determined that incident tooth loss was signifi cantly associated with pad, particularly among men with periodontal disease potentially implying an oral infection-infl ammation pathway (hund et al. 2003 ) . the same group of researchers used the population enrolled in the health professionals' follow-up study (41,000 men free of cardiovascular disease and diabetes at baseline) to assess the relationship between tooth loss and periodontal disease and ischemic stroke. controlling for a wide range of factors including smoking, obesity, and dietary factors, the researchers found a "modest" association between baseline periodontal disease history and ischemic stroke . as early as 1993 destefano and colleagues found that among 9760 subjects, those with periodontitis had a 25% increased risk of coronary heart disease relative to those without. the association was particularly high among young men. the authors questioned whether the association was causal or not, suggesting that it might be a more general indicator of personal hygiene and possibly health care practices (destefano et al. 1993 ) . in 2000 wu and colleagues used data from the first national health and nutrition examination survey and its epidemiologic follow-up study to examine the association between periodontal disease and cerebrovascular accidents. the study found that periodontitis was a signifi cant risk factor for total cva, in particular, for non-hemorrhagic stroke (wu et al. 2000 ) . in addition to diabetes and coronary artery disease, associations have been found between periodontal disease and rheumatoid arthritis and respiratory disease. this is not surprising given the role of periodontal disease in the production of infl ammation related proteins. dissick and colleagues conducted a pilot study of the associate ion between periodontitis and rheumatoid arthritis using multivariate regression and chi square tests. they found that periodontitis was more prevalent in patients with rheumatoid arthritis than in the control group and that patients who were seropositive for rheumatoid factor were more likely to have moderate to severe periodontitis than patients who were rf negative and also that patients who were positive for anti-cyclic citrullinated peptide antibodies were more likely to have moderate to severe periodontitis (redman et al. 2010 ) . paju and scannapeico investigated the association among oral biofi lms, periodontitis and pulmonary infections. they noted that periodontitis seems to infl uence the incidence of pulmonary infections, particularly nosocomial pneumonia in high-risk subjects and that improved oral hygiene has been shown to reduce the occurrence of nosocomial pneumonia. they found that oral colonization by potential respiratory pathogens, for possibly fostered by periodontitis and possibly by bacteria specifi c to the oral cavity contribute to pulmonary infections (paju and scannapeico 2007 ) . the implications for these fi ndings are profound. professionally, they suggest that managing patients with chronic illness and periodontal disease will require teamwork and a deeper knowledge base for dentists and for physicians (mealey and rose 2008 ) . dentists will need to be alert for early signs of chronic illness among their patients and physicians will need to be alert for signs of dental disease. both will need to consider wider treatment options than their specialty indicates. dentistry and medicine have operated as professional silos in the past. the relationship between dental disease and chronic medical conditions suggests that continued separation is detrimental to patient centered care. beyond treatment implications, there are extremely important health policy concerns. if treatment of periodontitis and other dental problems leads to reduced incidence of chronic illness, fewer complications from chronic diseases and reduced morbidity among chronically ill patients, increased access to dental services could signifi cantly reduce health care costs. the diseases associated with periodontitis are among the most common illnesses, the fastest growing and the most expensive diseases that we treat. a recent robert wood johnson report notes that approximately 141 million americans have one or more chronic conditions, that the number of people with chronic conditions is expected to increase by 1% per year for the foreseeable future and that the most common chronic conditions include hypertension, disorders of lipid metabolism, upper respiratory disease, joint disorders, heart disease, diabetes, cardiovascular disorders, asthma and chronic respiratory infections (anderson 2010 ) (see fig . 4 .1 ). one in four americans has multiple chronic conditions. ninety-one percent of adults aged 65 and older have at least one chronic condition and 73% have two or more of them (anderson 2010 ) . people with chronic conditions account for 84% of all healthcare spending. seventy eight percent of private health insurance spending is attributable to the 48% of privately insured persons with chronic conditions. seventy three percent of healthcare spending for the uninsured is for care received by the one third of uninsured people who have chronic conditions. seventy nine percent of medicaid spending goes to care for the 40% of non-institutionalized benefi ciaries who have chronic conditions (anderson 2010 ) (see fig. 4 .2 ). further, health care spending increases with the number of chronic conditions (anderson 2010 ) (see fig. 4 .3 ). more than three fi fths of healthcare spending (two thirds of medicare spending) goes to care for people with multiple chronic conditions. those with multiple chronic conditions are more likely to be hospitalized, fi ll more prescriptions, and have more physician visits (anderson 2010 ) . in 2002 the american diabetes association estimated direct medical expenditures for diabetes at $91.8 billion: $23.2 billion for diabetes care, $24.6 billion for chronic complications and $44.1 billion for excess prevalence of general medical conditions. approximately 52% of direct medical expenditures were incurred by people over 65. indirect expenditures included lost workdays, restricted productivity mortality and permanent disability -a total of $39.8 billion. all told, diabetes was found to be responsible for $160 billion of $865 billion in total expenditures. per capita medical expenditures totaled $13,000 annually for people with diabetes and $2600 for people without diabetes (hogan et al. 2002 ) . more recently, dall and colleagues estimated that the us national economic burden of prediabetes and diabetes had reached $218 billion in 2007, $153 million in higher medical costs and $65 billion in reduced productivity. annual cost per case was estimated at $2,900 for undiagnosed diabetes and 10,000 for type 2 diabetes (dall et al. 2010 ) . the costs of caring for people with diabetes have risen both because the numbers of diabetics has been increasing and because the per capita costs of care have increased. the number of diabetics increased from 5.8 million on 1980 to 14.7 million in 2004 (ashkenazy and abrahamson 2006 ) . a recent report by the unitedhealth group center for health reform & modernization provides a dire estimation -that more than 50% of adult americans could have diabetes (15%) or prediabetes (37%) by 2020 at a cost of $3.35 trillion over the decade. this compares with current estimates of 12% of the population with diabetes and 28% with prediabetes, or 40%. these estimates conclude that diabetes and prediabetes will account for 10% of total healthcare spending in 2020 at an annual cost of $500 billion, up from an estimated $194 billion in 2010 (unitedhealth center for health reform and modernization 2010 ) . average annual spending over the next decade by payer type is $103 billion for private health insurance, $204 billion for medicare, $11 billion for medicaid and $16.6 billion for the uninsured. what about cardiovascular disease and rheumatoid arthritis? among the top ten health conditions requiring treatment for medicare benefi ciaries in 2006 approximately 50% of benefi ciaries suffered from hypertension, 25% from heart conditions, 33% had hyperlipidemia 24% had copd, 23% had osteoarthritis and 22% had diabetes (thorpe et al. 2010 ) . the american heart association estimates the 2010 cost of cardiovascular disease and stroke to be $324 billion in direct expenditures and $41.7 billion for productivity losses due to morbidity and $137.4 billion in lost productivity due to mortality (present value of lost wages at 3%) (lloyd) . the centers for disease control estimates that during 2007-2009 50 million americans had selfreported doctor diagnosed arthritis, 21 million of them with activity limitations (cheng et al. 2010 ) . cisternas and colleagues estimated that total expenditures by us adults with arthritis increased from $252 billion in 1997 to $353 billion in 2005. most of the increase was attributable to people who had co-occurring chronic conditions (cisternas et al. 2009 ) . the cisternas study appears to aggregate all medical care expenditures by people with arthritis (which would include expenditures to treat diabetes and cardiovascular disease). an earlier cdc study focused on the direct and indirect costs in 2003 attributable to arthritis that estimated $80.8 billion in direct costs (medical expenditures) and $47 billion in indirect costs (lost earnings) (yelin et al. 2007 ) . in short, current cost estimates for direct health care expenditures (excluding productivity losses) related to diabetes are approximately $190 billion, for cardiovascular treatment, $324 billion, and for rheumatoid arthritis, approximately $111 billion (estimating that the $80.8 billion in 2003 costs have grown approximately 6% per year), a total of $625 billion of the $2.6 trillion that will be spent in the us in 2010. moreover, given current growth in the prevalence of diabetes, the unitedhealth estimate of $500 million in 2020 spending for diabetes alone is not unreasonable. if health care costs attributable to diabetes, cardiovascular disease and rheumatoid arthritis only increase by 100% over the next decade (even given added demand produced by the aging baby boomer population), annual costs of these chronic diseases will exceed $1.2 trillion in 2020. if we use the unitedhealth estimates for the proportions of diabetes costs paid by private insurance (48%), medicare (38%), medicaid (6%) and the uninsured (8%) and estimate total costs based on the 2010 studies projecting a 50% increase in 5 years and a 100% increase in 10 years we can obtain an estimate of future costs for treating diabetes, cardiovascular disease and arthritis. table 4 .1 set forth below, summarizes these cost estimates. by 2020 medicare costs for these chronic illnesses would be approximately $475 billion. the estimated costs to medicaid will be approximately $75 billion. the costs for the uninsured will be approximately $100 billion. any intervention that has the potential to substantially reduce these costs will produce meaningful results. unfortunately, even though there had been a substantial numbers of studies that show relationships between dental disease and chronic illness that are have been very few studies that actually test whether improved dental treatment reduces the incidence of chronic illness and complications due to chronic illness. the potential for large health care cost savings through an active and aggressive program of dental care is so large that such studies are clearly indicated. suppose, for example, that 10% of all medical care costs required to treat diabetes, cardiovascular disease and arthritis could be avoided through an active aggressive program of dental care. 4 what this would mean is that in 2020 private health insurers could see a $60 billion reduction in healthcare costs, medicare would see a $47.5 billion reduction and medicaid pay $7.5 billion reduction. recent health reform has provided for the issuance of health insurance to the uninsured by state exchanges. aggressive dental care that saved 10% of costs attributable to diabetes, cardiovascular disease and arthritis could save the exchanges $10 billion per year. and, if greater proportions of costs can be saved or if the 2020 estimates of costs are low, potential benefi ts will be even larger. once again, it would be important to know whether aggressive dental care could produce such savings and how much. ide and colleagues found that people who were treated for periodontitis incurred 21% higher health care costs than those who were free of periodontal disease (ide et al. 2007) . similarly, albert, et al., found medical costs associated with diabetes, cardiovascular disease and cerebrovascular disease were signifi cantly higher for enrollees who were treated for periodontitis than for other dental conditions (albert et al. 2006) . additional studies of this nature would be important to support a measured approach to expanding dental coverage. so what do we mean by an aggressive dental treatment plan? suppose we were to provide dental insurance to all medicare benefi ciaries at the level of current private dental insurance coverage and strongly encourage benefi ciaries to receive dental treatment. suppose we were to provide for medicaid payment for all benefi ciaries at the level of current private dental insurance coverage. suppose health care insurers provided dental coverage in order to reduce their costs and that such coverage was consistent with current private dental insurance coverage. suppose health insurance companies, understanding the benefi ts from dental care, were to require their private employer customers to cover the costs of dental care. how much would all of this cost? how would it compare to the benefi ts that may be available? in order to estimate the potential costs of providing enhanced coverage for dental care we start use the cms estimates of national health care spending for dental services and statistical abstract of the us estimates for medicare enrollment, medicaid enrollment, private health insurance enrollment and uninsured persons. based on the estimate that half of private employers with health insurance provided dental insurance coverage we estimate that of the private health insurance enrollment one half would have dental insurance coverage and one half would not. table 4 .2 sets forth the national health care expenditures for dental services in millions and enrollment in private dental plans, medicare, medicaid, the uninsured without health insurance and dental insurance, the uninsured with health insurance and dual eligibles. from this we derive a cost per enrollee for private dental insurance, medicare dental benefi ts and medicaid dental benefi ts. in order to estimate the annual cost of providing full dental coverage to medicare benefi ciaries we subtracted dual eligibles (who receive some dental insurance) from total medicare enrollees to determine the number of persons who would need coverage. in our 2009 example there were 43 million medicare benefi ciaries including 9 million dual eligibles. accordingly, the estimates would cover the 34 million medicare benefi ciaries that are not dual eligible at a cost equal to the per capita cost of private dental insurance ($494.66) less amounts that medicare is already paying for dental services ($6.73 per person). the result provides an estimate of the cost of covering all medicare benefi ciaries for dental services at a level equivalent to private health insurance. using the 2009 example the cost of providing full dental insurance coverage to medicare benefi ciaries would have been $16.6 billion. in addition, we used the cms national health expenditure fi gures to determine administrative costs for private health insurance, medicare and medicaid as a percentage of program expenditures for medical care. we found that the administrative costs of the medicare program were 6.2% on average for 1966-2009. in order to fully estimate the cost of medicare dental coverage we added 6.2% to the cost health insurers will be in the same position as medicare and medicaid regarding dental coverage. if quality dental coverage saves health care costs attributable to diabetes, cardiovascular disease and rheumatoid arthritis then the exchanges will have an incentive to provide quality dental coverage to reduce costs. accordingly, we estimated the cost of providing dental coverage equivalent to private dental insurance coverage through the exchanges. again we assume that the costs of such coverage will be equivalent to the number of uninsured persons multiplied by the annual per capita cost of coverage. 5 for the 2009 example, this would refl ect coverage for 52 million people at $494.66 per person, a total of $24.9 billion. with administrative costs, the cost of providing dental insurance coverage to the uninsured at a level equivalent to private dental coverage would be $26.8 billion. finally, given the evidence that improved dental care has the potential to reduce health care costs private health insurers may wish to expand health insurance to cover dental care. 6 here, we estimate the cost of providing dental insurance to the 50% of the workforce whose employers currently do not provide dental insurance benefi ts. once again, we multiply the number of covered lives by the estimated annual per capita cost. for the 2009 example we estimate 101 million adults will receive dental coverage at $495 per person: $50 billion for dental services and $3.8 billion for administrative costs or a total of $53.8 billion. of course, as noted a number of times above, these estimates are based on providing full "universal" dental insurance coverage at levels equivalent to current benefi t levels for private dental insurance. it may be that an appropriate package of dental services that deals specifi cally with periodontitis can be provided for less than the full cost of private dental insurance. once again, further research should provide better information. 7 5 the health reform law does not attempt to provide coverage to all 52 million people without health insurance. estimates are that only 31 million people will be covered by the bill. even though this is the case we prepare our estimates using all 52 million uninsured americans. 6 indeed, the failure of 50% of employers to cover dental services may well constitute a classic externality in the market for health insurance. internalizing this externality may well provide better effi ciency. 7 it is also possible that dental care for persons with greater incidence of chronic illness as is the case with medicare benefi ciaries may require even higher levels of spending per benefi ciary. again, it would be good to know scientifi cally if this is the case. as noted in sect. 6 above, 2010 costs for diabetes, cardiovascular disease and arthritis will be $300 billion for private health insurance, $238 billion for medicare, $38 billion for medicaid and $50 billion for the uninsured. costs of providing "full" dental coverage will be $17.6 billion for medicare, $18.2 billion for medicaid, $26.8 billion for the uninsured and $53.8 billion for private health insurance. given this, if 7.4% or more of the medicare costs can be "saved" through improved dental care, medicaid dental insurance will pay for itself and will provide a positive return on investment. see table 4 .3 . similarly, private health insurers could justify providing dental insurance coverage to employees who do not have it so long as they spend 17.9% or more of their chronic care costs for diabetes, cardiovascular disease and arthritis. on the other hand, it would appear that medicaid expansion would require cost savings of approximately 48% and that health care insurance coverage of the uninsured would require savings of approximately 54% in order to justify coverage. while it is possible, it may not be likely that full dental coverage would be justifi ed for these programs. of course, these estimates do not consider indirect costs in the form of lost wages or premature death. these costs are externalities to the health insurance programs. to the extent that they represent a social benefi t that a national dental insurance program might internalize, it would be appropriate to consider their impact in the cost-benefi t analysis. in any event, better understanding of the potential for deriving savings in health insurance costs related to chronic diseases like diabetes, cardiovascular disease and arthritis would be crucial to any determination whether to expand insurance coverage for dental care. heretofore the case for expanding medicare coverage to include dental care has taken the form of "benefi t" to patients rather than benefi t to health insurance programs and society and has been cast in emotional and political terms. for example, oral health america grades "america's commitment to providing oral health access to the elderly" (oral health america 2003 ) . in truth, there is no american commitment to providing oral health access to any age group, much less the elderly. rubenstein notes that "at least one commentator has suggested that the dental profession should join with senior citizen groups when the time is right to ask congress to expand medicare to cover oral health" (rubenstein 2005 ) . rubenstein emphasizes that "calls for action" are "mere words" unless they are accompanied by political actions that health policy professionals and the dental profession must help promote (rubenstein 2005 ) . another commentator has suggested that "as soon as the debate over medicare prescription drug coverage and, the debate to provide dental care coverage for the elderly may soon begin" (manski 2001 ) . rubenstein, again suggests that "the dental community must convince americans, and particularly aging boomers, that oral health is integral to all health, and for that reason, retiree dental benefi ts are an important issue". in truth, a decade of defi cit spending and public distaste for out of control program costs in the medicare and medicaid programs as well as the unpopularity of the process that was used to provide medicare prescription drug coverage (with perceived abuses by the health insurance and drug lobbies) and national health reform makes it unlikely that the public would be willing to approve expansions in insurance coverage for dental care "for its own sake" or "as the right thing" or to "benefi t seniors." what this political climate has produced is an arena in which a good idea that could provide appropriate return on investment for society might well be rejected out of hand based on political history of health insurance coverage. as a result, it is incumbent on policymakers, medical and dental research scientists and health economists to investigate and confi rm the potential savings that expansion of dental insurance coverage has the potential to produce and to develop hard evidence regarding potential costs of the expansion prior to, not as a part of, political efforts aimed at dental coverage expansion. a responsible, well informed effort to expand dental coverage may well go far to restore public confi dence in the health policy process. joseph kilsdonk and kelly boggs the adage of "putting your money where your mouth is" is often referenced when being challenged about public statements or claims. in this instance, we use it literally. in 2008 health care costs in us were $2.2 trillion. there have been numerous reports on health disparities, the burden of chronic diseases, increasing healthcare costs and the need for change. long-term economic benefi ts associated with the cost of care are dependent upon integrating oral health with medicine. this is particularly true as it relates to the management of those conditions which impact the economics of healthcare the most. as examples, 96% of medicare costs and 83% of medicaid costs are in managing chronic health conditions (partnership for solutions national program offi ce 2004 ) . more than 40% of the u.s. population has one or more chronic condition (cartwright-smith 2011 ) and in 2006, 76% of medicare spending was on patients with fi ve or more chronic diseases (swartz 2011 ) . effective management of health care resources and information are critical to the economic well-being of our healthcare system. we can no longer afford to manage care in isolation. integration of care between medicine and dentistry holds much promise in terms of reducing the cost of care and an integrated medical-dental electronic healthcare record (iehr) is the vehicle that will lead to downstream cost savings. in the united states the center for medicare & medicaid services (cms) has conducted demonstration projects around chronic disease management. section 121 of the benefi ts improvement and protection act of 2000 mandated cms to conduct a disease management demonstration project. april 1, 2005, as an effort to reduce the cost of care and improve quality associated with chronic diseases, cms partnered with ten premier health systems to effectively manage chronic diseases in a medicare physician group practice demonstration (pgp). it was the fi rst pay-for-performance initiative for physicians under the medicare program (center for medicare and medicaid services 2010 ) . it involved giving additional payments to providers based on practice effi ciency and improved management of chronically ill patients. participants included ten multispecialty group practices nationwide, with a total of more than 5,000 physicians, who care for more than 200,000 medicare benefi ciaries (frieden 2006 ) . the chronic diseases that were targeted were based on occurrence in the population and included diabetes, heart failure, coronary artery disease, and hypertension (frieden 2006 under the pgp, physician groups continued to be paid under regular medicare fee schedules and had the opportunity to share in savings from enhancements in patient care management. physician groups could earn performance payments which were divided between cost effi ciency for generating savings and performance on 32 quality measures phased in during the demonstration as follows: year 1, 10 measures, year 2, 27 measures and years 3 and 4 having 32 quality measures. for each of the 4 years only the university of michigan faculty group practice and marshfi eld clinic, earned performance payments for improving the quality and cost effi ciency of care. a large part of the success of this project was attributed to being able to extract, evaluate, and monitor key clinical data associated with the specifi c disease and to manage that data through an electronic health record (table 4 .4 ). during the third year of the demonstration project marshfi eld clinic, using a robust electronic health record succeeded in saving cms $23 million dollars; that's one clinic system in 1 year. as a result of such demonstration projects and as of this writing, cms is looking to establish accountable care organization's as the medical front runners to new care delivery methods for quality and cost control. accountable care organization (aco) is a term used to describe partnerships between healthcare providers to establish accountability and improved outcomes for the patients. in a cms workshop on october 5, 2010, don berwick, the administrator of cms, stated "an aco will put the patient and family at the center of all its activitiesâ�¦" an emerging model of an aco is the patient-centered medical home (pcmh). pcmh is at the center of many demonstration projects. acos were derived from studies piloted by cms. since funds provided by cms, do not cover routine dental care as part of the patient management or quality and cost objectives cms aco studies are limited if they become models for the pcmh, due to the exclusion of dental. more recently, organizations representing the major primary care specialtiesthe american academy of family practice, the american academy of pediatrics, the american osteopathic association, and the american college of physicianshave worked together to develop and endorse the concept of the "patient-centered medical home," a practice model that would more effectively support the core functions of primary care and the management of chronic disease (fisher 2008 ) . in 2011 geisinger health system, kaiser permanente, mayo clinic, intermountain healthcare and group health cooperative announced they will be creating a project called the care connectivity consortium. this project is intended to exchange patient information. although progressive in their approach their project does not include dental. these benefi ts however, are yet to be adapted in the arena of oral health. as of this writing, dentistry remains largely separate from medical reimbursement mechanisms such as shared billing, integrated consults, diagnosis, shared problem lists, and government coverage. for example, cms does not cover routine dental care. dentistry is also working to establish its own "dental home" with patients. however to reap the economic benefi ts of integrated care, a primary care "medical-dental" home is what needs to be created. according to an institute of oral health report ( 2010 ) it is widely accepted across the dental profession that oral health has a direct impact on systemic health, and increasingly, medical and dental care providers are building to bridge relationships that create treatment solutions. the case for medical and dental professionals' comanaging patients has been suggested for almost the past century, in 1926 william gies reported that "the frequency of periodic examination gives dentists exceptional opportunity to note early signs of many types of illnesses outside the domain of dentistry" (gies 1926 ) . as described by dr. richard nagelberg, dds "the convergence of dental and medical care is underway. our patients will be the benefi ciaries of this trend. for too long, we have provided dental care in a bubble, practicing -to a large degree -apart from other health-care providers. even when we consulted with our medical colleagues, it was to fi nd out if premedication was necessary, get clearance for treatment of a medically compromised patient, or fi nd out the hba1c level of a diabetic individual, rather than providing true patient co-management. we have made diagnoses and provided treatments without the benefi t of tests, reports, metrics, and other information that predict the likelihood of disease development and progression, as well as favorable treatment outcomes. we have practiced in this manner not due to negligence, but because of the limitations of tools that were available to us" (nagelberg 2011 ) . integrated medical/dental records need to be a tool in a providers' toolbox. in the case of marshfi eld clinic, dental was not included in their past cms demonstration project as dental is not a cms covered benefi t, and thus not part of the demonstration. however, as a leader in healthcare, the marshfi eld clinic recognizes the importance of data integration for both increased quality and cost savings. "marshfi eld clinic believes the best health care comes from an integrated dental/medical approach," said michael murphy, director, business development for cattails software. integration enhances communication between providers and can ultimately lead to better management of complex diseases with oral-systemic connection, avoidance of medical errors, and improved public health. while the cms pgp and other demonstration projects along with independent studies have shown to improve quality and reduce costs through integration, greater results may be afforded if studies are not done in isolation from dental data. in fact, if healthcare does not fi nd a way to manage the systemic nature of the 120 pathogens known to the oral cavity the economic impact and cost savings around chronic disease management will hit a ceiling. the economic opportunity of having clinical data for integrated decision making is readily identifi ed by the insurance industry. the effective management of clinical data around chronic and systemic oral and medical disease as part of an iehr is the greatest healthcare cost savings opportunity associated with such a tool. the insurance industry sustains itself through risk management [obtaining best outcomes] using actuarial analysis [data] and controlling costs [reduction of costs] in order to ensure coverage [profi tability]. as such they have pursued the economic and outcome benefi ts of integrated medical -dental clinical decision making. as an example, in 2009 there was a study conducted by the university of michigan, commissioned by the blue cross blue shield of michigan foundation ( 2009 ) , the study included 21,000 blue cross blue shield of michigan members diagnosed with diabetes who had access to dental care, and had continuous coverage for at least 1 year. with regular periodontal care, it was observed diabetes related medical costs were reduced by 10%. when compounding chronic health complications were also examined, the study showed a 20% reduction in cost related to the treatment of cardiovascular disease in patients with diabetes and heart disease. a 30% reduction in cost related to treatment of kidney disease for patients with diabetes and kidney disease. and a 40% reduction in costs related to treating congestive heart failure for patients with diabetes and congestive heart failure. according to a joint statement by lead researchers, and blue cross blue shield of michigan executives, "our results are consistent with an emerging body of evidence that periodontal diseaseâ�¦it addresses quality of care and health care costs for all michigan residents." also, at the institute for oral health conference in november 2007 joseph errante, d.d.s., vice president, blue cross blue shield of ma reported that 2003 blue cross blue shield of massachusetts claims data showed medical costs for diabetics who accessed dental care for prevention and periodontal services averaged $558/month, while medical costs for diabetics who didn't get dental care were about $702/month (errante 2007 ) . similarly insured individuals with cardiovascular diseases who accessed dental care had lower medical costs, $238/month lower than people who did not seek dental treatment (errante 2007 ) . the cost is $144 less per visit for those diabetics who accessed prevention and periodontal services. those savings could be translated into access to care or additional benefi ts for more individuals. in the case of neonatal health there is similar research. over 12% of all births in the u.s. are delivered preterm, with many infants at risk of birth defects ( martin et al. 2009 ) . according to a january 2006 statement issued by cigna, announcing their cigna oral health maternity program, "the program was launched in response to mounting research indicating an increased probability of preterm birth for those with gum disease. these research-based, value-added programs are designed to help improve outcomes and reduce expense" (cigna 2006 ) . the program was initially designed to offer extended dental benefi ts free of charge to members who were expecting mothers, citing "research supporting the negative and costly impact periodontal disease has on both mother and baby." according to research cited by cigna, expecting mothers with chronic periodontal disease during the second trimester are seven times more likely to deliver preterm (before 37th week), and the costs associated with treating premature newborns is an average of 15 times more during their fi rst year, and premature newborns have dramatically more healthcare challenges throughout their life. cigna also cited the correlation between periodontal disease and low birth weight, pre-eclampsia, gestational diabetes as additional rationale to support extended dental benefi ts to expecting mothers. six months later cigna initiated well aware for better health, an extended benefi ts free of charge program for diabetic and cardiovascular disease patients aimed at "turning evidence into action by enhancing dental benefi ts for participants in disease management" programs. it is interesting to note, not only does cigna offer extended dental benefi t to targeted groups, they also reimburse members for any out-of-pocket expenses associated to their dental care (co-pays, etc.) in 2006, columbia university researchers conducted a 2-year retrospective study of 116,306 aetna ppo members with continuous medical and dental insurance, exhibiting one of three chronic conditions (diabetes mellitus, coronary artery disease, and cerebrovascular disease) (aetna 2008 ) . researchers found members who received periodontal treatments incurred higher initial per member per month medical costs, but ultimately achieved signifi cantly lower health screening (episode risk group/erg) risk scores than peers receiving little or no dental care. convinced by the data and understanding lower risk scores ultimately leads to healthier people and cost savings, aetna initiated the dental/medical integration (dmi) program in 2006. aetna's dmi program offers enhanced benefi ts in the form of free-of-charge extended benefi t dental care to aetna's 37.2 million indemnity, ppo and managed choice medical plan members, specifi cally targeting members deemed at-risk, including those who are pregnant, diabetic, and/or have cardiovascular disease and have not been to a dentist in 1 year as a result of various outreach methods during the pilot, 63% of at-risk members who had not been to a dentist in the previous 12 months, sought dental care (aetna 2008 ) . "the fi ndings from this latest study we conducted continue to show that members with certain conditions who are engaged in seeking preventive care, such as regular dental visits, can improve their overall health and quality of life," said alan hirschberg, head of aetna dental (aetna 2008 ) . delta dental of wisconsin understands the connection between oral and systemic health and has created a program that is designed to offer members with certain chronic health conditions the opportunity to gain additional benefi ts. more than 2,000 groups now offer delta dental of wisconsin's evidence-based integrated care plan (ebicp) option (delta dental of wisconsin 2011 ) . ebicp provides expanded benefi ts for persons with diseases and medical conditions that have oral health implications. these benefi ts include increased frequency of cleanings and/or applications of topical fl uoride. they address the unique oral health challenges faced by persons with these conditions, and can also play an important role in the management of an individual's medical condition. ebicp offers additional cleanings and topical fl uoride application for persons who are undergoing cancer treatment involving radiation and/or chemotherapy, persons with prior surgical or nonsurgical treatment of periodontal disease and persons with suppressed immune systems. the ebic offers additional cleanings for persons with diabetes and those with risk factors for ie, persons with kidney failure or who are on dialysis and for women who are pregnant. the iehr provides the insurance industry in partnership with the healthcare industry an integrated tool to facilitate these health and subsequently economic outcomes across medicine and dentistry. in addition to the anticipated savings through better outcomes using integrated clinical data, an example of a positive economic outcome associated with an integrated record as related to increased effi ciency and patient safety is found in the united states veterans administration (va) hospitals and clinics. the va is one of the few institutions that have implemented the shared electronic medical-dental record successfully. the va has the ability to be the "one stop shop" for their patients. an april 2010 press release published on the department of veterans affairs website highlighted the success of va's health information technology in terms of cost reductions and "improvements in quality, safety, and patient satisfaction" (department of veterans affairs 2010 ) . the press release spotlighted a recent study conducted by the public health journal, health affairs, which focused on va's health it investment from 1997 to 2007. the study confi rmed that while va has spent $4 billion on their technology initiative, a conservative estimate of cost savings was more than $7 billion. after subtracting the expense of the it investment, there was a net savings of $3 billion for the va during the 10 years covered by the study (mcbride 2011 ) . furthermore, the study estimated that "more than 86 percent of the savings were due to eliminating duplicated tests and reducing medical errors. the rest of the savings came from lower operating expenses and reduced workload." independent studies show that the va system does better on many measures, especially preventive services and chronic care, than the private sector and medicare. va offi cials say "its [integrated] technology has helped cut down hospitalizations and helped patients live longer" (zhang 2009 ) . recently, the journal of obstetrics and gynecology reported on a tragic loss of life due to the systemic nature of oral health. a study found oral bacteria called fusobacterium nucleatum was the likely culprit in infecting a 35-year-old woman's fetus through her bloodstream (carroll 2010 ) . the doctors determined that the same strain of oral bacteria found in the woman's mouth was in the deceased baby's stomach and lungs. integrated records would provide critical data to the obstetrician including oral health issues and when the patient had her last dental exam. how does one measure the economic impact of a life not lived and another derailed by such tragedy? in a randomized controlled study, lopez et al. ( 2005 ) determined that periodontal therapy provided during pregnancy to women with periodontitis or gingivitis reduced the incidence of preterm and of low birth weight. the institute of medicine and national academies estimate that preterm births cost society at least $23billion annually . data integration of the iehr enables the effective management between the dentist and obstetrician to ensure proper periodontal therapy has been provided during pregnancy. such management based on the lopez et al. study, will have direct impact in reducing the prevalence per preterm births leading to reduced health care costs. there have also been studies indicating a correlation between poorer oral hygiene or defi cient denture hygiene and pneumonia or respiratory tract infection among elderly people in nursing homes or hospitals (rosenblum 2010 ; ghezzi and ship 2000 ; scannapieco 2006 ) . one such study of 141 elderly persons in two nursing homes in japan (adachi et al. 2002 ) concluded that "the number of bacteria silently aspirating into the lower respiratory tract was lower in the group who received professional oral care, which resulted in less fatal aspiration pneumonia in that group." over the 24 month period of the study, of the 77 patients receiving professional oral care, 5% died of pneumonia versus 16.7% of the 64 patients that died of the same cause who maintained their own oral hygiene. lack of access is certainly a key factor to consider. however, lack of available data respective to the interrelationship between oral health and systemic health also contributed to the apathy in these cases. as identifi ed above, complications are correlated to cost. as conditions compound, costs go up. marshfi eld clinic, as part of their iehr is creating a shared problem list that identifi es both oral and medical conditions and history to recent visits and medication lists for monitoring at point of care [be it a medical or dental visit], such cross access to clinical data and care management milestones serves as a tool to prevent conditions from compounding and escalating costs such as those described above. several other areas of economic impact will be seen as iehr's become broadly deployed. some of these are listed as follows: medication management. a great deal of provider and allied support time is â�¢ spent obtaining medication information between dentistry and medicine [and vice versa] including current medications, contraindications, tolerances, etc. marshfi eld clinic cattails software has created a dashboard that readily identifi es this for both the medical and dental providers. not is time saved but chances for complications or escalation of conditions is reduced [both of which impact cost]. for example an integrated record allows medical providers treating respiratory infections to include or exclude oral fl ora as the possible source of the infection which would lead to more knowledgeable prescribing decision on the antibiotic used. coordination of care has a direct impact on cost for the system and the patient. â�¢ for example, in 2008 55.6% of the us population aged 2 years and older that was diagnosed diabetes had been to the dentist in the past year (healthy people 2020 (2010 )). the us government's program healthy people 2020 includes an initiative to increase the proportion of people with diagnosed diabetes who have at least an annual dental examination. the american diabetes association recommends that diabetic patients be seen semi-annually and more if bleeding gums or other oral issues are present. the american diabetes association also recommends the consultation between the dentist and doctor to decide about possible adjustments to diabetes medicines, or to decide if an antibiotic is needed before surgery to prevent infection. the target from the healthy people 2020 is a 10% improvement at 61.2%. integrated medical/dental records could allow for the coordination of efforts between providers to include communication of treatment plan and services leading to quicker resolution, increased patient compliance, and less patient time away from work or home and potentially less travel. similarly, integrated records also create a platform to integrate clinical appointing â�¢ between medicine and dentistry. as such, combative patients or severely disabled patients needing anesthesia in order for care to be delivered can be treated with one hospital sedation vs. multiple sedations. family health center of marshfi eld, inc. (fhc) dental clinics shares an iehr with marshfi eld clinic and uses it integrated scheduling feature to complete dental care, lab work, ent care, woman's health, preventive studies, all in one visit. follow up care management can be more focused and coordinated. for example, â�¢ without the knowledge or dental conditions, medical providers could spend months attempting to control diabetes with periodontal disease. however, with access to an iehr, the practitioner or allied care manager can determine patient's oral health status immediately to determine possible infl uence of periodontal disease. similarly an iehr with a shared patient data dashboard brings to light history â�¢ and physical examination data without having to have patients be the historian to their physician on their last dental visit or for the dentist to have to rely on the patient's recall of medications or medical diagnosis. for example, if an integrated record saved providers 5 min per hour of patient care, that would be 40 min per day. imagine giving a physician or dentist 40 min more a day. in a capitated system, this allows for more patients to be seen in a day for roughly the same amount of expenditure. in a production based clinic this allows more patients to be seen and more charges per day. in either case, the investment into informatics is covered. in an underserved area, more patients get care quicker, which creates the opportunity for quicker resolution, which can lead to a healthier society, which in turn may lead them back to a productive livelihood sooner. an iehr results in one system for acquisition, orientation, training and support. â�¢ pc based owners who also own a mac and mac owners who also have to operate a pc can relate. need we say more? imagine if your pc function just like a mac [or your mac function just a pc]. no cross learning of software quirks. not having to purchase two separate units to begin with. reduced costs, increased space. not having to jump from one computer to the other computer to get data from one data from another to create a report. not having to call two separate computer companies for service or updates. third party coordination. having an iehr creates a platform for interfacing â�¢ with third party payers. a common system and language for timely reimbursement. in part, the result of an iehr is driving the diagnostic coding for dentistry. such an integrated interface provides a tool to bridge with healthcare payors that historically kept payment as segregated as the oral and medical health professions. the iehr overcomes that limitation. timely payment, consolidation of payment, expansion of covered patient and provider benefi ts based on clinical integration, and a viable system for interfacing are all potential economic benefi ts of iehr clinical data. the iehr creates new horizons for research that will lead to cost saving discov-â�¢ eries. as example, knowing the benefi ts of research, marshfi eld clinic research foundation (mcrf) has created an oral and systemic health research project (oshrp). the creation of oshrp, led by dr. murray brilliant, will allow mcrf to capitalize on its existing and growing strengths in the areas of complex disease interactions and personalized health care (phc) to advance oral health and the health of the rest of the body. the oshrp has three specifi c goals: understand the connections between oral and systemic health (diabetes, heart disease, pre-term births) understand the causes of oral diseases and determine the effect of genetics, diet, water source (well/city + fl uoridation) and microbiome. understand how improving oral health aids systemic health (comparative effectiveness) and bring personalized health care (phc) to the dental arena. the oshrp research resource will be unique in the nation. as mcrf has done â�¢ with other projects, it will share this resource with qualifi ed investigators at other academic institutions both within and outside of wisconsin. oshrp will advance scientifi c knowledge, improve healthcare and prevention, reduce the cost of oral healthcare, and create new economic opportunities. such knowledge will have a direct economic impact on the cost of care and care management. the iehr creates an ability to have an integrated patient portal to comprehen-â�¢ sively maintain their health. portals are becoming more and more popular in the healthcare industry as a means to helping maintain compliance with care management recommendations and preventative procedures. portals provide patents a tool to stay up to date on their care and recommendations. portals can take iehr clinical data, adapt it through programming, and provide creative visual reinforcement for patients as they monitor their health status. the more patients engage in owning their health status, the more preventative services are followed through with. the more medicine and dentistry can leverage the prevention potential [which insurance companies have come to realize] the more likely costly conditions can be avoided. the link between oral health and systemic health is well documented. the separation of dental and medical is not a sustainable model in modern healthcare delivery. a new model of integrated care is necessary. aristotle said, "the whole is greater than the sum of its parts." increased access to combined medical and dental histories and diagnosis at the providers' fi ngertips makes vital information available. shared diagnosis between physicians and dentists could aid in formulating interventions and to accelerate decision making abilities by allowing for prioritizing of medical/ dental procedures. clinical management and treatment of the patient would be expedited with immediate access to both records. quality could be improved through a complete picture of the patient through the dashboard. all of which have a direct or indirect economic benefi t. the iehr will be the tool that facilitates such delivery and the studies and scenarios described in these pages point to signifi cant economic benefi ts to patients, payors, and providers. if increased access, multi-provider monitoring, shared problems lists with enhanced decision making abilities from iehr could reduce healthcare costs. the greatest cost reduction will be with using the iehr to manage chronic disease. a combined dental-medical electronic record with a shared data informatics platform is most likely to yield the best long-term economic solution while maintaining or enhancing positive patient outcomes. this section reveals viewpoints from a variety of medical and dental providers. one section focuses on optimal use of ophthalmic imaging, which should show how that the challenges of clinical data integration go beyond those encountered in the effort to bring oral health and systemic health together. wendy e. mouradian , suzanne boulter , paul casamassimo , and valerie j. harvey powell oral health is an important but often neglected part of overall health. historically separate systems of education, financing and practice in medicine and dentistry fuel this neglect, contributing to poorer health outcomes for vulnerable populations such as children, while increasing costs and chances for medical error for all patients. advances in understanding the impact of oral health on children's overall health, changing disease patterns and demographic trends strengthen the mandate for greater integration of oral and overall healthcare, as reviewed in two recent institute of medicine reports (iom 2011a, b ) . the pediatric population could realize substantial benefit from oral disease prevention strategies under a coordinated system of care enhanced by integrated electronic health records (ehr). this approach would benefit all children but especially young children and those from low socioeconomic, minority and other disadvantaged groups who are at higher risk for oral disease and difficulties accessing dental care. this section focuses on the pediatric population and the need for close collaboration of pediatric medical and dental providers. first we consider how a child's developmental position and their parents' level of understanding might affect oral health outcomes. next we address the importance of children's oral health and the urgency of seizing missed opportunities to prevent disease. we then briefl y outlines some steps to preventing early childhood oral disease utilizing some of the many health providers that interact with families. finally we examine one pediatric hospital's approach to choosing an integrated ehr technology. children have unique characteristics which distinguish their needs from those of adults. children's developmental immaturities may increase their risks for poor oral health outcomes ( fig. 4 all children, but especially young children, are limited in their ability to care for their own health and must depend upon adults. a child's parent/caregiver may also lack basic oral health knowledge and an awareness of their child's oral health needs, and/or suffer from poor oral health themselves. low oral health literacy is prevalent among patients and health professionals alike in america; individuals of low socioeconomic status or from ethnically diverse backgrounds may be at particular risk for low oral health literacy (iom 2011a ) . without appropriate education, a parentâ�¦. may not correctly interpret a child's symptoms or signs of oral disease â�¢ may not know that caries is an infectious disease that can be spread to a child by â�¢ sharing spoons, for example, may not know the potential value of chewing gum with xylitol, â�¢ may not fully grasp the importance of good oral health hygiene habits, â�¢ may not grasp the consequences of a child consuming quantities of sugared â�¢ foods or beverages, may have diffi culty controlling the child's consumption of sugared foods or bev-â�¢ erages in or out of the home, may not realize the consequences of chronic use of sugared medications, â�¢ may not know the potential for systemic spread of disease from a toothache, or â�¢ for liver damage due to overuse of acetaminophen or other analgesics, may not grasp the long-term consequences of early childhood caries, â�¢ may live in a community without fl uoride in the tap water and not know about â�¢ alternative sources of fl uoride, may overlook oral health due to the stress of living in poverty, â�¢ may be fearful of dentists or oral health care due to their own experiences, â�¢ may have diffi culty locating a dental provider accepting public insurance, or â�¢ have other problems navigating the health care system. parents in turn depend on access to medical and dental providers with current understanding of the most effective ways to prevent caries and promote the child's oral and overall health. an important element in helping families is the provision of culturally-sensitive care to a diverse population. children are the most diverse segment of the population with 44% from minority backgrounds compared with 34% of the overall population (us census bureau 2010 ) . the separation of medical and dental systems and the lack of shared information can create additional barriers for families, especially for those with low health literacy or facing linguistic or cultural barriers. all pediatric health professionals have increased ethical and legal responsibilities to promote children's health, including advocacy for them at the system level (mouradian 1999 ) . although many factors can infl uence children's oral health outcomes, caries is largely a preventable disease. despite this, national trends and other data on 4 broader considerations of medical and dental data integration children's oral health attest to this persistent national problem ) . some important facts include the followingâ�¦. caries is the most prevalent chronic disease of childhood, â�¢ caries is a preventable disease unlike many chronic diseases of childhood, â�¢ yet according to (nicdr 2011) 42% of children 2-11 have had dental caries in â�¢ their primary teeth; 23% of children 2-11 have untreated dental caries. further, "21% of children 6-11 have had dental caries in their permanent teeth; 8% of children 6-11 have untreated decay." overall "[c]hildren 2-11 have an average of 1.6 decayed primary teeth and 3.6 decayed primary surfaces," the latest epidemiologic evidence shows increasing rates of caries for young-â�¢ est children, reverse from the healthy people 2010 goal of decreasing caries. according to (nicdr 2011), overall "dental caries in the baby teeth of children 2-11 declined from the early 1970s until the mid 1990s. from the mid 1990s until the most recent (1999) (2000) (2001) (2002) (2003) (2004) ) national health and nutrition examination survey, this trend has reversed: a small but signifi cant increase in primary decay was found. this trend reversal was more severe in younger children." disparities in children's oral health and access to care persist by age, income â�¢ level, race and ethnicity, and parental education level (edelstein and chinn 2009 ) . of concern, the latest increase was actually in a traditionally low-risk group of young children (dye and thornton-evans 2010 ) . the human and economic costs of early childhood caries are substantial â�¢ (casamassimo et al. 2009 ) . according to catalanotto ( 2010 ) , health consequences includeâ�¦ extreme pain, spread of infection/facial cellulitis, even death (otto 2007 ) diffi culty chewing, poor weight gain falling off the growth curve (acs et al. 1999 ) risk of dental decay in adult teeth (broadbent et al. 2005 ; li and wang 2002 ) crooked bite (malocclusion) -children with special health care needs (cshcn) may be at higher risk for oral â�¢ disease and diffi culties accessing care. analyzing data from the national survey of children with special health care needs, (lewis 2009 ) found that "cshcn are more likely to be insured and to receive preventive dental care at equal or higher rates than children without special health care needs. nevertheless, cshcn, particularly lower income and severely affected, are more likely to report unmet dental care need compared with unaffected children." children who were both low-income and severely affected had 13.4 times the likelihood of unmet dental care needs, dental care is the highest unmet health care need of children; 4.6 million children â�¢ had unmet dental care needs because families could not afford care compared with 2.8 million with unmet medical needs for the same reasons (cdc 2008 ) , according to the national survey of children's health, children are 2.6 times as â�¢ likely to lack dental as medical insurance (lewis et al. 2007 ) , there is evidence that children who get referred to a dentist early may have lower â�¢ costs of care and disease. savage et al. ( 2004 ) reported that children "who had their fi rst preventive visit by age 1 were more likely to have subsequent preventive visits but were not more likely to have subsequent restorative or emergency visits" and concluded that preschool "children who used early preventive dental care incurred fewer dentally related costs," ramosgomez and shepherd ( â�¢ 1999 ) , in their "cost-effectiveness model for prevention of early childhood caries," conclude that preventive ecc interventions could reduce ecc by 40-80% for a particularly vulnerable population of children, and that part of the costs of interventions will be offset by savings in treatment costs. as these facts convey, and the deaths of more than one child from consequences of untreated caries make painfully clear, there is an urgent need for more attention to the oral health needs of children. a more coordinated system for oral health care including integrated ehr would be an important advance. a glance at table 4 .5 , an ideal model, reveals that intervention should begin before birth and that a range of medical and oral health professionals can contribute to the child's oral health. early intervention is necessary because of the transmissibility of cariogenic bacteria from mother/caregiver to infant, and importance of oral health practice in preventing disease. the following professionals may be involved: â�¢ pediatric medical provider family physician pediatrician pediatric nurse nurse practitioner in pediatric/family practice physician assistant in pediatric /family practice other appropriate allied health professionals â�¢ the availability of some of these professionals can be affected socioeconomic status, health insurance, place of residence, or by a child's special health care need. one obvious limitation on developing a "relay" as in table 4 .5 , with a "hand-off" from family care to obstetric care to pediatric care is the education of the medical providers. as part of pre-conception and perinatal healthcare, providers should address oral health, but may lack the knowledge to do so. additionally, as noted by ressler-maerlaender et al. ( 2005 ) , "some women may believe that they or their table 4 .5 timeline of some oral health interventions to prevent early childhood caries (ecc) -birth to 3 years age (marrs et al. 2011 ; lannon et al. 2008 ; han et al. 2010 ; ezer et al. 2010 ; aap 2008 ; mouradian et al. 2000 ) child's age intervening professional(s) planning conception, prenatal and perinatal family physician the physician and/or obstetric provider educates mother-to-be about good maternal oral hygiene and infant oral health issues, including transmissibility of caries. mother's dentist assesses and treats caries, gingivitis or other oral health problems and educates the mother-to-be obstetrician/nurse midwife obstetric nurse general dentist obstetric nurse obstetric nurse advises new mother to chew xylitol gum, limit salivary contact between mother and infant, and help child avoid sugar intake (exposure) while asleep and from common sugar sources (medicines, sugared water, bottle feeding on demand at night with fl uid other than water -following tooth eruption, certain foods), and to schedule dental exam at 1 year age 6 months pediatric medical provider first dental examination recommended by aapd when the fi rst tooth comes in, usually between 6 to 12 months pediatric/general dentist educate mother about optimal fl uoride levels 1. assess the woman's oral health status, oral health practices, and access to a dental home; 2. discuss with the woman how oral health affects general health; 3. offer referrals to oral health professionals for treatment; 4. educate the woman about oral health during pregnancy, including expected physiological changes in the mouth and interventions to prevent and relieve discomfort; and 5. educate the woman about diet and oral hygiene for infants and children and encourage breastfeeding a combination of anticipatory guidance, with continuity from prenatal and perinatal care to pediatric care, can help move infant oral health from "missed opportunities" to "seized opportunities." others who may be of assistance to families in closing these gaps are professionals at the women, infants and children's (wic) supplemental nutrition program, early head start/head start and neurodevelopmental/birth to three programs. together medical, dental and community professionals can help create a system of care to improve maternal and child oral health. for the envisioned model in table 4 .5 to be realized, the mother requires access to a general dentist with accurate information on her oral health during pregnancy and on her infant's oral health, including the need for an early dental visit. the mother and child then need access to a pediatric medical provider who will provide oral health screening/counseling, and who will guide the family to establishing the child's dental home by age 1. success in dental referral requires access to a pediatric or general dentist willing and able to provide infant oral health. (dela cruz et al. 2004) , in a discussion of the referral process mentioned that among the factors in assessing the likelihood of a dental referral were the medical providers' "level of oral health knowledge, and their opinions about the importance of oral health and preventive dental care." since young children are much more likely to access medical than dental care, the medical provider plays an important role in promoting children's oral health. (catalanotto 2010 ) recommends, as part of a pediatric well child checkup: an oral screening examination, â�¢ a risk assessment, including assessment of the mother's/caregiver's oral health, â�¢ application of fl uoride varnish â�¢ anticipatory guidance (parental education) including dietary and oral hygiene â�¢ information, attempted referral to a dental home. â�¢ the aap recommends that child healthcare providers be trained to perform an oral health risk assessment and triage all infants and children beginning by 6 months of age to identify known risk factors for early childhood caries (ecc). the oral health component of pediatric care is integrated into the aap's "recommendations for preventive pediatric health care (periodicity schedule)" (aap 2008 ) . to what extent are medical and dental and providers aware of recommendations for a fi rst dental visit for a child by age one, as recommended by the aap, the american academy of pediatric dentistry (aapd), and the american dental association? (wolfe et al. 2006 ) reported that 76% of licensed general dentists in iowa were familiar with the aapd age 1 dental visit recommendation and that most obtained the information through continuing education; 11% believed that the fi rst dental visit should occur between 0 and 11 months of age. however, according to (caspary et al. 2008 ) , when pediatric medical residents were asked the age for the fi rst dental visit, the average response was 2.4 years, while 35% reported received no oral health training during residency. in a national survey of pediatricians ) reported that less than 25% of had received oral health education in medical school, residency, or continuing education. finally (ferullo et al. 2010 ) surveyed allopathic and osteopathic schools of medicine and found that 69.3% reported offering less than 5 h of oral health curriculum, while 10.2% offered no curriculum at all. other workforce considerations relevant to preventing early childhood caries include the training of dentists in pediatric oral health (seale et al. 2009 ) , the number and diversity of the dental workforce, the number of pediatric dentists, and the use of alternative providers such as dental therapists, expanded function dental assistants and dental hygienists (mertz and mouradian 2009 ; nash 2009 ) . examples of integrated care models do exist, such as that presented by (heuer 2007 ) involving school-linked and school-based clinics with an "innovative health infrastructure." according to heuer, "neighborhood outreach action for health (noah)" is staffed by two nurse practitioners and a part-time physician to provide "primary medical services to more than 3,200 uninsured patients each year" in scottsdale, arizona. heuer counts caries among the "top ten" diagnoses every year. mabry and mosca ( 2006 ) described community public health training of dental hygiene students for children with neurodevelopmental/intellectual disabilities. they mentioned that the dental hygiene students had worked together with school nurses and "felt they had impacted the school nurses' knowledge of oral disease and care." the decision to acquire an integrated ehr as pediatric clinicians (both medical and dental) work more closely together, they require appropriate ehr systems that integrate a patient's medical and dental records. following is a set of local "best practices" from nationwide children's hospital in columbus, ohio, which may help other children's hospitals in planning acquisition of an integrated pediatric ehr system. integrated (medical-dental) ehr technologies are becoming more widely available outside the federal government sector (see integrated models e1 and e2 in fig. 1.3 ). nationwide children's 'drivers' for the acquisition process were, in 2011: 1. minimize registration and dual databases . patient registration takes time and requiring both a stand-alone dental and a medical patient registration inhibits cost-effective fl ow of services. integration allows for the use of single demographics information for all clinics in the comprehensive care system serving the patient. clinicians always have an updated health history on patients, if they have been a patient of record. if not, and for a dental clinic that sees walk-ins, a brief "critical" dental health history can be completed on paper by a parent and scanned into the emr. in designing an integrated medical-dental record for patients of record, the system can sort essential health history elements into a brief focused dental history without the detail needed by other medical specialty clinics. kioskdriven electronic health histories for those children who are new to clinic similar to those used in airline travel could be considered if feasible in busy clinics. 2. for charting, no more key/mouse strokes than with paper . some commercial dental record products try to accomplish too much. moving from paper to electronics should be driven in part by effi ciencies. the tooth chart, which is an essential part of any dental record, must be such that examination fi ndings can be transferred quickly and accurately to either paper or electronic capture. a helpful exercise is visualization of the functionality of the charting process, including both the different types of entries (caries, existing restorations and pathology) and how these are entered in the paper world. if charting will be able to be used for research the system should be able to translate pictures to numerical values, often a complex programming function. dental practitioners and faculty may want to use drawings of teeth or graphics of surfaces because that is their current comfort level. a true digital charting is possible with no images of teeth, but some habits are hard to change. 3. maximizing drop downs with drop down building possible . duplication of paper chart entries using drop downs which can be upgraded as more clinical entities are found is a staple of an emr. the paper process usually relies on a clinician's wealth of medical-dental terms since inclusion of every possible, or even the most common fi ndings, is prohibitive on a paper chart. the emr drop down requires front-end loading of the most common clinical fi ndings with opportunity for free-hand additions. being able to add terms to any drop down is a needed capability. 4. don't design a system for uncommon contingencies, but for your bulk of work . a pediatric dental record should be primarily designed around dental caries, with secondary emphases on oral-facial development (orthodontics) and a lesser capability to record traumatic injuries and periodontal fi ndings. these second and third level characteristics can be hot-buttoned and should not drive the design of the basic system which is caries charting for 98% of our patients. sadly in most dental schools, the chart is slave to every teaching form, few of which ever exit with the dds into practice! these forms may have little relationship to patient care and only create "signature black holes" that need to be addressed, usually after treatment is completed. 5. progress notes should be designed for the routine entries with free-hand modification possible . student learners tend to write too much and a carefully crafted progress note format with standard entries in required fi elds helps patient fl ow and record completion. in federally funded clinics and residencies, attending reconciliation of student/resident service delivery is a compliance requirement. a well-designed emr system can "stack" required co-signing tasks on a computer screen, offer standard entries as well as free-hand options, and create a process far faster than paper records for an attending's validation (same as reconciliation?). 6. tie examination results to treatment planning and treatment planning into billing . a good system allows easy transfer of clinical fi ndings needing treatment into some problem "basket" and ideally in a tabulated format. an alternative is a split screen that allows a clinician to visualize clinical fi ndings, radiographic fi ndings while compiling a treatment plan. again, in clinical settings where compliance to medicaid/medicare regulations is required, the design of the record should give attention to auditing principles and security. a good emr system allows portals of entry for billing and compliance personnel. 7. plan for users of different skill levels and different periods of exposure . the teaching hospital or dental school environment often involves learners and attendings with varying skill levels and computer experience who may be there for brief periods of time. this reality adds signifi cant security and userfriendliness issues. some medical record systems are far too complex for shortterm or casual users. a well-integrated medical-dental emr allows navigation of the depths of the medical side should a user want to explore, but should focus on the dental portion. some suggestions in design: initial opening or logging into the dental portion for dental users, rather than â�¢ opening into the medical portion, clearly indicated options for exploration of medical portions, â�¢ orientation of major dental component (examination, radiographs, treatment â�¢ plan) in a logical dental treatment fl ow to replicate the way dentistry works rather than trying to reshape dentistry's normal fl ow to the record, minimization of seldom-used functions on the main dental screen, such as â�¢ specialty medical clinics, old laboratory tests and hyperfunctionalities like letter writing, clear identifi cation of existing non-caries dental portions like orthodontics or â�¢ trauma, so a novice user need not randomly search to see if a patient has any of these records. unfortunately, many pediatric hospitals do not yet have an ehr system that supports convenient communication among a pediatric patient's medical and dental providers. evidence of this state of affairs was provided unintentionally by (fiks et al. 2011 ) . some pediatric hospitals may have an awkward mix of systems serving physicians, dentists, and orthodontists and their shared patients. this section demonstrates how closely medical and dental professionals must collaborate to deliver appropriate oral health care for infants and children. such collaboration is especially important given the developmental vulnerabilities of children and the urgency of the oral health needs of many children, especially those from underserved populations. collaboration is made more diffi cult by the long-standing separation of medical and dental systems and poor oral health literacy of parents and medical professionals alike. teamwork in the delivery of pediatric care requires appropriate electronic patient record technology to facilitate sharing of patient information, to avoid patient record discrepancies between systems, and to create effi ciencies by maintaining only a single repository for patient demographics. only comparatively recently have appropriate integrated systems become available to support a range of clinical sites from pediatric special needs clinics to the largest children's hospitals. nationwide children's has given practical examples of effi cient decision-making in identifying an integrated system to acquire. much more work will be needed to develop the means to move towards integrating offi ce and community-based care for children through the sharing of electronic health records. oral health is an oft neglected area in the care of patients who have chronic kidney disease. furthermore, the provision of care by dentists and physicians to the same patient is fragmented as communication between the two health care providers is scant. emerging data suggesting the periodontal disease is closely linked to chronic kidney disease highlights the importance of proper oral health and the importance of communication between dentists and physicians in the care of the patient. investigators used data from nhanes iii, including information on 11, 211 adults who had an oral examination by a dentist who categorized each patient as having no periodontal disease, periodontal disease or edentulous to examine the relationship between numerous risk factors for moderate to severe chronic kidney disease, as determined by calculation of estimated gfr through use of the mdrd formula (fisher et al. 2011 ) . no chronic kidney disease was defi ned as an estimated gfr of 60 ml per min per 1.73 m 2 . three percent of the patients had ckd, 22.5% were hypertension and 4.4% had diabetes (2.4% with glycated hemoglobin of 7% or higher). four models were constructed to examine the potential relationship between periodontal disease and ckd. in model one adults with either periodontal disease or edentulous had an adjusted odds ratio of 1.83 (with 95% confi dence intervals of 1.31-2.55) of having ckd, independent of the other risk factors for ckd including of age above 60 years, ethnicity, hypertension, smoking status, female gender and c-reactive protein elevation. the fourth model contained 15 potential risk factors including the periodontal disease score and for every 1-unit increase in the score, the risk of having ckd increased by 1% controlling for the other risk factors. the authors hypothesized from their results that the relationship between periodontal disease and ckd was bidirectional in that ckd may increase the risk of periodontal disease which in turn increases the risk of ckd. grubbs et al. ( 2011 ) also used nhanes data to look more closely at the relationship between periodontal disease and ckd, using dental examinations obtained from 2001 to 2004 (n = 6,199 adults, 21-75 years) (grubbs v, et al. 2011 ) . in this analysis edentulous subjects were excluded and those with albuminuria were included in the defi nition of ckd. in the entire population ckd was present in 10.6%, but in those with moderate to severe periodontal disease this increased to 21.6%. other associations with moderate to severe periodontal disease were being older, male, nonwhite, less educated and poor. there was a strong relationship between periodontal disease and ckd (2.5 unadjusted odds ratio). when adjusted for age, gender, tobacco use, hypertension, diabetes, ethnicity, poverty and educational attainment, the odds ratio for the association of periodontal disease and ckd was still signifi cant (1.55). in some groups (mexican american, poor, and poorly educated) dental care was not received on an annual basis in the majority of this segment of the population. periodontal disease has been associated with an increased risk of death in hemodialysis patients (kshirsagar et al. 2009 ). this relationship has been poorly studied in peritoneal dialysis patients. this requires further study but it appears possible that periodontal disease might hasten loss of residual kidney function and perhaps contribute to atherosclerosis in dialysis patients and therefore, contribute to the high mortality in this population. patients who desire a kidney transplant are required to undergo a thorough evaluation beforehand including an oral examination by a dentist. some patients on dialysis have inadequate insurance which does not cover dental care, leading to a situation in which a kidney transplant is denied because the patient cannot afford the dental examination. communications between dentists and physicians in the care of the patient is scant. if oral surgery is required in a dialysis patient, the surgeon generally requires a brief summary from the nephrologist with recommendations. these might include suggestions for prophylactic antibiotics, avoidance of vasoconstrictor agents to an excess locally (which can elevate blood pressure) and the increased risk of bleeding of a dialysis patient. for more routine dental examinations no information is requested which could potentially lead to drug interactions or a dangerous situation. most nephrologists and health care providers in the dialysis unit do not inquire of the patient concerning dental health and examination of the mouth is quite uncommon. although the dialysis patient is seen monthly at a minimum, there is little conversation or documentation of oral health. connecting the electronic health records of in-patient care, the out-patient dialysis unit and the dentists' offi ce could potentially have a large impact in improving the care of those with end stage kidney disease. integrating medical and dental records in ehr's may or may not be the "golden ring." first, we need to integrate the clinical thinkingâ�¦something we both realize is important, but not likely to be solved by an inert computer. i also think that integrated records will be very cumbersome, given the fact that the language used by the separate disciplines is so different, and the kind of detail required to support good decisions and good work is so different. it could be doneâ�¦but for many professionals on either "side," they would never open the other module. to me, a more sensible solution may be to have a condensed "nugget" of information that could cross populate. "moderate periodontal disease" may be what the medical doctor needs to know, plus know what a treatment plan may include. she won't need to know the number of the teeth with the deepest pockets and erosions but will need to support the patient's determination to follow through. on the other hand, if the patient has shown remarkable initiative in gum care and has successfully migrated to a lower severity index, that would be important for congratulation and reinforcementâ�¦and also to encourage similar diligence in managing, let's say, the hypertension that is not optimally controlled. in the other direction, the dentist should know that a patient has been erratic in clinical follow up, does not self-test blood glucose, uses hypoglycemic drugs only intermittently, and has failed several appointments for eye exams. this would lead to a rather different set of approaches from a highly motivated grandmother who is enrolled in a community cultural center's senior exercise club, and is learning to become a lay community teacher for diabetes. right now, i don't think even this superfi cial degree of information is exchanged. we need to support each other's efforts, but we probably do not need to share minute details. the benefi ts of an electronic health record are well described. ehrs allow for legible standardized documentation and easier sharing of patient data between providers at multiple locations. they are less prone to loss and require much less space to store. they have the potential to result in a reduction in the cost of health care. a distinct disadvantage of the ehr, in its current confi guration, is the problem of information overload. simply put, there is often too much information presented in a way that is diffi cult to review and digest. the ehr equivalent of thumbing through a chart quickly is not yet available. as a result we frequently see practitioners look only at the last note or two as they review a patient's history. we require a way to communicate information directly relevant to patient diagnosis, treatment and prognosis among subspecialists and primary care providers. we require a way to identify subclinical cerebrovascular disease in a patient, independent of blood pressure and other traditional risk factors. we require a way to recognize which patients with cerebrovascular disease are two to four times more likely than average to develop a stroke in the next 3 years. we have a way -retinal imaging. the eye is the one place in the body we can directly observe arteries, veins and a cranial nerve in a noninvasive manner. routine imaging of the retina and optic nerve could allow primary care providers to assess retinal, and by proxy systemic, end organ damage from atherosclerosis in an effi cient manner. the key to optimal use of the medical record and effi cient yet effective communication among providers may lie with the familiar adage; a picture is worth a 1,000 words. traditionally, when ophthalmologists communicate with primary care providers they send brief letters regarding the fi ndings seen during a yearly dilated examination and the presence, absence or progression of diabetic retinopathy. these letters end by exhorting the virtues of improved blood sugar, blood pressure and lipid control, a sentiment that the primary care provider likely shares. this system of communication does not provide particularly useful information for the primary care provider, except to serve as a notice that the standard of care screening guidelines have been met. the box has been checked. if primary care providers, cardiologists, nephrologists had access to routine ophthalmic imaging, they would be able to directly visualize the effect that suboptimal blood sugar control is having on their diabetic patients. as importantly, they would be equipped with information directly predictive of congestive heart failure, stroke, and cardiovascular mortality for their patient with hypertension, hyperlipidemia and for those who smoke. large clinical studies have shown that assessment of retinal vascular changes such as retinal hemorrhages, microaneurysms and cotton wool spots provides important information for vasculopathy risk stratifi cation. as an example, wong et al. showed that the presence of retinopathy indicates susceptibility to and onset of preclinical systemic vascular disease, independent of and qualitatively different from measuring blood pressure or lipids (wong and mcintosh 2005 ) . in the atherosclerosis risk in communities (aric) study, individuals with hypertensive retinopathy signs such as cotton wool spots, retinal hemorrhages and microaneurysms were two to four times more likely to develop a stroke within 3 years, even when controlling for the effects of blood pressure, hyperlipidemia, cigarette smoking and other risk factors (wong et al. 2001 ) . in a recent study by werther et al., patients with retinal vein occlusions were found to have a two-fold increased risk of stroke compared to controls (werther et al. 2011 ) . in addition, the aric study group reported that individuals with retinopathy were twice as likely to develop congestive heart failure as individuals without retinopathy, even after controlling for pre-existing risk factors (wong et al. 2005a ) . interestingly, even among individuals without pre-existing coronary artery disease, diabetes or hypertension, the presence of hypertensive retinopathy was associated with a three-fold increased risk of congestive heart failure events (wong et al. 2005a ) . in the beaver dam eye study, cardiovascular mortality was almost twice as high among individuals with retinal microaneurysms and retinal hemorrhages as those without these signs ( wong et al. 2003a, b ) . the aric and beaver dam eye studies have also shown that, independent of other risk factors, generalized retinal arteriolar narrowing predicts the incidence of type ii diabetes among individuals initially free of the disease (wong et al. 2002a (wong et al. , 2005b . a primary care provider with access to patients' retinal photographs may therefore have the evidence needed to suggest which patient with either established systemic vascular disease or preclinical systemic vascular disease requires a more aggressive treatment and risk factor modifi cation. they could do this without wading through the electronic equivalent of piles of records. one photograph could refl ect both acute changes in blood pressure (retinal hemorrhages, microaneurysms and cotton wool spots) and chronic changes resulting from cumulative damage from hypertension (av nicking and generalized arteriolar narrowing) (sharrett et al. 1999 ; wong et al. 2002a ; leung et al. 2004 ) . in brown et al. 23 out of 24 patients, excluding those with known diabetes, that presented with a single cotton wool spot or a predominance of cotton wool spots on examination of the retina were found to have underlying systemic disease (brown et al. 1985 ) . systemic work-up revealed diagnoses including previously undiagnosed diabetes, hypertension, cardiac valvular disease, severe carotid artery obstruction, leukemia, metastatic carcinoma, systemic lupus erythematosus, aids and giant cell arteritis (brown et al. 1985 ) . these fi ndings illustrate the importance of retinal fi ndings on a systemic level. the utilization and integration of ophthalmic imaging may serve to achieve more effective communication among subspecialists and primary care providers and ultimately to provide improved diagnosis and treatment for delivery of optimal quality of patient care. moreover, the improved integration and maximal use of resources may serve to reduce overall health care cost and perhaps decrease provider frustration with the electronic health record (fig. 4 .5 ). there are cotton wool spots, exudates, intraretinal dot-blot hemorrhages and microaneurysms. av nicking is also present especially along the superior arcade just as the vessel leaves the optic nerve ( fig. 4.6 ) . av nicking, tortuosity of vessels, intraretinal hemorrhages and dry exudates are seen ( fig. 4.7 ) . there is edema of the optic nerve head, with cotton wool spots and fl ame shaped hemorrhage along the disc margin. there are several cotton wool spots along the vascular arcades and scattered dot hemorrhages throughout the posterior pole and periphery ( fig. 4.8 ) . notice the cholesterol plaque in the vessel just as it exits the optic nerve head and the pallor in the superior macula corresponding to retinal ischemia and edema ( fig. 4.9 ). the cholesterol embolus has resulted in lack of blood fl ow to the superior arcade ( fig. 4.10 ) . there is pooling of subretinal blood just superior to the optic disc with a central fi brin clot and associated vitreous hemorrhage (fig. 4.11 ) . optic disc edema, fl ame hemorrhages and venous congestion are seen in a patient with severe hypertension. biju cheriyan in clinical practice, an otolaryngologist often needs a dental consult not only because of the topographically adjacent nature of the structures but also because most structures are supplied by the same neurovascular bundle and therefore there is overlapping of symptoms. the converse scenario can also apply. apart from this, there are many systemic medical conditions (for example: bleeding diatheses, diabetes) a hypertensive optic neuropathy dentist encounters throughout his or her practice which can determine the outcome of a successful treatment. sometimes, providers may observe a cluster of diagnostic criteria which may have to a single source. in the sections below, i will explore a few of these scenarios and conditions, and indicate where and how an integrated electronic health record (ehr) could optimize delivery of health care by dentists and otolaryngologists. cleft palate/cleft lip : cleft lip and cleft palate (cl/cp) are congenital conditions that require multidisciplinary management by dentists, oral and maxillofacial surgeons, orthodontists, otolaryngologists, speech pathologists and plastic surgeons a number of studies report that a multidisciplinary approach is essential for better treatment outcomes (wangsrimongkol and jansawang 2010 ) and for post operative rehabilitation (furr et al. 2011 ). these multidisciplinary approaches may lead to new ways to manage and treat cl/cp patients (salyer et al. 2009 ). hutchinson's teeth : notching of the upper two incisors is typically seen in individuals infl icted with congenital syphilis. macroglossia refers to enlarged tongue in relation to oral cavity. macroglossia is an important sign. it can indicate important systemic diseases like systemic amyloidosis, congenital hypothyroidism, acromegaly, or down syndrome. a common complaint that dentists and otolaryngologists encounter in their practice is the common headache. because of the special nature of the neurovascular bundle of the head and neck this symptom can be presented to both dentists and otolaryngologists (ram et al. 2009 ). any sinus pathology can present as a headache to an otolaryngology practice. since the maxillary sinus fl oor is in close proximity to the maxillary premolars and molars, it is imperative to obtain a dental evaluation in persistent cases of headache. there are a number of causes for headache from the dental and otolaryngology perspective. a mal-aligned denture patient with chronic headache, whom i saw in my practice was shuttled between departments and an array of investigations only to fi nd at the end that an ill-fi tting denture caused the intractable headache. in these cases, an integration of fi ndings is extremely important in providing quality treatment to the patient and also saves money and time for the whole health care system. hence it is important to have an integrated patient record for this particular symptom alone. trigeminal neuralgia is facial pain of neurogenic origin experienced along the distribution of the trigeminal nerve(fi fth cranial nerve). it can present as a dental pain and can also be triggered by brushing teeth among other trigger factors. as a result, patients with dental pain without obvious causes are required to have a physicians' consultation to rule out this obscure condition. sometimes it is diagnosed by omission (aggarwal et al. 2011 ; rodriguez-lozano et al. 2010 ; spencer et al. 2008 ). any tumor of the nasal sinuses (specifi cally maxillary and ethmoids) can erode the lower bony wall and present in the oral cavity (usually the maxillary arch) as dental pain, loose tooth, etc. therefore, these are areas of interest to both dentists and otolaryngologists. such tumors most commonly present fi rst to a dentist or could also be an accidental fi nding. cancers of the naso/oro/laryngo pharynx can also present as toothache to a dentist as these structures have a common nerve supply from cranial nerves 5,7 and 9. therefore, an integration of the patient record may even help in early diagnosis of the tumor. the same principle applies to all oral tumors, tumors of the nasopharynx, the oropharynx etc. this is especially true of malignant lesions of the oral cavity as these may help in early detection and treatment of cancer. in these cases, an early biopsy and histopathology can save the life of the patient. therefore, it is imperative to say that a collaborative patient record can save patients' lives. ulcers of the oral cavity from aphthous ulcers to carcinomas can present both to a dentist and an otolaryngologist. oral ulcers can be of dental origin. contact ulcers from sharp edges of a mal-aligned tooth can result in intractable ulcers, where a simple smoothing of sharp edges may eradicate the ulcer and terminate it as a chronic condition and can even prevent the ulcer turning into a malignancy. if you have an integrated electronic health record (ehr) these problems are immediately addressed and managed. otherwise, the condition will consume valuable time of both the patient and the physician concerned. in addition to this, there are a few conditions which require special attention: aphthous stomatitis (canker sore), which may indicate oral manifestation of defi ciencies of iron, vitamin b12, folate deficiency and oral candidiasis, which can be a sign of diabetes mellitus or of an immunocompromised patient (e.g. aids). temperomandibular joint (tmj) disorders can present in a variety of symptoms to both dentists and otolaryngologists. they can present as a headache, earache, toothache, or as facial pain. there can be a number of causes for this including osteoarthritis of the tmj, recurrent dislocation, bruxism, or even an ill fi tting denture. there have been cases where patients have been subjected to removal of teeth for chronic toothache only to discover at the end that the symptom was a referred pain from tmj! therefore, an integrated ehr can prevent misdiagnoses and resulting impairment or disability to patients. trismus (lock jaw) can indicate important diagnoses such as tetanus and rabies.it is due to a spasm of muscles of mastication, which is an important oral manifestation of widespread muscle spasm. apart from these conditions, other causes of trismus are peritonsillar abscesses, and scleroderma. other problems dentists and otolaryngologists encounter in clinical practice are concurrent systemic diseases (patients with multiple problems): patients with bleeding diatheses, diabetes mellitus and a hidden primary malignancy. a non-healing ulcer in the oral cavity may hide a primary malignancy behind it. in these cases, you have to look for it specifi cally. similarly, one has to be aware of oral manifestations of internal pathology. some of them are crohn's disease, ulcerative colitis and gastro-intestinal tract malignancies. often dentists see patients after a tooth extraction with intractable bleeding to fi nd that they have a bleeding diathesis. so, this may be the fi rst presentation of these patients' bleeding disorder. when this patient undergoes any elective procedure in future, it will be a great help to surgeons to be aware of this information to prevent any inadvertent complications. therefore an integrated ehr can prevent unwanted complications where a patient's life may be in jeopardy. the source of otalgia or earache can be from a number of sites other than ear itself. technically ear lobe and ear canal are supplied by four different cranial nerve branches (5th, 7th, 9th, 10th). therefore, an area with a common nerve supply can present as earache. common dental problems which present as referred otalgia are (1) dental caries (2) oro-dental diseases or abscesses (3) an impacted molar tooth (which is a common cause) (4) malocclusion (5) benign and malignant lesions of oral cavity and tongue (kim et al. 2007 ) . therefore, it is essential these two departments collaborate with each other in diagnosing and treating these diseases, and one way of facilitating it is through an integrated ehr system. there is a lot of overlap between dentists and otolaryngologists in the diagnosis and treatment of patients with halitosis (delanghe et al. 1999 ; bollen et al. 1999 ) . poor oral hygiene is the most common cause for this common complaint. oral causes include tooth caries, oral ulcers, periodontal diseases, unhealthy mucosa of the oral cavity. it is interesting to note that a simple oral ulcer can form an abcess eroding the fl oor of mouth and becoming a life-threatening oral cellulitis (ludwig angina). once the cellulitis has developed, it becomes a medical emergency. therefore, it is essential to prevent it before it can progress into a life-threatening condition, which of course is possible. causes pertaining to otolaryngologists include: chronic sinusitis or mucociliary disorder, chronic laryngitis or pharyngitis, pharyngeal pouches-related pathology, tumors or ulcers of naso/oro/laryngopharynx, diseases or conditions that impair normal fl ow of saliva such as salivary gland diseases or stones preventing fl ow of saliva, medications which cause dryness of mouth: antihistamines, antidepressants; local manifestation of systemic disorders: auto immune disorders, sjã¶gren syndrome, dehydration from any cause, diabetes mellitus and gastro esophageal refl ux disorder (gerd). gerd is caused by improper neuro-autonomy of the lower esophageal sphincter (les). the les does not close tightly after food intake which causes gastric content to enter the esophagus. over time this can erode mucosa and cause various diseases even becoming cancerous (friedenberg et al. 2010 ). this disorder is attributed to life style. fast food consumption habits (oily fried foods) and eating habits (swallowing food without properly chewing) are partly responsible for this disorder (lukic et al. 2010 ; al-humayed et al. 2010 ) . here again an early diagnosis can manage the disease process before it is fully developed. at present there are no integrated ehr systems serving these specialties (dentistry and otolaryngology). an integrated ehr would facilitate effi cient communication between a dentist and an otolaryngologist who are providing care to the same patient and addressing a problem with a shared focus between the two disciplines. such integrated communication, may only require consulting the available medical or dental record of the patient, based on the particular circumstance. even enabling this simple communication would avoid duplication of effort, clarify the context of certain symptoms and reduce stress endured by the patient. it also has the potential to reduce healthcare delivery costs, and in some cases, even contribute to saving the patient's life. henry hood, allan g. farman, and matthew holder in this chapter, the authors attempt to put forth a justifi cation for precisely this kind of collaborative approach through a summary and discussion of a series of actual clinical cases. the protocols discussed in the management of each of these clinical cases illustrate the value in providing whole-person, interdisciplinary health care to this complex patient population. there is arguably no single patient population for whom the provision of collaborative, interdisciplinary health care is more challenging than for patients with neurodevelopmental disorders and intellectual disabilities (nd/id). in planning and delivering the generally-accepted standard of health care to this unique population, myriad biomedical, psychosocial and sociopolitical realities converge to create a landscape that is, at best, daunting for patients with these disorders, and for the clinicians who are charged with their care. anecdotal and scientifi c evidence suggest that this landscape has produced a paucity of physicians and dentists who are willing and able to provide care to patients with nd/id, and that american medical and dental schools are providing little training focused on their care (holder et al. 2009 ; wolff et al. 2004 ) . in february of 2002, 16th surgeon general david satcher issued a report, which documented that americans with nd/id experience great diffi culty accessing quality health care (thompson 2002) . in that same report, former health and human services secretary tommy thompson said, "americans with mental retardation and their families face enormous obstacles in seeking the kind of basic health care that many of us take for granted." (thompson 2002) the disparities identifi ed by dr. satcher and secretary thompson require that physicians and dentists approach this population in a spirit of collaboration, compassion, and teamwork in order to produce positive health outcomes for them. perhaps, an even greater imperative driving the need for collaboration between medicine and dentistry in this arena is the fact that many patients with intellectual disabilities have developed this cognitive impairment as the result of an underlying neurodevelopmental disorder that is often undiagnosed. and it is this neurodevelopmental illness and the constellation of potentially devastating complications associated with that illness that create a biomedical fragility and a vulnerability that neither begins nor ends at the oral cavity, and that leaves these patients at risk in almost every aspect of their daily lives. when, for example, patients with nd/id are dependent upon publicly-funded programs for their health care, and when these systems fail to provide the health services that biomedically complex cases require because they fail to account for and accommodate the link between medical and dental pathologies, the risk of a negative outcome is greatly enhanced. such was the case for an intellectually disabled woman in michigan who, in october of 2010, was unable to access dental services through the state's public medical assistance program, and who fatally succumbed to a systemic bacteremia resulting from an untreated periodontal disease (mich. dent. assoc. 2009 ). the american academy of developmental medicine and dentistry (aadmd) defi nes a neurodevelopmental disorder as a disorder involving injury to the brain that occurs at some point between the time of conception and neurological maturationapproximately age 21 or 22 (zelenski et al. 2008 ). examples of frequently-encountered neurodevelopmental disorders would include fragile x syndrome, a genetically acquired neurodevelopmental disorder caused by a mutation at the distal end of the long arm of the x chromosome (see fig. 4 .12 ), trisomy 21, another genetic disorder, which features extra genetic material at the chromosome 21 site (see fig. 4 .13 ), and cerebral palsy, a prenatal or perinatal, acquired neurodevelopmental disorder (see fig. 4 .14 ). patients with neurodevelopmental disorders tend to present clinically with one or more of fi ve frequently-encountered, objective symptom complexes or primary complications. these fi ve, classic primary complications include intellectual disability (aka: mental retardation), neuromotor impairment, seizure disorders, behavioral disturbances, and sensory impairment (aadmd). additionally, multiple secondary health consequences can derive from the fi ve primary complications; and any one of these secondary health consequences, or a combination of them, can produce profound morbidity. an example of a common secondary health consequence seen in patients with nd/id, which is derived from intellectual disability and / or neuromotor impairment, is the patient who is unable to care for his or her own mouth, and who develops ubiquitous caries and advanced periodontal disease as a result (see: fig. 4.15 ). another example would be the patient who suffers from the secondary health consequence of gastroesophageal refl ux disease (gerd) as a result of the neuromotor impairment associated with multiple neurodevelopmental disorders; and whose tooth enamel and dentinal tissues become chemically eroded as a result of the chronic intraoral acidity produced by gerd (see: fig. 4.16 ) . the diagnosis and management of these secondary health consequences provide dentists and physicians with a unique opportunity to work together to improve the quality of health and quality of life for their patients by implementing a team approach, which crosses the traditional interdisciplinary lines of communication, and which expands each clinician's ability to make meaningful treatment options available. indeed, it is often the case that quality primary care provided in one discipline will provide potentially valuable information to an attending clinician from another discipline. such is the case with the patients featured in figs. 4.16 and 4.17 . the patient whose intraoral photograph is featured in fig. 4.14 is a 19 year-old male patient who presented to a special needs dental clinic accompanied by his mother. the mother indicated that her son was exhibiting hand-mouthing behaviors that she believed suggested he was experiencing mouth pain. a comprehensive radiographic and intraoral exam revealed, among other maladies, notched incisors, multiple diastemas, grossly decayed mulberry molars, and advanced periodontal disease. the patient also exhibited moderate to severe intellectual disability. these fi ndings were all consistent with a diagnosis of congenital syphilis. 6 however, in developing the medical history with the mother, it was learned that no previous diagnosis of syphilis had been discussed with the mother, nor was it included in the health history. in cases like this, a comprehensive dental treatment plan should always include consultation with the primary care physician for purposes of moving forward with confi rmation of the clinical diagnosis by serologic testing, and consultation with a cardiologist to assist in the management of potential cardiovascular sequelae. as the dental treatment plan is being developed, consideration should also be given to human immunodefi ciency virus (hiv) testing for this patient, as coinfection is a common fi nding 7 . this issue could easily be attended to by a primary care physician, an internist or an infectious disease specialist. in the absence of any of these team members, the dentist should feel entirely comfortable ordering hiv testing. the primary care physician and the developmental dentist should continue to advise each other and their respective consultant specialists of any signifi cant developments or new information, which could in any way impact either the medical or the dental treatment plan. as treatment progresses, both the physician and the dentist should expect improvement in the patient's periodontal status, which will likely be refl ected in a decrease in the frequency of immune-related illnesses, and in the maladaptive behaviors produced by chronic oral pain. it is quite often the case in this patient population that, with a reduction in maladaptive behaviors, comes a reduction of the use of psychotropic medications prescribed in a frequently futile attempt to manage behaviors that were born of an undiagnosed medical or dental illness. gerd is defi ned as the refl ux of gastric contents into the esophagus. gerd is primarily associated with incompetence of the lower esophageal sphincter; however there are numerous co-contributors, which may predispose a patient to gerd or exacerbate an existing refl ux problem. these co-contributors include a diet high in fat, neuromotor impairment associated with functional abnormalities such as dysphagia, neuromotor impairment associated with impaired ambulation and prolonged periods of recumbence, and the use of multiple medications including anxiolytics, calcium channel blockers, and anticholinergics. gerd is thought to affect approximately 25-35% of the general us population. it has been established in the literature that the incidence of gerd in patients with intellectual disabilities is signifi cantly higher than in the neurotypical population, and that the relative number of unreported cases of gerd is much higher in patients with a neurodevelopmental diagnosis, as well. patients who have gastric refl ux as a function of a neurodevelopmentally-derived neuromotor impairment and a coexisting intellectual disability are impaired in their ability to voice the complaint that would, in the neurotypical patient, commonly lead to an encounter with either a family physician or a gastroenterologist and, ultimately, to a diagnosis. this inability to voice a complaint can be problematic in that, left untreated, gerd can produce maladaptive and sometimes aggressive behaviors in this population. and, of even greater concern, is the fact that undiagnosed esophageal refl ux can lead to more complex conditions that can produce signifi cant morbidity or even mortality -maladies such as barrett's esophagus or adenocarcinoma of the esophagus. chronic gerd can also produce an acidic intraoral environment, which can lead to the chemical erosion of the enamel and dentinal tissues of the teeth. ali et al. have established a link between erosion of the enamel and dentinal tissues of the teeth and gerd. there is additional anecdotal evidence suggesting a link between tooth enamel erosion and gerd, and related maladies. a special needs dental clinic in the eastern united states serving 1,000 patients with nd/id, has reported that, of nine patients referred to gastroenterology who presented for dental exam with a fi nding of either tooth enamel erosion or ubiquitous caries, two cases were diagnosed with gerd, two with barrett's esophagus, three with gastritis, and one with duodenitis. in all cases, medical treatment was required. in light of all that is known about the incidence of gerd and of the gerdrelated risks unique to this patient population; and in light of the link between tooth enamel erosion and gerd, it is incumbent upon any dentist encountering tooth enamel erosion in a patient with an intellectual disability to immediately refer that patient to gastroenterology for a work up, which should include esophagogastroduodenoscopy (egd) and ph monitoring. a dentist encountering gerd in a patient with an intellectual disability must be aware that he or she may be the fi rst and only link between that patient and the diagnosis of a potentially life-threatening illness. phenytoin-induced gingival enlargement can appear as either an infl ammatory lesion or a more dense, fi brotic hyperplastic lesion. the infl ammatory lesion is one in which the gingival tissues are swollen and bleeding, and in which pain is often a component. this type of gingival enlargement is the more acute lesion, frequently seen in patients who are currently taking phenytoin. in advanced cases of infl ammatory gingival enlargement, the tissues can appear botryoid, with a characteristic grape-cluster appearance. in advanced cases of phenytoin-induced gingival enlargement, the lesion can sometimes shroud entire sections of the dentition. phenytoin has long been a common medication used to treat seizure disorders in patients with neurodevelopmental disorders and intellectual disabilities. however, the gingival enlargement it produces, and the obstacle this lesion can pose to effective oral hygiene -especially in a population in which oral hygiene is typically compromised -can, over time, lead to periodontal disease, edentulism, and in advanced cases, systemic bacteremias. gingivectomy performed to reduce phenytoin-induced gingival enlargement will typically fail unless the patient is weaned off the offending medication, and another anti-seizure medication is titrated to effect. multiple alternative anti-seizure medications are currently available, which do not have the side effect profi le of phenytoin, and most patients who are weaned off phenytoin will demonstrate a virtual 100% resolution of the infl ammatory lesion within a matter of 3 or 4 months. the image in fig. 4 .18 is of a 22 year-old, microcephalic african-american male with intellectual disability, neuromotor impairment, and a seizure disorder. figure 4 .17 illustrates the appearance of this patient's gingival tissues while he was currently on phenytoin. figure 4 .18 features the same patient 4 months after being weaned off phenytoin and placed on topiramate. these images illustrate the dramatic result that can be achieved when a dentist and a physician work in collaboration in the best interests of the patient. it is worth noting that this particular collaboration required only one intervention to achieve this result: the patient was weaned off phenytoin and was placed on a safer alternate anti-seizure medication. any dentist caring for a patient with an intellectual disability who presents with phenytoin induced gingival enlargement should immediately contact either the primary care physician or neurologist managing the patient's seizure disorder, and strongly urge that the patient be weaned off phenytoin and placed on a safer alternative anti-seizure medication. edentulism and bacteremia need not be a side-effect of a seizure management protocol. the patient seen in fig. 4 .19 is a 20 year old male patient with idiopathic intellectual disability who presented to an outpatient dental clinic for comprehensive dental evaluation and treatment. he was accompanied by his father. his father was referred to the clinic by the staff at his son's day program workshop. the day program staff had observed hand-mouthing behaviors, and they had voiced concern that the patient may be in pain. in the waiting room, the patient exhibited behaviors consistent with neurodevelopmental dysfunction. he was non-communicative, and his gaze aversion and tactile defensiveness were suggestive of autism. he was resistant and somewhat combative when directed to the dental chair, and effective behavior management in both the waiting room and operatory required the combined efforts of his father and two staff fig. 4.19 the adult patient suspected of having fragile x syndrome members. the patient's health history was positive for attention defi cit hyperactivity disorder (adhd), and there was no history of seizure or neuromotor impairment. the father indicated that, at age ten, the patient was admitted to an inpatient psychiatric unit for evaluation of his uncontrollable behavior. the following day, the parents were told that managing the patient's behavior was beyond the ability of the psychiatric unit staff, and the parents were asked to take the child home. the father also indicated that the psychiatric unit staff described the child's behavior as overwhelming. the patient was last seen by a dentist 12 years prior to presentation; examination and treatment at that time were carried out in the operating room under general anesthesia. effective oral examination of this patient required utilization of papoose board and molt mouth prop. multiple options for behavior management, including utilization of general anesthesia in the operating room, were discussed with the father, and informed consent to utilize medical immobilization techniques for purposes of this examination was obtained and documented prior to taking the patient into the operatory. in the operatory a dental examination was performed, and a baseline panel of digital radiographs was obtained. the head and facial features of this patient were suggestive of fragile x syndrome (see: fig. 4.20 ) . the body of the mandible was somewhat elongated; the nose was prominent; the head had somewhat of a triangular shape, and the patient readily averted his gaze. upon further inquiry, the father reported that the patient also exhibited macroorchidism, although he indicated that no physician or dentist had ever suggested a work up for fragile x. fragile x syndrome is a disorder with which many clinicians are unfamiliar. yet it is the second leading genetic cause of intellectual disability in the united states, and it is the leading known cause of autism in the u.s. in addition to the phenotypic fi ndings noted in this case, there are other frequently-encountered physical characteristics consistent with fragile x that may move a clinician toward this diagnosis. they include pectus excavatum or funnel chest (see fig. 4 .21 ) and joint laxity (see fig. 4.22 ) . gaze aversion, as previously mentioned, is a typical fi nding in autism and in fragile x syndrome. indeed, in conjunction with non-verbal behaviors, gaze aversion is often the fi nding that initially alerts the clinician to the possibility of a neurodevelopmental diagnosis featuring autism as a complication. figure 4 .22 features a photograph of fi ve children at a school for children with special needs. four of the children have been diagnosed with autism, and a fi fth child is a neurotypical child who was visiting his brother on the day the photograph was taken. the reader is left to decide which child is the neurotypical child. any physician or dentist who encounters a patient with an obvious intellectual disability, who does not have an established underlying neurodevelopmental diagnosis, and who presents with additional fi ndings, which may include gaze aversion, shyness, a prominent chin, pectus excavatum, a large nose or large ears, should suspect a possible fragile x diagnosis. the primary care clinician -physician or dentist -should discuss with the guardian or family member the importance of establishing a neurodevelopmental diagnosis. the family member or guardian should be informed that genetic counseling should be made available to all members of the extended family, since fragile x syndrome is a genetic disorder that can be passed from parents to offspring. once this discussion has taken place, a referral to a geneticist for a complete genetic work up is indicated. both the dentist and physician should feel entirely comfortable making this referral. in remote areas where the services of a geneticist may not be available, the attending physician or dentist may order a high resolution chromosomal analysis and a fragile x dna test, and have those results sent to a remote location for interpretation by a geneticist. consultation with a psychiatrist or a clinical psychologist may also be advisable, as patients with fragile x can sometimes experience enhanced social integration as a benefi t of behavioral therapy. the healthcare access problem for americans with neurodevelopmental disorders and intellectual disabilities is, at its core, a healthcare education problem -an education problem resulting from a long-standing defi ciency in professional training focused on the care of this patient population. and it is clear that the medical and dental professions share equally in responsibility for these defi ciencies. eighty-one percent of america's medical students will graduate without ever having rendered clinical care to a single patient with a neurodevelopmental disorder or intellectual disability; and the graduates of 90% of america's medical residency programs will graduate from those residencies having had no formal training whatsoever -didactic or clinical -in the care of this patient population. 1 additionally, 50% of graduating dentists have never treated a single patient with a disability. 2 it is no wonder that patients like those whose cases were discussed in earlier sections of this chapter have such diffi culty accessing quality health care. as robert uchin, dean of nova southeastern university college of dental medicine observed in a speech in 2003 to his faculty, "not only do we not have enough doctors to care for these patients; we don't have enough teachers to teach them how to care for them." as a result of these defi ciencies in professional education, few clinicians with any expertise in developmental medicine or developmental dentistry are to be found in communities across america. the experts in developmental medicine and dentistry, for the most part, tend to be physicians and dentists who work at the few remaining intermediate care facilities, and at special needs outpatient clinics, psychiatric hospitals, and nursing homes. these physicians and dentists possess the knowledge and expertise in these disciplines because they are the physicians and dentists with the clinical experience. unfortunately for the patients with neurodevelopmental disorders who are clamoring for quality care, there are too few of these clinicians. national experts in developmental medicine and dentistry, however, have begun to collaborate in the creation of patient care protocols; and they have produced multidisciplinary curricula in both dvd and online format. the aadmd has made available 9 hours of online curriculum in developmental medicine, developmental dentistry, and developmental psychiatry (see: list of urls). the curriculum program is entitled, the continuum of quality care , and it teaches collaborative patient care in three disciplines through an interdisciplinary format. the aadmd, through a grant from the wal mart foundation and the north carolina developmental disabilities council, and in collaboration with the north carolina mountain area health education center and the family medicine education consortium, has also established the national curriculum initiative in developmental medicine. this initiative, which is scheduled for completion in 2012, will develop curriculum standards for physicians in the primary care of adults with nd/id. the curriculum stresses the importance of a collaborative approach, which includes medicine, dentistry, podiatry, optometry, and multiple ancillary health professions. if the disparities in access to healthcare for americans with nd/id are to be resolved, physicians and dentists must be willing to cross professional boundaries and work together to plan and deliver whole-person healthcare to their patients with nd/id. interdisciplinary protocols in the diagnosis of neurodevelopmental disorders and in the management of the secondary health consequences associated with these disorders must be established. additionally, clinicians with expertise in these arenas must be willing to work and teach in our nation's medical and dental schools. the clinicians with expertise must be willing to develop predoctoral and postdoctoral curricula, and the deans of america's professional schools must be willing to include these curricula as part of their larger programs in primary and specialized care. the clinicians with expertise in developmental medicine and dentistry must also be willing to conduct patient-focused, interdisciplinary, clinical research in an effort to solve the myriad problems that create obstacles to the delivery of the standard of care for patients with nd/id. they must be willing to obtain institutional review board approval for this research, and they must be willing to make this research available to their colleagues through publication in peer-reviewed journals and text books, and in professional lecture forums. the patient featured in figs. 4.23 and 4.24 is a man named james. he is a 48 year old patient with idiopathic intellectual disability who presented to a dental clinic for evaluation of a painful facial swelling. a comprehensive intraoral exam revealed a cellulitis resulting from multiple grossly decayed teeth, and a generalized advanced periodontitis. no fewer than fi ve clinicians became involved in this patient's care. they included a general dentist, two oral surgeons, a family practice physician, and a geneticist. over the course of several months, as the treatment plan was completed, and as the chronic dental and periodontal infections were eliminated, james experienced signifi cant improvement in his overall state of health. a comparison of these two photographs reveals not only signifi cant improvement in his aesthetic appearance, but also in his skin turgor and color. these improvements in the patient's health translated to improvements in his daily life. he found gainful employment, and his caregivers now report that he smiles constantly -at work and at home. these photographs were entered into evidence in 2007 before a congressional subcommittee investigating the death of a young african-american boy who died as a result of an untreated dental abscess. the photographs were intended to make the point that patients with intellectual disabilities need not die as a result of medical illnesses derived from untreated dental disease. this patient's case illustrates that, when physicians and dentists are willing to work together toward a common goal of whole-person health for their patients, profoundly positive outcomes can be achieved. in a larger context, if our nation's medical and dental professions are willing to commit to a shared agenda, one which promotes the idea of collaborative, interdisciplinary care as a foundational concept, signifi cant improvements in quality of health and quality of life can be realized, not just for americans with neurodevelopmental disorders, but for every patient seeking quality care. in light of the events of 2001, bioterrorism has become subject of increased attention from all members of society. government agencies, professional associations, academia, etc. have expressed their determination to wage war on such threats by all means available. dentists can also participate in this effort by providing assistance at interested groups and the general public (flores et al. 2003 ) . in this chapter we will examine the elements and components that may play a role in the establishment of an electronic network for the dental profession for supporting the fi ght against bioterrorism. in this section we review the threats, the public health system, current electronic surveillance systems, regulations and ethical issues, the computerization of dentistry, and how dentistry can serve in improving biosurveillance efforts. the aftermath of september 11 and the anthrax incidents in october 2001 ( lane and fauci 2003 ) , made the us government reorganize its priorities and reform its current structure (white house offi ce of the press secretary 2003 ) . in response to these incidents, president bush proposed the "health security initiative" (white house letter 2002 ) in february 2nd of 2003. this effort labeled the "bioshield initiative," (white house letter 2002 ) has the purpose to stimulate research and development of medical countermeasures against bioterrorism attacks. however, despite all these efforts, terrorist attacks are likely to happen in the future and even the best work from intelligence and security agencies will be unable to prevent such events (betts and richard 2002 ; council on foreign relations 2003; baker and koplan 2002 ) . to cope with this threat, a report published by an independent task force sponsored by the council on foreign relations "america-still unprepared, still in danger" (council on foreign relations 2003 ) , suggested a series of steps to assist the government in preparing to better protect the country. one of these suggestions is the bolstering of the "public health systems". baker et al. defi ne the u.s. public health system as a system that consists of a broad range of organizations and partnerships needed to carry out the essential public health services, such as hospitals, voluntary health organizations, other non-governmental organizations and the business community (baker and koplan 2002 ) which can collaborate with local, state and federal public health entities. after the unfortunate incidents in 2001 the public health system was revisited and the realization that "the nation's public health infrastructure is not fully prepared to meet this growing challenge" (frist 2002 ) became clear. to address this need, congress and president bush enacted the public law (p.l.) 107-188 titled "public health security and bioterrorism preparedness and response act of 2002" (frist 2002 ; 107th congress 2002 ) . the main purpose of this law was to improve the public health capacity by means of increasing funding and fostering other measures. frist ( 2002 ) , described the law as a "good start" and that "to be prepared for bioterrorism, it is imperative that we develop a cohesive and comprehensive system of ongoing surveillance and case investigations for early detection". in this way, several early detection systems have been implemented with different levels of success among different geographic regions in the us. one of the most important initiatives over the years has been the establishment of the national electronic disease surveillance system (nedss) (baker and koplan 2002 ; nedss 2001 ) . the national electronic disease surveillance working group establishes that the "nedss is a broad initiative focused on the use of data and information systems standards to advance the development of effi cient, integrated, and interoperable surveillance systems at the state and local levels. the long-term objectives for nedss are the ongoing automatic capture and analyses of data needed for public health surveillance". the purpose of this system is to take into consideration and integrate the information of current public health systems implemented at different health department levels: county, state and fi nally at the centers for disease control and prevention (cdc). another initiative spearheaded by the cdc is biosense (looks 2004 ) . the purpose of this program is to develop advance detection capabilities of health related events including disease outbreaks. in addition, its emphasis is to improve situational awareness by integrating advanced analytics to process data generated by different health providers and other entities in the us. now that we have examined the general aspects, we will continue our background review focusing on the aspects that pertain to the specifi cs of the dental profession. this section will provide some perspective of the structure of the dental profession in comparison with its medical counterpart. "there are approximately 150,000 active dentists in the united states" (mertz and o'neil 2002 ) . in 1990 the dentistto-population ratio was of 60-100,000. and it is expected that by the year 2020 the ratio will be 52.7, which translates into one dentist for every 1,898 people. " in contrast, the physician-to-population ratio has been increasing for the past 40 years and now stands at 286 per 100,000, about one physician for every 349 people." eighty percent of the dentists are in general practice. during march 27 and 28 of 2003, the american dental association and the us public health service sponsored the conference "dentistry's role in responding to bioterrorism and other catastrophic events" (palmer 2003 ; national institute of dental and craniofacial research 2004 ) . this meeting reviewed several aspects of bioterrorism and the dental profession: the nature of biological pathogens and its oral manifestations, what needed to be communicated, how dentists should participate, etc. dr. michael c. alfano described the diffi culties that biological pathogens create for clinicians because "they are so insidious." while discussing the anthrax mailings after september 11th he pointed out that: "â�¦ early symptoms appeared so they resembled the aches, fever, and malaise of fl u so those affected delayed seeking treatment, a delay that has proven fatal in some cases". lieutenant colonel ross h. pastel of the us army medical research institute of infectious disease (usamriid) listed the "category a" pathogens as defi ned by the centers for disease control and prevention, and those are: smallpox, anthrax, plague, botulinum toxin, tularemia and viral hemorrhagic fever. he also described an outbreak of smallpox in yugoslavia in 1972 and the measure that had to be taken to control it. dr. michael glick described the oral manifestations of smallpox showing "signs 24 hours before skin rash. these oral signs include tongue swelling, multiple mucosa vesicles, ulceration, and mucosal hemorrhaging. oral signs are also evident in inhalation and gastro-intestinal anthrax. in oropharyngeal anthrax the mucosa appears edematous and congested; there may be neck swelling, fever, and sore throat" . dr. ed thompson, deputy director of the centers for disease control and prevention mentioned that "none of the new counter-bioterrorism measures can be effective unless local health practitioners are vigilant in observing and reporting a possible disease outbreak. such surveillance-knowing what to look for and whom to report to-is critical and applies not only to suspected bioterrorist agents, but to a list of reportable diseases which has grown to include such entities as west nile virus and sever acute respiratory syndrome (sars)." dr. sigurs o. krolls presented the response at the local level and he "stressed the importance of communication and the need for redundant systems", "to keep all the parties informed". he also posed the question "can dentists recognize signs and systems of contagious diseases?", and emphasized that education can be essential. dr. louis depaola made several connotations that can be key in the scope of this paper by saying "dentists can contribute to bioterrorism surveillance by being alert to clues that might indicate a bioterrorism attack. such surveillance would note if there is an infl ux of people seeking medical attention with non-traumatic conditions and fl ulike or possibly neurological or paralytic symptomsâ�¦ or even specifi c signs of a bioterrorist agent. patterns of school of work absence, appointment cancellations or failures to appear, could also be indicators." dr. depaola made clear that in cases of limited release of bioterrorist agents, dentists "have little to offer" but "a widespread attack can certainly tap into dental professional skills in recognition, isolation and management". in addition, dr. guay ( 2002 ) lists all the possible roles in which dentists can participate including "education, risk communication, diagnosis, surveillance and notifi cation, treatment, distribution of medications, decontamination, sample collection and forensic dentistry." dental informatics must pay attention to these and other recommendations, in order to develop integrated systems that take these recommendations into consideration. it is also important to understand that informatics has to work with technologies already in place like the computer-based oral health record and current standards. the fi nal recommendation from the meeting stated that to play an important role in biodefense, a serious amount of coordination and preparation will be required, not only from dentists but from other groups, most likely requiring medical and dental data integration. the cohr as described by rhodes ( 1996 ) "can provide a structure for documentation that goes beyond the concept of a blank form on a page, it includes a glossary of dental terminology for the entire content of the form as well as knowledge bases and expert systems that can enhance the practitioner's diagnostic and treatment planning decisions". he also acknowledges that one of the advantages of this type of documentation is that it "is much more transportable". he also recognizes the need for standardized methods for collecting information from dentists. schleyer and eisner ( 1997 ) defi ned several scenarios where the cohr is used in a "shared" environment where several healthcare providers interact and information is seamless communicated, improving the decisions made by clinicians. delrose and steinberg ( 2000 ) discuss how the "digital patient record" enhances clinical practice by providing "better quality information" to the clinician. although all of these benefi ts sound promising and encouraging some still express concern of the lack of standards among different information systems, which translates in communication breakdowns (schleyer 2003 ) . on the other hand, heid and colleagues ( 2002 ) mention a list all the steps that are currently being taken by different organizations such as the ada in order to produce a standardized cohr. other examples of standardization can be found in a paper presented by narcisi ( 1996 ) where ada's participation as a voting member in the american national standards institute has allowed edi or the cohr to be discussed and improved at a national level. additional infl uences in the standardization of the cohr are the security regulations mandated by hipaa, the health insurance portability and accountability act of 1996. dentists are required to "adopt practices necessary for compliance" (sfi kas 2003 ; chasteen et al. 2003 ) . these and other regulations (szekely et al. 1996 ) will encourage the homogeny among different system vendors. computer ownership, on the other hand, has increased steadily during the last 25 years. according to schleyer et al. ( 2003 ) in 1976 only 1% of dental professionals used computers in their practices compared to 85% in the year 2000. additionally similar trends in internet connectivity where described. the issues mentioned above describe the issues that have to be considered in order to create surveillance system against bioterrorism for the dental profession. this review has tried to be inclusive by covering different aspects starting with the current state of affairs and environment, treats, technology, law, etc. next we present a blueprint for developing a biosurveillance system. the purpose of developing an electronic health surveillance system is to gather information from patients directly ( wagner et al. 2006 ) by detecting signs and/or symptoms, or indirectly by obtaining other types of information such as over the counter medication sales, patients' no-shows, usage of internet search engines keywords, etc. in this particular case, the proximity of contact between the dentist and the patient is equivalent to a medical inspection in terms of immediacy and/or closeness. such signs and symptoms can be easily detected if the dentist is properly prompted to search for them. this is just one example of ways how a system could provide assistance in the detection of a bioterrorism incident. but, before describing our proposed system, it would be important to address the fact that current syndromic surveillance systems have certain advantages in terms of its particular technological implementation . the rods laboratory obtains data directly from chief complains in the emergency departments from hospitals. the advantage of this surveillance system is that the implementation has to be made with only a limited number of parties (hospitals, clinics, health systems, etc.). on the other hand, our system would have to deal with thousands of different implementations (one in each dental offi ce). this and other challenges have to be considered when designing the proposed system: the proposed system should work at multiple levels: the system would have to provide a mechanism to alert the dentist if there is â�¢ suspicion that a bioterrorist attack may be happening. the mechanism would increase the dentist's awareness in case of fi nding suspicious signs or symptoms in a patient. this can be triggered by the patient's characteristics such as geographic location of residence, etc. automated collection of information from the patient's oral health record. the â�¢ system would report to a central database signs or symptoms of interest. the aggregation of this data could generate information that would eventually identify the presence of patterns that may lead to the early detection of such events. collection of additional information, which combined with other sources, can be â�¢ useful in terms of detecting or tracing some incident. patients' "no-shows" is the primary example, that, if combined with others such as work or school absenteeism can provide a relevant pattern for public health offi cials. dr. x, who practices in a community 20 min away from capitol city, installed a new clinical management system 2 months ago. among the features that were included in this new clinical management system (cms), a bioterrorism detection module was added. she felt curious because of recent news she read in the newspaper about possible attacks against the us and decided to install such feature. he read about how the module would work in combination with the cms she just bought. the educational information provided with the software instructed dr. x, that in case that a patient victim of a bioterrorism attack happens to be seen in her practice, the software would collect information and would send it to public health offi cials. when installing the software, dr. x was asked if she agreed to share such information with authorities. she was provided the option to receive notifi cation in case some information was sent but she decided not to enforce it. during the last week a patient walked into dr. x's dental offi ce. the patient presented some signs that indicated the presence of a disease; still its origin was not clear. an epidemiologic study later would show that the patient was present at the football stadium when an infectious agent was released (fig. 4.25 ) . although, at that time his medical history showed no indication of a systemic disease, the presence of multiple oral vesicles prompted the dentist to make an annotation into the cohr. the system, by using a natural language processing engine, detected such sign and sent this information to a central database. the patient was discharged and instructed to take some support medication to treat the oral ulcers. the next day, the central database pinpointed the presence of an out of the ordinary increase in the number of cases with the same signs and symptoms around that region. when the presence of this peak was detected, the central server sent a request to the dentist computer for additional information. one of the requested elements was if there was any use of medication for treating oral ulcers. fortunately this information was available. the central database crossed this with the information of other surveillance systems together with the information from other patients that happen to have similar clinical signs and/or symptoms. dr. x received an email from a public health offi cial asking her to communicate to the local health department to discuss information about one her patients. the case depicted above simulates the release of smallpox during a football game. in the case of smallpox oral symptoms include tongue swelling, multiple oral mucosal vesicles, ulceration, and mucosal hemorrhaging (national institute of dental and craniofacial research 2004 ) . dentists could be alerted by an electronic system to search for such signs or they can be detected automatically. in case of a high incidence within a group of patients, in a confi ned area, public health offi cials get to be notifi ed. in our hypothetical case there are issues that need to be addressed in order to make such detection system feasible: as described by schleyer et al. ( 2006 ) , 85% of dentists in the us use a computer in their practices. this fi gure would generate an estimate of 127,500 computers in dental practices. this prevalence of computers represents an opportunity for public health data collection. the creation of a software application for surveillance purposes must rely on existing technology. currently there are approximately 20 major clinical management software packages in the market (dentistry today 2003 ) . out of these 20, 17 clearly permit direct database manipulation. this characteristic can easily allow the creation of a "querying" application that would look for specifi c information within the data stored by those packages. additionally, a natural language processing engine could be embedded into the application in order to detect variations in data input on the computer oral health record. nevertheless, it is necessary to obtain a detailed list of the oral manifestations of diseases that are likely to be found on patients. successful implementations of similar systems have been shown to work successfully (chapman et al. 2001 ; ivanov et al. 2002 ) and using the same approach for our system seems technically feasible. this collected information later would be send to a central server in order to be analyzed and interpreted. the components of our system would be as follows (fig. 4.26 ) : thin client: a software application distributed for data collection. it would be â�¢ conformed of a "querying" mechanism, combined with a natural language processing engine and a communication module. this software client should be as thin as possible to reduce the work load on the dentist's equipment and should be embedded as a plug-in for current clinical management systems. vendors should be contacted to ask for their collaboration in the development of such application to ensure maximum compatibility and integrity of data collection. central servers: server software in charge of integrating all the data collected â�¢ from dental offi ces. it has to be capable of handling simultaneous requests from multiple users. this server would integrate all the data and would perform an analysis with the intention of detecting anomalies. it would be recommended that redundant servers should be located in different data centers with mirroring capabilities to guarantee their survivability in case of technical diffi culties. communication network: the transmission of information should be done using â�¢ the internet. this, of course, would essentially depend on the practitioner's current connectivity. if that is not available, backup connection to the central servers should be established. dentistry uses several standards for transmission of health related information. clinical management systems use standard-based technology to transmit information (narcisi 1996 ; chasteen et al. 2003 ; szekely et al. 1996 ; dentrix dental systems 2011 ) . dentists are aware of these standards and use them in a day-to-day basis to transmit information to insurers. additionally, in order to interact with other surveillance systems such as the nedss, our application should rely on the same standards. the software both client and server should be thoroughly verifi ed to be secure in terms of being safe against hacker attacks. on the server side, redundancy should be provided so downtime is reduced from design. the system should be developed so mirrored servers are always up and running. data integrity mechanism should also be considered. privacy and confi dentiality are important issues that need to be incorporated as part of a robust biosurveillance system and distinct regulations such as hipaa require protecting patient information (frist 2002 ; chasteen et al. 2003 ; bayer and colgrove 2002 ; etzioni 2002 ; ivanov et al. 2002 ) . in our hypothetical case we describe the use of several sources of information for detecting a bioterrorist attack. we described how syndromic information is transmitted to a central database which initially should be de-identifi ed. later, after the suspicion a bioterrorist attack more information is requested (medications) and more inferences are made. this, although technically possible, would require changing our processes and also the will to share clinical information. this leads to the discussion mentioned in the background section about "individual rights" vs. "common good". although hipaa addresses public health , some other implications may arise and the health professionals including dentists, physicians, public health offi cials and patients should discuss and address such issues. as discussed earlier, legislators face a diffi cult task in terms of determining what is best on behalf of the individuals they were asked to represent. legislation may have to be passed in order to guarantee the functioning of such a system. individual freedom and privacy are important values which may pose a confl ict when collecting individuals' information even for their own good. in any case, careful consideration has to be given to which information is required to detect a bioterrorist attack and also, by keeping in mind that it is always important to reduce, as much as possible, the collection and transmission of patients' information over the internet or any other network. a detection algorithm has to be created or adapted in order to determine the presence of a bioterrorist attack. some algorithms have proven their effectiveness (wong et al. 2003a, b ) and it is likely that from these, a new analysis should be done in order to select or create one that addresses the particular needs of our system. a study was conducted to assess the feasibility of using oral manifestations in order to detect disease outbreaks (torres-urquidy et al. 2009 ) . it was found that for diseases such as botulism and smallpox it would be feasible to gather data that contains oral manifestations that would allow creating a detection signal using natural language processing followed by the use of statistical methods such as moving average to serve as part of a detection algorithm. the system should also be thoroughly evaluated, before and after implementation. to perform the evaluation before the system implementation computer simulation can be used to assess the effectiveness and likelihood of detection. simulation and modeling techniques (reshetin and regens 2003 ) have been used to estimate the effects of a bioterrorist attack. the same techniques can be used to evaluate our system. in case of the study by torres-urquidy ( 2009 ) , the investigators utilized synthetic outbreaks to test the performance of different signals. from their evaluation process, they learned, for instance, how many cases would be necessary to occur for the system to reach certain detection thresholds. several dental organizations have shown publicly their support of measures against bioterrorism. the american dental association and the national institute of dental and craniofacial research are two organizations who could play an important role in the development, deployment and ongoing support for our system. local dental societies also would also play an important role in the deployment of the proposed system. similarly, local, state and federal public health agencies should engage in activities that could make these mechanisms for health surveillance feasible. if dentists want to play an active role in the fi ght against bioterrorism, they should commit to collaborate with public health entities as well as to seek a way to integrate their information with the rest of electronic biosurveillance systems. professional organizations such as the american dental association can also participate by endorsing such efforts and by collaborating in the educational process of the dental professionals and their patients. as mentioned by dr. depaola (national institute of dental and craniofacial research 2004 ) dentists "have little to offer" in the current biosurveillance state of affairs. however, the integration of different technologies can change this perception. goldenberg et al. ( 2002 ) described over-the-counter medication sales as a technique for discovering disease outbreaks and stated that their approach may be "more timely" than traditional medical or public health approaches. medical cases that result from bioterrorism attacks do not produce symptoms until they have fully developed, so it is likely that different patterns can be detected before the patients start reaching the emergency department. as stated earlier (torres-urquidy et al. 2009 ) , it may be possible to have dentists participating of biosurveillance efforts, if we solve the proper organizational and technical challenges. dr. john r. lumpkin ( 2001 ) states that "hippocrates noted the health of the community was dependent on characteristics of a community and the habits of the people who lived there." dr. krolls (nidcr 2003) in his fi nal remarks during his presentation at the dentistry's role conference against bioterrorism, said, "dentists may pick up telltale information about what is happening in the community. after all, dentists spend more time with their patients than any other health specialty". kass-hout t, zhang x. biosurveillance: methods and case studies. muhammad f. walji maintaining patient records are essential for both clinical care and research. clinical research often occurs in the context of also providing patient care, yet the systems that are used for each are different and often cannot exchange data. the lack of data exchange between systems pose signifi cant barriers to effi ciently treating patient and conducting clinical research in dentistry. the purpose of this section is to review the benefi ts and challenges of integrating electronic health record (ehr) used for patient care and electronic data capture (edc) which is used for clinical research such as clinical trials. an increasing number of dentists routinely use ehrs (schleyer et al. 2006 ) . most dental schools in north america also use ehrs. benefi ts of using ehrs include increased legibility, portability, and improved patient safety (buntin et al. 2011) . recent federal incentives, although not directly benefi cial to dentists, will also likely spur the adoption of ehr (blumenthal and tavenner 2010 ) . clinical researchers, especially those conducting clinical trials, are also discovering benefi ts of using electronic data capture compared to paper. a clinical trial is a process in which new treatments, medications and other innovations are tested to evaluate safety and effi cacy. a standard part of health care, clinical trials are often lengthy and costly due to myriads of regulatory oversight. recent estimates set the cost of drug development in excess of $800 million (grabowski et al. 2002 ) . accurately documenting data with suffi cient detail is critical for providing patient care and conducting clinical research. while the medical record is the foundation for patient care, the case report form is the foundation in a clinical trial. not all clinical research is clinical trials. clinical trials whose data will be submitted to the fda as a new therapy or device have additional requirements relating to the collection and transmission of the data. similarly for patient care data, ehrs need to meet the privacy and security requirements of hipaa. case report forms (crf) are a medium in which research study sites collect subject data in pre-defi ned formats for communication with clinical trial sponsors (rondel and webb 2000 ) many clinical trials data are collected on paper (rondel and webb 2000 ) . data measurement, collection, and recording are considered the "most crucial stage" in the data management process (hosking et al. 1995 ) . traditionally, study coordinators often record information in a case report form and subsequently mail or fax the crf to the centralized coordinating center. there, data entry staff, sometimes with the aid of optical character recognition systems, input crf data into a computer. errors made during this second data entry process are diffi cult to detect and correct (hosking et al. 1995 ) . lengthy guidelines in literature discuss methods for developing paper case report forms to reduce data entry mistakes (hosking 1995 ) . a well-designed crf may allow a user to effi ciently collect and record pertinent data. however, forms are often revised and redesigned during a clinical trial due to changes in protocol, unforeseen outcomes, or oversight (singer and meinert 1995 ) . there has been a recent drive to use electronic case report forms (ecrf). direct data entry at a study site shortens time to analysis and provides opportunities to audit data at time of entry. this could reduce data errors that might otherwise be caught weeks after submission. for quality control purposes, some studies require double data entry using computers and paper (day et al. 1998 ) , though alternative solutions have been explored including the use of data sampling (king and lashley 2000 ) and probability statistics to select only those forms likely to contain errors (kleinman 2001 ) . ecrfs may also facilitate data collection from existing electronic information systems such as lab systems. however, ecrfs are almost always reside in a separate system that is not linked to a patients record. although many clinical research studies are still being conducted using paper, an increasing number of studies are using ecrfs and electronic data capture (edc). for example, a review of canadian clinical trials found that 40% use edc (el emam et al. 2009 ). studies that are sponsored by a pharmaceutical company and are multicenter appear to use edc at a higher rate than those sponsored by government or a university. the cost of a commercial edc is substantial. recently a freely available edc has become popular amongst universities called redcap. a tool originally developed at vanderbilt university, it is now being used at over a 100 institutions worldwide (harris et al. 2009 ). however, such tools are generally not integrated with the institutions ehr. although moving from paper to electronic will afford benefi ts there is a great need to allow data exchange between the patient care and clinical research components of information systems. although ehr and edc are similar, several challenges remain unresolved that prevent integration. one of the major barriers is likely to be different workfl ows for patient care purposes and to collect data for research. research is needed in defi ning an optimal workfl ow that can streamline the tasks associated with patient care and research, while at the same time providing a unifi ed information system that support these activities. also, the data that are collected for care and research are likely to differ. a researcher may require far more granularity of an oral health measurement than a clinician seeking to provide care. in cases when conducting a double blind placebo controlled clinical trial, the investigator may not even know the type of treatment that has been delivered to the patient. due to complexities of each domain, and large differences in goals, to date mutually exclusive workfl ows have arisen. a clinician investigator who sees a patient for both care and research, will likely need to enter data on this same patient twice; once in the ehr and once in the edc system. despite the availability of electronic systems, a major barrier is the integration and compatibility of disparate health information systems to converse with one another. the languages are important because they can help data sharing. clinical trials are not usually conducted in isolation, but are part of conventional medical care. therefore sharing data by clinical trials, patient care and laboratory systems becomes especially important with the adoption of ehrs in dentistry. in biomedical informatics, standardized terminologies are recognized as a critically important area to help better represent and share data for use in electronic systems (cimino 2000 ) . the systematized nomenclature of medicine clinical terms (snomed-ct), developed by the college of american pathologists, is the most comprehensive medical terminology (strang et al. 2002 ; chute et al. 1996 ) and is used in a number of health informatics applications. the us department of health and human services ( 2010 ) has also licensed snomed-ct, allowing access throughout the us at no charge. therefore snomed-ct is even more likely to be the vocabulary used in electronic formats of patient records in the future. the medical dictionary for regulatory activities (meddra) is terminology used by the fda and drug development industry to classify, retrieve, present, and communicate medical information throughout the medical product regulatory cycle (brown et al. 1999 ) . in particular it is used to record and report adverse drug event data. therefore standard languages are essential in sharing clinical trials data between sites, and also with regulatory agencies. no one single terminology is suited for all tasks. snomed-ct is likely to be more comprehensive to code clinical encounters, while meddra is more suited to help adverse event reporting. however, it is important that terminologies are widely adopted and used for similar purposes. even when standard terminologies are agreed upon, such information needs to be interchanged in standard formats. health level 7 (hl7) is an important organization whose standards are widely adopted in healthcare to exchange information between computer systems. the clinical data interchange standards consortium (cdisc) is also an important group that helps to defi ne different data standards specifi cally for clinical trials research, such as clinical trials or regulatory submissions. one particular challenge in oral health has been the lack of a standardized terminology to describe diagnoses. although icd contains oral health concepts, they are often not granular enough to be useful for some patient care or research purposes. recently a dental diagnostic terminology has been developed by a group of dental schools, and has already been adopted by several institutions and used within dental ehrs (kalenderian et al. 2010 ) . the american dental association (ada) has also been developing snodent, but is not yet publically available for clinical use (goldberg et al. 2005 ). another link between ehr data and clinical research is the potential to fi nd human subjects. recruiting suffi cient numbers of patients that meet eligibility requirements within an allotted time frame for clinical trials is challenging. as ehrs contain detailed information about patients, they can be used to fi nd patients that meet specifi c inclusion and exclusion criteria. informatics for integrating biology and the bedside (i2b2), an open source data warehousing platform, has been found to be a useful tool for cohort selection especially if the source data from an ehr is represented in a structured format (deshmukh et al. 2009 ). further, with health information increasingly available to patients through the internet, it is possible interested patients will be more effective in fi nding clinical trials than investigators looking for patients. many clinical trial registers are now available online. the national institutes of health (nih) have made available their database of nih funded research (mccray 2000 ) . there is currently no single repository for patients to fi nd all trials studying a health condition. a recent study assessed the comprehensiveness of online trial databases concerning prostate and colon cancer and found that online trial registries are incomplete, especially for industry-sponsored trials (manheimer and anderson 2002 ) . a more collaborative effort between government and industry-sponsored research groups to compile and standardize information may be a mutually benefi cial effort. it is not clear how many patients now enroll in clinical trials through online discovery. ehr data originally collected for patient purposes can be potentially used for research. aggregating data from multiple sources can provide a large dataset that could otherwise not be available. electronic health records (ehr) contain a wealth of information and are a promising source to conduct research. data extracted from ehrs differ from other sources such as population surveys or data obtained from payers, as they provide a more detailed and longitudinal view of patients, symptoms, diseases, treatments, outcomes, and differences among providers. therefore ehr data in dentistry can potentially provide valuable insight into oral health diseases, and treatments performed on a large cohort of subjects. ehrs also play an important role in enhancing evidence-based decision-making in dentistry (ebd) and improving clinical effectiveness through decision support (atkinson et al. 2002 ; walji et al. 2007 ; valenza and walji 2007 ; taylor et al. 2007 ; spence et al. 2007 ; chambers et al. 2007 ; langabeer 2nd et al. 2008 ; walji mf et al. 2009 ). the consortium of oral health related informatics (cohri) provides an example of how dental ehrs are used for research purposes (schleyer et al. 2006; stark et al. 2010 ) . cohri was formed in 2007 by a group of dental schools who used the same ehr platform and who are interested in sharing clinical and education data. through funding from the national library of medicine, four dental schools are participating in a pilot project to develop an inter-university oral health research database by extracting and integrating data from ehrs. one promising area where data repositories derived from ehr data can be used for new discoveries is in the area of comparative effectiveness research. comparative effectiveness research is defi ned as "a rigorous evaluation of the impact of different options that are available for treating a given medical condition for a particular set of patients." (congressional budget offi ce 2007 ) further, such research includes focusing on the clinical benefi ts and risks of each option (clinical effectiveness), and an analysis on the costs and benefi ts (cost effectiveness analysis). comparative effectiveness research (cer) is also likely to reduce costs of dental care and increase access to the majority of the population who currently receive no dental care. unfortunately many recent systematic reviews focusing on cer questions in dentistry have been inconclusive due to the lack of existing evidence in the scientifi c literature. secondary analysis of the data that reside in dental electronic health records (ehr) is a particularly appealing approach to facilitate cer and generate new knowledge. ehr data has the potential to provide a comprehensive picture of patients' histories, treatments, and outcomes, and if integrated with similar data from other dental clinics can include a large and diverse set of patients. however, numerous challenges must be solved before ehrs can be used for cer. first, data suitable for cer must actually be collected from ehr systems. second, this data, which often resides in proprietary systems, must be accessible and retrievable. and lastly, this data should be structured in a format that can be integrated with data from other sources or institutions. practice-based research networks (pbrn) are groups of primary care clinicians and practices working together to answer community-based health care questions and translate research fi ndings into practice. pbrns engage clinicians in quality improvement activities and an evidence-based culture in primary care practice to improve the health of all americans. in 2005, the national institute of dental craniofacial research funded three such research networks. the dental pbrn's to date have been conducting both prospective and retrospective research. for example, barasch et al. conducted a case controlled study to investigate risk factors associated with osteonecrosis of the jaws . many prospective studies conducted as part of pbrns still require separate data collection systems for the research data. ehr data contained in practices as part of pbrns are beginning to be used for secondary purposes. for example fellows et al. conducted a retrospective analysis of data contained in electronic health records to estimate incidence rates of osteonecrosis of the jaws ( fellows et al. 2011 ) . pbrns provide great promise of how ehr and clinical research data can be used effectively to promote both patient care and new discoveries. another area that intersects both the patient care and research realm are patient registries. patient registries are ways to track groups of patients who have had specifi c diseases or have had certain treatments. while ehr data would contain information on all types of patients, their diseases, and treatments, registries would allow focus on specifi c diseases or treatments of interest. registries would not be as costly in terms of resource requirements like a traditional clinical trial, but would require specifi c eligibility criteria, informed consents, and collection addition to that collected as part of routing care. dentistry has lagged far behind in forming data registries, primarily because dentistry is practiced in small offi ces and not in large hospitals making the process of integrating data very diffi culty. however, dental schools which themselves house large clinical operations are ideally positioned to create disease specifi c registries that can potentially use data collected for patient care and extend for research purposes. there is great potential for providing new insight in oral health by the integration of patient records and clinical research from both a workfl ow and information systems perspective. the technology challenges of developing systems that can exchange data, and use standardized terminologies appear solvable. however, the socio-technical issues such as determining how to incorporate optimal workfl ows for conducing both patient care and research with minimal additional overhead appear to be the greatest challenge before widespread adoption. similarly, there appears to be great potential in using ehr data originally collected for patient care for the secondary use of research and discovery. this will require collaboration between patients, providers and researchers from all healthcare disciples, and institutions with friendly policies for sharing data to improve both patient care and drive new discoveries. amit acharya , andrea mahnke , po-huang chyou , and franklin m. din more recently there has been a strong push from the united states federal government for the adoption of the electronic health record (ehr) within the healthcare industry. as a result, $19.2 billion is made available to incentivize the physicians, dentists and hospitals for the adoption of the ehr through the health information technology for economic and clinical health (hitech) act. as the nation head towards adoption of the ehrs, there has also been a growing interest with the majority of the u.s. dental schools to implement ehrs within the educational setting. fifty of the fi fty-six u.s. dental schools, as well as dental schools in canada and europe, are either using or in the process of adopting some aspects of a common dental ehr framework (white et al. 2011 ) . a group of dental schools known as consortium for oral health-related informatics (cohri) was formed in 2007 which used this common dental ehr framework -axium (stark et al. 2010 ) . currently there are about 20 dental schools within cohri. the ehr will not only support clinical care, but will also result in training the next generation dental students and to conduct innovative research that was not possible earlier. however, not much is known about how many of these dental schools' electronic dental records are integrated with their respective university's electronic medical record. a common medical-dental ehr model at healthcare universities would enable a holistic approach to providing patient care and provide the much needed electronic infrastructure to study interrelationship between the various oral-systemic diseases. recently a group of researchers from marshfi eld clinic in wisconsin, us conducted a survey to investigate the current states of health information technology and informatics within the dental school in the us. list of us dental schools were identifi ed through the american dental education association (adea) web site. dental schools were contacted to determine who the most appropriate person to take the survey would be. once the list of contact was developed from each dental school, an email was sent to 55 us dental schools with a link to a survey created in surveymonkey. the survey was administered on tuesday march 1, 2011. reminder survey emails were sent to all recipients on march 9 and march 17. the survey was closed on march 31. the anonymous survey was at most 23 questions, depending on how questions were answered. the survey focused on topics such as presence of dental informaticians within the dental schools, use of fi nancial and clinical information systems, interest in federal stimulus support for ehr adoption provided through american recovery and reinvestment act and meaningful use of ehr, relationships with health care entities and bidirectional nature of the dental and medical ehrs. the study was approved as exempt from the marshfi eld clinic institutional review board under section 45 cfr 46.101(b) and waived requirement for an authorization. thirty out of the fi fty fi ve dental schools responded to the survey (response rate of 55%). however, fi ve of the thirty dental schools representative did not complete the survey and hence their response was not included in the analysis. regarding the question about the presence of a dedicated department or a center for information technology (it) or informatics within the dental school in us, 80% (n = 20) of the responding dental schools had a dedicated it/informatics department or center (p-value of 0.0027). the it or the informatics department size (in terms of the number of personnel) at the 20 dental schools is illustrated in fig. 4 .27 . thirty fi ve percent (n = 7) of the us dental schools that housed an it / informatics departments had personnel with not only it training but also dental informatics training. while 40% (n = 8) of the dental schools were considering integration of dental informatics personnel within their department or center. twenty fi ve percent (n = 5) of the dental schools did not have any plans of integrating personnel with dental informatics personnel within their department or center (see fig. 4 .28 ). partial responses to additional questions in the section 1 of the survey is provided under table 4.6 . the majority of the responding dental schools were currently using financial electronic systems (fes) (p-value of <0.0001) and electronic dental records (edr) (p-value of 0.0001). the use of fes outnumbered the use of edrs in the dental schools (see fig 4.29 ) . about 77% of the dental schools that were currently utilizing the edrs used it in all the clinical modules (p-value of 0.0105), while 23% of the dental schools used the edrs in some of the clinical modules. when asked about the commercial edr system that the dental schools were using, axium (exan group, canada) was by far the most implemented edr system. two dental schools had salud (two-ten health limited, ireland) implemented and two dental schools had gsd academic (general systems design group, iowa, us) implemented. combinations of two ehr systems (home grown and dentrix) were implemented at two dental schools. one school had a dentrix only implementation, while another had developed its own edr system (home grown) (see fig. 4 .30 ). there were 13 dental schools which had implemented an edr fi ve or more years ago, 3 dental schools 3-4 years ago, 4 dental schools 1-2 years ago and 2 dental schools less than a year ago (see fig. 4 .31 ) (p value of 0.0029). when the dental schools were asked the question as to whether they were expecting to apply for the medicaid meaningful use incentive program, majority (52%) of the dental schools did not know and only 28% of the dental schools were expecting to apply within the next 4 years (fig. 4 .32 ) (p-value of 0.0044). challenges or barriers identifi ed by some of the dental schools in complying with the meaningful use objectives were (a). lack of certifi ed edr and information regarding it, (b). issues with getting auxium certifi ed and (c). qualifi cations of the edr as many of the meaningful use objectives do not apply to dentistry and lack of specifi c information about it. only 44% of the responded dental schools were part of a health system. fifty two percent (n = 13) of the responded dental schools had a formal relationship with other health care delivery entities in terms of sharing facilities, patient transfer, training programs. some of the types of relationship mentioned by the dental schools that had a formal relationship with other health care delivery entities included: (a). a gpr program and an emergency dental unit in the hospital, (b). affi liated hospital, (c). affi liation agreements, (d). oral and maxillofacial surgery (omfs), anesthesia and pedodontics all have some portion of education in medical health center, (e). omfs residents are also residents of medical health center, (f). residents providing care under contract with area hospitals, (g). sharing patients wand facilities with the health center, (h). students rotating in the community health centers and (i) collaborative grand programs. eighty fi ve percent of the dental schools that had a formal relationship with the health care delivery entities had routine interaction with them because of their existing relationship (p-value of 0.0015). their usual method for exchanging information was through informal medium such as phones, emails and faxes and formal medium such as memorandums, letters and contracts. when the dental schools were asked about the communication between the health systems' emr and the school's edr, majority of the dental schools did not have any communication (60%) or did not know is such a communication existed (25%) (p-value of <0.0001) (see fig. 4 .33 ). out of the 60% (n = 15) of the responded dental schools who's edr did not communicate with the health system's emr, 47% (n = 7) of the dental schools stated that they did not need to exchange patient information electronically as a reason for the non-communication, while 33% (n = 5) dental schools states that they would like to exchange patient information electronically but there were barriers that prevent them from doing so. some of the barriers identifi ed by these dental schools were (a). the hospitals and the dental school are not part of the same medial system and hipaa concerns prevent sharing data, (b). the dental school currently neither did have an edr nor the infrastructure to support one and (c). hospital is not interested and has high and perhaps unrealistic security standards. the remaining 20% (n = 3) of the dental schools expected to exchange patient information electronically in the near future (next 5 years). some of the information categories that were shared between the edr and emr in the small number of dental schools are illustrated in fig 4. 34 . finally when asked about any research projects under way in their dental school to investigate discrepancies between medical and dental records for the same patient, only 1 (4%) dental school was currently undertaking such project. in all common diseases, including those that affect the oral cavity, both the environment and genetics are pathogenic conspirators. unfortunately, we currently know little about the specifi c mechanisms underlying any common disease; and oral diseases are among the least understood. elucidating the etiology of chronic oral diseases will involve a synthesis of results from careful experiments of environmental exposures such as diet and tobacco use, the oral microbiome, co-morbidities, largescale, well-designed genetic studies, and the various interaction effects. with regard to genetics, the past few decades have witnessed transformative developments in our ability to interrogate the entire genome for genes that contribute to disease. while dramatic advances in experimental designs, statistical approaches, and clinical insights have greatly aided this scientifi c campaign, the central driver of this progress has been the development of high-throughput, inexpensive genetic technologies. following initial molecular studies using variant forms of enzymes, or allozymes, a major breakthrough was the use of highly informative dna-based markers throughout the genome (botstein et al. 1980 ) . this idea of directly assaying existing dna variation to conduct linkage and association studies in genetics began a revolution in disease gene mapping. recent interest from commercial entities has produced a feverish pace of technological innovation, markedly reducing cost and expanding the depth of inquiry. previously unfathomable, the testing of over one million single nucleotide polymorphisms (snps) in thousands of patients and controls is now commonplace (wellcome trust case control consortium 2007 ; schaefer et al. 2010 ) ; and very recently, next generation sequencing technologies have progressed to the point where sequencing of the entire protein-coding portion of the genome (exome) or even the entire genome is a costeffective method to examine disease traits across the entire spectrum of genetic variants in small numbers of affected individuals (ng et al. 2010 ) . there is little doubt that soon whole genome sequencing will be applied to nuclear family-based designs, studies among distantly-related affected individuals in extended pedigrees, and case/control studies involving thousands of individuals. this unprecedented scope of inquiry made possible by large-scale genetics, has begun to yield fascinating resulting into predisposition to oral cancers, caries, and periodontal disease that will molecularly redefi ne these pathologies, explicate unique biological connections with related diseases, give impetus to the development of directed therapeutics, and indeed personalize medicine. still, much more genetic focus on oral disease phenotypes is required if we are to realize this medical impact in a timely fashion. as genetic technologies have allowed the progression of interrogating single protein variants to single dna markers to entire genes to markers across the genome, and now to the entire genome sequence, the promises of these large-scale genetic studies have understandably undergone monumental expansion. it may be reasonable to expect the results from whole genome sequencing to decidedly revolutionize medicine within the next two decades. however, this new scientifi c capacity comes at a cost. as genetics, and biology in general, transitions to a data-rich science, practitioners have found themselves woefully unprepared to store and analyze the volume of data generated. once analyzed, interpretation and integration of these abundant and multifaceted results into medical practice will also be an appreciable challenge. insuffi cient assimilation of genetic fi ndings into merged dental and medical records will severely limit the ability of clinicians to appropriately treat patients. inadequately addressing these informatics issues will severely derail efforts in the basic sciences efforts as well as the translational and clinical sciences. this chapter explores the current state of genomics studies, what we have learned from genetic investigations into oral diseases, and where we may be headed. genetic studies have much to offer investigations of disease etiology. why do some acquire diseases and others do not? for those affected, why do some progress more rapidly than others? what causes some patients to respond to therapies, while others suffer from adverse reactions? these are all fundamental questions in both biology and medicine, whether the focus is on the gastrointestinal tract, the hippocampus, the lymphatic system, metabolic disorders, or oral diseases. speaking generally across disease areas, a portion of the answers to these questions often lies in described environmental effects. in numerous chronic diseases, infectious agents are likely contributors to the disease process -periodontitis, for example, is initiated by gram negative anaerobes in susceptible individuals (holt and ebersole 2005 ) . surely, unique and latent environmental exposures provide a random component to common disease susceptibility and progression. through twin studies, studies of risk in close relatives, and quantitative traits experiments, it is well-understood that heritable factors, including but not limited to dna variation, are typically responsible for 30-90% of the phenotype variability for common diseases. this section will attempt to cover, at least at a cursory level, the major salient developments affecting genetic insights into chronic and aggressive periodontitis, with some comment on genetic factors infl uencing susceptibility to caries and oral cancers. while it would be extremely naã¯ve to view genetic studies as an immediate panacea for our ills, the discovery of disease-causing genes does illuminate hitherto unknown biological pathways and molecular mechanisms, draws unforeseen connections with other traits, may improve prognostic models applicable for individuals, and suggests specifi c therapeutics. industrialization has brought forth increased lifespan and wellness through vaccination, modern sanitation practices, public health policies, and advances in medical science translated into practice. however, the accompanying physical inactivity coupled with a high calorie diet are probable contributors to an extremely common, chronically infl amed metabolic syndrome (hotamisligil 2006 ) that is thought to give rise to a multitude of intimately related disease traits: insulin resistance, compromised insulin signaling, hyperglycemia, obesity, dyslipidemia, hypertension, impaired kidney function, elevated liver enzymes and steatohepatitis, poor wound healing, neurodegeneration, vascular disease, pregnancy complications, accelerated immunosenescence, and periodontal disease (ford et al. 2002 ; ferrannini et al. 1991 ; eaton et al. 1994 ; holvoet et al. 2008 ; speliotes et al. 2010 ; eckel et al. 2005 ; d'aiuto et al. 2008 ) . these diseases often co-occur within the same patient and could be considered variable expression complications arising from a state of aberrant caloric fl ux that induces metabolic dysfunction and chronic, systemic infl ammation. these features constitute a disruption in a fundamental homeostatic mechanism with intensifying pathogenic consequences. the rapidly increasing incidence and decreasing age of onset for this pathophysiological state have generated a major source of mortality and morbidity in modern cultures (ford et al. 2002 ; ferrannini et al. 1991 ; weiss et al. 2004 ) . it is becoming increasing clear that many chronic diseases have an infectious component. there is relatively convincing evidence that many systemic, t-cell mediated autoimmune disorders may be initiated by infections. for example, from archaeological data, it is believed that an infectious agent -currently unknown -is necessary for rheumatoid arthritis (firestein 2003 ) , and both guillain-barre syndrome and rheumatic fever have well-described pathogeneses triggered by specifi c infections in susceptible individuals (bach 2005 ) . in many instances, oncogenesis and tumor progression can be traced to pro-infl ammatory responses at the site of chronic infection (coussens and werb 2002 ) , although it is not known whether these effects are mediated through the actions of the immune system, the infectious agents, or a combination thereof. several cancers fall into this category including gastric adenocarcinoma (uemura et al. 2001 ) , cervical cancer (walboomers et al. 1999 ) , hepatocellular carcinoma (saito et al. 1990 ) , and kaposi's sarcoma (dictor 1997 ) , all having unequivocal infectious agent etiologies. recent fi ndings of antiinfl ammatory pharmaceuticals, particularly those that inhibit cox-1 and cox-2, reduce the incidence of certain classes of cancers are consistent with this view (dannenberg and subbaramaiah 2003 , rothwell rothwell et al. 2010 ) . in addition, there is moderate evidence that several bacteria -the most studied is chlamydia pneumoniae -play a role in atherosclerosis and myocardial infarction (saikku et al. 1988 ; watson and alp 2008 ) , however the studies are not conclusive and antibiotic treatment does not appear to be effective (andraws et al. 2005 ) . chronic periodontal disease is fi rmly footed at the intersection of infection, chronic infl ammation, and metabolic dysfunction. chronic periodontitis is characterized by infl ammation of the periodontal membrane, slowly causing gingival recession and eventual bone loss. the proximate cause of periodontitis is the virulent oral microbiome. the involvement of gram negative anaerobes has been fi rmly established for the disease. aside from the known oral pathogenic species p. gingivalis , t. denticola , and t. forsythensis , the so-called "red complex" (holt and ebersole 2005 ) , new bacterial species associated with chronic periodontitis have also been described (kumar et al. 2003 ) . the advent of an extensive database covering the oral microbiome will surely propel such investigations . numerous studies have shown that periodontal disease covaries with many diseases, presumably due to overlapping molecular etiologies. compelling meta-analyses demonstrate a highly signifi cant synchronicity of obesity and periodontal disease (chaffee and weston 2010 ) . in addition, the correlation between periodontal diseases/ alveolar bone loss and frank metabolic syndrome is repetitively observed (nesbitt et al. 2010 ; andriankaja et al. 2010 ) . extensive work has also shown a strong role for both infl ammation-related genes and circulating infl ammatory markers in periodontal disease (nikolopoulos et al. 2008 ; bretz et al. 2005a, b ) . treatment studies further support the link between periodontal disease and immuno-metabolic syndrome. these experiments have demonstrated a signifi cant improvement in intermediate molecular markers of infl ammation when chronic periodontitis in the presence of metabolic syndrome (acharya et al. 2010 ) or type 2 diabetes (iwamoto et al. 2001 ) was treated. conversely, treatment of periodontal disease with reduction of bacterial load leads to greater glycemic control among diabetic patients (simpson et al. 2010 ; stewart et al. 2001 ) . given the high prevalence of periodontitis and the co-morbidity of metabolic syndrome with periodontal disease, these treatment experiments appear to suggest that the virulent oral microbiome could play an important role in the pathogenesis of systemic infl ammatory metabolic syndrome, and is exacerbated by the syndrome. certainly, further studies are needed to defi nitively answer this question. as chronic periodontal disease seems to be a critical feature of sustained, systemic dysfunction of both metabolic and infl ammatory networks, uncovering the genetic variants carried by susceptible individuals would not only provide much needed insight into the molecular pathogenesis of chronic periodontal disease, but would also markedly aid our understanding of the infl ammatory metabolic syndrome and how it drives related co-morbidities. such genetic studies may also shed light on the specifi c mechanisms that appear to improve cardiovascular, infl ammatory, and diabetic outcomes when periodontal disease is treated, potentially leading to therapies and medical/dental intervention with greater effectiveness. such studies may also provide clues to which subsets of individuals respond more effectively than others and why they do so. periodontal disease can also present in a rapid manner with aggressive bone loss and early-onset. this is termed aggressive periodontitis (lang et al. 1999 ) . in contrast to chronic periodontitis, there is often a greater degree of familial aggregation with aggressive periodontitis, and it is hypothesized that most aggressive cases may affl ict individuals with one or more defective immune genes (zhang et al. 2003 ; amer et al. 1988 ; machulla et al. 2002 ; carvalho et al. 2010 ; toomes et al. 1999 ; hart et al. 2000 ; hewitt et al. 2004 ) . mutations in the lysosomal protease, cathepsin c, have been shown to be responsible for some forms of aggressive periodontitis, along with complications associated with other infl ammatory diseases (laine and busch-petersen 2010 ) . the specifi c hla variants thought to play a role in aggressive periodontitis, are also involved in infectious disease susceptibility and autoimmunity; and, interestingly, two of the non-mhc-linked regions, fam5c and a locus on chromosome 9p21, have been implicated in myocardial infarction (connelly et al. 2008 ) and may have action as a tumor suppressor in tongue squamous cell carcinoma (kuroiwa et al. 2009 ) . as with chronic periodontal disease, an infectious microbiome is heavily involved. however, in general, microbiome differences could not explain the presence of chronic versus aggressive forms of the disease, although in some aggressive periodontitis patients, a highly leukotoxic a. actinomycetemcomitans strain may contribute to the disease process (mombelli et al. 2002 ) . we currently do not fully know the differences between the genetic susceptibility factors for the chronic and aggressive forms of the disease. the most prevalent chronic disease in both children and adults is dental caries (national institute of dental and craniofacial research) . caries formation is a complex disease with several interacting components form the environment and host genetics. similar to gingivitis and periodontitis, caries have an infection-initiating etiology with acidifi cation leading to localized demineralization. epidemiological studies have long shown that diet is a strong predictor of caries formation; and the reduction in ph is exacerbated by high consumption of carbohydrates. the principal pathobacterial species are streptococcus mutans and lactobacillus ( van houte 1994 ) . there are also several reports of positive correlations of caries with infl ammatory diseases, although the association is not always repeatable. it is also not clear what proportion of the putative association with infl ammatory disease is due to innate upregulation of immune networks in contrast to the immuno-modulating pharmaceuticals prescribed to those with infl ammatory disease (steinbacher and glick 2001 ) . much of the effect is reported to result from lack of saliva volume (steinbacher and glick 2001 ) . interestingly, the presence of epilepsy may be associated with higher caries rates (anjomshoaa et al. 2009 ) . fluoride is an effective antimicrobial agent that interferes with bacterial growth and metabolism (wiegand et al. 2007 ) . hence, topical fl uoride administration as well as ingestion of fl uoridated water inhibits cariogenesis and caries progression (ripa 1993 ) . amelogenesis is a key process involved in modifying the rate of caries formation. both common variation and rare mutations in enamel formation genes such as amelogenin and enamelin are involved in caries rates (patir et al. 2008 ; kim et al. 2004 ; crawford et al. 2007 ) , the molecular actions of which are beginning to be revealed (lakshminarayanan et al. 2010 ) . over 35,000 new cases of cancers affecting the oral cavity and pharynx were expected in the united states for 2009, with deaths numbering 11,000 (jemal et al. 2009 ) . the majority of these malignancies involved solid tumors originating from cancerous changes in squamous cells of the mouth. again, oral cancers have a complex etiology existing of entangled genetic, epigenetic, infectious, and dietary causes, further modifi ed by tobacco, alcohol and other environmental exposures. as with most cancers, it is reasonable to expect that both germline and somatic genetic changes will be involved in carcinogenesis, tumor growth, and metastasis. promoter hypermethylation of genes central to cellular growth, differentiation, dna fi delity, apoptosis, and metabolic stability is an important facet of these cancers (poage et al. 2010 ) . indeed, methylation-mediated silencing of genes involved in tumor suppression (e.g. the cyclin-dependent kinase inhibitor 2a), detoxifi cation (e.g. mgmt ), and apoptosis (e.g. the death-associated protein kinase-1) are commonly found in oral squamous cell carcinoma samples (ha and califano 2006 ) . to quantify the proportion of the variance in a phenotypic trait that is due to variance in genetic factors, population geneticists defi ned the concept of heritability (visscher et al. 2008 ; falconer and mackay 1996 ) . researchers subsequently developed several methods for estimating heritabilities using the measure of a trait (e.g. occurrence of disease/not-disease) in combinations of relatives (e.g. parentoffspring, or monozygotic-dizygotic twins). in general, the higher the measured heritability of a variable phenotype, the larger the contribution of genetic factors is in comparison to environmental effects. it is fallacious to assume that the heritable variation is composed entirely of alleles residing in the dna sequence, for heritability studies simply examine the covariance between relatives without comment on specifi c molecular mechanisms. hence, any heritable variation such as methylation patterns, vertically-transmitted infectious agents, as well as dna variation can contribute to the heritability measure. heritability results are important because they not only give a rough estimate of the collective effects of heritable factors, but also can provide a measure to quantify how much of the total genetic effect is accounted for by specifi c loci examined. for periodontal disease, four twin-based studies of heritability have been performed (michalowicz et al. 1991 ; corey et al. 1993 ; michalowicz et al. 2000 ; mucci et al. 2005 ) . although varying in sample size and methodological details, all four arrived at consistent results, with 30-50% of the variance in periodontal disease being attributed to genetic variability for chronic periodontitis. given the segregation patterns described in the literature, it is reasonable to assume that aggressive periodontal disease exhibits a higher heritability. therefore, given the prevalence of periodontal disease, heritable factors within the population at large are likely appreciable. using 314 twin pairs, bretz and colleagues reported substantial heritability values for multiple traits related to caries ranging from 30% to 56% (bretz et al. 2005a, b ) . lastly, mutagen sensitivity studies of head and neck cancer patients suggest a signifi cant effect of genetic factors for the carcinogenesis of oral cancers (cloos et al. 1996 ) . hence, there is every reason to believe that a sizable pool of genetic and/or epigenetic factors await discovery for oral diseases. once the development of pcr (saiki et al. 1985 ) was applied to the idea of using naturally-occurring dna variation (botstein et al. 1980 ) , large-scale dna-based studies of disease underwent a substantial acceleration (schlotterer 2004 ) . genotyping of short, tandem repeated sequences (weber and may 1989 ) -microsatellites -spurred on a wave of genome-wide linkage studies, which evaluate the co-segregation of disease state with microsatellite markers, for both rare mendelian disorders as well as more common diseases with complex inheritance patterns. while the rarer traits with more coherent transmission patterns generally relinquished their genetic secrets to linkage analysis, more common diseases did not. in the mid-to late 1990s, several theoretical studies had shown that the power to detect disease-causing alleles is higher with association-based designs such as a case/control experiment or association in the presence of a linkage signal as in the transmission/disequilibrium test if the frequency of those alleles is high and the effects are moderate (kaplan et al. 1995 ; risch and merikangas 1996 ; jones 1998 ; long and langley 1999 ) . however, to conduct genomewide association studies presented an ominous obstacle for the genetic technologies at the time. the number of markers required to effectively cover the genome was prohibitively large as the chromosomal blocks in population-based samples used in association designs were expected to be small. even within large extended families, the limited number of recombination events generates substantial chromosomal blocks passed through the pedigree, but researchers had both theoretical and empirical evidence that the blocks in population-based samples were on the order of 50 k base pairs for most large human populations. as the reader can imagine, the mean length of blocks that are shared by descent is inversely related to the product of recombination rate, the number of affected individuals and the number of meioses separating the affected individuals. in practice, even very large extended families segregate regions shared by affected members on the order of several million base pairs in length. however, once geneticists seriously considered large-scale studies using a case/control design where individuals are separated by say 5,000 meioses, it became clear that to adequately cover the much smaller shared regions across the entire genome, hundreds of thousands of markers would be required (kruglyak 1999 ) . utilizing the human genome sequence (venter et al. 2001 ; lander et al. 2001 ) , a number of studies at celera diagnostics provided an intermediate solution, where approximately 30,000 putative functional snps primarily located in genes were assayed through allelespecifi c pcr in a number of common diseases using a staged case/control design. these studies were successful in identifying several gene-centric polymorphisms associated with common diseases (begovich et al. 2004 ; cargill et al. 2007 ) (fig. 4.35 ) . concurrently, several groups had performed extensive sequencing and genotyping across the genome to produce a genome-wide map of haplotype structure (hinds et al. 2005 ) , useful in linkage disequilibrium mapping. within 2 years, technology for snp hybridization arrays had advanced so as to enable genome-wide association studies capable of capturing most of the common genetic variation in the genome either through direct genotyping or indirect interrogation using linkage disequilibrium -the term linkage disequilibrium is a measure of the correlation of alleles at closely-linked sites (see fig. 4 .36 ). these investigations were met with numerous successes (klein et al. 2005 ; kathiresan et al. 2008 ; graham et al. 2008 ; gudmundsson et al. 2009 ) . inexpensive genotyping platforms and urging from theoreticians ensured that these genome-wide association studies were, in general, highly powered to detect all but very mild effects from high frequency alleles. these efforts, led by large academic consortia such as the wellcome trust, the international multiple sclerosis genetics consortium, and the broad institute and commercial entities such as decode genetics and perlegen have greatly expanded our understanding of the basic biology of common diseases: we now know, for example, that (i) autophagy-related genes are involved in crohn's disease (rioux et al. 2007 ) , (ii) there are a number of genes such as the protein tyrosine phosphatase, ptpn22 and the interleukin-23 receptor, il23r , that exhibit ample pleiotropic effects among autoimmune conditions (lopez-escamez 2010 ; safrany and melegh 2009 ) , (iii) in the case of age-related macular degeneration, predictive models using the genetic results enable fairly accurate prognosis of individuals who are at high risk of disease (seddon et al. 2008 transcription factor tcf7l2 plays a role in type 2 diabetes (grant et al. 2006 ) , and (v) aberrant il-7 signaling likely contributes to multiple sclerosis susceptibility (gregory et al. 2007 ) . the plot shows the tremendous progress in genotyping technology where, a decade ago, very little of the genome was accessible for disease studies using association designs through the current wave of viable sequencing-based whole exome studies (2010) (2011) and whole genome studies (2012) (2013) . in fig. 4 .37 , the average distance between adjacent genetic markers is plotted as a function of year of introduction to the disease mapping community. impressively, the total number of genetic markers has increased a million-fold over the past decade. although successful in uncovering numerous pathogenic pathways for common diseases, results from the current wave of genome-wide association studies, with a few exceptions, explain little of existing disease heritability. the reasons for this are cryptic and the subject of heavy debate (manolio et al. 2009 ) . multiple rare sequence variants generating high levels of allelic heterogeneity, functional de novo mutations, structural mutations such as copy number variants and large deletions, and epigenetic effects constitute four of several possible disease models that could account for the heritability discrepancy. the answer will almost certainly consist of a conglomeration of these and other effects. bringing forth the new genome-wide technologies that illuminate these previously non-or under-interrogated properties of the genome to bear on this enigma is a reasonable next step for all complex traits including oral diseases. the most commonly used measure of ld in a sample of chromosomes is linkage disequilibrium (ld) is a measure of the correlation between alleles at two sites in a sample of chromosomes. for two biallelic sites, if the a1 allele is always paired with the b1 allele, and the a2 allele is always on the same haplotype as the b2 allele, then the two sites are said to be in perfect ld. successive recombination diminishes ld. interrogating one site for disease association allows investigators to indirectly interrogate other sites in sufficiently high ld with the interrogated site. a key feature explicitly studied in molecular population genetics and implicitly used in disease gene mapping studies is the site frequency spectrum; that is, the distribution of allele frequencies at single sites in the genome that vary in the human population studied. from both diffusion models (kimura 1970 ) and coalescent theory (hudson 1991 ) in theoretical population genetics, we know that the vast majority of realistic models generate many more rare variants compared to common polymorphisms. this is particularly true for expanding populations. are these rare variants the source of much of the missing heritability? recently, with the application of high-throughput sequencing technology to human studies over the past decade, empirical studies have clearly verifi ed these predictions -the large majority of variants have low frequencies (the international hapmap 3 consortium 2010 ) . the distribution of deletions appears to be skewed toward more rare frequencies, presumably due to the deleterious effects of such variants. individual mutations appearing de novo typically are extremely rare events per locus, but collectively are numerous. other types of genetic variability, such as copy number repeats, span both ends of the frequency spectrum with the preponderance of the markers being rare. thus, there is a sizable pool of low-frequency variants in human populations that have yet to be thoroughly investigated. over the past few years it has become increasingly clear that structural variants exist in the human genome at a far higher rate than previously thought. structural variants can exist in a multitude of forms including deletions, copy number variants, and inversions among others. due to the nature of these genetic changes, many are considered to be highly disruptive of molecular function if they lie in functional motifs. indeed, there are several mendelian diseases are caused by fully-penetrant structural variants impacting a chromosomal region (lupski 2007 ) . numerous structural variants have recently been reported to be associated with common diseases, particularly in the neurological fi eld (sebat et al. 2007 ; stefansson et al. 2008 ; elia et al. 2010 ) , infectious disease susceptibility (gonzalez et al. 2005 ) , and drug metabolism (zackrisson et al. 2010 ) . although they have improved dramatically over the past few years, algorithms using snp-based data from hybridization arrays to infer copy number variants have had high error rates, perhaps explaining the rather low rates of replication of structural variation association results for common diseases. nevertheless, given the high frequency of structural variants, their pathogenic potential, and that we are on the precipice of a sequencing revolution in genome-wide studies, examination of these variants should be a high priority for new sequencing-based studies in oral disease susceptibility, progression, and related pharmacogenetic applications. as different technologies examine different portions of the site frequency spectrum (i.e. genome-wide snp scans interrogate variation that is common in the hapmap populations, whereas sequencing-based studies typically interrogate the entire frequency spectrum), where one believes genetic causation is harbored should infl uence the selection of genotyping technology. if common genetic variation contains the vast majority of heritable effects on disease phenotypes, then an investigator would be wise to employ a snp-based experimental design. if, however, there is reason to believe that a signifi cant portion of the genetic load of the disease studied exists in the highly populated portion of the distribution -the rare variants -then a sequencing-based study may be better suited to unravel causative alleles. the studies of heritability discussed previously show that there is heritable variation underlying a substantial portion of the variance observed in oral diseases. as discussed above, sequencing technologies may address many aspects of dna variation including copy number loci, rare haplotypes, inversions, and insertions/deletions, but it is also worthwhile to repeat that the molecular mechanisms for disease heritability are not necessarily limited to variation at the dna level. for a disease state, the covariance between relatives could be driven by co-inherited chromosomal regions or other phenomena. chief alternative heritable mechanisms include dna methylation (hammoud et al. 2009 ) , modifi cations to the histones (bestor 2000 ) , complex rna zygotic transfer (rassoulzadegan et al. 2006 ) , and vertical transmission of infectious agents. additionally, transgenerational effects offer an intriguing class of epigenetic mechanisms (nadeau 2009 ) . in a thorough review on epigenetics and periodontitis, gomez et al. make a strong argument for consideration of both cpg dinucleotide methylation and deacetylation actions on cytokine expression as a credible avenue for further investigation in periodontal disease etiology (gomez et al. 2009 ) . genome-wide epigenetic studies have been successfully conducted for oral cancers (poage et al. 2010 ) . the scale of this study on head and neck squamous cell carcinomas allowed these researchers to show a global pattern of tumor copy number changes signifi cantly correlated with methylation profi les that was not detectable at the individual gene promoter level. with advanced chromatin immunoprecipitation and new methods to study dna methylation, efforts to apply highthroughput epigenetic methods to oral diseases should be accelerated. numerous studies have been conducted in oral disease traits using a candidate gene approach. there are two large reviews of the existing candidate gene results (nikolopoulos et al. 2008 ; ) . laine and colleagues have recently put together a comprehensive review article covering gene polymorphisms. there are some suggestive fi ndings for cyclooxygenase-2 gene, cox-2 , the cytokineencoding genes, il6 and il1b , the vitamin d receptor, vdr , a polymorphism immediately upstream of cd14 , and the matrix metalloproteinase-1 gene, mmp1 . however, these initial results will require further confi rmation, for the association patterns are inconsistent across independent studies, the statistical signifi cance is moderate, and the posterior probability of disease is decidedly bland. the striking pattern that emerges from the laine et al. summary data is the lack of coherent replication of genetic association for the vast majority of polymorphisms examined. the situation is reminiscent of genetic association studies prior to large-scale snp studies where poor repeatability of results plagued the fi eld. in a pivotal study from 2002, hirschhorn and colleagues (hirschhorn et al. 2002 ) examined the state of genetic association studies, fi nding that "of the 166 putative associations that had been studied three or more times, only six have been consistently replicated." the dearth of robust results was largely remedied when large-scale genetic studies were applied to very substantial numbers of well-characterized patients and geneticallymatched controls and stringent statistical criteria enforced. one can only suspect that a similar state of affairs is operating in genetic studies of chronic periodontitis. perhaps efforts to (1) reduce the heterogeneity of the disease state through detailed clinical and laboratory assessments, (2) drastically increase sample sizes, and (3) expand the scope of inquiry to larger numbers of genes/regions, and examine a more comprehensive set of variants/epigenetic effects will improve the current situation. the second large study is a meta-analysis of 53 studies, where nikolopoulos and colleagues analyzed six cytokine polymorphisms linked to il1a , il1b , il6 , and tnf-alpha (nikolopoulos et al. 2008 ) . two of these, an upstream snp in il1a and a snp in il1b , exhibited signifi cant association with chronic periodontal disease risk. although the results were not particularly strong, as is typical with complex diseases, the results do suggest the importance of infl ammation-response variability in chronic periodontitis predisposition. perhaps the strongest, most replicable genetic association fi nding with coronary heart disease and myocardial infarction is centered on the short arm of chromosome 9 (9p21.3) (mcpherson et al. 2007 ; helgadottir et al. 2007 ) . two studies of periodontal disease showed that the same alleles at the 9p21.3 locus confer risk for aggressive periodontitis (schaefer et al. 2009 ; ernst et al. 2010 ) . the discovery of such a pleiotropic locus may explain a portion of the aggregation of periodontal disease with other co-morbid conditions. further studies investigating overlapping genetic susceptibility factors between periodontitis and cardiovascular disease, diabetes mellitus, metabolic syndrome, rheumatoid arthritis, and other related diseases may be a fruitful strategy for honing in on shared genes affecting these immuno-metabolic disorders. using patients from 46 families from the philippines, the fi rst genome-wide linkage study for caries was completed in 2008 (vieira et al. 2008 ) . the study identifi ed fi ve loci which exhibit suggestive statistical evidence (lod scores exceeding 2.0): 5q13.3, 14q11.2, xq27.1, 13q31.1, and 14q24.3. the latter of which overlapped with a quantitative trait locus discovered from mapping work in the mouse. further work is necessary to refi ne these signals and localize the variants that may be driving these linkage signals. aggressive periodontal disease and rarer dental diseases have also been subjected to linkage analysis. results from linkage studies for dentinogenesis imperfecta type i, for example, have gone on to produce the novel gene fi ndings of the dentin sialophosphoprotein-encoding gene on 4q21.3 being responsible (song et al. 2008 ; crosby et al. 1995 ) . a linkage study in african american families examining localized aggressive periodontitis found a strong linkage signal in a region covering approximately 26 megabases on chromosome 1 (li et al. 2004 ) . several interesting genes are in this region. in a study earlier this year further mapping from carvalho et al. in brazilian families identifi ed haplotypes in this region on 1q in fam5c which were associated with aggressive periodontitis (carvalho et al. 2010 ) . the function of the fam5c protein is not fully understood. fam5c is localized in the mitochondria and it appears to play a role in vascular plaque dynamics and risk of myocardial infarction (laass et al. 1997 ) . it should also be noted here that other types of mapping analyses such as homozygosity mapping to identify have yielded gene discoveries. for example, the lysosomal protease cathepsin c gene for the recessively-inherited papillon-lefevre syndrome which is characterized by aggressive and progressive periodontitis was effectively mapped using homozygosity mapping (fischer et al. 1997 ; connelly et al. 2008 ) . cathepsin c is highly expressed in leukocytes and macrophages and is a key coordinating molecule in natural killer cells (rao et al. 1997 ; meade et al. 2006 ) . although sparse, these linkage results are undoubtedly encouraging. employing very large extended families subjected to genome-wide genotyping or sequencing will surely shed much needed light on chromosomal regions and genes relevant to oral disease research (fig. 4.38 ). for periodontitis, a single study has employed a genome-wide association design in an effort to uncover aggressive periodontal variants (schaefer et al. 2010 ) . this study by schaefer and colleagues discovered and replicated an intronic snp, rs1537415, in the glycosyltransferase glt6d1 which is signifi cantly correlated with aggressive periodontal disease in both german and dutch samples. often, seemingly signifi cant results from large studies are due to the effect of reporting the top result from a great many statistical tests -this is called the multiple testing problem. in this situation, the strength of the fi nding, along with the replication across three case/control studies, argues for true association with aggressive periodontal susceptibility. the snp may modulate the binding affi nity of gata-3. the association with glt6d1 is currently one of strongest genetic associations for aggressive periodontal disease, testifying to the power of genome-wide studies to generate novel, relevant molecular pathophysiology for complex diseases. it seems unlikely that glt6d1 would be extremely high on a candidate gene list, and it was only through a genome-wide scan that it appeared. like many excellent studies, the fi nding by schaefer et al. raises more questions than it answers and will undoubtedly provide fertile ground for ensuing molecular work. after a somewhat sluggish start, due to a lack of critical mass of investigators aiming to collect large numbers of patient samples and bring high throughput genetic technologies to caries susceptibility, gingivitis, and periodontal disease traits, the future of genetic studies in oral health is bright. scientifi c progress in revealing the molecular pathogenesis of oral diseases is dependent on genome-wide genetic studies; and i have argued that progress in related immuno-metabolic diseases is also dependent on these large-scale genetic studies in periodontal disease. to study sporadic disease, substantial patient collection efforts are required for the application of these technologies. this may involve a combination of new recruitment and consortiumrelationships with existing collections. the beginning of such a collection for sporadic aggressive periodontitis in europe has shown extremely intriguing initial results, but more patients are needed to examine rare variants of moderate effect. both the german/dutch collection of aggressive periodontitis and the brazilian collection have begun to revolutionize the study of periodontal disease susceptibility with the discovery of glt6d1 snps and fam5c -linked haplotypes. there is little doubt that subsequent molecular work on these two genes will uncover novel mechanisms for the predisposition to aggressive periodontal disease. focus should also be placed on the collection of extended families segregating these diseases. applying sequencing technologies to large pedigrees can be an effective method of identifying rare variants and structural variants in a highly-refi ned phenotype. furthermore, applying these methods to the entire genome would make for a comprehensive genetic study. several trends in large-scale genomics science hold promise to signifi cantly advance our understanding of oral disease pathogenesis: the sociology of biological sciences has changed over the past 15 years so as to â�¢ become more collaborative. essential for association-based designs, consortiumbased genetic research has blossomed over that time period, increasing sample sizes and therefore the power to detect disease-causing variants. there currently is consortium-based research in periodontal disease and oral cancer. further expanding these efforts will enhance subsequent studies, particularly those investigating rare alleles and/or rare epigenetic effects. through over a century of laboratory work, the collective knowledge of bio-â�¢ chemical pathways, signal transduction, cell physiology, regulatory mechanisms, and structural biochemistry is weighty. incorporation of this information into etiological models may substantially advance oral disease work as well as the fi eld of complex disease genetics in general. sophisticated analysis techniques are needed to perform this task. recent advances merging results from network science with probability theory within the context of computer science have produced the fi eld of machine learning. this rigorous framework can be used to identify those factors responsible for disease status and can also be used to develop robust predictive models using known biological networks and genetic data. the output from such models, typically the probability of disease, an estimate of disease progression rate, or a probability of adverse reaction, can be used by physicians and dentists to personalize medical care. until relatively recently, population genetics did not contribute a great deal to â�¢ human genetics research. that has changed in the past decade where effort spent on association studies surpassed that spent on family-based studies. those investigating disease gene mapping began to collaborate with population geneticists and population geneticists took up a wide-spread interest in fi nding disease alleles. incorporation of population genetics theory into such studies markedly improved association studies on several levels: confounding by population stratifi cation was effectively treated using population genetics, linkage disequilibrium patterns. use of population genetics theory in large-scale oral disease mapping studies may accelerate discoveries. sequencing technology has rapidly progressed over the past decade. currently, â�¢ sequencing studies across the exome can be accomplished at reasonable cost and yield data for all known genes in the genome. within the next few years, sequencing costs will depreciate to a point where whole-genome sequencing studies will be commonplace, using both family-based and population designs. application of these technologies to oral disease studies is imperative for comprehensive studies of etiology. high-throughput dna methylation and chromatin immunoprecipitation studies â�¢ will enable large-scale epigenetic studies in oral diseases (meade et al. 2006 ; ehrich et al. 2005 ; bibikova et al. 2006 ; ren et al. 2000 ; pokholok et al. 2005 ) . these have already started to play an important role in delineating mechanisms responsible for oral cancers (poage et al. 2010 ) . additional application of these techniques to studies of gingivitis, caries, and periodontal diseases may generate novel fi ndings. molecular biologists and pharmacologists have increasingly become able to â�¢ develop and evaluate highly targeted pharmaceuticals based on genetic discoveries. the use of such genetic information may improve the chances of developing effi cacious therapies. geneticists and disease researchers are beginning to realize that oral diseases â�¢ both impact and are intrinsically tied to susceptibility and progression of other common diseases. a synthesis of genetic fi ndings from immuno-metaboliclinked disorders would seem to greatly increase the knowledge of these diseases and better pinpoint their respective etiologies. as the new high-throughput genomics and epigenomics technologies become â�¢ implemented in oral disease research, the storage, management, analysis, and interpretation of the ensuing colossal amounts of data will be critical to enable clinicians to use these results in daily practice. advances in dental and medical informatics will facilitate these steps. we are in exciting times where advances in genetic technologies will uncover the genetic causes of diseases, including those that affect the oral cavity. with more focus in the area of oral disease genomics and the harnessing of new high-throughput sequencing and epigenetic technologies, novel insights into the pathways driving these diseases are imminent. these discoveries will, in turn, motivate directed therapies, aid in illuminating the molecular etiology of related disorders such as diabetes, and increase the level of personalized medicine. joseph kilsdonk the title of this section reinforces a 1995 institute of medicine (iom) report titled "dental education: at the crossroads." to quote yogi berra, a baseball sage: "when you come to a fork in the road, take it." the implication being that dental education must take action and move beyond its crossroads. these crossroads are described in the fi rst third of the section. it includes a summary and recommendations of the iom report and three transitional reports that followed: the 2000 surgeon general's report identifying oral health as a silent epidemic, the josiah macy foundation report, and a "pipeline" study funded by both the robert wood johnson and the california foundations. having been at the crossroads for a decade or so, the middle portion of the section highlights educational models that may lead to a more promising future. the later third of this section describes an alternative path of action for dental education which emphasizes the central roles of clinic-based education and dental informatics in dental education curriculum. it is unknown how traditional dental educators may view this model; however, it is effectively a logical conclusion and responsive to the reports. in 1995 the institute of medicine (iom) published "dental education at the crossroads" (field 1995 ) . the title was apropos as the authors' analysis concluded: (1) economics surrounding dental education were unsustainable ; (2) student service learning opportunities and access to care for patients were limited; and (3) new dental schools were not replacing those forced to close due to the economic climate. the iom report additionally proposed key recommendations to reform dental education and service delivery. fifteen years later, we remain at "the crossroads" as these issues remain largely unresolved. furthermore, these recommendations have retained their validity. their implementation would directly impact structures and services for contemporary models of dental education in the future. the following iom recommendations (field 1995 ) are intrinsic to the proposed dental education reform: recommendation 2 : to increase access to care and improve the oral health status of underserved populationsâ�¦ recommendation 3 : to improve the availability of dental care in underserved areas and to limit the negative effects of high student debtâ�¦ recommendation 5 : to prepare future practitioners for more medically based modes of oral health care and more medically complicated patients, dental educators should work with their colleagues in medical schools and academic health centers to: move toward integrated basic science education for dental and medical â�¢ students; require and provide for dental students at least one rotation, clerkship or â�¢ equivalent experience in relevant areas of medicine and offer opportunities for additional elective experience in hospitals, nursing homes, ambulatory care clinics and other settings; continue and expand experiments with combined md-dds programs and â�¢ similar programs for interested students and residents; increase the experience of dental faculty in clinical medicine so that they, â�¢ and not just physicians, can impart medical knowledge to dental students and serve as role models for them. recommendation 6 : to prepare students and faculty for an environment that will demand increasing effi ciency, accountability, and evidence of effectiveness, the committee recommends that dental students and faculty participate in effi ciently managed clinics and faculty practices in which the following occurs: patient-centered, comprehensive care is the norm; â�¢ patients' preferences and their social, economic, and emotional circumstances â�¢ are sensitively considered; teamwork and cost-effective use of well-trained allied dental personnel are â�¢ stressed; evaluations of practice patterns and of the outcomes of care guide actions to â�¢ improve both the quality and the effi ciency of such care; general dentists serve as role models in the appropriate treatment and referral â�¢ of patients needing advanced therapies; larger numbers of patients, including those with more diverse characteristics â�¢ and clinical problems, are served. recommendation 17 : because no single fi nancing strategy exists, the committee recommends that dental schools individually and, when appropriate collectively evaluate and implement a mix of actions to reduce costs and increase revenues. potential strategies, each of which needs to be guided by solid fi nancial information and projections as well as educational and other considerations, include the following: increasing the productivity, quality, effi ciency, and profi tability of faculty â�¢ practice plans, student clinics, and other patient care activities; pursuing fi nancial support at the federal, state, and local levels for patient-â�¢ centered predoctoral and postdoctoral dental education, including adequate reimbursement of services for medicaid and indigent populations and contractual or other arrangements for states without dental schools to support the education of some of their students in states with dental schools; rethinking basic models of dental education and experimenting with less â�¢ costly alternatives; raising tuition for in or out-of-state students if current tuition and fees are low â�¢ compared to similar schools; developing high-quality, competitive research and continuing education â�¢ programs; consolidating or merging courses, departments, programs, and even entire â�¢ schools. in summary, the iom report identifi ed that: (1) an outdated curriculum continues to be retained which refl ects past dental practice rather than current and emerging practice and knowledge; (2) clinical education does not suffi ciently incorporate the goal of comprehensive care, with instruction focusing too heavily on procedures; (3) medical care and dentistry are not integrated; and (4) the curriculum is crowded with redundant material, often taught in disciplinary silos. the 1995 iom's report was followed by the surgeon general's report on oral health in 2000 and a subsequent supplement by the surgeon general in 2003 called "the national call to action" (u.s. department of health and human services 2003 ) . five signifi cant fi ndings and recommendations from the surgeon general's report(s) that have implications pertaining to the envisioned structure and services of new models for dental education include: changing the perception of oral health so that it will no longer be considered â�¢ separate from general health; improving oral health care delivery by reducing disparities associated with popu-â�¢ lations whose access to dental treatment is compromised by poverty, limited education or language skills, geographic isolation, age, gender, disability, or an existing medical condition; encouraging oral health research, expanding preventive and early detection pro-â�¢ grams, and facilitating the transfer of knowledge about them to the general population; increasing oral health workforce diversity, capacity, and fl exibility to overcome â�¢ the underrepresentation of specifi c racial and ethnic groups in the dental profession. in this regard, the national call to action urged the development of dental school recruitment programs to correct these disparities and to encourage parttime dental service in community clinics in areas of oral health shortage; increasing collaboration between the private sector and the public sector to cre-â�¢ ate the kind of cross-disciplinary, culturally sensitive, community-based, and community-wide efforts to expand initiatives for oral health promotion and dental disease prevention. spurred by the 1995 iom report and the 2000 surgeon general's report, the josiah macy foundation ( 2004 ) conducted a study entitled "new models of dental education." the study was prompted by concerns about declines in dental school budgets and the diffi culties experienced by schools in meeting their educational, research, and service missions. the macy study concluded that: financial problems of dental schools are real and certain to increase. â�¢ current responses of schools to these economic challenges are not adequate. â�¢ most promising solutions require new models of clinical dental education. â�¢ macy study lead researcher dr howard bailit, and his team recently concluded in reference to points one and two above, that: "if current trends (to aforementioned) continue for the next 10 years, there is little doubt that the term crisis will describe the situation faced by dental schools. further, assuming that it will take at least ten or even more years to address and resolve these fi nancial problems, now is the time for dental educators, practitioners, and other interested parties from the private and public sectors to come to a consensus on how to deal with the coming crisis. clearly, these fi nancial problems will not be solved by minor adjustments to the curriculum, modest improvements in the clinical productivity of students or faculty, or even signifi cant increases in contributions from alumni. the solutions 'must involve basic structural changes in the way dental education is fi nanced and organized' (bailit et al. 2008 ) ." this statement is supported by the fact that in the past 25 years more dental schools have closed than opened. specifi cally eight schools have closed, whereas to date a couple has opened and a handful is pending. curriculum relevance was also a focus of the study. findings concluded that "changing the curriculums in dental schools to allow students to spend more time in community venues would be highly benefi cial to both society and student. society benefi ted from having underserved patients cared for while students were assessed as being fi ve to ten times more productive, more profi cient, more confi dent, more technically skilled and more competent in treating and interacting with minority patients" (brodeur 2008 ) . macy study (formicola et al. 2005 ) outcomes represented signifi cant and foundational guideposts for assessing and planning any future models for dental education. their report led to the robert wood johnson foundation pipeline study ( 2007 ) , a major research study funded by the robert wood johnson foundation and the california endowment (tce). the goal of the dental pipeline program was to reduce disparities in access to dental care. the pipeline study provided over $30 million for the start up or expansion of 15 schools and student clinical programs that incorporated services to underserved extramural clinical settings (primarily community health centers). the following recommendations from the surgeon general's report structured â�¢ the goals of the pipeline's initiative: increase the number of under-represented minority and low-income students enrolled in the dental schools participating in the pipeline program so that there would be a voice of minority and low-income students at all the funded schools. provide dental students with courses and clinical experience that would prepare â�¢ them for treating disadvantaged patients in community sites. have senior dental students spend an average of 60 days in community clinics â�¢ and practices treating underserved patients. increasing the community experience of dental students was expected to have an immediate impact on increasing care to underserved patients (brodeur 2008 ) . this third point is pivotal to future success of dental curricula and dental education economics. recently published in a supplemental volume to the journal of dental education , february, 2009, the pipeline study reported the following outcomes: minority recruitment of low-income students increased by 27%; â�¢ the rate of recruitment for under-represented populations was almost twice that â�¢ of non-pipeline schools; the length of time dental students spent in extramural rotations increased from a â�¢ mean of 16 days to a mean of 39 days over a period of 4 years. procedural profi ciency increased compared to that of their non-extramural peers. of the 15 pipeline-funded programs, only four schools achieved the goal of â�¢ 60 days of extramural rotations; through extra funding from tce, the four schools extended extramural rotations to an average of 198 days; based on this publication, it appears that only a handful of pipeline schools defi -â�¢ nitely plan to sustain their extended extramural rotations. financial concerns were highlighted as the major problem in sustaining future recruitment and placement of students beyond the timeframe of the study; a survey of program seniors indicated a mean of 26% [range of 14-60% by â�¢ school] were planning to devote â³ 25% of their practice to serving minority patients. only 9% [range of 3-22% by school] were planning to practice at community clinics. in the context of these outcomes, discussion indicated that the unwillingness of students to practice in underserved settings was based on several factors: students that participated were already enrolled in traditional programs and were â�¢ not necessarily seeking a pipeline experience or a future in community service. concern over future reimbursement as a provider in a community setting; â�¢ limited time spent in underserved settings; â�¢ limited loan forgiveness scholarship opportunities. â�¢ the fact that the large majority of pipelines were unsustainable was attributed to lack of productivity in the school clinics while the students were on rotation at community based clinics. schools generate meager, yet necessary revenue streams on intramural student clinical activity to support the costly clinical and faculty infrastructure. currently, similar economic constraints involved with outsourcing students to serving rural and underserved populations impacts the ability of tradition dental schools to participate in sustained outreach programs. most recently, the pew center on the states national academy for health policy ( 2009 ) released "help wanted: a policy makers guide to new dental providers". this report provided an excellent summary outlining workforce needs, access issues, and strategies for dental-related services to help states and institutions develop creative ways to solve oral health access and care issues. the guide proposes the following relevant components and trends for consideration in development of future sustainable school models: dental colleges are willing to bear a large and disproportionate share of the burden â�¢ in terms of access to care, particularly during a time of incredibly scarce resources. expanded, extensive, and/or creative extramural rotations have been developed â�¢ in recent years under the conceptual umbrella of service-learning. these often involve clinics providing direct or indirect payment to dental schools or clinics managed in some way by dental schools. dental education has certain obligations. first, education must adhere to accreditation standards with the goal of producing competent practitioners. second, education must remain responsive and impact the societal need for care. lastly, the delivery of dental education must be economically sustainable. the macy, rwjf, and iom reports note that improved oral health, sustainable dental education economic models, and competent workforce pipelines converge around community health centers (chc). university of michigan researchers fitzgerald and piskorowski ( 2009 ) reaffi rm this conclusion in an evaluation of an ongoing 7-year program, stating that: (the chc model) is self-sustaining and can be used to increase service to the underserved and increase the value of students' clinical educational experiences without requiring grant or school funding, thus improving the value of dental education without increased cost. self-sustaining contracts with seven federally qualifi ed health centers (fqhcs) have resulted in win-win-win-win outcomes: win for the underserved communities, which experienced increased access to care; win for the fqhcs, which experienced increased and more consistent productivity; win for the students, who increased their clinical skills and broadened their experience base; and win for the school in the form of predictable and continuing full coverage of all program costs (fitzgerald and piskorowski 2009 ) (fig. 4.39 ). however, unlike medicine that outsources their students to clinical sites, dental education programs retain the majority of the student time within their own "clinical laboratories" as documented by the aforementioned studies, this limits students' exposure to extramural experiences. costs to operate such intramural clinical programs are ever increasing and many schools' clinical operations run defi cits. if that component can be outsourced to community-based resources such as a chc, then the burden of cost is shifted away from the school. an example would be a.t. still university's arizona school of dentistry and oral health (asdoh) which matriculated its fi rst class in 2003. at the prompting of the state's community based clinics, asdoh designed a program that placed students into community-based settings for up to 6 months, an unprecedented length of time for an extramural rotation. they also saw this as an opportunity to use an adjunct centric faculty that signifi cantly reduced traditional education overhead. through this innovation, the school was able to develop a program that was sustained by "fair market" value tuition and trained students where community needs were greatest for up to 6 months (which was then unprecedented). conversely, if the chc can rely on student service-learning to care for patients, the cost of care is reduced. other schools are also advancing with innovative education and care delivery. adea's charting progress (valachovic 2009 ) fig. 4.39 the synergy between access to care, student competency, and fi nancially sustainable dental education converge around chc/fqhcs little rock, arkansas; and the university of southern nevada in south jordan, utah. western university is planning placement of 50% of their fourth year class in community health centers, while east carolina is seeking to set up rural clinical campuses as well as clinical partnerships with the state's fqhc. at the time of this publication, several existing schools are expanding or looking to expand including the university of north carolina, marquette university, midwestern university in downer's grove, il. such expansions will contribute to solving the existent access supply and demand issues. however, it was observed even with all the start ups and expansions, graduation numbers will not approach the output of schools in the late 1970s and early 1980s. these creative models establish the foundation for a sustainable clinic structure by generating self-sustaining revenue through student service-learning, which, unlike medical student services, are billable. simultaneously these new models provide access to care for the needy while student exposure to clinical experiences that are often not available in academic patient pools. these models also shift some of the cost of providing clinical education from the dental college to community-based clinics. however, this innovation is not without criticism. schools are dependent on the success of their clinics and clinic partnerships. one author cautions: "however, these creative models also may present potential political strategic risk or confl ict: private practitioners may organize and protest higher than normal reimbursement schemes. potentially, such protests could even jeopardize the very existence of such models (dunning et al. 2009 ) ." notably, community health centers have historically received strong bipartisan support. for example, during the bush administration, fqhc funding was doubled and most recently expanded through health reform legislation by the obama administration. according to the institute for oral health, "the group practice of the future is the dentist working with the physician" (ryan 2007 ) . the ada reported "multidisciplinary education must become the norm and represent the meaning and purposes of primary care as it applies to dentistry. educational sequences should include rotation strategies across discipline specialties in medicine and dentistry, clerkships and hospital rotations, and experience in faculty and residency clinics." (barnett and brown 2000 ) the models alluded to, were school-based attempts at improving educational outcomes. perhaps the proverbial fork in the road regarding the future of dental education leaves two paths for consideration. is it better to travel down a road that leads a school to develop and operate a clinic? or is the road less traveled, where a clinic becomes a school, the better of the two options? the answer, perhaps, is that a combination of both will accomplish the desired outcome. for example, didactic knowledge is measured by examination whereas competency as a practitioner is measured by clinical demonstration. at a minimum, the result must achieve learner competency, quality, and sustainability. however, the road less traveled has not been taken yet. william gies, in his revered report written 85 years ago on the state of american dental education, wrote "dental faculties should show the needâ�¦. for integrated instruction in the general principles of clinical dentistry and in its correlations with clinical medicine" (gies 1926 ) . basic sciences aside, could a clinicalbased educational training center have an advantage over a school-based clinical center? soon-to-be-implemented new commission on dental accreditation (coda) standards will require schools to demonstrate competency in patient-centered care (valachovic 2010 ) . might an enterprise profi cient at running a successful clinical business model have an advantage running a professional, patient-centered clinical training program as compared to a pedagogical business model attempting to run a clinical training model? these questions should challenge us to reexamine why our thinking about educational models should be limited to schools being the starting point for the development of a profession that demands clinical competency, patientcenteredness, and integration as outcomes. the clinic based model may serve as an equivalent starting point and, have some distinct advantages for achieving responsiveness to recommendations and directions cited in this section. beginning in november 2002 through august 2010, the family health center (fhc) of marshfi eld, inc, marshfi eld, wisconsin, launched of a broad network of developing dental clinics, targeting dental professional shortage areas with the provision of dental services to the underserved communities whose dental needs were not being adequately met by the existing infrastructure. fhc-marshfi eld is a federally qualifi ed health center (fqhc). as an fqhc, fhc receives cost-based reimbursement for its dental services to medicaid populations. along with the cost-based reimbursement, fqhcs are obligated to provide care to anyone regardless of their ability to pay. presently, fhc is the nation's largest federally qualifi ed dental health center. to date, this network of dental clinics has served over 58,000 unique patients, 85% of whom were under 200% of poverty. notably, service was provided to a signifi cant number of cognitively and developmentally disabled patients in special stations developed for serving patients with special needs. these patients frequently travel the furthest to get to our dental centers for care. beginning in 2008, fhc stepped up the pace of dental clinic expansion, constructing two new dental centers in 2009, two in 2010, and two more are slated to open in 2011. when fully operational, this will establish capacity to serve 66,000 patients annually. each site has proactively included dedicated clinical and classroom training space for dental residents or students, thus laying the framework for clinic-based training of new dental professionals. the plan is to continue to stand up new dental centers until they have the capacity to serve 158,000 patients annually or approximately 50% of the 300,000 underserved patients in the rural service area. in addition to the capacity for training residents and students, a dental post-baccalaureate program is being considered in partnership with regional 4 year under graduate campuses. the post-baccalaureate program is aimed at preparing students from rural and underserved areas who desire to practice in rural and underserved areas for acceptance and success in dental schools. presently fhc in partnership with marshfi eld clinic is moving forward with plans to develop dental residencies at these sites and a dental post baccalaureate training program to better prepare pre-doctoral students from rural and/or underserved backgrounds to be successful in dental school as a means to create a dental academic infrastructure responsive to rural environments which have been classically underserved. marshfi eld clinic has a long-standing history in medical student education and multiple medical residency programs. creating access for the underserved population was the major motivational force driving the establishment of the dental clinic network back in 2002. the fi ndings of the iom, macy, and rwjf reports became the foundational framework for developing the vision of a dental education model that would realize the major recommendations found in the reports. by establishing clinical campuses in regional underserved dental health professional shortage areas, access to care where care is needed most was provided. sustainment of a work force for provision of care across the dental clinic network is accomplished by schools contracting with fqhc's for service learning, thus circumventing challenges associated with releasing dental students at traditional dental schools to distant extramural training sites as discussed previously. this model is however not without its own set of challenges including calibration of faculty, supervision and evaluation of students in training, and achieving accreditation acceptance. however, through video connectivity and iehr technology curriculum, learning plans, competency assessment, progression, performance, faculty development, and learner evaluations can be centrally calibrated. additionally, this dental service-learning model based in a community health center setting offers students unique state-of-the-art exposures to alternative access models, cutting-edge informatics (including access to a combined dental-medical record) and a quality-based outcomes-driven practice. given the novelty of such an extended extramural dental clinical training model, there is limited data on the success of rural placement leading to retention to practice in a rural setting. the pipeline study piloted a model for getting students into underserved communities. however, that experiment was limited to 60-day rotations. outcome driven programs may provide a predictive surrogate for purposes of comparative analysis. for example, the rural medical education "rmed" program of the university of illinois medical school at rockford, has sustained a longstanding program in illinois. over 17 years in duration with over 200 student participants of whom 70% have been retained as primary care medicine practitioners in rural illinois. rabinowitz et al. ( 2008a ) further reinforced that medical school rural programs have been highly successful in increasing the supply of rural physicians, with an average of 53-64% of graduates choosing to practice in rural areas. they also noted rural retention rates of 79-87% among the programs (rabinowitz et al. 2008 a) . recently, the university of wisconsin school of medicine and public health (uwsmph) launched the wisconsin academy for rural medicine (warm program). the warm program places medical students in rural academic medical centers during their third and fourth years in medical school. marshfi eld clinic is one of those sites. warm students affi liating with marshfi eld clinic's system would ultimately share learning experiences with dental students, clinical rotations, team-based rounding, lectures, and exposure to a combined medical-dental patient record. in an analogous manner, the marshfi eld clinic dental education model will incorporate a curriculum that embeds students in rural clinical practice for up to 2 years. a secondary but not insignifi cant outcome of placing residents and students in clinical campuses focused on developing competency and providing care where needs are often greatest is the cost savings to taxpayers associated with the public care of patients. these savings are accomplished through the "service-learning" of the student. for example, in the model described where clinical training is embedded within the fhc clinics, the stipend resident or unpaid student learner provides the patient care as part of their service learning training while requiring oversight from one paid faculty per four to six learners. as a result, an academic based clinical partnership creates a model that reduces the cost for care provided to underserved patients. an additional benefi t to the community based clinic might be realized through tuition assistance by the academic program to help support patient procedures that develop learner competencies. in educational quality and infl uence, dental schools should equal medical schools, for their responsibilities are similar and their tasks analogous (william gies 1926 ) . the commission on dental accreditation (coda) notes that one of the learning objectives of an advanced education general dentistry (aegd) residency is to have the graduate function as a "primary care provider". to function competently in this role, the graduate needs to have a strong academic linkage to primary care medicine. at a 2010 dental deans forum, 84 years after the gies report, dr polverini made the statement "dentistry has never been linked to the medical network but unless dentistry becomes part of the solution to the challenge of providing comprehensive patient care, it will be looked on as part of the problem, and ultimately, all dental schools will be called into question." (polverini 2010 ) the use of dental informatics and an integrated record are elements essential to this competency. on april 1, 2010, fhc and marshfi eld clinic successfully transitioned all of their dental centers to a new practice management and electronic health record system that fully integrates medical and dental; one of the fi rst such systems in the nation. along with the benefi ts derived in fig. 4 .39 , chc placement also exposes students to an integrated medical-dental care setting where learners can develop skills in system-based practice to include the interdependence of health professionals, systems, and the coordination of care. on the administrative side, dental and medical appointments can be coordinated to enhance convenience for patients and improve compliance with preventive dental visits. in 2009, marshfi eld clinic's research foundation biomedical informatics research center hired their fi rst dental informatician, dr. amit acharya, bds, ms, phd. with dedicated biomedical informatics and research resource centers, the marshfi eld clinic has laid the groundwork for true medical/dental integration with appropriate electronic health record decision support and is positioned to develop a dental education curriculum capable of implementing the iom recommendations. downstream benefi ts of using such a curriculum are the ability of future practitioners to use informatics to improve quality of care and reduce the burden of disease. according to an institute of oral health report ( 2010 ) it is widely accepted across the dental profession that oral health has a direct impact on systemic health, and increasingly, medical and dental care providers are building to bridge relationships to create treatment solutions. as early as 1926, william gies recognized that "the frequency of periodic examination gives dentists exceptional opportunity to note early signs of many types of illnesses outside the domain of dentistry" (gies 1926 ) . the following examples show how integration of dental and medical care can impact patient outcomes, underlining the importance of this concept in dental curriculum design. a 2009 study of 21,000 blue cross blue shield of michigan (bcbs) members with diabetes, who had access to dental care lead researchers, and bcbs executives to conclude that treatment of periodontal disease signifi cantly impacts outcomes related to diabetes care and related costs (blue cross blue shield of michigan 2009 ) . another example is found in the context of preterm delivery and miscarriage. according to research cited by cigna ( 2006 ) , expecting mothers with chronic periodontal disease during the second trimester are seven times more likely to deliver preterm (before 37th week), and have dramatically more healthcare challenges throughout their life. cigna also cites the correlation between periodontal disease and low birth weights, pre-eclampsia, gestational diabetes. equally important is the opportunity to develop and implement the team-based curriculum that trains future dentists and physicians in the management of chronic disease as an accountable care organization (aco) in a patient-centered environment. as an example, joseph errante, d.d.s., vice president, blue cross blue shield of ma, reported that medical costs for diabetics who accessed dental care for prevention and periodontal services were signifi cantly lower than those who didn't get dental care (errante 2007 ) . these data suggest that team based case management of prevalent chronic health conditions have considerable cost savings opportunities for government payers, third party payers, employers and employees (errante 2007 ) . these economic benefi ts to integration as it relates to the iehr are discussed elsewhere in this book, but begin with the ability of providers to function in a team based environment and as such, underscore the importance of training in such an environment. dentists trained in a fqhc iehr integrated educational model will be well positioned to function successfully within an aco model. an aco is a system where providers are accountable for the outcomes and expenditures of the insured population of patients they serve. the providers within the system are charged with collectively improving care around cost and quality targets set by the payor. within this system, care must be delivered in a patient-centered environment. the patient-centered environment according to the national committee for quality assurance (ncqa), is a health care setting that cultivates partnerships between individual patients and their personal physicians and, when appropriate, the patient's family. care is facilitated by registries, information technology, health information exchange and other means to assure that patients receive defi ned, timely and appropriate care while remaining cognizant of cultural, linguistic and literacy needs of the patient being served. the model includes the opportunity to deliver patient care that is patient-centric, incorporates the patient in the care planning, considers the patient's beliefs and views, and incorporates the patient's families as needed. the model allows providers to deliver care that is inclusive of needs, attentive, and accessible. the model equips payers to purchase high quality and coordinated care among teams of providers across healthcare settings. while this describes the medical home, most dental practices also follow this process. many dental practices function in this regard with insured populations and refl ect elements of the model that medicine is creating. william gies would be proud. training in the delivery of accountable and patient-centered medical-dental care must be done purposefully. commenting on the inadequate training relative to the integration of medical and dental education, baum ( 2007 ) stated that "we need to design new curricula with meaningful core competencies for the next generation of dentists rather than apply patches to our existing ones." while this statement was made in reference to the basic sciences, the same holds true for patient-centered system-based practice competencies. utilizing state-of-the-art electronic medical records as a tool and the fhc infrastructure as the service venue, meaningful patient-centered system-based practice core competencies achievement becomes possible in a manner highly responsive to societal needs. by defi nition, fqhcs must provide primary medical care, dental care, and behavioral health. fqhc have also historically been utilized as healthcare workforce training centers and the affordable care act of 2010 reinforced their role as healthcare training centers. specifi cally, this legislation serves to promote fqhcs as the entity through which the primary care workforce (including dental) will be developed and expanded. in combination, fqhcs and primary care centers are positioned to be the front runners in a medical/-dental home training model which will be essential to preparing future practitioners for practice in an aco. critical to this success is the ability to train these practitioners on an integrated medical-dental record and informatics platform. use of this platform imprints most strongly during the learner's formative years of training; instructing and guiding disease management, decision making, patient care coordination, prevention, and both outcome-based and comprehensive care. training in this hybrid academically orientated clinically integrated setting moves dental education off its crossroads and creates the highway to its future. concerns with the new models extend to their ability to integrate medical and dental disciplines at the clinical and informatics level. while the 1995 iom report identifi ed the need to integrate medical and dental curriculum, success at the curricular and technological level within schools, has been limited. three major factors have contributed to the limited progress: access priorities. creating access to care has outranked the need to integrate â�¢ care. in part, this refl ects societal need for care and public demand to reduce the burden of the "silent epidemic." schools play an important role as a safety net to care for the uninsured and underinsured through intramural clinical service learning. even though "dental colleges seem to be willing to bearing a large and disproportionate share of the burden in terms of access to care" (dunning et al. 2009 ) , schools were challenged as part of iom, surgeon general, and macy reports, to expand that role. while these reports have prompted creative educational solutions to increase access, the reports understate the tremendous opportunity, quality and cost benefi ts that could result from an integration of medicine and dentistry. it is diffi cult to change the culture and structure of existing schools. this is not â�¢ unique to dentistry. however, the 1995 iom report specifi cally recommended that schools "eliminate marginally useful and redundant courses and design an integrated basic and clinical science curriculum". the challenges with this are many. examples include: some schools may not have other disciplines to draw from to create an inte-grated curriculum; a number of schools use a faculty senate to determine curriculum. this can result in curriculum that preserves the current faculty structure; changing curriculum is associated with expense and can be fi nancially pro-hibitive to some schools physical changes may be needed and represent an expense and/or may, in some instances, may not be practicable based on structure of existing facilities. public school programs may direct the fi nal curriculum, as boards or regent's one or two steps removed from the curriculum often have fi nal authority conversely, private schools may specify business or mission objectives that determine fi nal design. perhaps most germane to this text is the lack of a common technology plat-â�¢ form between disciplines in a learning environment. an integrated curriculum requires an integrated platform to accomplish delivery and evaluation. this is particularly essential to clinical management of the patient by professionals in training as part of a healthcare delivery team. some progress in establishing shared basic sciences curricula has been documented in the literature. to date, no single integrated electronic health (medical-dental) record has been meaningfully adapted for educational purposes, including incorporation of assessment of the learner relative to integrated competencies, integrated case-based and problem-based curriculum, and integrated evaluation and assessment. another concern with new educational programs emerging in response to these reports and relative to creating a transformational integrated curriculum is that some of the programs are focusing primarily on creating clinicians with no value or emphasis on integrating training with research and/or scholarly activity. integrated training models counter such concerns. research will be fundamental to measuring the relative benefi ts and outcomes associated with treatment of patients in a shared curriculum setting and will be the catalyst for the development of integrated medical-dental informatics incorporating educational capabilities. additionally, accreditation will also need to evaluate its response to such models. presently it is unclear how accrediting bodies will view an integrated crossdisciplinary curriculum. further, due to its integrated nature, such a curriculum would lie outside of the expertise of a single traditional accrediting body focused on one particular discipline. it has yet to be determined how accrediting bodies will review and appraise such cross-disciplinary competencies. lastly, it is important to recognize that a successful education model with innovative informatics is only successful if its focus is patient care. graduating learners with competency only in the use of informatics will be limited unless adapted to training and delivery programs that result in patient centric care. research and reports over the past 15 years support the need to reform dental education. first steps have been taken and lead the way for continued innovation around clinic-based education and integrated curriculum. the models identifi ed point to a strong partnership and interrelationship with chcs for creative, cost saving, effective and sustainable delivery methods. moreover, chc's must be more involved in a training curriculum integrated with informatics. chcs, in turn, benefi t from residents and students through service-learning to help meet a societal and workforce need, while the learners benefi t from increased competency. in order to train an evidence-based, patient-centered, medical-dental workforce, it is imperative that medical and dental data and record accessibility be incorporated into these training and care delivery initiatives. in order to keep moving away from the crossroads, such integration must become the pathway on which curriculum is developed and implemented. public law 107-188 cardioprotective effect of periodontal therapy in metabolic syndrome: a pilot study in indian subjects effect of professional oral health care on the elderly living in nursing homes. oral surg oral med oral pathol study shows preventive dental care may help reduce risk of preterm birth dentists' and specialists' knowledge of chronic orofacial pain: results from a continuing professional development survey. prim dent care an examination of periodontal treatment and per member per month (pmpm) medical costs in an insured population an examination of periodontal treatment, dental care, and pregnancy outcomes in an insured population in the united states the changing pattern of upper gastro-intestinal lesions in southern saudi arabia: an endoscopic study association between hla antigens and periodontal disease chronic care: making the case for ongoing care effects of antibiotic therapy on outcomes of patients with coronary artery disease: a meta-analysis of randomized controlled trials association between metabolic syndrome and periodontal disease caries is associated with asthma and epilepsy medicare coverage for patients with diabetes infections and autoimmune diseases state-supported dental schools: fi nancial projections and implications strengthening the nation's public health infrastructure: historic challenge, unprecedented opportunity dental health policy analysis series: issues in children's access to dental care under medicaid. chicago: american dental association inadequate training in the biological sciences and medicine for dental students a missense single-nucleotide polymorphism in a gene encoding a protein tyrosine phosphatase ( ptpn22 ) is associated with rheumatoid arthritis the dna methyltransferases of mammals fixing intelligence, foreign affairs high-throughput dna methylation profi ling using universal bead arrays blue cross blue shield of michigan. study links good oral care to lower diabetes care costs halitosis: a multidisciplinary problem construction of a genetic linkage map in man using restriction fragment length polymorphisms longitudinal analysis of heritability for dental caries traits systemic infl ammatory markers, periodontal diseases, and periodontal infections in an elderly population attention to dental coverage lacking in health-care debate. from st. petersburg times community based dental education: the pipeline, profession and practice program cotton-wool spots a large-scale genetic association study confi rms il12b and leads to the identifi cation of il23r as psoriasis-risk genes mother's gum disease linked to infant's death chronic disease management. health reform gps: navigating the implementation process fam5c contributes to aggressive periodontitis us government printing offi ce centers for disease control. dental service use and dental insurance coverage-united states, behavioral risk factor surveillance system association between chronic periodontal disease and obesity: a systematic review and meta-analysis evaluation of negation phrases in narrative clinical reports the health insurance and portability and accountability act: practice of dentistry in the united states: privacy and confi dentiality the human oral microbiome database: a web accessible resource for investigating oral microbiome taxonomic and genomic information prevalence of doctor diagnosed arthritis and arthritis attributable activity limitation -united states cigna dental oral health maternity program trends in medical care expenditures of us adults with arthritis and other rheumatic conditions 1997 to genetic susceptibility to head and neck squamous cell carcinoma genetic and functional association of fam5c with myocardial infarction self-reported periodontal disease in a virginia twin population infl ammation and cancer genetic mapping of the dentinogenesis imperfecta type ii locus association of the metabolic syndrome with severe periodontitis in a large u.s. population-based survey the economic burden of diabetes targeting cyclooxygenase-2 in human neoplasia: rationale and promise an inventory of patients' response to treatment at a multidisciplinary breath odor clinic the clinical signifi cance of the digital patient record evidence-based integrated care plan oral and craniofacial data resource center of the national institute of dental and craniofacial research hipaa department of veterans affairs. va health information technology improves quality of health care while reducing costs dental disease and risk of coronary heart disease and mortality human herpesvirus 8 and kaposi's sarcoma strategic management and organizational behavior in dental education: refl ections on key issues in an environment of change prevalence of hypertension, dyslipidemia, and dyslipidemic hypertension the metabolic syndrome quantitative high-throughput analysis of dna methylation patterns by base-specifi c cleavage and mass spectrometry rare structural variants found in attention-defi cit hyperactivity disorder are preferentially associated with neurodevelopmental genes replication of the association of chromosomal region 9p21.3 with generalized aggressive periodontitis (gagp) using an independent casecontrol cohort integration of medical and dental benefi ts for improved overall health. institute for oral health public health law: a communitarian perspective introduction to quantitative genetics hyperinsulinaemia: the key feature of a cardiovascular and metabolic syndrome dental education at the crossroads: challenges and change. an institute of medicine report evolving concepts of rheumatoid arthritis mapping of papillon-lefevre syndrome to the chromosome 11q14 region building a medical neighborhood for the medical home bidirectional relationship between chronic kidney and periodontal disease: a study using structural equation modeling clinical outreach: seven years of a self-sustaining model dentistry and bioterrorism prevalence of the metabolic syndrome among us adults the macy study: a framework for consensus cms projects focus on chronic illnesses: three demonstration projects seek to improve effi ciency and care. caring for ages prevalence and risk factors for gastroesophageal refl ux disease in an impoverished minority population public health and national security: the critical role of increased federal support extending multidisciplinary management of cleft palate to the developing world automated syndromic surveillance for the 2002 winter olympics systemic diseases and their treatment in the elderly: impact on oral health dental education in the united states and canada early statistical detection of anthrax outbreaks by tracking over-the-counter medication sales epigenetics and periodontal disease: future perspectives the infl uence of ccl3l1 gene-containing segmental duplications on hiv-1/aids susceptibility genetic variants near tnfaip3 on 6q23 are associated with systemic lupus erythematosus variant of transcription factor 7-like 2 ( tcf7l2 ) gene confers risk of type 2 diabetes interleukin 7 receptor a chain ( il7r ) shows allelic and functional association with multiple sclerosis powe nr, for the centers for disease control and prevention ckd surveillance team. vulnerable populations and the association between periodontal and chronic kidney disease dentistry's response to bioterrorism: a report of a consensus workshop genome-wide association and replication studies identify four variants associated with prostate cancer susceptibility promoter methylation and inactivation of tumour-suppressor genes in oral squamous-cell carcinoma distinctive chromatin in human sperm packages genes for embryo development localisation of a gene for prepubertal periodontitis to chromosome 11q14 and identifi cation of a cathepsin c gene mutation the electronic oral health record a common variant on chromosome 9p21 affects the risk of myocardial infarction the role of cathepsin c in papillon-lefevre syndrome, prepubertal periodontitis, and aggressive periodontitis whole-genome patterns of common dna variation in three human populations a comprehensive review of genetic association studies economic cost of babies in the us in 2002 preparing health professionals to provide care to individuals with disabilities porphyromonas gingivalis, treponema denticola, and tennerella forsythia: the "red complex", a prototype polybacterial pathogenic consortium in periodontitis association between circulating oxidized low-density lipoprotein and incidence of the metabolic syndrome infl ammation and metabolic disorders gene genealogies and the coalescent process oral health and a referral arterial disease the effect of periodontal disease on medical and dental costs in a middle-aged japanese population: a longitudinal worksite study institute for oral health. focus groups on oral health in healthcare reform -focus group #2 accuracy of three classifi ers of acute gastrointestinal syndrome for syndromic surveillance the effect of antimicrobial periodontal treatment on circulating tumor necrosis factor-alpha and glycated hemoglobin level in patients with type 2 diabetes the relative power of linkage and association studies for the detection of genes involved in hypertension periodontal disease, tooth loss and incidence of ischemic stroke new models of dental education kaiser family foundation. the uninsured likelihood methods for locating disease genes in nonequilibrium populations six new loci associated with blood lowdensity lipoprotein cholesterol, high-density lipoprotein cholesterol or triglycerides in humans amelogenin p.m1t and p.w4s mutations underlying hypoplastic x-linked amelogenesis imperfecta stochastic processes in population genetics, with special reference to distribution of gene frequencies and probability of gene fi xation complement factor h polymorphism in age-related macular degeneration prospects for whole-genome linkage disequilibrium mapping of common disease genes periodontal disease adversely affects the survival of patients with end-stage renal disease new bacterial species associated with chronic periodontitis expression of the fam5c in tongue squamous cell carcinoma localisation of a gene for papillon-lefevre syndrome to chromosome 11q14-q21 by homozygosity mapping inhibitors of cathepsin c (dipeptidyl peptidase i) gene polymorphisms in chronic periodontitis perturbed amelogenin secondary structure leads to uncontrolled aggregation in amelogenesis imperfecta mutant proteins initial sequencing and analysis of the human genome bioterrorism on the home front a new challenge for american medicine consensus report: aggressive periodontitis impact of current and past blood pressure on retinal arteriolar diameter in an older population localized aggressive periodontitis is linked to human chromosome 1q25 heart disease & stroke statistics -2010 update the power of association studies to detect the contribution of candidate genetic loci to variation in complex traits biosense -a national initiative for early detection and quantifi cation of public health emergencies periodontal therapy reduces the rate of preterm low birth weight in women with pregnancy-associated gingivitis a variant of ptpn22 gene conferring risk to autoimmune diseases may protect against tuberculosis air, water, places, and data-public health in the information age the role of the nutrition in the pathogenesis of gastroesophageal refl ux disease, barrett's oesophagus and oesophageal adenocarcinoma structural variation in the human genome /5, dqb1 in german patients suffering from rapidly progressive periodontitis (rpp) and adult periodontitis (ap) finding the missing heritability of complex diseases dental insurance: design, need and public policy births: fi nal data for va uses integrated health informatics to produce $3 billion in savings. daily break a common allele on chromosome 9 associated with coronary heart disease a family with papillon-lefevre syndrome reveals a requirement for cathepsin c in granzyme b activation and nk cell cytolytic activity diabetes mellitius and infl ammatory periodontal diseases the growing challenge of providing oral health care services to all americans periodontal fi ndings in adult twins evidence of a substantial genetic basis for risk of adult periodontitis the relationship between reduction in periodontal infl ammation and diabetes control: a report of 9 cases can presence or absence of periodontal pathogens distinguish between subjects with chronic and aggressive periodontitis? a systematic review employer survey on health insurance in montana environmental and heritable factors in the etiology of oral diseases -a population-based study of swedish twins medicaid coverage and utilization of adult dental services transgenerational genetic effects on phenotypic variation and disease risk the convergence of dental and medical care the american dental association's commitment to electronic data interchange dentistry's role in responding to bioterrorism and other catastrophic events association of periodontitis and metabolic syndrome in the baltimore longitudinal study of aging exome sequencing identifi es the cause of a mendelian disorder cytokine gene polymorphisms in periodontal disease: a meta-analysis of 53 studies including 4178 cases and 4590 controls keep america smiling: oral health in america. chicago: oral health america oral biofi lms, periodontitis and pulmonary infections partnership for solutions national program offi ce. chronic conditions: making the case for ongoing care enamel formation genes are associated with high caries experience in turkish children genetic and epigenetic somatic alterations in head and neck squamous cell carcinomas are globally coordinated but not locally targeted genome-wide map of nucleosome acetylation and methylation in yeast the ann arbor dental deans forum: crafting a response to the emerging tiered system of dental education global. health nexus, college of dentistry medical school programs to increase the rural physician supply: a systematic review and projected impact of widespread replication human dipeptidyl-peptidase i clinical characteristics and diagnosis of atypical odontalgia: implications for dentists rna-mediated non-mendelian inheritance of an epigenetic change in the mouse association of periodontitis with rheumatoid arthritis: a pilot study genome-wide location and function of dna binding proteins simulation modeling of anthrax spore dispersion in a bioterrorism incident the computer-based oral health record genome-wide association study identifi es new susceptibility loci for crohn disease and implicates autophagy in disease pathogenesis a half-century of community water fl uoridation in the united states: review and commentary the future of genetic studies of complex human diseases foundation pipeline study. evaluation of pipeline, profession and practice: community-based dental education neuropathic orofacial pain after dental implant placement: review of the literature and case report. oral surg oral med oral pathol oral radiol endod oral hygiene can reduce the incidence of and death resulting from pneumonia and respiratory tract infection effect of daily asprin on long-term risk of death due to cancer: analysis of individual patient data from randomized trails access to oral health care for elders: mere words or action? reconnecting the head to the body. dental considerations for the optimal medical management of people with diabetes. seattle: institute for oral health functional variants of the interleukin-23 receptor gene in non-gastrointestinal autoimmune diseases enzymatic amplifi cation of b -globin genomic sequences and restriction site analysis for diagnosis of sickle cell anemia serological evidence of an association of a novel chlamydia , twar, with chronic coronary heart disease and acute myocardial infarction hepatitis c virus infection is associated with the development of hepatocellular carcinoma the severity of periodontal disease is associated with the element of glucose intolerance in non-diabetics skeletal facial balance and harmony in the cleft patient: principles and techniques in orthognathic surgery periodontal disease and mortality in type 2 diabetes pneumonia in nonambulatory patients identifi cation of a shared genetic susceptibility locus for coronary heart disease and periodontitis a genome-wide association study identifi es glt6d1 as a susceptibility locus for periodontitis integrating dental offi ce technology -the next frontier the computer-based oral health record: an essential tool for cross-provider quality management the technologically well-equipped dental offi ce clinical computing in general dentistry the evolution of molecular markers -just a matter of fashion? strong association of de novo copy number mutations with autism prediction model for prevalence and incidence of advanced age-related macular degeneration based on genetic, demographic, and environmental variables hipaa security regulations protecting patients' electronic health information retinal arteriolar diameters and elevated blood pressure: the atherosclerosis risk in communities study treatment of periodontal disease for glycaemic control in people with diabetes relationship between periodontal disease and c-reactive protein among adults in the atherosclerosis risk in communities study social security and medicare boards of trustees. actuarial publications, status of the social security and medicare programs: a summary of the 2011 annual reports dentin phosphoprotein frameshift mutations in hereditary dentin disorders and their variation patterns in normal human populations fatty liver is associated with dyslipidemia and dysglycemia independent of visceral fat: the framingham heart study toothache or trigeminal neuralgia: treatment dilemmas consortium for oral health-related informatics: improving dental research, education, and treatment large recurrent microdeletions associated with schizophrenia the dental patient with asthma: an update and oral health considerations the effect of periodontal treatment on glycemic control in patients with type 2 diabetes mellitus projected costs of chronic disease. health care cost monitor legal issues of the electronic dental record: security and confi dentiality the international hapmap 3 consortium. integrating common and rare genetic variation in diverse human populations national electronic disease surveillance system (nedss): a standards-based approach to connect public health and clinical medicine help wanted: a policy maker's guide to new dental providers. national academy for state health policy, wk kellogg foundation chronic conditions account for rise in medicare spending from medical status and complications in relation to periodontal disease experience in insulin-dependent diabetics loos-of-function mutations in the cathepsin c gene result in periodontal disease and palmoplantar keratosis detection of disease outbreaks by the use of oral manifestations technical description of rods: a real-time public health surveillance system department of health and human services. oral health in american; surgeon general's call to action. rockville: usdhhs, national institute of dental and craniofacial research, nation institutes of health helicobacter pylori infection and the development of gastric cancer unitedhealth center for health reform & modernization. the united states of diabetes: challenges and opportunities in the decade ahead. minnetonka: unitedhealth group healthy people 2020 how will we seize them? american dental education association charting progress holding ourselves to the highest standard: doing what's best for patients. american dental education association charting progress role of micro-organisms in caries etiology the sequence of the human genome genome-wide scan fi nd suggestive caries loci heritability in the genomics era -concepts and misconceptions handbook of biosurveillance human papillomavirus is a necessary cause of invasive cervical cancer worldwide the assessment of treatment outcome by evaluation of dental arch relationships in cleft lip/palate role of chlamydia pneumoniae in atherosclerosis abundant class of human dna polymorphisms which can be typed using the polymerase chain reaction obesity and the metabolic syndrome in children and adolescents wellcome trust case control consortium. genome-wide association study of 14,000 cases of seven common diseases and 3,000 shared controls myocardial infarction and cerebrovascular accident in patients with retinal vein occlusion fact sheet project bioshield evaluating a dental diagnostic terminology in an electronic health record review on fl uoride-releasing restorative materials -fl uoride release and uptake characteristics, antibacterial activity and infl uence on caries formation dental students' experiences with and attitudes toward people with mental retardation hypertensive retinopathy signs as risk indicators of cardiovascular morbidity and morality retinal microvascular abnormalities and incident stroke: the atherosclerosis risk in communities study retinal microvascular abnormalities and blood pressure in older people: the cardiovascular health study retinal arteriolar narrowing and risk of diabetes in middle-aged persons retinal microvascular abnormalities and ten-year cardiovascular abnormality: a population-based casecontrol study wsare: what's strange about recent events? retinopathy and risk of congestive heart failure retinal arteriolar narrowing, hypertension and subsequent risk of diabetes mellitus periodontal disease and risk of cerebrovascular disease national and state medical expenditures and lost earnings attributable to arthritis and other rheumatic conditions -united states high frequency of occurrence of cyp2d6 gene duplication/multiduplication indicating ultrarapid metabolism along suicide cases the digital pioneer evaluation of human leukocyte n-formylpeptide receptor (fpr1) snps in aggressive periodontitis patients integration of pediatric medical and pediatric dental care the effect of dental rehabilitation on the body weight of children with early childhood caries recommendations for preventive pediatric health care (periodicity schedule) american academy of pediatric dentistry. dental care for your baby does caries in primary teeth predict enamel defects in permanent teeth? a longitudinal study california dental association. oral health during pregnancy and early childhood: evidence-based guidelines for health professionals. sacramento: california dental association beyond the dmft: the human and economic cost of early childhood caries perceptions of oral health training and attitudes toward performing oral health screenings among graduating pediatric residents role of the medical team in preventing early childhood caries trends in oral health by poverty status as measured by hp 2010 objectives update on disparities in oral health and access to dental care for america's children early childhood caries: the dental disease of infants teaching oral health in u.s. medical schools: results of a national survey electronic medical record use in pediatric primary care term stillbirth caused by oral fusobacterium nucleatum integrated medical and dental health in primary care advancing oral health in america improving access to oral health care for vulnerable and underserved populations the bright futures training project: implementing systems to support preventive and developmental services in practice dental care and children with special health care needs: a population-based perspective preventive dental care for children in the us: a national perspective oral health and pediatricians: results of a national survey predicting caries in permanent teeth from caries in primary teeth: an eight-year cohort study interprofessional educational partnerships in school health for children with special oral health needs early childhood caries: determining the risk factors and assessing the prevention strategies for nursing intervention addressing children's oral health in the new millennium: trends in the dental workforce making decisions for children disparities in children's oral health and access to dental care executive summary: progress in children's oral health since the 2000 surgeon general's report adding dental therapists to the health care team to improve access to oral health care for children new york state department of health. oral health care during pregnancy and childhood cost-effectiveness model for the prevention of early childhood caries oral health during pregnancy: current research at the division of oral health, centers for disease control and prevention early preventive dental visits: effects on subsequent utilization and costs dental education's role in improving children's oral health and access to care survey of iowa general dentists regarding the age 1 dental visit integration of patient records and clinical research: lack of integration as a barrier to needed research electronic patient records for dental school clinics: more than paperless systems risk factors for osteonecrosis of the jaws: a case-control study from the condor dental pbrn the "meaningful use" regulation for electronic health records the medical dictionary for regulatory activities (meddra) the benefi ts of health information technology: a review of the recent literature shows predominantly positive results assessing user perceptions of an electronic patient record (epr) before implementation the content coverage of clinical classifi cations. for the computer-based patient record institute's work group on codes & structures from data to knowledge through concept-oriented terminologies: experience with the medical entities dictionary research on the comparative effectiveness of medical treatments: issues and options for an expanded federal role pub. no. 2975 double data entry: what value, what price? evaluating the informatics for integrating biology and the bedside system for clinical research the use of electronic data capture tools in clinical trials: web-survey of 259 canadian trials the signifi cance of snodent. stud health technol inform returns on research and development for 1990s new drug introductions research electronic data capture (redcap)-a metadata-driven methodology and workfl ow process for providing translational research informatics support data collection and transcription the development of a dental diagnostic terminology a quantifi able alternative to double data entry adaptive double data entry: a probabilistic tool for choosing which forms to reenter economic outcomes of a dental electronic patient record survey of public information about ongoing clinical trials funded by industry: evaluation of completeness and accessibility better access to information about clinical trials clinical data management clinical computing in general dentistry format-independent data collection forms documentation of clinical workfl ow: a key step in a plan to facilitate implementation of an electronic patient record consortium for oral health-related informatics: improving dental research, education, and treatment which coding system for therapeutic information in evidencebased medicine integrating electronic patient records into a multimedia clinic-based simulation center using a pc blade platform: a foundation for a new pedagogy in dentistry creating a searchable digital dental radiograph repository for patient care, teaching and research using an online photo management and sharing application a human-centered design of a dental discharge summary (dds) for patients factors infl uencing implementation and outcomes of a dental electronic patient record system continuum of quality care series, american academy of developmental medicine and dentistry key: cord-146850-5x6qs2i4 authors: gupta, abhishek; lanteigne, camylle; heath, victoria; ganapini, marianna bergamaschi; galinkin, erick; cohen, allison; gasperis, tania de; akif, mo; institute, renjie butalid montreal ai ethics; microsoft,; university, mcgill; commons, creative; college, union; rapid7,; global, ai; university, ocad title: the state of ai ethics report (june 2020) date: 2020-06-25 journal: nan doi: nan sha: doc_id: 146850 cord_uid: 5x6qs2i4 these past few months have been especially challenging, and the deployment of technology in ways hitherto untested at an unrivalled pace has left the internet and technology watchers aghast. artificial intelligence has become the byword for technological progress and is being used in everything from helping us combat the covid-19 pandemic to nudging our attention in different directions as we all spend increasingly larger amounts of time online. it has never been more important that we keep a sharp eye out on the development of this field and how it is shaping our society and interactions with each other. with this inaugural edition of the state of ai ethics we hope to bring forward the most important developments that caught our attention at the montreal ai ethics institute this past quarter. our goal is to help you navigate this ever-evolving field swiftly and allow you and your organization to make informed decisions. this pulse-check for the state of discourse, research, and development is geared towards researchers and practitioners alike who are making decisions on behalf of their organizations in considering the societal impacts of ai-enabled solutions. we cover a wide set of areas in this report spanning agency and responsibility, security and risk, disinformation, jobs and labor, the future of ai ethics, and more. our staff has worked tirelessly over the past quarter surfacing signal from the noise so that you are equipped with the right tools and knowledge to confidently tread this complex yet consequential domain. these past few months have been especially challenging, and the deployment of technology in ways hitherto untested at an unrivaled pace has left the internet and technology watchers aghast. artificial intelligence has become the byword for technological progress and is being used in everything from helping us combat the covid-19 pandemic to nudging our attention in different directions as we all spend increasingly larger amounts of time online. it has never been more important that we keep a sharp eye out on the development of this field and how it is shaping our society and interactions with each other. with this inaugural edition of the state of ai ethics we hope to bring forward the most important developments that caught our attention at the montreal ai ethics institute this past quarter. our goal is to help you navigate this ever-evolving field swiftly and allow you and your organization to make informed decisions. this pulse-check for the state of discourse, research, and development is geared towards researchers and practitioners alike who are making decisions on behalf of their organizations in considering the societal impacts of ai-enabled solutions. we cover a wide set of areas in this report spanning agency and responsibility, security and risk, disinformation, jobs and labor, the future of ai ethics , and more. our staff has worked tirelessly over the past quarter surfacing signal from the noise so that you are equipped with the right tools and knowledge to confidently tread this complex yet consequential domain. to stay up-to-date with the work at maiei, including our public competence building, we encourage you to stay tuned on https://montrealethics.ai which has information on all of our research. we hope you find this useful and look forward to hearing from you! wishing you well, abhishek gupta the state of ai ethics, june 2020 the debate when ethicists ask for rights to be granted to robots is based on notions of biological chauvinism and that if robots display the same level of agency and autonomy, not doing so would not only be unethical but also cause a setback for the rights that were denied to disadvantaged groups. by branding robots as slaves and implying that they don't deserve rights has fatal flaws in that they both use a term, slave, that has connotations that have significantly harmed people in the past and also that dehumanization of robots is not possible because it assumes that they are not human to begin with. while it may be possible to build a sentient robot in the distant future, in such a case there would be no reason to not grant it rights but until then, real, present problems are being ignored for imaginary future ones. the relationship between machines and humans is tightly intertwined but it's not symmetrical and hence we must not confound the "being" part of human beings with the characteristics of present technological artifacts. technologists assume that since there is a dualism to a human being, in the sense of the mind and the body, then it maps neatly such that the software is the mind and the robot body maps to the physical body of a human, which leads them to believe that a sentient robot, in our image, can be constructed, it's just a very complex configuration that we haven't completely figured out yet. the more representative view of thinking about robots at present is to see them as objects that inhabit our physical and social spaces. objects in our environment take on meaning based on the purpose they serve to us, such as a park bench meaning one thing to a skateboarder and another to a casual park visitor. similarly, our social interactions are always situated within a larger ecosystem and that needs to be taken into consideration when thinking about the interactions between humans and objects. in other words, things are what they are, because of the way they configure our social practices and how technology extends the biological body.our conception of human beings, then, is that we are and have always been fully embedded and enmeshed with our the state of ai ethics, june 2020 designed surroundings, and that we are critically dependent on this embeddedness for sustaining ourselves. because of this deep embedding, instead of seeing the objects around us merely as machines or on the other end as 'intelligent others', we must realize that they are very much a part of ourselves because of the important role they play in defining both our physical and social existence. some argue that robots take on a greater meaning when they are in a social context like care robots and people might be attached to them, yet that is quite similar to the attachment one develops to other artifacts like a nice espresso machine or a treasured object handed down for generations. they have meaning to the person but that doesn't mean that the robot, as present technology, needs to be granted rights. while a comparison to slaves and other disenfranchised groups is made when robots are denied rights because they are seen as 'less' than others, one mustn't forget that it happens to be the case that it is so because they are perceived as instruments and means to achieve an end. by comparing these groups to robots, one dehumanizes actual human beings. it may be called anthropocentric to deny rights to robots but that's what needs to be done: to center on the welfare of humans rather than inanimate machines. an interesting analogue that drives home the point when thinking about this is the milgram prison experiment where subjects who thought they had inflicted harms on the actors, who were a part of the experiment, were traumatized even after being told that the screams they heard were from the actors. from an outside perspective, we may say that no harm was done because they were just actors but to the person who was the subject of the experiment, the experience was real and not an illusion and it had real consequences. in our discussion, the robot is an actor and if we treat it poorly, then that reflects more so on our interactions with other artifacts than on whether robots are "deserving" of rights or not. taking care of various artifacts can be thought of as something that is done to render respect to the human creators and the effort that they expended to create it. discussion of robot rights for an imaginary future that may or may not arrive takes away focus and perhaps resources from the harms being done to real humans today as part of the ai systems being built with bias and fairness issues in them. invasion of privacy, bias against the disadvantaged, among other issues are just some of the few already existing harms that are being leveled on humans as intelligent systems percolate into the everyday fabric of social and economic life. the state of ai ethics, june 2020 from a for-profit perspective, such systems are poised and deployed with the aims of boosting the bottom line without necessarily considering the harms that emerge as a consequence. in pro-social contexts, they are seen as a quick fix solution to inherently messy and complex problems. the most profound technologies are those that disappear into the background and in subtle ways shape and form our existence. we already see that with intelligent systems pervading many aspects of our lives. so we're not as much in threat from a system like sophia which is a rudimentary chatbot hidden behind a facade of flashy machinery but more so from roomba which impacts us more and could be used as a tool to surveil our homes. taking ethical concerns seriously means considering the impact of weaving in automated technology into daily life and how the marginalized are disproportionately harmed. in the current dominant paradigm of supervised machine learning, the systems aren't truly autonomous, there is a huge amount of human input that goes into enabling the functioning of the system, and thus we actually have human-machine systems rather than just pure machinic systems. the more impressive the system seems, the more likely that there was a ton of human labor that went into making it possible. sometimes, we even see systems that started off with a different purpose such as recaptcha that are used to prevent spam being refitted to train ml systems. the building of ai systems today doesn't just require highly skilled human labor but it must be supplemented with mundane jobs of labeling data that are poorly compensated and involve increasingly harder tasks as, for example, image recognition systems become more powerful, leading to the labeling of more and more complex images which require greater effort. this also frames the humans doing the low skilled work squarely in the category of being dehumanized because of them being used as a means to an end without adequate respect, compensation and dignity. an illustrative example where robots and welfare of humans comes into conflict was when a wheelchair user wasn't able to access the sidewalk because it was blocked by a robot and she mentioned that without building for considering the needs of humans, especially those with special needs, we'll have to make debilitating compromises in our shared physical and social spaces. ultimately, realizing the goals of the domain of ai ethics needs to reposition our focus on humans and their welfare, especially when conflicts arise between the "needs" of automated systems compared to those of humans. what happens when ai starts to take over the more creative domains of human endeavour? are we ready for a future where our last bastion, the creative pursuit, against the rise of machines is violently snatched away from us? in a fitting start to feeling bereft in the times of global turmoil, this article starts off with a story created by a machine learning model called gpt-2 that utilizes training data from more than 8 million documents online and predicts iteratively the next word in a sentence given a prompt. the story is about "life in the time of coronavirus" that paints a desolate and isolating picture of a parent who is following his daily routine and feels different because of all the changes happening around them. while the short story takes weird turns and is not completely coherent, it does give an eerie feeling that blurs the line between what could be perceived as something written by a human compared to that by a machine. a news-styled article on the use of facial recognition systems for law enforcement sounds very believable if presented outside of the context of the article. the final story, a fictional narrative, presents a fractured, jumpy storyline of a girl with a box that has hallucinatory tones to its storytelling. the range of examples from this system is impressive but it also highlights how much further these systems have to go before they can credibly take over jobs. that said, there is potential to spread disinformation via snippets like the second example we mention and hence, something to keep in mind as you read things online. technology, in its widest possible sense, has been used as a tool to supplement the creative process of an artist, aiding them in exploring the adjacent possible in the creative phasespace. for decades we've had computer scientists and artists working together to create software that can generate pieces of art that are based on procedural rules, random perturbations of the audience's input and more. off late, we've had an explosion in the use of ai to do the same, with the whole ecosystem being accelerated as people collide with each other serendipitously on platforms like twitter creating new art at a very rapid pace. but, a lot of people have been debating whether these autonomous systems can be attributed artistic agency and if they can be called artists in their own right. the author here argues that it isn't the case because even with the push into using technology that is more the state of ai ethics, june 2020 automated than other tools we've used in the past, there is more to be said about the artistic process than the simple mechanics of creating the artwork. drawing on art history and other domains, there is an argument to be made as to what art really is -there are strong arguments in support of it playing a role in servicing social relationships between two entities. we, as humans, already do that with things like exchanging gifts, romance, conversation and other forms of social engagement where the goal is to alter the social relationships. thus, the creative process is more so a co-ownership oriented model where the two entities are jointly working together to create something that alters the social fabric between them. as much as we'd like to think some of the ai-enabled tools today have agency, that isn't necessarily the case when we pop open the hood and see that it is ultimately just software that for the most part still relies heavily on humans setting goals and guiding it to perform tasks. while human-level ai might be possible in the distant future, for now the ai-enabled tools can't be called artists and are merely tools that open up new frontiers for exploration. this was the case with the advent of the camera that de-emphasized the realistic paint form and spurred the movement towards modern art in a sense where the artists are more focused on abstract ideas that enable them to express themselves in novel ways. art doesn't even have to be a tangible object but it can be an experience that is created. ultimately, many technological innovations in the past have been branded as having the potential to destroy the existing art culture but they've only given birth to new ideas and imaginings that allow people to express themselves and open up that expression to a wider set of people. the state of ai ethics, june 2020 ranking and retrieval systems for presenting content to consumers are geared towards enhancing user satisfaction, as defined by the platform companies which usually entails some form of profit-maximization motive, but they end up reflecting and reinforcing societal biases, disproportionately harming the already marginalized. in fairness techniques applied today, the outcomes are focused on the distributions in the result set and the categorical structures and the process of associating values with the categories is usually de-centered. instead, the authors advocate for a framework that does away with rigid, discrete, and ascribed categories and looks at subjective ones derived from a large pool of diverse individuals. focusing on visual media, this work aims to bust open the problem of underrepresentation of various groups in this set that can render harm on to the groups by deepening social inequities and oppressive world views. given that a lot of the content that people interact with online is governed by automated algorithmic systems, they end up influencing significantly the cultural identities of people. while there are some efforts to apply the notion of diversity to ranking and retrieval systems, they usually look at it from an algorithmic perspective and strip it of the deep cultural and contextual social meanings, instead choosing to reference arbitrary heterogeneity. demographic parity and equalized odds are some examples of this approach that apply the notion of social choice to score the diversity of data. yet, increasing the diversity, say along gender lines, falls into the challenge of getting the question of representation right, especially trying to reduce gender and race into discrete categories that are one-dimensional, third-party and algorithmically ascribed. the authors instead propose sourcing this information from the individuals themselves such that they have the flexibility to determine if they feel sufficiently represented in the result set. this is contrasted with the degree of sensitive attributes that are present in the result sets which is what prior approaches have focused on. from an algorithmic perspective, the authors advocate for the use of a technique called determinantal point process (dpp) that assigns a higher probability score to sets that have higher spreads based on a predefined distance metric. how dpp works is that for items that the individual feels represents them well, the algorithm clusters those points closer together, for points that they feel don't represent them well, it moves those away from the ones that represent them well in the embedding space. optimizing for the triplet loss helps to achieve the goals of doing this separation. but, the proposed framework still leaves open the question of sourcing in a reliable manner these ratings from the individuals about what represents and doesn't represent them well and then encoding them in a manner that is amenable to being learned by an algorithmic system. while large-scale crowdsourcing platforms which are the norm in seeking such ratings in the machine learning world, given that their current structuring precludes raters' identities and perceptions from consideration, this framing becomes particularly challenging in terms of being able to specify the rater pool. nonetheless, the presented framework provides an interesting research direction such that we can obtain more representation and inclusion in the algorithmic systems that we build. in maryland, allstate, an auto insurer, filed with the regulators that the premium rates needed to be updated because they were charging prices that were severely outdated. they suggested that not all insurance premiums be updated at once but instead follow recommendations based on an advanced algorithmic system that would be able to provide deeper insights into the pricing that would be more appropriate for each customer based on the risk that they would file a claim. this was supposed to be based on a constellation of data points collected by the company from a variety of sources. because of the demand from the regulators for documentation supporting their claim, they submitted thousands of pages of documentation that showed how each customer would be affected, a rare window into the pricing model which would otherwise have been veiled under privacy and trade secret arguments. a defense that is used by many companies that utilize discriminatory pricing strategies using data sourced beyond what they should be using to make pricing decisions. according to the investigating journalists, the model boiled down to something quite simple: the more money you had and the higher your willingness to not budge from the company, the more the company would try to squeeze from you in terms of premiums. driven by customer retention and profit motives, the company pushed increases on those that they knew could afford them and would switch to save dollars. but, for those policies that had been overpriced, they offered less than 0.5% in terms of a discount limiting their downsides while increases were not limited, often going up as high as 20%. while they were unsuccessful in getting this adopted in maryland where it was deemed discriminatory, the model has been approved for use in several states thus showing that opaque models can be deployed not just in high-tech industries but anywhere to provide individually tailored pricing to extract away as much of the consumer surplus as possible based on the purportedly undisclosed willingness of the customer to pay (as would be expressed by their individual demand curves which aren't directly discernible to the producer). furthermore, the insurers aren't mandated to make disclosures of how they are pricing their policies and thus, in places where they should have offered discounts, they've only offered pennies on the dollar, disproportionately impacting the poorest for whom a few hundred dollars a year can mean having sufficient meals on the table. sadly, in the places where their customer retention model was accepted, the regulators declined to answer why they chose to accept it, except in arkansas where they said such pricing schemes aren't discriminatory unless the customers are grouped by attributes like race, color, creed or national origin. this takes a very limited view of what price discrimination is, harkening back to an era where access to big data about the consumer was few and far between. in an era dominated by data brokers that compile thick and rich data profiles on consumers, price discriminaton extends far beyond the basic protected attributes and can be tailored down to specificities of each individual. other companies in retail goods and online learning services have been following this practice of personalized pricing for many years, often defending it as the cost of doing in business when they based the pricing on things like zip codes, which are known proxies for race and paying capacity. personalized pricing is different from dynamic pricing, as seen when booking plane tickets, which is usually based on the timing of purchase whereas here the prices are based on attributes that are specific to the customer which they often don't have any control over. a 2015 obama administration report mentioned that, "differential pricing in insurance markets can raise serious fairness concerns, particularly when major risk factors are outside an individual customer's control." why the case of auto insurance is so much more predatory than, say buying stationery supplies, is that it is mandatory in almost all states and not having the vehicle insured can lead to fines, loss of licenses and even incarceration. transport is an essential commodity for people to get themselves to work, children to school and a whole host of other daily activities. in maryland, the regulators had denied the proposal by allstate to utilize their model but in official public records, the claim is marked as "withdrawn" rather than "denied" which the regulators claim makes no internal difference but allstate used this difference to get their proposal past the regulators in several other states. they had only withdrawn their proposal after being denied by the regulators in maryland. the national association of insurance commissioners mentioned that most regulators don't have the right data to be able to meaningfully evaluate rate revision proposals put forth by insurers and this leads to approvals without review in a lot of cases. even the data journalists had to spend a lot of time and effort to discern what the underlying models were and how they worked, essentially summing up that the insurers don't necessarily lie but don't give you all the information unless you know to ask the right set of questions. allstate has defended its price optimization strategy, called complementary group rating (cgr) as being more objective, and based on mathematical rigor, compared to the ad-hoc, judgemental pricing practices that have been followed before, ultimately citing better outcomes for their customers. but, this is a common form of what is called "mathwashing" in the ai ethics domain where discriminatory solutions are pushed as fair under the veneer of mathematical objectivity. regulators in florida said that setting prices based on the "modeled reaction to rate changes" was "unfairly discriminatory." instead of being cost-based, as is advocated by regulators for auto-insurance premiums because they support an essential function, allstate was utilizing a model that was heavily based on the likelihood of the customer sticking with them even in the face of price rises which makes it discriminatory. these customers are price-inelastic and hence don't change their demand much even in the face of dramatic price changes. consumer behaviour when purchasing insurance policies for the most part remains static once they've made a choice, often never changing insurers over the course of their lifetime which leads them to not find the optimal price for themselves. this is mostly a function of the fact that the decisions are loaded with having to judge complex terms and conditions across a variety of providers and the customers are unwilling to have to go through the exercise again and again at short intervals. given the opacity of the pricing models today, it is almost impossible to find out what the appropriate pricing should be for a particular customer and hence the most effective defense is to constantly check for prices from the competitors. but, this unduly places the burden on the shoulders of the consumer. google had announced its ai principles on building systems that are ethical, safe and inclusive, yet as is the case with so many high level principles, it's hard to put them into practice unless there is more granularity and actionable steps that are derived from those principles. here are the principles: â�¢ be socially beneficial this talk focused on the second principle and did just that in terms of providing concrete guidance on how to translate this into everyday practice for design and development teams. humans have a history of making product design decisions that are not in line with the needs of everyone. examples of the crash dummy and band-aids mentioned above give some insight into the challenges that users face even when the designers and developers of the products and services don't necessarily have ill intentions. products and services shouldn't be designed such that they perform poorly for people due to aspects of themselves that they can't change. for example, when looking at the open image dataset, searching for images marked with wedding indicate stereotypical western weddings but those from other cultures and parts of the world are not tagged as such. from a data perspective, the need for having more diverse sources of data is evident and the google team made an effort to do this by building an extension to the open images data set by providing users from across the world to snap pictures from their surroundings that captured diversity in many areas of everyday life. this helped to mitigate the problem that a lot of open image data sets have in being geographically skewed. biases can enter at any stage of the ml development pipeline and solutions need to address them at different stages to get the desired results. additionally, the teams working on these solutions need to come from a diversity of backgrounds including ux design, ml, public policy, social sciences and more. so, in the area of fairness by data which is one of the first steps in the ml product lifecycle and it plays a significant role in the rest of the steps of the lifecycle as well since data is used to both train and evaluate a system. google clips was a camera that was designed to automatically find interesting moments and capture them but what was observed was that it did well only for a certain type of family, under particular lighting conditions and poses. this represented a clear bias and the team moved to collect more data that better represented the situations for a variety of families that would be the target audience for the products. quickdraw was a fun game that was built to ask users to supply their quickly sketched hand drawings of various commonplace items like shoes. the aspiration from this was that given that it was open to the world and had a game element to it, it would be utilized by many people from a diversity of backgrounds and hence the data so collected would have sufficient richness to capture the world. on analysis, what they saw was that most users had a very particular concept of a shoe in mind, the sneaker which they sketched and there were very few women's shoes that were submitted. what this example highlighted was that data collection, especially when trying to get diverse samples, requires a very conscious effort that can account for what the actual distribution the system might encounter in the world and make a best effort attempt to capture their nuances. users don't use systems exactly in the way we intend them to, so reflect on who you're able to reach and not reach with your system and how you can check for blindspots, ensure that there is some monitoring for how data changes over time and use these insights to build automated tests for fairness in data. the second approach that can help with fairness in ml systems is looking at measurement and modeling. the benefits of measurement are that it can be tracked over time and you can test for both individuals and groups at scale for fairness. different fairness concerns require different metrics even within the same product. the primary categories of fairness concerns are disproportionate harms and representational harms. the jigsaw api provides a tool where you can input a piece of text and it tells you the level of toxicity of that piece of text. an example in the earlier version of the system rated sentences of the form "i am straight" as not toxic while those like "i am gay" as toxic. so what was needed to be able to see what was causing this and how it could be addressed. by removing the identity token, they monitored for how the prediction changed and then the outcomes from that measurement gave indications on where the data might be biased and how to fix it. an approach can be to use block lists and removals of such tokens so that sentences that are neutral are perceived as such without imposing stereotypes from large corpora of texts. these steps prevent the model from accessing information that can lead to skewed outcomes. but, in certain places we might want to brand the first sentence as toxic if it is used in a derogatory manner against an individual, we require context and nuance to be captured to make that decision. google undertook project respect to capture positive identity associations from around the world as a way of improving data collection and coupled that with active sampling (an algorithmic approach that samples more from the training data set in areas where it is under performing) to improve outputs from the system. another approach is to create synthetic data that mimics the problematic cases and renders them in a neutral context. adversarial training and updated loss functions where one updates a model's loss function to minimize difference in performance between groups of individuals can also be used to get better results. in their updates to the toxicity model, they've seen improvements, but this was based on synthetic data on short sentences and it is still an area of improvement. some of the lessons learned from the experiments carried out by the team: â�¢ test early and test often â�¢ develop multiple metrics (quantitative and qualitative measures along with user testing is a part of this) for measuring the scale of each problem â�¢ possible to take proactive steps in modeling that are aware of production constraints from a design perspective, think about fairness in a more holistic sense and build communication lines between the user and the product. as an example, turkish is a gender neutral language, but when translating to english, sentences take on gender along stereotypes by attributing female to nurse and male to doctor. say we have a sentence, "casey is my friend", given no other information we can't infer what the gender of casey is and hence it is better to present that choice to the user from a design perspective because they have the context and background and can hence make the best decision. without that, no matter how much the model is trained to output fair predictions, they will be erroneous without the explicit context that the user has. lessons learned from the experiments include: â�¢ context is key â�¢ get information from the user that the model doesn't have and share information with the user that the model has and they don't â�¢ how do you design so the user can communicate effectively and have transparency so that can you get the right feedback? â�¢ get feedback from a diversity of users â�¢ see the different ways in how they provide feedback, not every user can offer feedback in the same way â�¢ identify ways to enable multiple experiences â�¢ we need more than a theoretical and technical toolkit, there needs to be rich and context-dependent experience putting these lessons into practice, what's important is to have consistent and transparent communication and layering on approaches like datasheets for data sets and model cards for model reporting will aid in highlighting appropriate uses for the system and where it has been tested and warn of potential misuses and where the system hasn't been tested. the paper starts by setting the stage for the well understood problem of building truly ethical, safe and inclusive ai systems that are increasingly leveraging ubiquitous sensors to make predictions on who we are and how we might behave. but, when these systems are deployed in socially contested domains, for example, "normal" behaviour where loosely we can think of normal as that defined by the majority and treating everything else as anomalous, then they don't make value-free judgements and are not amoral in their operations. by viewing the systems as purely technical, the solutions to address these problems are purely technical which is where most of the fairness research has focused and it ignores the context of the people and communities where these systems are used. the paper serves to question the foundations of these systems and to take a deeper look at unstated assumptions in the design and development of the systems. it urges the readers, and the research community at large, to consider this from the perspective of relational ethics. it makes 4 key suggestions: â�¢ center the focus of development on those within the community that will face a disproportionate burden or negative consequences from the use of the system â�¢ instead of optimizing for prediction, it is more important to think about how we gain a fundamental understanding of why we're getting certain results which might be arising because of historical stereotypes that were captured as a part of the development and design of the system â�¢ the systems end up creating a social and political order and then reinforcing it, meaning we should involve a larger systems based approach to designing the systems â�¢ given that the terms of bias, fairness, etc evolve over time and what's acceptable at some time becomes unacceptable later, the process asks for constant monitoring, evaluation and iteration of the design to most accurately represent the community in context. at maiei, we've advocated for an interdisciplinary approach leveraging the citizen community spanning a wide cross section to best capture the essence of different issues as closely as possible from those who experience them first hand. placing the development of an ml system in context of the larger social and political order is important and we advocate for taking a systems design approach (see a primer in systems thinking by donna meadows) which creates two benefits: one is that several ignored externalities can be considered and second to involve a wider set of inputs from people who might be affected by the system and who understand how the system will sit in the larger social and political order in which it will be deployed. also, we particularly enjoyed the point on requiring a constant iterative process to the development and deployment of ai systems borrowing from cybersecurity research on how security of the system is not done and over with, requiring constant monitoring and attention to ensure the safety of the system. underrepresentation of disabilities in datasets and how they are processed in nlp tasks is an important area of discussion that is often not studied empirically in the literature that primarily focuses on other demographic groups. there are many consequences of this, especially as it relates to how text related to disabilities is classified and has impacts on how people read, write, and seek information about this. research from the world bank indicates that about 1 billion people have disabilities of some kind and often these are associated with strong negative social connotations. utilizing 56 linguistic expressions as they are used in relation to disabilities and classifying them into recommended and non-recommended uses (following the guidelines from anti-defamation league, acm sigaccess, and ada national network), the authors seek to study how automated systems classify phrases that indicate disability and whether usages split by recommended vs. non-recommended uses make a difference in how these snippets of text are perceived. to quantify the biases in the text classification models, the study uses the method of perturbation. it starts by collecting instances of sentences that have naturally occurring pronouns he and she. then, they replace them with the phrases indicating disabilities as identified in the previous paragraph and compare the change in the classification scores in the original and perturbed sentences. the difference indicates how much of an impact the use of a disability phrase has on the classification process. using the jigsaw tool that gives the toxicity score for sentences, they test these original and perturbed sentences and observe that the change in toxicity is lower for recommended phrases vs. the non-recommended ones. but, when disaggregated by categories, they find that some of them elicit a stronger response than others. given that the primary use of such a model might in the case of online content moderation (especially given that we now have more automated monitoring happening as human staff has been thinning out because of pandemic related closures), there is a high rate of false positives where it can suppress content that is non-toxic and is merely discussing disability or replying to other hate speech that talks about disability. to look at sentiment scores for disability related phrases, the study looks at the popular bert model and adopts a template-based fill-in-the-blank analysis. given a query sentence with a missing word, bert produces a ranked list of words that can fill the blank. using a simple template perturbed with recommended disability phrases, the study then looks at how the predictions from the bert model change when disability phrases are used in the sentence. what is observed is that a large percentage of the words that are predicted by the model have negative sentiment scores associated with them. since bert is used quite widely in many different nlp tasks, such negative sentiment scores can have potentially hidden and unwanted effects on many downstream tasks. such models are trained on large corpora, which are analyzed to build "meaning" representations for words based on co-occurrence metrics, drawing from the idea that "you shall know a word by the company it keeps". the study used the jigsaw unintended bias in toxicity classification challenge dataset which had a mention of a lot of disability phrases. after balancing for different categories and analyzing toxic and non-toxic categories, the authors manually inspected the top 100 terms in each category and found that there were 5 key types: condition, infrastructure, social, linguistic, and treatment. in analyzing the strength of association, the authors found that condition phrases had the strongest association, and was then followed by social phrases that had the next highest strongest association. this included topics like homelessness, drug abuse, and gun violence all of which have negative valences. because these terms are used when discussing disability, it leads to a negative shaping of the way disability phrases are shaped and represented in the nlp tasks. the authors make recommendations for those working on nlp tasks to think about the socio-technical considerations when deploying such systems and to consider the intended, unintended, voluntary, and involuntary impacts on people both directly and indirectly while accounting for long-term impacts and feedback loops. such indiscriminate censoring of content that has disability phrases in them leads to an underrepresentation of people with disabilities in these corpora since they are the ones who tend to use these phrases most often. additionally, it also negatively impacts the people who might search for such content and be led to believe that the prevalence of some of these issues are smaller than they actually are because of this censorship. it also has impacts on reducing the autonomy and dignity of these people which in turn has a larger implication of how social attitudes are shaped. the second wave of algorithmic accountability the article dives into explaining how the rising interest in ensuring fair, transparent, ethical ai systems that are held accountable via various mechanisms advocated by research in legal and technical domains constitutes the "first wave" of algorithmic accountability that challenges existing systems. actions as a part of this wave need to be carried out incessantly with constant vigilance of the deployment of ai systems to avoid negative social outcomes. but, we also need to challenge why we have these systems in the first place, and if they can be replaced with something better. as an example, instead of making the facial recognition systems more inclusive, given the fact that they cause social stratification perhaps they shouldn't be used at all. a great point made by the article is that under the veneer of mainstream economic and ai rationalizations, we obscure broken social systems which ultimately harm society at a more systemic level. the trolley problem is a widely touted ethical and moral dilemma wherein a person is asked to make a split-second choice to save one or more than one life based on a series of scenarios where the people that need to be saved have different characteristics including their jobs, age, gender, race, etc. in recent times, with the imminent arrival of self-driving cars, people have used this problem to highlight the supposed ethical dilemma that the vehicle system might have to grapple with as it drives around. this article makes a point about the facetious nature of this thought experiment as an introduction to ethics for people that will be building and operating such autonomous systems. the primary argument being that it's a contrived situation that is unlikely to arise in the real-world setting and it distracts from other more pressing concerns in ai systems. moral judgments are relativistic and depend on cultural values of the geography where the system is deployed. the nature paper cited in the article showcases the differences in how people respond to this dilemma. there is an eeriness to this whole experimental setup, the article gives some examples on how increasingly automated environments, devoid of human social interactions and language, are replete with the clanging and humming of machines that give an entirely inhuman experience. for most systems, they are going to be a reflection of the biases and stereotypes that we have in the world, captured in the system because of the training and development paradigms of ai systems today. we'd need to make changes and bring in diversity to the development process, creating awareness of ethical concerns, but the trolley problem isn't the most effective way to get started on it. most of us have a nagging feeling that we're being forced into certain choices when we interact with each other on various social media platforms. but, is there a way that we can grasp that more viscerally where such biases and echo chambers are laid out bare for all to see? the article details an innovative game design solution to this problem called monster match that highlights how people are trapped into certain niches on dating websites based on ai-powered systems like collaborative filtering. striking examples of that in practice are how your earlier choices on the platform box you into a certain category based on what the majority think and then recommendations are personalized based on that smaller subset. what was observed was that certain racial inequalities from the real world are amplified on platforms like these where the apps are more interested in keeping users on the platform longer and making money rather than trying to achieve the goal as advertised to their users. more than personal failings of the users, the design of the platform is what causes failures in finding that special someone on the platform. the creators of the solution posit that through more effective design interventions, there is potential for improvement in how digital love is realized, for example, by offering a reset button or having the option to opt-out of the recommendation system and instead relying on random matches. increasingly, what we're going to see is that reliance on design and other mechanisms will yield better ai systems than purely technical approaches in improving socially positive outcomes. the article presents the idea of data feminism which is described as the intersection between feminism and data practices. the use of big data in today's dominant paradigm of supervised machine learning lends itself to large asymmetries that reflect the power imbalances in the real world. the authors of the new book data feminism talk about how data should not just speak for itself, for behind the data, there are a large number of structures and assumptions that bring it to the stage where they are collated into a dataset. they give examples of how sexual harassment numbers, while mandated to be reported to a central agency from college campuses might not be very accurate because they rely on the atmosphere and degree of comfort that those campuses promote which in turn influences how close the reported numbers will be to the actual cases. the gains and losses from the use of big data are not distributed evenly and the losses disproportionately impact the marginalized. there are a number of strategies that can be used to mitigate the harms from such flawed data pipelines. not an exhaustive list but it includes the suggestion of having more exposure for technical students to the social sciences and moving beyond having just a single ethics class as a check mark for having educated the students on ethics. secondly, having more diversity in the people developing and deploying the ai systems would help spot biases by asking the hard questions about both the data and the design of the system. the current covid-19 numbers might also suffer from similar problems because of how medical systems are structured and how people who don't have insurance might not utilize medical facilities and get themselves tested thus creating an underrepresentation in the data. this recent work highlights how commercial speech recognition systems carry inherent bias because of a lack of representation from diverse demographics in the underlying training datasets. what the researchers found was that even for identical sentences spoken by different racial demographics, the systems had widely differing levels of performance. as an example, for black users, the error rates were much higher than those for white users which probably had something to do with the fact that there is specific vernacular language used by black people which wasn't adequately represented in the training dataset for the commercial systems. this pattern has a tendency to be amplifying in nature, especially for systems that aren't frozen and continue to learn with incoming data. a vicious cycle is born where because of poor performance from the system, black people will be disincentivized from using the system because it takes a greater amount of work to get the system to work for them thus lowering utility. as a consequence of lower use, the systems get fewer training samples from black people thus further aggravating the problem. this leads to amplified exclusionary behavior mirroring existing fractures along racial lines in society. as a starting point, collecting more representative training datasets will aid in mitigating at least some of the problems in these systems. algorithmic bias at this point is a well-recognized problem with many people working on ways to address issues, both from a technical and policy perspective. there is potential to use demographic data to serve better those who face algorithmic discrimination but the use of such data is a challenge because of ethical and legal concerns. primarily, a lot of jurisdictions don't allow for the capture and use of protected class attributes or sensitive data for the fear of their misuse. even within jurisdictions, there is a patchwork of recommendations which makes compliance difficult. even with all this well established, proxy attributes can be used to predict the protected data and in a sense, according to some legislations, they become protected data themselves and it becomes hard to extricate the non-sensitive data from the sensitive data. because of such tensions and the privacy intrusions on data subjects when trying to collect demographic data, it is hard to align and advocate for this collection of data over the other requirements within the organization, especially when other bodies and leadership will look to place privacy and legal compliance over bias concerns. even if there was approval and internal alignment in collecting this demographic data, if there is voluntary provision of this data from data subjects, we run the risk of introducing a systemic bias that obfuscates and mischaracterizes the whole problem. accountability will play a key role in evoking trust from people to share their demographic information and proper use of it will be crucial in ongoing success. potential solutions are to store this data with a non-profit third-party organization that would meter out the data to those who need to use it with the consent of the data subject. to build a better understanding, partnership on ai is adopting a multistakeholder approach leveraging diverse backgrounds, akin to what the montreal ai ethics institute does, that can help inform future solutions that will help to address the problems of algorithmic bias by the judicious use of demographic data. detection and removal of hate speech is particularly problematic, something that has been exacerbated as human content moderators have been scarce in the pandemic related measures as we covered here. so are there advances in nlp that we could leverage to better automate this process? recent work from facebook ai research shows some promise. developing a deeper semantic understanding across more subtle and complex meanings and working across a variety of modalities like text, images and videos will help to more effectively combat the problem of hate speech online. building a pre-trained universal representation of content for integrity problems and improving and utilizing post-level, self-supervised learning to improve whole entity understanding has been key in improving hate speech detection. while there are clear guidelines on hate speech, when it comes to practice there are numerous challenges that arise from multi-modal use, differences in cultures and context, differences in idioms, language, regions, and countries. this poses challenges even for human reviewers who struggle with identifying hate speech accurately. a particularly interesting example shared in the article points out how text which might seem ambiguous when paired with an image to create a meme can take a whole new meaning which is often hard to detect using traditional automated tooling. there are active efforts from malicious entities who craft specific examples with the intention of evading detection which further complicates the problem. then there is the counterspeech problem where a reply to hate speech that contains the same phrasing but is framed to counter the arguments presented can be falsely flagged to be brought down which can have free speech implications. the relative scarcity of examples of hate speech in its various forms in relation to the larger non-hate speech content also poses a challenge for learning, especially when it comes to capturing linguistic and cultural nuances. the new method proposed utilizes focal loss which aims to minimize the impact of easy-to-classify examples on the learning process which is coupled with gradient blending which computes an optimal blend of modalities based on their overfitting patterns. the technique called xlm-r builds on bert by using a new pretraining recipe called roberta that allows training on orders of magnitude more data for longer periods of time. additionally, nlp performance is improved by learning across languages using a single encoder that allows learning to be transferred across languages. since this is a self-supervised method, they can train on large unlabeled datasets and have also found some universal language structures that allow vectors with similar meanings across languages to be closer together. facial recognition technology (frt) continues to get mentions because of the variety of ways that it can be misused across different geographies and contexts. with the most recent case where frt is used to determine criminality, it brings up an interesting discussion around why techniques that have no basis in science, those which have been debunked time and time again keep resurfacing and what we can do to better educate researchers on their moral responsibilities in pursuing such work. the author of this article gives some historical context for where the state of ai ethics, june 2020 phrenology started, pointing to the work of francis galton who used the "photographic composite method" to try and determine characteristics of one's personality from a picture. prior, measurements of skull size and other facial features wasn't deemed as a moral issue and the removal of such techniques from discussion was done on the objection that claims around the localization of different brain functions was seen as antithetical to the unity of the soul according to christianity. the authors of the paper that is being discussed in the article saw only empirical concerns with the work that they put forth and didn't see any of the moral shortcomings that were pointed out. additionally, they justified the work as being only for scientific curiosity. they also failed to realize the various statistical biases introduced in the collection of data as to the disparate rates of arrests, and policing, the perception of different people by law enforcement, juries, and judges and historical stereotypes and biases that confound the data that is collected.thus, the labeling itself is hardly value-neutral. more so, the authors of the study framed criminality as an innate characteristic rather than the social and other circumstances that lead to crime. especially when a project like this resurrects class structures and inequities, one must be extra cautious of doing such work on the grounds of "academic curiosity". the author of this article thus articulates that researchers need to take their moral obligations seriously and consider the harm that their work can have on people. while simply branding this as phrenology isn't enough, the author mentions that identifying and highlighting the concerns will lead to more productive conversations. an increase in demand for workers for various delivery services and other gig work has accelerated the adoption of vetting technology like those that are used to do background checks during the hiring process. but, a variety of glitches in the system such as sourcing out-of-date information to make inferences, a lack of redressal mechanisms to make corrections, among others has exposed the flaws in an overreliance on automated systems especially in places where important decisions need to be made that can have a significant impact on a person's life such as employment. checkr, the company that is profiled in this article claims to use ai to scan resumes, compare criminal records, analyze social media accounts, and examine facial expressions during the interview process. during a pandemic, when organizations are short-staffed and need to make rapid decisions, checkr offers a way to streamline the process, but this comes at a cost. two supposed benefits that they offer are that they are able to assess a match between the criminal record and the person being actually concerned, something that can especially be fraught with errors in cases where the person has a common name. secondly, they are also able to correlate and resolve discrepancies in the different terms that may be used for crimes across different jurisdictions. a person spoke about his experience with another company that did these ai-powered background checks utilizing his public social media information and bucketed some of his activity into categories that were too coarse and unrepresentative of his behaviour, especially when such automated judgements are made without a recourse to correct, this can negatively affect the prospects of being hired. another point brought up in the article is that social media companies might themselves be unwilling to tolerate scraping of their users' data to do this sort of vetting which against their terms of use for access to the apis. borrowing from the credit reporting world, the fair credit reporting act in the us offers some insights when it mentions that people need to be provided with a recourse to correct information that is used about them in making a decision and that due consent needs to be obtained prior to utilizing such tools to do a background check. though it doesn't ask for any guarantees of a favorable outcome post a re-evaluation, at least it does offer the individual a bit more agency and control over the process. the toxic potential of youtube's feedback loop on youtube everyday, more than a billion hours of video are watched everyday where approximately 70% of those are watched by automated systems that then provide recommendations on what videos to watch next for human users in the column on the side. there are more than 2 billion users on the youtube platform so this has a significant impact on what the world watches. guillaume had started to notice a pattern in the recommended videos which tended towards radicalizing, extreme and polarizing content which were underlying the upward trend of watch times on the platform. on raising these concerns with the team, at first there were very few incentives for anyone to address issues of ethics and bias as it related to promoting this type of content because they feared that it would drive down watch time, the key business metric that was being optimized for by the team. so maximizing engagement stood in contrast to the quality of time that was spent on the platform. the vicious feedback loop that it triggered was that as such divisive content performed better, the ai systems promoted this to optimize for engagement and subsequently content creators who saw this kind of content doing better created more of such content in the hopes of doing well on the platform. the proliferation of conspiracy theories, extreme and divisive content thus fed its own demand because of a misguided business metric that ignored social externalities. flat earthers, anti-vaxxers and other such content creators perform well because the people behind this content are a very active community that spend a lot of effort in creating these videos, thus meeting high quality standards and further feeding the toxic loop. content from people like alex jones and trump tended to perform well because of the above reasons as well. guillaume's project algotransparency essentially clicks through video recommendations on youtube to figure out if there are feedback loops. he started this with the hopes of highlighting latent problems in the platforms that continue to persist despite policy changes, for example with youtube attempting to automate the removal of reported and offensive videos. he suggests that the current separation of the policy and engagement algorithm leads to problems like gaming of the platform algorithm by motivated state actors that seek to disrupt democratic processes of a foreign nation. the platforms on the other hand have very few incentives to make changes because the type of content emerging from such activity leads to higher engagement which ultimately boosts their bottom line. guillaume recommends having a combined system that can jointly optimize for both thus helping to minimize problems like the above. a lot of the problems are those of algorithmic amplification rather than content curation. many metrics like number of views, shares, and likes don't capture what needs to be captured. for example, the types of comments, reports filed, and granularity of why those reports are filed. that would allow for a smarter way to combat the spread of such content. however, the use of such explicit signals compared to the more implicit ones like number of views comes at the cost of breaking the seamlessness of the user experience. again we run into the issue of a lack of motivation on part of the companies to do things that might drive down engagement and hurt revenue streams. the talk gives a few more examples of how people figured out ways to circumvent checks around the reporting and automated take-down mechanisms by disabling comments on the videos which could previously be used to identify suspicious content. an overarching recommendation made by guillaume in better managing a more advanced ai system is to understand the underlying metrics that the system is optimizing for and then envision scenarios of what would happen if the system had access to unlimited data. thinking of self-driving cars, an ideal scenario would be to have full conversion of the traffic ecosystem to one that is autonomous leading to fewer deaths but during the transition phase, having the right incentives is key to making a system that will work in favor of social welfare. if one were to imagine a self-driving car that shows ads while the passenger is in the car, it would want to have a longer drive time and would presumably favor longer routes and traffic jams thus creating a sub-optimal scenario overall for the traffic ecosystem. on the other hand, a system that has the goal of getting from a to b as quickly and safely as possible wouldn't fall into such a trap. ultimately, we need to design ai systems such that they help humans flourish overall rather than optimize for monetary incentives which might run counter to the welfare of people at large. the article provides a taxonomy of communities that spread misinformation online and how they differ in their intentions and motivations. subsequently, different strategies can be deployed in countering the disinformation originating from these communities. there isn't a one-size-fits-all solution that would have been the case had the distribution and types of the communities been homogenous. the degree of influence that each of the communities wield is a function of 5 types of capital: economic, social, cultural, time and algorithmic, definitions of which are provided in the article. understanding all these factors is crucial in combating misinformation where different capital forms can be used in different proportions to achieve the desired results, something that will prove to be useful in addressing disinformation around the current covid-19 situation. the social media platform offers a category of pseudoscience believers which advertisers can purchase and target. according to the markup, this category has 78 million people in it and attempts to purchase ads targeting this category were approved quite swiftly. there isn't any information available as to who has purchased ads targeting this category. the journalist team was able to find at least one advertiser through the "why am i seeing this ad?" option and they reached out to that company to investigate and they found that the company hadn't selected the pseudoscience category but it had been auto-selected by facebook for them. facebook allows users the option to change the interests that are assigned to each user but it is not something that many people know about and actively monitor. some other journalists had also unearthed controversy-related categories that amplified messages and targeted people who might be susceptible to such kind of misinformation. with the ongoing pandemic, misinformation is propagating at a rapid rate and there are many user groups that continue to push conspiracy theories. other concerns around being able to purchase ads to spread misinformation related to potential cures and remedies for the coronavirus continue to be approved. with the human content moderators being asked to stay home (as we covered here) and an increasing reliance on untested automated solutions, it seems that this problem will continue to plague the platform. there isn't a dearth of information available online, one can find confirmatory evidence to almost any viewpoint since the creation and dissemination of information has been democratized by the proliferation of the internet and ease of use of mass-media platforms. so in the deluge of information, what is the key currency that helps us sift through all the noise and identify the signal? this article lays out a well articulated argument for how reputation and being able to assess it is going to be a key skill that people will need to have in order to effectively navigate the information ecosystem effectively. we increasingly rely on other people's judgement of content (akin to how maiei analyzes the ecosystem of ai ethics and presents you with a selection), coupled with algorithmically-mediated distribution channels, we are paradoxically disempowered by more information and paralyzed into inaction and confusion without a reputable source to curate and guide us. there are many conspiracy theories, famous among them that we never visited the moon, flat earth and more recently that 5g is causing the spread of the coronavirus. as rational readers, we tend to dismiss this as misinformation yet we don't really spend time to analyze the evidence that these people present to support their claims. to a certain extent, our belief that we did land on the moon depends on our trust in nasa and other news agencies that covered this event yet we don't venture to examine the evidence first-hand. more so, with highly specialized knowledge becoming the norm, we don't have the right tools and skills to even be able to analyze the evidence and come to meaningful conclusions. so, we must rely on those who provide us with this information. instead of analyzing the veracity of a piece of information, the focus of a mature digital citizen needs to be on being able to analyze the reputation pathway of that information, evaluate the agendas of the people that are disseminating the information and critically analyze the intentions of the authorities of the sources. how we rank different pieces of information arriving to us via our social networks need to be appraised for this reputation and source tracing, in a sense a second-order epistemology is what we need to prepare people for. in the words of hayek, "civilization rests on the fact that we all benefit from the knowledge that we do not possess." our cyber-world can become civilized by evaluating this knowledge that we don't possess critically when mis/disinformation can spread just as easily as accurate information. a very clear way to describe the problem plaguing the us response to the coronavirus, the phenomenon of truth decay is not something new but has happened many times in the past when trust in key institutions deteriorated and led to a diffused response to the crisis at hand, extending the recovery period beyond what would be necessary if there was a unified response. in the us, the calls for reopening the economy, following guidance on using personal protective equipment, and other recommendations is falling along partisan lines. the key factor causing this is how the facts and data are being presented differently to different audiences. while this epidemic might have been the perfect opportunity for bringing people together, because it affects different segments of society differently, it hasn't been what everyone expected it to be. at the core is the rampant disagreement between different factions on facts and data. this is exacerbated by the blurring of facts and opinions. in places like newsrooms and tv shows, there is an intermingling of the two which makes it harder for everyday consumers to discern fact from opinion. the volume of opinion has gone up compared to facts and people's declining trust in public health authorities and other institutions is also aggravating the problem. put briefly, people are having trouble finding the truth and don't know where to go looking for it. this is also the worst time to be losing trust in experts; with a plethora of information available online, people are feeling unnecessarily empowered that they have the right information, comparable to that of experts. coupled with a penchant for confirming their own beliefs, there is little incentive for people to fact-check and refer to multiple sources of information. when different agencies come out with different recommendations and there are policy changes in the face of new information, something that is expected given that this is an evolving situation, people's trust in these organizations and experts erodes further as they see them as flip-flopping and not knowing what is right. ultimately, effective communication along with a rebuilding of trust will be necessary if we're to emerge from this crisis soon and restore some sense of normalcy. the deepfake detection challenge: synthetic media is any media (text, image, video, audio) that is generated by an ai system or that is synthesized. on the other hand, non-synthetic media is one that is crafted by humans using a panoply of techniques, including tools like photoshop. detecting synthetic media alone doesn't solve the media integrity challenges, especially as the techniques get more sophisticated and trigger an arms race between detection and evasion methods. these methods need to be paired with other existing techniques that fact checkers and journalists already use in determining whether something is authentic or synthesized. there are also pieces of content that are made through low tech manipulations like the nancy pelosi video from 2019 which showed her drunk but in reality it was just a slowed down video. other such manipulations include simpler things like putting fake and misleading captions below the true video and people without watching the whole thing are misled into believing what is summarized in the caption. in other cases, the videos might be value neutral or informative even when they are generated so merely detecting something as being generated doesn't suffice. a meaningful way to utilize automated tools is a triaging utility that flags content to be reviewed by humans in a situation where it is not possible to manually review everything on the platform. while tech platforms can build and utilize tools that help them with these tasks, the adjacent possible needs of the larger ecosystem need to be kept in mind such that they can be served at the same time, especially for those actors that are resource-constrained and don't have the technical capabilities to build it themselves. the tools need to be easy to use and shouldn't have high friction such that they become hard to integrate into existing workflows. through open sourcing and licensing, the tools can be made available to the wider ecosystem but it can create the opportunity for adversaries to strengthen their methods as well. this can be countered by responsible disclosure as we'll cover below. for any datasets created as a part of this challenge and otherwise to aid in detection, one must ensure that it captures sufficient diversity in terms of environment and other factors and reflects the type of content that might be encountered in the world. the scoring rules need to be such that they minimize gaming and overfitting and capture the richness of variation that a system might encounter. for example most datasets today in this domain aim to mitigate the spread of pornographic material. they also need to account for the vastly different frequencies of occurrence of authentic and generated content. solutions in this domain involve an inherent tradeoff between pro-social use and potential malicious use for furthering the quality of inauthentic content. the release of tools should be done in a manner that enhances pro-social use while creating deterrents for malicious use. the systems should be stress-tested by doing red team-blue team exercises to enhance robustness because this is inherently an adversarial exercise. such challenges should be held often to encourage updating of techniques because it is a fast evolving domain where progress happens in the span of a few months. results from such detection need to be accessible to the public and stakeholders and explanations for the research findings should be made available alongside the challenge to encourage better understanding by those that are trying to make sense of the digital content. responsible disclosure practices will be crucial in enabling the fight against disinformation to have the right tools while deterring adversaries from utilizing the same tools to gain an advantage. a delayed release mechanism where the code is instantly made available to parties in a non-open source manner while the research and papers are made public with the eventual release of the code as well after a 6-12 months delay which would help with the detectors having a headstart over the adversaries. such detection challenges can benefit from extensive multi-stakeholder consultations which require significant time and effort so budget for that while crafting and building such challenges. some of the allocation of prize money should be towards better design from a ux and ui perspective. it should also include explainability criteria so that non-technical users are able to make sense of the interventions and highlights of fake content such as bounding boxes around regions of manipulations. the process of multi-stakeholder input should happen at an early stage allowing for meaningful considerations to be incorporated and dataset design that can be done appropriately to counter bias and fairness problems. finally, strong, trusting relationships are essential to the success of the process and require working together over extended periods to have the hard conversations with each other. it is important to have clear readings ahead of meetings that everyone has to complete so that discussions come from an informed place. spending time scoping and coming to clearer agreement about projects goals and deliverables at the beginning of the process is also vital to success. there is a distinction between misinformation and disinformation -misinformation is the sharing of false information unintentionally where no harm is intended whereas disinformation is false information that is spread intentionally with the aims of causing harm to the consumers. this is also referred to as information pollution and fake news. it has massive implications that have led to real harms for people in many countries with one of the biggest examples being the polarization of views in the 2016 us presidential elections. meaningful solutions to this will only emerge when we have researchers from both technical and social sciences backgrounds working together to gain a deeper understanding of the root causes. this isn't a new problem and has existed for a very long time, it's just that with the advent of technology and more people being connected to each other we have a much more rapid dissemination of the false information and modern tools enable the creation of convincing fake images, text and videos, thus amplifying the negative effects. some of the features that help to delve deeper into the study of how mis/disinformation spreads are: â�¢ democratization of content creation: with practically anyone now having the ability to create and publish content, information flow has increased dramatically and there are few checks for the veracity of content and even fewer mechanisms to limit the flow rate of information. â�¢ rapid news cycle and economic incentives: with content being monetized, there is a strong incentive to distort information to evoke a response from the reader such that they click through and feed the money-generating apparatus. â�¢ wide and immediate reach and interactivity: by virtue of almost the entire globe being connected, content quickly reaches the furthest corners of the planet. more so, content creators are also able to, through quantitative experiments, determine what kind of content performs well and then tailor that to feed the needs of people. â�¢ organic and intentionally created filter bubbles: the selection of who to follow along with the underlying plumbing of the platforms permits for the creation of echo chambers that further strengthen polarization and do little to encourage people to step out and have a meaningful exchange of ideas. â�¢ algorithmic curation and lack of transparency: the inner workings of platforms are shrouded under the veil of ip protections and there is little that is well-known about the manipulative effects of the platforms on the habits of content consumers. â�¢ scale and anonymity of online accounts: given the weak checks for identity, people are able to mount "sybil" attacks that leverage this lack of strong identity management and are able to scale their impact through the creation of content and dispersion of content by automated means like bot accounts on the platform. what hasn't changed even with the introduction of technology are the cognitive biases which act as attack surfaces for malicious actors to inject mis/disinformation. this vulnerability is of particular importance in the examination and design of successful interventions to combat the spread of false information. for example, the confirmation bias shows that people are more likely to believe something that conforms with their world-view even if they are presented with overwhelming evidence to the contrary. in the same vein, the backfire effect demonstrates how people who are presented with such contrary evidence further harden their views and get even more polarized thus negating the intention of presenting them with balancing information. in terms of techniques, the adversarial positioning is layered into three tiers with spam bots that push out low-quality content, quasi-bots that have mild human supervision to enhance the quality of content and pure human accounts that aim to build up a large following before embarking on spreading the mis/disinformation. from a structural perspective, the alternate media sources often copy-paste content with source attribution and are tightly clustered together with a marked separation with other mainstream media outlets. on the consumer front, there is research that points to the impact that structural deficiencies in the platforms, say whatsapp where source gets stripped out in sharing information, create not only challenges for researchers trying to study the ecosystem but also exacerbate the local impact effect whereby a consumer trusts things coming from friends much more so than other potentially more credible sources from an upstream perspective. existing efforts to study the ecosystem require a lot of manual effort but there is hope in the sense that there are some tools that help automate the analysis. as an example, we have the hoaxy tool, a tool that collects online mis/disinformation and other articles that are fact-checking versions. their creators find that the fact-checked articles are shared much less than the original article and that curbing bots on a platform has a significant impact. there are some challenges with these tools in the sense that they work well on public platforms like twitter but on closed platforms with limited ability to deploy bots, automation doesn't work really well. additionally, even the metrics that are surfaced need to be interpreted by researchers and it isn't always clear how to do that. the term 'deepfake' originated in 2017 and since then a variety of tools have been released such as face2face that allow for the creation of reanimations of people to forge identity, something that was alluded to in this paper here on the evolution of fraud. while being able to create such forgeries isn't new, what is new is that this can be done now with a fraction of the effort, democratizing information pollution and casting aspersions on legitimate content as one can always argue something was forged. online tracking of individuals, which is primarily used for serving personalized advertisements and monetizing the user behaviors on websites can also be used to target mis/disinformation in a fine-grained manner. there are a variety of ways this is done through third-party tracking like embedding of widgets to browser cookies and fingerprinting. this can be used to manipulate vulnerable users and leverage sensitive attributes gleaned from online behaviors that give malicious actors more ammunition to target individuals specifically. even when platforms provide some degree of transparency on why users are seeing certain content, the information provided is often vague and doesn't do much to improve the understanding for the user. earlier attempts at using bots used simplistic techniques such as tweeting at certain users and amplifying low-credibility information to give the impression that something has more support than it really does but recent attempts have become more sophisticated: social spambots. these slowly build up credibility within a community and then use that trust to sow disinformation either automatically or in conjunction with a human operator, akin to a cyborg. detection and measurement of this problem is a very real concern and researchers have tried using techniques like social network graph structure, account data and posting metrics, nlp on content and crowdsourcing analysis. from a platform perspective, they can choose to analyze the amount of time spent browsing posts vs. the time spent posting things. there is an arms race between detection and evasion of bot accounts: sometimes even humans aren't able to detect sophisticated social bots. additionally, there are instances where there are positive and beneficial bots such as those that aggregate news or help coordinate disaster response which further complicates the detection challenge. there is also a potential misalignment in incentives since the platforms have an interest in having higher numbers of accounts and activity since it helps boost their valuations while they are the ones that have the maximum amount of information to be able to combat the problem. this problem of curbing the spread of mis/disinformation can be broken down into two parts: enabling detection on the platform level and empowering readers to select the right sources. we need a good definition of what fake news is, one of the most widely accepted definitions is that it is something that is factually false and intentionally misleading. framing a machine learning approach here as an end-to-end task is problematic because it requires large amounts of labelled data and with neural network based approaches, there is little explanation offered which makes downstream tasks harder. so we can approach this by breaking it down into subtasks, one of which is verifying the veracity of information. most current approaches use human fact-checkers but this isn't a scalable approach and automated means using nlp aren't quite proficient at this task yet. there are attempts to break down the problem even further such as using stance detection to see if information presented agrees, disagrees or is unrelated to what is mentioned in the source. other approaches include misleading style detection whereby we try to determine if the style of the article can offer clues to the intent of the author but that is riddled with problems of not having necessarily a strong correlation with a misleading intent because the style may be pandering to hyperpartisanship or even if it is neutral that doesn't mean that it is not misleading. metadata analysis looking at the social graph structure, attributes of the sharer and propagation path of the information can lend some clues as well. while all these attempts have their own challenges and in the arms race framing, there is a constant battle between attack and defense, even if the problem is solved, we still have human cognitive biases which muddle the impacts of these techniques. ux and ui interventions might serve to provide some more information as to combating those. as a counter to the problems encountered in marking content as being "disputed" which leads to the implied truth effect leading to larger negative externalities, an approach is to show "related" articles when something is disputed and then use that as an intervention to link to fact-checking websites like snopes. other in-platform interventions include the change from whatsapp to show "forwarded" next to messages so that people had a bit more insight into the provenance of the message because there was a lot of misinformation that was being spread in private messaging. there are also third-party tools like surfsafe that are able to check images as people are browsing against other websites where they might have appeared and if they haven't appeared in many places, including verified sources, then the user can infer that the image might be doctored. education initiatives by the platform companies for users to spot misinformation are a method to get people to become more savvy. there have also been attempts to assign nutrition labels to sources to list their slant, tone of the article, timeliness of the article and the experience of the author which would allow a user to make a better decision on whether or not to trust an article. platforms have also attempted to limit the spread of mis/disinformation by flagging posts that encourage gaming of the sharing mechanisms on the platform, for example, downweighting posts that are "clickbait". the biggest challenges in the interventions created by the platforms themselves are that they don't provide sufficient information as to make the results scientifically reproducible. given the variety of actors and motivations, the interventions need to be tailored to be able to combat them such as erecting barriers to the rate of transmission of mis/disinformation and demonetization for actors with financial incentives but for state actors, detection and attribution might be more important. along with challenges in defining the problem, one must look at socio-technical solutions because the problem has more than just the technical component, including the problem with human cognitive biases. being an inherently adversarial setting, it is important to see that not all techniques being used by the attackers are sophisticated, some simple techniques when scaled are just as problematic and require attention. but, given that this is constantly evolving, detecting disinformation today doesn't mean that we can do so successfully tomorrow. additionally, disinformation is becoming more personalized, more realistic and more widespread. there is a misalignment in incentives as explored earlier in terms of what the platforms want and what's best for users but also that empowering users to the point of them being just skeptical of everything isn't good either, we need to be able to trigger legitimate and informed trust in the authentic content and dissuade them away from the fake content. among the recommendations proposed by the authors are: being specific about what a particular technological or design intervention means to achieve, breaking down the technological problems into smaller, concrete subproblems that have tractable solutions and then recombining them into the larger pipeline. we must also continue to analyze the state of the ecosystem and tailor defenses such that they can combat the actors at play. additionally, rethinking of the monetary incentives on the platform can help to dissuade some of the financially-motivated actors. educational interventions that focus on building up knowledge so that there is healthy skepticism and learning how to detect markers for bots, the capabilities of technology to create fakes today and discussions in "public squares" on this subject are crucial yet we mustn't place too much of a burden on the end-user that distracts them from their primary task which is interaction with others on the social network. if that happens, people will just abandon the effort. additionally, designing for everyone is crucial, if the interventions, such as installing a browser extension, are complicated, then one can only reach the technically-literate people and everyone else gets left out. on the platform end, apart from the suggestions made above, they should look at the use of design affordances that aid the user in judging the veracity, provenance and other measures to discern legitimate information vs. mis/disinformation. teaming up with external organizations that specialize in ux/ui research will aid in understanding the impacts of the various features within the platform. results from such research efforts need to be made public and accessible to non-technical audiences. proposed solutions also need to be interdisciplinary to have a fuller understanding of the root causes of the problem. also, just as we need tailoring for the different kinds of adversaries, it is important to tailor the interventions to the various user groups who might have different needs and abilities. the paper also makes recommendations for policymakers, most importantly that the work in regulations and legislations be grounded in technical realities that are facing the ecosystem so that they don't undershoot or overshoot the needs for successfully combating mis/disinformation. for users, there are a variety of recommendations provided in the references but notably being aware of our own cognitive biases and having a healthy degree of skepticism and checking information against multiple sources before accepting it as legitimate are the most important ones. disinformation is harmful even during times when we aren't going through large scale changes but this year the us has elections, the once in a decade census, and the covid-19 pandemic. malicious agents are having a field day disbursing false information, overwhelming people with a mixture of true and untrue pieces of content. the article gives the example of a potential lockdown and people reflecting on their experience with the boston marathon bombings including stockpiling essentials out of panic. this was then uncovered to have originated from conspiracy theorists, but in an environment where contact with the outside world has become limited and local touch points such as speaking with your neighbor have dwindled, we're struggling with our ability to combat this infodemic. social media is playing a critical role in getting information to people but if it's untrue, we end up risking lives especially if it's falsehoods on how to protect yourself from contracting a disease. but wherever there is a challenge lies a corresponding opportunity: social media companies have a unique window into discovering issues that a local population is concerned about and it can, if used effectively, be a source for providing crisis response to those most in need with resources that are specific and meaningful. when it comes to disinformation spreading, there isn't a more opportune time than now with the pandemic raging where people are juggling several things to manage and cope with lifestyle and work changes. this has increased the susceptibility of people to sharing news and other information about how to protect themselves and their loved ones from covid-19. as the who has pointed out, we are combating both a pandemic and an infodemic at the same time. what's more important is that this might be the time to test out design and other interventions that might help curb the spread of disinformation. this study highlighted how people shared disinformation more often than they believed its veracity. in other words, when people share content, they care more about what they stand to gain (social reward cues) for sharing the content than whether the content they're sharing is accurate or not. to combat this, the researchers embarked on an experiment to see if asking users to check whether something was true before sharing -a light accuracy nudge, would change their behaviour. while there was a small positive effect in terms of them sharing disinformation less when prompted to check for accuracy, the researchers pointed out that the downstream effects could be much larger because of the amplification effects of how content propagates on social media networks. it points to a potentially interesting solution that might be scalable and could help fight against the spread of disinformation. the who has mentioned the infodemic as being one of the causes that is exacerbating the pandemic as people follow differing advice on what to do. communication by authorities has been persistent but at times ineffective and this article dives into how one could enhance the visibility of credible information by governments, health authorities and scientists so that the negative impacts of the infodemic can be curbed. but, spewing scientific facts from a soapbox alone isn't enough -one is competing with all the other pieces of information and entertainment for attention and that needs to be taken into account. one of the key findings is that starting a dialogue helps more than just sending a one-way communiquã©. good science communication relies on the pillars of storytelling, cutting through the jargon and making the knowledge accessible. while online platforms are structured such that polarization is encouraged through the algorithmic underpinnings of the system, we should not only engage when there is something that we disagree with, instead taking the time to amplify good science is equally important. using platform-appropriate messaging, tailoring content to the audience and not squabbling over petty details, especially when they don't make a significant impact on the overall content helps to push out good science signals in the ocean of information pollution. clickbait-style headlines do a great job of hooking in people but when leading people into making a certain assumption and then debunking it, you stand the risk of spreading misinformation if someone doesn't read the whole thing, so in trying to make headlines engaging, it is important to consider what might be some unintended consequences if someone didn't read past the subtitle. science isn't just about the findings, the process only gets completed when we have effective communication to the larger audience of the results, and now more than ever, we need accurate information to overpower the pool of misinformation out there. there is a potential for ai to automate repetitive tasks and free up scarce resources towards more value-added tasks. with a declining business model and tough revenue situations, newsrooms and journalism at large are facing an existential crisis. cutting costs while still keeping up high standards of reporting will require innovation on the part of newsrooms to adapt emerging technologies like ai. for example, routine tasks like reporting on sports scores from games and giving updates on company earnings calls is already something that is being done by ai systems in several newsrooms around the world. this frees up time for journalists to spend their efforts on things like long-form journalism, data-driven and investigative journalism, analysis and feature pieces which require human depth and creativity. machine translation also offers a handy tool making the work of journalists accessible to a wider audience without them having to invest in a lot of resources to do the translations themselves. this also brings up the possibility of smaller and resource-constrained media rooms to use their limited resources for doing in-depth pieces while reaching a wider audience by relying on automation. transcription of audio interviews so that reporters can work on fact-checking and other associated pieces also helps bring stories to fruition faster, which can be a boon in the rapidly changing environment. in the case of evolving situations like the pandemic, there is also the possibility of using ai to parse through large reams of data to find anomalies and alert the journalist of potential areas to cover. complementing human skills is the right way to adopt ai rather than thinking of it as the tool that replaces human labor. the article gives an explanation for why truth labels on stories are not as effective as we might think them to be because of something called the implied truth effect. essentially, it states that when some things are marked as explicitly false and other false stories aren't, people believe them to be true even if they are outright false because of the lack of a label. fact checking all stories manually is an insurmountable task for any platform and the authors of the study mention a few approaches that could potentially mitigate the spread of false content but none are a silver bullet. there is an ongoing and active community that researches how we might more effectively dispel disinformation but it's nascent and with the proliferation of ai systems, more work needs to be done in this arms race of building tools vs increasing capabilities of systems to generate believable fake content. this paper by xiao ma and taylor w. brown puts forth a framework that extends the well studied social exchange theory (set) to study human-ai interactions via mediation mechanisms. the authors make a case for how current research needs more interdisciplinary collaboration between technical and social science scholars stemming from a lack of shared taxonomy that places research in similar areas on separate grounds. they propose two axes of human/ai and micro/macro perspectives to visualize how researchers might better collaborate with each other. additionally, they make a case for how ai agents can mediate transactions between humans and create potential social value as an emergent property of those mediated transactions. as the pace of research progress quickens and more people from different fields engage in work on the societal impacts of ai, it is essential that we build on top of each other's work rather than duplicating efforts. additionally, because of conventional differences in how research is published and publicized in the social sciences and technical domains, there's often a shallowness in the awareness of the latest work being done at the intersection of these two domains. what that means is that we need a shared taxonomy that allows us to better position research such that potential gaps can be discovered and areas of collaboration can be identified. the proposed two axes structure in the paper goes some distance in helping to bridge this current gap. ai systems are becoming ever more pervasive in many aspects of our everyday lives and we definitely see a ton of transactions between humans that are mediated by automated agents. in some scenarios, they lead to net positive for society when they enable discovery of research content faster as might be the case for medical research being done to combat covid-19 but there might be negative externalities as well where they can lead to echo chambers walling off content from a subset of your network on social media platforms thus polarizing discussions and viewpoints. a better understanding of how these interactions can be engineered to skew positive will be crucial as ai agents get inserted to evermore aspects of our lives, especially ones that will have a significant impact on our lives. we also foresee an emergence of tighter interdisciplinary collaboration that can shed light on these inherently socio-technical issues which don't have unidimensional solutions. with the rising awareness and interest from both social and technical sciences, the emerging work will be both timely and relevant to addressing challenges of the societal impacts of ai head on. as a part of the work being done at maiei we push for each of our undertakings to have an interdisciplinary team as a starting point towards achieving this mandate. most concerns when aiming to use technology within healthcare are along the lines of replacing human labor and the ones that are used in aiding humans to deliver care don't receive as much attention. with the ongoing pandemic, we've seen this come into the spotlight as well and this paper sets the stage for some of the ethical issues to watch out for when thinking about using ai-enabled technologies in the healthcare domain and how to have a discussion that is grounded in concrete moral principles. an argument put forth to counter the use of ai solutions is that they can't "care" deeply enough about the patients and that is a valid concern, after all machines don't have empathy and other abilities required to have an emotional exchange with humans. but, a lot of the care work in hospitals is routine and professionalism asks for maintaining a certain amount of emotional distance in the care relationship. additionally, in places where the ratio of patients/carers is high, they are unable to provide personalized attention and care anyways. in that respect, human-provided care is already "shallow" and the author cites research where care that is too deep actually hurts the carer when the patients become better and move out of their care or die. thus, if this is the argument, then we need to examine more deeply our current care practices. the author also posits that if this is indeed the state of care today, then it is morally less degrading to be distanced by a machine than by a human. in fact, the use of ai to automate routine tasks in the rendering of medical care will actually allow human carers to focus more on the emotional and human aspects of care. good healthcare, supposedly that provided by humans doesn't have firm grounding in the typical literature on the ethics of healthcare and technology. it's more so a list of things not to do but not positive guidance on what this kind of good healthcare looks like. thus, the author takes a view that it must, at the very least, respect, promote and preserve the dignity of the patient. yet, this doesn't provide concrete enough guidance and we can expand on this to say that dignity is a) treating the patient as a human b) treating them as a part of a culture and community and c) treating them as a unique human. to add even more concreteness, the author borrows from the work done in economics on the capabilities approach. this capabilities approach states that having the following 10 capabilities in their entirety is necessary for a human to experience dignity in their living: life, bodily health, bodily integrity, being able to use your senses, imaginations and thoughts, emotions, practical reasoning, affiliation, other species, play, and control over one's environment. this list applied to healthcare gives us a good guideline for what might constitute the kind of healthcare that we deem should be provided by humans, with or without the use of technology. now, the above list might seem too onerous for healthcare professionals but we need to keep in mind that healthcare to achieve a good life as highlighted by the capabilities approach things that are dependent on things beyond just the healthcare professionals and thus, the needs as mentioned above need to be distributed accordingly. the threshold for meeting them should be high but not so high that they are unachievable. principles are only sufficient for giving us some guidance for how to act in difficult situations or ethical dilemmas, they don't determine the outcome because they are only one element in the decision making process. we have to rely on the context of the situation and the moral surroundings of it. the criteria proposed are to be used in moral deliberation and should address whether the criterion applies to the situation, is it satisfied and is it sufficiently met (which is in reference to the threshold). with the use of ai-enabled technology, privacy is usually cited as a major concern but the rendering of care is decidedly a non-private affair, imagine a scenario where the connection facilitated by technology allows for meeting the social and emotional needs of a terminal patient, if there is a situation where the use of technology allows for a better and longer life, then in these cases there can be an argument for sacrificing privacy to meet the needs of the patient. ultimately, a balance needs to be struck between the privacy requirements and other healthcare requirements and privacy should not be blindly touted as the most important requirement. framing the concept of the good life with a view of restoring, maintaining and enhancing the capabilities of the human, one mustn't view eudaimonia as happiness but rather the achievement of the capabilities listed because happiness in this context would fall outside of the domain of ethics. additionally, the author proposes the care experience machine thought experiment that can meet all the care needs of a patient and asks the question if it would be morally wrong to plug in a patient into such a machine. while intuitively it might seem wrong, we struggle when trying to come up with concrete objections. as long as the patient feels cared for and has, from an objective standpoint, their care needs met, it becomes hard to contest how such virtual care might differ from real care that is provided by humans. if one can achieve real capabilities, such as the need to have freedom of movement and interaction with peers outside of their care confinement and virtual reality technology enables that, then the virtual good life enhances the real good life -a distinction that becomes increasingly blurred as technology progresses. another moral argument put forward in determining whether to use technology-assisted healthcare is if it is too paternalistic to determine what is best for the patient. in some cases where the patient is unable to make decisions that restore, maintain and enhance their capabilities, such paternalism might be required but it must always be balanced with other ethical concerns and keeping in mind the capabilities that it enables for the patient. when we talk about felt care and how to evaluate whether care rendered is good or not, we should not only look at the outcomes of the process through which the patient exits the healthcare context but also the realization of some of the capabilities during the healthcare process. to that end, when thinking about felt care, we must also take into account the concept of reciprocity of feeling which is not explicitly defined in the capabilities approach but nonetheless forms an important aspect of experiencing healthcare in a positive manner from the patient's perspective. in conclusion, it is important to have an in-depth evaluation of technology assisted healthcare that is based on moral principles and philosophy, yet resting more on concrete arguments rather than just the high-level abstracts as they provide little practical guidance in evaluating different solutions and how they might be chosen to be used in different contexts. an a priori dismissal of technology in the healthcare domain, even when based on very real concerns like breach of privacy in the use of ai solutions which require a lot of personal data, begets further examination before arriving at a conclusion. the article brings up some interesting points around how we bond with things that are not necessarily sentient and how our emotions are not discriminating when it comes to reducing loneliness and imprinting on inanimate objects. people experience surges in oxytocin as a consequence of such a bonding experience which further reinforces the relationship. this has effects for how increasingly sentient-appearing ai systems might be used to manipulate humans into a "relationship" and potentially steer them towards making purchases, for example via chatbot interfaces by evoking a sense of trust. the article also makes a point about how such behaviour is akin to animism and in a sense forms a response to loneliness in the digital realm, allowing us to continue to hone our empathy skills for where they really matter, with other human beings. with more and more of our conversations being mediated by ai-enabled systems online, it is important to see if robots can be harnessed to affect positive behaviour change in our interactions with each other. while there have been studies that demonstrate the positive impact that robots can have on influencing individual behaviour, this study highlighted how the presence of robots can influence human to human interactions as well. what the researchers found was that having a robot that displayed positive and affective behavior triggered more empathy from humans towards other humans as well as other positive behaviors like listening more and splitting speaking time amongst members more fairly. this is a great demonstration of how robots can be used to improve our interactions with each other. another researcher pointed out that a future direction of interest would be to see how repeated exposure to such robot interactions can influence behaviour and if the effects so produced would be long-lasting even in the absence of the robot to participate in the interactions. since time immemorial there has been a constant tussle between making predictions and being able to understand the underlying fundamentals of how those predictions worked. in the era of big data, those tensions are exacerbated as machines become more inscrutable while making predictions using ever-more higher-dimensional data which lies beyond intuitive understanding of humans. we try to reason through some of that high-dimensional data by utilizing techniques that either reduce the dimensions or visualize into 2-or 3-dimensions which by definition will tend to lose some fidelity. bacon had proposed that humans should utilize tools to gain a better understanding of the world around them -until recently where the physical processes of the world matched quite well with our internal representations, this wasn't a big concern. but a growing reliance on tools means that we rely more on what is made possible by the tools as they measure and model the world. statistical intelligence and models often get things right but often they are hostile to reconstruction as to how they arrived at certain predictions. models provide for abstractions of the world and often don't need to follow exactly the real-world equivalents. for example, while the telescope allows us to peer far into the distance, its construction doesn't completely mimic a biological eye. more so, radio telescopes that don't follow optics at all give us a unique view into distant objects which are just not possible if we rely solely on optical observations. illusions present us with a window into the limits of our perceptual systems and bring into focus the tension between the reality and what we think is the reality. "in just the same way that prediction is fundamentally bounded by sensitivity of measurement and the shortcomings of computation, understanding is both enhanced and diminished by the rules of inference." in language models, we've seen that end-to-end deep learning systems that are opaque to our understanding perform quite a bit better than traditional machine translation approaches that rest on decades of linguistic research. this bears some resemblance to searle's chinese room experiment where if we just look at the inputs and the outputs, there isn't a guarantee that the internal workings of the system work in exactly the way we expect them to. "the most successful forms of future knowledge will be those that harmonise the human dream of understanding with the increasingly obscure echoes of the machine." abhishek gupta (founder of the montreal ai ethics institute) was featured in fortune where he detailed his views on ai safety concerns in rl systems, the "token human" problem, and automation surprise among other points to pay attention to when developing and deploying ai systems. especially in situations where these systems are going to be used in critical scenarios, humans operating in tandem with these systems and utilizing them as decision inputs need to gain a deeper understanding of the inherent probabilistic nature of the predictions from these systems and make decisions that take it into consideration rather than blindly trusting recommendations from an ai system because they have been accurate in 99% of the scenarios. with increasing capabilities of ai systems, and established research that demonstrates how human-machine combinations operate better than each in isolation, this paper presents a timely discussion on how we can craft better coordination between human and machine agents with the aim of arriving at the best possible understanding between them. this will enhance trust levels between the agents and it starts with having effective communication. the paper discusses how framing this from a human-computer interaction (hci) approach will lead to achieving this goal. this is framed with intention-, context-, and cognition-awareness being the critical elements which would be responsible for the success of effective communication between human and machine agents. intelligibility is a notion that is worked on by a lot of people in the technical community who seek to shed a light on the inner workings of systems that are becoming more and more complex. especially in the domains of medicine, warfare, credit allocation, judicial systems and other areas where they have the potential to impact human lives in significant ways, we seek to create explanations that might illuminate how the system works and address potential issues of bias and fairness. however, there is a large problem in the current approach in the sense that there isn't enough being done to meet the needs of a diverse set of stakeholders who require different kinds of intelligibility that is understandable to them and helps them meet their needs and goals. one might argue that a deeply technical explanation ought to suffice and others kinds of explanations might be derived from that but it makes them inaccessible to those who can't parse well the technical details, often those who are the most impacted by such systems. the paper offers a framework to situate the different kinds of explanations such that they are able to meet the stakeholders where they are at and provide explanations that not only help them meet their needs but ultimately engender a higher level of trust from them by highlighting better both the capabilities and limitations of the systems. ai value alignment is typically mentioned in the context of long-term agi systems but this also applies to the narrow ai systems that we have today. optimizing for the wrong metric leads to things like unrealistic and penalizing work schedules, hacking attention on video platforms, charging more money from poorer people to boost the bottomline and other unintended consequences. yet, there are attempts by product design and development teams to capture human well-being as metrics to optimize for. "how does someone feel about how their life is going?" is a pretty powerful question that gives a surprising amount of insight into well-being distanced from what might be influencing them at the moment because it makes them pause and reflect on what matters to them. but, capturing this subjective sentiment as a metric in an inherently quantitative world of algorithms is unsurprisingly littered with mines. a study conducted by facebook and supported by external efforts found that passive use of social media triggered feelings of ennui and envy while active use including interactions with others on the network led to more positive feelings. utilizing this as a guiding light, facebook strove to make an update that would be more geared towards enabling meaningful engagement rather than simply measuring the number of likes, shares and comments. they used user panels as an input source to determine what constituted meaningful interactions on the platform and tried to distill this into the well-being metrics. yet, this suffered from several flaws, namely that the evaluation of this change was not publicly available and was based on the prior work comparing passive vs. active use of social media. this idea of well-being optimization extends to algorithmic systems beyond social media platforms, for example, with how gig work might be better distributed on a platform such that income fluctuations are minimized for workers who rely on it as a primary source of earnings. another place could be amending product recommendations to also capture environmental impacts such that consumers can incorporate that into their purchasing decisions apart from just the best price deals that they can find. participatory design is going to be a key factor in the development of these metrics; especially given the philosophy of "nothing about us without us" as a north star to ensure that there isn't an inherent bias in how well-being is optimized for. often, we'll find that proxies will need to stand in for actual well-being in which case it is important to ensure that the metrics are not static and are revised in consultation with users at periodic intervals. tapping into the process of double loop learning, an organization can not only optimize for value towards its shareholders but also towards all its other stakeholders. while purely quantitative metrics have obvious limitations when trying to capture something that is inherently subjective and qualitative, we need to attempt something in order to start and iterate as we go along. in a world where increasing automation of cognitive labor due to ai-enabled systems will dramatically change the future of labor, it is now more important than ever that we start to move away from a traditional mindset when it comes to education. while universities in the previous century rightly provided a great value in preparing students for jobs, as jobs being bundle of tasks and those tasks rapidly changing with some being automated, we need to focus more on training students for things that will take much longer to automate, for example working with other humans, creative and critical thinking and driving innovation based on insights and aggregating knowledge across a diversity of fields. lifelong learning serves as a useful model that can impart some of these skills by breaking up education into modules that can be taken on an "at will" basis allowing people to continuously update their skills as the landscape changes. students will go in and out of universities over many years which will bring a diversity of experiences to the student body, encouraging a more close alignment with actual skills as needed in the market. while this will pose significant challenges to the university system, innovations like online learning and certifications based on replenishment of skills like in medicine could overcome some of those challenges for the education ecosystem. individual actions are powerful, they create bottom-up change and empower advocates with the ability to catalyze larger change. but, when we look at products and services with millions of users where designs that are inherently unethical become part of everyday practice and are met with a slight shrug of the shoulders resigning to our fates, we need a more systematized approach that is standardized and widely practiced. ethics in ai is having its moment in the spotlight with people giving talks and conferences focusing on it as a core theme yet it falls short of putting the espoused principles into practice. more often than not, you have individuals, rank and file employees who go out of their way, often on personal time, to advocate for the use of ethical, safety and inclusivity in the design of systems, sometimes even at the risk of their employment. while such efforts are laudable, they lack widespread impact and awareness that is necessary to move the needle, we need leaders at the top who can affect sweeping changes to adopt these guidelines not just in letter but in spirit and then transmit them as actionable policies to their workforce. it needs to arrive at a point where people advocating for this change don't need to do so from a place of moral and ethical obligations which customers can dispute but from a place of policy decisions which force disengagement for non-adherence to these policies. we need to move from talk to action not just at a micro but at a macro scale. the wrong kind of ai? artificial intelligence and the future of labor demand do increasing efficiency and social benefits stand in exclusion to each other when it comes to automation technology? with the development of the "right" kind of ai, this doesn't have to be the case. ai is a general purpose technology that has wide applications and being offered as a platform, it allows others to build advanced capabilities on top of existing systems creating an increasingly powerful abstraction layer with every layer. according to the standard approach in economics, a rise in productivity is often accompanied with an increase in the demand for labor and hence a rise in wages along with standards of living. but, when there is a decoupling between the deployment of technology and the associated productivity accrual, it can lead to situations where we see more output but not a corresponding increase in the standards of living as the benefits accrue to capital owners rather than wage-earning labor which is distanced from the production lifecycle. this unevenness in the distribution of gains causes losses of jobs in one sector while increasing productivity in others, often masking effects at an aggregate level through the use of purely economic focused indicators like gdp growth rates. the authors expound on how the current wave of automation is highly focused on labor replacement driven by a number of factors. when this comes in the form of automation that is just as good as labor but not significantly better, we get the negative effects as mentioned before, that is a replacement of labor without substantial increases in the standards of living. most of these effects are felt by those in the lower rungs of the socio-economic ladder where they don't have alternate avenues for employment and ascent. a common message is that we just have to wait as we did in the case of the industrial revolution and new jobs will emerge that we couldn't have envisioned which will continue to fuel economic prosperity for all. this is an egregious comparison that overlooks that the current wave of automation is not creating simultaneous advances in technology that allow the emergence of a new class of tasks within jobs for which humans are well-suited. instead, it's increasingly moving into domains that were strongholds of human skills that are not easily repeatable or reproducible. what we saw in the past was an avenue made available to workers to move out of low skill tasks in agriculture to higher skill tasks in manufacturing and services. some examples of how ai development can be done the "right" way to create social benefits: â�¢ in education, we haven't seen a significant shift in the way things are done for close to 200 years. it has been shown that different students have different learning styles and can benefit from personalized attention. while it is infeasible to do so in a traditional classroom model, ai offers the potential to track metrics on how the student interacts with different material, where they make mistakes, etc., offering insights to educators on how to deliver a better educational experience. this is accompanied by an increase in the demand for teachers who can deliver different teaching styles to match the learning styles of students and create better outcomes. â�¢ a similar argument can be made in the field of healthcare where ai systems can allow medical staff to spend more time with patients offering them personalized attention for longer while removing the need for onerous and drudgery in the form of menial tasks like data entry. â�¢ industrial robots are being used to automate the manufacturing line often cordoning off humans for safety reasons. humans are also decoupled from the process because of a difference in the level of precision that machines can achieve compared to humans. but we can get the best of both worlds by combining human flexibility and critical thinking to address problems in an uncertain environment with the high degree of preciseness of machines by creating novel interfaces, for example, by using augmented reality. an important distinction that the authors point out in the above examples is that they are not merely the job of enablers, humans that are used to train machines in a transitory fashion, but those that genuinely complement machine skills. there are market failures when it comes to innovation and in the past, governments have helped mitigate those failures via public-private partnerships that led to the creation of fundamental technologies like the internet. but, this has decreased over the past two decades because of smaller amount of resources being invested by the government in basic research and the technology revolution becoming centered in silicon valley which has a core focus on automation that replaces labor, and with that bias and their funding of university and academic studies, they are causing the best minds of the next generation to have the same mindset. markets are also known to struggle when there are competing paradigms and once one pulls ahead, it is hard to switch to another paradigm even if it might be more productive thus leading to an entrenchment of the dominant paradigm. the social opportunity cost of replacing labor is lower than the cost of labor, pushing the ecosystem towards labor replacing automation. without accounting for these externalities, the ecosystem has little incentive to move towards the right kind of ai. this is exacerbated by tax incentives imposing costs on labor while providing a break on the use of capital. additionally, areas where the right kind of ai can be developed don't necessarily fall into the cool domain of research and thus aren't prioritized by the research and development community. let's suppose large advances were made in ai for health care. this would require accompanying retraining of support staff aside from doctors, and the high level bodies regulating the field would impose resistance, thus slowing down the adoption of this kind of innovation. ultimately, we need to lean on a holistic understanding of the way automation is going to impact the labor market and it will require human ingenuity to shape the social and economic ecosystems such that they create net positive benefits that are as widely distributed as possible. relying on the market to figure this out on its own is a recipe for failure. the labor impacts of ai require nuance in discussion rather than fear-mongering that veers between over-hyping and downplaying concerns when the truth lies somewhere in the middle. in the current paradigm of supervised machine learning, ai systems need a lot of data before becoming effective at their automation tasks. the bottom rung of this ladder consists of robotic process automation that merely tracks how humans perform a task (say, by tracking the clicks of humans as they go about their work) and ape them step by step for simple tasks like copying and pasting data across different places. the article gives an example of an organization that was able to minimize churn in their employees by more than half because of a reduction in data drudgery tasks like copying and pasting data across different systems to meet legal and compliance obligations. economists point out that white-collar jobs like these and those that are middle-tier in terms of skills that require little training are at the highest risk of automation. while we're still ways away from ai taking up all jobs, there is a slow march starting from automating the most menial tasks, potentially freeing us up to do more value-added work. with a rising number of people relying on social media for the news, the potential for hateful content and misinformation spreading has never been higher. content moderation on platforms like facebook and youtube is still largely a human endeavor where there are legions of contract workers that spend their days reviewing whether different pieces of content meet the community guidelines of the platform. due to the spread of the pandemic and offices closing down, a lot of these workers have been asked to leave (they can't do this work from home as the platform companies explained because of privacy and legal reasons), leaving the platforms in the hands of automated systems. the efficacy of these systems has always been questionable and as some examples in the article point out, they've run amok taking down innocuous and harmful content alike, seeming to not have very fine-tuned abilities. the problem with this is that legitimate sources of information, especially on subjects like covid-19, are being discouraged because of their content being taken down and having to go through laborious review processes to have their content be approved again. while this is the perfect opportunity to experiment with the potential of using automated systems for content moderation given the traumatic experience that humans have to undergo as a part of this job, the chasms that need to be bridged still remain large between what humans have to offer and what the machines are capable of doing at the moment. workplace time management and accounting are common practices but for those of us who work in places where schedules are determined by automated systems, they can have many negative consequences, a lot of which could be avoided if employers paid more attention to the needs of their employees. clopening is the notion where an employee working at a retail location is asked to not only close the location at the end of the day but also arrive early the next day to open the location. this among other practices like breaks that are scheduled down to the minute and on-call scheduling (something that was only present in the realm of emergency services) wreak havoc on the physical and mental health of employees. in fact, employees surveyed have even expressed willingness to take pay cuts to have greater control over their schedules. in some places with ad-hoc scheduling, employees are forced to be spontaneous with their home responsibilities like taking care of their children, errands, etc. while some employees try to swap shifts with each other, often even that becomes hard to do because others are also in similar situations. some systems track customer demand and reduce pay for hours worked tied to that leading to added uncertainty even with their paychecks. during rush seasons, employees might be scheduled for back to back shifts ignoring their needs to be with families, something that a human manager could empathize with and accommodate for. companies supplying this kind of software hide behind the disclaimer that they don't take responsibility for how their customers use these systems which are often black-box and inscrutable to human analysis. this is a worrying trend that hurts those who are marginalized and those who require support when juggling several jobs just to make ends meet. relying on automation doesn't absolve the employers of their responsibility towards their employees. while the dominant form of discussion around the impacts of automation have been that it will cause job losses, this work from kevin scott offers a different lens into how jobs might be created by ai in the rust belt in the us where automation and outsourcing have been gradually stripping away jobs. examples abound of how entrepreneurs and small business owners with an innovative mindset have been able to leverage advances in ai, coupling them with human labor to repurpose their businesses from areas that are no longer feasible to being profitable. precision farming utilizes things like drones with computer vision capabilities to detect hotspots with pests, disease, etc. in the big farms that would otherwise require extensive manual labor which would limit the size of the farms. self-driving tractors and other automated tools also augment human effort to scale operations. the farm owners though highlight the opaqueness and complexity of such systems which make them hard to debug and fix themselves which sometimes takes away from the gains. on the other hand, in places like nursing homes that get reimbursed based on the resource utilization rates by their residents, tools using ai can help minimize human effort in compiling data and let them spend more of their effort on human contact which is not something that ai succeeds on yet. while automation has progressed rapidly, the gains haven't been distributed equally. in other places where old factories were shut down, some of them are now being utilized by ingenious entrepreneurs to bring back manufacturing jobs that cleverly combine human labor with automation to deliver high-quality, custom products to large enterprises. thus, there will be job losses from automation but the onus lies with us in steering the progress of ai towards economic and ethical values that we believe in. the state of ai ethics, june 2020 what's next for ai ethics, policy, and governance? a global overview in this ongoing piece of work, the authors present the landscape of ethical documents that has been flooded with guidelines and recommendations coming from a variety of sectors including government, private organizations, and ngos. starting with a dive into the stated and unstated motivations behind the documents, the reader is provided with a systematic breakdown of the different documents prefaced with the caveat that where the motivations are not made explicit, one can only make a best guess based on the source of origin and people involved in its creation. the majority of the documents from the governmental agencies were from the global north and western countries which led to a homogeneity of issues that were tackled and the recommendations often touted areas of interest that were specific to their industry and economical make up. this left research and development areas of interest like tourism and agriculture largely ignored which continue to play a significant role in the global south. the documents from the former category were also starkly focused on gaining a competitive edge, which was often stated explicitly, with a potential underlying goal of attracting scarce, high-quality ai talent which could trigger brain drain from other countries that are not currently the dominant players in the ai ecosystem. often, they were also positioning themselves to gain an edge and define a niche for themselves, especially in the case of countries that are non-dominant and thus overemphasizing the benefits while downplaying certain negative consequences that might arise from widespread ai use, like the displacement and replacement of labor. for documents from private organizations, they mostly focused on self and collective regulation in an effort to pre-empt stringent regulations from taking effect. they also strove to tout the economic benefits to society at large as a way of de-emphasizing the unintended consequences. a similar dynamic as in the case of government documents played out here where the interests of startups and small and medium sized businesses were ignored and certain mechanisms proposed would be too onerous for such smaller organizations to implement this further entrenching the competitive advantage of larger firms. the ngos on the other hand seemed to have the largest diversity both in terms of the participatory process of creation and the scope, granularity, and breadth of issues covered which gave technical, ethical, and policy implementation details making them actionable. some documents like the montreal declaration for responsible ai were built through an extensive public consultation process and consisted of an iterative and ongoing approach that the montreal ai ethics institute contributed to as well. the ieee document leverages a more formal standards making approach and consists of experts and concerned citizens from different parts of the world contributing to its creation and ongoing updating. the social motivation is clearly oriented towards creating larger societal benefits, internal motivation is geared towards bringing about change in the organizational structure, external strategic motivation is often towards creating a sort of signaling to showcase leadership in the domain and also interventional to shape policy making to match the interests of those organizations. judging whether a document has been successful is complicated by a couple of factors: discerning what the motivations and the goals for the document were, and the fact that most implementations and use of the documents is done in a pick-and-choose manner complicating attribution and weight allocation to specific documents. some create internal impacts in terms of adoption of new tools, change in governance, etc., while external impacts often relate to changes in policy and regulations made by different agencies. an example would be how the stem education system needs to be overhauled to better prepare for the future of work. some other impacts include altering customer perception of the organization as one that is a responsible organization which can ultimately help them differentiate themselves. at present, we believe that this proliferation of ethics documents represents a healthy ecosystem which promotes a diversity of viewpoints and helps to raise a variety of issues and suggestions for potential solutions. while there is a complication caused by so many documents which can overwhelm people looking to find the right set of guidelines that helps them meet their needs, efforts such as the study being done in this paper amongst other efforts can act as guideposts to lead people to a smaller subset from which they can pick and choose the guidelines that are most relevant to them. the white paper starts by highlighting the existing tensions in the definitions of ai as there are many parties working on advancing definitions that meet their needs. one of the most commonly accepted ones frames ai systems as those that are able to adapt their behavior in response to interactions with the world independent of human control. also, another popular framing is that ai is something that mimics human intelligence and is constantly shifting as a goal post as what was once perceived as ai, when sufficiently integrated and accepted in society becomes everyday technology. one thing that really stands out in the definitions section is how ethics are defined, which is a departure from a lot of other such documents. the authors talk about ethics as a set of principles of morality where morality is an assemblage of rules and values that guide human behavior and principles for evaluating that behavior. they take a neutral stand on the definition, a far cry from framing it as a positive inclination of human conduct to allow for diversity in embedding ethics into ai systems that are in concordance with local context and culture. ai systems present many advantages which most readers are now already familiar given the ubiquity of ai benefits as touted in everyday media. one of the risks of ai-enabled automation is the potential loss of jobs, the authors make a comparison with some historical cases highlighting how some tasks and jobs were eliminated creating new jobs while some were permanently lost. many reports give varying estimates for the labor impacts and there isn't yet a clear consensus on the actual impacts that this might have on the economy. from a liability perspective, there is still debate as to how to account for the damage that might be caused to human life, health and property by such systems. in a strict product liability regime like europe, there might be some guidance on how to account for this, but most regimes don't have specific liability allocations for independent events and decisions meaning users face coverage gaps that can expose them to significant harms. by virtue of the complexity of deep learning systems, their internal representations are not human-understandable and hence lack transparency, which is also called the black box effect. this is harmful because it erodes trust from the user perspective, among other negative impacts. social relations are altered as more and more human interactions are mediated and governed by machines. we see examples of that in how our newsfeeds are curated, toys that children play with, and robots taking care of the elderly. this decreased human contact, along with the increasing capability of machine systems, examples of which we see in how disinformation spreads, will tax humans in constantly having to evaluate their interactions for authenticity or worse, relegation of control to machines to the point of apathy. since the current dominant paradigm in machine learning is that of supervised machine learning, access to data is crucial to the success of the systems and in the state of ai ethics, june 2020 cases where there aren't sufficient protections in place for personal data, it can lead to severe privacy abuses. self determination theory states that autonomy of humans is important for proper functioning and fulfillment, so an overreliance on ai systems to do our work can lead to loss of personal autonomy, which can lead to a sense of digital helplessness. digital dementia is the cognitive equivalent where relying on devices for things like storing phone numbers, looking up information, etc. will over time lead to a decline in cognitive abilities. the echo chamber effect is fairly well studied, owing to the successful use of ai technologies to promulgate disinformation to the masses during the us presidential elections of 2016. due to the easy scalability of the systems, the negative effects are multiplicative in nature and have the potential to become run-away problems. given that ai systems are built on top of existing software and hardware, errors in the underlying systems can still cause failures at the level of ai systems. more so, given the statistical nature of ai systems, behaviour is inherently stochastic and that can cause some variability in response which is difficult to account for. flash crashes in the financial markets are an example of this. for critical systems that require safety and robustness, there is a lot that needs to be done for ensuring reliability. building ethics compliance by design can take a bottom-up or top-down approach, the risk with a bottom-up approach is that by observing examples of human behaviour and extracting ethics principles from that, instead of getting things that are good for people, you get what's common. hence, the report advocates for a top-down approach where desired ethical behavior is directly programmed into the system. casuistic approaches to embedding ethics into systems would work well in situations where there are simple scenarios, such as in healthcare when the patient has a clear directive of do-not-resuscitate. but, in cases where there isn't one and where it is not possible to seek a directive from the patient, such an approach can fail and it requires that programmers either in a top-down manner embed rules or the system learns from examples. though, in a high-stakes situation like healthcare, it might not be ideal to rely on learning from examples because of skewed and limited numbers of samples. a dogmatic approach would also be ill-advised where a system might slavishly follow a particular school of ethical beliefs which might lead it to make decisions that might be unethical in certain scenarios. ethicists utilize several schools of thought when addressing a particular situation to arrive at a balanced decision. it will be crucial to consult with a diversity of stakeholders such that the nuances of different situations can be captured well. the wef is working with partners to come up with an "ethical switch" that will imbue flexibility on the system such that it can operate with different schools of thought based on the demands of the situation.the report also proposes the potential of utilizing a guardian ai system that can monitor other ai systems to check for compliance with different sets of ai principles. given that ai systems operate in a larger socio-technical ecosystem, we need to tap into fields like law and policy making to come up with effective ways of integrating ethics into ai systems, part of which can involve creating binding legal agreements that tie in with economic incentives.while policy making and law are often seen as slow to adapt to fast changing technology, there are a variety of benefits to be had, for example higher customer trust for services that have adherence to stringent regulations regarding privacy and data protection. this can serve to be a competitive advantage and counter some of the negative innovation barriers imposed by regulations. another point of concern with these instruments is that they are limited by geography which leads to a patchwork of regulation that might apply on a product or service that spans several jurisdictions. some other instruments to consider include: self-regulation, certification, bilateral investments treaties, contractual law, soft law, agile governance, etc. the report highlights the initiatives by ieee and wef in creating standards documents. the public sector through its enormous spending power can enhance the widespread adoption of these standards such as by utilizing them in procurement for ai systems that are used to interact with and serve their citizens. the report also advocates for the creation of an ethics board or chief values officer as a way of enhancing the adoption of ethical principles in the development of products and services. for vulnerable segments of the population, for example children, there need to be higher standards of data protection and transparency that can help parents make informed decisions about which ai toys to bring into their homes. regulators might play an added role of enforcing certain ethics principles as part of their responsibility. there also needs to be broader education for ai ethics for people that are in technical roles. given that there are many negative applications of ai, it shouldn't preclude us from using ai systems for positive use cases, a risk assessment and prudent evaluation prior to use is a meaningful compromise. that said, there are certain scenarios where ai shouldn't be used at all and that can be surfaced through the risk or impact assessment process. there is a diversity of ethical principles that have been put forth by various organizations, most of which are in some degree of accordance with local laws, regulations, and value sets. yet, they share certain universal principles across all of them. one concern highlighted by the report is on the subject of how even widely accepted and stated principles of human rights can be controversial when translated into specific mandates for an ai system. when looking at ai-enabled toys as an example, while they have a lot of privacy and surveillance related issues, in countries where there isn't adequate access to education, these toys could be seen as a medium to impart precision education and increase literacy rates. thus, the job of the regulator becomes harder in terms of figuring out how to balance the positive and negative impacts of any ai product. a lot of it depends on the context and surrounding socio-economic system as well. given the diversity in ethical values and needs across communities, an approach might be for these groups to develop and apply non-binding certifications that indicate whether a product meets the ethical and value system of that community. since there isn't a one size fits all model that works, we should aim to have a graded governance structure that has instruments in line with the risk and severity profile of the applications. regulation in the field of ai thus presents a tough challenge, especially given the interrelatedness of each of the factors. the decisions need to be made in light of various competing and sometimes contradictory fundamental values. given the rapid pace of technological advances, the regulatory framework needs to be agile and have a strong integration into the product development lifecycle. the regulatory approach needs to be such that it balances speed so that potential harms are mitigated with overzealousness that might lead to ineffective regulations that stifle innovation and don't really understand well the technology in question. ai is currently enjoying a summer of envy after having gone through a couple of winters of disenchantment, with massive interest and investments from researchers, industry and everyone else there are many uses of ai to create societal benefits but they aren't without their socio-ethical implications. ai systems are prone to biases, unfairness and adversarial attacks on their robustness among other real-world deployment concerns. even when ethical ai systems are deployed for fostering social good, there are risks that they cater to only a particular group to the detriment of others. moral relativism would argue for a diversity of definitions as to what constitutes good ai which would depend on the time, context, culture and more. this would be reflected in market decisions by consumers who choose products and services that align with their moral principles but it poses a challenge for those trying to create public governance frameworks for these systems. this dilemma would push regulators towards moral objectivism which would try and advocate for a single set of values that are universal making the process of coming up with a shared governance framework easier. a consensus based approach utilized in crafting the ec trustworthy ai guidelines settled on human rights as something that everyone can get on board with. given the ubiquity in the applicability of human rights, especially with their legal enshrinement in various charters and constitutions, they serve as a foundation to create legal, ethical and robust ai as highlighted in the ec trustworthy ai guidelines. stressing on the importance of protecting human rights, the guidelines advocate for a trustworthy ai assessment in case that an ai system has the potential to negatively impact the human rights of an individual, much like the better established data protection impact assessment requirement under the gdpr. additional requirements are imposed in terms of ex-ante oversight, traceability, auditability, stakeholder consultations, and mechanisms of redress in case of mistakes, harms or other infringements. the universal applicability of human rights and their legal enshrinement also renders the benefits of established institutions like courts whose function is to monitor and enforce these rights without prejudice across the populace. but they don't stand uncontested when it comes to building good ai systems; they are often seen as too western, individualistic, narrow in scope and abstract to be concrete enough for developers and designers of these systems. some arguments against this are that they go against the plurality of value sets and are a continued form of former imperialism imposing a specific set of values in a hegemonic manner. but, this can be rebutted by the signing of the original universal declaration of human rights that was done by nations across the world in an international diplomatic manner. however, even despite numerous infringements, there is a normative justification that they ought to be universal and enforced. while human rights might be branded as too individual focused, potentially creating a tension between protecting the rights of individuals to the detriment of societal good, this is a weak argument because stronger protection of individual rights has knock-on social benefits as free, healthy and well-educated (among other individual benefits) creates a net positive for society as these individuals are better aware and more willing to be concerned about societal good. while there are some exceptions to the absolute nature of human rights, most are well balanced in terms of providing for the societal good and the good of others while enforcing protections of those rights. given the long history of enforcement and exercises in balancing these rights in legal instruments, there is a rich jurisprudence on which people can rely when trying to assess ai systems. while human rights create a social contract between the individual and the state, putting obligations on the state towards the individual but some argue that they don't apply horizontally between individuals and between an individual and a private corporation. but, increasingly that's not the case as we see many examples where the state intervenes and enforces these rights and obligations between an individual and a private corporation as this falls in its mandate to protect rights within its jurisdiction. the abstract nature of human rights, as is the case with any set of principles rather than rules, allows them to be applied to a diversity of situations and to hitherto unseen situations as well. but, they rely on an ad-hoc interpretation when enforcing them and are thus subjective in nature and might lead to uneven enforcement across different cases. under the eu, this margin of appreciation is often criticized in the sense that it leads to weakening and twisting of different principles but this deferment to those who are closer to the case actually allows for a nuanced approach which would be lost otherwise. on the other hand we have rules which are much more concrete formulations and thus have a rigid definition and limited applicability which allows for uniformity but it suffers from inflexibility in the face of novel scenarios. yet, both rules and principles are complementary approaches and often the exercise of principles over time leads to their concretization into rules under existing and novel legal instruments. while human rights can thus provide a normative, overarching direction for the governance of ai systems, they don't provide the actual constituents for an applicable ai governance framework. for those that come from a non-legal background, often technical developer and designers of ai systems, it is essential that they understand their legal and moral obligations to codify and protect these rights in the applications that they build. the same argument cuts the other way, requiring a technical understanding of how ai systems work for legal practitioners such that they can meaningfully identify when breaches might have occurred. this is also important for those looking to contest claims of breaches of their rights in interacting with ai systems. this kind of enforcement requires a wide public debate to ensure that they fall within accepted democratic and cultural norms and values within their context. while human rights will continue to remain relevant even in an ai systems environment, there might be novel ways in which breaches might occur and those might need to be protected which require a more thorough understanding of how ai systems work. growing the powers of regulators won't be sufficient if there isn't an understanding of the intricacies of the systems and where breaches can happen, thus there is more of a need to enshrine some of those responsibilities in law to enforce this by the developers and designers of the system. given the large public awareness and momentum that built up around the ethics, safety and inclusion issues in ai, we will certainly see a lot more concrete actions around this in 2020. the article gives a few examples of congressional hearings on these topics and advocates for the industry to come up with some standards and definitions to aid the development of meaningful regulations. currently, there isn't a consensus on these definitions and it leads to varying approaches addressing the issues at different levels of granularity and angles. what this does is create a patchwork of incoherent regulations across domains and geographies that will ultimately leave gaps in effectively mitigating potential harms from ai systems that can span beyond international borders. while there are efforts underway to create maps of all the different attempts of defining principle sets, we need a more coordinated approach to bring forth regulations that will ultimately protect consumer safety. in containing an epidemic the most important steps include quarantine and contact tracing for more effective testing. while before, this process of contact tracing was hard and fraught with errors and omissions, relying on memories of individuals, we now carry around smartphones which allow for ubiquitous tracking ability that is highly accurate. but such ubiquity comes with invasion of privacy and possible limits on freedoms of citizens. such risks need to be balanced with public interest in mind while using enhanced privacy preserving techniques and any other measures that center citizen welfare in both a collective and individual sense. for infections that can be asymptomatic in the early days, like the covid-19, it is essential to have contact tracing, which identifies all people that came in close contact with an infected person and might spread the infection further. this becomes especially important when you have a pandemic at hand, burdening the healthcare system and testing every person is infeasible.an additional benefit of contact tracing is that it mitigates resurgence of the peaks of infection. r0 determines how quickly a disease will spread and is dependent on three factors (period of infection, contact rate and mode of transmission) out of which the first and third are fixed so we're only left with control over the contact rate.with an uptake of an application that facilitates contact tracing, the amount of reduction in contact rate is an increasing return because of the number of people that might come in contact with an infected person and thus, we get a greater reduction of r0 in terms of percentage compared to the percentage uptake of the application in the population. ultimately, reducing r0 to below 1 leads to a slowdown in the spread of the infection thus helping the healthcare system cope up with the sudden stresses that are brought on by pandemic peaks. one of the techniques that governments or agencies responsible for public health use is broadcasting in which the information of diagnosed carriers is made public via various channels but it carries severe issues like exposing private information of individuals and businesses where they might have been which can trigger stigma, ostracization and unwarranted punitive harm. it also suffers from the problem of people needing to access this source of information of their own volition and then self-identify (and remember correctly) if they've been in recent contact with a diagnosed carrier. selective broadcasting is a more restricted form of the above where information about diagnosed carriers is shared to a select group of individuals based on location proximity in which case the user's location privacy would have to be compromised and in another vector of dissemination, messages are sent to all users but filtered on device for their specific location and is not reported back to the broadcaster. but, the other second-order negative effects remain the same as broadcasting. both though require the download of an application which might decrease the uptake of it by people. unicasting is when messages are sent tailored specifically to each user and they require the download of an app which needs to be able to track timestamps and location and has severe consequences in terms of government surveillance and abuse. participatory sharing is a method where diagnosed carriers voluntarily share their information and thus have more data control but it still relies on individual action both on the sender and receiver and its efficacy is questionable at best. there is also a risk of abuse by malicious actors to spread misinformation and seed chaos in society via false alarms. private kit: safe paths is an open-source solution developed by mit that allows for contact tracing in a privacy preserving way. it utilizes the encrypted location trail of a diagnosed carrier who chooses to share that with public health agencies and then other users who are also using the solution can pull this data and via their own logged location trail get a result of they've been in close contact with a diagnosed carrier. in the later phases of development of this solution, the developers will enable a mix of participatory sharing and unicasting to further prevent possible data access by third parties including governments for surveillance purposes. risks of contact tracing include possible public identification of the diagnosed carrier and severe social stigma that arises as a part of that. online witch hunts to try and identify the individual can often worsen the harassment and include spreading of rumors about their personal lives. the privacy risks for both individuals and businesses have potential for severe harm, especially during times of financial hardship, this might be very troublesome. privacy risks also extend to non-users because of proximal information that can be derived from location trails, such as employees that work at particular businesses that were visited by a diagnosed carrier. it can also bring upon the same stigma and ostracization to the family members of these people. without meaningful alternatives, especially in health and risk assessment during a pandemic, obtaining truly informed consent is a real challenge that doesn't yet have any clear solutions. along with information, be it through any of the methods identified above, it is very important to provide appropriate context and background to the alerts to prevent misinformation and panic from spreading especially for those with low health, digital and media literacy. on the other hand, some might not take such alerts seriously and increase the risk for public health by not following required measures such as quarantine and social distancing. given the nature of such solutions, there is a significant risk of data theft from crackers as is the case for any application that collects sensitive information like health status and location data. the solutions can also be used for fraud and abuse, for example, by blackmailing business owners and demanding ransom, failing to pay which they would falsely post information that they're diagnosed carriers and have visited their place of business. contact tracing technology requires the use of a smartphone with gps and some vulnerable populations might not always have such devices available like the elderly, homeless and people living in low-income countries who are at high risk of infection and negative health outcomes. ensuring that technology that works for all will be an important piece to mitigating the spread effectively. there is an inherent tradeoff between utility from the data provided and the privacy of the data subjects. compromises may be required for particularly severe outbreaks to manage the spread. the diagnosed carriers are the most vulnerable stakeholders in the ecosystem of contact tracing technology and they require the most protection. adopting open-source solutions that are examinable by the wider technology ecosystem can engender public trust. additionally, having proper consent mechanisms in place and exclusion of the requirement of extensive third party access to the location data can also help allay concerns. lastly, time limits on the storage and use of the location trails will also help address privacy concerns and increase uptake and use of the application in supporting public health measures. for geolocation data that might affect businesses, especially in times of economic hardship, information release should be done such that they are informed prior to the release of the information but there is little else in current methods that can both protect privacy and at the same time provide sufficient data utility. for those without access to smartphones with gps, providing them with some information on contact tracing can still help their communities. but, one must present information in a manner that accounts for variation in health literacy levels so an appropriate response is elicited from the people. alertness about potential misinformation and educational awareness are key during times of crises to encourage people to have measured responses following the best practices as advised by health agencies rather than those based on fear mongering by ill informed and/or malicious actors. encryption and other cybersecurity best practices for data security and privacy are crucial for the success of the solution. time limits on holding data for covid-19 is recommended at 14-37 days, the period of infection, but for an evolving pandemic one might need it for longer for more analysis. tradeoffs need to be made between privacy concerns and public health utility. different agencies and regions are taking different approaches with varying levels of efficacy and only time will tell how this change will be best managed. it does present an opportunity though for creating innovative solutions that both allow for public sharing of data and also reduce privacy intrusions. while the insights presented in this piece of work are ongoing and will continue to be updated, we felt it important to highlight the techniques and considerations compiled by the openmined team as it is one of the few places that adequately capture, in a single place, most of the technical requirements needed to build a solution that respects fundamental rights while balancing them with public health outcomes as people rush to make ai-enabled apps to combat covid-19. most articles and research work coming out elsewhere are very scant and abstract in the technical details that would be needed to meet the ideals of respecting privacy and enabling health authorities to curb the spread of the pandemic. the four key techniques that will help preserve and respect rights as more and more people develop ai-enabled applications to combat covid-19 are: on-device data storage and computation, differential privacy, encrypted computation and privacy-preserving identity verification. the primary use cases, from a user perspective, for which apps are being built are to get: proximity alerts, exposure alerts, information on planning trips, symptom analysis and demonstrate proof of health. from a government and health authorities perspective, they are looking for: fast contact tracing, high-precision self-isolation requests, high-precision self-isolation estimation, high-precision symptomatic citizen estimation and demonstration of proof of health. while public health outcomes are at the top of the mind for everyone, the above use cases are trying to achieve the best possible tradeoff between economic impacts and epidemic spread. using the techniques highlighted in this work, it is possible to do so without having to erode the rights of citizens. this living body of work is meant to serve as a high-level guide along with resources to enable both app developers and verifiers implement and check for privacy preservation which has been the primary pushback from citizens and civil activists. evoking a high degree of trust from people will improve adoption of the apps developed and hopefully allow society and the economy to return to normal sooner while mitigating the harmful effects of the epidemic. there is a fair amount of alignment in the goals of both individuals and the government with the difference being that the government is looking at aggregate outcomes for society. some of the goals shared by governments across the world include: preventing the spread of the disease, eliminating the disease, protecting the healthcare system, protecting the vulnerable, adequately and appropriately distributing resources, preventing secondary breakouts, minimizing economic impacts and panic. the need for digital contact tracing is important because manual interventions are usually highly error prone and rely on human memory to trace how the person might have come in contact with. the requirement for high-precision self-isolation requests will avoid the need for geographic quarantines where everyone in an area is forced to self-isolate which leads to massive disruptions in the economy and can stall the delivery of essential services like food, electricity and water. the additional benefits of high-precision self-isolation is that it can help create an appropriate balance between economic harms and epidemic spread. high-precision symptomatic citizen estimation is a useful application in that it allows for more fine-grained estimation of the number of people that might be affected beyond what the test results indicate which can further strengthen the precision of other measures that are undertaken. a restoration of normalcy in society is going to be crucial as the epidemic starts to ebb, in this case, having proof of health that helps to determine the lowest risk individuals will allow for them to participate in public spaces again further bolstering the supply of essential services and relieving the burden from a small subset of workers who are participating. to service the needs of both what the users want and what the government wants, we need to be able to collect the following data: historical and current absolute location, historical and current relative position and verified group identity, where group refers to any demographic that the government might be interested in, for example, age or health status. to create an application that will meet these needs, we need to collect data from a variety of sources, compute aggregate statistics on that data and then set up some messaging architecture that communicates the results to the target population. the toughest challenges lie in the first and second parts of the process above, especially to do the second part in a privacy-preserving manner. for historical and current absolute location, one of the first options considered by app developers is to record gps data in the background. unfortunately, this doesn't work on ios devices and even then has several limitations including coarseness in dense, urban areas and usefulness only after the app has been running on the user device for some time because historical data cannot be sourced otherwise. an alternative would be to use wi-fi router information which can give more accurate information as to whether someone has been self-isolating or not based on whether they are connected to their home router. there can be historical data available here which makes it more useful though there are concerns with lack of widespread wi-fi connectivity in rural areas and tracking when people are outside homes. other ways of obtaining location data could be from existing apps and services that a user uses -for example, history of movements on google maps which can be parsed to extract location history. there is also historical location data that could be pieced together from payments history, cars that record location information and personal cell tower usage data. historical and current relative data is even more important to map the spread of the epidemic and in this case, some countries like singapore have deployed bluetooth broadcasting as a means of determining if people have been in close proximity. the device broadcasts a random number (which could change frequently) which is recorded by devices passing by close to each other and in case someone is tested positive, this can be used to alert people who were in close proximity to them. another potential approach highlighted in the article is to utilize gyroscope and ambient audio hashes to determine if two people might have been close together, though bluetooth will provide more consistent results. the reason to use multiple approaches is the benefit of getting more accurate information overall since it would be harder to fake multiple signals. group membership is another important aspect where the information can be used to finely target messaging and calculating aggregate statistics. but, for some types of group membership, we might not be able to rely completely on self-reported data. for example, health status related to the epidemic would require verification from an external third-party such as a medical institution or testing facility to minimize false information. there are several privacy preserving techniques that could be applied to an application given that you have: confirmed covid-19 patient data in a cloud, all other user data on each individual's device, and data on both the patients and the users including historical and current absolute and relative locations and group identifier information. private set intersections can be used to calculate whether two people were in proximity to each other based on their relative and absolute location information. private set intersection operates similarly to normal set intersection to find elements that are common between two sets but does so without disclosing any private information from either of the sets. this is important because performing analysis even on pseudonymized data without using privacy preservation can leak a lot of information. differential privacy is another critical technique to be utilized, dp consists of providing mathematical guarantees (even against future data disclosures) that analysis on the data will not reveal whether or not your data was part of the dataset. it asserts that from the analysis, one is not able to learn anything about your data that they wouldn't have been able to learn from other data about you. google's battle-tested c++ library is a great resource to start along with the python wrapper created by the openmined team. to address the need for verified group identification, one can utilize the concept of a private identity server. it essentially functions as a trusted intermediary between a user that wants to provide a claim and another party that wants to verify the claim. it functions by querying a service from which it can verify whether the claim is true and then serve that information up to the party wishing to verify the claim without giving away personal data. while it might be hard to trust a single intermediary, this role can be decentralized to provide for obtaining a higher degree of trust by relying on a consensus mechanism. building on theory from management studies by christensen et al. the authors of this article dive into how leaders of tech organizations, especially upstarts that are rapid in the disruption of incumbents should approach the accompanying responsibilities that come with a push into displacing existing paradigms of how an industry works. when there is a decoupling of different parts of the value chain in how a service is delivered, often the associated protections that apply to the entire pipeline fall by the wayside because of distancing from the end user and a diffusion of responsibility across multiple stakeholders in the value chain. while end users driven innovation will seek to reinforce such models, regulations and protections are never at the top of such demands and they create a burden on the consumers once they realize that things can go wrong and negatively affect them. the authors advocate for the leaders of the companies to proactively employ a systems thinking approach to identify different parts that they are disrupting, how that might affect users, what would happen if they become the dominant player in the industry and then apply lessons learned from such an exercise to pre-emptively design safeguards into the system to mitigate unintended consequences. many countries are looking at utilizing existing surveillance and counter-terrorism tools to help track the spread of the coronavirus and are urging tech companies and carriers to assist with this. the us is looking at how they can tap into location data from smartphones, following in the heels of israel and south korea that have deployed similar measures. while extraordinary measures might be justified given the time of crisis we're going through, we mustn't lose sight of what behaviors we are normalizing as a part of this response against the pandemic. russia and china are also using facial recognition technologies to track movements of people, while iran is endorsing an app that might be used as a diagnosis tool. expansion of the boundaries of surveillance capabilities and government powers is something that is hard to reign back in once a crisis is over. in some cases, like the signing of the freedom act in the usa reduced government agency data collection abilities that were expanded under the patriot act. but, that's not always the case and even so, the powers today exceed those that existed prior to the enactment of the patriot act. what's most important is to ensure that decisions policy makers take today keep in mind the time limits on such expansion of powers and don't trigger a future privacy crisis because of it. while no replacement for social distancing, a virus tracking tool putting into practice the technique of contact tracing is largely unpalatable to western democracies because of expectations of privacy and freedom of movement. a british effort underway to create an app that meets democratic ideals of privacy and freedom while also being useful in collecting geolocation data to aid in the virus containment efforts. it is based on the notion of participatory sharing, relying on people's sense of civic duty to contribute their data in case they test positive. while in the usa, discussions between the administration and technology companies has focused on large scale aggregate data collection, in a place like the uk with a centralized healthcare system, there might be higher trust levels in sharing data with the government. while the app doesn't require uptake by everyone to be effective, but a majority of the people would need to use it to bring down the rate of spread. the efficacy of the solution itself will rely on being able to collect granular location data from multiple sources including bluetooth, wi-fi, cell tower data, and app check-ins. a lot of high level cdc officials are advising that if people in the usa don't follow best practices of social distancing, sheltering in place, and washing hands regularly, the outbreak will not have peaked and the infection will continue to spread, especially hitting those who are the most vulnerable including the elderly and those with pre-existing conditions. on top of the public health impacts, there are also concerns of growing tech-enabled surveillance which is being seriously explored as an additional measure to curb the spread. while privacy and freedom rights are enshrined in the constitution, during times of crisis, government and justice powers are expanded to allow for extraordinary measures to be adopted to restore the safety of the public. this is one of those times and the us administration is actively exploring options in partnership with various governments on how to effectively combat the spread of the virus including the use of facial recognition technology. this comes shortly after the techlash and a potential bipartisan movement to curb the degree of data collection by large firms, which seem to have come to a halt as everyone scrambles to battle the coronavirus. regional governments are being imbued with escalated powers to override local legislations in an effort to curb the spread of the virus. the article provides details on efforts by various countries across the world, yet we only have preliminary data on the efficacy of each of those measures and we require more time before being able to judge which of them is the most effective. that said, in a pandemic that is fast spreading, we don't have the luxury of time and must make decisions as quickly as possible using the information at hand, perhaps using guidance from prior crises. but, what we've seen so far is minimal coordination from agencies across the world and that's leading to ad-hoc, patchy data use policies that will leave the marginalized more vulnerable. strategies that use public disclosure of those that have been tested positive in the interest of public health are causing harm to the individuals and other individuals that are close to them such as their families. as experienced by a family in new york, online vigilantes attempted to harass the individuals while their family pleaded and communicated measures that they had taken to isolate themselves to safeguard others. unfortunately, the virus might be bringing out the worst in all of us. an increasing number of tools and techniques are being used to track our behaviour online and while some may have potential benefits, for example, the use of contact tracing to potentially improve public health outcomes, if this is not done in a privacy-preserving manner, there can be severe implications for your data rights. but, barring special circumstances like the current pandemic, there are a variety of simple steps that you can take to protect your privacy online. these range from simple steps like using an incognito browser window which doesn't store any local information about your browsing on your device to using things like vpns which protect snooping of your browsing patterns even from your isp. when it comes to using the incognito function of your browser, if you're logged into a service online, there isn't any protection though it does prevent storing cookies on your device. with vpns, there is an implicit trust placed in the provider of that service to not store logs of your browsing activity. an even more secure option is to use a privacy-first browser like tor which routes your traffic requests through multiple locations making tracking hard. there is also an os built around this called tailsos that offers tracking protection from the device perspective as well not leaving any trace on the host machine allowing you to boot up from a usb. the eff also provides a list of tools that you can use to get a better grip on your privacy as you browse online. under the children's online privacy protection act, the ftc levied its largest fine yet of $170m on youtube last year for failing to meet requirements of limiting personal data collection for children under the age of 13. yet, as many advocates of youth privacy point out, the fines, though they appear to be large, don't do enough to deter such personal data collection. they advocate for a stronger version of the act while requiring more stringent enforcement from the ftc which has been criticized for slow responses and a lack of sufficient resources. while the current act requires parental consent for children below 13 to be able to utilize a service that might collect personal data, there is no verification performed on the self-declared age provided at the time of sign up which weakens the efficacy of this requirement. secondly, the sharp threshold of 13 years old immediately thrusts children into an adult world once they cross that age and some people are advocating for a more graduated approach to the application of privacy laws. given that such a large part of the news cycle is dominated by the coronavirus, we tend to forget that there might be censors at work that are systematically suppressing information in an attempt to diminish the seriousness of the situation. some people are calling github the last piece of free land in china and have utilized access to it to document news stories and people's first hand experiences in fighting the virus before they are scrubbed from local platforms like wechat and weibo. they hope that such documentation efforts will not only shed light on the reality and on the ground situation as it unfolds but also give everyone a voice and hopefully provide data to others who could use it to track the movement of the virus across the country. such times of crisis bring out creativity and this attempt highlights our ability as a species to thrive even in a severely hostile environment. there is a clear economic and convenience case to be made (albeit for the majority, not for those that are judged to be minorities by the system and hence get subpar performance from the system) where you get faster processing and boarding times when trying to catch a flight. yet, for those that are more data-privacy minded, there is an option to opt-out though leveraging that option doesn't necessarily mean that the alternative will be easy, as the article points out, travelers have experienced delays and confusion from the airport staff. often, the alternatives are not presented as an option to travelers giving a false impression that people have to submit to facial recognition systems. some civil rights and ethics researchers tested the system and got varying mileage out of their experiences but urge people to exercise the option to push back against technological surveillance. london is amongst a few cities that has seen public deployment of live facial recognition technology by law enforcement with the aim of increasing public safety. but, more often than not, it is done so without public announcement and an explanation as to how this technology works, and what impacts it will have on people's privacy. as discussed in an article by maiei on smart cities, such a lack of transparency erodes public trust and affects how people go about their daily lives. several artists in london as a part of regaining control over their privacy and to raise awareness are using the technique of painting adversarial patterns on their faces to confound facial recognition systems. they employ highly contrasting colors to mask the highlights and shadows on their faces and practice pattern use as created and disseminated by the cvdazzle project that advocates for many different styles to give the more fashion-conscious among us the right way to express ourselves while preserving our privacy. such projects showcase a rising awareness for the negative consequences of ai-enabled systems and also how people can use creative solutions to combat problems where laws and regulations fail them. there is mounting evidence that organizations are taking seriously the threats arising from malicious actors geared towards attacking ml systems. this is supported by the fact that organizations like iso and nist are building up frameworks for guidance on securing ml systems, that working groups from the eu have put forth concrete technical checklists for the evaluating the trustworthiness of ml systems and that ml systems are becoming key to the functioning of organizations and hence they are inclined to protect their crown jewels. the organizations surveyed as a part of this study spanned a variety of domains and were limited to those that have mature ml development. the focus was on two personas: ml engineers who are building these systems and security incident responders whose task is to secure the software infrastructure including the ml systems. depending on the size of the organization, these people could be in different teams, same team or even the same person. the study was also limited to intentional malicious attacks and didn't investigate the impacts of naturally occurring adversarial examples, distributional shifts, common corruption and reward hacking. most organizations that were surveyed as a part of the study were found to primarily be focused on traditional software security and didn't have the right tools or know-how in securing against ml attacks. they also indicated that they were actively seeking guidance in the space. most organizations were clustered around concerns regarding data poisoning attacks which was probably the case because of the cultural significance of the tay chatbot incident. additionally, privacy breaches were another significant concern followed by concerns around model stealing attacks that can lead to the loss of intellectual property. other attacks such as attacking the ml supply chain and adversarial examples in the physical domain didn't catch the attention of the people that were surveyed as a part of the study. one of the gaps between reality and expectations was around the fact that security incident responders and ml engineers expected that the libraries that they are using for ml development are battle-tested before being put out by large organizations, as is the case in traditional software. also, they pushed upstream the responsibility of security in the cases where they were using ml as a service from cloud providers. yet, this ignores the fact that this is an emergent field and that a lot of the concerns need to be addressed in the downstream tasks that are being performed by these tools. they also didn't have a clear understanding of what to expect when something does go wrong and what the failure mode would look like. in traditional software security, mitre has a curated repository of attacks along with detection cues, reference literature and tell-tale signs for which malicious entities, including nation state attackers are known to use these attacks. the authors call for a similar compilation to be done in the emergent field of adversarial machine learning whereby the researchers and practitioners register their attacks and other information in a curated repository that provides everyone with a unified view of the existing threat environment. while programming languages often have well documented guidelines on secure coding, guidance on doing so with popular ml frameworks like pytorch, keras and tensorflow is sparse. amongst these, tensorflow is the only one that provides some tools for testing against adversarial attacks and some guidance on how to do secure coding in the ml context. security development lifecycle (sdl) provides guidance on how to secure systems and scores vulnerabilities and provides some best practices, but applying this to ml systems might allow imperfect solutions to exist. instead of looking at guidelines as providing a strong security guarantee, the authors advocate for having code examples that showcase what constitutes security-and non-security-compliant ml development. in traditional software security there are tools for static code analysis that provide guidance on the security vulnerabilities prior to the code being committed to a repository or being executed while dynamic code analysis finds security vulnerabilities by executing the different code paths and detecting vulnerabilities at runtime. there are some tools like mlsec and cleverhans that provide white-and black-box testing; one of the potential future directions for research is to extend this to the cases of model stealing, model inversion, and membership inference attacks. including these tools as a part of the ide would further make it naturalized for developers to think about secure coding practices in the ml context. adapting the audit and logging requirements as necessitated for the functionality of the security information and event management (siem) system, in the field of ml, one can execute the list of attacks as specified in literature and ensure that the logging artifacts generated as a consequence are traced to an attack. then, having these incident logs be in a format that is exportable and integratable with siem systems will allow forensic experts to analyze them post-hoc for hardening and analysis. standardizing the reporting, logging and documentation as done by the sigma format in traditional software security will allow the insights from one analyst into defenses for many others. automating the possible attacks and including them as a part of the mlops pipeline is something that will enhance the security posture of the systems and make them pedestrian practice in the sdl. red teaming, as done in security testing, can be applied to assess the business impacts and likelihood of threat, something that is considered best practice and is often a requirement for supplying critical software to different organizations like the us government. transparency centers that allow for deep code inspection and help create assurance on the security posture of a software product/service can be extended to ml which would have to cover three modalities: ml platform is implemented in a secure manner, ml as a service meets the basic security and privacy requirements, and that the ml models embedded on edge devices meet basic security requirements. tools that build on formal verification methods will help to enhance this practice. tracking and scoring ml vulnerabilities akin to how they are done in software security testing done by registering identified vulnerabilities into a common database like cve and then assigning it an impact score like the cvss needs to be done for the field of ml. while the common database part is easy to set up, scoring them isn't something that has been figured out yet. additionally, on being alerted that a new vulnerability has been discovered, it isn't clear how the ml infrastructure can be scanned to see if the system is vulnerable to that. because of the deep integration of ml systems within the larger product/service, the typical practice of identifying a blast radius and containment strategy that is applied to traditional software infrastructure when alerted of a vulnerability is hard to define and apply. prior research work from google has identified some ways to qualitatively assess the impacts in a sprawling infrastructure. from a forensic perspective, the authors put forth several questions that one can ask to guide the post-hoc analysis, the primary problem there is that only some of the learnings from traditional software protection and analysis apply here, there are many new artifacts, paradigmatic, and environmental aspects that need to be taken into consideration. from a remediation perspective, we need to develop metrics and ways to ascertain that patched models and ml systems can maintain prior levels of performance while having mitigated the attacks that they were vulnerable to, the other thing to pay attention is that there aren't any surfaces that are opened up for attack. given that ml is going to be the new software, we need to think seriously about inheriting some of the security best practices from the world of traditional cybersecurity to harden defenses in the field of ml. all technology has implications for civil liberties and human rights, the paper opens with an example of how low-clearance bridges between new york and long island were supposedly created with the intention of disallowing public buses from crossing via the underpasses to discourage the movement of users of public transportation, primarily disadvantaged groups from accessing certain areas. in the context of adversarial machine learning, taking the case of facial recognition technology (frt), the authors demonstrate that harm can result on the most vulnerable, harm which is not theoretical and is gaining in scope, but that the analysis also extends beyond just frt systems. the notion of legibility borrowing from prior work explains how governments seek to categorize through customs, conventions and other mechanisms information about their subjects centrally. legibility is enabled for faces through frt, something that previously was only possible as a human skill. this combined with the scale offered by machine learning makes this a potent tool for authoritarian states to exert control over their populations. from a cybersecurity perspective, attackers are those that compromise the confidentiality, integrity and availability of a system, yet they are not always malicious, sometimes they may be pro-democracy protestors who are trying to resist identification and arrest by the use of frt. when we frame the challenges in building robust ml systems, we must also pay attention to the social and political implications as to who is the system being made safe for and at what costs. positive attacks against such systems might also be carried out by academics who are trying to learn about and address some of the ethical, safety and inclusivity issues around frt systems. other examples such as the hardening of systems against doing membership inference means that researchers can't determine if an image was included in the dataset, and someone looking to use this as evidence in a court of law is deterred from doing so. detection perturbation algorithms permit an image to be altered such that faces can't be recognized in an image, for example, this can be used by a journalist to take a picture of a protest scene without giving away the identities of people. but, defensive measures that disarm such techniques hinder such positive use cases. defense measures against model inversion attacks don't allow researchers and civil liberty defenders to peer into black box systems, especially those that might be biased against minorities in cases like credit allocation, parole decision-making, etc. the world of security is always an arms race whether that is in the physical or cyberspace. it is not that far-fetched to imagine how a surveillance state might deploy frt to identify protestors who as a defense might start to wear face masks for occlusion. the state could then deploy techniques that bypass this and utilize other scanning and recognition techniques to which the people might respond by wearing adversarial clothing and eyeglasses to throw off the system at which point the state might choose to use other biometric identifiers like iris scanning and gait detection. this constant arms battle, especially when defenses and offenses are constructed without the sense for the societal impacts leads to harm whose burden is mostly borne by those who are the most vulnerable and looking to fight for their rights and liberties. this is not the first time that technology runs up against civil liberties and human rights, there are lessons to be learned from the commercial spyware industry and how civil society organizations and other groups came together to create "human rights by design" principles that helped to set some ground rules for how to use this technology responsibly. researchers and practitioners in the field of ml security can borrow from these principles. we've got a learning community at the montreal ai ethics institute that is centered around these ideas that brings together academics and others from around the world to blend the social sciences with the technical sciences. recommendations on countering some of the harms centre around holding the vendors of these systems to the business standards set by the un, implementing transparency measures during the development process, utilizing human rights by design approaches, logging ml system uses along with possible nature and forms of attacks and pushing the development team to think about both the positive and negative use cases for the systems such that informed trade-offs can be made when hardening these systems to external attacks. in this insightful op-ed, two pioneers in technology shed light on how to think about ai systems and their relation to the existing power and social structures. borrowing the last line in the piece, " â�¦ all that is necessary for the triumph of an ai-driven, automation-based dystopia is that liberal democracy accept it as inevitable.", aptly captures the current mindset surrounding ai systems and how they are discussed in the western world. tv shows like black mirror perpetuate narratives showcasing the magical power of ai-enabled systems, hiding the fact that there are millions, if not billions of hours of human labor that undergird the success of modern ai systems, which largely fall under the supervised learning paradigm that requires massive amounts of data to work well. the chinese ecosystem is a bit more transparent in the sense that the shadow industry of data labellers is known, and workers are compensated for their efforts. this makes them a part of the development lifecycle of ai while sharing economic value with people other than the tech-elite directly developing ai. on the other hand, in the west, we see that such efforts go largely unrewarded because we trade in that effort of data production for free services. the authors give the example of audrey tang and taiwan where citizens have formed a data cooperative and have greater control over how their data is used. contrasting that, we have highly-valued search engines standing over community-run efforts like wikipedia which create the actual value for the search results, given that a lot of the highly placed search results come from wikipedia. ultimately, this gives us some food for thought as to how we portray ai today and its relation to society and why it doesn't necessarily have to be that way. mary shelly had created an enduring fiction which, unbeknownst to her, has today manifested itself in the digital realm with layered abstractions of algorithms that are increasingly running multiple aspects of our lives. the article dives into the world of black box systems that have become opaque to analysis because of their stratified complexity leading to situations with unpredictable outcomes. this was exemplified when an autonomous vehicle crashed into a crossing pedestrian and it took months of post-hoc analysis to figure out what went wrong. when we talk about intelligence in the case of these machines, we're using it in a very loose sense, like the term "friend" on facebook, which has a range of interpretations from your best friend to a random acquaintance. both terms convey a greater sense of meaning than is actually true. when such systems run amok, they have the potential to cause significant harm, case in point being the flash crashes the financial markets experienced because of the competitive behaviour of high frequency trading firm algorithms facing off against each other in the market. something similar has happened on amazon where items get priced in an unrealistic fashion because of buying and pricing patterns triggered by automated systems. while in a micro context the algorithms and their working are transparent and explainable, when they come together in an ecosystem, like finance, they lead to an emergent complexity that has behaviour that can't be predicted ahead of time with a great amount of certainty. but, such justifications can't be used as a cover for evading responsibility when it comes to mitigating harms. existing laws need to be refined and amended so that they can better meet the demands of new technology where allocation of responsibility is a fuzzy concept. ai systems are different from other software systems when it comes to security vulnerabilities. while traditional cybersecurity mechanisms rely heavily on securing the perimeter, ai security vulnerabilities run deeper and they can be manipulated through their interactions with the real world -the very mechanism that makes them intelligent systems. numerous examples of utilizing audio samples from tv commercials to trigger voice assistants have demonstrated new attack surfaces for which we need to develop defense techniques. visual systems are also fooled, especially in av systems where, according to one example, manipulating stop signs on the road with innocuous stripes of tape make it seem like the stop sign is a speed indicator and can cause fatal crashes. there are also examples of hiding these adversarial examples under the guise of white noise and other imperceptible changes to the human senses. we need to think of ai systems as inherently socio-technical to come up with effective protection techniques that don't just rely on technical measures but also look at the human factors surrounding them. some other useful insights are to utilize abusability testing, red-teaming, white hacking, bug bounty programs, and consulting with civic society advocates who have deep experience with the interactions of vulnerable communities with technology. reinforcement systems are increasingly moving from applications to beating human performance in games to safety-critical applications like self-driving cars and automated trading. a lack of robustness in the systems can lead to catastrophic failures like the $460m lost by knight capital and the harms to pedestrian and driver safety in the case of autonomous vehicles. rl systems that perform well under normal conditions can be vulnerable to adversarial agents that can exploit the brittleness of the systems when it comes to natural shifts in distributions and more carefully crafted attacks. in prior threat models, the assumptions for the adversary are that they can modify directly the inputs going into the rl agent but that is not very realistic. instead, here the authors focus more on a shared environment through which the adversary creates indirect impact on the target rl agent leading to undesirable behavior. for agents that are trained through self-play (which is a rough approximation of nash equilibrium), they are vulnerable to adversarial policies. as an example, masked victims are more robust to modifications in the natural observations by the adversary but that lowers the performance in the average case. furthermore, what the researchers find is that there is a non-transitive behavior between self-play opponent, masked victim, adversarial opponent and normal victim in that cyclic order. self-play being normally transitive in nature, especially when mimicking real-world scenarios is then no doubt vulnerable to these non-transitive styled attacks. thus, there is a need to move beyond self-play and apply iteratively adversarial training defense and population based training methods so that the target rl agent can become robust to a wider variety of scenarios. vehicle safety is something of paramount importance in the automotive industry as there are many tests conducted to test for crash resilience and other physical safety features before it is released to people. but, the same degree of scrutiny is not applied to the digital and connected components of cars. researchers were able to demonstrate successful proof of concept hacks that compromised vehicle safety. for example, with the polo, they were able to access the controller area network (can) which sends signals and controls a variety of aspects related to driving functions. given how the infotainment systems were updated, researchers were able to gain access into the personal details of the driver. they were also able to utilize the shortcomings in the operation of the key fob to gain access to the vehicle without leaving a physical trace. other hacks that were tried included being able to access and influence the collision monitoring radar system and the tire-pressure monitoring system which both have critical implications for passenger safety. on the focus, they found wifi details including the password for their production line in detroit, michigan. on purchasing a second-hand infotainment unit for purposes of reverse-engineering the firmware, they found the previous owner's home wifi details, phone contacts and a host of other personal information. cars store a lot of personal information including tracking information which, as stated on the privacy policy, can be shared with affiliates which can have other negative consequences like changes in insurance premiums based on driving behaviour. europe will have some forthcoming regulations for connected car safety but those are currently slated for release in 2021. we've all experienced specification gaming even if we haven't really heard the term before. in law, you call it following the law to the letter but not in spirit. in sports, it is called unsportsman-like to use the edge cases and technicalities of the rules of the game to eke out an edge when it is obvious to everyone playing the game that the rules intended for something different. this can also happen in the case of ai systems, for example in reinforcement learning systems where the agent can utilize "bugs" or poor specification on the part of the human creators to achieve the high rewards for which it is optimizing without actually achieving the goal, at least in the way the developers intended them to and this can sometimes lead to unintended consequences that can cause a lot of harms. "let's look at an example. in a lego stacking task, the desired outcome was for a red block to end up on top of a blue block. the agent was rewarded for the height of the bottom face of the red block when it is not touching the block. instead of performing the relatively difficult maneuver of picking up the red block and placing it on top of the blue one, the agent simply flipped over the red block to collect the reward. this behaviour achieved the stated objective (high bottom face of the red block) at the expense of what the designer actually cares about (stacking it on top of the blue one)". this isn't because of a flaw in the rl system but more so a misspecification of the objective. as the agents become more capable, they find ever-more clever ways of achieving the rewards which can frustrate the creators of the system. this makes the problem of specification gaming very relevant and urgent as we start to deploy these systems in a lot of real-world situations. in the rl context, task specification refers to the design of the rewards, the environment and any other auxiliary rewards. when done correctly, we get true ingenuity out of these systems like move 37 from the alphago system that baffled humans and ushered a new way of thinking about the game of go. but, this requires discernment on the part of the developers to be able to judge when you get a case like lego vs. move 37. as an example in the real-world, reward tampering is an approach where the agent in a traffic optimization system with an interest in achieving a high reward can manipulate the driver into going to alternate destinations instead of what they desired just to achieve a higher reward. specification gaming isn't necessarily bad in the sense that we want the systems to come up with ingenious ways to solve problems that won't occur to humans. sometimes, the inaccuracies can arise in how humans provide feedback to the system while it is training. ''for example, an agent performing a grasping task learned to fool the human evaluator by hovering between the camera and the object." incorrect reward shaping, where an agent is provided rewards along the way to achieving the final reward can also lead to edge-case behaviours when it is not analyzed for potential side-effects. we see such examples happen with humans in the real-world as well: a student asked to get a good grade on the exam can choose to copy and cheat and while that achieves the goal of getting a good grade, it doesn't happen in the way we intended for it to. thus, reasoning through how a system might game some of the specifications is going to be an area of key concern going into the future. the ongoing pandemic has certainly accelerated the adoption of technology in everything from how we socialize to buying groceries and doing work remotely. the healthcare industry has also been rapid in adapting to meet the needs of people and technology has played a role in helping to scale care to more people and accelerate the pace with which the care is provided. but, this comes with the challenge of making decisions under duress and with shortened timelines within which to make decisions on whether to adopt a piece of technology or not. this has certainly led to issues where there are risks of adopting solutions that haven't been fully vetted and using solutions that have been repurposed from prior uses that were approved to now combat covid-19. especially with ai-enabled tools, there are increased risks of emergent behavior that might not have been captured by the previous certification or regulatory checks. the problems with ai solutions don't just go away because there is a pandemic and shortcutting the process of proper due diligence can lead to more harm than the benefits that they bring. one must also be wary of the companies that are trying to capitalize on the chaos and pass through solutions that don't really work well. having technical staff during the procurement process that can look over the details of what is being brought into your healthcare system needs to be a priority. ai can certainly help to mitigate some of the harms that covid-19 is inflicting on patients but we must keep in mind that we're not looking to bypass privacy concerns that come with processing vast quantities of healthcare data. in the age of adversarial machine learning (maiei has a learning community on machine learning security if you'd like to learn more about this area) there are enormous concerns with protecting software infrastructure as ml opens up a new attack surface and new vectors which are seldom explored. from the perspective of insurance, there are gaps in terms of what cyber-insurance covers today, most of it being limited to the leakage of private data. there are two kinds of attacks that are possible on ml systems: intentional and unintentional. intentional attacks are those that are executed by malicious agents who attempt to steal the models, infer private data or get the ai system to behave in a way that favors their end goals. for example, when tumblr decided to not host pornographic content, creators bypassed that by using green screens and pictures of owls to fool the automated content moderation system. unintended attacks can happen when the goals of the system are misaligned with what the creators of the system actually intended, for example, the problem of specification gaming, something that abhishek gupta discussed here in this fortune article. in interviewing several officers in different fortune 500 companies, the authors found that there are 3 key problems in this domain at the moment: the defenses provided by the technical community have limited efficacy, existing copyright, product liability, and anti-hacking laws are insufficient to capture ai failure modes. lastly, given that this happens at a software level, cyber-insurance might seem to be the way to go, yet current offerings only cover a patchwork of the problems. business interruptions and privacy leaks are covered today under cyber-insurance but other problems like bodily harm, brand damage, and property damage are for the most part not covered. in the case of model recreation, as was the case with the openai gpt-2 model, prior to it being released, it was replicated by external researchers -this might be covered under cyber-insurance because of the leak of private information. researchers have also managed to steal information from facial recognition databases using sample images and names which might also be covered under existing policies. but, in the case with uber where there was bodily harm because of the self-driving vehicle that wasn't able to detect the pedestrian accurately or similar harms that might arise if conditions are foggy, snowy, dull lighting, or any other out-of-distribution scenarios, these are not adequately covered under existing insurance terms. brand damage that might arise from poisoning attacks like the case with the tay chatbot or confounding anti-virus systems as was the case with an attack mounted against the cylance system, cyber-insurance falls woefully short in being able to cover these scenarios. in a hypothetical situation as presented in a google paper on rl agents where a cleaning robot sticks a wet mop into an electric socket, material damage that occurs from that might also be considered out of scope in cyber-insurance policies. traditional software attacks are known unknowns but adversarial ml attacks are unknown unknowns and hence harder to guard against. current pricing reflects this uncertainty, but as the ai insurance market matures and there is a deeper understanding for what the risks are and how companies can mitigate the downsides, the pricing should become more reflective of the actual risks. the authors also offer some recommendations on how to prepare the organization for these risks -for example by appointing an officer that works closely with the ciso and chief data protection officer, performing table-top exercises to gain an understanding of potential places where the system might fail and evaluating the system for risks and gaps following guidelines as put forth in the eu trustworthy ai guidelines. there are no widely accepted best practices for mitigating security and privacy issues related to machine learning (ml) systems. existing best practices for traditional software systems are insufficient because they're largely based on the prevention and management of access to a system's data and/or software, whereas ml systems have additional vulnerabilities and novel harms that need to be addressed. for example, one harm posed by ml systems is to individuals not included in the model's training data but who may be negatively impacted by its inferences. harms from ml systems can be broadly categorized as informational harms and behavioral harms. informational harms "relate to the unintended or unanticipated leakage of information." the "attacks" that constitute informational harms are: behavioral harms "relate to manipulating the behavior of the model itself, impacting the predictions or outcomes of the model." the attacks that constitute behavioral harms are: â�¢ poisoning: inserting malicious data into a model's training data to change its behavior once deployed â�¢ evasion: feeding data into a system to intentionally cause misclassification without a set of best practices, ml systems may not be widely and/or successfully adopted. therefore, the authors of this white paper suggest a "layered approach" to mitigate the privacy and security issues facing ml systems. approaches include noise injection, intermediaries, transparent ml mechanisms, access controls, model monitoring, model documentation, white hat or red team hacking, and open-source software privacy and security resources. finally, the authors note, it's important to encourage "cross-functional communication" between data scientists, engineers, legal teams, business managers, etc. in order to identify and remediate privacy and security issues related to ml systems. this communication should be ongoing, transparent, and thorough. beyond near-and long-term: towards a clearer account of this paper dives into how researchers can clearly communicate about their research agendas given ambiguities in the split of the ai ethics community into near and long term research. often a sore and contentious point of discussion, there is an artificial divide between the two groups that seem to take a reductionist approach to the work being done by the other. a major problem emerging from such a divide is a hindrance in being able to spot relevant work being done by the different communities and thus affecting effective collaboration. the paper highlights the differences arising primarily along the lines of timescale, ai capabilities, deeper normative and empirical disagreements. the paper provides for a helpful distinction between near-and long-term by describing them as follows: â�¢ near term issues are those that are fairly well understood and have concrete examples and relate to rãªvent progress in the field of machine learning â�¢ long term issues are those that might arise far into the future and due to much more advanced ai systems with broad capabilities, it also includes long term impacts like international security, race relations, and power dynamics what they currently see is that: â�¢ issues considered 'near-term' tend to be those arising in the present/near future as a result of current/foreseeable ai systems and capabilities, on varying levels of scale/severity, which mostly have immediate consequences for people and society. â�¢ issues considered 'long-term' tend to be those arising far into the future as a result of large advances in ai capabilities (with a particular focus on notions of transformative ai or agi), and those that are likely to pose risks that are severe/large in scale with very long-term consequences. â�¢ the binary clusters are not sufficient as a way to split the field and not looking at underlying beliefs leads to unfounded assumptions about each other's work â�¢ in addition there might be areas between the near and long term that might be neglected as a result of this artificial fractions unpacking these distinctions can be done along the lines of capabilities, extremity, certainty and impact, definitions for which are provided in the paper. a key contribution aside from identifying these factors is that they lie along a spectrum and define a possibility space using them as dimensions which helps to identify where research is currently concentrated and what areas are being ignored. it also helps to well position the work being done by these authors. something that we really appreciated from this work was the fact that it gives us concrete language and tools to more effectively communicate about each other's work. as part of our efforts in building communities that leverage diverse experiences and backgrounds to tackle an inherently complex and muti-dimensional problem, we deeply appreciate how challenging yet rewarding such an effort can be. some of the most meaningful public consultation work done by maiei leveraged our internalized framework in a similar vein to provide value to the process that led to outcomes like the montreal declaration for responsible ai. the rise of ai systems leads to an unintended conflict between economic pursuits which seek to generate profits and value resources appropriately with the moral imperatives of promoting human flourishing and creating societal benefits from the deployment of these systems. this puts forth a central question on what the impacts of creating ai systems that might surpass humans in a general sense which might leave humans behind. technological progress doesn't happen on its own, it is driven by conscious human choices that are influenced by the surrounding social and economic institutions. we are collectively responsible for how these institutions take shape and thus impact the development of technology -submitting to technological-fatalism isn't a productive way to align our ethical values with this development. we need to ensure that we play an active role in the shaping of the most consequential piece of technology. while the economic system relies on the market prices to gauge what people place value on, by no means is that a comprehensive evaluation. for example, it misses out on the impact of externalities which can be factored in by considering ethical values as a complement in guiding our decisions on what to build and how to place value on it. when thinking about losses from ai-enabled automation, an outright argument that economists might make is that if replacing labor lowers the costs of production, then it might be market-efficient to invest in technology that achieves that. from an ethicist's perspective, there are severe negative externalities from job loss and thus it might be unethical to impose labor-saving automation on people. when unpacking the economic perspective more, we find that job loss actually isn't correctly valued by wages as price for labor. there are associated social benefits like the company of workplace colleagues, sense of meaning and other social structural values which can't be separately purchased from the market. thus, using a purely economic perspective in making automation technology adoption decisions is an incomplete approach and it needs to be supplemented by taking into account the ethical perspective. market price signals provide useful information upto a certain point in terms of the goods and services that society places value on. suppose that people start to demand more eggs from chickens that are raised in a humane way, then suppliers will shift their production to respond to that market signal. but, such price signals can only be indicated by consumers for the things that they can observe. a lot of unethical actions are hidden and hence can't be factored into market price signals. additionally, several things like social relations aren't tradable in a market and hence their value can't be solely determined from the market viewpoint. thus, both economists and ethicists would agree that there is value to be gained in steering the development of ai systems keeping in mind both kinds of considerations. pure market-driven innovation will ignore societal benefits in the interest of generating economic value while the labor will have to make unwilling sacrifices in the interest of long-run economic efficiency. economic market forces shape society significantly, whether we like it or not. there are professional biases based on selection and cognition that are present in either side making its arguments as to which gets to dominate based on their perceived importance. the point being that bridging the gap between different disciplines is crucial to arriving at decisions that are grounded in evidence and that benefit society holistically. there are also differences fundamentally between the economic and ethical perspective -namely that economic indicators are usually unidimensional and have clear quantitative values that make them easier to compare. on the other hand, ethical indicators are inherently multi-dimensional and are subjective which not only make comparison hard but also limit our ability to explain how we arrive at them. they are encoded deep within our biological systems and suffer from the same lack of explainability as decisions made by artificial neural networks, the so-called black box problem. why is it then, despite the arguments above, that the economic perspective dominates over the ethical one? this is largely driven by the fact that economic values provide clear, unambiguous signals which our brains, preferring ambiguity aversion, enjoy and ethical values are more subtle, hidden, ambiguous indicators which complicate decision making. secondly, humans are prosocial only upto a point, they are able to reason between economic and ethical decisions at a micro-level because the effects are immediate and observable, say for example polluting the neighbor's lawn and seeing the direct impact of that activity. on the other hand, for things like climate change where the effects are delayed and not directly observable (as a direct consequence of one's actions) that leads to behaviour where the individual prioritizes economic values over ethical ones. cynical economists will argue that there is a comparative advantage in being immoral that leads to gains in exchange, but that leads to a race to the bottom in terms of ethics. externalities are an embodiment of the conflict between economic and ethical values. welfare economics deals with externalities via various mechanisms like permits, taxes, etc. to curb the impacts of negative externalities and promote positive externalities through incentives. but, the rich economic theory needs to be supplemented by political, social and ethical values to arrive at something that benefits society at large. from an economic standpoint, technological progress is positioned as expanding the production possibilities frontier which means that it raises output and presumably standards of living. yet, this ignores how those benefits are distributed and only looks at material benefits and ignores everything else. prior to the industrial revolution, people were stuck in a malthusian trap whereby technological advances created material gains but these were quickly consumed by population growth that kept standards of living stubbornly low. this changed post the revolution and as technology improvement outpaced population growth, we got better quality of life. the last 4 decades have had a mixed experience though, whereby automation has eroded lower skilled jobs forcing people to continue looking for jobs despite displacement and the lower demand for unskilled labor coupled with the inelastic supply of labor has led to lower wages rather than unemployment. on the other hand, high skilled workers have been able to leverage technological progress to enhance their output considerably and as a consequence the income and wealth gaps between low and high skilled workers has widened tremendously. typical economic theory points to income and wealth redistribution whenever there is technological innovation where the more significant the innovation, the larger the redistribution. something as significant as ai leads to crowning of new winners who own these new factors of production while also creating losers when they face negative pecuniary externalities. these are externalities because there isn't explicit consent that is requested from the people as they're impacted in terms of capital, labor and other factors of production. the distribution can be analyzed from the perspective of strict utilitarianism (different from that in ethics where for example bentham describes it as the greatest amount of good for the greatest number of people). here it is viewed as tolerating income redistribution such that it is acceptable if all but one person loses income as long as the single person making the gain has one that is higher than the sum of the losses. this view is clearly unrealistic because it would further exacerbate inequities in society. the other is looking at the idea of lump sum transfers in which the idealized scenario is redistribution, for example by compensating losers from technology innovation, without causing any other market distortions. but, that is also unrealistic because such a redistribution never occurs without market distortions and hence it is not an effective way to think about economic policy. from an ethics perspective, we must make value judgments on how we perceive dollar losses for a lower socio-economic person compared to the dollar gains made by a higher socio-economic person and if that squares with the culture and value set of that society. we can think about the tradeoff between economic efficiency and equality in society, where the level of tolerance for inequality varies by the existing societal structures in place. one would have to also reason about how redistribution creates more than proportional distortions as it rises and how much economic efficiency we'd be willing to sacrifice to make gains in how equitably income is distributed. thus, steering progress in ai can be done based on whether we want to pursue innovation that we know is going to have negative labor impacts while knowing full well that there aren't going to be any reasonable compensations offered to the people based on economic policy. given the pervasiveness of ai and by virtue of it being a general-purpose technology, the entrepreneurs and others powering innovation need to take into account that their work is going to shape larger societal changes and have impacts on labor. at the moment, the economic incentives are such that they steer progress towards labor-saving automation because labor is one of the most highly-taxed factors of production. instead, shifting the tax-burden to other factors of production including automation capital will help to steer the direction of innovation in other directions. government, as one of the largest employers and an entity with huge spending power, can also help to steer the direction of innovation by setting policies that encourage enhancing productivity without necessarily replacing labor. there are novel ethical implications and externalities that arise from the use of ai systems, an example of that would be (from the industrial revolution) that a factory might lead to economic efficiency in terms of production but the pollution that it generates is so large that the social cost outweighs the economic gain. biases can be deeply entrenched in the ai systems, either from unrepresentative datasets, for example, with hiring decisions that are made based on historical data. but, even if the datasets are well-represented and have minimal bias, and the system is not exposed to protected attributes like race and gender, there are a variety of proxies like zipcode which can lead to unearthing those protected attributes and discriminating against minorities. maladaptive behaviors can be triggered in humans by ai systems that can deeply personalize targeting of ads and other media to nudge us towards different things that might be aligned with making more profits. examples of this include watching videos, shopping on ecommerce platforms, news cycles on social media, etc. conversely, they can also be used to trigger better behaviors, for example, the use of fitness trackers that give us quantitative measurements for how we're taking care of our health. an economics equivalent of the paper clip optimizer from bostrom is how human autonomy can be eroded over time as economic inequality rises which limits control of those who are displaced over economic resources and thus, their control over their destinies, at least from an economic standpoint. this is going to only be exacerbated as ai starts to pervade into more and more aspects of our lives. labor markets have features built in them to help tide over unemployment with as little harm as possible via quick hiring in other parts of the economy when the innovation creates parallel demands for labor in adjacent sectors. but, when there is large-scale disruption, it is not possible to accommodate everyone and this leads to large economic losses via fall in aggregate demand which can't be restored with monetary or fiscal policy actions. this leads to wasted economic potential and welfare losses for the workers who are displaced. whenever there is a discrepancy between ethical and economic incentives, we have the opportunity to steer progress in the right direction. we've discussed before how market incentives trigger a race to the bottom in terms of morality. this needs to be preempted via instruments like technological impact assessments, akin to environmental impact assessments, but often the impacts are unknown prior to the deployment of the technology at which point we need to have a multi-stakeholder process that allows us to combat harms in a dynamic manner. political and regulatory entities typically lag technological innovation and can't be relied upon solely to take on this mantle. the author raises a few questions on the role of humans and how we might be treated by machines in case of the rise of superintelligence (which still has widely differing estimates for when it will be realized from the next decade to the second half of this century). what is clear is that the abilities of narrow ai systems are expanding and it behooves us to give some thought to the implications on the rise of superintelligence. the potential for labor-replacement in this superintelligence scenario, from an economic perspective, would have significant existential implications for humans, beyond just inequality, we would be raising questions of human survival if the wages to be paid to labor fall below subsistence levels in a wide manner. it would be akin to how the cost of maintaining oxen to plough fields was outweighed by the benefits that they brought in the face of mechanization of agriculture. this might be an ouroboros where we become caught in the malthusian trap again at the time of the industrial revolution and no longer have the ability to grow beyond basic subsistence, even if that would be possible. authors of research papers aspire to achieving any of the following goals when writing papers: to theoretically characterize what is learnable, to obtain understanding through empirically rigorous experiments, or to build working systems that have high predictive accuracy. to communicate effectively with the readers, the authors must: provide intuitions to aid the readers' understanding, describe empirical investigations that consider and rule out alternative hypotheses, make clear the relationship between theoretical analysis and empirical findings, and use clear language that doesn't conflate concepts or mislead the reader. the authors of this paper find that there are 4 areas where there are concerns when it comes to ml scholarship: failure to distinguish between speculation and explanation, failure to identify the source of empirical gains, the use of mathematics that obfuscates or impresses rather than clarifies, and misuse of language such that terms with other connotations are used or by overloading terms with existing technical definitions. flawed techniques and communication methods will lead to harm and wasted resources and efforts hindering the progress in ml and hence this paper provides some very practical guidance on how to do this better. when presenting speculations or opinions of authors that are exploratory and don't yet have scientific grounding, having a separate section that quarantines the discussion and doesn't bleed into the other sections that are grounded in theoretical and empirical research helps to guide the reader appropriately and prevents conflation of speculation and explanation. the authors provide the example of the paper on dropout regularization that made comparisons and links to sexual reproduction but limited that discussion to a "motivation" section. using mathematics in a manner where natural language and mathematical expositions are intermixed without a clear link between the two leads to weakness in the overall contribution. specifically, when natural language is used to overcome weaknesses in the mathematical rigor and conversely, mathematics is used as a scaffolding to prop up weak arguments in the prose and give the impression of technical depth, it leads to poor scholarship and detracts from the scientific seriousness of the work and harms the readers. additionally, invoking theorems with dubious pertinence to the actual content of the paper or in overly broad ways also takes away from the main contribution of a paper. in terms of misuse of language, the authors of this paper provide a convenient ontology breaking it down into suggestive definitions, overloaded terminology, and suitcase words. in the suggestive definitions category, the authors coin a new technical term that has suggestive colloquial meanings and can slip through some implications without formal justification of the ideas in the paper. this can also lead to anthropomorphization that creates unrealistic expectations about the capabilities of the system. this is particularly problematic in the domain of fairness and other related domains where this can lead to conflation and inaccurate interpretation of terms that have well-established meanings in the domains of sociology and law for example. this can confound the initiatives taken up by both researchers and policymakers who might use this as a guide. overloading of technical terminology is another case where things can go wrong when terms that have historical meanings and they are used in a different sense. for example, the authors talk about deconvolutions which formally refers to the process of reversing a convolution but in recent literature has been used to refer to transpose convolutions that are used in auto-encoders and gans. once such usage takes hold, it is hard to undo the mixed usage as people start to cite prior literature in future works. additionally, combined with the suggestive definitions, we run into the problem of concealing a lack of progress, such as the case with using language understanding and reading comprehension to now mean performance on specific datasets rather than the grand challenge in ai that it meant before. another case that leads to overestimation of the ability of these systems is in using suitcase words which pack in multiple meanings within them and there isn't a single agreed upon definition. interpretability and generalization are two such terms that have looser definitions and more formally defined ones, yet because papers use them in different ways, it leads to miscommunication and researchers talking across each other. the authors identify that these problems might be occurring because of a few trends that they have seen in the ml research community. specifically, complacency in the face of progress where there is an incentive to excuse weak arguments in the face of strong empirical results and the single-round review process at various conferences where the reviewers might not have much choice but to accept the paper given the strong empirical results. even if the flaws are noticed, there isn't any guarantee that they are fixed in a future review cycle at another conference. as the ml community has experienced rapid growth, the problem of getting high-quality reviews has been exacerbated: in terms of the number of papers to be reviewed by each reviewer and the dwindling number of experienced reviewers in the pool. with the large number of papers, each reviewer has less time to analyze papers in depth and reviewers who are less experienced can fall easily into some of the traps that have been identified so far. thus, there are two levers that are aggravating the problem. additionally, there is the risk of even experienced researchers resorting to a checklist-like approach under duress which might discourage scientific diversity when it comes to papers that might take innovative or creative approaches to expressing their ideas. a misalignment in incentives whereby lucrative deals in funding are offered to ai solutions that utilize anthropomorphic characterizations as a mechanism to overextend their claims and abilities though the authors recognize that the causal direction might be hard to judge. the authors also provide suggestions for other authors on how to evade some of these pitfalls: asking the question of why something happened rather than just relying on how well a system performed will help to achieve the goal of providing insights into why something works rather than just relying on headline numbers from the results of the experiments. they also make a recommendation for insights to follow the lines of doing error analysis, ablation studies, and robustness checks and not just be limited to theory. as a guideline for reviewers and journal editors, making sure to strip out extraneous explanations, exaggerated claims, changing anthropomorphic naming to more sober alternatives, standardizing notation, etc. should help to curb some of the problems. encouraging retrospective analysis of papers is something that is underserved at the moment and there aren't enough strong papers in this genre yet despite some avenues that have been advocating for this work. flawed scholarship as characterized by the points as highlighted here not only negatively impact the research community but also impact the policymaking process that can overshoot or undershoot the mark. an argument can be made that setting the bar too high will impede new ideas being developed and slow down the cycle of reviews and publication while consuming precious resources that could be deployed in creating new work. but, asking basic questions to guide us such as why something works, in which situations it does not work, and have the design decisions been justified will lead to a higher quality of scholarship in the field. the article summarizes recent work from several microsoft researchers on the subject of making ai ethics checklists that are effective. one of the most common problems identified relate to the lack of practical applicability of ai ethics principles which sound great and comprehensive in the abstract but do very little to aid engineers and practitioners from applying them in their day to day work. the work was done by interviewing several practitioners and advocating for a co-design process that brings in intelligence on how to make these tools effective from other disciplines like healthcare and aviation. one of the things emerging from the interviews is that often engineers are few and far between in raising concerns and there's a lack of top-down sync in enforcing these across the company. additionally, there might be social costs to bringing up issues which discourages engineers from implementing such measures. creating checklists that reduce friction and fit well into existing workflows will be key in their uptake. for a lot of people who are new to the field of artificial intelligence and especially ai ethics, they see existential risk as something that is immediate. others dismiss it as something to not be concerned about at all. there is a middle path here and this article sheds a very practical light on that. using the idea of canaries in a coal mine, the author goes on to highlight some potential candidates for a canary that might help us judge better when we ought to start paying attention to these kinds of risks posed by artificial general intelligence systems. the first one is the automatic formulation of learning problems, akin to how humans have high-level goals that they align with their actions and adjust them based on signals that they receive on the success or failure of those actions. ai systems trained in narrow domains don't have this ability just yet. the second one mentioned in the article is achieving fully autonomous driving, which is a good one because we have lots of effort being directed to make that happen and it requires a complex set of problems to be addressed including the ability to make real-time, life-critical decisions. ai doctors are pointed out as a third canary, especially because true replacement of doctors would require a complex set of skills spanning the ability to make decisions about a patient's healthcare plan by analyzing all their symptoms, coordinating with other doctors and medical staff among other human-centered actions which are currently not feasible for ai systems. lastly, the author points to the creation of conversation systems that are able to answer complex queries and respond to things like exploratory searches. we found the article to put forth a meaningful approach to reasoning about existential risk when it comes to ai systems. a lot of articles pitch development, investment and policymaking in ai as an arms race with the us and china as front-runners. while there are tremendous economic gains to be had in deploying and utilizing ai for various purposes, there remain concerns of how this can be used to benefit society more than just economically. a lot of ai strategies from different countries are thus focused on issues of inclusion, ethics and more that can drive better societal outcomes yet they differ widely in how they seek to achieve those goals. for example, ai has put forth a national ai strategy that is focused on economic growth and social inclusion dubbed #aiforall while the strategy from china has been more focused on becoming a global dominant force in ai which is backed by state investments. some countries have instead chosen to focus on creating strong legal foundations for the ethical deployment of ai while others are more focused on data protection rights. canada and france have entered into agreements to work together on ai policy which places talent, r&d and ethics at the center. the author of the article makes a case for how global coordination of ai strategies might lead to even higher gains but also recognizes that governments will be motivated to tailor their policies to best meet the requirements of their countries first and then align with others that might have similar goals. reproducibility is of paramount importance to doing rigorous research and a plethora of fields have suffered from a crisis where scientific work hasn't met muster in terms of reproducibility leading to wasted time and effort on the part of other researchers looking to build upon each other's work. the article provides insights from the work of a researcher who attempted a meta-science approach to trying to figure out what constitutes good, reproducible research in the field of machine learning. there is a distinction made early on in terms of replicability which hinges on taking someone else's code and running that on the shared data to see if you get the same results but as pointed out in the article, that suffers from issues of source and code bias which might be leveraging certain peculiarities in terms of configurations and more. the key tenets to reproducibility are being able to simply read a scientific paper and set up the same experiment, follow the steps prescribed and arrive at the same results. arriving at the final step is dubbed as independent reproducibility. the distinction between replicability and reproducibility also speaks to the quality of the scientific paper in being able to effectively capture the essence of the contribution such that anyone else is able to do the same. some of the findings from this work include that having hyperparameters well specified in the paper and its ease of readability contributed to the reproducibility. more specification in terms of math might allude to more reproducibility but it was found to not necessarily be the case. empirical papers were inclined to be more reproducible but could also create perverse incentives and side effects. sharing code is not a panacea and requires other accompanying factors to make the work really reproducible. cogent writing was found to be helpful along with code snippets that were either actual or pseudo code though step code that referred to other sections hampered reproducibility because of challenges in readability. simplified examples while appealing didn't really aid in the process and spoke to the meta-science process calling for data-driven approaches to ascertaining what works and what doesn't rather than relying on hunches. also, posting revisions to papers and being reachable over email to answer questions helped the author in reproducing the research work. finally, the author also pointed out that given this was a single initiative and was potentially biased in terms of their own experience, background and capabilities, they encourage others to tap into the data being made available but these guidelines provide good starting points for people to attempt to make their scientific work more rigorous and reproducible. the push has been to apply ai to any new problem that we face, hoping that the solution will magically emerge from the application of the technique as if it is a dark art. yet, the more seasoned scientists have seen these waves come and go and in the past, a blind trust in this technology led to ai winters. taking a look at some of the canaries in the coal mine, the author cautions that there might be a way to judge whether ai will be helpful with the pandemic situation. specifically, looking at whether domain experts, like leading epidemiologists endorse its use and are involved in the process of developing and utilizing these tools will give an indication as to whether they will be successful or not. data about the pandemic depends on context and without domain expertise, one has to make a lot of assumptions which might be unfounded. all models have to make assumptions to simplify reality, but if those assumptions are rooted in domain expertise from the field then the model can mimic reality much better. without context, ai models assume that the truth can be gleaned solely from the data, which though it can lead to surprising and hidden insights, at times requires humans to evaluate the interpretations to make meaning from them and apply them to solve real-world problems. this was demonstrated with the case where it was claimed that ai had helped to predict the start of the outbreak, yet the anomaly required the analysis from a human before arriving at that conclusion. claims of extremely high accuracy rates will give hardened data scientists reason for caution, especially when moving from lab to real-world settings as there is a lot more messiness with real-world data and often you encounter out-of-distribution data which hinders the ability of the model to make accurate predictions. for ct scans, even if they are sped up tremendously by the use of ai, doctors point out that there are other associated procedures such as the cleaning and filtration and recycling of air in the room before the next patient can be passed through the machine which can dwindle the gains from the use of an unexplainable ai system analyzing the scans. concerns with the use of automated temperature scanning using thermal cameras also suffers from similar concerns where there are other confounding factors like the ambient temperature, humidity, etc. which can limit the accuracy of such a system. ultimately, while ai can provide tremendous benefits, we mustn't blindly be driven by its allure to magically solve the toughest challenges that we face. offering an interesting take on how to shape the development and deployment of ai technologies, mhlambi utilizes the philosophy of ubuntu as a guiding light in how to build ai systems that better empower people and communities. the current western view that dominates how ai systems are constructed today and how they optimize for efficiency is something that lends itself quite naturally to inequitable outcomes and reinforcing power asymmetries and other imbalances in society. embracing the ubuntu mindset which puts people and communities first stands in contrast to this way of thinking. it gives us an alternative conception of personhood and has the potential to surface some different results. while being thousands of years old, the concept has been seen in practice over and over again, for example, in south africa, after the end of the apartheid, the truth and reconciliation program forgave and integrated offenders back into society rather than embark on a kantian or retributive path to justice. this restorative mindset to justice helped the country heal more quickly because the philosophy of ubuntu advocates that all people are interconnected and healing only happens when everyone is able to move together in a harmonious manner. this was also seen in the aftermath of the rwanda genocide, where oppressors were reintegrated back into society often living next to the people that they had hurt; ubuntu believes that no one is beyond redemption and everyone deserves the right to have their dignity restored. bringing people together through community is important, restorative justice is a mechanism that makes the community stronger in the long run. current ai formulation seeks to find some ground truth but thinking of this in the way of ubuntu means that we try to find meaning and purpose for these systems through the values and beliefs that are held by the community. ubuntu has a core focus on equity and empowerment for all and thus the process of development is slow but valuing people above material efficiency is more preferable than speeding through without thinking of the consequences that it might have on people. living up to ubuntu means offering people the choice for what they want and need, rooting out power imbalances and envisioning the companies as a part of the communities for which they are building products and services which makes them accountable and committed to the community in empowering them. ethics in the context of technology carries a lot of weight, especially because the people who are defining what it means will influence the kinds of interventions that will be implemented and the consequences that follow. given that technology like ai is used in high-stakes situations, this becomes even more important and we need to ask questions about the people who take this role within technology organizations, how they take corporate and public values and turn them into tangible outcomes through rigorous processes, and what regulatory measures are required beyond these corporate and public values to ensure that ethics are followed in the design, development and deployment of these technologies. ethics owners, the broad term for people who are responsible for this within organizations have a vast panel of responsibilities including communication between the ethics review committees and product design teams, aligning the recommendations with the corporate and public values, making sure that legal compliance is met and communicating externally about the processes that are being adopted and their efficacy. ethical is a polysemous word in that it can refer to process, outcomes, and values. the process refers to the internal procedures that are adopted by the firm to guide decision making on product/service design and development choices. the values aspect refers to the value set that is both adopted by the organization and those of the public within which the product/service might be deployed. this can include values such as transparency, equity, fairness, privacy, among others. the outcomes refer to desirable properties in the outputs from the system such as equalized odds across demographics and other fairness metrics. in the best case, inside a technology company, there are robust and well-managed processes that are aligned with collaboratively-determined ethical outcomes that achieve the community's and organization's ethical values. from the outside, this takes on the meaning of finding mechanisms to hold the firms accountable for the decisions that they take. further expanding on the polysemous meanings of ethics, it can be put into four categories for the discussion here: moral justice, corporate values, legal risk, and compliance. corporate values set the context for the rest of the meanings and provide guidance when tradeoffs need to be made in product/service design. they also help to shape the internal culture which can have an impact on the degree of adherence to the values. legal risk's overlap with ethics is fairly new whereas compliance is mainly concerned with the minimization of exposure to being sued and public reputation harm. using some of the framing here, the accolades, critiques, and calls to action can be structured more effectively to evoke substantive responses rather than being diffused in the energies dedicated to these efforts. framing the metaphor of "data is the new oil" in a different light, this article gives some practical tips on how organizations can reframe their thinking and relationship with customer data so that they take on the role of being a data custodian rather than owners of the personal data of their customers. this is put forth with the acknowledgement that customers' personal data is something really valuable that brings business upsides but it needs to be handled with care in the sense that the organization should act as a custodian that is taking care of the data rather than exploiting it for value without consent and the best interests of the customer at heart. privacy breaches that can compromise this data not only lead to fines under legislation like the gdpr, but also remind us that this is not just data but details of a real human being. as a first step, creating a data accountability report that documents how many times personal data was accessed by various employees and departments will serve to highlight and provide incentives for them to change behaviour when they see that some others might be achieving their job functions without the need to access as much information. secondly, celebrating those that can make do with minimal access will also encourage this behaviour change, all being done without judgement or blame but more so as an encouragement tool. pairing employees that need to access personal data for various reasons will help to build accountability and discourage intentional misuse of data and potential accidents that can lead to leaks of personal data. lastly, an internal privacy committee composed of people across job functions and diverse life experiences that monitors organization-wide private data use and provides guidance on improving data use through practical recommendations is another step that will move the conversation of the organization from data entitlement to data custodianship. ultimately, this will be a market advantage that will create more trust with customers and increase business bottom line going into the future. towards the systematic reporting of the energy and carbon climate change and environmental destruction are well-documented. most people are aware that mitigating the risks caused by these is crucial and will be nothing less than a herculean undertaking. on the bright side, ai can be of great use in this endeavour. for example, it can help us optimize resource use, or help us visualize the devastating effects of floods caused by climate change. however, ai models can have excessively large carbon footprints. henderson et al.'s paper details how the metrics needed to calculate environmental impact are severely underreported. to highlight this, the authors randomly sampled one-hundred neurips 2019 papers. they found that none reported carbon impacts, only one reported some energy use metrics, and seventeen reported at least some metrics related to compute-use. close to half of the papers reported experiment run time and the type of hardware used. the authors suggest that the environmental impact of ai and relevant metrics are hardly reported by researchers because the necessary metrics can be difficult to collect, while subsequent calculations can be time-consuming. taking this challenge head-on, the authors make a significant contribution by performing a meta-analysis of the very few frameworks proposed to evaluate the carbon footprint of ai systems through compute-and energy-intensity. in light of this meta-analysis, the paper outlines a standardized framework called experiment-impact-tracker to measure carbon emissions. the authors use 13 metrics to quantify compute and energy use. these include when an experiment starts and ends, cpu and gpu power draw, and information on a specific energy grid's efficiency. the authors describe their motivations as threefold. first, experiment-impact-tracker is meant to spread awareness among ai researchers about how environmentally-harmful ai can be. they highlight that "[w]ithout consistent and accurate accounting, many researchers will simply be unaware of the impacts their models might have and will not pursue mitigating strategies". second, the framework could help align incentives. while it is clear that lowering one's environmental impact is generally valued in society, this is not currently the case in the field of ai. experiment-impact tracker, the authors believe, could help bridge this gap, and make energy efficiency and carbon-impact curtailment valuable objectives for researchers, along with model accuracy and complexity. third, experiment-impact-tracker can help perform cost-benefit analyses on one's ai model by comparing electricity cost and expected revenue, or the carbon emissions saved as opposed to those produced. this can partially inform decisions on whether training a model or improving its accuracy is worth the associated costs. to help experiment-impact-tracker become widely used among researchers, the framework emphasizes usability. it aims to make it easy and quick to calculate the carbon impact of an ai model. through a short modification of one's code, experiment-impact-tracker collects information that allows it to determine the energy and compute required as well as, ultimately, the carbon impact of the ai model. experiment-impact-tracker also addresses the interpretability of the results by including a dollar amount that represents the harm caused by the project. this may be more tangible for some than emissions expressed in the weight of greenhouse gases released or even in co2 equivalent emissions (co2eq). in addition, the authors strive to: allow other ml researchers to add to experiment-impact-tracker to suit their needs, increase reproducibility in the field by making metrics collection more thorough, and make the framework robust enough to withstand internal mistakes and subsequent corrections without compromising comparability. moreover, the paper includes further initiatives and recommendations to push ai researchers to curtail their energy use and environmental impact. for one, the authors take advantage of the already widespread use of leaderboards in the ai community. while existing leaderboards are largely targeted towards model accuracy, henderson et al. instead put in place an energy efficiency leaderboard for deep reinforcement learning models. they assert that a leaderboard of this kind, that tracks performance in areas indicative of potential environmental impact, "can also help spread information about the most energy and climate-friendly combinations of hardware, software, and algorithms such that new work can be built on top of these systems instead of more energy-hungry configurations". the authors also suggest ai practitioners can take an immediate and significant step in lowering the carbon emissions of their work: run experiments on energy grids located in carbon-efficient cloud regions like quebec, the least carbon-intensive cloud region. especially when compared to very carbon-intensive cloud regions like estonia, the difference in co2eq emitted can be considerable: running an experiment in estonia produces up to thirty times as much emissions as running the same experiment in quebec. the important reduction in carbon emissions that follows from switching to energy-efficient cloud regions, according to henderson et al., means there is no need to fully forego building compute-intensive ai as some believe. in terms of systemic changes that accompany experiment-impact-tracker, the paper lists seven. the authors suggest the implementation of a "green default" for both software and hardware. this would make the default setting for researchers' tools the most environmentally-friendly one. the authors also insist on weighing costs and benefits to using compute-and energy-hungry ai models. small increases in accuracy, for instance, can come at a high environmental cost. they hope to see the ai community use efficient testing environments for their models, as well as standardized reporting of a model's carbon impact with the help of experiment-impact-tracker. additionally, the authors ask those developing ai models to be conscious of the environmental costs of reproducing their work, and act as to minimize unnecessary reproduction. while being able to reproduce other researchers' work is crucial in maintaining sound scientific discourse, it is merely wasteful for two departments in the same business to build the same model from scratch. the paper also presents the possibility of developing a badge identifying ai research papers that show considerable effort in mitigating carbon impact when these papers are presented at conferences. lastly, the authors highlight important lacunas in relation to driver support and implementation. systems that would allow data on energy use to be collected are unavailable for certain hardware, or the data is difficult for users to obtain. addressing these barriers would allow for more widespread collection of energy use data, and contribute to making carbon impact measurement more mainstream in the ai community. the paper highlights four challenges in designing more "intelligent" voice assistant systems that are able to respond to exploratory searches that don't have clear, short answers and require nuance and detail. this is in response to the rising expectations that users have from voice assistants as they become more familiar with them through increased interactions. voice assistants are primarily used for productivity tasks like setting alarms, calling contacts, etc. and they can include gestural and voice-activated commands as a method of interaction. exploratory search is currently not well supported through voice assistants because of them utilizing a fact-based approach that aims to deliver a single, best response whereas a more natural approach would be to ask follow up questions to refine the query of the user to the point of being able to provide them with a set of meaningful options. the challenges as highlighted in this paper if addressed will lead to the community building more capable voice assistants. one of the first challenges is situationally induced impairments as presented by the authors highlights the importance of voice activated commands because they are used when there are no alternatives available to interact with the system, for example when driving or walking down a busy street. there is an important aspect of balancing the tradeoff between smooth user experience that is quick compared to the degree of granularity in asking questions and presenting results. we need to be able to quantify this compared to using a traditional touch based interaction to achieve the same result. lastly, there is the issue of privacy, such interfaces are often used in a public space and individuals would not be comfortable sharing details to refine the search such as clothing sizes which they can discreetly type into the screen. such considerations need to be thought of when designing the interface and system. mixed-modal interactions include combinations of text, visual inputs and outputs and voice inputs and output. this can be an effective paradigm to counter some of the problems highlighted above and at the same time improve the efficacy of the interactions between the user and the system. further analysis is needed as to how users utilize text compared to voice searches and whether one is more informational or exploratory than the other. designing for diverse populations is crucial as such systems are going to be widely deployed. for example, existing research already highlights how different demographics even within the same socio-economic subgroup utilize voice and text search differently. the system also needs to be sensitive to different dialects and accents to function properly and be responsive to cultural and contextual cues that might not be pre-built into the system. differing levels of digital and technical literacy also play a role in how the system can effectively meet the needs of the user. as the expectations from the system increase over time, ascribed to their ubiquity and anthropomorphization, we start to see a gulf in expectations and execution. users are less forgiving of mistakes made by the system and this needs to be accounted for when designing the system so that alternate mechanisms are available for the user to be able to meet their needs. in conclusion, it is essential when designing voice-activated systems to be sensitive to user expectations, more so than other traditional forms of interaction where expectations are set over the course of several uses of the system whereas with voice systems, the user comes in with a set of expectations that closely mimic how they interact with each other using natural language. addressing the challenges highlighted in this paper will lead to systems that are better able to delight their users and hence gain higher adoption. the paper highlights how having more translation capabilities available for languages in the african continent will enable people to access larger swathes of the internet and contribute to scientific knowledge which are predominantly english based. there are many languages in africa, south africa alone has 11 official languages and only a small subset are made available on public tools like google translate. in addition, due to the scant nature of research on machine translation for african languages, there remain gaps in understanding the extent of the problem and how they might be addressed most effectively. the problems facing the community are many: low resource availability, low discoverability where language resources are often constrained by institution and country, low reproducibility because of limited sharing of code and data, lack of focus from african society in seeing local languages as primary modes of communication and a lack of public benchmarks which can help compare results of machine translation efforts happening in various places. the research work presented here aims to address a lot of these challenges. they also give a brief background on the linguistic characteristics of each of the languages that they have covered which gives hints as to why some have been better covered by commercial tools than others.in related work, it is evident that there aren't a lot of studies that have made their code and datasets public which makes comparison difficult with the results as presented in this paper. most studies focused on the autshumato datasets and some relied on government documents as well, others used monolingual datasets as a supplement. the key analysis of all of those studies is that there is a paucity in the focus on southern african languages and because apart from one study, others didn't make their datasets and code public, the bleu scores listed were incomparable which further hinders future research efforts. the autshumato datasets are parallel, aligned corpora that have governmental text as its source. they are available for english to afrikaans, isizulu, n. sotho, setswana, and xitsonga translations and were created to build and facilitate open source translation systems. they have sentence level parallels that have been created both using manual and automatic methods. but, it contains a lot of duplicates which were eliminated in the study done in this paper to avoid leakage between training and testing phases. despite these eliminations, there remain some issues of low quality, especially for isizulu where the translations don't line up between source and target sentences. from a methodological perspective, the authors used convs2s and transformer models without much hyperparameter tuning since the goal of the authors was to provide a benchmark and the tuning is left as future work. additional details on the libraries, hyperparameter values and dataset processing are provided in the paper along with a github link to the code. in general the transformer model outperformed convs2s for all the languages, sometimes even by 10 points on the bleu scores. performance on the target language depended on both the number of sentences and the morphological typology of the language. poor quality of data along with small dataset size plays an important role as evidenced in the poor performance on the isizulu translations where a lowly 3.33 bleu score was achieved. the morphological complexity of the language also played a role in the state of the performance as compared to other target languages. for each of the target languages studied, the paper includes some randomly selected sentences to show qualitative results and how different languages having different structures and rules impacts the degree of accuracy and meaning in the translations. there are also some attention visualizations provided in the paper for the different architectures demonstrating both correct and incorrect translations, thus shedding light on potential areas for dataset and model improvements. the paper also shows results from ablation studies that the authors performed on the byte pair encodings (bpe) to analyze the impact on the bleu scores and they found that for datasets that had smaller number of samples, like for isizulu, having a smaller number of bpe tokens increased the bleu scores. in potential future directions for the work, the authors point out the need for having more data collection and incorporating unsupervised learning, meta learning and zero shot techniques as potential options to provide translations for all 11 official languages in south africa. this work provides a great starting point for others who want to help preserve languages and improve machine translations for low resources languages. such efforts are crucial to empower everyone in being able to access and contribute to scientific knowledge of the world. providing code and data in an open source manner will enable future research to build upon it and we need more such efforts that capture the diversity of human expression through various languages. presence of media cards, total interactions, history of engagement with the creator of the tweet, the user's strength of connection with the creator and the user's usage pattern of twitter. from these factors, one can deduce why filter bubbles and echo chambers form on the platform and where designers and technologists can intervene to make the platform a more holistic experience for users that doesn't create polarizing fractions which can promote the spread of disinformation. for the first time, there's a call for the technical community to include a social impact statement from their work which has sparked a debate amongst camps that are arguing to leave such a declaration to experts who study ethics in machine learning and those that see this as a positive step in bridging the gap between the social sciences and the technical domains. specifically, we see this as a great first step in bringing accountability closer to the origin of the work. additionally, it would be a great way to build a shared vernacular across the human and technical sciences easing future collaboration. we are impressed with all the work that the research and practice community has been doing in the domain of ai ethics. there are many unsolved and open problems that are yet to be addressed but our overwhelming optimism in the power of what diversity and interdisciplinarity can help to achieve makes us believe that there is indeed room for finding novel solutions to the problems that face the development and deployment of ai systems. we see ourselves as a public square, gathering people from different walks of life who can have meaningful exchanges with each other to create the solutions we need for a better future. let's work together and share the mic with those who have lived experiences, lifting up voices that will help us better understand the contexts within which technology resides so that we can truly build something that is ethical, safe, and inclusive for all. see you here next quarter, the state of ai ethics, june 2020 suckers list: how allstate's secret auto insurance algorithm squeezes big spenders algorithmic injustices towards a relational ethics social biases in nlp models as barriers for persons with disabilities the second wave of algorithmic accountability the unnatural ethics of ai could be its undoing this dating app exposes the monstrous bias of algorithms ( arielle pardes data is never a raw, truthful input -and it is never neutral racial disparities in automated speech recognition working to address algorithmic bias? don't overlook the role of demographic data ai advances to better detect hate speech algorithms associating appearance and criminality have a dark past beware of these futuristic background checks go deep: research summaries 30 the toxic potential of youtube's feedback loop study: facebook's fake news labels have a fatal flaw research summaries capabilities, and ai assistive technologies go wide: article summaries 50 ancient animistic beliefs live on in our intimacy with tech humans communicate better after robots show their vulnerable side at the limits of thought engineers should spend time training not just algorithms, but also the humans who use them using multimodal sensing to improve awareness in human-ai interaction different intelligibility for different folks aligning ai to human values means picking the right metrics why lifelong learning is the international passport to success (pierre vandergheynst and isabelle vonã¨che cardia you can't fix unethical design by yourself research summaries the wrong kind of ai? artificial intelligence and the future of labor demand ai is coming for your most mind-numbing office tasks tech's shadow workforce sidelined, leaving social media to the machines here's what happens when an algorithm determines your work schedule automation will take jobs but ai will create them research summaries what's next for ai ethics, policy, and governance? a global overview (daniel schiff a holistic approach to implement ethics in ai beyond a human rights based approach to ai governance: promise, pitfalls and plea go wide: article summaries 69 this is the year of ai regulations apps gone rogue: maintaining personal privacy in an epidemic maximizing privacy and effectiveness in covid-19 apps article summaries who's allowed to track my kids online? chinese citizens are racing against censors to preserve coronavirus memories on github can i opt out of facial scans at the airport? with painted faces, artists fight facial recognition tech research summaries adversarial machine learning -industry perspectives article summaries the deadly consequences of unpredictable code adversarial policies: attacking deep reinforcement learning specification gaming: the flip side of ai ingenuity doctors are using ai to triage covid-19 patients. the tools may be here to stay the future of privacy and security in the age of machine learning research summaries 95 towards a clearer account of research priorities in ai ethics and society integrating ethical values and economic value to steer progress in ai machine learning scholarship go wide: article summaries 104 microsoft researchers create ai ethics checklist with ml practitioners from a dozen tech companies why countries need to work together on ai quantifying independently reproducible machine learning be a data custodian, not a data owner research summaries towards the systematic reporting of the energy and carbon footprints of machine learning challenges in supporting exploratory search through voice assistants a focus on neural machine translation for african languages radioactive data: tracing through training using deep learning at scale in twitter's timeline (nicolas koumchatzky neurips requires ai researchers to account for societal impact and financial conflicts of interest in modern ai systems, we run into complex data and processing pipelines that have several stages and it becomes challenging to trace the provenance and transformations that have been applied to a particular data point. this research from the facebook ai research team proposes a new technique called radioactive data that borrows from medical science where compounds like baso4 are injected to get better results in ct scans. this technique applies minor, imperceptible perturbations to images in a dataset by causing shifts within the feature space making them "carriers".different from other techniques that rely on poisoning the dataset that harms classifier accuracy, this technique instead is able to detect such changes even when the marking and classification architectures are different. it not only has potential to trace how data points are used in the ai pipeline but also has implications when trying to detect if someone claims not to be using certain images in their dataset but they actually are. the other benefit is that such marking of the images is difficult to undo thus adding resilience to manipulation and providing persistence. prior to relevance based timeline, the twitter newsfeed was ordered in reverse chronological order but now it uses a deep learning model underneath to display the most relevant tweets that are personalized according to the user's interactions on the platform. with the increasing use of twitter as a source of news for many people, it's a good idea for researchers to gain an understanding of the methodology that is used to determine the relevance of tweets, especially as one looks to curb the spread of disinformation online. the article provides some technical details in terms of the deep learning infrastructure and the choices made by the teams in deploying computationally heavy models which need to be balanced with the expediency of the refresh times for a good experience on the platform. but, what's interesting from an ai ethics perspective are the components that are used to arrive at the ranking which constantly evolves based on the user's interaction with different kinds of content.the ranked timeline consists of a handful of the tweets that are the most relevant to the user followed by others in reverse chronological order. additionally, based on the time since one's last visit on the platform, there might be an icymi ("in case you missed it") section as well. the key factors in ranking the tweets are their recency, key: cord-023364-ut56gczm authors: nan title: education day monday: plenary session 1 monday: parallel sessions date: 2005-06-08 journal: vox sang doi: 10.1111/j.1423-0410.2005.00651.x sha: doc_id: 23364 cord_uid: ut56gczm nan from the perspective of causal inference, there is a hierarchy of evidence, ranging from case-series to large randomized controlled trials (rcts). in addressing a particular clinical or policy problem, clinicians or policy-makers can base their decisions on the types of clinical reports that have been published, along with an assessment of the strengths and weaknesses of each study. rcts are controlled clinical experiments in which patients are randomly allocated by the investigators to receive a treatment under study. if randomization is used, all participants are equally likely to be allocated to either the treatment or the control arm of the study. rcts should be distinguished from observational studies in which investigators passively observe patients who happen to receive a treatment under study. because the allocation of subjects to the treatment and control arms of an rct is random, the play of chance should distribute all confounding factors equally between these two arms. thus, the treatment and control arms of an rct should be equivalent with respect to all confounding factors except for the treatment under study. randomization removes selection bias, because neither the investigator nor the participant knows what the treatment allocation will be before a patient enters a study. moreover, if an rct is double-blind, observation bias is also removed, because preconceived notions about benefit from the treatment cannot color the reporting of symptoms by a patient or the assessment of disease activity by an investigator. when the undertaking of rcts is deemed unlikely, meta-analyses of individual patient data, that is, of the data recorded previously on subjects enrolled in published, small rcts may allow investigators to address issues of possible biases 2ed-01-01 standardizing blood components would allow better monitoring of the effectiveness of transfusion d davenport university of michigan, ann arbor, mi, usa in routine transfusion of red blood cells (rbc) or platelet concentrates (pc), we have only a very rough idea of the dose we administer. the minimum expected hemoglobin content of rbc should be 50 g (fda criteria). however, actual measurements have found a mean hemoglobin content of 46.6 ± 11.7 g per unit, with variability between manufacturers. 25 percent of units may contain less than 34.9 g of hemoglobin while 25 percent may contain more than 56.3 g. the effect of storage senescence can magnify these differences. the resulting hematocrit increase, depending on size of recipient and age of a unit, may be 1.5 to 9 percent. apheresis collection of rbc can help standardized unit contents, but this technology is relatively expensive and time consuming. knowledge of actual hemoglobin content can be used to optimize red cell transfusion. one trial, using a simple formula incorporating the desired hemoglobin and estimated blood volume, showed that nearly 60 percent of transfusion orders could be met with one unit while achieving a mean target hemoglobin of 9.3 g/dl. for pc transfusion, about 66 percent of transfusions achieve the targeted dose of 3-6 ¥ 10 11 platelets in actual practice, with considerable variability between institutions. without knowledge of the actual content of pc, determination of refractoriness and response to matched pc is problematic. standardization and labeling of rbc and pc units for content will permit accurate dosage and quantification of transfusion practice. are transfusion guidelines evidence-based? jp aubuchon dartmouth-hitchcock medical center, lebanon, nh, usa application of the results of randomized, controlled clinical trials to similar clinical situations should increase the predictability of the outcomes of transfusions. unfortunately, too few such trials have been conducted to investigate all the potential situations where transfusion might be applied. however, the ones that have been reported indicate that, in general, transfusion is unlikely to provide substantial benefit in many of the situations where it is routinely used. in the intensive care setting, transfusing red cells at a trigger point of 7 g/dl rather than 10 g/dl not only did not lead to increased anemic morbidity but was actually associated with a reduction in overall mortality. subgroup analyses indicated transfusion at the higher trigger point did not decrease morbidity in patients with cardiac disease nor decrease the time until weaning from a ventilator. following cardiac surgery, a transfusion trigger of a hematocrit of 25% yielded the same outcomes as one of 32%. an observational study suggested that patients with peripheral vascular disease and a post-operative hematocrit below 28% were more likely to suffer a morbid cardiac event, but this group of patients had more evidence of anemia and cardiac ischemia preoperatively, illustrating the pitfalls of observational studies. most would agree that transfusion is necessary below a hb of 7 g/dl and unlikely to be necessary at a hb above 10 g/dl, but most patients fall between these limits, and there are insufficient data in the and residual confounding factors, and may permit them to make a judgment of a causal relationship. sackett proposed that the evidence generated from meta-analyses of individual patient data be regarded as being equivalent in strength to that generated from rcts. meta-analysis is the structured and systematic integration of information from different studies of a given problem. when the results of individual studies are discrepant, the purpose of an overview is to investigate reasons for disagreements among studies. when the results are concordant, the goal of meta-analysis is to derive, through application of a number of quantitative techniques, a measure of the effect of the intervention across combined investigations. this measure is referred to as the 'summary' effect of the treatment under study. meta-analysis differs from the traditional, narrative reviews of the literature, in that: (1) all completed investigations of the efficacy of an intervention that meet specific, eligibility criteria are retrieved and included in the overview; (2) the quality of retrieved studies is assessed systematically; (3) the degree of agreement among studies is evaluated, both conceptually and based on statistical criteria, and the synthesis of the findings proceeds only if the variation in reported results is sufficiently modest to be attributed to chance; and (4) quantitative methods are used to calculate the summary effect of the intervention and to test that effect for statistical significance. 2ed-02-04 biology of stem cell mobilization th papayannopoulou university of washington, seattle, wa, usa at baseline hematopoiesis, the majority of developing hematopoietic cells (precursors, progenitors and stem cells) is actively retained in bone marrow (bm), whereas fully mature cells emigrate to peripheral circulation. a small number of stem/progenitor cells are also present in blood, serving presumed physiologic roles for the repopulation of remote damaged areas. however, in several hematopoietic perturbations, i.e. post irradiation, post chemotherapy or by using empiric treatments, a heightened emigration (mobilization) of progenitor/stem cells was noted over 30 years ago. the introduction of g-csf as an efficient mobilizing agent not only had a major clinical impact, but has triggered a flurry of studies exploring the mechanisms of mobilization. from these studies, significant insight has been gained about the molecular pathways leading to mobilization, especially by studying the altered environment within bm post mobilization, and less so by studying cells mobilized in blood. several attractive scenarios have been proposed and their importance was further bolstered by studying genetic mouse models. a prominent role of the sdf-1/cxcr4 pathway has been emphasized, either by down regulation of cxcr4, changes in sdf-1 gradients, or by disruption of sdf-1/cxcr4 signaling. whether this pathway is disrupted by a proteolytic mechanism prevailing within bm post g-csf or by other protease-independent mechanisms has not yet been settled. in addition to sdf-1/cxcr4, disruption of other pathways responsible for the retention of primitive cells in bm can lead to mobilization. for example, disengagement of the vla4/vcam-1 pathway by anti-functional antibodies or its genetic deficiency in mice results in egress of stem/progenitor cells. mobilization is also seen following the use of other cytokines (i.e. kit-l, flt-3 l, il-3) or chemokines (i.e. il-8, gro?), complement activation, etc., but the detailed mechanisms or the interdependence of these other pathways with the ones already proposed have not been worked out. finally, efforts to improve mobilization efficiency in man has led to the use of combination treatments with g-csf, either by adding another cytokine (i.e. growth hormone), agonistic sdf-1 molecules (i.e. ctce0021), or cxcr4 antagonists (i.e. amd3100) which have yielded synergistic increments, and these will be discussed. a full understanding of the principles of mobilization, as well as the bm homing characteristics of mobilized cells after their i.v. infusion in transplantation, should lead to targeted, more efficient experimental protocols in the future. the possibility of deriving all kinds of mature cell types from embryonic stem (es) cells for the purpose of cell replacement therapies will probably face a major obstacle; a limited supply of available hla types for an ever growing population in demand. one possible solution is to use adult sources of stem cells such as the haematopoietic stem cells (hsc) that can repopulate the entire haematopoietic system after transplantation in a myeloablated host. however cells with the repopulating ability of hscs are still elusive for tissues such as muscle, heart, cns and pancreas. it was therefore exciting news when it was shown that hscs could repopulate other non-haematopoietic tissues arguing for a general role of bmderived cells in tissue regeneration. the term plasticity was thus coined to describe the phenomenon whereby cells of one lineage could trans-differentiate into cells of another tissue. this in turn implied that a certain degree of developmental plasticity was still available in the adult hsc, or their derived progeny, that allowed them to present with novel phenotypes. how exactly this was accomplished was a matter of speculation. in order to address the mechanism we used an animal model of liver failure where bmt with normal (wild type, wt) hsc was shown to rescue the liver failure; the repopulating hepatocytes apparently differentiated from the sole source of wt cells, the bm compartment. by studying the genetic composition of the regenerating liver nodules we observed that both wt and mutant dna was present in the nodules, a finding that was consistent with bm-derived cells fusing with host hepatocytes. the mechanism of plasticity was therefore directly related to the exposure of the donor bm-derived wt nucleus to the transcriptional environment of the hepatocyte and the expression of hepatocyte-specific genes. this fusion mechanism was also shown to underlie perceived cases of haematopoietic cell transdifferentiation to purkinje cells in the cerebellum and to cardiomyocytes. however, there are carefully executed studies that strongly support the alternative transdifferentiation mechanism in tissues such as epithelia or the epidermis. what these observations may imply is that in tissues with high rates of cell turnover, there may be a potent stem cell niche that can reprogram incoming cells to follow relevant cell lineages. if this hypothesis is correct, then the major research effort should focus on what makes the niche and whether it can be recreated faithfully in vitro so as to educate hscs to diverse lineages opening the way for realistic cell replacement therapies. collection and distribution of blood and its products to meet the medical needs of industrialized countries are typically managed through large-scale national or nationally-supported blood programs. the development, maintenance, and ultimate success of such programs are all components of a process that involves the delicate balance of continuously competing pressures, e.g. social, economic, ethical, political, regulatory/legislative. as with all endeavors that directly affect human life, safety is a central, unifying theme of this process. because transactions of blood inherently involve risks at both donation and reception/transfusion ends, efforts to enhance and maintain an acceptable level of safety generally rely on a focused program of risk management (rm). rm involves three equally important elements: identification/evaluation of risk factors in a process, control of exposure to them, and continuous monitoring to assess the effectiveness of countermeasures and the emergence of new risk factors. specific approaches to rm and quality assurance in transfusion medicine will be presented. examples from blood programs currently in effect in selected countries will be discussed. rm efforts within the corresponding blood program in greece will then be examined. the lack of data to adequately assess the status of this program suggests that at least one of the elements of rm -monitoring -can still be improved. a preliminary research effort aims to survey blood donors, transfusion recipients, and physicians in order to build an understanding of the specific factors that drive the perception of risk in the greek population. the findings form important stepping points upon which specific recommendations can be made for the development and maintenance of a comprehensive, effective rm program in greece. 2ed-03-08 the use of haemovigilance data to increase safety in transfusion medicine haemovigilance is a surveillance of all the procedures in the transfusion chain. the intension is to collect and assess information on unexpected or undesirable effects in bleeding of donors and transfusion of blood components. in europe haemovigilance was introduced as a concept in the middle of the nineties. the first national reports on haemovigilance appeared in the late nineties, and were only dealing with complications related to transfusion. later on other kind of events like 'near miss' events were added. nowadays national reports are dealing with all steps in the transfusion line, and to some extend also with complications in blood donors. the state of the art is haemovigilance with retrospective registration of unwanted events that has happened, but also a more active prospective part with an early warning in a rapid alert system about new threats in the transfusion world. the aim of the different national haemovigilance systems has been to improve safety in the transfusion line. after the first 10 years with haemovigilance in the european countries, what has been the outcome of this big effort? data from national reports do not signify major improvements, but safety is suggested to have improved due to many minor changes of procedures and awareness of dangerous situations. however, in most countries nothing really effective has been done to avoid failures, the most important cause of serious complications in transfusion of patients. systems which can prevent most of the failures are on the market. the cost of these equals the cost of nat screening for virus introduced in the same period of time. the common perception of transfusion risks is still much more focused on the hypothetical risk of virus transmission than to the demonstrated magnitude of severe complications related to bleeding of donors and transfusion of patients. therefore, to increase safety in transfusion medicine the nature and occurrence of the risks should be revealed by haemovigilance and not less important the results should be published with the aim to get a realistic common perception of the transfusion risks. the role of diagnostic testing to identify congenital vs acquired disorders of hemostasis and to optimize the management of perioperative bleeding g despotis washington university school of medicine, st louis, mo, usa excessive bleeding with trauma or after surgery can result in hypoperfusion and anemia related end-organ dysfunction and mortality as well as transfusion-related complications. several large studies have demonstrated that if bleeding is excessive after cardiac surgery to the point that reexploration is required, that overall mortality increases by 3-4 fold. transfusion related complications include the following potentially lethal complications: disease transmission of pathogens, acute hemolytic reactions, allergic reactions, transfusion associated acute lung injury, allo-immunization related disease (e.g. post-transfusion purpura, platelet refractoriness, transfusion-associated graft-vs-host disease) and other potential complications (e.g. increased perioperative infection or multi-organ system failure) related to transfusion associated immune modulation. in addition, blood shortages related to increasing consumption (i.e. expanding geriatric population) and/or a shrinking supply (i.e. related to exclusion of donors related to donor exclusion criteria designed to prevent disease transmission) may limit our ability to adequately manage our anemic and bleeding patients. this highlights the relative importance of accurate diagnosis and optimal management of excessive bleeding to minimize bleeding related complications and conserve our blood supply. patients at risk for excessive bleeding include those with an established hereditary disorder (e.g. vwd, hemophilia, connective tissue disorders), end-stage hepatic or renal disease as well as patients with acquired defects of the hemostatic system. in specific, patients with platelet (i.e. platelet refractoriness) or coagulation factor inhibitors at increased risk, extracorporeal circulation related defects or as related to certain pharmacologic agents). patients who require longer periods of extracorporeal circulation for cardiac surgical procedures (e.g. repeat, combined procedures or use of deep hypothermic circulatory arrest) are at a greater risk of developing excessive bleeding (e.g. cpb-related abnormalities). in addition, patients receiving one or more longacting anti-thrombotic medications in the immediate preoperative period (e.g. plavix, reopro, low molecular weight heparin etc) are at increased risk for bleeding. clinicians in the past have resorted to empiric and 'shot gun' approaches when managing excessive bleeding due lack of immediate availability of results from laboratory-based coagulation tests. on this basis, the use of point-of-care (poc) tests of hemostatic function to facilitate the optimal management of excessive bleeding, help differentiate between microvascular vs surgical bleeding and reduce transfusion have been investigated. five of six recently published studies have demonstrated that implementation of a standardized approach to manage bleeding (e.g. algorithm) which when coupled with either point-ofcare or laboratory tests of hemostatic function can optimize the management of bleeding and reduce total donor exposures by 50%. ddavp has bee shown to be beneficial with uremia-induced platelet dysfunction and with type i von willbrand's disease. although previous studies have not been able to conclusively show that ddavp can reduce bleeding and transfusion when administered prophylactically, more recent evidence indicates that this agent may be useful in preventing excessive bleeding when a test (point-of-care) reveals platelet dysfunction. recombinant activated factor vii (rfviia) is licensed for use in bleeding episodes in hemophiliac patients with inhibitors. although, only anecdotal reports and results from small clinical studies have shown that this agent can reverse life-threatening bleeding after major surgery, other reports indicate that there is variability in the effectiveness of rfviia as well as highlight lifethreatening thrombotic complications in a subset of high risk patients (i.e. patients with congenital or acquired thrombotic disorders or systemic activation of the hemostatic system such as with dic or after cardiac surgery). therefore, large clinical trials evaluating the efficacy and safety of rfviia are needed before any widespread use can be recommended. in recent years, molecular methods of blood grouping have become routine diagnostic procedures in many laboratories throughout the world. they have proved especially valuable for the determination of fetal blood groups. the ability to detect rhd in pregnancies where the fetus is at risk from haemolytic disease of the newborn represents a significant advance in obstetric care. furthermore, the occurrence of fetal dna in the mothers' peripheral blood has allowed this diagnostic procedure to be carried out without the risks that accompany amniocentesis (lo ymd. (1999) ann med 31:308-312). determination of the coding sequence of all the genes giving rise to antigens within the 29 blood group systems currently recognised is virtually complete. by correlating the coding sequence of these genes with the phenotype of red cells from individuals with different blood groups it has been possible to infer the molecular bases of most of the antigens comprising each blood group system (daniels gl (2002) human blood groups, 2nd ed. blackwell science). the polymorphic antigens of most systems other than abo and rh, are defined by single nucleotide substitutions (snps) which effect a single amino acid sequence change in the gene product. numerous methods, both manual and automated, are available to determine snps and these have been applied to the determination of blood groups. useful applications of snps detection methods include, determination of the blood group phenotype of patients who have been transfused recently and still have donor blood in their circulation (rozman p, dove t, gassner c et al. (2000) transfusion 40:936-942), and donor screening to find compatible units for patients with multiple blood group antibodies or for the management of patients with diseases like the haemoglobinopathies who are going to be transfusion-dependent over many years (castilho l, rios m, bianco c et al. (2002) transfusion 42:232-238). other applications of molecular methods include zygosity determination for rhd, determination of the blood group phenotype of patients with a positive direct antiglobulin test, selection of donors with rare blood group phenotypes, and as aids to the solution of complex blood grouping problems in the reference laboratory. the molecular bases of abo and rh antigens are complex and cannot be determined reliably by a single snp. nevertheless, methodologies are available that allow the comprehensive sequence analysis of individual genes and could be applied to abo and rh typing. whether or not this will ever be a cost-effective procedure is a moot point. it is clear that molecular methods are a useful addition to the range of diagnostic procedures available to transfusion medicine practitioners but it is important to remember that methods based on dna analysis determine phenotype by inference and not by direct measurement. the results of molecular tests should be interpreted with this caveat in mind. 2ed-06-18 the hla system g stavropoulos general hospital 'g. gennimatas', athens, greece since its discovery in the mouse in 1936 the major histocompatibility complex (mhc) has become one of the most important region in the vertebrate genome with respect to infection, autoimmunity and transplantation. mhc primary function is to provide protection against pathogens. this is achieved through sophisticated pathways in which mhc class i molecules present endogenous antiges to cd8+ t cells and class ii molecules present exogenous antigens to cd4+ t cells. an increasing number of other proteins are being found that support these two pathways; many of these proteins, together with the class iii complement proteins, also map to the mhc. the mhc molecules were originally studied for their ability to cxonfer tolerance (histocompatibility) following tissue grafts or later, organ transplants. nowadays, the success of unrelated hematopoetic cell transplantation is influenced by the degree of mhc compatibility between the donor and patient. thus, for patients who lack matched donors, the rules that govern permissibility of mhc mismatching still need to be identified. for patients with high risk disease who lack matched donors, use of donors with a single mhc mismatch may permit early treatment before disease progression. scientific evidence to fully answer the questions 'when are platelet components clinically effective' and 'what do we really know?' is limited. despite this limitation and the level of uncertainty it generates with regard to treatment options and policies, it is reassuring to note that current prophylactic platelet transfusion protocols -mostly derived from empirical observations collected during the 1960's and 1970's -protect oncology patients from clinically relevant bleeding in more than 95% of thrombocytopenic days spent in hospital or at home, even in aggressive conditions such as leukemia, lymphoma and other severe blood diseases. this evidence seems to justify the prevalent policy of transfusing platelets when the patient's platelet count falls below 10 000 per microliter (or 20 000 in 'unstable' patients). this policy is based on positive outcomes of prospective and retrospective studies performed during the 1990's including some hundred non-surgical patients and on the desire of balancing the will of reducing patient exposure to limited, expensive, potentially infectious and immunogenic blood products with the objective of preventing clinically relevant hemorrhage. several national and international organizations endorse the above policy. although the role of platelet support in surgery or in specific clinical situations requiring invasive procedures or in patients affected by multi-organ and system co-morbidity suffers from even more limited evidence, the latter has been carefully collected and summarized in the guidelines for platelet transfusion published in j clin oncol 2001;19:1519-1538. additional data on lumbar puncture are reported in ann hematol 2003;82:570-573. another important topic where there is room for improvement and need for further investigation is platelet refractoriness, a condition developed by a proportion of chronic platelet recipients, which causes high hemorrhage risk if compatible platelets are not provided and in which consensus on the diagnosis and treatment is lacking. with regard to the type of platelet product, it will be necessary to determine the clinical impact of buffy-coat derived platelets, consistently used in europe and current object of increased interest in canada, as compared to the platelet-rich plasma method traditionally used in the us, of viral inactivation procedures and of laboratory methods for detection of bacterial contamination of platelet concentrates. in addition, ongoing studies on the clinical impact of high versus low platelet doses will provide novel elements to determine the relative merits of apheresis versus whole-blood derived platelets. as far as the established procedure of white cell reduction by filtration, which shows high technical efficiency and has been implemented as a standard by a number of institutions, debate is still ongoing on its equivalence with serologic screening for the prevention of cmv transmission, whereas general consensus supports its pre-storage use to prevent febrile, non hemolytic transfusion reactions. finally, although the high cost of filters do not allow a generalized use of this technology in many settings, white cell reduced platelets have been unequivocally shown to reduce alloimmune refractoriness from about 15% to about 5% of patients. 2ed-07-20 ffp: appraisal of the evidence for the clinical use of ffp although the indications for transfusion of plasma (fresh frozen plasma; ffp) are limited, the use of ffp continues to rise in the united kingdom and has risen by over 20% in the past few years. local uk audits continue to document that a significant proportion of ffp transfusions are not consistent with indications reported in guidelines. a systematic review of the evidence base for the effectiveness of ffp was therefore undertaken to identify, select and appraise all relevant randomised controlled trials, as the most robust form of study to assess effectiveness of ffp. in the systematic review of ffp, the criteria for inclusion of full-published studies were: there must have been at least two groups in the study; • allocation to the groups must have been either by formal randomisation or by a quasi random method e.g. alternation; • one of the arms of trial must include ffp or plasma as an intervention; results on the relevant clinical or laboratory outcome must be presented. the main analysis was qualitative, and differentiated between: 1. studies of interventions comparing ffp with no ffp; 2. studies of interventions comparing ffp with a non-blood product e.g. solutions of colloids and/or crystalloids; 3. studies of interventions comparing ffp with a different blood product; 4. studies comparing different formulations of ffp, e.g. solvent detergent and methodine blue treated. an evaluation of studies comparing ffp with no ffp would be expected to provide the clearest direct evidence for a positive effect of ffp. studies comparing ffp with colloids or crystalloids were separately appraised because these latter products may have variable effects on coagulation tests. studies comparing different formulations of ffp do not directly evaluate effectiveness of ffp but were included because there is now a uk national policy to use these products in younger patients and therefore these trials might identify negative outcomes. the search strategy identified a total 57 publications. although it may appear that this number of randomised trials might provide a reasonable evidence base to help inform clinical policy and decision making, the review identified a number of important concerns about the published trials. few of the identified studies included details of the study methodology (method of randomisation, blinding of patients and study personnel). the sample size of many included studies was very small (range 8-78 patients per arm). few studies took adequate account of the extent to which adverse events might negate the clinical benefits of treatment with ffp. many of the identified trials in groups such as cardiac, neonatal, and other clinical conditions, evaluated a prophylactic transfusion strategy. however, when these trials evaluating prophylactic usage were assessed together as a single grouping in the review, it appeared there was no consistent support for a beneficial effect of prophylactic ffp, irrespective of clinical setting. there is a pressing need to develop new trials to determine the effectiveness of ffp, including for those clinical situations in which it has become an accepted part of current transfusion practice. a law decided upon by the riksdag (the swedish parliament) often sets the general standards as a framework and authorises the central government authority concerned to elaborate and decide upon the more detailed regulations. laws and regulations define the level of quality and safety that has to be reached and state what must be done, while establishments and their managers are kept responsible for fulfilling the requirements and how this is done. guidelines (non-binding) present detailed instructions on ways how to fulfil the requirements. making a law is complicated and time-consuming. the ministry draws up a legislative proposal, refers the proposal to relevant bodies for consideration and comments, and then drafts a bill which is referred to the council on legislation for consideration before it is submitted to the riksdag. a parliamentary committee deals with the bill before it is put to the chamber of the riksdag for approval. when adopted, the bill becomes law. regulations elaborated by central government authorities are handled in a principally similar way. however, regulations are more readily adjusted to scientific and technical progress, and when legislation need requirements for technical details, these are preferably presented in a regulation. the ministry of health and social affairs is responsible for elaborating the bill to be submitted to the riksdag on the blood safety law. two central government authorities, designated as competent authorities, are responsible for preparing the appropriate complimentary regulations as well as for inspecting and licensing the blood establishments: • the national board of health and welfare (nbhw), responsible for requirements related to blood components intended for transfusion, and • the medical product agency (mpa), responsible for requirements related to blood components intended for the manufacture of medicinal products. sweden became a member of the eu ten years ago. since then, experts in transfusion medicine have been appointed by the ministry, nbhw and mpa for advice on scientific and technical issues and for representing sweden at expert meetings arranged by the commission. some of these experts are also members of the handbook committee of the national society for transfusion medicine, preparing and revising the national guidelines. consequently, the requirements of the directives are readily implemented in the daily work of the blood establishments before (due to legal and technical reasons) the new blood safety law and the revised nbhw and mpa regulations can be promulgated. to a great extent, requirements of the blood directives are covered by existing swedish legislation. no major problems or obstacles seem to arise when implementing new requirements into law and regulations and into the blood establishments' daily service. a few detailed requirements have been found difficult to follow precisely in the routine work. these minor difficulties, however, will not compromise the high quality and safety of blood components prepared according to the standards set by the blood directives. the landscape for blood collection and distribution includes availability and safety. true international availability of blood has not been reached. the landscape of safety has been mountainous, if viewed by the degree of frustration among the transfusion specialists. the reasons for frustration have varied from serological problems to those of transmissible infections, to which no end is in sight. mistrust in the safety of blood taken by others is one reason for lack of international landscape in blood collection and distribution. increasing demands on safety, availability and economy force the health care providers to reorganise blood services. national systems are winning ground. this may make it easier to achieve international collaboration. the council of europe has pioneered in creation of international recommendations for blood collection and distribution. in 1958 a european agreement on the exchange of therapeutic substances of human origin, including human blood, was published. its purpose was to make blood available to other parties of the agreement in case of urgent need. many countries ratified the agreement rapidly, some did it first in the nineties. in practice little exchange of blood components has taken place. the council of europe recommendations lack legal power. the european union is different, and it has taken on its agenda activities aiming at improving confidence in the safety of the blood transfusion chain in the community (commission communication 1994) . the agenda is based on an agreement reached at a high level eu meeting in adare, ireland in 1994. first in the commission agenda was a recommendation on donor selection criteria, given in 1998. then came the european blood directive 2002/98/ec, the aim of which is to improve the safety of blood and blood components within the union so that enough mutual trust between member countries can be achieved to make the exchange of blood components possible. being a legally binding document the directive is undoubtedly helpful in reaching a harmonised standard in europe. hopefully the european commission has the resources to follow its implementation. there are concerns, however. the epidemiological differences among the eu member countries and frequent appearance of new infectious agents potentially transmitted by blood make it difficult to foresee that even effective viral inactivation of blood components could totally erase the national differences in donor approval. blood collection and preparation of components are already the same in eu member countries and the directive helps in their further standardisation. there has not been much distribution of blood components internationally, with the exception of export of red cell concentrates to the us from switzerland and the netherlands. the european legislation opens new horizons and widens the european landscape as its purpose is to simplify the crossing of borders between the member states, but before true movement of blood components is achieved more work is needed on the eu commission blood agenda. the eu directive implemented into the legal act for polish blood transfusion service blood transfusion service (bts) in poland is an integral part of the public polish health service. in the country of nearly 39 million people, approximately one million units of blood and plasma are collected every year from voluntary, nonremunarated donors, which is statistically over 25 donations per 1000 inhabitants. blood and plasma are collected in strictly appointed centers and no private collection sites are permitted. the legal basis for the activity of polish bts is polish blood transfusion act of 22nd august 1997 which came into force as of january 1st 1999. this act was then updated in november 2003 according to eu directive 2002/98/ec and came into force as of january 13th 2004. this act introduced the principles for organization, collection, processing, storage, transport and quality assurance in bts. it specified -among others -the system of accreditation, haemovigilence, as well as requirements for bts employees. according to this act the polish bts is obliged to monitor and supervise immunohematology testing and transfusion procedures in all hospitals where blood and blood products are transfused. polish bts consists of 21 regional blood transfusion centers (rbtc), one military center and one ministry of internal affairs and administration center as well as the institute of hematology and blood transfusion (ihbt) which acts as supervisor. the 21 rbtcs have a uniform organization structure, uniform quality assurance system and act according to uniform guidelines issued by ihbt. they have two financing sources -the central budget and hospital reimbursement for distributed blood and blood products. the polish blood transfusion act of 22nd august 1997, in force since january 1st 1999, has been supplemented by 8 decrees: 1. procedures for external bts audits; 2. requirements for donor selection; 3. requirements and procedures for organization and safe management of blood transfusion in hospitals; 4. requirements for implementing of national and regional donor registers; 5. employment criteria for bts personnel; 6. training requirements for hospital personnel involved in blood and blood product administration; 7. national, uniform price list for blood and blood products; 8. organization requirements for setting up of a national committee for blood and blood transfusion. introduction: several technical aspects must be considered in pediatric apheresis due to the size of the patient. factors that must be evaluated are extracorporeal circuit volume, blood flow rates, type of anticoagulant and vascular access. adverse events are mainly related either to vascular access or to metabolic or hemodynamic changes. aim of the study: in this study we show our experience using fresenius hemocare com.tec for pbsc collection in children. methods: twelve pediatric patients (median age 8 years, range 2-16; median weight 38 kg, range 12.2-60.5 kg) with solid tumors at onset or on relapse underwent collections with the p1y kit of the fresenius hemocare com.tec blood cell separator. our cd34+ cells target was 10 ¥ 10 e 6 /kg. collections were started if a peak of at least 0.02 10e9/l cd34+ cells (20 per microlitre) were reached in the peripheral blood. in all the patients, leukapheresis were performed through a central venous catheter and temporary peripheral venous access. acd ratio 1 : 14-1 : 16 was combined with heparin 40 u/kg. in 3 children with <15 kg the separator was initialized with a compatible filtered and irradiated red blood cell unit, suspended in 4% albumin, up to the patient's hematocrit, to avoid transient hypovolemia, due to the volume sequestered in the separator. results: twenty-five procedures were performed. a median blood volume of 5400 ml (range 2.100-12.400 ml) was processed in a separation time of 270 min (range 180-330 min). the median product weight was 108 g (range 37-258 g) and yield of cd34+ cells was 3.2 ¥ 10e 6 /kg body weight (range 0.7-12.5 ¥ 10e 6 /kg body weight). three poor mobilizing patients (peripheral blood cd34+ peak of 20-26 cells per microlitre) underwent more than two apheresis to collect the desired transplantation dose (3.5 and 4). all collection procedures were well tolerated. children never required sedation to perform the leukapheresis. only mild hypocalcemia-related symptoms, promptly responding to small i.v. boluses of calcium gluconate, were reported. no circulatory side effects were observed. blood flow alarms occurred in every procedures but no collection had to be terminated due to insufficient flow. conclusion: in summary, leukapheresis in children can be safely and effectively performed with the fresenius hemocare com.tec separator with minimal technical difficulties even in patients under 15 kg. m-pa-007 alternative methods for prevention of infection transmission (pathogen inactivation etc.), cost-benefit considerations of the procedure itself. however, one must also take the loss of plasma into consideration -and more difficult: calculate the effects of changes in product quality, as reduced content of factor viii, protein s and other labile proteins. for methods involving pooling, there are also concerns about the risks due to the pool sizes. the major benefit of the methods will be the potential reduction of infectious transmission, but also possible advantages as reduction of allergic transfusion reactions and trali must be evaluated. the study types involved in cost-benefit considerations are costeffectiveness analysis where the cost and effects of an intervention and an alternative are presented in a ratio of incremental cost to incremental effect and cost-utility analysis, where quality-adjusted life years (qaly) are used as the effectiveness endpoint. qualityadjusted life years is a method that assigns a preference weight to each health state and estimates life-expectancy as the sum of these products of each preference weight and time spent for each state. a complete glossary of terms is found at http://www.hsph.harvard.edu/cearegistry. in literature, there are several publications on cost-benefit considerations within the field of transfusion medicine. concerning plasma products, the costeffectiveness of solvent-detergent plasma has been the major focus. however, the conclusions of different authors have been conflicting. in 1994, aubuchon and birkmeyer published a paper (jama 1994; 272(15) :1210-1214) where they concluded that the cost was usd 340 000 per qaly, which is far above the 'acceptable limit' of usd 50 000. this estimate was adjusted to usd 9.7 mill. per qaly in a 1999 letter to jama (jackson, jama 282). in 2003 , riedler et al. published (vox sang 2003 85:88-95 ) that the discounted cost/life year saved for sd-ffp use in the uk was gbp 22 278 for neonates and gbp 98 465 for patients aged 70. the main reason for the differences between the two papers (and others) was considered to be different calculation of non-infectious complications. the papers cited above underline the difficulties of the cost-benefit considerations. the age of the patient, the outcome of the treatment, the quality of life in an infected patient and the cost of side effects will differ. in addition, how much are we willing to pay for protection against emerging viruses? this paper will not provide the answers, but introduction: the photodynamic treatment of therapeutic plasma using methylene blue (mb) in combination with visible light is a well established procedure for the inactivation of blood borne viruses. aim of the study: evaluation of the quality and stability of mb/light-treated plasma (mb plasma) prepared under worst case conditions for routine processing. methods: single donor units (n = 12) were treated using the macopharma theraflex mb-plasma system which includes plasma membrane filtration (plas4) and addition of mb prior to illumination followed by mb and photoproduct filtration (blueflex). samples were taken before treatment and from the final product. additionally mb-treated plasma was prepared from four different plasma pools and stored for up to 9 months. treatment was done under worst case conditions for the preservation of coagulation factors: maximum mb concentration during illumination (1.15 mmol/l), maximum storage time of whole blood before separation (4°c, 17 h), maximum storage time of mb plasma before freezing (1 h). several plasma parameters and the concentration of mb and its photoproducts (azure a, azure b, azure c and thionine) were determined. results: mb/light treatment had a significant influence on thrombin time (+20.5%), fibrinogen (clauss) (-20 .4%), factor v (-15.6%), factor viii (-22.3%), factor xi (-13.3%) and protein c (-10.3%). no significant changes were detected for at iii, vwf : rco, vwf cleaving protease, plasmin inhibitor and alpha-1-antitrypsin. the entire virus inactivation procedure including the filtration steps for leukocyte depletion and mb and photoproduct depletion had no significant effect on activation markers (prothrombin fragment 1 + 2, thrombin-antithrombin-complex, d-dimers). no further essential loss of coagulation factor activity was observed during storage of the plasma at 30°c for 9 months. mb and its photoproducts (azure a, azure b, azure c) were depleted to a final concentration of <0.1 mmol/l. thionine was undetectable in all samples. conclusion: photodynamic treatment of fresh frozen plasma (ffp) using the theraflex mb-plasma system leads to only moderate decreases in the activities of different coagulation factors even under worst case conditions for routine production. mb and its photoproducts were effectively removed from the plasma by the blue-flex-filter integrated in the theraflex-system. the quality of mb plasma is well preserved during storage. pulmonary complications, particularly transfusion-related acute lung injury and circulatory overload, are the most common causes of transfusion-associated morbidity and mortality in the developed world. trali is a syndrome characterized by acute respiratory distress, hypoxemia, hypotension and pulmonary edema, occurring within 6 hours (usually 1-2 hours) of transfusion of a plasmacontaining blood product. other signs, including hypertension, leucopenia and hypocomplementemia, are less frequent. all blood products, except for albumin and solvent/detergent plasma, have been associated with trali, but red blood cells, platelets and ffp are the most common. the incidence is unknown, but 1 : 5000 plasma-containing transfusions is the most commonly cited figure. in 80% of patients, recovery is well underway within 96 hours, and leads to complete resolution. death occurs in 6-23%. the profile of the at-risk recipient has not been identified. recurrent cases have been infrequently described. there are two prevailing theories of pathogenesis: (1) antibody-mediated and (2) 2-hit hypothesis. evidence supports both concepts and neither is mutually exclusive. more than 60% of reported cases are associated with blood components containing either hla-specific (class i or ii) or hnaspecific antibodies. in 50% these antibodies correspond to at least one epitope in the recipient. most implicated components are donated by multiparous women. five percent of patients have hla or hna antibodies in their pre-transfusion serum. treatment requires prompt, assertive respiratory intervention, frequently necessitating mechanical ventilation. because trali has a much better prognosis than ards, it is an important diagnosis. some blood collectors are diverting plasma from multiparous donors away from ffp production or routinely screening for hla antibodies. the most effective method for identifying 'high risk' components has not been identified. introduction: trali is a life threatening adverse reaction of blood transfusion. it is characterized by noncardiogenic pulmonary edema developed soon after blood transfusion. in japan, we built hemovigilance system in 1993 and have been collecting the voluntary reports of severe adverse reactions of blood transfusion including trali cases since 1997. since the diagnostic criteria of trali have not been established until recently and no specific diagnostic markers for trali have been discovered so far, it is very difficult to make proper diagnosis of trali in each reported case. we have been collecting the cases with respiratory distress and pulmonary edema developed after blood transfusion as suspected case of trali. we reevaluated each report whether it meet the recommended diagnostic criteria for trali published in transfusion journal in dec. 2004 . aim of the study: purpose of this study is to select trali cases which met internationally recognized criteria so that it will reveal the possible causes of trali with laboratory testing for anti-leukocyte antibodies in donors and recipients. methods: the cases with respiratory failure and pulmonary edema are selected for evaluation from the adverse reaction case reports voluntarily reported to japanese red cross. in order to make a proper diagnosis of trali, we have been utilizing the respiratory distress questionnaire which has recently revised. this helps us eliminate other adverse reactions such as circulatory overload, cardiac failure, anaphylaxis and bacterial contamination, which results in selecting out the internationally recognized trali cases properly. for laboratory testing, flowpra and labscreen for hla antibodies and gift-fcm for hna antibodies are performed. the cross-matching test is also performed if possible. results: during past 8 years, 95 cases of trali and 29 cases of possible trali have been confirmed by critical review of each questionnaire. of 95 cases of definite trali, donor specimens were obtained in 84 cases. of 84 cases, anti-leukocyte antibodies were detected in 43 cases (51%) of donors' blood, which was significantly higher than the positive rate of anti-leukocyte antibodies in donors' blood of other adverse transfusion reactions (<10%). of 43 cases of antibody positive donors, anti hla antibodies were detected in 33 cases, anti hna antibodies were detected in 18 cases, and both were detected in 8 cases. of 33 cases of positive anti hla antibodies, class i antibodies were detected in 22 cases, class ii antibodies were detected in 28 cases, and both were detected in 17 cases. on the other hand, the anti-leukocyte antibodies were detected in 38% of trali recipients, and this rate is almost the same with that of positive rate of other adverse reactions of blood transfusion (39%). these results indicate anti-leukocyte antibodies in the blood donors are one of the prerequisites for developing trali from the antibody-hypothesis-oriented point of view. other cases with no detectable antibodies should be investigated in more detail in the future. thus, reevaluating trali cases based on recommended trali criteria will allow us to reveal new information about trali. of 2087 adverse events analysed by the serious hazards of transfusion (shot) scheme (1996) (1997) (1998) (1999) (2000) (2001) (2002) (2003) , 556 (27%) were haemolytic transfusion reactions (htrs). 303 were due to incorrect blood component transfused (ibct): 226/303 were abo incompatible and 77/303 caused by other red cell antibodies. a further 40 cases were reported as acute htrs (ahtrs; i.e. occurring within 24 hours of transfusion) whilst 213 were recognised more than 24 hours after transfusion and reported as delayed htrs (dhtr). 21/556 (4%) patients died and 58 suffered major morbidity. htrs associated with ibct result from clinical or laboratory errors and are all preventable. it has been assumed that other htrs are unavoidable. closer scrutiny reveals that this may not always be the case, though review is hampered by incomplete investigations. 9/40 ahtrs occurred in group a (8/9) or group b (1/9) patients given group o platelets. of the 31 ahtrs related to red cell transfusion, 4 were due to errors, 9 were in patients with auto-antibodies, in only 4 of whom alloantibodies had been adequately excluded/identified. at least 24/213 dhtrs were potentially avoidable; in 18 cases the antibody was detectable retrospectively in the pre-transfusion sample; in 6 cases the presence of a previous antibody was not communicated to the laboratory. two patient deaths related to dhtr might have been avoided by earlier diagnosis and clinical involvement. 14% htrs reported to shot would have been avoided by compliance with pretransfusion testing guidelines and provision of group a platelets for all group a recipients. htrs can be clinically overlooked and inadequately investigated. national guidelines are needed for the investigation and management of htr, with focus on the identification of underlying causes to guide the choice of future component therapy. reference laboratories can provide valuable support in elucidating complex serological problems. patients treated with high dose chemotherapy and autologous blood progenitor cell (bpc) support may get malignant cells reinfused together with stem cells. we analyzed bpc products collected from patients suffering from germ cell cancer measuring malignant contamination and followed these patients for up to 98 months after transplantation also with the question if transplanted malignant cells influence survival. aliquots of stem cell apheresis products containing one million mononuclear cells were sedimented on glass slides and by immunocytochemistry quantitation of cytokeratin expressing cells was performed manually with light microscopy and by automated image analysis. in 11 of 35 patients (31%) cytokeratin expressing cells were detected in bpc apheresis products of patients treated for germ cell cancer. we followed the activity of the malignant disease of patients for more than eight years in median 60 months after transplantation. no significant difference in survival was demonstrated for our two patient groups. background: the use of adequate number of peripheral blood stem cells (pbsc), collected by mnc apheresis is essential for effective treatment of hematological malignancies, solid tumors, and other disorders by transplantation. aims: the goals of this study were: (a) to obtain an effective apheretic protocol for mnc harvesting, and (b) to compare the hematopoietic reconstitution after hspc transplantations in different clinical settings. methods: in this study, pbsc transplantations -60 allogeneic from matched sibling healthy donors and 60 autologous -were performed in the management of patients with severe aplastic anemia, leukemias (all, anll, cml), multiple myeloma, hodgkin's and non-hodgkin's lymphoma, breast and ovarial cancer, and extragonadal non-seminal germ cell tumor. pbsc mobilization was achieved with rhg-csf (5-16 g/kgbm/day). mnc-apheresis procedures (generally one and occasionally two) were performed using blood cell separator cobe-spectra. the first mnc-apheresis was accomplished when the leukocyte cont was 5-10 ¥ 10e9/l (autologous setting) or on the 5th day 4-5 hour after the last rhg-csf administration (allogeneic setting). the processed blood volume during one mnc-apheresis was 12. 7-22.2 l, and 24 .3 l for one pbsc transplantation in average. results: using a minimal target dose of cd34+ cell count (3 ¥ 10e6/kgbm), performing one mnc-apheresis procedure for 86% recipients sufficient number of pbscs were obtained. the mnc yield was 10.1 ¥ 10e8/kgbm in allogeneic and 8.1 ¥ 10e8/kgbm in autologous setting in average. the mean cd34+ yields for allogeneic and autologous transplantations were 12.1 ¥ 10e6/kgbm and 6.5 ¥ 10e6/kgbm, respectively. hematopoietic reconstitution was achieved on the 9.4th day for leukocytes and the 13.05th day for platelets when pbsc transplantation was applied. summary/conclusion: improved mnc and cd34+ cell yield, as well as rapid hematopoietic reconstitution were observed when: (a) the intention of auditing any clinical practice is, ostensibly, to improve patient care. the term 'audit' implies comparison against a standard. although absolute indications for transfusion have not been defined by clinical trials applicable to most clinical situations, many institutions have established their own transfusion triggers based on their reading of the literature and common practice at that hospital. assuming these represent prudent guidelines, comparison of actual practice to them may allow physicians to re-assess their pattern of hemotherapy and bring it into conformance with the guideline. there are several common ways of performing transfusion audits. retrospective analysis of transfusions allows appreciation of the clinical situation (and its ultimate outcome) but reviews the event through a 'retrospectoscope' that assesses clinical information in a different manner than that available to the clinician making the decision to transfuse. furthermore, the time lapse between the decision to transfusion and feedback about that decision may render the feedback from the reviewing body (e.g., a transfusion committee or blood utilization review committee) of little practical import. to speed the provision of feedback and emphasize educational rather than any punitive outcomes of the audit, some facilities have abolished attempts at determining whether the decision to transfuse was supportable and have used electronic means to feed back to clinicians a non-judgmental informational message summarizing the literature regarding the indications for transfusion. this appears to be at least as effective as the traditional retrospective audit system. prospective review of requests for blood components have the potential to redirect practice in a manner that immediately helps patients. however, such interactions with clinicians may come at inopportune times, may require considerable (unscheduled) time, and are most likely to be fruitful if a knowledgeable transfusion medicine expert can serve as the intermediary. both approaches (or a combination) have been shown to be beneficial in altering practice, but efforts must be diligent and sustained. providing data comparing a physician's practice to colleagues in the same specialty may prompt additional introspection and practice change, particularly if physician leadership of the institution supports the effort as a quality improvement tool. extending the comparison to a group of physicians and contrasting their transfusion habits with benchmark data from other institutions may also be helpful, but one needs to be ready to counter arguments that differences in patient groups are the reason for the differences in practice (studies have shown that, in general, practice patterns are primarily related to training and habit rather than large differences in patient acuity). increased focus on the performance of hospitals as expressed in outcome data may soon extend to transfusion practices as well. the public or governmental institutions may ask to see data illustrating the transfusion practice of an institution and its improvement over time. carefully conducted, diligent, and ongoing transfusion audits are an integral part of an institution's quality improvement program. informed consent: the term informed consent, appeared for the first time in the late 40s but it was only in the 70s that it attracted attention with regard to health care. numerous discussions and publications have attempted to define the meaning and the justification of informed consent in recent years. initially it consisted in the obligation of the physician to disclose information to the patient, regarding the procedure he was to undergo, but more recently, ethicists have emphasized the need to ensure the patient's understanding and his autonomous decision to consent. current institutional rules of ethics demand that the physician must obtain the informed consent of a patient 'prior to any substantial intervention' . what is however the meaning of informed consent? is it a mutual decision making between physician and patient? in 'principles of biomedical ethics' beauchamp and childress claim that' it is critically important to distinguish informational exchanges through which patients elect medical interventions, from acts of approving and authorizing those interventions. the elements of consent include: disclosure, voluntariness, decision and authorization. when applying the concept of informed consent in transfusion medicine one can distinguish it into donors' and patients' consent. donor informed consent: information to blood donors constitutes a sensitive issue. it refers to their protection from side-effects of the donation, as well as to protection of the recipient. with regard to whole blood donors a detailed history and information as to side effects are necessary for first-time donors. for repeat donors one needs mainly an updated history. because of time-pressure, whole blood donors are usually given written information and are asked to answer written questions. donors however differ in literacy and even literate ones do not always understand medical terminilogy; so, during the interview one should probe the degree of understanding of each donor. with first time apheresis donors more time is needed in order to explain the procedure and potential side effects. since granulocyte and stem-cell donors require premedication with growth factors and or corticosteroids, the responsibility for detailed information is even greater. the fact that all donors sign the informed consent does not mean that they are all adequately informed! interviewers must be familiar with side effects, their frequency and sequelae and must pass on this information. misses and near-misses, (serious) adverse events and failures in medical practice seem to be not preventable. in medical interventions, preparing and prescribing of medication, assistance by doctors and nurses, medical treatment and follow-up, unwanted and unexpected events occur (http://www.mederrors.com.). these events happen also in transfusion medicine and focus on safety is not unique. haemovigilance which is defined in the eu blood directive as 'a set of organised surveillance procedures relating to serious adverse or unexpected events or reactions in donors or recipients, and the epidemiological follow-up of donors' (eu directive 2002/98/ec off. j. european union.8.2.2003:l33/30-l33/40) is established to help in trying to identify and minimise the misses and (serious) adverse events in the chain from donor to recipient of blood components. the causes or reasons should be studied in order to prevent re-occurrence. adverse event reporting in blood transfusion and transfusion medicine is complex. it depends on the cooperation between blood establishments with clinicians and hospitals. it implies knowledge of blood banking, transfusion medicine and routine clinical care of all gender and ages, of potential hazards of transfusion, of immune-haematology, of microbiology, and of epidemiology. an adverse event may have its cause in every single part of the blood chain and reference may take place to a proven problem, a potential problem, or to a justified doubt. in almost all blood transfusion centres, a single donation will be processed into a number of different products, and these units might be divided or processed into more products. blood components are produced from whole blood or apheresis donations, and depending of the blood drawing and processing techniques, a high number of products with different specifications is prepared. the products' shelf life is not equal and therefore the moment in time of actual use of each unit prepared from the same donation may differ. in case the unit of platelets harms the recipient, a rapid alert can warn in order not to issue or to transfuse the unit of red cells or the unit of ffp prepared from the same donation because of the potential adverse reaction, which was detected during or after the transfusion of the first unit used. haemovigilance is not only important to blood establishments and to patients and prescribers, but it is also to clinical scientists, and to the public at large. it should provide a basis for minimising adverse reactions on blood components, and it should enable the therapeutic potential of new or established treatments with components to be maximised, since demonstrations of safety during widespread use may lead to extended usage, and wider availability. it is quite worrisome that underreporting is a general problem in medical care. it might be expected that in transfusion medicine the same rates of underreporting can be found, but also that the same mechanisms for improvement are applicable. although medical misses occur and do not seem to be preventable, the handling of these misses is often quite poor. many patients like to hear a detailed explanation, and the majority expects even apologies from the treating physician. it seems desirable to look for new routes in the prevention of unwanted events of transfusion medicine. for problem solving, analysis and improvement of working methods, where needed and possible, are often the most effective methods. attention should be focused on improvement and not on identification of the person who caused the problem ('bad apple'). lack of communication and insufficient insight in each other work are often the causes of problems and unwanted events. it should be recognised that advices given by the haemovigilance officer or the blood transfusion committee about prevention without the input and commitment of the direct responsible persons will lead in most cases to advices which are not effective or which will not be accepted. setting up a haemovigilance system or appointing of haemovigilance officers or installation of blood transfusion committees will not be sufficient. it will be necessary to develop ways of registration, data collection and analysis, but more importantly to support by giving advice and training to prevent reoccurrence of the adverse events or not optimal use of blood components. the confidentiality of the information should be guarded sufficiently. for a physician-patient relation, even after a medical failure, a 'blame-free culture' with a central role for openness and transparency is necessary. for blood establishments and hospitals, there is an important role in the right assistance and help of the physicians concerned both on a practical and on an emotional way. m-pa-032 8 years of shot data 1996-2004: a view of transfusion safety in the uk and reactions. this will require investment in infrastructure, for which there must be a trade-off in improved transfusion safety. transfusion-transmitted infections and serious immunological reactions are rare; shot has highlighted the need for blood services to implement strategies to minimise bacterial contamination and transfusion related acute lung injury and will monitor their effectiveness. from the inception of shot it has been clear that the most frequent transfusion hazard is 'incorrect blood component transfused' i.e. a patient receiving a blood component intended for another person or not meeting appropriate requirements. only a minority of these events results in patient harm and is reportable under the terms of the directive. haemovigilance schemes such as shot, that analyse no-harm errors and near-misses, can reveal clues as to the root causes of 'wrong blood', which contributed to 16 deaths and 85 cases of major morbidity in the uk between 1996 and 2003 . analysis of such events shows that most errors occur in clinical areas, the most frequent being failure of the 'bedside check' . clinical audit data indicates that 10% of patients are transfused without a wristband or other form of identification, whilst anecdotal reports suggest that urgent clinical situations, massive transfusions and nocturnal transfusions are particularly error-prone. strategies aimed at reducing errors include structured education and competency testing, and methodologies, both high and low-tech, to ensure accurate patient identification in all circumstances. onethird of errors occurs in hospital laboratories; denominator data on laboratory workload shows that work done outside of 'core hours' accounts for 20% of all pre-transfusion testing but 40% of errors, suggesting that biomedical scientists 'on-call' or on shift work are working under pressure and beyond their competency. 85% of hospitals reported that they participated in shot in 2003, but only 47% of eligible hospitals reported adverse events suggesting that transfusion hazards remain under-recognised and under-reported. however, benchmarking of 'wrong blood' incidents against transfusion activity shows that the number of observed incidents is roughly proportional to blood use. if haemovigilance data is to contribute to improved transfusion safety, clinicians must be encour-aged to report all events, thus contributing to an evidence base that can be used to effect change and facilitate learning. barriers to reporting include cumbersome systems, lack of time and resource, lack of feedback and fear of blame. transfusion practitioners have a vital role in the recognition and reporting of adverse events, education and clinical audit, but must be adequately resourced and supported by senior clinicians and managers through an active hospital transfusion committee. *provisional. m-pa-033 passing the borders: when, how and where d pirc-tiljak croatian institute of transfusion med., zagreb, croatia there may come that moment in professional life when you have to follow a strong professional and ethical need and confront your evidence-based statements with the leadership, passing the border of your own small society. in order to protect patient's health and respect human right to be informed about all possible consequences of irregular medical therapy, insisting on professional dignity and truth, you feel responsible and follow-up processing of your serious error report. once you pass the border, trying to warn authorities and find ethical resonance and critical confirmation of your professional fears, you are 'persona non grata' . methods/results: reality checking, personal experiences and observations studying the path of serious error report. although the qc system functions, yet omissions happen . . . the possible reasons could be: lack of knowledge, lack of experience, lack of independency, personal confront of interest, immature leadership, political influence, even corruption . . . how strict do the authorities manage a fault, bearing in mind the responsibility toward the patients under the risk. there is a need to create an available, effective international expert's board which will react and give professional counselling support-asylum for endangered professionals who found enough power to blow the whistle. who will hear it? transfusion transmitted infections (tti) are a major source of concern given the repercussions of hiv, hepatitis c, bacteria and vcjd transmission by blood components. large amounts of resource have been expended in making products safer and in maintaining public confidence in the blood supply. identifying emerging infections of concern is a major activity for many transfusion services. of the long list of emerging infections identified by disease control agencies around the world, identifying those responsible for tti requires, amongst other things, that: • the agent is identifiable. • it is present in blood • it causes a disease of concern. • it is transmitted by transfusion. • it is present at relatively low frequency. • if a test is available (nat, serology or immunoassay) what the infection window period is. once an agent has been identified various approaches are possible, including: • donor selection by testing, geography or lifestyle e.g. wnv; • product selection e.g. erythrovirus (b19) antibody or bacterial testing; • product treatment e.g. pathogen inactivation; • patient selection e.g. cmv matching, immune status. against this background where should our attentions focus? some agents of initial concern are now known to be ubiquitous and have minimal disease association (ttv, gbv) although transmitted by transfusion. for others (coronavirus -sars or the possible kawasaki disease agent, dengue, flavivirus encephalopathies, avian flu, etc.) this is less certain, with agents arising from species crossover being of particular concern (avian flu, vcjd, hiv). • enhancement of automation/computerisation; • process control to provide an 'error-free pathway'; • (national) surveillance and trend analysis of results, preferably based on national working standards; • significantly increased sensitivity, especially from development of antigen/antibody 'combi' assays (e.g. for hiv, and recently, for hcv); • awareness of hbsag vaccine-escape mutants and design of assays to cope with this; • extension of range of agents and markers tested for (varies in different countries); • increasing range of assays available for testing donors with a relevant history of exposure to malaria or chagas' disease infection (for retrieval of otherwise wasted blood); • european union's in vitro diagnostics directive: this has caused some problems and reduced flexibility. nucleic acid testing (nat): nat continues to increase in blood service usage world wide, although not (as yet) to replace serological methods. trends include: • reduction in sample pool size; • increased automation (and process control); • increased multiplexing to detect 2 or more agents in the same assay; • increased number of agents being tested by nat (varies in different countries); • introduction of rapid and flexible nat to detect west nile virus, in north america. bacterial screening of platelet preparations: several countries have introduced (or will introduce) routine screening of platelet concentrates either with biomerieux, bactalert or pall ebds (02 depletion assessment). other bacterial testing methods are under active assessment, some rapid enough for possible 'point of use' testing. m-pa-036 evaluation of in vivo red blood cell recovery after processing with a new filter designed to reduce prions e nelson*, h taylor † , p whitley † and t lieu* *pall medical, covina, ca, † american red cross and evms, norfolk, va, usa background: a filter, called the leukotrap affinity prion reduction filter (prf1b filter, pall medical), has been developed to reduce the level of infectious prions, associated with several fatal neurodegenerative diseases including variant creuztfeldt-jakob disease (vcjd), from leukocyte-reduced red cell products. aim: the objective of this study was to evaluate the quality of leukocyte-reduced red cells (lr-rbc) processed through this filter and stored for 42 days. red cell quality was determined by measuring the in vivo red blood cell recovery 24 hours after re-infusion of the 42-day stored red cells. storage hemolysis and atp were also determined. methods: units of blood (450 ml) were collected from normal volunteers into the leukotrap wb system containing cp2d/as-3 anticoagulant/preservative solutions (pall medical). units were either processed to lr-rbc within 8 hours at room temperature (rt units), or after 24 hours at 1-6°c (cold units). the prion filter set was sterilely connected to the units on day 1, and the units were filtered and stored for 42 days. samples were taken pre-and post-prion filtration and post-storage for plasma hemoglobin and atp determinations. post-storage samples were taken for labeling with 51-cr radioisotope, re-infusion, and determination of the 24-hour in vivo rbc recovery. a donor sample was also labeled with 99m-tc to allow for red cell mass determination. thus, both single-and double-label 24-hour recovery values were calculated. results: twelve units were collected. in vitro testing was completed on all units. in vivo testing was completed on 11 units. the mean single-label 24-hour recoveries were 84.4% and 85.7% for the rt and cold units, respectively. the mean double-label recoveries were 81.9% and 84.1% for the rt and cold units, respectively. the overall combined mean in vivo and in vitro results are shown in the table. conclusion: the 24-hour in vivo red cell recovery means are well above the fda and council of europe's requirement of achieving a mean post-transfusion survival of no less than 75% of the transfused red cells, and they are comparable to this center's previous results of red cells filtered using the licensed leukotrap rc system with cp2d/as-3 (pall medical introduction: to reduce the risk of platelet transfusion-associated sepsis (tas), methods to routinely screen for bacterial contamination have been implemented. pathogen inactivation treatment of labile blood components provides an alternative means to prevent tas. the intercept blood system for platelets (baxter healthcare) has received the ce mark and has been introduced into clinical practice. aims: this study compared the efficacy of bacterial screening using a culture method (bact/alert system, biomerieux) with pathogen inactivation (intercept blood system) for prevention of transfusion of platelet components contaminated with bacteria. methods: seven strains of bacteria associated with tas, including gram-positive staphylococcus epidermidis, streptococcus agalactiae, and staphylococcus aureus, gram-negative escherichia coli, and klebsiella pneumoniae, and the anaerobes propionibacterium acnes and clostridium perfringens were studied. for each strain, three double-dose platelet concentrates (~6 ¥ 10e 11 platelets in 600 ml of 35% plasma and 65% intersol) were collected using the amicus® cell separator. on day of collection, calibrated stocks of bacteria (20, 200, 2000 cfu) were added to the double units. each double unit was divided into two identical products containing 10, 100, or 1000 cfu of bacteria and stored overnight under conventional blood bank conditions. the control platelet concentrate was not treated. the test platelet concentrate was treated with the intercept process (150 mm amotosalen + 3 j/sq cm uva). both units were cultured using the bact/alert system at the time of bacterial inoculation and on days 1, 2 and 5 of storage. samples (10 ml) were taken for both the aerobic and anaerobic cultures. a platelet sample was considered contaminated with bacteria if a positive signal was registered within 120 hours of culture. results: for control platelet concentrates, cultures failed to detect low-dose inocula. the time to positive culture varied with the bacterial strain, contamination level, and time of sampling. at 10 and 100 cfu per product, 4 strains (s. epidermidis, e. coli, c. perfringens, s. agalactiae) and 2 strains (e. coli, c. perfringens) tested negative after 5 days of platelet storage, respectively. k. pneumoniae tested positive after 7-8 hours of culture when sampled on day 2 of platelet storage for both 10 and 100 cfu per product. at 1000 cfu per product, p. acnes tested negative in aerobic culture and c perfringens tested negative in anaerobic culture after 5 days of platelet storage. the anaerobic cultures of p. acnes became positive after 50 hours of culture when sampled on day 5 of platelet storage. of the 7 strains studied, only s. aureus consistently tested positive after 7-16 hours of culture. in contrast, all test platelet concentrates treated with intercept remained negative by bact/alert cultures throughout the entire 5-day observation period regardless of the strain and the contamination level. conclusions: bacterial detection using cultures may fail to detect low levels of bacteria typically associated with platelet contamination at time of collection and processing. failure to detect bacteria will result in the release of contaminated platelet products with 'test negative-to-date' status. in contrast, inactivation of bacteria is capable of preventing release of contaminated platelet components. background: since nat implementation for hiv-1 and hcv rna in france, the residual risk (rr) of transfusion-transmitted infections (tti) has dramatically decreased. the rr estimates, for a threeyear period from 2001 to 2003 showed a significant decrease from 1/1 700 000 and 1/1 560 000 before nat implementation to 1/3 150 000 and 1/10 000 000 after nat implementation for hiv and hcv respectively. as for hbv, the serological screening is only based on both hbsag and anti-hbc assays. for the same period, the rr estimate for hbv is 1/640 000, five times higher than hiv one and 16 times higher than hcv one. aims: as the overall rr of tti is mainly related to hbv, and given the availability of hbv nat assays, a study was conducted to determine whether hbv nat has the ability to further reduce the hbv rr and then should be implemented in blood donor screening in france. we have estimated the wp reduction by nat in comparison with one of the most sensitive hbsag screening assays, on 10 commercial seroconversion panels (bioclinical partners, franklin, ma, usa). the nat test was the procleix ultrio assay (genprobe/chiron, san diego, usa). the hbs ag test was the prism hbsag (abbott, france). the comparison was performed on both neat samples and diluted samples 1/8, 1/16, and 1/24, in order to simulate minipools of different sizes. then, we have calculated the yield of hbv-infected donations detected by nat relative to prism hbsag assay. results: on the basis of a window period (wp) of 56 days, ultrio assay is projected to close the wp by an average of 15 days on undiluted samples, 5 days in minipools of 8 samples, 4 days in minipools of 16 samples and only 2 days in minipools of 24 samples. the projected yield calculated on the basis of 2.4 million donations collected per year in france, would be 0.65 unit per year for minipool-nat and 2 to 3 units per year for individual donation nat. conclusion: introduction of minipool-nat will offer only a little added benefit to transfusion safety relative to current serological screening strategies based on both hbsag and anti-hbc assays. hbv minipool-nat is then unsuitable for hbv screening in french blood donors. single-sample nat or minipool-nat with smaller pool sizes and/or modified procedures (genome enrichment or test improvement) would be more relevant. automation when technologically and practically feasible is a prerequisite for single-donation nat. therefore, decision has been made not to implement hbv nat in the french transfusion network until fully automated systems will be available. however, as the prevalence of hbv infections is higher in the overseas territories than in continental france, and as nat is performed on individual donations in these sites, hbv-nat has been implemented since december 2004 in these territories. combined detection of hepatitis c virus core antigen and antibody as an alternative to nucleic acid testing in blood screening grating the capillary cytometer with a robotic workstation and a small footprint centrifuge. significantly, there was no decrement in system performance following automation: 474 of 501 clinical samples (94.6%) typed identically with this system and cat, and 25 of the 27 discrepant results were eventually resolved in favor of the automated cytometry method. testing showed high-throughput capabilities (currently 280 samples/day) and was inexpensive. to demonstrate the flexibility of this testing platform, we also developed a method to perform completely automated counting of residual wbcs (rwbc) following leukoreduction of blood components. there were no significant differences in accuracy and precision when rwbc in analytical controls and authentic clinical samples were quantitated by the automated capillary cytometry method or the leucocount method performed manually. given the flexibility of this system, it is very likely that additional blood bank assays could be modified for high performance automated testing on this platform. noninvasive prenatal genotyping on cell free fetal dna in maternal plasma ce van der schoot sanquin research, amsterdam, netherlands in 1997 lo et al. demonstrated that in the maternal circulation small amounts of cell free fetal dna are present, concentrations ranging from on average 25 genome equivalents(geq)/ml early in pregnancy to about 100 geq/ml at the end of pregnancy. most likely this dna is derived fom apoptotic syncitiorophoblasts. the human placenta is hemichorial, which means that the syncitiotrophoblast is in direct contact with the maternal blood flow, and apoptotic nuclei are directly released into the maternal circulation. the cell free dna is very rapidly cleared from the circulation, the t1/2 being only 15 minutes. in 1998 we have shown that this cell free fetal dna could be used for rhd genotyping. in the last 5 years many groups have shown the successful application of different prenatal genotyping assays such as fetal sexing, thalassemia, achondroplasia, duchenne's disease, adrenogenital syndrome etc. on this source of dna. importantly, no false positive result have been described due to the presence of fetal dna from previous pregnancies, the main draw back of prenatal diagnostics on circulating fetal cells. at present prenatal rhd genotyping has been introduced in routine diagnostics in the united kingdom, france and the netherlands. in large scale high throughput studies it has been shown that the diagnostic accuracy of prenatal rhd genotyping is over 99%, and it is to be expected that in the netherlands this screening will soon be introduced to restrict the antenatal anti-d immunoprophylaxis to women carrying rhd-positive fetuses. in a large european project (safe, co-ordinator maj hulten, warwick uk) many researchers collaborate to further explore the possibilities of cell free fetal dna for future diagnostics. standard operating procedures for the isolation of plasma dna have been established. control pcrs for the presence of fetal dna have been developed. recent findings on differences in methylation status of placental genes in fetal dna opens new possibilities. the main technical problem that hampers wide application of prenatal genotyping is the impurity of the fetal dna, only 1-5% of the cell free dna in plasma is from fetal origin. this makes diagnostic assays on numerical chromosomal abnormalities impossible. and also for many single nucleotide polymorphisms (snps) such as almost all blood group antigens, assays are hampered by aspecific amplification from maternal dna. our own preliminary results indicate that this latter problem can be solved by pna clamping. the addition of a pna probe specific for the k-allele partial d feature d antigen alteration, often identified as distinct 'partial' d epitope loss. the clinical impact of partial ds is due to the ability of their carriers to form anti-d antibodies upon confrontation with regular d after transfusion, or pregnancy. this leads to the -naively spoken -contradictory finding of an allo anti-d antibody in a d positive individual in connection with a negative autocontrol. the antibodies themselves include the same fatal clinical potential as anti-d antibodies of d negative individuals, but may be even more hazardous since unexpected in d positive individuals a priori. d categories (ii to vii) represent a nomenclatorily defined subgroup of partial ds. the molecular cause of partial d lies within single (caused by point mutation in the respective rhd gene sequence), or multiple amino acid exchanges (caused by gene conversion events leading to rhd-rhce-rhd hybrid genes) which determines a qualitative d antigen alteration, rendering them distinguishable from regular d by a partial d carriers immune system. nowadays, transfusion specialists and gynaecologists are more or less aware of these facts and are taking them into consideration in the clinical setting. most partial d exhibit decreased d antigen density, enabling principal recognition of them. however, routine serological methods may not properly recognise all partial ds and will identify their carriers after immunisation only, which represents a reactive diagnostic/therapeutic attitude second best to an actively prognostic one. this actively prognostic proceeding with respect to early detection of partial ds became widely feasible by rhd dna typing techniques. currently, routine rhd dna typing techniques offer an affordable, accurate and fast approach to an unambiguous identification of partial ds and their reliable discrimination from weak d types, not at risk for allo anti-d immunisation. a reasonable proactive proceeding could e.g. demand for (once in a lifetime) routine rhd dna typing of all weakly expressed ds as defined by serology, since most partial ds also meet this phenotype. rhd allele frequencies and their geographical and regional prevalence will certainly have an important impact on dna typing strategies and their (mandatory) specificities. fluorescence cytometry for completely automated immunohematology testing d roback*, b barclay † and d hillyer † *emory university school of medicine, atlanta, † transfusion & transplantation technologi, decatur, ga, usa we previously described a methodology for accurate immunohematology testing by fluorescence cytometry [roback, j.d. et al. (2004) transfusion 44 (2), 187]. this system utilized low-speed centrifugation of 96-well filter plates for red cell staining, and a smallfootprint capillary cytometer for data acquisition. when authentic clinical samples from hospitalized patients were tested for abo group, the presence of d antigen, and red cell alloantibodies, the results were well-correlated with those obtained by commerciallyavailable column agglutination technology (cat). this system determined the correct abo group and d type for 98.7% of 520 samples, compared to 98.8% for cat (p > 0.05). when samples were tested for unexpected alloantibodies, fc determined the correct result for 98.6% of 215 samples, as compared to 96.3% for cat (p > 0.05). this novel method was better than cat at detecting weak anti-a (p < 0.0001) and alloantibodies. based on these promising results, we sought to completely automate this method by inte-prevents the aspecific amplification of the k-allele, and makes it possible to detect the fetal k-allele in the presence of excess of maternal k-alleles. furthermore, it has been shown that fetal dna is in the plasma present in shorter fragments (<300 bp) than maternal dna. size separation of cell free fetal dna can therefore be used to increase the relative concentration of fetal dna, which will help the development of new genotyping assays. in conclusion, cell free fetal dna in maternal plasma is nowadays routinely used for prenatal rhd typing and fetal sexing. new technical developments will make it possible to extend these indications to other blood group antigens in the near future. more insight in the characteristics of fetal dna might finally lead to wider applications, including numerical chromosomal aberrations. furthermore, it might become possible to apply genomic dna microarrays for the screening on many different inherited diseases, including hemoglobinopathies. determination of the affinity of anti-d present in the serum of immunized subjects and in anti-d ig preparations by a method using unlabeled antibodies p lambin*, m debbia* and y brossard † *institut national de la transfusion, † chp hopital saint antoine, paris, france introduction: few data are available concerning the affinity of maternal anti-d responsible for the hemolytic disease of the fetus and the newborn (hdn), and the affinity of anti-d immunoglobulin used for the prophylaxis of that disease. we recently described a method to measure the affinity (ka) of untagged anti-d monoclonal antibodies. aims of the study: in this work, a similar method was applied to determine the affinity constant (ka) of polyclonal anti-d present in the serum from d-immunized mothers and donors and from anti-d ig preparations. methods: a constant amount of o r1r rbcs was sensitized with increasing concentrations of anti-d present in the sera from immunized subjects, and in anti-d ig preparations. at equilibrium, the amount of anti-d bound to rbcs was measured by elisa. the scatchard equation (linear regression) and the langmuir equation (hyperbolic regression) were used to determine the ka of anti-d. the experimental data fitted well with the scatchard equation (mean r † = 0.95) but a better correlation was observed with the langmuir equation (mean r † = 0.99). in 11 maternal sera, the mean ka of anti-d was 5.6 ¥ 10 to the 8 m-1 (from 2.8 to 12 ¥ 10 to the 8 m-1). in the sera from 6 immunized donors, the mean ka was 3.9 ¥ 10 to the 8 m-1 (from 1.5 to 6.8 ¥ 10 to the 8 m-1) and in 5 lots of anti-d ig, the mean ka was 3.4 ¥ 10 to the 8 m-1 (from 3.1 to 4.2 ¥ 10 to the 8 m-1). the comparison of anti-d affinity measured in 5 cases of hdn in which infants presented a fetal anemia and in 6 cases of hdn in which infants presented only a postnatal anemia showed no significant difference. the mean value of ka in the cases of fetal anemia was 5.4 ¥ 10 to the 8 m-1 whereas in the cases of postnatal anemia the mean value of ka was 5.8 ¥ 10 to the 8 m-1. conclusion: the method previously described for monoclonal anti-d was applied to polyclonal anti-d present in the serum of d-immunized subjects and in ig preparations. the experimental data fitted well with the langmuir equation, and the affinity of polyclonal of anti-d was measured with accuracy. in addition, no significant difference was observed (at least in the 11 cases of this study) between the affinities of anti-d measured in the most severe cases of hdn (fetal anemia) and in the less severe cases of hdn (post-natal anemia). introduction: cryopreservation of platelets is widely used in platelet immunology to ensure the availability of well characterised panel cells for the detection of hpa antibodies. but recovered platelets do not express the hpa-15 alloantigens. aim of the study: here we describe a method for the successful preservation of platelets by lyophilization. we report the value of this new reagent for the detection of hpa alloantibodies and especially anti hpa-15 alloantibodies. methods: rehydrated lyophilised platelets (lyo p) were tested for their reactivity with monoclonal antibodies against gpiibiiia, gpibix, gpiaiia and cd109 by flow cytometry. the levels of reactivity were comparable with the ones obtained with fresh platelets. the rehydrated platelets were used in the maipa with a panel of hpa antibodies (anti-hpa-1a, 30; anti-hpa-1b, 3; anti-hpa-3a, 3; anti-hpa-5a, 3; anti-hpa-5b, 50; anti-hpa-15a, 2 and anti-hpa-15b, 4). results: all hpa antibodies showed the expected pattern of reactivity and in several cases absorbance reading were well above those obtained with fresh platelets. absorbance values produced by inert sera were comparable with those obtained with fresh platelets (ranges 0.001-0.008). interestingly, we used lyophilized platelet with a high expression of cd 109 bearing the hpa-15 system and we have detected 24 anti hpa 15 antibodies among 1000 sera previously negative with fresh platelets. nineteen sera concerned patients suffering from hematological diseases and 5 from pregnancy women. conclusion: lyophilized platelets are possibly an ideal reagent for the platelet immunologist to be used for the detection of hpa antibodies. moreover, this work bring new insights on the hpa-15 system in platelet transfusion. we are now pursuing more extensive validation studies with a larger number of samples representing all known hpa specificities and several diseases. the diagnosis and treatment of sick infants and children requires a broad knowledge of physiology, biochemistry, genetics and the application of sophisticated testing and treatment options. one of these options is transfusion of blood and blood products. transfusion of the infant, especially the premature infant, and sick child, especially those with major organ dysfunction, requires careful consideration of their unique metabolic, hepatic and renal clearance mechanisms. guidelines that direct the indications for transfusion differ from those in adults. non-invasive measures of oxygen delivery and oxygen offloading may assist in guidelines for red blood transfusion. metabolic complications from massive transfusion and/or the manipulation of blood products must also be considered. evidence from a high quality randomised controlled trial suggests that anaemia is also well tolerated by critically ill patients. a restrictive approach to rbc transfusion that maintained the hb concentration between 70 and 90 g/l was found to be as effective as and possibly superior to a more liberal strategy of maintaining the hb concentration between 100 and 120 g/l. there are concerns that some groups of critically ill patients, such as those with cardiovascular disease and patients who are difficult to wean from mechanical ventilation, may benefit from higher hb levels. rbcs also have a role in primary haemostasis and higher triggers may be appropriate in coagulopathic patients. it is important to realise that blood is not a uniform product and the clinical efficacy of rbc transfusion may vary. one factor that may have a considerable effect on the quality of the rbc product is the storage time. rbcs undergo marked changes during refrigerated storage. the implications of these changes on tissue oxygenation are not known but these concerns have led some clinicians to request 'fresh' blood for critically ill patients. there is insufficient evidence to support such practice. it is of great practical importance to determine if, or when, fresh rbcs could be superior to stored rbcs. background: with a decreasing blood donor base, fully tested, fresh unrefrigerated whole blood (fuwb) has been found to be a more efficient and effective use of a limited resource in place of, or as an adjunct to, traditional blood component therapy in surgical situations associated with massive blood loss. aims: to outline the use of fuwb in situations where there is potential for intractable bleeding associated with major surgery, and evaluate platelet function in fuwb versus platelet components. methods: outcomes of fuwb and traditional blood component use were examined for 20 cases of cardiac bypass surgery. in addition, exclusive use of fuwb for 23 burns debridement cases was analysed. an evaluation of platelet function in whole blood compared to platelet components was also performed by measuring platelet aggregation and activation parameters. results: there was a decreased requirement for blood components following administration of whole blood post cardiac surgery. whole blood usage for burns debridement surgery eliminated the requirement for additional blood components. platelet activation was markedly reduced in whole blood compared to component platelets, and this may be one reason for the increased efficacy of whole blood in these clinical settings. conclusion: fuwb appears to have a role in minimising blood product requirements and consequent donor exposure in situations associated with massive blood loss. m-pa-051 transfusion practice for coronary artery bypass surgery in greece s lakoumenta, m vassili, g hatzidimitriou, t asteri, p stratigi, s kanellas and g palatianos hellenic society of blood transfusion, athens, greece cardiac surgery is associated with a demand for allogenic blood and blood product availability as well as a considerable consumption. the impact of consensus guidelines for allogenic blood transfusion during coronary artery bypass graft surgery (cabg) in us attracted great attention 1. the present study is conducted in order to reveal the transfusion practice in greece on a similar population i.e. patients undergoing cabg operations. methods: five participating centers collected data concerning transfusion of allogenic blood and blood products in patients undergoing elective first time cabg procedures, as well as parameters that may influence blood loss, such as duration of the operation, cardiopulmonary bypass time (cpb) etc. the total estimated blood loss was calculated as the sum of red blood cell volume reduction [(body weight in kg ¥ 60 ml/kg) ¥ (admission haematocrit-discharge haematocrit)]+(red blood cell volume transfused). results: results are shown in table 1 : means, standard deviations, and p-values of the wilcoxon test comparisons between the hospitals. a preliminary analysis of data from 5 centres (268 patients) showed no difference in patient characteristics (age, body weight, male to female ratio). there is a statistically significant difference (p < 0.0001) between the five centers in duration of the operation, cpb, estimated blood loss and volume of transfused plasma. red cell use showed also a variation which however did not reach statistical significance (p < 0.02). the center with the highest figure for blood loss has the lowest volume of allogeneic red cell transfusion because of the use of cell salvage. conclusions: although variations, as those observed in greek cardiac surgery centers, have been documented in other countries, the variation in the use of plasma is striking and we are in the process of trying to identify the reasons. our study is in progress and additional data are being collected and will be presented. introduction: premature infants and at term newborns have an higher circulating blood volume per kilogram than the adults (100 ml/kg in premature; 80 ml/kg in at term), for this reason, in case of neonatal thrombocytopenia, a specific hemocomponent, with a very high platelet concentration, needs for transfusion therapy. the laboratory criteria for platelet transfusion are the following: (a) a plt count < 150 ¥ 10 9 cells/l if bleeding is observed; (b) a plt count < 20 ¥ 10 9 cells/l without bleeding; (c) a plt count < 50 ¥ 10 9 cells/l in newborns showing critical clinical conditions. aim of this study: in this study, we have monitored the plt transfusion therapy in our neonatal intensive care unit (nicu) in the last four years. methods: effects of plt transfusions have been followed in 37 children (31 premature infants and 6 at term newborns). the weight of premature infants ranged from 600-1800 g and at term newborn from 1800-4000 g. gestation age of premature infants ranged from 26-36 weeks and of at term ones, of course, from 37-42 weeks. for every platelet transfusion in these newborns, the volume of platelet concentrate has been of 10-20 ml/kg, with a plt count < 700 ¥ 10 9 cells/l. results : in the study period, 77 plt transfusions have been performed: 23 children have been only transfused one time, while multiple plt transfusions (ranged 2-10) needed for 14 children according to clinical conditions. the observed clinical indications for transfusions have been the sepsis with haemorrhagic syndrome (26 cases) , haemorrhagic syndrome without sepsis (7 cases) and neonatal alloimmune thrombocytopenia without haemorrhagic syndrome (4 cases). after 24 hours from transfusion therapy, the absolute plt count and the correct count increment have increased in all 37 little patients. the highest increase in plt count was 45 ¥ 10 9 cells/l, while the lowest 5 ¥ 10 9 cells/l. no difference in the efficacy of therapy has been detected between premature group and at term group. 94% of children have been discharged from hospital in good general conditions without complications in following controls. in conclusion, we can affirm that plt transfusion in premature infants and in at term newborns is an efficient and safe treatment of severe haemorrhagic conditions. however a collaboration between nicu and transfusion center is necessary to choice the adequate platelet concentrate's volume for transfusion and the best plt donor for the newborn. developing transfusion strategies 27 fusion society of turkey (bbtst) in 2004 with contribution of 253 blood transfusion centers. according to these figures 51% of centers attended operates apheresis procedures. two centers informed us that apheresis in the hospital was carried out at blood bank. there was not enough information from one center, so it was excluded. of the 51 blood banks performing apheresis, 30 were university hospital blood banks. another 38 blood banks were producing both productive and therapeutic, 10 produce only productive and 2 produce only therapeutic procedures. one center did not respond. all centers reported to prepare and separate erythrocyte and plasma. however only 48 centers reported to prepare random platelets as well. each center had apheresis machines between 8-15. a total of 19 centers was carrying out around <500 procedures, 12 around 501-1000, at 9 centers about 1001-2000, at a further 10 centers around 2001-5000 procedures a year (one center was excluded). of the responders to the survey 36, all procedure were done at blood banks, whereas at 6 of them all were carried out by the hematology clinics. at 8 other centers, productive procedures were conducted by the blood bank, and therapeutics were performed by the hematology division. a total of 48 blood banks stated that they have not kept the platelet suspensions produced and used them straightaway. productive apheresis center capacities were as shown: 13 centers < 5000, 16 centers 5000-10000, 13 centers 10001-20000 and 6 centers > 20000 units have donations a year. around 68% of all apheresis procedures were carried out by large well run blood banks. conclusion: as the use and production of random platelets increase, and settle of apheresis devices in big centers will eventually decrease the demand of apheresis procedures and keep the welltrained staff at big centers, decrease the cost thereafter. • planning of resources for the financing of the bts, adoption of a methodology for creating and adjusting the price list of products, adoption of the yearly plan of needs for blood/products and services of health institutions which use blood/products. • achieving recognition of real costs of products and services from the health insurance fund and ministry of health. • harmonization of low level of acclaimed costs and real costs of basic transfusion activities. results: acclaimed costs for activities in transfusion practice (collection, testing, processing, storage, distribution and transport) as a reflection on the price of the products are 60% lower than real costs. the prices of health services in the official price list are much lower than the proportion of costs of material resources needed for the realization of these services. this especially affects the management of independent blood establishments (bti's in serbia) with core blood transfusion activities as their basic field of work, in comparison with the 44 hospital based transfusion services, which are financed within the budget of the whole hospital. the 44 hospitals with hospital based transfusion services involved partly in core transfusion activities are completely financed by the health insurance fund, while independent blood establishments are financed through the price of products and services they provide. conclusion: in order to provide adequate quantities of safe blood/products for the end user -the patient, it is elementary to create stabile and equal financial management conditions for the whole blood transfusion service in serbia. this can be achieved only by continuous cooperation of the health insurance fund, ministry of health and independent blood establishments. sion centers (rbtc). the activities on promotion and organization of voluntary, nonremunerated blood donation, blood collection and patients' services are carried out in the rbtc and in 24 departments of blood transfusion (dbt), part of the district hospitals. the collected units in dbt are transported by special cars to the ncht and the rbtc for processing, testing and control. the same transport is used for the requested by dbt blood components for storage and distribution to hospital departments. thus the issued components are with an equal quality and safety for all patients throughout the country. lbbdbt introduces hemovigilance as a mandatory system, covering the whole chain of the blood transfusion process. it includes as well the creation of registries at a national, regional and district level of blood donors, recipients of blood products and all activities of the blood transfusion service. . seventy hospitals are exclusively users of blood, blood products and services. the current organization of blood transfusion services faces the following problems: fragmented transfusion service, lack of a national blood policy, the blood program is not nationally coordinated, limited knowledge on quality management, inadequately distributed human resources, limited material resources, lack of it system, lack of planned, continuous skill upgrading. as a direct consequence we have: suboptimal blood collection activities, inadequate blood supplies significantly vary between seasons, high percentage of replacement donors, outdated methodologies, old, even obsolete equipment, the quality of blood products is not standard, there is a lack of traceability. aim of study: to reorganize blood collection activities in serbia to increase collection of safe blood up to 4% (304 000 blood units). methods: division of responsibilities between blood establishments and hospital based transfusion services by: • optimizing organizational structure • implementing blood collection standards to enhance blood safety and donor care • gradually replacing family donors with a network of voluntary non-remunerated blood donors from low risk population groups • creating and implementing a training strategy. results: through the eu funded project support to a national blood transfusion service in serbia, we are in the effort of integrating the services and standardizing their work. the blood collection working group began by dividing serbia into 3 blood collection regions: north, central, and south. each region is divided into sub regions covering approximately half a million population (4 in the north, 8 in the central and 4 in the south region). each sub region will have one standard mobile blood collection team to collect 80 blood units daily, i.e. 19 000 annually. the 19 000 blood units per 16 teams provide the 304 000 blood units (4%) to cover hospital needs in serbia. to this effect, the following has been achieved: • blood collection activities in serbia analyzed • performance analysis of bte and hbts mobile teams in place • two model standard mobile teams tested in the field • national blood collection sop's written • national donor questionnaire form prepared • national set of blood collection standards prepared • list of donor deferral criteria prepared • blood collection equipment renewed • regional reorganization plans in progress. the objectives and results can be achieved by the participation and mutual cooperation of all institutions involved in blood transfusion within an integrated, standardized system with clearly delegated responsibilities. p-005 60 years of the national blood transfusion institute in serbia n nedeljkovic national blood transfusion institute, belgrade, yugoslavia nbti was founded in 1944 . since 1953 and unpaid blood donation is mandatory, organized in cooperation with red cross. blood donation is regulated by the law in 1974, 1993/94. codex of voluntary blood donation and health care staff has also been established; 300 000 blood donors donates blood annually. in the past 60 years, there was over 3 million blood donations, performed in accordance with who regulations. over 250 transfusion medicine specialists and 700 technicians specially trained for the work in blood transfusion service (n = 53), perform transfusion medicine doctrine of rational labile blood component and stable blood derivatives therapy, based on the selfsufficiency concept in fr yugoslavia with 10.3 million inhabitants in serbia, montenegro and kosovo. plastic blood containers and tests are imported or given as humanitarian aid gift and from 2003. now, they have been regulated by tender. in 1951, test to lues was introduced, to hbsag in 1971, to a-hiv in 1985 and to a-hcv in 1993. information system was introduced in 1987. nbti includes: national haemovigilance coordinatoin center, center for medical care of haemophiliacs, tissue typing center, center for prenatal and perinatal protection of pregnant women and newborns. activities of nbti are organized through: center for planning, organization and development of blood transfusion service, center for blood collection, preparation and distribution, center for immunology and immunochemistry, plasma fractionation center for plasma in 8 west balkan countries, center for diagnostics means, center for quality control of drugs and medical and diagnostic means, center for education and training and scientific research work. nbti is the third year of gmp, sop, yus iso 9002 implementation. in the current reform of transfusiology system we are aiming for 4 percent of voluntary blood donation. nbti is the publisher of the national bulletin of transfusion medicine and it is included in the education system of the belgrade university medical faculty and the estm in belgarde 2003. the problems of blood service in russia ea selivanov and t danilova russian inst. of hematol. & transfusiol., st. petersburg, russian federation background: the blood transfusion service (bts) development as a platform for providing the hospitals with blood and blood derivatives is an important national problem. aims: russian blood service assessment with international comparison. methods: a study was conducted on the base of the reports from all regions of russia followed by a computer statistical analysis. results: blood and blood components were collected in the russian federation in 2003 in 190 stations of blood transfusion and in 1065 blood transfusion departments at big hospitals. amount of donors in 2003 was equal to 2 202 853, voluntary donors being 87.2% of them. the average number of whole blood donations in relation to the general population is 25 per 1000 inhabitants, and on average 28 percent of the donor base consists of first time donors. the average number of blood collected in relation to the general population and health care system is 11.4 ml per inhabitant and 1240 ml per one bed. an average volume of one blood donation is 396 ml. blood was collected into plastic bags containing domestic or foreign anticoagulants. about 93.2% of collected blood is used for procurement of blood components and preparations, 0.8% of banked blood is used for transfusions. amount of donors and the volume of whole blood have been significantly decreased for the last 15 years. at present in russia all donations are tested for abo blood group, rh(d) type, anti-hiv-1/2, hbsag, anti-hcv and syphilis. the total percentage of blood discarded after testing for transfusion-transmissible infections is 4.5%. 32% of plasma is obtained by plasmapheresis. blood components collected are as ffp, rbc, frozen rbc, 1eukocyte-and platelet-depleted rbc, rbc suspension, and preparations: 10% albumin, immunoglobulins, and cryoprecipitate. as to blood safety measures -implementation of blood components leucodepletion and ffp and rbc quarantine in process. the new national strategy of bts reorganization has been developed. it includes the following: increasing the visibility and resource commitment to blood issues at the national, regional and municipal levels; the national voluntary donor programme promoting; blood safety increasing; blood collection, testing and pro-cessing concentration in federal and regional bts establishments, appropriate blood and blood components usage. calculating the cost of blood in turkey n solaz, s kemahli and s cin ankara university, ankara, turkey background: like other fields of the medicine cost efficacy is gaining importance in blood banking and transfusion medicine since last few years. since last 5 years even the most developed countries started to discuss about the cost of blood. in turkey ministry of health determines the cost of blood annually. aim: to establish a safe, cost effective and reliable prices for blood components. methods: turkish ministry of health (moh) started to determine the cost of blood components as 'all inclusive' principle. this means that cost of a unit of blood component will cover all conventional expenses such as; blood typing, infectious screening, labour, consumables, etc. this system has provided uniformity to blood component costs but if the system is not controlled and followed properly it will cause serious risks. there might be some blood banks which will not respect the safety regulations and may modify the test standards for decreasing the cost of tests. conclusion: current blood product pricing system looks generally reasonable and reliable but moh should establish close follow up systems for avoiding any abuse on the safety of blood. background: a positive direct antiglobulin test occasionally occurs in normal blood donors, and is often discovered when the donor's red cells are found incompatible in a compatibility test. the incidence of a positive dat was expected to increase since more sensitive techniques (gel test) were installed. the aim of our study was to examine whether dat positive otherwise healthy donors presented any clinical or laboratory abnormalities. methods: in the first 19.000 cross-matches last year (in 10 months) 38 were found incompatible due to dat positivity of blood donors' red cells (0.02%). dat positive [(1+)-(3+)] samples were only igg positive in 32 cases, only c3d positive in 5 and igm positive and c3d positive in 1 case. all blood donors were notified and thirty two of them responded to a request for a further sample. a complete blood count, a reticulocyte count, bilirubin (total, direct, indirect), transaminases, serologic immunological tests (ana, anti-dna, anti-ena, rf, anticardiolipin antibodies), quantitative assessment of immunoglobulins, aptt and lupus anticoagulant were performed, as well as serologic tests for markers of viral infections. dat and iat were performed by gel test (id-diamed) according to the manufacturer's instructions. dat were performed with polyvalent and monovalent reagents (anti-igg, -igm, -iga, -c3c, -c3d). the blood donors were also examined clinically. the donors who had positive immunological tests were referred to a rheumatologist for further investigation. results: among the thirty one blood donors eight had received medication the last 24 hours before blood donation, two had been vaccinated for hepatitis b recently, four presented signs of a viral infection soon after blood donation, three had evidence of an allergic condition, five had positive tests for anticardiolipin antibodies and ana, two were positive for anticardiolipin antibodies only and two had a positive ana test only. in six blood donors we did not find any abnormality that might be interrelated to dat positivity. conclusions: all blood donors with positive dat should be requested to undergo further investigation. some of them are possibly candidates to long medical follow-up, especially those with other immunologic abnormalities such as positive ana and/ or anticardiolipin antibodies. the eligibility of such donors for future donation of whole blood, platelets or plasma needs to be elucidated. tions: usefulness, frequency and sincerity in answering questions. donors could choose one of the offered answers and elaborate in writing the answer they have chosen. results: of the 1000 donors that participated in the survey 947 (97.4%) answered the questionnaire, 820 (86.6%) men and 127 (13.4%) women. that the survey was useful thought 91% and 9% that it was not. opinions were elaborated by 61.1%. that the questionnaire should be completed before each blood donation was the opinion of 81.2%, 17% thought it should be filled out only the first time blood is donated and 1.8% that the questionnaire should not be completed at all. the answers given were sincere in 95.4% of blood donors, 2% were not and 2.6% were given automaticallywithout comprehension. conclusion: most donors believe that completing the questionnaire before each blood donation is an effective way to increase safety by preventing potentially infected individuals from donating blood. they are also aware of the importance of answering questions truthfully because the end result is protecting the wellbeing of both blood donors and receivers. analysis of blood donor's deferral in national institute for transfusion medicine -skopje for the last five years (2000) (2001) (2002) (2003) (2004) p blagoevska*, i nikolovska † and r grubovik* *national institute for transfusion medic, skopje, † medical center, prilep, macedonia introduction: safety of blood and blood products depends on many different factors, starting with selection of blood donors. the aim of this study is to analyze the number of deferred blood donors and the reasons for their deferral, as well as the total number of blood donors in nitm and their correlation (voluntary/family donors). materials and methods: this is a retrospective, epidemiological study and data were taken from the blood donor's registry in nitm from 01.01.2000 till 31.12.2004. statistical mass includes blood donors who came to nitm to donate blood in the mentioned period. results: there were total 14 229 donors in nitm and 541 (3.8%) deferrals. 71.5% of deferred ones are male, as well as in the group with donated blood (males are predominant). the most common reason for deferral is low hb level in 232 (42.9%) blood donors, use of drugs -78 (14.4%), low blood pressure -47 (8.7%), high blood pressure -43 (7.9%), infections -30 (5.5%), cardiovascular diseases -21 (3.9%) and others. relation voluntary/family donors is almost equal (49. 6 : 50.4 ). in the last two years the number of voluntary blood donors is increasing (60 : 40), which is good sign. conclusion: percentage of deferred blood donors in first three years is ~2%, which is result of insufficient data and it is increasing in the last two years (>6%). reasons for deferral are predominantly from temporary character (98.9%). permanent deferrals are only 6 (1.1%), which is probably due to good education of the population and self-deferral. we should establish the national registry for deferred donors, as well as for the donors with positive markers for tti. we should design a strategy for returning of temporary deferred donors. regruting blood donors in multiethnical environment p blagoevska*, r grubovik* and k elezi † *national institute for transfusion medic, skopje, † medical center, gostivar, macedonia introduction: population in r.macedonia consists of 75% macedonians, 22% albanians and 3% others (serbs, gypsies, turks). over 80% of blood donors are voluntary non-remunerated and ~20% are family donors. transfusion service and red cross should recognize the values and cultural differences of minors groups and recruiters should developed methods for reaching and motivating them to donate blood. the aim of the study is to present the ethnical structure of our donors and to develop strategy for their regrutation and retention. the study reviews the results from the blood donation actions among the high schools and university students in west part of the country (multiethnical environment) from 2002 till 2004. results: there were 3704 blood donations for the mentioned period. predominant blood donors are employed and high school students in 75%. family blood donors are ~20%; between them 70% are from albanian population. the ratio between blood donors macedonians vs. albanians is 80 : 20. woman blood donors are presented with 14%. first time blood donors are 53%, and regular donors arẽ 13%. conclusion: first step in planning the blood donation in multiethnic society is creation of special teams of important and devoted volunteers, such as religious leaders, teachers, doctors and businessman. for a successful campaign it is necessary to design special promotional material and address personally to the target population on their mother language. background: pursuit of pharmaceutical purity of the blood in the bag has led to a shrinking donor base and a significantly more expensive product. decisions regarding new infectious marker testing and donor deferrals have typically been made emphasizing decreasing one specific risk without considering the effect the intervention will have on the overall safety of blood transfusion. regulations have been formulated by governmental agencies with limited input from the medical community. the decision making process has lacked risk benefit analyses and has not had the robustness associated with spirited discussions. policies made in this manner may result in certain risks being decreased but can also have adverse unintended consequences. discussion: in the u.s., the fda's implementation of donor exclusions to prevent possible transfusion transmitted vcjd has reduced the donor base by more than 2%. given the demographics of the deferred donors, the impact on plateletpheresis donations has been even greater. to compensate for the loss of donors, blood services will have to persuade present donors to donate more frequently, to recruit new donors, or both. one study has indicated that two-thirds of donors have no intention of donating more frequently. new donors have higher rates of infectious disease markers with positivity for hiv and hcv twice as high as repeat donors. despite sensitive testing techniques, window periods still exist and not all potentially infectious donors will be excluded. another area of concern is the aggressive use of inducements to attract new donors. some blood services are offering lavish incentives such as enrolling donors into drawings to win automobiles. most donors entering the lottery will be low risk; however, it is reasonable to worry that such extreme tactics might also attract persons who should not be donatconclusion: (a) blood donors who were patients' relatives were many more than volunteers as well as more were men than women. also people of young ages were more than those from older ages. (b) the frequency of the diseases for which the blood units were tested was found to be in low levels in the population of the area. specifically as concerns hcv, it seems that transmission frequency has been reduced after the obligatory testing of hcv in blood transfusion centres and stations. genotype 1b of hepatitis c virus is the most frequent in blood donors 2d, from 3a to 3f, from 4a to 4k, 5a and 6a. these are differently distributed in the world: types 1 and 3 are the most common in europe and in usa. aims: considering that, in our region, anti-hcv antibody positivity is variable from 0.87 to 4% of general population, aim of this study has been to evaluate the prevalence of hcv genotypes in blood donors. methods: in period from may 2003 to december 2004, 83 362 blood units were analyzed by nat for viral rna research. nat has been performed on single sample by tma technique. on rna-positive samples, the hcv genotype has been identified by reverse hybridisation with line probe assay. results: 143 blood donors have resulted hcv-rna positive with identification of the following genotypes: 1a = 11 cases (7.7%); 1b = 89 (62.3%); 1a + 1b = 3 (2.1%); 2a/2c = 35 (24.4%); 3 = 1 (0.7%); 4 = 4 (2.8%); none was 5a or 6a. we have also analyzed the differences between the two sexes in hcv-genotypes distribution. hcv-1a has showed a double prevalence in men (8 cases, 5.6%) respect in women (3 cases, 2.1%), while genotype 1b is more frequent in women (48 cases, 33.6%) than in men (41 cases, 28.7%), moreover genotypes 3 and 4 do not compare in women. although an accurate pre-donation selection, discharging all subjects with alt >40 iu, our results show that 1.7 : 1000 donors, apparently healthy and without risk factors, have resulted hcv-positive. analyzing our data, the genotype 1b has resulted the most frequent in blood donors' population, followed by type 2, while the others have showed a very low prevalence. the high frequency of genotype 1 in blood donors is explained by the observation that hcv 1 is usually associated with low alt levels, for this reason affected subjects may escape to donor's screening only based on dosage of alt. on the contrary subjects affected by other hcv types, associated with high alt levels, may be deferred increasing the hcv 1b relative prevalence. at the end, the different distribution of hcv genotypes between men and women and between age's classes probably reflects differences in the pathogenic characteristics of the virus, in the transmission way and in the risk factors. in fact, it has been demonstrated that genotype 1 is principally linked to a not transfusion transmission way; genotype 2 is linked to old age, to female sex and to post-transfusion transmission; genotypes 3 and 4 are associated to young age and to an history of drugs abuse, respectively with high and low viral load; genotypes 5 and 6 are still little known because extremely rare in europe. p-023 kell blood group system and rare blood donors v fakitsa*, p karyda*, s giannoulea † , c antoniou*, j flesiopoulou*, e haliou*, m papakonstantinou*, h dessilla † , e katsadorou*, g lyrakos* and k sofroniadou* *general hospital of nikea, pireas, † blood transfusion center, athens, greece background: the kell blood group system is a compound antigen system exclusively of red blood cells. some of the kell antigens are highly immunogenic. the commoner kell antibody is anti-kel1. the kel2 (cellano) antigen is a high frequency antigen and the blood donors lacking this antigen are quite rare. the blood donors who have not factor cellano are classified in the rare blood donors. rare blood by its very nature is required rarely, but when needed that blood has to be ensured to specified patients. there are other blood donors in their family -74 (35.57%) students, but the number of persons that donate blood from their neighborhood and close environment is much bigger -175 (84.13%). motives for their donation are the following: their wish to help the ones that need blood -142 (68.26%), concern that some day everyone can be a potential recipient of blood -38 (18.26%), because of offered benefits -27 (12.98%), for a friend or relative -23 (11.05%), care for their health -16 (7.69%), because of citizen duty -8 (3.84%), because the others donate -6 (2.88%), curiosity -1 (0.48%). they want to be invited every 6 months -68 (%) students, every 12 months -38 (18.26%), every 3 months -16 (7.69%) and 16 ( the mean age of case group was 30/55 ± 9/33 and the mean weight of them was 71/23 ± 9/3, 72/6% was male and the mean number of blood donation was 2/34 ± 2/19. the mean age of control group was 35/27 ± 10/72 and the mean weight of them was 78/61 ± 12/77. 90/3% of them was male and the mean number of blood donation was 6/58 ± 7/08. the blood donors who were female, first time blood donor low wt the rate of vasovegal rx was higher in female, first time, low weight, younger blood donors (p < 0.005). the rate of vasovegal rx was higher in blood donor (p < 0.005) who were fatigue or first time blood donor, low wt blood donation, fatigue of them and starvation of them had higher absolute donation reaction than other donors. when each variable was adjusted for other variable by regression analysis. young age, first time blood donation, anxiety, fatigue, starvation were significant (p < 0.005) and the others were not. conclusion: donation -related vasovegal syncopal reactions are a multi factorial process. these reaction are more prevalent in blood donors who are young, first time donor, anxiety, fatigue, starvation. these reactions might be predicted vasovegal reaction and these some facth donors need more care. with better donation care, syncopal reaction may be decrease this would be improved donor safety, better donor retention, higher donor satisfaction, and reduce cost and increase regular blood donors. to avoid iron deficiency in blood donors, iron compensation is necessary in most females and males who donate more than 1-2 and 3-4 whole blood units per year, respectively. we present 3 studies dealing with different dose and duration of iron compensation. in the first randomized placebo controlled study iron decreased continuously in males and females at donation intervals of two (males) and three months (females) without iron compensation. 40 mg and 20 mg daily combined with 400 mg ascorbic acid over 2 months (males) or 3 months (females) compensated for iron loss or even overcompensated in females. in the second open study we reduced iron dose to 20 mg daily over one month for both genders. this iron dose was sufficient for compensation of iron loss. a further reduction of iron dose to 20 mg daily over half a month led to negative iron balance in the majority of donors. in all three studies donors with exhausted iron stores profit more from iron compensation, whereas donors with high ferritin values (>50 mg/ml) tend to loose storage iron. aim of the study: one of our campaign strategy how to increase blood donation among adolescents are periodical seminars and excursions for students of secondary schools (more than 40 per year). the aim of this study is to analyze impact of our campaign educational system on adolescents in period 2000-2004. methods: the donation of whole blood and aphaeresis platelets from donors of age from 18 to 20 (max. 3 years for each class) were count for the period of five years (2000) (2001) (2002) (2003) (2004) . the percentage of the man´s donation was calculated for each target class (1982) (1983) (1984) (1985) (1986) . results please see tables 1 and 2. in the tables there is shown observed data in relation to the total number of births in the czech republic in reviewed years. the study showed that number of donation from donors of age from 18 to 20 decreased during objected years. unfavourable state of total number of births in the czech republic (153 801 birth in 1980 republic (153 801 birth in , 133 942 birth in 1986 ) and its decreasing tendency (92 786 birth in 2002!) is with high probability a major demographic factor affected number of young donors. despite energy invested in our campaign educational system our recruitment efforts should be intensified to decrease influence of demographic factors. we should find new ways and methods to attract new blood donors and keep the regular ones, too. the aim of the research was to investigate women's attitudes towards blood donation in cyprus. a statistical sample was selected using stratified sampling and consisted of 369 women from the district of limassol (the second largest urban center of cyprus) between the ages of 19 and 60. using linear logistic regression, the analysis of the data collected revealed that there is a greater probability for a woman to be a blood donor if she is of a higher educational level, a member of an organized group or association, or if she is acquainted with other blood donors. the percentage of female blood donors is higher in rural areas than in urban centers. 40% of women do not donate blood and attribute their reluctance to do so to health-related problems, while about 25% of those who have never donated blood claim to fear the blood donation procedure. in addition, more than half of the women who have stated they would never donate blood again have attributed their denial to healthrelated problems. the research revealed that there could be an increase of up to 30% of the percentage of female blood donors if they were given time off work for a few hours or one or two days afterwards. even though very few female blood donors expressed a preference for the blood donation to take place on a particular weekday, half of them prefer the donation to take place on the discussion: it is about small group of students. the impression is that the altruistic behaviour is present at most of the questioned students. the fact about free school days is not underestimated because it is one of the most important motives of blood donoring of the young population. families where the blood donoring is a tradition have a great influence for young children because the children in these families are better informed for blood donoring. conclusion: including the children in the process of education for young children is of particular importance because the altruistic behaviour as a higher feeling is from an early age of the child and it is under the influence of the environment (family and friends). active participation of the department for transfusion medicine in the educational process, especially in the education of young children, is a guarantee to achieve longlasted positive results. adverse reactions in 2 blood donors taking betablocking antihypertensive medications l paesano*, m d'onofrio*, s misso † , g fratellanza* and e d'agostino* *university federico ii, naples, † hospital san sebastiano, caserta, italy one aim of blood donor's selection is to avoid an adverse reaction to phlebotomy (as vasovagal reaction, syncope and/or hypovolemic cardiac insufficiency). blood donation is surely contraindicated in various pharmacologic therapies, but not in all. in fact a certain degree of discretionarily exists about the assumption, or the period of suspension, relative to a numerous pharmaceutical products, as the antihypertensive agents. according to literature, the deferral of donors taking antihypertensive medication is not indicated when blood pressure is normal, symptoms are absent, and diuretics or similar agents are the only drugs used. on the contrary, it is a common opinion that an antihypertensive therapy by betablockers is not compatible with blood donation for its cardiac effects. nevertheless, in our daily activity, the observation of a blood donor taking beta-blocking drugs may occur for various causes. a possible error is a superficial pharmacological anamnesis, as it can occur in donations on autohemotheca, for a too fast medical visit (due to a large number of donors), or for the inexperience of the selector (often a not specialist of transfusion medicine young doctor). another possibility of observation is constitute by patients, undergoing to elective surgery, included in a program of autologous blood donation, suffering hypertension treated with betablockers. in fact, in this last case, the risk/benefit balance justify the blood letting procedure. in the last year we have just observed two severe post-donation reactions in donors suffering hypertension treated with atenolol. the reactions have been similar, in fact both donors showed lypotimia followed by convulsions about past half hour by the end of phlebotomy. no prodromic symptoms have been observer or referred. cardiac frequencies (cf) before donation were respectively 60 and 64 beat per minutes and blood pressures (bp) were both in the normal range (130/80 and 120/80 mmhg). after donation, during adverse reaction, cf showed no substantial variations, while bp have been decreased respectively to 80/45 and 60/30 mmhg. immediate treatment has consisted in putting the donors in the trendeleburg's position and in applying a dolorous stimulation. in the first case this treatment has been sufficient to report the bp to 100/65 mmhg (with disappearing of all symptoms) in only half hour time. in the second one, the marked hypotension showed a very slow remission, for this reason the subministration of a plasma expander needed, with the complete resolution of the symptoms after two hours. these two donors were not deferred from donation because they were periodic donors that had modified their antihypertensive therapy, without referring it neither in the questionnaire nor during anamnesis. our experience confirms that the blood donation don't must be permitted to subjects taking betablocking antihypertensive drugs. in fact these medications act on cardiac pump decreasing the cardiac rhythm and limiting the postdonation cardiac recover. this effect is very dangerous because it appears relatively in retard respect to the end of donation, when donor may have just leaved the transfusion center. introduction and aim of the study: in society under transition privatisation and marketisation probe all areas of life. transition to market economy is extremely important and sensitive issue in health and welfare services in general, and specifically in the case of blood transfusion service. the aim of the study was to analyze effects of confusing publicity which introduced possible ways of transforming blood transfusion service in serbia (ideas about privatization of some parts of national blood transfusion institute, buying blood from blood donors, selling blood from voluntary blood donors to private clinics, exporting blood from vbd, stories about tradition of paid blood donations in some european countries). publicity was restricted to a small number of sporadic outbreaks concerted in a limited period of time. table. conclusion: surveillance of adverse reactions and injuries or accidents during or after blood donation is essential for maintaining the well being of active blood donors, as well as for the safety and quality of the donated blood components. information on other activities and parameters affecting the quality of blood including materials, reagents and equipment should be collected and any serious deviations from standard operating procedures should be notified to the competent authority using haemovigilance infrastructures. skae has built up such procedures working along the lines of the european haemovigilance network. improvement of existing national haemovigilance systems is expected to follow from the implementation of the eu directive. although inevitable, blood donor deferrals lead to losses in donated blood supply and may affect donor-return rates and subsequent blood donations. to estimate the scope of blood donor deferrals and their causes, we analyzed the 2003-2004 data from regional blood centers using standardized criteria for temporary and permanent blood donor deferrals. within this period (2003) (2004) , 14.6 percent of persons who presented for donation were deferred; 85.9% were temporary deferrals (50% due to laboratory test results, among others low hemoglobin, 6.4% due to risk of acquiring a transfusiontransmissible infection) and 14.1% were permanent (20% due to the infectious diseases markers, 8.7% due to cardiovascular diseases). for regional blood centers the temporary deferral rates varied widely (see the table below ). in the case of individual regional centers, the differences as well as the most common causes were often difficult to explain. according to our analysis, some blood centers have a more restrictive approach to donor acceptance than others and this results in increased donated-blood loss. to some extent such losses could be avoided. further studies are recommended to elucidate the problem and eliminate unnecessary deferrals. caption 1: percentage of deferrals aims: from our experience in selecting blood donors, a certain number of issues have been noticed that remain obscure and need to clarification since those seem to 'haunt' the whole process of blood donation. methods: many first time blood donors and especially volunteers think that rejection reasons are permanent and they are completely incapable of donating blood their entire life. this is a 'tragic' misunderstanding since the doctor did not explain that the reason of the rejection is only temporary and in the future this man is capable of donating blood. those potential donors will never even approach again blood donation centre and when in the future they are asked why they do not donate blood, they repeat the cause of the past rejection. results: one of these rejection reasons is for example low blood pressure (12.54% of total causes of rejection). as we all know blood pressure must be determined according to age, sex, weight and from other factors as sleep, emotional status, food and liquid intake. therefore blood pressure is very important but should be evaluated with all the above factors and must not be alone the only reason for rejection. even when one blood donor is rejected it should be made clear to him that this is only temporary and if in the future he is in better physical condition, he could donate blood. in fact 85-90% of those donors rejected for hypotension are readmitted in blood donation after meeting the above mentioned criteria. another matter of equal importance is anemia (25.04% of total causes of rejection), especially concerning young women. since most of those women tend to develop anemia due to depletion of iron stores, they should be advised to donate blood at longer periods than regular, to receive proper medication and diet according to their needs. the doctor must explain the donor the reasons for iron depletion, so blood donation should not be considered as the only cause for this situation from the donor. there are many factors contributing to anemia, menses, specific diets, overwhelming stress and exercise, not to mention other medical reasons. it is the duty of the doctor to correct those factors that resulted in iron depletion or anemia and readmits those donors in blood donation in the future (75-80% of those rejected are readmitted in our centre). summary/conclusions: at our blood centre we have created a program of regular tests (blood tests-physical examination) for all our blood donors. our experienced and well taught personnel offers advice and provides useful information in every aspect of blood donation and more. we have created a friendly environment for all our volunteers with love, understanding and appreciation and believe that this is the only way to keep a constant 'flow' of blood in our region. introduction: an innovative perception for blood donation in a new and evolving environment must focus on specific matters and ideas and adopt in a certain level lifestyles and concerns of society. aims: the purpose of this study is to find methods and ideas that can help blood donation centers throughout our country to create new blood donors, give a motive and inspiration for blood donation by adopting new trends of society and finally accomplish national need. methods: by having a personal interview with many volunteers about their feelings for healthier life, their nutritional habits, daily physical activity, sports, vitamins, smoking, weight, cholesterol levels. we investigated whether they believe that blood donation has, if any role towards a more hygienic life. results: we divided blood donor volunteers according to their age, educational level, and number of blood donations per year. our results indicated that there is a tendency among young educated people to adopt a personal lifestyle that includes consuming healthier food, keeping their weight low close to the ideal, having some kind of personal activity, not smoking, watching cholesterol levels, following doctors advice and concerning seriously about their health. this dynamic group of blood donor volunteers considers blood donation as a contributing factor to well being and donates blood at specific intervals. besides the yearly run lab tests that are done by our blood centre they also seek advice and discuss any matter concerning their health with the blood centre doctor. it appears that they are extremely sensitive in those matters and they seem to appear well informed about issues concerning their health, they also believe that blood donation is part of the plan they have to keep fit and being well. in our blood centre we encourage this belief and we also provide information concerning this new trend towards healthier habits. summary/conclusions: this approach has already shown some positive results in our blood centre as many people especially young educated women have joined our blood donorship program and donate blood at scheduled intervals. in order to achieve our goal which is to raise the percentage of blood donors in the region we have to be flexible, innovative according to new habits and lifestyles. we have to move with society and modernize the way we attract various groups of people. blood donation against prejudice as saltamavros*, s dimitrakopoulos † , v zacharaki*, p giannaros*, s markou* and p tseliou* *st. andrews hospital patras greece, patras, † pyrgos general hospital, pyrgos, greece introduction: in order to achieve a greater population to be admitted in blood donation we have to provide information concerning any obscure issues that presents in selecting donors. to examine the accuracy of hb measurements obtained by the noninvasive clinical device, as compared to values detected by standard methods, (cell-dyn 1600, abbott laboratories, usa), in a blood donor setting. methods: the nbm-100 device utilizes a finger base sensor using occlusion red/near-infrared spectroscopy (o-rnirs) to detect and analyze the hb/hct levels. the clinical trials were conducted at two blood donor centers (israel and usa). studies were carried out on a group of 159 subjects (86 females, 73 males) aged 19-64. subjects were healthy volunteers who had come to donate whole blood or aphaeresis components. after obtaining informed consent, hb/hct levels of all the study volunteer participants was tested non-invasively, using the nbm-100 device, followed by a venous blood sample. additionally, the usa center tested a capillary blood sample using the hematastat hct measurement device (71 donors). hb levels were considered normal when readings were equal to or >12.5 g/dl. results: venous hb measurements ranged from 9.9-16.2 g/dl. the mean nbm-100 hb level was 13.20 ± 1.35 g/dl, only 0.28 g/dl lower than the mean hb result obtained by venous sampling, which reached 13.48 ± 1.22 g/dl. the standard deviation of the difference between the invasive and noninvasive hb readings was found to be ±1.04 g/dl. the mean absolute error (mae) of their difference was 0.81 g/dl. when checked against the cell-dyn in the usa center, where 21 subjects had hb of 12.5 g/dl or lower, the nbm-100 and hematastat devices showed comparable sensitivity results. the nbm-100 using o-rnirs is a promising noninvasive technique for hb screening in blood donors. the device is easy to use and agreeable for both blood donors and personnel. the technique reduces the need for the invasive finger prick or venous blood sampling, thereby enhancing safety, reducing costs, and improving the experience of blood donation. the effect of short-term, temporary deferral on blood donor return rates and subsequent blood donations background: blood donors are deferred for numerous reasons. some deferrals like intravenous drug use, male homosexual contact or certain positive test results are permanent. the majority of donor deferrals, however, are short-term temporary deferrals (sttds) that are resolved in a matter of days, weeks or months, after which time the person is again an eligible blood donor. the effect of sttds on blood donor return rates and subsequent blood donations is studied. materials and methods: donors given sttds during the 15 december 1999 to 15 march 2000 were computer-matched with non deferred donors on the basis of age, sex, and donation date (case group: 804 donors -control group: 295 donors). computer records were evaluated during the next 3 years (21 march 2000 to 21 march 2003 to determine donor return rates. significance for comparison between the two groups was based on chi-square analysis. results: the most common reasons sttds were elevated blood pressure (52%), deferred for medication (29%) and colds and/or sore throats (4.2). non deferred donors were a little more likely than donors with sttds to return over the next 3 years (36.6% vs. 34.8% pv = 0.0001) and non deferred donors donated more whole blood units. . according to ethnic structure, women -ethnic macedonians donate blood in largest numbers -1096 (14%), while all other ethnic groups are present with only 3%. the most prevalent is the group of adults aged 41-50 (358 -27.5%), with high school education -800 (61.4%) and mostly those who donated blood 1-5 times (719 -55%). conclusion: having in mind that 50% of the population in macedonia is female, the obtained results reveal a significant, yet insufficient participation of women in blood donation with 17% in relation to the total number of blood donors surveyed in the period 1999-2003. this is due to insufficient motivation and education of women from all ethnic groups especially those from the younger population and with elementary education. incorporating them in education and organization would contribute to their more extensive participating in blood donation. comparison of serum beta 2-microglobulin (b2-mg) between hbsag positive donor and healthy control f tarabadi*, m shaeigan*, g babaee † , a talabiean* and m khadir* *iranian blood transfusion center, † tarbiat modarrs university, tehran, iran background: beta 2-microglobulin (b2-mg) is a low molecular weight protein (11 800 daltons) and found in all biological fluids it is light chain of histocompatibility class1-human present on the most membranes of cells. in the hepatitis infection the viral antigen presentation on the hepatocyte in the presence of class 1-hla antigen plays a role in the elimination of the virus. method & samples: beta 2-micro globulin was measured in serum drawn from 45 hbs ag positive blood donors include 5 (11.1%) female and 40 (89.9%) male in age between 17-56 years, and 50 healthy 16 (32%) female and 34 (68%) male in the same age we detected serumic b2-mg by enzyme immunoassay (ela). results: our studies showed b2 mg level increased in 7 (15.6%) hbs ag positive donor that was significant differences with healthy control (p = 0.0001). conclusions: it seems that serum b2mg is a good marker for hbs ag replication. the role of b2mg in monitoring of response therapy needs to be more evaluated. and 1635 (0.8%) were contributed by vd, rd and dd respectively. over the last 5 1 / 2 years, voluntary donations have shown a rising trend from 54.3% to 60.9%, where as rd (44.9% to 38.4%) and dd (0.9% to 0.8%) have shown a declining trend. the percentage of female donors was maximum in voluntary group as compared to rd and dd (11.01% vs. 2.02% vs. 6.17%) respectively. the rates of all tti markers reactivity were significantly higher in rd as compared to others donors. the hbsag and anti hcv reactivity in vd and dd is comparable (0.84% vs. 0.85% and 0.45% vs. 0.49%). hiv antibodies was found more frequently in vd as compared to dd [0.15% vs. 0.06% (p < 0.05)] whereas, vdrl reactivity was lower in formal as compared to latter [0.26% vs. 0.37% (p < 0.05)]. conclusion: voluntary blood donation has shown a rising trend over a last few years, thus highlighting efficient donor motivational strategies. these strategies need to be strengthened to increase the female donor base. the safety of dd is equivalent to vd when the rates of tti are compared. thus, dd should be advised to donate blood regularly as voluntary blood donors. blood safety depends on a number of factors. the chain of safe blood starts with the donor. one of the procedures for obtaining safe blood for transfusion is the medical selection based on the completed questionnaire and the possibility of self-exclusion from the process of blood donation, the medical history of the potential donor and the medical examination. donor selection consists of two sets of information necessary for protection of the blood recipient as well as the donor himself. aim: to present the most frequent reasons for declining volunteer blood donors. material: the materials used for analysis were the questionnaires completed by all the potential blood donors at the transfusion department of the medical center in strumica as well as the record books of the blood donors which contain the results of the analysis we make for the potential donors. these donors donated blood in the period between 2001 and 2003. results: during this period 4129 people volunteered to donate blood, out of which 3940 were allowed to donate blood, while 189 were declined. out of the total number of blood donors 3313 were male and 627 female donors. the reasons for declining potential donors were the following: 42.3% had low levels of hb, 22.36% were taking antibiotics, 9.37% were ill, 7.83% had low blood pressure, 6.75% had high blood pressure, 11.39% for other reasons. conclusion: donor selection and their care on one side and obtaining safe blood for transfusion on the other side entails obligatory organized medical control. the obligatory completion of questionnaires, the medical examination of the potential donor and their self-exclusion as a result of the feeling of personal responsibility as well as the obtained information are very important for the selection of quality blood donors and obtaining safe blood for transfusion. questionnaire on 2700 subjects-students, their knowledge and motivation on blood donation f vladareanu, a bugner and s sirian national institute of heamatology transf, bucharest, romania the research theme of this questionnaire is as follows: 'what is the level of knowledge and of motivation in the non-remunerated and voluntary blood donation at students?' we also tried to see the practical implications that this study will have and how it will influence the knowledge in this area. the purpose of this questionnaire was not dissimulated. the general theme of the knowledge and motivation on blood donation had been studied before through two big questionnaires applied in 1987 and 1992, but the general population was their target. students had never been an investigated lot up to now. the hypothesis referring to this problem is as follows: students are not informed either on the act of donation, or on the crisis of blood. 1. the lack of information is a first cause of the indifference of the studied lot towards the idea of donation. 2. the lack of motivation of the studied lot is another cause. the questionnaire was applied on a 2700 lot of students from seven different cities: bucharest, iasi, constanta, cluj, sibiu, brasov, timisoara. the number of the questions was limited to 22, which we consider best for a questionnaire applied on the street or at college. as a conclusion, we can say that a passive-defensive attitude towards the blood donation was revealed after this questionnaire. not knowing the issue caused by their lack of information sometimes determines indifference at the statement of the subject. on a general dissolution environment of the responsibility of the youth, the donation problem is not in their aria of preoccupations, the general attitude being of non-involvement for the moment, at this idea which is not yet in every individual conscience and which is normally administrated at an institutional level. the donor data and the details of blood application of the north west transdanubian region of hungary k vörös*, c bercsényi † , o petró † , r jáger † and e miskovits ‡ *hungarian national blood transfusion s., györ, † blood bank, tatabánya, ‡ headquarters hungarian n.b.s., budapest, hungary the ongoing fundamental reorganization of the blood service began on the 01.07.1998 in hungary. as the consequence of reorganization till 01.07.2000, 28 blood banks had been established instead of 63 existing before, under direction of the hhnbts. the working profile of the 6 regional blood centers and 22 local blood banks will be changed step by step. virus screening, blood group serology and processing will be made in the 6 regional centers. one of the regions is the 'north west transdanubian region' (nwtr, city györ as the center, with about 930 000 inhabitants and 8000 hospital beds). 3 local blood banks (tatabánya, sopron, and szombathely) are belonging to nwtr. the regional center and the local blood banks provide the labile blood products and high level clinical-transfusion service (cross-matching, antibody screening, outpatient immunhematology investigations, etc.) for the hospitals. annually 56 000 donors donate blood in this region. this donation activity covers about the 6% of all inhabitants. the acceptance ratio of the donors is good (6-8% of the donors were deferred). there are 14 hospitals in our region. the regional demand on rbcc is 30-45.000 u/year, on ffp is 5.500-7.000 u/year and on pc is 8-10.000 u/year. the poster shows the donor data and the details of blood application of this region since 1999. p-054 implementation of 2rbc collection using haemonetics mcs ® +: medical staff training, donor recruitment and acceptance g woimant, c fretz, d puydupin, e pélissier and jl beaumont efs ile de france, paris, france background: single donor 2rbc collection is an approved apheresis technique in france. aims: our goal was to evaluate the implementation of 2rbc collection in our center in terms of donor recruitment and acceptance, as well as medical staff training and adaptation. methods: donors were selected according to the french requirements for 2rbc collection (weight ≥ 65 kg, height ≥ 165 cm, hb ≥ 13.5 g/dl, ferritin ≥ 20 ng/ml for repeat 2rbc donors). all personnel were trained on adequate communication with donors. eligible donors were contacted by mail, by phone or during pre-donation interview. among the 102 recruited donors, all donors were male, 63% were regular whole blood donors, 36% were regular whole blood or apheresis donors and 3% were new donors. the medical staff was trained on 2rbc collection with the sdr protocol and disposable set ln 948pf on the mcs ® +. most of the medical staff was already used to autologous rbc donation with similar apheresis devices. blood samples were taken from donors pre-and post-donation, as well as 2 to 4 months later for those returning for a subsequent donation. donors were asked to fill out a post-donation survey for assessing donor comfort and information. results: donor profile and clinical follow-up are summarized in table 1 . six percent of the donors had a ferritin level below 20 ng/ml; these donors were regular whole blood donors. the collections were well tolerated and no changes in vital signs were noted. four reactions were reported: 2 hematomas and 2 citrate reactions. no reaction was observed post-donation and hemoglobin levels measured before next donation were back to normal. the technique was easily implemented by the medical staff and fitted well in the existing blood center processes. the medical staff as well as the donors found collection duration short (average of 35 min). the results of the survey were very favorable as more than 95% of the donors considered their donation and the information they received as satisfying. most of them agreed to donate again and several actually donated twice during the evaluated period. conclusion: the implementation of 2rbc collection in our center, using haemonetics mcs ® +, was successful in terms of ease of use of the technique, as well as user and donor acceptance. we now plan to evaluate donor loyalty in the longer term. risk from first-time blood donors e zhiburt, s golosova and p reizman federal blood center, moscow, russian federation introduction: each third dose of whole blood in russia is donated by first-time blood donor. there are two reasons for attention to this kind of donors: (1) possible risk of infectious disease in seronegative study; (2) possible risk of donation for person with contraindication. aim of the study: we investigated role of regional deferred donors registry (rddr) in by first-time donor selection. methods: moscow rddr includes parts: hiv, viral hepatitis, syphilis, tuberculoses, malaria, drug users, psychiatry, 60 days after blood donation. rddr was complete and our center began actively work with it since last year. each donor has to be registered in rddr and automatically checked for deferral reason. effectiveness of rddr was investigated. results: first-time donors donate less than 10% blood in our center. about a quarter of them are deferred before possible donation. part of donors deferred by rddr has been significantly increase in 2003 (c2 = 19.6; p < 0.001) at the expense of seropositive people. conclusion: rddr is effective for blood donor selection and decreases necessity in laboratory screening. first-time blood donors have to be examined before blood donation. if they have not contraindications, donation can be performed up to 10 days before examination and screening. the double unite platelet production is important especially if the relatives of patient find the donors. we evaluated the effectiveness two apheresis machine for platelet collection. in our blood bank, one fenvall amicus and one cs3000+ apparatus were used for platelet apheresis. 1531 apheresis were performed between 2/12/2002 and 13/2/2005. including criteria of donors are that estimated process time is smaller than 90 minute and estimated postapheresis platelet count is higher than 100 ¥ 10 9 /l. donors firstly was enrolled to amicus. if amicus was busy, then it was enrolled to cs. the properties of our donor populations were given in blood and plasma cell components are obtained either by traditional manual method from whole blood or by apheresis. modern medical treatment is based on transfusion of deficient components such as erythrocytes, leukocytes or plasma proteins. this involves new solutions to achieve higher yields and better quality of such components. the aim of our study was to estimate the efficacy of blood cell separator cobe trima in obtaining platelet concentrates (pcs) as compared to older-generation cobe spectra blood separator. apheresis procedures were performed on both these blood cell separators. the quality of platelet concentrates was tested during 5 day storage period (see table below ). we have tested the effect of apheresis procedure on donors and estimated the operating comfort of both separators. the tolerance of both separators was satisfactory except for more frequent hypocalcemia when trima separator was used. most donors were more satisfied with trima procedure because of single venipuncture although it involved special donor selection (good vein access). in general we may say that trima is undoubtedly a more modern and more friendly separator. however, cobe spectra may continue to be used with success especially when a more versatile cell separator is necessary (leukocyte concentrates, peripheral blood stem cells or therapeutic apheresis). methods and results: tls (587 procedures on 67 patients) were used successfully in patients with acute or chronic leukemia with hyperleukocytosis (white cell count >100 ¥ 10e9/l or blast count >50 ¥ 10e9/l) when high cell count would promote leukostasis with vascular occlusion in the microcirculation. performed tl procedures were rapidly reduced both the white cell count and the whole blood viscosity. average fall in white cell count after treatment was 73.3%. tp-treatments (186 procedures on 32 patients with symptomatic thrombocythemia and/or platelet count higher than 1000 ¥ 10e9/l) were applied in order to prevent the development of 'thrombotic-hemorrhagic syndrome' . the tps performed resulted with rapid platelet counts reduction (84.3% in average) and with clearly noted clinical improvements, subsequently. tes (429 procedures on 196 patients) were performed using manually technique in patients with 'cellular hyperviscosity syndrome' induced by high red blood cell count. it was shown that te procedures resulted to red blood cell number lowering and decreasing of blood hyperviscosity. average fall in hemoglobin and red blood cell concentrations after te treatments was from 20.5% till 35.7%. rbcx treatment (8 procedures on five patients with malaria and two with severe aiha crysis) was performed on an urgent basis, particularly when clinical symptoms indicate life-threatening situations and resulted with rapid and significant reduction of concentration of unwanted pathogen affected rbcs and summary/conclusion: the effects of tcs depended on the nature and stage of the basic hds, of adequate selection of patients and of timely applied apheresis. rapid cytoreduction is obtained justly in patients with excessively high cell count, and this effect did not associated with bone marrow remission. thus, tc should be looked upon as adjunct to the standard treatment of different cithemias, but not as replacement therapy. the present study indicates that the best therapeutic effects were obtained by rbcx. were carried out with continuous flow blood cell separator cobe spectra and all patients underwent large volume leukapheresis (lvl). in all procedures, a blood warmer was connected to the return line and a continuous calcium infusion was administered preventively. six patients, who were under 25 kg body weight, had the extracorporeal circuit primed with irradiated, filtered packed red cells diluted with 5% albumin solution. seven children had vital signs and ecg continuously monitored during the procedure. results: each patient underwent a median of 2 collections (range 1-6). the inlet blood flow ranged between 16.0 and 95.4 ml/min (median 54.5 ml/min). the median blood volume processed was 11 754 ml (range 2700-22 500). leukapheresis lasted a median of 240 min (range 120-270). the median total nucleated cell yield was 11.86 ¥ 10e8/kg (range 1.94-21.21), mononuclear cell (mnc) yield was 6.01 ¥ 10e8/kg (range 0.97-12.73) and cd34+ cell yield was 3.5 ¥ 10e6/kg (range 0.19-28.01). the median of mnc collection efficiencies was 56.11% (range 12.34-158.09). in 9 (39.1%) patients, in only one apheresis procedure more than 2 ¥ 10e6 cd34+ cell/kg were collected. during 6 (9.09%) procedures patients had experienced apheresis-related side effects. the citrate-induced reactions were most commonly observed. the reactions were mild and cessation of collection was required only in one case, because of catheter related complication. mild sedation was required only in few very small children. post-donation platelet count was less than 30 ¥ 10e9/l in 3 cases and these patients required platelet transfusion before subsequent procedure. our results show that lvl in pediatric patients is relatively safe procedure, well tolerated and with a very low risk of serious adverse events. close monitoring of blood counts, especially platelets, between pbsc collections is necessary. the cessation of procedure was required in only one case and no life threatening side effects occurred. neonatal alloimmune thrombocytopenia (natp) caused by fetomaternal mismatch for human platelet (plt) alloantigens (hpas) worsens approximately 1/2000 pregnancies and can lead to a serious bleeding diathesis, intracranial hemorrhage (ich) and sometimes death of the fetus or newborns. we describe the successful management of a 37-year-old pregnant woman, alloimmunized to the hpa-1a (p1a1, zwa) antigen, with a history of two previously children with severe thrombocytopenia and ich. the pregnant woman was at her terminal pregnancy and was suddenly admitted. to evaluate the risk of ich in the fetus, cordocente was performed to demonstrate fetal thrombocytopenia (plt 4.000/mmc). to ensure a rapid provision of compatible negative-antigen platelets, we decide to collect platelets from the mother using apheresis. plateletapheresis was performed using com.tec separator, fresenius. blood processed was 1.100 ml in a short time procedure (35 minutes). no significant adverse effects were observed in the mother and fetus, during and after the procedure. platelets collected (1.5 ¥ 10e11) were transferred to the preparation set and plasma was removed after centrifugation to resuspend the platelets in octaplas ab. then we separated the platelets into two units containing 0.7 ¥ 10e11 each. the day after the donation, the mother gave birth to a girl by caesarean section. after the transfusion, the plt account increased from 4.000/mmc to 35.000/mmc and after a week the child had plt 75.000/mmc without hemorrhagic complication. according to the literature data and our observations of the patients, there are changes of the hemostasis system indexes in the most patients with the endogenous intoxication syndrome and immune disturbances. in the number of cases medicamentous therapy appears to be not enough to normalize the changes, but it is especially important for pregnant women and women in childbirth, because on the background of these disturbances different complications of pregnancy and postnatal period take place. the aim of our study was the substantiation of plasmapheresis using in complex therapy of purulent inflammatory complications in obstetrics and immunoincompatible pregnancy with hemostasiologic disturbances. 10 patients with hemostasis system disturbances: one woman in childbirth with exacerbation of chronic pyelonephritis, who had in the first 24 hours some signs of hypocoagulation on the background of permissible blood loss (prolonged coagulation time up to 20-30 minutes with episodes of its absence on the background of the normal indexes of general coagulogramm, quantity and function of thrombocytes and the dilute fibrin monomer complex level in 7 times higher than the norm) and nine pregnant women with the perinatal losses in anamnesis severed by the pregnancy (threat of abortion, places of fetal egg detachment). these women were examined, the following was revealed: the high antibody titer to chorionic gonadotropin, parameters of partially activated thrombin time were higher than the norm (47-52 seconds), thrombin time (18-20 seconds), the dilute fibrin monomer complex (8-12 mg%), coagulation time (15-20 min). in all these cases the conservative methods of treatment (antibacterial, hemostatic, hormonal therapy) were effective for a short period of time and they didn't succeed to correct the given parameters of hemostasiogramm. the discrete centrifugation plasmapheresis was included in the complex of medical treatment. the woman in birth 5 operations were done, in the programme of plasma replacement during the first two plasmapheresis procedures donor fresh-frozen plasma was included. six pregnant women on the given stage one course consisting of 3 plasmapheresis procedures for plasma replacement with crystalloids was done, the volume of the removed plasma was 25-30% of the circulating plasma volume. three pregnant women before delivery were required two courses of plasmapheresis more consisting of 3-4 procedures each. the system heparin was not used. in all patients already after the first procedure of plasmapheresis the normalization of hemostasis indexes was marked, that allowed to prolong pregnancy, to prevent the coagulopathy bleeding and the development of disseminated intravascular syndrome. four women are discharged from the hospital, the other patients are observed with progressive pregnancy. thus, the using of discrete centrifugation plasmapheresis is effective at the signs of hypocoagulation in patients with isoimmunisation with fetal antigens and infectious pathology, and is the reserve in prevention and treatment of obstetric complications. extracorporeal photochemotherapy: an alternative therapeutic approach to control graft versus host disease after allotransplant with reduced intensity conditioning regimen c del fante*, c perotti*, gl viarengo*, p bergamaschi*, p pedrazzoli † and l salvaneschi* *irccs policlinico s. matteo, pavia, † ospedale niguarda cà granda, milano, italy background: extracorporeal photochemotherapy (ecp) can be defined as an immunomodulatory therapy that demonstrated to be efficacious in treating patients affected with graft versus host disease (gvhd) after allotransplants for oncohematological diseases. reduced intensity conditioning regimen (ricr) for allotransplant is a relatively new practice in patients (pts) ineligible for a conventional myeloablative conditioning regimen. the use of immunosuppressive therapy (ist) to control gvhd is limited for the high risk of developing infections and disease relapse due to the strong reduction of graft versus tumor (gvt) effect. aims: to evaluate the effectiveness and safety of ecp in treating pts affected with gvhd post rcr and the possibility to taper, at the same time, the ist. methods: 6 pts (5 females, 1 male), median age 47.5 years (25-63), affected with agvhd grade ii (1) and extensive cgvhd (5) gtx with median total granulocyte doses of 55 (21-150) ¥ 10 9 per gtx corresponding to 1.46 ¥ 10 9 granulocytes/kg in children and 0.58 ¥ 10 9 granulocytes/kg in adults. the wbc counts increased from baseline values of 0.2 (0-0.5 ¥ 10 12 ) g/l for both pediatric and adult patients to peak values of 3.8 (0.4-18.2) ¥ 10 12 g/l (children) and 0.9 (0.3-9.4) ¥ 10 12 g/l (adults) at one hour after gtx and to 1.4 (1.1-13.6) ¥ 10 12 g/l (children) and 0.4 (0.3-8.6) ¥ 10 12 g/l (adults) at 24 hours after gtx. in 42 out of 54 patients (80%), the crp levels significantly declined (60 (11-91)%; p£0.001) during the granulocyte transfusion period; in almost all cases (39/42; 90%) after the initial or 2nd transfusion. thirty-eight patients (71%) were alive at day +30 after termination of neutropenia and gtx. patients without crp response to gtx (6/9, 66%) and patients with severe viral infections 6/7 (86%) were not among the day +30 survivors. background: in recent years, the use of platelet concentrates obtained from single donors by automated apheresis has grown steadily. plateletpheresis donation is considered to be a safe procedure with modern instruments. so far, no studies have identified donor or procedure specific factors that may be associated with serious adverse events. aim: to evaluate the incidence of adverse events during plateletpheresis procedure, over a five-year period in our hospital. materials and methods: eight hundred single-needle plateletpheresis collections were performed by using two automated intermittent-flow cell separators: 700 of them with mcs3p and 100 with mcsplus (haemonetics), according to automatic standard protocols a3p and ldplp, respectively (with the collection of an additional plasma unit). acd-a was used as the anticoagulant in all apheresis procedures (acd-a: blood ratio was 1 : 11). most of the donors (84% men and 16% women) were patient´s relatives. half-hour before the initiation of the procedure, 250 mg of calcium (1 tablet cal-c-vita) were administered to each donor. the mean platelet yield was 3.2e11 /unit. the overall rate of the donor related adverse events was 3.4%. feeling faint was the most frequent event, which was occurred in 1.62% of donations. hypotension and citrate related rates were 0.77% and 0.73%, respectively. all citrate related symptoms were only transient perioral paresthesias, which were relieved by slowing the i.v. rate, without additional administration of oral calcium. donor unconsciousness was the only observed severe event, the rate of which was 0.25%. other adverse events were venipuncture related (2.37%), machine related (2.7%) and miscellaneous complications (1.25%). (1) plateletpheresis using the mcs3p and the mcsplus automated cell separators is a safe procedure, with a low risk of serious adverse effects. (2) with the used acd-a-to-blood ratio (1 : 11) satisfactory platelet concentrates were obtained with very low incidence of citrate-related events. (3) the peros administration of calcium before the initiation of the procedure, probably lowers the rate and the severity of hypocalcemia symptoms. quality assessment of ffp collected as a byproduct of plateletpheresis 1. from the donors immediately with the initiation of the procedure (citrated whole blood) and 2. from the final platelet concentrates after one hour rest at room temperature without agitation. in vitro platelet response to the aggregation-inducing agonists adp, collagen, ristocetin and arachidonic acid was investigated by means of an aggregometer (pap-4c, bio/data). results: there were no significant differences between the groups of donors with respect to age, sex, smoking habits, preapheresis wbc and plt counts and hemoglobin concentration, as well as in the harvesting time between the two cell separators. our findings are shown in the following table. mildly decreased response to all agonists was observed (mainly to adp and arachidonic acid) in the samples taken right after the initiation of the procedure, in both groups. platelets from the final component showed a further slight decrease in response to adp, which was more prominent in the mcs3p device (p = 0.025). on the contrary, an increase in platelet response to the other three agonists was observed in both devices, which, however, was statistically significant upon collagen and ristocetin stimulation. conclusions: reduced response to aggregation stimuli is possibly caused immediately with the initiation of the apheresis process. literature reports regarding further platelet traumatisation due to the procedure, are rather conflicting. in our study, such traumatisation was observed only in the case of adp in the mcs3p obtained collections and this could be correlated with the technological differences between the two devices. recovery of platelet aggregability, as it was expressed by the upregulation in platelet response in the other stimuli, could be attributed to the resting period and seems not to be affected by the timing of the leucodepletion procedure. background: lupus erythematosus often is accomplished with severe symptoms, such as polyarthritis, nephritis, pericarditis or dermal alterations. in pregnancy cytostatic therapy affects gestation. on the other hand the course of disease can be refractory to corticosteroid therapy. elimination of autoantibody and immuncomplexes by plasmapheresis could be an efficient way to amend the severity of symptoms. a 24 year old pregnant woman in the 24th gestation week with systemic lupus erythematosus showed severe symptoms like polyarthritis, nephritis and pericarditis. treatment was initially 1 mg/kg bw prednisolone for 3 weeks and subsequently 8 mg/kg bw for 2 weeks. plasmapheresis was applied daily in the beginning and continued depending on the condition of the patient ( table 1) . the eliminated plasma was substituted by fresh frozen plasma. the medium volume was 2.951 ml per apheresis. after day 11 plasmapheresis treatment was suspended to avoid problems with coagulation and was followed by a cycle of 3 immunoabsorptions to eliminate circulating immuncomplexes. results: prednisolone therapy alone brought no effect even after changing to high-dose treatment. a significant amelioration of all symptoms could be observed after the first plasmapheresis. good condition of the patient remained stable over the period of daily plasmapheresis for 4 days. intermitting apheresis treatment for one day lead to a significant aggravation of symptoms. apheresis no. 6 again lead to a recovery of the patient which held on until day 11. conclusions: treatment of systemic lupus erythematosus in pregnancy especially in combination with resistance to corticosteroid therapy, is an effective therapy to ease severe symptoms such as polyarthritis, pericarditis and nephritis. exposure to cytostatic drugs can be avoided and therefore the impairment of the fetus can be reduced. background: the collection of mnc represents the first step of photopheresis procedures and could be of critical importance in achieving a therapeutic goal. in this work we compare the cell yield of two collection programs on cobe spectra device: the mnc versus the autopbsc program using the ecp procedure modified by andreu. methods: 39 procedures were carried out with mnc program and 19 procedures with autopbsc on 4 patients with cgvhd. both hemoglobin increased from 35.9 ± 5.7 to 204 ± 100 mg/tu, k+ from 1.7 ± 0.7 to 44.8 ± 13.9 mmol/l, level of glucose decreased from 26.65 ± 2.06 to 9.75 ± 2.06 to 9.75 ± 0.91 mmol/l, ldh from 1.81 ± 0.48 to 6.24 ± 1.89 ukat/, lactate from 2.77 ± 0.47 to 22.72 ± 14.02 mmol/l, ph from 7.50 ± 0.14 to 6.74 0.08. the volume of apheresis units was lower than wb-rbc, the leucocyte count was normal in all units. the rbc loss by filtration was 24.9 ± 3.1 ml/tu and was lower than at wb-rbc. in apheresis rbc there were the differences in hb and ht value between the day of storage 0 and 40, in wb-rbc there were no differences. during the storage period we found no differences in k+ increasing value and no change in ph value between apheresis rbc and wb-rbc, the increasing of lactate was higher in wb -rbc, increasing of ldh correlated to hemolysis. the plasma hb value increase was higher at apheresis rbc in contradistinction to literature. hb and ht correlation in apheresis units according to predonate value in donors was lower than at wb -rbc. the method is a useful alternative to conventional whole blood donation, we get rbc units with high standard of quality and low correlation according to predonate hb and ht value in donors. acknowledgement: the study is supported by grant iga ministry of healthy cr n. nr/8015-3. background: in life-threatening exacerbations of sle a satisfying efficient therapy is lacking. despite intensive immunosuppressive therapy some patients are resistant or contraindicated to conventional treatment. in particular circulating antibodies and immune complexes play an important role in the pathogenesis of sle and mctd. an extracorporeal removal of these pathological substances may be effective in the treatment of active disease. methods: five patients with severe therapy-resistant sle/mctd underwent immunoadsorption onto protein a. blood was drawn from patients by using a jugular catheter or a peripheral intravenous catheter. anticoagulation was performed with acd-a and heparine or acd-a and r-hirudine. plasma was separated by centrifugation. the 1.5 to 2-fold total plasma volume was treated in every immunoadsorption. the columns were floated with a maximal plasma flow of 20 ml/min. the procedure was carried out every second day. additionally supplementary intravenous immunglobulin therapy was given only once. results: remission of the disease was achieved in four patients. see table below . conclusion: pa-ia is highly effective regarding the elimination of autoantibodies and circulating immune complexes, might induce a remission in patients with sle/mctd. it is an acceptable alternative treatment option in patients when other therapies are ineffective or contraindicated. background: purification of bone marrow from erythrocytes is used to prevent early hemolysis in major abo incompatible allogeneic hemopoietic cell transplantations. erythrocyte depletion is strongly recommended to reduce product volume and stem cell purification before storing autologous and even allogeneic bone marrow in order to prevent early hemolysis and dmso toxicity that might develop after thawing. centrifugation, sedimentation with hes, and cell separating devices are methods for erythrocytes depletion. aim: in our center, we prefer to use cell separation device, since it is a reliable method and has a high-yield and risk of contamination with erythrocytes is low. success of the process is retrospectively analyzed for high and low volumes. method: erythrocytes depletion of bone marrow harvest was done in hemapharesis unit with cobe spectra device in the last five years in 20 cases with bone marrow volume over 750 ml, and 17 cases with bone marrow volume under 750 ml. fifteen of these cases were allogeneic, and 22 were autologous procedures; a software uploaded with cobe pbsc coll vers 5.1 and (catalog no: 777-006-000) set was used in the procedure, and at the same time, double bag system with intermediate connectors were used to prevent re-circulation (catalog no: 777-006-300). results: the mean volume reduction was 90.48% (83.81-93.54) for volumes over 750 ml, and 89.79% (84.03-94.02) for volumes less than 750 ml. regarding the success of the procedure no statistically significant difference was found between procedures with high and low volumes. no complication developed related to the device or product, and waste bag never had to be re-used. in none of the patients early massive intravascular hemolysis was observed. conclusion: erythrocyte depletion and volume reducing with cell separation device is a reliable method. this process is successfully applied with high volumes (over 750 ml); and in low volumes as well for reducing erythrocytes, and gain of mononuclear cells and cd34+ cells. platelet concentrates obtained by apheresis procedure-correlation between the initial count and the final concentration v srejic*, g bogdanovic*, z garic*, n vavic* and b balint † *national blood transfusion institute, † military medical academy, belgrade, serbia apheresis team of the national blood transfusion institute processed and classified data of 100 donors who donated platelets by apheresis procedure from january till april 2004. procedures were performed in accordance with the ldplp protocol, using haemonetics mcs+. initial donors' platelet count and the absolute platelet concentration in the final preparation were followed, as well as red blood cell and leukocyte contamination and the volume of the processed blood. donors' initial platelet count was not less than 191 ¥ 10 9 /l and the volume of the processed blood was not less than 2300 ml. according to histogram, the most frequent donors' initial platelet value ranged from 210 ¥ 10 9 /l to 308 ¥ 10 9 /l (70%). final concentration of the samples of 100 tested donors ranged from 3.9 ¥ 10 11 to 6.1 ¥ 10 11 in the average volume of 400 ml. regression analysis demonstrated that there was a correlation between the initial donors' platelet count and the obtained final concentrate. student's t test showed p < 0.0011. leukocyte contamination of the final concentrate prepared without the filter ranged from 0.0 ¥ 10 9 /l to 0.4 ¥ 10 9 /l. presence of red blood cells in the final concentrate ranged from 0.01 ¥ 10 12 /l to 0.02 ¥ 10 12 /l. p-077 therapeutic apheresis (ta) in croatian hospitalsadherence to respectable guidelines z zivkovic*, b jeren strujic*, s boras † , i bojanic † , b golubic cepulic † and z ivankovic † *clinical hospital dubrava, † clinical hospital center zagreb, zagreb, croatia introduction: besides considerable resources, ta requires high costs and risk for patients. therefore, indication for ta often considers interests of patient, hospital and requesting physician. the most respectable guidelines for the implementation of ta were defined by aabb and asfa, classifying total of 58 diseases into 4 categories (ctg), ranging from 'standard therapy' to 'lack of efficacy' . the objective of this study was to determine indications for ta performed in 6 croatian hospitals in the period 2001-2004, respecting aabb/asfa guidelines. results: during the observed period in croatia, ta was performed in 777 patients suffering from 26 various diseases. in 514 (66%) patients ta was performed by membrane filtration, while in 263 (34%) separation by centrifugation was used (table 1) . according to the 4 ctg, s of the aabb/asfa guidelines, ta was performed in 573 (74%) diseases from ctg i, 126 (17%) ctg ii, 54 (6%) ctg iii, and 25 (3%) ctg iv of patients. the most frequent indications included in ctg i were: myasthenia gravis (32%), collection of pbpcs (31%), sy. guillain-barré (17%), and plasmacytoma (6%). in ctg ii frequent indications were: poisonings (35%), systemic lupus erythematodes (30%), and rapidly progressing glomerulonephritis (14%), and in ctgs iii and iv: cytoreduction-polycythaemia (52%), thyroid storm (15%), gvhd (10%), and reumatoid arthritis (8%). (1) the time spent for resolving h 52/40, (2) mtp 0/0, (3) discarded blood units 10/4. iii group: wrong data input 5/4, donor replacement 4/1, marking errors 3/10, error in determining blood group at the first blood taking 28/13, errors in input medical consulting 9/12 and disregard of prohibitions 2/13. the consequences are: (1) the time spent for resolving h 35/40, (2) mtp 2.5/3.5, (3) discarded blood units 9/18. conclusion: the analysis of errors has showed that the number of errors can be decreased by implementation of corrective/preventative action based on continual education of the staff, appropriate sop, effective organization, qmp for equipment. the conclusion of our study is that reducing the rate of work errors will decrease the waste of material and time, also that will decrease the number of discarded blood units. an iso standard for blood transfusion? background: in our search for an independent, objective assessment at western province blood transfusion service, we were unable to find one single model that met the specific requirements of blood transfusion. we therefore resorted to developing our own model but ask the question: why not have one international standard for blood transfusion? aims: our aim was to have a standardised system for the independent, objective assessment of our blood transfusion service with audits carried out by an internationally recognised body. we wanted a formalised, professional system of accreditation with inspection checklists, reports, certificates etc. methods: having moved away from accreditation by the american association of blood banks in mid 1990's, we evaluated various other options such as inspection by our government department of health or the world health organisation but neither organisation had trained inspectors or systems in place. we also investigated iso 9000 certification but, although this was acceptable on the quality management side, it did not cover the technical parameters relating to blood transfusion. results: we therefore developed our own model for accreditation that consists of three parts: • a quality management section incorporating iso 9000 principles; • a technical section incorporating specifications from the south african standards of practice (we also consulted the european, american, canadian and australian guides); • a laboratory section incorporating iso 17025 parameters (soon to be updated with iso 15189). we then chose to be accredited by the south african national accreditation system, sanas, an internationally recognised institution. once we had written a national accreditation checklist, sanas submitted this to two international accreditation bodies (iaf and ilac) for approval. the system has been in place for three years now during which time we have had four successful assessments. summary/conclusions: in developing our system, we reviewed what was being done elsewhere in the world and it became evident that, although there are great similarities between countries, there p-078 software for the management of the scansystem bacterial detection method the scansystem tm was developed for bacterial detection in blood products. to be implemented in blood banks, a specific software is now available in compliance with blood bank regulation in order to manage sample traceability and data file transfer. the software is divided in 3 main levels: an administrator level to create an application configuration in compliance with customer needs (product bar code characteristics, frequency of the positive controls, manual or automatic data file transfer . . .), a technical level to manage the operators (password, id) and to validate some specific results, an operator level for routine testing. the software assess sample traceability when testing pools of samples from 1 to more than 20. indeed, bacterial detection is performed for pools of 1 to 3 platelet samples and 1 to 20 red cell samples. each sample in the pool is traced through its barcode until the final result. the system is compatible with most of the barcode standard including isbt 128. in addition, the system checks each barcode protecting the sample against duplicate testing. the software assists and monitors the bacterial detection process from the sampling to the end of the test (final result), each step of the procedure is identified through its barcode and at each time, it is possible to know the test status for each sample. for a pool of samples, 2 results can be obtained: 'negative' or 'on hold' for a positive result. for a 'negative' pool, each sample constituting the pool are determined as 'negative' . for an 'on hold' pool each sample constituting the pool must be tested as a single sample and the final result is 'negative' or 'positive' . data transfer may be manual or automatic. a final technical validation is necessary before the transfer through an active selection of results to download. final results are provided in a compliant format for an easy import into the blood bank database. all necessary information are displayed: 'machine id' 'product/sample barcode id' 'date' 'time of transfer' 'operator id' 'result' ('negative' or 'positive'). the main advantage of this software is a continuous check of each step reducing the risk of error in testing. it makes the scansystem tm test compatible with a routine use in blood banks according to the current regulations and quality assurance programs. is no overall consistency. we feel it would be of benefit to establish an international working group to investigate the feasibility of writing an iso standard for blood transfusion. the standard would harmonise quality management parameters based on iso principles and technical/laboratory parameters specific to blood transfusion. minimum technical specifications would need to be agreed upon based on the various standards and guidelines available around the world. this iso standard could then be used for the purposes of certification/accreditation or government inspections. this would ensure global standardisation of world-class best practices. first world countries would be able to achieve compliance and a subsequent step could be the establishment of an international forum to assist developing countries to work towards compliance in the longer term. residual leukocytes in leukoreduced cellular blood products -evaluation by flow cytometry web-based outcome review: do you know how productive your trima® can be? background: web-based outcome review is a new software tool developed by gambro bct for the management and the interpretation of data from the trima and trima accel tm automated blood collection systems. aim: does the interpretation of reports obtained through outcome review lead to an increase in the number of products per run and the overall productivity of the apheresis center? method: run data files (rdf) from trimaᮀ were collected and transferred onto the outcome review server. these rdf do not contain any donor related data that can lead to possible donor identification. the reports were generated on the outcome review website (gambro bct intranet), interpreted by a gambro bct employee and presented and discussed with the management of blood centers. results: a total of 21 different reports can be generated on the website as a pdf file. for this study, 9 reports were investigated. they are: doses per collection, doses per collection trend, platelet collection trend, platelet procedure performance, platelet procedure performance trend, product distribution, average procedure time, procedure time, machine productivity trend. the results obtained for a center can be compared to word-wide, national, regional or to other individual trima devices in the centre (benchmarks). this benchmarking allows the management of an apheresis center to compare the results, to draw the right conclusions and to develop and implement corrective actions. implementing successfully these corrective action plans will lead over time to productivity results that are more in line with the figures that are generally accepted to be the optimal production capabilities of trima. the reports also help to monitor the effects of the corrective action plan over time and to adjust this plan if the results are not in line with the expectations. reaching and maintaining the optimal production capabilities of trima will also increase the net revenue by procedure or decrease the cost by procedure for the blood centre. conclusion: web-based outcome review allows getting more products from the existing donor base by interpretation of the multiple reports and implementing the required corrective actions until optimal production capabilities of trima are reached and maintained. introduction: to examine the cell vitality of packed rbc's during storage several parameters like atp, free hb or 2,3-dpg are used. less kits for the determination of atp are available and they need either a large sample volume and/or are time consuming. here we present the modification of a commercial testkit for a time-and cost saving detection of atp. methods: samples were analysed with (a) 3-phosphoglyceratekinase reaction according to bergmeyer, h. methods of enymatic analysis 2nd. edition. academic press, new york 1965; (b) detection via hplc and c) using a commercial testkit (r. greiner bio-chemica, germany). the atp-kit were minimized from 3500 ml to a total volume of 200 ml and tests were performed in microtiterplates. results: 245 samples were analysed in hplc and modificated commercial testkits, another 20 samples have been examined in all tests. comparison of the results showed no discrepancies in the above mentioned methods. standard curves have been performed (range 5-1000 mm atp) and statistical analysis demonstrated a given linearity (r = 9.97). variability has been calculated as 1.2% (intraassay; n = 25) and 4.6% (inter-assay; n = 70). the hand-on-time calculated for 96 samples has been decreased from 4.5 hours to 30 minutes. at least the costs of atp-determination have been reduced from €3.45 to €0.22 per sample. conclusion: performance of test kits in microtiter format is a fast and rapid method, reliable for high-throughput determination of atp in packed rbc's. background: in order to preserve both blood safety and availability it is mandatory that a minimal amount of blood units would be discarded due to defects in the materials and supplies used for blood collection, or to deviations in blood processing or storage. aims: (1) to monitor the derangements of different materials and disposables used during blood collection and processing, and to study the suppliers' responses and corrective actions taken. (2) to asses the relative contribution of different defective materials (dm) to the need to discard valuable blood components. materials and methods: about 831 000 whole blood units were collected and processed by mda national blood services in [2002] [2003] [2004] . as part of the routine quality control activities, derangements of the dm used were recorded and analyzed. some 40 different types of dm were defined. out of 68 reports sent to the corresponding manufacturers for investigation, 45 responses (66%) were received and analyzed. about 70% of dm were detected during blood collection. manufacturer defects of different materials were the reason for components discard in nearly 25% of cases. conclusion: defective materials are one of the major causes of the infringements of blood collection and blood component preparation processes. analysis and monitoring of the different defects and of the suppliers' responses and corrective actions are essential to improve products' safety and availability. establishment of a network for the exchange of information among international blood centers would enable the blood banking community to compare between different suppliers and to use the documented cases for training of personnel of both the blood services and the manufacturers. such a system may contribute to the improvement in quality of materials used and might lower the discard rate of valuable blood units. results: table 1 analysis and characteristics of t (1) comprehension of the blood bank's processes and the interaction between them and between the processes of the whole hospital. (2) monitor, measure and analyze these processes, in order to improve their effectiveness continuously. (3) implementation of internal and external quality controls for blood and blood products and implementation of appropriate statistical techniques for monitoring their results. (4) identification of interested parties (doctors, donors, patients) satisfaction and taking up the necessary preventive or corrective actions to improve their satisfaction. the qms of our blood bank was certified by tuv rheinland in 16/11/2004 and the scope of the certification is: 'blood collection, testing of infections markers, production of blood components, compatibility screening for blood transfusion and other immunological tests and implementation of therapeutic schemes in thalassaemia patients' . the implementation of the qms based on iso 9001:2000 standards ensures the improvement of services provided by the blood bank and the increase in customer satisfaction, whether donors or patients are concerned. the former enjoy the respect and recognition of their social contribution, while the latter are assured of very high levels of service and health protection. finally, we shall not underestimate the positive impact of qms in the motivation of blood bank personnel. quality becomes integrated both in their professional and personal attitude and allows for achieving increased satisfaction from their work. equipment management in the national blood transfusion service in serbia introduction: new equipment was urgently needed in three blood transfusion establishments (bte) in serbia. equipment was mostly inadequate for core blood transfusion activities, placed in inappropriate facilities, very old without routine maintenance or calibration. also, technical documentation for most of the equipment did not exist, and procedures for equipment management and responsibilities were not defined. further more, coordination on equipment issues with the qa department was not recognized. service funded by the european agency for reconstruction, provided various equipment for the three blood transfusion establishments. the new equipment includes blood collection equipment, centrifuges, refrigerators, incubators, automated testing equipment, genetic analyzer and it equipment of 1.6 million euros value. before the new equipment is installed the bte's agreed to have the national procedures on installation, validation, calibration and preventative maintenance in place. this will ensure that the equipment can be properly installed and validated before use. the project has provided training on validation to the working group (wg) on quality. the wg has created national procedures related to the equipment, including quite new term validation. the same problems in implementation of procedures were present in all three bte's. significant efforts are made to explain to the staff how the equipment has an impact on quality, how to ensure that the equipment does what it is supposed to do, how to be confident that the results obtained are accurate and how important it is to generate records. qa managers played an important role in the preparation of facilities for equipment installation, making plans for equipment layouts, creating documents (master cards, instruction for use) and designing the validation protocols. the same procedures and records enable an exchange of results, comparability, sharing information on what works and what does not work between bte's. the qa managers also prepared an introduction of equipment requirements to the heads of departments, with special attention to the validation process so that they are able to fully understand what is required and why to validate their equipment. results: national standards in equipment management in serbia are set and are being implemented. qa managers carefully managed that the new, numerous equipment, delivered in the short period of 2 months was correctly installed, validated, obtained with necessary documentation, followed by previously trained staff, so that equipment can be considered as controlled. the additional, positive effect is that validation is performed in one establishment for all bte in the country, allowing a more prompt response to problems and presenting of joint request to suppliers, as well as an easier way of monitoring equipment performance of the three bte's. organized equipment management has affect on every aspect of blood activities and finally to the quality of blood and blood components. background: the vista information system (vista tm ) is used in centers in europe as an apheresis management system. with vista tm it is possible to increase productivity, donor comfort and loyalty and therefore simultaneously improving the overall process in the center. aim: a calculation tool (microsoft excel) was developed to evaluate the added value of vista tm . three blood centers in different european countries completed a questionnaire using their local data. summarizing these figures gives us an idea about the impact of vista tm on the daily work and budget of a blood centre. method: the excel calculation tool that was used investigated 3 major areas where vista tm could show added value: improvement of regulatory compliance, increase the efficiency in operations and improve productivity. results: regulatory compliance: the number of infringements decreased, causing a considerable direct financial gain because these events are very expensive to deal with. the time spent on regulatory reviews decreased with a mean value of 35%. operational efficiency: the number of reports is very site dependent: sometimes a report is made for every procedure together with the printout of a blood loss history form. because vista tm tracks all procedure related data, some sites decided to stop printing these types of reports and to go completely paperless. the percent time reduction in reporting is therefore very variable. however the % of errors related to these reports decreased considerably with a mean value of 30%. efficiency in operations was also obtained because of the number of reports that are available in vista tm . productivity, management and process reports allow verifying and correcting the daily operations of the blood center. increased productivity: depending on the center, also an increase in the number of platelet and plasma products collected was detected. the number of product discards caused by infiltration reduced with a mean value of 50%, mainly due to the possibility to have customized and more donor adapted trima settings. the percentage of whole blood donors targeted for conversion was very site dependent (min 0.5%-max 4%). but because with vista any procedure brings between 2.0 and 3.0 products in general, the financial gain was considerable when donors could be converted from whole blood to apheresis. the use of vista allows the apheresis center to work with a reduced error rate and to increase the operational efficiency and the productivity. the financial impact of this has been estimated by the centers between 4€ and 26€ (mean value 10€) per procedure. establishment of national quality system in blood transfusion service in serbia introduction: the production of blood products is a semiautomated process in which the manual steps may be difficult to control and standardize. aim of the study: we introduced a specialised team for the blood production to test if this improved the control of the quality of the blood products. methods: the blood products tested for statistical process control were red cells in additive solution, buffy coat removed, and leukodepleted (ld) platelet pools prepared from 4 buffy coats. the products were collected in t&b triple opti-pac from baxter and the platelet pools were ld using plx-5 filters from asahi and stored in platelet bags from baxter. using control charts, namely x-mrchart, exponentially weighted moving average ewma chart and for autocorrelated stationary data the ewmast chart, we examined if time series of quality control values were in statistical control. if not we examined if autocorrelation and/or differences between the technologists producing the blood products could explain the lack of control. data included approximately biweekly measurements of volume, haemoglobin (hb) concentration, hb/unit, haematocrit and log leukocyte count (wbc)/unit of 352 units of red cells, measurements of volume, platelet concentration and platelet count/pool of 79 ld platelet pools produced by a team of 13 technologists and of 79 ld platelet pools produced by a specially trained team of four technologists. results: log wbc/unit was out of statistical control due to systematic differences between technologists. apparent lack of control of volume, hb-concentration, hb/unit caused by autocorrelation disappeared when the ewmast chart was used. platelet concentration and volume of the platelet pools produced by the 13 technologists were out of control. in that some technologists systematically produced low values. this could be explained by inappropriate handling of the platelet product between centrifugation and separation. systematic differences between the four specially trained technologists could not be demonstrated and they produced platelet pools with a significantly higher platelet count/pool. however, standard deviations of the four technologists differed significantly causing occasional outlying values. conclusion: training and routine in blood production or process automation, and also importantly, feed back to the technologists based on control chart quality control data, is recommended. background: one important principle of the use of blood and blood products is the ability to trace the units from donor to the recipient. this study set out to establish whether or not there was sufficient reporting on transfusions from the hospitals supplied by fort portal regional blood bank in western uganda as a means of establishing sound haemovigilance and look back systems. were reported with no unit number and could not be traced to the patients. there was sufficient reporting on the data requested by the blood bank. these results suggest that it is possible to establish effective 'look back' and haemovigilance systems. capture of data on outcomes and adverse effects will be necessary to fully establish the system. further efforts are required to educate those involved in transfusing blood about the need for adequate and accurate documentation. external quality assessment of blood grouping were misinterpreted as rhd-positive samples without the use of control reagent. rbc phenotyping was made correctly by 79.7% of participants. the remaining 20.3% of participants carried out the phenotyping incorrectly, while false-positive and false-negative results were derived in 8.91% and 11.39% of cases correspondingly. polyspecific human sera and monoclonal antibodies were used for abo, rh and antigens typing. the reason of errors in antigen detection was low quality of reagents. antibodies identification was carried out in six distributed exercises. 75% of participants detected anti-d-k-c alloantibody correctly. the rest of participants did not found alloantibodies or detected their specificity incorrectly. results of testing depended on quality of screening cells. thus, the participants using homemade pooled screening cells had a significant lower detection rate of antibodies comparing with those using diamed ag cell panel. consequently, the results of the first federal external quality assessment scheme show the necessity of improving the quality of red cell reagents produced in russia. in addition, the more appropriate training of staff is required. the importance of iso 15189-quality system in increasing safety of blood transfusion introduction: hadassah hospital transfusion medicine department received on 3/2004 iso 15189-quality system accreditation. an essential element of this standard is the development of a reliable system to identify, document, analyze and correct actual and near miss events and assess the effectiveness of corrective and preventive actions. aim of the study: assessment of events and corrective actions following implementation of iso 15189 quality system. methods: events in the blood bank were identified by the staff, by internal or external audits and by complaints from the wards. all events were recorded and classified into two categories; quality system and technical. the latter were further classified into preanalytical (sample receipt), analytical (abo rh typing, antibody screen and identification, cross matching and phenotyping) and post-analytical (issue of components to wards). all events were graded into 4 levels; 1-most severe, potentially harmful to patient. 2-severe, damage to process and result. 3-moderatly severe, damage to process only. 4-benign, no harm. events were corrected and effectiveness of corrective actions was assessed by monitoring recurrence of the event. results: during the years 2003-2004, 248 events were detected and recorded in the blood bank, they comprised 0.072% of all tests performed (343 595). most of the events were technical. all events were detected before causing harm to the patients. results are summarized in the table. *the percent analytical value is a summary of rates of events per test types included in this category. events detected in the quality system were mainly of severity level 2&4, whereas technical events were mainly of severity levels 2&1. analysis of event recurrence in the quality system revealed that 97% of events were resolved, whereas only 14%-31% of technical problems were completely solved. the main source of event identification and documentation, in the quality system were audits whereas in the technical system, staff members revealed most events. the implementation of iso 15189 quality system provided a powerful means for recognition, analysis and study of patterns of near misses and actual events. understanding the root causes of events enables to choose the most effective corrective and preventive action to control event recurrence. evaluation of the frequency of events confined to the blood bank revealed a very low rate of 0.072%. these results are in agreement with data in the literature. creating a non-punitive, non-stressing open environment, motivates personnel to identify and document events, which are regarded as opportunities for improvement and serve as important tools for upgrading transfusion safety. the explosive use of information technology and the speed with which it has spread into all life activities has created vulnerabilities for all organizations. those vulnerabilities are compounded by the complexity of information technology, limited time to market, development constraints, and constantly changing relationships between organizations and suppliers. growth in the sophistication of security threats makes it imperative that organizations remain equally competent in identifying vulnerabilities and mitigating security risks. aim: the goal of this document is to explain how isbt intends to provide guidance to the blood banking community on implementation of effective information security policy. when using information for critical activities, blood banks should consider information security as an important aspect of their management policies. evaluation of existing standards, such as iso 17799 and hipaa, allows us to establish a framework for information security without regard to the type of organization. it remains very difficult however, due to the complexities involved, to establish an information security policy without guidance. method: the isbt information task force was created to provide guidelines on information security for blood banking organizations of all sizes. the intent is to help them understand existing standards as well as provide tools for implementing information security policy. these guidelines are based on existing standards that are followed by most worldwide countries: iso 17999 and hipaa. results: information security can be defined as the 'protection of systems, information and services from accidental and deliberate threats to confidentiality, integrity and availability' . understanding existing information security standards was the first step for establishing a structure for the guidelines. the core is organized within an implementation framework and presented under the three following layers: 1. administrative for defining the it security organization, the information security policy, and information security awareness and training. 2. physical for providing solutions that relate to physical environment protection and access, equipment and it infrastructure security, and control for accessing computerized equipment. 3. technical for maintaining confidentiality of electronic information and ensuring that authorized access to information systems is maintained (technology relating to identification and authentication, logical access, operating system, network management, application access, etc.). strategy guidance is also included for senior managers in charge of establishing organization policy, including responsibilities and methods to successfully implement policy. further, the task force is addressing both risk analysis and management including identification of potential dangers to information systems (threat-source) and existing controls (risk description), as well as a plan to address identified vulnerabilities and mitigation of specific risks. conclusions: information security standards are prerequisite to understanding the issues involved when considering information vulnerability. international guidelines for information security, specifically directed to the blood banking community, are equally necessary if we are to identify, plan for, and mitigate risks associated with vulnerabilities to critical blood banking information. the isbt task force is committed to providing such guidelines. introduction: the important part of quality planning and quality assurance within production of blood components is measurement system analysis (msa). measurement system analysis was performed on microscopic counting of blood cells in our study. aim of the study: aim of this study is determination if microscopy counting of residual elements in whole blood plasma is suitable for quality control of blood components. methods: we practiced measurement system analysis in counting of residual elements (leukocytes and erythrocytes) in whole blood plasma. the counting performed two lab technicians in ten samples of plasma from whole blood. leukocytes and erythrocytes were measured in naggeotte counting chamber. we used the method of mean and range for the determination of reproducibility and repeatability (r&r) and analysis of variance for complex measurement system analysis. regulation diagrams were applied for the graphic statement. we determined the value of repeatability, reproducibility, coefficient r&r, variability among samples of plasma and total variability of measurement system. the important conclusion was to determinate if the microscopic counting of samples of blood components is sufficient with regard to quality parameters specified in guide to the preparation of blood components (erythrocytes: <6.0 ¥ 10 9 /l, leukocytes: <0.1 ¥ 10 9 /l). our results show that microscopy counting of residual elements in whole blood plasma is suitable for quality control of blood components. aim of the study: to provide transfusion services with a tool for proper qms implementation, an international collaborative study on qms applications, employing the 'process approach', has been undertaken by a group of transfusion services of varying sizes and structures with experience of qms, in collaboration with a university institute offering a master in qms implementation in health services, and an expert from a quality association. the 'process approach' serves as a tool to manage transfusion activities as a system based upon a network of processes and their interactions. guide-lines have been produced based upon this principle and will be published (volume and cd-rom) and distributed at no cost to all transfusion services of nations participating in the study. the main contents of the guide-lines' chapters are: through definition/analysis of single processes and the correlation network amongst these, the 'process approach' methodology renders the transfusion centre's functioning units completely interdependent, eliminates process interface barriers, provides personnel with a unified focus on the main transfusion objectives, and lays the basis for improvement of transfusion service quality, organization and performance through efficient control of processes' interactions. the blood screening system 400 automated high-throughput nat system for simultaneous screening of hcv, hbv and hiv nucleic acids: full process surveillance e pfeifer*, b alessandri † , hr bachmann † , t barker † , y ohhashi*, c parkhouse*, j pinsl-ober ‡ , p wenzig ‡ and g ziegler ‡ *roche molecular systems, inc., pleasanton, usa, † roche instrument center ag, rotkreuz, switzerland, ‡ roche diagnostics gmbh, penzberg, germany introduction: the blood screening system 400 combines on one deck both automation of dna/rna extraction from blood/plasma samples and multiplex pcr amplification and detection of nucleic acid targets. the system is designed for high-throughput single unit testing and pooled specimen processing. a data management system supervises and controls the complete process from initial sample pipetting through to result compilation and reporting. the objective of this project was to present the system 400 assay and device built-in quality control measures that guarantee process safety and reliability. the study shows how internal control, external batch controls, pipetting sensors, validation & maintenance procedures, and a controlled development & manufacturing process yield an optimal test method and system stability. method: failure modes and effects criticality analysis (fmeca) was used to evaluate a mathematically derived safety metric for optimizing risk reduction. this method makes use of a system risk objective-function (srof), which provides a multivariate description of sample processing, amplification and detection steps. the method for analyzing system behaviour first employs product classification into risk domains, followed by ranking of process steps that are determined to be linked to known system hazards. this provides objective means for directing system design leading to risk minimization. the srof responses associated with redundant liquid sensing channels were shown to substantially reduce risk during sample or reagent transfer steps. the system 400 liquid flow, airpressure-based (plld) and capacity-coupled liquid (clld) sensors detect aspiration and dispensing inaccuracies. sensor signal tolerance band widths studied for fluid classes representative of system 400 reagents and for plasma samples from lipemic, icteric, and hemolized sample sources were shown to correlate with srof multivariate modelling. the impact of surveillance design elements on the srof response demonstrates that risk is functionally dependent on design elements. additional surveillance examples are presented that describes temperature sensing, robotic positioning and motion control. treatment of residual risk is addressed by introducing external controls that are configurable to specific workflow scenarios. the negative external control (nc) is used to check for contamination of reagents. five low concentration armored external controls, i.e. hiv-1 group m arna, hiv-1 group o arna, hiv-2 arna, hcv arna, and protected hbv dna are run at the beginning of a batch as a run-control measure. in addition to these roche controls the system 400 supports running of user-defined external controls for co-validating the batch. the internal control (ic) is based on armored hiv-1 group m rna that is co-extracted and co-amplified with the external controls and the target nucleic acids potentially present in the sample. lastly, the system 400 resource management monitors the status of samples, ready-to-use reagents and disposables via rack sensors and bar-coded bottles and tubes. the fmeca methodology provides a risk-minimized, comprehensive system design. redundant sensors with internal and external controls are evaluated through comparison of modelled versus actual run results. the system 400 brings a new level of surveillance and throughput for automated pcr testing, and emphasizes roche's commitment to increasing the safety of the global blood supply. technical standards for safe storage and transport of blood components and blood samples objective: to implement standardization of technical specifications for safe storage, transport and distribution of blood samples and blood components intended for transfusion, as part of a quality system in blood transfusion medicine. methods: in the course of implementing a quality system in our blood establishment (distributing 10% of the national blood supply), we have developed standard operating procedures (sops) for temperature and hygienic conditions to maintain and control storage of blood components during their shelf life and to ensure their safe distribution to other blood services in the country and abroad, in compliance with eu directives 2002/98/ec and 2004/33/ec and the recommendations of the council of europe and who. procedures are validated and relevant records are kept. a statistical process control is also in place to monitor deviations from specified temperature and time range throughout the period of transportation. blood components collected and prepared for specific purposes (e.g. directed donations, irradiated units, hla-typed units, anti-cmv negative blood components and blood for neonates) are stored separately, and alarms and warning systems are in place. packing and transport conditions of red cells, platelets and ffp are submitted to the tests and criteria of adr (european agreement concerning the international carriage of dangerous goods by road). in greece, the adr legal framework has applied since 1999. blood samples are classified according to adr in division 6.2 under un 3373 as diagnostic specimens, and the criteria for safe carriage include packaging, specific labelling and vehicle requirements as well as carrier obligations and personnel training. our blood establishment has a contract with biotrans, a private company accredited for packaging, storing and transporting blood, organs, tissues and cells as well as potentially infectious biologic substances and blood samples for diagnostic purposes. assurance system). the approach is specified in the form of requirements of art. 4.1 of the standard: the organization shall: (a) identify the process needed for the quality management system and their application throughout the organization, (b) determine the sequence and interactions of these processes, (c) determine criteria and methods needed to ensure that both the operation and control of these processes are effective. regional centre for transfusion medicine in biaĺystok was the first transfusion service in poland which was certified according to iso 9001:2000 standard. our expected profits from the implementation qms were: possibility to overview organization's pathways of operation and to inspire corrective and improvement actions, emphasis on the role of staff in the system, focus on self-control and responsibility for one's own work as a factor of staff's mentality creation/modification. our one year of experience with iso 9001:2000 standard proved the system to be handy tool of management for the organization collecting, processing, testing blood and releasing of blood products for the hospitals and to be well accepted by the staff. the implementation and certification of the internationally nor-malized quality management system simplify and shorten all accreditation and registration procedures required for legal activities of transfusion service as well as for any supplemental medical activities which may be performed by the centre. the implementation of qms facilitates the implementation of other quality systems and simplifies procedures required for the ce certification for the products. red blood cells stored in blood banks, normally undergo a series of chemical alterations, or storage lesions. the ultimate consequence of these lesions is a decrease in the viability of the red cells following transfusion. the chemical alterations are mainly changes in levels of na+, k+, cl-concentrations, ph and 2,3 dpg levels and they affect the electrical impedance of blood. the electrical impedance, cole-cole parameters, is determined mainly by the resistance of the red cell extracellular fluid (re), the resistance of the intracellular fluid (ri) and the capacitance of the cell membranes (cm). in this study we aimed to investigate the relation between blood parameters and electrical impedance changes, and their further clinical implication. all parameters were measured on 51 erythrocyte suspension (es) samples during 42 days of storage at 4oc on days 0, 10, 21, 35 and 42. for 31 whole blood (wb) samples during 21 days of storage, same parameters were measured on days 0, 10, and 21. the measurement of the complex impedance of blood samples were performed in the frequency range from 100 khz to 1 mhz. by using the 4284a hp lcr meter, the impedance z, and the phase angle a for each sample were read. these values were corrected according to the gain and phase characteristics of the amplifier; and the resistance r and the reactance x were calculated. each data was first fit to the cole-cole model; hence the cole-cole parameters, intracellular resistance ri, extracellular resistance re, characteristic frequency fc and phase angle a were obtained by using lms software. afterwards, cm was calculated by using ri, re, a and fc. whereas ri and cm decreased progressively with time on both wb and es, re changes showed some differences. the electrical impedance alterations were explained by measurements of na+, k+, clconcentrations, ph and 2,3 dpg by indicating days. storage of red cells resulted in a rise in extracellular k+ and a fall in extracellular na+, cl-, ph and 2,3 dpg. anova was used to evaluate differences in blood measures in relation to storage time. the results were presented as the mean ± sd. according to the regression analysis in spss, the intracellular resistance (ri) on both es and wb was affected more efficiently from all blood parameters among all other electrical parameters. although ri and re were correlated with na+, k+, cl-, ph and 2,3 dpg more significantly, cm measurements were failed to show correlations with blood parameters because of the intervening parameters, a and fc. the best relationship between the parameters mentioned above on both es and wb was ri and k+. ph changes were the same for ri and re both for es and wb. the correlation between parameters on es was better than those on wb, because whole blood consists of several particles that may affect our measurements. our study showed that 2,3 dpg has an effect on ri and re as efficiently as other blood parameters and therefore electrical impedance measurements may serve future implications. background: issue of quality blood products and donor safety are the main aims of blood transfusion services. a comprehensive quality system should be in place to fulfill these aims, which can be attained through strict adherence to the established standard operating procedures (sops). the drugs and cosmetics act of india, which controls the licensing of blood transfusion services, does not provide clear guidelines regarding plateletpheresis procedure. aim: we therefore established our own sop and operational flow chart for plateletpheresis that can be easily followed by other centers in india. methods: a total of 100 plateletpheresis procedures performed using two cell separators (cs3000 baxter, usa, mcs3p, hemonetics, usa) were evaluated following our established sop. the mean platelet yield in cs3000 was 2.9 ± 0.84 ¥ 10 11 and in mcs3p, it was 2.88 ± 0.75 ¥ 10 11 per unit, however, only 4-7% of sdps showed wbc levels <5 ¥ 10 6 . six of 100 donors complained of hypocalcemic symptoms. the operational flow chart designed in this study was found to be simple and easy to adapt by blood transfusion services in this country. the first advanced quality management training course for blood transfusion services in the western pacific region mk tan*, jp yu † and d teo* *health sciences authority, singapore, † who regional office for wpr, manila, singapore background: blood transfusion is a key part of modern medicine. a well-organised blood transfusion service (bts) is a prerequisite for the safe and effective use of blood and blood products. in 2000, the world health organization (who) introduced a new initiative of quality management project (qmp) to achieve the goal of safe and adequate global supply of blood. through qmp, regional training centers were identified and quality management training (qmt) courses were established. in 2001, centre for transfusion medicine (ctm) of health sciences authority singapore was appointed as a collaborating center for qmt in the western pacific region (wpr background: spectrophotometric method according to harboe was traditionally used at our institute for determination of free haemoglobin in supernatants of rbc blood components at the end of storage time. in the year 2002 hemocue plasma low hb system (hemocue, sweden) was introduced as a replacement method. aim: the aim of the study was to examine reliability and suitability of the hemocue method for measurement of free haemoglobin in supernatants of rccs. methods: hemocue plasma low hb method was validated and compared with harboe method. supernatants of 36 rbc products were tested by two methods and results compared using regression analysis. additional testing was performed to investigate precision of hemocue method (within-run and between-day variation), trueness using reference material and to define optimal sample handling. results: regression analysis showed high correlation between two methods (r = 0.98), with higher values obtained with hemocue method. immediately after centrifugation one supernatant was measured 6 times in order to determine within-run imprecision. coefficient of variation (cv) calculated from 6 consecutive measurements was 6.1%. to investigate between-day imprecision of the hemocue method one sample was divided in 5 aliquots and frozen. one sample was thawed each day and measured. cv of five measurements was 1.95%. during the period of validation 37 measurements of reference material (low, medium and high) were performed. cv calculated for these measurements were 7.86% (low), 1.88% (medium) and 1.04% (high). in order to investigate possibility of batch testing, supernatants of 5 different rbc products were divided in aliquots and measured periodically during 1-month period. cvs calculated from 7 measurements of each sample were in range 3.1-9.9%. conclusion: hemocue plasma low hb method appears to be an excellent replacement for the harboe method. it is consistent, easy to use, measurements are performed in short time, and errors are minimized by eliminating dilutions and manual calculations. background: determination of haemolysis in red cell concentrates (rccs) at the end of storage time is routine method in quality control of blood components at our institute. for this purpose, haemoglobin concentration in supernatant of rccs is measured using hemocue plasma/low hb system (hemocue, sweden). according to manufacturer recommendation, visually turbid samples should be filtered before analysis with a 0.2 mm filter. because visual estimation of turbidity is highly subjective and unreliable, filtration of all supernatants before analysis using appropriate filters is possible solution for standardisation of the method. aim: the aim of the study was to investigate the effect of filtration of visually non-turbid samples on results of free haemoglobin measurement. methods: haemoglobin concentrations were measured in 42 visually non-turbid supernatants before and after filtration using hemocue plasma/low hb photometer (7 wb negative controls were used for each blood component. in all blood components tested bacterial contamination was detected using both types of bottles. in comparison with sa/sn bottles, nearly all enrolled microorganisms were detected faster in bpa/bpn bottles (see table 1 ). all negative controls were negative (no false positive). the results of the study performed support the use of bact/alert bpa and bpn plastic bottles in quality control testing of different blood components. objective: management of returned blood products is important part of quality assurance activities in transfusion medicine. blood products are returned most frequently because of routine rotation of stock and because of nonconformities discovered after the product has been received. methods: qa data about returned blood products were retrospectively analysed for the 5-year period (2000-2004) . only blood products returned because of nonconformities were taken into consideration. data about the reasons for returns are presented and discussed. the top 4 reasons for returns were positive dat, labelling errors, blood bag defects and visual appearance of blood units (see table 1 ). in all cases positive dat was confirmed in our institute, and blood donors managed accordingly. nonconformities related to visual appearance of blood component and other nonconformities related to the quality of blood products were investigated and corrective actions conducted. in case of blood components returned because of damaged bag, significant problem is to investigate the cause of nonconformity (usually inappropriate transport conditions). conclusion: management of returned blood products is important tool in improving the quality of blood products. introduction: hemovigilance is the systematic monitoring of the blood transfusion chain for side effects and adverse incidents from the moment of blood collection until after administration of the unit to the recipient, and comprises all activities that can lead to a safer and more effective use of blood components. attempts to achieve a more safe and effective use of blood components constitute a huge task given the number of interventions and array of medical and not-medical personnel involved in the transfusion process. registration of the impact of all participants is a tremendous job as such. information management may enhance the chances for a successful hemovigilance. aim: the aim is, to establish a basis for all the information related to the chain, such as standard operating procedures, (inter)national guidelines, local transfusion protocols and forms, and procedures to direct the process. additional factors that contribute to the final results are the profile and role of employees, incidents, points of care, product information, prices, budget, contracts, etc. by clarifying and explaining the basis to all participants by means of an existing infra structure, everyone will gain access to identical, consistent and up-to-date information at all times. the primary activity is to create the basis by an outline of every individual link in the transfusion chain from donor to recipient. secondly, the resources, responsibilities, actions, and internal controls (what task, who is performing, why, which help, where, when) are documented. by creating distinct databases for these 6 'w's' in combination with a separate database for the hemovigilance process itself, it is possible to establish relations, hyperlinks, between the different databases. all the information collected in the foundation, complete with all the resources, responsibilities, actions, and internal controls is made available to target groups by means of the intranet in our hospital as well as in a printed handbook format. collecting and displaying the information on hemovigilance this way, creates a transparent and more easy-to-manage process. it establishes a basis, which incorporates all resources, responsibilities, actions, and internal controls to all participants and enables the organisation to operate both efficiently and effectively. it allows us to show auditors (both internal and external) which processes are related to which guidelines as well as the results in daily. in addition, it reveals which factors determine the genuine application of the guidelines in clinical practice. conclusion: by outlining the process, followed by defining, analysing, securing, on the 'w6' method, the hemovigilance process offers a stable basis and framework for all participants and may stimulate a more safe and effective use of blood components. background: transfusion medicine is a basic post-graduate specialty for medical doctors with a specific national curriculum in bulgaria. in order to improve the post-graduate training of medical doctors in transfusion medicine, a new curriculum was elaborated in 2004. purpose of the new program: independent work of transfusion medicine specialists on all levels of the blood transfusion service (bts) -organization of bts, promotion of voluntary, nonremunerated blood donation, collection, processing, testing, storage and transport of blood and blood components, immunohematologic testing of patients, laboratory hematology, clinical experience, assistance and advice on diagnostic and therapeutic problems of patients, requiring treatment with blood products, teaching transfusion medicine to bts and clinical personnel. admittance and duration to training -medical doctors are admitted to post graduate specialization after a successful state examination. the overall duration of training is 4 years, of which at least 2 are obligatory for training in the national or regional blood transfusion centers. main sections of the curriculum: 1. organization of blood donation and blood transfusion, including promotion of voluntary, nonremunerated blood donation; organization, planning and information systems; organization of blood transfusion; quality management. 2. collection, processing, storage and transport of blood and blood components 3. laboratory hematology and specialized methods (general laboratory methods, immunology, immunohematology, transmissible infections, hemostaseology, nat techniques, flowcytometry) 4. clinical use of blood components and plasma products (treatment with blood products, adverse events and reactions, alternatives to blood transfusion). the new curriculum of transfusion medicine guarantees a better training of medical doctors, thus leading to safe and effective blood products their proper clinical use. introduction: transfusion medicine is integrated medical discipline based on clinical and laboratory practice. it is closely connected with molecular biology, genetics, as well as with apheresis, automatically techniques and transplantation. the aim of studies in transfusion medicine is proper education, skills, attitudes and knowledge of students at the medical faculty and of doctors on residency in transfusion medicine and other specialties about blood and its usage. material and methods: there will be presented how transfusion medicine is implemented in educational and health sector in r. results: there is 3-year specialization in transfusion medicine, established 30 years ago (over 90 specialists finished this specialization till now). we have postgraduate studies in tm started 15 years ago. transfusiology, as subject at the medical faculty, is now included in new curricula of medical student, consists of 15 theoretical and 15 practical lectures. also, medical doctors on specialization in surgery, anesthesiology, obstetrics and gynecology, pediatrics, internal medicine, maxillo-facial surgery and others have obligatory 1-month education in tm. transfusion medicine is integral part in education of nurses and laboratory technicians; all personnel that work in transfusion field in our country finished 6-mounths course in tm and get certificate. conclusion: although we have already done so much in educational field, we will continue with our efforts to improve our collaboration with clinicians and to involve ourselves more in clinical disciplines. introduction: in hospitals of saint petersburg donor blood, its components and preparations, autoblood and its components, hemocorrectors are applied with the medical purpose at 20-30% of hospital patients. the service of blood and the existing organization transfusion therapy in hospitals should provide maximal immunological and infectious safety and also its high medical efficiency. it demands special preparation for all doctors: general physicians on clinical transfusiology and doctors of blood service on all sections of the general, industrial and clinical transfusiology. clinical gemostasiology, pharmacology of hemotransfusion means and hemocorrectors, the organization of the blood service, the donor service; the organization, technique and technics of preparation of blood, its components and preparations, quality assurance, transfusion therapy programs, technique and technics of transfusion medicine, a problem of maintenance of immunological and infectious safety, preventive maintenance, diagnostics and treatment of posttransfusion complications, feature transfusion therapy in pediatrics and medicine of accidents). clinical physicians master all basic questions of clinical transfusiology, thus the special attention is given questions of clinical immunohematology and clinical gemostasiology, programs and a technique of transfusion therapy, the prevention of posttransfusion complications. employment are carried out in departments of city blood bank, hospitals blood departments. final examination under the test program and at interview shows sufficient mastering a theoretical and practical material (right answers of 80-98%). the described system of postgraduate studies provides an opportunity of successful independent work of doctors on the workplaces both regular increase and qualifications not less often than 1 time in 3-5 years. conclusion: the existing system of postgraduate education for doctors on transfusiology provides the blood services and hospital by the qualified staff. introduction: nowadays in hospitals of saint-petersburg more and more it is frequently applied the extracorporal hemacorrection (haemapheresis, haemasorbtion, plasmasorbtion, krhyoplasmasorbtion, ultrafiltration etc.) and photohemotherapy (optical radiation influence on blood) at various diseases and traumas. they are used at treatment almost 6% from the general number of patients of hospitals with positive results at 85% from them. for this purpose in hospitals there are specialized branches or cabinets with staff of the doctors -transfusiologists. therefore an actual problem is organization of postgraduate special education for them. because there is no specific blood banking and transfusion medicine training in either under or post graduate medical education those general practitioners have limited knowledge about this field. aim: to give basic knowledge and practice to those general practitioners moh has established a special training programme with the collaboration of medical schools for 5 years. methods: this is a 6 months programme which has 2 basic components; theoretical education, laboratory and blood banking practice. the trainees are evaluated by a final examination. each group has limited cadets and the training is provided in selected blood banks with the collaboration of universities and moh training hospitals. conclusion: almost 100 general practitioners are trained by this programme in the last 5 years and those doctors helped to increase the quality of blood banking and transfusion service at their hospitals. background: even the history of public blood banking in turkey has dates back to 1957, whole blood was comprising the majority of transfused units (more then 96%) in turkey until the last few years. aim: the main target was to decrease the whole blood use in a short time and establish countrywide effective blood transfusion practice. methods: there were no specific blood banking and transfusion education either at undergraduate and postgraduate medical education in turkey until the last few years. blood banks and transfusion society of turkey (bbtst) was established at 1997 with the main aim of education of the related people about blood banking and transfusion medicine. bbts has organized 35 symposiums, 13 national courses, 1 national and 1 international congresses since 1997. due to increased knowledge about the blood components and improved infrastructure of blood banks whole blood consumption has decreased to national average to 60%. in most of the university hospitals and training hospitals this is around 19%. conclusion: like most of the public based behaviours dedicated efforts of civil initiative has changed the traditional habit of blood consumption in turkey by the direct affect of well organized educational activities. training of general practitioners in blood banking and transfusion medicine n solaz*, b keskinkilic † and c oruc † *ankara university, ankara, † ministry of health, ankara, turkey background: turkish ministry of health runs majority of the hospital blood banks in turkey. most of the moh hospital blood banks give service under the medical supervision of general practitioners. because there is no specific blood banking and transfusion medicine training in either under or post graduate medical education those general practitioners have limited knowledge about this field. aim: to give basic knowledge and practice to those general practitioners moh has established a special training programme with the collaboration of medical schools for 5 years. methods: this is a 6 months programme which has 2 basic components; theoretical education, laboratory and blood banking practice. the trainees are evaluated by a final examination. each group has limited cadets and the training is provided in selected blood banks with the collaboration of universities and moh training hospitals. conclusion: almost 100 general practitioners are trained by this programme in the last 5 years and those doctors helped to increase the quality of blood banking and transfusion service at their hospitals. background: it has been accepted that during cardiac surgery the complement-system is activated which enhances ischemia-reperfusion injury, leading to postoperative complications as infections and organ failure. the lectin pathway is one of the mechanisms that can activate the complement-system, this pathway can be mediated by mannose-binding-lectin (mbl). the level of mbl in serum shows a wide variation. a low level of mbl can lead to more infections in some conditions and a high level to tissue damage after ischemiareperfusion injury. the effect of transfusions of erythrocyteconcentrates and plasma on the mbl levels and thereby on complications after cardiac surgery are not known. aim of the study: the role of pre-and postoperative mbl levels and the effect of transfusions on postoperative complications after cardiac surgery. in a randomized controlled trial 474 cardiac surgery patients were included and blood samples were taken pre-and postoperatively. mbl measurements were performed by elisa assays. the data were linked with postoperative complications as mortality, infections and multiple-organ-dysfunction-syndrome-mods and with peri-operative erythrocyte and plasma transfusions. results: the mean pre-operative mbl-level was 837 ± 796 and postoperative 456 ± 347 mg/ml. there were no differences in preand post-operative mbl levels between patients with infections or mods compared with non-infected and non-mods patients. the difference in pre-operative mbl between non-survived (672 ± 587) and survived patients (853 ± 812) was not significant (p = 0.20). postoperative mbl levels between survived and non-survived patients was smaller. conclusions: pre-and postoperative mbl levels in cardiac surgery are not related to postoperative infections, mods and hospitalmortality. whether large amount of blood transfusions are affecting the mbl-levels and thereby the patients outcome is not known. introduction: patients undergoing cardiac surgery are receiving high amount of blood transfusions and are at risk for the development of infections and multiple-organ-dysfunction-syndrome (mods), these complications are influencing the survival of the patients. during cardiac surgery pro-and anti-inflammatory cytokines are released by different mechanisms. we found in a randomized trial in cardiac surgery a significant reduction in infections and mortality due to mods by transfusion of leukocytedepleted erythrocyte concentrates (ld) compared to leukocyte-containing, buffy-coat depleted erythrocytes (pc). aim of the study: the effect of ld on the concentration of proinflammatory cytokine il-12 and anti-inflammatory cytokine il-10 in relation to clinical outcome and complications after cardiac surgery. methods: in 474 participating patients blood samples were taken before and after surgery. using elisa il-10 and il-12 were measured in these samples. the results were linked with the endpoints of the randomized trial: postoperative infections, mods and in-hospital mortality. results: all pre-operative concentrations of il-10 and il-12 were low. mean postoperative level for il-10 was 70 ± 121 and for il-12 57 ± 74. compared with patients without mods and without infections and survived patients only the il-10 was significant higher in patients who died and in patients with mods. there were no differences in mean levels related to ld and pc. the levels of il-10 were higher in patients receiving more then 10 units blood transfusions compared to 0 transfusions. conclusion: there is an association between mods and mortality and il-10, not with il-12. ld has no influence on mean levels of il-10 and il-12. there is a correlation between transfusion of more then 10 units and il-10, not with il-12. these cytokine-profiles are not associated with the beneficial effect of ld on the postoperative outcome in cardiac surgery patients. anemia and blood transfusion practice in critically ill patients e grouzi*, e tsigou*, p evagelopoulou † , g baltopoulos † and i spiliotopoulou* *kat general hospital, transfusion service, athens, † athens university school of nursing icu, athens, greece background: anemia is a common problem in critically ill patients admitted to intensive care unit (icu), but the consequences of anemia on mortality and morbidity in the critically ill is poorly defined. aim: to define the incidence of anemia and red blood cell (rbc) transfusion practice in critically ill patients in the icu) of our hospital, and to examine the relationship of anemia and rbc transfusion to clinical outcome. patients and methods: the period study was from july 2003 to december 2004. patients were enrolled within 48 h of icu admission. follow-up time was 30 days, hospital discharge or death, whichever occurred first. results: a total of 169 patients (116 male, 53 female, mean age 57.6 ± 21.1 years, range 15-96) were included in the study. the mean hemoglobin (hb) level at baseline was 11.2 ± 2.1, which level was descending during the study. overall 63.9% (108/169) of the patients received one or more rbc units while in the icu stay (mean 3.1 ± 3.8 units per patients). the mean pretransfusion hb was 8.7 ± 1.8 g/dl and the mean time to first icu transfusion was 2.6 ± 4.1 days. more rbc transfusions were given in the first week of the icu stay (331 units vs 110, 61, 36 units in the second, third and forth week respectively). the number of rbc units which a patient received during the study was positive associated with longer icu length of stay and an increase in mortality (r = 0.19, p < 0.05 and r = 0.26, p < 0.05 respectively). baseline hb level was significantly related to the number of rbc transfusion (r = 0.55, p < 0.001), but was not an independent predictor risk factor of length of stay or mortality (r = 0.16, p > 0.05 and r = 0.13, p > 0.05 respectively). the mean baseline apache ii and saps scores were 19.6 ± 7.4 and 49.8 ± 17.4 respectively. furthermore both baseline apache ii and saps scores were significantly higher for patients with a baseline hb level of <10 g/dl (23.2 ± 7.9 vs 18.3 ± 6.7 and 54.6 ± 18.8 vs 48.1 ± 16.5 respectively), while the apache ii values were positive associated with a significantly increased likelihood of rbc transfusion (pearson correlation p < 0.005). conclusions: anemia is common in the critically ill patients, it appears early in the icu course and persists throughout the duration of the icu stay. rbc transfusion seems to be associated with worse clinical outcome. despite the intensive research for the transfusion practice, which taken place worldwide during recent years, data from our country is limited. the results of our study suggest that approaches to reduce rbc transfusion would be desirable. however, further well designed prospective studies with large number of patients are required to efficiently explore the risk of anemia, optimal transfusion hb threshold and the risk and benefit of rbc transfusion in the critically ill. consumption of blood products in a large, general hospital he heier*, j pillgram-larsen † , m hestnes ‡ , b gran*, a krog* and no skaga ‡ *ullevaal university hospital, oslo, † ullevaal university hospital, dept. of thoracic surgery, oslo, ‡ ullevaal university hospital, dept. of anestesiology, oslo, norway uuh is the largest general hospital in norway, and trauma referral centre for half of the norwegian population (2.5 mill) the trauma team performed initial assessment and resuscitation of 323 patients, 75% males, during the first six months of 2002. the aim of our study was to analyze transfusion practice at uuh with specific focus on trauma. materials and methods: blood consumption during this period was recorded. clinical data of trauma patients were collected from our trauma registry and anonymized before analysis. results: 7012 units of erythrocytes (er), 914 of thrombocytes (thr) (buffy coat preparations from 4 donors) and 903 of s/d-treated whole plasma (op) (octaplasò) were transfused in uuh during this period. 56.6% of er were given to surgical, 21.3% to medical and 12.2% to gynaecological and obstetric patients. 62% of er were given to patients above 45 years. er units were given to 1475 patients (mean 4.75 units/patient; range 1-63). mean age of trauma patients was 33 ± 18, median 30, range 1-89 years. for transfusion of this group local guidelines state that haemodynamically unstable patients should receive er if hgb <9 g/dl, irrespective of age and sex. eighty-eight patients (27.8%) received er, 15 of them as massive transfusion ( 3 10 er units in <24 hours) (17.0%), 6 of these died (40%). altogether trauma patients received 851 units of er (12.1% of total uuh consumption), 51.5 thr (6.1% of total) and 150 units of op (16.5% of total). massive transfusion consumed 340 er (40%), 29.5 thr (57.3%) and 110 op (73.3%) units. fourteen adult trauma patients (15.9% of those transfused) received 1 or 2 er units only. lowest pre-transfusion hgb was 3.3-11.3 g/dl, median 7.6, mean 7.5 ± 1; highest post-transfusion hgb 7.8-14.3 g/dl, median 9.5, mean 9.8 ± 1. five patients received er transfusion at higher pretransfusion hgb level than 9 g/dl, but in 4 er were given because of a hyperacute clinical situation. fifty-five% of issued er units had been stored for >15 days, while only 10% were issued before 10 days of storage. discussion: life-saving effect of transfusion would seem evident in the massively transfused survivors, while in the other transfused patients documentation of clinical effect is inadequate. transfusion practice in trauma at uuh seems fairly well in accordance with internationally accepted guidelines. the trauma unit consumed a surprisingly small part of total uuh transfusion resources. trauma patients deviate from the general uuh patient population by sex and age; transfusion is otherwise mainly given to more elderly patients. further analysis is needed on transfusion indications and results to optimize the total use of blood products in uuh. special focus should be on transfusion to non-bleeding patients without haematological disease. it seems that only a small part of transfusion efforts results in the saving of life. in croatia national guidelines for the use of rhd gamma globulin were laid down in the year 2000. in accordance with the guidelines, rhd gamma globulin is administered intramuscularly in doses of 250-300mg and should be given to rhd negative women after delivery of rh positive children, after abortions, in week 28 of their first pregnancy, as well as in cases pregnancies with an increased risk of fetomaternal hemorrhage. as to the latter, detection and measurement of fetomaternal hemorrhage are recommended. three years after the issuance of the guidelines a survey was conducted regarding the use of rhd gamma globulin that involved 33 health institutions across the country. as many as 38 601 deliveries and 10 974 abortions were reported in 2003. the institutions reported the usage of 5103 doses of 250 mg rhd gamma globulin or 110 doses/1000 deliveries + abortions. we compared our data with those obtained from similar surveys conducted in 1999, i.e. before the issuance of the guidelines. in that year 4407 deliveries and 1400 abortions were reported, and 6125 doses of rhd gamma globulin or 100 doses/1000 deliveries + abortions were administered. institutions were then divided into four categories: clinical hospitals, general hospitals with the capacity of 400-600 beds, general hospitals with the capacity of 100-400 beds, and institutions of rather limited capacity with gynaecology department. the range of the consumption of rhd gamma globulin/1000 deliveries + abortions in the first category was 73-167 with the median being 119; in the second category there were 73-115 doses with the median being 92, in the third category 55-128 doses with the median being 88, while in the fourth category the range was 10-191 doses with the median of 79 doses. the number of pregnancies which should have been protected with rhd immunization in 2003 year was obtained by the following formula: (frequency of rhd negative subjects) ¥ (frequency of the r gene) = 0.17 ¥ 0.59 = 0.10. if a complete antenatal and postnatal preventive measures involving rhd immunization had been taken in 2003, 12 287 doses of rhd gamma globulin or 250 mg/1000 deliveries + abortions should have been given. our research has shown that, despite of the national guidelines adopted in 2000, the prevention of the rhd immunization in pregnant women is still not adequate in croatia. in fact, it has not improved since a year prior to the adoption of the guidelines. it has also been established that the category of the institution is a factor influencing the implementation of the protective measures. we consider that much more should be done on informing both women and gynaecologists about the importance of prophylaxis of the rhd immunization. consumption of plasma derivates in croatia g jaklin*, b golubic-cepulic † and m dondur* *general hospital, varazdin, † clinical hospital center zagreb, zagreb, croatia a increasing consumption of plasma derivates, their limited supply and insufficient national reserves are problems that croatia is faced with nowadays, as well as many other countries. in 2003 a study was carried out regarding the usage of plasma derivates. as many as 34 health institutions took part having the capacity of 15 487 acute beds out of a total of 16 279. the data regarding the use of plasma derivates were collected as follows: albumin, i.v. gamma globulin, and concentrate of coagulation factor viii in two periods of time. we divided institutions into three categories: clinical hospitals, general hospitals with the capacity of 400-600 beds and general hospitals with the capacity of 100-400 beds. institutional practice patterns regarding the use plasma derivates were compared among them. the parameters considered were the total consumption of plasma derivates, consumption per bed and consumption per inhabitant. data on the use of plasma derivates was obtained from hospital transfusion departments and pharmacies across the country. we compared our data with similar study carried out in 1997. the consumption of albumin was 441.44 kg or 98 kg/million inhabitants in 2003 and consumption per bed for the first category institutions was in range 14.27-72.27 kg with the median being 39.42, for the second category the range was 1.24-44.64 kg with the median being 4.36, while for the third category the range was 0.32-23.13 kg with the median being 6.63. in 1997 in croatia the consumption of albumin was 436.50 kg or 97 kg/million inhabitants. the consumption of i.v. gamma globulin was 59.92 kg or 13.32 kg/million inhabitants in 2003 and the consumption per bed for the first category institutions was in range 0.03 g-8.89 g. with the median being 2.78, for the second category was in range 0.03-3.95 g. with the median of 1.56 and for the third category was in range 0.00-12.75 with the median 1.56 g. in 1997 in croatia the consumption of i.v. gamma globulin was 22.20 kg or 4.93/million inhabitants. the consumption of the concentrate of factor viii was 8807.000 iu or 1.96 iu per inhabitant in 2003. 1539.500 iu, i.e. 17.48%, was a recombinant factor viii. in 1997 the consumption of factor viii was 4876.350 iu or 1.08 iu per inhabitant. discussion: the use of albumin showed stagnation. however, the use of i.v. gamma globulin increased 2.6 times and the use of the concentrate of factor viii increased 1.8 times if compared with the results in 1997. self-sufficiency has not been reached in plasma derivates in croatia we needed 20 000 l plasma for gamma globulin and 58 000 l plasma for concentrate of factor viii for the level of usage of these plasma derivates in 2003. significant differences in the level of usage among hospitals have been observed. these reflected a considerable difference in hospital policy regarding the use of these products in defined clinical settings. therefore, we would recommend that the national society sets out guidelines for the use of the products. background: blood bank good practice requires avoidance of rh alloimmunization. several studies report a probability of immunization of more than 80% following transfusion with rhd+ rbc's and as many as 19% in the case of platelets. aims: the purpose of this study was to investigate the development of anti-d and the causes of alloimmunization in a university hospital (1300 beds), reviewing our practice on the use of rhd+ blood components in rhd-recipients. method: a retrospective study was performed whereby 15.872 rhd-patients, from 1990 to 2004, were evaluated for the development of anti-d analysing the data on the blood bank information system. results: from the 15.872 rhd-patients we found out 274 identified anti-d, 77% (211) detected before any transfusion in our hospital. of the 63 left, nine were excluded because no information was available during some years. 5 had history of pregnancy related to the immunization. 36 patients were exposed to rhd+ blood components: 12 were transfused only with rhd+ platelets (including two women of childbearing age); 24 with rhd + rbc's. the remaining 13 had been exposed exclusively to rhd -rbc's suggesting that some units could be mistyped. this idea was corroborated in three patients where there was a common donor (he was called for investigation). conclusions: transfusion services avoid as far as possible administration of rhd+ blood components to rhd-patients, although there are situations in which such transfusions are necessary. the retrospective review of records revealed the need for specific recommendations regarding the use of rh immunoglobulin to prevent anti-d immunization. the possibility of mistyping blood components also appeared in this review, emphasizing the role of these studies in the evaluation of methodologies used at blood banks. the purpose of the work: to present the usage of whole blood (wb) and blood products (bp) in the treatment of patients who are hospitalized in the internal disease department in gevgelija. material and methods: retrospective analysis is done according the data which were analysed in five years period (2000) (2001) (2002) (2003) (2004) . statistical methods which were taken from the history of the hospitalized patients in the internal department were used for analysis and the data of transfused wb units and units of bp were taken from the department of transfusion medicine. results: from the total 4285 hospitalized patients who have been analyzed, 759 (17.71%) received wb and bp, and there have been transfused 1411 units wb and bp. every patient, on an average, has been transfused with 1.86 units wb and bp. at the same time 326 (23.1%) units have been transfused as a wb, 928 (65.77%) have been transfused as red blood cells (rbcs) and 157 (11.13%) units have been transfused as a fresh frozen plasma (ffp). the data for every year particularly will be presented in our paper to the congress. the collaboration between doctors of transfusion medicine and health workers from the other medical branches is the most important thing in the medical practice. our aim is to raise the awareness among the health workers for the importance of blood safety and their role though adequate clinical usage of wb and rbcs, minimizing the non-useful transfusions, evaluation of reflexive information from undesirable reactions in the usage of blood and blood products, use the alternatives etc. the necessity of direct involvement of the doctors in transfusion medicine in the process of healthy workers' education from the other branches for regularly transfusion therapy via meetings and lectures is an imperative for regular function of the department of transfusion medicine. femoral neck fracture repair is one of the commonest orthopedic procedures. it mainly concerns intracapsular or intratrochanteric fractures and it may be considerably hemorrhagic, requiring blood transfusion perioperatively. the aim of our study was to investigate blood transfusion requirement in patients with femoral neck fractures repair at katerini general hospital during 2002-2003 and to compare them with other studies. one hundred sixty five (165) unselected patients of whom 116 (70%) were women and 49 (30%) were men with a mean age of 76 years (range 26-94 years) were studied retrospectively. seventy six (46%) patients had intracapsular fracture and 89 (54%) intratrochanteric fracture. the mean hb concentration on admission was 12.25 g/dl (range 6.8-17 g/dl) and the mean hb concentration at discharge was 10.78 g/dl (range 7.2-14.3 g/dl). a total of 297 units of rbc were transfused (a mean of 1.8 units per patient). blood transfusion occurred in 125 patients (75.8%), (45-69% in other studies) with a mean of 2.38 units per patient (range 1-10 units), (1.47-2.6 in other studies). patients with preoperative hb values <10 g/dl were transfused more often than those with hb values >10 g/dl (100% versus 73%) p: 0.017. women were transfused more often men (80.2% versus 65.3%) p: 0.042. patients aged >70 years were transfused more often than those aged <70 years (81% versus 60%) p: 0.008. finally patients with intratrochanteric fractures were transfused more often than those with intracapsular fractures (88.65% versus 60%) p: 0.001. conclusions: in our study blood transfusion requirement for femoral neck fracture repair are similar with these reported in other studies. blood transfusion frequency is greater in patients with hb <10 g/dl, in patients older than 70 years, in women and in intratrochanteric fractures repair. fresh frozen plasma guidelines and practices as saltamavros*, g talampouka*, c koumoundourou*, s dimitrakopoulos † and p tseliou* *st. andrews hospital patras greece, patras, † pyrgos general hospital, pyrgos, greece introduction: fresh frozen plasma transfusions should be done according to specific standards and guidelines. aims: we have reviewed the ffp transfusion practices followed in our hospital in an attempt to provide a set of guidelines and principles that will assist physicians and health care workers to make the right decisions for appropriate use of ffp transfusions. methods: retrospective review of ffp transfusions practices during the entire year of 2004. we classified transfusion practices according to the diagnosis of the patient and also of the clinical depart-ment that demanded transfusion. then we analyzed our results by comparing the requests with the internationally approved guidelines for ffp transfusion and counted the percentage of ffp to rbc units. finally we discussed with our fellow physicians the proper use of ffp and informed them about the accepted guidelines. as we can see from the underneath table, diagnoses and departments with high consumption were: internal medicine 22%, plasmapheresis to patients suffering from guillain barre and ttp (thrombotic thrombopenic purpura) 21.04% and trauma/surgery 20.68%, cancer 13.60%. summary/conclusions: the use of ffp corresponds to 27.88% of the rbc units transfused in our hospital. plasma units should be used properly and according to internationally accepted standards. plasma use should be justified by the physician, in order to avoid unnecessary risks on behalf of the patient for effective treatment and care. to show gradual discontinuation of using whole blood and increased use of red blood cells (rbc) in the management of patients in the transfusion department of the clinical hospital 'zvezdara' . methods: data of wb and rbc use from 1982 to 2004 in our hospital. results: our data are presented in table 1 . conclusion: after 1982, wb usage rates systematically decreased from 51% to 1.3%. however, in the period of 1991-2004, the wb usage rates increased slightly, and we arrived at a generally very low rate of wb use, namely only in about 3% of the cases. education about transfusion medicine and rational use of blood in the medical curriculum was generally insufficient and very poor. therefore, we started an education program and we established a hospital transfusion committee (htc) with the task of medical control and monitoring the quality of clinical transfusion practice in our hospital. members of the htc are a specialist in transfusion medicine, an internal medicine specialist, an anesthesiologist, surgeon and gynecologist. we have been organizing courses for the medical staff, doctors and medical technicians since 1995. background: platelet transfusions are given mainly to thrombocytopenic patients with malignant haematological diseases. currently the decision to give a prophylactic platelet transfusion is based almost exclusively on the number of circulating platelets in the patient although it is known that various clinical factors might influence the bleeding tendency. by free oscillating rheometry (for), using a reorox® 4 instrument, it is possible to monitor the coagulation over time in whole blood and obtain information about clotting time and coagulum elasticity. aim of the study: the aim of this study was to find out if for using the reorox® 4 instrument could be used to evaluate the hemostatic status of thrombocytopenic patients. methods: the change in elasticity over time in non-anticoagulated whole blood from leukaemia/lymphoma patients with thrombocytopenia was measured in the reorox® 4 pre and post a platelet transfusion (n = 10) and was compared with healthy controls (n = 40). the effect of platelet concentration on coagulation was studied by diluting platelet rich plasma from healthy subjects with autologous plasma to various platelet counts whereafter the coagulation was monitored with for (n = 8). the change in elasticity per minute (g'max slope) and maximum elasticity (g'max) were evaluated from the elasticity curves. results: the g'max, g'max slope and platelet count increased after transfusion for all patients. the thrombocytopenic patients had significantly lower g'max and g'max slope values both pre and post transfusion compared with healthy control subjects (p < 0.01). the measurement in platelet rich plasma showed that g'max and g'max slope increased with increasing platelet concentration. however patients with similar platelet count developed different clot elasticity. the reorox ® 4 instrument responds to changes in haemostatic function in form of the clot elasticity. the fact that patients with similar platelet concentration developed different elasticity shows that clot elasticity is a function of not only platelet concentration but also of functional properties. the for method seems promising to evaluate platelet function. quality control of pre-storage leukodepleted red cell concentrates vu urlep salinovic, k perbil lazic and l lokar teaching hospital maribor, maribor, slovenia background: the system of quality assurance including quality control (qc) in transfusion medicine is most important for safe blood supply. the safety and quality of blood components are increased by pre-storage leukodepletion of whole blood (wb). aim: the qc of leukodepleted red cell concentrates (rcc), prepared from pre-storage filtration of wb (quadruple blood bag systems imuflex wb-rp terumo) is performed with the aim to be sure that the quality of leukodepleted rcc is in accordance with the guidelines of council of europe. in three years (2002) (2003) (2004) , 35 559 units of wb were collected, among them 7765 (21.6%) units were collected for pre-storage filtration and in 376 (4.8%) of these qc was done. within 4 hours after collection, wb was filtered at room temperature. filtered wb was centrifuged at 4000 g for 20 minutes and separated in rcc and plasma. the samples for qc were collected before and after filtration. for each unit, volume, hemoglobin, hematocrit, % of hemolysis and residual white blood cells (wbc) count were measured. the number of residual wbc after filtration was determined in the nageotte chambre. for all parameters the mean value and standard deviation were calculated. the results of qc for the following parameters were: volume 320 ± 25.1 ml (89% corresponds with the guidelines of council of europe), hematocrit 0.61 ± 0.06 (96%), hemoglobin 60.9 ± 8.0 g/unit (99%), number of residual leukocytes 0.028 ± 0.16 ¥ 10 6 (100%), hemolysis 0.30 ± 0.19% (100%), the test of sterility was 100% negative. during filtration of wb, wbc were removed in approximately 99.99%, the duration of filtration was 16 ± 6 minutes and the loss of hemoglobin was 8.2 ± 4.3%. the pre-storage filtration of wb is highly efficient, 99.99% of wbc were removed and the loss of hemoglobin was small. background: the patients of cardiosurgery use big amount of blood components. there is no uniform approach by treatment with blood components in these patients. the safest and the most rational use of blood is based on the individual treatment of a patient and the evaluation of the most clinical and laboratory factors. aim: the aim of our study is to analyse the use of blood components from 1998, when the cardiosurgery department was founded in our hospital, to 2004. the data about use of red cell concentrates (rcc), platelet concentrates (pc) and fresh frozen plasma (ffp) in the period from 1998 to 2004 were collected from information system datec. the average use of all three blood components per patient is presented. we were interested, if the average use per patient was diminished in the analysed period. results: the use of three blood components and average use of all blood components are presented in the table. the data from the table shows, that the number of patients increased more than three times, but the average use of blood components per patient was not essentially diminished. conclusion: during seven years period the use of blood components per patient in cardiosurgery was not diminished. for more rational use it is necessary to apply all methods of autologous blood transfusion because of the increasing number of cardiac operations and decreased number of voluntary blood donors. background: the presence of leucocytes in blood components is cause for appearance of various adverse posttransfusion reactions such as nhfptrs, urticary, anaphylactic shock, alloimunization and platelet refractorines, infection with bacteria and leucothropyc viruses (cmv, htlv). the removal of leucocytes from blood components through filtration is especially important in the treatment of patients with malignant diseases who need frequent transfusions of blood and blood components. this is because of their lowered immunologic status which is a result of the disease and received immunosuppressive chemotherapy and radiotherapy. aim: to show the prevention of posttransfusion reactions and the positive effect from transfusion of leucoreduced er. concentrates, produced with filtration in therapy of anaemia in patients with malignant diseases, treated in our daily transfusion hospital at medical center -stip. methods: 123 patients with malignant diseases have been treated in our daily transfusion hospital in the last four years. most of these patients suffer from ca pulmonum, ca collonis, ca uteri, ca mammae. also, because of the secondary anaemia, the same patients were transfunded with at least two doses of leucoreduced er. concentrates. the blood donored by voluntary repeated blood donors was filtrated the same day after the donation, separation and the control of the same one. the blood was collected in baxter and terumo bags, and it was filtrated with baxter -sepacell rs -2000 and pall -purecell rn filters. the analyses of leucoreduction were made for every filtrated and transfunded unit before and after filtration. the analyses samples were taken from the tubing system before and after the filter. haemathologic parameters were automatically made in the central clinic laboratory. results: er. concentrates poor with the leucocytes for about 98-99.9% were produced with the use od these filters, and platelets from 88-100% with which side effects from frequent transfusions at these patients were prevented. with the routine monitoring of all patients none posttransfusion reactions were registered. the therapeutic effect is also important because of the fact that the number of er and the level of hg and hct remain almost unchanged. the aim of the blood banks is to help and to increase the safety of the blood components. the leucoreduction through of er. concentrates poor with leucocytes is a regular procedure in the therapy of malignant patients with remarkable clinical picture of anaemic syndrome and prevention the cancer recurrence end infection. the time of filtration is also very important which has to be shorter after collection of blood, because the leucocytes should be removed before they become disintegrated and relapse potentially dangerous substances end metabolites in the blood components. we have been applying so called ' prestorage filtration' because it has been proved that it is more efficient that bed -side filtration. background: the effect of leukocyte reduction of rbcs by filtration has been the topic of several rcts. these rcts investigated the effect of leukocyte reduction on mortality; post-operative infections and hospital stay in different patient populations. some rcts came up with answers that conflicted with the results of other rcts. aim of the study: with including the individual patient datasets of several rcts in one database, combined analyses are possible that may explain the differences in the reported results. using this technique, we may come up with answers (or questions) that cannot be obtained by performing standard meta-analyses of these rcts. methods: we coordinated several rcts comparing the perioperative use of buffy-coat depleted rbcs with the use of filtered rbcs. cardiac surgery patients were included in three rcts (1¥ cabg and/or valve; 1¥ re-cabg and/or valve; 1¥ valve with or without cabg). oncologic surgery patients were also included in three rcts (1¥ colorectal cancer; 1¥ gi-oncology or vascular surgery; 1¥ gi-oncology, vascular surgery or orthopedic surgery). the electronic data files of these rcts were uniformly recoded and entered in a single database. as different primary endpoints were investigated in the rcts, we focused on common endpoints, recorded in 4 or more of the rcts. multivariate analyses were performed on: in-hospital mortality, 30-day mortality, hospital stay, stay on icu, postoperative infections, and mods. results: in the rcts, individual datasets from 3875 surgery patients were collected (1630 onco; 1272 cardiac; 569 vascular; 352 orthopedic, 52 other). in the multivariate analyses, patients in the buffycoat depleted trial arm showed a higher mortality rate both in-hospital (p = 0.01) and at 30 days post-surgery (p = 0.03). no association between randomization and stay in hospital (p = 0.10) or stay on icu (p = 0.56) was seen in the combined study population. also, no association of randomization with the incidence or duration of mods was seen. the analyses of post-operative infections in the total population showed the trial arm to be associated (p = 0.016), and 'hospital' to be far stronger associated (p < 0.001). however, when the oldest rct, that had not yet used our standard definition list for scoring post-operative infections, was excluded, the association with the hospital was lost (p = 0.34) and the trial arm became more strongly associated with infections (p = 0.002). in the analyses of 3875 surgery patients, the use of filtered rbcs, compared to the use of buffy-coat depleted rbcs, resulted in reduced mortality (both in-hospital and at 30 days postsurgery) and a reduction in post-operative infections. the association of the variable 'hospital' with post-operative infections, as is frequently reported in literature, was initially confirmed in our analyses. however, when a standard definition for post-operative infections was used (excluding the datasets from one of six studies), this association was lost. background/aims: uk neqas for blood transfusion laboratory practice (btlp) operates an eqa service for 472 uk laboratories (including eire) and participants throughout europe, including major groups in denmark (n = 52) and portugal (n = 32). in november 2004 a questionnaire was distributed to determine the criteria used for selecting phenotyped blood for different patient groups, including pre-menopausal women (pmfs). results were analysed by country to establish any variation in practice relating to the selection of k negative (k-) and rhc negative (c-) blood, since antibodies to these antigens are now the major cause of hdn. results: overall return rate was 84% (uk) and 64% (non-uk), although only centres treating pmfs were included in this analysis. k-blood was selected for pmfs by 81% in wales (n = 16) and 71% in england (n = 291), but only 6% in scotland (n = 32), 8% in northern ireland (n = 12) and 10% in eire (n = 31). in mainland europe, variation was also observed: 12% in denmark (n = 17), 75% in portugal (n = 20) and 85% in other countries (n = 27 from 10 countries). fewer laboratories selected c-blood for pmfs: 8% in england and northern ireland, 0% in scotland and eire, rising to 81% in wales. mainland europe showed similar variation: 12% in denmark, 75% in portugal (all ccee matched) and 51% in other countries. there are no guidelines requiring selection of k-and c-blood for pmfs, but in the uk d negative blood is required for d negative females aged <60 years. trend analysis of questionnaire data in the uk, using theoretical clinical scenarios, shows a decrease in the number of laboratories that would select k + blood for a 30 year old female (with pre-existing antibodies other than anti-k), from 58% (1996), 42% (1999) to 26% in 2004. in this survey, k + blood was selected for a female aged 30 (no antibodies) by 31%, whilst 81% selected a k + unit for a female aged 54 (no antibodies), despite this patient being treated as a pmf for provision of d negative blood. a similar distinction was made between the 30 and 54 year old females in portugal and mainland europe. conclusions: variation in practice may at least in part be due to the availability of blood routinely labelled for rh and k. in the uk all donations are labelled, except for those from new donors, as are most units in portugal. uk questionnaire data (1999) suggested that >50% laboratories would change to selecting k-blood for pmfs if all units were labelled. however, labelling alone does not account for the differences seen within the uk, and perhaps the trend towards providing k-(and to a lesser extent c-) blood for pmfs is influenced by advice from transfusion services. it would be of great interest to monitor and compare the incidence of hdn due to anti-k and anti-c in different countries to measure the outcome of differing practices for provision of blood to pmfs and to thereby inform future policy. there is no ultimate in ex-vivo assay described, which can predict the outcome of plt transfusion in vivo. current in ex-vivo assays and animal studies are rather very complicated to carry out, cost effective and time consuming. objective: we hypothesized that the quantitative measurement of gpib expression by facs can be used to predict the outcome of platelet survival post transfusion. in our previous studies we demonstrated that our phagocytosis assay can predict the plt survival sensitively (blood dec-2004) . we isolated human washed plts by centrifugation and labelled with mepacrine and then incubated with pma-matured thp-1 cells (37°c). binding was measured by facs analysis of cd42b/cd14 positive particles, and phagocytosis by counting mepacrine/cd14 positive particles. we measured gpib expression before and after plt-macrophages interaction by facs flowcytometry. results: gpib expression at surface of c22 fresh showed 90 ± 5 and after 48 hours storage 50 ± 15% expression. the gpib expression at surface of plt decreased and showed three populations with different densities high (gpib-1), low (gpib-2) and in-between (gpib-3). the binding and phagocytosis of plt showed an increase of 40 ± 10% which implicates an indirect and negative relation to gpib-1, and direct relation to gpib-2 expression. anti-human pselectin (cd62p) delayed 45 ± 10% the binding and annexing v 60 ± 12% the phagocytosis of stored plts, after 48-hour storage. these results show that gpib expression is rather easy, reproducible test which can be standardised and be used as a very sensitive in vitroassay to predict platelet survival posttransfusion. transfusion and lung injury jd dodig*, d sovic † , m tomicic † and h sager* *university hospital 'sestre milosrdnice', zagreb, † institute for transfusion med, zagreb, croatia background: the respiratory tree has been viewed as an infrequent site of injury arising as serious complication of transfusion. in recent years, this view has changed as investigators have shown that two complications-circulatory overload and transfusion related acute lung injury-are relatively frequent events. case report: a 59-year-old men was admitted due to chronic macrohematuria. he had no history or current evidence of cardiac failure. the hb level was measured 36 g/l. he received 500 ml 0.9% nacl and 500 ml ringer solutions. after that, he was transfused with 2 units of rbcs. during transfusion second unit he developed following symptoms: tachycardia, dyspnea, hypertension. massive pulmonary edema was noted. he was treated with mechanical ventilation, oxygen, diuretics, aminophillin, antihistaminic and corticosteroides. the patient recovered after being on ventilation for 6 hours. two days after the patient was operated. after surgery he was transfused with 4 units of rbcs. all transfusions were regularly performed. results: chest x-ray confirmed bilaterally pulmonary edema. the samples (patient and donors of first two units rbcs) tested were negative for the presence of hla specific and granulocyte antibodies. granulocyte agglutination and lymphocytotoxicity test were negative. tnf-a in recipient serum was slightly increased. conclusion: our patient is the first reported case suspected of trali, but all of the investigations didn't give us the answer. against neutrophils using flowcytometric detection of cell surface cd11b expression and l-selectin shedding. methods: a hundred microl of volunteers' heparinized whole blood were incubated for 15 minutes at 37°c with fmlp, lps or pma. monoclonal antibodies against hna1a and hla-i, or patient serum were incubated with whole blood for 30 min. surface cd11b and l-selectin were detected using facscalibur. results: neutrophil activation was detected after fmlp, lps or pma stimulation. p38 map kinase inhibitor reduced activation induced by fmlp and lps. anti-hna1a monoclonal antibodies induced neutrophil activation, which were also inhibited by p38 map kinase inhibitor. ten serum samples obtained from patients or donors who caused transfusion reactions were evaluated. five sera out of 8 samples having anti-neutriphil and/or hla antibodies exhibited neutrophil activation, while two samples without leukocyte antibodies had no effect on any activation. conclusion: these findings indicate that neutrophils activation is regulated through mak kinase and detection of neutrophil activation may be useful to predict transfusion-related reactions. results: 38 out of 50 transfusion reactions were febrile, 19 were anaphylactic, 1 were due to circulatory overload, 1 out of 50 transfusion reactions concerned the transfusion of incorrect blood component. 1 out of 50 transfusion reactions concerned acute haemolytic reaction. post transfusion purpura or suspected trali was not seen. fatal complication was not seen. the reactions to plasma were predominately anaphylactic. we detected no case of bacterial contamination among the cultures of transfusion bags. conclusions: the incidence of reactions among patients malignancy was high, while among surgical patients was lower. the incidence of transfusion reactions during the months had no statistically significant difference. we suggest that the improvement in prevention of transfusion reactions require a continual vigilance system for rapid recognition and information regarding these complications. measurements of ige immunoglobulin in thalassemic patients, before and after blood transfusion background: many studies have reported the implications of proinflammatory cytokines including interleukin (il)-1 beta, il-8 and tumor necrosis factor alpha (tnf-alpha) in febrile nonhemolytic transfusion reactions. il-8 has been shown to accumulate in packed rbcs even after the procedure of filtration, which is explained by the release of il-8 from rbc receptors into the packed rbc super-natant. stress induced elevation in tnf-alpha levels was demonstrated in healthy individuals. aim: to assess the level of proinflammatory cytokines in peripheral blood of blood donors. material and methods: immediately upon blood collection, plasma was separated from postdonation blood samples obtained from 75 blood donors and frozen at -80°c. upon thawing, the level of the il-1 beta, il-8 and tnf-alpha cytokines was determined in plasma samples by elisa method using commercial kits for cytokine determination (roche molecular biochemicals). the level of il-1 beta was at the test detection limit in all donor plasma samples. in 1 (1.3%) bd, the level of il-8 was 36.8 pg/ml, exceeding the test sensitivity limit of 6.2 pg/ml. in 12 (16.6%) bd, the level of tnf-alpha was within the range of 14-60 pg/ml, with a test sensitivity limit of 12 pg/ml. tnf-alpha levels >800 pg/ml were measured in plasma samples of 3 (4.1%) blood donors. the increased activity of blood donor's immune cells, indicated by elevated levels of the proinflammatory cytokines il-8 and tnf-alpha in peripheral circulation some blood donors, may lead to the occurrence of febrile nonhemolytic transfusion reactions in the recipients of the blood products manufactured from the blood of these blood donors. this hypothesis will be thoroughly investigated in our future studies. background: transfusion-related acute lung injury (trali) is a life-threatening complication of transfusion, under-recognized and underreported possibly lacking a consensus definition. aim: we report here a 'probable' case of trali syndrome in an elderly female patient. case presentation: an eighty-two year old female patient was transferred to the intensive care unit on the third postoperative day (pod) following the abrupt onset of acute pulmonary insufficiency (pao 2 = 49 mmhg, o 2 saturation = 79%), hypotension (bp = 90/50 mmhg) and fever (38°c). this occurred ten minutes after initiation of an infusion of a unit of packed red blood cells (prc). the patient had no history of any cardiac or pulmonary disease. she was intubated and placed on mechanical respiration and supported hemodynamically. the cvp (12 cm h 2 o) ruled out fluid overload. the chest x-ray revealed bilateral pulmonary oedema, the echo cardiogram and blood cultures ruled out cardiac and infectious aetiology of the episode. after treatment for 48 hours the patient improved significantly, was extubated and returned to the surgical unit on the 6th pod. reviewing the transfusion history we discovered that the patient did not receive any blood or blood component prior or during the correction of her ileum, but she was transfused a unit of ffp (fresh frozen plasma) five hours prior to the incident. the donor review revealed that the unit of ffp was from a 29-year old female with a history of multiple abortions whereas the prc unit was from a 52-year old male, a volunteer of several years who had no history of transfusions. there are some prerequisites for the implementation of a haemovigilance network and some of them have been met, so we can present the first results. methods: traceability of blood components is possible because there is unique information system in place in the whole country, identifying the donor, donation, each single blood component and identification of the recipient, but feed back information of the performed transfusion is still missing. cooperation between blood transfusion service and hospitals was established by introduction of hospital transfusion committees; the intensity and quality of their work is very different. homogeneity of reporting is achieved by the introduction of unique reporting form. a reporting route was defined: patients physician sends notification of atr to the local blood transfusion service. atr reporting form is prepared there and sent to the national blood transfusion service, where the data is collected for the centre for haemovigilance, which is going to be established very soon. data analysis will be responsibility of the centre for haemovigilance at the governmental level. type of adverse transfusion reactions and events is defined. education and information was passed to the health personnel by inclusion of haemovigilance in under and postgraduate programmes as well as seminars, scientific meetings and some publications. results: in the years 2002-2004 there were 410.000 blood components issued in slovenia and 333 atrs were reported (1 in 1232 blood components issued), in 25 of them the severity grade was 2 (1 in 16.412 blood components issued). in the year 2004 there were considerable differences between the number of atr reports compared to the number of blood components issued among slovenian hospitals, ranging from 1 in 3375 to 1 atr in 490 blood components issued. 9.6% of all atr were not classified. conclusions: although the number of reported atrs was increased by 24% and 31% a year in 2003 and 2004 respectively, it can be assumed that the collected data is not complete. feed back reports, much more information and cooperation is needed, especially in some hospitals, which should contribute to a better registration of atrs and events in the future. the slovenian haemovigilance system still needs upgrading, but despite this, some important work has been done in building a national haemovigilance system. . considering the multiplicity groups in cattle and since there was no research on repeated blood transfusions reactions in iran's native cattle, we decided to consider crossmatching in different processes of repeated blood transfusion from a head of blood donor to five recipients and observe the clinical and hematological alterations. six healthy iran's native cow, 2.5 years old, with average weight of 210 kg were used. the animals were dewormed by albendazole (15 my/kg bw) and were kept for two weeks under uniform managemental condition. three days prior to blood transfusion, vital signs registration (temperature, heart rate and respiratory rate) blood collection via jugular vein was done to indicate the baseline of research parameters. after that, blood transfusions were performed from one donor cow to five recipients three times at one-week intervals. cross matching was done at each transfusion. after each transfusion the research parameters do determined. results indicated that one of the recipients cows experienced anaphylactic shock, in the first step of blood transfusion and another cows in the second step and finally in the third steps two other cows showed the serious shock. this is in the contrary of this opinion that the first transfusion can be given safely without crossmatching in cattle practice ( van der valt, et al. (1969) background and objectives: blood transfusion may lead to the manifestation of anti-hla and platelet-specific antibodies that may in turn bring about different problems like platelet refractoriness. it appears that the study of antibodies against hla-class i and platelet-specific antigens are useful for the selection and success of the appropriate treatment protocol. the aim of this study was to detect anti-hla and anti-platelet-specific antibodies by flowcytometry in patients with hematologic disorders (including acute leukemia, aplastic anemia) and patients with itp. in this descriptive study, anti-hla and platelet-specific antibodies were detected by flowcytometric technique, using 62 sera drawn from patients with different haematological disorders who showed a poor response to platelet transfusion and 20 from patients with itp. the results of anti-hla antibodies were then compared by panel reactive antibodies (pra). results: our results showed 44 (53.7%) out of 82 (53.7%) patients had anti-hla class-i antibodies in their sera. the frequency of each antibody isotype was found to be as follows: igm (51.2%), igg (32.9%) and iga (1.2%). 36 (43.9%) out of 82 patients had platelet specific antibodies and the frequency of each antibody isotype was found to be as follows: igm (40.2%), igg (30.5%) and iga (12.2%). 27 (31.7%) out of 82 patients had both antibodies. no difference was found between the two groups in platelet specific antibodies. despite significant correlation between flowcytometry and pra methods, pra can only detect antibodies which react with complement. conclusions: with increase in the number of platelet transfusion, immunization to hla antigens occurs; moreover, immunization against platelet specific antigens may also occur during autoimmunity. the presence of these antibodies may be one of the reasons of poor response to platelet transfusion and platelet refractoriness in patients under study. conducting similar studies with higher number of samples, platelet cross-match, and the use of hlamatched platelets for these patients are recommended. post transfusion purpura (ptp) is a rare, severe thrombocytopenia that results from alloimunization to platelet specific alloantigens, following blood transfusion. in this condition, the patient's own platelets are destroyed by the alloantibody even though they are not supposed to carry the 'guilty ' antigen. the disease is rare occurring mainly in multiparous women. the majority of reported cases involved antibodies against the platelet specific alloantigen hpa-1a in a homozygous hpa-1b patient. in a minority of cases the offending antibodies were directed against hpa-1b, -3a, -3b, 4a, -5a, -5b. although self-limiting, the syndrome is characterized by severe bleeding with high morbidity and mortality; therefore prompt diagnosis and appropriate therapy is crucially important. ptp is a challenging diagnosis, because the patients are often critically ill or post-surgery, and have alternative explanations for thrombocytopenia such as infections or drugs. we present three patients with severe thrombocytopenia initially misdiagnosed. the first patient, a 54-year old women, had a past history of systemic lupus erythematosus and coombs positive autoimmune hemolytic anemia. at the present hospitalization, after antibiotic therapy for endocarditis, a severe hemolytic episode occurred and she need blood transfusion. when severe thrombocytopenia appeared, she was wrongly diagnosed as evans syndrome. the second patient, a 73-year old man, suffered from sepsis after vascular surgery and revealed clinical and laboratory picture of dic. the third patient, a 58-year old women, had end-stage renal failure and received heparin during hemodialysis, thus heparin-induced thrombocytopenia was first suspected. history of recent blood transfusion rose the suspicion of ptp in all this cases, and appropriate therapy with high dose iv immunoglobulin was started. adequate laboratory work-up confirmed the diagnosis. three different anti hpa-antibodies were identified: anti hpa-3a, anti hpa-1b and anti hpa-3b, respectively. the platelets genotype of the first patient was hpa-3b/3b, of the second hpa-1a/1a and of the third patient, hpa-3a/3a. the reported cases emphasized the importance of keeping in mind the possibility of ptp. incorrect diagnosis may lead to wrong treatment and fatal outcome. health sciences authority, singapore, singapore introduction: the haemovigilance programme in singapore was started by the centre for transfusion medicine (ctm) in 2002. the system covers registration of collected, produced and transfused blood components, and monitors adverse transfusion reactions (atr). the programme runs on a voluntary, non-punitive and confidential basis. aims of the study: (1) to gather and analyse reports of all adverse and untoward events occurring during transfusion of blood and components. (2) to use the information acquired to determine the morbidity of transfusion. (3) to provide guidance on corrective measures to prevent the recurrence of some accidents, and to improve transfusion safety. (4) to improve public confidence by demonstrating to public, patients and professionals the safety of the existing transfusion system. methods: (1) a common report form is used and made available to all participating hospitals. within the reporting system, the identification of the patient and staff involved are not required, to ensure confidentiality and protection of information belonging to the hospital. (2) reportable events include immediate reactions during transfusion (haemolysis, non-haemolytic febrile transfusion reaction, urticaria, anaphylactic shock, bacterial contamination, trali), delayed untoward effects after transfusion (haemolysis, post-transfusion purpura, acute gvhd), transfusion-transmitted infections, incorrect components transfused, and near misses. (3) within the hospitals, a responsible person ensures that all adverse events and untoward effects of transfusion are reported on the haemovigilance forms and provided to ctm for collation. within the ctm, the haemovigilance coordinator is designated to assist hospitals in investigating serious adverse events and advise on the reporting formats. results: (1) the total number of reported cases has steadily increased since the introduction of the programme. (2) the number of participating healthcare institutions has also increased to 100% (n = 12). please refer to table entitled 'summary of the haemovigilance report 2002-2004' . (1) the implementation of the haemovigilance programme in singapore is feasible with respect to the asian setting, and can significantly contribute to blood safety. (2) there has been very good participation from the 12 participating healthcare institutions, signifying greater awareness and willingness to partake in the programme. (3) the results obtained from the programme have given rise to initiatives and recommendations aimed at reducing (71%) were rated for seriousness. of these, 50 (6.5%) were rated as grade 2 (moderate to serious morbidity) or worse. 793 (72.6%) were rated for imputability to the blood transfusion. of these, a relationship to the transfusion was graded as 'certain' or 'probable' in 470 (59.3%) and as 'possible' in 245 (30.9%). overall relatively few errors were reported in comparison to other systems. a small number of reports concern (possibly) infected blood components, and imputability was deemed probable or certain only in a minority of these reports. autologous blood components gave rise to five reports (errors as well as mild transfusion reactions) which shows a relatively high risk associated with their use (9.98 per 1000 the rate of post transfusion hepatitis (pth) in israel in unknown. this information is important in order to learn about the residual infection risk in blood recipients. aim: to summarize the data on reported cases of pth. methods: suspected cases of pth are reported to mda blood services. the investigation procedure includes follow up testing of implicated donors and retesting of an archive sample of the transfused unit, if available. donors involved in suspected pth-b are tested for hbsag and anti-hbc. anti-hbc+ donors are tested for anti-hbs. hbv-dna testing is done if anti-hbs is less than 10 miu/ml. donors involved in suspected pth-c, are tested for anti-hcv and alt. since 2000 hcv-ag or hcv-rna are performed, when appropriate. investigation is considered complete if all the involved donors are retested >6 months following the implicated unit. results: between 1993 between -2004 suspected pth cases were reported: 74 (57%) were pth-b, 54 were pth-c (42%) and in 1 patient both hbv and hcv infections were reported. investigation was completed in 53/129 (41%) cases, with 70% of the involved donors (1222/1749) being retested >6 months after the implicated donation. hbsag was not detected in any of the 773 retested donors. anti-hbc was detected in 26 donors involved in 7 pth-b cases of which 21 were also positive for anti-hbs. pcr for the detection of hbv-dna was performed on the 5 'anti-hbc+ only' donors, and none was found positive. only in 1/449 donors suspected to be involved in pt-hcv, anti-hcv antibodies were subsequently detected. this donation was collected and transfused before the introduction of anti-hcv testing in israel, which was implemented in 1991. in another pth-c case, the implicated donor is still negative for anti-hcv, in follow up samples of up to 28 months, but was found positive for hcv-ag and hcv-rna. pth investigation of all the donors involved was completed in 49% (52/105) of the cases where the patient received up to 10 blood components. conclusion: in the past 12 years an average of 11 cases/year of suspected pth were reported to the israeli national blood services. investigation was completed in 41% of the reported cases. so far, there was no clear evidence of hbv transmission. in 2 cases (out of 2.99 million blood units collected nationwide during 1993-2004) hcv seemed to be associated with blood transfusion: one caseprior to the implementation of anti-hcv testing and the otherprior to the implementation of hcv-ag testing in a donor that did not develop anti-hcv antibodies. these findings suggest that other modes of hbv and hcv transmission should be sought in blood recipients in israel. 3-2.9, p < 0.05). the seroprevalence for a-hiv in the bd population was 0.096% (se: 0.028; ci: 0.034-0.153; p < 0.01) whereas in the a-hbc positive bd was 1.08% (p < 0.01) and in the a-hbc negative bd was 0.07%. from 113 a-hiv positive donations, 33 were also positive for a-hbc, that means that the sensitivity of the a-hbc in the detection of a donation a-hiv positive was 29.2%. the relative prevalence (percentaje of positive donations for a-hiv in the a-hbc positive population divided the percentage of this marker in the donations a-hbc negative: rp) was 15.43, which indicates that the number of bd a-hiv positive is 15.43 times higher in the a-hbc positive that in the a-hbc negative population of bd. as regards a-htlv, the seroprevalence in the bd population was 0.042% (se: 0.019; ci: 0.01-0.084, p < 0.01), in the a-hbc positive bd was 0.42% (p < 0.01) and in the a-hbc negative bd was 0.031%. from 49 a-htlv positive donations, 13 were also positive for a-hbc, that means that the sensitivity of the a-hbc in the detection of a donation a-htlv positive was 26.5%. the rp for a-hbc as regards a-htlv was 13.54, which indicates that the number of bd a-htlv positive is 13.54 times higher in the a-hbc positive that in the a-hbc negative population of bd. our results suggest that, in our bd population the screening with a-hbc would be useful to prevent other infections transmissible by blood transfusion, like retroviruses, because of the high sensitivity and the relative prevalence. 1. despite the high specificity of currently available elisas, the positive predictive value is lower in blood donors. therefore, immunoblot tests and polymerase chain reaction (pcr) have been adopted for routine testing in elisa +ve blood donors, by our service. 2. our data show that the real anti-hcv prevalence of our donor population is very low (0.04%). 3. the selection and evaluation of appropriate assays of all donated blood for hcv infection ensure good laboratory practice and accurate post-notification counselling of infected donors. 4. given that donors who are elisa positive but persistently negative or indeterminate probably do not represent a risk for transmission, their deferral from donation increases the problem of availability of blood supply. 5. donor re-entry in the pool of donors is an issue for further discussion. 6. the introduction of nat technology may elicit more accurate responses and improve the screening process. background: the introduction of hcv antibody screening of all donor blood in 1992 represented a major step in the prevention of transfusion-associated hcv hepatitis and in identification of infected donors. the study of infected individuals provides a unique opportunity to define behavioral factors associated with infection. evaluating risk factors in hcv infected blood donors is essential for monitoring blood supply safety, donor screening effectiveness and developing appropriate prevention programs objectives: 1. to recognize the epidemiology of hepatitis c and how it differs geographically; 2. to investigate the risk factors for presence of anti-hcv antibody in blood donors; 3. to evaluate the effectiveness of our donor selection program in local level. methods: serological testing for hcv was performed according to standard procedures. initial screening was performed using secondgeneration eia and, after january 1996, using third-generation eia. our study included a confirmation test (riba) a questionnaire was used to collect data concerning demographic, social and sexual behaviors, and number and type of donations of blood donors. the study also included testing of sexual partners and family members. changes in rates of hcv infections were evaluated by comparing yearly prevalence estimates. the overall prevalence of anti-hcv (eia) was 0.11% out of 168 374 blood donations. the average prevalence of hcv infection by riba was 0.04%, which reaffirms the very low risk of transfusion-transmitted disease. the cumulative number of hcv infected donors was 68, with 54 cases in males and 14 cases in females. most infections were found among older persons (32% were aged 22-39, and 68% aged 40-59). the seropositivity was higher in family/replacement donors (63%) than in volunteers (37%). the annual prevalence decreased throughout years. the relative importance of risk factors for hepatitis c was: transfusion 10%, hospitalization 8%, immigrants 8%, occupational 5%, sexual transmission 6%, injection drug use 3%, household contacts 5%, other 3%, tattooing 2%, unknown 50%. according to the criteria for blood donation, certain donors should have been excluded in the predonation interview but these donors had denied risky behaviour when questioned. the importance of sexual activity in the transmission of hcv has not been well-established as we tested 30 sexual partners and 14 family members and none of them was found positive. conclusions: our results suggest that major improvement in the safety and quality of our blood supply has been made in our area. introduction: apart from immuno-haematological complications, blood transfusion recipients are exposed to the risk of viral and bacterial contamination of donor blood. the latter infectious risks are generally associated with the number of donations that are needed in the production of blood products: the pooling effect. one measure recently being discussed is the generalisation of the use of trombocytapheresis for the production of trombocyte concentrates. normally, the trombocyte product is a concentration of trombocyte extractions from a pool of 5 buffy coats: pooled platelet concentrates (ppc). in case of trombocytapheresis sufficient trombocytes for one transfusion can be collected from one single donor. aim of the study: in this presentation the effect of using 100% trombocytapheresis for the production of single donor platelets (sdp) instead of pooled platelet concentrates (ppc) on contamination risks will be assessed. these risks can be divided in 1) the risk of bacterial contamination, 2) the risk of viral contamination (e.g. hcv, hbv, hiv) resulting from window period donations, and 3) the risk of contamination with tse or emerging infections for which no screening test exists. the contamination probability of sdp versus ppc is assessed on the basis of the production characteristics of both products, e.g. the presumption that the contamination risk per trombocyte product will be reduced by a factor equal to the number of pooled donations (five in our case). reduction of the bacterial contamination risk was estimated using the results of the bacterial testing of pooled and apheresis platelet products. as patients are likely to obtain multiple blood products during treatment, the contamination risk reduction through sdp is not only dependent on the reduction of risk in trombocyte products, but also will also dependent on the total number of blood products transfused and their associated contamination risks. the platelet recipient risk reduction was calculated on basis of the distribution of blood products received by the patient population of the university medical center utrecht (umcu) in the year 2001. results: in the attached table the estimated risk reduction through 100% trombocytapheresis is shown for the general blood recipient patient and for the trombocyte recipient patients only. our analysis indicate that in platelet recipients, general application of sdp instead of ppc will reduce the risk for transfusion acquired tse infections by 49%, the risk of known viral infections by 56%, and transfusion acquired bacterial infections by 59%. the confidence intervals surrounding the results were obtained by bootstrapping. the large confidence intervals surrounding the reduction of bacterial infection risk is caused by the fact that only a limited set of 4300 apheresis trombocyte products were tested. conclusion: our analysis indicates that general application of sdp instead of ppc will not reduce the risk of transmitting infections to platelet recipients as linearly (1 : 5) as expected. whether it is a costeffective precautionary measure will have to be evaluated by a costbenefit analyses consideration clinical benefits and additional costs and risks of apheresis donations. introduction: hepatitis b is serious health problem world wide. its prevention, particularly in the population of blood donors is essential for providing good health care and protection. aim: the aim of this study is to present the distribution of hbsag(+) and hbsag(-) blood donors according to their profession. methods: specially designed questionnaires are used for interviewing the blood donors who has previously given signed consent for participation in this study. results: table 1 shows the distribution of the blood donors in different professions. administrative clerks with 43 (34.12%) and the workers with 40 (31.73%) registered in the group of hbsag(+) blood donors, as well as workers with 50 (50%) and administrative clerks with 30 (30%) from the hbsag(-) group of blood donors are dominantly more frequent than the other categories of professions. health care professionals, housewives and farmers in both groups of blood donors are least frequent. taking in consideration the distribution of blood donors by the given professions in both groups, for u = 21 and p > 0.05 there is no significant difference found. the analysis of the differences among the different frequencies, in distribution of blood donors according the profession, for d = 0.436 and p < 0.05 shows a significant difference, where the workers with 90 (39.82%) are the most dominantly represented. in relation to the issue of whether the type of profession of the blood donors is production or non production the results are presented on table 2 . in the group of hbsag(+) blood donors the number of those who work in production profession-92 (73.01%), is dominant over the number of those that has non-production profession-34 (26.98%). in the group of hbsag(-) blood donors there is no significant difference between the types of professions. conclusion: having in consideration the professions of blood donors in both groups, for c 2 = 14.8 and p < 0.01 there is a significant difference in the presented distribution. according to our study, which shows the horizontal transmission of hbv infection in the family in which there is an index case, the biggest number of participants in the study is workers, and the least number of participants are the farmers. introduction: blood donors, as part of the healthy population are tested for hbsag with each blood unit they give. therefore, they can be an epidemiological model for exploring the appearance of hbv infection in general population. aim: this study aims to show the distribution of hbv infection in blood donors in relation with their living conditions, space and facilities. the material needed for the study consists of the data obtained from 126 confirmed hbsag(+) blood donors and 100 confirmed hbsag(-) blood donors as control group. results: the table 1 shows almost equal number of hbsag(+) blood donors that live in houses or flats. in the hbsag(-) group 64 (64%) donors that live in a flat dominate compared to 36 (36%) of those that live in a house. having in consideration the presented distribution (table 10) for c 2 = 3.96 and p < 0.05 there is a significant difference, that comes from the bigger number of blood donors (128) that live in flat. as for the distribution of blood donors according the living space they use by member of the family (table 2) , we can show that 24 (19.04%) of the hbsag(+) donors have less than 10 m 2 living space, in compared to 5 (5%) from the hbsag(-) group. the bigger number of hbsag(+) blood donors with small living space gives bigger possibility for transmission of hbv infection in the family. the differences in the two groups for donors that have between 10 and 30 m 2 , and between 31 and 40 m 2 of living space per person, obviously are not very big. for u = 19 and p > 0.05 there is no significant difference in the number of the donors in the two groups, when the available living space in m 2 is discussed. introduction: when one of the sexual partners has hbv infection the other is also infected in 1 from 3 cases. aim: the aim of this study is to outline that the risk from transmission of hbv infection between sexual partners is smaller if they use condom as protection. methods: two groups of blood donors-hbsag(+) and hbsag(-) have been interviewed whether they are using condoms as protection, or not. results: table1 shows the distribution of blood donors concerning the use of condoms. table1: in the group of hbsag(+) blood donors those who have not used condoms dominate with number of 81 (64.28%), in compared to those 45 (35.71%) who used condoms. these data are in favor of eventually possible sexual transmission of hbv infection in hbsag(+) blood donors. in the group of hbsag(-) blood donors dominate those who have used condom-57 (57%), compared to 43 (43%) who have not. the given distribution of blood donors concerning the use of condoms for c 2 = 10.2 and p < 0.01, shows significant difference, which is due to the prevailing of the number of blood donors (124), who have not used condom. of er -concentrates are leukodepleted. the choice of bacteriological control of empty bags for blood, bags with er -concentrates in additive solution, universal and iso group plasma, as well as the systems for taking of blood are taken on free choice. the control of the erytrocyte concentrates is performed on the first day after the preservation and dekanting, and again between the 15th-21st day and 30th-35th day after the preservation. the pulled plasma is controlled on the day of pouring (spreading), and the control of the iso group plasma on the day of deplasming. three months later the iso group and the universal plasma kept on the temperature of -30°c is bacteriologically controlled again. bacteriological control is performed with standard procedures in the institute for health protection in stip. the transfusion transmitted infections are potentially dangerous complications of transfusion therapy in immunocompromised patients. the aim of this study was to determine the prevalence of transmissible infections in blood donor population in kashan, iran. a total of 600 consecutive sera were tested for cmv-igm antibody, hbsag, hepatitis b core (hbc) antibody, hepatitis c (hcv) antibody, and hiv antibody with standard methods. of the sera tested, 14 specimens (2.3%) were cmv-igm positive. the frequency of seropositive revealed no significant differences between male and female donors. the frequency rates of cmv-igm seropositive tests tend to decline with increasing the age. there was no relation between the frequency rates of cmv-igm seropositive with the educational level, socioeconomic status, marital status, urban dweller and rural resident patients. the prevalence of hbv, hcv, and hiv antibody were 0.5%, 0.5%, and 0%, respectively. these findings implied important clinical applications because detection of cmv positive sera may reduce the risk for transmission of cmv in blood transfusion and thereby decrease the risk on cmv-induced complications. introduction: the worlds problem, aids, steel can't be said that is a problem in these three centers in r. macedonia, in which blood is collected, controlled, and distributed. found negative and 5 of them were found positive for one of the three viruses (hiv-1, hcv, hbv). with the elisa/axsym assay 29 of the 992 blood units which were negative by the procleix ultrio assay were positive for anti-hbcag and negative for anti-hbsag and hbsag. from those 29 blood units 23 units were given for transfusion following our blood centre protocol and the remaining 6 units were discarded. the protocol consists of a good medical history, liver enzymes (ast, alt, ggt). we must take into consideration that from those 5 that were found positive by the procleix ultrio assay 1 was positive for anti-hcv and 4 were positive for anti-hbsag. summary/conclusions: despite the fact of the short period of time we perform this method, the ability of the nat technique for rapid use, reliability and sensitivity in detecting three viruses simultaneously, indicates the need for immediate use in blood donation as a screening method. in spite of the high cost of the method, it is clear that this assay is a valuable tool in our blood centre to provide fast and safer blood. bacterial contamination of blood products is a persistent, but often overlooked, problem in transfusion medicine. in greece it is recommended that platelets (plt) must be used or discarded within five days post-collection. recent reports from europe have advocated the use of bacterial culturing of platelets on day 2 or 3 and, in case of negative result, prolongation of their storage time to 7 days. aim of the study: to assess the prevalence of bacterial contamination of standard platelet units from whole blood, and to provide evidence that with the use of bacterial culturing it is feasible to extend the self life of platelets to 7 days. materials and methods: eligible blood donors were bled according to standard operating procedures used in greece. plt were prepared from whole blood, solely for the purpose of the present study, by the platelet-rich plasma method. plts were stored for up to 7 days at 20 to 24°c with end-over-end agitation. other 111 plts prepared from blood collected in triple-pack container system also provided with a predonation sampling device were also tested. plts were sampled in the bacteriology laboratory. plts were sampled on day 2, 5 and 7. both aerobic and anaerobic culture bottles were inoculated with a 7-ml platelet sample. culture bottles were incubated at 35°c in an automated microbe -detection system (bact/alert system) until a positive reaction was detected or for 10 days. all samples that were reactive were confirmed by routine culture. each reactive sample with bacteria growth on the routine culture was sub cultured for identification of the bacteria. results: a total of 1889 plt concentrates were cultured and bacterial contamination was assessed in each unit at day 2, 5 and 7 after collection. on of storage day 2 two out of 1889 (0.1%) plt units were found to be positive for bacterial growth. 7 cases of unconfirmed positive results were noted at the beginning of the study. out of the other 1887 units which were negative on day 2 and continued to be cultured for the next days, the assessment at day 5 found no other positive. after further storage, at day 7, defined as the end of the prolonged incubation period, 5 out of the 1887 plt concentrates (0.26%) grew bacteria although testing of the same units on day 2 and 5 gave no signal. from the 111 platelets units that were prepared from blood collected with a predonation sampling device, none of the plt concentrates gave a positive signal although 3 pouches were found to be positive, and subculture showed bacterial growth of coagulase -negative staphylococcus. despite the relative small number of tested platelet concentrate units, our findings discourage specialists in attempting platelet storage time prolongation to 7 days. bacterial contamination testing on day 2 and a storage time of maximum 5 days seems to be still the safest practice. bacterial screening of platelet concentrates using bact background: bacterial screening of blood components is a routine measure in the evaluation of blood product quality. at our institute bacterial screening is performed using bact/alert system. sampling and culturing of blood products is performed according to paul-erlich institute recommendations. methods: quality control data on the bacterial screening of platelet concentrates performed from 1999-2004 were retrospectively analysed. an initially positive (ip) and true positive (cp) rate, organisms isolated and time of detection are presented. results: a total of 5359 platelet products were tested during the 6year period. thirty (0.56%) were found initially positive by bact/alert. the cultures screening positive were subjected to bac-terial identification to distinguish false positive from real positive signals. bacterial contamination was confirmed in 14 (0.26%) plt concentrates. positive cultures were confirmed and identified in an independent laboratory ( hbsag, anti-core, anti-hbs, anti-hcv, anti-hiv i/ii, anti-htlv i/ii, rpr. these patients were transfused with 5-131 units of concentrated red cells, depending on their problem. a total 530 units were delivered from the beginning of their problem until december 2003. the control were done using last generation enzyme-linked immunoassay (dade behring, ortho, biomeurieux), as also using automated enzyme-linked immunoassay (axgym). the same patients were checked by their physicians before the initiation of transfusions for the same diseases. results: we found: 12 patients anti-hbc (+) and anti-hbs (+). 5 patients anti-hbc (-) and ánti-hbs (-) 3 patients anti-hbc (-) and anti-hbs (+) 1 patient anti-hbc (+) and anti-hbs (-) 1 patient hbsag (+), anti-hbc (+) and anti-hbs (-). all patients were negative for hcv, hiv, htlv, and rpr. the same results were found also from patients' physicians. conclusions: we conclude that the blood supply for blood transfusion-transmitted diseases is 100% safe in our centre. these results are in accordance with current international literature. this is due to careful selection of blood donors, to high quality of corporation between departments as also to internal and external quality control. all these factors contribute to safety of transfusions, the quality of life of the patients and the protection of patient's environment. neither in the pipetor nor in the extractor runs was found contamination. no false positive were detected and all the positive samples confirmed. (see tables 1 and 2) . for hiv and hcv the specificity was 99.99%. the validation criterion were met, so the system was implemented routinely in our laboratory. the purpose of our study was to analyse the applicability of the pall enhanced bacterial detection system (ebds) in the routine of our transfusion unit which is totally focused on apheresis platelet collection. methods: apheresis pcs, obtained by trima (cobe) and amicus (baxter) separators and re-suspended in 30% plasma and 70% ssp solution (macopharma), were submitted to microbiologic control using pall ebds system which uses oxygen percentage decrease as a surrogate marker of bacterial growth. the working steps were the following: 16-24 hour after donation, about 3 ml of pc were sampled into the ebds collection pouch and then incubated at 35°c under continuous agitation (incubator helmer 900) for 24 hours. after this period, oxygen percentage was measured using an oxygen analyser (pall bdso2). the test is based on the 'pass/fail' principle. in case of 'fail' result the microbiology department has drawn up the procedure to follow in order to confirm the data and to allow the micro-organism to be identified. the incidence of post transfusion hepatitis has been reduced by blood donor screening for hbsag, but the hbv infection is still responsible for a certain cases of post-transfusion hepatitis in world-wide. in this study the hbsag negative blood units were evaluated for anti-hbc and hbv dna by pcr method. an extra sample was collected from 2000 hbsag, anti-hcv, anti-hiv and rpr-negative blood donors. all of samples were examined by approved anti-hbc assay. all of anti-hbc positive samples were tested by hbsab assay and evaluated for hbv dna (pcr). the sensitivity of the hbv dna (pcr) assay was estimated 50 geq/ml according to vqc proficiency and run control panels. 212 (10.6%) out of 2000 samples were positive for anti-hbc.166 (78.3%) out of 212 anti-hbc positive samples were hbsab positive, and 46 (21.7%) were hbsab negative. all of 212 samples were assayed for hbv dna (pcr) single and all of them were negative for hbv dna (pcr). further study for evaluation of anti-hbc test as a screening assay for blood unites in high hbv infection prevalence area strongly recommended. early detection of hepatitis b surface antigen: a comparison of ten assays hbsag detection is the corner stone of detection of hepatitis b virus infection in blood donors and patients with hbv infection. one of the most challenges is sensitivity of the technique and kit. in this study ten different assays evaluated by seroconversion and performance panels. some of them can not be used as screening assay due to low sensitivity. the sensitivity of ten hbsag assays from biorad, dade behring, biomeriux, diasorn, radim, diesse, thermo. biokit, gb and shanghai companies were evaluated by two or three seroconversion and two performance panels from boston biomedica ink. seroconversion panel is a series of samples that collected over a period of time from individual developing antibodies due to a primary infection. for evaluation of the assay sensitivity who and other notified body in the world-wide recommended the seroconversion and performance panels. the hbsag assays are two groups. group one with high sensitivity included six assays. they can detect ad and ay subtypes from 0.1 ng/ml bbi to 0.4-0.5 ng/ml bbi respectively. low sensitivity group included four assays and they can detect ad and ay subtypes more than 0.7 and 0.5 ng/ml bbi respectively. for blood safety, the high sensitivity hbsag assays recommended for blood screening and all assays should be evaluated by seroconversion and performance panels. diagnosis of chronic hdv infection is usually by antibody testing and hbv dna detected by pcr method. it is rare to find patients with two replicating hepatotropic viruses and if the accompanying hbv is replicating, prognosis will be very poor. to clarify the correlation between hepatitis delta virus infection and hepatitis b virus dna positivity, sensitive hbv dna (pcr) assay was used. the presence of hbv dna was investigated in 274 patients referred during the 21 aug. to 28 dec. 2003. all of them were hbsag positive. all samples were evaluated for hbv dna (pcr). the sensitivity of the hbv dna (pcr) assay was estimated 50 geq/ml according to vqc proficiency panel and run control. anti-hdv was tested by commercial available enzyme immunosorbent assay. 179 (65.3%) were hbv dna positive and 95 (34.6%) were negative. 8 (4.4%) out of 171 patients had evidence of delta infection and 13 (13.6%) samples of hbv dna (pcr) negative patients were positive for delta agent. the serum alanine aminotransferase (alt) levels in 5 out of 8 hbv dna (pcr) and anti-hdv positive patients were higher than reference interval, but only in 2 out of 13 hbv dna (pcr) negative and anti-hdv positive samples were higher than reference interval. the present data indicate that 7.7% of patients with chronic hepatitis b have hepatitisdelta infection. patients with hbv dna (pcr) negativity had a significantly higher prevalence of delta marker (13.6%) than those with hbv dna (pcr) positivity (4.4%). delta rna testing in positive hbv dna and anti-hdv patients is recommended. introduction: reduction of the window period of hepatitis c virus (hcv) infection represents an important goal in the transfusional and diagnostic setting and nucleic acid technology-based tests have been introduced in some developed countries to reduce the potential risk of transfusion-associated infection. a prototype assay designed to simultaneously detect circulating hcv antigen and anti-hcv has been developed by biorad (biorad laboratories limited, marnes la coquette, france). aim of the present study: to define the cut-off (co) value of the assay and to evaluate the specificity and sensitivity of this new assay in the detection both of antibody and antigen comparing its efficacy with commercial assays. methods: in order to establish the co value and to evaluate the specificity of the assay, we tested 400 sera samples from the general population and 100 'difficult' sera from haemodialysis patients (n conclusion: the new assay shows high sensitivity and specificity and could be a useful tool not only in the diagnostic setting, where procedures to reduce the window period, such as antigen or hcv-rna detection, are not currently recommended, but also in the screening of blood donations, when nucleic acid technologies is not feasible due to costs, organization, emergency and/or logistic difficulties. introduction: in recent years the concern with the blood safety regarding the transmission of blood-borne viruses has been improved. this safety has been achieved with the combining of different strategies, such as a careful selection of donors, the screening for relevant virological markers and the viral inactivation/ removal methods. more recently, the implementation of the nucleic acid amplification technologies for the detection of hiv-1, hcv and hbv, has increase this aim by reducing the 'window period' of the infections. other viruses, such as parvovirus b19 (pb19) and hepatitis a virus (hav), can raise problems to the blood safety. these infections could provoke serious complications in some risk groups, like pregnant women, patients with haematological problems, children and patients with immunodeficiency. material and methods: an observational study was performed to determine the prevalence of pb19 and hav in portuguese blood donors. we gather, during four months, 5025 plasma donations and joined them into 505 pools, with no more than 10 donations each. [1999] [2000] [2001] [2002] [2003] in voluntary donors the anti-hcv prevalence ranged from 0.3% to 0.7%, in family replacement donors from 0.4% to 1.2%, autologous donors from 1% to 2.02%. we observed that the anti-hcv prevalence has a decline tendency during years in blood donors. according to sex the anti-hcv prevalence in men is 0.7% and women 0.6% (p = 0.004). over the 5 year periods the prevalence in men has a decline tendency (1.2% to 0.4%; p = 0.033) and increasing tendency in women (0.4% to 0.9%; p = 0.007). according to age group the anti-hcv is 0.6% in 17-29 age group, 0.65% in 30-39 age group, 0.8% in 40-49 age group, 0.86% in 50-60 age group (p = 0.001). the prevalence of anti-hcv is higher in fds than vds, but not statistical significant. [1999] [2000] [2001] [2002] [2003] . a total 14627 ftd and 684 ad have been tested for hbsag. a sample was considered as hbsag positive when found repeatedly reactive by 3rd generation. immunoassay method (elisa). the chi-square test was used for statistical analysis. results: the 5-year overall hbsag prevalence among first time blood donors was 8.9%. and ad 6.7%. among autologous blood donors was observed a decreasing hbv prevalence from 6.3% to 3.7% in 2003. according to age the prevalence was higher in 17-29 year group 7.7%, while according to sex was higher in man (8.8%) than female 6.2% (p < 00.5). among ad, a decreased hbsag prevalence according to age was observed in men and women. the same trends by sex and age were observed in ftd. the prevalence of hbsag in ad was lower than in ftd. however, from 1999-2003 hbsag prevalence has decreased in the same proportion in both population. this decreased can explain by to main factor: the improvement hbsag screening method in blood donors and decreased the hbsag prevalence in general population. (1 : 30 250) . the hbv dna-emia in hbsag negative samples was 1.14 ¥ 10 2 -9.55 ¥ 10 4 copies/ml. in two donors anti-hbc total was positive and in one anti-hbe was also detected. in one donor the glycin 145 alanin mutation in the s region was identified. the frequency of hbv dna pos/hbsag neg donors in poland is high (0.002%) therefore the decision to introduce routine hbv nat screening is justified. (1/1001) with stored apheresis and whole blood derived platelet concentrates. of these 118 failed results there were 23 confirmed positives (presence of bacteria in both the ebds pouch and the platelet mother bag by culture) representing 1/5133. the bacteria detected were staphylococcus or streptococcus sp. 76 of the 118 fail results were false positives (no presence of bacteria in the ebds pouch and the platelet mother bag by culture) representing 0.064% or 1/1554, and 18 were not confirmed initial positives (no bacteria in the mother bag by culture, ebds pouch not tested) representing 1/6559. there was one reported case of a missed detection with confirmed presence of bacteria (staphylococcus epidermidis) in the mother bag by culture. subsequently, the bacteria strain was isolated and inoculated into platelet units in our laboratory at levels as low as 5 cfu/ml. in all cases the pall ebds was able to detect. this supports the hypothesis that this missed detection was the result of a statistical sampling error rather than a system failure. the results from blood centers routinely using pall ebds demonstrated effective detection of bacteria in platelet products stored under routine conditions with a true positive rate of 1/5133, and with a low false positive rate (<0.1%). this is comparable to a recent survey result with other culture based systems. summary/conclusions: the minority group of pregnant women who come to labor without prenatal testing of hepatitis b and c revealed essentially similar prevalence of anti-hcv with healthy bd even if definitive confirmation is probably increased in this minority group. there is however markedly higher prevalence of hbv infection in the pw so that screening for hbv is essential for the prevention of vertical transmission. the systematic screening of bd with anti-hbc serves as further assurance for the prevention posttransfusion hepatitis eliminating only 1.21% of the possibly infectious, a percentage which can be restored to the blood pool after proving their immunity. methods: blood samples were screened for the presence of hbsag, hcv and hiv antibodies using enzyme immune assay and for syphilis using the tpha test. the results were analysed retrospectively. all samples with results at or above the minimum positive value were considered reactive. the tests for hbsag, anti-hiv and anti-hcv were repeated in duplicate in all reactive donations. blood units that were reactive in the primary or secondary assays were discarded. hiv positivity was confirmed by western blot analysis using hiv blot 2.2 (genelab diagnostics) results: results from a total of 77 903 screened donors were analysed. hepatitis b surface antigen rates was 1.99%; anti-hcv seropositivity was 0.54%; anti-hiv seropositivity was 0.01% and tpha seropositivity was 0.06%. one study calculated this risk to be one in 63 000 for hbv, one in 493 000 for hiv and one in 103 000 for hcv. it is therefore important to take a careful history from blood donors to eliminate those at high risk of infection. in view of the high infectivity of hiv positive blood, it is important not only to screen donated blood but also to exclude donations from high-risk individuals, such as males who have engaged in homosexual activity and intravenous drug users. a careful history should identify those who should not give blood. in turkey, among blood donors the average hbsag prevalence in 1985-1998 was 5.1%. but it had decreased to approximately 2.1% in 2003. anti-hcv positivity has been reported to be 0.6% between 1992 and 1999. but it was approximately 0.5% in 2003. rpr positivity in blood donors in turkey was reported to be <0.1% in 1973 and 0.0026% in 1990. in 2003, the rpr rates was 0.09%. in our study these rates are 1.99%, 0.54%, 0.09% and 0.06% respectively. anti-hiv seropositivity was found around 0. introduction: the serological detection of specific antibodies to treponema pallidum (tp) is an effective means of diagnosing syphilis, and an automated chemiluminescent assay is ideally suited to testing large numbers of specimens for the laboratory diagnosis of the disease. aims of the study: to develop a qualitative syphilis assay for the detection of tp immunoglobulin m (igm) and g (igg) antibodies. the assay will be used for the serological diagnosis of syphilis using the architect platform, which has the capacity to test 200 specimens/hour. the two-step assay is based on paramagnetic microparticle chemiluminescent technology, utilising microparticles coated with three recombinant tp antigens (tpn15, tpn17 and tpn47) and acridinium labelled anti-human igg and igm monoclonal antibodies as conjugates. in the first step, specimens, microparticles and diluent are incubated together, prior to a wash step; in the second step, acridinium labelled antibodies are added and after washing, pre-trigger and trigger are added to produce chemiluminescence, which is measured as relative light units (rlu). specimens yielding rlus less than the cut-off are considered negative, while those yielding rlus greater than the cut-off are considered positive. the sensitivity of architect syphilis tp was determined to be 100%, after testing 426 specimens that were previously screened as syphilis positive in fujirebio tppa; no prozoning was observed with high positive specimens (over 2 16 titer by tppa). the specificity generated from testing 564 hospitalised patients previously screened as tppa negative, was 99.6%. testing a mixture of sera and plasma from 5000 random donor specimens, generated donor specificity figures of 99.8%. the precision (cv%) with a positive control was 5.8% (95% confidence interval: 4.9-7.0%) by the standard 20-day nccls analysis (ep5a2). in a study conducted at asahikawa medical college hospital, in which, 81 positive and 82 negative specimens were tested, concordance with fujirebio tppa was determined to be 100%. no significant interference to the assay was observed from bilirubin (conjugated type and free type), haemoglobin or lipid. the architect syphilis tp assay is an automated, specific and sensitive test for the detection of antibodies to t. pallidum. background: hcv exposure of blood donors is serologically determined by the detection of anti-hcv antibodies in serum or plasma. however a 'window' period of 30-70 days after exposure exists during which specific antibodies to hcv antigens cannot be detected. hcv rna detection and/or hcv core protein testing result in dramatic reductions in the preseroconversion window period. the new bio-rad test, based on the simultaneous detection of hcv core antigen and anti-hcv (core, ns3, ns4) antibodies, improves the detection of hcv infection in the early phase. aims: the aim of this study is to assess the performance characteristics of this new screening microplate immunoassay, monolisa hcv ag-ab ultra, by using the bio-rad evolis automated microplate processor system. methods: this two-step elisa assay is based on the combination of an indirect test for the detection of antibodies (core, ns3, ns4) and a sandwich test for core antigen detection. results are available within 2.5 hours, with sample addition monitoring and color coded reagents. no specimen pretreatment is required. evolis is a self-contained microplate processor designed for full automation of microplate-based eia techniques. the walkaway system can process four microplates at a time with continuous loading of samples and reagents. positive identification of samples, reagents and microplates, usage of disposable tips with clot detection, integrated quality control and complete traceability provide a high level of safety management. the monolisa hcv ag-ab ultra/evolis system performance is evaluated for clinical sensitivity on 30 commercially available and well-documented seroconversion panels. the results are compared to viral rna detection and conventional hcv ab screening assays. specificity is evaluated by using random blood donor samples. results: among the seroconversion panels that begining with samples negative for hcv rna and anti-hcv antibodies, the monolisa hcv ag-ab ultra assay detects exposure to hcv an average of 25 days earlier than the monolisa hcv plus v2 test. the mean delay of the monolisa hcv ag-ab assay in detecting hcv infection compared to hcv rna testing is around 2.5 days. the monolisa hcv ag-ab ultra/evolis system allows simultaneous detection of hcv core antigen and anti-hcv (core, ns3, ns4) antibodies, thus significantly reducing the time gap between the initial detection of hcv rna and the first appearance of detectable anti-hcv antibodies. the fully automated system combines high degree of assay performance with optimization of laboratory workflow and safety management. operational evaluation of pall ebds bacterial detection system l larrea gonzalez, ma soler and rj roig centro de transfusion, valencia, spain introduction: regulatory bodies are increasingly mandating the use of bacterial detection systems for platelet products (22 ed standards for blood banks and transfusion services). one system currently available is the pall ebds bacterial detection system which utilises percentage oxygen as a surrogate marker for bacterial growth. aims: to evaluate the pall ebds in routine use in our blood centre. in particular, to assess feasibility and adaptability to daily labour routines. the orbisac (gambro bct) system was used to produce leucocyte depleted buffy coat (bc) platelet pools (4 bc/pool) stored in platelet additive solution (ssp macopharma). mean platelet count was 3.2 ¥ 10e 11 /pool with mean leucocyte count 0.09 ¥ 10e 6 /pool. ebds installation and training occurred over a 2 day period. 500 platelet pools were tested for bacterial contamination over the subsequent 5 weeks. ebds pouches were sterile connected onto platelet pools 22 hours after blood donation. platelet samples were taken into the pouches and then the pouches were incubated for 24 hours on a shaking agitator at 35°c. after this time, percentage oxygen was measured. no positive results were found in this study. this was as expected due to the relatively low number of platelet pools tested and it also highlights the absence of false positive results. minimal training was required to use the ebds. the system was easy to use and did not require the use of a laminar flow cabinet to take samples. it was quick and simple to take samples and perform oxygen measurement. after pouch incubation, 1 technician was able to make 20 oxygen measurements in less than 13 minutes. the data management system allowed full traceability of product and work flow. results were very easy to interpret. conclusion: the pall ebds was found to adapt perfectly to a routine blood centre environment. (2004) was 8470. the percentage collected from volunteer blood donors was 56% (n = 4783) and the rest 44% (n = 3687) was given from patient-related donors. the age of donors ranged from 18 to 65 years old. the assay used for the detection of hbsag, hbeag, anti-hbc igg/total, anti-hbc igm, anti-hbe and anti-hbs was the automated microparticle enzyme immunoassay (axsym) of the abbot company. all the units were tested for hbsag, and anti-hbc igg. if the anti-hbc igg was detected, the specimens were automatically tested for anti-hbs. the units were wasted if the anti-hbs was negative, and the specimens were manually programmed for the testing of the anti-hbc igm, hbeag, and anti-hbe. from the total of 8470 tested units, 1195 of them were found to be positive to at least one marker of hbv infection, that means the 14.11% of the health adult population was infected in the past by the hbv. the 13.69% (n = 1160) was previously infected and now immunized with hbsag(-) and anti-core igg(+) and 0.41% (n = 35) were chronic carriers of the hbv with hbsag(+). the 68.57% (n = 24) of the positive donors were patient related donors and 31.42% (n = 11) were volunteer donors. in other words, 24 of the 3687 not volunteers (0.65%) and 11 of the 4783 volunteers (0.23%) were detected to be infectious. the combinations of the serologic markers for hbv are illustrated in the table attached. these results indicate that the incidence of hbv infection in the northeastern department of greece is equivalent to the incidence of hbv in other greek regions (0.42%) as it is referred to the national haemovigilance data and moreover, the percentage of infectious donors is bigger among replacement donors, 0.65% compared with the 0.23% of voluntary donors. as a consequence, the best source for safe blood collection is the population of volunteers. earlier detection of human immunodeficiency type 1, hepatitis c and hepatitis b viruses using the procleix® ultrio tm assay on the procleix® system and the study objective was to assess the ability of the ultrio assay and associated discriminatory assays to reduce the detection windows for hiv-1, hcv, and hbv. commercially available seroconversion panels were used for testing. methods: hiv-1 (n = 13), hcv (n = 12), and hbv (n = 16) seroconversion panels were tested neat and diluted (1 : 8 and 1 : 16) in the ultrio assay. panels were tested neat in the appropriate discriminatory assay. times to detection of hiv-1, hcv, and hbv nucleic acids in seroconversion panels were compared to the vendor's historical data on time to detection of antibody and/or antigen using licensed or validated serologic tests. p-210 effectiveness and limitations of methods for platelet bacteria screening -how to apply which screening method? the successful concept of virus safety in transfusion medicine is not suitable in bacterial contamination. bacteria can grow up in blood components to enormous amounts, whereas the initial number of contaminating bacteria is typically very low. therefore, sample drawing for bacteria screening must not be done immediately after blood donation. the established concept of relevance of clinical microbiology (pathogenic, non-pathogenic, facultative pathogenic species) is not valid for bacteria contaminating blood. here, the currently discussed criterion of clinical relevance is the ability of bacteria strains (not species!) to grow up in blood components. the paul ehrlich institute (pei) developed pei bacteria standards, which are characterized concerning their behavior in blood components. they contain a defined number of living bacteria, they are deep frozen, ready to use and shippable. there are two strategies to improve bacteria safety of blood: screening and pathogen reduction. neither of them is perfect, but screening methods are successfully established since several years in routine (belgium, the netherlands), and represent the current state of the art. further development and collecting of experience will produce the basis for assessments in the future. it is of high importance to apply the screening methods in dependence on their properties. methods implying an incubation/cultivation step ('early methods') have to be distinguished carefully from 'rapid methods' . for example, it is unreasonable to compare (or to advertise with) different sensitivities of methods not considering their detection principle or their informative value. both principles, cultivation methods as well as rapid methods, show advantages and disadvantages. selection of the method has to consider the respective conditions of the given blood service (including logistics up to time frame between issue and transfusion). results from the procleix hiv-1/hcv and hiv-1/hcv/hbv (procleix ultrio) assays for the detection of hiv-1 rna, hcv rna and hbv dna in blood donors of two blood transfusion centers of sw greece in discriminatory assay testing, 3 out of 5 (60% of the positive, 0.020% of total) were reactive for hcv rna only and 2 out of 5 (40% of the positive, 0.013% of total) were reactive for hiv-1 rna only. none were positive for both hiv-1 and hcv. the standard serological assays gave the same results for the above positive samples. two samples that tested positive by the standard serological assays tested negative in the procleix hiv-1/hcv assay. of the 4151 samples tested by the ultrio assay, 21 (0.5%) tested reactive for hiv-1/hcv/hbv. in discriminatory assay testing, 1 out of 21 (4.76% of the positive, 0.024% of total) was reactive for hiv-1 rna, 4 out of 21 (19% of the positive, 0.096% of total) were reactive for hcv rna, and 16 out of 21 (76.2% of the positive, 0.38% of the total) were reactive for hbv dna. all were single positive i.e. none tested positive for more than 1 virus. three out of 16 positive samples for hbv dna tested negative by the standard serological tests. the opposite was not observed. the procleix ultrio assay is a definite improvement over the procleix assay in a region with a high incidence of hbv carriers. up until its use, it is obvious that hbv positive blood with very low antibody titers was transfused into patients. more results will show whether procleix ultrio can eventually replace the standard serological tests. the introduction: patients with hemophilia represent a high-risk group for post-transfusion hepatitis whose frequency is closely linked with the number and quantity of blood products used. in albania, the frequency of hepatitis is also linked with hbsag testing with elisa (introduced in1993), and hcv testing (introduced in 1997). aim of the study: evaluation of the prevalence of the markers of hepatitis b, c, and d in patients with hemophilia. methods: our study included 145 patients with hemophilia treated with cryoprecipitate and commercial clotting factors. blood testing for anti-hcv, anti-hdv, and hbsag was performed with elisa -gen. iii. results: of 145 patients tested, 26 cases (17%) were hbsag positive, 87 cases (60%) were anti-hcv positive, and 7 cases (5%) were anti-hdv positive. co-infection of hbsag and hcv was found in 16 cases (11%), whereas co-infection of hcv, hdv, and hbv was found in 4 persons (3%). the highest rates of infections and coinfections were found in patients above 30 years of age. conclusion: mandatory blood testing has decreased the levels of post-transfusion hepatitis. in albania, hemophilia is also still treated with cryo-precipitation, thus patients are at a particularly high risk during the 'window period' . results: 2/16 760 (0.012%) samples from rbd were anti hiv 1 + 2 nonreactive and rr for p24 ag both being nonreactive in the neutralization test, they were interpreted as false positives. 1/134 617 (0.0007%) sample from fbd was rr for p24ag/anti hiv 1 + 2 nonreactive and it was confirmed positive by neutralization. this bd had been autoexcluded himself after blood donation. he showed seroconvertion 21 days later: p24ag nonreactive, anti hiv 1 + 2 reactive and western blot positive. the only bd p24ag positive/anti hiv 1 + 2 nonreactive during the analized period, was an first time donor and the post donation autoexclusion was effective en this case. although a larger populations of bd is necessary to be studied and in spite of the low prevalence we have found, we consider p24ag screening is an alternative up to implementation of nucleic acid testing and simultaneously we should increase the quantity of altruist repeat blood donors, undoubtedly, the best population to give blood. owing to the rather short interval between successive donations (~70 days), this suggests that some 260-310 infectious units escape the screening annually. to these, one has to add the (now unknown) proportion of potentially hbsag negative + hbv dna positive ftbds. hcv: since the introduction of the screening in 1995, the general incidence in rbd has dropped from 13.1‰ to 0.001‰, suggestive of a 1 : 10 000 escape rate. the prevalence in ftbd has stabilized at 12 ± 2‰. based on reasons similar to these employed for hbv, the residual incidence in rbd suggests that 1 potentially infectious donation in 10 000 rbd escapes the screening (= to a total of aprox. 40, annually). a limited investigation using hcv-antigen eia evidenced a 1‰ escape rate in ftbds (= to a total of aprox. 50, annually table 1 and 2 are concerned to the fist two months of the implementation, where we had to adjust the volume of the eluate. conclusion: these system adjusts to the laboratory daily routine in the blood bank, with the pools released after first analysis in less than 18 hours. background: the hbsag, anti-hcv, anti-hiv1/2, p24 antigen, alt and syphilis tests are performed for blood donations in czech republic. no nat tests are mandatory in czech republic. the aim of this pilot study was: 1. hcv rna pcr testing in anti-hcv negative blood donations; 2. correlation between hcv nat and anti-hcv testing results. methods: blood samples (anti-hcv serologically negative, alt not elevated) were pooled using the guardian plus spii into pools of 24 samples. pools of 1 ml were tested using the cobas ampliscreen hcv test v. 2.0 (roche). results: 891 pools of 24 samples from 21 384 a-hcv serologically negative donations were tested from october 2002 to july 2004. no one pool was initially reactive. invalid tests: 14 (1.6%) run failures were observed, due to: invalid internal controls 10 (1.1%) and invalid positive controls 4 (0.5%). invalid tests were repeated. in none of 891 pools a positive hcv nat result was observed. conclusions: no discrepancy between hcv nat and a-hcv results was observed in our study. all of the nat tested donors in our study were regular voluntary whole blood or plasma donors who were repeatedly a-hcv serologically negative. the hcv incidence in the czech republic blood donor population is low but it is slightly growing up in general population. hcv nat testing could improve the safety of blood supply by reducing the window period for hcv. introduction: parvovirus b19 is the only parvovirus known to be a human pathogen. most commonly, it causes a mild childhood rash, erythema infectiosum, but in some cases more serious symptoms can be linked to b19, such as acute or persistent arthropathies, critical failures of red cell production, hydrops fetalis, fetal loss, myocarditis or hepatitis. inactivation of the non-enveloped virus has proven difficult. as a consequence, manufacturers of blood products have implemented screening measures to reduce the load of parvovirus b19 in manufacturing plasma pools by the use of nucleic acid amplification techniques (nat). in our institute all blood donations were screened for human parvovirus b19 by nat since april 2000. methods: over the last 5 years 5.5 million donations were screened for b19 by nat. samples with a virus load over 105 iu/ml were defined as positive, whereas samples with a virus load between the detection limit (103 iu/ml) and 105 iu/ml were defined as weak positive. weak positive products were released, whereas positive products were discarded. in addition infection markers of 50 b19 positive donors (case group) were determined over a time period of one year. virus load and b19 antibody status was compared with 100 b19 negative donors (randomised control group). b19 antibodies (igg vp2, igm vp2, ns1) were analysed by two commercial antibody tests. results: overall 496 b19 nat-positive donors were identified with a virus load over 105 iu/ml out of 5.5 million tested. there was a seasonal accumulation during spring and summer, whereas a large epidemic occurred throughout the last year. vp2 igg was detected in 90.9% and 74% of the case and control group, respectively (p = 0.01). these data demonstrated statistically significance (p = 0.01). all donor samples which were b19 nat positive for more than three months developed neutralizing vp2 antibodies. in contrast, ns1 antibodies were observed in 83% of the case group and in 15% of the control group (p < 0.01). ns1 antibodies were detected more frequently in samples, which were b19 nat positive for more than six months. conclusion: b19 nat could be implemented in blood donor screening as release criterion without causing a shortage in blood supply. all b19 positive donors of the case group developed neutralizing antibodies within three months and virus load was dropped rapidly below 105 iu/ml. these data support our testing algorithm all components of high positive donations (virus load over 105 iu/ml) were discarded. donors with ns1 antibodies showed more often signs of a chronic disease with detectable levels of parvovirus b19 longer than six months. background: on recent years, the syphilis screening of blood donors has become increasingly important not only because of the transmission risk of this infection but also due to the risk behavior that this implies. on account of the importance of this screening the tests used are becoming more and more sensitive. aims: to evaluate an elisa screening test (the tmpa test recombinant is based on the sandwich principle, an immunoenzymatic technology in solid phase, for the measure of anti-treponema pallidum in serum or plasma). methods: in this study 1696 samples from blood donors were tested by the rotine 'cardiolipidic reagent for syphilis screening on microplates' -diagast laboratories as well as with 'hdtmpa recombinant' -hoslab diagnostics. positive samples were then confirmed with fta abs/tpha. results: using the mentioned tests we obtained the following results: 1. 1666 (98.23%)cases turned out negative with both technologies; 2. 3 (0.17%) cases were positive in both methods; 3. 27 cases were positive only using tmpa recombinant [of which 23 (1.36%) were confirmed positive by tpha/fta abs. as seen we found 23 samples (1.36%) that were only positives by tmpa recombinant test and that were confirmed by tpha/fta abs. we concluded that tmpa recombinant seems to be a suitable test for a quick and automated syphilis screening of blood donors and provides maximum safety for the recipients. background: in recent years, there has been substantial evidence indicating that typing and subtyping for hcv is clinically important in understanding hcv disease and its therapeutycal options. 'naive' viral load also seems to influence disease severity and responsiveness to therapy. therefore, viremia and genotype identification have been done routinely in molecular biology laboratory units. aims: the university hospital of coimbra studies and tests his own patients and patients from 11 other hospitals in the central portugal. we also collect and test blood donor candidates from this region. we proposed to analyse the distribution of hcv genotypes in this region, among 1578 patients with cronic hcv infection. we have simultaneously analysed the viremia and correlated it with age and severity of liver disease. methods: nucleic acid extraction was done using the semiautomatic 'xstractor' from biomerieux laboratories (boom method). the genotyping used reverse hybridization and was performed using probes from the 5¢ non-coding region (innulipa introduction: bacterial contamination of blood products remains a persistent problem. various techniques for the detection of bacteria in blood products exist but none of them has been widely accepted. bacterial detection systems could be divided into culture systems and rapid technologies. hemosystem has developed a rapid and sensitive technology for bacteria detection named scansystem tm . bacterial contamination of platelet concentrates is a rare event with an incidence between 1 : 2000 to 1 : 3000 per donation. therefore hemosystem developed a positive control in order to validate the scansystem tm platelet kit before use. aim of the study: the current study was designed to evaluate the performance of the scansystem tm positive control. the scansystem tm positive control is a capsule containing lyophilised lactobacillus casei subsp rhamnosus. the bacteria concentration per capsule is at least 8 ¥ 10 8 cfu. the positive control has to be stored at room temperature and is stable for 2 years. after dilution in pbs, the preparation has to be used within 1 hour. two capsules were tested for ten consecutive days with scansystem tm platelet kit as well as with optimised scansystem tm platelet kit. in an independent experiment three capsules were diluted in platelets stored in additive solution and were tested each with scansystem tm platelet kit and optimised scansystem tm platelet kit. results: 25 microscopic fields were analysed for bacteria specific fluorescence for each sample. the ratio between bacteria specific fluorescence signals and analysed signals was 1.0 in all samples for both scansystem tm platelet kit and optimized scansystem tm platelet kit. therefore by definition all tested capsules were positive. the lyophilized positive control capsules enable the user to validate the scansystem tm platelet kit before use. because bacterial contamination of platelet products occurs rarely, the routine use of positive controls improves safety of the screening method. scansystem tm is currently the only method that provides this safety measure. introduction: whereas implementation of nat for blood donor screening reduced the risk for transfusion transmitted hiv and hcv infections currently below one per 23 million transfusions, the risk for bacterial infections is estimated to be 1 : 200 to 1 : 3000. especially platelet products, which are stored at room temperature, are prone to bacterial contamination. aim of the study: several methods are currently developed to prevent the transfusion of bacterial contaminated platelet concentrates. the study investigates a new rapid bacterial detection method. material/methods: pool platelet concentrates were spiked with seven transfusion relevant bacteria strains under sterile conditions at concentrations of 100 cfu/ml to 106 cfu/ml. bacterial concentration was verified on blood agar plates immediately after spiking. five millilitres of spiked platelet concentrates were centrifuged, stained with thiazole orange dye and analysed directly by facs within five minutes after staining. aliquots of pool platelets spiked with concentration of 102 cfu/ml and 101 cfu/ml of each bacteria strain were incubated for two to eight hours in special bouillon at 37°c and were analysed by facs immediately after incubation. results: sensitivity of facs analysis differed between 103 cfu/ml for e. coli and 105 cfu/ml for klebsiella pneumoniae without preincubation and was enhanced to 101 cfu/ml when a pre-incubation step of two to four hours was included. conclusion: bacteria detection by facs analysis combined with a short pre-incubation (2-4 h) at 37°c is a quick and simple method with sensitivity comparable to other commercially available detection systems. the advantage of this new method is the rapid analysis, easy handling, high sensitivity and less expensive price. introduction: detection of bacterial contamination of platelet concentrates represents a major challenge in transfusion medicine. for blood transfusion services the method must have a high sensitivity, an easy performance and a low price. aim of the study: in this spiking study we evaluated the new optimised scansystem tm platelet kit detection method for use in apheresis platelets. methods: apheresis platelet concentrates (apcs) were spiked with strains of ten different bacteria species. after different incubation periods, apcs spiked with 10 cfu/ml were analysed by the optimised scansystem tm platelet kit. the number of bacteria was monitored by plating on blood agar. results: all bacteria strains were detected with the optimised scansystem tm platelet kit when the sample was collected 24 h after spiking. identity of the spiked bacteria was confirmed by gram staining and dna fingerprints. conclusion: in summary, the optimised scansystem tm platelet kit was able to reliably detect ten transfusion relevant bacteria species in apheresis platelet concentrates within 70 minutes when the sample was taken 24 hours after spiking. background: since year 2000 our laboratory started routine screening of hcv-rna in plasma minipools for all plasma intended for fractionation. although nat testing is not yet mandatory all blood products are released depending upon nat results. aim: to test and compare two different methods of rna extraction in order to make all the necessary adjustments to the test procedures while preserving the availability of blood products. methods: plasma minipools of 24 donations are prepared either on a tecan genesis robot or on a hamilton at plus. hcv-rna is isolated from 2 ml plasma by using either the qiagen biorobot 9604 and qiamp virus biorobot 960 kit or the manual extraction with cobas ampliscreen hcv pcr kit v 2.0. results: between march 2000 and december 2004 a total of 238 000 seronegative donations (9916 pools) were tested for the presence of hcv-rna. four pools were found to be positive for hcv-rna. of the four nat-positive pools with no eia-positive donor, four were confirmed as true positive by donor follow-up testing and/or testing of an independent sample from the index donation. all the positive donations were detected independently of the extraction method used (manual or automated). our experience shows that although the automated extraction method is 'off label' and it has to be validated, the use of biorobot 9604 does not pose a detectable contamination risk and it is possible to achieve a detection level for hcv less than 50 iu/ml. the advantage of the manual method is that it has better recovery of nucleic acids than the qiagen extraction. concerning the time needed for the extraction process the automated method runs 96 samples in 2 hours where the manual method needs 3 hours for 24 samples, needing, prior to extraction, an extra centrifugation step for one hour. the automated extraction method results in an assay with a high sample throughput, fast time, sufficiently sensitive, that can be successfully introduced into routine use in laboratories which have more than 800 samples/day while preserving the availability of blood products. anti-hcv similarly was high till 2002 (0.5-0.8%), but in trend to decrease afterwards (0.6%). anti-hiv reflected the low endemicity of the disease in public setting and was 0% through the mentioned years. rpr test for syphilis was around 0.1%. directed donors were 95% of all and volunteer donors consisted nearly 5%. donors in our blood center are being informed about donation prior to giving their blood and donor questionnaire forms (dqf) are filled out by the donor candidates. using dqfs have been mandatory at all blood banks in turkey by law since 1996. from that time infectious disease marker rates were dramatically reduced at all centers. donor information about the risks of transfusion and the importance of safe blood supply were detailed by the donation staff and physicians, consequently self-exclusion by the donor candidates who have risky behaviors was encouraged at our center. the interviewing staff was trained specifically for this topic. this steps were particularly emphasized in the last three years and the infectious screening results were displayed the outcome of this efforts. conclusion: education of the prospective donors, and recruit the voluntary, non-remunerated and regular donors will be the utmost goal of all blood banks. rigorous donor selection will contribute this ultimate success. we should spend more efforts to maximize enrolling voluntary donors to lower the serological marker results, consequently achieve safe blood. background: human t cell lymphotropic virus type i is endemic in japan, the caribbean, southeastern united states and parts of south america and africa. in non-endemic areas such as europe, htlv-i is less common and most infections are identified in immigrants. the epidemiology of htlv-ii is different, being predominantly found among indigenous american-indian populations and among ivdus, but the routes of transmission are the same. aim: our study's aim was to ascertain the prevalence of htlv i/ii in blood donors in order to understand the epidemiology of htlv in greece and initiate discussions of an acceptable level of risk and appropriate level of screening for rare transfusion-transmitted diseases. overall, anti-htlv seroprevalence levels among blood donors, are low. although the number of annual donations in this study is relatively small, the data for htlv indicate that rates of this infection are low and that infected donors will be seen infrequently. as all blood donations are screened for htlv i/ii during the last six years, a national survey is necessary in order to define the epidemiology of htlv in greece. introduction: toxoplasma gondii is the causative organism of toxoplasmosis. the disease transmitted by ingestion of either oocysts (in the feces of cats) or bradyzoites (in raw or undercooked meat). the parasite can also be acquired transplacentally by organ transplantation or from blood transfusion. the purpose of this study was survey of toxoplasma antibodies in some iranian blood donors at tehran blood center. blood samples were randomly collected for detecting of igg and igm antibodies (by elisa technique).the total numbers of donors was 217 of 52 (%24) were female and 165 (%76) male in age ranged from 18 to 55 years. results: 217 sera tested, 128 (59%) were found to be positive for toxoplasma igg antibodies and 6 (2.8%) were igm antibodies positive and 2 of them (0.9%) were borderline for igm antibodies. among males the frequency of positivity was higher than woman but this different was not significant. the most frequency of seropositivity was found in age group 41 to 50 years. conclusions: diagnosis of toxoplasmosis can be aided by serologic or histocytologic examination. the acute infection in healthy individuals is generally asymptomatic and not associated with any morbidity but in an immunocompromised host, toxoplasmosis be a very serious disease, and this can occur if a person is infection with toxoplasmosis before or after his/her immunosystem is compromised. in spite of the progress in the development of diagnostic, therapeutic and prophylactic methods, virus hepatitis still presents a serious global health problem. the possibility of transmission of these infections through transfusion of blood and blood derivates implies obligatory control of the donated blood. post-transfusion hepatitis is an important health problem in everyday practice, especially in patients who have to receive transfusion of erythrocyte concentrates as the only possible treatment for many years. objective: to show the prevalence of hepatitis b (hbsag) and hepatitis c (anti hcv antibodies) in multitransfused thalassemic patients. in our region there are 5 patients suffering from thalassemia major who are aged between 9 and 17, and who have been receiving erythrocytic transfusion 1-2 times a month since the age of one or two. they receive washed red blood cells, and in certain periods filtered red blood cells, controlled for viral markers and they mostly receive blood from voluntary, periodic and regular donors. the patients are tested periodically for the presence of viral markers (hbsag, anti hcv antibodies), using tests for hbsag (abbott auxyme monoclonal eia) and for anti hcv (abbott hcv eia 3.0). the presence of markers for hepatitis b and hepatitis c has not been detected in any of these multitransfused thalassemic patients who receive at least 20 transfusions a year. the tests in all 5 patients were negative. the blood used for transfusion must be tested for viral markers, and for the patients who have to receive blood for their whole life, the blood should be from voluntary, regular and periodic donors who donate blood at least three times a year, because then the risk of transfusion transmissible infections is very small. introduction: we observe yearly the prevalence of transfusion transmitted diseases following instructions of skae (national coordination haemovigilance centre). aims: to investigate the prevalence of the most important blood borne infections in our blood transfusion centre in the state achaia during the last five years (2000) (2001) (2002) (2003) (2004) . materials and methods: the detection of hbsag, anti-hcv, anti-hiv 1/2 was made by automated microparticle enzyme immunoassay (axsym, abbott) and by enzyme-linked immunoassay methods (dade behring, ortho, biomerieux) syphilis tests were made by using rpr kits. confirmation for anti-hcv positive samples was made riba or inno-lia, while the confirmation of anti-hiv1/2 positive samples was made by 'st. andrews' general hospital of patras reference centre. results: all the seropositive donors were first time donors. conclusion: (1) we observe that there is a decrease in all four infections. (2) the absence of anti-hiv seropositive donors is due to the high percentage of volunteer blood donation which approaches 70% in our centre during the last four years. methods: a prospective, one-year study has been set up in order to enrol at least 40 000 out of the estimate of 200 000 first-time donors, involving 21 blood transfusion centres from 9 of the 21 italian regions. each centre was required to enrol all first-time donors born before december 31st, 1978, and thus not included in the hbv mass vaccination campaign. the selected donors were tested for hbsag (mandatory by law) and for anti-hbc by commercial assays. all hbsag and/or anti-hbc positive specimens were stored frozen and sent to a reference laboratory for additional serological testing (anti-hbs, anti-hbe, anti-hbc/igm and anti-hbc avidity index by an experimental procedure) and for the determination of hbv-dna (both qualitative and quantitative) by real-time pcr. results: in the first 9 months of the study the 21 sites saw almost 29 000 first-time donors, of whom 70.5% belonged to the required age groups. among eligible donors, 0.4% were both hbsag and anti-hbc positive, and 10.3% were hbsag negative/anti-hbc positive. hbv positivity rates were higher in southern than in northern regions, although a high variability in hbv prevalence was observed between neighbouring areas in the north. hbsag positives were mostly males, 93% were positive for anti-hbe, 6% had raised alt and 8% were concurrently positive for anti-hbs. among hbsag negative/anti-hbc positive donors, 18% were negative and 82% were positive for anti-hbs. among anti-hbs positives, 35% showed values <100 miu/ml and 65% > 100 miu/ml. the avidity index results suggested that approximately 30% of anti-hbc positive individuals were recently infected. conclusions: our preliminary data indicate that approximately 70% of the italian first-time donors are older than 28 years of age and thus not belonging to the age groups who underwent to the mandatory vaccination against hbv, and that 10.7% of them have serological markers of ongoing or past hbv infection. anti-hbc alone was detected in nearly 2% of the study population. hbv-dna testing is underway at the time of this writing. in our country mandatory tests for each blood donations are: hbsag, anti-hcv, anti-hiv1/2 and tp ab. to c + ns3 + ns4, 9 (18.4%) to c + ns3, 4 (8.2%) to c + ns3 + ns5, 2 (4%) to ns3 + ns4 + ns5, 2 (4%) to c + ns4 + ns5, 2 (4%) to ns3 + ns5. the use of the hcv core ag elisa test system may provide substantially earlier identification of hcv infection than it is possible with current serological assays. although all of six anti-hcv assays are very sensitive and specific screening assays, they didn't detect hcv infection in one patient. majority of anti-hcv positive patients (77.5%) had anti-hcv ab for 3 or more different epitopes of hcv. international comparison of performance of abbott prism assays used for blood donor screening background: the national serology reference laboratory, australia (nrl), coordinates a quality control (qc) programme for laboratories that screen for anti-hiv 1&2, anti-hcv, hbsag and anti-htlv i/ii using the abbott prism assays. nineteen laboratories from australia, belgium, canada, ireland, israel, the netherlands, new zealand, norway, singapore, south africa and thailand have submitted data for this programme. aims: to determine the accuracy and precision of results from laboratories, individual prism instruments and different reagent lots by analysing data accumulated between october 2001 and january 2005. the multi-marker qc sample 'pelispy s2058 type 7' (s2058), produced by viral quality control (now acrometrix-viral quality control), was provided to participants. laboratories tested s2058 in each calibration run, in addition to the manufacturer's controls, on each sub-channel of the instrument. pelispy was used as a 'go/nogo control' and results were required to be reactive (s/co > 1) for a test run to be deemed valid. data were collected and analysed using the nrl's internet-based application edcnet (https://www.nrlqa.net). after submission, laboratories were able to compare their results with those submitted by other laboratories and investigate differences in results from reagent lots and instruments. data for five different s2058 lots were exported from edcnet and analysed. results: nearly 95 000 results were submitted: all results were reactive (s/co > 1). fifty of these results (0.0005%) were excluded from analyses because they were reported from invalid test runs [due to pipetting, aspiration or sampling error (n = 48) or due to unacceptable results (n = 2)]. a further 16 results were excluded because data provided by laboratories were inconsistent or incorrect. a total of 94 189 results, reported using 332 different prism reagent lots (145 for anti-hiv, 79 for anti-hcv, 55 for anti-htlv and 53 for hbsag), were analysed. results from prism hbsag and anti-hiv showed the least variation with coefficient of variations (cv) of <10% for all s2058 lots. results from prism anti-hcv and anti-htlv produced cvs between 9.17% and 15.38% for all s2058 lots. data reported for s2058 lot ps030618 (n = 38 662, range 8080 for anti-htlv to 10 225 for hbsag) were analysed further to review performance of prism reagent lots. hbsag showed the least variability between prism reagent lots with <8% bias for the 14 prism hbsag reagent lots used (bias: the difference between the mean ratio for the reagent lot and the weighted mean ratio for all reagent lots, expressed as a percentage of the weighted mean ratio for all reagent lots). prism anti-htlv showed greater variability between reagent lots with a single reagent lot generating a +63% bias. prism anti-hcv showed the greatest variability within reagent lot with results from 12 of 21 reagent lots showing a cv between 10% and 13%. conclusion: in 94 255 results in a qc sample distributed to 19 laboratories the abbott prism performance was found to be consistent over four assays. edcnet was robust in supporting laboratories' abilities to follow precision and accuracy of the assays in real time. introduction: since hcv rna testing of all blood donors started in finland in 2000, the nat screening process has continuously been improved. investments in process automation have made the work more efficient and blood safety has further increased since hiv-1 rna screening of all blood donors started late 2004. aim of the study: to implement hiv-1 rna testing in the nat screening program cost effectively, without increasing the throughput time of the samples and delay in the result reporting. to study if the sensitivity for both hiv-1 rna and hcv-rna will be sufficient when a single extraction is used and when the pool size of 96 donations and the sample volume are kept unchanged. methods: the nucleic acids were isolated from plasma samples of 1 ml with the magna pure lc instrument using the magna pure lc total nucleic acid isolation large volume kit. the internal controls from the cobas ampliscreen multiprep specimen preparation and control kit and the cobas amplicor hcv specimen preparation kit were added to the lysis/binding buffer (5 ml/ml of each). from the final volume of the nucleic acid eluate (100 ml) 50 ml was used for the detection of hiv-1 rna (roche cobas ampliscreen) and 30 ml for the detection of hcv rna (roche cobas amplicor). a run control containing both hiv-1 rna (200 iu/ml) and hcv rna (50 iu/ml) was included in all extraction runs. sensitivity of the both assays was assessed by testing dilution series of the who standards for hiv-1 rna and hcv rna. specificity was evaluated by testing fractionation plasmapool samples (n = 35) and minipool samples (n = 68). results: detection limits of the hiv-1 and hcv assays (95% hit rate) were calculated to be 100.1 iu/ml and 18.7 iu/ml respectively. specificity for both assays was 100% and during the validation phase also the robustness was good. the sensitivity of both assays with a pool size of 96 was below the recommendations by the council of europe for blood donor screening (for hiv-1 rna 10 000 iu/ml and for hcv 5000 iu/ml per individual donation). specificity of the assays was excellent, false reactive results were not observed. implementation of the hiv-1 nat assay in the screening program did not increase the throughput time of the donor samples when the pool size of 96 donations, 1 ml sample volume and a single extraction for two assays were used. the the very substantial increase in the number of industry-sponsored clinical trials has created challenges for medical schools, academic hospitals, faculty members of these institutions, and the journals that publish the results of these trials. in many cases, authors of reports of industry-sponsored clinical trials are paid consultants to the sponsor, have been paid by the sponsor to lecture on behalf of its products, or have equity in the sponsoring company. these ties to industry create a tension that actually is or can be perceived as • work internationally; • send young volunteers to international youth forums; • employ young people in your organisation; why use volunteers in blood donor recruitment? • they have networks to scout-groups, sports-organizations, tradeunions, rotary, staff of large companies etc. • they bring in fellow volunteers -with different prospects of society; • often paid recruiters are underpaid (!) and tend not to remain • you can not recruit by telephone! • 2 out of 3 donors are recruited by personal contact! • so you need direct personal contact = need many people (e.g. young ambassadors); • a large number of volunteer recruiters is a gift from heaven! paid donation gives the act of blood donation low status. the act of blood donation should be respected, and praised by role models, queens and presidents. efficient work and close cooperation of blood bank staff and volunteer organisations is the key to success in blood donor recruitment and retention! with such a prospect, it was important to evaluate the practices of blood donor selection in the eu. material and methods: a questionnaire was designed and sent in 2003 to the relevant institutions of the 15 eu countries plus switzerland. the questionnaire included questions on the interviewing practices before homologous blood donations, regarding non-specific risks to donors and recipients, identified risks to donors, infectious, bacterial, viral, parasitic and prionic risks to recipients and non-infectious risks to recipients. the questionnaire also inquired about each country's exclusion period for each contraindication (ci) to donation. results: predonation interviews were prepared, in all countries, by circulating informative documents to blood donors. they were supported by written questionnaires in nearly all countries. in half of the countries, those interviews had to be led by physicians (nurses or technologists in the others). the 18-65 age limits for blood donation (18-60 for a first donation) were the rule in 11 countries. in other countries the age limit could be brought forward to 17 and extended to 70 years old. the time interval between donations was identical for men and women in 11 countries, and varied from 8 to 16 weeks according to country. the questions of the questionnaires were very similar as regards the identification of risks to donors and recipients, and very close to the requirements that appeared later in the 2004/33/ec directive. this particularly concerned how to meet the expectations of european donors, so that they come back to your blood center! know your donors: make regular donor surveys. age, gender, number of donations, number of first time donors, media consumption, education, job situation, income brackets. use local donor organisations let volunteers help! they work for free, but donor recruitment and -retention costs money. each blood center should have a local donor organisation, run by volunteers. the donor organisation should receive a payment for each bag collected. the reputation of the blood system tell the donors, what the blood is used for. that all blood is tested. and that blood provides safe medical treatment safety of the donor. insurance is a must good quality and efficiency in blood services decentralized blood collection no waste, and minimal outdating efficient service: • a friendly environment, • donor friendly opening hours, • pleasant rooms, beds and well equipped waiting rooms, • parking-spaces, transport, • beverages and food, • letters with correct data etc. donors expect to be serviced by trained, medical professionals and that the medical check-up is taken seriously. donors should be recognized continuously. use directed press-coverage to higher the self-esteem of the donors. donors should be well informed: • leaflets, posters and questionnaires should be 100% correct; • use e-mail and web-sites for quick up-date of donor information. be visible! • have an offensive and comprehensive media approach; • have a yearly national campaign 14 june up to world blood donor day; • have an attractive home-page, constantly updated; • streamline your lay-out; • mail a donor magazine to all regular donors: • send newsletters regularly to volunteers and the press. • use recruitment cards. • easy phone-and fax numbers, e-mail addresses. directed campaign towards young people: • young ambassadors group; • special training sessions for young volunteers; • advertisements in media catering to young people; • poster competitions; • book and leaflets on blood addressed directly to young people; minimum bodyweight, blood pressure, pulse limits, questions involving viral risks, either sexually transmitted or linked to drug abuse, questions investigating risks of malaria transmission, questions aimed at identifying risk of prionic disease transmission. analyzing ineligibility times, on the other hand, revealed wide differences. for example, ineligibility for current multiple sexual partners, sexual relations with risk individuals, tattooing or body piercing, endoscopy, general anesthesia or invasive surgery, could vary from 3 to 12 months. previous transfusion history could not be a ci or could be one varying from 6 months to indefinite ci (this point recently changed in several countries). the results of that survey have revealed some differences between countries in the questions asked and especially in the ineligibility times. however, the conditions under which donor selection interviews are conducted were similar in all countries. the enquiry tool used in this study proved to be well adapted to evaluate the donor selection practices throughout eu. a next step will be to use it to appreciate their evolutions and especially the impact of the 2004/ec/33 directive on these practices in the 25 eu countries and furthermore to evaluate the results of this selection (rates and motives of deferral) which is a major factor of patients' transfusional safety. background: in europe on average 43 whole-blood donations are performed per 1000 inhabitants and year. whole-blood donation comprises a puncture of a venous vessel and letting of blood (usually 450 ml), which may be repeated several times a year. like other invasive procedures, blood donation has a range of effects on the individual who is subjected to it. aim: the aim of this paper is to review some aspects of the present state of knowledge on effects and complications of whole-blood donations. results: most studies of the effects of whole-blood donations on the donor have focused on negative or unpleasant events and on time in rather close association to the donation. complications related to percutaneous needle insertion (bruise assessed by inspection 0.66% -bruise assessed using post-donation interview 22.7%, sore arm 0-10.0%, nerve irritation 0.02%, arterial puncture/pseudoaneurysm/arteriovenous fistula 0.003-0.009% etc) are most commonly reported, while negative systemic reactions (vasovagal reactions 2.5-7.8%, syncope 0.08-0.34%) occurring in connection to blood donation are less frequent (newman bh: blood donor complications after whole-blood donation. curr opin hematol 2004; 11: 339-345.) serious complications requiring hospitalization (myocardial infarction, stroke) are extremely rare (0.0005%). fatigue (7.8%-10.0%) and diminished physical working capacity (7.0%) are reported to occur during days after the donation. a recent study of a consecutive sample of 528 swedish blood donors (nilsson sojka b, sojka p: the blood-donation experience: perceived physical, psychological and social impact of blood donation on the donor. vox sang 2003; 84: 120-128.) (with a selfadministered questionnaire with an open-ended question: how does blood donation affect you? physically-bodily/psychologicallyspiritually/ethically-morally/socially during or after blood donation?) revealed that perceived negative effects (fatigue, diminished physical working capacity, vertigo/dizziness, susceptibility to infections etc) were less common (25% of the donors) than positive effects (feeling of satisfaction, being more alert, feeling generally better, less migraine, higher physical working capacity, respect from environment, feeling of relaxation etc; 35% of the donors). the duration of positive effects was regularly reported to be weeks, while negative effects lasted only days. investigations on the long-term effects of blood donation are scarce. yet, they may indicate that the donation of blood is associated with e.g. lower blood pressure and a reduced risk of myocardial infarction (nilsson sojka b, sojka p: the blood-donation experience: perceived physical, psychological and social impact of blood donation on the donor. vox sang 2003; 84: 120-128). conclusion: both the panorama and the frequency of occurrence of the different effects and complications of whole-blood donations vary as a function of how the information was gathered (openended questions, observations, interviews etc). serious reactions to whole-blood donations are extremely rare. more studies are needed in particular with respect to the long-term effects of regular wholeblood donations. t-pa-056 establishing a national adverse event reporting system for blood donors -a prospective study of 1. there was no national system and significant regional variation showed that the data was scientifically unsound. a coincidental initiative to remove the mandatory post donation rest period for regular donors further emphasised the lack of reliable, retrospective data to monitor and compare the impact of this new policy. aims: 1. to develop and implement a high quality, reliable national donor adverse events reporting (daer) system; 2. to define and categorise adverse events; 3. to record data systematically and prospectively using the existing computerised donor database. methods: from summer 2002, a small project team of senior clinical and operational staff took 9 months to agree a detailed policy for capturing and recording all donor adverse events, including precise definitions for grades of vasovagal reactions and bruising. detailed training material was written in may 2003 and the new protocol was validated in one region. from july to december a formal one day training programme was delivered to over 3000 staff working on 98 mobile collection teams, 19 static sites and 11 blood centres. daer was fully implemented by january 2004. adverse events are assessed by health care professionals on session and the relevant code entered onto the donor's computer record by clerical staff. information received after the session is entered by centre based doctors using the same system. the database is interrogated monthly for statistics. results: in the first 9 months 1.86 million donors attended sessions throughout england and north wales. results were very consistent month on month. 25 175 donors (1 : 75) had vasovagal symptoms but only 14% of these suffered syncope. 71% of all vasovagal reactions occurred in women. 21% occurred in donors aged 17-20 and a further 30% in donors aged 21-30. (donors aged 17-30 represent only 25% of the total donor base.) 831 donors (1 : 2250) reported a delayed reaction, 52% of whom did lose consciousness. nerve injury, unrelated to haematoma, occurred in 109 donors (1 : 17 000) and, more rarely, arterial puncture was diagnosed in 46 donors (1 : 40 000). bruising was reported after the session by 1 : 1300 donors. summary: a robust system has been developed and successfully implemented across a large, national blood service. based on the data already accumulated our next phase is to develop strategies to minimise adverse events, confident that any intervention will be effectively monitored. the community role in enhancement of voluntary, non-remunerated blood donation in the new millennium introduction: in the developing countries, about 80% of the blood supply comes from paid or replacement donors, where a high number of infected persons are in the donors population. only 16% of the global blood supply is donated as voluntary nonremunerated blood donors in countries with low and medium human development indices. and around 80% of the global blood population has access to only 20% of a safe blood supply. conclusion: blood is a national resource, it is the responsibility of governments through it's communities to ensure that the blood supply is safe and adequate to meet the needs of patients population and available to all who needs it. background: in response to a documented increase in the average age of donors, a survey was conducted to explore if young people had more unfavourable attitudes towards becoming blood donors. aim: to identify if the increasing difficulty in recruitment and retention of young people as donors, is linked to a low level of motivation for donating blood in this age group. methods: a national telephone-survey was conducted among a cross-sectional sample of the adult norwegian population (1000 participants). the survey was performed in november 2004. results: five percent reported being active donors (had donated during the last 12 months), 17% were passive donors (had not donated during the last 12 months), 22% were non-donors with a positive attitude towards becoming donors, and 55% non-donors with no intentions ever to donate blood. in the youngest age group (age 18-29), 1% reported being active donors and 8% were passive donors. however, 36% of the young non-donors reported having intentions of becoming a blood donor. fifty-five percent of young non-donors had a negative attitude towards ever donating blood. all non-donors were asked why they did not donate. thirty-six percent of all non-donors reported health related reasons for not donating blood. thirty-one percent of all non-donors claimed that they did not donate because no one had requested them to do so personally, and 4% reported they did not care about blood donation. in comparison only 28% of young non-donors reported medical reasons for not donating. thirty-eight percent claimed lack of personal request, and 7% reported of lack of interest as the main reason for not donating. summary/conclusion: although the youngest age group was under-represented among active donors, we found that a great proportion (36%) of young non-donors had a positive attitude towards becoming blood donors. the most important reason why young people do not donate was the lack of a personal request. indifference regarding donation was not very widespread. a relatively high proportion of young people considered themselves as not medically disqualified to donate. in light of these findings, efforts to recruit young people as blood donors are strongly recommended. background: the ever-increasing demand for blood, coupled with emerging new threats to blood safety, motivate the strengthening of the blood banking infrastructure. aim: employing new technology as an instrument for building relationships of trust between blood bank and blood donors. material: 1. a new software module supporting the management of magnetic cards was added to e-aima blood bank management application. the magnetic card supports the following information: • front-side: donor's photograph, surname, name and blood group (including rhesus phenotype & kell) and sign of blood collection centre. • magnetic stripe where data concerning donor's serial number, medical history (risk factors), test results for infections and the number of donations is stored. 2. specific hardware allowing reading from and writing to magnetic cards is integrated to the software module: • magnetic card scanners were added to pcs serving to donation collection, including laptop for mobile team collection. • web cameras to capture the photograph of the donor to be printed on the card. • card printer was deemed necessary to produce magnetic cards for donors on a need basis. 3. consumables: blank plastic magnetic cards, ink cartridges for printer. methods: in order to evaluate the performance of magnetic cards compared to the paper-based system, a questionnaire was distributed to 300 first-time and 200 repeat blood donors, in order to be used as an indicant of donor's satisfaction. 1. first-time donors increased 7.8% in the 6 months of application. among them, 4.3% were donors 'for relatives or friends' turned into volunteer donors and 3.5% were first-time volunteer donors. the questionnaire analysis further revealed: • 96% were motivated by the use of magnetic card. • 92% appreciated the presence of their photo on the card and they confessed that they had used it as a spill for recruiting their friends as donors. • 100% were persuaded that employing new technology would result in safer and more trustworthy procedures combined with reduced waiting time. • 100% considered magnetic cards more practical compared to paper cards because of their compact size and improved durability. 2. the turnover of repeat donors also increased 1.3% after replacing their plain old paper cards with new ones. further analysis revealed that: • 100% appreciated the quick cross-checking of donor's identity. • 100% were satisfied with the effectiveness and efficiency of magnetic cards in managing donor's data. conclusions: in 2004, greek health policy provided the legal basis for establishing the electronic national health card. the introduction of the national donor's magnetic card is another step towards this direction, being aligned with the modern national health strategy. apart from the positive impact on the number of both firsttime and repeat blood donors, it should be also pointed out that the use of a unique donor serial number on country level results in less error-prone procedures due to the reduction of administrative process overhead and facilitates interoperability between national blood banks using compatible technological infrastructure. t-pa-060 emerging technologies in transfusion. dna based assays until the late 1990s, mandatory blood screening for transmissible infectious agents depended entirely on antigen/antibody-based detection assays. recent emergence of nucleic acid technologies (nat) has revolutionized viral diagnosis by not only increasing the sensitivity level but also facilitating the detection of several viruses in parallel, by multiplexing specific primers. however, in more complex biological situations when a broad spectrum of pathogens must be screened, the limitations of these first generation technologies became apparent. high throughput systems such as dna arrays permit a conceptually new approach. these miniaturized microsystems allow the detection of hundreds of different targets simultaneously, inducing a dramatic decrease in reagent consumption, in additional confirmation tests and simplify data interpretation. however, the microsystems actually available require additional instrumentation and reagents for sample preparation and target-amplification prior to detection on the dna array. future technologies such as 'lab-on-a-chip' include channels, fluidics and thermal zones allowing extraction, amplification and detection. another major challenge in the area of dna detection is the development of methods that do not rely on target-amplification systems. almost all blood group antigens are bi-allelic and encoded by single nucleotide polymorphisms (snps). to facilitate the direct availability of typed red cells and platelets, we develop a high-throughput technique to genotype by dna microarray the whole donor cohort for all clinically relevant red cell and platelet antigens. methods: a multiplex pcr was developed to both amplify and fluorescently label 28 gene fragments of red cell and platelet antigens in one reaction. each array contains spots of short (17-27 nt) allelespecific oligonucleotides to discriminate between the two alleles of an antigen system. results: two blinded panels encompassing 94 donors were genotyped for hpa-1 through -5 and 15; no discrepancies were found. currently, arrays are prepared for the red cell systems. the fya/fyb, fy-gata1 mutation, jka/jkb, k/k, kpa/kpb, m/n, rhc/c, rhe/e, rhdpseudogen, rhdvi negative, rs, doa/dob, genotypes can be determined. the set up of genotyping assays for rare genotypes is difficult because of lack or insufficient amount of dna. the latter can be overcome by phi29 dna polymerase-mediated isothermal genomic dna amplification, from minute amounts of dna present in stored red cell fractions or antiserum. the results show that the blood group typing dna microarray will provide a reliable and fast genotyping procedure. the method can be further improved to obtain the necessary automated throughput for typing of large donor cohorts. and 15, all other weak d types should be regarded as potential anti-d immunizers. for correct determination of weak d both serological typing (polyclonal and monoclonal), as rhd dna typing are mandatory. when serology indicates weak d, more anti-d antibodies are tested (9 epitope model) to distinguish partial d from weak d. in addition, an rhd mpx pcr is performed to detect the presence of rhd exons 3, 4, 5, 6, 7 and 9. in all known weak d types, all six rhd specific exons are amplified (except for weak d type 4 which lacks rhd exons 4 and 5), whereas partial d phenotypes usually show aberrant patterns. aim: the aim of this study was to evaluate the diagnostic scheme for weak d typing. methods: between 2002 and 2004, 24 samples were investigated for weak d characteristics. four pcr-ssp assays were developed for identification of weak d types 1 (809t > g), 2 (1154g > c), 3 (8c > g) and 5 (446c > a). weak d type 4 was identified by the combination of serology and absence of exons 4 and 5 by rhd mpx pcr. rhd-specific exon sequencing was performed when serology and molecular typing were inconsistent. results: all 24 samples were subjected to the rhd mpx pcr and 1 sample showed absence of rhd exons 4 and 5, indicative of a weak d type 4 when combined with serology. the remaining 23 samples were analyzed by the weak d pcr-ssps, resulting in 12 weak d type 1 samples, 4 weak d type 2 samples, 4 weak d type 3 samples and 1 weak d type 5 sample. two samples remained undetermined and were sequenced for all 10 rhd exons and the rhd promotor region. one sample showed the mutations corresponding to the dau 2 partial d phenotype (209g>a, 998g>a and 1136c>t). the other sample had only one, not previously known mutation (1193a>t), which is located intracellularly at the coohtail. extensive serology using the 37 epitope model showed a pattern matching weak d. this new weak d variant was registered as weak d type 41. conclusions: based on these results it may be concluded that weak d phenotypes should be confirmed on molecular level to avoid misinterpretation of partial d that cannot be detected by rhd mpx pcr analyses. patients with weak d phenotypes, except for types 1, 2 and 3 should be regarded as being at risk for anti-d immunization after transfusion of rhd-positive blood products and should therefore be treated with rhd-negative bloodproducts. in this evaluation, 4 out of 24 patients carried such alleles. introduction: although kell antigens are expressed very early during erythropoiesis and a 0.1% incidence of anti-kel1 is found in obstetric patients, this is a relatively rare cause of hdn. anemia is produced by immune destruction of fetal rbcs and suppression of erythropoiesis. maternal antibody titers or amniotic/cord blood bilirubin levels are not relevant indicators of the severity of the disease, and the measurement of the fetal haemoglobin by cordocentesis is a procedure with risks of miscarriage and sensitization. pcr techniques for the determination of blood groups using fetal dna isolated from maternal plasma, allows the application of noninvasive methods. clinical cases: we describe two cases of pregnancies in women with anti-kel1 acquired by transfusion/previous pregnancies: 1st case: in july 2000, a 30-year-old woman (gravida 2, para 0), rhdnegative, kel1-negative was referred at 20 weeks gestation. the father's phenotype was rhd-positive, kel1-positive. a maternal antibody screen revealed d and kel1 alloantibodies. dna was extracted from amniotic liquid. the kel genotype was determined by pcr-rflp using the bsm i. pcr-ssp was used to studied intron 4 and exon 7 of the rhd gene. the results showed that the fetus was positive for rhd sequences and showed kel2 homozygosity; 2nd case: in august 2004, a 36-year-old woman (gravida 2, para 1) was referred at 28 weeks gestation. she had a history of transfusion with 2 rbcs units in 1996 -one of the donors was kel1positive. the woman typed rhd-negative and her husband typed rhd-positive. rbcs from both were kel1-negative. the maternal antibody screen revealed anti-kel1. doubts existed about the putative father of this child. dna was extracted from maternal plasma using the magna pure lc (roche). real-time pcr was applied to analyse: sequences of intron 4, exons 4, 6, 7 and pseudogene of the rhd gene and the sry gene by sybr green; and the alleles kel1/kel2 by hybridization probes. all rhd sequences were detected (with the exception of the pseudogene) and the kel genotype gave a kel2/kel2 result. in both cases the doctors choose not to use any invasive method to monitoring the fetuses regarding a hdn due to anti-kell antibodies, and the results of the molecular analysis were confirmed by testing the cord rbcs after birth. discussion: these cases illustrate the reliability of the molecular biology results, based on the collection of simple peripheral blood samples. a determination that the fetus lacks the relevant antigen obviates the need for expensive and invasive monitoring throughout the pregnancy. evidence-based medicine (ebm) is defined as: 'the conscientious, explicit and judicious use of current best evidence in making decisions about the care of individual patients. the practice of ebm requires the integration of individual clinical expertise with the best available external clinical evidence from systematic research and our patient's unique values and circumstances. ' otherwise healthy individuals without cardiopulmonary dysfunction (cdf) tolerate acute reduction of haemoglobin concentration to about 5 g/dl, provided that blood volume is kept normal by a volume expander. however, individuals experience physical fatigue, and there is faint reduction of perception as measured by neurophysiological tests. symptoms are reversed upon retransfusion of fresh, autologous erythrocytes. acute, normovolemic anemia seems to be progressively less tolerated with increasing age and cdf. controversy has existed on whether or not to correct hypoalbuminemia in asb or icp by infusion of albumin. recently a large trial showed no outcome differences between icp patients treated with albumin or saline. thus in general there is no indication for albumin in asb or icp. however, albumin may yet be advantageous in e.g. patients with head injuries. furthermore, fractionated albumin is not equivalent to native albumin, since fractionation stabilizers remain bound to the albumin molecule. thus more refined albumin preparations may carry advantages still to be investigated. erythrocytes are given to increase the total oxygen transportation capacity of the organism. the effect of blood bank stored erythrocytes may differ from that of fresh, autologous erythrocytes, since changes of presumably important erythrocyte properties occur during storage. in the only large trial available, a transfusion trigger of 7.0 g/dl was found to be favourable to one of 10 g/dl in icp, except possibly in icp with unstable angina pectoris or heart infarction. however, the erythrocyte concentrates given were not leukocyte filtered, and side effects of infused leukocytes may have hampered the conclusion. on the other hand, a metanalysis showed transfusion as an independent indicator of unfavourable outcome in coronary bypass patients, but again, leukocyte filtered erythrocyte preparations were not applied. the effect on morbidity and mortality of 'top up' transfusions given e.g. to mobilize patients postoperatively has not been studied by trials, although this effect seems evident to many clinicians. grave anemia may reduce the haemostatic effect of thrombocytes because changes of blood rheology reduces the pressure forcing thrombocytes against the walls of small vessels. the transfusion trigger for thrombocytes in asb or icp remains to be established by clinical trials, however. the same applies for fresh frozen plasma, which is infused as a source of coagulation factors. on the other hand, the haemostatic effect of various fibrinolysis inhibitors is well established in asb and icp, but many clinicians appear hesitant to use them. another interesting haemostatic agent is recombinant fviia, the use of which to control asb in blunt trauma is supported by one well controlled clinical trial. evidence by systematic research is insufficient to decide what is optimal transfusion practice. the procurement of such evidence is one of the greatest current challenges to transfusion medicine research. . concerns about the transfusion-related complications, such as infections, tumour behaviour and immuno-modulatory effects, and the costs, necessitated a re-evaluation of the transfusion practice. aims: the goal of this study is to evaluate if a restrictive transfusion policy (hb transfusion trigger <4.5 mmol/l) reduces the amount of red cell transfusion compared to a liberal transfusion trigger (hb < 6.0 mmol/l) without a decrease in hrqol. because of concerns about the feasibility of this study early results were analysed and are presented in this abstract. material and methods: after a run in period of 3 months (hb transfusion trigger of hb < 6.0 mmol/l) patients are randomised for the restrictive or the liberal transfusion policy. patients are followed then 12 months. hrqol is measured after inclusion, after randomisation, 6 weeks, 3, 6, 9, and 12 months after randomisation. also anaemia related complications and red cell antibodies are scored. hb values were blinded for the patients during the study period. results: from july 2002 till june 2004 15 patients were included (4 ra, 5 rars, 4 rcmd, 1 raeb, 1 cmmol) in 2 general hospitals and 1 university hospital. two patients died in the run in period. eight patients were randomised for the restrictive transfusion policy and 5 patients for the liberal transfusion policy. the mean follow up period in the liberal group was 6.2 months (inclusive run in period) and 7.4 months for the restrictive group. two patients in the liberal group died after randomisation. one patient received growth factors. in the restrictive group 2 patients finished the study, 1 received growth factors and 1 patient withdrew informed consent. the mean hb level was lower in the restrictive group and after randomisation about 55% reduction in amount of transfused red cells was found (1.8 units per pt per month in the liberal group vs 0.8 in the restrictive group). no anaemia related complications were found, e.g. cardiac failure and cerebro vascular ischemia nor a decrease in activity performance. conclusion: there were some concerns after introduction of the restrictive transfusion policy. this preliminary results show, however, that a restrictive transfusion policy leads to a diminished use of red cell transfusion without an increase of cardiac complications or a decrease in activity performance. this study will be continued to compare hrqol scores in both groups. introduction: strong evidence supports the efficacy of blood conservation strategies such as autologous blood donation (abd) and erythropoietin (epo) for reducing exposure to allogeneic blood. however, use of these interventions is highly variable among institutions and frequently sub-optimal. the program to reduce orthopedic blood exposure (probe) evaluated a blood conservation program in patients undergoing total hip joint arthroplasty (thja) at ontario hospitals. aim of the study: the objective of probe was to determine whether a comprehensive blood conservation algorithm (bca) was more effective than usual care (uc) for reducing exposure to allogeneic blood in patients undergoing thja. methods: we randomized 30 hospitals that perform high volume elective primary thja to implement either a bca or to continue with uc. the bca consisted of three components: physician and patient education, blood conservation interventions (use of abd or epo), and transfusion guidelines. t-pa-071 table) . mortality for non-transfused patients was significantly lower than for patients receiving either lr-or s-prbc at all time points (p < 0.0001). t-pa-074 thalassaemia: the impact on blood transfusion services thalassaemia major is a genetically determined disease that causes severe chronic anaemia and further complications. it can be managed successfully in the vast majority of cases, so long as public health and other scientific and organizational infrastructures are adequate. although the progress achieved in the field of bone marrow transplantation and other disciplines promises cure of the genetic defect, regular blood transfusion from early childhood remains the cornerstone of treatment of patients with thalassaemia major. this presents national health authorities with the formidable task of assuring an adequate blood supply of high quality and safety for these patients and ensuring that it is transfused in the appropriate way. the basic principle in the modern management of thalassaemia patients is that of a global approach to care. within this approach, a standardized protocol for regular blood transfusions is a prerequisite for the patient's long survival and quality of life. if thalassaemia patients are not transfused effectively, the severe anaemia and over-expansion of bone marrow due to ineffective erythropoesis can lead to poor growth, bone deformities, organomegaly and impairment of normal physical activities. in countries or regions with large numbers of thalassaemic patients, the organizational and technical aspects of meeting their blood requirements represents a heavy additional workload for the blood transfusion services responsible for providing blood for this group of multi-transfused patients. the acquisition and preparation of blood, genotyping the patients' blood group (including at least rh, kell, kidd and duffy systems) preventing the transmission of infectious diseases and other transfusion associated complications, and assessing the patients' blood transfusion indices all have a tremendous impact on blood transfusion and treatment units. blood transfusion services are thus confronted with major challenges that can only be met if appropriate national transfusion policies are in place, both in the laboratory and the clinical setting of blood transfusion. the availability of safe blood is related to the effectiveness of donation programmes aimed at recruiting and retaining voluntary unpaid blood donors who are at low risk for the transmission of infectious diseases. sufficiency is further related to resources, organization and management of the blood transfusion service and continuous education of its staff. high technical standards for the transfused product and quality management systems are required to ensure that the product meets these requirements, as well as pre-transfusion, transfusion and haemovigilance systems and other more stringent quality measures in the whole chain of blood donation and transfusion. additional measures and continuous care are specifically required for the optimal transfusion therapy of the thalassaemic patient. the patients should be transfused with red cell concentrates, (rccs) preferably not more than one week's old and leucodepleted. other processes i.e washing of rccs, use of nutrient additive solutions, irradiation etc may be used to improve the quality and the safety of the transfused product, while other advances in red cell transfusion are expected to improve blood safety by preventing adverse reactions and reducing exposure of the patient to donor blood. should patients with thalassemia intermedia be regularly transfused? thalassemia major (tm) and thalassemia intermedia (ti) share mostly a common basic molecular mechanism, that is the reduced synthesis of the * globin chains. 1 the consequences of the resulting chronic hemolytic anemia are also common and include growth retardation, bone marrow expansion, extramedular hematopoiesis, splenomegaly, increased intestinal iron absorption, susceptibility to infections and hypercoagulability. what differentiates the two forms of the disease is the severity of the clinical phenotype, which in turn depends on a particularly heterogeneous molecular background and imposes diverse therapeutic strategies. the consequences of the genetic defect as well as the effect of the applied therapy seem to be mainly responsible for the clinical course of the disease. in untreated tm cases, the aforementioned consequences occur fast and patients die early in life mainly due to high output heart failure. over the past decades, the gradual adoption of the current transfusion and iron chelation strategies and the patients' compliance with this therapy have resulted in a significant improvement of survival, that according to recent statistics reaches 68% at age 35. this rate is even better in well-treated patients, almost 80% of whom survive at age 40. regular therapy extends survival mainly by preventing early development of cardiac complications. in addition, a multi-organ improvement is accomplished while patients' physical appearance is almost indistinguishable from that of the general population, hence permitting a normal social behavior with a high overall quality of life. bone marrow expansion and extramedular hematopoiesis are prevented; hepatosplenomegaly is substantially restricted and usually there is no need for splenectomy, while thromboembolic complications are rare and pulmonary hypertension is practically absent. patients with ti remain as a rule without regular therapy until a number of severe complications arise. the consequences of chronic anemia develop slowly compared to untreated tm cases and dominate patients' clinical picture usually by the third decade of life. at this time, all patients have developed hepatosplenomegaly and most of them have been splenectomized. bone marrow expansion results to bone deformities and fractures often occur. extramedular hemaotpoietic masses and bone deformities may lead to various complications depending on their bulk and location, such as neurological symptoms from masses arising in the paraspinal area or dyspnea from lung restriction. hypercoagulability, resulting from defects of native erythrocyte membrane phospholipids, together with the coexistent thrombocytosis in splenectomized patients lead to a wide spectrum of thromboembolic events. pulmonary involvement with respiratory dysfunction and hypoxemia as well as pulmonary hypertension leading to congestive heart failure are well documented in ti patients. nowadays, the beneficial effects of regular transfusion and chelation therapy in tm are beyond any doubt. the occasional application of transfusions in ti has a transient effect and does not seem to inhibit the consequences of chronic hypoxia. intensive and regular transfusion and chelation therapy in ti has proved effective in ameliorating the established complications such as spinal cord compression, hypercoagulability and pulmonary hypertension, without however reversing them. given the 30-year experience on intensive therapy in tm and the first encouraging data in ti, the earlier application of such treatment seems to be crucial in ti. the timing however of therapeutic intervention in ti in order to prevent anemia-related complications still remains an open issue that needs to be properly addressed. the impact of prestorage leucodepletion on the immediate transfusion adverse events of patients with thalassaemia major backround: regular blood transfusion therapy in patients with bthalassaemia major decreases the complications of anemia but it is associated to many immediate and delayed side effects. febrile non haemolytic transfusion reactions (fnhtr) are common complications due to alloimmunization of recipients against hla and/or specific antigens on donor's wbcs or to the accumulation during storage of biologic response modifiers (bmrs) that are directly pyrogenic or indirectly by stimulating recipients' white cells to produce pyrogenic mediators. post storage leucoreduction (lr) has reduced the fnhtr in these patients from 13% to 0.5% per unit. it is unknown whether introduction of prestorage leucodepletion (ld) has reduced the incidence of nhftr further. we analyzed the immediate transfusion reactions of 175 adult patients with b-thalassaemia major transfused with a total of 32.990 rbc units from january 1998 to december 2003. all units were fresh, stored less than 7 days. 11.950 units were lr-rbc, 8.370 units were ld-rbc and 12.670 were washed lr-rbc. results: the incidence of fnhtr and allergic reactions in patients receiving lr-rbc was 0.16% and 0.35%/per unit respectively, in those receiving ld-rbc was 0.10% and 0.36%/per unit respectively, while in those receiving washed lr-rbcs was 0.10% and 0.25%/per unit respectively. the relative risk (rr) of fnhrt and allergic reactions following transfusion of ld-rbc and washed lr-rbc compared to lr-rbc is shown in table. conclusion: prestorage leucodepletion and washing of rbcs reduced the risk of fnhrt in regularly transfused b-thalassaemia patients 1.6 times compared to poststorage filtration. these findings show that fnhtr after rbc transfusions are due not only to alloimmunization but also to accumulation of bmrs even in patients transfused with fresh rbcs. washing is as effective as prestorage leucodepletion in reducing fnhtr. prestorage leucodepletion has no effect on allergic reactions or other immediate adverse events in these patients. t-pa-078 viral inactivation/elimination of plasma derived medicinal products the safety of medicinal plasma products (mpps) relies on a whole range of measures from the quality of the source material to the release of the products after manufacturing under cgmp conditions. viral safety relies on careful donor selection, viral testing of the source material and viral inactivation and/or elimination during the manufacturing process. mpp manufacturing processes must include viral safety steps capable of inactivating, and/or eliminating, a large range of viruses covering the known blood borne viruses as well as anticipating possible future pathogens. it is recognized that one single step is often not sufficient to satisfy this requirement and manufacturing processes very often include two or even three complementary viral safety steps. very efficient methods have been implemented by manufacturers, for two decades, for the inactivation of major blood borne enveloped viruses (hiv, hcv and hbv). additional safety steps have also been introduced to provide a second step for enveloped viruses and to extend the efficacy to nonenveloped viruses (hav and parvovirus b19). pasteurisation (liquid heat treatment at 60°c) which has been historically used for viral inactivation of albumin solutions has been applied to some other plasma products. solvent-detergent (sd) treatment which is specific to enveloped viruses is used primarily for coagulation factors. since the introduction of sd-treated products, no hiv, hcv or hbv transmission has been reported. viral inactivation of coagulation factors can also be achieved using various conditions of dry-heating. acidic treatment is also an efficient means of inactivating viruses in igg products. nanofiltration using filters of less than 50 nm pore size was introduced in the early 1990s. this technique for viral elimination is based on the size of the agent and is independent of their resistance to other treatments. this property could be helpful in cases of new emerging pathogenic agents. new inactivation tech-niques are currently under development such as uv treatment or gamma irradiation with efficacy reported on enveloped as well as non-enveloped viruses. these new techniques can complement existing methods after careful validation that they do not have harmful effects on proteins in the product. the efficacy of existing techniques is well documented in controlled clinical studies and pharmacovigilance records. their application to each product is extensively validated at laboratory scale, according to international regulations and then carefully evaluated by health authorities. in this context, a recent european guideline established a viral risk assessment model to quantitatively estimate the theoretical safety margins of mpps, by taking into account the different safety measures, such as viral testing of plasma and the efficacy of viral inactivation/elimination steps. whilst technical limitations and some lack of scientific data lead to very conservative estimates, this model gives an overall assessment of the efficacy of the measures in the manufacture of a given product. developments in viral inactivation/elimination methods, in plasma testing as well as in evaluation procedures have together given mpps an excellent level of safety never previously achieved. to date the important safety measures needed to ensure a high safety margin to pooled plasma products are well understood by the plasma fractionation industry. safety nets rely on carefully done: donor selection to exclude high-risk donors, serological and nat viral testing of single donations and, pooled plasma testing using sensitive validated methods, and most particularly, efficient viral reduction treatments that must be validated and implemented at a large-scale following good manufacturing practices. over the last 25 years, successive key breakthrough in plasma product viral safety have included the use of solvent-detergent treatment to inactivate lipid-enveloped viruses, and nat testing of starting plasma pools and viral nanofiltration of products to reduce the risks associated to small non-enveloped viruses. the excellence of the system currently in place is illustrated by the demonstration that these safety barriers have virtually stopped the transmission of known viruses and avoided that of 'emerging' agents, such as west nile virus (wnv). however, multiple viral reduction treatments have generally decreased product recovery. in addition, although the implementation of viral reduction treatments have forced fractionators to introduce significant changes to product manufacturing methods, this period has been understandably followed by a period of relative conservatism of the plasma fractionation industry against further process changes. as time evolves and market dynamics changes struggle for improved economic balance of the plasma product industry is putting product recovery and diversified product portfolio at the forefront of r&d objectives. these developments in the plasma fractionation scene of the western world have been taking place in a context where many patients in the developing world are still treated with sub-standard, non-virally inactivated crude plasma fractions. in this specific area, one can expect that the developing world will bring innovative thoughts and take actions to find ways to improve the quality and safety of their own local plasma product supply. safety strategies adapted to the infrastructure and economy of less solvable countries may have to be considered. the intercept blood system for plasma uses a synthetic psoralen, amotosalen hcl, and long-wavelength ultraviolet light to photochemically inactivate a broad spectrum of bloodborne pathogens in plasma intended for transfusion (intercept plasma, i-ffp). phase 3 clinical trials have shown that i-ffp retains proteins necessary for hemostasis in the treatment of acquired and inherited coagulopathies, and in support of therapeutic plasma exchange for ttp. a prototype plasma processing set was used for the clinical trials. for commercialization, a new processing set has been developed to improve productivity. the prototype set accommodated approximately 250 ml of plasma, whereas the improved set accommodates up to 635 ml of plasma, resulting in up to three i-ffp doses per treatment. aims: this study was designed to characterize pro-and anti-thrombotic proteins in i-ffp prepared using the improved processing set. proteins of interest included components of the intrinsic and extrinsic coagulation cascade, the fibrinolytic pathway, the contact factor pathway, and the complement system, the vonwillebrand complex, endogenous inhibitors, and markers of thrombin generation. methods: six fresh jumbo (600 ml) apheresis plasma units, collected using the haemonetics pcs device (gambro), were photochemically treated. sodium citrate was used as the anticoagulant. plasma samples for analysis were collected before and after photochemical treatment, and were frozen below -60°c until batch analysis. standardized clinical assays were used for all analyses. results: (results in the table below are expressed as the percent activity in i-ffp in proportion to the activity in plasma before treatment [mean ± sd]). retention of procoagulant factors in i-ffp plasma ranged from 77% to 92%. factor viii and vonwillebrand factor activity, antigen, cleaving protease activity (vwf : cp, adamts-13), and multimeric composition remained within normal ranges after treatment. endogenous inhibitors of coagulation were retained 93% to 100%. plasminogen and alpha 2-antiplasmin were retained 94% and 78%, respectively. retention of contact factors was variable; some factors were below the reference range prior to pct. with the exception of tat, all markers of coagulation activation were well within normal ranges. the tat level in one i-ffp unit was slightly above the normal range; all other units had tat levels that were well within the normal range. the significance of this is unclear. cept plasma is similar to conventional plasma. the improved processing set, intended for commercialization, allows up to 3 doses of i-ffp to be produced from a single photochemical treatment. background: guidelines of the european directive 2004/33/ec require that fresh plasma prior to freezing contains <6109 residual rbcs per litre. this rbcs content is below the sensitivity limit of the automated cell counters used in routine laboratories. aim of the study: it was therefore essential to make available an alternative method to detect and quantify rbcs in plasma. we implemented a method by flow cytometry using a pe conjugated anti-glycophorin a (gpa) monoclonal antibody that recognises rbcs and erythroid precursors. to quantify residual rbcs in fresh plasma, the method uses the same trucount test tubes (becton dickinson) as those used to quantify wbcs and that contain a known number of fluorescent beads. after addition of plasma and pe-gpa antibody, cell counting is performed on flow cytometer (bd facscalibur). validation of the method: assessment of accuracy, linearity, and reproducibility with 2 different pe-gpa antibodies (immunotech and pharmingen) application of the method: quantification of rbcs in 105 fresh plasmas divided into 3 groups: group 1: 45 plasmas from leucoreduced whole blood, group 2: 30 plasmas from packed cells after removal of buffy coat and specific filtration, group 3: 30: apheresis plasmas. results: validation of the method: for both anti gpa antibodies, detection threshold is 0.05 ¥ 10 9 residual rbcs/l; linearity study with concentrations of 0.1, 0.25, 0.50, 1.0, 1.5, 3.0, and 6.0 ¥ 10 9 rbcs/l showed excellent correlation between observed and expected values (r > 0.99); reproducibility study showed c.v of respectively 5.7% and 6%. for values >2 ¥ 10 9 rbcs/l, it appeared necessary to introduce a correction factor of 1.3 for the anti gpa pharmingen. quantification of rbcs in plasma: group 1 (plasma from leucoreduced whole blood): 0.39 10 9 ± 0.23 rbcs/l; group 2 (plasma from packed cells after removal of buffy coat and filtration): <0.05°¥ 10 9 rbcs/l in all the cases; group 3 (apheresis plasma): <0.05°¥ 10 9 rbcs/l in all the cases. conclusion: quantification of rbcs in plasma by flow cytometry is a precise, quick and reproducible test. in addition this study shows that even if there are differences in residual rbcs counts according to the origin of plasma, the obtained values are much lower than regulatory requirements. 1. improving basic transfusion knowledge amongst health workers; 2. improving pre-operative preparation for surgery 3. strategies such as cell salvage autotransfusion combined with a conservative transfusion strategy for the use of allogeneic blood. my presentation will outline the approach adopted to ensure that the use of cell salvage autotransfusion both improves the use of allogeneic blood and preserves allogeneic stores. well-organised training can both minimise the risk of using such advanced techniques and decrease the overall risk involved in undergoing surgery where blood loss may be a significant factor in increasing morbidity and mortality. the various training methods employed to improve knowledge in this area will be described. the increasing current perception that the safety of allogeneic blood transfusion has dramatically been improved during the last decade is challenging autologous haemotherapy methods. in addition, growing concern about the unfavourable cost-effectiveness of most autologous haemotherapy methods requires a refinement of the application of these measures to well defined circumstances. in contrast, newly emerging transfusion-transmissible infections or periods of blood shortage might revive interest in these blood sparing techniques. the first two cases of transfusion transmitted vcjd provide a paradigm for this scenario; not so much with respect to a public fear of infection but rather a waning donor population due to more rigorous recruitment criteria. preoperative autologous blood donation (pabd) still plays a significant role in settings with high individual benefit for the patient, high transfusion probabilities and when all opportunities of cost minimization can be applied. adjustment to the individual situation of the patient is the main aim of a medically reasonable and economic use of autologous haemotherapy. this implies consideration of the patient's haematocrit, blood volume, tolerable blood loss, expected blood loss, etc. in order to choose the optimal method in the individual case. in this respect, double red cell apheresis may play a significant role. with this approach, donation schedules assumed to enhance erythropoiesis can be adopted. moreover, inconveniencies caused by long distances between patient home and donation service can be facilitated by withdrawing two red cell units during one session in selected patients. in conclusion, red cell apheresis can be used to promote the proposed approach towards individualized autologous haemotherapy preoperative plasmapheresis is considered to be a sensible adjunct if intraoperative retransfusion of salvaged and washed red cells is planned. acute normovolaemic haemodilution is valuable when the patient's tolerability of the haemodilution and the expected blood loss are carefully examined beforehand. intraor postoperative salvage of wound blood can also be regarded as useful measures to prevent allogeneic transfusions as long as the specific advantages and disadvantages of the different methods are taken into account. finally, alternative and supplemental measures such as iron or erythropoietin administration should always be considered in order to optimize the efficacy and effectiveness of autologous haemotherapy methods. the goal of a 'bloodless medicine' might not be reached but is supposed to be approached closely with an integrated concept exploiting all measures available. however, in times of restricted health care resources, regular sound costeffectiveness analyses, taking the availability and the cur-rent safety profile of allogeneic blood products into account, are always warranted and needed. compensatory fluid replacement of surgical blood losses: the transfusion of allogeneic blood is expensive and -although safer than ever before -still associated with potential complications. to reduce both, costs and immanent risks, allogeneic transfusion should either be completely avoided or at least minimized during surgical procedures. as a consequence an intraoperative blood loss is initially not replaced by red blood cells, but by erythrocyte-free, i.e. cristalloidal or colloidal solutions. when normovolemia is maintained the resulting dilutional anemia is compensated by an increase of cardiac output and enhanced arterial o2 extraction. however, once the hb has dropped to values recommended as the lower intraoperative limit, or once compensatory mechanisms of acute anemia become exhausted, as a rule transfusion of red blood cells (rbc) is initiated to increase arterial oxygen content (cao2) and to preserve a margin of safety for tissue oxygenation and organ function. as an alternative to immediate rbc transfusion, ventilation with pure o2 (hyperoxic ventilation) can be employed to rapidly raise cao2 by increasing the amount of physically dissolved o2 in plasma (hyperoxia). however, molecular o2 causes vasoconstriction, mediated by products of the arachidonic acid metabolic pathway. as a consequence hyperoxia has been shown to increase systemic vascular resistance and to decrease cardiac output and o2 consumption in subjects with normal hemoglobin concentration ( properly scheduled, three women who did not reach the necessary hct level after the first donation and consequently they got out of the protocol, and one woman who had unexpected intraoperative bleeding and received 5 homologous units in addition. the 3 patients undergoing tkr and the 11 patients undergoing removal of implants predeposited 9 and 21 units respectively, but finally 3 and 12 of them have been used. according to our patients data, the 55.5% of unused autologous blood units belongs to patients with tkr and implant removal. therefore a better schedule is needed for these type of surgery. all the autologous donors were supported by oral iron supplementation throughout the predonation and 1 month past surgery. fourteen of our cases were supported by erythropoietin s.c. in a dose of 300 iu/kg every other day. the majority of these patients was female, only one was male with multiple alloantibodies and was scheduled to predonate 5 autologous units. all of them underwent thr except one woman who also had a tkr 6 months later. the autologous blood donation was well tolerated by all patients and only one woman had a reaction during predonation. furthermore a group of 98 patients matched for age, sex and type of surgery, who did not predeposit blood, received a mean of 3.2 homologous units per patient, that is more than the patients on pabd program. our results show that autologous transfusion can be used in scheduled orthopedic surgical procedures and can reduce the need for homologous blood. however, every effort should be made to render the practice of pabd more efficient and to minimize its costs. colloid solutions and their establishment in clinical practice background: various situations like trauma, critical ill patients, sepsis, major surgical procedures and anaphylactic reactions are associated with disturbances in fluid homeostasis. this disturbance is related with reduced oxygen delivery, subsequent lactic acidosis and imbalance in oxidative status. the final result will most likely be an increased mortality and morbidity. aim: the important issue from clinical aspect is to define the optimal volume and type of fluid therapy. the debate for the ideal resuscitation solution lasts a couple of decades due to inconclusive and conflicting results. method: we searched the literature for clinical trials and major met analyses concerning patients undergoing scheduled surgical procedures, trauma patients and critical ill who received resuscitation fluids. results: dextrans reduce blood viscosity and von willebrand factor levels more, for the same degree of hemodilution, compared to other plasma expanders. in clinical setting they are effective in reducing the incidence of deep vein thrombosis and pulmonary embolism. after the initiation of dextran 1 for prophylaxis against anaphylactic reactions, they are considered the safest plasma substitutes, except maybe during pregnancy. dextran 40 10% is the most like to cause the 'hyperoncotic acute renal failure' syndrome. gelatins also cause a decrease in circulating levels of vwf : ag, vwf r : co, thrombin-antithrombin complexes and f1+2. in clinical aspect however, there are contradicted results about the effect of gelatins in bleeding diathesis, although they appear to exert a greater effect on rbcs protection from mechanical stress. in respect to anaphylactoid reactions, they have the greatest relative risk. hes has the same effectiveness in volume expansion with albumin and has the advantage of remaining intravascular even if there is an increased capillary permeability. in addition, it may improve splanchnic blood flow and tissue oxygenation. hes130/0.4 subsides the inflammatory response in patients undergoing major surgery, compared to a crystalloid-based volume therapy, but has conflicting results about it's effects on neutrophil respiration burst. clinical trials have so far failed to have a unanimous conclusion about the bleeding diathesis after hes administration, especially. caution must be held when administering hes during renal transplantation. albumin became the scapegoat of transfusion strategy during the past 20 years. resent met analysis have contradicted results about the safety of albumin infusion in variouw settings. a positive effect seems to have the early administration of hypertonic solutions in trauma patients, especially in combination with dextrans. conclusions: although some minor conclusions can be extracted, there is still a great lack of large scale multicentre randomized prospective clinical trials for extracting evidence based criteria. instead, we try to extract conclusions through met analysis. results show no evidence that resuscitation with colloids reduces the risk of death compared with crystalloids in patients with trauma, burns, major surgery or sepsis. also, there is lack of evidence that one colloid solution is safer -in clinical aspect -than any other. recombinant human erythropoietin therapy in critically ill patients -a dose response study* objective: the aim of our study was to assess the efficacy of two dosing schedules of recombinant human erythropoietin (rhuepo) in increasing hemoglobin (hb) level and reducing the exposure to red blood cells (rbc) transfusion in critically ill patients. design: a prospective, randomized, multicenter trial. patients: a total of 148 patients who met eligibility criteria were enrolled. intervention: patients were randomly assigned to receive intravenous (i.v.) iron saccharate alone (control group), i.v. iron saccharate and subcutaneous rhuepo 40 000 units once per week (group a) and i.v. iron saccharate and subcutaneous rhuepo 40 000 units three times per week (group b). rhuepo was given for a minimum of 2 weeks or until icu discharge or death. the maximum duration of therapy was 3 weeks. the requirement for rbc transfusions was significantly higher in control group than that in group a and b. no significant difference was observed between group a and b. the mean increase in hematocrit (dhct) and hb (dhb) from baseline to final measurement were significantly higher in group b than these in control group. dhct was significantly higher in group b than that in group a. dhct in group a was significantly higher than that in controls, whereas dhb did not differ significantly between control and group a. conclusion: administration of rhuepo in critically ill patients significantly reduced the need for rbc transfusions. the magnitude of the reduction did not differ between the low and high dose of rhuepo, whereas there was a dose response of hct and hb to rhuepo in these patients. in transfusion medicine, antibodies to antigens in the platelet membrane have traditionally been regarded as less significant compared with antibodies towards red cell antigens. there is an increasing awareness of antibodies towards platelet antigens. detection of autoantibodies to platelets can be a diagnostic challenge, but is seldom a problem in transfusion medicine because patients with such antibodies rarely are candidates for platelet transfusions. also, severe foetal thrombocytopenia is seldom present in pregnancies with autoantibodies to platelet antigens. the real challenge in transfusion medicine is related to patients with severe thrombocytopenia who are refractory to platelet transfusion due to alloantibodies towards platelet antigens. in our department, flow cytometry is used for compatibility testing and the choice of compatible blood donor is done without knowledge of antibody specificity. if crossmatch negative random donors cannot be identified, antibody specificity testing is performed and donors are chosen based on the specificity of the antibodies determined by a modified maipa procedure and with hla class i beads (flowpra from one lambda, usa) in flow cytometry. in some cases both hla class i and human platelet antigen (hpa) specific antibodies are detected and hla class i, hpa matched donors are chosen for crossmatch. if the crossmatch is negative, there is >90% chance of successful transfusions. in some cases drug induced anti-platelet antibodies are suspected and flow cytometry based antibody tests are performed in the presence and absence of the drug. in the case of suspected heparin induced antibodies, a beads assay is performed (diamed, switzerland). two percent of caucasian women have the platelet type hpa 1bb. ten percent of these women make anti-hpa 1a antibodies in their first hpa 1a incompatible pregnancy. 1 in 1000-2000 new-born has thrombocytopenia due to maternal alloantibodies which have crossed the placenta (neonatal alloimmune thrombocytopenia, naitp). results from a screening study covering the outcome of 100 000 pregnancies show that only 2 babies were born with intracranial haemorrhage (ich) and there was no still-born babies in the study. pregnant women with a-hpa 1a antibodies were diagnosed, received careful clinical follow-up and the delivery was performed by caesarean section in week 38 of the pregnancy with immediate transfusion of hpa compatible platelets if the new-born had platelet count <35 ¥ 10 e 9 /l. in previous studies, it is reported the ich appears in 10-30% of the pregnancies where antibodies are present and that 50% of the babies with ich, die. our results are different from what is reported from other studies and this may reflect the prospective approach and the clinical interventions. naitp represent a challenge in transfusion medicine both diagnostically, but also related to compatible blood products for the thrombocytopenic new-born and the mother who may have high level of antibodies towards platelet antigens. methods: apheresis platelets (4 ¥ 10e 11 mean) from donors with same blood group were pooled and divided equally into two bags, po-80 and control (pl2410, baxter), which have 2660 and 2024 ml/m 2 *day*atm of oxygen permeability, respectively. on days 0, 1, 3, 5, 7, and 9 of storage, swirling, mean platelet volume, po2, pco2, ph, glucose, lactate, aggregation, and p-selectin expression were evaluated. six experiments were performed. results: the swirling pattern was preserved better for up to 9 days in po-80 (6/6) than in control (3/6) bags. dropped ph less than 6.2 on day 9 was observed 1/6 in po-80 whereas 3/6 in the control. aggressive drop of glucose (0 mmol/l) with prominent lactate accumulation (327 mg/l) was also observed on day 9 in 1 of control bags. the po2 level in the control dropped more significantly by 45% (46.6 mmhg) on day 3 than in po-80 (65.8 mmhg) compared with the initial level (84.9 mmhg) (p < 0.001). these results suggest that aerobic metabolism of higher concentration platelets was maintained better in a container with higher oxygen permeability. and less lactate generation with slower glucose consumption is also suggested in po-80 bags than in control bags. the %hsr and aggregation decreased gradually in a similar manner in both bags until day 7, and became a detrimental defect in 2 of 6 control bags on day 9. p-selectin expression was higher in control bags than in po-80 on days 7 and 9 with no statistical difference. in two control bags p-selectin expression reached >84% and was accompanied by a loss of swirling. these functional and biochemical characteristics of platelets at a higher concentration were kept better for 7-9 days when stored in a container with higher oxygen permeability than in the best of marketed containers. t-pa-096 background: maintenance of a neutral ph in the range of 6.8-7.4 is essential for preservation of platelet function and viability during storage. furthermore, studies have also indicated that the presence of glucose in the platelet suspending medium is important for maintenance of platelet quality. however, a platelet additive solution (pas) containing glucose having a ph of 6.8-7.2 cannot be manufactured by steam sterilization due to caramelization of glucose. in order to have optimal ph, the currently available pas such as t-sol does not contain any glucose. this study describes a novel twostep approach to provide a glucose containing additive solution (pas-g) by using an acid, glucose containing electrolyte solution (ph 5.5) for resuspension and processing of the pooled buffy coats (bc), followed by transfer of the processed platelet concentrate (pc) into a storage bag containing bicarbonate for ph neutralization and maintenance during extended storage. aim: compare the platelet quality of pooled bc pc stored in pas-g with pooled bc pc stored in t-sol. methods: a paired study design was used, where a pool of 8 bcs obtained from standard day 1-old cpd-wb units, was divided into two equal parts: one part was resuspended and processed with a pall leukoreduction system (atsbc) using t-sol, the other part was processed in a similar manner with the acid part of pas-g. percentage plasma carryover ranged from 20-35%. both processed pc products were transferred and stored in elx tm bags with the pas-g pc elx bag containing a bicarbonate tablet. ten replicate studies were performed. results: the yields (3.2 ± 0.3 vs. 3.2 ± 0.4°¥ 10e 11 ) were similar (pags vs t-sol). statistically significant (p < 0.05 with paired ttest) improved platelet quality at days 5, 7, and 9 of storage was observed with platelets stored in pas-g as compared to t-sol. the table below shows results at 7 and 9 days of storage for ph, extent of shape change (esc) and hypotonic shock response (hsr). the results for t-sol stored platelets correlated highly with initial glucose (% plasma carry over) level (r = 0.91 for esc, and r = 0.68 for hsr at 7 days storage), while no significant correlations were found for pas-g stored platelets. conclusion: this study demonstrated the practicality of using a two step procedure to store bc pc in a glucose containing additive solution with neutral ph during storage, and confirmed the importance of glucose in the storage medium as nutrient for optimal platelet storage quality. the effect of irradiation on white cell reduced platelet concentrates, stored for 7 days background: the storage of white cell (wbc)-reduced platelet concentrates (pcs) can be extended from 5 to 7 days provided the quality has been validated and bacterial screening is performed. irradiation up to 50 gray (gy) does not affect platelet quality, but the effect of pre storage irradiation with subsequent storage up to 7 days is not known. method: two wbc reduced pcs, each made from 5 buffy coats and a unit of plasma, were pooled and divided into control group 'a' and study group 'b' . pcs in group 'b' were irradiated immediately after preparation with 25 gy. pcs in both groups 'a' and 'b' were then stored on a continuous flat bed shaker at 20-24°c. swirl, ph and cd62p expression were determined on day 1, 6 and 8. twelve experiments were performed and compared with a paired t-test, p < 0.05 was considered significant. results: see table (day 8 values; mean ± sd; n = 12). pooling and dividing of the pcs was successful with respect to volume and platelet number. on day 8, the ph in group 'b' was slightly lower than in group 'a', but the difference is not significant. in group 'a', ph on day 8 was <6.8 in 3/12 pcs, versus 5/12 in group 'b' (not significant). the cd62p expression in irradiated pcs is not significantly higher than in non-irradiated pcs. conclusion: irradiation had no significant effect on platelet quality when stored for up to 7 days after blood collection. -1b, -3a, -5b, pra 63%, 12 donors. in patient 3 -three transfu-sions were effective (two crossmatches neg by lct and pift, one pos lct, neg pift) but later on, when the patient was in severe clinical status (shortly before his death) two transfusions were ineffective in spite of neg crossmatches. it is very likely that for the same reason patient 4 and 5 were refractory to two and one hpa compatible platelet units respectively. in patient 6 and 7 compatible platelets were not transfused because they died before the whole procedure (diagnosis and finding a proper donor) was completed. conclusions: 1. the frequency of occurrence of anti-hpa antibodies in transfused patients was: -5b, -1b, -2b, -3a, -1a; in three patients they were monospecific, in four polyspecific. 2. in 2 patients who developed anti-hpa alone, transfusions of platelets without relevant hpa antigens were successful. 3.the effectiveness of compatible platelets in patients with both anti-hpa and -hla was more difficult to assess because of their severe clinical status, which might have been responsible for transfusion failure. in one of these patients, however, the transfusions of compatible platelets were successful when he was in relatively good clinical status, but shortly before death transfusions were ineffective. t-pa-099 the successful implementation of nucleic acid testing (nat) for hiv, hbv, hcv and further viruses as well as improved donor selection led to a dramatic risk reduction for viral transmission via blood transfusion over the last years. today, other risks get into the focus of haemovigilance. bacterial contamination of blood products can occur via the donor, suffering from a (clinically unapparent) bacterial infection, or via the donation process itself, storage and handling of the blood product. particularly platelet concentrates (pc) are vulnerable to bacterial growth due to their storage conditions. patients receiving such products have a potential risk of severe complications or even death. modern hygiene regimes, e.g. improved disinfection of the donors´ skin or preparation of blood products in fully closed systems as well as diversion, led to a significant reduction of bacterial contamination risk in the past. however, a small risk remains. therefore, two possible ways of further reducing the risk of bacterial contamination of blood products are feasible: (a) testing and/or (b) inactivation. testing for bacterial contamination is possible by different methods: direct detection methods for bacteria (microscopy, flow cytometry) have disadvantages regarding sample size and detection limit. bacteria might rapidly grow in a contaminated pc, so testing should be performed as close as possible to transfusion to the recipient. biochemical methods like oxygen consumption might not detect anaerobic germs. automated culture methods are still the most sensitive technique, but they have their downsides as well (e.g. time and size of aliquot drawn). novel molecular genetic test methods for detection of bacterial nucleic acid are in different states of development, but still have to proof their suitability for routine use. three different principles of pathogen inactivation can be distinguished: photodynamic reactions produce oxygen radicals which in turn inactivate bacterial structures by oxidation processes. examples for these chemicals are phenothiazines like methylene blue and thionin as well as vitamins like riboflavin (vitamin b2). photochemical reactants penetrate cell boundaries and irreversibly inhibit nucleic acid, thus blocking replication and proliferation of pathogens. chemicals of this group are psoralens like amotosalen as well as pen-110 or s-303. the third method, the solvent detergent (sd) method, is used for pooled plasma only and consists of the combination of both solvents and detergents, which interact with membranes and destroy bacteria. methods for inactivation of bacterial contaminants have to proof, that they effectively inhibit bacterial growth while maintaining full functionality of the blood product at the same time. these two qualities have to be fulfilled up to the end of the storage period. toxic or mutagenic compounds must not remain in the final product. the technology must be easily integrated into the existing work cycle of a blood bank. finally, costs per product must be acceptable. in summary, both testing and inactivation have their advantages and disadvantages, which have to be weighed up against costs and benefits of both procedures. pros and cons of introduction of inactivation methods in a blood donor service producing 1 600 000 blood components per year will be discussed. bacterial contamination of blood components, particularly platelets, is now recognized as a serious adverse reaction that is preventable. there are many studies that have documented that these reactions occur from platelets stored at room temperature, most commonly arising from a skin contaminant, but originating from donors with asymptomatic bacteremia in about 1/3 of cases. the problem is intensified for patients receiving pools of platelets compared to single donor platelets collected by apheresis. reactions are more commonly noted and more severe with platelets stored for greater lengths of time. previous studies at johns hopkins described a series of 23 reactions in 12 years with reactions more common in platelet pools (1 : 1600 transfusions) than with single donor platelets (1 : 15 000 transfusions). these reactions caused fatalities in 4 of 23 cases. although our data suggests that these reactions are more common than other studies using hemovigilance systems, our case definition requiring culture of all transfusion reactions to platelets led to a more reliable estimate of the incidence of sepsis from platelets, many potential solutions have been proposed to prevent these reactions. improved skin antisepsis should always be sought but will never eliminate the 1/3 of reactions due to asymptomatic bacteremia. the same limitation applies to methods that divert potential skin plugs from the collection bag. antibiotics in the bag would lead to manufacturing concerns or problems for patients with drug allergies. although cold storage of platelets is currently being revisited, it is not yet a practical solution. pathogen eradication systems have been developed but they remain unapproved in most of the world and have led to concerns about toxicity of additives, damage to treated cells, and cost. as a result of increasing recognition of the problem, there has been increased interest in bacterial screening to prevent sepsis from platelets. in march 2004, the aabb standards required testing of platelets for bacterial contamination. testing programs have been implemented in the us widely as a result of the aabb action. licensed systems based upon bacterial culture or growth characteristics are available for single donor platelets and have been commonly employed. although these systems have some difficulty with false positive reactions, the evolving national data suggest an incidence of 1 : 5000 true positive reactions. these data suggest that a number of serious reactions have been averted, although some cases have persisted due to incomplete adoption, problems with slow growing bacteria, or the use of inferior testing methods with inadequate sensitivity. whole blood derived platelets have become a more difficult issue, since the approved testing methods are limited. the use of ph monitoring, gram stain, glucose measurements, and inspection for swirling have all been attempted. these methods are not sufficiently sensitive or specific to interdict many contaminated units, so that screening for bacteria in pooled platelets is less effective. it is anticipated that new methods may become available to make screening of whole blood derived platelets easier to perform in a reliable manner. it is also hoped that bacterial screening of platelets may form the basis to permit seven day storage and prestorage pooling in the us. results: twenty four hcv rna (+)/anti-hcv(-) repeat donors were previously tested in routine hcv rna in mini-pools and were negative. twenty available look back samples were individually tested for hcv rna and in one the virus was detected. to make sure that the failure of hcv rna detection in routine nat was not due to the pooling procedure, the hcv rna was tested in undiluted look back sample and dilutions of this sample by hcv negative plasma: 2/2 repeats of 48x dilution and 2/2 repeats of 24x dilution were hcv rna negative, whereas 1/1 repeat of 8x dilution and 2/2 repeats of undiluted sample were positive. the results of cobas amplicor monitor (sensitivity 600 iu/ml) were negative, which means that viremia in hcv rna mini-pool negative donation was below 600 iu/ml. in the recipient of red blood cell concentrate from this donation hepatitis c was diagnosed. however, the possibility of pretransfusion hcv infection cannot be excluded as no hcv marker tests were performed before transfusion. the patient and the donor were infected with genotype 3a. the low hcv viremia (below 600 iu/ml) in the preseroconversion window period was responsible for no hcv rna detection in routine mini-pool hcv rna testing. introduction and aim of the study: bacterial contamination is a life threatening risk of blood transfusion, especially with platelet transfusions. bacterial culturing (bc) of platelets as well as pathogen reduction (pr) reduce the likelihood of such contamination. where the costs of bacterial contamination are far less than the costs of pathogen reduction, the latter will reduce not only the risk of bacterial contamination but also risks of other pathogens. therefore, the question arises whether this additional expenditure can be justified in the light of the additional effect achieved. this question we will answer by cost-effectiveness and sensitivity analyses. methods: the balance between costs and effects of preventing adverse events due to platelet transfusion is assessed using a mathematical model and assuming optimal effectiveness of pr. model parameters and valuations of health states were obtained from literature and information from dutch sanquin blood banks. . while the estimates in comparison to the situation without bc or pr are surrounded with large uncertainties, the conclusion that pr is not cost-effective in comparison to bc is very robust. the cost-effectiveness of bc and pr are very sensitive to the estimates concerning sepsis probability and associated complication rate, the cost-effectiveness of pr relative to bc is not. this conclusion is also insensitive to a wide range of assumptions regarding residual risks and costs associated with hiv, hcv and hbv. the estimates indicate that culturing in the netherlands is cost-effective, even with the deviation bag in place. the estimates however appear to be very sensitivity to the probability of sepsis. a decision to use pr will, after the introduction of bc and the use of a deviation bag, never meet cost-effectiveness criteria. even when assuming perfect protection, the conclusion that it is not cost-effective in comparison to bc is very robust and does not alter when varying underlying parameters within their margins of uncertainty. table) . of cb collection. two collections have been transplanted to date and this represents a 2.5% take-up rate (3.5% where a sibling is alive). this compares favourably with the numbers transplanted from unrelated cb banks. dcb collection is therefore at least as efficient a method as unrelated cb collection for transplantation albeit in the limited number of cases where a dcb collection is possible. dcb collection has the benefit of a possible immediate transplant combined with the availability of a sibling donor for future donation of both stem cells and lymphocytes. it is therefore a useful service to provide and complements the work of unrelated cord blood banks. increased yield of mature platelets in cultures of cd34-enriched cord blood cells maintained at 39°c introduction: the future use in transplantation of ex vivo expanded hematopoietic stem (hsc) and progenitors cells will facilitate the transplantation of adult patients and speed up hematologic recovery. also ex vivo cultures of hscs may eventually permit to produce donor-free blood components such as platelets for transfusion. culture of animal cells is routinely done at 37°c. however there is previous clinical evidence suggesting that hematopoiesis may be more active in hyperthermic patients. we have therefore compared the effect of hyperthermia on the ex vivo expansion and differentiation of cord bloodderived hsc in megakaryocytes (mk) and mature platelets. the cord blood-derived cd34 cells were cultured continuously at 37°c or 39°c for 14 days in cytokine conditions optimized for mk development and maturation. the cultures were regularly monitored for various parameters. results: compared to 37°c, the cultures maintained at 39°c produced significantly more total cells (4.9 fold) and total mks (7 fold), and showed accelerated and enhanced mk maturation with increased yield of proplatelets and mature platelets (16.6 fold). accordingly, the cells cultured at 39°c contained an increased frequency of cfc-mk (8 fold) at day 14. cultures done at 38°c and 40°c were also more efficient than at 37°c but less than at 39°c. platelets produced in 39°c cultures could be normally activated by thrombin. as expected, the cells cultured at 39°c contained an increased amount of the heat shock protein hsp70. control experiments showed that the culture of several cell lines was inhibited or unaffected by the 39°c temperature. the unexpected resistance of hematopoietic cells to the deleterious effects of heat and the stimulatory effect of >37°c temperatures on hsc proliferation and differentiation indicate that the routine culture of normal human cells at 37°c is a paradigm that needs to be revised. the responsible molecular mechanisms remain to be identified but the observation will facilitate the ex vivo expansion of the progenitors of the mk and possibly other lineages. the synchronous generation of a significant number of mature platelets in vitro will facilitate the study of the mechanisms of platelet formation and ageing and could eventually have important applications in transfusion medicine. it remains to be seen if the stimulatory effects of higher than 37°c temperatures represent a protective response against sustained body fever that is specific to the hematopoietic system. several countries have, in the past few years, included human tissue banking within a regulatory framework similar to that of blood. indeed, tissue safety has come to the forefront of the preoccupations of regulatory agencies after several well publicised morbidity and mortality cases have been reported in the press. tissue safety has many features similar if not identical to blood safety and a review of those common elements will be reported. as well, arguments in favor of integrating tissue banking within a blood system will be discussed, one of the more important aspect of which being the expertise of the blood centre staff with cgmps. the experience of a blood establishment (héma-québec) with the integration of tissue banking such as bone, skin, heart valves within its operations will be reported, emphasizing the medical as well as the management aspects of such an integration. finally, tissue banking is an activity which brings more expertise to a blood centre, expands its knowledge of its customers and gives more opportunities to its personnel. umbilical cord blood (cb) is an important source of stem cells for clinical transplantation and may cause less gvh disease than non-t-depleted bone marrow (bm). the relatively low numerical cell dose available from cb has usually restricted its use for transplantation in adults. only 25-30% of patients have an hla matched sibling and for others an unrelated bm or a stored unrelated cb donation may also not be available. for some children the collection of cb following the birth of a sibling may be the only opportunity for a transplant. directed cb (dcb) donations from matched siblings have been shown to give better long-term overall results than matched unrelated cb or bm. dcb collection is however not as easy to control as cb for banking where dedicated hospitals and trained staff are used. here we review dcb banking in oxford over a 2.5-year period. requests were received for 88 deliveries from 84 mothers and of these, 81 collections were successful including 4 pairs of twins. failed collection was most often due to a damaged cord at delivery. 61 collections were made for 57 siblings possibly requiring transplant (median age 4) with 24 for leukaemia, 16 for erythroid disorders, 13 for immune deficiency, 5 for enzyme deficiency and 3 others. the remaining 20 collections were mostly requested where there was a family history of an inherited disorder (majority scid). three collections tested positive for anti-hcv antibody but negative for hcv by pcr. collections were not excluded on the basis of volume or cell number. mean volume was 80 ml (range 22-174, 86% exceeded 40 mls) and mean tnc count was 1.0 ¥ 10 9 (range 0.2-2.7, 58% exceeded 0.8 ¥ 10^9). the mean cd34+ve count was 3.1 ¥ 10 6 (range 0.2-19.0). all collections were cryopreserved within 24 hours using dmso/dextran/saline without volume reduction. the mean tnc viability prior to freezing was 98% (range 86-100%) and the mean cd34+ve viability post freezing was 82% (range 71-96%). the reliance on the goodwill of midwives and the logistical difficulties that arise when organising collections from many different hospitals do not appear to reduce the success . rhd and rhce typing was performed by multiplex-pcr with 24 fluorescent primer pairs. positive results were obtained for rhd-exons 2-7, 9, 10 and polymorphisms associated with antigens c, c, cw, e and e. sequencing of rhce-sequences, including exons 1 to 10 and intron/exon borders, were done by direct taq cyclesequencing using bigdye-terminators v.1.1 in an abi310 (applied biosystems). background: a number of adverse immune reactions associated with blood transfusion result from contamination of blood products by donor white blood cells. among these reactions, transfusionassociated graft-versus-host disease (ta-gvhd) has a mortality of greater than 90%. mirasol ® pathogen reduction technology (prt) has been developed for the reduction of viruses, bacteria, parasites and white blood cells loads in blood products. the technology is based on light and riboflavin photochemistry. this study was performed in order to evaluate the effectiveness of the mirasol ® prt process for inactivation of human pbmncs. methods: human pbmncs were collected from trima platelet apheresis disposable sets, purified by ficoll-hypaque discontinuous gradient centrifugation and divided into test and control samples. the test cells were treated with mirasol ® prt in autologous plasma on day 0. both test and control samples (n = 3) were tested on day 1 for cellular immunophenotype, t-cell activation using flow cytometry, proliferation in response to mitogen or allogeneic stimulator cells, ability to stimulate the proliferation of allogeneic responder cells and cytokine synthesis in response to lps stimulation was measured using a cba assay kit. results: although mirasol ® prt treatment did not significantly change the distribution of cd3+, cd3+cd4+, cd3+cd8+, cd19+ and cd16+cd56+ human lymphocyte subpopulations there were significant functional change. the expression of the activation marker, cd69, was observed in 65.4% (sd = 15.6%) of control t cells upon activation with pma, while only a 1.7% (sd = 1.3%) of the test t cells increased cd69 expression. proliferation assays showed that 3h-thymidine incorporation did not increase in the test cells in response to either pha or allogeneic stimulator pbmnc compared to the significant increase in thymidine incorporation levels observed with control cells. the test cells, when compared to the controls cells, demonstrated an inability to stimulate allogeneic responder pbmnc proliferation. the release of il-10, il-6, il-1b and il-8 cytokines after 24-h incubation in culture media increased significantly to 128 pg/ml (sd = 73), >5000 pg/ml, 404 pg/ml (sd = 301) and >5000 pg/ml for control cells, respectively. under the same conditions, these cytokines in test samples remained at background levels of 0.4 pg/ml (sd = 0.7) for il-10, 2.4 pg/ml (sd = 1.2) for il-6, 11 pg/ml (sd = 3) for il-1b and 1022 pg/ml (sd = 286) for il-8. addition of lps further stimulated the release of tnf-a, il-10, il-6, il-1b and il-8 in the control samples, but not in the test cell samples. in vitro studies demonstrate that mirasol ® prt treatment does not change lymphocyte immunophenotype, inhibits tcell activation by pma, abolishes pbmnc proliferative activity, eliminates pbmnc stimulatory activity for responder cell proliferation and suppresses the production of cytokines by pbmnc in both the absence or presence of lps. introduction: it has been discovered that vaccination of dendritic cells (dcs) with tumor antigens is a potential strategy to induce tumor-specific immunity in tumor-bearing patients. aim of the study: the purpose of the study was to investigate whether human monocyte-derived dendritic cells (dcs) were able to present p210bcr-abl protein and induce antigen-specific ctl responses in vitro after transfected with total rna of k562 cells (k562-rna). methods: dcs were derived from human pbmncs, which were incubated for 5 days in the presence of gm-csf and il-4, and then were transfected with k562-rna using electroporation or dotap lipofection. to verify the successful transfection of dcs with k562-rna, bcr-abl fusion genes expression of dcs was detected by rt-pcr and western blot. the immune phenotypes of the dcs were analyzed by flow cytometry. the cytotoxicity of ctl was assayed by propidium iodide (pi) staining and flow cytometry. results: it was shown that the bcr-abl fusion gene was detected in the dcs immediately after the transfection, but disappeared 24 hours later, while the cells were expressing p210bcr-abl protein and expressing increased cd80, cd83, cd86, hla-dr. moreover, the transfected dcs could significantly promote the t lymphocytes to kill the target k562 cells. conclusion: human dendritic cells transfected with total rna of k562 cells in vitro could induce effective p210bcr-abl proteinspecific immune responses and be used to induce tumor-specific immunity, which implies potential application of immunotherapy to tumors. appear to be relevant to the clinical response. ivig has a remarkably good safety record for long term administration, however the following side effects have been observed: mild, infusion-rate related reactions such as headaches, myalgia or fever; moderate but inconsequential events, such as aseptic meningitis and skin rash; and severe, but rare, complications such as thromboembolic events and renal tubular necrosis. judicial use of ivig based on results from controlled studies is recommended. t-pl3-09 donor-lymphocyte infusion: transfusion immunotherapy following allogeneic hematopoietic transplantation the notion that bone marrow containing immunocompetent cells is capable of mediating an antitumor effect was determined experimentally almost 50 years ago. subsequently, pooled leukocytes from patients with cml were found to effect responses in patients with advanced leukemia. response correlated with cell dose and with severity of gvhd. in the 1970's, the graft-versus-leukemia (gvl) effect was defined in the transplant setting using a lethallyirradiated mouse model and splenocyte infusions. such studies suggested that gvl could be enhanced without causing severe gvhd. the era of adoptive immunotherapy in the transplant setting began in the 1990's with reports of donor lymphocyte infusions (dli) for relapsed acute and chronic leukemias after bone marrow transplant. it is now clear that chronic myelocytic leukemia (cml) in chronic phase is highly susceptible to gvl effects mediated by dli which induce durable remission in 70-80% of relapsed patients. the success rate is 30% or less in patients with accelerated phase or blast crisis. since dli cell dose appears to be important in this setting, strategies of escalating dose infusions have been investigated to enhance gvl without exacerbating gvhd. unfortunately, the response to dli in relapsed acute leukemia and myeloma is less favorable (<30%) and less durable. dli have been used successfully to treat viral infections and virus-associated malignancies following transplant. both unfractionated dli and ex vivo-generated tcell clones have suppressed reactivated cytomegalovirus and eradicated epstein-barr virus-induced lymphoproliferative disease, a polyclonal proliferation of donor-origin b cells that occurs after transplant. where tumor-specific antigens have been defined, efforts to target dli have been undertaken and donor and patient immunization has been investigated. acute or chronic gvhd develops in approximately 60% of patients receiving dli for relapsed hematologic malignancies and for related, but not unrelated transplants, correlates with the donor t-cell dose. dli-induced pancytopenia occurs in approximately 20% to 50% of patients, is generally mild, and transient, but in <5% of patients, aplasia is severe and prolonged. complications of aplasia include infection, bleeding, increased transfusion requirements. efforts to limit the adverse effects of dli while retaining the therapeutic effects include insertion of 'suicide genes, ' selection of lymphocyte subpopulations, and targetting lineage-specific minor histocompatibility antigens. available clinical and experimental evidence suggests, that in addition to primary and secondary immune deficiencies, a wide spectrum of immune-mediated conditions could benefit from intravenous immunoglobulin (ivig), including acute and chronic/relapsing diseases, autoimmune diseases mediated by pathogenic autoantibodies or by autoaggressive t cells and inflammatory disorders e.g. an imbalance in cytokine networks. trimar-collected apheresis platelet concentrates (pcs) were exposed to 17.2 j/ml uv light in the presence of 50 um riboflavin, followed by storage under blood bank conditions with various concentrations of 2-deoxyglucose from 0 to 20 mm for 5 days. the control platelets were not stressed by uv light exposure and were stored under the same conditions without 2-dog presence. all test and control platelets were measured for in vitro cell quality including rates of glycolysis, morphology score and activation levels at days 0, 1, 3 and 5. results: lactate production and glucose consumption increased from 0.0378 mmol/1012 cells/h (sd = 0.0097) and 0.0268 mmol/1012 cells/h (sd = 0.0114) for control samples to 0.1371 (sd = 0.0281) and 0.0724 (sd = 0.0151) for uv-treated platelets, respectively. uv treatment also caused a decrease in ph from 7.50 (sd = 0.07) for controls to 6.55 (sd = 0.26) for treated platelets at day 5, hsr from 75% (sd = 12.4) to 33% (sd = 25.1), esc from 24.3% (sd = 3.9) to 3.8% (sd = 3.6), swirl from 2.8 (sd = 0.7) to 1.0 (sd = 1.0), and increased p-selectin expression from 21.2% (sd = 7.3) to 85.2% (sd = 9.4). addition of 2-dog up to 20 mm significantly reduced lactate production rate to 0.0515 mmol/1012 cells/h (sd = 0.0045) and glucose consumption rate to 0.0293 mmol/1012 cells/h (sd = 0.0060), and maintained ph above 7.35 (sd = 0.09) for 5 days of storage. the effect of 2-dog exhibited a dose-dependent response. however, the addition of 2-dog had no effects on hsr (28.1 + 11.4% at day 5), esc (2.86 + 2.75% at day 5), swirl (0.8 + 0.5 at day 5) and p-selectin expression (69.9 + 6.4% at day 5) during platelet storage. atp contents in both treated and control groups were maintained at a relatively constant level above 87% of the value seen in fresh platelets. furthermore, an exaggeration of uv-stressed platelet aggregation by addition of 2-dog was also observed. conclusions: increased glycolytic flux is not a direct cause for platelet morphology changes and spontaneous activation incurred during the development of the storage lesion. the results also suggest that a reduction in glucose utilization may foster an increase in platelet loss during storage. aim of the study: was to evaluate analytical sensitivity, sensitivity and inclusivity for subtypes and genotypes of hiv, hcv and hbv, the assay's effectiveness in closing the pre-seroconversion window period, clinical specificity as well as the effect of endogenous substances and microorganisms on the sensitivity and specificity of the assay. methods: secondary standard traceable to who international standard for hiv-1 (97/ 656), international standards for hcv (96/798) and hbv (97/746) were used to determine the analytical sensitivity. sensitivity and inclusivity for hiv-1 subtypes other than hiv-1b, for hiv-2 and for hepatitis b and c genotypes as well as specificity was evaluated with >1000 specimens. results: results from this study indicate that high analytical sensitivities (39 iu/ml hiv-1m, 114 cp/ml hiv-1o and 1.1 cp/ml hiv-2, 7 iu/ml hcv and 3 iu/ml hbv) and a specificity of >99.4% are accomplishable for the mpx test. the 95% detection rate for hiv-1m subtype isolates (a through h) was between 10 to 50 iu/ml, for hcv genotype isolates (1a through 6) between 2 to 10 iu/ml and for hbv genotype isolates (a through g and precore mutant) between 2 to 5 iu/ml. investigating seroconversion panels, hiv-1 rna was detected an average of 6 and 5 days earlier than hiv-1 antigen with abbott hivag-1 monoclonal and coulter p24 antigen tests, respectively, hcv rna an average of 31 or 21 days earlier than hcv antibody with the abbott hcv eia 2.0 or ortho eia 3.0 tests, hbv dna an average of 18 days earlier than hbsag with the abbott hbsag eia imx test. for all targets, no interference was detected with 17 microorganisms tested as well as elevated levels of triglycerides, albumin, hemoglobin, human dna or bilirubin. conclusion: automated pooling, sample preparation, and real time pcr using the blood screening system 200 taqscreen mpx test is an efficient and sensitive method to simultaneously screen for five important viruses in human plasma. the mpx test is another evolution step in the development of pcr automation by roche molecular diagnostics, and further represents roche's commitment to increasing the safety of the global blood supply. aim of the study: a prospective hemovigilance plan was set up in order to establish a registry for future reference, and to detect any unexpected side effect of ip that may occur with significant frequency in populations and indications that were not studied before and outside of a formal trial environment. methods: this plan is proposed to blood establishments and transfusion prescribers who have already decided to implement ip. this is an observational, non randomized, non controlled plan. no patient selection, inclusion or exclusion criteria are required. all ip transfusions are documented using an internet form, whether or not a reaction is observed. patient population data are collected anonymously, for epidemiological purposes. results: between october 2003 and september 2004, 2512 apheresis ip units have been transfused in 2 sites and registered in the database. ip platelets were considered leucocyte inactivated and were not irradiated, but were antigen matched as indicated (3.9%). the population of patients receiving at least one transfusion (n = 271) included 58.3% of males, 40.6% of females, the median age was 64 (range 0-89). the most frequent broad diagnostic categories were hematology-oncology (64.9%) and cardiovascular surgery (19.9%). the patients received their transfusions either in regular hospital wards (63.5%), intensive care units (27.3%) or as outpatients (9.2%). the number of transfusions by patient ranged from 1 to 106 (mean 9.3 ± 16.0, median 2). half of the patients (49.8%) had previous transfusion experience and 5.9% had previous history of transfusion reaction. transfusion reactions, defined as any deterioration of the patient's state of health observed following transfusion, were observed in 1.2% (n = 31) of the transfusions (95% ci 0.84-1.75), and 8.5% of patients. only 2 (0.1%) were considered serious. after further causality analysis including biological and clinical investigations by the transfusion physician, 0.9% (95% ci 0.58-1.37) of the transfusions were confirmed as having caused reactions in 6.3% of patients, none of them serious. the most often reported symptoms were chills (0.9%) and fever (0.6%). itching, skin rash or urticaria was observed in 0.5% of transfusions. of the 2 serious reactions, one was hypotensive shock in a patient with liver cirrhosis and haemorrhage, and one was septic shock, in which the platelet unit bacterial culture was negative. none of the reactions occurred in cardiovascular surgery patients. patients were more likely to experience reactions if they had previous transfusion history (odd ratio 2.17, p = 0.15). the active hemovigilance plan is a valid and feasible method to collect epidemiological data on transfusion safety. the risk profile of ip transfusions appears favorable. quality of theraflex mb-plasma during storage and treatment s reichenberg* and n müller † *maco pharma international gmbh, langen, † inst. for transfusion medicine, essen, germany background: although in the last decades thanks to the implementation of several methods like donor selection and testing procedures the risk of virus transmission from plasma has decreased, infection of patients still exists. additionally new viruses like west nile virus enter the transfusion chain. therefore, the treatment of therapeutic plasma with methylene blue (mb) is a technique used in several european countries for pathogen inactivation. macopharma has developed the proprietary theraflex mb-plasma bag system including a mb pill and a final mb filtration step. aims: aim of the study is to show the quality of the mb plasma during the preparation procedure and during storage using the theraflex system. methods: for the preparation process every single step was evaluated using 18 single donor plasma units. for the evaluation of the plasma factors 5 ml were drawn at different stages (before treatment, after plasma filtration with plas4, after dissolution of the mb pill, after illumination, after treatment). because the sample volume for single sample measurements would be too low the samples were pooled after drawing and measured for the specified factors. six samples of each stage were pooled at three days. a whole panel of plasma factors was measured for the resulting three pools. global tests: quick, inr, aptt, thrombin time coagulation factors: fibrinogen, factor ii, factor v, factor viii:c, factor ix, factor x, factor xi inhibitors: at iii, protein c, protein s fibrinolysis: plasmin inhibitor, alpha1-antitrypsin complement: ch50 activation: tat, factor xiia, d-dimer stability data were generated using three plasma pools. six plasmas were pooled and afterwards divided into six aliquots. each was treated as single unit and then each was divided into six storage samples. the same plasma factors as for the manufacturing process were evaluated. results: a moderate reduction for some coagulation factors during the preparation was found in the illumination step but not in the other preparation stages. this was mainly fibrinogen (17.5%), factor viii (22.2%), and factor x (13.4%). despite this reduction the values were within the ranges found in non-treated plasma. all investigated plasma factors remained stable during the investigated storage time. summary/conclusions: the investigation showed that plasma treated with the theraflex procedure showed slight reduction during treatment and no reduction during storage. all plasma factors remained within the threshold values. the treatment of therapeutic plasma with mb is a valid technique of pathogen inactivation. validation of intercept treatment of pooled platelets g santos, c silva, f pereira and g sousa lisbon regional blood centre, lisbon, portugal background: intercept blood system for platelets uses amotosalen hcl and uva light to inactivate viruses, bacteria, protozoa and leucocytes that may contaminate platelet products. aims: the purpose of the study was to assess the feasibility of introducing this technology in the routine of lisbon regional blood center (crsl) and validate the procedure in our center. material and methods: whole blood units of 450 ml were collected from volunteer blood donors in quadruple top and bottom blood bags (optipure rc soft t& b baxter), kept in n-butanodiol plates; buffy coats with a volume of 55 ml were obtained in the opipress ii and kept overnight at room temperature before pooling. five buffy coats were pooled with 280 ml intersol using the octopus system intercept buffy coat pooling set with an integrated filter. the pools were treated using the intercept. samples were taken before treatment, after cad remotion, on days 2, 5 and 7. the following tests were performed: platelet count, mean platelet volume, ph, swirling. results: all pools met the intercept guardbands. platelet yield pre inactivation was 3.93 ¥ 10 11 (2.95-4.72 ¥ 10 11 ; sd-0.41). platelet pool volume was 362.3 ml (sd-10.7). plasma % was within 32.9% and 36.5%. all pools had leucocytes within council of europe specifications. after photoinactivation the platelet concentrates had 3.58 ¥ 10 11 (±0.37). the average platelet loss was 0.35 ¥ 10 11 . the ph was within specifications during all the storage period. conclusions: intercept treatment of pooled buffy coat platelets is feasible in the routine of crsl and in vitro parameters do not show significant changes, allowing us to proceed to clinical use. shown ip and conventional platelets (cp), stored for up to 5 days, exhibit comparable hemostatic efficacy and safety. extension of platelet storage duration to 7 days has the potential to improve platelet availability and reduce outdating and inventory shortages. clinical efficacy and safety of ip stored for 7 days were investigated. methods: a randomized, controlled, single-center, crossover, noninferiority design (pilot) study evaluated efficacy and safety of buffy coat ip vs buffy coat cp, each stored for 7 days. patients were randomized to receive one 7-day ip transfusion and one 7-day cp transfusion in random order. after each study transfusion, the 1hour platelet count, ci, and cci; time to next transfusion; bleeding response; transfusion reactions; and serious adverse events (saes) were assessed. the primary endpoint, 1-hour cci, was analyzed by a one-sided non-inferiority test for the per protocol population (patients with both transfusions and no major protocol deviations interfering with efficacy evaluation). the per protocol population included 20 patients, 9 randomized to the ip-cp sequence and 11 to the cp-ip sequence. more patients received allogeneic stem cell transplant in the cp-ip sequence than the ip-cp sequence (64% vs 33%; p = 0.37). mean platelet dose (¥10e11) was 2.8 for ip and 3.0 for cp (p = 0.23). there was a significant period by treatment interaction (p = 0.07) at the 0.10 significance level; therefore, the first period only was also analyzed for the primary endpoint. including both treatment periods, mean (±sd) 1-hour cci (¥10e3) was 6.6 ± 4.5 for ip vs 8.9 ± 5.5 for cp. the mean paired difference for both sequences was 2.4 ¥ 10e3 (p = 0.55 by non-inferiority test; upper bound of the 95% confidence interval = 4.0). for the first period only, mean 1-hour cci (¥10e3) was 8.7 ± 3.8 for ip vs 7.4 ± 5.4 for cp) the mean paired difference for the first period sequences was 2.4 ¥ 10e3 (p = 0.06 by non-inferiority test; upper bound of the 95% confidence interval = 2.4). the non-inferiority margin for the study was 2.2 ¥ 10e3 for mean treatment difference in cci (cp-ip). median time to next transfusion was 25 h for ip vs 24 h for cp following the first transfusion (p = 0.87 log-rank test; data censored at 7 days after transfusion) and 16 h ip vs 34 h cp after the second transfusion (p = 0.02). bleeding pre-or post-transfusion was uncommon, usually mucocutaneous, and grade 2 or lower, and responded similarly to ip and cp. no significant transfusion reactions or saes were reported. in this double-blinded, two-treatment crossover study the primary endpoint regarding 1-hour cci was not met. however, transfusion with 7-day-old platelets treated by the intercept blood system showed only a marginally and probably clinically insignificantly lower 1-hour cci compared to 7-day-old conventional platelets. methods: new zealand white rabbits were transfused with syngeneic blood (10 ml/kg), across a major antigen (hgd) mismatch. high anti-s-303 ab titers (≥1 : 1000) were induced after repeated immunization (days 1, 8, 15, 36, 64) with klh-(s-303) hapten (klhhapten) in complete freund's adjuvant. ab titers against srbc were determined by gel card agglutination, or by facscan with fitc-goat_anti-rabbit_igg. survival of infused rbc (4 ml/kg) treated with different methods was assessed by rbc biotinylation. blood samples were taken 1, 3, 7, 15, 21 and 28 days after transfusion, analyzed using streptavidin_pe and facscan to determine the proportion of circulating biotinylated rbc. results: groups (g) of rabbits were transfused with control rabbit rbc (crbc; n = 4, g1), or o-srbc (n = 6, g2). no ab against o-srbc developed after biweekly transfusions over 24 weeks in g2 rabbits. high ab titers to o-srbc could however be induced by klh-hapten immunization in a different group of animals (n = 2; g3). transfused rabbits (g1 & g2) exhibited no change in hematocrit or body weight and maintained good vital signs. high titer anti-s-303 abs were then induced by klh-hapten immunization in rabbits from g1 (n = 2) and g2 (n = 4), and in a group (n = 6, g4) of naïve rabbits. non-immunized rabbits (g1, n = 2), and (g2, n = 2) were maintained on the biweekly transfusion schedule of crbc and o-srbc, respectively. all rabbits treated with klh-hapten developed comparably high ab titers. klh-hapten immunization did not affect the viability of crbc in g1 rabbits. transfusion of o-srbc demonstrated reduced viability in hyper-immune g4 rabbits, but not any of the g2 rabbits. g2 rabbits exposed to 12 o-srbc transfusions prior to hyper-immunization with klh-hapten, had viability of o-srbc comparable to crbc, suggesting induction of immune tolerance by repeated exposure to o-srbc. after depletion of labeled o-srbc from circulation, g2 and g4 were transfused with m-srbc. viability of m-srbc in all g2 rabbits (hyper-immune or not) and the hyperimmune g4 rabbits was equivalent to crbc circulation in g1 rabbits. in pre-immunized rabbits with high titer anti-s-303 ab, o-srbc are cleared faster than control. in contrast, m-srbc survive normally in rabbits with high anti-s-303 ab titers. repeated transfusion of o-srbc does not result in alloimmunization of naive rabbits. the modified s-303 rbc process offers the potential for pathogen inactivation with elimination of immunoreactivity and retention of rbc viability. introduction: the bombay phenotype is extremely rare and characterized by complete absence of abh activity both on erythrocytes and in secretions. those individuals can produce anti-h, which is active over a wide thermal range. method: using liss indirect antiglobulin technique, the patient's serum showed +4 reaction by panel of eleven cells at room temperature phase as well as indirect phase while the auto reaction is negative. a cold adsorption using rabbit erythrocyte stroma was done to remove the cold antibodies from the serum; +2 reaction of an antibody was detected in the patient serum after five folds of rabbit erythrocyte stroma adsorption. introduction: immunohematology reference laboratory in kuwait central blood bank receives samples from all hospitals in kuwait both governmental and private sector. the laboratory performs the tests according to international standards and it is monitored by internal and external quality assessments on periodic basis. material and method: a tube and gel cards are two methods in the reference laboratory for antibody identification. the laboratory can identify the most commonly encountered clinically significant antibodies and investigates causes of positive direct antiglobulin test that occur mostly in autoimmune hemolytic anemia. there are facilities to phenotype most of rare red blood cell antigens. results: records of all patients investigated in the laboratory since the year 1985 are kept in computerized system that has 7528 patient's records. central blood bank has the potential to identify rare phenotypes such as kpb-, jsb-, lan-, bombay, rzr2, rzr1, r¢r¢, r¢r≤, r≤r≤, ge-2,3,4 and rare red blood cell antibodies such as high frequency antibodies anti-k, anti-ge2, anti-h, anti-lan, anti-kpb, anti-jsb, anti-wrb, anti-ena, anti-csa and low frequency antibodies anti-kpa-, anti-jsa-, anti-dia, anti-lua, anti-cob as well as hightiter-low-avidity antibodies such as anti-chido. samples of rare red blood cells and rare serums are kept frozen either by glycerol or liquid nitrogen technique to be used for pre-transfusion compatibility testing and continuing educational program. purpose of the work: to present the substitution of the blood groups o, a, b, ab in abo blood group system and rh (d) blood group in rh (d) blood group system in the population in the gevgelija-valandovo region. material and methods: a retrospective analysis was done on the data of following the blood groups o, a, b, ab and d in the blood group system abo and rh in the gevgelija-valandovo region. the asked population are voluntary blood donors, candidates for drivers, patients, pregnant women and newborn children. the period (1989) (1990) (1991) (1992) (1993) (1994) (1995) (1996) (1997) (1998) (1999) (2000) (2001) (2002) (2003) (2004) was analysed. the examinations were done with two standard methods (on the plate and in tube), to define blood groups from the most represented blood group systems abo and rh in the population. tests were done with different series of commercial anti serum tests from domestic and foreign origin. results: totally 43.395 examinees were typified. with o blood group were 15.761 (36.31%); with blood group a were 17.675 (40.73%); b blood group 7.119 (16.4%) and ab blood group were 3.019 (7.52%) examinees. totally 37.365 (86.11%) were d positive and 6.029 (13.89%) were d negative. discussion: the given results from our examinations for the frequency of o, a, b, ab and rh (d) blood groups from abo and rh (d) blood group systems in the region gevgelija-valandovo show that the most present is the blood group a from abo blood group system 17.675 (40.73%) and d blood group in rh system with 37.365 (86.11) form examined population. the results are in correlation with data from the literature for other european nations. introduction: the policy of our center is to transfuse all heamatologic multitransfused patients with their own rhesus and kell phenotype. in addition, thalassemic and young leukemic patients are being transfused with compatible phenotype of the most clinical important duffy and kidd systems while all the other patients receive blood compatible only with abo and rhesus system. nevertheless, it is observed a significant positivity of the indirect antiglobulin test, due to alloimmunization. aim of the study: in this study we tried to evaluate the prevalence of alloimmunization in patients of our region. the most frequent detectable alloantibody remains the anti-d, with high prevalence 88.9% of anti-d in females versus 11.1% in males, due to alloimmunization during the pregnancy. as a consequence, the high incidence of anti-d is not transfusion related and anti-e is evidenced to be the most frequent transfusion related alloantibody, followed by anti-kell. care must be taken in order to transfuse as more patients as possible with their own phenotype regarding, at least the most immunogenic antigens, like anti-e and anti-kell. severe hemolytic reaction due to anti-j k3 background: red blood cell alloantibodies directed against antigens of the kidd system are notorious for causing delayed hemolytic transfusion reactions. the antibodies are formed because of pregnancy or transfusion. blood donors with the red blood cell (rbc) phenotype jk(a-b-) are extremely rare in the white population and exhibit a frequency of less than 0.1%. however, the rare phenotype jk(a-b-) is more common in polynesians (1.4%). individuals with jk(a-b-) phenotypes typically form anti-jk3 with inseparable anti-jka and anti-jkb activity. some jk(a-b-) patients' sera may show an additional distinct anti-jka or anti-jkb component when examined with adsorption studies. case report: a 63 years-old caucasian female with a negative antibody screen, no prior history of transfusion, presented with gastrorrhagia. it is reported four pregnancies with no history of haemolytic disease of the newborn (hdn). on admission, her haemoglobin was 5.5 g/dl. she was given 4 units of crossmatchcompatible rbc. on day 6 her haemoglobin was 12.1 g/dl, with a total bilirubin of 0.72 mg/dl and lactate dehydrogenase of 141 u/l. on 10th day an unexpected fall in hb (5.6 g/dl) occurred with an increase of bilirubin to 1.9 mg/dl and of lactate dehydrogenase to 1042 u/l. a new blood sample obtained for antibody screening and additional crossmatches showed a pan-agglutination and incompatible crossmatch. anti-jk3 antibody high titer was detected in the plasma by gel-test using liss/coombs cards (id-diamed). the dat was negative and the antibody reacted equally with jk(a-b+), and jk(a-b+) panel cells (jka:1/16 384 and jkb:1/16 384). other alloantibodies could not excluded, because jk(a-b-) cells are not available. she was started with erythropoietin-a (epo), folic acid, fe iv and high dose intravenous immunoglobulin (ivig). the epo was discontinued after four week of therapy when the haemoglobin was 12 g/dl. two months later her haemoglobin was 13.5 g/dl and anti-jk3 was present in the same titer. a year later her blood cell count was normal and the anti-jk3 was detected in a lessened titer (1/16). no additional distinct anti-jka or anti-jkb component was shown after two adsorptions at 37°c using carefully selected phenotyped red cell compatible with patient's rh, fy, mnss, lu, le system and jka(+) and jkb(-), but two additional alloantibodies anti-c and anti-e of low titer (1/4) were revealed. the rare anti-jk3 alloantibody found in this case displayed the erratic nature of many kidd system antibodies. although anti-jk3 may cause mild hemolytic disease of newborn, she did not have a history of hdn. our patient was sensitized to a kidd antigen during pregnancy, but showed no serologically detectable antibody until challenged with a massive transfusion following a gastrorrhagia. the use of epo and high dose intravenous immunoglobulin succeeded to avoid transfusion with incompatible rbc unit. background: differential warm adsorption is used in the investigation of patients with red cell autoantibodies for searching of underlying alloantibodies, but it is also useful in the detection of clinically significant alloantibodies in patients with alloantibodies to high frequency antigens such as k, kpb, lub and inb. this technique is especially useful in cases when patients when patient's phenotype cannot be identified due to recent transfusion. purpose: differential warm adsorption is performing on cases presented with an antibody reacting with all red cells of the panel and having a negative auto control test. in these cases, even rare cells panels, which allow the identification of a pan antibody, are available, other more common clinical significant antibodies cannot be excluded. methods and result: case 1. a 63 years-old caucasian female with preexisting myeloproliferative disorder (polycythemia) presented with pancytopenia. anti-k was detected in the plasma. it is reported two pregnancies and no history of transfusion. the dat was negative and the plasma did not react with one k-cell present on the red cell panel in use. the anti-k specificity was confirmed using additional k-cells. the patient's red cell were group a, d+, k+, k-, c-, e-, fy(a-), s-, le(a-), kp(a-), cw-. she was transfused with two units rbc k-. ten days after the first transfusion the dat became positive and the one k-cell present on the red cell panel reacted with her plasma. two adsorptions were carried out at 37°c using carefully selected phenotyped red cell (compatible with patient's rh, fy, jk, mnss, lu, le system and k positive). an anti-fya was identified in the presence of ant-k. . an antik (titer 1/16) was suspected. because k-red cell was not present in the panel in use, adsorptions were carried out at 37°c using carefully selected phenotyped red cell (compatible with patient's rh, fy, jk, mnss, lu, le system and k positive). after the anti-k antibody was totally removed, no additional alloantibodies were revealed. conclusion: differential adsorption in cases with alloantibodies to high frequency antigens represents a useful application of the technique and helps in the identification of clinical significant antibodies present, allowing a more accurate decision for transfusion. evaluation of validity of the expired enzymetreated 0.8% red cells in antibody identification gel tests using nacl cards p chalkia, s intzepeli, v avgoloupi, a tsoukala, e ntinopoulou and p didoudi ahepa hospital, thssaloniki, greece background: expired red blood cells of required phenotypic profile is often used to identify antibody specificities in patients with multiple anti-erythrocytes antibodies. accurate results depend on the integrity of the antigens. purpose: to validate the expired enzyme-treated 0.8% red cells for use in antibody identification gel tests using nacl cards. the serum of nineteen patients with common specificities antibodies in rhesus, kell, duffy, kidd, and mnss systems tested with commercially prepared 0.8% enzyme treated cells rbc panel (id-diamed). gel tests were performed according to the manufacturer's instructions on in-date rbc and simultaneously on rbc 1 month to 5 months past expiration. reactivity of the expired antigen positive and antigen cells was compared to in-date cells. results: twenty antibodies detected with enzyme treated red cells in neutral gel cards [d(4), c(3), e(2), k(7), cw(4)] and were tested with enzyme treated red cells in use and rbc 1 to 5 months post expiration. seventeen antibodies tested with enzyme treated cells gave acceptable results with antigen positive cells 1 to 5 months post-expiration, except 3 anti-k antibodies (negative with k positive cells 2-4 months post expiration). conclusion: most rbc antigens studied were detectable 5 months after rbcs expiration date. tests with 0.8% cells were valid in gel test (nacl/enzyme) for at least 5 months after manufacturer assigned expiration date and may be helpful for complex identification studies. studies for more antigens specificities are needed to testify the validity of the expired enzyme-treated 0.8% red cells. background: wr(a) is a low-incidence blood group antigen (1 : 1000) in the caucasian population. despite that anti-wr(a) is a common antibody type, it may cause severe transfusion reactions, haemolytic disease of the new-born. anti-wr(a) may occur as an autoantibody or arise without immune stimulus. we report a case of a naturally occurring anti-wr(a) antibody. case report, methods and results: 56-year-old non-transfused male patient with acute pancreatitis and severe anaemia had been transferred to surgery from a county hospital. the serological status identified by their blood bank was: b rhd positive, with anti-wr(a) antibody in the serum (cellbind card method). our results: the patient's cells were group b rhd positive (microplate method), dat negative (tube and gel test method). the antibody identification showed positive antibody reaction with all enzyme treated test cells but negative reactions in liss iat (tube test) and gel iat (scangel and diamed). discussion: our routine tests for antibody detection didn't detect any specific antibody in patient's serum. he was transfused several units of blood, that was wr(a) negative and showed negative crossmatch reactions. the patient had no transfusion reactions. 10 days after the transfusion anti-wr(a) specificity was confirmed in the serum with cellbind test. only few test cell panels contain wr (a) positive cells, which are usually not present in commercial screening cells. in our case the cross-match was only performed for this patient because of the detected nonspecific antibody reaction in enzyme. the risk of transfusion reactions caused by rare antigens are particularly high in the type and screen cases. background: according to requirements of the french committee for accreditation (comité français pour l'accréditation cofrac, iso 17 025 standards), it is essential to use validated and standardised methods in immunohematology. this imposes, among various requirements, the knowledge of metrological tolerances for all the techniques. aim: a multicentre study was carried out to define the maximal acceptable deviations concerning incubation temperature and time, volumes of patient plasma and of tests cells for antibody screening using indirect antiglobulin test (iat) in filtration technique. the antibody screenings were performed manually in 3 blood centres using 3 different filtration systems: id diamed, biovue ortho and scangel biorad, the same tests cells, a standard 20 ng/ml anti rh1 (provided by cnrgs), a positive control anti kel1 and a negative control. all equipment used (oven, chronometer, pipettes) were calibrated according to cofrac standards. each antibody sample was tested under the following combined conditions (243 tests/sample): results: all the tests of antibody screenings from the multiples combinations of the above parameters gave the same results with a 2+ intensity agglutination for positive samples and the absence of agglutination for the negative control. conclusion: this study allowed us to define a range of tolerance for 4 critical physical parameters involved in the antibody screening in iat using commercial filtration systems maryvonne. the authors present a retrospective study involving 12 091 blood donors from the university hospital of coimbra during the year 2004. the incidence of weak d and rh (d) phenotype was determined in 1729 individuals who were rh (d) negative. the ab0 and rh(d) blood grouping was performed using a column gel agglutination card (diamed). the rh (d) typing was done using a anti-d polyclonal and a anti-d monoclonal antibodies. all donors that gave negative or poor agglutination results were tested for weak d with an indirect antiglobulin test, with anti-igg (gel matrix card) plus anti-d serum (diamed). within our target group of blood donors the rh (d) negative represented 14.3% of the total sample. we also describe ab0, rh(d) phenotype (c, c, d, e, e) group frequencies and establish reliable estimates frequency for weak d and rhesus haplotypes. the background: in 80s and 90s several authors tried to assess the relationship between the number od igg molecules per rbc and in vivo haemolysis, but determinations usually concerned small groups of tested patients. some of the investigators suggested that the number of igg autoantibody molecules per rbc was a major determinant of the severity of the haemolysis, whereas others found aiha patients with severe haemolysis and undetectable autoantibodies. aim: presentation of our experience with the quantitative elat performed on a large group of aiha patients during long-term observation. material and methods: six hundred fifty eight blood samples from 268 warm-type aiha patients were randomly tested for the number of igg molecules per rbc. eighty six of the patients were tested periodically from 3 to 15 times at one-month intervals. autoantibodies on rbcs were detected by the direct antiglobulin test (microcolumn technology) and measured by the enzyme-linked antiglobulin test (elat). results: in about 1/3 of tested samples the number of igg molecules per rbc was small (<190) and the laboratory signs of haemolysis were present in 65.7% of them as well as in 70.4% samples with moderately coated red cells (200-980 igg/rbc). the large number of igg molecules per rbc (>1000) was significantly associated with high frequency (87.9%) of severe haemolysis and it was also associated with presence of multiple igg subclasses on rbcs and c3d. in 79% of patients tested periodically, the number of igg molecules per rbc decreased and it significantly correlated with improvement of haemolysis parameters. in 21% of aiha patients the number of igg fluctuated and it was a poor prognostic factor. conclusion: in aiha patients the dynamics of the changing number of igg autoantibody molecules per rbc is a more helpful diagnostic and prognostic parameter than the number of igg molecules per rbc evaluated in one test. -b+) , -h-negative (using the anti-h lectin). antibody work-up showed a positive antibody screen (liss and peg-tube methods) reacting 4+ with o rbcs at all phases and 2+ with a1 rbcs. the direct antiglobulin test (dat) was positive with polyspecific ahg as well as anti-c3b,-c3d (table 1) . prewarming of test system did not change the reactivity (4+ at antiglobulin phase). a treatment with dithiothreitol (dtt) was performed and abolished all reactivity of the serum ( table 2 ). autoabsorption of the patient's plasma was performed. the absorbed plasma showed a decrease in reactivity from 4+ to 2+ when tested with o red cells, as well as a significant reduction in antibody titer from 1 : 256 to 1 : 2 tested at immediate spin (table 3 ). the patient remained crossmatch incompatible with o and a rbcs, but was compatible with oh rbcs. summary: we report an unusually strong igm anti-h antibody in this patient, who may require oh phenotype units. the patient is not a para-bombay since her red cells type strongly as group a. the cause for the auto-anti-h remains unknown at this time. if a thermal amplitude test shows that the antibody appears to be clinically significant the patient should receive h-units if transfusion is required. introduction: fetomaternal haemorrhage may determine an alloimmunization, in fact the transplacental passage of antibodies may cause the haemolytic disease of newborn. for this reason, in pregnant women, a screening for irregular antibodies research is routinely performed. however the indirect antiglobulin test (iat) may result falsely positive or negative for various causes, as operative mistakes or low specificity/sensitivity of the used techniques. aim of the study. in this study we have retrospectively evaluated the real incidence of alloimmunizations occurred in women screened by private laboratories. methods: we have studied 1.560 women, 19-43 years old, resulted iat positive at the first screening and successively assisted by our two hospitals. all women were re-tested, using gel-agglutination technique, for both direct antiglobulin test and iat. results: a positive iat was confirmed only in 64 cases; moreover a rbc autoimmunization was found in 7 women. anti-d (16 cases), e (10), c (8), k (8), c (6), s (5), d + jka (1), d + s + e (2), d + c + k (2), m (3), c + e (1), d + c + g (2) were the identified alloantibody specificities. anti-s, -e, -k, -c and -jka were the specificities in autoimmunized women. conclusion: in conclusion, a real alloimmunization is occurred only in 4.1% of screened women, while in the remaining cases iat resulted falsely positive: this observation forces us to affirm that, in order to minimize errors and alarmisms, the screening for antibody research in pregnant women must be performed only by immunohematology qualified center. background: one of the problems of the rbc transfusion is the alloimmunisation and the delayed haemolytic reactions (dhtr). besides rhesus and kell systems the antibodies against kidd antigens cause both dhtr and difficulties in their detection. aim and methods: the exact recording of all blood units according to abo rhesus kell and kidd systems. the abo-rh-kell systems are identified through automated microcolumn method (autovue, ortho), while kidd antigens are identified manually using microcolumn gel (diamed). results: the percentage of jka+ and jkb+ found in our department (75.8%) and (70%) respectively is similar to that of the caucasian population. conclusions: given the fact that there is lack of available freezing rbc system in greece, detailed recording of all units to the above antigenic systems can be proved extremely useful under circumstances of incompatibility. in the latter case suitable donors can be called and cover the shortage. the identification of all antigentic systems of the donated rbc units is underway. background: in many countries transfusion recipients are currently typed and transfused d-positive, if their red cells are agglutinated by 2 igm monoclonal anti-d that do not react with dvi. the transfusion strategy in weak d patients is not clear defined and it depends on the chosen monoclonal reagents and methods. patients who are carrying dw types 1, 2 and 15 were prone to develop anti-d. aim: the aim of this pilot study was to estimate capability of commercially available monoclonal anti-d reagents to recognize this weak d types as rhd positive. material and methods: edta anticoagulant blood samples were collected from 50 blood donors, previously typed as weak d positive by indirect antiglobulin test. molecular genotyping of rhd gene and weak d alleles by cde-ssp and d weak-ssp kits (inno-train, germany) were performed. direct agglutination was tested in a tubes and microplates using the following antibodies: rum-1, th-28, ms-201 (bioscot/serologicals) and d415 1e401/175-2 (immucor). results: out of 50 samples molecular typing results were as follows: in 5 samples dw were not determined, in 45 samples, 20 weak d type 1; 12 weak d type 2; 8 weak d type 3, 1 weak d type 11; 3 weak d type 14 and 1 weak d type 15 were determined. by all monoclonal reagents 95% weak d type 1, 100% weak d type 2, 10% weak d type 3 and 100% weak d type 15 negative results were given. by all monoclonal reagents weak d type 11 and weak d type 14 positive results were given. conclusion: according to weak d types, which were known to be at risk for anti-d immunization further advances may be brought by improved patient's monoclonal typing reagents with a low and donor's monoclonal typing reagents with high affinity for weak d type 1, type 2 and type 15. such improved typing strategies with novel reagents would enhance the transfusion safety. background: vel is a high-incidence antigen found in >99% of the population. anti-vel can be igm or igg and reacts optimally at iat, although it can also react at immediate spin and 37c. anti-vel may or may not cause severe hemolytic transfusion reactions and mild to severe hdn. autoanti-vel has also been reported. the aabb technical manual 14th edition states that the vel antigen is unaffected by protease and sulfhydryl treatment. we have reason to believe that sulfhydryl treatment may have an effect on the vel antigen as evidenced by a recently referred case. case report: a 67 year-old caucasian female presented with symptoms of anemia and renal vascular hypotension. transfusion history indicated multiple red cell transfusions in 1959. according to the patient, previous attempts to locate compatible units were unsuccessful. the case was referred to our laboratory. the patient's red cells (rbcs) typed as group o, d+ with a negative dat. the serological picture revealed an antibody reacting 2 + -3 + s at 37 c/liss, as well as at the antiglobulin phase. the antibody reacted with all rbcs tested and the autocontrol was negative. further characterization of the antibody showed similar reactivity using enzyme-treated rbcs (0.1% ficin) and no reactivity using 0.2 m dithiothreitol-(dtt)treated rbcs. a high incidence negative red cell panel (untreated) was selected that lacked antigens reported to be destroyed by dtt. the antibody reacted with all rbcs tested. additional rbcs were tested that lacked high-incidence antigens, including vel. the antibody did not react with four vel-rbcs tested using liss and peg methods. the patient's rbcs typed as vel-negative. all other clinically significant antibodies were ruled out using vel-or dtt-treated rbcs. based on the unusual reactivity demonstrated by the anti-vel, we tested 12 different examples of anti-vel (frozen in our rare sera inventory) against two sets of known vel+ and vel-rbcs. one rbc set was dtt-treated; the other set was tested neat. liss enhancement was used to test both sets. one of the 12 antisera failed to react with the positive control and one reacted with the negative control. both were disqualified from the study. four of ten remaining antisera demonstrated a decrease in reactivity >1 grade, between the neat and the dtt-treated rbcs. the remaining six antisera showed no change in reactivity. conclusion: contrary to the statement in the aabb technical manual, we discovered that sulfhydryl treatment (0.2 m dtttreatment) can have an effect on the vel antigen. our experience has demonstrated that in some cases anti-vel may not react or may show reduced reactivity when tested with dtt-treated rbcs. therefore, the presence of anti-vel should not be ruled out if negative reactivity with dtt-treated rbcs is encountered. additionally, dtt treatment may be a useful tool obtaining rule-outs of other clinically significant antibodies in the presence of anti-vel. additional data is needed to confirm these findings. but with no identified specific antibodies were investigated by repeated screening/crossmatch, papainized panel identification, hla antibody screening by lymphocytotoxicity test (lct) and elisa in some cases. patients' age, sex, department, diagnosis, previous transfusions/pregnancies, techniques, reactions' strength, number of positive cells, urgency, subsequent antibody tests, identification and lct were noted. antibody tests were performed: at pretransfusion testing (pt) by liss-coombs (diamed) and at blood grouping (bg) by biovue polyspecific (ortho) microcolumns -manually in urgency and routinely by sampler iif (diamed) and mitis (ortho) systems. results: investigated reactivity was recorded in 241 samples from 186 patients; 29 (15.6%) patients had > 1 episode. these findings comprised 15.1% of 1598 unexpected results found at pt and bg. incidences were 0.18% at routine and 0.21% at urgent bg (37 339 and 16 003 bgs, respectively), and 0.35% both at routine and urgent pt (15 803 and 23 705 pts, respectively). 56.5% patients were female, 44.6% over 60, but 19.9% < 30 years, coming mostly from surgery (19.4%), internal medicine (15.1%), hematology (10.8%), ginecology (10.2%), cardiac diseases (9.1%) and cardiac surgery (8.6% patients). frequent diagnosis were solid tumors (16.1%), cardiac diseases (13.4%), hematologic malignancies (8.6%), uraemia (4.8%), orthopedic surgery (4.3%) and hepatic diseases (3.2% patients). 43.0% patients were previously transfused, with only 9.1% patients proved as not transfused or pregnant. subsequently positive antibody test during the study had 27.6% tested patients. at pt positive crossmatch was found in 38.8%, antibody screening in 43.9% and both tests in 17.3% cases. majority of reactions were '1+' (50% at pt and 38.6% at bg); reactions '3+' or '4+' were found in only 5.1% cases at pt, compared to 17% at bg. one crossmatch only was positive in 75.2% positive crossmatches, with 38/43 patients having > 1 crossmatched unit. ahg identification was positive in 27.6% tested patients; in 26% of them papainized panel was also positive. lymphocytotoxic antibodies were found in 45.5% tested patients; 72.6% (8%-100%) of lymphocytes were reactive. finally, the cause of reactivity in antibody tests was determined as 'laboratory mistake' in 42.5%, hla lymphocytotoxic antibodies in 9.7%, 'igg antibodies of unknown specificity' in 7.5% (hla noncytotoxic antibodies in 2/2 elisa tested samples!), contaminated sample in 5.9%, anti-bga in 4.8%, non-specific cold antibodies in 3.8%, subsequently recognized specific antibodies in 3.2% (2 lua, 2 m, 1 kpa, 1 yka), non-specific autoantibodies in 3.2%, carry-over of dat-positive cells and antibody to reagent in 2.2% cases each, while in 4.3% cases antibody screening and in 4.8% cases crossmatch was repeatedly positive without confirmation in panels. discussion: after introducing of sensitive microcolumns, positive antibody tests without detectable specific antibodies require significant laboratory activities, particularly in older patients with malignancies or surgery. such reactivity was frequently caused by laboratory mistake, but often hla and sometimes specific antibodies were later recognized, or reactivity continued without confirmation in panels. relationships that may be helpful are further discussed in abstract part ii. results: significant differences (p < 0.05) and relationships of interest were noted. sex. in female vs male patients frequent features were: crossmatch as only reactivity at pt (45.8% vs 28.2%), positive lct (59.4% vs 26.1%), lymphocytotoxic hla antibodies (lytxab) (13.3% vs 4.9%) and reactivity 'positive screening, negative panels' (5.7% vs 2.5%); in males non-specific cold antibodies (7.4% vs 1.0%) and antibodies to reagents (4.9% vs 0) were noted. age. in patients > 60 vs < 30 reactivity was often found at pt (65.1% vs 35.8%), caused by lytxab (21.7% vs 5.4%), anti-bga (8.4% vs 2.7%) or 'positive crossmatch, negative panels' (9.6% vs 0), but rarely by laboratory mistake (37.3% vs 59.5%) or later recognized antibody (1 of 6 patients). subsequent antibody tests: tests were subsequently positive more often if reactivity was found at pt (79.3% vs 20.7% at bg), as positive antibody screening (41.7% vs 29.2% if positive crossmatch), with positive panels (48.3% vs 12.2% if negative). subsequent tests were positive in only 43.8% patients with lytxab, 50% with anti-bga, 1 of 3 with antibodies to reagent and in no case with 'positive crossmatch, negative panels' . techniques. lct was positive in 60% and 45.5% tested samples found at urgent and routine pt (diamed), vs 0 at bg. all 18 cases due to lytxab, 8 of 9 anti-bga and 6 of 8 'positive antibody screening, negative panels' were found by diamed. at bg (ortho) 71.4% cold antibodies and 64.6% laboratory mistakes were found. positive antibody test: identification was negative in 78.6% screening-only cases; 69% of them were caused by laboratory mistake. lytxab were found in 67.9% crossmatch-only cases; 77.8% reactivities caused by lytxab were crossmatch-only. identification. ahg panel was positive in 60% cases with lytxab (in 2 with papainized panel) and often due to 'igg antibodies of unknown specificity' (29.7%), anti-bga (24.3%), contaminated sample (16.2%), but also to later recognized specific antibody (10.8% cases). strength of reaction: laboratory mistake was noted in 61.6% of 'w', 41.3% of '1+', 46.4% of '2+' and 58.8% of '3+ and 4+' antibody screenings (ns). diagnosis. in patients with solid and hematologic malignancy reactivity was often found at pt (80% and 81.3% of patients, respectively), due to positive crossmatch (40% and 50%; 0 and 4.3% patients with liver and cardiac diseases) and caused by lytxab (16.7% and 31.3%) or 'crossmatch/screening positive, panels negative' (16.7% patients with solid tumors). in patients with liver and cardiac diseases reactivity was often found at bg (83.3% and 68.0% of patients), due to laboratory mistake (50% and 60%) or cold antibodies (16.7% and 12%), respectively. discussion: features of non-specific reactivity depended on sex, age, positive antibody test, diagnosis and, moreover, used techniques, sometimes in very distinctive manner. this analysis might be of considerable help in planning of laboratory tests, but also in quick analysis of unexpected results and choice of further testing, particularly in urgent situations. background: worldwide screen and type is a very usual method for pre-transfusional testing. the ultimate objective is to prevent not only the clinically expressed delayed hemolytic transfusion reactions but also the serologically revealed ones. aim: the aim of this study was to determine the frequency of red blood cell (rbc) alloantibodies in patients undergoing cardiac surgery or cardiac procedure, during the pre-transfusion screening. materials and methods: blood samples of 16 291 patients (12 628 male and 3663 female) were evaluated. the mean age of the patients was 57 years. pre-transfusion samples were examined for clinically significant alloantibodies, using antibody screening with gel test (liss -enzyme). in case of a positive result, identification was performed (panel with autologous control). in addition, the serological testing included cold agglutinins´ detection (tube test), as well as titration (tube test) and identification (gel test) in case of a positive result. when the result was marginal (1/32) a new test was carried out after a seven days period. in the presence of a positive autologous control or an autoantibody, samples were examined with direct antiglobulin test (dat). results: alloantibodies were detected in 580 patients with the incidence of 3.56%. antibodies were registered more frequently in females (190/3663, 5.18%) than in males (390/12 628, 3.09%). 183 patients (31.49%) developed single antibody with anti-kell being the most frequent. the incidence and the specificity of the detected antibodies are summarized in the following table (table 1 ). in 23 patients (3.96%) multiple antibodies were detected, with most frequent the anti-d and anti-c combination. 35 patients (6.03%) were dat positive. autoantibodies were found in 10 patients (1.72%), all of which had specificity to rhesus system. cold agglutinins were positive in 53 patients (0.32%). no specificity could be assigned in 245 patients (42.24%), while in 110 patients (18.96%) non specific reactions in enzyme treated rbcs, were observed. one patient developed delayed haemolytic reaction 15 days post-transfusion, due to anti-jka. the antibody, however, was not detected in the pretransfusion sample re-testing. the frequency of the pre-transfusion detection of red blood cell alloantibodies in our center, was 3.56%. the most frequently identified were the anti-kell and anti-rh. the high rates of unidentifiable antibodies and non specific reactions in enzyme treated rbcs are probably attributed to the kind of medication that most of these patients receive, as well as to the degree of inflammatory process which usually accompanies such diseases. the high frequency of unidentifiable antibodies indicates that a larger and more complex erythrocyte panel would be useful for routine testing. the routine pre-transfusion screening for alloantibodies probably assures the prevention of dhtrs and provides sufficient time for blood selection for transfusion. introduction: in the united kingdom, about 1% of women form red cell allo-antibodies in pregnancy and 0.07% of all pregnant women produce anti-c. before the introduction of prophylactic anti-d, it was reported that 3% of the total haemolytic disease of the newborn (hdn) cases were due to anti-c. currently, cases of hdn due to anti-c are half as frequent as anti-d and 14% of the uk population are rhc negative. we present two unusual cases of pregnant women who are d and c negative and have anti-c and anti-d detected in their serum. case studies and results: case 1: a 38-year-old asian woman had three previous uneventful pregnancies. in her 4th pregnancy she presented with miscarriage at 19 weeks gestation. she was group b, d and c negative. her serum contained anti-c and anti-d. anti-d was detected by liss tube iat and anti-c was only detected by manual polybrene technique (0.9 iu/ml by quantification using r2r2 cells). in a 5th pregnancy, no antibodies were detected until 34 weeks gestation when this patient presented in early labour. anti-c was then detected by two-stage papain technique only as well as anti-d. case 2: a 33-year-old asian woman had a positive antibody screen post caesarean section in jan 2005. no antibody had been detected during the pregnancy. standard prophylactic anti-d was given at 28 and 34 weeks gestation as this patient was d neg. anti-c and anti-d were confirmed in her serum. the anti-c level was 0.7 iu/ml using rr cells and the anti-d level was <0.1 iu/ml using r1r1 cells (prophylactic anti-d ig). she was phenotyped as o r¢r¢. at delivery the baby was found to have a negative dat and was phenotyped as r¢r. discussion: cde/cde (r¢r¢) is an uncommon phenotype in the uk population with a frequency of 1/10 000. in routine antenatal testing, abo/d grouping is only performed for pregnant women at booking and 28 weeks gestation according to bcsh guidelines. full rh phenotyping is not carried out unless the pregnant women has a positive antibody screen. in routine testing of the above cases, this extremely rare phenotype is missed. prophylactic anti-d was given to both patients and immunisation due to anti-d was prevented. there is currently no prophylactic regime developed to prevent anti-c allo-immunisation by the fetus in pregnancy. antenatal management of patients with anti-c and anti-d during pregnancy can be problematic: (i), problem in antibody identification; (ii) monitoring of antibody level (i.e. quantitation by auto analyser for anti-c and anti-d) with two different cells and (3) provision of blood during pregnancy, at labour and post delivery for both mother and newborn. study on the frequency of red cell phenotypes (e.g. duffy, kidd and mns blood group system) in our local population l leou, yf wong, mbc koh and d teo health sciences authority, singapore, singapore background: the frequency of various red cell antigens in the caucasian population has been well studied. to date, the frequency of these antigens in the local population composed of a multi-racial mixture of chinese, malay, indian and others is still unclear, especially in the malays with paucity of data in the literature. aims: to investigate the frequency of clinically significant red cell antigens duffy, kidd and mns across the local ethnic groups. to investigate the occurrence of rare phenotypes. to be aware of these rare phenotypes so as to facilitate planning of blood inventories and supplies. typing for the duffy, kidd and ss antigen on blood donors was performed using monoclonal as well as polyclonal anti-sera by manual tube method. a total of 1427 blood donor samples were tested using specific anti-sera that will agglutinate red blood cells that have the corresponding antigen. agglutination is demonstrated by the indirect antiglobulin technique. result and discussion: table 1 -a higher frequency of the fy(a+b-) phenotype is seen in chinese, malay and others in contrast to the indian and caucasian population. the clinically significant allo-antibody anti-fya is rarer in our local population. it occurs predominantly in malays and indians and usually in combination with other antibodies. it means that provision of antigen negative blood may be difficult. table 2 -all groups show similarity of distribution of the kidd phenotype with the caucasians and distinct from the american blacks. the jk(a+b-) and jk(a+b+) phenotypes are relatively equal in frequency and there should be no problem looking for such a phenotype in the local population. table 3 -the s-s+ phenotype is most common amongst all races. the indians are more similar to the caucasians with a relatively high frequency of s+s+ phenotype. the chinese and malay distribution are unique with > 99% being s+. conclusion: there is a unique distribution of red cell antigen groups in the 4 races and the data on malays is especially useful. this data will allow the national blood service in its inventory planning and the potential difficulties of providing antigen negative products due to clinically significant allo-antibodies. miltenberger phenotypes among taiwanese table 1 . conjointly, in order to obtain the frequency of mi.v phenotype, we screened 543 samples among 1230 with anti-hil and four additional cases of mi.v were found. conclusion: in this study significant miltenberger polymorphism was seen among the taiwanese population. besides previously described mi.iii phenotype (1.9%), there were also mi.i/ii phenotype (0.4%), mi.v phenotype (0.7%), mi.vi phenotype (0.7%), mi.x phenotype (0.1%), and most interestingly two miltenberger related not yet classified variants (1%). related variant a (0.5%) was phenotypes as mia+, anek+ and hil+. related variant b (0.5%) was phenotyped as mia+, anek+. interestingly, both variants were mur-(negative). the total estimated frequency of miltenberger variants in taiwanese population (including related variants a and b) is therefore 4.8% (table1). the discovery of 2 unclassified variants (most likely not yet described in the literature) is of great interest in the field of immunohaematology and warrant further molecular genetic study. introduction: the use of column technologies for the detection of rbc antibodies improved significantly the screen test sensitivity. each column-based method has its advantages and disadvantages. aim: to compare antibody detection by two column agglutination tests; the fully automated ortho auto vue tm method and the manual diamedᮀ id-micro typing system. material and methods: during the study period 57 375 patient samples were screened, 414 of the positive results were evaluated. 377 blood samples with positive screen tests by the ortho auto vue tm method (av) performed with 3% cell suspension, and 37 patient samples with antibodies identified by the diamedᮀ system (dm), were reciprocally re-screened, respectively. positive samples were tested by diamed panels for antibody identification. sera samples were divided into 6 categories according to antibody specificity; 1. rh system antibodies (n = 150), 2. clinically significant non rh system antibodies (n = 85), 3. clinically non significant antibodies (n = 28), 4. auto antibodies (n = 13), 5. not identified (ni) antibodies (n = 48), 6. negative screening results by the diamed technique (n = 90). the 6 categories were divided, according to the intensity of agglutination in the screen test, into those exhibiting stronger reactions by the auto vue (av > dm), those exhibiting equal strength reactions (av = dm) and those with weaker reactions by the auto vue (av < dm). statistical analysis was carried out using the wilcoxon signed ranks matched-pairs test. results: a total of 414 samples were compared, 31.2% of them gave stronger reactions by av, 45.4% gave equal reaction strength and 23.4% of them gave weaker reactions by av. positive screen tests by av only were detected in 90 samples, no specific antibodies were identified. in contrast to that, positive screen tests by dm only were detected in 9 samples, 5 were rh system related, 3 kell system related and 1 not identified, results are summarized in the table. discussion and summary: the antibodies detected by dm only, are anti-d and antibodies directed to low frequency antigens. the failure of av to detect rh system antibodies is further sustained by the fact that statistically significant weaker reactions for this antibody system were obtained by av technique. recently ortho-clinical diagnostics modified the screening reagent red blood cells to 0.8% suspension, in order to improve the sensitivity of the method (unpublished data). the possible explanation for the failure to detect low frequency antibodies is that ortho screen cells do not consistently carry the low frequency antigens cw and kpa. positive screen tests detected by av only, can be explained either by the fact that antibody identification was carried out on dm panels, or these are false positive reactions. in order to clarify this question we recently repeated these av only positive samples by the manual ortho bio vue technique. preliminary results indicate that no specific antibodies were detected. it can be assumed that these results are false positive. further study of this issue is required. aim of the study: we have detected blood donor with rohar variant which was mistaken as d-and his donations used for d-recipients. we tested these patients in order to evaluate possible anti-d or anti-lfa immunization. methods: rohar variant was tested serologically (commercial and workshop moabs) and on dna level (pcr-ssp). involved recipients were tested by diamed column agglutination (gliat with normal and enzyme treated rbcs) with commercial rbcs and with rh33 and rh50 rbcs. results: rohar variant was confirmed on phenotype and genotype levels. in four d-and two d+ recipients of rohar positive units no anti-d not anti-rh33 or -rh50 antibodies were detected. in one case anti-le(a) antibody was found. conclusion: in our cases massive exposition of recipients (whole transfusion unit) by rohar red cells did not lead to production of detectable anti-d or anti-lfa. the immunogenic potential of this variant seems to be low. unusual ab0 grouping discrepancy -inhibition of anti-b reagent by isolated increase of plasmatic b substance in a patient with group ab and pancreatic cancer m pisacka*, k petrtylova † , m kralova* and h flidrova* *uhkt, prague 2, † blood bank, faculty hospital m, prague 5, czech republic introduction: in rare pathological conditions excess amount of blood-group specific substances can be observed and can cause neutralization of grouping reagents. changes in abh and related histo-blood group antigens in malignant tissues were described but there are few information about similar changes in secreted bloodgroup specific substances. aim of the study: we describe a case of isolated increase of b group substance in plasma of a group ab patient with pancreatic cancer. methods: ab0 grouping was performed with registered immucor reagents (immuclone: anti-a birma 1, anti-b lb2) by slide and tube tests. neutralizing effect was quantified by (i) inhibition od anti-b reaction by titrated patient's serum; and (ii) inhibition of titrated anti-a and anti-b reagents by patient's serum, compared to ab serum of a healthy donor. results: slide test: unwashed rbcs: group a, washed rbcs: ab. tube test (washed rbcs): ab. titrated patient's serum when added in aliquot to anti-b reagent inhibited agglutination up to titre 64. titration of reagents /+ aliquot of serum added/: anti-a: titre 4096 (both patient's serum and control); anti-b + patient's serum: titre 4, anti-b + control: titre 4096. conclusion: pancreas is known as rich source of blood group specific substances. malignant transformation is known to be associated with either loss or re-expression of cell-bound abh antigens. reported excessive increase of b substance in group ab patient could be either due to loss of a-transferase activity in malignant pancreas cells or isolated increase of b-transferase activity. further studies on larger groups of pancreatic cancer patients will help to understand this observation. weakened abh reactions of unwashed rbcs could be of diagnostic importance, because in other case this observation preceded several years the pancreatic cancer clinical manifestation. implementation of the autovue innova, an upgraded column agglutination technology (cat) for pre-transfusion testing in a large blood establishment c politis, k armyros, a antypas, v malamou and p katsea g. gennimatas general hospital, athens, greece objective: automated cat testing in a blood transfusion laboratory aims at standardization and savings in labour as well as reagents. a new technology is evaluated in comparison to standard methods. materials and methods: we used column agglutination technology with the innova autovue system, ortho, ratrian n.j. according to the manufacturers this system provides priority to the management of the stat samples while the random access feature of the system enhances the system throughput. it provides an extended test menu as well as automated antibody identification with the red cells panels. the autovue innova system supports the bi-directional communication with the lis interface for all the tests including the crossmatches and it provides a continuous traceability and notifying of the instrument's status concerning either the required and available resources or the proper function of the system's submodules. in this study we performed 1423 tests including forward and reverse abo group, rh type and phenotype and kell in randomly selected blood donors, as well 895 tests in haematological patients for antibody screening using an untreated three-cell panel and autocontrol in the indirect antiglobulin test (iat). the results and the time performance (specimen handing, operation of testing and recording of results) were compared with those obtained by the semiautomatic id-diamed gel agglutination microtyping system and by standard manual methods. results: test results showed 100% agreement between all three methods. 68 samples were tested per 60 min with the autovue innova, compared to 90 min required for the same number of tests performed with manual testing and 70 min with the semi-automated method. the technical execution was easy with the autovue innova procedure and it appears that the consumption of testing reagents is smaller with the automated system comparing with the other methods. computerization of the test results with the autovue innova provides an important advantage in record keeping in the blood establishment. conclusion: standardization of sample collection and tests performance in pre-transfusion testing, as well as computerized records and time saving are advantages offered by the autovue innova, a new automated column agglutination technology, comparing with a semi-automated and the classical manual methods. a heavy workload is expected to significantly decrease time performance. clearance of senescent erythrocytes in young and old individuals al racca, a ensinck, c cotorruelo, s garcía borrás, l racca and cs biondi universidad nacional de rosario, rosario, argentina introduction: after a lifespan of 120 days, human red blood cells (rbc) are captured and phagocytized by monocytes/macrophages. the accumulation of autologous igg on rbc membrane provides a direct mechanism for the removal of senescent (se) rbc. an alternative pathway, immunoglobulin-independent, with participation of sialic acid, has been proposed. the physiological elimination of serbc might be modified by individual's age. aim of the study: to investigate in young and old individuals, the interaction between monocytes and different erythrocytes suspensions: serbc, rbc stored with or without serum and desialinized rbc. methods: healthy individuals blood samples (20-40 years old, n = 50 and > 70 years old, n = 49) were studied. different suspensions from each sample were obtained: (i) se and young (y) rbc by differential centrifugation; (ii) rbc stored with its own serum (rbcs) and without serum (rbcws); and (iii) rbc desialinized with neuraminidase (ne) and tripsine (t). the suspensions were subjected to the erythrophagocytosis assay: peripheral blood monocytes were incubated with the different erythrocyte suspensions for 3 h at 37°c. two hundred cells were analyzed to determine the percentage of active monocytes (am) with phagocytosed and adherent red cells. non sensitized rbc (nrbc) and ex vivo sensitized rbc (srbc) were used as negative and positive controls respectively. results: the% of am obtained with old individuals were: serbc: 21.9 + 1.0, yrbc: 2.9 + 1.2, rbcs: 16.2 + 1.4, rbcws: 3.1 + 0.8, nerbc: 11.1 + 1.3, trbc: 3.2 + 1.1, nrbc: 3.0 + 1.2; srbc: 31.2 + 1.8. the values of am obtained with young individuals were: serbc: 17.1 + 1.5, yrbc: 3.1 + 0.9, rbcs: 11.1 + 1.1, rbcws: 2.8 + 1.2, nerbc: 10.8 + 1.4, trbc: 3.5 + 1.0. nrbc: 2.9 + 1.3; srbc: 30.5 + 1.7. conclusions: no differences in the% of am were found when compared to positive and negative controls, indicating that this assay would not detect variations in the phagocytic activity of monocytes from young and old donors. the values of am with serbc were higher (p < 0.001) than those obtained with yrbc in both populations analysed. the rate of erythrophagocytosis with serbc in old individuals was significantly higher (p < 0.001) than that obtained in young donors. the increase observed may be due to agedependent changes of rbc that occur with human ageing. the% of am with rbcs were higher (p < 0.001) in old individuals. no modifications were observed with rbcws. the significant increase in the rate of erythrophagocytosis with serbc and rbcs show the involvement of autologous igg in the selective removal of erythrocytes. these values were higher in old individuals indicating that this process would increase in aged donors. the tripsine activity was not enough to modify the% am. the values of am obtained with neuraminidase treated rbc were higher than those observed with yrbc (p < 0.001). however these values were similar between young and old individuals, suggesting that the desialylation would not participate in the increased removal of erythrocytes observed in old donors. cell-cell adhesion is a crucial phenomenon for the relationship between the cell and its environment. therefore, the development of experimental methods to obtain quantitative parameters of cellular adhesion is important. erythrocytes are widely available and their membrane properties are well known, so these cells are used as an ideal model for studying the cellular interaction mechanisms. the study of the formation and break-up of receptor-ligand bonds in sheared erythrocyte suspensions is a subject of considerable importance in the blood circulation, where formation and break-up of blood cell aggregates occur in a variety of physiological and pathological conditions. the main two objectives of this work were to study the cellular adhesion phenomenon using erythrocyte adhesion mediated by monoclonal anti-a antibody as a model and to achieve quantitative values of the parameters involved in the intercellular binding and its possible relationship with cellular deformability parameters. agglutinates of two a erythrocytes (doublets) induced by specific monoclonal antibodies anti-a were used. adhesion energy was indirectly quantified by the study of doublet dissociation under the effect of a given shear stress using a controlled flow chamber system. the chamber was installed on the stage of an optical inverted microscope (union optical, magnification 100¥) in such a way that the cover glass was the floor of the microchannel. a ccd (charge coupled device) camera was placed in the ocular tube of the microscope and connected to a digital image processor (ipplus system) to digitize, record and analyze microscopic images. the doublets were initially immobilized inside the chamber, and then put under different shear stress values by a controlled laminar flow during a definite time. in this way, a shear stress parallel to the contact surface between both cells was applied, observing that the upper cell detached progressively with time and also with a gradual rise of the shear stress. the sequential microscopical images were registered and digitally processed, measuring the geometrical dimensions that the cells acquire during the deformation process, and the dissociation of the antigen-antibody bond. digital image processing allows the analysis and the quantification of the red blood cells doublets dissociation phenomenon. these results show that, while the shear stress applied is raised, the contact area between both cells diminishes. the obtained parameters give important information that lead to the estimation of the value of adhesion energy between two red blood cells agglutinated by monoclonal antibodies. as consequence, they will allow the characterization of the antibodies used, since it would evaluate their association capacity with cellular antigens. glycophorins (gp) a, b and c are abundant transmembrane integral proteins in red blood cell (rbc). their highly glycosylated nature and high sialic acid content account for the net negative charge of mature rbc membrane, which is physiologically important because it impedes any tendency to stick together in the circulation. in this study, we have tested anti-gp specific mouse monoclonal antibodies (moab) as primary antibody to directly agglutinate rbc. fret technique has been developed to characterize the hemoagglutination based on the interaction of fluorophores (alexa488tm, dio and dii) located in the rbc membrane or combined to secondary antibody directed against the primary agglutining moab. combined intensity (spectral) and lifetime (flim) imaging was used to discriminate the fret signal of molecules on their different lifetimes whereas their emission spectra overlap (alexa488tm or dio/dii) as independent phenomena of the fluorophore concentration and photobleaching. furthermore, gp-cytoskeleton interactions were analyzed by 3d-fluorescence microscopy after lipid extraction with triton x-100 in red cell. all the antibody used was found to directly agglutinate the human rbc. in view of the fluorescence properties depicted in rbc for the pair of fluorophore alexa-488tm (donor) and dii (acceptor), it may be expected that upon agglutination of rbc, effective fret should be observed. flim in dynamic-state provides a discrimination of molecules in their fluorescence lifetime, which allows to evaluate the underlying mechanism of energy transfer process in the agglutinated erythrocytes. the results demonstrate that's upon excitation at 780 nm the flim-fret from alexa488tm to dii for the anti-gpb moabs only with a lifetime distribution in the picosecond range. similarly, effective fret was not observed for the anti-gpa moabs. the contrast in measured lifetime image is a reliable indicator for spatial variations in donor-acceptor association. 3d-fluorescence microscopy images showed interactions between gpc or gpb and cytoskeleton and did not show interactions between gpa and cytoskeleton. all together, these results only revealed by fret-flim and 3d-fluorescence microscopy strongly support the importance of the specific reactivity with glycophorin a or b of agglutining moab. introduction: the frequency of alloimmunization to red blood cell antigens in transfused sickle cell patients can range from 10 to 40%, but the development of autoantibodies is much less recognized and descried. aim of the study: in order to evaluate the autoantibody formation and observe the blood transfusion association, we analyzed the direct antiglobulin test (dat) as well as the antibody screening results in 612 transfused sickle cell patients. methods: all the patients included in the study had received at least 1 unit of red blood cell concentrate, on the majority of the cases matched for the c, c, e, e, k antigens. the transfusion range was 1-94 units. the dat performed was a polyspecific gel test. in cases of positive dat, was performed the monospecific gel test (igg, iga, igm, c3c, c3d) . in almost all of cases an acid glicin elution was performed and the eluate was tested against a red cell panel (liss/coombs and papain). an antibody screening by gel test (liss/coombs and papain) was performed in all the 612 patients. the dat was positive in 98 (16%) of the 612 patients. in 96 cases (98%), the dat was igg type, in 1 case (1%) igg + c3d and in 1 case (1%) igg + c3c + c3d. the acid elution was performed in 97 cases. the eluate was positive in 38 cases (39%). in 32 (84%), of the 38 cases, autoantibodies were pointed out whose specificity were 25 specificity public, 5 anti-e (rh5), 1 anti-c (rh2), 1 anti-ce (rh7). in 6 cases (16%) we found alloantibodies whose specificity were 2 anti-e(rh3), 2 anti-k(k1), 1 anti-c (rh2) and 1 anti-jka (jk1). in the 59 cases (61%) no antibody was identified on the eluate and the cause of positive dat is unclear. we could correlate the positive dat with a presence of autoantibodies in 32 (5.2%) of the 612 patients. of these, 13 (40%) had a blood transfusion association. the global frequency of red cell alloimmunization in this set of 612 patients was 15%. seventeen patients (53%) of the 32 patient who had autoantibodies had also alloantibodies associated. conclusion: the mechanism by which erythrocyte antibodies form in association with blood transfusion are not well understood, but even though the medical literature indicates strong association with blood transfusion as well erythrocyte alloantibody formation, we could not find support for this association. the loss of splenic to sickle cell patients could be important because experimental studies suggest that the spleen is involved in the regulation of autoantibody formation. forward and reverse blood grouping with lateral flow based assays p schwind*, i aebischer*, k loester † and p monod* *medion diagnostics gmbh, duedingen, switzerland, † prisma diagnostika gmbh, berlin, germany background: recently, a lateral flow assay for simultaneous typing of abod, rhesus subgroups and kell with stable end-point and without a centrifugation step was presented (loester k, fleischhauer s, schwind p: lateral flow assay for simultaneous typing of of abo, rhesus subgroups and kell. vox sang 2004, 87 (suppl. 3), 40). in many countries, the determination of isoagglutinins in addition to the red cell antigens is mandatory for abo grouping. aims: to develop a lateral flow test for reverse grouping, supplementing the lateral flow typing assay. a lateral flow device was constructed with a separation membrane equipped in a cassette housing having 4 distinct incubation wells, application zones and detection areas. 25 microliters of 5% suspensions of reagent red cells for reverse grouping (reverse-cyte a1, a2, b, 0, medion diagnostics, switzerland) are mixed in each incubation well with 100 microliters of plasma. the resulting suspensions are incubated for 2 min, followed by the transfer of 50 microliters each to the application zones, where migration starts immediately. results can be read after 3 min in the detection areas. a positive result is recognized as a distinct red dot, a negative result is monitored by the absence of a dot. results: the plasmas of 80 donors, previously determined for the respective blood groups and isoagglutinins by the tube technique, have been tested with this method. the results for both methods were in full agreement. conclusions: a simple, rapid and flexible lateral flow method for reverse grouping is presented, allowing now for the determination of forward and reverse typing in similar formats. both methods give results after 5 min with stable end-points without the need of a centrifugation step and are easily applicable to non-laboratory environments. the performance of the autovuetm system for red cell antibody screening of blood donors during background: in israel every donation is tested for the presence of red cell antibodies (rbc abs). screening is performed on the autovuetm system, using ortho 2 screening rbc since the year 2000. aim: (i) to summarize the performance of the autovuetm system for rbc abs, during 4 years (2000) (2001) (2002) (2003) (2004) . (ii) to evaluate if an initial low positive result, with two negative repeats, could be considered a negative result. methods and results: rbc abs screening was performed using igg cassettes. positive results were confirmed by diamed gel cards, with 3 screening rbc, followed by diamed panels for abs identification. results: summary of the results is presented in the table. (i) in 0.57% of 1358,769 blood donations that were screened, during 2000-2004, using the autovuetm system, a positive initial result for rbc abs was detected, which was confirmed in 40.2% (0.22% of the donations). an increased percentage of confirmed tests is noted, since 2003, probably due to an improvement in the manufacturing of the cassettes by ortho. (ii) during the validation, 83 samples with low positive results (agglutination degree of 0.5) were retested twice on the autovuetm. 57/83 (69%) gave negative results in both repeats. only 1/57 was confirmed positive and anti-m was identified by diamed gel test. the remaining 26 samples had at least one positive repeat. 5/26 (19.2%) of those samples were confirmed positive. summary: autovuetm can be used as a competent method for rbc abs screening, in blood services which require high thoughput automated systems. samples with a low positive agglutination, which were negative in two repeats, can be considered negative. retesting these samples on the autovuetm, can reduce the number of tests sent for confirmation and allow early release of blood components. rk tagi-zadeh*, aa karimov*, ar hasanov † , ly novruzova* and si donskov ‡ *hematology and transfusiology, baku, † blood transfusion centre, ganja, azerbaijan, † centre for hematology, moscow, russian federation background: the main method of treatment of severe homozygous thalassemia forms at the moment is still an anemia control with regular rbc transfusions. the use of adequate transfusion regime for these patients not only prolongs their lives but also promotes normal physical development of the children and improves the quality of their lives. however, necessity to do multiple transfusions increases a risk of post-transfusion reactions and complications due to alloimmunization to rbc antigens. in order to prevent posttransfusion reactions it is essential to know the frequency of blood group distribution in the region and then implement organizational procedures to enhance the transfusion services for these patients. objective: examine the distribution of rbc antigens among thalassemic patients and blood donors. methods: 108 blood samples of homozygous betta-thalassemia patients (who stayed at the daytime inpatient wards of scientific research institute of hematology and transfusiology baku, azerbaijan) and 1690 blood donors of azeri nationality were examined. 108 patients' blood samples were typed on rbc rh (c, c, d, e, e) and kell (k, k) antigens. gel test bio-rad (france) was used to detect rbc antigens. results: phenotyping of the patients' rbc rh antigens (d, c, c, e, e, cw) revealed that frequency of occurrence of the antigens in general was higher than in the donors. studying of rh phenotype distribution showed that the patients' ccdee (44.1%) •• ccdee (30.4%) phenotypes were twice as much higher than the occurrence of those in the donors (20% and 16.5% respectively). phenotype ccdee occurs more frequently in the donors (32%), whereas it is significantly rare in the patients (8.8%). the similar picture was observed in relation to ccdee and ccdee phenotypes, which occurred more frequently among donors (11.8% and 10.6%) than in thalassemic patients (6.86% and 2.94%). additionally, the results demonstrated that k antigen of the kell system was not detected in the thalassemic patients whereas k antigen was detected in all the patients. the same picture was observed with two other antigens of this system. thus among the patients typed on rbc antigens kpa antigen was detected in just one case whereas kpb was detected in the rest the patients. the results of the analysis showed that for thalassemic patients with phenotypes ccdee and ccdee it is easier to find compatible blood than for patients with phenotypes ccdee, ccdee and ccdee. furthermore, it is known, that some congenital diseases are genetically connected with the specific group systems, for example, the mcleod syndrome is genetically associated with the sharp suppression of the kell alloantigen expression (absence of gene kx). the fact of the discovered absence of antigen k in homozygous bthalassemic patients makes it possible to assume, that this group of patients suffer from scarcity along the kell system, like the scarcity in mcleod syndrome. all the mentioned above make it possible for us to conclude, that prior to blood transfusion to thalassemic patients phenotyping of rbc rh and kell antigens must be carried out. hyperhaemolysis in sickle cell disease due to complement activation. tessa thorp rci national blood service manchester uk tm thorp national blood service, manchester, uk introduction: sickle haemoglobin is caused by a genetic mutation in codon 6 of the beta globin gene, resulting in the conversion of glutamic acid to valine. when critical amounts of polymer accumulate within the sickled erythrocyte cellular injury results. clinically sickle cell disease is characterised by chronic haemolysis and intermittent vaso-occlusion. mold et al. demonstrated that deoxygenation and sickling of erythrocytes is related to membrane phospholipid changes and these changes result in the activation of the alternative complement pathway. aim: the objective of this study was to measure the amount of c3c and c3d bound in vivo to red cells of homozygous and heterozygous sickle cell patients. these levels were then compared to 'normal' sickle negative blood donors. haemoglobin levels and red cell morphology were also examined for signs of active haemolysis. the hypothesis was that an increase in red cell bound complement in vivo could provide an indication of hyperhaemolysis syndrome is sickle patients. method: flow cytometric analysis using rabbit anti-c3c or anti-c3d and fitc labelled goat anti-rabbit was developed using a beckman epics xlmcl flow cytometer. control samples were prepared using a fruitstone buffer technique of complement coating. results: a total of 16 heterozygous sickle patients and 11 homozygous patients were analysed. of the 11 homozygous patients analysed only one was undergoing a sickle crisis. a positive result was indicated if the mean trait or homozygous sample was coated with complement to a greater degree that the mean normal donor plus two standard deviations. the results obtained in this research project indicate an increase in c3c levels bound to red cells of homozygous sickle patients in vivo. statistical analysis of results obtained suggest that there was no increase in c3d levels in either sickle trait or homozygous sickle patients. conclusion: these findings support research carried out by mold et al. (1995) and are present in more than 90% of caucasian population. it has been reported that anti-chido and anti-rogers don't cause hemolitic reaction but may be responsible for life-threatening anaphylactic reaction during transfusion of plasma proteins. case report: positive iat and dat were detected in a 34 years old swedish woman, a rh negative, at 34th week of pregnancy. previous iat and dat determinations were negative. at time of detection dat wsa weakly positive and igg subclasses identified. antibody identification revealed the presence of high titre (1 : 64) anti-chido and anti-rogers antibodies. a slight decrease in platelet count (from 210.000/ul to 80.000/ul) was observed. this pattern showed no variation until the end of the pregnancy. at 39th week a healthy baby was delivered, dat negative. the mother dat and iat remained positive for four months after delivery. the platelets count raised again to 200.000/ul. the same laboratory findings were detected in a previuos pregnancy in sweden. the patient reported the presence of antibodies at 35th week that disappeared few months after delivery. a healthy baby, dat negative was delivered in this case too. conclusions: this is the first report of the presence of chido and rogers as autoantibodies during the last months of pregnancy. the association with decrease in platelets count and the lack of evident clinical symptoms need further investigation. p-289 rbc alloantibody frequency and their prevalence within chinese, malay and indian community in singapore e widjaja, mbc koh and d teo health science authority, singapore, singapore background and aim: there is a recognised existence of different alloantibodies in different ethnic groups. while this has been studied in the caucasian population, their frequencies remain less well documented in the asian population. the frequency of different alloantibodies and their distribution in terms of age, sex in singapore population is studied, as were their prevalence within the chinese, malay and indian races in singapore. design and method: we conducted a retrospective study for the frequency of 28 alloantibodies on 1856 blood samples over 2004. these blood samples were largely sent by hospitals where preliminary antibody screening had been done and positive result obtained. they were sent to the centre for transfusion medicine for antibody identification. small number of these samples came from hospitals where preliminary antibody testing was not done. the antibody distribution across different ethnic groups (chinese, malays, indians) age and sex were studied. results: the most frequent alloantibodies in the population is mia (40.7%), e (20%), le a (17.5%), le b (14.8%), p1 (6.1%), m (5.4%), d (4.5%), c (2.7%), jka (1.9%) and c (1.4%). within the chinese community, the most frequent alloantibodies were similar: mia (51.9%), e (24.1%), le a (11%), le b (8.8%) and p1 (6.1%). in the malays, the most frequent alloantibodies were le a (35%), le b (26.4%), mia (24.2%), e(13.4%) and p1 (8.2%); while in the indians, these were mia (28.9%), le b (28.9%), le a (27.8%), d (17.5%), and e (12.4%), with the anti-d reflecting the higher incidence of rh d negativity in the indians race. for the lower incidence antibodies, anti-c was more common in the malays and indians (4%) compared to chinese (1.9%). anti jka tended to occur mainly in the malay race and anti-c was rare in all (<2%) reflecting the high prevalence of c in the singapore population (r1r1 phenotype). the ratio of alloimmunised male to female (m : f) is 1 : 3. most alloantibodies demonstrated significant skewing towards to the female, although relatively less so for mia where m : f ratio is almost equal at 1 : 1.9. alloimmunisation increased with age for mia, e, k, p1, jka and fyb while the frequency of alloimmunisation to lea, leb, d, m and c decresed with age. the prevalence of patients with multiple alloantibodies (2 or more) within the alloimmunised subjects is 21.7%. conclusion: anti mia is very common within the asian population especially in the chinese. anti d is common in the indians. most antibodies show increased frequency with age except for anti lea + b, d and m. the majority of alloimmunised patients are females. study aim: to identify the present antibodies in newborns, after a positive direct antiglobulin test (dat). material and method: during a 7 years period, from 1/1/1998-12/31/2004, we studied 2652 newborns and their mothers. each newborn was examined for abo group, rhesus with phenotype, kell and dat. each mother was also tested for abo group, rhesus with phenotype, kell and indirect antiglobulin test (iat). after each positive dat, the study was continued with the elution test, in order to identify the present antibody. dat test was performed with dc screening i-diamed sa, switzerland. for the laboratorial analysis of newborns the sample used was cord blood and in certain cases venous blood. results: the dat test was positive in 132 (5%) of newborns and the antibodies found were igg type immunoglobulin. anti-a antibody was detected in 89 (67.4%) (newborns of group a with mothers of group o), anti-b antibody in 25 (19%) (newborns of group b with mothers of group o), anti-d antibody in 2 (1.5%) (newborns d+ from d-mothers), anti-e in one case and anti-jka in another one. the eluate test was found negative in 3 newborns and in the rest 11, no special antibody could be identified. the results are presented in the following table. conclusion: the majority of antibodies in newborns with a positive dat test, is due to abo incompatibility (the mother belongs to group o and the newborn to group a or b). anti-d (2), anti-e (6), anti-k (7), anti-fya (5), anti-s (2), anti-jkb (1), anti-n (1), anti-kpb (1). in two patients 2 antibodies were identified, while in 24/97 (24.7%) no antibody was identified (unspecific). it is remarkable that only in 10 out of 30 patients with both dat and iat positive, an irregular antibody was identified, while the rest 20 patients had unspecific antibodies. in 19 patients with only iat positive, 15 had an irregular antibody and 4 had unspecific antibodies. in 3 out of 31 patients with both dat and iat negative the cause of incompatibility was the positive dat in the corresponding sample of the blood donor, while in the rest 28 patients the reasons were technical problems that include the inappropriate blood sample of the patient, patients under medication and errors during the crossmatching procedure. the results show that the incidence of red cell incompatibility in our hospital is 2.96% and the most common antibodies are anti-k, anti-e, anti-fya, while anti-d is important for d negative patients. 24.7% of the detected antibodies were unspecific and this is still a problem that possibly was due to the lack of additional panels of reagent red cells or antibodies to low-incidence antigens. finally, in some cases the reasons for incompatibility are due to factors affecting the blood donor or to technical problems in the crossmatching procedure. multiple isoforms excluding normal rhd mrna detected in rh blood group del phenotype with rhd 1227a allele introduction: del phenotype is very common in rh-negative chinese. the rates in hans (more than 91% in china) were reported from 26% to 30%. moreover all del individuals in this population were found mainly carrying a same allele, rhd 1227a, through genomic dna analysis. those individuals always possess one or two of this allele with ccee or ccee phenotypes. aim and the study: we focused on the mrna investigations of del individuals carrying rhd 1227a alleles, in chinese, to expect it could be explained that why a silent mutation is associated with del phenotype. the full-length rhd mrna was analyzed in 2 rh-positive donors with cde/cde and cde/cde genotypes, respectively, and 4 del phenotype individuals carrying rhd 1227a allele with cde/cde, cde/cde, cde/cde and cde/cde genotypes, respectively, through reversed-trancriptase pcrs and cdna direct or cloning sequencing. results: five transcripts and 6 isoforms were detected in rh-positive and del, respectively. among them, 4 isoforms have identical sequences, which are transcripts with exon 9, exons 8 and 9, exons 7 and 9, and exons 7 to 9 spliced out. the normal rhd mrna was only observed in rh-positive, but not in del individuals. in stead, two additional transcripts were found in del individuals. its exon 9 or exons 8-9 were spliced out, but both possess a 170 bp segment of sequence from intron 7 of rhd. through 2 additional reversedtrancriptase pcrs, which amplified exon 8 to 3¢-region and exon 9 to 3¢-region, the results showed that exon 9 did not exist in del anyway. conclusion: (i) a normal rhd protein does not exist in a del individual with rhd 1227a allele since the exon 9 was always spliced out in all isoforms. all 6 transcripts in del maintain a normal open reading frame and encode 6 proteins with different numbers of amino acid residues and different c-terminals (genbank ay751491, ay751492, ay751493, ay751494, ay751495, ay751496). among them, the sequence of del9 (isoform with exon 9 spliced) transcript was the most similar as normal rhd mrna. this isoform was first described by chang et al. in taiwan in 1998 . it encodes 463 amino acid residues and has 46 amino acids more than normal rhd. it is different from rhd after codon 384. in normal rhd protein, the amino acids after 384 (33 residues) are mainly the trans-membrane and intracellular regions. therefore a further study on if a del red cell possesses all epitopes of normal d antigen may be significative. (ii) a normal rh-positive individual has also the transcript of del9 that was found in del. (iii) there is only one polymorphism in the region of 170 bp segment between rhd intron 7 and 2 of the del transcripts, which indicated that other polymorphisms may exist in intron 7 of rhd 1227a allele compared rhd to explain that this situation was not happened in normal rh-positive individuals. total wbc were enumerated by flow cytometry and cell counting. wbc subsets were analyzed by flow cytometry with three-color fluorescence. in this study, the third generation bags and filters are used. results: before filtration, the total number of wbc, was significantly higher in fresh units compared with stored units, whereas in postfiltration samples the number of white cells was significantly lower in the fresh compared with the stored units. although absolute numbers were significantly reduced, filtration also induced significant changes in the proportions of subsets in hoth fresh and storod units, the percentage of t cells was decreased, whereas the percentage of b cells and monocytes was increased after filtration. in conclusion, both pre and post storage wbc filtration affect the proportions of wbc in the final product but pre storage wbc filtration of platelet concentrates is superior than post storage wbc filtration. the effect of pre-and post-storage filtration on platelet rich plasma: derived platelet concentrations background and objective: the white blood cells (wbc) within transfusion products are a major stimulus for a number of detrirmental biological reactions, including febrile nonhemolytic transfusion reactions, alloimmunization against hla antigens and cytomegalovirus transmission. in this work, our objective was to study the effect of storage time on the filtration of platelet concentrates (pcs). the total number of white blood cells as well as the distribution of wbc subsets, in units filtered before and after storage were compared. materials and methods: platelet rich plasma -derived pcs were filtered either fresh (5 pooled we reported earlier that metabolic arrest followed by incubation at 4°c reduces the platelet storage lesion (badlou et al. transfusion 2005) . here we report that this treatment also reduces binding and phagocytosis by macrophages. metabolic suppressed platelets (msp) were prepared by incubation in glucose-free, antimycin a containing medium (40 min, 37°c) followed by storage (48 h, 4°c) and recovery with glucose (1 h, 37°c). controls were (i) platelets in glucose-rich medium stored for 48 h at 22°c and recovery with glucose (c22) and (ii) platelets stored for 48 h at 0°c (c0) with rewarming. platelets were labelled with mepacrine and incubated with pma-matured thp-1 cells (37°c). binding was measured by facs analysis of cd42b/cd14 positive particles, and phagocytosis by counting mepacrine/cd14 positive particles. binding of msp, c22, c0 was 30 ± 4, 39 ± 10, 50 ± 12% of total platelets. phagocytosis of msp, c22 and c0 was 32 ± 9, 40 ± 8, 50 ± 9% of total macrophages (means ± sem, n = 4). before recovery of msp, binding/phagocytosis was 30% higher than thereafter, revealing energy-dependent control of the mechanisms that trigger plateletmacrophage interaction. these data show that metabolic suppression prior to cold storage attenuates binding and phagocytosis by phagocytes and may help to develop means to improve platelet survival post-transfusion. platelet compatibility testing and alloimunization in multiply transfused hematologic patients purpose: multiply transfused patients with heamotological malignancy often become refractory to platelets due to alloimmunization. refractoriness is usually defined as an insufficient platelet increment after consecutive platelet transfusions. two major causes of a decreased platelet increment can be distinguished, immune and nonimmune factors. alloimmunization occurs most frequently against the hla, and rarely against the hpa system. nonimmune factors been identified are splenomegaly, fever, sepsis, and disseminated intravascular coagulation as well as the quality of the transfused platelet concentrates. we performed this study in order to investigate platelet crossmatching, compatibility, and antibody determination among thrombocytopenic patients multiply transfused. we performed 164 crossmatchingcompatibility tests of single donor leucodepleted, abo compatible, platelet concentrates been transfused in 23 patients with leukemia and lymphoma, 18 males and 5 females with mean age 50 ± 25 years old. we also obtained samples from the patients for platelet antibody detection. we evaluated the cci (corrected count increment) 18 h after the transfusion. the solid phase rbc adherence assay (modified capture p system/immucor) was used for platelet compatibility and antibody detection. a total of 164 compatibility tests were performed, of which 84 were compatible. twenty five compatible platelet concentrates out of 84 were clinically evaluated. twenty from 25 compatible crossmatches (80%) were resulted in successful transfusion while only 5 from 25 (20%) in unsuccessful. the 80 incompatible platelets been transfused was resulted in unsuccessful transfusion. we found statistically significant difference among patients successfully transfused with compatible and incompatible platelets (p~0.01). additionally 14 patients out of 23 (60.8%) had been alloimmunized against multiple hla antibodies. three patients transfused with compatible platelets, during the study, developed alloantibodies. we found a large number of incompatible platelet concentrates that result in unsuccessful transfusion and clinical response. the platelet compatibility testing as well as alloantibody determination of multiply transfused patients is necessary for the identification and selection of compatible, with the patients, donors in order to result in succesful transfusion and clinical outcome. furthermore compatible platelet concentrates provide optimal support for refractory patients and it is known that they are acceptable as an alternative component. naitp is a rare clinical syndrome characterized by marked thrombocytopenia shortly after birth. it is caused by maternal immunization against paternally inherited antigens present on foetal platelets. screening and identification of antibodies in the maternal blood sample is the main support in the diagnosis and management of naitp. we have evaluated the frequency of maternal alloimmunization, the role of the antibodies involved (hpa and/or hla systems) and the pertinent risk of naitp in neonates using a fully automated system with a solid phase red cell adherence methodology (sprc-capture p) and paternal or random donors (indirect test). screening started in june 2003 and is still in course: in january 2005, 2300 blood samples were examined. identification of antibodies in maternal serum was carried out using elisa methodologies: maipa and commercial kits (pak-plus and quick screen-gti). of the 2300 blood samples analysed, 43 were reactive and the specificity of the antibodies were: anti hpa1a: 8, anti hpa1b: 2, anti hpa1a + hla: 2, anti hpa5b. 6, anti hla: 24, auto hpa-5b: 1. specificity of hpa antibodies was confirmed by determination of parents' hpa genotype (hpa-1,2,3,4,5,6) using pcr-ssp or pcr-rflp. the infants with hpa 1 immunization suffered from severe (plt count 5-103/ml) and symptomatic naitp (bleeding and petechiae were present), therefore they were treated with platelet transfusion and administration of high doses of intravenous immunoglobulin. we confirm that naitp due to hpa-5 and hla immunization is clinically less severe: all neonates had mild and self limiting thrombocytopenia at birth; no therapy was administered. it would be advisable to carry out pre-natal screening, at reasonable cost, using maternal serum versus paternal platelets and to proceed to the identification of antibodies only in presence of positive results. background: fetal or neonatal alloimmune thrombocytopenia (fmait) results from a maternal alloimmunization against fetal platelet antigens. it is the commonest cause of severe thrombocytopenia in the neonatal period. the diagnosis of fmait is made initially on clinical grounds, depending on exclusion of other causes of neonatal thrombocytopenia. in caucasians, hpa-1a is the most frequently implicated antigen. other antigens such as hpa-3a, or hpa-4a are less often implicated. during the past few years fmait has been reported associated with rare or private antigens. the diagnosis is straightforward when a maternal alloantibody with a corresponding parental antigen incompatibility is present. however it could be equivocal in the absence of such an antibody or difficult when a private antigen is implicated. if the father is heterozygous for the considered antigen, the infant's platelet typing should be performed to confirm the diagnosis. due to the risk of hemorrhage, particularly intracranial hemorrhage (ich), during the course of severe thrombocytopenia, specific therapy is mandatory. because subsequent siblings may be more severely affected, accurate diagnosis will allow better management of subsequent pregnancies. study design and methods: since the first documented case of feto-maternal alloimmune thrombocytopenia (fmait) due to anti hpa-9bw (maxa+), no additional cases have been reported. we present here a retrospective analysis of the cases referred to our laboratories in recent years. since 1999 we have screened for rare or private antigens in suspected cases of fmait when there is no incompatibility for the most frequently implicated antigens. the diagnosis was performed by genotyping and identification of the maternal alloantibody by the maipa technique. results: parental genotyping showed hpa-9bw (maxa+) mismatch as the sole antigenic incompatibility in 7 out of 8 families. in the last one, incompatibility was found for hpa-3 without anti hpa-3b maternal alloantibody. as the father was found to be hpa-9bw (maxa+) heterozygous in all the cases, the infant or fetus was genotyped to ascertain the diagnosis. the maternal alloantibody was identified in the maipa technique. however, our data strongly suggest that recognition of the hpa-9bw (maxa+) epitope is not uniform. the neonatal thrombocytopenia was severe in most cases with bleeding. the outcome was good in all the cases but one. conclusion: this analysis confirms that anti hpa-9bw (maxa+) fmait is not uncommon and was found to be around 2% of our confirmed fmait cases with parental incompatibilities and presence of maternal alloantibodies. it is a clinically severe syndrome which requires prompt diagnosis, albeit difficult, and maternal platelet transfusion therapy. laboratory investigation of a suspected fmait case should be carried out in a specialist laboratory wellexperienced in optimal testing. therapy requires strict collaboration between clinicians and blood bank services. appropriate management and antenatal therapy should be considered for successive pregnancies to prevent fetal bleeding. introduction: the human platelet (plt) antigen (hpa) system is independent of the hla system. therefore, host-or donor derived alloimmune thrombocytopenia can develop after allogeneic haematopoietic stem cell transplantation (hsct) even in hlamatched donor-recipient pairs. we report the first case on a stem cell recipient developing thrombocytopenia due to host-derived hpa-1a antibodies after non-myeloablative allogeneic hsct. a 52year-old male patient was diagnosed with multiple myeloma in 6/2003. treatment consisted of 3 cycles vincristin, adriamycin and dexamethason followed by tandem autologous stem cell transplantation. because of progressive disease he received 4 cycles of bortezomib, and after complete remission a stem cell allograft (7.99 ¥ 106/kgbw cd34 + cells) from his histocompatible (hla a,b,c,dr identical) brother after reduced intensity conditioning regimen with fludarabine (3 ¥ 30 mg/m 2 ) and alkeran (100 mg/m 2 ). he had received only twice packed red blood cell concentrates and one plt concentrate before allogeneic hsct. stable bilinear engraftment occurred around d12 but was accompanied with a continuous decrease of plt counts. between d10 and d19 the patient received seven plt transfusions, containing a median of 2.96 ¥ 1011 plt/unit (range 2,7-3,21 ¥ 1011 plt/unit) from random donors. the corrected plt count increments at 12 to 24 h after these transfusions were <2500/ml. therefore, and because of even a further decline of platelet counts to 1000/ml on d19 we investigated the presence of plt antibodies. methods: the patient's serum was tested by antigen capture elisa assays (pakplus® and pak1®, gti) and a solid phase assay (capture-p®, immucor). the maipa assay was used to confirm the results obtained by the above mentioned assays. in addition, we tested the patient's serum by the maspat kit (clb) against plt from the donor and against homozygous hpa-1a plt obtained from our donor pool. stored recipient's dna from the time before hsct was used for genotyping. genotyping for hpa-1, -2, -3 and -5 of the donor and the recipient was performed by pcr-ssp (hpa, protrans). the patient's serum obtained on d19 after hsct reacted strongly with the donor's plt due to anti-hpa-1a antibodies and antibodies against hla class i antigens. the patient's genotype before transplantation was hpa-1bb, -2aa, -3ab, and -5aa; the donor was hpa-1ab, -2aa, -3aa, and -5aa. thus, the antibodies were host derived and directed against the donor's plt. serum samples obtained on d38, d45 and d65 after hsct contained antibodies against hla class i antigens but hpa-1a antibodies were not anymore detectable. no hla antibodies were detectable on d149 after hsct. the severe thrombocytopenia was caused by hostderived hpa-1a antibodies. fortunately, plt counts started to increase on d20 spontaneously and the patient could be discharged at d27 (plt 51.000/ml) with a complete donor chimerism. the decrease of the serum antibodies parallel to the increase of the plt count strongly suggests a progressive elimination of residual host cells. we conclude that the hpa mismatch between recipient and host affected thrombopoietic engraftment and the success of plt transfusions. severe neonatal alloimmune thrombocytopenia with anti-hpa-5b antibodies: case report p moncharmont, m vignal, y mérieux and d rigal efs rhone alpes site de lyon, lyon cedex 07, france usually, in case of feto-maternal incompatibility, the platelet (plt) specific anti-hpa-5b antibodies (ab) induce only sometimes a mild neonatal alloimmune thrombocytopenia (nait). contrary to this observation here is reported the case of a severe nait. a 34-year old mother, gestation 2/partum 2, gave birth to a male neonate by caesarean section at 34 weeks of gestation because of intra-uterine growth retardation (iugr) and anamniotic fetus. five years before, she had had a first pregnancy with iugr of the fetus but no nait. the second neonate weighted 1.350 g, was 41.0 cm tall and had a head circumference of 27.5 cm. the apgar score was 9,10,10,10 at 1, 3, 5 and 10 min respectively after birth. no bleeding, hepatomegaly, splenomegaly or infectious signs were noted. five hours after birth, a respiratory distress syndrome appeared and an oxygenotherapy was performed during 44 h. the plt count which was 82.0, 79.0 and 73.0 giga/l at day (d) 0, d1 and d2 respectively dropped dramatically at 9.0 giga/l at d5. simultaneously, an intracranial hemorrhage grade ii was diagnosed on ultrasound scan. because of the clinical signs and of the decreasing plt count the mother's serum was tested for plt-specific ab by immunocapture and the ab identified by the monoclonal ab-specific immobilization of plt antigen (maipa) assay. a plt genotyping was performed in the neonate and his parents by sequence-specific primers polymerase chain reaction. the mother was hpa-5a/a and anti-hpa-5b ab were detected in her serum. the baby was heterozygous, hpa-5a/b. plt were transfused to the baby and the plt count rose to 80.0, 115.0 and 315.0 giga/l at d6, 10 and 18 respectively. no further transfusion was needed and the development of the baby was satisfactory with a normal electroencephalogram. in conclusion, when a mild thrombocytopenia with iugr and hypoxia but without bleeding signs is present in a neonate immediately after birth, a maternal plt specific ab screening must be performed in case the thrombocytopenia became severe during the newborn monitoring. anti-hpa-5b ab can be detected. partial results of the incidence of heparin induced thrombocytopenia type ii osc oliveira*, ra rached*, c cavalheiro-filho*, jc nicolau*, shgl pasqualucci*, daf chamone † and sp bydlowski † *heart institute, university of são paulo, † university of são paulo, são paulo, brazil heparin induced thrombocytopenia type ii (hit) is a severe side effect of heparin, associated with heparin-platelet factor 4 antibodies. hit type ii occurs in up to 5% of patients who are exposed to unfractionated heparin (ufh). in our institution patients that are under heparin treatment are mostly cardiac patients. the purpose of this study is to determine the incidence of hit type ii in these patients. material and methods: 111 patients from the intensive care unit and cardiac care unit treated with ufh or low molecular weight heparin (lmwh) for 5 or more days were studied. known causes of thrombocytopenia were excluded. platelet count was monitored pre and post heparin therapy. all selected patients were tested for detection of anti heparin/pf4 antibody test (diamed id-card). results: from the studied patients, 63 (56.8%) developed thrombocytopenia (determined by a decrease in the platelet count below 30%, after the introduction of heparin therapy); 48 (43.2%) did not show decrease in the platelet count. six (5.4%) out of 63 thrombocytopenic patients were positive for anti-heparin/pf4 antibody. three (2.7%) out of 48 non thrombocytopenic patients were positive for anti-heparin/pf4 antibody. the results demonstrate that 9 (8.1%) patients were positive for anti-heparin/pf4 antibody and they were no different from those described in the literature regarding the frequency of heparin induced thrombocytopenia. moreover, a higher frequency of patients with heparin/pf4 antibody was noted without the presence of thrombocytopenia, indicating that other factors should be considered. introduction: neonatal alloimmune thrombocytopenia (natp) due to maternal immunization against fetal platelet antigens affects 0.8-1 in 1000 live births. although it is usually a self-limiting condition, a major complication in cases of severe thrombocytopenia is the occurrence of intracranial haemorrhage leading to death (in up to 10% of reported cases). the commonest antibodies are anti-hpa-1a. treatment consists of ivig, compatible donor platelet concentrates or washed maternal platelets. the administration of random donor platelet transfusions is controversial but has been used successfully in some urgent cases when compatible platelets were not available. case report: a baby born in week 40 of gestation to a healthy mother after first uneventful pregnancy; birth weight 3000 g, apgar score 8. immediately after birth, severe thrombocytopenia (7 ¥ 109/l) and signs of haemorrhagic diathesis (generalized petechiae and ich gr. ii-iii) were observed. coagulation tests were abnormal and k-vitamin, fresh frozen plasma and random donor platelet concentrate (retrospectively genotyped as hpa-1a/a) were given. twenty-four hours later platelet count rose to 26 ¥ 109/l and no new petechiae were observed. on third day of life the blood platelet count was 13 ¥ 109/l and the newborn received ivig 2 g/kg and corticosteroides. twenty-four hours later the platelet count rose to 107 ¥ 109/l and further clinical course was uneventful. natp due to hpa-1a was serologically confirmed. conclusion: optimal therapy for an infant with severe thrombocytopenia during the first 24 h of life is the transfusion of platelets that will not be destroyed by the maternal alloantibody in the infant circulation. random donor platelet concentrates are controversial in a setting where optimal treatment is not available, however, in this case they led to a significant platelet count improvement in spite of hpa-1a incompatibility. accordingly, random donor platelets may be considered appropriate in emergency situations. background: rhd is the most immunogenic blood group antigen, and its correct identification is essential in the blood bank and in the prevention of the haemolytic disease of the newborn. the weak d phenotype is the most common d variant, with a frequency of 0.2% to 1% in caucasian individuals. there are several weak d types, with different frequencies in european countries, which may pose serologic problems and have the potential for alloimmunization. the objective of the study was to determine the frequency of the principal weak d types in portugal. study design and methods: lisbon regional centre of the portuguese blood institute and oporto são joão university hospital selected samples from blood donors and patients. rhd was tested by two (oporto) or three (lisbon) distinct anti-sera, in direct agglutination tests, at room temperature. when discrepant results were observed, the samples were tested with panels of monoclonal anti-d by liss-iat. samples that reacted weakly with igm anti-d but positive with igg anti-d were sent to the molecular biology centre. pcr with sequence-specific primers was performed using two commercially available kits (inno-train and bagene). real-time pcr, carried out on a light cycler, was applied when the interpretation was dubious. results: 101 samples were referred after being characterized as weak d. in 99 cases we obtained a positive result, with a preponderance of weak d type 2 (63.6%) over type 1 (16.2%), 3 (14.1%) and 4 (6.1%). two samples were not categorized. the high incidence of weak d type 2 in our population is in marked contrast to studies performed in other european populations where weak d type 1 was the most frequent. this might be due to our sample selection criteria or ethnic variation in the causes of weak d. there are advantages in genotyping serologically depressed d samples: to avoid the waste of d-negative rbc units and the use of immunoglobulin in pregnant women, who have no risk of alloimmunization. analysis of rhd zygosity in different rh phenotypes cal except for a 4-bp t insertion. the deletion of the rhd gene, found in most rhd-negative caucasians, was theoretically due to recombination of the upstream and downstream rhesus boxes resulting in the formation of a hybrid rhesus box. thus, the detection of a hybrid rhesus box in an rhd-positive individual denotes an rhd heterozygous status. aim of the study: to determine the rhd zygosity in different rh phenotypes. methods: blood samples from 106 white trios (father, mother and child) were studied. the rh phenotype was performed by hemmaglutination and the rhd zygosity was inferred in each member of the family groups. the rhd deleted allele was determined by a pcr strategy using a forward primer complementary to 5¢ end of the identity region of the upstream and hybrid rhesus boxes and a reverse primer complementary to the 3¢ end of identity region of the downstream and hybrid rhesus boxes. these primers selectively amplify a 1980-bp segment of the hybrid rhesus box in rhdnegative and rhd-positive heterozygous samples. serological and pcr inconsistencies were studied by a pcr-rflp method to detect another polymorphic site of the hybrid rhesus box. frequencies were obtained analysing only unrelated individuals (fathers and mothers, n = 212). results: 179 (84.4%) rhd-positive and 33 (15.6%) rhd-negative samples were phenotyped. of the rhd-positive donors, 75 (41.9%) were rhd homozygous and 104 (58.1%) were rhd heterozygous according to pcr. pcr-rflp analysis confirmed the results of pcr in serological and molecular discrepancies. these results were coherent within each family group and did not differ from those published in the literature for caucasians based on the most probable genotype method. however, the homozygosity indexes were significantly higher in the dccee (20.0% vs 6.6%) and dccee (16.6% vs 3.3%) phenotypes due to an increase of the dce haplotype. in all samples with the dce haplotype the rhces allele, frequent in individuals of african descent, was investigated by pcr-ssp. this allele was found in 33.0% of the dce haplotypes. . rhd and rhce typing was performed by multiplex-pcr with 24 fluorescent primer pairs. positive results were obtained for rhd-exons 2-7, 9, 10 and polymorphisms associated with antigens c, c, cw, e and e. sequencing of rhce-sequences, including exons 1 to 10 and intron/exon borders, were done by direct taq cycle-sequencing using bigdye-terminators v.1.1 in an abi 310 (applied biosystems). results: see table 1 . the substitutions of rhce-specific nucleotides in exons 3, 5 and 6 with their rhd-specific counterparts lead to 3 different new rhc-antigens with weakened expression. since one amino acid change in allele 3 lie in extracellular loop of the antigen suggested that this antigen may be involved in allo-immunization. inheritance of a rhd cat vi type ii in a twin pregnancy: a case report introduction: determination of hdn-relevant fetal blood groups in amniocentic fluid with pcr is a routinely used method. misinterpretation of test results -e.g. overlooking rhd category -decreases depending on the number of examinated rhd-specific exons. case report: a 28-year old mother (w.o.g. 23) pregnant with twins was regularly tested for irregular antibodies and was shown to have an anti-d. after amniocentesis of both foetuses tests for the occurrence of rhd intron 4, exons 7 and 10 were performed in the hospital's lab. the sample of fetus i showed pcr-products for intron 4, exon 7 and exon 10 while sample of fetus ii lacked rhd-specific intron 4. therefore we investigated blood samples from the parents as well as amniocentic fluid of both foetuses. methods: total dna was amplified in a multiplex pcr with fluorogenic primers for rhd exons 2-7, 9 & 10; polymorphisms for dweak type 1-5, d-vii, d-hmi and rhce polymorphisms for c, c, cw, e, e. capillary electrophoresis for size fractionation and fluorigeneic analysis was done in an abi 310. rhd zygosity was determined by quantitative real-time pcr with co-amplification of rhdand rhce-specific exon 3 and subsequent calculation of d ct value (ct rhce minus ct rhd). results: while maternal dna sample has been genotyped as rhdnegative, amplification of paternal dna for rhd-specific exons 2, 3, 7, 9 and 10 were possible and failed only in exon 4-6. determination of rhd-zygosity revealed a homozygous constellation of the rhd-gen. investigation of amniocentic fluid from both foetuses resulted in a rhd-wildtyp for fetus i and a rhd cat. vi type ii for fetus ii which was the reason for missing amplification in rhd intron 4 pcr. results: weak d type 3 was not identified in our research population. weak d type 1 was identified in 2 cn, weak d type 2 was identified in 1 cn and weak d type 4 was found in 1 cn, 3 bsa, 4 be, 1 bc and 1 saa. conclusions: although weak d types 1 to 3 represent the majority of all weak d phenotypes in caucasian populations, none of these weak d alleles were found in black populations. since none of the frequent weak d types were identified in non-caucasians one might not expected to find type 4 in all populations. however, regarding rhd phylogeny, the weak d type 4 mutations (602c > g and 667t > g) form the basis of a cluster of aberrant alleles that are predominantly observed in blacks. therefore, it is not surprising that weak d type 4 was identified in non-caucasians. based on these results it may be concluded that weak d phenotypes have evolved relatively recently since they are present in caucasian and asian methods: dna was extracted from 9 a, 14 b, 11 ab subgroups, and 21 provisional cis-ab after serological abo typing. allelespecific pcr, rflp, direct sequencing of exon 6 and 7, and allele separation were performed on these samples. results: abo*a205 allele was observed in an aint subgroup. two new a alleles that showed 784g > a base change and 990c > t of intron 6 and a polymorphism of 532c > t in a(pro) intron 5 were discovered. o04 and o20 alleles were also observed. in b subgroups, a silent substitution 255c > t (leu85leu) was observed as a new b allele. another new b allele which showed 547g > a was also found in 4 b subgroups. conclusion: we discovered new abo alleles and polymorphisms in korean populations. many other polymorphisms and alleles already reported in japanese were also observed in koreans. evaluation of a multiplex snapshot-pcr method for red blood cell marker genotyping c jungbauer*, c hobel*, em dauber † , pk rabitsch*, dwm schwartz* and wr mayr* *austrian red cross, † medical university vienna, vienna, austria once conventional reagents for identification of rare red blood cell phenotypes are scarce, methods using current nucleic acid testing techniques to identify the patient's genotype and possibly to screen for donors would be desirable. the approach of multiplexgenotyping using pcr-ssp has apparently technical constraints so we are currently analyzing whether a modification using snapshot pcr-technique could provide a routine application for such purposes. compared to ssp-technique, the snapshot pcr only requires one single primer (labeling reaction) for the detection of two (or more) alleles instead of one pair of primers for every single allele. briefly, we perform snapshot pcr as follows: in a first step, dna segments covering the essential polymorphic regions for the abo, rhd, kel, jk and fy genes are amplified using a standard pcr step. after purification treatment of the pcr products (roche high pure pcr product purification kit or shrimp alkaline phosphatase (sap)/exo1 treatment according to the abi prism snapshot multiplex kit protocol) the labeling reaction is performed using the abi prism snapshot multiplex kit. after a second purification step the products are analyzed on a abi prism 310 genetic analyser in fragment analysis mode. our preliminary results show the feasibility of this approach as reliable typing results can be obtained for all tested single nucleotide polymorphisms corresponding to alleles. generally a better signal quality from controls and samples is obtained compared to the ssp-technique with consecutive gel electrophoresis. we also consider the possibility of the automated interpretation of the results as an important improvement, especially when aiming for an application for a large number of samples (donors) or markers (patients). in contrast, the method involves more manual steps and higher costs. we may conclude that the implementation of snapshot-pcr techniques for red cell marker genotyping is a promising alternative to pcr-ssp. the obvious quality improvements compared to pcr-ssp might be critical for a routine application in blood banks (donor screening) or complex questions in clinical laboratories. quantification and quality control of monoclonal antibody using an optical sensor based on the laser reflectometry technique the monoclonal antibodies (moab) are biological reagents of homogeneous activity. they are generally used in the recognition and quantification of very small amounts of biological substances (hormones, enzymes) present in a solution, ag identification, blood group determination, oncology, organs transplant, etc. moab can be characterized by its specificity and affinity. affinity may be expressed as the equilibrium association constant (k). several techniques are available to determine the equilibrium constant of the ag-ab interaction. in this work, is shown an optical sensor for monoclonal antibody quantification by reflectometry technique. the laser reflectometry technique (null ellipsometry technique) can give information about the kinetic of the interactions, stoichiometry of molecular binding and the concentration of molecules in a solution, and also offers detailed and accurate determinations of real-time adsorption kinetics of protein without labeling. a silicon wafer was chosen as reflectant surface. once fixed the principal angle of incidence, an amount of anti-ab is added to the sample. the reflected laser intensity is registered in real time as the protein is being adsorbed onto the wafer. the mathematical analysis of the results verifies that the antibodies adsorption follows langmuir's kinetics. from the curve analysis, the parameters related to the anti-ab concentration are extracted. from them, the calibration curve is constructed. this curve allows the desired commercial monoclonal antibody quantification. the developed technique shows to be sensible and precise. the obtained graphics are very well approximated (r > 0.94) verifying that the monoclonal anti-ab associa study aim: to prevent the sensitization to rh0(d), to a d-patient who was transfused with d+ blood. material and method: on september 2003, (9/28/2003), we admitted to our hospital through air-carriage, a female of 22 years old, badly injured after a car-accident. the patient was on an olighemic shock (ht: 16%) due to retroperitoneal, paracolic and procystic hematomas, had multiple fractures on the left feet, the main important of which was an acetabulum one. she had a blood group: o, d-and her phenotype was ccdee with du-. after her arrival she was urgently admitted to the surgery room and our blood bank was asked for condensed red cells. initially she was transfused with blood of the same group (o, d-) but when we were short out of dblood, we were asked for 7 more units. the necessity of blood was imperative, the patient was on a critical condition and the mechanism of blood transport, from another blood bank would take some time to be put in motion, and so it was decided that the patient would be provided with d+ blood. the indirect antiglobulin test (iat) and papaine were negative, so there would be no problem with the transfusion with d+ blood. the patient received finally 3 units (850 ml) of d+ condensed red cells, out of 7 that were initially asked. before the 48h from the surgery had passed, it was decided that human anti-d immunoglobulin (rhesogamma p) should be provided, in order to prevent the sensitization of the patient to rh0(d). the indicted dose was 500-1250 iu/10 ml of the transfused blood, provided piecemeal during a several days period. analyzed by real-time pcr amplification. based on a published report, we selected 12 primer pairs targeting insertion/deletion polymorphisms which are located on different chromosomes, unrelated to each other and not associated with immunocompatibility. we optimized the amplification conditions for all 12 primer pairs using our sybr green real-time quantitative pcr protocols, and investigated analytical sensitivity for each primer pair by performing spiking studies, in which a single copy of positive dna was added into 100 000 copies of negative dna followed by allele-specific pcr amplification. we also created a theoretical panel of donor-recipient pairs (n = 73) to evaluate the clinical sensitivity for detection of ta-mc using both hla-dr and indel panels. results: for the short-term samples, 10 additional mc cases was identified in non-lr group using indel panel; and one additional mc case was detected in lr group (table 1) . for the long-term follow-up samples, 4 additional mc cases were found. when evaluating analytical sensitivity, we were able to detect a single copy of positive dna mixed with 100 000 copies of negative dna in a single amplification tube for all 12 primer pairs. we were also able to calculated the clinical sensitivity that using 73 donor-recipient pairs. 99.5% of donor-recipient had at least one informative allele for detection of ta-mc if we consider both hla-dr and indel panels. conclusion: using our new indel panel, we were able to detect more instances of mc in this cohort of patients. we conclude that the dr assay underestimates the presence of mc. moreover, the tandem use of both panels provides a powerful tool for the detection of mc with 99.5% of recipients having at least one informative allele. background: we reported severe immunesuppression and longterm transfusion-associated microchimerism (ta-mc) in transfused trauma patients. we have also reported, in a murine transfusion model, that sensitivity to 1-chloro-2-4 dinitrobenzene could be transferred, albeit transiently, by transfusion of fresh blood from a sensitized donor to a naïve immunecompetent recipient. in order to mimic the immunecompromised status of trauma patients and further investigate the mechanism underlying ta-mc, we established an animal model using immunedeficient knock-out mice. aim: our objective was to test virus-specific immune functionality of the chimeric donor leukocytes in a murine ta-mc model. material and methods: female rag-2/common gamma double knock-out mice were transfused with fresh blood collected from male balb/c mice, which were either not infected (non-primed, np) or infected twice (primed, p) with 100 million viral particles of murine cmv (mcmv). at different post-transfusion time-points (1 h, 2 weeks, 4 weeks, 6 weeks, 8 weeks), different female recipients plus non-transfused female knock-out controls were challenged with 100 million viral particles of mcmv intra-peritoneally, and then monitored weekly for the concentrations of male donor cells as well as mcmv viral load in recipient's circulation. each female knock-out received only one challenge of mcmv. if the subject died, we quantitated mcmv viral load in the brain, spleen, lung and liver. we used real-time quantitative pcr targeting murine y-chromosome, h2k and mcmv to quantitate male donor cells, transfused recipient cell dna input, and mcmv vrial load, respectively. the number of recipient cell dna input served as a denominator to calculate the concentration of male donor cells and the mcmv viral load. results: results of overall mortality are summarized in the table. all female knock-out recipients transfused with primed donor blood, except for the post-transfusion 6 weeks, are able to survive mcmv infection. all non-transfused control and recipients transfused with non-primed donor blood died after mcmv infection; these two groups also had higher mcmv viral load in blood than the recipients transfused with primed donor blood. when the subject died, we were able to detect mcmv in all four organs we analyzed, with liver having the highest mcmv viral load. there was no significant difference for the concentration of donor cells in recipients' blood between recipients transfused with non-primed donor blood and recipients with primed donor blood. the preliminary data of our study showed that chimeric primed donor cells, but not non-primed donor cells, are able to protect immune compromised knock-out recipients from murine cmv infection. the time-point of 'post-transfusion 6 weeks' might represent a weak window for the functionality of chimeric donor cells, which requires further investigation and confirmation. aims: to compare the effectiveness and the cost of epoetin-a and darbepoetin in patients undergoing pabd. methods: seven adult patients scheduled for operations were administered aranesp (300 mg sc once or q3 weeks if needed) for pabd (aranesp group) and they were compared with a historical epoetin-a group of seven age-matched adults (eprex 300 iu/kg biweekly). the two groups were matched according to the ht, ferritin levels, number of the predonated units and type of the operation performed. cbc count and reticulocytes were measured weekly during the donation period, the day before, day 1 and day 6 after surgery while ferritin and biochemical indices were measured during the first visit. erythropoiesis-stimulating factor was administered when ht £ 36% during blood donations and blood donation was not performed if ht < 34%. results: there was no statistical significant difference in hematological parameters during the donation period, the pre-operation day and after surgery between the two groups. five of seven patients from both groups received one or two autologous blood units. both factors were well tolerated without any side effects. the cost per patient was 609.09€ in the aranesp group and 414.2€ in the epoetin group. conclusion: despite the small number of patients and the limitations of this preliminary retrospective trial we believe that subcutaneous darbepoetin-a is equally effective with epoetin-a in patients undergoing pabd. darbepoetin has the advantage of less frequent administration and it is possibly superior that epoetin-a in terms of patient compliance. however smaller doses should be examined in order to reduce the cost. larger prospective randomized trials are needed to estimate the cost-effectiveness of the use of darbepoetin-a in pabd. background: incompatibility with many blood units is a major problem in transfusion therapy. in selective operations, preoperative autologous blood donation could solve many problems, when of course the patient's condition and his haemoglobin levels are appropriate. we present here the experience of our blood transfusion centre from 4 operations in 3 patients with anti-erythroid antibodies. materials: three patients (1 male and 2 females), aged between 67-80 years old, had to undergo selective operations, 3 total hip replacement surgery and 1 aortic aneurysm. introduction: because of improvements in surgical techniques, preoperative autologous blood donation (pabd) in patients undergoing radical retropubic prostatectomy (rp) is contested. aim of the study: we wanted to develop and validate an algorithm to determine the patients who probably do not benefit from pabd. methods: we calculated the perioperative hb-loss of 153 consecutive patients (group 1) who donated two red cell units (rbc) of autologous blood 5 and 4 weeks before undergoing rp: hb-loss (g) = preop-hb ¥ bv + (n rbc ¥ 65) -(postop-hb ¥ bv) (bv = blood volume (l) = body weight (kg) ¥ 0.08; postop-hb = hb (12-18 h after rp); n rbc = number of transfused autologous and allogeneic rbc). hb of rbc was taken as 65 g. rbc requirement is probably if initial-hb -(hb-loss: bv) -trigger-hb (taken as 80 g/l) < 0 (initial-hb = hb at the first contact with the pabd-unit). this assumption was validated by the next 125 patients who were also assigned for pabd (group 2). pabd was refused if the probability of rbc requirement (prr) was <25%. between 25-50% one rbc was taken after considering the patient's individual risk of pabd. if prr exceeded 50% two donations were planned. results: both groups did not significantly differ in age or initial hb. preop-and postop-hb were significantly lower in group 1 (141 vs 151 and 98 vs 111 g/l). 79% of autologous blood of group 1 were discarded, 3/153 patients needed additional allogeneic rbc. hb-loss caused by rp was 269 ± 111 g. mean prr in group 2 was 8.5%. 5/125 patients donated one rbc, which was later discarded, and no patients donated two rbc. 7/125 of group 2 needed allogeneic rbc. mean prr of these patients was 12% (range 0.65-33.3). conclusion: postop-hb were lower in rp-patients with pabd because of the lower preop-hb and the restrictive indication for transfusion of autologous blood. the individual calculation of prr, for which only body-weight and initial-hb of the patient are necessary, shows that pabd in patients undergoing rp is indicated only in rare cases. the algorithm also may be used in other major operations, if hb-loss is known. use of darbepoetin-alpha in preoperative autologous blood donation: preliminary results background: preoperative autologous blood donation (pabd) is an alternative practice to eliminate complications of allogeneic blood transfusion although its cost-effectiveness has been questioned. darbepoetin-a (aranesp, genesis pharma sa) is a novel erythropoiesis-stimulating factor that it has been shown to be equivalent to epoetin-a (eprex, janssen-cilag) in patients with chronic renal failure and cancer. darbepoetin-a has a longer serum half-life and higher relative potency than epoetin-a. this property leads to less frequent administration and may reduce drug cost. so far, no clinical trials with darbepoetin have been published in patients with surgical anemia. other 44 (15.3%) patients who required autologous and homologous blood, had average predonation hb level of 13 509 (sd ± 9.01) g/l. we found a significant relationship between the need for postoperative transfusion and the predonation hb level (p = 0.003), predonation htc values (p = 0.006), weight (p = 0.003) and gender (p = 0.002): female patients and patients with lower predonation hb and htc, as well as patients with lower body weight more often needed additional homologous blood transfusion. no relationship was found between age of patients and the need for transfusion (p = 0.121). 104 (80.6%) patients with ptka were transfused with autologous blood only, and had average predonation hb level of 13 867 (sd ± 9.06) g/l. other 25 (19.4%) patients transfused with autologous and homologous blood had average predonation hb level of 13 588 (sd ± 8.93) g/l. the significant relationship was found between the need for postoperative transfusion and weight (p = 0.012): patients with lower body weight more often needed additional homologous blood transfusion. no relationships were found between predonation hb level (p = 0.099) predonation htc values (p = 0.243), gender (p = 0.241) and age (p = 0.276) of patients and the need for postoperative transfusion. conclusions: our results show that over 80% of patients needed only autologous blood. in our patients with ptha predonation hb was significant predictive factor for additional transfusion therapy, while in ptka it was not observed. in both groups of patients body weight was significant predictive factor, thus this feature seems important for planning of transfusion therapy in patients with ptha and ptka. aim: prevention results of loosen anastomoses on colon, with fibrin sealent (fs) application and influence on colagen production. materials and methods: investigations were done on rats, weight 350-500 g. in control group, after partial resection of left half of colons termino-terminal anastomosis was derivated. fs was applied in examined group. concentration of colagen was done indirectly, with quantitative l-hydroxyproline determination. place of anastomosis, 1 cm proximal and 1 cm distal of anastomosis, was analyzed iii, v, vii and xiii day postoperatively. results: analysis of hydroxyproline on the place of anastomosis showed higher hydroxyproline value in group with fs application. the highest approximate value of hydroxyproline was registered v day postoperatively. distal, 1 cm of anastomosis, the quantity of hydroxyproline is higher iii day postoperatively in control group but v postoperative day value is intensively growing in group with fs application. electronic microscopical was done v postoperative day in control group at the place of anastomosis detected a defect with detritus and absence of larger colagen fibres. in group with fs application on the place on anastomosis, in the shape of bundle, colagen fibres were grouped and completely fills the place of anastomosis. conclusion: fs application accomplish higher concentration of colagen in all segments of isolated colon, that enables better healing of anastomosis. the study of the use of the safest blood (autologous blood transfusion) through preoperative blood donation (pbd) in 100 surgery patients introduction: the use of the autologous blood is already under consideration in developed countries. thus, it is probable that autologous blood donation would be effective in one way or the other in reducing blood transfusion complications. in this study, pbd as the easiest method to use and the most cost-effective one was selected. aim of the study: it aims at improving blood safety and raising blood inventory. methods: in this study, 100 patients, including 75 males and 25 females, intended to undergo elective surgery were selected as subjects to donate their autologous blood. the subjects with hematocrite level of about 30-33 percent as ordered by their physician donated their blood by this method. blood collection procedure was followed at 3-7 day intervals. the blood volume taken from patients in every collection differed from 100-450 ml according to their weight. results: this study showed that in all patients undergoing plastic, gynaecological, jaw, and ent surgeries autologous blood transfusion was used with no need for allogenic transfusion. in other surgeries, including orthopaedics, the need for allogenic transfusion was estimated to be at about 50 percent of cases. to avoid the complications of allogenic blood transfusion, the safest way is the use of autologous blood which involves low cost and is easy to perform. the introduction: the purpose of this study is to describe a technique to perform labelling of autologous platelet-gel with 111in-oxine and to evaluate its usefulness, after in vivo graft implant, as a marker of bone osteoinduction by means of scintigraphy, in patients with jaws bone defects following the enucleation of cystic lesions and cystic lesion derived from extraction of deeply impacted lower third molar. methods: agp made. consent was obtained by patient to conduct hiv and hbv testing. briefly, 40 cc to 60 cc of blood is withdrawn from the patient. the blood was separated, by means of successive step of centrifugation, in to platelet-rich plasma (prp) , platelet-poor plasma (ppp), and red blood cells (rbc). the red blood cells were discarded. the prp [comprises of approximately 10% of the total blood volume withdrawn] had platelet counts of 500 000-3 000 000/mm 3 . the procedures of agp labelling were performed in laminar flow chamber. to 3 seconds the solution will assume a gel-like consistency forming platelet gel. imaging: the scintigraphy was performed 2 h after application of labelled agp (early scan) and at 24, 48, 72, 96 h (delayed scan) by means of a gamma camera equipped with medium energy collimator. a later scan was performed at 10 days after graft. the platelet uptake index (pui) was then calculated by dividing the cpm/pixel in the graft roi (recognized in and planar and trans-axial slice) for cpm/pixel in a mirror background roi. in vitro sampling: the radioactivity of the plasma samples collected at 2, 24 h and at lapse time = 24 h for 7 days, were used for the plasma clearance determinations and for in vitro studies of the platelet loss from the gel. results: all patients presented early high concentration of 111in oxine agp, at site of the graft, that was easy recognized at scintigraphy performed as in anteroposterior and lateral planar projection of the jaw as in spet slices reconstructions. all labelled agp was well confined within area of original implant and no activity was seen in the surrounding tissues or in the distant organ. conclusion: all patients studied well tolerate the implant of agp; no adverse reactions were observed and follow up -performed 3 months later -showed bone remodelling activity in the site of the graft. serial blood donations in a ko pregnant woman with the use of recombinant erythropoietin for intrauterine transfusions of severe hemolytic disease of the newborn due to anti-ku biweekly) were administered to the mother to ensure an adequate supply of compatible rbcs for intrauterine transfusions and possible perinatal haemorrhage as well. results: intrauterine transfusions were repeated every 1-3 weeks. by the 35th week of gestation the patient had donated four units of blood, her hematocrit was 40%, anti-ku titre was 1/2048 and four intrauterine transfusions had been performed. cesarian section was decided and the apgar of the newborn were 8 and 10 at 1 and 5 min. the newborn was treated with phototherapy but without exchange transfusions and two weeks later he was discharged. by the 15th day of life rh-epo was administrated to him due to anemia. the maternal red cells completely disappeared from the child's blood by the day 100. the experience of the use of erythropoietin in pregnancy is minimal. as illustrated by this case treatment with rh-epo and iv fe has effectively increased mother's capacity to donate rbcs' for autologous use and intrauterine transfusions as well, with no adverse effects to the mother or the child. however, further research is necessary to evaluate if rh-epo crosses the placenta. introduction: blood components should be transported by a system which has been validated. the containers used for transport should be well insulated, some form of temperature indicator should be used to monitor the in-transit temperature. whole blood should be stored at different temperatures above 2°c. aim of the study: bags with whole blood collected in a collection site are transported in containers without active cooling. we tested temperature of blood in containers put into the extreme weather conditions (+50°c, -20°c) during loading test for transport. methods: the container without active cooling was filled with the exact number of bags with blood and the exact number of passive cooling elements (frozen water cubes in plastic) placed in the exact positions without close contact with the blood bags. the bags with blood of the temperature +4°c, +20°c and +37°c were used. temperature indicators were situated in the bottom, centre and top of the container. filled container was placed into thermostat (+50°c) or freezer (-20°c). the temperature was observed in 10 min intervals for three hours, first measurement was 30 min after putting into freezer or termostat. results: (see table 1 ) nt, non tested; tmp, temperature. in the table there are shown minimum and maximum temperature parametres observed during tested time including increasing or decreasing trend. conclusion: loading test for transport of the bags with collected whole blood helps us to optimize transporting system, especially number of cooling elements in relation to the season and its place in the container. in the light of presented data we corrected transporting system to maintain the recommended temperature during transport. background: since 1999, we have produced pooled and filtered platelet concentrates out of four buffy coats in tsol platelet additive solution and have stored them in pall autostop clx bags made out of pvc/totm. the residual plasma content is between 30-40%, the mean volume about 260 ml and the mean platelet-content is 3.1 ¥ 10 11 per unit. for pathogen inactivation or bacterial screening it is necessary to extent the storage time from 5 to 7 days. new foliage like polyolefin is supposed to maintain a good quality environment for prolonged storage of platelets. aims: storage bags made out of polyolefin (pall autostop elx) were tested to prove their suitability for prolonged storage of platelets. methods: 18 twins made out of pools from 8 buffy coats were produced with the standard method, one twin was stored in the conventional bag (cb) the other in the new foliage bag (nfb). the platelet pools were stored on flatbed shakers at 22°c, and sampled at day 1, day 5 and day 7. ph, glucose, lactate, hypotonic shock response (hsr) and p-selectin expression were measured by standard in-house methods. results: mean ph on day 7 with cb was 7.21, with nfb 7.35; glucose with cb 4.4 mmol/l, with nfb 6.0 mmol/l; lactate with cb 17.4 mmol/l, and with nfb 14.8 mmol/l, hsr with cb 79%, with nfb 85%; p-selectin with cb 29% and with nfb 18%. the new platelet storage bag showed better results of in vitro quality markers, especially after day 5 of storage. prolonging storage time will make it easier to introduce bacterial screening or pathogen inactivation techniques into platelet transfusion. the possibility to filter rbc either at 4°c or rt simplifies the preparation process. filtration at +4°c enables to achieve a better leukoreduction performance. the nbs has successfully implemented this project which has the potential for improvement in patient safety and is predicated upon practical application and risk reduction rather than elimination. the impact of this work on the incidence of trali will require detailed, long term analysis of hemovigilance data using existing mechanisms. active communication, a team approach, perceived value of the initiative and the hard work of all staff involved were key success factors. quality assessment of buffy coat-derived platelets prepared from leucoreduced whole blood background: whole blood can be separated into; plasma, buffy coat and red-cell conc (rcc) by differential centrifugation and separation on a separation device. because of the high hematocrit of the rcc, 60% of the process time is needed for expression of the rcc. by increasing the internal diameter of the tubing at the bottom of a t&b system by 0.7 mm. a decrease of the process time is expected. methods: 220 units of whole blood were collected with the new t 3988 'wide boring' blood pack and separated on a routine base. quality control parameters were checked and the whole process time was monitored. free hemoglobine was measured up to 42 days. results: process time of a 'wide boring' bag is significant shorter compared to a standard blood bag. average decrease: 86 s. slightly increase in free hemoglobine is measured probably due to the increased express rate of the red cells. bloodproducts produced with the new t 3988 meet european guidelines. no significant increase of free hemoglobine due to the faster expression is measured. an significant decrease in process time is measured with the wide boring bloodpack. the new fresenius hemocare rcc in-line system: t 3988 can be used for routine production which will speed up the production process considerably. introduction: leukoreduction of blood components is required to prevent several transfusion-associated complications. aim: the aim of this study was the full process validation of the pall leukotrap wb system for the preparation of leukoreduced blood components. we collected 60 whole blood units from donors suitable for donation using a quadruple blood-bag, which includes an wbf3 in-line filter (pall) for the removal of leukocytes and platelets. mixer balances (baxter) were used and donation occurred within 12 min in all cases. after donation whole blood units were stored at room temperature for 2 h. subsequently, whole blood filtration was performed by gravity at a standard height of 150 cm using a blood leukoreduction cart (baby leuko cart, itl-corporation). filtered units were centrifuged at 5000 g ¥ 10 min by an heraeus cryofuge 6000i. an automatic extractor (bag press plus-bioelettronica) was used to prepare red cell concentrates in sag-m solution and fresh plasma units. air in the system was automatically expelled by the extractor. complete cell counts and hemoglobin concentration were evaluated in pre-filtration samples and at the end of the blood components preparation using an automated cell counter (pentra dx120-abx). we enumerated residual leukocytes in red cell units by flow cytometry (becton dickinson-leucocount kit). results: pre-filtration data of whole blood and end-of-process data of red cell and plasma filtered units, are summarized in table 1 . results are given as mean and standard deviation. whole blood filtration was completed within 10 min in all cases. red cell units were transfused after a mean of 6 days to 57 patients affected from transfusion dependent (45%), post-surgery (30%), and post chemotherapy anemias (25%). no cases of transfusion reaction were observed. the pall leukotrap wb system was easily introduced in our setting. all blood components prepared by the system fulfilled the council of europe requirements with regards to hemoglobin content in red cell units and post-filtration residual leukocytes. future studies are needed to evaluate its cost-effectiveness in the setting of routine blood component preparation. background: during an evaluation of the compodock (fresenius hemocare) sterile connection device (scd), we observed irregularities on the inside of the tubing at the site of the weld. it was our aim to investigate the effect of these observations on the quality of blood products. methods: three leukoreduced red cell concentrates (rccs) were pooled and divided over 3 bag systems: one without weld in the connecting tubing, one with a compodock-weld, and one with a weld made with the terumo scd. the rcc was transferred 10 times over this tubing to have maximum result if the weld had deleterious effects. the rccs were stored in pvc containers, and sampled on day 1, 28, 35 and 42, and free hemoglobin (hb) was measured. the same procedure was also performed using platelet concentrates (pcs), but these were stored in polyolefin containers, sampled on day 1, 6 and 8, and cd62 expression was measured. ten experiments were performed per blood component. according to who standards processing of blood into labile components are considered an expression of quality of transfusion service. in our practice, modern transfusion principles are successfully applied. they cover blood collection, serological processing of blood units, technological preparation of blood products (gmp, sop) and rational utilization of blood components and blood derivatives. in the past four years (2001, 2004.) aberrations from these principles have taken place (self-sufficiency). nbti collected x = 62 926 (249 778) blood units into blood bags. in serbia x = 230 537 (691 611) blood units. retrospective analysis: ldpc-bc was administered x = 97% with the satisfactory haemostatic effect. increase of the cyta and plasmapheresis-manual procedures was also noted (100%). increase of the use of leukocyte poor red blood cells was also registered (95% introduction: according to the relevant recommendations of the council of europe, whole blood is a source material used for the preparation of blood components and blood products. basic concept of the therapeutic use of blood components is the compensation of the lacking or deficient blood component. in that way, a possibility of the infusion of unrequired or deleterious components of whole blood is eliminated. objective of this presentation is to analyze the reasons of non-utilization of certain blood units, the actual quantity and ratio. aim of the study: the purpose of our in vitro study is to compare storage of platelet concentrates at 4°c with platelets stored at 22°c, and to determine the in vitro-effects of pre-incubation at 37°c for 1h prior to analysis on the basis of the maintenance of platelet metabolic and cellular integrity. methods: platelets concentrates (pcs) were prepared from pooled buffy coats (bc) for paired studies to be stored into different conditions. (i) at 20-24 degree on a flat bed agitator; (ii) at 20-24 degree on a flat bed agitator and pre-incubated for 1 h prior to analysis; (iii) at 4°c; and (iv) at 4°c and pre-incubated for 1 h prior to analysis. this paired in vitro study (n = 8) over 21 days include volume, platelet counts, mpv, volume, ph, po 2 , pco 2 , bicarbonate, glucose, lactate, swirling, leucocytecount, hsr, esc, atp, ldh and release of a-granule content (rantes, ß-thromboglobulin and pf4). results: platelet count (day 5 and 21; p < 0.05), mpv (day 5; p < 0.01), ph (day 14 and 21; p < 0.01), pco 2 (day 14 and 21; p < 0.01), bicarbonate (day 21; p < 0.01), glucose (day 5, 7, 10, 14 and 21; p < 0.01), atp (day 14 and 21; p < 0.01) was significantly higher in platelets stored at 4°c and platelets stored at 4°c with preincubation. ldh (day 21; p < 0.01), bicarbonate (day 5 and 7; p < 0.01), lactate (day 5, 7, 10, 14 and 21; p < 0.01), ph (day 5 and 7; p < 0.01), esc (day 5, 7, 10, and 14; p < 0.05), hsr (day 5, 7 and 14; p < 0.05) was significantly lower in platelets stored at 4°c and platelets stored at 4°c with pre-incubation. the concentration of rantes, ß-thromboglobulin and pf4 was significantly higher in platelets stored at 22°c than in platelets stored at 4°c (day 5, 7, 10, 14 and 21; p < 0.01). hsr (day 5 and 10; p < 0.05) and esc (day 5, 7, 10 and 14; p < 0.01) was significantly higher in preincubated platelets stored at 22°c compared with platelets stored at 22°c. conclusion: platelets stored at 4°c maintain metabolic and cellular characteristics to a great extent during 21 days of storage. we confirm the loss of platelet discoid shape and have shown that loss of discoid shape in platelets stored at 4°c is associated with decreased metabolic rate and decreased release of a-granule content. aim: as reference centre of the swiss blood transfusion service for new materials and blood products we evaluated that system for routine use and official registration in switzerland. method: 20 whole blood donations were collected in a whole blood filtration set with cpda-1 and stored at room temperature for 1 h before filtration at room temperature. the leucodepleted whole blood was stored for 42 days. following parameters were analysed on day 0, 35, 42: free haemoglobin in%, k. in addition leucocyte count was performed on day 0 and a blood culture on day 49 (see table) . blood cultures on day 49 remained negative and all counts of residual leucocytes were below 1 ¥ 10 (exponential) 6/unit. summary and conclusion: as expected there was a clear increase in k and free haemoglobin after day 35. however the results were within the required specifications from the european and swiss guidelines up to day 42. we conclude that autologous leucodepleted whole blood can be stored in cpda-1-for 42 days without loss of stability of the red cells. we will introduce the system to the offi-cial material list of the swiss blood transfusion service and then implement the procedure to our daily routine. results of ffp production from whole blood, and of ffp and pc produced by use of cell separator over a 5-month period before and after the introduction of measures for trali prevention are presented. the following measures were undertaken: (4) blood of female donors was not used for ffp production, and plasma was only used for fractionation (5) plasma of female donors was not used for kt-bc pools (6) platelets and plasma were produced on a cell separator only from the female donors without a history of pregnancy. female donors of whole blood: 16%*; 16%** ffp produced by plasmapheresis: 2%*; 2.5%** female donor units on cell separator: 9.4%*; 4.5%** ffp from total plasma units: 63%*; 58%** plasma units used for bc-pc pools: 4%*; 6%** *period before and **after the introduction of measures for trali prevention the exclusion of female donors had no major impact on the production of ffp and bc-pc pools from whole blood because of the very low rate of female subjects in the croatian blood donor population. the amount of plasma and pc collected from female donors by use of cell separator was significantly lower (~50%), however, without any major impact on total ffp store because of the small rate of plasma and platelets obtained by apheresis. background: platelet concentrates (pcs) are currently stored for a maximum of 5 days. extended storage to 7 days would increase the supply and reduce the waste of pcs. transfusion-associated graftversus-host-disease (ta-gvhd) is a severe transfusion reaction caused by t-lymphocytes in the transfusion product. the risk of developing ta-gvhd can be prevented by gamma irradiation of the pcs. various in vitro tests can be used to study the quality of pcs such as inspection of the swirling phenomenon, hypotonic shock response (hsr), detection of platelet surface markers (e.g. cd62p and cd42b), metabolic parameters and blood gases. free oscillation rheometry (for) using the instrument reorox® 4 can be used to monitor the coagulation over time in whole blood, pcs and plasma samples, and to obtain information about clotting time and coagulum elasticity. aim of the study: the purpose of this study was to evaluate the quality of pcs obtained by apheresis technique during storage for 7 days and to study the effect of gamma irradiation by using several in vitro methods including for. methods: platelets were collected from healthy donors (n = 12) using apheresis technique. the pc from each donor was divided in 2 units, one served as control and the other was gamma irradiated with 25 gy. the pcs were stored on a flatbed agitator at 22°c for 7 days. samples were taken on day 0 (= day of collection) for analysis of blood gases, metabolic parameters (glucose and lactate), platelet count and swirling. samples taken on day 1, 5 and 7 were also analysed for hrs, cd62p (p-selectin) and cd42b (gpib) expression utilising flow cytometry. evaluation of coagulation by for was performed on day 1, 5 and 7. the maximum elasticity (g'max) and the time to g' were evaluated from the for elasticity curves. results: there was no difference between irradiated and nonirradiated pcs regarding any of the tested parameters during the storage period. swirling, hsr, platelet count and percentage of cd42b expressing cells were well maintained for 7 days of storage. glucose decreased and lactate increased significantly during the storage period, from 2.5 mmol/l to 16.2 mmol/l for lactate and from 16.5 mmol/l to 8.9 mmol/l for glucose. the percent cd62p expressing cells increased significantly during storage from 15% on day 1 to 32% on day 7. po 2 was well maintained but ph increased and pco 2 decreased significantly between day 0 and 1 whereafter ph decreased and pco 2 continued to decrease. the for parameters g'max and time to g'max increased significantly between day 1 and 5 and the time to g'max continued to increase significantly between day 5 and 7. the results indicate a well preserved platelet quality after storage for 7 days. gamma irradiation did not affect the platelet quality. cytokine release during storage of buffy coat platelet concentrates produced manually and automatically background: transfusion reactions following platelet transfusion are still a problem even when leukoreduction is included in the production process. platelet derived cytokines released during storage upon activation or lysis, accumulate in the platelet products and have been suggested to be involved in transfusion reactions. rantes (regulated upon activation, normal t cell expressed and presumably secreted) is a chemokine playing an important role in the inflammatory immune response and causes degranulation of eosinophiles and release of histamines from basophiles, which again can cause allergic reactions. tgf-b1 (transforming growth factor b1) has been shown to be immunosuppressive, inhibits the proliferation of t-and b-lymphocytes and decreases the secretion of igg and igm from b-lymphocytes. aims: as part of our quality control program, we aimed to quantify the amounts of rantes and tgf-b1 released during storage in platelet concentrates produced from pooled buffy coats by our manual routine method (m-pcs) and by an automated method using the orbisac system (gambro) (a-pcs). methods: pcs were produced from 5 buffy coats. following overnight storage at 20-22°c, buffy coats were pooled with 300 ml t-sol (baxter). forty-two pcs were produced either manually (n = 21) using the imugard iii s-pl set (terumo) with integrated soft leuko-reduction filter or by the automated procedure (n = 21) using the orbisac validation bc set (gambro) equipped with the lrp6 leuko-reduction filter (pall). swirling was scored visually, platelet count and mpv were measured on a cell counter (cobas argos, roche), and blood gas analyses, glucose as well as lactate were measured on an abl700 series analyser (radiometer). samples for testing of cytokines were centrifuged for 15 min at 4800 g, 20°c; supernatants were harvested and frozen at -86°c until analysis. cytokines were quantified using quantikine human rantes immunoassay (r&d systems) and human tgf-b1 elisa immunosorbent assay (bender medsystems gmbh). all analyses were performed on days 1, 4 and 7. results: platelet concentrate volume (mean): m-pcs: 316 ml, a-pcs: 328 ml. platelet yield was found to be 3.15 ¥ 10 11 for m-pcs and 3.43 ¥ 10 11 for a-pcs (p < 0.0001). in all pcs ph levels were between 6.9-7.2. glucose consumption and lactate production from days 1-4 and days 4-7 did not differ significantly. rantes levels (pg pr 10 9 plts) were significantly higher in a-pcs than in m-pcs (p = 0.04, repeated measures analysis of variance), but no significant difference was found in tgf-b1 levels (pg pr 10 9 plts). summary and conclusions: preparation of buffy coat platelet concentrates by the automated orbisac system improves platelet yield compared to our manual processing procedure, but the levels of the chemokine rantes were significantly highest in the automatically produced products. the clinical importance of these findings is still unclear, but may be related to the shear stress the platelets are subjected to during the automated production process. the quality of cryopreserved vs liquid stored platelets: a comparative study table 1 . the mismatches can be divided into the two categories. the first of them is characterized by differences in allelic groups, i.e. at low-resolution level. 22 allelic group differences were detected in the group with one mismatch, most of them in hla-c locus (this locus was not concluded in primary donor search). in the other category there are differences in alleles within the same group, i.e. at high-resolution level only. 12 differences within the same group in all tested loci were detected in the group with one mismatch. the mismatches described above were heterogeneous and a correlation of specific mismatch with transplantation outcome was not possible in this group. conclusion: the use of high-resolution dna methods makes the identification of hla match/mismatch more accurate and can affect the outcome of unrelated hsct. this work was supported by the grant iga mz, no. nr/7984-3. pre-freezing and post-thawing quality controls in umbilical cord blood assigned for transplantation p bergamaschi, c perotti, g viarengo, c del fante, c parisi, a marchesi, l bellotti and l salvaneschi irccs policlinico 'san matteo', pavia, italy background and aims: nowadays umbilical cord blood (ucb) represents a well established source of haematopoietic stem cells for unrelated transplantation in children affected with haematological and inherited diseases. thanks to the large-scale banking of unrelated units and the preliminary encouraging results, ucb employ in adults is quickly growing up. in this context, total nucleated cells (tnc) count of the graft is considered the main predictor for clinical outcome; however, other indicators of the haematopoietic potential, such as cd34+ cell content and short-term culture clonogenic assay, are recommended in accordance to netcord-fact stan-dards. in order to guarantee the safety and the prompt availability of a ucb unit assigned to a matched recipient, a pattern of rigorous quality controls should be carried out not only at the time of cryopreservation but also before the release for transplant. we report the results of the quality controls performed on thawed cryovials referring to the 26 units delivered by our ucb bank compared to the pre-freezing values. methods: every ucb unit stored in our bank is accompanied by 3 satellite cryovials available for subsequent controls. for each unit issued for transplantation, one cryotube was thawed in 37°c water bath with gentle agitation without washing out dmso. tnc and mononucleated cells (mnc) were estimated by an automated cell counter; viability and cd34+ cell count were evaluated by flow cytometry with a no-wash, single-platform technique and 7aminoactinomycin d. cfu assay was performed using commercial reagents (methocult gf h4434, stemcell technologies) and colonies were counted after 14 days. the same tests were performed before cryopreservation, taking a sample from each fresh unit. moreover, before the delivery for transplant, a second cryotube was thawed to investigate the bacterial contamination by direct microbial culture, whereas the sterility test before freezing was performed by inoculum into 30 ml media (bact/alert®fa/fn, biomérieux inc). results: the ucb characteristics before freezing and after thawing are detailed in the tables 1, 2 and 3. post-thawing tnc and mnc, as well as cd34+ cells, showed no significant difference in comparison to the pre-freezing values. despite of the expected decrease of the overall viability after thawing, we observed a highly satisfactory viability referred to the cd34+ cells. the colony forming units (cfu) growth after thawing was documented and was always lower as respect to the pre-freezing assay. finally, the results of microbial cultures were negative for all the 26 units on both fresh and thawed specimens. conclusions: in our experience, well standardized evaluation of ucb content could be obtained with regard to tnc, mnc and cd34+ cell. concerning the results of short-term cultures, the presence of dmso as inhibiting factor may be advocated to explain the discrepancies between fresh and thawed samples. finally, rigorous quality controls documented that the procedures of manipulation and cryopreservation did not affect the quality of ucb to be infused for transplant and provided to the physician all the parameters necessary for a safe transplant in a close and appropriate time. bone marrow transplantation or bmt transplantation of progenitor blood cells to regenerate blood normal cells in patients with blood disorders. bone marrow has an organized and structured architecture in which close relationships exist between a regulatory microenvironment and primitive hematopoietic cells. in fact, normal hematopoietic cells depends on critical interactions that occur between stem cells and their microenvironment. this microenvironment is a complex meshwork composed of growth factors, stromal cells, and extracellular matrix. marrow injury can occur as a consequence of a variety of diseases. some diseases could be due to a microenvironment that fails to support hematopoiesis. a possibility is that aplasia and leukemia share a common etiology such as drug, chemical, radiation, virus or other environmental hazards. we can say that microenvironmental abnormalities in interactions between stromal cells and hematopoietic progenitors may be important in the pathogenesis and clinical expression of hematopoietic malignancies in humans. background: intrauterine growth retardation, with associated low birth weight, represents one of the most important cause of baby mortality and morbidity. understanding the genetic bases of this adverse event is still an open goal. there is evidence that motherchild hla compatibility and hla-drb1 foetal genotype are associated with a reduced placental growth and a low birth weight. the recent institution of cord blood banks, with their huge amount of hla types, offers an unique opportunity to look inside the molecular bases of normal birth weight. aims: we investigated whether the baby-linked immunogenetic profile, i.e. hla gene frequencies and homozygosity rate, affects the physiological variance of the size at birth. methods: 1868 cord blood units (961 from males and 907 from females) were hla typed with pcr-ssp and/or reverse pcr-sso techniques and recorded in the cord blood bank database of pavia-italy. all were defined at low resolution level for hla-a and b genes and at high resolution for hla-drb1. 686 blood units were also randomly typed for hla-dqa1 and dqb1 at high resolution. results: mean birth weight was 3430 g and mean relative birth weight (i.e. corrected for gestational age according to the gender) was 0.19 g. 70 babies were <5th centile (2782 g) and 70 were >95th centile (4068 g). comparing the hla allele distribution in these extreme bands we found that hla drb1*08 was significantly associated with high relative birth weight: 5.1% in 95th centile vs 0.7% in 5th centile, p = 0.032. on the contrary, hla-drb1*14 and dqb1*05 were associated with low relative birth weight: 7.9% and 30.4% respectively in 5th centile vs 2.2% and 9.6% in 95th centile p = 0.035 and p = 0.019. all infants were analysed as to the effect of the above mentioned alleles. we confirmed the positive association of hla-drb1*08 and higher relative birth weight (mean 0.17 vs 0.08; p = 0.033) as well as the association of hla-drb1*14 with lower relative birth weight (mean 0.03 vs 0.27, p = 0.04). no significant association was found as far as hla homozygosity was concerned. conclusions: the present findings confirm the role of foetal hla-drb1 gene in the intrauterine growth. about the specific involved alleles, one possible explanation comes from the studies of crystallography and amino acid sequencing of hla-dr binding groove. it has been demonstrated that hla-drb1*08 and hla-drb1*14 genes encode for different amino acid sequences in the pocket 4 of the molecule (aa 70, 71, 74). this implies distinct functional restriction patterns. the sequence motif of hla-dr14 is characteristic of some autoimmune conditions, such as hashimoto's thyroiditis, and preeclampsia which is associated with intrauterine growth retardawhich provides a high yield and excellent purity without lymphocyte and erythrocyte contamination. in a 3 month period, we studied blood samples from bone marrow transplant patients and from normal subjects. the extraction of leukocyte polymorphonuclear was obtained with a 6%-7% dextran solution in 0.8% saline. after incubation at room temperature with lymphopre solution, the mixture was centrifuged. two clear and separate rings of mononuclear and pmn leukocytes were obtained. to eliminate any red blood cells, pmnl ring was separated and washed three times with cold ammonium chloride. after a short period of incubation 4°c, mixture was centrifuged and the pmnls were isolated. the purity and viability of total leukocyte population was counted and the percentage of pmnl obtained was established. the total blood samples studied were divided in two groups, i.e., bone marrow transplant patients and normal subjects. in both cases the pmns isolated were of high purity and viability. the overall percentage of pmnls obtained from both groups under study was 98% to 99% when stained with gimsa or wright staining method. the viability of isolated pmnls was also 98% too, which is excellent for numerous immunological or molecular studies. the pmnls isolated by this method were highly pure and viable in comparison with standard methods used to isolate human pmnls. generation a high amount of pmnls is another advantage of the suggested method. this method to separate pmnls is recommended for in vitro studies of different subjects. et al. 1950 .) the object of exchange transfusion (et) is to remove bilirubin already present in the plasma or remove alloantibody which can cause hemolytic disease (in order anti-d, anti-c and anti-k are easily the most important) or remove anti-d positive red cells. we have studied exchange transfusion in our hospital in neonatal intensive care unit, during 1997 to 2004. at delivery cord samples were taken for determination blood group, rhesus, hb and ht have been counted. also direct antiglobulin test (dat) has been performed. in cases of positive dat, hb and bilirubin levels were monitored. newborn body-weight were weighted (ranged 710 g to 3100 g). the blood for et was of group o, d negative and kell negative and was compatible with the serum of mothers; it was less than 7 days old. the blood was screened for hbs and for anti-cmv as well as being submitted to all usual tests. the method has been determined by using the umbilical vein; the multi-way tap makes it possible to draw blood from the infant into the syringe, to discard the blood into a sterile empty vessel, then to draw blood from a donor unit into the syringe and inject this into the infant. results: 17 exchange transfusions were carried out. four out of 17 et due to abo incompatibility mother-newborn. five out of 17 due to rhesus incompatibility mother-newborn and eight out of 17 due to jaundice undetermined origin and immaturity. the last two years only three et were carried out due to immaturity. conclusions: phototherapy, when applied early enough and with sufficient intensity, can avoid the need for exchange transfusion in many infants. phototherapy alone or phototherapy plus high dose igg therapy has been used to minimize exchange transfusions in this population. detection abo blood group system antibodies of neonatal using fully automated column agglutination technology (auto-vue) background: the abo blood group system remains the most important in transfusion practice. this is because of the regular occurrence of the antibodies anti-a, anti-b and anti-a, b reactive at 37°c, in persons whose red cells lack the corresponding antigens. the regular presence of anti-a and anti-b is used in the routine determination of abo blood groups; in addition to testing red cells for a and b antigens, the group is checked, in serum or reverse grouping, by testing the serum against red cells of known abo groups. methods: 100 samples were taken from 100 newborns at first 24 h of life for abo blood group typing during 2001-2004. simultaneously the presence of anti-a and anti-b antibodies has been studied using fully automated column agglutination technology (auto vue ortho diagnostic systems) with bio-vue cassettes aborh/reverse. the column agglutination technology is based upon the ability of glass beads to form a physical barrier between agglutinated and unagglutinated red cells. to determine the abo serum group, test serum and abo reagent red cells were added to the top of column containing diluents. the abo grouping columns were centrifuged and examined for agglutination. the presence or absence of agglutination has been recorded. results: in 68 out of 100 newborns were found detected antibodies anti-a, anti-b. in 24 out of 100 newborns no antibodies were found. in 8 out of 100 were found antibodies maternal origin. the automated reader detected all positive reactions. positive results were recorded on a scale from +0.5 to +4. conclusions: the technical performance of device allows objectivity and precision to detect abo blood group antibodies of newborn. the origin and the type of antibodies and also factors that influence their presence are to be studied. introduction: diagnosis, management and prevention of red blood cell immunization have improved, so hemolytic disease of the newborn (hdn) has changed from a common to a rare pathology. aim of the study. in this study we have retrospectively evaluated the benefits of the immunohematological screening for the management of pregnancies with alloimmunization. methods: in the last 3 years, we have performed an immunohematological screening on all pregnant women assisted by our hospitals. ab0 and rh typing, antibody screening and, eventually, identification and titration were performed on maternal specimens by microcolumn technique. results: not considering ab0 incompatibilities, we have discovered 64 alloimmunized women with the following specificities: 10 anti-d, 9 anti-c, 7 anti-c, 9 anti-e, 3 anti-jka, 1 anti-d + anti-jka, 1 anti-d + anti-s, 3 anti-d + anti-c, 1 anti-d + anti-c + anti-k, 5 anti-s, 10 anti-k, 2 anti-m, 1 anti-c + anti-e and 2 anti-d + anti-c + anti-g. the most severe hdn were the 2 d + c + g, the c + e and 2 out of 7 c newborns, with mean hemoglobin between 5 and 6 g/l, bilirubin = 20.5 g/l, reticulocyte count = 10%. in these exchange transfusion needed at the delivery. other newborns were only treated with phototherapy. conclusion. thanks to the immunohematological monitoring, the diagnosis of alloimmunization, the correct management of pregnancy and the adequate neonatal therapy were possible. in fact all newborns survived and showed no neurological lesions in the following controls. conclusion: in order to provide a highly specialized perinatal care, immunohematologist, obstetric and pediatric should provide a good antenatal and perinatal screening. this is an interesting case of rhd immunisation in rhd negative woman despite the application of rhd immunoprophylaxis. case report: a blood sample of pregnant woman, 30 years of age, in 12th gestation week, was sent to our laboratory for serological analysis. her blood group was o, ccddee and she had an anti-d antibody reactive only with enzyme treated panel of test erythrocytes. her husband was a, ccdee, and two children were both a, ccddee. on the next visit, she gave the data of one arteficial and one missed abortion before the 12th gestation week covered with 50 mg of rhd immunoprophylaxis, but without the measurement of fetomaternal haemorrhage (fmh). both of abortions were after the deliveries. until the end of the pregnancy, detailed serological analysis showed anti-d specificity of antibody in her sera which remained reactive only with enzyme treated red blood cells. the fetus was under permanent ultrasound control. she delivered a mature, rhd positive, ccdee male child, without any sign of haemolytic disease. the proper personal history, measurement of the size of fmh, distinguishing the anti-d and anti-g specificity of the antibody, administration of rhd immunoprophylaxis and cooperation between transfusion medicine specialists, gyneacologists and neonatologists still remain major principles of prenatal and perinatal care concerning haemolytic deisease of the fetus and newborn caused by anti-d antibody. anti d antibody, reactive only with enzyme treated red blood cells is usually harmless for fetus and the newborn. introduction: red blood cell (rbc) transfusion is widely used in neonatal intensive care units for acute or chronic pathological conditions. clinical indications for rbc transfusion are shock, sepsis and/or anemia with the following laboratory criteria: a) hematocrit (hct) < 20% or hemoglobin (hb) < 7 g/dl and reticulocytes < 4%; b) hct < 30% or hb < 10 g/dl in these conditions: o2 required < 35%, recurrent apnoea and bradicardia, cardiac rate > 180 bpm and respiratory rate > 80 bpm for more 24 h; c) hct < 35% or hb < 12 g/dl with severe respiratory distress. aim of the study. aim of this study has been to evaluate the effectiveness of rbc transfusion therapy in premature and at term newborns independently of initial pathological conditions. methods: our therapeutic objective has been to achieve an hct of 50% after the whole cycle of transfusion therapy. for each little patient, the volume of transfused rbc unit has been calculated, according to international guide lines, using the following criteria: weight (kg) ¥ blood volume (100 ml per kg if premature or 80 ml per kg if at term newborn) ¥ (hct desired -hct observed)/hct of unit transfused. particularly we have considered that premature infants (with a gestation age of 26-36 weeks) show a range of weight from 600 to 1.800 g, while at term newborns from 1.800 to 4.000 g. in order to avoid a circulatory overload, the indicated hemocomponent has been always packed rbc with higher possible hematocrit. for the same reason, the rate of infusion has been always 3-10 ml/kg/hour. methods: this is a descriptive study. the name of the neonates who received transfusion was obtained from the blood bank of beheshti hospital. information concerning the type of blood product, frequency and indication of transfusion, sex, gestational age and weight of infants was recorded in questionnaire and analyzed. results: out of 541 neonates admitted during one year, 118 (68 male, 50 female) received blood components. fifty four percent received one, 35% two and 11% received three types of blood components. the frequency of transfusions were 311 times. the most common used blood products were fresh frozen plasma (49%), red blood cell (33%), whole blood (14%) and platelets (4%). all the blood products except whole blood were used more common in premature and low birth weight infants. appropriateness of transfusion of red cells, fresh frozen plasma, platelets and whole blood were 92%, 88%, 100% and 91% respectively. (hdn) . in accordance to current regulations, this study is carried out in all pregnant women attending in our service. according to our protocol, when an alloantibody of any specificity is detected through the liss-coombs gel technique, the same determination is made using papain-treated screen cells to detect any association with other antibodies which could be of clinical relevance for a hdn. there are scientific evidences that the use of enzymatic techniques increase the test's sensitivity, though clinical relevance of 'enzyme only antibodies' may be questioned. aims: demonstrate the importance of a routine identification of irregular antibodies in pregnant by two methods (liss-coombs -enzymatic) and the prevalence of anti-d associated antibodies, only detected by using enzymatic technique. analyze the need to carry out a follow up of sensitized patients to determine if the associated antibodies only detected with in an enzymatic medium can be detected with a liss-coombs medium during pregnancy, thus acquiring clinical significance for hdn. materials and methods: between january 2000 and december 2004 we studied 1970 d-negative pregnant women. the studies performed were: abo grouping, d and weak d, rh phenotype, direct antiglobulin test and irregular antibody detection (iad) against commercial screen cells with liss-coombs (diamed) ® gel-medium technique, according to the manufacturer's specifications. when iad were positive, an antibody identification using two commercial cell panels with gel techniques (liss-coombs-enzymatic) was performed. along the pregnancy, periodic controls were carried out to determine the exact moment when antibodies, previously only identified in an enzymatic medium, could be detected in a liss-coombs medium (clinically significant antibodies). results: out of 1970 d-negative studied samples, 278 (14.1%) had a positive dai and it was only showed anti-d specificity in a liss-coombs medium. after analyzing this specificity against enzymetreated erythrocytes, it was possible to determine that 79 patients (24.8%) had in their serum other anti-d associated alloantibodies: 5 anti-k (1.8%), 63 anti-c (22.7%), 10 anti-e (3.6%) and 1 anti-c + e (0.3%). during the immunohematologic follow up, it was determined that in 4/79 patients some of the antibodies which were previously only detected in an enzymatic medium, could be identified in a liss-coombs medium and later they were identified in the red cell elution of the newborn. conclusions: these results confirm the relevance of a screening for irregular antibodies of clinical importance by means of a conven-tional technique and one of increased sensitivity in all pregnant women. the detection of an association of antibodies provides information for the undertaking of diagnostic and therapeutic measures, both by the obstetrician, as for any eventual transfusional requirements for hdn. it was also concluded that, although antibodies detected in an enzymatic medium are considered of low clinical significance, its investigation and follow up is suggested in pregnant women to determine the moment in which they can be detected in an antiglobulinic medium, thus revealing their clinical significance. background: fibrin glue is one of the most complex human plasma derivatives both in terms of composition and clinical applications. this product mimics the last step of coagulation cascade through activation of fibrinogen by thrombin, leading to the formation of a fibrin clot in the presence of factor xiii. in contrast to synthetic adhesives, the significant advantage of this plasma-derived sealant is its biocompatibility and biodegradability as well as the fact that it does not induce inflammation, foreign body reaction or extensive fibrosis. readsorption of the fibrin clot is achieved during wound healing within days/weeks following application, depending upon the type of surgery, the amount and type of product used or the proteolytic activity of the treated site. the risk of virus transmission by commercial fibrin glue products is still debated and investigators are looking for alternative fibrinogen sources. many of these studies rely on autologous on single donor cryoprecipitate as source of fibrinogen. aims: the aim of this study was to compare single and double methods of cryoprecipitation of fibrin glue. the influence of different plasma preparation methods and plasma storage temperatures (-30°c and -80°c) on the quality of fibrinogen concentrate was examined. methods: whole blood was collected by standard phlebotomy technique and centrifuged at 4000 ¥ g for 5 min within 2 h of collection. plasma was removed. four units of plasma were pooled into a 2000 ml bag, mixed, divided into 4 parts (aprox. 219.7 ml) and immediately frozen. two of these units were stored at -30°c and 2 units of ffp at -80°c. after one month the plasma was thawed at 4°c during 16-18 h. the fibrinogen concentrates (21-25 ml) were received by single and double cryoprecypitation. to compare single and double methods of cryoprecipitation, the levels of fibrinogen, fibronectin, plasminogen and factor xiii were determined. results and conclusion: the levels of fibrynogen were significantly higher in fibrynogen concentration obtained by double cryoprecypitation from plasma stored at -30°c. there were no significant differences in the level of plasminogen in both tested groups. double cryoprecipitation of a single unit of plasma (stored -30°c) is an efficient, simple and safe method of obtaining fibrin glue. background: talking about professional risk we generally consider the risk of acquiring some kind of infective disease through accidental injury. in this article i would like to point out another side of the problem-the risk of making preventable medical error. with all its consequences. aim: how blood transfusion errors are among the most serious types of medical errors, the final goal is to initiate nationwide, regular, mandatory error reporting. information obtained, distributed and openly discussed at professional meetings will contribute to improving the patient safety. at the same time it would contribute to avoidance of blaming and shaming of many health care providers. prevent what preventable could be! method: retrospective analyze was conducted at the middle size blood transfusion center (10 000 donations per year and utilization of approximately 25 000 of components). results: after clearly distinguishing adverse events due to underlying patient condition from preventable medical error we fined out that: -great majority of adverse events resulted from medical error -every part of blood transfusion center, from blood donation ward, through laboratory testing to component issuing has it' weak points' or vulnerable places -any educational level is equally liable to error there is no significant difference about occurrence time: -working day/holiday -emergency/routine request -routine h/out of routine h -main error cause were as follows: -donor sample misidentification -rhd typing error -abo typing error -incorrectly performed cross match -recipient misidentification -wrong component prepared -sample confusion during freezing preparation conclusion: the truth incidence of transfusion medical errors is underestimated. mandatory report of fatal or 'only' harmful errors to the referent institution and its periodical announcement is the step ahead in preventing errors. those reports should be discussed at professional meetings (not at the 'yellow pages') and served as educational tool. but, as the most of the errors are system related, the key to reduce them is to focus on improvement of the system and nil for plasma. wastage rate was highest for plasma components. the influence of local practices on such discarding and whether avoidable shall be discussed. audit for blood discarding and corrective actions to minimize discarding is essential for all transfusion services and blood centers. designed technical and economic support. options include importing finished products and/or procuring products made from locally collected plasma. one approach is to consider local fractionation of plasma by building and operating a plasma fractionation facility, which may produce, finished products, or may produce intermediate products that are further manufactured in another facility. an alternative approach is the implementation of a plasma fractionation program where local plasma is sent to an established fractionator, and the plasma is fractionated following preagreed terms. the end products are returned to the country of the plasma supplier. in 1954 the national center for the production of blood products was established, under the direction of elias politis and 9 years later in 1963 begun the production of dried plasma from greek donors. by the year 1965 the center started the production of fibrinogen and by the year 1967 the production of antihaemophilc factor. in 1992 all the activities of the center settled down due to administrative aspects. at the beginning of 1990s a contract fractionation program was instituted (under the direction of k. sofroniadou) concerning the fractionation of liquid plasma and production of albumin, which by the end of year 2000 stopped and was replaced with a new contract for the fractionation of source plasma and the production of albumin. the challenge of adapting to the new and more stringent regulations governing the manufacture of blood products was great and brought a lot of changes in the structure of our center. a new bar-coding system ensuring the traceability of blood donations was instituted together with complex software for packaging and preparation of plasma shipments to the fractionation center together with all necessary paper work. a close collaboration with the medicines regulatory authority in order to be able to fulfill all the requirements that regulate issues associated to the quality and safety of human derived medicinal products. collaboration with blood collection establishments was promoted in order to increase the amount of plasma produced. there is a continuous effort from all the implicated parts in order to follow defined quality assurance procedures as highlighted by international guidelines for the blood donor selection, collection procedures, testing methods, donation handling, storage and transportation of plasma. the plasma contract fractionation program may serve, as an initial step prior to switching production to a locally built facility. this lapse of time may be used to expand the plasma collection potential, and to permit appropriate design, qualification and validation of the facility as well as training of local personnel. background: fibrin glue became a reality in the early 1970s, when techniques for the isolation and concentration of clotting factors were improved. in 1972, matras et al. described successful application of fibrin glue for peripheral nerve repair. this encouraging report prompted the use of fibrin glue in wound closure, skin grafting and bone union of osteotomies. the fibrinogen component of fibrin glue is produced from single unit donations of fresh frozen plasma. such procedure helps to reduce the risk of transfusion transmitted infections encountered by exposure to pools from large numbers of donors or by use of fibrinogen prepared from autologous blood prior to surgery. the second component, a mixture of thrombin and cacl2, is commercially available. thrombin is applied to the operation site simultaneously and in equal volume to the fibrinogen but from a separate syringe. there are many methods of fibrinogen concentrate preparation but none of them has been described in detail. aims: the aim of this study was to choose/select the most effective, simple and safe method of obtaining fibrinogen concentrate (basic component of fibrinogen glue) which would also be easy to prepare in blood transfusion centers. methods of precipitation of fibrinogen by polyethylene glycol (peg), ammonium sulphate, ethanol or cryoprecipitation were compared. methods: plasma was obtained after centrifugation (4000 ¥ g for 5 min) of whole blood. four units of plasma were pooled into a 2000 ml bag, mixed, divided into 4 parts and immediately frozen. one of them was stored at -30°c and after one month the plasma was thawed at 4°c during 16-18 h. fibrinogen was obtained by cryprecipitation and each of the three remaining units was precipitated with ethanol, peg and ammonium sulphate. the levels of fibrinogen, fibronectin, plasminogen and factor xiii were determined in each fibrinogen concentrate. results and conclusion: the level of fibrynogen ratio in fibrinogen concentration, obtained by peg and ammonium sulphate was significantly higher. cryoprecipitation is a simple, economic and reproducible procedure with the advantage of being performed in a closed system. plasma fractionation program in greece: an unknown history the provision of safe and sufficient plasma derivatives to meet the needs of local population requires special consideration and a well(light cycler, roche diagnostic systems, nj) was used for the identification of the c282y, h63d and the s65c point mutations of the hemochromatosis gene, and were based on protocols developed, for c282y by the unidad de medicina molecular (ingo, santiago de compostela, espanha), and for the other two mutations by bolhalder m et al. the primers and probes were designed by tib molbiol (berlin, germany). results: the analysis of the percentages of genotypes and allele frequencies of the hemochromatosis gene mutations are described in the table. no differences were found between the patients and the controls. when we compared subgroups of patients based on their hepatitis c genotypes, a higher value for the c282y allele was obtained (9.0%) in individuals with genotype 1, however without statistical significance. discussion: hereditary hemochromatosis is a common disorder and is associated, in some studies, with a worst prognosis in patients with viral hepatitis. follow-up studies are necessary in order to evaluate if the presence of these mutations can cause a more severe course of the illness (greater risk to develop fibrosis or cirrhosis) and a different outcome when treated with antiviral drugs. also, it will be important to evaluate if aggressive phlebotomies will modify their clinical evolution. introduction: portugal has a higher prevalence of viral hepatitis, with probably more than 250.000 patients chronic infected with hepatitis b and/or c. hereditary hemochromatosis (hfe) is one of the most common causes of known hereditary illnesses with hepatic repercussion. hfe mutations are also found in linkage desiquilibrium with particular hla haplotypes, conferring, eventually, a different response to viral agents and antiviral drugs. in this study we evaluated the prevalence of the main mutations c282y, h63d and s65c for the hfe in a population with chronic hepatitis b and/or c and in a cohort control. background: haemolytic disease of the newborn (hdn) is the destruction of the red blood cells of the fetus and neonate by antibodies produced by the mother. although postpartum rhig prophylaxis reduced the incidence of alloimmunization from pregnancy from 16% to 1-2%, the doubt subsists if it is appropriate to use it as routine antenatal prophylaxis. material and methods: a total of 1836 samples (918 mothers and 918 newborns), from 01/05/2003 and 30/04/2004, were studied. all abo, rh typing, antibody tests and dat were carried out in column agglutination tests. results: from the 918 cases studied it was found 778 cases without incompatibility (85%). from the 140 incompatibilities, 88.6% were abo, 8.6% were rhd, 1.4% were other rh incompatibilities, and 1.4% were due to auto-antibodies. 80% of the mothers were rhd+ and 20% rhd-. conclusion: of the 918 pregnant women studied, only 184 were rhd-. from this group 83 (45%) delivered rhd-newborns, what revealed that the antenatal prophylaxis they were submitted was unnecessary. from the 184 pregnant women rhd-, 13% had incompatibility abo, which decreases to near 2% the risk of development of rhd immunization. being anti-d immunoglobulin a product that has the potential risk of infection transmission, is it appropriate to use indiscriminately as a routine antenatal prophylaxis? the introduction of molecular methods to determine the fetal rhd genotype could rationalize the use of antenatal anti-d immunoglobulin prophylaxis. introduction: the frequency of hla a201 haplotype expression has been found about 44-46% in caucasian and in greek population particularly, 44.3%. because of the high frequency, it is used widely in anticancer immunization. the immune system plays an important role in the defense against neoplastic disease and immune response show temporal chances related to circadian variation of antibodies and total lymphocytes in the peripheral blood. aim: the probable difference in the frequency of hla a201 expression and their lymphocyte phenotype into a group of cancer patients and a group of healthy donors, during screening of immunization with hla-combined peptides. materials and methods: 323 healthy donors who proceeded in the department of transfusion medicine, university hospital of heraklion crete were tested for the hla a201 expression. in 11 of these donors the expression of cd3, cd4, cd8, cd2, cd19, cd20, hla dr, cd3+cd4+, cd3+cd8+, cd16-cd56+, cd3+cd16-cd56+, cd3-cd16-cd56+, cd3-cd16-cd56+ was examined. meanwhile, 132 patients with metastatic cancer who were hospitalized in the department of medical oncology, university hospital of heraklion crete, were tested for their hla a201 expression, while in 50 of them for their lymphocyte phenotype. the antigens expression was examined in flow cytometry. the hla a201 expression in healthy donors was 44.8% and in cancer patients 45% (p > 0.05). in table 1 the mean, standard error, t-test and p of the two groups are included. (see table 1 ). the two groups (healthy donors and cancer patients) revealed no statistical significant difference on lymphocyte phenotype, except of the cd20 expression, which was higher in cancer patients. summary and conclusion: the expression of hla a201 in cancer patients and in healthy donors was comparable. also, the lymphocyte phenotype among the two groups has not statistical significant difference, except of the cd20 (total b-cells). the significance of this result has to be investigated. in the course of original documents research i found out that dr. kalic, head of the first organized blood transfusion institution in the balkan region (at beograd, serbia, in 1936), set himself a professional goal: blood should be awaiting all patients and transfusion should not be a privilege of large city inhabitants only. dr. kalic's idea was that blood transfusion should be administered according to clearly given instructions and using simple blood sets. encouraged by the conclusions of the congress held in paris in 1937, dr. kalic started preparations for the transport of blood to the inland. he concluded bravely that citrated blood could be sent by regular mail, as an ordinary parcel, without particular protection from the outside temperature. he advised his colleagues to use blood as an intravenous injection. blood was taken from voluntary female donor in belgrade (capital), march 1938. after keeping it for 10 days at storage, blood was forwarded on a two-day journey to a small town, 150 kilometres away from belgrade. there it was kept on a room temperature before its final use for a treatment of a patient suffering from secondary anaemia. the patient underwent the procedure without side effects and responded to the transfusion with blood sent in this manner much better in comparison to earlier methods of direct blood transfused. reminding ourselves of the courage of our ancestors to implement their professional knowledge and personal original ideas in a new way with the desire to help the patient as successfully as possible, we pay them the deserved respect and gratitude for inspiring and encouraging us in this way to try the same. conclusions: automation leads to increased standardization, faster specimen processing and reporting, elimination of manual specimen identification, uniform interpretation of serological reaction patterns and objective reading of haemagglutination endpoints. using auto-vue allowed the staff uninterrupted time to perform quality assurance duties, extended antibody identifications, preventative maintenance, inventory control. the instrument allowed us to leverage current staff to a more productive, less stressful level. introduction: exosomes are 60-100 nm secreted vesicles produced by antigen-presenting cells (apcs). the finding that exosomes from dc pulsed with tumor-derived peptides elicited potent antitumor tcell responses and tumor regression in mice has led to the proposal that human exosomes could be effective vectors for antigen delivery in the context of cancer immunotherapy. aim of the study: to establish the method of producing a new kind of tumor vaccine -exosomes secreted by dc, pulsed with tumor peptides. methods: exosomes used in this study were generated from monocyte-derived dc pulsed with peptides from k562 tumor cell lines. exosomes were purified by the methods of ultrafiltration and ultracentrifugation. the methods of dynal magnetic beads, flow cytometry and western-blotting were used to determine the surface molecules of the exosomes. the function of the exosomes was deterobjective: to develop an immunoheatological technique for the study of erythrocyte hyaluronic acid sodium salt (cd44) receptor expression in red blood cells (rbcs) from adults and newborns. materials and methods: samples of anticoagulated blood from adults (n = 30) and umbilical cordon (n = 30) were used. several dilutions oh hailuronic acid sodium salt solution 2% (sigma l-77h0544) were confronted with 2% erythrocyte suspension in phosphate saline buffer (pbs) ph 7.4. the rbcs were previously treated with an enzymatic solution of 5% bromeline in pbs ph 7.4 (sigma l60h0168). agglutination readings' were been by slow sharking after of 12 h incubation at 4°c. the results were expressed through the sensibility parameter which involves titer and score. this is defined by a mathematical expression a = à6 si. di-1. 10-3 (i = 1, 2, 3 . . .) where si represent the score and di-1 is dilution inverse. the adult' rbcs showed a = 26 ± 8, while en the newborn the parameter was a = 3 ± 1. our results showed significant differences between both groups. conclusions: in this work, we present a simple immunohematological technique for the hyaluronic acid sodium salt (cd44) receptor expression in red blood cells, which could be a useful tool to evaluate the alterations of the receptor's expression in rbc. a new technology for crossmatching tests adapted to a fully automated system l gaillard, v desvigne, a boulet, l fauconnier and jm pelosin diagast, loos, france we have developed a new automated technology for crossmatching (compatibility) test suitable for automation and high throughput. the method does not require centrifugation steps thanks to the use of magnetised red blood cells (rbc). all the steps described are performed on the fully automated qwalys system. this methodology requires washing steps under magnetic field and is based on the fixation of sensitised rbc on the surface of a well coated with monoclonal anti-human globulins. in a first step, the red blood cells from target blood bags were magnetised during 10 min. then the patient plasma is distributed on a microplate and incubated with the previously magnetised rbc during 20 min at 37°c. excess of unbound immunoglobulins is removed by washing steps. in a third step, sensitised magnetised rbc were transferred in the antiglobulins coated plate and placed 5 min on a magnet plate. wells in which antigen-antibody interactions have occurred display a confluent layer of rbc (positive reaction). the negative reaction appeared as a pellet in the middle of the well. the test can be read by an automatic reader or by naked eye. the patterns in the well are stable for at least 24 h at room temperature. the plasma samples are provided by the laboratory of haematology of the chru of lille. the red blood cells are collected from segment of tubing of blood bags coming from the laboratory of blood donors of the efs (french blood services) nord de france-lille. the results are obtained in 40 min. comparative studies showed that our new technology, without any centrifugation steps, is reliable and sufficiently sensitive and specific enough to perform cross matching tests using a high throughput automated system. the mechanisms of p53-dependent apoptosis involve a set of genes that possess the ability to modulate oxidative stress. one of them pig3, is induced by p53 through a microsatellite in its promoter region. this microsatellite has been proposed to represent an evolutionary adaptation of tumor suppressor mechanisms. microsatellite instability and genetic constitution, comprising the presence of the low repetition allele (10 tgycc repeats), at this locus have been hypothesized to provide an increased risk for cancer development. aim: in the present analysis we examined this polymorphism in blood samples from voluntary health donors and compared it with human lung cancer samples, employing two different ethnic groups, greek and british. results: analysis of this locus in both types of samples showed: (i) the homozygous presence of the 10 repeats allele only in the samples from healthy blood donors; (ii) a very low frequency of microsatellite instability (<1%) and no loss of heterozygosity in matched normal-tumor tissues; and (iii) a non-significant increase of the most frequent allele (15 repeats) in the cancer groups as compared to samples from healthy blood donors. the last two observations were found in both greek and british populations. conclusion: taken together, these data do not support the notion that this pig3 polymorphism is associated with an increased risk for cancer susceptibility. background: blood group determinations are routinely performed by the sensitive technique 'gel test' for the last few years. many weak d and partial d phenotypes which react as d negative or weak d by slide test, are assigned the rh d + status by gel test. this is most desirable in the case of blood donors but creates concern in case of patients and antenatal women with a partial d phenotype. case report: we report a female patient (blood group o, c+, c+, e-, e+) whose red blood cells gave a positive reaction of different strength and speed with different anti-d antibodies in slide tests. we were asked to type the patient and provide the appropriate blood units. the patient's cells gave a 4+/3+ reaction in the standard screening procedure for the rh d in gel test micro-typing system that contains a polyclonal reagent of human origin (which allows a direct detection of most weak ds), a 4+ reaction in a test with monoclonal anti-d and a 4+/3+ reaction in the gel test micro-typing system destined to detect du and which contains polyclonal anti-d of human origin. however, since the slide test gave a rather slow onset of agglutination with one commercial reagent (made up of a blend of polyclonal and monoclonal anti-d) we tested the patient's red cells against 6 anti-d reagents in the id-partial d typing system. one of these (number 4) gave negative reactions and the remaining five gave positive reactions (ranging from 3+/2+ to 4+), indicative of a partial d category vii phenotype. the patient's red cells were also tested in the id-card 'diaclon abo/d' . this card provides the complete profile for abo/rh d in one single procedure step, including the confirmation of rh d. it contains two different anti-d reagents within the gel matrix in two consecutive microtubes. the first anti-d (polyclonal human) is expected to give a positive result with d+ red cells and partial d category vi, while the second (monoclonal rabbit) is expected to give a negative result with dvi+ red cells. our patient's cells gave a negative reaction with the first and a 3+/2+ reaction with the second anti-d in this system, indicating a d variant other than dvi. finally the patient was assigned the partial d category vii phenotype (according to the pattern of the reactions obtained with the id-partial d typing set) and rhesus d negative blood units were issued. this case illustrates the diversity of reagents used for rhesus typing in different laboratories. failure to disclose some d variants is a disadvantage when typing patients. a combination of techniques is often needed to reveal the real rh d phenotype. the only single system that could have revealed a d variant in our patient from the beginning, is the id-card 'diaclon abo/d' with two different anti-d reagents in two consecutive microtubes as described above. a cost-benefit analysis should be undertaken to show whether it should replace other screening tests for abo and rh d when typing patients. who cares about the quality of life of the chronic patients treated with blood products? d ilcenco*, e hanganu-turtureanu † , c burcoveanu † , c vartolomei ‡ and d azoicai § *blood transfusion center, † hospital 'sfantul spiridon', ‡ institute of hygiene, § university of medicine, iasi, romania quality of life is one of the methods used to appreciate the quality of the health system. romania is going to join soon the european union, so there must be a concern regarding the improvement of the national health system. blood receiver's life quality never been researched before in romania. we have been chosen a batch of chronic ill patients who have been received blood transfusion with blood or blood components, and asked them to complete two types of questionnaires regarding their life. we used nottingham health profile and beck's depresion index. results shows that this kind of patients need special care, because they all (with one single exception) feel frustrated and feel like a burden to the other normal persons. evolution of the pain index, mobility index, energy index, emotions index, sleep index and social isolation index was in concordance with the depression index. in conclusion, this type of patients needs special attention and medical authorities should make more efforts to assure their life quality support. transfusion medicine practice in surgically treated urology patients: our experience il ilincic*, bm bozovic* and ts tadic † *clinical center dr dragisa misovic, † natio. blood transfusion inst., belgrade, serbia objective: multiple studies demonstrate that the use of blood/blood products in patients undergoing elective urology surgeries, as well as the actual needs assessment, present the issue of numerous debates. method: using the retrospective method, utilization of blood/blood products was analyzed, as well as the ratio of prepared/used blood units in urology patients in the surgical ward, in the intensive care unit (icu) and at the urology center within the cc dr dragisa conclusion: due to a rather liberal use of primarily ffp in certain cases (cystectomiae in the first place), and a discrepancy between the prepared and actually used blood units, hospital transfusion committees should be an imperative in order to solve current dilemmas regarding justified use and proper administration of blood and blood products. background: the safe collection, production, distribution and application of blood and blood products in a high quality needs logistic on a high level. since the seventies computers, special software and barcode are used in transfusion medicine and improved the safety of processing data. in the last years a new technology was developed for industrial use, the radio frequency identification (rfid). aim: the aim of our studies was to check whether rfid can use reasonable in transfusion medicine. methods: at first we developed a flow chart, where we can use the technology and where are the problems by introduction. so we tested in the red cross donation centre in saxony about 1000 passive rfid smart label under real conditions. in cooperation between the akh vienna and novatech research a new handheld pc software 'labelview' for all steps around the transfusion was developed, including the identification of the patient and the processing of the haemovigilance data, and tested in first time. results: passive and semiactive (with temperature control) rfid labels survives all hard steps during the working up of the whole blood (e.g. centrifugation by 5000 g, separation, etc.). as a result of the contactless identification they are help to make easier the documentation of all processing steps according good manufacturing practice. in clinical practice they are a good supplement to bed side transfusion software. conclusion: for all lot of problems by the logistic and the safe identification around the transfusion existing various single point solutions such as patient-wristband, bed-side test, double check of blood group typing and donor -donation registry in software, etc. the lecture will deal with new developments in logistics and data management, which can help to reduce the problems associated with documentation, safe identification and reporting of haemovigilance data. our experiences with the immunohaematological analyser olympus pk 80 applied conventional and no conventional (hemolytic medium time) techniques in 23 sera of patients with ascariasis. results: the ai and hk tests showed: b epithopes in 4 ae from b patients and in 3 ae from ab patients; a epithopes in 1 ae from ab patient and in 3 ae from a patients; p and p1 epithopes in 18 ae and only p1 epithopes in 3 ae. these 21 patients had both epithopes in their erythrocytes. the hemolytic techniques showed: anti b immune antibodies in 8 sera and anti a immune antibodies in 8 sera. the presence of abo and p epithopes in ae and immune antibodies in patients with ascariasis show a relation about blood groups and ascariasis. the fact of to find the same abo and p antigens in a. umbricoides and in its hosts suggests that the parasite might absorb them during its life cycle. these epithopes would be involved in the molecular mimicry. the use of filters for leucocyte depletion in anemic patients on maintenance hemodialysis g poposki*, s kovaceski*, b krstanoski*, s mena* and n solaz † *institute of nephrology, struga, macedonia, † ankara university, faculty of medicine, ankara, turkey introduction: renal anemia is one of the major chronic complications in end stage renal disease. it is caused by reduced production of erythropoietin (epo) due to uremic toxin effects, reduced halflife of rbc, iron deficiency, aluminum intoxication, blood loss during hemodialysis, gastrointestinal hemorrhage, epistaxis, infections etc. allogenic blood transfusion is transplantation of certain or all cell types. however, allogenic blood transfusion can contribute to many immune system disturbances with clinical side effects. besides erythrocytes, mononuclear, t and b-lymphocytes, are also transfused, which cause immunomodulatory disturbances in immune system of recipient. leukocytes are responsible for frequent febrile non-hemolytic transfusion reactions, alloimmunization toward leukocytes and hla antigen and transmission of cmv. anti-le antibodies, forming of immune-complexes, complement activation with pirogenic c3a and c5a immunoinflamatoric citokines cause febrile reactions. commercial use of filters for leukocyte depletion with removal of leukocytes and degraded products of microagregates and cytokines, cause minimum harmful immunomodulatory effects and prevent transmission of cmv. aim: the aim of the study was to present the effects of transfusion of erythrocytes with residual number of leukocytes in anemic patients on chronic hemodialysis at institute of nephrology in struga. matherial and methods: during 1997-2004 period all anemic patients on hemodialysis were divided in 4 groups. the first group 34 pts with febrile non-hemolitic transfusion reaction. the second group-25 pts immunized toward leukocyte and hla antigen. the third group 57 young candidates for kidney transplantation for prevention of hla immunization. the fourth group 16 pts with sle (for immune-complexes and autoantibodies). total 132 patients (65 males and 67 females) received 319 units of rbc with residual number of leukocytes. commercial filters of baxterô (lekostop 4 lds) and terumoô (imugard iii rc) of second and third generation with microagregate filter and synthetic polyurethane fibers, with 20-40 microns pores that remove leukocytes, platelets, microagregates and fibrin were used. erythrocyte concentrates are filtered until 5 days of collection. result: aabb permits maximum <5 ¥ 106 wbcs/unit for prevention of febrile non-hemolytic reaction. the filters we used reach residual leukocyte number of 2 ¥ 10 5 the le reduction of 99-99.99%. the number of rbc after filtration is minimum 90% -40 g hb per unit. in none of the patients who have received the leuco-filtered blood, no adverse post transfusion reactions were noticed. conclusion: the used filters for leucocyte depletion are characterized with superior biocompatibility, excellent elimination of all types of leucocytes and high 'recovery' of erythrocytes. the use of filters for le depletion reduces and minimizes the side effects of allogenic blood transfusion in patients on chronic hemodialysis who are alloimmunized, in patients with sle, and particularly in young patients candidates for kidney transplantation. background: fv leiden, prothrombin g20210a, mthfr c677t are three most common and important prothrombotic inherited mutations. aims: the aim of the case-control study was to assess the prevalence of mutations and their single or combined effects as risk factors for thrombosis. methods: the study included 103 thrombotic patients (venous thromboembolism, chronical venous diseases, different etiology) and 200 asymptomatic healthy individuals as control group. extraction of genomic dna was followed with genotyping of fvl by pcr-ssp, prothrombin and mthfr mutation by pcr-rflp. results: a statistically significantly higher prevalence of fvl mutation was found in thrombotic patients (32.0% heterozygous, 1.9% homozygous) compared to controls (3.0% heterozygous), p < 0.0001. the or for heterozygous carriers was 15.24 (95% ci 6.13-37.94), confirming the association of fvl mutation with the risk of thrombosis. there was no statistically significant difference in the prevalence of the prothrombin mutation in patients (5.8%) and controls (3.5%), or 1.71 (95% ci 0.56-5.21), p = 0.3441. although the group of thrombotic patients showed a higher prevalence of homozygous carriers of c677t mthfr than the control group (17.5% vs 10.5%), or was not significant (1.81, 95% ci 0.91-3.57), p = 0.0859. analysis of combined effects of mutations showed an additional thrombotic risk for carriers of fvl mutation and both mutated alleles of c677t mthfr gene (tt and ct) (or 9.40, 95% ci 3.41-25.80), p < 0.0001. conclusions: fv leiden mutation was detected as significant single risk factor for thrombosis in studied patients group. additional prothrombotic risk have carriers of fvl mutation and c677t mthfr gene mutation. a female patient in a high fever due to urinary tract infection does not respond being given antibiotics. on the contrary, leukocytes rose (to 30 ¥ 10 9 /l), anaemia became even deeper, as well as thrombocytopenia. hemocultures were negative. hematologist decided to search for hematological disease. the first citology results of bone marrow aspirate suggested lymphoproliferative disease (15% atipical plasma cells). to treat heavy anaemia (hgb 53 g/l) hematologist asked for red blood cell concentrate. pretransfusion testing revealed warm autoantibodies in the patient serum and on red blood cells. antibodies had no apparent specificity. biochemical parameters (bilirubin, ldh, haptoglobin) suggested mild hemolitic process. electroforesis revealed polyclonal hypergamaglobulinaemia. the 9th day of hospital treatment, the therapy with corticosteroids was introduced (solu-medrol 125 mg per day). coagulation parameters were tested: pt 1.75 inr, aptt 41s, fibrinogen 0.5 g/l, trb 66 ¥ 109/l, d-dimer 251 mg/l, atiii 52%. dic was suspected. liver enzymes showed mild liver dysfunction (normal ast, alt, elevated ggt, low che). substitution therapy started with 1 dose of cryoprecipitate, 1 dose of fresh frozen plasma, 1000 iu atiii, 2 doses of red blood cells and vitamin k 10 mg. two days after the substitution therapy we saw pt 1.28 inr, aptt 31s, fibrinogen <0.5 g/l, trb 75 ¥ 10 9 /l, atiii 71%. during the next few days erythrocytes and thrombocytes rose, but due only to corticosteroid therapy and not to substitution therapy. the patient had neither signs of con-sumptive coagulopathy, nor hypoproduction of coagulation factors, except for fibrinogen. till 17th day of therapy, fibrinogen was below 0.5 g/l. there was no hemorrhagic diathesis. after that, fibrinogen rose, and on the 21st day the patient was recovered, in both clinical and laboratory terms. the results of immunological tests, collected later, confirm the diagnosis of systemic lupus erythematosus. we did not have any specific test to confirm antibody mediated hypofibrinogenaemia, but in the setting of sle, without any specific treatment except corticosteroids, fibrinogen recovered. we assume it is quite enough for highly suspected immunological hypofibrinogenaemia. results: twenty-four-year-old male patient with severe hemophilia type a suffering from low incoercible digestive bleeding secondary to ischemic colitis caused by autoimmunity (vasculitis) without response to current management. treatment was initiated with 90 mg/kg/dose of rfviia (*) for 7 days, after which there was clinical and endoscopic recovery, and an inh decrease to 1.0 ub/ml (fviii dosage 19%) . he began to take meprednisone (1 mg/kg/day) for 45 days, after which the inh titre was 12.0 ub/ml. (table 1a ,b) the patient underwent surgery the following year (correction of equinus foot). he entered the operating room with an inh of 26.7 ub/ml and was treated with 120 mg/kg/dose of rfviia (*) for 10 days, obtaining an excellent hemostatic response. he had two autologous blood units, but it was not necessary to be administered. the inh titre decreased again (down to 5.7 ub/ml) during the intratreatment stage. thirty-five days after rfviia, the inh titre was 7.5 ub/ml. (table 2a ,b) the presence of high titre inh against fviii is a critical problem in cases of bleeding or surgery need due to the inefficacy of the available therapeutic options and the severity of the events. in this case, we have observed that, apart from inducing hemostasia through the activation of the coagulation extrinsic path, rfviia could reduce the inh titre in sequential dosages. we have introduced the case of a 24-year-old patient with hemophilia complicated by a high titre inh against fviii. in this case, we have observed that, apart from inducing an effective hemostasia through the activation of the coagulation extrinsic path, rfviia could reduce the inh titre in sequential dosages. there was also a decrease in the inh titre concomitantly with an increase of the plasma fviii level during its use. this phenomenon suggests that rfviia could produce a modulation in the immune response. evaluation of an automated blood collection system with standard ratio of anti-coagulation and integrated filter for whole blood leucodepletion l dadiotis, a kolokytha, m dimou, a perdiou, c alepi, p spyropoulou, e igoumenides, c velidou, v panagopoulou and s matsagos tzaneion general hospital, pireas, greece automated blood collection system (abc) is a device manufactured by macopharma which collects by gravity a preset volume of blood and mixes it with anticoagulant (ac) in standard ratio (7 : 1). this is managed by passing the ac, which is stored in a special bag, anti-erythrocyte antibodies are immunoglobulins that belong to the igg, igm and iga classes. their common characteristic is a specific reaction with antigens that are located on the erythrocyte surface. they can emerge as auto antibodies and alloantibodies. the blood transfusion in patients may induce a post-transfusion hemolytic reaction (pthr). in order to avoid or reduce the danger of the pthr it is necessary to examine whether there are irregular anti-erythrocyte antibodies in the patient's serum as well as in the serum of the voluntary blood donors. all the irregular anti-erythrocyte antibodies are not clinically relevant. the experience shows that the clinically significant antibodies most often belong to abo, rh, kell, kidd, duffy and ssu blood groups. in the period from april, 2003 to november, 2004, we monitored and examined, at the institute for blood transfusion, clinically significant antibodies in the serum of the patients who are treated with blood transfusions as well as in the serum of the voluntary blood donors. we used the following tests for detecting irregular anti-erythrocyte antibodies: enzyme test, indirect coombs test, screening test by the commercial test erythrocytes and gel filtration method. the detected irregular antierythrocyte antibodies are identified by means of the commercial test erythrocytes for identification. our results are the following: voluntary blood donors: anti d, anti c + d and anti-leb antibodies. patients: anti-d, anti-k, anti-fya, anti-c and anti-e antibodies. in nine patients, anti-erythrocyte antibodies were discovered, namely, those that react at the temperature higher than 30°b ut whose specificity we could not discover with the existing techniques. improved predictive factors of response for myelodysplastic syndrome patients treated by the combination of erythropoietin and g-csf s park*, c kelaidi † , s grabar ‡ , v bardet ‡ , d vassilieff ‡ , f picard ‡ , m guesnu ‡ , mc quarre ‡ , p fenaux § and f dreyfus ‡ *service hématologie, hopital cochin, † hématologie, hopital avicenne, ‡ service hématologie, hopital cochin, § hématologie, hopital avicenne, paris, france it has previously been shown that serum epo level and number of previous red blood cell transfusions are predictive factors of response to epo + g-csf treatment of myelodysplastic syndromes (mds). in a subgroup of patients with mds having sepo <500 ui/l, known to be good responders to epo + g-cscf, the gfm group wanted to refine the model predicting the response to epo + g-csf, especially with cytology (who classification with dysplasia and percentage of erythroblasts and blasts). in a population of 64 patients (ra, rars and raeb <10% blasts) receiving epo ± gcsf between 2000 and 2004 and having serum epo <500 ui/l, the response rate at week 12 (iwg criteria) was 60%. six variables were associated with response to epo ± g-csf for mds: age >70 years (p = 0.02), number of prior red blood cell transfusions <2 packs/months (p = 0.005), serum epo level <200 ui/l (p = 0.02), percentage of blasts <5% (p = 0.05), percentage of erythroblasts >30% (p = 0.02) and low ipss score (p = 0.001). we did not found any influence of dysplasia, type of rhepo (darbopoietin alfa or epoietin alfa) and karyotype on response rate. in multivariate analysis, age through a rotating pump. the abc can be used with all types of p-417 pan-european blood safety alliance the pan-european blood safety alliance is a unique alliance of patient organizations, formed to promote the highest level of blood safety for all in europe. it was formerly established on 11 february 2005, during the course of the first general meeting of the pbsa, which comprised of 8 founding patient organizations. the objectives of the pbsa are: 1. to promote the fundamental right and duty to safety of all patients in need of blood transfusions and blood products. 2. to ensure the availability of sufficient amounts of safe blood, to meet all treatment need through: -the education of all staff handling blood components, to reduce human error. -the implementation of and access to, proactive blood safety technologies, for each patient across europe. -haemovigilance -the adequate access to blood transfusion services, which should be provided free of charge to the patient. other objectives are to raise awareness on a local and european level regarding blood safety, to promote eu legislation that improves safety standards of blood transfusion services, including stem cell preparation and storage across europe and to lobby for increased patient influence on eu health policy makers. very importantly, the alliance aims at providing a forum for patients, healthcare professionals, health policy makers and relevant industry, as well as acting as a point of reference to the national health authorities, the european commission and other european institutions, when seeking the opinions of patients on blood safety. cerns of insertional mutagenesis and the safety of some viral vectors that randomly insert genes through the genome have been recently resurfaced following the development of a haematological malignancy in a child treated with a retroviral vector. particularly questions also remain as to, whether gene therapy and the production of ectopic factor viii and ix will be a risk for inhibitor development or indeed whether it might promote tolerance in those patients with inhibitors. w-pl4-11 gene therapy for thalassemia: will it become reality? university of washington, seattle, wa, usa experiments aimed to develop gene therapy approaches for the beta chain hemoglobinopathies, sickle cell disease and beta thalassemia started about 25 years ago. in the beginning results were dismal because of the extremely low and variable expression of globin genes contained in the therapeutic vectors. a major development occurred in 1987 with the discovery of powerful regulatory elements that could guarantee high level of globin gene expression. these elements when incorporated into viral vectors allow expression of therapeutic levels of the transferred globin genes. a second major progress was achieved with the development of safe lentiviral vectors that can efficiently infect the human pluripotent repopulating hemopoietic stem cells. as a result of this progress, today beta thalassemia and sickle cell disease can be cured in murine models of these disorders. considerable effort is already being devoted into further improvement of lenti viral vectors with emphasis on incorporating elements which will decrease the probability of insertional mutagenesis and leukemogenesis. the major challenge for the clinical application of stem cell gene therapy of thalassemia is the need for genetic correction of large numbers of mutant stem cells. in vivo selection of corrected stem cells is being investigated but there are questions about its safety because of the possibilities of clonal expansion of stem cell lines carrying undesirable integrants. other major challenges have to do with logistics: production of therapeutic vectors, infrastructure required for stem cell gene therapy delivery, and sponsoring and funding of the clinical trials. gene therapy trials on limited number of patients are expected to be initiated relatively soon. if these trials are successful and cures of beta thalassemia ensue, the major challenge will be the delivery of this molecular therapy in the context of medical practice. w-pl4-10 gene therapy for haemophilia haemophilia is an ideal target for gene therapy because only a small rise in factor levels to 1-2 u/dl would achieve the goals of prophylaxis without regular infusions of concentrate and deliver a substantial improvement in lifestyle for patients with severe haemophilia. gene therapy for haemophilia today relies upon addition of normal factor viii or ix genes. with present technology gene therapy can offer the prospect of a true 'cure' for haemophilia in animal models, although this may not be currently realizable in man. more than 30 patients with haemophilia have now been treated in phase 1 gene therapy protocols. all studies have failed to conclusively show that therapeutic levels of factor viii and ix can be reliably obtained. the first trial reported used im injection of a factor ix containing recombinant adeno associated virus (raav) in adult patients with severe haemophilia b. only very modest increases in factor ix level, <2 u/dl rise, in 4/8 patients enrolled were observed, although less factor ix concentrate was needed in 3/6 subjects. a similar study using the same raav vector via intrahepatic artery infusion has been conducted. this has been complicated by the observation of aav vector in the semen of subjects. in six patients enrolled, no durable levels of ix above 1 u/dl were seen. further development of this raav vector is suspended. for haemophilia a, three systems are have been tried. the first study was an ex vivo addition of factor viii gene to autologous fibroblasts and then laparoscopic reimplantation. preclinical assessments demonstrated durable expression of factor viii (>5% of normal) for >1 year in mice following a single treatment. in 4/6 patients treated repeated factor viii rises (0.5-3.5 u/dl) were seen, but no improvements lasted beyond 10 months. the second protocol used a murine leukemia retrovirus containing factor viii, injected intravenouslya development of preclinical data in rabbits and haemophilic dogs. 0/13 patients enrolled sustained levels of factor viii >1 u/dl. the third study, using a modified, 'gutless', adenovirus containing factor viii gene has recruited one patient. this patient demonstrated transient liver toxicity and thrombocytopenia at doses lower than those that cause toxicity in primates. sustained levels of factor viii of ~1 u/dl have been observed over a number of months. accrual to the study has been poor. haemophilia remains a prime target for gene therapy. however, haemophilia is no longer a life threatening disease with current therapy that is both safe and efficacious. a balance between the benefits and theoretical risks must be borne in mind when considering gene-based approaches to therapy. conreference: petz ld, garratty g. immune hemolytic anemias. 2nd ed. philadelphia: churchill livingstone, 2004, pp 375-400. w-pa-114 autoimmune neutropenia introduction: autoimmune neutropenia (ain), a granulocytic disorder due to the presence of anti-neutrophil antibodies, may present as neutropenia of varying degree with or without recurrent infections un previously healthy individuals (primary or idiopathic ain) or in patients with a known underlying disease such as lupus erythematosus, lymphoid malignancies, etc (secondary ain). the condition affects more frequently infants of small ages while it is rare in adults [1] [2] [3] [4] [5] [6] [7] . in some patients, diagnosis is established in occasion of a respiratory, urinary or cutaneous infection, but in many cases is simply a finding of cell blood counting performed for unrelated reasons [6] . clinical and laboratory findings: in general, physical examination is negative. laboratory investigation reveals the existence of isolated neutropenia. association of the disorder with autoimmune hemolytic anemia or autoimmune thrombocytopenia is rarely seen [8] . blood biochemistry is normal while serologic tests for bacterial, viral or other pathogens may be positive depending on the underlying infection. bone marrow is hypercellular without maturation arrest of granulocytic series. hemopoietic stem cell reserves and function are normal or increased, and stromal cell function is within the range of the normality [9]. methods for the detection of granulocyte-specific antibodies: serology for the detection of granulocyte-specific antibodies has been marred, compared to erythrocyte serology, because the target cell here, the granulocyte, is short-lived, fragile and becomes easily activated. the former two of these difficulties require absolutely freshly (<6 h old) isolated neutrophils from a panel of donors to be used every day to run the tests with sera from patients, while the third difficulty is more important since spontaneous cell clumping in vitro is very common and nay mimic the specific aggregation caused by cross-linkage of surface bound antibodies in the granulocyte agglutination test (gat). in order to overcome these problems, the second international granulocyte serology workshop [10] recommended a combination of two tests as the best screening procedure for the detection of granulocyte autoantibodies in patient sera, gat and granulocyte immunofluorescence test (gift). gat is mainly mediated by igm antibodies and is positive in about 2% of cases. gift detects igg antibodies and is positive in about 98% of cases. it is to be noted that flow-cytometry fluorescence may arise not only from the surface but also from the cytoplasm of neutrophils, necessitating assessment of membrane fuorescence by microscopy. a good direct anti-granulocyte test is not available today. this is due to the fact that too few neutrophils can be obtained from the blood of neutropenic patients, and also to the observation that neutrophils are often activated in vivo because if an underlying infection or other inflammatory process, thus expressing fcgrii and fcgriiib to which nonspecific binding of w-pa-113 practical approach to transfusion in autoimmune hemolytic anemia (aiha) g garratty american red cross blood services, pomona, ca, usa a major problem when transfusing patients with aiha is that often all units are incompatible. this may be due to autoantibodies (autoab) and/or alloantibodies (allo-ab). if the incompatibility is due to only auto-ab, then transfusion of incompatible blood will not usually result in a clinically significant reaction, but if due to alloab, the result may be similar to that seen in any other patient (i.e. a hemolytic transfusion reaction ranging from mild to severe). thus, it is essential (as in any other patient) to exclude the presence of allo-ab. it is wise to phenotype all patients, for as many antigens as possible, before the patient receives transfusion. there are two popular approaches to determine if allo-abs are present but being masked by 'warm' auto-ab activity. the preferred method is to remove the auto-ab by adsorbing the patient's serum with autologous rbcs treated with enzymes, or preferably, with zzap reagent. the latter reagent contains an enzyme leading to optimal adsorption of auto-ab, and dtt. these two chemicals will destroy significant antigens other than rh and kidd (e.g. mns, duffy, kell, lutheran, dombrock, cromer, lw, some yta and ge, inb, jmh, ch, rg, pr antigens), thus will not adsorb alloantibodies to these antigens. if autoadsorption is not possible (e.g. patient has been transfused recently or there are too few rbcs), one has to perform adsorption with enzymes or zzap-treated allogeneic rbcs. one does not have to be concerned with covering any antigens destroyed by zzap (e.g. kell and duffy systems). we use a rough guide relating the strength of the indirect antiglobulin test to the number of adsorptions needed to remove auto-ab (1+ = 1 adsorption; 2+ = 2 adsorptions; 3+ = 3 adsorptions; 4+ = 3 or more adsorptions). if there is no activity left after the adsorptions, then one can suspect that the incompatibility was due to auto-ab, but on rare occasions one can be wrong and an allo-ab to a high-frequency antigen has been removed. this is a major disadvantage of using allogeneic adsorptions, and is why adsorptions with autologous rbcs are preferred. if time (2-8 h) does not allow for adsorptions, one can dilute the patient's serum (e.g. 1 in 4, or 1 in 5) and test the dilution against a panel to see if any alloantibody specificity becomes obvious. another approach is to select units matching the patient's phenotype as closely as possible. when dealing with cold agglutinin syndrome one can usually exclude allo-ab activity by testing strictly at 37c. this can be helped by performing adsorptions with enzyme-treated autologous rbcs at 4c, but it is difficult to adsorb all of the powerful cold autoagglutinin activity. it is reported that 30-40% of aiha have allo-abs; the incidence is even higher in patients who have received multiple transfusions. thus, we feel that procedures such as those discussed above must be performed before transfusing incompatible blood if time allows. one should always negotiate with the attending physician regarding the time it will take to perform adsorptions. a decision may be made not to perform adsorptions if the patient has life-threatening hemolysis, and especially if the patient has never been transfused, or pregnant. serum igg may occur (naig). the presence of immune-comlexes in the serum, such as in patients with felty's syndrome, lupus erythematosus and other diseases, as well as the presence of immune aggregates formed in sera stored frozen for long time, may give false-positive tests given that they may bind to fcgriiib molecules expressed on the surface of neutrophils. elimination of immunecomplexes and immune aggregates can be easily obtained by ultracentrifugation [11] . another cause of false-positive results may be the presence of anti-hla antibodies because of allo-immunization. these allo-antibodies react with hla molecules found not only on neutrophils but also on the surface of many other cells including lymphocytes. these allo-antibodies can be eliminated by platelet absorption. it seems that the best method in the search of true antigranulocyte antibodies is the monoclonal antibody-specific immobilization of granulocyte antigens (maiga) [12] . with this method one can specify anti-granulocyte antibodies using a panel of known granulocyte antigenic specificity. finally, it is notable that the levels of serum antibodies to neutrophils may vary considerably over the time. one negative test does not exclude ain. usually, two to three tests have to be run over a period of months [13] . antigenic specificity: human neutrophil antigens (hna) are classified according to an international granulocyte antigen working party [14] . three glycoproteins have been found to be involved in the determination of antigenic specificity, fcgriiib, gpnb1 (cd177) and gp70-95. the respective antigens, frequencies and alleles are illustrated in table 1 . antigenic specificity can also be studied by using methods applied in molecular biology. a promising approach is transfection of mammalian cells by cdna derived from granulocyte antigen specific mrna. cell lines have been established with cells expressing the respective human granulocyte antigen, making the detection of anti-granulocyte antibodies more easier. genotyping of hna antigens can also be stydied with the pcr technique [15] . references are available from the author upon request. w-pa-115 a rare case of 'coombs negative' autoimmune haemolytic anaemia due to red cell autoantibodies of iga class warm autoimmune haemolytic anaemia (waiha) is usually associated with red cell auto-antibodies of the igg class, which can be detected by polyspecific direct antiglobulin test (dat). routine polyspecific direct antiglobulin tests contain anti-igg and anti-c3d components, and are not standardized to react with iga-or igmsensitized red blood cells. haemolytic anaemia caused by warmreacting auto-antibodies solely of the iga class is exceedingly rare. those cases of autoimmune haemolytic anaemia can be difficult to diagnose because of the negative polyspecific coombs' test, which is a standard in investigation of possible causes of haemolysis. we present a case of severe warm autoimmune haemolytic anaemia caused by iga class autoantibodies. a 40-yr old male patient was admitted with anaemia, haemoglobinuria, and other signs of severe haemolytic disease. he received multiple transfusions but haemoglobin level did not rise above 7 g/dl. the initial polyspecific direct antiglobulin test, containing an anti-igg and anti-c3d antiserum, was negative. tests for cold agglutinins and other possible causes of haemolysis were negative. only by using a monospecific, anti-iga antiserum could we show that the warm iga auto-antibodies against red blood cells were present on patient's erythrocytes. we have not detected signs of complement activation by iga autoantibodies in this patient. the patient received corticosteroids with good initial effect. his haemoglobin level stabilized and he did not require more transfusions. anti-iga direct antiglobulin test became negative about to weeks after the therapy was initiated. however, in spite for the initial effect of steroid therapy haemolysis continued, and splenectomy was performed 6 months after diagnosis was made. it has been shown that human lymphocytes, granulocytes and monocytes contain specific fc receptors for iga, and both monocyte-mediated phagocytosis and antibody-dependent cellular cytotoxicity due to iga auto-antibodies has been demonstrated. there is also increasing evidence that iga auto-antibodies can activate complement, both via the classical and the alternative pathway. a phenomenon of 'reactive haemolysis', which involves c3-independent binding of c5b6 complexes to 'bystander' red blood cells, has also been described. we emphasize the importance of performing additional testing in cases of apparent 'coombs' negative' haemolytic anaemia due to iga, igm or 'low affinity' igg autoantibodies, and serological aids that are available for that purpose. described. both siblings were born on term, in good general clinical status, free from any signs of infection, and with isolated severe neutropenia (170 and 123 neutrophils/ml). the diagnosis of annanti hna-2a was made upon exclusion of other possible causes of neonatal neutropenia, and confirmed by serological testing of granulocyte antigens and antigranulocyte antibodies. in both cases, the course of the disease was mild, with bacterial omphalitis on day 15 and 5, respectively. omphalitis was successfully treated with 7-day antibiotic therapy according to antibiotic sensitivity report. the first neonate received standard dosage of intravenous gammaglobulins for 5 days without success. this was followed by an attempt at neutrophil count increase with 2-week corticosteroid therapy, also without response. the second neonate received no specific therapy for neutrophil count increase. the children were discharged for home care with clinical and laboratory control examinations at 2-week intervals. in spite of prolonged neutropenia (2 and 6 months, respectively), no other infections were recorded. discussion and conclusion: in our patients, the therapeutic approach to ann was individualized, based on standard antibiotic therapy, intravenous gammaglobulins, corticosteroids, available literature data, and our own clinical experience. although in the last few years rh-gcsf is successfully used in patients with neutropenia, we decided to postpone its use in case the neonatal sepsis developed. the reasons for such decision were: (1) the fact that both neonates were in good general clinical status, with a mild course of the disease with only short-term umbilical infection successfully managed with antibiotic therapy; (2) literature reports suggesting the unexpected failure to respond to rh-gcsf therapy in patients with neutropenia induced by anti hna-2a immunization, and (3) the unknown effect of rh-gcsf on developing tissues of the neonate. the choice and efficacy of specific therapy for neutrophil count increase in the management of alloimmune neonatal neutropenia have not yet been fully defined and require additional evaluation in the majority of cases. male donors for the production of fresh frozen plasma: a special issue for trali patients trali is a significant cause of transfusion associated morbidity and mortality, and has been reported as the third most common cause of fatal transfusion reactions. there is no good evidence on which to base transfusion support policy for patients who have experienced trali. the hypothesis that there may be patient associated factors that contribute to the risk of trali is generally accepted. for this reason it seems reasonable to try to avoid further transfusion during the period of illness. if this is unavoidable the next best solution to reduce the risk of recurrence seems to be the avoidance of using plasma containing blood components (especially ffp) as there is a high chance of positivity for leucocyte antibodies especially for those coming from female donors. as fresh frozen plasma transfusion accounts for up to half of all trali cases and as our center is the only in greece responsible for the testing and processing of blood from blood donors representing military recruits, the last two years we tried to set up a project in order to provide components from male donors on request. our donor base consists predominantly from males donors (98.7%), aged between 18 and 25 years old, with a small chance of having a positive history for transfusion the difficulty of the project consisted on the fact that these donors are assigned to military camps throughout greece, which makes difficult the on time arrival of the units to our establishment in order to be processed for the production of ffp conform the european council quality requirements. this was the main reason why, till now, all plasma produced from these donations was regarded as plasma for fractionation. the first step for implementing the new project was to evaluate the number of donations that, by minor changes on the time of arrival, could be processed for ffp production. the next step was to re-schedule the shifts of the personnel for the on time production of ffp. during 2003, 25% of donations were fulfilling the specifications for the production of ffp and with the flexibility of the schedule the 97% of them were successfully processed to ffp. during 2004, 30% of the donations were fulfilling the specifications and 97.8% of them were processed to ffp. so it is feasible to increase the proportion of male ffp by organizing better the transportation of blood from the donation sites to the blood establishment and by retaining available specialized personnel to cover the extra shifts. maximising the blood supply chain in times of shortage shortages in the blood supply chain may occur for a variety of reasons. they may be temporary e.g. due to a flu epidemic or prolonged e.g. due to the exclusion of a high proportion of donors due to new pre-donation tests or because of a lack of volunteer donors. increasing awareness of the possibility of blood shortages mainly related to increased precautions associated with the possible transmission of vcjd by transfusion has been the driver for the development of blood shortage contingency plans in the uk. in england and north wales, hospitals and the national blood service (nbs) have worked together to develop an integrated blood shortage plan (ibsp) designed to ensure that hospitals and the nbs work together within a consistent, integrated framework giving patients equal access to available blood on the basis of need. an essential element of the plan is the principle that shortages can, in most cases be avoided by reducing the current usage of blood through appropriate use programmes. the impetus for hospitals to implement these programmes was a government circular (hsc 2002/009). hospitals have embraced the circular and have recruited specialist hospital transfusion practitioners, introduced lower hb triggers, cell salvage and hospital transfusion teams and are participating in the blood stocks management scheme (bsms). audits of compliance with the circular have taken place, and a web based tool kit is available. the demand for blood has declined for the last three years, with a decrease of about 5% during 2004-05, suggesting that the drive for improvement has been successful. the shortage plan introduced in england and north wales has two key aims: that the national pool of blood is available for all essential transfusions for all patients and that overall usage is reduced to ensure the most urgent cases receive blood. the plan is structured to provide actions for the nbs and hospitals in three phases, 'normal' circumstances, reduced availability and severe prolonged shortage. hospitals should have documented emergency blood management arrangements for each of the phases. the national plan is activated when the nbs red cell stock level falls to pre-defined levels, hospitals are informed by fax that they should reduce their normal stock holding levels according to guidance in the ibsp and comply with the daily hospital usage budget. the bsms has used its knowledge of hospital inventory levels and demand to provide guidance on appropriate inventory levels for normal and reduced status, it also provides the daily hospital budget. to monitor progress against the recommendations in hsc 2002/009 hospitals will be benchmarked against a number of performance indicators. these include the presence of emergency blood management arrangements, median red cell usage for a number of surgical procedures and percentage wastage of blood. there have been no shortages within the nbs for more than six years, it is hoped that the implementation of the ibsp will help to ensure that in the unlikely event of reduced availability blood will be available to the maximum number of patients requiring a blood transfusion. w-pa-120 transfusion during disaster g klein nih, bethesda, md, usa publicity given to blood donation during wartime has created a powerful association between the need for blood and occurrence of a disaster. blood is rarely needed in excessive quantities at the moment a disaster occurs. the outpouring of blood donors, especially at the site of a disaster, often proves counterproductive. the terrorist attacks on the world trade center on september 11, 2001, with almost 3000 deaths and more than 4000 injuries, provides an instructive model. more than a million potential donors contacted blood-collection centers. hundreds of thousands of prospective blood donors crowded collection facilities and many waited for hours, often to be turned away. qualified staff were in short supply and screening errors occurred as minimally qualified staff were recruited and as collection personnel fatigued. supplies and storage capabilities were pushed to their limit. some blood was inadequately processed and stored. resources were diverted from needed apheresis collections and component preparation to whole blood collection. in the aftermath of the disaster, blood outdated and volunteer donors became disillusioned as their 'gift of life' was refused or unused. similar responses have occurred numerous times over the 60-year period since blood-donor programs were introduced. in virtually every civilian disaster in the u.s. during the past century, all the blood that was needed was immediately available from blood inventory. in only four cases were more than 100 units of blood used in the first 24 to 30 h. in 2001 in new york, the five hospitals closest to the disaster site admitted only 139 disaster victims. the new york blood center, which supplies 80 percent of blood for the city's hospitals, added 600 units to routine inventory at hospitals. the center received 12 000 telephone calls and collected more than 5000 units of blood in the first 12 h. in the area of the pentagon, the chesapeake and potomac red cross blood center supplemented hospital blood inventories within h of the disaster. meanwhile, spurred by well-meaning media and federal officials, lines of blood donors were being processed at local hospitals, makeshift collection centers, the small research hospital at nih, and at a building next to the white house. in the week after september 11, america's blood centers collected 167 000 more units of blood, and the american national red cross collected 287 000 more units than in the same period the previous year. more than 475 000 units were collected for the disaster victims, but only 258 units were used. u.s. blood collectors and federal agencies have created a disaster plan that acknowledges the need for altruistic people to volunteer for blood donation in the time of disaster and speaks with a single voice to avoid needless collection activity while harnessing the good will of well-intentioned people to supplement the ongoing need for volunteer blood donation. rehabilitation of blood transfusion service in azerbaijan cd asadov, ga huseynov and ab hagiyev institute of hematology and transfusiolo, baku, azerbaijan at the end of 80th years of the last century in azerbaijan as well as in other republics of the former ussr began process of progressive deterioration of blood service parameters. in result there was an essential reduction of prepared blood and blood components quantity, manufacture of preparations from blood's plasma has completely stopped. it is connected by that our republic experiences a heavy transition period from scheduled to market economy. after reception by azerbaijan of the sovereignty on development of a national policy the big work has been lead to areas blood transfusion and development of national rules and the standards regulating functioning of establishments of blood service. in 1996 the law about ' the donorship of blood and its components in the azerbaijan republic' has been accepted, instructions on physical examination of donors and preparations of blood and its components are authorized, and also the new speciality transfusiology has been entered into the nomenclature of medical specialities. now the national program of blood service development is developed. at drawing up of the program social and economic conditions of the country, ethnic both cultural traditions and a mental potential of the nation are considered. within the framework of this program is planned to refuse gradually a paid blood donation during the certain period of time to reach 100%s' voluntary unpaid blood donorship. however in connection with limitation of resources, the state is not capable to allocate enough of means for its realization. the big work on attraction of the international organizations has been carried out. now the project of the united nations development program (undp) 'rehabilitation of blood transfusion service in azerbaijan' is carried out at sponsor's support of the norwegian government. realization of this project will lead to reorganization of blood transfusion service in our country according to practice of the european countries. within the framework of project realization it is planned to make changes and additions to a existing law about a blood and its components donorship to bring it into accord with recommendations of the europe council. updating of the russian law 'concerning the donation of blood and blood components' on june 9, 1993, a law, 'concerning the donation of blood and blood components, ' was signed by the first russian president, boris eltsin. now, after more than ten years of market economy and democratic evolution in russia, this law was significantly changed on august 22, 2004, as shown in the following sections: 1. the development of a voluntary blood donor system. 2. removal of the upper age limit for blood donors. 3. funding for blood donations. from january 1, 2005, each level of the state power budgets for a blood donor service to supply blood products for federal, regional, or municipal hospitals. costs of these drugs and the need of prolonged growth factor treatment in these disorders. w-pa-127 can iron administration reduce peripartum blood transfusion c breymann university of zurich, zurich, switzerland the prevalence of iron-deficiency anemia in different regions of the world ranges from 12 to 43%. the increased iron requirement in pregnancy and the puerperium carry with it an increased susceptibility to iron deficiency and iron-deficiency anemia and perioperative or peripartal blood transfusion. however, if ever possible administration of blood transfusion should be avoided for several reasons which will be pointed out in the talk. infections: it is well known that various pathogens such as bacteria and virus can be transmitted by administration of blood. around 0.03% of are contaminated by bacteria such as yersinia or pseudomonas species but are not screened routinely for bacteria. in addition there is no donor screening for hepatitis a, herpes species (cmv, ebv, hhv 7, hhv 8), parvovirus b19, hepatitis g (3.4%) and tt (transfusion transmitted) virus (1.9%). numbers for positive testings for 'classic' virus such as hiv, hep. b and hep. c vary from country to country and lie around 1 : 30 000 to 1 : 5000 000 depending on quality of donor screening programs, pcr sensitivity etc. recently there is increasing evidence that even prions which cause the jakob creutzfeld disease variation ('mad cow disease') might be transmitted by transfusions. therefore the fda has determined that blood donors from countries with high prevalence of prion positive persons are not permitted to give blood in the us (e.g. donors from uk). beside infections, other well known effects of transfusion are problems due to incorrect blood or components transfused, post transfusion purpura, acute and delayed lung injury, graft versus host disease and other acute and delayed allergic reactions. beside these negative effects it was also shown that patients who receive blood transfusion liberally after operations or in icu show higher morbidity and mortality compared to patients with restrictive transfusion policy. this might be due to negative effects on immune functions and inflammatory reactions and lack of stored blood to efficiently improve organ oxygenation. for example it is known that stored blood has worse capillary perfusion and worse viscosity properties compared to fresh blood. taken together there is increasing scientific evidence that blood transfusion is not the gold standard for anaemia management and alternatives such as endogenous blood pooling and efficient treatment of any anaemia must be enforced in the clinical settings. prevention and correction presuppose reliable laboratory parameters and a thorough understanding of the mechanisms of iron therapy. in order to correctly diagnose the type and degree of anaemia, a prerequisite for selection of the proper therapy, one must first of all correctly differentiate between the relative, i.e. the physiological anaemia of pregnancy due to the normal plasma volume increase during pregnancy, and 'real anaemias' with various different pathophysiological causes. when defining the hb cutoff value for anaemia in pregnancy, the extent of the plasma volume changes with respect to the gestational age must be taken into consideration. it has been found that haemoglobin values <11.0 g/dl in the first and third trimesters, and <10.5 g/dl in the second trimester may point to an anaemic situation which should be further clarified. the first important steps for diagnosing anaemia in a pregnant patient include a thorough check of her medical w-pa-126 impact of epo treatment on transfusion requirements in myelodysplasia c gardin and p fenaux hopital avicenne, aphp, university of paris 13, bobigny, france myelodysplastic syndromes (mds) are clonal disorders of hematopoeisis, associated with bone marrow failure and an increased risk of evolution to acute myeloid leukemia (aml). despite an normal or increased bone marrow cellularity in most cases, cytopenias worsen with time due to increased apoptosis and defective differentiation of blood lineage precursors. incidence of mds increases with age and reach 15/100 000 above 70 years of age. bone marrow cytogenetics number of cytopenia and percentage of bone marrow blasts are strong predictors of survival and evolution to aml. a composite international prognosis scoring system (ipss) is used in everyday practice to guide the management of these diseases. these disorders are heterogeneous and include 'low risk' patients (less than 10% bone marrow blasts) with a prolonged evolution marked by chronic anemia, and 'high-risk' patients (excess of bone marrow blasts >10%) evolving in a short timespan with severe cytopenias, and to aml in approximately 50% of cases. at diagnosis, 80% of mds patients are anemic, with an hemoglobin level less than 100 g/l, and 80% of them will require chronic blood components transfusion, during the evolution of their disease. chronic anemia and multiple blood products transfusions are associated with an altered quality of life, clinical iron overload, and important health care costs. although transfusion practices and patient's transfusion need are variable, elderly mds patients require a mean of 10-20 units/year of follow-up, in recent surveys. therapies able to diminish or abolish the need for rbc transfusion have therefore a major role in the management of mds, as allogeneic bone marrow transplantation, the only curative therapy of these diseases, is limited to a small subset of mds patients. high-doses of recombinant erythropoetin (epo) (150-300 u/kg tiw, or a 40 000-60 000 u as single weekly dose) are typically used in low-risk mds. the response rate to epo is 25-40%, including major responses (suppression of rbc dependency or rise of hemoglobin level of more than 20 g/l). absent or low rbc transfusion needs and a serum epo level less than 500 u/l are strongly predictive of response to epo, in patients with low-risk mds. the duration of response is variable (12-20 months) in most studies, with some long-term responders. the use of higher doses of epo or its prolonged administration may be associated with higher response rates, although no randomized studies are available combination of epo and low-dose granulocyte-colony stimulating factor (g-csf) increases the response rate to 40-60%, including in patients not responding to several weeks of treatment with epo alone. two randomized trials published in 2004, compared g-csf-epo to rbc transfusions and confirmed the efficacy of this combination, and a longer survival of epo-g-csf responding patients. studies are ongoing in mds, including with darbepoetin, a modified erythropoetin with longer half-life, administered once a week. two such studies have been recently reported, (darbepoetin 150 or 300 ug/week) with response rates varying from 35% to 60% in low-risk mds. in both studies, a response to darbepoetin was observed in some patients, who failed to respond to previous treatments with alpha or beta epoetin. further assessment of the optimal dosage, administration schedule of these drugs, and validation of their likely impact on qol are required, in order to epo and its derivatives to gain acceptance in mds, due the high history and a medical examination. this procedure often lays the basis for a correct diagnosis. the current gold standard to detect iron deficiency remains the serum ferritin value. to be reliable, this requires the ruling out of an infection (chronic or acute) as a cause of the anaemia. we recommend a complete laboratory test for the exact haematological status as well as the assessment of specific chemical laboratory parameters. these should the hb level alone is insufficient to guide management. a complete work-up (ferritin, transferrin saturation) is essential, preferably with haematological indices such as hypochromic and microcytic red cells and reticulocytes, classified by degree of maturity, in particular, before parenteral therapy is given. since ferritin acts as both an iron-storage and acute-phase protein, it cannot be used to evaluate iron status in the presence of inflammation. a high ferritin level thus requires the presence of an inflammatory process to be eliminated before it can be taken at face value. if the c-reactive protein level is also raised, the soluble tfr concentration can be used, since it is unaffected by inflammation. inadequate understanding of the complex chemistry of parenteral iron administration was previously responsible for serious side effects, such as toxic and allergic reactions, and even anaphylactic shock, in particular with dextran preparations. however, the current type ii iron complexes that release iron to the endogenous iron-binding proteins with a half-life of about 6 h are not only effective but carry a minimal risk of allergic accident and overload, especially after a comprehensive pretreatment work-up. after correct diagnosis, major emphasis should be put on safe and effective treatment of anemia which again depends on severity of anemia, time for restoration and patients characteristics. today effective alternatives to oral iron only or blood transfusion such as parenteral iron sucrose complex and in selected cases also recombinant erythropoietin have been investigated and show promising results concerning effective treatment of anemia during pregnancy and postpartum. our departmental data collected over 10 years and backed by postmarketing experience in 25 countries indicate that iron sucrose complex therapy is a valid first-line option for the safe and rapid reversal of iron-deficiency anemia. w-pa-128 iron therapy in orthopaedic surgery surgery of the vertebral column, hip or knee is considered a bloody procedure (blood loss >1 l) and as a consequence represents the main indication for red blood cell transfusion in orthopaedics. because of the non-negligible residual risk of transmission of infectious agents by transfusion, but mainly because of immunologic complications induced by the administration of foreign proteins and cells, an alternative solution has been actively sought. studies have clearly shown that in patients undergoing such surgery, transfusion risk correlates inversely with pre-operative hemoglobin level. correction of even slight preoperative anemia is thus mandatory. in the elderly, iron and vitamin deficiency (b12 and/or folic acid) should be looked for as a matter of routine. we recommend the use of iron + epo whenever a rapid correction (<4 weeks) of the anemia is desirable in cases with transferrin saturation <30% and ferritin levels <200 mg/l. with this regime it is possible to collect up to 6 autologous blood units in cases of increased perioperative blood loss (e.g. double hip replacement). in the post-operative period, anemia worsens because of the existing inflammatory state. this inhibits iron absorption from the intestine and iron release from the macrophages while it affects epo function and production. there is increasing evidence that i.v. iron combined with epo induces a rapid correction of post-operative anemia. it is thus recommended to stimulate erythropoiesis by i.v. iron and epo starting on the first post-operative day and to avoid transfusions in asymptomatic patients even in cases with hb as low as 70 g/l. background: hereditary hemochromatosis is one of the most common inherited disorders in which an excessive amount of iron is absorbed from the diet and then deposited in organs. the effective treatment is the regular whole blood removal which causes erythropoesis activation and leads to decrease of iron stores. red cell apheresis is an optional method for removing of higher amount of erytrocytes in one session. we performed 262 red cell apheresis in 15 patients with diagnosis of hereditary hemochromatosis (14 ¥ c282y homozygotes, 1 ¥ c282y + h63d heterozygote) using haemonetics mcs 3p cell separator (protocol tae) in which red cells are removed from patients in 2-5 cycles; plasma and buffy-coat are reinfused. collection time, donor convenience, side effects and red cell yield were recorded and analysed. samples for hematology and iron studies in patients were drawn, analyzed and compared to baseline levels. background: the collection of 2 units of red blood cells by apheresis (drbc) has been reported to be safe and effective in increasing the yield of rbc units from a donor population. however several reports demonstrated the risk of inducing iron depletion when the interval between a drbc donation and a subsequent rbc donation is shorter than 180 days. aims: to evaluate the recovery from anaemisation and iron stores depletion after drbc donation. methods: donors who underwent drbc donation between december 01, 2002 and february 28, 2003 have been enrolled in a follow up program to monitor haemoglobin (hb), htc, serum iron and ferritin values. these parameters have been assessed on the day of donation and, thereafter 7 and 180 days after drbc procedure. donors suitable to drbc apheresis had to have: age between 18 and 60 years, weight > 70 kg, hb > 15.5 g/dl and serum ferritin between 50 and 400 ng/ml. a written informed consent about the collection procedure and the follow-up program has been obtained from all the enrolled donors. drbc collection procedures have been performed by using a mcs + (haemonetics) cell separators. results: out of 130 donors who donated drbc during the study period, only 14 males completed the follow up program and have been analysed. baseline haematological values and iron metabolism parameters were: mean hb 16.3 ± 0.5 g/dl, ferritin 85 ± 31 ng/ml, serum iron 121 ± 42 microg/dl. on day 7 mean hb was 14.3 ± 0.5 g/dl (p < 0.001). on day 180 mean hb was 15.3 ± 0.8 g/dl (p < 0.001), ferritin 53 ± 22 ng/ml (p < 0.05), serum iron 111 ± 27 (p ns). only 6 out of 14 donors (43%) had a ferritin value > 50 ng/ml. in the studied donors the collection of 2 units of rbcs induced an expected reduction of about 2 grams of hb, however only 50% of this reduction was recovered after 180 days (p < 0.001). similarly, also iron stores have not been restored after 6 months from donation, as shown by a 38% reduction in mean serum ferritin value. according to these data it appear that the amount of iron 'lost' with the donation of 2 units of rbcs (approximately 320-420 mg of elemental iron) could not be completely compensated by iron absorption from the diet intake. further data are necessary to define the risk of iron depletion after the donation of a drbc, however, at least in areas where iron intake by diet is not very high, the opportunity to prolong the interval between a drbc and a subsequent rbcs donation beyond six months or to provide adequate iron supplementation therapy should be carefully considered. background: increased transferrin saturation and/or serum ferritin have been observed in italy in approximatively 5% of subjects at first blood donation and, in these subjects, hfe mutations prevalence was 0.11 for c282y and 0.26 for h63d (velati et al., 2003) . aims: the role of the c282y mutation is well known in the patho-genesis of iron overload, whereas the role of the h63d mutation remains uncertain. the aims of the present study were first to study the main hfe mutations prevalence in a random group of repeat blood donors and second to evaluate iron parameters and iron depletion in repeat blood donors heterozygous for the h63d mutation in comparison to a population of blood donors wt/wt for the h63d mutation. methods: a total of 1165 repeat blood donors were examined in 10 italian transfusion centers (6 in northern italy and 4 in southern) for c282y and h63d mutations. out of those, 50 blood donors heterozygous for the h63d mutation and 31 wt/wt for the same hfe mutation, both groups wt/wt for the c282y, were enrolled to evaluate iron parameters and iron depletion. these two groups were similar for number of blood donations (expressed as iron loss) and for sex distribution. serum ferritin (sf) was the iron index recorded at first and second observation. results: table 1 summarizes the allelic frequencies in the 900 blood donors. table 2 reports the haematological evaluation in the 50 subjects heterozigous for h63d mutation and the 31 wt/wt for the same mutation. conclusions: these data suggest that subjects with h63d mutation of the hfe gene have, at first observation, a higher ferritin levels than subjects wt/wt. this seems to be more evident in blood donors of southern italy than in northern. blood donation induces significant reduction of the iron stores both in h63d heterozygous and in wt/wt subjects. although our observation is preliminary and restricted to a limited number of subjects, it seems worthwhile to extend the follow-up of blood donors h63d heteroxygotes or even homozygotes when available, in order to get further insights on the h63d role in iron metabolism. background: cd 43 is a sialylated glycoprotein expressed on the surface of most hematopoietic cells and has been implicated in cell adhesion and signaling. consequently the levels of soluble cd43 as well as the expression on the cell surface is a marker of cell activation. furthermore, downregulation of this molecule has been correlated with increased susceptibility to infections. the myelodysplastic syndromes (mds) are a group of stem cell disorders characterized by ineffective hematopoiesis, refractory cytopenias and an increased risk of leukemic transformation. the mds patients are often introduced to transfusions for anemia improvement and present increased susceptibility to infections. aims: we studied cd43 expression in transfusion-dependent and non-transfused mds patient in an effort to investigate mechanisms of regulation of this molecule. we also studied other activationassociated antigens in the absence of manifest infection. material and methods: forty-two patients were included in the study suffering from refractory anaemia (ra). thirty-one were males and 11 females aged 33 to 85 (median 75). twenty of them had never been transfused (group a) and 22 were regularly transfused (group b). nineteen age matched healthy individuals were used as controls (group c). cell surface antigens were detected by direct immuno-fluorescence evaluated by flow cytometer. the following mouse monoclonal antibodies were tested: anti-cd11b, anti-cd18, anti-cd43, and anti-cd45. leukocytes were gated according to cd45. we used a sensitive sandwich enzyme linked immunoassay to measure the level of soluble vascular adhesion molecule as an indicator of endothelial cell activation. the r&d elisa kit was used according to the manufacturer's instructions. results: the cd43 was found down-regulated in the transfusiondependent mds patients compared with the non-transfused ones (p < 0.001) and controls (p = 0.012). this downregulation concerned the proportion of cd43 + cells, that was lower in the transfused patients than the non-transfused (p < 0.01) and controls (p = 0.006), and the rfi (relative fluorescence intensity) value that was also lower in the group a compared to the group b (p < 0.01) and group c (p = 0.001). negative correlation was observed between the cd43 expression and cd11b (p = 0.001) and cd18 (p = 0.001). cd11b was found up-regulated in the transfused patients. the rfi value was significantly elevated in the transfused patient compared with the non-transfused and controls (p = 0.001 and 0.001 respectively) while the percentage of cd11b cells did not differ significantly between the various groups. increased expression of cd 18 was also found in the group a compared to group b (p < 0.01) and c (p = 0.008). the proportion of cd18 + cells did not differ between the various groups. the levels of immuno-reactive svcam-1 as determined by elisa were found 512.02 + 35.56 in group a, 3.42 + 65 in group b and 194.11 + 45.48 in the control group. conclusions: activated hemopoietic and endothelial cells are found in mds that may be associated to the vascular disorders found in these patients. cd43 downregulation may also be associated to increased susceptibility to infections in these patients. despite improved safety of the blood supply, allogeneic blood transfusion continues to be associated with risks that can be eliminated or reduced by autologous transfusion. preoperative autologous blood donation (pad) prevents transfusion-transmitted viral infection, red cell alloimmunization, and some adverse transfusions reactions. it may decrease the risk of postoperative wound infection because immunosuppression as a result of allogeneic blood transfusion is avoided. pad also supplements the blood supply, provides compatible blood for patients with alloantibodies and rare red cell phenotypes, accelerates erythropoiesis, and provides peace of mind to patients. as any medical intervention, pad has both advantages and disadvantages. with proper patient selection and dedicated attention to process control and quality assurance, the advantages outweigh. background: prestorage pooling of whole blood derived (wbd-pc's) buffy coat platelet concentrates (pc) is common practice in europe event-free survival was significantly better in patients who responded to epo + g-csf. we have reviewed data in 2 centers and the gfm has the intention to extend the study to a larger population in at least 10 centers in france blood components and preparations. the new law prohibits the mixing of different blood products, i.e. blood components and blood fractions. different methods are necessary for the quality control of blood components and blood preparations privileges for blood donors include: -a paid day off work on the day of blood donation and medical examination for blood donation additional paid day off work after blood donation an extra paid day off work if blood is given during vacation or on a holiday this award will be given to non-remunerated donors after 40 blood donations or 60 plasma donations. before 2005, each 'honoured donor of russia' or 'honoured donor of the ussr' had three privileges: free use of public transportation, receipt of certain pharmaceuticals free of charge, and a discount on apartment utilities previously, municipalities also could have their own blood establishments. this resulted in more than 1500 blood establishments in the russian federation. from both administrative and financial points of view, many of these are too small to be costeffective, and should be discontinued. services, and wider implementation of modern technology for blood collection, testing, processing, storage, and distribution acknowledgements: we thank ksw microtec ag, dresden/ germany for sponsoring the rfid-labels and novatech research gmbh, vienna/austria for developing the clinic-software. background: the national preparation human immunoglobulin g 5% for intravenous use (ivig) that is produced at the serbian institute for blood transfusion is used in therapy of neurological, heartand haemolytic diseases and on patients that have undergone surgery. aims: it is our aim to prove the impact of this national medical preparation human immunoglobulin g 5% for intravenous use on patients that have been infected with sepsis as a consequence of surgery. material and methods: human immunoglobulin g 5% for intravenous use (ivig) has been used in the study. the preparation is liquid, 5% stabilised with glucose of a ph value of 4.25 ± 0.25. it is used in those cases where sepsis developed after surgery. both an ivig group (n = 40) and a control group (n = 40) were viewed; the control group not being treated with ivig. the number of specimens with the ivig therapy cholecystitis is (n = 3), and the control group (n = 2); pancreatitis (n = 8) control group (n = 7); intestinal obstruction (n = 7) control group (n = 8); abdominal organ perforation (n = 5) control group (n = 8); abdominal perforate injuries (n = 12) control group (n = 13); serious abdominal interventions (n = 5) control group (n = 6). the period of hospitalisation of the patients in the ivig group was 25 ± 9 days while the period of hospitalisation in the control group was 28 ± 12 days. the mortality rate in the ivig group was 40% counter 63.7% in the control group. summary: toxic gram -negative bacteria caused synergistic damage of human tissues and generalized inflammatory responsesepsis. by using human immunoglobulin g 5% for intravenous use, in cases of severe disease, the mortality rate is significantly lowered, depending of course on the anamnesis of the patient prior to surgery and the presence of other diseases such as diabetes mellitus, neoplasma, cardiac diseases etc. background: manual production pc from buffy coats (bc) is a procedure with some consecutive manipulations. the orbisac system (gambro bct) automates the steps and we assessed its performance. material and methods: 160 pc were produced by this device and some parameters were studied. for the preparation of pc, 5 bc were pooled using the orbisac set, with an integrated filter (pall lrp6). bc pool was resuspended in the additive solution t-sol in order to obtain a final ratio plasma/t-sol 35/65. the pc was stored in a gambro elp bag. results: the average platelet count per unit was 3.41 ¥ 10e11. the platelet recovery from pooled bc was 85.12% (range 79.75%-93.21%). all products of the tested pc containing <1 ¥ 10e6 wbc (by flow cytometry). the values of ph on day 1 and 5 of storage were 7.45 and 7.43. the swirling phenomenon was good until day 5°. the average loss of haemoglobin per bc was 6.8 g.conclusions:the orbisac system is very suitable for routine pc preparation and it allows increased productivity and better standardization method for pc preparation. platelet concentrates met the requirements for leucodepleted product. increased production of plasma components from male donors background: we routinely separate whole blood (wb) after hard centrifugation into a red cell concentrate (rcc), a buffy coat (bc) and plasma (pl) by an automated expresser (compomat, fresenius). the bcs are subsequently processed into platelet concentrates (pcs) by soft centrifugation and an additional (manual) expression step. the atreus 3c system (gambro bct) eliminates several of those hand-on steps by combining them into one integrated process. a processing 'circular' bag is placed in the device and filled with the wb. while the bag is centrifuged, the system expresses pl, pc and rcc into separate containers. the rccs are subsequently leukoreduced (manually) with a filter (lr-rccs). this study was designed to evaluate the storage characteristics of the rccs obtained with a prototype of the atreus system in comparison to rccs obtained by routine procedure. methods: whole blood (500ml) was collected in top-and-bottom bags, and randomly selected to be processed by either (1) current routine or (2) atreus 3c. rccs were leukoreduced with the integrated inline filter: fresenius (routine group) or pall rc2d (atreus). lr-rccs were stored at 4°c and sampled until day 42. various in vitro measurements were performed (n = 20 per group).results: see table (mean ± sd). the lr-rccs contained significantly more leukocytes in the atreus group. despite the rbc loss in the bc, hemoglobin (hb) content was 6% lower in the atreus group, but met the requirements. in vitro storage characteristics for the rccs were similar in both groups. the atreus pcs contained 73 ± 28 ¥ 10 9 platelets in 78 ± 6 ml. although plasma volume was higher in the routine group, subsequent preparation of pcs would have resulted in an additional loss of 50 ml per unit in the control group. atreus plasma had extremely low levels of residual wbc and rbc. 0.02 ± 0.02 4.0 ± 4.2 <30 <0.001 aims: the aim of this study was to examine platelet quality of prestorage pooled prp-derived pc's for up to 7 days storage. methods: pc's were manufactured from wbd-pc's using in-line filtration of prp on day 0. on day 1, either 4, 5, 6, or 8 pc's were pooled into an elx® container using a sterile connecting device. studies were performed on days 1, 5 and 7 for the following measure of platelet quality. ph, morphology score (ms), extent of shape change (esc), hypotonic shock response (hsr), percent in surface expression of p-selectin (p-sel), phosphatidyl serine (ps), glycoprotein 1b (gp1b) and by thromboelastography of the prp (maximum aplitude, ma). results: a total of 20 pools were studied, 5 each of 4, 5, 6 and 8 pc's. the mean platelet yield was 4.3 ¥ 10e11 with a range of 2.4-7.0 ¥ 10e11. the five 8 pc's had a mean yield of 6.0 ¥ 10e11 and all maintained a ph > 7.0 on day 7. all products had less than 1 ¥ 10e6 residual wbc. platelet quality data is presented in the table. data are the mean ± 1 sd, n = 20. conclusion: platelet pools manufactured from pc's produced by inline filtered prp and stored in elx® containers show good quality preservation to day 7 over a range of platelet yields. introduction: the big progress in treatment of critically ill children significantly increases the need for blood and blood products. loss of blood (lowering of the total erythrocyte mass), as well as decreasing of oxygen capacity of blood that can influence cardiovascular function, is main indication for the erythrocyte transfusion. aim of the study: to present the number of erythrocyte concentrates (ek) that were issued to the pediatric clinic in skopje, as well as to point out how they were distributed. material and method: this is a retrospective study performed in nitm-skopje from january till may 2004. the following criteria were followed: hemoglobin (hb), hematocrit (htc), as well the clinical evaluation, and then final decision for transfusion was made.results: there were 254 blood units (ek) issued for the mentioned period to pediatric clinic for 73 pediatric patients (~3, 5% units/per child). the biggest consumers are children at intensive care unit and at the hematology-oncology unit. one unit of leukodepleted erythrocytes (er) was split equally to 3-5 bags. for small and prematurely born children and for some other selected patients er unit was filtered and irradiated. the dosage was 25-100 ml er (depends on age and body weigh). 173 ek was issued as washed concentrates, 15 ek were filtered and 25 ek were resuspended in ab plasma. distribution among abo system was the following: conclusion: gynecologic patients consumed rbc more than 4 times than obstetric ones (159 vs 44) and the number of given transfusions is high. the a blood group is the most needed one. we should insist on using the who guidelines for the proper clinical use of blood and try to minimize the percentage of given transfusion. and z. cermakova university hospital, ostrava, czech republic background: fully automated system olympus pk 80 is an immunohaematological analyser for detection of red blood cells antigens of ab0, rh (d, c, c, e, e) and kell systems without centrifugation by mam (microplate agglutination method) on unique terraced microplate olympus. in the czech republic analyser pk 80 is used only in blood center ostrava. aim: to evaluate the validity of results, sensitivity of microplate agglutinaton method, cause of abortive tests, requirements for analyst, capacity and reliability. methods: 95 880 blood samples of donors were tested between july 2002 and january 2005. all samples were analysed for ab0 blood group. 69 052 samples were tested for rhd antigen and 15 220 ones for rh (c, c, e, e) and k antigen. the validity of the results was evaluated for ab0 with parallel testing antigens and antibodies, while for rh (d, c, c, e, e) and k using two diagnostic serums. sensitivity of mam i.e. occurrence false negative or positive results were found out when results were confronted with previous ones in our data bank acquired testing classical manual tube or microplate methods. requirements for analyst were evaluated in according to demands for needful knowledge for new analyst, necessity of control pk during testing and maintenance. capacity were evaluated as a number of samples tested per day. reliability determine by occurrence disorders. results: ab0, rh (d, c, c, e, e) and k were investigated truly by first testing at 99.5% samples. two diagnostic serums anti-d olymp igm and totem differentiate directly rhd negative and rhd positive donors. false negative or positive results were not founded out due to mam or quality of diagnostic serums. about 0.5% samples with abortive tests were analysed next time the same testing or manual technique. causes of abortive tests were microagglutination several samples except for anticoagulative edta, weak solution of red blood cells prepared by analyser, damage of microplate, hemolysis due to impurity of microplate. in one case analyser evaluated false ab blood group due to hemolysis. analyser has friendly software, simple maintenance and sound control during testing, capacity about 400 samples per day and minimal occurrence of weighty disorders. conclusion: analyser olympus pk 80 is an effective alternative full automation for medium serological laboratory and together with mam easy and truly proves blood groups of majority samples with minimal necessity repetition due to abortive tests. introduction and aim of the study: the purpose of this study is to establish nested-pcr for the detection of hepatitis b virus (hbv) in blood and blood products. methods: the primer pair set was designed to amplify 513 bp in sregion of hbv genome in the first pcr and 233 bp of first pcr amplicon with rubisco (internal control) in the second pcr. to assess the specificity of pcr results, all the samples were tested cross-reactivity or interference in the assay. results: in case of hbv spiked blood products such as immunoglobulin and coagulation factors, this method could detect hbv dna up to 62.5 iu/ml. nested-pcr was compared with pcr-elisa and hybrid capture ii (hc-ii), the pcr-elisa showed a sensitivity of 96% (hc-ii; 77%) and a specificity of 98% (hc-ii; 100%) (p < 0.005). the results of the study show that nested-pcr and pcr-elisa could be used equally in the management for hbv detection in blood and blood products. p-398 blood component therapy: slow improvement a mrdja health center subotica, subotica, serbia background: transfusion department at general hospital was founded in 1953. since that time till now it has answered to all demands in blood and blood components. aim: the aim is to present development of the transfusiology department in the last 20 years, so that we could see how much of scientific knowledge we have adopted and in which direction our department goes at the moment. method: retrospective analysis of blood/component utilization in period from 1. january 1984 to 31. december 2004. results: in 1984 whole blood participated in the consumption with 84.18%, packed red blood cells with (rbc) only 5.94%, washed rbc were used in 9.87% of the cases. in 2004 whole blood participated in the consumption with 31.5%, packed rbc with 61.65%, washed rbc with 0.14% and rbc in additive solution with 6.71%. as far as plasma preparations are concerned, there has been, since 1984, a great consumption of plasma -witch was separated from whole blood in period up to five day in 97.1% cases, and small consumption of fresh frozen plasma (ffp) only 2.9%. since 2003, there has completely been cancelled the production of five day old plasma, only ffp is being used. from 1984 to 2002 for the patients who needed platelets, platelet rich plasma (prp) was prepared and applied right after preparation. the consumption rate was from 35 units to 103 units per year. in 2002, after the purchase of platelet shaker, began the production of platelet concentrates (pc) and consumption suddenly rose from 58 units in 2002 to 462 units in 2004. conclusions: it is obvious from the analysis that irrational consumption of whole blood was reduced to more acceptable values and therefore the use of component therapy got increased. variation in blood consumption and its slight increase is obvious though application red blood cells was conducted according to strict indications. in 2003 the production of plasma old up to five days was cancelled and instead we produced only ffp. pc we prepared for patients only in agreement of treating physician. although very slow progress in development of transfusion therapy in our department can be seen in accepting scientific knowledge. transfusion specialist are active participants in patient treatment and by accepting scientific achievement are able to set standards and help our colleagues, clinics, in successful hemotherapy. introduction: blood groups may act as receptors of parasites, bacteria and viruses. there is evidence that they perform a function and play a biological role. objective: the aim was to detect abo and p epithopes in ascaris lumbricoides extracts (ae) and to study the presence of immune antibodies in patients with ascariasis. materials and methods: 50 ae were prepared by refrigerated mechanical rupture of adult specimens. agglutination inhibition (ai) and haemogglutination kinetics (hk) tests were made with the ae. the patients´ abo and p blood groups were determined. we 1998 1999 2000 2001 2002 2003 2004 total febrile non haemolitic 2 3 2 3 2 4 2 5 2 4 2 4 2 9 4 34 16 male transfusion reaction 2 1 1 2 3 2 2 5 1 8 f e m a l e alloimunisation on le/hla 3 1 2 2 1 3 1 3 3 4 1 5 2 25 6 male antigens 2 2 1 2 3 3 3 3 1 9 f e m a l e kandidates of renal 4 3 4 4 5 4 6 4 6 5 10 8 12 8 10 7 57 43 male transplantation 1 1 2 1 2 4 3 14 female lupus nephritis 3 3 2 3 2 1 1 1 1 6 male 3 3 2 3 2 1 1 1 1 6 f e m a l e total 12 12 12 16 16 18 21 25 132 65 female 67 male introduction: irradiation of blood product has been in routine use to prevent graft-versus-host disease (gvhd) in certain recipients for many yeas. gamma irradiation can abrogate the ability of lymphocytes to proliferate in vitro, 1500 cgy of gamma radiation reduce lymphocyte response to mitogens by 90%.the aim of the study: 1. to estimate potassium level increment in stored irradiation blood units. 2. to compare the increment in potassium level between leucodepleted and non leucodepleted, irradiated stored blood units. 3. to evaluate the expiratory date of blood units post irradiation. the study included 40 units of blood collected in cpd-adsol (as-1). in twenty units the blood collection bag was with inline leucodepletion, while the other 20 units were non leucodepleted. all the 40 units were irradiated using caesium 137 as a source of irradiation, with a dose of 1500-2500 cgy. baseline samples from the bags were obtained for measuring of extra cellular potassium (k+). control samples included. results: there is statistically significant increment in potassium level in the irradiated samples compared to the non irradiated samples starting from 1st day post irradiation and continues to day 35 post irradiation. comparing the group of irradiated leucodepleted, with irradiated non leuconondepleted, for potassium level estimation during the days of storage post irradiation. there is no statistically significant difference between the two groups during all the days of storage, starting from base line samples and other samples post irradiation until day 35, p value of more than 0.05. 1. gamma irradiation of bloods units can cause cell damage that the use of such components needs to be modified. 2. there is a significant increment in the extra cellular potassium level in irradiated blood units that shows doubling value within 48 h post irradiation. 3. there is no significant difference in extra cellular potassium level increment post irradiation when prestorage leucodepleted units are compared with non leucodepleted units. 4. an out date of 28 days post collection (unless they expired before) for irradiated red blood units seems reasonable to ensure transfusion of irradiated units without serious complications, except in neonates and massive transfusion cases where irradiated blood units should be fresh and used within 48-72 h post irradiation. 5. the percentage of irradiated blood units requested by our physicians (0.09%) is very less that reflects the needs of physicians awareness of the indications for requesting irradiated components that can prevent serious post transfusion complications. the use of whole blood and blood components in treatment of surgical patients in ten years period was analyzed in iran. in accordance with world trend of using blood component therapy, in medical centers throughout the country in ten years period, there are decreasing trend of using whole blood from 26% (1994) to 9.37% (2003) and increasing trend of using packed red cells component therapy from 70.3% (1994) to 88. 54% (2003) . there is also increasing trend of using fresh frozen plasma (ffp) from 26.4% (1994) to 55. 2% (2003) . comparing 1994 and 2003 year, in use of blood therapy related to hospitalized patients at surgical department who received blood and patients who did not received blood; it appears that there is statistically significant difference between these two years. results: during 1 year period, a total of 5310 units of blood and 867 units of f.f.p were used. more specifically, the results can be shown in the following table 1 . a high rate of f.f.p usage is observed both in surgery and pathology clinics. the main causes of its usage are: haemodynamic disorders -volume depletion, and coagulation disorders and low blood protein, for the two clinics respectively. conclusion: the only way for rational usage of f.f.p is the regular reminding of plasma transfusion indications to the clinical doctors, so that undesirable side-effects caused by plasma transfusion will be reduced and the percentage of plasma used for fractionation will increase. acquired factor v inhibitor is extremely rare and is associated with diverse clinical symptomatology that varies from asymptomatic forms of the disease to very severe hemorrhagic episodes with a potentially lethal outcome. it may occur spontaneously or as a result of various clinical conditions. a 50-year-old man was admitted to our hospital with a diagnosis of left-sided periscrotal abscess and scheduled for an incision procedure. during the routine preoperative procedure screening coagulation tests showed pathologic values: aptt 41s, pt 35%, fibrinogen 2.8 g/l, fv 15% (other factors were in normal range), platelet count 189 ¥ 10 9 /l. factor v inhibitor was detected by a modified bethesda assay. the assay showed a low level of inhibitor of about 1.30 bethesda units (bu). the patient's medical history showed no major morbidity except appendectomy performed 22 years ago. the patient was prepared for operative procedure, with preventive preoperative administration of fresh frozen plasma (ffp) in a dose of 15 mg/kg (~1500 ml). upon ffp transfusion, repeated determination of the factor v plasma was unchanged from the initial finding (15%), indicating a failure of therapeutic response. as the measured level of factor v activity was at the borderline hemostatic level, and the operative procedure was not associated with a high risk of hemorrhage, the patient underwent abscess incision. the procedure and postoperative course were uneventful and without major hemorrhage. laboratory testing for the possible systemic autoimmune disorder produced normal findings. control examination performed two years later revealed no major clinical or laboratory variation, while a low factor v level persisted (17%) along with the presence of factor v inhibitor at a level of 1.15 bu. we have evaluated two groups of rcc's, one we routinely use (quadruple leucoflex lcr5 t/t cpd/sagm) (macopharma) and one using an automated collection device which gives the ability to collect whole blood in cpd with a rate of 7 : 1 respectively during the whole donation. the mentioned system has been evaluated using the suitable, quadruple leu-coflex lcr5 t/t cpd/sagm (macopharma). whole blood 450 ml in cpd was collected from random donors. in both groups the whole system was stored at scaled r.t. 20 ± 2°c for 2 to 5 h. after component separation (beckman coulter j6mi-optipress i-baxter), the red cell concentrates were filtered immediately at r.t. 20 ± 2°c. sampling was done after filtration and wbc measurements were determinated using nageotte champer (bright line-detection limit 0.05 wbc/ml) with leucoplate solution (sobioda). the other parameters were measured with (celldyne 1700 abbott). conclusion: all products met the accordance of national and european norms for blood components quality. leucodepletion with leucoflex lcr5 and abc leucoflex lcr5 (5.040 and 5.015) is highly efficient. the use of abc leucoflex lcr5 showed better scaled donation in terms of collection (statistical analysis mann-whitney, minitab p-value = 0.0392 < 0.05). additionally less hb-loss occurred, due to the filtration process, most probably due to the total absence of clots (analysis man-whitney, minitab, p-value = 0.0382 < 0.05). this new generation of collection gives the ability in blood services to collect well calibrated donations, indoors or outdoors. smaller quantities of donations theoretically can be valid because of the stable 7 : 1, rate of donation. the abc system gives the ability of fully traceability during donation. macopharma's blood collection bags, with or without integrated filters, provided they have this modified system of storing the ac. the device can keep records for many parameters and can transfer them to the data base server of the blood center. to evaluate the performance of the abc, we conducted a comparative study between abc leucoflex lst1 system and leucoflex lst1 system we currently use. the later is a well known macopharma's system for collection of whole blood with integrated whole blood filter and the final production of one unit of leucodepleted crcs and one unit of leucodepleted plasma. the abc was handled with the same system modified with the storage bag for the ac. issues of comparison were the accuracy in donation volume, the duration of filtration, the loss of blood in the filter and the residual wb cells after filtration. we performed 24 donations with the lst1 system and 28 donations with the abc lst1 system. all the donors were random male volunteers and they were meant to donate 450 mls of whole blood. our results analysed by mann-whitney, minitab statistical analysis have as follows:1. no significant difference was found between the two systems concerning the whole blood volume, but there was broader distribution of the values in the lst1 system compared to the abc lst1 system. 2. the duration of filtration has been found without statistically significant differences between the two systems. 3. the loss of volume in the filter of lst1 is higher than in the abc lst1 (p = 0.007 < 0.05, which is statistically significant). 4. there was very good leucodepletion with the lst1 systems (median reduction of the wb cells 4.7 log) but there was a superiority of the abc lst1 over the lst1 concerning the leucodepletion per litter and per unit (p: 0.0368 and p: 0.0398 respectively). 5. the personnel after a very short period of training accepted fully the abc procedure.in conclusion abc is an easy to handle device which provides with high quality blood products in combination with leucoflex lst1. evaluation of a post-storage filter for wbcs with an incorporated waste bag for washing rccs leucolab lcg4 is a system manufactured by macopharma for poststorage filtration of a rcc unit (with or without an incorporated waste bag for further washing of the filtered product). to evaluate the efficiency and reliability of the above system we conducted a study with twenty three random units of rccs stored in cpd/sag-m, aged 2-6 days, filtered and consecutively washed with 200 ml of normal saline, span down in the regular way and the supernatant extracted in the waste bag. issues for evaluation were:1. the duration of priming and filtering the rccs. 2. the loss of volume in the filter.3. the efficiency of the filtration. 4. the acceptance of the personnel of a new (to them) filtration system.our results have as follows:1. we counted the duration of priming and filtration. median time of priming was 90 s (range 40-300) and of filtration was 12 min (range 8-20). 2. the median volume lost in the filter (as calculated) was 23.4 ml (ranging from 22.4 to 28.1). this narrow range is apparently due to the non-flexible cell of the filter. 3. the efficiency of the leucodepletion was counted by flow cytometry. the median wbc counted per lt was 0.9 ¥ 106 (range 0.3-2.1 ¥ 106) and per unit was 0.17 ¥ 106 (range 0.05-0.5 ¥ 106). the median reduction of the wbc count was 4.2 log (range 3.9-4.7). 4. the personnel involved in the procedure found the system easy to handle, even without specific training. in conclusion the lcg4 is a reliable and easy to handle system, for leucodepleting (and washing) rccs, very efficient in removing wbcs with negligible loss of volume. objective: standardization of blood banks and establishing quality assurance are important landmarks in the new era of transfusion medicine. as the number of blood banks grows and the capacities of them changes, centralization need arises and trends to nationally coordinated blood services eventually appear. aim: to investigate the types and capacities of blood bank in the country and evaluate the statistics of them. material and method: blood banks and transfusion society of turkey conducted a nation-wide survey of a comprehensive questionnaire. this is a preliminary report of this investigation and illustrates the capacities of the most blood banks in turkey. it also guides the nationally planned renewing structure of blood banks. results: there are nearly 340 blood banks and blood stations in whole country. the overall blood collected at those centers is about 1.800.000 units. nearly one in third is being collected at government hospitals (31.5%), nearly the same is collected at university hospital blood banks (29.6%). the third major group is the red crescent society blood centers-rcsbc (23.5%), followed by the social security hospitals (12.9%). blood collecting capacities are not appearing in the same order. the major blood banks belong to the rcsbcs, whereas the small ones are mostly government hospitals. the only donor recruitment organization is run by the rcsbcs. yearly blood collecting capacity blood banks (%) >50.000 0.4 20.000-50.000 4.8 10.000-20.000 8.7 5.000-10.000 13.8 <5.000 72.3conclusion: there are many steps for improving blood safety in a country, and the prior ones are structuring the blood transfusion system and donor organization on a national basis, and then establishing good manufacturing practices. these are only possible after centralization of all existing blood banks. in our country, we should first arrange all small capacity blood banks and standardize them. controversial clinical questions considered in a medical opinion forum by physicians for the advancement of transfusion medicine (patm) patm is a newly formed group of close to 300 physicians drawn from pathology and hematology, transfusion services, hospital blood banks and blood centers. its mission is to address the concern that patient oriented medical opinion and influence has been diminished in transfusion medicine (tm). they believe that a patient oriented voice should be distinct from institutional, commercial or regulatory weight and have a common focus on patients and the therapeutics of transfusion and related therapies. therefore, their first objective is to create a new medical, patient oriented voice that weighs in on national policy pertaining to treatments related to tm. as such, patm held its first medical opinion forum where members of the group debated important questions pertaining to tm clinical practice. the forum was held just prior to the aabb annual meeting and attracted 58 physicians. the questions debated were preselected by patm membership via an email survey. respondents were asked to select their top four preferences from among 17 topics in five broad categories. the top two topics were selected for the forum from the 80 completed surveys. the subjects selected and debated were (i) what are the medical considerations for reducing the rate of mistransfusion? and (ii) what are the medical considerations for managing a limited blood inventory? the participants were divided into four groups, with each topic assigned to two groups. all the groups were given two h to debate and arrive at consensus on their topic. each group then presented a summary of their discussions along with specific recommendations for addressing these clinical practice issues. there was remarkable consensus between the groups debating the same issue. the conclusions and recommendations on these two topics will be presented in detail. patm is a new organization that will add an important medical voice and opinion on current topics in tm. at its first meeting, two topics were successfully discussed and debated with broad consensus achieved on current issues confronting the field. key: cord-008777-i2reanan authors: nan title: ecb12: 12th european congess on biotechnology date: 2005-07-19 journal: j biotechnol doi: 10.1016/j.jbiotec.2005.06.005 sha: doc_id: 8777 cord_uid: i2reanan nan in the last 20 years biotechnology has made tremendous progress in its different application fields: red biotechnology, the use of biological methods for medical purposes, is firmly established in the development of new drugs. the use of plant or green biotechnology is under controversial discussion in politics and public. nevertheless, genetically modified herbicide and insect resistant crops are cultivated to a large extent. industrial biotechnology, now often named white biotechnology, seems widely underestimated in the public perception. it includes all industrial processes for the production of chemical products and enzymes, which fully or partly rely on the biological toolbox of nature. white biotechnology processes are carried out in a contained environment, typically in a bioreactor in a dedicated industrial plant. well-known examples are the fermentative productions of antibiotics, amino acids, vitamins and enzymes, products related to medical, food and feed applications. many products like the amino acids glutamic acid, lysine, threonine and tryptophane are exclusively produced using microbes in large scale industrial processes. in other cases, like the water soluble vitamin b2, biotechnological processes successfully replaced chemical productions, due to lower costs and improved ecoefficiency. in contrast to this, most industrial chemicals and polymers are produced by chemical synthesis based on oil and gas. however, there are some examples for bioproducts among industrial chemicals. the solvents acetone and butanol, for instance, were manufactured by fermentation for several decades in the last century. since the 1950s these fermentations have been replaced by more efficient and cheaper chemical synthesis. recently, new pilot and production processes for biopolymers like pha or biomonomers like 1,3-propanediol or lactic acid were announced by different companies. currently, ethanol is by far the largest white biotech product by volume. in brazil, where bioethanol is used as liquid transportation fuel, the annual production is in the range of 15 mio m 3 . bioethanol is of growing importance also in the united states. business consultants predict a tremendous growth of biotechnological products within the chemical industry. high prices for crude oil, dropping prices for renewable resources, and scientific progresses nourish the expectation that industrial biotechnology will replace many bulk chemicals. is this realistic? will we switch from a petrochemical industry to a biobased chemistry within the next years? based on economic considerations it can be stated that this is a long term goal. to achieve this it remains a scientific challenge to make renewable raw materials available for competitive bioproduction of bulk chemicals at low costs. conversion of lignocellulosic material to fermentation sugar may be a solution. also green biotechnology can contribute to the supply of cheap fermentation raw materials. innovative ideas for downstream processing or further chemical conversion of fermentation products are required to enter the chemical value chains. furthermore, the identification of new higher value bioproducts is a chance for short term successes in white biotechnology. enzyme and protein engineering has the potential to create new biomolecules, metabolic engineering can contribute to develop new metabolic pathways, may be even for unnatural compounds. by continuously increasing the efficiency and throughput of dna sequencing we, together with colleagues, have sequenced the human genome and the genomes of all the major model organisms. the challenge now centers on understanding these vast instruction sets. our ability to read these instructions must be enhanced through collection of key additional data sets. one productive path for delineating the functional sequences and inferring their function is comparative sequence. the mouse genome sequence, for example, led to estimates that only 5% of the human genome is functional. sequencing of an extensive set of additional mammalian genomes promises to define these functional sequences with a resolution of less than 10 base pairs. on a different course, we have sequenced the chimpanzee genome to learn what has changed in the evolution of humans. beyond providing for the first time a catalog of the differences between the two genomes, the comparison of the chimpanzee and human genomes reveals the patterns of neutral mutation and regions that deviate from that. the talk will summarize these and related findings. the sequence of additional primate genomes will help delineate what has changed specifically in humans and add power to the analysis. ultimately, capturing human sequence variation and correlating with phenotypic variation will be required to understand function. but learning what these functional elements do requires new sets of experimental data. for this, we have turned to the nematode c. elegans. in this simple system most of the ∼20k genes have been defined and experimentally confirmed. beyond the hundreds of genes with already known mutants, two centers are systematically producing gene knockouts or rnai can be used to inhibit any gene temporarily. sequences of three caenorhabditis species are already available, and two more are underway. expression data has been collected for all the genes under many conditions and time points through development. to enhance the resolution of expression data and to simplify phenotypic analysis of embryonic mutants, we are developing a system that will automatically trace the cell lineage and assign gene expression to precise cells with high temporal resolution. the latest results with the system will be described. in the longer term, this and similar datasets should provide an understanding of how the genome specifies the form and behavior of the worm. uhlen department of biotechnlogy, albanova university center, royal institute of technology (kth), stockholm, sweden here, we present a new protein atlas database (www.proteinatlas.org) showing the expression and localization of human protein in normal and cancer tissues. the atlas is based on the use of antibodies (agaton et al., 2003) to generate high-resolution immunohistochemistry images representing 48 normal tissues and 20 different cancer types (uhlen and ponten, 2005) . each antibody is used to generate more than 500 individual images and each image has been annotated by a pathologist (kampf et al., in press) . the database has been created by the swedish human proteome resource (hpr) and the program has been set-up to allow the exploration of the human proteome with antibody-based proteomics (nilsson et al., in press) . the basic concept is to generate, in a systematic and high-throughput manner (uhlen and ponten, 2005) , specific antibodies to all human proteins, and subsequently used these for functional analysis of the corresponding proteins in a wide range of assay platforms, including (i) a protein atlas for tissue profiles (kampf et al., in press) , (ii) specific probes to evaluate the functional role of individual proteins, and (iii) affinity reagents for purification of the specific proteins and their associated complexes for structural and biochemical analyses. ments, most effective source of variation was perturbation in growth medium, followed by perturbation in growth rate. effect of gene deletion on data variation was found to be less apparent when compared to other perturbations. a significant similarity in variation of metabolome and mrna data was observed, which may be used as the key point for integration of these two sets of data in functional analysis of genes. projection to latent structures (partial least squares, pls) is used for integration of transcriptome and metabolome data. comparison of pca and pls shows that linear model constructed via pls to predict the metabolome data does not make use of all the variation in transcriptome data. thus, pls allows the discrimination between the portion of gene expression change that affects the metabolome profile and the portion that is not directly effective on metabolome. both pca and pls can be used to detect the open reading frames (orfs) which are the main sources of variation in transcriptome data and/or effective on metabolome profile. extracellular metabolomics to accelerate the discovery of key genes involved in fibre degradation silas g. villas-bôas, geoffrey lane, graeme attwood, adrian cookson agresearch limited, grasslands research centre, tennent drive, private bag 11008, palmerston north, new zealand. e-mail: silas.villas-boas@agresearch.co.nz (s.g. villas-bôas) the genome of the hemicellulose-degrading microbe clostridium proteoclasticum is been sequenced and an array of candidate genes with diverse activity relevant to fibre degradation have been identified by automated gene annotation methods. c. proteoclasticum falls within the butyrivibrio-pseudobutyrivibrio assemblage of rumen bacteria which are though to play an important role in the degradation of plant hemicellulose-lignin complexes which limit fibre degradation in the rumen. for new zealand it makes strategic sense to invest in microbial genomics efforts applied to agriculture where the country holds a strong competitive advantage and where ruminants constitute the vast majority of farmed animals. in conjunction with dna sequencing, proteomics and transcriptomics (micro-array analysis) we are using metabolomics as an additional functional genomics tool for gene discovery. we have established a footprinting approach for microbial metabolome analysis focused mainly on metabolic intermediates of polysaccharide degradation to provide quantitative information on end products of fibre-degrading enzymes. a gc-ms method has been developed that is able to resolve complex biological mixtures containing mono-, di, and oligosaccharides, in addition to a series of organic acids. we are currently phenotyping a series of c. proteoclasticum mutants to validate our analytical methodology and we are going to fully characterize the fibrolytic ability of c. proteoclasticum to be compared with other fibre-degrading microbes. we believe that our metabolomics data will complement current proteomic analysis of fibre-degrading enzymes and micro-array analysis of gene expression from a series of mutants by providing direct evidence of the metabolic function of key genes involved in fibre-degradation processes. many gram-negative bacteria utilize cell-to-cell communication systems that rely on diffusible n-acyl homoserine lactone (ahl) signal molecules to monitor the size of the population in a process known as quorum sensing (qs). in human pathogens this form of gene regulation ensures that the cells remain invisible to the immune system of the host until the pathogen has reached a critical population density sufficient to overwhelm host defenses and to establish the infection. the qs regulon of pseudomonas aeruginosa and burkholderia cepacia, two important pathogens for patients suffering from cystic fibrosis, has been studied by proteome analyses. comparative twodimensional gelelectrophoresis of pre-fractionated protein mixtures (extra-, surface-, and intracellular proteins) coupled to mass spectrometry analysis or n-terminal sequencing has been employed to recognize and identify qs-controlled proteins. our findings strongly support the importance of ahl-mediated cell-cell-communication as a global regulatory system and suggest that qs control also operates via post-translational mechanisms. as qs has been proven to be a central regulator for the expression of pathogenic traits and biofilm formation in opportunistic human pathogens it represents a highly attractive target for the development of novel anti-infective compounds. functional genomics technologies (transcriptomics and proteomics) have been exploited to validate the target specificity of natural and synthetic qs inhibitors, thus having a great potential as alternative therapeutics for the treatment of bacterial infections. modeling cell cycle complex formation from high-throughput data sets lars juhl jensen european molecular biology laboratory, meyerhofstrasse 1, 69117 heidelberg, germany. e-mail: jensen@embl. de to analyze the dynamics of protein complexes during the mitotic cell cycle, we integrated data on protein interactions and gene expression. the resulting time-dependent interaction network for the first time places both periodically and constitutively expressed proteins in a temporal cell cycle context, thereby revealing novel components and modules. we discover that most complexes consist of both periodically and constitutively expressed subunits, suggesting that the former control complex activity by a mechanism of just-in-time assembly. consistent with this, we show that additional regulation through targeted degradation and phosphorylation by cdk1 (cdc28p) specifically affects the periodically expressed proteins. alessandra luchini, andrea callegaro, silvio bicciato department of chemical engineering processes, university of padova, padova, italy. e-mail: alessandra.luchini@unipd.it (a. luchini) since transcriptional control is the result of complex networks, analyzing dynamical states of gene expression is of paramount importance to detect the multivariate nature of biological mechanisms. although hundreds of studies fully demonstrated the relevancy of microarrays in describing different physiological conditions, to reconstruct complex interaction pathways it is necessary to analyze the temporal evolution of transcriptional states. however, a robust experimental design for identifying differentially expressed genes over a temporal window would require large amounts of microarrays. unfortunately, replicates for each time point and experimental condition are not always available, because of cost limitations and/or biological samples scarcity. in addition, common data analysis tools, like anova, require replicates and disregard correlation structure among times. we present a method for the identification of differentially expressed genes in un-replicated time-course experiments. the procedure does not assume any model or distribution function, takes into account the correlation of data, and does not require sample replicates at the various time points, other than the presence of an initial time point for all analyzed conditions. the identification of differentially expressed genes as the result of a system perturbation is formally stated as a hypothesis testing problem in which a defined statistic is used to rank transcripts in order of evidence against the null hypothesis. specifically, (i) data are structured so that measurements are correlated in time, within the same biological condition; (ii) the null hypothesis is formulated so that changes in expression levels at different time points are equivalent; (iii) time point t0 represents the system before the perturbation. therefore, modulated genes are detected testing the statistical significance of expression differences between physiological states at each time point, once corrected by the variability at t0, and given an empirical null distribution constructed using permutations. statistical significance is assessed by the q-value. the method has been tested on time-course microarray experiments aimed at studying the temporal changes of gene expression in: (i) skeletal muscle cells treated with a histone deacetylase inhibitor (iezzi et al., 2004) and (ii) immature mouse dendritic cells (dc) exposed to larval and egg stages of s. mansoni (trottein et al., 2004) . differentially expressed genes, identified using the proposed algorithm, have been compared with results obtained from anova model and sam paired test. the biological significance and soundness of selected transcripts was also verified using global functional profiling by means of ontotools. results demonstrate that this novel procedure allows the identification of biologically relevant genes using half of the replicates required by standard model-based approaches. carbon sources. interesting data on the expression profile of the sty and paa genes in pseudomonas sp. y2 have been obtained, and have raised new questions on styrene and paa degradation by this bacterium. the calcium-dependent antibiotic (cda) is a lipopeptide synthesised non-ribosomally and produced by streptomyces coelicolor a3(2). cda contains several non-proteinogenic amino acid residues. hydroxyphenylglycine (4-hpg) is one of the unusual amino acids in the structure of the cda and vancomycin groups of antibiotics. for the members of the vancomycin group of antibiotics, the 4-hpg residue plays crucial roles in the structure and function of the final glycopeptide antibiotic. to reveal the putative biosynthetic pathway of this amino acid in cda, a standard "double crossover replacement strategy" was used to delete 4-hydroxymandelic acid synthase (4-hmas, encoded by hpd) from s. coelicolor mt1110 and 2377, using the delivery plasmid pzmh3. there was no cda production in the disrupted strains. plates containing a gradient of hydroxymandelic acid were used to restore cda production in both s. coelicolor mt1110 hpd and 2377 hpd. exogenous supply of 4-hydroxyl phenylglyoxylate and 4-hydroxyphenylglycine reestablished cda production by the hpd mutant. feeding analogs of these precursors to the mutant resulted in the directed biosynthesis of novel lipopeptides with modified arylglycine residues (mutasynthesis). a cxcl2 tandem repeat promoter polymorphism is associated with susceptibility to severe sepsis in the spanish population n. maca-meyer 1 , c. flores 1 , l. pérez-méndez 1 , r. sangüesa 2 , e. espinosa 2 , j. villar 1 : 1 research institute, hospital universitario n.s. de candelaria, s/c tenerife 38010, spain; 2 department of anesthesiology, hospital universitario n. s. de candelaria, s/c tenerife 38010, spain. e-mail: nmacame@ull.es (n. maca-meyer) sepsis describes a complex clinical syndrome resulting from a systemic inflammatory response to bacteria, and remains an important cause of mortality in the intensive care unit. cxcl2 chemokine (or mip-2) exhibits a pivotal role in the immune response, and several functional studies in animal models of sepsis have catalogued cxcl2 as a candidate gene for the development of sepsis. we have performed a case-control association study of cxcl2 gene variants and susceptibility to severe sepsis in 179 hospitalised patients and 364 healthy individuals. after the examination of linkage disequilibrium in the region, we analysed whether two promoter polymorphisms (snp rs3806792 and a newly described polymorphic short tandem repeat d4s3454) were associated with the syndrome. we found a significant association of common variants at d4s3454 with the development of severe sepsis (heterozygote carriers or 2.82; 95% ci 1.10-7.24, and homozygote carriers or 3.65; 95% ci 1.41-9.43; mantel-haenszel χ 2 test for linear trend p = 0.0006). the risks remained significant even after a genomic control adjustment, based on 20 additional genotyped polymorphisms not linked to the candidate gene. these preliminary results suggest that cxcl2 gene variants may contribute to the development of severe sepsis. kasper møller 1 , ana paula oliveira 1 , jens nielsen 2 , mark johnston 1 : 1 center for microbial biotechnology, biocentrum, technical university of denmark, denmark; 2 department of genetics, school of medicine, washington university, st. louis, usa glucose is the preferred carbon and energy source for most cells. in saccharomyces cerevisiae, a complex regulatory network ensures that s. cerevisiae ferments glucose to ethanol even in the presence of oxygen. to obtain a better understanding of this crabtree effect and the logic of the glucose signalling network in s. cerevisiae, we are analyzing glucose sensing and signalling in the related species saccharomyces kluyveri, which exhibits much less of a crabtree effect (it prefers not to ferment glucose when oxygen is available). we show that there are only two major glucose transporters in s. kluyveri, and that these are regulated in response to the availability of glucose via a glucose sensor and a signalling pathway similar to the glucose induction (rgt2/snf3-rgt1) pathway in s. cerevisiae. we have used dna-microarrays for s. kluyveri to find targets of the s. kluyveri glucose induction pathway, as well as to evaluate the global response to a change in environment from growth on ethanol to growth on glucose. this study identifies a number of differences in the regulation of glucose uptake and global responses to glucose between s. kluyveri and s. cerevisiae, which may contribute to their different glucose metabolism. detection and analysis of microrna using lna probes nana jacobsen, christian lomholt, peter mouritzen, peter stein nielsen, mikkel noerholm exiqon a/s, bygstubben 9, dk-2950 vedbaek, denmark. e-mail: mouritzen@exiqon.com (p. mouritzen) micrornas are a class of short endogenous rnas that act as post-transcriptional modulators of gene expression. growing evidence suggest that micrornas exhibit a wide variety of regulatory functions and exert significant effects on cell growth, development, and differentiation. recent studies have shown that human microrna genes are frequently located in cancer associated genomic regions and perturbed microrna expression patterns have been observed in many malignant tumors. we have exploited the significantly improved hybridization properties of lna oligonucleotides against rna targets to design lna-modified dna probes for detection of different micrornas in animal and plants by northern blot analysis, microarray hybridization and in situ hybridization. we will describe the results obtained from detection and analysis of different micrornas in c. elegans, zebrafish, mouse, and plants. in addition, we will describe a novel lna-based method for expression profiling of mature micrornas by quantitative rt-pcr. expression profile of the sty and paa genes in pseudomonas sp. y2 by means of dna microarrays david bartolomé-martín 1 , david juck 2 , m a teresa del peso-santos 1 , charles w. greer 2 , julián perera 1 : 1 departamento de bioquímica y biología molecular i, facultad de ciencias biológicas, universidad complutense de madrid, 28040 madrid, spain; 2 environmental microbiology group, biotechnology research institute, national research council canada, montréal, que., canada h4p 2r2. e-mail: perera@bio.ucm.es (j. perera) dna microarrays are a new and powerful tool to study gene expression in very diverse systems. environmental biotechnology and biodegradation are some of the fields of research where this technology may be very promising. pseudomonas sp. y2 is a bacterium able to grow in minimal medium plus either styrene (sty) or phenylacetic acid (paa) as the sole carbon and energy sources. this bacterium is the only organism where the genes that code for both the upper (sty genes) and the lower (paa genes) catabolic pathways for the styrene degradation have been described till now. it is unique in having two active copies of the genes encoding the lower pathway (paa1 and paa2 gene clusters). we have designed a dna microarray with the sty and paa genes in order to analyse their expression in the wild type pseudomonas sp. y2, in p. sp. y2 t2 (a paa2 deletion mutant) and in p. sp. y2 c1 (a crc gene mutant). this analysis has been performed on bacterial cultures grown in media with different carbon sources. interesting data on the expression profile of the sty and paa genes in pseudomonas sp. y2 have been obtained, and have raised new questions on styrene and paa degradation by this bacterium. dynamics in induced repression of phosphomannose isomerase pmi40 gene of saccharomyces cerevisiae anssi törmä 1,2 , juha-pekka pitkänen 1,2 , laura huopaniemi 2 , risto renkonen 2 : 1 medicel ltd., haartmaninkatu 8, 00290 helsinki, finland; 2 rational drug design program, department of bacteriology and immunology, haartman institute and biomedicum, university of helsinki, p.o. box 63, 00014 helsinki, finland. e-mail: juhapekka.pitkanen@medicel.com (j.-p. pitkänen) gdp-mannose is the precursor of cell wall biosynthesis in s. cerevisiae. to understand the system level role of gdp-mannose, we studied a conditional knock-out strain of the key enzyme in its synthesis; pmi40. the experimental procedure allowed us to study the order of mechanisms the cells launch in order to adjust to a sudden malfunction in the metabolic machinery. we collected 100 samples from continuous cultivations over 80 h and measured genome-wide gene expression levels, 10 enzyme activities, and concentrations of 30 intracellular metabolites. for sampling we have built a sample robot, which automatically takes and preserves the samples. in order to carry out this magnitude of experimentations and generated data, we have constructed a proprietary software platform to handle all the phases from project management in wet-lab to workflow and pathway management in in silico. after normalization and clustering, significantly changed genes and metabolites were searched for enrichment in biological processes and molecular complexes. further, gene expression levels, metabolite concentrations, and enzyme activities were searched against each other for causality over time. overall, we focused on thorough analysis of our own data and known database data in order to reward our efforts with knowledge. at the transcriptome level, repression of pmi40 led to two major types of activation profiles, one peaking at the time when pmi40p activity and gdp-mannose were depleted and the other later during recovery from the perturbation. the primary response was most enriched with genes known to play roles in mating and filamentous growth and associated with the transcription factors ste12p, tec1p, dig1p, and mcm1p, whereas the secondary response consisted of genes involved in carbon metabolism and associated with the general stress response regulators msn2p and msn4p. skn7p, a high-level transcription factor was associated with both the primary and the secondary response, consistent with its suggested role of coordinating environmental responses and developmental processes. transcriptome of pig ovarian cells: discriminant genes involved in follicular development bonnet a., le cao k.a., low-so g., san cristobal m., tosser-klopp g., hatey f. laboratoire de génétique cellulaire, centre inra de toulouse, castanet-tolosan 31326, france in order to identify genes and gene networks involved in pig ovarian follicular development, we built subtractive suppressive hybridization libraries (ssh) from granulosa cells of healthy follicles (small, medium or large). the rna isolated from these cells was used to hybridize cdna nylon micro-arrays. data analysis using a gaussian linear mixed model showed that 83% of the variability is due to the genes. two hundred fifty one regulated genes (from the 956 expressed) were identified and clustered into three groups according to the follicle size. moreover, we found previously identified genes such as aromatase, igfbp2 which supported the validity of our experimental model. ramdom forest analysis put forward the most discriminant 11 genes between the three follicle classes. this study put forward gene sets such as those involved in cell modeling, regulation of transcription, apoptosis during follicle growth. the next step will be to describe more precisely the spatio-temporal expression patterns at the mrna levels of the genes identified by these experiments. microalgae constitute a significant source of valuable natural products, e.g. sulfated polysaccharides, polyunsaturated fatty acids, and phycobiliproteins that find applications in wide range of industries, including food, pharmaceutical, agricultural and cosmetics. however, genomic and molecular genetic studies of microalgae lag far behind those of higher plants. in order to accelerate red microalgal genomic studies by taking advantage of current genomics and post-genomic technologies, we have generated expressed sequence tag (est) databases of two red microalgae porphyridium sp. and dixoniella grisea grown under various physiological conditions. to date we have sequenced 7210 and 6231 ests of porphyridium sp. and d. grisea, respectively. the sequence assembly resulted into, ca. 2000 non-redundant unigenes for each microalga, only 40% of which were identified by similarity to sequences in the public databases. porphyridium sp. and d. grisea unigenes were compared with the whole-genome predicted proteomes of three microalgae and those of representative eukaryotic and prokaryotic organisms. both microalgae have highest similarity to the red microalga cyanidioschyzon merolae. the order of sequence similarity to other organisms examined was arabidopsis thaliana, oryza sativa, chlamydomonas reinhardtii, thalassiosira pseudonana (diatom), saccharomyces cerevisiae, caenorhabditis elegans, archaea and cyanobacteria. although red microalgae are considered as phylogenetic bridge between prokaryotes and eukaryotes, our data show that the red microalgae have strong similarity to eukaryotes and only distant similarity to prokaryotes. gene expression profiles were studied by analyzing cdna and subtraction libraries constructed from algae grown under various physiological conditions. we observed that top three most abundant ests in the stationary phase of porphyridium sp. were adp ribosylation factor like-1, flavohemoglobin and adp ribosylation factor-1. in addition, we have identified several genes which were specific to nitrate-and sulfate starvation. the sarco(endo)plasmic reticulum ca 2+ -atpase (serca, "the calcium pump"), is responsible for pumping the ca 2+ released into the cytoplasm during muscle contraction back into the sarcoplasmic reticulum store while proton are pumped the opposite way as counter-cations. these transport processes go against the concentration gradients and are therefore energy consuming. the energy is derived from atp hydrolysis via formation and break-down of a phospho-enzyme intermediate. over the last year a number of new crystal structures have been published which have added to our understanding of how this task is accomplished, which provides an impressive insight to the mechanism of a molecular pump. rhomboids are a family of intramembrane serine proteases that are widely conserved throughout evolution. among diverse functions discovered so far, rhomboids participate in intercellular signalling, parasite invasion, membrane dynamics and bacterial quorum sensing, making them potentially valuable therapeutic targets. the identification of physiological substrates and of selective inhibitors will be key towards their evaluation as drug targets. we have developed an in vitro cleavage assay to monitor rhomboid activity in the detergent solubilised state, enabling the first isolation of a highly pure rhomboid with catalytic activity. analysis of purified mutant proteins suggests that rhomboids use a serine protease catalytic dyad instead of the previously proposed triad, and gives insights into subsidiary functions like ligand binding and water supply. this work was supported principally by embo and the mrc. structure and target-specificity of thioredoxin h kenji maeda 1 , anette henriksen 2 , per hägglund 1 , christine finnie 1 , birte svensson 1 : 1 biochemistry and nutrition group, biocentrum-dtu, technical university of denmark, dk-2800 kgs. lyngby, denmark; 2 biostructure group, carlsberg laboratory, dk-2500 valby, denmark. e-mail: kenji@biocentrum.dtu.dk (k. maeda) thioredoxins are ubiquitous small proteins with protein disulphide reductase activity. thioredoxins can alter the structures and the activities of various target proteins by reducing their disulphide bonds. seeds of several plants are abundant in cytosolic thioredoxins referred as h-type. in barley, two thioredoxin h isoforms, hvtrxh1 and hvtrxh2 that share 51% sequence identity but differ in temporal and spatial distributions were previously identified and characterised. in the present study, the relationship between structures and targetspecificities of h-type thioredoxins are analysed. the 3d-structures of hvtrxh1 and hvtrxh2 are determined by x-ray crystallography as the first crystal structures of thioredoxin h. comparison of the structures shows that the majority of solvent exposed residues near the active sites are conserved between the two isoforms. this is in agreement with previously observed similarity in target-specificity of the two isoforms. thioredoxins from organisms distantly related to barley, such as e. coli, have highly similar folds but different surface charge distributions compared to barley thioredoxins. a comparison of the target-specificities of hvtrxh1, hvtrxh2, e. coli thioredoxin and several thioredoxin mutants will be attempted to reveal the structural features that influence specificity of barley thioredoxin h isoforms. enbrel is a dimeric fusion protein consisting of the extracellular ligand binding portion of the human 75 kda (p75) tumor necrosis factor receptor (tnfr) linked to the fc portion of human igg1. the cho-expressed molecule contains both n-and o-linked oligosaccharides with a total carbohydrate content of 20% by mass. the o-linked oligosaccharides were released by hydrazinolysis and their structure determined by exoglycosidase sequencing and maldi-tof mass spectrometry. to locate precisely the o-linked sites, the glycosylation heterogeneity of tnfr:fc was simplified by treatment with n-acetyl neuraminidase and n-glycanase. the remaining molecule, which only carries core o-linked glycan structures, was cleaved by trypsin and analyzed by lc-ms. precise localization of o-glycosylation sites was determined based on the concept of a specific modification of the o-glycosylated serine into 2aminopropenoic acid and o-glycosylated threonine into 2-amino-2butenoic acid. the deficit in mass resulting from this transformation was the marker used to localize the modified residues on the peptides by tandem mass spectrometry sequencing (ms-ms). ms-ms spectrum of enbrel glycopeptides were interpreted based on the presence of 2-aminopropenoic acid and 2-amino-2-butenoic acid, resulting in a complete map of o-linked glycans precisely located at 10 different sites. anu mursula, beatrix fahnert, sari krapu, eija-riitta hämäläinen, ritva isomäki, peter neubauer bioprocess engineering laboratory and biocenter oulu, university of oulu, oulu, finland. e-mail: anu.mursula@oulu.fi (a. mursula) wnt proteins form a highly conserved family of secreted glycoproteins important in cell-cell signaling events during embryogenesis and adult tissue maintenance. impairments within this complex signaling pathway can lead for example to developmental defects in embryos, degenerative diseases and cancer. respectively, wnt proteins can be used as tools in basic research concerning wnt function, developmental biology, screening for interacting compounds, and for medical applications (e.g. therapeutics, stem cells). hence, recombinant wnts provide a valuable basis for these purposes. however, production of recombinant wnt proteins is challenging, because they contain multiple disulfide bonds making the folding very difficult. a process for production of murine wnt-1 in e. coli has been developed in our laboratory. the knowledge obtained from this research has also been applied to the expression of other wnts, namely wnt-4 and wnt-6, and can be used to approach other cysteine-rich proteins as well. since the expression level of wnt proteins is rather low so far, tools for monitoring and optimizing the production process have been established. by means of this sandwich hybridization method the level of target (wnt) mrna can be measured. the technique has already been applied to analyzing wnt-1 mrna levels. probes also for wnt-4 and wnt-6 have been generated. thus, transcription of wnt genes in all kind of cells (e.g. tissue, recombinant hosts) in general as well as kinetics of transcription can be studied using these tools. in growth factor signaling, stimulation of cell-surface receptors first triggers activation of the receptor itself and then of a large number of intracellular effector molecules. the stimulus is integrated with a host of other cellular processes, leading to cytoskeletal changes, activating transcriptional programs in the nucleus and ultimately resulting in cell proliferation, differentiation or motility. classical signaling pathways and networks depict potential protein-protein interactions only in a static form. in the cell, these interactions are dynamic and occur in an ordered fashion. here, we apply a mass spectrometric method that converts temporal changes to differences in peptide isotopic abundance in order to study the global dynamics of signaling events. briefly, three cell populations are metabolically labeled with either normal arginine or a 13 c substituted form, or a 13 c 15 n variant (stable isotope labeling by amino acids in cell culture, silac). each population was then stimulated with egf for a different time period and tyrosine phosphorylated proteins were affinity purified with anti-phosphotyrosine antibodies. the proteins from the precipitated complexes were quantitatively analyzed and identified using lc-ms/ms. arginine containing peptides occurred in three forms, directly indicating protein activation at the corre-sponding time point. combination of two experiments sharing one common time point of activation then generated five-point dynamic profiles. from the 202 proteins quantified, we identified 81 signaling proteins, including virtually all known egfr substrates and 31 novel effectors, and the time course of their activation upon egf stimulation. discriminating proteins involved in the signaling network from unspecific binders was straightforward as these presented an activation profile. we have now further extended this study by directly measuring in vivo phosphorylation sites in response to growth factor stimulation and monitoring the time evolution of the phosphorylation events. finally, we determined and quantitatively compared the global egf and pdgf tyrosine phosphoproteomes in human mesenchymal stem cells and revealed a control point in their differentiation into bone-forming cells. such global activation profiles provide a novel perspective in cell signaling and will be crucial to model the highly dynamic signaling networks in a systems biology approach. klaus schneider 1 , dave g smith 2 , steven skaper 2 , alastair d. reith 1 : 1 discovery research, glaxosmithkline, coldharbour road, harlow, essex, uk; 2 neurology & gastrointestinal cedd, glaxo-smithkline, coldharbour road, harlow, essex, uk over the last 15 years, progress in signal transduction research has revealed an astonishing degree of complexity in cell signalling which is manifested in positive and negative regulations and feedback loops within signalling pathways and by cross-talks between pathways, all of which are highly cell-type dependent. it has become evident that protein phosphorylation by protein kinases plays a major role in this complex regulation of cell signalling (hunter, 2000) . due to the importance of signal transduction in disease processes, many protein kinases may constitute key targets for disease intervention. yet, the lack of a full understanding of the regulation, the activation and, importantly, of downstream substrates of particular protein kinases requires often more detailed studies before initiation of resource-intensive efforts to find disease-modifying molecules. technologies for the study of protein kinase signalling include 32 p labelling, mutational and knock-out studies and more recently rna interference. these tools are complemented by approaches that are based on proteomic technologies developed over the course of the last 10 years. in this presentation, the scope of proteomics technologies to contribute to an understanding of kinase signalling will be discussed. an overview of available technologies will be given and results will be presented from a proteomic study of glycogen-synthase kinase 3 (gsk3) signalling (coghlan et al., 2000) . novel findings will be presented on a study of gsk3 inhibition in a primary neuronal cell line by differential 2d gel electrophoresis, which resulted in the identification of more than 40 proteins that were significantly regulated. proteome analysis is typically done by nanospray lc/ms in order to achieve higher sensitivity and thus a greater number of protein identifications. however, nano-scale lc systems can be more challenging to use and maintain. to obtain the best chromatographic performance, connections must be made carefully to minimize band broadening. improved chromatographic performance can enhance the mass spectrometric results by tandem ms as a greater number of peptides can be detected. a microfluidic chip-based system has been developed (yin et al., 2004) that minimizes the number of connections and the delay volumes. this work evaluates the performance of this device against the traditional nanospray approach. a yeast extract sample was separated by sds-page and bands were excised from the gel for further analysis. after in-gel digestion, the sample was analyzed by both traditional nanospray and the microfluidicbased chip device. after protein database searching, the identified proteins and the protein sequence coverage's were compared for the two approaches. the microfluidic device was demonstrated to be equivalent or better compared to the traditional approach. yin, h., killeen, k., brennen, r., et al., 2004. anal. chem. 77, 527-533 . plant cytochromes p450 (p450s) play key roles in the biosynthesis of most bioactive compounds with agronomic and therapeutic applications. a collection of about 120 plant p450s was expressed in yeast. the cdnas were isolated from the model plant with a sequenced genome arabidopsis thaliana, some others from wheat, helianthus tuberosus or vicia sativa. they were expressed under the control of a galactose-inducible promoter in an engineered strain of saccharomyces cerevisiae in which the gene of the native p450 reductase was replaced with the gene of a p450 reductase from a. thaliana under the control of the same galactose-inducible promoter, in order to provide an optimal context for plant p450 expression and activity (pompon et al., 1996) . an original procedure was designed for the high-throughput functional screening of this enzyme collection. it is based on the detection of oxygen consumed during the catalytic reaction by a fluorochrome embedded in the bottom of the microwell plates. this method was validated using several recombinant p450s of known activity. it also allows for a very efficient screening for enzyme inhibitors. the advantages and limits of the method will be discussed. this work was carried out with the support of génoplante programme (no 2001004) . reference pompon, d., louerat, b., bronine, a., urban, p., 1996. methods enzymol 272, 51-64. 3 folding of a bacterial membrane protein studied by protein engineering daniel e.otzen, pankaj sehgal, peter a. christensen department of life sciences, aalborg university, sohngaardsholmsvej 49, dk -9000 aalborg. e-mail: dao@bio.aau.dk (d.e. otzen) we have carried out a kinetic analysis of the folding of the 4-helix transmembrane protein dsbb in a mixed micelle system consisting of varying molar ratios of sodium dodecyl sulfate and dodecyl maltoside. this analysis incorporates both folding and unfolding rates, making it possible to determine both the stability of the native state and the process by which the protein folds. the analysis also takes into account the composition of the mixed micelles, which is different from the bulk detergent composition. refolding and unfolding are consistent with a three-state folding scheme involving the sdsdenatured state, the native state and an unfolding intermediate that accumulates only under unfolding conditions at high mole fractions of sds. the temperature-dependence of the folding reaction displays an unusual decrease in heat capacity accompanying unfolding, which probably reflects the amphiphilic environment of the membrane protein. destabilization of dsbb by different short-chain alcohols correlates very well with the alcohols' respective hydrophobicities. data from a series of ala-scanning mutants tentatively identify a nucleus for folding, which is relatively diffuse and involves all four helices. we are currently complementing this work with studies of the association of peptides corresponding to individual transmembrane segments of dsbb. the reca protein of e. coli plays a crucial role in homologous recombination and dna repair. the recombination process takes place in a filamentous complex, in which the protein monomers are arranged in a helical manner around a single-stranded dna (ss-dna). in the presence of atp the filament can accommodate a second, double-stranded dna (ds-dna) and the strand exchange reaction can occur. the three-dimensional structure of reca itself and its complex with adp have been determined by x-ray crystallography. the active nucleoprotein filament, however, has only been studied at low resolution. both electron microscopy (em) and small-angle neutron scattering (sans) indicate significant differences between the structures of the active nucleoprotein filament and the compressed, inactive filament of only reca. we have presented a structural model of the reca protein in its active filament with ss-dna, using data obtained by linear dichroism (ld) polarized-light spectroscopy, based on a technique, we call "site-specific linear dichroism", which allows the orientation of a set of amino acids to be determined from ld data by systematic modification of the protein. here, we show that ld data of the nucleoprotein filament with ds-dna is over all similar to the data of the complex with ss-dna, indicating that the orientation as well as internal structure of reca in the active filament is not significantly altered when the bound dna is changed from single-stranded to double-stranded. this result supports the idea that the strand exchange reaction occurs without large conformational change of the reca protein. the choline-binding modules: a powerful biotechnological tool jesús m. sanz instituto de biología molecular y celular, universidad miguel hernández, elche, spain choline-binding modules (chbms) are present in some virulence factors of streptococcus pneumoniae (pneumococcus). the most extensively studied chbm is c-lyta, the carboxy-terminal domain of the pneumococcal cell-wall amidase lyta. the three-dimensional structure of choline-ligated c-lyta is built up from six loop-hairpin structures ("choline binding repeats", chbrs) forming a left-handed ␤-solenoid with four choline binding sites. although the structure of the ligand-free form is not yet known, our folding studies suggest that it is more loosely packed, with a partially unfolded amino-terminal region and a stable carboxy-terminal moiety that is extremely resistant to chemical denaturation (maestro and sanz, 2005) . the affinity of c-lyta for choline and other structural analogues allows its use as an efficient affinity tag for overexpression, immobilization and single-step purification of proteins of biomedical interest (c-lytag fusion protein purification system). this system presents many advantages when compared to current commercial methods, namely simplicity, compatibility with buffers and robustness. the availability of multiple supports that specifically bind chbms (such as multiwell plates) has recently allowed the development of a new procedure for the immobilization of c-lyta-containing hybrid proteins that may be used in proteomics, diagnostics and peptide display. in this communication, we present our last results about the stability, folding and engineering of c-lyta, together with a compendium of the current biotechnological potential of this protein, and highlight the productive link between basic molecular studies and their application. many of the modern approaches for studying disease compare steady state functions, such as repair, growth, and regulated gene expression within the various biological compartments organised by specialized function, be it mitochondria or blood vessels. the assignment of protein identities, which are linked to key biological mechanisms, which are associated with disease processes and disease progressions are an important area of this work (marko-varga and fehniger, 2004) . today, the technology available for studying proteome expression and resolving exact protein and peptide identities in complex mixtures of biological samples allows global protein expression within cells, fluids, and tissue to be approached with confidence. this confidence is due in part to reproducible repetitive sampling and analysis technologies including robotics data acquisition and high level mass spectrometry including both laser-desorbtion and electro spray ionisation. the precision in defining differences between normal and diseased steady states is aided by the creation of compiled reference and master data sets and by new methods for multiplexing the analysis of samples in groups. the establishment of key representative reference proteome systems representing the dynamic changes in protein expression during disease will be vital to the interpretation of changes observed in specific samplings of disease states and specific cells obtained from these samples. the creation of reference databases of proteins linked to disease pathways will play an important role in furthering our understanding of the "proteome of disease". examples will be given where protein expression patterns have been generated from compartments within tissue sections. marko-varga, g., fehniger, t.e., 2004. j. proteome res. 3, 167178. 2 adaptation of the saccharomyces cerevisiae proteome to nutrient limitations studied by metabolic stable isotope labeling and mass spectrometry albert j.r. heck netherlands proteomics centre and utrecht university, the netherlands. e-mail: heck@npc.genomics.nl. url: www.netherlandsproteomicscentre.nl one of the major aims of proteomics is to provide quantitative data on differential protein expression levels. recently, mass spectrometry-based methods have been introduced that can provide quantitative data on differential protein expression, mostly using stable isotope labeling (goshe and smith, 2003) . we opted for metabolic labeling as this provides efficient means to quantify differential protein expression, and has the advantage that all proteins are labeled universally (romijn et al., 2003) . in their natural habitat microorganisms encounter non-optimal growth conditions and often growth is limited by one nutrient. microorganisms need to respond rapidly to changes in the environment in order to survive. in the present study, we investigate the proteome response of chemostat cultivated wildtype saccharomyces cerevisiae to two different nutrient limitations, namely carbon and nitrogen limitation. yeast was metabolically labeled in well-controlled chemostat cultures. 14 n and 15 n labeled proteins were separated using 1d gel electrophoresis followed by rp-lc-esi-ms on a lc-q. relative quantification was performed by using relex software (maccoss et al., 2003) . we quantified 759 proteins, using on average 8 peptide peak pairs per protein. this analysis revealed that 419 proteins showed a significant increase/decrease in expression level. the functional annotation of these proteins revealed that the yeast cells change expression levels of enzymes involved in metabolism of the growth-limiting compound. the protein expression ratios were compared with corresponding transcript levels. moreover, we compared the accuracy of quantifica-profiles mainly reflected differences in cellular origins in addition to different functional roles. mass spectrometric analysis identified 82 proteins pertaining to several functional classes, i.e. acute phase proteins, antioxidant proteins and proteins involved in protein synthesis/maturation/degradation, cytoskeletal (re)organization and in lipid metabolism. several proteins not previously implicated in nerve regeneration were identified, e.g. translationally-controlled tumor protein, annexin a9/31, vitamin d-binding protein, ␣-crystallin b, ␣-synuclein, dimethylargininases and reticulocalbin. real-time pcr analysis of selected genes showed which were expressed in the nerve versus the dorsal root ganglion neurons. in conclusion, this study highlights the complexity and temporal aspect of the molecular process underlying nerve regeneration and points to the importance of glial and inflammatory determinants. yeasts plasma membrane macromolecular components involved in stress resistance paola branduardi, paola paganoni, danilo porro dipartimento di biotecnologie e bioscienze, università degli studi di milano-bicocca, piazza della scienza, 2-20126 milano, italy. e-mail: paola.branduardi@unimib.it (p. branduardi) the plasma membrane is a universal structure of living cells constituting an essential barrier dividing and defining the intracellular from the extracellular environment. it is consequently easy to deduce the crucial role played by said structure for any cell of any living organism, and especially for unicellular organisms, since all the information deriving from the external environment as well as many of the consequent cellular responses have to pass through this barrier. unicellular organisms, thanks to easy manipulation and cultivation techniques, can represent a very useful model for studying the plasma membrane function and response. in addition microorganisms, and among them yeasts, can be considered advantageous cell factories for recombinant productions. in this contest, the implementation of any process of production has to take into account, among others, the response and the tolerance of the host to the external environment. from these considerations derives the interest of our group to analyse the main macromolecular components of yeasts plasma membranes (proteins, lipoproteins and lipids), isolated from cells grown under different stress conditions, with particular attention to acidic environments. here, we present our recent data about separation and identification (by sequencing analyses) of lipoproteins isolated from the conventional yeast saccharomyces cerevisiae as well as from the non-conventional and acid tolerant yeast zygosaccharomyces bailii cell cultures grown in different conditions. in parallel, the protein fraction is under evaluation through a differential 2d proteomic approach and consequent analyses. effect of fungal polysaccharides on the expression of pancreatic proteins in streptozotocin-induced diabetic rats sang woo kim 1 , hye jin hwang 1 , kwang bon koo 2 , jang won choi 2 , jong won yun 1* : 1 department of biotechnology; 2 department of bioindustry, daegu university, kyungsan, in an attempt to search novel biomarkers for monitoring diabetes prognosis, we examined the influence of the hypoglycemic fungal extracellular polysaccharides (eps) on the differential expression of pancreatic proteins in streptozotocin-induced diabetic rats. the results of diabetic study revealed that orally administrated eps exhibited excellent hypoglycemic effect, lowering the average plasma glucose level of the diabetic rats to 52.3%. pancreatic proteome were analyzed by 2-de system, which separated more than 2000 individual spots. the 2-de analysis demonstrated that thirty-four proteins from a total of about 500 matched spots were differentially expressed, of which 26 spots were identified as the proteins whose expression has previously been associated with diabetes. twenty-two overexpressed and twelve underexpressed proteins were significant (p < 0.05) between the healthy and diabetic rats, and the altered proteins were restored (p < 0.05) upon eps treatment. it was first found that carbonyl reductase (18.6-fold, p < 0.001) and mawdbp (31.4-fold, p < 0.01) were surprisingly upregulated upon diabetes induction, and then those two protein concentrations were completely restored by eps treatment. moreover, we obtained eight unidentified proteins that have not been reported to be related with diabetes mellitus. these results evidenced the effect of fungal eps on searching potential markers for diagnosis and therapeutic manipulation of diabetes mellitus. although molecular basis of protein modulation after eps administration in diabetic rats was not verified in this study, the results of the proteomic analysis provide impetus for further studies of mining the biomarkers for diabetic therapy. the model established in our experiment is expected to mimic human diabetic status, which will help us to interpret the roles of biomarkers in diabetic state. the use of polyol-responsive monoclonal antibodies in immunoaffinity chromatography and as a probe for unfolding of wild-type and altered (t103i) amidase from pseudomonas aeruginosa s. martins 1 , j. andrade 1 , a. karmali 1 , a.i. custódio 2 , m.l. since immunoaffinity chromatography is a powerful protein purification technique of interest in proteomics, monoclonal antibodies (mabs) against mutant (t103i) amidase from p. aeruginosa were raised by hybridoma technology. in order to identify mabs that bind t103i amidase tightly but release under gentle conditions, hybridoma clones secreting polyol-responsive mabs (pr-mabs) were previously screened. nearly 10% of elisa assay-positive hybridoma produced clones secreting pr-mabs with potential application as ligands for immunoaffinity chromatography. to select the optimal conditions for amidase elution, an elisa-elution assay was carried out, with two of these clones (f6g7; e2a6). the dissociation of ag-ab complex required 10% of propylene glycol and either 0.25 m (nh 4 ) 2 so 4 or 0.25 m nacl. the binding of purified mab of igm class (e2a6) to wild-type and mutant amidases was investigate by direct elisa, which revealed that it recognised specifically a common epitope on both amidases. conformational changes on antigen molecule were studied. mab e2a6 showed a higher affinity for heat denatured forms than for native forms as revealed by affinity constants suggesting that the mab recognizes a cryptic epitope. the effect of mab e2a6 on amidase activity was also investigated. the binding of mab to wild-type and mutant amidases exhibited an inhibition and activation of 60% as a function of time, respectively. this pr-mab is useful as a probe to detect conformational changes in native and denatured amidases as well as a ligand in immunoaffinity chromatography, which is of great interest in protein purification and proteomics. fragility and solubility of non-classical inclusion bodieš s. peternel 1 , a. ristič 1,2 , v. gaberc-porekar 1 , v. menart 1,2 : 1 national institute of chemistry, ljubljana, si-1000; 2 lek pharmaceuticals d.d., ljubljana, si-1000. e-mail: spela.peternel@ki.si (š. peternel) human granulocyte colony stimulating factor (g-csf) is a pharmaceutically important cytokine. when overexpressed in escherichia coli, it is usually accumulated in the form of inclusion bodies (ibs) . when produced at 42 • c classical insoluble ibs are formed while at 25 • c non-classical ibs containing a high amount of correctly folded g-csf are formed. as higher fragility and solubility of non-classical ibs were noticed, we decided to check whether bacterial cell disruption method has any influence on their mechanical stability and solubility. enzymatic lysis, sonication and homogenization, methods often used for disruption of bacterial cells during the isolation of ibs were compared. lysozyme treatment of bacterial cells appears to be mild enough disruption method not influencing the integrity of ibs. homogenization of bacterial cells at high pressure (100.000-120.000 kpa) shows no impact on classical ibs while some loss of target protein from the non-classical ibs is observed. sonication seems to be most harmful as even at rather low sonication altitudes, noticeable disassembling and solubilization of non-classical ibs occurs while no effect on classical ibs is perceived. our studies show that non-classical ibs are much more fragile and soluble than classical ones. therefore, one should extremely carefully choose the method for cell disruption to avoid undesirable loss of the target protein. the danish tick ixodes ricinus parasitize three different hosts both mammals and birds during the 3-year life cycle. the aim of this study was to identify the last blood host being the host, which the nymph had parasitized before molting to the adult instar. the reason for the study was to reveal the origin of the host contributing the most to the life cycle of the tick and thereby the maintenance of tick-borne diseases in denmark. the most common tick-borne diseases are lyme borreliosis and tick-borne encephalitis (tbe) causing illness in both animals and humans. we analyzed adult ticks, which were collected from known hosts. the analysis was performed at different heat stable proteins, which could be detected during the off host period by elisa. we found that heat stable proteins could be used as identification markers for host recognition. mushroom polysaccharides alter the expression of diabetesassociated proteins in the liver of streptozotocin-induced diabetic rats hye-jin hwang 1 , sang-woo kim 1 , kwang-bon koo 2 , jang-won choi 2 , jong-won yun 1* : 1 department of biotechnology, daegu university, kyungsan, kyungbuk 712-714, korea; 2 department of bioindustry, daegu university, kyungsan, kyungbuk 712-714, korea. e-mail: jwyun@daegu.ac.kr (j.-w. yun) in the present study, we investigated the influence of the hypoglycemic fungal extracellular polysaccharides (eps) on the differential expression of liver proteins in streptozotocin (stz)-induced diabetic rats. the results of diabetic study revealed that orally administrated eps exhibited an excellent hypoglycemic effect, lowering the average plasma glucose level in eps-fed rats to 52.3%. in the next step, we analyzed the differential expression patterns of rat liver proteins from each group, to discover potent candidates for diabetesassociated proteins. a total of 69 proteins of the 2-de gel were expressed differentially between diabetic and healthy rats. among them, 34 proteins were upregulated and 35 proteins were downregulated upon diabetes induction. many of these changes were in accordance with observations in previously published studies. surprisingly, the altered levels of most proteins in diabetic group were fully or partially restored to those of non-diabetic control group by eps treatment. moreover, we obtained 13 unidentified proteins that have not been reported to be related with diabetes mellitus. although molecular basis of protein modulation after eps administration in diabetic rats was not verified in this study, the results of the proteomic analysis provide impetus for further studies of mining the biomarkers for diabetic therapy. potential is still limited by the differences observed in the structure of plant and mammalian n-glycans. indeed, theses differences and particularly the presence of ␤1,2-xylose and ␣1,3-fucose glycoepitopes are responsible for the immunogenicity of plant n-glycans. in order to reduce the structural differences between plant and mammalian n-glycans, current strategies are to knock out plant-specific glycosyltransferases or to humanize plant n-glycans by expression of mammalian glycosyltransferases in plants. in the present study, we have expressed a human ␤1,4-galactosyltransferase in alfalfa. in order to further increase the efficiency of the human ␤1,4-galactosyltransferase in the plant golgi apparatus, we have exchanged the endogenous targeting signal of this human glycosyltransferase for the ones from plant glycosyltransferases recently characterized in our laboratory. we will illustrate this approach of targeted expression with the results obtained by fusion of the catalytic domain of human ␤1,4-galactosyltransferase with the n-terminal sequence of a plant glycosyltransferase that targets the fusion to the very early compartments of the golgi apparatus. the efficiency of natural versus targeted expression of human ␤1,4galactosyltransferase in alfalfa will be compared in term of n-glycan humanization. altogether, our results clearly illustrate that we are now on the way to get perfect copy of mammalian glycoproteins in alfalfa plants. construction of recb-recd gene fusion and analysis of fusion enzyme activities oytun portakal 1 , gerald r. smith 2 , pakize dogan 1 : 1 department of biochemistry, hacettepe university medical school, 06100, ankara, turkey; 2 divisions of basic sciences, fred hutchinson cancer research center, seattle, wa 98109-1024, usa. e-mail: oytun@hacettepe.edu.tr (o. portakal) protein folding is a fundamental process to gain protein function. in an oligomeric protein, the interaction between polypeptides affects the folding process and assembly to the holoenzyme. recbcd is a heterotrimeric and multifunctional enzyme that plays an essential role for major pathway of homologous recombination in e. coli. it is composed of recb, recc and recd gene products. recd is the fast motor unit of the recbcd enzyme. recd also plays a role for high affinity dsdna binding, nuclease activity and chidependent regulation. this study was designed to test the hypothesis that recd polypeptide regulates the essential reca loading activity. the approaching of the study was to fuse recd gene to subsequent recb gene and to observe the changes in enzyme activity and structure. for these purpose two genetic fusion mutations, two-nucleotide deletion and three-codon substitution were created at the overlap sites (ta) of recb and recd genes. fusion mutations were constructed by phage-mediated recombination system, which is called recombineering. this technology requires red function but not host reca protein function. here, we showed the recbd fusion polypeptides in crude extracts. genetic characterization tests were revealed that both fusion enzymes are recombination proficient and have wild-type phenotype. biochemical assays demonstrated that recbdc fusion heterotrimers have dsdna exonuclease, unwinding and chi cutting activities. they were also resistant to dna damaging agents. western blot analysis also detected a wild type length recd polypeptide together with recbd fusion polypeptides and a decreased heterotrimer compared to wild type. our findings suggest that recb-recd genetic fusions may affect recd assembling to the heterotrimer, but not affect it's native folding. sandwich immunoassay-a simple strategy for enhancement of the sensitivity and the specificity in prostate specific antigen detection based on surface plasmon resonance cuong cao, sang jun sim department of chemical engineering sungkyunkwan university, 300 chunchun-dong, jangan-gu suwon, prostate cancer is a deadly disease in men. prostate specific antigen (psa) has been proved to be the most reliable and specific biomarker in preoperative diagnosis, monitoring and followup of patients with prostate cancer. in this study, a biochip based on surface plasmon resonance was fabricated to detect psa at concentrations ranging from 1 to 1000 ng/ml. to reduce nonspecific binding, the chemical surface of sensor was constructed by using various ethyleneglycol mixtures of different molar ratios of hs(ch 2 ) 11 (och 2 ch 2 ) 6 cooh and hs(ch 2 ) 11 (och 2 ch 2 ) 3 oh. we also biotinylated the sams surface to enhance the orientation of protein immobilization. by using this surface, spr-based psa detection gave a positive ru value at the fist response in the whole range of psa concentrations. however, this ru value could get better and more reliable by simply applying a secondary interactant, the psa polyclonal antibody, in sandwich immunoassay. the results shown this approach could satisfy our purpose without modify the secondary interactant, which has usually been done by the other report. expression of epitopic domains of human coagulation factor viii in escherichia coli amir amiri yekta 1,2 , naser amirizadeh 3 , alireza zomorodipour 1* , fariba ataei 1 : 1 department of mol genet. national institute for genet eng & biotechnol tehran-iran p.o. box: 14155-634, tehran, iran; 2 islamic azad university of jahrom, jahrom, iran; 3 department of hematol, faculty of med, tarbiat modarres university, tehran, iran. e-mails: amir amiriyekta@yahoo.com (a.a. yekta), * zomorodi@nrcgeb.ac.ir (a. zomorodipour) human factor viii (hfviii) plays major role in the intrinsic pathway of blood coagulation and is used to treat individuals with hemophilia a for bleeding episodes. many researches have been focused on the molecular aspects of this protein. in this regard, epitopes of hfviii as well as their corresponding antibodies have many important applications. bacterially produced fviii-epitopes are capable to neutralize the alloantibodies that inhibit hfviii activity. the purpose of present study was to over-express two epitope-containing fragments of fviii in e. coli under t7 promoter (novagen). two dna fragments from light-and heavy-chains of hfviii (942bp-c1c2 and 1644bp-a1a2, respectively) were subcloned in the expression vector. the use of his 6 -tagged tail was also considered for detection and purification purposes. in each of the examined clones, a protein of expected size was detectable. in the c1c2-expressing clone the specificity of the over-expressed protein was confirmed by its reaction with the rabbit serum directed against native hfviii as well as anti-his-tag antibody. in the heavy chain-related-expressing clone, the expression level was low, but it was detectable by immunoblotting experiments. manipulations of the growth as well as induction may be required. the over-expression of the other epitopes reported in the heavy chain may be achievable by the expression of (a) sub-fragment(s) of this region. the over-expressed his-tagged c1c2-related protein was appeared to be trapped in the cell as non-soluble inclusion bodies. therefore, after homogenizing of the induced recombinant cells, the nonsoluble fraction was dissolved in a solution of denaturant (guanidine hydrochloride) and subjected for the purification, using a ni-nta resin (qiagen) followed by protein measurement. accordingly, an expression level of 5 mg/l (of culture) of the purified c1c2-related peptide was obtained. the recombinant hfviii c1c2-derived peptide has provided useful mean for further experimental and medical applications. isotachophoresis has almost exclusively been applied for contracting and stacking samples ions before zone electrophoretic separation of proteins. this study attempts to apply microfluidic isotachophoresis (itp) as a high resolution analytical method for proteins. beta-lactoglobulin and other milk proteins with slightly different pi were labelled with fluorescent red 646 and analysed by the micralyne tk system using microfluidic glass chips, either with simple cross (sc) or double cross (tt) injection or designed 2d-itp-cze chips with double tt injection and sc for transfer to the second dimension cze channel, efficiently non-covalently coated with 0.6% w/v epoxy-polydimethylacrylamide to lower electroendoosmosis. capillary zone electrophoresis (cze) in borate or phosphate buffer was reproducibly perform for more than 75 consecutive runs using upchurch tm reservoirs glued to the wells to enable larger buffer volumes and greater run-to-run stability. finally, isotachophoretic anionic separation of the proteins were done using phosphate (ph 8.1) or chloride (ph 8.5) as leading ion and -amino-caproic acid (ph 8.9) as terminating ion. the effect of narrow cut ampholytes as spacers needs further investigations. the perspective aim is to combine the migrating itp separated zones with second dimension capillary zone electrophoresis as a new microfluidic proteomic 2danalysis. development of strategies for heterologous expression of glucose dehydrogenase from the halophilic archaeon halobacterium sp. nrc-1 juan carlos cruz-jiménez 1 , lorenzo saliceti-piazza 1 , rafael montalvo 2 : 1 chemical engineering, university of puerto rico-mayagüez campus, mayagüez 00680, puerto rico; 2 biology, university of puerto rico-mayagüez campus, mayagüez 00680, puerto rico. e-mail: juancruzj@hotmail.com (j.c. cruz-jiménez) halophilic archaea are excellent model organisms and valuable for biotechnology applications; they are easy to culture in the lab, genetically tractable, and exhibit a variety of interesting and useful characteristics. most halophilic archaea require 1.5 m nacl to sustain growth and structural integrity. among the 2.630 genes in halobacterium, we are studying the gene encoding a glucose dehydrogenase, gene id is 446, located between the 345205 and 346272 bases (halobacterium sp. nrc-1 genome project). this extremozyme is bioengineerable, and its use as a model for studying biocatalysis in aqueous/organic and nonaqueous media has not been explored to date. the utilization of enzymes in organic solvents has several potential advantages over aqueous systems. a major benefit is the increased solubility of many substrates, resulting in higher concentrations of reactant and products, hence reducing and purification costs and simplifying recovery protocols. cells were grown aerobically during seven days at 37 • c in a complex medium, harvested by centrifugation and their genomic dna extracted. for cloning of the gene, primers were designed based on the sequence recently published by the halobacterium sp. nrc-1 genome project. the forward (5 -ccgcatgcgcc cacagtccc-3 ) and reverse (5 -ccggcctctagaacggcctgg-3 ) primers were designed to incorporate restriction sites for sph i and xba i, respectively (in bold). we are pcr amplifying the genomic dna and developing methods for the heterologous expression using the mesophilic escherichia coli, as well as purifying the enzyme. the purification procedure will be carried out using high resolution methods based on the protein's halophilicity. bioinformatics methods will be used to facilitate conforming of protein function and for comparison with a native enzyme. quantitative measurements by mass spectrometry of hundreds of proteins simultaneously using the new proteinchip systemseries 4000 p. iversen, e. fernvik ciphergen biosystems inc., symbion research park, fruebjergvej 3, dk-2100 copenhagen, denmark. e-mail: piversen@ciphergen.com (p. iversen) most mass spectrometry methods used in proteomics allow for the identification of multiple proteins in a limited number of complex samples, but lack the ability to assess the quantity of the proteins and their modifications. however, mounting evidence shows specific cleavage of well-known proteins as being strong candidates for specific biomarkers, and in order to discover these biomarkers one has to be able to monitor the quantity and mass of hundreds of proteins from hundreds of complex samples reproducibly. the new series 4000 instrument in connection with proteinchip arrays ® from ciphergen biosystems enables this. the new 4000 series instrument is optimized for sensitivity, reproducibility and quantitation. new ion optics allows the use of higher acceleration voltages thus increasing the sensitivity, but without lowering the resolution. a new method of blanking the detector in connection with a non-linear gain of the detector also increases the sensitivity to the effect that igg can be detected down to 0.2 fmol. furthermore, the unique design of the instrument permits the detection of proteins with great variation in both mass and concentration and thus making it ideal for proteomics studies. a unique feature of the 4000 series instrument is the possibility to normalize the output by controlling the laser and detector so that results can be read with equal precision on different instruments, which is not often possible in mass spectrometry where individual instruments yield different results. this feature is vital in the validation of research results beyond individual laboratories. the coupling of liquid chromatography with mass spectrometry is now firmly established as a routine method for the identification of proteins that have been subjected to enzymatic digestion. in an on-line lc-ms experiment, the column eluent is coupled to the electrospray source via an emitter and any tryptic peptides present in the mixture are mass analyses as they elute from the hplc column. should there be any co-eluting species in the eluent, these will be separated in the mass analyser by their mass-to-charge ratio. it has become increasingly clear that relative quantification of protein expression changes is important in modern biology and medicine. several current approaches have been developed that utilise stable isotope labelling of samples in combination with separation and subsequent analysis by mass spectrometry. however, we have recently described an lc-ms strategy where quantification is achieved via normalisation of the ms datasets and comparison of the peptide intensities (of the observed tryptic peptides) across samples is performed. in this case, it is desirable to perform replicate injections and hence reduce statistical errors. this approach places a requirement upon good chromatography, especially in terms of retention time reproducibility. in addition exact mass measurement of the eluting ions is required as well as the ability to generate reproducible and reliable peak intensity, or area, calculations for the eluting tryptic peptides. the ability to measure the mass to charge ratios of ions accurately, across injections and across samples, increases confidence that the same ions have been matched from each sample injection. in this presentation our current strategy for the relative quantification of proteins will be discussed using, as examples, complex protein mixtures from salmonella enterica, eschericia coli and human serum. proteomic analysis for the production of rhctla4ig in transgenic rice cell cultures using dige ji-suk cho, song-jae lee, inha university, difference in gel electrophoresis (dige) technology using fluorescent dyes is a novel method, which simplifies the process of detecting and matching proteins between multiple gels by allowing the separation of up to three separate protein samples in the same gel. it provides accurate quantitative and reproducible differential expression values for proteins in several samples. recombinant human cytotoxic t lymphocyte-associated antigen 4-immunoglobulin (rhctla4ig) was produced in the transgenic rice suspension cell cultures using ␣-amylase promoter system. this system is efficient for the production of recombinant proteins, as it secretes target proteins into culture medium under sugar-depleting condition. in this study, the intracellular proteins expressed at both growth and induction stages of culture were separated and analyzed using 2-d dige. each sample from different conditions and internal standard were labeled with n-hydroxy succinimidyl ester-derivatives of cy2, cy3 and cy5 dyes and run within a single dige gel. using decyder tm software, 2218 spots were detected with two-fold thresholds with 95% confidence and it was found that 60 proteins underwent significant change during the production of rhctla4ig with normalization method improving data distribution. a pooled sample mixture for the picking gel was prepared with deep purple staining and analyzed with mass spectrometry. in addition, the intracellular rhctla4ig spots were identified with western blot analysis using goat anti-human igg (fc) antibody after dige gel was transferred to pvdf membrane. study of substrate specificity of rnr-exoribonucelases using hybrid proteins ana barbas, mónica amblar, cecília m. arraiano instituto de tecnologia química e biológica, ean, 2784-505 oeiras, portugal. e-mail: ab@itqb.unl.pt (a. barbas) the ribonucleases are essential enzymes responsible for the regulation of gene expression and have shown to be important for biotechnology purposes. for instance, commercial mutants deficient in ribonucleases have been quite relevant for the over-production of recombinant proteins. escherichia coli rnase ii is a processive 3 -5 exoribonuclease prototype of the rnr family that has homologues widespread in the majority of the sequenced genomes. by sequence alignment it has been proposed for the rnr type proteins the existence of three different domains: an n-terminal cold shock nucleotide binding domain (csd), a rnb catalytic domain, and a c-terminal s1 nucleotide binding domain. we have constructed several rnase ii deletion mutants to enable the characterization of each domain. these studies have allowed us to determine that both csd and s1 are involved in the binding of the enzyme to the rna substrate, being the s1 domain the most important. in rna-binding proteins it has been shown that the s1 domain's conformation is highly conserved. however, it is not known whether the substrate specificity is s1-dependent. in order to characterize the s1 domain and verify if it is directly related to substrate specificity, we have constructed rnase ii hybrid proteins in which the s1 domain was substituted by the s1 of two other exoribonucleases, rnase r (rnii-rnr) and pnpase (rnii-pnp). preliminary results have demonstrated that both quimeric proteins are capable of binding and degrading various rna substrates. in addition, studies are currently being carried out to verify the possibility that s1 domain of pnp in the hybrid protein might be involved in multimerization and/or interaction with other proteins. the murine monoclonal antibody igg1, anti-digoxin was produced in a rolling bottle fermentor. purification was performed on a protein g column. cd spectra were recorded on a jasco-810 spectropolarimeter. protein concentrations of 20-50 g/ml and path length of 1 cm were used for measurements in a far uv region. all measurements were performed in a cell holder thermostand with an accuracy of ±0.2 at 25 • c. at this temperature the predominance of ␤-strands is indicated. large conformational changes occur at 78 • c. at this temperature the spectra tense to irregular ␤-strands and unordered structures. these evidences confirming temperaturedependent conformational changes of protein and also high thermal stability of mentioned monoclonal antibody. generation of monoclonal antibodies for the assessment of protein purification by recombinant ribosomal coupling janni kristensen, kim kusk mortensen, hans peter sørensen 1 laboratory of biodesign, department of molecular biology, aarhus university, gustav wieds vej 10 c, dk-8000 aarhus c, denmark. e-mail: hans.peter.sorensen@teknologisk.dk (h.p. sørensen) we recently described a conceptually novel method for the purification of recombinant proteins with a propensity to form inclusion bodies in the cytoplasm of escherichia coli. recombinant proteins were covalently coupled to the e. coli ribosome by fusing them to ribosomal protein 23 (rpl23) followed by expression in an rpl23 deficient strain of e. coli. this allowed for the isolation of ribsomes with covalently coupled target proteins which could be efficiently purified by centrifugation after in vitro proteolysis at a specific site incorporated between rpl23 and the target protein. to assess the efficiency of separation of target protein from ribosomes, by site specific proteolysis, we required monoclonal antibodies directed against rpl23 and gfp. we therefore purified rpl23-gfp-his, rpl23-his and gfp from e. coli recombinants using affinity, ion-exchange and hydrophobic interaction chromatography. these proteins could be purified with yields of 150, 150 and 1500 g per gram cellular wet weight, respectively. however, rpl23-gfp-his could only be expressed in a soluble form and subsequently purified, when cells were cultivated at reduced temperatures. the purified rpl23-gfp-his fusion protein was used to immunize balb/c mice and the hybridoma cell lines resulting from in vitro cell fusion were screened by elisa using rpl23-his and gfp to select for monoclonal antibodies specific for each protein. this resulted in 20 antibodies directed against rpl23 and 3 antibodies directed against gfp. antibodies were screened for isotypes and their efficiency in western immunoblots. the most efficient antibody against rpl23 and gfp were purified by protein g sepharose affinity chromatography. the purified antibodies were used to evaluate the separation of ribosomes from gfp, streptavidin, murine interleukin-6, a phagedisplay antibody and yeast elongation factor 1a by centrifugation, when ribosomes with covalently coupled target protein were cleaved at specific proteolytic cleavage sites. proteomic analysis for the production of rhctla4ig in transgenic rice cell cultures using dige ji-suk cho, song-jae lee, inha university, difference in gel electrophoresis (dige) technology using fluorescent dyes is a novel method, which simplifies the process of detecting and matching proteins between multiple gels by allowing the separation of up to three separate protein samples in the same gel. it provides accurate quantitative and reproducible differential expression values for proteins in several samples. recombinant human cytotoxic t lymphocyte-associated antigen 4-immunoglobulin (rhctla4ig) was produced in the transgenic rice suspension cell cultures using ␣-amylase promoter system. this system is efficient for the production of recombinant proteins, as it secretes target proteins into culture medium under sugar-depleting condition. in this study, the intracellular proteins expressed at both growth and induction stages of culture were separated and analyzed using 2-d dige. each sample from different conditions and internal standard were labeled with n-hydroxy succinimidyl ester-derivatives of cy2, cy3 and cy5 dyes and run within a single dige gel. using decydertm software, 2218 spots were detected with two-fold thresholds with 95% confidence and it was found that 60 proteins underwent significant change during the production of rhctla4ig with normalization method improving data distribution. a pooled sample mixture for the picking gel was prepared with deep purple staining and analyzed with mass spectrometry. in addition, the intracellular rhctla4ig spots were identified with western blot analysis using goat anti-human igg (fc) antibody after dige gel was transferred to pvdf membrane. similarity searches and multiple alignment of s 1 and s 2 protein of sars-cov for modeling 3d structure and its evolution (origin) mohammad soltany rezaee rad, iman tavassoly, negar mottaghi, banafsheh rezaee. e-mail: mohammad.soltany@gmail.com (m.s.r. rad) aims: the exact origin of the cause of severe acute syndrome (sars) is still an open question. nowadays 8 recombinant origins for this virus have been found. s 1 and s 2 subunit of spike protein of this virus are the most important proteins responsible for severe acute respiratory syndrome. in fact they are glycoproteins of this virus exist on its surface. they are responsible for mediating fusion of viral and cellular membrane. the classification and modeling 3d structure of this virus can help us to suggest new ideas about its charististics and function, which may lead to new therapeutic and preventing modalities. methods: we used nucleotide sequence of s 1 and s 2 subunit of s (spike) protein for multiple alignments. we have done multiple alignments with different bioinformatics software (clusterx, entrez) for comparing the sequence with the other viruses and, we used weblab view software for modeling and identifying 3d structure of these proteins. findings: the similarity searches on nucleotide sequence of this protein with the 30 single strands rna (ssrna) shows the virus belong to a known classification named coronaviridae. these 3d structures show the responsibility of s protein in this syndrome. another findings based on these alignments is an important similarity between these subunits and genome of hiv-1 showing they have familiar mechanism in pathogenesis. discussion: multiple alignments are powerful tool in classification of new recombinational virus and emerging infection. 3d structure model of this virus is an important guide to understand the mechanism of this virus. the shape of glycoprotein that modeled with bioinformatics software can help us in understanding mechanism of binding this virus to human cells. this fact can be used in designing drug and vaccine to cure and prevent the sars. blocking these origins and sites leads to inhibiting the virus attachment. also the similarity between this virus and hiv-1 shows us that both of them have similar proteins that cause pathogenesis of these viruses. the simulated moving bed (smb) technology is a continuous countercurrent chromatographic separation technique that has been applied successfully in the last years to a number of significant problems. an smb consists of a series of fixed bed chromatographic columns connected in a loop, and outperforms column chromatography in terms of productivity and solvent consumption. the use of smb instead of batch processes for bioseparations, i.e. separations involving large and rather complex molecules with multiple 3d configurations depending on parameters such as ph, temperature, etc., is becoming of greater and greater interest. examples of these are therapeutic proteins, antibodies and plasmid dna among others. for all chromatographic processes in this field, one of the most crucial issues is the cleaning of the chromatographic media with a special solvent system, an operation usually referred to as cleaning in place (cip). in single column chromatography this is easily done off-line, but this is not compatible with standard smb operation. in order to overcome this limitation, the standard smb configuration has been modified by adding a dedicated section plus an additional section for the re-equilibration of the freshly cleaned column with the working solvent before it is re-inserted into the smb loop. in such a way, cip according to gmp criteria can be incorporated into the smb unit and operation, which is then called cip-smb. this new smb configuration is also related to the three fraction separation unit called 3f-smb that has been recently introduced and applied to the separation of nucleosides. in this work we apply cip-smb using a size exclusion stationary phase to the separation of plasmid dna from the filtered cell lysate solution. plasmid purification has become a key issue in the last years as a result of the advances in gene therapy, whereas traditional laboratory methods are not always suitable for therapeutic purposes. we report about separation performances, which are then discussed in the light of smb design criteria and compared to column chromatography performance. computer guided optimization of adsorptive bioseparation processes bernt nilsson department of chemical engineering, lund university, 221 00 lund, sweden. e-mail: bernt.nilsson@chemeng.lth.se separation processes like chromatography can be highly nonlinear and the behavior can sometimes be hard to predict. optimization of preparative chromatography is often done experimentally, which is both time consuming and expensive. a model-based approach to optimization is therefore an attractive and challenging way to overcome some drawbacks in the traditional working procedure in biotechnical industry. efficient model-based optimization for industrial needs requires three parts; models, methods and tools. model-based methodology requires a set of chromatography column model structures, which can capture the phenomenon of interest. for instance they have to capture column load variations, elution profile changes, operation condition disturbances, column configurations and stationary phase properties. to derive a reliable model for optimization it has to be calibrated and validated to experimental data, which requires an efficient calibration procedure. different calibration procedures are discussed and compared. after validation the model can be used in the design of a separation step. to do a robust design a set of requirements have to be fulfilled. the column size and operation conditions are used to optimize the performance of the step, which requires a constraint nonlinear optimization method. the choice of objective function for optimization and corresponding constraints are not obvious and the resulting operation conditions are often not robust. therefore there have to be additional methods available for analysis of the performance, like sensitivity and robustness analysis. optimization of the purification of antibodies is discussed and exemplified. the work with mathematical models and numerical methods has to be supported by a set of computer tools of different kinds in order to solve industrial problems effective. there is a need for different kinds of tools; customized tool to solve specific problems by a non skilled user and general toolbox for the expert. an example of a toolbox is presented. tina tarmann, alois jungbauer department of biotechnology, university of natural resources and applied life sciences, vienna, austria plasmids and viruses are the contemporary vehicles for genetherapy and genetic vaccination. extremely promising results have been reported from in-vitro, in-vivo and clinical studies. currently a lot of these compounds are manufactured with a technology which has been directly transferred from laboratory to pilot scale without further engineering. membrane based separations, chromatographic separations and precipitation have been employed for separation of plasmids and viruses. chromatographic separation have been designed with aim of protein separation. thus such processes suffer from either mass transfer limitations or low capacity. monoliths without intraskeleton mesopores and chromatography particles with giga pores are excellently suited for adsorption and separation of plasmids and viruses. low mass transfer resistance and high capacity compared to conventional beaded materials can be observed. adsorption kinetics were derived from infinite and finite bath methods and isotherms were constructed. these data also suggest that a conformational change of the plasmids takes place upon adsorption. discussion of the mass transfer properties and an example of scale up of a chromatographic separation process using these novel materials will be shown and discussed in respect to already existing processes. the recent developments in molecular therapies such as non-viral gene therapy and dna vaccination have fostered the development of efficient plasmid dna (pdna) purification processes. the separation of supercoiled (sc) and open circular (oc) isoforms is one of the key steps in the large scale purification of pdna vectors intended for a therapeutic use. although escherichia coli produces mainly the more compact sc pdna isoform, oc, linear and denatured pdna isoforms are usually present and are likely to be less efficient in transferring gene expression. for this reason, regulatory agencies specify that more than 90% of pdna in a therapeutic product is in the sc isoform. in this work histidine-base recognition is explored as a mean to separate pdna isoforms. the agarose gel used here combines the mild hydrophobic characteristics of an epoxy spacer arm with a pseudo-affinity histidine ligand. chromatographic profiles were obtained by injection of native plasmid (sc + oc) samples in the histidine-agarose support showing an efficient and baseline separation of both isoforms. the high resolution obtained with this support indicates that the method is potentially applicable to the separation of pdna at preparative and analytical scale. affinity ligand development with a novel encoded bead screening technology ib johannsen, versamatrix a/s, gamle carlsberg vej 10, dk-2500 valby, denmark. e-mail: www.versamatrix.com the presentation describes a new invention for fast development of affinity ligands, where up to 20,000 ligands can be screened onbead and identified in a few hours. combinatorial synthesis by the split and mix procedure is a powerful technique for generating vast numbers of diverse chemical compounds on polymer beads with relatively little effort. traditionally, the technique is hampered by the laborious spectroscopic and chemical analysis, needed to determine the exact structures of the ligand on selected beads. in this way, 6-12 month analysis time could easily be spent just to analyze a tiny fraction of the library. in the versaffin tm technology each bead is encoded, individually tracked, and identified during the synthesis and screening. this decreases the whole ligand development time from months to weeks and increases the amount of information significantly. the bead code further enables evaluation of the ligandprotein binding under varying binding and elution conditions. the instrument for reading the encoded beads and for quantifying the amount of bound protein is presented. the encoded beads we use are based on functional cross-linked polyethylenglycol (peg), which is compatible with water as well as most organic solvents. thus, the combinatorial synthesis can be carried out in organic solvents and the resulting compounds can be evaluated, still bound to the parent beads, under aqueous conditions. a further advantage of using peg based beads for on-bead screening is the fact that peg is biologically inert and therefore does not interfere in a bioassay. in the biopharmaceutical industry, pressure is mounting to shorten development times and thereby time to market, e.g. in the field of monoclonal antibodies, generic processes have been established which allow for more rapid development from gene to production of pre-clinical and proof of concept/phase i material. for non-mab products from various expression systems, productspecific approaches still prevail. however, for most product types similar issues like clearance of process-and product-related impurities, overall purity and yield, or manufacturing issues have to be dealt with in downstream process development. integrated and timely approaches based on process science and developed orthogonal analytical tools are often hampered by tight time frames and limited resources available. on the other hand, thorough understanding and analytical characterization of product characteristics but also of (process-related) impurities are pre-requisites for fully exploiting separation power and for achieving final purities way above 95% in a robust and cost-effective manner. from primary separation to polishing steps, we have made several attempts to improve the efficiencies of process steps themselves but also of ways to develop them. the strategies applied comprise implementation of innovative processing tools, rational streamlining and optimization of a sequence of unit operations, tech transfer and scale up considerations. also in this context, the applicability of scale down and ultra scale down models for process development and optimization purposes, their potential for speeding up and their limitations will be discussed. chromatographic monoliths are rather new chromatographic stationary phases. they consist of a single piece of a highly porous material. the pores are interconnected forming a network of channels. since the transport mechanism is predominantly based on convection, mass transfer between mobile and stationary phase is significantly enhanced resulting in short separation times. because of that they seem to be an ideal support for separation and purification of extremely large molecules like proteins, dna or even viruses. in this talk, various features of the monoliths like high porosity, fast mass transfer, surface accessibility and dynamic binding capacity will be described. effect of each feature on the separation and purification efficiency will be discussed in terms of molecular size and properties. while chromatographic monoliths are already widely accepted in microchip fluid devices, capillary columns as well as analytical columns, very few reports about preparative monolithic columns can be found. reasons for lack of preparative chromatographic columns will be elucidated and preparation strategy for construction of several liter volume methacrylate based monoliths will be presented. finally, several examples of biomolecule purification like large proteins, plasmid and genomic dna and viruses on cim convective interaction media ® monolithic columns will be given. further, their application as bioreactors and supports for solid state synthesis will be demonstrated. hubbuch institute of biotechnology, forschungszentrum juelich, 52425 juelich, germany. e-mail: m.schroeder@fz-juelich. de (m. schroeder) the intraparticle diffusion coefficient is an important parameter for modeling of chromatographic separation processes. a new method based on dynamic measurements of intraparticle concentration profiles of proteins in process chromatographic media with a confocal laser scanning microscope is presented. the diffusion coefficient is determined by fitting experimental data to a spherical diffusion model. excellent agreement of experimental data with simulation results is obtained. the diffusion coefficient is measured for seven proteins in sepharose 6 ff, spanning molecular weights from 14.3 to 160 kda. in addition, multicomponent diffusion processes for combination of differently sized proteins are analyzed and the influence of adsorbed proteins on the diffusion coefficient is measured in sp or q sepharose ff. taken together the presented method allows measuring the diffusion coefficient of proteins in process chromatographic media in a packed column. use of automated docking for predicting chromatographic behavior of proteins in hydrophobic interaction chromatography andrea mahn, m. elena. lienqueo contreras university of chile, santiago, chile in the present work, we have extended and automated the methodology proposed by mahn et al., 2005 for predicting protein behavior in hydrophobic interaction chromatography (hic). this methodology is based on the good correlation level between the average surface hydrophobicity of the interfacial zone (local hydrophobicity lh) and protein retention time in hic, for only three different ribonucleases. for determining the lh it is necessary to select the most probable protein-ligand conformation. in this work, we have determined the most probable conformation, of more than 12 proteins, using (i) first, the module insight ii affinity by accelrys for providing automated docking (grid method) of ligands (phenyl) to the proteins (100 conformations); (ii) then, the different probable docked protein-ligand conformations were automatically scored using the module insight ii ludi by accelrys; (iii) after that, each conformation was clustered and each cluster was scored by using the average score of each cluster (iv) finally, the most probable conformation was selected using a function based on the number of cluster components, and the average score value. then, when the most probable conformation was selected, the local hydrophobicity (lh) was calculated using the graphical representation and analysis of structural properties (grasp) program. the results have shown an acceptable correlation level (r > 0.90) between lh and the experimental dimensionless retention time (drt). in view of these results, we consider that this methodology could be used to adequately represent the chromatographic behavior in hic for all kinds of proteins (with a heterogeneous and homogeneous surface hydrophobicity distribution) and without a large number of tedious experiments, but only using computational simulation and adequate score criterion. potato tuber proteins are nutritious and show potential as functional ingredient in food systems. however, the present bulk processing technology can only recover byproduct protein for animal feed use. an expanded bed adsorption (eba) process for isolating functional food-grade protein from crude potato starch effluent was previously developed. moderate capture efficiency (20-25%) of the total crude protein was most likely caused by diffusion limitations and aggregated protein, inaccessible for adsorption. we employed the same adsorption ligand attached to agarose-tungsten carbide beads to create stable beds of 2.0-2.7× expansion using flow rates at 400-750 cm h −1 . a pilot scale eba process was run in a commercial processing plant over a three month campaign of starch production from potatoes of mixed variety. fresh crude effluent (150-300 l/cycle) was applied to a column (20 cm × 2 m) containing 20 l of resin. protein capture by eba was reliable in operation, producing a refined protein material, which after dewatering and gentle drying, showed improved functionality over heat-coagulated protein produced at the same plant. overall productivity increased. however, finding a robust operating window of predictable productivity is challenging since the potato fruit water is complex and deteriorates easily. from breakthrough curves, it is observed that the major bulk protein, patatin, displays non-langmuir adsorption behavior. this may indicate a range of interactions for different species of the same protein. chlorogenic acid (ca), the main polyphenolic substance in potato tuber, causes enzymatic browning and undesirable flavor changes, but polyphenols can also react with protein. assessing the effects of interacting cell components therefore applies to the bioprocessing of plant material. at present the acceptance of biochip technology for on site use, e.g. diagnosis or environmental control is hindered by rather expensive and complex instrumental systems. there is a need to provide reliable and cost-effective systems that can be operated with minimal training. the construction of electronic biochip microarrays using semiconductor technology enables the construction of compact systems with high integration at acceptable production costs. the key feature of the fully electrical biochip technology are micro arrays made in advanced si-technology and carrying several array positions with interdigitated nanometer gold electrodes on its surface. the chips are fabricated by standard silicon fabrication methods allow-ing high volume production and to minimise the cost per chip. the advantage of fully electronic microarrays is the intrinsic high spatial resolution and direct signal coupling of the biosensing element and the transducer. the function of fully electronic biochips is also based on the electrochemical transduction and quantification of the formation of affinity complexes on the chip surface. a portable device for field applications and point of care diagnosis have been designed and manufactured. the amperometric device enables the recognition of biomolecular interactions by measuring the redox recycling products of elisa enzyme labels. the highly sensitive signal transduction is achieved with a 16-channel interdigitated ultramicroelectrode array. one major advantage of fully electronic microarrays is the direct signal coupling of the biosensing element and the resulting robustness and opportunity for miniaturisation. those electrical biochip arrays have been adapted for the detection of all types of affinity complexes, such as for dna, rna, proteins and haptens. self assembling of capture oligonucleotides via thiol-gold coupling have been used to construct the array chip nucleic acid interface. thus e.g. pathogenic micro organisms have been identified and quantified via their genomic dna or ribosomal rna respectively. another application based on immobilized antibodies is shown to sense extreme low concentration of bioagent toxins. for processing the assay formats and the electrical read out of the detection of affinity complexes a modular fully automated measurement system has been developed. it is manufactured in industrial lines and available at market. dynamics and self-assembly of organic molecules on surfaces revealed by high-resolution, fast-scanning stm flemming besenbacher interdisciplinary nanoscience center (inano), university of aarhus, dk-8000 aarhus c, denmark. e-mail: fbe@inano.dk in the interdisciplinary area of nanoscience and nanotechnology, the adsorption and self-assembly of organic molecules on singlecrystal surfaces have attracted much attention lately due to the potential applications in fields ranging from molecular electronics to biocompatible interfaces. the supramolecular structures formed upon deposition of molecular species on solid surfaces depend on the molecular architecture and the distribution of functional groups on one hand, which determines the thermodynamically stable molecular arrangement, and on the other hand, on kinetic factors like thermal diffusion, spontaneous rotations and conformational dynamics. i will show how the unique aspect of our aarhus stm and the time-resolved, high-resolution stm imaging can be used to obtain important new insight into the dynamics, and can provide very important new information on the atomic-scale realm and on the dynamics of molecular nanostructures. the time-resolved stm data are visualized in the form of stm movies (see www.inano.dk/spm) which can subsequently be analyzed in order to extract quantitative information on the activation energy, the prefactors and the adsorbate-promoted diffusion. i will specifically discuss: (i) the self-assembly of guanine quartets on au(1 1 1) and the influence of cooperative hydrogen bonds, and (ii) the molecular recognition in binary mixtures of dna bases. g molecules are found to self-assemble into a hydrogen-bonded network of g-quartets, whose structure corresponds perfectly with the quartet structure of telomeric dna determined by x-ray crystallography. the strong preference of g molecules to form quartets can be explained by a cooperative effect that strengthens the hydrogen bonds within the g-quartet network over the hydrogen bonds in isolated dimers. by means of a combination of stm experiments and dft calculations we compare the 2d molecular networks formed on deposition of the binary mixtures g-c (purine-pyrimidine pair of complementary bases) and a-c (purine-pyrimidine pair of non-complementary bases). we find that the non-complementary bases segregate into islands of pure a and a network of pure c, whereas the complementary bases g and c form a network that cannot be separated by annealing up to the desorption temperature for c. high-resolution stm images allow us to identify the structures for the enhanced thermal stability as structures that contain g-c bonds, possibly with the same structure as the watson-crick pairs in dna molecules. kühnle, r., et al., 2002 . nature 415, 891. otero, r., et al., 2004 . angewandte chemie int. ed. 43, 2092 . otero, r., et al., 2004 . nat. mater. 3, 779. otero, r., et al., 2005 . angewandte chemie int. ed. 44, 2. rosei, f., et al., 2002 . science 296, 328. schunack, m., et al., 2002 . biomedical and pharmaceutical companies are using an increasing number of carbohydrate polymers in the formulation of drugs. one such polymer with highly attractive features is chitosan that can be produced from crustacean shells. chitosan is non-toxic, biocompatible and biodegradable. chitosan can be formulated as nanoparticles or membranes and have enhanced several bioprocesses. among the well-documented features are enhanced drug uptake by tight junction relaxation and enhanced in vivo uptake and protection of nucleic acid formulations. chitosan research has increased throughout this decade. research programs are addressing the potential of chitosan applications but preparations with variable molecular size and charge are not easily available. specifically companies working with the development of new drugs and enhancement of drug functionality are in need of formulation technology that provides well characterized biocompatible material. in the chitosan innovation consortium the danish companies, coloplast, novozymes biopolymers, pipeline biotech, and zgene, together with the research center inano (aarhus university) and bioneer a/s have developed a series of chitosan preparations suitable for research of biopharmaceutical applications (www.chitosan.dk). the ability to obtain functional formulations is currently being tested in both in vitro and in vivo experiments. the consortium participants have established a platform from which chitosan processing, characterization and formulation technology can be extracted. by providing specified chitosan preparations the polymer feature can be adjusted to fit specialized biopharmaceutical applications. high throughput bioprocessing govind rao center for advanced sensor technology, umbc, baltimore, md 21250, usa the post genome era holds a great deal of promise. an enormous number of new proteins await study. these will require sophisticated culture techniques, as cells will have to be grown under large numbers of environmental conditions to elucidate expression triggers. unfortunately, unlike molecular biology, bioreactor technology is little changed since its inception. the primary reason has been a lack of sensor technology that can be readily employed to monitor the cellular environment. we will take a look at the current status of the technology and report on promising optical sensor technology that permits low-cost high throughput cell culture and fermentation. noninvasive sensors that monitor ph, po 2 and pco 2 and high sensitivity solutions for glucose and glutamine measurements will be presented. in addition, the mixing characteristics that determine bioreactor performance will be examined at the small scale and their relevance to the large scale will be demonstrated. on-line monitoring and fed-batch operation in shake flask and micro titre plate cultures jochen büchs 1 , frank kensy 1 , markus jeude 1 , tibor anderlei 2 , barbara dittrich 3 , doris klee 3 : 1 biochemical engineering, rwth aachen university, aachen, germany; 2 ac biotec, jülich, germany; 3 textile chemistry and macromolecular chemistry, rwth aachen university, aachen, germany although methods of molecular biology has led to rational design of micro-organisms to suit our requirements, screening of large numbers of strains and media is still one of the most important tasks in biotechnology. batch operation of shaken bioreactors and absence of on-line monitoring is still the general state of the art for that purpose. it is also a very common practice in screening projects that only the final product titre is measured at the end of the culture for the evaluation of the "best performers". in the recent years several approaches were introduced to follow microbial cultures also in shaken bioreactors like shake flasks or micro titre plates (mtp's). it became obvious that the cultures can behave quite unexpected and most relevant and essential information is lost, if only the final product titre at the end of the cultures is utilised for evaluation. as a result, the screening may be directed to an unknown and non-intended direction or may even fail. this is demonstrated with some examples in this contribution. new methods and techniques are introduced to measure the oxygen and carbon dioxide transfer rate and the respiratory quotient in shake flasks and the optical density, nadh fluorescence, ph and do 2 in mtp's. if the desired product can be fused to a fluorescence protein, like gfp or yfp, also the product formation can be monitored on-line in mtp's. it is of utmost importance that the operating conditions of the applied shaking bioreactors are suitable and shaking motion is not stopped during the measurement. otherwise, e.g. power input, mixing and oxygen supply is interrupted and the micro-organisms will ongoingly rearrange their metabolism to cope with these disturbances of their environmental conditions. another problem of screening is the commonly applied batch operation mode. a lot of microbial systems display an overflow metabolism, substrate or osmotic inhibition or are characterised by a catabolite repressed product formation. in all these cases, batch operation is not the preferred operation mode and, therefore, these cultures are run in fed-batch in larger scales. in particular, it is nearly impossible to screen systems, which are catabolite repressed by the carbon source, in batch mode in defined mineral media. after initial growth has led to a nearly complete consumption of the carbon source, the product formation is derepressed. but then no more carbon source is available to continue with production. it is quite questionable whether in batch operation mode suitable strains can be selected for later fed-batch operation or not. this consideration has resulted in the development of a new technique which allows to run the screening in fed-batch operation mode. this technique is applicable in shake flasks as well as in mtp's. dramatic increases in product titre between 4-and 400-folds were observed under these conditions compared to conventional batch screenings. mikkel nordkvist, john villadsen center for microbial biotechnology, technical university of denmark, dk-2800 lyngby. e-mail: mnq@biocentrum.dtu.dk (m. nordkvist) efficient mixing and mass transfer are highly important in the chemical industry and in the fermentation industry. poor mixing can result in low yield and variable product quality in a number of cultivation processes, and mass transfer can easily become the limiting step in aerobic cultivations, especially at high cell density. we have tested a new tank reactor system, where liquid is withdrawn from the bottom of a tank, rapidly circulated, and injected back into the bulk liquid through the nozzles of rotary jet heads. liquid feed as well as gas is added in the recirculation loop and thereby distributed via the rotary jet heads. solid feed in powder form can also be added in the loop with advantage, and heat is efficiently removed in a plate-type heat exchanger, which is part of the loop. the system has a very simple design with no internal baffles or heat exchange area, and between batches the rotary jet heads are used for cleaning in place. a number of applications ranging from dispersion of liquid and powder to mass transfer will be presented. mass transfer applications include baker's yeast cultivation and oxidation of lactose to lactobionic acid by a carbohydrate oxidase. fast and accurate analytical information that can be used to rapidly evaluate the interactions between biological systems and bioprocess operations is essential for optimization of biological production processes. we have researched and developed a multiplexed microbioreactor system for the parallel operation of multiple microbial fermentations. the microbioreactors have working volumes from 5 to 150 l, and are instrumented for real-time monitoring of dissolved oxygen, ph and optical density. the growth profiles obtained with escherichia coli compare favorably to results obtained from conventional 500 ml batch bioreactors. we also demonstrate the use of our microbioreactors coupled to dna microarray analy-sis, as a tool for accelerated discovery and elucidation of metabolic pathways and gene expression profiles. the multiplexed system represents a significant step towards high-throughput data acquisition and has the potential to replace current instrumented bioreactors, which are bulky, expensive to run, and require many mechanical manipulations. design of a laboratory scale bioreactor to study solid-state tobacco fermentation m. di giacomo, l. nappi, d. silvestro, m. paolino, d. parente r&d biology department, british american tobacco italia, naples 80126, italy italian toscano cigar production is based on the fermentation of dark fire-cured tobacco. the process starts with the rise of leaf moisture to levels of water activities assuring development of the wild phylloplane microflora in the absence of free water. the intense growth of microorganisms modifies leaf characteristics (ph rise from acidic to alkaline condition) contributing to define the toscano typical smoke profile. tobacco fermentation takes place in great bulks of 500 kg which cause considerable amount of heat evolution as a function of the metabolic activities of the microorganisms. this heat accumulates and temperature can rise to as high as 70 • c. a laboratory cylindrical packed-bed bioreactor was designed to work under isothermal conditions. the reactor was ideal to ferment small quantities of tobacco (200 g) and was made up of a column aerated from the bottom with humidified air and placed in a thermoregulated room. experiments were conducted with constant temperature and air flow. moreover, bioenrichment experiments were conducted in the presence of different microbial starter cultures. fermentation courses were monitored by measuring microbial counts and chemical/physical modification of the substrate. with this laboratory scale system we obtained different kinds of information on the role and dynamics of the microorganisms involved in the fermentation process and on the influence of different environmental conditions. for the future, the design of an adiabatic device for tobacco fermentation is planned. on-line liquid chromatography as a process analytical technology for monitoring and control of biotech processes rick e. cooley 1,2 : 1 process analytics center of excellence, dionex corporation, sunnyvale, ca, usa; 2 eli lilly and company, indianapolis, in, usa (retired) biotech processes, used to produce an active pharmaceutical ingredient (api), generally differ from small molecule api manufacturing processes in that the starting materials tend to be more variable, more complex, and the product of interest in lower concentration due to the fact that they originate from a biological rather than a chemical process. this complexity and low starting concentration has generated a high level of interest in developing technologies that can be utilized to increase yield and reduce variability in the initial bioreactor phase, as well as, downstream isolation and purification operations. the use of process analytics, or on-line analytical measurements, is a technology approach that can contribute to increased process understanding and control. this presentation will provide examples of how various analytical measurement technologies have been utilized to monitor typical bioprocess unit operations leading to increased automation and control. examples of the use on-line liquid chromatography to monitor bioreactors, process scale chromatography columns, and enzymatic reactions will be presented in more detail. application of advanced monitoring strategies for recombinant protein production karl bayer department of biotechnology, university of natural resources and applied life sciences, vienna a-1190, austria high yield in combination with the required quality are the key objectives of large-scale production of recombinant proteins using different host/vector systems. however, in the past the efficient production of recombinant proteins was frequently limited due to inadequate exploitation of the cell factory and deficiencies in process design. to achieve optimal exploitation of microbial cell factories the key requirement is to enhance the monitoring capabilities to improve the insight into the host metabolism dynamics and to cope with limited understanding of the interaction of recombinant protein synthesis with host cell metabolism. since each protein exerts an individual influence on the host/vector system, the selection of appropriate analytical methods is even more important. in order to overcome these problems high throughput technology platforms, such as dna microarrays for transcriptome and differential 2-d electrophoresis (dige) for proteome analysis provide extensive data to screen for significant analytes and provide an appropriate basis for in silico modelling and reverse engineering of regulatory networks. taking advantage from such an iterative process experimental design will be improved and further aid to increase the performance of modelling. in addition, transcriptome data are used to screen fast stress responsive promoters to set up gfp based reporter gene fusions for in-situ monitoring of the metabolic load due to recombinant gene expression. moreover chemometric methods are frequently applied to model complex data from easily obtainable on-line data sets to overcome the limited monitoring capabilities due to the high complexity and nonlinearity of biological reactions and reaction networks. this strategy has been successfully applied to model bdm (bacterial dry matter), pcn (plasmid copy number) and the amount of recombinant protein using data sets acquired from off-gas analysis (o 2 consumption, co 2 evolution), alkaline consumption rate, in-situ capacitance and multi-wavelength fluorescence measurements. at-line monitoring of bioprocess-relevant marker genes using an electric dna-chip britta jürgen 1 , daniel pioch 1 , le thi hoi 1 , jörg albers 2 , rainer hintsche 2 , stefan evers 3 , karl-heinz-maurer 2 , michael hecker 4 , thomas schweder 1 : 1 institut für pharmazie, ernst-moritz-arndt-universität, 17487 greifswald, germany; 2 ebiochipsystems, 25524 itzehoe, germany; 3 vtb-enzymtechnologie, henkel kgaa, 40191 düsseldorf, germany; 4 institut für mikrobiologie, ernst-moritz-arndt-universität, 17487 greifswald, germany. e-mail: britta.juergen@uni-greifswald.de (b. jürgen) the gram-positive bacteria bacillus licheniformis and bacillus subtilis represent important industrial hosts for the production of enzymes (e.g., proteases and amylases) or antibiotics (e.g., bacitracin). both organisms are attractive for this purpose because of their apathogenity and their classification as gras organisms (generally regarded as save). moreover, their easy cultivation and their high natural capacity to secrete proteins into the growth medium qualify them for the industrial overproduction of homologous or heterologous proteins (simonen and palva, 1993) . for the control of the physiological state and the productivity of the production cells efficient analysis tools are of great interest. a prerequisite for the evaluation of the physiological state of cells during industrial fermentation processes is the analysis of so-called process-relevant marker genes, the expression of which indicates unfavorable growth and production conditions (schweder and hecker, 2004) . by means of proteome and transcriptome analyses we have identified critical process-relevant genes of b. licheniformis and b. subtilis cells under different nutrient limitation conditions and during industrialclose bioprocesses. dna-chips with probes for such process-relevant marker genes could be valuable diagnostic tools for the monitoring of the cellular physiology during microbial bioprocesses. in order to provide reliable tools for the monitoring of the cell physiology during microbial bioprocesses, we have developed a fast mrna analytical approach, which allows an at-line monitoring of the transcriptional activity of selected marker genes during bioprocesses. this approach is based on an easy, fast and reliable rna isolation procedure and the measurement of specific mrnas by means of an electric dna-chip barken et al., 2004 a robust bioprocess is crucial to ensure the consistent process performance and provide the high quality of product for drug manufacturing in biopharmaceutical industries. existing methodologies for bioprocess design do not involve establishing mechanisms to achieve the desirable robust bioprocesses and have low capacity in handling uncertainty in the product manufacturing. also, the solutions are often obtained step wise and do not account for interactions between the steps. despite its importance, the robustness of a bioprocess has not been properly defined and studies carried out in statistic sense are often retrospective. in addition, the computational cost is expensive due to using a line search algorithm for finding an optimal operating solution. finally, the existing methodologies are difficult to apply to the whole bioprocess in biopharmaceutical industries. this paper attempts to define rigorously a measure for process robustness and presents a new methodology for evaluating the robustness of bioprocess operations and their performance. the methodology is based on the concept of 'windows of operation' which shows the whole range of possible operating regions. the methodology also establishes a lower bound for the largest variation of a design variable to ensure the performance. these bounds are achieved by min-max optimization techniques. a direct search algorithm has been developed and its computational cost is much lower than the line search algorithm. results include visualization of robust operating regions and a set of indices which compare the performance of different operating strategies. the capabilities and efficiency of this methodology are illustrated by applying it to the centrifuge selection for the clarification of high solids density cell broths. the research work will impact considerably upon robust bioprocess operation. when grown on methanol, pichia pastoris is able to synthesize proteins to high titres as well as secreting and glycosylation, thereby making this organism a very interesting host for the production of recombinant drugs at large scale. the methanol residual concentration has been reported to strongly influence the specific productivity, the optimum concentration being around 3 g/l. a suitable monitoring and control technique is therefore necessary to study and improve the productivity of p. pastoris fermentations. the current research aims at showing how a mid-infrared spectrometer (atr-ftir) can be calibrated in-situ in order to monitor and control p. pastoris fermentations. this method is simple and fast, and eliminates the need of both standards preparation and off-line calibration. it is based on the observation that during fed-batch processes, only substrate and biomass concentrations vary significantly. the method therefore consists in adding a known amount of methanol at the beginning of the process, just after inoculation, and subsequently calibrating the instrument. financial support for the swiss national science foundation is gratefully acknowledged. implantable biomaterials are subjected to inflammatory responses mediated by adherent phagocytes such as monocytes and macrophages. these cellular responses and behavior have been shown to be dependent on the type of protein that adsorbs to the surface. surface modification is necessary to control and prevent protein adsorption, and thus modulate the inflammatory responses. hydrophilic surfaces that adsorb minimal amounts of protein are considered useful for minimizing the inflammatory reactions to biomaterials. in this study we have used two routes to modify polyethylene terephthalate (pet) films: (1) a wet-chemical method for attachment of linear polyethylene glycol chains (mpeg); and (2) gas-phase plasma polymerisation of diethylene glycol vinyl ether (degve) to generate peg-like surfaces. the surface chemistry was assessed by x-ray photoelctron spectroscopy (xps), fourier transform infrared spectroscopy (ftir) and time-flight secondary ion mass spectrometry (tof-sims). the two pegylated surfaces were compared for their ability to minimise both fibrinogen adsorption and the adhesion and activation of macrophage-like human leukocytes. adsorbed fibrinogen has been shown to be one of the key proteins in stimulating inflammatory responses to biomaterials. adsorption was investigated quantitatively using 125 i-radiolabeled human fibrinogen. in addition, the conformation of the adsorbed protein was tested using an antifibrinogen monoclonal antibody in an enzyme-linked immunosorbent assay. the results showed that pegylated surfaces adsorbed up to 90% less fibrinogen, and that unfolding of adsorbed fibrinogen was more pronounced on the linear mpeg layers than on the peg-like plasma polymer surfaces. adhesion of in-vitro differentiated macrophage-like u937 cells was reduced on both the peg-like plasma polymer surfaces and the linear mpeg layers compared to the unmodified pet surface, but cells adhering to the peg-like plasma polymer surfaces secreted less tumor necrosis factor-␣ (tnf-␣) than cells adhering to the linear mpeg layers. thus, the linear mpeg surface is relatively efficient at reducing adhesion of macrophage-like cells, but those cells that do attach are in a more activated and proinflammatory state. analysis of ceramides from biological samples m. budvytiene 1 , j. liesiene 1 , b. niemeyer 2 , n. ceramides in human skin play an important role in the regulation of cell growth, differentiation and apoptosis. moreover, they are involved in the numerous signaling pathways. the growing interest in the investigations of ceramides physiological functions requires efficient separation methods of ceramides from biological resources and sensitive analytical methods. in this work some sensitive and selective methods, involving thin layer chromatography (tlc), high performance liquid chromatography (hplc), mass spectrometry (ms) and nuclear magnetic resonance spectroscopy (nmr) were employed for the separation and characterization of ceramides from human foreskin. epidermal lipids were extracted from human foreskin for 24 h at room temperature using three solvent mixtures (chloroform/ethanol/water 1:2:0.5, v/v/v); (chloroform/ethanol 1:1, v/v), and (chloroform/ethanol 2:1, v/v). ceramides retention char-acteristics in tlc and hplc were compared with the retention of commercial standards. the best separation was obtained using normal phase column packed with hilic silica 3 m. the elution was performed using mixture of chloroform and ethanol 50/50 (v/v) as an eluent. two commercial standards n-stearoyl-sphingosine cer(ns), r f = 0.51, and n-palmitoyl-sphingosine cer(np), r f = 0.50, were selectively separated with hplc system in above mentioned conditions. the retention time of cer(np) and cer(ns) was 4.31 and 5.19 min, respectively. the same lipids were detected by hplc in the human foreskin extracts. the structure of the lipids from collected fractions was confirmed by means mass spectrometry and nmr. the physiological functions of the separated ceramides are investigated. production recombinant human thymosin-␣ 1 overexpressed as intein fusion protein in e. coli roman s. esipov, vasily n. stepanenko, anatoly i. miroshnikov, shemyakin-ovchinnikov institute of bioorganic chemistry, russian academy of sciences, ul. miklukho-maklaya 16/10, moscow, 117997 russia. e-mail: esipov@ibch.ru (roman s. esipov) medicines based on polypeptides consisting of 30 and more amino acid residues are widely spread in pharmaceutical market at the present time. practically all polypeptide medicines known are prepared by general chemical synthesis that caused high cost of their production. that's why biotechnological way of polypeptide medicines preparation using recombinant gene expression in bacteria seems to be promising. during our work we design hybrid construction allowing solving a problem of specific cleavage of target polypeptide from the hybrid protein using system of protein splicing from new england biolabs. in case of thymosin-␣ 1 production system intein mediated purification with affinity chitin (impac system)-binding tag has been used, where the modified sce vma from s. cerevisiae has been applied as intein. in contrast to other systems, impact allows the preparation of target protein without using of serine protease and other factors that may cleave the hybrid protein. in the presence of thiol reagents, such as dithiothreitol, mercaptoethanol, or cystein the hybrid protein can be site-specifically cleaved to give intein, the target protein and small fragment of nextein. using of this system does not allow to obtain the target protein with ser, cys or thr on n-terminal of protein, because in those cases target protein and n-extein ligation product will be formed. ser is n-terminal amino-acid in thymosin-␣ 1 . it was recently found that some metal ions essentially affect the splicing. we tried to use zncl 2 and we have found that, in case of intein-thymosin-␣ 1 , the maximal yield of target polypeptide and the minimal yield of splicing products are observed in the absence of dithithreitol and in the presence of 0.5-1 mm zinc chloride in buffers on all stages of thymosin ␣ 1 isolation. the structure of recombinant thymosin-␣ 1 of human was confirmed by the determination of n-and c-terminal amino-acid sequences and by maldi tof mass-spectrometry. we present a microbioreactor with thermoelectric cooling to inactivate cellular metabolism by cell culture freezing. small-scale cultivation methods have gained increased importance and their development has been supported by advances in bioprocess monitoring methods. yet efficient sampling methods for off-line analysis remain important where in-situ real-time measurements are difficult, for example for intracellular metabolite concentration or enzyme activity measurements, and for all methods which are invasive by nature, such as protein purification. freeze-stop measurements of metabolite levels are ideal, because they are inert, i.e. do not require the addition of a chemical. in large systems, the chilling time is often the limiting factor, and alternative methods for cell metabolism inactivation are required, such as the spraying of the cell suspension in 60% methanol at a temperature of −40 • c, or the use of boiling buffered ethanol. due to the small thermal mass of microsystems, shorter chilling times can be expected. sample cooling to 4 • c in a microbioreactor has been presented previously. in this contribution, we investigate sample freezing to completely inactivate the cell metabolism from microbioreactor working volumes of approximately 100 l. the use of multi-parameter flow cytometry for characterisation and monitoring of insect cell-baculovirus fermentations in a mechanically-agitated bioreactor bojan isailovic 1 , alvin w. nienow 1 , ian w. taylor 2 , ryan hicks 2 , christopher j. bacteria and mammalian cells have been traditionally used as hosts for commercial recombinant protein production. however, in recent years, the insect cell-baculovirus system has emerged as a potentially attractive recombinant protein expression vehicle. although flow cytometry has been used widely for analysis of mammalian and microbial cells, there is very little information on applications of this powerful technique in insect cell culture. here we compared cell ratiometric counts and viability (propidium iodide and calcein am) of sf-21 cell cultures using flow cytometry to those determined by more traditional methods using a haemocytometer and the trypan-blue exclusion dye. flow cytometry has also been used to monitor various parameters during cultures of sf21 infected with the recombinant autographa californica nuclear polyhedrosis virus (acnpv) containing the inserted nucleic acid sequence amfp486 coding for am-cyan coral protein, which emits natural green fluorescence. the optimization of a fermentation process requires the organism to be cultivated under desirable conditions, which depends on how well the fermentation process is controlled. inadequate mixing and mass transfer are responsible for the heterogeneous environment at large scale in terms of nutrient concentration and ph profile, resulting in lower product yields. these have been associated with, inadequate control of ph and the production of acetate or formate in response to over-feeding of glucose and oxygen deficiency. rapid analyses of substrates and indicator metabolites in a fermentation process is critical for optimal control. this can be achieved in real time with nir spectroscopy. in this study, nir-spectroscopy has been applied to monitor the concentrations of glucose, acetate, formate, ammonium hydroxide and biomass in the cultivation of e. coli (w3110). a comparison of partial least square models built using water standards, synthetic medium standards and fermentation samples has been made. the control of the brewing process is important for improving product quality, and lowering costs. infrared spectroscopy is a technique that can be used at-line and on-line to rapidly measure component concentrations of unprocessed whole broth samples in real time. in this study, both mid -infrared (mir) and near-infrared (nir) spectroscopy have been used and compared for the monitoring of ethanol, flavour components, wort sugars, biomass and specific gravity during the brewing. partial least-squares regression (pls) was used to model relationships between component concentrations and spectra. the performance of these models was evaluated in terms of the standard error of prediction (sep), number of pls factors and the correlation coefficient (r). calibration models were constructed using spectra acquired for multi-component mixtures, intended to simulate brewing fermentation conditions, and actual brewing fermentation samples. chemometric results indicated that nir is a powerful tool for accurately measuring sugar, ethanol and biomass concentrations. when choosing an expression host for production of a specific recombinant protein, one can essentially select from a multiplicity of different systems. while e. coli bacterium is usually the starting point for any cloning and expression effort, there is no universal expression host that would work optimally for all proteins. practical issues to consider include e.g. need for post-translational modifications and protease activity of the host. we have produced recombinant hiv-1 nef in different host cell systems: e. coli, p. pastoris yeast and stable drosophila s2 insect cells. using strain/cell line development, production and purification data from practical experiments, we were able to conduct a techno-economical comparison of the different host cell systems. the annual production goal was set at 100 mg of high-purity nef. this was supposed to be produced campaign-wise in 1-2 batches using laboratory-scale bioreactors and other equipment. in this study, it was shown that although the production costs of the different systems were in the same range, the production in e. coli was most inexpensive, and the s2 cell system was the most expensive. regardless of the selected host system, the labour costs incurred the most expenses. when comparing different stages of the work (strain/cell line development, bioreactor production and down-stream processing), the strain development, the most man-hours demanding stage, involved approximately half of the costs of every production system. although e. coli was the most inexpensive host system for producing nef, it has some definite disadvantages: e.g. the production of endotoxins and the disability to perform post-translational modifications. if these disadvantages are of importance, the production must be done using more expensive system. modelling and control of industrial fermentation j.k. rasmussen, s.b. jørgensen capec, department of chemical engineering, technical university of denmark, dk-2800 lyngby, denmark. e-mail: jkr@kt.dtu.dk (j.k. rasmussen) fed-batch processes play a very important role in chemical and biochemical industry. fermentations in biochemical industry are most often carried out as fed-batch processes. present control schemes do not utilize the full potential of the production facilities and may fail to achieve uniform product quality and optimal productivity. the introduction of model based control strategies is considered difficult because suitable models are not readily available. first principle engineering models can be used but the usually limited knowledge of the regulatory network in the micro-organism makes model development very time consuming. another strategy is to use a purely data-driven approach where only limited prior knowledge of the process is required. a framework for generation of such blackbox models is used in this project. this framework is called "grid of linear models" (golm), it uses a large number of linear models which each describes the behaviour of the process within a certain time interval. the combination of these models results in a model which covers the entire time span of the fermentation and approximates the nonlinear time varying behaviour. a procedure for deriving golm models from operational data has been developed and because they consist of a large set of linear models it makes them suitable for model predictive control implementation with iterative learning capabilities from batch to batch. iterative learning model predictive control (ilmpc) based on a golm model is being implemented on a fermentor at novozymes a/s. the results will be evaluated in terms of the controller's capability to ensure uniform product quality and reject both intra and inter batch process disturbances. the model based approach renders optimization of the process recipe possible by using the ilmpc capability. this opportunity provides a great potential for increase of productivity and reduction of cost. j.m. viader-salvadó, j.a. fuentes-garibay, l.j. galán-wong, m. guerrero-olazarán institute of biotechnology, biological science school, autonomous university of nuevo león, san nicolás de los garza, n.l., méxico, the production of recombinant trypsin and trypsinogen has been reported as difficult due to a probably toxicity on host or its instability. in an effort to attain high-level production of shrimp trypsin for aquaculture applications, we have evaluated shrimp trypsinogen production by recombinant pichia pastoris strains in 5-l bioreactors. a p. pastoris gs115 mut+ containing in its genome the litopenaeus vannamei trypsinogen cdna fused in frame to saccharomyces cerevisiae ␣-factor secretion signal, previously constructed in our laboratory, was used. four three-step fermentations (glycerol batch, glycerol and methanol fed-batch) were carried out. the glycerol batch step (2 l of basal salts medium, 50 g/l glycerol, 8.8 ml biotin 0.02%, 8.8 ml ptm1, 250 l 289 antifoam, ph 5 adjusted to with 28% nh 4 oh) was carried out until glycerol was completely exhausted (21 h). the glycerol fed-batch step was carried out feeding with 50% glycerol (12 ml/l biotin 0.02% and ptm1) at 0.8 ml/min by 9 h and 45 min of posterior starvation. the methanol fed-batch step was carried out feeding with 100% methanol (12 ml/l biotin 0.02% and ptm1) by 133 h using a methanol concentration on/off feedback control to maintain constant the methanol concentration in the culture medium to 1 g/l. in all the fermentations the air flow rate and the agitation were set at 5 l/min and 800-1000 rpm, respectively. with the four fermentation assays, the influence of the ph and temperature in the production phase to the recombinant shrimp trypsinogen production were evaluated. in the four fermentations, at the end of the second step a biomass of 250 g/l wet weight were obtained (od600 230). the methanol demand in the four fermentations surprisingly was not increasing rather initially it was 0.37 ml/min, after 32 h decreased 2.5 times for 29 h, increased to 0.49 ml/min for 23 h and afterwards it was decreased manually to a constant value of 0.3 ml/min for that the dissolved oxygen will not decrease to values less than 20% (last 48 h). the total protein amount in the culture medium supernatant increased during the production step until values of 1.6 g/l (assay at ph 6), 6.5 times more than the worst assay (ph 3) recombinant shrimp trypsinogen production was confirmed by sds-page (about 500 mg/l) and trypsin enzymatic activity was detected using bapna as substrate after trypsinogen activation with shrimp hepatopancreas extract. large conformational change on giant dna induced by ascorbic acid: a nobel scheme on its antioxidative activity yuko yoshikawa, emi sakai, yoshiko oda department of food and nutrition, nagoya bunri college, nagoya 451-0077, japan ascorbic acid is often regarded as an antioxidant in vivo, where it protects against dna damage by scavenging reactive oxygen species. in the present, we will show another potent scenario on the protective effect of ascorbic acid through a significant structural change of giant dna. recently, we examined the effect of ascorbic acid on the higher order structure of dna through single molecular observation with fluorescence microscopy, and found that ascorbic acid generates a pearling structure in giant dna molecules, where elongated and compact parts coexist along a molecular chain. the results of observations with atomic force microscopy indicate that the compact parts assume a loosely packed conformation. as the extension, here we study the protective effect against double-strand breaks by reactive oxygen at different concentrations of ascorbic acid, in relation to the change of the higher order structure of giant dna. we have performed a real time observation on the doublestrand breaks on individual dna molecules by use of fluorescence microscopy. we have found that the double-strand break is markedly protected when ascorbic acid exists over millimolar concentrations. it is found that such a protective effect of ascorbic acid corresponds well to the above mentioned change on the higher order structure of dna. it has been reported that human circulating immune cells, such as neutrophils, monocytes and lymphocytes, accumulate ascorbic acid in millimolar concentrations. therefore, it is expected that the ascorbic acid concentration that induces the large conformational change on dna may be of physiological significance. , y., et al., 2004 . febs lett. 566, 39-42. yoshikawa, y., et al., 2003 . plant proteins are increasingly being used as an alternative to proteins from animal sources and substantially contribute to the human diet in several developing countries. there are many process both industrial and food based which employ protein hydrolysis and hydrolytic products have a wide variety of applications from industrial fermentation media to food additives. traditionally, proteins are hydrolysed by chemical means. acid hydrolysates of protein are used to produce food ingredients and flavour compounds. however, hydrolysis by chemical reagents produce potentially hazardous byproducts and these non-selective hydrolysis products cannot easily be defined. the use of enzymes allows for selective hydrolysis of protein and thus produces a potentially safer and more defined material. the present investigation describes the effects of substrate, enzyme and hydrolysate concentration on the hydrolysis of corn gluten. the corn gluten was hydrolysed by a commercial protease preparation neutrase. the protein hydrolysis reactions were carried out in 0.2 l of aqueous solutions at the temperature of 50 • c and ph 7 and were monitored by using ph-stat method. the degree of hydrolysis (%) and soluble protein concentration depending on time were investigated by using 1, 2, 4, 6, 8 and 10% (w/v) substrate concentrations; and 0.25, 0.4, 0.5, 0.6 and 0.75, 1% (v/v) enzyme concentrations; and 25, 50, 75 and 100% (v/v) this paper describes medium optimization for urease production by aspergillus niger ptcc 5011 by one-factor-at-a-time and orthogonal array design methods. the one-factor-at-a-time method was used to study the effects of carbon and nitrogen sources on urease production. among various carbon and nitrogen sources used, sucrose and yeast extract were the most suitable for urease production, respectively. subsequently, the concentration of sucrose, yeast extract and mineral sources were optimized using the orthogonal array method in two stages. the effects of nutritional components for urease production by a. niger ptcc 5011 in the first and second stages were in order of urea > niso4 > sucrose >kh 2 po 4 > k 2 hpo 4 > cacl 2 > yeast extract > mgso 4 and yeast extract > sucrose > k 2 hpo 4 > kh 2 po 4 > urea > cacl 2 > niso 4, respectively. the optimal concentrations of nutritional components for improved urease production were determined as 20g/l sucrose, 0.85 g/l urea, 3.4 g/l yeast extract, 0.03g/l niso 4 ·6h 2 o, 0.5 g/l mgso 4 ·7h 2 o, 0.04 g/l cacl 2 , 0.35 g/l kh 2 po 4 , and 0.35 g/l k 2 hpo 4 . these results showed that urea, niso 4, yeast extract and sucrose had significant effect on urease production by a. niger ptcc 5011. tween 80 and mgso 4 had negligible effect on urease production by this strain. the subsequent confirmation experiments determined the validity of the models. maximum urease activity in optimized media by onefactor-at-a-time and orthogonal array methods were about 1.14 and 2.74 times greater than with the basal medium, respectively. carbon sources create fingerprint fermentation characteristics pınar ç alık 2 , güzide ç alık 1 , tunçer h.özdamar 1 : 1 bre lab, department of chemical engineering, ankara university, 06100 ankara, turkey; 2 ib lab, department of chemical engineering, metu, 06531 ankara, turkey. e-mail: calik@eng.ankara.edu.tr (g. ç alık) this work reports on a systematic investigation of the interactions between the single-carbon sources, i.e. glucose and citric acid, and complex-medium components, i.e., carbon and nitrogen sources, in enzyme fermentation processes, i.e. serine alkaline protease (sap; ec 3.4.21.62),) and ␤-lactamase (ec 3.5.2.6), with the oxygentransfer and ph conditions to demonstrate the influences of carbon sources that create the fingerprint fermentation characteristics, moreover, their influences on the product and by-product formations and the intracellular reaction rates. the influence of the medium composition i.e. citric acid-, glucose-, molasses-and soybean-based media together with the oxygen transfer (ot)-and ph-conditions applied, on the product and by-product distributions and ot characteristics were investigated in batch bioreactors. for sap, in general, under uncontrolled-ph operations the variation of the medium ph in sap fermentation process has a tendency to increase in the sap production phase; however, depending on the carbon source used, its behaviour changes in the early stages of the fermentation as the consequences of the directed functioning of the intracellular bioreaction network. the loci of the dissolved oxygen (do) curves also strongly depend on the carbon source(s) utilised, in addition to the applied ot conditions. the complex media profiles are significantly different compared to the defined media as the ph and do profiles are interrelated owing to the bioreactor operation conditions affecting the metabolic reaction network. the highest volumetric oxygen uptake rates were obtained with soybean-based medium that was ca. three-fold higher than the values reported in citrate-based and glucose based media, and ca. 1.5-2-fold higher than the values reported in molasses-based medium. the significant changes, moreover, the drastic change observed with the use of soybean-based complex medium are due to the compositions of the fermentation media used, and its influence on the intracellular bioreaction network. thus, we conclude that the change in medium composition based on the carbon source changes the fermentation characteristics under the designed bioreactor operation conditions that appear as the fingerprints of the bioprocess. effects of oxygen transfer on ␣-amylase production by b. amyloliquefaciens nurhan güngör, güzide ç alık bre lab, department of chemical engineering, ankara university, 06100 ankara, turkey ␣-amylase (e.c. 3.2.1.1) a commercially important enzyme used in food, textile, detergent, brewing, paper, and animal feed industries; hydrolyses ␣-1,4 glucosydic bonds in amylose, amylopectin, and related polysaccharides. optimum medium composition and influence of bioreactor operation parameters on ␣-amylase production with high yield and selectivity were determined together with the metabolic flux distributions using b. amyloliquefaciens (nrrl b-14396) which is found to be a good producer of the enzyme. systematic investigation of oxygen transfer in relation with the metabolic fluxes for ␣-amylase is not available in the literature. shake-flask experiments were conducted in 0.5 dm 3 airfiltered bioreactors in orbital shakers with agitation and heating controls (b. braun, certomat-bs1). laboratory-scale bioreactors were composed of agitation, heating, foam, dissolved-oxygen and ph-controlled 1.0 and 3.5 dm 3 systems (b. braun, biostat q; and chemap). after separation of the cells with a sorval rc28s ultracentrifuge, ␣-amylase activity was measured by the dns method (bernfeld, 1955) . amino acid concentrations were determined with a hplc (waters), protein and organic acid concentrations were measured with a hpce (waters, quanta 4000e) (ç alık et al., 1998) . oxygen transfer characteristics in the bioreactor systems were calculated using the dynamic method (rainer, 1990) . in the mass flux balance-based analyses, a pseudo-steady state approximation for the intracellular metabolites and the accumulation rates of the extracellular metabolites measured throughout the fermentations in considerations of the biochemical feature of the system were used to acquire the flux distributions. in laboratory scale, the effects of different c sources i.e., glucose, fructose, maltose, lactose and soluble starch; n sources i.e., (nh 4 ) 2 hpo 4 , (nh 4 ) 2 so 4, and nh 4 cl and/or their concentrations; and the operation parameters, ph and temperature on cell growth, substrate utilization, ␣-amylase and byproduct concentrations, and ␣-amylase activity were investigated. in pilot scale, the fermentation and oxygen transfer characteristics of the bioreaction system together with the metabolic fluxes were determined. oxygen transfer regulates benzaldehyde lyase production in e. coli pınar ç alık iblab, department of chemical engineering, metu, 06531 ankara, turkey. e-mail: pcalik@metu.edu.tr the effects of oxygen transfer rate on benzaldehyde lyase (bal) production by puc18::bal carrying recombinant e. coli on a defined medium with 8.0 kg/m 3 glucose were investigated in order to finetune the bioreactor performance, in v = 3 dm 3 batch bioreactors at five different conditions with the parameters at, i.e. q 0 /v r = 0.5 vvm and n = 250, 375, 500 and 750 min −1 and; q 0 /v r = 0.7 vvm and n = 750 min −1 . the concentrations of the product and by-products amino acids and organic acids were determined in addition to bal activities. medium oxygen transfer rate conditions and uncontrolled ph operation at ph 0 7.25 are optimum for maximum bal activity, i.e. 860 u/cm 3 at 12 h, and productivity and selectivity. on the bases of the data, response of the intracellular bioreaction network of r-e. coli to oxygen transfer conditions were analysed using a mass-flux balance based stoichiometric model that contains 102 metabolites and 133 reaction fluxes. the results reveal that metabolic reactions are intimately coupled with the oxygen transfer conditions. oxygen transfer rate showed diverse effects on the product formation by influencing metabolic pathways and changing metabolic fluxes. metabolic flux analysis was helpful to describe the interactions between the cell and the bioreactor by predicting the changes in the fluxes and the rate controlling step(s) in the metabolic pathways. therefore, knowing the distribution of the metabolic fluxes during the growth, and bal and by-product formations provide new information for understanding physiological characteristics of the r-e.coli, and reveals important features of the regulation of the bioprocess and opens new avenues to successful application of metabolic engineering. the detection and diagnosis of pathogenic bacteria causing many diseases to the human body is an area of important research to public welfare. food is the most important energy source to humans, but it can give rise to disease caused by pathogenic bacteria not performing adequate detection tests. oligonucleotide-based microarrays are becoming increasingly useful for the analysis of expression profiles and polymorphisms among interested genes. here, we checked the possibility of development of oligonucleotide-based microarrays for detection and diagnosis of foodborne pathogenic bacteria. the oligonucleotide chip technology was applied to one control strain and seven foodborne pathogenic bacteria strains. it was designed repeated spots of eight hyperspecific and two highly conserved (control) capture probes from 16s rdna sequences. in order to validate experimental quality and to certificate specificities among specific spots at a glance by 2d and 3d views, quantitative visualization tool was developed. using the proposed oligonucleotide chip, we could classify and diagnose species and even subtypes of some pathogens. induction of in vitro neuro-muscular junctions using neuroblastoma and fibroblast cell lines for facilitating receptor-binding studies with botulinum toxin arindam chaudhury, bal ram singh department of chemistry and biochemistry, university of massachusetts dartmouth, 285 old westport road, north dartmouth, ma 02747-2300, usa. e-mail: g achaudhury@umassd.edu (a. chaudhury) botulinum toxin (bont), the most potent biological toxin known, is responsible for botulism, a fatal paralytic disease of the neuromuscular transmission. it blocks the release of acetylcholine at the neurotransmitter junction of the synapse. the objective of the current study was to induce in vitro neuro-muscular junctions through co-culturing of nerve and precursor-muscle cell lines. presently no known primary cultures or cell lines are available for nerve-muscle co-culture, thus validating the current work. j2-3t3 fibroblast cell line was first adapted to grow in media conducive for growth of sh-sy5y neuroblastoma cell line. the two cell lines were then splitted and co-cultured and observed for junction formations. light and fluorescent microscopic studies revealed en plaque (twitch-type) and en grappe (bulbous nerve endings) nerve-muscle junctions. growth rate of j2-3t3 cells decreased substantially when the media was initially changed. structurally they were more spindle-shaped than the normal reticular shapes of j2-3t3 cells, when grown in a media tailor-made for them. the formation of nerve-muscle junctions were confirmed using markers specific for each cell type. future work is focusing on receptor identification for the botulinum toxin in the established in vitro neuro-muscular junctions and also the transcellular translocation of the toxin. fourier-transform infrared (ftir) spectrometers have recently enjoyed widespread popularity in bioprocess monitoring applications due to their non-invasiveness and in-situ sterilizability. their online applicability creates an interesting opportunity for process control and optimization. however, the precision and accuracy of the predicted analyte concentration values directly depend on the quality of the measured signal and the robustness of the calibration model. instability and time drift in the measured spectra are currently one of the main obstacles in ftir monitoring. the intensity of the detected signal is influenced both by random noise and structural drifts and offsets. as a result, it is often necessary to scale the measured spectrum with respect to a constant reference spectrum, a technique similar to the internal standard approach used in analytical assays, such as hplc. applying this technique has lead to a noticeable decrease in the standard error of prediction in the monitoring of an anaerobic s43 fermentation of the saccharomyces cerevisiae yeast. in order to test the robustness of the calibration model and to increase its resistance to signal instability, random spikes of known amounts of analytes were introduced into the measured medium. this approach can be used to fine-tune the calibration model on-line and is currently one of the aspects investigated in this laboratory. the effect of the stringent response induction on l-valine biosynthesis by corynebacterium glutamicum ilze denina 1,2 , longina paegle 2 , liga zala 1,2 , maija ruklisha 2 : 1 faculty of biology, university of latvia, kronvalda blvd. 4, riga lv-1586, latvia; 2 institute of microbiology and biotechnology, university of latvia, kronvalda blvd. 4, riga lv-1586, latvia. e-mail: ilzede@hotmail.com (i. denina) the present study was focused on methods of the stringent response induction and on investigation of its effect on valine overproduction by isoleucine auxotrophs of corynebacterium glutamicum. the intracellular level of guanosine 5 -diphosphate 3diphosphate (ppgpp) increased and bacterial growth rate (µ) decreased during the short-term experiments when the exponentially growing cells were exposed to isoleucine limited conditions. the induction of the cellular stringent response resulted in a drastic increase in the activity of acetoxydroxy acid synthase (ahas), also by a significant increase in valine production. in contrast, an increase in ahas activity and valine synthesis by c. glutamicum was not achieved when bacterial growth was down-regulated in a ppgpp-independent manner. these results demonstrated that induction of the ppgpp-mediated stringent response might be significant in order to increase valine overproduction by c. glutamicum. infections with human cytomegalovirus (hcmv) continue to be an important health problem in certain patient populations, such as newborns, graft recipients of solid organs, or bone marrow and aids patients. in these groups, hcmv is a major cause of morbidity and mortality. the complex biology of hcmv necessarily begins with an initial interaction between the envelope of the infectious virus and the host cell. glycoprotein b (gb) is the major antigen, on the envelope of hcmv, for the induction of neutralizing antibodies. the region between aa 552 and 635 of hcmv gb (termed antigenic domain 1, ad-1) has been identified as the immunodominant target for the humoral immune response following natural infection. screening methods for detection of neutralizing antibodies have not been used because they are costly and labor intensive and thus far are not feasible for use on a large scale. for the development of reliable and inexpensive serodiagnostic tests the ad-1 of hcmv glycoprotein gp58, which are known to bind neutralizing antibodies, was expressed in prokaryotic systems. in this work, one prokaryotically expressed fusion protein which codifies ad-1 with ␤-galactosidase was used. the influence of different process conditions, on the production of the fusion protein containing the ad-1 as well as sugars addition to the fermentation medium was investigated. in order to analyze the expression of fusion protein, the ␤-galactosidase activity was followed throughout the fermentation. lysis process was also optimized and some final confirmation tests about protein antigenicity were performed. polyenzymic systems for the preparation of drugs based on modified nucleosides d. chuvikovsky, r. esipov, t. muravyova, a. miroshnikov shemyakin-ovchinnikov institute of bioorganic chemistry, russian academy of sciences, ul. miklukho-maklaya 16/10, moscow 117997, russia. e-mail: esipov@ibch.ru (r. esipov) considerable progress in the preparation of nucleoside analogues was achieved by combination of chemical and biochemical transformations. enzyme-catalyzed chemical transformation is now widely recognized as practical alternative to traditional organic synthesis in pharmaceutical and chemical industries. pentofuranosyltransfer reaction catalyzed by nucleoside phosphorylases was successfully employed for the synthesis of a variety of base-and sugar-modified nucleosides. enzymes involved in the metabolism of ribose phosphate and deoxyribose phosphate, such as ribokinase and phosphopentomutase, were used for the preparation of sugar-modified nucleosides. nucleosides phosphorylases (thymidine phosphorylase (tp), uridine phosphorylase (up) and purine nucleoside phosphorylase (pnp)), ribokinase and phosphopentomutase from e. coli have been cloned and overexpressed in e. coli. fast and efficient methods for the purification of nucleosides phosphorylases have been developed. the amount of purified protein was about 140 mg/l of cell culture, corresponding to 6300, 16,800 and 4200 units, respectively, of the up, tp and pnp. synthesis of medicinal drugs ribavirin (1-(␤-d-ribofuranosyl)-1,2,4-triazole-3-carboxamide), cladribine (2-chloroadenine-9-␤-d-2 -deoxyribofuranoside) and fludarabine (2-fluoroadenine-9-␤-darabinofuranoside) with the use of recombinant enzymes were studied. several important factors affecting the modified nucleosides production (ph, temperature, enzyme concentration, donor/acceptor ratio) were investigated and optimized. under optimum conditions ribavirin, cladribine and fludarabine produced in the reaction mixture in yields of 84, 85 and 80%, referred to 1,2,4-triazole-3-carboxamide, 2-chloroadenosine and 2-fluoroadenosine, respectively. aggregation and adsorption of fibroin molecules in aqueous solution won hur school of biotechnology and bioengineering, kangwon national university, chunchon 200-701, korea. e-mail: wonhur@kangwon.ac.kr fibroin, the structural protein from bombyx mori, is composed of heavy chain (generally called 'fibroin') and light chain polypeptides of about 370 and 25 kda, respectively. this study investigated the aggregation of fibroin and the adsorption between fibroin and surfaces. the variations of particle size and zeta potential were investigated by electrophoretic light scattering spectrophotometer (els). the adsorption of fibroin on surface was investigated in a continuous flow system by biacore applied surface plasmon resonance(spr) technique. the particle size and zeta potential range of aqueous fibroin were 140 nm, ±20 mv, respectively. iso-electric point(ph iep )of fibroin was ph 4.29. the amount of fibroin adsorbed on a gold surface was less than 0.1 g/ml even in the presence of high concentration of fibroin. the modification of gold surface was accomplished by applying chemicals known to form self-assembled monolayer, those are carrying nh 3 + , coo − , benzene ring and peptide that similar structure with fibroin. the adsorbed amount of fibroin on the self-assembly monolayers (sams) increased in the following order: nh 3 + > benzene ring > coo − > peptide surface. the deposition of fibroin in aqueous solution on non-waven fabric was affected by nacl and high temperature. ph influences metabolite profiling of ␤-lactamse producing b. licheniformis nazari̇leri 1 , pınar ç alık 1 , ali şengül 2 : 1 ib lab, department of chemical engineering, metu, 06531 ankara, turkey; 2 gülhane sch med, dept immunol, 06018 ankara, turkey. e-mail: e115715@metu.edu.tr (n.i̇leri) ph conditions in the bioreactor affect product and by-product formations by influencing metabolic pathways and changing metabolic fluxes, based on its influence on, i.e. dna transcription, protein synthesis, transport mechanism, atp generation and cellular energetics. whereupon, some fermentation processes favours uncontrolled-ph conditions while others favours controlled-ph conditions. on the bases of the interactions between the cell and the bioreactor through a process, carried out at either uncontrolled-or controlled-ph conditions, intracellular ph can be widely different and variable during the fermentation process. consequently, if one aims towards a quantitative understanding of the cell metabolism, one has to take into account the time variations of the intracellular ph and its effects on the in-vivo kinetics of the metabolic steps involved. moreover, since the presence of dormant or dead cells in the cultivation medium have negative effect on the synthesis of the production of desired product; the physiological state of the culture has great importance. in this context, the effects of ph on the regulation of intracellular ph, transport mechanism, and metabolic activity of b. licheniformis during production of ␤-lactamase (ec 3.5.2.6), an industrial enzyme catalyzing the hydrolysis of beta-lactam ring in beta-lactam antibiotics, was investigated. in addition, the physiological state of the organism and its effect on the production were observed. the optimal controlled-ph operation was found to be ph 6.75 with 54 u/cm 3 enzyme activity. the intracellular and extracellular na + , k + ion concentrations increased significantly throughout the process with increasing ph. on the other hand, the intracellular nh 4 + ion concentration was relatively constant. isolation and characterization of angiotensin-i converting enzyme inhibitory peptides by use of anti-peptide antibody fida hasan 1 , megumi kitagawa 2 , yoichi kumada 1 , naoya hashimoto 1 , masami shiiba 2 , shigeo katoh 1 , masaaki terashima 2 : 1 graduate school of science and technology, kobe university, nada, kobe 657-8501, japan; 2 department of human science, kobe college, okadayama, nishinomiya 662-8505, japan inhibitory peptides against angiotensin-i converting enzyme can be promising bio-functional peptides as natural alternatives for the non-peptide ace inhibitory drugs. these peptides are inactive within sequences of parent proteins and can be released during enzymatic digestion or food processing. immunointeraction is very effective for the purification of proteins and peptides with high purity. in this study, ace inhibitory peptides from hydrolysate of bonito meat were isolated by an anti-peptide antibody column and hplc, and kinetics of production of these ace inhibitory peptides was studies. an anti-peptide antibody against an ace inhibitory peptide, which was found by kohama et al. from tuna was obtained by immunization of the antigen peptide pc-iace (kkpthikwgd). water extract of bonito meat was digested at 37 • c in a modified gastric juice, 1.76 mg/ml nacl containing 312 g/ml pepsin (ph 2). peptides in hydrolysates were purified by use of an affinity column coupled with the antipeptide antibody and separated by hplc equipped with a reverse phase column (cosmosil 5c18-ms-ii, 4.6 cm × 150 cm). amino acid sequences and ic 50 values of the potent ace inhibitory peptides were determined. sds-page and western blotting experiments clarified that bonito protein contained peptides having similar sequence to the antigen peptide. a fraction of retention time 18-28 min in hplc purification samples showed high inhibitory activity, and several peptides in this fraction were separated. after 48 h digestion, two major inhibitory peptides, herdpthikwgd and pthikwgd, were found to be relatively stable in the gastric juice. kluyveromyces marxianus physiology on several levels of carbon, nitrogen sources and oxygenation during inulinase production silva-santisteban yépez, o. bernardo, francisco maugeri department of food eng./unicamp, 13081-970, campinas, sp-brazil. email: maugeri@fea.unicamp.br (f. maugeri) inulinase produced by yeasts is an interesting alternative compared with the one produced by filamentous molds, as culture conditions can be better controlled. during the assays, it was observed that inulinase production levels varied with nutritional conditions in batch culture. kluyveromyces marxianus atcc 16045 culture is described by two main phases, the first one being the growth phase, where substrate consumption and basal inulinase production were performed, and the second one being the phase where some metabolites are uptaken and high inulinase production is observed. the metabolic fluxes analyses were used to describe the cell physiology in the first phase, in a variety of conditions of sucrose and ammonium sulfate concentration and aeration condition. the metabolic network included the main metabolic pathways such as glycolisis, pentose phosphate pathway, krebs cycle, oxidative phosphorylation and biomass biosynthesis. the physiology in this phase was correlated with high inulinase production in the second phase. it was also noticed that inulinase production diminished when sucrose was in high concentration, leading, additionally, to ethanol production. in these terms, it was unveiled a kind of crabtree effect performed by this strain. forward extraction of l-aspartic acid from fermentation broths by reverse micelles and backward extraction by gas hydrate methodö. aydogan 1 , e. bayraktar 1 ,ü. mehmetoglu 1 , m. parlaktuna 2 , t. mehmetoglu 2 : 1 department of chemical engineering, faculty of engineering, ankara university, tandogan, ankara 06100, turkey; 2 department of petroleum and natural gas engineering, middle east technical university, ankara 06531, turkey. e-mail: mehmet@eng.ankara.edu.tr (ü. mehmetoglu) recently gas hydrate method has been applied as a technique for backward extraction of amino acids from reverse micelle systems. in this study, backward extraction of l-aspartic acid was investigated by gas hydrate method. at the first stage, production of l-aspartic acid was carried out using 50 ml of 0.5 m ammonium fumarate (ph 9.5) as substrate at 37 circc in an orbital shaker at 150 rpm for 4 h. e. coli (atcc 11303) was used as biocatalyst. at the end of reaction excess fumaric acid was extracted in reverse micelle phase. then forward extraction of l-aspartic acid was carried out with injection method in reverse micelle phase. for back extraction, co 2 is used to form gas hydrates crystalline structure. back extraction experiments were carried out between 35-37 bar g pressure and at 2 circc. at the end of the back extraction l-aspartic acid was obtained in crystalline form. the results indicate that recovery of l-aspartic acid from reverse micelles by forming gas hydrate can be achieved with a yield of 55.3%. consequently, gas hydrate method can be used as a new technique for backward extraction of amino acids from reverse micelles. aerobic and anaerobic cultivations of aspergillus niger on different nitrogen sources susan meijer, gianni panagiotou, lisbeth olsson and jens nielsen center of microbial biotechnology, biocentrum-dtu, technical university of denmark, dk-2800 lyngby, denmark in this study, we aim at creating a succinic acid producing strain of a. niger. a. niger is known to be a strictly aerobic organism, meaning it is not able to use the reductive part of the tca cycle to produce succinate. during aerobic growth a. niger uses oxygen as electron acceptor in the respiratory chain, thereby reoxidizing the produced nadh to nad + . however, under anaerobic conditions other compounds than oxygen, such as no 3 − are required as electron acceptor (denitrification). this process consists of no 3 − reduction to nh 4 + coupled to substrate-level phosphorylation that supports growth under anaerobic conditions. in the present study, our aim was to investigate the effect of different nitrogen sources on the physiology of a. niger during growth under aerobic and anaerobic conditions. aerobic growth experiments on three different nitrogen sources; ammonium, nitrate and nitrite, showed that ammonium and nitrate could be consumed by the filamentous fungus. nitrite on the other hand could not facilitate growth, indicating the absence of a nitrite uptake system. however, under anaerobic conditions notable growth was only observed on nitrate. these data support the hypothesis of the existence of an alternative electron acceptor that might facilitate anaerobic growth of a. niger. among the therapeutic proteins derived from mammalian cells, recombinant antibodies received a great deal of attention as a prominent product through biotech pipelines toward the marketplace. they now occupy about 25% of the estimated medicines in clinical development and many more antibodies which lead the value of the market going forward are reported. there are various environmental factors affecting rcho cell cultures such as medium components, temperature, ph, and byproducts (ammonia, lactate, and, etc.) . because most of mammalian cells are very sensitive to their environmental change, appropriate control of environmental parameters is a very important consideration to enhance cell growth and production of target proteins. balanced addition of limiting medium components plays an essential role on improvement of cell density and product concentration. temperature and ph are easily adjustable process parameters, being reported to influence cell growth and recombinant protein production. ammonia and lactate are well-known byproducts which have an inhibitory effect on cell growth when their concentrations exceed a specific level. in this work, effects of various environmental factors including temperature, ph, amino acids, vitamins, hormones, and metabolic byproducts on cell growth and recombinant antibody production were investigated in the cultivation of recombinant chinese hamster ovary cells. the most suitable condition of each environmental condition was proposed for enhancement of the cell growth and the productivity of recombinant antibody. the present study was carried out in order to assess the protective effects of calycosin-7-o-␤-d-glucopyranoside isolated from astragali radix (ar) on hyaluronidase (haase) and the recombinant human interleukin-1␤ (il-1␤) induced matrix degradation in human articular cartilage and chondrocytes. we isolated the active component from the n-butanol soluble fraction of ar as haase inhibitor and structurally identified as calycosin-7-o-␤-d-glucopyranoside by lc-ms, ir, 1 h nmr, and 13 c nmr analyses. the protective effect of arbu on the matrix gene expression of immortalized chondrocyte cell line c-28/i2 treated with haase was investigated using a reverse transcription polymerase chain reaction (rt-pcr). its effect on haase and il-1␤-induced matrix degradation in human articular cartilage was determined by using a staining method and calculating the amount of degraded glycosaminoglycan (gag) from the cultured media. pretreatment with calycosin-7-o-␤-d-glucopyranoside effectively protected against matrix degradation of the human chondrocytes and articular cartilage. therefore, it would appear that calycosin-7-o-␤-d-glucopyranoside from ar is a potential natural ant-inflammatory or anti-osteoarthritis agent and can be effectively used to protect against proteoglycan (pg) degradation. catechol-o-methyltransferase (comt) is an enzyme that catalyses a variety of endogenous and exogenous catechol substrates by transferring a methyl group from s-adenosylmethionine (sam) to either the meta-or the para-hydroxyl group of the catechol ring. the enzyme has a physiological role in the metabolism of the catechol estrogens, inactivation of the catecholamine neurotransmitters such as dopamine and epinephrine and detoxification of a variety of xenobiotic catechols. comt activity has been identified in various tissues; however with the developments in molecular biology and gene technology, the production and purification of large amounts of recombinant comt is a good option for biochemical, pharmacological and structural studies. in this work, cultures of recombinant e. coli harbouring a model plasmid were grown in a 500 ml shake-flask containing 125 ml of complex medium. the influence of medium composition and induction time on comt production, recovery and clarification by sonication, ammonium sulphate precipitation and purification by hydrophobic interaction chromatography onto a butyl-sepharose column will be presented and discussed. bioactive bacterial exopolysaccharides corinne sinquin 1 , karim senni 2 , jacqueline ratiskol 1 , farida guéniche 2 , jean guézennec 1 , gaston godeau 2 , sylvia colliec-jouault 1 : 1 ifremer, 44311 nantes cedex 3, france; 2 ea2496 université rené descartes, 92120 montrouge, france. e-mail: corinne.sinquin@ifremer.fr (c. sinquin) interest in mass culture of microorganisms from the marine environment has increased considerably, representing an innovative approach to the biotechnological use of under-exploited resources. marine bacteria associated with deep-sea hydrothermal conditions have demonstrated their ability to produce in an aerobic carbohydrate-based medium, unusual extracellular polymers (guezennec, 2002; colliec-jouault et al., 2004) . these exopolysaccharides (eps) present original structural features that can be modified to design innovative bioactive compounds and improve their specificity. with the aim of promoting biological activities, chemical modifications (depolymerization and substitution reactions) of one eps produced by vibrio diabolicus have been undertaken (raguenes et al., 1997) . the structure of the native eps has been described (rougeaux et al., 1999) the potential of the eps derivatives as therapeutical agents will be presented. physiological responses of e. coli to glucose and oxygen shifts in fed-batch fermentations jaakko soini 1 , christina saarimaa 1 , arne matzen 2 , peter neubauer 1 : 1 bioprocess engineering laboratory, department of process and environmental engineering, university of oulu, oulu fi-90014, finland; 2 sanofi-aventis, germany. e-mail: jaakko.soini@oulu.fi (j. soini) in high-cell density fermentations e. coli cells are often subjects of transient changes in microenvironment around them. these changes can be, for example, medium component gradients or differences in oxygen availability. we have studied the physiological response of e. coli w3110 cells to simultaneous oxygen limitation and overfeeding of glucose. the aim is to obtain more information of physiological changes for better understanding of the bottlenecks in such processes. the response of the cells for glucose and oxygen shifts was studied by analyzing key metabolites and proteins and mrna transcript levels. the transcript levels were measured using a sandwich hybridization technique (rautio et al., 2003) proteomic analysis was carried out by 2d-electrophoresis and the metabolite analysis by hplc. the main focus of this study is to monitor the expression patterns of marker genes involved in mixed acid fermentation, glycolytic pathway and tricarbonic acid cycle. rautio et al., 2003 . sandwich hybridisation assay for quantitative detection of yeast rnas in crude cell lysates. microb. cell fact. influence of ner on genetic instability of the (ctg/cag) tracts in bacterial chromosome sylwia szwarocka 1 , paweł parniewski 2 : 1 department of microbiology and immunology, university of łódź, 90-237 łódź, banacha 12/16, poland; 2 centre for medical biology, polish academy of sciences, 93-232 łódź, lodowa 106, poland many human hereditary neurological diseases, including fragile x syndrome, myotonic dystrophy and friedreich's ataxia, are associated with expansions of triplet repeat sequences (trs) (cgg/ccg, ctg/cag and gaa/ttc) in or near specific genes. mechanisms that mediate the expansions and deletions of trs include dna replication, repair and recombination. many investigations suggest that the structural properties of the trs play a consequential role in their genetic instabilities. nucleotide excision repair (ner) is the major cellular system in both prokaryotes and eukaryotes and recognises damages due to distortion of the dna helix. involvement of ner in the hairpin loop repair that can form within ctg tracts has been reported. the participation of this repair systems in the trs instability was investigated in e. coli only on multicopy plasmids. the results showed that deficiency of some ner functions dramatically affects the stability of long (ctg/cag) inserts. in this work we present a chromosomal model to study the instability of the trs in e. coli. we introduced the (ctg/cag)n tracts into the chromosome of e. coli and used strains with some deficiency of the ner and investigated genetic stability of these tracts after multiple recultivations. in general, our results show that the (ctg/cag)n repeats are much more stable in the chromosome than in plasmids. these data may suggest that instability of trs in plasmids is associated with interaction between repetitive tracts on different plasmid molecules inside the cell. however, mutations of ner genes may increase (uvra and uvrb mutants) or decrease (uvrc and uvrd mutants) stability of the trs in the e. coli chromosome. this study was partially funded by the kbn grant 2 p05a 01927. performance analyses of a multi-stage integrated fermentation process for lactic acid production hsun-tung lin, feng-sheng wang department of chemical engineering, national chung cheng university, chia-yi 621-02, taiwan. e-mail: chmfsw@ccu.edu.tw (f.s. wang) in this work, we considered a multi-stage integrated continuous fermentation process for producing lactic acid. each stage consists of a mixing tank, a fermenter, a cell recycle unit and an extractor. the generalized kinetic model is first applied to formulate the integrated process. we have compared the overall productivity and conversion of the integrated process with those of two simplified processes. from the design equations, we obtain that three processes have the identical overall conversion. however, the proposed process has the greatest overall productivity. the specific kinetic model for lactic production (youssef et al., 2000) was applied to the integrated process in order to find the maximum overall productivity. two optimization problems are respectively considered to determine the optimal stages, operating conditions and design variables. the first problem supposes that the integrated process has the equal working volume ratio for each fermenter. such a process requires four stages to yield the maximum overall productivity and the nearly complete overall conversion. however, if the working volume ratio for each stage is considered as the decision variables in the second optimization problem, three stages is enough to achieve the identical overall productivity. youssef, c.b., guillou, v., olmos-dichara, a., 2000. contr. eng. pract. 8, 1297-1307. modelling of the binding of ligands to macromolecules jørgen m. mollerup department of chemical engineering, building 229, dtu, 2800 lyngby, denmark a variety of factors that govern the properties of proteins are utilized in the development of chromatographic processes for the recovery of biological products including the binding and release of protons, the non-covalent association with non-polar groups (often hydrophobic interactions), the association of small ions (ion exchange) and the highly specific antigen-antibody interaction (affinity interactions). the fulcrum point in the understanding and modelling a chromatographic separation is the adsorption isotherm that determines the peak shape at preparative load. to enable an efficient chromatographic process development strategy it is necessary to conduct theoretical and experimental investigations of the adsorptive behaviour of proteins. thermodynamically consistent models for ion exchange chromatography and hydrophobic interaction chromatography have been developed and can be utilised in the simulation of a chromatographic separation. besides, measurements on hic media can be utilised to determine the cohn salting-out coefficient. the lig-and binding process can frequently be coupled to associated structure changes in the protein, the ligand or both. this gives rise to nonlinear adsorptive behaviour known as cooperativity which cannot be modelled using conventional models which displays convex behaviour. examples of cooperative behaviour are the reversible binding of oxygen and carbon monoxide to haemoglobins and the binding of nad + to yeast glyceraldehydes 3-phosphate dehydrogenase. in the paper we discuss the modelling of reversible binding of mobile as well as immobilised ligands to macromolecules and compare modelling to experiment. comparative analysis of the temperature policy for processes with a deactivating native enzyme i. grubecki, m. wojcik department of chemical and biochemical engineering, university of technology and agriculture, 85-326 bydgoszcz, ul. seminaryjna 3, poland a comparative analysis of the temperature policy for an enzymatic reaction with michaelis-menten kinetics in a batch reactor has been carried out. both isothermal and optimal temperature policies for processes with deactivating native enzyme have been considered. in the model, the thermal deactivation was described by a first-order reaction, and the arrhenius-type dependence between rate parameters and temperature was assumed. as an indicator for a direct comparison between the isothermal and optimal temperature policies the quotient of conversions under identical initial and final condition was used. a method was presented to calculate this indicator, which is based on the analytical and numerical solutions. this method can be of great importance for the industrial practice. application of changeable temperature policy could result in significant increase in conversion when ratio of activation energy for deactivation and activation energy for reaction is high. studies on the impact of mixing during brewing using near and mid-infrared spectroscopy georgina mcleod 1 , alvin w. nienow 1 , graham poulter 2 , reg wilson 3 , henri tapp 3 , christopher j. the control of the brewing process is important for improving product quality, and lowering costs. infrared spectroscopy is a technique that can be used at-line and on-line to rapidly measure component concentrations of unprocessed whole broth samples in real time. in this study, both mid -infrared (mir) and near-infrared (nir) spectroscopy have been used and compared for the monitoring of ethanol, flavour components, wort sugars, biomass and specific gravity during the brewing. partial least-squares regression (pls) was used to model relationships between component concentrations and spectra. the performance of these models was evaluated in terms of the standard error of prediction (sep), number of pls factors and the correlation coefficient (r). calibration models were constructed using spectra acquired for multi-component mixtures, intended to simulate brewing fermentation conditions, and actual brewing fermentation samples. chemometric results indicated that nir is a powerful tool for accurately measuring sugar, ethanol and biomass concentrations. the optimization of a fermentation process requires the organism to be cultivated under desirable conditions, which depends on how well the fermentation process is controlled. inadequate mixing and mass transfer are responsible for the heterogeneous environment at large scale in terms of nutrient concentration and ph profile, resulting in lower product yields. these have been associated with, inadequate control of ph and the production of acetate or formate in response to over-feeding of glucose and oxygen deficiency. rapid analyses of substrates and indicator metabolites in a fermentation process is critical for optimal control. this can be achieved in real time with nir spectroscopy. in this study, nir-spectroscopy has been applied to monitor the concentrations of glucose, acetate, formate, ammonium hydroxide and biomass in the cultivation of e. coli (w3110). a comparison of partial least square models built using water standards, synthetic medium standards and fermentation samples has been made. template refolding utilizing biospecific interactions shigeo katoh 1 , yoichi kumada 1 , nanae maeshima 1 , daisuke nohara 2 : 1 graduate school of science and technologykobe university, kobe 657-8501, japan; 2 department of biomolecular sciencegifu university, gifu 501-1193, japan. e-mail: katoh@kobe-u.ac.jp (s. katoh) recombinant proteins over-expressed in e. coli are often accumulated as insoluble particles called inclusion bodies. proteins in inclusion bodies must be solubilized by a denaturing agent, such as urea and guanidine hydrochloride, and refolded to recover their native structures having biological activities. in bioprocesses it is important to obtain high refolding efficiencies and high throughputs at high protein concentrations. in refolding operation, a denatured protein solution is usually added batch-wise into a large volume of a refolding buffer in order to start refolding by reducing the concentration of a denaturant and to prevent aggregate formation of renaturing molecules. thus, a large volume of a stirred tank is required, and the concentration of protein after renaturation becomes low. biointeractions between a pair of biomolecules, such as enzyme-inhibitor, antigen-antibody and hormone-receptor, are highly specific and have been used for detection and separation of biomolecules. these interactions may be used as templates for refolding of target molecules, which can be captured with the templates and are prevented from aggregate formation and, in the case of proteases, from autoproteolysis. the specific interaction might promote refolding of the target molecules. these might improve the refolding efficiency. the biointeractions between antigen-antibody and enzyme-inhibitor were used for efficient refolding in packed columns, in which template ligands (antibody, inhibitor) were coupled on gel support. denatured solutions of target molecules (carbonic anhydrase and s. griseus trypsin) were mixed with refolding buffer and supplied to the affinity column coupled with the template ligands for refolding. with refolding in the column, higher refolding efficiencies were obtained than those by the batch dilution method with relatively low concentrations of denaturants. by increasing the adsorption capacity of the column, throughput of refolding can be increased without decrease in the refolding efficiency. the use of multi-parameter flow cytometry for characterisation and monitoring of insect cell-baculovirus fermentations in a mechanically-agitated bioreactor bojan isailovic 1 , alvin w. nienow 1 , ian w. taylor 2 , ryan hicks 2 , christopher j. bacteria and mammalian cells have been traditionally used as hosts for commercial recombinant protein production. however, in recent years, the insect cell-baculovirus system has emerged as a potentially attractive recombinant protein expression vehicle. although flow cytometry has been used widely for analysis of mammalian and microbial cells, there is very little information on applications of this powerful technique in insect cell culture. here we compared cell ratiometric counts and viability (propidium iodide and calcein am) of sf-21 cell cultures using flow cytometry to those determined by more traditional methods using a haemocytometer and the trypan-blue exclusion dye. flow cytometry has also been used to monitor various parameters during cultures of sf21 infected with the recombinant autographa californica nuclear polyhedrosis virus (acnpv) containing the inserted nucleic acid sequence amfp486 coding for am-cyan coral protein, which emits natural green fluorescence. carbon sources create fingerprint fermentation characteristics pınar ç alık 2 , güzide ç alık 1 , tunçer h.özdamar 1 1 bre lab, department of chemical engineering, ankara university, 06100 ankara, turkey; 2 ib lab, department of chemical engineering, metu, 06531 ankara, turkey. e-mail: calik@eng.ankara.edu.tr (g. ç alık) this work reports on a systematic investigation of the interactions between the single-carbon sources, i.e., glucose and citric acid, and complex-medium components, i.e., carbon and nitrogen sources, in enzyme fermentation processes, i.e., serine alkaline protease (sap; ec 3.4.21.62),) and ␤-lactamase (ec 3.5.2.6), with the oxygentransfer and ph conditions to demonstrate the influences of carbon sources that create the fingerprint fermentation characteristics, moreover, their influences on the product and by-product formations and the intracellular reaction rates. the influence of the medium composition i.e. citric acid-, glucose-, molasses-and soybean-based media together with the oxygen transfer (ot)-and ph-conditions applied, on the product and by-product distributions and ot characteristics were investigated in batch bioreactors. for sap, in general, under uncontrolled-ph operations the variation of the medium ph in sap fermentation process has a tendency to increase in the sap production phase; however, depending on the carbon source used, its behaviour changes in the early stages of the fermentation as the consequences of the directed functioning of the intracellular bioreaction network. the loci of the dissolved oxygen (do) curves also strongly depend on the carbon source(s) utilised, in addition to the applied ot conditions. the complex media profiles are significantly different compared to the defined media as the ph and do profiles are interrelated owing to the bioreactor operation conditions affecting the metabolic reaction network. the highest volumetric oxygen uptake rates were obtained with soybean-based medium that was ca. three-fold higher than the values reported in citrate-based and glucose based media, and ca. 1.5-2-fold higher than the values reported in molasses-based medium. the significant changes, moreover, the drastic change observed with the use of soybean-based complex medium are due to the compositions of the fermentation media used, and its influence on the intracellular bioreaction network. thus, we conclude that the change in medium composition based on the carbon source changes the fermentation characteristics under the designed bioreactor operation conditions that appear as the fingerprints of the bioprocess. kinetic resolution of racemic benzoin with different lyophilized microorganisms ç . babaarslan 1 ,ü. mehmetoglu 1 , a.s. demir 2 : 1 ankara university, faculty of engineering, department of chemical engineering, 06100, ankara, turkey; 2 middle east technical university, department of chemistry, 06531 ankara, turkey. e-mail: barslan@eng.ankara.edu.tr (ç . babaarslan) the biocatalytic resolution of racemates is valuable tool for enantioselective synthesis and proved to be a convenient method for obtaining optically enriched compounds from their racemic form. in this work, enantiomerically pure benzoin which is one of the 2-hydroxy ketones was synthesized by kinetic resolution of racemic benzoin using different lyophilized microorganisms as lipase sources. the effect of lyophilized microorganism type, solvent type and acyl donor type on enantioselectivity were studied. in kinetic resolution experiments, lyophilized microorganism was resuspended in the media containing solvent, racemic benzoin and acyl donor at 30 • c and 150 rpm on orbital shaker. the reaction was followed by tlc during the experiment and the enantiomeric ratio of benzoin was determined by hplc analysis using chiral cell ob column. the best enantioselectivity value was obtained with lyophilized rhizopus orayzae cbs 112-07 as ee s = 16% and ee p = 30% (conversion = 35%) using thf as solvent and vinyl acetate as acyl donor. otimizing the fermentation broth for tanase production by a new isolated strain paecilomyces variotii vania battestin, gláucia pastore, gabriela macedo department of food science, unicamp, p.o. box 6121, campinas, cep 13083-862, são paulo, brazil tannase is an inducible enzyme that catalyses the breakdown of ester linkages in hydrolysable tannins, resulting in gallic acid and glucose. the fermentation broth can use by-products as wheat bran, rice or oats, adding tannic acid. the use of by products or residues rich in carbon source for fermentation purposes an alternative to solve pollution problems that can be caused by an incorrect environmental disposal. in the present study we have optimized the production of an extracellular tannase by a new isolated paecilomyces variotii using response surface methodology. the first step was identify the variables having a significant effect on enzyme production. the variables evaluated were temperature, residues ratio (coffe: wheat bran), concentration of tannic acid, salt solution during 3, 5 and 7 days of fermentation time. results showed that temperature, residues ratio (coffe: wheat bran) and tannic acid had significant effects on tannase production. commercial wheat bran (cwb) and coffe rusk residues (cr) were used as solid substrate. for fermentation the mediun was composed by, cwb:cr were mixed with distilled water and transferred into 250 ml capacity erlenmeyers flasks and autoclaved at 120 • c for 20 min. the medium was then inoculated with spores (5.0 × 10 7 ) and the flaks were incubated at 32 • c. tannase was assayed according to the methodology of mondal et al. (2001) . acording to the statist analyses, the optimum conditions to produce tannase was the range of temperature (29-34 • c); tannic acid (8.5-14%); residues % (coffe: wheat bran) (50:50) and 5 days fermentation time. the enzyme production increased 8.6 times more enzyme production than that was obtained before this optimization. how to cope with fda's pat-initiative with respect to fermentation process monitoring and control marco jenzsch 1 , andreas luebbert 1 , rimvydas simutis 2 : 1 institute of bioengineering, martin-luther-university halle-wittenberg, halle (saale), germany; 2 process control department, kaunas university of technology, kaunas, lithuania with its pat initiative, fda forces drug manufacturers to increase their activities in innovative manufacturing techniques, and, more than previously, to focus on quality assurance. the agency particularly places emphasis on making use of modern process supervision and control techniques such as up-to-date process analytics, multivariate data acquisition and analysis tools in order to improve process monitoring and control. in this contribution we show by means of practical examples how this guidance can be applied to cultivations of genetically modified microorganisms. a comparison of different multivariate state estimation techniques will be presented and compared with more knowledge-based techniques such as the extended kalman filter. the comparison was made for the model system gfp expressed from e. coli bacteria (bl21/de3/gfp) for which more than 40 full data sets are available. all these techniques have already been used during real protein formation at productionscale fermenters, with the same success. hence, the requirements expressed in the pat initiative can immediately be put into practice. feedback control of the recombinant protein production processes based on such estimations is show for several cultivation systems. simple parameter adaptive controllers are compared with model supported controllers, for instance, generic model controllers and model predictive controllers. the results clearly show that we have at hand a rather extended arsenal of feedback control procedures that can be used successfully to tightly control the processes even along set-point profiles of physiological variables such as the specific growth rate (µ). again, fda's suggestion with respect to "control in the engineering sense" can be applied immediately to reduce batch-to-batch variances and thus to increase process quality. extending life by alternative respiration? alexander kern, franz hartner, anton glieder institute of biotechnology, graz university of technology, a-8010 graz, austria. e-mail: a.kern@tugraz.at (a. kern) alternative oxidase transfers electrons directly from the ubiquinol pool in mitochondria to oxygen, allowing cell respiration in presence of complexs iii and iv inhibitors like antimycin a or cyanide. electron transfer by alternative oxidase is not coupled with proton transfer across the mitochondrial membrane, thereby uncoupling the supply of small metabolic intermediates by the central metabolic pathway from energy production in the cell. alternative oxidase is present in mitochondria of plants, many fungi and a few, mostly crabtree-negative yeasts, but not in p. angusta (hansenula polymorpha) and s. cerevisiae. alternative oxidase has multiple functions in different organisms. it is involved in stress answers, in programmed cell death, maintenance of the cellular redox balance, and also citric acid accumulation in a. niger. we isolated the alternative oxidase gene from the methylotrophic yeast p. pastoris in order to study its effects on the cellular energy content, respiratory activity, its protective role against oxidative stress. our results indicate the importance of an exact regulation of the alternative oxidase due to its impact on many cellular functions. new types of energy efficient fermenters with better mass transport, mixing and cooling properties than the current crop of rushton turbine derived tank bioreactors are likely to be required in the future. such fermenters will be needed in order to meet the increasing pressure on costs for low price commodity type products such as single cell protein or food and technical grade enzymes, and to meet the demands of the new wave of white biotech, in which bio-produced chemicals must be made at prices competitive with those of the traditional chemical industry. with this in mind, a prototype pilot scale (500 l) u-loop fermenter has recently been commissioned at biocentrum-dtu. in this fermenter, liquid circulation is driven by a propeller pump through a vertical u-shaped pipe, which is connected at the top with a de-gassing tank. we present here a study of liquid mixing and dispersion in the prototype u-loop fermenter. sub-sequently we show that the results can be described with the tanks in series model. mixing was characterised using pulses of nacl tracer, which were detected with a conductivity probe in various parts of the fermenter. bodenstein numbers (bo) were determined for flow rates corresponding to a linear fluid velocity of 1.05 m/s in the 'legs' of the reactor and showed that the majority of the mixing occurred in the top degassing part (bo = 29) rather than in the u-loop section (bo = 422). it was also observed that the time for mixing to 90% homogeneity after tracer pulse addition was a function of the number of cycles through the reactor (3-3.5) within the range of flow velocities (u) studied (u = 0.41 m/s to u = 1.74 m/s). the mixing time to 90% homogeneity was between 44.6 s (at u = 1.74 m/s) and 181 s (at u = 0.41 m/s). today many biotechnological processes are operated at suboptimal conditions and according to best practice. however, the current industrial development is towards analyzing more parameters and in particular there is a large interest in analysis of biological/biochemical variables. the quality of products and also the possibility to optimize production in submerged cultivations would be greatly enhanced if more on-line/real-time information were at hand. the present investigation was undertaken with the aim of evaluating the potential in using multi-wavelength fluorescence for monitoring and control of filamentous fungi fed-batch cultivations. a recombinant a. oryzae expressing a heterologous lipase was applied as model system. spectra of multi-wavelength fluorescence were collected every five minutes with the bioview ® system (delta, denmark) and both explorative and predictive models, correlating the fluorescence data with the important biological parameters cell mass and lipase activity, were built. the models will be presented, furthermore, advantages and disadvantages of multiwavelength fluorescence for monitoring of cultivation processes will be discussed. moving from r&d to pharmaceutical development is a costly process. it is therefore of paramount importance to design a manufacturing process that combines robust and well-documented technological platforms. therapeutic recombinant proteins designed for human administration should be as close to the authentic product as possible. here, the use of a scalable process and an economically sound affinity tag can be a relevant choice. the tagzyme tm system has been designed to allow for the precise removal of amino terminal affinity tags. the system is based on the use of recombinant aminopeptidases including dipeptidyl peptidase i (dapase tm ). dapase tm is currently produced under cgmp providing a suitable strategy for its use in pharmaceutical production. the tagzyme tm system is superior to other methods since: (1) it is based on exopeptidases, precluding, e.g., unspecific protein cleavage reported when using so-called site-specific endoproteases. (2) it has been tested for production of more than 200 recombinant proteins. (3) it is easily scalable from lab scale to kg of processed protein. (4) it allows the use of his-tags for commercial production without patent infringment, due to our ipr position. (5) the commercial use of tagzyme tm does not require any licensing, only purchase of the enzyme(s). (6) the use of aminopeptidases for pharmaceutical production has been extensively documented for approved drugs. (7) a number of therapeutics is currently being developed using tagzyme tm . (8) unizyme can assist in the optimization of the dsp to enable further cost reduction in the process. these aspects will be discussed and illustrated in the presented poster. website sphingolipids are biologically active molecules involved in the regulation of a large quantity of biological responses. they function in cell proliferation, survival and death (apoptosis) as messengers. dysregulation of apoptosis has significance in numerous pathological conditions including cancer. several anticancer agents act by increasing tumor cell ceramide (a kind of sphingolipid) content. so, a novel approach to cancer therapy would be the pharmacological manipulation of sphingolipid metabolism. in this study, sphingolipid metabolism in baker's yeast s. cerevisiae is used as a model system as many of its sphingolipid related genes and proteins have been characterized. gepasi-biochemical kinetics simulator was used for metabolic control analysis (mca) of the above-specified system. the concentration control coefficients (ccc), flux control coefficients (fcc) and elasticity coefficients were calculated, and their significance in identification of anticancer drug targets is determined. elementary flux modes were also identified and metabolic pathway analysis (mpa) was performed. quantitatively, control effective flux (cef) values were used for potential drug target identification. the results from mca and mpa indicate almost the same potential drug targets: serine palmitoyl transferase, ceramide synthase and ceramidase. drugs against these targets are in preclinical and clinical development. for the identification of new potential drug targets, the cccs, fccs, cefs and elasticity coefficients were examined with an objective function of maximizing the cell ceramide concentrations. it was found that manipulation of inositol-1-phosphate synthase and phosphoinositide kinase activities have considerable effects on ceramide concentrations. if a drug targeting the two enzymes at the same time is designed, it might give a better outcome in terms of cancer therapy. in recent years, there is a growing interest in utilization of airlift reactors (alrs) to biotechnological processes. nevertheless, their industrial application still remains limited because of a lack of reliable studies on transfer phenomena and mixing enabling a suggestion of suitable scale-up procedure. the way to more widely utilization of alrs to biological processes lies in experimental research (on a model medium as well as on a real fermentation medium) followed by mathematical modelling and scaling-up of the processes. this paper deals with a modelling of a glucose-gluconic acid fermentation by a. niger in an internal loop airlift reactor. knowledge of the stoichiometric relationship in the key reaction provides a good opportunity for estimation of substrate and product concentration. the model is based on material balance equations and has been adjusted to experimental data obtained from three internal loop airlift reactors (10.5, 40 and 200 l) . in the model, the alr is divided into ideal stirred tanks in series. in each zone (tank) of the alr the material balance is calculated in two phases (the gas and the liquid phase). this work was supported by the slovak scientific grand agency, grant number vega 1/0066/03 alkaline phosphatase (ap, e.c. 3.1.3.1) is a thermolabile enzyme which is indigenous to all dairy products. it has an inactivation temperature slightly above the value that is required to destroy the most resistant pathogenic microorganism likely to be found in milk. due to that feature, this enzyme is used as an indicator of proper pasteurization. the effect of temperature treatment on the activity of ap was investigated in raw cow's and goat's milk. the stability of alkaline phosphatase in raw milk was compared with the stability of this enzyme in a 0.1 m potassium phosphate buffer with ph 6.6. the ph value of the buffer was approximately the same as that of raw milk. the inactivation curves were measured in the temperature range from 54 to 69 • c. ap in cow's milk was completely inactivated at 69 • c during 60 s but approximately 30% of activity remained at 54 • c after 100 min of treatment. the time required for a complete inactivation of the enzyme in the raw cow's milk was reduced from 90 to 1 min as the temperature increased by 11 • c. heat treatment of goat's milk caused the decrease of activity of the enzyme in the same temperature range as in the case of cow's milk. the increase of temperature from 58 to 68 • c reduced the inactivation time from 35 min to 40 s. the study of thermal stability of the alkaline phosphatase in the buffer solution showed that the time required for inactivation of enzyme was significantly shorter than in milk. milk thus had a protective effect on the activity of alkaline phosphatase. the experi-mental curves were fitted simultaneously using kinetic models where the initial heating period was considered. this work was supported by a grant of 6th framework program of eu, project foodpro, no. sme-2003-1-508374. during the process of separation, purification and concentration of monoclonal antibodies (mabs) at industrial scale, the chromatographic unit operations have an important role. three different protein-binding modes are employed: ion-exchange, hydrophobic and affinity binding. two adsorbent properties are of uppermost importance: a high selectivity and adsorption capacity. in the case of ion-exchange/hydrophobic chromatography, the binding of charged proteins can be affected by ph and ionic strength. in this work, the adsorption capacity of eight commercially available adsorbents designed for separation of mabs (mabselect, rprotein a sepharose ff, poros 50a, prosep-va, fractogel emd se hicap (m), sp sepharose ff, mep hypercel, s ceramic hyperd f) was measured as a function of ph. as a model mab and contaminant proteins, human immunoglobulin (igg), human serum albumin (hsa) and horse skeletal muscle myoglobin (myo) were used. the resin properties were investigated within the range of ph 4-8. the experiments were conducted in a batch-mode, individually for each model protein. the results showed that ion-exchange and hydrophobic resins provided the best selectivity for igg at ph 6. the selectivity of affinity adsorbents was essentially unaffected by ph, however, the highest capacity for igg was at ph 7. another investigated aspect was the dynamics of protein binding. the solution of individual protein in contact with tested adsorbent was circulated through an uv spectrophotometer, what enabled the measurement of time-dependent decrease of protein concentration. the results indicated that affinity adsorbents with a rigid matrix needed approximately four times shorter time to reach the adsorption equilibrium with igg in comparison with a gel. the gels, however, provided higher adsorption capacity. at ion-exchange resins, the time necessary to adsorb 99% of total amount of igg was about 0.5-2 h. the affinity adsorbents were highly selective and therefore they adsorb very small amount of tested contaminant proteins (hsa, myo). the adsorption capacity was saturated by 50% in less than 25 min in all cases of dynamic adsorption measurements. this work was supported by a grant of 6th framework program of eu, project aims, no. nmp3-ct-2004-500160. microtechnology has for several years been applied within chemical reaction engineering. the advantages of microtechnology are that it makes it possible to develop light weight and compact systems, and the systems enable large surface-to-volume ratio, which results in low mass-transfer distances. in addition, parameters like pressure, temperature, residence time, and flow rate are more easily controlled. the use of microtechnology is also beginning to find its ways into the field of biotechnology. what we are aiming at is the development of a microreactor that can be applied as a production tool in industry as an alternative to conventional enzymatic reactors. our strategy is to use a small plate of a suitable material with microchannels fabricated into its surface, and the approach is to covalently couple enzymes into the microchannels. substrate can then be pumped through the channels and the enzymatic conversion will take place within the channels. as model enzyme in the development of the microreactor we are applying celb, a thermostable ␤-glycosidase from pyrococcus furiosus. kinetic resolution of racemic benzoin with different lyophilized microorganisms ç . babaarslan 1 ,ü. mehmetoglu 1 , a.s. demir 2 : 1 ankara university, faculty of engineering, department of chemical engineering, 06100, ankara, turkey; 2 middle east technical university, department of chemistry, 06531, ankara, turkey. email: barslan@eng.ankara.edu.tr (ç . babaarslan) the biocatalytic resolution of racemates is valuable tool for enantioselective synthesis and proved to be a convenient method for obtaining optically enriched compounds from their racemic form. in this work, enantiomerically pure benzoin which is one of the 2-hydroxy ketones was synthesized by kinetic resolution of racemic benzoin using different lyophilized microorganisms as lipase sources. the effect of lyophilized microorganism type, solvent type and acyl donor type on enantioselectivity were studied. in kinetic resolution experiments, lyophilized microorganism was resuspended in the media containing solvent, racemic benzoin and acyl donor at 30 • c and 150 rpm on orbital shaker. the reaction was followed by tlc during the experiment and the enantiomeric ratio of benzoin was determined by hplc analysis using chiral cell ob column. the best enantioselectivity value was obtained with lyophilized rhizopus orayzae cbs 112-07 as ee s = 16% and ee p = 30% (conversion = 35%) using thf as solvent and vinyl acetate as acyl donor. chromatography is one of the most important unit operations at separation, purification and concentration of monoclonal antibodies (mabs) at industrial scale. since these proteins belong to the group of immunoglobulins, their molecular weight (about 150,000 g/mol) or hydrodynamic radius (10.7 nm), respectively, is relatively large. the adsorbents used in ion-exchange/affinity chromatography of these biomolecules should thus provide a high pore accessibility coupled with a high value of specific surface area in order to ensure a sufficient ligand density and a high binding capacity. in this study, the pore accessibility of eight commercially available adsorbents designed for separation of mabs (mabselect, rprotein a sepharose ff, poros 50a, prosep -va, fractogel emd se hicap (m), sp sepharose ff, mep hypercel, s ceramic hyperd f) was investigated via size exclusion of standard-sized molecules (glucose, sucrose and dextrans with molar weight range 1200-40 × 10 6 g/mol) at nonbinding conditions. the experiments were conducted in a batch and column-mode. the batch experiments provided absolute partition coefficients, which were calculated from a mass balance and represent the fraction of pore water accessible to a solute. it was found that several adsorbents contained a small fraction of very small pores (less than 1 nm), whereas some adsorbents contained a significant fraction of pores larger than 100 nm. the column measurements provided relative partition coefficients, which were calculated from the retention volumes of solutes and represented the relative accessibility of pores, scaled between the accessibility of the smallest and largest solute used. when the absolute partition coefficients were recalculated into the relative form, it was found that the coefficients obtained by both methods correlated very well. the relative partition coefficients of solutes with the hydrodynamic radius of 10 nm (corresponding to mabs) was about 0.2-0.4 at ion-exchange and hydrophobic adsorbents and 0.6-0.8 at affinity adsorbents. the relation between hydrodynamic radius of the solutes and their partition coefficients was successfully described with the giddings random plane model. this work was supported by a grant of 6th framework program of eu, project aims, no. nmp3-ct-2004-500160. the transfer of laboratory results to a larger scale is often a critical step in process development to industrial application. the objective of this study was to scale-up the bioreactor for the fructosyltransferase production based on the results obtained in a 3 dm 3 stirred bioreactor. the investigations made in this bioreactor provided a clear picture of the effect of medium composition on the obtained fructosyltransferase (ftase) activity but the influence of mixing intensity was unequivocal. the increase of agitation rate had a positive effect up to a certain level where both fructosyltransferase and biomass production increased. the final optimal yield factor of ftase per dry cell mass obtained in the laboratory bioreactor was 10,300 u g −1 . we studied the effect of oxygen transfer on the process of ftase production at a larger scale, in 12 and 100 dm 3 mechanically stirred bioreactors and in air-lift bioreactors, 20 and 60 dm 3 whilst the medium composition was kept constant. the yield factors of ftase were comparable in both mechanically stirred bioreactors and they were about 7500 u g −1 . this decrease compared to that in the laboratory bioreactor could be explained by a slower cell growth. this fact was also confirmed by that glucose was not depleted till the end of fermentation and free fructose concentration was also lower. the yield factor of ftase was 7400 u g −1 in the 60 dm 3 air-lift reactor and 6100 u g −1 in the 20 dm 3 air-lift bioreactor. the lower yield of ftase in 20 dm 3 bioreactor was caused by a better biomass growth. this work was supported by slovak scientific grand agency, grant numbers vega 1/0066/03 and 1/0065/03 and by science and technology assistance agency, grant number apvt-20-025704. the poster gives an overview of the objectives and achieved results of an interdisciplinary project on direct product isolation from crude feedstocks using magnetic micro adsorbents in combination with suitable magnet technology. the project was funded by the deutsche bundesstiftung umwelt and was running between august 2002 and november 2004. in the course of the project several milestones could be met, which can be looked at as critical key points on a route towards an industrial realization of the process. among these milestones are: (i) the production of magnetic micro adsorbents with high capacity and selectivity in batches up to 50-100 g; (ii) the proof that the micro adsorbents can be reused many times; (iii) generation of a variety of recombinant tagged, active enzymes; and (iv) the design, assembly and operation of a fully automated pilot plant capable of generating approx. 5 g/h (≈95% purity) protein. the process was also simulated by help of the software tool superpro designer and simple mass balance and sorption equilibrium approaches were used to derive rules for estimating optimum process parameters and productivities. finally an environmental performance evaluation was conducted externally by the german dechema. in this study the effect of fed-batch cysteine addition to a culture of a high-gsh-accumulating yeast strain on the metabolism of glutathione was investigated. it is known that cysteine is the rate limiting amino acid in the biosynthesis of gsh. the influence of the consumption rate of cysteine on glutathione metabolism and growth of s. cerevisiae mt-32 was determined. the results show that for rates of consumption below a critical value the microorganism growth is similar to a culture without feed of cysteine, but glutathione production is increased two-fold. on the other hand, if cysteine consumption rate is above the critical value the changes of cell metabolism implies ethanol accumulation in the extracellular media which diminishes biomass synthesis. the maximum specific glutathione production in this case is maintained at two-fold; however, gamma-glutamylcysteine accumulation is increased. cysteine present in culture media directs cell metabolism to a greater synthesis of ammonia and amino acids. hydrophobic interaction chromatography (hic) exploits the hydrophobic properties of protein surfaces for separation and purification by performing interactions with chromatographic sorbents of hydrophobic nature. in contrast to reversed phase chromatography this methodology is less detrimental to the protein and is therefore more commonly used in industrial scale as well as in bench scale when the conformational integrity of the protein is important. hydrophobic interactions are promoted by salt and thus proteins are retained in presence of a cosmotropic salt. when proteins are injected on hic columns with increasing salt concentrations under isocratic conditions only, a fraction of the applied amount is eluted. the higher the salt concentration the lower is the amount eluted protein. the rest can be desorbed with a buffer of low salt concentration or water. it has been proposed that the stronger retained protein fraction has partially changed the conformation upon adsorption. this has been also corroborated by physicochemical measurements. the retention data of five different model proteins and 10 different stationary phases were evaluated. partial unfolding of proteins upon adsorption on surfaces of hic-media were assumed and a model describing the adsorption of native and partial unfolded fraction was developed. furthermore we hypothesize that the surface acts as catalyst for partial unfolding, since the fraction of partial unfolded protein is increasing with length of the alkyl chain. stationary phases for bioseparation of glycoproteins j. aniulyte 1 , j. liesiene 1 , b. niemeyer 2 : 1 department of chemical technology, kaunas university of technology (ktu), radvilenu pl. 19, 50254 kaunas, lithuania; 2 institute of thermodynamic, helmut-schmidt-university/university of the federal armed forces hamburg, holstenhofweg 85, hamburg, germany d-22043 nowadays glycomics raise new challenges for affinity chromatography related with an abundance of glycoconjugates in living organisms and with scaling-up of the preparative processes. economics, efficiency and practicality dictate the search of novel chromatographic biospecific adsorbents that could contribute to enhancing the productivity of the affinity separation process. the purpose of the work was to prepare cellulose-and silica-based biospecific adsorbents with immobilized lectins and to evaluate them for the affinity chromatography of glycoproteins. cellulose-based matrix granocel and silica coated with hydrophilic polymers were used as a support. the effect of support characteristics, such as pore size, chemistry of active groups and their density on the support' surface on lectin immobilization and on the efficiency of adsorbents obtained were evaluated.three different methods were used for the activation of the support: oxidation with sodium periodate, modification with pentaethylenhexamine (spacer arm of 18 atoms) and carbonyldiimidazole activation.cona and wga two lectins of different molecular weight and shape were selected to notice differences resulting from the size and diffusion behaviour. chromatographic performances of the adsorbents were studied applying two different glycoproteins (god and fetuin) carrying specific terminal glycomoieties of mannose (god), and n-acetylglucosamine (fetuin) for specific interaction with cona and wga, respectively. the adsorbents demonstrated high affinity to glycoproteins with a sorption capacity in the column up to 7.4 mg per ml support and a high recovery (up to 93%). it was shown that spacer arm affected ligand coupling kinetic as well as the chromatographic behavior of the adsorbents obtained. the adsorption isotherms of god onto cona adsorbents reveal an adsorption behavior with high and low affinity binding sites. the dissociation constant k d of the ligand-sorbate complex is approximately 1 × 10 −6 m, and 0.4 × 10 −5 m, respectively. it was supposed that the second step is related to the sorption of solvated god onto already adsorbed god forming sorbate dimers. cell disruption and chromatography are key unit operations in the downstream processing of an intracellular product. the cost involved in the extraction and purification of intracellular products can be reduced by selective release of proteins and reduction in the number of steps involved in the purification. the extent of disruption can be varied to provide a selective release, limiting the release of the contaminant proteins. the particle size distribution of the cell debris in the resulting suspension depends on the extent of disruption. expanded bed adsorption chromatography allows for the direct capture of the proteins from an unclarified suspension. this technique allows for the integration of solid-liquid separation, concentration and preliminary purification in one unit operation. a perfectly stable expanded bed can be obtained by choosing the appropriate flow conditions and a suitable adsorbent. the difference in the density between the adsorbent and the cell debris in the suspension, permits the cell debris to flow through the column without blocking, whilst the protein molecules in the suspension are adsorbed onto the adsorbent. after sample application, the bed is washed with buffer and the proteins eluted from the column in the packed bed mode. the presence of the cell debris in the feedstock influences the expansion of the bed and the adsorption of protein molecules. the physical properties of the suspension obtained after cell disruption depends on the extent of disruption. the particle size distribution of the cell debris, the viscosity and the release of soluble proteins and other intracellular components are influenced by the extent of disruption. the influence of the extent of disruption of e. coli on the expansion of the bed and the adsorption of ß-galactosidase is presented in the current study. e.coli cells were disrupted at different operating pressure using a high pressure homogenizer. the resulting crude homogenate is subjected to expanded bed adsorption chromatography using streamline deae as adsorbent. the disrupted suspension was characterised in terms of viscosity, density, particle size distribution of the cell debris and the extent of protein and ß-galactosidase released. the interaction between cell debris and adsorbent was quantified as the cell transmission index (ratio of the amount of cells present in the sample before and after passing through the bed). the expansion of the bed at a constant settled bed height and flow rate was measured. the influence of the cell debris on the extent of adsorption of ß-galactosidase has been quantified in terms of dynamic binding capacity (dbc) at 10% of the inlet concentration. the dbc of ß-galactosidase that was released by disruption at 7500 psi (5%, w/v, w/w, 1 pass) was found to be 100 u/ml of adsorbent while dbc of samples disrupted at 2500 psi (5% w/v, w/w, 1 pass) was 67 u/ml of adsorbent. the extent of disruption of e. coli over a wide range and its effect on the expansion and adsorption will be presented. study of dna binding during expanded bed adsorption and factors affecting adsorbent aggregation ayyoob arpanaei 1 , niels mathiasen 1 , timothy hobley 1 , owen rt thomas 1,2 : 1 center for microbial biotechnology, building 223, biocentrum-dtu, technical university of denmark, 2800, lyngby, denmark; 2 department of chemical engineering, university of birmingham, edgbaston, b15 2tt, uk. e-mail: aa@biocentrum.dtu.dk (a. arpanaei) the adsorption of sonicated calf thymus dna (as a model dna molecule) to biosepra q hyper z adsorbents was evaluated in batch and expanded bed modes. stability of the expanded bed during feedstock loading was also studied. two batches of prototype q hyper z (batch 1 and 2) were examined, which had ionic capacities measured to be 122 and 147 mmol cl − /ml support respectively. in all adsorption experiments a 50 mm tris-hcl ph 8 buffer was used. maximum static binding capacities of adsorbent batches 1 and 2 were determined to be 20.9 and 23.8 mg dna/ml particle, respectively. dynamic binding capacity at 10% breakthrough (dbc 10% ) was measured in a 1-cm diameter eba column containing 6.7±0.5 cm settled bed with a feed of 20 g/ml dna. dbc 10% of the adsorbents were 7.4 and 12.7 mg dna/ml support for batches 1 and 2, respectively in buffer containing no salt. however, the maximum dbc 10% for batch 1 (10.1 mg dna/ml support) and 2 (18.9 mg dna/ml support) were obtained in buffers containing 0.25 and 0.35 m nacl, respectively. further increases in salt concentration led to a decrease in dbc 10% for both adsorbent batches. the bed compression during loading that was observed in experiments at high conductivities (achieved by adding salt) was less than that seen with low conductivity (2 ms/cm) solutions. aggregation of adsorbent particles and channeling of flow were not observed in the presence of salt concentrations more than 0.1 m. the effect of different concentrations of dna during loading in the presence of 0.15 m nacl was studied. it was found that increasing dna concentrations in the feed from 20 to 40 g/ml, 60 to 80 g/ml resulted in a decrease of dbc 10% by 16, 24 and 30%, respectively. the bed compressed slower during loading of feedstock with low dna concentrations compared to that for higher concentrations. the expanded bed showed a partly reversible compression behavior during feedstock loading. this is attributed to the electrostatic interaction between dna adsorbed on the particles surface and rearrangements of dna strands as the number of free ligands on the adsorbent surfaces decrease during loading. large-scale production of plasmid dna for gene therapy and dna vaccination applications has become necessary as a result of the increasing number of approved protocols using non-viral vectors for gene delivery. a major challenge of large-scale plasmid production is to establish a robust cgmp manufacturing capable of producing hundreds of milligrams or grams of a pharmaceutical grade product. alcohol and salt precipitation are operations largely used in the early steps of plasmid downstream processes. however, there are few systematic studies on the influence of these precipitation agents in the final plasmid recovery and purity. in this work, alcohol and salt precipitation steps used in a plasmid purification process developed by our group have been optimized aiming at large-scale production. the optimization of alcohol precipitation indicated that almost 100% of the pdna precipitated when 0.6 vol. of isopropanol were used. the studies also indicated that the precipitation profile was strongly influenced by pdna initial concentration. finally, the final plasmid recovery and purity after a sequential alcohol and salt precipitation were strongly dependent on the concentrations of these precipitation agents. thus, a commitment between high recovery and purity level should be made during the development of the downstream processes. comparison of novel and conventional processes for protein refolding and initial purification h. ferré 1,2 , u. jørgensen 1 , l. scale down of downstream processing unit operations is convenient for assessing process alternatives, particularly if feedstock is scarce. in this study it was imperative to use the smallest possible scale for comparison of a new system for continuous protein refolding and direct expanded bed adsorption (eba) capture with a traditional process composed of discrete operations of batch renaturation, centrifugation, microfiltration and packed bed chromatography (pbc). minimisation of the scale was restricted by the eba step: the smallest practical scale being a 1cm diameter column with 5-6 cm of settled bed, expanded two fold. in order to permit a fair comparison a similar column diameter and adsorbent volume was used in the packed bed process. in both alternatives, chelating media charged with cu 2+ was used and a feedstock of denatured hat-tagged human beta-2 microglobulin (hat-h␤2m). following batch refolding and clarification, the performance of the packed column was severely hampered due to fouling of the top adapter. reducing the protein loaded to the packed bed to 50% of dbc working lead to a recovery of 68.5% at a purity of 87% and 4.6-fold concentration. the eba-based process performed unimpeded and productivity was calculated to be 8% higher than for that employing a packed bed. however, due to the severe scale restrictions placed on the eba process, which limited optimisation, significant productivity improvements of eba over packed bed are expected at larger scale. high gradient magnetic filtration has the potential for rapid processing of large volumes of crude bioprocess liquors when magnetic adsorbents are employed. the binding of a protein to a superparamagnetic solid support provides a unique selective 'handle'. typically the focus is placed on using the magnetic handle for direct capture of a protein from a fermentation broth. however, magnetic adsorbents may provide solutions to a range of downstream processing problems and in this presentation we illustrate this with a number of case studies. using whey as a model system, we show that the extent of the tryptic hydrolysis (ca. 0.2 mg/ml added enzyme) of proteins could be controlled by adding benzamidine-linked magnetic adsorbents after a given period (4-15 min), followed by removal of the loaded adsorbents using a magnetic filter. hydrolysis was stopped effectively and approx. 50% of the added trypsin could be recovered. a coupled process was devised for the refolding and purification of inclusion body proteins. solubilised (in 8 m urea) inclusion bodies of recombinant histidine affinity tagged human beta 2 microglobulin (hat-h-beta2m), were refolded by dilution in a pipe reactor (14 s), then captured directly on cu(ii) charged magnetic immobilised metal affinity adsorbents in a second pipe reactor (10 s residence time). loaded adsorbents were retained in a magnetic filter, then washed and the protein eluted. a generic framework for the prediction of scale-up when using compressible chromatographic packings r. tran 1 , j. joseph 1 , a. sinclair 2 , y. zhou 1 , n. packed bed chromatography is the pre-eminent technique in the downstream purification of many biological products. the aspect ratio of a packed bed has a significant effect on the column pressure drop by virtue of wall support which is reduced at low aspect ratios. this can result in unexpectedly high pressures during manufacturing caused by the compression of the matrix via drag forces due to fluid flow through the bed. the need for an accurate model to predict flow conditions at increasing scale is essential for the scaling-up of chromatographic processes and for avoiding bed compression during operation so that maximum throughput can be achieved. several studies have generated correlations which allow for the prediction of column pressure drops but have either been mathematically complex, which makes their practical use unfeasible, or they have used highly specific empirical constants and hence require a large amount of experiments to be performed before they can be used. in this study, we have established relationships to link the critical velocity of operation, to bed height (l), column diameter (d c ), feed viscosity (µ) and also to the matrix rigidity through the level of agarose cross-linking (a%). the correlation is straight forward to use and involves very few system-specific constants thus significantly reducing the need for any preceding laboratory-scale experimentation. this paper describes the series of experiments that were performed to establish the correlation, using a range of cross-linked agarose matrices (2-10%), at various aspect ratios, fluid flow rates and varying viscosities (0.9-1.85 mpas). a mathematical model was developed where parameter estimation for multi-variables was achieved by least squares optimisation. the model can be used to predict the extent of compression in industrial chromatography applications and will be useful in the development of chromatographic operations and for column sizing. institute of process engineering, swiss federal institute of technology, zurich. e-mail: makart@ipe.mavt.ethz.ch (s. makart) simulated moving bed (smb) technology receives increasing attention in biotechnology and in the biopharmaceutical industry as it enables an increase in productivity per unit mass of stationary phase, reduced solvent consumption, and fast and reliable scale-up. combining continuous chromatographic separation unit and reactor should enable the production of biopharmaceuticals and fine chemicals with high purity and yield at the same time. due to the increasing demand of enantiopure intermediates in the pharmaceutical industry, biocatalytic processes gain more and more importance because of their excellent enantioselectivity. yet the application of biocatalytic carbon-carbon bond formation on process scale is often hampered by an unfavourable equilibrium position and difficult downstream processing due to substrate/product mixtures. coupling a continuous separation unit to such a process would improve the feasibility by driving the reaction to completion and thus increasing the overall yield. we will discuss the design of such an integrated biocatalytic/smb process, taking the formation of l-allo-threonine from glycine and acetaldehyde, catalysed by the glycine-dependent aldolase glya from e. coli, as a model reaction. the enzyme exhibits absolute stereoselctivity at the c-alpha atom, whereas selectivity is less strict at c-beta. in situ product removal, by the integration of an smb unit, would aid to maintain a high diastereomeric excess as it shortens the residence time of the products in the reactor, in addition to shifting the reaction to the product side. the in-line coupling of the chromatographic unit to the enzyme reactor requires the use of the same solvents for reaction and separation, so the choice is limited to aqueous solutions close to physiological ph, limiting in turn the possible stationary phase materials. in a screening of different cation exchangers, amberlite cg-120 ii gave promising results: threonine is more retained than glycine, acetaldehyde is poorly and the cofactor plp is not retained. adsorption isotherms were determined by the retention time method and a smb under process conditions was simulated. by improving the packing of the column, i.e. achieving a more even particle size distribution, we tried to further increase the efficiency of the separation step. the application of enzymes for the synthesis of optically active substances is nowadays of growing importance in the pharmaceutical industry. this requires a proper cultivation of the microorganism as well as a posterior isolation process yielding a constant catalyst quality at high purity. goal of this project is the development of an integrated process for the production and isolation of a lipase from trichosporon beigilie and its posterior application for the enantioselective synthesis of pharmaceutical products. the cultivation of the microorganism is optimised in a laboratory and pilot-scale fermenter in a fed-batch mode. parameters like media composition, temperature, ph and aeration rate are set up. taking advantage of the localisation of the enzyme (covalently attached to the cell membrane) the first step of product isolation consists of a continuous cell disruption. optimal results are achieved with the continuous bead mill disruption process (68% enzyme release with a specific activity of 0.8 u/mg of protein). the non disrupted cells are recycled as inoculums for a new cultivation, increasing the yield of the overall process (by 5-times in the pilot-scale fermenter). in order to isolate the product two different process sequences are considered. the first one consists of an extraction (peg and phosphate buffer) coupled to an ion-exchange chromatography (q-sepharose ff). the second one applies a precipitation step with ammonia sulphate followed by a hydrophobic interaction chromatography (sepharose-hic) provid-ing a lipase yield of 72% (8-times higher than the one provided by combining extraction-chromatography). an ultrafiltration process is used in order to concentrate the lipase and its final properties (molecular weight, isoelectric point, activity, stability and kinetic data) are studied using p-nitrophenylacetate as model substrate. the relevance of the obtained product for its application in the pharmaceutical industry is proven by transforming (r,s)-naproxen-methylester into (s)-naproxen acid with an enantiomeric excess of >99% (after 24 h). biotensides (sugar fatty acid esters, sfaes) find nowadays a wide range of applications in pharmaceutical, personal care and food industry because of their biocompatibility, biodegradability and special surfactant properties. goal of this project is the development and optimisation of an integrated process for the enzymatic synthesis of sfaes from renewable sources to be used in cosmetic formulations. the following figure shows the scheme of the overall process. commercial and also new screened lipases are applied in the reaction between sugar and fatty acid. the mixture grade of the initial reaction system is increased by ultrasounds taking into account the influence on the catalyst characteristics and also the necessity of an organic solvent as adjuvant. the reaction takes place in an enzymatic membrane reactor (emr) equipped with an ultrafiltration membrane which retains the catalyst. the separation of the by-product (water) from the rest of the components can be achieved by means of a pervaporation unit which coupling to the emr allows the semi-batch process. in order to separate the esters from the fatty acid a stepwise elution chromatography method is developed using silica as adsorbent and ethyl acetate and methanol as eluents. with this system 91% of the dimer is isolated with purity (hplc) of 93%. the application of a dialysis membrane technique allows the separation of 80% of the fatty acid by building ester micelles changing the polarity of the organic solvent used as eluent. solubility and crystallisation properties of recombinant bacillus halmapalus ␣-amylase cornelius faber centre for microbial biotechnology, biocentrum dtu, building 223, 2800 lyngby, denmark a comprehensive knowledge of solubility properties is a prerequisite for the efficient design and operation of bulk enzyme recovery processes, however, complete phase diagrams are only available for very few proteins, in particular lysozyme of high purity. here, we present the results of detailed solubility studies in aqueous solutions of an industrially relevant ␣-amylase of technical grade. experiments were conducted in small scale batch mode (working volume of 1 ml). the influence of selected cations and anions from the hofmeister series on the stability of the ␣-amylase was examined. the hofmeister series for anions was followed in the correct order at all salt concentrations studied, i.e. from 0 to 1 m, whereas the series was reversed for monovalent cations at concentrations up to 0.5 m, with the exception of lithium. to further investigate why the position for lithium was different to the hofmeister series established for lysozyme, the zeta potential of protein solutions at low concentrations of selected salts was determined. the results of these measurements indicate a pronounced effect of lithium on the zeta potential, as compared to other salts. in particular, the ph of zero zeta potential (i.e. the pi) was shifted approximately 0.5 ph units towards alkaline conditions in the presence of lithium, whereas the pi stayed almost constant for sodium and potassium. since the solubility exhibits a minimum at ph-values at or near the protein's pi, shifts in ph caused by salt addition are important to identify and quantify to avoid uncontrolled phase separation. the measurement of the zeta potential of proteins in solution holds significant promise as an attractive tool for understanding and controlling processes that are operated close to the solubility limit and which are often plagued by uncontrolled precipitation or crystallisation and thus rely on carefully chosen operating conditions. polyphenolic interactions with potato proteins during industrial expanded bed adsorption processing sissel løkra 1 , knut olav straetkvern 1 , bjørg egelandsdal 2 , gerd vegarud 2 : 1 department of natural science & technology, hedmark university college, n-2317 hamar, norway; 2 norwegian university of life sciences, 1432 as, norway. e-mail: sissel.lokra@lnb.hihm.no (s. løkra) in plant extracts it has been shown that polyphenols have a tendency to react with proteins, either by covalent or non-covalent interactions. these reactions can induce changes in the surface properties of the proteins, and, e.g. cause proteins to be insoluble and precipitate at ph-values below their isoelectric point. potato proteins have a high nutritional quality and show interesting functional properties in food systems. moreover, chlorogenic acid (ca) and caffeic acid constitute about 90% of the total polyphenol content of potato tuber. we have experienced expanded bed adsorption (eba) chromatography to be a method well suited for recovering industrial proteins from potato starch effluent. the process separates proteins from polyphenolic pigments, fiber and minerals. during the adsorption step, patatin, the major potato tuber protein shows complex binding kinetics demonstrated by breakthrough curves. in addition to diffusion limitations in the eba resin, changes in protein structure and surface properties probably are likely to affect this adsorption behavior. reactions between ca and patatin might result in a range of interactions for different species of the same protein. this project therefore aims to assess the interactions between ca, patatin and other major tuber protein fractions and how these changes affect the protein capture in eba. changes in size and charge are screened in 2-d electrophoresis and analyzed further. samples of different protein fractions are taken from breakthrough curves and dynamic binding capacity experiments in model systems with real feedstock. sandwich hybridisation assay for analysis of brewery contaminants s. huhtamella 1 , m. leinonen 1 , t. nieminen 1 , a. breitenstein 2 , p. neubauer 1 : 1 bioprocess engineering laboratory, university of oulu, finland; 2 scanbec gmbh, halle, germany. email: peter.neubauer@oulu.fi (p. neubauer) here we describe the development of a sensitive, cultivationindependent analytical method for the analysis of brewery contaminants which can be performed within three hours in crude sample extracts. the method is based on 16s rrna detection by a paramagnetic bead based sandwich hybridization assay (sha) with two oligonucleotide probes designed to either detect the species or a group of contaminants. the signals were read out either by a fluorimeter (rautio et al., 2003; leskelä et al., 2005) or potentiometrically with an electric biochip instrument (ebiochip systems) . this assay is advantageous over rt-pcr becasue it only detects viable cells and the method can be directly applied to crude cell extracts without prior purification. we describe the principle of designing and evaluating a series of groupspecific lactobacillus probes and the optimisation towards effective cell lysis and high assay sensitivity. the applicability of the sha was evaluated with real brewery samples and the results were compared to routine tests. in all steps of the evaluation the reliability and usability of the method was prioritised. the optimised method combined with a 24 h pre-enrichment period gave reliable results, had a detection limits of about 10 4 -10 5 cells per assay and was easily applicable in a brewery environment. biodesulfurization is one of the possibilities studied by the researchers to attain the maximum sulfur levels imposed for a near future by governments (european directive, 2003) . rhodococcus erythropolis igts8 is a natural and strictly aerobic microorganism able to remove the sulfur atom from dibenzothiophene (dbt) in a selective way (4s route (oldfield et al., 1997)), obtaining 2hydroxibifenyl (hbp) and sulfate. growth is carried out using the experimental procedure performed in previous works dealing with the inoculum built up, media composition and operational conditions (del olmo et al., 2005a (del olmo et al., , 2005b . this work is focused to determine the oxygen uptake rate during the production of the biocatalyst. four experiments were carried out at a biostat b fermentor (braun biotech.) using as only variable the constant stirrer speed used: 150, 250, 400 and 550 rpm. oxygen uptake rate have been determined by means of two methods: dynamic technique at different times during growth for few seconds to ovoid influences and from oxygen profile when the term dealing with oxygen transfer rate is known (predicted by the model proposed in a previous work (garcía-ochoa and gómez, 2004)). our values obtained from the techniques used present the same tendency in all the runs carried out: our values from dynamic technique is always lower than our values obtained from the oxygen profile. these values are modeled and the difference observed is explained due to the cellular economy principle: during the seconds employed in the dynamic technique determinations microorganism do not produce 4s route enzymes. it was studied different methods to recover a p. salmonis antigenic protein from recombinant e. coli cells. this protein has shown be highly effective in vivo vaccine. it has the ability to stimulate salmon immune system protecting them against of aggressive disease salmonid rickettsial syndrome resolving by this way a great problem of salmonid aquaculture. biomass obtained from iptg induced e. coli bl21 (de3) codon plus culture was used for soluble and insoluble antigenic protein recovery. it was evaluated recuperation by glass bead mill, freezing and thawing, osmotic shock and lisozyme/edta treatments, all of them applied in single or combined way. biomass was measured by dry weight of cells, soluble protein concentration was quantified according to bradford method, and antigenic protein was identified by sds-page and western-blot analysis. cells treated with lisozyme/osmotic shock and then glass bead mill the soluble protein was a 62.9% of the dry weight cell mass whereas using lisozyme/edta and glass bead mill as a single treatment only a 24.4 and 22.6% were obtained respectively. the freezing and thawing disruption treatment released less than 5% of soluble protein, as well as the osmotic shock procedure too. the sds-page and west-ernblot analysis revealed that the antigenic protein must be purified from the insoluble cell fraction when physical or mechanical disruption methods were employed and from the soluble cell fraction when chemical or enzymatic treatments were used. we propose investigate in further studies the inclusion bodies formation to design an efficient purification procedure for the target protein. the iso-peroxidase pox2 from garlic bulb allium sativum that represented the major peroxidase activity was purified to homogeneity. the enzyme is monomeric and has a molecular mass of 36 kda, and a pi around 9. the optimum temperature ranged between 25 and 40 • c, while optimum ph was around 5. pox2 appeared remarkably thermostable since it retained 50% of its activity at 50 • c for at least 6 h. in addition, the enzyme was stable at a ph range from 3, 5 to 11. kinetic constants were calculated, the apparent k m values were 500 and 150 m for gaïacol and h 2 o 2 , respectively. the high thermostability of pox2 may represent clear advantages in a number of processes including immobilizing peroxidase and use it as a biosensor to detect oxidant component as h 2 o 2 and other peroxides. immobilization of pox2 was achieved by binding covalently the enzyme to a sepharose matrix (bead and membrane va epoxy). the immobilized peroxidase showed great stability at heat and storage than the soluble enzyme. the native enzyme retained 55% of its activity at 60 • c for 10 mn while the immobilized pox2 retained full activity for 35 mn at the same temperature. in other side, the free enzyme retained full activity for at least one month and a half during storage at 25 • c, and lost 50 m of its activity after 2 months. the immobilized form of pox2 retained complete activity for 2 months at the same temperature. the immobilized enzyme was used to detect h 2 o 2 in some food components such as milk and fruit juices. in a second study, same experiments were performed in order to detect the smaller quantity of added h 2 o 2 to the farming milk. purpose: a new research field has been created to begin to address protein function at level of regulation of enzyme activity. this new area has been given the name chemical proteomics, or activity-based proteomics (abps), and makes use of small molecules that can covalently attach to catalytic residues in an enzyme active site. the selectivity of the chemically reactive group allows specific proteins or protein subset to be tagged, purified and analyzed. methods: this molecule (abps) has three subsets: tag, linker, and warhead. warhead is a nucleophile and attach to active site. linker is a polypeptide that makes a simple connection between warhead and tag. tag is fluorcent or radioactive material that facilitates the detection of drugs. findings: several diseases such as cancer, rheumatoid arthritis and osteoporosis are associated with elevated levels of protease activity. serine hydrolyses abps have been used to profile enzyme activity in a diverse range of cancer cell lines. in studies comparing metastatic and non-metastatic human breast cancer models, it was shown that the former exhibited a higher activity of a ␥-glutathione-s-transferase, an enzyme that has not previously been associated with breast cancer. discussion: additionally, abps can be used to develop robust screens for small molecule inhibitors of a specific enzyme target within a large family of related enzymes. this method of inhibitor screening allows compounds to be assayed for both potency and selectivity against a set of related in complex biological samples. this technique is able to identify novel enzymatic proteins and drugs and has the potential to accelerate the discovery of new drug target. a cyclodextrin glycosyltransferase (cgtase) from a new isolated strain from bacillus clausii e16, was purified through q-sepharose, gel filtration chromatography and deae-sephadex a-50. the mw of the pure enzyme was 75 kda with sds-page. the enzyme displayed optimum ph value and ph stability at ph 6.0 and in range of 6.0-11.0, respectively. the optimum temperature and thermostability were at 55 • c and up to 60 • c by 1 h, respectively. the k m and v max were 2.85 mg/ml and 80.0 mol/min mg and 0.83 mg/ml 13.45 mol/min mg using maltodextrin and soluble starch, respectively. the isoeletric point was 4.8 and the n-terminal region of the pure enzyme was sequencing by maldi-tof-ms. the ratio of ␣-, ␤and ␥-cd was 0.29:1.00:0.79 and 0:1:0 with maltodextrin and soluble starch at 2.5%. application of magnetic separation technology for recovery of immobilised lipases nadja schultz 1 , anke neumann 1 , george metreveli 3 , matthias franzreb 2 , christoph syldatk 1 1 chair of technical biology, university of karlsruhe, engler bunte ring 1, d-76131 karlsruhe; 2 forschungszentrum karlsruhe, institute of technical chemistry, water-and geotechnology; 3 inst. für wasserchemie, engler bunte ring 1,76131 ka. e-mail: nadja.schultz@ciw.uni-karlsruhe.de (n. schultz). url: www.fzk.de/itc-wgt (m. franzreb) first results on the development of the magnetic separation technology for the recovery of immobilised lipase from a 2-phase-system, which should be suitable for a large scale use in future, known as high gradient magnetic separation (hgms) are presented. the application of immobilised lipases makes the reuse of the enzyme in a process possible and is therefore interesting for industrial applications. in this study immobilised lipase is used in a 2-phase-system. here the new approach to recycle and reuse the lipase, immobilised on magnetic particles, from a 2-phase-system with the help of the new high gradient magnetic separator (hgms) is examined in 30 ml scale. as model enzyme for the immobilisation on magnetic microparticles (polyvinyl alcohol (pva), 1-2 m) the commercially available (novonordisc) lipase a (cala) from candida antarctica was used. necessary for screening of immobilisation methods and characterisation of the immobilised lipase (candida antarctica) was the development of a robust, simple and rapid chromophoric activity assay. therefore the pnpp-lipase assay was optimised for direct application on immobilised lipases (in preparation schultz et al., 2005) . further more a ph-stat-assay for measuring the activity of free and immobilised cala in a 2-phase-system of tributyrate and buffer was optimized for this system. another important basis for the realisation of the recovery of immobilised lipases was to optimise the immobilization technique of the lipase. furthermore approaches for the explication of generally empirical based immobilization techniques on insoluble support were made. hereby we successfully applied the zeta potential measurement on the immobilization behaviour of the lipase cala. for to determine the operating temperature for the biomagnetic separation procedure we studied stability analysis of free and immobilised lipase cala at different temperatures (37, 25, 4, −20 • c) and at different ph values (ph 6, 7 and 8). the optimal temperature and ph value for the free and immobilised lipase was determined. presently and constructively on the so far developed methods and techniques we intensively work on the demonstration and feasibility of the recovery of immobilized lipase from a 2-phase-system. challenging approaches and first results on the recovery of immobilized lipase from a 2-phase-system in 30 ml scale were shown already. nowadays glycomics raise new challenges for affinity chromatography related with an abundance of glycoconjugates in living organisms and with scaling-up of the preparative processes. economics, efficiency and practicality dictate the search of novel chromatographic biospecific adsorbents that could contribute to enhancing the productivity of the affinity separation process. the purpose of the work was to prepare cellulose-and silica-based biospecific adsorbents with immobilized lectins and to evaluate them for the affinity chromatography of glycoproteins. cellulose-based matrix granocel and silica coated with hydrophilic polymers were used as a support. the effect of support characteristics, such as pore size, chemistry of active groups and their density on the support' surface on lectin immobilization and on the efficiency of adsorbents obtained were evaluated. three different methods were used for the activation of the support: oxidation with sodium periodate, modification with pentaethylenhexamine (spacer arm of 18 atoms) and carbonyldiimidazole activation. cona and wga two lectins of different molecular weight and shape were selected to notice differences resulting from the size and diffusion behaviour. chromatographic performances of the adsorbents were studied applying two different glycoproteins (god and fetuin) carrying specific terminal glycomoieties of mannose (god), and n-acetylglucosamine (fetuin) for specific interaction with cona and wga, respectively. the adsorbents demonstrated high affinity to glycoproteins with a sorption capacity in the column up to 7.4 mg per ml support and a high recovery (up to 93%). it was shown that spacer arm affected ligand coupling kinetic as well as the chromatographic behavior of the adsorbents obtained. the adsorption isotherms of god onto cona adsorbents reveal an adsorption behavior with high and low affin-ity binding sites. the dissociation constant k d of the ligand-sorbate complex is approximately 1 × 10 −6 , and 0.4 × 10 −5 m, respectively. it was supposed that the second step is related to the sorption of solvated god onto already adsorbed god forming sorbate dimers. quantitative methods in high throughput screening of aqueous two phase systems matthias bensch, björn selbach, jürgen hubbuch institute of biotechnologie 2, forschungszentrum jülich, germany purification of biopharmaceuticals is one of the most expensive and at the same time least understood steps in bioprocesses. during the process development for protein production, short time to market and the demand for cheap processes dominate today's process development. one way of reducing process costs is to implement integrative processes. aqueous two phase systems (atps) combine the advantages of removing cell debris and simultaneously purifying and concentrating the target protein, however, to the cost of highly complex systems which are difficult to predict and optimize. using high throughput screening techniques in the development of atps processes thus seems to be an ideal candidate for achieving both a reduced development time and an economical process without the need for preliminarily well characterized systems. in this study, we use the robotic system tecan freedom evo tm as an automation platform for the evaluation of aqueous two phase systems. central to this workstation are the integrated hardware as liquid handler, gripper, reader and centrifuge. we have created high throughput methods for rapid parameter estimations. as a first step, pipetting and mixing had to be calibrated for the use of highly viscous polymer solutions which are common in atps. the focus of the current work lies on the integration of the automatic preparation and analysis of two phase systems in microtiter plate scale. the robotic platform can now automatically create aqueous two phase systems and measure characteristic values such as binodal curves, protein concentrations and protein distributions between the two phases. the major bottleneck of hts processes, namely the rapid analysis of impure systems, is tackled by using automated elisa tools. depending on the intended use, the high number of measured partition coefficients and yields can be used for modelling or rapid process design. today, most optimisations of chromatography separations are based on experimental work and rule of thumb. the pat initiative has opened up for a model-based approach for downstream processing of pharmaceutical substances. this work uses a nonlinear chromatography model to optimize an ion exchange separation step. the general rate model with langmuir mpm kinetics described the behaviour of the components in the column. the optimal operating points using both productivity and yield as objective functions were found. the optimizations were run with both igg and bsa as target proteins respectively to compare their optimal operating points. the requirement on the optimal operating point was a purity of at least 99%. this requirement was added to the optimization problem as a nonlinear inequality constraint. flow rate, loading volume, start salt concentration in elution, elution gradient and cut points were used as decision variables in the optimization. the more retained component, bsa, was much easier to separate from igg with a gradient elution than igg from bsa while still retaining a high productivity and yield. the higher load volume at the optimal operating point, with bsa as target protein, causes a displacement of igg and thereby improving the separation. a high productivity at the yield optimum was still possible with bsa as target protein. both a lower productivity and yield was obtained with igg as target protein. optimisation and robustness analysis of a hydrophobic interaction chromatography step niklas jakobsson, marcus degerman, bernt nilsson department of chemical engineering, lund university, p.o. box 124, se-221 00 lund, sweden process development, optimisation and robustness analysis for chromatography separations are often entirely based on experimental work and generic knowledge. the present study proposes a method of gaining process knowledge and assisting in the robustness analysis and optimisation of a hydrophobic interaction chromatography step using a model-based approach. factorial experimental design is common practice in industry today for robustness analysis. the method presented in this study can be used to find the critical parameter variations and serve as a basis for reducing the experimental work. in addition, the calibrated model obtained with this approach is used to find the optimal operating conditions for the chromatography column. the methodology consists of threes consecutive steps. firstly, screening experiments are performed using a factorial design. secondly a kinetic-dispersive model is calibrated using gradient elution and column load experiments. finally the model is used to find optimal operating conditions and a robustness analysis is conducted at the optimal point. the process studied in this work is the separation of polyclonal igg from bsa using hydrophobic interaction chromatography. department of biochemistry and microbiology, ict prague, technicka 3, prague cz-166 28, czech republic the display of novel metal binding sites on the surface of the biosorbent represents potent tool to increase its binding capacity and improve selectivity. in this study, the 15.8 kda transcriptional regulator merr of mercury-inducible mer operon of tn21 exhibiting high affinity and selectivity towards hg 2+ , was displayed on the surface of s. cerevisiae. to achieve this, merr was genetically fused with gene encoding c-terminal domain of ␣-agglutinin which resulted in covalent attachment of the of the fusion protein on the cell wall glucan via glycosylphosphatidylinositol anchor. to evaluate the performance of such modified whole-cell biosorbent with specific regard to hg 2+ , we constructed a new biosensor e. coli strain, which utilizes kanamycine resistance gene as a reporter under the control of mer promoter. it allowed determination of hg 2+ in a range of 5-100 nm by simply monitoring the growth in the hg 2+ /kanamycine-containing media. the effect of genetic engineering of s. cerevisiae surface by merr became significantly pronounced in biosorption experiments with solutions containing 10 m hg 2+ when modified cells accumulated 2.5-fold more hg 2+ than the control strain expressing mere anchoring domain. sensitivity analysis of amino acids in simulated moving bed chromatography ju weon lee, chong ho lee, yoon mo koo center for advanced bioseparation technology, inha university, inchon 402-751, korea. e-mail: ymkoo@inha.ac.kr (y.m. koo) the difficulty of simulated moving bed (smb) design is that the optimization of the operation conditions relies on the determination of accurate adsorption isotherms. most smb chromatograph is carried out under nonlinear conditions, and the nonlinear behavior should be considered properly in the equilibrium isotherms. the other difficulty is the smb operation which has the characteristics of continuous process, all flow rates and switching time of valves should be maintained during the operation of smb. if the disturbances of operating conditions and isotherm parameters are occurred, it affects the zone flow rates and the migration velocity of the solutes, and these effects change the internal profiles of the solutes. therefore, it is the reason of decreasing the purity and the yield of products the objective of this work is to consider the sensitivity of isotherm parameters and operating parameters in smb chromatography process. two amino acids, phenylalanine and tryptophan, separation by smb process is selected as control system. application of ph and po 2 probes during bacillus caldolyticus fermentation: an additional approach in improving a feeding strategy johannes bader 1 , boris neumann 2 , karima schwab 1 , milan popovic 1 , rakesh bajpai 3 : 1 studiengang biotechnologie, fachbereich v, tfh-berlin, seestr., 13347 berlin, germany 2 proteome factory ag, dorotheenstr. 94, 10117 berlin, germany; 3 department of chemical engineering, university of missouri-columbia, w2061 ebe, columbia, mo, usa. e-mail: popovic@tfh-berlin.de (m. popovic) bacillus caldolyticus,a thermophilic microorganism, is a good producer of thermostable liquefying ␣-amylase. during optimisation of initial and feeding media for fed-batch fermentation a two component feeding containing starch and casitone was found advantageous. to approach the optimal feeding rate the method published by akesson et al., 2001 was extended to two component feeding. the key idea, discussed in this presentation, was using the po 2 and ph probing signals to determine if the feeding of one or the other component should be increased or decreased. each of the probes offers information of different areas of feeding condition. to prevent excessive feeding of starch the ph probe is preferable. in case of excessive casitone feeding the po 2 probe responds in very authentic way enabling together with the ph signal reliable and reproducible evaluation of feeding strategy. however a congruent response of po 2 and ph probes means the approaching of the optimum feeding rate for both components. akesson, m., hagander, p., axelsson, j.p., 2001. probing control of fed-batch cultivations: analysis and tuning. contr. eng. pract. 9, 709-723. antibody immobilization by using the plasma polymerized acrylic acid r. jafari, m.tatoulian, f. arefi-khonsari laboratoire de génie des procédés plasmas et traitement de surface, enscp, upmc, 11 rue pierre et marie curie, 75005 paris, france the objective of this work is therefore to produce a surface containing a high density of cooh functions on the polymer beads (ps) for the covalent immobilization of antibodies. we have investigated the plasma polymerization of acrylic acid in a fluidized bed reactor the polystyrene (ps) beads. for such application, there is a strong need to obtain stable plasma polymerized acrylic acid (ppaa) coating, resistant to washing with water. different physico-chemical analyses have been used (water contact angle measurements (wca), xps and sem analysis) to characterize the ppaa coating deposited on ps beads under different experimental conditions. the xps results showed that the pretreatment of surface of the beads before deposition of acrylic acid plays an important role on the stability of ppaa layer. the instability of the coating is partially due the fact that under certain conditions the coatings are soluble in water and secondly due to the bad adhesion of the polymer beads which are hydrophobic to the growing ppaa coatings. xps as well as tof-sims gives evidence of the immobilization of the antibody. xps results as well as static sims allows to detect nitrogen on the surface of the treated beads which proves the presence of the immobilized antibodies. under optimum condition the ppaa coatings provides the possibility to show a nitrogen uptake which varies between 6.5 and 9% of the apparent stoicheiometry of the surface. holst department of medical physiology, the panum institute, university of copenhagen, dk-2200 copenhagen, denmark glp-1 (glucagon-like peptide-1), a peptide of 30 amino acids secreted by endocrine cells in the gut in response to meal ingestion, was discovered during a systematic search for gut factors capable of enhancing insulin secretion. it turned out to be the most efficacious insulin releaser known, and unlike other factors, was shown to retain its insulinotropic activity also in patients with type 2 diabetes. subsequent research has documented that the peptide not only releases insulin from the beta cells, but also enhance all steps of insulin biosynthesis, up-regulates beta cell gene transcription, and has trophic effects on the beta cells. the latter includes both proliferation of existing cells, neogenesis from ductal precursor cells, and inhibition of apoptosis. the peptide also inhibits glucagon secretion, reduces gastric emptying and reduces appetite and food intake. because of these actions, glp-1 administered to patients with type 2 diabetes dramatically lowers blood glucose as well as glycated hemoglobin levels, and reduces body weight. however, natural glp-1 is extremely rapidly metabolized in the body, and the problem has been how to convert the unstable peptide into a clinically useful agent. the two main problems are its susceptibility to enzymatic degradation by ubiquitous dipeptidylpeptide peptidase iv (dpp-iv) and its rapid renal elimination. a related peptide, isolated from the saliva of a lizard, exendin-4, was found to be a full agonist of the glp-1 receptor, to be resistant to dpp-iv and to be cleared more slowly by the kidneys. this peptide was highly effective in clinical studies and has now (30/4) been approved for diabetes treatment by the fda. other approaches include acylation of glp-1 whereby it attaches to albumin in the body and acquires resistance to dpp-iv as well as a slow renal elimination. also this analogue (liraglutide) has favourable clinical effects. fusion proteins of glp-1 and larger, slowly eliminated proteins in the body are currently being evaluated. small molecule, orally available inhibitors of dpp-iv have been demonstrated to protect endogenous glp-1 from degradation and to be efficacious in both experimental and clinical diabetes, and numerous inhibitors are currently in clinical development. the incretin hormones are released from gut endocrine cells upon meal ingestion. they enhance glucose-induced insulin secretion and nay be responsible for up to 70% of postprandial insulin secretion. the incretin hormones are glucagon-like peptide-1 (glp-1) and glucosedependent insulinotropic polypeptide (gip). in patients with type 2 diabetes (2dm) the incretin effect is severely reduced or absent. in 2dm patients the secretion of gip is normal, but its effect on insulin secretion is almost completely lost. glp-1 secretion, on the other hand, may be impaired, but its insulinotropic actions are preserved and it may restore insulin secretion to near normal levels. substitution therapy with glp-1 might therefore be possible. glp-1 is a product of the glucagon gene and its actions include: (1) potentiation of glucose-induced insulin secretion; (2) stimulation of the expression of ␤-cell genes essential for insulin secretion, including the insulin gene; (3) stimulation of ␤-cell proliferation and neogenesis (by enhancing endocrine differentiation of duct cells) and inhibition of ␤-cell apoptosis; (4) inhibition of glucagon secretion; (5) inhibition of gastrointestinal secretion and motility, notably gastric emptying; and (6) inhibition of appetite and food intake. these actions make glp-1 particularly attractive as a therapeutic agent for 2dm. thus, continuous subcutaneous administration of glp-1 for 6 weeks resulted in a 5 mmol/l reduction in mean plasma glucose and a reduction in hgba1c of 1.3%; a weight loss of 2 kg; improved insulin sensitivity; improved ␤-cell function; and the treatment was associated with no significant side effects. unfortunately, glp-1 is rapidly destroyed in the body by the ubiquitous enzyme, dipeptidylpeptidase iv (dpp-iv). clinical strategies therefore include: (1) the development of metabolically stable analogues of glp-1 viz. activators of the glp-1 receptor; and (2) inhibition of dpp-iv. orally active dpp-iv inhibitors have proven successful in experimental diabetes and several companies are now trying to develop clinically suitable inhibitors. so far the clinical experience is limited, but recent clinical studies have provided proof of concept. metabolically stable analogues/activators include the structurally related lizard peptide, exendin-4 or analogues thereof, as well as glp-1 derived molecules that bind to albumin and thereby assume the pharmacokinetics of albumin. these molecules are effective in animal experimental models of type 2 diabetes, and have been employed in clinical studies of up to 52 weeks' duration. on the basis of these studies it can be concluded that a therapy of type 2 diabetes mellitus based on stimulation of glp-1 receptors is likely to be effective and to become a clinical reality within the not too distant future(1-4). recombinant activated coagulation factor vii (rfviia) was developed to treat bleedings in hemophilia patients, who have developed inhibitors against fviii or fix, and has been demonstrated to have an efficacy rate of 80-90% in major surgery as well as in serious bleedings in such patients. to use rfviia as a hemostatic agent in severe hemophilia is a new concept of treatment, not being a substitution therapy, but using a pharmacological dose of exogeneous rfviia to compensate for the lack of fviii or fix. the administration of extra rfviia has been found not only to bind to tissue factor (tf), but also to the negatively charged phospholipids surface of thrombin activated platelets. hemostasis occurs on surfaces being initiated on the tf-expressing cells as a result of exposure of tf, not normally exposed to the circulating blood, following an injury to the vessel wall. tf is a true receptor protein with an intramembraneous part and an intracellular tail. its ligand is fvii/fviia. as soon as tf is being exposed to the blood, it forms complexes with fviia already present in the circulation. these complexes activates fx and provide the initial limited amount of thrombin molecules activating the co-factors, fviii and fv, as well as fxi and platelets. following the thrombin activation of platelets, negatively charged phospholipids are being exposed on the outer surface of the platelets. on this surface most coagulation proteins bind tightly, facilitating the conversion of fx into fxa and the full thrombin burst, necessary for the formation of a tight fibrin hemostatic plug resistant against premature lysis. in hemophilia patients the initiation of hemostasis is essentially normal, but, since they lack fviii or fix, they do not form the fviiia-fixa complex necessary for full thrombin generation on the activated platelet surface. as a consequence the fibrin plug formed in hemophilia is loose, fagile and easily dissolved resulting in continuous bleeding. pharmacological doses of rfviia have been demonstrated to mediate direct binding of rfviia to the negatively charged thrombin activated platelet surface, thereby generating thrombin formation in the absence of fviii/fix. through this mechanism hemostasis is generated in hemophilia patients independent of fviii/fix. furthermore, by generating more thrombin at an increased rate the formation of stable, tight fibrin hemostatic plugs are facilitated. such fibrin plugs are more resistant against premature lysis and help not only to initiate but also to maintain hemostasis. based on its capacity of enhancing thrombin generation locally on the activated platelet surface, rfviia has been used to ensure hemostasis also in other situations than hemophilia, such as platelet defects including thrombocytopenia. recently, rfviia was shown to be hemostatically effective in patients with profuse bleedings as a result of vast trauma and tissue damage. in these patients with a complex hemostasis pattern including a host of changes leading to an impaired hemostatic function, extra rfviia seems to help generate a burst of thrombin resulting in the formation of a stable hemostatic plug more resistant against the ongoing lysis. in patients with intracerebral haemorrhage, one single dose of rfviia recently was found to limit the expansion of the haemorrhage and thereby leading to improved functional outcome. institute for medical microbiology and immunology, panum 18.3.12, blegdamsvej 3, dk-2200 copenhagen n, denmark. email: s.buus@immi.ku.dk complete genomes from several species including many pathogenic microorganisms are rapidly becoming available along with the corresponding "proteomes". even at the peptide level, the diversity of proteome is enormous and easily represents a unique imprint of the originating organism. it is perhaps not surprising that the immune system considers peptides as key targets. recent immunological advances have shown that mhc molecules act as peptide selectors for immune recognition. we have proposed to generate accurate predictions of peptide binding to mhc and used these to identify immunogenic epitopes directly from genomic data. we have developed an iterative data-driven immunobioinformatics approach where data is used to generate predictors, and predictors are used to select new and complementary data for the next iteration. we have demonstrated the superior performance of this approach compared to a random data selection approach. we have also developed an efficient approach to select the most informative mhc molecules to investigate. the resulting, immunobioinformatics resource represents an immediate and powerful application and interpretation of genomic data, and will enable a rational approach to immunotherapy in the future. allergen specific immunotherapy is a causal treatment for igemediated allergic diseases such as hay-fever, and it has relied traditionally on preparations derived from aqueous extracts of various natural allergenic source materials. the cloning and production of an increasing number of allergens through the use of dna technology has not only facilitated the characterisation and analysis of the allergenic proteins, but also provided the opportunity to use these recombinant proteins instead of natural allergen extracts for the diagnosis and therapy of allergic disease. detailed physicochemical, biochemical and immunological characterisation are essential for the comparison of natural and recombinant proteins, and also provide a basis for developing derivatives. chemically modified allergens with attenuated ige-reactivity are currently used for immunotherapy in order to enable high doses to be achieved with a minimized risk of inducing allergic side reactions. dna technology provides the opportunity to develop and produce hypoallergenic allergen variants using strategies including gene mutation. the design of such variants must ensure that t cell reactivity and immunogenic activity are retained in order to preserve therapeutic potential. the recombinant allergens and their derivatives have several advantages over natural allergen extracts. they are relatively easy to produce in consistent pharmaceutical quality; the problems of natural extract standardisation can be avoided completely; the relative concentrations of the individual allergens can be controlled to obtain optimal dosages; nonallergenic proteins are excluded; the possible risks of contamination are avoided. the first clinical trials with grass pollen allergens and birch pollen hypoallergenic variants have yielded very encouraging results. the use of recombinant polyclonal antibodies (pabs) may improve the treatment of disease caused by complex targets such as infectious agents, when compared to monoclonal antibody therapy. symphogen has developed a method for reproducible production of target-specific fully human pab compositions, so-called symphobodies. the antibody genes are first isolated from donors with an immune response against the target and antibodies are screened for specificity. subsequently, the pabs are expressed in mammalian cells using the sympress technology, which is based on site-specific integration. this procedure ensures that each of the expression constructs encoding the antibody genes are stably integrated at the same site in each of the host cells, thereby eliminating genomic position effects and differential growth and production rates. further, the sympress technology comprises the generation of a polyclonal working cell bank (pwcb) which is used as inoculation material for the manufacturing. these cells display sufficient genetic stability to enable a controlled gmp production of recombinant polyclonal antibodies. symphogen's first product, sym001, is a recombinant human polyclonal rhesus d-specific symphobody preparation consisting of 25 different anti-rhesus d antibodies. this product is intended to be used for treatment of idiopathic thrombocytopenia purpura and prevention of hemolytic disease in newborns. recombinant anti-rhesus d symphobodies were produced and shown to be biologically active against rhesus d. the expression technology provided a compositional reproducibility between batches which is sufficient for manufacturing of such a polyclonal product for clinical use. scaledup production for clinical trials is currently ongoing. stem cells play an important role in renewing tissues such as skin and cornea. they are responsible for the continuous generation of the differentiated epithelium. we have characterized stem cells of the skin and cornea in situ and their fate in vitro in human skin reconstructed by tissue engineering using keratin (k)19. in the outer root sheath of the hair follicle, stem cells (label-retaining cells) present in the basal layer of the bulge area express k19 and present a loosely arranged keratin filament network and low levels of k14 protein in their cytoplasm. in addition, another stem cell population (also labelretaining) is present in the first suprabasal layers. these cells exhibit a very dense keratin network and express k17. three-dimensional tissue constructs (dermis and epidermis) obtained by the self-assembly approach of tissue engineering allow the preservation of k19 positive stem cells in the basal layer of the epithelium. in the eye, the stem cells are located in the limbal part but not in central cornea and they express k19. the epithelium of reconstructed cornea is thinner compared to reconstructed skin and more transparent. the characterization of stem cells in reconstructed tissue is essential to evaluate the long-term survival of these tissues in vitro but also after grafting. these human reconstructed tissues are developed for fundamental (physiological, toxicological studies) and clinical applications such as transplantation for the permanent replacement of damaged organs. lg is holder of the canadian research chair (cihr) on stem cells and tissue engineering. alessandra gliozzi physical department, university of genoa, 16146 genoa, italy hollow nanometer-sized capsules can be prepared by means of different techniques. first "nanocapsules" were liposomes, however they are too unstable for many medical or pharmaceutical applications. in contrast, recently developed polyelectrolyte capsules prepared by means of the layer-by-layer technique are much more stable and seem to be a very promising way for coating living cells or tissues in order to prevent or reduce their immune rejection after implantation. several observations on single living cells encapsulated by the alternative adsorption of oppositely charged polyelectrolytes will be presented. the most relevant result is that cell preserve their metabolic activity, are still capable of dividing and performing specific functions. moreover, a technique to immobilize in defined arrays coated cells expressing green fluorescent protein by using a microcontact printing of polyelectrolytes will be presented. finally, tests performed to study the induction of fibrosis and vascularization by nanocapsules implanted in rat kidney and liver will be presented. over the past decade we have developed methods to generate spontaneously and synchronously beating tissue equivalents from neonatal rat heart cells in the culture dish. these tissue equivalents display the key morphological and functional features of intact myocardium and have been termed engineered heart tissue (eht). to generate ehts, heart cells are mixed with freshly neutralized, liquid collagen i, matrigel and growth supplements and grown in a circular casting mold around a central cylinder, which subjects the cells to a continuous mechanical load. this process is enforced by cyclic mechanical stretch. we use eht mainly for two purposes, as a test bed for the effects of pharmacological or genetic manipulations and for cardiac repair. as a cell culture model, ehts compare with standard 2d monolayer cultures of neonatal rat cardiac myocytes and freshly isolated adult cardiac myocytes. advantages of ehts are their functional similarities with intact heart muscles, the ability to easily measure force of contraction under mechanical load, the pos-sibility to transfect cardiac myocytes inside ehts with adenovirus at high efficiency and the reproducibility in large series. a disadvantage is that contractile function as measured at the end of the culture period also integrates influences on tissue development, cell-cellconnections, extracellular matrix production and on non-myocytes. at present we are working on downscaling the eht method to a 96well format for screening purposes. to use ehts for cardiac repair we created multi-looped ehts from five circular ehts large enough to cover the infarct scar 14 days after coronary artery ligation in rats. ehts survived and formed a layer of muscle tissue on top of the infarct scar. ehts restored undelayed anterograde impulse propagation over the scar, prevented further ventricular dilatation, normalized enddiastolic pressure and relaxation, and partly restored contraction of the scar. thus, the study provides evidence that implanting ehts onto infarcted hearts can improve cardiac contractile function after myocardial infarction. the goal of tissue engineering is the development of skin, bones and even organs to restore, maintain and improve tissue function within the body. the current paper focuses on the investigation of invitro growth of osteoblast cells in different types of scaffolds. three of the scaffolds were made of pcl (polycaprolactone) 10% glycerol and 10% hca(hydroxylapetite). two of the scaffolds were made by compression molding, and one was made by fused deposition modeling utilizing the stratasys. the fourth cerabio was a commercially available product totally ceramic. the pores in compression molding were obtained by putting in 75% volume of sugar either 150 and 595 m which was later removed by leaching. the fused deposition scaffold was made by placing the filaments in a predetermined arrangement. the stratasys system was a computer designed model. the scaffolds were seeded with hfob 1.19 human fetal osteoblast cell line with vigorous shaking overnight and incubating at 37 • c and 5% co 2 . observation of the seeded scaffolds was made after 3 days and 9 days of incubation. the seeded cells were stained with bcip/nbp at 37 • c overnight. the cell proliferation in the 595 and 150 m scaffolds appeared approximately the same with a possible advantage of 595 m. the cerabio sample demonstrated the greatest proliferation among the four scaffolds studied and the stratasys sample exhibited a different type of cell adhesion with the cells were clustered in the interstices of the structure. denise freimark, ruth freitag, valérie jérôme chair of process biotechnology, university of bayreuth, d-95448 bayreuth, germany. e-mail: denise.freimark@uni-bayreuth. de (d. freimark) tissue engineering is emerging as an alternative to bone grafts for the regeneration of defects that do not heal spontaneously. ultimately, the development of an optimum carrier and the identification of ideal inductive factors and cells may enable tissue engineering to provide an improvement over bone grafts in the future. bone formation and repair require a complex cascade involving growth factors, cytokines and angiogenesis. at present the complexity of the molecular mechanisms that control gene expression in bone forming cells in embryo as well as in adult is not fully understood. several factors like bone morphogenetic proteins (bmps), transforming growth factor beta (tgf-␤), vascular endothelial growth factor (vegf) and insuline-like growth factor (igf) have been identified and their ability to stimulate bone formation in vitro and in vivo has been investigated. while much is known about these factors per se, less is known about genetic regulation of artificially stimulated osteogenesis. interestingly, some in vitro investigations showed that only optimal growth factors concentrations lead to effective bone formation whereas higher concentrations had deleterious effects which suggest some variation in the activated regulation pathways. therefore, one of our goals is to analyze kinetics, dose-dependence and synergistic effects of growth factors and cytokines on regulation pathways of bone formation. moreover, the optimal vascularization of the scaffold is a major hurdle in the development of engineered bone. it is well known that: (i) vascular invasion precedes bone growth and (ii) osteogenesis takes place in the vicinity of newly formed vessels. thus, inadequate bone vascularization is associated with decreased bone formation. further analysis of the intercommunication between endothelial cells and osteoprogenitors in co-culture systems could provide key information that could be thereafter used to solve this problem. we propose to add some new knowledge to this complicated puzzle. a first step in our investigation is the production of some of the growth factors mentioned above in recombinant form. these factors are expressed in a novel vector (ptriex tm ; novagen) which allows recombinant protein production in prokaryotic or in eukaryotic systems with a single plasmid. afterwards, we analyze potential synergistic effects of these factors as well as kinetic and dose-depend parameters on the genetic regulation of downstream pathways in osteoblasts culture. in parallel, we develop an in vitro system allowing us to investigate the intercommunication between endothelial cells and osteoprogenitors. there has been significant interest in the therapeutic and scientific potential of human embryonic stem (es) cells since they were first isolated in 1998. if human es cells could be differentiated into suitable cell types, stem cells might be used in cell replacement therapies for degenerative diseases such as type i diabetes and parkinson's disease, or to repopulate the heart following myocardial damage. however, there is a significant shortage of high quality human es cell lines and few research groups have experience in the propagation and manipulation of these cells. we are addressing this important issue using the combined expertise of the stem cell biology laboratory and the assisted conception unit at king's college, london. with local ethical approval and under licence from the uk human fertilisation and embryology authority, we have been establishing high quality human es cell lines from a novel source of human embryos. to date, we have derived three human es cell lines and are now focused on the generation of therapeutically important cell populations, including cells that may have clinical application in degenerative and traumatic injury to the brain and spinal cord, heart, retina and other target organs. wallenberg neuroscience center, department of physiological sciences, lund university, bmc a11, s-221 84 lund, sweden cell replacement therapy for parkinson's disease is based on the idea that implanted dopamine neurons may be able to substitute for the lost nigrostriatal neurons. in rodent and primate models of parkinson's disease it has been shown that transplanted dopamine neuroblasts can re-establish a functional innervation and restore dopaminergic neurotransmission in the area of the striatum reached by the outgrowing axons; that the grafted neurons are spontaneously active and release dopamine in an impulse-dependent manner, at both synaptic and non-synaptic sites; and that they can reverse or ameliorate some of the parkinson-like motor impairments induced by damage to the nigrostriatal system. clinical trials in patients with advanced parkinson's disease have shown that dopamine neuroblasts obtained from fetal human mesencephalic tissue can survive and function also in the brains of pd patients, restore striatal dopamine release, and ameliorate impairments in motor behavior. the principal limitation of this approach is the problems associated with the use of tissue derived from aborted human fetuses, and the large numbers of donors needed to obtain good therapeutic effects. until now, transplantation of dopamine neurons has focused primarily on differentiated neuroblasts and young postmitotic neurons, at the stage of neuronal development that is optimal for survival and growth of the grafted cells. however, progenitors taken at earlier stages of development might prove more effective. efforts are now made to expand multipotent neural stem-or progenitor cells in vitro, and control their phenotypic differentiation into a dopaminergic neuronal fate. initial results suggest that in vitro expanded cells can survive and function after transplantation to the striatum in the rat pd model, but the overall yield of surviving dopamine neurons has been very low. with further development, expanded progenitors or dopamine neuron precursors, possibly in combination with cell engineering techniques, may offer new sources of cells for replacement therapy in pd. stem cell therapy has been very much in vogue for several years now. like gene therapy before it, it has raised unrealistic hopes of cures being available imminently. unlike gene therapy, it can cite proof of principle in the well established practice of bone marrow transplantation which is actually a good example of stem cell therapy. however, most of the uses that are now touted as targets for stem cell therapy, envisage the conversion of the stem cells into lineage restricted progenitor cells or more commonly, the final differentiated cell type. such conversions are extremely difficult to initiate and control. the procedures involve the manipulation, differentiation, and expansion of cell cultures in the laboratory, with unknown long term effects on the genetics and physiology of the cells. these issues are compounded when one considers as source material, human embryonic stem cells, where not only the final cell product requires significant scrutiny, but also there are safety issues surrounding the persistence of undifferentiated cells. on top of all these challenges are commercial (for stem cell companies), clinical, and regulatory pressures which will impact heavily on the pace of progress. nonetheless, despite all these hurdles, various academic groups and companies are making significant progress and examples of such developments in diabetes and cardiovascular repair will be given stem cells have the unique ability to perpetuate themselves while continually replenishing tissues throughout the life of an organism. the era of cellular and tissue regeneration for the treatment of disease and the effects of aging has indeed begun. it has been known that mechanical factors play an important role in the regulation of cell physiology. it is therefore reasonable to believe that mechanical factors also play a significant role in the metabolic activity and differentiation of mscs. in this study, we investigated the viscoelasticity of individual bone marrow-derived adult human mesenchymal stem cells (hmscs), and the role of specific cytoskeletal component -f-actin microfilaments on the mechanical properties of individual hmscs. the mechanical properties of hmscs were determined using the micropipette aspiration technique coupled with a viscoelastic solid model of the cell. for the hmscs under control conditions the instantaneous young"s modulus e 0 was found to be 886 ± 289 (pa), the equilibrium young"s modulus e ∞ 372 ± 125 (pa), and the apparent viscosity 2714 ± 1626 (pas). after exposed to 2 m of chemical agent-cytochalasin d that disrupt the f-actin microfilaments, the young's moduli of hmscs decreased by up to 72% and the apparent viscosity increased by 167%. these findings suggest that microfilaments are crucial in providing the viscoelastic properties of the hmscs, and changes in the structure and properties of them may influence the mechanical properties of hmscs significantly. pharmacologic transcription control of desired transgenes is essential for gene-function analysis, drug discovery, biopharmaceutical manufacturing, design of complex artificial regulatory networks, precise and timely reprogramming of key cell characteristics for gene therapy and engineering of preferred cell phenotypes for tissue engineering. capitalizing on our recent advances in the design of small molecule-responsive transcription control modalities we have used conditional molecular interventions for (i) improvement of specific productivity in biopharmaceutical manufacturing, (ii) transdifferentiation of therapeutically relevant cell phenotypes and (iii) design of artificial microtissues. we will also report on a completely new dimension of transgene control as well as engineering of hysteretic and epigenetic gene networks in mammalian cells. chronic diseases are a growing burden for the individual and society alike. one key factor driving this increase is an ageing population. currently, there are 600 million persons aged 60 years or over and this number is predicted to triple by the middle of the 21st century. effective prevention of irreversible damage to major organ systems requires early diagnosis and treatment yielding significant quality of life to the individual and sparing valuable health care resources. even when safe and effective medicines are available, a remaining problem to successful therapy are issues of patient compliance. historically, vaccines have been one of the major advances towards the longevity we enjoy today, with compliance rates close to 100%. hence, vaccines for early treatment of chronic diseases are ideally positioned for long-term therapy, and will take away the burden of self-medication associated with orally active drugs. here we will discuss a new generation of therapeutic vaccines based on virus-like particles (vlps). by displaying target molecules in a highly repetitive manner on vlps, it is possible to break b cell unresponsiveness in experimental animals as well as in humans. using such vaccines in animals, chronic diseases such as hypertension, alzheimer's disease, obesity and rheumatoid arthritis could be treated. furthermore, vaccination against nicotine resulted in high nictotine-specific antibody titers in humans, greatly facilitating smoking cessation in immunized individuals. interdependence of the impact of methanol and oxygen supply on protein production with recombinant pichia pastoris n.k. khatri, f. hoffmann martin-luther-university halle-wittenberg, institute for biotechnology, halle d-06120, germany. e-mail: f.hoffmann@biochemtech.uni-halle.de (f. hoofmann) the methylotrophic yeast pichia pastoris is a potent expression system for secretion of recombinant proteins. methanol as inductor of the foreign gene expression is also a substrate with high oxygen demand, which can lead to sudden oxygen depletion upon induction. thus, supply rates of methanol and oxygen are major process parameter during protein production with recombinant pichia pastoris. limiting dosage of methanol allowed maintenance of oxygen sufficient conditions during production of a single chain antibody fragment, but the product was degraded from the c-terminal end. in contrast, full-length product accumulated with controlled methanol concentrations despite oxygen limitation. the volumetric methanol uptake rate are limited by the oxygen transfer capacity of the reactor. higher methanol concentrations decreased the biomass yield and thereby increased the specific methanol uptake rate. this enabled prolonged production and yielded fivefold higher product concentrations. at the same time, the accumulation of small molecular weight contaminants was reduced. dosage of pure oxygen accelerated methanol uptake, grow and production. switching to dostat mode upon oxygen depletion led to an arrest of product accumulation, in contrast to persistent methanol feeding. the productivity was tenfold higher than without oxygen. combined with high methanol concentrations, however, fast methanol uptake led to toxication of the cells and early stop of production. flow cytometry revealed that perturbation of oxygen metabolism was followed by partial lysis of the culture. recombinant production of therapeutic proteins poses severe challenges due to their complexity (cystines, subunits, size). formation of the correct disulphide bridges is a prerequisite for activity but difficult to achieve in a prokaryotic host. there proteins mainly fold post-translationally as opposed to eukaryotic co-translational folding. thus the recombinant products are often not soluble and/or active. that is why different production parameters (e.g. host, induction conditions, temperature, compartment, proteinaceous fusion partners, co-expression of chaperones, foldases) are applied in order to gain functional recombinant proteins. the impact of all these strategies cannot be predicted and every problem of the production (expression, solubility, activity) might need to be solved for every target protein separately. nevertheless, much effort has been put into this for almost 3 decades, because they are important targets for the pharmaceutical industry. human growth factors influencing cellular proliferation and/or differentiation are one example. this case study gives an overview of strategies tested within the development of two processes leading to an optimised yield of active protein. murine wnt-1 (wnt family) possesses 23 conserved cysteines (most likely all involved in disulphide bridge formation and one in posttranslational modification) and could only be successfully produced in e. coli (fahnert, 2004) recently despite various attempts for many years. the other target protein is human collagen prolyl-4-hydroxylase being a heterotetramer consisting of two ␣-subunits and ␤-subunits each. the ␣-subunit strongly aggregates if produced separately whereas the ␤-subunit is pdi and is suggested to have a chaperone function. therefore a sequential induction strategy was proposed for this protein . the moss physcomitrella patens has been recently recognized as an ideal producer of recombinant proteins with respect to glycosylation. due to the elaborated post-translational capabilities of moss cells, the glycosylation patterns have been manipulated to obtain similar proteins to those found in animal cells. the protein expression using moss in suspension offers important advantages in comparison to other systems e.g. cho cells. the recombinant proteins can be targeted into the mineral medium, simplifying the down stream processing. moreover, there are neither known moss viruses nor plant viruses that are pathogenic for humans. the moss are cultivated axenically in a filamentous stage, the so called protonema. a pilot 30 l tubular photoreactor is used to characterize the response of p. patens to variations on the culture conditions. the phototrophic culture in bioreactors is systematically investigated, where light quantity and quality, stress, concentration of phytohormones, and moss morphology influence the differentiation, growth, and protein expression. a tight control of the moss morphology in suspension, quantified by image analysis, has shown to be advantageous in order to delay the cell differentiation and maintain the carbon dioxide uptake in long bioreactor runs. the introduced perfused culture system by means of cross flow filtration allowed for a continuous product separation and concentration, and feed back of the productive cells. the characterization of this highly controlled culture system is presented and the potential of p. patens as an alternative tool for molecular farming is discussed. (rnai) is an evolutionarily conserved, endogenous mechanism for sequence-specific gene silencing that uses small double-stranded rnas (called short interfering rnas or sirnas) to direct cleavage or prevent translation of homologous mrnas. harnessing rnai for therapy presents an opportunity for potentially treating a wide variety of diseases. the main obstacle is delivering sirnas into the cytosol of target cells in vivo. although we were able to protect mice from autoimmune hepatitis by hydrodynamic tail vein injection of sirnas targeting fas, this delivery method is unlikely to be adaptable for human use. alternate strategies to deliver sirnas in vivo as small molecule drugs using currently available, clinically acceptable injection methods that have shown promise in mouse models will be discussed. these include local delivery to mucosal surfaces and delivery into specific cells via cell surface receptors using an antibody fragment fused to protamine. these sirna complexes silence gene expression in vivo only in cells bearing the targeted receptor. experiments showing efficient, effective and cell-specific delivery in a mouse tumor model will be discussed. in addition, encouraging preliminary data using rnai for a microbicide to prevent sexually transmitted infection will be presented. rna interference (rnai) holds significant progress as a therapeutic approach to siolence disease-causing genes, particularly those that encode "non-druggable" targets. the key hurdle for rnai therapeutics is in vivo delivery. a critical requirement for achieving systemic rnai in vivo is the introduction of "drug-like" properties, such as stability, cellular delivery and tissue biodistribution, into synthetic sirnas. our progress in achieving in vivo silencing of endogenous genes with chemically modified sirnas will be discussed. hiv-1 replication in human t cells can be inhibited by stable expression of a short hairpin rna targeting the viral nef gene (shrna-nef). however, hiv-1 escape variants emerge after prolonged culturing, and all but one escape mutant acquire a mutation in the shrna-nef target sequence. we observed single and multiple nucleotide substitutions, but also partial or complete deletion of the target sequence. these results demonstrate the sequencespecificity of this antiviral approach. we observed an inverse correlation between the level of resistance and the stability of the shrna/target-rna duplex for most of the escape mutants. however, two escape variants did not follow this pattern, including an escape mutant with a single point mutation at position −7 upstream of the target sequence. these mutants provide a much higher level of resistance than expected based on duplex stability, which is obviously not affected in the −7 mutant. we demonstrate that these mutants adopt an alternative rna secondary structure that occludes the target sequence. this results in reduced shrna-nef binding and provides a novel mechanism for rnai-resistance. to avoid viral escape, one should ideally target hiv-1 with multiple effective shrnas against conserved genome sequences. we performed a large scale screening to identify such targets, and we have identified at least nine genome segments that can be targeted effectively with shrnas. these potent antivirals are currently being assembled in a lentiviral vector for gene therapy applications in hiv-infected individuals. furthermore, we will describe approaches to forecast viral escape routes and to effectively block such evolutionary paths with additional rnai measures. in this study we analyzed the effect of antibodies against electronegative ldl on the development of atherosclerotic lesions in low-density receptor-deficient (ldlr −/− ) mice. two groups of (ldlr −/− ) mice (eight females) fed 0.5% cholesterol-enriched chow were used. the first group received a monoclonal antibody against electronegative ldl (100 g) and the second one received pbs (controls). additionally, other two groups (eight males) of (ldlr −/− ) mice were treated with a polyclonal antibody against electronegative ldl (100 g) or pbs (controls). antibodies were administered by intravenous route one week before starting the hypercholesterolemic diet and then every week over an experimental time of 21 days. afterwards, quantification of atherosclerotic plaque area of heart and aortic arch was done by analysis of the slices stained with oil red/hematoxolin/light green with the image propus software. the passive immunization with either monoclonal or polyclonal antibodies against electronegative ldl significantly reduced the atherosclerotic plaque areas in atherosclerosis-prone ldlr −/− mice. in conclusion, antibodies against electronegative ldl administered by intravenous route may play a protective role in atherosclerosis. supported by fundação de amparoà pesquisa do estado de são paulo (fapesp, scholarships to d.m.g., l.s. and a.b. and grants to m.h.k. and d.s.p.a.). the enzyme asparaginase from the procaryote escherichia coli or erwinia crysanthemi is used for the treatment of lymphoblastic leukaemia. the drug causes immunological reactions in despite of the treatment efficiency. asparaginase may also be obtained from saccharomyces cerevisiae and this enzyme could provide an alternative to its bacterial counterparts. in this study, the periplasmic nitrogen regulated asparaginase ii from s. cerevisiae, that is coded by the asp3 gene, was cloned and expressed in the methylotrophic yeast pichia pastoris under the control of the aox1 gene promoter. the recombinant p. pastoris strain was cultured in shake flasks and in a 2 l instrumented bioreactor. in both cases it was observed specific enzyme yields seven fold higher in comparison to that using a nitrogen derepressed strain of s. cerevisiae, reaching 800 u/g dry cell mass. high cell density cultures carried out in the 2 l bioreactor, in which it was attained 107 g dry cell mass/l, resulted in a dramatic improvement in asparaginase fermentation. as such, it was measured enzyme yields of 85,600 u/l and productivities of 1083 u/l h. department of biochemistry and food chemistry, biotechnology, university of turku, tykistokatu 6, biocity 6th floor, 20540 turku, finland. e-mails: lorenzo.galluzzi@utu.fi; deadoc@libero.it; deadoc@aliceposta.it (l. galluzzi) the bacterial luciferase operon from the bacterium photorhabdus luminescens has been used, since its first description, for exceptionally different applications. these ranged from the environmental monitoring to the cell tagging, from the analysis of cellular metabolism to the high-throughput screening of novel compounds. the wild-type luxcdabe operon has been engineered in countless ways (for instance by changing the order of the constituent genes, by optimizing the codon usage and by coupling it to many promoters) and has been expressed in prokaryotic and eukaryotic organisms in order to meet precise research and commercial needs. upon the operon expression light is emitted as the side product of a chemical reaction catalyzed by the luciferase enzyme, an ␣␤ heterodimer encoded in luxa and luxb genes. the reaction involves the oxidation of a long-chain aliphatic aldehyde and reduced flavin mononucleotide (fmnh 2 ) with the liberation of excess free energy in the form of a blue-green light at 490 nm. the luxcde genes code for the polypeptides (transferase, synthetase, and reductase) forming the fatty acid reductase complex that catalyzes the conversion of fatty acids into the long-chain aldehyde required for the luminescent reaction. noteworthy is that for the production of the substrates for the luciferase both atp and nadph are required, while neither is involved in the actual light emitting reaction (wilson and hastings, 1998) . recently, the coupling of the luciferase operon to regulated promoters lead to the construction of genetically modified bacteria able to sense the presence of chemicals and to respond, in a dosespecific manner, with light emission. this approach has been applied to the detection of antibiotics in samples from the food industry as well as to the detection of heavy metal ions in environmental samples (kurittu et al., 2000; bechor et al., 2002) . in addition, it opened the possibility of screening wide libraries of new compounds looking for molecules with pre-determined features, able to induce the bioluminescent response by de-repressing the lux operon transcription when incubated with the appropriate bacterial sensor. the high throughput and low costs are the main advantages of this system, which shows as well a certain degree of specificity (galluzzi and karp, 2003; galluzzi et al., 2004) . nevertheless, since the in vivo bioluminescence relies upon a complex network of biochemical reactions, under certain circumstances the light emission is not a direct consequence of the lux operon transcriptional induction but it more likely originates at a posttranslational stage. as a matter of fact, one can suppose that a change in the light emission will be observed whenever the concentration of one or more substrates for the lux␣␤ reaction occurs. consequently, all the molecules sharing the ability to impair the delicate chemical equilibrium regarding the compounds involved in bioluminescence will be sensed by the bacteria as inducing compounds. this, in turn, will result in a loss of specificity of the assay. we investigated this aspect of the whole-cell assays based upon the bacterial luciferase operon for drug discovery by means of a reporter plasmid in which the luxabcde genes, rearranged and optimized for the after 60 min (black downward arrow) of incubation at 37 • c under vigorous shaking the following concentrations of trimethoprim were added to the growing cells: 25 g/ml (triangles), 50 g/ml (circles) and 250 g/ml (rumbles). water was administered to control cultures (squares). light emission was quantified every 30 min by means of the wallac victor 2 multilabel counter (perkin-elmer, turku, finland) and normalized to the absorbance, measured at 600 nm with the same device. multi-96 white-walled transparent-bottomed plates were used for the assays (nalge nunc, usa). between the measurements the plates were kept at +37 • c under vigorous shaking. filled symbols refers to the absorbance values (left side y-axis); open symbols to the normalized count per seconds (right side y-axis). expression in gram + organisms, are under the control of the qacr regulatory region from staphylococcus aureus . non-pathogenic s. aureus rn4220 cells bearing the pqaclux plasmid (cultivated in lb broth supplemented with 0,5% d-glucose and 10 g/ml chloramphenicol) emit light upon the specific transcriptional induction with quaternary ammonium compounds, widely used as surface disinfectants and in many over-the-counter drugs . however, the incubation of the same cells with an inhibitor of the dihydrofolate reductase enzyme, trimethoprim (sigma-aldrich chemie, steinheim, germany), enhanced in a dosedependant manner the light emission observed upon the induction with the optimal concentration (1 g/ml) of benzalkonium chloride (bc). the extent of this increase in luminescence varied from 5-10% to more than 200%, according to the trimethoprim concentration and to the measurement time. interestingly, when the same plasmid is carried by escherichia coli xl1 cells, the lux operon is expressed constitutively (since the qacr regulatory region is not functional in xl1 cells) and at much higher levels than in induced rn4220 cells. also in this experimental system the incubation with trimethoprim results in a dramatic increase of the luminescent signal from the cultures. the explanation for these observations can be found in the molecular mode of action of trimethoprim. the inhibition of dihydrofolate reductase, indeed, directly leads to the accumulation of its substrates, among which is nadph, deeply entangled in the biochemical network of reactions centred on the light emission from the lux operon. nadph provides the reducing power to restore the reduced flavin mononucleotide pool and it is as well involved in fig. 2 . xl1/pqaclux growing cells were incubated with the following concentration of trimethoprim: 25 g/ml (triangles), 50 g/ml (circles) and 250 g/ml (rumbles). water was administered to control cultures (squares). light emission and absorbance measurements were performed as previously described for rn4220 cells. filled symbols refers to the absorbance values (left side y-axis); open symbols to the normalized count per seconds (right side y-axis). the diverse level of growth observed for rn4220 and xl1 cells can be accounted by the different antimicrobial activity exerted by trimethoprim towards gram− and gram+ cells and by the lower activity of the promoter which in pqaclux plasmid drives the transcription of the selection marker (chloramphenicol acetyl transferase). the production of the long-chain aldehyde, both substrates of the lux␣␤ heterodimer (wilson and hastings, 1998) . in conclusion, here we demonstrate that the use of light emission from the bacterial luciferase as a transcriptional reporter has to be very carefully controlled, since some molecules (here trimethoprim) could mimic to some extent a specific promoter activation by impairing the delicate intracellular biochemical equilibrium. on the reverse side of the coin, fig. 3 . simplified scheme depicting the bacterial folate metabolic pathway. only part of the reactions and compounds are reported. trimethoprim inhibits the nadph-dependant reduction of dihydrofolic acid into tetrahydrofolic acid catalyzed by dihydrofolate reductase. this results in the accumulation of both substrates, which become available for other reactions, and in the depletion of tetrahydrofolate, the major c 1 carrier in the synthesis of purines, thymidine, glycine, methionine, and pantothenate in bacteria. for antimicrobial purposes trimethoprim is often associated with sulfonamides, with which it displays a synergistic activity (since they act on the same pathway but at an earlier reaction) (scholar and pratt, 2000) . however, it has to be noted that the lux reporter system could be used in many different in vivo experimental setups, where the change in the intracellular concentration of nadph, atp or other metabolites would be sensibly detected. we have carried out the construction of five pichia pastoris strains harboring in their respective genome three different growth hormones cdna's (22 and 20 kda human growth hormones and bovine growth hormone), a shrimp (litopenaeus vannamei) trypsinogen cdna and a bacterial (bacillus subtilis) phytase gene. in all the cases, the same kind of expression vector and the same transformation technique were used. each dna was fused in frame to saccharomyces cerevisiae alpha-factor secretion signal to lead the secretion of the foreign protein into the culture medium. the induction of each recombinant strain, all with his + and mut + phenotype, was carried out in shake flaks using methanol buffered minimal medium (bmm) and growth rates on methanol determined. production level of secreted proteins was evaluated by protein analysis by sds-page. furthermore, the expression with three of the constructed strains was performed in a 5-l bioreactor and the level of secreted protein determined in each case. both human growth hormones (hghs) were secreted into the culture medium with a high degree of purity obtaining up to 64% of hghs of total proteins in crude fermentation medium and 3-18 mg/l of hghs. neither bovine growth hormone, shrimp trypsinogen or bacterial phytase were detected in methanol induced cultures of the respective strains, in spite of the same culture conditions were used for all p. pastoris strains. the growth rates on methanol were different between some strains. in fermentor cultures the hghs production were increased and shrimp trypsinogen was produced and secreted into the culture medium after improving the culture conditions. bovine growth hormone was detected only when the culture conditions were modified. the protein production level for each strain was affected by the proprieties of each recombinant protein produced. competitive advantages of the diagnostic method for invasive amoebiasis using preserved antigenic extracts without using enzymatic inhibitors m.s. flores 1,2* , e. tamez we have patented a method to diagnose invasive amoebiasis using a novel assay that preserves antigenicity of extracts with high protease content without using enzymatic inhibitors (ic:mc). the available tests for serologic diagnosis of invasive amoebiasis like elisa and indirect haemaglutination (iha) do not have consistent results in endemic zones. here we show the advantages of the assay and the validation of this diagnostic test for invasive amoebiasis. we demonstrated the reduction of proteolytic activity of ic:mc compared with the proteolytic activity of crude extract and crude extract with enzymatic inhibitors using assays. we displayed the ic:mc sds-page pattern and the western blot (wb) pattern useful for diagnosis. to search the clinical utility of this test we examined the wb obtained with sera from patients with different liver diseases; 90 patients had invasive amoebiasis and 45 patients had other liver diseases. the results were compared with those of iha test. also we have tested the accuracy of wb using sera from people with multiple intestinal parasites, like giardia lamblia, hymenolepis nana, blastocystis hominis, entamoeba coli, etc. the sensibility of the wb using the preserved amoebic antigens was 99%, specificity was 100%, positive predictive value was 100%, negative predictive value was 98% and accuracy was 99%. the wb did not exhibit cross reactions with sera from persons with intestinal parasites. our test was better than the (iha) test commonly used in endemic zones. these results show the improvement of using the preserved amoebic antigens in diagnostic tests. also they prove the diagnostic accuracy of our new wb test. antibacterial and antifungal activity of heracleum sphondylium subsp. artvinense yasemin kaçar 1 , sema tan 2 , aysun ergene 2 , perihan güler 2 , semra mirici 3 , ergin hamzaoglu 2 , ahmet duran 4 , sinem yildirim 2 : 1 mersin university, faculty of science and literature, department of biology, 33800 mersin, turkey; 2 kırıkkale university, faculty of science and literature, department of biology, 71450 yahsihan-kırıkkale, turkey; 3 akdeniz university, faculty of education, department of biology education, 07400 antalya, turkey; 4 selcuk university, faculty of education, department of biology education, 42300 konya, turkey turkey is covered yearly with a huge number of plant species. about 9222 species are condenced on the region that between asia and europe. many plant species have been used in folkloric medicine to treat various ailments. heracleum l (apiaceae) is include over than 70 species on the world. this variety is represent with 17 species in turkey that seven species are endemic. heracleum sphondylium subsp. artvinense is endemic species for turkey. ethanolic and aquous extract of heracleum sphondylium subsp. artvinense were investigated for their antimicrobial activities against six bacterial species (e. feacalis, e. coli, s. aureus, p. aeruginosa, l. monocytogenes, shigella) and two yeast (c. albicans, c. krusei). both ethanolic and aqueous extract of heracleum sphondylium subsp. artvinense showed antimicrobial activity against the gram negative bacterium (shigella) and gave the best activity against c.albicans. to develop artificial vectors allowing nucleic acid to transfect into mammalian cells are crucial for extending gene therapy. synthetic vectors based on lipid molecules are particularly attractive because of their potential safety. however, the low encapsulation efficiency of nucleic acid is one of the problem to be solved. recent advances in dna-lipid complex have improved this drawback, and now some lipid molecules are used as transfection agents. in particular, cationic lipids interact with negatively-charged cell surfaces and nucleic acids. the former interaction results in delivery of the nucleic acids directly across the cell membrane. on the other hand, the latter interaction improves the efficiency of nucleic acids entrapment. in this study, we developed a preparation method of "nanovesicle" containing nucleic acids by using reverse micellar solubilization. since dna interacts spontaneously with cationic amphiphiles, complete extraction of the dna molecules into an organic phase using dimethyl distearyl ammonium bromide (2c18ab), an oil-soluble cationic surfactant, is achieved. the re-encapsulation of the reverse micellar droplets solubilizing dna by water-soluble surfactants facilitates the formation of nanovesicles. a high salt concentration at the re-encapsulation step promotes the production of nanovesicles containing dna molecules. eventually, more than 80% of dna was encapsulated in this nanovesicles under the condition of 6m nacl and 5% (w/v) cetyltridecyl ammonium bromide (ctab). in addition, the nanovesicles prepared at 45 • c was smaller than that prepared at room temperature. the resultant 2c18ab/ctab nanovesicle was ca. 16 nm. asymmetric and chemically-modifiable vesicle surface are effective to design gene delivery system. moreover, such a small dna (or rna) carrier has a potential for novel gene therapy application from skin. the key players in clinically important inflammatory diseases, endothelial cells and leukocytes, communicate through membranebound cell adhesion molecules (cam's). obvious strategies for therapeutic intervention include specific means to affect the expression of cam's on the cells involved. rna-interference (rnai) is a well known means to achieve specific gene-inhibition. for this study, two cam's were chosen, namely vascular cell adhesion molecule-1 (vcam-1) as a target for inhibition, and intercellular adhesion molecule-1 (icam-1) as a non-target reference. we designed short interfering rna (sirna) oligos for vcam-1 in order to selectively inhibit the expression of this cam in cultured human vascular endothelial cells (huvec). real-time rt-pcr showed an 80% down-regulation of vcam-1 mrna while the expression of icam-1 remained unaffected. neither cam was affected by non-specific sirna. in order to further substantiate the potential use of sirna for therapeutic purposes, we have set out to investigate two things: (1) does vcam-1-specific sirna affect the amount of vcam-1 on the surface of huvec? (2) is adhesion between leukocytes and endothelial cells affected by vcam-1-specific sirna? the manufacturing of plasmid dna (pdna) is crucial to obtain a consistent product for gene therapy applications. although flowsheets for pdna production are established on the basis of experience, simulation tools provide a valuable help for evaluating alternatives. a process designed to produce 23 kg pdna/year is analysed with the superpro designer tool. the target pdna is amplified in escherichia coli. after harvest, alkaline lysis is used to disrupt cells and release pdna and impurities. precipitations with isopropanol and ammonium sulphate are performed to concentrate/pre-purify pdna prior to hydrophobic interaction chromatography. experimental data is used as input for simulation. inventory analysis identified water, yeast extract, tryptone, isopropanol and ammonium sulphate as the major raw materials. major raw material costs (50%) are related to fermentation components. economic analysis indicates a unit production cost of $ 375/g pdna. for a selling price of $ 1667/g, the payback time was 1.2 years and the roi was 88.6%. an environmental analysis highlighted the replacement of the isopropanol precipitation for a microfiltration step as a benefit which would: (i) reduce the cost of raw materials (13.8%), (ii) reduce the environmental impact associated with isopropanol (70%) and (iii) reduce costs associated with the treatment/disposal of liquid waste (32.3%). preparation of chitosan microspheres for controlled release of somatotropin s. simsek 1 , j. introduction: proteins and peptides have received extensive interest for their therapeutic applications in clinical applications. in order to achieve high administration efficacy of proteins, polymeric particulate carriers have been developed as an effective way to control the drug release profile and to protect the protein molecules from degradation. somatotropin also known growth hormone is a protein hormone of about 190 amino acids. growth hormone is of considerable interest as a drug used in both humans and animals. chitosan a natural linear biopolyaminosaccharide is obtained by alkaline deacetylation of chitin. properties such as biodegradability, low toxicity and good biocompatibility make it suitable for use in biomedical and pharmaceutical formulations. the aim of this study was to prepare chitosan microspheres containing somatotropin and to investigate these microsphere formulations in-vitro release properties. methods: somatotropin-chitosan microspheres were prepared as follows: chitosan was dissolved in acidic solution (2%) containing polysorbate 80. sodium sulphate solution (20% w/v) containing somatotropin was added into the chitosan solution and mixed 500 rpm for a hour. resulting suspension was centrifuged 15,000 rpm 15 min at 4 • c. the formed microspheres were freeze-dried and sieved. in-vitro release studies were performed in ph 7.4 phosphate buffer and time interval samples were removed and analysed by bradford protein assay method. results: microspheres were obtained by using chitosan. protein encapsulation efficiency was between 95 and 99%. average particle size of microspheres was between 48 and 57 m. during to in-vitro release studies burst effect was observed with chitosan microspheres. for decreasing the burst effect gluteraldehit, betacyclodextrin and poly ethylene oxide were added to formulations. conclusion: according to our results modified chitosan microspheres are promising vehicles for controlled release somatotropine delivery. cancer immunotherapy using hyperthermia with magnetic nanoparticles and dendritic cells k. tanaka, a. ito, t. kobayashi, t. kawamura, s. shimada, k. matsumoto, t. saida, h. honda department of biotechnology, school of engineering, nagoya university, nagoya, aichi 464-8603, japan. e-mail: h041306d@mbox.nagoyau.ac.jp (k. tanaka) our hyperthermia system utilizes magnetic nanoparticle covered with lipid layer including cationic lipid (magnetite cationic liposomes, mcls) as a heating mediator and necrotic cell death is induced by means of locally generating heat. in this process, heat shock proteins (hsps) are strongly induced and released. dendritic cells (dcs) are potent antigen-presenting cells (apcs) that play a pivotal role in regulating immune responses in cancer, which is in carrying antigens to apcs and in the maturation of dcs by acting as a danger signal. in the present study, we investigated the therapeutic effects of dc therapy combined with mcl-induced hyperthermia on b16 melanoma. in an in vitro study, when immature dcs were pulsed with b16 cells heated at 43 • c for 30 min, mhc class i/ii, costimulatory molecules cd80/cd86, and chemokine receptor ccr7 in the dcs were up-regulated, thus resulting in dc maturation. c57bl/6 mice bearing a b16 melanoma nodule were subjected to combination therapy using hyperthermia and dc immunotherapy. mice were divided into four groups: group i (control), group ii (hyperthermia), group iii (dc therapy), group iv (hyperthermia + dc therapy). complete regression of tumors was observed in 60% of mice in group iv, while no tumor regression was seen among mice in the other groups. increased ctl and nk cell activity was observed on in vitro cytotoxicity assay using splenocytes in the cured mice treated with combination therapy, and the cured mice rejected a second challenge of b16 melanoma cells. this study has important implications for the application of mcl-induced hyperthermia plus dc therapy in patients with advanced malignancies as a novel cancer therapy. fermentation of a marine bacterium for the production of cytotoxic compounds vicky webb 1 , els maas 1 , eiichi akaho 2 , hiroto kambara 2 , debbie hulston 1 , anna kilimnik 1 : 1 marine biotechnology, national institute for water and atmospheric research ltd, kilbirnie, wellington new zealand; 2 faculty of pharmaceutical sciences, kobe gakuin university, kobe 651-2180, japan marine bacteria are a potential source of novel compounds for the pharmaceutical industry. new zealand marine bacteria were isolated from a variety of sources and initially bacterial supernatants were screened using a cell based mtt cytotoxic assay. two bacteria were chosen for further fermentations in different media to optimise cytotoxic compound production. the media used were selected for their diverse ingredients. they were either carbon rich, nitrogen rich, starch rich or a basic seawater medium. the fermentations were extracted using ethyl acetate and methanol prior to assaying. the results showed that cytotoxic compound production was enhanced ten fold in the starch rich medium compared to the other media. the levels of cytoxicity appeared to be depended on the cell line used, with the epithelial lung carcinoma cell line a549 being more sensitive to the cytotoxic compounds than the human fibroblast cell line mrc-5. isolation and identification of marine bacteria from deep-sea sediments els maas 1 , cara brosnahan 1 , vicky webb 1 , helen neil 2 , phil sutton 2 : 1 marine biotechnology, national institute for water and atmospheric research ltd., kilbirnie, wellington new zealand; 2 oceanography, national institute for water and atmospheric research ltd., kilbirnie, wellington, new zealand polystyrene micro plate activated by utilizing a common co-60 gamma source with a crotonic acid. the well surface have been studied in term of optical quality, protein (h-igg) binding capacity and stability. a significantly enhanced total capacity and strength of binding to grafted surfaces was demonstrated as compared to passive adsorption of the proteins to untreated surfaces.the majority routine laboratory tests for the measurement of rheumatoid factor (rf) are semi quantitative and in order to achieve accurate, sensitive and specific rf assay, we are developed enzyme linked immuno sorbent assay (elisa) for human igm-rf kit. stable and reproducible binding of antigen (human-igg) to well of micro titer plate is a prerequisite for rf-elisa kit. the principle of the assay was that the antibodies in serum of patients with rheumatoid arthritis (ra), commonly rheumatoid factor (rf) are directed to the fc part of human igg. in most cases rf belong to the igm class, the well of micro titer plate are coated with antigen (h-igm), and antibodies (h-igg) binding to immobilized antigen is detected by adding enzyme conjugate (anti human-igm-hrp-enzyme) to the wells, substrate was used for color reaction.the performance characteristics of the assay was, the coefficient of variation (c.v) of intra and inter assay was 4.4% and 2.2% respectively, the linearity is ranging from 86 to 112%, and the recovery for three different sera is ranging from 89 to 106%. the data presented in this paper indicated that the activation of polystyrene micro titer plate by the gamma rays could be use for preparation of igm-rf kit and may be others immunoassay techniques. overcoming the nuclease barrier to gene expression during trafficking of plasmid dna vectors a.r. azzoni 1 , a. tavares 2 , g.a. monteiro 1 , d.m.f. prazeres 1 : 1 centro de engenharia biológica e química (cebq), instituto superior técnico, 1049-001 lisboa, portugal; 2 instituto gulbenkian de ciência, rua da quinta grande 6, 2780-156 oeiras, portugal. e-mail: azzoni@ist.utl.pt (a.r. azzoni) inefficient nuclear delivery of plasmid dna (pdna) vectors is thought to be a bottleneck to gene transfer in gene therapy and dna vaccination utilizing non-viral delivery systems. one of the main barriers found by pdna vectors during trafficking to the nucleus is degradation by a population of endo/exo-nucleases. this barrier may be partially circumvented by shielding the pdna from the nucleaserich cell environment with adjuvants or by using nuclease inhibitors. a different approach that has been studied at the cebq is the generation of pdna vectors that are more resistant to nuclease action a priori. in this work, the nuclease barriers to gene expression are being studied aiming at the generation of pdna with improved resistance to nucleases and thus higher transfection efficiency. by engineering the plasmid labile sequences, new plasmid vectors with an improved resistance to physical-chemical and biological degradation are being generated. this was indicated by an extended half-life of the supercoiled isoforms during storage and when the plasmids were exposed to nucleases present at eukaryotic cell lysates and mice plasma. the intracellular trafficking of the new plasmid vectors through the cytosol of mammalian cells was then assessed by fluorescence in situ hybridisation (fish) and the expression of the reporter protein (egfp) was detected by fluorescence microscopy. identification and evaluation of antibacterial phytochemicals of fishbone fern (nephrolepis cordifolia) rikhia chakraborty 1,2 , promod kumar verma 2 : 1 department of cancer biology, lerner research institute, 9500 euclid avenue cleveland, oh 44195, usa, 2 department of biotechnology, guru nanak dev university, amritsar, punjab 143005, india. e-mail: riar5400@rediffmail.com (r. chakraborty) in this study, different aqueous and organic extracts from the fern, nephrolepis cordifolia, were used for screening tests for antibacterial effects. protein and lipid extracts were first tested for antibacterial activity. subsequently, crude extracts of leaves, roots, and stems were prepared in methanol, ethanol, chloroform, hexane, petroleum ether, diethyl ether, and water using optimized standard protocols. each fraction was tested for anti-microbial effect through agar well-diffusion assay, and paper disc method. the antibacterial spectrum against which the fern is active was thus determined. dosagedetermination for optimum activity was also determined for each of the extracts. bacillus and staphylococcus were used as the indicator test-organisms. the results were very encouraging; being effective even at the 54th day, thus showing that the antibacterial properties were not due to any changes in external factors and physiological effects. ethanol, methanol and chloroform extracts from the subaerial portions had strong anti-microbial properties. agar-well diffusion assay and paper-disc diffusion assay done for different solvent fractions were giving comparable results. 2.5 mg was adequate for maximum effect against b. circulans, s. aureus, s. epididermis, and streptococcus sp. 7.5 mg was adequate as effective dosage for kleb-siella pneumoniae. 10 mg was required for mycobacterium bovis, e. coli. pseudomonas was not showing any susceptibility. given the broad spectrum of activity, especially towards the gram-negative bacteria, nephrolepis cordifolia is definitely a promising plant having pharmacological importance. for a full interpretation of the present results further investigations are necessary to elucidate the different physical and chemical parameters of the active principles and also to determine the mechanism of action. the present work highlights n. cordifolia as a plant having a broad spectrum of antimicrobial activity, a phenomenon very rarely observed in the plant kingdom. bacteria (escherichia, salmonella, proteus, staphylococci and bacillus) were isolated from hospital soil using selective enrichment and growth on selective and differential medium, viz. macconkey's agar, clyed medium and baird parkers medium. confirmation and species identification was carried out by biochemical and serological tests. from these isolates, three different pathogens were used to study multiple antibiotic resistances. e. coli bj 83 showed resistance to ampicillin, streptomycin and cefurixime. s. typhi and s. aureus showed resistance to ampicillin and cefuroxime. assay using octadisc using e. coli and s. typhi showed a broader resistance pattern to antibiotics including amoxicillin, clavulanic acid, cephalexin, chloramphenicol, ciprofloxacin, and cotrimoxazole. the r plasmid profile was studied to understand the mechanism of drug resistance. to combat the problem of drug resistance, a strategy of combined antibiotic response of cultures were studied. such a synergistic combination would possibly have the effect of overcoming multiple antibiotic resistances. for e.g. kanamycin resistance strains were inhibited in presence of kanamycin and cefotaxime. further, the bactericidal activities of antimicrobials in honey and garlic were also tested. the mic of honey was observed to be 4%, while that of garlic was between 0.1 and 1%. honey and garlic were also found to inhibit the growth of organisms in the presence of antibiotics. kanamycinresistant e. coli was unable to grow in presence of kanamycin and 0.08% garlic. these traditional agents, long used in ayurvedic system of medicine in india, could be further explored for potent antimicrobial properties. hepatitis b virus (hbv) infection is s global health problem. assays for hbv antigens and antibodies are widely available and standardized. extremely sensitive qualitative pcr kits are also available for detection of hbv in serum. hbv pcr kit may be useful in assessment of occult hepatitis b in hbcab positive alone subjects and carriers. a positive pcr results show presence of virus articles in serum without considering serologic results. but there are differences in efficiency of hbv dna amplification kits. in this study we compare two commercially available hbv pcr kits for evaluation viremia of hbsag positive carriers and hbcab alone positive subjects. material and methods: of the 368 randomly selected subjects serologically examined for hbv, 49 and 43 were positive for hbsag and hbcab alone respectively. both later groups were tested for hbv dna by two commercial kits, hbv pcr detection kit (cinnagen, iran) and hbv pcr test (pazhohesh azma, iran). dna extraction kits recommended by each manufacturer were used for hbv dna extraction. amplicons in both kits were a highly overlapped fragment in 5 conserved sequence of viral genome. results: of the 49 hbsag positive carriers, hbv dna was detected in 37.4 and 65.3% using kit1 and kit2 respectively. only 28.6% were positive in both kits. in hbcab positive subjects (n = 43), 23.3% were positive by kit 2 and all samples were negative when tested by kit 1. there was a significant difference between two kits. sensitivity of kit 1 and kit 2 were 48.6 and 91.4%, respectively. overall, kit 2 increased the detection rate of hbv dna by 88.2%. discussion: our study show there is a significant variation between these two commercial kits especially in hbcab positive subjects that the copy of virus is very low. from these results, it can be concluded that the unstandardized kits have not compatible results, and pcr test interpretation should be done with great care. . the antibacterial activity of the extracts was evaluated based on the inhibition zone using plate diffusion method. most of the extracts were active against both gram positive and gram-negative bacteria, but pseudomonas aerugenosa, bacillus subtilis and sarcina marcescens, were more susceptible to almost all the extracts. finally, further research will be done to elucidate the nature of the active compound and investigate for peptides by using protein gel immobilization bioassay. short interfering rna delivery and gene silencing using polymeric nanocarrier systems k.a. howard 1 , x. liu 1 , d. oupicky 2 , f. besenbacher 1 , j. kjems 1 : 1 inano, university of aarhus, denmark, 2 department of pharmaceutical sciences, wayne state university, detroit, usa the effectiveness of a drug is determined by the ability to migrate through the body and reach target sites in therapeutically relevant levels. nanocarriers for delivery of bioactive agents are being developed at inano to maximise drug payload at target sites. the inclusion of "biological triggers" into the nanocarrier design is used for modulation of cellular nucleic acid trafficking and increased target interaction. chitosan and peptide-based polymers were used to formulate nanocarriers in the size range 30-250 nm containing small interfering rnas (sirnas) for gene silencing applications. page analysis showed the structural integrity of the sirna was maintained during particle formation. in systems composed of bioresponsive polymers, nanocarrier disassembly and sirna release under cellular conditions were shown, using atomic force microscopy. the time course for sirna uptake into nih cells was visualised using confocal microscopy. in addition, sirna localisation within cells could be modulated by the composition of the polymer used. the ability of the nanocarrier system to mediate gene expression was investigated in a cell line stably expressing enhanced green fluorescent protein (egfp). furthermore, the various delivery systems were tested in a mouse model stably expressing the egfp protein using both nasal and intravenous delivery routes. the use of animals as a source of organs and tissues for xenotransplantation can overcome the growing shortage of human organ donors. however, the presence of xenoreactive antibodies in humans directed against swine gal antigen present on the surface of xenograft donor cells leads to the complement activation and immediate xenograft rejection as a consequence of hyperacute immunological reaction. the graft of genetically modified organ of a swine depleted of enzyme ␣1,3-galactosyltransferase that is responsible for gal antigen origin, would be tolerated with simultaneous administration of medicines decreasing other less severe immunological reactions. to prevent hyperacute rejection it is also possible to modify swine genome by human genes controlling enzymatic cascade of complement or modifying the set of donor's cell surface proteins. for this purpose genetic constructs containing inactivated ␣1,3-galactosyltransferase gene, human cd59, cd55 and cd46 genes controlling complement activation and human genes encoding ␣1,2-fucosyltransferase and ␣-galactosidase enzymes modifying cell surface proteins were prepared. these genetic constructs were transfected into the pig foetal fibroblast using lipofection method. after selection, molecular and cytogenetic characteristic of cells with transgene integrated into the host genome were performed. introduction: protease 2a(2a-pro) of coxsackievirus b3 (cvb3) plays major role in viral replication. in case of infection, viral proteins are being synthesized from viral mrna using host biosynthesis machinery. 2a-pro of virus, after being synthesized, exhibit two critical functions, cleavage of viral proteins and breaking eif4g (eukaryotic initiation factor 4g-formerly called p220) which leads to host cell translational system shot-off. the enzyme plays essential role in viral replication and cellular damage. to understand pathogenicity of infection and also developing potent and selective inhibitors against picornavirus infection, it is necessary to prepare pure 2apro enzyme. in this study an expression system with efficient and high yields was obtained. methods: cdna of 2apro was synthesized using in vitro infection of permissive host through reverse transcription process and was cloned in pet22b(+) and since 2a-pro is a toxic product, naturally before induction its expression will act on the host and damage the cells. for this different hosts were checked and finally, blr(de3) plyss which carries an extra-plasmid for lysozyme expression that minimizes unwanted target protein production (leakage) was selected. on the other hand for biological activity assay, polyclonal antibodies against antigenic sites of p220 was prepared by synthesizing small peptides, corresponding to antigenic site of p220 coupling to klh and injecting subcutanously to rabbit. then, the enzyme and its substrate (hela cells lysate that contain p220) were incubated together for different times intervals. results: the recombinant product was confirmed by sds polyacrylamide gel electrophoresis and immunoblot analysis. also p220 cleavage by 2apro was assessed by sds-page and western blot analysis. cleavage of p220 by r2a-pro was prominent after 24 h. so recombinat 2a-pro with good activity was prepared. application of bombyx mori nuclear polyhedrosis virus (bmnpv) bacmid system on production of glycoprotein in larvae of silkworm ayano kageshima, tatsuya kato, misun kwon, enoch y. park department of applied biological chemistry, shizuoka university, ohya 836, suruga-ku, shizuoka 422-8529, japan bombyx mori nuclear polyhedrosis virus (bmnpv) bacmid system was applied on production of glycoprotein in larvae of silkworm. the bacmid system of autographa californica nuclear polyhedrosis virus (acnpv) has already been established and widely used. since the acnpv does not have a potential to infect silkworm we developed the first practical bombyx mori nuclear polyhedrosis virus (bmnpv) bacmid system directly applicable for the protein expression of silkworm. by using this system, the green fluorescence protein and glycoprotein, human ␤1,3-n-acetylglucosaminyltransferase 2 were successfully expressed in silkworm larvae not only by infection of its recombinant virus but also by direct injection of its bacmid dna. three different kinds of signal sequences were tested for the secretion of glycoprotein into hemolymph of silkworm. signal peptides of prophenoloxidase-activating enzyme and bombyxin: insulin-like brain secretory peptide showed the highest secretion ratio, 99% of total expressed ␤1,3-n-acetylglucosaminyltransferase 2 was secreted into hemolymph of silkworm. using bacmid system 50 mu/ml of ␤1,3-n-acetylglucosaminyltransferase 2 was expressed in hemolymph of silkworm, which was 2-three times higher than that of insect cell. silkworm is one of the most attractive hosts for large-scale productions of eukaryotic proteins as well as recombinant baculoviruses for gene transfer to mammalian cells. this method provides the rapid protein production in silkworm, is free from biohazard, thus will be a powerful tool for the future production factory of recombinant eukaryotic proteins and baculoviruses. we have established an efficient system for foreign gene expression in lily (lilium longiflorum) pollen in a transient mode. pollen was transformed using agrobacterium via vacuum filtration for 20 min. the pollen germinated for 24 h was analyzed to confirm its foreign gene expression in molecular analysis. mouse fed the transgenic pollen culture for 8 weeks showed immune reaction specific for pollen-derived recombinant protein. and the igg level was highly elevated by one time boosting injection afterwards. the lily pollen system may be suggested as a novel type of biofactory for producing edible vaccine with rapidity. anti-apoptosis engineering with the 30kc6 gene obtained from silkworm shin sik choi, won jong rhee, tai hyun park school of chemical and biological eng., seoul national university, seoul 151-744, korea the chinese hamster ovary (cho) cell line producing recombinant human erythropoietin (epo) was manipulated to express the 30kc6 gene, which was originally obtained from a silkworm. the expression of 30kc6 inhibited serum deprivation-induced apoptosis and increased the cell density and epo expression level per unit cell by five-and two-folds, respectively. an increase in these two factors resulted in a 10-fold increase in the volumetric productivity of epo. compared with the controls, the oligosaccharide structures of the epo synthesized by the cells expressing 30kc6 showed greater homogeneity. the terminal sialylation of the glycans of epo were promoted by the expression of 30kc6. the positive effects of 30kc6 expression on the cell viability and productivity were attributable to the stable maintenance of the mitochondrial activity. these results demonstrate that the cho cell line genetically engineered with the 30kc6 gene has a great potential for use in the production of therapeutic proteins. evaluation of fucoidan-chitosan hydrogels on superficial dermal burn healing in rabbit: an in vivo study a.d. sezer 1 , f. hatipoglu 2 , z. ogurtan 3 , a.l. baş 4 , j. introduction: healing of dermal wounds with macromolecular agents such as natural polymers is one of the research areas of the pharmaceutical biotechnology. fucoidan is a sulphated polysaccharide which is commonly obtained from seaweeds. although a great number of studies on the different pharmacological properties of fucoidan are present, there is very limited information on the fucoidan-based system used in dermal burns. the aim of this study was to prepare chitosan hydrogel containing fucoidan and to investigate this hydrogel formulation for treatment of dermal burns on rabbits. methods: fucoidan-chitosan hydrogels were prepared as follows: the polymers were dissolved in acidic solution and sonicated for removing the air-bubbles then the gels were stored at +4 • c for in vivo studies. seven adult male new zealand white rabbits (mean weight, 3.8 ± 0.6 kg) were used for the evaluation of the gels on superficial dermal burns. the back of the rabbits were depilated and sedated. the wounds were made by circular stamp aluminium caps (3.8 cm 2 ) at 80 • c. four wounds were formed for each rabbit; (a) was treated with fucoidan-chitosan gel, (b) was treated with fucoidan solution, (c) was treated with chitosan gel (without fucoidan) and (d) as a negative control group. biopsy samples were taken at 7, 14 and 21st days at the beginning of the study and each wound site was macroscopically observed and evaluated histopathologically. results: oedema was not observed in all groups after 3 days treatment except controls. after 7 days treatment, fibroplasia and scar were observed on wounds treated with fucoidan-chitosan gel and fucoidan solution. the best regenerate dermal papillary formation and the fastest closure of wounds were observed in group a after 14 days treatment. the wound epithel elongation and thickness values were measured; a (5566 and 179 m), b (3586 and, 146 m), c (3666 and, 162 m), d (3533 and, 134 m) at the end of the study. conclusion: re-epithelization and contraction of the wound area which was treated with fucoidan-chitosan hydrogel were faster than the other groups. the fucoidan-chitosan hydrogel formulations can be suitable for the treatment of dermal burns. aim: to analyze the neutralizing -related activity of antibodies against e1 region of hcv, specific polyclonal antibody was raised by immunized rabbits with synthetic peptide that had been derived from e1 region of hcv with the amino acid sequence e1 antibody [ghrmawdmm). materials and methods: hyper-immune hcv e1 antibodies were incubated over night at 4 • c with serum samples from patients positive for hcv rna, with different viral load, ranged 7-11 million copes/ml, then incubated 90 min to hepg2 cells. rt-pcr and flow cytometry were used to study the inhibition binding and entry effect of e1 antibody. direct immunostaining of e1 antibody conjugated with fitic and flow cytometry analysis showed reduced the mean fluorescence intensity in the samples pre-incubated with e1 antibody compared with samples without e1. of 18 positive serum samples, 13 (72%) samples showed completely inhibition of infectivity as detected by rt-pcr. conclusion: in house produced e1 antibody, blocks binding and entry of hcv virion infection to target cells suggesting the involvement of this epitope in virus binding and entry. isolation of these antibodies that block virus binding and entry will be useful in providing potential therapeutic reagents and for vaccine development. tumor necrosis factor beta (tnf␤) is a pleiotropic cytokine mediating its activity through the same receptors as the structurally related tnf␣. shortening of the n-terminal part has been reported to enhance its cytotoxic activity, with the explanation that removal of the flexible n-terminus reduces steric interferences in the receptor binding process. for studies of relationship between the n-terminal protein structure and physicochemical properties, three different forms mettnf␤, his7tnf␤ and n19tnf␤ were expressed in e. coli. high solubility of n19tnf␤ was expected, however, both analogs his7tnf␤ and n19tnf␤ were predominantly obtained in the form of inclusion bodies (ibs). on the other hand, mettnf␤ was equally distributed between the soluble and insoluble fraction. most probably, the composition of the n-terminal part, such as the exposure of hydrophobic residues in the case of n19tnf␤, or a mildly hydrophobic stretch of seven histidines appended to the natural hydrophobic n-terminus in the case of his7tnf␤, lead to incorrect folding and force aggregate formation. solubilization of ibs was performed under native and denaturing conditions using various concentrations of nls, urea and gndhcl. mettnf␤ ibs were easily dissolved with 0.2% nls, while his7tnf␤ and n19tnf␤ demanded denaturing conditions. structural differences in the nterminal part of various tnf␤ proteins were also reflected in protein refolding characteristics and chromatographic behavior. dilution, ultrafiltration and dialysis were used for refolding, and imac was chosen as the main chromatographic step. our results suggest that not only the length of the n-terminal part of tnf␤ but also the composition and exposure of certain amino acid residues affect its physicochemical properties. enzymatic modification of sphingomyelin long zhang, lars hellgren, xuebing xu biocentrum-dtu. e-mail: lz@biocentrum.dtu.dk (l. zhang) due to its major role in maintaining the water-retaining properties of the epidermis, ceramide is of great commercial potential in cosmetic and pharmaceuticals such as hair and skin care products. currently, chemical synthesis of ceramide is a costly process, and developments of alternative cost-efficient, high yield production methods are of great interest. in the present study, the potential of producing ceramide through enzymatic hydrolysis of sphingomyelin (sm) have been studied. sm is a ubiquitous membrane-lipid and rich in dairy products or by-products. in present study, we have optimized the production of ceramide from sm using phospholipase c from clostridium perfringens. water and enzyme amount had the biggest influence on sm hydrolysis in the system. botulinum neurotoxins constitute a family of bacterial toxins for botulism syndrome in human. the toxins bind with high affinity to nerve cells where they cause a complete inhibition and release of neurotransmitters and thereby produce flaccid paralysis. in this work we have reported isolation of the binding domain of type e neurotoxin by pcr and expressed in a proper expression vector. the output of this investigation can be used as a tool to study the mechanism of binding of holotoxins and also can be useful to study the antibody production against botulism syndrome. the synaptobrevin (vamp2) a protein which play a key role in the fusion and exocytosis of the vesicle of mammalian nerves terminals this protein is substrate different serotypes of botulinum neurotoxins. light chain of clostridium botulinum type b, d, f and g cleave end of neurons. due to intraction of clostridium botulinum neurotoxin with vamp2, this protein can be used as one of the toolsin the detection of poisings cause by this bacteria in the clinical laboratory using the enzyme with proof reading activity the above gene amplified by pcr technique for the production of the recombinant protein, the prokaryotic expression vectors (pet system) was used. a recombinant protein developed on poly acrylamide gel analyzed by western blotting and eliza. most children with down syndrome (ds) are born to younger mothers (<35 years). recent reports linking down syndrome (ds) to maternal polymorphisms at the methylenetetrahydrofolate reductase (mthfr) gene locus have generated great interest among investigators in the field. in this study, forty mothers with their affected outcomes and 100 control mothers were included. all mothers were subjected to complete medical and nutritional history with special emphasis on folate intake through food or oral supplementation. estimation of blood homocysteine level was done. also we examined the two polymorphisms in genes encoding the folate metabolizing enzyme methylenetetrahydrofolate reductase (mthfr), namely, 677c > t and 1298a > c. folic acid intake from food and from vitamin supplements was significantly low (below the recommended daily allowance) in the group of mothers with ds children compared to control mothers (p < 0.01) using student t-test. blood homocysteine was normal in both control and ds mothers. the prevalence of the two polymorphisms, namely, 677c > t and 1298a > c in mothers of ds children (case mothers) (n = 40) was compared with controls (n = 100). frequencies of mthfr genotypes (cc, ct, and tt) at position 677 demonstrated no difference between the case and control groups. genotype frequencies of mthfr at position 1298a (aa, ac, and cc) were different among the case and control mothers. we here report the first study on a possible relation between ds with mthfr 1298a > c genotypes in egypt. our results showed that mthfr 1298a > c polymorphism is a remarkable genetic entity among egyptian females with d.s. children. sufficient folate intake and supplementation is an important preventive strategy in overcoming the risk of nondisjunction. homologous recombination (hr) is the mechanism that permits the creation of genetically engineered strains through gene targeting. in order to further develop gene targeting techniques, notably for higher eukaryotes such as filamentous fungi, it is of crucial importance to fully understand the molecular mechanisms behind mitotic hr. in saccharomyces cerevisiae, a dna double strand break (dsb) is an essential intermediate in meiotic hr. however, as hr occurs at a low rate in mitotic cells, it has been difficult to determine the nature of the event(s) that triggers it. rad52 is a key protein involved in hr and is evolutionarily conserved from yeast to human. to shed light on the molecular events in hr, we have generated a large collection of defined rad52 mutant strains in the yeast s.cerevisiae. a screen of these mutants led to the identification of strains that fail to repair dna dsbs, yet are proficient for homologous recombination. this result strongly suggests that dsbs may not be the major cause of spontaneous mitotic hr and gives new perspectives in respect to novel potential gene targeting substrates. we have analyzed these separation of function mutants in a variety of new assays to obtain a more detailed understanding of their controversial phenotype. our latest results will be presented. the outcome of interferone plus ribavirine treatment of hepatitis c virus (hcv) genotype 4 is unfortunately poor. development of alternative therapy for this genotype is of a paramount importance. inhibition of hcv gene expression in vitro by the use of antisense phosphorothioate oligodeoxynucleotides (s-odn) against internal ribosomal entry site (ires) elements were associated with favorable results. to assess s-odn activity, previous studies utilized viral subgenomic or full cdna fragments linked to reporter genes and transfected into adhered cells or in a cell free system. in the present study we utilized hepg2 cells infected with native hcv rna of genotype 4. the culture system presented herein was shown to support hcv replication on the following bases (1) consistent detection of both plus and minus rna strands for 4 weeks in cells and in fresh culture supernatent, (2) ability of supernatent to infect naive hepg2 cells (3) consistent expression of core and e1 envelope proteins in infected cells throughout the 4 week culture. s-odns against aug translation start site (s-odn-1, nt 326-348) of the viral polyprotein precursor and stem loop iiid within the ires region (s-odn2, nt 264-282) were added to infected cells. intracellular viral replication was monitored by nested rt-pcr of plus and minus strands. the results of these experiments demonstrated that intracellular replication of hcv genotype 4 was completely arrested after 48 h in culture using either s-odn molecule (with more efficacy of s-odn1 than s-odn2) at concentrations as low as 1 m. the inhibitory effect of s-odn appeared to be specific to hcv replication since equal levels of human glyceralehyde 3-phosphate dehydrogenase (gapdh) gene expression were noted pre and post supplementation of s-odns. in conclusion, the present study provides evidence antisense phosphorothioate oligonucleotides have potent inhibitory effect on genomic replication of hcv genotype 4, the most common type in egypt. a sensitive and specific pcr-elisa was developed to detect shigella dysentery in food. the assay was based on the incorporation of degoxigenin-labeled dutp and a biotin-labeled primer specific for shiga toxin genes during pcr amplification. the labeled pcr product were bound to streptoavidin-coated wells of a microtiter plat and detected by an elisa. the elisa detecting system was able to increase the sensitivity of the pcr assay by up to 100-fold, compared with a conventional gel electrophoresis. the detection limit of the pcr-elisa was 0.1-10 cfu dependent upon shigella dysentery serotypes and genotypes of shigatoxin. the entire procedure took about 4 h. isolation, cloning, expression and purification of snap-25 as a substrate of botulinum neurotoxin type a and e m.l. mossavi 1 , f. ebrahimi 1 , j. amani 1* , h. basiry 1 , z. ahmadi 2 : 1 department of biology, faculty of science, imam hussein university, tehran, iran; 2 department of biology, faculty of medicine, bageatallah university, tehran, iran. e-mail: kpjamani@ihu.ac.ir (j. amani) clostridial neurotoxin inhibit neurotransmitter relase by selective and specific intracellular proteolysis of synaptosomal associated protein of 25 kda (snap-25); synaptobrevin/vamp-2 and syntaxin. snap-25 is one of the components that form docking complex in synaptic ends. this protein is substrate for botulinum neurotoxins type a and e. each of these toxin serotypes specifically cleave snap-25 in particular position and there by block docking and synaptic vesicle membrane fusion and finally prevent neurotransmitter exocytosis and transition of neurotic signals. in order to use the protein as a substrate for detection of different type of clostridium neurotoxin in vitro test, the protein was produced by recombinant technique. the dna encoding snap-25 was isolated from rat brain by pcr using the two primer the amplified fragment colonel into expression vector pet32a.the expression protein was purified by affinity chromatography. confirm by different method the his tag fusion protein was digested with entrokinase. the present work deals with the preparation of pure alpha-1antitrypsin (aat) protein from healthy subjects, which can be used in preparing its corresponding monospecific antibody in albino rabbits. this antibody was found very useful in the immuno-diagnosis of pulmonary emphysema. this study has been also concerned with the biochemical changes associated with aat deficiency in pulmonary diseases. to fulfill this work, a group of healthy blood donors was selected for separation of pure aat antigen from blood. the pure aat was used for the preparation of anti-aat, the purity and potency of antibody was checked by titration methods. the biochemical changes were studied in thirty subjects clinically divided into three groups including, control, heavy cigarette smokers with pulmonary emphysema, and non-smoking subjects with pulmonary emphysema. the activity of elastase and hydroxyproline (hp) level as a marker of elastin and collagen breakdown were assayed in bronchoalveolar lavage (bal) fluid. the activity of aat and to inhibit proteases as represented by tryptic inhibitory capacity (tic) was evaluated. serum ceruloplasmin, transferrin and iga level as well as thiobarbituric acid reactive substances (tbars) were estimated in all groups. our data revealed that aat and its tic showed very highly significant decreased levels in all patients with emphysema as compared to control, while the elastase activity and hp level in bal fluid were significantly increased in these patients. serum tbars was significantly increased in such patients associated with increasing level of both ceruloplasmin and transferrin. while, serum iga was significantly increased. furthermore, the biochemical changes were markedly changed in smokers with emphysema than non-smoking subjects. in conclusion, the preparation of anti-aat on the local level is very important where less expensive and less time consuming and can be useful in immuno-diagnosis and prognosis of pulmonary diseases. a new method combined with boosting and projective adaptive resonance theory for analysis of gene expression data from cancer patients hiro takahashi, yasuyuki murase, hiroyuki honda department of biotechnology, school of engineering, nagoya university, nagoya 464-8603, japan. e-mail: h041305d@mbox.nagoyau.ac.jp (h. takahashi) an optimal and individualized treatment protocol based on accurate diagnosis is urgently required for the adequate treatment of patients. for this purpose, it is important to develop that a sophisticated algorithm that can manage large amount of data, such as gene expression data from dna microarray, for optimal and individualized diagnosis. in our previous study, we developed the projective adaptive resonance theory (part) as a gene filtering method and boosted fuzzy classifier with sweep operator (bfcs) as a modeling method. in the present study, we applied the combination of part and bfcs (part-bfcs method) to microarray data of brain tumor (central nervous system tumor) obtained from the website. the method enabled the selection of 14 important genes related to the prognosis of the tumor, i.e., sensitivity for combined therapy with surgery, radiotherapy, and chemotherapy mainly with vincristine, cisplatin, and cytoxan. the constructed model showed about 20% higher accuracy than that of the conventional method. genomic signal analysis of hiv variability based on rt gene p.d. cristea, rodica tuduce d. otelea biomedical engineering center, university "politehnica" of bucharest, romania. e-mail: pcristea@dsp.pub.ro (p.d. cristea) dna sequences genotyped from 60 clade f hiv-1 isolates from romanian patients in the laboratory of the national institute of infectious diseases "matei bals", bucharest, romania, have been studied. the symbolic sequences have been converted into digital genomic signals by using a complex quadrantal representation of the nucleotides described earlier. the cumulated phase and unwrapped phase of a complex genomic signal reflect the statistical distribution of bases and base-pairs, respectively. independent component analysis of the genomic signals has been used to characterize the variability of the f subtype hiv strains isolated in romania. the sequenced segment is of (about) 1302 base pairs, approximately aligning with the standard sequence of hiv-1 (accession nc001802 in genbank) over the interval 1799-2430 bp. this segment, which is currently used for the standard identification and assessment of hiv-1 strains, comprises the protease (pr) gene and two thirds of the reverse transcriptase (rt) gene. only results referring to the analysis of the rt gene region are presented here and used for extracting features of virion isolates and for establishing phylogenetic trees of the studied strains. the analyzed rt encoding segment has the length 1005 bp and is located in the second interval (298-1302 bp) of the analyzed dna segment, respectively along the 2096-3100 bp region of nc001802. taking into account the mutations identified in these sequences, the samples were classified in three groups from the point of view of their resistance to current antiretroviral compounds: sensitive, resistant and multiresistant. the paper presents results for the isolates in which mutations leading to multiple drug resistance have been identified. over expression of secb protein in e. coli enhances the periplasmic expression of human growth hormone m. ghafari 1,2 , a. zomorodipour 2 : 1 islamic azad university of jahrom, tehran, iran; 2 national institute for genet eng and biotechnol., tehran, iran. email: maryamghafari2001@yahoo.com (m. ghafari) among several proteins involved in the secretion pathway of proteins in e. coli, secb plays a key-important role in solubilization of preproteins before processing. in order to increase processing of a human growth hormone precursor (pelb::hgh) which appeared to have problem in processing efficiency, as a possible solution a regulated co-expression of a secb gene was considered. in this regard, we designed an arabinose-regulated secb expressing plasmid compatible with an iptg/lactose-regulated pelb::hgh expressing plasmid. for the construction of the secb expressing plasmid the origin of replication and antibiotic resistant gene (amp) of a pbad vector was replaced by a p15a-ori and a kanamycin resistant gene, respectively. the expression and processing of pelb::hgh preprotein in the two-plasmid containing bacteria in a secb over-expression state was compared to that of the pelb::hgh expression in normal bacteria. although a decline in total expression level of hgh during the overexpression of secb was observable, probably due to presence of two different expressing plasmids, but both the processing efficiency of pelb::hgh and the transport of mature protein into the periplasmic space was enhanced during prolonged arabinose induction. current diagnostics and therapeutics for cat allergy are based on cat epithelial extracts. natural allergen extracts contain a mixture of allergenic and non-allergenic components that are difficult to standardize. recombinant allergens can improve diagnosis and de-sensitization against single component. major cat allergen is a heterodimer composed of disulfide linked 7.8 and 10.1 kda polypeptide chains. both chains of feldi protein were obtained in e.coli system. purification of recombinant proteins was performed in denaturing conditions using immobilized metal affinity chromatography specific for proteins with histidine tag. from 1000 ml culture approximately 13 mg protein for feldi chain 1 and 43 mg of feldi chain 2 were obtained. after purification histidine tag was removed by hydrolysis with thrombin. immunological activity of feldi against serum of patients allergic to cat was narrowed to subgroup of patients allergic to feldi protein by surface plasmon resonance. immunological activity of each chain and renatured heterodimer was also tested using immunoprecipitation techniques against serum of population group. subdoligranulum variabile -a novel member of the human gut micro flora with a high prevalence kim holmstrøm, trine møller bioneer a/s, hørsholm, dk-2970, denmark in 2003 we isolated and cultured for the first time a bacterium from a human fecal sample representing a hitherto unknown member of the clostridium leptum rrna supra generic cluster. the c. leptum rrna supra generic cluster represents one of the 3 major phylogenetic lineages within the human gut microbiota, and is characterized by having only a small proportion of its members actually identified by cultivation compared to the estimated numbers of bacteria contained in this group from culture-independent gut flora analyses. s. variabile is an obligate anaerobe gram negative bacterium with a characteristic pleiomorphic coccoid-droplet-like cellular morphology. its closest previously cultivated relative based on a 16s rdna phylogenetic analysis is faecalibacterium prausnitzii, a rod-shaped and therefore easily distinguishable gram negative bacterium present in high numbers in the human fecal micro flora. based on 16s rdna sequence we designed a s. variabile specific oligonucleotide probe for use in fish analysis to estimate the prevalence of this "new" bacterium in fecal samples collected from healthy human beings. interestingly, we observed a high proportion of s. variabile present in all tested samples, and in some instances we observed a higher prevalence than the more well-known group of bifidobacteria equally estimated by fish analysis. documentation of these results will be presented. several species of sea cucumbers, long an incumbent of traditional medicines were selected as the source of animal based antibiotic compounds. swabs of the inner surface and coelomic fluid (inner fluid) samples from sea cucumber (holothuria atra jaeger) were taken. thirty strains of bacteria were isolated. these strains were grown in different antibiotic production media. nine human pathogenic bacterial species were used as test agents and they are, k. pneumoniae, mrsa, m. luteus, s. thyphimurium, s. epidermitis, s. saprophyticus, b. subtillis, p. aeruginosa and s. pyogenes; only four bacterial strains showed mild antibiotic activity against s. pyogenes and s. thyphimurium. similar testing on two other species, h. scabra and s. variegatus will be carried out. different media, especially antibiotic production enrichment media will also be used. characterization will be done upon obtaining an antibiotic compound, which shows moderate to high activity against at least one of the nine human pathogens used. for plant based medicines, three rhizomes, "cekor," "jerangau" and "bonglai" were analyzed. solvent extraction using ethanol, methanol and acetone was carried out, at a concentration of 20-50 mg per ml solvent. the filtrates were used for the antibiotic testing stage. all the three plant species showed moderate antibiotic activity against m. luteus, s. epidermitis, s. pyogenes and s. saprophyticus. interestingly, the antibiotic activity increased when combinations of the herbal extracts were used. cell-based therapy continues to be a promising avenue for the treatment of duchenne muscular dystrophy, an x-linked skeletal muscle-wasting disease. recently, we have demonstrated that freshly isolated myogenic progenitors contained within the adult skeletal muscle side population (sp) can engraft into dystrophic fibers of non-irradiated mdx 5cv mice after intravenous transplantation. engraftment rates, however, have not been therapeutically significant, achieving at most 1% of skeletal muscle myofibers expressing protein from donor-derived nuclei. to improve the engraftment of transplanted myogenic progenitors, an intra-arterial delivery method was adapted from a previous procedure. cultured, lentivirus transduced skeletal muscle sp cells were transplanted into the femoral artery of non-injured mdx 5cv mice. based on the expression of microdystrophin and gfp transgenes in host muscle, sections of the recipient muscles exhibited 5% to 8% of skeletal muscle fibers expressing donor-derived transgenes. further, donor muscle sp cells, which did not express any myogenic markers prior to transplant, express the satellite cell transcription factor pax7 and the musclespecific intermediate filament desmin after extravasation into host muscle. the expression of these muscle-specific markers indicates that progenitors within the side population can differentiate along a myogenic lineage after intra-arterial transplantation and extravasation into host muscle. given that femoral artery catheterization is a common, safe clinical procedure and that the transplantation of cultured adult muscle progenitor cells has proven to be safe in mice, our data may represent a step towards the improvement of cell-based therapies for dmd and other myogenic disorders. metabolic engineering design of an extracellular hgh synthesis system birgül şentürk 1 , pınar ç alık 2 , güzide ç alık 1 , tunçer h.özdamar 1 : 1 bre lab, department of chemical engng, ankara university, 06100 ankara, turkey; 2 iblab, department of chemical engineering, metu, 06531 ankara, turkey. e-mail: ozdamar@eng.ankara.edu.tr (t.h.özdamar) metabolic engineering design of an extracellular human growth hormone (hgh) synthesis system is based on cloning the dna encoding human therapeutic protein together with the signal dna sequence of an extracellular enzyme gene into a host-vector system. in this context, extracellular protease (subc) signal dna sequence, i.e. pre-signal dna sequence, was fused into the frame in front of hgh mature dna sequence, by the use of four primers designed using pcr-based gene splicing by overlap extension method. b. licheniformis chromosomal dna and plasmid carrying hgh cdna were used as templates in pcrs, respectively, for the amplification of the subc signal dna sequence and hgh mature peptide sequence. for the fusion of two target genes, i.e. mature peptide sequence of hgh and, signal dna sequences were amplified separately by pcrs. the primers used at the ends to be joined were designed as complementary to one another by including nucleotides at their 5 ends that are complementary to 3 portion of the other primer. these products were mixed in the next pcr reaction, where one strand from each fragment contains the overlap sequence at 3 end. extension of this overlap by dna polymerase has yielded the recombinant hybrid-gene; and hybrid-gene serve as template for the continuation of reactions for the increase of the concentration in the microreactors. the hybrid gene fragment was first cloned into puc19, and then sub-cloned to pmk4 e.coli-bacillus shuttle plasmid. thus, a new expression vector with high stability and high copy-number was obtained and transferred into host b. subtilis 168 (spo − ). the metabolic flux distributions calculated by the mass balance based stoichiometric model based on the proposed metabolic reaction network for r-b.subtilis were determined by using time profiles of the substrate, dry-cell, hgh, amino acids and organic acids concentrations as the constraints. on the basis of the intracellular bioreaction rates and the interactions with the bioreactor operation parameters, an in-depth insight will be provided for further metabolic engineering design for the extracellular hgh production in r-b.subtilis. medicines based on polypeptides consisting of 30 and more amino acid residues are widely spread in pharmaceutical market at the present time. practically all polypeptide medicines known are prepared by general chemical synthesis that caused high cost of their production. that's why biotechnological way of polypeptide medicines preparation using recombinant gene expression in bacteria seems to be promising. during our work we design hybrid construction allowing solving a problem of specific cleavage of target polypeptide from the hybrid protein using system of protein splicing from new england biolabs. in case of thymosin ␣ 1 production impac system (intein mediated purification with affinity chitin-binding tag) has been used, where the modified sce vma from s. cerevisiae has been applied as intein. in contrast to other systems, impact allows the preparation of target protein without using of serine protease and other factors that may cleave the hybrid protein. in the presence of thiol reagents, such as dithiothreitol, mercaptoethanol, or cystein the hybrid protein can be site-specifically cleaved to give intein, the target protein and small fragment of nextein. using of this system does not allow to obtain the target protein with ser, cys or thr on n-terminal of protein, because in those cases target protein and n-extein ligation product will be formed. ser is n-terminal amino-acid in thymosin ␣ 1 . it was recently found that some metal ions essentially affect the splicing. we tried to use zncl 2 and we have found that, in case of intein-thymosin ␣ 1 , the maximal yield of target polypeptide and the minimal yield of splicing products are observed in the absence of dithithreitol and in the presence of 0.5-1 mm zinc chloride in buffers on all stages of thymosin ␣ 1 isolation. the structure of recombinant thymosin ␣ 1 of human was confirmed by the determination of n-and c-terminal amino-acid sequences and by maldi tof mass-spectrometry. mature embryos of five t. aestivum and five t. durum cultivars formed embryogenic callus on two different media. embryos were removed from surface sterilised seeds and placed with the scutellum upwards on a solid agar medium containing the inorganic components of murashige skoog and 2 mg/l 2,4-dichlorophenoxyacetic acid (2,4-d) or 1 mg/l naphtalenacetic acid (naa). the developed calli and regenerated plants were maintained on 2,4-d or naa free ms medium. wheat plants can be regenerated via two different systems. there were significant differences in percentage of callus induction and regeneration capacity on the different initiation medium. among the t. aestivum cultivars, yakar had the highest regeneration capacity in both induction medium. in t. durum cultivars, kiziltan gave the highest regeneration capacity in ms + 2,4 d medium and yilmaz gave the highest regeneration capacity in ms + naa medium. a strong genotypic effect on the culture responses was found for both induction medium. tumor necrosis factor beta (tnf␤) is a pleiotropic cytokine mediating its activity through the same receptors as the structurally related tnf␣. shortening of the n-terminal part has been reported to enhance its cytotoxic activity, with the explanation that removal of the flexible n-terminus reduces steric interferences in the receptor binding process. for studies of relationship between the n-terminal protein structure and physicochemical properties, three different forms mettnf␤, his7tnf␤ and n19tnf␤ were expressed in e. coli. high solubility of n19tnf␤ was expected, however, both analogs his7tnf␤ and n19tnf␤ were predominantly obtained in the form of inclusion bodies (ibs). on the other hand, mettnf␤ was equally distributed between the soluble and insoluble fraction. most probably, the composition of the n-terminal part, such as the exposure of hydrophobic residues in the case of n19tnf␤, or a mildly hydrophobic stretch of seven histidines appended to the natural hydrophobic n-terminus in the case of his7tnf␤, lead to incorrect folding and force aggregate formation. solubilization of ibs was performed under native and denaturing conditions using various concentrations of nls, urea and gndhcl. mettnf␤ ibs were easily dissolved with 0.2% nls, while his7tnf␤ and n19tnf␤ demanded denaturing conditions. structural differences in the nterminal part of various tnf␤ proteins were also reflected in protein refolding characteristics and chromatographic behavior. dilution, ultrafiltration and dialysis were used for refolding, and imac was chosen as the main chromatographic step. our results suggest that not only the length of the n-terminal part of tnf␤ but also the composition and exposure of certain amino acid residues affect its physicochemical properties. high level expression of recombinant growth hormones in e.coli faces common problems such as protein aggregation and inclusion body formation. discussions are raised whether it is more beneficial to obtain soluble protein but to loose expression rates. here we describe an experiment based on the hypothesis that slower expression should result in at least partially soluble recombinant protein. experiments were performed on bovine, chicken and mink growth hormones. expression rate was controlled externally by adjusting cultivation temperature, media, inducer amount, and both induction and cultivation times. another approach to the problem was performed through genetic manipulation. we changed strong t7 promoter to e. coli promoter consensus sequence thus reducing expression rate. recombinant growth hormone was still found to form aggregates, even when expressed at extremely low levelsseveral (2-8) percent of total intracellular protein. we developed optimization scheme of insoluble protein production and showed that expression rate minimization is not influencing recombinant growth hormone solubility in vivo thus suggesting an idea of sequence specific aggregation. to optimize the recombinant protein production in small-scale shake-flask system and high cell density fermentation, new tools have been developed at our laboratory which helps to get knowledge about the physiological state of the cell culture. these tools include (i) a quantitative monitoring system for cellular mrnas based on a sandwich hybridization technique, and (ii) a wireless online monitoring tool (senbit), applicable for standard sensors such as ph, po 2 and temperature for the continuous data collection from shake flasks. the senbit system is a new tool supplying valuable information for the optimisation of the expression of recombinant genes in shake flasks and allowing conclusions towards the reproducibility of shake flask cultures. the presentation will focus on use of the sensitive sandwich hybridization technology for the quantitative analysis of process relevant marker genes in different kind of microbial cell cultures with a focus on the production of recombinant proteins. samples from shake flask cultures and high cell density fed-batch fermentations of the yeast pichia patoris, have been analysed. additionally the mrna analysis was combined with the application of the senbit wireless system to study the production of a recombinant protein in shake-flask cultures of p. pastoris. aside from p. pastoris, mrna sandwich hybridization also was used to monitor product expression in fed-batch fermentation of e. coli for the production of a protein with two subunits by sequential induction. quantitative rna analysis as a tool for optimization of tetrameric collagen prolyl 4-hydroxylase production in e. coli a. neubauer 1 , m. bollok 2 , j. myllyharju 1 , p. collagen prolyl 4-hydroxylase (p4h) involved in the biosynthesis of collagens is an ␣ 2 ␤ 2 tetramer. recombinant expression of p4h in e. coli was described recently . the construct for cytoplasmic expression contains the genes of both subunits in one plasmid under control of different promoters. the ␣ subunit forms inactive aggregates, when expressed separately. in mammalian cells the ␤ subunit is available in large excess and keeps the ␣ subunit in a soluble active form. to mimic this in the bacterial system, we induced both genes sequentially. after induction of the ␤ subunit with iptg, expression of the ␣ subunit was initiated with anhydrotetracycline. here we use the analysis of the product mrnas with a bead based sandwich hybridisation assay (sha) (rautio et al., 2003) for optimization of the fermentation procedure. a high p4h activity was obtained if a high mrna level of the ␣ subunit could be maintained over a longer time. the obtained results illustrate the importance of the second induction for a high level expression of the p4h tetramer. the cells need to be in a "healthy state" with low metabolic load to react efficiently to the second induction. the data illustrate the optimization of a fermentation process by monitoring mrna levels which is of general interest for optimization of products which are difficult to detect. cell-based therapy continues to be a promising avenue for the treatment of duchenne muscular dystrophy, an x-linked skeletal muscle-wasting disease. recently, we have demonstrated that freshly isolated myogenic progenitors contained within the adult skeletal muscle side population (sp) can engraft into dystrophic fibers of non-irradiated mdx 5cv mice after intravenous transplantation. engraftment rates, however, have not been therapeutically significant, achieving at most 1% of skeletal muscle myofibers expressing protein from donor-derived nuclei. to improve the engraftment of transplanted myogenic progenitors, an intra-arterial delivery method was adapted from a previous procedure. cultured, lentivirus transduced skeletal muscle sp cells were transplanted into the femoral artery of non-injured mdx 5cv mice. based on the expression of microdystrophin and gfp transgenes in host muscle, sections of the recipient muscles exhibited 5-8% of skeletal muscle fibers expressing donor-derived transgenes. further, donor muscle sp cells, which did not express any myogenic markers prior to transplant, express the satellite cell transcription factor pax7 and the muscle-specific intermediate filament desmin after extravasation into host muscle. the expression of these muscle-specific markers indicates that progenitors within the side population can differentiate along a myogenic lineage after intra-arterial transplantation and extravasation into host muscle. given that femoral artery catheterization is a common, safe clinical procedure and that the transplantation of cultured adult muscle progenitor cells has proven to be safe in mice, our data may represent a step towards the improvement of cell-based therapies for dmd and other myogenic disorders. collagen and its derived product gelatin are attractive mammalian proteins to be used as model for the production of complex heterologous proteins in plants. the availability of a recombinant product will provide a safer, more homogeneous product than the current animal-derived material. the aim of the project is to investigate the feasibility of a production system for the accumulation of recombinant collagen for conversion to gelatin using barley. the 5 -end of the cocksfoot mottle virus (cfmv; genus sobemovirus) genomic rna sequence, called cfmv -element, has been shown to enhance recombinant protein synthesis in barley (wo 01/55298, mäkinen et al., 1995) . the -element will be used to study whether accumulation levels of complex mammalian proteins can be further increased, using collagen as a model that will serve as basis for exploring the expression of other complex proteins. this system can be study the production of barley-derived recombinant collagen for conversion to gelatin. classical swine fever virus (csfv) is an animal pestivirus which can be used as a surrogate model to elucidate the role of envelope glycoproteins of closely related human hepatitis c virus (hcv). the necessity to use the surrogate models for hcv is due to the fact that this virus cannot be grown in vitro cultures. csfv genome codes for three major antigenic glycoproteins which are located in the same cluster of genes; they are designated as e2 and e0 (e rns ) and e1. glycoproteins form heterodimeric and homodimeric complexes on the external part of viral particles. it is generally accepted that envelope glycoproteins play a major role in the initial stages of viral infection both for csfv and hcv. formation of complexes is needed to effectively infect host cells. we have investigated the formation of glycoprotein dimers by immunoperoxidase monolayer assay and by immunoblotting (western blotting). immunoblotting is a very useful technique in these studies because the complexes are formed via cysteine-cysteine disulphide bonds and they are retained during sds-page under non-reducing conditions. by modifying the glycoprotein genes and by arresting n-glycosylation of e2 and e0 we have investigated which factors influence the formation of complexes. it has been found that some glycosylation inhibitors which act at the early stages of glycan chain processing influence, not only glycosylation, but also the stability of e2 protein, effectively inhibiting the formation of glycoprotein complexes and the yield of the virus. these inhibitors are potential agents for arresting the multiplication and spread of csfv, and its relative -human hcv. recombinant proteins have been produced in a variety of heterologous protein expression systems. eukaryotic unicellular algae have distinct advantages, e.g. it can synthesize complex protein that requires post-translational modification. furthermore, microalgae can be grown in confined environment and thus prevents leakage of genes to the environment. our group has developed a platform technology for the production of recombinant proteins in red microalga porphyridium sp. we have constructed algal transformation plasmid vectors containing a camv 35s promoter and polya signal site. a streptoalloteichus hindustanus bleomycin-resistant gene was used as the selective marker. we have expressed ovalbumin and hepatitis-b surface antigen (hbsag) as model proteins. transformation was carried out by agitating algal cells and vector dna with glass bead. transgenic lines were selected by growing algal cells on agar plate containing 6 g/ml zeocin. positive transgenic lines were selected by screening the colonies by pcr and confirmed by dna sequencing. expression of ovalbumin and hbsag protein was examined by western blot analysis. ovalbumin was found to be expressed inside the algal cells while small hbsag was secreted into the medium due to presence of signal peptide. these findings indicate that red microalgae are capable of producing heterologous proteins. life-material exhibition as ethical interpretation chang shih-lung biotechnology industry study center, taiwan institute of economic research, taipei 106, taiwan. email: schang@tier.org.tw in this thesis we aim to explore the medical-related life-material exhibitions within ntu hospital-the humanity building, and taipei mackay hospital-the historical showroom as abecedarian clues to understand the burgeoning phenomenon-medical museums in taiwan. following these clues, we treat the whole context of medical museums, which transform values through situational construction, as the background to interpret the ethical implications of group val-ues that are transformed in the medical profession. in this thesis, we see medical museums, as the social prescription transforming medical profession, format the ritual context of situation ethics with the cultural construction of life-material. multi-disciplinary interactions and visiting itinerary can be transferred to the exploring horizon of research approach through description and interpretation. among them, we observe that humanistic elements have become essential equipment (or mat'eriel) of medical profession. though humanistic equipment (or mat'eriel) has its bottleneck in the museum situation, it can also unblock new possibilities for museum exhibitions, ethical practice or life ethics. as part of a wide research program aimed at developing new antitumoral agents, we present herein a series of stereoisomeric derivatives of fused tetrahydrofuranes (fthf) substituted with diverse protecting groups either at the primary or secondary hydroxyl groups. unprotected derivatives were also synthesised to investigate the influence of substituents on the in vitro activity of fthf. data on the synthesis, chemosensitivity and apoptosis induction by this series of fthf will be correlated to substitution pattern, stereochemistry and protecting groups, as an aid to the rational design of novel antitumour drugs. establishment of in vitro test systems for pulmonary edema resorption by peptide drug candidates dominik geiger, aswin mangerich, rudolf lucas, klaus p. schäfer, inge mühldorfer 1 department of biotechnology, altana pharma ag, konstanz, germany; 2 imc university of applied sciences, krems, austria tnf-␣ was found to up-regulate the rate of lung liquid clearance (llc) in several animal models by the activity of its lectin-like tip domain. this activity can be mimicked by a circular peptide designated tip. tip was shown to induce llc and consequently pulmonary edema resorption in different animal models. in order to study the mechanism of action of tip, we established two in vitro test systems for edema resorption with the human lung epithelial cell line calu-3: (1) the ability of calu-3 cells for spontaneous dome formation within confluent monolayers was utilized for quantitative examination of tip's activity on active transepithelial fluid transport ("dome assay"). (2) the effects of tip on bioelectrical properties of polarized cell monolayers were studied by using the transepithelial electrical resistance (teer) technology ("teer assay"). dome assay experiments confirmed that dome formation is a sodium dependent process and that tip is able to increase this process. teer assay experiments proofed that tip acts in a polarized and dose dependent manner. in conclusion, there is strong evidence that the dome and the teer assays are suitable systems for in vitro activity testing of tip and other anti-edema peptide drug candidates and are useful for studies on their mechanism of action. increasing safety concerns in gene therapy result in more stringent regulatory requirements. those cover the complete process chain of cell banking, fermentation, and purification. a wide range of applications for pdna requires gram to kilogram amounts for clinical trials and market supply. economic, productive and robust processes are a prerequisite for low cost of goods (cogs). therefore manufacturer of biopharmaceuticals need to address these considerations by developing new production processes meeting the new standards. boehringer ingelheim austria developed a novel pdna production process suitable for large-scale cgmp production. the process is based on e. coli fermentation. the process contains no components from animal origin. the optimized fermentation process yields up to 1 g pdna/l fermentation volume. for isolation of pdna from e. coli alkaline lysis in glass bottles or stirred tanks is commonly used. cell wall structure is destroyed by a combination of alkaline ph and detergents. in our process alkaline lysis is operated in a closed, continuous system directly connected to clarification without using enzymes. we developed a scalable process for pdna purification based on 3 different chromatographic principles. the capture step is carried out by hydrophobic interaction chromatography followed by anion exchange chromatography as intermediate step. final polishing is carried out by conventional size exclusion chromatography in a group separation mode providing also buffer exchange and desalting for the final formulation. the process results in a pdna drug substance of highest quality containing a low level of impurities (genomic dna, rna, proteins endotoxines) suitable for therapeutic applications. depending on the conditions during fermentation and the used host homogeneities of greater than 95% or even 98% are possible, while a high over all yield can be achieved (∼50%). during the development monolithic chromatography supports (cim ® ) were compared with conventional resins and evaluated as potential alternatives. the complete process is monitored by a set of analysis covering cell banking to final purification. new sensitive methods based on hpce, hpiex and fluorometric measurement were developed. whereas many natural amino acids are currently produced by very cost efficient biological processes, the manufacture of methionine is still performed by traditional chemical synthesis. this was mainly due to the poor performances of the currently available producing strains that inhibited the development and commercialization of a biological process to l-methionine. metabolic explorer has recently reinvestigated and developed an efficient biological process that employs an engineered microorganism and utilizes a renewable starting material (corn sugar) as its feedstock and converts glucose into l-methionine. we will describe (a) the general scheme for the engineering of the host organisms and (b) the general approach to maximize carbon and reducing equivalent throughput to l-methionine. to highlight this effort, we will present the global approach developed to improve the process combining metabolic flux analysis, traditional protein biochemistry, molecular biology and fermentation optimisation. saccharomyces cerevisiae is an established 'work horse' of the fermentation industry and modern biotechnology has led to a spectacular expansion of the range of products that can be produced by this yeast. however, for the large-scale sustainable production of chemicals, it is equally important that the range of carbohydrate feedstocks be expanded. especially relevant in this respect is the ability to consume the pentose sugars d-xylose and l-arabinose, which make up a substantial part of plant carbohydrates. wild-type s. cerevisiae strains cannot metabolise d-xylose, but are capable of slowly metabolising its keto-isomer, d-xylulose. therefore, efficient conversion of d-xylose into d-xylulose has long been a key issue in yeast metabolic engineering. non-saccharomyces yeasts that is capable of growing on d-xylose use two enzymes, xylose reductase and xylitol dehydrogenase for this purpose. while both enzymes have been successfully expressed in s. cerevisiae, this is not always compatible with efficient product formation. for example, in the case of ethanol production, the different cofactor specificities of these two oxidoreductases cause massive byproduct formation. theoretically, introduction of a xylose isomerase, which catalyses the interconversion of xylose and xylulose, might circumvent these problems. however, it is notoriously difficult to express bacterial and archaeal xylose isomerases in s. cerevisiae and, until recently, activities of heterologous xylose isomerases expressed in s. cerevisiae were vanishingly low, at least under physiological conditions. a breakthrough was reached when, in 2003, a xylose isomerase gene from the anaerobic fungus piromyces was expressed in s. cerevisiae. while this led to high activities of xylose isomerase, these were not enough to enable fast growth or product (ethanol) formation. in this presentation, we will discuss how a combination of metabolic and evolutionary engineering led to fast and efficient xylose utilization by engineered saccharomyces cerevisiae strains under aerobic as well as anaerobic conditions. furthermore, we will illustrate how evolutionary approaches can be applied to facilitate the utilization of mixed substrates. hyaluronic acid (ha) is a natural and linear polymer composed of ␤-1,3-n-acetyl glucosamine and ␤-1,4-glucuronic acid repeating disaccharide units with a molecular weight (mw) up to 6 mda. it is a major constituent of the extracellular matrices and the synovial fluid. in the last decades, various fields of application including cosmetics, ophthalmology, rheumatology, tissue engineering and drug delivery have been explored, owing to the many important biological functions of ha and its unique physico-chemical properties. however, for some specific applications, the relatively high mw of ha is a limiting factor and the availability of low mw fractions would be highly desired. for food applications, low mw ha has been shown to penetrate the gastrointestinal barrier, thereby increasing the ha bioavailability. moreover, low mw ha fractions are able to re-establish the ha content in the skin and can thus be used in cosmetics as anti-aging and anti-wrinkle agents. finally, low mw ha has shown to prevent oxygen free radical damage in granu-lation tissue during wound healing. a range of methods has been described for the depolymerization of ha to low mw fractions. these techniques involve heat treatment, ultrasonication, uv/gamma irradiation, chemical and enzymatic degradation. we present results on a degradation process of ha originating from bacillus subtilis fermentation into well-defined low mw fractions. the process developed in lab scale is safe, well-controlled and produce low mw ha fractions with narrow polydispersity. moreover, the process is readily up-scalable. the low mw ha fractions are being evaluated in various cosmetic applications. metabolic pathway manipulation for improving the properties and productivity of microorganisms is becoming an established concept. metabolic engineering can be defined as the directed improvement of product formation or cellular properties through the modification of specific biochemical reactions or introduction of new ones with the use of recombinant dna technology. a detailed analysis of the physiological means of the different pathways is needed to be able to introduce modifications aimed to the production of not only important metabolites, but also to understand the fundamentals of cell biology. aimed to produce single compounds, metabolic engineering necessarily includes the modification of the cellular pathway(s) as well as the redirection of the energy toward the production itself. the existing metabolic engineering applications are the culmination of more than two decades of global experience developing processes for the production of fine chemicals, vitamins, nutraceuticals and animal nutritional aids such as amino acids. based on the relative low complexity, the first biotechnological applications have been developed from microorganisms. our laboratory has been engaged in this field since different years. yeasts like saccharomyces cerevisiae, kluyveromyces lactis and zygosaccharomyces bailii have been developed for the production of fine chemicals like lactic and ascorbic acids from d-glucose. in this contribution, we will present the last data obtained. since 40 years amino acid production is in the focus of industrial microbiology. l-glutamate and l-lysine are produced with corynebacterium glutamicum, while escherichia coli is used for l-threonine production. the worldwide market of threonine drastically increases: in 1996 the amount of threonine produced worldwide was 15,000 t and raised to 30,000 t in 2000 and 45,000 t in 2004. concerning predictions of experts the demand of threonine will rise with a two-digit rate of economic growth within the next few years. meanwhile the prices declined. these conditions enforce very efficient production processes. beside the strain development and an optimised downstream procedure the fermentation process is an important target for productivity improvement. strain development is dependent on detailed knowledge of the production strains. with innovative methods we are able to get a close look inside the cells under different culture conditions. these methods have been called 'omics' in recent literature. knowledge about genome, transcriptome, proteome, phosphoproteome, metabolome and fluxome leads to new ideas for strain improvements. data generated by these methods must be based on clearly defined culture conditions. therefore, highly parallel fermentations have to be performed to generate biological parallel samples. cutting-edge technical equipment is the basic requirement for experiments like this. other requirements are optimised sampling for different analysis, technical parallels of analytical steps and a detailed statistical analysis of data. these procedures guarantee distinction between real data and data noise. integration of all these data to a holistic model of the cell is the challenge for the future. by combination of a new strain, process and downstream improvements the plant productivity was increased drastically. we ended up in an optimized, fast and high yield process to scope challenges worldwide market comes up with. rieping, m., hermann, t. (2002); fermentation process for the preparation of l-threonine; wo/0218543. ) are potentially quite useful biocatalysts, as they allow for the regioselective and stereoselective hydroxylation of activated as well as non-activated carbon atoms. in addition, the large number of members of the p450 superfamily exhibits a wide diversity of specificities from which a useful biocatalyst may be selected. from a technical point of view, however, they have significant drawbacks. thus, they usually cannot be produced in large quantities nor recovered or stored without a severe loss in activity. their catalytic activity is mostly quite low, and their operational stability leaves much to be desired. most p450 enzymes require a complex protein/phospholipid machinery for activity, and the final electron donor in the reaction cascade, usually nadph, does require extensive recycling to arrive at a commercially satisfactory process. recently, the use of bacterial cytochromes as hydroxylation biocatalysts has received considerable attention. some of them are natural fusion proteins, which contain the heme and the reductase domains on a single polypeptide chain. they are catalytically much more active compared to, e.g. cytochromes occurring in human tissue. we thus have set out to further improve the technical applicability of these enzymes, and have centered our activities around several bacterial cytochromes. it proved very useful to apply rational mutagenesis and directed evolution to these enzymes, leading to a surprising compatibility of mutant enzymes with a wide variety of substrates. mechanisms for the limited stability of the enzymes were explored, leading to hybrid enzymes with enhanced stability, and the cofactor problem was alleviated using auxiliary enzymes or mediator-based technologies. as a result, a bioreactor based on microbial cytochromes was built and operated for several days. baeyer-villiger monooxygenases represent useful biocatalytic tools as they can catalyze reactions, which are difficult to achieve using chemical means. however, so far only a limited number of these monooxygenases were available in recombinant form kamerbeek et al. (2003) . using a recently described protein sequence motif fraaije et al. (2002) and the available genome sequence information, we were able to identify and overexpress a number of novel bacterial bvmos. one of the overexpressed bvmos was found to be relatively stable as it originates from thermobifida fusca, which grows at ∼60 • c. the enzyme was shown to be active on a broad range of substrates, preferring aromatic ketones fraaije et al. (2005) . the best substrate discovered so far is phenylacetone, hence its name: phenylacetone monooxygenase. we have solved the crystal structure of phenylacetone monooxygenase, which represents the first structure of a bvmo malito et al. (2004) . the crystal structure provides insight into the complex mechanism of catalysis mediated by fadcontaining bvmos. by site-directed mutagenesis we have probed the role of several active-site residues. a crucial role is played by an arginine residue. as phenylacetone monooxygenase shares significant sequence identity (>40%) with all known nadph-dependent bvmos, many of the observed structural features seem to be conserved within this class of atypical monooxygenases. by homology modeling using the phenylacetone monooxygenase structure, catalytic properties of other baeyer-villiger monooxygenases can be explained or predicted. screening for fungal baeyer-villiger monooxygenases l. butinar 1 , j. friedrich 1 , v. alphand 2 : 1 laboratory of biotechnology, national institute of chemistry, ljubljana si-1001, slovenia; 2 groupe biocatalyse et chimie fine cnrs fre2712, université de la méditerranée, marseille, france the asymmetric form of the baeyer-villiger (bv) oxidation (transformation of ketones into lactones) is an important challenge for organic chemistry since the obtained lactones are valuable building blocks for synthesis of countless biologically active products. to date, enzymatic or microbial bv oxidations appears as more successful than their chemical counter-parts. (ten brink et al.) whereas most active bv monooxygenases are produced by bacteria (among which the well-studied enzyme of acinetobacter calcoaceticus), only a few fungal strains expressing bvmo were described (alphand et al., carnell and willetts) . in order to increase the number of available biocatalysts which perform such an asymmetric biotransformations, a screening of fungi belonging to major groups of zygo-, ascoand basidiomycetes was conducted using bicycloheptenone as testsubstrate. surprisingly, a large number of the tested fungi were able to transform the substrate into one to four different lactone isomers. the yields, the enantio-and regio-selectivity of the reaction depended on the fungal strain. alphand, v., furstoss, r., 2000. j. mol. catal. b 9, 209-17. carnell, a., willetts, a.,1992 . biotechnol. lett. 14, 17-21. ten brink, g.j., et al., 2004 . pyranose oxidase (p2ox) is a periplasmic enzyme that widely occurs in basidiomycetes. it catalyses the c-2 oxidation of several aldopyranoses to the respective 2-keto derivatives, transferring electrons to molecular oxygen to yield h 2 o 2 . p2ox is of interest for carbohydrate conversions, as its reaction products (2-keto sugars) can be attractive intermediates in the production of food ingredients. we cloned the gene encoding p2ox, and subsequently amplified a cdna clone by rt-pcr. the cdna was inserted into a bacterial expression vector and successfully expressed in e. coli. properties of the heterologous protein were compared to those of the native enzyme showing that they are essentially identical. both the native as well as the recombinant enzyme were used in biotransformations of sugars. recently, the 3d-structure of this tetrameric enzyme was elucidated. based on structural information, several enzyme variants containing point mutations were constructed and further characterised. two of these variants (e542k and e542r) displayed improvements in stability and certain kinetic properties thus making them attractive for biocatalytic applications. lactones are important compounds for the fragrance and flavour industry. right now the production of lactones is dependant on the import of crude materials from tropical countries. in this project, we want to tackle the manufacture of lactones via a biocatalytic route using p450 monooxygenases. cytochrome p450 monooxygenases catalyse the oxyfunctionalization of non-activated c-atoms. cyp102a1 from bacillus megaterium, cyp102a2 and cyp102a3 from bacillus subtilis are soluble fusion proteins comprising p450 monooxygenase and fad/fmn reductase domains in one polypeptide chain. all three enzymes are highly homologous fatty acid hydroxylases. especially, cyp102a1 also known as p450 bm-3 is well characterized and shows high activity compared to other p450 monooxygenases. the aim of the work is to change selectivity but conserve the high activity that is typical for those enzymes. using methods of structure modelling, rational protein design and directed evolution new mutants of these enzymes with changed regioselectivity are obtained. products of conversion with monooxygenases are intermediates in the production of lactones. the interface of biology and materials science has led to new materials with unique structural and functional properties, and new process technologies with the ability to produce, from "bottoms up", a wide range of biomimetic structures. these materials and their designs have broad application as catalysts, sensors, and devices for use in synthesis, cell and tissue engineering, bioanalysis and screening, and nanoelectronics. we have focused on the generation of sugar-based nanostructures, complete with tailored selectivities and biocatalytic activities at the molecular and nanoscales. these include biocatalytically-generated carbohydrate derivatives that selfassemble with high precision to give novel architectures with functional and responsive properties. izumoring: a strategy for total production of rare sugars ken izumori rare sugar research center, kagawa university, kagawa 761-0795, japan. e-mail: izumori@ag.kagawa-u.ac.jp we found a new enzyme, d-tagatose 3-epimerase (dte), that epimerize all ketohexoses at c-3 position. this epimerase catalyze not only between d-tagatose and d-sorbose, but also d-fructose = dpsicose, l-sorbose = l-tagatose, and l-psicose = l-fructose. this new enzyme offered us a useful key tool to connect all ketohexoses using hexitols as intermediates. the figure shows that all eight ketohexoses can be connected with dte and polyol dehydrogenases (pdh) in a ring. using this ring, we can easily find the pathway to transfer d-fructose to d-tagatose via d-psicose using dte and pdh. various aldose isomerases transform ketohexoses to the corresponding aldohexoses. so, we can connect all 16 aldohexoses with 8 ketohexoses using the enzyme. finally, all hexoses, 8 ketohexoses, 10 hexitols and 16 aldohexoses are connected using enzyme reactions in a ring structure (not shown). this kind of strategy is effective also on transformation of tetroses and pentoses. now, we can produce all monosaccharides; tetroses, pentoses and hexoses by enzyme reac-tions. the bioproduction strategy of all rare sugars (monosaccharides that are rare in nature) is illustrated using ring form structures named as izumoring. we have already succeeded to produce d-psicose in large scale and are now in the progress of mass production of various rare sugars from natural and cheap sugars using izumoring. bioprocess development for chiral intermediates christian wandrey institute of biotechnology, forschungszentrum jülich gmbh, jülich d-52425, germany chiral alcohols, diols, amino alcohols and chiral acids (e.g. hydroxy acids and amino acids) play an important role in pharma and agro synthesis. in the past such chiral intermediates were obtained by racemic resolution via chiral reductions using prochiral precursors. here the problem of cofactor regeneration arises. this problem could be solved by enzyme-coupled or substrate-coupled cofactor regeneration using formate or isopropanol as reducing agent. alternatively, whole cell bioreductions were developed where glucose is used as the reducing agent. in recent years "designer microorganisms" were developed in which oxidoreductases (e.g. alcohol dehydrogenases) were over-expressed in escherichia coli. in such cases, cofactor regeneration was achieved intracellularly with isopropanol as the reducing agent or by coexpression of a formate dehydrogenase, so that once again format could be used for reduction. another route to obtain chiral intermediates is a fermentative approach using classical pathways (like the aromatic amino acid pathway). here, the pathway is interrupted after the intracellular production of chorismate. new chiral intermediates can be obtained by over expression of additional genes, which catalyzed the production of chorismate derivatives leading to cyclohexadiene-transdiols and the corresponding amino cyclitols. the last example can be regarded as an example of industrial biotechnology where glucose is used as starting material (white biotechnology). here bioprocess development is carried out in an integrated approach, in which molecular biochemical engi-neering cares for the optimal intracellular metabolic network and the classical biochemical engineering cares for the optimal environment of the cell in a fermenter. examples will be given which reach (in cooperation with industrial partners) up to kilogram scale. biotransformations are usually involved in just one or very few separate reactions in organic syntheses. the development of a cell-free "system of biotransformations" (sbt), in which a set of enzymes acts in a coordinated fashion in a one-pot synthesis, lead to increased catalytic complexity, selectivity and yield, as well as facilitated operation at reduced costs. the example chosen to prove the usefulness of the sbt-approach is the production of dihydroxyacetone phosphate (dhap). dhap, a c3-compound from glycolysis, is an important precursor for asymmetric c-c-bond formation. so far, the production of dhap is difficult and expensive. for the construction of the dhap-producing sbt, e. coli's glycolysis is isolated from the metabolism to an as large as possible extent by the construction of a multi-ko-mutant. a culture is grown in an appropriate medium, homogenized in the production buffer, and used as the catalytic system. high production yields can be achieved since the production pathway is almost completely isolated from the metabolic network. the employed dhap-producing sbt provides not only a path from glucose to the product, but also an integrated atp-regeneration and nad-recycling system. in first experiments with a tpi-ko-mutant, a dhap-production yield on glucose of 32% could be achieved, without optimizing the system. the system remained active for more than 24 h. up to now, atp cannot be applied in catalytic concentrations, but has to be present in equimolar amounts to glucose. the production yield could be increased by 10% through the addition of phosphate ions as substrate to the reaction, enabling the system to utilize atp more efficiently. these experiments indicate that the sbt-approach is viable and a large potential remains to improve the dhap-producing sbt. for some years novozymes have manufactured a pectate lyase for scouring of textile as an ecological alternative to the traditional harsh chemical treatment, and recently, we at novozymes discovered additional applications for pectate lyases. however, to be commercially attractive more robust pectate lyses had to be developed. in this paper, we will demonstrate how we for two different pectate lyases have improved their stability significantly. as the two enzymes are quite similar in sequence and structure, it was a new discovery for us to find that different concepts of protein engineering had to be used in our attempt to stabilize each individual pectate lyase. the stability of one enzyme was improved by substitutions in the internal of the structure whereas the stability of the other pectate lyase was primarily improved by changing surface residues. starting with knowledge from structural analysis, we have applied rational based protein engineering resulting in few selected variants. also random based protein engineering combined with screening of hundreds of thousands variants was used. in conclusion: the project team showed that by synergistic use of the two approaches, we were able to move faster towards a solution and eventually we succeeded finding new stabilized pectate lyase variants, applicable for new business areas. the importance energy independence as a national goal equals or exceeds that of the moon landing in 1990. the development of a new industry to produce fuel ethanol from woody biomass would increase national security, improve employment and the environment, and provide substantial relief from the debt of imported petroleum. costs associated with the rapid development of this new industry (∼$1.4 billion per year) could be paid by re-assigning 1 cent per gallon from existing federal gasoline taxes, a small price to pay for future energy independence. the corn-to-ethanol industry continues to make a remarkable contribution to our liquid fuel needs through expansion. today, one row of every six rows of corn is converted into fuel ethanol in the u.s. however, this expansion will be limited to 3-4% of total automotive fuel by the economics of corn costs and production. corn can do more! corn stover is the single most abundant agricultural residue in the us and can be used as a feedstock to produce 60-80 gallons of ethanol per dry ton. further expansion with other biomass feedstocks such as agricultural and municipal residues (lignocellulose, woody biomass) could produce over 100 billion gallons of fuel ethanol annually according at a recent joint report by the usda and doe (april, 2005) . current technology has been demonstrated at pilot scale for the production of fermentable sugars from hemicellulose by dilute acid hydrolysis and for the hydrolysis cellulose using fungal cellulose enzymes. biocatalysts such as recombinant escherichia coli have been developed and demonstrated for the efficient conversion of all sugar constituents of biomass to ethanol. a national goal for the full-scale deployment of current technology to produce biomass-based fuel ethanol will allow the us to reduce imported petroleum by 50%. together with increased efficiencies of hybrid vehicles, energy independence could be achieved within 10-20 years. similar gains could be realized by many nations around the world to provide new manufacturing and employment, redistributing wealth and ensuring a cleaner, healthier environment. bioethanol production using thermophilic bacteria marie just mikkelsen, birgitte k. ahring emab, biocentrum-dtu, 2800 lyngby, denmark the industry of bioethanol production is facing the challenge of redirecting the process from fermentation of relatively easily convertible but expensive starchy materials, to complex but inexpensive lignocellulosic biomass. on lignocellulosic hydrolysates, gram-positive thermophilic bacteria have unique advantages over the conventional ethanol production strains. the primary advantages are their natural broad substrate specificities, and in some strains, a high tolerance to lignocellulosic hydrolysates. moreover, ethanol fermentation at high temperatures also has the advantages of high productivities and substrate conversions, low risk of contamination and facilitated product recovery. some thermophilic bacteria naturally produce primarily ethanol from most sugar monomers present in lignocellulosics, but modifications are still necessary to increase ethanol yields. the release of useable sugars from lignocellulose biomass for industrial fuel-ethanol fermentation is often facilitated by a weak acid hydrolysis step. as a consequence, inhibitors such as furfural and 5-hydroxymethylfurfural (hmf) are formed as degradation products of xylose and glucose, respectively. moreover, the fermentative end-product of ethanol is also inhibitory. these, and other inhibitors present an environment, which elicits the expression of stress-related genes in saccharomyces cerevisiae. recently, 65 s. cerevisiae genes have been identified as important in furfural stress tolerance. when furfural is present, yeast with these genes disrupted grows poorly compared to wild-type yeast. a sub-class of these genes suggests that yeast grown under furfural-induced stress may rely upon similar pathways as cells grown under various other stresses, including oxidative, heat, and sorbate. to investigate this link further, we analyzed stress-induced phenotypes such as ros activity, dna damage, and membrane damage in wild-type and mutant yeast exposed to furfural or hmf stress. moreover, we investigated whether overexpression of this sub-class of genes would provide protection from furfural-induced stress and oxidative damage. micro-organism to be used in fermentation of lignocellulose hydrolyzates should preferably have three characters: (a) high ethanol tolerance, (b) resistance to inhibitors found in the hydrolyzate, and (c) a broad substrate utilization range, since the hydrolyzate contains several sugars. in addition to the possibility of controlling the level of potential inhibitors, fed-batch fermentations also permit the parallel uptake of several different monomeric sugars. two strains of saccharomyces cerevisiae, cbs 8066 (a commonly used laboratory strain) and tmb 3000 (a strain isolated from a spent sulfite liquor fermentation plant), were characterized in batch and fed-batch fermentation of a dilute-acid hydrolyzate from spruce. the strains had different abilities to ferment spruce hydrolyzate. the study suggests that the furan reduction capacity of a yeast strain is a key factor for its performance in fermentation of lignocellulosic hydrolyzate. polyketides constitute a structurally highly diverse group of natural products that possess broad ranges of pharmacological properties and represent a major source for novel cancer therapeutics. however, these compounds may be sub-optimal in regard of activity, selectivity, availability and unwanted side effects. in addition, the sustainable production of these valuable metabolites can be a challenge. studying the molecular basis of the biosynthetic pathways may set the basis for improving the production and for rationally engineering derivatives with altered bioactivity profiles, e.g. through targeted knockouts, mutasynthesis ziehl et al. (2005) , and swapping of pathway genes. our results in elucidating and manipulating the biosynthesis of selected antitumoral polyketide metabolites from bacteria (aureothin, chartreusin) and fungi (cytochalasines, rhizoxin) are presented. analyses at the genetic and biochemical levels provided new insights into several unusual biosynthetic features, e.g. non-linear polyketide assembly for the nitroaryl-substituted polyketide aureothin he and hertweck (2003, 2005) , an oxidative rearrangement cascade in the chartreusin pathway xu et al. (2005) , and a fungal iterative pks-nrps hybrid synthase schuemann and hertweck (2005) involved in cytochalasin biosynthesis. the most surprising result was obtained from elaborating the biogenesis of the antimitotic agent rhizoxin from rhizopus sp., which allowed for a significant improvement in large-scale production partida-martinez and hertweck (2005). he, j., hertweck, c., chem. biol. 2003 , 10, 1225 -1232 chem. bio. chem. 2005, 6, glycopeptides such as vancomycin and teicoplanin are the drugs of last resort for the treatment of severe infections caused by antibiotic resistant gram-positive bacteria. glycopeptides inhibit the peptidoglycan biosynthesis by binding as dimers to the d-ala-d-ala termini of the cell wall precursors. amycolatopsis balhimycina synthesizes the vancomycin-type glycopeptide balhimycin, whose structure and biological properties greatly resemble vancomycin and which only differs by its glycosylation pattern. using a "reverse genetics" approach we have identified the 66-kb gene cluster encoding the biosynthesis of balhimycin. by a combination of genetics, biochemistry and analytical organic chemistry, we were able to elucidate the biosynthetic pathway and to assign functions to almost all genes of the cluster. the biosynthesis starts with the pathway-specific provision of the non-proteinogenic amino acids ␤-hydroxytyrosine (␤-ht), hydroxyphenylglycine (hpg) and dihydroxyphenylglycine (dpg) which form together with (n-methyl)-leucine and asparagine the heptapeptide backbone of balhimycin. dpg is synthesized via a polyketide synthase mechanism (pksiii) similar to that known from plant chalcon/stilben synthases (pfeifer et al., 2001) . for the ␤-ht synthesis three genes are essential which form an operon (puk et al., 2004) : bpsd, an nrps binds a tyrosine molecule, which is then hydroxylated by the p450 monooxygenase oxyd. the perhydrolase bhp is required for the release of ␤-ht. subsequently bhaa, a nadh/fad-dependent halogenase catalyzes the chlorination of ␤-ht to form chloro-␤-hydroxytyrosine (puk et al., 2002) , which is needed to stabilize the dimerization. the amino acids are linked by non-ribosomal peptide synthetases (recktenwald et al., 2002) , and the aromatic side chains are interconnected by p450 monooxygenases; a series of reactions which lead to the first antibiotically active intermediate. inactivation of the oxygenase genes revealed the order of the cyclization steps (bischoff et al., 2001) : the oxygenases act in a stepwise fashion in the sequence oxyb, oxya and oxyc. the resulting cross-linked heptapeptide is then finally modified by methylation and glycosylation. the biosynthesis is regulated by the strr-type regulator bbr, which was shown to bind in front of different operons of the balhimycin gene cluster. this ensures the coordinated expression of the biosynthetic genes. the non-producing mutants, defective in the supply of the non-proteinogenic amino acids, were used as recipients in cloning experiments as well as in approaches of precursor-directed biosynthesis by feeding chemically synthesized alternative precursors. thus, novel balhimycin derivatives were generated (weist et al., 2002 (weist et al., , 2004 . bischoff et al., 2001 . angew. chem. int. ed. 40, 4688-4691. pfeifer et al., 2001 . j. biol. chem. 276, 38370-38377. puk et al., 2004 . j. bacteriol. 186, 6093-6100. puk et al., 2002 . chem. biol. 9, 225-235. recktenwald et al., 2002 . microbiology 148, 1105 -1118 . weist et al., 2002 . angew. chem. int. ed. 41, 3383-3385. weist et al., 2004 spectroscopy guided discovery of novel bioactive microbial natural products thomas ostenfeld larsen, michael edberg hansen cmb biocentrum-dtu, technical university of denmark, 2800 lyngby, denmark the task of finding novel bioactive natural products is usually bioassay driven. often a certain type of compound (e.g. polyketide, alkaloid) turns out to be active in an assay. when having generated a promising hit in a bioassay the normal procedure in the drug discovery process usually is to produce a large number of structurally analogous compounds either by traditional chemical synthesis or by combinatorial chemistry in order to study structure activity relation-ships and to find even more active lead compounds. alternatively to chemical synthesis of analogues nature can be explored for structurally similar compounds by uv-spectroscopy guided screening. this work will present a new method for the systematic and automated computer assisted search of full uv spectra in large number of datafiles for both dereplication of known and discovery of new natural products based on the use of the new mathematical algorithm x-hitting. exploring the substrate spectrum of the antibiotic producing bacteria saccharopolyspora erythraea p. krabben, p. oliveira, f. baganz, j. ward department of biochemical engineering, university college london, london wc1e 7je, uk knowledge of substrate utilisation capabilities play an important role in the development of genome scale metabolic models (borodina et al., 2005) and refinement of first generation annotations. furthermore, knowledge of the product formation during catabolism of different substrates provides valuable information about the distribution of metabolic fluxes and thereby forms a basis for rational strain improvement. we present here, data on the substrate utilisation capabilities and the corresponding product formation of s. erythraea. this analysis will help in improving the production of erythromycin and provide clues to the activation of the cryptic secondary metabolic pathways present in the s. erythraea genome. reference borodina, i., et al., (2005) . genome-scale analysis of streptomyces coelicolor a3 (2) modeling of growth and product formation on complex media containing multiple substitutable substrates is a challenge. complex media offers the organism multiple choices of carbon and nitrogen substrates including free amino acids, peptides, soluble and insoluble proteins in addition to the defined sources such as glucose and ammonium sulfate. we present a structured model that accounts for growth and product formation kinetics of rifamycin b fermentation in a multi-substrate complex medium. the model considers the organism to be an optimal strategist with a mechanism to regulate the uptake of the substrate combinations. further, we assume that the uptake of a substrate depends on the level of a key enzyme, which may be inducible. the model also considers control parameters as fraction of flux through a given metabolic branch. the control parameters are obtained using a simple multi-variable constrained optimization. the model parameters were rigorously estimated via a specifically designed experimental plan. the model correctly predicts the experimentally observed growth and product formation kinetics and the regulated simultaneous uptake of the substitutable substrates under different fermentation conditions. the model and the model parameters provide useful insights into the growth and product formation strategy of this industrially important process. this presentation will describe the experimental results, the model development and the relevant model parameters for a. mediterranei s699. the recent surge in oil price and the increasing concern on our environment have generated much interest in the production of chemicals from renewable resources. succinic acid, also called as amber acid, is a four-carbon dicarboxylic acid, which can be used as a precursor of numerous products including biodegradable polymers, green solvents, pharmaceuticals, and bulk and fine chemicals. a new capnophilic bacterium named mannheimia succiniciproducens mbel55e was isolated from the rumen of korean cow. this bacterium can produce large amounts of succinic acid along with some other metabolites such as lactic, formic and acetic acids. we have completely sequenced the genome of m. succiniciproducens and charaterized its genome content in the context of metabolic pathways. we then constructed the genome scale in silico metabolic network for metabolic flux analyses, and carried out metabolic flux analysis under varying environmental conditions. based on the in silico analyses results, we selected several target genes to be manipulated for enhanced succinic acid production. detailed results of metabolic engineering based on genome-scale information will be reported. we have been developing tools for inverse metabolic engineering in order to identify gene targets that improve the phenotype of industrial strains and cells for medical applications. to this end, we create genomic fragment libraries from a source organism and use it to transform the host organism. cells are properly selected in environments that favor the phenotype of interest and genes enriched in these cells are sequenced and used in follow up transformations of cells with specific genetic backgrounds. this overall strategy is complemented with additional tools for modulating gene over-expression, gene deletion, and high throughput clone isolation. we will demonstrate applications of this strategy to the identification of gene targets for improved xylose assimilation in recombinant saccharomyces cerevisiae and improved lycopene production in escherichia coli. based on assumed reaction network structures, nadph availability has been proposed to be a key constraint in ␤-lactam production by penicillium chrysogenum. in this study, nadph metabolism was investigated in glucose-limited chemostat cultures of an industrial p. chrysogenum strain. enzyme assays confirmed the nadpspecificity of the dehydrogenases of the pentose-phosphate pathway and the presence of nadp-dependent isocitrate dehydrogenase. pyruvate decarboxylase/ nadp-linked acetaldehyde dehydrogenase and nadp-linked glyceraldehyde-3-phosphate dehydrogenase were not detected. although the nadph requirement of penicillin-gproducing chemostat cultures was calculated to be 1.5-1.7-fold higher than that of non-producing cultures, activities of the major nadph-providing enzymes were the same. isolated mitochondria showed high rates of antimycin a-sensitive respiration of nadph, thus indicating the presence of a mitochondrial nadph dehydrogenase that oxidizes cytosolic nadph. the presence of this enzyme in p. chrysogenum has important implications for stoichiometric modelling of central carbon metabolism and ␤-lactam production and may provide an interesting target for metabolic engineering. bacteria and mammalian cells have been traditionally used as hosts for commercial recombinant protein production. however, in recent years, the insect cell-baculovirus system has emerged as a potentially attractive recombinant protein expression vehicle. although flow cytometry has been used widely for analysis of mammalian and microbial cells, there is very little information on applications of this powerful technique in insect cell culture. here we compared cell ratiometric counts and viability (propidium iodide and calcein am) of sf-21 cell cultures using flow cytometry to those determined by more traditional methods using a haemocytometer and the trypan-blue exclusion dye. flow cytometry has also been used to monitor various parameters during cultures of sf21 infected with the recombinant autographa californica nuclear polyhedrosis virus (acnpv) containing the inserted nucleic acid sequence amfp486 coding for am-cyan coral protein, which emits natural green fluorescence. complete elucidation of the genetic control of a metabolic flux requires the availability of fine-grained expression levels of the gene(s) of interest. we developed a collection of promoters of varying strength for tuning gene expression in the yeast s. cerevisiae. engineered promoters were obtained through random mutagenesis of the constitutive tef1 promoter. eleven mutated promoters were selected by fluorescence-activated cell sorting (facs) spanning gradually increasing activities between about 8 and 120% compared to the native tef1 promoter. data were also confirmed at the level of mrna via rt-pcr. by introducing selectable markers in front of the different tef1 promoter mutations, we provided plasmid collections, which can be directly used to amplify promoter replacement cassettes for genomic integration of the fine-grained promoter collection in front of any yeast gene. l-arabinose, widely distributed in plant kingdom, is a component of plant cell wall. l-arabinose does not abundantly exist in free state in plants, but usually in corn hull, sugar beet pulp, gum arabic, mesquite gum, as the polysaccharide such as arabinoxylan and arabinogalactan. to produce arabinose from agricultural wastes, we screened arabinogalactan degradable strain from compost. thereafter, putative arabinase gene from this strain was cloned (b1029 ts2-8). as a result of spectrometric assay using -nitrophenyl ␣l arabinofuranoside, recombinant showed 3-4-fold higher activity than wild type e. coli strain. after enzymatic reaction with corn fiber, b1029 ts2-8 produced 2.15 g/l of l-arabinose, which was detected on hplc. however, the enzyme activity was very low. so, we are transferring the gene into expression vector system. further characterization study and enzyme engineering to enhance the activity toward corn fiber will be presented in poster. there are only a limited number of hypersaline areas all over the world, which include several locations in turkey such as van lake located in eastern region of turkey. isolation and identification of halophilic and hyperhalophilic microorganisms from such locations is essential for the determination of biodiversity in turkey. high-level production of extremozymes from these microorganisms has also many economical advantages due to their stability at extreme reaction conditions. proteolytic enzymes are the most important group of enzymes produced commercially. of these, proteases produced by alkalophilic microorganisms are investigated not only in scientific areas such as protein chemistry and protein engineering but also find wide application in food, pharmaceutical, leather and detergent industries. in this study, 24 microorganisms isolated from van lake were screened for the presence of extracellular alkaline protease activity. the optimum screening temperature and ph were determined as 37 • c and ph 9.5, respectively. one of the isolates that could grow at 0-20% salinity reached highest levels of extracellular alkaline protease activity. this best producer, which was identified as the halotolerant bacillus pumilus, was found to produce alkaline protease both in the presence and absence of nacl. to improve enzyme production yields, culture conditions such as medium composition, growth ph and temperature were optimized. the effect of different carbon sources, organic and inorganic nitrogen sources on the production of alkaline protease was studied. whereas a mixture of inorganic and organic nitrogen sources induced high protease production, use of only an organic nitrogen source supported poor enzyme production. halotolerant bacillus pumilus produced maximum alkaline protease activity when maltose, yeast extract and sodium nitrate were used as carbon source, organic and inorganic nitrogen sources, respectively. this project was supported by tubitak through project tbag 2321-103t069. in the market of biochemical products a very important role is played by heterologous proteins production, and despite recent advances in mammalian cells exploitation, yeasts can still present advantages as host systems. among them, the spoilage yeasts belonging to the zygosaccharomyces genus have become, due to some peculiar properties, significantly attractive. in particular, z. bailii is characterized by acid resistance, osmotolerance to high sugar and ethanol concentration combined with high biomass yield. despite still little is known about its genetics and cellular biology, our group is working on its development and exploitation for recombinant productions with an integrated approach coupling physiological study with the creation of molecular tools for heterologous proteins production. we previously described and did a patent application regarding the first techniques necessary to transform this yeast and to express and secrete different proteins derived from different sources. here we present and discuss the last advances in optimization of heterol-ogous protein expression in particular, on one side we present a reproducible strategy for target gene deletion, leading to the first z. bailii auxotrophic mutant, and on the other we show the improvement of gene dosage and plasmid stability by building a set of multicopy expression vectors based on the sequences of the z. bailii 2 -like endogenous plasmid psb2 and an integrative plasmid. all the known ␥-butyrolactone autoregulator receptors are highly conserved in the dna binding motif present in their n-terminal portions and have been proposed to play roles as transcriptional regulators in antibiotic production and/or morphological differentiation. previously, kim et al. reported that the cloned scar in streptomyces clavuligerus has several characteristics of the autoregulator receptors in the genus streptomyces. in this study, to clarify the in vivo function of scar, a scar-disrupted strain was constructed by means of homologous recombination after introducing a scar-disruption construct via transconjugation from e. coli. no difference in morphology was found between the wild-type strain and the scar disruptant. however, the scar disruptant showed a 1.8-fold higher production of clavulanic acid than the wild-type strain. the phenotype was restored to the original wild-type phenotype by complementation with intact scar. therefore, the autoregulator receptor, scar, acts as a negative regulator of biosynthesis of clavulanic acid but plays no role in cytodifferentiation of s. clavuligerus. lactate dehydrogenase catalyses the production of lactate from pyruvate. it is the first target for many researches on lactic acid producer microorganisms like rhizopus oryzae. in the present study based on the known sequences of r. oryzae ldha and ldhb genes skory (2000), they were cloned and expressed in a citric acid producer fungus aspergillus niger. the aspergillus niger strains expressing ldha or ldhb gene resulted in increased production of lactate in aspergillus niger. among 50 transformants tested 4 ldha and 5 ldhb expressing strains were found to have higher lactate dehydrogenase activity compared to wild type in the conditions tested. the highest specific activity obtained with ldha transformants was only 2.5 times of the wild type while this was 10 times for one of the transformants expressing ldhb. in addition to increased lactate production citric acid production was also increased. however, gluconic acid production ceased in the ldha or ldhb expressing a. niger strains. the production of lactic acid in a. niger transformants and lactate dehydrogenase a and lactate dehydrogenase b enzymes are being investigated in the chosen strains. selection of n source suitable for production of rhodococcus sp. biomass for the purposes of microbial transformation of 5␣h-epoxypregnanolone (5␣h) and 5 -epoxy-pregnenolone ( 5 ) into their 9␣-hydroxy-derivatives was carried out. three dehydrated and three non-dehydrated n sources were tested. the transformation reaction was carried out in phosphate buffered medium containing 1 g l −1 of the steroid substrate and 0.1 g l −1 cells. the steroids were determined by hplc. the transformation resulted in formation of three derivatives appearing in the reaction medium in the sequence: 4 -3-keto-; 9␣-hydroxy-and 9␣,20␤-hydroxy-epoxy-pregnenolone. a strong influence of the n source on the hydroxylating activity of the biomass was observed. triptose (difco) gave a cell depot actively hydroxylating 5␣h without any significant accumulation of the 4 -3-keto-derivative. the most effective accumulation of hydroxylated derivatives of 5 was observed with biomass grown on freshly prepared meat extract while the commercial products triptose (difco), meat extract (difco) and lactalbumin (flika) gave valuable information about the dynamics of the transformation process. biobleaching of kraft cellulose pulp by poliporus versicolor aysun ergene 1 , nazif kolankaya 2 : 1 kırıkkale university, faculty of science and literature, department of biology, yahsihan, kırıkkale, turkey; 2 hacettepe university, faculty of science, department of biology, beytepe, ankara, turkey the suitability of culture supernatant from poliporus versicolor for use in the biobleaching of kraft cellulose pulp was investigated. p. versicolor was found to grow on mycological broth (1% soytone, 4% d-glucose and 0.5% cellulose pulp). maximal extracellular ligninase production was detected after7 day (7 nkat). the optimum biobleaching conditions are 30 • c and ph: 4.8, with 10 days. in this condition p. versicolor decreased the kapa number from 38.55 to 19.42 and increased brigthness from 28 to 32.7 in 10 day treatment. xylanase production, purification and characterization from a soil isolate, bacillus m-13 ayşegül ersayin 1 , aytaç kocabaş 1 , b. zümrüt ogel 2 , ufuk bakir 3 : 1 biotechnology department, middle east technical university, ankara, turkey; 2 food engineering department, middle east technical university, ankara, turkey; 3 chemical engineering department, middle east technical university, ankara, turkey, 06531. e-mail: ubakir@metu.edu.tr (u. bakir) xylan is a major component of the plant cell wall, representing up to 35% of the dry weight. xylan molecule is a complex polymer consisting of a ␤-d-1,4-linked xylanopyranoside backbone substituted with acetyl, arabinosyl and glucuronosyl side chains. hydrolysis of the xylan backbone is mainly catalysed by endo-␤-1,4-xylanases (ec 3.2.1.8). many bacterial and fungal species are able to utilize xylan as a carbon source. interest in the enzymology of xylan hyrdolysis has increased because use of xylanases in bioconversion of agricultural wastes to valuable products like single cell protein, xylo-oligosaccharides and fuel, in bio-bleaching processes, food and animal feed industries. in this study, xylanolytic nature of a soil isolate bacillus spp., bacillus m-13, has been shown. bacillus m-13 produced multiple xylanases when grown on a liquid medium containing agricultural wastes as the sole carbon source. various agricultural wastes including corn-cobs and cotton waste, with and without pretreatments were used to maximize enzyme production. the major xylanase having molecular weight of 20 kda upon sds-page and a pi of 9.1 upon ief was partially purified by liquid chromatographic techniques 150-fold with 40% recovery, including gel filtration, ion exchange and hydrophobic interaction chromatography. enzymes are important constituents in the laundry detergents due to their contribution to shortening washing times, reduction of energy and water consumption by lowering washing temperatures, provision of environmentally friendlier wash-water effluents and fabric care. however, they can loose a significant part of their activity in the chemically-hostile detergent matrix over a time period of several weeks. therefore, improving the storage stability of enzyme granulates is the main challenge in the development of a new product. the complexity of the detergent matrix implies the presence of a complicated mechanism involved in the inactivation of the enzymes. a combination of factors, such as oxidation by h 2 o 2 released by the bleaching agents, humidity, high temperature, autolysis of enzymes, high local ph in a granule, oxygen, and other detergent components, plays a role in the activity loss. an experimental investigation on the inactivation of the solid-state enzyme during storage has been initiated. the release rate of h 2 o 2 from the bleaching agent, sodium percarbonate, was determined using a simple and accurate method for measuring the gas phase h 2 o 2 concentration. the deactivation kinetics of pure enzyme was determined as a function of gas phase h 2 o 2 concentration and humidity. the preliminary results indicate that humidity plays a significant role in the inactivation mechanism of the detergent enzyme due to a possible increase in the mobility of the enzyme molecule and the surface area exposed to destructive agents. the effect of main granulate ingredients on the stability of the enzyme was investigated and the extent of the protection of each component was estimated. the study is important for the revealing of the phenomena occurring in the detergent matrix during storage. understanding the inactivation mechanism provides a valuable tool for the development of more effective protective coatings and stabilizers. the use of biosurfactants in cosmetic industry has attracted great attention of biotechnological researchers because they consist of two inexpensive, renewable and easily accessible starting agricultural materials: sugar and oil/fat. carbohydrate based products are non-toxic, biocompatible and biodegradable. in addition, the enzymatic processes present many advantages in comparison with the chemical methods, which employ high temperatures in the presense of alkalin catalysts, high-energy consuption and low selectivity of products. sugar esters present vast application, such as for antibiotics, biomaterials, surfactants, cosmetics and so on. we investigated the synthesis of sugar vinyl esters, using protease-catalysed transesterefication method applying protease from bacillus subtilis. sucrose 0.125 m and vinyl ester 0.5 m has been mixed in dimethylformamide at 160 rpm of agitation. at first, we have studied the effects of protease from bacillus subtilis concentrations (5, 10, 20 and 40 mg/ml) as catalyst. afterwards, the influence of the temperature (30 and 50 • c). after that, the influence of the molar ratios (1:1; 1:2; 1:4 m/m) between vinyl laurate (ch3(ch2)10cooch ch2) and sucrose. subsequently, we investigated the effects of water amount, using 0, 5, 10 and 20% of water in dmf. the conversion ratios of sucroseto-sucrose esters were determined decreasing sucrose measurement with hplc. the results showed that the best conditions to produce high activity on the enzymatic reaction was by using 40mg/ml of protease from bacillus subtilis at 30 • c, molar ratio of 1:4 (vinyl laurate:sucrose) and adding 10% of water in dmf. finally, we succeeded in the characterization of vinyl sugar ester, which was produced after 25 hours of reaction by 13 c nmr. the results confirmed the c1substituted sugar mono-ester (1 -o-vinyl lauroyl sucrose). effects of oxygen transfer on ␣-amylase production by b. amyloliquefaciens nurhan güngör, güzide ç alık bre lab, department of chemical engng, ankara university, 06100 ankara, turkey ␣-amylase (e.c. 3.2.1.1) a commercially important enzyme used in food, textile, detergent, brewing, paper, and animal feed industries; hydrolyses ␣-1,4 glucosydic bonds in amylose, amylopectin, and related polysaccharides. optimum medium composition and influence of bioreactor operation parameters on ␣-amylase production with high yield and selectivity were determined together with the metabolic flux distributions using b. amyloliquefaciens (nrrl b-14396), which is found to be a good producer of the enzyme. systematic investigation of oxygen transfer in relation with the metabolic fluxes for ␣-amylase is not available in the literature. shake-flask experiments were conducted in 0.5 dm 3 air-filtered bioreactors in orbital shakers with agitation and heating controls (b. braun, certomat-bs1). laboratory-scale bioreactors were composed of agitation, heating, foam, dissolved-oxygen and ph-controlled 1.0 and 3.5 dm 3 systems (b. braun, biostat q; and chemap). after separation of the cells with a sorval rc28s ultracentrifuge, ␣-amylase activity was measured by the dns method bernfeld (1955) . amino acid concentrations were determined with a hplc (waters), pro-tein and organic acid concentrations were measured with a hpce (waters, quanta 4000e) ç alik et al. (1998) . oxygen transfer characteristics in the bioreactor systems were calculated using the dynamic method rainer (1990) . in the mass flux balance-based analyses, a pseudo-steady state approximation for the intracellular metabolites and the accumulation rates of the extracellular metabolites measured throughout the fermentations in considerations of the biochemical feature of the system were used to acquire the flux distributions. in laboratory scale, the effects of different c sources, i.e. glucose, fructose, maltose, lactose and soluble starch; n sources, i.e. (nh 4 ) 2 hpo 4 , (nh 4 ) 2 so 4, and nh 4 cl and/or their concentrations; and the operation parameters, ph and temperature on cell growth, substrate utilization, ␣-amylase and by-product concentrations, and ␣-amylase activity were investigated. in pilot scale, the fermentation and oxygen transfer characteristics of the bioreaction system together with the metabolic fluxes were determined. bernfeld, p., 1955 . methods in enzymol. 1:149-159. ç alık, p., ç alık, g.,özdamar, t.h., 1998 . enzyme microb. technol. 23, 451-461. rainer, b.w., 1990 . geldanamycin is a benzoquinone ansamycin produced as a secondary metabolite by the actinomycete, streptomyces hygroscopicus var. geldanus, in submerged culture. it is a broad-spectrum antibiotic and exhibits an anti-tumour activity through its interaction with the heat shock protein 90 family of chaperone proteins. the optimal recovery of geldanamycin from fermentation broths is the focus of the presented work. the application of adsorbent resins was assessed and the viability of developing a solid phase extraction process for geldanamycin was determined. it was found that recovery of geldanamycin from fermentation broth was possible using adsorbent resins and the use of resins facilitated the recovery of a product stream of high purity. the composition of the fermentation broth had an impact on the performance of the resins and it was found that assessing performance on the basis of experimentally derived data was more apt than studying the kinetics of adsorption alone. adsorptive processes are, by their nature, difficult to optimise and this was found to be the case when optimising the recovery of geldanamycin from partially clarified fermentation broth. considerable effort was required to optimise geldanamycin adsorption, via examining the effect of environmental conditions and process system configuration, and geldanamycin desorption, via examining the effect of environmental conditions and investigating selective elution patterns. it is well known that halophilic eubacteria synthesize compatible solutes in order to face the high ionic strength environment in which they proliferate. these biomolecules are gaining more and more importance as biotechnological tools in a wide array of applications, and the recently developed novel bioprocesses enabled large-scale production of these compounds and therefore commercial distribution. however, there is still interest in the optimization of the production process of sole hydroxyectoine that was demonstrated to have a superior stabilization capacity. in this research project, we optimized growth conditions of marinococcus m52 to obtain high yield of hydroxyectoine. their production proved faster in the batch experiments at a higher oxygen supply, even if the stationary phase was comparable in all cases. the mf experiments showed a final biomass which was 5-fold that obtained in the corresponding batch process. in addition the monitoring of compatible solutes production showed that in the last 24h experiment hydroxyectoine accounted for 80-90% of the total content, accumulating up to 13-15% of the cell dry weight. studies for improving downstream process for ectoine and hydroxyectoine recovery showed that short permeabilization cycles in water are effective in a temperature range between 45 • c and 55 • c using a ratio 1:4/biomass:water. moreover, we evaluated the ability of ectoine to stabilize lactic acid bacteria during freeze-drying and to protect human cells from heat stress. in particular, the compatible solutes were added to the medium of confluent keratinocytes before subjecting the cells to heat stress, or lps insult. rt-pcr and western blot analysis demonstrated the hsp70b' gene over-expression in heat stressed human keratinocytes treated with ectoine. finally, we demonstrated that even at low concentration (50 mm) these compatible solutes are able to diminish cell death in lactic acid bacteria due to lyophilization procedure. among all existing alternative energy sources, biomass-derived bioethanol is especially advantageous since it is clean, sustainable and potentially inexpensive. the actual production of bioethanol is divided into a pre-treatment step, an enzymatic hydrolysis step and a fermentation step. while fermentation has been practiced by humans for centuries, our knowledge of enzymatic hydrolysis is still limited. nevertheless, it is well accepted that hydrolysis is a synergism among three classes of enzymes, ␤-glucosidase, endoglucanase and cellobiohydrolase. furthermore, a complete and efficient hydrolysis is only achieved when the enzymes are in the correct proportion. the common enzyme proportions have so far been based on the natural enzyme abundance as produced by the microorganism or mainly been determined by a trial-and-error approach. in this study, however, we used metabolic control analysis (mca) as a modelling tool to gain fundamental knowledge about enzymatic hydrolysis and to design an optimal enzyme mixture. using gepasi, a free software, the degree of control of each reaction step or each enzyme towards the overall hydrolysis can be calculated. our hypothesis is that the amount of each enzyme used for hydrolysis should be proportional to the degree of control of the enzyme. with mca, a significant amount of time, labour and reagents can be saved on developing hydrolysis enzyme mixture. furthermore, this study should demonstrate the usefulness of mca on understanding enzyme-catalyzed reactions outside the cell. process optimization for fed-batch fermentation of bacillus thuringiensis subsp. israelensis arindam chaudhury, gopinathan c department of biotechnology, university of calicut, calicut, kerala 673635 india. e-mail: g achaudhury@umassd.edu (a. chaudhury) bacillus thuringiensis (bt) is a desirable biopesticide because of its low cost and lack of toxicity. the use of bt in developing countries is limited due to process complications and economic non-feasibility of the fermentation process. in the present study, we have shown how regional production, using inexpensive alternatives for carbon and protein sources, can effectively reduce the cost of mass production of bt. while using alternative media supplements, the biomass production, nor the larvicidal activity was hampered. in addition, the positive effects of sparged aeration and the indispensable role of yeast extract were also proved. this work provides the first experimental proof of delineating the sporulation process and delta-endotoxin production. the role of various buffering agents and additives in increasing biomass production and early sporulation were also investigated. for the production of coenzyme q 10 (coq 10 ), an electron carrier in the respiration chain with antioxidant activity. with decrease of dissolved oxygen level from 20 to 5%, the intracellular coq 10 content increased about 4-fold, yielding 2 mg per g-dry cell weight at 5% dissolved oxygen level. azide significantly increased the intracellular coq 10 content, with the highest value of 5.3 mg per g dry cell weight in the presence of 0.45 mm of sodium azide. however, dnp (up to 200 m) and h 2 o 2 (up to 10 m) did not affect the intracellular coq 10 content, indicating proton gradient release and oxidative stress do not affect the synthesis of coq 10 . these results show that restricted electron flux by limited oxygen supply and the addition of azide increases the intracellular coq 10 content. fourier transform infrared spectroscopy (ft-ir), combined with in situ heat sterilizable attenuated total reflection (atr) probes, constitutes a promising and versatile technique for on-line monitoring of bioprocesses. the ft-ir enables rapid determinations of the medium composition without the requirement of sample withdrawal and preparation. in this work the concentration levels of the substrates glycerol and methanol were monitored on-line in pichia pastoris cultivation. partial least squares (pls) models were used for obtaining the concentration readings. the glycerol concentration measurement proved to be very reliable and reproducible in the glycerol batch phase. however, the on-line information regarding the glycerol concentration was not utilized for any process control purposes. on the other hand, the availability of on-line information about the methanol concentration proved to be crucial for the successful implementation of the cultivations. the temperature strategy in the methanol fed-batch phase utilized temperatures as low as 10 • c. in order to keep the metabolic activity at a reasonable level the culture was therefore pushed towards the maximal substrate consumption rate, rather than being a conventional substrate limited fed-batch. as a consequence methanol accumulation occurred on occasions. without on-line information about the concentration this accumulation, if sustained, would have resulted in a poisoning of the culture, either from methanol itself, or perhaps more importantly from formaldehyde. therefore, it can be concluded that the ft-ir/atr instrument was very useful in this application. jørgensen department of chemical engineering, denmark technical university, building 229, dk-2800 lyngby, denamrk. e-mail: fpd@kt.dtu.dk (f.p. davidescu) modeling biochemical reaction network in microorganisms still represents a challenge due to the very large number of enzyme catalyzed biochemical reactions, to the very complex system and to the many feed-forward and feedback regulation mechanisms. the presented approach to model such a system is based on the stochastic grey-box modeling framework proposed by kristensen et al. (2003) . this methodology consists of parameters estimation based on a prediction error method followed by different statistical tests for parameter significance and for model (in-) validity. the methodology furthermore allows estimation of unknown functional relations, e.g. kinetic rates. a set of experimental data zangirolami (1998) obtained during continuous cultures of a high enzyme producing aspergillus oryzae strain. the oxygen concentration was decreased stepwise and the substrate concentration was modified from one experiment to other. a model proposed by agger et al. (1998) is investigated on these data. the primary interest is to develop a physiologically feasible model, also at the low oxygen concentrations often found in industrial practice. microbially produced secondary metabolites such as antibiotics have tremendous economic importance. streptomyces spp. have long been identified as sources of antibiotics and chemotherapeutic compounds, synthesising over 4000 bioactive compounds. geldanamycin is a novel chemotherapeutic agent produced by streptomyces hygroscopicus var. geldanus in submerged fermentation. initial studies have focused on optimisation of media design through understanding and controlling metabolic routes of biosynthesis within the cell. geldanamycin is a by-product of the shikimate or aromatic amino acid biosynthesis pathway. stimulation of this pathway and concomitant production of geldanamycin is achievable through amino acid control. increasing concentrations of primary carbon source greatly influence biomass generation and product formation, as does the inclusion of cations such as magnesium and calcium to the fermentation media. optimisation of production media through balancing minerals, nitrogen, and carbon sources has significantly improved antibiotic yields in shake flask cultures and the development process will be extended into pilot scale through the use of bioreactors. microbiology and biotechnology research group, school of life sciences, napier university, edinburgh, eh10 5dt, scotland. email: m.el-mansi@napier.ac.uk (m. el-mansi) synopsis: during growth of corynebacterium glutamicum on glucose or other glycolytic intermediates, pep carboxylase fulfils an anaplerotic function as it replenishes intermediary metabolism with biosynthetic precursors that are essential for growth and glutamate production. under these conditions, pep carboxylase plays a central role and this in turn is characterised by a high flux control coefficient thus rendering this enzyme an ideal target for metabolic interventions. further analysis in silico revealed that any increases in the concentration of the enzyme was accompanied by increases in flux through the enzyme itself as well as glutamate formation, presumably as a consequence of sustaining a high intracellular level of ␣-ketoglutarate; the immediate precursor for glutamate biosynthesis. a combined approach to enhance periplasmic expression of human growth hormone in escherichia coli, using a modified signal peptide from alpha amylase gene of bacillus licheniformis s.k. falsafi 1,2 , a. zomorodipour 2 : 1 islamic azad university of jahrom, iran; 2 department of mol genet. national inst for genet eng & biotechnol., tehran, iran. e-mail: soheil falsafi@yahoo.com (s.k. falsafi) the alpha amylase gene signal peptide, originated from a strain of bacillus licheniformis, was shown to be able to transport its native protein, when expressed in e. coli the competence of the fusion protein being processed and translocated through the inner membrane is highly dependent on the amino acid sequences in the signal peptide. therefore, in order to increase the expression efficiency of bla signal peptide, we reconstructed the bla signal peptide coding fragment with the following modifications. two rare codons of arg 6 (cgg) and arg 10 (cga) and codons for leu 15 (tta) and pro 23 (cct), in the signal peptide were substituted with their corresponding e. coli major codons. two other changes, including phe 20 (ttc) → leu 20 (ctg) and ala 28 (gcg) → met 28 (atg), were also introduced to increase the processing efficiency. the hgh-expressing plasmid equipped with the modified bla (blaf2) was subjected for further expression analysis in a t7-based expression system. the results obtained from the protein patterns of the induced bacteria indicates in high expression level of hgh preprotein (hgh::blaf2) followed by efficient transfer of the mature hgh to e. coli periplasm. (ip6) has been demonstrated to have a wide range of health benefits such as prevention and therapy of various cancers, amelioration of heart disease, and prevention of renal stone formation as well as complications from diabetes. on the hand, lower phosphorylated forms of inositol, especially inositol trisphosphate (ip3) and inositol tetrakisphosphate (ip4) are important signal transduction molecules within the cells both in plants and the animal kingdom. it has been hypothesized that at least the anticancer function of ip6 is mediated via these lower inositol phosphates. the diversity and practical unavailability of the individual myo-inositol phosphates preclude their investigation. phytases, which catalyze the sequential hydrolysis of phytate, render production of defined myoinositol phosphates in pure form and sufficient quantities. different phytases may result in different positional isomers of myo-inositol phosphates and therefore different biochemical properties. phytases differing in ph optima, substrate specificity, and specificity of hydrolysis have been identified in plants and microorganisms. in this paper the dephosphorylation pathway of the novel phyfauia1 was compared to other bacterial phytate degrading enzymes. preliminary results have shown that phyfauia1 converted ip6 into ip5 (myoinositol 1,2,3,5,6 pentakisphosphates) and another isomer, which is yet to be elucidated. characterization of the novel ␤-peptidyl aminopeptidase (bapa) from sphingomonas sp. 3-2w4 that cleaves synthetic ␤peptides birgit geueke, hans-peter e. kohler environmental microbiology, eawag, 8600 duebendorf, switzerland. e-mail: birgit.geueke@eawag.ch (b. geueke) non-natural peptides, which are capable of evoking a specific biological response, are currently receiving much attention. oligomers of ␤-amino acids (␤-peptides) are representing a group of pharmaceutically interesting peptides because of their very high stability towards enzymatic degradation and their ability to mimic the structure of naturally occurring biologically active peptides. the pharmaceutical potential on the one hand and the high stability on the other hand aroused interest for studies on the environmental fate and the degradation behaviour of this class of compounds. a novel bacterial strain (sphingomonas sp. 3-2w4) that was capable of degrading short ␤-peptides was isolated from an enrichment culture. the ␤peptide degrading enzyme was purified and its gene sequence was determined (bapa). the gene encodes a ␤-peptidyl aminopeptidase (bapa) of 402 amino acids that is synthesized as preprotein with a signal sequence of 29 amino acids. it belongs to the n-terminal nucleophile (ntn) hydrolase superfamily and is the first peptidase that is capable of cleaving amide bonds in ␤-peptides composed of synthetic ␤-amino acids. the biochemical properties of recombinant bapa were investigated regarding its substrate specificity and possible application in the synthesis of ␤-peptides. to produce efficient strains of agaricus bitorquis (quel.) saccardo, which are resistant to high temperatures p. guler, a. ergene, s. tan kirikkale university, faculty of science and literature, department of biology, yahsihan-kirikkale in this study, the culture mushroom agaricus bitorquis (quel.) sacc. the growth of the mycelium and the fructifications under high temperature is examined. the spores taken from the mushrooms that were collected from nature were grouped as a, b, c, d, e. the spores were inoculated into malt extract agar and incubated at 30 • c and primer mycelium was produced. the mycelium discus taken from primer mycelium in 8 mm diameter were inoculated into the center of malt extract agar and incubated at 30, 32, 34, 36 and 38 • c separately. during the incubation period the growth of the mycelium were measured. during the growing period the radial growth speed of the mycelium were taken as criteria. the best mycelium growth for all groups was seen at 30 • c. at 36 • c the e group mycelium and at 38 • c other group's mycelium did not grow. these temperatures were determined as thermal lethal point for the groups. from all the mycelium produced from all temperatures spawn was prepared and with the results taken from these, spawn calendar is prepared. in this research, the spawn was inoculated to compost with mixing system and separately put in culture rooms, temperatures as 30 and 32 • c. at this level the culture mushroom production techniques were used. the harvested mushrooms were inspected morphologically. at this morphological inspections the cap width, cap tissue thickness, stalk thickness and stalk long ness was taken as criteria. in the study the best growth was seen at d group mushrooms and this group mushrooms tyrosinase's activities were measured and graphics were made. introduction: viral contamination of biological products; cause many problems in viral diagnostic laboratories, blood transfusion organizations, and biological producers. bovine viral diarrhea virus (bvdv), from the pestivirus genus, is the most common viral contamination in (fetal) bovine serums (fbs). also, bvdv used as a module, for study hepatitis c virus inactivation due to its similarity in structure and genome. pulsed uv lights (puvls) have this potential to inactivate known and unknown or reemerging viruses as well as prions. two puvl with the wavelengths of 355 and 266 nm, were produced by q-switched nd 3+ :yag laser in its third and forth harmonic, respectively. the energy of each pulse for 355 nm was 12.7 mj/cm 2 and for 266 nm was 35.2 mj/cm 2 . bvdv were produced and titrated in mdbk cell line. mdbk and fbs were already checked for non-cytopathic or cytopathic pestiviruses, using related ag-elisa kit. bvdv suspended in solution with the dilution of 1:2 before exposure. the quartz tube with the minimum uv-absorption in compare with air, used as a container for exposed solutions. calculation of the virus titer, 10 4.3 tcid 50 /ml, was done based on the reed and muench method. bvdv suspended in pbs was exposed into the 3.52-352 j/cm 2 of puvls with the wavelength of 355 nm and also, was exposed into the 1.27-92.25 j/cm 2 of puvls with the wavelength of 266 nm. furthermore, bvdv suspended in fbs was exposed into the 88, 176, 352 and 704 j/cm 2 of puvls with the wavelength of 355 nm and also, was exposed into the 6. 35, 25.4, 63.5, 127 and 190 .5 j/cm 2 of puvls with the wavelength of 266 nm. results: the minimum dose for inactivation of bvdv suspended in pbs with the 355 and 266 nm wavelengths of puvls, were 352 and 92.25 j/cm 2 , respectively. also, the minimum dose for inactivation of bvdv suspended in fbs with the 355 and 266 nm wavelengths of puvls, were 704 and 127 j/cm 2 , respectively. to evaluate the fbs quality to support cell culture, treated fbs with the dose of 190.5 j/cm 2 of 266 nm puvls was used to grow vero cell line in 12 successive passages. the viability of cells in two study groups was identical. the statistical evaluation of two treated groups showed no significant difference, in 12 passages. conclusion: because inexpensive equipment can be used to produce puvls capable of handling different volumes of biologics with operational ease, this viral inactivation technique is cost effective for relevant industries. the procedure has the potential to be combined synergically with other inactivation method. puvls offer a new, nonadditive and chemically safe alternative for the treatment of fbs to inactivate adventitious viruses and to preserve the biological activity necessary for the propagation of cell culture. characterization and gene cloning of the g-resorcylic acid decarboxylase for application to selective production of g-resorcylic acid y. iwasaki 1 , y. ishii 2 , k. kino 1 , k. kirimura 1 : 1 dept. appl. chem., sch. sci. eng., waseda univ., tokyo, japan; 2 bme, asmew, waseda univ., tokyo, japan. e-mail: iwasaki@moegi.waseda.jp (y. iwasaki) for selective production of ␥-resorcylic acid (␥-ra, 2,6-dihydroxy-benzoic acid) from resorcinol (re, 1,3dihydroxybenzene) under mild conditions, we screened various microorganisms and found the reversible ␥-ra decarboxylase (rdc) as a novel enzyme applicable to carboxylation of re to form ␥-ra, in a bacterial strain rhizobium radiobacter wu-0108 1) . rdc catalyzed the decarboxylation of ␥-ra, and regio-selective carboxylation of re to form ␥-ra, without formation of ␣-ra and ␤-ra. the molecular weight of rdc was estimated to be 130 kda by gel-filtration, and that of the subunit was determined to be 34 kda by sds-page, suggesting that rdc is a homotetrameric structure. the gene encoding rdc was sequenced, and a site-directed mutagenesis study revealed that the two histidine residues at positions of 164 and 218 in rdc are essential for the catalytic activity of rdc. through the reactions using e. coli cells highly expressing rdc, 6.7 mm ␥-ra was produced from 15 mm re at 30 • c for 16 h, with a yield of 45%. ishii, y. et al., 2004. biochem. biophys. res. commun., 324, 611-620. laccase biosynthesis in stirred fermenters teresa jamroz, stanislaw ledakowicz, barbara sencio department of bioprocess engineering, technical university, lodz pl 90-924, poland industrial applicability of enzymes is closely related to development of efficient methods of their production. currently, significant interest in lignolytic enzymes, including laccase, has been observed. laccase is an enzyme applied in various industrial branches and environmental processes. broad laccase applicability induces researchers to develop urgently efficient methods for its commercial production. laccase (ec.1.10.3.2. p-diphenol oxidase) is produced by cap mushrooms from the class basidomycetes. this is the so-called white rot fungi which in natural conditions appears on both living and dead wood. as shown in the practice of biotechnological processes, high-efficient strains have low resistance to destructive factors in bioreactors. hence, to preserve a proper morphology and physiological state of an organism, strictly determined culture conditions must be obeyed. this is very important in the case of basidomycetes for which submerged culture in the liquid phase is not a natural habitat. results of studies on laccase production from cerrena unicolor family are discussed. cultivation of active biomass was carried out in stirred tank and rotating disc bioreactors of different volume (b. braun of working volume 12 dm 3 ; fas-01 of working volume 3.5 dm 3 ). experiments in both fermenters were made at impeller revolutions 200 and 300 min −1 , on a modified lindberg substrate. significant differences in the rate and yield of laccase production were reported. an almost three times higher values of laccase activity were obtained in the b. braun fermenter, at rotational speed 300 min −1 . to retain suspended cells in bioreactor a filtration process can be used. the biomass is concentrated by withdrawing cell-free culture broth. if the desired product is dissolved in the broth (extracellular production), the procedure enables the continuous harvest in the cellfree permeate. an application test of filtration system for suspended biomass of aspergillus niger in submerged single stage continuous culture was presented in this report. the system is easy to construct and there is a possibility of its sterile exchange during cultivation. the culture medium contained the following substances (g/dm 3 ): white sugar, 150.0; nh 4 no 3 , 1.5; mgso 4 ·7h 2 o, 0.2; kh 2 po 4 , 0.2; feso 4 ·7h 2 o, 0.05. fermentations were carried out in the lab bioreactor biomer 10. the bioreactor was a standard cstr (continuous stirred tank bioreactor) with working volume of 5 dm 3 . high citric acid concentration in culture medium (p = 108.9 g/dm 3 ), high yield of citric acid (y p/s = 72.6%) and high efficiency coefficient (k ef = 71.1) were observed in single stage continuous culture with biomass retention. oxygen transfer regulates benzaldehyde lyase production in e. coli pınar ç alık, iblab, department of chemical engineering, metu, 06531 ankara, turkey. e-mail: pcalik@metu.edu.tr the effects of oxygen transfer rate on benzaldehyde lyase (bal) production by puc18::bal carrying recombinant escherichia coli on a defined medium with 8.0 kg/m 3 glucose were investigated in order to fine-tune the bioreactor performance, in v = 3 dm 3 batch bioreactors at five different conditions with the parameters at, i.e. q o /v r = 0.5 vvm and n = 250, 375, 500, 750 min −1 and; q o /v r = 0.7 vvm and n = 750 min −1 . the concentrations of the product and by-products amino acids and organic acids were determined in addition to bal activities. medium oxygen transfer rate conditions and uncontrolled ph operation at ph o = 7.25 are optimum for maximum bal activity, i.e. 860 u/cm 3 at 12 h, and productivity and selectivity. on the bases of the data, response of the intracellular bioreaction network of r-e. coli to oxygen transfer conditions were analysed using a mass-flux balance based stoichiometric model that contains 102 metabolites and 133 reaction fluxes. the results reveal that metabolic reactions are intimately coupled with the oxygen transfer conditions. oxygen transfer rate showed diverse effects on the product formation by influencing metabolic pathways and changing metabolic fluxes. metabolic flux analysis was helpful to describe the interactions between the cell and the bioreactor by predicting the changes in the fluxes and the rate controlling step(s) in the metabolic pathways. therefore, knowing the distribution of the metabolic fluxes during the growth, and bal and by-product formations provide new information for understanding physiological characteristics of the r-e. coli, and reveals important features of the regulation of the bioprocess and opens new avenues to successful application of metabolic engineering. saprophytic mycobacterium strains belong to the best known microorganisms which have been applied to the pharmaceutical industry for the production of steroid drugs. the mycobacterial cell wall is the permeation barrier to chemical compounds, including lipophiles. using isoniazid (inh), the inhibitor of the mycolic acids biosynthesis, we were able to demonstrate increased ad production and susceptibility to antimicrobial agents. the process of sterol transformation and products accumulation was monitored using gas chromatography. isoniazid was shown to intensify ␤-sitosterol side-chain degradation by mycobacterium sp., and accumulation of 4-androstene-3,17-dione (ad) and 1,4-androstadien-3,17-dione (add), which are the starting materials in the biotechnology of medically important steroids. to confirm these results, the sensitivity of the bacteria to antimycobacterial drugs was performed. the minimum inhibitory concentration mic 50 of rifampicin and erythromycin decreased markedly in the presence of inh. this work was supported by grant nr 3p04c 06923 of the committee for scientific research. for the purpose of high-throughput screening and to reduce experiments with animals in pharma biotechnology biosensor systems gain importance. the principle of a biosensor is the combination of cultured cells and a sensorchip device, which allows the monitoring of cellular activity. in contrast to traditional analytics with a biosensor you can measure on-line cellular activity change caused by an effector as well as the restored activity after privation of the effector (re-native activity). cmos technology can be used for the realisation of various biological sensorchips such as adhesion sensorchips, metabolical sensorchips and electrophysiological sensorchips. the standard cmos technology allows a high reproducibility of the chips, the integration of electronic components on the chip, which reduces the amount of external devices and the combination of different sensors on one chip. in cooperation with the semiconductor company micronas and the biotech company bionas we have realised different types of cmos sensorchips to measure adhesion of a cellular monolayer with interdigitated electrodes (ides), metabolical activity via acidification with ion-sensitive field effect transistors (isfet) and sponteneous neuronal network activity with passive palladium electrodes. microbial agents have been applied to the different stages of pulp and paper processing. the work presented describes a study on the effect of applying ligninolytic enzymes, such as a laccase plus mediator system, on a variety of different types of pine and eucalyptus pulps and subsequently subjecting these to different ageing processes. industrial pulps were obtained from different portuguese pulp and paper companies. the pulps used were (1) unbleached pine pulp from portucel tejo; (2) unbleached eucalyptus pulp from portucel setúbal; (3) bleached eucalyptus pulp from portucel setúbal; and (4) pulp made from recycled paper from renova s.a. several types of handsheets were produced with 2 different grammage namely, 60 and 180 g/m 2 . the prepared handsheets were subject to an aging sequence in three different chambers: ultraviolet radiation (wavelength of 280 nm), temperature (19 • c) and moisture (70%); and thick saline fog at a concentration of 1% and temperature of 35 • c. in order to evaluate the effect of moisture cycles and temperature, two aging sequences were used for each type of handsheet. in the first, the moisture varied (60, 80 and 100%), while the temperature was held constant (25 • c); in the second the temperature varied (60, 70 and 80 • c) and the moisture was held constant (50%). following the aging phase, the handsheets were subject to several chemical (viscosity and index kappa) and physico-mechanical (colour, tensile breaking strength, stretch and the bursting strength) tests in order to characterize the effect of the aging conditions. results will be presented describing the effect of application of the laccase-mediator system on the optical and mechanical properties of the prepared handsheets. fundação para a ciência e a tecnologia, project pocti/ agr/47309/02. aspergillus niger is a filamentous fungi widely used in industry. its growth as freely dispersed hyphae leads to an increase in the medium viscosity and to problem of mass transfer, especially oxygen transfer. oxygen acts both as final electron acceptor in the mitochondrial chain and as nutrient for the biosynthesis of unsaturated fatty acids and sterols. thereby, a lack of oxygen affects the nadh/nad ratio, the atp production, the growth and has a strong influence on the physiology of the microrganism. in the present study, the metabolic changes of a. niger in response to a lack of oxygen was investigated using oxygen limited chemostats combined with nitrogen pulse. under these conditions, the main consequence of a sudden decrease of oxygen availability is an increase in the mannitol production. this work showed that the mannitol biosynthesis, involving the enzyme mannitol-1-p dehydrogenase, helps the reoxidation of nadh when the final electron transport acceptor, oxygen, is limiting. investigation of the lipase activity of the bacteria isolated from olive mill wastewater sevgi ertugrul 1 , nur koçberber 1 , gönül dönmez 1 , serpil takaç 2 1 department of biology faculty of science ankara university 06100 beşevler ankara turkey; 2 department of chemical engineering faculty of engineering ankara university 06100 tandogan, ankara, turkey the bacteria that could grow on media containing olive mill wastewater (omw) were isolated and their lipase production capacities were investigated. the strain possesing the highest lipase activity among 17 strains grown on tributryin agar medium was identified as bacillus sp. the effect of ph on the lipase activity of the strain was investigated in tributryin medium and ph 6 was found to be the optimal. the liquid medium composition was improved by adding different carbon sources and fatty acids into tributryin medium -omitted tributryin -to increase the enzyme activity. the cultivations were performed at 30 • c and ph 6. lipase activity of the bacillus sp. was measured spectrophotometrically through the hydrolysis of p-nitrophenol palmitate. among the media containing different compositions of tricapryn, trimyristin, tributyrin, triacetin, tween 80, glycerol-trioleate, glycerol-trioctanoate, glycerol-tridodecanoate, omw, glucose, and whey; the medium consisted of 20% whey + 1% glycerol-trioleate was found to give the highest lipase activity. cultivation of bacillus sp. in the optimum medium at ph = 6 and 30 • c for 64 h was resulted in the extracellular and intracelluar lipase activities of 15 and 168u/ml, respectively. this study was supported by ankara university biotechnology institute (project no: 2004-151 and 2005-164) . under different abiotic stresses, cell growth and metabolic activity are highly influenced in all types of living organisms medium osmolality is usually one of those factors affecting different types of biological systems in different ways. however, even in the same organ of higher eukaryotes the degree of osmoregulation mechanism is highly variable in different types of cells. therefore, studying the effect of osmotic stress on mammalian cell is very important subject for particular cell line. the effect of hyperosmotic pressure on the kinetics of cell growth of and metabolic activity of mesenchymal stem cells (mscs) and two industrially important cell lines, hybridoma cells and human embryonic kidney cell (hek) were investigated in batch cultures at different osmotic pressures in the range from 325 to 500 mosm kg −1 . in case of mscs cells, the maximal specific growth rate [] of 0.029 [h −1 ] associated with the highest specific glucose consumption rate [-q gluc ] of 0.1129 × 10 −6 [mol cells −1 h −1 ] was obtained in medium of 375 mosm kg −1 . in case of hybridoma cells, osmotic pressure showed not only influence on the kinetics of cell growth and metabolism but also on the monoclonal antibody production. the maximal mab production was obtained in case of cells cultivated under osmotic pressure of 375 mosm kg −1 . further increase in osmotic pressure resulted in significant reduction in growth rate as well as mab production. on the other hand, hek cells were more sensitive to osmotic pressure in industrially used serum free medium and the addition of serum decreased the inhibitory effect of high osmotic pressure on the cells. gustavo g. fonseca 1,2 , andreas k. gombert 2 , elmar heinzle 1 , christoph wittmann 1 : 1 biochemical engineering, saarland university, saarbrücken, germany; 2 chemical engineering, são paulo university, brazil kluyveromyces marxianus cbs 6556 is a potentially interesting yeast strain characterized by a high capacity of conversion of substrate into biomass. however, this yeast has been only marginally studied so far. therefore, we performed a metabolic characterization in batch and chemostat cultures at dilution rates of 0.10, 0.25 and 0.5 h −1 . the specific rate of o 2 consumption (qo 2 ) increased with dilution rate from 2.87 to 11.09 mmol (g dw) −1 h −1 . the respiratory coefficient remained almost stable around 1.0 for all metabolic states investigated. even at the dilution rate of 0.5 h −1 , which is close to the maximum growth rate of the strain of 0.56 h −1 , no significant overflow metabolism was observed. the concentration of extracellular metabolites increased with the dilution rate, but remained below 6% of the carbon consumed as glucose. all carbon balances closed near 100% underlining the consistency of the data. in contrast to s. cerevisiae the respiratory capacity of k. marxianus cbs 6556 is not strongly influenced by the dilution rate in aerobic chemostat or batch cultures, indicating its high potential for biomass-directed applications. a thermostable l-arabinose isomerase for enzymatic production of d-tagatose o. hansen, f. jørgensen, p. stougaard department of enzyme technology, bioneer a/s, kogle allé 2, dk-2970 hørsholm, denmark. e-mail: och@bioneer.dk (o. hansen) d-tagatose, an isomer of d-fructose, is a low-calorie bulk sweetener with a sweetness equivalent to sucrose. d-tagatose has obtained gras approval for use as a food ingredient, and is currently produced by chemical isomerization of d-galactose, which may readily be obtained by hydrolysis of lactose. structurally, d-galactose is closely related to l-arabinose, and it has previously been shown that some variants of l-arabinose isomerase (araa) may catalyze the conversion of d-galactose to d-tagatose, in addition to the metabolic conversion of l-arabinose to l-ribulose. we have screened a number of bacterial araa enzymes for their ability to catalyze the isomerization of d-galactose to d-tagatose. the best enzyme was found in the thermophilic bacterium thermoanaerobacter mathranii (dsm11426). the araa gene of t. mathranii was cloned, sequenced and expressed in e. coli. amino acid sequence comparisons of the t. mathranii sequence and other known araa sequences showed a relatively low sequence identity of about 30%, indicating a distant phylogenetic relationship to the other members of the l-arabinose isomerase group. the t. mathranii enzyme was thermostable with optimal activity at 65 • c and it required manganese ions. unlike other araa variants, the t. mathranii enzyme showed k m values in the same order of magnitude for l-arabinose and d-galactose, suggesting that this enzyme is a versatile isomerase capable of isomerizing structurally related aldoses. the enzyme was immobilized by chemical cross-linking of a crude e. coli cell homogenate, and the immobilized enzyme efficiently converted d-galactose into d-tagatose. currently, we are developing an enzymatic method for industrial production of d-tagatose using the immobilized enzyme. the agricultural production can be negatively affected by different pest insects (pi) and the use of chemical insecticides (chi) has been the traditional method for controlling pi during decades. nevertheless, there are various ecological implications due to the extensive application of chi. a viable alternative for the use of chi in certain agro-systems, is the use of entomopathogenic nematodes (epn) of the genera steinernema and heterorhabditis that are natural pathogens for different pi; besides, the presence of a symbiotic bacterium is necessary for an effective entomopathogenic activity can take place. the nematode/bacterium complex does not represent a risk for the environment. different authors propose that the best alternative for the massive production of epn is the submerged culture within bioreactors; nevertheless, more research is required to have really robust processes. particularly, information regarding the actual hydrodynamics during epn production and its relation with the epn productivities are scarce, among other aspects. the present study deals with the hydrodynamic characterisation during the production of the epn steinernema carpocapsae and its symbiont bacterium xenorhabdus nematophilus, in submerged monoxenic culture in an internal-loop air-lift bioreactor (v l = 4.5 l) using two culture media: one of them containing whey and the other one, agave juice, aguamiel, (agave spp.). process viscosity of the culture broth was determined along the time, exhibiting a maximum value of 20 mpa s. moreover, it was determined that the hydrodynamic conditions were always located within the laminar region (re < 500). at the experimental conditions tested, it can be inferred that the epn productivity are more sensitive to changes in the culture medium composition than on the prevailing hydrodynamic conditions during the fermentations. bacillus thuringiensis is a gram-positive bacterium used as a biological pest control agent. moreover, it is able to produce, several biologically active molecules such as bacteriocins and hydrolytic enzymes among which chitinases that play double roles, fungicide and improving the insecticidal effect of b. thuringiensis deltaendotoxins. a newly isolated b. thuringiensis subsp. kurstaki strain bupm4, was shown to produce a novel bacteriocin named bacthuricin f4. the highest bacteriocin activity was found in the growth medium and evidenced in the late exponential growth phase. upon purification of bacthuricin f4, the specific activity was increased 100-fold. this bacteriocin was heat-stable up to 70 • c and resisted up to ph 3.0. its molecular mass, determined by mass spectrometry was 3160.05 da. direct n-terminal sequencing of bacthuricin f4 revealed the following sequence: dwtxwsxl. the latter was unique in the databases. bacthuricin f4 was active against bacillus species while it had little or no effect on gram-negative bacteria. the bacteriocin produced by the b. thuringiensis strain bupm4 respond to both criteria of thermostability and stability to low phs. thus, it could be used as a source of bacteriocin active against related species of bacillus harmful for agricultural products and as food preservative. the other example of antimicrobial compound produced by b. thuringiensis is a chitinase. we describe the selection of b. thuringiensis high chitinase-producing strain bupm255, and the characterization and the heterologous expression of a novel chitinase encoding gene. the cloning and sequencing of the corresponding gene named chi255 showed an open reading frame of 2031 bp, encoding a 676 amino acid residue protein. both nucleotide and amino acid sequences similarity analyses revealed that the chi255 is a new chitinase gene, presenting several differences from the published chi genes of b. thuringiensis. the identification of chitin hydrolysis products resulting from the activity, exhibited by chi255 through heterologous expression in e. coli revealed that this enzyme is a chitobiosidase. the addition of the sequence of chi255 to the few sequenced b. thuringiensis chi genes might contribute to a better investigation of the chitinase "structure-function" relation. cloning and characterization of s-adenosyl-l-methionine synthetase from pichia ciferrii dscc 7-25 kwon-hye ko 1 , gee-sun yoon 1 , gi-sub choi 1 , joo-won suh 2 , yeon-woo ryu s-adenosyl-l-methionine(sam) has an important role for dna methylation and cell signaling. sam was synthesized from methionine and atp by sam synthetase and play an pivotal function in the primary and secondary metabolism of cells. recent studies have revealed in the effect of sam in case of morphological differentiation in both eukaryotes and prokaryotes. the p. ciferrii produces large quantities of sphingoid base. tetraacetylphytosphingosine(taps), which is a precursor of sphingolipid, could be used for the production of pharmaceuticals and cosmetics. we isolated sam gene from p. ciferrii and cloned it into expression vector for e. coli and p. pastoris, respectively. an 1.2 kb sam-s gene fragment was isolated by low-strigency pcr using degenerated primer. by the analysed primary sequence deduced from dna sequence, this gene included conserved domains similar with other well-known sam synthetase. first of all, sam synthetase gene cloned pgem-t vector and subcloned into histidine tagging system to purify the expressed protein using metal chelating resin. typical characteristic analysis of this enzyme is underway. metabolic networks offer a large variety of different synthesis pathways starting from cheap substrates and leading to interesting high-value compounds, i.e. metabolites. in case an interesting pathway can be disconnected from the remaining metabolic network, the perforated cell or the crude extract could be used for a one-pot multi-step synthesis of the desired compound. pathway isolation, achieved by deletion of genes encoding gene products enabling side reactions, interferes with the viability of the organism, which is a requisite for the production of the system of biotransformation (sbt). in this work, a rational systems biology-derived approach is presented for the design of a sbt. it is illustrated for a sbt allowing for production of dihydroxyacetone phosphate. the design procedure comprises three steps: (i) a production pathway is identified in the metabolic network of e. coli. the e. coli pathway is complemented by two additional enzymes in order to obtain a fully energy and redox balanced production pathway. (ii) an optimal combination of gene knockouts is designed and a suitable growth medium composition is identified, both by a model-based approach: flux balance analysis of a genome-scale metabolic network is used to predict enzyme expression within the wild-type organism on different media, while a mixed-integer optimisation is employed to identify viable mutants as this approach strongly depends on the quality of the fba prediction, available regulatory information on the usage of metabolic pathways and thermodynamic constraints were taken into account. (iii) thermodynamic analysis of the obtained, partially branched sbt reaction cascade revealed the extent of loss in yield by the remaining side reactions. in summary, this systems biology-driven approach potentially enables the substitution of a elaborative multi-step synthesis process by a one-pot enzyme reaction cascade. institute of industrial biotechnology, inha university, incheon 402-751, korea. e-mail: leecg@inha.ac.kr (c.-g. lee) algal biotechnology is drawing increasing interest due to its potential as a source of valuable pharmaceuticals, pigments, carbohydrates, and other fine chemicals. currently, its application is being extended to the areas of wastewater treatment and agriculture. however, lack of suitable photobioreactors (pbrs) makes the cost of algally-derived compounds higher than those derived by chemical synthesis and thus has prevented widespread use of algal cultures. the culture of algae prior to the late 1940s was apparently restricted to laboratory scale operations. experiments on outdoor algal mass production began in the late 1940s with nearly concurrent development of experimental culture facilities in germany and the united states. for the next two decades, outdoor mass culture of algae was undoubtedly the hottest topic in the algal biotechnology area. recent developments of high-density pbrs enable the production of valuable biologically active compounds by algal mass cultures. however, light is almost always the limiting factor in high-density photobioreactors. key factors for successful photobioreactors will be discussed and various photobioreactors will be analyzed and compared for their advantages and disadvantages. the new techniques, such as pigment redcution and application of flashing light and lumostatic operation, will be discussed for possible solutions to overcome the light limitation in high-density microalgal cultures. a quick and reliable method for screening fungal transformants for specific genetic modifications is essential for many molecular applications, for example when one tries to develop a transformation system for a new fungal host. southern analysis is laborious and time consuming. several colony hybridisation methods have been developed for the analysis of a large number of transformants. unfortunately these methods suffer from different disadvantages such as non-specific binding, a limited usability to screen for specific integration and the fact that these procedures always take a few days (van zeijl et al., 1998) . recently, methods for pcr-based analysis of fungal transformants have been described. most of these methods require high quality dna. many methods for dna extraction from fungi have been described in the past few years. these methods often are tedious, time consuming, costly or limited to a small number of samples each time. most of the available protocols include the growth of mycelium in a liquid culture, followed by freeze-drying or maceration in liquid nitrogen and grinding of the frozen material to break the cell walls (cassago et al., 2002) . lately a few methods have been described to isolate dna from fungi suitable exclusively for pcr and appropriate for the simultaneous treatment of a large number of samples. some methods also describe the direct use of mycelium (cooke et al., 1997) in the pcr-reaction mixture. we compared the applicability of a few rapid dna extraction methods for myrothecium gramineum and tested the resulting dna samples on there suitability for pcr-applications. myrothecium gramineum is a filamentous ascomycete used in ongoing research as a new cloning and expression host. five methods were tested. in four of these methods dna was extracted from mycelium (goodwin et al., 1993 and aljanabi et al., 1997) or spores (ferreira et al., 1997 and xu et al., 1995) prior to pcr. a fifth assay used mycelium straight in the pcr-reaction mixture. only this last method seemed useful for myrothecium to isolate dna suitable for pcr. fragments up to 2000bp were amplified. cheese whey is a liquid effluent from cheese-making processes. there is an increased interest in the economic utilization of whey produced by the dairy industries, because the whey is a pollutant, due mainly to its lactose content. the goal of this work was to find the most suitable values of some fermentation parameters for lactic acid production from whey by a lactic acid bacterium, lactobacillus helveticus (atcc 15009). the effects of lactose content, temperature, ph and the supplementation with yeast extract were investigated using surface response methodology. a composite central design was used with three center points, making a total of 27 operational conditions. the region of maximum production is outlined by the following intervals: temperature around 40 • c; lactose concentration between 70 and 85 g/l; concentration of yeast extract between 20 and 25 g/l; ph between 7 and 7.5. fermentation studies on a continuous fermentative process coupled to a vacuum flash evaporator were carried out in lab scale equipment. the phases of this work consisted in an assembly and instrumentation of the prototype and elaboration of a supervisory system coded in labview 6.1, which allows the data acquisition and control through personal computers. the experiments in continuous fermentation used saccharomyces cerevisiae and sugar cane molasses as substrate. the analytical follow up was done through analysis of total reducing sugars, ethanol, glycerol, dry mass and viable cells. the system worked for months uninterruptedly, producing an alcoholic solution at the condenser with 50 • gl. the fermentation operated with concentrations of ethanol at 5 • gl, which is a weakly inhibitory value for the yeast of the process, even when fed with concentrated cane molasses, containing up to 330 g/l of sugar. the result meets the initial goal, which was to operate the system with low level of ethanol and to guarantee high productivity, even in high concentrations of sugar in the feeding. the results showed that system productivity was superior to that of the conventional continuous process. lactic acid (la) is a versatile chemical, used as an acidulant, flavor and preservative in the food, pharmaceutical, leather and textile industries, and for production of biodegradable poly lactic acid (pla). l(+)lactic acid is the only optical isomer for use in pharmaceutical and food industries because human body is only adapted to assimilate this form. in this research, lactic acid production was improved on 20 l fermentor. in our experience, among six strains of lactobacillus were examined for the production of l(+) lactic acid, lactobacillus casei ssp. casei atcc 39392 was selected as a highest l(+) lactic acid producer. optimized medium used for lactic acid production contained (per l) 80 g glucose, 50 g whey powder and 20 g corn steep powder. for a homofermentative process, ph 6.0 was found to be optimal. in order to avoid product inhibition, the produced lactic acid was neutralized using calcium hydroxide. maximum production and productivity of lactic acid in batch system, were 81 g and 1.35 g/lh, but in fed batch system, after 3 feeds of glucose, production and productivity increased up to 360 g and 4 g/lh. saleh a. mohamed molecular biology dept., national research centre, cairo, egypt an extracellular polygalacturonase (pgii) from trichoderma harzianum was purified to homogeneity by two chromatography steps using deae-sepharose and sephacryl s-200. the molecular weight of t. harzianum pgii was 31,000 da by gel filtration and sds-page. pgii had isoelectric point of 4.5 and optimum ph of 5.0. pgii was very stable at the ph 5.0. the extent of hydrolysis of different pectins by enzyme was decreased with increasing of degree of esterification (de). pgii had very low activity toward nonpectic polysaccharides. the apparent km value and kcat value for hydrolyzing polygalacturonic acid (pga) were 3.4 mg/ml and 592 s −1 , respectively. pgii was found to have temperature optimum at 40 • c and was approximately stable up to 30 • c for 60 min of incubation. all the examined metal cations showed inhibitory effects on the enzyme activity. 1, 10 phenanthroline, tween 20, tween 80, triton x-100 and sds had no effect on the enzyme activity. the rate of enzyme catalyzed reduction of viscosity of solutions of pga or pectin was higher three times than the rate of release of reducing sugars indicating that the enzyme had an endo-action. the storage stability of the enzyme in liquid and powder forms was studied, where the activity of the powder form was stable up to one year. these properties of t. harzianum pgii with appreciable activity would be potentially novel source of enzyme for food processing. tarek m. mohamed, biochemistry division, chemistry department, tanta university, tanta, egypt preparation of peroxidase from horseradish, which could be used for commercial applications such as diagnostic kits, was occurred through a simple reproducible method consisting of extraction, ammonium sulphate precipitation, filtration through non-binding protein filter and lyophilization. the purification method was developed allow the preparation of 33 mg of enzyme from 1 kg of horseradish roots. the one mg of enzyme contains 900 units of peroxidase. this value is similar to that produced by sigma (50-1000 unit mg −1 powder). the final preparation is salt free reddish brown powder with free ammonia content less than 0.01 g −1 units. the rz value (a400/a280) of the enzyme, which is a good criterion of purity and heme content, was 2.6. the lyophilized enzyme was stable at −20 • c for at least one year. the liquid form of the enzyme in presence of 0.1% sodium azide was stable up to 25 days at 4 • c, while it lost most of activity at room temperature in the same period. the properties of horseradish peroxidase including km, optimal ph and temperature, activation energy, thermal stability and effect of different compounds were studied. the applicability of this enzyme in determination of serum glucose was performed. the analysis of glucose in human sera gave results using the kit containing the prepared peroxidase similar to those obtained with a commercial glucose kit. lactobionic acid production using lactose oxidase: from laboratory to 600 l scale mikkel nordkvist 1 , per munk nielsen 2 , peter budtz 3 , john villadsen 1 : 1 center for microbial biotechnology, technical university of denmark, dk-2800 lyngby, denmark; 2 novozymes a/s, dk-2880 bagsvaerd, denmark; 3 chr. hansen a/s, dk-2970 hørsholm, denmark. e-mail: mnq@biocentrum.dtu.dk (m. nordkvist) currently, lactobionic acid is mainly a high-price specialty product used, e.g. in solutions for organ stabilization. however, lactobionic acid can also be used as a biodegradable cobuilder in detergents, and it has several applications in food technology. with lower production costs it has the potential to become a bulk chemical. the kinetics for the oxidation of lactose to lactobionic acid by a new carbohydrate oxidase was studied in a 1 l bio-reactor with control of ph, temperature, and dissolved oxygen. the byproduct hydrogen peroxide has a negative influence on the lactose oxidase enzyme, and hence additional experiments were made with addition of catalase to remove hydrogen peroxide, thereby also providing extra oxygen. on the basis of the experiments in 1 l scale, experiments were performed in a 600 l reactor equipped with a new system for dispersion of air to supply the necessary oxygen for the oxidation. the aeration system in the large scale reactor was able to supply oxygen sufficiently fast to give the same production rate, at low values of the air flow rate and the energy input, as was obtained in the high-performance laboratory reactor. the non-characterized gene previously proposed as d-tagatose 3-epimerase from agrobacterium tumefaciens was cloned and expressed in escherichia coli. the expressed enzyme was purified by affinity chromatography on histrap hp, desalting chromatography on hiprep 16/60, and gel filtration chromatography on sephacryl s-300 hr with a final specific activity of 8.89 u/mg. using maldi-tof-ms, the native protein was estimated to have a molecular mass of 32,600 da and a monomeric structure. the purified enzyme exhibited maximal activity at 50 • c and ph 7.5 without the addition of metal ions and at 60 • c and ph 7.0 with 1.0 mm mn 2+ . among various metal ions, mn 2+ was the most effective divalent cation for d-fructose epimerization activity. the addition of mn 2+ significantly increased the thermal stability and the epimerization activity with other ketoses such as d-psicose, d-tagatose, d-ribulose, d-sorbose, and d-xylulose. the activity, substrate affinity, maximum velocity, and catalytic efficiency (k cat /k m ) of the enzyme for d-psicose were higher than those for d-tagatose, which suggests that the enzyme is not d-tagatose 3-epimerase but d-psicose 3-epimerase. the equilibrium ratio between d-psicose and d-fructose was 37:63 at 60 • c with 1.0 mm mn 2+ . when the enzyme was used at 14 u/ml, dpsicose was produced at 211 g/l from 700 g/l d-fructose containing 1 mm mn 2+ after 120 min, corresponding to a conversion yield of 30.2%. the role of ammonium ions in glucosamine formation during the citric acid fermentation process by aspergillus niger m. papagianni 1 , f. wayman 2 , m. mattey 2 : 1 department of hygiene and technology of food of animal origin, school of veterinary medicine, university of thessaloniki, thessaloniki 54006, greece; 2 department of bioscience, university of strathclyde, glasgow, g1 1xw, uk. e-mail: mp2000@vet.auth.gr (m. papagianni) stoichiometric modeling of the citric acid fermentation process by aspergillus niger, in 12-l stirred tank reactor, indicates that nh 4 + ions combine with a c-containing metabolite inside the cell to form a nitrogen compound which is then excreted by the mycelium. the close correlation between calculated and experimental profiles indicates that this metabolic process is rapid and takes place before the c-structure of the glucose has been greatly altered by glycolysis or the pentose phosphate pathway. hplc analysis identified glucosamine as the product of this relationship. a clear effect of medium concentration of nh 4 + on glucosamine formation was observed when fermentations carried out with optimal and sub-optimal ammonium concentrations. (nh 4 ) 2 so 4 addition when medium nitrogen was depleted, enhanced the formation of new cells from the tips of fragmented hyphae and led to glucosamine accumulation in amounts depending to pulse concentration. the fungus reacts in excess ammonium by converting it to glucosamine, to be utilized later when a regeneration process takes place with fragmentation of vacuolated hyphae and subsequent regrowth, depending always on the culturing conditions. however, depending on carbon and ammonium concentration in medium, glucosamine can be secreted in concentrations as high as 50 g/l. about 1000 microorganisms originated from traditional korean food origin were screened for efficient palatinose production. an isolate designated fmb1 was exceptionally efficient in sucrose-palatinose conversion activity. conversion of sucrose into palatinose by fmb1 was much faster than a reference strain of erwinia rhapontici. fmb1 is a gram negative, facultatively anaerobic, motile, noncapsulate, and straight rod-shaped bacterium producing acid from glucose. based on api and 16s rdna analyses, fmb1 was determined to be enterobacter sp. the maximum conversion of 10% sucrose to palatinose and trehalulose by enterobacter sp. fmb1 was achieved within 6 h. the preliminary dna sequencing result of the gene corresponding to sucrose isomerase of enterobacter sp. fmb1 revealed that it showed 87% similarity to that of klebsiella sp. (??). within the scope of an r&d project developed in collaboration with leather tanning portuguese industrial partners a screening of new proteases to be used in the industrial process was performed. a bacillus subtilis strain isolated from alkaline spent purge liquor was shown to be a promising protease producer. microorganism growth was studied for optimisation of temperature, agitation, ph and medium composition either for biomass or proteases production. optimal growth temperature is different for maximum biomass growth (40 • c) and optimal proteolytic activity (43 • c) yielding biomass specific growth rate of 1.6 and 1.4 h −1 , respectively. the achieved proteolytic activities were 5.7 and 7.2 u/ml of protease, respectively. optimised medium composition (7 g/l beef extract, 4 g/l yeast extract, 5 g/l peptone and 0.4 g/l cacl 2 ) yielded a specific growth rate of 1.5 h −1 and 13.9 ku/l of protease, in shake flask experiments. bioreactor experiments (from 1 to 16 l) with the selected medium were performed at 43 • c in order to test aeration rate (1 and 2 vvm), stirring (300-700 rpm) and ph (uncontrolled, controlled at 7 and 8). best protease activity was 64 u/ml in 2 l bioreactor without ph control at 500 rpm and 2 vvm. the proteolytic extract was characterized and compared to commercial bates. results indicate that these proteases can be employed in the purge phase of the leather tanning process in industry. the gram positive bacterium bacillus megaterium is known for its capacity to produce exoenzymes including amylases, proteinases, and penicillin amidase at industrial scale. here, we describe the development of various vectors for the production and export of recombinant heterologous proteins employing b. megaterium signal peptides. the target gene can be cloned directly adjacent to the signal peptide coding sequence (bart et al., 2005) . this arrangement allows for a correct n-terminal sequence of the mature protein after processing by the signal peptidase sipm. using this newly developed protein production and export system lactobacillus reuteri 121 levansucrase (van hijum et al., 2001) was secreted in significant amounts (∼4 mg/l) into the growth medium. fusion of the recombinant levansucrase to affinity tags allowed one-step purification of the recombinant protein from the growth medium. however, fused peptide tags resulted in a decreased secretion of the fusion protein. 1.4 mg his 6 -tagged levan-sucrase were purified per litre of culture. the system was further enhanced via coexpression of a gene for the signal peptidase sipm (malten et al., 2005a) and deletion of the gene for the extracellular protease nprm. developed new tools allow for various strategies of integrated high level production, export and purification of heterologous proteins in b. megaterium. methods for high-throughput screening of secreted enzymes are under development. the determined sequence of the b. megaterium genome, studies using high-cell density cultivations (malten et al., 2005b) and proteome data from batch fermentations implicate new targets for directed genetic optimization of b. megaterium production and secretion strains. novel strong and inducible promoters are currently under investigation. toru matsui 1 , naoya shinzato 1 , hisashi saeki 2 , hitoshi matsuda 2 : 1 center of molecular biosciences, university of the ryukyus, okinawa 903-0213, japan; 2 japan energy co., japan. e-mail: tmatsui@comb.u-ryukyu.ac.jp (t. matsui) optically active epoxides are considered as the potential intermediate for chiral drugs synthesis. although s-styrene oxide (so) have been extensively investigated using styrene monooxygenase from pseudomonas sp., microbial production of r-so with high enantiomeric excess was hardly examined. in this study, r-so producing bacteria from styrene was screened using various alkene assimilating bacteria. r-so with the highest ee (ca. 100%ee) was obtained using ethene utilizing bacteria, identified as mycobacteirum sp., while produced relatively lower at around 70%ee when using propene utilizing bacteria. the alkene monooxygenase gene homologue sequence amplified from the genomic dna revealed a significant similarity to that of etnabc. these bacteria also showed stereoselective degradation of racemic so, suggesting that the produced so might be further stereoselectively degraded to increase the ee. the ethene utilizing bacteira produced not only r-so but also s-epichrolhydrin at high ee.when using arylchloride as the substrate. this research was supported by nagase science and technology foundation. the secretion efficiency of the escherichia coli sec pathway is dependent on the growth phase but not on protein size f.j.m. mergulhão, g.a. monteiro centro de engenharia biológica e química, instituto superior técnico, av. rovisco pais, 1049-001 lisbon, portugal. e-mail: filipem@alfa.ist.utl.pt (f. mergulhão) the secretion efficiency of the escherichia coli sec pathway was evaluated through the expression of green fluorescent protein and human proinsulin fusion proteins. translocation to the periplasm is dependent on the growth phase of the bacterial culture and the highest secretion efficiency is attained in mid-exponential phase. secretion performance is independent of protein size (17-42 kda) and even when the amino acid composition of the secreted proteins is very similar, the amino acid distribution within the protein can affect translocation. in silico prediction analysis suggests that proteins that are prone to form ␣-helix structures are more efficiently translocated. culture medium composition plays an important role on secretion performance with the highest secretion results being obtained in minimal medium. streptokinase is a common fibrinolytic drug. that is used in thrombolytic therapy for long time. to compare with another thermbolytic drugs like tpa, it has lot of advantages. in this present research dna was extracted from s. equisimilis h46a for the first time in iran. streptokinase gene was amplified by using two forward primers and one reverse primer. a common restriction enzyme, bamh-i, was used for cloning. both ends of the pcr products (full length: 1323 bp and mature section: 1245 bp) and the restriction site on mcs of pqe-30 vector were digested. in this study, pqe-30 expression vector was used with high level expression ability for production of recombinant fusion streptokinase with simplifying the purification by employing affinity-metal chromatography method. in addition, the cloning results were controlled by double digestion and sequencing. takesono oxidation of short-chain iso-alkanes was studied with propanegrown resting mycelia of scedsporium sp. a-4. isobutane was oxidized to tert-butanol, but not to isobutanol. isobutanol was used for growth, but both isobutene and tert-butanol were not used for growth. isopentane was oxidized to 3-methyl-1-butanol, 2-methyl-2-butanol, and 3-methyl-2-butanol but not to 2-methyl-1-butanol. 2-methylpentane was oxidized to 4-methyl-1-pentanol, 2-methyl-2pentanol, and 4-methyl-2-pentanol but not to 2-methyl-1-pentanol or 2-methyl-3-pentanol. 3-methylpentane was not oxidized. oxidation of branched alcohols was also studied. application of nadph-dependent 2.5-diketo-gluconic acid reductase for production of l-ascorbic acid claudia pacher 1,2 : 1 division of food biotechnology, department of food sciences and technology, boku, university of natural resources and applied life sciences, muthgasse 18, a-1190 vienna, austria; 2 research centre applied biocatalysis, petersgasse 14, a-8010 graz, austria. e-mail: claudia.pacher@boku.ac.at ascorbic acid is an organic acid with various applications in the food and pharmaceutical industries. at present, the majority of commercially manufactured vitamin c is synthesized via the reichstein process, which is highly energy-consuming, involves considerable quantities of organic solvents and gives an overall yield of about 50%. therefore, during the past decades a lot of research was done to develop biotechnological alternatives for the synthesis of reichstein intermediates by enzymatic or fermentative means, which show some advantages regarding costs and environmental friendliness. one of the fermentation routes runs via 2.5-diketo-d-gluconic acid (2.5-kdg), produced by pectobacter (erwinia) cypripedii. this compound has been reduced by a nadph-dependent 2.5-diketogluconic acid reductase (dkr) from corynebacterium glutamicum to the key intermediate 2-keto-l-gulonic acid (2-klg) before chemical rearrangement leads to the final product. for economical reasons we wanted to express dkr heterologously. based on our long term experience with coenzyme regeneration we wanted also to perform the reaction in homogeneous solution. the spent coenzyme of nadph-dependent dkr has been regenerated by a second isolated nadp-dependent enzyme like glucose dehydrogenase. we describe here the recombinant production, purification and characterization of dkr and the results of enzymatic 2-klg formation by using the recombinant enzyme in a homogeneous system with conjugated coenzyme regeneration. grp78 residing in endoplasmic reticulum (er) functions as a molecular chaperon by associating transiently with incipient proteins as they traverse the er and aiding in their folding and transport. furthermore, the protein can also be induced under various stress condition such as glucose starvation, inhibition of protein glycosylation by tunicamycin, blockage of vesicular trafficking by brefeldin a and er-calcium-atpase pump inhibition by thapsigargin. thus, substances that directly down and up-regulate grp78 transcription are expected to be useful for treatment of cancer and alzheimer's disease, respectively. in the course of our screening program to obtain substances, which regulate grp78 expression, we first constructed an assay system monitored by the expression of a reporter gene. hela cells, which are transformed with luciferase gene under the control of grp78 promoter designated as hela 78c6 cells, respond sensitively to luciferase grp78 induction by er stress such as treatment of tunicamycin. by using this screening system, we isolated pyrisulfoxin as an up-regulator of grp78, and valinomycin, citreoviridin and alternariol as down-regulators. detailed studies on other biological activities were now under way. pdh is an enzyme that was described only several years ago in a number of ecologically related litter-decomposing fungi (agaricales, gasteromycetales). it catalyzes the c-3 and/or c-2 oxidation of several aldopyranoses to the respective keto sugar derivates. pdh shows a very broad substrate range, oxidizing almost all major sugar components of wood polysaccharides, and is implicated to play a role in lignocellulose degradation. agaricus bisporus, the white button mushroom, is an economically significant agricultural crop. the cultivation, which is done by solid-substrate fermentation on straw-and hay-based composted substrate, is sometimes seen as one of only few economically feasible methods for bioconversion of lignocellulosic agricultural waste material. deeper insight in the physiological role of pdh may provide help for mushroom growers to increase yield, improve quality or make new sources of raw materials utilizable. we amplified a fragment of the pdh gene with degenerated primers derived from internal peptide sequences. the screening of a genomic library led to the isolation of the pdh gene. subsequently we amplified a cdna clone by rt-pcr and investigated the transcriptional regulation by different carbon sources on a defined minimal medium. optimization of monoclonal antibody production processes with simulation and scheduling tools demetri petrides, charles siletti intelligen inc., scotch plains, nj 07076, usa. e-mail: dpetrides@intelligen.com (d. petrides) this presentation will review the state of the art in batch process simulation and scheduling tools and their applications in the design and debottlenecking of integrated biopharmaceutical processes. a systematic methodology will be presented for identifying and eliminating size, time, and throughput bottlenecks that limit production in single and multi-product facilities. the methodology will be illustrated with an industrial case study dealing with the optimization of a multi-product facility that produces therapeutic monoclonal antibodies (mabs). mab processes are characterized by a long bio-reaction time (e.g., 1.5-2 weeks for fed-batch operation and 1-2 months for perfusion operation). the cycle time for processing a lot in the recovery and purification train typically takes 3-4 days. consequently, one way of increasing throughput is by installing extra bioreactors that operate in staggered mode and utilize the same recovery train. the result is that multiple batches may be at different stages of completion at any given time. since cleaning equipment (e.g., cip skids) and buffer preparation and holding tanks are shared by multiple steps across many batches, this type of operation leads to time/scheduling bottlenecks that constrain the cycle time and the throughput of a process. the problem becomes more challenging in the context of multi-product facilities and when constraints imposed by the limited availability of resources are considered. our methodology and its computer implementation will illustrate how to systematically identify and eliminate such bottlenecks. the industrial case study will provide a real world example of the methodology. application of two stage continuous cultures of aspergillus niger for citric acid biosynthesis jerzy j. pietkiewicz, malgorzata janczar, wladyslaw lesniak food biotechnology department, university of economics, wroclaw 53-345, poland. e-mail: jerzy.pietkiewicz@ae.wroc.pl (j.j. pietkiewicz) the aim of the work was application test of submerged two stage single stream continuous cultures of aspergillus niger for citric acid production from sucrose. studies were carried out in lab fermenters with working volume of 5 dm 3 . the bioreactors were standard cstrs. in two stage continuous cultures (tscc) high mycelium growth and high citric acid production were observed in the first bioreactor. there was almost four times lower growth of biomass rate and about three times lower citric acid production rate in the second bioreactor. studies on influence of dilution rate in race from 0.0098 to 0.0230 dm 3 /(dm 3 h) on course and efficiency of tscc showed, that the highest citric acid yield (y p/s = 86.3%), high volumetric rate of its production (r pc = 0.958 g/(dm 3 h)) and the highest biosynthesis efficiency coefficient (k ef = 82.7) were obtained with dilution rate d = 0.0148 dm 3 /(dm 3 h). there was also high citric acid concentration (p = 129.5 g/dm 3 ) and low residual sugar concentration (s k = 6.9 g/dm 3 ) in the medium flowing out the second bioreactor in those cultures. the beta-galactosidases in commercial use are of different origins and yeast and fungal lactases present the greatest interest. the yeast lactases present neutral optima ph and are suitable for the hydrolysis of lactose in milk. in this work, the aim was to study the influence of aeration in the production of beta-galactosidase in batch fermentations with kluyveromyces marxianus atcc 46537. the medium composition for culture was as follows (in g/l): lactose pa 50, yeast extract 5, (nh 4 ) 2 so 4 4 and kh 2 po 4 2. the fermentations was carried out at 30 • c, ph 5.5, at 200 rpm starting with an initial cellular concentration of 1 × 10 7 cels/ml, with different aeration rates. the cells were disruped with chloroform 2% (v/v) as solvent. the enzymatic activity was determined as initial rate of lactose hydrolysis at defined conditions. the studies have revealed the importance of aeration on kluyveromyces marxianus in the growth and beta-galactosidase synthesis. the enzymatic activity of fermented medium with 0.5 vvm was 50% higher than one without aeration. furthermore, the cellular growth was faster in the aerobic fermentation than in the anaerobic one. the aeration has taken an important place in the enzymatic synthesis and in the cellular growth, however the results have shown that the aeration rate increase of 0.5-1.5 vvm has not implied a increase in the cellular growth neither in the enzymatic reached activity. the lactose presents in the milk has a solubility of only 20% at 30 • c, and a high percentage of the world population presents intolerance to this sugar, due to the low or absence of the activity of the lactase enzyme in the organism. to minimize such problems, the most viable alternative for nourishing dairy products is the enzymatic hydrolysis of milk, although it is an expensive process due to the high cost of the beta-galactosidase enzyme. an alternative that has been greatly studied is the immobilization of this enzyme, originated from many different sources. there are several immobilization procedures for this enzyme, however, a procedure considered ideal was not obtained yet. the objective of this work was to study the immobilization process of beta-galactosidase from aspegillus oryzae in sodium alginate with commercial gelatin. in the immobilization process was studied the glutaraldehyde influence for 1, 3 and 5%, in the presence of commercial gelatin at the concentration of 2% at the immobilization medium. the activities of the immobilized enzymes were obtained in a stirred micro-reactor, at the temperature 30 • c, ph 4.5 with a 50 gl −1 lactose solution in acetate buffer. the experimental results showed that the immobilized biocatalyst that presented the larger initial activity was the one obtained at the immobiliza-tion medium that contained 5% of glutaraldehyde. after 20 daily determinations of enzymatic activities, a fall of 30, 40 and 24% was verified in the enzymatic activities for the immobilized biocatalysts using glutaraldehyde at 1, 3 and 5%, respectively, however, in all cases, the enzymatic activity reached the half of their initial activity after 10 determinations. hydrolysis of sucrose by immobilized beta-fructofuranosidase in silica eloízio júlio ribeiro, ubirajara coutinho filho faculdade de engenharia química, universidade federal de uberlândia, uberlândia 38400-902, brazil. e-mail: ejribeiro@ufu.br (e.j. invertase, known as beta-fructofuranosidase (ec 3.2.1.26), plays a catalytic role in the conversion of sucrose into glucose and fructose. it is largely used in the food industry to prevent the crystallization of sucrose in sugar mixtures and can be used in enzyme reactors for hydrolysis of sucrose. the objective of this work was to study the kinetic of sucrose hydrolysis by immobilized betafructofuranosidase in a continuous recirculation reactor, evaluated the enzyme stability and determine the effective half-life of immobilized enzyme. invertase was covalently immobilized on sillanized controlled pore silica. nonlinear fitting were used to determine the kinetic parameters for substrate and product inhibition observed in the enzymatic hydrolysis of sucrose. the kinetics studies of immobilized invertase were carried out in a continuous recirculating reactor. the half-time of enzyme inactivation (t 1/2 ) was calculated from the initial rates of the remaining enzyme activity. the model of inhibition by substrate and product adequately represented the enzymatic hydrolysis. the fructose effect was competitive inhibition (k f = 3.1022.10 −4 mol/ml) and the glucose effect was noncompetitive inhibition (k g = 2.2521.10 −4 mol/ml). the effective diffusivity of sucrose into the support was shown to be the same as for sucrose in dilute solution (0.75 × 10 −5 cm 2 /s at 40 • c). the half-time of enzyme inactivation (t 1/2 ) was 1656 h. controlled pore silica showed to be an excellent immobilizing support. the immobilized invertase was very stable at temperatures lower than 50 • c. the intrinsic parameters (k i , k f , k g and v m ) were shown to be similar to the apparent values. the low permeability of mycobacterial cell wall envelopes is a result of the unique composition and organization of the cell wall lipids. the permeability of mycobacterial cell wall can be changed by means of partial disintegration of its compounds. the aim of present work was to characterize the changes in the cell wall skeleton (cws) and non covalently bound free lipids under the influence of isoniazid, the inhibitor of mycolic acids biosynthesis. fatty acid (fames) and mycolic acid methyl esters (mames) obtained from all tested preparations were analyzed by gc/ms analysis. the analysis of free lipids and cws revealed distinct changes in the composition of the frac-tions obtained from the cells exposed to action of the isoniazid. the changes in the quantity of fatty acids in the inh-treated cells indicates that inh interferes with the synthesis of lipidic compounds of the mycobacterial cell wall also. the decreased amount of covalently bound mycolic acids in the cws is responsible for the enhanced penetration of hydrophobic compounds through the cell wall. this work was supported by grant nr 3p04c 06923 of the committee for scientific research. barbara sencio, teresa jamroz, stanislaw ledakowicz department of bioprocess engineering, technical university, lodz, poland the enzyme laccase (ec.1.10.3.2. p-diphenol oxidase) is a subject of research in many centres dealing with improvement of bioprocesses with the use of different white rot fungi species. most strains that produce this enzyme in vitro require inductors initiating its biosynthesis. when cerrena unicolor was applied, it was found that the strain produced laccase very efficiently without additional toxic compounds. to specify optimum conditions for laccase production in a submerged culture, research was undertaken to obtain the most efficient inoculum c. unicolor. the goal of this research was to determine the effect of form and incubation time of inoculum on enzymatic activity of the laccase producing strain. the experimental inoculum was the mycelium prepared on a solid and liquid substrate. basing on results obtained, it was found that the laccase yield was the highest in the cultures where the mycelium was grown on a solid substrate. maximum activity of the c. unicolor strain was achieved on the 16th day of culture, and the amount of laccase produced was higher by, ca. 30% as compared to the mycelium obtained from the liquid substrate. results of these experiments were used to continue studies on the impact of inoculum age. experiments were carried out using an inoculum incubated for 1-3 weeks at the temperature 30 • c in a certomat bs1 shaker at 110 rpm. the best results in the c. unicolor strain culture were achieved using a 7-day-old inoculum. effect of alcohol treatment on hydrolytic activity of candida rugosa lipase serpil takaç, a. ezgiünlü department of chemical engineering, institute of biotechnology, ankara university, 06100 tandogan, ankara, turkey candida rugosa lipase (crl) was treated with 20, 40, and 60% concentrations of methanol (m), ethanol (e), 2-propanol (2p) and 1-butanol (1b) to investigate the changes in its hydrolytic activity toward p-nitrophenylacetate. the treatment included the following steps at +4 • c: (i) stirring crl with phosphate buffer for 24 h; (ii) treating the solutions with alcohols; (iii) stirring treated-crl for 24 h; (iv) centrifugation at 10,000 rpm for 30 min; (iv) dialysis the supernatant against bidistilled water for 39 h. the activity of crls was followed for 120 h at 37 • c in the presence and absence of isooctane. the enzyme activity was measured spectrophotometrically and the protein concentration was measured by lowry's method. it was found that the recovered protein did not change considerably with the type of alcohol; however, decreased with alcohol concentration. in the presence of isooctane, specific activities of the untreated and treated-crls increased compared with those obtained in the absence of isooctane. 1b-crls and e-crls showed higher activities than m-crls and 2p-crls whereas untreated-crl exhibited higher activity than m-crls, e-crls and 2p-crls. the highest and the lowest activities were obtained with 20% 1b-crl and 60% 2p-crl, respectively. the changes occur in the structure of crl after treatments were investigated by electrophoretic analysis. this study was supported by ankara university biotechnology institute (project no: 89) . different genera, species and strains of microorganisms were found to posses different cryoresistance. optimal ways for cryopreservation of microorganisms-producers of antibiotics, microorganisms, used in food industry, agriculture and veterinary have been developed. it was demonstrated, that non-lethal damages could occur in cryopreserved microorganisms after their returning to physiological culture conditions, which were manifested in streptomyces' hypha fragmentation, that of cyanobacteria's, streptococci's chains as a result there was an increase in a number of colony-forming units, a reversible inhibition of microorganisms' proliferative activity in bacillus thuringiensis and lactic streptococci, stimulation of the enzyme processes and antibiotic production. non-lethal damages are repaired during microorganism culturing in the first passage. the cause of non-lethal damages is a reversible inhibition of biosyntheses of protein and nucleic acids respiratory activity. the repairing of non-lethal damages is accompanied by the production of stressproteins, different from heat shock proteins. effect of ph in the 2-propanol treatment of candida rugosa lipase on its enantioselectivity in the hydrolysis of racemic naproxen methyl ester serpil takaç, a. ezgiünlü department of chemical engineering, ankara university, 06100 tandogan, ankara, turkey candida rugosa lipase (crl) was treated with 2-propanol (2p) at the ph values of 1.5, 4, 6, 7.5, 9 and 12 to investigate the changes in its enantioselectivity in the hydrolysis of racemic naproxen methyl ester. the treatment included the following steps at +4 • c: (i) stirring crl with different buffer solutions to maintain the desired ph values for 24 h; (ii) treating the solutions with 40% 2p; (iii) stirring treated-crls for 24 h; (iv) centrifugation at 10,000 rpm for 30 min; (iv) dialysis the supernatant against bidistilled water for 39 h. hydrolyses of racemic naproxen methyl ester to form s-naproxen were performed in shaking flasks at 200 rpm and 37 • c for 192 h in isooctane-phosphate buffer solution biphasic system using treated-crls with the activity of 7.75 u. the concentrations of the enantiomers of naproxen methyl ester and naproxen were determined with hplc. it was found that the treatment ph has an important role on the enantioselectivity and conversion. the highest enantiomeric excess for the substrate, for the product, enantiomeric ratio, and con-version were obtained with crl treated at ph 1.5 as 39, 98, 181 and 29%, respectively. these values were followed with 2p treated crl at ph 12 as 35, 98, 121 and 27%. however, lower enantiomeric excesses, conversions and enantiomeric ratios were obtained at the treatment ph values between 1.5 and 12. the effects of fatty acids, nitrogen (as nh 4 no 3 ), phosphorus (as kh 2 po 4 ), ph value, manganese (mn 2+ ), iron (fe 2+ ) and methanol concentration on growth and production of oxalic acid from post refining fatty acids by a mutant of aspergillus niger xp in submerged fermentation experiments was studied. of the a. niger strains screened, a. niger xp was identified as the best oxalate producer on lipids. the influence of the ph on oxalic acid formation shows that the maximum production rate and higher concentration of product are observed at the ph ranging from 4 to 5. with a medium containing 50 g fatty acids/l, the production reached a maximum of 68 g oxalic acid/l after 7 days. the addition of 1.5% (w/v) methanol to seed culture increased the product yield and concentration of oxalic acid but decreased the amount of an undesired by-product (citric acid). under this condition, the maximum oxalate productivity (14-18 g/l days) was maintained for 2-4 days of fermentation. other results of the experiments show that supplementation of the production medium with manganese and iron enhances oxalate production. fatty acids proved to be a very good substrate for oxalic acid production by a. niger xp giving excellent yields and productivity at low ph. the results are very promising as they may lead to cheap alternative processes for oxalic acid production from renewable lipid resources. department of food engineering, middle east technical university, ankara 06531, turkey. e-mail: banuy@metu.edu.tr (b. yalcindag) laccase (e.c. 1.10.3.2, p-benzenediol:oxygen oxidoreductase), which is an enzyme belonging to the multi-copper oxidase family, catalyzes the oxidation of a broad variety of polyphenols with a preference for p-isomers, which are converted to p-quinones. fungi generally contain several laccases which have been found to be involved in delignification, melanin synthesis and pathogenesis. laccase has also important potential application areas especially in food and chemical industries. after aspergillus fumigatus genome data were released, research on functional analysis of laccases has been initiated in our laboratory. laccase genes of aspergillus nidulans, ya and tila, and laccase and multi-copper oxidase genes of aspergillus fumigatus, abr2 and abr1, were used to analyze a. fumigatus genome for laccases. this sequence analysis resulted in 4 probable laccase genes, one of which was the previously cloned abr2 gene. in this study, one of these genes (aflac1) was further characterized. after sequence alignment and characterization studies, aflac1 was predicted to have 2128 bp having six introns, which makes the protein 606 amino acid long, and the predicted protein sequence showed 63% homology with the dihydrogeodin oxidase of aspergillus terreus and 38% homology to the laccase 2 of botryotinia fuckeliana. aflac1 gene is found within an uncharacterized gene cluster containing genes with homology to glutathione-s-transferase, polyketide synthase, o-methyl transferase, and others. the information obtained from sequence analysis was employed in designing pcr-primers to amplify the aflac1 gene, followed by cloning onto pan52-1 and pan52-4 vectors for heterelogous expression in aspergillus sojae. in addition, by the use of rt-pcr, aflac1 cdna will be cloned and expressed in escherichia coli. furthermore, gene silencing studies will be performed to enlighten the function of aflac1 and associated gene cluster. stability of growth rate of photosynthetic cells is an important factor in designing of effective photobioreactors especially in long term operations. in our experiments, in order to keep operational parameters almost constant, a semi-continuous culture method was developed. in this method, a part of culture broth containing grown cells was repeatedly replaced by fresh medium at a predetermined time interval. the replacement of broth with fresh media could keep the cell concentration, volume of broth and distribution of light intensity constant at initial values throughout the cultivation. it was shown that in one side illumination with a halogen lamp, if the ratio of the light intensity at the front side of a flat plate photobioreactor to that at the rear side was kept lower than 4, the growth rates was sustained in constant levels. however, at higher ratios the growth was followed by rapid decrease after 5-6 h. supplemental illumination with a fluorescent lamp from the rear side of the flat plate photobioreactor could sustain almost stable growth rate. beside of the illumination conditions, increased ferrous ion concentrations in medium could keep the stability of growth rate even in unstable illumination conditions, while consumed ferrous ion was slight. glutathione (gsh) plays a pivotal role in protecting cells from by-products generated by oxidative metabolism. these characteristics make this active tripeptide an important drug for the treatment of liver diseases and is of interest in the food additive industry, therapeutics and sport nutrition. in the first part of the research, a screening was carried out among 48 yeast strains, to find out those able to accumulate higher gsh intracellular levels. two saccharomyces cerevisiae strains proved to be the best gsh producers (1.3%dw), in every samples the presence of s-adenosyl-methionine in traces (0.2% dw) was also evidenced. s-adenosyl-methionine (sam) plays a role in the immune system, maintains cell membranes, participates in detoxification reactions and in the manufacture of brain chemicals and cartilage. the second part of the research was aimed at increasing, in a post fermentative procedure, gsh levels present inside the cells at the end of the growth phase. moreover, time course of sam intracellular levels, to be related with accumulated gsh, was also monitored. cells were then suspended in an appropriate solution containing mineral salts, glucose and the aminoacids, precursors of the two studied molecules. according to this procedure, gsh intracellular levels reached 4.6% dw after 48 h incubation. moreover, gsh levels can be related to sam production (up to 1.3% dw). the presence, in several samples, of intermediate metabolites, such as cystathionine and omocysteine, proved the establishement of an intracellular equilibrium between gsh and sam; this behaviour represents a promising starting point for the set-up of a microbial process for the simultaneous production of the two studied molecules. three acetate mutants of y. lipolytica yeasts, which varied in colony morphology (rough and smooth), were employed for continuous citric acid production from glucose and fructose syrup in a membrane reactor with cell recycle. the strains were compared for their product yields, specific acid production rates and ratios of citric acid to isocitric acid. experiments shoved that glucose syrup was a better substrate for citric acid production by y. lipolytica. citric acid concentration in the effluent ranged from 80 to 120 g/l, depending on the yeast strain used. all y. lipolytica strains produced very low amounts of isocitric acid. its concentration did not exceed 3 g/l. based on the results of these experiments, smooth strain awg-7 was found to be the most suitable for citrate production both from glucose and fructose syrup during long time continuous processes (500 h). in the steady state, the highest citrate productivity (1.3 g/lh) was obtained with this strain, when the feed medium contained 200 g/l of glucose and dilution rate (d) was d = 0.013 1/h. supplementation of the feed medium with bacto-pepton improved the productivity, citric acid yield and stability of the continuous process in the cell recycle fermentation system. for textile dyeing with natural dyes, indigo has an almost unique position as the most blue natural dye. due to the importance of indigo, considerable research has been conducted to replace the chemical synthesis of the dye by an application of biotechnological methods. therefore, we investigated several characteristics of natural indigo derived from polygonum tinctorium and its dyeing properties using silk fabrics, such as washing, perspiration, and light fastnesses. this work was financially supported by program for cultivating graduate students in regional strategic industry from korea industrial technology foundation. glycine oxidase is the product of the yjbr gene of bacillus subtilis that was predicted by sequence homology to be a flavoprotein similar to sarcosine oxidase. glycine oxidase catalyzes the oxidative deamination of various primary and secondary amino acids (e.g. sarcosine, n-ethylglycine, and glycine) and d-amino acids to form the corresponding ␣-keto acids and hydrogen peroxide. previous investigations reported on the cloning and production of the glycine oxidase gene in escherichia coli was up to 1 u/g cell. the present works has improved the expression of the recombinant his-tagged glycine oxidase by 15-fold by using pet28a and rosetta cells under the optimal iptg, temperature and time of induction. the protein obtained represented 30% of total soluble proteins in crude extract. the enzyme was purified to near homogeneity using imac with a 95% recovery and with and specific activity of 1.21 u/mg protein. the enzyme was active towards glycine, sarcosine and different d-amino acids, having in general, a basic ph optimum. the kinetic parameters were also studied, showing a km range from 0.3 to 300 mm. the enzyme was immobilized, and used to obtain pyruvic acid (␣-keto acid) from d-alanine with a good yield. the enzymatic synthesis of lipophilic derivatives of various natural antioxidants including flavonoid glycosides, as well as derivatives of cinnamic acid, was performed using various immobilized lipases in ionic liquids such as 1-butyl-3-methylimidazolium tetrafluoroborate (bmim-bf 4 ) and 1-butyl-3-methylimidazolium hexafluorophosphate (bmim-pf 6 ). the influence of various reaction parameters on the catalytic behavior and the selectivity of lipases was pointed out. a response surface methodology was applied for the optimization of the yield and the productivity of the biocatalytic process. the antioxidant activity of the biocatalytically prepared lipophilic derivatives of natural antioxidants, as expressed on cu 2+ -induced oxidation of low-density lipoprotein (ldl) and total serum, was investigated. process strategy for reduction of proteolysis in pichia pastoris fermentations jan weegar 1 , john dahlbacka 1 , noora sirén 2 , niklas von weymarn 2 , kaj fagervik 1 : 1 faculty of chemical engineering,åbo akademi university, finland; 2 laboratory of bioprocess engineering, helsinki university of technology, finland. e-mail: jan.weegar@abo.fi (j. weegar) the yeast pichia pastoris is a popular host organism for production of recombinant proteins. it is, however, common that the products are degraded by proteases towards the end of the fermentation, resulting in productivity and purity decreases. proteolysis of recombinant proteins in p. pastoris fermentations is affected by the temperature and ph of the growth medium. in this study, it was shown that decreasing the temperature from 30 to 10 • c during the induction phase effectively prohibited proteolysis. on the other hand, the temperature decrease resulted in a reduced maximal methanol consumption rate, which subsequently resulted in a culture highly sensitive to residual methanol. the temperature was slowly decreased according to a predetermined trajectory. as the temperature reached values below 12 • c, the methanol concentration had to be closely monitored and the substrate feed rate adjusted in order to prohibit methanol poisoning as well as to maintain the culture as a substrate limited fed-batch. measurement of protease concentrations revealed that proteases were present at 10 • c, but at this temperature the proteolysis rate was evidently effectively reduced. the recombinant protein produced could almost totally be recovered (i.e. high purity) with this process strategy compared to a constant high temperature culture (30 • c) where severe breakdown of the product was observed. with the growing of an ecological conscience in the public opinion, more and more industrial processes are analyzed for a possible ecologically beneficial alternative. for the production of ascorbic acid, which is nowadays done by the reichstein process, a lot of research was done to develop biotechnological alternatives for the synthesis of reichstein intermediates by enzymatic means, which show some advantages regarding costs and environmentalfriendliness. besides of two-stage fermentation, our approach is to design a tailor-made organism that produces 2-keto-l-gulonic acid, which is the direct precursor of ascorbic acid from glucose or gluconic acid and which can easily be converted to the final product by conventional methods. erwinia (pectobacter) cypripedii, which is a natural producer of 2,5-diketo-d-gluconic acid, was selected as a suitable host for a 2,5-diketo-d-gluconate reductase from corynebacterium glutamicum. to increase the yield of the desired compound we investigate two 2-keto-reductases in the host organism that diminish the yield of 2-keto-l-gulonate by reducing the compound to l-idonic acid, or by metabolisation of intermediates. these two enzymes were investigated with vari-ous biochemical and molecular biological methods, which will be presented. overproduction of bioinsecticides by heat and salt stress and control of dissolved oxygen in cheap media of bacillus thuringiensis nabil zouari, dhouha ghribi, samir jaoua laboratoire des biopesticides, centre of biotechnology of sfax, tunisia, bp:k, 3038 sfax, tunisia bioinsecticides based on preparations of spores and insecticidal crystal-proteins (icps) produced by the bacterium bacillus thuringiensis (bt) proved to be a high tool for fighting some agricultural pests and vectors of diseases. however, the use of bt preparations as commercial insecticides would be prohibitively expensive because it is not easy to reach cheap overproduction of icps during large-scale fermentation. here, we report possibilities to improve delta-endotoxins production as a consequence of responses of bt strains to low levels of heat and salt stress. each stressor results differently in the improvement of delta-endotoxins production, but both were shown to be most efficient at the beginning or the midexponential phase of the cultures which become resistant at the stationary or the sporulation steps. heat stress caused increase of 84% of synthesis yields of the sporulating cells, in contrast, salt caused increase of 25% of spores counts, corresponding to 28% toxins production improvement. combined effects of both stressors lead to toxins production improvement of 66%, yield improvement of 40%. we focused on the overcome of carbon repression catabolite, closely related to oxidative metabolism, by an adequate control of dissolved oxygen in the cheap media we formulated for bt insecticides production. we showed that an equilibrium between the high density of vegetative cells and their ability to synthesize toxins during their sporulation was necessary to take into account. 40% increase of icps production was reached into 3 l fermenter combination of mutagenesis, heat and salt stress and oxidative metabolism control allowed more than 100% improvement of delta-endotoxins. these results are of great importance in practical point of view, since high bioinsecticides concentrations could be produced without decrease of the yields of their production. mechanism and function of the intramembrane-cleaving protease rhomboid marius lemberg 1 , javier menendez 2 , christopher koth 2 , matthew freeman 1 : 1 mrc laboratory of molecular biology, cambridge, uk; 2 ontario center for structural proteomics, toronto, canada rhomboids are a family of intramembrane serine proteases that are widely conserved throughout evolution. among diverse functions discovered so far, rhomboids participate in intercellular signalling, parasite invasion, membrane dynamics and bacterial quorum sensing, making them potentially valuable therapeutic targets. the identification of physiological substrates and of selective inhibitors will be key towards their evaluation as drug targets. we have developed an in vitro cleavage assay to monitor rhomboid activity in the detergent solubilised state, enabling the first isolation of a highly pure rhomboid with catalytic activity. analysis of purified mutant proteins suggests that rhomboids use a serine protease catalytic dyad instead of the previously proposed triad, and gives insights into subsidiary functions like ligand binding and water supply. oligosaccharides and 3-keto-glycosides. availability of the enzyme is, however, hampered by the very slow growth and low production rates of the fungus. cloning of the encoding gene and production of the protein by heterologous expression are therefore a prerequisite not only for any biotechnological application, but further scientific investigations as well. on the basis of peptide sequences degenerated primers were designed, and the resulting pcr fragment was used as a probe to isolate the gene from a genomic library. two very similar genes encoding previously uncharacterized proteins were also found, and flanking regions were amplified using rage-pcr. furthermore cdna clones of all genes were isolated by rt-pcr. for textile dyeing with natural dyes, indigo has an almost unique position as the most blue natural dye. due to the importance of indigo, considerable research has been conducted to replace the chemical synthesis of the dye by an application of biotechnological methods. therefore, we investigated several characteristics of natural indigo derived from polygonum tinctorium and its dyeing properties using silk fabrics, such as washing, perspiration, and light fastnesses. this work was financially supported by program for cultivating graduate students in regional strategic industry from korea industrial technology foundation. the behavior of phytate degrading enzymes isolated from malaysian zea mays root in rice bran media anis shobirin meor hussin 1 , abd-elaziem farouk 1 , hamzah mohd salleh 1 , ralf greiner 2 : 1 biomolecular engineering research group, department of biotechnology engineering, kulliyyah of engineering, international islamic university malaysia, jalan gombak, 53100 kuala lumpur, malaysia; 2 centre for molecular biology federal research centre for nutrition and foods, haid-und-neu-straße 9, d-76131 karlsruhe, germany phytate degrading enzymes catalyze the step-wise release of phosphate from phytate, the principle storage form of phosphorus in plant seeds and pollen. they are widespread in nature, occurring in plants and microorganisms, as well as in some animal tissues. phytate-degrading enzymes have been studied intensively in recent years because of the great interest in such enzymes for phytate degradation and their application for animal feed and human health. from over isolate 140 screened isolates of phytate degrading enzymes, three isolates from the root malaysian maize plantation have shown phytate degrading activity. the production of phytate degrading enzyme was studied using different concentrations [%, w/v] of rice bran media during different stages of cultivation. the dephosphorylation of phosphate from rice bran phytate has shown regulatory effect on the secretion of bacterial phytases. in this conference, we will present data for the characterization of the enzymes. in this paper we present the properties of a phytase purified from a bacterium isolated from malaysian wastewater, which might find application as an animal feed supplement. the phytase described herein is a periplasmic enzyme. the phytase was purified about 180fold to apparent homogeneity using ion-exchange chromatography and gel-filtration with a recovery of 10% referred to the phytatedegrading activity in the crude extract. the enzyme exhibits an activity of about 1106 u mg −1 . gel filtration of the native enzyme on a calibrated sephacryl s-200 column gave a molecular mass of the phytase of 42,000 ± 1500 da with elution position being measured by determination of enzyme activity. lower molecular mass species or higher molecular mass aggregates were not observed. the phytase appeared homogeneous by polyacrylamide gel electrophoresis under non-denaturing conditions at ph 8.3 and 4.8 and gave a single protein band upon sds gel electrophoresis after coomassie staining of the gels. these results indicate that the phytase could be regarded as homogeneous. the estimated molecular mass after sds-page indicated that the phytase having a molecular mass of 41,500 ± 2500 da. consequently, this enzyme is a monomeric protein. the purified enzyme had a single ph optimum at ph 4.5 and was virtually inactive above ph 7.0. at ph 3.0, 40% and at ph 2.5, 20% of the activity at optimal ph was observed. the effect on enzyme stability was studied in the ph range 1.0-9.0 at 4 • c. within 14 days the phytase did not lose any activity in the ph range from 3.0 to 8.0, but at ph values below 2.0 a rapid decline in activity was observed. at ph 1.5, 72% and at ph 9.0, 65% of the initial activity was lost during 24 h. in the range of temperatures studied, 10-80 • c, the optimum temperature for the enzyme was found to be 65 • c. the apparent activation energy was estimated at ph 4.5 from the slope of log v max versus 1/t. the data showed excellent linearity from 15 to 65 • c. the arrhenius activation energy for the hydrolysis of phytate was calculated to be 37.5 kj/mol. in order to check thermal stability, the purified enzyme was incubated at different temperatures, cooled to 4 • c and assayed using the standard phytase assay. the enzyme lost no activity in 10 min at temperatures up to 65 • c. when exposed for 10 min at 70 • c, it retained 50% and at 80 • c 12% of the initial activity. in order to determine the substrate selectivity of the purified phytase, several phosphorylated compounds in addition to phytate, were used for k m and v max estimation by detecting the release of the phosphate ion during hydrolysis using formation of a soluble phospho-molybdate complex in an acidic water-acetone mixture. only phytate was identified as a substrate. the kinetic parameters for the hydrolysis of phytate were determined to be k m = 0.15 mmol l −1 and k cat = 1164 s −1 at ph 4.5 and 37 • c. like other bacterial phytatedegrading enzymes, the purified enzyme showed substrate inhibition. the activity of the purified enzyme was inhibited at substrate concentrations >7 mm. the study of the effect of metal ions on enzyme activity showed that none of them had an activating effect when used at a concentration between 10 −4 and 10 −3 m. mg 2+ , ca 2+ , mn 2+ , co 2+ , ag + , hg 2+ , and cu 2+ had little or no effect on enzyme activity, while zn 2+ , fe 2+ , and fe 3+ showed strong inhibitory effects. the reduced phytate-degrading activity in the presence of fe 2+ and fe 3+ is attributed to a lower phytate concentration in the enzyme assay because of the appearance of a fe-phytate precipitate. when compounds which tend to chelate metal ions, such as o-phenanthroline, edta, oxalate, citrate or tartrate, were tested for their effect on enzyme activity, it was noted that none of them was inhibitory at a concentration from 10 −4 to 10 −3 m. fluoride, a known inhibitor of different phytate-degrading enzymes from bacteria and the hydrolysis product phosphate as well as its structural analogs molybdate, wolframate and vanadate were found to be strong inhibitors of the purified enzyme. flouride inhibited the hydrolysis of phytate with a k i value of 112 mol l −1 . several fusion strategies have been developed for the expression and purification of small antimicrobial peptides (amps) in recombinant bacterial expression systems. in the present work, we investigated the use of the baculoviral polyhedrin (polh) protein as a novel fusion partner for production of a model amp (halocidin 18 subunit; hal18) in escherichia coli. the recombinant hal18 amp could then be hydroxylamine cleaved from the fusion protein and easily recovered by simple dialysis and centrifugation. this was facilitated by the fact that polh was soluble in the alkaline cleavage reaction but became insoluble during dialysis at a neutral ph. importantly, recombinant and synthetic hal18 peptides showed nearly identical antimicrobial activities against e. coli and staphylococcus aureus, which were used as representative gram-negative and -positive bacteria, respectively. these results demonstrated that baculoviral polh can provide an efficient and facile platform for production or functional study of target amps. extensive industrial and food additive applications of succinate have attracted much effort towards finding an environment-friendly alternative to the petrochemical production processes. it is very attractive to engineer s. cerevisiae for succinate production because of its generally regarded as safe (gras) status, ease of genetic manipulation and fermentation. we approached this metabolic engineering problem with a two-step methodology combining modern computational as well as molecular biology tools. in the first step we identified potential metabolic engineering targets leading to high succinate yield and productivity, with the aid of genome scale metabolic model and a bi-level optimization framework using flux balance analysis and quadratic programming. in the next step, various deletion mutants are being constructed and characterized for physiology and succinate production. results from these experiments then will be used to improve the predictions in computational models. so far, we have constructed a saccharomyces cerevisiae mutant deleted in sdh3, which encodes a major subunit of sdh-complex converting succinate to fumarate in mitochondria. the physiology of sdh3 mutant has been characterized in aerobic and anaerobic batch cultivations and in glucose limited chemostat at dilution rate as low as 0.027 h −1 . in aerobic batch fermentations, the mutant showed reduced maximum specific growth rate as compared to the wild-type, and it was incapable of growing on ethanol as sole carbon source, as predicted from the model. interestingly, the mutant showed much higher specific growth rate in anaerobic conditions, close to the wildtype strain. moreover, the chemostat cultivations indicate that the critical dilution rate of the mutant is below 0.027 h −1 . this opens further opportunities to investigate interesting behavior of the mutant and the underlying regulatory processes to improve our understanding of yeast mitochondrial metabolism. department of chemical engineering, yıldız technical university, davutpaşa campus, 34210 esenler/istanbul, turkey. e-mail: dkilic@yildiz.edu.tr (d.k. apar) lactose is the dominant carbohydrate in milks which are, in turn, the only significant natural sources of lactose. a large number of people do not digest lactose properly due to a lack or inactivity of the intestinal beta-galactosidase and they suffer from intestinal dysfunctions -gas, abdominal pain and diarrhea -if their diet contains lactose. moreover, lactose is a sugar with a high bod, low sweetness, low solubility, when compared to the products of its hydrolysis (glucose and galactose) and being a hygroscopic sugar has a strong tendency to adsorb flavours and odours. the hydrolysis of this sugar is very attractive towards the improvement of processes for the production of ice cream and other refrigerated dairy products and it could be very interesting for the development of additives for animal and human alimentation. the enzymatic hydrolysis of lactose is carried out by beta-galactosidases, enzymes that are widely distributed in nature, appearing in micro-organisms, plants and animal tissues. the present investigation describes the effects of the sonication process parameters on enzymatic hydrolysis of milk lactose and enzyme stability. bandelin sonopuls sonicator was used for the lactose hydrolysis experiments. ␤-galactosidase enzyme used is produced from kluyveromyces marxianus. the reactions were carried out in 250 ml of milk. the process variables for the sonicator are duty cycle, acoustic power and enzyme concentration. the amount of residual lactose concentration (g/l) and residual enzyme activity (%) against time were investigated versus process variables. beside of this; the mathematical models depending on the operating conditions were also derived by using the experimental data of lactose concentration and enzyme activity. kinözbek department of chemical engineering, yıldız technical university, davutpaşa campus, 34210 esenler/istanbul, turkey. e-mail: dkilic@yildiz.edu.tr (d.k. apar) over the last decade, the use of plant protein hydrolysates alternative to animal protein hydrolysates in human nutrition has broadly expanded. protein hydrolysates are often used in different nutritional formulations, such as supplementation of drinks to enhance their nutritional and functional properties, or special medical diets. there are many processes which employ protein hydrolysis and hydrolytic products. among these processes, the use of enzymes allows selective hydrolysis of protein and produces a potentially safer and more defined material. in the present study, the effect of the temperature, ph and viscosity on the hydrolysis of corn gluten was investigated using a stirred batch reactor system. the corn gluten was hydrolysed by using neutrase enzyme, a bacterial protease produced by a selected strain of bacillus amyloliquefacien. the reactions were carried out in 0.2 l of aqueous solutions containing 1% (w/v) corn gluten and 0.4% (v/v) enzyme. the degree of hydrolysis (%) and soluble protein concentration depending on the time were investigated at the temperatures 40, 45, 50, 55, and 60 • c; and at the ph values 6.5, 7, 7.5, and 8. to investigate the effect of viscosity, the various amounts of glycerol was added to the reaction solutions to produce viscosities in the range of 1.415-13.43 cp. the degree of hydrolysis (dh) was computed by using ph-stat method. for the soluble protein determination in the hydrolysates samples, the folin-lowry method (1951) was used. polyhydroxyalkanoates (pha), one of the most promising bioplastics for the partial replacement of synthetic polymers like polypropylene, are polyesters produced by bacteria as intracellular storage reserves of carbon and energy. the industrial production of pha is achieved by pure cultures in its natural state or using genetically engineered organisms. the main obstacle to the replacement of synthetic plastics by biopolymers is their great cost difference. research on the field of biopolymers synthesis using mixed cultures and waste organic carbon sources as substrates prove to decrease substantially the production costs of pha. the optimization of pha production under aerobic feeding conditions (adf) was achieved recently in our group, obtaining the highest value of pha content stored by mixed cultures (79.2% of cell dry weight). in this work only a homopolymer of polyhydroxybutyrate (phb) was obtained and since it is a highly crystalline and brittle material its application field is limited. the mechanical and thermal properties of pha can be varied to a great extend by adjusting the monomer composition. the incorporation of different monomeric units, other than hb, in the polymer chain, originates copolymers with improved mechanical properties. optimization of pha production from propionate by a mixed culture was studied varying the carbon and ammonia concentrations. propionate only, acetate alone or a mixture of acetate and propionate were tested. copolymers of hydroxybutyrate and hydroxyvalerate, p(hb/hv), with different compositions were obtained. consequently polymer properties could be manipulated by feeding the selected volatile fatty acid composition. the pharmaceutically important plant species of glycyrrhiza sp. (called licorice) is an important commercial product used as a natural sweetener, anti-inflammatory, anti-cancer and anti-diabetic agents. agrobacterium rhizogenes transformation system was used for the hairy root cultures of licorice g. uralensis. after inoculation of aseptic stem segments the ability of hairy root formation was scored for a period of 6 weeks. mean transformation frequency ranged from 27% (for 8196 up to 31% (for 15834). some transformed genotypes showed significant differences in roots weight, flavonoids and glycyrrhizin (gl) production. the cotransformation rate of licorice intact explants cultivars with lba 9402 tl-dna and the 35s gus gene showed an average of more than 35%. these obtained root cultures were additionly elicited with extracts of biotic elicitor acremonium sp. (endomycorhizal fungus), and were used as an in vitro system to metabolites production. the transformed and elicited hairy roots of g.uralensis were obtained by infection of a. rhizogenes 8196 have produced gl at an yield of 4.5% dry weight on the period of culture as a 30 days. according to tentative analyses the hairy roots cultures of glycyrrhiza species produced flavonoids (liquiritigenin and liquiritigen). more high levels (3.42 g/l) of the total flavonoids production have been identificated on the strains which transformed by lba 9402. this study involved any difference among elicitor treatments and incubation periods for the optimal meabolites production. clearly, the selection of an effective agrobacterium strain for the production of transformed root cultures is highly dependent on the plant species, and must be determined empirically. production, purification and characterization of scytalidium thermophilum phenol oxidases didem sutay 1 , ufuk bakir 1 , zumrut b. ogel 2 : 1 chemical engineering department, middle east technical university, inonu bulvari, 06531 ankara, turkey; 2 food engineering department, middle east technical university, inonu bulvari, 06531 ankara, turkey. e-mail: ubakir@metu.edu.tr (u. bakir) phenol oxidases (pos) are a group of enzymes which are responsible for oxidation of various phenolic compounds in the presence of molecular oxygen. there are different types of pos present in nature and three major groups of these enzymes are laccases (e.c. 1.10.3.2, p-benzenediol: oxygen oxidoreductase), catechol oxidases (e.c. 1.10.3.1, o-diphenol oxidoreductase) and tyrosinases (e.c. 1.14.18.1, monophenol monooxygenase). another group of enzymes, peroxidases (e.c. 1.11.1.7), can also be considered as a member of po family. pos have very wide substrate range and final oxidation products of these substrates are quinones, which are highly reactive molecules and polymerize into brown, red or black waterinsoluble compounds. pos are very common in nature, they can be found in almost all plants, animals and microorganisms. pigmentation and protection from the environment are main functions of these enzymes. pos have different applications in food, pharmaceutical, textile industries and waste-water treatment systems. the objective of this study was po production by the thermophilic fungus, scytalidium thermophilum, purification and characterization of the enzyme. for this purpose, enzyme production was performed either in a shaker-incubator or a temperature, ph and dissolved oxygen controlled 2 l bioreactor (probiotem) to optimize enzyme production medium composition and bioreactor parameters. as the carbon and nitrogen sources, 4% glucose and 0.4% yeast extract were determined as the optimal concentrations, respectively. copper, gallic acid and tannic acid were determined to increase enzyme production. purification was performed by using membrane and chromatographic techniques. hydrophobic, ion exchange and gel filtration columns were used by using a fplc system;äkta prime (amersham biosciences). especially the phenyl sepharose tm high performance column appeared to be very efficient for po purification from scytalidium thermophilum. purified po have been characterized by electrophoretic techniques and kinetic studies. isolation of lipolytic microorganisms from subtropical soils. cloning, purification and characterization of a novel esterase from strain pseudomonas sp. cr-611 núria prim, cristian ruiz, cristina bofill, f.i. javier pastor, pilar diaz department microbiology, university of barcelona. av. diagonal 645, microorganisms or their enzymes are used in a wide range of biotechnological activities such as polymer hydrolysis, synthesis of added-value compounds, sample decontamination, etc. thus, there is an increasing interest for isolating new enzymes and new enzymeproducing organisms for their use in industrial conversions (cherry and fidantsef, 2003) . among these enzymes, lipases, esterases, cellulases, xylanases and pectinases play an important role in many biotechnological processes like those related to pulp and paper processing. three samples of subtropical forest soil from puerto iguazú (argentina) were used for the isolation of autoctonous microorganisms growing in an organic matter-rich environment. a total of 724 pure cultures of bacteria and fungi were obtained and their hydrolytic activities on polysaccharide and lipidic substrates were assayed using olive oil, tributyrin, cholesterol esters, xylan, cellulose and pectin as substrates. among the isolates analysed, 449 were active on one or more of the substrates evaluated, and 43 of them degraded all substrates. nearly half of the strains displayed lipolytic activity, whereas the number of strains active on xylan, cellulose, pectin and cholesterol esters, was much lower. the 76 strains bearing the highest hydrolytic activities were selected and stored for further characterization (ruiz et al., 2005) . among them, strain cr-611, one of the most active isolates on tributyrin, was selected for identification and characterization of its lipolytic system. lipolytic strain cr-611 was identified by morphological, physiological and phylogenetic tests, as a pseudomonas sp., closely related to p. fluorescens. sds-page and zymogram analysis (diaz et al., 1999) of cell extracts and supernatants from the strain revealed a complex lipolytic system consisting of at least two lipolytic enzymes. sequence alignment and clustering of previously described pseudomonas lipases and esterases allowed the design of different sets of primers for the isolation of the lipase/esterase coding genes. a gene coding for an esterase with homology to family vi bacterial lipases (arpigny and jaeger, 1999) was isolated and cloned in escherichia coli. the cloned enzyme was further purified and characterized, showing preference for short fatty acid esters and displaying a typical michaelis-menten kinetics, with no interfacial activation. the substrate profile, together with the kinetic behaviour and sequence similarity of the cloned enzyme to family vi bacterial esterases, allowed to identify this enzyme as an esterase and was named esta6. maximum activity was achieved on muf-butyrate at 55 • c and ph 8.5, suggesting that it could be of interest for biotechnological purposes. microbial xylitol production from agricultural wastes has recently attracted much attention from industries because it has potentials to realize the cheaper production of xylitol with low environmental impact (tada et al., 2004) . in order to realize the effective xylitol production by a xylose utilizing yeast, the oxygen supply is a key for maximizing xylitol yield over consumed xylose (y xl ) because the intracellular xylitol metabolism is strongly influenced by the amount of available oxygen. in the present work, we tried to apply a metabolic reaction model in order to determine the optimal oxygen transfer rate (otr) in a fermentor for maximizing xylitol yield. corn cob hydrolysates containing 25 g-xylose/l was employed as medium for xylitol production by computer-controlled batch cultures using candida magnoliae (ferm p-16522, aist). a metabolic reaction model considering main xylitol metabolisms including glycolysis, pentose-phosphate pathway, tca cycle and cell synthesis was developed. the model allows to estimate various intracellular metabolic flux distributions including a xylitol production rate. the oxygen uptake rate to maximize the ratio of xylitol production rate over xylose consumption rate corresponds to the otr condition to maximize a xylitol yield over xylose consumed. based on the metabolic reaction model, the otr was optimized by a linear programming, the optimal otr and the maximum xylitol yield were estimated as 0.5 mmol o 2 /l h and 0.81 g-xylitol/g-xylose, respectively. the experimental verification using the optimal otr demonstrated that the xylitol yield was greatly improved to 0.75 g-xylitol/g-xylose from 0.6 g-xylitol/g-xylose in our previous study. expression of a bacterial sugar phosphate transporter in s. cerevisiae to release l-glycerol 3-phosphate accumulated by metabolic engineering almut popp, huyen thi thanh nguyen, ulf stahl, elke nevoigt department of microbiology and genetics, university of technology, 13353 berlin, germany. e-mail: a.popp@lb.tu-berlin. de (a. popp) in contrast to glycerol, its phosphorylated precursor l-glycerol-3phosphate (l-g3p) is retained by the plasma membrane. therefore, engineered yeast strains accumulate l-g3p in the cytosol resulting in low overall yield of the desired product and laborious downstream processing. a suitable sugar phosphate transporter in the yeast plasma membrane would overcome these limitations. the glycerol-3-phosphate transporter (glpt) of e.coli is an antiporter and naturally mediates the uptake of l-g3p in exchange with inorganic phosphate. we assume that this transporter would also mediate the excretion of accumulated l-g3p into a phosphate-rich medium if it was present in the plasma membrane of engineered yeast. despite many inconsistencies in codon usage, we were able to express the bacterial glpt gene in yeast. expression was monitored by a c-myc tag added to the c-or n-terminal hydrophilic tail, respectively. the quantity of the construct with n-terminal tag clearly exceeds the quantity of the construct with c-terminal tag. both gene products are located in the endoplasmic reticulum, as shown by immunofluorescence microscopy. obviously, yeast's transmembrane protein sorting machinery does not recognise it as a substrate for the secretory pathway. metabolic flux analysis of c-and p-limited shikimic acid producing e. coli gaspard lequeux 1 , louise johansson 2 , jo maertens 1 , peter vanrolleghem 1 , gunnar lidén 2 : 1 biomath, ghent university, coupure links 653, 9000 gent, belgium; 2 department of chemical engineering, lund university, p.o. box 124, 22100 lund, sweden. e-mail: gaspard.lequeux@biomath.ugent.be (g. lequeux) metabolic flux analysis (mfa) was applied to decipher why plimited e. coli fermentations are more optimal for shikimic acid production in comparison with glucose-limited fermentations. as mfa allows obtaining insight in the intracellular flux distribution over different pathways by only measuring net production and consumption rates of metabolites, under the condition that parallell pathways are removed. to this end a detailed metabolic network model was created and checked for consistency, dead-ends, and parallell pathways. several fermentations were performed at different dilution rates (ranging from 0.05 to 0.3 h −1 ) and different limitations (phosphate and glucose). the e. coli strain used was w3110 with genetic modifications in the aromatic amino acid pathway to enhance shikimic acid production. for each dilution rate, a metabolic model was solved. this way, the evolution of each flux can be followed with respect to the dilution rate. the p-limited cultures showed a better yield which can be explained by the diminished excretion of dehydro-shikimic acid (as is known from literature) and a reduction of the hydrolysis of atp. takasumi hattori, kuniki kino, kohtaro kirimura department of applied chemistry, school of science and engineering, waseda university, tokyo, japan. e-mail: takasumi@suou.waseda.jp (t. hattori) alternative oxidase is a terminal oxidase in respiration chain, which is a branched chain of cytochrome pathway, and inhibited by salicylhydroxamic acid (sham), but not by cyanide. the citric acid-producing fungus aspergillus niger wu-2223l has a cyanide-insensitive and sham-sensitive respiration catalyzed by the alternative oxidase (kirimure et al., 1999) and did not produce citric acid when cultivated with sham (kirimure et al., 2000) . in this study, the transcript levels of alternative oxidase gene (aox1) (kirimure et al., 1999) and activities of alternative oxidase under the conditions of citric acid production were examined during 9 days-cultivation. the amount of aox1 mrna was determined by northern blot analysis, and the specific activity of alternative oxidase as that of duroquinol oxidase. the transcript level and the activity were highest at 2 days at log-phase, decreased during 2 and 4 days, and thereafter maintained at low levels. however, the transcript and alternative oxidase activity was constitutively detected during whole the cultivation periods under the conditions of citric acid production. the sequence analysis of aox1 chromosomal dna revealed the presence of potential binding site of cyclic amp responsive element (cre), stress responsive element (stre) and heat shock factor (hsf) in its upstream region. these results indicated that the expression of alternative oxidase was regulated in the transcription level and alternative oxidase contributes as the main respiration chain during citric acid production. kirimura, k., et al., 1999 . curr. genet. 34, 472-477. kirimura, k., et al., 2000 . biosci. biotechnol. biochem. 64, 2034 -2039 . trichoderma strains are considered to be among the most useful fungi in industrial enzyme production, agriculture and bioremediation. metabolic versatility displayed by these fungi makes it a very amenable source of new gene products. functional analysis of candidate genes goes by two complementary ways: gene overexpression and loss-of-fuction mutants generation. a few expression systems are available mainly to direct constitutive gene expression in catabolite repression conditions. following a genomic approach, we have recently cloned some gene promoters with high expression in glucose in trichoderma harzianum cect2413. a main goal in a gene functional analysis is the generation of knock-out mutants. up to date, there is no reference about successful gene disruptions in t. harzianum mainly due to a very low homolog recombination frequency. rna-mediated gene silencing has been shown as an efficient tool to diminish or totally abolish specific gene expression. especially those strategies based on the use of hairpin constructs allow rapid and easy generation of strains with reduced levels of mrna from genes of interest. we have used the t. harzianum cect2413 tss1 promoter to direct the expression of a hairpin dna construct that induced the appearance of small-interfering rnas (sirnas) and the silencing of the uida reporter gene in a previous uida overexpressing strain. reduced levels of mrna and gus activity correlated with the presence of sirnas. this is the first report on rna-mediated gene silencing in trichoderma and constitutes a useful and promising tool for functional genomic studies in fungal systems. analysis of the metabolic response of escherichia coli to quantitative modulations of the glucose-6-phosphate dehydrogenase based on 13 c-labelling experiments cécile nicolas, fabien létisse, stéphane massou, philippe soucaille, jean-charles portais. e-mail: fabien.letisse@insa-toulouse.fr (f. létisse) microorganisms have an efficient capacity for adapting their metabolism in response to genetic or environmental changes, and understanding metabolic robustness has become an emergent issue. part of the robustness originates from the network organization of metabolic systems, where the interplay between all available biochemical reactions provides alternative mechanisms for compensating the perturbations. recently, 13 c-metabolic flux analysis ( 13 c-mfa) has been applied to escherichia coli knock-out mutants lacking key enzymes to determine the phenotypic effects of structural changes in the metabolic network, providing further evidences for compensatory phenomena. the aim of our on-going work is to understand how the central metabolism in e. coli responds to quantitative alterations at a specific key-point of the metabolic network. the glucose-6-phosphate dehydrogenase (g6pdh), a key enzyme in the central metabolism for which the effects of deleting the gene (zwf) has been already described (zhao et al., 2004) , was chosen as the target. to this aim we have generated a set of expression mutants, i.e. mutants having each a fixed level of expression of the zwf gene. four different levels of expression, leading respectively to g6pdh activity 2; 2.9; 5.7 and 14 times higher than in the wt strain, have been obtained. for each mutant, transcriptomics analysis will be carried out and compared to both the zwf-and wt strains to detect changes in the network structure, and the distribution of fluxes will be measured using 13 c-mfa. the flux maps obtained for the various strains will be compared to evaluate the quantitative response of the central metabolic network to imposed and increased g6pdh activity. metabolic control analysis will be applied to provide insights onto the sensitivity of the measurable metabolic fluxes to g6pdh activity. combination of transcriptomics and fluxomics approaches will provide information on the nature and extent of the compensatory mechanisms. because the activity of a single enzyme is tuned at different levels in knock-out and expression mutants, this investigation provides a situation that mimics gene-level regulation of metabolism. , j., et al., 2004, metab. eng., 6, 164 . with the depletion of the world's petroleum supply, there has been an increasing worldwide interest in ethanol as an alternative, nonpetroleum source of energy. this fact caused increased interest in the new ethanol technology fermentation process research. as reported before, bacteria zymomonas mobilis possesses more advantages than saccharomyces cerevisiae, microorganism used for ethanol production in industrial scale. for that reason, we have focused on the fermentation studies of this facultative bacterium in free and immobilized form. the immobilization of the cells into polyvinylalcohol (pva) hydrogel lens-shaped capsules lentikats, improved the batch fermentation process efficiency nine times. starch, the substrate considered as one of the best of renewable energy source is considered as fuel ethanol feedstock. due to z. mobilis disability of maltose, maltotriose and dextrin utilization, the starch has to be converted into glucose monomers. this pre-fermentation step can overcharged whole ethanol production process. this ineffective part of the process was resolved with immobilization of glucoamylase into lentikats. the system with immobilized enzyme and cell was stabile in continuous mode for 60 days without any significant change in the system efficiency. the combination of cell and enzyme immobilization can significantly improve the efficiency and the cost of ethanol production in industrial scale. fermentation of an inhibitory dilute acid-hydrolysate from spruce using a fed-batch procedure combined with cell-reuse andreas rudolf, gunnar lidén department of chemical engineering, box 124, lund university, se-221 00 lund, sweden. e-mail: andreas.rudolf@chemeng.lth.se (a. rudolf) a well-controlled addition of hydrolysate to the fermentation has proved very efficient in reducing yeast inhibition due to compounds formed during lignocellulose hydrolysis. furthermore, using high cell mass concentrations has been another way of avoiding the negative impact of the inhibitory compounds. if possible, the yeast should therefore be re-used in the process. in the present work a dilute-acid hydrolysate from spruce was fermented using a fed-batch procedure with reutilization of yeast. the fermentation procedure worked satisfactorily, with more than 98% of fermentable sugars consumed in each of the four consecutive fed-batch fermentations performed. the ethanol yields on fermentable sugars reached 0.45 g/g. there was continued cell growth in the repeated fed-batch experiments, with an average cell yield on fermentable sugars of 0.06 g/g. in contrast, only about 20% of the fermentable sugars were consumed within 24 h, when the fermentation of the hydrolysate was run in a batch process. the work shows the potential to re-use the yeast in a suitably designed process. metabolic engineering is defined by bailey in his seminal 1991 paper as "the improvement of cellular activities by manipulation of enzymatic, transport, and regulatory functions of the cell with the use of recombinant dna technology". the manipulation of these functions ultimately results in the manipulation of metabolism, which is the purpose of many biotechnological processes. metabolic engineering has sought its methods and tools in mathematics and the physical sciences, and later in information technology (it) leading to the proliferation of bioinformatics. in this work we propose a novel approach to metabolic engineering that regards it as a business process reengineering (bpr) endeavour. hammer and champy in their celebrated 1993 book define bpr as "the fundamental rethinking and radical redesign of business processes to achieve dramatic improvements in critical contemporary measures of performance, such as cost, quality, service and speed". our thesis is that metabolic engineering with its goal of reengineering the metabolism of the microorganism, is equivalent to business process reengineering (bpr) in business and management. indeed this is essentially what metabolic engineering does to the cell through the use of recombinant dna technology, which can be viewed as a radical redesign of the metabolic process. after all, it causes changes that cannot be attained otherwise, and whose purpose is to achieve dramatic improvements in cellular activities such as the increase in production of some metabolites by orders of magnitude. the cost incurred by the process, which is metabolism in this case, can be, for example, energy requirements or change to a cheaper substrate. in this study we elucidate this parallelism between the two with emphasis on modelling of metabolism and how the concepts of business process modelling can be applied to it. the purpose is not to produce a model, but rather to introduce the modelling methodology and demonstrate its utilisation and benefits and outline its limitations and challenges. we believe that the novelty of our work lies in applying a new paradigm in approaching metabolic engineering that has not been considered previously. thermophilic ethanol production from wheat straw hydrolysate in continuous culture tania i. georgieva, birgitte k. ahring biocentrum-dtu, technical university of denmark, building 227, dk-2800 lyngby, denmark. e-mail: tig@biocentrum.dtu.dk (t. georgieva) ethanol production from lignocellulosic biomass has attracted widespread attention as an unlimited low cost renewable source of energy to transportation fuels due to increasing petroleum use and air pollution towards greenhouse gases. wheat straw available as agricultural residue has been considered as a potential lignocellulosic substrate for industrial bioethanol production. a major technical obstacle to commercialize bioethanol production form lignocellulose (such as wheat straw) is associated with a lack of microorganism able to rapidly and efficiently ferment both hexose and pentose sugars into ethanol and to tolerate the inhibitors present in undetoxified hydrolysates. currently used industrial mesophilic microorganisms (saccharomyces cerevisiae and zymomonas mobilis) are excellent ethanol producer from glucose, however, they are not able to ferment other sugars such as xylose, which is the second most abundant sugar in lignocellulose. thermophilic anaerobic bacteria have been considered for ethanol production from lignocellulosic biomass as an alternative to mesophilic ethanol producing strains, predominantly because of their abilities naturally to ferment the whole diversity of sugars found in lignocellulosic biomass. an increase attention to thermophilies for ethanol production have also arise from broad spectrum of advantages regarding industrial scale ethanol fermentation such as high growth and metabolic rates, low oxygen solubility, reduced risk of reactor contamination, and cost savings via mixing, cooling and facilitated product recovery. in addition, simultaneous co-fermentation of glucose and xylose in a single operation unit could substantially reduced the ethanol production cost. research has been attempted to study the potential of using a thermophilic anaerobic bacterium for continuous ethanol fermentation of lignocellulosic biomass, with particular emphasis on effectiveness of our strain to ferment undetoxified wet oxidized wheat straw hydrolysate with respect to sugar (glucose and xylose) conversion and ethanol yield. the experiment was carried out in a lab-scale reactor operated at 70 • c with wheat straw hydrolysate as a substrate in concentrations from 20 to 80 wt.% equivalent to total sugar mixture of 11-38 g/l. wheat straw hydrolysate (woh) [200 g/l wheat straw, 92.4% dry matter (dm)] was prepared using wet oxidation pretreatment process followed by enzymatic saccharification with commercial enzymes mixture of celluclast1.5, and novozym188 (novozymes, denmark). both xylose and glucose sugars were simultaneously converted to ethanol. the sugar utilization was higher than 90%, and high ethanol yields were achieved. reactor shows good long-term performance (124 days) in terms of operation stability and reactor contamination. maltotriose is the second most abundant fermentable sugar in wort and due to incomplete fermentation, residual maltotriose in beer may cause problems in the brewing industry. to study genes that might improve utilization of maltotriose we used a library with dna from brewer's strains and a laboratory strain and identified a new transporter encoded by mtt1. mtt1 gave lager strain a15 the ability to grow on yp/2% maltotriose in the presence of 3 mg/l of the respiratory inhibitor antimycin a. this transporter gene shares 74% similarity with mph2 and mph3, 62% similarity with agt1 and 91% similarity with mal61 and mal31. moreover, mtt1 shares even higher similarity (98%) with the s. pastorianus mty1 gene (m. salema-oom, unpublished, ncbi accession number aj491328). purified radiolabeled maltotriose and radiolabeled maltose were used to study sugar uptake of lager strains a15 and ws34/70, and of a15 containing mtt1 or mtt1alt, a more efficient, altered version of this gene lacking the 66 basepairs from the 3 end and containing 57 base-pairs of vector sequences. these transport studies show that mtt1 and, especially, mtt1alt encode maltose transporters with relatively high activity towards maltotriose compared to maltose. this study is part of a multi-disciplinary project, funded by the european union (contract no. qlk1-ct-2001-01066) focusing on the development of high-gravity resistant brewer's yeast strains. metabolic engineering of l-phenylalanine pathway in bacillus subtilis yasemin demirci 1 , pınar ç alık 2 , güzide ç alık 1 , tunçer h.özdamar 1 : 1 bre laboratory, department of chemical engineering, ankara university, 06100 ankara, turkey; 2 iblab, department of chemical engineering, metu, 06531 ankara, turkey. e-mail: ozdamar@eng.ankara.edu.tr (t.h.özdamar) metabolic engineering design of a recombinant l-phenylalanine (phe) production system is based on coordinated overexpression of the flux-controlling genes in the aromatic-amino acid pathway. based on the insights gained by the work carried out in our laboratories (özçelik et al., 2004) , aroh for the reaction r96 at the branch-point chorismate that connects the preceding reactions of the aromatic group amino acid pathway to the proceeding reactions towards phe, was predicted as the first-, and aroa for dahp synthase (r89) predicted as second-metabolic engineering sites. aroa gene was cloned next to aroh, by the use of four primers designed using pcr-based gene splicing by overlap extension method. the genes were amplified separately by pcrs. the primers used at the ends to be joined were designed as complementary to one another by including nucleotides at their 5 ends that are complementary to the 3 portion of the other primer. these products were mixed in the next pcr reaction, where one strand from each fragment contains the overlap sequence at 3 end. extension of this overlap by dna polymerase has yielded the recombinant two-gene product; and the two-gene product serve as template for the continuation of reactions for the increase of the concentration in the micro-reactors. the two-gene fragment was first cloned into puc19, and then sub-cloned to pmk4 e. coli -bacillus shuttle plasmid. the new expression vector with high stability and high copy-number was obtained and transferred into host b. subtilis. the metabolic flux distributions calculated by the mass balance based stoichiometric model based on the metabolic reaction network for r-b.subtilis were determined by using time profiles of the substrate, dry-cell, phe and other amino acids, and organic acids concentrations as the constraints. on the bases of calculated intracellular fluxes of recombinant b. subtilis carrying pmk4::aroa::aroh, an in-depth analyses of the metabolic engineering design will be presented. ozçelik,i̇., ç alık, p., ç alık, g.,özdamar,t.h., 2004. metabolic engineering of aromatic group amino acid pathway in bacillus subtilis for l-phenylalanine production. chem. eng. sci. 59 (22-23), 5019-5026. the 100% respiratory-deficient nuclear petite amylolytic saccharomyces cerevisiae npb-g strain capable of excreting a hybrid protein possessing both ␣-amylase and glucoamylase enzyme activities was generated and its employment for direct fermentation of starch into ethanol was investigated under both shake flask and controlled bioreactor cultivation conditions. when compared with a standard host strain, higher ethanol concentrations and yields were achieved with the nuclear petite strain under both cultivation conditions. further improvement in ethanol production was achieved by the use of an initial glucose supplement. comparison of the ethanol fermentation performances of the respiratory-deficient npb-g and the parental respiratory-sufficient wtpb-g strain showed an increase of, ca. 48% in both ethanol production yields and ethanol productivities with the respiratory-deficient strain. response surface methodology (rsm) was used as a statistical tool to optimize the initial yeast extract and starch contents of the medium, which resulted in a substantial increase in the stability of the expression plasmid in both strains with concomitant improvement in their amylolytic potentials. high ethanol yields on substrate values of the bioreactor cultures, that were very close to the theoretical yield, indicated that the amylolytic respiratory-deficient strain developed in this study was very effective in the direct fermentation of starch into ethanol. establishing a biotechnology educational framework to support a knowledge-based economy in puerto rico rosa buxeda, lorenzo saliceti-piazza industrial biotechnology program, university of puerto rico, mayagüez campus, mayaguez 00680, puerto rico. e-mail: rbuxeda@uprm.edu (r. buxeda) industrial biotechnology has been identified as a major thrust area of economic development within the past five years for the island of puerto rico. the portfolio of biotechnology manufacturing investments in the island has passed the two billion dollar mark. world known companies like amgen, abbott and eli-lilly lead these investments, which have catalyzed a strong technology transfer to the island. a strong collaboration between industry and academia was needed to provide a well-trained workforce for these company startups. as a result, the university of puerto rico, mayagüez campus (upr-m) developed four initiatives which are part of the educational pipeline in biotechnology. these are: (i) an industrial biotechnology program, a 5-year bs degree which contains a novel curriculum with courses from science and engineering with undergraduate research and industrial internships as part of the degree requirements; (ii) an industrial biotechnology learning center, which provides customized biotechnology and bioprocessing training modules, including lectures and hands-on experiences to train and develop the workforce needed in the biotechnology manufacturing plants; (iii) a biotechnology summer camp, which addresses the high school student population and its main purpose is to educate and advise on the different career paths that can be followed in the field of biotechnology; and (iv) a biotechnology center for research and training in bioprocessing, that will address the development of corporatesponsored research projects to strengthen links between industry and academia in order to build up a knowledge-based economy. our paper will describe each initiative in detail as well as its outcomes and impact on puerto rico's knowledge-based economy goals. isolation of acid phosphatase from sweet potato and immobilization using different adsorbent d. omay, y. güvenilir, n. deveci istanbul technical university, department of chemical engineering, maslak 34769, istanbul, phosphatase enzymes occur in a wide range of plant and animal tissues. they catalyze the hydrolysis of phosphate bonds in organic phosphates, between the phosphate group and rest of the molecule. immobilization of enzymes and biological compounds is currently gaining importance due to its wide variety of applications in the food and pharmaceutical industries and also its biomedical applications. it was reported that enzymes can be activated by complexation with polysaccharides such as chitin or chitosan. the aim of this experimental study was determined as partial purification and isolation of acid phosphatase enzyme and its immobilization. the purification was realized by applying centrifugation, ammonium sulfate precipitation and dialysis respectively. the specific activity of the supernatant was 0.1 u/mg and after 80% saturation this value increased 0.64 u/mg. furthermore, acid phosphatase was investigated using different adsorbent (chitin, chitosan, synthetic zeolite and raw zeolite) and evaluated the storage stability and re-usability of the immobilized acid phosphatase. it was estimated that, acid phosphatase activity was shielded the ratio of 94, 96, 99, and 92% by using raw zeolite, synthetic zeolite, chitin and chitosan respectively under 12 h operation condition. øyvind m. jakobsen 1,2 , michael c. flickinger 3 , svein valla 1 , trond e. ellingsen 2 , trygve brautaset 1 : 1 department of biotechnology, norwegian university of science and technology, norway; 2 sintef applied chemistry, sintef, norway; 3 biotechnol. institute, department of biochemistry, molecular biology and biophysics, university of minnesota, usa aerobic methylotrophs can utilize one-carbon (c 1 ) compounds such as methane and methanol as a sole c-source for growth and energy. the majority of research on these organisms has focused on their biochemical novelity and commercial viability. for the industrial production of bulk products such as the amino acids lysine and glutamate raw material costs and abundance are important, and c 1 sources are thus attractive compared to sugars. bacillus methanolicus can secrete up to 55 g/l of glutamate upon methanol growth at 50 • c (thermotolerant and methylotroph) and mutants producing 35-40 g/l of l-lysine have been selected. we study the genetics for conversion of methanol into biosynthesis of glutamate and lysine, and in the present report we unravel the regulation of genes impor-tant for the consumption and tolerance level for c 1 compounds by b. methanolicus. b. methanolicus has a methanol dehydrogenase gene (mdh) for oxidation of methanol into formaldehyde and a ribulose monophosphate (rump) pathway for assimilation of formaldehyde. we recently discovered that mdh and five rump genes are carried by natural plasmid pbm19 in this bacterium and this represented the first documentation of plasmid-dependent methylotrophy in any microorganism. we here use real-time pcr to analyse the regulation of plasmid-and chromosomally located rump genes, in cells upon methylotrophic and non-methylotrophic growth. high methanol concentrations in the growth medium is cell toxic and the mechanisms for this sensitivity of b. methanolicus is poorly understood. our results indicate that plasmid pbm19 plays a fundamental role for this trait as well and the impact of our results on the biotechnological applications of this bacterium is discusssed. department, national research centre, dokki, cairo, egypt adding a proteolytic enzyme extraction from jack fruit (artocarpus integrifolis) in combination of fermentation process in low fat yogurts manufacture was tried to improve yogurt flavour and rheological properties. experimental yogurts milk contained control, 3.9 (t1), 7.8 (t2) and 11.7 (t3) units/ml milk from crude extracts of plant proteinase. the ph of the product treated with crude proteinase was lower than the control. however, the rate of acidity development during storage slightly increased with increasing the addition of crude proteinase level and progress of storage period of yogurt. the proteolytic activity of all yogurts gradually increased until the end of storage period (15 days). yogurts made from milk treated with crude proteinase preparations were less firm compared with control at all storage periods, where t3 showed more less firm after 15 days of storage being 20.17 g/100 g. generally, increasing units of plant proteinase preparations decreased the firmness. on the other hand, yogurt made from milk pretreated with plant proteinase had higher syneresis, and apparent viscosity than the untreated product. the greatest viscosity was found in t2 and t3 of 433 and 479 mpa s respectively, compared with control of 299 mpa s at 15 days storage. the results indicated that there is an inverse relationship between the amount of units of crude proteinase preparations and susceptibility of yogurt to syneresis. the t2 gained the highest scores (85 points) followed by the control (81.5 points) after 15 days of storage, while yogurt of t3 showed a low scoring being 75. from the foregoing results, it is recommend to use jack fruit (artocarpus integrifolis) as a source of plant proteinases and utilize it to develop a high quality yogurt at a level of 7.8 units of plant proteinases/ml milk. the penicillium chrysogenum oat1 gene encoding a class iii omega-aminotransferase has been cloned and characterized. this enzyme that converts lysine into 2-aminoadipic semialdehyde is important in providing 2-aminoadipic acid, a precursor of penicillin and other ␤-lactam antibiotics. the enzyme is related to ornithine-5-aminotransferases and to lysine-6-aminotransferases encoded by the lat gene located in the bacterial cephamycin gene clusters. expression of oat1 is induced by lysine, ornithine and arginine and repressed by ammonium ions. area-binding consensus sequences and an 8-bp direct repeat associated with arginine induction in emericella (aspergillus nidulans) have been found in the oat1 promoter region. deletion of the oat1 gene resulted in the loss of omegaaminotransferase activity. the deletion mutants were unable to grow on ornithine or arginine as sole nitrogen sources and showed a reduced growth on lysine. complementation of the deleted mutant with the oat1 gene restored growth on ornithine, arginine and lysine to the levels of the parental strain and omega-aminotransferase activity. the strong expression of oat1 gene after induction by the basic amino acids may provide additional 2-aminoadipic acid for the formation of the 2-aminoadipyl-cysteinyl-valine tripeptide for ␤-lactam biosynthesis. morphological characterisation of two high producing strains of penicillium chrysogenum carrying a disruption in the nadph-dependent glutamate dehydrogenase k. rueksomtawin, j. thykaer, h. noorman, j. nielsen center for microbial biotechnology, biocentrum-dtu, technical university of denmark, dk-2800 lyngby, denmark. e-mail: kr@biocentrum.dtu.dk (k. rueksomtawin) metabolic engineering has proven to be useful in optimisation of ␤-lactam production, e.g. constructing superior strains with multiple copies of the ␤-lactam gene cluster. it is however, of equal importance to gain insight into other aspects of the metabolism to establish a general overview in order to apply metabolic engineering for further improvement of the production strains. in that context, the redox metabolism is essential, as it functions as a tightly controlled connection between the different parts of the metabolism. in order to investigate this role of the redox metabolism in more detail, the gdha-gene, encoding the nadph-dependent glutamate dehydrogenase, was disrupted in two industrial strains of penicillium chrysogenum. during physiological characterisation of the two strains it became apparent that considerable changes in the morphology had occurred due to the genetic alteration. since the morphology is an important parameter in process optimisation, an examination of the morphology of the two strains was undertaken. in this work, the morphological differences between the gdha-disrupted strains and the reference strains were comprehensively investigated both during growth on solid media and submerged growth in a flow-through growth chamber. with the advance development of computerized image analysis techniques, the key morphological properties of the individual hyphal elements were quantified. in comparison to the reference strains, the disruption of the gdha gene resulted in a morphological change from short hyperbranched hyphal elements to long elongated hyphal elements with less branches. in the parallel studies with aspergillus nidulans and its corresponding gdha-deleted strain, no difference in the morphology was observed. polyketides (pk) represent one of the largest groups of natural products and are found in fungi, bacteria and plants. since many useful polyketides either originate from sources that are difficult or even impossible to cultivate or are produced in inadequate amounts, we are interested in expressing polyketide synthases (pkss) in heterologous hosts. saccharomyces cerevisiae, aspergillus niger and aspergillus nidulans were chosen as initial hosts, because the techniques necessary to cultivate and manipulate these strains genetically are well established. 6-methylsalicylic acid synthase (6-msas) was chosen as a model pks. replicative plasmids carrying the genes encoding 6-msas from penicillium patulum and phosphopantetheinyl transferase (pptase), respectively, were transformed into s. cerevisiae. in addition, an integrative vector was designed and the gene encoding 6-msas was integrated in the yeast chromosome. batch cultivations on galactose minimal media were performed. the results are presented and in particular the effect of expression mode and type of pptase (bacterial versus fungal) is discussed. furthermore, the progress of the work on expressing 6-msas in a. niger and a. nidulans using an integrative vector system is presented and discussed. the valorisation of functionalized chemicals from biomass resources compared to the conventional fossil production route ben brehmer, wageningen ur agrotechnology & food sciences, workgroup: valorisation of plant production chains, p.o. box 17, 6700 aa wageningen, the netherlands. e-mail: ben.brehmer@wur.nl at some undisclosed point in the foreseeable future, cheap fossil fuels resources will become depleted and the industry will be forced to pursue more difficult reserves with increasingly high extraction costs. most alternatives available and under research do not consider price as the main motivation for replacement, but focus solely on sustainability and environmental benefits. sustainability is an interesting word as there are enough fossil resources scattered around the world to be sustainable in quantity but not sustainable in price. seeing that fossil fuels are derived from prehistoric biomass it is not at all presumptuous to assume that every application and product can be replaced by the biomass of today. in fact, many highly specialised pharmaceutical chemicals already have a biomass origin. yet, not all of the uses of fossil fuels need to be replaced by a comparable carbon based source, such as biomass. energy and transportation in particular do not necessary need to rely on carbon cleavage, whereas practically all of the petrochemicals contain a carbon backbone. the main stipulation in substituting fossil-based chemicals with bio-based chemicals is availability and cost. it is proposed that already today, by utilising existing, recently developed and developing technology, it is economically advantageous for many chemicals to derive from biomass, in particular the functionalized chemicals. the only way to validate this conjecture is to develop a complete comparative life cycle analysis. as opposed to a traditional lca, the "multicriterion" developed here will revolve around energy flows and process efficiency in terms of exergy. the aim is to assess the optimum route with the best production options along the whole production chain while determining any possible limiting factors. using this tool, a systematic production matrix relating several logical source crops and a few key chemicals of varying derivative levels can be created and compared to the conventional fossil routes. combined with economic considerations and some unambiguous environmental factors, the investigation will provide all the information relevant to the industry. the goal is to create an objective and reliable simulation system ratifying the economic and environmental feasibility of exploiting biobased chemicals today and indicate the steps necessary for further improvement. biosynthesis of multi-enzymatic preparation from aspergillus niger ibt-90 useful in textile fabric treatment rita pyc 1 , jadwiga sojka-ledakowicz 2 , tadeusz antczak 1 , joanna lichawska 2 : 1 institute of technical biochemistry, the technical university of lodz, lodz 90-924, poland; 2 textile research institute, lodz 92-103, poland among many methods of producing enzymatic preparations, i.e. by liquid surface fermentation -lsf, submerged fermentation -smf or solid state fermentation -ssf, this last is most advantageous. cultivation in solid state means fermentation on a matrix formed by industrial and agricultural wastes. most often filamentous fungi -due to the lack of available water in the foundation -are the efficient, competitive microorganisms applied in solid state bio-conversion. the aim of research works carried out at the institute of technical biochemistry of the technical university of lodz and at textile research institute, lodz was defining optimal conditions for biosynthesis and testing the possibility of applying multi-enzymatic preparation from aspergillus niger ibt-90 in the treatment of woven fabrics made of natural cellulose fibres. as the result of biosynthesis optimization process, malt sprouts, wheat barn and beet pulp were selected as the best media to obtain enzymes of high pectinolytic activity maintaining at the same time high activity of cellulolytic enzymes and xylanase. the highest obtained activities reached: 2000 0 pm for total pectinolytic activity, 7 j/ml for endoglucanase and 527 j/ml for endoxylanase and they were 2.4-3.0 times higher than those achieved before optimizing process. performed research works demonstrated that the optimum activity of applied enzymatic system is obtained in the range of ph 4.6-4.8. woven fabrics made of flax and cotton fibre blends, subjected to bio-pre-treatment, were evaluated with reference to their sorption properties. comparative evaluation of liquid sorption by woven fabrics allowed to notice efficient enhancement of fibres' sorption capabilities after pre-treatment using enzymes system from aspergillus niger ibt-90. this offers the possibility of substitution of alkali scouring of linen-cotton woven fabrics before their bleaching by bio-treatment. the phylum actinobacter includes many bacteria of industrial importance both for accumulation of primary and secondary metabolites. both primary and secondary metabolites are dependent on precursors and cofactors that are provided by the central carbon metabolism of microorganisms. there are alternative pathways for catabolism of carbon either via the embden meyerhof parnas (emp) and pentose phosphate (pp) pathway or through the entner doudoroff (ed) pathway. the emp pathway is energetically more favorable and has therefore been presumed to be the dominating route for carbon metabolism in bacteria producing secondary metabolites. however, primary metabolism is poorly studied for most actinobacter species as focus traditionally has been on secondary metabolism. with the aim to gain more knowledge about the diversity of central carbon metabolism within the phylum actinobacter, 17 different strains were collected from various sources and strain collections. the strains were grown in minimal medium with supplement of standard vitamin solution and [1-13 c] glucose as carbon source. the 13 c-labeling patterns of proteinogenic amino acids were determined by gc-ms analysis. through this method, the fluxes in the central carbon metabolism during balanced growth were estimated and pathways for carbon metabolism were determined. in particular the labeling patterns of alanine and valine were of interest as they are derived from pyruvate and therefore can be used to distinguish between whether glucose is metabolized through the ed pathway or the emp pathway. nobacter, amycolatopsis balhimycina produces the glycopeptide balhimycin. the balhimycin aglycone is identical to the aglycon of vancomycin, which is a commercial glycopeptide. as a. balhimycina appears to be accessible to genetic modifications and the biosynthetic cluster responsible for balhimycin production is published, this genetically well-characterised academic strain can serve as a platform strain for production of vancomycin analogues derived through combinatorial biosynthesis. the understanding of the physiology of this microorganism is essential for the efficient accumulation of potentially commercial secondary metabolites. the bacterium is capable of growth in a fully defined minimal medium, with the production of balhimycin. the strain was grown at either nitrogen or phosphate limitation and balhimycin accumulation was followed at these conditions. flux analysis of balhimycin production by amycolatopsis balhimycina based on 13 c labelling experiments was performed. the zygomycetes blakeslea trispora and phycomyces blakesleeanus accumulate beta-carotene, ubiquinone (coenzyme q), various different sterols, and other terpenoids, all of them produced via the mevalonate pathway. these fungi are used or could be used for the industrial production of these terpenoids, edible oil, chitosan, and various organic acids. by measuring the specific radioactivity of terpenoids made from radioactive mevalonate, leucine or acetate in the presence of excess glucose in wild types and mutant strains we have concluded that these fungi have separate subcellular compartments for the production of carotene, sterols and triacylglycerols. the terpenoid moiety of ubiquinone is synthesized in the same compartment as ergosterol. these compartments contain separate pools of all their common metabolites, beginning from acetyl-coa. mevalonate carbon atoms do not find their way back to general metabolism, i.e., these fungi lack the "shunt" pathway. the compartments are regulated independently. the very large variations in carotene content caused by many environmental and genetic changes are not accompanied by variations in the ubiquinone content. the ubiquinone content increases when the cultures grow on leucine or acetate as carbon sources and is not affected by illumination. phycomyces, but not blakeslea, increases the production of ubiquinone in presence of oligomycin. lincomycin, produced by streptomyces lincolnensis, is important, clinically used antibiotic. its gene cluster consists of 27 putative open reading frames with biosynthetic or regulatory functions (lmb genes) and three resistance genes (lmra, lmrb, lmrc). the organization of transcription units was determined. the analysis of the lincomycin biosynthetic gene transcripts in various cultivation stages revealed the genes with putative regulatory functions which are transcribed in early stages of cultivation. previous analysis of biosynthetic pathways of lincomycin and functionally different anthramycin antibiotics (anthramycin, sibiromycin, tomaymycin, mazetharmycin and porothramycin) indicates that the genetic information on the lincomycin and anthramycin biosynthesis should share common elements (genes), both biosynthetic and regulatory. hybridization experiments demonstrated presence of several analogues of lmb genes involved in the biosynthesis of anthramycin produced by streptomyces refuineus and porothramycin produced by streptomyces albus. effect of various calcium salts on the erythromycin production by saccharopolyspora erythraea m. rostamza 1 , a. noohi 1 , j. hamedi 2* : 1 department of biology, faculty of science, science and research branch, islamic azad university, tehran, iran; 2 microbial biotechnology lab., department of biology, faculty of science, university of tehran, tehran, iran. e-mail: jhamedi@khayam.ut.ac.ir (j. hamedi) calcium carbonate has the positive effect on the erythromycin, however, because of its low water solubility, caused to clogging the spargers of fermenters and fouling the microfilters. in this research, various soluble calcium salts was added to the fermentation medium and their effect the growth of saccharopolyspora erythraea and erythromycin production was studied in the complex medium consisted soy bean meal, dextrin and starch as major ingredients. the fermentation conditions were 220 rpm, 8 days at 30 • c. the results obtained showed that there is no significant difference between erythromycin concentrations in the medium containing calcium lactate and calcium carbonate. however, erythromycin concentrations in the other calcium salts containing media were less than to calcium carbonate containing medium. optimum concentration of calcium lactate for erythromycin production was 10 g/l. lincosamides and its derivatives are clinically important antibiotics. comparison of gene cluster coding for lincomycin biosynthesis and newly identified cluster of genes for celesticetin biosynthesis revealed new information on functions of several genes common for both biosynthetic pathways. the celesticetin gene cluster was identified by screening the cosmid library of streptomyces caelestis with heterologous probes based on lincomycin biosynthetic genes involved in a part of biosynthesis shared by both antibiotics. sequence analysis of the cluster revealed 24 putative orfs, out of which 18 are lincomycin biosynthetic genes analogues, four are specific for celesticetin biosynthesis and one codes for resistance. the gene cluster is bounded with transposase genes on both sides. in order to clarify function of three putative regulatory genes of lincomycin biosynthesis, the insertional inactivation with the pcr targeting system in streptomyces lincolnensis was done and resulted in differently reduced production of lincomycin. larsen cmb biocentrum-dtu, technical university of denmark, 2800 kgs. lyngby, denmark we present a new algorithm called "x-hitting" for automatic identification of novel bioactive compounds based on full spectroscopic characters of highly complex mixtures of natural product. one of the most dramatic advances in recent drug discovery has been the increase in screening capacity throughput and data handling. therefore, analysis has become the bottleneck in the drug discovery process. the algorithm presented here is investigated and demonstrated on identifying potentially new bioactive compounds. in addition method is shown to have a high performance for automatic identification of known structures. these tasks are referred to as new-hitting and cross-hitting, respectively. finally, the receiver operating characteristics (roc) is introduced to the research field as an important tool for evaluating the performance of the "compound predictor". through examples it is shown, that known cross-hits are identified with high proficiency, and that the new-hitting works on finding new targets represented by analogues and structurally new compounds. a gene encoding a ␥-butyrolactone autoregulator receptor that have a common activity as dna-binding transcriptional repressors, controlling secondary metabolism and/or morphological differentiation in streptomyces was cloned from a natamycin producer, streptomyces natalensis, and its function was evaluated by in vivo. pcr using the primers designed from two highly conserved regions of streptomyces autoregulator receptors gave a 102-bp band, the sequence of which revealed high similarity to the expected region of a receptor gene. by genomic southern hybridization with 102-bp insert as a probe, a 687-bp intact receptor gene (sngr) was obtained from s. natalensis. in vivo to clarify the function of sngr, a sngrdisrupted strain was constructed, and a phenotype was compared with the wild-type strain. the sngr disruptant started natamycin production 6 h earlier and showed a 4.6-fold-higher production of natamycin than the wild-type strain. in addition, sporulation was earlier and 10-fold abundance. the phenotype indicates that the autoregulator receptor protein of s. natalensis acts as a primary negative regulator of the biosynthesis of natamycin and is related to regulation of sporulation. exploring the biocatalytic potential of the novel thermostable baeyer-villiger monooxygenase: phenylacetone monooxygenase daniel e. torres pazmiño 1 , gonzalo de gonzalo 2 , gianluca ottolina 2 , giacomo carrea 2 , dick b. janssen 2 , marco w. fraaije 1 , 1 biochemical laboratory, groningen biomolecular sciences and biotechnology institute, university of groningen, nijenbaeyer-villiger monooxygenases (bvmos) represent useful biocatalytic tools as they can catalyze reactions which are difficult to achieve using chemical means. however, only a limited number of these atypical monooxygenases are available in recombinant form. using a recently described protein sequence motif, a putative bvmo was identified in the genome of the thermophilic actinomycete thermobifida fusca. the nadph-dependent and fad-containing monooxygenase is active with a wide range of aromatic and aliphatic ketones and sulfides. genetic and kinetic data suggest that phenylacetone is the physiological substrate of the enzyme. previously, it was reported that this bvmo exhibits only a moderate enantioselectivity with (r,s)-␣-methylphenylacetone. this poster we will show an overview of the biocatalytic potential of the enzyme as explored so far. interestingly the enzyme has been found to perform highly enantioselective oxidations with a range of ketones and sulfides. this again indicates that this novel thermostable oxidative biocatalyst can be a useful tool for the synthesis of chiral building blocks. the enzyme s-adenosylhomocysteine hydrolase (adohcyase) catalyzes hydrolysis of s-adenosylhomocysteine (adohcy), an inhibitor of transmethylation reactions, into adenosine (ado) and homocysteine (hcy). the catalysed reaction is reversible, and the equilibrium is strongly displaced in direction of the synthesis of adohcy when reaction occurs in vitro. nevertheless, its biotechnological application resides in the synthesis of antivirals. for it, we selected between different producing microorganisms of the enzyme, the gram positive bacterium corynebacterium glutamicum. after designing the specific oligonucleotides, the gene was expressed in escherichia coli using the expression system pet28a+ with iptg. the electrophoretic analysis under denaturing conditions, shows a clear induction and over-expression of a protein with a mw of 49 kda. on the other hand, the immobilization of this recombinant enzyme in a solid support allows to use it as a catalyst for the synthesis of adohcy. the enzyme was purified by imac thanks to the presence of n-terminal 6 × his tag end, and immobilized in eupergit c for the optimization of the production of adohcy, a product of high value. sequencing, cloning, expression, purification and characterization of a novel cytochrome p450 monooxygenase from rhodococcus rubber luo liu, rolf d. schmid, vlada b. urlacher institute of technical biochemistry, stuttgart university, allmandring 31, 70569 stuttgart, germany. e-mail: itbvur@itb.unistuttgart.de (l. liu) the cytochrome p450 monooxygenases are heme-containing proteins, which catalyze a wide range of oxidative reactions (werck-reichhart and feyereisen, 2000) . a monooxygenation activity was observed for the strain rhodococcus ruber dsm 44319. a p450-like gene fragment was amplified by pcr using degenerated primers. for identification of regions that flank this p450-like dna fragment, the method "directional genome walking using pcr" was applied (mishra et al., 2002) . the full size gene encoding a cytochrome p450 enzyme was amplified by pcr from genomic dna and cloned into the vector pet28a(+) for heterologous expression in escherichia coli bl21(de3) cells. the enzyme was purified using metal affinity chromatography. the primary protein structure suggests, that this enzyme is a natural self-sufficient fusion protein consisting of a ferredoxin, a reductase and a p450 monooxygenase. the reductase activity was determined using an exogenous electron acceptor cytochrome c. the reductase domain of this p450 monooxygenase showed a strong preference for nadph over nadh. the substrate spetrum was investigated. in the presence of nadph the p450 enzyme shows hydroxylation activity towards 7-ethoxycoumarin, naphthalene, indene, acenaphthene, toluene and fluorene. mishra, r.n., singla-pareek, s.l., nair, s., sopory, s.k., reddy, m.k., 2002 . directional genome walking using pcr. biotechniques 33, 830-834. werck-reichhart, d., feyereisen, r., 2000. cytochromes p450: a success story. genome biol. 1 (6), 3003.1-3003.9. selective production of monoglyceride consisted of conjugated linoleic acid by penicillium lipase yomi watanabe 1,2 , yoshie yamauchi-sato 3 , toshihiro nagao 1 , satoshi negishi 3 , tadamasa terai 4 , takashi kobayashi 1 , rolf d. schmid 2 , yuji shimada 1 : 1 osaka municipal technical research institute, osaka, japan; 2 stuttgart university, stuttgart, germany; 3 the nisshin oillio group, ltd, yokosuka, japan; 4 osaka institute of technology, osaka, japan conjugated linoleic acid (cla) is a group of c18 fatty acid (fa) containing two conjugated double bonds. it is expected to prevent cancer, adipositas, atherosclerosis etc, and is commertially available in the primary form of free fa, containing almost equal amounts of 9cis,11trans-and 10trans,12cis-cla. it is therefore desired to be converted to a palatable form. for this purpose, we have previously proposed two enzymatic ways to convert cla to monoglyceride, an emulsifier, with penicillium camembertii lipase; (1) sequencial esterification-glycerolysis, (2) esterification at low tem-perature. these methods, however, are time-and energy-consuming. esterification of cla with glycerol under ambient pressure by the lipase produces equal amounts of mono-and diglycerides. in contrast, it was newly found that the reaction under reduced pressure supressed the formation of diglycerides and achieved to produce 90% monoglyceride at 95% esterification. improving the thermal stability of cellobiohydrolases i (cel7a) from t. reesei by site directed evolution frits goedegebuur 1 , lydia dankmeyer 1 , peter gualfetti 2 , brad kelemen 2 , edmundo larenas 2 , paulien neefe 1 , pauline teunissen 1 , colin mitchinson 2 : 1 genencor international bv, archimedesweg 30, 2333cn leiden, the netherlands; 2 genencor international inc., 925 page mill road, palo alto, ca 94304, usa genencor international has been working to produce improved enzyme products for economic conversion of ligno-cellulosic biomass to fermentable sugars. most of this work was performed under a subcontract with the u.s. department of energy for cellulose cost reduction for biomass conversion. cellulolytic biomass conversion is performed in nature by a complex mixture of enzymes. cellobiohydrolases play a key role and all effective cellulase mixtures contain a large excess of cellobiohydrolases over endoglucanases, suggesting that it is the exoglucanase activity that is limiting. the fundamental dependence of reaction rate on temperature predicts that large increases in performance, and decreased enzyme cost, would be achieved if the enzymatic conversion could be operated at elevated temperatures. industrial strains of trichoderma reesei produce cellulases at very high levels and low cost. however, t. reesei cbh1 (hypocrea jecorina cel7a) does not have sufficient stability to survive and perform at high temperatures. this poster shows the thermal stability improvement in t. reesei cbh1 by site directed evolution. sites with increased thermal stability properties were combined and evolved in high temperature stable cbhi variants. the evaluation of lipases as biocatalysts for organic chemistry can be carried out, at laboratory scale, by using soluble enzymes for biotransformations in aqueous media. however, the industrial exploitation of such an enormous potential should require a suitable protocol for immobilization of lipases. the binding of lipases on suitable pre-existing supports should greatly improve the performance of industrial reactors allowing us a continuous use or re-use of such interesting biocatalysts. in addition, lipases, like most enzymes, are not perfect chemical catalysts. lipases may be unstable and they may not have the optimal activity nor the optimal enantio or regioselectivities. in this way, immobilization of lipases, together with its relevance for the performance of each different industrial reactor, could be also used as a tool to improve and optimize some of these parameters. that is, immobilization of lipases, far from an already solved problem, constitutes an exciting field of research in the promising area of industrial bio-organic chemistry. in this work we would like to present useful immobilization methods of several lipases. the lipase immobilised were used for enzymatic hydrolysis of peptidomimetics of structure a. type of immobilzation used can changed the enantioselectivity of biocatalyst prepared. the absolute configuration of products b and c obtained in enzymatic reactions were assigned by chemical correlation. two-component flavin-dependent monooxygenases form an interesting class of flavoenzymes. they consist of two separate proteins; a monooxygenase component, which catalyses an oxygenation reaction in the presence of reduced flavin, and a flavin reducing component, which reduces flavin (fad or fmn) using nad(p)h as an electron donor. a well-known example of this class of monooxygenases is styrene monooxygenase (otto et al., 2004) . due to the ability to form enantiopure epoxides, which are relevant building blocks for the pharmaceutical industry, styrene monooxygenases form a valuable class of enzymes for biocatalysis. while screening a metagenomic library for oxidative enzymes, an indigo-producing clone was found. sequencing the particular clone revealed an inserted fragment of environmental dna encoding a two-component monooxygenase ( many investigations over the recent years have been directed to the production of natural aroma compounds. through biotransformation and bioconversion, aroma compounds considered as "natural" can be produced starting from monoterpenes, generating high value products as rose oxide. rose oxide is found in small amounts in some essential oils such as bulgarian rose oil and geranium oil. (−)-rose oxide is an impacting flavor compound and has a small threshold: 0.5 ppb. application of agro-industrial residues in bioprocess on one hand provides alternative substrates, and on the other hand helps solving pollution problems, that might be caused by the disposal of this residue in nature. the liquid cassava waste, is originated by the pressing of cassava roots. it is considered as a "harmful" pollutant waste due to its high organic charge and presence of cyanide. on the other hand, can be considered rich in nutrients that can be used in other applications. it was found that sporulated surface cultures of penicillium sp. were able to convert citronellol into cis-and trans-rose oxides. other bioproducts were 3,7-dimethyl-5-octen-1,7diol and 3,7-dimethyl-6,7-epoxy-1-octanol. no chemical oxidation or auto-oxidation products were detected in liquid control broths. the experiments were conducted at 30 • c and 160 rpm. when the medium was cassava, the production of rose oxide, 3,7-dimethyl-5octen-1,7-diol and 3,7-dimethyl-6,7-epoxy-1-octanol were insignificant reaching trace amounts. but when the mycelium developed in cassava medium and than transferred to mineral medium (citronellol as c-source) the concentrations of rose oxide increased dramatically, reaching 70 mg/l for the cis-isomer and 30 mg/l for trans-isomer. a mechanistic mathematical model of enzymatic degradation of avicel and phosphoric acid swollen cellulose (pasc) has been proposed. the model is based on the degree of polymerization (dp) of starting substrate, and follows its decline with time, to the final end product -glucose. three enzyme classes, namely, endoglucanase (eg), cellobiohydrolase (cbh) and ␤-glucosidase (bg) are all individually incorporated in the model. the model is, additionally, taking into account cooperative action of the involved enzymes, as well as effects of enzyme inhibition by end-products, cellobiose and glucose. to be able to describe the complex process of enzymatic hydrolysis with a set of differential equations certain assumptions needed to be introduced. those assumptions represent the simplification of an up-to-date knowledge of both substrate and enzyme structure, but also enzyme mode of action. for example, one of the often asked questions is: "what is happening with shorter cellooligosacharides (dp 7 and up) laying on the surface of cellulose chain? are they being adsorbed to the core cellulose chain or partly solubilized to a hydrolysis broth?" to give answers to these questions and confirm mathematical modeling real enzyme hydrolysis data are needed. in this work, four well characterized, highly purified mono-component enzymes from humicola insolens (two eg and two cbh) and one bg from aspergillus niger were used to hydrolyze avicel and pasc. by careful choice of catalyst, some enzyme specific characteristics like presence or absence of cellulose binding domain will also be incorporated into the model. hydrolysis experiments were initially performed by distinct mono-component enzymes, to confirm the basic characteristics of each of the enzyme classes. soluble hydrolysis products (dp 1-6) were analyzed by hplc and detection of non-soluble, higher-dp polysaccharides was performed by technique of polysaccharide analysis using carbohydrate gel electrophoresis. optimisation of halogenase enzyme activity k. muffler 1 , m. retzlaff 2 , k.-h. van pée 3 , r. ulber 1 : 1 technische universität kaiserslautern, germany; 2 technische universität münchen, germany; 3 technische universität dresden, germany. e-mail: muffler@rhrk.uni-kl.de (k. muffler) halogenases provide the opportunity of a regioselective and stereospecific halogenation of organic subtrates in contrast to the class of haloperoxidases. these enzymes allow a gentle synthesis of halogenated organic molecules and are capable to halogenate in specific positions (hammer et al., 1997; keller et al., 2000) , whereas traditional organic synthesis often failures or mainly leads to byproducts (hasegawa et al., 1999) , e.g. halogenation of tryptophan in other positions than position 3. our current research is focussed on tryptophan-5-halogenases, because the 5-br/cl-tryptophan could be applied as a pharmacologically attractive precursor of serotonin. in our work we describe the optimization of an enzyme assay respectively the enzyme activity. for this purpose we use a genetic algorithm. the responsible gene of the fadh 2 dependent enzyme was cloned from streptomyces sp. origin into a pseudomonas fluorescens strain. however, the optimization procedure was done with the purified his-tagged tryptophan-5-halogenase, which was easily obtained from the crude extract of the lysed cells by application of immobilized metal affinity chromatography. the application of algorithms allows an optimization of the multidimensional search problem leading to a global optimum in the search space in contrast to the traditional used one-factor-at-a-time method. latter often failures, because this method does not reflect possible influences the parameters can have on each other. effect of organic solvent type on the enantioselectivity of candida rugosa lipase in the hydrolysis of racemic naproxen methyl ester in biphasic reaction system serpil takaç, deniz mutlu department of chemical engineering, institute of biotechnology, ankara university, 06100 tandogan, ankara, turkey hydrolysis of racemic naproxen methyl ester to produce s-naproxen was carried out in the biphasic system using isooctane, cyclohexane, hexane and toluene with candida rugosa lipase after stirring in the phosphate buffer (ph 7.5, 0.02 m) for 2 h at +4 • c and centrifuging. the hydrolyses were carried out in shaking flasks for 120 h at 200 rpm and 37 • c with the initial substrate concentration of 0.034 m. the concentrations of the enantiomers of racemic naproxen methyl ester in organic solvents and those of naproxen in buffer solution were determined with hplc. it was found that the enantiomeric excess for substrate (ee s ), enantiomeric ratio (e) and conversion (x) decreased in the following order: isooctane > cyclohexane > hexane > toluene. the enantiomeric excess for product (ee p ) was found to be the same for isooctane, cyclohexane and hexane where the lowest ee p was obtained in toluene. the highest ee s , ee p , e and x values achieved in isooctane at the residence time of 120 h were 91, 95, 130, and 49%, respectively. this study was supported by ankara university biotechnology institute (project no: 89). fusarium fujikuroi (gibberella fujikuroi mating group c) produces multiple secondary metabolites such as gibberellins and bikaverin. gibberellins are terpenoid hormones that induce growth and regulate various stages of development in plants. they have numerous applications in agriculture industry. bikaverins are polyketides that have toxicity against different organisms because they inhibit respiration. regulation of polyketide and gibberellin synthesis by nitrogen has been intensively studied in fusarium but little is known about their regulation by carbon source. our main interest is to understand the regulation of biosynthesis of these compounds in f. fujikuroi imi 58289. to investigate this regulation the organism was grown in high nitrogen medium under submerged conditions and then transferred to nitrogen-free media having various concentrations of different carbon sources. the gibberellin production was not affected significantly. on the other hand bikaverin was synthesized enormously when sucrose was used as the only carbon source. high production in sucrose required a minimal amount of the sugar, but did not change appreciably above this threshold along a wide range of concentration. the bikaverin synthesis was repressed when glucose coexisted with sucrose in the medium. the effect of the c source on the expression of key genes, cps/ks (copalyl diphosphate synthase/kaurene synthase) for gibberellin and pks4 (polyketide synthase) for bikaverin biosynthesis is currently under investigation. ment of dormancy and loss of viability in seeds with the passage of time, it lacks any systematic propagation from seeds and is typically propagated through rhizomes. this restricts large scale cultivation of this plant. in vitro propagation of plants is an effective means for rapid multiplication of species, in which conventional methods have limitations. in the pesent study we have analysed the role of various growth promoters and the effects of dark and light incubation on germination of n. alba seeds. the results indicate that in vitro propagation of n. alba from seeds can be applied as an efficient method of multiplication. the study was funded by the state planning commisson (dpt) turkey and the university of ankara vide projects no. 120640. this research was performed for developing of biological treatment process of odor gas such as mek, h 2 s, and toluene, which is generated from the food waste recycling process. to establish the operational conditions of odor gas removal by small-scale biofiltration equipment, it was continuously operated by using toluene as a treating odor object. when the odor treating microorganisms were adhered to fibril form biofilter, high removal efficiency over 93% was obtained by biofilm formation. at 400 ppm of inlet odor gas concentration and 10 s of retention time, the removal efficiency was 76 and 93% in first stage reactor and second stage reactor, respectively. however, the removal efficiency remained over 97% at the operational conditions above 15 s of retention time. post soviet countries are going through the transition stage and are extremely sensitive to new technology, economics or social changes and globalization processes. that is why decision-making and system of regulation of the use of gmo's are very sensitive to number of factors. three levels of factors, the most influential to decision-making: global, regional and local; are identified at the research paper. global level depends on external policy of leaders of gmo regulations. usa and eu have the biggest influence on transition countries, though their positions completely differ. as usa was leader in inventing gmos, it is lobbing newly created biotechnological industry. in eu and other european countries lobbing of biotechnological industry was not as strong as in usa. thus, their national law is stricter. world trade organization and international agreements are also part of global level. regional level. geographical position of country is also very important because every regulation system depends a lot on regulation that is implemented in neighboring countries. countries do not exist in vacuum; they are linked territorially, politically, economically and socially with neighboring states. regulation systems of the transition countries in the eastern europe can be divided into three types: those who have no system of regulation of gmo (belarus, romania, hungary and ukraine); who approved some variety that are treated as safe to the market (poland, moldavia and georgia) and who approved all of the gmos (bulgaria, croatia and russia [before 2004]). but even if a country declares not to use gmo it is rather difficult to control import of such products, because of the lack of the testing laboratories, corruption of the state employees, agreements on intellectual property, and institutional country problems. in spite of global and regional tendency of gmos related regulation the most important part is the local level, namely the national regulation system. depending on national level we are choosing priorities at higher levels. as an example of post soviet countries ukraine is taken, as ukraine is one of the largest countries and one of the biggest exporters of agricultural products in europe. research includes the analysis of attitude to gm product, their potential risks and benefits of three categories that influence decision-making the most: farmers, gm experts and non-governmental organizations. recombinant microorganism development for extracellular benzaldehyde lyase production hande kaya 1 , pınar ç alık 1 , tunçer h. ozdamar 2 : 1 iblab, department of chemical engineering, metu, 06531 ankara, turkey; 2 bre laboratory, department of chemical engng, ankara university, 06100 ankara, turkey. e-mail: e119497@metu.edu.tr (h. kaya) in this study, the extracellular production of the benzaldehyde lyase (bal, ec 4.1.2.38) that catalyses the synthesis the enantiopure 2-hydroxy ketones for drug syntheses, by bacillus sp., was aimed. for this purpose, the signal dna sequence of an extracellular bacillus enzyme, i.e., serine alkaline protease, was fused in front of the bal gene (accession number ax349268) from pseudomonas fluorescens biovar i, using pcr-based gene splicing by overlap extension (soe) method. b. licheniformis (dsm 1969) chromosomal dna was used as sap gene (accession number x03341) template for the synthesis of sap signal sequence. bal gene was amplified by using the plasmid carrying bal gene, puc18::bal. thereafter, the signal peptide of sap with its own promoter was fused in front of the bal gene by soe method. the hybrid gene first cloned into puc19 plasmid, thereafter sub-cloned into pbr373, pmk4 and phv1431 shuttle vectors. the escherichia coli-bacillus plasmids carrying the hybrid gene pre(subc)-bal was transferred into bacillus subtilis npr− apr−, and bacillus licheniformis. the influence of the host bacillus species on bal production on a defined medium with glucose was investigated in bioreactor systems. for each of the recombinant (r-) bacillus species, effects of initial glucose concentration on cell growth and bal production were investigated; and, physiological differences and similarities between the wild-type and r-bacillus species are discussed. thereafter, the benzaldehyde lyase production capacities of recombinant e. coli and b. subtilis are compared in terms of cell concentration and bal volumetric and specific activities. for the comparison bal gene was cloned into prseta vector which is under the control of strong t7 promoter and expressed in e. coli bl21 (de3) plyss strain. the variations in by-product distributions with each recombinant organism and yields are also discussed. phosphoketolases (ec 4.1.2.9) are thiamine diphosphate (thdp)-dependent enzymes, that play a crucial role in the pentose phosphate pathway (ppp) of heterofermentative and facultative homofermentative lactic acid bacteria, and of the d-fructose 6phosphate shunt of bifidobacteria. reports affirm that cellulomonas flavigena can use the ppp when is cultured under anaerobic conditions. a genomic library of c. flavigena constructed in -zap express vector was screened. four positive clones were isolated, in vivo excised and the resulting pbk-cmv phagemids, each containing a 4.0 kb insert, were characterized by restriction analysis and dna sequencing. the open reading frame (orf) of the dxylulose 5-phosphate/d-fructose 6-phosphate phosphoketolase gene, xpkl, was located from nucleotide 54 to 2466. the xpkl orf encoded a 804 amino acids-residue polypeptide (xpkl) with a calculated molecular mass of 89,000 da, a value coincident with that estimated by comparative sds-page (about 90,000 da). a putative ribosome binding site (gggagc) is present 11-5 nucleotide upstream of the translational start of the xpkl polypeptide. the c. flavigena xpkl polypeptide sequence was 66% identical to dxylulose 5-phosphate/d-fructose 6-phosphate phosphoketolase from bifidobacterium sp., bifidobacterium gallinarum, gloeobacter violaceus and bifidobacterium adolescentis. this analysis also revealed highly conserved regions. lactococcus lactis is a main diacetyl-producing bacteria by citrate metabolism in dairy products. the transport of citrate in these bacteria is dependent on citrate permease that is encoded by citp gene. previous studies of the citqrp operon in escherichia coli mutants showed that citp message is considerably stabilized in rnase iii mutant. so, in the context of the citrate metabolism research, the characterization of the lactococcal rnase iii enzyme is very important for the dairy industry. rnase iii is an endoribonuclease which has an important role in rrna processing and control of gene expression. with the aim of studying lactococcal rnase iii we have cloned the rnc gene from l. lactis ssp lactis il1403 in the broad host range pls1rgfp vector. this plasmid includes the gfp gene, encoding the green fluorescent protein (gfp), cloned under the control of the pm promoter, that is inducible by maltose. maltose induction of the lactococcal rnc expression showed a 5-fold increase of rnc transcription from this plasmid. activity assays for lactococcal rnase iii were standardized using crude extracts and a substrate specific for b. subtillis rnase iii. the results showed that this substrate was specifically cleaved by lactococcal rnase iii and its activity induced by maltose. lac-rnc was cloned in pet15 vector and the corresponding six-histidine-tagged rnase iii protein was overproduced in e. coli bl21 (de3) strain by iptg induction. the protein was purified by affinity chromatography using hplc system and was shown to be active by in vitro activity assays using the lac-rnase iii specific substrate mentioned above. we have also cloned lactococcal rnc gene and studied its expression in an e. coli rnc deletion mutant ( rnc). complementation assays performed in e. coli demonstrate that the lactococcal rnase iii (lac-rnase iii) is able to process rrnas and to regulate the levels of polynucleotide phosphorylase (pnpase). these results demonstrate that the lactococcal enzyme is able to substitute the ec-rnase iii not only in the rrna processing, but also in the processing of mrnas. the amount of lactococcal rnc transcript in an e. coli rnc strain was 3.3-fold higher than in the wild type strain, suggesting that the e. coli rnase iii triggers the degradation of the heterologous rnc mrnas. the results obtained have shown that lac-rnase iii is an interesting enzyme for biotechnological purposes. objectives: the pharmaceutical and food industry has an increasing demand for selectively glycolized active agents. in our application isomaltose can be synthesized by immobilized dextransucrase, which transfers a glycosyl residue from sucrose (substrate) to glucose (acceptor). as the reaction proceeds, isomaltose can act as an acceptor and is converted into undesired follow-up products called isomalto-oligosaccharides, imos. we investigate on two approaches to avoid imo formation, selective adsorption of isomaltose (ergezinger et al., 2005) and the co-entrapment of dextranase adsorbate, which breaks imos down to isomaltose. results and conclusions: the first part of our research concerns the adsorption of dextranase on bentonite, which complies with langmuir model. at complete saturation (0.8 g g −1 ) our immobilisate exhibits an activity of 16,000 u g −1 . a kinetic analysis does not reveal significant differences between the adsorbed and free form of enzyme (k m,bentonit : 14.1 ± 0.7 m versus k m,free : 13.0 ± 0.7 m). thus, bentonite displays a high binding capacity paired with favorable kinetic properties. beyond that we investigate the activity of dextransucrase in co-immobilisates, which is reduced during coimmobilization due to interactions with the adsorbate. among various co-immobilisates, the one containing dextranase bound to preblotted bentonite imparts the highest activity (40% as compared to control: immob. dextransucrase). the molar yield coefficient of coimmobilisates y isomaltose/sucrose surpasses coefficient of control by 13%. further on we will characterize mass transfer of dextranase substrate into alginate matrix as well as bentonite-dextransucrase interactions. and kefir. a second approach is to use yeast as a production organism to produce natural folates for fortification. here we investigate and discuss the folate content in skq2n, a diploid strain of saccharomyces cerevisiae, when cultured in different media and at different stages of growth. the aim is to gain a basal knowledge of the folate production profile, forms of folate produced and degree of leakage to the surrounding medium, in relation to the culturing medium and physiological state of the cells. danisco innovation, danisco a/s, langebrogade 1, po box 17, dk 1001 copenhagen k, denmark we at danisco a/s (copenhagen, denmark) have revealed a new starch degrading pathway by the discovering several new enzymes and metabolites in fungi and algae. these new enzymes include glucan lyases, dehydratases and tautomerases, which proved to be useful in biocatalysis. these new metabolites proved to be useful as both antioxidants and antimicrobials for food and non-food applications. this pathway is named as anhydrofructose pathway of starch and glycogen degradation. this technology is referred to as the anhydrofructose technology. diet is evolving from nourishing populations via providing essential nutrients to improving health of individuals through nutrition. modern nutritional research focuses on health promotion and disease prevention, on protection against toxicity and stress, and on performance improvement. as a consequence of these ambitious objectives, the disciplines "nutrigenetics" and "nutrigenomics" have evolved. nutrigenetics asks the question how individual genetic disposition, manifesting as single-nucleotide polymorphisms (snps), copy-number polymorphisms (cnps) and epigenetic phenomena, affects susceptibility to diet. nutrigenomics addresses the inverse relationship, i.e. how diet influences gene transcription, protein expression and metabolism. the mid-term objective of nutrigenomics is integrating genomics (gene analysis), transcriptomics (gene expression analysis), proteomics (global protein analysis) and metabolomics (metabolite profiling) to define a "healthy" phenotype. the long-term deliverable of nutrigenomics is personalised nutrition for maintenance of individual health and prevention of disease. the major challenges for -omics in nutrition and health still lie ahead of us, some of which apply to -omic disciplines in general while others are specific for -omic discovery in the food context: (i) the integration of gene-and protein expression profiles with metabolic fingerprints is still in its infancy as we need to understand how to (a) select relevant sub-sets of information to be merged, and (b) resolve the issue of the different time-scales, at which transcripts, proteins and metabolites appear and act; (ii) the definition of health and comfort is less of a clear-cut case than the one of disease; (iii) -omics in nutrition must be particularly sensitive: it has to reveal rather many subtle than a few abundant signals to detect early deviations from normality; (iv) in the food context, health cannot be uncoupled from pleasure, that is, food preference and nutritional status are interconnected. transcriptomics serves to put proteomic and metabolomic markers into a larger biological perspective and is suitable for a first "round of discovery" in regulatory networks. metabolomics, the comprehensive analysis of metabolites, is an excellent diagnostic tool for consumer classification. the great asset of this platform is the quantitative, non-invasive analysis of easily accessible human body fluids like urine, blood and saliva. this feature also holds true to some extent for proteomics, with the constraint that proteomics is more complex in terms of absolute number, chemical properties and dynamic range of compounds present. proteomics in the context of nutrition and health has the potential to (a) deliver biomarkers for health and comfort, (b) reveal early indicators for disease disposition, (c) assist in differentiating dietary responders from non-responders, and, last but not least, (d) discover bioactive, beneficial food components. independent of the context of application, proteomics represents the only platform that delivers not only markers for disposition or condition but also targets of intervention: the only way to intervene in a biological condition and to modulate its outcome is interfering with the proteins involved. it is evident that not only comprehensive analyses with one discovery platform (lateral integration of information) are required but also vertical integration between different -omic levels are indispensable for a deeper understanding of disposition, health, environment and diet (desiere, 2004) . a major "vertical integration issue", to date unresolved, is given by different timescales of transcript production, protein expression and metabolite generation (nicholson et al., 2004) . the transcript machinery usually responds fast to an external stimulus (seconds to minutes), the proteins may be expressed within minutes to hours (and have a halflife from minutes to even months) and metabolites vary significantly during the day and depend on latest dietary input. this means that data, which seem to correlate qualitatively (e.g. reflecting the same pathway), may not necessarily be related time-wise. rather, they may represent different responses at different time points and, possibly, to different stimuli. comprehensive -omic analyses is an essential building block of "systems biology", which can be defined as follows (clish et al., 2004) : systems biology is the comprehensive analysis of the dynamic functioning of a biological system (cell, organ, organism or even ecosystem) at gene, protein and metabolite (or higher organizational) level, achieved by comparison of two defined biological states of this system, typically before and after perturbation. while a comprehensive list of components (genes, proteins, metabolites) of a given biological system is a pre-requisite for this kind of research, the main reasoning for the "system view" is that only information on the interactions between the components gives clues to function of the entire network. a systems biology approach has recently demonstrated the power of proteomics to dissect immunity and inflammation. toll-like receptor recognition and signalling was elucidated and showed, how bacterial "barcodes" are read and interpreted in order to trigger an adapted immune response (aderem and smith, 2004) . in order to address some of the challenging objectives of -omics-driven nutritional research, we have addressed (a) the effect of early antibiotic administration on the maturation of intestinal tissues, (b) protein discovery in human milk, (c) the effects of polyunsaturated fatty acids on gene expression and lipid profile in the liver, and (d) biomarkers for intestinal stress. (a) antibiotics and gut maturation: the effects of early administration of antibiotics on intestinal maturation were assessed at the gene expression level in a rat model. (b) human milk: rapid enrichment and iterative, consolidated identification of immunologically relevant milk proteins was achieved through the employment of restricted-access media and a tailored proteomic strategy (labéta et al., 2000; lebouder et al., 2003; panchaud et al., 2005) . (c) fatty acids and liver transcriptome/lipidome: epidemiological studies have correlated higher intakes of poly-unsaturated fatty acids (pufas) with lower incidence of chronic metabolic disease. the molecular mechanisms regulated by pufa consumption were examined assaying the liver transcriptome and lipid metabolome of mice fed a control and a pufa-enriched diet (mutch et al., 2005) . (d) gut stress markers: as a first step, we catalogued protein expression along the jejunum, ileum and colon of the rat intestine and found gut segment-specific proteins (marvin-guy et al., 2005) . the innovative combination of a neonatal separation model with proteomic analysis allowed us to study, whether early life psychological stress may impact the adult gut neuromuscular protein expression and the approach revealed specific protein biomarkers. omics for engineering lactic acid bacteria willem m. de vos wageningen center for food sciences, wageningen university, the netherlands. e-mail: willem.devos@wur.nl. url: http://www.wcfs.nl/ lactic acid bacteria (lab) are high at-rich gram-positive bacteria that have a well-established record in industrial food fermentations where they contribute to conservation, flavour and texture. in addition, several lab are used as food-grade hosts for the production of enzymes, peptides or metabolites. finally, lab are exploited in functional foods that contribute to the health and well-being of the consumer. a variety of metabolic engineering approaches have allowed for the improvement of many attributes of lab. these approaches have been facilitated by the possibility of uncoupling of growth and metabolite production in lab, the wealth of genetic tools that allow modulation of gene expression in a dynamic range, and the determination of several complete lab genomes (de vos et al., 2005) . we have developed lactobacillus plantarum as a paradigm for lab engineering by experimental and modelling approaches, the application of functional and comparative genomics, and the implementation of other post-genomics avenues (kleerebezem et al., 2003; smid et al., 2005; de vos et al., 2004) . examples of optimizing the production of vitamins and other cofactors, the impact of these engineering approaches on the global transcription and metabolite profiles, and determining the l. plantarum activity in the human host will be discussed. solanum tuberosum (potato) is the fourth major crop worldwide and used for food, feed and biotechnological applications. to fully realize the biosynthetic potential for production of starch, protein and metabolites, we conducted an extensive quantitative profiling of the expressed genes of mature potato tuber. a total of 58,322 sage (serial analysis of gene expression) tags of 19 nt representing 22,235 different tags were analyzed. the 695 tags seen 10 or more times were assigned a tentative function by comparison to homologous genes. contrary to the transcript profile of rice seedlings (gibbings et al., 2003) the storage organ of potato is not dominated by transcripts encoding storage proteins. transcripts for four types of protease inhibitors, a metallothionein and a lipoxygenase were more prominent than patatin isoforms. the lactic acid bacterium lactococcus lactis is used extensively in the production of fermented milk products. during cheese production the bacterium experiences many changes in its immediate environment, as a result of its own reactions. the most severe change is the accumulation of lactic acid, which changes the ph of the medium until growth is totally inhibited. we have focused upon a survey of these dairy related stress responses, as a means of constructing more robust strains. when l. lactis starter cultures are produced in rich media, they will experience an initial period with purine limitation after being added to the milk substrate, a stress condition that in several studies have been found to induce cross resistance towards a number of other stresses. we have analyzed both general purine nucleotide (atp and gtp) and specific gtp limitation in chemi-cally defined medium, using both proteomics and transcriptomics. the differential expression analyses were performed with a custom designed dna microarray of pcr amplified probes. the two stress conditions resulted in very different stress responses, both at the transcriptomic and proteomics level. from a new study on the temporal expression pattern of l. lactis during growth in milk, we present preliminary data showing differential expression of genes and proteins of the purine stress stimulon as well as other stress stimulons. cell physiology of the yeast saccharomyces cerevisiae glucose repression mutants ∆snf1, ∆snf4 and ∆snf1∆snf4 was studied in batch and glucose limited chemostat cultivations. detailed physiological studies were performed on cells grown in batch using glucose, galactose, or glucose-galactose mixture as a carbon source. during growth on glucose-galactose mixtures it was shown that after glucose was consumed, galactose consumption remained repressed for about 15 h in ∆snf1 or ∆snf4 mutants, and for more then 40 h in ∆snf1∆snf4 mutant, whereas it only lasted 6 h in wild-type cells. the global transcriptional response in the glucose repression mutants was studied using chemostat cultures. s. cerevisiae wild type and the mutants were grown in glucose limited aerobic chemostats at a dilution rate of 0.1 h −1 . biological triplicates were performed for each strain. to identify transcriptional responses of the glucose repression mutants, statistical tests, clustering method and a model-driven analysis method were used. the global transcription data analysis experiments showed that genes involved in hexose transport, carbohydrates metabolism, respiration, and signal transduction were differently expressed in ∆snf1 and ∆snf4 mutants comparing to wild type cells. combination of gene expression data and gene-scale metabolic model indicated changes in the metabolic sub-networks among studied glucose repression mutants. genomics technologies have recently been introduced into food and nutrition science for identifying targets of molecular actions of nutrients as well as non-nutrient components of foods. changes in the transcriptome, proteome and metabolome have been determined for assessing the molecular actions of zinc as an essential micronutrient and of flavonoids in processes such as colon carcinogenesis and atherosclerosis. zinc is essential for the structural and functional integrity of cells and plays a pivotal role in the control of gene expression. zinc deficiency effects in human cells and an animal model (rats) were analyzed by microarrays and showed that a low intracellular zinc concentration caused major alterations in the steady-state mrna levels of several hundred target genes-dependent on the tissues studied including liver, brain, muscle, intestine and kidney. proteome analysis from the same samples by 2d-page followed by peptide mass fingerprinting via maldi-tof-ms identified similarly a large set of proteins with altered expression level but allowed a common theme of action to be identified. although pleiotropic in first view, the obtained pattern of zinc-affected genes/proteins may represent a reference for defining the zinc regulon in mammalian cells. flavonoids occurring in large number in plant species are considered protective agents in a variety of processes including inflammation and cancer development. we have studied the effects of around 80 selected flavonoids in a screening program and identified for compounds such as flavon, genistein or quercetin by genomics technologies their putative mode of action in colon cancer models and endothelial cells. as part of a collaborative effort employing human endothelial cells and blood mononuclear cells from a human intervention trial with soy isoflavones (genistein/daidzein) the effects of the flavonoids on the stress-response to oxidized ldl and homocystein was analyzed. a set of markers of anti-inflammatory and anti-apoptotic activity was identified for genistein and daidzein and cell biological studies confirmed that both compounds prevented programmed cells death in stressed endothelial cells. in the food processing industry, unwanted presence of extremely heat-resistant bacterial endospores creates major problems due to their capability to survive classical thermal treatments and their ability to subsequently germinate and form actively growing vegetative cells. screening of spoilage isolates using genomic typing techniques to visualise putative genome-based biomarkers allowed us to classify strains according to the degree of thermal resistance of their spores. in addition, we showed that sporulation in the presence of ingredients rich in calcium ions promotes thermal resistance of developing spores and correlates with the expression of specific (marker) genes (see oomes and brul, 2004) . finally, the molecular program that forms the basis of spore germination has been assessed using genome-wide expression analysis. noticeably genes involved in dna-repair were transiently expressed in germinating wild-type spores. also, surprisingly, it was found that spores contain significant levels of ribosomal and messenger rnas. degradation of these rna molecules upon spore thermal injury was found to be characteristic for their thermal resistance and predictive for their subsequent outgrowth behaviour. this finding is currently being patented. the information on spore presence, predictions of their thermal resistance and process survival chances, is used to structure a process management system to facilitate optimal food quality assurance and allow for real time analysis in case of the need for quality control. the information on spore presence, predictions of their thermal resistance and process survival chances is now being integrated. this is used to formulate the conditions for a process management system with state of the art food production quality assurance, which allows for real time analysis in case of the need for quality control. the interplay of the pectinase spectrum of aspergillus niger as revealed by dna microarray studies elena martens, jac benen, johan van den berg, peter schaap fungal genomics group, laboratory of microbiology, wageningen university, dreijenlaan 2, 6703 ha wageningen, the netherlands. e-mail: elena.martens@wur.nl (e. martens) the saprobic fungus aspergillus niger is an efficient producer of extracellular enzymes many of which show carbohydrate modifying activities. these enzymes have gras status and therefore are widely used in the food and feed industry. after determination of the genomic sequence of a. niger by dsm, it was estimated that only a fraction of the potential of secreted enzymes is currently characterised. database mining using the proprietary genome sequence has resulted in the identification of more then 80 genes encoding enzymes involved in the depolymerisation of the back bone and the site chains of the complex polysaccharide pectin. additional enzymatic activities required to remove methyl and acetyl esters, present in pectin were also observed. by using dna microarrays we have sought to gain insight into the complex regulation of all the genes involved in pectin degradation. a. niger was cultivated on sugar beet pectin and on the monomeric constituents of pectin, viz. galacturonic acid, rhamnose and xylose. subsequently the corresponding transcriptomes were analysed. we will report on our findings concerning the regulation of the expression of the genes involved in the degradation of pectin and its main constituent-galacturonic acid and the consequences for the interplay of the encoded (novel) enzymes. since reactive oxygen species (ros) are formed in all living organisms a wide range of antioxidative enzyme systems are present to keep the system in balance. when an animal is slaughtered the cellular anti-oxidative capability is reduced, resulting in an accumulation of ros followed by an increased oxidation of dna, lipids and proteins. generally lipid oxidation is a well-known problem, causing increased rancidity during prolonged storage, of especially fatty fish. the implications of protein oxidation are, however, more unclear also in respect to quality decay of fish. protein oxidation causes a wide variety of amino acids modifications, where of the most studied is carbonylation of proline, argenine, lysine or threonine. these carbonyl groups can be labelled with 2,4-dinitro-phenylhydrazine. combining two-dimensional gel electrophoresis with immunoblotting enables the detection of carbonyl groups for each single protein. the results presented here, reveal that both during frozen storage and tainting of rainbow trout protein oxidation/carbonylation increases, furthermore there is an increase in oxidation/carbonylation for distinct proteins. anne-marie neeteson european forum of farm animal breeders, benedendorpsweg 98, 6862 wl oosterbeek, the netherlands society is concerned about food, animal welfare, food safety, new technologies, scientists and industry. these elements are all present in genomics for farm animals. therefore, it is important to build awareness in scientists and industry, start a dialogue with stakeholders and society, and to be transparent in a pro-active way. this paper will address the issues at stake for scientists and industry, when it comes to genomics and animal health. it will combine the results of imperical, ethical and sociological efforts in three eu funded projects. (c) the proper use of genomics in relation to infectious diseases in production animals, and the role of the scientist in the development in new technologies in this field, are being addressed in european animal disease genomics network of excellence (ead-gene, http://www.eadgene.org/). some observations are that: (1) genomics does not concern changing the gene. however, acceptability of any discovery dealing with living beings and edible products, is not obvious just like that! animals have a symbolic and emotional load. (2) genes are still related to eugenics, in the mind of people. genes as such cause reluctance, but it is seen as positive if the use of medication can be reduced, and if animals will be better resistant to disease. (3) consumers are in favour of consumer education, compulsory labelling and the imposition of minimum standards. the inclination to pay more for foods produced according to desired standards relates closely to income level. (4) animal welfare is the major issue citizens mention as a concern. the focus of breeding organisations on productivity should be counterbalanced by serious attention to the animal's needs in order to avoid unnecessary negative impact on the welfare of the animals. (5) when technical specialists and lay people communicate, they tend to use different languages: they use the same words, but with rather different interpretations. so transparency of breeding practices and clear definitions of terminology will be essential for effective communication among all stakeholders. (6) food safety and human health are the major concern for most people, when it comes to making a choice. during the latest decades research within the field of animal genomics has in general been following the same strategies as those used within the field of human genomics, although with much less resources. the porcine genome has been characterized intensively through the development of linkage maps, comparative maps and physical maps. until a few years ago it had not been anticipated that it would be possible to embark on whole genome sequencing of animals genomes. however, because of technological developments and much lower costs for sequencing, several animal genomes have now been assembled/are on the way to being assembled. the initial step towards sequencing the porcine genome was taken by the sino-danish pig genome project. the efforts within this project have now generated approximately 3.84 million genomic shotgun sequences and 700.000 expressed sequence tags (ests). the shotgun sequences have been included in a three-species alignment to make an initial evolutionary analysis. the results show that pig is much closer to human than mouse is. the ests represent 5 -end sequences from a total of 98 non-normalized cdna libraries. based on assembly and annotation of the ests the structure of the porcine transcriptome has been analysed. the relevance of assembling the porcine genomic sequence is justified both from the perspective of sustainable animal breeding and from the fact that the porcine model is an important research platform because of the anatomical, physiological, biochemical and metabolically similarities with man. examples of functional genomic studies both aimed at sustainable animal breeding and aimed at exploiting the pig as a model for medical studies will be discussed. genomics refers to global, systematic and high throughput approaches that allow collecting large amount of data and thus offer new possibilities for analysis and understanding biological processes. we will present some new knowledge related to reproduction in farm animals resulting from three different strategies. (1) functional analysis of gene and protein expression: the transcriptome and the proteome analysis allowed to identify new genes and proteins whose expression is associated with processes of ovarian follicular growth and atresia as well as oocyte maturation in bovine and porcine species, maturation of spermatozoa in the different compartments of epididymis. farm animals produce food as cost effectively as possible, however this may have negative side effects for their health and welfare. trade off processes between production on one hand and reproduction and health on the other hand play a crucial role. the principles of selective breeding for the best of naturally occurring variation has proven to be able to balance an increased level of production and quality of life for the animal. every year, the economic value of the genetic gain achieved by the breeders and carried over to the producers is 1.5% of the economic value of eu farm animal production. consequently, a conservative estimate of the gain from animal breeding is, every year d 1.2 billion in europe. recent developments, such as the sequencing of the genomes of the human, chicken and cow, together with high throughput laboratory techniques, means that there are new opportunities to enhance quality of life. the goal of this paper is to give an overview of the options offered by genomics for enhanced quality of life with focus on identifying relevant gene variants and technologies for large scale tracking and tracing. selective breeding for the best of naturally occurring variation remains the same as in traditional systems, but by pinpointing the relevant gene variants along genomics it is possible to identify directly the animals best selected for high production without comprising health and welfare. the combination of full genome sequences, software tools, study of functional physiological processes cost-effective high-throughput snp genotyping and comparative mapping have the (proven) potential to identify relevant gene variants, e.g. pork color, boar taint, general disease resistance. functional mutations have direct option of application in breeding programs. unfortunately this is not the case for genetic markers due to cost of genotyping and inconsistent phenotypic effects. new technologies for snp genotyping are cost effective and enable large scale genotyping (1.000 of animals/day). a selection of the best technology and strategic use of these opportunities enable tracking and tracing. the application of this technology offers new opportunities for quality of life, both for animal and humans. background: studies have shown that prebiotic and probiotic consumption alters the gastrointestinal flora, modulates the immune system, inhibits genotoxicity and has a protective effect on colon carcinogenesis. however, the effect of synbiotic consumption on these parameters in subjects at risk of colon cancer has not until now been investigated. aim: to determine if a synbiotic (prebiotic and probiotics together) modulates cancer risk biomarkers in human subjects at risk of colon cancer. methods: a 12-week randomised, double blind, placebo controlled, ethically approved trial of a food supplement containing lactobacillus gg, bifidobacterium bb-12 and raftilose synergy 1 (prebiotic) was performed in 37 colon cancer subjects who had undergone 'curative resection'. faecal and blood samples were obtained before (t1, week 0) midway through (t2, 6 weeks) and following intervention (t3, 12 weeks). rectal biopsies were obtained at t1 and t3. the effect of synbiotic consumption on the faecal flora was assessed using standard plate count techniques. genotoxic damage was measured in single cells derived from biopsies using the comet assay. fw was prepared by diluting faeces 1:1 in dmem, ultracentrifugation and sterile filtration. the genotoxic (comet assay) and cytotoxic potential (almar blue assay) of fw was determined. peripheral blood mononuclear cells were isolated from blood and cytokine production in vitro assayed by elisa. natural killer cell cytotoxic activity and the phagocytic and respiratory burst activity of monocytes and granulocytes in whole blood were determined by flow cytometry. results: in the synbiotic group faecal numbers of bifidobacteria significantly increased (p < 0.001) and lactobacilli increased although not significantly (p = 0.0674) while coliforms decreased (p < 0.05). enterococci, clostridium perfringens and bacteroides were unaffected. in the placebo group bifidobacteria decreased (p < 0.001), the other bacterial groups were unaffected. in biopsies genotoxic damage was increased in the placebo group at t3 versus t1 (p = 0.0301) but was unchanged in the synbiotic group. the genotoxic and cytotoxic potential of fw was unaltered. synbiotic consumption significantly increased (p < 0.05), ifn-␥ production by pbmcs but il-2, il-10, il-12, and tnf-␣ production was unaffected. natural killer cell, phagocytic and respiratory burst activities were unaltered. conclusion: synbiotic consumption did not have a strong immunomodulatory effect on the systemic immune system in this study, nor did it influence the genotoxic and cytotoxic potential of fw. however, synbiotic consumption altered the composition of the gut flora to a more beneficial composition as well as protecting against genotoxic damage in vivo, suggesting a protective effect of synbiotics against colon carcinogenesis. emmaårsköld, malin svensson, halfdan grage, peter rådström, ed w.j. van niel applied microbiology, lund institute of technology, lund university, p.o. box 124, sweden lactobacillus reuteri is used today in a variety of dairy products as a probiotic bacterium. several lactic acid bacteria have the ability to produce different kinds of exopolysaccharides (eps), which have the potential to be used as an alternative biothickener. however, the yield of eps is too low to be profitable in the food industry. to optimise the environmental conditions for eps formation a l. reuteri strain was chosen for a factorial design study. the factors used in this experiment were temperature (30-43 • c), ph (4.5-6.5) and sucrose concentration (50-150 g/l); for each factor three different values were chosen. the strain was grown in batch mode using a semi-defined medium at constant ph and sucrose as the carbon source. the results obtained with 27 fermentations revealed that the highest eps formation was found at 37 • c, ph 4.5 and 100 g/l of sucrose. sucrose did not further affect the eps formation above a concentration of 100 g/l. temperature and ph were significant for the eps formation, but only temperature was significant for growth. a central composite design study was chosen for further optimization of the ph and sucrose concentration for maximum eps formation. also the gene expression of the sucrase enzyme responsible for eps formation was investigated using qrt-pcr. the data were used to develop a model for growth and eps formation. dendritic cells (dc) play a pivotal immune regulatory role in the th1, th2 and treg cell balance. dc are present in the gut mucosa and may thus be target for modulation by gut microbes. here, we screened a large panel of human gut-derived lactobacillus and bifidobacterium spp. for dc polarizing capacity: bone marrow-derived murine dc were exposed to lethally irradiated bacteria and cytokine and dc surface markers were analyzed. substantial differences were found among strains in their capacity to induce proinflammatory cytokines, while the differences for anti-inflammatory cytokines were less pronounced. bifidobacteria were weak il-12, il-23 and tnf-␣inducers, while both strong and weak cytokine-inducers were found among the strains of lactobacillus. remarkably, strains weak in il-12 induction inhibited il-12, il-23 and tnf-␣ production induced by otherwise strong cytokine-inducing strains, while il-10 production remained unaffected. those lactobacilli with greatest capacity to induce il-12 were also most effective in up-regulating surface markers. surface marker up-regulation was however reduced in the presence of weak il-12-inducing strains. in conclusion, human lactobacillus and bifidobacterium spp. polarize differentially dc maturation. thus, the potential exists for th1/th2/treg-driving capacities of the gut dc to be modulated according to composition of gut flora including ingested probiotics. cell surface-associated glycolytic enzymes from lactobacillus plantarum 299v mediate adhesion to human epithelial cells and extracellular matrix proteins s.m. madsen, j. glenting, a. vrang, p. ravn, h.k. riemann, h. israelsen, m.r. nørrelykke, a.m. hansen, m. antonsson, s. ahrné, h.c. beck bioneer a/s, hørsholm dk-2970, denmark among the main selection criteria of lactic acid bacteria for probiotic use, the ability to adhere to intestinal epithelial cells, mucus, or extracellular matrix proteins is considered important. using a proteom based approach we identified a group of novel surface proteins that are non-covalently bound to the cell wall of the probi-otic bacterium lactobacillus plantarum 299v. surface proteins were extracted and analysed by gel electrophoreses followed by mass spectrometry analysis. the surface proteins included glycolytic enzymes like, e.g glyceraldehyde 3-phosphate dehydrogenase, which usually is a typical intracellular enzyme. a collection of lactobacillus species was screened and the phenomenon of surface-associated glycolytic enzymes was found in many of the analyzed species. this is to our knowledge the first example of surface-associated glycolytic enzymes in probiotic bacteria. however, in pathogenic bacteria these enzymes are well known and their surface localization is involved in adhesion to human epithelial cells and invasion. we suspect that the presence of these enzymes on the surface of probiotic bacteria could prevent adhesion of pathogenic bacteria and possibly also involve other probiotic activities such as immune modulation. binding studies showed that the surface-associated glycolytic enzymes of lb. plantarum 229v were able to bind to caco-2 intestinal cells and extracellular matrix proteins like fibronectin. scientific and industrial interest on exopolysaccharides (eps) synthesized by microorganisms and on their chemico-physical properties has been quickly growing in the last years. many strains of lactobacilli produce eps allowing them to adhere to human mucosae and therefore to have probiotic effects such as the stimulation of immune response and even antitumoral activity of these molecules has been claimed. futhermore prebiotic actions of eps beneficially affect the human host health improving the properties of indigenous microflora. in this research we have studied two novel interesting lactobacilli strains: lactobacillus plantarum dsmz 12028 and a particular human isolated strain of lactobacillus crispatus that have probiotic potentialities and are good eps and l(+)-lactic acid producers. the aim of this work has been to characterize these strains and their metabolites (eps, organic acid and bacteriocins), to state them as probiotics and to study their adhering ability on human cells. we have studied the physiology of l. plantarum and l. crispatus in shaking flasks as well as their optimal fermentation conditions to obtain high cell density cultures suitable for use as starters in the food industry and eventually for probiotic preparations. fermentation experiments have been performed in a bioreactor equipped with microfiltration (mf) modules using a semidefined medium and various carbohydrates in different culture conditions (aeration, temperature). the kinetic of eps production has been followed and according to results both strains have shown a growth-related production ranging from 200 to 400 mg/l. in vitro studies concerning the ability of these strains to adhere to human mucosae are in progress as well as the structural characterization of the exopolisaccharides. previous studies have shown that compression coating improves the storage stability of freeze-dried lactobacillus acidophilus, although this stability is related to the degree of cell injury, which in turn is related to the compression pressure used. compression coating has also been found to improve the survival of freeze dried l. acidophilus during exposure to simulated gastric fluid (sgf). the aim of the present work is to create a compression coated l. acidophilus formulation, with targeted release at the terminal ileum and beginning of the colon in the human gastrointestinal tract. dissolution studies were performed using a phosphate buffer with a ph of 2 and 6.8, to simulate gastric fluid and intestinal fluid (sif), respectively. cell viability was monitored using multi-parameter flow cytometry (mpfc), together with traditional cfu/ml counts. mpfc was used to identify live, dead and stressed cell populations, using the fluorescent stains propidium iodide (pi), 3,3 -dihexylocarbocyanine iodide (dioc 6 (3)) and to-pro-3. results show that an enteric coating material, eudragit l100-55, is both suitable for compression coating, and enhancing the survival of cells when exposed to sif. pectin usp 100 has also been shown to promote targeted release of the cells. the opium poppy papaver somniferum contains more than 80 tetrahydrobenzylisoquinoline-derived alkaloids. it is the source of the narcotic analgesics codeine and morphine, which accumulate in specialized internal secretory cells called laticifers. in the aerial parts of the plant, the laticifer cells are anastomosed, forming an articulated network. laticifers are found associated with the vascular bundle in all plant parts. the morphinan alkaloids morphine, codeine and thebaine are found both in roots and in aerial plant parts and specifically accumulate in vesicles within laticifers. the benzo[c]phenanthridine alkaloids sanguinarine and 10-hydroxysanguinarine are found in root tissue. the syntheses of sanguinarine and of the tetrahydrobenzylisoquinoline latex alkaloid laudanine are completely understood at the enzyme level. nearly all enzymes of morphine biosynthesis have also been described. in more recent years, cdnas encoding enzymes of alkaloid biosynthesis in p. somniferum have been isolated and characterized. the cell-specific localization of several of the enzymes of morphine, sanguinarine and laudanine biosynthesis has also been described. with knowledge of many of the gene of alkaloid formation and their sites of expression, the metabolic engineering of p. somniferum for tailored alkaloid profiles is now being undertaken. an agrobacterium tumefaciens-based transformation and a regeneration protocol have recently been developed specifically for narcotic tasmanian cultivars. the various cdnas encoding genes of alkaloid biosynthesis in p. somniferum are being systematically reintroduced into the plant to achieve engineered plants with altered alkaloid profiles. the first results have now been obtained with sense and antisense genes stably expressed in a regenerated tasmanian cultivar. the ultimate goal of exploiting the genes of alkaloid biosynthesis is to produce transgenic medicinal plants of specific alkaloid content that would facilitate commercial production and improve our understanding of the factors that regulate biosynthesis as well as provide experimental systems with which to investigate the ecological role of alkaloids in planta. integrated transcript and metabolite profiling for gene discovery in plant natural product pathways richard a. dixon, lahoucine achnine, bettina deavours, mohammed farag, marina naoumkina, lloyd w. sumner plant biology division, samuel roberts noble foundation, ardmore, ok 73401, usa the rich diversity of chemical structures found in the plant kingdom arises in large part from a limited number of basic chemical scaffolds (e.g. terpene, polyketide) that are modified by a limited number of chemical substitution types (hydroxylation, glycosylation, acylation, prenylation, o-methylation, etc.) . much of the diversity is brought about by the substrate-and/or regio-specificities of the substitution enzymes. in contrast to the large collections of gene sequence and transcript level data available on-line, little detailed information exists on the plant (secondary) metabolome. promiscuity of substrate specificity in vitro may complicate attempts to assign functions to genes of secondary metabolism accessible to researchers through various cdna library collections. using the isoflavonoid and triterpene pathways in medicago species as examples, we describe how integrated metabolite and transcript profiling approaches can aid functional genomics, help explain metabolic regulation, and provide tools for assessing the impacts of genetic modifications in plant secondary metabolism. focused and non-targeted approaches were used to assess the impact associated with introduction of new high flux pathways in arabidopsis thaliana by genetic engineering. transgenic a. thaliana plants expressing the entire biosynthetic pathway for the tyrosine derived cyanogenic glucoside dhurrin as accomplished by insertion of cyp79a1, cyp71e1, and ugt85b1 from sorghum bicolor accumulated 4% dry-weight dhurrin with marginal inadvertent effects on plant morphology, free amino acid pools, transcriptome and metabolome. in a similar manner, plants expressing only cyp79a1 accumulated 3% dry-weight of the novel tyrosine derived glucosinolate, p-hydroxybenzylglucosinolate with no morphological pleitropic effects. in contrast, insertion of cyp79a1 plus cyp71e1 resulted in stunted plants, transcriptome alterations, accumulation of numerous new glucosides derived from detoxification of intermediates in the dhurrin pathway, and in loss of the brassicaceae specific uv protec-tants sinapoyl glucose and sinapoyl malate as well as kaempferol glucosides. the accumulation of new glucosides in the plants expressing cyp79a1 and cyp71e1, was not accompanied by induction of glycosyltransferases, demonstrating that plants are constantly prepared to detoxify novel xenobiotics. the pleiotrophic effects observed in plants expressing sorghum cyp79a1 and cyp71e1 were complemented by retransformation with s. bicolor ugt85b1. accordingly, insertion of high flux pathways directing synthesis and intracellular storage of high amounts of natural products is achievable in transgenic plants with marginal inadvertent effects. arabidopsis thaliana-distinct function in gene transcription dynamics? jeppe madura larsen, brian stougaard vad, søren mølgaard, kell andersen, mads n. davidsen, klaus d. grasser department of life sciences, aalborg university, 9000 aalborg, denmark in recent years it has been shown that introns to some extent can regulate expression in the eukaryotic cell. insertion of introns in the 5 utr in arabidopsis genes has shown to increase gene expression at both rna and protein level. in order to investigate if 5 utr introns have distinct characteristics, we analyse these in the well annotated arabidopsis thaliana genome published by the arabidopsis genome initiative. 12,898 loci annotated with full-length cdnas were analysed and 1989 loci (15.4%) containing 5 utr introns were isolated. we studied if the genes containing these introns showed different patterns in alternative splicing and gene function (gene ontology classification) compared to the remaining genes not containing this intron type. 1802 of the isolated loci (90.6%) contained only one 5 utr intron and these were used for further analysis, where it was investigated if the 5 utr introns had a characteristic size distribution. genes containing transcripts with 5 utr introns were more subjected to alternative splicing (9.63% versus 2.39%) and had a tendency to be more involved in cell regulatory functions compared to genes without this intron type. it was also found, that 5 utr introns was characteristically size-distributed. we identified thee predominant sizes of approximate 110, 270 and 390 bp compared to only one for orf introns. this suggests widespread multiple splicing events in 5 utr introns. the results presented here suggest that 5 utr introns have distinct characteristics and function in gene transcription dynamics. cytochrome p450 monooxygenases appear to be involved in the biosynthetic pathways of a large variety of primary and secondary metabolites in microbial, animal and plant cells. in particular, cytochrome p450 scc catalyzes the conversion of cholesterol into pregnenolone-the precursor of all steroid hormones in mammalian steroidogenic tissues. cytochromes p450 are also involved in the biosynthesis of different plant steroid derivates that play important role in regulation of plant growth and development. therein, investigation of possible influence of cytochrome p450 scc expression on plant regulatory system is of a great interest. this report devoted to the investigation of transgenic tobacco plants, which have been generated by the transformation with recombinant plasmid pgbp450f constitutively expressing cyp11a1 cdna of the bovine cytochrome p450 scc . the transgenic state of the plants was confirmed by southern blot analysis. transgenic plants are phenotypically different from the control ones. in particular, they obtained a substantially higher growth rate and are larger than wild type plants. we have demonstrated that incubation of fragments of the transgenic plants leaves in [ 14 c]-labeled cholesterol containing medium results in formation of the radioactively labeled product with chromatographic mobility corresponding to pregnenolone. the presence of this metabolite in the steroid fraction of lipid extracts obtained from the transgenic plants leaves was confirmed by gas chromatography mass spectrometry (gc-ms) method. the data obtained indicate that cytochrome p450 scc synthesized in transgenic plants displays its specific catalytic activity. biotechnological production of glucose isomerase enzyme with streptomyces olivochromogenes for production of fructose syrup from hydrol m. hashemiravan, a. sadat barikani department of food science and technology, azad university (pishva, varamin unit), institute of food science and agriculture, tehran, iran the use of glucose isomerase for isomerization and production of fructose syrup was performed by selected industrial strain of streptomyces olivochromogenes ptcc 1457. growth of microorganism and production of enzyme in different culture media was studied, and effects of different parameters such as phosphate and aeration was evaluated. the growth of microorganism at 28 • c, caused a production of high amount of enzyme. the production of enzyme was considered in two culture media (a and b). medium (a) was selected for the higher production amount of enzyme. the highest amount of enzyme production was seen in medium a, which was 36.4 giu/ml, after 80 h. the use of baffles in culture flasks, increased the amount of enzyme production, four times more. the production of enzyme was increased, 1.25 times more, in phosphate deficient medium. the cells containing enzyme (intra cellular glucose isomerase) was separated by centrifuge, and extraction and release of enzyme was performed by ultra sonication, that is a physical-mechanical method. this method released about 89.9% of total intracellular enzyme. the best length of time for sonication was found to be 4 min. experiments showed that optimum ph and temperature of the enzyme were 7.5-8 and 80 • c, respectively. the highest activity of the enzyme was observed at ph 5.8 for up 120 min. at the time of isomerization reaction the existence of magnesium ions showed to be necessary and omission of this ion cause a decrease of enzyme activity in isomerization process, but this effect was not necessary for the enzyme activity results showed that treatment of glucose syrup at temperatures of 40, 60 and 80 • c, by the enzyme, caused 48%, 50% and 52% of isomerization, respectively. efficiency increase of high acetic acid production with the use of acetobactereace iranian native strains mutation m. hashemiravan 1 , a. alirezasadat barikani 2 : 1 department of food science and technology, azad university (pishva, varamin unit) institute of food science and agriculture, iran; 2 young iranian researchrer's club, tehran, iran the first step in the present research is the isolation of acetobactereace native strains from fruits (such as grape or apple) and fresh vinegar. this separation has been done with the use of effective isolation methods and optimized mediums. ten strains were isolated effectively, the bio chemicals test were performed, each of them were detected, classified and nominated with a special code. two methods have been used for mutation: (a) mutation with the ultraviolet radiation; (b) mutation with nitrous acid. in the first method the microbial cells were treated with us radiation for different periods of 15, 30, 45 and 60 s, and the effect of the uv mutation was assessed. as a result, the period of 45 s was determined as the optimum mutation periods. in the second method, the microbial cells were first washed with the acetate buffer 0.2 m with the ph of 4.5, then 10 ml nitrous acid 0.07 m was added and was mixed for 2-10 min. finally the sampling was done in the periods of 2, 4, 6, 8 and 10 min and was transferred to a plate containing the medium of ethanol-phenol red-agar. the two methods have been compared with each other. each method has its own advantages and disadvantages. the mutation pathway in method (b) is more stable and conducted, while mutation with the uv radiation method, change the position of thiamine-cytosine bases absolutely randomly. finally, the best mutant site was inoculumed with the medium containing alcohol 16%, acetozyme gz 0.6 g/l, acetozyme d 1 g/l, acetic acid 1.5% and 1 l double distilled water. the acetic acid was produced in the rate of 16 g/100 ml. also the mutant strains were detected with scanning electron microscope (sem) and interesting photographs were taken from mutant cell. the performed experiments were planned with the use of taguchi statistical method (qualitek 4). this research has been performed in the irost as the thesis of ph.d. in food biotechnology industry. isotachophoresis has almost exclusively been applied for contracting and stacking samples ions before zone electrophoretic separation of proteins. this study attempts to apply microfluidic isotachophoresis (itp) as a high resolution analytical method for proteins. beta-lactoglobulin and other milk proteins with slightly different pi were labelled with fluorescent red 646 and analysed by the micralyne tk system using microfluidic glass chips, either with simple cross (sc) or double cross (tt) injection or designed 2d-itp-cze chips with double tt injection and sc for transfer to the second dimension cze channel, efficiently non-covalently coated with 0.6% (w/v) epoxy-polydimethylacrylamide to lower electroendoosmosis. capillary zone electrophoresis (cze) in borate or phosphate buffer was reproducibly perform for more than 75 consecutive runs using upchurch tm reservoirs glued to the wells to enable larger buffer volumes and greater run-to-run stability. finally, isotachophoretic anionic separation of the proteins were done using phosphate (ph 8.1) or chloride (ph 8.5) as leading ion and -amino-caproic acid (ph 8.9) as terminating ion. the effect of narrow cut ampholytes as spacers needs further investigations. the perspective aim is to combine the migrating itp separated zones with second dimension capillary zone electrophoresis as a new microfluidic proteomic 2danalysis. genotypical differences affecting the response of pisum sativum to differing boron/iron applications e.e. hakki, u. zeynep, m. hamurcu, a. tamkoc, m.b. babaoglu, s. gezgin department of field crops, faculty of agriculture, selcuk university, kampus, konya 42079, turkey. e-mail: eehakki@selcuk.edu.tr (e.e. hakki) boron and iron are among the microelements required for the proper development of the vegetative and generative tissues of plants. though iron is present in high amounts in almost all soil types, its bioavailability to crops is extremely reduced, hence most of the plants face an iron defficiency problem and while on one side crop productions effected, on the other hand the nutrition problems come up to human through contagious nutritional chains. boron is also among the most problematic micronutrients of the major crop plantation areas of turkey. both defficiency and toxicity problems exist in a total of about 50% of the central anatolian soil where pea is among the legumes cultivated. application of varying levels of boron and iron combinations in greenhouse and the analysis of plant aquisition via icp-aes as well as determination of the effects of the element combinations both in morphological and molecular levels are the aim of our studies. the genetic bases of the response differences of plant genotypes to b and/or fe, were investigated through the applications of molecular marker techniques. considerable growth rate and stem size differences were detected within the parents (wild-type versus cultivar) and the f9 plants. the presence of efficient genotypes to high micronutrient levels are expected to help us increase the cultivation of the crop in problematic areas as well as in exploring the molecular bases of the microelement uptake mechanisms. increase in sulfite production by accelerating sulfate uptake in brewing yeast t. fujimura, y. kodama, y. nakao, n. nakamura, w. miki institute for advanced technology, suntory ltd., mishima-gun, osaka 618-8503, japan sulfite plays a role as an antioxidant, which stabilizes beer flavor. therefore, it is important to control the sulfite concentration during fermentation. sulfite is produced as an intermediate in the sulfate assimilation pathway in yeast. we have already reported that over-expression of a lager yeast-specific ssu1 gene, encoding a sulfite efflux pump, leads to increase of sulfite production (fujimura et al., 2003) . in the present work, we have clarified that there are two types of sul2 genes (scsul2 and non-scsul2) each encoding a high affinity sulfate permease in lager brewing yeast. eighty percent and 86% identity are found by comparing the dna sequences and the deduced amino acid sequences, respectively. a comparative functional analysis of the two genes has been performed aimed at achieving further increases in sulfite production by accelerating sulfate uptake. over-expression of scsul2 and non-scsul2 has been achieved by transformation of lager brewing yeast, saccharomyces pastorianus. experiments have been done with and without expression of non-scssu1. the resultant transformants have been evaluated by fermentation tests in wort. over-expression of either scsul2 or non-scsul2 failed to show significant effect on sulfite formation. a combination of over-expression of non-scsul2 and non-scssu1 resulted in two-fold higher sulfite production compared with overexpression of only non-scssu1 and four-fold higher compared with the parental strain. these results suggest that the non-sc-gene types significantly contribute to sulfite production in lager brewing yeast. , t., et al., 2003. functional phytases (myo-inositol hexakisphosphate phosphohydrolase) catalyse the release of phosphate from phytate (myo-inositol hexakisphosphate), the predominant form of phosphorus in cereal grains, oilseeds and legumes. possible applications of phytases have been suggested in animal nutrition to increase mineral bioavaliability and to decrease phosphate pollution in area of intensive life stock management and in human health. zea mays is one of the cereals that contain high amount of phytate as the major phosphate storage compound. over 200 bacteria were isolated and screened for phytases from the halosphere, rhizosphere and endophyte of malaysian maize plantation. the phytase activity of the isolates was screened by a modification of the ammonium molybdate method. the highest extracellular phytase activity was detected from bacteria that isolated from the endophyte of the maize root. in this paper, results for 24 isolates chosen for media, temperature and ph optimization will be presented. production of plant proteinase from jack fruit seeds (artocarpus integrifolis) and its influence on rheological and sensory characteristics of low fat yogurt el-sayed el-tanboly dairy sciences department, national research centre, dokki, cairo, egypt adding a proteolytic enzyme extraction from jack fruit (artocarpus integrifolis) in combination of fermentation process in low fat yogurts manufacture was tried to improve yogurt flavour and rheological properties. experimental yogurts milk contained control, 3.9 (t1), 7.8 (t2) and 11.7 (t3) units/ml milk from crude extracts of plant proteinase. the ph of the product treated with crude proteinase was lower than the control. however, the rate of acidity development during storage slightly increased with increasing the addition of crude proteinase level and progress of storage period of yogurt. the proteolytic activity of all yogurts gradually increased until the end of storage period (15 days). yogurts made from milk treated with crude proteinase preparations were less firm compared with control at all storage periods, where t3 showed more less firm after 15 days of storage being 20.17 g/100 g. generally, increasing units of plant proteinase preparations decreased the firmness. on the other hand, yogurt made from milk pretreated with plant proteinase had higher syneresis, and apparent viscosity than the untreated product. the greatest viscosity was found in t2 and t3 of 433 and 479 mpa s, respectively, compared with control of 299 mpa s at 15 days storage. the results indicated that there is an inverse relationship between the amount of units of crude proteinase preparations and susceptibility of yogurt to syneresis. the t2 gained the highest scores (85 points) followed by the control (81.5 points) after 15 days of storage, while yogurt of t3 showed a low scoring being 75. from the foregoing results, it is recommend to use jack fruit (artocarpus integrifolis) as a source of plant proteinases and utilize it to develop a high quality yogurt at a level of 7.8 units of plant proteinases/ml milk. an effective process for the chemical-biotechnological utilization of distilled white lees was studied. a first treatment with hydrochloric acid allowed the solubilisation of tartaric acid. the influence of temperature, amount of hcl and reaction time were considered through an experimental design. under the optima conditions 77 g/l from white distilled lees and 45.6 g/l from red distilled lees were recovered. the tartaric acid was precipitated as calcium tartrate so that it can be isolated from the rest of the raw material compounds. the solid residue was used as an economic nutrient for lactic acid production by lactobacillus pentosus using trimming wastes as substrate. the lactic acid concentrations and volumetric productivities achieved were similar to those obtained using distilled lees without tartaric acid recovery as nutrient. toasted wine was traditionally produced in galicia, northwest of spain. nowadays this technique is being recovered. grapes after harvesting are air dried in order to concentrate sugars, acids and flavor compounds. raisings are pressed to obtain a must with high sugars concentrations. two different grape wines were prepared concentrating the sugars up to 37 and 57 brix, respectively. in order to get a better knowledge of the problems involved, synthetic media simulating the grape musts were prepared. theses musts were used to optimize the initial sugar concentration, the amount of nutrients required, the optimum temperature to carry out the fermentation and the influence of the type and amount of yeast. under the best conditions some fermentations with grape must were carried out to produce wines with intense aroma and flavor notes and high residual sugar concentrations. in this studies, bacillus sp. e1 strain was isolated from koreanstyle fermented soybean paste and it was producing the biological response modifier (brm). the brm activated the b cell selectively. it was identified the bacillus licheniformis e1. the brm was purified by ion-exchange chromatography and gel filtration. chemical properties of brm: molecular weight of brm was estimated to be about 1,594,000 da. sugar content of brm was 33.0% (w/w) and glucosamine (35.1 mol%) was the high level. protein content of brm was 4.3% (w/w) and serine (17.2 mol%) was the high level. infra-red absorption spectrum was showed the characterization of glycoprotein. biological properties of brm: the brm which isolated from fermented soybean paste was similar to that of bacillus licheniformis e1 by immuno-fluorescence assay. we confirmed that the brm was capsular substance of b. licheniformis e1. potato nitrogen concentrate (pnc) is a highly viscous liquid with high complex nitrogen content produced from the protein-fraction in potato starch extraction. the concentrated extract is rich in minerals and ␣-amino nitrogen. although ␣-amylase nowadays is mainly produced exploiting bacillus production systems there is still considerable demand for fungal ␣-amylase from aspergillus oryzae origin. the aim of the experiments to be reported here was to investigate, if pnc can replace commonly used complex nitrogen sources in the production of fungal ␣-amylase. the following data have been measured in pnc pretreated by diluting to 1/2 and clarifying by centrifugation. total-n: 8.4 g n/l; ␣-amino-n: 2.8% (w/v) (as glycine); soluble protein (bradford): 51.2 mg/l (as bsa); total carbohydrates: 110.0 g/l; reducing sugars: 5.6 g/l; dry weight: 57.3% (w/w). in the following experiments nitrogen sources were replaced on the basis of their ␣-amino nitrogen content. the carbon source for all experiments was maize starch. the formation of ␣-amylase by a. oryzae atcc 1011 in shake flasks -using pnc (centrifuged or not), yeast extract, malt extract, casein hydrolysate or meat extract -was compared to "standard" cultivation with corn steep liquor. the experiments showed only small differences in ␣-amylase titers using complex nitrogen sources. no remarkable differences were observed in the resulting biomass. in general no differences in enzyme productivity and biomass formation could be seen after 50 h of incubation. especially the bench top bioreactor experiments indicated an optimal fermentation time of about 100 h. cultivations of a. oryzae atcc 1011 were carried out in bench top bioreactors. comparing cultivations in a medium with pnc as the sole complex nitrogen source to one containing csl as such no significant differences both in the formation and amount of ␣-amylase and the fungal growth were observed. thus pnc might be able to replace complex nitrogen sources such as csl or even the more expensive yeast extract and casein hydrolysate in fungal amylase production systems. 1.19) is involved in the metabolism of inositol and catalyzes the conversion of d-glucuronic acid to l-gulonic acid with nadph as a cosubstrate posterior to the oxidation of inositol to glucuronic acid by the enzyme inositol oxygenase. although the yeast sporobolomyces oryzicola (nakase and suzuki, 1986) is not able to grow on inositol as the sole carbon source, intracellular glucuronate reductase can be found in cells grown in a medium containing d-glucuronic acid. the enzyme could be a useful tool in the design of a specific quantitative assay for glucuronic acid, e.g. in so called energy drinks. the organism was grown in media containing either glucose and glucuronic acid or only glucuronic acid and difco yeast nitrogen base. whereas growth on both media was similar in shake flask culture, hardly any growth in either medium was observed in bench top bioreactors. the influence of dissolved oxygen tension was investigated and the relevant data will be shown. the formation of intracellular glucuronate reductase activity by sp. oryzicola is inducible by media containing glucuronic acid. no activity is found in cells grown in a medium containing only glucose as the carbon source. besides the activity against d-glucuronic acid, activities against 5-ketogluconate and -at very low levelsagainst galacturonic acid and the lactone of glucuronic acid were detected. the enzyme activity is stable up to 35 • c. the ph has relatively low influence on the activity against glucuronate, whereas the reduction rate of 5-ketogluconic acid is optimal at ph 7.0-7.5 with significantly lower values at ph 6.0 and 8.0, respectively. data on the kinetics of the conversion of both glucuronate and 5-ketogluconate will be shown. nakase, t., suzuki, m., 1986. j. gen. appl. microbiol. 32, 149-155 . the multiple nutritional and functional impacts of food fermentation on human health have been widely accepted (reddy and pierson, 1994; hugenholtz et al., 2002) . however, the related role of the involved microorganisms to the nutritional effect from the fermented food is still not well defined and the mechanisms involved are still largely unknown. the present study was to investigate iron bioavailability in carrot juice fermented by two selected lab strains, l. pentosus fsc1 and ln. mesenteroides fsc2. after digestion by gi enzymes, the juice was supplied to fully differentiated caco-2 cells to study iron uptake and transepithelial transport by caco-2 cells from the digested juice. our data revealed strain specified changes in iron bioavailability in carrot juice fermented by these two strains. after in vitro digestion with pepsin and pancreatic-bile enzymes, the best yield of soluble iron was from ln. mesenteroides fsc2 fermented juice. surprisingly, the l. pentosus fsc1 fermented juice yielded about five times higher uptake iron as compared to fresh juice, while ln. mesenteroides fsc2 fermented juice was not significantly different from the fresh juice. interestingly, the transepithelial transferred iron across the cell line was however better from ln. mesenteroides fsc2 fermented juice than from l. pentosus fsc1 fermented juice. to summarise, our study showed that level of soluble iron after in vitro digestion does not necessary indicate iron absorption, especially in the case of lab fermented food. data on improved iron uptake from l. pentosus fsc1 fermented juice indicated exiting of promoter(s) for iron absorption in such juice that is not related to the production of organic acids and lowering ph effect. peng zhang, herve vanderschuren, martin stupak, wilhelm gruissem institute of plant sciences, 8092 zurich, the tropical root crop cassava (manihot esculenta crantz) is a major source of food for approximately 1 billion people worldwide. in sub-saharan africa, more than 200 million people rely on cassava as their major source of dietary energy. in many parts of africa and latin america, cassava leaves are a vegetable source for daily uptake. cassava is grown mostly by poor farmers under marginal environmental conditions and in areas where few other crops can sustain competitive yields. the crop is therefore fundamental for subsistence farming and food security, but it is also very susceptible to stresses common in the areas and conditions where it grows. in many parts of africa, reliable cassava production is strongly impacted by infections with the african cassava mosaic geminiviruses (cmgs), a rapidly spreading disease that causes large yield losses. in the coastal areas of east africa, cassava production now is threatened by another devastating disease, cassava brown streak disease (cbsd). cassava plants are also frequently attacked by many pests, such as cassava hornworm and stemborers. several reports also indicate that greater leaf longevity, especially under drought conditions, could be important for increasing yields and/or the stability of production in cassava, as well as improve the access to an important nutrient source. conventional breeding efforts have attempted to address the constraint to cassava production, but with limited success. the new tools of biotechnology can change this situation by offering new approaches to the challenges of cassava. these new technologies have the potential to make cassava much more productive, a better source of nutrients, and profitable to grow, hence, greatly contributing on the sustainable development of tropical agriculture. recently we have developed biotech cassava with value-add traits, including resistance to cassava mosaic virus, prolonged leaf life and insect resistance. new strategies are also explored to increase protein content of cassava storage roots. we are currently undertaking pilot studies with two teams of leading scientists and experts for projects to test acmv-resistant transgenic cassava lines in africa and lines with extended leaf retention at ciat, colombia under field conditions. this development of substantially equivalent improved transgenic cassava lines is part of a larger study to analyze the need, effectiveness and biosafety of biotech cassava for agricultural production. the goal of the pilot studies will be the development and coordination of a broader project that produces important and novel scientific results, valuable information on the need and impact of biotechnology at the subsistence farming level, and a sound scientific basis for the development of guidelines for biosafety assessments and release of transgenic organisms into the environment and agricultural production in africa and latin american countries. this study was conducted to reveal the effects of different pretreatments on obtaining haploid plants by using the anther culture in pepper capsicum annum l. cultivars demre sivrisi and sirena. buds were collected at uninucleate microspore stage. anthers collected from buds were cultured in ms medium containing different hormones and hormone concentrations. experiment results revealed that when sirena anthers were pre-treated cold at +4 • c for 24 h and kept in darkness at 25 • c for a period of 1 week gave good results. in the case of demre sivrisi anthers were pre-treated cold at +4 • c for 48 h and kept in darkness at 25 • c for a period of 1 week gave good results. on the other hand no cold pretreatment to anthers resulted with low embryo formation. similar results were also observed on the anthers kept at 35 • c for 1 week as callus was produced in some petri dishes but no regeneration was observed. as a conclusion, since no cold pretreatment to anthers resulted with low embryo formation it is possible to say that cold pretreatment should be applied to anthers in pepper another culture studies. the yeast d. hansenii ufv-170 was tested in this work in batch experiments in synthetic media at constant initial substrate concentration (100 g l −1 ) under variable oxygenation conditions. to get additional information on its fermentative metabolism, a stoichiometric network was proposed on the basis of the general knowledge available in the literature on xylose metabolism in pentose-fermenting yeasts and the specificities of xr and xdh activities in d. hansenii and checked through a bioenergetic study performed using the experimental data of product and substrate concentrations. it can be stressed that under strongly oxygen-limited conditions xylitol production was negligible, whereas under semi-aerobic conditions maximum xylitol production (p max = 76.6 g l −1 ) and yield (y p/s = 0.73 g g −1 ) were obtained. a progressive decrease in these parameters was observed under fully aerobic conditions, suggesting that xylitol-producing yeasts require limited oxygen conditions, which is species-dependent. the proposed model, which utilizes the experimental specific rates of substrate consumption and product formations, allows estimating the main bioenergetic parameters. besides, it proved to be an effective tool to investigate different metabolic situations and showed how they can influence the flux distribution of the carbon source and the bioenergetics of this biosystem. the effect of disinfectants on fungi anne svendsen, pernille skouboe bioneer a/s, hørsholm dk-2970, denmark prevention of mould spoilage of foods can only be carried out successfully, if the species, which are actually spoiling the food product, are known. a very limited number of fungal species has been associated with the spoilage of each food category. proper disinfection of production facilities is very important to avoid mould spoilage. resistance of moulds to disinfectant treatments are known and different species have shown different response to the same disinfectant. to obtain proper disinfection it is important to know the resistance of the spoilage fungi against different disinfectants. in this study the effect of disinfectants on the spoilage fungi of cheese, rye bread, liver paté and fruit juice was investigated. in collaboration with five food companies the dominating species responsible for spoilage of each food product were isolated and used for testing. commercial disinfectants and disinfectants "under development" were tested. tests were performed in suspension and on surfaces, the methods used were modified after en 1650 and en 13697. considerable variability in fungicidal effect among the species was observed. some disinfectants were ineffective at low temperature. some disinfectants showed different effect in suspension and on surfaces, resulting in an effective kill in suspension and almost no effect on surface. the identification of effective disinfectants in the food industry includes: (1) testing against the specific spoiling species of the food product, (2) testing on surface, not only in suspension, (3) test parameters adapted to the food manufacturing plant. intensified research efforts in recent years confirm the major importance of the microbial flora in the gastro-intestinal tract for human health. ingestion of prebiotic oligosaccharides increases the number of the desirable bacteria like bifidobacteria and lactobacilli in the colon. we are looking at beta-galactosidases from lacto-bacillus spp. for the production of galacto-oligosaccharides (gos) because we speculate that the enzymes of probiotics will form gos with high prebiotic potential. in this present study, purified betagalactosidases of selected lactobacillus strains were used for the production of gos from lactose. different enzyme reactor set-ups, both discontinuous and continuous, were tested and compared. temperatures up to 37 • c and ph values between 6 and 6.5 were required for satisfactory enzyme stability during the process. enzyme source, substrate concentration and the level of substrate conversion were found to be critical process parameters for gos yields and composition. yields of up to 40% (w/w) of total sugars were achieved when the initial lactose concentration was 200 g/l. capillary electrophoresis (ce) and hplc with pulsed amperometric detection were the analytical tools for investigating the influence of reactor type, enzyme source and conversion level on gos composition. the prebiotics market is increasing rapidly and is expected to more than double until 2010 to about 180 million d world-wide. therefore, the development of enzymatic processes on an industrial scale is a high priority goal of our research. starter addition does not always succeed in improving standardisation and quality of the complex sensory properties of traditional fermented foods. in many cases the added strains do not grow as well as the environmental strains present in the production plant. here, a method of geometric simplification (by dichotomy) of a complex ecosystem found on a raw milk livarot (82 strains) was tested on cheese curd. by a limited number of cultures, successively, 40 out of 82, 20 out of 40 and 10 out of 20 strains were selected on the basis of two criteria (i) respect of the taxonomic proportion, (ii) generation by the daughter ecosystems of an odour close to the one of the mother ecosystem. finally a sub-ecosystem of 10 strains gave an odour similar to the one of the more complex mixture. the use of molecular methods (pcr-sscp) permitted to follow the main species growing. mother and daughter ecosystems were characterized by sensory analysis and gc-ms. probably because of an important redundancy of the strain functions, the method was very efficient. this method may permit to improve a lot the set up of mixture of strains and species used in fermented food industry. effect of the dilution rate on the exopolysaccharide production by bifidobacterium longum atcc 15707 c. shene, m. rubilar, s. bravo universidad de la frontera, chemical engineering, av. francisco salazar 01145, casilla 54-d temuco, chile exopolysaccharides (eps) producing lactic acid bacteria are used in dairy industry (cheese and yogurt) due to the rheological properties that these compounds confer to the products. preliminary results also suggest the use of eps as health-promoting (anti-tumor and immunostimulatory actions) ingredients. bifidobacteria are grampositive bacteria natural inhabitants of the gut of warm-blooded animals and man. a number of investigations have shown that bifidobacteria promote host health mainly because of the reduction proliferation of some pathogenic bacteria through acid synthesis. in this work results obtained in the experiments carried out to test the capability of b. longum atcc 15707 to synthesize eps are presented. continuous culture fermentations were carried out at dilution rates between 0.04 and 0.44 h −1 . composition of the culture media was that of the mrs broth. biomass concentration presents higher values (2.9-3.2 g l −1 ) at dilution rates between 0.1 and 0.2 h −1 . biomass growing at these rates is difficult to pellet and adheres to the fermentor walls behavior that was not observed at other growth conditions. eps from cultures grown at these rates were preparated and fractionated. authors wish to thank the chilean conicyt for the economical assistance given through the project fondecyt 1050602. high pressure-low temperature (hplt) inactivation processes were performed on bacillus subtilis vegetative cells at various conditions. at atmospheric pressure, lowering the temperature to as low as −45 • c was found to have minor anti-microbial effects. upon application of high pressure various phase transitions occurred in the microbial suspensions under study. after pressure treatment at 150-450 mpa, cells were plated under optimal conditions to assess cell viability. treatments at 250-450 mpa and −25 • c were the most effective in inactivation. in these cases, ice i-iii solid-solid phase transition was observed. in addition, we hypothesised that intracellular thawing (solid-liquid phase transition) had already occurred while the extracellular surrounding was undergoing solid-solid phase transition. this double effect is suggested to be key in mediating the observed large drop in viability. we speculate that more cells survived after treatment at −45 • c compared to the same treatment at −25 • c because both the extra-and intracellular surrounding remained fully frozen. at −45 • c a solid-solid phase transition was observed when pressure was higher than 350 mpa. a metastable state of ice i was observed at 250 mpa treatment. results from the current study will be presented (see also shen et al., ifset in press) . the data call for a mechanistic evaluation of the effects of hplt as an anti-microbial treatment. such data are currently being gathered and will be used in defining optimal hplt process conditions for the food industry. the influence of saccharomyces cerevisiae, kloeckera apiculata and candida pulcherrima mixed cultures on the selected alcohols formation during model fermentation pawel satora, tadeusz tuszynski department of fermentation technology and technical microbiology, food technology faculty, agricultural university, cracow, poland. e-mail: psatora@ar.krakow.pl (p. satora) for the study five yeast species were chosen, isolated from successive stages of plum fruits spontaneous fermentation: from the beginning (candida pulcherrima, kloeckera apiculata, saccharomyces cerevisiae w4), middle (s. cerevisiae w54) and final fermentation (s. cerevisiae k1). to characterize the potential influence of yeast mixed cultures on the selected alcohols formation, wick-erham synthetic medium (10% glucose) was fermented by mixed cultures of two and three yeast species. after distillation, ethanol, propanol, isobutanol, isoamyl alcohols, hexanol and 2-phenylethanol were determined using gas chromatography. findings were compared with the results obtained after monoculture fermentations. the use of mixed cultures resulted in increasing of glucose utilization rate, ethanol and fusel alcohols formation (except propanol) and decreasing of methanol synthesis. the samples fermented using two yeast species characterized higher (about 10%) amount of volatile compounds in relation to monocultures. it takes note of especially high level of ethanol (av. 44.3 g/dm 3 ), methanol (16.7 mg/dm 3 ) and isoamyl alcohols (47.6 mg/dm 3 ). the positive feature of triple cultures using was limitation of methanol and fusel alcohols synthesis that was accompanied by relatively high ethyl alcohol production (av. 41.5 g/dm 3 ). the consumption of sugar syrup becomes increasingly significant in industrial processes due to economic advantages and the easy of use. the production of sucrose syrup using enzymatic hydrolysis represents the safest alternative, once the reaction does not produce any toxic or undesirable substance. this work consists on the production of sugar syrup by immobilized inulinase from kluyveromyces marxianus, with two alternatives process: (a) syrup enriched with fructooligosaccharides or solely with glucose and fructose. the process is comprised by the following stages: production and purification of the enzyme in optimized conditions, immobilization of the enzyme in solid support and the conversion of sucrose in a fixed bed bioreactor with the immobilized enzyme. the final composition of the product can be a mixture of glucose, fructose, sucrose and fructooligosaccharides or a mixture of fructose and glucose, according to the operational conditions. the bioreactor can be operated continually for approximately 5 months with the same biocatalyst. the product from this process is ideal for applications in the food products such as sweet, candies, chocolates, yogurts, etc. besides, the prebiotics properties of the fructooligosaccharides, is a beneficial stimulant of the intestinal flora, which gives to the product a functional property. studies on plant microbial interactions using azotobacter sp. as bio-inoculants towards soil fertility baljeet singh saharan faculty of biotechnology, jcdm college of engineering, sirsa 125055, india. e-mail: baljeet.saharan@gmx.de, baljeet br@yahoo.co.uk (b.s. saharan) high nitrogen fixing, phytohormone producing isolates of azotobacter, azospirillum, acetobacter and pseudomonas were used as inoculants on wheat and cotton with varying doses of nitrogen under field conditions. bio-inoculants were selected on the basis of yield, dry weight and survival rate of bacteria under field conditions. seeds of wheat variety wh 711 were treated with different biofertilizers using nitrogen level of 90, 120 and 150 kg ha −1 and one level of p, i.e. 60 kg ha −1 in field along with control. under field conditions, maximum yield was obtained with azotobacter chroococcum e 12 at 90 (2506 ± 0.04 kg ha −1 ) as well as 120 kg ha −1 (2817 ± 0.07 kg ha −1 ) followed by a. chroococcum ht 57 (2482 ± 0.16 kg ha −1 ) and avk 51 (2474 ± 0.37 kg ha −1 ). whereas, with 120 kg ha −1 highest yield was observed with mac 27 (2833 ± 2.59 kg ha −1 ) followed by e12 (2817 ± 0.91 kg ha −1 ) and avk 51 (2804 ± 0.16 kg ha −1 ). maximum height at 90 kg ha −1 was observed with mac 27 inoculation (71.1 ± 5.24 cm) followed by avk 51 (70.8 ± 4.70 cm) and ht 57 (70.3 ± 1.76 cm). various chosen strains were tested with desi (hd 123) and american cotton (h 1098) under similar pot and field conditions as for wheat in the following season. plant height and yield were determined at the time of harvesting whereas survival rate was monitored at various intervals of time. survival rate of inoculated bacteria was determined after 30, 80, and 135 days. highest survival rate was observed in mac 68 ((3.34 ± 2.56) × 10 6 ), which decreased after 80 and 135 days, respectively. (3.38 ± 1.48) × 10 5 , (1.53 ± 0.92) × 10 5 with mac 68 and (2.97 ± 2.01) × 10 5 and (1.26 ± 3.01) × 10 5 with ht 54, respectively. maximum boll weight was with avk 51 (76.2 ± 2.34 g boll wt. plant −1 ) followed by pseudomonas (71.3 ± 1.77 g), ac 18 (61.5 ± 1.73 g) and ala27 (61.4 ± 2.79 g) boll no. plant −1 was maximum with ala 27 and avk 51 (46 ± 2.59 plant −1 ) followed by pseudomonas (34 ± 0.07 plant −1 ). maximum height and dry matter was obtained with pseudomonas (179.7 ± 1.97 cm) and avk521 (146.7 ± 3.49 cm) with variety hd 123 under field conditions. net saving of 20% nitrogen was observed using a. chroococcum (e 12 and avk 51) bioinoculants for wheat and cotton, respectively. to characterize the antioxidative properties of tempeh-fermented food prepared from vicia faba (l. kontu) with the use of rhizopus oligosporus, the sulfhydryl groups content and surface aromatic hydrophobicity of albumins were investigated. the results obtained for tempeh albumins were compared with raw vicia faba and bovine serum albumin (bsa). these results indicate that tempeh fermentation increased antioxidative activity of albumins. the measurements of antioxidative activity were carried out with the use of 1,1-diphenyl-2-picrylhydrazyl (dpph) and 2,2 -azinobis-(3ethylbenzothiazoline-6-sulfonic acid (abts). the albumins of faba bean-tempeh have possessed much higher activity for scavenging free radicals as measured with the dpph and abts (49.4% and 41.1%) than raw seeds (27.9% and 23.2%) and bsa (5.8% and 13.4%), respectively. it has been also found that tempeh fermentation process increased 2.5 times sulfhydryl groups content (43.2 m/mg of albumins) as compared to raw seeds (17.5 m/mg of albumins). the tempeh albumins have possessed lower surface aromatic hydrophobicity than raw seeds (352.3 fi and 692.1 fi, respectively). orange peel characterization and generation of fermentable sugars solutions for the biotechnological production of food additives b. rivas 1 , j.m. domínguez 1 , p. torre 2 , j.c. parajó 1 : 1 department of chemical engineering, vigo university (campus of ourense), polytechnic building, as lagoas, 32004 ourense, spain; 2 department of chemical and process engineering, genoa university, via opera pia 15, 16145 genoa, italy. e-mail: brivas@uvigo.es (b. rivas) the citrus processing industry generates in mediterranean area around 3 millions tonnes of orange peel as byproduct from the extraction of citrus juices in industrial plants. in order to avoid ecological problems and provide an extra profit, this residue was studied in order to generate a suitable substrate for the fermentation process oriented to the production of food additives. orange peels were characterized and the data collected allowed quantifying a 97% of this waste. soluble sugars (21.2%), cellulose (17.0%) and pectin (42.5%) were identified as more important fractions. this material was submitted to two hydrolysis techniques, prehydrolysis (with diluted sulfuric acid) and autohydrolysis (with water) under different experimental conditions. autohydrolysis was selected as the most appropriate technique for the production of suitable fermentation media. finally, the liquors obtained at 130 • c and liquid:solid ratio of 8 g/g, containing 38.2 g/l of sugars, without additional nutrients, were employed to citric acid production by aspergilus niger cect 2090 (atcc 9142, nrrl 599) . the influence of the addition of calcium carbonate and methanol were studied. under the best conditions an effective conversion of sugars into citric acid was attained, showing the viability of the production of fermentable solutions from this industrial waste. today there is an increasing interest in using high gravity fermentation in brewing. high-gravity fermentation involves production of beer wort of up to 18 • p or even higher and results in beer that has more consistent product quality. the main aim of this study is an increased understanding of how brewer's yeast respond to the various stress factors imposed during high gravity beer fermentation and the consequences these stress factors have on the gene regulation and its consequences on the metabolite levels (both intra-and extracellular). higher attenuation of the wort will be achieved by two different techniques: by the addition of highly fermentable adjuncts such as sucrose or glucose syrups and by mashing with addition of microbial enzymes such as pullulanases and glucoamylases. in the first part of the study model fermentation conditions are established, where the sugar uptake and product formation can be studied in details. characterization of the carbohydrate profile is analyzed by hplc. as flavour changes may occur at higher gravities, it is important to study changes in formation of secondary metabolites, especially esters. transcriptome and metabolome analysis will be used to establish how the stressful conditions prevailing under high gravity fermentations may influence the secondary metabolism in saccharomyces cerevisiae. furthermore, analytical aroma characterization of final beer will be studied by spme and gc-ms. detailed analysis of the effect of different stress factors on the cellular response using dna arrays and metabolite profiling will be carried out. dna arrays will be employed to evaluate if specific metabolic pathways are up-regulated or down-regulated as a consequences of the stress factors. naringin, a bitter compound that occurs in citrus fruit juices, may be converted to a nonbitter form by enzyme hydrolysis. the enzymatic complex naringinase was produced in aspergillus niger cect2088 cultures with naringin as inducer (pérez-mateos et al., 2004) . crude extracts from a. niger and purified naringinase from penicillium decumbens were immobilized into a polymeric matrix of polyvinyl alcohol (pva) hydrogel cryostructured in liquid nitrogen. the operating stability of the pva-naringinase beads was tested using synthetic citric juice (gray and olson, 1981) . immobilized enzymes reduced 40% the naringin content at 20 • c and ph 3.2. furthermore, immobilized preparations from aspergillus and penicillium could be re-used through six cycles (144 h) remaining 70% and 38% catalytic efficiency, respectively. financial support from "ministerio de ciencia y tecnología" and feder (no. agl2003-08006/ali). gray and olson, 1981 . j agric. food chem. 29, 1298 -1301 . pérez-mateos, et al., 2004 (1), 230. otimizing the fermentation broth for tanase production by a new isolated strain paecilomyces variotii vania battestin, gláucia pastore, gabriela macedo department of food science, unicamp, p.o. box 6121, campinas, cep 13083-862 são paulo, brazil tannase is an inducible enzyme that catalyses the breakdown of ester linkages in hydrolysable tannins, resulting in gallic acid and glucose. the fermentation broth can use by-products as wheat bran, rice or oats, adding tannic acid. the use of by products or residues rich in carbon source for fermentation purposes an alternative to solve pollution problems that can be caused by an incorrect environmental disposal. in the present study we have optimized the production of an extracellular tannase by a new isolated paecilomyces variotii using response surface methodology. the first step was to identify the variables having a significant effect on enzyme production. the variables evaluated were temperature, residues ratio (coffe: wheat bran), concentration of tannic acid, salt solution during 3, 5 and 7 days of fermentation time. results showed that temperature, residues ratio (coffe: wheat bran) and tannic acid had significant effects on tannase production. commercial wheat bran (cwb) and coffe rusk residues (cr) were used as solid substrate. for fermentation the medium was composed by, cwb:cr were mixed with distilled water and transferred into 250 ml capacity erlenmeyers flasks and auto-claved at 120 • c for 20 min. the medium was then inoculated with spores (5.0 × 10 7 ) and the flaks were incubated at 32 • c. tannase was assayed according to the methodology of mondal et al. (2001) . according to the statist analyses, the optimum conditions to produce tannase was the range of temperature (29-34 • c); tannic acid (8.5-14%); residues percent (coffe: wheat bran) (50:50) and 5 days fermentation time. the enzyme production increased 8.6 times more enzyme production than that was obtained before this optimization. yeast and lactic acid bacteria are two major microbial groups of the most fermented products. a large variety of fermented foods and beverage are made by the activities of both yeast and lactic acid bacteria, simultaneously or successively. during the spontaneous mixed fermentation of lactic acid bacteria and yeast population, it is extremely difficult to control microbial species due to the complexity of the microorganism involved. therefore, we have compared the antimicrobial activity of chitosan against two lactic strains, lactobacillus plantarum and lb. brevis, and yeast strains, saccharomyces cerevisiae to investigate the possible use of non toxic biopolymer chitosan for selective control in mixed culture. the lactobacilli were more sensitive to the inhibitory activity of chitosan than s. cerevisiae. the results suggest the possible use of low molecularweight-chitosan for the control of food fermentation in which both groups of organisms frequently occur together. the effect of vegetable oils on astaxanthin production of phaffia rhodozyma and xanthophyllomyces dendrorhous csaba vágvölgyi, gyöngyi lukács, miklós takó, árpád csernetics, tamás papp department of microbiology, faculty of sciences, university of szeged, p.o. box 533, astaxanthin (3,3 -dihydroxy-␤,␤-carotene-4,4 -dione) is one of the most important carotenoid product. it is used primarily as food colorant and animal feed additive. their effective antioxidant properties linked to a preventive action on various types of cancer and an enhancement of the immune response could lead to expanded commercial applications. among the natural microbial source available, the closely related red pigmented yeasts phaffia rhodozyma and xanthophyllomyces dendrorhous are of great biotechnological interest. these yeasts have desirable properties as biological sources of pigment, including rapid metabolism and producing high cell densities in fermentor, but the commercial production of astaxanthin is limited by the relatively low content in wild-type strains. the purpose of this study was to determine whether the different vegetable oils had an effect on the carotenoid production in p. rhodozyma. effects of media supplemented with corn germ oil, wheat germ oil, sesame-seed oil, palm oil, pumpkin-seed oil, coconut grease, olive oil (extra virgin), olive oil (sanza), sunflower-seed oil and cottonseed oil were tested. studies were performed on both a phaffia and a xanthophyllomyces strain. yeast was grown in yeast-pepton-glucose liquid medium complemented with the appropriate vegetable oil in different concentrations (0.5-2, v/v, %) . after four days the total carotenoid production was determined spectrophotometrically, and it was referred to dry cell mass. palm oil increased significantly the carotenoid production of the phaffia strain, while a similar effect on the xanthophyllomyces strain could be observed with coconut grease. composition of carotenoid compounds in the strains was determined by thin layer chromatography. lutein is considered a nutraceutic compound that has developed an increasing interest since it is one of the two carotenoids that are located in the macula of the human eye. its consumption is associated with the prevention of age related macular disease (amd). industrially, lutein may be produced using a saponification step of a mixture of lutein diesters that are previously extracted with hexane from natural sources. our proposal is to improve the process by catalyzing the same reaction using microbial lipases during the extraction step with hexane. additionally, the use of supercritical fluids represents an extension of enzymology in non-conventional media with process and environmental advantages. this work was developed using extracts from marigold flower (tagetes erecta) in hexane and supercritical carbon dioxide (sc-co 2 ) where the lutein esters were hydrolyzed by two commercial lipases: lipase b from candida antarctica (novozym 435) and lipase from mucor miehei (lipozyme rm 1m). in particular, we focused our interest in the role of water in the system. interestingly our results show an inverse dependence of the initial reaction rate with respect to the initial water activity (awi) for both lipases, a phenomena that seems to be related to the partition of substrates and products in the solid support)and the hexane phase as a function of water. when sc-co 2 was used as solvent an increase in the consumption rate of lutein diesters occurred, reaching conversions of 70% in 24 h. for hexane, the same conversion was reached after 160 h. this result suggests a significant effect of the media on the reaction that can be related to shifts in the partition of compounds that bring the substrates in closer contact with the enzyme. this work also demonstrates that lutein hydrolysis seems to be another potential application of commercial immobilized lipases in the food/nutraceutical market. the enzyme of interest in this work is ␤-galactosidase from lactobacillus sp. (ec 3.2.1.23). ␤-galactosidases catalyze the hydrolysis and transgalactosylation of ␤-d-galactopyranosides (such as lactose). an attractive biocatalytic application is found in the transgalactosylaction potential of these enzymes which is based on the catalytic mechanism of ␤-galactosidases. the products of transgalactosylation, galacto-oligosaccharides, are non-digestible carbohydrates which meet the criteria of 'prebiotics' and therefore have attracted increasing attention. to produce these 'prebiotic' galactooligosaccharides, an inexpensive and efficient process is desired. immobilization of the enzyme ␤-galactosidase on an insoluble support is an attractive tool to make the process of lactose conversion more economical because the enzyme can be recovered and reused during continuous operation. in this present study, we aimed at immobilizing ␤-galactosidase from lactobacillus sp. by covalent linkages on two solid supports which are commonly used for protein immobilization: chitosan and eupergit c. the protein-binding capacity, the immobilization yield, ph and temperature dependency of activity and stability, and the kinetic parameters of immobilized enzymes were studied. higher activity retention of the immobilized enzymes over a broader ph range and at higher temperatures compared to those of the free enzyme was observed. the immobilized enzymes were evaluated in terms of transgalactosylation activity and stability for a to introduce of foreign genes for the important crop plants such as rice, we need a reproducible efficient procedure for regeneration of the calli through somatic embryogenesis. for this intention, we established the best callus induction medium for tarom mahalli and deilamani cultivars and created the method that the regeneration frequency was reached to 48%. calli were induced from scutellar tissues of mature seeds on ms medium supplemented with three level of 2,4-d (2, 2.5 and 3 mg l −1 ) and n6 medium supplemented with five level of 2,4-d (1.5, 2, 2.5, 3 and 3.5 mg l −1 ). for deilamani cultivar the best medium was n6 with 1.5 mg l −1 2,4-d and for tarom mahalli the same medium with 2 mg l −1 were the best. in subculture media, sucrose was used instead of maltose. for regeneration analysis of plantlets, we used two-factorial experiment in base of crd; one factor was regeneration media with six levels (ms medium supplemented with five amount of kinetin and: naa (mg l −1 ) [(6:2), (4:2), (2:0.5), (3:1), (2:1), respectively] and 0.2 mg l −1 2,4-d and 2 mg l −1 bap). other factor was dehydration process with three levels (without dehydration, dehydration with two layers of filter paper for 30 min [prior to transfer to the regeneration medium], and third factor was factor of 2 with substitution of sucrose with maltose [after 2 weeks; 2 sucrose:1 maltose]). we conclude that maltose due to changing in osmolarity proceeding can elevate the regeneration frequency to 48%. therefore, type of carbon source is critical in callus induction and regeneration. márová ivana, hrdličková jana, kubešová jitka, kočí radka, vidláková tereza faculty of chemistry, brno university of technology, purkyňova 118, 612 00 brno, carotenoids are the most widespread natural pigments with important biological activities and applications mainly in food and feed industry. at present many ways including genetic engineering are developed to reach higher production of naturally formed carotenoids using microbial producers. in this work cloning and expression of crt gene cluster from pectobacterium carotovorum in recipient bacterial strain e. coli dh5␣ as well as in yeast strain s. cerevisiae was tested. plasmid vector phsg298 with inserted crt genes was used for transformation of chemically competent e. coli dh5␣ cells, while in s. cerevisiae shuttle vector paur135 was used. transformants were selected based on resistance to antibiotics, formation of orange-coloured transformant colonies, analysis of recombinant plasmid size and lc/ms analysis of carotenoids produced by recombinant cells. the yield of individual carotenoids (lutein, beta-carotene, lycopene) obtained from various bacterial transformants was several fold higher than in natural producer (lutein: 0.2-1.2 g/g of d.w., beta-carotene: 0.1-1.4 mg/g of d.w.). the highest yield obtained in transformed strain was 63.5 g/g of lutein and 15.4 g/g of beta-carotene. the yield of biomass and carotenoids in. transgenic s. cerevisiae was comparable to some industrial red yeast strains (4.5 mg of total carotenoids + 32 mg ergosterol/l; 36 g/l of biomass). so, transgenic yeasts could be suitable for large scale production of carotenoids and/or enriched biomass, while transgenic bacterial producers are perspective above al for high production of rare carotenoids as lutein or lycopene using transformation by specific genes of crt gene cluster. this work was supported by the project msm 0021630501 of the czech ministry of education, youth and sports. two forms of grape seeds, whole and powdered forms, were heated at four different temperatures-50, 100, 150 and 200 • c. after heating, grape seeds were extracted with 70% ethanol (0.1 g grape seed/10 ml of 70% ethanol), and total phenol contents (tpc), radical scavenging activity (rsa) and reducing power of the extracts were determined. thermal treatment of grape seed increased the antioxidant activity of extracts. the maximum tpc and rsa of whole grape seed extract (wgse) were achieved when the seeds were heat-treated at 150 • c for 40 min, while that of powdered grape seed extract (pgse) were at 100 • c for 10 min, and were greater than that of the non-treated control. according to the gc-ms analysis, several low-molecular-weight phenolic compounds were newly formed in the wgse heated at 150 • c for 40 min. these results indicated that antioxidant activity of gse was affected by heating conditions (temperature and time) and physical conditions of grape seeds at the time of heat treatments. analysis of the unexpected phenotypic consequences associated with plant transformation jonathan latham, allison wilson, ricarda steinbrecher econexus, 6, canon frome court, ledbury hr8 2td, uk. e-mail: jrlatham@gn.apc.org (j. latham) transgenic plants often exhibit unexpected phenotypes. such phenotypes could arise from pleiotropic effects associated with the transgene, or they could arise from other sources. a recent econexus report underlined the potential for the process of plant transformation to result in genetic damage to the transformed plant (genome scrambling-myth or reality? transformation-induced mutations in transgenic crop plants: http://www.econexus.info/). the report showed that mutations arising at the site of transgene insertion are often substantial, frequently resulting in loss or rearrangement of chromosomal dna and insertion of multiple superfluous dna fragments. unintended mutations were also documented at other locations in the plant genome. such transformation-induced mutations could provide an explanation for unexpected phenotypes in transgenic plants. we decided to survey regulatory documents and the scientific literature for instances of unexpected phenotypic consequences arising in transgenic plants. this poster documents the preliminary results of our survey. it is intended to assess the range and frequency of unexpected consequences and to examine whether there is sufficient data available to determine their origin. it is our belief that investigating the origin of these unexpected phenotypes should be a principal aim of biosafety research. biotechnological production of metabolites such as carotenoids could be of high interest because of their antioxidative and antimutagenic activities in human body. production of these metabolites by microbial cells is dependent on cultivation conditions. so, presence of exogenous stress in cultivation environment could stimulate biosynthetic pathways of desired metabolites. two non-conventional yeast strains, rhodotorula glutinis and sporidiobolus salmonicolor, were chosen for study of carotenoid production useful in feed industry. hydrogen peroxide, sodium chloride and/or their combinations were used as exogenous stimulators of carotenoid pathway. presence of exogenous stress led to important overproduction of pigments as well as of supplementary studied substances (ergosterol, glycerol). higher adaptability of yeast cells was observed not only in cultivations with one type of stress. combination of stress factors in cultivation media induced significant increase of pigment formation. moreover, under controlled conditions in laboratory fermentor s. salmonicolor produced about eight-fold amount of ␤-carotene (230 g/g) in medium with 2% nacl and 5 mm h 2 o 2 than in control sample. similar result was observed in r. glutinis cultivated in presence of 2% nacl in inoculation medium only (200 g/g of ␤-carotene). the use of stressed biomass of red yeasts in feed industry could have positive effect not only in animal and fish feeds because of high content of physiologically active substances, but it could influence nutritional value and organoleptic properties of final products for human nutrition. this work was supported by the project msm 0021630501 of czech ministry of education. the culture ph significantly affects mycelial growth and morphology, exopolysaccharide (eps) formation, and their molecular properties during submerged cultures of a medicinal mushroom ganoderma lucidum. when the culture ph shifts from 3 to 6, mycelial growth (12.5 g/l) and eps production (4.7 g/l) were favorable compared with other ph-control strategies. the mycelial morphology was also significantly varied upon culture ph: a feather-like pellets were found when the ph was controlled shifting from 6 to 3 at day 4, which was regarded as undesirable morphological form for eps production. compositional analyses revealed that the ratios and chemical compositions of the eps formed in bottom or top fractions of ethanolic precipitates were significantly different upon culture ph. the molecular characteristics of the eps were further investigated using a size exclusion chromatography/multi-angle laser light scattering (sec/malls) system. plant ␤-n-acetyl-hexosaminidase (hex) (ec 3.2.1.52) is reported to have diverse physiological roles like fruit ripening, degradation of reserved glycoproteins in germinating seed and chitin-elicited lignification. in this paper we report the purification and characterization of hex from korean ginseng roots. after extraction with citrate-phosphate buffer, hex was purified to homogeneity using ion exchanger chromatography, hydrophobic interaction chromatography and gel filtration. its molecular weight was determined using gel filtration and mass spectrometer. enzymatic parameters were studied with 4-methyl-umbelliferyl-n-acetylglucosaminide as substrate. the effect of heat stress and weak organic acids on escherichia coli and a comparison of its recovery by the plate count method and flow cytometry monica s. talsania due to the importance of microbiology for human health, methods have been developed to enumerate viable bacteria. dilution plating is seen to be the 'gold standard' for proof of a cells viability. however, the success of this method relies on post sampling growth, which is limited by our ability to grow cells in the laboratory. additionally, stressed or sub-lethally damaged cells, remain undetected. single cell measurements can provide rapid detailed physiological information, and the assessment of population heterogeneity. this work compared the recovery of stressed e. coli as measured by the number of cfu/ml and by multi-parameter flow cytometric analysis. weak organic acids and high temperature-short time processing (htst) were used to stress the cells both methods commonly used during food preservation. it was shown here that flow cytometry is a powerful tool for the enumeration and detailed analysis of any non-culturable microbial population, which is important because cytotoxic compounds and heat stresses used in food preservation often have a growth inhibiting effect but not necessarily a lethal one. paul g. kovalenko molecular biology & genetics nasu, zabolotnogo str. 150, 031473 kyiv, ukraine the pharmaceutically important plant species of glycyrrhiza sp. (called licorice) is an important commercial product used as a natural sweetener, anti-inflammatory, anti-cancer and anti-diabetic agents. agrobacterium rhizogenes transformation system was used for the hairy root cultures of licorice g. uralensis. after inoculation of aseptic stem segments the ability of hairy root formation was scored for a period of 6 weeks. mean transformation frequency ranged from 27% (for 8196 up to 31% (for 15,834). some transformed genotypes showed significant differences in roots weight, flavonoids and glycyrrhizin (gl) production. the cotransformation rate of licorice intact explants cultivars with lba 9402 tl-dna and the 35s gus gene showed an average of more than 35%. these obtained root cultures were additionally elicited with extracts of biotic elicitor acremonium sp. (endomycorhizal fungus), and were used as an in vitro system to metabolites production. the transformed and elicited hairy roots of g. uralensis were obtained by infection of a. rhizogenes 8196 have produced gl at an yield of 4.5% dry weight on the period of culture as a 30 days. according to tentative analyses the hairy roots cultures of glycyrrhiza species produced flavonoids (liquiritigenin and liquiritigen). more high levels (3.42 g/l) of the total flavonoids production have been identificated on the strains which transformed by lba 9402. this study involved any difference among elicitor treatments and incubation periods for the optimal meabolites production. clearly, the selection of an effective agrobacterium strain for the production of transformed root cultures is highly dependent on the plant species, and must be determined empirically. ayse gul nasircillar akdeniz university, biology, akdeniz univ. faculty of art-science, biological department, 07058 antalya, turkey mature embryos of five t. aestivum and five t. durum cultivars formed embryogenic callus on two different media. embryos were removed from surface sterilised seeds and placed with the scutellum upwards on a solid agar medium containing the inorganic components of murashige skoog and 2 mg/l 2,4-dichlorophenoxyacetic acid (2,4-d) or 1 mg/l naphthalenacetic acid (naa). the developed calli and regenerated plants were maintained on 2,4-d or naa free ms medium. wheat plants can be regenerated via two different systems. there were significant differences in percentage of callus induction and regeneration capacity on the different initiation medium. among the t. aestivum cultivars, yakar had the highest regeneration capacity in both induction medium. in t. durum cultivars, kiziltan gave the highest regeneration capacity in ms + 2,4-d medium and yilmaz gave the highest regeneration capacity in ms + naa medium. a strong genotypic effect on the culture responses was found for both induction medium. the glycolytic enzyme triosephosphate isomerase (tpi), which catalyses the interconversion of the triosephosphates dihydroxyacetone phosphate (dhap) and glyceraldehyde-3-phosphate (gap), was studied for its control on glycolysis and mixed acid production in lactococcus lactis il1403. we constructed a number of l. lactis strains in which the tpi activity was modulated from 3% to 217% of the wild-type level. the enzyme was found to be present in high excess with 3% tpi activity supporting 30% of the wildtype glycolytic flux, and with 24% of the wildtype tpi activity the glycolytic flux was essentially unchanged. measurements of the upstream metabolites glucose-6-phosphate (g6p), fructose-1,6bisphosphate (fbp) and dhap were essentially unchanged for tpi activities from 24% to 217%, and only in the strain with 3% tpi activity we observed a significant increase in the intracellular dhap concentration. homolactic product formation was preserved throughout the interval of tpi activity studied, though a small increase in the amount of acetate and formate production was observed in the strain expressing tpi at the lowest level (3% tpi activity). the finding of an increased mixed acid pattern under intracellular conditions with a high dhap concentration is in contrast to earlier data from literature, which indicated that the triosephosphates play an important role in regulation of pyruvate metabolism in l. lactis with a negative effect on the mixed acid flux. we have recently shown that alcohols induce the adhesion of l. monocytogenes at low temperatures, presumably accompanied by enhanced exopolysaccharide (eps) production. however, little is known about the mechanisms involved in the formation of biofilm and eps by l. monocytogenes. in the present project, we show that deletion of selected regulatory and up-regulated genes did not abolish attachment, though the degree of alcohol-induction in some cases was affected. we are applying bioinformatics to search for homologues in l. monocytogenes of known eps genes from various gram positive bacteria. this has revealed candidate genes involved in the synthesis of eps, such as genes encoding glycosyltransferases. moreover, we are at present performing dna microarray analysis for the egde strain grown at 10 • c in the presence of 2.5% isopropanol. this data should, combined with the bioinformatic results, give us a good indication of the genes involved in alcohol-induced surface attachment. repetitive-pcr (rep-pcr) was applied in research on non-starter lactic acid bacteria (nslab) in cheese. we first showed that strains previously differentiated by pulsed field gel electrophoresis (pfge) also could be differentiated by rep-pcr. this was partially due to slight changes in the pcr conditions that allowed reproducible amplification of 7-9 kb bands. more than 20 bands were obtained for most strains. a clear differentiation was also obtained between lactobacillus paracasei, lactobacillus plantarum, lactobacillus curvatus and lactobacillus danicus (a new species found in danish and estonian cheeses and traditional starter cultures). we found that this technique is highly reproducible, e.g. identical profiles in three different pcr-machines, two different dna isolation procedures, and different trained personnel. we applied the developed rep-pcr technique to confirm that survivors after heat treatment, were the actual strains introduced and not due to post-pasteurization contamination. we also showed that when we added a cocktail combination of five strains as protecting cultures to cheese, two to three members of this cocktail was dominating the cheese nslab microflora. in control cheeses without the cocktail in most cases other strains dominated, but in a few cases we were able to show cross-contamination between cheese vats. these data indicate that the rep-pcr will be useful to follow development of adjunct cultures as well as provide a reproducible subspecies (e.g. strain) differentiation. rep-pcr is a much quicker and less labour requiring procedure than pfge, and is apparently a much more reproducible technique than what has been seen for rapd. dynamic modeling of lactococcus lactis metabolism and its dynamic behavior for lactate secretion and regulatory characteristics jinwon lee, ui sub jung, hye won lee department of chemical and biomolecular engineering, sogang university, seoul, south korea, 121-741. e-mail: jinwonlee@sogang.ac.kr (j. lee) dynamic metabolic model for lactococcus lactis has been developed in order to analyze a time-dependent behavior of lactate secretion mechanism and probe its regulatory roles. the model was used to compare and analyze the lactate metabolism through in silico simulation and in vitro experimental measurements most of all pyruvate branch point seems to play a major role in producing lactate, and the results of metabolic control coefficient analysis recommend to increase lactate dehydrogenase activity and to decrease nadh oxidase activity. for obtaining more realistic data, we have added some measured flux data including some intermediate metabolites. by combining the simulation results and experimental measurements, we could establish more reliable and robust systematic lactate secretion model. in addition, an efficient parameter estimation method was used to test the exactness of the reported kinetic parameters. what to choose -the fast or the detailed -strategy to get informative profiles of secondary metabolite produced by fungi in culture. chemo-diversity and lead discovery calls for high throughput techniques, but do we need columns will direct infusion esi-ms (dims) do the job. the latter may give matrix effects and lacks resolution resulting in loss of information, while lc-ms analysis takes time and challenge the data processing. results from nano-esi dims and lc-ms analyses of the same extracts important penicillium species are compared. these results illustrate advantages and problems using these techniques for rapid profiling of fungal secondary metabolites, reviling that matrix effects in dims do not seriously hampers detection of important metabolites while the specificity and certainty, for e.g. de-replication is much higher in lc-ms. phenotypic classification of fungi is essential in food biotechnology ulf thrane center for microbial biotechnology, biocentrum-dtu, søltofts plads 221, technical university of denmark, dk-2800 kgs. lyngby, denmark. e-mail: ut@biocentrum.dtu.dk fungi are of great importance in food and food production. the intended use of fungi as cell factories for production of food ingredients is an upcoming issue in food biotechnology; however, this brings up a possible contamination with mycotoxins as a major issue. a reliable identification of the producer strains is crucial as a correct identification at species level following an updated taxonomy is the key to information on functional characters, e.g. useful metabo-lites and potential mycotoxins, growth conditions, resistance, etc. unfortunately, many mycological reports do not specify the taxonomy used or do not pay sufficient attention to taxonomical systems based on classification by functional characters-in contrast they are using a nucleotide sequence based phylogeny, which conveys little -if anything -about function of the organism. this situation is a major challenge for biotechnologists and mycologists in the years to come and will be highlighted by illustrative examples. the commercial interest in functional foods containing sufficient amounts of living probiotics is paralleled by the increasing scientific attention to the beneficial effect in the digestive tract. a daily intake of viable cells is proposed to ensure probiotic effect on consumer's health. one of the approaches which seems to be feasible to enhance probiotic viability and stability is to improve the fermentation conditions. during batch fermentation the viability of lactobacillus gasseri 5714 decreases after reaching a maximal value apparently indicating cell death. in this work, the apparent loss of viability can be avoided during fed-batch fermentation. a three-fold increase in viability is obtained when nutrient concentration was controlled compared with the viability reached in batch cultures. as a consequence, higher biomass concentration and lower specific lactic acid production were obtained. a mathematical model was developed to simulate and describe the effect of nutrient limitation on growth, viability, glucose consumption and lactic acid production. contribution to the metabolic adaptation to food restriction in rabbits (preliminary results) s. van harten 1 , s. borges 2 , p. cravo 2 , l.a. cardoso 1 : 1 instituto de investigação científica tropical, cvz, lisboa, portugal; 2 instituto de higiene e medicina tropical, lisboa, portugal. e-mail: svharten@gmail.com (s. van harten) in order to understand metabolic differences between two breeds of rabbits (halop ab and oryctolagus cuniculus algirus) during food restriction, the activities and expression of key enzymes and hormones of the rabbit were studied. animals from each breed were divided in two groups (ad libitum and restricted), revealing the results a similar difference in glycemic levels between fed and underfed rabbits, with a restriction of 50% of ad libitum feeding in the wild animals (decrease of 23% lw) and 16% of that ingestion in the halop breed (decrease of 32% lw). the activities of glutamine synthetase and glutaminase show a higher reduction of these enzymes in the wild animals superior to that of the halop breed, compromising, in this way, the ammonium detoxification and the entry of residual carbonated groups of the protein catabolism into the krebs cycle. in the latter animals, a rapid mobilization capacity of triacylglycerols (tga) appears to exist, with a rapid catabolism of fatty acids leading to their oxidation. the wild breeds' results reveal a rise of circulating tga, reflecting difficulties in the lipolysis and mobilization of nefa for oxidation. in these underfed animals, phosphoenolpyruvate and pyruvate suffered a large increase and oxaloacetate a decrease. the halop breed revealed results that indicate a diminution of glycolisis, being glucoses' energy substituted by carbonated chains of lipolysis and protein catabolism. hormone results showed a higher decrease in insulin, t3 and igf-1 in the underfed halop animals. in order to confirm the biochemical results, relative quantification of enzyme expression was studied by real time-pcr. since the introduction of genetically modified (gm) crops in 1996, the area under their cultivation has globally increased from 1.7 million hectares in 1996 to 67.7 in 2003. the number of countries adopting gm crops also rose from one country, the usa, in 1996 to 20 in 2003. despite numerous successes public opinion still questions the ecological, moral, ethical considerations and issues concerning altering the natural state of the organisms. in this study, a survey of food shoppers' knowledge, attitudes and perceptions of gm foods was carried out in food outlets in nairobi. the food outlets were determined by simple random sampling. using systematic sampling, shoppers were interviewed at targeted imported food products. focus group discussions were also conducted with farmers at city markets. the survey reflected views of a systematic sample of 387 shoppers in seven food outlets between november and december of 2003. it revealed knowledge at 20%, with positive attitudes and good perceptions towards gm foods (χ 2 = 42.873, d.f. 9, p < 0.001). seventy nine percent of shoppers were willing to buy and consume gm foods (χ 2 = 61.321, d.f. 2, p < 0.001). cross-tabulation of shopper's position on various issues raised in the survey showed a strong correlation between the respondents' respective knowledge, attitudes, and perceptions (r = 0.84). nineteen percent of food sampled tested positive for gms. poisson statistics were used to calculate the number of sample sequences. the statistical tools were obtained from spss version 11.5. the results of this study will be of great interest in determining the use and adoption of gm crops in kenya. it will also guide the development of national foreign food policy on gm foods. the technology should be embraced as soon as it is acceptable to alleviate, drought, famine and hunger estimated to be affecting 3.3 million kenyans today, mostly children. consumers and gm foods: the case of turkeyözlenözgen 1 , mustafa yildiz 2 : 1 department of family and consumer sciences, school of home economics, university of ankara, ankara 06130, turkey; 2 department of field crops, faculty of agriculture, university of ankara, 06110 ankara, turkey the future development of food biotechnology depends on consumer acceptance. scientists are aware that consumer attitudes will have a crucial impact on the process of the food biotechnology. because, food is one of the central features in human life. consumers' attitudes and trusts in the institutions will determine how gene technology will be used in food sector, in the future. recently, research concerned with consumer aspects of gm foods accelerated. but in turkey, the literature that deals with this subject is very limited and sparse. therefore, this research was carried out on the turkish consumers with the purpose of analyzing the consumers' awareness, assessments about benefits-risks, market place and labelling, and trusts in institutions, towards gm foods. this study was based on interviews with consumers who have recently purchased from major malls, during shopping hours. of the four major malls, voluntary male and female consumers were included in the research if they had main or secondary responsibility for household shopping. the questionnaire form was applied to subjects through face-to-face individual interview. the data were analyzed by using statistical methods according to explanatory variables, including age, gender and educational level. findings indicated that consumers' awareness and views about gm foods were connected to selected demographic characteristics. the results of this study can be important for consumer educators, marketing managers and policy makers. benefit-risk perceptions and moral beliefs of turkish consumers towards transgenic productsözlenözgen 1 , haluk emiroglu 2 , mustafa yildiz 3 , ayşe sezen taş 1 : 1 department of family and consumer sciences, school of home economics, university of ankara, 06130 ankara, turkey; 2 faculty of law, university of bilkent, 06590 ankara, turkey; 3 department of field crops, faculty of agriculture, university of ankara, 06110 ankara, turkey the use of biotechnology in production has generated considerable debate involving the benefits-risks and moral beliefs associated with its use. consumer acceptance of genetically modified product is a critical factor will affect the future of this technology. this study was planned and conducted to determine the relationship between product/process related benefit perceptions, product/process related risk percentions and moral beliefs of consumers towards transgenic products. a total of 400 university educated consumers, consisting of 200 males and 200 females, employed at the ministries selected by random sampling method in ankara, were included into study. interview techniques were used in the gathering the research materials. the interview instrument had been prepared considering previous research and literature. answers given to sentences typed likert were scored, used "varimax analysis technique" for validity. in order to test the reliability of questionnaire were calculated "cronbach alpha" as inner consistency coefficient. the t-test were performed for determining the differences dependent on gender and age variables between product related benefit perceptions, process related benefit perceptions, product related risk perceptions, process related risk perceptions and moral beliefs of consumers. the examination of relationships between product/process related benefit perceptions, product/process related risk perceptions and moral beliefs of consumers was made by correlation analysis technique. it is thought that the results of this study are important both for scientists and social scientists. the application of the dna recombinant technology for food production is generating a great debate in our society with the participation of scientists trying to explain the way of obtaining these new foods and which are their implications; environmentalist groups and anti-biotechnology associations that systematically are against to the application of this technology; legislatives bodies and the public in general, represented by consumers' organizations that expresses their right to be informed. considering that university students will be the future professionals and consumers their opinion on this topic will be decisive in its success or failure, this research is aimed to performed a global and comparative study of the agrobiotechnology perception by students from different areas of knowledge and studies. this study was carried out during the academic years 2000-2004, being analyzed a total of 1516 valid surveys. the designed questionnaire included 30 questions relatives to: evaluation of the own knowledge and interest on the topic; evaluation of the information sources mainly used by university students to obtain nutritional information; the opinion about gm food labelling; risks/benefits perception; purchasing intention and support of biotechnology. results obtained showed that 37% of the university students interviewed have a clear positive perception of biotechnology, mainly the students of the health sciences area. these students understand the scientific terminology and they use the university as the main source of information. they support the development of the biotechnology and they consider that in a future it will report them benefits. other group (11%) has a clear negative perception, they are mainly students from law and art history. they do not understand the scientific terminology, they consider that biotechnology will cause them risks, and as a consequence they don't have intention of buying these foods. the technology of the dna recombinant can be also used to introduce in the plants genome the gene that codes a protein of interest for their use as antigen. the application of agrobiotechnology has allowed the development of a new generation of vaccines that try to reduce or to eliminate the inconveniences of the classic ones. for the new vaccines design, the detailed knowledge of the biology of the pathogen is considered. with this knowledge the genes implied in virulence can be inactivated or modified selectively. the term "edible vaccines" it is usually applies to the use of edible parts of the plants (tubers, fruits, leaves, etc.) genetically modified with the purpose to produce specific components (antigens) of a pathogen (virus, bacteria, etc.) against which is wanted to protect a person or animal. however, oral is not the best vaccination route since the quantity of antigen for an efficient immunization it is usually high, being also needed, the co administration of an adjuvant that stimulates the immune answer. on the other hand, it is also important to highlight that the levels of antigen accumulation in transgenic plants is usually lower than the necessary ones. another problem is the irregular accumulation of the antigen in the different parts of the plants, thus difficult the appropriate control of the doses. tannin is polyphenolic component having some antioxidant properties and exists in many plants and fruits. in pomegranate juice this component causes turbidity and haze. during fruit juice clarification by conventional gelatin method, all poly phenolic substances which are responsible for antioxidant activity are removed and as a result the quality of the product is reduced. in the present study tannase enzyme (tannin acyl hydrolase; ec 3.1.1.20) was used to decompose tannin to gallic acid and glucose and as the result the amount of turbidity of the juice is decreased, however, the antioxidant properties remain unchanged since tannin is not decomposed and not separated in the juice as it occurs in the gelatin method. the amount of gallic acid in pomegranate juice samples before and after addition of tannase was measured using hplc tests and the optimum temperature, the enzyme and juice contact time, ph, and solvent concentration for clarification of pomegranate juice were obtained as 45 • c, ph = 5.5, 2 h and 50 mm citrate buffer, respectively. the potential benefits of enzymatic clarification of pomegranate juice, that is preservation of antioxidant activity and hence increasing the quality of the fruit product, in comparison to that of conventional clarification method by gelatin introduce a new technique in turbidity and haze removed in tannin containing fruit juices. the objectives of this study are to investigate the inhibitory effect of low molecular weight chitosan for its use as biopreservatives of foods. the inhibitory activity of chitosan against escherichia coli and staphylococcus aureus was investigated by determining the effect of chitosan treatment on bacterial growth during culture and its effect on the viability of non-growing cells. the activity of chitosan was decreased considerably for both strains when sucrose was added to ts broth containing chitosan. the addition of ethanol affected little on the inhibition of chitosan against s. aureus. the influence of nacl on the activity of chitosan was similar to the influence of sucrose and ethanol. however, the experiments with non-growing cells showed that the ethanol enhance drastically the antibacterial activity of chitosan on s. aureus. the results presented in this paper demonstrate that its antibacterial activity may be affected considerably by common food additives such as sucrose, ethanol and nacl. in lactococcus lactis the enzymes phosphofructokinase (pfk), pyruvate kinase (pk) and lactate dehydrogenase (ldh) are uniquely encoded in the las operon. we have applied metabolic control analysis to study the role of this organisation. earlier work showed that ldh at wildtype level has zero control on glycolysis and growth rate but high negative control on formate production (c j formate ldh = −1.3). we find that pfk and pk have zero control on glycolysis and growth rate at the wildtype enzyme level but both enzymes exert strong positive control on the glycolytic flux at reduced activities. pk has high positive control on formate (c j formate pk = 0.9 − 1.1) and acetate production (c jacetate pk = 0.8 − 1.0), whereas pfk has no control on these fluxes. decreased expression of the entire las operon resulted in a strong decrease in growth rate and the glycolytic flux. increased las expression resulted in a slight decrease in the glycolytic flux. at the wildtype level the control was close to zero on both glycolysis and the pyruvate branches. the sum of control coefficients for the three enzymes individually was comparable to the control coefficient found for the entire operon at the wildtype level; the strong positive control by pk almost cancels out the negative control by ldh on formate production. the analysis suggests that co-regulation of pfk and pk provides a very efficient way to regulate glycolysis, and co-regulating pk and ldh allows the cells to maintain homolactic fermentation during regulation of glycolysis around wildtype level. bovine chymosin is used extensively in cheese production because of its specificity and low proteolytic activity. we are interested in the caprine chymosin as an alternative because in the canary islands cheese has traditionally been made using goats milk with extract from the abomasum of newborn goats as coagulating factor. we isolated and characterized the prochymosin cdna from the abomasum of milk-fed kid goats. this cdna predicts a polypeptide of 381 amino acid residues, with a signal peptide and a proenzyme region of 16 and 42 amino acids, respectively. the caprine preprochymosin has 99% and 94% identity with the corresponding lamb and calf sequences. the cdna fragment encoding prochymosin was fused in frame to the killer toxin signal sequence in a constitutive vector, and to the ␣-factor signal sequence-flag in an inducible expression vector. kluyveromyces lactis pm3-5c, k. lactis sel1, characterized by a "supersecreting" phenotype, and saccharomyces cerevisiae bj3505 were transformed with the recombinant plasmids. activated culture supernatants of yeast transformants showed milk-clotting activity. the flag-prochymosin fusion was purified from bj3505 culture supernatants by affinity chromatography. after activation at acid ph, proteolytic activity assayed toward casein fractions showed that the recombinant caprine chymosin specifically hydrolyzed -casein. the recombinant caprine enzyme could be an alternative milk coagulant in cheese making. lipid accumulation in schizochytrium g13/2s was studied under batch and continuous culture. different glucose and glutamate source concentrations were supplemented in a defined medium. during batch cultivation, lipid accumulation occurred towards the end of the growth phase but ceased when cell proliferation stopped. under continuous culture, as dilution rate decreased from 0.08 to 0.02 h −1 , both cell dry weight and total fatty acid content (tfa) of the cell increased. with a constant dilution rate of 0.04 h −1 , nitrogen limitation induced lipid synthesis (28% tfa) as described for other lipid-accumulating organisms. however, with carbon-limited conditions, some lipid accumulation was still possible, the tfa being 22%. finally, the batch and continuous culture methods are compared for docosahexaenoic acid (22:6, n − 6) production. the objectives of this study is to investigate the inhibitory effect of low molecular weight chitosan for its use as biopreservatives of foods. the inhibitory activity of chitosan against e. coli and staphylococcus aureus was investigated by determining the effect of chitosan treatment on bacterial growth during culture and its effect on the viability of non-growing cells. the activity of chitosan was decreased considerably for both strains when sucrose was added to ts broth containing chitosan. the addition of ethanol affected little on the inhibition of chitosan against s. aureus. the influence of nacl on the activity of chitosan was similar to the influence of sucrose and ethanol. however, the experiments with non-growing cells showed that the ethanol enhance drastically the antibacterial activity of chitosan on s. aureus. the results presented in this paper demonstrate that its antibacterial activity may be affected considerably by common food additives such as sucrose, ethanol and nacl. nitrite-oxidizing bacteria catalyze an essential step of nitrogen elimination in biological wastewater treatment. recently, novel and yet uncultured nitrite-oxidizing nitrospira-like bacteria were found to be abundant in municipal and industrial wastewater treatment systems where they outcompete nitrobacter, which has long been considered as the organism responsible for nitrite oxidation in bioreactors. despite the importance of nitrospira-like bacteria for wastewater treatment and for nitrogen fluxes in natural ecosystems, little is known about their ecophysiology and interactions with other organisms. cultivation-independent molecular techniques were applied to investigate the diversity, distribution, and physiological and genetic features of nitrospira-like bacteria in nitrifying activated sludge and biofilm. a surprisingly high diversity of these organisms was found to exist in these engineered and in natural habitats. moreover, significant physiological differences could be identified among various phylogenetic sublineages in the genus nitrospira. quantitative co-localization analyses performed by novel image analysis software revealed that these metabolic features are reflected by the spatial organization of nitrifiers living in biofilm and activated sludge flocs. based on an environmental genomics approach the genome of a nitrospira-like bacterium found in activated sludge is being analyzed. results obtained so far point at unexpected physiological capabilities of this organism, and allow us to propose that nitrospira-like bacteria may also play roles in the bioremediation of (per)chlorate and chlorite. the activated sludge process is the most common way to remove organic matter, nitrogen and phosphorus from wastewater by microbiologically means. knowledge about the microorganisms involved is fundamental for optimisation of existing plants and development of new plants and process designs. many of the bacteria believed to be involved in nitrification, denitrification, biological phosphorusremoval, and removal of organic matter in full scale plants are now identified by use of molecular methods. recent developments in experimental approaches have allowed the study of the ecophysiology of these uncultured and potentially important bacteria, thus providing a better understanding of their function in full-scale activated sludge ecosystems. relatively few dominant species in each functional group (e.g. denitrifiers and polyphosphate accumulating organisms) seems to be present. some species appear to be very specialized regarding nutrient requirements while others are more versatile. a new method for mercury remediation from industrial wastewater based on the enzymatic reduction of mercury by live mercury resistant bacteria immobilized on the pumice particles has been developed in gbf, germany, and implemented in the industrial scale (unknown). the experience gained during operation of this instalation led to the idea, that the process of bioremediation may be integrated in one bioreactor with the sorption of mercury from wastewater, by immobilization of the bacteria directly on the activated carbon. for this it was necessary to define several significant parameters of the activated carbon used and the sorption process itself. the paper presents results of the equilibrium and kinetics investigations of the process of mercury sorption from aqueous solutions onto seven different types of activated carbon. the effective diffusion coefficients in the particles were obtained from the transient-state experiments and the sorption isotherms, saturation capacity of the sorbents and its dependence on the temperature and ph were identified. then the hydrodynamic and sorption characteristics of the activated carbon bed in a laboratory-scale fixed-bed bioreactor were investigated in different process conditions (mercury concentration, volumetric flow rate, temperature, ph). the results (effective capacity of the bed, dispersion and diffusion coefficients, mass transfer coefficient) enable implementation of this bioreactor for modified, integrated process of mercury bioremediation from industrial wastewaters. research supported by the grant kbn 4 t09c 013 25. bacterial cr(vi) reductases convert the very mobile toxic cr(vi) to the less toxic and less mobile cr(iii). the ability to reduce cr(vi) was studied on cell extracts of ochrobactrum tritici strain 5bvl1 and microbacterium sp. strain 3a. both microorganisms were isolated from the same sample of chromium-contaminated sludge, taken from a wastewater treatment plant. while in the first case activity was found to be associated with the intracellular soluble extract, in the second case it was a process occurring extracellularly. cr(vi) reduction by the intracellular soluble extracts of strain 5bvl1 required the presence of nadh or nadph as electron-donor, while the extracellular fraction of strain 3a only used nadph. several studies were made on strain 5bvl1 intracellular soluble extracts. a k m of 26.11 m cr(vi) and a v max of 5.75 ± 0.13 nmol cr(vi) min −1 mg −1 protein were estimated from the lineaweaver-burk plot and michaelis-menten non-linear regression. the temperature and the ph optima for cr(vi) reduction were 37.5 • c and 5.0, respectively. hyperthermus butylicus is an anaerobic hyperthermophilic crenarchaeon, isolated from the solfataric sea floor off sáo migel island, azores (zillig et al., 1990) . h. butylicus grows at up to 108 • c (optimally between 95 and 106 • c) at ph 7. it can utilize peptides, polysaccharides, and other substrates, as carbon sources to produce acetate, butyrate, and n-butanol. the capability to produce enzymes (e.g. hydrolases, dna and rna polymerases, etc.) that can tolerate and function at temperatures 20 • c higher than most other thermophilic archaea, renders h. butylicus of particular interest to the biotechnology industry. the complete genome sequence of h. butylicus was determined and it contains 1,667,186 bp on a single circular chromosome. 1695 protein encoding genes were identified which use a high level of uug and gug start codons. many of these were assigned functions on the basis of sequence comparisons. our analyses revealed some unusual metabolic properties in h. butylicus. several sugar transporters were identified, although the set of genes required for glycolysis is incomplete. moreover, genes encoding enzymes converting glucose to trioses are absent and no genes encoding enzymes of the pentose phosphate cycle or the kdpg pathway were detected. the h. butylicus genome encodes many proteases and peptidases although the lon proteases, encoded in all other archaeal genomes, are absent. although it was reported that h. butylicus does not utilize free amino acids in the media, genes for amino acid transporters were identified, and several proteins involved in di-or oligo-peptide transport are encoded. genes encoding signal peptidases are absent. we will summarize gene products of special biotechnological interest. reference zillig et al., 1990. j. bact. 172, 3959-3965. 2 hot genomics: insights in the thermophilic lifestyle of thermus thermophilus from its complete genome holger brüggemann 1,2 , anke henne 1 , gerhard gottschalk 1 : 1 göttingen genomics laboratory, institute of microbiology and genetics, university of göttingen, germany; 2 institut pasteur, unité de génomique des microorganismes pathogènes, paris, france thermus thermophilus is an extremely thermophilic, halotolerant bacterium, which was originally isolated from a natural thermal environment. recently completed genome sequences of two strains, hb27 and hb8, provide a solid foundation for investigating many aspects of thermophilic lifestyle; these range from molecular stability determinants to key elements of organismic physiology. in addition, the species has considerable biotechnological potential; many thermostable proteins isolated from members of the genus thermus are indispensable in research and in industrial applications. the closely related genera thermus and deinoccoocus belong to a distinct branch of bacteria called the deinococcus-thermus group. genome comparison of t. thermophilus and d. radiodurans, a mesophilic organism, which exhibits high resistant to radiation, oxidative stress, and desiccation, is of particular interest for the identification and exploration of thermophilic determinants. a large number of orthologs with a high degree of sequence identity are shared between the two species. this opens the opportunity for comparative studies of conformational and chemical thermostability of proteins, as well as for the identification of specific traits for each organism, explaining their unique physiological properties and their intriguing differences in stress tolerance. although strains hb27 and hb8 share a highly conserved chromosome, striking differences can be found between their megaplasmids, which encode a huge proportion of genes not found in the genome of d. radiodurans. possible contributions made by the megaplasmids to a thermophilic lifestyle will be discussed. microorganisms that can live in high temperatures, extreme ph and high salt concentration are called extromophiles. extromophilic microorganisms have extended our knowledge and understanding of fundamental questions such as the origin of life. the ability to grow in extreme conditions and to produce stable proteins makes extremopliles very attractive for the researchers and also for the industry. extremozymes from extremophiles have a great economic potential in many industrial processes, including agricultural, chemical and pharmaceutical applications. concurrent development of protein engineering will increase the application of enzymes from extremophiles in industry. turkey has vast and various ecologi-cal areas, and so it has a broad microbial diversity. based on the extremophilles which defined in the scope of this project, halophilic microorganisms produced industrially important proteins were isolated from ç amaltı saltern area in izmir, turkey. in this work, growth of isolates at different temperature, salinity and ph values were investigated to determine the effects of various growth conditions. eight isolates grow at ph between 6.50 and 8.50 and two isolates at 6.50-7.50. they grow at temperature between 37 and 55 • c and salt concentration between 3% and 25%. the results of some phenotypic characters showed that they are gram (−) and oxidase,ürease, dnase and nitrate reduction are (−), and catalase (+). they used d(+) glucose, maltose, lactose, sucrose, l(+) arabinose, d(+) mannose, glycerol and four of isolates used d(+) xylose as a carbon source. the isolates resistant to erythromycine, ampicilin sulbactam, cefoxitin, penicillin, bacitracin, novabiocin, amikacin and sensitive to ceftazidine, ciprofloxacin, amoxycillin/clavulanic acid, imipenem, chloromphenicol, ceftazidime/clavulanic acid, aztreonam, cefepime, cefotaxime, cefoperazone amoxicillin. this project was supported by tubitak through project tbag 2321-103t069. the technology of producing renewable energy sources such as ethanol, methane and hydrogen from biomass holds the potential of creating in-house energy resources while lowering the emission of greenhouse gasses as demanded by the kyoto protocol. recently, goals were defined for the european union determining that 5.75% of the transportation fuel has to come from biofuels in year 2010. a large-scale implementation of biofuels into the transportation sector will demand that lignocellulosic biomass, which is found in a surplus throughout the world is used as the raw material for the production process. the presentation will include a comprehensive description of the special bio-refinery concept developed in denmark for production of biofuels and other valuable products from straw. the concept includes several innovative steps such as a pre-treatment method using wet oxidation, on-site production of enzymes and a continuous fermentation process using a genetic modified thermophilic bacterium. by co-producing several biofuels in the plant optimal use of the biomass has been assured and the price of for instance of bioethanol is getting close to conventional oil-based fuels. optimizing each step in the bio-refinery, while having the full integration in mind, will be the way to make an economical viable biofuel production. in the presentation we will present our road map for achieving this goal in the nearest future. replacement of gasoline by liquid fuels produced from renewable sources is a high-priority goal in many countries worldwide. one such fuel, which has been found well suited, is ethanol. it may be produced from various lignocellulosic materials, such as forest and agricultural residues, which are fairly inexpensive. to compete with gasoline the production cost must be substantially lowered. ethanol production from lignocellulose comprises the following main steps: hydrolysis of hemicellulose, hydrolysis of cellulose, fermentation, separation of lignin, recovery and concentration of ethanol and wastewater handling. the enzymatic hydrolysis and fermentation can either be run separately (shf) or combined into a simultaneous saccharification and fermentation (ssf). the latter has been shown to result in higher ethanol yields than shf. some of the most important factors to reduce the cost are: efficient utilisation of the raw material by high ethanol yields, high productivity, high ethanol concentration in the feed to distillation and process integration in order to reduce capital cost and energy demand. in the last years we have performed several studies on the hydrolysis and fermentation of various forest and agricultural residues in a mini-pilot to improve the overall yield of ethanol and to reduce the energy demand and production cost. steam pretreatment, with small addition of acid catalyst, has resulted in sugar yields close to 90% of the theoretical for various types of raw materials, e.g. spruce, salix and corn stover. the ssf has been developed and optimized to give high yield of ethanol. for spruce an ethanol yield of about 80% of theoretical based on the composition of the raw material has so far been obtained using a two-stage steam-pretreatment of so 2 impregnated raw material followed by ssf. improvements of the ssf step, in the form of high dry matter content, recirculation of process streams and adapted yeast have resulted in ethanol concentrations around 45 g/l leading to substantial reduction in energy demand and production cost. these improvements have been assessed by techno-economic evaluation to determine the effect on the ethanol production cost. the process has been further optimised by process integration to further reduce the energy demand. the ethanol production cost was estimated to be around 0.38-0.46 euro/l ethanol assuming a yearly capacity of 200 000 tonnes raw material (dry matter). production of bioethanol from spent grain, a by-product of beer production sho shindo, tadanori tachibana, akita research institute of food and brewing, akita-city, akita 010-1623, japan. e-mail: shindo@arif.pref.akita.jp (s. shindo) the breweries generate one million tons of spent grain every year, and about 20% of the spent grain is recycled in japan. therefore, it is environmentally and economically significant to consider the production of ethyl alcohol as biomass energy using the spent grain from the breweries industry. ethyl alcohol production from spent grain with immobilized yeast cells was investigated. spent grains were liquefied by a steam explosion treatment to obtain liquefied sugar. when 1 kg of wet spent grain was treated under the 30 kg/cm 2 pressure for 1 min using a 5 l steam explosion reactor, 60 g of total sugar was obtained from the liquefied spent grain. furthermore, 1.3% (w/v) of glucose, 0.4% (w/v) of xylose, and 0.1% (w/v) of arabinose were produced when the liquefied spent grain was treated with glucoamylase, cellulase, and hemicellulase enzymes. ethyl alcohol production was carried out by immobilized sacchromyces cereviseae and immobilized yamadazyma stipitis simultaneously from liquefied spent grain. both yeast cells were immobilized on the glass beads carrier. xylose and arabinose were consumed after glucose was consumed completely during ethyl alcohol production. 5.8% (v/v) ethyl alcohol was produced from liquefied spent grain that was adjusted 17% of initial sugar concentration after 2 days. the vegetable oils constitute a resource of renewable potential for the production of fuels, becoming a viable alternative when compared to the diesel from petroleum. among the vegetable oils, the extracted oil of the castor plant seeds is a promising alternative source because it is constituted mainly of the ricinoleic acid (12-hydroxy-9octadecenoic) that represents 90% of the total constitution of the oil approximately. the biodiesel obtained from castor oil can be defined chemically as being a mixture of methyl esters or ethyl esters of carboxylic acids synthesized by transesterification reaction of the existent triglyceride and an alcohol of little chain through the use of alkaline or enzymatic catalysts. in this work, we described the results found in castor oil with different degrees of purity. initially, it was made a rheological characterization followed by structural characterization (rmn 13c, rmn 1h and infrared) and thermal characterization (dtg, dta and dsc) of the crude and refined castor oil. it has been also measured the hydroxyl tenor, acidity index, saponification index and iodine index in different oils. later, these results were used to evaluate possible differences in the quality of the biodiesel (ethyl esters) produced in the enzymatic alcoholysis of the castor oil catalyzed by lipases (novozym 435, liposyme rm im and lipozyme tl im). the degree of substitution of castor oil derivative was performed by titration with 0.1n hcl and confirmed by tlc analysis and the results showed conversion rates about 90%. has been demonstrated to have a wide range of health benefits such as prevention and therapy of various cancers, amelioration of heart disease, and prevention of renal stone formation as well as complications from diabetes. on the hand, lower phosphorylated forms of inositol, especially inositol trisphosphate (ip3) and inositol tetrakisphosphate (ip4) are important signal transduction molecules within the cells both in plants and the animal kingdom. it has been hypothesized that at least the anticancer function of ip6 is mediated via these lower inositol phosphates. the diversity and practical unavailability of the individual myo-inositol phosphates preclude their investigation. phytases, which catalyze the sequential hydrolysis of phytate, render production of defined myoinositol phosphates in pure form and sufficient quantities. different phytases may result in different positional isomers of myo-inositol phosphates and therefore different biochemical properties. phytases differing in ph optima, substrate specificity, and specificity of hydrolysis have been identified in plants and microorganisms. in this paper the dephosphorylation pathway of the novel phyfauia1 was compared to other bacterial phytate degrading enzymes. preliminary results have shown that phyfauia1 converted ip6 into ip5 (myoinositol 1,2,3,5,6-pentakisphosphates) and another isomer, which is yet to be elucidated. in a denitrifying pilot plant reactor, a new obligately anaerobic ammonium oxidation (anammox) process with great potential for nitrogen removal for high strength wastewater was discovered. after transfer of the complex microbial community to a laboratory sbr system, a highly enriched population, dominated by a single anaerobic chemolithoautotrophic bacterium related to the planctomycetes was obtained. the bacterium was purified via percoll centrifugation and characterized as 'candidatus brocadia anammoxidans'. survey of different wastewater treatment plants using anammox specific 16s rrna gene primers and anammox specific oligonucleotide probes revealed the presence of at least four other anammox bacteria, tentatively named 'candidatus kuenenia stuttgartiensis', 'candidatus brocadia fulgida', 'candidatus scalindua wagneri' and 'candidatus scalindua brodae'. a close relative of the two scalindua species, 'candidatus scalindua sorokinii' was found to be responsible for about 50% of the nitrogen conversion in the anoxic zone of the black sea and in the benguela upwelling system along the namibian coast, making anammox an important player in the global nitrogen cycle. electron microscopic studies of all five anammox bacteria showed that several prokaryotic membrane-bounded compartments are present inside the cytoplasm, which are surrounded by unique ladderane lipids. hydroxylamine oxidoreductase, a key anammox enzyme, was present exclusively inside one of these compartments, named the 'anammoxosome'. unique peptides fragments of the purified hao were used to locate the hao gene in genome assembly of 'candidatus kuenenia stuttgartiensis'. the implementation of the anammox process in the treatment of wastewater with high ammonium concentrations was started at the treatment plant in rotterdam, the netherlands, where it is combined with the partial nitrification process sharon. the estimated price for nitrogen removal with partial nitrification and anammox is about 0.75 euro/kg n. gas lift reactors could sustain the highest anammox capacity at 8.9 kg n removed/m 3 reactor per day. an alternative configuration of anammox is the oxygen-limited canon process in which aerobic ammonium-oxidizing bacteria protect anammox bacteria from oxygen and produce the necessary nitrite. maximum nitrogen removal with canon in gas lift reactors was 1.5 kg n/m 3 reactor per day. using several different conditions and parameters, the competition and co-existence of aerobic and anaerobic ammonium-oxidizing bacteria were modeled. in addition to ammonia, urea was also converted after a 2-week adaptation in the canon system. recently it was shown that anammox bacteria can use organic acids as additional energy source. murray moo-young, wa anderson department of chemical engineering, university of waterloo, waterloo, ont., canada n2l 3g1 bioreactors are central to the bioremediation of contaminated environments of water, air or soil. in all three areas of application, bioreactor design is critical to the development of new or improved processes. this overview focuses on the physical limitations of bioreactors caused by biological requirements. the information is based on our own research findings. the need for more applicationsoriented bioremediation research becomes apparent. for technoeconomic reasons, the airlift type has often been the bioreactor of choice for most bioremediations. however, lack of adequate understanding of the quantitative effects of operating conditions on its performance has been an ongoing concern. these effects have been characterized for engineering implementation. to enhance productivity, innovative pretreatment techniques of the polluted sources have also been developed using photocatalytic and chemical oxidation methods. case studies on petrochemical-contaminated water and soil reveal significant enhancement potentials. other studies on microbial biofilters for air bioremediation indicate that the active mass of the biological consortia is not sufficiently understood for rational design. analysis and retrofit design of wastewater treatment facilities using process simulation tools demetri petrides, alexandros koulouris, intelligen, inc., scotch plains, nj 07076, usa. email: dpetrides@intelligen.com (d. petrides) process simulators have been used in the petroleum and chemical industries for over four decades to facilitate the design of new processes and optimize the performance of existing ones. similar benefits can be derived from the use of such tools in the environmental arena, particularly in the field of physical and biological treatment of municipal and industrial wastewater. specifically, process simulators can be used to evaluate and improve options for: (1) more efficient removal of nutrients (e.g., organic nitrogen and phosphorous) that cause eutrofication, (2) estimation and control of volatile organic compound (voc) emissions from open tanks, and (3) more efficient removal and control of hazardous compounds. the potential benefits will be illustrated with cases studies involving both municipal and industrial wastewater facilities. the microbial reduction of metals has showed recent interest as these transformations can play crucial roles in the cycling of both inorganic and organic species in a range of environments and, if harnessed, may offer the basis for a wide range of innovative biotechnological processes. under certain conditions, however, microbial metal reduction can also mobilise toxic metals with potentially calamitous effects on human health. some effluents present heavy metals as soluble compounds, several microorganisms have the capacity to precipitate these metals as insoluble compounds, and this fact allows the collection and separation of these metallic precipitates from contaminated medium. sulfate-reducing bacteria (srb), under anaerobic conditions, oxidize simple organic compounds (such as acetic acid and lactic acid) by utilizing sulfate as an electron acceptor and generate hydrogen sulfide. hydrogen sulfide reacts with heavy metal ions to form insoluble metal sulfides that can be easily separated from a solution. the purpose of this work was study the capacity of desulfovibrio sp. cultures to reduce mixtures of the heavy metals in presence or not of petroleum. for it the experimental design 2 k (k = 5) was carried out. the five studied factors were cr, cu, mn, zn and petroleum. the study was carry out with desulfovibrio sp. batch studies were performed in 50 ml sealed bottles with different concentrations (cr(iii)-10 ppm, cu(ii)-5 ppm, mn(ii)-10 ppm, zn(ii)-15 ppm) of metal sulfate and 2 g l −1 of petroleum. during batch incubation the dissolved concentration of metal studied in supernatant were decreased to undetectable levels for zn (70-100%), however with cu (40-60%), mn (40-70%) and cr (50-80%). the development of continuous process with sulfatereducing bacteria seems to be a suitable alternative to reduce metals in solution from contaminated media such as industrial or mine effluents. after these preliminary results, some experiments in course are focused to study that purpose. reduction of odour emissions from livestock buildings using a bioscrubbing system morten øgendahl, nawaf abu-khalaf, jens jørgen lønsmann iversen department of biochemistry and molecular biology, university of southern denmark, dk-5230 odense m, denmark. e-mail: tvede@bmb.sdu.dk (m. øgendahl) a bioscrubbing system for reducing odour emissions from livestock buildings is presented. the bioscrubbing system consists of two separate units; an absorption column and a water purification module. the absorption column is mounted in the ventilations stacks in the livestock buildings absorbing odorants in the effluent air flow. the odorants are absorbed in a spray of droplets formed by a grid of high pressure nozzles in the inlet of the absorption column. the spray of droplets is extracted from the air flow and pumped to a centrally located water purification module, an inverse three phase fluidised bed bioreactor, where the bio-degradation of the absorbed odorants occurs. the bioreactor features a split sparging system for maximum mixing and aeration. the cleaned water is recirculated to the absorption column. an electronic tongue will quantify key odorants in the bioreactor. the absorption column is designed to be retrofitted into existing livestock building ventilation systems. the water purification module is constructed in standard size units simplifying scaling to match the requirements of individual applications. the total bioreactor volume is increased by increasing the number of standard bioreactors. this work describes a "light off" toxicity bioassay sensor based on whole cell genetically modified bioluminescent bacteria. the biosensor was constructed by mating between the environmentally isolated phenol-degrading acinetobacter sp. strain df4 and the plasmid putk2 that is an inc p␤ plasmid with the bioluminescence genes luxcdabe inserted into a genetic region involved in plasmid replication and transfer. subsequently, the bioreporter designated df4/putk2 and used to investigate phenolics toxicity. among examined phenolics, pentachlorophenol, catechol and nitrophenol recorded the fastest effect on the bioluminescence of bioreporter df4/putk2 over incubation period of 350 min. the effect of various concentrations of phenol and its derivatives either in an individual, duple or triple mixture forms on the bioluminescence response of the constructed bioreporter df4/putk2 were also examined. significant reduction of the bioluminescence was observed whenever a mixture contained pentachlorophenol, catechol and nitrophenol, respectively. to develop a system appropriate to commercialize, the constructed bioreporter df4/putk2 was subjected for immobilization in microtiter plates using several entrapment gels. after a selection of materials was tried, lb/agar was chosen as the most suitable candidate material. characterization of key odour compounds in an air wet scrubber is presented. the key odour compounds represent five chemical groups, i.e. sulphide, alcohol, volatile fatty acids (vfas), phenol and indole. direct aqueous injection (dai) and solid phase extraction (spe) methods were used before injection of key odorants into the gas chromatography-flame ionisation detection (gc-fid). the dai and spe methods were efficient in the identification of odour compounds in the wet scrubber. the spe method had a high recovery and can be more effective in the identification of compounds at low initial concentration. however, dai showed a better linearity and a lower limit of detection (lod) than the spe method. the dai method was the method of choice for characterization, as it is cheaper, easier to handle and highly applicable. at least two odorants, phenol and 1-butanol, were quantified successfully using the dai method. their lod was less than their odour detection limit in the wet scrubber. dai method can be used as a reference measurement method for any further analytical application, e.g. electronic tongue. recent developments in biotechnology enabled the widespread use of microbial enzymes in textile, detergent, food and dairy industries and also in various environmental applications. microorganisms which live at extremes of temperature, ph and salinity, produce extremozymes that offer many exciting opportunities for their use in clean production. in this study, microorganisms were isolated from camaltı saltern area ini̇zmir, turkey. effect of medium salinity on the growth of these microorganisms was determined. seven out of 10 isolates required salt for growth. the salinity ranges at which growth was detected were: 5-25% for two isolates, 6-25% for one isolate, 7-25% for two isolates and 8-25% for one isolate. the isolates were also screened for their capability of producing industrially important enzymes such as amylase, protease, lipase, xylanase and cellulose which are widely used not only in textile, detergent, food and dairy industries but also in various environmental applications. all of the isolates were found to be producers of both amylase and xylanase enzymes at varying salinity array within 5-30% salt concentration range at ph 7.0. extracellular protease activity was detected in the medium of all isolates grown at 5, 10, 15, 20 and 25% salinity at both ph 7.0 (optimum growth ph) and ph 9.0. out of 10 isolates, 9, 10 and 9 were found to produce cellulase enzyme when the salt concentrations were 5, 10 and 15%, respectively. at 20% salt concentration, only one isolate was found to be cellulase enzyme producer. none of the isolates were found to produce lipase enzyme at 5-30% salt concentration range. this project was supported by tubitak through project tbag 2321-103t069. chemical engineering department, middle east technical university, ankara 06531, turkey. e-mail: ubakir@metu.edu.tr (u. bakir) glass and ceramic tiles are very widely used industrial materials. in most cases, periodical cleaning is required to maintain their optical properties such as transparency and visual aspects. because of the ever-growing demand for healthy living, there is a keen interest in materials capable of killing harmful microorganisms. the application of these tiles in care facilities to reduce the spread of infections, in public and residental places to improve hygienic conditions are of general interest. in this study the aim is developing methods to apply thin film coatings on glass tiles to make them anti-bacterial by utilizing photocatalysis and investigating their anti-bacterial properties. semiconductors because of their reasonable band gap energies find great attraction through this purpose. the photocatalytic property of semiconductors are used in this process. oxidising radicals are formed on the coated surfaces and these radicals attacks the organic pollutants and bacteria on contact with the surface. titanium dioxide (tio 2 ) coated surfaces are considered to be very effective against organic and inorganic materials, as well as against bacteria. in the experimental procedure coating solution is prepared by sol-gel technique. after pretreatment of surfaces, the coating solution is applied on the surfaces by dip-coating method. after appropriate thermal treatments, to achieve thin, dense and strong coatings, indicator microorganism is directly applied on the coated surfaces and illuminated under solar simulater light source. finally, the number of surviving microorganisms are determined. in this study, the effects of titanium dioxide (tio 2 ), tin oxide (sno 2 ) solutions and metal doping to these coating solutions on anti-bacterial function were investigated. as a result of this study, the number of escherichia coli that is used as indicator microorganism, on tio 2 and sno 2 coated glasses with respect to the control glass reduced by 80-85% and 40-45%, respectively. doping with metals increased the activity of the coatings, hence the number of surviving microorganisms decreased. activity of a methanogenic ecosystem during the primary contact with a solid support s. michaud, n. bernet, p. buffière, j.p. delgenès inra-lbe, avenue des etangs, f-11100 narbonne, france in this paper, the biological activity during the first initial contact between a methanogenic sludge and a solid support was investigated in batch experiments, at different solid concentrations, using two different granular solid materials and with glucose as the main organic substrate. in all cases, the introduction of a solid material in a methanogenic suspended biomass induced a response of the anaerobic microorganisms, after a lag phase during which biological activity was not detected. this lag phase could be the consequence of a physical stress induced by the first contact between microbial cells and the solid surface. this lag phase was not observed when the biomass used originated from a biofilm reactor, i.e. using a biomass previously exposed to a solid material. a change in the metabolism of organic matter from catabolism and methane production toward production of other compounds could be observed, characterised by a sharp decrease of the methane yield in the anaerobic system. analyses of the gas and liquid phases did not show the production of any new gaseous or soluble compound as the biological end product of this activity. this suggests the production of non-soluble compounds by an anabolic pathway, which could indicate the initiation of biofilm formation. this metabolic activity was shown to be directly correlated to the ratio between the solid surface introduced and the microorganism concentration in the anaerobic culture (m 2 g vs −1 ). from kinetic observations, it could be observed that acetogenic methanogenesis recovered more rapidly than syntrophic propionate and butyrate degradation. evaluating microbial diversity of hydrocarbon degrading bacteria cleantis braithwaite, howard rosser, tawfiq al-ibrahim, hussain, al-bandi research and development center, saudi aramco, dhahran, saudi arabia the analysis of microbial diversity with molecular methods is central to isolating and identifying new and potential biocatalysts resources for research and industry. the ability to degrade hydrocarbon components of petroleum is widespread among bacteria, and is an effective method for remediation of a variety of ecosystems. due to the high carbon content of oil and the low levels of other nutrients essential for microbial growth, treatment of oil with phosphorus and nitrogen is generally required to enhance the growth of hydrocarbon-degrading bacteria and to stimulate oil sludge degradation. in this research study, three types of oily sludges from a gas plant, refinery, and terminal facilities were treated with nutrients. to assess the microbial diversity, both biolog culture method and culture independent polymerase chain reaction (pcr,) denaturing gradient gel electrophoresis (dgge) methods were used. nutrient addition significantly improved oil sludge degradation. we identified and characterized several hydrocarbon degrading bacterial strains that have the ability to convert petroleum. these bacteria included representatives both gram positive and gram-negative genera. there were slight difference in the quantity and type of hydrocarbon degrading bacteria found in the three sites. this is the first molecular analysis of hydrocarbon degrading microbial population in saudi arabian operations. mussel adhesive proteins, including the 20-plus variants of foot protein type 3 (fp-3), have been suggested as potential environmentally friendly adhesives for use in aqueous conditions and in medicine. here we report the novel production of a recombinant mytilus galloprovincialis foot protein type 3 variant a (mgfp-3a) fused with a hexahistidine affinity ligand in escherichia coli, and its ∼99% purification with affinity chromatography. recombinant mgfp-3a showed a superior purification yield and better apparent solubility compared to those of the previously reported recombinant m. galloprovincialis foot protein type 5 (mgfp-5). the adsorption abilities and adhesion forces of purified recombinant mgfp-3a were compared with those of cell-tak (a commercial mussel extract adhesive) and mgfp-5 using qcm analysis and modified afm, respectively. these assays showed that the adhesive ability of recombinant mgfp-3a was comparable to that of cell-tak but lower than that of recombinant mgfp-5. collectively, these results indicate that recombinant mgfp-3a may be useful as a commercial bioadhesive or an adhesive ingredient in medical or underwater environments. cresol, a monomethylated phenol, is an aromatic compound. the environmental protection agency (epa) has determined the carcinogenic potential of cresol. various options are being examined for the degradation of cresol because of their unavoidable large scale production and toxicity. many aromatic hydrocarbons can be used as electron donors aerobically by species of pseudomonas, thus leading to the ring cleavage of these compounds. in the present study, pseudomonas strains were isolated from activated sludge collected from sewage treatment plant. it was repeatedly transferred onto nutrient agar plate to check the purity of the culture. the organism was grown aerobically in an inorganic medium with p-cresol as the solitary carbon source. pseudomonas was confirmed by the expression of green pigment, gram staining and biochemical tests including koh, catalase, nitrate reduction and carbohydrate fermentation reaction. inoculation status was used to determine the rate of degradation of p-cresol. the effect of temperature on p-cresol degradation was studied. moreover, the effect of different concentrations of the aromatic compounds on pseudomonas as well as substrate variability was also documented. phenolic intermediates were estimated colorimetrically using 4-aminoantipyrene, folin-lowry method, uv spectrophotometry and hplc. the results indicated that pseudomonas could degrade up to 300 mg/l of p-cresol within 10 h. pseudomonas sp. exhibited good metabolic versatility and degraded other aromatic compounds including m-cresol and p-hydroxybenzoic acid. we conclude that this strain of pseudomonas has excellent potential for bioaugmenting the degradation of p-cresol-containing waste water treatment units. a considerable amount of waste cooking oil is produced by the restaurant industry worldwide. this poses a significant environmental and economic problem, since high oil and grease concentrations in the sewage system could lead to pipes occlusion and decreased efficiency in water treatment operation plants. therefore, sending these wastes to recycling companies or hazardous waste processors is usually required. yarrowia lipolytica, a well-known lipase producer, requires the presence of lipidic compounds (i.e. vegetable oils) to boost enzyme biosynthesis. in this work, the suitability of waste cooking oil as lipase inducer in submerged cultures of this yeast has been assessed. if successful, this procedure could allow both the degradation of an abundant waste and its valorisation as a raw material for the production of a high added value product. the microorganism was grown in a liquid medium to which various amounts of waste cooking oil were added. biodegradation degrees up to 80% (measured as decrease in cod) were obtained after 3 days of treatment. also, initial glucose concentration in the basal medium seemed to influence the efficiency of the process. on the other hand, addition of waste oil led to a significant increase in lipase production (more than two-fold), compared to oil-free cultures. moreover, chain-length specificity of the produced enzymes was significantly different: high activity towards medium chain length esters was found, which hinted to the occurrence of both lipases and esterases. biodesulfurization: a documental review j. ferrer, simon bolivar university, environmental engineering lab. caracas, venezuela a documental review about larger interest aspects in biodesulfurization technique is showed. especifically, the investigation is related to general framework and the justification of this technique, degradatives pathways elucidated up to now, involved microorganism, important elements in development of bacterial desulfurization and progress areas, and future tendency. in situ bioremediation of a p-nitrophenol contaminated site and assessment of its community structure debarati paul, gunjan pandey, sumeet labana, rakesh k. jain institute of microbial technology, sector 39a, chandigarh 160036, india. e-mail: rkj@imtech.res.in (r.k. jain) biodegradation of p-nitrophenol (pnp), a priority pollutant, was studied as a model system for bioremediation of sites contaminated with nitroaromatic/organic compounds. bioremediation studies were carried out in pnp-spiked soil in small plots under natural field conditions using arthrobacter protophormiae rkj100. role of carrier material was examined by immobilizing the bacteria on corncob powder prior to adding them to soil. these studies demonstrated successful removal of pnp by immobilized cells that were able to deplete pnp completely in 5 days, whereas free cells were able to deplete 75% pnp in the same time period. monitoring the fate of released bacteria revealed fairly stable population of the cells when they were immobilized on corncob powder throughout the period of study. on the other hand, there was a decrease of 2.7 log units in colony forming units of free cells at the end of the study (30 days). bacterial community structure and diversity was also studied for the pesticidecontaminated site wherein the effect of addition of an exogenous strain on the existing soil community structure and on soil functionality was determined using molecular techniques. as revealed by restriction fragment length polymorphism (rflp) studies 45 different phylotypes could be identified on the basis of similar banding patterns. sequencing of representative clones of each phylopyte showed that the community structure of the pesticide-contaminated soil mainly constituted of proteobacteria and actinomycetes. terminal fragment length polymorphism (t-rflp) analysis showed only subtle changes in community structure during the process of bioremediation. bacteriocins encompass an array of structurally different molecules produced by a number of phylogenetically distinct bacterial groups and trigger the killing of the same or closely related species. the recombined escherichia coli strain harboring a bacterocin coding region of xanthomonas campestris pv glycines 8ra was disrupted to obtain cell homogenate. peptidic xanthomonas bacteriocins (pxb) were separated by lowering ph and adding salt. the resulting pxb's were partially purified using ion exchangers, gel filtration. two final active fractions, a and b, were obtained with a yield of 0.005% and 500-1000-fold purification. the activity of pxb was stable at the ph ranging from 7.0 to 10.0. andreja kresal, vanja kokol, vera golob textile department, university of maribor, 2000, slovenia wastewater from textile dyeing industries is characterized by high chemical and biological oxygen demands (cod and bod) and intense color due to the extensive use of synthetic dyes. as dyes of complex aromatic structures are resistant to removal by the typical microbial population and may be toxic to the microorganisms present in the treatment plants, discharge of the wastewater to the treatment plants may lead to its failure. beside, direct discharge of these effluents into municipal wastewater plants and/or environment may cause the formation of toxic carcinogenic and/or unhealthy breakdown products. different chemical and physical methods (adsorption, coagulation-flocculation, oxidation, filtration and electrochemical treatments) for color removal have been proposed, but due capital costs and slow operating speed as well as huge amounts of sludge creation there is still a great need to develop an economic and effective method. the use of lignin degrading white-rot fungi and their enzymes (laccase, lignin peroxidase, manganese peroxidase) has attracted increasing scientific attention due their ability to oxidative degrade a wide range of recalcitrant organic compounds. in the contribution, the decolorization efficacy of different commercial textile reactive dyes (anthraquinone, azo, triphenylmethane) will be investigated after the treatment by laccase from trametes versicolor. in order to examine the reuse of enzymatically decolorized liquors, the ecological suitability and the toxicity of the degradation products after different time of enzyme exposure will be studied. this work was carried out within the scope of research project e! 3100 cawab. influence of heavy metals on growth and extracellular enzyme production of a trichoderma harzianum strain with biocontrol potential l. hatvani 1 , l. kredics 2 , a. szekeres 1 , z. antal 2 , l. manczinger 1 , a. nagy 3 , c. vágvölgyi 1 : 1 department of microbiology, university of szeged, p.o. box 533, h-6701 szeged, hungary; 2 hungarian academy of sciences, university of szeged, microbiological research group, hungary; 3 pilze-nagy ltd. kecskemét, p.o. box 407, hungary. e-mail: kredics@bio.u-szeged.hu (l. kredics) trichoderma species are common soil inhabiting asexual filamentous fungi with teleomorphs belonging to the hypocreales order of the ascomycota division. besides the industrial and clinical importance of the genus, certain strains have been found to cause great losses in mushroom cultivation while other strains are well known to possess high antagonistic activity against several plant pathogenic fungi and therefore used as biocontrol agents. important mechanisms of antagonism include competition and mycoparasitism, which -among others -can be related to the fast growth of trichoderma strains and the production of several extracellular enzymes. the influence of certain, soil-occurring heavy metals on mycelial growth and the secretion of extracellular enzymes involved in competition and mycoparasitism was examined in this study regarding an effective, potential biocontrol isolate of trichoderma harzianum. the metal ions zinc, manganese, copper, iron, lead and mercury were applied at the concentrations of 8, 16, 24, 32, 40, 60 and 80 m, and dry mycelial weight as well as the activities of extracellular ß-glycosidase, cellobiohydrolase, trypsinand chymotrypsin-like protease and n-acetyl-glucosaminidase enzymes were determined. it was found that mercury totally blocked mycelial growth, while other metal ions exerted a much lower influence on growth. the presence of heavy metals did not have a significant effect on the activity of the examined extracellular enzymes with the exception of trypsin-like protease, which showed a four-to six-fold rise in activity in the presence of certain sublethal concentrations of copper. based on these results, our further aim is to develop copper-resistant derivatives by mutagenesis from trichoderma strains with biocontrol potential. since proteases play an important role in mycoparasitism, these strains could be applied within the frames of integrated pest management in combination with copper-containing fungicides, resulting in an enhanced level of crop protection even with reduced amounts of fungicides. this work was supported by grants f037663 of the hungarian scientific research fund and grant omfb-00219/2002 of the hungarian ministry of education. the significance of biocontrol agents (bcas) is that some of them possess good antagonistic abilities against plant pathogenic fungi. a significant number of the most prominent fungi for the purposes of agricultural application belong to the genus trichoderma. in previous studies, in vitro assays on agar plates were reported as the generally used method for the evaluation of antagonistic abilities, as the results of these assays are well transferable to the practical application. the aim of the present study was to develop an accurate, image analysis-based method for the evaluation of the biocontrol characters of bcas. randomly selected trichoderma isolates were tested against fusarium culmorum. in the currently developed method, the areas of the fungal colonies were calculated on petri dishes by measuring the occupied surface of the medium on digital images. the inhibition effect was recorded as the value of biocontrol index (bci), which was calculated from the ratio of the area of the trichoderma colony and the total area occupied by the colonies of trichoderma and the plant pathogen. the proposed method was tested for numerous parameters, and the results revealed that bci proves to be capable for the accurate measurement and scale of the biocontrol abilities of fungal isolates. this work was supported by grants f037663 of the hungarian scientific research fund and grant omfb-00219/2002 of the hungarian ministry of education. the effect of advanced oxidation processes and recirculation on biodegradation of leachates from aerobic landfills liliana krzystek, anna zieleniewska-jastrzębska, stanisław ledakowicz department of bioprocess engineering, technical university of lodz, 90-924 lodz, poland modern landfills are built and operated in a way which allows us to treat them as a special type of bioreactor. simulation of municipal waste biodegradation in lysimeters provides knowledge on basic processes that take place in an aerated landfill. the aim of aeration is to stabilise mainly biodegradable and nitrogen containing components and to reduce methanogenic potential. stabilised leachates from old landfills contain big quantities of refractive carbon compounds that cannot be removed by biological methods. in such case most advantageous is to apply advanced oxidation processes (aops). the objective of this study is an experimental simulation of a landfill aerobic stabilisation and the impact of aops and recirculation of leachate on the reduction of organic load. the performance of the processes was monitored by the reduction in time of basic indices of organic load (bod 5 , cod, toc, vfa, tkn, n-nh 4 + ) and changes in biogas composition. the simulation of aerobic landfill processes was carried out in lysimeters with a fixed bed of household solid waste stabilised during 8 months in anaerobic conditions. leachates taken from the lysimeters were recirculated and subjected to advanced oxidation processes, i.e. ozonation and uv radiation with the addition of h 2 o 2 . experimental studies showed that the aerobic waste stabilisation was a very quick process. during a month the bed was stabilised, reaching a significant reduction of organic load indices. aeration of the lysimeters caused a quick reduction of mainly degradable organic substance (in terms of bod 5 ) and n-nh 4 + and vfa. the reduction of methanogenic potential of the landfill was even faster. the composition of gas at the outlet from the lysimeter changed and after one day already its content was similar to atmospheric air. a more frequent recirculation of leachates enhanced greatly the aerobic biodegradation. it was found that application of advanced oxidation processes (especially ozonation) contributed to a growing reduction of the organic load in the leachates from aerated lysimeters. the application of leachate ozonation resulted in a very high degree of reduction of organic compounds (up to 77%). the objective of the experimental study was to assess the effect of temperature on the extent of aerobic batch biodegradation of potato stillage with a mixed culture of bacteria of the genus bacillus. the experiments were performed at 20, 30, 35, 40, 45, 50, 55, 60, 63 and 65 • c, at ph 7, in five l l working volume stirred tank reactor (str) (biostat ® b, b. braun biotech international). the duration of the process was 120 h. initial cod of the stillage amounted to 51.9 g o 2 /l, the main carbon sources being reducing substances (18.7 g/l), organic acids (determined as their sum) (12.2 g/l) and glycerol (3 g/l). at 65 • c, no cod reduction or biomass increment was found to occur. at the other investigated temperatures, the reduction in cod measured after suspended solids (ss) separation varied from 77.6% (55 • c) to 89.1% (35 • c). without ss separation, cod reduction ranged between 55.6% (20 • c) and 75.1% (35 • c). this indicates that, in terms of the extent of cod reduction, the optimal process temperature was 35 • c and that there was a local optimum at about 63 • c. according to the temperature applied, the content of reducing substances decreased by 84.3-96%, that the organic acids by 91.7-99.6%, and that of glycerol by 91.5-96%. the experiments also produced the following two findings: (1) the rise in temperature brought about a decrease of biomass concentration in the str (measured as ss and bacterial number), and (2) temperature was a factor affecting the demand for ammonia nitrogen (n-nh 4 ), which was the highest at 20 and 60 • c. the high n-nh 4 demand observed both over the higher and lover ranges of the investigated temperature should be attributed to the release of n-nh 4 and to the large amounts of the biomass produced, respectively. the results obtained imply that the extent of potato stillage biodegradation with a mixed bacterial culture was high over a wide range of the investigated temperature. polychlorinated compounds such as tetrachloroethylene (pce) have become serious environmental pollutants. considerable attention has been paid to these organochlorine compounds. this paper describes the molecular analysis of dechlorinating gene in halorespirating bacterium and efficient bioremediation process. an anaerobic bacterium, that dechlorinates pce to tce, was isolated and identified as a species of the genus desulfitobacterium. a novel pce reductive dehalogenase (prda) gene from the desulfitobacterium sp. strain kbc-1 was identified. these prd genes, including membrane anchor protein, were classified as a novel type of pce reductive dehalogenase (approximately 40% homology with the general pce dehalogenase). according to the substrate utility of this strain kbc-1 and phylogenetic analysis of prda, the type of this microorganism may be expected to play the role of a primary degrader of pce in the environment. high efficient bioremediation process so called the restricted aeration system which means microaerobic/aerobic reciprocal bioremediation process was developed. strong modifications take place, as ammonia production with a subsequent rise of the ph value and a rapid heat evolution leading to temperatures of up to 70 • c. little is known about the microbial community in the toscano cigar fermentation and its development as fermentation proceeds. the aim of this study is to investigate the microbial community composition, its dynamic and its influence on the toscano cigar production process. our results show that the fermentation could be divided into three different phases: initially yeasts are the predominant microorganisms while bacterial growth is partially inhibited; the middle phase is characterized by exponential growth of bacteria while yeasts disappear. in the final phase the microorganism population is mostly represented by sporigen microbial species. the occurrence of yeasts in the first phase could be attributed to their ability to grow at low temperature and low ph levels. the bacterial population flourishes after the yeast cells have reached a stationary phase and probably grows on residual nutrients and autolysing yeast cells. yeasts and bacteria involved in the fermentation process were isolated and characterized. the microbial community was investigated by a combination of phenotypic and molecular approaches. the phenotypic characterization was based on both colony and cell morphology. the isolates were then identified by rrna genes sequence analysis. finally, in order to clarify the role of the identified microorganisms in the production process, a preliminary biochemical characterization was carried out. biosensors have undergone rapid development over the last few years; in particular, in environmental field many biosensors using microorganisms and purified enzymes as biological component, were recently studied. benzene is present everywhere with high levels in the cities and sometimes, in petroleum processing plants. it is classified as carcinogenic compound of first class able to cause leukaemia. because the evaluation of benzene requires complex instruments and quite long analysis times, it is required to study alternative systems for benzene detection simple, fast and highly sensitive, such as biosensors. from pseudomonas putida mst, strain previously isolated in our laboratory and able to degrade benzene, we isolated genes encoding for benzene 1,2-dioxygenase and cis-1,2-dihydrodiolbenzene dehydrogenase to use in the development of two different hydrocarbon biosensors based on microorganisms and on purified enzymes. the genes isolated were cloned in pvlt33 and we developed three microbial systems carrying: (1) benzene dioxygenase, (2) dihydrodiol dehydrogenase and (3) benzene dioxygenase-dehydrogenase modified by pcr to obtain enzymes with histidine tag. the cloning was planned to construct recombinant strains able to overproduce the enzymes; the enzymatic activities will be evaluated both using whole cells and purified enzymes. study of operation condition of biofilter using fibril-form matrix for odor gas removal don-hee park, chonnam national university, this research was performed for developing of biological treatment process of odor gas such as mek, h 2 s, and toluene, which is generated from the food waste recycling process. to establish the operational conditions of odor gas removal by small-scale biofiltration equipment, it was continuously operated by using toluene as a treating odor object. when the odor treating microorganisms were adhered to fibril form biofilter, high removal efficiency over 93% was obtained by biofilm formation. at 400 ppm of inlet odor gas concentration and 10 s of retention time, the removal efficiency was 76% and 93% in first stage reactor and second stage reactor, respectively. however, the removal efficiency remained over 97% at the operational conditions above 15 s of retention time. ozonated water is produced using an ozone generator in a container filled with cold water. it is useful for sanitizing the surfaces of various products for which heat or chemical treatment is inappropriate, such as fresh food products. in this study, we investigated the antimicrobial effects of ozonated water and electrolyzed ozonated water against escherichia coli, s. aureus, bacillus subtilis and yeast, saccharomyces cerevisiae for practical use in sanitizing various products. the results demonstrated that the electrolyzed ozone water was effective for the reduction of microbial population at relatively low concentration of ozone. also, the electrolyzed and the ozonated water showed synergistic antimicrobial effects. many xenobiotics can react spontaneously with thiol moieties of glutathione (gsh), forming gsh-conjugates, or via glutathione s-transferases (gst). these enzymes participate in detoxification of potentially harmful compounds from endo or xenobiotic origin. using saccharomyces cerevisiae as experimental model, we observed that cells mutated in the gtt1 or gtt2 genes showed twice as much cadmium absorption than the control strain. we proposed that the formation of the cadmium-glutathione complex is dependent on those transferases, since it was previously demonstrated that the cytoplasmic levels of this complex affect cadmium uptake. the addition of glutathione monoethyl ester (gme), a drug that mimics glutathione (gsh), to gtt1∆ cells restored the levels of metal absorption to those of the control strain. however, with respect to gtt2∆ cells, addition of gme did not alter the capacity of removing cadmium from the medium. taken together, these results suggest that gtt1p and gtt2p play different roles in the mechanism of cadmium detoxification. by analyzing the toxic effects of this metal, we verified that gtt2∆ and gsh1∆ cells showed, respectively higher and lower tolerance to cadmium stress than control cells, suggesting that although gsh plays a relevant role in cell protection, formation of the gsh-cd 2+ conjugate is deleterious to the mechanism of defense. furthermore, analyzing the harmful effects of other xenobiotic, menadione (2-methyl-1,4-napthoquinone), we have also observed that gtt1p and gtt2p isoforms play distinct functions in the process of cell protection as well as in drug remove, since both strains showed lethal phenotypes after direct exposure to 20 mm menadione. however, after adaptive treatments (mild-heat or exposure to a lower menadione concentration), cells acquired tolerance to menadione stress, although the gtt2∆ mutant had still shown a higher sensitivity against drug toxicity. by analyzing the malondialdehyde (mda) produced in response to menadione, we observed that gtt2∆ cells exhibited increased levels of lipid peroxidation, indicating that, during menadione exposure, gsh-conjugates are formed by the same transferase isoform, gtt2p, involved in cadmium stress. financial support: stint (sweden), cnpq and faperj (brazil). polycyclic aromatic hydrocarbons (pahs) are ubiquitous and persistent throughout the environment. they are generally distributed from both natural and industrial sources. many pahs can have a detrimental effect on the flora and fauna of affected habitats through uptake and accumulation in food chains, and in some instances, they induce serious health problems and/or genetic defects in humans. many research efforts have been expended to find a suitable method for remediation of soil and water environments contaminated with pahs. amongst them, the use of ligninolytic fungi is particularly suitable for the development of such processes, since they produce extracellular lignin-degrading enzymes (mnp, lip, laccase, . . .) which degrade a wide range of organic pollutants. coriolopsis rigida has been reported to produce extracellular laccase as the sole ligninolytic enzyme. this makes this fungus particularly suitable for the study of xenobiotics degradation by laccase. the purpose of this research was to obtain high laccase activities by c. rigida in solid state cultures and to determine their ability to degrade anthracene (typical pah). both in vivo and in vitro assays were performed. the former led to 60-80% degradation in 3 days depending on the culture conditions, whereas the latter showed a degradation percentage above 90% in 2 days when low mediator concentration (hbt) was added to the reaction mixture. focus will be given on pressure-driven membrane bioreactors, gastransfer membrane bioreactors and the novel ion exchange membrane bioreactor (iemb). the latter concept has been developed and currently studied by our group. this process, based on integration of donnan dialysis with bioconversion of one or more target pollutants to harmless products, has been modeled and experimentally verified for the removal of various charged inorganic pollutants such as nitrate, perchlorate and bromate by mixed microbial cultures under anoxic conditions. tests of up to 3 months showed a very good operational stability. the essential role of the microbial membraneattached biofilm, which develops naturally in this type of systems, will be also demonstrated and discussed. poly(lactic acid) (pla), which is one of biodegradable plastics, is depolymerized by hydrolysis and releases soluble monomer or oligomer of lactic acid. many bacteria can use the monomer and oligomer as an energy source or a carbon source. in this study, we applied pla to an electron donor for denitrification process of the previously developed bioreactor, which could remove ammonia from wastewater by simultaneously carrying out two biological processes, aerobic nitrification and anaerobic denitrification. a bench-scale bioreactor was constructed with a gel-plate containing pure-cultured cells of nitrosomonas europaea and paracoccus denitrificans and a pla-plate. the pla-plate was prepared by mixing three kinds of plas with different molecular weight and tricalcium phosphate to keep the constant release of the electron donor for a long term. batch treatment experiment with the bioreactor was repeated with an artificial wastewater containing ammonia for 100 days. the bioreactor could remove nitrogen from the artificial wastewater at nitrogenremoval rate of approximately 4 g n/day per square meter of gel-plate surface during the experiment period without an additional electron donor. the performance was equivalent to that obtained with our bioreactor using ethanol as electron donor for denitrification. the bioreactor using pla dose not need an additional pump for serving an electron donor (e.g., ethanol) and a hollow space for serving. therefore, the concept using solid electron donors like a pla would be effective our bioreactor to compact and simplify, and would be possible to develop a portable or disposable bioreactor. leucosporidium antarcticum as a source of enzymes for biotechnology arkadiusz wojtasik 1,2 , marianna turkiewicz 2 , jaroslaw dziadek 1 , pawel parniewski 1 : 1 centre for medical biology pas, 106 lodowa street, 93-232 lodz, poland; 2 faculty of biotechnology and food sciences technical university of lodz, stefanowskiego 4/10 street, 90-924 lodz, poland. e-mail: awojtasik@cbm.pan.pl (a. wojtasik) leucosporidium antarcticum is a psychrophilic yeast able to growth at low temperature. these microorganisms live in antarctic marine waters and are endemic to that cold environment. furthermore, l. antarcticum is also isolated from the digestive tract of antarctic krill euphausia superba. enzymes isolated from coldadapted microorganisms such as l. antarcticum having a specific activity at low temperatures ranging from 0 to 30 • c are considered for utilization at biotechnological applications such as bioremediation, production of polyunsaturated fatty acids of dietary significance and might be a source of industrially useful enzymatic proteins. the main goal of this study was to construct a cdna library of l. antarcticum. the partial cdna library was obtained and some of the clones were analysed. the sequencing analyses allowed us to find an approximately 450 base pair nucleotide sequence which displayed a very high homology to disulfide bond chaperone belonging to the hsp33 family from psychrobacter sp. high similarity of that heat shock protein was found on an amino acid sequence level and was reaching nearly 85%. the main object of our further research is to clone hsp33 family protein gene and to obtain its expression in a mezophilic host strain. also, further clones will be analysed to find other interesting genes encoding the psychrophilic proteins. this work was partially funded by the kbn grant i29/205/05. out of 9000 plant species found in the flora of turkey, about 3000 are endemic. beautiful flowering (geophytes) bulbous plants form an important part of this rich biodiversity. besides use as ornamental plants, these have great potential in perfume and pharmaceutical industry. genera of fritillaria, ornithogalum, muscari, bellevalia, tulipa, galanthus, sternbergia, crocus, arum and biarum have important and critically endangered species with high export potential that enters into this group. most of these are endangered and their collection from wild and export has been banned to conserve them. large scale production and conservation of these species could also be achieved by in vitro techniques. therefore bulb scale and immature embryo explants of sternbergia candida, s. fischeriana, muscari muscarimi, fritillaria imperialis and f. persica were cultured on different nutrient media supplemented with various concentrations of plant growth regulators using different culture applications. large numbers of bulblets were produced (over 100 bulblets/explants) from single immature embryos on nutrient media in most species tested after 12 months of culture initiation. regenerated bulblets were kept at 5 • c for 5 weeks and then transplanted to soil successfully. to our knowledge the present study is the first report for in vitro bulblet production from immature embryos of geophytes. the procedure described here provides a prolific bulblet production system that may form the basis of bioreactor culture and conservation of endemic and endangered geophytes. the commercial use of organofluorine compounds in industrial, pharmaceutical and pest-control applications has dramatically increased over the past few years, resulting in the introduction of numerous new organic compounds into the environment. organofluorine compounds are chemically very stable and are assumed to be resistant to biological degradation. given the chemical inertness of fluorinated organics, their bioactivity, and their potential for accumulation in the environment, it is important to understand their environmental fate and the mechanisms by which they might be degraded. examples of the biodegradation of fluorinated compounds in literature are scarce, being fluorobenzoic acids the most commonly reported. information on the cleavage of carbon-fluoride bonds in synthetic compounds is limited to fluoroacetate dehydrogenase. in this project we try to obtain more insight in the defluorination mechanisms by investigating the diversity of degradation routes for these compounds in several soil bacteria by making use of modern genetic tools. a gram-positive strain capable of aerobic biodegradation of 4-fluorophenol (4-fp) as the sole source of carbon and energy was isolated by selective enrichment from soil samples collected near an industrial site. batch cultures were set up and substrate consumption, accumulation of intermediates and product formation were monitored. the consortium was able to use 4-fp up to concentrations of 448.4 mg l −1 and was able to utilize a range of other organic compounds. stoichiometric release of fluoride ions was measured in batch cultures suggesting that there is no formation of dead-end products during 4-fp metabolism. biobleaching of kraft cellulose pulp by poliporus versicolor aysun ergene 1 , nazif kolankaya 2 : 1 kırıkkale university, faculty of science and literature, department of biology, yahsihan, kırıkkale, turkey; 2 hacettepe university, faculty of science, department of biology, beytepe, ankara, turkey the suitability of culture supernatant from poliporus versicolor for use in the biobleaching of kraft cellulose pulp was investigated. p. versicolor was found to grow on mycological broth (1% soytone, 4% d-glucose and 0.5% cellulose pulp). maximal extracellular ligninase production was detected after 7 days (7 nkat). the optimum biobleaching conditions are 30 • c and ph 4.8, with 10 days. in this condition p. versicolor decreased the kapa number from 38.55 to 19.42 and increased brightness from 28 to 32.7 in 10-day treatment. boron and iron are among the microelements required for the proper development of the vegetative and generative tissues of plants. though iron is present in high amounts in almost all soil types, its bioavailability to crops is extremely reduced, hence most of the plants face an iron defficiency problem and while on one side crop productions effected, on the other hand the nutrition problems come up to human through contagious nutritional chains. boron is also among the most problematic micronutrients of the major crop plan-tation areas of turkey. both defficiency and toxicity problems exist in a total of about 50% of the central anatolian soil where pea is among the legumes cultivated. application of varying levels of boron and iron combinations in greenhouse and the analysis of plant acquisition via icp-aes as well as determination of the effects of the element combinations both in morphological and molecular levels are the aim of our studies. the genetic bases of the response differences of plant genotypes to b and/or fe, were investigated through the applications of molecular marker techniques. considerable growth rate and stem size differences were detected within the parents (wild-type versus cultivar) and the f9 plants. the presence of efficient genotypes to high micronutrient levels are expected to help us increase the cultivation of the crop in problematic areas as well as in exploring the molecular bases of the microelement uptake mechanisms. butachlor is one of the selective systemic herbicide toxins that are act by inhibition of protein synthesis. this toxin is used exclusively in the rice, barely, cotton and wheat farmlands. butachlor is belonging to chloroacetanilide herbicide group, which are consisting of butachlor, alachlor, acetochlor, metolachlor and poropachlor. in the view of bioenvironmental, butachlor is degraded in the soil by microbial activity. its stability is about 6-10 weeks. it is converted to the water-soluble derivatives in soil or water, with a slow evolution of carbon dioxide. because of butachlor is one of the herbicide toxin, it is inhibitor factor against growth of bacteria and microorganisms. microorganisms can be continuing their activities in the limited concentration of butachlor. therefore treatment of industrial wastewater consist of concentrated butachlor by the biological treatment is impossible and it is necessary chemical or physico-chemical treatment are used. in this research, biological treatment methods are used. in the biological treatment, an activated sludge system with volume of 6.5 l is used. in this method, butachlor with concentration of 5 mg/l are treated. removal percent of butachlor for concentration of 2.5 and 3 mg/l are calculated to 41.20% and 41.67%, respectively. removal percent of cod is also calculated to 86%. olive oil mill wastewater (omw) as the effluent of the concern of olive industry has high organic load. the conventional biological treatments despite of their simplicity and rather suitable performance are ineffective for the omw treatment since phenolics possess antimicrobial activity. in order to carry out a proper treatment on omw, use of microorganism able to degrade the phenolics thus, is necessary. the ability of phanerochaete chrysosporium immobilized purification and downstream process of xylitol obtained biotechnologically from hemicellulosic hydrolyzate of corncobs b. rivas 1 , p. torre 2 , j.m. domínguez 1 , j.c. parajó 1 , a. converti 2 : 1 department of chemical engineering, vigo university (campus of ourense), polytechnic building, as lagoas, 32004 ourense, spain; 2 department of chemical and process engineering, genoa university, via opera pia 15, 16145 genoa, italy. e-mail: brivas@uvigo.es (b. rivas) biotechnological production of xylitol from lignocellulosic materials has been widely studied in the recent years with promising results that confirming the possible industrial application of this technology. xylitol purification from fermented broth is the limiting stage of this process. previous works suggest crystallization procedures in order to recovery xylitol from fermented synthetic solutions. the complexity of fermented hydrolyzate not allows direct crystallization. in this work, corncobs hydrolyzate obtained with autohydrolysis-posthydrolysis techniques, detoxificated with activated charcoal and concentrated was fermented to xylitol by d. hansenii. the fermented broth composition was 64.9% of xylitol (68 g/l), 13.3% of other sugars and 21.8% of other compounds that interferes in the crystallization process (as dry matter of the liquor). the fermented media was submitted to an absorption process with activated charcoal and concentrated until a xylitol concentration of 340 g/l. the liquor was then submitted to a second step of precipitation with ethanol, the best results achieved in this study were obtained with an ethanol/liquor ratio of 4. in these conditions this treatment allows to remove a 56.9% of the impurities. the resulting solution was evaporated and crystallized containing 60% of ethanol and a xylitol concentration of 443 g/l. crystallization was performed at t = 5 • c with slightly agitation. after 36 h were separated xylitol crystals with a recovery yield of 13% and a purity degree of 90%. numerous publications have documented that only a minor number of the indigenous prokaryotic organisms found in complex environments such as the human intestine, biogas reactors, and soil are known, and probably only a fraction of this diversity can be accessed using traditional culturing techniques. some of the reasons for this are the lack of knowledge of specific growth conditions, specific nutrients, and obligate coculture requirements. also growth on a solid surface directly exposed to the atmosphere puts a very strong selective pressure on single cells supposed to develop into visible colonies. therefore, the knowledge of these microorganisms is scarce and generally limited to the 16s rrna genes that have been extracted from different environments and cloned for phylogenetic analyses. an obvious approach to circumvent these problems was the development of techniques based upon micromanipulation for isolation of single cells from complex mixtures. continuous development of modern microscopes in combination with the precision of a servo-powered micromanipulator and the development of the modern microscopic micro injectors used in ivf techniques has further aided the manipulation of single cells. this technique, however, does not solve the problems of the non-culturable cells, and other approaches are needed to gain more information about these organisms. an approach to the non-culturable cells could be genomic analysis of isolated single cells without preceding cultivation. this pcr-based technique is widely used for genetic analysis of human cells, but due to the small amounts of dna present in prokaryotic cells it has so far not been possible to produce identifiable amounts of dna from single cell amplification using conventional polymerases. a promising alternative used for amplification of small amounts of dna is the f29 dna polymerase operating under isothermic conditions. applying random hexamer primers, this polymerase carries out a multiple displacement amplification (mda) of high molecular weight dna template. in this study we demonstrate the successful application of mda for selective amplification of genomic dna from a single prokaryotic cell. the yield was >20 mg of amplified genomic dna corresponding to about a 5 billion-fold amplification from a single cell. the technique was used to approach a large group of non-thermophilic archaea found in agricultural soil. our results show that combining mda with fluorescent in situ hybridization and cell isolation by capillary micromanipulation enables an unprecedented ability to investigate new species without cultivation. also this combination of techniques opens for studies of genetic heterogeneity within populations and processes such as horizontal gene transfer. precipitation of zn 2+ , cu 2+ and pb 2+ at bench scale using biogenic hydrogen sulphide produced from the utilization of volatile fatty acids by sulphate reducing bacteria maria teresa alvarez 1,2 , carla crespo 2 , bo mattiasson 1 : 1 department of biotechnology, center for chemistry and chemical engineering, lund university, p.o. box 124, s-22100 lund, sweden; 2 instituto de investigaciones fármaco bioquímicas, universidad mayor de san andrés, la paz, bolivia biological production of hydrogen sulphide (h 2 s) from sulphate using sulphate reducing bacteria (srb) is popular within environmental biotechnology. srb require absence of oxygen, presence of nutrients required for growth and oxidizable organic substrates (to supply hydrogen atoms for reduction of sulphate). many organic wastes have been used as electron donors for the sulphate-reducers in the treatment of acid mine drainage (amd) including straw, hay, sawdust, peat, spent mushroom compost and whey, however, other wastes such as municipal organic waste can be used. the aim of this work was to study the possibility of using srb for the treatment of amd at bench-scale. this process involved three stages: the volatile fatty acid (vfa) production by hydrolytic bacteria from the degradation of vegetables and fruits, the production of h 2 s through the utilization of the produced vfas by sulphate reducing bacteria and the precipitation of metals by using the biologically produced h 2 s. the substrates used for vfa production consisted of tomato, papaya, apple and banana. the h 2 s produced from the degradation of vfas was utilised for the precipitation of an artificial effluent simulating the heavy metal concentrations of a mine located at bolivian andean region, containing approximately 9 mg/l of zn +2 , 8 mg/l of cu +2 and 4 mg/l of pb +2 . the maximum concentration of hydrogen sulphide obtained was approximately 17 mm. removal efficiencies of 97%, 98% and 100% for zinc, cooper, and lead, respectively, were achieved in the present work. at 30 • c during 14 days. bacterial population was determined by counting in a neubauer chamber with optical microscope. sulfate concentration was measured by turbidity method and metal concentrations in the filtered supernatant were measured by icp-aes. the first part of study consists of determine the maximum concentration of each metal at which d. vulgaris and desulfovibrio sp. grow in similar way than control culture (without metal). both cultures tolerate: cr(iii) 15 ppm, ni(ii) 8.5 ppm, zn(ii) 20 ppm. the maximum precipitation percentages were approximately: 25% (15 ppm cr(iii)), 96% (8.5 ppm ni(ii)) and 99% (for d. vulgaris-10 ppm zn(ii) and desulfovibrio sp.-15 ppm de zn(ii)). time to reach the highest precipitation was minor for mixed culture (desulfovibrio sp.) y all the cases. the next part was focused to study the precipitation percentage when metals are present in combination in the same metal levels (cr(iii)-ni(ii), cr(iii)-zn(ii), ni(ii)-zn(ii) and cr(iii)-ni(ii)-zn(ii)). the combination of metals does not affect significantly the bacterial growth and precipitation percentage of metals. this fact supposes an importance advantage so metals are commonly found together in the environment. future experiments are focused in development of this process in continuous operation mode. biosolubilisation and depolymerisation of coal has potential to produce a clean energy source or high value organic products from low rank coals such as lignite or sub-bituminous coal. these complex soluble phenolic compounds are of value as starting materials for biotransformation to value-added compounds such as antioxidants and flavourants. the bioprocess is carried out at ambient temperature and pressure and is perceived to be environmental benign. in the evaluation of coal solubilisation an important quantity for the assessment of process feasibility is the yield, i.e. the determination of the mass of product obtained per unit mass of coal solubilised. to date, results for coal biosolubilisation reported in the literature are qualitative or at best semi-quantitative, indicating trends with operating variables. the process kinetics has not been determined rigorously because measurement of fungal growth during coal solubilisation is hindered by the presence of the solid coal substrate. knowledge of the profile of biomass growth is required for the rigorous determination of the kinetic parameters necessary for process design and optimisation. in this paper, the use of an indirect method for the estimation of the growth and metabolism of fungal biomass by measuring co 2 evolution and o 2 consumption using an off-gas analyser is reported in the study of fungal coal solubilisation. coal determined rigorously because measurement of fungal growth during coal solubilisation is hindered by the presence of the solid coal substrate. knowledge of the profile of biomass growth is required for the rigorous determination of the kinetic parameters necessary for process design and optimisation. biosolubilisation was carried out in a stirred tank slurry bioreactor with working volume of 1.0 l. complete suspension of the coal particles of 650-800 m mean diameter was achieved at an agitation rate of 560 rpm. growth yield coefficients based on coal and oxygen as well as maintenance coefficients were calculated from growth of the fungus under the same conditions using a non-coal carbon source such as glucose. these data were used to determine the stoichiometric coefficients for biomass growth, enabling the biomass production rate to be quantified in terms of co 2 production rate and o 2 consumption rate. a dna-chip platform for parallel detection of microorganisms related to biofilm in industrial systems and drinking water systems pernille skouboe, dorte lauritsen, kim holmstrøm bioneer a/s, kogle allé 2, dk-2970 hørsholm, denmark. e-mail: psk@bioneer.dk (p. skouboe) an oligonucleotide microarray for simultaneous detection and identification of pathogenic bacteria related to technical water systems as well as drinking water has been developed. the approach is based on the use of a tandem hybridization technique with two ribosomal 16s rdna-pcr products, 1000 bp and 500 bp long, generated from two consensus pcr reactions using conserved ribosomal primers end-labeled with cy3 and cy5, respectively. the tandem hybridization technique implies an internally quality control for discrimination between target and non-target signals. the current prototype of the dna-chip platform includes 20 oligonucleotide probes representing 11 different genera (and subgroups of species), e.g. legionella, mycobacterium, aeromonas, campylobacter, vibrio and enterococcus. the platform has been used for detection and identification of species from pure cultures, and initial experiments with water samples from industrial systems have been performed. the potential as well as the limitations of using a dna-chip based detection format in its present form will be documented. particularly, its potential application as a rapid method for initial screening of environmental or food samples will be addresses. the aim is to reduce and optimize the number of samples required for traditional microbiological identification tests. in the course of a project for the development of a novel kind of a mycotoxin inactivating feed additive, the aim of this study was to isolate and characterize microorganisms with the specific ability to enzymatically break down and detoxify fumonisins, a group of structurally related fungal toxins, with fumonisin b 1 (fb 1 ) being the most abundant and -with respect to toxicology -also the most important representative of this group. these toxins are produced as secondary metabolites by some fusarium species such as fusarium verticillioides and f. proliferatum and are naturally occurring contaminants of cereal grains worldwide. they are found especially in maize and maize based products, and are known to be hazardous to human as well as to animal health. a natural feed additive, based on microorganisms and/or enzymes, should ensure the detoxification of fumonisins during feed uptake and digestion via microbial or enzymatic break down of these compounds, by that protecting the animal from the harmful effects of these mycotoxins. besides an extensive screening of microbial strains derived from strain collections, various different natural habitats were investigated for the presence of fb 1 degrading microbial activity, such as intestinal contents of pigs, soil samples, and naturally fumonisin contaminated maize. while testing of nearly 150 organisms from strain collections did not show positive results, fumonisin transforming activity could be detected in one soil sample and a number of maize samples. trials in order to isolate the respective fumonisin degrading microorganisms resulted in a number of strains, whose fb 1 degrading activity could be proven. the most promising bacterial and yeast strains were further characterized with regard to a general taxonomic description, and to different aspects of their toxin degradation behaviour. approaching a more relevant in vivo situation, fb 1 degradation trials in food-and feed-stuffs were conducted. further on, the applicability of the respective organisms as stabilized lyophilisates was investigated. arsenic is one of the most important global environmental pollutants and the toxicological effects are related to its chemical form and oxidation state. arsenite [as(iii)] is reported to be on average 100 times more toxic than arsenate[as(iv)]. this work shows the ability of one strain of the species ochrobactrum tritici to grow in presence of several metals including arsenite, arsenate, selenite, selenate, tellurite and antimonite. its arsenite mic was determined as 50 mm, whereas for arsenate, this bacterium could resist to concentrations upper than 200 mm. we report the identification of two loci involved in high-level arsenic resistance. sequencing of the first locus identified four complete genes in the following order: arsr, arsd, arsa, arsb. the second locus containing genes for arsenic resistance was also characterized. each sequence has been compared with nucleotide and protein databank (blast programs) and significant homology with known orfs coding for arsenic resistance has been found. it is also possible that the phenomenon of high-level arsenic resistance in o. tritici could evolve other genes or loci. the ability of the ␣-proteobacterium o. tritici to tolerate high levels of arsenic in addition to other oxyanions has considerable potential for detoxification and bioremediation of contaminated environments. this work is based on the mathematical modeling of kinetics of a thermophilic bacteria cultivation system. the cultivation proceeded by way of batch and continuous on the synthetic medium with the main carbon source-lactose. this medium simulated an industrial waste approximately. mixed thermophilic aerobic bacteria popula-tion, applied to the wastewater treatment (sludge v&k bystrice pod hostynem), was used to the inoculation. the cultivation system consisted of the laboratory fermentor biostat b (b. braun biotech) with working volume 2 l and the control unit connected with a computer. it is possible the temperature, ph, aeration, stirring and foaming regulation. physical and chemical cultivation conditions were optimized. the chemical oxygen demand (cod), generally expressive the impurity level, was selected as the main parameter for the cultivations run classification. but into the mathematical model also the kinetics of biomass growth, lactose consumption, production of choice metabolites (acetate, lactate, succinate) and dissolved oxygen concentration was included. the modeling was located to two head distinguishable growth phases of the microorganisms. an optimization and identification of mathematical model parameters was practised in the software language psi/c. the difference between simulated curves and experimental data is not statistically significant on the relevancy level 0.05 (f-test). it took place the cod degradation at 74.0% with the average yield coefficient y chsk/x 3.8 g cod/g biomass in the batch process with the air aeration only. there is a better way to the cod elimination (>90.0%)-the aeration of air enriched by the pure oxygen. in experiments with the continuous system was obtained the 69.0% cod decrease after the steady state stabilization. this work was supported by project msm 0021630501. fluorescence in situ hybridization (fish) of whole cells using oligonucleotide probes was applied to study the influence of low temperature and temperature reduction on the bacterial community of biofilm reactors for the removal of chlorophenols (cps). two packed bed reactors were set up for degradation of a mixture of 2-cp, 4-cp, 2,4-dicp, and 2,4,6-tricp as sole source of carbon and energy at 14 • c (ra) and 24 • c (rb) and were inoculated with bacterial consortia adapted to these respective initial temperatures. the performance of the reactors was studied under different conditions of pollutant loading, aeration rate, and hydraulic retention times over 7 months. total chlorophenol removal capacities of 1240 and 1420 mg l −1 day −1 were achieved in the bioreactors ra and rb, respectively, under a total pollutant load of 1440 mg l −1 day −1 . the population of ␤-proteobacteria was the major bacterial community of the biofilm (35-47%) followed by the ␥-proteobacteria (12-6.5%). two bacteria with the ability to mineralize 50 mg chlorophenols l −1 were isolated from the bioreactors and characterized as ralstonia basilensis and alcaligenes sp., both belonging to ␤-proteobacteria. decreasing the temperature by 10 • c (in two steps of 5 • c each) resulted in an increase in the population of ␥-proteobacteria and a decrease in the population of ␤-proteobacteria in both reactors. application of genus specific probes showed an increase in the pseudomonas population from 25% of the ␥-proteobacteria at 14 • c to 59% at 4 • c. the pollutant removal capacity decreased to 548 and 833 mg l −1 day −1 in ra (4 • c) and rb (14 • c), respectively. the ␣and ␦-proteobacteria, cytophaga-flavobacteria and actinobacteria the survey was carried out in urban and rural areas of two cities (ankara and isparta). this paper is only analysing urban people by excluding villagers. urban sample is consisted of 400 urban consumers and 200 professionals. professionals were selected amongst pharmacists, doctors, agricultural engineering's and industrialists that is thought are affective in the process of developing new technologies and also the development of biotechnology in the society. basic data was gathered by a questionnaire including both structured and open-ended questions besides deep interviewed. workforce development for life sciences-the scottish experience carol booth scottish entreprise, uk the presentation will look at, the background and definition of workforce development for scottish enterprise, examine information available to support scottish enterprises economic intervention and where and when to intervene. conclusions emerging from the evidence base will be used to outline scottish enterprises approach to workforce development and look at which actions might be required to address identified issues. moving on to reasons for integrating workforce development into business development and how the life sciences cluster team at scottish enterprise, stakeholders and partners in scotland have approached their current and future contributions to workforce development for life sciences using a variety of projects. the national institute for bioprocessing research and training (nibrt) in ireland is a proposal that will be a state-of-the-art training, research and pilot plant service facility that brings together institutions with complementary expertise and state-of-the-art research technology, and industry partners. these include university college dublin, trinity college dublin, institute of technology, sligo and dublin city university. nibrt is an innovative collaboration between academic institutions at the forefront of biotechnology, cell biology, engineering and pharmacy and industry. for training, two separate training labs, for upstream and downstream training, in addition to 5 research labs are planted. it will also include a state-of-the-art pilot plant fermentation facility for fermentation optimisation, fermentation scale-up, product separation and purification, regulatory aspects and automation. by aligning with industrial demands, the new institute will tailor its training programmes while remaining on the cutting edge of biotechnology research and technologies. the fermentation facility will offer hands-on training workshops and educational modules for outside researchers and companies. these workshops cover the fundamentals of small-scale fermenta-tion, scale-up considerations, and fermentor design and set-up. the training and educational philosophy underpinning the nibrt will focus on the needs of industry with an emphasis on providing training for accreditation of existing industry staff and prepare technicians and graduates for the technical, business, regulatory and professional aspects of the industry. the strategy is to provide specialised modules in nibrt in support of courses established in the higher education institutions, which will provide the certificates, diplomas and degrees. modules will be offered for all categories of students and will be given credits respected by other third level institutions in ireland. the role of professional graduate degrees in meeting current and future biotechnology industry workforce needs a. stephen dahms san diego state university, usa the presentation will review the status of new graduate training models designed to meet the unique needs of the biotechnology industry as it transitions to commercialization. emphasis will be on professional master's degree programs in biotechnology and their various versions, with a focus on operational and funding strategies and industry acceptance. discussion will also centre upon the creation and operation of industry-validated, specialized and highly targeted professional masters degrees in various refined aspects of the drug development process, including regulatory affairs, biomedical quality systems, clinical affairs, management of drug development, management of reimbursement affairs, bioinformatics, etc. the eurodoctorate in biotechnology, new combined mba/phd combined degrees in the molecular life sciences, the u.s. professional doctorate in chemistry and the proposed u.s. professional doctorate in biotechnology will be also discussed. data will also be presented on the current workforce and the industry's projected needs. genetic studies show that mankind is a rapidly expanded population of closely related individuals with very similar disease sensitivity. bad nutrition and infections dominate among the main health problems in the world. apart from malnutrition, the overeating habits of the developed world are now creating problems in the developing world as well. infectious diseases are also a global problem since new contagious agents like hiv, sars and avian flew do not recognize borders. thus, the global responsibilities of modern health care are obvious. many research scientists from and in developing countries find it nearly impossible to use their talents for the benefit of their own countries. some struggle to develop research and education programmes with poor facilities, some leave science completely, and others migrate to more developed countries. the talents of such people are either being wasted or lost completely to their home countries just at the time they are most needed to combat the great humanitarian challenges of hunger, illness and lack of knowledge. europe must strengthen programmes which allow third world scientists to work to their full potential in their home countries or regions. yang beijing genomics institute, chine academy of sciences, beijing, china europe has all the reasons to be proud of being the cradle of modern science and of its achievements and resources in life sciences. as a model of having solved many of the problems that many other counties are now facing, europe is expected by the whole world to make its further contribution to a better future of mankind and to play a more important role in the international community of life sciences. tropical diseases and public health basilio valladares director of the university institute of tropical diseases and, public health, university of la laguna, 38200 la laguna, tenerife, spain. e-mail: bvallada@ull.es the presentation will look at the main research interests of the university institute of tropical diseases. these are the following: 1. immunology and molecular biology of parasites. we express and purify recombinant proteins from leishmania sp. which have been shown to act as immunomodulators and protect against disease such as l25, hsp70, hsp83. the study of acanthamoeba pathogenic factors has also resulted in the isolation and silencing of extracellular proteins related to their pathogenecity, which has a great potential in the development of novel chemotherapeutics. 2. diagnosis of parasites. the immunological diagnosis of leishmaniasis has been one of the main research interests in our laboratory for several years. as a result, we have identified peptides which could be used to develop kits for the immunological diagnosis of leishmaniasis such as hsp70 c-end, l25 n-end, etc. we have also developed some dna based methods for the identification of acanthamoeba species from biological and environmental sources. 3. water quality. biological parameters. our water research group has the expertise to identify and characterise bacterial, viral and parasitic indicators of faecal contamination in diverse water sources including tap water, rivers, reservoirs, sea, etc. this research area has been developed in collaboration with the local sewage treatment plant and reservoir managing authorities. currently, we are establishing a conjoined project with the center for disease control and prevention (cdc) in atlanta, usa for the identification of water-borne emerging pathogens. 4. development and formulation of chemotherapeutic antiparasitic agents. in this field, we evaluate the leishmanicidal activity both in vivo and in vitro of natural and synthetic drugs and synthetic peptides. in a later stage, the drugs which have shown the highest antiparasitic activity have been subjected to cytotoxicity assays and their molecular targets dissected. some of the drugs tested in the last few years have been submitted to patent due to their outstanding activity. finally, in order to allow the commercialization of these drugs, both in vivo and in vitro assays are being carried out to predict their chemical stability and degradation pathways. this will be followed by the use of liofilization and controlled crystallisation strategies for the development of efficient and safe treatments. 5. human and population genetics. tachykinins and their receptors in different tissues and groups of patients and their association with molecular polymorphisms is another one of our research interests. the knowledge of ligand and receptor sequences and their similarities will allow the rational development of drugs with specific activity against these receptors. furthermore, we are also interested in the interspecific variation along the evolutionary scale of these markers. 6. nitrate assimilation group. research in our group is focused on understanding nitrate assimilation in the yeast hansenula polymorpha. several biotechnological companies use this yeast to produce heterologous proteins (hepatitis b vaccine). genetic manipulation techniques for h. polymorpha are available in our laboratory. head of the department of biotechnology, technological institute of canary islands, pozo izquierdo 35119 santa lucía, las palmas, spain single cell analysis by flow cytometry has proved to be a tool to perform simultaneous and rapid measurements related to cell morphology and physiological state. previous studies showed the possibility of quantifying neutral and polar lipids spectrofluorometrically using a lipid specific fluorescent dye, nile red (nr), however the existence of inter and intraspecific variations in the fluorescent response had not been clearly established. in this work, two strains of marine microalgae: crypthecodinium cohnii and tetraselmis suecica, characterized both by high contents of polyunsaturated long chain fatty acids (dha and epa, respectively) and an hypersaline microalgae: dunaliella salina, characterized by a high ␤-carotene production, were grown under different conditions and collected at different growth phases to be used for in vivo lipid quantification with nr by flow cytometry. our results showed a high correlation between the mean fluorescence signal of nr stained cells and the neutral and polar lipid content measured by gravimetry for each strain. in this respect, these data make feasible the development of a rapid method for lipid quantification in monoalgal cultures. however, differences in the dye uptake related to specificity were detected. in this communication we also assess the possibility of use this cytometric technique to select microalgal strains with high lipid and polyunsaturated long chain fatty acids content from mixed samples. performance of such technique would be a good alternative to the time-consuming traditional screening protocols based on gravimetry and gas chromatography and would optimise the search of new commercial strains of microalgae. claverie-martín head of research unit, biomedical research institute, hospital universitario, de n.s. de candelaria, santa cruz de tenerife, spain our group is involved in the cloning and production of proteins of interest to the food and pharmaceutical industry. we have recently expressed in yeast the cdna that encodes the precursor of caprine chymosin. chymosin is the enzyme responsible for the coagulation of milk in the abomasum of unweaned calves. this enzyme is secreted by gastric mucosa cells as an inactive precursor, known as prochymosin. in the acidic conditions of the lumen, prochymosin is converted into the active form by autocatalytic cleavage of the n-terminal prosequence. chymosin is used extensively in cheese production because it cleaves -casein in a specific manner with low proteolytic activity. several biotechnology companies are producing the bovine recombinant enzyme for commercial use in the process of cheese making. we are interested in the caprine chymosin as an alternative because in the canary islands cheese has traditionally been made using goats milk with extract from the abomasum of newborn goats as coagulating factor. it is well known that the activity of these types of extracts varies depending on the age of the animal and the type of food ingested. these difficulties should be overcome using a recombinant caprine chymosin. the caprine mrna used for the synthesis of the cdna was obtained from the abomasum of milkfed kid goats. the cdna fragment encoding the mature portion of caprine prochymosin was fused in frame to a signal sequence in yeast expression vectors. culture supernatants of yeast cells transformed with the recombinant plasmids showed milk-clotting activity after activation at acid ph. proteolytic activity assayed toward casein fractions indicated that the recombinant caprine chymosin specifically hydrolysed -casein (patent 200402025). the recombinant caprine chymosin could be an alternative milk coagulant in cheese making. work is underway to optimise the expression of the new recombinant prochymosin for further purification and characterization. smart molecules for health victor martín head of research, university institute of bio-organics "antonio gonzález", avda. astrofísico francisco sánchez 2, 38206 la laguna, tenerife, spain the instituto universitario de bio-orgánica "antonio gonzález" (iubo) is a multidisciplinary research centre that belongs to the university of la laguna. the iubo is located at the town of la laguna, inscribed on unesco's world heritage list in 1999, and former capital of the canary island of tenerife. the geographical location in addition to its mild climate has made the canary islands to posses a variety of ecosystems with unique plants and animals. the institute was started up in the 1960s with the need to study the natural products and secondary metabolites produced by those marine and terrestrial organisms, thus providing a new source of bioactive products. the main research lines that are being developed at iubo are summarised in the following paragraphs: anticancer agents from natural sources: several natural products and their semisynthetic derivatives are produced at the iubo in diverse joint projects for the development of new antitumour drugs with novel mechanisms of action. as an example we can mention natural products from the mevalonic, shikimic or polyketide pathways. some products have recently shown in vitro reversion of the resistance in multidrug resistant (mdr) tumour cell lines. genetic engineering: in vitro cultures of the plants atropa baetica, maytenus amazonica and m. macrocarpa are developed in order to manipulate their biosynthetic pathways and induce the production in large scale of the secondary metabolites for diverse applications, including arthritis, rheumatism, and back pain. marine organisms and toxins: dinoflagellates are marine organisms responsible for the red tides and food poisoning episodes. among others, okadaic acid and yessotoxin are the most common toxins present in european shellfish. the isolation of these products is best done from the microorganism cultures, since they are present in very low amounts in the natural sources. at the iubo we develop culture systems to provide us with amounts of toxins large enough to perform biological, metabolic and bioactive studies. insecticide and repellents: natural products are being isolated for their use against plagues, specially those affecting agriculture. these projects are run in collaboration with a number of public institutions and agrochemical companies throughout europe and latin america. fine chemicals and pharmachemicals: our institute possesses large expertise in the field of organic synthesis devoted to the synthesis of medicinal substances, with special focus on asymmetric processes. of particular interest is the development of new methodologies for the total synthesis of biologically active substances like polyether toxins, (un)natural amino acids, sphingosine analogs, alkaloids, etc. with an annual source of 100s of new compounds, the fine chemicals and medicinal chemistry branch at iubo have recently started and anticancer screening program in collaboration with the biomedical research unit at the hospital universitario n.s. de la candelaria. the program is committed to the discovery of novel drugs for application in cancer treatment. the outcome of this project in its first year has been outstanding, leading to the finding of several leads that form the basis for current and future projects. institute of canary islands, biotechnological department, playa de pozo izquierdo s/n, 35119 santa lucía, gran canaria, spain the presentation will look at the main research interests of the biotechnological department of the technological institute of canary islands. these are the following: nutrition and feeding in aquaculture: we conduct studies on digestion, absorption, transport, and utilization of the different nutrients applying also different techniques such as histology, enzymology, genetic or immunology among others. aquaculture feeding is the main important cost in fish farms, being higher that the prize of fries, personnel cost or energetic costs. thus, studies conducted on the improvement of diet formulation and the use of different dietary ingredients is one of our main research lines (such as vegetable oils and meals to be used as alternative to fish meal and oil, or carotenoid sources to improve fish colour). not only the use of the different ingredient is studied, but also the effect of these ingredients on fish health, flesh quality, flesh healthy aspects related with human consumption, are being studied. different formulae are being developed and patents of different diets are being obtained. studies on nutritional requirements are also conducted allowing us to patent different formulae in larvae studies, developing microdiets to substitute the high-cost processes associated with live prey in larval nutrition. development of immunostimulants, anti-stress diets, immune techniques to be applied as bio-indicators of fish health and welfare, as well as use of dietary ingredients derived from bio-reactors are other research lines in our group. genetics: genetic techniques applied to aquaculture are being an important tool. microsatellites are being used to determine genealogy of the fish, allowing to decreases important problems in aquaculture such as fish deformities. genetic techniques such as micro-arrays and gene expression are being applied to obtain indicators of stress and health in fish. these technologies also permit to make different selective breeding programs, increasing the accuracy to estimate genetic parameters and evaluating brood-stock. furthermore, this technology allows to obtain a procedure to increase the productivity and quality of fish hatcheries. new species for aquaculture. development of new culture techniques: the diversification of species cultured is one of the main objectives of european aquaculture, since nowadays only four marine species are commercially produced: gilthead sea bream. european sea bass, turbot and salmon. we have been developed rearing techniques for new species such as red porgy or canarian abalone and also we are conducting studies on different new species, such as different sparids species, octopus or yellowtail. new rearing technology: the election of adequate systems for fish growth for each species and site of production and the localization of more appropriate sites for farms, using gis technology are also of special importance in our research team. besides, new larval rearing techniques such as semi-extensive hatchery (mesocoms) were development and nowadays is being used to increase fry quality (survival, no-deformities, better growth). this technology is being exported to other countries in order to offer new technology easy to manage to be implanted in developing countries. alejandro cañeque project officer, canary islands special zone, ministry of finance, c/leon y castillo 431 -4 a planta, edificio urbis, 35007 las palmas, spain. e-mail: acaneque@zec.org the canary islands special zone is the newest tax instrument within the canary islands economic and fiscal regime (ref). it offers a reduced tax rate of between 1 and 5% of corporate income tax for companies setting up a business. the companies must cover the following minimum requirements: create employment and make a minimum investment. the zec offers other tax advantages such as the exemption from paying transfer tax and stamp duty and the canary islands general indirect tax (igic). this tax scheme particularly fosters the biotechnology and pharmaceutical sectors. references fahnert references bechor the antimicrobial drugs high-level production of human collagen prolyl 4-hydroxylase in sandwich hybridisation assay for quantitative detection of yeast rnas in crude cell lysates microbial processes and products press. biotech. bioeng. 218. van hijum microbial xylitol production from corn cobs using candida magnoliae the role of bacillus methanolicus citrate synthase gene, city, in regulatiing the secretion of glutamate in lysine-secreting mutants plasmid-dependent methylotrophy in thermotolerant bacillus methanolicus 1,5-anhydrod-fructose; a versatile chiral building block: biochemistry and chemistry detailed dissection of a new mechanism for glycoside cleavage: the ␣-1,4-glucan lyase ␣-1,4-glucan lyase, a new class of starch and glycogen degrading enzyme ␣-1,4-glucan lyase, a new starch processing enzyme for production of 1,5-anhydro-d-fructose efficient purification, characterization and partial amino acid sequencing of two ␣-1,4-glucan lyases from fungi ␣-1,4-glucan lyases producing 1,5-anhydro-d-fructose from starch and glycogen have sequence similarity to alpha-glucosidases enzymatic description of the anhydrofructose pathway of glycogen degradation i. identification and purification of anhydrofructose dehydratase, ascopyrone tautomerase and ␣-1,4-glucan lyase in the fungus anthracobia melaloma a systems approach to dissecting immunity and inflammation integrative biological analysis of the apoe*3-leiden transgenic mouse innate recognition of bacteria in human milk is mediated by a milk-derived highly expressed pattern recognition receptor, soluble cd14 soluble forms of toll-like receptor (tlr)2 capable of modulating tlr2 signaling are present in human plasma and breast milk proteomics of the rat gut: analysis of the myenteric plexuslongitudinal muscle preparation an integrative metabolism approach identified stearoyl-coa desaturase as a target for an arachidonate-enriched diet the challenges of modeling mammalian biocomplexity rapid enrichment of bioactive milk proteins and iterative, consolidated protein identification by mudpit technology post-genomics of lactic acid and other food-grade bacteria to discover gut functionality genetics, metabolism and application of lactic acid bacteria. fems microbiol complete genome sequence of lactobacillus plantarum wcfs1 functional ingredient production: application of global and metabolic models metabolic engineering of lactic acid bacteria for the production of nutraceuticals reduction in antinutritional and toxic components in plant foods by fermentation the authors gratefully acknowledge the financial supports given to this work by ankara university, biotechnology institute (project no 2001k120240-41). the authors gratefully acknowledge the financial supports given to this work by ankara university, biotechnology institute (project no. 2001k120240-40) and the microorganisms given to this work by prof. dr. francesco molinari at university of milano, and prof. dr. leyla aç ikel at gazi university. the authors gratefully acknowledge the financial supports given to this work by ankara university, biotechnology institute (project no 2001k120240-40) and the microorganisms given to this work by prof. dr. francesco molinari at university of milano, and prof. dr. leyla aç ikel at gazi university. mrs. lynnette fernandez, johanna mäkeläinen, m.sc. and olli rämö, m.sc. are gratefully acknowledged for their help. this work was supported by the health science council, the academy of finland and the tekes-neobio program. supported by the mec of spain (ppq2002-04361-c04-02) and the canary islands government. jmp thanks icic for a postdoctoral fellowship. frpc thanks cajacanarias for a fpi fellowship. tm thanks the spanish mcyt-fse for a ramón y cajal contract. this work was supported by the korean systems biology research grant from the ministry of science and technology, lg chem chair professorship, ibm sur program and bk21 program. this study was supported by ankara university biotechnology institute (project no: 89). this work was partially supported by mec and feder (bio2004-00439), and fundación séneca carm (00842/pi/01). this work was supported by grants f037663 of the hungarian scientific research fund and grant omfb-00219/2002 of the hungarian ministry of education. keto sugars have long been implicated as attractive intermediates or substrates for further chemical or enzymatic reactions, to generate a number of synthetic sugar derivatives and fine chemicals. the quinone-dependent pyranose dehydrogenase (pdh) purified of the basidiomycete fungus agaricus meleagris catalyzes with high specificity the oxidation of the c-3 of glycosidically bound d-glucose, whereas in contrast it oxidizes simultaneously the c-2 and c-3 of free d-glucose. considering the broad substrate tolerance, pdh provides a new convenient tool for high yield production of 3-ketothis work was partially financed by the scientific and technological research council (cicyt, spain), grant ren2001-3224, by the iii pla de recerca de catalunya (generalitat de catalunya), grant 2001sgr-00143, and by the generalitat de catalunya to the "centre de referència en biotecnologia" (cerba). this work was supported by mega a.s. (czech republic) (www.mega.cz) and following vega grants: 1/2391/05 and 1/2390/05. this work was partially supported by mec and feder (bio2004-00439), and fundación séneca carm (00842/pi/01). financed by a marie curie re-integration grant merg-ct-2004-006378 and consejería de educación y ciencia de la junta de andalucía. this project is part of the collaborative research centre sfb 578 "development of biotechnological processes by integrating genetic and engineering methods", which is supported by the german research foundation dfg. this research was supported in part by grants from the hungarian scientific research fund (otka t37471, f46658 and d48537) and the hungarian-spanish intergovernmental s & t cooperation programme (omfb00103/2005). this research was supported in part by grants from the hungarian scientific research fund (otka t37471, f46658 and d48537) and the hungarian-spanish intergovernmental s & t cooperation programme (omfb00103/2005). this research was supported in part by grants from the hungarian scientific research fund (otka t37471, f46658, d48537) and gvop-3.1.1.-2004-05-0471. the authors thank dr. hamid narjiss (director of morocco inra) for the instruction to identify nadorcott mandarin by molecular markers and helpful discussions regarding this paper. this work was partially funded by the program for scientific cooperation cnrst (morocco) and iccti (portugal), the international foundation for science (ifs) support in stockholm (sweden). this work has been financed by xunta de galicia (pgidt03pxib30103pr). sevgil sadettin, gönül dönmez department of biology, faculty of science, ankara university 06100 beşevler, ankara, turkey this study was supported by ankara university biotechnology institute. demet ç etin 1 , sedat dönmez 2 , gönül dönmez 1 : 1 department of biology, faculty of science, ankara university, 06100 beşevler, ankara, turkey; 2 department of food engineering, faculty of engineering, ankara university, 06110 dışkapı, ankara, turkey sulfate-reducing bacteria (srb) that could grow on modified postgate c medium (pc) containing chromium(vi) were isolated from industrial wastewater and their chromium(vi) reduction capacities were investigated as a function of changes in the initial ph values, chromium, sulfate, nacl concentrations and carbon source. the optimum ph value at 50 mg l −1 initial chromium(vi) concentrations was determined as 8. chromium(vi) reduction by srb was investigated at 22.7-98.4 mg l −1 initial chromium(vi) concentrations. at the end of the experiments, the mixed cultures of srb were found to reduce more than 99% of the initial chromium(vi) levels which ranged from 22.7 to 74.9 mg l −1 within 2-6 days period. the effects of initial 0-9.0 g l −1 concentrations of sulfate and 0-6% (w/v) concentrations of nacl to chromium reduction were showed that, the lowest concentrations of sulfate and nacl, were the best for chromium reduction in the pc media including 50 mg l −1 chromium(vi). when the 25% whey was used as carbon source in the pc medium, 99.6% of the 65.9 mg l −1 initial chromium(vi) concentration was reduced within this study was supported by ankara university biotechnology institute. this study was carried out as a part of the project for analyzing and controlling the mechanism of biodegrading and processing entrusted by the new energy and industrial technology development organization (nedo). this work has been financed by xunta de galicia (pgidt03pxib30103pr). lead ions are considered a high pollutant of different waters. in our previous work were selected seven potential sorbent-strains rhodotorula mucilaginosa 1776, rh. aurantiaca 1195, rhodotorula sp. 4, williopsis californica 248, candida krusei 61t, cryptococcus sp. wt of lead ions. it was determined their stability to high concentration (up to 750 mg/l) of lead ions in medium. the ph changes and yeast physiologies of growth were studied in medium with these heavy metal ions. the influences of environmental factors such as ph of solution, age of microbial culture, biosorbent concentration in suspension, alive or living state of biosorbent, time of contact on sorption were investigated. the levels of maximal sorption ability and biomass affinity to heavy metal ions were established by experimentally received sorption isotherms with mathematical modeling of biosorption process separately for everyone researched yeast strain. the sorption isotherms obtained in these experiments for non-living yeast biomass showed that the maximal sorption capacity was 225 and 195 × 10 −6 mol (g sorbent) −1 for rh. aurantiaca 1195 and s. cerevisiae 1968, respectively. in the case of living biomass, the highthis work has been financed by the spanish ministry of science and technology and european feder (project ctm2004-01539). the authors wish to thank dra. m.j. martínez (cib, csic, madrid, spain) for providing coriolopsis rigida. this work was supported principally by embo and the mrc. fed-batch cultivation of haematococcus pluvialis under illumination with leds for production of astaxanthin abdolmajid lababpour, tomohisa katsuda, shigeo katoh department of molecular science and material engineering, kobe university, kobe, hyogo 657-8501, japan photosynthetic microalga haematococcus pluvialis is a most promising microorganism for production of astaxanthin, which has powerful antioxidant activity and is used both for human being as a food supplement and cosmetic; as well as animal farming such as salmon and poultry. the deficiency of nutrients in batch culture of h. pluvialis decreases the growth of cells and increases the induction of astaxanthin as an induction factor. therefore, it is impossible to reach to high cell concentrations in batch cultures. in previous experiments, the medium replacement increased the cell concentration, while accumulation of astaxanthin was not induced without other factors. in this work, the effects of fed-batch addition of culture medium on the cell growth and astaxanthin production in h. pluvialis cultures were studied. h. pluvialis was cultivated in 50 cm 3 culture medium containing sodium acetate, yeast extract, l-asparagines, mgcl 2 ·6h 2 o, feso 4 ·7h 2 o and cacl 2 ·2h 2 o (ph 6.8). light was supplied by panels of blue or red led lamps. the temperature was kept at 20 • c and the culture was mixed with a magnetic stirrer. in fed-batch cultures of h. pluvialis, the cell concentration and production of astaxanthin increased in comparison with those in batch culture. in addition, the operation in feb-batch manner is easier than medium replacement from industrial viewpoints for production of astaxanthin. simvastatin and similar compounds are wide used as antihypercholesterolemic agents. simvastatin is obtained by c-methylation of side chain of lovastatin. this process is not perfect and some unreacted lovastatin is present in the reaction mixture. simvastatin is separated from lovastatin using fungi clonostachys compactiuscula. using of living microbials has some disadvantages such as high cost, difficult product separation from the reaction mixture. we work on overcome these difficulties by separation and immobilization of enzyme that hydrolyses lovastatin ammonium salt in presence of simvastatin ammonium salt. our results of purification and immobilization lovastatin hydrolase will be presented. investigation of peptide antibiotics produced by trichoderma strains isolated from winter wheat rhizosphere a. szekeres 1 , l. kredics 2 , l. hatvani 1 , z. antal 2 , l. manczinger 1 , a. nagy 3 , c. vágvölgyi 1 : 1 department of microbiology, university of szeged, p.o. box 533, h-6701 szeged, hungry; 2 hungarian academy of sciences, university of szeged, microbiological research group, hungry; 3 pilze-nagy ltd. , kecskemét, p.o. box 407, species of the imperfect filamentous fungal genus trichoderma with teleomorphs belonging to the hypocreales order of the ascomycota division are of great economic importance as sources of enzymes, antibiotics, as plant growth promoters, decomposers of xenobiotics, and as commercial biofungicides. peptaibols and related peptaibiotics (prps) are secondary metabolites constituting a family of fungal peptide antibiotics which is constantly growing since alamethicin was isolated from cultures of trichoderma viride. these compounds are linear, amphipathic polypeptides composed of 5-20 amino acids and usually containing several non-proteinogenic amino acid residues, which are representing characteristic building blocks of the structure. one hundred and twenty trichoderma strains were isolated from roots of winter wheat grown in agricultural fields of southern hungary. the identity of species was examined based on morphological and molecular characters. the presence of prps-producing strains among the isolated trichoderma strains was detected by biological tests and the antibiotics were partially purified using a multistep chromatography procedure involving exclusion chromatography, adsorption chromatography and thin-layer chromatography. about 20% of the isolates proved to be able to produce prps. the antibacterial activity of the compounds was tested against staphylococcus aureus, bacillus subtilis, micrococcus luteus and escherichia coli, while the antifungal effect was recorded against fusarium oxysporum, f. culmorum, rhizoctonia solani and pythium debaryanum. ergezinger, m., bohnet, m., berensmeier, s., buchholz, k., 2005. integrierte enzymatische synthese und adsorption von isomaltose in einem mehrphasenbioreaktionsreaktor. cit 77, 167-171. glucansucrases from family 70 of glycoside-hydrolases are transglucosidases that produce ␣-glucans from sucrose, a very cheap substrate, without any use of nucleotide activated sugars. based on sequence analyses, these enzymes have been classified in two families, the family 70 and the family 13 of glycoside hydrolases. among the natural diversity existing in family 70 in which are found the glucansucrases produced by lactic acid bacteria, three enzymes have been selected for their distinctive specificities: dextransucrase from l. mesenteroides nrrl b-512f (dsr-s), which catalyses almost essentially the synthesis of ␣-1,6-linkages, alternansucrase from l. mesenteroides nrrl b-1355 (asr), which produces alternan polymer formed of ␣-1,6and ␣-1,3-alternated linkages and finally dextransucrase from l. mesenteroides nrrl b-1299 (dsr-e), which is responsible for the synthesis of a branched dextran composed of about 70% of ␣-1,6-linkages in the main chain and 30% of ␣-1,2-branched linkages. for all these enzymes, the natural polymerase activity can be shifted towards oligosaccharide production or gluco-conjugate syntheses by introducing acceptors in the reaction medium. a number of sugar acceptors have been successfully glucosylated with the view of developing new functional food products. acceptor glucosylation yield as well as acceptor reaction product structures were shown to be highly dependant on the enzyme specificity. consequently, using glucansucrases of distinctive specificities and varying the acceptors give access to a large variety of applications. amylosucrase, the sole glucansucrase found in family 13 of glycoside-hydrolases is also of great interest for functional food applications. this enzyme is able to synthesize highly resistant amylose from sucrose. again the reaction conditions can be used to modulate the yield and the size of amylose. the aim of our work is to further develop the applications of these enzymes via rational and combinatorial engineering. the most recent results obtained in this field will be discussed. novel food structure engineering concepts with enzymes johanna buchert vtt biotechnology, espoo, finland food structure is a very important quality attribute in food choice, since it affects not only the sensory perception of texture, but also release of flavour. enzymes offer specific means to engineer food structure by creating cross-links to food biopolymers, i.e. to proteins and/or carbohydrates. enzymatic cross-linking of food biopolymers can be exploited to create novel types of structures to foods without any need of added food ingredients. laccases and peroxidases can be used to crosslink ferulic acid containing carbohydrates, such as sugar beet pectin or arabinoxylan. proteins can be crosslinked by different oxidative or transferase type of enzymes. transglutaminases can crosslink protein via formation of isopeptide bond between glutamine and lysine residues. laccase and peroxidase can oxidize tyrosine residues to corresponding radicals, which in turn can further react with different groups in proteins. tyrosinases, on the other hand, oxidize tyrosine to a quinone, which can further react with aromatic ring, amine and thiol groups present in proteins. the biopolymer networks formed can be further engineered by combining adequate processing to the enzyme treatment. in this work the potential of enzymatic food structure engineering is reviewed. asparaginase-mediated reduction of acrylamide formation in baked, fried, and roasted products hanne vang hendriksen, beate kornbrust, steffen ernst, mary stringer, hans peter heldt-hansen, peter østergaard novozymes a/s, dk-2880 bagsvaerd, denmark. e-mail: hvhe@novozymes.com (h.v. hendriksen) in 2002, it was discovered that acrylamide is formed in several potato and grain-based foods (e.g. chips, french fries, toasted bread, biscuits, cereals) and in coffee, all of which have been prepared at high temperatures. the level of this potential carcinogen in the final food appears to range from 50 to 4000 ppb. later that year, the mechanism of acrylamide formation was unraveled, demonstrating that asparagine and reducing sugars are the precursors for acrylamide. this pointed to several potential enzymatic approaches to remove the root cause of the problem by degrading the precursors in situ. here, we demonstrate that asparaginase treatment leads to a more efficient reduction in acrylamide than alternative enzymatic treatments. asparaginase from aspergillus oryzae is used to reduce acrylamide formation significantly in laboratory models of a range of common food products. examples are french fries, biscuits, crisp bread, and fabricated chips. the sensory qualities appear to be constant. the implications for scaling up the processes for industrial food production are discussed. effect of cultivation conditions on folate content in yeast: exploring the potential of yeast as a bio-enrichment vehicle for folate in foods sofia hjortmo 1 , johan patring 2 , jelena jastrebova 2 , thomas andlid 1 : 1 department of chemical and biological engineering, chalmers university of technology, po box 5401, 402 29 gothenburg, sweden; 2 department of food science, swedish university of agricultural sciences, po box 7051, 750 07 uppsala, sweden. e-mail: sh@fsc.chalmers.se (s. hjortmo) over the past 10 years, the interest in health benefits of the b vitamin folate has increased considerably. a good folate status may hinder progression of several diseases such as neural tube defects and downs syndrome in foetus, as well as cancer, dementia, alzheimer's disease and cardiovascular disease in adults. it is however not easy to reach the recommended intake and new strategies have to be developed to increase the folate status. in this project we explore the use folate producing microorganisms for this purpose. many yeasts have the ability to synthesise folate de novo and can thus serve as a source for humans. folate enrichment in fermented foods could be much improved by using starter cultures better at producing folate compared to traditional strains. this is, e.g. applicable to bread making fb32 over-expression of isoprene biosynthetic enzymes in the ␤-carotene producer zygomycete mucor circinelloides tamás mucor circinelloides has been involved to study the carotene biosynthesis genesis of fungi. this fungus is more amenable to molecular techniques than the others traditionally used in carotenogenic studies (e.g. blakeslea trispora and phycomyces blakesleeanus). moreover, mucor has a great advantage: it is a dimorphic organism. this type of morphology is preferred by the fermentation industry because yeast-like growth allows the submerged culture, when usually higher biomass production can be achieved and cells can be more easily separated from the media. β-carotene is a terpenoid-type chemical compound likewise to sterols, quinones or chlorophylls. the production can be increased by improving the non-carotene specific terpenoid biosynthesis. this can be carried out by the overexpression of the genes responsible for the ratelimiting steps of these pathways. in this study, polyethylene glycol mediated transformations of m. circinelloides protoplasts were performed with autoreplicative expression vectors containing the known terpenoid genes of m. circinelloides (e.g. isoa encoding farnesyl pyrophosphate synthase and carg encoding geranylgeranyl pyrophosphate synthase). carotene production of the transformants and the wild-type strains were analysed by high-performance liquid chromatography (hplc). transformants harbouring plasmids with isoa or carg produce about 1.5 times more carotene than the recipient strain, while carotene production increased about two times in the co-transformants containing both type of plasmids. members of the genus rhizopus are important from biotechnological aspects in consequence of their effective extracellular enzyme, alcohol and organic acid production. moreover, rhizopus strains are used for fermentation of various foods, because they are capable of transforming soybeans into edible products. the high affinity iron permease (ftr1) contains both highly conservative and variable regions applicable for phylogenetic comparisons. the aim of this study was the comparative analysis of this gene of different rhizopus species in order to elaborate a simple and fast method to identify these fungi at a species and subspecies level. conserved regions of candida albicans and rhizopus oryzae ftr1 genes (fu et al., 2004) have been analysed to design degenerate primers for polymerase chain reaction. they were used to amplify the homologous regions from different strains of r. oryzae, r. microsporus, r. stolonifer and r. niveus. isolates of the similarly thermophilic rhizomucor miehei and r. pusillus, as well as a strain of m. rouxii were involved in the study as outgroups. deduced protein sequences were aligned and phylogenetic analysis was performed. surprisingly the r. oryzae isolates formed a group completely different with a significant distance from the r. microsporus isolate. r. niveus is currently not distinguished from r. stolonifer var. stolonifer because of morphological considerations. however, phylogeny of ftr1 gene sequences, in agreement with earlier results based on rapd data (vágvölgyi et al., 2004) , raise the need to handle r. niveus as a separate species. sequences and pcr primers useful for identification of all tested rhizopus strains were elaborated. potential application in a lactose conversion process. the prebiotics market is at high demand therefore the development of the process to produce 'prebiotic' galacto-oligosacharides efficiently and inexpensively is our particular interest. citrus, particularly mandarins and clementines, are among the most economically important fruit crops in morocco. besides morphological traits multiple molecular markers have been used for the caracterisation of citrus germplasm. the main aim of this study was to evaluate the moroccan mandarin germplasm and to identify specific polymorphisms among accessions sharing identical name. eighty mandarin and two sweet orange varieties were analyzed by dna markers. issr markers were amplified using 3 anchored primers and analysed by agarose gel electrophoresis. aflp markers analyses were performed using three primer combinations. the dice coefficient was used to estimate genetic similarities and the upgma algorithm was utilised to generate a phenogram depicting the genetic relationships among the acessions. the selection of 9 primers out of the 31 issr primers primarily assayed, allowed us to maximize the average number of amplified fragments analyzed per reaction (7.3), and the percentage of informative polymorphisms (49%). the three combinations of ecori/msei primers revealed 73 reliable aflp markers, 35 (47%) of witch were polymorphic. the range of fragment sizes varied from 100 to 650 bp. contrasting with the phenotypic diversity for agronomic and fruit quality traits, very low variability at the dna level has found among mandarins, which always showed a high (s > 0.81) coefficient of genetic similarity. the molecular marker analyses allowed the clarification of ambiguous denominations and the establishment of phenological relationships. the mandarin cultivars have been clustered into several different sub groups. this study allowed the identification of one issr marker, distinct and specific for the clementine sidi aissa and some aflp markers specific to maroc late and w. navel. many hybrids used in this study presented high coefficient of the similarity with one of their parents, such as siamelo and king of siam (s = 0.92), fortuna and clementine (s = 0.93), kara and king of siam (s = 0.92). this work was partially funded by the program for scientific cooperation cnrst (morocco) and iccti (portugal), the international foundation for science (ifs) support in stockholm (sweden). the new morocco mandarin variety nadorcott became very important in the international market because of high quality, good size, easy peeling and absence of seeds. also known as afourer or w. murcott, this variety was selected in 1981-1982 at the afourer experimental station, inra, located near to beni mellal city, as an original tree among several 18-year-old murcott honney (c. reticulata × c. sinensis) seedling trees. in order to shed additional light on the genetic origin of this variety, we have carried out isozyme and dna fingerprinting analyses. for better interpretation of the nadorcott molecular profiles, others mandarin cultivars, among which murcott honney, were also analyzed by molecular markers. three enzymatic systems (idh, pgm and pgi) permitted the discrimination between nadorcott and his female parent murcott honney. the molecular patterns displayed by these cultivars point out the sexual origin of nadorcott and discard the previously assumed hypothesis for its origin as a mutation of a nucellar zygote. the issr and rapd markers analyses allowed the identification of kinnow; du japon, vietnam; and swett lime as the genetically most closely related mandarins (s ∼ 0.95) to nadorcott. strong genetic similarity was also found with the clementine group, a possible male parent of nadorcott. the analysis by aflp markers confirmed the hybrid origin of nadorcott and the high genetic relatedness (s = 0.93) of this mandarin to its putative female parent murcott honey, and other cultivars as kinnow (s = 0.94) and clementine (s = 0.93). the possibility of an accurate molecular identification of nadorcott by specific molecular markers is of paramount importance for the protection and management of this original moroccan citrus variety. an effective process for the chemical-biotechnological utilization of distilled white lees was studied. a first treatment with hydrochloric acid allowed the solubilisation of tartaric acid. the influence of temperature, amount of hcl and reaction time were considered through an experimental design. under the optima conditions 77 g/l from white distilled lees and 45.6 g/l from red distilled lees were recovered. the tartaric acid was precipitated as calcium tartrate so that it can be isolated from the rest of the raw material compounds. the solid residue was used as an economic nutrient for lactic acid production by lactobacillus pentosus using trimming wastes as substrate. the lactic acid concentrations and volumetric productivities achieved were similar to those obtained using distilled lees without tartaric acid recovery as nutrient. thermophilic cyanobacterial strains that could grow in the bg 11 media was isolated from hot springs and their reactive dye bioaccumulation was studied under thermophilic conditions in a batch system, in order to determine the optimal conditions required for the highest dye accumulation. in the experiments performed with newly isolated synechocystis sp. and phormidium sp., the optimum ph values at about 25 mg l −1 initial reactive dye concentrations was determined as 8. lipases are extremely versatile enzymes, that catalyze both hydrolysis and synthesis reactions. they have a wide range of industrial applications, among which the manufacture of detergents, pharmaceuticals and fine chemicals are outstanding. the fungus rhizopus oryzae has been reported to synthesize a number of commercially interesting enzymes. in this work, its ability to produce extracellular lipases when grown in solid state culture has been assessed. cultures were carried out in erlenmeyer flasks, using a complex medium and several supports, both synthetic (nylon sponge) and natural (barley bran, ground walnut or peanut). the latter appeared to be more suitable for lipase production. since the best results were initially obtained with lipid-containing supports, barley bran cultures were supplemented with a vegetable oil, in an attempt to optimise lipase production and design an efficient procedure for reusing this agroindustrial waste. surprisingly, this strategy did not improve enzyme synthesis. however, when a surfactant (triton x-100) was added to the basal medium, a dramatic increase in extracellular activity was detected (up to 20-fold). the results agreed with those previously obtained in submerged cultures of r. oryzae, in which addition of olive oil did not increase lipase production, while the presence of triton x-100 had a remarkably beneficial effect. also, enzyme concentration in solid state cultures was up to two-fold that of the submerged ones. est maximal sorption capacity was found for yeasts cryptococcus sp. wt and rh. aurantiaca 1195 . these cultures also demonstrated the high sorption affinity, which makes them especially efficient biosorbents at low concentrations of lead ions. the high efficiency of lead elution was shown with 0.1n edta. isolation and identification of marine bacteria from deep-sea sediments els maas 1 , cara brosnahan 1 , vicky webb 1 , helen neil 2 , phil sutton 2 : 1 marine biotechnology, national institute for water and atmospheric research ltd., kilbirnie, wellington, new zealand; 2 oceanography, national institute for water and atmospheric research ltd., kilbirnie, wellington, new zealand marine sediments were obtained using a piston corer with associated trigger core (0.5 m, 0.06 m diameter). cores were collected from depths of 270 to 3911 m, along norfolk ridge and across challenger plateau. sediments ranged from coarse carbonate sands in the north to sandy and silty hemipelagic mud with increasing depth and latitude. all samples sites underlie subtropical surface water masses associated with, and south of, the tasman front. sediment samples were aseptically taken from the triggers cores upon recovery. samples were stored in sterile tubes at 4 • c on board the vessel for 3-17days. the core samples were plated on several different agar types and incubated aerobically for 4 weeks at 16 • c. individual colonies were sub-cultured and purified using standard microbiological techniques. morphological and molecular taxonomy revealed that the bacteria isolated from the sediments were closely related to novosphingomonas, halomonas, stappia, glaciecola, pseudoalteromonas and leeuwenhoekiella. phylogenetic trees constructed using 16s rrna gene sequence data showed that two other isolates were unrelated to known genera. the bacterial isolates are currently being investigated for their biotechnological potential. olive oil mill wastewater (omw) as the effluent of the concern of olive industry has high organic load. the conventional biological treatments despite of their simplicity and rather suitable performance are ineffective for the omw treatment since phenolics possess antimicrobial activity. in order to carry out a proper treatment on omw, use of microorganism able to degrade the phenolics thus, is necessary. the ability of phanerochaete chrysosporium immobilized on loofa was studied. the basal mineral salt solution along with glucose, ammonium sulfate and yeast extract were used to dilute omw properly. the fungus did not grow on the concentrated omw. therefore, omw diluted by 20% was used thought this study. the extent of removal in this biotreatment, of total phenolics (tp) and cod were 90 and 50%, respectively. while the color and aromatocity decreased by 60 and 95%, respectively. the kinetic behavior of the loofa immobilized fungus was found to follow monod equation. the maximum growth rate was 0.045 h −1 while the monod constant based on the consumed tp and cod (mg/l) were 370 and 6900, respectively. the control of water pollution has become of increasing importance in recent years. the release of dyes into the environment constitutes only a small proportion of water pollution, but dyes are visible in small quantities due to their brilliance. many dyes are difficult to decolourise due to their complex structure and synthetic origin. the adsorption of reactive dye remazol brilliant blue r (rbbr) on polyelectrolyte complex (pec) was studied in a batch systems. the adsorption parameters determined were: effect of the different values of ph on the adsorption of dye by pec, and the effect of contact time on the amount of rbbr adsorbed (in mg g −1 ). the data indicates that the adsorption capacity of rbbr by pec is dependent on ph. the maximum adsorption at 50 ppm was 88.52% equal to 11.8 mg of dye/g of polymer. the results show a tendency towards greater adsorption for reactive dyes (ph range of 8-12). the effect of contact time was studied at initial concentration (50 ppm) of dye, the amount of rbbr adsorbed for these pec increased and reached a constant value with the increase in contact time. the increase in the extent of removal of dye after 15 min of contact time is less and hence it is fixed as the optimum contact time. the pec show their capacity to remove rbbr to aqueous solutions by adsorption. flocculation of saccharomyces cerevisiae (diastaticus) ifo1958 was studied. cells of ifo 1958 did not flocculate even in the stationary phase without mg 2+ ("mg 2+ -deficient cells") although they began to flocculate strongly 18h after inoculation in the presence of mg 2+ ("complete cells"). cycloheximide completely inhibited induction of floc-forming ability of "mg 2+ -deficient cells". co-flocculation between "complete cells" and "mg 2+ -deficient cells" was investigated by chemical modification. treatment of "mg 2+ -deficient cells" by proteolytic enzymes did not affect the co-flocculation with "complete cells". photo-oxidation or mercaptoethanol-reduction of "mg 2+ -deficient cells" failed to weaken the co-flocculation with "complete cells" while treatment of "mg 2+ -deficient cells" by periodate brought about a significant loss of the co-flocculation. on the contrary, "complete cells" deflocculated by proteolysis or chemical modification of proteinaceous component failed to co-flocculate with "mg 2+ -deficient cells". these findings suggest that "mg 2+ -deficient cells" are non-flocculent because of lack of proteinaceous component essential for flocculation of cells of ifo 1958. the industrial toscano cigar production starts with the dark firecured kentucky tobacco fermentation process. during this phase the present work is a trial to study the portal serum factors which stimulate the cell proliferation of the schistosomules, aiming to find ways to block or inhibit their effects. our previous studies showed that portal serum of human and hamster (highly susceptible hosts) and a 1-50 kd fraction separated from human portal sera by ultrafiltration stimulate cell proliferation in immature schistosomules (20 days old) in vitro. for further identification of the portal serum factors in the range of 1-50 kd that stimulate cell proliferation, schistosomules were incubated in vitro in medium containing 10% fetal calf serum, 10% portal human serum or 10% peripheral human serum or their fractions separated by native electrophoresis followed by electroelution, incubations were performed in presence of bromodeoxyuridine (brdu) in order to measure differences in cell proliferation. the results showed that human portal sera enhanced cell proliferation of schistosomules compared to the peripheral serum. this stimulatory effect was substantially reproduced by fraction separated from human portal serum with molecular weight 20.8 kd. these results may help in designing a drug or antibody therapy to block the stimulating effect of the portal serum fraction and subsequently to disturb the life cycle of the parasite at early stage of development. center of molecular biosciences, university of the ryukyus, okinawa 903-0213, japan. e-mail: naoya-s@comb.u-ryukyu.ac.jp (n. shinzato)oil strage tank sludge, mainly composed of water and solid hydrocarbons (waxes) needs to be treated when harvesting the stored oil. although the sludge treatment by microbial surfactant or microbial cracking are considered as the feasible method, microbial degradation of the waxes (ca. solid n-alkanes) have been reported in a very limited species such as acinetobacter. in addition, long-chain n-alkanes (so called paraffin waxes) are one of the major components of oil, and their resistant properties to biological attack hold up the recovery of oil-polluted environments. in this report, we have screened n-tetracosane (c24) degrading bacteria from soils in okinawan island, an unique sub-tropical area in japan, to know the bacterial diversity and their degrading mechanism.16srdna phylogenetic analysis of the isolates, totally ca 40, showed they were not only acinetobacter and pseudomonas, but also other proteobacteria (alcaligenes), actinomycetes (gordonia, nocardia, and leifsonia), bacillus, staphylococcus, and unidentified ones. they also grew not only the solid n-alkanes but also iso-alkanes, mid-chain n-alkanes as the sole carbon source. results for the biosurfactant production will also be shown. the properties of 188 environmental enterococci were studied. the strains were isolated mainly from surface and waste waters and several strains from sheep manure were also included. species identification was provided by combination of phenotypic (micronaut system, merlin) and molecular detection methods (automated its-pcr, ddl-pcr). several discrepancies were observed when comparing molecular and biochemical identification. six enterococcal species were overall identified; e. faecium and e. hirae were the most abundant ones, almost 80% of isolates belonged to these two species. the distribution of selected genes conferring virulence to enterococci (cyla, gele and esp) was investigated, the positive signal was obtained mainly for e. faecalis strains. the strains were also characterized for the possession of enterocin genes (enta, entb, entp, ent31, entl50ab) and high frequency of enterocins was observed. biosorption of three different dyes (reactive black 5, cibacron brilliant yellow, cibacron brilliant red) onto immobilized scenedesmus obliquus a microalga was investigated in a batch sys-tem. the immobilized alga exihibited the highest dye uptake capacity at the initial ph value of 2.0 for all dyes. the effect of temperature on equilibrium sorption capacity indicated that maximum was obtained at 25 • c for rb5, cby and cbr biosorption. the freundlich, and langmuir adsorption models were used for the mathematical description of the biosorption equilibrium and isotherm constants were evaluated. biocontrol properties of microbially-treated sugar beet wastes in presence of rock phosphate n. vassilev, i. nikolaeva, m. vassileva department of chemical engineering, faculty of sciences, university of granada, c/fuentenueva s/n, granada-18071, spain. e-mail: nbvass@yahoo.com (n. vassilev) the effect of soil application of sugar beet wastes (sb) treated with aspergillus niger in the presence of rock phosphate (rp) on the control of fusarium wilt of tomato were studied. two treatments and a control were used: inoculation with glomus intraradices (am), further inoculation with a. niger grown on sb + rp medium, and the control (c). application of the am fungus increased plant growth, p and n uptake and reduced disease caused by fusarium oxusporum f. sp. licopersici (fol) as compared to non-mycorrhizal control plants. soil amendment with sb + rp + a. niger resulted in 347% and 467% (versus c) higher plant shoot biomass in plant-soil experiments contaminated or not with fol, respectively. in this case, disease severity and number of fol cfu reached the lowest levels while soil phosphatase and beta-glucosidase activities increased compared to all other treatments. fol negatively affected plant root mycorrhization determined in the am treatment while the difference between the mycorrhization of plants grown in the presence and absence of f. oxysporum in sb + rp + a. niger-amended soil was insignificant (53% versus 59%, respectively). in conclusion, the fermentation mixture containing mineralized organic matter, partially solubilized rp, and a. niger biomass could be efficiently used not only in improving plant growth, nutrient uptake and properties of degraded and polluted soils, as previously reported (vassilev and vassileva, 2003) , but also in environmentally-mild management of fusarium wilt. vassilev, n., vassileva, m., 2003. appl. microbiol. biotechnol. 61, 435-440 . biological treatment processes allow for the effective elimination of charged inorganic micropollutants, e.g. a number of oxyanions, heavy metals, etc. from contaminated drinking water supplies. however, dedicated technologies have to be implemented in order to eliminate the target pollutants without changing the quality of treated water, avoiding its secondary pollution by cells, nutrients and metabolic by-products. some innovative technologies, which combine the use of membranes with the bioconversion of charged micropollutants in order to deal with the secondary water contamination problem, will be presented and critically compared. a special on loofa was studied. the basal mineral salt solution along with glucose, ammonium sulfate and yeast extract were used to dilute omw properly. the fungus did not grow on the concentrated omw. therefore, omw diluted by 20% was used thought this study. the extent of removal in this biotreatment, of total phenolics (tp) and cod were 90 and 50%, respectively. while the color and aromaticity decreased by 60 and 95%, respectively. the kinetic behavior of the loofa immobilized fungus was found to follow monod equation. the maximum growth rate was 0.045 h −1 while the monod constant based on the consumed tp and cod (mg/l) were 370 and 6900, respectively. advanced start-up strategy of an anaerobic three-phase turbulent bed reactor treating winery wastewaters r. cresson, h. carrère, n. bernet, j.p. delgenès laboratoire de biotechnologie de l'environnement, institut national de la recherche agronomique (inra), avenue des etangs, 11100 narbonne, france. e-mail: cresson@ensam.inra.fr (r. cresson)the objective of our study was to compare two start-up strategies for an anaerobic biofilm process, to create an effective biofilm and increase the organic loading rate (olr) as quickly as possible. two methanogenic three-phase biofilm reactors have been started, using the same operational parameters (solid hold-up ratio, gas velocity of 1 mm s −1 ), in order to test two different strategies:• maximal load strategy (reactor a): the olr is increased as long as the global amount of removed cod (biogas production) increased. • maximal removal strategy (reactor b): the olr is increased stepwise as soon as the cod removal rate reaches 80%.both reactors have been operated for 90 days, until a volumetric olr of 20 g cod l −1 j −1 , with more than 90% of carbon removal. the total amount of cod removed and methane produced were higher in reactor b (19.6 and 32.2%, respectively). in both reactors, the short hydraulic retention time (hrt) applied during all the experiment caused a rapid wash-out of planktonic bacteria and an exclusive use of the substrate by the attached micro-organisms, which accelerates the biofilm growth. the lag-phase was reduced to approximately 7 days. the reactor submitted to repetitive disturbance by the maximal removal strategy appeared to be more robust when confronted to perturbation like organic overload or nutritional deficiency. experiments have demonstrated capability and the efficiency of the aggressive strategy for controlling anaerobic bioreactor start-up. sustainability is the generally accepted paradigm for future industrial development. the re-integration of waste products into production processes is a major aspect of environmental sustainability. in this study the use of sugar cane molasses is being investigated for the production of bioplastics by mixed microbial cultures, with the added possibility of parallel biohydrogen production. polyhydroxyalkanoates (phas) are polyesters synthesized by bacteria and accumulated as granules in the cytoplasm. studies conducted by this group have shown that mixed microbial cultures subjected to dynamic feeding conditions may accumulate phas up to 80% cell dry weight, a value close to that obtained for pure cultures. volatile fatty acids are good substrates for the production of phas by mixed cultures. on the other hand, sugar molasses, with a very high sugar content (about 50% dry weight), can produce organic acids by fermentation. the two-stage process being implemented in this study includes a molasses fermentation step, in which the high sugar content of the molasses is converted into volatile fatty acids (vfas), and a pha production step, in which the vfas serve as the precursors to the formation of phas under dynamic feeding conditions. moreover, hydrogen can be produced by anaerobic bacteria from carbohydrate-rich substrates giving organic fermentation end products, h 2 and co 2 . to optimize the production of both high-value products, design of experiments (doe) is being used to elaborate a set of experiments to study the effect of ph, hydraulic retention time and organic loading on both the organic acids distribution (which will serve as precursors for pha production in the second step) and h 2 production in the acidogenic fermentation reactor (a 1 l cstr). preliminary results show that the effluent of the acidogenic reactor fed with 10 g/l total sugars and operated at ph 7 and d = 0.1 h −1 (composed mainly of acetate and propionate) can be successfully fed to a polymer-accumulating mixed culture. under these conditions, the h 2 production yield has been estimated at 3.9 mol h 2 /mol sucrose. vanillin is a flavour compound used in food industry, fragrances and pharmaceutical preparations, which is nowadays mainly produced by chemical synthesis. the increased demand of natural products for the food industry as well as the high cost of natural vanillin extracted from vanilla pods has recently stimulated the research for alternatives to produce this compound by a natural way. the microbial transformation of ferulic acid, a phenolic compounds from lignin degradation, is recognized as being the most interesting alternative to produce natural vanillin. the combined effects of initial ferulic acid concentration (s 0 ) and biomass concentration (x 0 ) on vanillin production by resting cells of escherichia coli strain were investigated using response surface methodology. e. coli jm109/pbb1 a recombinant strain producing key enzymes of ferulate catabolic pathway from p. fluorescens bf13 (feruloyl-coa synthetase and feruloyl-coa hydratase/aldolase) was utilized in this work. a 3 2 full-factorial design was employed for experimental design. the results shown a possible inhibition phenomena at a vanillin concentration of about 0.1 g l −1 leading to the accumulation in the fermentation media of secondary compounds like vanillic acid and vanillin alcohol. removal of dissolved nutrients from wastewater using a microalgae biofilter line christensen, suvina sooknandan, jens jørgen lønsmann iversen department of biochemistry and molecular biology, university of southern denmark, odense, a microalgae biofilter can be used for treatment of wastewater from landbased fish farms in order to remove excess amounts of dissolved nutrients such as nitrate, ammonium and phosphate. a bubble column bioreactor has been developed for cultivation and characterization of microalgae. this type of bioreactor is equipped with a control system that enables online determination of the photosynthetic quotient and optimization of light intensity. furthermore the bioreactor has a dualsparging system simultaneously allowing adequate mixing and high gas-liquid mass transfer coefficients. different species of microalgae have been cultivated in batch and fed batch cultures to characterize growth and ability to take up the different dissolved nutrients. the specific growth rate and substrate uptake rate have been determined to compare and select the algal species most suited for use in a biofilter. additionally the composition of lipid, protein and carbohydrates has been measured to determine the nutritional quality of the algae when used as animal feed. at present, biological nitrogen-removal is mostly carried out through several complicated steps. to simplify the present systems for nitrogen-removal, we have investigated a new nitrogenremoval bioreactor using packed gel envelopes capable of simultaneous nitrification and denitrification. the envelope consists of two plate polymeric gels with a spacer in between. ammonia oxidizer, nitrosomonas europaea and denitrifier, paracoccus denitrificans are co-immobilized in the plate gels. when the envelopes are exposed to wastewater containing ammonia, the immobilized n. europaea oxidizes ammonia to nitrite in the outer aerobic surfaces of envelopes. at the same time, as ethanol solution is injected into the internal anaerobic spaces of envelopes, the immobilized p. denitrificans reduces the nitrite to nitrogen gas using the ethanol solution as an electron donor for denitrification. in this way, the envelopes can remove ammonia from wastewater in a single step. we have already reported advantages of our bioreactor in laboratory-scale experiments. in this study, we show our large-scale bioreactor (water volume 1.8 m 3 ) could treat three kinds of wastewater derived from coal power plants. ammoniacontaining wastewater that occurred regularly in a coal power plant was continuously treated with the bioreactor using thirty envelopes for over a year. the bioreactor could remove more than 90% of total nitrogen at hydraulic retention time (hrt) of 24 h. at hrt of 4 h, the bioreactor accomplished a maximum rate (the transformation of nh 4 + to n 2 ) of 6.0 g n/day m 2 of the envelopes' surface. the performance was equivalent to that obtained in the laboratory-scale experiments. furthermore, our bioreactor showed similar nitrogen-removal performances when it treated nitrate-containing wastewater occurring regularly and condensed ammonia-containing wastewater occurring at irregular intervals in coal power plants. these results show that our bioreactor can treat various wastewater containing nitrogen in coal power plants. thus, our concept is effective to simplify the large-scale systems in coal power plants and the other plants. in order to establish an environmentally friendly process for the treatment of metal containing waste, in a portuguese refinery a process involving sulphur oxidizing acidophilic microbes is being considered. bioleaching of metal containing bottom ash, from fluidised bed incineration of sludge resulting from the refinery water treatment station, was performed using a sulphur oxidising acidophilic culture isolated from an acid pool resulting from the weathering of sulphur piles from the claus plant. this sample served as inoculum for liquid medium cultures with 1% sterile sulphur flowers as source of energy. application of monod kinetics to adapted culture growth of free cells presented a value of µ = 0.124 day −1 . yield of sulphur conversion to sulfate after 17 days was η = 78%. in the presence of bottom ash from the incineration of refinery sludges µ = 0.141 day −1 and the yield of sulphur conversion was η = 67.5%. a η fe = 90% removal of iron is obtained from the treated ash. x-ray fluorescence spectroscopy of the solid residue revealed a total removal of metals namely, v, cu, ni, zn and most of the fe after 15 days of bioleaching. the present of heavy metals in the environment is a serious problem. they are commonly present in effluents from mining and industrial activities. usually, chemical conventional methods are very expensive and they have limitations when heavy metals are in low concentrations. at the moment the interest increases for processes that involve microorganisms as alternative method. some effluents present heavy metal sulphates which are soluble compounds. sulphate-reducing bacteria (srb), under anaerobic conditions, oxidize simple organic compounds (such as acetic acid and lactic acid) by utilizing sulphate as an electron acceptor and generate hydrogen sulphide. hydrogen sulphide reacts with heavy metal ions to form insoluble metal sulphides that can be easily separated from a solution. the purpose of this work was to evaluate the ability of srb to reduce cr(iii), ni(ii) and zn(ii) in artificial contaminated solution. desulfovibrio vulgaris and desulfovibrio sp. strains has been tested in this study. batch cultures was carried out in 50 ml sealed bottles with different concentrations of studied metals (1-20 mg/l), with 10% of inoculum bacterial and adapted to postgate's medium c. a gaseous nitrogen current was employed to purge oxygen and obtain anaerobic conditions. the assays were incubated statically represented very low portion (less than 4-5%) of the total bacterial community at all temperatures tested. schistosomules of schistosoma mansoni (20 days old) were incubated in rpmi 1640 medium containing 10% fetal calf serum, 10% hamster portal venous or 10% hamster peripheral venous serum (highly susceptible host) or 10% rat portal venous or 10% rat peripheral venous serum (poorly susceptible host) in presence of bromodeoxyuridine (brdu) in order to measure differences in cell proliferation. also the rate of cell proliferation of s. mansoni were assessed in vivo in hamster to study the cell proliferation in the natural ontogeny of the organism. the rates of cell proliferation as expressed by brdu labeling indices (blis) were determined as a function of time of incubation by immunohistochemistry using monoclonal antibody to brdu. compared to schistosomules cultured in presence of rpmi plus 10% fetal calf serum, blis were increased by 41% in the presence of hamster portal, but not in peripheral serum. while in case of rat, no significant changes were observed in the blis in both portal and peripheral sera. the experiment was repeated using hamster portal and peripheral sera containing different schistosomal igg antibody titres. the results showed decreased values of blis compared to sera which did not contain the schistosomal antibody(ies). the in vivo results revealed that there was no cell proliferation of s. mansoni schistosomules (6 days old) in the lungs. cell proliferation was detected in schistosomules of 17 days old and the results revealed a significant decrement in the brdu labeling indices (blis) with the increase of the age of schistosomules in vivo. the results indicated that hamster portal venous serum (highly susceptible host) could have stimulating factor(s) for schistosomule cell proliferation which is not found in rat (poorly susceptible host) and the presence of antibody(ies) greatly inhibit the cell proliferation. this could be due to the blocking of some portal serum factors, which stimulate the cell proliferation by the antibody(ies). the release of dyes into the environment constitutes only a small proportion of water pollution, but dyes are visible in small quantities due to their brilliance. many dyes are difficult to decolourise due to their complex structure and synthetic origin. the adsorption of reactive dye remazol brilliant blue r (rbbr) on polyelectrolyte complex (pec) was studied in a batch systems. the adsorption parameters determined were: effect of the different values of ph on the adsorption of dye by pec, and the effect of contact time on the amount of rbbr adsorbed (in mg g −1 ). the data indicates that the adsorption capacity of rbbr by pec is dependent on ph, the maximum adsorption at 50 ppm was 88.52% equal to 11.8 mg dye/g of polymer. the results show a tendency towards greater adsorption for reactive dyes (ph range of 8-12). the effect of contact time was studied at initial concentration (50 ppm) of dye, the amount of rbbr adsorbed for these pec increased and reached a constant value with the increase in contact time. the increase in the extent of removal of dye after 15 min of contact time is less and hence it is fixed as the optimum contact time. the pec show their capacity to remove rbbr to aqueous solutions by adsorption. compared to other european nations, the austrian population shows a low level of knowledge in biosciences and a strong denial of gene technology (1). the austrian non-profit organisation dialog<>gentechnik, a scientific society, organizes various activities to raise awareness for the "hot topics" in the life sciences. according to its principle of independence, all activities are funded publicly. projects on behalf of the austrian authorities and international cooperations demonstrate credibility and trust in dialog<>gentechnik (2). a few examples will be presented. dialogue with the public: on the occasion of the first anniversary that the gmo labelling rules became effective, the action "gene technology on my plate" is performed austrian wide in shopping centres. here, consumers are informed about health and labelling aspects of gm food. two days of open discussions were organized in the context of the austrian genome research program gen-au (3): topics were "gene diagnosis" (2002) and "genome research-what is in it for me?" (2004) . an open lab is currently set up in vienna to offer "hands on" experience in life sciences for everybody. motivating students: in the very successful gen-au summer school (3), high school students spend 3-4 weeks in the lab and work with scientists. the best documentations are awarded. in an innovative project, student groups (age 16-18) work on the topics stem cells and cloning and develop units of an elearning course which will be accessible for all austrian schools in the near future. engaging stakeholders: dialog<>gentechnik manages interdisciplinary wor king groups that develop leaflets, brochures and questionnaires on various aspects of gene diagnosis. four products are currently distributed to the public and to health services. (1) european commission. eurobarometer 55.2, december 2001; (2) www.dialog-gentechnik.at; (3) www.genau.at. the aim of this paper is to compare the attitudes of the urban consumers with professionals towards to new technologies, especially to biotechnology. it was tried to find out in which area, medical, agriculture or industry, people can accept biotechnological developments and in which area not accept. key: cord-004584-bcw90f5b authors: nan title: abstracts: 8th ebsa european biophysics congress, august 23rd–27th 2011, budapest, hungary date: 2011-08-06 journal: eur biophys j doi: 10.1007/s00249-011-0734-z sha: doc_id: 4584 cord_uid: bcw90f5b nan o-003 structure determination of dynamic macromolecular complexes by single particle cryo-em holger stark max-planck-institute for biophysical chemistry, goettingen, germany macromolecular complexes are at the heart of central regulatory processes of the cell including translation, transcription, splicing, rna processing, silencing, cell cycle regulation and repair of genes. detailed understanding of such processes at a molecular level requires structural insights into large macromolecular assemblies consisting of many components such as proteins, rna and dna. single-particle electron cryomicroscopy is a powerful method for threedimensional structure determination of macromolecular assemblies involved in these essential cellular processes. it is very often the only available technique to determine the 3d structure because of the challenges in purification of complexes in the amounts and quality required for x-ray crystallographic studies. in recent years it was shown in a number of publications that it is possible to obtain near-atomic resolution structures of large and rigid macromolecules such as icosahedral viruses. due to a number of methodological advances there are now also great perspectives for high-resolution single particle cryo-em studies of large and dynamic macromolecules. successful high-resolution structure determination of dynamic complexes requires new biochemical purification strategies and protocols as well as state of the art electron microscopes and high-performance computing. in the future cryo-em will thus be able to provide structures at near-atomic resolution and information about the dynamic behavior of macromolecules simultaneously. detection and rapid manipulation of phosphoinositides with engineered molecular tools tamas balla section on molecular signal transduction, program for developmental neuroscience, nichd, nih, bethesda, md 20892, usa polyphosphoinositides (ppis) are ubiquitous lipid regulators of a variety of cellular processes serving as docking sites and conformational switches for a large number of signaling proteins. the localization and dynamic changes in ppis in live cells have been followed with the use of protein domain gfp chimeras. in this presentation we will show experimental systems that allow rapid manipulation of the levels of ppis in specific membrane compartments. we are also actively pursuing strategies that will allow us to map the distribution and possible functional diversity of the phosphatidylinositol (ptdins) pools within intact cells since they are the precursors of ppis. we will show our most recent progress in addressing this question: the use of a ptdins specific plc enzyme isolated from listeria monocytogenes together with a highly sensitive diacylglycerol sensor to determine the distribution and also to alter the level of ptdins in living cells. these studies reveal that a significant metabolically highly active ptdins pool exists associated with tiny mobile structures within the cytoplasm in addition to the known er and pm ptdins pools. we will show our most recent data on the consequences of ptdins depletion within the various ptdins pools on ppi production and on the morphology and functions of various organelles. the functionality of proteins is known to be intimately related to the motion of their constituents on the atomic/molecular level. the study of microscopic motion in complex matter is often reduced to the observation of some average mean square atomic displacement, a first, very partial characterization of the dynamics. the marked crossover in the temperature dependence of such quantities in hydrated proteins around 200 k, the so called ''dynamic transition'' has been originally observed a quarter of century ago. the origin, nature and the key characteristics of the atomic motions behind this remarkable evolution of the mean square displacement in proteins remained controversial over the past decades. recent analysis of mö ssbauer, dielectric relaxation and neutron scattering spectroscopic data provide unambiguous evidence that this phenomenon is caused by the temperature dependence of a relaxation process spread over several orders of magnitude in the time domain, similarly to the b relaxation process observed in glasses. the review and critical analysis of the available data highlights the inherent ambiguities of commonly used data fitting approaches. emerging evidence from model independent observations tend to exclude some of the proposed mechanisms. microbial rhodopsins: light-gated ion channels and pumps as optogenetic tools in neuro-and cell biology e. bamberg, c. bamann, r.e. dempski, k. feldbauer, s. kleinlogel, u. terpitz, p. wood department of biophysical chemistry, max-planck-institute of biophysics, frankfurt, germany microbial rhodopsins are widely used in these days as optogenetic tools in neuro and cell biology. we were able to show that rhodopsins from the unicellar alga chlamydomonas reinhardtii with the 7 transmembrane helix motif act as light-gated ion channels, which we named channelrhodopsins(chr1,2). together with the light driven clpump halorhodopsin chr2 is used for the non-invasive manipulation of excitable cells and living animals by light with high temporal resolution and more important with extremely high spatial resolution the functional and structural description of this new class of ion channels is given (electrophysiology, noise analysis,flash photolysis and 2d crystallography). new tools with increased spatial resolution and extremely enhanced light sensitivity in neurons are presented. a perspective for basic neurobiology and for medical applications is given. extracellular signals consists of the induction of specific gene expression patterns and the re-organization in space and time of stereo-specific macromolecular interactions that endow the cell with its specific morphology. we develop quantitative experimental and computational approaches to derive and conceptualize physical principles that underlie these dynamics of signal processing and cellular organization. we have an experimental emphasis on functional microscopic imaging approaches at multiple resolutions to study the localization and dynamics of protein reactions/ interactions, maintaining the inherent spatial organization of the cell. we have a strong recursion between computation of molecular dynamics in realistic cell geometries as sampled by microscopy, and experiments that reveal the dynamic properties of networks in living cells. we investigate the cellular topography of activities that transmit signals from receptors at the cell surface. here we ask, how spatial partitioning of intracellular signalling activities is achieved by the causality structure of the signalling network, and how this partitioning affects signal response. this entails the experimental elucidation of connections between reactions and the determination of enzyme kinetic parameters in living cells. o-008 molecular photovoltaics mimic photosynthesis michael grä tzel laboratory of photonics and interfaces, institute of chemical science and engineering, station 6, ecole polytechnique fé dé rale, ch-1015 lausanne, switzerland e-mail: michael.graetzel@epfl.ch the field of photovoltaic cells has been dominated so far by solid state p-n junction devices made e.g. of crystalline or amorphous silicon, profiting from the experience and material availability of the semiconductor industry. however, there is an increasing awareness of the possible advantages of devices referred to as ''bulk'' junctions due to their interconnected three-dimensional structure. their embodiment departs completely from the conventional flat p-n junction solid-state cells, replacing them by interpenetrating networks. this lecture focuses on dye sensitized mesoscopic solar cells (dscs), which have been developed in our laboratory. imitating natural photosynthesis, this cell is the only photovoltaic device that uses a molecular chromophore to generate electric charges from sunlight and that accomplishes the separation of the optical absorption from the charge separation and carrier transport processes. it does so by associating the molecular dye with a film constituted of tiny particles of the white pigment titanium dioxide. the dsc has made phenomenal progress, present conversion efficiencies being over 12 percent for single junction and 16 percent for tandem cells, rendering the dsc a credible alternative to conventional p-n junction devices. molecularly single-molecule imaging and tracking techniques that are applicable to living cells are revolutionizing our understanding of the plasma membrane dynamics, structure, and signal transduction functions. the plasma membrane is considered the quasi-2d non-ideal fluid that is associated with the actinbased membrane-skeleton meshwork, and its functions are likely made possible by the mechanisms based on such a unique dynamic structure, which i call membrane mechanisms. my group is largely responsible for advancing highspeed single molecule tracking, and based on the observations made by this approach, i propose a hierarchical architecture of three-tiered meso-scale (2-300 nm) domains as fundamental organizing principles of the plasma membrane. the three tiers i propose are the following. [tier 1] 30-200 nm compartments made by partitioning the entire plasma membrane by the membrane-associated actinbased meshwork (membrane skeleton: fences) and its associated transmembrane proteins (pickets). since the entire plasma membrane is partitioned by these structures, and the membrane skeleton provides important platforms for the molecular interactions and pools, membrane compartments are the most basic tier for the plasma membrane organization. [tier 2] meta-stable 1-5 nm raft domains that can be turned into stable *10-20-nm domains (receptor-cluster rafts), based on ligand-induced homo-dimers of glycosylphosphatidylinositol (gpi)-anchored receptors (coupling with [tier 3]) and facilitated by raft-lipid interactions. [tier 3] protein complexes of various sizes (3-10 nm) and lifetimes. i will also talk about how domains of tiers 2 and 3 are coupled to the membrane partitioning (tier 1). the concept of the three-tiered domain architecture of the plasma membrane and the cooperative interactions of different tiers provides a good perspective for understanding the mechanisms for signal transduction and many other functions of the plasma membrane. introduction: in the present study we investigate the effects of electromagnetic fields (emf) on the binding of norfloxacin (nrf) to human serum albumin (hsa) by fluorescence, three-dimensional fluorescence and uv-visible spectroscopic approaches. hsa is the most abundant protein in human blood plasma which works as a carrier that transports different materials in the body. nrf is used to treat variety of bacterial infections. it works by stopping the bacterial growth. methods: hsa, nrf and potassium phosphate buffer were purchased from sigma. fluorescence spectrofluorometer, uv-vis spectrophotometer, three-dimensional fluorescence and a home-built emf generator apparatuses were used. results: results obtained from this study indicated that nrf has a strong ability to quench hsa in 280 nm. in addition, there was a slight blue shift, which suggested that the microenvironment of protein became more hydrophobic after addition of nrf. moreover, synchronous fluorescence demonstrated that the microenvironment around tyrosine (tyr) had a trivial increase. these, and the results of hsa-nrf in the presence of emf with 1 khz, illustrates the same results inferred from quenching and blue shift. however, there was a significant decrease in k sv of nrf with hsa in presence of emf exposure. moreover, the binding parameters including the number of binding sites and the binding constant were calculated form hill equation. conclusion: it was shown that nrf could induce conformational changes in hsa both in the absence and presence of emf with no significant difference. yet, the affinity is decrease significantly in the presence of emf. the clinical implications are discussed in detail. characterization of the biochemical properties and biological function of the formin homology domains of drosophila we characterised the properties of drosophila melanogaster daam-fh2 and daam-fh1-fh2 fragments and their interactions with actin and profilin by using various biophysical methods and in vivo experiments. the results show that while the daam-fh2 fragment does not have any conspicuous effect on actin assembly in vivo, in cells expressing the daam-fh1-fh2 fragment a profilindependent increase in the formation of actin structures is observed. the trachea specific expression of daam-fh1-fh2 also induces phenotypic effects leading to the collapse of the tracheal tube and lethality in the larval stages. in vitro, both daam fragments catalyze actin nucleation but severely decrease both the elongation and depolymerisation rate of the filaments. profilin acts as a molecular switch in daam function. daam-fh1-fh2, remaining bound to barbed ends drives processive assembly of profilin-actin, while daam-fh2 forms an abortive complex with barbed ends that does not support profilinactin assembly. both daam fragments also bind to the sides of the actin filaments and induce actin bundling. these observations show that the drosophila melanogaster daam formin represents an extreme class of barbed end regulators gated by profilin. electron spin echo studies of free chain-labelled stearic acids interacting with b-lactoglobulin rita guzzi, luigi sportelli, rosa bartucci dipartimento di fisica, università della calabria, 87036 rende (cs), italy b-lactoglobulin (blg) binds non-covalently fatty acids within its central calyx, a cavity in the barrel formed by the strands ba-bh. we present results of pulsed electron paramagnetic resonance (epr) spectroscopy on the interaction of blg with stearic acids spin-labelled at selected positions, n, along the acyl chain (n-sasl, n = 5,7,10,12,16). d 2 o-electron spin echo envelope modulation (eseem) fourier transform spectra indicate that all segments of the bound chains in the protein binding site are accessible to the solvent. the extent of water penetration decreases progressively on moving from the first segments toward the terminal methyl end of the chain. about 50% of the nitroxides in the upper part of the chain (n = 5,7) are h-bonded by a single water molecule and this fraction reduces to 30% at the chain terminus (n = 12,16). a lower fraction of the nitroxides are h-bonded by two water molecules, and it decreases from about 15% to a vanishingly small value on going down the chain. echo-detected ed-epr spectra reveal subnanosecond librational motion of small amplitude for both 5-and 16-sasl in the protein cavity. the temperature dependence of the librations is more marked for 16-sasl and it arises mainly from an increase in librational amplitude with increasing temperature. fusion peptides (fp) pertaining to the spike glycoprotein from severe acute respiratory syndrome (sars) coronavirus are essential for the fusion between viral and host cellular membranes. here we report a biophysical characterization of the interaction of two putative fps with model membranes. fluorescence and dsc experiments showed that both peptides bind stronger to anionic than to zwitterionic lipid membranes. esr spectra showed that toac-sars ifp rotational dynamics is modulated by lipid composition and ph as compared to the spectrum of this peptide in solution. however, stearic acid spin labels reported no changes on the dynamic structure of zwitterionic micelles, whereas the whole chain of anionic surfactants was perturbed by the peptides. finally, cd data revealed a predominant b-strand structure for sars fp and an a-helix for sars ifp in the presence of micelles, in contrast to their disordered structures in buffer. overall the results point out that electrostatic and hydrophobic interactions are both important to the energetic behavior of peptide membrane interaction. these findings might provide a useful rationale for the elucidation of one of the steps involved in the fusion process, and thus help understanding the more general way of action of fps at a molecular level. interaction of filamentous actin and ezrin within surface modified cylindrical nanopores daniela behn 1,2 and claudia steinem 1 1 institute for organic and biomolecular chemistry, university of gö ttingen, tammannstraße 2, 37077 gö ttingen, germany, 2 ezrin is a member of the ezrin-radixin-moesin (erm) protein family that acts as a dynamic linker between the plasma membrane and the actin cytoskeleton and is hence involved in membrane organization, determination of shape and surface structures and other cellular processes. the protein is highly enriched in microvilli of polarized epithelial cells, where it binds filamentous actin (f-actin) with its c-terminal domain, while the n-terminal domain is connected to the plasma membrane via specific binding to l-a-phosphatidylinositol-4,5-bisphosphate (pip 2 ). nanoporous anodic aluminum oxide (aao) films provide similar dimensions as microvilli and are thus a versatile template to investigate the interaction of ezrin with f-actin within spationally confined areas. owing to their optical transparency, functionalized aaos can be used to measure the binding process of ezrin to a pip 2 containing solid supported membrane by means of time resolved optical waveguide spectroscopy (ows). confocal laser scanning microscopy (clsm) will elucidate, whether f-actin binding to ezrin takes place within or atop the nanopores. furthermore, elasticity mapping of f-actin filaments by means of atomic force microscopy will allow determining binding forces and the lateral tension of the actin cytoskeleton. in vitro application of porphyrin photosensitisers on mcf7, hela and g361 tumour cell lines binder s., kolarova h., bajgar r., tomankova k., daskova a. deparment of medical biophysics, faculty of medicine of palacky university, olomouc, czech republic tumour treatment presents a challenge to all scientists and clinicians. contemporary methods like radiotherapy, chemotherapy or surgery have many undesirable side effects. photodynamic therapy (pdt) seems to be one of alternatives which can be helpful in malignant cell therapy. pdt is not only limited to cancer treatment but is also used as an alternative for cardiovascular, skin and eye disease treatment. pdt employs photosensitive agents which need to be activated by light which is not harmful to a patient. the activated photosensitive agent provokes a formation of reactive oxygen species leading to cell damage or death. the phototoxicity of the two porphyrin photosensitizer (tmpyp, zntpps 4 .12h 2 o) on the malignant cell lines (g361, hela, mcf7) irradiated with the 1 jcm -2 doses was evaluated by ros production assay, mtt assay and comet assay. our results indicate higher efficiency of tmpyp over zntpps 4 .12h 2 o. as for the photodynamic effectiveness of the used photosensitizers on chosen cell lines we found that hela cell line is the most sensitive to phototoxic damage induced by tmpyp. p-018 nmr analysis of the respiratory syncytial virus m2-1 protein structure and of its interaction with some of its targets c. sizun 1 the respiratory syncytial virus (rsv) is a major cause of acute respiratory tract infections (bronchiolitis, pneumonia) in human and a leading cause of viral death in infants and immunocompromised patients. rsv genome is formed of a single non-segmented negative strand rna which transcription and replication is ensured by a specific rna-dependent rna polymerase complex formed of the large (l) polymerase subunit and of several cofactors. this complex has no cellular counterpart and represents an ideal target for antiviral drugs. among the cofactors, m2-1 acts as an antitermination factor and increases the polymerase processivity. its central domains has been shown, in vitro, to bind the phosphoprotein p and genomic rna in a competitive manner. here we report the nmr structure of this central domain and its interaction with p and rna fragments. m2-1 shares structural similarity with vp30, a transcription factor of ebola virus. the binding surfaces for rna and p are distinct but overlapping. rna binds to a basic cluster located next to residues found to be critical for transcription both in vitro and in vivo by mutational analysis. we speculate that m2-1 might be recruited by p to the transcription complex, where interaction with rna takes place, stabilized by additional elements. force spectroscopy at the membrane-cytoskeleton interface: interactions between ezrin and filamentous actin julia a. braunger 1,2 , ingo mey 1 and claudia steinem 1 1 institute for organic and biomolecular chemistry, georg-august-university of gö ttingen, tammannstraße 2, 37077 gö ttingen, germany, 2 ggnb doctoral program: imprs ezrin, a member of the erm (ezrin/radixin/moesin) protein family, provides a regulated linkage between the plasma membrane and the actin cytoskeleton. it contributes to the organization of structurally and functionally distinct cortical domains participating in adhesion, motility and fundamental developmental processes. ezrin is negatively regulated by an intramolecular interaction of the terminal domains that masks the f-actin binding site. a known pathway for activation involves the interaction of ezrin with phosphatidylinositol 4,5bisphosphate (pip 2 ) in the membrane, followed by phosphorylation of the threonine 567 residue in the c-terminal domain. to date, it is unclear to what extent both regulatory inputs contribute to the activation. we developed an in vitro system that facilitates the specific analysis of the interaction forces between ezrin and f-actin by means of atomic force spectroscopy (afm). applying ezrin wild type and the pseudophosphorylated mutant protein ezrin t567d, respectively, permits to monitor the individual influence of phosphorylation on the f-actin-ezrin interaction. thus, a thorough characterization of the acting forces at the ezrin-actin interface will elucidate the activation mechanism of ezrin. delivery system even more efficient, we have constructed nano-carrier by coating of ldl by polyethylene glycol (peg) . the hydrophilicity of peg should reduce the interaction of ldl with other serum proteins and consequently decrease the redistribution of loaded drug from ldl to the (lipo)proteins. dynamic light scattering was used for determination of hydrodynamic radius of ldl-peg particles. cd spectroscopy measurements didn't reveal structural changes of apoliprotein b-100 (ligand for ldl receptors on cell surface), after conjugation of peg with ldl. interaction of ldl-peg complexes with hypericin (hyp) a natural photosensitizer was studied by fluorescence spectroscopy. we have demonstrated accumulation of higher number of hyp in ldl-peg than ldl particles. however, the kinetics of hyp redistribution from hyp/ldl-peg complex to free ldl have similar parameters as those for the kinetics of hyp transfer between non-modified ldl molecules. we suggest that hyp molecules are mostly localized in the vicinity of the surface of the ldl-peg particles and they are prone to redistribution to other serum proteins. grant support: lpp-0072-07, vega-0164-09. modification of the head-group of aminophospholipids by glycation and subsequent lipid oxidation affect membrane's structure causing cell death. 1 these processes are involved in the pathogenesis of aging 2 and diabetes. 3 non-enzymatic glycation forms in the first step a schiff base (sb), which rearranges to a more stable ketoamine, amadori product, which leads to the formation of a heteregenous group of compounds (ages). although several studies have been focused on identification of aminophospholipid glycation products, 4 less attention has been paid to kinetic mechanism of the reaction. for that reason, in the present work, we compare the kinetic reactivity of polar head-group of phosphatidylethanolamine (pe) and phosphatidylserine (ps), the two target phospholipids components of mammalian cell membranes. the reaction of pe and ps's head-group with glycating compounds (glucose and arabinose) was studied in physiological conditions by using nmr spectroscopy. the obtained formation rate constants for sb are lower than those determined for the sb of the peptide ac-phe-lys with the same carbohydrates. 5 it suggests that the phosphate group may delay the glycation process. moreover, the ps's head-group has a carboxilic group in the structure, which affects the stability of the sb. we developed ultrasensitive, elisa-like nanoimmuno assays suitable for proteomics/interactomics studies in low sample volumes. we exploit the approach of dna microarray technologies applied to proteomics [1] , in combination with atomic force microscopy (afm) to generate functional protein nanoarrays: semisynthetic dna-protein conjugates are immobilized by bioaffinity within a nanoarray of complementary ssdna oligomers produced by afm nanografting (ng). a nanoarray of different antibodies or synthetic molecular binders can be generated in a single operation, once the dna nanoarray is produced. moreover, ng allows adjusting the packing density of immobilized biomolecules to achieve optimum bio-recognition. afm-based immunoassays with these nanoarrays were shown to achieve detection limit of hundreds of femto molar, in few nanoliters volumes, with very high selectivity and specificity [2] . to detect the hybridization efficiency of our devices, we run a combined experimental-computational study that provides quantitative relations for recovering the surface probe density from the mechanical response (afmcompressibility measurements) of the sample. nucleoside analogues used as anticancerous drugs can be rapidly degraded within treated cells, constituting a major obstacle of their therapeutic efficiency. among the enzymes responsible for this degradation, the cytosolic 5'nucleotidase ii (cn-ii) catalyses the hydrolysis of some nucleoside monophosphates. in order to improve the efficacy of anticancerous drugs and to define the precise role of cn-ii, new original inhibitors have been developed against cn-ii. virtual screening of chemical libraries on the crystal structure has allowed us to identify very promising candidates that turned to be competitive inhibitors of cn-ii. one molecule was included in the anticancerous treatment of tumoral cell lines in order to evaluate the potential benefit and could induce in fine a sensitization of certain anticancerous drugs. we also explore other inhibitors targeting the allosteric sites of this enzyme using a strategy that takes into account the dynamics of cn-ii. the chemical structures of the newly identified allosteric inhibitors as well as the atomic interactions with enzyme residues will be presented. the final goal of this study is to find molecules that can freeze the enzyme in a conformation for which its dynamics is severely limited and therefore its function. native mass spectrometry to decipher interactions between biomolecules sarah cianferani laboratoire de spectromé trie de masse bio-organique, université de strasbourg, iphc, 25 rue becquerel 67087 strasbourg, france. cnrs, umr7178, 67037 strasbourg, france mass spectrometry is generally understood as ''molecular mass spectrometry'' with multiple applications in biology (protein identification using proteomic approaches, recombinant protein and monoclonal antibody characterization). an original and unexpected application of mass spectrometry emerged some twenty years ago: the detection and the characterization of intact biological noncovalent complexes. with recent instrumental improvements, this approach, called native ms, is now fully integrated in structural biology programs as a complementary technique to more classical biophysical approaches (nmr, crystallography, calorimetry, spr, fluorescence, etc.). native ms provides high content information for multiprotein complexes characterization, including the determination of the binding stoichiometries or oligomerization states, sitespecificities and relative affinities. recent developments of ion mobility / mass spectrometry instruments (im-ms) provide a new additional level for ms-based structural characterization of biomolecular assemblies allowing size and shape information to be obtained through collisional cross section measurements. these different aspects of native ms for structural characterization of biomolecular assemblies will be illustrated through several examples, ranging from multiprotein-complexes to protein/nucleic acid assemblies. complex coacervation is a process which may result by electrostatic interaction between charged polysaccharides. it depends essential on ph, ionic strength and biopolymers properties like ratio, concentration and charge density. in this case, the main work was to study the structural properties of a colloidal system of opposite charge -chitosan and gum-arabic by atomic force microscopy (afm). according to some of complexes show tendency to agglomerate. this depends on the molar ration of the macromolecules and their relative molecular weights. afm micrographs show, too, that some formation of irregular aggregates by both polymers were due to presence of noncharged polar monomers in chitosan molecule. at higher gum-arabic/chitosan ratios biopolymer concentrations, coacervates appear like a core-shell miccelar structure composed of hydrophobic core (charge neutralized segments) stabilized by the excess component (positive zeta potential) and non-charged segments of gum arabic. interaction of human serum albumin with rutin theoretical and experimental approaches ícaro p caruso 1 human serum albumin (hsa) is the principal extracellular protein with a high concentration in blood plasma and carrier for many drugs to different molecular targets. flavonoids are a large class of naturally occurring polyphenols widely distributed in plants. rutin (quercetin-3-rutinoside) is the glycoside between flavonoids quercetin and disaccharide rutinose. like other flavonoids, rutin displays anti-inflammatory and anti-oxidant properties. the interaction between hsa and rutin was investigated by fluorescence spectroscopy, ab initio and molecular modeling calculations. fluorescence titration was performed by keeping the hsa concentration (4 lm) constant and stoichiometrically varying the rutin concentration (1-4 lm) . the emission spectra were obtained in the range of 305 to 500nm, with the excitation wavelength at 295nm. the obtained fluorescence data were corrected for background fluorescence and for inner filter effects. the stern-volmer quenching constant values were 3.722 9 10 5 and 1.868 9 10 5 m -1 at 298 and 303 k, respectively. from the modified stern-volmer association constants 2.285 9 10 5 (at 298 k) and 2.081 9 10 5 m -1 (at 303 k) were calculated the thermodynamic parameters dh = 14.048 kj mol -1 , dg 298k = -30.557 kj mol -1 and dg 303 k = -30.834 kj mol -1 , and ds = 55.4 kj mol -1 k -1 . fluorescence quenching method was used also to study the binding equilibria thus determining the number of binding sites 1.085 and 1.028, and binding constant 1.094 9 10 6 m -1 and 0.255 9 10 6 m -1 at 298 and 303 k, respectively. the efficient quenching of the trp214 fluorescence by rutin indicates that the binding site for the flavonoid is situated within subdomain iia of hsa. the distance r = 2.397 nm between the donor (hsa) and the acceptor (rutin) was obtained according to fluorescence resonance energy transference (fret). wavelength shifts in synchronous fluorescence spectra showed the conformation of hsa molecules is changed in the presence of rutin. the structure of rutin utilized in molecular modeling calculation was obtained by gaussian 98 program. the optimization geometry of rutin was performed in its ground states by using ab initio dft/b3lyp functional with 6-31g(d,p) basis set used in calculations. the molecular electrostatic potential (mep) was calculated to provide the molecular charge distribution of rutin. the gap energy value between the homo and lumo of the rutin molecule was about 4.22 ev which indicates that rutin is classified as a reactive molecule. from molecular modeling calculation the interaction between hsa and rutin was investigated using the autodock program package. the three-dimensional coordinates of human serum albumin were obtained from the protein data bank (entry pdb code 1ao6) and of rutin were obtained from output optimization geometry of dft. the best energy ranked result shows that rutin is localized in the proximity of single tryptophan residue (trp214) of hsa that is in agreement with the fluorescence quenching data analysis. the effect of toxofilin on the structure of monomeric actin lívia czimbalek, veronika kollá r, roland kardos, gá bor hild university of pé cs, faculty of medicine, department of biophysics, pé cs, hungary actin is one of the main components of the intracellular cytoskeleton. it plays an essential role in the cell motility, intracellular transport processes and cytokinesis as well. toxoplasma gondii is an intracellular parasite, which can utilise the actin cytoskeleton of the host cells for their own purposes. one of the expressed proteins of t. gondii is the 27 kda-sized toxofilin. the long protein is a monomeric actinbinding protein involved in the host invasion. in our work we studied the effect of the actin-binding site of toxofilin 69-196 on the g-actin. we determined the affinity of toxofilin to the actin monomer. the flourescence of the actin bound e-atp was quenched with acrylamide in the presence or absence of toxofilin. in the presence of toxofilin the accessibility of the bound e-atp decreased, which indicates that the nucleotide binding cleft is shifted to a more closed conformational state. the results of the completed experiments can help us to understand in more details what kind of cytoskeletal changes can be caused in the host cell during the invasion of the host cells by intracellular parasites. t7 bacteriophage, as a surrogate on non-enveloped viruses was selected as a test system. both tmpcp and bmpcp and their peptide conjugates proved to be efficient photosensitizers of virus inactivation. the binding of porphyrin to phage dna was not a prerequisite of phage photosensitization, moreover, photoinactivation was more efficiently induced by free than by dna bound porphyrin. mechanism of photoreaction (type i. versus type ii) and the correlation between dna binding, singlet oxygen production and virus inactivation capacity was also analyzed. dna binding reduced the virus inactivation due to the reduced absorbance and singlet oxygen production of bound photosensitizer, and altered mechanism of photoinactivation. as optical melting studies of t7 nucleoprotein revealed, photoreactions of porphyrin derivatives affected the structural integrity of dna and also of viral proteins, even if the porphyrin did not bind to np or was selectively bound to dna. anesthesia is a medical milestone (friedman & friedland, medicine's 10 greatest discoveries, 2000) and local anesthetics (la) are the most important compounds used to control pain in surgical procedures. however, systemic toxicity is still a limitation for la agents as well as low solubility, as for tetracaine (ttc). approaches to improve la effects include macrocyclic systems formation, such as in cyclodextrins (cd). we have studied complexes formed between ttc and b-cd or hydroxylpropyl (hp)-b-cd through nmr and other (uv-vis, fluorescence, dsc and x-ray diffraction) techniques. at ph 7.4 a 1:1 stoichiometry of complexation was detected for both complexes, with association constants of 777 m -1 and 2243 m -1 for ttc:b-cd and ttc:hp-b-cd, respectively. the nuclear overhauser nmr data disclosed trough the space proximities between hydrogens h h and h iat the aromatic ring of ttc -and hydrogens from the inner cavity of the cyclodextrins, allowing us to propose the topology of ttc:cd interaction. complex formation did not curb ttc association with model (liposomes) and biological membranes since the total analgesic effect (infraorbital nerve blockade in rats) induced by 15mm ttc increased 36% upon complexation. supported by (fapesp # 06/121-9, 03838-1) brazil. p-033 itc as a general thermodynamic and kinetic tool to study biomolecule interactions philippe dumas 1 , dominique burnouf 1 , eric ennifar 1 , sondes guedich 1 , guillaume bec 1 , guillaume hoffmann 1 isothermal titration calorimetry (itc) is a powerful technique for thermodynamic investigations that is little used to obtain kinetic information. we have shown that, in fact, the shape of the titration curves obtained after each ligand injection is strictly governed by the kinetics of interaction of the two partners. a simple analysis allowed us to explain several facts (e.g. the variation of time needed to return to equilibrium during a titration experiment). all simplifications were further released to obtain a very realistic simulation of an itc experiment. the method was first validated with the binding of the nevirapine inhibitor onto the hiv-1 reverse transcriptase by comparison with results obtained by biacore tm . importantly, for more complex systems, the new method yields results that cannot be obtained in another way. for example, with the e. coli transcription-regulator thiamine pyrophosphate riboswitch, we could resolve kinetically and thermodynamically the two important successive steps: (1) the binding of the tpp ligand and (2) the subsequent rna folding. our results show that initial tpp binding is controlled thermodynamically by tpp concentration, whereas the overall transcription regulation resulting from rna folding is kinetically controlled. gfps, due to their tendency to dimerize at high concentration. we have characterized for the first time the selfassociation properties of cfp (cyan fluorescent protein), the fluorescent protein mostly used as fret donor. we found that the fluorescence quenching observed at high expression level in the cell cytoplasm and the fluorescence depolarization measured at high concentration in vitro are insensitive to the a206k mutation, shown to dissociate other gfp dimers. both phenomena are satisfactorily accounted for by a model of non-specific homo-fret between cfp monomers due to molecular proximity. modeling the expected contributions to fluorescence depolarization of rotational diffusion, homo-fret within a hypothetical dimer and proximity homo-fret shows that cfp has a homo-affinity at least 30 times lower than gfp. this difference is due to an intrinsic mutation of cfp (n146i), originally introduced to increase its brightness and that by chance also disrupts the dimers. biomolecular recognition typically proceeds in an aqueous environment, where hydration shells are a constitutive part of the interacting species. the coupling of hydration shell structure to conformation is particularly pronounced for dna with its large surface to volume ratio. conformational substates of the phosphodiester backbone in b-dna contribute to dna flexibility and are strongly dependent on hydration. we have studied by rapid scan ftir spectroscopy the isothermal b i -b ii transition on its intrinsic time scale of seconds. correlation analysis of ir absorption changes induced by an incremental growth of the dna hydration shell identifies water populations w 1 (po 2 --bound) and w 2 (non-po 2 --bound) exhibiting weaker and stronger h-bonds, respectively, than those in bulk water. the b ii substate is stabilized by w 2 . the water h-bond imbalance of 3-4 kj mol -1 is equalized at little enthalpic cost upon formation of a contiguous water network (at 12-14 h 2 o molecules per dna phosphate) of reduced !(oh) band width. in this state, hydration water cooperatively stabilizes the b i conformer via the entropically favored replacement of w 2 -dna interactions by additional w 2 -water contacts, rather than binding to b i -specific hydration sites. such water rearrangements contribute to the recognition of dna by indolicidin, an antimicrobial 13-mer peptide from bovine neutrophils which, despite little intrinsic structure, preferentially binds to the b i conformer in a water-mediated induced fit. in combination with cd-spectral titrations, the data indicate that in the absence of a bulk aqueous phase, as in molecular crowded environments, water relocation within the dna hydration shell allows for entropic contributions similar to those assigned to water upon dna ligand recognition in solution. segmental-labeling expression of sh3 domains of cd2ap protein to study interaction with their ligand i.f. herranz-trillo 1 , j.l. ortega-roldan 1 , n.a.j. van transient and low affinity interactions within the cell can be enhanced by the combination of more than one domain. up to now most of the effort has been put on the study of the regulation in the affinity and specificity of the binding to isolated single domains but little is known about the effect of the presence of a second or third domain. multiple examples of proteins containing tandem domains exist in the genome like the cin85/cms family of adaptor proteins. in this family all three n-terminal sh3 domains are involved in a wide variety of different interactions, they share higher similarity among themselves than to any other sh3 domains, suggesting an overlapping specificities in binding. cd2 associated protein (cd2ap) is an adaptor protein and belongs to this family, its n-terminus consists of three sh3 domains and the interaction of each one of them with its target(-s) might be ultimately modulated by the presence of its next-door-neighbor. in this work we present the expression and purification of the tandem cd2ap-sh3a/ sh3b produced by segmental labeling techniques that allow us to express the domains with different isotopic label, improving the nmr signal and facilitating to study the interaction of the natural ligand in the presence of nextdoor-neighbor domain. there are plenty of molecules that exert their effects at the cell membrane. the evaluation of these interactions, frequently quantified by the nernst lipid/water partition constant (kp), helps to elucidate the molecular basis of these processes. we present here a recently derived and tested method to determine kp for single solute partitions using fpotential measurements. the concept was then extended to the interaction of supramolecular complexes with model membranes. a simultaneous double partition with an aqueous equilibrium is considered in this partition model. the results were validated by dynamic light scattering -dls, f-potential, fluorescence spectroscopy and laser confocal microscopy experiments. we evaluated the interaction of supramolecular complexes (peptides derived from dengue virus proteins with oligonucleotides) with luv to study our biophysical models. dengue virus (dv) infects over 50-100 million people every year and may cause viral hemorrhagic fever. no effective treatment is available and several aspects of its cellular infection mechanism remain unclear. the extension of the interactions of these complexes with biomembranes helps to elucidate some steps of dv life cycle. the aggregation of amphotericin b in the lipid membranes induced by k + and na + ions: langmuir monolayers study marta arczewska, mariusz gagoś department of biophysics, university of life sciences in lublin, poland the polyene antibiotic amphotericin b (amb) is currently the drug of choice in the treatment of fungal infections despite its undesirable side effects. according to the general conviction, the biological action of the drug is based on the formation of transmembrane channels which affect physiological ion transport, especially k + ions. this work reports the results of langmuir monolayers study of the effect of k + and na + ions on the molecular organizations of amb in the model lipid membrane. the two-component monolayers containing amb and phospholipid (dppc) have been investigated by recording surface pressure-area isotherms spread on aqueous buffers containing physiological concentration of k + and na + ions. the strength of the amb-dppc interactions and the stability of the mixed monolayers were examined on the basis of surface pressure measurements, the compressional modulus and the excess free energy of mixing. the obtained results proved a high affinity of amb towards lipids in the presence of k + than na + ions. the most stable mixed monolayers were formed with the 1:1 and 2:1 stoichiometry in the presence of k + and na + ions, respectively. this research was financed by ministry of education and science of poland within the research project n n401 015035. microcalorimetric study of antibiotic amphotericin b complexes with na + , k + and cu 2+ ions arkadiusz matwijczuk, grzegorz czernel, mariusz gagoś department of biophysics, university of life sciences in lublin, poland amphotericin b (amb) as a metabolite of streptomyces nodosus is one of the main polyene antibiotics applied in the treatment of deep-seated mycotic infections. we presented microcalorimetric (dsc) study of molecular organization of amphotericin b in lipid membranes induced by na + , k + and cu 2+ ions. the analysis of dsc curves indicates the influence of na + and k + ions on the main phase transition of pure dppc lipid. for the molar fractions of 3, 5, 10, 15 mol% amb in dppc we observed the thermal shift towards higher temperatures in respect to pure lipid, both in the presence of na + and k + ions. this result may be connected with the changes in dynamic properties of the model membrane system. in case of amb-cu 2+ complexes in aqueous solution at two ph values, 2.0 and 10.6, the dsc measurements reported endothermic heat effect. this phase transition was related to the dissociation process of amb-cu 2+ complexes. the formation of amb-cu 2+ complexes are accompanied by changes of the molecular organization of amb especially disaggregation. these all observed effects might be significant from a medical point of view. this research was financed by ministry of education and science of poland within the research project n n401 015035. membrane proteins and peptides are acting in an environment rich in other proteins or peptides. aim of our study was to understand how such molecular crowding and resulting intermolecular interactions can influence the behavior of membrane proteins, using various antimicrobial peptides and membrane proteins as examples. in the case of antimicrobial peptides we have previously described a change in their alignment in the membrane at a characteristic threshold concentration. to understand whether this change is due to unspecific crowding or specific peptidepeptide interactions, we tested if this re-alignment depends on the presence of additional peptides. in most cases we found a similar re-orientation behavior irrespective of the added peptide type, indicating unspecific crowding. when pairing pgla and magainin-2, however, we observed a distinctly different sequence of pgla re-orientation in the membrane, indicating a specific interaction between these two peptides, which correlates well with their known synergistic activity. a rather different effect of crowding was observed for the larger channel protein mscl, which was found to form clusters of functionally active proteins in the membrane. we propose that this clustering is caused by lipid-mediated protein-protein interactions. water, hydrophobic interaction, and protein stability j. raul grigera and c. gaston ferrara instituto de física de líquidos y sistemas bioló gicos (iflysib), conicet-unlp, la plata, argentina although there are several forces maintaining protein structure, it is well know that hydrophobic interaction is the dominant force of protein folding. then, we can infer that any factor that alters hydrophobic interaction will affect the protein stability. we have study by computer simulation a model system consisting in solution of lenard-jones particles in water (spc/e model) at different pressures and temperatures and analyzed the solubility i.e. the aggregation properties, of such a system. from the obtained data we are able to build up the phase surface determining the critical point. the computing results where compared with experimental data of binary mix of non polar substance in water and of protein denaturation, finding high coincidence on the critical point. since the behavior of our model system can only be due to hydrophobic effects, the coincidences with the denaturation of proteins allow us to conclude that the dominant factor that determine temperature and pressure denaturation of proteins is the hydrophobic interaction. the temperature and pressures at which the denaturation, as well the disaggregation of simple non-polar particles, starts agree with what we could expect based on the cross over line of the low to high density structure water transition. the functional reconstitution of a mitochondrial membrane protein into a lipid bilayer was studied using a quartz crystal microbalance. the 6xhis-tagged protein was immobilised via specific binding to a cu 2+ terminated sensor surface, with a change in frequency indicating approximately 75% coverage of the sensor surface by the protein. a lipid bilayer was reconstituted around the protein layer, with a final change in frequency that is consistent with the remaining area being filled by lipid. incubation with a specific ligand for the protein resulted in a significant change in frequency compared to the interaction with the surface or lipid alone. the change is greater than expected for the mass of the ligand, indicating a possible conformational change of the protein, such as the opening of a channel and increased water content of the layer. electrical impedance measurements on the same system have provided additional evidence of protein-lipid bilayer formation, and it is intended that this system will be studied with neutron reflectometry to characterise potential ligand induced channel formation. valuable functional and structural information about this membrane protein was obtained by using surface sensitive techniques to study the protein in a biomimetic lipid bilayer. visualizing and quantifying hiv-host interactions with fluorescence microscopy jelle hendrix 1, *, zeger debyser 2 , johan hofkens 3 and yves engelborghs 1 1 laboratory for biomolecular dynamics, university of leuven, belgium, 2 laboratory for molecular virology and gene therapy, university of leuven, belgium, 3 laboratory for photochemistry and spectroscopy (*present address), university of leuven, belgium protein-chromatin interactions are classically studied with in vitro assays that only provide a static picture of chromatin binding. fluorescence correlation spectroscopy (fcs) is a non-invasive technique that can be used for the same purpose. being applicable inside living cells it provides dynamic real-time information on chromatin interactions. transcriptional co-activator ledgf/p75 has well characterized protein and chromatin interacting regions. we studied ledgf/p75 in vitro and inside living cells with fcs and other techniques (luminescent proximity assay, spot/half-nucleus fluorescence recovery after photobleaching, continuous photobleaching). protein-protein interactions in living cells can be monitored with fluorescence cross-correlation spectroscopy (fccs) using fluorescent proteins as genetic labels. advantages over using fö rster resonance energy transfer (fret) are the independence from intermolecular distance and knowledge of absolute protein concentrations. we characterized fccs with fluorescent proteins in vitro and then studied the intracellular complex of ledgf/p75 and the hiv-1 integrase (in) enzyme both with fret and fccs. nucleus and its compartment nucleolus are a seat of enormous biosynthetic activity in human cancer cells. nucleolar proteins, e.g. b23 or c23, play an important role in regulation of cell division and proliferation. one of the strategies how to intermit malignant cell proliferation is affecting, e.g. by drug treatment, a net of intracellular protein interactions to bring the cell on a way of apoptosis. a cytostatic agent actinomycin d initiates apoptosis in human cancer cells, as well as in normal peripheral blood lymphocytes. at the same time, translocation of b23 and c23 into nucleoplasm is observed in the treated cells. therefore interaction between nucleolar and apoptotic proteins comes into a question. co-immunoprecipitation, fluorescence microscopy and yeast two hybrid analysis are used to answer it. in co-immunoprecipitation experiments, tumor suppressor p53 showed up to be a promising candidate for the interaction. fluorescence deposits mostly constituted by variants of transthyretin (ttr), a homotetrameric plasma protein implicated in the transport of thyroxine and retinol [1] . nowadays, the only effective therapy for ttr amyloidosis is liver transplantation. new therapeutic strategies are being developed taking advantage of our current understanding of the molecular mechanisms of amyloid formation by ttr [2] . a significant effort has been devoted to the search and rational design of compounds that might decrease ttr tetramer dissociation, for example, through ligand binding at the thyroxine binding sites of ttr [3, 4] . here, we use isothermal titration calorimetry (itc) to characterize the thermodynamic binding signature of potential ttr tetramer stabilizers, previously predicted by computerassisted methods [3] . itc allows the measurement of the magnitude of the binding affinity, but also affords the characterization of the thermodynamic binding profile of a protein-ligand interaction. high affinity/specificity ttr ligands, enthalpically and entropically optimized, may provide effective leads for the development of new and more effective drug candidates against ttr amyloidosis. we have established a set of vectors to promote easy cloning of ecfp and eyfp fusions with any protein of interest. we exploit these fluorescent fusion proteins to study protein-protein interactions by fluorescence lifetime of ecfp. the decrease of ecfp lifetime reveals fret between ecfp and eyfp and hence the interaction between proteins in question. groel-groes chaperonin complex is required for the proper folding of eschericia coli proteins. bacteriophage t4 and its distant relative coliphage rb49 encode co-chaperon proteins (respectively gp31 and coco) that can replace groes in the chaperonin complex. gp31 is also required in the folding of the major capsid protein of the phage. prd1 is a large membrane-containing bacteriophage infecting gram-negative bacteria such as e. coli and salmonella enterica. it has 15 kb long linear dsdna genome and the capsid has an icosahedral symmetry. the groel-groes chaperonin complex is needed in the assembly of prd1. we have found evidence that prd1 protein p33 can work similar way as other viral co-chaperones and substitute groes in chaperonin complex. fluorescence lifetime studies between proteins groel and p33 reveals an interaction that backs up the theory. structural modification of model membranes by fibrillar lysozyme as reaveled by fluorescence study a.p. kastorna v.n. karazin kharkiv national university, 4 svobody sq., kharkiv, 61077, ukraine recent experimental findings suggest that protein aggregation, leading to the formation and depositions of amyloids play a central role in the neurodegenerative diseases, type ii diabetes, systemic amyloidosis, etc. in the present study we focused our efforts on investigation of the influence of fibrillar lysozyme on the structural state of model lipid membranes composed of phosphatidylcholine and its mixtures with cardiolipin (10 mol %) and cholesterol (30 mol %). to achieve this purpose, two fluorescent probes with different bilayer location, 1,6-diphenyl-1,3,5-hexatriene (dph) distributing in membrane hydrocarbon core and 6-lauroyl-2-dimethylaminonaphthalene (laurdan) locating at lipid-water interface, have been employed. the changes in membrane viscosity under the influence of amyloid lysozyme were characterized by fluorescence anisotropy of dph. this fluorescence parameter was not markedly affected by fibrillar protein in all types of model membranes. the changes in emission spectra of laurdan were analysed by the generalized polarization value (gp). it was found that adding of amyloid lysozyme resulted in the increment of gp value. our data suggest that lysozyme fibrils cause reduction of bilayer polarity and increase of lipid packing density. isothermal titration calorimetry (itc) is the gold standard for the quantitative characterisation of protein-ligand and protein-protein interactions. however, reliable determination of the dissociation constant (k d ) is typically limited to the range 100 lm [ k d [ 1 nm. nevertheless, interactions characterised by a higher or lower k d can be assessed indirectly, provided that a suitable competitive ligand is available whose k d falls within the directly accessible window. unfortunately, the established competitive itc assay requires that the high-affinity ligand be soluble at high concentrations in aqueous buffer containing only minimal amounts of organic solvent. this poses serious problems when studying protein binding of small-molecule ligands taken from compound libraries dissolved in organic solvents, as is usually the case during screening or drug development. here we introduce a new itc competition assay that overcomes this limitation, thus allowing for a precise thermodynamic description of highand low-affinity protein-ligand interactions involving poorly water-soluble compounds. we discuss the theoretical background of the approach and demonstrate some practical applications using examples of both high-and low-affinity protein-ligand interactions. interaction of myoglobin with oxidized polystyrene surfaces studied using rotating particles probe m. kemper 1,2 , d. spridon 1 , l.j. van ijzendoorn 1 , m.w.j. prins 1,3 1 eindhoven university of technology, department of applied physics, eindhoven, the netherlands, 2 dutch polymer institute, eindhoven, the netherlands, 3 philips research, eindhoven, the netherlands the interaction of proteins with polymer surfaces is of profound importance for the sensitivity of biosensors. polymer surfaces are often treated in order to tune their chemical and physical properties, for example by oxidation processes. to get a better understanding of the association of proteins to treated polymer surfaces, we use the rotating particles probe (x.j.a. janssen et al., colloids and surfaces a, vol. 373, p. 88, 2011). in this novel technique, protein coated magnetic particles are in contact with a substrate and the binding is recorded for all individual particles using a rotating magnetic field. we investigate the interaction of myoglobin coated magnetic particles to spincoated polystyrene surfaces that have been oxidized with a uv/ozone treatment. the surfaces have been characterized by xps, afm and water contact angle measurements. we will demonstrate a clear influence of polystyrene oxidation on the binding fractions of the myoglobin coated particles. we interpret the results in terms of dlvo-theory: electrostatic as well as electrodynamic properties of the surfaces will be influenced by the oxidation. interact with monomeric and/or filamentous actins. twinfilin is a 37-40 kda protein composed of two adf-homologue domains connected by a short linker. in our work we studied the effects of the mouse twinfilin-1 (twf1) on the monomeric actin. we determined the affinity of twf1 to the atp-actin monomer with fluorescence anisotropy measurement (k d = 0.015 lm). the fluorescence of the actin bound e-atp was quenched with acrylamide in the presence or absence of twf1. in the presence of twinfilin the accessibility of the bound e-atp decreased, which indicates that the nucleotide binding cleft is shifted to a more closed conformational state. it was confirmed with stopped-flow experiments that the kinetics of nucleotide-exchange of actin decreased in the presence of twf1. we determined the thermodynamic properties of twf1 and investigated the effect of twinfilin on the stability of actin monomer with differential scanning calorimetry. the twf1 stabilized the stucture of the g-actin. our results can help us to understand the regulation of actin cytoskeleton in more details. magnetic np have attracted attention due to their potential of contrast enhancement of magnetic resonance imaging and targeted drug delivery, e.g. tumor magnetic hyperthermia therapy. potential nephrotoxicity of single i.v. administration of fe 3 o 4 np was studied in female wistar rats i.v. administered either placebo (10% v/v rat serum in 0.9% nacl), suspension of tio 2 np (positive control, bimodal 84/213 nm distribution), or fe 3 o 4 np (bimodal 31/122 nm distribution) in doses of 0.1, 1.0 or 10.0 mg/kg. rats were sacrificed 24h, 7-, 14-and 28-days after np injection (n=9-10/each group). administration of np did not alter kidney size significantly; renal function of np administered rats as monitored by plasma creatinine and urea concentrations, creatinine clearance and protein excretion rate did not differ significantly in either interval from rats administered placebo. one week after administration significant rise in plasma ca, its urinary and fractional excretion was observed in rats administered 10 mg fe 3 o 4 /kg. plasma mg levels rose in this group 1 and 2 weeks after administration. no significant changes in the expression of tnf-a, tgf-b, and collagen iv genes in renal cortex were revealed. no obvious nephrotoxic effects were observed in rats after a single i.v. dose of fe 3 o 4 np. study was supported by 7fp ec eu: nanotest (development of methodology for alternative testing strategies for the assessment of the toxicological profile of nanoparticles used in medical diagnostics.), grant no.: 201335. biomimetic supramolecular assemblies for studying membrane interactions in vitro and in vivo s. kolusheva, r. jelinek ben-gurion university, beer-sheva, israel we designed a novel biomimetic sensor, composed of conjugated polydiacetylene (pda) matrix embedded within lipid vesicles. the system is capable of detecting various compounds occurring within lipid membranes through rapid colorimetric as well as fluorescent transitions. the colorimetric response of the sensor is correlated to the extent of compound-membrane binding and permeation and quantified binding sensitivity to lipid composition. we describe a new disease diagnostic approach, denoted ''reactomics'', based upon reactions between blood sera and an array of vesicles comprising different lipids and polydiacetylene (pda), a chromatic polymer. we show that reactions between sera and such a lipid/pda vesicle array produce chromatic patterns which depend both upon the sera composition as well as the specific lipid constituents within the vesicles. through attachment of chromatic polydiacetylene (pda) nanopatches onto the plasma membrane, real-time visualization of surface processes in living cells is possible. the ras protein is mutated in 30% of human tumors. ras acts as a switch, transmitting a growth signal in an active gtp-bound form and turning the signal off in an inactive gdp-bound form. the switch off is accomplished by gtp hydrolysis, which is catalyzed by ras and can be further accelerated by gtpase activating proteins (gaps). mutations which prevent hydrolysis cause severe diseases including cancer. we investigate the reaction of the ras gap protein-protein complex by time-resolved ftir spectroscopy. 1 detailed information on the mechanism and the thermodynamics of the reaction was revealed: 2 first, the catalytic arginine-finger of gap has to move into the gtp binding pocket, then cleavage of gtp is fast and h 2 po 4 hydrogen-bonded in an eclipsed conformation to the b-phosphate of gdp is formed. further, we performed for the first time atr-ftir spectroscopy of ras in its native environment, a lipid membrane. 3 in this setup we are able to do difference spectroscopy of the immobilized protein. interactions with other proteins can be determined in a similar way as in spr experiments but with the additional information from the infrared spectra. galectins are a family of animal lectins that specifically bind b-galactosides and have gained much attention due to their involvement in several biologic processes such as inflammation, cell adhesion and metastasis. in such processes, several issues are still not clear including the mechanisms of interaction with different carbohydrates. galectin-4 (gal-4) is a tandem-repeat type galectin that contains two carbohydrate recognition domains (crd-i and crd-ii) connected by a linking peptide. in this study, we performed spectroscopic studies of the carbohydrate-recognition domains from human gal-4. our goals are two-fold: (1) to monitor conformational changes in each domain upon its binding to specific ligands and then to correlate the observed changes with structural differences between the crds and (2) to investigate the interaction between the crds and lipid model membranes. to achieve such objectives we used a combined approach of spectroscopic techniques involving circular dichroism and electron spin resonance. overall the results obtained so far show that crd-i and crd-ii have distinct behaviors in terms of carbohydrate recognition and membrane binding. this may be due to specific differences in their structures and certainly suggests a non-equivalent role in protein function. hemoglobin influence on lipid bilayer structure as revealed by fluorescence probe study o.k. kutsenko, g.p. gorbenko, v.m. trusova v.n. karazin kharkov national university, kharkov, ukraine hemoglobin (hb) is a red blood cell protein responsible for the oxygen transport. its affinity for lipid bilayers represents interest for gaining insight into protein biological function as well as for some applied aspects such as development of blood substitutes or biosensors. hb influence on lipid bilayer structure was investigated using fluorescent probes pyrene and prodan. model membranes were prepared of phosphatidylcholine (pc) and its mixtures with phosphatidylglycerol (pg) and cholesterol (chol). hb penetration into membrane interior is followed by the increase of relative intensity of pyrene vibronic bands and decrease of prodan general polarization value suggesting an enhancement of bilayer polarity. this implies that hb incorporation into membrane interior decreases packing density of lipid molecules, promoting water penetration into membrane core. chol condensing effect on lipid bilayer prevents protein embedment into bilayer, thus decreasing membrane hydration changes as compared to pc bilayers. in the presence of anionic lipid pg hb-induced increase of bilayer polarity was found to be most pronounced, pointing to the modulatory role of membrane composition in hb bilayer-modifying propensity. we present optimized sialic acid-based mimics binding in the low nanomolar range. molecular interactions were determined with surface plasmon resonance (spr), characterizing the affinity and the kinetics of binding. furthermore, isothermal titration calorimetry (itc) was applied to dissect the standard free energy of binding (dg°) into the standard enthalpy of binding (dh°) and the standard entropy of binding (ds°). in order to pass the cell membranes, most of these medicines has to be administrated to patients as nucleoside pro-drugs and not directly as triphosphorylated forms. because of the poor phosphorylation of the nucleoside analogues used in therapy, it is important to understand and to optimize their metabolism. our aim is to understand how compounds of chirality l turn away 3-phosphoglycerate kinase (pgk) from its normal glycolytic function to be converted into the triphosphate forms. in order to elucidate pgk mechanism and substrate specificity, we have measured the kinetics of the different steps of the enzymatic pathways by rapid mixing techniques and studied the influence of the nature of the nucleotide substrate thereon. we first performed an extensive study with d-and l-adp (see poster by p. lallemand). we are now extending the studies to other nucleotide diphosphates (some of them used in therapies). changes in the nature of the nucleobase or deletion of hydroxyl group of the sugar affect the efficiency of phosphorylation by pgk, either by decreasing dramatically their affinity or by altering the phospho-transfer step itself. structural explanations are given based on docking data. probing drug/lipid interactions at the molecular level represents an important challenge in pharmaceutical research, drug discovery and membrane biophysics. previous studies showed that enrofloxacin metalloantibiotic has potential as an antimicrobial agent candidate, since it exhibits antimicrobial effect comparable to that of free enrofloxacin but a different translocation route. these differences in uptake mechanism can be paramount in counteracting bacterial resistance. in view of lipids role in bacterial drug uptake, the interaction of these compounds with different e. coli model membranes were studied by fluorescence spectroscopy. partition coefficients determined showed that lipid/antibiotic interactions were sensitive to liposomes composition and that the metalloantibiotic had a higher partition than free enrofloxacin. these results corroborate the different mechanism of entry proposed and can be rationalized on the basis that an electrostatic interaction between the metalloantibiotic positively charged species, present at physiological ph, and the lipids negatively charged head groups clearly promotes the lipid/antimicrobial association. oligomerization and fibril assembly of amyloid b peptide amyloid b peptide (ab) forms a large amount of extracellular deposits in the brain of alzheimer's disease (ad) patients and it is believed that this peptide is related to the pathogenesis of that disease. the most abundant monomeric form of physiological ab (*90%) is constituted by 40 amino acids and is benign, but by an unknown mechanism this endogenous material becomes aggregated and neurotoxic. increasing evidence suggests that membrane interaction plays an important role in ab neurotoxicity. in this work it will be studied the interactions of ab(1-40) with ctac (cationic), sds (anionic), pfoa (anionic with fluorine atoms) and og (nonionic) amphiphiles in monomeric and micellar forms. the results demonstrated that ab(1-40) forms fibrils with different morphologies in the presence of micelles. in addition, the presence of micelles accelerates the formation of fibrils and decreases the lifetime of oligomers. we present here the exploitation of the powerful approach of surface plasmon resonance imaging and mass spectrometry coupling for protein fishing in biological fluids such as human plasma at the same sensitivity. on one hand, multiplex format spri analysis allows direct visualization and thermodynamic analysis of molecular avidity, and is advantageously used for ligand-fishing of captured bio-molecules on multiple immobilized receptors on a spri-biochip surface. on the other hand, maldi mass spectrometry is a powerful tool for identification and characterization of molecules captured on specific surface. therefore, the combination of spri and ms into one concerted procedure, using a unique dedicated surface, is of a great interest for functional and structural analysis at low femtomole level of bound molecules. to reach these goals, particular surface engineering has been engaged to maintain a high level of antibody grafting and reduce non-specific adsorption. thus, various chemistries have been tested and validated towards biological fluids such plasma, keeping in mind the capacity of the in situ investigation by ms. finally, signal to noise ratio was magnified leading to the characterization of protein lag3, a potential marker of breast cancer, in human plasma. atenolol incorporation into pnipa nanoparticles investigated by isothermal titration calorimetry mihaela mic, ioan turcu, izabell craciunescu, rodica turcu, national institute for r&d of isotopic and molecular technologies, 400293 cluj-napoca, romania e-mail: mihaela.mic@itim-cj.ro poly(n-isopropylacrylamide) (pnipa) is a thermo-sensitive hydrogel undergoing a volume phase transition at about of 32°c close to the body temperature. this volume phase transition is envisaged as a key property for drug binding and release. the purpose of our research is the thermodynamic characterization of the binding of atenolol by pnipa polymeric nanoparticles. the thermodynamic parameters which characterize the binding process are obtained using the isothermal titration calorimetry (itc) as the main investigation technique. when polymeric nanoparticles bind drug molecules, heat is either generated or absorbed depending on the amount of bond molecules and also on the exothermic or endothermic character of the binding process. the heat measurement allows the determination of binding constants, reaction stoichiometry and the thermodynamic profile of the interaction. itc technique has been used to investigate the binding properties of nanoparticles which shrink from the swollen to the collapsed state. the capacity of such nanogels to bind atenolol molecules is directly related to relevant differences between the binding properties in the swollen and in the collapsed state respectively. aggregation study of x-(alkyldimethylamonium)alkylaldonamide bromides p. misiak 1 , b. ró _ zycka-roszak 1 , e. woźniak 1 , r. skrzela 1 , k.a. wilk 2 1 department physics and biophysics, wrocław university of environmental and life sciences, wrocław, poland, 2 department of chemistry, wrocław university of technology, wrocław, poland sugar-based surfactants are of considerable research interest because they have improved surface and performance properties, reduced environmental impact, and have potential pharmaceutical and biomedical applications. x-(alkyldimethylamonium)alkylaldonamide bromides (c n gab) with different chain lenghs (n = 10, 12, 14, 16) belonging to cationic sugar-based surfactants were newly synthesised. the aggregation processes of c n gabs were studied by means of isothermal titration calorimetry (itc), electric conductance method and molecular modelling methods. the critical micelle concentrations (cmc), the degree of micelle ionization (b), the enthalpies (dh m ) and the entropies (ds m ) of micellization as well as the contributions of the headgroups to the gibbs free energies (dg m 0 (hy)) were calculated. the obtained values were compared with those for dodecyldimethylethylammonium bromide and literature data for analogical glucocationic surfactants. the latest compounds differ from c n gab surfactants by substitution of sugar chain by gluco ring. molecular modelling methods were used to relate the molecular properties of the compounds with their experimentally studied properties in solution. this work was supported by grant n n305 361739. every year over 50 million people are infected with dengue virus (denv), transmitted by a mosquito (aedes aegypti). this enveloped virus, member of the flaviviridae family, has four distinct serotypes. it has a single stranded positive rna molecule with a single open reading frame that encodes a single poliprotein, which, after appropriate processing by viral and host proteases, gives rise to three structural proteins (c, prm and e) and seven non-structural proteins (ns1, ns2a, ns2b, ns3, ns4a, ns4b and ns5) [1] . the surface of the immature virion is composed of e and prm heterodimers that are arranged as trimers protruding from the membrane [2] . the virus is thought to enter the host cell via a receptor-mediated endocytosis, although, if any, the specific dengue receptors have not been described. once inside the cell, the acidified environment inside the endocytic vesicle triggers an irreversible trimerization of the envelope (e) protein, inducing the release of the nucleocapsid (composed of viral rna and multiple copies of c protein) to the cytoplasm, thus starting the infection process, where the poliprotein is translated and processed, originating all viral proteins. considering the structural proteins c and e, these are essential for the viral infection, specifically, protein c is thought to be involved in the viral assembly and specific encapsidation of the genome and protein e (a class ii fusion protein) plays a major role in the fusion process. as recently described by some studies [3] , protein c is composed of four a helices connected by four short loops and has a highly hydrophobic region forming a concave groove that could interact with lipid membranes and a region with an increased concentration of positive charges, possibly interacting with the viral rna. as for protein e, it is composed of three b stranded domains. it is proposed that the fusion loop is located in domain ii of this protein and the putative receptor binding sites, considered essential for the viral entry, are supposedly located in domain iii. in this work, we describe the identification of the membrane active regions of both these proteins, considering both theoretical studies, hydrophobic moments, hydrophobicity and interfaciality values as well as experimental ones, namely fluorescence spectroscopy, where a fluorescent probe is encapsulated in model membrane systems, and differential scanning calorimetry [4] . we have found one region in protein c and four regions in protein e with membranotropic activity. this is the first work describing experimentally the putative membrane interacting zones of both these proteins. this work was funded by grant bfu2008-02617-bm from ministerio de ciencia y tecnologia, spain, granted to jose villalaín. investigation of membrane-membrane interaction mediated by coiled coil lipopeptides gesa pä hler, andreas janshoff georg-august-university, tammannstrasse 6, 37077 gö ttingen, germany e-mail: gpaehle@gwdg.de specific cellular membrane interaction and fusion are crucial points in vivo which are in eukaryotic cells mediated by snare proteins. the definite mechanism behind these processes is still poorly understood, but the coiled coil formation of a snare core complex consisting of four a-helices seems to generate a fusogenic driving force. this offers the possibility to design a straightforward experimental setup to mimic the complex protein-mediated membrane-membrane interaction by using mere protein fragments or peptides attached to artificial lipid bilayers which self-assemble to a coiled coil structure. in our approach, two artificial three heptad repeat coiled coil forming peptides were synthesized and attached to maleimide functionalized membranes via an in situ-coupling reaction. thus, secondary structure changes, kinetic characteristics and binding energetics were monitored during coiled coil formation with real time ellipsometry, ir and cd spectroscopy. the lipopeptide mediated membrane-membrane interaction itself is investigated by colloidal probe spectroscopy and tirfm. these techniques and the setup of our model system allow screening the energetic and structural properties of variable coiled coil forming peptides, i.e. linker-modified or biologically inspired sequences. enzymatic reactions in nanostructured surfaces: unzipping and cutting the double helix pietro parisse 1 , matteo castronovo 2 , bojana lucic 3 , alessandro vindigni 3 , giacinto scoles 2 and loredana casalis 1 1 sincrotrone trieste s.c.p.a., trieste, italy, 2 temple university, philadelphia, usa, 3 protein-dna interactions are vital for living organisms. from viruses to plants and humans, the interactions of these two different classes of biopolymers control processes as important and diverse as the expression of genes and the replication, rearrangement, and repair of dna itself. to understand these processes at the molecular level, and to follow changes in cellular pathways due to different kinds of perturbations and/or diseases it is necessary the identification and quantification of proteins and their complex network of interactions. we have exploited the high spatial resolution given by atomic force microscopy to generate dna arrays of variable density by means of nanografting. on such nanostructures, we investigate the mechanism of different enzymatic reactions (from restriction enzymes to helicases). registering with high precision the height variation due to the action of the enzyme onto the engineered dna sequences (in the case of restriction enzymes) or taking advantage of the different mechanical properties of single and double stranded dna (in the case of helicases, where for the first time kinetic data were obtained on recq1 human helicase), we were able to monitor either the activity and/or the action mechanisms of these two important classes of enzymes. in this study an attempt has been made to investigate the structure, dynamics and stability of cyclic peptide nanotubes (cpnts) formed by the self-assembly of cyclic peptides (cps) using classical molecular dynamics (md) simulation and semiempirical quantum chemistry calculation employing pm6. the structure and energetics of monomer and various oligomeric cpnts have been investigated by considering the (cyclo-[(d-ala-l-ala) 4 ]) peptide as the model for cp. various geometrical parameters extracted from the md simulation reveal that the terminal residues are loosely hydrogen bonded to the inner subunits regardless of degree of oligomerization. the hydrogen bonds present in the inner core regions are stronger than the terminal residues. as the degree of oligomerization increases, the stability of the tube increases due to the hydrogen bonding and stacking interactions between the subunits. the results show that the binding free energy increases with the extent of oligomerization and reaches saturation beyond cpnt5. in addition, hydrophobic and electrostatic interactions play crucial roles in the formation of cpnts. analysis of both structural and energetics of formation of cpnts unveils that the selfassembly of dimer, trimer and tetramer cpnts are the essential steps in the growth of cnpts. monolayers on a langmuir trough constitute a great biomimetic model to characterize protein-protein or protein-lipid interaction, where the physical state of the interfacial layer is completely controlled. we present here three studies performed on monolayers, with a wide panel of experimental (optical, spectroscopical, rheological) techniques. i) surface properties and conformation of nephila clavipes spider recombinant silk proteins (maspi and masp2) was studied at the air-water interface: we show that the mechanism of assembly of both proteins is different, although both proteins share the same sequence pattern and a close hydrophobicity. they both exhibit a certain propensity to form b-sheets that may be important for the efficiency of the natural spinning process. ii) the dystrophin molecular organization and its anchoring in a lipidic environment depend on the rod fragment used and on the lipid nature. moreover the interaction is guided by the lateral surface pressure. this lipid packing variation is essential to understand the role of the dystrophin during compression-extension cycle of the muscle membrane. iii) we evidence that non additive behavior of mixtures of food globular proteins leads to enhanced foaming properties or to self assembled objects. nucleolar-targeting peptides (nrtps) were designed by structural dissection of crotamine, a toxin from the venom of a south-american rattlesnake. at lm concentration, nrtps penetrate different cell types and exhibit exquisite nucleolar localization. the aim of this work was to pursue with the study of nrtps molecular mechanism for translocation, as well as to determine the ability of nrtp to delivery large molecules into cells. for the translocation experiments, rhodamine b-labeled nrtps were used and tested with giant multilamellar vesicles. confocal microscopy results show that there is an efficient translocation across model membranes. high levels of intracellular peptide were also seen in different cell lines and pbmc, soon after incubation with nrtp. furthermore, a conjugate of nrtp (nrtp6c) bound to b-galactosidase was prepared by chemical synthesis and tested in hela cells. this conjugate maintains enzymatic activity and is stable at 4°c for several days. the work done so far with this new family of cell-penetrating peptides revealed strong interaction and translocation with lipid model systems. moreover, successfully cellular delivery of bgalactosidase was observed and quantified. interaction of zinc phthalocyanine with ionic and non ionic surfactants: uv-vis absorption and fluorescence spectroscopy for application in photodynamic therapy m. p. romero, s. r. w. louro physic department, pontifícia universidade cató lica de rio de janeiro puc-rio, brazil among the second-generation photosensitizer (ps) developed for the treatment of neoplastic diseases by photodynamic therapy (pdt), metallo-phthalocyanines (mpc) have been proposed as an alternative to the currently used ps in clinical application. unsubstituted mpc are not soluble in physiological solvents and their in vivo administration relies upon their incorporation into carriers or their chemical conversion into water-soluble dyes by the attachment of selected substituents. in this work, uv-vis absorption and fluorescence spectroscopy were used to study the ability of different micelles for dispersing zinc phthalocyanine (znpc). the following surfactants were tested: sds, ctab, hps, tween 80, tween 20, and pluronic f127. znpc has low solubility in virtually all solvents, but dmf and dmso are observed to dissolve znpc in concentrations of the order of 0.9 and 0.2 mmol/l, respectively. stock solutions of znpc in dmf and dmso were prepared. micelles of the different surfactants containing znpc were prepared by dissolving in aqueous medium (milli-q water or phosphate buffer ph 7.4) small amounts of the stock solutions previously mixed with each surfactant. the amounts of each surfactant were calculated to give an average ratio of one znpc molecule per micelle in the final solution. the absorption and fluorescence spectra of znpc in the micellar systems were obtained, and were observed to change in time. immediately after dissolution the spectra are characteristic of monomeric znpc, suggesting formation znpccontaining nanoemulsions with the mixture of znpc-organic solvent in the hydrophobic region of the micelle. since dmso and dmf are miscible with water, the solvent diffuses out of the micelle and znpc stays inside the micelle in a monomeric or aggregated form. the different surfactants lead to different time evolution of znpc aggregation. aggregation lifetimes vary from one hour, in the case of pluronic f127, to more than twelve hours, in the case of ctab and hps. it was observed that the ionic surfactants were more efficient than non ionic ones for monomeric delivery of znpc . work partially supported by cnpq, inami and faperj. nucleobase-containing peptides are an important class of molecules comprising both artificial (synthetic nucleopeptides) and natural (peptidyl nucleosides and willardiine-containing peptides) compounds characterized in many cases by interesting biological properties. 1, 2 in this work, we report a spectroscopic study on the properties of a chiral nucleobase-bearing peptide obtained by chemical synthesis starting from commercial sources. the findings of this research strongly encourage further efforts in the field of the use of nucleopeptides as supramolecular assembling systems and open the way to novel drug delivery approaches based on nucleobase recognition. conformational plasticity. their structure depends tremendously on their local environment and confinment, and may accommodate several unrelated conformations, that are a strong challenge for the traditional characterizations of structure, supramolecular assembly and biorecognition phenomena. atomic force microscopy (afm) has been successfully exploited for both highly controllable nanolithography of biomolecules and for biorecognition studies, such as oriented prion protein -antibody interaction (sanavio et al., acs nano (2010) 4(11): 6607, bano et al. nano lett (2009) 9(7): 2614-8). here, we report different strategies for selective, oriented confinement of alphasynuclein at the nanoscale for sensitive and accurate direct detection, via precise topographic measurements on ultraflat surfaces, of biomolecular interactions in confined assemblies. a new class of cell penetrating peptides (cpps) was generated by splicing the (1-9) and (38-42) segments of crotamine, a toxin from crotalus durissus terrificus venom [1] . as they localize preferably on the nucleolus, these novel cpps were named nucleolar-targeting peptides (nrtps). the extent of nrtp partition to zwitterionic (popc; popc:cholesterol 67:33) and anionic (popg; popc:popg 70:30) lipid vesicles was studied following the intrinsic tyr or trp fluorescence of the peptides. the partition curves into popc zwitterionic vesicles were characterized by downward slopes and higher partition coefficients (k p *10 4 -10 5 ). for pure popg, an upward curve and smaller partition coefficient point out for a different type of membrane-peptide interaction. popc:popg membranes present characteristics of both types of interaction. from red edge excitation shift and quenching experiments similar conclusions were attained. leakage assays ruled out lipid vesicle disruption by crotamine or nrtps. further studies on nrtp cellular translocation mechanism and large molecule delivery are currently in progress. dystrophin is essential to skeletal muscle function and confers resistance to the sarcolemma by interacting with cytoskeleton and membrane. we characterized the behaviour of dys r11-15, a five spectrin-like repeats from the central domain of human dystrophin, in the presence of liposomes and monolayers as membrane models. interaction of dys r11-15 depends on the lipid nature, anionic or zwitterionic, with suvs, and on the lipid packing when comparing luvs to suvs. lateral pressure of lipid monolayers modifies the protein organization and leads dys r11-15 to form a regular network as revealed by afm. trypsin proteolysis assays show that the protein conformation is modified following its binding to monolayer and suvs. label free quantification by nano-lc/ms-ms allowed identifying the helical amino acid sequences in repeats 12 and 13 that are involved in the interaction with anionic suvs. results indicate that dys r11-15 constitutes a part of dystrophin that interacts with anionic as well as zwitterionic lipids and adapts its interaction and organization depending on lipid-packing and lipid nature. we provide here strong experimental evidence for a physiological role of the central domain of dystrophin on sarcolemma scaffolding through modulation of lipid-protein interactions. extracellular matrix proteins. overexpression of the mmps has been associated with a variety of diseases ranging from periodontal disease and arthritis to tumor invasion and metastasis. the majority of the more powerful synthetic inhibitors of mmps incorporate a hydroxamate group, but exhibit low selectivity and are toxic. in a recent modeling study, astaxanthin (ast), a carotenoid with potent antioxidant property, has been shown to be a potential inhibitor of mmp-13 function by occupying a binding site near the active center of the enzyme (bika 0 di et al. 2006). in our ongoing project, we investigate the binding of ast to the catalytic domain of mmps using biochemical and ultimately crystallization to validate the proposed action of ast. along these lines, the catalytic domain of mmp-13 (cdmmp-13) was expressed in e.coli bl21(de3) codon-plus and refolded using a novel effective refolding method. our results reveal that ast has a potent inhibitory effect on cdmmp-13 activity, however, determination of ic 50% or k i is difficult due to fast oxidation and structural instability of ast. ongoing work aims at optimizing the inhibition conditions and improving the refolding yield to allow analyzing structure and function of the ast-bound mmp-13 in more detail. hyaluronic acid (hyaluronan, ha) is a linear polysaccharide with a molar mass in the range of 10 5 to 10 7 da and is built from alternating units of glucuronic acid and n-acetylglucosamine. synthesized in the cellular plasma membrane, it is a network-forming and space-filling component in the extracellular matrix of animal tissues. here, we create hyaluronic acid films atop a porous alumina substrate, where they act as a barrier for macromolecular transport depending on their length and geometry. the geometry of the hyaluronic acid switches between a fully stretched and a mushroomlike state and is dependent on the concentration of hyaluronic acid. to bind hyaluronic acid selectively atop the nanoporous anodic aluminum oxide (aao), the aao is orthogonally functionalized by silane chemistry. by means of time resolved optical waveguide spectroscopy (ows) the transport of macromolecules, e.g. avidin, across the hyaluronic acid barriers can be recorded as a function of the pore diameter and hyaluronic acid concentration in a time resolved and label free manner. confocal laser scanning microscopy (clsm) provides an alternative method to investigate the orthogonal functionalization of the pores and to elucidate whether a molecule can cross the barrier at the pore entrance. we functionalized gold surfaces with a hydroxy-terminated self-assembled thiol monolayer exposing an adjustable fraction of biotin moieties. [1] by in-situ acetylation or fluorination, surface properties could be fine-tuned to different protein immobilization scenarios. using streptavidin as a linker protein, immobilization of human abcc3 [2] in liposomal and planar bilayer systems was possible. abcc3-containing proteoliposomes doped with a biotinylated anchor lipid were successfully tethered to our streptavidin-coated surfaces. biotinylation of the extracellular glycosylation of abcc3 allowed direct immobilization with inside-up orientation and subsequent assembly of a lipid bilayer. outside-up orientation was achieved by exploiting the c-terminal histidin tag of recombinant abcc3 for immobilization via ni 2+ and biotinnitrilotriacetate. all systems were thoroughly characterized by quartz crystal microbalance, atomic force microscopy and surface plasmon resonance techniques with respect to monitoring abcc3mediated substrate transport in real time. because of its role in the apoptotic pathway, conformational transitions of cytochrome c (cyt c) have gain on interest. in native cyt c, met 80 and his 18 residues serve as heme axial ligands. cyt c interaction with the membrane causes the disruption of the iron-met80 bond. this allows the binding of others endogenous ligands forming alternative low spin species 1,2 or induces peroxidase activity through the formation of a five coordinated high spin iron specie. acquisition of this peroxydase activity by cyt c has been shown to be a key stage before its release from the mitochondria 3 . in order to study these non native low spin species by checking the possible amino acids able to bind the human cyt c heme, different mutants have been designed and produced: h26q, h33n, and the double one h26q/h33n. sds in countries where seafood is an integrate part of the diet, fish are among the most common food allergen sources. the major fish allergen parvalbumins are abundant in the white muscle of many fish species. parvalbumin belongs to the family of ef-hand proteins and has a globular shape containing six helical parts. high pressure is known to unfold proteins. we performed high pressure ftir experiments, to explore the p-t phase diagram of cod parvalbumin, gad m 1, to test the possibility of its inactivation by high pressure treatment. the infrared spectrum of parvalbumin is characteristic for the helical conformation, in agreement with the crystal structure. a marked transition in the structure of the parvalbumin was observed with the central point of 0.5 gpa (at room temperature). the amide i position shifts to a wavenumber which is between the helical and the unfolded position. we assign this change to a native-molten globule transition. it was reversible as seen from the infrared spectra. it has been proven in the past that reflectometric interference spectroscopy (rifs) is a powerful measurement system for the detection of protein-protein interactions 1 . we present here the development of a reflectometric sensor which allows for the detection of membrane-protein interactions in the micromolar regime. in this study we employ two different instrumental assemblies. the first installation enables direct detection and quantification of the interaction of membrane proteins with solid supported lipid bilayers. in the second assembly the original instrument is combined with an upright fluorescence microscope. the advantages of this installation are the direct optical control of the experiment as well as a smaller sensing area. the set-up allows for the detection of interactions on lipid-patches of just several micrometers in diameter. the aim of this work is an experimental system that enables the measurement of transport processes through lipid membranes. we attempt to achieve this by covering a closed porous substrate with a lipid membrane. the first steps to reach this goal were done by spanning membranes over anodized aluminum oxide substrates. initiation of actin polimerization in cells requires nucleation factors. a pointed-end-binding protein of f-actin -the lei-omodin2-acts as a strong filament nucleator in muscle cells. the dynamical, structural and kinetic properties of a protein can provide important information to understand the intramolecular events underlying its function. we are interested in how does the leiomodin2 regulate the actin polimerization. our aim is to determine the dissociation constant of the actin-leiomodin2 complex, and study a possible side-binding effect of the leiomodin2. the cardiac leiomodin2 of rattus norvegicus is a 50 kda molecular weight protein, which contains a 17 kda n-terminal, a 18 kda leucin reach repeat (lrr) and a 15 kda c-terminal region. the n-term and the lrr regions are together tropomodulin homologues. we expressed the wild type the n-term+lrr the lrr+c-term and the c-term protein fragments by using a ptyb1 vector that contains an amplicillin resistance gene. the expression of the proteins was carried out with the twin-cn (ne bio-labs) kit, which is a chitin-intein self-cleavage and purification system. the nucleation activity of leiomodin and the polimerization speed of actin in the presence of tropomyosin and leiomodin were studied by using pyrene-actin polimerization assay. we measured the stoichiometric, conformational and kinetic properties of the leiomodin-actin complexes with co-sedimentation assay, fluorescence spectroscopic and rapid-kinetic methods. the results showed that the rate of actin polimerization depended on the leiomodin2 concentration. the nucleator activity of leiomo-din2 was ionic strength dependent. the data also confirmed that leiomodin2 is a side-binding and pointed-end binding protein of f-actin. the binding of leiomodin2 to the sides of the actin filaments was slower than to the pointed-end of the f-actin. the structure of f-actin was changed by the sidebound leiomodin2. these observations will contribute to the better understanding of the development and function of thin filaments in cardiac and other muscle tissues. leukemias are one of the most common malignancy worldwide. there is a substantial need for new chemotherapeutic drugs effective against these diseases. doxorubicin (dox), used for treatment of leukemias and solid tumors is poorly efficacious when administered systemically at conventional doses. therefore, in our study, to overcome these limitations, we used transferrin (trf) as a drug carrier. we compared the effect of dox and doxorubicin-transferrin conjugate (dox-trf) on human leukemic lymphoblasts (ccrf-cem). the in vitro growth-inhibition test, xtt assay, indicated that dox-trf was more cytotoxic for leukemia cells than dox alone. in our researches we also evaluated the alternations of mitochondrial transmembrane potential (dw m) , and production of reactive oxygen species (ros). we monitored the dw m using dye jc-1 (5,5',6,6'-tetrachloro-1,1',3,3'-tetraethylbenzimidazolcarbocyanine) . the level of ros was studied using the fluorescent probe dcfh 2 -da (2', 7'dichlorodihydrofluorescein diacetate). the results demonstrate that dox-trf induced, decrease of mitochondrial membrane potential and significantly higher production of ros compared with dox treated cells. moreover, all these results seem to be correlated with dna fragmentation analyzed by dna ladder. the tested processes were partially inhibited by the antioxidant, n -acetylocysteine (nac). the changes induced by dox-trf conjugate and free drug were suggest the different mechanism of action of dox alone and conjugated with transferrin. time-resolved detection of protein-protein interaction masahide terazima kyoto university, kyoto, 606-8502, japan revealing molecular mechanism of a protein reaction has been a central issue in biophysics. for that purpose, a variety of time-resolved spectroscopic methods have been developed. however, most of them can monitor only dynamics associated with an optical transition and it has been very difficult to trace processes without optical transition. we used the pulsed laser induced transient grating (tg) method to study spectrally silent reactions of various proteins in time-domain. here we will show studies on pixd. pixd is a 17 kda short protein which consists of the bluf domain and additional short helices, and is involved in phototactic movement. the photochemical reaction studied by absorption spectroscopy revealed that this protein exhibits typical photochemistry of the bluf proteins. the red-shifted intermediate is generated within a 100 ps. the spectrum does not change after this initial reaction, and returns back to the dark state with a time constant of 12 s at room temperature. we studied the reaction of this protein by our method and found that the proteinprotein interaction is drastically changed during the reaction. the details and the biological meaning will be presented. human ileal bile acid-binding protein (i-babp) plays a key role in the enterohepatic circulation of bile salts. previously we have shown that the protein has two binding sites and triand dihydroxy bile salts bind with strong and moderate positive cooperativity, respectively. positive cooperativity is thought to be related to a slow conformational change in the protein. our current study is directed at the structural and dynamic aspects of molecular recognition in human i-babp using nmr spectroscopy and other biophysical techniques. as a first step in the investigation, 15 n relaxation nmr experiments have been employed to characterize the backbone motion in the apo and holo protein on a wide range of timescales. our results show a moderately decreased ps-ns flexibility in the ligated protein, with most significant ordering near the portal region. in addition, the measurements indicate a slow ls-ms fluctuation at four distinct segments in the apo protein, a motion not observed in the doubly-ligated form at room temperature. our studies support the hypothesis of an allosteric mechanism of binding cooperativity in human i-babp. to shed more light on the molecular details of the binding mechanism, a site-directed mutagenesis study is in progress. cationic porphyrin-peptide conjugates were recently shown to enhance the delivery of peptide moiety to the close vicinity of nucleic acids but their interaction with dna is not yet studied. we synthesized two cationic porphyrin-peptide conjugates: tetra-peptides were linked to the tri-cationic meso-tri(4-n-methylpyridyl)-mono-(4-carboxyphenyl)porphyrin and bi-cationic meso-5,10-bis(4-n-methylpyridyl)-15,20di-(4-carboxyphenyl)porphyrin. dna binding of porphyrins, and their peptide conjugates was investigated with comprehensive spectroscopic methods. evidences provided by the decomposition of absorption spectra, fluorescence decay components, fluorescence energy transfer and cd signals reveal that peptide conjugates of di-and tricationic porphyrins bind to dna by two distinct binding modes which can be identified as intercalation and external binding. the peptide moiety does not oppose the interaction between the dna and cationic porphyrins. we compared the effect of complexation on structural stability of dna and nucleoprotein complex : hela nucleosomes and t7 phage. uv and cd melting studies revealed that the porphyrin binding increases the melting temperature of dna and destabilizes the dna protein interaction in the nucleosomes but not in the t7 phage. the wide nucleotide specificity of 3-phosphoglycerate kinase (pgk) allows its contribution to the effective phosphorylation (activation) of nucleotide-based pro-drugs against hiv. here the structural basis of the nucleotide-pgk interaction is characterised in comparison to other kinases, namely pyruvate kinase (pk) and creatine kinase (ck) by enzyme kinetic and structural modelling studies. the results evidenced favouring the purine vs. pyrimidine base containing nucleotides for pgk rather than for pk or ck. this is due to the exceptional ability of pgk in forming the hydrophobic contacts of the nucleotide rings that assures the appropriate positioning of the connected phosphate chain for catalysis. the unnatural l-configurations of the nucleotides (both purine and pyrimidine) are better accepted by pgk than either by pk or ck. further, for the l-forms the absence of the ribose oh-groups with pgk is better tolerated for the nucleotides with purine rather than pyrimidine base. on the other hand, positioning the phosphate chain of both purines and pyrimidines with l-configuration is even more important for pgk, as deduced from the kinetic studies with various nucleotide-site mutants. these characteristics of the kinase-nucleotide interactions can provide a guideline in drug-design. the role of the enzyme types atp-ases in the muscle contraction g. vincze-tiszay 1 , j. vincze 1 , e. balogh 2 1 hheif, budapest, hungary, 2 nové zá mky, slovakia the myofibrilla assuring muscle contraction gains energy to the slipping in mechanisms and the degree of efficiency of this process will decisively be determined by the velocity of recombination of the atp molecule. in this there play a particular part the na + -k + -atp-ase and mg ++ -atp-ase enzymes. chemical reactions taking place in the living organism are catalyzed by enzymes, so the recombination from adp to atp, too. this transport process can be modelled from the energetic point of view on the basis of the general transport theorem through the following formula: grad a x dx dt: from the point of view of muscle contraction it is of interest that, dependent from the type of the motions whether the length of time is very short, some seconds, or we can speak about a long lasting process. in the first case one can compare the decomposition of the atp with the avalanche effect while in the spot. its degree of efficiency is determined by the migration and linkage velocity of the ions. conclusion: the degree of efficiency of the muscle contraction is determined by the quantities of the two enzymes (na + -k + -atp-ase and mg ++ -atp-ase) as related to each other. [1] . experiments with deletion mutants have shown that the aminoterminal domain contains a beta sheet with an ordered array of acidic residues, which mediates the attachment to basic calcium phosphates [2, 3] . the inhibition is based on the formation of nanometer-sized, spherical mineral-fetuin-a colloids, denoted as calciprotein particles (cpps) [2, 4] . the initially formed cpps show hydrodynamic radii in the range of 50 nm and are only transiently stable. after a distinct lag time, they are subject to a morphological change towards larger prolate ellipsoids with hydrodynamic radii of 100-120 nm [5] . in this context, we studied the role of fetuin-a in the formation and ripening of cpps. on the one hand, dynamic light scattering (dls) was used to study the influence of temperature, fetuin-a concentration and mineral ion supersaturation on the kinetics of cpp ripening [6] . on the other hand, the protein fetuin-a was investigated by means of small angle x-ray scattering (saxs) and fluorescence correlation spectroscopy (fcs degradation of the mrna cap (mrna 5' end) by dcps (decapping scavenger) enzyme is an important process of the gene expression regulation, but little is known about its mechanism. the biological role of dcps and its potential therapeutic applications, e.g. as a novel therapeutic target for spinal muscular atrophy, make it an interesting object for biophysical investigations. the ability of dcps to act on various short capped mrnas will be presented. we have examined the substrate specificity and binding affinity of the enzyme in a quantitative manner, employing experimental physics' resources, such as atomic force microscopy (afm) and fluorescence spectroscopy for enzyme kinetics and timesynchronized-titration method (tst 2007) . in this study we extended the application of mqd-ihc to investigate potential biomarkers associated with prostate cancer (pca) invasiveness and lethal progression. objectives: to establish a mqd-ihc protocol using qd light-emitting nanoparticles 1) to detect the expression/activation of critical cell signaling proteins at the single cell level; 2) to image the plasticity and lethal progression of human pca with specific emphasis on emt and c-met signaling; and 3) to examine the utility of mqd-ihc in clinical pca specimens to determine its invasion ability and predict its metastatic capability. results: we analyzed the co-expression and activation of osteomimicry associated biomarkers: b2-microlgobulin (b2-m), phosphorylated cyclic amp responsive element binding protein (pcreb) and androgen receptor (ar) in 2,100 cells from 14 localized pca tissue areas (gleason 3 and 4) of 10 patients with known metastatic status. the overall median % triple positive for b2-m + /pcreb + /ar + cells was 51.5%. the median triple positive for the samples with metastatic potential was 61% compared with those without metastatic potential (median = 0%); p = 0.01 by a wilcoxon rank sum test. the results were confirmed in 11 pca bone metastatic specimens. we also investigated the c-met signaling in castration-resistant human pca model or crpc xenografts and the clinical pca specimens and found that the downstream signal components including pakt and mcl-1 were activated. conclusion: to validate our findings, additional clinical specimens with confirmed survival data will be analyzed and the cell-signaling-network-based mqd-ihc will be automated by vectra image analysis system in a high throughput manner with the hope to predict the lethal progression of pca prior to clinical manifestation of distant metastases. protein ligand binding is an important field of biopharmaceutical research. there are many techniques for quantitative determination of the ligand binding. the combination of isothermal titration calorimetry (itc) and thermal shift assay provides a robust estimate of the binding constant. many binding reactions are coupled to the absorption or release of protons by the protein or the ligand, conformational changes of the protein and other processes. to correlate the structural features of binding with the energetics of binding one needs to carry out a detailed thermodynamic study of the binding reaction and to determine dependencies such as ph, buffer and temperature. here we present a detailed thermodynamic description of radicicol binding to human heat shock protein hsp90 and determined proton linkage contributions to observed binding thermodynamics. we calculated the pk a of the group responsible for proton linkage, the protonation enthalpy of this group and intrinsic thermodynamic parameters for radicicol binding. the intrinsic enthalpy of radicicol binding to hsp90 is one of the largest enthalpies observed for any protein -ligand binding. the structural features responsible for such large binding enthalpy and very favorable intrinsic binding gibbs free energy are discussed. neuronal systems and modelling o-111 optogenetic electrophysiology walther akemann, amelie perron, hiroki mutoh, and thomas knö pfel laboratory for neuronal circuit dynamics, riken brain science institute, japan the combination of optical imaging methods with targeted expression of protein-based fluorescent probes enables the functional analysis of selected cell populations within intact neuronal circuitries. we previously demonstrated optogenetic monitoring of electrical activity in isolated cells, brain slices and living animals using voltage-sensitive fluorescent proteins (vsfps) generated by fusing fluorescent proteins with a membrane-integrated voltage sensor domain. however, several properties of these voltage reporters remained suboptimal, limiting the spatiotemporal resolution of vsfpbased voltage imaging. a major limitation of vsfps had been a reduced signal-to-noise ratio arising from intracellular aggregation and poor membrane targeting upon long-term expression in vivo. to address this limitation, we generated a series of enhanced genetically-encoded sensors for membrane voltage (named vsfp-butterflies) based on a novel molecular design that combines the advantageous features of vsfp2s and vsfp3s with molecular trafficking strategies. the new sensors exhibit faster response kinetics at subthreshold membrane potentials and enhanced localization to neuronal plasma membranes after long-term expression in vivo, enabling the optical recording of action potentials from individual neurons in single sweeps. vsfp-butterflies provide optical readouts of population activity such as sensoryevoked responses and neocortical slow-wave oscillations with signal amplitudes exceeding 1% dr/r 0 in anesthetized mice. vsfp-butterflies will empower optogenetic electrophysiology by enabling new type of experiments bridging cellular and systems neuroscience and illuminating the function of neural circuits across multiple scales. opsin molecules are a burgeoning new tool for temporally precise neuronal stimulation or inhibition. opsin properties are commonly characterized in cell culture or acute brain slice preparations using whole cell patch clamp techniques, where neuronal membrane voltage is fixed at the resting potential. however, in vivo, where neurons are firing action potentials, opsins are exposed to large fluctuations in membrane voltage and transmembrane ionic concentrations which can influence opsin function. in the case of implanted light delivery devices, stimulation light power varies as a function of brain tissue volume. we therefore investigated the stability of opsin properties across a variety of in vivo-like stimulation conditions. we find that off-kinetics of excitatory opsins vary significantly with holding membrane potential; channelrhodopsin (chr2) slowing with depolarisation and chief (chr1/chr2 hybrid) in contrast, accelerating. new chr2 point mutation variants demonstrate stability across all membrane potentials. we additionally explore responses to initial and subsequent light pulses and find that chief has the unique property of accelerating kinetics after the first light stimulation. inhibitory opsins vary in sensitivity to light in a manner which correlates with their off-kinetics. slower opsins, such as mac (leptosphaeria maculans), have higher sensitivity at low light power densities, saturating early relative to fast inhibitory opsins such as arch (archaeorhodopsin) and nphr (halorhodopsin). we discuss the relative merits of stability versus versatility of opsins under variable stimulation conditions. it has been previously shown that overexpression of ndm29 ncrna in a sknbe2-derived neuroblastoma (nb) cell line leads to cell differentiation, with a decrease of malignant potential. here we use the patch-recording technique to characterize the ionic channel apparatus of nb cells expressing ndm29 at its basal level (mock cells) or at 5.4 fold higher levels (s1 cells). the two cell lines shared very similar pools of functional k channels, but s1 cells displayed larger ttx-sensitive na currents and were able to generate action potentials, while mock cells were not. in addition, while mock cells barely express functional gaba receptors, in the majority of s1 rapid application of gaba elicited a current with a ec 50 =11.4 lm; this current was antagonized by bicuculline (10 lm) and potentiated by zaleplon (ec 50 = 35 nm). in mock cells, real time pcr evidenced a high level of gaba a a 3 subunit, while in s1 cells a significant expression of a 1 and a 4 was detected, whereas a 3 mrna was downregulated by 70%, confirming the development of functional gaba a receptors. in the same cell lines, the presence of specific markers and the secretion of specific cytokines confirmed that ndm29 expression leads to a differentiation process toward a neuron-like, rather than glial-like, phenotype. was planned therefore to reconstitute a model of brain tumors in rats by orthotopic implantation of xenogenic transformed human cells. iron is important element used for chemical reaction catalysis and physiological cell functions. the reason of iron deposition is still unknown. under conditions prevailing in human brain it is expected the formation of an amorphous or minute crystalline phase. we used light, scanning (sem) and transmission electron microscopy (tem), energy-dispersive microanalysis, electron diffraction and electron paramagnetic resonance (epr) for investigation of iron deposits in globus pallidus of human brain. sem revealed iron rich particles with na, si, p, s, cl, ca and cu around glial cells. tem revealed bumpy, solid particles of platy and sometimes rounded shape with the size of 2 lm to 6 lm. these ones were identified as hematite. epr measurements showed the presence of fe(iii) and cu(ii), but little amount of fe(ii) can not be excluded. we consider low-temperature process of hematite formation in human globus pallidus in aqueous environment influenced by organic and inorganic factors. chemical processes leading to nanoparticles formation can be associated with neurodegenerations such as alzheimer or parkinson disease. over the past 50 years our understanding of the basic biophysical mechanisms governing the spatio-temporal dynamics of neuronal membrane potentials and synaptic efficacies has significantly expanded and improved. much research has focussed on how ionic currents contribute to the generation and propagation of action potentials, how subthreshold signals propagate along dendritic trees, how the active properties of dendrites shape the integration of incoming signals in a neuron, and how pre-and postsynaptic activities â and potential heterosynaptic effects a determine the way synaptic efficacies change on the short-and long-term. yet, despite these advances, there have been no systematic efforts to relate the basic dynamical repertoire of neurons to the computational challenges neural circuits face, and in particular to explain systematically how the biophysical properties of neurons are adapted to process information efficiently under the constraints of noise and uncertainty in the nervous system. as an initial step in this direction, i will show how various biophysical properties of neurons, in particular short-term synaptic plasticity and dendritic non-linearities, can be seen as adaptations to resolve an important bottleneck in neuronal information processing: the loss of information entailed by the conversion of analogue membrane potentials to digital spike trains. the optogenetic toolbox has greatly expanded since the first demonstration of genetically-targeted optical manipulation of neural activity. in addition to the cation channel channelrhodopsin-2 (chr2), the panel of excitatory opsins now includes an array of chr2 variants with mutations in critical residues, in addition to other, related cation channels, and channel hybrids. the inhibitory opsin panel has similarly expanded beyond the first-described halorhodopsin (nphr), a chloride pump, to include trafficking-enhanced versions of nphr as well as the proton pumps mac and arch. while the expansion of available opsins offers researchers an increasingly powerful and diverse selection of tools, it has also made it increasingly difficult to select the optimal tool for a given experiment. one cannot extract a comparison of opsins from the current literature, since studies differ across multiple variables known to contribute to opsin performance (e.g. expression method, light power density, stimulation protocols, etc.). here, we provide the first empirical comparison of both excitatory and inhibitory opsins under standardized conditions. furthermore, we identify the set of parameters that describe the properties of an opsin in a way that is maximally relevant for biological application. o-120 subcellular compartment-specific distribution of voltage-gated ion channels zoltan nusser institute of experimental medicine, hungarian academy of sciences, budapest, hungary voltage-gated na + (nav) channels are essential for generating the output signal of nerve cells, the action potential (ap). in most nerve cells, aps are initiated in the axon initial segment (ais). in vitro electrophysiological and imaging studies have demonstrated that dendritic nav channels support active backpropagation of aps into the dendrites, but the subunit composition of these channels remained elusive. here, i will present evidence for the somato-dendritic location of nav channels in hippocampal pyramidal cells (pcs). using a highly sensitive electron microscopic immunogold localization technique, we revealed the presence of the nav1.6 subunit in pc proximal and distal dendrites, where their density is 40-fold lower than that found in aiss. a gradual decrease in nav1.6 density along the proximo-distal axis of the dendritic tree was also detected. we have also investigated the subcellular distribution of kv4.2 voltage-gated k + channel subunit and found a somato-dendritic localization. in contrast to that of nav1.6 channels, the density of kv4.2 first increases then decreases as a function of distance from the somata of pcs. such subcellular compartment-specific distribution of voltage-gated ion channels increases the computational power of nerve cells. keywords: memory, extra cellular matrix, random walk we expose first a biological model of memory based on one hand of the mechanical oscillations of axons during action potential and on the other hand on the changes in the extra cellular matrix composition when a mechanical strain is applied on it. due to these changes, the stiffness of the extra cellular matrix along the most excited neurons will increase close to these neurons due to the growth of astrocytes around them and to the elastoplastic behavior of collagen. this will create preferential paths linked to a memory effect. in a second part, we expose a physical model based on random walk of the action potential on the array composed of dendrites and axons. this last model shows that repetition of the same event leads to long time memory of this event and that paradoxical sleep leads to the linking of different events put into memory. myelinated nerve fibres were studied with fluorescent microscopy and laser interference microscopy. ca 2+ redistribution during prolonged stimulation, changes in morphology and rearrangement of cytoplasmic structures were compared in normal conditions and after membrane modification by lysolecithin and methyl-b-cyclodextrin. lysolecithin is a detergent known to provoke demyelination, and methyl-b-cyclodextrin extracts cholesterol from membranes. cholesterol extraction could lead to disruption of membrane caveolae-like microdomains or ''rafts'' and solubilisation of different proteins connected to them. our data suggest that methyl-b-cyclodextrin and lysolecithin lead to different changes in morphology and distribution of cytoplasmic structures. the effect was different for different regions of the nerve (node of ranvier, paranodal and internodal regions). the agents also altered the kinetics of ca 2+ response to stimulation in myelinated fibres. extracellular carbonic anhydrase contributes to the regulation of ca2+ homeostasis and salivation in submandibular salivary gland nataliya fedirko, olga kopach, nana, voitenko lviv national university, human and animal physiology, lviv, ukraine the maintenance of ph in the oral cavity is important for the oral heath since even a minor drop in ph can result in dental caries and damage to the teeth. submandibular salivary gland (smg) is main source of fluid and electrolytes enriched saliva therefore its core for oral ph homeostasis. smg secretion is activated by acetylcholine (ach) in [ca 2+ ] idependend manner and accompanied with oral ph acidic shifts. ph shifts could be due to changes in buffering capacity that is regulated by carbonic anhydrase (ca). despite the expression of different subtypes of ca in smg the role of ca in the regulation of smg function is unclear yet. we found that ca inhibition by benzolamide (bz) decreased of fluid secretion in vivo extracellular na 2+ concentration in situ. the latter confirm the ability of ca to modify both primarily and final saliva secretion. we also found correlation between the secretion and ca 2+ -homeostasis since bz-induced decrease of: in striated muscle ca 2+ release from the sarcoplasmic reticulum (sr) occurs when ryanodine receptors (ryr-s) open either spontaneously or upon the stimulation from dihydropyridine receptors that are located in the adjacent transverse-tubular membrane and change their conformation when the cell is depolarized. recent observations demonstrated that muscles from animal models of ptdinsp phosphatase deficiency suffer from altered ca 2+ homeostasis and excitation-contraction coupling, raising the possibility that ptdinsp-s could modulate voltage-activated sr ca 2+ release in mammalian muscle. the openings of a single or a cluster of ryr-s can be detected as ca 2+ release events on images recorded from fibres loaded with fluorescent ca 2+ indicators. to elucidate the effects of ptdinsp-s on ca 2+ release events, images were recorded from skeletal muscle fibers enzymatically isolated from the m. flexor digitorum breavis of mice utilizing a super-fast scanning technique. a wavelet-based detection method was used to automatically identify the events on the images. three different ptdinsp-s (ptdins3p, ptdins5p, and ptdins(3,5)p) were tested. all these ptdinsp compounds decreased the frequency of spontaneous ca 2+ release events. supported by the hungarian national science fund (otka 75604), té t. calcium sparks elicited by 1 mmol/l caffeine and by a depolarization to -60 mv were recorded at high time resolution on both x-y (30 frames/s) and line-scan images (65 lines/ ms) on intact skeletal muscle fibers of the frog. while a typical spark appeared in one frame only, 17.3 and 26.0% of spark positions overlapped on consecutive frames following caffeine treatment or depolarization, respectively. while both caffeine and depolarization increased the frequency of sparks, as estimated from x-y images, the morphology of sparks was different under the two conditions. both the amplitude (in df/f 0 ; 0.49 ± 0.025 vs. 0.29 ± 0.001; n = 22426 vs. 23714; mean ± sem, p \ 0.05) and the full width at half maximum (in lm; parallel with fiber axis: 2.33 ± 0.002 vs. 2.21 ± 0.005; perpendicular to fiber axis: 2.07 ± 0.003 vs. 1.88 ± 0.004) of sparks was significantly greater after caffeine treatment than on depolarized cells. these observations were confirmed on sparks identified in line-scan images. in addition, x-t images were used to analyze the time course of these events. calcium sparks had significantly slower rising phase under both conditions as compared to the control. on the other hand, while the rate of rise of signal mass was decreased after depolarization, it increased in the presence of caffeine. prolonged depolarisation of skeletal muscle cells induces entry of extracellular calcium into muscle cells, an event referred to as excitation-coupled calcium entry. skeletal muscle excitation-coupled calcium entry relies on the interaction between the 1,4 dihydropyridine receptor on the sarcolemma and the ryanodine receptor on the sarcoplasmic reticulum membrane. in this study we exploited tirf microscopy to monitor with high spatial resolution excitationcoupled calcium entry (ecce) in primary coltures of human skeletal muscle cells harbouring mutation in the ryr1 gene linked to malignant hyperthermia and central core disease. we found that excitation-coupled calcium entry is strongly enhanced in cells from patients with central core disease compared to individuals with malignant hyperthermia and controls. in addition, excitation-coupled calcium entry induces generation of reactive nitrogen species and causes the nuclear translocation of nfatc1. the activation of nfatc1 dependent genes is consistent with an increase of the il6 secretion from primary coltures human myotubes from ccd patients and with fibre type 1 predominance of skeletal muscle of ccd patients. membrane lipids, microdomains & signalling p-132 ftir and calorimetric investigation of the effects of trehalose and multivalent cations on lipid structure sawsan abu sharkh, jana oertel, and karim fahmy division of biophysics, institute of radiochemistry, helmholtz-zentrum dresden-rossendorf, germany e-mail: s.sharkh@hzdr.de the structure of membrane lipids is of fundamental importance for the integrity of cell and organelle membranes in living organisms. membrane lipids are typically hydrated and their headgroup charges counter-balanced by solvated ions. consequently, water loss can induce severe structural changes in lipid packing (lyotropic transitions) and can lead to the damage of lipid membranes even after rehydration. this can be one out of several factors that affect the viability of organisms undergoing desiccation. many organisms, however, are resistant to even extreme water loss. some of them synthesize trehalose which has been shown to be associated with survival of desiccation in phylogenetically diverse organisms (yeast, nematodes, brine shrimp, insect larvae, resurrection plants, and others). here we have studied hydration sensitive transitions in model lipids to determine the effect of trehalose and electrostatics on lipid order. hydration pulse-induced time-resolved fourier-transform infrared (ftir) difference spectroscopy was used to address hydration-dependent lipid structure as a function of trehalose. in combination with differential scanning calorimetry and studies of langmuir-blodget films we arrive at a structural and energetically consistent picture of how trehalose can affects lipidic phase behaviour and support a native lipid structure under water loss. experiments were performed on model lipids with different headgroups and native lipids from desiccation-tolerant organisms. controlled self-assembly and membrane organization of lipophilic nucleosides and nucleic acids: perspectives for applications martin loew 1 , paula pescador 3 , matthias schade 1 , julian appelfeller 1 , jü rgen liebscher 2 , oliver seitz 2 , daniel huster 3 , andreas herrmann 1 , and anna arbuzova 1 1 humboldt universitä t zu berlin, institute of biology/ biophysics, berlin, germany, 2 humboldt universitä t zu berlin, institute of chemistry, berlin, germany, 3 universitä t leipzig, department of medical physics and biophysics, leipzig, germany lipophilic conjugates of nucleosides and nucleic acids such as dna, rna, and peptide nucleic acid (pna) -combining assembly properties of amphiphiles and specific molecular recognition properties of nucleic acids -allow numerous applications in medicine and biotechnology. we recently observed self-assembly of microtubes, stable cylindrical structures with outer diameters of 300 nm and 2-3 lm and a length of 20-40 lm, from a cholesterol-modified nucleoside and phospholipids. morphology and properties of these microtubes and functionalization with lipophilic dna will be characterized. we also observed that lipophilic nucleic acids, pna and dna differing in their lipophilic moieties, partition into different lipid domains in model and biological membranes as visualized by hybridization with respective complementary fluorescently-labeled dna strands. upon heating, domains vanished and both lipophilic nucleic acid structures intermixed with each other. reformation of the lipid domains by cooling led again to separation of membrane-anchored nucleic acids. by linking specific functions to complementary strands, this approach offers a reversible tool for triggering interactions among the structures and for the arrangement of reactions and signaling cascades on biomimetic surfaces. conformational dependent trafficking of p-glycoprotein with rafts zsuzsanna gutayné tó th, orsolya bá rsony, katalin goda, gá bor szabó and zsolt bacsó university of debrecen, mhsc, department of biophysics and cell biology, debrecen, hungary p-glycoprotein (pgp), an abc-transporter playing a prominent role in multidrug resistance, demonstrate conformationdependent endocytosis on the surface of 3t3-mdr1 cells. these cell surface transporters have a uic2 conformationsensitive-antibody-recognizable subpopulation, which is about one-third of the rest persisting long on the cell surface and perform fast internalization via rafts. we have identified that the rapid internalization is followed by quick exocytosis, in which the other subpopulation is not or only slightly involved. the exocytosis presents a cholesterol depletion dependent intensification, in contrast to the internalization, which is inhibited by cyclodextrin treatment. this continuous recycling examined by total internal reflection (tirf) microscopy increases the amount of the raft associated subpopulation of pgps in the plasma membrane, and it might have a role in restoring the cholesterol content of the membrane after cholesterol depletion. our presentation will summarize related endocytotic, exocytotic and recycling processes and that how does our data fit into our current notions regarding to the cholesterol and sphingomyelin trafficking. membrane nanodomains based on phase-segregating lipid mixtures have been emerged as a key organizing principle of the plasma membrane. they have been shown to play important roles in signal transduction and membrane trafficking. we have developed lipid-like probes carrying multivalent nitrilotriacetic acid (tris-nta) head groups for selective targeting of histagged proteins into liquid ordered or liquid disordered phases. in giant unilamellar vesicles strong partitioning of tris-nta lipids into different lipid phases was observed. for a saturated tris-nta lipid, at least 10-fold preference for the liquid ordered phase was found. in contrast, an unsaturated nta lipid shows a comparable preference for the liquid disordered phase. partitioning into submicroscopic membrane domains formed in solid supported membranes was confirmed by superresolution imaging. single molecule tracking of his-tagged proteins tethered to solid supported membranes revealed clear differences in the diffusion behavior of the different nta-lipids. by using bsa as a carrier, multivalent nta lipids were efficiently incorporated into the plasma membrane of live cells. based on this approach, we established versatile methods for probing and manipulating the spatiotemporal dynamics of membrane nano domains in live cells. il-9 is a multifunctional cytokine with pleiotropic effects on t cells. the il-9r consists of the cytokine-specific a-subunit and the c c -chain shared with other cytokines, including il-2 and -15, important regulators of t cells. we have previously shown the preassembly of the heterotrimeric il-2 and il-15r, as well as their participation in common superclusters with mhc glycoproteins in lipid rafts of human t lymphoma cells. integrity of lipid rafts was shown to be important in il-2 signaling. we could hypothesize that other members of the c c cytokine receptor family, such as the il-9r complex, may also fulfill their tasks in a similar environment, maybe in the same superclusters. co-localization of il-9r with lipid rafts as well as with the il-2r/mhc superclusters was determined by clsm. molecular scale interactions of il-9ra with il-2r and mhc molecules were determined by microscopic and flow cytometric fret experiments. the role of lipid rafts in il-9r signaling was assessed by following the effect of cholesterol depletion on il-9 induced stat phosphorylation. our results suggest the possibility that preassembly of the receptor complexes in common membrane microdomains with mhc glycoproteins may be a general property of c c cytokines in t cells. to unravel the molecular processes leading to fas clustering in lipid rafts, a 21-mer peptide corresponding to the single transmembrane domain of the death receptor was reconstituted into model membranes that display liquid-disordered/ liquid-ordered phase coexistence, i.e. mimicking cells plasma membranes. using the intrinsic fluorescence of the peptide two tryptophans residues (trp 176 and trp 189 ), fas membrane lateral organization, conformation and dynamics was studied by steady-state and time-resolved fluorescence techniques. our results show that the fas has preferential localization to liquid disordered membrane regions, and that it undergoes a conformational change from a bilayer inserted state in liquid-disordered membranes to an interfacial state in liquidordered membranes. this is a result of the strong hydrophobic mismatch between the (hydrophobic) peptide length and the hydrophobic thickness of liquid-ordered membranes. in addition, we show that ceramide, a sphingolipid intimately involved in fas oligomerization and apoptosis triggering, does not affect fas membrane organization. overall, our results highlight ceramide role as an enhancer of fas oligomerization, and unravel the protective function of fas transmembrane domain against non-ligand induced fas apoptosis. organization and dynamics of membrane-bound bovine a-lactalbumin: a fluorescence approach arunima chaudhuri and amitabha chattopadhyay centre for cellular and molecular biology, hyderabad 500 007, india e-mail: amit@ccmb.res.in many soluble proteins are known to interact with membranes in partially disordered states, and the mechanism and relevance of such interactions in cellular processes are beginning to be understood. interestingly, apo-bovine alactalbumin (bla), a soluble protein, specifically interacts with negatively charged membranes and the membranebound protein exhibits a molten globule conformation. we have used the wavelength-selective fluorescence approach to monitor the molten globule conformation of bla upon binding to negatively charged membranes as compared to zwitterionic membranes. tryptophans in bla exhibit differential red edge excitation shift (rees) upon binding to negatively charged and zwitterionic membranes, implying differential rates of solvent relaxation around the tryptophan residues. our results utilizing fluorescence anisotropy, lifetime and depth analysis by the parallax approach of the tryptophans further support the differential organization and dynamics of the membrane-bound bla forms. in addition, dipole potential measurements and dye leakage assays are being used in our ongoing experiments to explore the mechanism of bla binding to membranes. these results assume significance in the light of antimicrobial and tumoricidal functions of a-lactalbumin. role of long-range effective protein-protein forces in the formation and stability of membrane protein nano-domains nicolas destainvill laboratoire de physique thé orique, université paul sabatier toulouse 3 -cnrs, toulouse, france we discuss a realistic scenario, accounting for the existence of sub-micro-metric protein domains in plasma membranes. we propose that proteins embedded in bio-membranes can spontaneously self-organize into stable small clusters, due to the competition between short-range attractive and intermediate-range repulsive forces between proteins, specific to these systems. in addition, membrane domains are supposedly specialized, in order to perform a determined biological task, in the sense that they gather one or a few protein species out of the hundreds of different ones that a cell membrane may contain. by analyzing the balance between mixing entropy and protein affinities, we propose that protein sorting in distinct domains, leading to domain specialization, can be explained without appealing to preexisting lipidic micro-phase separations, as in the lipid raft scenario. we show that the proposed scenario is compatible with known physical interactions between membrane proteins, even if thousands of different species coexist. lipid rafts are cholesterol and sphingolipid-enriched functional microdomains present in biomembranes. rafts have been operationally defined as membrane fractions that are detergent insoluble at low temperature. here we have characterized drms from erythrocytes treated with the nonionic detergents brij58 and brij98, at 4°c and 37°c, and compared them to drms obtained with triton x-100 (tx100). we have also investigated the effect of cholesterol depletion in drms formation. brij drms were enriched in cholesterol as well as tx100 drms. hptlc analysis showed a very similar distribution of phosphatidylcholine-pc, phosphatidylethanolamine-pe and sphingomyelin-sm in brij drms to that found in ghost membranes. sm-enriched drms were obtained only with tx100 while pe content was decreased in tx100 drms, in comparison to brij drms. immunoblot essays revealed that rafts markers (flotillin-2 and stomatin) were present in all drms. contrary to tx100 drms, analysis of electron paramagnetic resonance spectra (with 5-doxyl stearate spin label) revealed that brij drms are not in the liquid-ordered state, evincing the differential extraction of membrane lipids promoted by these detergents. supported by fapesp/cnpq (brazil). several biological membrane mimics have been built to investigate the topology of molecules in membranes. among them ''bicelles'', i.e., mixtures of long-chain and short-chain saturated phospholipids hydrated up to 98%, became very popular because they orient spontaneously in magnetic fields. disk-shaped systems of 40-80 nm diameter and 4-5 nm thickness have been measured by electron microscopy and solid state nmr and can be oriented by magnetic fields with the disc-plane normal perpendicular to the field. we have been developing recently lipids that contain in one of their chains a biphenyl group (tbbpc) affording an orientation parallel to the magnetic field, in the absence of lanthanides. a large number of hydrophobic molecules including membrane proteins have been successfully embedded and static nmr afforded finding the orientation of protein helices in membranes; mas nmr provided the 3d structure of peptides in bicelles. biphenyl bicelles keep their macroscopic orientation for days outside the field, thus leading to combined nmr and x-rays experiments. tbbpc allows also construction of lm vesicles showing a remarkable oblate deformation in magnetic fields (anisotropy of 3-10) and opens the way to applications for structural biology or drug delivery under mri. imaging membrane heterogeneities and domains by super-resolution sted nanoscopy christian eggeling, veronika mueller, alf honigmann, stefan w. hell department of nanobiophotonics, max planck institute for biophysical chemistry, am fassberg 11, 37077 gö ttingen, germany cholesterol-assisted lipid and protein interactions such as the integration into lipid nanodomains are considered to play a functional part in a whole range of membrane-associated processes, but their direct and non-invasive observation in living cells is impeded by the resolution limit of [200nm of a conventional far-field optical microscope. we report the detection of the membrane heterogeneities in nanosized areas in the plasma membrane of living cells using the superior spatial resolution of stimulated emission depletion (sted) far-field nanoscopy. by combining a (tunable) resolution of down to 30 nm with tools such as fluorescence correlation spectroscopy (fcs), we obtain new details of molecular membrane dynamics. sphingolipids or other proteins are transiently (* 10 ms) trapped on the nanoscale in cholesterol-mediated molecular complexes, while others diffuse freely or show a kind of hopping diffusion. the results are compared to sted experiments on model membranes, which highlight potential influences of the fluorescent tag. the novel observations shed new light on the role of lipid-protein interactions and nanodomains for membrane bioactivity. ca2+ controlled all-or-none like recruitment of synaptotagmin-1 c2ab to membranes sune m. christensen, nicky ehrlich, dimitrios stamou bio-nanotechnology laboratory, department of neuroscience and pharmacology, nano-science center, lundbeck foundation center biomembranes in nanomedicine, university of copenhagen, 2100 copenhagen, denmark e-mail: ehrlich@nano.ku.dk & stamou@nano.ku.dk synaptotagmin-1 (syt) is the major ca2+ sensor that triggers the fast, synchronous fusion of synaptic vesicle with the presynaptic membrane upon ca2+-mediated membrane recruitment of the cytosolic c2ab domain. the ca2+-dependent recruitment of syts c2ab domain to membranes has so far been investigated by ensemble assays. here we revisited binding of wild type c2ab and different c2ab mutants of syt to lipid membranes using a recently developed single vesicle assay. the hallmark of the single vesicle approach is that it provides unique information on heterogeneous properties that would otherwise be hidden due to ensemble averaging. we found that c2ab does not bind to all vesicles in a homogenous manner, but in an all-or-none like fashion to a fraction of the vesicles. the fraction of vesicles with bound c2ab is regulated by the amount of negatively charged lipids in the membrane as well as by [ca2+] . this ca2+ controlled all-ornone like recruitment of syt to membranes provides a possible explanation for the strongly heterogeneous behavior of the in vitro model system for neuronal membrane fusion. furthermore, heterogeneity in release probability among synaptic vesicles is a critical property in determining the output of a neuronal signaling event. new insights in the transport mechanism of ciprofloxacin revealed by fluorescence spectroscopy mariana ferreira, sílvia c. lopes, paula gameiro requimte, faculdade de ciê ncias da universidade do porto, porto, portugal keywords: fluoroquinolones; liposomes; proteoliposomes; ompf; ciprofloxacin; fluorescence; anisotropy. fluoroquinolones are antibiotics that have a large spectrum of action against gram negative and some gram positive bacteria. the interaction between these species and liposomes has been cited as a reference in the understanding of their diffusion through phospholipid bilayer and can be quantified by the determination of partition coefficients between a hydrophobic phase (liposomes) and an aqueous solution. it is also known that some porins of the bacterial membranes are involved in transport mechanism of many fluoroquinolones. ompf, a well characterized membrane protein characteristic of the outer membrane of gram negative bacteria assumes the conformation of homo-trimer, whose monomers have two tryptophan residues (one located at the interface of monomers and the other at the interface lipid/protein). thus, we proceeded to study the interaction of ciprofloxacin, a second generation fluoroquinolone, with unilamellar liposomal vesicles and ompf proteoliposomes of pope/popg, pope/popg/cardiolipin and e. coli total. partition coefficients (kp's) and the association with ompf proteoliposomes were determined by steady state fluorescence spectroscopy under physiological conditions (t=37°c; ph 7.4). the membrane mimetic systems used were characterized by dls and fluorescence anisotropy. motivation is whether there exist differences in the patternforming capabilities of two adhesion molecules of different roles: cd44, mediating ,,dynamic'' adhesion in cell rolling and icam-1, mediating ,,static'' adhesion during the formation of immune-synapse. homo-and hetero-associations of cd44, icam-1 and the mhci is investigated on the nm-and lmdistance levels on ls174t colon carcinoma cells in two different conditions of lymphocyte homing: (1) with ifnc and tnfa, both lymphokines up-regulating the expression level of mhci and icam-1 and down-regulating that of cd44. (2) crosslinking of cd44 and icam-1 representing receptor engagement. the observations are explained by assuming the existence of a kinase cascade-level crosstalk between the cd44 and icam-1 molecules which manifests in characteristic complementary changes in the properties of cell surface receptor patterns. for the characterisation of cluster morphology new colocalization approaches were developed: (i) ,,number of first neighbours'' distribution curves, (ii) ,,acceptor photobleaching fret-fluorescence intensity fluctuation product'' correlation diagrams, and (iii) ,,random gradient-kernel smoothing assisted decay'' of pearson-correlations, and (iv) k-function formalism. analyzing janus kinases in living cells with single-color fcs using confocal and tir-illumination thomas weidemann 1 , hetvi gandhi 1 , remigiusz worch 1,3 , robert weinmeister 1 , christian bö kel 2 , and petra schwille 1 1 biophysics research group, technische universitä t dresden, germany, 2 crtd, center for regenerative therapies dresden, technische universitä t dresden, germany, 3 institute of physics, polish academy of science, warsaw, poland cytokine receptors of the hematopoietic superfamily transduce their signal through non-covalently bound janus kinases. there are only 4 such kinases in humans (jak1, 2, 3 and tyk2), which associate to 46 different cytokine receptor chains. here we study the dynamics of gfp-tagged jak1 and jak3 in epithelial cells with fluorescence correlation spectroscopy (fcs). jak1 and jak3 behave differently in various aspects: in the absence of receptors, jak1 still binds the membrane, whereas jak3 diffuses homogeneously in the cytoplasm. we used fcs under total internal reflection illumination (tir-fcs) and determined the membrane binding affinity of jak1 to be 60±36 nm. the association of jak3 with the common gamma chain (c c ) is very tight as shown by fluorescence recovery after photobleaching (frap). molecular brightness analysis of single-point fcs shows that jak1 diffuses as a monomer in the rather small cytoplasmic pool, whereas jak3 diffuses as dimers, which undergo a defined oligomerization. the degree of oligomerization decays at higher concentrations, indicating that some unknown, saturable scaffold is involved. characterizing the binding and mobility schemes of the janus kinases may be important to further elucidate their specific and redundant effects in signal transduction. plasma membrane (pm)-enriched fraction obtained through subcellular fractioning protocols are commonly used in studies investigating the ability of a compound to bind to a receptor. however, the presence of mitochondria membranes (mi) in the pm-enriched fraction may compromise several experimental results because mi may also contain the interest binding proteins. aiming to analyze the subcellular fractioning quality of a standard sucrose density based protocol, we investigated (a) the na + k + -atpase (pm marker) and succinate dehydrogenase -sd (mi marker) activities; (b) the immunocontent of the adenine nucleotide translocator (ant -mi membrane marker) in both pm-and mi-enriched fractions. since several binding protocols may require long incubation period, we verified the quality of both fractions after 24 hours of incubation in adequate buffer. our results show that pmand mi-enriched fractions exhibit contamination with mi or pm, respectively. we did not observe any effect of incubation on na + k + -atpase activity and ant content in both fractions. surprisingly, sd activity was preserved in the pm-but not in mi-enriched fraction after incubation. these data suggest the need of more careful use of pm-enriched fraction preparation in studies involving pm proteins characterization. human neutrophil peptide 1 (hnp1) is a human cationic defensin that present microbial activity against various bacteria (both gram-positive and negative), fungi and viruses. hnp1 is stored in the cytoplasmic azurophilic granules of neutrophils and epithelial cells. in order to elucidate the mode of action of this antimicrobial peptide (amp), studies based on its lipid selectivity were carried out. large unilamellar vesicles (luv) with different lipid compositions were used as biomembranes model systems (mammal, fungal and bacterial models). changes on the intrinsic fluorescence of the tryptophan residues present in hnp1 upon membrane binding/insertion were followed, showing that hnp1 have quite distinct preferences for mammalian and fungal membrane model systems. hnp1 showed low interaction with glucosylceramide rich membranes, but high sterol selectivity: it has a high partition for ergosterol-containing membranes (as fungal membranes) and low interaction with cholesterolcontaining membranes (as in mammalian cells). these results reveal that lipid selectivity is the first step after interaction with the membrane. further insights on the hnp1 membrane interaction process were given by fluorescence quenching measurements using acrylamide, 5-doxylstearic acid (5ns) or (16ns). nanoparticles (np) are currently used in many industrial or research applications (paints, cosmetics, drug delivery materials…). recent papers demonstrate clearly their activity with biological membranes (nanoscale holes, membrane thinning, disruption). different studies of the np-membrane interaction suggest that parameters are particularly important, such as the np size, their surface properties or their aggregation state. composition of biological membranes being particularly complex, supported lipid bilayers (slb) composed of a restricted number of lipids are usually used as simplified membrane model. moreover, these two-dimensional systems are convenient for surface analysis techniques, such as atomic force microscopy (afm), giving information on the morphology of the slb and its mechanical properties. in this work, we study the behaviour of slbs made of lipids representative of the membrane fluid phase (popc) or of the raft phase (sphingomyelin). these slbs are deposited on planar surfaces (mica or glass) previously recovered with silica beads (10 or 100 nm in diameter) in order to mimick the np-membrane interaction. we will present in this work our first results obtained by afm and fluorescence microscopy. it is well known that the eukaryotic nuclei are the sphere of lipids active metabolism. the investigations demonstrated the existence of numerous enzymes in nuclei which modulate the changes of nuclear lipids during different cellular processes. although the nuclear membrane is accepted as the main place of the lipids localization, nearly 10% of nuclear lipids are discovered in chromatin fraction. the ability of chromatin phospolipids to regulate dna replication and transcription was already demonstrated. the chromatin phospolipids seems to play an important role in cell proliferation and differentiation as well as in apoptosis. it seems also possible that chromatin phospholipids may be participated in realization of cisplatin antitumor effects. the 24-hour in vivo effect of cisplatin on rat liver chromatin phospholipids was investigated. the phospholipids of rat liver chromatin were fractionated by microtlc technique. the quantitative estimation of fractionated phospholipids was carried out by computer program fugifilm science lab. 2001 image gauge v 4.0. the alteration of total phospholipids content as well as the quantitative changes among the individual phospholipids fractions in rat liver chromatin after in vivo action of cisplatin was established. the total content of chromatin phospholipids was significantly decreased after the cisplatin action. four from five individual phospholipids fractions were markedly changed after the drug action. two cholin-content phospholipids, particularly phosphatidylcholine and sphingomyelin exhibit diversity in sensitivity to this drug: the increase of sphingomyelin content accompanied by quantitative decrease of phosphatidylcholine. the quantity of cardiolipin was markedly increased while the amount of phosphatidylinositol was decreased after the cisplatin treatment. the phosphatidylethanolamin content remained unchanged after the drug action. it seems that high sensitivity of chromatin phospholipids exhibited to cisplatin action may play an important role in antitumor effects of this drug. membrane lipids and drug resistance in staphylococci r. d. harvey institute of pharmaceutical science, king's college london, 150 stanford street, london se1 9nh, uk staphylococci express numerous resistance mechanisms against common antimicrobials, including peptide components of the innate immune system which have been trumpeted as being likely candidates to replace our increasingly ineffective antibiotics. the membrane phospholipid lysylphosphatidylglycerol (l-pg) appears to play a key role in staphylococcal drug resistance, since its absence in mutant bacteria renders them susceptible to a range of cationic antimicrobials. the current assumption about the role l-pg plays in drug resistance is that of facilitating charge neutralisation of the plasma membrane, leading to loss of affinity towards cationic moieties. we have investigated this phenomenon using a range of model membrane systems composed of both synthetic lipids and reconstituted natural lipid extracts, using such techniques as stopped-flow fluorescence, circular dichroism and neutron scattering. our conclusions indicate that the initial assumptions about the role of l-pg in drug resistance are over-simplistic and certainly do not tell the whole story of the physical and biological properties of this fascinating moderator or membrane behaviour. our findings show that l-pg does not inhibit antimicrobial drug action by charge dampening, hinting at a different protective mechanism. modulation of a-toxin binding by membrane composition m. schwiering, a. brack, c. beck, h. decker, n. hellmann institute for molecular biophysics, university of mainz, mainz, germany although the alpha-toxin from s. aureus was the first poreforming toxin identified, its mode of interaction with membranes is still not fully understood. the toxin forms heptameric pores on cellular and artificial membranes. the present hypothesis is that the initial binding to the membrane occurs with low affinity, and that an efficient oligomerisation, relying on clusters of binding sites, is the reason for the overall high affinity of the binding process. in order to separate the effects of increasing concentration of binding sites from this topological effect, we investigated the oligomer formation based on pyrene-fluorescence for a series of lipid compositions, where the fraction of toxin binding lipids (egg phospatidylcholine (epc) or egg sphingomyelin (esm)) was varied while their concentration remained constant. the results indicate that an increased local density of toxin binding sites occurring due to phase separation facilitates oligomer formation. furthermore, the change in local environment (number of neighboring cholesterol molecules) upon domain formation also enhances oligomer formation.we thank the dfg (sfb 490) for financial support, s. bhakdi and a. valeva for production of the toxin and helpful discussions. we explored quercetin effects on lipid bilayers containing cholesterol using a spectrofluorimetric approach. we used the fluorescent probe laurdan which is able to detect changes in membrane phase properties. when incorporated in lipid bilayers, laurdan emits from two different excited states, a non-relaxed one when the bilayer packing is tight and a relaxed state when the bilayer packing is loose. this behavior is seen in recorded spectra as a shift of maximum emission fluorescence from 440 nm at temperatures below lipids phase transition to 490 nm at temperatures above lipids phase transition values. emission spectra of laurdan were analyzed as a sum of two gaussian bands, centered on the two emission wavelengths allowing a good evaluation of the relative presence of each population. our results show that both laurdan emission states are present with different shares in a wide temperature range for dmpc liposomes with cholesterol. quercetin leads to a decrease in the phase transition temperature of liposomes, acting in the same time as a quencher on laurdan fluorescence. this paper is supported by the sectorial operational programme human resources development, financed from the caveolins are essential membrane proteins found in caveolae. the caveolin scaffolding domain of caveolin-1 includes a short sequence containing a crac motif (v 94 tkywfyr 101 ) at its c-terminal end. to investigate the role of this motif in the caveolin-membrane interaction at the atomic level, we performed a detailed structural and dynamics characterization of a cav-1(v94-l102) nonapeptide encompassing this motif and including the first residue of cav-1 hydrophobic domain (l102), in dodecylphosphocholine (dpc) micelles and in dmpc/dhpc bicelles, as membrane mimics. nmr data revealed that this peptide folded as an amphipathic helix located in the polar head group region. the two tyrosine sidechains, flanked by arginine and lysine residues, are situated on one face of this helix, whereas the phenylalanine and tryptophan side-chains are located on the opposite face (le lan c. et al., 2010, eur. biophys. j., 39, 307-325). to investigate the interactions between the crac motif and the lipids, we performed molecular dynamics simulations in two different environment: a dpc micelle and a popc bilayer. the results obtained are in good agreement with nmr data and the comparison between both systems provided insight into the orientation of the crac motif at the membrane interface and into its interactions with lipids. this work was partially supported by the strategic grant posdru/21/1. our study suggests that conformation and positioning of hydroxyl groups significantly affects thermotropic properties of sphingolipids and sterol interaction. the polymorphism of a new series of bolaamphiphile molecules based on n-(12-betainylamino-dodecane)-octyl b-d-glucofuranosiduronamide chloride is investigated. the length of the main bridging chain is varied in order to modify the hydrophilic/lipophilic balance. the other chemical modification was to introduce a diacetylenic unit in the middle of the bridging chain to study the influence of the pp stacking on the supramolecular organisation of these molecules. dry bolaamphiphiles self-organize in supramolecular structures such as lamellar crystalline structure, lamellar fluid structure and lamellar gel structure. the thermal dependence of these structures, as well as the phase transition is followed by smallangle and wide-angle x-ray scattering. once the thermal cycle is accomplished, the system remains in the kinetically stabilized undercooled high-temperature phase at temperature of 20°c. subsequently, the time dependence of the relaxation to the thermodynamically stable phase is followed and very slow relaxation on the order of hours or days is observed. the study of polymorphism and the stability of various phases of this new series of bolaamphiphiles is interesting for potential application in health, cosmetics or food industry, is undertaken in this work. alkylphospholipids have shown promising results in several clinical studies and among them perifosine (opp) is promising for breast cancer therapy. antitumor effect was much better in estrogen receptor negative (er-) than in estrogen receptor positive (er+) tumors in vivo. it is believed that apl do not target dna, but they insert in the plasma membrane and ultimately lead to cell death. liposomes made of opp and different amount of cholesterol (ch) showed diminished hemolytic activity as compared to micellar opp, but in most cases cytotoxic activity was lower. in order to find optimal liposomal composition and to understand better the difference in the response of er+ and er-cells the interaction opp liposomes with er+ and er-cells was studied. for liposomes with high amount of ch both cell types showed slow release of the liposome entrapped spin probe into the cytoplasm. liposmes with low amount of ch interact better with cells but the release is faster for er-as for er+ cells at 37°c. experiments with nitroxide-labeled opp (sl-opp) liposomes suggest that the exchange of sl-opp between liposomes and cellular membranes is fast. however, translocation of sl-opp across the plasma membrane is slow, but seems to be faster for opp resistent, er+ cells as for er-cells at 37°c. estimation of a membrane pore size based on the law of conservation of mass krystian kubica 1 , artur wrona 1 and olga hrydziuszko 2 1 institute of biomedical engineering and instrumentation, wroclaw university of technology, wroclaw, poland, 2 centre for systems biology, university of birmingham, birmingham, uk the size of biomembrane pores determines which solutes or active compounds may enter the cell. here, using a mathematical model of a lipid bilayer and the law of conservation of mass, we calculate the radius of a membrane pore created by rearranging the lipid molecules (the pore wall was formed out of the lipid heads taken from the membrane regions situated directly above and below the pore, prior its formation). assuming a constant number of lipid molecules per bilayer (with or without the pore) and based on the literature data (60% decrease in the area per chain for a fluid-to-gel transition and a matching change of one chain volume not exceeding 4%) we have shown that the pore radius can measure up to 4.7nm (for a 7nm thick lipid bilayer) without the lipid molecules undergoing a phase transition. a further assumption of area per chain being modified as a consequence of the lipids conformational changes has resulted in an increase of the calculated radius up to 7.1nm. finally, a comparison of the pore volume with the corresponding volume of the lipid bilayer has led to a conclusion that for the system under consideration the membrane pore can only be created with the lipids undergoing fluidto-gel conformational changes. the key signaling pathway involves tyrosine phosphorylation of signal transducers and activators of transcription (stat1 and stat2) by receptor-associated janus kinases. we aim to unveil of the very early events of signal activation including ligand-induced receptor assembly and the recruitment of the cytoplasmic effector proteins stat1 and stat2 in living cells. to this end, we have explored the spatiotemporal dynamics of stat recruitment at the membrane on a single molecule level. highly transient interaction of stats to membrane-proximal sites was detected by tirf-microscopy, allowing for localizing and tracking individual molecules beyond the diffraction limit. thus, we obtained a pattern of the spatio-temporal recruitment of stat molecules to the plasma membrane revealing distinct submicroscopic structures and hotspots of stat interaction with overlapping recruitment sites for stat1 and stat2. strikingly, these stat binding sites were independent on receptor localization and expression level. simultaneous superresolution imaging of the cytoskeleton revealed the organization of stat recruitment sites within the cortical actin skeleton. characterization of molecular dynamics on living cell membranes at the nanoscale level is fundamental to unravel the mechanisms of membrane organization andcompartmentalization. we have recently demonstr ated the feasibility of fluorescence correlation spectroscopy (fcs) based on the nanometric illumination of near-field scanning optical microscopy (nsom) probes on intact living cells [1] . nsom-fcs was applied to study the diffusion of fluorescent lipid analogs on living cho cells. the experiments allowed to reveal details of the diffusion hidden by larger illumination areas and associated with nanoscale membrane compartmentalization. the technique also offers the unique advantages of straightforward implementation of multiple color excitation, opening the possibility to study nanoscale molecular cross-correlation. furthermore, the nsom probe evanescent axial illumination allows to extend diffusion study to the membrane-proximal cytosolic region. as such, nsom-fcs represents a novel powerful tool to characterize the details of many biological processes in which molecular diffusion plays a relevant role. the growing interest in supported lipid bilayers (slbs) on conductive substrates, such as gold, is due to the possibility of designing lipid-based biosensor interfaces with electrochemical transduction. due to the hydrophobicity of gold it is still a challenge to deposit planar and continuous bilayers without previous surface modification. most studies on gold concern single-phase slbs without cholesterol or gangliosides, two vital components of biomembranes. in this work the experimental conditions suitable for the formation of complex slbs with phase-separation directly on gold are exploited. the mixtures dopc/dppc/cholesterol (4:4:2) with 0 or 10 mol % of ganglioside gm1, which should yield lipid raft-like domains according to reported phase diagrams, were studied. slb with lipid rafts were successfully formed onto bare au (111), although surface modification with 11-mercapto-undecanoic acid sam stabilized the slbs due to its charge and hydrophilicity. the different experimental conditions tested had an impact on nano/microdomains organization observed by atomic force microscopy in buffer solution. surface characterization through the combined use of ellipsometry, cyclic voltammetry and afm allowed to optimize the conditions for the formation of more planar and compact slbs. it is widely accepted that the conversion of the soluble, nontoxic amyloid b-protein (ab) monomer to aggregated toxic ab rich in b-sheet structures is central to the development of alzheimer's disease. however, the mechanism of the abnormal aggregation of ab in vivo is not well understood. we have proposed that ganglioside clusters in lipid rafts mediate the formation of amyloid fibrils by ab, the toxicity and physicochemical properties of which are different from those of ab amyloids formed in solution [1, 2] . in this presentation, we report a detailed mechanism by which ab-(1-40) fibrillizes in raft-like lipid bilayers composed of gm1/cholesterol/sphingomyelin. at lower concentrations, ab formed an a helix-rich structure, which was cooperatively converted to a b sheet-rich structure above a threshold concentration. the structure was further changed to a seed-prone b structure at higher concentrations. the seed recruited ab in solution to form amyloid fibrils. [ hepatitis c virus (hcv) has a great impact on public health, affecting more than 170 million people worldwide since it is the cause of liver-related diseases such as chronic hepatitis, cirrhosis and hepatocarcinoma. hcv entry into the host cell is achieved by the fusion of viral and cellular membranes, replicates its genome in a membrane associated replication complex, and morphogenesis has been suggested to take place in the endoplasmic reticulum (er) or modified er membranes. the variability of the hcv proteins gives the virus the ability to escape the host immune surveillance system and notably hampers the development of an efficient vaccine. hcv has a single-stranded genome which encode a polyprotein, cleaved by a combination of cellular and viral proteases to produce the mature structural proteins (core, e1, e2, and p7) and non-structural ones (ns2, ns3, ns4a, ns4b, ns5a and ns5b), the latter associated with the membrane originated from the er in the emerging virus. the ns4b protein, a fundamental player in the hcv replicative process and the least characterized hcv protein, is a highly hydrophobic protein associated with er membranes. it has recently been shown that the c-terminal is palmitoylated and the n-terminal region has potent polymerization activity. the expression of ns4b induces the formation of the so called membranous web, which has been postulated to be the hcv rna replication complex. thus, a function of ns4b might be to induce a specific membrane alteration that serves as a scaffold for the formation of the hcv replication complex and therefore has critical role in the hcv cycle. due to the high hydrophobic nature of ns4b, a detailed structure determination of this protein is very difficult. the ns4b protein is an integral membrane protein with four or more transmembrane domains. the c-terminal region of ns4b is constituted by two a helices, h 1 (approximately from amino acid 1912 to 1924) and h 2 (approximately from amino acid 1940 to 1960), which have been studied as potential targets for inhibiting hcv replication. previous studies from our group, based on the study of the effect of ns4b peptide libraries on model membrane integrity, have allowed us to propose the location of different segments in this protein that would be implicated in lipid-protein interaction. additionally, the h 1 region could be an essential constituent in the interaction between protein and membrane. in this study we show that peptides derived from the c-terminal domain of ns4b protein of hcv are able to interact with high affinity to biomembranes, significantly destabilizing them and affecting their biophysical properties. there were also differences in the interaction of the peptide depending on the lipid composition of the membranes studied. we have also applied fluorescence spectroscopy, infrared spectroscopy and differential scanning calorimetry which have given as a detailed biophysical study of the interaction of the peptide with model biomembranes. this work was supported by grant bfu2008-02617-bmc (ministerio de ciencia y tecnología, spain) to j.v. a semi-quantitative theory describing the adhesion kinetics between soft objects as living cells or vesicles was developed. the nucleation-like mechanism has been described in the framework of a non-equilibrium fokker-planck approach accounting for the adhesion patch growth and dissolution (a. raudino, m. pannuzzo, j. chem. phys. 132, 045103 (2010)). a well known puzzling effect is the dramatic enhancement of the adhesion/fusion rate of lipid membranes by water-soluble polymers that do not specifically interact with the membrane surface. we extend the previous approach by molecular dynamics simulations in the framework of a coarse-grained picture of the system (lipid+polymer+ions embedded in an explicit water medium) in order to test and support our previous analytical results. simulations show that the osmotic pressure due to the polymer exclusion from the inter-membrane spacing is partially balanced by an electrostatic pressure. however, we also evidenced an interesting coupling between osmotic forces and electrostatic effects. indeed, when charged membranes are considered, polymers of low dielectric permittivity are partially excluded from the inter-membrane space because of the increased local salt concentration. the increased salt concentration means also a larger density of divalent ions which form a bridge at the contact region (stronger adhesion). the overall effect is a smaller membrane repulsion. this effect disappears when neutral membranes are considered. the model could explain the fusion kinetics between lipid vesicles, provided the short-range adhesion transition is the rate-limiting step to the whole fusion process. the role of ceramide acyl chain length and unsaturation on membrane structure sandra n. ceramide fatty acid composition selectively regulates distinct cell processes by a yet unknown mechanism. however, evidence suggests that biophysical processes are important in the activation of signalling pathways. indeed, ceramide strongly affects membrane order, induces gel/fluid phase separation and forms highly-ordered gel domains. the impact of ceramide n-acyl chain in the biophysical properties of a fluid membrane was studied in popc membranes mixed with distinct ceramides. our results show that: i) saturated ceramide has a stronger impact on the fluid membrane, increasing its order and promoting gel/fluid phase separation, while their unsaturated counterparts have a lower (c24:1 ceramide) or no (c18:1 ceramide) ability to form gel domains at physiological temperature, ii) differences between distinct saturated species are smaller and are related mainly to domain morphology, and iii) very long chain ceramide induces the formation of tubular structures probably associated with interdigitation. these results suggest that generation of different ceramide species in cell membranes has a distinct biophysical impact with acyl chain saturation dictating membrane lateral organization, and chain asymmetry governing interdigitation and membrane morphology. extra high content of cholesterol (chol) in fiber-cell membranes in the eye lens leads to chol saturation and formation of cholesterol bilayer domains (cbds). it is hypothesized that high enrichment in cholesterol helps to maintain lens transparency and protect against cataractogenesis. in model studies, the cbd is formed in a phospholipid bilayer when cholesterol content exceeding the cholesterol solubility threshold, thus, the cbd is surrounded by phospholipid bilayer saturated with cholesterol. in the present study, we carried out molecular dynamics (md) simulation of two bilayers: a palmitoyloleoylphosphatidylcholine (popc) bilayer (reference) and a 1:1 popc-chol bilayer, to investigate the smoothing effect of the saturating amount of cholesterol on the bilayer. to our knowledge, this effect has not been studied so far so this study is certainly providing new results. our results indicate that saturation with cholesterol significantly narrows the distribution of vertical positions of the center-of-mass of the popc molecules and the popc atoms in the bilayer and smoothes the bilayer surface. we hypothesize that this smoothing effect decreases light-scattering and helps to maintain lens transparency. the phospholipid content of staphylococcus aureus membranes displays a high degree of variability (1-3). the major phospholipids found in s. aureus are phosphatidylglycerol (pg), cardiolipin (cl) and lysylphosphatidylglycerol (lpg), (1) the concentrations of which are environment dependent and see fluctuations on exposure to high concentrations of positively charged moieties (4) . up regulation of lpg has a suspected role in neutralisation of the plasma membrane in response to cationic threats. studies have been conducted to probe biomimetic models of this theory however our focus is to look at atomic details of membrane extracts in the presence of magaininf5w. s. aureus 476 lipid extracts from cells grown at ph 5.5 and 7.0, studies by neutron diffraction with and without peptide at two contrasts. d-spacings were assessed by vogt area fitting and bragg's law. bilayer separation at low ph was *1-2 å less than ph 7.0. with peptide, bilayer separations of ph 5.5 and 7.0 extracts were reduced by *2 å and *4 å , respectively. reduced pg content of low ph extracts is suggested to reduce d-spacing, however presence of peptide further reduces this, possibly by an anion neutralisation effect. abnormal d-spacing on increased humidity may be due to the breakdown of lpg. activation of neutrophils releasing hocl and apoptosis of vein endothelial cells are the events documented to occur in the course of atherosclerosis. as lipid chlorohydrins, which are the key products of the reaction between hocl and unsaturated fatty acid residues, were found in atherosclerotic plaques, we decided to check their biological activity in the context of their ability to act as the mediators of hoclinduced oxidative stress and apoptosis in the culture of immortalized human umbilical vein endothelial cells (hu-vec-st). the concentration of reactive oxygen species was found to be elevated after 1 h cell incubation with phospahtidylcholine chlorohydrins. this effect was at least partially caused by the leakage of superoxide anion from mitochondria and followed by depletion of gsh and total thiols. the significant decrease of antioxidant capacity of cell extracts was also observed. the intracellular red-ox imbalance was accompanied by the increase of the ratio between phosphorylated and dephosphorylated forms of p38 map kinase. after longer incubation a significant number of apoptotic cells appeared. summing up, phosphatidylcholine chlorohydrins may be regarded as signaling molecules, able to initiate signalling pathways by induction of oxidative stress. giant unilamellar vesicles (guvs) are a valuable tool in the study of lateral distribution of biological membrane components. guv dimensions are comparable to typical cell plasma membranes and lipid phase separation can be observed through fluorescence microscopy. guv studies frequently require immobilization of the vesicles, and several methods are available for that effect. one of the most common methodologies for vesicle immobilization is the use of avidin/streptavidin coated surfaces and biotin labeled lipids at very low concentration in the vesicles. here, we analyze the effect of using this methodology on lipid domain distribution for different lipid compositions. we show that as a result of non-homogeneous distribution of biotin labeled lipids between liquid disordered, liquid ordered and gel phases, distribution of lipid domains inside guvs can be dramatically affected. monitoring membrane permeability: development of a sicm approach christoph saßen 1,2 and claudia steinem 1 1 institute for organic and biomolecular chemistry, university of gö ttingen, tammannstraße 2, 37077 gö ttingen, germany, 2 ggnb doctoral program: imprs, physics of biological and complex systems scanning ion conductance microscopy (sicm) utilises a nanopipette containing an electrode as a probe for surface investigations with resolutions of 1/3 of the inner pipette diameter. experiments are conducted under physiological conditions, in situ and without mechanical contact of probe and sample. hence, sicm serves as a well-suited technique for the investigation of soft objects such as cells or artificial lipid membranes. using pore-suspending membranes (psm) as a model system, interactions of melittin as an example for cell penetrating peptides (cpps) and lipid membranes are investigated by means of sicm. formation of a range of solvent free psm from lipid vesicles has been achieved as confirmed by means of fluorescence microscopy and sicm. application of melittin results in rupturing of the lipid bilayer. putative insights gained from this assay are critical concentrations of membrane permeabilising ccps and answers to mechanistic questions, e.g. whether ccps translocate only or form pores within the lipid bilayer. positioning of the z-ring in escherichia coli prior to cell division is regulated by intracellular pole-to-pole oscillation and membrane binding of min proteins, allowing assembly of ftsz filaments only at the center plane of the cell. in order to investigate the influence of membrane geometry on the dynamic behavior of membrane binding of min proteins, we combined concepts of synthetic biology and microfabrication technology. glass slides were patterned by a gold coating with microscopic windows of different geometries, and supported lipid bilayers (slb) were formed on these microstructures. on slbs, min-proteins organize into parallel waves. confinement of the artificial membranes determined the direction of propagation.min-waves could be guided along curved membrane stripes, in circles and even along slalom-geometries. in elongated membrane structures, the protein waves always propagate along the longest axis. coupling of protein waves across spatially separated membrane patches was observed, dependent on gap size and viscosity of the aqueous media above the bilayer. this indicates the existence of an inhomogeneous and dynamic protein gradient above the membrane. minimal systems for membrane associated cellular processes petra schwille bio technology center biotec, technical university of dresden, germany the strive for identifying minimal biological systems, particularly of subcellular structures or modules, has in the past years been very successful, and crucial in vitro experiments with reduced complexity can nowadays be performed, e.g., on reconstituted cytoskeleton and membrane systems. in this overview talk, i will first discuss the virtues of minimal membrane systems, such as guvs and supported membranes, in quantitatively understand protein-lipid interactions, in particular lipid domain formation and its relevance on protein function. membrane transformations, such as vesicle fusion and fission, but also vesicle splitting, can be reconstituted in these simple subsystems, due to the inherent physical properties of selfassembled lipids, and it is compelling question how simple a protein machinery may be that is still able to regulate these transformations. as an exciting example of the power of minimal systems, i show how the interplay between a membrane and only two antagonistic proteins from the bacterial cell division machinery can result in emergence of protein self-organization and pattern formation, and discuss the possibility of reconstituting a minimal divisome. quantitative microscopic analysis reveals cell confluence regulated divergence of pdgfr-initiated signaling pathways with logically streamlined cellular outputs á rpá d szö } or, lá szló ujalky-nagy, já nos szö ll} osi, gyö rgy vereb university of debrecen, department of biophysics and cell biology, debrecen, hungary platelet derived growth factor receptors (pdgfr) play an important role in proliferation and survival of tumor cells. pdgf-bb stimulation caused a redistribution of pdgf receptors towards gm1 rich domains, which was more prominent in confluent monolayers. pdgf-bb stimulation significantly increased relative receptor phosphorylation of the ras / mapk pathway specific tyr716 residues and the pi3-kinase / akt pathway specific tyr751 residues in nonconfluent cultures. tyr771 residues that serve as adaptors for ras-gap which inactivates the mapk pathway and tyr1021 residues feeding into the plc-gamma / camk-pkc pathway were the docking sites significantly hyperphosphorylation following ligand stimulation in confluent cells. we found that p-akt facilitated cell survival and pmapk dependent proliferation is more activated in dispersed cells, while phospholipase c-gamma mediated calcium release and pkc-dependent rhoa activation are the prominent output features pdgf stimulus achieves in confluent cultures. these observations suggest that the same stimulus is able to promote distinctly relevant signaling outputs, namely, cell division and survival in sparse cultures and inhibition of proliferation joined with promotion of migration in confluent monolayers that appear contact inhibited. a thermodynamic approach to phase coexistence in ternary cholesterol-phospholipid mixtures jean wolff, carlos m. marques and fabrice thalmann institut charles sadron, université de strasbourg, cnrs upr, 22, 23 rue du loess, strasbourg cedex, f-67037, france e-mail: thalmann@ics-cnrs.unistra.fr we present a simple and predictive model for describing the phase stability of ternary cholesterol-phospholipid mixtures. assuming that competition between the liquid and gel organizations of the phospholipids is the main driving force behind lipid segregation, we derive a phenomenological gibbs free-energy of mixing, based on the calorimetric properties of the lipids main transition. gibbs phase diagrams are numerically obtained that reproduces the most important experimental features of dppc-dopc-chol membranes, such as regions of triple coexistence and liquid orderedliquid disordered segregation. based on this approach, we present a scenario for the evolution of the phase diagram with temperature. results for other phospholipid species, such as popc or psm will also be presented. interleukin-2 and -15 receptors play a central role in the activation, survival and death of t lymphocytes. they form supramolecular clusters with mhc i and ii glycoproteins in t cells. in damaged or inflamed tissues the extracellular k+ concentration increases, which can depolarize the membrane. the common signaling beta and gamma chains of il-2/15r are phosphorylated upon cytokine binding and get a permanent dipole moment, thus their conformation, interactions, mobility and activity may be sensitive to the membrane potential. we induced depolarization on ft 7.10 t lymphoma cells by increasing the ec. k+ level or by blocking kv 1.3 voltage gated k+ channels with margatoxin. fcs measuremens showed that the lateral mobility of fab-labeled il-2/ 15r and mhc i and ii decreased upon depolarization, while that of gpi-linked cd48 did not change. fret efficiency measured between some elements of the il-receptor/mhc cluster increased, which may reflect an increase of cluster size. il-2-induced receptor activity, as monitored by measuring stat5-phosphorylation, increased upon depolarization, whereas il-15 induced phosphorylation did not change. our results may reveal a novel regulatory mechanism of receptor function by the membrane potential. cytokines play an important role in t cell activation and immunological memory, whereas mhcs are known for the role in antigen presentation. we applied rnai to silence the expression of mhc i in order to study its possible role in receptor assembly and function. fret data indicated that the association of il-2r and il-15r with mhc i as well as between il-2r and il-15r weakened. fcs indicated an increase of receptor mobility also suggesting the partial disassembly of the clusters. mhc i gene silencing lead to a remarkable increase of il-2/il-15 induced phosphorylation of stat5 transcription factors. in search for the molecular background of this inhibition of signaling by mhc i we checked il-2 binding and the formation of the receptor complex (il-2r alpha -il-2r beta association), but we did not find a difference as compared to the control. our results suggest that mhc i plays an organizing role in maintaining supramolecular receptor clusters and inhibits il-2r signaling, revealing a nonclassic new function of mhc i beyond its classical role in antigen presentation. interleukin-4 (il-4) is an important cytokine involved in adaptive immunity. il-4 binds with high affinity the singlepass transmembrane receptor il-4ra. the occupied complex, il-4/il-4ra then engages either il-2rc or il-13ra1, to form an activated type i or ii receptor, respectively. this formation of heterodimers is believed to trigger cross-activation of intracellular janus kinases. here we follow a fluorescently labeled ligand through various stages of receptor activation in hek293t: using fluorescence correlation spectroscopy (fcs), we see that the receptor chains diffuse as monomers within the plasma membrane. using dual-color fccs provides direct evidence for ligand induced co-diffusion of occupied il-4ra and il-13ra1. in contrast, type i complexes containing il-2rc could not be observed. however, ectopic expression of gfp-tagged il-2rc/jak3 induced stable fluorescent speckles in or close to the plasma membrane. we identified these structures as early sorting endosomes by colocalization of surface markers like eea1 and rab gtpases. the il-4ra chain is continuously trafficking into these compartments. these observations suggest that the formation of a type i il-4r heterodimer may require internalization and that early endosomes serve as a platform for il-4 signaling. among the membrane associated proteins, the ras family, which is lipid-anchored g protein, plays a key role in a large range of physiological processes and, more importantly, is deregulated in a large variety of cancer. in this context, plasma membrane heterogeneity appears as a central concept since it ultimately tunes the specification and regulation of ras-dependent signaling processes. therefore, to investigate the dynamic and complex membrane lateral organization in living cells, we have developed an original approach based on molecule diffusion measurements performed by fluorescence correlation spectroscopy at different spatial scales (spot variable fcs, svfcs) (1). we have shown in a variety of cell types that lipidbased nanodomains are instrumental for cell membrane compartmentalization. we have also observed that thesenanodomains are critically involved in the activation of signaling pathways and are essential for physiological responses (2-3). more recently, weextend the application of svfcs to characterizethe dynamics of k-rasproteinat the plasma membrane. as major result, we demonstrated that the rate of k-ras association/dissociation from the membrane is fast but vary as a functional of the activation state of the molecule as well as of specific intracellular protein interactions. we have so demonstrated that an helical lid sub-domain in the sbd is essential for monomeric as binding, but not for the anti-aggregation activity of the chaperone, suggesting that hsp70 is able to interact with pre-fibrillar oligomeric species formed during as aggregation and that, then, the mechanism of binding for these species is different from that of the monomeric protein. aggregation of the acylphosphatase from sulfolobus solfataricus (sso acp) into amyloid-like protofibrils is induced by the establishment of an intermolecular interaction between a 11-residue unfolded segment at the n-terminus and the globular unit of another molecule. we have used data from hydrogen/deuterium exchange experiments, intermolecular paramagnetic relaxation enhancements and isothermal titration calorimetry measurements on an aggregation-resistant sso acp variant lacking the 11-residue n-terminus to characterize the initial steps of the aggregation reaction. under solution conditions that favour aggregation of the wild-type protein, the truncated protein was found to interact with a peptide corresponding to the n-terminal residues of the full length protein. this interaction involves the fourth strand of the main b-sheet structure of the protein and the loop following this region and induces a slight decrease in protein flexibility. we suggest that the amyloidogenic state populated by sso acp prior to aggregation does not present local unfolding but is characterized by increased dynamics throughout the sequence that allow the protein to establish new interactions, leading to the aggregation reaction. amyloid-like aggregates alter the membrane mobility of gm1 gangliosides martino calamai and francesco pavone university of florence, lens -european laboratory for non-linear spectroscopy, sesto fiorentino, florence, italy neuronal dysfunction in neurodegenerative pathologies such as alzheimer's disease is currently attributed to the interaction of amyloid aggregates with the plasma membrane. amongst the variety of toxic mechanisms proposed, one involves the binding of amyloid species to gm1 gangliosides. gm1 takes part into the formation of membrane rafts, and exerts antineurotoxic, neuroprotective, and neurorestorative effects on various central neurotransmitter systems. in this study, we investigated the effects of amyloid-like aggregates formed by the highly amyloidogenic structural motif of the yeast prion sup35 (sup35nm) on the mobility of gm1 on the plasmamembrane of living cells. preformed sup35nm aggregates were incubated with cells and gm1 molecules were subsequently labeled with biotinylated ctx-b and streptavidin quantum dots (qds). single qds bound to gm1 were then tracked. the mobility of gm1 was found to decrease dramatically in the presence of sup35nm aggregates, switching from brownian to mainly confined motion. the considerable interference of amyloid-like aggregates with the lateral diffusion of gm1 might imply a consequent loss of function of gm1, thus contributing to explain the toxic mechanism ascribed to this particular interaction. insights into the early stages of fibrillogenesis of insulin using mass spectrometry harriet l. insulin is a vital hormone in metabolic processes as it regulates the glucose levels in the body. insulin is stored in the b cells of the pancreas as a hexamer, however its biologically active form is the monomer. the formation of fibrillar aggregates of insulin rarely occurs in the body; however localised amyloidosis at the site of injection for diabetes patients and aggregation of pharmaceutical insulin stocks present problems. in the current study oligomers formed early in the process of fibril assembly in vitro are observed by mass spectrometry (ms). ms is the only technique which allows early species to be characterised as it can identify different oligomeric orders by mass to charge ratio and show protein abundance and aggregation propensity. on mobility ms is used to examine rotationally averaged collision cross sections of oligomers in the aggregating solution. a wide array of oligomers is observed and the stability of specific species is remarked. the presence of multiple conformations for the highly charged oligomers is particularly noted and their assignment confirmed using fourier transform ion cyclotron resonance ms and collision induced dissociation. molecular modelling has been used to further explore the conformational space the oligomers inhabit. amyloid fibrils consisting of different proteins have been recognized as an accompanying feature of several neurodegenerative diseases. many proteins without known connection to any diseases have been found to form amyloid fibrils in vitro, leading to suggestion that the ability to form fibrils is the inherent property of polypeptide chain. the observed common character of protein amyloid formation enables to seek further clues of fibrillation mechanism by studying generalized sequenceless polypeptide models, e.g. polylysine. we have studied conformational transitions of polylysine, with different chain length at various ph, ionic strength and temperature by means of novel approachviscometric method. this polypeptide undergoes alfa-helix to beta-sheet transition and forms amyloid fibrils in special conditions. temperature induced a-helix to b-sheet transitions occurs at ph interval form 10 to 11.8 and with increasing chain length is slightly shifted to the lower ph. we have found narrow ph interval, in which the thermal transition is fully reversible, suggesting the high sensitivity of polypeptide conformation on subtle changes in charge on its side chains. this work was supported within the projects vega 0079,0038 and 0155, cex sas nanofluid and by esf project no. 26220120033. understanding the mechanisms of the conversion from the native state of a protein to the amyloidal state represents a fundamental step in improving the purification, storage and delivery of protein-based drugs and it is also of great relevance for developing strategies to prevent in vivo protein aggregation. amyloid fibrils have a structural arrangement of cross b-sheet but they can also experience different packing into three dimensional superstructures, i.e. polymorphism. it is well known that, among others, both the geometric confinement of the molecules and shear forces can affect the final morphology of the aggregates. importantly, due to the complexity and crowding of the cellular region, such parameters also play a crucial role in in vivo processes. we present an experimental approach to study in vitro amyloid aggregation in a controlled and uniform shear force field and within microscale environments. in particular we focus on the effect of these two parameters on the formation of spherical aggregates, known as spherulites. using micro channels of different cross-sections from 5 to1000 lm x 12 lm and flow rates in the range of hundreds of ll/min, the number and diameter of spherulites within the channels have been characterized using crossed polarizers optical microscopy. inhibition of insulin amyloid fibrillization by albumin magnetic fluid k. insulin amyloid aggregation causes serious problems for patient with insulin dependent diabetes undergoing long-term treatment by injection, in production and storage of this drug and in application of insulin pumps. recent studies indicate that protein amyloid aggregation causes the cell impairment and death; however, the prevention of amyloid aggregation is beneficial. we have investigated ability of albumin magnetic fluid (amf) to inhibit insulin amyloid aggregation by spectroscopic and microscopic techniques. albumin magnetic fluid consists of magnetic fe 3 o 4 nanoparticles sterically stabilized by sodium oleate and functionalized with bovine serum albumin (bsa) at various weight ratios bsa/fe 3 o 4 . we have found the positive correlation between inhibiting activity of afm and nanoparticle diameter and zeta potential. the ability of amf to inhibit formation of amyloid fibrils exhibits concentration dependence with ic 50 values comparable to insulin concentration. the observed features make amf of potential interest as agents effective in the solving of problems associated with insulin amyloid aggregation. (this work was supported within the projects vega 0079, 0155 and 0077, cex sas nanofluid, apvv-0171-10, sk-ro-0012-10 and esf project 26220220005). amyloid formation of peptides causes diseases like alzheimer's and parkinson's disease. however, the conditions for the onset of the neurotoxic beta-sheet formation are poorly understood. we focus on aggregation triggers and their interplay: interactions with hydrophobic-hydrophilic interfaces, orientation of peptides in 2d, metal ion complexation and lipid layers. the tailor-made model peptides exhibit defined secondary structure propensities and metal ion binding sites. the interactions of the peptide with the air-water interface and with metal ions are studied using surface sensitive methods connected to film balance measurements. x-ray diffraction, x-ray reflection, infrared reflection-absorption spectroscopy and total reflection x-ray fluorescence were applied to reveal the layer structure, peptide conformations and metal ion binding at the interface. we found that amyloid formation in 2d is dominated by the hydrophobic-hydrophilic interface and not comparable to the bulk behaviour. the interface can enhance or inhibit betasheet formation. the effect of metal ion complexation depends on the arrangement of the binding sites in the peptide and the preferred metal complexation geometry. the two triggers interface and metal ion complexation, can oppose each other. effect of apoe isoform and lipidation status on proteolytic clearance of the amyloid-beta peptide hubin, e. alzheimer's disease (ad) is the most common type of dementia in the elderly. the most important genetic risk factor identified for ad is the isoform, e2, e3 or e4, of apolipoprotein e (apoe), a lipid-carrying protein. one hallmark of ad is the accumulation of amyloid-beta peptide (ab) in the brain which is thought to result from an imbalance between the production of ab and its clearance. previous studies report an important role for apoe in ab degradation. we sought to determine the effect of apoe isoform and lipidation status on the degradation of soluble ab by proteinases such as insulin-degrading enzyme and neprilysin. in this study an in vitro ab clearance assay based on the competition between ab and a fluorogenic peptide substrate is developed to quantify ab degradation. to elucidate the proteolytic clearance mechanism, the fragments resulting from cleavage are identified by mass spectrometry and further analyzed to identify the interacting stretch of the ab sequence with the different apoe isoforms. the results suggest that apoe influences the rate of ab degradation. the aggregation of proteins into fibrillar nanostructures is a general form of behaviour encountered for a range of different polypeptide chains. the formation of these structures is associated with pathological processes in the context of alzheimer's and parkinson's diseases but is also involved in biologically beneficial roles which include functional coatings and catalytical scaffolds. this talk focuses on recent work directed at understanding the kinetics of this process through the development and application of experimental biophysical methods and their combination with kinetic theories for linear growth phenomena. lbs contains not only as, but also other proteins including 14-3-3 proteins. 14-3-3 proteins exist mainly as a dimer and its exact functions are remain unclear. however, recent work has shown that the association of 14-3-3(eta) with as in lbs. herein we show how 14-3-3(eta) can modulate as in vitro aggregation behavior, by rerouting it toward the formation of stable non-fibrillar aggregates. we also show that the resulting populations of fibrillar and pre-fibrillar aggregates exhibit a modified toxicity in vivo with respect to the unperturbed aggregates. interestingly, 14-3-3(eta) does not show any binding affinity for monomeric as, nor for the mature fibrillar aggregates. we provide evidence that it acts on the oligomeric species which form during the amyloidogenesis process of aggregation. since 14-3-3(eta) can influence the toxicity of amyloidogenesis without perturbing the functional as monomers, we are convinced that once fully understood, its mode of action could represent a promising model to mimic with synthetic drugs and peptides. what makes an amyloid toxic: morphology, structure or interaction with membranes? more than 40 human diseases are related to amyloids. in order to understand why some amyloids may become toxic to their host and some others are not, we first developed a genetical approach in the yeast saccharomyces cerevisiae. we have chosen the amyloid/prion protein het-s prion forming domain (218-289) from the fungi podospora anserina, which is not toxic in yeast. some toxic amyloids mutants were generated by random mutagenesis. in vitro the most toxic mutant called ''m8'' displays very peculiar nanofibers, which polymerized mainly in amyloid antiparallel b-sheets whereas the non-toxic wt exhibits a parallel polymerization. we further established the dynamic of assembling of the m8 toxic amyloid, in comparison to the wt non-toxic amyloid, and showed the presence of specific oligomeric intermediates also organized in antiparallel b-sheet structures. a more global structure/toxicity study on more than 40 mutants clearly identified an antiparallel b-sheet signature for all the toxic mutants. therefore size, intermediates and antiparallel structures may account for amyloid toxicity in yeast but we still wonder what their cellular targets are. recently, we established the first evidences that toxic mutants may specifically bind in vitro to lipids, particularly negatively charged. interconnected mechanisms in abeta (1-40) we present an experimental study on the fibril formation of ab(1-40) peptide at ph 7.4. the kinetics of this process is characterized by the occurrence of multiple transient species that give rise to final aggregates whose morphology and molecular structure are strongly affected by the growth conditions. to observe in details the aggregation pathway as a function of solution conditions, we have used different experimental techniques as light scattering, thioflavin t fluorescence, circular dichroism and two-photon fluorescence microscopy. this approach gives information on the time evolution of conformational changes at molecular level, on the aggregates/fibrils growth and on their morphologies. the selected experimental conditions allowed us to highlight the existence of at least three different aggregation mechanisms acting in competition. a first assembly stage, which implies conformational conversion of native peptides, leads to the formation of small ordered oligomers representing an activated conformation to proceed towards fibril growth. this process constitutes the rate limiting step for two distinct fibril nucleation mechanisms that probably implicate spatially heterogeneous mechanisms. the formation of amyloid fibrils of amylin 10-29 was studied by means of molecular dynamics (md) and energy partition on three peptide ß-sheet stack systems with the same amino acid composition: wild type amylin 10-29 (amyl 10-29), reverse amylin 10-29 (rev-amyl 10-29) and scrambled amylin version scr-amyl 10-29. the results show that for amylin10-29 peptides, amino acid composition determines the propensity of a peptide to form amyloid fibrils independent of their sequence. the sequence of amino acids defines the shape and the strength of amyloid protofibril, which conforms with the atom force microscope (afm) data [1] . md show that the 6x6revamyl-10-29 stack has looser selfassembly than the 6x6amyl-10-29 stack, which conforms with the results of fourier transform infrared spectroscopy (ftir) measurements for the peptides studied [1] . the results of md show that 6x6amyl-10-29 could have a turn, which consists with ftir data [1] . data on ab aggregation kinetics have been characterized by a large spread between experiments on identical samples that contain so many molecules that stochastic behaviour is difficult to explain unless caused by uncontrolled amounts of impurities or interfaces. we have therefore spent considerable effort to eliminate sources of inhomogeneity and reached a level of reproducibility between identical samples and between experiments on separate occasions that we can now collect data that can lead to mechanistic insights into the aggregation process per se, and into the mechanism of action of inhibitors. data on ab42 aggregation will be shown that give insight into the influence of physical parameters like peptide concentration, shear and ionic strength, as well as the effect of inhibitory proteins, model membranes and the effects of sequence variations. monte carlo simulations of amyloid formation from model peptides corroborate the finding from experiments and underscore that the very high level of predictability and reproducibility comes from multiple parallel processes. negatively-charged membranes were reported to catalyze ''amyloid-like'' fiber formation by non-amyloidogenic proteins [1] . our study aims to elucidate the factors that govern the formation of these amyloid-like fibers. lysozyme was selected as a model of non-amyloidogenic protein and was fluorescently-labeled with alexa fluor 488 (a488-lz). first, a488-lz partition towards phosphatidylserine-containing liposomes was characterized quantitatively using fluorescence correlation spectroscopy (fcs), in order to calculate the protein coverage of liposomes. secondly, the interaction between a488-lz and negatively-charged lipid membranes was studied using both steady-state and time-resolved fluorescence techniques. this interaction was found to switch from a peripheral binding to the anionic headgroups, at high lipid/ protein molar ratio (l/p), to a partial insertion of protein into the hydrophobic core of the membrane, at low l/p. finally, the lipidprotein supramolecular complexes formed at low l/p were characterized by fluorescence lifetime imaging microscopy (flim). the mean lifetime of a488-lz in these supramolecular structures is much lower compared to the values obtained for the free and bound a488-lz at high l/p. the fiber characterization will be complemented by fcs studies. [ the conversion of normal prp c to its pathological isoform prp sc is a key event in prion diseases and is proposed to occur at the cell surface or more probably in acidic late endosomes. a convergence of evidence strongly suggests that the early events leading to the structural conversion of the prp seem to be in relation with more or less stable soluble oligomers, which could mediate neurotoxicity. as commonly shared by other amyloidogenic proteins, membrane-bound monomers undergo a series of lipid-dependent conformational changes, leading to the formation of oligomers of varying toxicity rich in b-sheet structures (annular pores, amyloid fibrils). here, we have used a combination of biophysical techniques (dynamic and static light scattering, fluorescence techniques, and quartz crystral microbalance) to elucidate the interaction of native monomeric prp and that of purified b-rich oligomeric prp on model lipid membranes. under well established conditions, three b-sheet-rich oligomers were generated from the partial unfolding of the monomer in solution, which were found to form in parallel. from single mutation and/or truncation of the full length prp, the polymerization pathway is strongly affected, revealing the high conformational diversity of prp. in our previous work, we identify the minimal region of the prp protein leading to the same polymerization pattern of the full length prp. soluble 12-subunits and 36-subunits oligomers were obtained depending on the single mutation or truncation and purified. we compare their structural properties (ftir, cd) when associated with anionic lipid bilayers and study their propensities to permeabilize the membrane. fluorescence kinetics suggest different mechanisms of membrane perturbation for the monomer and the prp oligomers. deciphering this complex network of lipid interactions and conformational diversity of the prp protein will help for understanding of how amyloidogenic proteins induce neurotoxicity. the traditional view of the lipid bilayer described as a ''sea'' of lipids where proteins may float freely, is going to be inadequate to describe the increasingly large number of complex phenomena which are known to take place in biological membranes. membrane-assisted protein-protein interactions, formation of lipid clusters, protein-induced variation of the membrane shape, abnormal membrane permeabilities and conformational transitions of membrane-embedded proteins are only a few examples of the variegated ensemble of events whose tightly regulated cross-talk is essential for cell structure and function. experimental work on the above mentionated problems is very difficult and some time not accessible, especially when the studied systems have a fast dynamics. due to the large size of the systems usually involved in this multifaceted framework, a detailed molecular description of these phenomena is beyond the possibilities of conventional amyloid aggregation, a generic behavior of proteins, is related to incurable human pathologies -amyloid-related diseases, associated with formation of amyloid deposits in the body. all types of amyloid aggregates possess rich bsheet structural motif. the recent data confirm the toxic effect of aggregates on the cells, however, it was found that reduction of amyloid aggregates plays important role in prevention as well as therapy of amyloidosis. we have investigated effect of phytoalexin derivatives on amyloid fibrillization of two proteins, human insulin and chicken egg lysozyme, by tht and ans fluorescence assays. we have found that amyloid aggregation of both studied proteins was significantly inhibited by phytoalexin derivates cyclobrassinin and benzocamalexin. for most effective phytoalexins the estimated ic 50 values were at low micromolar concentration. the observed inhibiting activity was confirmed by transmission electron microscopy. our data suggest the potential therapeutic use of the most effective phytoalexins in the reduction of amyloid aggregation. (this work was supported within the projects vega 0079, 0155, cex sas nanofluid, apvv-0171-10, sk-ro-0012-10 and esf project 26220220005). the amyloid pore hypothesis suggests that interactions of oligomeric alpha-synuclein (as) with membranes play an important role in parkinson's disease. oligomers are thought to permeabilize membranes and interfere with ca 2+ pathways. permeabilization by as requires the presence of negatively charged phospholipids. whether as can bind and permeabilize membranes with physiologically relevant lipid compositions has not been extensively explored. here we report on the binding of as to giant unilamellar vesicles (guvs) with physiologically relevant lipid compositions. comparing different protocols of oligomer preparation, leakage assays on both large unilamellar vesicles (calcein release) and guvs (hpts efflux assay) show that as is not able to permeabilize these membranes. the presence of cholesterol has a stabilizing effect on these membrane systems. in agreement with these findings, we do not observe concentration dependent as toxicity using in vivo mts assays. however, in the calcein release assay, different as preparations show differences in kinetics and as concentrations that cause 100% leakage. these results motivate us to critically reassess the amyloid pore hypothesis, and suggest that membrane permeabilization may be attributable only to a very specific as species. alpha-synuclein oligomers impair membrane integrity-a mechanistic view martin t. stö ckl, mireille m. a. e. claessens, vinod subramaniam nanobiophysics, mesa+ institute for nanotechnology, university of twente, enschede, the netherlands one of the most prevalent neurodegenerative diseases is parkinson's disease (pd), which is accompanied with the loss of dopaminergic neurons. although the mechanisms leading to the death of these cells are still unclear, the protein alpha-synuclein (as) is one of the pivotal factors. previous studies indicate that especially oligomeric forms of as show a detrimental effect on membrane integrity. as an intact membrane is crucial to many cellular processes, the impairment of the membrane integrity is a likely pathway for neuronal death. we use different phospholipid bilayer model systems to investigate the mechanisms underlying this process. atomic force microscopy in combination with suspended asymmetric phospholipid bilayers, which closely mimic the plasma membrane, allows the identification of the binding sites, the measurement of penetration depths of the as oligomers into the phospholipid bilayer, and the detection of membrane thinning or creation of membrane defects. using an approach based on phospholipid vesicles we were able to observe for the first time that as oligomers cause an enhanced lipid-flip flop. suggesting that the loss of lipid asymmetry is a novel mechanism which may contribute to or trigger neuronal death in pd. amyloid protein-membrane interactions and structuretoxicity relationship study h.p. ta (toxic) has a much higher and more specific effect on negatively charged phospholipids (dops, dopi and dopg) than in the case of wt (non-toxic). therefore the insertion of protein into phospholipid monolayers, which occurred similarly for both wt and m8, is not a key factor for these effects (h.p. ta et al. langmuir, in press). we are now using unilamellar vesicles as a membrane model to investigate the amyloid protein (toxic and nontoxic) -phospholipid interactions. results confirmed the high specific and strong effects of m8 on negatively-charged membrane. in this project, we study the chemical, physical and biological properties of fibrillar networks. the formation and the network mechanics are investigated by combining droplet-based microfluidics with optical microscopy and small angle x-ray scattering (saxs). the chosen system, fibrin network formation, plays an important role in blood coagulation processes. crosslinking of fibrinogen induced by an enzymatic reaction with thrombin leads to 3d fibrin network formation. the fibrillar networks are formed within picoliter droplets of aqueous solutions in an continuous oil phase. droplets containing fibrinogen and thrombin can be produced of different sizes and stored for fibrin network formation. the formation of the fibrillar networks is imaged by fluorescence microscopy. to analyze the elastic properties of the networks, the droplets flow through a microchannel device of alternating width in order to squeeze and stretch the networks. additionally, saxs experiments will give structural information about the molecular dimensions of the networks. the amyloid beta peptide (ab), implicated in alzheimer's disease (ad), is released from the amyloid precursor protein (app) by secretase-induced cleavage. this process results in the release of a range of ab peptides varying in length. the brains of ad patients often contain longer ab peptides while the total concentration of ab is unaffected. longer peptides are more hydrophobic having far-reaching consequences for their toxicity and aggregation. as ab is necessary for normal neuronal function, research activities into ad therapeutic development currently explore the possibilities of modulating c-secretase activity to produce short ab peptides. whether such an approach effectively ameliorates the toxic effect of ab has not been explored yet. to answer this question, we studied the impact of heterogeneity in ab pools in an in vitro biophysical and in cellulo context using microelectrode array to assay the synaptic activity of primary neurons. we show that various lengths of the ab peptide and mixtures thereof aggregate with distinct kinetics and notoriously affect synaptotoxic and cytotoxic response. we also show that small amounts of less abundant peptides ab38 and ab43 induce aggregation and toxicity of ab40 while the behavior of ab42 is unaffected. one of the most important irreversible oxidative modifications of proteins is carbonylation, a process of introducing the carbonyl group in reaction with reactive oxygen species. importantly, carbonylation increases with the age of cells and is associated with the formation of intracellular protein aggregates and the pathogenesis of age-related disorders such as neurodegenerative diseases and cancer. however, it is still largely unclear how carbonylation affects protein structure, dynamics and aggregability on the atomic level. here, we use classical molecular dynamics simulations to study structure and dynamics of the carbonylated headpiece domain of villin, a key actin-organizing protein. we perform an exhaustive set of molecular dynamics simulations of native villin headpiece together with every single combination containing carbonylated versions of its seven lysine, arginine and proline residues, the quantitatively most important carbonylable amino acids. surprisingly, our results suggest that high levels of carbonylation, far above those associated with cell death in vivo, may be required to destabilize and unfold protein structure through the disruption of specific stabilizing elements, such as salt bridges or proline kinks, or tampering with the hydrophobic effect. on the other hand, by using thermodynamic integration and molecular hydrophobicity potential approaches, we quantitatively show that carbonylation of hydrophilic lysine and arginine residues is equivalent to introducing hydrophobic, chargeneutral mutations in their place, and, by comparison with experimental results, demonstrate that this by itself significantly increases intrinsic aggregation propensity of both structured, native proteins and their unfolded states. finally, our results provide a foundation for a novel experimental strategy to study the effects of carbonylation on protein structure, dynamics and aggregability using site-directed mutagenesis. septins are an evolutionarily conserved family of gtp-binding proteins involved in important cellular processes, such as cytokinesis and exocytosis, and have been implicated in neurological diseases, such as alzheimer's and parkinson's diseases. the focus of this study was two septins of schistosoma mansoni, (the causative agent of schistosomiasis in south america) named smsept5 and smsept10, which were produced in a recombinant system. our objective was to verify if these septins from a simpler organism display similar characteristics to human septins. analysis of protein structure by circular dichroism showed that both recombinant smseptins produced were folded. the gtpase activity assay showed that smsept5 was able to hydrolyze gtp, whereas smsept10 was not. aggregation studies for amyloid fibril detection by right angle light scattering and thioflavin t fluorescence assay were performed. both proteins showed a temperature dependent increase in light scattering and fluorescence emission of tht probe. this indicated that s. mansoni septins tend to aggregate into amyloid-like fibers in high temperatures, with thresholds of 30°c for smsept5 and 37°c for smsept10. these results are in accordance to that previously reported for human septins. in our work we investigated the response to standard chemotherapy of blood lymphocytes of patients suffering with melanoma. dna single and double strand breaks were determined using comet assay; intracellular levels of marker proteins were detected using immunocytochemistry. ultimately this set of parameters allows to characterize two mechanisms of dna repair (base excision repair, ber and mismatch repair, mmr) which together with apoptosis proneness underlie response of tumor cells to chemotherapy. cell death caused by o 6 mg adducts is promoted by mmr system by inducing unrepaired double strand breaks in dna. there was a linear correlation between the level of dsdna breaks in lymphocytes after 1-st cycle of chemotherapy and mmr efficiency in them. the level of double strand breaks in dna after 1-st cycle of chemotherapy is predictive of clinical outcome. otherwise damage at the level of ssdna (ap-sites and single strand breaks) and ber mechanism associated with it couldn't be a good prognostic factor of chemotherapy. high level of double strand breaks in dna in blood lymphocytes of melanoma patients 48 hours after 1-st cycle of chemotherapy appears to be a marker of a good prognosis. self-assembly and stability of g-quadruplex: counterions, pressure and temperature effects e. baldassarri jr., p. mariani, f. spinozzi, m. g. ortore saifet dept. & cnism, marche polytechnic university, ancona, italy the important role of g-quadruplex in biological systems is based on two main features: composition and stability of telomeres, and activity of telomerase. the g-quadruplex structures are formed by supramolecular organization of basic units called g-quartets that are planar rings constituted by four guanosines linked by hoogsten hydrogen bonds. gquadruplex requires the presence of monovalent cations playing a key role in stabilizing these structures, since they give rise to coordination bonds needed for the stacking of more tetrads. we performed x-ray diffraction experiments at different pressures (ranging from 1 to 2000 bar), and small angle x-ray scattering (saxs) changing the temperature (between 20-60°c retinoic acid receptor (rar) is a member of the nuclear receptor superfamily. this ligand-inducible transcription factor binds to dna as a heterodimer with the retinoid x receptor (rxr) in the nucleus. the nucleus is a dynamic compartment and live-cell imaging techniques make it possible to investigate transcription factor action in real-time. we studied the diffusion of egfp-rar by fluorescence correlation spectroscopy (fcs) in order to uncover the molecular interactions determining receptor mobility. in the absence of ligand we identified two distinct species with different mobilities. the fast component has a diffusion coefficient of d 1 = 1.8-6 lm 2 /s corresponding to small oligomeric forms, whereas the slow component with d 2 = 0.06-0.12 lm 2 /s corresponds to interactions of rar with the chromatin or other large structures. the rar ligand binding domain fragment also has a slow component probably as a result of indirect dna-binding via rxr, with lower affinity than the intact rar:rxr complex. importantly, rar-agonist treatment shifts the equilibrium towards the slow population of the wild type receptor, but without significantly changing the mobility of either the fast or the slow population. by using a series of mutant forms of the receptor with altered dna-or coregulator-binding capacity we found that the slow component is probably related to chromatin binding, and that coregulator exchange, specifically the binding of the coactivator complex, is the main determinant contributing to the redistribution of rar during ligand activation. formation of inactive nuclear with high level of dna compaction in sperm cells is accompanied by a substitution of linker histones h1 by a number of other proteins. among them sperm-specific histones (ssh), which are characterized by elongated arginine-rich polypeptide chain compared to the somatic h1. the secondary and tertiary structure of the ssh and their interactions with dna were studied using spectroscopic and thermodynamic approaches. the histones were isolated from sperm of marine invertebrates and rat thymus. all studied ssh demonstrate no considerable compaction of dna in solutions of low ionic strength. however, at physiological conditions, ssh h1 from s.intermedius and a.japonica compact dna more intensively than other ssh. the somatic h1 from rat thymus revealed a minimal ability to compact the dna. we suggest that the ssh h1 are able to interact with dna not only in the major groove but also in the minor groove of the double helix inducing considerable structural changes in dna and facilitating the formation of the supercompact sperm chromatin. the authors are grateful for the financial support from the russian foundation for basic research (grants § 10-04-00092 and 09-08-01119) and from administration of saint-petersburg. ionizing radiation causes modification and destruction of nitrogenous bases in dna molecule. there are also local breakages of hydrogen bonds both in the lesion sites mentioned above and in other sites of the macromolecule. to reveal the amount of some of these damages we applied cd and uv absorption spectroscopy. radiation-induced changes in dna structure influence its uv absorption spectrum in different ways: partial denaturation causes hyperchromic effect, while destruction of the bases results in hypochromic shift. at the same time both of them result in the same changes in dna cd spectra: the decrease in intensity. we attempted to segregate the described damages in dna structure and studied the influence of dna ionic surroundings on the radiation effect. it is shown that the radiation efficiency of base destruction and partial denaturation increases with decreasing concentration of nacl in irradiated solution. udu (ugly duckling) has been first identified from a zebrafish mutant and shown to play an essential role during erythroid development; however, its roles in other cellular processes remain largely unexplored. facs analysis showed that the loss of udu function resulted in defective cell cycle progression and comet assay indicated the presence of increased dna damage in udu mutants. we further showed that the extensive p53-dependent apoptosis found in udu mutants is a consequence of activation in the atm-chk2 pathway. udu appears not to be required for dna repair, because both wild-type and udu embryos similarly respond to and recover from uv treatment. yeast two-hybrid and coimmunoprecipitation data demonstrated that pah-l repeats and sant-l domain of udu interacts with mcm3 and mcm4. furthermore, udu was colocalized with brdu and heterochromatin during dna replication, suggesting a role in maintaining genome integrity. recently, we started to work on the second zebrafish homolog, udu2, and its mammalian counterpart, gon4l. preliminary data showed that udu2 and gon4l mrna injection can rescue zebrafish udu mutant phenotypes. furthermore, pah-l and sant-l domains of udu2 and gon4l can bind to mcm3 and mcm4 and they are localized in the nucleus. these data suggest that udu2 and gon4l are functionally equivalent to zebrafish udu. their molecular mechanism leading to udu phenotypes is currently under investigation. chromatin condensation: general polyelectrolyte association and histone-tail specific folding nikolay korolev, nikolay berezhnoy, abdollah allahverdi, renliang yang, chuan-fa liu, james p. tam, lars nordenskiö ld school of biological sciences, nanyang technological university, 60 nanyang drive, 637551, singapore the major component of chromatin, dna, is a densely charged polyanion. electrostatic interactions between dna and dnapackaging proteins contribute decisively to formation of its elementary unit, the nucleosome, and are also important in chromatin folding into higher-order structures. we investigate condensation of dna and chromatin and find that electrostatics and polyelectrolyte character of dna play dominant role in both dna and chromatin condensation. by comprehensive experimental studies and using novel oligocationic ligands, we suggest simple universal equation describing dna condensation as a function of oligocation, dna and monovalent salt concentrations and including the ligand-dna binding constant. we found that similar dependence was also observed in condensation of the nucleosome arrays. next, we studied how general electrostatic and specific structural alterations caused by lysine acetylations in the histone tails influence formation of 30-nm chromatin fibre and intermolecular nucleosome array association. for the first time, a structural model is suggested which explains critical dependence of chromatin fibre folding on acetylation of the single lysine at position 16 of the histone h4. exceptional importance of the h4 lys 16 acetylation in general and gene specific transcriptional activation has been known for many years but no structural basis for this effect has yet been proposed. detection of specific dna sequences is central to modern molecular diagnostic. ultrasensitive raman measurements of nucleic acids are possible through exploiting the effect of surface-enhanced raman scattering (sers). in this work, the sers spectra of genomic dnas from leaves of different apple trees grown in the field, have been analyzed [1] . a detailed comparative analysis of sers signatures of genomic dnas is given. sers wavenumbers (cm -1 ) are reported here for all types of vibrations of plant genomic dnas, including bands assigned to localized vibrations of the purine and pyrimidine residues, localized vibrations of the deoxyribosephosphate moiety, etc. proposed band assignments are given. a strong dependence of the sers spectra on dna concentration and on time have been observed. in biochemical fields, nucleic acids might be used to explore the interaction between dna and small molecules, which is important in connection with probing the accurate local structure of dna and with understanding the natural dnamediated biological mechanisms [2] . the ph-dependent structure of dna studied by fourier transform infrared spectroscopy the region of the infrared spectrum studied covered the wave number range from 4000 cm -1 to 700 cm -1 . ir spectra show that in ph 8.0-9.0 interval carbonyl (c=o) band at 1689 -1 cm (assigned to guanine) is reduced in intensity and slightly shifted to lower frequencies. at the ph 10.0 significantly decreases band intensity at 1712 cm -1 due to unbounded c2=o of thymine and shifts to lower frequencies, indicating at the transition of this group in bounded form, supposedly by means of excess polarized hydroxyl ions. together this, in basic region a new intense absorption band has been observed in 1500-1300cm -1 frequency interval, corresponding to o-h group in-plane bending vibration (1410-1310cm -1 ). as for acidic conditions, it was observed that under the extreme ph (*2.8) value carbonyl absorption region shifts to higher frequencies and absorption intensity significantly increased, indicated at releasing of c=o groups from h-bonding between base pairs. moreover, bands intensity at 1418cm -1 and 1022cm -1 corresponding to out-of-plan deformation of nh 2 groups increased due to rupture of connections between the dna strands. during the last decade it was found that in many cases specific structural organization of multi-molecular protein and dna-protein complexes determines their functioning in living cells. although these functioning structures are usually unique, it is often possible to identify their common structural elements. one of the interesting examples of such universal elements are hmgb domains: structurally conservative functional domains of non-histone proteins hmgb1/2 also identified in many nuclear proteins. using afm, thermodynamic approaches, circular dichroism and molecular absorption spectroscopy in far-uv and mid-ir regions we have studied structural organization of the complexes between dna and different proteins, including hmgb1, hmgb-domain recombinant proteins and linker histone h1. we have demonstrated, that interaction with dna leads to increasing both a-helicity of the proteins and thermal stability of dna. also, this interaction may result in formation of highly ordered supramolecular complexes facilitated by hmgb-domains. the c-terminal sequence of hmgb1/2 regulates affinity of the proteins to dna and can be ''inactivated'' by interaction with histone h1. based on the data obtained a model of the interaction of multy-domain hmgb-proteins with dna is suggested. darmstadt, germany, 4 lmu biozentrum; munich, germany *these authors contributed equally to this work chromatin in living cells displays considerable mobility on a local scale. this movement is consistent with a constrained diffusion model, in that individual loci execute multiple, random jumps. to investigate the connection between local chromatin diffusion (lcd) and the changes in nuclear organization, we established a stable hela cell line expressing gfp-pcna. this protein, a core component of the replication machinery, serves as a cell-cycle marker and allows us to visualize sites of ongoing dna synthesis within the nucleus. to monitor lcd, we labeled discrete genomic loci through incorporation of cy3-dutp. this experimental system, in conjunction with particle tracking analysis, has enabled us to quantitatively measure chromatin mobility throughout the cell cycle. our results demonstrate that lcd is significantly decreased in s-phase. to explore the connection between dna replication and reduced chromatin movement, we undertook a more detailed examination of lcd in s-phase nuclei, correlating chromatin mobility with sites of replication. our results demonstrate that labeled chromatin in close proximity to gfp-pcna foci exhibit significantly decreased mobility. we therefore conclude that presence of active replication forks constrains the movement of adjacent chromatin. single-molecule studies of dna replication antoine m. van oijen zernike institute for advanced materials, groningen university, nijenborgh 4. 9747 ag groningen, the netherlands e-mail: a.m.van.oijen@rug.nl advances in optical imaging and molecular manipulation techniques have made it possible to observe individual enzymes and record molecular movies that provide new insight into their dynamics and reaction mechanisms. in a biological context, most of these enzymes function in concert with other enzymes in multi-protein complexes, so an important future direction will be the utilization of single-molecule techniques to unravel the orchestration of large macromolecular assemblies. we are applying a single-molecule approach to study dna replication. i will present recent results of single-molecule studies of replication in bacterial and eukaryotic systems. by combining the stretching of individual dna molecules with the fluorescence observation of individual proteins, we visualize the dynamic interaction of replication factors with the fork. in the bacteriophage t7 replication system, we show that dna polymerases dynamically associate with and dissociate from the fork during replication. further, i will present new data from single-molecule replication studies in x. laevis oocyte extracts. we have developed a novel imaging scheme that permits single-molecule fluorescence experiments at concentrations of labeled protein that were hitherto inaccessible. using this method, we visualize, in real time, origin firing and fork movement. in force-extension diagrams of reference models of naked dna (freely jointed chain, wormlike chain) as well as extensionrotation diagrams of naked dna have been successfully recovered. of note, plectonemic structures are most efficiently simulated thanks to ode's collision detection code. new insights into nucleosome and chromatin fiber structure and dynamics will be presented. the study of the pkm.101 plasmid effect on the repair of dna j. vincze 1 , i, francia 2 , g. vincze-tiszay 1 1 hheif, budapest, hungary, 2 univ. debrecen, debrecen, hungary in our experiments was studied the effect of pkm.101 plasmid on repair of single strand breaks in dna induced by 60cogamma irradiation in e.coli k 12 ab 1157 (wild type) and its different rec mutant cells. the pkm.101 resistant-factor in case of the control decreases the sensitivity of radiation, which as we suppose, is reached by the help of dna conformation change. it can be supposed from the well known effect of radiation biology that by the effect of pkm.101, the ratio of dna radiation sensitive volumes by appearing its new conformation decreases. the pkm.101 r-factor in rec mutants in case of gamma irradiation shows effects in two ways. one is the ''chemical'' connection between the r-factor and dna, though the other relate to positive and negative ''induced'' radiation resistance from the local type effect of the connection of an r-factor and a rec mutant, and the two radiation resistant effects are added algebraically. as a result from the view of biology we have to categorize the radiation resistance and the connected repair processes as two different classes according to the change either in the chemical or in the induced radiation resistant effect. recent studies have indicated that two trimethylated peptides (k6, k7), derived from the parental hybrid peptide ca(1-7)m(2-9), strongly interact with a bacterial membrane model (mixture of zwitterionic and negatively charged lipids), but not with a membrane model of mammalian erythrocytes (zwitterionic lipids) [1] . a reduction of the cytotoxicity effect and an improvement of the therapeutic index have also been reported for the derivatives when compared with the parental ca(1-7)m(2-9) [2] . in this work, with the aim of providing insight on the interaction phenomena of the indicated peptides with zwitterionic and negatively charged membrane models, a systematic molecular dynamics study was carried out. full hydrated bilayers of dmpc:dmpg (3:1) and pope:popg(3:1) were studied in the presence of each peptide, and results analyzed in terms of peptide structure and membrane composition. lipid-water and lipid-lipid interactions at the membrane/water interface play important role in maintaining the bilayer structure, however, this region is not easily available for experimental studies. we performed molecular dynamics simulations of two bilayers composed of two different types of lipids: (1) dioleoylphosphatidylcholine (dopc); (2) galactolipid monogalactosyldiacylglycerol (mgdg). to investigate the properties of the membrane/water interface region, we performed analysis of lipid-lipid interactions: direct, via charge pairs (dopc) and hydrogen bonds (mgdg) as well as indirect, via water bridges. we also examined water-lipid interactions. existence of well-defined entities (lipids) linked by different types of interactions (hydrogen bonds, charge pairs, water bridges) makes the analysis of the membrane/ water interface region a suitable for a graph theoretical description. we applied a network analysis approach for comparative analysis of simulated systems. we note a marked difference between the organization as well as the dynamics of the interfacial region of the two bilayers. l-nucleoside analogues form an important class of antiviral and anticancer drug candidates. to be pharmacologically active, they need to be phosphorylated in multiple steps by cellular kinases. human phosphoglycerate kinase (hpgk) was shown to exhibit low specificity for nucleotide diphosphate analogues and its catalytic efficiency in phosphorylation was also affected. to elucidate the effect of ligand chirality on dynamics and catalytic efficiency, molecular dynamics simulations were performed on four different nucleotides (d-/l-adp and d-/l-cdp) in complex with hpgk and 1,3-bisphospho-d-glycerate (bpg). the simulation results confirm high affinity for the natural substrate (d-adp), while l-adp shows only moderate affinity for hpgk. the observed short residence time of both cdp enantiomers at the active site suggests very weak binding affinity which may result in poor catalytic efficiency shown for hpgk with d-/l-cdp. analysis of the simulations unravels important dynamic conditions for efficient phosphorylation replacing the single requirement of a tight binding. using the van der waals density functional based on the semilocal exchange functional pw86 together with a longrange component of the correlation energy [1] implemented in the siesta program code, we have calculated the band structure of the double stranded dna. the unit cell was built by taking together 10 gc (or at) homogenous base pairs and we have considered the translational symmetry as the periodic boundary condition. the results obtained are compared with the oligomer calculations taking up to seven base pairs. the band structure obtained with this van der waals density functional is also compared with results obtained with other exchange-correlation functionals as well as with band structure obtained by the hartree-fock crystal-orbital method taking into account the helical symmetry of the double stranded dna. the role of different parts of dna (base pairs, sugar-phosphate backbone, na ions) is also presented. transmembrane (tm) proteins comprise some 15% to 30% of the proteome but owing to technical difficulties, relatively few of these structures have been determined experimentally. computational modeling techniques can be used to provide the essential structural data needed to shed light on structurefunction relationships in tm proteins. low-resolution electrondensity maps, obtained from cryo-electron microscopy (cryo-em) or preliminary x-ray diffraction studies, can be used to restrict the search in conformational space. at the right resolution, the locations of tm helices can be roughly determined even when the amino acids are not visible. when these data are combined with physicochemical characteristics of amino acids (such as their hydrophobicity) and with evolutionary conservation analysis of the protein family, the location of the amino acids can be modeled. the modelstructure may provide molecular interpretations of the effects of mutations. moreover, it can be used to suggest molecular mechanisms and to design new mutations to examine them. the overall approach will be demonstrated using two human proteins: copper transporter 1 (ctr1), which is the main copper transporter in the human cell, and the 18 kda translocator protein (tspo) of the outer mitochondria. modelstructures of these proteins and their functional implications in health and disease will be discussed. calcium channels play a crucial role in many physiological functions and their selectivity mechanism is still an unresolved question and a subject of debate. a physical model of selective ''ion binding'' in the l-type calcium channel is constructed, and consequences of the model are compared with experimental data. this reduced model treats only ions and the carboxylate oxygens of the eeee locus explicitly and restricts interactions to hard-core repulsion and ion-ion and ion-dielectric electrostatic forces. according to the charge/space competition mechanism, the charge of structural ions attracts cations into the filter, while excluded volume effects are trying to keep them out. this is a competition between energy and entropy, where the balance of these terms minimizes free energy and determines selectivity. experimental conditions involving binary mixtures of alkali and/or alkaline earth metal ions are computed. the model pore rejects alkali metal ions in the presence of biological concentrations of ca2+ and predicts the blockade of alkali metal ion currents by micromolar ca2+. conductance patterns observed in varied mixtures containing na+ and li+, or ba2+ and ca2+, are predicted. ca2+ is substantially more potent in blocking na+ current than ba2+. in apparent contrast to experiments sing buffered ca2+ solutions, the predicted potency of ca2+ in blocking alkali metal ion currents depends on the species and concentration of the alkali metal ion, as is expected if these ions compete with ca2+ for the pore. these experiments depend on the problematic estimation of ca2+ activity in solutions buffered for ca2+ and ph in a varying background of ulk salt. equilibrium binding affinity (expressed as the occupancy of the selectivity filter by various ions) is computed by equilibrium grand canonical monte carlo (gcmc) simulations. the conductivity of the channel is estimated from the equilibrium concentration profiles using the integrated nernst-planck equation. our simulations show that the selectivity of l-type calcium channels can arise from an interplay of electrostatic and hard-core repulsion forces among ions and a few crucial channel atoms. the reduced system selects for the cation that delivers the largest charge in the smallest ion volume. we have also performed dynamic monte carlo (dmc) simulations for a model ca channel to simulate current directly and present our results for the dynamical selectivity (expressed as the flux carried by various ions). we show that the binding affinity of ca2+ versus na+ is always larger than the dynamical selectivity because ca2+ ions are tightly bound to the binding site of the selectivity filter of the channel and, at the same time, their mobility and drift velocity is smaller in this region. carotenoids are used in light-harvesting complexes with the twofold aim to extend the spectral range of the antenna and to avoid radiation damages. the effect of the polarity and conformation of the environment is supposed to be responsible for the tuning of the electronic, optical and vibrational properties of peridinin carotenoid both in solution and in protein matrix. we investigate the effect of vibrational properties of peridinin in different solvents by means of vibrational spectroscopies and qm/mm molecular dynamics simulations 1 . the shift of vibrational fingerprints in the 1500-2000 cm -1 frequency region, due to the solvent polarity and proticity, is studied in three cases: cyclohexane (apolar/aprotic), deuterated acetonitrile (polar/aprotic) and methanol (polar/ protic). the frequencies and vibrational modes of the carbonyl, the allene, and the polyene chain were identified using effective normal mode analysis 2 and compared with the present and previous experimental data 3 . on the basis of our calculations and experiments in different solvents, we propose a classification of the four peridinins of the high-salt pcp form. the controlled self-assembly of functional molecular species on well defined surfaces is a promising approach toward the design of nanoscale architectures. by using this methodology, regular low-dimensional systems such as supramolecular clusters, chains, or nanoporous arrays can be fabricated. small biological molecules such as amino acids represent an important class of building blocks that are of interest for molecular architectonic on surfaces because they inherently qualify for molecular recognition and selfassembly [1] . the interaction between amino acids and solid surfaces is decisive for the development of bioanalytical devices or biocompatible materials as well as for a fundamental understanding of protein-surface bonding. we investigate the adsorbtion mechanism of the cysteine on au(111) surfaces by means of the dft [2] . our main concern is to describe the molecule-metal bonding mechanism. therefore we present a complex study, including the full determination of the density of states for the free and adsorbed molecule, the determination of molecule-surface bonding energy. the method of crystal orbital overlap populations is used in order to determine the contribution of specific atomic orbitals to the molecule-metal bond. it is now widely accepted that myoglobin (mb) is not simply an o 2 storage/delivery system but, depending on oxygen concentration, it exerts other fundamental physiological roles. recent studies revealed a widespread expression and, in particular, an over-expression in response to hypoxia, in various non-muscle tissues, including tumor cells. in human five different mb isoforms are present. the two most expressed ([90%) differ only at the 54 th position, k54 (mb-i) and e54 (mb-ii) respectively. since high-altitude natives from tibet are characterized by a higher mb concentration and locomotion efficiency, together with the observation that the mb overexpression is totally attributable to mb-ii, the idea that the latter might be one of the responses to high-altitude evolutionary adaptation, i.e. hypoxic environment, started to emerge. however, this is not yet supported by any structure/function investigation. we performed hundred nanoseconds md simulations on human mbs to investigate the structure and dynamics of both protein and surface water. important differences have been protein kinases play key roles in cell signaling and constitute crucial therapeutic targets. in normal cell, upon substrate binding, tyrosine kinase receptor kit undergoes extensive structural rearrangement leading to receptor dimerization and activation. this process is initiated by the departure of the juxta membrane region (jmr) from the active site, allowing the activation loop (aloop) deployment. the deregulation of kit activity is associated with various forms of cancer provoked by abnormalities in signal transduction pathways. mutations v560g (jmr) and d816h/v (a-loop) have been reported as oncogenic and/or drug-resistant. to contribute further in the understanding of kit activation/ deactivation mechanisms, we applied a multi-approach protocol combining molecular dynamics (md), normal modes analysis (nma) and pocket detection. disturbing structural effects, both local (a-loop) and long-range (jmr), were evidenced for kit d816h/v in the inactive state. nma showed that jmr is able to depart its position more easily in the mutants than in the wild type. pockets analysis revealed that this detachment is sufficient to open an access to the atp binding site. our results provided a plausible conception of mutant dimerization and a way to explore putative allosteric binding sites. transmembrane association of leukocyte integrin heterodimer might be mediated by a polar interaction choon-peng chng 1 and suet-mien tan 2 1 biophysics group, a*star institute of high performance computing, and, 2 school of biological sciences, nanyang technological university, republic of singapore the lateral association of transmembrane (tm) helices is important to the folding of membrane proteins as well as a means for signaling across the cell membrane. for integrin, a hetero-dimeric protein important for cell adhesion and migration, the association of its a-and b-subunits' tm helices plays a key role in mediating bi-directional mechanical signaling across the membrane. we found evidence from experiment and simulation for a polar interaction (hydrogenbond) across leukocyte integrin alb2 tm that is absent in the better-studied platelet integrin aiibb3 [1] . our coarse-grained molecular dynamics simulations of tm helix-helix selfassembly showed more native-like packing achieved by alb2 within the simulation timescale as compared to its 'lossof-function' b2t686g mutant or aiibb3 [2] . association free energy profiles also showed a deeper minimum at a smaller helix-helix separation for alb2, suggestive of tighter packing. the likely conservation of this polar interaction across the b2 integrin family further reinforces its importance to the proper functioning of leukocyte integrins. active extrusion of drugs through efflux pumps constitutes one of the main mechanisms of multidrug resistance in cells. in recent years, large efforts have been devoted to the biochemical and structural characterization of rnd efflux pumps in gram-negative bacteria, in particular the acrb/a-tolc system of e.coli. specific attention has been addressed to the active part of the efflux system, constituted by the acrb unit. despite the presence of several data, crucial questions concerning its functioning are still open. the understanding of the structure-dynamics-function relationship of mexb, the analogous transporter in p. aeruginosa, encounters even more difficulties, because of the lack of structural data of the transporter in complex with substrates. to shade some light on the activity of mexb, we performed computational studies on mexb interacting with two compounds, meropenem and imipenem, the first known to be a good substrate, and the second a modest one. several techniques were used in the present work, ranging from flexible docking [1] to standard and targeted molecular dynamics (md) simulations. starting from the published crystal structure [2] we identified the most probable poses of the two compounds in both the original experimental and in the md-equilibrated structures. we used information from acrb binding pocket in order to find relevant binding sites of the two compounds in the analogous binding pocket of mexb. meropenem frequently lies with appropriate orientation in a pocket similar to the one identified for doxorubicin in acrb [3] , while the occurrence of imipenem poses in the same pocket is very scarce. additionally, when present in the pocket, imipenem is located in a position that renders very unlikely its extrusion toward the oprm docking domain during the simulation of the functional peristalsis. the analysis of the trajectories has provided a complete inventory of the transporter and antibiotic hot spots, which is key information in terms of screening and design of antibiotics and inhibitors. clathrins are three-legged proteins with the intriguing ability to self-assemble into a wide variety of polyhedral cages. the nucleation and growth of a clathrin lattice against the cytosolic face of a cell membrane enables the endocytosis of membrane proteins and various external molecules, by wrapping the membrane around the cargo to produce a coated transport vesicle within the cell. clathrins can also self-assemble, in slightly acidic solutions devoid of auxiliary proteins, into empty cages. our simulations of this process, using a highly coarsegrained model, indicate that the key to self-assembly is neither calthrin's characteristic puckered triskelion shape, nor the alignment of four legs along all cage edges, but an asymmetric distribution of interaction sites around the leg's circumference. based on the critical assembly concentration, the binding strength in these cages is estimated at 25 to 40 k b t per clathrin. the simulations also answer the long-standing conundrum of how flat patches of purely hexagonal clathrin lattices, which in cryo electron microscopy are frequently seen to decorate cell membranes, can produce highly curved cages containing twelve pentagonal faces interdispersed between hexagonal faces. we present experimental evidence supporting this pathway. in eukaryotic cells, the exchange of macromolecules between the cytoplasm and the nucleus is mediated by specialized transport factors. by binding to these transporters, cargo molecules, which are otherwise excluded from entering the nucleus, can traverse the nuclear pore complex efficiently. most of the proteins mediating nuclear import and export exhibit a characteristic _-solenoid fold, which provides them with an unusual intrinsic flexibility. crm1 is an essential nuclear export receptor, which recognizes a very broad range of export cargoes. crm1-dependent nuclear export is ran-gtpase-driven, and recognition of rangtp and cargo is highly cooperative. however, recent crystal structures show that the binding sites for export cargos and rangtp are located at distant parts of crm1 [1] [2] [3] . we have used a combined approach of all-atom molecular dynamics simulations and small-angle x-ray scattering to study rangtp and cargo binding to crm1. we have found that the allosteric effect in crm1-dependent nuclear export arises from a combination of subtle structural rearrangements and changes in the dynamic properties of crm1. light-induced phototactic responds in green algae chlamydomonas reinhardtii are mediated via microbial-type rhodopsins, termed channelrhodopsin-1 (chr1) and channelrhodopsin-2 (chr2)1, which carry the chromophore retinal covalently linked to lysin via a schiff base and were shown to be directly light-gated ion-channels2. the n-terminal putative seven-transmembrane region of chr2 was shown to be responsible for the generation of photocurrents and exhibits sequence similarity to the well understood proton pump bacteriorhodopsin (br) and the sensory rhodopsin anabaena sensory rhodopsin (asr)3. as for the majority of membrane proteins, there is no 3d-structural data for chr2 available yet. here we present homology models of chr2 using two high-resolution x-ray template structures of br (1qhj4) respectively asr (1xio5) in order to get structural and functional insights into chr2. with both homology models we performed molecular dynamics (md) simulations in a native membrane/solvent environment using gromacs 4.0.36. comparison of energetic and structural results revealed obvious advantages of the br-based homology model of chr2. here we show that the br-based homology model is a reliable model of chr2 exhibiting structural features already found experimen-tally7. our br-based homology model of chr2 allows predictions of putative crucial residues within chr2. so we proposed several mutations within the chr2 sequence which are already accomplished. electrophysiologic and spectroscopic studies of these mutations are underway in order to confirm the functional relevance of these residues and to contribute to an optimized usage of chr2 as a powerful tool in optogenetics. ( neuroglobin is a recently discovered globin protein predominantly expressed in brain. its biological function is still elusive. despite the fact that neuroglobin shares very little sequence homology to the well-known globins as mioglobin and hemoglobin, they all have a characteristic globin fold with heme molecule bound to the distal pocket. the structural investigations and co binding kinetics revealed existence of cavities and tunnels within the protein matrix, where small ligands can be stored even for hundreds of microseconds [1] . in human neuroglobin there is one internal disulfide bond possible which existence is proved to have significant effect on ligand affinity [2] . in this study effects of temperature, ph, distal histidine mutation and presence of disulfide bond on co rebinding to neuroglobin are investigated by flash photolysis experiments. in parallel, the molecular dynamics simulations are performed in corresponding conditions in order to investigate structural change of neuroglobin and especially changes in distribution of internal tunnels and cavities able to bind diatomic ligands in response to different physical conditions listed above. the thrombospondin family, being extracelluar proteins, is known to be implicated in various physiological processes such as wound healing, inflammation, angiogenesis and neoplasia. the signature domain of thrombospondins shows high sequence identities and thus allows us to transfer results obtained, studying this complex calcium reach part of the proteins, from one member of the family to the other. the domain is known to play a key role in hereditary diseases such as psach or med. in this part of thrombospondins lies a binding site to integrins, important for cell attachment. it is further known that the lectin like globe binds to cd-47, a feature known to be important in cancer research. as the theoretical unit we are trying to resolve these problems by numerical means and are constantly challenged by the size, where thrombospondin can be a huge trimeric protein as one strand can measure 430 kda, and the large variety of subdomains found in this proteins. we are thus facing a multiscale problem which can range from solving, by means of quantum mechanics a specific ion binding site, to large scale abstraction by continuum mechanics. in our talk we will show you our newest results that we obtained by simulating calcium rich c-terminal domain which is known to be conserved across the entire family, and give you an outlook into the future of our research. the process of swift heavy ions energy deposition while penetrating a solid or scattering on its surface can result in a strong and nonequilibrium excitation of matter. an extremely localized character of this excitation, meanwhile, can make possible both selective changes in chemistry of matter 1 and its surface nanomodifications 2 . since possible applications have been found in bio-and it-technologies (cancer curing and nanostructuring respectively) in the last decade, the heavy ion bombardment technique has attracted a lot of scientific interest 3, 4 . the processes of fast energy deposition into the solid and its further dissipation, however, are essentially perturbed with highly excited and nonequilibrium state of both lattice and electron systems. at such conditions therefore, the precision in treatment of processes of electron thermalization, fast electron heat conduction, and phase transformation of the overheated solid becomes crucial. having several physical models to handle the mentioned processes, it is nevertheless difficult to describe all of them within a scale of a single computational approach. our work is aimed on elaboration of the atomistic-continuum model 5 of heavy ion bombardment of solids. in particular, the model will be applied to study the formation of nanohillocks in the experiments on swift heavy ion xe + scattering on srtio 3 surface 2 . [3] g. aquaporins are protein channels located across the cell membrane with the role of conducting water or other small sugar alcohol molecules (aquaglyceroporins). the presence of the human aquaporin 5 (hsaqp5) in cells proximal to airinteracting surfaces (eyes, lacrimal glands, salivary glands, lungs, stomach etc.) suggest its potentially important role in ''wetting'' these surfaces. the high-resolution x-ray structure of the hsaqp5 tetramer (pdb code 3d9s) exhibits two important features: (i) lack of the four fold symmetry, common in most of the aquaporins, and (ii) occlusion of the central pore by a phosphatidylserine lipid tail. in this study we investigate the importance of these two features on the transport properties of the human aqp5 by means of molecular dynamics simulations. we found that the asymmetry in the tetramer leads to a distribution of monomeric channel structures characterized by different free energy landscapes felt by the water molecules passing through the channel. furthermore, the structures' distribution is influenced both by the presence/absence of the lipid tail in the central pore, and by the lipid composition of the bilayer that solvates the hsaqp5 tetramer. elucidating the modular structure of the protein g c2 fragment and human igg fc domain binding site using computer simulations hiqmet kamberaj faculty of technical sciences, international balkan university, skopje, r. of macedonia protein-protein recognition plays an important role in most biological processes. although the structures of many protein-protein complexes have been solved in molecular detail, general rules describing affinity and selectivity of proteinprotein interactions break down when applied to a larger set of protein-protein complexes with extremely diverse nature of the interfaces. in this work, we will analyze the non-linear clustering of the residues at the interface between proteins. the boundaries between clusters are defined by clustering the mutual information of the protein-protein interface. we will show that the mutations in one module do not affect residues located in a neighboring module by studying the structural and energetic consequences of the mutation. to the contrary, within their module, we will show that the mutations cause complex energetic and structural consequences. in this study, this is shown on the interaction between protein g c2 fragment and human igg fc domain by combining molecular dynamics simulations and mutual information theory, and computational alanine scanning technique. the modular architecture of binding sites, which resembles human engineering design, greatly simplifies the design of new protein interactions and provides a feasible view of how these interactions evolved. the results test our understanding of the dominant contributions to the free energy of protein-protein interactions, can guide experiments aimed at the design of protein interaction inhibitors, and provide a stepping-stone to important applications such as interface redesign. membrane proteins can form large multimeric assemblies in native membranes that are implicated in a wide range of biological processes, from signal transduction to organelle structure. hydrophobic mismatch and membrane curvature are involved in long range forces largely contributing to such segregation. however, the existing assembly specificity is thought to be coded in the atomic details of protein surface and topology. these are best described in high resolution structures and atomistic molecular dynamics simulations. in order to explore more systematically such forces and energetics arising at intermediate time scales and resolution, we use coarse grained molecular dynamics simulations applied to 20 membrane systems spanning over 5 to 15us. as a first glimpse we study how proteins induce lipid perturbations using a previously developed conformational entropy estimator. we show that in the model membrane where hydrophobic mismatch is present, lipid perturbations extend up to *40a from the protein surface. however, significant variations in perturbation profiles are seen. parameters such as protein shape, surface topology, and amino acid physicochemical properties are studied to discover the parameters governing such perturbations. crossing energy barriers with self-guided langevin dynamics gerhard kö nig, xiongwu wu, bernard brooks national institutes of health, national heart, lung and blood institute, laboratory of computational biology, rockville, md, usa even with modern computer power, the applicability of molecular dynamics simulations is restricted by their ability to sample the conformational space of biomolecules. often high energy barriers cause normal molecular dynamics simulations to stay trapped in local energy minima, leading to biased results. to address this problem, self-guided langevin dynamics (sgld) were developed. it enhances conformational transitions by accelerating the slow systematic motions in the system. this is achieved by calculating the the local average of velocities and adding a guiding force along the direction of systematic motions. thus, the efficiency of sgld is governed by three factors: a.) the friction constant involved in the langevin dynamics b.) the local averaging time and c.) the guiding factor that determines the guiding force. however, the guiding force also causes deviations from the original ensemble that have to be corrected by reweighting the data, thus decreasing the efficiency. here, we explore the three-dimensional parameter space of sgld for several benchmark systems with particularly rough energy surfaces. based on our data, we supply guidelines for the optimal selection of sgld parameters, to allow the extension of our method to other biological problems of interest. propagation of d816v/h mutation effects across kit receptor e. laine, i. c. de beauchê ne, c. auclair and l. tchertanov lbpa, cnrs -ens de cachan, france receptor tyrosine kinases (rtks) regulate critical biological processes. constitutive activation of rtks provokes cancers, inflammatory diseases and neuronal disorders. biological data evidenced that oncogenic mutations of the rtk kit, located either in the juxtamembrane region (jmr) or in the activation loop (a-loop) -as is the case of d816v/h, displace the equilibrium of conformational states toward the active form. we present a multi-approach study that combines molecular dynamics (md), normal modes (nm) and pocket detection to characterize and compare the impact of d816v/h on the structure, dynamics and thermodynamics of kit. we have evidenced a local structural destabilization of a-loop induced by the mutation and a long-range effect on the structure and position of jmr. we have further correlated these observations with experimental data and decipher some details about the activation mechanisms of the mutants, involving leverage of the jmr negative regulation and release of an access to the catalytic site. through the identification of ''local dynamic domains'' and the recording of interactions within the protein, we propose a model of the mutational effects propagation, which highlights the importance of both structural distortion and local conformational fluctuations. investigation of biological active azolidinones and related heterocycles refer to one of the most successful scientific projects of dh lnmu. it is based on three strategic vectors: organic synthesis, pharmacological research, rational design of ''drug-like'' molecules (including in silico approaches). while applying the research strategy we succeeded in gaining a number of interesting results that make possible to extend the field, especially in the scope of ''drug-like'' molecules design, notably it has focused on the search of new anticancer agents. anticancer activity screening was carried out for more than 1000 compounds (us nci protocol (dtp) based on obtained directed library over 5000 new compounds, among them 167 compounds showed high activity level. for the purpose of optimization and rational design directions of highly active molecules with optimal ''drug-like'' characteristics and discovering of possible mechanism of action sar, compare analysis, molecular docking and qsa(p)r were carried out. obtained results allowed to form main directions of possible anticancer activity mechanisms, which probable are apoptosis-related. nowadays the investigation of cellular and molecular aspects of anticancer effects is in progress. regulation of (bio)chemical reactions by mechanical force has been proposed to be fundamental to cellular functions [1, 2, 3] . atomic force microscopy experiments have identified the effect of mechanical force on the reactivity of thiol/disulfide exchange, a biologically important reaction [4] . in order to understand the influence of the force at an atomistic level, we have performed hybrid quantum mechanicsmolecular mechanics (qm/mm) transition path sampling simulation of the reaction under external forces. the results of the simulations supported the experimental findings and demonstrated that the location of the transition state on the free energy surface was shifted due to force [5] . in contrast to our findings, however, a recent experimental study suggests only a weak coupling between the mechanical force and the reaction rate [6] . in this study, the reactants were covalently linked to a stilbene molecule. in this system a force can be applied by photo-isomerization from the relaxed trans to the strained cis configuration. a drawback of this system is that one cannot easily determine the forces that acting on the reaction coordinate. therefore, we have developed a force distribution analysis method for quantum mechanical molecular dynamics simulations. the results of the analysis show how isomerization of stilbene alters the forces acting on the reacting atoms. the force distribution is essential for understanding how chemistry is controlled by external forces. [ conformational space modelling (csm) is a promising method for membrane protein structure determination. it is based on the concept of the side-chain conformational space (sccs), which is formed by the allowed side-chain conformations of a given residue. each sccs can be calculated from a 3d structure or measured via epr-sdsl experiments. for structure determination a set of singly spinlabelled mutants is needed. the final structure is obtained by altering an initial (possibly random) 3d structure until the best fit between the calculated and measured sccs for the whole set is found. such optimization is computationally intensive; therefore csm includes several empirical approximations. one of them describes the effect of the lipid tails on the sccs. the implementation is not trivial as lipids diffuse in the plane of the membrane and the lipid tails behave differently at different membrane depths. to unravel this relationship adaptive biasing force md simulations were used. an alanine peptide helix was made in silico, spin-labelled at the middle and inserted perpendicularly into a dmpc membrane. the free energy of the spin-label orientation at various membrane depths was calculated. a 3d free energy surface describing the membrane ''depth'' effect was obtained. it is known that b-cyclodextrins (bcds) are able to modify the cholesterol content of cell or model membranes. however the molecular mechanism of this process is still not resolved. using molecular dynamics simulations, we have been able to study the bcd-mediated cholesterol extraction from cholesterol monolayers and lipid-cholesterol monolayer mixtures. we have investigated many conditions that would affect this process (e.g. lipid-cholesterol ratio, lipid chain unsaturation level) our results can be summarized as follow: i) dimerization of bcds, ii) binding of the dimers to the membrane surface assuming either a tilted (parallel to the membrane normal) or untilted (90°respect to the normal of the membrane) configuration, iii) the latter configuration is suitable to extract cholesterol at a reasonable computational time scale (100-200 ns), however, this process may be affected by unfavorable energy barriers (from 10 to 80 kj mol -1 ), iv) desorption of the complex brings cholesterol in solution, v) the bcd-cholesterol complex is able to transfer cholesterol to other membranes. with a clearer understanding of the basic molecular mechanism of the bcd mediated process of cholesterol extraction, we can begin to rationalize the design of more efficient bcds in numerous applications. the mechanism of the complex formation of biopolymers with ligands including the solvent molecules is an actual problem of modern biophysical and biological science. polypeptides form a secondary structure and mimic the motifs of the protein architecture. the study of complexation between polypeptides and solvent molecules leads to deeper understanding of the basic interaction of proteins with environment at atomic level. besides polypeptides are promising for the development of applications which encompass some of the following desirable features: anti-fouling, biocompatibility, biodegradability, specific biomolecular sensitivity. on this account polypeptides have a great significance for a variety modern applications ranging from the nanoscale medicine devises up to food technology and others. we compare the results of calculations of complexes between helical polypeptides (polyglutamic acid in neutral form and poly-c-benzyl-l-glutamate) and water molecules at dft pbe level and the results of ftir-spectroscopic study of the film of wetted polypeptides. vibrational spectroscopy is one of the most useful experimental tools to study non-covalent bonded complexes, and calculated spectra in comparison with experimental data are reliable test for reality of simulated complexes. platelet aggregation at the site of vascular injury is vital to prevent bleeding. excessive platelet function, however, may lead to thrombus formation after surgery. therefore, an accurate measure and control of platelet aggregation is important. in vitro platelet aggregometry monitors aggregate formation in platelet rich plasma triggered by agonists such as adp, epinephrine or collagen. the fraction of aggregated platelets is plotted versus time, and platelet function is assessed by analyzing the plot's morphology. we propose new measures of platelet function based on a compartmental kinetic model of platelet aggregation induced by adp. our model includes three compartments: single, aggregated and deaggregated platelets. it is simpler than earlier models and agrees with experimental data. the model parameters were determined by non-linear least squares fitting of published data. we associated the kinetic parameters with the activity of the adp receptors p2y1 and p2y12. to this end, we studied published data obtained in the presence and in the absence of specific antagonists of these receptors. comparison of kinetic parameters of healthy subjects with those of patients with myeloproliferative disorder (mpd) shows that the function of p2y12 is significantly reduced in mpd. coarse-grained modeling of drug-membrane interactions manuel n. melo, durba sengupta, siewert-jan marrink groningen biomolecular sciences and biotechnology institute, university of groningen, groningen, the netherlands the martini coarse-grained (cg) forcefield was used to simulate the actions of the antimicrobial peptide alamethicin and of the anti-tumoral drug doxorubicin. both drugs were shown to interact strongly with a fluid phospholipid bilayer, and aggregate there, in agreement with experimental results. because doxorubicin may establish intermolecular h-bonding, and thus lower its dipole moment, the cg representation of a doxorubicin dimer was adjusted. this less polar dimer was then tested for translocation and/or pore formation. contrary to results of atomistic simulations, alamethicin aggregates did not spontaneously open pores. they did so, however, when the size of the water beads was decreased. several small independent pores could then be observed. the magnitude of the permeability of these pores is analyzed and compared to experimental values. the occurrence of multiple small pores could indicate that the different conductance levels experimentally observed for alamethicin might simply result from the association of different numbers of these small pores. polarization refers to the asymmetric changes in cellular organization in response to external or internal signals. neuronal polarization begins with the growth of a single neurite shortly after cell division, followed by the growth of a second neurite at the opposite pole. this early bipolar shape is critical for brain function, as it defines axis of migration and consequently proper three dimensional organization and nerve circuitry. however, it is not known if a direct relationship exists between the formation of the second, opposite, neurite and the mechanisms involved in the formation of the first. we tackled this issue through mathematical modeling, based on membrane traffic (exocytosis-endocytosis), and lateral diffusion. with this approach, we demonstrated that a single pole of molecular asymmetry is sufficient to induce a second one at the opposite side, upon induction of growth from the first pole. our work gives mathematical proof that the occurrence of a single asymmetry in a round cell is sufficient to warrant morphological bipolarism. trypsin is one of the best characterized serine proteases. enzyme acylation process is required for substrate degradation. there is a lot of information about how this process undergoes. however, in order to obtain a more detailed description of the catalytic triad mechanism, a qm/mm approach was used. we used the hybrid qm/mm potential implemented in amber11 package. in the qm calculations a dft hamiltonian was used. we develop an approach based on the adaptively biased md in order to obtain the free energy surface of the conformational space defined by the reaction coordinates. this approach presents some characteristics of steered md and umbrella sampling procedures. our results offer information about the lowest energy trajectory, the barrier profile of the reaction, and the geometry of the transition state. this method also provides a further insight into the role of specific residues in the reaction. substituting asp102, member of the catalytic triad, for ala we were able to detect an increase of the barrier profile. this was due to the loss of the interaction of carbonyl group of asp102 with nd of his57, which make ne of this residue a worst proton acceptor. this results show our approach as a valuable method in the study of enzymatic mechanisms. the intracellular media comprise a great variety of macromolecular species that are not individually concentrated, but being preset in the same compartment they exclude each other's volume and produce crowding. crowding has a profound impact on protein structure and determines conformational transitions and macromolecular association. we investigated macromolecular association on a 50% w/w bovine serum albumin (bsa) solution by time-domain terahertz (thz) spectroscopy and molecular modeling. molecular crowding was simulated by including two bsa molecules in the same water box. we generated *300 such dimeric models, computed their thz spectra by normal modes analysis and compared them with the experimental data. the best bsa dimer model was selected based on the agreement with the experiment in the lowest frequencies domain of up to 1 thz. symmetry constraints improve accuracy of ion channels models. application to two-pore-domain channels adina l. ion channels are important drug targets. structural information required for structure-based drug design is often filled by homology models (hm). making accurate hm is challenging because few templates are available and these often have substantial structural differences. besides, in molecular dynamics (md) simulations channels deviate from ideal symmetry and accumulate thermal defects, which complicate hm refinement using md. we evaluate the ability of symmetry-constrained md simulations to improve hm accuracy, using an approach conceptually similar to casp competition: build hm of channels with known structure and evaluate the efficiency of various symmetry-constrained md methods in improving hms accuracy (measured as deviation from x-ray structure). results indicate that unrestrained md does not improve accuracy, instantaneous symmetrization improves accuracy but not stability during subsequent md, while gradually imposing symmetry constraints improves both accuracy (by 5-50%) and stability. moreover, accuracy and stability are strongly correlated, making stability a reliable criterion in predicting hm accuracy. we further used this method to refine hm of trek channels. we also propose a gating mechanism for mechanosensitive channels that was further experimentally confirmed. nucleotide modifications and trna anticodon-mrna codon interactions on the ribosome olof allné r and lennart nilsson karolinska institutet, stockholm, sweden molecular dynamics simulations of the trna anticodon and mrna codon have been used to study the effect of the common trna modifications cmo 5 u34 and m 6 a37. in trna val these modifications allow all four nucleotides to be successfully read at the wobble position in a codon. previous data suggest entropic effects are mainly responsible for the extended reading capabilities but detailed mechanisms have remained unknown. the aim of this paper is to elucidate the details of these mechanisms on an atomic level and quantify their effects. we have applied: extensive free energy perturbation coupled with umbrella sampling, entropic calculations of trna (free and bound to the ribosome) and thorough structural analysis of the ribosomal decoding center. human neuroserpin (hns) is a serine protease inhibitor (serpin) of a tissue-type plasminogen activator (tpa). the conformational flexibility and the metastable state of this proteins underlies to misfolding and to dysfunctional mutations causing a class of rare genetic diseases which share the same molecular basis. the conformational transition of the native form, triggered upon the cleavage at reactive center loop (rlc), releases a complex of the cleaved form bound to the inactivated target protease. without rlc cleavage a stable inactive latent form can be obtained by intra/inter molecular loop insertion leading to polymerization. this work concerns the study of the three above mentioned forms of hns by md simulations to investigate the relation between their conformational stability and. the starting native and cleaved configurations are based on the x-ray structure, while the latent form is here modelled. the results of the simulation reveal a striking conformational stability along with the intrinsic flexibility of selected regions of the fold.the analysis of the essential collective modes of the native hns shows that the initial opening of the b-sheet a coincides with several changes in the local pattern of salt bridges and of hydrogen bonds. regulation of ubiquitin-conjugating enzymes: a common mechanism based on a pattern of hydrophobic and acidic residues enzyme temperature adaptation generally involves a modulation of intramolecular interactions, affecting proteins dynamics, stability and activity [1] [2] . in this contribution, we discuss studies of different classes of extremophilic enzymes, focusing on cold-adapted variants, as well as their mesophilic-like mutants, performed by all-atom molecular dynamics simulations with particular attention to structural communication among residues within the threedimensional architecture [3] [4] . common adaptation strategies turned out to be based on improved local flexibility in the proximity of the functional sites, decrease in interconnected electrostatic interactions, and modulation of correlated motions and networks of communicating residues. specific differences related to the diverse protein folds can also be detected. bneurexins and neuroligins are cell adhesion molecules and play important role in synapse junction formation, maturation and signal transduction between neurons. mutations in genes coding these proteins occurs in patients with cognitive diseases like autism disorders, asperger syndrome and mental retardation [1] . it has been found that the complex bneurexin-neuroligin has also an important role in angiogenesis [2] . herein we will present molecular foundations of bneurexin-neuroligin interactions obtained from all-atom molecular dynamics simulations of bneurexin, neuroligin and their complex (3b3q) [3] . 50 ns md trajectories (charmm force field) were analyzed and roles of ca2+ and n-actetyl-d-glucosamine posttranslational modifications in intermolecular interactions were scrutinized. advances in hardware and software have enabled increasingly long atomistic molecular dynamics simulations of biomolecules, allowing the exploration of processes occurring on timescales of hundreds of microseconds to a few milliseconds. increasing the length of simulations beyond the microsecond time scale has exposed a number of limitations in the accuracy of commonly employed force fields. such limitations become more severe as the size of the systems investigated and the length of the simulations increase. here i will describe the force field problems that we have encountered in our studies, how we identified and addressed them, and what we have learned in the process about the biophysics of the systems we are investigating. while the quest for a ''perfect'' force field is not over (and may never be), our work has greatly improved the accuracy and range of applicability of simple physics-based force fields, to the point that reliable predictions can now be obtained from millisecond-timescale simulations of biomolecules. local anesthetics (la) are pain-relief drugs, widely used in medicine and dentistry. the relatively short duration of analgesia still restricts their clinical use for the treatment of chronic pain. nowadays, intensive research is focused on anesthetics entrapped into liposomes to enhance their activity and pharmacokinetic properties [1] . in this work, we investigated the encapsulation of prilocaine (plc), an aminoamide local anesthetic, into a small unilamellar liposome. on the line of our previous work [2] , we have carried out molecular dynamics (md) simulations using a coarse grain model up the microsecond time scale. in this way, we compare the effects of the concentration of la at fisiological ph. we were able to capture important features of the plc-vesicle interactions. the behavior of plcs at fisiological ph is essentially a combination of high and low ph: we found that all neutral plc molecules rapidly diffuse into the hydrophobic region of the vesicle adopting an asymmetric bimodal density distribution. protonated plc molecules (pplc) initially placed in water were instead only found on the external monolayer, with a high rate of exchange with the water phase and no access to the inner part of the liposome in a concentration dependent way. we focus on applications of molecular and mesoscale simulation methodologies to the cellular transport process of endocytosis, i.e., active transport mechanisms characterized by vesicle nucleation and budding of the cell membrane orchestrated by protein-interaction networks, and functionalized carrier adhesion to cell surfaces. we discuss theoretical and computational methodologies for quantitatively describing how cell-membrane topologies are actively mediated and manipulated by intracellular protein assemblies. we also discuss methods for computing absolute binding free energies for carrier adhesion. we present rigorous validation of our models by comparing to diverse range of experiments. the importance of delta-opioid receptors as target of a large number of drugs is well recognized, but the molecular details of interaction and action of the compounds are largely unknown. in an effort to shade some light on this important issue we performed an extensive computational study on the interaction of two compounds, clozapine and desmetilclozapine, with a delta-opioid receptor. according to experiments, the lacking of a single methyl group in desmetilclozapine with respect of clozapine makes the former more active than the latter, providing a system well suited for a comparative study. we investigated stable configurations of the two drugs inside the receptor by simulating their escape routes by metadynamics, an algorithm that allows the simulation of events that are otherwise out of range for standard molecular dynamics simulations. our results point out that the action of the compound is related to the spatial distribution of the affinity sites it visits during its permanency. desmetilclozapine has a larger distribution of residues, which is interacting with, than clozapine. however, large conformational changes of the receptor were not observed in the presence of both compounds. thus, a more dynamical picture of ligand-receptor affinity is proposed on the basis of the results obtained, involving the competition among different stable states as well as the interaction with the solvents. such information might be useful to provide hints and insights that can be exploited in more structure-and-dynamics-oriented drug design. the coupling between the mechanical properties of enzymes and their biological activity is a well-established feature that has been the object of numerous experimental and theoretical works. in particular, recent experiments show that enzymatic function can be modulated anisotropically by mechanical stress. we study such phenomena using a method or investigating local flexibility on the residue scale, which combines a reduced protein representation with brownian dynamics simulations. we performed calculations on the enzyme guanylate kinase in order to study its mechanical response when submitted to anisotropic deformations. the resulting modifications of the protein's rigidity profile can be related to the changes in substrate binding affinities that were observed experimentally. further analysis of the principal components of motion of the trajectories shows how the application of a mechanical constraint on the protein can disrupt its dynamics, thus leading to a decrease of the enzyme's catalytic rate. eventually, a systematic probe of the protein surface led to the prediction of potential hotspots where the application of an external constraint would produce a large functional response both from the mechanhiv-1 protease autocatalyses its own release from gag and gagpol precursor polyproteins into mature functional proteins. as it is functional in the dimeric form, whilst initially, only a single monomer is embedded within each gagpol chain, the question arises as to what cut's the cutter. two individual monomers in different gagpol chains are known to come together to form an embedded-dimer precursor protease. mature-like protease activity is concomitant with n-terminal intramolecular cleavage of this transient embedded-dimer precursor, but how this crucial maturation-initiating step is physically achieved, has remained unknown. here, we show via 400 all-atom explicit solvent molecular dynamics simulation runs of 400 ns each that the n-terminal of an immature-like protease, with the n-terminal initially unbound as in the gagpol polyprotein, can self-associate to the active site and therefore be cleaved under conditions of thermodynamic equilibrium, identifying possible binding pathways at atomic resolution, in agreement with previous indirect experimental evidence [1] . the binding pathway predominantly makes use of the open conformation of the beta-hairpin flaps characterised by us previously [2] , and the n-terminal binds across the entire active site in good agreement with crystal structures of a cleavage-site peptidebound protease. the n-terminus serves two roles, firstly in the maturation of the protease itself by self-associating to the protein and then as a stabilizing component of the dimer interface in a mature protease. targeting the prior mechanism could be the focus of a novel therapeutic strategy involving immature protease inhibition. [ knotted proteins are the object of an increasing number of experimental and theoretical studies, because of their ability to fold reversibly in the same topologically entangled conformation. the topological constraint significantly affects their folding landscape, thus providing new insight and challenges into the funnel folding theory [1] . recently developed experimental methods to trap and detect knots have suggested that denaturated ensembles of the knotted proteins may be knotted [2] . we present numerical simulations of the early stage of folding of the knotted proteins belonging to protein families mtase (methyltransferase) and sotcase (succinyl-ornithine transcarbamylase), and of their unknotted homologues [3] . our results show that native interactions are not sufficient to generate the knot in the denaturated configurations. however, when non-native interactions are included we observe formation of knots only in the chains whose native state is knotted. in addition, we find that the knots are formed through a universal mechanism. such a knot formation mechanism correctly predicts the fraction of the knotted proteins found in nature and can be used to make qualitative prediction on their folding times. shape and motility and also for numerous signaling processes. adhesion is based on non-covalent interactions between transmembrane proteins and the extracellular matrix. cells are able to create two-dimensional assemblies of integrins, so called focal adhesions, which they use to stick to the substrate and collect information about the environmental properties. the goal of this work is a deeper understanding of the formation and the stability of these adhesion clusters. bond cluster formation and disintegration is dynamically modeled with the aid of monte carlo simulations. in the model, a membrane is attached to a flat surface via a variable number of adhesion bonds. the spacial configuration of these adhesion points subjected to an inhomogeneous stress field maps into a distribution of local membrane/ surface distances. we introduce a model which explicitly accounts for the membrane elasticity and demonstrate that such models are able to explain the spontaneous formation of adhesion bond clusters. structure based models are successful at conjugating the essence of the energy landscape theory of protein folding [1] with an easy and efficient implementation. recently their realm expanded beyond single protein structure, been used profitably to widely study large conformational transitions [2] [3] . still, when dealing with conformational transition between two well-defined structures an unbiased and realistic description of the local backbone and sidechain interactions is necessary. the proposed model merges a precise description of these interactions with a structure-based long range potential that takes into account different conformers. we present the results of the activation of the catalytic domain of human csrc tyrosine kinase for which we reconstructed the transition free energy and the description of the activation loop flexibility. the excellent model performances in terms of speed and the satisfactory accuracy of the description of the system and its flexibility are promising for a more systematic study of the tyrosine kinase family activation mechanisms. [ we introduce a previously undescribed technique for modelling the kinetics of stochastic chemical systems. we apply richardson extrapolation, a sequence acceleration method for ordinary differential equations, to a fixed-step tau-leaping algorithm, to produce an extrapolated tau-leaping method which has weak order of accuracy two. we prove this mathematically for the case of linear propensity functions. we use four numerical examples, two linear and two nonlinear, to show the higher accuracy of our technique in practice. we illustrate this by using plots of absolute error for a fixed-step tau-leap and the extrapolated tau-leap. in all cases, the errors for our method are lower than for a fixedstep tau-leap; in most cases they are second order of accuracy. the major tripartite efflux pump acrab-tolc is responsible for the intrinsic and acquired multidrug resistance in escherichia coli. at heart of the extrusion machinery there is the homotrimeric transporter acrb, which is in charge of the selective binding of structurally and chemically different substrates and energy transduction. the effects of conformational changes, which have been proposed as the key features of the extrusion of drugs, are investigated at molecular level using different computational methods like targeted molecular dynamics. simulations, including almost half a million atoms, have been used to assess several hypotheses concerning the structure-dynamics-function relationship of the acrb protein. the results indicate that, upon induction of conformational changes, the substrate detaches from the binding pocket and approaches the gate to the central funnel. in addition, we provide evidence for the proposed peristaltic transport involving a zipper-like closure of the binding pocket, responsible for the displacement of the drug. using these atomistic simulations the role of specific amino acids during the transitions can be identified, providing an interpretation of sitedirected mutagenesis experiments. additionally, we discuss a possible role of water molecules in the extrusion process. virus inhibitory peptide (virip), a 20 amino acid peptide, binds to the fusion peptide (fp) of human immunodeficiency virus type 1 (hiv-1) gp41 and blocks viral entry. molecular dynamics (md) and molecular mechanics/poisson-boltzmann surface area (mm/pbsa) free energy calculations were executed to explore the binding interaction between several virip derivatives and gp41 fp. a promising correlation between antiviral activity and simulated binding free energy was established thanks to restriction of the flexibility of the peptides, inclusion of configurational entropy calculations and the use of multiple internal dielectric constants for the mm/pbsa calculations depending on the amino acids sequence. bases on these results, a virtual screening experiment was carried out to design enhanced virip analogues. a selection of peptides was tested for inhibitory activity and several improved virip derivatives were identified. these results demonstrate that computational modelling strategies using an improved mm/pbsa methodology can be used for the simulation of peptide complexes. as such, we were able to obtain enhanced hiv-1 entry inhibitor peptides via an mm/pbsa based virtual screening approach. an essential step during the hiv life cycle is the integration of the viral cdna into the human genome. hiv-1 integrase mediates integration in a tight complex with the cellular cofactor: ledgf/p75 [1] . disruption of the interaction interferes with hiv replication and therefore provides an interesting new drug target for antiretroviral therapy [2, 3] . here we present the structure based discovery and optimization of a series of small molecule inhibitors that bind to hiv-1 integrase and block the interaction with ledgf/p75. the work flow was set up according to a funnel principle in which a series of virtual screening tools were applied in such way to discard at each step molecules unlikely to be active against the desired target (including 2d filtering, pharmacophore modelling and molecular docking) the activity and selectivity of the selected molecules were confirmed in an alpha screen based assay, that measure protein-protein interaction in vitro, and furthermore by in vivo experiments. active compounds proceeded towards crystallographic soaking into the receptor protein crystals. these crystal structures not only validated the binding mode and activity of the hit compounds, but furthermore were used as a platform for structure based drug design which resulted in the rational discovery of new hit compounds and optimized lead compounds. in vitro and in vivo experiments validated the mechanism of action of these compounds and show that they are a novel class of antiretroviral compounds with in vivo inhibitory activity by targeting the interaction between ledgf/p75 and hiv-1 integrase. cross resistance profiling indicate that these compounds are active against current resistant viral strains. [4] currently the most potent inhibitors show an in vivo ic 50 of 55nm. these compounds are promising for future pharmaceutical optimizations to be used in the clinic as new antiretroviral agents. crystallography was used to validate the binding mode of the discovered inhibitors. insights in the interaction of the ligand-protein complex allowed for rational design of optimized inhibitors. ligand-induced structural changes are small, thermal fluctuations can play a dominant role in determining allosteric signalling. in thermodynamic terms, the entropy change for subsequent binding is influenced by global vibrational modes being either damped or activated by an initial binding event. one advantage of such a mechanism is the possibility for long range allosteric signalling. here, changes to slow internal motion can be harnessed to provide signalling across long distances. this paper considers homotropic allostery in homodimeric proteins, and presents results from a theoretical approach designed to understand the mechanisms responsible for both cooperativity and anticooperativity. theoretical results are presented for the binding of camp to the catabolite activator protein (cap) [1] , where it is shown that coupling strength within a dimer is of key importance in determining the nature of the allosteric response. results from theory are presented along side both atomistic simulations and simple coarse-grained models, designed to show how fluctuations can play a key role in allosteric signalling in homodimeric proteins. [ reversibly switchable fluorescent proteins (rsfps) can be switched between a fluorescent (on) and a nonfluorescent (off) state which is accompanied by a cis-trans isomerization of the chromophore on the molecular level 1,2. this unique property has already provided new aspects to various microscopy techniques, such as high resolution microscopy, fcs or monochromatic multicolor microscopy 3-5. despite of their established potential, rsfps still have a major drawback: the wavelength for fluorescence excitation is always one of the two switching wavelengths. the imaging process thus inevitably results in the switching of a small fraction of the rsfps, which might hinder or complicate some experiments. we developed a new reversibly switchable fluorescent protein which eliminates the problem of the coupling between switching and fluorescence excitation. this fluorescent protein follows an unusual and currently unknown mechanism of switching between a fluorescent and a nonfluorescent state. it is brightly fluorescent and exhibits an excellent signal to noise ratio. in parallel studies [2] , qd-based ligands (egf, mabs) were targeted to egfr in gliomas. cell-cultures, animal models and ex vivo biopsies of human high-grade as well as low-grade gliomas showed high probe specificity.. the aim is to define more precisely the tumor boundaries at the time of resection. we used the programmable array microscope designed for sensitive, high-speed optical sectioning, particularly of living cells. the pam is based on structured illumination and conjugate detection using a digital micromirror device (dmd) [3] located in a primary image plane. the unique feature is the rapid, (re)programmable adjustment of local excitation intensity, dynamic, on-the-fly optimization is thus achieved, e.g. multipoint frap [4] , light exposure minimization and object tracking [5] , or super-resolution strategies. the features and operation of the 3rd generation pam will be presented. contraction of muscle cells, motility of microorganisms, neuronal activity, and other fast cellular processes require microscopic imaging of a three-dimensional (3d) volume with a video-rate scanning. we present 3d video-rate investigations of structural dynamics in biological samples with the multicontrast third-and second-harmonic generation as well as fluorescence microscope. the multidepth scanning is achieved by two combined laser beams with staggered femtosecond pulses. each of the beams is equipped with a pair of deformable mirrors for dynamic wavefront manipulation enabling multidepth refocusing with simultaneous corrections for optical aberrations. combined, more than 250 frames per second lateral scanning with fast refocusing enables the 3d video-rate imaging of dynamically moving structures. in addition, combination of two laser beams is accomplished at two perpendicular polarizations enabling live imaging of sample anisotropy, which is important for structural studies particularly with the second harmonic generation microscopy. investigations of beating chick embryo hearts with the 3d video-rate scanning microscope revealed multidirectional cardiomyocyte contraction dynamics in myocardial tissue. intricate synchronization of contractions between different layers of myocytes in the tissue will be presented. the video-rate 3d microscopy opens new possibilities of imaging fast biological processes in living organisms. confocal fluorescence microscopy is an invaluable tool to study biological systems at cellular level also thanks to the synthesis of always new specific fluorescent probes, e.g. multiprobe labelling enables complex system characterization. however, only the recent employment of narrowband tunable filters overcomes the problems due to the use of the broadband ones. the possibility of acquiring the emission spectra in a spatially resolved way extends simple image intensity studies into characterization of complex probeenvironment relationship through the sensitivity of fluorescence spectra to the local molecular environment differences. consequently, fluorescence microspectroscopy (fms) is able to provide the spectral information in a well defined spatial region allowing the researcher to simultaneously obtain spatial and spectroscopic information. our instrument has been specially built to study live cells and their interaction with nanomaterials, drug carriers and modified cell environment. other main characteristics are reducing the bleaching effect and employing a white light source that does not limit the use to specific probes. graphical tools, such as colour coded images, have also been introduced to provide explicit and straightforward visual information. high speed fpga based multi-tau correlation for singlephoton avalanche diode arrays jan buchholz 1,2 , jan krieger we demonstrate the use of fret-imaging (forster resonance energy transfer) as an assay to directly monitor the dynamics of cross-bridge conformational changes in single fibres of skeletal muscle. we measured nm-distances of several fret pairs located at strategic positions to sense myosin head conformational changes: we focused our attention on the essential light chain, elc (specifically labelling a modified elc and exchanging it with the natural elc of the fibre) and we investigated its interaction with the sh1 helix, with the nucleotide binding pocket and with actin. we characterized fret in single rigor muscle fibres, determining distances in agreement with those from the crystallographic data. the results demonstrate the viability of the approach in sensing different fret efficiencies over a few nanometres, an essential requirement to follow the expected small fret variations in contracting muscle fibres. we are now performing dynamic experiments on rigor and active fibres by applying small stretch/release cycles to alter the interaction distances (estimated time resolution of nearly 20ms/frame). in this configuration, it will be possible to measure functional changes, shedding light on the myosin head dynamics during contraction. focal stimulation of cultured neurons is crucial since it mimics physiological molecules release. indeed, the nervous system finely tunes the activity of each synapse by regulating the secretion of molecules spatially and temporally. currently used techniques have some drawbacks such as a poor spatial resolution or a low flexibility. we propose a novel approach based on optical tweezers (ot) [1] to overcome these limitations. ot allow an ease manipulation with sub-micrometric precision of silica beads, which can be functionalized with any protein. for a proof-of-principle study we coated 1,5lm large beads with brain-derived neurotrophic factor (bdnf) or bovine serum album (bsa) as control. we showed that a single bead was able to activate the bdnf receptor trkb, inducing its phosphorylation. moreover bdnf beads but not control beads were able to induce c-fos translocation into the nucleus [2] , indicating that the whole pathway was activated. finally, we positioned vectors in proximity to the growth cones of cultured hippocampal neurons [3] . control beads didn't affect the normal development of these structures while bdnf beads significantly did. these findings support the use of the ot technology for long-term, localized stimulation of specific subcellular neuronal compartments. a key role of its photoactivity, due to the singlet oxygen production, which has a very short lifetime (ns-ls, depending of hyp environment). hyp sub-cellular localization depends on its concentration in the medium, incubation time and used delivery system. variations in activity of protein kinase c, (anti-apoptotic pkca and pro-apoptotic pkcd) in correlation with the activity of bcl-2 protein, cytochrome c release from mitochondria and decreasing of mitochondrial membrane potential after photodynamic action were monitored. the study was performed for two different delivery modes of hyp to u-87mg glioma cells-hyp alone (membrane diffusion) vs. hyp loaded in low density lipoprotein (ldl) (endocytosis). confocal fluorescence microscopy, flow-cytometry and specific fluorescence labeling were used as main experimental techniques. our results show that hyp photoaction strongly affects apoptotic response of the cells and that the dynamics of this action significantly depends on used delivery system. correlation analysis between the monitored parameters (see above) determined for both delivery system is presented and critically discussed. surface contamination by bacteria is a natural and spontaneous process occurring in any natural environment on biotic (mucosa, tissues…) and abiotic surfaces (medical equipments, food surfaces…). whatever the bacterial nature (gram-positive or -negative), the environmental fluid (air, water, blood…) and the receptor surface (implants, medical equipments, food surfaces…), the surface contamination initiated by the first adherent bacteria can evolve to a three dimensional structure named biofilm (cohesive bacteria assembly ensured by an autoproduced extracellular organic matrix). the mechanisms by which these biofilms offer protective environment to viral particles or hypertolerance to antimicrobial action are not yet elucidated. to reach a better understanding of biofilm reactivity, we reported for the first time successful applications of correlative time-resolved optical microscopy approach by time-lapse (tl), frap, fcs, flim, for real-time analysis of molecular mobility and reaction inside biofilms. by means of non-biological or biological (virus, biocides and antibiotics) reactive compounds, significant advances to understand the roles of the extracellular matrix and bacteria physiological properties were obtained, an important step to improve pathogenic biofilm inactivation. here we present a feasibility study to develop two-photon microscopy (2pm) into a standard diagnostic tool for noninvasive, skin cancer diagnosis. the goal is defining experimental parameters to maximize image quality of optical biopsies in human skin, while avoiding tissue damage. possible diagnostic indicators will be compared for healthy tissue, benign, and malignant melanocytic lesions. we report on preliminary results of a study on 2pm imaging of ''ex-vivo'' biopsy samples, were autofluorescence intensity and contrast between lesion and surrounding tissues were optimised varying excitation wavelength, detection band, and dispersion pre-compensation. moreover, we determined modulation functions for laser power and detector gain to compensate losses in deep tissue imaging. as the main process of photo-damage, thermo-mechanical modifications were quantified and damage threshold powers were determined. in order to image structural changes in ordered tissue like collagen fibres, second-harmonic generation signals were recorded and optimised. in-vivo two-photon imaging of the honeybee antennal we adapted a two-photon microscope for in-vivo imaging of the honeybee olfactory system, focusing on its primary centres, the antennal lobes. the setup allowed obtaining both 3d-tomographic measurements of the antennal lobe morphology and time-resolved in-vivo calcium imaging of its neuronal activity. the morphological data were used to precisely measure the glomerular volume in both sides of the brain, investigating the question of volumetric lateralization. functional calcium imaging allowed to record the characteristic glomerular response maps to external odour stimuli applied to the bees' antennae. compared to previous neural imaging experiments in the honeybee, this work enhanced spatial and temporal resolution, penetration depth, and it minimized photo-damage. final goal of this study is the extension of the existing functional atlases of the antennal lobe to 3d and into the temporal dimension by investigating the time-resolved activity pattern. the use of voltage sensitive fluorescent dyes (vsd) for noninvasive measurement of the action potential (ap) in blood perfused heart have been hindered by low interrogation depth, high absorption and auto-fluorescence of cardiac tissue. these limitations are diminished using new near infrared (nir) vsd (di-4-anbdqbs). here we validated toxicity and photo-toxicity of these dyes in guinea pig and human cardiac muscle slabs. application of nir vsd showed no effect on cardiac muscle contraction force or relaxation. optical action potentials closely tracked kinetics of microelectrode recorded aps in both field and electrode stimulated preparations. for phototoxicity assessment dye (50 lm) preloaded cardiac slabs were exposed to prolonged laser radiation of various power. microelectrode ap recordings show that exposure to prolonged laser radiation (10min.; 2mw/mm 2 ) of dye loaded tissue had no statistically significant effect on apd50 or conduction velocity, thus indicating no or weak photo-toxicity on the nir vsd. in contrast, exposure to 5 min. laser radiation of phototoxic dyes (mitotracker deep-red) preloaded tissue caused significant reduction in apd50 (by 13%) and conduction velocity (30%). thus, due to the low photo-toxicity, nir vsd are well suited for in vivo cardiac imaging. streptomycesetes are filamentous gram-positive soil bacteria well known for their complex morphological development and secondary metabolite production. during their life cycle spores germinate to form a network of hyphae, which later develops into aerial mycelium when cross-walls are generated and spores are formed. we have examined and compared the last stage of the differentiation process in a wild-type s. coelicolor (m145) and its dcabb mutant lacking a calmodulin-like calcium binding protein. the strains were grown on four kinds of media: smms, smms with 10 % saccharose, r5 and r5 with reduced calcium in order to study the effect of environment and osmotic stress on the sporulation of the two strains to assess the function of cabb protein. from the cultures pictures were taken at 48 hours and after 7 days using phase contrast, atomic force and confocal laser scanning microscope and the sizes of spores were measured. our results showed that the dcabb mutant made smaller spores, its differentiation and stress response were slower. we could conclude from it that the aberrant protein slows the metabolism, the signal transduction and has effects on sporulation, septation and air-mycelium formation. based on it we can tell that the cabb has a significant role in normal development. the mobility and reaction parameters of molecules inside living cells can be conveniently measured using fluorescent probes. typically fluorescence correlation spectroscopy (fcs) based on confocal microscopy is used for such measurements. this implies high time-resolution but only for a single spot at a time. in order to achieve a high time-resolution at multiple spots, we built a single plane illumination microscope (spim) equipped with high-speed image acquisition devices and a high-na detection optics. this allows us to do parallel fcs measurements in a thin plane (width * 2-3 lm) of the sample. our setup is equipped with a fast emccd camera (full frame time resolution 2000ls) and a 32932 pixel array of spads. the spad array has a full frame time resolution of 3-10ls, which is even fast enough to resolve the typical motion time-scale of small molecules (like egfp) inside living cells. the performance of the system is characterized by diffusion measurements of water-soluble fluorescent beads, as well as fcs measurements in living cells. our data acquisition system uses programmable hardware for some tasks and is fast enough to allow real-time correlation of 1024 pixels, as well as saving the complete dataset for later evaluation. electron cryo-microscopy (cryo-em) covers a larger size range than any other technique in structural biology, from atomic resolution structures of membrane proteins, to large noncrystalline single molecules, entire organelles or even cells. electron crystallography of two-dimensional (2d) crystals makes it possible to examine membrane proteins in the quasi-native environment of a lipid bilayer at high to moderately high resolution (1.8-8å ). recently, we have used electron crystallography to investigate functionally important conformational changes in membrane transport proteins such as the sodium/proton antiporters nhaa and nhap, or the structure of channelrhodopsin. ''single particle'' cryo-em is well suited to study the structure of large macromolecular assemblies in the 3.3 to 20å resolution range. a recent example is our 19å map of a mitochondrial respiratory chain supercomplex consisting of one copy of complex 1, two copies of complex iii, and one of complex iv. the fit of the x-ray structures to our map indicates short pathways for efficient electron shuttling between complex i and iii by ubiquinol, and between complex iii and iv by cytochrome c. electron cryo-tomography can visualize large protein complexes in their cellular context at 30-50å resolution, and thus bridges the gap between protein crystallography and light microscopy. cryo-et is particularly suitable for studying biological membranes and large membrane protein complexes in situ. we found that long rows of atp synthase dimers along ridges of inner membrane cristae are an ubiquitous feature of mitochondria from all 6 species we investigated (2 mammals, 3 fungi, 1 plant). the proton pumps of the respiratory chain appear to be confined to the flat membrane regions on either side of the ridges. this highly conserved pattern suggests a fundamental role of the mitochondrial cristae as proton traps for efficient atp synthesis. single-particle analysis: advanced fluorescence imaging, including subdiffraction microscopy, relies on fluorophores with controllable emission properties. chief among these fluorophores are the on/off photoswitchable and green-to-red photoconvertible fluorescent proteins. irisfp was recently reported as the first fluorescent protein to combine irreversible photoconversion from a green-emitting to a red-emitting state with reversible on/off photoswitching in both the green and red states. the introduction of this protein resulted in new applications such as super-resolution pulse-chase imaging, but the properties of irisfp are far from being optimal from a spectroscopic point of view and its tetrameric organization complicates its use as a fusion tag. we have demonstrated how four-state optical highlighting can be rationally introduced into photoconvertible fluorescent proteins by developing and characterizing a new set of such enhanced optical highlighters derived from meo-sfp and dendra2. one of these, which we called nijifp, was identified as a promising new multi-photoactivatable fluorescent protein with optical properties that make it ideal for advanced fluorescence-based imaging applications. introducing to medicine and biology concept of optical markers in tremendous way has changed the recent status of these two important disciplines. this was mainly due to strong development in imaging techniques which recently allow us to investigate both static as well dynamic properties of living cells, their components and their interactions with external factors. recently used molecular markers including organic dyes, fluorescent proteins or chelates containing lanthanide ions have several significant limitations. one of the alternatives for molecular markers are inorganic quantum dots (ie. cdse, cds) which are recently commonly used in many academic works. however, even if they are much better from physicochemical point of view, from the application point of view at this moment they are rather useless mainly because of their high risk of toxicity. one of the solution combining advantages of both concepts is to make nontoxic inorganic nanocrystals doped by lanthanide ions. in this work, we will present optical results obtained for nayf 4 nanocrystals doped by different lanthanide ions. the aim of this work was to design and to synthesize these markers and to understand physical processes responsible for their emission and to optimize these processes to the physical limits. intravital microscopy has fostered a full blown of publication regarding the behavior of cells in different tissues and physiological conditions. however, a few papers describe how the motility parameters can be used to understand whether an interaction is occurring and, on balance, the distinction between interacting and non interacting cells is performed visually on the image time stack. here we describe a multi-parameter approach that allows to discern among different cell behaviors on an objective ground and we demonstrate its effectiveness valuating the mutual fate of natural killer (nk) and dendritic (dc) cells at the draining lymph-nodes in inflammatory and stationary conditions. the method is time saving and allows a wide scale characterization of the lymphocyte tracks and to build up statistics of the cell-cell interaction duration. this has allowed the development of a numerical model of the nk-dc interaction, based on a molecular-stochastic dynamic approach, whose output can be directly compared to the data. hemozoin is formed during malaria infection of red blood cells, the malaria parasite cleaves hemoglobin, leaving free heme which is then toxic to the parasite. the free heme is then bio-crystalized to form hemozoin which allows the parasite to remain viable. the hemozoin is released during the breakdown of the red blood cells, is small and can be difficult to resolve spatially. since it contains an abundance of heme protein, which has a strong absorbance at 532 nm, it can be readily detected and tracked by using resonant raman scattering spectroscopy. here we use slit-scanning confocal raman imaging to detect the hemozoin and resolve it against the background molecules. inside a red blood cell, hemoglobin is the strongest background signal since it also contains large amounts of heme. nevertheless, the discrimination is possible, and the time-resolved observation of hemozoin is an important tool to understand effects of malaria since the hemozoin can trigger the immune response and cause inflammation in tissue. muscle performance at the molecular level is determined by the elementary displacement (working stroke) produced by the motor protein myosin ii, and its dependence on load. we developed a laser trap assay (the optical leash) capable of applying controlled loads to a single myosin head before the working stroke is initiated and probing actin-myosin interaction on the microsecond time scale. we found that the working stroke size depends both on load and on the detachment pathway followed by myosin. in a first pathway, myosin detaches very rapidly from actin (\1ms) without producing any movement. in a second pathway, myosin steps and remains bound to actin for a time inversely proportional to atp concentration; the working stroke remains constant (*5 nm) as the load is increased, until it suddenly vanishes as the isometric force is reached (5.7±0.6pn). a third dissociation pathway becomes more populated as the force is increased, due to premature unbinding of myosin from actin, resulting in a working stroke that decreases with load. taken together, these results give a new insight into the molecular mechanism of load dependence of the myosin working stroke, which is a primary determinant of skeletal muscle performance and efficiency. previously we have deleted either or both of these terminal helices genetically. surprisingly, all mutants rotated in the correct direction, showing that the shaft portion is dispensable. here we inquire if the rest of the c rotor, the globular protrusion that accounts for *70 % of the c residues, is also dispensable. keeping the n-and c-terminal helices that constitute the shaft, we have replaced the middle *200 residues with a short helix-turn-helix motif borrowed from a different protein. the protrusion-less mutant thus made retained a high atpase activity and rotated fast in the correct direction. this may not be unexpected because, in crystal structures, most of the removed residues do not contact with the a 3 b 3 ring. combined with the previous results, however, the present results indicate that none of the c residues are needed for rotation. the rotary mechanism of a molecular engine, the vacuolar proton-atpase, working in a biomembrane csilla ferencz 1 , pá l petrovszki 1 , zoltá n kó ta 1 the rotary mechanism of vacuolar proton-atpase (v-atpase) couples atp hydrolysis and trans-membrane proton translocation. we tested the effect of oscillating electric (ac) field on v-atpase activity in yeast vacuoles. the ac technique has several advantages over direct observations: it can be applied on native membranes, there are no labels and attachments involved, and the target protein is in its natural environment. this was/is the first of its kind of experiment on v-atpase, and we got strikingly different results from previous studies on other proteins: both low and high frequency ac field reduces atpase activity in a wide frequency range. a sharp resonance is seen at 88.3 hz, where the atpase activity reaches or exceeds the control (no ac) level. we think that the resonance happens at that of the 60 degrees rotor steps, meaning that the rotation rate of the rotor is around 15 hz, under the given conditions. synchronisation of individual atpases by slow or matching, but not fast ac is likely via a hold-and-release mechanism. we can explain the above observations by assuming that the ac field interacts with the proton movements, and if we consider the estimated geometry of the hydrophilic proton channels and the proton binding cites on the rotor. [2] . the ttss constitutes a continuous protein transport channel of constant length through the bacterial envelope [3] . the needle of the type three secretion system is made of a single small protein (protomer). we analyzed the assembly and the structure of the ttss needle using different biophysical methods including fourier transform-infrared spectroscopy, nmr spectroscopy and x-ray crystallography. we show that the ttss needle protomer refolds spontaneously to extend the needle from the distal end. the protomer refolds from a-helix into b-strand conformation to form the ttss needle [4] . regulated secretion of virulence factors requires the presence of additional protein at the ttss needle tip. x-ray crystal structure analysis of the tip complex revealed major conformational changes in both the needle and the tip proteins during assembly of the s. typhimurium ttss. our structural analysis provides the first detailed insight into both the open state of the ttss needle tip and the conformational changes occurring at the pathogen-host interface [5] . [ the membrane-bound component f o of the atp synthase works as a rotary motor and plays the central role of driving the f 1 component to transform chemi-osmotic energy into atp synthesis. we have conducted molecular dynamics simulations of the membrane-bound f o sector with explicit lipid bilayer, in which the particular interest was to observe the onset of helix motion in the c ring upon the change of the protonation state of asp61 of the c 1 subunit, which is the essential element of the boyer's binding-change mechanism. to investigate the influence of transmembrane potential and ph gradient, i.e., the proton motive force, on the structure and dynamics of the a-c complex, different electric fields have been applied along the membrane normal. correlation map analysis indicated that the correlated motions of residues in the interface of the a-c complex were significantly reduced by external electric fields. the deuterium order parameter (s cd ) profile calculated by averaging all the lipids in the f o -bound bilayer was not very different from that of the pure bilayer system, which agrees with recent 2 h solid-state nmr experiments. however, by delineating the lipid properties according to their vicinity to f o , we found that the s cd profiles of different lipid shells are prominently different. lipids close to f o formed a more ordered structure. similarly, the lateral diffusion of lipids on the membrane surface also followed a shell-dependent behavior. the lipids in the proximity of f o exhibited very significantly reduced diffusional motion. the numerical value of s cd was anti-correlated with that of the diffusion coefficient, i.e., the more ordered lipid structures led to slower lipid diffusion. our findings will not only help for elucidating the dynamics of f o depending on the protonation states and electric fields, but may also shed some light to the interactions between the motor f o and its surrounding lipids under physiological condition, which could help to rationalize its extraordinary energy conversion efficiency. this work has been published 1 in march 2010, and it was selected as one of the two featured articles of that issue. the research project is directed toward the construction of a synthetic bio-inorganic machine that consists in a single actin filament that interacts with a linear array of myosin ii motors regularly disposed on a nano-structured device. the motor array is intended to simulate the unique properties of the ensemble of motor proteins in the half-sarcomere of the muscle by providing the condition for developing steady force and shortening by cyclic interactions with the actin filament. the mechanical outputs in the range 0.5-200 pn force and 1-10,000 nm shortening will be measured and controlled the bacterial flagellar motor is a membrane-embedded molecular machine that rotates filaments, providing a propulsive force for bacteria to swim. the molecular mechanism of torque (turning force) generation is being investigated through the study of the properties and three-dimensional structure of the motor's stator unit. we are taking both topdown and bottom-up approaches, combining data from the molecular genetics studies, cross-linking, x-ray protein crystallography and molecular dynamics simulations. we have recently determined the first crystal structure of the protein domain that anchors the proton-motive-force-generating mechanism of the flagellar motor to the cell wall, and formulated a model of how the stator attaches to peptidoglycan. the work presented at the meeting will inform the audience on our latest work that establishes the relationship between the structure, dynamics and function of a key component of the bacterial flagellar motor, the motility protein b (motb). this work will be put in the perspective of the mechanism of rotation, stator assembly, anchoring to peptidoglycan and interaction with the rotor, and discussed in the light of the elementary events composing the cycle of electrochemical-to-mechanical energy conversion that drives flagellar rotation. members of the conserved kinesin-5 family fulfill essential roles in mitotic spindle morphogenesis and dynamics and were thought to be slow, processive microtubule (mt)-plusend directed motors. the mechanisms that regulate kinesin-5 function are still not well understood. we have examined in vitro and in vivo functions of the saccharomyces cerevisiae kinesin-5 cin8 using single-molecule motility assays and single-molecule fluorescence microscopy and found that cin8 motility is exceptional in the kinesin-5 family. in vitro, individual cin8 motors could be switched by ionic conditions from rapid (up to 50 lm/min) and processive minus-end, to bidirectional, to slow plus-end motion. deletion of the uniquely large insert of 99 amino acids in loop 8 of cin8 induced bias towards minus-end motility and strongly affected the directional switching of cin8 both in vivo and in vitro. we further found that deletion of the functionally overlapping kinesin-5 kip1 and of the spindle-organizing protein ase1 affected cin8 velocity and processivity, but directionality was not affected. the entirely unexpected finding of switching of cin8 directionality in vivo and in vitro demonstrates that the ''gear box'' of kinesins is much more complex and versatile than thought. many biological motor molecules move within cells using stepsizes predictable from their structures. myosin-vi, however, has much larger and more broadly-distributed stepsizes than those predicted from its short lever arms. we explain the discrepancy by monitoring qdots and gold nano-particles attached to the myosin vi motor domains using high-sensitivity nano-imaging. the large stepsizes were attributed to an extended and relatively rigid lever arm; their variability to two stepsizes, one large (72 nm) and one small (44 nm). these results suggest there exist two tilt-angles during myosin-vi stepping, which correspond to the pre-and post-powerstrokes states and regulate the leading head. the large steps are consistent with the previously reported hand-over-hand mechanism, while the small steps follow an inchworm-like mechanism and increase in frequency with adp. switching between these two mechanisms in a strain sensitive, adpdependent manner allows myosin-vi to fulfill its multiple cellular tasks including vesicle transport and membrane anchoring. http://www.fbs.osaka-u.ac.jp/labs/yanagida/, http:// www.qbic.riken.jp/. ferritin deposits iron in oxyhydroxide iron core surrounded by protein shell. the iron core structure may vary in different ferritins in both normal and pathological cases. to study iron core variations the mö ssbauer spectroscopy with a high velocity resolution was applied for comparative analysis of normal and leukemia chicken liver and spleen tissues, human liver ferritin and commercial pharmaceutical products imferon, maltoferò and ferrum lek as ferritin models. mö ssbauer spectra of these samples measured with a high velocity resolution at room temperature were fitted using two models: homogenous iron core (one quadrupole doublet) and heterogeneous iron core (several quadrupole doublets). the results of both fits demonstrated small variations of mö ssbauer hyperfine parameters related to structural variations of the iron cores. these small structural variations may be a result of different degree of crystallinity, the way of iron package, nonequivalent iron position, etc. obtained small differences for normal and leukemia tissues may be useful for distinguishing ferritins from normal and pathological cases. this work was supported in part by the russian foundation for basic research (grant # 09-02-00055-a). invasion of epithelial cells by salmonella enterica is mediated by bacterial ''effector'' proteins that are delivered into the host cell by a type iii secretion system (ttss). the collaborative action of these translocated effectors modulates a variety of cellular processes leading to bacterial uptake into mammalian cells. type iii effectors require the presence in the bacterial cytosol of specific tts chaperones. effectors are known to interact with their chaperone via a chaperone binding domain (cbd) situated at their n-terminus. this work focus on sopb, an effector with phosphoinositide phosphatase activity and particularly its interaction with the specific chaperone sige by biochemical, biophysical and structural approaches. we have co-expressed sopb with its specific chaperone sige and purified the complex, determined the limits of the cbd and purified the sopb cbd /sige complex. the structure of sige has been solved previously but no crystals could be obtained for structure determination of both complexes. we used saxs experiment combined with biophysical approach to analyse the interaction between sopb and its chaperone as well as the quaternary structure on the complex that will be described in this presentation. guanylate monophosphate kinase (gmpk) is a cytosolic enzyme involved in nucleotide metabolic pathways. one of the physiological roles of gmpks is the reversible phosphoryl group transfer from atp to gmp (its specific ligand), yielding adp and gdp. the gmpk from haemophilus influenzae is a small protein, with a number of 208 amino acids in the primary structure. in order to determine the secondary structure changes of this enzyme, as well as some physical characteristics of its complexes with gmp and atp ligands, circular dichroism (cd) and atr -ftir studies were performed. the enzyme and its ligands were dissolved in tris -hcl buffer, at ph 7.4 and 25°c. from the cd spectra the content of the secondary structure elements of gmpk and gmpk/gmp, gmpk/atp (with and without mg 2+ ) were determined. the major secondary structure elements of gmpk from haemophilus influenzae were a-helix (* 37 %) and b-sheet (* 36 %). atr -ftir experiments show that the amide i and amide ii bands of the gmpk are typical for a protein with great a-helix content. from the second derivative spectra, the content of the secondary structure elements were estimated. these data were in agreement with those obtained by cd. assembly of the mature human immunodeficiency virus type 1 capsid involves the oligomerization of the capsid protein, ca. the c-terminal domain of ca, ctd, participates both in the formation of ca hexamers, and in the joining of hexamers through homodimerization. intact ca and the isolated ctd are able to homodimerize in solution with similar affinity (dissociation constant in the order of 10 lm); ctd homodimerization involves mainly an a-helical region. in this work, we show that peptides derived from the dimerization helix (which keep residues energetically important for dimerization and with higher helical propensities than the wild-type sequence) are able to self-associate with affinities similar to that of the whole ctd. moreover, the peptides have a higher helicity than that of the wild-type sequence, although is not as high as the theoretically predicted. interesting enough, the peptides bind to ctd, but binding in all peptides, but one, does not occur at the dimerization interface of ctd (helix 9). rather, binding occurs at the last helical region of ctd, even for the wild-type peptide, as shown by hsqc-nmr. as a consequence, all peptides, but one, are unable to inhibit capsid assembly of the whole ca in vitro. the peptide whose binding occurs at the ctd dimerization helix has an val?arg mutation in position 191, which has been involved in dimer-dimer contacts. these findings suggest that event keeping the energetically important residues to attain ctd dimerization within a more largely populated helical structure is not enough to hamper dimerization of ctd. putp is an integral membrane protein located in the cytoplasmic membrane of escherichia coli, being responsible for the coupled transport of na + and proline. it belongs to the family of sodium solute symporters (sss). structural data for putp is not available, but secondary structure predictions together with biochemical and biophysical analyses suggest a 13 transmembrane motif. from a recent homology model based on the x-ray structure of the related na + /galactose symporter vsglt, previously published electron paramagnetic resonance (epr) studies, and recent crystallographic and epr studies on the cognate bacterial homolog of a neurotransmitter:na + symporter, leut, it has been proposed that helices viii and ix as well as the interconnecting ''loop 9'' region determine the accessibility of the periplasmic cavities which bind sodium and proline. we performed site-directed spin labeling of ''loop 9'' in combination with epr spectroscopy to investigate the structural features of this region and possible conformational changes induced by sodium and proline. analyses of spin label mobility and polarity as well as accessibility to paramagnetic quenchers allow us to refine this region in the present homology model. furthermore, our data suggest conformational changes in this region upon substrate binding including an overall motion of a helical segment. fatty acid-binding proteins (fabp) are a family of low molecular weight proteins that share structural homology and the ability to bind fatty acids. the common structural feature is a b-barrel of 10 antiparallel b-strands forming a large inner cavity that accommodates nonpolar ligands, capped by a portal region, comprising two short a-helices. b-fabp exhibits high affinity for the docosahexaenoic (dha) and oleic acid (oa). it is also postulated that b-fabp may interact with nuclear receptors from ppar family. in the present work, we used molecular biology and spectroscopic techniques to correlate structure, dynamics and function. site-directed mutagenesis was used to produce 5 mutants of b-fabp with a nitroxide spin probe (mtsl) selectively attached to residues located at the portal region. esr spectra of the labeled b-fabp mutants were sensitive to the location of the mutation and were able to monitor interactions in three cases: (1) shsp are ubiquitous proteins involved in cellular resistance to various stress (oxidative, heat, osmotic…). they are able to prevent aggregation of non-native proteins through the ability of forming large soluble complexes and preventing their nonspecific and insoluble aggregation. in consequence to this molecular chaperone function, they can regulate many processes (resistance to chemotherapy, modulation of the cellular adhesion and invasion, inflammatory response in skin), and the modulation of their expression has been found to be a molecular marker in cancers, spermatogenesis, or cartilage degeneration. furthermore, they are involved in several pathologies: myopathies, neuropathies, cancers, cataracts. among the 10 human members (hspb1-10), this study focused on hspb1 (hsp27 involved in some cancers), hspb4 (lens specific), hspb5 (lens, muscle, heart, lung) and hspb5-r120g (responsible for a desmin-related myopathy and a cataract). as shsp form large, soluble (but polydisperse in mammals) hetero-oligomers, molecular biology, biochemistry, biophysics and bioinformatics were successfully combined to compare the functional/dysfunctional assemblies in order to understand the critical parameters between shsp members depending upon their tissue and cellular localization. ionizing radiation is a type of radiation that contains enough energy to displace electrons and break chemical bonds. this can promote the removal of at least one electron from an atom or molecule, creating radical species, namely, reactive oxygen species (ros) [1, 2] . these are often associated with damages at cellular level, such as, dna mutations, cell cycle modifications and, in animal cells, cancer. to overcome this problem, organisms developed different protection/repair mechanisms that enable them to survive to these threats. dna glycosylases are enzymes that are part of base excision repair (ber) system, mainly responsible for dna repair. they can recognize a dna lesion and, in some cases, are able to remove the mutated base. here we propose to study one of those enzymes, endonuclease iii, which contains a [4fe-4s] cluster [3, 4] . samples were exposed to different doses of uv-c radiation and the effects were studied by electrophoretic and spectroscopic methods. [ na,k-atpase is an integral protein present in the plasma membrane of animal cells, and consists of two main subunits: the a and b. cholesterol is an essential constituent of the animal membrane cells. in order to study the interaction between na,k-atpase and cholesterol, we have used the dsc technique, and a proteoliposome system composed by the enzyme and dppc:dppe, with different percentage in mol of cholesterol. the heat capacity of purified na,k-atpase profile exhibits three transitions with 31, 189 and 60 kcal/mol at 55, 62 and 69°c. multiple components in the unfolding transition could be attributed either to different steps in the pathway or to independent unfolding of different domains. this denaturation of na,k-atpase is an irreversible process. for the proteoliposome, we also observed three peaks, with 180, 217 and 41 kcal/mol and 54, 64 and 72°c. this increase in dh indicates that the lipids stabilize the protein. when cholesterol was used (from 10 to 40 mol %), the first transition was shifted to a lower temperature value around 35°c. these results confirm that cholesterol has an influence on the packing and fluidity of lipid bilayer and changes in lipid microenvironment alter the thermostability as well as the activity of na,k-atpase. financial support: fapesp. we have undertaken to study the structure and function of peroxisomal multifunctional enzyme type 2 (mfe-2) from different organisms. mfe-2 is a key enzyme in long-and branched-chain fatty acids breakdown in peroxisomes. it contains two enzymes in the same polypeptide and also consists of differing amount of domains depending on the species. crystal structure and enzyme kinetics of drosophila melanogaster mfe-2 has revealed the domain assembly and raised a question about existence of a substrate channeling mechanism. small-angle x-ray scattering studies have further resolved the assembly of domains in the human mfe-2. mutations in the mfe-2-coding gene in humans may cause d-bifunctional protein deficiency -a metabolic disease characterized by accumulation of fatty acyl-coa intermediates due to inactive or residually active mfe-2 protein. we have also studied the structure, stability and dynamics of such mutant proteins both experimentally and in silico. the latest results on all these studies will be presented. ftsz is a protein that plays a key role in bacterial division, forming a protein ring directly related to the constriction of the membrane. this process has been observed to occur without the help of molecular motors. nonetheless, the details of the self-assembly and subsequent force generation of the septal ring are still obscure. afm observations allows the study of the behaviour of ftsz solutions on a substrate with unprecedented resolution, permitting the identification of individual protein filaments. the different resulting structures can be compared to monte carlo models for a 2d lattice accounting for the essential interactions between monomers. these include a strong longitudinal bond that allows a limited flexibility (i.e., curvature of the filaments) and a weaker lateral interaction. the work we present follows this approach, focussing on the latest experiments with ftsz mutants. by using these mutants, it is possible to choose the specific region of the monomer that will anchor to the substrate, thus generating new structures that provide an insight into monomer-monomer interactions. in this way, we explore the anisotropy of the lateral bond in ftsz, a factor that has not been taken into account before but may prove to be of importance in fstz behaviour in vivo. modeling protein structures and their complexes with limited experimental data dominik gront university of warsaw, pasteura 1, 02-093 warsaw, poland conventional methods for protein structure determination require collecting huge amounts of high-quality experimental data. in many cases the data (possibly fragmentary and/or ambiguous) on itself cannot discriminate between alternative conformations and a unique structure cannot be determined. small angle xray scattering is an example of such a ''weak'' experiment. the spectrum encodes only several independent degrees of freedom that provide a global description of a molecular geometry in a very synthetic way. in this contribution we utilized both local information obtained from nmr measurements and global description of a macromolecule as given by saxs profile combined with a knowledge-based bimolecular force field to determine tertiary and quaternary structure of model protein systems. saxs curve as well as various kinds of local nmr data such as isotropic chemical shifts and their tensors, j-couplings, rdc, backbone noe and redor from nmr in solid phase are parsed with the ''experimental'' module of bioshell toolkit and utilized by rosetta modeling suite to generate plausible conformations. obtained results show the new protocol is capable to deliver very accurate models. noenet-use of noe networks for nmr resonance assignment of proteins with known 3d structure dirk stratmann, carine van heijenoort and eric guittet structural genomics programs today yield an increasing number of protein structures, obtained by x-ray diffraction, whose functions remain to be elucidated. nmr plays here a crucial role through its ability to readily identify binding sites in their complexes or to map dynamic features on the structure. an important nmr limiting step is the often fastidious assignment of the nmr spectra. for proteins whose 3d structures are already known, the matching of experimental and back-calculated data allows a straightforward assignment of the nmr spectra. we developed noenet, a structure-based assignment approach. it is based on a complete search algorithm, robust against assignment errors, even for sparse input data. it allows functional studies, like modeling of protein-complexes or protein dynamics studies for proteins as large as 28 kd. almost any type of additional restraints can be used as filters to speed up the procedure or restrict the assignment ensemble. noenet being mainly based on nmr data (noes) orthogonal to those used in triple resonance experiments (jcouplings), its combination even with a low number of ambiguous j-coupling based sequential connectivities yields a high precision assignment ensemble. we observed, that t. thermophilus isopropylmalate dehydrogenase (ipmdh) has higher rigidity and lower enzyme activity at room temperature than its mesophilic counterpart (e. coli), while the enzymes have nearly identical flexibilities under their respective physiological working conditions, suggesting that evolutionary adaptation tends to maintain optimum activity by adjusting a ''corresponding state'' regarding conformational flexibility. in order to reveal the nature of the conformational flexibility change related to enzymatic activity, we designed a series of mutations involving non conserved prolines specific to thermophilic ipmdh. proline to glycine mutations substantially increased conformational flexibility and decreased conformational stability. the mutant enzyme variants did not show enhanced catalytic activity, but the non arrhenius temperature dependence of enzyme activity of the wild type was abolished. this phenomenon underlines the fact that the delicate balance between flexibility, stability and activity which is required for the environmental adaptation of enzymes can be easily disrupted with mutations even distant from the active site, providing further evidence that optimization of proper functional motions are also a selective force in the evolution of enzymes. the kinetoplastids trypanosoma brucei, t. cruzi and leishmania major are responsible for causing great morbidity and mortality in developing countries. the all a-helical dimeric dutpases from these organisms represent promising drug targets due to their essential nature and markedly different structural and biochemical properties compared to the trimeric human enzyme. to aid in the development of dutpase inhibitors we have been structurally characterizing the enzymes from these species. here we present the structure of the t. brucei enzyme in open and closed conformations, completing the view of the enzymes from the kinetoplastids. furthermore, we sought to probe the reaction mechanism for this family of enzymes as a mechanism has been proposed based on previous structural work but has not received any further verification. the proposed scheme is similar to that of the trimeric enzyme but differs in detail. using tryptophan fluorescence quenching in the presence of the transition state mimic alf 3 we have been able to identify which species is the likely transition state in the reaction. the crystal structure of t. brucei in complex with this transition state analogue confirms the nature of the nucleophilic attack clearly showing how it differs from trimeric enzymes. the structure of factor h-c3d complex explains regulation of immune complement alternative pathway circular dichroism (cd) spectroscopy is a widely used technique for studying the secondary structure (ss) of proteins. numerous algorithms have been developed for the estimation of the ss composition from the cd spectra. although, these methods give more or less accurate estimation for proteins rich in a-helical structure, they often fail to provide acceptable results on mixed or b-rich proteins. the problem arises from the diversity of b-structures, which is thought to be an intrinsic limitation of the technique. the worst predictions are provided for proteins of unusual b-structures and for amyloid fibrils. our aim was to develop a new algorithm for the more accurate estimation of ss contents for a broader range of protein folds with special interest to amyloid fibrils. using synchrotron radiation cd (srcd), we were able to collect high quality spectra of amyloid fibrils with good s/n ratios down to 175nm. the novel reference dataset with spectra that significantly differ from present reference sets, extends the information content for ss determination. our algorithm takes into account the diverse twist of the b-sheets that has a great influence on the spectral features. for the first time, we can reliably distinguish parallel and antiparallel b-structure using cd spectroscopy. monitoring the assembly of the membrane protein insertase alexej kedrov 1 , marko sustarsic 2 , arnold j.m. driessen 1 1 groningen biomolecular sciences and bioengineering institute, university of groningen, the netherlands, 2 university of oxford, uk molecular forces that govern membrane protein integration and folding remain a major question in current molecular biology and biophysics. each nascent polypeptide chain should acquire its unique three-dimensional folded state within a complex environment formed by the anisotropic lipid membrane and the membrane-water interface. secyeg translocase and members of a recently described yidc/oxa1/alb3 chaperone family are recognized as primary players in the membrane protein genesis. these proteins, so called insertases, serve as membraneembedded molecular pores where the newly synthesized protein is loaded prior its release into the bilayer. here we apply fluorescence correlation spectroscopy to monitor the assembly of the insertase:ribosome:nascent polypeptide chain complexes in solution and reconstituted into nanodiscs and model membranes. results provide insights on molecular mechanisms and dynamics of the insertase functioning. conformational changes during gtpase activity induced self-assembly of human guanylate binding protein 1 revealed by epr spectroscopy correct assembly and regulation of multi-component molecular machines is essential for many biological activities. the type iii secretion system (t3ss) is a complex molecular machine that is a key virulence determinant for important gram-negative pathogens including shigella, yersinia and salmonella species [1] [2] . the t3ss consists of multiple copies of *25 different proteins (totalling *7mda), spans both bacterial membranes and drives insertion of a contiguous pore into the host-cell membrane. virulence factors are secreted via this apparatus directly into the host cell. in all t3ss various levels of regulation occur with switching between secretion off and on states overlaid on control of which substrates are secreted. genes involved in a variety of these switches have been identified but the molecular mechanisms underlying these functions is poorly understood. we are studying the t3ss of shigella flexneri, the causative agent of dysentery and will present the structure of the so-called ''gatekeeper protein'' mxia. the diacylglycerolacyltransferase1 (dgat1) is an integral protein from the reticulum endoplasmic membrane that plays an essential role in triacylglyceride synthesis. in cattle, this enzyme is associated to the fat content regulation on milk and meat. in this study, synthetic peptides corresponding to both dgat1 binding sites (sit1 and sit2) were designed, purified and employed to investigate the enzyme interaction with substrates and membrane models. different binding specificities in the interaction with phospholipid vesicles and micelles were noted. sit1 showed to bind more strongly in nonpolar membrane models, while sit2 was electrostatically attracted to negative phospholipid surfaces. the binding of both peptides was followed by significant conformational changes (like unordered to helix transition) in circular dichroism spectra and a 20nm blue shift in fluorescence emission. the binding of sit1 and sit2 peptides to negative liposome gave dissociation constants (k d ) of 170 and 0.44lm, respectively, and a leakage action 24-fold higher to sit2. the difference in specificity is related to the features of the putative substrates (acyl-coas and diacylglycerol) and can be attributed to the distinct role of each dgat1 binding site during lipid synthesis. supported by fapesp. ftsz, the bacterial homologue of tubulin, assembles into polymers in the bacterial division ring. the interfaces between monomers include a gtp molecule, although the relationship between polymerization and gtpase activity is still controversial. a set of short ftsz polymers were modelled and the formation of active gtpase structures was monitored using molecular dynamics. only the interfaces nearest the polymer ends exhibited geometry adequate for gtp hydrolysis. conversion of a mid-polymer interface to a close-to-end interface resulted in its spontaneous rearrangement from an inactive to an active conformation. fluorescent proteins (fps) have become extremely valuable tools in the life sciences. due to the latest advances in the light microscopy, there is a steady need for fps with improved spectral properties. mirisfp is a monomeric fp that can be switched reversibly between a bright green fluorescent and a dark state by illumination with light of specific wavelengths [1] . structurally, this photo-switching is based on a cis-trans isomerization of the chromophore. upon illumination with violet light, mirisfp can be irreversibly photoconverted from the green-emitting to a red-emitting form. the red form can again be switched reversibly between a fluorescent and a dark state. to elucidate the mechanistic details of the photoinduced reactions, we have generated mirisgfp1. this variant can still undergo reversible photoswitching, but lacks the ability to photoconvert to the red state so that the photoinduced transitions of the green form can be studied without 'artifacts' due to green-to-red photoconversion. using uv/visible spectroscopy, we have characterized the on-and off-switching processes in great detail. several light-activated reaction pathways have been identified. they are highly intertwined so that the net effect achieved with light of a particular wavelength depends on the relative probabilities to photoinduce the various processes. phosducin (pd) is a g t bc-binding protein that is highly expressed in photoreceptors. pd is phosphorylated in dark-adapted retina and is dephosphorylated in response to light. dephosphorylated pd binds g t protein bc-heterodimer with high affinity and inhibits its interaction with g t a or other effectors, whereas phosphorylated pd does not. therefore pd down-regulates the light response in photoreceptors. phosphorylation of pd at s54 and s73 leads to the binding of the 14-3-3 protein. the 14-3-3 proteins function as scaffolds modulating the activity of their binding partners and their role in pd regulation is still unclear. the 14-3-3 protein binding may serve to sequester phosphorylated pd from g t bc or to decrease the rate of pd dephosphorylation and degradation. we performed several biophysical studies of the 14-3-3:pd complex. analytical ultracentrifugation was used to determine the complex stoichiometry and dissociation constant. conformational changes of pd induced both by the phosphorylation itself and by 14-3-3 binding were studied using the time-resolved fluorescence spectroscopy techniques. mö ssbauer spectroscopy with a high velocity resolution was used for comparative study of various oxyhemoglobins for analysis of the heme iron electronic structure and protein structure-function relationship. samples of pig, rabbit and normal human oxyhemoglobins and oxyhemoglobins from patients with chronic myeloleukemia and multiple myeloma were measured using mö ssbauer spectrometric system with a high velocity resolution at 90 k. mö ssbauer spectra were fitted using two models: one quadrupole doublet (model of equivalent iron electronic structure in a-and b-subunits of hemoglobins) and superposition of two quadrupole doublets (model of non-equivalent iron electronic structure in a-and b-subunits of hemoglobins). in both models small variations of mö ssbauer hyperfine parameters (quadrupole splitting and isomer shift) were observed for normal human, rabbit and pig oxyhemoglobins and related to different heme iron stereochemistry and oxygen affinity. small variations of mö ssbauer hyperfine parameters for oxyhemoglobins from patients were related to possible variations in the heme iron stereochemistry and function. the different types of silk produced by orb-weaving spiders display various mechanical properties to fulfill diverse functions. for example, the dragline silk produced by the major ampulate glands exhibits high toughness that comes from a good trade-off between stiffness and extensibility. on the other hand, flagelliform silk of the capture spirals of the web is highly elastic due to the presence of proline and glycine residues. these properties are completely dictated by the structural organization of the fiber (crystallinity, degree of molecular orientation, secondary structure, microstructure), which in turn results from the protein primary structure and the mechanism of spinning. although the spinning process of dragline silk begins to be understood, the molecular events occurring in the secretory glands and leading to the formation of other silk fibers are unknown, mainly due to a lack of information regarding their initial and final structures. taking advantage of the efficiency of raman spectromicroscopy to investigate micrometer-sized biological samples, we have determined the conformation of proteins in the complete set of glands of the orb-weaving spider nephila clavipes as well as in the fibers that are spun from these glands. domain of rgs3 at 2.3å resolution was solved. the stoichiometry of 14-3-3 f /rgs3 protein complex was elucidated using the analytical ultracentrifugation. to map the interaction between 14-3-3f and rgs3 protein we performed a wide range of biophysical measurements: h/d exchange and cross link experiments coupled to mass spectrometry, fret (fö rster resonance energy transfer) time-resolved fluorescence experiments, time-resolved tryptophan fluorescence spectroscopy and saxs (small angle x-ray scattering) measurements. based on all these results we build 3d model of 14-3-3 f /rgs3 protein complex. our model revealed new details on architecture of complex formed by 14-3-3 proteins. to date all known structure of 14-3-3 proteins complexes suggests that the ligand is docked in the central channel of 14-3-3 protein. our results indicate that the rgs domain of rgs3 protein is located outside the central channel of 14-3-3f protein interacting with less-conserved residues of 14-3-3f. the receptor for advanced glycation end-products (rage) is a multiligand cell surface receptor involved in various human diseases. the major alternative splice product of rage comprises its extracellular region that occurs as a soluble protein (srage). although the structures of srage domains were available, their assembly into the functional full length protein remained unknown. here we employed synchrotron small-angle x-ray scattering to characterize the solution structure of human srage. the protein revealed concentration-dependent oligomerization behaviour, which was also mediated by the presence of ca 2+ ions. rigid body models of monomeric and dimeric srage were obtained from the scattering data recorded at different solvent conditions. the monomer displays a j-like shape while the dimer is formed through the association of the two n-terminal domains and has an elongated structure. the results provided insight into the assembly of i) the heterodimer srage:rage, which is responsible for blockage of the receptor signalling, and ii) rage homodimer, which is necessary for signal transduction, paving the way for the design of therapeutical strategies for a large number of different pathologies. clpb is a hexameric aaa? atpase that extracts unfolded polypeptides from aggregates by threading them through its central pore. the contribution of coiled-coil m domains is fundamental for the functional mechanism of this chaperone, and its location within the protein structure in previous structural models is contradictory. we present cryo-electron microscopy structural analysis of clpb from e. coli in several nucleotide states. the study reveals a novel architecture for clpb and shows that m domains form an internal scaffold located in the central chamber of clpb hexamers. this inner structure transmits local signals due to atp binding and hydrolysis by aaa? domains. surprisingly, coiled-coil m domains are seen to bend significantly around a hinge region that separates two structural motifs. our results present a new framework to understand clpb-mediated protein disaggregation. streptomyces clavuligerus isoenzymes involved in clavulanic acid biosynthesis: a structural approach clavulanic acid (ca) is a potent b-lactamase inhibitor produced by streptomyces clavuligerus. n 2 -(2-carboxyethyl) arginine synthase (ceas) and proclavaminate amidino hydrolase (pah) catalyze the initial steps in the biosynthesis of ca. recently ceas1and pah1genes (paralogous of ceas and pah) were related to the ca biosynthesis but their products have not been studied yet. here we present the initial structural analysis of ceas1 and pah1 using biophysical techniques. pah1 and ceas1 genes were isolated from the genomic dna of s. clavuligerus and overexpressed in e. coli. the recombinant proteins were purified by affinity chromatography and analyzed by size exclusion chromatography, non-denaturing page, dynamic light scattering, far-uv circular dichroism (cd) and fluorescence spectroscopy. our results showed that pah1 and ceas1 were obtained as hexamer and dimer respectively. both proteins showed an a/b folding, being stable up to 35°c. above this temperature protein unfolding was observed but the complete unfolding was not observed, even at 100°c. moreover ceas1 and pah1 showed to be stable over a wide ph range (ph 5.5 -9.5). we are currently working on improving ceas1 crystals which are a promising step towards the elucidation of the ceas1 structure. supported by fapesp. • synchrotron radiation circular dichroism • mass spectrometry following vuv photoionisation • fluorescence imaging with lifetime and spectral measurements here we will present the srcd experiment. high photon flux of 10 10 photons / sec, improved detector performances as well as user-orientated software developments have proven to be the garants for successful data collections,which considerably increased the information content obtained. the exploration into the charge transfer region of the peptide bonds is adding specifically new insights. low sample volumes of as little as 2ll per spectra as well as convenient sample chamber handling allow for economic and efficient data collections. typical spectra acquisition from 280 to 170nm, last for 9 min for three scans with a 1nm step size. prior to high resolution based techniques, srcd spectrawill answer questions about folding states of macromolecules including dna, rna and sugar macromolecules as well as their complexes with proteins and specially membrane proteins. sporulation in bacillus subtilis begins with an asymmetric cell division producing a smaller cell called the forespore, which initially lies side-by-side with the larger mother cell. in a phagocytosis-like event, the mother cell engulfs the forespore so that the latter is internalised as a cell-within-a-cell. engulfment involves the migration of the mother cell membrane around the forespore until the leading edges of this engulfing membrane meet and fuse. this releases the forespore, now surrounded by a double membrane, into the mother cell cytoplasm. membrane migration during engulfment is facilitated by the interacting proteins spoiiq and spoiiiah that are membrane-associated and expressed in the forespore and the mother cell respectively 1 . they interact in the intercellular space and function initially as a molecular zipper and later they participate in a more elaborate complex in which spoiiq and spoiiiah are integral components of an intercellular channel. this channel is a topic of much current interest, having initially been proposed as a conduit for the passage from the mother cell to the forespore of a specific, but putative, regulator of the rna polymerase sigma factor, r g 2 and later as a gap junction-like feeding tube 3 through which the mother cell supplies molecules for the biosynthetic needs of the forespore. here we present data on the structure and interactions of spoiiq and spoiiiah gleaned from biophysical methods and protein crystallography. these data lead to a plausible model for the intercellular channel. the glycine receptor (glyr) is a chloride permeable ligand gated ion channel and that can mediate synaptic inhibition. due to a possible involvement in the pathophysiology of temporal lobe epilepsy, the different properties of glyrs containing alpha3l and k subunit isoforms are currently investigated. previous characterizations of homomeric receptors consisting of these isoforms have shown a difference in their electrophysiological properties and their membrane distribution as observed by diffraction-limited fluorescence microscopy. we studied these isoforms, when separately expressed in transfected hek 293t cells, by using single molecule tracking (smt) in living cells and direct stochastic optical reconstruction microscopy (dstorm) on fixated cells. for both techniques the glyrs are stained using a primary antibody directly labeled with alexa fluor dyes. the dstorm experiments support the observation that alpha3l glyr are clustered, while the alpha3k glyrs are more uniformly spread. the analysis of the short range diffusion coefficients obtained by smt reveals the presence of heterogeneous motion for both isoforms. the k-isoform has a higher fraction of fast diffusion. in contrast, the l-isoform is more associated with slow diffusion and appears to undergo hindered diffusion. since nanoparticles are suitable for tumor therapy due to their passive targeting to cancer cells by enhanced permeability and retention effect [1] , it is important to understand mechanisms of their delivery into the living cancer cells. in this respect we have developed a modular spectral imaging system based on a white light spinning disk confocal fluorescence microscope and a narrow tunable emission filter. firstly, interaction of polymer nanoparticles and cells labeled with spectrally overlapping probes was examined. the use of fluorescence microspectroscopy (fms) allowed co-localization, which showed that the size of polymer nanoparticles strongly influences their transfer across the cell plasma membrane. next, the delivery of liposomes (composed of cancerostatic alkylphospholipid (opp) and cholesterol) labeled with environment-sensitive fluorescent probe was monitored. we were able to detect a very small shift in emission spectra of cholesterol-poor opp liposomes inside and outside the cells, which would not be possible without the use of fms. this shift implies that the delivery of these liposomes into cancer cells is based on fusion with the cell membrane [2] . [ high-resolution optical imaging techniques make now accessible the detection of nanofeatures in bio-and soft-matter by non-ionizing visible radiation. however, high-resolution imaging is critically dependent by the fluorescent probes used for reporting on the nano-environment. on account of our long-standing interest in the development of fluorescent probes, we set out to design and engineer new fluorescent systems for nanoscale imaging and sensing of biological specimens and soft-matter. these fluorophores report on fast subtle changes of their nanoscale environment at excited state and are meant to fulfill these requirements: a) optical responses (intensity, wavelength-shift, lifetime, anisotropy) predictably related to the environmental polarity, viscosity, macromolecular structure, b) high brightness allowing for single-molecule detection, c) easily conjugable to biomolecules or macromolecules of interest. notably, we aim at conjugating these properties with the capability of nanoscopy imaging based on stimulated emission depletion or stochastic reconstruction optical microscopy. in this lecture the main features and applications of the engineered probes will be reviewed and future developments in this exciting field will be discussed. foamy virus (fv) is an atypical retrovirus which shares similarities with hiv and hepatitis b viruses. despite numerous biochemical studies, its entry pathway remains unclear, namely membrane fusion or endocytosis. to tackle this issue, dual color fluorescent viruses were engineered with a gfp labeled capsid and a mcherry labeled envelope. using high resolution 3d imaging and 3d single virus tracing, we followed the entry of the fluorescent viruses in living cells with a precision of 30nm in the plane and 40 nm along the optical axis. to distinguish between the two possible pathways, we developed a novel colocalization analysis method for determining the moment along every single trace where the colors separate, i.e. the fusion event. the combination of this dynamical colocalization information with the instantaneous velocity of the particle and its position within the reconstructed 3d cell shape allows us to determine whether the separation of capsid and envelope happens at the cell membrane or from endosomes. we then compared two types of fv and demonstrated, consistently with previous ph-dependency studies, that the prototype fv can enter the cell by endocytosis and membrane fusion, whereas the simian fv was only observed to fuse after endocytosis. phosphatidylinositol 4,5-bisphosphate (pi(4,5)p 2 ) is a minor component of the plasma membrane known to a critical agent in the regulation of synaptic transmission. clustering of pi(4,5)p 2 in synaptic active zones is important for synaptic transmission. however, pi(4,5)p 2 does not spontaneously segregate in fluid lipid membranes and another mechanism must be responsible for the lateral segregation of this lipid in active zones. clustering of pi(4,5)p 2 is expected to be associated with lipid-protein interactions and possibly partition towards lipid rafts in the plasma membrane. here we analyze the influence of protein palmitoylation on the formation of pi(4,5)p 2 clusters and on synaptic protein-pi(4,5)p 2 interaction by means of fö rster resonance energy transfer measurements by fluorescence lifetime imaging (fret-flim) and fret confocal microscopy. . during sporulation an entire chromosome is transferred into the forespore. this process starts by the formation of an asymmetrically located division septum that leads to the formation of two unequally sized compartments: a large mothercell and a smaller forespore. the septum traps about 30% of the chromosome to be transferred into the forespore. the remaining (*3mbp) are then translocated from the mother cell into the forespore by an active mechanism involving the spo-iiie dna translocase. the mechanisms of translocation, particularly the control of the directionality, still remains unknown and various models have been proposed so far. since each model predicts very different distribution of spoiiie proteins at the sporulation septum, we used palm microscopy (photoactivated localization microscopy) to investigate proteins localization in live-sporulating bacteria. using this technique, we showed that spoiiie proteins are forming a single tight focus at the septum with a characteristic size of around 30nm. more surprising, the focus is usually localized in the mother cell compartment and the mean distance between the spoiiie focus and the septum is 35nm. our data suggest that during the translocation process, spoiiie proteins are only forming stable complexes on the mother cell side, allowing then for a control of the chromosome translocation from the mother cell to the forespore. morphogenetic gradients determine cell identity in a concentration-dependent manner and do so in a way that is both incredibly precise and remarkably robust. in order to understand how they achieve this feat, one needs to establish the sequence of molecular mechanisms starting with morphogen gradient formation and leading to the expression of downstream target genes. in fruit flies, the transcription factor bicoid (bcd) is a crucial morphogen that forms an exponential concentration gradient along the embryo ap axis and turns on cascades of target genes in distinct anterior domains. we measured bcd-egfp mobility in live d. melanogaster embryos using fluorescence correlation spectroscopy and fluorescence recovery after photobleaching. we found that bcd-egfp molecules had a diffusion coefficient on the order of * 7 lm 2 /s during nuclear cycles 12-14, both in the cytoplasm and in nuclei. this value is large enough to explain the stable establishment of the bcd gradient simply by diffusion. on the other hand, in the context of the extremely precise orchestration of the transcription of the hunchback bcd target gene, it is too slow to explain how a precise reading of bicoid concentration could be achieved at each interphase without the existence of a memorization process. single molecule studies of key processes during the initiation of innate and adaptive immune response the two pillars of the vertebrate immune system are the innate and adaptive immune response, which confer resistance to pathogens and play a role in numerous diseases. here we exploit single molecule fluorescence imaging on live cells to study the key molecular processes that underpin these responses. the first project looks at the changes in the organisation of toll-like receptor 4 (tlr4) on the cell surface of macrophages upon activation via lipopolysaccharide (lps), as it is currently not known whether a higher level of tlr4 organisation is required for the signalling process. macrophages natively express tlr4 at a low level which allows for oligomerisation to be analysed in live cells by dynamic single molecule colocalisation (dysco) using data obtained by totalinternal reflection fluorescence (tirf) microscopy. the experiments of the second project aim at determining the critical initial events in t-cell triggering by labelling key proteins like the tcr receptor and cd45 on the surface of live t-cells and following how their spatial distribution changes following the binding of the t-cell to a surface. this enables us to distinguish between the different models of t-cell triggering which are based on aggregation, segregation or a conformational change of the tcr. the study of cells using scanning force microscopy the motility of unicellular parasites in mammalians seems very interesting, yet very complex. in a world, were inertia cannot be used for propulsion, in a world at low reynolds numbers, most of our everyday strategies of self-propulsion do not work. one class of parasites that know their way around, the flagellate trypansome, manage not only to survive in the blood stream, which is a lot faster than its own propulsion velocity and where the trypanosome is constantly attacked by its host's immune response, but also to penetrate the bloodbrain-barrier, which actually should be to tight to enter. even though trypanosomes are known for more than 100 years, their motility behaviour is not completely elucidated yet. now, using high-speed darkfield-microscopy in combination with optical tweezers in microfluidic devices and analyzing the recorded data, new light has been shed on the motility of these parasites. astonishing results show that trypanosomes are very well adapted to their hosts environment, they even can abuse red blood cells for their self-propulsion and use the bloodstream itself to drag antibodies bound to their surface to their cell mouth, where the antibodies are endocytosed and digested. the first part of the presentation will discuss nanoparticle (quantum dot, qd) biosensors and nanoactuators that exploit novel and unusual fret phenomena in the induction/detection of protein aggregation [1] , reversible on-off qd photoswitching [2] , and ph sensing [3] . the second part of the presentation will feature the application of an integrated chemical biological fret approach for the in situ (in/on living cells) detection of conformational changes in the ectodomain of a receptor tyrosine kinase (the receptor for the growth factor egf) induced by ligand binding [4] . the measurements were conducted with a two-photon scanning microscope equipped with tcspc detection; novel methods for lifetime analysis ad interpretation were employed to confirm the concerted domain rearrangements predicted from x-ray crystallography. the study of protein-protein interactions in vivo is often hindered by the limited acquisition speed of typical instrumentation used, for instance, for lifetime imaging microscopy. anisotropy polarization is altered by the occurrence of foerster resonance energy transfer (fret) and anisotropy imaging was shown to be comparatively fast and simple to implement. here, we present the adaptation of a spinning disc confocal microscope for fluorescence anisotropy imaging that allowed to achieve in vivo imaging at high spatial and temporal resolution. we demonstrate the capabilities of this system and in-house developed analysis software by imaging living caenorhabditis elegans expressing constitutive dimeric and monomeric proteins that are tagged with gfp. measuring intracellular viscosity: from molecular rotors to photodynamic therapy of cancer marina k. kuimova department of chemistry, imperial college london, exhibition road, sw7 2az, uk viscosity is one of the main factors which influence diffusion in condensed media and is crucial for cell function. however, mapping viscosity on a single-cell scale is a challenge. we have imaged viscosity inside live cells using fluorescent probes, called molecular rotors, in which the speed of rotation about a sterically hindered bond is viscosity-dependent [1] [2] [3] . this approach enabled us to demonstrate that viscosity distribution in a cell is highly heterogeneous and that the local microviscosity in hydrophobic cell domains can be up to 1009 higher than that of water. we demonstrated that the intracellular viscosity increases dramatically during light activated cancer treatment, called photodynamic therapy (pdt) [2] . we also demonstrated that the ability of a fluorophore to induce apoptosis in cells during pdt [4] , or to act as a benign molecular rotor, measuring viscosity, can be controlled by carefully selecting the excitation wavelength in viscous medium [5] . in the field of biophysics and nanomedicine, the cellular reaction and the kinetics of gene expression after transfection of live cells with plasmid dna or gene-silencing sirna is of great interest. in a previous study on the transfection kinetics of non-viral gene-transfer [1] we realised that the development of single-cell arrays would be a great step towards easy-to-analyse, high-throughput transfection studies. the regular arrangement of single cells would overcome the limitations in image-analysis that arise from whole populations of cells. in addition to that, the analysis of expression kinetics at the single-cell can help to identify the cell-to-cell variability within a cell population. in order to develop suitable single-cell arrays, we are currently adjusting the different parameters of such a microenvironment (e.g. size, shape, surface-functionalisation) in order to end up with a defined surrounding for single-cell transfection studies. in addition to that, we try to find the optimal uptake pathway for each of the different applications. the neurodegenerative disorder alzheimer's disease (ad) causes cognitive impairment such as loss of episodic memory with ultimately fatal consequences. accumulation and aggregation of two proteins in the brain -amyloid beta and tau -is a characteristic feature. these soluble proteins aggregate during the course of the disease and assemble into amyloid-like filaments. recently it was found that the toxicity of soluble amyloid beta oligomers must also be taken into account for the pathogenesis of cognitive failure in ad. if oligomers are the predominant toxic species it would be pertinent to determine how they disrupt and impair neuronal function. the prion protein (prp) receptor has been proposed to mediate amyloid beta binding to neuronal cells. we have characterised the interaction of the amyloid beta and the prp receptor expressed on hippocampal and neuroblastoma cells at the single-molecule level. we do not detect any colocalisation of either the 40 or 42 amino acids variants with the prp receptor. bacterial biofilms are of the utmost importance in the study of environmental bioremediation and the design of materials for medical applications. the understanding of the mechanisms that govern cell adhesion must be analysed from the physics point of view in order to obtain quantitative descriptors. the genus rhodococcus is widely spread in natural environments. the species are metabolically diverse and thus they can degrade a wide range of pollutants. due to their high hydrophobicity, these cells are very resistant to harsh conditions, are able to degrade hydrophobic substances (e.g. oil) and attach to high-contact angle surfaces. the hydrophobicity of several strains of rhodococcus is measured and mapped using chemical force microscopy (cfm) in the present study. cfm relies on the functionalisation of scanning force microscopy (sfm) tips using hydrophobic or hydrophilic groups. in cfm, the microscope is operated in the forcevolume mode, which combines adhesion data with topographic images. the careful control of the tip chemistry permits the study of interactions between the functional groups on the tip and the bacterial surface, thus allowing the assessment of hydrophobicity. in order to perform a cfm study, the cells need to be firmly anchored to a substrate under physiological conditions (i.e. under a nutrient media or a saline buffer). to this end, several adhesive surfaces have been tested in order to find the one that gives the best results. optical microscopy is arguably the most important technique for the study of living systems because it allows 3d imaging of cells and tissues under physiological conditions under minimally invasive conditions. conventional far-field microscopy is diffraction-limited; only structures larger than *200 nm can be resolved, which is insufficient for many applications. recently, techniques featuring image resolutions down to *20 nm have been introduced such as localization microscopy (palm, storm) and reversible saturable optical fluorescent transition microscopy (resolft, sted). these methods are well suited for live-cell imaging and narrow the resolution gap between light and electron microscopy significantly. we have used palm imaging to study the formation and disassembly of focal adhesions of live hela cells in a high resolution pulse-chase experiment using monomeric irisfp [1] . mirisfp is a photoactivatable fluorescent protein that combines irreversible photoconversion from a green-to a red-emitting form with reversible photoswitching between a fluorescent and a nonfluorescent state in both forms. in our experiments a subpopulation of mirisfp molecules is photoconverted to the red form by irradiating a specified region of the cell with a pulse of violet light. migration of tagged proteins out of the conversion region can be studied by subsequently localizing the proteins in other regions of the cell by palm imaging, now using the photoswitching capability of the red species. real-time image reconstruction developed in our lab [2] allowed instant control imaging parameters. live cell imaging of cancer cells is often used for in-vitro studies in connection with photodynamic diagnostic and therapy (pdd and pdt). especially in presence of a photosensitizer, this live cell imaging can only be performed over relatively short duration (at most 1 hour). this restriction comes from the light-induced cell damages (photodamages) that result from rapid fluorescence photobleaching of photosensitizer. while these studies reveal exciting results, it takes several hours to discover the detailed effects of the photosensitizer on cell damage. up to our knowledge, however, there is no general guideline for modification of excitation light dose to achieve that. in this paper, the relation between excitation light doses, photobleaching of photosensitizer (pvp-hyperycin) and cell vitality are investigated using human lung epithelial carcinoma cells (a549). the strategy of this paper is to reduce the excitation light dose by using a low-power pulsed blue led such that the structures are visible in time-lapse images. fluorescence signals and image quality are improved by labelling the cells with an additional non-toxic marker called carboxyfluorescein-diacetate-succinimidyl-ester (cfse). in total we collected 2700 time-lapse images (time intervals 2 min) of dual-marked a549 cells under three different light intensities (1.59, 6.34 and 14.27 mw/cm 2 ) and a variety of pulse lengths (0.127, 1.29, 13, 54.5 and 131 ms) over five hours. we have found that there is a nonlinear relationship between the amount of excitation light dose and cell vitality. cells are healthy, i.e. they commence and complete mitosis, when exposed to low light intensities and brief pulses of light. light intensities higher than 6.34 mw/cm 2 together with pulse durations longer than 13 ms often cause cell vesiculation, blebbing and apoptosis. in all other cases, however, we found no cell death. in the future, this striking nonlinearity will be studied in more detail. progressive advances in scanning ion conductance microscopy (sicm) [1] enabled us to convert ordinary scanning probe microscope (spm) in to versatile multifunctional technique. as an imaging tool, ion conductance microscopy is capable to deliver highest possible topographical resolution on living cell membranes among any other microscopy techniques [2] . also, it can visualize surfaces complexity of those makes them impossible to image by other spms [3] . ion conductance microscopy combined with a battery of powerful methods such as fluorescence resonance energy transfer (fret) [4] , patch-clamp, force mapping, localized drug delivery, nano-deposition and nano-sensing is unique among current imaging techniques. the rich combination of ion conductance imaging with other imaging techniques such as laser confocal and electrochemical [5] will facilitate the study of living cells and tissues at nanoscale. ) and coleoptiles of wheat (triticum aestivum l.) seedlings, which were growing in light and dark conditions, were used to determine fluorescence of whole cells. fluorescence emission spectrum was monitored by fluorescent microscopy using the spectrometer usb 4000. fluorescence intensity f490, f680, f710 and f740 was determined and data was statistically analyzed in annova. we observed that bgf, rf and frf intensity increased in the first leaves with the age of the seedlings. in the coleoptiles was observed great bgf intensity increase with the age of the seedlings. in the coleoptiles decreased rf intensity of the 144 and 196 hours old seedlings, and bgf intensity decreased of 196 hours old seedlings. it was found that emission spectrum and fluorescence intensity changes are induced by the lack of light and salt (nacl) stress. analysis of fluorescence spectrum can quickly and accurately indicate the outset of light and salt stress in plants. there are analogical changes in fluorescence emission spectrum of plant cells in senescence and stress conditions. it was assumed that environmental stress and senescence have common mechanisms in plants. this changes can be monitored by fluorescent microscopy. triple-colour super-resolution imaging in living cells markus staufenbiel, stephan wilmes, domenik lisse, friedrich roder, oliver beutel, christian richter and jacob piehler universitä t osnabrü ck, fachbereich biologie, barbarastraße 11, 49076, germany, markus.staufenbiel@biologie.uniosnabrü ck.de super-resolution fluorescence imaging techniques based on single molecule localisation has opened tremendous insight into the sub-micrometre organisation of the cell. live cell imaging techniques such as fluorescence photoactivation localization microscopy (fpalm) are currently limited to dual-colour detection due to the restricted availability of red-fluorescent photoswitchable proteins. we employed photoswitching of the oxazine dye atto655 under reducing conditions for super-resolution imaging in the cytoplasm of living cells. for efficient and specific covalent labelling of target proteins, we have made use of the halotag system. atto655 was coupled to the halotag ligand (htl) and fast reaction of htl-atto655 with the halotag enyzme was confirmed in vitro by solid phase binding assays. efficient labelling of the membrane cytoskeleton using lifeact fused to the halotag was observed and super-resolution imaging was readily achieved. based on this approach, we managed to follow the nanoscale dynamics of the actin cytoskeleton as well as clathrincoated pits using clathrin light chain fused to the halotag. we combined this technique with fpalm for triplecolour super-resolution imaging of the spatial distribution of membrane receptors in context of the membrane skeleton. the erbb family of receptor tyrosine kinases consists of four transmembrane proteins that transduce signals across the membrane to control cell fate. growth factor binding results in homo-and hetero-interactions between these receptors at the membrane. erbb receptors are implicated in many cancers, making them a target for therapeutic drugs. to date, studies of erbb interactions have been limited to individual family members or specific pairs, giving an incomplete picture of the highly complex behaviour controlling positive and negative feedback loops and signalling outcomes. to investigate erbb receptor interactions, we have developed tirf-based single molecule fluorescence microscopes capable of simultaneously imaging three, and soon five, fluorescence probes in live cells. we have also developed a catalogue of extrinsic fluorescent probes for 1:1 labelling of both endogenous and transfected erbb family members in mammalian cells, plus a bayesian approach to the analysis of single molecule data. this allows us to track active and inactive erbb family members at the basal surface of a model breast cancer cell line that expresses physiological levels of all four receptors. we present here initial characterisation of the entire erbb family together in the cell membrane. the human genome contains more than 800 g proteincoupled receptors (gpcrs); overall, 3-4% of the mammalian genome encodes these molecules. processes controlled by gpcrs include neurotransmission, cellular metabolism, secretion, and immune responses. however it is the stoichiometry of these receptors that is the most controversial. the starting point for understanding gpcr function was the idea that these receptors are monomeric. on the other hand a lot of recent studies favour the concept that gpcr form dimers and are not capable of signalling as independent monomers. recent single molecule studies try to solve this dilemma by suggesting that gpcrs form transient dimers with a lifetime of *100 ms. however questions remain about the physiological relevance of the preparations necessary for these studies, since they have not been performed on endogenous receptors. here, we directly image individual endogenous receptors using an equimolar mixture of two colour fluorescent fab fragments. we can then determine the receptors stoichiometry by quantifying its dynamic single molecule colocalisation (dy-sco) recorded by total-internal reflection fluorescence (tirf) microscopy. we have recently investigated the domain dynamics of pgk (1) . structural analysis by small angle neutron scattering revealed that the structure of the holoprotein in solution is more compact as compared to the crystal structure, but would not allow the functionally important phosphoryl transfer between the substrates, if the protein would be static. brownian large scale domain fluctuations on a timescale of 50 ns was revealed by neutron spin echo spectroscopy. the observed dynamics shows that the protein has the flexibility to allow fluctuations and displacements that seem to enable function. [ many physiological and pathological processes involve insertion and translocation of soluble proteins into and across biological membranes. however, the molecular mechanisms of protein membrane insertion and translocation remain poorly understood. here, we describe the ph-dependent membrane insertion of the diphtheria toxin t domain in lipid bilayers by specular neutron reflectometry and solid-state nmr spectroscopy. we gained unprecedented structural resolution using contrast-variation techniques that allow us to propose a sequential model of the membrane-insertion process at angstrom resolution along the perpendicular axis of the membrane. at ph 6, the native tertiary structure of the t domain unfolds, allowing its binding to the membrane. the membrane-bound state is characterized by a localization of the c-terminal hydrophobic helices within the outer third of the cis fatty acyl-chain region, and these helices are oriented predominantly parallel to the plane of the membrane. in contrast, the amphiphilic n-terminal helices remain in the buffer, above the polar headgroups due to repulsive electrostatic interactions. at ph 4, repulsive interactions vanish; the n-terminal helices penetrate the headgroup region and are oriented parallel to the plane of the membrane. the c-terminal helices penetrate deeper into the bilayer and occupy about two thirds of the acyl-chain region. these helices do not adopt a transmembrane orientation. interestingly, the t domain induces disorder in the surrounding phospholipids and creates a continuum of water molecules spanning the membrane. we propose that this local destabilization permeabilizes the lipid bilayer and facilitates the translocation of the catalytic domain across the membrane. the limited stability in vitro of mps motivates the search of new surfactants (1) (2) (3) (4) . fss with a polymeric hydrophilic head proved to be mild towards mps (1) .new fss were designed with chemically defined polar heads for structural applications. lac-derivative was efficient in keeping several mps water soluble and active but formed elongated rods (2) . the glu-family was synthesized, characterized in by sans and auc and for its biochemical interest. the formation of rods is related to the low volumetric ratio between the polar head and hydrophobic tail. the surfactant bearing two glucose moieties is the most promising one, leading to both homogeneous and stable complexes for both br and the b 6 f. it was also shown be of particular interest for the structural investigation of membrane proteins using sans (3). by combining elastic and quasi-elastic neutron scattering data, and by applying theory originally developed to investigate dynamics in glassy polymers, we have shown that in lyophilised apoferritin above t * 100 k the dynamic response observed in the pico-to nano-second time regime is driven by ch 3 dynamics alone, where the methyl species exhibit a distribution of activation energies. our results suggest that over the temporal and spatial range studied the main apoferritin peptide chain remains rigid. interestingly, similar results are reported for other smaller, more flexible lyophilised bio-materials. we believe this work elucidates fundamental aspects of the dynamic landscape in apoferritin which will aid development of complex molecular dynamic model simulations of super-molecules. a detailed appreciation of the relationships between dynamics and biological function will require analysis based on such models that realize the full complexity of macromolecular material. biological systems must often be stored for extended periods of time. this is done by lyophilisation in the presence of lyoprotectants, such as sugars, which results in stable products at ambient conditions. [1] in an effort to understand the mechanism of preservation and stabilization, the interactions between sugars and liposome vesicles, which serve as a simple membrane model, have been studied extensively. amongst the common sugars, trehalose has superior preservative effects [1] and accumulates to high concentrations in many anhydrobiotic organisms. despite many experimental and numerical studies three mechanisms are proposed: vitrification [2] , preferential exclusion [3] and water replacement [4] . to gain more insight into the stabilization mechanism we have recently investigated the effect of trehalose on the bending elasticity of fully hydrated unilamellar vesicles of 1,2-dipalmitoyl-phosphatidylcholine (dppc) in d 2 o at temperatures below and above the lipid melting transition (tm) using neutron spin-echo. the data was analyzed using the zilman-granek theory. at all temperatures measured, trehalose stiffens the bilayer suggesting strong interactions between trehalose and the lipid. trehalose appears to broaden the melting transition but does not change the tm. this agrees with observations using differential scanning calorimetry. influence of macromolecular crowding on protein stability sté phane longeville, clé mence le coeur laboratoire lé on brillouin, gif-sur-yvette, france cell interior is a complex environment filled with a variety of different objects with respect to shape and size. macromolecules are present at a total concentration up to several hundred grams per litre and the overall occupied volume fraction can reach ö & 0.3-0.4. under crowding environment protein-protein interaction play a fundamental role. the crowding environment can affect some physical, chemical, and biological properties of biological macromolecules [1, 2] . traditionally, protein folding is studied in vitro at very low concentration of proteins. under such conditions, small globular single chain proteins can unfold and refold quite rapidly depending mainly to the nature of the solvent. such processes have been very intensively studied, since folding of proteins into their native structure is the mechanism, which transforms polypeptide into its biologically active structure. protein misfolding is involved in a very high number of diseases [4] (e.g. alzheimer, parkinson, and kreuzfeld-jacob diseases, type ii diabetes, …). theoretically, the problem was studied by the introduction of the concept of excluded volume [5] . in recent papers [6, 7] , minton uses statistical thermodynamic models to address the question. he predicted that inert cosolutes stabilize the native state of proteins against unfolding state mainly by destabilizing the unfolded state and that the dimension of the unfolded state decreases with increasing the concentration of cosolute in a measurable way. small angle neutron scattering (sans) is a technique of choice for such study because, by using appropriate mixtures of light and heavy water, it is possible to match the scattering length density of the solvent to the one of the cosolute and thus to measure the conformation of a molecule at low concentration in a presence of a high concentration of another one. we will present a complete experimental study of the mechanism that leads to protein stabilization by macromolecular crowding [7, 8] . coupled dynamics of protein and hydration water studied by inelastic neutron scattering and molecular dynamics simulation hiroshi nakagawa 1,2 , mikio kataoka 1,3 1 japan atomic energy agency, tokai, japan, 2 juelich centre for neutron science, forschungszentrum juelich gmbh, 3 nara institute of science and technology, ikoma, japan proteins work in an aqueous environment at ambient temperature. it is widely accepted that the proteins are flexible and mobile. the flexibility and mobility, that is, protein dynamics are essential for protein functions. neutron incoherent scattering is one of the most powerful techniques to observe protein dynamics quantitatively. here i will talk about dynamics of protein and its hydration water. the structure of a soluble protein thermally fluctuates in the solvated environment of a living cell. understanding the effects of hydration water on protein dynamics is essential to determine the molecular basis of life. however, the precise relationship between hydration water and protein dynamics is still unknown because hydration water is ubiquitously configured on the protein surface. we found that hydration level dependence of the onset of the protein dynamical transition is correlated with the hydration water network. hydration water dynamics change above the threshold hydration level, and water dynamics control protein dynamics. these findings lead to the conclusion that the hydration water network formation is an essential property that activates the anharmonic motions of a protein, which are responsible for protein function. thermal motions and stability of hemoglobin of three endotherms (platypus -ornithorhynchus anatinus, domestic chicken -gallus gallus domesticus and human -homo sapiens) and an ectotherm (salt water crocodile -crocodylus porosus) were investigated using neutron scattering and circular dichroism. the results revealed a direct correlation between the dynamic parameters, melting -, and body temperatures. on one hand, a certain flexibility of the protein is mandatory for biological function and activity. on the other hand, intramolecular forces must be strong enough to stabilize the structure of the protein and to prevent unfolding. our study presents experimental evidence which support the hypothesis that the specific amino acid composition of hb has a significant influence on thermal fluctuations of the protein. the amino acid sequence of hb seems to have evolved to permit an optimal flexibility of the protein at body temperature. macromolecular resilience was found to increase with body and melting temperatures, thus regulating hb dynamics. where k is the trap spring constant, a is the subdiffusion exponent and e a is the mittag-leffler function. the parameters obtained by fitting this equation to the experimental msds are summarized in table 1 . at short lag times we have not found any difference between the two cell types, contrarily to the previous results obtained by afm 2 . for both cell lines the subdiffusion exponent, a was found close to , the value predicted by the theory of semiflexible polymers. but the crossover frequency x 3 , was found smaller for the cancerous cells for all datasets. it corresponds to passage to the confined regime at longer times. we attribute it to the bigger impact of molecular motors. we study the spatiotemporal evolution of fibrous protein networks present in the intracellular and extracellular matrices. here, we focus on the in vitro actin network dynamics and evolution. in order to study the hierarchical self-assembly of the network formation in a confined environment and provide external stimuli affecting the system minimally, we introduce a new microfluidic design. the microfluidic setup consists of a controlling channel to which microchambers of different shapes and sizes are connected through narrow channels. this design results in having mainly convective transport in the controlling channel and diffusive transport into the microchamber. rhodamine labeled actin monomers diffuse into the chamber. after polymerization is induced, they form a confined entangled network. cross-linking proteins can then be added to increase the network complexity. moreover, we can generate gradients of reactants across the microchambers and vasodilator-stimulated phosphoprotein (vasp) is a crucial regulator of actin dynamics. it is important in cellular processes such as axon guidance and migration, promoting assembly of filopodia and lamellipodia. vasp's multiple domain structure increases the range of interactions it has with actin monomers, filaments, and other proteins and it displays multiple binding modes both in vitro and in vivo, including barbed end elongation and filament bundling. however, it is not fully understood how vasp affects the structural and mechanical properties of actin networks. we characterize vasp-mediated bundling of actin networks in a simplified in vitro system using confocal microscopy and quantify mechanical properties with rheology measurements. we show that the network properties differ from other actin bundling proteins and reflect vasp's multiple domain structure, displaying a complex bundling phase space that depends upon solution conditions. we observe the formation of large bundle aggregates accompanied by a reduction in network elasticity at high protein ratios. in addition, we change vasp's actin binding mode and eliminate bundling by introducing free actin monomers. finally, we show preliminary results from a biomimetic system that extends the range of actin-vasp interaction. cell migration or proliferation? the go or grow hypothesis in cancer cell cultures tamá s garay, é va juhá sz, jó zsef tímá r, balá zs heged} us 2 nd department of pathology, semmelweis university, budapest, hungary background: cancer related death is constantly growing in the past decades. the mortality of solid tumors is mostly due to the metastatic potential of tumor cells which requires a fine adjustment between cell migration and cell proliferation. as the metabolic processes in the cell provide a limited amount of available energy (i.e. atp) the various biological processes like cell motility or dna synthesis compete for the atp available. the go or grow hypothesis postulates that tumor cells show either high migration or proliferation potential. in our study we investigate on a large series of tumor cell lines whether this assumption stands for malignant cells. materials and methods: twenty tumor cell lines derived from malignant mesothelioma (mesodermal origin) and malignant melanoma (neuroectodermal origin) were subjected to three-days-long time-lapse videomicroscopic recordings. cell motility and proliferation were characterized by the probability of cell division within 24 hours and the 24-hour migration distance of the cells. results: we found a wide range in both the cell migratory activity and the proliferation capacity in our series. the 24-hour migration distance ranged from 40 to 300 micron and from 10 to 130 micron in mesothelioma and melanoma cells, respectively. the lowest 24-hour cell division probability was found to be 0.21 in both the melanoma and mesothelioma series while the highest proliferation activity reached 1.1 and 1.4 in melanoma and mesothelioma, respectively. interestingly, in the melanoma cell lines we found a significant positive correlation (r=0,6909; p=0,0186) between cell proliferation and cell migration. in contrast our mesothelioma cell lines displayed no correlation between these two cellular processes. conclusions: in summary our findings demonstrate that the investigated tumor cells do not defer cell proliferation for cell migration. important to note the tumor cells derived from various organ systems may differ in terms of regulation of cell migration and cell proliferation. furthermore our observation is in line with the general observation of pathologists that the highly proliferative tumors often display significant invasion of the surrounding normal tissue. many cell types are sensitive to mechanical signals. one striking example is the modulation of cell proliferation, morphology, motility, and protein expression in response to substrate stiffness. changing the elastic moduli of substrates alters the formation of focal adhesions, the formation of actin filament bundles, and the stability of intermediate filaments. the range of stiffness over which different primary cell types respond can vary over a wide range and generally reflects the elastic modulus of the tissue from which these cells were isolated. mechanosensing also depends on the type of adhesion receptor by which the cell binds, and therefore on the molecular composition of the specific extracellular matrix. the viscoelastic properties of different extracellular matrices and cytoskeletal elements also influence the response of cells to mechanical signals, and the unusual non-linear elasticity of many biopolymer gels, characterized by strain-stiffening leads to novel mechanisms by which cells alter their stiffness by engagement of molecular motors that produce internal stresses. the molecular mechanisms by which cells detect substrate stiffness are largely uncharacterized, but simultaneous control of substrate stiffness and adhesive patterns suggests that stiffness sensing occurs on a length scale much larger than single molecular linkages and that the time needed for mechanosensing is on the order of a few seconds. to explore potential role of cytoskeletal component in cardiomyocyte for adaptation to extreme conditions was carried out the comparative study of expression of cytoskeletal sarcomeric protein titin in myocardium of ground squirrels during hibernation and gerbils after spaceflight. we have revealed a two-fold increase in content of long n2ba titin isoform as compared to short n2b titin isoform in different heart chambers of hibernating ground squirrels. the prevalence of the long titin isoform is known to determine the larger extensibility of heart muscle that promotes, according to frank-starling law, the increase in force of heart contractility for pumping higher viscous blood during torpor and adapting the myocardium to greater mechanical loads during awakening. moreover, titin mrna level showed seasonal downregulation in which all hibernating stages differed significantly from summer active level. it is possible that the decline of mrna and protein synthesis during hibernation may be regarded as the accommodation for minimization of energetic expenditures. we have not revealed differences in titin mrna levels between control gerbils and gerbils after spaceflight. but we have also observed the two-fold growth in the amount of n2ba titin isoform in left ventricle of gerbils after spaceflight that is likely to be directed to the restoration of the reduced heart contractility at zero-gravity. these results suggest that the increase of the content of the long n2ba titin isoform may serve as universal adaptive mechanism for regulating of heart function in response to the extreme conditions. nuclear migration is a general term for a non-random movement of the nucleus toward specific sites in the cell. this phenomenon has been described throughout the eukaryotes from yeast to mammals. the process is however still poorly understood in mammalian cells. by using microcontact printing we are able to regulate the geometry and spreading of cultured cells. adhesive micropatterns of fibronectin provide an attachment surface for the cells whereas the passivation of the surface by pll-peg prevents protein, thus cell adhesion. live cell imaging by time-lapse microscopy has shown that under these conditions cells gain a bipolar shape, and more interestingly, the nuclei of the cells showed auto-reversed motion. our research tries to understand the molecular cues and mechanisms behind the observed cellular and nuclear movement. we have already shown that the cytoskeleton plays an important role in this phenomena but the exact players and the detailed mechanism remain to be clarified. in order to identify the most important components and their relationship have drug treatments and sirna experiments have been applied. although our research focuses mainly on the motility of the nucleus, it may also help to get a better understanding of the general theme of cell migration. cell motility involves a number of strategies that cells use to move in their environments in order to seek nutrients, escape danger and fulfil morphogenetic roles. when these processes become uncontrolled, pathological behaviours, like cancer or metastasis of cancerous cells, can occur. here we present a new method for the contextual quantification of cellular motility, membrane fluidity and intracellular redox state, by using the ratiometric, redox-sensitive protein rxyfp and the ratiometric fluidity-sensitive probe laurdan. we provide evidence that dynamic redox and fluidity changes are correlated with signaling processes involved in cellular motility. these findings may pave the way to novel approaches for the pharmacological control of cell invasiveness and metastasis. manipulation of cellular mechanics anna pietuch, andreas janshoff georg-august university, tammanstraße 6, 37077 gö ttingen, germany, e-mail: anna.pietuch@chemie.unigoettingen.de rheological properties of cells determined by the underlying cytoskeleton (cortex) are key features in cellular processes like cell migration, cell division, and cell morphology. today it is possible to investigate local cellular elastic properties under almost physiological conditions using the afm. by performing force indentation curves on local areas on a cell surface the use of contact mechanic models provides the young's modulus, comprising information about the elastic properties of cells. the administration of cytoskeleton modifying substances into the cell is achieved by microinjection. we are also investigating morphological changes and rearrangements of the cytoskeleton in time resolved impedance measurements. electric cell-substrate impedance sensing is a label-free and minimal invasive technique which allows monitoring morphological changes of cells in real time. readout of the impedance is sensitive to changes in cell-substrate contacts as well as density of cell-cell contacts yielding important information about the integrity of the cell layer and changes in the properties of the cell membrane. we are studying the cellular response to modification of the cytoskeleton e.g. by introducing proteins which affect directly the organization of the actin structure like ezrin. mechanical characterization of actin gels by a magnetic colloids technique thomas pujol, olivia du roure, julien heuvingh pmmh, espci-cnrs-umpc-p7. paris, france the actin polymer is central in cell biology: it is a major component of cytoskeleton and it plays a fundamental role in motility, division, mechanotransduction…. its polymerization just beneath the cell membrane generates forces responsible for cell movement. actin filaments form a network whose architecture is defined by the nature of the binding proteins and depends on the location in the cell. for example, in the structure which leads cell migration, the lamellipodium, the gel is branched due to its interaction with arp2/3 protein complex. determining the mechanical properties of such actin network is a crucial interest to understand how forces are generated and transmitted in living cells. we grow a branched actin network from the surface of colloids using the arp2/3 machinery. the particles are super paramagnetic and they attract each other via dipole-dipole interaction to form chain. by increasing the magnetic field we apply an increasing force to the gel and we optically measure the resulting deformation. from those measurements we deduce a young modulus for a large amount of data. we are characterizing different networks by varying the concentration of the capping and branching proteins and we show how mechanics can be regulated by the different proteins. microtubules (mts) are central to the organization of the eukaryotic intracellular space and are involved in the control of cell morphology. in fission yeasts cells mts transport polarity factors to poles where growth is located, thus ensuring the establishment and maintenance of the characteristic spherocylindrical shape. for this purpose, mt polymerization dynamics is tightly regulated. using automated image analysis software, we investigated the spatial dependence of mt dynamics in interphase fission yeast cells. we evidenced that compressive forces generated by mts growing against the cell pole locally reduce mt growth velocities and enhance catastrophe frequencies. in addition, our systematic and quantitative analysis (in combination with genetic modifications) provides a tool to study the role of ?tips (plus-end tracking proteins) such as mal3 and tip1 in the spatial regulation of mt dynamics. we further use this system to decipher how the linear transport by mt interferes with the feedback circuitry that assures the correct spatial distribution of tea1, the main polarity factor in fission yeast cells. the dynamics of the cytoskeleton are largely driven by cytoskeletal motor proteins. complex cellular functions, such as mitotsis, need a high degree of control of these motors. the versatility and sophistication of biological nanomachines still challenges our understanding. kinesin-5 motors fulfill essential roles in mitotic spindle morphogenesis and dynamics and were thought to be slow, processive microtubule (mt)-plus-end directed motors. here we have examined in vitro and in vivo functions of the saccharomyces cerevisiae kinesin-5 cin8 using single-molecule motility assays and single-molecule fluorescence microscopy. in vivo, the majority of cin8 motors moved slowly towards mt plus-ends, but we also observed occasional minus-end directed motility episodes. in vitro, individual cin8 motors could be switched by ionic conditions from rapid (up to 50 lm/min) and processive minus-end, to bidirectional, to slow plus-end motion. deletion of the uniquely large insert in loop 8 of cin8 induced bias towards minus-end motility and strongly affected the directional switching of cin8 both in vivo and in vitro. the entirely unexpected in vivo and in vitro switching of cin8 directionality and speed demonstrate that kinesins are much more complex than thought. these results will force us to rethink molecular models of motor function and will move the regulation of motors into the limelight as pivotal for understanding cytoskeleton-based machineries. morphological and dynamical changes during tgf-b induced epithelial-to-mesenchymal transition david schneider 1 , marco tarantola 2 , and andreas janshoff 1 1 institute of physical chemistry, georg-august-university, gö ttingen, germany, 2 max planck institute for dynamics and self-organization, laboratory for fluid dynamics, pattern formation and nanobiocomplexity (lfpn), goettingen, germany the epithelial-to-mesenchymal transition (emt) is a program of cellular development associated with loss of cell-cell contacts, a decreased cell adhesion and substantial morphological changes. besides its importance for numerous developmental processes like embryogenesis, emt has also been held responsible for the development and progression of tumors and formation of metastases. the influence of the cytokine transforming growth factor 1 (tgf-b1) induced emt on structure, migration, cytoskeletal dynamics and long-term correlations of the mammalian epithelial cell lines nmumg, a549 and mda-mb231 was investigated by time-resolved impedance analysis and atomic force microscopy (afm) performing force-indentation measurements. the three cell lines display important differences in cellular morphology mirrored in changes of their elastic response (young modulus), as well as their dynamics upon tgf-b1 treatment. impedance based measurements of micromotility reveal a complex dynamic response to tgf-b1 exposure which leads to a transient increase in fluctuation amplitude and long-term correlation. additionally, the investigation of cellular elasticity via afm depicts the different cytoskeletal alterations depending on the metastatic potential of the used cell type. physics of cellular mechanosensitivity studied with biomimetic actin-filled liposomes bjö rn stuhrmann, feng-ching tsai, guido mul, gijsje koenderink fom institute amolf, amsterdam, the netherlands biological cells actively probe the mechanical properties of their tissue environment by exerting contractile forces on it, and use this information to decide whether to grow, migrate, or proliferate. the physical basis for cell contractility is the actin cytoskeleton, which transmits motor generated stresses to mechanosensitive adhesions sites that anchor the cell to the tissue. the origins of mechanosensing are far from understood due to the complex interplay of mechanical effects and biochemical signaling that occurs far from equilibrium. we use a quantitative biophysical approach based on biomimetic constructs to elucidate physical principles that underlie active mechanosensing in biological cells. we have built realistic in vitro models of contractile cells by encapsulating cross-linked actin networks together with myosin motors in cell-sized membranous containers (liposomes). our method has several advantages over prior methods, including high liposome yield, compatibility with physiological buffers, and chemical control over protein/lipid coupling. i will show contour fluctuation spectra of constructs and first data on mechanical response obtained by laser tweezers microrheology. our work will yield novel insights into stress generation and stiffness sensing of cells. setting up a system to reconstitute cytoskeleton-based protein delivery and patterning in vitro nú ria taberner, liedewij laan, marileen dogterom fom institute amolf, amsterdam, the netherlands keywords: microtubules, fission yeast, cell polarity, protein patterning, plus end binding proteins. many different cell types, from mobile fibroblasts [1] to fission yeast cells [2] , display non-homogenous protein patterns on their cell cortex. in fission yeast the cell-end marker protein tea1 that among others is responsible for recruiting the actin dependent cell-growth machinery, is specifically located at the growing cell ends. tea1 travels at the tips of growing microtubules and is delivered to the cell ends [2] . we aim to in vitro reconstitute a minimal microtubule plus-end tracking system that leads to cortical protein patterning in functionalized microfabricated chambers. our model will allow us to perturb microtubule-based transport and diffusion independently and evaluate the resulting protein patterns. [ the tropomyosins (tm) are dimeric actin-binding proteins that form longitudinal polymers along the actin filament groove. there is a great variety of isoforms, but the division of labour between the individual tms and their significance is poorly understood. as in most cell types, also in the neurons several isoforms are present, whose spatio-temporal localisation is differentially regulated. the neuron-specific brain 3 isoform (tmbr-3) can be found in the axon of the mature cells. we aimed to clone, express and charaterise this protein in terms of its effects on the kinetic parameters of the actin filament. using a pet28a construct we purified native, tag-free protein, and examined if it influences the rate of actin polymerisation or the stability of the filaments in the presence of either gelsolin or latrunculin-a, two depolymerising agents. in cosedimentation experiments the affinity of tmbr-3 to actin was* 3lm, about six times that of skeletal muscle tropomyosin. the net rate of actin polymerisation was reduced by 17% in the presence of tmbr-3. the depolymerisation induced by gelsolin or latrunculin-a was inhibited in a concentration-dependent manner. tmbr-3 seems to stabilise actin filaments against disassembly without significant effect on the net polymerisation. cell mobility and metastatic spreading: a study on human neoplastic cells using optical tweezers f. tavano the primary causes of death in cancer patients are local invasion and metastasis but their mechanisms are not yet completely understood. metastatization is accompanied by alterations of the cytoskeleton and membrane structure leading to changes in their biomechanical properties [1] . in this study we analyzed by means of optical tweezers the mechanical properties of two different breast adenocarcinoma cell lines corresponding to different metastatic potential. ot were used to grab the plasma membrane by a 1,5 um silica bead and form a plasma membrane tether. we measured the force exerted by the cell membrane on the bead and drew the force-elongation curves. fitting data in the kelvin body model [2] we found out the values for the viscoelastic parameters influencing the pulling of the membrane tethers. the first cell line analyzed, mcf-7, associated to a low metastatic potential showed tether stiffness of 153 pn/um in average. the second cell line, mda-mb 231, poorly differentiated with a high metastatic potential had a tether stiffness of 36pn/um in average, that is a four times lower value. these results seems to confirm the hypotesis that metastasis prone cells are softer than less aggressive cancer cells, and support the use of ot for these measurements for its sub-pn force resolution and because cells are manipulated without damage. [ tubulin polymerization promoting protein (tppp/p25) is a brain-specific protein that primary targets the microtubule network modulating its dynamics and stability. tppp/p25 is a disordered protein with extended unstructured segments localized at the n-and c-terminals straddling a flexible region. tppp/p25 is primarily expressed in oligodendrocytes where its multifunctional features such as tubulin polymerization promoting and microtubule bundling activities are crucial for the development of the projections in the course of oligodendrocyte differentiation enabling the ensheathment of axons with a myelin sheath that is indispensable for the normal function of the central nervous system. microtubule network, a major constituent of the cytoskeleton, displays multiple physiological and pathological functions in eukaryotic cells. the distinct functions of the microtubular structures are attained by static and dynamic associations of macromolecules and ligands as well as by post-translational modifications. tppp/p25 is actively involved in the regulation of microtubule dynamics not exclusively by its bundling activity, but also by its tubulin acetylation-promoting activity. atypical histone deacetylases, such as nad-dependent sirt2 and histone deacetylase-6, function outside of the nucleus and control the acetylation level of cytosolic proteins, such as tubulin. acetylation-driven regulation of the microtubule network during cellular differentiation is an ambiguous issue. tppp/p25 has been recently identified as an interacting partner and inhibitor of these deacetylases and their interaction decreased the growth velocity of the microtubule plus ends and the motility of the cells. we have established cell models for the quantification of the acetylation degree of microtubule network in correlation with its dynamics and stability as well as in relation to aggresome formation, that mimics the pathological inclusion formation. the intracellular level of tppp/p25 is controlled at posttranscription level by microrna and at protein level by the proteosome machinery. under pathological circumstances this disordered protein displays additional moonlighting function that is independent of its association with microtubule system or deacetylases; it enters aberrant proteinprotein interaction with a-synuclein forming toxic aggregates within the neuronal and glial cells leading to the formation of inclusions characteristic for parkinson's disease and multiple system atrophy, respectively. the cell membrane separates the intracellular from the extracellular environment while intimately interacting with the cytoskeleton in numerous cellular functions, including cell division and motility. cell shape changes are for a large part mediated by the contractile actomyosin network forming the cortex underneath the cell membrane. to uncover molecular mechanisms of cell shape control based on actin-membrane interactions, we built a novel biomimetic model system: a cell-sized liposome encapsulating an actively contracting actin-myosin network. our fabrication method is inspired by a recent report of liposome preparation by swelling of lipid layers in agarose hydrogel films. 1 we extensively characterize important liposomal properties, finding diameters between 7 and 20 lm, unilamellarity, and excellent and uniform encapsulation efficiency. we further demonstrate chemical control of actin network anchoring to the membrane. the resulting liposomes allow quantitative tests of physical models of cell shape generation and mechanics. in the cohesive structure of the cytoskeleton functionally distinct actin arrays orchestrate fundamental cell functions in a spatiotemporally controlled manner. emerging evidences emphasize that protein isoforms are essential for the functional polymorphism of the actin cytoskeleton. the generation of diverse actin networks is catalyzed by different nucleation factors, like formins and arp2/3 complex. these actin arrays also exhibit qualitative and quantitative differences in the associated tropomyosin (tm) isoforms. how the molecular composition and the function of actin networks are coupled is not completely understood. we investigated the effects of different tm isoforms (skeletal muscle, cytoskeletal 5nm1 and br3) on the activity of mdia1 formin and arp2/3 complex using fluorescence spectroscopic approaches. the results show that the studied tm isoforms have different effects on the mdia1-, and arp2/3 complexmediated actin assembly. the activity of the arp2/3 complex is inhibited by sktm and tm5nm1, while tmbr3 does not have any effect. all three tm isoforms inhibited the activity of mdia1. these results contribute to the understanding of the mechanisms by which tropomyosin isoforms regulate the functional diversity of the actin cytoskeleton. chronic thromboembolic pulmonary hypertension (cteph) is a dual pulmonary vascular disorder, which combines major vascular remodelling with small-vessel arteriopathy. the presence of fibroblasts in the clot, occluding the pulmonary arteries, and its composition create a microenvironment with increased collagen level, which might affect the local endothelial function. in this study, human pulmonary artery endothelial cells (hpaec) were exposed to collagen type i to address the effect of the thrombotic microenvironment on the vessel wall forming cells. the hpaecs, cultured under standard conditions were treated with 1, 10 and 100lg/ml of collagen type i for 6h and 24h. the changes in the endothelial cell barrier function were investigated by performing permeability and migration test as well as ve-cadherin staining. collagen type i treatment led to a decrease in ve-cadherin signal in hpaec. the loosening of cell-cell contacts could be proven with a significant increase in permeability after 6h of collagen treatment with different concentrations. besides the loosening of the cell-cell contacts, the hpaec migration was also dose dependently retarded by collagen application over time. our data show that collagen-rich microenvironment leads to a disruption of the junctional proteins in hpaecs, indicating an environmental induced possible alteration in the function of endothelial cells in the clots of cteph patients. the implementation of miniaturisation and high throughput screening has quickened the pace of protein structure determination. however, for most proteins the process still requires milligram quantities of protein with purity [ 95 %. these amounts are required as a result of unavoidable losses during purification and for the extensive screening of crystallisation space. for integral membrane proteins (imps), one of the initial steps in the structure determination procedure is still a major bottleneck -the over-expression of the target protein in the milligram quantity range. with a view of developing guidelines for over-expression of human imps, a systematic approach using the three most common laboratory expression systems (e. coli, s. cerevisiae, sf9 insect cells) was implemented. initial expression levels were determined by either partial purification using ni 2? -nta (e. coli), green fluorescence protein (gfp) fluorescence using a c-terminal gfp tagged protein (s. cerevisiae) or flag tagged partial purification (sf9 cells). the results show that e. coli is suitable for the over-expression of human imps in the required quantity range however protein size and complexity is an important factor. the yeast system is fast and affordable but, for the group of human imps tested, the expression levels were borderline. finally for the insect cell system, the timelines are slower and it is in comparison costly to run, however, it can produce relatively large quantities of human imps. the cu?-atpase copb of enterococcus hirae is a bacterial p-type atpase involved in resistance to high levels of environmental copper by expelling excess copper. the membrane protein copb was purified from an over-expressing strain and solubilized in dodecyl-maltoside. by uv circular dichroism the secondary structure is predicted to contain 40-50 % a-helices and 10-15% b-sheets in agreement with estimates based on homology with the ca atpase serca1. we present cd-spectroscopic data on thermal unfolding of the protein to address the influence of the binding of the atp analogs atpcs and the fluorescent analog mant-atp on the protein stability. such analogs are used to mimic functional states of the atpase but undergo different interactions with the binding site that are not well characterized. we propose a competition-based assay for nucleotide binding using cdspectroscopy to deduce the occupancy of the nucleotidebinding site by non-fluorescent nucleotides. alternatively, the change of intrinsic fluorescence of mant-atp upon binding to the atpase is exploited in these assays. finally, we show how the simultaneous measurement of protein cd and nucleotide fluorescence in thermal denaturation experiments may help to determine the stability of several functional conformational states of copb. showing the steady-state distribution of electric potential, ionic concentrations are obtained efficiently. channel current, a summation of drift and diffusive currents, can be further computed from the flux of ionic concentrations. the influence of finite size effect will be also addressed. effect of cholesterol and cytoskeleton on k v 10.1 membrane distribution jimé nez-garduño am 1,2 , pardo la 2 , ortega a 1 , stü hmer w 2 1 unam, mexico-city, mexico, 2 mpi-em, gö ttingen, germany the potassium channel k v 10.1 is expressed nearly exclusively in the central nervous system. besides its function as an ion channel, k v 10.1 has also been associated with non-canonical signaling functions. various membrane proteins associated with cholesterol-sphingolipids enriched microdomains are involved in signaling pathways. in this work we studied the membrane distribution of k v 10.1 in highly purified brain-tissue plasma membranes as a function of cholesterol content versus cytoskeletal proteins. the results show that one fraction of kv10.1 associates to cholesterol-rich domains or detergent resistant membranes (drm) and another fraction to non-drm domains. the kv10.1 fraction inserted in drm is dependent on cholesterol as well as on cytoskeleton proteins. depletion of cholesterol leads to a doubling of k v 10.1 current density. we suggest that k v 10.1 coexists in two different populations: one where the transmembrane domain fits cholesterol enriched membranes and another able to fit into a less packed lipid bilayer. the importance of this distribution on signaling processes needs to be further investigated. we use the reduced model of an axis-symmetric water-filled channel whose protein wall has a single charged site. the channel length, radius and fixed charge are selected to match experimental data for gramicidin a. the ion current, occupancy and escape rate are simulated by the 1d self-consistent bd technique with account taken of the electrostatic ion-ion interaction. the bath with non-zero ion concentration on one side of the channel is modelled via the smoluchowski arrival rate. it is shown that: a) the occupancy saturates with michaelis-menten kinetics. b) the escape rate starts from the kramers value at small concentrations and then increases with concentration due to the electrostatic amplification of charge fluctuations. the resulting dynamics of the current can be described by modified reaction rate theory accounting for ionic escape over the fluctuating barrier [1] . many membrane-protein functions are amenable to biophysical and biochemical investigation only after the protein of interest has been reconstituted from a detergent-solubilised state into artificial lipid bilayers. unfortunately, functional reconstitution has remained one of the main bottlenecks in the handling of numerous membrane proteins. in particular, gauging the success of reconstitution experiments has thus far been limited to trial-and-error approaches. to address this problem, we have established high-sensitivity isothermal titration calorimetry (itc) as a powerful method for monitoring the reconstitution of membrane proteins into liposomes. itc has previously been employed for characterising liposome solubilisation and reconstitution in the absence of protein. here we show that itc is also excellently suited for tracking the complex process of membrane-protein reconstitution in a non-invasive and fully automated manner. the approach is exemplified for the prokaryotic potassium channel kcsa, which we first purified in detergent micelles and then reconstituted into stable proteoliposomes at very high protein densities. electrophysiological experiments performed in planar lipid membranes confirmed that kcsa regained its functional activity upon itc-guided reconstitution. gating currents of low-voltage-activated t-type calcium channels family má ria karmažínová , ľ ubica lacinová institute of molecular physiology and genetics, sav, bratislava, slovak republic t-type calcium channels are distinguished by relatively low voltage threshold for an activation and steep voltage dependence of activation and inactivation kinetics just above the activation threshold. kinetics and voltage dependence of macroscopic inward calcium current through ca v 3 channels was described in a detail. in contrast, very little information is available on gating current of these channels. therefore we compared gating currents measured from all three ca v 3.1, ca v 3.2 and ca v 3.3 channels. voltage dependencies of charge movement differ dramatically from those for macroscopic current. first, their slope factors are several-fold bigger that slope factors of macroscopic current activation. second, activation mid-point for ca v 3.3 channels on-gating is shifted to more positive membrane potentials by about 20 mv compare to ca v 3.1 and ca v 3.2 channels, whose activation mid-points are similar. the same is truth for off-gating voltage dependences. kinetics of both onand off-gating is remarkably faster for ca v 3.1 and ca v 3.2 channels compare to ca v 3.3 channels. further, more charge is moved per unit of macroscopic current amplitude in ca v 3.3 channels compare to ca v 3.1 and ca v 3.2 channels. supported by apvv-0212-10 and vega 2/0195/10. the local anaesthetic lidocaine (lid) is generally believed to reach its binding site in the intracellular vestibule of the voltage-gated sodium channel via the cell membrane. qx222 (qx) is a permanently charged, quaternary amine analogue of lid, that can access this binding site via a hydrophilic route across the channel protein. the mutation i1575e of the adult rat muscle-type sodium channel (rna v 1.4) opens such a hydrophilic pathway. when bound to the internal vestibule, lid stabilizes both fast and slow inactivated states. we wondered whether qx, once bound to the internal vestibule, exerts a similar modulatory action on inactivated states as lid. the construct i1575e was expressed in tsa201 cells and studied by means of the patch-clamp technique. when applied from the extracellular side 500 lm qx stabilized the slow but not the fast inactivated state in i1575e. when applied internally, qx entered the channel, but stabilization of inactivated states could not be observed. these results suggest that binding site for use-dependent block is in the inner vestibule of the channel, fast inactivation is modulated only by the hydrophobic form of lid, and the binding site for modulation of slow inactivation by qx is only accessible from the extracellular side of the channel. we observed that lipophilicity (quantified by the logarithm of the calculated water-octanol partition coefficient, logp) is important in determining both kr and ki, but had a greater effect on ki. distribution coefficients (logd) discriminated better between kr and ki than partition coefficients (logp). the ratio of positively charged/neutral forms (quantified by the acidic dissociation constant, pka) was a significant determinant of resting affinity: predominantly charged compounds tended to be more potent against resting channels, while neutral compounds tended to be more state-dependent. aromaticity was more important for inactivated state affinity. the acidification of intracellular compartments is critical for a wide range of cellular processes. a recent candidate for ph regulation within early endosomes and tgn is the highly conserved intracellular na ? /h ? exchanger 7 isoform (nhe7), whose mutation leads to neurological syndromes in human patients. however, due to its intracellular localization, nhe7 biochemical features are still poorly characterized and its biological function remains elusive. we have developed somatic cell genetic techniques that enable the selection of variant cells able to resist h ? killing through plasma membrane expression of h ? extruders. this enabled us to obtain stable cell lines with forced plasma membrane expression of nhe7. we used them to measure its functional and pharmacological parameters with high accuracy, using fast transport kinetics. to summarize, this exchanger displays unique features within the nhe family, especially with respect to its affinity for its substrates, lithium, sodium and protons and for its guanidine-derived inhibitors. taken together with our results on the subcellular localization of the native nhe7, these unique biochemical features provide new insights on the biological function and pathological implications of this intracellular na ? /h ? exchanger. analysis of the collective behaviour of ion channels j. miśkiewicz, z. trela, s. przestalski wrocław university of environmental and life sciences, physics and biophysics department, ul. norwida 25, 50-375 wrocław, poland a novel approach to the analysis of the ion current recordings is proposed. the main goal of the standard patch clamp technique is to measure single channel activity (however the whole cell configuration is also used in various researches). in the presented study the ion channels time series recordings were several (up to four) ion channels were present are analysed and the collective behaviour of ion channels is investigated. the time ion current time series are converted into dwelltime series and the channel activity is analysed. the hypothesis of collective ion channels behaviour is verified and the influence of organolead compounds (met3pbcl) on collective ion channel activity is measured. the analysis is performed on the sv cation channels of vacuolar membrane of beta vulgaris. the aim of our computed study was to examine the possible binding site of primaquine (pq) using a combined homology protein modeling, automated docking and experimental approach. the target models of wild-type and mutant-types of the voltage-dependent sodium channel in rat skeletal muscle (rna v 1.4) were based on previous work by tikhonov and zhorov. docking was carried out on the p-loop into the structure model of rna v 1.4 channel, in the open state configuration, to identify those amino acidic residues important for primaquine binding. the threedimensional models of the p-loop segment of wild types and mutant types (w402. w756c, w1239c and w1531a at the outer tryptophan-rich lip, as well as d400c, e755c, k1237c and a1529c of the deka motif) helped us to identify residues playing a key role in aminoquinoline binding. in good agreement with experimental results, a 1000-fold inhibition loss was observed, tryptophan 756 is crucial for the reversible blocking effects of pq. as a result, w756c abolished the blocking effect of primaquine in voltage-clamp assays. hydrogen bond formation accelerates channel opening of the bacterial mechanosensitive channel mscl yasuyuki sawada and masahiro sokabe department of physiology, nagoya university graduate school of medicine, nagoya, japan the bacterial mechanosensitive channel mscl is constituted of homopentamer of a subunit with two transmembrane inner and outer (tm1, tm2) a-helices, and its 3d structure of the closed state has been resolved. the major issue of mscl is to understand the gating mechanism driven by tension in the membrane. to address this question, we performed molecular dynamics (md) simulations for the opening of mscl embedded in the lipid bilayer. in the closed state of mscl, neighboring tm1 inner helices are crossed each other near the cytoplasmic side and leu19 and val23 in the constricted part form a stable hydrophobic environment called gate. upon membrane stretch, phe78 in tm2 outer helices was dragged by lipids, leading to an opening of mscl. thus phe78 was concluded to be the major tension sensor. during opening, tm1inner helices were also dragged and tilted, accompanied by the outward sliding of the crossings. this led to a slight expansion of the gate associated with an exposure of oxygen atoms of the backbone to the inner surface of the gate. this allows water penetration in the gate and formation of hydrogen bonds between water and the exposed oxygen, which in turn weakened the hydrophobic interaction at the crossings, causing a further opening of the gate and water permeation. mitochondrial bk ca , mitobk ca has been proposed to be cardioprotective and formed by proteins of *50 to *125 kda. thus, we investigated the molecular characteristics of this channel in isolated mitochondria from murine heart. labeling of adult mouse cardiomyocytes with plasmalemma bk ca antibodies, mitotracker, and wheat germ agglutinin yielded remarkable mitochondrial but not plasma membrane localization. nanoscale fluorescence microscopy (stimulated emission depletion) revealed 7 to 15 of *40-50 nm bk ca clusters per mitochondria. further, western blot analysis of purified mitochondria showed the presence of a full length *125 kda protein. systematic rt-pcr exon scanning of isolated cardiomyocyte mrnas were consistent with a full length *125 kda alpha-subunit protein and revealed the expression of three splice inserts. insertless-bk ca robustly localized to the plasma membrane of cho cells but when a c-terminal splice insert was present bk ca was readily targeted to the mitochondria (protein proximity index was *1.0 indicating 100% colocalization). hence, cardiac mitobk ca is composed by full-length bk ca protein but with splice inserts which may facilitate its targeting to mitochondria. supported by nih and aha. patch-clamp technique was used to examine effect of trimethyl-lead and -tin on the sv channel activity in the red beet (beta vulgaris l.) taproot vacuoles. it was found that the addition of both investigated compounds to the bath solution inhibit, in a concentration-dependent manner, sv currents. when single channel properties were analyzed, only little channel activity can be recorded in the presence of 100 lm of organometal. compounds investigated decreased significantly (by about one order of magnitude) the open probability of single channels. the recordings of single channel activity obtained in the presence and in the absence of organometal showed that compounds only slightly (by ca. 10%) decreased the unitary conductance of single channels. it was also found that organometal diminished significantly the number of sv channel openings, whereas it did not change the opening times of the channels. taken together, these results suggest that organometal binding site is located outside the channel's selectivity filter and that the inhibitory effect of both compounds investigated on sv channel activity probably results from organometal-induced disorder in compatibility between membrane lipids and membrane proteins. the research was financed by polish ministry of science and higher education by grant no. nn305 336434. electrophysiological investigation of the hvdac1 ion channel in pore-spanning membranes conrad weichbrodt, claudia steinem georg-august-universitä t gö ttingen, iobc, tammannstr. 2, d-37077 gö ttingen, germany, e-mail: cweichb@gwdg.de the human voltage-dependent anion channel 1 (hvdac1) plays an important role in cell life and apoptosis since it is the main porin of the outer mitochondrial membrane. as hvdac1 is believed to play a pivotal role in apoptosis-related diseases such as stroke, alzheimer, parkinson and cancer, the alterations of its electrophysiological properties under different conditions are of great value. to perform different investigations, refolded hvdac1 is reconstituted in artificial membranes which typically consist of dphpc with a cholesterol fraction of 10-30%. they are prepared via the mü ller-rudin-technique on a functionalized porous alumina substrate containing pores with a diameter of 60 nm. the quality of these so-called nano-black-lipid-membranes (nano-blms) is verified via electrochemical impedance spectroscopy (eis), hvdac1 is reconstituted and single channel recordings are made. membranes are also prepared by spreading proteoliposomes on hydrophobized porous silicon nitride with pores of 1 lm diameter. information about altered gating-characteristics and related conductivities is gained by application of holding potentials up to ±100 mv and evaluation of the resulting currents. the hvdac1 was a kind gift of prof. c. griesinger, mpibpc, gö ttingen. pharmacological inhibition of cardiac herg k ? channels is associated with increased risk of arrhythmias. many drugs bind directly to the channel, thereby blocking ion conduction. ala-scanning mutagenesis identified residues important for drug block. two aromatic residues y652 and f656 were found to be crucial for block of most compounds. surprisingly, some cavity blocking drugs are only weakly affected by mutation y652a. in this study we provide a structural interpretation for this observation. md simulations on the y652a mutant suggest side chain rearrangements of f656 located one helical turn below y652. loss of p-p stacking induces reorientation of f656 from a cavity facing to a cavity lining conformation, thereby substantially altering the shape of the binding site. docking studies reveal that due to their rigid shape and compactness y652 insensitive drugs can still favorably interact with the reoriented f656 aryl groups, while molecules with more extended geometries cannot. the ankyrin transient receptor potential channel trpa1 is a transmembrane protein that plays a key role in the transduction of noxious chemical and thermal stimuli in nociceptors. in addition to chemical activation, trpa1 can be activated by highly depolarizing voltages but the molecular basis of this regulation is unclear. the transmembrane part of the tetrameric trpa1 is structurally related to the voltagegated k ? channels in which the conserved charged residues within the fourth transmembrane region (s4) constitute part of a voltage sensor. compared to these channels, the voltage-dependence of trpa1 is very weak (apparent number of gating charges * 0.4 versus 12 in k ? channels) and its putative voltage-sensing domain most likely lies outside the s4 because trpa1 completely lacks positively charged residues in this region. in the present study we used homology modelling and molecular dynamics to create models of the transmembrane part and the proximal cytoplasmic c terminus of trpa1. in combination with electrophysiological data obtained from whole cell patchclamp measurements we were able to point out several positively charged residues which mutation strongly alter the voltage sensitivity of trpa1 channel. these may be candidates for as yet unrecognized voltage sensor. photosynthesis p-511 action of double stress on photosystem 2 aliyeva samira a., gasanov ralphreed a. institute of botany, azerbaijan national academy of sciences, baku, azerbaijan the simultaneous effect of photoinhibitory illumination and toxic action of heavy metals ions (cd 2? and co 2? ) on activity of ps2 in vitro measuring by millisecond delayed fluorescence (ms-df) of chlorophyll a was studied. during action on chloroplasts only of cd 2? (10 -3 m) the fast component of ms-df, which originates via radiative recombination of reaction center with the camn 4 -cluster or y z on donor side of ps2, is inhibited stronger than at action of only co 2? . the steadystate level at cd 2? treatment is remain stable, while at co 2? action it is increased. simultaneous action of cd 2? and photoinhibitory illumination (4000 lmol photons m -2 s -1 ) have shown that fast component of ms-df was inhibited faster with time than in case of action of co 2? and excess light. result indicates that damage sites of action cd 2? and co 2? are donor and acceptor side of ps2, accordingly. we assume that binding site of cd 2? is y z or camn 4 -cluster, one of the recombination partners with p 680 ? on the donor side of ps2. thereby, action of cd ions on donor side of ps2 leads mainly to development of mechanism of donor-side photoinhibition. field instrument for determination of the photosynthetic capacity of intact photosynthetic bacteria e. asztalos 1 , z. gingl 2 and p. maró ti 1 1 department of medical physics and informatics, university of szeged, hungary, 2 department of experimental physics, university of szeged, hungary a combined pump and probe fluorometer and spectrophotometer with high power laser diodes has been constructed to measure fast induction and relaxation of the bacteriochlorophyll fluorescence yield and light-induced absorption changes in intact cells of photosynthetic bacteria. the construction is the upgraded version of our previous set up 1 with better time resolution (5 ls). the compact design of the mechanics, optics, electronics and data processing makes the device easy to use as outdoor instrument or to integrate into larger measuring systems. the versatility and excellent performance of the apparatus will be demonstrated on different fields: 1) organization and redox state of the photosynthetic apparatus of the whole cells under different growth conditions deduced from fluorescence characteristics including the lag phase, the amplitude and the rise time of the variable fluorescence, 2) electron transfer in the reaction center, cytochrome bc 1 complex and in between obtained from relaxation of the fluorescence and 3) re-reduction kinetics of the oxidized primary donor of the reaction center and energetization and relaxation of the intracytoplasmic membrane tracked by absorption changes at 798 and 525 nm, respectively. previous work has established that the iron stress induced protein a (isia) synthesized by cyanobacteria under stress conditions, has at least two functions: light harvesting [1] and photoprotection [2] . under prolonged iron starvation isia becomes the main chlorophyll-binding protein in the cell and occurs without a photosystem association. these isia aggregates have a strong ability to dissipate light energy and there is evidence of carotenoid participation in the quenching mechanism via downhill energy transfer from chlorophyll to the s1 state of a carotenoid [3] . in the present work we have measured the temperature dependence of the fluorescence of carotenoid depleted mutants (echinenone and/or zeaxanthin) and isia monomers in order to investigate the role of carotenoid and aggregation in the quenching process. pigment analysis confirms the absence of the carotenoid mutated in its biosynthesis but shows that it is mainly replaced. the monomers are lacking two carotenoids, echinenone and one of the two ß-carotenes found previously in isia aggregates. temperature dependent fluorescence shows that quenching properties are affected in the monomers and the mutants lacking zeaxanthin. soon exhausted oil resources and global climate change have stimulated research aiming at production of alternative fuels, ideally driven by solar energy. production of solar fuels needs to involve the splitting of water into protons, energized electrons and dioxygen. in photosynthetic organisms, solar-energy conversion and catalysis of water splitting (or water oxidation) proceed in an impressive cofactor-protein complex denoted as photosystem ii (psii). the heart of biological water-oxidation is a protein-bound manganese-calcium complex working at technically unmatched efficiency. in an attempt to learn from nature, the natural paragon is intensely studied using advanced biophysical methods. structural studies by x-ray spectroscopy with synchrotron radiation play a prominent role in this endeavor. time-resolved methods provide insights in the formation of intermediate states of the reaction cycle. an overview is presented focusing on (i) the efficiency of solar energy usage in psii, (ii) the interrelation between electron transfer and proton relocations, and (iii) the mechanism of water oxidation. as an outlook, new results on water oxidation by biomimetic manganese and cobalt oxides, which may become a key element in future solar-fuel systems, are presented. the peridinin-chlorophyll-protein (pcp) is a light-harvesting complex (lhc) that works as antenna in the photosynthetic process of dinoflagellates. the protein contains both chlorophylls and carotenoids molecules, the latter being responsible to extend the spectral range of captured light to regions where chlorophylls are transparent. pcp crystal structures [1] reveal that each chlorophyll is surrounded by 3 or 4 molecules of the carotenoid peridinin, located in non-equivalent positions. the different protein environment of the sites might be responsible of a spectral shift of the pigments with the functional role to extend the absorption spectra of the complex and enhance its light harvesting capabilities. high resolution x-ray diffraction data on a reconstructed pcp, the refolded peridinin-chlorophyll a-protein (rfpcp) [2] , and on the less common high salt-pcp (hspcp) opened the way to the mechanistic understanding of peridinin spectral tuning, peridinin chlorophyll energy transfer and photoprotective mechanism [3] . the two pcp forms differ in various features: spectral properties, molar mass, amino acid sequence and, above all, pigment stoichiometry, the peridinin:chlorophyll ratio being 4:1 for the rfpcp and 3:1 for the hspcp [4] . in the present work we perform classical molecular dynamics simulations of the rfpcp and the hspcp in explicit water solution. we analyse the structure and dynamics of the proteins and of their pigments to characterize the different peridinin sites in both pcp forms in terms of quantities that can affect the chromophore spectra, such as distorsion, fluctuations and nature of the protein environment. the comparison between the data suggests correspondences between the pigments of the two forms. quantum and mixed quantum/ classical molecular dynamics simulations are also under progress to investigate the effect of the protein environment on the electronic and optical properties of the pcp pigments. peculiar applications, like in optoelectronics, biosensors, photovoltaics. among the existing carrier matrices conductive metal oxides (e.g. indium tin oxide, ito), carbon nanotubes, graphenes, silicon (si) are the most frequently used materials because of their unique characteristics such as good conductivity, good optical properties and excellent adhesion to substrates. in our work we combined purified photosynthetic reaction center protein (rc) and porous silicon (psi) investigating the morphology and optoelectronic properties of the bio-nanocomposite material. ftir spectroscopy, scanning electron microscopy and energydispersive x-ray spectroscopy indicated the binding of the protein to the psi. specular reflectance spectra showed a red shift in the characteristic interference peak of the psi microcavity which was saturated at higher concentration of the protein. the binding was more effective if the functionalization was done by the si-specific oligopeptide compared to the classical covalent binding via aminopropyl-triethoxysilane (aptes). excitation by single saturation flashes indicated that the rc still exhibited photochemical turnover after the binding. the role of reactive oxygen species (ros) in plant stress, both as damaging agent and as potential signal molecule is often assessed in experiments using photosensitized elicitor dyes. for these studies, it is essential to know how efficiently these chemicals generate ros, whether they are specific ros sources, as well as their cellular localization and additional side effects. the present study addresses these issues using a variety of dyes known and traditionally applied as singlet oxygen ( 1 o 2 ) sources. rose bengal (rb), methylene violet (mvi), methylene blue (mb), neutral red (nr) and indigo carmine (ic) were studied as putative ros sources in tobacco leaves. ros products of photosensitized dyes were measured in vitro, using spin trapping epr spectroscopy. dye concentrations and irradiation concentration leading to equal absorbed excitation quanta were determined spectrophotometrically. in vivo studies were carried out using tobacco leaves infiltrated with water solutions of the putative 1 o 2 sources. cellular localizations were identified on the basis of the dyes' fluorescence. rb, nr and mvi reached into mesophyll cells and were used to study the effects of these dyes on photosynthesis. photochemical yields and quenching processes were compared before and after photosensitization of the elicitor dyes inside the leaf samples. chlorophyll-chlorophyll charge transfer quenching is the main mechanism of non-photochemical quenching in higher plants alfred r. holzwarth max-planck-institut fü r bioanorganischechemie, stiftstraße 34-36, d-45470 mü lheim a.d.ruhr, germany non-photochemical quenching (npq) in plants protects against photochemical destruction of the photosynthetic apparatus under excess light conditions.while one location of the npq process has been shown to be centered on the major light harvesting complex ii (lhcii) (q1 type or qequenching), an additional quenching center responsible for qi type (identical to q2 center) quenchinghas been suggested to be located on the minor light-harvesting complexes upon accumulation ofzeaxanthin (zx), in particularon cp24 and cp29 we have performed femtosecond transient absorption and time-resolved fluorescence measurements of npq quenching in intact leaves of higher plants, on isolated light harvesting complexes in the minor (non-aggregated)light harvesting complex cp29 reconstituted with violaxanthin (vx) or zx, and in the isolated major lhc ii complex in the aggregated state. in all of these situations we find the formation of chl-chl charge transfer (ct) states to be the dominant quenching mechanism. the yield of formation of carotenoid cation states and/or carotenoid s 1 state is either extremely low or absent, thus excluding their involvement in npq quenching as a major quenching mechanism. single-molecule spectroscopy (sms) is a powerful technique that allows investigation of fluorescence properties from single fluorescing systems. this technique enabled us to investigate the dynamics of the fluorescence intensity and spectral profiles of single, isolated light harvesting complex (lhc) on timescales of milliseconds to seconds, during continuous laser illumination. we were able to observe how each complex can rapidly switch between different emission states [1, 2] and to characterise the intensity and the spectral dynamics of major and minor antenna complexes from plants, in two different environments, mimicking the light harvesting and the light dissipating state, respectively. the results will be discussed with respect to the current models for nonphotochemical quenching (npq) mechanisms [3, 4, 5] , a vital photoprotection mechanism during which the lhcs of plants switch between a state of very efficient light utilisation and one in which excess absorbed excitation energy is harmlessly dissipated as heat. phaeodactylum tricornutum is one of the most utilized model organisms in diatom photosynthesis research mainly due to availability of its genome sequence (1). it's photosynthetic antennae are the fucoxanthin chlorophyll a/c binding proteins (fcps) which share a high degree of homology with lhcs of higher plants and green algae (2) . for detailed investigation of the antenna system of p.tricornutum, a transgenic strain expressing recombinant his-tagged fcpa protein was created which simplified the purification of a specific stable trimeric fcp complex consisting of fcpa and fcpe proteins. excitation energy coupling between fucoxanthin and chlorophyll a was intact and the existence of a chlorophyll a/fucoxanthin excitonic dimer was demonstrated (3). we investigated in detail the existence of specific antenna system for psi and psii in p.tricornutum as in case of higher plants. our studies indicated that at least the main light harvesting proteins fcpa and fcpe are most probably shared as a common antenna by both psi and psii. harvesting complex ii (lhciib) from spinach or in native thylakoid membranes by picosecond time-resolved fluorescence. the domain size was estimated by monitoring the efficiency of added exogeneous singlet excitation quenchers -phenyl-p-benzoquinone (ppq) and dinitrobenzene (dnb). the fluorescence decay kinetics of the systems under study were registered without quenchers and with quenchers added in a range of concentrations. stern-volmer constants, k 0 sv and k sv -for aggregates (membranes) and detergentsolubilized complexes, respectively, were determined from the concentration dependence of the ratio of the mean fluorescence lifetimes without/with quencher (s 0 , s). the ratio k 0 k sv • s 0 /s 0 0 was suggested as a measure of the functional domain size. values in the range of 15-30 were found for lhcii macroaggregates and 12-24 for native thylakoid membranes, corresponding to domain sizes of 500-1000 chlorophylls. although substantial, the determined functional domain size is still orders of magnitude smaller than the number of physically connected pigment-protein complexes; thus our results imply that the physical size of an antenna system beyond these numbers has little or no effect on improving the light-harvesting efficiency. the interaction between photosynthetic reaction center proteins (rcs) purified from purple bacterium rhodobacter sphaeroides r-26 and functionalized and non-functionalized (single (swnt) and multiple (mwnt) walled) carbon nanotubes (cnt-s) has been investigated. both structural (afm, tem and sem microscopy) and functional (flash photolysis and conductivity) techniques showed that rcs can be bound effectively to different cnts. both physical sorption and binding through -nh 2 or -cooh groups gave similar results. however, it appeared that by physical sorption some sections of the cnts were covered by multiple layers of rcs. after the binding the rcs kept their photochemical activity for a long time (at least for three months, even in dried form) and there is a redox interaction between the cnt and rcs. the attachment of rc to cnts results in an accumulation of positive and negative charges followed by slow reorganization of the protein structure after excitation. in the absence of cnt the secondary quinone activity decays quickly as a function of time after drying the rc onto a glass surface. the special electronic properties of the swnt/protein complexes open the possibility for several applications, e.g. in microelectronics, analytics or energy conversion and storage. the decay of the high fluorescence state generated by actinic illumination of different durations was measured in whole cells of various strains and mutants of photosynthetic purple bacteria. although similar method is used in higher plants, its application in photosynthetic bacteria is novel and highly challanging. the available data are restricted and only the re-oxidation of the reduced primary quinone (q a -? q a ) is usually blamed for the decay kinetics. here, we will analyse the complexity of the kinetics over a very broad time range (from 5 ls to 5 s) and show that the dark relaxation of the bacteriochlorophyll fluorescence reflects the overlap of several processes attributed to the intra-and interproteinous electron transfer processes of the reaction center (rc) and cytochrome bc 1 complex of the bacterium. in the shorter (\ 1 ms) time scale, the dominating effect is the re-reduction of the oxidized primary donor (p ? ? p) that is followed by the re-oxidation of the acceptor complex of the rc by the cytochrome bc 1 complex. as the life times and amplitudes of the components depend on the physiological state of the photosynthetic apparatus, the relaxation of the fluorescence can be used to monitor the photosynthetic capacity of the photosynthetic bacteria in vivo. circular dichroism (cd) spectroscopy is an indispensable tool to probe molecular architecture. at the molecular level chirality results in intrinsic cd, pigment-pigment interactions in protein complexes give rise to excitonic cd, whereas ''psitype'' cd originates from large densely packed chiral aggregates. it has been well established that the anisotropic cd (acd), measured on samples with defined orientation, carries specific information on the architecture of molecules. however, acd can easily be distorted by linear dichroism of the sample or instrumental imperfections -which might be the reason why it is rarely studied in photosynthesis research. here we present acd spectra of isolated intact and washed, unstacked thylakoid membranes, photosystem ii membranes (bby), and tightly-stacked lamellar macroaggregates of the main light-harvesting complex ii (lhcii). we show that the acd spectra of face-and edge-aligned stacked thylakoid membranes and lhcii lamellae exhibit profound differences in their psi-type cd bands. marked differences are also seen in the excitonic cd of bby and washed thylakoid membranes. thus acd provides an additional dimension to the light induced conformation changes of quinone depleted photosynthetic reaction centers (rcs) purified from the carotenoid-less rhodobacter sphaeroides r-26 were investigated by transient absorption (ta) and grating (tg) methods. surprisingly, the decay of the ta signal measured at 860 nm was divided into a 15 ns and a 40 ls components. the latter coincides with the life time of the tg signal, which was assigned earlier [nagy et al. (2008) febs lett. 582, 3657-3662] to spectrally silent conformational changes. the nature of the 40 ls phase was investigated further. although, the probability of chlorophyll triplet formation under our measuring conditions was small, possible contribution of the triplet states was also studied. the presence of carotenoid in the wild type rcs eliminated the 40 ls component indicating the role of carotenoid in the energy transfer within the rcs. there was no significant effect of the molecular oxygen on the ta. this fact may be explained if the chlorophyll triplets inside the protein have reduced accessibility to molecular oxygen. a differential effect of osmotic potential and viscosity on conformation changes accompanying the primary charge separation was measured by the effect of ficoll, glucose and glycerol as compared the ta to the tg signals. variable chlorophyll fluorescence: in part a yield change due to light-induced conformational change gert schansker 1 , szilvia z. tó th 1 , lá szló ková cs 1 , alfred r. holzwarth 2 and gy} oz} o garab 1 1 institute of plant biology, biological research center, hungarian academy of sciences, szeged, hungary, 2 max-planck-institut fü r bioanorganische chemie, mü lheim an der ruhr, germany on a dark-to-light transition the chlorophyll fluorescence rises from a minimum intensity (f 0 ) to a maximum intensity (f m ). conventionally, this rise is interpreted to arise from the reduction of the primary quinone acceptor, q a , of photosystem ii -although this cannot explain all presently available observations. in untreated leaves, at room temperature, the fluorescence rise follows the reduction of the electron transport chain (etc). once induced, *30-40 % of the variable fluorescence intensity relaxes within 100 ms in darkness and can be re-induced within 3 ms as long as the etc remains reduced. analyzing the fluorescence relaxation kinetics, ?/-dcmu, *30% of the amplitude cannot be explained by q a -re-oxidation. special properties of this phase determined on dcmu-inhibited samples: at cryogenic temperatures (below -40°c), where the q a -/s 2 recombination is blocked, it still relaxes and it exhibits a strong temperature dependence with an apparent e a & 12 kj/mol, whereas the reduction of q a is nearly temperature insensitive. a fluorescence yield change, driven by light-induced conformational change in the reaction center complex, can explain all these observations. tuning function in bacterial light-harvesting complexes katia duquesne 1 , edward o'reilly 2 , cecile blanchard 1 , alexandra olaya-castro 2 and james n. sturgis 1 1 lism, cnrs and aix-marseille university, marseille, france, 2 department of physics, university college, london, uk purple photosynthetic bacteria are able to synthesize a variety of different light-harvesting complexes, sometimes referred to as lh2, lh3 and lh4. here we have investigated the structural origins of these different forms and the manner in which the sequence tunes the absorption spectrum of the light-harvesting system. we then consider the functional consequences of this tuning for the organization of the light harvesting system and on the ecology of the organisms. specifically by spectroscopic techniques, in particular circular dichroism and resonance raman spectroscopy, we have been able to obtain information on the organization of the bacteriochlorophyll binding sites in the unusual lh4 of roseobacter denitrificans. this provides a picture of how different peripheral light-harvesting complexes are able to modulate the absorption spectrum. the structure and organization of this complex is the put in the context of the the recently published variability of the light-harvesting complexes. in particular the observation of their ability to form mixed complexes containing different polypeptides. we examine quantitatively the possible reasons for maintaining such variability by considering the transport properties of membranes containing either pure or mixed complexes and show that mixed complexes can permit light-harvesting to continue during adaptation. we then consider the different constraints that may be behind this type of adaptation in different bacteria and the conditions under which different types of antenna system might be optimal finally we integrate this into the evolutionary context of adaptation to variable light intensity and the ecological niches where such organisms are found. interaction between photosynthetic reaction centers and ito t. szabo 1 , g. bencsik 2 , g. kozak 1 , cs. visy 2 , z. gingl 3 , k. hernadi 4 , k. nagy 5 , gy. varo 5 and l. nagy 1 1 departments of medical physics and informatics, 2 physical chemistry and materials science, 3 technical informatics and of, 4 applied and environmental chemistry, university of szeged, hungary, 5 institute of biophysics, has biological research center, szeged, hungary photosynthetic reaction center proteins (rc) purified from rhodobacter sphaeroides purple bacterium were deposited on the surface of indium-tin-oxide (ito), a transparent conductive oxide, and the photochemical/-physical properties of the composite was investigated. the kinetics of the light induced absorption change indicated that the rc was still active in the composite and there was an interaction between the protein cofactors and the ito. the electrochromic response of the bacteriopheophytine absorption at 771 nm showed an increased electric field perturbation around this chromophore on the surface of ito compared to the one measured in solution. this absorption change is associated with the chargecompensating relaxation events inside the protein. similar life time, but smaller magnitude of this absorption change was measured on the surface of borosilicate glass. the light induced change in the conductivity of the composite as a function of the concentration showed the typical sigmoid saturation characteristics unlike if the chlorophyll was layered on the ito which compound is photochemically inactive. in this later case the light induced change in the conductivity was oppositely proportional to the chlorophyll concentration due to the thermal dissipation of the excitation energy. the supramolecular organization of photosystem ii in vivo studied by circular dichroism spectroscopy tü nde tó th 1,2 , herbert van amerongen 2,3 , gy} oz} o garab 1 , lá szló ková cs 1 1 institute of plant biology, biological research center, hungarian academy of sciences, hungary, 2 wageningen university, laboratory of biophysics, wageningen, the netherlands, 3 microspectroscopy centre, wageningen university, wageningen, the netherlands the light reactions of photosynthesis in higher plants take place in granal chloroplast thylakoid membranes, which contain chirally organized macrodomains composed of photosystem ii (psii) supercomplexes associated with light harvesting antenna complexes (lhciis). the physiological relevance of this hierarchic organization, which often manifest itself in semicrystalline assemblies, has not been elucidated but the diversity of the supramolecular structures and their reorganizations under different conditions indicates its regulatory role. the present work focuses on the structural and functional roles of different components of lhcii-psii supercomplexes. we used various growth conditions, influencing the protein composition, and different arabidopsis mutants (kocp24, kocp26, kopsbw, kopsbx, dgd1), with altered organization of the membranes, and measured their circular dichroism (cd) spectra as well as their chlorophyll fluorescence kinetics to characterize the chiral macro-organization of the chromophores and the functional parameters of the membranes, respectively. we show that the formation of chiral macrodomains require the presence of supercomplexes. our data also reveal specific functions of some of the protein or lipid compounds in the light adaptation processes of plants. excitation energy transfer and non-photochemical quenching in photosynthesis rienk van grondelle department of physics, vu university, de boelelaan 1081, 1081hv, amsterdam, the netherlands the success of photosynthesis relies on two ultrafast processes: excitation energy transfer in the light-harvesting antenna followed by charge separation in the reaction center. lhcii, the peripheral light-harvesting complex of photosystem ii, plays a major role. at the same time, the same light-harvesting system can be 'switched' into a quenching state, which effectively protects the reaction center of photosystem ii from over-excitation and photodamage. in this talk i will demonstrate how lhcii collects and transfers excitation energy. using single molecule spectroscopy we have discovered how lhcii can switch between this light-harvesting state, a quenched state and a red-shifted state. we show that the switching properties between the light-harvesting state and the quenched state depend strongly on the environmental conditions, where the quenched state is favoured under 'npq-like' conditions. it is argued that this is the mechanism of non-photochemical quenching in plants. photobiology in the soil: arrested chlorophyll biosynthesis in pea epicotyl sections beá ta vitá nyi, katalin solymosi, annamá ria kó sa, bé la bö ddi eö tvö s university, institute of biology, department of plant anatomy, pá zmá ny p. s. 1/c, h-1117 budapest, hungary the key regulatory step of chlorophyll (chl) biosynthesis is the nadph:protochlorophyllide oxidoreductase (por) catalyzed reduction of protochlorophyllide (pchlide)which is light activated in angiosperms. this process is usually described on artificially dark-grown plants. in this work, we studied epicotyl segments developed under the soil surface, which were dissected from pea plants grown under natural light conditions. using 77 k fluorescence spectroscopy, pigment analyses, electron microscopy and fluorescence microscopy, we found that upper segments showed transitional developmental stages, i.e. chl appeared besides pchl(ide) and etio-chloroplasts were typical. in regions under 5 cm depth, however, the characteristics of the segments were similar to those of plants germinated artificially in complete darkness, i.e. only pchl(ide) and etioplasts were present. the results of this work prove that these latter symptoms may occur in shaded tissues of fully developed, photosynthetically active plants grown under natural conditions. in this overview talk it will be shown how atomistic computations can complement experimental measurements in our quest to understand biological electron and proton transfer reactions. at first the molecular simulation methods for calculation of important electron transfer parameter such as reorganization free energy, electronic coupling matrix elements and reduction potentials will be explained. then three applications will be discussed where such computations help interpret experimental data on a molecular level. the first example concerns electron tunneling between heme a and heme a3 in the proton pump cytochrome c oxidase. this reaction is very fast, occurring on the nano-second time scale, and it is unclear if this is due to an unusually low reorganization free energy or due to high electronic coupling. carrying out large-scale all-atom molecular dynamics simulation of oxidase embedded in a membrane, we do not find evidence for unusually small values of reorganization energy as proposed previously, implying that the nanosecond tunneling rate between heme a and a3 is supported by very efficient electronic coupling. the second example is on electron transport in a deca-heme 'wire'-like protein, used by certain anaerobic bacteria to transport electrons from the inside of the cell to extracellular substrates. the crystal structure of such a protein has been solved recently for the first time. however, it is unclear if and in which direction the wire structure supports electron transport. here we present results of heme reduction potential calculations that help us reveal the possible electron flow in this protein. in a third example we explain how quantum mechanical/molecular mechanical methods (qm/mm) recently helped us understand why the catalase from h. pylori is prone to undergo an undesired protein radical migration reaction during catalysis. proton pumping activity of purple and brown membranes regenerated with retinal analogues k. bryl 1 , k.yoshihara 2 1 university of warmia and mazury, department of physics and biophysics, olsztyn, poland, 2 suntory institute for bioorganic research, wakayamadai, osaka 618, japan the retinal protein bacteriorhodopsin (br) acts as a light-driven proton pump in the purple membrane (pm) of halobacterium salinarium (h.s.). the aim of these studies was to clarify whether the specific crystalline structure of protein and protein-substrate interactions are significant for h ? transfer into the aqueous bulk phase. two membrane systems were prepared: purple membranes (br arranged in a two-dimensional hexagonal lattice) and brown membranes (br not arranged in a crystalline lattice) were regenerated with 14-fuororetinals. light-induced proton release and reuptake as well as surface potential changes inherent in the regenerated systems reaction cycles were measured. signals of optical ph indicators residing in the aqueous bulk phase were compared with signals of ph indicator covalently linked to the extracellular surface of proteins and with surface potential changes detected by the potentiometric probe. the energies of activation of proton transfer have been calculated. experimental results and thermodynamic parameters (energies of activation) suggest the different mechanism of proton transfer into the aqueous bulk phase in these two systems. the implications for models of localized-delocalized energy coupling by proton gradients will be discussed. iron regulation is a vital process in organisms and in most of them it is accomplished through the metal solubilisation and storage by ferroxidase enzymes of the ferritin family, which have the ability of sequester, oxidize and mineralize ferrous ions using oxygen or hydrogen peroxide as substrate. dnabinding proteins from starved cells (dps) belong to this ferritin's family. dps belongs to the sub-type designated as miniferritins and, besides iron storage and release capability, is responsible for hydrogen peroxide resistance showing the ability to form stable complexes with dna. the preferable cosubstrate of this enzyme is h 2 o 2 although the reaction can occur in the presence of oxygen with lower rate [1, 2] . in this work, the electrochemical behaviour of the recombinant dps from pseudomonas nautica, was assessed as a function of metal content in anaerobic environment with h 2 o 2 as co-substrate. the obtained electrochemical results together with spectroscopic studies allowed inferring some new hypothesis on the dps iron uptake mechanism. for myoglobin electrostatically immobilized on au-deposited mixed self-assembled monolayers (sams) of the composition: -s-(ch 2 ) 11 -cooh/-s-(ch 2 ) 11 -oh. our approach allows for a soft switching of the haem group charge state and accurate probing of the accompanying reorganizational dynamics of conformational (quasi-diffusional) and quantum (e.g. protonrelated) modes. the electron transfer rate constants were determined with h 2 o or d 2 o as solvent, under the variable temperature (288-328 k) or pressure (5-150 mpa) conditions, revealing the overall reorganization free energy of 0.5±0.1 ev, activation volume of -3.1±0.1 cm 3 mol -1 and inverse solvent kinetic isotope effect of 0.7±0.1 (25°c). on the grounds of an extended charge-transfer theory, we propose specific protoncoupled et mechanism additionally coupled to the slow conformational dynamics of the protein matrix accompanied by translocation(s) of haem-adjacent water molecule(s). proton gradients across pore spanning membranes: towards on-chip energy conversion daniel frese, claudia steinem institute for organic and biomolecular chemistry, georg-august-university goettingen, germany in cell organelles, chemiosmotic potentials resulting from proton gradients across membranes are widely used to fix chemical energy in forms of atp. the high efficiency of this protein-mediated energy conversion raises interest for artificial proton gradient setups. to investigate proton transport across artificial membranes, we prepared pore spanning membranes (psms) on porous silicon substrates via painting technique. this allowed us to trap aqueous content of well-defined composition and volume inside the substrates microcavities. nigericin, a peptide that acts as an h ? -k ? -antiporter and bacteriorhodopsin, a transmembrane protein which is well known to be a light driven proton pump, were reconstituted into the pre-formed psms to achieve proton transport from one aqueous compartment to the other. changes of proton concentrations inside the pores were monitored by means of confocal laser scanning microscopy (clsm). therefore, pores were filled with pyranine, a ph-sensitive fluorescence dye and variations in intensity were measured to analyze proton translocation. we were able to show that both, nigericin and bacteriorhodopsin are capable of building up a proton gradient across psms and plan to co-reconstitute atp synthases for on-chip energy conversion by formation of atp. application of the gibbs free energy profiles to sequential biochemical reactions pé ter farkas, tamá s kiss and eugene hamori department of biological physics, eö tvö s ló rá nd tudomá ny egyetem, budapest, hungary, and department of biochemistry, tulane university, new orleans, la., usa the full understanding of the energetic details of complex metabolic reaction sequences requires a step-by-step analysis of the gibbs free energy (g) changes of the ''parasystem'' (i.e., a collection of atoms comprising all the molecules participating in a given reaction) as it gradually changes from its initialreactants state to its final-products state along the reaction pathway. knowing the respective equilibrium constants of each of the participating reaction steps and also the actual in vivo concentrations of the metabolites involved, a free-energy profile can be constructed that will reveal important information about the progress of the reaction as driven by thermodynamic forces. this approach will be illustrated on some biochemical reactions including the glycolytic/gluconeogenetic pathways. furthermore, the often misleading text-book representation of enzymatic catalysis will be reexamined and explained in thermodynamic terms using the free-energy profiles of both the non-catalyzed and the enzyme-catalyzed reactions. redox-active proteins can be diversely functionalized at metaldeposited self-assembled monolayers (sams) of a widely variable composition and thickness. the voltammetric methodology in combination with the advanced data processing procedures allow for comprehensive kinetic data within the congruent series of nano-devices and the subsequent calculation of the key physical parameters, such as the medium reorganization energy of et, the donor-acceptor electronic coupling, effective relaxation time (related to the protein's and environment's fluctuational dynamics), etc. in our studies the ''model'' redox protein, cytochrome c (cytc), was either freely diffusing to sam terminal groups (mode 1), or attached to sams through the electrostatic interaction (mode 2), or specific ''wiring'' (mode 3). another redox-active protein, azurin, was confined at terminal sam groups through the hydrophobic interaction (mode 4). diverse experimental strategies including the variation of sam thickness, solution viscosity temperature and hydrostatic pressure, allowed for a severe demonstration of the full adiabatic and nonadiabatic control (thinner and thicker sams, respectively), and the intermediary regime, in a nice agreement with the major theoretical predictions. proton transfers in a light-driven proton pump j.k. lanyi dept. physiology & biophysics, university of california, irvine, usa illumination of bacteriorhodopsin causes isomerization of alltrans retinal to 13-cis,15-anti and a cyclic reaction ensues, in which the protein and the chromophore undergo conformational changes with an overall ten millisecond turn-over time and a proton is transported from one membrane side to the other. with crystal structures of six trapped intermediate states and plausible structural models for the remaining two intermediates, structures are now available for the initial bacteriorhodopsin state and all intermediates. they reveal the molecular events that underlie the light-induced transport: protonation of the retinal schiff base by asp85, proton release to the extracellular membrane surface, a switch event that allows reprotonation of the schiff base from the cytoplasmic side, side-chain and main-chain motions initiated in the cytoplasmic region, formation of a single-file chain of hydrogen-bonded water molecules that conducts the proton of asp96 to the schiff base, and reprotonation of asp96 from the cytoplasmic surface. the observed changes can be summarized as a detailed atomic-level movie in which gradual relaxation of the distorted retinal causes a cascade of displacements of water and protein atoms results in vectorial proton transfers to and from the schiff base. electron transfer (et) processes are fundamental in photosynthesis, respiration and enzyme catalysis. the relative importance of superexchange and sequential mechanisms in biological et is still a matter of debate. the identification of any ''stepping stones'' necessary for electron hopping is a key point in the understanding of long range et. hence, the study of a single event in the sequence of reactions occurring in these phenomena is a fundamental but formidable task. muon spin relaxation (lsr) has been shown to be sensitive to charge transport on a molecular lengthscale. the muon is a very sensitive probe of electron transport, as any changes to the electronic density sampled by the muon can change its spin polarization, which can easily be measured. in this context, a very useful tool is the detection of the so-called avoided level crossing (alc) resonances [1] . the enhancement in the loss of polarization of the muon's spin on these resonances dramatically increases sensitivity. we show that a laser pump -lsr probe technique can measure et processes at particular, and most importantly known, sites within the amino acids chain, and therefore track the time evolution of the electron over the molecule. keywords: photosynthesis, reaction center, electron transfer, proton transfer, fourier transform infrared, l210dn, isotopic labeling, band assignment, histidine, mechanism in photosynthesis, the central step in transforming light energy into chemical energy is the coupling of light-induced electron transfer to proton uptake. in the photosynthetic reaction center (rc) of rhodobacter sphaeroides, fast formation of the charge separated state p ? q a is followed by a slower electron transfer from the primary quinone q a to the secondary quinone q b and the uptake of a proton from the cytoplasm to q b . previous fourier transform infrared (ftir) measurements on rc suggested an intermediate x in the q a -q b to q a q b transition. mutation of the amino acid aspl210 to asn (l210dn mutant) slows down proton uptake and oxidation of q a -. using time-resolved ftir spectroscopy we characterized this rc mutant and proposed specific ir bands that belong to the intermediate x. to study the role of the iron-histidine complex located between q a and q b , we performed fast-scan ftir experiments on the l210dn mutant marked with isotopically labelled histidine. we assigned ir bands of the intermediate x between 1120 cm -1 and 1080 cm -1 to histidine vibrations. these bands show the protonation of a histidine, most likely hisl190, during the q a -q b to q a q b transition. based on these results we propose a new mechanism of the coupling of electron and proton transfer in photosynthesis. complex i of respiratory chains is an energy transducing enzyme present in most bacteria and in all mitochondria. it is the least understood complex of the aerobic respiratory chain, even though the crystallographic a-helical structures of bacterial and mitochondrial complexes have been recently determined [1] [2] . this complex catalyses the oxidation of nadh and the reduction of quinone, coupled to cation translocation across the membrane. rhodothermus marinus complex i, our main model system, is a nadh:menaquinone oxidoreductase and has been extensively characterized. we have made an exhaustive study in order to identify all the subunits present in the complex [3] . the nature of the coupling charge of r. marinus complex i was investigated using inside-out membrane vesicles, which were active with respect to nadh oxidation and capable of creating and maintain an nadh-driven membrane potential (dw) positive inside. it was observed that this bacterial complex i is able of h ? and na ? transport, although to opposite directions. the coupling ion of the system was shown to be the h ? being transported to the periplasm, contributing in this way to the establishment of the electrochemical potential difference, while na ? is translocated to the cytoplasm [4] . the sodium ion extrusion from the membrane vesicles was due to the activity of complex i, since it was sensitive to its inhibitor rotenone, and it was still observed when the complex i segment of the respiratory chain was isolated by the simultaneous presence of cyanide and external quinones. additional studies have shown that although neither the catalytic reaction nor the establishment of the dph requires the presence of na ? , the presence of this ion increased the proton transport. combining all these results, a model for the coupling mechanism of complex i was proposed, suggesting the presence of two different energy coupling sites, one that works only as a proton pump (na ? independent), and the other functioning as a na ? /h ? antiporter (na ? dependent) [4] . this model was reinforced by further studies performed in the presence of the na ? /h ? antiporter inhibitor, 5-(n-ethyl-n-isopropyl)-amiloride (eipa) [5] . a deeper inside into the coupling mechanism of this enzyme was provided by studying the influence of sodium ions on energy transduction by complexes i from escherichia coli and paracoccus denitrificans. it was observed that the na ? / h ? antiporter activity is not exclusive of r. marinus complex i, since the e. coli enzyme is also capable of such a transport, but is not a general property given that the p. denitrificans enzyme does not performed sodium translocation [6] . due to the fact that r. marinus and e. coli enzymes reduce menaquinone while p. denitrificans complex i reduce ubiquinone, it is suggested that the na ? /h ? antiporter activity may be correlated with the type of quinone used as substrate. under anaerobic conditions some bacteria can use nitrate instead of oxygen in a process called denitrification. during denitrification, the reduction of no to n 2 o is catalyzed by a membrane-bound enzyme nitric oxide reductase (nor). this enzyme is an important step in the evolution of a respiratory system: nor belongs to the superfamily of o 2 -reducing heme-copper oxidases and is assumed to be the evolutionary ancestor of cytochrome c oxidase. the understanding of nor functioning was limited by the lack of structural information, but recently the first structures (cnor type from ps. aerug. and qnor type from g. stearoth.) were solved [1] [2] . we will present results of the first computational studies of nor (both cnor and qnor types) [2] [3] . the studies include: (i) large-scale all-atom md simulations of proteins in their natural environment (i.e. embedded in membrane and solvent), which were performed to describe water dynamics inside the protein and to identify potential proton transfer pathways, and (ii) free-energy calculations by the empirical valence bond (evb) method [4] for the explicit proton translocations along the pathways established by md. among important findings are new proton pathways, which were not predicted from the x-ray structure and could be identified only by means of computer simulations. simulations also reveal that, despite a high structural similarity between cnor and qnor, these enzymes utilize strikingly different proton uptake mechanisms. our results provide insights into the functional conversion between no and o 2 reductases, and into evolution proton transfer mechanisms and respiratory enzymes in general. the genome of the bacterium geobacter sulfurreducens (gs) encodes for 111 c-type cytochromes (1). genetic studies using cytochrome deficient gs strains and proteomic studies identified cytochromes that were produced under specific growth conditions (2) (3) (4) . a putative outer-membrane cytochrome, omcf, is crucial for fe 3? and u 6? reduction and also for microbial electricity production (4). omcf is a monoheme c-type cytochrome with sequence similarity to soluble cytochromes c 6 of photosynthetic algae and cyanobacteria (4). the structure of oxidized omcf was determined (5) being the first example of a cytochrome c 6 -like structure from a nonphotosynthetic organism. the structural features of omcf hinted a different function compared to cytochromes c 6 from photosynthetic organisms, being an excellent example of how structurally related proteins are specifically design by nature to perform different physiological functions. in order to elucidate omcf structural-functional mechanism, isotopic labeled protein ( 15 n and 13 c) was produced and its structure in the reduced form determined by nmr. single point-mutations at key residues were produced by site-directed mutagenesis and their impact on the structural and functional properties of omcf will be presented. in the early 90s, the search for the source of nitrogen monoxide (no) production in mammals led to the discovery of three major isoforms of no-synthases (nos): the neuronal nos (nnos), the inducible nos (inos), and the endothelial nos (enos) ( (1)). 20 years later, based on genomic analysis, numerous nos-like proteins have been identified in the genome other organism and in particular of several bacteria (bacillus anthrax, staphylococcus aureus… (2)). in spite of superimposable 3d structures and the ability to catalyse no production, all these enzymes are carrying different (if not opposite) physiological activities including cgmp signalling, cytotoxic activities, anti-oxidant defence, metabolism… moreover, noss become increasingly associated to oxidative-stress related pathologies ranging from neurodegenerative disorders, cardiovascular and inflammatory diseases, diabetes, cancers (3)…. this apparent paradox seems related to the belief that the strong similarity of sequence and structure of no-synthases must lead to a unique and identical functioning (no production) for all isoforms. this is blatant for bacterial nos-like proteins that are lacking the essential components required for no biosynthesis but remain considered as genuine no synthases. this approach might remain an obstacle to the understanding of nos actual biological role and could prevent the design of efficient nos-targeted therapeutic strategies. to elucidate this ''nos paradox'' our group has initiated a multidisciplinary approach that aims to relate the wide diversity of nos biological activities to variations in the catalytic mechanism of noss, to modifications of their regulation patterns and to adaptations to their physiological environment. in this context we have been investigating the mechanism of bacillus subtilis nos-like proteins with a special focus on the features that are specific to nos mechanism: i) electron and proton transfers and the role of the substrate and the pterin cofactor ii) oxygen activation and the role of the proximal ligand iii) the very molecular mechanism and the variations in the nature of reaction intermediates. for that matter we have been using a combination of radiolytical techniques (cryoreduction with 60 co y-irradiation, pulse-radiolysis with the elyse electron accelerator), stateof-the-art spectroscopies (epr, atr-ftir and resonance raman, and picoseconds uv-visible absorption spectrosocopies), organic synthesis (synthesized substrates and cofactors analogues) biochemistry and molecular biology (site-directed mutagenesis). we will present our results on the coupling of electron and proton transfers, on the tuning of the proximal ''push effect'' and we will discuss the conditions that favour for each nos isoform no production versus other reactive nitrogen and oxygen species. photosynthetic iron oxidation (pio) is an ancient form of photosynthesis with relevant consequences in the shaping of the planet. this form of metabolism may have been involved in the deposition of geological structures known as banded iron formations, which hold key information regarding the co-evolution of photosynthesis and earth. rhodopseudomonas palustris tie-1 and rhodobacter ferroxidans sw2 both use ferrous iron as an electron donor to support photosynthetic growth (i.e. photoferrotrophy). the sw2 foxeyz operon can stimulate light-dependent iron oxidation by other bacteria. it codes a two-heme cytochrome, a pyrroloquinoline quinone protein and an inner membrane transporter, respectively. in tie-1 the pioabc operon is required for photoferrotrophy. it codes for a ten-heme cytochrome, an outer membrane beta-barrel and a high potential iron-sulfur protein (hipip) respectively. here we present functional and structural characterization of proteins involved in pio. this molecular characterization is essential for understanding this mode of bioenergetic metabolism, and may one day aid the development of biotechnological applications like microbial fuel cells and bioremediation. alongside classical, cytochrome respiratory pathway, phycomyces blakesleeanus possess alternative, cyanideresistant respiration (crr) facilitated by alternative oxidase (aox). in order to study role of oxygen in regulation of crr, the effects of cyanide on respiration of 24h old mycelia in aerated (control), hypoxic and anoxic conditions were measured. mycelium was incubated in these conditions for 1.5h, 3h and 5h. after 1.5h, aox activity was increased only in specimens incubated in anoxic conditions (13.6%). after 3h, increase in aox activity was significant in both hypoxic and anoxic specimens (18.9% and 18.8%, respectively), with even greater increase after 5h, 20.7% for hypoxic and 23.3% for specimens in anoxic conditions. mycelia treated for 5h was then oxygenated for 10 minutes. this induced decrease in aox activity of 17% in anoxic and even 23.9% in hypoxic mycelia. aox is recognized as one of the mechanisms for maintaining low levels of reduced ubiquione which can function in conditions in which cytochrome chain is disabled, such as anoxia. this is in concordance with results obtained on p. blakesleeanus, where aox levels rise in hypoxic and anoxic conditions and decrease close to control level shortly after introduction of oxygen into the system. influence of escherichia coli f 0 f 1 -atpase on hydrogenase activity during glycerol fermentation k. trchounian 1,2 , g. sawers 2 , a. trchounian 1 1 department of biophysics, yerevan state university, 0025 yerevan, armenia, 2 institute for microbiology, martin-luther university halle-wittenberg, 06120 haale, germany e. coli encodes four hydrogenases (hyd); only three of these, hyd-1, hyd-2 and hyd-3 have been well characterized. hyd-2 has been shown recently to reversibly evolve hydrogen during glycerol fermentation at ph 7.5 [1] . proton reduction was inhibited by n,n'dicyclohexylcarbodiimide suggesting a link with the proton-translocating f 0 f 1 -atpase. indeed, at ph 7.5 in an e. coli mutant (dk8) lacking f 0 f 1 overall hyd-activity was reduced to approximately 50% of the wild type activity; hyd-2, but not hyd-1, was detected in an in-gel activity assay. f 0 f 1 is therefore suggested to be required for hyd-1 activity. at ph 6.5 in glycerol medium hyd-activity in dk8 was *10% of wild type activity and hyd-1 and hyd-2 exhibited only weak activity. this indicated a significant f 0 f 1 contribution towards hyd-activity as ph decreased. furthermore, at ph 5.5 hydactivity was negligible and only a very weak activity band corresponding to hyd-2 could be observed. these results suggest that f 0 f 1 -atpase is essential for hydrogenase activity during glycerol fermentation at ph 5.5. taken together, the results suggest an interdependence between hyd-1, hyd-2 and f 0 f 1 -atpase activity. [ ion channels and transporters control many facets of cancer cell biology 1 and blocking the activity of these impairs tumor cell growth in vitro and in vivo. this new paradigm has opened new opportunities for pharmaceutical research in oncology 1,2 . we have contributed to this field showing that k v 11.1 (herg1) channels are aberrantly expressed in several human cancers where they control different aspects of the neoplastic cell biology such as proliferation and apoptosis, invasiveness and angiogenesis, the latter through the regulation of vegf secretion (reviewed in 1 ). the herg1dependent effects were shown in vitro and, more recently, in vivo. in preclinical models of both leukemia 1 and colorectal cancer 3 , herg1 overexpression confers a higher malignancy to neoplastic cells. moreover, herg1 blockers have therapeutic potential, since preclinical tests showed that treatment with specific herg1 blockers overcame chemoresistance in acute leukemias 4 as well as reduced gi cancer growth, angiogenesis and metastatic spread 3 . the overall message emerging from our data is that the herg1 protein represents a novel biomarker and drug target in oncology. up to now, herg1 was considered an ''antitarget'' due to the cardiac side effects that many (but not all) herg1 blockers produce and that result in lengthening the electrocardiographic qt interval. we report here recent studies on known and newly developed herg1 blockers that exhibit no cardiotoxicity and are more specific for the herg1 channels expressed in cancer cells. we reported previously that the increase of the cholesterol content of the cell membrane (in vitro) modified the biophysical parameters of the gating of kvl.3 k ? ion channels in human t-lymphocytes. in our present study we aimed to determine the effect of hypercholesterolemia on the biophysical parameters of kv1.3gating and the proliferation of t cells. t-lymphocytes were isolated from the peripheral blood of patients with cholesterol level considered normal (\5.2 mmol/l,control group) and patients with hypercholesterinaemia (hc). whole-cell k? currents were measured in patchclamped t cells and the kinetic (activation and inactivation kinetics) and equilibrium parameters (voltage-dependence of steady-state activation) of kv1.3 gating were determined. lymphocyte proliferation was measured using cfse staining with and without anti-cd3 and anti-cd28 stimulation. our results indicate that the biophysical parameters of kv1.3 gating are similar in the control group and in the 'hc' samples. the cfse-based assay showed that hypercholesterolemic t cells had higher spontenaous activation rate compared to control group. however, t cells from high cholesterol level patients challenged by anti-cd3 and anti-cd28 exhibited lower proliferation rate than control cells. generalized epilepsy with febrile seizures plus (gefs?, omim 604233) is a childhood genetic epilepsy syndrome correlated to mutations in the ancillary b-subunit of neuronal voltage-gated sodium channels (nachs). b1-subunit is non-covalently associated with nach a-subunits, serving as modulator of channel activity, regulator of channel cell surface expression and as cell adhesion molecule. the first and best characterized gefs? mutation is the c121w. this mutation changed a conserved cysteine residue into a tryptophan, disrupting a putative disulphide bridge that should normally maintain an extracellular immunoglobulinlike fold. in this study, we investigated the presence of this putative disulphide bond using 2-d-diagonal-sds-page, where the proteins were separated in the first dimension in absence of a reduction agent and in presence of it in the second dimension. this method allows to visualize the protein above the diagonal experimentally confirming that the disulphide bond is intramolecular. duchenne muscular dystrophy (dmd) is associated with severe cardiac complications. recent research suggests that impaired voltage-gated ion channels in dystrophic cardiomyocytes accompany cardiac pathology. it is, however, unknown if ion channel defects are primary effects of dystrophic gene mutations, or secondary effects of the developing cardiomyopathy. here, we studied na and ca channel impairments in dystrophic neonatal cardiomyocytes, derived from dmd mouse models, prior to cardiomyopathy development. dystrophin-deficiency reduced na current density. in addition, extra utrophin-deficiency altered na channel gating. moreover, also ca channel inactivation was reduced, suggesting that ion channel abnormalities are universal primary effects of dystrophic gene mutations. to assess developmental changes, we also studied na channel impairments in dystrophic adult cardiomyocytes, and found a stronger na current reduction than in neonatal ones. the described na channel impairments slowed the action potential upstroke in adult cardiomyocytes, and only in dystrophic adult mice, the qrs interval of the ecg was prolonged. ion channel impairments precede pathology development in the dystrophic heart, and may be considered cardiomyopathy triggers. supported by austrian fwf (p19352). it has been over 15 years since sensory neuron-specific sodium channel na v 1.8 was identified. since then na v 1.8 has been shown to play a crucial role in pain pathways, and it became a prominent drug target for novel pain killers. in contrast to myelinated neurons, mechanisms that target voltagegated sodium channels to the unmyelinated c-fibre axons are largely unknown. we investigated the localisation of na v 1.8 in unmyelinated primary sensory neurons. na v 1.8 was found to be clustered in lipid rafts on unmyelinated axons. when the lipid rafts are disrupted, remarkable reduction in both the conduction velocity and the number of cells responsive to mechanical stimuli to the unmyelinated axons were seen. using a compartment culture system, we also found that disruption of rafts in the middle region of the sensory axons caused a significant reduction in the responsiveness of the neurons to chemical stimuli to nerve endings. this is due to the failure of action potential propagation through the axons. these data suggest that clustering of na v 1.8 in lipid rafts of unmyelinated fibres is a key factor for the functional properties of the channel, which may due to a change in the voltage threshold. disruption of the na v 1.8 cluster and modifying lipid rafts in primary sensory neurons may be a useful new approach to control the excitability of nociceptive neurons. ion currents in are crucially important for activation of t-lymphocytes. our aim was to investigate how the blockage of various ion channels in isolation or in combination affects mitogen-dependent activation and proliferation of t-cells. we activated human peripheral blood lymphocytes using monoclonal antibodies against the tcr-cd3 complex and cd28. we applied specific channel blockers inhibiting the major ion channels of the t-cell: either the kv1.3 (tea or anuroctoxin), ikca1 (tram-34), or the crac channel (2-apb) alone or in combination. five days after the stimulus we measured the change in cell size and cellular granulation with flow cytometry along with the proportion of dividing cells, using cfse (carboxyfluorescein succinimidyl ester) dilution assay. our measurements indicated that the ion channel blockers suppressed the proportion of dividing cells in a dosedependent manner. increasing the strength of the stimulation reduced the potency of the blockers to inhibit cell proliferation and eventually the blockers were ineffective in decreasing lymphocyte proliferation. we experienced the greatest amount of inhibition using the combination of the blockers, which indicates synergy in the regulation pathway of the various ion channels. recently, sodium dependent phosphate transporter napi2b was revealed as potential marker for breast, thyroid and ovarian cancer. in vivo, napi2b is involved in maintenance of phosphate homeostasis and mutations or aberrant expressions of its gene (slc34a2) are associated with several diseases including cancer. however, data about napi2b mrna expression in different types of cancer and correspondent normal tissues are controversial and restricted. we investigated slc34a2 gene expression level in normal ovarian tissues and different histomorphological types of ovarian tumors using real-time pcr analysis. it was found that slc34a2 gene was highly expressed in well-differentiated endometrioid and papillary serous tumors, but was not expressed in low-differentiated tumors, benign tumors and in most normal tissues. mrna expression of slc34a2 in serouse and endometrioid ovarian tumors accurately correlated with protein expression that was detected in these tumor samples by western blot analysis and immunohistochemistry in our previous investigation. upregulation of slc34a2 gene expression in well-differentiated tumors may reflect cell differentiation processes during ovarian cancerogenesis and could serve as potential marker for ovarian cancer diagnosis and prognosis. in the present contribution a procedure for molecular motion characterization based on the evaluation of the mean square displacement (msd), through the self-distribution function (sdf), is presented. it is shown how msd, which represents an important observable for the characterization of dynamical properties, can be decomposed into different partial contributions associated to system dynamical processes within a specific spatial scale. it is shown how the sdf procedure allows us to evaluate both total msd and partial msds through total and partial sdfs. as a result, total msd is the weighed sum of partial msds in which the weights are obtained by the fitting procedure of measured elastic incoherent neutron scattering (eins) intensity. we apply sdf procedure to data collected, by in13, in10 and in4 spectrometers (institute laue langevin), on aqueous mixtures of two homologous disaccharides (sucrose and trehalose) and on dry and hydrated (h 2 o and d 2 o) lysozyme with and without disaccharides. the nature of the dynamical transition is highlighted and it is shown that it occurs when the system relaxation time becomes shorter than the instrumental energy time. finally, the bioprotectants effect on protein dynamics and the amplitude of vibrations in lysozyme are presented. we evaluated q for genotoxicity in mcf-7 breast cancer cells in the presence or absence of doxorubicin (dox), docetaxel (dtx) and paclitaxel (ptx), anticancer drugs commonly used in chemotherapy of different solid tumors. damage to dna was determinated by comet assay. the cells, after treatment with investigated compounds, were washed up and cultured in fresh medium for 0, 24, 48, 72 and 96 hours. we have found that q by itself caused significant dna damage. moreover flavonol enhanced genotoxic effect of anticancer drugs. the highest amount of dna in the comet tail was observed 48 h after treatment with combination of q with dox. similar changes were found in cells incubated with combination of q with taxanes -ptx or dtx. however, damage to dna in this case was considerably lower than damage caused by combination of q with dtx. our results confirmed anticancer and genotoxic activity of quercetin, which makes it a promising candidate for a potential use as a modulator of cytotoxicity and anticancer activity of anthracycline and taxane chemotherapeutics. although the health effects of low-frequency and intensity electromagnetic fields (lfi-emfs) are controversial, increasing evidence suggests that lfi-emfs are capable for initiating various healing processes. many (bio)physical ideas were suggested to explain the influence of lfi-emfs in living systems but the main effect of lfi-emfs on cell functions remains vague. however, some effects of lfi-emfs may be explained by redox and membrane processes. during diseases, cells not only demonstrate altered biochemical processes but also produce altered non-linear bioelectromagnetic complex patterns. thus, it is reasonable to use non-linear bioelectric and bioelectromagnetic signals from cells of the body for potential therapeutic applications that may be more effective than the artificial lfi-emfs signals. our novel emost (electromagnetic-own-signal-treatment) method is based on the utilization of the non-linear, bioelectric and bioelectromagnetic signals of the patients without any electromagnetic wave modulation and inversion of recorded output signals of subjects. here, we report our some restorative results after emost application. we also suggest that the possible effects of the emost may be achieved via redox-related processes. background or leak potassium conductances are a major determinant of resting potential and input resistance, two key components of cell excitability. these currents are not passive but finely tuned and adapted to cell specific functions. k 2p channels producing these currents are tightly regulated by a variety of chemical and physical stimuli including temperature, membrane stretch, free fatty acids, ph, ca 2? , neurotransmitters and hormones as well as protein partners. these different stimuli converge on gating mechanisms that show remarkable conservation between intracellular k2p channels (twik1 channels) and k2p channels located at the plasma membrane (trek1/2 channels). living at the edge: volume 2 the conduction system of interfacial forces into the alveolar type ii cell in previous investigations, using new microscopic approaches, we found that the presence of an a li leads to a paradoxical situation: it is a potential threat that may cause cell injury, but also a important stimulus: at ii cells respond promptly, and show sustained ca 2?signals that activate exocytosis. exocytosed surfactant, in turn, clearly prolonged the time to irreversible cell damage, and may be an adaptive and evolutionary defense mechanism against the harmful nature of surface forces. recently we published that at ii cells are sensing the a li but how this stimulus is conducted and converted into the cell, is still obscure. currently we are searching for potential calcium sources and it seems that the cells signal ca 2? by extracellular ca 2? entry probably through mechanosensitive channels. specialdesigned gene chips allowed a whole genome profiling of a li exposed single cells. these cells react with rapid changes on the transcriptional level: cellular pathways that are involved include e.g. defense response and lipid metabolism, and we identified genes associated with several lung diseases and injuries. we summarize, interface forces are strong, they are acting on the cells and triggering cellular events that are closely related with classical concepts in mechanotransduction, it is very plausible that those forces play a crucial role in the lung surfactant homeostasis. calorimetric method and instrumentation were worked out and applied for investigation aqueous solutions of proteins. thermal effects were analyzed by own construction heat-fluxtype dsc cell designed for temperature range from the boiling point of water down to 120 k. the achieved sensitivity of heat flow rate (hf) of the instrument is better than 50%w tested using 1 ll of 150 mm aqueous solution of nacl. from the integral value of hf -the total enthalpy change dh total , the enthalpies of transitions were separated from the heat capacities. using the method, several types of proteins (bsa, erd10, ubq, a-, b-casein, and wt, a53t, a-synuclein mutants) were investigated in that temperature range. the results are shown in detail as an illustrative example. potential applications are outlined, which include (i) the distinction between the solvent accessible surfaces of globular and intrinsically disordered proteins, (ii) the distinction between protein mutants, and (iii) the identification of monomer and polymer protein states. this method provides a possibility to study the polymerization process (amyloid formation) and to investigate in-situ the reason and circumstances of that. morphometry due to self gravity in living organism is an integral part in understanding self gravitation bio. computational studies of biological system, especially in ab-initio embryo as self gravitating mass, floating over amniotic fluid, as if maintaining anti -gravitation (extrinsic) mechanism, would be an interesting one to explore biomechanics of intrinsic gravity. since the work would be of exploratory nature, both from the point of view of identifying appropriate stage of development of embryological morulla and available computational logics, it was contemplated to initiate functional study with a few reported ultrasound evidential works on small animals like mice, grasshopper including compact human embryo as mathematical structure that may allow the formal definition of concepts such as convergence, mapping and continuity. as many of the finite-dimensional function analysis in topological vector spaces are available, we initiated our simulation and studies on concentrating locally compact banach spaces. 3 institute for x-ray physics, university of gö ttingen, germany we report on hard x-ray phase contrast imaging of black lipid membranes (blms), which are freely suspended over a micro machined aperture in an aqueous solution. this new way of membrane structure analysis allows investigating bio molecular and organic substances in aqueous environments by parallel and divergent beam propagation imaging, using partially coherent multi-kev x-ray radiation. the width of the thinning film is significantly smaller than the detector pixel size, but can be resolved from quantitative analysis of the intensity fringes in the fresnel diffraction regime down to its native thickness of about 5nm. to our knowledge this is the first time that such small features of a very weak phase object have been visualized by direct x-ray imaging techniques. we have put forward a simplified but extendable model, which enables the theoretical description of image formation and characterization of membrane thickness and its decrease during the thinning process from a bulk to a bimolecular film. on the basis of recent experiments, future investigations will be performed to study the interactions of membranes, as they are for example known from synaptic fusion, with high spatial resolution. shown that the acid incorporates mainly in the exterior part of the erythrocyte membrane, inducing creation of echinocytes. this suggests that it interacts predominantly with the outer part of the lipid layer of erythrocytes and liposomes. it was also shown that the cga decreases the packing order of the hydrophobic part of the membranes, without changing the anisotropic fluorescence of the hydrophobic part. one of the unique features of single molecule absorption/ emission is their anisotropy due to the well-defined transition dipoles for both processes allowing the determination of the molecule's 3d orientation. therefore, several techniques have been proposed in order to determine the full 3d orientation of dipole emitters on a single molecule level. we recently demonstrated a technique that combines emission distribution and polarization detection [1, 2, 3] . as the method is an intensity distribution technique and based on single photon detection in principle, one can extend the 3d orientation determination to fluorescence correlation spectroscopy (fcs) as well as dynamical anisotropy measurements. this allows for the determination of the dynamics in 3d orientation of single molecules down to a nanosecond timescales. the 3d orientation is particularly interesting in non-isotropic environments. a lipid membrane is such a non-isotropic environment of enormous importance in biological systems. we therefore use giant unilamellar vesicle (guv) labeled with dyes like dio as a model system. due to the defined curvature of such vesicles all possible dipole orientations can be achieved. this allows us to show the capabilities of our method on different timescales and to quantify the error in determination of 3d orientation dynamics in lipid membranes. [ the aim of the studies was to determine the changes that occur in a biological membrane and the model lipid membrane as a result of interaction with strawberry leaf extract. numerous studies conducted all over the world have documented a beneficial effect of polyphenolic compounds on the human organism. however, the mechanism of the interaction on the molecular and cell level is not yet known. in the work presented, the effect of strawberry leaf extract on the erythrocyte and black lipid membranes has been investigated. the applied methods -spectroscopic, fluorimetric and electric -allowed to determine the hemolytic and antioxidant activity, and the packing order in the erythrocyte membrane as well as the electric capacity of blms. the results obtained indicate that the extract is efficient in protecting membrane lipids against oxidation, does not induce hemolysis, increases osmotic resistance and decreases packing order in the hydrophilic region of the erythrocyte membrane. moreover, it increases stability and life-time of flat lipid membranes, without altering their specific capacity. supported lipid bilayers are an abundant research platform for understanding the behavior of cell membranes as they allow for additional mechanical stability and enable characterization techniques not reachable otherwise. however, in computer simulations these systems have been studied only rarely up to now. we present systematic studies on different length scales of the changes that a support inflicts on a phospholipid bilayer using molecular modeling. we characterize the density and pressure profiles as well as the density imbalance induced by the support. it turns out that the changes in pressure profile are strong enough that protein function should be impacted leading to a previously neglected mechanism of transmembrane protein malfunction in supported bilayers. we determine the diffusion coefficients and characterize the influence of corrugation of the support. we also measure the free energy of transfer of phospholipids between leaflets using the coarsegrained martini model. it turns out that there is at equilibrium about a 2-3% higher density in the proximal leaflet. these results are in agreement with data obtained by very large scale modeling using a water free model where flip-flop can be observed directly. we are additionally characterizing the intermediate states which determine the barrier height and therefore the rate of translocation. we also study the influence of surface roughness and curvature on the behavior. simulations in atomistic detail are performed for selected systems in order to confirm the findings. [ the inverse bar (i-bar) domain is part of the superfamily of the membrane-deforming protein is bin-amphiphysin-rvs (bar) proteins which induce either positive or negative membrane curvature both in vitro and in cells. generation of membrane curvature by these membrane deforming proteins often works together with actin dynamics. i-bar shares its function between actin bundling and membrane binding but it is still obscured what molecular mechanisms are responsible for these functions. the aim of our project is to investigate the detailed membrane binding properties of the i-bar of irsp53 and its relations to the actin cytoskeleton. in vitro fret experiments and fluorescence quenching studies were carried out between the i-bar and liposomes made up from different lipid constructs. we have found that the i-bar has preference to bind to the negatively charged lipids however it can also bind to the uncharged lipids. the fluorescence quenching studies reflected that the accessibility of the i-bar surface was higher toward the negatively charged lipids than for the uncharged ones. tns fluorescence assay reflected that the i-bar domain binds to the surface of the micells rather than penetrating into its core. lipid bilayers present a well-known order-disorder chain transition at ambient temperatures. this transition may become anomalous if the lipid head-group presents ionic dissociation at low ionic strength, as detected by several experimental techniques: between the gel and the liquid phases an intermediate phase appears as a shoulder in the specific heat, a deep in turbulence or a maximum in conductivity. we propose a statistical model which allows ionic dissociation of the polar group on the membrane surface and thus introduces competition between the hydrophobic interaction of hydrocarbonic chains, which favours the gel phase, at low temperatures, and the electrostatic interaction of charged head-groups, which favours the fluid phase, at higher temperatures. the model presents an intermediate fluid phase with higher dissociation and charge ordering on the membrane surface, beyond a sharp gel-fluid transition. the model presents increasing temperature of the main transition by addition of salt, as well as the shrinking of the anomalous region as chain length increases. model thermodynamic behavior is compared to results for pgs, phospholipids with a glycerol head-group. the well-programmed membrane fusion systems, operating in a weakly acidic environment, have attracted attention in the fields of biochemistry, biophysics, and pharmacy because these acidic conditions are generally observed in endosomal membranes or tumor tissues. we have reported a selective liposomal membrane fusion system toward a sugar-like cyclic cis-diol structure on the target liposome. this system consists of a lapidated phenyl boronic acid derivative as membrane -bound fusogen and phosphatidylinositol as target. here we report the preparation of a boronic acid / ph-responsive polypeptide conjugate as a novel membrane fusion device and the development of a target selective liposomal membrane fusion system with endosomal ph-responsiveness. during the course of lipid-mixing, inner-leaflet lipid-mixing, and contents-mixing assays to characterize membrane fusion behavior, we clearly observed a liposomal membrane fusion phenomenon when the ph of the experimental system was changed from 7.4 (physiological) to 5.0 (endosomal). our highly effective methods, which include a target selective liposomal membrane fusion, can be useful in the area of nanomedicine such as hybridoma technology and liposomebased drug or gene delivery. complete and reversible chemical denaturation of an a-helical membrane protein jana broecker, sebastian fiedler, sandro keller molecular biophysics, university of kaiserslautern, erwin-schrö dinger-str. 13, 67663 kaiserslautern, germany the question of how an unordered polypeptide chain assumes its native, biologically active conformation is one of the greatest challenges in molecular biophysics and cell biology. this is particularly true for membrane proteins. chemical denaturants such as urea have been used successfully for in vitro un-and refolding studies of soluble proteins and b-barrel membrane proteins. in stark contrast with these two protein classes, in vitro unfolding of a-helical membrane proteins by urea is often irreversible, and alternative denaturation assays using the harsh detergent sodium dodecyl sulphate suffer from a lack of a common reference state. here we present the complete and reversible chemical denaturation of the bacterial a-helical membrane protein mistic out of different micellar environments by urea. we applied multidimensional spectroscopy and techniques typically used in b-barrel membrane protein unfolding. mistic unfolds reversibly following a two-state equilibrium that exhibits the same unfolded reference state. this allows for a direct comparison of the folding energetics in different membrane-mimetic systems and contributes to our understanding of how a-helical membrane proteins fold as compared with both b-barrel membrane proteins and water-soluble proteins. in recent years, buckwheat has been of great interest in the world markets of healthy food, due to its high energy value, the content of unsaturated fatty acids, mineral constituents and vitamins. its seeds contain flavonoids which are natural, efficient antioxidants. the aim of the present studies was to investigate the effect of buckwheat extracts on the properties of biological membrane, which is the main site of the interaction between the substances buckwheat contains and the organism. the research was conducted on red blood cells and their isolated membranes, using the spectrophotometric, microscopic and fluorimetric methods. from the results obtained it follows that the compounds contained in buckwheat extracts increase the osmotic resistance of erythrocytes, making them less sensitive to the medium's osmotic pressure, induce changes in cell shape, producing increased number of echinocytes, and decrease the packing order of the polar heads of membrane lipids. it can thus be inferred that the compounds contained in the extracts penetrate the hydrophobic region of the erythrocyte membrane and alter its properties. .due to its small size, symmetric structure, amphipathicity, proteolytic stability and testable mode of activity, the gs backbone is a convenient model system to examine the structure-activity relationship of individual amino acid substitutions. we have previously reported the structure analysis of two gs analogues in which either the val or leu residues on the hydrophobic face of the molecule were substituted by the aliphatic 19 f-labeled amino acid 4f-phg. using 19 f-ssnmr in oriented lipid bilayers, we observed a re-alignment of the peptide that is compatible with the formation of a putative pore [top.curr.chem. 273:139]. here, we present novel analogs of gs with different 19 f-prolines in the b-turn region, and with cf 3 -bpg in place of leu. based on these 19 f-ssnmr results and supported by cd, dsc and activity tests, we could demonstrate that all analogues are structurally intact and antimicrobially active. we observe, however, differences in the re-alignment propensity when comparing these gs analogues in dlpc and dmpc bilayers. these differences can be rationalized in terms of molecular shape being changed upon incorporation of unnatural amino acids at various sites of the molecule. the beta-propiolactone (bpl) is an inactivating reagent commonly used to produce viral vaccine preparations (whole virions or split-virions). although bpl has been reported to inactivate nucleic acids, its mechanism of action on proteins and the outcome on viral infection remains ill-defined. in this work, h3n2/victoria/210/2009 influenza virus strain has been submitted to various bpl inactivation conditions (from 2lm to 1 mm). cell infection ability was progressively reduced and entirely abolished at 1 mm bpl. to clarify the bpl effect, we focused on membrane fusion infection steps using kinetic fluorescence molecule leakage from liposome and lipid fret assays combined with cryo electron microscopy. membrane fusion measured at ph 5 on gm3 liposomes was reduced in a dose-dependent manner. interestingly the fusion activity was partially restored using the proton-ionophore monensin as confirmed by cryoem images. in addition, a decrease of molecule leakage irrespective to bpl concentration was measured suggesting that the hemagglutinin affinity for gm3 was slightly modified even at low bpl concentration. altogether these results strongly suggest that bpl treatment impairs m2 protein activity likely by preventing proton transport and bring new light on the mechanism of action of bpl. cellular membranes have a heterogeneous lipid composition, potentially forming nano-domains or membrane rafts, believed to be platforms of altered fluidity involved in protein sorting and trafficking 1 . an alternative mechanism, potentially leading to protein sorting, has recently been proposed, suggesting that the curvature of membranes can also actively regulate protein localization 2 . recently we showed that a variety of protein anchoring motifs are membrane-curvature sensors and thus up concentrate in regions of high membrane curvature 3 . furthermore the curvature sensing ability of the anchoring motifs persisted independently of their structural characteristics. this leads us to speculate that curvature sensing might be an inherent property of any curved membrane and as a consequence, the lipid composition of the bilayer could potentially regulate this recruitment by membrane curvature. thus there might be an intimate, yet unrecognized, link between the way raft-like membrane domains and membrane-curvature promotes the localization of membrane-anchored proteins. we examined how changing the lipid composition of liposomes influenced the recruitment by membrane curvature of a model amphiphilic protein-anchoring motif. employing our single liposome curvature assay, we tested lipid mixtures with different ratios of dopc, sphingomyelin and cholesterol, giving rise to liposome populations of different phase-states. we found an amplified recruitment by membrane curvature for all raft-like l o phase-state mixtures when compared to the l d phase-state counterparts. based on these findings we suggest a synergetic effect when combining a raft-like lipid phase-state and high membrane curvature, resulting in a highly potent mechanism for selective localization of membrane-anchored proteins. keywords: non-lamellar lipid structure, phase transition, minerval, 2-hydroxylated fatty acid. minerval (2-hydroxyoleic acid), a potent antitumoral drug, is known to modulate the lipid membrane structure by decreasing the lamellar-to-non-lamellar phase transition temperature (t h ). a series of 2-hydroxy fatty acid derivatives, varying in acyl chain length and degree of unsaturation, have been analyzed in terms of their ability to stabilize the inverted hexagonal (h ii ) phase in palmitoyl-oleoyl-phosphatidylethanolamine membranes. differential scanning calorimetry and 31 p-nuclear magnetic resonance showed that mono-and polyunsaturated, but not saturated, 2-hydroxylated fatty acid molecules were able to decrease the t h . lipid vesicles mimicking the lipid composition of a cell membrane were solubilized at 4°c in the presence of triton x-100. the results demonstrated that the amount of detergent-resistant membranes, which are related to liquid ordered (lo) structures, decreased in the presence of 2-hydroxylated fatty acids. the so-called lipid membrane therapy focuses on the reversion of cell disfunction through the modulation of the membrane structure, thus altering the activity of membrane-associated proteins. the ability to modify the biophysical properties of a lipid membrane makes the studied 2-hydroxylated fatty acid molecules be prospective candidates for the use in the lipid membrane therapy. ]. however, a significant downward divergence occurs above 4 mol % of probe content, which might indicate deviations to ideal mixing in fluid phase. results for b-py-c 10 -hpc, in mixtures of popc with 10 and 20 mol % pops were indistinguishable from those obtained with pure popc vesicles; however excimer formation in pure pops bilayers appears to be appreciably higher. we also compared the excimer formation findings with quenching of the same probes by low concentrations of doxyl quencher groups labeled acyl phospholipid chain at the same depth of the pyrenyl group. the results are also scrutinized by the same two-dimensional kinetic formalism and good correlation was also found. derek marsh max-planck-institut fü r biophysikalische chemie, 37070 gö ttingen, germany the amassing of comprehensive data on the lipid composition of biological membranes by lipidomics initiatives provides a potent challenge to the membrane biophysicist interested in lipid structure. this resolves itself essentially into two aspects. the first systematises the dependence of membrane biophysical parameters on lipid molecular structure. lipid volumes, membrane dimensions, chain-melting temperatures and enthalpies, nonlamellar phase formation and structure, critical micelle concentrations and thermodynamics of membrane formation, membrane-membrane interactions and lipid transfer are amongst the properties of central biophysical interest. the relevant structural parameters are lipid chain length, degree of unsaturation, chain branching and headgroup configuration. the second, more complex and less well developed, aspect concerns the lipidlipid interactions that determine the membrane properties of lipid mixtures. in part, these can be obtained from binary phase diagrams, and the more limited number of ternary phase diagrams -notably with cholesterol -that are available. extrapolation to higher order mixtures lies in the future. i shall attempt to summarise some of the progress in these directions. the immediate aim is a second edition of my handbook of lipid bilayers, which, in addition to a vastly expanded database, will include interpretative features and will be available in the early part of next year. lateral diffusion dynamics in phosphatidylcholine/cholesterol bilayers has been mostly accessed by means of epr, nmr and fcs spectroscopic techniques. reliable stady-state florescence quenching analysis of diffusion-controlled processes has been ampered by the lack of a self-consistent kinetic formalism for the two-dimensional (2d) counterpart of the classical stern-volmer analysis for three-dimensional (3d) solvents. we studied the excimer formation of phospholipid-labeled pyrenyl probes (proportion of 4 mol %) in mixed popc/cholestrol mlv liposomes by combined steady-state and timeresolved fluorescence the findings are in very good agreement with the theoretical predictions of the kinetic formalism specific for fluorescence quenching processes occurring in the small hydrophobic head group, the closely packed acyl chains, and the capability of interfacial hydrogen bonding have been suggested to govern the characteristic membrane behavior of long chain saturated ceramides: the self-segregation, and the formation of hexagonal phases and highly ordered gel phases. while it has been shown that structural alterations of the ceramide acyl chains induce position dependent effects on their behavior, we wanted to study the effect of interfacial properties, including hydrogen bonding, on ceramide membrane properties. the h-bond donor functions of 2nh and 3oh in the sphingosine backbone of palmitoylceramide were disrupted either separately or simultaneously by replacing the hydrogen with a methyl-group. when the lateral phase behavior of mixed bilayers containing cholesterol/sphingomyelin-rich domains was studied in the presence of the ceramide analogs, the 3o-methylated ceramide appeared to form a thermally stable, sterol-excluding gel phase with sphingomyelin, whereas the 2o-methylated ceramide failed in both thermal stabilization and sterol displacement. the doubly methylated analog was the poorest ceramide mimic. together with the possible steric effects induced by the methylations, the lack of 2nh h-bond donor function impaired ceramide membrane behavior to a greater degree than the lack of 3oh h-bond donor function. sugar-based surfactants are made from renewable resources using the ''green chemistry'' methods, are easily biodegradable and used in washing agents, cosmetics, and drug carriers. besides, there are attempts to use them as nonviral vectors in gene therapy. we studied the influence of new x-(alkyldimethylamonium)alkylaldonamide bromides (c n gab) with different chain lengths (n = 10, 12, 14, 16) on the thermotropic phase behavior of dppc, and dppc/chol bilayers by means of differential scanning calorimetry. the surfactants were added either to the water phase or directly to the lipid phase (a mixed film was formed). we analyzed the changes in the temperatures, enthalpies and shapes of the main phase transitions as a function of concentration. molecular modeling methods were also used. cytotoxicicity of the c n gabs was determined in the cell line l929 and a549. for cytotoxicity test, the cells were seeded in 96-well plates 1 ml of 2á10 6 cells/ml in the culture medium eagle'a or dulbecco with 2% calf serum, penicillin and streptomycin was deposited into each plates. the cells were treated with various doses of surfactants and incubated. the minimal concentration which was toxic to approximately 50% of cells was taken as tccd 50 . this work was supported by grant n n305 361739. membrane fusion is ubiquitous in life requiring remodeling of two phospholipid bilayers. as supported by many experimental results and theoretical analyses, merging of membranes seems to proceed via similar sequential intermediates. contacting membranes form a stalk between the proximal leaflets which expand radially into a hemifusion diaphragm (hd) and subsequently open to a fusion pore. direct experimental verification of the hd is difficult due to its transient nature. using confocal fluorescence microscopy we have investigated the fusion of giant unilamellar vesicles (guvs) containing fluorescent membrane protein anchors and fluorescent lipid analogues in the presence of divalent cations. time resolved imaging revealed that fusion was preceded by displacement of peptides and lipid analogues from the guv-guv contact region being of several lm in size. a detailed analysis showed that this structure is consistent with the formation of an hd. a quantitative model of the hemifusion equilibrium and kinetics of the growing hd was developed. bilayer tension could be shown to drive hd expansion and interleaflet tension was found to act as a counterforce, because the outer leaflets are compressed upon hd growth. the model and its predictions fit nicely with observations above. concentration effects of trehalose in the equivalent polarity of fluid popc bilayers c. nobre 2 , d. arrais 1 , j. martins 1,2 1 ibb -cbme, faro, portugal, 2 dcbb -fct, universidade do algarve, faro, portugal trehalose is an important disaccharide, formed by two units of glucose linked by a a-1,1 glicosidic bond. it is capable of replacing water molecules in the hydration shell of the phospholipid headgroups, in cases of extreme dehydration, by establishing hydrogen bonds with their -co and -po groups, preserving this way the membrane structure. the polarity gradient is a significant feature of lipid bilayers and is influenced by the amounts of water within this medium. it is therefore important to understand the effects of different concentrations of trehalose in simple model membranes. using the pyrene empirical polarity scale, we monitorized changes in the polarity values when varying trehalose concentration in the bounding aqueous phase. for lower concentrations (until 0.25 m), we observed a decrease in polarity, comparing with popc bilayers in pure water. for higher trehalose concentrations (above 0.5 m), the polarity values are indistinguishable from those popc in water. using the freeze and thaw technique we obtained the same results, except for the lower trehalose concentrations. general anesthetics are indispensible tools of daily surgery. yet, their molecular mode of action remains elusive. while one school favors specific (direct) interactions with proteins of the central nervous system, another school adheres to a nonspecific modulation of biophysical membrane properties. one of the strongest arguments against lipid theories is the absence of stereo-specific effects in model membranes, as opposed to their detection by electrophysiological measurements on ionchannels. we have combined x-ray scattering and molecular dynamics simulations on palmitoyl-oleoyl-phosphatidylcholine bilayers with fluorescence microscopy on live cells to study the effects of the stereoisomers of ketamine on membrane properties. we find significant effects of both enantiomers on the distribution of lateral pressures at clinically relevant concentrations, being more pronounced for s-(?)-ketamine. we further calculated the effect of the lateral pressure profile changes on the opening probability of an ion-channel using crystallographic information. the observed channel inhibition compares remarkably well with clinically observed effects of the enantiomers. we thus provide first evidence for a stereo-specific, but indirect effect of general anesthetics on ion-channels. dependence of gramicidin a channel lifetime on membrane structure obtained from x-ray scattering measurements horia i. petrache department of physics, indiana university purdue university indianapolis, in 46202, usa the activity of ion channels, in particular the lifetime of their conducting (open) state depends on the physical properties of lipid bilayers [1, 2] which in turn depend on lipid headgroup and acyl chain composition. in order to investigate this dependence, we have performed measurements of gramicidin a (ga) channel lifetimes in three different lipid series. in each series, the lipid headgroups were phosphatidylcholine (pc), phosphatidylethanolamine (pe), and phosphatidylserine (ps), while the acyl chains consisted of symmetric monounsaturated di(18:1), mixed (16:0)(18:1), and methylated di(16:0-4me). in order to minimize the effect of headgroup electrostatics, measurements where performed in 1m kcl salts. we show how ga lifetimes depend on headgroup and acyl chain composition and on structural parameters determined by x-ray scattering. for the lipids considered, ga lifetimes cover a range from 0.7 seconds in the dope lipid to 18 seconds in dphps. in this range, we find a gaussian dependence of ga lifetime on bilayer thickness, consistent with hydrophobic matching models. we discuss different aspects of channel-lipid interactions and to what extent measurements of ga lifetime in binary mixtures are consistent with measurements in pure lipid systems. [ the aim of the studies was to determine the effect of chlorogenic acid (cga), which is the main constituent of plant extracts, on properties of the model membranes. its effect was studied on temperature of the main phase transition of various lipids with and without presence of cholesterol, using the differential scanning calorimetry (dsc) method and the fluorimetric method. in particular, the degree of packing order of the hydrophilic phase of liposomes was determined using the laurdan and prodan probes, and fluorescence anisotropy of the hydrophobic phase with the probes dph and tma-dph. it had also been studied the effect of chlorogenic acid on the structure and capacity of black lipid membranes (blms), formed of egg lecithin and lipids extracted from erythrocytes. the results obtained indicate that cga lowers the main phase transition temperature slightly, without changing the fluorescence anisotropy in the hydrophobic part of the bilyer, and causes a decease in the packing order of the hydrophilic phase. by monitoring the capacity during blm formation we have found that the presence of chlorogenic acid accelerates the process of lipid self-organization into a bilayer, and increases stability and life of the blms. however, the was no effect of cga on specific capacity of the membranes, and thus on thickness of the liposome membrane hydrophobic layer. this work was sponsored by the ministry of science and education, scientific project no. n n312 422340 and n n304 173840. many examples have recently been found where biological processes in the lipid bilayer are affected by the changes in the physicochemical properties of the membrane, e.g. the local curvature, the membrane tension and certainly the membrane structure. it has been shown that the activity of polyene antibiotics is strongly correlated to the phase diagram in a membrane composed of a mixture of popc and ergosterol or cholesterol (j membrane biol, 237:41-49, (2010)). it is known that polyene action is quite sensitive to the type of sterol in the membrane, which enables its medical use, mainly as antifungals. it has been proposed that this selectivity of the drug to fungi is related to structure modulation by the sterols (see for example, biophys. j., 85, 2323, (2003)) and therefore the correlation found could be due to structural differences between popc/ergosterol and popc/ cholesterol along the corresponding phase diagrams. to investigate this, molecular dynamics simulations of the above mixtures along their phase diagrams were performed. it was found that there are indeed marked differences in structure along the phase diagrams, but for the sterol-sterol distribution function. an analysis of the behavior of this observable and the implications on polyene action is discussed. acyl transfer from lipids without enzyme catalysis: a new paradigm for membrane protein ageing? john sanderson, catherine pridmore, jackie mosely, paul yeo durham university, department of chemistry, durham, uk membrane proteins are recycled in cellulo with half-lives ranging from minutes to days. in other systems, such as enveloped viruses, proteins may equally remain membranebound for periods of days. it is therefore of interest to examine the behaviour of proteins in model membranes over extended periods in order to determine the long-term stability of the mixed systems, both in kinetic terms (attainment of equilibrium states) and chemical terms (reactivity). the reactivity of proteins towards membranes has been examined using the peptide melittin as a model for membrane proteins. acyl transfer from phospholipids to the peptide was found to occur over a period of several days, in the absence of any enzyme catalysis. transfer was detectable after 2 days and reached 50% conversion in 8 days. using tandem mass spectrometry approaches, the sites of melittin modification were localised. these sites included the side chain of lysine, opening the possibility that this residue may be modified in any membrane protein where this residue has an appropriate disposition. these observations challenge preconceptions concerning the membrane as an inert medium and highlight potential new mechanisms for membrane protein ageing. interaction of poly(l-arginine) with negatively charged bilayers studied by ft-ir spectroscopy christian schwieger, alfred blume martin-luther-university, halle-wittenberg, institute of chemistry, van-dankelman-platz 3, 06108 halle / saale, germany e-mail: christian.schwieger@chemie.uni-halle.de oligoarginine residues attached to macromolecules are known to facilitate the transport through lipid membranes. since the mechanism of this transport is still unclear, the effect is often called ''arginine magic''. we studied the interaction of poly(larginine) (pla) of different molecular weight with negatively charged lipid bilayers. we have shown by calorimetric and monolayer techniques that the interaction is due to a combination of electrostatic and hydrophobic forces. now we present an ft-ir spectroscopic study to reveal the effect of pla binding on membrane organisation and peptide conformation. we will show that pla binding reduces the lipid miscibility of negatively charged (pg or pa) and zwitterionic (pc) lipids within the bilayer. from the shift of the c=o stretching vibration we deduce that arginine side chains penetrate into the hydrophobic/ hydrophilic interface and replace hydration water molecules. the binding reduces the rotational freedom of the lipid molecules, as could be shown by an analysis of the ch 2 -streching vibrations. pla binds in a b-sheet conformation to pg or pa gel phase membranes whereas its structure in bulk is random coil. the shift of the guanidyl vibration frequencies shows that also hydrogen bonds contribute to the pla -lipid interactions. neutron scattering studies of model membrane as a function of hydration and temperature federica sebastiani 1,2 , alessandra filabozzi 1 and giovanna fragneto 2 1 dipartimento di fisica, università degli studi di roma ''tor vergata'', roma, italy, 2 institut laue-langevin, grenoble, france cell membranes carry out highly specialised functions in living materials. the composition of bacterial membranes is essential to understand the mechanism of action of antimicrobial peptides. in order to understand the role of the various components contributing to the overall behaviour, we have reproduced the membrane of bacillus subtilis and carried out neutron diffraction studies on d16 (small momentum-transfer diffractometer) and d17 (reflectometer used as a diffractometer), at ill. an ordered and homogeneous sample has been obtained by using the widely studied dmpc. the measured d-spacing of dmpc as a function of the relative humidity (rh) is related to the physical and chemical conditions affecting the sample. consequently the reliability of the humidity chamber, which has been previously upgraded, has been stated. moreover, the most suitable preparation technique has been set up. in order to investigate the component roles within bacillus subtilis membrane, three samples of phospholipids were prepared (with pope, popg and cardiolipin). neutron diffraction measurements, performed at controlled rh and temperature, suggested the presence of interesting phase transitions or coexistence of phases. the rupture of membrane vesicles near solid surfaces annamá ria taká ts-nyeste, imre deré nyi department of biological physics, eö tvö s university, h-1117 budapest pazmany p. stny. 1/a, hungary the behavior of lipid membranes near solid surfaces has a great significance both in medicine and in technology. in spite of the widespread use and study of such membrane phenomena, their theoretical analysis is rather scarce. our main goal here is to understand the process during which membrane vesicles first adhere to solid surfaces, then rupture (or go through a series of transient ruptures) due to the mechanical tension induced by the adhesion, and finally spread along the surface forming a supported lipid bilayer. in our theoretical description we simultaneously consider the dynamics of spontaneous pore opening and closing; volume loss via leakage through the pores; and the advancement of the adhesion front. all these processes are supposed to follow an overdamped dynamics and coupled to each other through membrane tension. our numerical simulations reveal that the rupture process consists of three well distinguishable phases: a fast initial volume loss; followed by a slow volume loss; ending with a final burst and surface spreading. the second phase can be skipped if either the first phase advances far enough or the third phase sets in early enough. the smaller the vesicle, the further the first phase can advance. the third phase can start earlier if either the surface is smooth enough, or the adhesion energy is large enough, or the line tension is small enough. when the second phase is not skipped the time needed for the rupture process can take very long with a large variance. in the realistic range of the material properties (line tension, bending rigidity) the process is qualitatively always the same, so the most decisive parameter remains the size of the vesicle: the smaller the vesicle the faster and easier it ruptures. (3)). we chose four plant-derived polyphenols (flavonoids and stilbenes) of documented biological activity to study their influence on lipid domain number, area, shape, and borderlength. we found that resveratrol elevated the number of domains per vesicle, decreased their area and markedly increased the total length of domain border without affecting domains' circular shape. surprisingly, no such effect was observed for piceatannol differing from resveratrol by one hydroxyl group only. neither genistein nor 8-prenylnaringenin changed the morphology of lipid domains significantly. the possible mechanism of resveratrol-induced effect on lipid domains' morphology could be its selective accumulation in the interfacial regions between liquid ordered and liquid disordered domains. putative cholesterol recognition amino acid consensus (crac) motif in hiv coreceptors cxcr4 and ccr5 mikhail a. zhukovsky, albrecht ott biological experimental physics department, saarland university, 66041 saarbruecken, germany we identified a cholesterol recognition amino acid consensus (crac) motif in transmembrane domain 5 (tmd5) of two g protein-coupled receptors (gpcrs), human chemokine receptors cxcr4 and ccr5, coreceptors of human immunodeficiency virus (hiv). we suggest that residues belonging to this crac motif are involved in cholesterol binding to cxcr4 and ccr5 that is responsible for cholesterol requirement for cxcr4 and ccr5 conformation and function and for the role that cell cholesterol plays in the cell entry of cxcr4-using and ccr5-using hiv strains. putative crac sequences involve residues v 214 /l 216 -y 219 -k 225 in cxcr4 and l 208 /v 209 /v 211 -y 214 -k 219 in ccr5. in cxcr4, crac motif is highly conserved across chordata species, whereas in ccr5, crac motif is less conserved. t curve describe quantitatively the interfacial landscape around the protein molecules and can be used for the distinction between the globular and idp states. the behavior of the t 1 and t 2 data showed that there are two reorientation types present for every protein solutions below 0°c, irrespective for the nature of the protein or the solvent composition. local field fluctuation and the bpp models were applied, which failed for the buffered protein solutions and for the idps dissolved in water. a main cause of the failure is the changing h in the analyzed temperature range. this case is valid for the solutions of idps and for buffered solutions of both protein types. another cause can be the active relaxation channels other than dipolar when ions of quadrupolar nuclei are present. ligand-induced disorder-to-order transition plays a key role in the biological functions of many proteins that contain intrinsically disordered regions. this trait is exhibited by rtx (repeat in toxin) motifs found in more than 250 virulence factors secreted by gram-negative pathogenic bacteria. we investigated several cyaa rtx polypeptides of different lengths ranging from 111 to 706 residues. we showed that the rtx proteins exhibit the hallmarks of intrinsically disordered proteins in the absence of calcium: they adopt premolten globule conformations and exhibit a strong timeaveraged apparent hydration, due in part to the internal electrostatic repulsions between negatively charged residues, as revealed by the high mean net charge. calcium binding triggers a strong reduction of the mean net charge, dehydration and compaction, folding and stabilization of secondary and tertiary structures of the rtx proteins. we propose that the intrinsically disordered character of the rtx proteins may facilitate the uptake and secretion of virulence factors through the bacterial secretion machinery. these results support the hypothesis that the folding reaction is achieved upon protein secretion and, in the case of proteins containing rtx motifs, could be finely regulated by the calcium gradient across bacterial cell wall. occupational exposure to heavy metals has been recognized to be a risk factor for parkinson's disease via metaltriggered deposition of alpha-synuclein (as) 1,2 . in the present work, al 3? induced conformational change and instant oligomerization of as have been studied using fret and fcs as main techniques. donor and acceptor were labeled in the c-terminal at positions a107c and a140c. the average lifetime of donor in the presence of acceptor increases with the increase of al 3? concentration, indicating as adopts a more extended conformation upon al 3? binding. the intrinsic tyr fluorescence rises sharply within the mixing dead time, reflecting an enhanced hydrophobicity of the tyr environment and a fast conformational change of as. al 3? also induces an immediate oligomerization of as as monitored by fcs. the diffusion coefficient of as changes from 85 ± 5 lm 2 /s as monomer state to 23 ± 5 lm 2 /s as oligomer state. the oligomerization is supposed to be induced by the ligand bridging of trivalent al ions. nearly 2% of human genes encode protein-kinases (pk), enzymes involved in cellular signaling and several other vital biochemical functions, which transfer phosphate groups from atp to specific target molecules, modifying their activity. [1] deregulated pk have been linked to numerous diseases including cancer and diabetes, making them attractive targets for drug design. [2] conformational transitions play a central role in regulating the phosphorylation activity. pk adopt an on state that is maximally active and one or more inactive states that shows minimal activity. [3] the similarity of the relatively rigid and largely conserved atp binding site makes the design of selective inhibitors binding to the active state very difficult. indeed some of the best cancer therapies available are based on inhibitors, as imatinib, that bind to inactive states peculiar to a small subset of pk (abl, c-kit and pdgfr in the case on imatinib). thus, understanding the atomic details of the active to inactive transitions in kinases has a great importance. here we study a particular active-toinactive transition of c-src, a fundamental proto-oncogene involved in cancer and metastasis, by using multi-microsecond long fully solvated molecular dynamics simulations, metadynamics and ptmetad calculations [4, 5] . the results, validated by mutagenesis, x-ray crystallography and binding kinetics, are suggestive of a functional role for the conformational transition. moreover, we were able to single out the most important residues affecting the conformational transition and to show that even a very conservative amino-acid substitution can have a dramatic effect on the conformational free energy landscape. the time-scales of protein folding events range over many orders of magnitude. in order to understand the complex folding mechanisms, peptides with well-defined secondary structure are often used as model systems as they may be regarded as smallest folding units of proteins. the formation of secondary structure elements occur on the nanosecond to low microsecond time scale. thus, stopped-flow techniques are too slow whereas pulsed laser techniques are capable to trigger folding processes in nanoseconds and to analyze faster folding events. we study ns-to-ls peptide dynamics by temperature-jump infrared spectroscopy. after initiation of a nanosecond temperature jump, the spectral response is monitored at single wavelengths in the amide i region reflecting the dynamics of the peptide backbone. relaxation rates are obtained. the helix-to-coil relaxation of polyglutamic acid is a multi-step process and requires more complex models than two-state kinetics. however, there are kinetic steps that are well described by single-exponential behavior and a two-state model. we demonstrate how equilibrium and time-resolved infrared spectroscopic data can be combined to deduce folding rates. unfolding and refolding studies using chemical denaturants have contributed tremendously to our understanding of the thermodynamics and kinetics of protein folding and stability. however, a major limitation of this approach lies in the large uncertainty inherent in the extrapolation of the free energy of unfolding in the absence of denaturant from free energy values measured at finite denaturant concentrations. here we show that this limitation can be overcome by combining multiple spectroscopic signals-including fluorescence, circular dichroism, and absorbance-recorded in a quasi-simultaneous and fully automated way at different wavelengths. we have optimised the number of wavelength values used, the integration time per data point, the increment in the denaturant concentration, and the weighting scheme applied for global data fitting. compared with the traditional approach based on the use of a single or a few wavelengths, we could thus improve the precision of the free energy value by an order of magnitude. we exemplify and validate this novel approach using representative, well-studied globular proteins and explain how it can be exploited to quantify subtle changes in membrane-protein stability which have thus far remained elusive. the rates of protein conformational changes are usually not only limited by external but also internal friction, however, the origin and significance of this latter phenomenon is poorly understood. it is often found experimentally that a linear fit to the reciprocal of the reaction rate as a function of the viscosity of the external medium has a non-zero 1 , the physical basis of pressure unfolding is still largely unknown. we report here a specific study of cavities contributions to the volume difference between unfolded and folded states (dv u ), using four single point mutants of staphylococcus nuclease (snase). each mutation is localised in a strategic position on the protein structure and was designed to change a large buried hydrophobic side chain into alanine, thus opening tunable cavities in the snase 3d structure. measuring hsqcs peaks intensities up to 2500 bar monitored the equilibrium high pressure unfolding and leads us to precise estimations of dv u for more than two-thirds of the 143 residues of each mutant. so-fast hmqc experiments 2 were also performed to measure folding and unfolding rates from 200 bar pressure jumps. high-pressure fluorescence experiments were done on six additional alanine mutants to complement the nmr study, allowing a more complete exploration of the local pressure sensitivity along the protein 3d structure. all these highly reliable measurements shed light on the real signification of the thermodynamic parameter dvu, and bring an unprecedented complex and heterogeneous picture at a residue level of the apparent two-state folding process of snase. determination of contributing factors to the volume change magnitude between unfolded and folded states (dv u ) is a longstanding question in the high-pressure field. we provide here new experimental and computational data using two wellcharacterized model proteins: notch ankyrin repeat domain (nank) and staphylococcal nuclease (snase). the repetitive nature of the nank protein was used to study influence of the protein size on dv u in a systematic way with a set of deletion mutants. high-pressure fluorescence data provided new evidences that neither peptide bonds hydration nor side chains differential hydration could be considered as major contributor to the measured dv u value. additional molecular dynamics (md) simulations rather suggested that the heterogeneous distribution of void volume in the folded states structures could explain the dv u variations among the nank deletion mutants. the specific issue of the void volume contribution to dv u values was studied using 10 cavity mutants in snase, allowing a large structural mapping of the alanine mutations on this globular protein. combination of x-ray crystallography, highpressure fluorescence, high-pressure nmr and md simulations provided a first clear determination of the void volume contribution to the dv u values. these results also bring an unprecedented complex and heterogeneous picture at a residue level of the apparent two-state folding process of snase. we expressed an ig domain (i27) and a 170-residue-long fragment of the pevk domain in order to investigate the effect of temperature and pressure on their conformation. ftir spectroscopy is a useful method for investigating the secondary structure of proteins. we analyzed the amide i band to obtain information on protein structure. fluorescence labeling was also used in some experiments. to generate high pressures, a diamond anvil cell was employed. the ftir and fluorescence spectra of the protein fragments were recorded across the pressure and temperature ranges of 0-1 gpa and 0-100°c, respectively. moderate changes were observed in the conformation of the pevk fragments in the explored range of the t-p plane, suggesting that the domain is a highly flexible, random-coil across the entire studied t-p range. by contrast, the i27 domain showed quite stable secondary structure. intrinsically disordered proteins participate in important regulatory functions in the cell, including regulation of transcription, translation, the cell cycle, and numerous signal transduction events. disordered proteins often undergo coupled folding and binding transitions upon interaction with their cellular targets. the lack of stable globular structure can confer numerous functional advantages, including, paradoxically, both binding promiscuity and high specificity in target interactions. nmr is unique in being able to provide detailed insights into the intrinsic conformational preferences and dynamics of unfolded and partly folded proteins, and into the mechanism of coupled folding and binding. the function of intrinsically disordered protein domains in transcriptional regulation and signaling will be described, with particular reference to the general transcriptional coactivators cbp and p300, the tumor suppressor p53, and the adenovirus e1a oncoprotein. the globular domains of cbp/p300 are targets for coupled folding and binding of disordered transactivation motifs of numerous transcription factors and viral oncogenes, which compete for binding to limiting amounts of cbp/p300. many intrinsically disordered proteins contain multipartite interaction motifs that perform an essential function in the integration of complex signaling networks. the role of multipartite binding motifs and post translational modifications in regulation of p53-mediated signaling pathways will be discussed. the early vascular network is one of the simplest functioning organs in the embryo. its formation involves only one cell type and it can be readily observed and manipulated in avian embryos or in vitro explants. the early vascular network of warm-blooded vertebrates self-organizes by the collective motility of cell streams, or multicellular ''sprouts''. the elongation of these future vascular network segments depends on a continuous supply of cells, moving along the sprout towards its tip. to understand the observed self-organization process, we investigate computational models containing interactions between adherent, polarized and self-propelled cells. by comparing the simulations with data from in vivo or simplistic in vitro experiments, we explore the role of active migration, leader cells, invasion of the ecm, and cell guidance by micromechanical properties of adjacent cell surfaces. boron neutron capture therapy (bnct) is a promising method for treating the highly fatal brain tumor; glioblastoma multiform. it is a binary modality; in which use is made of two components simultaneously; viz. thermal neutrons and boron-10. the biophysics of bnct is very complicated; primarily due to the complexity of element composition of the brain. moreover; numerous components contributes to the over all radiation dose both to normal brain and to tumor. simple algebraic summation cannot be applied to these dose components, since each component should at first be weighed by its relative biological effectiveness (rbe) value. unfortunately, there is no worldwide agreement on these rbe values. thermal neutrons were formerly employed for bnct, but they failed to prove therapeutic efficacy. later on; epithermal neutrons were suggested proposing that they would be enough thermalized while transporting in the brain tissues. however; debate aroused regarding the optimum source neutrons energy for treating brain tumors located at different depths in brain. insufficient knowledge regarding the rbe values of different bnct dose components was a major obstacle. a new concept was adopted for estimating the optimum source neutrons energy appropriate for different circumstances of bnct. four postulations on the optimum source neutrons energy were worked out, almost entirely independent of the rbe values of the different dose components. four corresponding condition on the optimum source neutrons energy were deduced. an energy escalation study was carried out investigating 65 different source neutron energies, between 0.01 ev and 13.2 mev. mcnp4b monte_carlo neutron transport code was utilized to study the behavior of these neutrons in the brain. the deduced four conditions were applied to the results. a source neutron energy range of few electron volts (ev) to about 30 kev was estimated to be optimum for bnct of brain tumors located at different depths in brain. simulation of mutation induction by inhaled radon progenies in the bronchial epithelium balá zs g. madas, á rpá d farkas and imre balá shá zy hungarian academy of sciences kfki atomic energy research institute, konkoly-thege mikló s ú t 29-33., budapest, h-1121, hungary radon is considered as the second most important cause of lung cancer after smoking. to understand the mechanisms leading from radon exposure to cancer formation is of crucial importance. this study focuses on the description of mutation induction by radon progenies in the bronchial epithelium. computational fluid and particle dynamics approach was applied to determine the radio-aerosol deposition distribution in the central airways. a numerical replica of a small fragment of the bronchial epithelium was prepared based on experimental data. microdosimetric computations were performed to quantify the cellular radiation burdens at the very site of deposition accumulation. a mutagenesis model was applied supposing that radiation induces dna damages and enhances the cell turnover rate. the results show that both considered mutagenic effects of densely ionising radiation contribute significantly to mutation induction and mutation rate depends non-linearly on exposure rate. furthermore, simulations suggest that the local maintenance capacity of the bronchial epithelium can be exhausted by chronic exposure to radon progenies with activity concentration characteristic of some uranium mines. the present work demonstrates possible applications of numerical modelling in radon related carcinogenesis studies. the neural crest is a group of cells found in all vertebrate embryos. it forms in the neural folds at the border of the neural plate and gives rise to a huge variety of cells, tissues and organs. one of the astonishing characteristic of neural crest cells is that they are able to migrate very long distances in the embryo. the neural crest has been called the ''explorer of the embryo'' as it is one of the embryonic cell types that migrate most during development, eventually colonizing almost every tissue. in this talk i will discuss our recent finding about neural crest migration. we have shown that neural crest cells, classically described as mesenchymal cells, migrate in large clusters cytokinesis relies on tight regulation of the mechanical properties of the cell cortex, a thin acto-myosin network lying under the plasma membrane. although most studies of cytokinetic mechanics focus on force generation at the equatorial acto-myosin ring, a contractile cortex remains at the poles of dividing cells throughout cytokinesis. whether polar forces influence cytokinetic cell shape is poorly understood. combining cell biology and biophysics, we demonstrate that the polar cortex makes cytokinesis inherently unstable and that any imbalance in contractile forces between the poles compromises furrow positioning. we show that limited asymmetric polar contractions occur during normal cytokinesis, and that perturbing the polar cortex leads to cell shape oscillations and division failure. a theoretical model based on a competition between cortex turnover and contraction dynamics accurately accounts for the oscillations. we further propose that blebs, membrane protrusions that commonly form at the poles of dividing cells, stabilise the position of the cleavage furrow by acting as valves releasing cortical contractility. taken together, our findings show that the physical properties of the entire cell are integrated into a finetuned mechanical system ensuring successful cytokinesis. collective motion of individual cells marks the onset of the transition to multicellularity in many microorganisms. this transition is often mediated by intercellular communication signals between cells. here, we show, in contrast, that the transition from single cell to collective motion in an ensemble of gliding bacterial cells can be understood as a dynamical selfassembly process of self-propelled rods. experiments were carried out with a mutant of the bacterium myxococcus xanthus moving by means of the a-motility system only and without undergoing reversals. the collective motion phase is confined to a monolayer and is characterized by the organization of cells into larger moving clusters. a transition to collective motion is detected in experiments by image analysis, that reveals a qualitative change of the cluster-size distribution at a critical cell packing fraction around 17%. this transition is characterized by a scale-free power-law cluster size distribution with an exponent 0.88. we provide a theoretical model for cluster formation of self-propelled rods that reproduces the experimental findings for the cluster size distribution. our findings suggest that the interplay of selfpropulsion of bacteria and volume exclusion effects of the rodshaped cell bodies is sufficient to explain the onset of collective motion and the related changes in the cluster statistics. despite much speculation on the existence of structurally distinct oligomeric species associated with the conversion of certain monomeric proteins into amyloid fibrils, it has not previously been possible to observe them directly or to relate them to any key mechanistic steps involved in the interconversion process. we have developed a novel application of singlemolecule intermolecular fret to investigate in unprecedented detail the aggregation and disaggregation of alpha-synuclein, the protein whose pathogenic deposition as intracellular lewy bodies is a characteristic feature of parkinson's disease. our study reveals that a range of oligomers of different size and structure are formed, even at physiologically relevant concentrations. interestingly, the resistance to degradation of the aggregated state of alpha-synuclein, which is a wellwe focused on the structure-dynamics interplay and showed how the fractal-like properties of proteins lead to such anomalous dynamics. we used diffusion, a method sensitive to the structural features of the protein fold and them alone, in order to probe protein structure. conducting a large scale study of diffusion on over 500 pdb structures we found it to be anomalous, an indication of a fractal-like structure. taking advantage of known and newly derived relations between vibrational dynamics and diffusion, we demonstrated the equivalence of our findings to the existence of structurally originated anomalies in the vibrational dynamics of proteins. more specifically, the time dependent vibrational mean square displacement (msd) of an amino acid is predicted to be subdiffusive. the thermal variance in the instantaneous distance between amino acids is shown to grow as a power law of the equilibrium distance. the autocorrelation function in time of the instantaneous distance between amino acids is shown to decay anomalously. our analysis offers a practical tool that may aid in the identification of amino acid pairs involved in large conformational changes. more recently, we studied the effect of the hydrodynamic interaction between amino acids using a zimm-type model. we computed the time-dependent msd of an amino acid and the time-dependent autocorrelation function of the distance between two amino acids, and showed that these dynamic quantities evolve anomalously, similar to the rouse-type behavior, yet with modified dynamic exponents. we also studied the dynamic structure factor s(k,t) of proteins at large wavenumbers k, kr g [ [1, with r g the gyration radius, that are sensitive to the protein internal dynamics. we showed that the decay of s(k,t) is dominated by the spatially averaged msd of an amino acid. as a result, s(k,t) effectively decays as a stretched exponential. we compared our theory with recent neutron spin-echo studies of myoglobin and hemoglobin for the rouse and zimm models of hydrodynamic friction. in addition, i will mention two other projects currently underway: (i) a new elastic network model that accounts for the tensorial aspects of protein elasticity and is a combination of stretch-compress springs and bond-bending energies. (ii) the unfolding of a protein under the exertion of a large pulling force. allosteric regulation of enzymatic activity is crucial for controlling a multitude of fundamental cellular processes. yet the molecular level details underlying regulation often remain poorly understood. here we employed single molecule activity studies to dissect the mechanistic origin of enzymatic activity regulation. as a model system we employed a lipase and measured its activity as a function of accessibility to surface tethered liposomes (1), which are known regulators of its activity. our results surprisingly revealed that the lipase oscillates between 2 states of different activity. we accurately quantified for the first time both the interconversion rates between activity states and the inherent activity of these states. based on these we calculated the energetic landscape of the entire reaction pathway and identified that regulatory interactions redistributed the probability to reside on preexisting enzymatic activity states but did not alter the activity of these states. our findings provide the missing link between conformational and activity substates supporting and represent the first direct validation of the textbook hypothesis of conformational selection for regulation of enzymatic activity to identify the potential targets of cgmp in arabidopsis plants we adopted a proteomic approach to isolate possible cgmp-binding proteins. purification of soluble cgmp-binding proteins was performed using cgmp-agarose-based affinity chromatography procedure. next eluted proteins were analyzed by sds-page which revealed ten bands. we focused the subsequent analysis on low-molecular peptides of 15, 16 and18 kda which were bound cgmp more intensively. after 2d-ief-page of the proteins isolated by cgmp-agaroseaffinity chromatography eight most abundant protein spots in the low-molecular area were visualized. these spots of interest were excised from the gel and in gel digested by trypsin. then tryptic peptides were analyzed by maldi-tof mass spectrometry and identified as isoforms of nucleoside diphosphate kinase (ndpk) from arabidopsis. thus, our data suggest that ndpk is a potential target of cgmp signaling in arabidopsis. dual-color fluorescence-burst analysis (dcfba) was applied to measure the quaternary structure and high affinity binding of the bacterial motor protein seca to the protein-conducting channel secyeg reconstituted into lipid vesicles. dcfba is an equilibrium technique that enables the direct observation and quantification of protein-protein interactions at the single molecule level. seca binds to secyeg as a dimer with a nucleotideand preprotein-dependent dissociation constant. one of the seca protomers binds secyeg in a salt-resistant manner, while binding of the second protomer is salt-sensitive. since protein translocation is salt-sensitive we conclude that the dimeric state of seca is required for protein translocation. a structural model for the dimeric assembly of seca while bound to secyeg is proposed based on the crystal structures of the thermatoga maritima seca-secyeg and the escherichia coli seca dimer. • dcfba is a flurorescence based single molecule technique that allows assessment of the stoichiometry of ligands bound to membrane receptors • dimeric seca binds asymmetrically to the protein-conducting membrane channel secyeg • monomeric seca binds secyeg but dimeric seca is required for protein translocation • protein translocation depends on receptor cycling of the dimeric seca if the dna charge is sufficiently neutralized by counter-ions, electrostatic interactions between helical charge patterns can cause attraction [1] . helix specific interactions also cause tilt, in one direction, between two dna fragments [1] . in braids and supercoils, this impetus to tilt breaks positive-negative supercoil symmetry. we show that these effects may cause spontaneous braiding of two molecules, lowering the dna pairing energy [2] . the pairing is more energetically favourable for homologues (same base pair text) than for nonhomologous pairs. this might explain pairing between only homologues observed in nacl solution [3] . also, we construct a simple model for a closed loop supercoil, including chiral electrostatic interactions. there are very interesting effects, for sufficient charge neutralization and groove localization of counter-ions. i.) positive super-coils are more energetically favourable than negative ones. ii.) a transition between loosely and tightly wound supercoils as one moves from negative to positive values of the supercoiling density. iii.) in positive super-coils the chiral interaction underwinds dna. [ von willebrand factor (vwf) is a large multimeric protein that is crucial for the force sensing cascade triggering primary hemostasis. it mediates binding of activated thrombocytes to injured epithelial tissue and serves as a transporter for coagulation factor viii. while it was shown that the hemostatic activity of vwf is affected by shear stress [1] , the exact impact that shear forces have on the inflammatory cascade remains unclear. it is assumed that hydrodynamic forces lead to partial unfolding of vwf, which in consequence exposes more binding sites. in order to observe shear-induced changes of the protein's functionality, we measure conformational changes of vwf under flow with fluorescence correlation spectroscopy (fcs). we aim to measure the degree of uncoiling of vwf multimers under various buffer conditions, e.g. in the presence of colloids, vesicles or platelets. as only large multimers show significant hemostatic activity we intend to monitor the molecular weight distribution of vwf. shifts in this distribution indicate various pathological conditions making our multimer analysis a fast diagnostical tool for vwf-related diseases. this will serve as a basis for studies of vwf binding to collagen, fviii, gpib, vesicles and membranecoated nanoparticles under shear flow. [ data on mechanical properties of medically important proteins located in neural junctions are very limited. contactins (cntn) and paranodin proteins, located in extracellular part of ranvier nodes, are important for proper brain wiring. here we study a new series of fniii modules from human cntn-1 and -4 using a single molecule afm force spectroscopy and advanced, all-atom steered molecular dynamics (smd) computer simulations. mutations in cntns are responsible for numerous brain disorders including autism or pathological development of odor maps. perhaps mechanical properties of individual fniii mutated protein modules are compromised, thus we address this problem. a comparison of our afm force spectra with those of reference proteins will be presented [1] [2] , and the molecular level interpretation fniii nanomechanics, based on our smd data will be given. we believe that these data should help to understand a role of cntn in regulation of sodium ion channels in both normal and autistic subjects. supported in part by polish ministry of education and science, grant no. n202 262038, the computational center task in gdansk and license for accelrys software. recent achievements in rational dna-motors engineering demonstrate the possibility to design nano-motors and nanorobots capable of performing externally controlled or programmed tasks. a major obstacle in developing such a complex molecular machine is the difficulty in characterizing the intermediates, the final products and their activity. typically, non in-situ gel and afm and in-situ bulk fluorescence methods are used. i will present two dna-motors recently developed and studied using in-situ single-molecule fluorescence resonance energy transfer (smfret), alternating laser excitation (alex) and total internal reflection fluorescence spectroscopy (tirf), and will demonstrate that these methods can improve the way we design, construct, measure and understand highly complex dna-based machines. a motor made of bipedal dna-walker, which walks on a dna track embedded on a dna-origami, capable of long walking distance and maintaining structural stability, will be presented. the motor is non-autonomous; it receives ss-dna fuel/anti-fuel commands from outside (as in shin & pierce, jacs, 2004). the motors assembly stages and singlemotor's walking steps are monitored using smfret. the second motor is based on published bipedal autonomous dna-motor (seeman, science 2009). it is characterized by coordinated activity between the different motor domains leading to processive, linear and synchronized movement along a directionally polar track. to prove that the motor indeed walks, the authors chemically froze the motor at each step and use a complicated radioactive gel assay. i will demonstrate that using single-molecule approach, we are able to directly and in-situ measure single-motor's movements in few simple experimental steps, and measure its structural dynamics and kinetics. translation by a single eukaryotic ribosome using single molecule total internal reflection fluorescence microscopy, we observed translation of a short messenger rna (mrna) strand by single eukaryotic ribosomes. the ribosome-mrnas complexes are fixed to a microscope coverslip through the mrna, and mrnas are located through fluorescently labelled oligonucleotides hybridized to it downstream start codon. because of the ribosome helicase activity, the double strand formed by the oligonucleotide and the mrna is opened while the ribosome translates this region of the mrna. thus, the loss of the fluorescence signal allows us to measure the distribution of translation speed of single ribosomes. careful attention was given to photobleaching for the data analysis. this experiment opens the door to the study of eukaryotic translation at the single molecule level. erythrocyte hyperaggregation, a cardiovascular risk factor, has been associated to high plasma concentrations of fibrinogen. using atomic force microscopy (afm)-based force spectroscopy measurements, we have recently identified the erythrocyte membrane receptor for fibrinogen, an integrin with a a3 or a3-like subunit [1] . after this, we extended the study to the influence of erythrocyte aging on fibrinogen binding [2] . force spectroscopy measurements showed that upon erythrocyte aging, there is a decrease of the binding to fibrinogen by decreasing the frequency of its occurrence (from 18.6% to 4.6%) but not its force. this observation is reinforced by zeta-potential and fluorescence spectroscopy measurements. knowing that younger erythrocytes bind more to fibrinogen, we could presume that this population is the main contributor to the cardiovascular diseases associated with increased fibrinogen blood content, which disturbs the blood flow. our data also show that sialic acid residues on the erythrocyte membrane contribute for the interaction with fibrinogen, possibly by facilitating the binding to its receptor. antimicrobial peptides are usually polycationic and amphiphilic with high affinity for bacterial membranes. in order to characterize their therapeutic potential it is crucial to disclose which properties of the peptide/lipids are important for target selectivity, and to examine the peptide structure and its association with lipid bilayers. in this work, first experiments have been carried out on a promising peptide called sb056, which might represent the basis for developing a novel class of antibiotics. with the goal of enhancing the activity of a new semi-synthetic sequence, two identical peptides (wkkirvrlsa) were assembled via a lysine-linker, carrying also an octanoyl-lipid anchor. a highly active compound was obtained, but its structure and mode-of-action remain unexplored. this dendrimeric peptide and its linear deca-peptide counterpart are being studied in parallel to highlight the relevant properties and differences between dendrimeric structure and the sequence. monolayer intercalation is investigated with microtensiometry, fluorescence spectroscopy is applied to study thermodynamics and kinetics of the binding process. circular dichroism, nmr and md simulations are employed with the aim of elucidating the 3d structure in the membrane-bound state. the capability of proteins to build structures via self-organization is fascinating biophysicists since decades. with the advent of single-molecule methods, namely fluorescence correlation spectroscopy (fcs) and fluorescence resonance energy transfer (fret), the process of complex formation is becoming accessible to direct observation. coronaviruses (cov) are enveloped positive-stranded rna viruses. for sars-cov, it was shown that coronaviruses encode a rna-dependent rna-polymerase (rdrp) build from non-structural protein 7 (nsp7) and non-structural protein 8 (nsp8). this hexadecameric nsp7-nsp8 complex is a hollow, cylinder-like structure assembled from eight copies of nsp8 and held together by eight nsp7 molecules [1, 2] . we are aiming at understanding the assembly process and conformational changes of the complex for the related feline coronavirus. first results implicate that nsp8 alone forms a dimer, where interchain fret is more efficient than intrachain fret. for the complex the results indicate that nsp7-nsp8 form a heterodimer which is different from sars-cov. our experiments highlight the potential of single-molecule fret for the study of protein complex formation. diffracted x-ray tracking (dxt) has been considered as a powerful technique for detecting subtle dynamic motion of the target protein at single molecular level. in dxt, the dynamics of a single protein can be monitored through trajectory of the laue spot from the nanocrystal which was labeled on the objective protein. in this study, dxt was applied to the group ii chaperonin, a protein machinery that captures an unfolded protein and refolds it to the correct conformation in an atp dependent manner. a mutant group ii chaperonin from thermococcus strain ks-1 with a cys residue at the tip of the helical protrusion was immobilized on the gold substrate surface and was labeled with a gold nanocrystal. we monitored diffracted spots from the nanocrystal as dynamic motion of the chaperonin, and found that the torsional motion of the chaperonin in the presence of atp condition was 10 times larger than that in the absence of atp condition. and uv-light triggered dxt study using caged atp revealed that the chaperonin twisted counterclockwisely (from the top view of chaperonin) when the chaperonin closed its chamber, and the angular velocity from open to closed state was 10 % faster than that from closed to open state. peptides or proteins may convert (under some conditions) from their soluble forms into highly ordered fibrillar aggregates. in vivo such transitions can lead to neurodegenerative disorders such as alzheimer's disease. alzheimer's disease is characterised by the extracellular deposition of abeta peptide in amyloid plaques, and the intracellular formation of neurofibrillary tangle (nft) deposits within neurons, the latter correlating well with disease severity. the major constituent of nft deposits are paired helical filaments (phf) composed of a microtubule-associated protein known as tau. studying the process by which tau forms these large aggregates may be an essential step in understanding the molecular basis of alzheimer's disease and other tauopathies. we have applied a two-colour single molecule fluorescence technique, and single molecule intermolecular fret measurements to study the soluble oligomers of tau which are formed during the aggregation and disaggregation of phf's. the neuronal protein alpha-synuclein is considered to play a critical role in the onset and progression of parkinson's disease. fibrillar aggregates of alpha-synuclein are the main constituents of the lewy bodies that are found in the brains of parkinson patients. however, there is growing evidence suggesting that oligomeric aggregates are significantly more toxic to cells than fibrillar aggregates. very little is known about the structure and composition of these oligomeric aggregates. we present results using single-molecule photobleaching approaches to determine the number of monomeric subunits constituting the oligomers. our results show that the oligomers have a narrow size distribution, consisting of *13-20 monomers per oligomer. fluorescence correlation spectroscopy data confirm the narrow size distribution and additionally indicate a very loose packing of the oligomers. in combination with bulk fluorescence spectroscopy results of tryptophan containing mutants of alpha-synuclein, we present a structural model for the alpha-synuclein oligomer. gold colloids are widely used for in vitro and in vivo imaging. compared to the traditional optical tags sers-coded nanoparticles show a narrow emission bandwidth with structured spectra typical of the molecule used, a wider excitation bandwidth, higher emission intensity, a better photo-stability, and a lower toxicity. this is why in cancer therapy, besides being considered good tools for the delivery of anti-tumor drugs, aunp can be also good optical tags for the analyses of both np localization by laser scanning microscopy and the process of drug release inside the cells by raman. in our work we used 10 nm diameter aunp loaded with rhodamine 6g, a molecule with a high raman and fluorescence efficiency, and with a chemical structure similar to doxorubicin, the antitumoral drug used in our system. the data showed that aunp are internalized by cells and sers can be performed. 10 nm and 60 nm diameter aunp loaded with doxorubicin were incubated at different time points with a549 cell line (human adenocarcinomic alveolar basal epithelial cells). only 60 nm aunp showed intense raman emission typical of the doxorubicin phonon transitions. in recent years biomedical applications of diamond nanoparticles have become of significant interest, which rises questions of their biocompatibility and mechanisms of interactions with cells. the aim of this study was to compare the effect of nonmodified diamond nanoparticles (dnps) and dnps modified by the fenton reaction on human endothelial cells. dnps (\10 nm particle size, sigma) were modified by the fenton reaction introducing surface -oh groups. immortalized human endothelial cells (huvec-st) were incubated with 2-100 lg/ml dnps in the optimem medium. diamond nanoparticles modified by the fenton reaction had smaller hydrodynamic diameter estimated by dynamic light scattering and the surface potential (zeta potential) measured using laser-doppler electrophoresis. they were more cytotoxic as evaluated by the mtt reduction assay. dnps augmented generation of reactive oxygen species in the cells, estimated by oxidation of 2',7'-dichlorofluorescin, the effect being higher for the fenton-modified dnps after 48-h incubation. cellular production of nitric oxide, estimated with daf-fm, was also affected by dnps; after 72h, fentonmodified oh, in contrast to non-modified diamond, decreased no production. diamond nanoparticles affected also the cellular level of glutathione and activities of main antioxidant enzymes (superoxide dismutase, catalase, glutathione peroxidase, glutathione reductase and glutathione s-transferase). we aim at investigating how photoreactions of proteins can be controlled by means of intense thz radiation tuned in resonance to specific vibrational modes, much in analogy to coherent control experiments conducted by fs nir laser pulses [1] . for this we will combine a time-resolved ir difference spectroscopic setup with uniquely intense, tunable narrow bandwidth thz radiation (3 -280 lm) at the ps beamline of the thz free electron laser felbe. these experiments will be performed on bacteriorhodopsin (br) which is the sole protein of the purple membrane of the archaebacterium halobacterium salinarum [2] . upon illumination, the chromophore retinal isomerizes around the c 13 -c 14 double bond [3] and br pumps a proton from the cytoplasmic to the extracellular side. this proton gradient is used by the bacterium to drive photosynthetic atp production under low oxygen tension [4] . in our experiment, the photoreaction is initiated by a visible laser pulse as in standard experiments, but then the sample will be irradiated by a thz pulse from the free electron laser tuned into resonance with low-energy vibrational modes which is supposed to influence the photoreaction [1] . such vibrational control will be monitored by time-resolved ftir spectroscopy using the step-scan technique [5] . liposomes are increasingly studied as nanoscale drug delivery systems and biomembrane models. however the exact structure dynamics and mechanical behavior of liposomes is little known. atomic force microscopy (afm) is a powerful tool to characterize nanoscale morphology and enables the mechanical manipulation of submicron-sized vesicles. a drawback of afm, however, is that liposomes may flatten and rupture on substrates to form patches or supported planar bilayers (spb). our aim was to obtain better understanding of factors affecting liposomes on substrates and find experimental conditions at which liposomes preserve their structural integrity. in the presence of divalent cations dppc liposomes formed spb on mica. vesicles sedimented subsequently preserved their integrity and showed stronger attachment to spb. in addition to cross-bridging lipid head groups, divalent cations influence the surface charge of liposomes, thereby modulating liposome-substrate and liposome-liposome interfacial interactions. preserved vesicles stabilized by divalent cations may provide a unique experimental system for studying membrane-protein interactions. the influence of e.g. ph and ionic strength on various chromatographic bead -biomass combinations. to analyze the force curves, possible elastic contributions, e.g. from deforming cellular membranes, have to be decoupled from the interaction forces. then, bead-biomass interactions will be modeled using (extended) dlvo theory and resulting data can also be compared to real-life eba processes. the project aims for a better understanding of the interaction forces in chromatography and might help to improve the process quality of eba. long-term non-invasive in vivo monitoring of the survival, migration, homing and fate of transplanted cells is of key importance for the success of cell therapy and regenerative medicine. tools for in vivo magnetic resonance (mr) imaging of labeled cells are therefore being developed. we have prepared superparamagnetic iron oxide nanoparticles by the coprecipitation of fe(ii) and fe(iii) salts and oxidation. to stabilize the particles and to facilitate their internalization by the cells, the nanoparticles were coated with several novel low-and highmolecular weight compounds including d-mannose, poly(llysin), poly(n,n-dimethylacrylamide) and dopamine-hyaluronate conjugate. the surface-modified magnetic nanoparticles were thoroughly characterized by a range of physico-chemical methods, which proved the presence of the coating on the particles. the particles were then investigated in stem cell experiments in terms of real time cell proliferation analysis, viability, labeling efficiency and differentiation. the iron oxide concentration of the labeled cells was assessed using mr relaxometry. the advantages/disadvantages of particular iron oxide coatings will be discussed and the optimal coating suggested. excellent contrast was achieved by labeling the cells with dopamine-hyaluronate-coated nanoparticles. support of the as cr (no. kan401220801) is acknowledged. in the last decades the interest towards the fabrication of innovative bio-sensors with improved sensitivity and reliability for medical-diagnostics applications has been constantly risen. among the different techniques, microfluidic systems are playing a major role. in order to detect extremely low concentrations of biomolecules (pm and fm), attention should be placed on the controlled, selective functionalization of micro-and nano-channels. in this work we propose a new approach to functionalize gold patches inside fluidic channels. we start from self-assembled monolayers (sams) of thiolated molecules on a gold electrode deposited inside the channel. then, by using an electrochemical approach [1, 2] we remove molecules from the sam at selected locations, by applying a negative voltage to the electrode. the newly exposed gold surface can be re-functionalized by using a thiolated biomolecule (i.e an antibody) capable to bind specific proteins flowing inside the channel. the cycle can be applied to other electrodes in the microfluidic system, creating a multiplexing device which, as we will show, can differentially measure ionic current flows in different channels. optically actuated micromanipulation and micro-probing of biological samples are increasingly important methods in the today's laboratory. microbeads as probes are the most commonly used tools in this field, although the only manipulative motion they allow is translation. we present polymerized 3d microstructures which can also be used for optical micromanipulation with more degree of freedom than microbeads. the two-photon polymerization (tpp), based on focused fs laser beam into appropriate photopolymers is a powerful method to build structures of arbitrary complexity with submicrometer resolution. the presented tools have the advantage of being capable of twisting and rotational manipulative motion, and also that the position of biological manipulation and optical trapping is spatially separated. different manipulative interfaces, the positioning stability and surface activation of the manipulators will be discussed. the potential application of multiplexed quantum dot labeling, mqdl, in clinical detection, prognosis and monitoring therapeutic response has attracted high interests from bioengineers, pathologists and cancer biologists. mqdl is superior comparing with conventional organic dye staining in its narrow emission bandwidths, wide signal dynamic ranges, high detection sensitivity, and low noise to signal ratios. however, the majority of the mqdl application has been limited to identification of specific cell type or cancer subtype and the improvement in labeling methodology. in this study, we focused on simultaneous detection and analysis of 5 proteins in the c-met activation pathway, i.e. rankl, vegf, nrpln-1, p-c-met and mcl-1, which are known to be associated with human prostate cancer progression and metastasis. two experimental systems were analyzed: 1) fixed xenograft tissues from an established ltl313 castration resistance human prostate cancer or crpc model; and 2) clinical prostate tissue specimens from localized cancer and bone metastasis. in the presentation we will report our experience in 1) the mqdl protocol optimization for the sequential reactions of individual primary antibody, the biotinylated secondary antibody and streptavidin-coated qd conjugate with nuclear dapi staining; and 2) the multiplexed image catching, image unmixing, and subsequent per cell base quantification. for future multi-specimen analyses and validation, we will introduce a high throughput vectra image analysis system. carbon nanotubes (cnts) 1 are already quite popular among many scientific and technological disciplines 2 . in recent years they have been targeted for biotechnological and medical applications. in this work we have investigated the nanostructural self assemblies of biological lipid molecules in presence of cnts. advantage of using highly aligned cnts 3 for this purpose being the possibility of studying the interactions of lipid molecules on the macromolecular surface as well as in the confinement of aligned cnts. we have observed various lyotropic nanostructures that are found for corresponding lipids in the bulk under dry and hydrated conditions. 4 nanostructural studies were mainly performed using small and wide angle x-ray scattering techniques. this work is crucial for designing the nano-micro-fluidic architectures and supported model membranes where both -functionalization of cnts and nanostructural assembling of lipids, could be employed simultaneously. alternative of the commonly used measuring tools in protein research and medical diagnostics. thazolidone derivatives are novel synthetic compounds possessing various biological activities. we selected three such compounds les-3120, 3166, 3372 which passed national cancer institute in vitro tests. annexin v/pi and dapi staining, dna electrophoresis in agarose gel, and western-blot analysis using specific antibodies against 30 cellular proteins involved in apoptosis were applied to study molecular mechanisms of tumor cell death induced by these compounds. it was found that molecular targets of thiazolidones in target cells strongly depend on structure of their side groups: les-3120 containing isatine fragment, activated caspase-8 involved in receptor-mediated apoptosis, while les-3166 possessing benzthiazol residue, induced mitochondrial apoptosis mediated by caspase-9, and les-3372 which has a unique chlorine atom in side chain, also led to mitochondrial apoptosis mediated by aif (apoptosis-inducing factor). to increase anticancer potential of these molecules, in silico study was performed and most active groups of les-3120 and les-3372 were combined into one molecule. in vitro studies showed that such hybrid molecule called les-3661 possessed 10 times higher anticancer potential (ic 50 =1 lm) comparing with initial compounds. we report on the synthesis and characterization of vaterite microcontainers for controlled drug release. moreover, we present experiments on possible release strategies of encapsulated substances via recrystallization, ph controlled, or by desorption methods. vaterite spherical particles were fabricated with controllable average sizes from 400±12nm till 10±1hm. we considered two ways of functionalization of the containers: encapsulation of the substances during the vaterite synthesis or their adsorption onto the prepared particles. as model experiments, vaterite containers, encapsulating rhodamine 6g, were imaged by two-photon microscopy, showing dye release into the aqueous medium due to recrystallization to calcite within 3 days. differently, in ethanol only small amounts of the encapsulated markers were diffusion released after one week. the release mechanisms can be further controlled by covering the microcontainers with additional polymer layers to increase diffusion and recrystallization time. a change of the ph from neutral to acid conditions leads to the destruction of the vaterite matrix followed by a quick release of the encapsulated materials. these flexible control mechanisms make this system an interesting candidate for pharmaceutical applications. magnetic nanoparticles (np) in combination with therapeutic molecules represent one of most promising methods for targeted drug delivery. one of major current limitations of magnetic drug targeting is to achieve efficient concentration of magnetic carrier-drug complexes at the targeted sites due to poor mobility of nanoparticles in tissue structures. interstitial delivery is hindered by microscopic extracellular matrix, which represents a major barrier for nanoparticles motilities. in order to achieve efficient magnetic drug targeting it is crucial to know particle mobility in a given in vivo environment as well as to apply magnetic field having appropriate field gradient which drags magnetic nps. we used gel magnetophoresis in order to measure motilities of different magnetic nps (co-ferrite, c-fe 2 o 3 ) in agarose gel. numerical modeling using fem method was used to determine appropriate settings of magnets, which generate sufficient magnetic field gradient. further, we used the numerical modeling to evaluate the magnetic force on the nps for different geometries. we obtained that one of crucial factors which determines final mobility in tissue is formation of larger aggregates of nanoparticles under physiological conditions and interaction of nanoparticles with surrounding matrix. defining the forces required to gate mechanosensitive channels in mammalian sensory neurons kate poole and gary lewin department of neuroscience, max delbrueck center for molecular medicine, robert-roessle str 10, 13125, berlin-buch, germany our sense of touch and mechanical pain is based on mechano-electrical transduction (met) at the terminal endings of subsets of dorsal root ganglion (drg) neurons innervating the skin. to quantify the stimulus strengths required to gate mechanosensitive channels in these subsets of neurons, we developed an approach using microstructured surfaces. the drg neurons are grown on laminin-coated pdms pillar arrays, mechanical stimuli are applied by deflecting individual pili and the deflection is monitored using light microscopy. as the pili behave as light-guides, the center of each pilus can be determined from a fit of the intensity values, allowing detection of movements of a few nanometers. the response to such stimuli is monitored using whole-cell patch-clamp. pili deflections of 10nm can gate the rapidly adapting-current in mechanoreceptor cells, while deflections above 150nm are required for gating of slowly adapting-currents in nociceptors. smaller stimuli are required to generate currents via pili deflection (10nm) vs neurite indentation (70-100nm), suggesting that gating occurs at the cell-substrate interface. we have also characterized the met currents present in n2a cells which we show are modulated by the substrate to which the cells are attached. enhanced stimulation of toll-like receptor 9 via immunostimulatory nanoparticles jan rother, anna pietuch, andreas janshoff georg-august university gö ttingen, institute of physical chemistry, tammanstr. 6, d-37077 gö ttingen, germany e-mail: jrother@gwdg.de among the toll-like receptor family (tlrs), the tlr9 has been subject of intensive research because of its predominant localization in the lysosomes of immune cells and its ligand rendering it a potential candidate for immunotherapy of autoimmune diseases and cancer. additionally, a use as an adjuvant in vaccination is aimed using synthetic cpg-oligodeoxynucleotides (cpg-odn's). albeit, immunostimulatory cpg-odns already showed promising results in animal experiments and clinical trials, several groups found that tlr9 is also expressed by tumor cells. first experiments show that activation of tlr9 displayed by cancerous cells leads to a decreased apoptosis rate and proliferation posing unpredictable threat to tumor patients exposed to cpg-odns. therefore, detailed knowledge about the impact of cpg-odns on cancer cells is inevitable for a save use in pharmaceutics. herein, we describe a sophisticated way to address tlr9 in cancer cells using cpg-odn functionalized ''superparamagnetic'' mno-and c-fe 2 o 3 -nanoparticles (nps) to stimulate tlr9 in a549 cells. analysis of impedimetric measurements revealed a cytotoxic effect of the mno-nps. cells treated with immunostimulatory fe 2 o 3 -nps showed an increased micromotility as well as a higher long-term correleation of the impedance signal. biomedical diagnostics like high-sensitivity, single-molecule study, easy sample preparation. furthermore, sers allows to conduct non-invasive studies of conformations of the molecules without destruction of living cells, i.e. in vivo [1] . this work presents a sers study of cytosolic hemoglobin (hb c ) using silver nanoparticles (agnps). the hb c was isolated from cytoplasm of red blood cells taken from rat erythrocytes and diluted. agnps were prepared by developing leopold and lendl method [2] . three types of colloids were prepared at various temperatures (25, 40 and 60°c). the resulted agnps were characterized by uv-vis-, ftirspectroscopy, dls and tem. reduction of ag ions leads to the formation of predominantly spherical agnps but also silver nanorods, faceted and aggregated agnps in small quantities with a surface plasmon resonance band in the range of 413 -445 nm. for agnps synthesized at 25°c, for example, a bimodal size distribution was observed (about 7 and 45 nm medium sizes, respectively). sers measurements were optimized for each type of agnps. it was demonstrated that agnps gave strong raman enhancement from hb c and types of sers spectra differ from each other. nano-zno is characterized by unique properties, low toxicity and high biocompatibility instead of a lot of others nanomaterials. for this fact nanoparticles of zno have great potential for applications in biosystems, for example biolabeling, biosensoring, delivery systems and others, which can be used in genetics, pathology, criminology, safety of food and many other industries. for these bioapplications are necessary surface modifications, which can made to the nanostructures to better suit their integration with biological systems, leading to such interesting properties as enhanced aqueous solubility, bio-recognition or applicability for biological systems. for synthesis of zno nanoparticles in aqueous solution we used 11-mercapto-undecanoic acid (mua) as stabilizing agent. the coating of nanoparticles with mua could allow their solubility in the water and the binding through carboxyl groups present in its structure. we defined the optimal ph for mua modificated nano-zno solubility and their ability interaction with positive charges. we studied the optical properties of pure and surface modificated nanoparticles and their conjugates with cytochrome c and also the effect of ph on the interaction between nano-mua and horse cytochrome c. the permeation of water soluble molecules across cell membranes is controlled by channel forming proteins and particularly the channel surface determines the selectivity. an adequate method to study properties of these channels is electrophysiology and in particular analyzing the ion current fluctuation in the presence of permeating solutes provides information on possible interactions with the channel surface. as the binding of antibiotic molecules in the channels of interest is significantly weaker than that of preferentially diffusing nutrients in substrate-specific pores, the resolution of conductance measurements has to be significantly increased to be able to resolve the events in all cases. due to the limited time resolution, fast permeation events are not visible. here we demonstrate that miniaturization of the lipid bilayer; varying the temperature or changing the solvent may enhance the resolution. although electrophysiology is considered as a single molecule technique, it does not provide atomic resolution. molecular details of solute permeation can be revealed by combining electrophysiology and all atom computer modeling. [ novel functionalized nanocomposites (nc) were designed and synthesized on the basis of polymeric surface-active oligoelectrolytes. the developed technology permits controlling: 1) quality and quantity of structural blocks of nc, and size unimodality; 2) branching at specific sites in polymer chain of nc; 3) providing nc with reactive chemical groups; 4) covalent conjugation of specific bio-targeting molecules. provided bioactive elements were: a) specific anticancer drugs, antibiotics, alkaloids; b) dna and sirna; c) immunoglobulins and lectins; d) lipids and amino acids; e) polyethylene glycol. fluorescent, luminescent, super-paramagnetic, or x-ray detectable compounds were also incorporated in nc to make them detectable and measurable. biocompatible nc possessing low toxicity towards mammalian cells in vitro and in vivo (mice) were created. they were effective in delivery of: 1) drugs (doxorubicine and antibiotics) for chemotherapy in vitro and in vivo; 2) dna for transfection of mammalian, yeast and bacterial cells; 3) protein antigens for animal immunization and specific lectins for targeting apoptotic cells. these and other approaches in application of developed nc and nanobiotechnologies are considered. this work was supported by stcu grants #1930, #4140, #4953. in the anti-cancer drug delivery domain, nanotechnologies are a promising tool, providing a good tissue distribution and a low toxicity. drug delivery vehicles relying on solid nanoparticles have been proposed, among which diamond nanoparticle (size\20 nm) is a very promising candidate [1] . we have investigated the delivery of sirna by nanodiamonds (nd) into cells in culture, in the context of the treatment of a rare child bone cancer (ewing sarcoma), by such a gene therapy. sirna was bound to nds after nds coating by cationic polymers, so that the interaction is strong enough to pass the cell membrane without loss of the drug and does not prevent its subsequent release. the cellular studies showed a specific inhibition of the gene expression at the mrna and protein level by the nd vectorized sirna. we also uses the fluorescence of color center created in the nanodiamonds [2] to monitor the release of fluorescently-labeled sirna in the intracellular medium. this technique brings a quantitative insight in the efficiency of sirna to stop cell proliferation. considering the success of the cell model we recently started the drug delivery in tumor xenografted on nude mice. silica nanoparticles are stable aqueous suspension of condensed siloxane nanocomposites, having an average diameter between 10 and 100 nm. particles containing organic functional groups on their surface are called organically modified silica nanoparticles (ormosil). due to the various chemical and physical properties of the surface groups, ormosil nanoparticles may have an enormous variety of biological applications, such as in vivo bioimaging, non-viral gene delivery or targeted drug delivery. our aim was to synthesize both void and fluorescent dye doped amino functionalized ormosil nanoparticles through the microemulsion method and use them for gene delivery. the obtained nanoparticles have been characterized by transmission electron microscopy and dynamic light scattering. furthermore, the nanoparticles have been investigated to exploit their transfection efficiency and the possible toxicity caused by surfactants used in the synthesis. the transfection efficiency was tested on various cell cultures. our further aim is the in vivo transfection of salivary glands using ormosil nanoparticles. our work has shown that the nanomedicine approach, with nanoparticles acting as a dna-delivery tool is a promising direction for targeted gene therapy. in vivo amperoetric cells for detection of fast diffusing, physiologically important small molecules lívia nagy, bernadett bareith, tü nde angyal, erika pinté r, gé za nagy university of pé cs, pé cs, hungary h 2 s is a naturally occurring gas that is toxic in high concentration. it exists also in different tissues of living animals sometimes in concentrations as high as 20 lm. it is generally accepted, that h 2 s has important roles in modulating different, physiologically important biochemical processes similarly to other, fast diffusing molecules like no, co and h 2 o 2 . for investigation of the physiological effects of these species their local concentration in the studied biological media is important to know. this means methods needed for measuring the instantaneous concentration with high spatial resolution in living tissues without major invasion. electrometric micro, and ultramicro sensors are often gain application in experimental life sciences for measurement of local ion concentration or following neurotransmitter species in vivo measurements. in our work efforts are being carried out to improve the applicability of selective electrometric sensors in life science experiments. as a result of these work an improved h 2 s measuring cell and improved electrode and method was developed for measurement of electroactive small molecules like no or h 2 o 2 . in the poster to be presented the structure, the working principles and the performances of the different sensors mentioned will be described. bacteriorhodopsin (br) is the only protein in the purple membrane of the halophilic organism halobacterium salinarium. it is a light-driven proton pump converting light into a transmembrane proton gradient through isomerization of the covently bound retinal chromophore. its stability, as well as its photoactivity in dried films, has made br an attractive material for biomolecular devices. such studies, however, have used br within the membrane, on relatively large surfaces. here, conducting-probe atomic force microscopy (c-afm) analysis was performed after isolating the protein from its native membrane environment while keeping its basic trimeric structure, and demonstrated that the molecular conductance of br can be reversibly photoswitched with predictable wavelength sensitivity. intimate and robust coupling to gold electrodes was achieved by using a strategically engineered cysteine mutant located on the intracellular side of the protein which, combined with a 75% delipidation, generated protein trimers homogenously orientated on the surface. c-afm proximal probe analysis showed a reproducible 3 fold drop of br mean resistance over *5 cycles of interspersed illuminations at the same gold-br-gold junction when k[495 nm, while no shift was observed with other wavelengths. capture of circulating tumor cells with a highly efficient nanostructured silicon substrates with integrated chaotic micromixers shutao wang 1 ). this core technology shows significantly improved sensitivity in detecting rare ctcs from whole blood, thus provides an alternative for monitoring cancer progression. by assembling a capture-agent-coated nanostructured substrate with a microfluidic chaotic mixer, this integrated microchip can be applied to isolate ctcs from whole blood with superb efficiency. ultimately, the application of this approach will open up opportunities for early detection of cancer metastasis and for isolation of rare populations of cells that cannot feasibly be done using existing technologies. this technology helped to find a needle in a haystack and will open up the opportunity for single cell genomic and epigenetic sequencing and gene expression profiling. results from further development of this technology will assist the physicians in follow-up patients and testing vigorously the concept of personalized oncology with individualized therapy. this novel technology has recently been reviewed and highlighted by nature medicine the growing crisis in organ transplantation and the aging population have driven a search for new and alternative therapies by using advanced bioengineering methods. the formation of organized and functional tissues is a very complex task: the cellular environment requires suitable physiological conditions that, presently, can be achieved and maintained by using properly-designed bioreactors reproducing all specific functions and bioactive factors that assure viability/regeneration of cells cultured in an appropriate scaffold. the creation of biomimetic environment requires the use of biomaterials such as membranes with specific physico-chemical, morphological and transport properties on the basis of the targeted tissue or organ. tailor-made membranes (organic, functionalized with specific biomolecules, in hollow-fiber configuration), designed and operated according to well-defined engineering criteria are able to sustain specific biotransformations, to provide adequate transport of oxygen, nutrients and catabolites throughout the cellular compartment, and to supply appropriate biomechanical stimuli of the developing tissue. in this talk the author will show the development of membrane engineered constructs focusing on liver and neuronal systems. the role of membrane surface and transport properties in providing instructive signals to the cells for the guiding of proliferation and differentiation will be discussed. membrane bioreactors, which through the fluid dynamics modulation may simulate the in vivo complex physiological environment ensuring an adequate mass transfer of nutrients and metabolites and the molecular and mechanical regulatory signals, will be presented. here we present a novel but simple system for cell-based assays enabling simultaneous testing of multiple samples on a same tissue without cross-contamination between neighbouring assays, as well as sequenced or repeated assays at the same tissue location. the principle of this method lies in the spatially-controlled diffusion of test compounds through a porous matrix to the target cells. a simple microfabrication technology was used to define areas where diffusion processes are allowed or inhibited. we performed proof-of-principle experiments on madin-darby canine kidney (mdck) epithelial cells using hoechst nuclear staining and calcein-am cell viability assay. fluorescent staining superimposed properly on membrane pattern with a dose-dependent response, indicating that both compounds specifically and selectively diffused to the target cells. mdck cells similarly treated with cytochalasin b showed their actin network rapidly altered, thus demonstrating the suitability of this system for drug screening applications. such a well-less cell-based screening system enabling multiple compounds testing on a same tissue and requiring very small volumes of test samples appears interesting for studying potential combined effects of different biochemicals applied separately or sequentially. it is generally believed that all-optical data processing is the most promising direction to achieve serious improvements both in capacity and speed of internet data traffic. one of the bottlenecks of the state-of-the-art photonic integration technology is to find the proper nonlinear optical (nlo) materials that are supposed to serve as cladding media in waveguidebased integrated optical circuits performing light-controlled active functions. recently, the unique chromoprotein bacteriorhodopsin (br) has been proposed to be used as an active, programmable nlo material in all-optical integrated circuits. in integrated optical applications of br, its light-induced refractive index change is utilized. in this paper we exploit the refractive index changes of a dried br film accompanying the ultrafast transitions to intermediates i and k, which allows even sub-ps switching, leading beyond tbit/s communication rate. in the experiments direct pulses of a femtosecond laser system at 800 nm were used along with synchronized ultrafast laser pulses at 530 nm. we believe that the results may be the basis for the future realization of a protein-based integrated optical device, and represent the first steps to a conceptual paradigm change in optical communication technologies. last years, such autoantibodies attract an increasing attention of researchers as potential cancer biomarkers. since the sera of cancer patients typically contain a unique set of antibodies that reflect the tumor-associated antigens expressed in a particular malignant tissue, diagnosing and predicting the outcome of disease such as breast cancer based on serum autoantibody profiling is an attractive concept. to create a representative panel of antigens for detecting of breast cancer autoantibody profile we selected 18 breast cancer associated antigens. these antigens were identified by screening of tumor cdna libraries with autologous sera using serex (serological investigation of recombinantly expressed clones) approach. all antigens were cloned, expressed, purified in bacteria and tested with sera of breast cancer patients and healthy donors in large-scale allogenic screening using elisa. the utility of selected tumor associated antigens for detecting of autoantibody profile in different types of breast cancer was evaluated. (controlled by architectural software) is carried out according to a design template, consistent with the geometry and composition of the desired organ module. structure formation occurs by the post-printing fusion of the discrete bio-ink units. when the bio-ink units contain more than one cell type, fusion is accompanied by sorting of the cells into the physiologically relevant pattern. thus structure formation takes place through self-assembly processes akin to those utilized in early embryonic morphogenesis. we demonstrate the technology by detailing the construction of vascular and nerve grafts. spherical and cylindrical bio-ink units have been employed to build fully biological linear and branching vascular tubular conduits and multiluminal nerve grafts. upon perfusion in a bioreactor the constructs achieved desirable biomechanical and biochemical properties that allowed implantation into animal models. our results show that the printing of conveniently prepared cellular units is feasible and may represent a promising tissue and organ engineering technology. femtosecond lasers have become important tools for noncontact microprocessing of biological specimens. due to the short pulse length and intensity-dependent nature of the multiphoton ionization process, fs-laser pulses affect only a small volume of a treated cell, providing a high degree of spatial localization. we employed fs-laser to address topical bioengineering and biomedical problems such as cell fusion and embryo biopsy respectively. a tightly focused laser beam (cr:f seed oscillator and a regenerative amplifier, 620nm, 100 fs, 10 hz) was used for a fusion of blastomeres of two-cell mouse embryos and for a polar body (pb) biopsy. in order to fuse blastomeres the contact border of cells was perforated by a single laser pulse. the fusion process usually completed within *60 min. in order to perform a noncontact laser based pb biopsy we initially drilled an opening in the zona pellucida with a set of laser pulses, and then extracted the pb out of zygote by means of optical tweezer (cw laser, 1064 nm). the energy of laser pulses was thoroughly optimized to prevent cell damage and increase the fusion and biopsy rates. the proposed techniques demonstrate high efficiency and selectivity and show a great potential for using fs lasers as a microsurgical tool. new insights into mechanisms of electric field mediated gene delivery maš a kanduš er and mojca pavlin university of ljubljana, faculty of electrical engineering, si-1000 ljubljana, slovenia gene electrotransfer is widely used for transfer of genetic material in biological cells by local application of electric pulses and is currently the most promising non-viral delivery method for gene therapy for a series of diseases as well as for dna vaccination. current description of the process defines several steps: electropermeabilization, dna-membrane interaction, translocation, trafficking to nucleus and into nucleus. but the mechanisms of electrotransfer are still not fully understood. we present results of the systematic in vitro analysis using pegfp of all steps involved in electrotransfection from electropermeabilization, analysis of different pulsing protocols, theoretical analysis of plasmid mobility to visualization of the processes of dna-membrane interaction. we demonstrate that in order to translate in vitro results to tissue level sub-optimal plasmid concentrations have to be used. furthermore, sofar the method of dna entry into cytoplasm was only speculated. our results suggest that it is crucial that first, membrane is electropermeabilized, then sufficient electrophoretic force is crucial for insertion of dna into destabilized lipid bilayer followed by dna translocation into cytoplasm via a slow process. efficiency of electrotransfer depends also on the stage of of cell culture -cells in dividing phase are easer to electrotransfect. gentamicin interaction with b16f10 cell membrane studied by dielectrophoresis dielectrophoresis (dep) is the translational motion of polarizable particles due to an electric field gradient. positive-dep and negative-dep correspond to particle movement forward or backward the region of high field intensity, respectively. our study reveals some of the cell membrane modifications induced by gentamicin (gt), as they are reflected in the crossover frequency f co of b16f10 murine cells incubated with gt for different concentrations and durations. f co is the ac frequency when cells turn from positive-dep to negative-dep. gentamicin is a positively charged aminoglycosidic antibiotic, with concentration-dependent killing action; it is widely used because of its low cost and reliable bactericidal activity. gt drawbacks consist in high toxicity for renal and hearing cells; the molecular mechanisms of this toxicity are still unclear. for low external medium conductivities (& 0.0012 s/m), f co of control and gt-cells was found to range from 3 to 10khz. f co shifts to higher frequencies with the increase of gt concentration and incubation time. cells dielectrophoretic behavior is discussed using the cell singleshell based model. extracellular matrix (ecm) is a major obstacle for succesful delivery of genes. chitosan is a versatile and biocompatible polysaccharide derived from chitin and is a promising gene carrier. chitosan-dna interactions, and hence dna polyplexation and release can be controlled through chitosan de-acetylation degree, molecular weight and functionalization of chitosan cationic groups. grafting of poly(ethylene glycol) peg to gene delivery vectors increases circulation time of gene delivery systems in blood vessels and reduces polyplexes charge. diffusion and unpacking of pegylated and non-pegylated chitosan-dna polyplexes through articial ecms based on collagen and collagen-hyaluronic acid (ha) gels were compared using fluorescence correlation microscopy, confocal microscopy and colocalization analysis. non-pegylated polyplexes were immobilized in the gels whereas pegylated polyplexes were diffusing. the smaller charge of pegylated polyplexes seems to decrease interactions between polyplexes and ecm components. furthermore, ha might also screen collagen fibers-pegylated polyplexes interactions. pegylated polyplexes also showed a higher degree of unpacking in gels, probably due to a looser compaction of dna by pegylated chitosan compared to non-pegylated chitosan. fabrication of vesicles, a close membrane made of an amphiphile bilayer, has great potentiality for encapsulation and controlled release in chemical, food or biomedical industries but also from a more fundamental point of view for the design of biomimetic objects. methods based on lipid film hydratation 1 , inverse emulsion techniques 2 and more recently microfluidic techniques such as double emulsion 3 or jetting 4 method are limited either by a low yield, a low reproducibility, a poor control on the size, or by the presence of remaining solvent or defects. we propose a fast and robust method 5 easy to implement: continuous droplet interface crossing encapsulation (cdice), that allows the production of defect-free vesicles at high-yield with a control in size and content. the vesicles have controlled bilayer composition with a polydispersity in size lower than 11%. we have shown that solutions as diverse as actin, cells, micrometric colloids, protein and high ionic strength solutions can easily be encapsulated using this process. by adjusting the parameters of our set-up, we are able to produce vesicles in the range 4-100 lm in diameter, stable for weeks. we believe this method open new perspectives for the design of biomimetic systems and even artificial tissues. under appropriate conditions. the extremely variable d3 domain of flagellin subunits, comprising residues 190-283, protrudes at the outer surface of flagellar filaments. the d3 domain has no significant role in the construction of the filament structure. thus, replacement of d3 may offer a promising approach for insertion of heterologous proteins or domains without disturbing the self-assembly of flagellin subunits. our work aims at the construction of flagellinbased fusion proteins which preserve the polymerization ability of flagellin and maintain the functional properties of the fusion partner as well. in this work a fusion construct of flagellin and the superfolder mutant of green fluorescent protein (gfp) was created. the obtained gfp variant was highly fluorescent and capable of forming filamentous assemblies. our results imply that other proteins (enzymes, binding domains etc.) can also be endowed by polymerization ability in a similar way. this approach opens up the way for construction of multifunctional filamentous nanostructures. generation 5 polyamidoamine (pamam) dendrimer has been shown to be highly efficient nonviral carriers in gene delivery. however, their toxicity limits their applications. in this study, to improve their characteristics as gene delivery carriers, g5 pamam dendrimer was modified with anti-tag72 nanobody through hetrobifunctional peg, then complexed with t-bid coding pdna, yielding pamam-peg-anti-tag72 nanobody/pdna nanoparticles (nps). nuclear magnetic resonance (nmr) spectroscopy, zeta sizing and gel retardation assay results provided evidence that the nanovector was successfully constructed. the transfection efficiency of vector/pdna complexes were evaluated in vitro. real time pcr results also demonstrated that anti-tag72 nanobody modified nps are more efficient in t-bid killer gene expressing in colon cancer cell line than the unmodified nps. in conclusion, pamam-peg-anti-tag72 nanobody showed great potential to be applied in designing tumour-targeting gene delivery system. 3 dept of chemistry, faculty of science, national university of singapore, singapore, 4 dept of biochemistry, yong loo lin school of medicine, national university of singapore, singapore, 5 division of bioengineering, faculty of engineering, national university of singapore, singapore macromolecular crowding (mmc) is a biophysical tool which has been used extensively to enhance chemical reactions and biological processes by means of the excluded volume effect (eve). the in vivo stem cell microenvironment contains macromolecules which are crucial for stem cell selfrenewal and cell fate determination. in order to mimic this physiological microenvironment, crowders are included in cell culture medium. we have observed that the ex vivo differentiation of human mesenchymal stem cells (hmscs) into the adipogenic lineage is significantly amplified when a crowder mixture comprising ficoll 70 and ficoll 400 is added to the culture medium. stem cell differentiation is modulated by soluble chemical substances as well as interactions between cells and the extracellular matrix (ecm), and both these external influences may be affected by mmc. measurements we have performed by fluorescence correlation spectroscopy (fcs) show that ficoll additives cause anomalous subdiffusion within a crowder concentration range of 0 to 300 mg/ml. the diffusion of fluorophorelabelled molecules in artificial lipid bilayers and membranes of living cells is not changed by crowders, suggesting that these crowders do not directly alter membrane properties and cell surface signalling. however, we have data to suggest that crowders increase actin polymerization reaction rates in vitro. we have also observed that crowders are taken up by stem cells and that they localize to specific compartments. based upon our observations, we hypothesize that crowders can influence stem cell differentiation by influencing molecular kinetics. lignocellulose-based composites are becoming extremely important and perspective sustainable and renewable natural materials. fibre modification enhancing their existing properties can be obtained to broaden the application areas. in response to shortcomings of traditional chemical and physical methods, enzymes and chemo-enzymatic methods have emerged as eco-friendly catalysts working under mild conditions and enable tailoring of the material surface properties by substrate specificity and regional selectivity. recently, binding of different functional molecules to lignin-rich fibres by using an oxidative enzyme (e.g. laccase) has been reported leading to their functionalisation through free radical reactions. by the application of electron paramagnetic resonance spectroscopy (epr) laccase action was inspected. consumption of substrates was investigated and their polymerization traced. stable radical intermediates were detected with epr when substrate molecules were in contact with active enzymes. secondly, oxidation of mediators like nitroxides was determined via epr spectroscopy of stable water-soluble nitroxide radicals. finally, the generation of short-lived radicals as well as their reduction was measured via epr spin trapping using dmpo as sensitive water soluble spin trap. mammalian ovary hormone stimulation (ohs) is known to be an inalienable stage of reproductive biotechnology as well as human infertility treatment. the basic aim of the ohs is to receive a stock of valuable oocytes and early embryos for subsequent utilization in the reproductive technology, experimental work et al. however, it is known that ohs itself affects the character of ovulation and oocyte quality, which in its turn affects the development of embryos and even has distant consequences. the wideness of cell parameters and appropriate methods for investigation of gamete/embryo quality are very important. the aim of this study is determination of specific electric conductivity of mouse oocytes and early embryos which have been received after ohs in comparison with the ones that have been received in natural animal sex cycle. using techniques of electroporation the dependence of specific electric conductivity of mouse oocytes, zygotes, 2-cell and 8-cell embryos on the external electric field intensity has been studied. it is shown that the whole pool of oocytes that were obtained in the result of ohs consists of two groups of oocytes that don't differ from each other morphologically, but differ by their electric parameters and resistance to electric breakdown. at the zygote stage, dividing of embryos into two groups is preserved, but is less expressed. at the stage of 2-cell and 8-cell dividing of embryos into two groups on their electric conductivity disappeared but certain scattering of the parameters due to individual embryo peculiarities is observed. the obtained data show that ohs may lead to latent changes of oocyte state that in their turn affect embryo quality. many microbes synthesize and accumulate granules of polyhydroxyalkanoates (pha, biodegradable storage materials alternative to traditional plastics), which help them survive under stresses. in particular, the plant-growth-promoting rhizobacterium azospirillum brasilense, that is under investigation worldwide owing to its agricultural and biotechnological significance, can produce poly-3hydroxybutyrate (phb) [1] . in our work, phb synthesis in a. brasilense cells was studied under various stresses using diffuse reflectance ftir spectroscopy. phb in cells was determined from the band intensity ratio of the polyester m(c=o) at *1740 cm -1 to that of cell proteins (amide ii band at *1550 cm -1 ), showing a. brasilense to be able to produce phb up to over 60% of cells' dry weight. stresses induced phb accumulation, enhancing ir absorption in phb specific regions. analysis of a few structure-sensitive phb vibration bands revealed changes in the degree of intracellular phb crystallinity (related to its enzymatic digestion rate) at different stages of bacterial growth, reflecting a novel trait of the bacterial adaptability to an enhancing stress, which is of great importance to agricultural biotechnology. the aim of this work is to furnish enzymes with polymerization ability by creating fusion constructs with the polymerizable protein, flagellin, the main component of bacterial flagellar filaments. the d3 domain of flagellin, exposed on the surface of flagellar filaments, is formed by the hypervariable central portion of the polypeptide chain. d3 is not essential for filament formation. the concept in this project is to replace the d3 domain with suitable monomeric enzymes without adversely affecting polymerization ability, and to assemble these chimeric flagellins into tubular nanostructures. to test the feasibility of this approach, xylanase a (xyna) from b. subtilis was chosen as a model enzyme for insertion. with the help of genetic engineering, a fusion construct was created in which the d3 domain was replaced by xyna. the flic(xyna) chimera exhibited catalytic activity as well as polymerization ability. these results demonstrate that polymerization ability can be introduced into various proteins, and building blocks for rationally designed assembly of filamentous nanostructures can be created ( table 1) . the support of the hungarian national office for research and technology and the hungarian scientific research fund (otka) (grants ck77819, nk77978, nanoflag) is acknowledged. cluster phases of membrane proteins an alternative scenario for the formation of specialized protein nano-domains (cluster phases) in biomembranes dna fragmentation induced in human fibroblasts by accelerated 56 fe ions of differing energies swift heavy ion irradiation of srtio 3 under grazing incidence'' proc. natl. acad. sci. usa 105 forespore engulfment mediated by a ratchet-like mechanism a channel connecting the mother cell and forespore during bacterial endospore formation a feeding tube model for activation of a cell-specific transcription factor during sporulation in bacillus subtilis the scanning ion-conductance microscope imaging proteins in membranes of living cells by high-resolution scanning ion conductance microscopy nanoscale live-cell imaging using hopping probe ion conductance microscopy beta2-adrenergic receptor redistribution in heart failure changes camp compartmentation simultaneous noncontact topography and electrochemical imaging by secm/sicm featuring ion current feedback regulation timasheff in protein-solvent interactions the roles of water in foods on motor and electrical oscillations in urinary tract: computer evaluation daniele martin 1,2 , viktor foltin 1,2 , erich gornik 2 , rumen stainov 3,4 , tanya zlateva 4 nü rnberg. icsd e.v. postfach (pob) 340316, d-80100 mü nchen method: parameters: motor patterns (guinea-pig) -frequency/f, amplitudes/a (% init. length, isot. & intracell. rec.) of spontaneous phasic/spc & tonic/stc contractions, also electrical spikes/s, bursts/b, burst plateaus/bp (neu et al. biophys.j. 125/6a/jan2008 stretch (3-80mn), k-/ca-influence induced specific changes in motor/electrical parameters. special computer programme reflects exactly biophysical parameters. conclusion: acc. to earlier/recent results mechano-sensitive ca ?? -activated k ? -channels participate in electrical oscillations of detrusor/ureteral myocytes. further experiments/evaluations incl effect of hydrophobic mismatch on the light-induced structural changes in bacterial reaction centers s. s. deshmukh, h. akhavein, and lá szló ká lmá n department of physics mechanism of proton transfer in nitric oxide reductase: computational study andrei pisliakov riken advanced science institute, wako-shi proteins 74, 266. acknowledgments this work was supported by project grant ptdc/qui/70182/ 2006 (cas) and doctoral grants sfrh th anniversary conference aicr this work was supported by the italian association for cancer research (airc), the istituto toscano tumori and the associazione noi per voi differential hydration, void volume: which factor provides the main contribution to dv u ? inserm umr 1054 fr; roumestand@cbs.cnrs.fr introduction: globalization needs new organizational models also for biophysics. reports on necessity of int. institutes for biophysics (iib) c/o int. universities (proposed by british nobel laureate b.russell) are given conception: proposals for ebsa-discussion: 1. enlargement of executive committee by a. honorary & presidents (permanent 1-3: moral support & 1-3 fixed term), b. interdisciplinary commission: scientists from biology, medicine, physics, etc. (feps/iups, iuphar, iupab, etc). 2. implication of interdisciplinary topics to esba/iupab congressprogrammes, 3. also for biophysical journals. 4. organization of common interdisciplinary sessions not only to biophysical, but also to other congresses. 5. co-operation between esba/iupab with int. interdisciplinary organisations (waas, icsd/ias, eur. academies) for creation of iib by network of national ones: successive common personnel, possibility for whole life work, etc. conclusion: realization of proposals 1.-5. could increase scientific/political authority of ebsa/iupab, leading to model for renewal of scientific organizations collective migration of neural crest cells: a balance between repulsion and attraction roberto mayor university college london goodilin 2,3 , olga v single-molecule cut-and-paste surface assembly (smcp) has been also used to build up a biotin scaffold that streptavidin utilizing specific molecular interactions, for example between dna-binding proteins and dna or antibodies and antigens, this technique is capable of providing a scaffold for the controlled self-assembly of functional complexes. furthermore, this allows for the introduction of smcp into protein science. we aim to employ dna-binding zinc-finger variants and gfp-binding nanobodies as shuttle-tags fused to the proteins of interest. thus a fully expressible system that can be used for the step-wise assembly of individual building blocks to form single-molecule cut-and-paste surface assembly optically monitoring the mechanical assembly of single molecules nanoparticle self-assembly on a dna-scaffold written by single-molecule cut-and-paste torsional motion analysis of group ii chaperonin using diffracted x-ray tracking nanomechanical manipulation of mason-pfizer monkey retroviral rna fragment with optical tweezers melinda simon 1 , zsolt má rtonfalvi 1 , pasquale bianco 1 , beá ta vé rtessy 2 , mikló s kellermayer 1 micro-viscosimeter generated and manipulated by light andrá s buzá s 1 , lá szló oroszi 1 , ló rá nd kelemen 1 , pá l ormos 1 temesvá ri krt proc. natl. acad. sci. usa 105 neural signal recordings with a novel multisite silicon probe gergely má rton 1 , anita pongrá cz 1 , lá szló grand 2,3 , é va vá zsonyi 1 pé ter pá zmá ny catholic university, faculty of information technology, h-1083, 50/a prá ter st multiscale pattern fabrication for life-science applications francesco valle 1 , beatrice chelli 1 , michele bianchi 1 , eva bystrenova 1 , marianna barbalinardo 3 , arian shehu 1 , tobias cramer 1 , mauro murgia 1 , giulia foschi 1 miroslava kuricova 1 , jana tulinska 1 , aurelia liskova 1 , eva neubauerova 1 , maria dusinska 2,1, ladislava wsolova acceleration neuronal precursors differentiation induced by substrate nanotopography gianluca grenci 1 , jelena ban 2 , elisabetta ruaro 4 , massimo tormen 1 , marco lazzarino 1,3 and vincent torre 2 light-induced structural changes are reported near the primary electron donor of bacterial reaction centers (brc) dispersed in detergent micelles and in liposomes from lipids with different fatty acid chain lengths. in this study we present evidence for the correlation between the light-induced increase of the local dielectric constant, determined by the analysis of the electrochromic absorption changes, and the lifetime of the charge-separated state at physiologically relevant temperatures. the increase of the local dielectric constant induced a significant decrease of the oxidation potential of the primary electron donor and a slow proton release, which appears to be the rate limiting step in the overall process. systematic selection of the head group charges of detergents and lipids, as well as the thickness of the fatty acid chains of the liposome forming lipids can increase the lifetime of the charge-separated state by up to 5 orders of magnitude. such extensions of the lifetime of the charge-separated state were reported earlier only at cryogenic temperatures and can provide new opportunities to utilize the brc in energy storage. ontogenesis of photosynthetic bacteria tracked by absorption and fluorescence kinetics m. kis, e. asztalos, p. maró ti department of medical physics and informatics, university of szeged, hungarythe development of photosynthetic membrane of rhodobacter sphaeroides was studied by absorption spectroscopy and fast induction of bacteriochlorophyll fluorescence in different phases of the growth, under various growing conditions (oxygen content, light intensity etc.) and in synchronous cell population. the results are: 1) the newly synthesized components of the membranes were imbedded immediately into the proteinous scaffold independently on the age of the cell (no ,,transient'' membranes were observed). 2) under aerobic conditions, the pigments were bleached and under anaerobic conditions the pigment systems showed greening. the relative variable fluorescence (f v /f max ) had small age-dependent (but not cellcycle-related) changes. the fluorescence induction kinetics was sensitive marker of the aerobiosis: the f v /f max ratio dropped from 0.7 to 0.4 and the photochemical rate constant from 5á10 4 s -1 to 3á10 4 s -1 with an apparent halftime of about 4-5 hours after change from anaerobic to aerobic atmosphere.3) the electrogenic signal (absorption change at 525 nm) reflected the energetization of the membrane which showed cell-cycle dependent changes. that included periodic production and arrangement of protein-lipid components of the membrane synchronized to the cell division. interfacial water in b-casein molecular surfaces: wide-line nmr, relaxation and dsc characterization t. verebé lyi 1 , m. bokor 1 , p. kamasa 1 , p. tompa 2 , k. tompa 1 1 research institute for solid state physics and optics, hungarian academy of sciences, pob. 49, 1525 budapest,hungary, 2 institute of enzymology, biological research center, hungarian academy of sciences, pob. 7, 1518 budapest, hungarywide-line proton nmr fid, echoes, spin-lattice and spin-spin relaxation times were measured at 82.55 mhz frequency in the -70°c to ?40°c temperature range, in lyophilized bcasein and aqueous and buffered solutions, and dsc method were also applied. the motivation for the selection of b-casein is the uncertainty of structural order/disorder. naturally, the nmr and thermal characteristics were also evaluated. the melting of hydration water could be detected well below 0°c and the quantity of mobile water molecules (hydration) was measured. the hydration vs. melting temperature curve has informed us on the bonding character between the protein surfaces and water molecules. the generally used local field fluctuation model and the bpp theory were applied in the interpretation, and the limits of the models were concluded. the torsional properties of dna play an important role in cellular processes such as transcription, replication, and repair. to access these properties, a number of single-molecule techniques such as magnetic tweezers have been developed to apply torque to dna and coil it. i will briefly refer to investigations of dna-protein interactions using these techniques, and describe what has been learnt. i will then focus on the development of novel magnetic techniques that go beyond standard magnetic tweezers, such as the magnetic torque tweezers 1 and the freely-orbiting magnetic tweezers 2 . these approaches allow one to quantify conjugate variables such as twist and torque. for example, the magnetic torque tweezers rely on high-resolution tracking of the position and rotation angle of magnetic particles in a low stiffness angular clamp. we demonstrate the experimental implementation of this technique and the resolution of the angular tracking. subsequently, we employ this technique to measure the torsional stiffness c of both dsdna molecules and reca heteroduplex filaments. lastly, i will describe novel applications of the optical torque wrench 3, 4 . the optical torque wrench is a laser trapping technique developed at cornell capable of applying and directly measuring torque on microscopic birefringent particles via spin momentum transfer. we have focused on the angular dynamics of the trapped birefringent particle 4 , demonstrating its excitability in the vicinity of a critical point. this links the optical torque wrench to non-linear dynamical systems such as neuronal and cardiovascular tissues, non-linear optics and chemical reactions, which all display an excitable binary ('all-or-none') response to input perturbations. based on this dynamical feature, we devise a conceptually novel sensing technique capable of detecting single perturbation events with high signal-to-noise ratio and continuously adjustable sensitivity.for the first time we report a comparative approach based on surface enhanced raman spectroscopy (sers) and raman spectroscopy to study different types of haemoglobin molecules in living erythrocytes. in erythrocytes there are two fractions of haemoglobin: cytosolic (hb c ) and membranebound (hb m ). the concentration of hb m is less then 0,5% and therefore it is impossible to study hb m with traditional optical techniques. modifications of cellular membrane can affect conformation of hb m . therefore, it can be used as a sensitive marker of pathologies. firstly, we investigated enhancement of sers signal of hb m depending on ag nanoparticles' size. we found that the intensity of sers spectra of hb m and enhancement factor increase with the decrease in ag nanoparticles' size. secondly, we investigated the dependence of haemoporphyrin conformation in both hb c and hb m ion ph values. we observed different sensitivity of hb c and hb m to the ph and found that conformational movements of haemoporphyrin (vibrations of pyrrol rings and side radicals) in hb m are sterically hampered comparably with hb c . our observation is an evidence of a benefit of application of surface enhanced raman spectroscopy to investigate properties of the hb m in erythrocytes and provide new information about conformational changes and functional properties of hb m . rna nanotechnology is an emerging field with high potential for nanomedicine applications. however, the prediction of rna three-dimensional nanostructure assembly is still a challenging task that requires a thorough understanding the rules that govern molecular folding on a rough energy landscape. in this work, we present a comprehensive analysis of the free energy landscape of the human mitochondrial trna lys , which possesses two different folded states in addition to the unfolded one. we have quantitatively analyzed the degree of rna tertiary structure stabilization, firstly, for different types of cations, 1 and, secondly, for several naturally-occurring nucleotide modifications in the structural core of the trna lys . 2,3 thus, notable variations in the rna binding specificity was observed for the divalent ions of mg 2? , ca 2? and mn 2? , that can be attributed to their sizes and coordination properties to specific ligands. furthermore, we observed that the presence of m 2 g10 modification together with the principal stabilizing m 1 a9 modification facilitates the rna folding into the biologically functional cloverleaf shape to a larger extent than the sum of individual contributions of these modifications. in order to elucidate the mechanism of the recognition, we used diffracted x-ray tracking method (dxt) that monitors real-time movements of individual proteins in solution at the single-molecule level. we found that peptides move distinctly from i-a k , and the rotational motions of peptides correlate with the type b t cell activation. in the case of diabetogenic i-a g7 , immediately after peptide exchange, all the peptides moves magnificently but the motion ceased in a week, then new ordered motion appears; the rotational motion of peptides correlate to t cell activation, which is analogous to the peptide in i-a k . the rotational motion of peptides may create transient conformation of peptide/mhc that recognized by a population of t cells. dxt measurement of peptide/mhc complex well correlated to other biological phenomenon too. our finding is the first observation that fluctuations at the level of brownian motion affect to the functions of proteins.mason-pfizer monkey virus (mpmv) is an excellent model for the analysis of retrovirus assembly and maturation. however, neither the structure of the viral rna, nor its modulation by capsid-protein binding are exactly known. to explore the structure of the mpmv genome, here we manipulated individual molecules of its packaging signal sequence with optical tweezers. the 207-base-long segment of mpmv rna corresponding to the packaging signal, extended on each side with 1200-base-long indifferent gene segments for use as molecular handles, was cloned into a pet22b vector. rna was synthesized in an in vitro transcription system. rna/ dna handles were obtained by hybridization in a pcr with complementary dna initiated with primers labeled with either digoxigenin or biotin. the complex was manipulated in repetitive stretch and relaxation cycles across a force range of 0-70 pn. during stretch, transition occurred which increased the rna chain length and likely corresponds to unfolding. the length gain associated with the unfolding steps distributed across three main peaks at *13, 20, 32 nm, corresponding to *35, 57, 85 bases, respectively. often reverse transitions were observed during mechanical relaxation, indicating that refolding against force proceeds in a quasi-equilibrium process. structural investigation of gpcr transmembrane signaling by use of nanobodies jan steyaert 1,2 1 structural biology brussels, vrije universiteit brussel, pleinlaan 2, 1050 brussel, belgium, 2 department of structural biology, vib, pleinlaan 2, 1050 brussel, belgiumin 1993, scientists at the vrije universiteit brussel discovered the occurence of bona fide antibodies devoid of light chains in camelidae. the small and rigid recombinant antigen binding fragments (15kd) of these heavy chain only antibodies -known as vhhs or nanobodies -proved to be unique research tools in structural biology. by rigidifying flexible regions and obscuring aggregative surfaces, nanobody complexes warrant conformationally uniform samples that are key to protein structure determination by x-ray crystallography:• nanobodies bind cryptic epitopes and lock proteins in unique native conformations • nbs increase the stability of soluble proteins and solubilized membrane proteins • nbs reduce the conformational complexity of soluble proteins and solubilized membrane proteins • nbs increase the polar surface enabling the growth of diffracting crystals • nbs allow to affinity-trap active protein i will focus my talk on the use of nbs for the structural investigation of gpcr transmembrane signaling to illustrate the power of the nanobody platform for generating diffracting quality crystals of the most challenging targets including gpcrs and their complexes with downstream signaling partners. dynamics of the type i interferon receptor assembly in the plasma membrane stephan wilmes, sara lö chte, oliver beutel, changjiang you, christian paolo richter and jacob piehler university of osnabrü ck, division of biophysics, barbarastrasse 11, 49076 osnabrü ck, germanytype i interferons (ifn) are key cytokines in the innate immune response and play a critical role in host defense. all ifns bind to a shared cell surface receptor comprised of two subunits, ifnar1 and ifnar2. detailed structure-function analysis of ifns has established that the ifn-receptor interaction dynamics plays a critical role for signalling specificities.here we have explored the dynamics of receptor diffusion and ifn assembly in living cells. by using highly specific orthogonal posttranslational labelling approaches combined with tirf-microscopy we probed the spatio-temporal dynamics of receptor diffusion and interaction in the plasma membrane of live cells on the single molecule level. for this purpose, we employed posttranslational labelling with photostable organic fluorophores. this allowed us to map diffusion and lateral distribution of ifnar1 and ifnar2 with very high spatial and temporal resolution by using single particle tracking (spt) and single molecule localization imaging. observed events of ''transient confinement'' and co-localization with the membrane-proximal actin-meshwork suggest partitioning of ifnar1/2 in specialized microcompartments. this will be investigated in terms influence on receptor assembly and recruitment of cytoplasmic effector proteins. cytoplasmic dynein moves through uncoordinated action of the aaa1 ring domains ahmet yildiz department of physics, and department of molecular and cell biology, university of california, berkeley, ca 94720 usa cytoplasmic dynein is a homodimeric aaa? motor that moves processively toward the microtubule minus end. the mechanism by which the two catalytic head domains interact and move relative to each other remains unresolved. by tracking the positions of both heads at nanometer resolution, we found that the heads remain widely separated and move independently along the microtubule, a mechanism different from that of kinesin and myosin. the direction and size of steps vary as a function of interhead separation. dynein processivity is maintained with only one active head, which drags its inactive partner head forward. these results challenge established views of motor processivity and show that dynein is a unique motor that moves without strictly coordinating the mechanochemical cycles of its two heads.o-715 self-controlled monofunctionalization of quantum dots and their applicaitons in studying protein-protein interaction in live cells changjiang you, stephan wilmes, sara loechte, oliver beutel, domenik lisse, christian paolo richter, and jacob piehler universitä t osnabrü ck, fachbereich biologie, barbarastrasse 11, 49076 osnabrü ck, germanyindividual proteins labeled with semiconductor nanocrystals (quantum dots, qd) greatly facilitate studying protein-protein interactions with ultrahigh spatial and temporal resolution. multiplex single molecule tracking and imaging require monovalent quantum dots (mvqd) capable of orthogonally labeling proteins with high yield. for this purpose, we prepared monovalance qd-trisnta by a chemical conjugation method. our results indicated that monovalent qd-trisnta was obtained in high yield by restricting the coupling by means of electrostatic repulsion. monovalent functionalization of the qd-trisnta was confirmed by assays in vitro and in vivo. two-color qd tracking of interferon receptors ifnar1 and ifnar2 based on mvqd-trisnta were realized on live cell [1] .to broaden the multiplex toolbox of mvqds, we extended the electrostatic-repulsion induced self-control concept for mono-functionalizing quantum dots with different affinity moieties. as a first instance, we used negatively-charged biotin peptide to produce qd with biotin mono-functionalization. we confirmed our approach was a general method to rend qd monovalent by single molecule assays based on stepwise photobleaching. these mvqds facilitate obtaining spatiotemporal information of ifnars' organization in live cells. by orthogonal labeling u5a cells stably expressing ifnar2 at low level with biotin mvqd and mvqd-trisnta-ifn, we verified colocalization and colocomotion of individual ifn and ifnar2 at minute scale. combined with super-resolution imaging of ifnars' cytosolic effecter stat2, we observed the dynamic coming-and-going contact between the microcompartments of ifnar2 and stat2.a micron sized viscometer was fabricated using the couette type geometry that is capable of measuring the complex viscosity of fluids. the viscometer was produced by two photon polymerization of su8 photopolymer using a femtosecond laser system, a high na objective and a piezo translator stage. the viscometer was manipulated by holographic optical tweezers and operated in the 0.005-1 hz frequency range. video analysis algorithm was used to evaluate our measurements. we tested the viscometer with water-glycerol solutions. one of the main reasons for lack of reliability in protein analysis for disease diagnostics or monitoring is a lack of test sensitivity. this is because, for many tests, to be reliable, they need to be performed on a homogeneous, and therefore very small, sample. current in-vitro techniques fail in accurately identifying small differences in protein content, function and interactions starting from samples constituted of few or even single cells. a nanotechnology approach may overcome the current limits in low abundance protein detection. we aim at designing a microwell device for the trapping (in native environment) and the parallel characterization of rare cells (e.g. adult stem cells). such versatile device, based on soft and nanolithography, will promote cell adhesion and viability on differently functionalized bio-compatible materials, allowing for the morphological characterization of the cells, at a single cell level. in parallel, by facing our microwell device with a protein nanoarray, produced via atomic force microscopy nanolithography, we can run proteomic studies at a single/few cells level. moreover, we could foresee the possibility to deliver different stimuli to each cell, correlating the changes in chemistry/ morphology with the protein profile at a single cell level. using an electrophysiological assay the activity of nhaa was tested in a wide ph range from ph 5.0 to 9.5. forward and reverse transport directions were investigated at zero membrane potential using preparations with inside out and right side out oriented transporters with na ? or h ? gradients as the driving force. under symmetrical ph conditions with a na ? gradient for activation, both the wt and the ph-shifted g338s variant exhibit highly symmetrical transport activity with bell shaped ph dependencies, but the optimal ph was shifted 1.8 ph units to the acidic range in the variant. in both strains the ph dependence was associated with a systematic increase of the k m for na ? at acidic ph. under symmetrical na ? concentration with a ph gradient for nhaa activation an unexpected novel characteristic of the antiporter was revealed; rather than being down regulated it remained active even at ph as low as 5. these data allowed to advance a transport mechanism based on competing na ? and h ? binding to a common transport site and to develop a kinetic model quantitatively explaining the experimental results. in support of these results both alkaline ph and na ? induce the conformational change of nhaa associated with nhaa cation translocation as demonstrated here by trypsin digestion. furthermore, na ? translocation was found to be associated with the displacement of a negative charge. in conclusion, the electrophysiological assay allowed to reveal the mechanism of nhaa antiport and sheds new light on the concept of nhaa ph regulation. swimming motility is widespread among bacteria. however, in confined or structured habitats bacteria often come in contact with solid surfaces which has an effect on the swimming characteristics. we used microfabrication technology to quantitatively study the interaction of swimming cells with solid boundaries. we tracked bacteria near surfaces with various engineered topologies, including flat and curved shapes. we were able to study several surface related phenomena such as hydrodynamic trapping and correlated motion. we think that our results may help to understand how physical effects play a role in surface related biological processes involving bacteria such as biofilm formation. cell labeling efficiency of oppositely charged magnetic iron oxide nanoparticles-a comparative study raimo hartmann 1 , christoph schweiger 2 , feng zhang 1 , wolfgang. j. parak 1 , thomas kissel 2,# , pilar rivera_gil 1,# 1 biophotonics, institute of physics, philipps university of marburg, 2 pharmaceutical technology, institute of pharmacy, philipps university of marburg e-mail: kissel@staff.uni-marburg.de; pilar.riveragil@physik.uni-marburg.dethe interaction of nanomaterials with cells is a key factor when considering their translocation into clinical applications. especially an effective accumulation of nanoparticles inside certain tissues is beneficial for a great number of applications. predominantly size, shape and surface charge of nanoparticles influence their cellular internalization and distribution. to investigate this, two series of maghemite (c-fe 2 o 3 ) nanoparticles were synthesized either via aqueous coprecipitation or via thermal decomposition of organometallic precursor molecules. size and the spherical shape of both nanoparticle types were kept constant whereas the charge was changed by modifying the surface of the nanoparticles with polymers of opposite charge, in detail poly(ethylene imine) (pei) and a polymaleic anhydride derivative (pma). the positively and negatively charged c-fe 2 o 3 nanoparticles were characterized with respect to size, zeta potential, colloidal stability and magnetic properties. furthermore, the uptake rate and localization of both formulations into a549 carcinoma cells after fluorescent labeling of the carriers as well as the resulting alteration in mr-relaxation times were evaluated. membrane proteins are the target of more than 50% of all drugs and are encoded by about 30% of the human genome. electrophysiological techniques, like patch-clamp, unravelled many functional aspects of membrane proteins but usually suffer from poor structural sensitivity. we have developed surface enhanced infrared difference absorption spectroscopy (seidas) 1,2 to probe potential-induced structural changes of a protein on the level of a monolayer. a novel concept is introduced to incorporate membrane proteins into solid supported lipid bilayers in an orientated manner via the affinity of the his-tag to the ni-nta terminated gold surface 3 . full functionality of surface-tethered cytochrome c oxidase is demonstrated by cyclic voltammetry after binding of the natural electron donor cytochrome c. general applicability of the methodological approach is shown by tethering photosystem ii to the gold surface 4 . in conjunction with hydrogenase, the basis is set towards a biomimetic system for h 2 -production. recently, we succeeded to record ir difference spectra of a monolayer of sensory rhodopsin ii under voltage-clamp conditions 5 . this approach opens an avenue towards mechanistic studies of voltage-gated ion channels with unprecedented structural and temporal sensitivity. initial vibrational studies on the novel light-gated channelrhodopsin-2 will be presented 6 . probing biomass-chromatographic bead interactions by afm force spectroscopy gesa helms, marcelo ferná ndez-lahore, rami reddy vennapusa, and jü rgen fritz school of engineering and science, jacobs university bremen, 28759 bremen, germany e-mail: g.helms@jacobs-university.dein expanded bed adsorption (eba), bioproducts are purified from an unclarified fermentation broth by their adsorption on chromatographic beads in a fluidized bed. the unspecific deposition of biomass onto the adsorbent matrix can severely affect the process performance, leading to a poor system hydrodynamics which then decreases the success of this unit operation. to quantify the bead-biomass interactions different chromatographic beads are attached to afm cantilevers, and force spectroscopy experiments are performed with these colloidal probes on model surfaces and cells in solution. the experiments are conducted under varying conditions to study uncovering physiological processes at the cellular level is essential in order to study complex brain mechanisms. using multisite signal recording techniques in the extracellular space, functional connectivity between different brain areas can be revealed. a novel microfabrication process flow, based on the combination of wet chemical etching methods was developed, which yields highly reproducible and mechanically robust silicon-based multielectrode devices. the fabricated shaft of the probe is 280 lm wide, 80 lm thick, has rounded edges and ends in a yacht-bow like, sharp tip. its unique shape provides decreased invasivity. the sensor contains 24 platinum recording sites at precisely defined locations. murine in vivo experiments showed that the probes could easily penetrate the meninges. high quality signals, providing local field potential, multi-and single unit activities, were recorded. the interfaces between the tissue and the platinum contacts were further improved by electrochemical etching and carbon nanotube coating of the metal sites. the integrated optical mach-zehnder interferometer is a highly sensitive device, considered a powerful lab-on-a-chip tool for specific detection of various chemical and biochemical reactions. despite its advantages, there is no commercially available biosensor based on this technique. the main reason is the inherent instability of the device due to slight changes of environmental parameters. in this paper we offer a solution to this problem that enables the optimal adjustment of the working point of the sensor prior to the measurement. the key feature is a control unit made of a thin film of the lightsensitive chromoprotein bacteriorhodopsin deposited on the reference arm of the interferometer. after showing the transfer characteristics of such a device, we demonstrate its applicability to sensing of specific protein-protein interactions. we expect our method to become a rapid and cost-efficientthe combination of unconventional fabrication technology and biomaterials allows both to realize state-of-the-art devices with highly controlled lateral features and performances and to study the main properties of the biomolecules themselves by operating at a scale level comparable with the one crucial for their activity. soft lithography and microfluidic devices offer a tool-box both to study biomolecules under highly confined environments [1] and to fabricate in an easy way topographic features with locally controlled mechanical and chemical surface properties, thus leading to a finer control of the interplay of mechanics and chemistry. i will present an application of this technology to the control of cell fate that is becoming a key issue in regenerative medicine in the perspective of generating novel artificial tissues. patterns of extracellular matrix (ecm) proteins have been fabricated, by a modified lithographically controlled wetting (lcw), on the highly antifouling surface of teflon-af to guide the adhesion, growth and differentiation of neural cells (shsy5y, 1321n1, ne-4c) achieving an extremely accurate guidance [2] . local surface topography is also known to influence the cell fate [3] , thus, integrating this parameter in the substrate fabrication could increase the complexity of the signals supplied to the cells. in this perspective we have developed a novel fabrication technique, named lithographically controlled etching (lce), allowing, in one step, to engrave and to functionalize the substrate surface over different lengthscales and with different functionalities. i will conclude showing how we have been developing ultrathin film organic field effect transistors (ofets) as label-free biological transducers and sensors of biological systems. ofets are low dimensional devices where ordered conjugated molecules act as charge transport material. unconventional patterning techniques and microfluidics have been adapted to proteins and nucleic acids to dose the molecules on the ofet channel with a high control of the concentration. in another set of experiments, we have also been addressing the signalling from neural cells and networks grown on pentacene ultra-thin film transistosr [3, 4] .advances in nanotechnology are beginning to exert a significant impact in medicine. increasing use of nanomaterials in treatment of diseases has raised concerns about their potential risks to human health. in our study, the effect of poly(lactic-co-glycolic acid) (plga) and titanium dioxide (tio 2 ) nanoparticles (nps) on function of b-and t-lymphocytes was investigated in vitro. human blood cultures were treated with plga and tio 2 nps in concentrations: 0.12; 3 and 75 lg/cm 2 for 72h. lymphocyte transformation assay was used to assess the effect of nps on lymphocyte function. lymphocytes were stimulated with mitogens: concanavalin a, phytohaemmagglutinin (t-cell response) and pokeweed mitogen (b-cell response). our findings indicate immunomodulatory effect of plga nps. proliferative response of t-and b-lymphocytes exposed in vitro to the highest dose of plga for 72h was suppressed significantly (p.01, p.05). on the other hand, we observed stimulative effect of exposure to middle dose of plga nps on b-lymphocyte proliferation (p.05). no alteration was found in lymphocyte proliferation treated in vitro with tio 2 nps for 72h. in conclusion, proliferation of lymphocytes in vitro might be one of the relevant tests for evaluation of nps immunotoxicity.embryonic stem (es) cell differentiation in specific cell lineage is still a major challenge in regenerative medicine. differentiation is usually achieved by using biochemical factors (bf) which concentration and sides effects are not completely understood. therefore, we produced patterns in polydimethylsiloxane (pdms) consisting of groove and pillar arrays of sub-micrometric lateral resolution as substrates for cell cultures. we analyzed the effect of different nanostructures on differentiation of es-derived neuronal precursors into neuronal lineage without adding biochemical factors. neuronal precursors adhere on pdms more effectively than on glass coverslips but the elastomeric material itself doesn't enhance neuronal differentiation. nano-pillars increase both precursors differentiation and survival with respect to grooves. we demonstrated that neuronal yield was enhanced by increasing pillars height from 35 to 400 nm. on higher pillar neuronal differentiation reaches *80% 96 hours after plating and the largest differentiation enhancement of pillars over flat pdms was observed during the first 6 hours of culture. we conclude that pdms nanopillars accelerate and increase neuronal differentiation. key: cord-023346-8sqbqjm1 authors: nan title: monday: posters date: 2005-06-08 journal: vox sang doi: 10.1111/j.1423-0410.2005.00652.x sha: doc_id: 23346 cord_uid: 8sqbqjm1 nan from the perspective of causal inference, there is a hierarchy of evidence, ranging from case-series to large randomized controlled trials (rcts). in addressing a particular clinical or policy problem, clinicians or policy-makers can base their decisions on the types of clinical reports that have been published, along with an assessment of the strengths and weaknesses of each study. rcts are controlled clinical experiments in which patients are randomly allocated by the investigators to receive a treatment under study. if randomization is used, all participants are equally likely to be allocated to either the treatment or the control arm of the study. rcts should be distinguished from observational studies in which investigators passively observe patients who happen to receive a treatment under study. because the allocation of subjects to the treatment and control arms of an rct is random, the play of chance should distribute all confounding factors equally between these two arms. thus, the treatment and control arms of an rct should be equivalent with respect to all confounding factors except for the treatment under study. randomization removes selection bias, because neither the investigator nor the participant knows what the treatment allocation will be before a patient enters a study. moreover, if an rct is double-blind, observation bias is also removed, because preconceived notions about benefit from the treatment cannot color the reporting of symptoms by a patient or the assessment of disease activity by an investigator. when the undertaking of rcts is deemed unlikely, meta-analyses of individual patient data, that is, of the data recorded previously on subjects enrolled in published, small rcts may allow investigators to address issues of possible biases 2ed-01-01 standardizing blood components would allow better monitoring of the effectiveness of transfusion d davenport university of michigan, ann arbor, mi, usa in routine transfusion of red blood cells (rbc) or platelet concentrates (pc), we have only a very rough idea of the dose we administer. the minimum expected hemoglobin content of rbc should be 50 g (fda criteria). however, actual measurements have found a mean hemoglobin content of 46.6 ± 11.7 g per unit, with variability between manufacturers. 25 percent of units may contain less than 34.9 g of hemoglobin while 25 percent may contain more than 56.3 g. the effect of storage senescence can magnify these differences. the resulting hematocrit increase, depending on size of recipient and age of a unit, may be 1.5 to 9 percent. apheresis collection of rbc can help standardized unit contents, but this technology is relatively expensive and time consuming. knowledge of actual hemoglobin content can be used to optimize red cell transfusion. one trial, using a simple formula incorporating the desired hemoglobin and estimated blood volume, showed that nearly 60 percent of transfusion orders could be met with one unit while achieving a mean target hemoglobin of 9.3 g/dl. for pc transfusion, about 66 percent of transfusions achieve the targeted dose of 3-6 ¥ 10 11 platelets in actual practice, with considerable variability between institutions. without knowledge of the actual content of pc, determination of refractoriness and response to matched pc is problematic. standardization and labeling of rbc and pc units for content will permit accurate dosage and quantification of transfusion practice. are transfusion guidelines evidence-based? jp aubuchon dartmouth-hitchcock medical center, lebanon, nh, usa application of the results of randomized, controlled clinical trials to similar clinical situations should increase the predictability of the outcomes of transfusions. unfortunately, too few such trials have been conducted to investigate all the potential situations where transfusion might be applied. however, the ones that have been reported indicate that, in general, transfusion is unlikely to provide substantial benefit in many of the situations where it is routinely used. in the intensive care setting, transfusing red cells at a trigger point of 7 g/dl rather than 10 g/dl not only did not lead to increased anemic morbidity but was actually associated with a reduction in overall mortality. subgroup analyses indicated transfusion at the higher trigger point did not decrease morbidity in patients with cardiac disease nor decrease the time until weaning from a ventilator. following cardiac surgery, a transfusion trigger of a hematocrit of 25% yielded the same outcomes as one of 32%. an observational study suggested that patients with peripheral vascular disease and a post-operative hematocrit below 28% were more likely to suffer a morbid cardiac event, but this group of patients had more evidence of anemia and cardiac ischemia preoperatively, illustrating the pitfalls of observational studies. most would agree that transfusion is necessary below a hb of 7 g/dl and unlikely to be necessary at a hb above 10 g/dl, but most patients fall between these limits, and there are insufficient data in the and residual confounding factors, and may permit them to make a judgment of a causal relationship. sackett proposed that the evidence generated from meta-analyses of individual patient data be regarded as being equivalent in strength to that generated from rcts. meta-analysis is the structured and systematic integration of information from different studies of a given problem. when the results of individual studies are discrepant, the purpose of an overview is to investigate reasons for disagreements among studies. when the results are concordant, the goal of meta-analysis is to derive, through application of a number of quantitative techniques, a measure of the effect of the intervention across combined investigations. this measure is referred to as the 'summary' effect of the treatment under study. meta-analysis differs from the traditional, narrative reviews of the literature, in that: (1) all completed investigations of the efficacy of an intervention that meet specific, eligibility criteria are retrieved and included in the overview; (2) the quality of retrieved studies is assessed systematically; (3) the degree of agreement among studies is evaluated, both conceptually and based on statistical criteria, and the synthesis of the findings proceeds only if the variation in reported results is sufficiently modest to be attributed to chance; and (4) quantitative methods are used to calculate the summary effect of the intervention and to test that effect for statistical significance. 2ed-02-04 biology of stem cell mobilization th papayannopoulou university of washington, seattle, wa, usa at baseline hematopoiesis, the majority of developing hematopoietic cells (precursors, progenitors and stem cells) is actively retained in bone marrow (bm), whereas fully mature cells emigrate to peripheral circulation. a small number of stem/progenitor cells are also present in blood, serving presumed physiologic roles for the repopulation of remote damaged areas. however, in several hematopoietic perturbations, i.e. post irradiation, post chemotherapy or by using empiric treatments, a heightened emigration (mobilization) of progenitor/stem cells was noted over 30 years ago. the introduction of g-csf as an efficient mobilizing agent not only had a major clinical impact, but has triggered a flurry of studies exploring the mechanisms of mobilization. from these studies, significant insight has been gained about the molecular pathways leading to mobilization, especially by studying the altered environment within bm post mobilization, and less so by studying cells mobilized in blood. several attractive scenarios have been proposed and their importance was further bolstered by studying genetic mouse models. a prominent role of the sdf-1/cxcr4 pathway has been emphasized, either by down regulation of cxcr4, changes in sdf-1 gradients, or by disruption of sdf-1/cxcr4 signaling. whether this pathway is disrupted by a proteolytic mechanism prevailing within bm post g-csf or by other protease-independent mechanisms has not yet been settled. in addition to sdf-1/cxcr4, disruption of other pathways responsible for the retention of primitive cells in bm can lead to mobilization. for example, disengagement of the vla4/vcam-1 pathway by anti-functional antibodies or its genetic deficiency in mice results in egress of stem/progenitor cells. mobilization is also seen following the use of other cytokines (i.e. kit-l, flt-3 l, il-3) or chemokines (i.e. il-8, gro?), complement activation, etc., but the detailed mechanisms or the interdependence of these other pathways with the ones already proposed have not been worked out. finally, efforts to improve mobilization efficiency in man has led to the use of combination treatments with g-csf, either by adding another cytokine (i.e. growth hormone), agonistic sdf-1 molecules (i.e. ctce0021), or cxcr4 antagonists (i.e. amd3100) which have yielded synergistic increments, and these will be discussed. a full understanding of the principles of mobilization, as well as the bm homing characteristics of mobilized cells after their i.v. infusion in transplantation, should lead to targeted, more efficient experimental protocols in the future. the possibility of deriving all kinds of mature cell types from embryonic stem (es) cells for the purpose of cell replacement therapies will probably face a major obstacle; a limited supply of available hla types for an ever growing population in demand. one possible solution is to use adult sources of stem cells such as the haematopoietic stem cells (hsc) that can repopulate the entire haematopoietic system after transplantation in a myeloablated host. however cells with the repopulating ability of hscs are still elusive for tissues such as muscle, heart, cns and pancreas. it was therefore exciting news when it was shown that hscs could repopulate other non-haematopoietic tissues arguing for a general role of bmderived cells in tissue regeneration. the term plasticity was thus coined to describe the phenomenon whereby cells of one lineage could trans-differentiate into cells of another tissue. this in turn implied that a certain degree of developmental plasticity was still available in the adult hsc, or their derived progeny, that allowed them to present with novel phenotypes. how exactly this was accomplished was a matter of speculation. in order to address the mechanism we used an animal model of liver failure where bmt with normal (wild type, wt) hsc was shown to rescue the liver failure; the repopulating hepatocytes apparently differentiated from the sole source of wt cells, the bm compartment. by studying the genetic composition of the regenerating liver nodules we observed that both wt and mutant dna was present in the nodules, a finding that was consistent with bm-derived cells fusing with host hepatocytes. the mechanism of plasticity was therefore directly related to the exposure of the donor bm-derived wt nucleus to the transcriptional environment of the hepatocyte and the expression of hepatocyte-specific genes. this fusion mechanism was also shown to underlie perceived cases of haematopoietic cell transdifferentiation to purkinje cells in the cerebellum and to cardiomyocytes. however, there are carefully executed studies that strongly support the alternative transdifferentiation mechanism in tissues such as epithelia or the epidermis. what these observations may imply is that in tissues with high rates of cell turnover, there may be a potent stem cell niche that can reprogram incoming cells to follow relevant cell lineages. if this hypothesis is correct, then the major research effort should focus on what makes the niche and whether it can be recreated faithfully in vitro so as to educate hscs to diverse lineages opening the way for realistic cell replacement therapies. collection and distribution of blood and its products to meet the medical needs of industrialized countries are typically managed through large-scale national or nationally-supported blood programs. the development, maintenance, and ultimate success of such programs are all components of a process that involves the delicate balance of continuously competing pressures, e.g. social, economic, ethical, political, regulatory/legislative. as with all endeavors that directly affect human life, safety is a central, unifying theme of this process. because transactions of blood inherently involve risks at both donation and reception/transfusion ends, efforts to enhance and maintain an acceptable level of safety generally rely on a focused program of risk management (rm). rm involves three equally important elements: identification/evaluation of risk factors in a process, control of exposure to them, and continuous monitoring to assess the effectiveness of countermeasures and the emergence of new risk factors. specific approaches to rm and quality assurance in transfusion medicine will be presented. examples from blood programs currently in effect in selected countries will be discussed. rm efforts within the corresponding blood program in greece will then be examined. the lack of data to adequately assess the status of this program suggests that at least one of the elements of rm -monitoring -can still be improved. a preliminary research effort aims to survey blood donors, transfusion recipients, and physicians in order to build an understanding of the specific factors that drive the perception of risk in the greek population. the findings form important stepping points upon which specific recommendations can be made for the development and maintenance of a comprehensive, effective rm program in greece. 2ed-03-08 the use of haemovigilance data to increase safety in transfusion medicine haemovigilance is a surveillance of all the procedures in the transfusion chain. the intension is to collect and assess information on unexpected or undesirable effects in bleeding of donors and transfusion of blood components. in europe haemovigilance was introduced as a concept in the middle of the nineties. the first national reports on haemovigilance appeared in the late nineties, and were only dealing with complications related to transfusion. later on other kind of events like 'near miss' events were added. nowadays national reports are dealing with all steps in the transfusion line, and to some extend also with complications in blood donors. the state of the art is haemovigilance with retrospective registration of unwanted events that has happened, but also a more active prospective part with an early warning in a rapid alert system about new threats in the transfusion world. the aim of the different national haemovigilance systems has been to improve safety in the transfusion line. after the first 10 years with haemovigilance in the european countries, what has been the outcome of this big effort? data from national reports do not signify major improvements, but safety is suggested to have improved due to many minor changes of procedures and awareness of dangerous situations. however, in most countries nothing really effective has been done to avoid failures, the most important cause of serious complications in transfusion of patients. systems which can prevent most of the failures are on the market. the cost of these equals the cost of nat screening for virus introduced in the same period of time. the common perception of transfusion risks is still much more focused on the hypothetical risk of virus transmission than to the demonstrated magnitude of severe complications related to bleeding of donors and transfusion of patients. therefore, to increase safety in transfusion medicine the nature and occurrence of the risks should be revealed by haemovigilance and not less important the results should be published with the aim to get a realistic common perception of the transfusion risks. the role of diagnostic testing to identify congenital vs acquired disorders of hemostasis and to optimize the management of perioperative bleeding g despotis washington university school of medicine, st louis, mo, usa excessive bleeding with trauma or after surgery can result in hypoperfusion and anemia related end-organ dysfunction and mortality as well as transfusion-related complications. several large studies have demonstrated that if bleeding is excessive after cardiac surgery to the point that reexploration is required, that overall mortality increases by 3-4 fold. transfusion related complications include the following potentially lethal complications: disease transmission of pathogens, acute hemolytic reactions, allergic reactions, transfusion associated acute lung injury, allo-immunization related disease (e.g. post-transfusion purpura, platelet refractoriness, transfusion-associated graft-vs-host disease) and other potential complications (e.g. increased perioperative infection or multi-organ system failure) related to transfusion associated immune modulation. in addition, blood shortages related to increasing consumption (i.e. expanding geriatric population) and/or a shrinking supply (i.e. related to exclusion of donors related to donor exclusion criteria designed to prevent disease transmission) may limit our ability to adequately manage our anemic and bleeding patients. this highlights the relative importance of accurate diagnosis and optimal management of excessive bleeding to minimize bleeding related complications and conserve our blood supply. patients at risk for excessive bleeding include those with an established hereditary disorder (e.g. vwd, hemophilia, connective tissue disorders), end-stage hepatic or renal disease as well as patients with acquired defects of the hemostatic system. in specific, patients with platelet (i.e. platelet refractoriness) or coagulation factor inhibitors at increased risk, extracorporeal circulation related defects or as related to certain pharmacologic agents). patients who require longer periods of extracorporeal circulation for cardiac surgical procedures (e.g. repeat, combined procedures or use of deep hypothermic circulatory arrest) are at a greater risk of developing excessive bleeding (e.g. cpb-related abnormalities). in addition, patients receiving one or more longacting anti-thrombotic medications in the immediate preoperative period (e.g. plavix, reopro, low molecular weight heparin etc) are at increased risk for bleeding. clinicians in the past have resorted to empiric and 'shot gun' approaches when managing excessive bleeding due lack of immediate availability of results from laboratory-based coagulation tests. on this basis, the use of point-of-care (poc) tests of hemostatic function to facilitate the optimal management of excessive bleeding, help differentiate between microvascular vs surgical bleeding and reduce transfusion have been investigated. five of six recently published studies have demonstrated that implementation of a standardized approach to manage bleeding (e.g. algorithm) which when coupled with either point-ofcare or laboratory tests of hemostatic function can optimize the management of bleeding and reduce total donor exposures by 50%. ddavp has bee shown to be beneficial with uremia-induced platelet dysfunction and with type i von willbrand's disease. although previous studies have not been able to conclusively show that ddavp can reduce bleeding and transfusion when administered prophylactically, more recent evidence indicates that this agent may be useful in preventing excessive bleeding when a test (point-of-care) reveals platelet dysfunction. recombinant activated factor vii (rfviia) is licensed for use in bleeding episodes in hemophiliac patients with inhibitors. although, only anecdotal reports and results from small clinical studies have shown that this agent can reverse life-threatening bleeding after major surgery, other reports indicate that there is variability in the effectiveness of rfviia as well as highlight lifethreatening thrombotic complications in a subset of high risk patients (i.e. patients with congenital or acquired thrombotic disorders or systemic activation of the hemostatic system such as with dic or after cardiac surgery). therefore, large clinical trials evaluating the efficacy and safety of rfviia are needed before any widespread use can be recommended. in recent years, molecular methods of blood grouping have become routine diagnostic procedures in many laboratories throughout the world. they have proved especially valuable for the determination of fetal blood groups. the ability to detect rhd in pregnancies where the fetus is at risk from haemolytic disease of the newborn represents a significant advance in obstetric care. furthermore, the occurrence of fetal dna in the mothers' peripheral blood has allowed this diagnostic procedure to be carried out without the risks that accompany amniocentesis (lo ymd. (1999) ann med 31:308-312). determination of the coding sequence of all the genes giving rise to antigens within the 29 blood group systems currently recognised is virtually complete. by correlating the coding sequence of these genes with the phenotype of red cells from individuals with different blood groups it has been possible to infer the molecular bases of most of the antigens comprising each blood group system (daniels gl (2002) human blood groups, 2nd ed. blackwell science). the polymorphic antigens of most systems other than abo and rh, are defined by single nucleotide substitutions (snps) which effect a single amino acid sequence change in the gene product. numerous methods, both manual and automated, are available to determine snps and these have been applied to the determination of blood groups. useful applications of snps detection methods include, determination of the blood group phenotype of patients who have been transfused recently and still have donor blood in their circulation (rozman p, dove t, gassner c et al. (2000) transfusion 40:936-942), and donor screening to find compatible units for patients with multiple blood group antibodies or for the management of patients with diseases like the haemoglobinopathies who are going to be transfusion-dependent over many years (castilho l, rios m, bianco c et al. (2002) transfusion 42:232-238). other applications of molecular methods include zygosity determination for rhd, determination of the blood group phenotype of patients with a positive direct antiglobulin test, selection of donors with rare blood group phenotypes, and as aids to the solution of complex blood grouping problems in the reference laboratory. the molecular bases of abo and rh antigens are complex and cannot be determined reliably by a single snp. nevertheless, methodologies are available that allow the comprehensive sequence analysis of individual genes and could be applied to abo and rh typing. whether or not this will ever be a cost-effective procedure is a moot point. it is clear that molecular methods are a useful addition to the range of diagnostic procedures available to transfusion medicine practitioners but it is important to remember that methods based on dna analysis determine phenotype by inference and not by direct measurement. the results of molecular tests should be interpreted with this caveat in mind. 2ed-06-18 the hla system g stavropoulos general hospital 'g. gennimatas', athens, greece since its discovery in the mouse in 1936 the major histocompatibility complex (mhc) has become one of the most important region in the vertebrate genome with respect to infection, autoimmunity and transplantation. mhc primary function is to provide protection against pathogens. this is achieved through sophisticated pathways in which mhc class i molecules present endogenous antiges to cd8+ t cells and class ii molecules present exogenous antigens to cd4+ t cells. an increasing number of other proteins are being found that support these two pathways; many of these proteins, together with the class iii complement proteins, also map to the mhc. the mhc molecules were originally studied for their ability to cxonfer tolerance (histocompatibility) following tissue grafts or later, organ transplants. nowadays, the success of unrelated hematopoetic cell transplantation is influenced by the degree of mhc compatibility between the donor and patient. thus, for patients who lack matched donors, the rules that govern permissibility of mhc mismatching still need to be identified. for patients with high risk disease who lack matched donors, use of donors with a single mhc mismatch may permit early treatment before disease progression. scientific evidence to fully answer the questions 'when are platelet components clinically effective' and 'what do we really know?' is limited. despite this limitation and the level of uncertainty it generates with regard to treatment options and policies, it is reassuring to note that current prophylactic platelet transfusion protocols -mostly derived from empirical observations collected during the 1960's and 1970's -protect oncology patients from clinically relevant bleeding in more than 95% of thrombocytopenic days spent in hospital or at home, even in aggressive conditions such as leukemia, lymphoma and other severe blood diseases. this evidence seems to justify the prevalent policy of transfusing platelets when the patient's platelet count falls below 10 000 per microliter (or 20 000 in 'unstable' patients). this policy is based on positive outcomes of prospective and retrospective studies performed during the 1990's including some hundred non-surgical patients and on the desire of balancing the will of reducing patient exposure to limited, expensive, potentially infectious and immunogenic blood products with the objective of preventing clinically relevant hemorrhage. several national and international organizations endorse the above policy. although the role of platelet support in surgery or in specific clinical situations requiring invasive procedures or in patients affected by multi-organ and system co-morbidity suffers from even more limited evidence, the latter has been carefully collected and summarized in the guidelines for platelet transfusion published in j clin oncol 2001;19:1519-1538. additional data on lumbar puncture are reported in ann hematol 2003;82:570-573. another important topic where there is room for improvement and need for further investigation is platelet refractoriness, a condition developed by a proportion of chronic platelet recipients, which causes high hemorrhage risk if compatible platelets are not provided and in which consensus on the diagnosis and treatment is lacking. with regard to the type of platelet product, it will be necessary to determine the clinical impact of buffy-coat derived platelets, consistently used in europe and current object of increased interest in canada, as compared to the platelet-rich plasma method traditionally used in the us, of viral inactivation procedures and of laboratory methods for detection of bacterial contamination of platelet concentrates. in addition, ongoing studies on the clinical impact of high versus low platelet doses will provide novel elements to determine the relative merits of apheresis versus whole-blood derived platelets. as far as the established procedure of white cell reduction by filtration, which shows high technical efficiency and has been implemented as a standard by a number of institutions, debate is still ongoing on its equivalence with serologic screening for the prevention of cmv transmission, whereas general consensus supports its pre-storage use to prevent febrile, non hemolytic transfusion reactions. finally, although the high cost of filters do not allow a generalized use of this technology in many settings, white cell reduced platelets have been unequivocally shown to reduce alloimmune refractoriness from about 15% to about 5% of patients. 2ed-07-20 ffp: appraisal of the evidence for the clinical use of ffp although the indications for transfusion of plasma (fresh frozen plasma; ffp) are limited, the use of ffp continues to rise in the united kingdom and has risen by over 20% in the past few years. local uk audits continue to document that a significant proportion of ffp transfusions are not consistent with indications reported in guidelines. a systematic review of the evidence base for the effectiveness of ffp was therefore undertaken to identify, select and appraise all relevant randomised controlled trials, as the most robust form of study to assess effectiveness of ffp. in the systematic review of ffp, the criteria for inclusion of full-published studies were: there must have been at least two groups in the study; • allocation to the groups must have been either by formal randomisation or by a quasi random method e.g. alternation; • one of the arms of trial must include ffp or plasma as an intervention; results on the relevant clinical or laboratory outcome must be presented. the main analysis was qualitative, and differentiated between: 1. studies of interventions comparing ffp with no ffp; 2. studies of interventions comparing ffp with a non-blood product e.g. solutions of colloids and/or crystalloids; 3. studies of interventions comparing ffp with a different blood product; 4. studies comparing different formulations of ffp, e.g. solvent detergent and methodine blue treated. an evaluation of studies comparing ffp with no ffp would be expected to provide the clearest direct evidence for a positive effect of ffp. studies comparing ffp with colloids or crystalloids were separately appraised because these latter products may have variable effects on coagulation tests. studies comparing different formulations of ffp do not directly evaluate effectiveness of ffp but were included because there is now a uk national policy to use these products in younger patients and therefore these trials might identify negative outcomes. the search strategy identified a total 57 publications. although it may appear that this number of randomised trials might provide a reasonable evidence base to help inform clinical policy and decision making, the review identified a number of important concerns about the published trials. few of the identified studies included details of the study methodology (method of randomisation, blinding of patients and study personnel). the sample size of many included studies was very small (range 8-78 patients per arm). few studies took adequate account of the extent to which adverse events might negate the clinical benefits of treatment with ffp. many of the identified trials in groups such as cardiac, neonatal, and other clinical conditions, evaluated a prophylactic transfusion strategy. however, when these trials evaluating prophylactic usage were assessed together as a single grouping in the review, it appeared there was no consistent support for a beneficial effect of prophylactic ffp, irrespective of clinical setting. there is a pressing need to develop new trials to determine the effectiveness of ffp, including for those clinical situations in which it has become an accepted part of current transfusion practice. a law decided upon by the riksdag (the swedish parliament) often sets the general standards as a framework and authorises the central government authority concerned to elaborate and decide upon the more detailed regulations. laws and regulations define the level of quality and safety that has to be reached and state what must be done, while establishments and their managers are kept responsible for fulfilling the requirements and how this is done. guidelines (non-binding) present detailed instructions on ways how to fulfil the requirements. making a law is complicated and time-consuming. the ministry draws up a legislative proposal, refers the proposal to relevant bodies for consideration and comments, and then drafts a bill which is referred to the council on legislation for consideration before it is submitted to the riksdag. a parliamentary committee deals with the bill before it is put to the chamber of the riksdag for approval. when adopted, the bill becomes law. regulations elaborated by central government authorities are handled in a principally similar way. however, regulations are more readily adjusted to scientific and technical progress, and when legislation need requirements for technical details, these are preferably presented in a regulation. the ministry of health and social affairs is responsible for elaborating the bill to be submitted to the riksdag on the blood safety law. two central government authorities, designated as competent authorities, are responsible for preparing the appropriate complimentary regulations as well as for inspecting and licensing the blood establishments: • the national board of health and welfare (nbhw), responsible for requirements related to blood components intended for transfusion, and • the medical product agency (mpa), responsible for requirements related to blood components intended for the manufacture of medicinal products. sweden became a member of the eu ten years ago. since then, experts in transfusion medicine have been appointed by the ministry, nbhw and mpa for advice on scientific and technical issues and for representing sweden at expert meetings arranged by the commission. some of these experts are also members of the handbook committee of the national society for transfusion medicine, preparing and revising the national guidelines. consequently, the requirements of the directives are readily implemented in the daily work of the blood establishments before (due to legal and technical reasons) the new blood safety law and the revised nbhw and mpa regulations can be promulgated. to a great extent, requirements of the blood directives are covered by existing swedish legislation. no major problems or obstacles seem to arise when implementing new requirements into law and regulations and into the blood establishments' daily service. a few detailed requirements have been found difficult to follow precisely in the routine work. these minor difficulties, however, will not compromise the high quality and safety of blood components prepared according to the standards set by the blood directives. the landscape for blood collection and distribution includes availability and safety. true international availability of blood has not been reached. the landscape of safety has been mountainous, if viewed by the degree of frustration among the transfusion specialists. the reasons for frustration have varied from serological problems to those of transmissible infections, to which no end is in sight. mistrust in the safety of blood taken by others is one reason for lack of international landscape in blood collection and distribution. increasing demands on safety, availability and economy force the health care providers to reorganise blood services. national systems are winning ground. this may make it easier to achieve international collaboration. the council of europe has pioneered in creation of international recommendations for blood collection and distribution. in 1958 a european agreement on the exchange of therapeutic substances of human origin, including human blood, was published. its purpose was to make blood available to other parties of the agreement in case of urgent need. many countries ratified the agreement rapidly, some did it first in the nineties. in practice little exchange of blood components has taken place. the council of europe recommendations lack legal power. the european union is different, and it has taken on its agenda activities aiming at improving confidence in the safety of the blood transfusion chain in the community (commission communication 1994) . the agenda is based on an agreement reached at a high level eu meeting in adare, ireland in 1994. first in the commission agenda was a recommendation on donor selection criteria, given in 1998. then came the european blood directive 2002/98/ec, the aim of which is to improve the safety of blood and blood components within the union so that enough mutual trust between member countries can be achieved to make the exchange of blood components possible. being a legally binding document the directive is undoubtedly helpful in reaching a harmonised standard in europe. hopefully the european commission has the resources to follow its implementation. there are concerns, however. the epidemiological differences among the eu member countries and frequent appearance of new infectious agents potentially transmitted by blood make it difficult to foresee that even effective viral inactivation of blood components could totally erase the national differences in donor approval. blood collection and preparation of components are already the same in eu member countries and the directive helps in their further standardisation. there has not been much distribution of blood components internationally, with the exception of export of red cell concentrates to the us from switzerland and the netherlands. the european legislation opens new horizons and widens the european landscape as its purpose is to simplify the crossing of borders between the member states, but before true movement of blood components is achieved more work is needed on the eu commission blood agenda. the eu directive implemented into the legal act for polish blood transfusion service blood transfusion service (bts) in poland is an integral part of the public polish health service. in the country of nearly 39 million people, approximately one million units of blood and plasma are collected every year from voluntary, nonremunarated donors, which is statistically over 25 donations per 1000 inhabitants. blood and plasma are collected in strictly appointed centers and no private collection sites are permitted. the legal basis for the activity of polish bts is polish blood transfusion act of 22nd august 1997 which came into force as of january 1st 1999. this act was then updated in november 2003 according to eu directive 2002/98/ec and came into force as of january 13th 2004. this act introduced the principles for organization, collection, processing, storage, transport and quality assurance in bts. it specified -among others -the system of accreditation, haemovigilence, as well as requirements for bts employees. according to this act the polish bts is obliged to monitor and supervise immunohematology testing and transfusion procedures in all hospitals where blood and blood products are transfused. polish bts consists of 21 regional blood transfusion centers (rbtc), one military center and one ministry of internal affairs and administration center as well as the institute of hematology and blood transfusion (ihbt) which acts as supervisor. the 21 rbtcs have a uniform organization structure, uniform quality assurance system and act according to uniform guidelines issued by ihbt. they have two financing sources -the central budget and hospital reimbursement for distributed blood and blood products. the polish blood transfusion act of 22nd august 1997, in force since january 1st 1999, has been supplemented by 8 decrees: 1. procedures for external bts audits; 2. requirements for donor selection; 3. requirements and procedures for organization and safe management of blood transfusion in hospitals; 4. requirements for implementing of national and regional donor registers; 5. employment criteria for bts personnel; 6. training requirements for hospital personnel involved in blood and blood product administration; 7. national, uniform price list for blood and blood products; 8. organization requirements for setting up of a national committee for blood and blood transfusion. introduction: several technical aspects must be considered in pediatric apheresis due to the size of the patient. factors that must be evaluated are extracorporeal circuit volume, blood flow rates, type of anticoagulant and vascular access. adverse events are mainly related either to vascular access or to metabolic or hemodynamic changes. aim of the study: in this study we show our experience using fresenius hemocare com.tec for pbsc collection in children. methods: twelve pediatric patients (median age 8 years, range 2-16; median weight 38 kg, range 12.2-60.5 kg) with solid tumors at onset or on relapse underwent collections with the p1y kit of the fresenius hemocare com.tec blood cell separator. our cd34+ cells target was 10 ¥ 10 e 6 /kg. collections were started if a peak of at least 0.02 10e9/l cd34+ cells (20 per microlitre) were reached in the peripheral blood. in all the patients, leukapheresis were performed through a central venous catheter and temporary peripheral venous access. acd ratio 1 : 14-1 : 16 was combined with heparin 40 u/kg. in 3 children with <15 kg the separator was initialized with a compatible filtered and irradiated red blood cell unit, suspended in 4% albumin, up to the patient's hematocrit, to avoid transient hypovolemia, due to the volume sequestered in the separator. results: twenty-five procedures were performed. a median blood volume of 5400 ml (range 2.100-12.400 ml) was processed in a separation time of 270 min (range 180-330 min). the median product weight was 108 g (range 37-258 g) and yield of cd34+ cells was 3.2 ¥ 10e 6 /kg body weight (range 0.7-12.5 ¥ 10e 6 /kg body weight). three poor mobilizing patients (peripheral blood cd34+ peak of 20-26 cells per microlitre) underwent more than two apheresis to collect the desired transplantation dose (3.5 and 4). all collection procedures were well tolerated. children never required sedation to perform the leukapheresis. only mild hypocalcemia-related symptoms, promptly responding to small i.v. boluses of calcium gluconate, were reported. no circulatory side effects were observed. blood flow alarms occurred in every procedures but no collection had to be terminated due to insufficient flow. conclusion: in summary, leukapheresis in children can be safely and effectively performed with the fresenius hemocare com.tec separator with minimal technical difficulties even in patients under 15 kg. m-pa-007 alternative methods for prevention of infection transmission (pathogen inactivation etc.), cost-benefit considerations of the procedure itself. however, one must also take the loss of plasma into consideration -and more difficult: calculate the effects of changes in product quality, as reduced content of factor viii, protein s and other labile proteins. for methods involving pooling, there are also concerns about the risks due to the pool sizes. the major benefit of the methods will be the potential reduction of infectious transmission, but also possible advantages as reduction of allergic transfusion reactions and trali must be evaluated. the study types involved in cost-benefit considerations are costeffectiveness analysis where the cost and effects of an intervention and an alternative are presented in a ratio of incremental cost to incremental effect and cost-utility analysis, where quality-adjusted life years (qaly) are used as the effectiveness endpoint. qualityadjusted life years is a method that assigns a preference weight to each health state and estimates life-expectancy as the sum of these products of each preference weight and time spent for each state. a complete glossary of terms is found at http://www.hsph.harvard.edu/cearegistry. in literature, there are several publications on cost-benefit considerations within the field of transfusion medicine. concerning plasma products, the costeffectiveness of solvent-detergent plasma has been the major focus. however, the conclusions of different authors have been conflicting. in 1994, aubuchon and birkmeyer published a paper (jama 1994; 272(15) :1210-1214) where they concluded that the cost was usd 340 000 per qaly, which is far above the 'acceptable limit' of usd 50 000. this estimate was adjusted to usd 9.7 mill. per qaly in a 1999 letter to jama (jackson, jama 282). in 2003 , riedler et al. published (vox sang 2003 85:88-95 ) that the discounted cost/life year saved for sd-ffp use in the uk was gbp 22 278 for neonates and gbp 98 465 for patients aged 70. the main reason for the differences between the two papers (and others) was considered to be different calculation of non-infectious complications. the papers cited above underline the difficulties of the cost-benefit considerations. the age of the patient, the outcome of the treatment, the quality of life in an infected patient and the cost of side effects will differ. in addition, how much are we willing to pay for protection against emerging viruses? this paper will not provide the answers, but introduction: the photodynamic treatment of therapeutic plasma using methylene blue (mb) in combination with visible light is a well established procedure for the inactivation of blood borne viruses. aim of the study: evaluation of the quality and stability of mb/light-treated plasma (mb plasma) prepared under worst case conditions for routine processing. methods: single donor units (n = 12) were treated using the macopharma theraflex mb-plasma system which includes plasma membrane filtration (plas4) and addition of mb prior to illumination followed by mb and photoproduct filtration (blueflex). samples were taken before treatment and from the final product. additionally mb-treated plasma was prepared from four different plasma pools and stored for up to 9 months. treatment was done under worst case conditions for the preservation of coagulation factors: maximum mb concentration during illumination (1.15 mmol/l), maximum storage time of whole blood before separation (4°c, 17 h), maximum storage time of mb plasma before freezing (1 h). several plasma parameters and the concentration of mb and its photoproducts (azure a, azure b, azure c and thionine) were determined. results: mb/light treatment had a significant influence on thrombin time (+20.5%), fibrinogen (clauss) (-20 .4%), factor v (-15.6%), factor viii (-22.3%), factor xi (-13.3%) and protein c (-10.3%). no significant changes were detected for at iii, vwf : rco, vwf cleaving protease, plasmin inhibitor and alpha-1-antitrypsin. the entire virus inactivation procedure including the filtration steps for leukocyte depletion and mb and photoproduct depletion had no significant effect on activation markers (prothrombin fragment 1 + 2, thrombin-antithrombin-complex, d-dimers). no further essential loss of coagulation factor activity was observed during storage of the plasma at 30°c for 9 months. mb and its photoproducts (azure a, azure b, azure c) were depleted to a final concentration of <0.1 mmol/l. thionine was undetectable in all samples. conclusion: photodynamic treatment of fresh frozen plasma (ffp) using the theraflex mb-plasma system leads to only moderate decreases in the activities of different coagulation factors even under worst case conditions for routine production. mb and its photoproducts were effectively removed from the plasma by the blue-flex-filter integrated in the theraflex-system. the quality of mb plasma is well preserved during storage. pulmonary complications, particularly transfusion-related acute lung injury and circulatory overload, are the most common causes of transfusion-associated morbidity and mortality in the developed world. trali is a syndrome characterized by acute respiratory distress, hypoxemia, hypotension and pulmonary edema, occurring within 6 hours (usually 1-2 hours) of transfusion of a plasmacontaining blood product. other signs, including hypertension, leucopenia and hypocomplementemia, are less frequent. all blood products, except for albumin and solvent/detergent plasma, have been associated with trali, but red blood cells, platelets and ffp are the most common. the incidence is unknown, but 1 : 5000 plasma-containing transfusions is the most commonly cited figure. in 80% of patients, recovery is well underway within 96 hours, and leads to complete resolution. death occurs in 6-23%. the profile of the at-risk recipient has not been identified. recurrent cases have been infrequently described. there are two prevailing theories of pathogenesis: (1) antibody-mediated and (2) 2-hit hypothesis. evidence supports both concepts and neither is mutually exclusive. more than 60% of reported cases are associated with blood components containing either hla-specific (class i or ii) or hnaspecific antibodies. in 50% these antibodies correspond to at least one epitope in the recipient. most implicated components are donated by multiparous women. five percent of patients have hla or hna antibodies in their pre-transfusion serum. treatment requires prompt, assertive respiratory intervention, frequently necessitating mechanical ventilation. because trali has a much better prognosis than ards, it is an important diagnosis. some blood collectors are diverting plasma from multiparous donors away from ffp production or routinely screening for hla antibodies. the most effective method for identifying 'high risk' components has not been identified. introduction: trali is a life threatening adverse reaction of blood transfusion. it is characterized by noncardiogenic pulmonary edema developed soon after blood transfusion. in japan, we built hemovigilance system in 1993 and have been collecting the voluntary reports of severe adverse reactions of blood transfusion including trali cases since 1997. since the diagnostic criteria of trali have not been established until recently and no specific diagnostic markers for trali have been discovered so far, it is very difficult to make proper diagnosis of trali in each reported case. we have been collecting the cases with respiratory distress and pulmonary edema developed after blood transfusion as suspected case of trali. we reevaluated each report whether it meet the recommended diagnostic criteria for trali published in transfusion journal in dec. 2004 . aim of the study: purpose of this study is to select trali cases which met internationally recognized criteria so that it will reveal the possible causes of trali with laboratory testing for anti-leukocyte antibodies in donors and recipients. methods: the cases with respiratory failure and pulmonary edema are selected for evaluation from the adverse reaction case reports voluntarily reported to japanese red cross. in order to make a proper diagnosis of trali, we have been utilizing the respiratory distress questionnaire which has recently revised. this helps us eliminate other adverse reactions such as circulatory overload, cardiac failure, anaphylaxis and bacterial contamination, which results in selecting out the internationally recognized trali cases properly. for laboratory testing, flowpra and labscreen for hla antibodies and gift-fcm for hna antibodies are performed. the cross-matching test is also performed if possible. results: during past 8 years, 95 cases of trali and 29 cases of possible trali have been confirmed by critical review of each questionnaire. of 95 cases of definite trali, donor specimens were obtained in 84 cases. of 84 cases, anti-leukocyte antibodies were detected in 43 cases (51%) of donors' blood, which was significantly higher than the positive rate of anti-leukocyte antibodies in donors' blood of other adverse transfusion reactions (<10%). of 43 cases of antibody positive donors, anti hla antibodies were detected in 33 cases, anti hna antibodies were detected in 18 cases, and both were detected in 8 cases. of 33 cases of positive anti hla antibodies, class i antibodies were detected in 22 cases, class ii antibodies were detected in 28 cases, and both were detected in 17 cases. on the other hand, the anti-leukocyte antibodies were detected in 38% of trali recipients, and this rate is almost the same with that of positive rate of other adverse reactions of blood transfusion (39%). these results indicate anti-leukocyte antibodies in the blood donors are one of the prerequisites for developing trali from the antibody-hypothesis-oriented point of view. other cases with no detectable antibodies should be investigated in more detail in the future. thus, reevaluating trali cases based on recommended trali criteria will allow us to reveal new information about trali. of 2087 adverse events analysed by the serious hazards of transfusion (shot) scheme (1996) (1997) (1998) (1999) (2000) (2001) (2002) (2003) , 556 (27%) were haemolytic transfusion reactions (htrs). 303 were due to incorrect blood component transfused (ibct): 226/303 were abo incompatible and 77/303 caused by other red cell antibodies. a further 40 cases were reported as acute htrs (ahtrs; i.e. occurring within 24 hours of transfusion) whilst 213 were recognised more than 24 hours after transfusion and reported as delayed htrs (dhtr). 21/556 (4%) patients died and 58 suffered major morbidity. htrs associated with ibct result from clinical or laboratory errors and are all preventable. it has been assumed that other htrs are unavoidable. closer scrutiny reveals that this may not always be the case, though review is hampered by incomplete investigations. 9/40 ahtrs occurred in group a (8/9) or group b (1/9) patients given group o platelets. of the 31 ahtrs related to red cell transfusion, 4 were due to errors, 9 were in patients with auto-antibodies, in only 4 of whom alloantibodies had been adequately excluded/identified. at least 24/213 dhtrs were potentially avoidable; in 18 cases the antibody was detectable retrospectively in the pre-transfusion sample; in 6 cases the presence of a previous antibody was not communicated to the laboratory. two patient deaths related to dhtr might have been avoided by earlier diagnosis and clinical involvement. 14% htrs reported to shot would have been avoided by compliance with pretransfusion testing guidelines and provision of group a platelets for all group a recipients. htrs can be clinically overlooked and inadequately investigated. national guidelines are needed for the investigation and management of htr, with focus on the identification of underlying causes to guide the choice of future component therapy. reference laboratories can provide valuable support in elucidating complex serological problems. patients treated with high dose chemotherapy and autologous blood progenitor cell (bpc) support may get malignant cells reinfused together with stem cells. we analyzed bpc products collected from patients suffering from germ cell cancer measuring malignant contamination and followed these patients for up to 98 months after transplantation also with the question if transplanted malignant cells influence survival. aliquots of stem cell apheresis products containing one million mononuclear cells were sedimented on glass slides and by immunocytochemistry quantitation of cytokeratin expressing cells was performed manually with light microscopy and by automated image analysis. in 11 of 35 patients (31%) cytokeratin expressing cells were detected in bpc apheresis products of patients treated for germ cell cancer. we followed the activity of the malignant disease of patients for more than eight years in median 60 months after transplantation. no significant difference in survival was demonstrated for our two patient groups. background: the use of adequate number of peripheral blood stem cells (pbsc), collected by mnc apheresis is essential for effective treatment of hematological malignancies, solid tumors, and other disorders by transplantation. aims: the goals of this study were: (a) to obtain an effective apheretic protocol for mnc harvesting, and (b) to compare the hematopoietic reconstitution after hspc transplantations in different clinical settings. methods: in this study, pbsc transplantations -60 allogeneic from matched sibling healthy donors and 60 autologous -were performed in the management of patients with severe aplastic anemia, leukemias (all, anll, cml), multiple myeloma, hodgkin's and non-hodgkin's lymphoma, breast and ovarial cancer, and extragonadal non-seminal germ cell tumor. pbsc mobilization was achieved with rhg-csf (5-16 g/kgbm/day). mnc-apheresis procedures (generally one and occasionally two) were performed using blood cell separator cobe-spectra. the first mnc-apheresis was accomplished when the leukocyte cont was 5-10 ¥ 10e9/l (autologous setting) or on the 5th day 4-5 hour after the last rhg-csf administration (allogeneic setting). the processed blood volume during one mnc-apheresis was 12. 7-22.2 l, and 24 .3 l for one pbsc transplantation in average. results: using a minimal target dose of cd34+ cell count (3 ¥ 10e6/kgbm), performing one mnc-apheresis procedure for 86% recipients sufficient number of pbscs were obtained. the mnc yield was 10.1 ¥ 10e8/kgbm in allogeneic and 8.1 ¥ 10e8/kgbm in autologous setting in average. the mean cd34+ yields for allogeneic and autologous transplantations were 12.1 ¥ 10e6/kgbm and 6.5 ¥ 10e6/kgbm, respectively. hematopoietic reconstitution was achieved on the 9.4th day for leukocytes and the 13.05th day for platelets when pbsc transplantation was applied. summary/conclusion: improved mnc and cd34+ cell yield, as well as rapid hematopoietic reconstitution were observed when: (a) the intention of auditing any clinical practice is, ostensibly, to improve patient care. the term 'audit' implies comparison against a standard. although absolute indications for transfusion have not been defined by clinical trials applicable to most clinical situations, many institutions have established their own transfusion triggers based on their reading of the literature and common practice at that hospital. assuming these represent prudent guidelines, comparison of actual practice to them may allow physicians to re-assess their pattern of hemotherapy and bring it into conformance with the guideline. there are several common ways of performing transfusion audits. retrospective analysis of transfusions allows appreciation of the clinical situation (and its ultimate outcome) but reviews the event through a 'retrospectoscope' that assesses clinical information in a different manner than that available to the clinician making the decision to transfuse. furthermore, the time lapse between the decision to transfusion and feedback about that decision may render the feedback from the reviewing body (e.g., a transfusion committee or blood utilization review committee) of little practical import. to speed the provision of feedback and emphasize educational rather than any punitive outcomes of the audit, some facilities have abolished attempts at determining whether the decision to transfuse was supportable and have used electronic means to feed back to clinicians a non-judgmental informational message summarizing the literature regarding the indications for transfusion. this appears to be at least as effective as the traditional retrospective audit system. prospective review of requests for blood components have the potential to redirect practice in a manner that immediately helps patients. however, such interactions with clinicians may come at inopportune times, may require considerable (unscheduled) time, and are most likely to be fruitful if a knowledgeable transfusion medicine expert can serve as the intermediary. both approaches (or a combination) have been shown to be beneficial in altering practice, but efforts must be diligent and sustained. providing data comparing a physician's practice to colleagues in the same specialty may prompt additional introspection and practice change, particularly if physician leadership of the institution supports the effort as a quality improvement tool. extending the comparison to a group of physicians and contrasting their transfusion habits with benchmark data from other institutions may also be helpful, but one needs to be ready to counter arguments that differences in patient groups are the reason for the differences in practice (studies have shown that, in general, practice patterns are primarily related to training and habit rather than large differences in patient acuity). increased focus on the performance of hospitals as expressed in outcome data may soon extend to transfusion practices as well. the public or governmental institutions may ask to see data illustrating the transfusion practice of an institution and its improvement over time. carefully conducted, diligent, and ongoing transfusion audits are an integral part of an institution's quality improvement program. informed consent: the term informed consent, appeared for the first time in the late 40s but it was only in the 70s that it attracted attention with regard to health care. numerous discussions and publications have attempted to define the meaning and the justification of informed consent in recent years. initially it consisted in the obligation of the physician to disclose information to the patient, regarding the procedure he was to undergo, but more recently, ethicists have emphasized the need to ensure the patient's understanding and his autonomous decision to consent. current institutional rules of ethics demand that the physician must obtain the informed consent of a patient 'prior to any substantial intervention' . what is however the meaning of informed consent? is it a mutual decision making between physician and patient? in 'principles of biomedical ethics' beauchamp and childress claim that' it is critically important to distinguish informational exchanges through which patients elect medical interventions, from acts of approving and authorizing those interventions. the elements of consent include: disclosure, voluntariness, decision and authorization. when applying the concept of informed consent in transfusion medicine one can distinguish it into donors' and patients' consent. donor informed consent: information to blood donors constitutes a sensitive issue. it refers to their protection from side-effects of the donation, as well as to protection of the recipient. with regard to whole blood donors a detailed history and information as to side effects are necessary for first-time donors. for repeat donors one needs mainly an updated history. because of time-pressure, whole blood donors are usually given written information and are asked to answer written questions. donors however differ in literacy and even literate ones do not always understand medical terminilogy; so, during the interview one should probe the degree of understanding of each donor. with first time apheresis donors more time is needed in order to explain the procedure and potential side effects. since granulocyte and stem-cell donors require premedication with growth factors and or corticosteroids, the responsibility for detailed information is even greater. the fact that all donors sign the informed consent does not mean that they are all adequately informed! interviewers must be familiar with side effects, their frequency and sequelae and must pass on this information. misses and near-misses, (serious) adverse events and failures in medical practice seem to be not preventable. in medical interventions, preparing and prescribing of medication, assistance by doctors and nurses, medical treatment and follow-up, unwanted and unexpected events occur (http://www.mederrors.com.). these events happen also in transfusion medicine and focus on safety is not unique. haemovigilance which is defined in the eu blood directive as 'a set of organised surveillance procedures relating to serious adverse or unexpected events or reactions in donors or recipients, and the epidemiological follow-up of donors' (eu directive 2002/98/ec off. j. european union.8.2.2003:l33/30-l33/40) is established to help in trying to identify and minimise the misses and (serious) adverse events in the chain from donor to recipient of blood components. the causes or reasons should be studied in order to prevent re-occurrence. adverse event reporting in blood transfusion and transfusion medicine is complex. it depends on the cooperation between blood establishments with clinicians and hospitals. it implies knowledge of blood banking, transfusion medicine and routine clinical care of all gender and ages, of potential hazards of transfusion, of immune-haematology, of microbiology, and of epidemiology. an adverse event may have its cause in every single part of the blood chain and reference may take place to a proven problem, a potential problem, or to a justified doubt. in almost all blood transfusion centres, a single donation will be processed into a number of different products, and these units might be divided or processed into more products. blood components are produced from whole blood or apheresis donations, and depending of the blood drawing and processing techniques, a high number of products with different specifications is prepared. the products' shelf life is not equal and therefore the moment in time of actual use of each unit prepared from the same donation may differ. in case the unit of platelets harms the recipient, a rapid alert can warn in order not to issue or to transfuse the unit of red cells or the unit of ffp prepared from the same donation because of the potential adverse reaction, which was detected during or after the transfusion of the first unit used. haemovigilance is not only important to blood establishments and to patients and prescribers, but it is also to clinical scientists, and to the public at large. it should provide a basis for minimising adverse reactions on blood components, and it should enable the therapeutic potential of new or established treatments with components to be maximised, since demonstrations of safety during widespread use may lead to extended usage, and wider availability. it is quite worrisome that underreporting is a general problem in medical care. it might be expected that in transfusion medicine the same rates of underreporting can be found, but also that the same mechanisms for improvement are applicable. although medical misses occur and do not seem to be preventable, the handling of these misses is often quite poor. many patients like to hear a detailed explanation, and the majority expects even apologies from the treating physician. it seems desirable to look for new routes in the prevention of unwanted events of transfusion medicine. for problem solving, analysis and improvement of working methods, where needed and possible, are often the most effective methods. attention should be focused on improvement and not on identification of the person who caused the problem ('bad apple'). lack of communication and insufficient insight in each other work are often the causes of problems and unwanted events. it should be recognised that advices given by the haemovigilance officer or the blood transfusion committee about prevention without the input and commitment of the direct responsible persons will lead in most cases to advices which are not effective or which will not be accepted. setting up a haemovigilance system or appointing of haemovigilance officers or installation of blood transfusion committees will not be sufficient. it will be necessary to develop ways of registration, data collection and analysis, but more importantly to support by giving advice and training to prevent reoccurrence of the adverse events or not optimal use of blood components. the confidentiality of the information should be guarded sufficiently. for a physician-patient relation, even after a medical failure, a 'blame-free culture' with a central role for openness and transparency is necessary. for blood establishments and hospitals, there is an important role in the right assistance and help of the physicians concerned both on a practical and on an emotional way. m-pa-032 8 years of shot data 1996-2004: a view of transfusion safety in the uk and reactions. this will require investment in infrastructure, for which there must be a trade-off in improved transfusion safety. transfusion-transmitted infections and serious immunological reactions are rare; shot has highlighted the need for blood services to implement strategies to minimise bacterial contamination and transfusion related acute lung injury and will monitor their effectiveness. from the inception of shot it has been clear that the most frequent transfusion hazard is 'incorrect blood component transfused' i.e. a patient receiving a blood component intended for another person or not meeting appropriate requirements. only a minority of these events results in patient harm and is reportable under the terms of the directive. haemovigilance schemes such as shot, that analyse no-harm errors and near-misses, can reveal clues as to the root causes of 'wrong blood', which contributed to 16 deaths and 85 cases of major morbidity in the uk between 1996 and 2003 . analysis of such events shows that most errors occur in clinical areas, the most frequent being failure of the 'bedside check' . clinical audit data indicates that 10% of patients are transfused without a wristband or other form of identification, whilst anecdotal reports suggest that urgent clinical situations, massive transfusions and nocturnal transfusions are particularly error-prone. strategies aimed at reducing errors include structured education and competency testing, and methodologies, both high and low-tech, to ensure accurate patient identification in all circumstances. onethird of errors occurs in hospital laboratories; denominator data on laboratory workload shows that work done outside of 'core hours' accounts for 20% of all pre-transfusion testing but 40% of errors, suggesting that biomedical scientists 'on-call' or on shift work are working under pressure and beyond their competency. 85% of hospitals reported that they participated in shot in 2003, but only 47% of eligible hospitals reported adverse events suggesting that transfusion hazards remain under-recognised and under-reported. however, benchmarking of 'wrong blood' incidents against transfusion activity shows that the number of observed incidents is roughly proportional to blood use. if haemovigilance data is to contribute to improved transfusion safety, clinicians must be encour-aged to report all events, thus contributing to an evidence base that can be used to effect change and facilitate learning. barriers to reporting include cumbersome systems, lack of time and resource, lack of feedback and fear of blame. transfusion practitioners have a vital role in the recognition and reporting of adverse events, education and clinical audit, but must be adequately resourced and supported by senior clinicians and managers through an active hospital transfusion committee. *provisional. m-pa-033 passing the borders: when, how and where d pirc-tiljak croatian institute of transfusion med., zagreb, croatia there may come that moment in professional life when you have to follow a strong professional and ethical need and confront your evidence-based statements with the leadership, passing the border of your own small society. in order to protect patient's health and respect human right to be informed about all possible consequences of irregular medical therapy, insisting on professional dignity and truth, you feel responsible and follow-up processing of your serious error report. once you pass the border, trying to warn authorities and find ethical resonance and critical confirmation of your professional fears, you are 'persona non grata' . methods/results: reality checking, personal experiences and observations studying the path of serious error report. although the qc system functions, yet omissions happen . . . the possible reasons could be: lack of knowledge, lack of experience, lack of independency, personal confront of interest, immature leadership, political influence, even corruption . . . how strict do the authorities manage a fault, bearing in mind the responsibility toward the patients under the risk. there is a need to create an available, effective international expert's board which will react and give professional counselling support-asylum for endangered professionals who found enough power to blow the whistle. who will hear it? transfusion transmitted infections (tti) are a major source of concern given the repercussions of hiv, hepatitis c, bacteria and vcjd transmission by blood components. large amounts of resource have been expended in making products safer and in maintaining public confidence in the blood supply. identifying emerging infections of concern is a major activity for many transfusion services. of the long list of emerging infections identified by disease control agencies around the world, identifying those responsible for tti requires, amongst other things, that: • the agent is identifiable. • it is present in blood • it causes a disease of concern. • it is transmitted by transfusion. • it is present at relatively low frequency. • if a test is available (nat, serology or immunoassay) what the infection window period is. once an agent has been identified various approaches are possible, including: • donor selection by testing, geography or lifestyle e.g. wnv; • product selection e.g. erythrovirus (b19) antibody or bacterial testing; • product treatment e.g. pathogen inactivation; • patient selection e.g. cmv matching, immune status. against this background where should our attentions focus? some agents of initial concern are now known to be ubiquitous and have minimal disease association (ttv, gbv) although transmitted by transfusion. for others (coronavirus -sars or the possible kawasaki disease agent, dengue, flavivirus encephalopathies, avian flu, etc.) this is less certain, with agents arising from species crossover being of particular concern (avian flu, vcjd, hiv). • enhancement of automation/computerisation; • process control to provide an 'error-free pathway'; • (national) surveillance and trend analysis of results, preferably based on national working standards; • significantly increased sensitivity, especially from development of antigen/antibody 'combi' assays (e.g. for hiv, and recently, for hcv); • awareness of hbsag vaccine-escape mutants and design of assays to cope with this; • extension of range of agents and markers tested for (varies in different countries); • increasing range of assays available for testing donors with a relevant history of exposure to malaria or chagas' disease infection (for retrieval of otherwise wasted blood); • european union's in vitro diagnostics directive: this has caused some problems and reduced flexibility. nucleic acid testing (nat): nat continues to increase in blood service usage world wide, although not (as yet) to replace serological methods. trends include: • reduction in sample pool size; • increased automation (and process control); • increased multiplexing to detect 2 or more agents in the same assay; • increased number of agents being tested by nat (varies in different countries); • introduction of rapid and flexible nat to detect west nile virus, in north america. bacterial screening of platelet preparations: several countries have introduced (or will introduce) routine screening of platelet concentrates either with biomerieux, bactalert or pall ebds (02 depletion assessment). other bacterial testing methods are under active assessment, some rapid enough for possible 'point of use' testing. m-pa-036 evaluation of in vivo red blood cell recovery after processing with a new filter designed to reduce prions e nelson*, h taylor † , p whitley † and t lieu* *pall medical, covina, ca, † american red cross and evms, norfolk, va, usa background: a filter, called the leukotrap affinity prion reduction filter (prf1b filter, pall medical), has been developed to reduce the level of infectious prions, associated with several fatal neurodegenerative diseases including variant creuztfeldt-jakob disease (vcjd), from leukocyte-reduced red cell products. aim: the objective of this study was to evaluate the quality of leukocyte-reduced red cells (lr-rbc) processed through this filter and stored for 42 days. red cell quality was determined by measuring the in vivo red blood cell recovery 24 hours after re-infusion of the 42-day stored red cells. storage hemolysis and atp were also determined. methods: units of blood (450 ml) were collected from normal volunteers into the leukotrap wb system containing cp2d/as-3 anticoagulant/preservative solutions (pall medical). units were either processed to lr-rbc within 8 hours at room temperature (rt units), or after 24 hours at 1-6°c (cold units). the prion filter set was sterilely connected to the units on day 1, and the units were filtered and stored for 42 days. samples were taken pre-and post-prion filtration and post-storage for plasma hemoglobin and atp determinations. post-storage samples were taken for labeling with 51-cr radioisotope, re-infusion, and determination of the 24-hour in vivo rbc recovery. a donor sample was also labeled with 99m-tc to allow for red cell mass determination. thus, both single-and double-label 24-hour recovery values were calculated. results: twelve units were collected. in vitro testing was completed on all units. in vivo testing was completed on 11 units. the mean single-label 24-hour recoveries were 84.4% and 85.7% for the rt and cold units, respectively. the mean double-label recoveries were 81.9% and 84.1% for the rt and cold units, respectively. the overall combined mean in vivo and in vitro results are shown in the table. conclusion: the 24-hour in vivo red cell recovery means are well above the fda and council of europe's requirement of achieving a mean post-transfusion survival of no less than 75% of the transfused red cells, and they are comparable to this center's previous results of red cells filtered using the licensed leukotrap rc system with cp2d/as-3 (pall medical introduction: to reduce the risk of platelet transfusion-associated sepsis (tas), methods to routinely screen for bacterial contamination have been implemented. pathogen inactivation treatment of labile blood components provides an alternative means to prevent tas. the intercept blood system for platelets (baxter healthcare) has received the ce mark and has been introduced into clinical practice. aims: this study compared the efficacy of bacterial screening using a culture method (bact/alert system, biomerieux) with pathogen inactivation (intercept blood system) for prevention of transfusion of platelet components contaminated with bacteria. methods: seven strains of bacteria associated with tas, including gram-positive staphylococcus epidermidis, streptococcus agalactiae, and staphylococcus aureus, gram-negative escherichia coli, and klebsiella pneumoniae, and the anaerobes propionibacterium acnes and clostridium perfringens were studied. for each strain, three double-dose platelet concentrates (~6 ¥ 10e 11 platelets in 600 ml of 35% plasma and 65% intersol) were collected using the amicus® cell separator. on day of collection, calibrated stocks of bacteria (20, 200, 2000 cfu) were added to the double units. each double unit was divided into two identical products containing 10, 100, or 1000 cfu of bacteria and stored overnight under conventional blood bank conditions. the control platelet concentrate was not treated. the test platelet concentrate was treated with the intercept process (150 mm amotosalen + 3 j/sq cm uva). both units were cultured using the bact/alert system at the time of bacterial inoculation and on days 1, 2 and 5 of storage. samples (10 ml) were taken for both the aerobic and anaerobic cultures. a platelet sample was considered contaminated with bacteria if a positive signal was registered within 120 hours of culture. results: for control platelet concentrates, cultures failed to detect low-dose inocula. the time to positive culture varied with the bacterial strain, contamination level, and time of sampling. at 10 and 100 cfu per product, 4 strains (s. epidermidis, e. coli, c. perfringens, s. agalactiae) and 2 strains (e. coli, c. perfringens) tested negative after 5 days of platelet storage, respectively. k. pneumoniae tested positive after 7-8 hours of culture when sampled on day 2 of platelet storage for both 10 and 100 cfu per product. at 1000 cfu per product, p. acnes tested negative in aerobic culture and c perfringens tested negative in anaerobic culture after 5 days of platelet storage. the anaerobic cultures of p. acnes became positive after 50 hours of culture when sampled on day 5 of platelet storage. of the 7 strains studied, only s. aureus consistently tested positive after 7-16 hours of culture. in contrast, all test platelet concentrates treated with intercept remained negative by bact/alert cultures throughout the entire 5-day observation period regardless of the strain and the contamination level. conclusions: bacterial detection using cultures may fail to detect low levels of bacteria typically associated with platelet contamination at time of collection and processing. failure to detect bacteria will result in the release of contaminated platelet products with 'test negative-to-date' status. in contrast, inactivation of bacteria is capable of preventing release of contaminated platelet components. background: since nat implementation for hiv-1 and hcv rna in france, the residual risk (rr) of transfusion-transmitted infections (tti) has dramatically decreased. the rr estimates, for a threeyear period from 2001 to 2003 showed a significant decrease from 1/1 700 000 and 1/1 560 000 before nat implementation to 1/3 150 000 and 1/10 000 000 after nat implementation for hiv and hcv respectively. as for hbv, the serological screening is only based on both hbsag and anti-hbc assays. for the same period, the rr estimate for hbv is 1/640 000, five times higher than hiv one and 16 times higher than hcv one. aims: as the overall rr of tti is mainly related to hbv, and given the availability of hbv nat assays, a study was conducted to determine whether hbv nat has the ability to further reduce the hbv rr and then should be implemented in blood donor screening in france. we have estimated the wp reduction by nat in comparison with one of the most sensitive hbsag screening assays, on 10 commercial seroconversion panels (bioclinical partners, franklin, ma, usa). the nat test was the procleix ultrio assay (genprobe/chiron, san diego, usa). the hbs ag test was the prism hbsag (abbott, france). the comparison was performed on both neat samples and diluted samples 1/8, 1/16, and 1/24, in order to simulate minipools of different sizes. then, we have calculated the yield of hbv-infected donations detected by nat relative to prism hbsag assay. results: on the basis of a window period (wp) of 56 days, ultrio assay is projected to close the wp by an average of 15 days on undiluted samples, 5 days in minipools of 8 samples, 4 days in minipools of 16 samples and only 2 days in minipools of 24 samples. the projected yield calculated on the basis of 2.4 million donations collected per year in france, would be 0.65 unit per year for minipool-nat and 2 to 3 units per year for individual donation nat. conclusion: introduction of minipool-nat will offer only a little added benefit to transfusion safety relative to current serological screening strategies based on both hbsag and anti-hbc assays. hbv minipool-nat is then unsuitable for hbv screening in french blood donors. single-sample nat or minipool-nat with smaller pool sizes and/or modified procedures (genome enrichment or test improvement) would be more relevant. automation when technologically and practically feasible is a prerequisite for single-donation nat. therefore, decision has been made not to implement hbv nat in the french transfusion network until fully automated systems will be available. however, as the prevalence of hbv infections is higher in the overseas territories than in continental france, and as nat is performed on individual donations in these sites, hbv-nat has been implemented since december 2004 in these territories. combined detection of hepatitis c virus core antigen and antibody as an alternative to nucleic acid testing in blood screening grating the capillary cytometer with a robotic workstation and a small footprint centrifuge. significantly, there was no decrement in system performance following automation: 474 of 501 clinical samples (94.6%) typed identically with this system and cat, and 25 of the 27 discrepant results were eventually resolved in favor of the automated cytometry method. testing showed high-throughput capabilities (currently 280 samples/day) and was inexpensive. to demonstrate the flexibility of this testing platform, we also developed a method to perform completely automated counting of residual wbcs (rwbc) following leukoreduction of blood components. there were no significant differences in accuracy and precision when rwbc in analytical controls and authentic clinical samples were quantitated by the automated capillary cytometry method or the leucocount method performed manually. given the flexibility of this system, it is very likely that additional blood bank assays could be modified for high performance automated testing on this platform. noninvasive prenatal genotyping on cell free fetal dna in maternal plasma ce van der schoot sanquin research, amsterdam, netherlands in 1997 lo et al. demonstrated that in the maternal circulation small amounts of cell free fetal dna are present, concentrations ranging from on average 25 genome equivalents(geq)/ml early in pregnancy to about 100 geq/ml at the end of pregnancy. most likely this dna is derived fom apoptotic syncitiorophoblasts. the human placenta is hemichorial, which means that the syncitiotrophoblast is in direct contact with the maternal blood flow, and apoptotic nuclei are directly released into the maternal circulation. the cell free dna is very rapidly cleared from the circulation, the t1/2 being only 15 minutes. in 1998 we have shown that this cell free fetal dna could be used for rhd genotyping. in the last 5 years many groups have shown the successful application of different prenatal genotyping assays such as fetal sexing, thalassemia, achondroplasia, duchenne's disease, adrenogenital syndrome etc. on this source of dna. importantly, no false positive result have been described due to the presence of fetal dna from previous pregnancies, the main draw back of prenatal diagnostics on circulating fetal cells. at present prenatal rhd genotyping has been introduced in routine diagnostics in the united kingdom, france and the netherlands. in large scale high throughput studies it has been shown that the diagnostic accuracy of prenatal rhd genotyping is over 99%, and it is to be expected that in the netherlands this screening will soon be introduced to restrict the antenatal anti-d immunoprophylaxis to women carrying rhd-positive fetuses. in a large european project (safe, co-ordinator maj hulten, warwick uk) many researchers collaborate to further explore the possibilities of cell free fetal dna for future diagnostics. standard operating procedures for the isolation of plasma dna have been established. control pcrs for the presence of fetal dna have been developed. recent findings on differences in methylation status of placental genes in fetal dna opens new possibilities. the main technical problem that hampers wide application of prenatal genotyping is the impurity of the fetal dna, only 1-5% of the cell free dna in plasma is from fetal origin. this makes diagnostic assays on numerical chromosomal abnormalities impossible. and also for many single nucleotide polymorphisms (snps) such as almost all blood group antigens, assays are hampered by aspecific amplification from maternal dna. our own preliminary results indicate that this latter problem can be solved by pna clamping. the addition of a pna probe specific for the k-allele partial d feature d antigen alteration, often identified as distinct 'partial' d epitope loss. the clinical impact of partial ds is due to the ability of their carriers to form anti-d antibodies upon confrontation with regular d after transfusion, or pregnancy. this leads to the -naively spoken -contradictory finding of an allo anti-d antibody in a d positive individual in connection with a negative autocontrol. the antibodies themselves include the same fatal clinical potential as anti-d antibodies of d negative individuals, but may be even more hazardous since unexpected in d positive individuals a priori. d categories (ii to vii) represent a nomenclatorily defined subgroup of partial ds. the molecular cause of partial d lies within single (caused by point mutation in the respective rhd gene sequence), or multiple amino acid exchanges (caused by gene conversion events leading to rhd-rhce-rhd hybrid genes) which determines a qualitative d antigen alteration, rendering them distinguishable from regular d by a partial d carriers immune system. nowadays, transfusion specialists and gynaecologists are more or less aware of these facts and are taking them into consideration in the clinical setting. most partial d exhibit decreased d antigen density, enabling principal recognition of them. however, routine serological methods may not properly recognise all partial ds and will identify their carriers after immunisation only, which represents a reactive diagnostic/therapeutic attitude second best to an actively prognostic one. this actively prognostic proceeding with respect to early detection of partial ds became widely feasible by rhd dna typing techniques. currently, routine rhd dna typing techniques offer an affordable, accurate and fast approach to an unambiguous identification of partial ds and their reliable discrimination from weak d types, not at risk for allo anti-d immunisation. a reasonable proactive proceeding could e.g. demand for (once in a lifetime) routine rhd dna typing of all weakly expressed ds as defined by serology, since most partial ds also meet this phenotype. rhd allele frequencies and their geographical and regional prevalence will certainly have an important impact on dna typing strategies and their (mandatory) specificities. fluorescence cytometry for completely automated immunohematology testing d roback*, b barclay † and d hillyer † *emory university school of medicine, atlanta, † transfusion & transplantation technologi, decatur, ga, usa we previously described a methodology for accurate immunohematology testing by fluorescence cytometry [roback, j.d. et al. (2004) transfusion 44 (2), 187]. this system utilized low-speed centrifugation of 96-well filter plates for red cell staining, and a smallfootprint capillary cytometer for data acquisition. when authentic clinical samples from hospitalized patients were tested for abo group, the presence of d antigen, and red cell alloantibodies, the results were well-correlated with those obtained by commerciallyavailable column agglutination technology (cat). this system determined the correct abo group and d type for 98.7% of 520 samples, compared to 98.8% for cat (p > 0.05). when samples were tested for unexpected alloantibodies, fc determined the correct result for 98.6% of 215 samples, as compared to 96.3% for cat (p > 0.05). this novel method was better than cat at detecting weak anti-a (p < 0.0001) and alloantibodies. based on these promising results, we sought to completely automate this method by inte-prevents the aspecific amplification of the k-allele, and makes it possible to detect the fetal k-allele in the presence of excess of maternal k-alleles. furthermore, it has been shown that fetal dna is in the plasma present in shorter fragments (<300 bp) than maternal dna. size separation of cell free fetal dna can therefore be used to increase the relative concentration of fetal dna, which will help the development of new genotyping assays. in conclusion, cell free fetal dna in maternal plasma is nowadays routinely used for prenatal rhd typing and fetal sexing. new technical developments will make it possible to extend these indications to other blood group antigens in the near future. more insight in the characteristics of fetal dna might finally lead to wider applications, including numerical chromosomal aberrations. furthermore, it might become possible to apply genomic dna microarrays for the screening on many different inherited diseases, including hemoglobinopathies. determination of the affinity of anti-d present in the serum of immunized subjects and in anti-d ig preparations by a method using unlabeled antibodies p lambin*, m debbia* and y brossard † *institut national de la transfusion, † chp hopital saint antoine, paris, france introduction: few data are available concerning the affinity of maternal anti-d responsible for the hemolytic disease of the fetus and the newborn (hdn), and the affinity of anti-d immunoglobulin used for the prophylaxis of that disease. we recently described a method to measure the affinity (ka) of untagged anti-d monoclonal antibodies. aims of the study: in this work, a similar method was applied to determine the affinity constant (ka) of polyclonal anti-d present in the serum from d-immunized mothers and donors and from anti-d ig preparations. methods: a constant amount of o r1r rbcs was sensitized with increasing concentrations of anti-d present in the sera from immunized subjects, and in anti-d ig preparations. at equilibrium, the amount of anti-d bound to rbcs was measured by elisa. the scatchard equation (linear regression) and the langmuir equation (hyperbolic regression) were used to determine the ka of anti-d. the experimental data fitted well with the scatchard equation (mean r † = 0.95) but a better correlation was observed with the langmuir equation (mean r † = 0.99). in 11 maternal sera, the mean ka of anti-d was 5.6 ¥ 10 to the 8 m-1 (from 2.8 to 12 ¥ 10 to the 8 m-1). in the sera from 6 immunized donors, the mean ka was 3.9 ¥ 10 to the 8 m-1 (from 1.5 to 6.8 ¥ 10 to the 8 m-1) and in 5 lots of anti-d ig, the mean ka was 3.4 ¥ 10 to the 8 m-1 (from 3.1 to 4.2 ¥ 10 to the 8 m-1). the comparison of anti-d affinity measured in 5 cases of hdn in which infants presented a fetal anemia and in 6 cases of hdn in which infants presented only a postnatal anemia showed no significant difference. the mean value of ka in the cases of fetal anemia was 5.4 ¥ 10 to the 8 m-1 whereas in the cases of postnatal anemia the mean value of ka was 5.8 ¥ 10 to the 8 m-1. conclusion: the method previously described for monoclonal anti-d was applied to polyclonal anti-d present in the serum of d-immunized subjects and in ig preparations. the experimental data fitted well with the langmuir equation, and the affinity of polyclonal of anti-d was measured with accuracy. in addition, no significant difference was observed (at least in the 11 cases of this study) between the affinities of anti-d measured in the most severe cases of hdn (fetal anemia) and in the less severe cases of hdn (post-natal anemia). introduction: cryopreservation of platelets is widely used in platelet immunology to ensure the availability of well characterised panel cells for the detection of hpa antibodies. but recovered platelets do not express the hpa-15 alloantigens. aim of the study: here we describe a method for the successful preservation of platelets by lyophilization. we report the value of this new reagent for the detection of hpa alloantibodies and especially anti hpa-15 alloantibodies. methods: rehydrated lyophilised platelets (lyo p) were tested for their reactivity with monoclonal antibodies against gpiibiiia, gpibix, gpiaiia and cd109 by flow cytometry. the levels of reactivity were comparable with the ones obtained with fresh platelets. the rehydrated platelets were used in the maipa with a panel of hpa antibodies (anti-hpa-1a, 30; anti-hpa-1b, 3; anti-hpa-3a, 3; anti-hpa-5a, 3; anti-hpa-5b, 50; anti-hpa-15a, 2 and anti-hpa-15b, 4). results: all hpa antibodies showed the expected pattern of reactivity and in several cases absorbance reading were well above those obtained with fresh platelets. absorbance values produced by inert sera were comparable with those obtained with fresh platelets (ranges 0.001-0.008). interestingly, we used lyophilized platelet with a high expression of cd 109 bearing the hpa-15 system and we have detected 24 anti hpa 15 antibodies among 1000 sera previously negative with fresh platelets. nineteen sera concerned patients suffering from hematological diseases and 5 from pregnancy women. conclusion: lyophilized platelets are possibly an ideal reagent for the platelet immunologist to be used for the detection of hpa antibodies. moreover, this work bring new insights on the hpa-15 system in platelet transfusion. we are now pursuing more extensive validation studies with a larger number of samples representing all known hpa specificities and several diseases. the diagnosis and treatment of sick infants and children requires a broad knowledge of physiology, biochemistry, genetics and the application of sophisticated testing and treatment options. one of these options is transfusion of blood and blood products. transfusion of the infant, especially the premature infant, and sick child, especially those with major organ dysfunction, requires careful consideration of their unique metabolic, hepatic and renal clearance mechanisms. guidelines that direct the indications for transfusion differ from those in adults. non-invasive measures of oxygen delivery and oxygen offloading may assist in guidelines for red blood transfusion. metabolic complications from massive transfusion and/or the manipulation of blood products must also be considered. evidence from a high quality randomised controlled trial suggests that anaemia is also well tolerated by critically ill patients. a restrictive approach to rbc transfusion that maintained the hb concentration between 70 and 90 g/l was found to be as effective as and possibly superior to a more liberal strategy of maintaining the hb concentration between 100 and 120 g/l. there are concerns that some groups of critically ill patients, such as those with cardiovascular disease and patients who are difficult to wean from mechanical ventilation, may benefit from higher hb levels. rbcs also have a role in primary haemostasis and higher triggers may be appropriate in coagulopathic patients. it is important to realise that blood is not a uniform product and the clinical efficacy of rbc transfusion may vary. one factor that may have a considerable effect on the quality of the rbc product is the storage time. rbcs undergo marked changes during refrigerated storage. the implications of these changes on tissue oxygenation are not known but these concerns have led some clinicians to request 'fresh' blood for critically ill patients. there is insufficient evidence to support such practice. it is of great practical importance to determine if, or when, fresh rbcs could be superior to stored rbcs. background: with a decreasing blood donor base, fully tested, fresh unrefrigerated whole blood (fuwb) has been found to be a more efficient and effective use of a limited resource in place of, or as an adjunct to, traditional blood component therapy in surgical situations associated with massive blood loss. aims: to outline the use of fuwb in situations where there is potential for intractable bleeding associated with major surgery, and evaluate platelet function in fuwb versus platelet components. methods: outcomes of fuwb and traditional blood component use were examined for 20 cases of cardiac bypass surgery. in addition, exclusive use of fuwb for 23 burns debridement cases was analysed. an evaluation of platelet function in whole blood compared to platelet components was also performed by measuring platelet aggregation and activation parameters. results: there was a decreased requirement for blood components following administration of whole blood post cardiac surgery. whole blood usage for burns debridement surgery eliminated the requirement for additional blood components. platelet activation was markedly reduced in whole blood compared to component platelets, and this may be one reason for the increased efficacy of whole blood in these clinical settings. conclusion: fuwb appears to have a role in minimising blood product requirements and consequent donor exposure in situations associated with massive blood loss. m-pa-051 transfusion practice for coronary artery bypass surgery in greece s lakoumenta, m vassili, g hatzidimitriou, t asteri, p stratigi, s kanellas and g palatianos hellenic society of blood transfusion, athens, greece cardiac surgery is associated with a demand for allogenic blood and blood product availability as well as a considerable consumption. the impact of consensus guidelines for allogenic blood transfusion during coronary artery bypass graft surgery (cabg) in us attracted great attention 1. the present study is conducted in order to reveal the transfusion practice in greece on a similar population i.e. patients undergoing cabg operations. methods: five participating centers collected data concerning transfusion of allogenic blood and blood products in patients undergoing elective first time cabg procedures, as well as parameters that may influence blood loss, such as duration of the operation, cardiopulmonary bypass time (cpb) etc. the total estimated blood loss was calculated as the sum of red blood cell volume reduction [(body weight in kg ¥ 60 ml/kg) ¥ (admission haematocrit-discharge haematocrit)]+(red blood cell volume transfused). results: results are shown in table 1 : means, standard deviations, and p-values of the wilcoxon test comparisons between the hospitals. a preliminary analysis of data from 5 centres (268 patients) showed no difference in patient characteristics (age, body weight, male to female ratio). there is a statistically significant difference (p < 0.0001) between the five centers in duration of the operation, cpb, estimated blood loss and volume of transfused plasma. red cell use showed also a variation which however did not reach statistical significance (p < 0.02). the center with the highest figure for blood loss has the lowest volume of allogeneic red cell transfusion because of the use of cell salvage. conclusions: although variations, as those observed in greek cardiac surgery centers, have been documented in other countries, the variation in the use of plasma is striking and we are in the process of trying to identify the reasons. our study is in progress and additional data are being collected and will be presented. introduction: premature infants and at term newborns have an higher circulating blood volume per kilogram than the adults (100 ml/kg in premature; 80 ml/kg in at term), for this reason, in case of neonatal thrombocytopenia, a specific hemocomponent, with a very high platelet concentration, needs for transfusion therapy. the laboratory criteria for platelet transfusion are the following: (a) a plt count < 150 ¥ 10 9 cells/l if bleeding is observed; (b) a plt count < 20 ¥ 10 9 cells/l without bleeding; (c) a plt count < 50 ¥ 10 9 cells/l in newborns showing critical clinical conditions. aim of this study: in this study, we have monitored the plt transfusion therapy in our neonatal intensive care unit (nicu) in the last four years. methods: effects of plt transfusions have been followed in 37 children (31 premature infants and 6 at term newborns). the weight of premature infants ranged from 600-1800 g and at term newborn from 1800-4000 g. gestation age of premature infants ranged from 26-36 weeks and of at term ones, of course, from 37-42 weeks. for every platelet transfusion in these newborns, the volume of platelet concentrate has been of 10-20 ml/kg, with a plt count < 700 ¥ 10 9 cells/l. results : in the study period, 77 plt transfusions have been performed: 23 children have been only transfused one time, while multiple plt transfusions (ranged 2-10) needed for 14 children according to clinical conditions. the observed clinical indications for transfusions have been the sepsis with haemorrhagic syndrome (26 cases) , haemorrhagic syndrome without sepsis (7 cases) and neonatal alloimmune thrombocytopenia without haemorrhagic syndrome (4 cases). after 24 hours from transfusion therapy, the absolute plt count and the correct count increment have increased in all 37 little patients. the highest increase in plt count was 45 ¥ 10 9 cells/l, while the lowest 5 ¥ 10 9 cells/l. no difference in the efficacy of therapy has been detected between premature group and at term group. 94% of children have been discharged from hospital in good general conditions without complications in following controls. in conclusion, we can affirm that plt transfusion in premature infants and in at term newborns is an efficient and safe treatment of severe haemorrhagic conditions. however a collaboration between nicu and transfusion center is necessary to choice the adequate platelet concentrate's volume for transfusion and the best plt donor for the newborn. developing transfusion strategies 27 fusion society of turkey (bbtst) in 2004 with contribution of 253 blood transfusion centers. according to these figures 51% of centers attended operates apheresis procedures. two centers informed us that apheresis in the hospital was carried out at blood bank. there was not enough information from one center, so it was excluded. of the 51 blood banks performing apheresis, 30 were university hospital blood banks. another 38 blood banks were producing both productive and therapeutic, 10 produce only productive and 2 produce only therapeutic procedures. one center did not respond. all centers reported to prepare and separate erythrocyte and plasma. however only 48 centers reported to prepare random platelets as well. each center had apheresis machines between 8-15. a total of 19 centers was carrying out around <500 procedures, 12 around 501-1000, at 9 centers about 1001-2000, at a further 10 centers around 2001-5000 procedures a year (one center was excluded). of the responders to the survey 36, all procedure were done at blood banks, whereas at 6 of them all were carried out by the hematology clinics. at 8 other centers, productive procedures were conducted by the blood bank, and therapeutics were performed by the hematology division. a total of 48 blood banks stated that they have not kept the platelet suspensions produced and used them straightaway. productive apheresis center capacities were as shown: 13 centers < 5000, 16 centers 5000-10000, 13 centers 10001-20000 and 6 centers > 20000 units have donations a year. around 68% of all apheresis procedures were carried out by large well run blood banks. conclusion: as the use and production of random platelets increase, and settle of apheresis devices in big centers will eventually decrease the demand of apheresis procedures and keep the welltrained staff at big centers, decrease the cost thereafter. • planning of resources for the financing of the bts, adoption of a methodology for creating and adjusting the price list of products, adoption of the yearly plan of needs for blood/products and services of health institutions which use blood/products. • achieving recognition of real costs of products and services from the health insurance fund and ministry of health. • harmonization of low level of acclaimed costs and real costs of basic transfusion activities. results: acclaimed costs for activities in transfusion practice (collection, testing, processing, storage, distribution and transport) as a reflection on the price of the products are 60% lower than real costs. the prices of health services in the official price list are much lower than the proportion of costs of material resources needed for the realization of these services. this especially affects the management of independent blood establishments (bti's in serbia) with core blood transfusion activities as their basic field of work, in comparison with the 44 hospital based transfusion services, which are financed within the budget of the whole hospital. the 44 hospitals with hospital based transfusion services involved partly in core transfusion activities are completely financed by the health insurance fund, while independent blood establishments are financed through the price of products and services they provide. conclusion: in order to provide adequate quantities of safe blood/products for the end user -the patient, it is elementary to create stabile and equal financial management conditions for the whole blood transfusion service in serbia. this can be achieved only by continuous cooperation of the health insurance fund, ministry of health and independent blood establishments. sion centers (rbtc). the activities on promotion and organization of voluntary, nonremunerated blood donation, blood collection and patients' services are carried out in the rbtc and in 24 departments of blood transfusion (dbt), part of the district hospitals. the collected units in dbt are transported by special cars to the ncht and the rbtc for processing, testing and control. the same transport is used for the requested by dbt blood components for storage and distribution to hospital departments. thus the issued components are with an equal quality and safety for all patients throughout the country. lbbdbt introduces hemovigilance as a mandatory system, covering the whole chain of the blood transfusion process. it includes as well the creation of registries at a national, regional and district level of blood donors, recipients of blood products and all activities of the blood transfusion service. . seventy hospitals are exclusively users of blood, blood products and services. the current organization of blood transfusion services faces the following problems: fragmented transfusion service, lack of a national blood policy, the blood program is not nationally coordinated, limited knowledge on quality management, inadequately distributed human resources, limited material resources, lack of it system, lack of planned, continuous skill upgrading. as a direct consequence we have: suboptimal blood collection activities, inadequate blood supplies significantly vary between seasons, high percentage of replacement donors, outdated methodologies, old, even obsolete equipment, the quality of blood products is not standard, there is a lack of traceability. aim of study: to reorganize blood collection activities in serbia to increase collection of safe blood up to 4% (304 000 blood units). methods: division of responsibilities between blood establishments and hospital based transfusion services by: • optimizing organizational structure • implementing blood collection standards to enhance blood safety and donor care • gradually replacing family donors with a network of voluntary non-remunerated blood donors from low risk population groups • creating and implementing a training strategy. results: through the eu funded project support to a national blood transfusion service in serbia, we are in the effort of integrating the services and standardizing their work. the blood collection working group began by dividing serbia into 3 blood collection regions: north, central, and south. each region is divided into sub regions covering approximately half a million population (4 in the north, 8 in the central and 4 in the south region). each sub region will have one standard mobile blood collection team to collect 80 blood units daily, i.e. 19 000 annually. the 19 000 blood units per 16 teams provide the 304 000 blood units (4%) to cover hospital needs in serbia. to this effect, the following has been achieved: • blood collection activities in serbia analyzed • performance analysis of bte and hbts mobile teams in place • two model standard mobile teams tested in the field • national blood collection sop's written • national donor questionnaire form prepared • national set of blood collection standards prepared • list of donor deferral criteria prepared • blood collection equipment renewed • regional reorganization plans in progress. the objectives and results can be achieved by the participation and mutual cooperation of all institutions involved in blood transfusion within an integrated, standardized system with clearly delegated responsibilities. p-005 60 years of the national blood transfusion institute in serbia n nedeljkovic national blood transfusion institute, belgrade, yugoslavia nbti was founded in 1944 . since 1953 and unpaid blood donation is mandatory, organized in cooperation with red cross. blood donation is regulated by the law in 1974, 1993/94. codex of voluntary blood donation and health care staff has also been established; 300 000 blood donors donates blood annually. in the past 60 years, there was over 3 million blood donations, performed in accordance with who regulations. over 250 transfusion medicine specialists and 700 technicians specially trained for the work in blood transfusion service (n = 53), perform transfusion medicine doctrine of rational labile blood component and stable blood derivatives therapy, based on the selfsufficiency concept in fr yugoslavia with 10.3 million inhabitants in serbia, montenegro and kosovo. plastic blood containers and tests are imported or given as humanitarian aid gift and from 2003. now, they have been regulated by tender. in 1951, test to lues was introduced, to hbsag in 1971, to a-hiv in 1985 and to a-hcv in 1993. information system was introduced in 1987. nbti includes: national haemovigilance coordinatoin center, center for medical care of haemophiliacs, tissue typing center, center for prenatal and perinatal protection of pregnant women and newborns. activities of nbti are organized through: center for planning, organization and development of blood transfusion service, center for blood collection, preparation and distribution, center for immunology and immunochemistry, plasma fractionation center for plasma in 8 west balkan countries, center for diagnostics means, center for quality control of drugs and medical and diagnostic means, center for education and training and scientific research work. nbti is the third year of gmp, sop, yus iso 9002 implementation. in the current reform of transfusiology system we are aiming for 4 percent of voluntary blood donation. nbti is the publisher of the national bulletin of transfusion medicine and it is included in the education system of the belgrade university medical faculty and the estm in belgarde 2003. the problems of blood service in russia ea selivanov and t danilova russian inst. of hematol. & transfusiol., st. petersburg, russian federation background: the blood transfusion service (bts) development as a platform for providing the hospitals with blood and blood derivatives is an important national problem. aims: russian blood service assessment with international comparison. methods: a study was conducted on the base of the reports from all regions of russia followed by a computer statistical analysis. results: blood and blood components were collected in the russian federation in 2003 in 190 stations of blood transfusion and in 1065 blood transfusion departments at big hospitals. amount of donors in 2003 was equal to 2 202 853, voluntary donors being 87.2% of them. the average number of whole blood donations in relation to the general population is 25 per 1000 inhabitants, and on average 28 percent of the donor base consists of first time donors. the average number of blood collected in relation to the general population and health care system is 11.4 ml per inhabitant and 1240 ml per one bed. an average volume of one blood donation is 396 ml. blood was collected into plastic bags containing domestic or foreign anticoagulants. about 93.2% of collected blood is used for procurement of blood components and preparations, 0.8% of banked blood is used for transfusions. amount of donors and the volume of whole blood have been significantly decreased for the last 15 years. at present in russia all donations are tested for abo blood group, rh(d) type, anti-hiv-1/2, hbsag, anti-hcv and syphilis. the total percentage of blood discarded after testing for transfusion-transmissible infections is 4.5%. 32% of plasma is obtained by plasmapheresis. blood components collected are as ffp, rbc, frozen rbc, 1eukocyte-and platelet-depleted rbc, rbc suspension, and preparations: 10% albumin, immunoglobulins, and cryoprecipitate. as to blood safety measures -implementation of blood components leucodepletion and ffp and rbc quarantine in process. the new national strategy of bts reorganization has been developed. it includes the following: increasing the visibility and resource commitment to blood issues at the national, regional and municipal levels; the national voluntary donor programme promoting; blood safety increasing; blood collection, testing and pro-cessing concentration in federal and regional bts establishments, appropriate blood and blood components usage. calculating the cost of blood in turkey n solaz, s kemahli and s cin ankara university, ankara, turkey background: like other fields of the medicine cost efficacy is gaining importance in blood banking and transfusion medicine since last few years. since last 5 years even the most developed countries started to discuss about the cost of blood. in turkey ministry of health determines the cost of blood annually. aim: to establish a safe, cost effective and reliable prices for blood components. methods: turkish ministry of health (moh) started to determine the cost of blood components as 'all inclusive' principle. this means that cost of a unit of blood component will cover all conventional expenses such as; blood typing, infectious screening, labour, consumables, etc. this system has provided uniformity to blood component costs but if the system is not controlled and followed properly it will cause serious risks. there might be some blood banks which will not respect the safety regulations and may modify the test standards for decreasing the cost of tests. conclusion: current blood product pricing system looks generally reasonable and reliable but moh should establish close follow up systems for avoiding any abuse on the safety of blood. background: a positive direct antiglobulin test occasionally occurs in normal blood donors, and is often discovered when the donor's red cells are found incompatible in a compatibility test. the incidence of a positive dat was expected to increase since more sensitive techniques (gel test) were installed. the aim of our study was to examine whether dat positive otherwise healthy donors presented any clinical or laboratory abnormalities. methods: in the first 19.000 cross-matches last year (in 10 months) 38 were found incompatible due to dat positivity of blood donors' red cells (0.02%). dat positive [(1+)-(3+)] samples were only igg positive in 32 cases, only c3d positive in 5 and igm positive and c3d positive in 1 case. all blood donors were notified and thirty two of them responded to a request for a further sample. a complete blood count, a reticulocyte count, bilirubin (total, direct, indirect), transaminases, serologic immunological tests (ana, anti-dna, anti-ena, rf, anticardiolipin antibodies), quantitative assessment of immunoglobulins, aptt and lupus anticoagulant were performed, as well as serologic tests for markers of viral infections. dat and iat were performed by gel test (id-diamed) according to the manufacturer's instructions. dat were performed with polyvalent and monovalent reagents (anti-igg, -igm, -iga, -c3c, -c3d). the blood donors were also examined clinically. the donors who had positive immunological tests were referred to a rheumatologist for further investigation. results: among the thirty one blood donors eight had received medication the last 24 hours before blood donation, two had been vaccinated for hepatitis b recently, four presented signs of a viral infection soon after blood donation, three had evidence of an allergic condition, five had positive tests for anticardiolipin antibodies and ana, two were positive for anticardiolipin antibodies only and two had a positive ana test only. in six blood donors we did not find any abnormality that might be interrelated to dat positivity. conclusions: all blood donors with positive dat should be requested to undergo further investigation. some of them are possibly candidates to long medical follow-up, especially those with other immunologic abnormalities such as positive ana and/ or anticardiolipin antibodies. the eligibility of such donors for future donation of whole blood, platelets or plasma needs to be elucidated. tions: usefulness, frequency and sincerity in answering questions. donors could choose one of the offered answers and elaborate in writing the answer they have chosen. results: of the 1000 donors that participated in the survey 947 (97.4%) answered the questionnaire, 820 (86.6%) men and 127 (13.4%) women. that the survey was useful thought 91% and 9% that it was not. opinions were elaborated by 61.1%. that the questionnaire should be completed before each blood donation was the opinion of 81.2%, 17% thought it should be filled out only the first time blood is donated and 1.8% that the questionnaire should not be completed at all. the answers given were sincere in 95.4% of blood donors, 2% were not and 2.6% were given automaticallywithout comprehension. conclusion: most donors believe that completing the questionnaire before each blood donation is an effective way to increase safety by preventing potentially infected individuals from donating blood. they are also aware of the importance of answering questions truthfully because the end result is protecting the wellbeing of both blood donors and receivers. analysis of blood donor's deferral in national institute for transfusion medicine -skopje for the last five years (2000) (2001) (2002) (2003) (2004) p blagoevska*, i nikolovska † and r grubovik* *national institute for transfusion medic, skopje, † medical center, prilep, macedonia introduction: safety of blood and blood products depends on many different factors, starting with selection of blood donors. the aim of this study is to analyze the number of deferred blood donors and the reasons for their deferral, as well as the total number of blood donors in nitm and their correlation (voluntary/family donors). materials and methods: this is a retrospective, epidemiological study and data were taken from the blood donor's registry in nitm from 01.01.2000 till 31.12.2004. statistical mass includes blood donors who came to nitm to donate blood in the mentioned period. results: there were total 14 229 donors in nitm and 541 (3.8%) deferrals. 71.5% of deferred ones are male, as well as in the group with donated blood (males are predominant). the most common reason for deferral is low hb level in 232 (42.9%) blood donors, use of drugs -78 (14.4%), low blood pressure -47 (8.7%), high blood pressure -43 (7.9%), infections -30 (5.5%), cardiovascular diseases -21 (3.9%) and others. relation voluntary/family donors is almost equal (49. 6 : 50.4 ). in the last two years the number of voluntary blood donors is increasing (60 : 40), which is good sign. conclusion: percentage of deferred blood donors in first three years is ~2%, which is result of insufficient data and it is increasing in the last two years (>6%). reasons for deferral are predominantly from temporary character (98.9%). permanent deferrals are only 6 (1.1%), which is probably due to good education of the population and self-deferral. we should establish the national registry for deferred donors, as well as for the donors with positive markers for tti. we should design a strategy for returning of temporary deferred donors. regruting blood donors in multiethnical environment p blagoevska*, r grubovik* and k elezi † *national institute for transfusion medic, skopje, † medical center, gostivar, macedonia introduction: population in r.macedonia consists of 75% macedonians, 22% albanians and 3% others (serbs, gypsies, turks). over 80% of blood donors are voluntary non-remunerated and ~20% are family donors. transfusion service and red cross should recognize the values and cultural differences of minors groups and recruiters should developed methods for reaching and motivating them to donate blood. the aim of the study is to present the ethnical structure of our donors and to develop strategy for their regrutation and retention. the study reviews the results from the blood donation actions among the high schools and university students in west part of the country (multiethnical environment) from 2002 till 2004. results: there were 3704 blood donations for the mentioned period. predominant blood donors are employed and high school students in 75%. family blood donors are ~20%; between them 70% are from albanian population. the ratio between blood donors macedonians vs. albanians is 80 : 20. woman blood donors are presented with 14%. first time blood donors are 53%, and regular donors arẽ 13%. conclusion: first step in planning the blood donation in multiethnic society is creation of special teams of important and devoted volunteers, such as religious leaders, teachers, doctors and businessman. for a successful campaign it is necessary to design special promotional material and address personally to the target population on their mother language. background: pursuit of pharmaceutical purity of the blood in the bag has led to a shrinking donor base and a significantly more expensive product. decisions regarding new infectious marker testing and donor deferrals have typically been made emphasizing decreasing one specific risk without considering the effect the intervention will have on the overall safety of blood transfusion. regulations have been formulated by governmental agencies with limited input from the medical community. the decision making process has lacked risk benefit analyses and has not had the robustness associated with spirited discussions. policies made in this manner may result in certain risks being decreased but can also have adverse unintended consequences. discussion: in the u.s., the fda's implementation of donor exclusions to prevent possible transfusion transmitted vcjd has reduced the donor base by more than 2%. given the demographics of the deferred donors, the impact on plateletpheresis donations has been even greater. to compensate for the loss of donors, blood services will have to persuade present donors to donate more frequently, to recruit new donors, or both. one study has indicated that two-thirds of donors have no intention of donating more frequently. new donors have higher rates of infectious disease markers with positivity for hiv and hcv twice as high as repeat donors. despite sensitive testing techniques, window periods still exist and not all potentially infectious donors will be excluded. another area of concern is the aggressive use of inducements to attract new donors. some blood services are offering lavish incentives such as enrolling donors into drawings to win automobiles. most donors entering the lottery will be low risk; however, it is reasonable to worry that such extreme tactics might also attract persons who should not be donatconclusion: (a) blood donors who were patients' relatives were many more than volunteers as well as more were men than women. also people of young ages were more than those from older ages. (b) the frequency of the diseases for which the blood units were tested was found to be in low levels in the population of the area. specifically as concerns hcv, it seems that transmission frequency has been reduced after the obligatory testing of hcv in blood transfusion centres and stations. genotype 1b of hepatitis c virus is the most frequent in blood donors 2d, from 3a to 3f, from 4a to 4k, 5a and 6a. these are differently distributed in the world: types 1 and 3 are the most common in europe and in usa. aims: considering that, in our region, anti-hcv antibody positivity is variable from 0.87 to 4% of general population, aim of this study has been to evaluate the prevalence of hcv genotypes in blood donors. methods: in period from may 2003 to december 2004, 83 362 blood units were analyzed by nat for viral rna research. nat has been performed on single sample by tma technique. on rna-positive samples, the hcv genotype has been identified by reverse hybridisation with line probe assay. results: 143 blood donors have resulted hcv-rna positive with identification of the following genotypes: 1a = 11 cases (7.7%); 1b = 89 (62.3%); 1a + 1b = 3 (2.1%); 2a/2c = 35 (24.4%); 3 = 1 (0.7%); 4 = 4 (2.8%); none was 5a or 6a. we have also analyzed the differences between the two sexes in hcv-genotypes distribution. hcv-1a has showed a double prevalence in men (8 cases, 5.6%) respect in women (3 cases, 2.1%), while genotype 1b is more frequent in women (48 cases, 33.6%) than in men (41 cases, 28.7%), moreover genotypes 3 and 4 do not compare in women. although an accurate pre-donation selection, discharging all subjects with alt >40 iu, our results show that 1.7 : 1000 donors, apparently healthy and without risk factors, have resulted hcv-positive. analyzing our data, the genotype 1b has resulted the most frequent in blood donors' population, followed by type 2, while the others have showed a very low prevalence. the high frequency of genotype 1 in blood donors is explained by the observation that hcv 1 is usually associated with low alt levels, for this reason affected subjects may escape to donor's screening only based on dosage of alt. on the contrary subjects affected by other hcv types, associated with high alt levels, may be deferred increasing the hcv 1b relative prevalence. at the end, the different distribution of hcv genotypes between men and women and between age's classes probably reflects differences in the pathogenic characteristics of the virus, in the transmission way and in the risk factors. in fact, it has been demonstrated that genotype 1 is principally linked to a not transfusion transmission way; genotype 2 is linked to old age, to female sex and to post-transfusion transmission; genotypes 3 and 4 are associated to young age and to an history of drugs abuse, respectively with high and low viral load; genotypes 5 and 6 are still little known because extremely rare in europe. p-023 kell blood group system and rare blood donors v fakitsa*, p karyda*, s giannoulea † , c antoniou*, j flesiopoulou*, e haliou*, m papakonstantinou*, h dessilla † , e katsadorou*, g lyrakos* and k sofroniadou* *general hospital of nikea, pireas, † blood transfusion center, athens, greece background: the kell blood group system is a compound antigen system exclusively of red blood cells. some of the kell antigens are highly immunogenic. the commoner kell antibody is anti-kel1. the kel2 (cellano) antigen is a high frequency antigen and the blood donors lacking this antigen are quite rare. the blood donors who have not factor cellano are classified in the rare blood donors. rare blood by its very nature is required rarely, but when needed that blood has to be ensured to specified patients. there are other blood donors in their family -74 (35.57%) students, but the number of persons that donate blood from their neighborhood and close environment is much bigger -175 (84.13%). motives for their donation are the following: their wish to help the ones that need blood -142 (68.26%), concern that some day everyone can be a potential recipient of blood -38 (18.26%), because of offered benefits -27 (12.98%), for a friend or relative -23 (11.05%), care for their health -16 (7.69%), because of citizen duty -8 (3.84%), because the others donate -6 (2.88%), curiosity -1 (0.48%). they want to be invited every 6 months -68 (%) students, every 12 months -38 (18.26%), every 3 months -16 (7.69%) and 16 ( the mean age of case group was 30/55 ± 9/33 and the mean weight of them was 71/23 ± 9/3, 72/6% was male and the mean number of blood donation was 2/34 ± 2/19. the mean age of control group was 35/27 ± 10/72 and the mean weight of them was 78/61 ± 12/77. 90/3% of them was male and the mean number of blood donation was 6/58 ± 7/08. the blood donors who were female, first time blood donor low wt the rate of vasovegal rx was higher in female, first time, low weight, younger blood donors (p < 0.005). the rate of vasovegal rx was higher in blood donor (p < 0.005) who were fatigue or first time blood donor, low wt blood donation, fatigue of them and starvation of them had higher absolute donation reaction than other donors. when each variable was adjusted for other variable by regression analysis. young age, first time blood donation, anxiety, fatigue, starvation were significant (p < 0.005) and the others were not. conclusion: donation -related vasovegal syncopal reactions are a multi factorial process. these reaction are more prevalent in blood donors who are young, first time donor, anxiety, fatigue, starvation. these reactions might be predicted vasovegal reaction and these some facth donors need more care. with better donation care, syncopal reaction may be decrease this would be improved donor safety, better donor retention, higher donor satisfaction, and reduce cost and increase regular blood donors. to avoid iron deficiency in blood donors, iron compensation is necessary in most females and males who donate more than 1-2 and 3-4 whole blood units per year, respectively. we present 3 studies dealing with different dose and duration of iron compensation. in the first randomized placebo controlled study iron decreased continuously in males and females at donation intervals of two (males) and three months (females) without iron compensation. 40 mg and 20 mg daily combined with 400 mg ascorbic acid over 2 months (males) or 3 months (females) compensated for iron loss or even overcompensated in females. in the second open study we reduced iron dose to 20 mg daily over one month for both genders. this iron dose was sufficient for compensation of iron loss. a further reduction of iron dose to 20 mg daily over half a month led to negative iron balance in the majority of donors. in all three studies donors with exhausted iron stores profit more from iron compensation, whereas donors with high ferritin values (>50 mg/ml) tend to loose storage iron. aim of the study: one of our campaign strategy how to increase blood donation among adolescents are periodical seminars and excursions for students of secondary schools (more than 40 per year). the aim of this study is to analyze impact of our campaign educational system on adolescents in period 2000-2004. methods: the donation of whole blood and aphaeresis platelets from donors of age from 18 to 20 (max. 3 years for each class) were count for the period of five years (2000) (2001) (2002) (2003) (2004) . the percentage of the man´s donation was calculated for each target class (1982) (1983) (1984) (1985) (1986) . results please see tables 1 and 2. in the tables there is shown observed data in relation to the total number of births in the czech republic in reviewed years. the study showed that number of donation from donors of age from 18 to 20 decreased during objected years. unfavourable state of total number of births in the czech republic (153 801 birth in 1980 republic (153 801 birth in , 133 942 birth in 1986 ) and its decreasing tendency (92 786 birth in 2002!) is with high probability a major demographic factor affected number of young donors. despite energy invested in our campaign educational system our recruitment efforts should be intensified to decrease influence of demographic factors. we should find new ways and methods to attract new blood donors and keep the regular ones, too. the aim of the research was to investigate women's attitudes towards blood donation in cyprus. a statistical sample was selected using stratified sampling and consisted of 369 women from the district of limassol (the second largest urban center of cyprus) between the ages of 19 and 60. using linear logistic regression, the analysis of the data collected revealed that there is a greater probability for a woman to be a blood donor if she is of a higher educational level, a member of an organized group or association, or if she is acquainted with other blood donors. the percentage of female blood donors is higher in rural areas than in urban centers. 40% of women do not donate blood and attribute their reluctance to do so to health-related problems, while about 25% of those who have never donated blood claim to fear the blood donation procedure. in addition, more than half of the women who have stated they would never donate blood again have attributed their denial to healthrelated problems. the research revealed that there could be an increase of up to 30% of the percentage of female blood donors if they were given time off work for a few hours or one or two days afterwards. even though very few female blood donors expressed a preference for the blood donation to take place on a particular weekday, half of them prefer the donation to take place on the discussion: it is about small group of students. the impression is that the altruistic behaviour is present at most of the questioned students. the fact about free school days is not underestimated because it is one of the most important motives of blood donoring of the young population. families where the blood donoring is a tradition have a great influence for young children because the children in these families are better informed for blood donoring. conclusion: including the children in the process of education for young children is of particular importance because the altruistic behaviour as a higher feeling is from an early age of the child and it is under the influence of the environment (family and friends). active participation of the department for transfusion medicine in the educational process, especially in the education of young children, is a guarantee to achieve longlasted positive results. adverse reactions in 2 blood donors taking betablocking antihypertensive medications l paesano*, m d'onofrio*, s misso † , g fratellanza* and e d'agostino* *university federico ii, naples, † hospital san sebastiano, caserta, italy one aim of blood donor's selection is to avoid an adverse reaction to phlebotomy (as vasovagal reaction, syncope and/or hypovolemic cardiac insufficiency). blood donation is surely contraindicated in various pharmacologic therapies, but not in all. in fact a certain degree of discretionarily exists about the assumption, or the period of suspension, relative to a numerous pharmaceutical products, as the antihypertensive agents. according to literature, the deferral of donors taking antihypertensive medication is not indicated when blood pressure is normal, symptoms are absent, and diuretics or similar agents are the only drugs used. on the contrary, it is a common opinion that an antihypertensive therapy by betablockers is not compatible with blood donation for its cardiac effects. nevertheless, in our daily activity, the observation of a blood donor taking beta-blocking drugs may occur for various causes. a possible error is a superficial pharmacological anamnesis, as it can occur in donations on autohemotheca, for a too fast medical visit (due to a large number of donors), or for the inexperience of the selector (often a not specialist of transfusion medicine young doctor). another possibility of observation is constitute by patients, undergoing to elective surgery, included in a program of autologous blood donation, suffering hypertension treated with betablockers. in fact, in this last case, the risk/benefit balance justify the blood letting procedure. in the last year we have just observed two severe post-donation reactions in donors suffering hypertension treated with atenolol. the reactions have been similar, in fact both donors showed lypotimia followed by convulsions about past half hour by the end of phlebotomy. no prodromic symptoms have been observer or referred. cardiac frequencies (cf) before donation were respectively 60 and 64 beat per minutes and blood pressures (bp) were both in the normal range (130/80 and 120/80 mmhg). after donation, during adverse reaction, cf showed no substantial variations, while bp have been decreased respectively to 80/45 and 60/30 mmhg. immediate treatment has consisted in putting the donors in the trendeleburg's position and in applying a dolorous stimulation. in the first case this treatment has been sufficient to report the bp to 100/65 mmhg (with disappearing of all symptoms) in only half hour time. in the second one, the marked hypotension showed a very slow remission, for this reason the subministration of a plasma expander needed, with the complete resolution of the symptoms after two hours. these two donors were not deferred from donation because they were periodic donors that had modified their antihypertensive therapy, without referring it neither in the questionnaire nor during anamnesis. our experience confirms that the blood donation don't must be permitted to subjects taking betablocking antihypertensive drugs. in fact these medications act on cardiac pump decreasing the cardiac rhythm and limiting the postdonation cardiac recover. this effect is very dangerous because it appears relatively in retard respect to the end of donation, when donor may have just leaved the transfusion center. introduction and aim of the study: in society under transition privatisation and marketisation probe all areas of life. transition to market economy is extremely important and sensitive issue in health and welfare services in general, and specifically in the case of blood transfusion service. the aim of the study was to analyze effects of confusing publicity which introduced possible ways of transforming blood transfusion service in serbia (ideas about privatization of some parts of national blood transfusion institute, buying blood from blood donors, selling blood from voluntary blood donors to private clinics, exporting blood from vbd, stories about tradition of paid blood donations in some european countries). publicity was restricted to a small number of sporadic outbreaks concerted in a limited period of time. table. conclusion: surveillance of adverse reactions and injuries or accidents during or after blood donation is essential for maintaining the well being of active blood donors, as well as for the safety and quality of the donated blood components. information on other activities and parameters affecting the quality of blood including materials, reagents and equipment should be collected and any serious deviations from standard operating procedures should be notified to the competent authority using haemovigilance infrastructures. skae has built up such procedures working along the lines of the european haemovigilance network. improvement of existing national haemovigilance systems is expected to follow from the implementation of the eu directive. although inevitable, blood donor deferrals lead to losses in donated blood supply and may affect donor-return rates and subsequent blood donations. to estimate the scope of blood donor deferrals and their causes, we analyzed the 2003-2004 data from regional blood centers using standardized criteria for temporary and permanent blood donor deferrals. within this period (2003) (2004) , 14.6 percent of persons who presented for donation were deferred; 85.9% were temporary deferrals (50% due to laboratory test results, among others low hemoglobin, 6.4% due to risk of acquiring a transfusiontransmissible infection) and 14.1% were permanent (20% due to the infectious diseases markers, 8.7% due to cardiovascular diseases). for regional blood centers the temporary deferral rates varied widely (see the table below ). in the case of individual regional centers, the differences as well as the most common causes were often difficult to explain. according to our analysis, some blood centers have a more restrictive approach to donor acceptance than others and this results in increased donated-blood loss. to some extent such losses could be avoided. further studies are recommended to elucidate the problem and eliminate unnecessary deferrals. caption 1: percentage of deferrals aims: from our experience in selecting blood donors, a certain number of issues have been noticed that remain obscure and need to clarification since those seem to 'haunt' the whole process of blood donation. methods: many first time blood donors and especially volunteers think that rejection reasons are permanent and they are completely incapable of donating blood their entire life. this is a 'tragic' misunderstanding since the doctor did not explain that the reason of the rejection is only temporary and in the future this man is capable of donating blood. those potential donors will never even approach again blood donation centre and when in the future they are asked why they do not donate blood, they repeat the cause of the past rejection. results: one of these rejection reasons is for example low blood pressure (12.54% of total causes of rejection). as we all know blood pressure must be determined according to age, sex, weight and from other factors as sleep, emotional status, food and liquid intake. therefore blood pressure is very important but should be evaluated with all the above factors and must not be alone the only reason for rejection. even when one blood donor is rejected it should be made clear to him that this is only temporary and if in the future he is in better physical condition, he could donate blood. in fact 85-90% of those donors rejected for hypotension are readmitted in blood donation after meeting the above mentioned criteria. another matter of equal importance is anemia (25.04% of total causes of rejection), especially concerning young women. since most of those women tend to develop anemia due to depletion of iron stores, they should be advised to donate blood at longer periods than regular, to receive proper medication and diet according to their needs. the doctor must explain the donor the reasons for iron depletion, so blood donation should not be considered as the only cause for this situation from the donor. there are many factors contributing to anemia, menses, specific diets, overwhelming stress and exercise, not to mention other medical reasons. it is the duty of the doctor to correct those factors that resulted in iron depletion or anemia and readmits those donors in blood donation in the future (75-80% of those rejected are readmitted in our centre). summary/conclusions: at our blood centre we have created a program of regular tests (blood tests-physical examination) for all our blood donors. our experienced and well taught personnel offers advice and provides useful information in every aspect of blood donation and more. we have created a friendly environment for all our volunteers with love, understanding and appreciation and believe that this is the only way to keep a constant 'flow' of blood in our region. introduction: an innovative perception for blood donation in a new and evolving environment must focus on specific matters and ideas and adopt in a certain level lifestyles and concerns of society. aims: the purpose of this study is to find methods and ideas that can help blood donation centers throughout our country to create new blood donors, give a motive and inspiration for blood donation by adopting new trends of society and finally accomplish national need. methods: by having a personal interview with many volunteers about their feelings for healthier life, their nutritional habits, daily physical activity, sports, vitamins, smoking, weight, cholesterol levels. we investigated whether they believe that blood donation has, if any role towards a more hygienic life. results: we divided blood donor volunteers according to their age, educational level, and number of blood donations per year. our results indicated that there is a tendency among young educated people to adopt a personal lifestyle that includes consuming healthier food, keeping their weight low close to the ideal, having some kind of personal activity, not smoking, watching cholesterol levels, following doctors advice and concerning seriously about their health. this dynamic group of blood donor volunteers considers blood donation as a contributing factor to well being and donates blood at specific intervals. besides the yearly run lab tests that are done by our blood centre they also seek advice and discuss any matter concerning their health with the blood centre doctor. it appears that they are extremely sensitive in those matters and they seem to appear well informed about issues concerning their health, they also believe that blood donation is part of the plan they have to keep fit and being well. in our blood centre we encourage this belief and we also provide information concerning this new trend towards healthier habits. summary/conclusions: this approach has already shown some positive results in our blood centre as many people especially young educated women have joined our blood donorship program and donate blood at scheduled intervals. in order to achieve our goal which is to raise the percentage of blood donors in the region we have to be flexible, innovative according to new habits and lifestyles. we have to move with society and modernize the way we attract various groups of people. blood donation against prejudice as saltamavros*, s dimitrakopoulos † , v zacharaki*, p giannaros*, s markou* and p tseliou* *st. andrews hospital patras greece, patras, † pyrgos general hospital, pyrgos, greece introduction: in order to achieve a greater population to be admitted in blood donation we have to provide information concerning any obscure issues that presents in selecting donors. to examine the accuracy of hb measurements obtained by the noninvasive clinical device, as compared to values detected by standard methods, (cell-dyn 1600, abbott laboratories, usa), in a blood donor setting. methods: the nbm-100 device utilizes a finger base sensor using occlusion red/near-infrared spectroscopy (o-rnirs) to detect and analyze the hb/hct levels. the clinical trials were conducted at two blood donor centers (israel and usa). studies were carried out on a group of 159 subjects (86 females, 73 males) aged 19-64. subjects were healthy volunteers who had come to donate whole blood or aphaeresis components. after obtaining informed consent, hb/hct levels of all the study volunteer participants was tested non-invasively, using the nbm-100 device, followed by a venous blood sample. additionally, the usa center tested a capillary blood sample using the hematastat hct measurement device (71 donors). hb levels were considered normal when readings were equal to or >12.5 g/dl. results: venous hb measurements ranged from 9.9-16.2 g/dl. the mean nbm-100 hb level was 13.20 ± 1.35 g/dl, only 0.28 g/dl lower than the mean hb result obtained by venous sampling, which reached 13.48 ± 1.22 g/dl. the standard deviation of the difference between the invasive and noninvasive hb readings was found to be ±1.04 g/dl. the mean absolute error (mae) of their difference was 0.81 g/dl. when checked against the cell-dyn in the usa center, where 21 subjects had hb of 12.5 g/dl or lower, the nbm-100 and hematastat devices showed comparable sensitivity results. the nbm-100 using o-rnirs is a promising noninvasive technique for hb screening in blood donors. the device is easy to use and agreeable for both blood donors and personnel. the technique reduces the need for the invasive finger prick or venous blood sampling, thereby enhancing safety, reducing costs, and improving the experience of blood donation. the effect of short-term, temporary deferral on blood donor return rates and subsequent blood donations background: blood donors are deferred for numerous reasons. some deferrals like intravenous drug use, male homosexual contact or certain positive test results are permanent. the majority of donor deferrals, however, are short-term temporary deferrals (sttds) that are resolved in a matter of days, weeks or months, after which time the person is again an eligible blood donor. the effect of sttds on blood donor return rates and subsequent blood donations is studied. materials and methods: donors given sttds during the 15 december 1999 to 15 march 2000 were computer-matched with non deferred donors on the basis of age, sex, and donation date (case group: 804 donors -control group: 295 donors). computer records were evaluated during the next 3 years (21 march 2000 to 21 march 2003 to determine donor return rates. significance for comparison between the two groups was based on chi-square analysis. results: the most common reasons sttds were elevated blood pressure (52%), deferred for medication (29%) and colds and/or sore throats (4.2). non deferred donors were a little more likely than donors with sttds to return over the next 3 years (36.6% vs. 34.8% pv = 0.0001) and non deferred donors donated more whole blood units. . according to ethnic structure, women -ethnic macedonians donate blood in largest numbers -1096 (14%), while all other ethnic groups are present with only 3%. the most prevalent is the group of adults aged 41-50 (358 -27.5%), with high school education -800 (61.4%) and mostly those who donated blood 1-5 times (719 -55%). conclusion: having in mind that 50% of the population in macedonia is female, the obtained results reveal a significant, yet insufficient participation of women in blood donation with 17% in relation to the total number of blood donors surveyed in the period 1999-2003. this is due to insufficient motivation and education of women from all ethnic groups especially those from the younger population and with elementary education. incorporating them in education and organization would contribute to their more extensive participating in blood donation. comparison of serum beta 2-microglobulin (b2-mg) between hbsag positive donor and healthy control f tarabadi*, m shaeigan*, g babaee † , a talabiean* and m khadir* *iranian blood transfusion center, † tarbiat modarrs university, tehran, iran background: beta 2-microglobulin (b2-mg) is a low molecular weight protein (11 800 daltons) and found in all biological fluids it is light chain of histocompatibility class1-human present on the most membranes of cells. in the hepatitis infection the viral antigen presentation on the hepatocyte in the presence of class 1-hla antigen plays a role in the elimination of the virus. method & samples: beta 2-micro globulin was measured in serum drawn from 45 hbs ag positive blood donors include 5 (11.1%) female and 40 (89.9%) male in age between 17-56 years, and 50 healthy 16 (32%) female and 34 (68%) male in the same age we detected serumic b2-mg by enzyme immunoassay (ela). results: our studies showed b2 mg level increased in 7 (15.6%) hbs ag positive donor that was significant differences with healthy control (p = 0.0001). conclusions: it seems that serum b2mg is a good marker for hbs ag replication. the role of b2mg in monitoring of response therapy needs to be more evaluated. and 1635 (0.8%) were contributed by vd, rd and dd respectively. over the last 5 1 / 2 years, voluntary donations have shown a rising trend from 54.3% to 60.9%, where as rd (44.9% to 38.4%) and dd (0.9% to 0.8%) have shown a declining trend. the percentage of female donors was maximum in voluntary group as compared to rd and dd (11.01% vs. 2.02% vs. 6.17%) respectively. the rates of all tti markers reactivity were significantly higher in rd as compared to others donors. the hbsag and anti hcv reactivity in vd and dd is comparable (0.84% vs. 0.85% and 0.45% vs. 0.49%). hiv antibodies was found more frequently in vd as compared to dd [0.15% vs. 0.06% (p < 0.05)] whereas, vdrl reactivity was lower in formal as compared to latter [0.26% vs. 0.37% (p < 0.05)]. conclusion: voluntary blood donation has shown a rising trend over a last few years, thus highlighting efficient donor motivational strategies. these strategies need to be strengthened to increase the female donor base. the safety of dd is equivalent to vd when the rates of tti are compared. thus, dd should be advised to donate blood regularly as voluntary blood donors. blood safety depends on a number of factors. the chain of safe blood starts with the donor. one of the procedures for obtaining safe blood for transfusion is the medical selection based on the completed questionnaire and the possibility of self-exclusion from the process of blood donation, the medical history of the potential donor and the medical examination. donor selection consists of two sets of information necessary for protection of the blood recipient as well as the donor himself. aim: to present the most frequent reasons for declining volunteer blood donors. material: the materials used for analysis were the questionnaires completed by all the potential blood donors at the transfusion department of the medical center in strumica as well as the record books of the blood donors which contain the results of the analysis we make for the potential donors. these donors donated blood in the period between 2001 and 2003. results: during this period 4129 people volunteered to donate blood, out of which 3940 were allowed to donate blood, while 189 were declined. out of the total number of blood donors 3313 were male and 627 female donors. the reasons for declining potential donors were the following: 42.3% had low levels of hb, 22.36% were taking antibiotics, 9.37% were ill, 7.83% had low blood pressure, 6.75% had high blood pressure, 11.39% for other reasons. conclusion: donor selection and their care on one side and obtaining safe blood for transfusion on the other side entails obligatory organized medical control. the obligatory completion of questionnaires, the medical examination of the potential donor and their self-exclusion as a result of the feeling of personal responsibility as well as the obtained information are very important for the selection of quality blood donors and obtaining safe blood for transfusion. questionnaire on 2700 subjects-students, their knowledge and motivation on blood donation f vladareanu, a bugner and s sirian national institute of heamatology transf, bucharest, romania the research theme of this questionnaire is as follows: 'what is the level of knowledge and of motivation in the non-remunerated and voluntary blood donation at students?' we also tried to see the practical implications that this study will have and how it will influence the knowledge in this area. the purpose of this questionnaire was not dissimulated. the general theme of the knowledge and motivation on blood donation had been studied before through two big questionnaires applied in 1987 and 1992, but the general population was their target. students had never been an investigated lot up to now. the hypothesis referring to this problem is as follows: students are not informed either on the act of donation, or on the crisis of blood. 1. the lack of information is a first cause of the indifference of the studied lot towards the idea of donation. 2. the lack of motivation of the studied lot is another cause. the questionnaire was applied on a 2700 lot of students from seven different cities: bucharest, iasi, constanta, cluj, sibiu, brasov, timisoara. the number of the questions was limited to 22, which we consider best for a questionnaire applied on the street or at college. as a conclusion, we can say that a passive-defensive attitude towards the blood donation was revealed after this questionnaire. not knowing the issue caused by their lack of information sometimes determines indifference at the statement of the subject. on a general dissolution environment of the responsibility of the youth, the donation problem is not in their aria of preoccupations, the general attitude being of non-involvement for the moment, at this idea which is not yet in every individual conscience and which is normally administrated at an institutional level. the donor data and the details of blood application of the north west transdanubian region of hungary k vörös*, c bercsényi † , o petró † , r jáger † and e miskovits ‡ *hungarian national blood transfusion s., györ, † blood bank, tatabánya, ‡ headquarters hungarian n.b.s., budapest, hungary the ongoing fundamental reorganization of the blood service began on the 01.07.1998 in hungary. as the consequence of reorganization till 01.07.2000, 28 blood banks had been established instead of 63 existing before, under direction of the hhnbts. the working profile of the 6 regional blood centers and 22 local blood banks will be changed step by step. virus screening, blood group serology and processing will be made in the 6 regional centers. one of the regions is the 'north west transdanubian region' (nwtr, city györ as the center, with about 930 000 inhabitants and 8000 hospital beds). 3 local blood banks (tatabánya, sopron, and szombathely) are belonging to nwtr. the regional center and the local blood banks provide the labile blood products and high level clinical-transfusion service (cross-matching, antibody screening, outpatient immunhematology investigations, etc.) for the hospitals. annually 56 000 donors donate blood in this region. this donation activity covers about the 6% of all inhabitants. the acceptance ratio of the donors is good (6-8% of the donors were deferred). there are 14 hospitals in our region. the regional demand on rbcc is 30-45.000 u/year, on ffp is 5.500-7.000 u/year and on pc is 8-10.000 u/year. the poster shows the donor data and the details of blood application of this region since 1999. p-054 implementation of 2rbc collection using haemonetics mcs ® +: medical staff training, donor recruitment and acceptance g woimant, c fretz, d puydupin, e pélissier and jl beaumont efs ile de france, paris, france background: single donor 2rbc collection is an approved apheresis technique in france. aims: our goal was to evaluate the implementation of 2rbc collection in our center in terms of donor recruitment and acceptance, as well as medical staff training and adaptation. methods: donors were selected according to the french requirements for 2rbc collection (weight ≥ 65 kg, height ≥ 165 cm, hb ≥ 13.5 g/dl, ferritin ≥ 20 ng/ml for repeat 2rbc donors). all personnel were trained on adequate communication with donors. eligible donors were contacted by mail, by phone or during pre-donation interview. among the 102 recruited donors, all donors were male, 63% were regular whole blood donors, 36% were regular whole blood or apheresis donors and 3% were new donors. the medical staff was trained on 2rbc collection with the sdr protocol and disposable set ln 948pf on the mcs ® +. most of the medical staff was already used to autologous rbc donation with similar apheresis devices. blood samples were taken from donors pre-and post-donation, as well as 2 to 4 months later for those returning for a subsequent donation. donors were asked to fill out a post-donation survey for assessing donor comfort and information. results: donor profile and clinical follow-up are summarized in table 1 . six percent of the donors had a ferritin level below 20 ng/ml; these donors were regular whole blood donors. the collections were well tolerated and no changes in vital signs were noted. four reactions were reported: 2 hematomas and 2 citrate reactions. no reaction was observed post-donation and hemoglobin levels measured before next donation were back to normal. the technique was easily implemented by the medical staff and fitted well in the existing blood center processes. the medical staff as well as the donors found collection duration short (average of 35 min). the results of the survey were very favorable as more than 95% of the donors considered their donation and the information they received as satisfying. most of them agreed to donate again and several actually donated twice during the evaluated period. conclusion: the implementation of 2rbc collection in our center, using haemonetics mcs ® +, was successful in terms of ease of use of the technique, as well as user and donor acceptance. we now plan to evaluate donor loyalty in the longer term. risk from first-time blood donors e zhiburt, s golosova and p reizman federal blood center, moscow, russian federation introduction: each third dose of whole blood in russia is donated by first-time blood donor. there are two reasons for attention to this kind of donors: (1) possible risk of infectious disease in seronegative study; (2) possible risk of donation for person with contraindication. aim of the study: we investigated role of regional deferred donors registry (rddr) in by first-time donor selection. methods: moscow rddr includes parts: hiv, viral hepatitis, syphilis, tuberculoses, malaria, drug users, psychiatry, 60 days after blood donation. rddr was complete and our center began actively work with it since last year. each donor has to be registered in rddr and automatically checked for deferral reason. effectiveness of rddr was investigated. results: first-time donors donate less than 10% blood in our center. about a quarter of them are deferred before possible donation. part of donors deferred by rddr has been significantly increase in 2003 (c2 = 19.6; p < 0.001) at the expense of seropositive people. conclusion: rddr is effective for blood donor selection and decreases necessity in laboratory screening. first-time blood donors have to be examined before blood donation. if they have not contraindications, donation can be performed up to 10 days before examination and screening. the double unite platelet production is important especially if the relatives of patient find the donors. we evaluated the effectiveness two apheresis machine for platelet collection. in our blood bank, one fenvall amicus and one cs3000+ apparatus were used for platelet apheresis. 1531 apheresis were performed between 2/12/2002 and 13/2/2005. including criteria of donors are that estimated process time is smaller than 90 minute and estimated postapheresis platelet count is higher than 100 ¥ 10 9 /l. donors firstly was enrolled to amicus. if amicus was busy, then it was enrolled to cs. the properties of our donor populations were given in blood and plasma cell components are obtained either by traditional manual method from whole blood or by apheresis. modern medical treatment is based on transfusion of deficient components such as erythrocytes, leukocytes or plasma proteins. this involves new solutions to achieve higher yields and better quality of such components. the aim of our study was to estimate the efficacy of blood cell separator cobe trima in obtaining platelet concentrates (pcs) as compared to older-generation cobe spectra blood separator. apheresis procedures were performed on both these blood cell separators. the quality of platelet concentrates was tested during 5 day storage period (see table below ). we have tested the effect of apheresis procedure on donors and estimated the operating comfort of both separators. the tolerance of both separators was satisfactory except for more frequent hypocalcemia when trima separator was used. most donors were more satisfied with trima procedure because of single venipuncture although it involved special donor selection (good vein access). in general we may say that trima is undoubtedly a more modern and more friendly separator. however, cobe spectra may continue to be used with success especially when a more versatile cell separator is necessary (leukocyte concentrates, peripheral blood stem cells or therapeutic apheresis). methods and results: tls (587 procedures on 67 patients) were used successfully in patients with acute or chronic leukemia with hyperleukocytosis (white cell count >100 ¥ 10e9/l or blast count >50 ¥ 10e9/l) when high cell count would promote leukostasis with vascular occlusion in the microcirculation. performed tl procedures were rapidly reduced both the white cell count and the whole blood viscosity. average fall in white cell count after treatment was 73.3%. tp-treatments (186 procedures on 32 patients with symptomatic thrombocythemia and/or platelet count higher than 1000 ¥ 10e9/l) were applied in order to prevent the development of 'thrombotic-hemorrhagic syndrome' . the tps performed resulted with rapid platelet counts reduction (84.3% in average) and with clearly noted clinical improvements, subsequently. tes (429 procedures on 196 patients) were performed using manually technique in patients with 'cellular hyperviscosity syndrome' induced by high red blood cell count. it was shown that te procedures resulted to red blood cell number lowering and decreasing of blood hyperviscosity. average fall in hemoglobin and red blood cell concentrations after te treatments was from 20.5% till 35.7%. rbcx treatment (8 procedures on five patients with malaria and two with severe aiha crysis) was performed on an urgent basis, particularly when clinical symptoms indicate life-threatening situations and resulted with rapid and significant reduction of concentration of unwanted pathogen affected rbcs and summary/conclusion: the effects of tcs depended on the nature and stage of the basic hds, of adequate selection of patients and of timely applied apheresis. rapid cytoreduction is obtained justly in patients with excessively high cell count, and this effect did not associated with bone marrow remission. thus, tc should be looked upon as adjunct to the standard treatment of different cithemias, but not as replacement therapy. the present study indicates that the best therapeutic effects were obtained by rbcx. were carried out with continuous flow blood cell separator cobe spectra and all patients underwent large volume leukapheresis (lvl). in all procedures, a blood warmer was connected to the return line and a continuous calcium infusion was administered preventively. six patients, who were under 25 kg body weight, had the extracorporeal circuit primed with irradiated, filtered packed red cells diluted with 5% albumin solution. seven children had vital signs and ecg continuously monitored during the procedure. results: each patient underwent a median of 2 collections (range 1-6). the inlet blood flow ranged between 16.0 and 95.4 ml/min (median 54.5 ml/min). the median blood volume processed was 11 754 ml (range 2700-22 500). leukapheresis lasted a median of 240 min (range 120-270). the median total nucleated cell yield was 11.86 ¥ 10e8/kg (range 1.94-21.21), mononuclear cell (mnc) yield was 6.01 ¥ 10e8/kg (range 0.97-12.73) and cd34+ cell yield was 3.5 ¥ 10e6/kg (range 0.19-28.01). the median of mnc collection efficiencies was 56.11% (range 12.34-158.09). in 9 (39.1%) patients, in only one apheresis procedure more than 2 ¥ 10e6 cd34+ cell/kg were collected. during 6 (9.09%) procedures patients had experienced apheresis-related side effects. the citrate-induced reactions were most commonly observed. the reactions were mild and cessation of collection was required only in one case, because of catheter related complication. mild sedation was required only in few very small children. post-donation platelet count was less than 30 ¥ 10e9/l in 3 cases and these patients required platelet transfusion before subsequent procedure. our results show that lvl in pediatric patients is relatively safe procedure, well tolerated and with a very low risk of serious adverse events. close monitoring of blood counts, especially platelets, between pbsc collections is necessary. the cessation of procedure was required in only one case and no life threatening side effects occurred. neonatal alloimmune thrombocytopenia (natp) caused by fetomaternal mismatch for human platelet (plt) alloantigens (hpas) worsens approximately 1/2000 pregnancies and can lead to a serious bleeding diathesis, intracranial hemorrhage (ich) and sometimes death of the fetus or newborns. we describe the successful management of a 37-year-old pregnant woman, alloimmunized to the hpa-1a (p1a1, zwa) antigen, with a history of two previously children with severe thrombocytopenia and ich. the pregnant woman was at her terminal pregnancy and was suddenly admitted. to evaluate the risk of ich in the fetus, cordocente was performed to demonstrate fetal thrombocytopenia (plt 4.000/mmc). to ensure a rapid provision of compatible negative-antigen platelets, we decide to collect platelets from the mother using apheresis. plateletapheresis was performed using com.tec separator, fresenius. blood processed was 1.100 ml in a short time procedure (35 minutes). no significant adverse effects were observed in the mother and fetus, during and after the procedure. platelets collected (1.5 ¥ 10e11) were transferred to the preparation set and plasma was removed after centrifugation to resuspend the platelets in octaplas ab. then we separated the platelets into two units containing 0.7 ¥ 10e11 each. the day after the donation, the mother gave birth to a girl by caesarean section. after the transfusion, the plt account increased from 4.000/mmc to 35.000/mmc and after a week the child had plt 75.000/mmc without hemorrhagic complication. according to the literature data and our observations of the patients, there are changes of the hemostasis system indexes in the most patients with the endogenous intoxication syndrome and immune disturbances. in the number of cases medicamentous therapy appears to be not enough to normalize the changes, but it is especially important for pregnant women and women in childbirth, because on the background of these disturbances different complications of pregnancy and postnatal period take place. the aim of our study was the substantiation of plasmapheresis using in complex therapy of purulent inflammatory complications in obstetrics and immunoincompatible pregnancy with hemostasiologic disturbances. 10 patients with hemostasis system disturbances: one woman in childbirth with exacerbation of chronic pyelonephritis, who had in the first 24 hours some signs of hypocoagulation on the background of permissible blood loss (prolonged coagulation time up to 20-30 minutes with episodes of its absence on the background of the normal indexes of general coagulogramm, quantity and function of thrombocytes and the dilute fibrin monomer complex level in 7 times higher than the norm) and nine pregnant women with the perinatal losses in anamnesis severed by the pregnancy (threat of abortion, places of fetal egg detachment). these women were examined, the following was revealed: the high antibody titer to chorionic gonadotropin, parameters of partially activated thrombin time were higher than the norm (47-52 seconds), thrombin time (18-20 seconds), the dilute fibrin monomer complex (8-12 mg%), coagulation time (15-20 min). in all these cases the conservative methods of treatment (antibacterial, hemostatic, hormonal therapy) were effective for a short period of time and they didn't succeed to correct the given parameters of hemostasiogramm. the discrete centrifugation plasmapheresis was included in the complex of medical treatment. the woman in birth 5 operations were done, in the programme of plasma replacement during the first two plasmapheresis procedures donor fresh-frozen plasma was included. six pregnant women on the given stage one course consisting of 3 plasmapheresis procedures for plasma replacement with crystalloids was done, the volume of the removed plasma was 25-30% of the circulating plasma volume. three pregnant women before delivery were required two courses of plasmapheresis more consisting of 3-4 procedures each. the system heparin was not used. in all patients already after the first procedure of plasmapheresis the normalization of hemostasis indexes was marked, that allowed to prolong pregnancy, to prevent the coagulopathy bleeding and the development of disseminated intravascular syndrome. four women are discharged from the hospital, the other patients are observed with progressive pregnancy. thus, the using of discrete centrifugation plasmapheresis is effective at the signs of hypocoagulation in patients with isoimmunisation with fetal antigens and infectious pathology, and is the reserve in prevention and treatment of obstetric complications. extracorporeal photochemotherapy: an alternative therapeutic approach to control graft versus host disease after allotransplant with reduced intensity conditioning regimen c del fante*, c perotti*, gl viarengo*, p bergamaschi*, p pedrazzoli † and l salvaneschi* *irccs policlinico s. matteo, pavia, † ospedale niguarda cà granda, milano, italy background: extracorporeal photochemotherapy (ecp) can be defined as an immunomodulatory therapy that demonstrated to be efficacious in treating patients affected with graft versus host disease (gvhd) after allotransplants for oncohematological diseases. reduced intensity conditioning regimen (ricr) for allotransplant is a relatively new practice in patients (pts) ineligible for a conventional myeloablative conditioning regimen. the use of immunosuppressive therapy (ist) to control gvhd is limited for the high risk of developing infections and disease relapse due to the strong reduction of graft versus tumor (gvt) effect. aims: to evaluate the effectiveness and safety of ecp in treating pts affected with gvhd post rcr and the possibility to taper, at the same time, the ist. methods: 6 pts (5 females, 1 male), median age 47.5 years (25-63), affected with agvhd grade ii (1) and extensive cgvhd (5) gtx with median total granulocyte doses of 55 (21-150) ¥ 10 9 per gtx corresponding to 1.46 ¥ 10 9 granulocytes/kg in children and 0.58 ¥ 10 9 granulocytes/kg in adults. the wbc counts increased from baseline values of 0.2 (0-0.5 ¥ 10 12 ) g/l for both pediatric and adult patients to peak values of 3.8 (0.4-18.2) ¥ 10 12 g/l (children) and 0.9 (0.3-9.4) ¥ 10 12 g/l (adults) at one hour after gtx and to 1.4 (1.1-13.6) ¥ 10 12 g/l (children) and 0.4 (0.3-8.6) ¥ 10 12 g/l (adults) at 24 hours after gtx. in 42 out of 54 patients (80%), the crp levels significantly declined (60 (11-91)%; p£0.001) during the granulocyte transfusion period; in almost all cases (39/42; 90%) after the initial or 2nd transfusion. thirty-eight patients (71%) were alive at day +30 after termination of neutropenia and gtx. patients without crp response to gtx (6/9, 66%) and patients with severe viral infections 6/7 (86%) were not among the day +30 survivors. background: in recent years, the use of platelet concentrates obtained from single donors by automated apheresis has grown steadily. plateletpheresis donation is considered to be a safe procedure with modern instruments. so far, no studies have identified donor or procedure specific factors that may be associated with serious adverse events. aim: to evaluate the incidence of adverse events during plateletpheresis procedure, over a five-year period in our hospital. materials and methods: eight hundred single-needle plateletpheresis collections were performed by using two automated intermittent-flow cell separators: 700 of them with mcs3p and 100 with mcsplus (haemonetics), according to automatic standard protocols a3p and ldplp, respectively (with the collection of an additional plasma unit). acd-a was used as the anticoagulant in all apheresis procedures (acd-a: blood ratio was 1 : 11). most of the donors (84% men and 16% women) were patient´s relatives. half-hour before the initiation of the procedure, 250 mg of calcium (1 tablet cal-c-vita) were administered to each donor. the mean platelet yield was 3.2e11 /unit. the overall rate of the donor related adverse events was 3.4%. feeling faint was the most frequent event, which was occurred in 1.62% of donations. hypotension and citrate related rates were 0.77% and 0.73%, respectively. all citrate related symptoms were only transient perioral paresthesias, which were relieved by slowing the i.v. rate, without additional administration of oral calcium. donor unconsciousness was the only observed severe event, the rate of which was 0.25%. other adverse events were venipuncture related (2.37%), machine related (2.7%) and miscellaneous complications (1.25%). (1) plateletpheresis using the mcs3p and the mcsplus automated cell separators is a safe procedure, with a low risk of serious adverse effects. (2) with the used acd-a-to-blood ratio (1 : 11) satisfactory platelet concentrates were obtained with very low incidence of citrate-related events. (3) the peros administration of calcium before the initiation of the procedure, probably lowers the rate and the severity of hypocalcemia symptoms. quality assessment of ffp collected as a byproduct of plateletpheresis 1. from the donors immediately with the initiation of the procedure (citrated whole blood) and 2. from the final platelet concentrates after one hour rest at room temperature without agitation. in vitro platelet response to the aggregation-inducing agonists adp, collagen, ristocetin and arachidonic acid was investigated by means of an aggregometer (pap-4c, bio/data). results: there were no significant differences between the groups of donors with respect to age, sex, smoking habits, preapheresis wbc and plt counts and hemoglobin concentration, as well as in the harvesting time between the two cell separators. our findings are shown in the following table. mildly decreased response to all agonists was observed (mainly to adp and arachidonic acid) in the samples taken right after the initiation of the procedure, in both groups. platelets from the final component showed a further slight decrease in response to adp, which was more prominent in the mcs3p device (p = 0.025). on the contrary, an increase in platelet response to the other three agonists was observed in both devices, which, however, was statistically significant upon collagen and ristocetin stimulation. conclusions: reduced response to aggregation stimuli is possibly caused immediately with the initiation of the apheresis process. literature reports regarding further platelet traumatisation due to the procedure, are rather conflicting. in our study, such traumatisation was observed only in the case of adp in the mcs3p obtained collections and this could be correlated with the technological differences between the two devices. recovery of platelet aggregability, as it was expressed by the upregulation in platelet response in the other stimuli, could be attributed to the resting period and seems not to be affected by the timing of the leucodepletion procedure. background: lupus erythematosus often is accomplished with severe symptoms, such as polyarthritis, nephritis, pericarditis or dermal alterations. in pregnancy cytostatic therapy affects gestation. on the other hand the course of disease can be refractory to corticosteroid therapy. elimination of autoantibody and immuncomplexes by plasmapheresis could be an efficient way to amend the severity of symptoms. a 24 year old pregnant woman in the 24th gestation week with systemic lupus erythematosus showed severe symptoms like polyarthritis, nephritis and pericarditis. treatment was initially 1 mg/kg bw prednisolone for 3 weeks and subsequently 8 mg/kg bw for 2 weeks. plasmapheresis was applied daily in the beginning and continued depending on the condition of the patient ( table 1) . the eliminated plasma was substituted by fresh frozen plasma. the medium volume was 2.951 ml per apheresis. after day 11 plasmapheresis treatment was suspended to avoid problems with coagulation and was followed by a cycle of 3 immunoabsorptions to eliminate circulating immuncomplexes. results: prednisolone therapy alone brought no effect even after changing to high-dose treatment. a significant amelioration of all symptoms could be observed after the first plasmapheresis. good condition of the patient remained stable over the period of daily plasmapheresis for 4 days. intermitting apheresis treatment for one day lead to a significant aggravation of symptoms. apheresis no. 6 again lead to a recovery of the patient which held on until day 11. conclusions: treatment of systemic lupus erythematosus in pregnancy especially in combination with resistance to corticosteroid therapy, is an effective therapy to ease severe symptoms such as polyarthritis, pericarditis and nephritis. exposure to cytostatic drugs can be avoided and therefore the impairment of the fetus can be reduced. background: the collection of mnc represents the first step of photopheresis procedures and could be of critical importance in achieving a therapeutic goal. in this work we compare the cell yield of two collection programs on cobe spectra device: the mnc versus the autopbsc program using the ecp procedure modified by andreu. methods: 39 procedures were carried out with mnc program and 19 procedures with autopbsc on 4 patients with cgvhd. both hemoglobin increased from 35.9 ± 5.7 to 204 ± 100 mg/tu, k+ from 1.7 ± 0.7 to 44.8 ± 13.9 mmol/l, level of glucose decreased from 26.65 ± 2.06 to 9.75 ± 2.06 to 9.75 ± 0.91 mmol/l, ldh from 1.81 ± 0.48 to 6.24 ± 1.89 ukat/, lactate from 2.77 ± 0.47 to 22.72 ± 14.02 mmol/l, ph from 7.50 ± 0.14 to 6.74 0.08. the volume of apheresis units was lower than wb-rbc, the leucocyte count was normal in all units. the rbc loss by filtration was 24.9 ± 3.1 ml/tu and was lower than at wb-rbc. in apheresis rbc there were the differences in hb and ht value between the day of storage 0 and 40, in wb-rbc there were no differences. during the storage period we found no differences in k+ increasing value and no change in ph value between apheresis rbc and wb-rbc, the increasing of lactate was higher in wb -rbc, increasing of ldh correlated to hemolysis. the plasma hb value increase was higher at apheresis rbc in contradistinction to literature. hb and ht correlation in apheresis units according to predonate value in donors was lower than at wb -rbc. the method is a useful alternative to conventional whole blood donation, we get rbc units with high standard of quality and low correlation according to predonate hb and ht value in donors. acknowledgement: the study is supported by grant iga ministry of healthy cr n. nr/8015-3. background: in life-threatening exacerbations of sle a satisfying efficient therapy is lacking. despite intensive immunosuppressive therapy some patients are resistant or contraindicated to conventional treatment. in particular circulating antibodies and immune complexes play an important role in the pathogenesis of sle and mctd. an extracorporeal removal of these pathological substances may be effective in the treatment of active disease. methods: five patients with severe therapy-resistant sle/mctd underwent immunoadsorption onto protein a. blood was drawn from patients by using a jugular catheter or a peripheral intravenous catheter. anticoagulation was performed with acd-a and heparine or acd-a and r-hirudine. plasma was separated by centrifugation. the 1.5 to 2-fold total plasma volume was treated in every immunoadsorption. the columns were floated with a maximal plasma flow of 20 ml/min. the procedure was carried out every second day. additionally supplementary intravenous immunglobulin therapy was given only once. results: remission of the disease was achieved in four patients. see table below . conclusion: pa-ia is highly effective regarding the elimination of autoantibodies and circulating immune complexes, might induce a remission in patients with sle/mctd. it is an acceptable alternative treatment option in patients when other therapies are ineffective or contraindicated. background: purification of bone marrow from erythrocytes is used to prevent early hemolysis in major abo incompatible allogeneic hemopoietic cell transplantations. erythrocyte depletion is strongly recommended to reduce product volume and stem cell purification before storing autologous and even allogeneic bone marrow in order to prevent early hemolysis and dmso toxicity that might develop after thawing. centrifugation, sedimentation with hes, and cell separating devices are methods for erythrocytes depletion. aim: in our center, we prefer to use cell separation device, since it is a reliable method and has a high-yield and risk of contamination with erythrocytes is low. success of the process is retrospectively analyzed for high and low volumes. method: erythrocytes depletion of bone marrow harvest was done in hemapharesis unit with cobe spectra device in the last five years in 20 cases with bone marrow volume over 750 ml, and 17 cases with bone marrow volume under 750 ml. fifteen of these cases were allogeneic, and 22 were autologous procedures; a software uploaded with cobe pbsc coll vers 5.1 and (catalog no: 777-006-000) set was used in the procedure, and at the same time, double bag system with intermediate connectors were used to prevent re-circulation (catalog no: 777-006-300). results: the mean volume reduction was 90.48% (83.81-93.54) for volumes over 750 ml, and 89.79% (84.03-94.02) for volumes less than 750 ml. regarding the success of the procedure no statistically significant difference was found between procedures with high and low volumes. no complication developed related to the device or product, and waste bag never had to be re-used. in none of the patients early massive intravascular hemolysis was observed. conclusion: erythrocyte depletion and volume reducing with cell separation device is a reliable method. this process is successfully applied with high volumes (over 750 ml); and in low volumes as well for reducing erythrocytes, and gain of mononuclear cells and cd34+ cells. platelet concentrates obtained by apheresis procedure-correlation between the initial count and the final concentration v srejic*, g bogdanovic*, z garic*, n vavic* and b balint † *national blood transfusion institute, † military medical academy, belgrade, serbia apheresis team of the national blood transfusion institute processed and classified data of 100 donors who donated platelets by apheresis procedure from january till april 2004. procedures were performed in accordance with the ldplp protocol, using haemonetics mcs+. initial donors' platelet count and the absolute platelet concentration in the final preparation were followed, as well as red blood cell and leukocyte contamination and the volume of the processed blood. donors' initial platelet count was not less than 191 ¥ 10 9 /l and the volume of the processed blood was not less than 2300 ml. according to histogram, the most frequent donors' initial platelet value ranged from 210 ¥ 10 9 /l to 308 ¥ 10 9 /l (70%). final concentration of the samples of 100 tested donors ranged from 3.9 ¥ 10 11 to 6.1 ¥ 10 11 in the average volume of 400 ml. regression analysis demonstrated that there was a correlation between the initial donors' platelet count and the obtained final concentrate. student's t test showed p < 0.0011. leukocyte contamination of the final concentrate prepared without the filter ranged from 0.0 ¥ 10 9 /l to 0.4 ¥ 10 9 /l. presence of red blood cells in the final concentrate ranged from 0.01 ¥ 10 12 /l to 0.02 ¥ 10 12 /l. p-077 therapeutic apheresis (ta) in croatian hospitalsadherence to respectable guidelines z zivkovic*, b jeren strujic*, s boras † , i bojanic † , b golubic cepulic † and z ivankovic † *clinical hospital dubrava, † clinical hospital center zagreb, zagreb, croatia introduction: besides considerable resources, ta requires high costs and risk for patients. therefore, indication for ta often considers interests of patient, hospital and requesting physician. the most respectable guidelines for the implementation of ta were defined by aabb and asfa, classifying total of 58 diseases into 4 categories (ctg), ranging from 'standard therapy' to 'lack of efficacy' . the objective of this study was to determine indications for ta performed in 6 croatian hospitals in the period 2001-2004, respecting aabb/asfa guidelines. results: during the observed period in croatia, ta was performed in 777 patients suffering from 26 various diseases. in 514 (66%) patients ta was performed by membrane filtration, while in 263 (34%) separation by centrifugation was used (table 1) . according to the 4 ctg, s of the aabb/asfa guidelines, ta was performed in 573 (74%) diseases from ctg i, 126 (17%) ctg ii, 54 (6%) ctg iii, and 25 (3%) ctg iv of patients. the most frequent indications included in ctg i were: myasthenia gravis (32%), collection of pbpcs (31%), sy. guillain-barré (17%), and plasmacytoma (6%). in ctg ii frequent indications were: poisonings (35%), systemic lupus erythematodes (30%), and rapidly progressing glomerulonephritis (14%), and in ctgs iii and iv: cytoreduction-polycythaemia (52%), thyroid storm (15%), gvhd (10%), and reumatoid arthritis (8%). (1) the time spent for resolving h 52/40, (2) mtp 0/0, (3) discarded blood units 10/4. iii group: wrong data input 5/4, donor replacement 4/1, marking errors 3/10, error in determining blood group at the first blood taking 28/13, errors in input medical consulting 9/12 and disregard of prohibitions 2/13. the consequences are: (1) the time spent for resolving h 35/40, (2) mtp 2.5/3.5, (3) discarded blood units 9/18. conclusion: the analysis of errors has showed that the number of errors can be decreased by implementation of corrective/preventative action based on continual education of the staff, appropriate sop, effective organization, qmp for equipment. the conclusion of our study is that reducing the rate of work errors will decrease the waste of material and time, also that will decrease the number of discarded blood units. an iso standard for blood transfusion? background: in our search for an independent, objective assessment at western province blood transfusion service, we were unable to find one single model that met the specific requirements of blood transfusion. we therefore resorted to developing our own model but ask the question: why not have one international standard for blood transfusion? aims: our aim was to have a standardised system for the independent, objective assessment of our blood transfusion service with audits carried out by an internationally recognised body. we wanted a formalised, professional system of accreditation with inspection checklists, reports, certificates etc. methods: having moved away from accreditation by the american association of blood banks in mid 1990's, we evaluated various other options such as inspection by our government department of health or the world health organisation but neither organisation had trained inspectors or systems in place. we also investigated iso 9000 certification but, although this was acceptable on the quality management side, it did not cover the technical parameters relating to blood transfusion. results: we therefore developed our own model for accreditation that consists of three parts: • a quality management section incorporating iso 9000 principles; • a technical section incorporating specifications from the south african standards of practice (we also consulted the european, american, canadian and australian guides); • a laboratory section incorporating iso 17025 parameters (soon to be updated with iso 15189). we then chose to be accredited by the south african national accreditation system, sanas, an internationally recognised institution. once we had written a national accreditation checklist, sanas submitted this to two international accreditation bodies (iaf and ilac) for approval. the system has been in place for three years now during which time we have had four successful assessments. summary/conclusions: in developing our system, we reviewed what was being done elsewhere in the world and it became evident that, although there are great similarities between countries, there p-078 software for the management of the scansystem bacterial detection method the scansystem tm was developed for bacterial detection in blood products. to be implemented in blood banks, a specific software is now available in compliance with blood bank regulation in order to manage sample traceability and data file transfer. the software is divided in 3 main levels: an administrator level to create an application configuration in compliance with customer needs (product bar code characteristics, frequency of the positive controls, manual or automatic data file transfer . . .), a technical level to manage the operators (password, id) and to validate some specific results, an operator level for routine testing. the software assess sample traceability when testing pools of samples from 1 to more than 20. indeed, bacterial detection is performed for pools of 1 to 3 platelet samples and 1 to 20 red cell samples. each sample in the pool is traced through its barcode until the final result. the system is compatible with most of the barcode standard including isbt 128. in addition, the system checks each barcode protecting the sample against duplicate testing. the software assists and monitors the bacterial detection process from the sampling to the end of the test (final result), each step of the procedure is identified through its barcode and at each time, it is possible to know the test status for each sample. for a pool of samples, 2 results can be obtained: 'negative' or 'on hold' for a positive result. for a 'negative' pool, each sample constituting the pool are determined as 'negative' . for an 'on hold' pool each sample constituting the pool must be tested as a single sample and the final result is 'negative' or 'positive' . data transfer may be manual or automatic. a final technical validation is necessary before the transfer through an active selection of results to download. final results are provided in a compliant format for an easy import into the blood bank database. all necessary information are displayed: 'machine id' 'product/sample barcode id' 'date' 'time of transfer' 'operator id' 'result' ('negative' or 'positive'). the main advantage of this software is a continuous check of each step reducing the risk of error in testing. it makes the scansystem tm test compatible with a routine use in blood banks according to the current regulations and quality assurance programs. is no overall consistency. we feel it would be of benefit to establish an international working group to investigate the feasibility of writing an iso standard for blood transfusion. the standard would harmonise quality management parameters based on iso principles and technical/laboratory parameters specific to blood transfusion. minimum technical specifications would need to be agreed upon based on the various standards and guidelines available around the world. this iso standard could then be used for the purposes of certification/accreditation or government inspections. this would ensure global standardisation of world-class best practices. first world countries would be able to achieve compliance and a subsequent step could be the establishment of an international forum to assist developing countries to work towards compliance in the longer term. residual leukocytes in leukoreduced cellular blood products -evaluation by flow cytometry web-based outcome review: do you know how productive your trima® can be? background: web-based outcome review is a new software tool developed by gambro bct for the management and the interpretation of data from the trima and trima accel tm automated blood collection systems. aim: does the interpretation of reports obtained through outcome review lead to an increase in the number of products per run and the overall productivity of the apheresis center? method: run data files (rdf) from trimaᮀ were collected and transferred onto the outcome review server. these rdf do not contain any donor related data that can lead to possible donor identification. the reports were generated on the outcome review website (gambro bct intranet), interpreted by a gambro bct employee and presented and discussed with the management of blood centers. results: a total of 21 different reports can be generated on the website as a pdf file. for this study, 9 reports were investigated. they are: doses per collection, doses per collection trend, platelet collection trend, platelet procedure performance, platelet procedure performance trend, product distribution, average procedure time, procedure time, machine productivity trend. the results obtained for a center can be compared to word-wide, national, regional or to other individual trima devices in the centre (benchmarks). this benchmarking allows the management of an apheresis center to compare the results, to draw the right conclusions and to develop and implement corrective actions. implementing successfully these corrective action plans will lead over time to productivity results that are more in line with the figures that are generally accepted to be the optimal production capabilities of trima. the reports also help to monitor the effects of the corrective action plan over time and to adjust this plan if the results are not in line with the expectations. reaching and maintaining the optimal production capabilities of trima will also increase the net revenue by procedure or decrease the cost by procedure for the blood centre. conclusion: web-based outcome review allows getting more products from the existing donor base by interpretation of the multiple reports and implementing the required corrective actions until optimal production capabilities of trima are reached and maintained. introduction: to examine the cell vitality of packed rbc's during storage several parameters like atp, free hb or 2,3-dpg are used. less kits for the determination of atp are available and they need either a large sample volume and/or are time consuming. here we present the modification of a commercial testkit for a time-and cost saving detection of atp. methods: samples were analysed with (a) 3-phosphoglyceratekinase reaction according to bergmeyer, h. methods of enymatic analysis 2nd. edition. academic press, new york 1965; (b) detection via hplc and c) using a commercial testkit (r. greiner bio-chemica, germany). the atp-kit were minimized from 3500 ml to a total volume of 200 ml and tests were performed in microtiterplates. results: 245 samples were analysed in hplc and modificated commercial testkits, another 20 samples have been examined in all tests. comparison of the results showed no discrepancies in the above mentioned methods. standard curves have been performed (range 5-1000 mm atp) and statistical analysis demonstrated a given linearity (r = 9.97). variability has been calculated as 1.2% (intraassay; n = 25) and 4.6% (inter-assay; n = 70). the hand-on-time calculated for 96 samples has been decreased from 4.5 hours to 30 minutes. at least the costs of atp-determination have been reduced from €3.45 to €0.22 per sample. conclusion: performance of test kits in microtiter format is a fast and rapid method, reliable for high-throughput determination of atp in packed rbc's. background: in order to preserve both blood safety and availability it is mandatory that a minimal amount of blood units would be discarded due to defects in the materials and supplies used for blood collection, or to deviations in blood processing or storage. aims: (1) to monitor the derangements of different materials and disposables used during blood collection and processing, and to study the suppliers' responses and corrective actions taken. (2) to asses the relative contribution of different defective materials (dm) to the need to discard valuable blood components. materials and methods: about 831 000 whole blood units were collected and processed by mda national blood services in [2002] [2003] [2004] . as part of the routine quality control activities, derangements of the dm used were recorded and analyzed. some 40 different types of dm were defined. out of 68 reports sent to the corresponding manufacturers for investigation, 45 responses (66%) were received and analyzed. about 70% of dm were detected during blood collection. manufacturer defects of different materials were the reason for components discard in nearly 25% of cases. conclusion: defective materials are one of the major causes of the infringements of blood collection and blood component preparation processes. analysis and monitoring of the different defects and of the suppliers' responses and corrective actions are essential to improve products' safety and availability. establishment of a network for the exchange of information among international blood centers would enable the blood banking community to compare between different suppliers and to use the documented cases for training of personnel of both the blood services and the manufacturers. such a system may contribute to the improvement in quality of materials used and might lower the discard rate of valuable blood units. results: table 1 analysis and characteristics of t (1) comprehension of the blood bank's processes and the interaction between them and between the processes of the whole hospital. (2) monitor, measure and analyze these processes, in order to improve their effectiveness continuously. (3) implementation of internal and external quality controls for blood and blood products and implementation of appropriate statistical techniques for monitoring their results. (4) identification of interested parties (doctors, donors, patients) satisfaction and taking up the necessary preventive or corrective actions to improve their satisfaction. the qms of our blood bank was certified by tuv rheinland in 16/11/2004 and the scope of the certification is: 'blood collection, testing of infections markers, production of blood components, compatibility screening for blood transfusion and other immunological tests and implementation of therapeutic schemes in thalassaemia patients' . the implementation of the qms based on iso 9001:2000 standards ensures the improvement of services provided by the blood bank and the increase in customer satisfaction, whether donors or patients are concerned. the former enjoy the respect and recognition of their social contribution, while the latter are assured of very high levels of service and health protection. finally, we shall not underestimate the positive impact of qms in the motivation of blood bank personnel. quality becomes integrated both in their professional and personal attitude and allows for achieving increased satisfaction from their work. equipment management in the national blood transfusion service in serbia introduction: new equipment was urgently needed in three blood transfusion establishments (bte) in serbia. equipment was mostly inadequate for core blood transfusion activities, placed in inappropriate facilities, very old without routine maintenance or calibration. also, technical documentation for most of the equipment did not exist, and procedures for equipment management and responsibilities were not defined. further more, coordination on equipment issues with the qa department was not recognized. service funded by the european agency for reconstruction, provided various equipment for the three blood transfusion establishments. the new equipment includes blood collection equipment, centrifuges, refrigerators, incubators, automated testing equipment, genetic analyzer and it equipment of 1.6 million euros value. before the new equipment is installed the bte's agreed to have the national procedures on installation, validation, calibration and preventative maintenance in place. this will ensure that the equipment can be properly installed and validated before use. the project has provided training on validation to the working group (wg) on quality. the wg has created national procedures related to the equipment, including quite new term validation. the same problems in implementation of procedures were present in all three bte's. significant efforts are made to explain to the staff how the equipment has an impact on quality, how to ensure that the equipment does what it is supposed to do, how to be confident that the results obtained are accurate and how important it is to generate records. qa managers played an important role in the preparation of facilities for equipment installation, making plans for equipment layouts, creating documents (master cards, instruction for use) and designing the validation protocols. the same procedures and records enable an exchange of results, comparability, sharing information on what works and what does not work between bte's. the qa managers also prepared an introduction of equipment requirements to the heads of departments, with special attention to the validation process so that they are able to fully understand what is required and why to validate their equipment. results: national standards in equipment management in serbia are set and are being implemented. qa managers carefully managed that the new, numerous equipment, delivered in the short period of 2 months was correctly installed, validated, obtained with necessary documentation, followed by previously trained staff, so that equipment can be considered as controlled. the additional, positive effect is that validation is performed in one establishment for all bte in the country, allowing a more prompt response to problems and presenting of joint request to suppliers, as well as an easier way of monitoring equipment performance of the three bte's. organized equipment management has affect on every aspect of blood activities and finally to the quality of blood and blood components. background: the vista information system (vista tm ) is used in centers in europe as an apheresis management system. with vista tm it is possible to increase productivity, donor comfort and loyalty and therefore simultaneously improving the overall process in the center. aim: a calculation tool (microsoft excel) was developed to evaluate the added value of vista tm . three blood centers in different european countries completed a questionnaire using their local data. summarizing these figures gives us an idea about the impact of vista tm on the daily work and budget of a blood centre. method: the excel calculation tool that was used investigated 3 major areas where vista tm could show added value: improvement of regulatory compliance, increase the efficiency in operations and improve productivity. results: regulatory compliance: the number of infringements decreased, causing a considerable direct financial gain because these events are very expensive to deal with. the time spent on regulatory reviews decreased with a mean value of 35%. operational efficiency: the number of reports is very site dependent: sometimes a report is made for every procedure together with the printout of a blood loss history form. because vista tm tracks all procedure related data, some sites decided to stop printing these types of reports and to go completely paperless. the percent time reduction in reporting is therefore very variable. however the % of errors related to these reports decreased considerably with a mean value of 30%. efficiency in operations was also obtained because of the number of reports that are available in vista tm . productivity, management and process reports allow verifying and correcting the daily operations of the blood center. increased productivity: depending on the center, also an increase in the number of platelet and plasma products collected was detected. the number of product discards caused by infiltration reduced with a mean value of 50%, mainly due to the possibility to have customized and more donor adapted trima settings. the percentage of whole blood donors targeted for conversion was very site dependent (min 0.5%-max 4%). but because with vista any procedure brings between 2.0 and 3.0 products in general, the financial gain was considerable when donors could be converted from whole blood to apheresis. the use of vista allows the apheresis center to work with a reduced error rate and to increase the operational efficiency and the productivity. the financial impact of this has been estimated by the centers between 4€ and 26€ (mean value 10€) per procedure. establishment of national quality system in blood transfusion service in serbia introduction: the production of blood products is a semiautomated process in which the manual steps may be difficult to control and standardize. aim of the study: we introduced a specialised team for the blood production to test if this improved the control of the quality of the blood products. methods: the blood products tested for statistical process control were red cells in additive solution, buffy coat removed, and leukodepleted (ld) platelet pools prepared from 4 buffy coats. the products were collected in t&b triple opti-pac from baxter and the platelet pools were ld using plx-5 filters from asahi and stored in platelet bags from baxter. using control charts, namely x-mrchart, exponentially weighted moving average ewma chart and for autocorrelated stationary data the ewmast chart, we examined if time series of quality control values were in statistical control. if not we examined if autocorrelation and/or differences between the technologists producing the blood products could explain the lack of control. data included approximately biweekly measurements of volume, haemoglobin (hb) concentration, hb/unit, haematocrit and log leukocyte count (wbc)/unit of 352 units of red cells, measurements of volume, platelet concentration and platelet count/pool of 79 ld platelet pools produced by a team of 13 technologists and of 79 ld platelet pools produced by a specially trained team of four technologists. results: log wbc/unit was out of statistical control due to systematic differences between technologists. apparent lack of control of volume, hb-concentration, hb/unit caused by autocorrelation disappeared when the ewmast chart was used. platelet concentration and volume of the platelet pools produced by the 13 technologists were out of control. in that some technologists systematically produced low values. this could be explained by inappropriate handling of the platelet product between centrifugation and separation. systematic differences between the four specially trained technologists could not be demonstrated and they produced platelet pools with a significantly higher platelet count/pool. however, standard deviations of the four technologists differed significantly causing occasional outlying values. conclusion: training and routine in blood production or process automation, and also importantly, feed back to the technologists based on control chart quality control data, is recommended. background: one important principle of the use of blood and blood products is the ability to trace the units from donor to the recipient. this study set out to establish whether or not there was sufficient reporting on transfusions from the hospitals supplied by fort portal regional blood bank in western uganda as a means of establishing sound haemovigilance and look back systems. were reported with no unit number and could not be traced to the patients. there was sufficient reporting on the data requested by the blood bank. these results suggest that it is possible to establish effective 'look back' and haemovigilance systems. capture of data on outcomes and adverse effects will be necessary to fully establish the system. further efforts are required to educate those involved in transfusing blood about the need for adequate and accurate documentation. external quality assessment of blood grouping were misinterpreted as rhd-positive samples without the use of control reagent. rbc phenotyping was made correctly by 79.7% of participants. the remaining 20.3% of participants carried out the phenotyping incorrectly, while false-positive and false-negative results were derived in 8.91% and 11.39% of cases correspondingly. polyspecific human sera and monoclonal antibodies were used for abo, rh and antigens typing. the reason of errors in antigen detection was low quality of reagents. antibodies identification was carried out in six distributed exercises. 75% of participants detected anti-d-k-c alloantibody correctly. the rest of participants did not found alloantibodies or detected their specificity incorrectly. results of testing depended on quality of screening cells. thus, the participants using homemade pooled screening cells had a significant lower detection rate of antibodies comparing with those using diamed ag cell panel. consequently, the results of the first federal external quality assessment scheme show the necessity of improving the quality of red cell reagents produced in russia. in addition, the more appropriate training of staff is required. the importance of iso 15189-quality system in increasing safety of blood transfusion introduction: hadassah hospital transfusion medicine department received on 3/2004 iso 15189-quality system accreditation. an essential element of this standard is the development of a reliable system to identify, document, analyze and correct actual and near miss events and assess the effectiveness of corrective and preventive actions. aim of the study: assessment of events and corrective actions following implementation of iso 15189 quality system. methods: events in the blood bank were identified by the staff, by internal or external audits and by complaints from the wards. all events were recorded and classified into two categories; quality system and technical. the latter were further classified into preanalytical (sample receipt), analytical (abo rh typing, antibody screen and identification, cross matching and phenotyping) and post-analytical (issue of components to wards). all events were graded into 4 levels; 1-most severe, potentially harmful to patient. 2-severe, damage to process and result. 3-moderatly severe, damage to process only. 4-benign, no harm. events were corrected and effectiveness of corrective actions was assessed by monitoring recurrence of the event. results: during the years 2003-2004, 248 events were detected and recorded in the blood bank, they comprised 0.072% of all tests performed (343 595). most of the events were technical. all events were detected before causing harm to the patients. results are summarized in the table. *the percent analytical value is a summary of rates of events per test types included in this category. events detected in the quality system were mainly of severity level 2&4, whereas technical events were mainly of severity levels 2&1. analysis of event recurrence in the quality system revealed that 97% of events were resolved, whereas only 14%-31% of technical problems were completely solved. the main source of event identification and documentation, in the quality system were audits whereas in the technical system, staff members revealed most events. the implementation of iso 15189 quality system provided a powerful means for recognition, analysis and study of patterns of near misses and actual events. understanding the root causes of events enables to choose the most effective corrective and preventive action to control event recurrence. evaluation of the frequency of events confined to the blood bank revealed a very low rate of 0.072%. these results are in agreement with data in the literature. creating a non-punitive, non-stressing open environment, motivates personnel to identify and document events, which are regarded as opportunities for improvement and serve as important tools for upgrading transfusion safety. the explosive use of information technology and the speed with which it has spread into all life activities has created vulnerabilities for all organizations. those vulnerabilities are compounded by the complexity of information technology, limited time to market, development constraints, and constantly changing relationships between organizations and suppliers. growth in the sophistication of security threats makes it imperative that organizations remain equally competent in identifying vulnerabilities and mitigating security risks. aim: the goal of this document is to explain how isbt intends to provide guidance to the blood banking community on implementation of effective information security policy. when using information for critical activities, blood banks should consider information security as an important aspect of their management policies. evaluation of existing standards, such as iso 17799 and hipaa, allows us to establish a framework for information security without regard to the type of organization. it remains very difficult however, due to the complexities involved, to establish an information security policy without guidance. method: the isbt information task force was created to provide guidelines on information security for blood banking organizations of all sizes. the intent is to help them understand existing standards as well as provide tools for implementing information security policy. these guidelines are based on existing standards that are followed by most worldwide countries: iso 17999 and hipaa. results: information security can be defined as the 'protection of systems, information and services from accidental and deliberate threats to confidentiality, integrity and availability' . understanding existing information security standards was the first step for establishing a structure for the guidelines. the core is organized within an implementation framework and presented under the three following layers: 1. administrative for defining the it security organization, the information security policy, and information security awareness and training. 2. physical for providing solutions that relate to physical environment protection and access, equipment and it infrastructure security, and control for accessing computerized equipment. 3. technical for maintaining confidentiality of electronic information and ensuring that authorized access to information systems is maintained (technology relating to identification and authentication, logical access, operating system, network management, application access, etc.). strategy guidance is also included for senior managers in charge of establishing organization policy, including responsibilities and methods to successfully implement policy. further, the task force is addressing both risk analysis and management including identification of potential dangers to information systems (threat-source) and existing controls (risk description), as well as a plan to address identified vulnerabilities and mitigation of specific risks. conclusions: information security standards are prerequisite to understanding the issues involved when considering information vulnerability. international guidelines for information security, specifically directed to the blood banking community, are equally necessary if we are to identify, plan for, and mitigate risks associated with vulnerabilities to critical blood banking information. the isbt task force is committed to providing such guidelines. introduction: the important part of quality planning and quality assurance within production of blood components is measurement system analysis (msa). measurement system analysis was performed on microscopic counting of blood cells in our study. aim of the study: aim of this study is determination if microscopy counting of residual elements in whole blood plasma is suitable for quality control of blood components. methods: we practiced measurement system analysis in counting of residual elements (leukocytes and erythrocytes) in whole blood plasma. the counting performed two lab technicians in ten samples of plasma from whole blood. leukocytes and erythrocytes were measured in naggeotte counting chamber. we used the method of mean and range for the determination of reproducibility and repeatability (r&r) and analysis of variance for complex measurement system analysis. regulation diagrams were applied for the graphic statement. we determined the value of repeatability, reproducibility, coefficient r&r, variability among samples of plasma and total variability of measurement system. the important conclusion was to determinate if the microscopic counting of samples of blood components is sufficient with regard to quality parameters specified in guide to the preparation of blood components (erythrocytes: <6.0 ¥ 10 9 /l, leukocytes: <0.1 ¥ 10 9 /l). our results show that microscopy counting of residual elements in whole blood plasma is suitable for quality control of blood components. aim of the study: to provide transfusion services with a tool for proper qms implementation, an international collaborative study on qms applications, employing the 'process approach', has been undertaken by a group of transfusion services of varying sizes and structures with experience of qms, in collaboration with a university institute offering a master in qms implementation in health services, and an expert from a quality association. the 'process approach' serves as a tool to manage transfusion activities as a system based upon a network of processes and their interactions. guide-lines have been produced based upon this principle and will be published (volume and cd-rom) and distributed at no cost to all transfusion services of nations participating in the study. the main contents of the guide-lines' chapters are: through definition/analysis of single processes and the correlation network amongst these, the 'process approach' methodology renders the transfusion centre's functioning units completely interdependent, eliminates process interface barriers, provides personnel with a unified focus on the main transfusion objectives, and lays the basis for improvement of transfusion service quality, organization and performance through efficient control of processes' interactions. the blood screening system 400 automated high-throughput nat system for simultaneous screening of hcv, hbv and hiv nucleic acids: full process surveillance e pfeifer*, b alessandri † , hr bachmann † , t barker † , y ohhashi*, c parkhouse*, j pinsl-ober ‡ , p wenzig ‡ and g ziegler ‡ *roche molecular systems, inc., pleasanton, usa, † roche instrument center ag, rotkreuz, switzerland, ‡ roche diagnostics gmbh, penzberg, germany introduction: the blood screening system 400 combines on one deck both automation of dna/rna extraction from blood/plasma samples and multiplex pcr amplification and detection of nucleic acid targets. the system is designed for high-throughput single unit testing and pooled specimen processing. a data management system supervises and controls the complete process from initial sample pipetting through to result compilation and reporting. the objective of this project was to present the system 400 assay and device built-in quality control measures that guarantee process safety and reliability. the study shows how internal control, external batch controls, pipetting sensors, validation & maintenance procedures, and a controlled development & manufacturing process yield an optimal test method and system stability. method: failure modes and effects criticality analysis (fmeca) was used to evaluate a mathematically derived safety metric for optimizing risk reduction. this method makes use of a system risk objective-function (srof), which provides a multivariate description of sample processing, amplification and detection steps. the method for analyzing system behaviour first employs product classification into risk domains, followed by ranking of process steps that are determined to be linked to known system hazards. this provides objective means for directing system design leading to risk minimization. the srof responses associated with redundant liquid sensing channels were shown to substantially reduce risk during sample or reagent transfer steps. the system 400 liquid flow, airpressure-based (plld) and capacity-coupled liquid (clld) sensors detect aspiration and dispensing inaccuracies. sensor signal tolerance band widths studied for fluid classes representative of system 400 reagents and for plasma samples from lipemic, icteric, and hemolized sample sources were shown to correlate with srof multivariate modelling. the impact of surveillance design elements on the srof response demonstrates that risk is functionally dependent on design elements. additional surveillance examples are presented that describes temperature sensing, robotic positioning and motion control. treatment of residual risk is addressed by introducing external controls that are configurable to specific workflow scenarios. the negative external control (nc) is used to check for contamination of reagents. five low concentration armored external controls, i.e. hiv-1 group m arna, hiv-1 group o arna, hiv-2 arna, hcv arna, and protected hbv dna are run at the beginning of a batch as a run-control measure. in addition to these roche controls the system 400 supports running of user-defined external controls for co-validating the batch. the internal control (ic) is based on armored hiv-1 group m rna that is co-extracted and co-amplified with the external controls and the target nucleic acids potentially present in the sample. lastly, the system 400 resource management monitors the status of samples, ready-to-use reagents and disposables via rack sensors and bar-coded bottles and tubes. the fmeca methodology provides a risk-minimized, comprehensive system design. redundant sensors with internal and external controls are evaluated through comparison of modelled versus actual run results. the system 400 brings a new level of surveillance and throughput for automated pcr testing, and emphasizes roche's commitment to increasing the safety of the global blood supply. technical standards for safe storage and transport of blood components and blood samples objective: to implement standardization of technical specifications for safe storage, transport and distribution of blood samples and blood components intended for transfusion, as part of a quality system in blood transfusion medicine. methods: in the course of implementing a quality system in our blood establishment (distributing 10% of the national blood supply), we have developed standard operating procedures (sops) for temperature and hygienic conditions to maintain and control storage of blood components during their shelf life and to ensure their safe distribution to other blood services in the country and abroad, in compliance with eu directives 2002/98/ec and 2004/33/ec and the recommendations of the council of europe and who. procedures are validated and relevant records are kept. a statistical process control is also in place to monitor deviations from specified temperature and time range throughout the period of transportation. blood components collected and prepared for specific purposes (e.g. directed donations, irradiated units, hla-typed units, anti-cmv negative blood components and blood for neonates) are stored separately, and alarms and warning systems are in place. packing and transport conditions of red cells, platelets and ffp are submitted to the tests and criteria of adr (european agreement concerning the international carriage of dangerous goods by road). in greece, the adr legal framework has applied since 1999. blood samples are classified according to adr in division 6.2 under un 3373 as diagnostic specimens, and the criteria for safe carriage include packaging, specific labelling and vehicle requirements as well as carrier obligations and personnel training. our blood establishment has a contract with biotrans, a private company accredited for packaging, storing and transporting blood, organs, tissues and cells as well as potentially infectious biologic substances and blood samples for diagnostic purposes. assurance system). the approach is specified in the form of requirements of art. 4.1 of the standard: the organization shall: (a) identify the process needed for the quality management system and their application throughout the organization, (b) determine the sequence and interactions of these processes, (c) determine criteria and methods needed to ensure that both the operation and control of these processes are effective. regional centre for transfusion medicine in biaĺystok was the first transfusion service in poland which was certified according to iso 9001:2000 standard. our expected profits from the implementation qms were: possibility to overview organization's pathways of operation and to inspire corrective and improvement actions, emphasis on the role of staff in the system, focus on self-control and responsibility for one's own work as a factor of staff's mentality creation/modification. our one year of experience with iso 9001:2000 standard proved the system to be handy tool of management for the organization collecting, processing, testing blood and releasing of blood products for the hospitals and to be well accepted by the staff. the implementation and certification of the internationally nor-malized quality management system simplify and shorten all accreditation and registration procedures required for legal activities of transfusion service as well as for any supplemental medical activities which may be performed by the centre. the implementation of qms facilitates the implementation of other quality systems and simplifies procedures required for the ce certification for the products. red blood cells stored in blood banks, normally undergo a series of chemical alterations, or storage lesions. the ultimate consequence of these lesions is a decrease in the viability of the red cells following transfusion. the chemical alterations are mainly changes in levels of na+, k+, cl-concentrations, ph and 2,3 dpg levels and they affect the electrical impedance of blood. the electrical impedance, cole-cole parameters, is determined mainly by the resistance of the red cell extracellular fluid (re), the resistance of the intracellular fluid (ri) and the capacitance of the cell membranes (cm). in this study we aimed to investigate the relation between blood parameters and electrical impedance changes, and their further clinical implication. all parameters were measured on 51 erythrocyte suspension (es) samples during 42 days of storage at 4oc on days 0, 10, 21, 35 and 42. for 31 whole blood (wb) samples during 21 days of storage, same parameters were measured on days 0, 10, and 21. the measurement of the complex impedance of blood samples were performed in the frequency range from 100 khz to 1 mhz. by using the 4284a hp lcr meter, the impedance z, and the phase angle a for each sample were read. these values were corrected according to the gain and phase characteristics of the amplifier; and the resistance r and the reactance x were calculated. each data was first fit to the cole-cole model; hence the cole-cole parameters, intracellular resistance ri, extracellular resistance re, characteristic frequency fc and phase angle a were obtained by using lms software. afterwards, cm was calculated by using ri, re, a and fc. whereas ri and cm decreased progressively with time on both wb and es, re changes showed some differences. the electrical impedance alterations were explained by measurements of na+, k+, clconcentrations, ph and 2,3 dpg by indicating days. storage of red cells resulted in a rise in extracellular k+ and a fall in extracellular na+, cl-, ph and 2,3 dpg. anova was used to evaluate differences in blood measures in relation to storage time. the results were presented as the mean ± sd. according to the regression analysis in spss, the intracellular resistance (ri) on both es and wb was affected more efficiently from all blood parameters among all other electrical parameters. although ri and re were correlated with na+, k+, cl-, ph and 2,3 dpg more significantly, cm measurements were failed to show correlations with blood parameters because of the intervening parameters, a and fc. the best relationship between the parameters mentioned above on both es and wb was ri and k+. ph changes were the same for ri and re both for es and wb. the correlation between parameters on es was better than those on wb, because whole blood consists of several particles that may affect our measurements. our study showed that 2,3 dpg has an effect on ri and re as efficiently as other blood parameters and therefore electrical impedance measurements may serve future implications. background: issue of quality blood products and donor safety are the main aims of blood transfusion services. a comprehensive quality system should be in place to fulfill these aims, which can be attained through strict adherence to the established standard operating procedures (sops). the drugs and cosmetics act of india, which controls the licensing of blood transfusion services, does not provide clear guidelines regarding plateletpheresis procedure. aim: we therefore established our own sop and operational flow chart for plateletpheresis that can be easily followed by other centers in india. methods: a total of 100 plateletpheresis procedures performed using two cell separators (cs3000 baxter, usa, mcs3p, hemonetics, usa) were evaluated following our established sop. the mean platelet yield in cs3000 was 2.9 ± 0.84 ¥ 10 11 and in mcs3p, it was 2.88 ± 0.75 ¥ 10 11 per unit, however, only 4-7% of sdps showed wbc levels <5 ¥ 10 6 . six of 100 donors complained of hypocalcemic symptoms. the operational flow chart designed in this study was found to be simple and easy to adapt by blood transfusion services in this country. the first advanced quality management training course for blood transfusion services in the western pacific region mk tan*, jp yu † and d teo* *health sciences authority, singapore, † who regional office for wpr, manila, singapore background: blood transfusion is a key part of modern medicine. a well-organised blood transfusion service (bts) is a prerequisite for the safe and effective use of blood and blood products. in 2000, the world health organization (who) introduced a new initiative of quality management project (qmp) to achieve the goal of safe and adequate global supply of blood. through qmp, regional training centers were identified and quality management training (qmt) courses were established. in 2001, centre for transfusion medicine (ctm) of health sciences authority singapore was appointed as a collaborating center for qmt in the western pacific region (wpr background: spectrophotometric method according to harboe was traditionally used at our institute for determination of free haemoglobin in supernatants of rbc blood components at the end of storage time. in the year 2002 hemocue plasma low hb system (hemocue, sweden) was introduced as a replacement method. aim: the aim of the study was to examine reliability and suitability of the hemocue method for measurement of free haemoglobin in supernatants of rccs. methods: hemocue plasma low hb method was validated and compared with harboe method. supernatants of 36 rbc products were tested by two methods and results compared using regression analysis. additional testing was performed to investigate precision of hemocue method (within-run and between-day variation), trueness using reference material and to define optimal sample handling. results: regression analysis showed high correlation between two methods (r = 0.98), with higher values obtained with hemocue method. immediately after centrifugation one supernatant was measured 6 times in order to determine within-run imprecision. coefficient of variation (cv) calculated from 6 consecutive measurements was 6.1%. to investigate between-day imprecision of the hemocue method one sample was divided in 5 aliquots and frozen. one sample was thawed each day and measured. cv of five measurements was 1.95%. during the period of validation 37 measurements of reference material (low, medium and high) were performed. cv calculated for these measurements were 7.86% (low), 1.88% (medium) and 1.04% (high). in order to investigate possibility of batch testing, supernatants of 5 different rbc products were divided in aliquots and measured periodically during 1-month period. cvs calculated from 7 measurements of each sample were in range 3.1-9.9%. conclusion: hemocue plasma low hb method appears to be an excellent replacement for the harboe method. it is consistent, easy to use, measurements are performed in short time, and errors are minimized by eliminating dilutions and manual calculations. background: determination of haemolysis in red cell concentrates (rccs) at the end of storage time is routine method in quality control of blood components at our institute. for this purpose, haemoglobin concentration in supernatant of rccs is measured using hemocue plasma/low hb system (hemocue, sweden). according to manufacturer recommendation, visually turbid samples should be filtered before analysis with a 0.2 mm filter. because visual estimation of turbidity is highly subjective and unreliable, filtration of all supernatants before analysis using appropriate filters is possible solution for standardisation of the method. aim: the aim of the study was to investigate the effect of filtration of visually non-turbid samples on results of free haemoglobin measurement. methods: haemoglobin concentrations were measured in 42 visually non-turbid supernatants before and after filtration using hemocue plasma/low hb photometer (7 wb negative controls were used for each blood component. in all blood components tested bacterial contamination was detected using both types of bottles. in comparison with sa/sn bottles, nearly all enrolled microorganisms were detected faster in bpa/bpn bottles (see table 1 ). all negative controls were negative (no false positive). the results of the study performed support the use of bact/alert bpa and bpn plastic bottles in quality control testing of different blood components. objective: management of returned blood products is important part of quality assurance activities in transfusion medicine. blood products are returned most frequently because of routine rotation of stock and because of nonconformities discovered after the product has been received. methods: qa data about returned blood products were retrospectively analysed for the 5-year period (2000-2004) . only blood products returned because of nonconformities were taken into consideration. data about the reasons for returns are presented and discussed. the top 4 reasons for returns were positive dat, labelling errors, blood bag defects and visual appearance of blood units (see table 1 ). in all cases positive dat was confirmed in our institute, and blood donors managed accordingly. nonconformities related to visual appearance of blood component and other nonconformities related to the quality of blood products were investigated and corrective actions conducted. in case of blood components returned because of damaged bag, significant problem is to investigate the cause of nonconformity (usually inappropriate transport conditions). conclusion: management of returned blood products is important tool in improving the quality of blood products. introduction: hemovigilance is the systematic monitoring of the blood transfusion chain for side effects and adverse incidents from the moment of blood collection until after administration of the unit to the recipient, and comprises all activities that can lead to a safer and more effective use of blood components. attempts to achieve a more safe and effective use of blood components constitute a huge task given the number of interventions and array of medical and not-medical personnel involved in the transfusion process. registration of the impact of all participants is a tremendous job as such. information management may enhance the chances for a successful hemovigilance. aim: the aim is, to establish a basis for all the information related to the chain, such as standard operating procedures, (inter)national guidelines, local transfusion protocols and forms, and procedures to direct the process. additional factors that contribute to the final results are the profile and role of employees, incidents, points of care, product information, prices, budget, contracts, etc. by clarifying and explaining the basis to all participants by means of an existing infra structure, everyone will gain access to identical, consistent and up-to-date information at all times. the primary activity is to create the basis by an outline of every individual link in the transfusion chain from donor to recipient. secondly, the resources, responsibilities, actions, and internal controls (what task, who is performing, why, which help, where, when) are documented. by creating distinct databases for these 6 'w's' in combination with a separate database for the hemovigilance process itself, it is possible to establish relations, hyperlinks, between the different databases. all the information collected in the foundation, complete with all the resources, responsibilities, actions, and internal controls is made available to target groups by means of the intranet in our hospital as well as in a printed handbook format. collecting and displaying the information on hemovigilance this way, creates a transparent and more easy-to-manage process. it establishes a basis, which incorporates all resources, responsibilities, actions, and internal controls to all participants and enables the organisation to operate both efficiently and effectively. it allows us to show auditors (both internal and external) which processes are related to which guidelines as well as the results in daily. in addition, it reveals which factors determine the genuine application of the guidelines in clinical practice. conclusion: by outlining the process, followed by defining, analysing, securing, on the 'w6' method, the hemovigilance process offers a stable basis and framework for all participants and may stimulate a more safe and effective use of blood components. background: transfusion medicine is a basic post-graduate specialty for medical doctors with a specific national curriculum in bulgaria. in order to improve the post-graduate training of medical doctors in transfusion medicine, a new curriculum was elaborated in 2004. purpose of the new program: independent work of transfusion medicine specialists on all levels of the blood transfusion service (bts) -organization of bts, promotion of voluntary, nonremunerated blood donation, collection, processing, testing, storage and transport of blood and blood components, immunohematologic testing of patients, laboratory hematology, clinical experience, assistance and advice on diagnostic and therapeutic problems of patients, requiring treatment with blood products, teaching transfusion medicine to bts and clinical personnel. admittance and duration to training -medical doctors are admitted to post graduate specialization after a successful state examination. the overall duration of training is 4 years, of which at least 2 are obligatory for training in the national or regional blood transfusion centers. main sections of the curriculum: 1. organization of blood donation and blood transfusion, including promotion of voluntary, nonremunerated blood donation; organization, planning and information systems; organization of blood transfusion; quality management. 2. collection, processing, storage and transport of blood and blood components 3. laboratory hematology and specialized methods (general laboratory methods, immunology, immunohematology, transmissible infections, hemostaseology, nat techniques, flowcytometry) 4. clinical use of blood components and plasma products (treatment with blood products, adverse events and reactions, alternatives to blood transfusion). the new curriculum of transfusion medicine guarantees a better training of medical doctors, thus leading to safe and effective blood products their proper clinical use. introduction: transfusion medicine is integrated medical discipline based on clinical and laboratory practice. it is closely connected with molecular biology, genetics, as well as with apheresis, automatically techniques and transplantation. the aim of studies in transfusion medicine is proper education, skills, attitudes and knowledge of students at the medical faculty and of doctors on residency in transfusion medicine and other specialties about blood and its usage. material and methods: there will be presented how transfusion medicine is implemented in educational and health sector in r. results: there is 3-year specialization in transfusion medicine, established 30 years ago (over 90 specialists finished this specialization till now). we have postgraduate studies in tm started 15 years ago. transfusiology, as subject at the medical faculty, is now included in new curricula of medical student, consists of 15 theoretical and 15 practical lectures. also, medical doctors on specialization in surgery, anesthesiology, obstetrics and gynecology, pediatrics, internal medicine, maxillo-facial surgery and others have obligatory 1-month education in tm. transfusion medicine is integral part in education of nurses and laboratory technicians; all personnel that work in transfusion field in our country finished 6-mounths course in tm and get certificate. conclusion: although we have already done so much in educational field, we will continue with our efforts to improve our collaboration with clinicians and to involve ourselves more in clinical disciplines. introduction: in hospitals of saint petersburg donor blood, its components and preparations, autoblood and its components, hemocorrectors are applied with the medical purpose at 20-30% of hospital patients. the service of blood and the existing organization transfusion therapy in hospitals should provide maximal immunological and infectious safety and also its high medical efficiency. it demands special preparation for all doctors: general physicians on clinical transfusiology and doctors of blood service on all sections of the general, industrial and clinical transfusiology. clinical gemostasiology, pharmacology of hemotransfusion means and hemocorrectors, the organization of the blood service, the donor service; the organization, technique and technics of preparation of blood, its components and preparations, quality assurance, transfusion therapy programs, technique and technics of transfusion medicine, a problem of maintenance of immunological and infectious safety, preventive maintenance, diagnostics and treatment of posttransfusion complications, feature transfusion therapy in pediatrics and medicine of accidents). clinical physicians master all basic questions of clinical transfusiology, thus the special attention is given questions of clinical immunohematology and clinical gemostasiology, programs and a technique of transfusion therapy, the prevention of posttransfusion complications. employment are carried out in departments of city blood bank, hospitals blood departments. final examination under the test program and at interview shows sufficient mastering a theoretical and practical material (right answers of 80-98%). the described system of postgraduate studies provides an opportunity of successful independent work of doctors on the workplaces both regular increase and qualifications not less often than 1 time in 3-5 years. conclusion: the existing system of postgraduate education for doctors on transfusiology provides the blood services and hospital by the qualified staff. introduction: nowadays in hospitals of saint-petersburg more and more it is frequently applied the extracorporal hemacorrection (haemapheresis, haemasorbtion, plasmasorbtion, krhyoplasmasorbtion, ultrafiltration etc.) and photohemotherapy (optical radiation influence on blood) at various diseases and traumas. they are used at treatment almost 6% from the general number of patients of hospitals with positive results at 85% from them. for this purpose in hospitals there are specialized branches or cabinets with staff of the doctors -transfusiologists. therefore an actual problem is organization of postgraduate special education for them. because there is no specific blood banking and transfusion medicine training in either under or post graduate medical education those general practitioners have limited knowledge about this field. aim: to give basic knowledge and practice to those general practitioners moh has established a special training programme with the collaboration of medical schools for 5 years. methods: this is a 6 months programme which has 2 basic components; theoretical education, laboratory and blood banking practice. the trainees are evaluated by a final examination. each group has limited cadets and the training is provided in selected blood banks with the collaboration of universities and moh training hospitals. conclusion: almost 100 general practitioners are trained by this programme in the last 5 years and those doctors helped to increase the quality of blood banking and transfusion service at their hospitals. background: even the history of public blood banking in turkey has dates back to 1957, whole blood was comprising the majority of transfused units (more then 96%) in turkey until the last few years. aim: the main target was to decrease the whole blood use in a short time and establish countrywide effective blood transfusion practice. methods: there were no specific blood banking and transfusion education either at undergraduate and postgraduate medical education in turkey until the last few years. blood banks and transfusion society of turkey (bbtst) was established at 1997 with the main aim of education of the related people about blood banking and transfusion medicine. bbts has organized 35 symposiums, 13 national courses, 1 national and 1 international congresses since 1997. due to increased knowledge about the blood components and improved infrastructure of blood banks whole blood consumption has decreased to national average to 60%. in most of the university hospitals and training hospitals this is around 19%. conclusion: like most of the public based behaviours dedicated efforts of civil initiative has changed the traditional habit of blood consumption in turkey by the direct affect of well organized educational activities. training of general practitioners in blood banking and transfusion medicine n solaz*, b keskinkilic † and c oruc † *ankara university, ankara, † ministry of health, ankara, turkey background: turkish ministry of health runs majority of the hospital blood banks in turkey. most of the moh hospital blood banks give service under the medical supervision of general practitioners. because there is no specific blood banking and transfusion medicine training in either under or post graduate medical education those general practitioners have limited knowledge about this field. aim: to give basic knowledge and practice to those general practitioners moh has established a special training programme with the collaboration of medical schools for 5 years. methods: this is a 6 months programme which has 2 basic components; theoretical education, laboratory and blood banking practice. the trainees are evaluated by a final examination. each group has limited cadets and the training is provided in selected blood banks with the collaboration of universities and moh training hospitals. conclusion: almost 100 general practitioners are trained by this programme in the last 5 years and those doctors helped to increase the quality of blood banking and transfusion service at their hospitals. background: it has been accepted that during cardiac surgery the complement-system is activated which enhances ischemia-reperfusion injury, leading to postoperative complications as infections and organ failure. the lectin pathway is one of the mechanisms that can activate the complement-system, this pathway can be mediated by mannose-binding-lectin (mbl). the level of mbl in serum shows a wide variation. a low level of mbl can lead to more infections in some conditions and a high level to tissue damage after ischemiareperfusion injury. the effect of transfusions of erythrocyteconcentrates and plasma on the mbl levels and thereby on complications after cardiac surgery are not known. aim of the study: the role of pre-and postoperative mbl levels and the effect of transfusions on postoperative complications after cardiac surgery. in a randomized controlled trial 474 cardiac surgery patients were included and blood samples were taken pre-and postoperatively. mbl measurements were performed by elisa assays. the data were linked with postoperative complications as mortality, infections and multiple-organ-dysfunction-syndrome-mods and with peri-operative erythrocyte and plasma transfusions. results: the mean pre-operative mbl-level was 837 ± 796 and postoperative 456 ± 347 mg/ml. there were no differences in preand post-operative mbl levels between patients with infections or mods compared with non-infected and non-mods patients. the difference in pre-operative mbl between non-survived (672 ± 587) and survived patients (853 ± 812) was not significant (p = 0.20). postoperative mbl levels between survived and non-survived patients was smaller. conclusions: pre-and postoperative mbl levels in cardiac surgery are not related to postoperative infections, mods and hospitalmortality. whether large amount of blood transfusions are affecting the mbl-levels and thereby the patients outcome is not known. introduction: patients undergoing cardiac surgery are receiving high amount of blood transfusions and are at risk for the development of infections and multiple-organ-dysfunction-syndrome (mods), these complications are influencing the survival of the patients. during cardiac surgery pro-and anti-inflammatory cytokines are released by different mechanisms. we found in a randomized trial in cardiac surgery a significant reduction in infections and mortality due to mods by transfusion of leukocytedepleted erythrocyte concentrates (ld) compared to leukocyte-containing, buffy-coat depleted erythrocytes (pc). aim of the study: the effect of ld on the concentration of proinflammatory cytokine il-12 and anti-inflammatory cytokine il-10 in relation to clinical outcome and complications after cardiac surgery. methods: in 474 participating patients blood samples were taken before and after surgery. using elisa il-10 and il-12 were measured in these samples. the results were linked with the endpoints of the randomized trial: postoperative infections, mods and in-hospital mortality. results: all pre-operative concentrations of il-10 and il-12 were low. mean postoperative level for il-10 was 70 ± 121 and for il-12 57 ± 74. compared with patients without mods and without infections and survived patients only the il-10 was significant higher in patients who died and in patients with mods. there were no differences in mean levels related to ld and pc. the levels of il-10 were higher in patients receiving more then 10 units blood transfusions compared to 0 transfusions. conclusion: there is an association between mods and mortality and il-10, not with il-12. ld has no influence on mean levels of il-10 and il-12. there is a correlation between transfusion of more then 10 units and il-10, not with il-12. these cytokine-profiles are not associated with the beneficial effect of ld on the postoperative outcome in cardiac surgery patients. anemia and blood transfusion practice in critically ill patients e grouzi*, e tsigou*, p evagelopoulou † , g baltopoulos † and i spiliotopoulou* *kat general hospital, transfusion service, athens, † athens university school of nursing icu, athens, greece background: anemia is a common problem in critically ill patients admitted to intensive care unit (icu), but the consequences of anemia on mortality and morbidity in the critically ill is poorly defined. aim: to define the incidence of anemia and red blood cell (rbc) transfusion practice in critically ill patients in the icu) of our hospital, and to examine the relationship of anemia and rbc transfusion to clinical outcome. patients and methods: the period study was from july 2003 to december 2004. patients were enrolled within 48 h of icu admission. follow-up time was 30 days, hospital discharge or death, whichever occurred first. results: a total of 169 patients (116 male, 53 female, mean age 57.6 ± 21.1 years, range 15-96) were included in the study. the mean hemoglobin (hb) level at baseline was 11.2 ± 2.1, which level was descending during the study. overall 63.9% (108/169) of the patients received one or more rbc units while in the icu stay (mean 3.1 ± 3.8 units per patients). the mean pretransfusion hb was 8.7 ± 1.8 g/dl and the mean time to first icu transfusion was 2.6 ± 4.1 days. more rbc transfusions were given in the first week of the icu stay (331 units vs 110, 61, 36 units in the second, third and forth week respectively). the number of rbc units which a patient received during the study was positive associated with longer icu length of stay and an increase in mortality (r = 0.19, p < 0.05 and r = 0.26, p < 0.05 respectively). baseline hb level was significantly related to the number of rbc transfusion (r = 0.55, p < 0.001), but was not an independent predictor risk factor of length of stay or mortality (r = 0.16, p > 0.05 and r = 0.13, p > 0.05 respectively). the mean baseline apache ii and saps scores were 19.6 ± 7.4 and 49.8 ± 17.4 respectively. furthermore both baseline apache ii and saps scores were significantly higher for patients with a baseline hb level of <10 g/dl (23.2 ± 7.9 vs 18.3 ± 6.7 and 54.6 ± 18.8 vs 48.1 ± 16.5 respectively), while the apache ii values were positive associated with a significantly increased likelihood of rbc transfusion (pearson correlation p < 0.005). conclusions: anemia is common in the critically ill patients, it appears early in the icu course and persists throughout the duration of the icu stay. rbc transfusion seems to be associated with worse clinical outcome. despite the intensive research for the transfusion practice, which taken place worldwide during recent years, data from our country is limited. the results of our study suggest that approaches to reduce rbc transfusion would be desirable. however, further well designed prospective studies with large number of patients are required to efficiently explore the risk of anemia, optimal transfusion hb threshold and the risk and benefit of rbc transfusion in the critically ill. consumption of blood products in a large, general hospital he heier*, j pillgram-larsen † , m hestnes ‡ , b gran*, a krog* and no skaga ‡ *ullevaal university hospital, oslo, † ullevaal university hospital, dept. of thoracic surgery, oslo, ‡ ullevaal university hospital, dept. of anestesiology, oslo, norway uuh is the largest general hospital in norway, and trauma referral centre for half of the norwegian population (2.5 mill) the trauma team performed initial assessment and resuscitation of 323 patients, 75% males, during the first six months of 2002. the aim of our study was to analyze transfusion practice at uuh with specific focus on trauma. materials and methods: blood consumption during this period was recorded. clinical data of trauma patients were collected from our trauma registry and anonymized before analysis. results: 7012 units of erythrocytes (er), 914 of thrombocytes (thr) (buffy coat preparations from 4 donors) and 903 of s/d-treated whole plasma (op) (octaplasò) were transfused in uuh during this period. 56.6% of er were given to surgical, 21.3% to medical and 12.2% to gynaecological and obstetric patients. 62% of er were given to patients above 45 years. er units were given to 1475 patients (mean 4.75 units/patient; range 1-63). mean age of trauma patients was 33 ± 18, median 30, range 1-89 years. for transfusion of this group local guidelines state that haemodynamically unstable patients should receive er if hgb <9 g/dl, irrespective of age and sex. eighty-eight patients (27.8%) received er, 15 of them as massive transfusion ( 3 10 er units in <24 hours) (17.0%), 6 of these died (40%). altogether trauma patients received 851 units of er (12.1% of total uuh consumption), 51.5 thr (6.1% of total) and 150 units of op (16.5% of total). massive transfusion consumed 340 er (40%), 29.5 thr (57.3%) and 110 op (73.3%) units. fourteen adult trauma patients (15.9% of those transfused) received 1 or 2 er units only. lowest pre-transfusion hgb was 3.3-11.3 g/dl, median 7.6, mean 7.5 ± 1; highest post-transfusion hgb 7.8-14.3 g/dl, median 9.5, mean 9.8 ± 1. five patients received er transfusion at higher pretransfusion hgb level than 9 g/dl, but in 4 er were given because of a hyperacute clinical situation. fifty-five% of issued er units had been stored for >15 days, while only 10% were issued before 10 days of storage. discussion: life-saving effect of transfusion would seem evident in the massively transfused survivors, while in the other transfused patients documentation of clinical effect is inadequate. transfusion practice in trauma at uuh seems fairly well in accordance with internationally accepted guidelines. the trauma unit consumed a surprisingly small part of total uuh transfusion resources. trauma patients deviate from the general uuh patient population by sex and age; transfusion is otherwise mainly given to more elderly patients. further analysis is needed on transfusion indications and results to optimize the total use of blood products in uuh. special focus should be on transfusion to non-bleeding patients without haematological disease. it seems that only a small part of transfusion efforts results in the saving of life. in croatia national guidelines for the use of rhd gamma globulin were laid down in the year 2000. in accordance with the guidelines, rhd gamma globulin is administered intramuscularly in doses of 250-300mg and should be given to rhd negative women after delivery of rh positive children, after abortions, in week 28 of their first pregnancy, as well as in cases pregnancies with an increased risk of fetomaternal hemorrhage. as to the latter, detection and measurement of fetomaternal hemorrhage are recommended. three years after the issuance of the guidelines a survey was conducted regarding the use of rhd gamma globulin that involved 33 health institutions across the country. as many as 38 601 deliveries and 10 974 abortions were reported in 2003. the institutions reported the usage of 5103 doses of 250 mg rhd gamma globulin or 110 doses/1000 deliveries + abortions. we compared our data with those obtained from similar surveys conducted in 1999, i.e. before the issuance of the guidelines. in that year 4407 deliveries and 1400 abortions were reported, and 6125 doses of rhd gamma globulin or 100 doses/1000 deliveries + abortions were administered. institutions were then divided into four categories: clinical hospitals, general hospitals with the capacity of 400-600 beds, general hospitals with the capacity of 100-400 beds, and institutions of rather limited capacity with gynaecology department. the range of the consumption of rhd gamma globulin/1000 deliveries + abortions in the first category was 73-167 with the median being 119; in the second category there were 73-115 doses with the median being 92, in the third category 55-128 doses with the median being 88, while in the fourth category the range was 10-191 doses with the median of 79 doses. the number of pregnancies which should have been protected with rhd immunization in 2003 year was obtained by the following formula: (frequency of rhd negative subjects) ¥ (frequency of the r gene) = 0.17 ¥ 0.59 = 0.10. if a complete antenatal and postnatal preventive measures involving rhd immunization had been taken in 2003, 12 287 doses of rhd gamma globulin or 250 mg/1000 deliveries + abortions should have been given. our research has shown that, despite of the national guidelines adopted in 2000, the prevention of the rhd immunization in pregnant women is still not adequate in croatia. in fact, it has not improved since a year prior to the adoption of the guidelines. it has also been established that the category of the institution is a factor influencing the implementation of the protective measures. we consider that much more should be done on informing both women and gynaecologists about the importance of prophylaxis of the rhd immunization. consumption of plasma derivates in croatia g jaklin*, b golubic-cepulic † and m dondur* *general hospital, varazdin, † clinical hospital center zagreb, zagreb, croatia a increasing consumption of plasma derivates, their limited supply and insufficient national reserves are problems that croatia is faced with nowadays, as well as many other countries. in 2003 a study was carried out regarding the usage of plasma derivates. as many as 34 health institutions took part having the capacity of 15 487 acute beds out of a total of 16 279. the data regarding the use of plasma derivates were collected as follows: albumin, i.v. gamma globulin, and concentrate of coagulation factor viii in two periods of time. we divided institutions into three categories: clinical hospitals, general hospitals with the capacity of 400-600 beds and general hospitals with the capacity of 100-400 beds. institutional practice patterns regarding the use plasma derivates were compared among them. the parameters considered were the total consumption of plasma derivates, consumption per bed and consumption per inhabitant. data on the use of plasma derivates was obtained from hospital transfusion departments and pharmacies across the country. we compared our data with similar study carried out in 1997. the consumption of albumin was 441.44 kg or 98 kg/million inhabitants in 2003 and consumption per bed for the first category institutions was in range 14.27-72.27 kg with the median being 39.42, for the second category the range was 1.24-44.64 kg with the median being 4.36, while for the third category the range was 0.32-23.13 kg with the median being 6.63. in 1997 in croatia the consumption of albumin was 436.50 kg or 97 kg/million inhabitants. the consumption of i.v. gamma globulin was 59.92 kg or 13.32 kg/million inhabitants in 2003 and the consumption per bed for the first category institutions was in range 0.03 g-8.89 g. with the median being 2.78, for the second category was in range 0.03-3.95 g. with the median of 1.56 and for the third category was in range 0.00-12.75 with the median 1.56 g. in 1997 in croatia the consumption of i.v. gamma globulin was 22.20 kg or 4.93/million inhabitants. the consumption of the concentrate of factor viii was 8807.000 iu or 1.96 iu per inhabitant in 2003. 1539.500 iu, i.e. 17.48%, was a recombinant factor viii. in 1997 the consumption of factor viii was 4876.350 iu or 1.08 iu per inhabitant. discussion: the use of albumin showed stagnation. however, the use of i.v. gamma globulin increased 2.6 times and the use of the concentrate of factor viii increased 1.8 times if compared with the results in 1997. self-sufficiency has not been reached in plasma derivates in croatia we needed 20 000 l plasma for gamma globulin and 58 000 l plasma for concentrate of factor viii for the level of usage of these plasma derivates in 2003. significant differences in the level of usage among hospitals have been observed. these reflected a considerable difference in hospital policy regarding the use of these products in defined clinical settings. therefore, we would recommend that the national society sets out guidelines for the use of the products. background: blood bank good practice requires avoidance of rh alloimmunization. several studies report a probability of immunization of more than 80% following transfusion with rhd+ rbc's and as many as 19% in the case of platelets. aims: the purpose of this study was to investigate the development of anti-d and the causes of alloimmunization in a university hospital (1300 beds), reviewing our practice on the use of rhd+ blood components in rhd-recipients. method: a retrospective study was performed whereby 15.872 rhd-patients, from 1990 to 2004, were evaluated for the development of anti-d analysing the data on the blood bank information system. results: from the 15.872 rhd-patients we found out 274 identified anti-d, 77% (211) detected before any transfusion in our hospital. of the 63 left, nine were excluded because no information was available during some years. 5 had history of pregnancy related to the immunization. 36 patients were exposed to rhd+ blood components: 12 were transfused only with rhd+ platelets (including two women of childbearing age); 24 with rhd + rbc's. the remaining 13 had been exposed exclusively to rhd -rbc's suggesting that some units could be mistyped. this idea was corroborated in three patients where there was a common donor (he was called for investigation). conclusions: transfusion services avoid as far as possible administration of rhd+ blood components to rhd-patients, although there are situations in which such transfusions are necessary. the retrospective review of records revealed the need for specific recommendations regarding the use of rh immunoglobulin to prevent anti-d immunization. the possibility of mistyping blood components also appeared in this review, emphasizing the role of these studies in the evaluation of methodologies used at blood banks. the purpose of the work: to present the usage of whole blood (wb) and blood products (bp) in the treatment of patients who are hospitalized in the internal disease department in gevgelija. material and methods: retrospective analysis is done according the data which were analysed in five years period (2000) (2001) (2002) (2003) (2004) . statistical methods which were taken from the history of the hospitalized patients in the internal department were used for analysis and the data of transfused wb units and units of bp were taken from the department of transfusion medicine. results: from the total 4285 hospitalized patients who have been analyzed, 759 (17.71%) received wb and bp, and there have been transfused 1411 units wb and bp. every patient, on an average, has been transfused with 1.86 units wb and bp. at the same time 326 (23.1%) units have been transfused as a wb, 928 (65.77%) have been transfused as red blood cells (rbcs) and 157 (11.13%) units have been transfused as a fresh frozen plasma (ffp). the data for every year particularly will be presented in our paper to the congress. the collaboration between doctors of transfusion medicine and health workers from the other medical branches is the most important thing in the medical practice. our aim is to raise the awareness among the health workers for the importance of blood safety and their role though adequate clinical usage of wb and rbcs, minimizing the non-useful transfusions, evaluation of reflexive information from undesirable reactions in the usage of blood and blood products, use the alternatives etc. the necessity of direct involvement of the doctors in transfusion medicine in the process of healthy workers' education from the other branches for regularly transfusion therapy via meetings and lectures is an imperative for regular function of the department of transfusion medicine. femoral neck fracture repair is one of the commonest orthopedic procedures. it mainly concerns intracapsular or intratrochanteric fractures and it may be considerably hemorrhagic, requiring blood transfusion perioperatively. the aim of our study was to investigate blood transfusion requirement in patients with femoral neck fractures repair at katerini general hospital during 2002-2003 and to compare them with other studies. one hundred sixty five (165) unselected patients of whom 116 (70%) were women and 49 (30%) were men with a mean age of 76 years (range 26-94 years) were studied retrospectively. seventy six (46%) patients had intracapsular fracture and 89 (54%) intratrochanteric fracture. the mean hb concentration on admission was 12.25 g/dl (range 6.8-17 g/dl) and the mean hb concentration at discharge was 10.78 g/dl (range 7.2-14.3 g/dl). a total of 297 units of rbc were transfused (a mean of 1.8 units per patient). blood transfusion occurred in 125 patients (75.8%), (45-69% in other studies) with a mean of 2.38 units per patient (range 1-10 units), (1.47-2.6 in other studies). patients with preoperative hb values <10 g/dl were transfused more often than those with hb values >10 g/dl (100% versus 73%) p: 0.017. women were transfused more often men (80.2% versus 65.3%) p: 0.042. patients aged >70 years were transfused more often than those aged <70 years (81% versus 60%) p: 0.008. finally patients with intratrochanteric fractures were transfused more often than those with intracapsular fractures (88.65% versus 60%) p: 0.001. conclusions: in our study blood transfusion requirement for femoral neck fracture repair are similar with these reported in other studies. blood transfusion frequency is greater in patients with hb <10 g/dl, in patients older than 70 years, in women and in intratrochanteric fractures repair. fresh frozen plasma guidelines and practices as saltamavros*, g talampouka*, c koumoundourou*, s dimitrakopoulos † and p tseliou* *st. andrews hospital patras greece, patras, † pyrgos general hospital, pyrgos, greece introduction: fresh frozen plasma transfusions should be done according to specific standards and guidelines. aims: we have reviewed the ffp transfusion practices followed in our hospital in an attempt to provide a set of guidelines and principles that will assist physicians and health care workers to make the right decisions for appropriate use of ffp transfusions. methods: retrospective review of ffp transfusions practices during the entire year of 2004. we classified transfusion practices according to the diagnosis of the patient and also of the clinical depart-ment that demanded transfusion. then we analyzed our results by comparing the requests with the internationally approved guidelines for ffp transfusion and counted the percentage of ffp to rbc units. finally we discussed with our fellow physicians the proper use of ffp and informed them about the accepted guidelines. as we can see from the underneath table, diagnoses and departments with high consumption were: internal medicine 22%, plasmapheresis to patients suffering from guillain barre and ttp (thrombotic thrombopenic purpura) 21.04% and trauma/surgery 20.68%, cancer 13.60%. summary/conclusions: the use of ffp corresponds to 27.88% of the rbc units transfused in our hospital. plasma units should be used properly and according to internationally accepted standards. plasma use should be justified by the physician, in order to avoid unnecessary risks on behalf of the patient for effective treatment and care. to show gradual discontinuation of using whole blood and increased use of red blood cells (rbc) in the management of patients in the transfusion department of the clinical hospital 'zvezdara' . methods: data of wb and rbc use from 1982 to 2004 in our hospital. results: our data are presented in table 1 . conclusion: after 1982, wb usage rates systematically decreased from 51% to 1.3%. however, in the period of 1991-2004, the wb usage rates increased slightly, and we arrived at a generally very low rate of wb use, namely only in about 3% of the cases. education about transfusion medicine and rational use of blood in the medical curriculum was generally insufficient and very poor. therefore, we started an education program and we established a hospital transfusion committee (htc) with the task of medical control and monitoring the quality of clinical transfusion practice in our hospital. members of the htc are a specialist in transfusion medicine, an internal medicine specialist, an anesthesiologist, surgeon and gynecologist. we have been organizing courses for the medical staff, doctors and medical technicians since 1995. background: platelet transfusions are given mainly to thrombocytopenic patients with malignant haematological diseases. currently the decision to give a prophylactic platelet transfusion is based almost exclusively on the number of circulating platelets in the patient although it is known that various clinical factors might influence the bleeding tendency. by free oscillating rheometry (for), using a reorox® 4 instrument, it is possible to monitor the coagulation over time in whole blood and obtain information about clotting time and coagulum elasticity. aim of the study: the aim of this study was to find out if for using the reorox® 4 instrument could be used to evaluate the hemostatic status of thrombocytopenic patients. methods: the change in elasticity over time in non-anticoagulated whole blood from leukaemia/lymphoma patients with thrombocytopenia was measured in the reorox® 4 pre and post a platelet transfusion (n = 10) and was compared with healthy controls (n = 40). the effect of platelet concentration on coagulation was studied by diluting platelet rich plasma from healthy subjects with autologous plasma to various platelet counts whereafter the coagulation was monitored with for (n = 8). the change in elasticity per minute (g'max slope) and maximum elasticity (g'max) were evaluated from the elasticity curves. results: the g'max, g'max slope and platelet count increased after transfusion for all patients. the thrombocytopenic patients had significantly lower g'max and g'max slope values both pre and post transfusion compared with healthy control subjects (p < 0.01). the measurement in platelet rich plasma showed that g'max and g'max slope increased with increasing platelet concentration. however patients with similar platelet count developed different clot elasticity. the reorox ® 4 instrument responds to changes in haemostatic function in form of the clot elasticity. the fact that patients with similar platelet concentration developed different elasticity shows that clot elasticity is a function of not only platelet concentration but also of functional properties. the for method seems promising to evaluate platelet function. quality control of pre-storage leukodepleted red cell concentrates vu urlep salinovic, k perbil lazic and l lokar teaching hospital maribor, maribor, slovenia background: the system of quality assurance including quality control (qc) in transfusion medicine is most important for safe blood supply. the safety and quality of blood components are increased by pre-storage leukodepletion of whole blood (wb). aim: the qc of leukodepleted red cell concentrates (rcc), prepared from pre-storage filtration of wb (quadruple blood bag systems imuflex wb-rp terumo) is performed with the aim to be sure that the quality of leukodepleted rcc is in accordance with the guidelines of council of europe. in three years (2002) (2003) (2004) , 35 559 units of wb were collected, among them 7765 (21.6%) units were collected for pre-storage filtration and in 376 (4.8%) of these qc was done. within 4 hours after collection, wb was filtered at room temperature. filtered wb was centrifuged at 4000 g for 20 minutes and separated in rcc and plasma. the samples for qc were collected before and after filtration. for each unit, volume, hemoglobin, hematocrit, % of hemolysis and residual white blood cells (wbc) count were measured. the number of residual wbc after filtration was determined in the nageotte chambre. for all parameters the mean value and standard deviation were calculated. the results of qc for the following parameters were: volume 320 ± 25.1 ml (89% corresponds with the guidelines of council of europe), hematocrit 0.61 ± 0.06 (96%), hemoglobin 60.9 ± 8.0 g/unit (99%), number of residual leukocytes 0.028 ± 0.16 ¥ 10 6 (100%), hemolysis 0.30 ± 0.19% (100%), the test of sterility was 100% negative. during filtration of wb, wbc were removed in approximately 99.99%, the duration of filtration was 16 ± 6 minutes and the loss of hemoglobin was 8.2 ± 4.3%. the pre-storage filtration of wb is highly efficient, 99.99% of wbc were removed and the loss of hemoglobin was small. background: the patients of cardiosurgery use big amount of blood components. there is no uniform approach by treatment with blood components in these patients. the safest and the most rational use of blood is based on the individual treatment of a patient and the evaluation of the most clinical and laboratory factors. aim: the aim of our study is to analyse the use of blood components from 1998, when the cardiosurgery department was founded in our hospital, to 2004. the data about use of red cell concentrates (rcc), platelet concentrates (pc) and fresh frozen plasma (ffp) in the period from 1998 to 2004 were collected from information system datec. the average use of all three blood components per patient is presented. we were interested, if the average use per patient was diminished in the analysed period. results: the use of three blood components and average use of all blood components are presented in the table. the data from the table shows, that the number of patients increased more than three times, but the average use of blood components per patient was not essentially diminished. conclusion: during seven years period the use of blood components per patient in cardiosurgery was not diminished. for more rational use it is necessary to apply all methods of autologous blood transfusion because of the increasing number of cardiac operations and decreased number of voluntary blood donors. background: the presence of leucocytes in blood components is cause for appearance of various adverse posttransfusion reactions such as nhfptrs, urticary, anaphylactic shock, alloimunization and platelet refractorines, infection with bacteria and leucothropyc viruses (cmv, htlv). the removal of leucocytes from blood components through filtration is especially important in the treatment of patients with malignant diseases who need frequent transfusions of blood and blood components. this is because of their lowered immunologic status which is a result of the disease and received immunosuppressive chemotherapy and radiotherapy. aim: to show the prevention of posttransfusion reactions and the positive effect from transfusion of leucoreduced er. concentrates, produced with filtration in therapy of anaemia in patients with malignant diseases, treated in our daily transfusion hospital at medical center -stip. methods: 123 patients with malignant diseases have been treated in our daily transfusion hospital in the last four years. most of these patients suffer from ca pulmonum, ca collonis, ca uteri, ca mammae. also, because of the secondary anaemia, the same patients were transfunded with at least two doses of leucoreduced er. concentrates. the blood donored by voluntary repeated blood donors was filtrated the same day after the donation, separation and the control of the same one. the blood was collected in baxter and terumo bags, and it was filtrated with baxter -sepacell rs -2000 and pall -purecell rn filters. the analyses of leucoreduction were made for every filtrated and transfunded unit before and after filtration. the analyses samples were taken from the tubing system before and after the filter. haemathologic parameters were automatically made in the central clinic laboratory. results: er. concentrates poor with the leucocytes for about 98-99.9% were produced with the use od these filters, and platelets from 88-100% with which side effects from frequent transfusions at these patients were prevented. with the routine monitoring of all patients none posttransfusion reactions were registered. the therapeutic effect is also important because of the fact that the number of er and the level of hg and hct remain almost unchanged. the aim of the blood banks is to help and to increase the safety of the blood components. the leucoreduction through of er. concentrates poor with leucocytes is a regular procedure in the therapy of malignant patients with remarkable clinical picture of anaemic syndrome and prevention the cancer recurrence end infection. the time of filtration is also very important which has to be shorter after collection of blood, because the leucocytes should be removed before they become disintegrated and relapse potentially dangerous substances end metabolites in the blood components. we have been applying so called ' prestorage filtration' because it has been proved that it is more efficient that bed -side filtration. background: the effect of leukocyte reduction of rbcs by filtration has been the topic of several rcts. these rcts investigated the effect of leukocyte reduction on mortality; post-operative infections and hospital stay in different patient populations. some rcts came up with answers that conflicted with the results of other rcts. aim of the study: with including the individual patient datasets of several rcts in one database, combined analyses are possible that may explain the differences in the reported results. using this technique, we may come up with answers (or questions) that cannot be obtained by performing standard meta-analyses of these rcts. methods: we coordinated several rcts comparing the perioperative use of buffy-coat depleted rbcs with the use of filtered rbcs. cardiac surgery patients were included in three rcts (1¥ cabg and/or valve; 1¥ re-cabg and/or valve; 1¥ valve with or without cabg). oncologic surgery patients were also included in three rcts (1¥ colorectal cancer; 1¥ gi-oncology or vascular surgery; 1¥ gi-oncology, vascular surgery or orthopedic surgery). the electronic data files of these rcts were uniformly recoded and entered in a single database. as different primary endpoints were investigated in the rcts, we focused on common endpoints, recorded in 4 or more of the rcts. multivariate analyses were performed on: in-hospital mortality, 30-day mortality, hospital stay, stay on icu, postoperative infections, and mods. results: in the rcts, individual datasets from 3875 surgery patients were collected (1630 onco; 1272 cardiac; 569 vascular; 352 orthopedic, 52 other). in the multivariate analyses, patients in the buffycoat depleted trial arm showed a higher mortality rate both in-hospital (p = 0.01) and at 30 days post-surgery (p = 0.03). no association between randomization and stay in hospital (p = 0.10) or stay on icu (p = 0.56) was seen in the combined study population. also, no association of randomization with the incidence or duration of mods was seen. the analyses of post-operative infections in the total population showed the trial arm to be associated (p = 0.016), and 'hospital' to be far stronger associated (p < 0.001). however, when the oldest rct, that had not yet used our standard definition list for scoring post-operative infections, was excluded, the association with the hospital was lost (p = 0.34) and the trial arm became more strongly associated with infections (p = 0.002). in the analyses of 3875 surgery patients, the use of filtered rbcs, compared to the use of buffy-coat depleted rbcs, resulted in reduced mortality (both in-hospital and at 30 days postsurgery) and a reduction in post-operative infections. the association of the variable 'hospital' with post-operative infections, as is frequently reported in literature, was initially confirmed in our analyses. however, when a standard definition for post-operative infections was used (excluding the datasets from one of six studies), this association was lost. background/aims: uk neqas for blood transfusion laboratory practice (btlp) operates an eqa service for 472 uk laboratories (including eire) and participants throughout europe, including major groups in denmark (n = 52) and portugal (n = 32). in november 2004 a questionnaire was distributed to determine the criteria used for selecting phenotyped blood for different patient groups, including pre-menopausal women (pmfs). results were analysed by country to establish any variation in practice relating to the selection of k negative (k-) and rhc negative (c-) blood, since antibodies to these antigens are now the major cause of hdn. results: overall return rate was 84% (uk) and 64% (non-uk), although only centres treating pmfs were included in this analysis. k-blood was selected for pmfs by 81% in wales (n = 16) and 71% in england (n = 291), but only 6% in scotland (n = 32), 8% in northern ireland (n = 12) and 10% in eire (n = 31). in mainland europe, variation was also observed: 12% in denmark (n = 17), 75% in portugal (n = 20) and 85% in other countries (n = 27 from 10 countries). fewer laboratories selected c-blood for pmfs: 8% in england and northern ireland, 0% in scotland and eire, rising to 81% in wales. mainland europe showed similar variation: 12% in denmark, 75% in portugal (all ccee matched) and 51% in other countries. there are no guidelines requiring selection of k-and c-blood for pmfs, but in the uk d negative blood is required for d negative females aged <60 years. trend analysis of questionnaire data in the uk, using theoretical clinical scenarios, shows a decrease in the number of laboratories that would select k + blood for a 30 year old female (with pre-existing antibodies other than anti-k), from 58% (1996), 42% (1999) to 26% in 2004. in this survey, k + blood was selected for a female aged 30 (no antibodies) by 31%, whilst 81% selected a k + unit for a female aged 54 (no antibodies), despite this patient being treated as a pmf for provision of d negative blood. a similar distinction was made between the 30 and 54 year old females in portugal and mainland europe. conclusions: variation in practice may at least in part be due to the availability of blood routinely labelled for rh and k. in the uk all donations are labelled, except for those from new donors, as are most units in portugal. uk questionnaire data (1999) suggested that >50% laboratories would change to selecting k-blood for pmfs if all units were labelled. however, labelling alone does not account for the differences seen within the uk, and perhaps the trend towards providing k-(and to a lesser extent c-) blood for pmfs is influenced by advice from transfusion services. it would be of great interest to monitor and compare the incidence of hdn due to anti-k and anti-c in different countries to measure the outcome of differing practices for provision of blood to pmfs and to thereby inform future policy. there is no ultimate in ex-vivo assay described, which can predict the outcome of plt transfusion in vivo. current in ex-vivo assays and animal studies are rather very complicated to carry out, cost effective and time consuming. objective: we hypothesized that the quantitative measurement of gpib expression by facs can be used to predict the outcome of platelet survival post transfusion. in our previous studies we demonstrated that our phagocytosis assay can predict the plt survival sensitively (blood dec-2004) . we isolated human washed plts by centrifugation and labelled with mepacrine and then incubated with pma-matured thp-1 cells (37°c). binding was measured by facs analysis of cd42b/cd14 positive particles, and phagocytosis by counting mepacrine/cd14 positive particles. we measured gpib expression before and after plt-macrophages interaction by facs flowcytometry. results: gpib expression at surface of c22 fresh showed 90 ± 5 and after 48 hours storage 50 ± 15% expression. the gpib expression at surface of plt decreased and showed three populations with different densities high (gpib-1), low (gpib-2) and in-between (gpib-3). the binding and phagocytosis of plt showed an increase of 40 ± 10% which implicates an indirect and negative relation to gpib-1, and direct relation to gpib-2 expression. anti-human pselectin (cd62p) delayed 45 ± 10% the binding and annexing v 60 ± 12% the phagocytosis of stored plts, after 48-hour storage. these results show that gpib expression is rather easy, reproducible test which can be standardised and be used as a very sensitive in vitroassay to predict platelet survival posttransfusion. transfusion and lung injury jd dodig*, d sovic † , m tomicic † and h sager* *university hospital 'sestre milosrdnice', zagreb, † institute for transfusion med, zagreb, croatia background: the respiratory tree has been viewed as an infrequent site of injury arising as serious complication of transfusion. in recent years, this view has changed as investigators have shown that two complications-circulatory overload and transfusion related acute lung injury-are relatively frequent events. case report: a 59-year-old men was admitted due to chronic macrohematuria. he had no history or current evidence of cardiac failure. the hb level was measured 36 g/l. he received 500 ml 0.9% nacl and 500 ml ringer solutions. after that, he was transfused with 2 units of rbcs. during transfusion second unit he developed following symptoms: tachycardia, dyspnea, hypertension. massive pulmonary edema was noted. he was treated with mechanical ventilation, oxygen, diuretics, aminophillin, antihistaminic and corticosteroides. the patient recovered after being on ventilation for 6 hours. two days after the patient was operated. after surgery he was transfused with 4 units of rbcs. all transfusions were regularly performed. results: chest x-ray confirmed bilaterally pulmonary edema. the samples (patient and donors of first two units rbcs) tested were negative for the presence of hla specific and granulocyte antibodies. granulocyte agglutination and lymphocytotoxicity test were negative. tnf-a in recipient serum was slightly increased. conclusion: our patient is the first reported case suspected of trali, but all of the investigations didn't give us the answer. against neutrophils using flowcytometric detection of cell surface cd11b expression and l-selectin shedding. methods: a hundred microl of volunteers' heparinized whole blood were incubated for 15 minutes at 37°c with fmlp, lps or pma. monoclonal antibodies against hna1a and hla-i, or patient serum were incubated with whole blood for 30 min. surface cd11b and l-selectin were detected using facscalibur. results: neutrophil activation was detected after fmlp, lps or pma stimulation. p38 map kinase inhibitor reduced activation induced by fmlp and lps. anti-hna1a monoclonal antibodies induced neutrophil activation, which were also inhibited by p38 map kinase inhibitor. ten serum samples obtained from patients or donors who caused transfusion reactions were evaluated. five sera out of 8 samples having anti-neutriphil and/or hla antibodies exhibited neutrophil activation, while two samples without leukocyte antibodies had no effect on any activation. conclusion: these findings indicate that neutrophils activation is regulated through mak kinase and detection of neutrophil activation may be useful to predict transfusion-related reactions. results: 38 out of 50 transfusion reactions were febrile, 19 were anaphylactic, 1 were due to circulatory overload, 1 out of 50 transfusion reactions concerned the transfusion of incorrect blood component. 1 out of 50 transfusion reactions concerned acute haemolytic reaction. post transfusion purpura or suspected trali was not seen. fatal complication was not seen. the reactions to plasma were predominately anaphylactic. we detected no case of bacterial contamination among the cultures of transfusion bags. conclusions: the incidence of reactions among patients malignancy was high, while among surgical patients was lower. the incidence of transfusion reactions during the months had no statistically significant difference. we suggest that the improvement in prevention of transfusion reactions require a continual vigilance system for rapid recognition and information regarding these complications. measurements of ige immunoglobulin in thalassemic patients, before and after blood transfusion background: many studies have reported the implications of proinflammatory cytokines including interleukin (il)-1 beta, il-8 and tumor necrosis factor alpha (tnf-alpha) in febrile nonhemolytic transfusion reactions. il-8 has been shown to accumulate in packed rbcs even after the procedure of filtration, which is explained by the release of il-8 from rbc receptors into the packed rbc super-natant. stress induced elevation in tnf-alpha levels was demonstrated in healthy individuals. aim: to assess the level of proinflammatory cytokines in peripheral blood of blood donors. material and methods: immediately upon blood collection, plasma was separated from postdonation blood samples obtained from 75 blood donors and frozen at -80°c. upon thawing, the level of the il-1 beta, il-8 and tnf-alpha cytokines was determined in plasma samples by elisa method using commercial kits for cytokine determination (roche molecular biochemicals). the level of il-1 beta was at the test detection limit in all donor plasma samples. in 1 (1.3%) bd, the level of il-8 was 36.8 pg/ml, exceeding the test sensitivity limit of 6.2 pg/ml. in 12 (16.6%) bd, the level of tnf-alpha was within the range of 14-60 pg/ml, with a test sensitivity limit of 12 pg/ml. tnf-alpha levels >800 pg/ml were measured in plasma samples of 3 (4.1%) blood donors. the increased activity of blood donor's immune cells, indicated by elevated levels of the proinflammatory cytokines il-8 and tnf-alpha in peripheral circulation some blood donors, may lead to the occurrence of febrile nonhemolytic transfusion reactions in the recipients of the blood products manufactured from the blood of these blood donors. this hypothesis will be thoroughly investigated in our future studies. background: transfusion-related acute lung injury (trali) is a life-threatening complication of transfusion, under-recognized and underreported possibly lacking a consensus definition. aim: we report here a 'probable' case of trali syndrome in an elderly female patient. case presentation: an eighty-two year old female patient was transferred to the intensive care unit on the third postoperative day (pod) following the abrupt onset of acute pulmonary insufficiency (pao 2 = 49 mmhg, o 2 saturation = 79%), hypotension (bp = 90/50 mmhg) and fever (38°c). this occurred ten minutes after initiation of an infusion of a unit of packed red blood cells (prc). the patient had no history of any cardiac or pulmonary disease. she was intubated and placed on mechanical respiration and supported hemodynamically. the cvp (12 cm h 2 o) ruled out fluid overload. the chest x-ray revealed bilateral pulmonary oedema, the echo cardiogram and blood cultures ruled out cardiac and infectious aetiology of the episode. after treatment for 48 hours the patient improved significantly, was extubated and returned to the surgical unit on the 6th pod. reviewing the transfusion history we discovered that the patient did not receive any blood or blood component prior or during the correction of her ileum, but she was transfused a unit of ffp (fresh frozen plasma) five hours prior to the incident. the donor review revealed that the unit of ffp was from a 29-year old female with a history of multiple abortions whereas the prc unit was from a 52-year old male, a volunteer of several years who had no history of transfusions. there are some prerequisites for the implementation of a haemovigilance network and some of them have been met, so we can present the first results. methods: traceability of blood components is possible because there is unique information system in place in the whole country, identifying the donor, donation, each single blood component and identification of the recipient, but feed back information of the performed transfusion is still missing. cooperation between blood transfusion service and hospitals was established by introduction of hospital transfusion committees; the intensity and quality of their work is very different. homogeneity of reporting is achieved by the introduction of unique reporting form. a reporting route was defined: patients physician sends notification of atr to the local blood transfusion service. atr reporting form is prepared there and sent to the national blood transfusion service, where the data is collected for the centre for haemovigilance, which is going to be established very soon. data analysis will be responsibility of the centre for haemovigilance at the governmental level. type of adverse transfusion reactions and events is defined. education and information was passed to the health personnel by inclusion of haemovigilance in under and postgraduate programmes as well as seminars, scientific meetings and some publications. results: in the years 2002-2004 there were 410.000 blood components issued in slovenia and 333 atrs were reported (1 in 1232 blood components issued), in 25 of them the severity grade was 2 (1 in 16.412 blood components issued). in the year 2004 there were considerable differences between the number of atr reports compared to the number of blood components issued among slovenian hospitals, ranging from 1 in 3375 to 1 atr in 490 blood components issued. 9.6% of all atr were not classified. conclusions: although the number of reported atrs was increased by 24% and 31% a year in 2003 and 2004 respectively, it can be assumed that the collected data is not complete. feed back reports, much more information and cooperation is needed, especially in some hospitals, which should contribute to a better registration of atrs and events in the future. the slovenian haemovigilance system still needs upgrading, but despite this, some important work has been done in building a national haemovigilance system. . considering the multiplicity groups in cattle and since there was no research on repeated blood transfusions reactions in iran's native cattle, we decided to consider crossmatching in different processes of repeated blood transfusion from a head of blood donor to five recipients and observe the clinical and hematological alterations. six healthy iran's native cow, 2.5 years old, with average weight of 210 kg were used. the animals were dewormed by albendazole (15 my/kg bw) and were kept for two weeks under uniform managemental condition. three days prior to blood transfusion, vital signs registration (temperature, heart rate and respiratory rate) blood collection via jugular vein was done to indicate the baseline of research parameters. after that, blood transfusions were performed from one donor cow to five recipients three times at one-week intervals. cross matching was done at each transfusion. after each transfusion the research parameters do determined. results indicated that one of the recipients cows experienced anaphylactic shock, in the first step of blood transfusion and another cows in the second step and finally in the third steps two other cows showed the serious shock. this is in the contrary of this opinion that the first transfusion can be given safely without crossmatching in cattle practice ( van der valt, et al. (1969) background and objectives: blood transfusion may lead to the manifestation of anti-hla and platelet-specific antibodies that may in turn bring about different problems like platelet refractoriness. it appears that the study of antibodies against hla-class i and platelet-specific antigens are useful for the selection and success of the appropriate treatment protocol. the aim of this study was to detect anti-hla and anti-platelet-specific antibodies by flowcytometry in patients with hematologic disorders (including acute leukemia, aplastic anemia) and patients with itp. in this descriptive study, anti-hla and platelet-specific antibodies were detected by flowcytometric technique, using 62 sera drawn from patients with different haematological disorders who showed a poor response to platelet transfusion and 20 from patients with itp. the results of anti-hla antibodies were then compared by panel reactive antibodies (pra). results: our results showed 44 (53.7%) out of 82 (53.7%) patients had anti-hla class-i antibodies in their sera. the frequency of each antibody isotype was found to be as follows: igm (51.2%), igg (32.9%) and iga (1.2%). 36 (43.9%) out of 82 patients had platelet specific antibodies and the frequency of each antibody isotype was found to be as follows: igm (40.2%), igg (30.5%) and iga (12.2%). 27 (31.7%) out of 82 patients had both antibodies. no difference was found between the two groups in platelet specific antibodies. despite significant correlation between flowcytometry and pra methods, pra can only detect antibodies which react with complement. conclusions: with increase in the number of platelet transfusion, immunization to hla antigens occurs; moreover, immunization against platelet specific antigens may also occur during autoimmunity. the presence of these antibodies may be one of the reasons of poor response to platelet transfusion and platelet refractoriness in patients under study. conducting similar studies with higher number of samples, platelet cross-match, and the use of hlamatched platelets for these patients are recommended. post transfusion purpura (ptp) is a rare, severe thrombocytopenia that results from alloimunization to platelet specific alloantigens, following blood transfusion. in this condition, the patient's own platelets are destroyed by the alloantibody even though they are not supposed to carry the 'guilty ' antigen. the disease is rare occurring mainly in multiparous women. the majority of reported cases involved antibodies against the platelet specific alloantigen hpa-1a in a homozygous hpa-1b patient. in a minority of cases the offending antibodies were directed against hpa-1b, -3a, -3b, 4a, -5a, -5b. although self-limiting, the syndrome is characterized by severe bleeding with high morbidity and mortality; therefore prompt diagnosis and appropriate therapy is crucially important. ptp is a challenging diagnosis, because the patients are often critically ill or post-surgery, and have alternative explanations for thrombocytopenia such as infections or drugs. we present three patients with severe thrombocytopenia initially misdiagnosed. the first patient, a 54-year old women, had a past history of systemic lupus erythematosus and coombs positive autoimmune hemolytic anemia. at the present hospitalization, after antibiotic therapy for endocarditis, a severe hemolytic episode occurred and she need blood transfusion. when severe thrombocytopenia appeared, she was wrongly diagnosed as evans syndrome. the second patient, a 73-year old man, suffered from sepsis after vascular surgery and revealed clinical and laboratory picture of dic. the third patient, a 58-year old women, had end-stage renal failure and received heparin during hemodialysis, thus heparin-induced thrombocytopenia was first suspected. history of recent blood transfusion rose the suspicion of ptp in all this cases, and appropriate therapy with high dose iv immunoglobulin was started. adequate laboratory work-up confirmed the diagnosis. three different anti hpa-antibodies were identified: anti hpa-3a, anti hpa-1b and anti hpa-3b, respectively. the platelets genotype of the first patient was hpa-3b/3b, of the second hpa-1a/1a and of the third patient, hpa-3a/3a. the reported cases emphasized the importance of keeping in mind the possibility of ptp. incorrect diagnosis may lead to wrong treatment and fatal outcome. health sciences authority, singapore, singapore introduction: the haemovigilance programme in singapore was started by the centre for transfusion medicine (ctm) in 2002. the system covers registration of collected, produced and transfused blood components, and monitors adverse transfusion reactions (atr). the programme runs on a voluntary, non-punitive and confidential basis. aims of the study: (1) to gather and analyse reports of all adverse and untoward events occurring during transfusion of blood and components. (2) to use the information acquired to determine the morbidity of transfusion. (3) to provide guidance on corrective measures to prevent the recurrence of some accidents, and to improve transfusion safety. (4) to improve public confidence by demonstrating to public, patients and professionals the safety of the existing transfusion system. methods: (1) a common report form is used and made available to all participating hospitals. within the reporting system, the identification of the patient and staff involved are not required, to ensure confidentiality and protection of information belonging to the hospital. (2) reportable events include immediate reactions during transfusion (haemolysis, non-haemolytic febrile transfusion reaction, urticaria, anaphylactic shock, bacterial contamination, trali), delayed untoward effects after transfusion (haemolysis, post-transfusion purpura, acute gvhd), transfusion-transmitted infections, incorrect components transfused, and near misses. (3) within the hospitals, a responsible person ensures that all adverse events and untoward effects of transfusion are reported on the haemovigilance forms and provided to ctm for collation. within the ctm, the haemovigilance coordinator is designated to assist hospitals in investigating serious adverse events and advise on the reporting formats. results: (1) the total number of reported cases has steadily increased since the introduction of the programme. (2) the number of participating healthcare institutions has also increased to 100% (n = 12). please refer to table entitled 'summary of the haemovigilance report 2002-2004' . (1) the implementation of the haemovigilance programme in singapore is feasible with respect to the asian setting, and can significantly contribute to blood safety. (2) there has been very good participation from the 12 participating healthcare institutions, signifying greater awareness and willingness to partake in the programme. (3) the results obtained from the programme have given rise to initiatives and recommendations aimed at reducing (71%) were rated for seriousness. of these, 50 (6.5%) were rated as grade 2 (moderate to serious morbidity) or worse. 793 (72.6%) were rated for imputability to the blood transfusion. of these, a relationship to the transfusion was graded as 'certain' or 'probable' in 470 (59.3%) and as 'possible' in 245 (30.9%). overall relatively few errors were reported in comparison to other systems. a small number of reports concern (possibly) infected blood components, and imputability was deemed probable or certain only in a minority of these reports. autologous blood components gave rise to five reports (errors as well as mild transfusion reactions) which shows a relatively high risk associated with their use (9.98 per 1000 the rate of post transfusion hepatitis (pth) in israel in unknown. this information is important in order to learn about the residual infection risk in blood recipients. aim: to summarize the data on reported cases of pth. methods: suspected cases of pth are reported to mda blood services. the investigation procedure includes follow up testing of implicated donors and retesting of an archive sample of the transfused unit, if available. donors involved in suspected pth-b are tested for hbsag and anti-hbc. anti-hbc+ donors are tested for anti-hbs. hbv-dna testing is done if anti-hbs is less than 10 miu/ml. donors involved in suspected pth-c, are tested for anti-hcv and alt. since 2000 hcv-ag or hcv-rna are performed, when appropriate. investigation is considered complete if all the involved donors are retested >6 months following the implicated unit. results: between 1993 between -2004 suspected pth cases were reported: 74 (57%) were pth-b, 54 were pth-c (42%) and in 1 patient both hbv and hcv infections were reported. investigation was completed in 53/129 (41%) cases, with 70% of the involved donors (1222/1749) being retested >6 months after the implicated donation. hbsag was not detected in any of the 773 retested donors. anti-hbc was detected in 26 donors involved in 7 pth-b cases of which 21 were also positive for anti-hbs. pcr for the detection of hbv-dna was performed on the 5 'anti-hbc+ only' donors, and none was found positive. only in 1/449 donors suspected to be involved in pt-hcv, anti-hcv antibodies were subsequently detected. this donation was collected and transfused before the introduction of anti-hcv testing in israel, which was implemented in 1991. in another pth-c case, the implicated donor is still negative for anti-hcv, in follow up samples of up to 28 months, but was found positive for hcv-ag and hcv-rna. pth investigation of all the donors involved was completed in 49% (52/105) of the cases where the patient received up to 10 blood components. conclusion: in the past 12 years an average of 11 cases/year of suspected pth were reported to the israeli national blood services. investigation was completed in 41% of the reported cases. so far, there was no clear evidence of hbv transmission. in 2 cases (out of 2.99 million blood units collected nationwide during 1993-2004) hcv seemed to be associated with blood transfusion: one caseprior to the implementation of anti-hcv testing and the otherprior to the implementation of hcv-ag testing in a donor that did not develop anti-hcv antibodies. these findings suggest that other modes of hbv and hcv transmission should be sought in blood recipients in israel. 3-2.9, p < 0.05). the seroprevalence for a-hiv in the bd population was 0.096% (se: 0.028; ci: 0.034-0.153; p < 0.01) whereas in the a-hbc positive bd was 1.08% (p < 0.01) and in the a-hbc negative bd was 0.07%. from 113 a-hiv positive donations, 33 were also positive for a-hbc, that means that the sensitivity of the a-hbc in the detection of a donation a-hiv positive was 29.2%. the relative prevalence (percentaje of positive donations for a-hiv in the a-hbc positive population divided the percentage of this marker in the donations a-hbc negative: rp) was 15.43, which indicates that the number of bd a-hiv positive is 15.43 times higher in the a-hbc positive that in the a-hbc negative population of bd. as regards a-htlv, the seroprevalence in the bd population was 0.042% (se: 0.019; ci: 0.01-0.084, p < 0.01), in the a-hbc positive bd was 0.42% (p < 0.01) and in the a-hbc negative bd was 0.031%. from 49 a-htlv positive donations, 13 were also positive for a-hbc, that means that the sensitivity of the a-hbc in the detection of a donation a-htlv positive was 26.5%. the rp for a-hbc as regards a-htlv was 13.54, which indicates that the number of bd a-htlv positive is 13.54 times higher in the a-hbc positive that in the a-hbc negative population of bd. our results suggest that, in our bd population the screening with a-hbc would be useful to prevent other infections transmissible by blood transfusion, like retroviruses, because of the high sensitivity and the relative prevalence. 1. despite the high specificity of currently available elisas, the positive predictive value is lower in blood donors. therefore, immunoblot tests and polymerase chain reaction (pcr) have been adopted for routine testing in elisa +ve blood donors, by our service. 2. our data show that the real anti-hcv prevalence of our donor population is very low (0.04%). 3. the selection and evaluation of appropriate assays of all donated blood for hcv infection ensure good laboratory practice and accurate post-notification counselling of infected donors. 4. given that donors who are elisa positive but persistently negative or indeterminate probably do not represent a risk for transmission, their deferral from donation increases the problem of availability of blood supply. 5. donor re-entry in the pool of donors is an issue for further discussion. 6. the introduction of nat technology may elicit more accurate responses and improve the screening process. background: the introduction of hcv antibody screening of all donor blood in 1992 represented a major step in the prevention of transfusion-associated hcv hepatitis and in identification of infected donors. the study of infected individuals provides a unique opportunity to define behavioral factors associated with infection. evaluating risk factors in hcv infected blood donors is essential for monitoring blood supply safety, donor screening effectiveness and developing appropriate prevention programs objectives: 1. to recognize the epidemiology of hepatitis c and how it differs geographically; 2. to investigate the risk factors for presence of anti-hcv antibody in blood donors; 3. to evaluate the effectiveness of our donor selection program in local level. methods: serological testing for hcv was performed according to standard procedures. initial screening was performed using secondgeneration eia and, after january 1996, using third-generation eia. our study included a confirmation test (riba) a questionnaire was used to collect data concerning demographic, social and sexual behaviors, and number and type of donations of blood donors. the study also included testing of sexual partners and family members. changes in rates of hcv infections were evaluated by comparing yearly prevalence estimates. the overall prevalence of anti-hcv (eia) was 0.11% out of 168 374 blood donations. the average prevalence of hcv infection by riba was 0.04%, which reaffirms the very low risk of transfusion-transmitted disease. the cumulative number of hcv infected donors was 68, with 54 cases in males and 14 cases in females. most infections were found among older persons (32% were aged 22-39, and 68% aged 40-59). the seropositivity was higher in family/replacement donors (63%) than in volunteers (37%). the annual prevalence decreased throughout years. the relative importance of risk factors for hepatitis c was: transfusion 10%, hospitalization 8%, immigrants 8%, occupational 5%, sexual transmission 6%, injection drug use 3%, household contacts 5%, other 3%, tattooing 2%, unknown 50%. according to the criteria for blood donation, certain donors should have been excluded in the predonation interview but these donors had denied risky behaviour when questioned. the importance of sexual activity in the transmission of hcv has not been well-established as we tested 30 sexual partners and 14 family members and none of them was found positive. conclusions: our results suggest that major improvement in the safety and quality of our blood supply has been made in our area. introduction: apart from immuno-haematological complications, blood transfusion recipients are exposed to the risk of viral and bacterial contamination of donor blood. the latter infectious risks are generally associated with the number of donations that are needed in the production of blood products: the pooling effect. one measure recently being discussed is the generalisation of the use of trombocytapheresis for the production of trombocyte concentrates. normally, the trombocyte product is a concentration of trombocyte extractions from a pool of 5 buffy coats: pooled platelet concentrates (ppc). in case of trombocytapheresis sufficient trombocytes for one transfusion can be collected from one single donor. aim of the study: in this presentation the effect of using 100% trombocytapheresis for the production of single donor platelets (sdp) instead of pooled platelet concentrates (ppc) on contamination risks will be assessed. these risks can be divided in 1) the risk of bacterial contamination, 2) the risk of viral contamination (e.g. hcv, hbv, hiv) resulting from window period donations, and 3) the risk of contamination with tse or emerging infections for which no screening test exists. the contamination probability of sdp versus ppc is assessed on the basis of the production characteristics of both products, e.g. the presumption that the contamination risk per trombocyte product will be reduced by a factor equal to the number of pooled donations (five in our case). reduction of the bacterial contamination risk was estimated using the results of the bacterial testing of pooled and apheresis platelet products. as patients are likely to obtain multiple blood products during treatment, the contamination risk reduction through sdp is not only dependent on the reduction of risk in trombocyte products, but also will also dependent on the total number of blood products transfused and their associated contamination risks. the platelet recipient risk reduction was calculated on basis of the distribution of blood products received by the patient population of the university medical center utrecht (umcu) in the year 2001. results: in the attached table the estimated risk reduction through 100% trombocytapheresis is shown for the general blood recipient patient and for the trombocyte recipient patients only. our analysis indicate that in platelet recipients, general application of sdp instead of ppc will reduce the risk for transfusion acquired tse infections by 49%, the risk of known viral infections by 56%, and transfusion acquired bacterial infections by 59%. the confidence intervals surrounding the results were obtained by bootstrapping. the large confidence intervals surrounding the reduction of bacterial infection risk is caused by the fact that only a limited set of 4300 apheresis trombocyte products were tested. conclusion: our analysis indicates that general application of sdp instead of ppc will not reduce the risk of transmitting infections to platelet recipients as linearly (1 : 5) as expected. whether it is a costeffective precautionary measure will have to be evaluated by a costbenefit analyses consideration clinical benefits and additional costs and risks of apheresis donations. introduction: hepatitis b is serious health problem world wide. its prevention, particularly in the population of blood donors is essential for providing good health care and protection. aim: the aim of this study is to present the distribution of hbsag(+) and hbsag(-) blood donors according to their profession. methods: specially designed questionnaires are used for interviewing the blood donors who has previously given signed consent for participation in this study. results: table 1 shows the distribution of the blood donors in different professions. administrative clerks with 43 (34.12%) and the workers with 40 (31.73%) registered in the group of hbsag(+) blood donors, as well as workers with 50 (50%) and administrative clerks with 30 (30%) from the hbsag(-) group of blood donors are dominantly more frequent than the other categories of professions. health care professionals, housewives and farmers in both groups of blood donors are least frequent. taking in consideration the distribution of blood donors by the given professions in both groups, for u = 21 and p > 0.05 there is no significant difference found. the analysis of the differences among the different frequencies, in distribution of blood donors according the profession, for d = 0.436 and p < 0.05 shows a significant difference, where the workers with 90 (39.82%) are the most dominantly represented. in relation to the issue of whether the type of profession of the blood donors is production or non production the results are presented on table 2 . in the group of hbsag(+) blood donors the number of those who work in production profession-92 (73.01%), is dominant over the number of those that has non-production profession-34 (26.98%). in the group of hbsag(-) blood donors there is no significant difference between the types of professions. conclusion: having in consideration the professions of blood donors in both groups, for c 2 = 14.8 and p < 0.01 there is a significant difference in the presented distribution. according to our study, which shows the horizontal transmission of hbv infection in the family in which there is an index case, the biggest number of participants in the study is workers, and the least number of participants are the farmers. introduction: blood donors, as part of the healthy population are tested for hbsag with each blood unit they give. therefore, they can be an epidemiological model for exploring the appearance of hbv infection in general population. aim: this study aims to show the distribution of hbv infection in blood donors in relation with their living conditions, space and facilities. the material needed for the study consists of the data obtained from 126 confirmed hbsag(+) blood donors and 100 confirmed hbsag(-) blood donors as control group. results: the table 1 shows almost equal number of hbsag(+) blood donors that live in houses or flats. in the hbsag(-) group 64 (64%) donors that live in a flat dominate compared to 36 (36%) of those that live in a house. having in consideration the presented distribution (table 10) for c 2 = 3.96 and p < 0.05 there is a significant difference, that comes from the bigger number of blood donors (128) that live in flat. as for the distribution of blood donors according the living space they use by member of the family (table 2) , we can show that 24 (19.04%) of the hbsag(+) donors have less than 10 m 2 living space, in compared to 5 (5%) from the hbsag(-) group. the bigger number of hbsag(+) blood donors with small living space gives bigger possibility for transmission of hbv infection in the family. the differences in the two groups for donors that have between 10 and 30 m 2 , and between 31 and 40 m 2 of living space per person, obviously are not very big. for u = 19 and p > 0.05 there is no significant difference in the number of the donors in the two groups, when the available living space in m 2 is discussed. introduction: when one of the sexual partners has hbv infection the other is also infected in 1 from 3 cases. aim: the aim of this study is to outline that the risk from transmission of hbv infection between sexual partners is smaller if they use condom as protection. methods: two groups of blood donors-hbsag(+) and hbsag(-) have been interviewed whether they are using condoms as protection, or not. results: table1 shows the distribution of blood donors concerning the use of condoms. table1: in the group of hbsag(+) blood donors those who have not used condoms dominate with number of 81 (64.28%), in compared to those 45 (35.71%) who used condoms. these data are in favor of eventually possible sexual transmission of hbv infection in hbsag(+) blood donors. in the group of hbsag(-) blood donors dominate those who have used condom-57 (57%), compared to 43 (43%) who have not. the given distribution of blood donors concerning the use of condoms for c 2 = 10.2 and p < 0.01, shows significant difference, which is due to the prevailing of the number of blood donors (124), who have not used condom. of er -concentrates are leukodepleted. the choice of bacteriological control of empty bags for blood, bags with er -concentrates in additive solution, universal and iso group plasma, as well as the systems for taking of blood are taken on free choice. the control of the erytrocyte concentrates is performed on the first day after the preservation and dekanting, and again between the 15th-21st day and 30th-35th day after the preservation. the pulled plasma is controlled on the day of pouring (spreading), and the control of the iso group plasma on the day of deplasming. three months later the iso group and the universal plasma kept on the temperature of -30°c is bacteriologically controlled again. bacteriological control is performed with standard procedures in the institute for health protection in stip. the transfusion transmitted infections are potentially dangerous complications of transfusion therapy in immunocompromised patients. the aim of this study was to determine the prevalence of transmissible infections in blood donor population in kashan, iran. a total of 600 consecutive sera were tested for cmv-igm antibody, hbsag, hepatitis b core (hbc) antibody, hepatitis c (hcv) antibody, and hiv antibody with standard methods. of the sera tested, 14 specimens (2.3%) were cmv-igm positive. the frequency of seropositive revealed no significant differences between male and female donors. the frequency rates of cmv-igm seropositive tests tend to decline with increasing the age. there was no relation between the frequency rates of cmv-igm seropositive with the educational level, socioeconomic status, marital status, urban dweller and rural resident patients. the prevalence of hbv, hcv, and hiv antibody were 0.5%, 0.5%, and 0%, respectively. these findings implied important clinical applications because detection of cmv positive sera may reduce the risk for transmission of cmv in blood transfusion and thereby decrease the risk on cmv-induced complications. introduction: the worlds problem, aids, steel can't be said that is a problem in these three centers in r. macedonia, in which blood is collected, controlled, and distributed. found negative and 5 of them were found positive for one of the three viruses (hiv-1, hcv, hbv). with the elisa/axsym assay 29 of the 992 blood units which were negative by the procleix ultrio assay were positive for anti-hbcag and negative for anti-hbsag and hbsag. from those 29 blood units 23 units were given for transfusion following our blood centre protocol and the remaining 6 units were discarded. the protocol consists of a good medical history, liver enzymes (ast, alt, ggt). we must take into consideration that from those 5 that were found positive by the procleix ultrio assay 1 was positive for anti-hcv and 4 were positive for anti-hbsag. summary/conclusions: despite the fact of the short period of time we perform this method, the ability of the nat technique for rapid use, reliability and sensitivity in detecting three viruses simultaneously, indicates the need for immediate use in blood donation as a screening method. in spite of the high cost of the method, it is clear that this assay is a valuable tool in our blood centre to provide fast and safer blood. bacterial contamination of blood products is a persistent, but often overlooked, problem in transfusion medicine. in greece it is recommended that platelets (plt) must be used or discarded within five days post-collection. recent reports from europe have advocated the use of bacterial culturing of platelets on day 2 or 3 and, in case of negative result, prolongation of their storage time to 7 days. aim of the study: to assess the prevalence of bacterial contamination of standard platelet units from whole blood, and to provide evidence that with the use of bacterial culturing it is feasible to extend the self life of platelets to 7 days. materials and methods: eligible blood donors were bled according to standard operating procedures used in greece. plt were prepared from whole blood, solely for the purpose of the present study, by the platelet-rich plasma method. plts were stored for up to 7 days at 20 to 24°c with end-over-end agitation. other 111 plts prepared from blood collected in triple-pack container system also provided with a predonation sampling device were also tested. plts were sampled in the bacteriology laboratory. plts were sampled on day 2, 5 and 7. both aerobic and anaerobic culture bottles were inoculated with a 7-ml platelet sample. culture bottles were incubated at 35°c in an automated microbe -detection system (bact/alert system) until a positive reaction was detected or for 10 days. all samples that were reactive were confirmed by routine culture. each reactive sample with bacteria growth on the routine culture was sub cultured for identification of the bacteria. results: a total of 1889 plt concentrates were cultured and bacterial contamination was assessed in each unit at day 2, 5 and 7 after collection. on of storage day 2 two out of 1889 (0.1%) plt units were found to be positive for bacterial growth. 7 cases of unconfirmed positive results were noted at the beginning of the study. out of the other 1887 units which were negative on day 2 and continued to be cultured for the next days, the assessment at day 5 found no other positive. after further storage, at day 7, defined as the end of the prolonged incubation period, 5 out of the 1887 plt concentrates (0.26%) grew bacteria although testing of the same units on day 2 and 5 gave no signal. from the 111 platelets units that were prepared from blood collected with a predonation sampling device, none of the plt concentrates gave a positive signal although 3 pouches were found to be positive, and subculture showed bacterial growth of coagulase -negative staphylococcus. despite the relative small number of tested platelet concentrate units, our findings discourage specialists in attempting platelet storage time prolongation to 7 days. bacterial contamination testing on day 2 and a storage time of maximum 5 days seems to be still the safest practice. bacterial screening of platelet concentrates using bact background: bacterial screening of blood components is a routine measure in the evaluation of blood product quality. at our institute bacterial screening is performed using bact/alert system. sampling and culturing of blood products is performed according to paul-erlich institute recommendations. methods: quality control data on the bacterial screening of platelet concentrates performed from 1999-2004 were retrospectively analysed. an initially positive (ip) and true positive (cp) rate, organisms isolated and time of detection are presented. results: a total of 5359 platelet products were tested during the 6year period. thirty (0.56%) were found initially positive by bact/alert. the cultures screening positive were subjected to bac-terial identification to distinguish false positive from real positive signals. bacterial contamination was confirmed in 14 (0.26%) plt concentrates. positive cultures were confirmed and identified in an independent laboratory ( hbsag, anti-core, anti-hbs, anti-hcv, anti-hiv i/ii, anti-htlv i/ii, rpr. these patients were transfused with 5-131 units of concentrated red cells, depending on their problem. a total 530 units were delivered from the beginning of their problem until december 2003. the control were done using last generation enzyme-linked immunoassay (dade behring, ortho, biomeurieux), as also using automated enzyme-linked immunoassay (axgym). the same patients were checked by their physicians before the initiation of transfusions for the same diseases. results: we found: 12 patients anti-hbc (+) and anti-hbs (+). 5 patients anti-hbc (-) and ánti-hbs (-) 3 patients anti-hbc (-) and anti-hbs (+) 1 patient anti-hbc (+) and anti-hbs (-) 1 patient hbsag (+), anti-hbc (+) and anti-hbs (-). all patients were negative for hcv, hiv, htlv, and rpr. the same results were found also from patients' physicians. conclusions: we conclude that the blood supply for blood transfusion-transmitted diseases is 100% safe in our centre. these results are in accordance with current international literature. this is due to careful selection of blood donors, to high quality of corporation between departments as also to internal and external quality control. all these factors contribute to safety of transfusions, the quality of life of the patients and the protection of patient's environment. neither in the pipetor nor in the extractor runs was found contamination. no false positive were detected and all the positive samples confirmed. (see tables 1 and 2) . for hiv and hcv the specificity was 99.99%. the validation criterion were met, so the system was implemented routinely in our laboratory. the purpose of our study was to analyse the applicability of the pall enhanced bacterial detection system (ebds) in the routine of our transfusion unit which is totally focused on apheresis platelet collection. methods: apheresis pcs, obtained by trima (cobe) and amicus (baxter) separators and re-suspended in 30% plasma and 70% ssp solution (macopharma), were submitted to microbiologic control using pall ebds system which uses oxygen percentage decrease as a surrogate marker of bacterial growth. the working steps were the following: 16-24 hour after donation, about 3 ml of pc were sampled into the ebds collection pouch and then incubated at 35°c under continuous agitation (incubator helmer 900) for 24 hours. after this period, oxygen percentage was measured using an oxygen analyser (pall bdso2). the test is based on the 'pass/fail' principle. in case of 'fail' result the microbiology department has drawn up the procedure to follow in order to confirm the data and to allow the micro-organism to be identified. the incidence of post transfusion hepatitis has been reduced by blood donor screening for hbsag, but the hbv infection is still responsible for a certain cases of post-transfusion hepatitis in world-wide. in this study the hbsag negative blood units were evaluated for anti-hbc and hbv dna by pcr method. an extra sample was collected from 2000 hbsag, anti-hcv, anti-hiv and rpr-negative blood donors. all of samples were examined by approved anti-hbc assay. all of anti-hbc positive samples were tested by hbsab assay and evaluated for hbv dna (pcr). the sensitivity of the hbv dna (pcr) assay was estimated 50 geq/ml according to vqc proficiency and run control panels. 212 (10.6%) out of 2000 samples were positive for anti-hbc.166 (78.3%) out of 212 anti-hbc positive samples were hbsab positive, and 46 (21.7%) were hbsab negative. all of 212 samples were assayed for hbv dna (pcr) single and all of them were negative for hbv dna (pcr). further study for evaluation of anti-hbc test as a screening assay for blood unites in high hbv infection prevalence area strongly recommended. early detection of hepatitis b surface antigen: a comparison of ten assays hbsag detection is the corner stone of detection of hepatitis b virus infection in blood donors and patients with hbv infection. one of the most challenges is sensitivity of the technique and kit. in this study ten different assays evaluated by seroconversion and performance panels. some of them can not be used as screening assay due to low sensitivity. the sensitivity of ten hbsag assays from biorad, dade behring, biomeriux, diasorn, radim, diesse, thermo. biokit, gb and shanghai companies were evaluated by two or three seroconversion and two performance panels from boston biomedica ink. seroconversion panel is a series of samples that collected over a period of time from individual developing antibodies due to a primary infection. for evaluation of the assay sensitivity who and other notified body in the world-wide recommended the seroconversion and performance panels. the hbsag assays are two groups. group one with high sensitivity included six assays. they can detect ad and ay subtypes from 0.1 ng/ml bbi to 0.4-0.5 ng/ml bbi respectively. low sensitivity group included four assays and they can detect ad and ay subtypes more than 0.7 and 0.5 ng/ml bbi respectively. for blood safety, the high sensitivity hbsag assays recommended for blood screening and all assays should be evaluated by seroconversion and performance panels. diagnosis of chronic hdv infection is usually by antibody testing and hbv dna detected by pcr method. it is rare to find patients with two replicating hepatotropic viruses and if the accompanying hbv is replicating, prognosis will be very poor. to clarify the correlation between hepatitis delta virus infection and hepatitis b virus dna positivity, sensitive hbv dna (pcr) assay was used. the presence of hbv dna was investigated in 274 patients referred during the 21 aug. to 28 dec. 2003. all of them were hbsag positive. all samples were evaluated for hbv dna (pcr). the sensitivity of the hbv dna (pcr) assay was estimated 50 geq/ml according to vqc proficiency panel and run control. anti-hdv was tested by commercial available enzyme immunosorbent assay. 179 (65.3%) were hbv dna positive and 95 (34.6%) were negative. 8 (4.4%) out of 171 patients had evidence of delta infection and 13 (13.6%) samples of hbv dna (pcr) negative patients were positive for delta agent. the serum alanine aminotransferase (alt) levels in 5 out of 8 hbv dna (pcr) and anti-hdv positive patients were higher than reference interval, but only in 2 out of 13 hbv dna (pcr) negative and anti-hdv positive samples were higher than reference interval. the present data indicate that 7.7% of patients with chronic hepatitis b have hepatitisdelta infection. patients with hbv dna (pcr) negativity had a significantly higher prevalence of delta marker (13.6%) than those with hbv dna (pcr) positivity (4.4%). delta rna testing in positive hbv dna and anti-hdv patients is recommended. introduction: reduction of the window period of hepatitis c virus (hcv) infection represents an important goal in the transfusional and diagnostic setting and nucleic acid technology-based tests have been introduced in some developed countries to reduce the potential risk of transfusion-associated infection. a prototype assay designed to simultaneously detect circulating hcv antigen and anti-hcv has been developed by biorad (biorad laboratories limited, marnes la coquette, france). aim of the present study: to define the cut-off (co) value of the assay and to evaluate the specificity and sensitivity of this new assay in the detection both of antibody and antigen comparing its efficacy with commercial assays. methods: in order to establish the co value and to evaluate the specificity of the assay, we tested 400 sera samples from the general population and 100 'difficult' sera from haemodialysis patients (n conclusion: the new assay shows high sensitivity and specificity and could be a useful tool not only in the diagnostic setting, where procedures to reduce the window period, such as antigen or hcv-rna detection, are not currently recommended, but also in the screening of blood donations, when nucleic acid technologies is not feasible due to costs, organization, emergency and/or logistic difficulties. introduction: in recent years the concern with the blood safety regarding the transmission of blood-borne viruses has been improved. this safety has been achieved with the combining of different strategies, such as a careful selection of donors, the screening for relevant virological markers and the viral inactivation/ removal methods. more recently, the implementation of the nucleic acid amplification technologies for the detection of hiv-1, hcv and hbv, has increase this aim by reducing the 'window period' of the infections. other viruses, such as parvovirus b19 (pb19) and hepatitis a virus (hav), can raise problems to the blood safety. these infections could provoke serious complications in some risk groups, like pregnant women, patients with haematological problems, children and patients with immunodeficiency. material and methods: an observational study was performed to determine the prevalence of pb19 and hav in portuguese blood donors. we gather, during four months, 5025 plasma donations and joined them into 505 pools, with no more than 10 donations each. [1999] [2000] [2001] [2002] [2003] in voluntary donors the anti-hcv prevalence ranged from 0.3% to 0.7%, in family replacement donors from 0.4% to 1.2%, autologous donors from 1% to 2.02%. we observed that the anti-hcv prevalence has a decline tendency during years in blood donors. according to sex the anti-hcv prevalence in men is 0.7% and women 0.6% (p = 0.004). over the 5 year periods the prevalence in men has a decline tendency (1.2% to 0.4%; p = 0.033) and increasing tendency in women (0.4% to 0.9%; p = 0.007). according to age group the anti-hcv is 0.6% in 17-29 age group, 0.65% in 30-39 age group, 0.8% in 40-49 age group, 0.86% in 50-60 age group (p = 0.001). the prevalence of anti-hcv is higher in fds than vds, but not statistical significant. [1999] [2000] [2001] [2002] [2003] . a total 14627 ftd and 684 ad have been tested for hbsag. a sample was considered as hbsag positive when found repeatedly reactive by 3rd generation. immunoassay method (elisa). the chi-square test was used for statistical analysis. results: the 5-year overall hbsag prevalence among first time blood donors was 8.9%. and ad 6.7%. among autologous blood donors was observed a decreasing hbv prevalence from 6.3% to 3.7% in 2003. according to age the prevalence was higher in 17-29 year group 7.7%, while according to sex was higher in man (8.8%) than female 6.2% (p < 00.5). among ad, a decreased hbsag prevalence according to age was observed in men and women. the same trends by sex and age were observed in ftd. the prevalence of hbsag in ad was lower than in ftd. however, from 1999-2003 hbsag prevalence has decreased in the same proportion in both population. this decreased can explain by to main factor: the improvement hbsag screening method in blood donors and decreased the hbsag prevalence in general population. (1 : 30 250) . the hbv dna-emia in hbsag negative samples was 1.14 ¥ 10 2 -9.55 ¥ 10 4 copies/ml. in two donors anti-hbc total was positive and in one anti-hbe was also detected. in one donor the glycin 145 alanin mutation in the s region was identified. the frequency of hbv dna pos/hbsag neg donors in poland is high (0.002%) therefore the decision to introduce routine hbv nat screening is justified. (1/1001) with stored apheresis and whole blood derived platelet concentrates. of these 118 failed results there were 23 confirmed positives (presence of bacteria in both the ebds pouch and the platelet mother bag by culture) representing 1/5133. the bacteria detected were staphylococcus or streptococcus sp. 76 of the 118 fail results were false positives (no presence of bacteria in the ebds pouch and the platelet mother bag by culture) representing 0.064% or 1/1554, and 18 were not confirmed initial positives (no bacteria in the mother bag by culture, ebds pouch not tested) representing 1/6559. there was one reported case of a missed detection with confirmed presence of bacteria (staphylococcus epidermidis) in the mother bag by culture. subsequently, the bacteria strain was isolated and inoculated into platelet units in our laboratory at levels as low as 5 cfu/ml. in all cases the pall ebds was able to detect. this supports the hypothesis that this missed detection was the result of a statistical sampling error rather than a system failure. the results from blood centers routinely using pall ebds demonstrated effective detection of bacteria in platelet products stored under routine conditions with a true positive rate of 1/5133, and with a low false positive rate (<0.1%). this is comparable to a recent survey result with other culture based systems. summary/conclusions: the minority group of pregnant women who come to labor without prenatal testing of hepatitis b and c revealed essentially similar prevalence of anti-hcv with healthy bd even if definitive confirmation is probably increased in this minority group. there is however markedly higher prevalence of hbv infection in the pw so that screening for hbv is essential for the prevention of vertical transmission. the systematic screening of bd with anti-hbc serves as further assurance for the prevention posttransfusion hepatitis eliminating only 1.21% of the possibly infectious, a percentage which can be restored to the blood pool after proving their immunity. methods: blood samples were screened for the presence of hbsag, hcv and hiv antibodies using enzyme immune assay and for syphilis using the tpha test. the results were analysed retrospectively. all samples with results at or above the minimum positive value were considered reactive. the tests for hbsag, anti-hiv and anti-hcv were repeated in duplicate in all reactive donations. blood units that were reactive in the primary or secondary assays were discarded. hiv positivity was confirmed by western blot analysis using hiv blot 2.2 (genelab diagnostics) results: results from a total of 77 903 screened donors were analysed. hepatitis b surface antigen rates was 1.99%; anti-hcv seropositivity was 0.54%; anti-hiv seropositivity was 0.01% and tpha seropositivity was 0.06%. one study calculated this risk to be one in 63 000 for hbv, one in 493 000 for hiv and one in 103 000 for hcv. it is therefore important to take a careful history from blood donors to eliminate those at high risk of infection. in view of the high infectivity of hiv positive blood, it is important not only to screen donated blood but also to exclude donations from high-risk individuals, such as males who have engaged in homosexual activity and intravenous drug users. a careful history should identify those who should not give blood. in turkey, among blood donors the average hbsag prevalence in 1985-1998 was 5.1%. but it had decreased to approximately 2.1% in 2003. anti-hcv positivity has been reported to be 0.6% between 1992 and 1999. but it was approximately 0.5% in 2003. rpr positivity in blood donors in turkey was reported to be <0.1% in 1973 and 0.0026% in 1990. in 2003, the rpr rates was 0.09%. in our study these rates are 1.99%, 0.54%, 0.09% and 0.06% respectively. anti-hiv seropositivity was found around 0. introduction: the serological detection of specific antibodies to treponema pallidum (tp) is an effective means of diagnosing syphilis, and an automated chemiluminescent assay is ideally suited to testing large numbers of specimens for the laboratory diagnosis of the disease. aims of the study: to develop a qualitative syphilis assay for the detection of tp immunoglobulin m (igm) and g (igg) antibodies. the assay will be used for the serological diagnosis of syphilis using the architect platform, which has the capacity to test 200 specimens/hour. the two-step assay is based on paramagnetic microparticle chemiluminescent technology, utilising microparticles coated with three recombinant tp antigens (tpn15, tpn17 and tpn47) and acridinium labelled anti-human igg and igm monoclonal antibodies as conjugates. in the first step, specimens, microparticles and diluent are incubated together, prior to a wash step; in the second step, acridinium labelled antibodies are added and after washing, pre-trigger and trigger are added to produce chemiluminescence, which is measured as relative light units (rlu). specimens yielding rlus less than the cut-off are considered negative, while those yielding rlus greater than the cut-off are considered positive. the sensitivity of architect syphilis tp was determined to be 100%, after testing 426 specimens that were previously screened as syphilis positive in fujirebio tppa; no prozoning was observed with high positive specimens (over 2 16 titer by tppa). the specificity generated from testing 564 hospitalised patients previously screened as tppa negative, was 99.6%. testing a mixture of sera and plasma from 5000 random donor specimens, generated donor specificity figures of 99.8%. the precision (cv%) with a positive control was 5.8% (95% confidence interval: 4.9-7.0%) by the standard 20-day nccls analysis (ep5a2). in a study conducted at asahikawa medical college hospital, in which, 81 positive and 82 negative specimens were tested, concordance with fujirebio tppa was determined to be 100%. no significant interference to the assay was observed from bilirubin (conjugated type and free type), haemoglobin or lipid. the architect syphilis tp assay is an automated, specific and sensitive test for the detection of antibodies to t. pallidum. background: hcv exposure of blood donors is serologically determined by the detection of anti-hcv antibodies in serum or plasma. however a 'window' period of 30-70 days after exposure exists during which specific antibodies to hcv antigens cannot be detected. hcv rna detection and/or hcv core protein testing result in dramatic reductions in the preseroconversion window period. the new bio-rad test, based on the simultaneous detection of hcv core antigen and anti-hcv (core, ns3, ns4) antibodies, improves the detection of hcv infection in the early phase. aims: the aim of this study is to assess the performance characteristics of this new screening microplate immunoassay, monolisa hcv ag-ab ultra, by using the bio-rad evolis automated microplate processor system. methods: this two-step elisa assay is based on the combination of an indirect test for the detection of antibodies (core, ns3, ns4) and a sandwich test for core antigen detection. results are available within 2.5 hours, with sample addition monitoring and color coded reagents. no specimen pretreatment is required. evolis is a self-contained microplate processor designed for full automation of microplate-based eia techniques. the walkaway system can process four microplates at a time with continuous loading of samples and reagents. positive identification of samples, reagents and microplates, usage of disposable tips with clot detection, integrated quality control and complete traceability provide a high level of safety management. the monolisa hcv ag-ab ultra/evolis system performance is evaluated for clinical sensitivity on 30 commercially available and well-documented seroconversion panels. the results are compared to viral rna detection and conventional hcv ab screening assays. specificity is evaluated by using random blood donor samples. results: among the seroconversion panels that begining with samples negative for hcv rna and anti-hcv antibodies, the monolisa hcv ag-ab ultra assay detects exposure to hcv an average of 25 days earlier than the monolisa hcv plus v2 test. the mean delay of the monolisa hcv ag-ab assay in detecting hcv infection compared to hcv rna testing is around 2.5 days. the monolisa hcv ag-ab ultra/evolis system allows simultaneous detection of hcv core antigen and anti-hcv (core, ns3, ns4) antibodies, thus significantly reducing the time gap between the initial detection of hcv rna and the first appearance of detectable anti-hcv antibodies. the fully automated system combines high degree of assay performance with optimization of laboratory workflow and safety management. operational evaluation of pall ebds bacterial detection system l larrea gonzalez, ma soler and rj roig centro de transfusion, valencia, spain introduction: regulatory bodies are increasingly mandating the use of bacterial detection systems for platelet products (22 ed standards for blood banks and transfusion services). one system currently available is the pall ebds bacterial detection system which utilises percentage oxygen as a surrogate marker for bacterial growth. aims: to evaluate the pall ebds in routine use in our blood centre. in particular, to assess feasibility and adaptability to daily labour routines. the orbisac (gambro bct) system was used to produce leucocyte depleted buffy coat (bc) platelet pools (4 bc/pool) stored in platelet additive solution (ssp macopharma). mean platelet count was 3.2 ¥ 10e 11 /pool with mean leucocyte count 0.09 ¥ 10e 6 /pool. ebds installation and training occurred over a 2 day period. 500 platelet pools were tested for bacterial contamination over the subsequent 5 weeks. ebds pouches were sterile connected onto platelet pools 22 hours after blood donation. platelet samples were taken into the pouches and then the pouches were incubated for 24 hours on a shaking agitator at 35°c. after this time, percentage oxygen was measured. no positive results were found in this study. this was as expected due to the relatively low number of platelet pools tested and it also highlights the absence of false positive results. minimal training was required to use the ebds. the system was easy to use and did not require the use of a laminar flow cabinet to take samples. it was quick and simple to take samples and perform oxygen measurement. after pouch incubation, 1 technician was able to make 20 oxygen measurements in less than 13 minutes. the data management system allowed full traceability of product and work flow. results were very easy to interpret. conclusion: the pall ebds was found to adapt perfectly to a routine blood centre environment. (2004) was 8470. the percentage collected from volunteer blood donors was 56% (n = 4783) and the rest 44% (n = 3687) was given from patient-related donors. the age of donors ranged from 18 to 65 years old. the assay used for the detection of hbsag, hbeag, anti-hbc igg/total, anti-hbc igm, anti-hbe and anti-hbs was the automated microparticle enzyme immunoassay (axsym) of the abbot company. all the units were tested for hbsag, and anti-hbc igg. if the anti-hbc igg was detected, the specimens were automatically tested for anti-hbs. the units were wasted if the anti-hbs was negative, and the specimens were manually programmed for the testing of the anti-hbc igm, hbeag, and anti-hbe. from the total of 8470 tested units, 1195 of them were found to be positive to at least one marker of hbv infection, that means the 14.11% of the health adult population was infected in the past by the hbv. the 13.69% (n = 1160) was previously infected and now immunized with hbsag(-) and anti-core igg(+) and 0.41% (n = 35) were chronic carriers of the hbv with hbsag(+). the 68.57% (n = 24) of the positive donors were patient related donors and 31.42% (n = 11) were volunteer donors. in other words, 24 of the 3687 not volunteers (0.65%) and 11 of the 4783 volunteers (0.23%) were detected to be infectious. the combinations of the serologic markers for hbv are illustrated in the table attached. these results indicate that the incidence of hbv infection in the northeastern department of greece is equivalent to the incidence of hbv in other greek regions (0.42%) as it is referred to the national haemovigilance data and moreover, the percentage of infectious donors is bigger among replacement donors, 0.65% compared with the 0.23% of voluntary donors. as a consequence, the best source for safe blood collection is the population of volunteers. earlier detection of human immunodeficiency type 1, hepatitis c and hepatitis b viruses using the procleix® ultrio tm assay on the procleix® system and the study objective was to assess the ability of the ultrio assay and associated discriminatory assays to reduce the detection windows for hiv-1, hcv, and hbv. commercially available seroconversion panels were used for testing. methods: hiv-1 (n = 13), hcv (n = 12), and hbv (n = 16) seroconversion panels were tested neat and diluted (1 : 8 and 1 : 16) in the ultrio assay. panels were tested neat in the appropriate discriminatory assay. times to detection of hiv-1, hcv, and hbv nucleic acids in seroconversion panels were compared to the vendor's historical data on time to detection of antibody and/or antigen using licensed or validated serologic tests. p-210 effectiveness and limitations of methods for platelet bacteria screening -how to apply which screening method? the successful concept of virus safety in transfusion medicine is not suitable in bacterial contamination. bacteria can grow up in blood components to enormous amounts, whereas the initial number of contaminating bacteria is typically very low. therefore, sample drawing for bacteria screening must not be done immediately after blood donation. the established concept of relevance of clinical microbiology (pathogenic, non-pathogenic, facultative pathogenic species) is not valid for bacteria contaminating blood. here, the currently discussed criterion of clinical relevance is the ability of bacteria strains (not species!) to grow up in blood components. the paul ehrlich institute (pei) developed pei bacteria standards, which are characterized concerning their behavior in blood components. they contain a defined number of living bacteria, they are deep frozen, ready to use and shippable. there are two strategies to improve bacteria safety of blood: screening and pathogen reduction. neither of them is perfect, but screening methods are successfully established since several years in routine (belgium, the netherlands), and represent the current state of the art. further development and collecting of experience will produce the basis for assessments in the future. it is of high importance to apply the screening methods in dependence on their properties. methods implying an incubation/cultivation step ('early methods') have to be distinguished carefully from 'rapid methods' . for example, it is unreasonable to compare (or to advertise with) different sensitivities of methods not considering their detection principle or their informative value. both principles, cultivation methods as well as rapid methods, show advantages and disadvantages. selection of the method has to consider the respective conditions of the given blood service (including logistics up to time frame between issue and transfusion). results from the procleix hiv-1/hcv and hiv-1/hcv/hbv (procleix ultrio) assays for the detection of hiv-1 rna, hcv rna and hbv dna in blood donors of two blood transfusion centers of sw greece in discriminatory assay testing, 3 out of 5 (60% of the positive, 0.020% of total) were reactive for hcv rna only and 2 out of 5 (40% of the positive, 0.013% of total) were reactive for hiv-1 rna only. none were positive for both hiv-1 and hcv. the standard serological assays gave the same results for the above positive samples. two samples that tested positive by the standard serological assays tested negative in the procleix hiv-1/hcv assay. of the 4151 samples tested by the ultrio assay, 21 (0.5%) tested reactive for hiv-1/hcv/hbv. in discriminatory assay testing, 1 out of 21 (4.76% of the positive, 0.024% of total) was reactive for hiv-1 rna, 4 out of 21 (19% of the positive, 0.096% of total) were reactive for hcv rna, and 16 out of 21 (76.2% of the positive, 0.38% of the total) were reactive for hbv dna. all were single positive i.e. none tested positive for more than 1 virus. three out of 16 positive samples for hbv dna tested negative by the standard serological tests. the opposite was not observed. the procleix ultrio assay is a definite improvement over the procleix assay in a region with a high incidence of hbv carriers. up until its use, it is obvious that hbv positive blood with very low antibody titers was transfused into patients. more results will show whether procleix ultrio can eventually replace the standard serological tests. the introduction: patients with hemophilia represent a high-risk group for post-transfusion hepatitis whose frequency is closely linked with the number and quantity of blood products used. in albania, the frequency of hepatitis is also linked with hbsag testing with elisa (introduced in1993), and hcv testing (introduced in 1997). aim of the study: evaluation of the prevalence of the markers of hepatitis b, c, and d in patients with hemophilia. methods: our study included 145 patients with hemophilia treated with cryoprecipitate and commercial clotting factors. blood testing for anti-hcv, anti-hdv, and hbsag was performed with elisa -gen. iii. results: of 145 patients tested, 26 cases (17%) were hbsag positive, 87 cases (60%) were anti-hcv positive, and 7 cases (5%) were anti-hdv positive. co-infection of hbsag and hcv was found in 16 cases (11%), whereas co-infection of hcv, hdv, and hbv was found in 4 persons (3%). the highest rates of infections and coinfections were found in patients above 30 years of age. conclusion: mandatory blood testing has decreased the levels of post-transfusion hepatitis. in albania, hemophilia is also still treated with cryo-precipitation, thus patients are at a particularly high risk during the 'window period' . results: 2/16 760 (0.012%) samples from rbd were anti hiv 1 + 2 nonreactive and rr for p24 ag both being nonreactive in the neutralization test, they were interpreted as false positives. 1/134 617 (0.0007%) sample from fbd was rr for p24ag/anti hiv 1 + 2 nonreactive and it was confirmed positive by neutralization. this bd had been autoexcluded himself after blood donation. he showed seroconvertion 21 days later: p24ag nonreactive, anti hiv 1 + 2 reactive and western blot positive. the only bd p24ag positive/anti hiv 1 + 2 nonreactive during the analized period, was an first time donor and the post donation autoexclusion was effective en this case. although a larger populations of bd is necessary to be studied and in spite of the low prevalence we have found, we consider p24ag screening is an alternative up to implementation of nucleic acid testing and simultaneously we should increase the quantity of altruist repeat blood donors, undoubtedly, the best population to give blood. owing to the rather short interval between successive donations (~70 days), this suggests that some 260-310 infectious units escape the screening annually. to these, one has to add the (now unknown) proportion of potentially hbsag negative + hbv dna positive ftbds. hcv: since the introduction of the screening in 1995, the general incidence in rbd has dropped from 13.1‰ to 0.001‰, suggestive of a 1 : 10 000 escape rate. the prevalence in ftbd has stabilized at 12 ± 2‰. based on reasons similar to these employed for hbv, the residual incidence in rbd suggests that 1 potentially infectious donation in 10 000 rbd escapes the screening (= to a total of aprox. 40, annually). a limited investigation using hcv-antigen eia evidenced a 1‰ escape rate in ftbds (= to a total of aprox. 50, annually table 1 and 2 are concerned to the fist two months of the implementation, where we had to adjust the volume of the eluate. conclusion: these system adjusts to the laboratory daily routine in the blood bank, with the pools released after first analysis in less than 18 hours. background: the hbsag, anti-hcv, anti-hiv1/2, p24 antigen, alt and syphilis tests are performed for blood donations in czech republic. no nat tests are mandatory in czech republic. the aim of this pilot study was: 1. hcv rna pcr testing in anti-hcv negative blood donations; 2. correlation between hcv nat and anti-hcv testing results. methods: blood samples (anti-hcv serologically negative, alt not elevated) were pooled using the guardian plus spii into pools of 24 samples. pools of 1 ml were tested using the cobas ampliscreen hcv test v. 2.0 (roche). results: 891 pools of 24 samples from 21 384 a-hcv serologically negative donations were tested from october 2002 to july 2004. no one pool was initially reactive. invalid tests: 14 (1.6%) run failures were observed, due to: invalid internal controls 10 (1.1%) and invalid positive controls 4 (0.5%). invalid tests were repeated. in none of 891 pools a positive hcv nat result was observed. conclusions: no discrepancy between hcv nat and a-hcv results was observed in our study. all of the nat tested donors in our study were regular voluntary whole blood or plasma donors who were repeatedly a-hcv serologically negative. the hcv incidence in the czech republic blood donor population is low but it is slightly growing up in general population. hcv nat testing could improve the safety of blood supply by reducing the window period for hcv. introduction: parvovirus b19 is the only parvovirus known to be a human pathogen. most commonly, it causes a mild childhood rash, erythema infectiosum, but in some cases more serious symptoms can be linked to b19, such as acute or persistent arthropathies, critical failures of red cell production, hydrops fetalis, fetal loss, myocarditis or hepatitis. inactivation of the non-enveloped virus has proven difficult. as a consequence, manufacturers of blood products have implemented screening measures to reduce the load of parvovirus b19 in manufacturing plasma pools by the use of nucleic acid amplification techniques (nat). in our institute all blood donations were screened for human parvovirus b19 by nat since april 2000. methods: over the last 5 years 5.5 million donations were screened for b19 by nat. samples with a virus load over 105 iu/ml were defined as positive, whereas samples with a virus load between the detection limit (103 iu/ml) and 105 iu/ml were defined as weak positive. weak positive products were released, whereas positive products were discarded. in addition infection markers of 50 b19 positive donors (case group) were determined over a time period of one year. virus load and b19 antibody status was compared with 100 b19 negative donors (randomised control group). b19 antibodies (igg vp2, igm vp2, ns1) were analysed by two commercial antibody tests. results: overall 496 b19 nat-positive donors were identified with a virus load over 105 iu/ml out of 5.5 million tested. there was a seasonal accumulation during spring and summer, whereas a large epidemic occurred throughout the last year. vp2 igg was detected in 90.9% and 74% of the case and control group, respectively (p = 0.01). these data demonstrated statistically significance (p = 0.01). all donor samples which were b19 nat positive for more than three months developed neutralizing vp2 antibodies. in contrast, ns1 antibodies were observed in 83% of the case group and in 15% of the control group (p < 0.01). ns1 antibodies were detected more frequently in samples, which were b19 nat positive for more than six months. conclusion: b19 nat could be implemented in blood donor screening as release criterion without causing a shortage in blood supply. all b19 positive donors of the case group developed neutralizing antibodies within three months and virus load was dropped rapidly below 105 iu/ml. these data support our testing algorithm all components of high positive donations (virus load over 105 iu/ml) were discarded. donors with ns1 antibodies showed more often signs of a chronic disease with detectable levels of parvovirus b19 longer than six months. background: on recent years, the syphilis screening of blood donors has become increasingly important not only because of the transmission risk of this infection but also due to the risk behavior that this implies. on account of the importance of this screening the tests used are becoming more and more sensitive. aims: to evaluate an elisa screening test (the tmpa test recombinant is based on the sandwich principle, an immunoenzymatic technology in solid phase, for the measure of anti-treponema pallidum in serum or plasma). methods: in this study 1696 samples from blood donors were tested by the rotine 'cardiolipidic reagent for syphilis screening on microplates' -diagast laboratories as well as with 'hdtmpa recombinant' -hoslab diagnostics. positive samples were then confirmed with fta abs/tpha. results: using the mentioned tests we obtained the following results: 1. 1666 (98.23%)cases turned out negative with both technologies; 2. 3 (0.17%) cases were positive in both methods; 3. 27 cases were positive only using tmpa recombinant [of which 23 (1.36%) were confirmed positive by tpha/fta abs. as seen we found 23 samples (1.36%) that were only positives by tmpa recombinant test and that were confirmed by tpha/fta abs. we concluded that tmpa recombinant seems to be a suitable test for a quick and automated syphilis screening of blood donors and provides maximum safety for the recipients. background: in recent years, there has been substantial evidence indicating that typing and subtyping for hcv is clinically important in understanding hcv disease and its therapeutycal options. 'naive' viral load also seems to influence disease severity and responsiveness to therapy. therefore, viremia and genotype identification have been done routinely in molecular biology laboratory units. aims: the university hospital of coimbra studies and tests his own patients and patients from 11 other hospitals in the central portugal. we also collect and test blood donor candidates from this region. we proposed to analyse the distribution of hcv genotypes in this region, among 1578 patients with cronic hcv infection. we have simultaneously analysed the viremia and correlated it with age and severity of liver disease. methods: nucleic acid extraction was done using the semiautomatic 'xstractor' from biomerieux laboratories (boom method). the genotyping used reverse hybridization and was performed using probes from the 5¢ non-coding region (innulipa introduction: bacterial contamination of blood products remains a persistent problem. various techniques for the detection of bacteria in blood products exist but none of them has been widely accepted. bacterial detection systems could be divided into culture systems and rapid technologies. hemosystem has developed a rapid and sensitive technology for bacteria detection named scansystem tm . bacterial contamination of platelet concentrates is a rare event with an incidence between 1 : 2000 to 1 : 3000 per donation. therefore hemosystem developed a positive control in order to validate the scansystem tm platelet kit before use. aim of the study: the current study was designed to evaluate the performance of the scansystem tm positive control. the scansystem tm positive control is a capsule containing lyophilised lactobacillus casei subsp rhamnosus. the bacteria concentration per capsule is at least 8 ¥ 10 8 cfu. the positive control has to be stored at room temperature and is stable for 2 years. after dilution in pbs, the preparation has to be used within 1 hour. two capsules were tested for ten consecutive days with scansystem tm platelet kit as well as with optimised scansystem tm platelet kit. in an independent experiment three capsules were diluted in platelets stored in additive solution and were tested each with scansystem tm platelet kit and optimised scansystem tm platelet kit. results: 25 microscopic fields were analysed for bacteria specific fluorescence for each sample. the ratio between bacteria specific fluorescence signals and analysed signals was 1.0 in all samples for both scansystem tm platelet kit and optimized scansystem tm platelet kit. therefore by definition all tested capsules were positive. the lyophilized positive control capsules enable the user to validate the scansystem tm platelet kit before use. because bacterial contamination of platelet products occurs rarely, the routine use of positive controls improves safety of the screening method. scansystem tm is currently the only method that provides this safety measure. introduction: whereas implementation of nat for blood donor screening reduced the risk for transfusion transmitted hiv and hcv infections currently below one per 23 million transfusions, the risk for bacterial infections is estimated to be 1 : 200 to 1 : 3000. especially platelet products, which are stored at room temperature, are prone to bacterial contamination. aim of the study: several methods are currently developed to prevent the transfusion of bacterial contaminated platelet concentrates. the study investigates a new rapid bacterial detection method. material/methods: pool platelet concentrates were spiked with seven transfusion relevant bacteria strains under sterile conditions at concentrations of 100 cfu/ml to 106 cfu/ml. bacterial concentration was verified on blood agar plates immediately after spiking. five millilitres of spiked platelet concentrates were centrifuged, stained with thiazole orange dye and analysed directly by facs within five minutes after staining. aliquots of pool platelets spiked with concentration of 102 cfu/ml and 101 cfu/ml of each bacteria strain were incubated for two to eight hours in special bouillon at 37°c and were analysed by facs immediately after incubation. results: sensitivity of facs analysis differed between 103 cfu/ml for e. coli and 105 cfu/ml for klebsiella pneumoniae without preincubation and was enhanced to 101 cfu/ml when a pre-incubation step of two to four hours was included. conclusion: bacteria detection by facs analysis combined with a short pre-incubation (2-4 h) at 37°c is a quick and simple method with sensitivity comparable to other commercially available detection systems. the advantage of this new method is the rapid analysis, easy handling, high sensitivity and less expensive price. introduction: detection of bacterial contamination of platelet concentrates represents a major challenge in transfusion medicine. for blood transfusion services the method must have a high sensitivity, an easy performance and a low price. aim of the study: in this spiking study we evaluated the new optimised scansystem tm platelet kit detection method for use in apheresis platelets. methods: apheresis platelet concentrates (apcs) were spiked with strains of ten different bacteria species. after different incubation periods, apcs spiked with 10 cfu/ml were analysed by the optimised scansystem tm platelet kit. the number of bacteria was monitored by plating on blood agar. results: all bacteria strains were detected with the optimised scansystem tm platelet kit when the sample was collected 24 h after spiking. identity of the spiked bacteria was confirmed by gram staining and dna fingerprints. conclusion: in summary, the optimised scansystem tm platelet kit was able to reliably detect ten transfusion relevant bacteria species in apheresis platelet concentrates within 70 minutes when the sample was taken 24 hours after spiking. background: since year 2000 our laboratory started routine screening of hcv-rna in plasma minipools for all plasma intended for fractionation. although nat testing is not yet mandatory all blood products are released depending upon nat results. aim: to test and compare two different methods of rna extraction in order to make all the necessary adjustments to the test procedures while preserving the availability of blood products. methods: plasma minipools of 24 donations are prepared either on a tecan genesis robot or on a hamilton at plus. hcv-rna is isolated from 2 ml plasma by using either the qiagen biorobot 9604 and qiamp virus biorobot 960 kit or the manual extraction with cobas ampliscreen hcv pcr kit v 2.0. results: between march 2000 and december 2004 a total of 238 000 seronegative donations (9916 pools) were tested for the presence of hcv-rna. four pools were found to be positive for hcv-rna. of the four nat-positive pools with no eia-positive donor, four were confirmed as true positive by donor follow-up testing and/or testing of an independent sample from the index donation. all the positive donations were detected independently of the extraction method used (manual or automated). our experience shows that although the automated extraction method is 'off label' and it has to be validated, the use of biorobot 9604 does not pose a detectable contamination risk and it is possible to achieve a detection level for hcv less than 50 iu/ml. the advantage of the manual method is that it has better recovery of nucleic acids than the qiagen extraction. concerning the time needed for the extraction process the automated method runs 96 samples in 2 hours where the manual method needs 3 hours for 24 samples, needing, prior to extraction, an extra centrifugation step for one hour. the automated extraction method results in an assay with a high sample throughput, fast time, sufficiently sensitive, that can be successfully introduced into routine use in laboratories which have more than 800 samples/day while preserving the availability of blood products. anti-hcv similarly was high till 2002 (0.5-0.8%), but in trend to decrease afterwards (0.6%). anti-hiv reflected the low endemicity of the disease in public setting and was 0% through the mentioned years. rpr test for syphilis was around 0.1%. directed donors were 95% of all and volunteer donors consisted nearly 5%. donors in our blood center are being informed about donation prior to giving their blood and donor questionnaire forms (dqf) are filled out by the donor candidates. using dqfs have been mandatory at all blood banks in turkey by law since 1996. from that time infectious disease marker rates were dramatically reduced at all centers. donor information about the risks of transfusion and the importance of safe blood supply were detailed by the donation staff and physicians, consequently self-exclusion by the donor candidates who have risky behaviors was encouraged at our center. the interviewing staff was trained specifically for this topic. this steps were particularly emphasized in the last three years and the infectious screening results were displayed the outcome of this efforts. conclusion: education of the prospective donors, and recruit the voluntary, non-remunerated and regular donors will be the utmost goal of all blood banks. rigorous donor selection will contribute this ultimate success. we should spend more efforts to maximize enrolling voluntary donors to lower the serological marker results, consequently achieve safe blood. background: human t cell lymphotropic virus type i is endemic in japan, the caribbean, southeastern united states and parts of south america and africa. in non-endemic areas such as europe, htlv-i is less common and most infections are identified in immigrants. the epidemiology of htlv-ii is different, being predominantly found among indigenous american-indian populations and among ivdus, but the routes of transmission are the same. aim: our study's aim was to ascertain the prevalence of htlv i/ii in blood donors in order to understand the epidemiology of htlv in greece and initiate discussions of an acceptable level of risk and appropriate level of screening for rare transfusion-transmitted diseases. overall, anti-htlv seroprevalence levels among blood donors, are low. although the number of annual donations in this study is relatively small, the data for htlv indicate that rates of this infection are low and that infected donors will be seen infrequently. as all blood donations are screened for htlv i/ii during the last six years, a national survey is necessary in order to define the epidemiology of htlv in greece. introduction: toxoplasma gondii is the causative organism of toxoplasmosis. the disease transmitted by ingestion of either oocysts (in the feces of cats) or bradyzoites (in raw or undercooked meat). the parasite can also be acquired transplacentally by organ transplantation or from blood transfusion. the purpose of this study was survey of toxoplasma antibodies in some iranian blood donors at tehran blood center. blood samples were randomly collected for detecting of igg and igm antibodies (by elisa technique).the total numbers of donors was 217 of 52 (%24) were female and 165 (%76) male in age ranged from 18 to 55 years. results: 217 sera tested, 128 (59%) were found to be positive for toxoplasma igg antibodies and 6 (2.8%) were igm antibodies positive and 2 of them (0.9%) were borderline for igm antibodies. among males the frequency of positivity was higher than woman but this different was not significant. the most frequency of seropositivity was found in age group 41 to 50 years. conclusions: diagnosis of toxoplasmosis can be aided by serologic or histocytologic examination. the acute infection in healthy individuals is generally asymptomatic and not associated with any morbidity but in an immunocompromised host, toxoplasmosis be a very serious disease, and this can occur if a person is infection with toxoplasmosis before or after his/her immunosystem is compromised. in spite of the progress in the development of diagnostic, therapeutic and prophylactic methods, virus hepatitis still presents a serious global health problem. the possibility of transmission of these infections through transfusion of blood and blood derivates implies obligatory control of the donated blood. post-transfusion hepatitis is an important health problem in everyday practice, especially in patients who have to receive transfusion of erythrocyte concentrates as the only possible treatment for many years. objective: to show the prevalence of hepatitis b (hbsag) and hepatitis c (anti hcv antibodies) in multitransfused thalassemic patients. in our region there are 5 patients suffering from thalassemia major who are aged between 9 and 17, and who have been receiving erythrocytic transfusion 1-2 times a month since the age of one or two. they receive washed red blood cells, and in certain periods filtered red blood cells, controlled for viral markers and they mostly receive blood from voluntary, periodic and regular donors. the patients are tested periodically for the presence of viral markers (hbsag, anti hcv antibodies), using tests for hbsag (abbott auxyme monoclonal eia) and for anti hcv (abbott hcv eia 3.0). the presence of markers for hepatitis b and hepatitis c has not been detected in any of these multitransfused thalassemic patients who receive at least 20 transfusions a year. the tests in all 5 patients were negative. the blood used for transfusion must be tested for viral markers, and for the patients who have to receive blood for their whole life, the blood should be from voluntary, regular and periodic donors who donate blood at least three times a year, because then the risk of transfusion transmissible infections is very small. introduction: we observe yearly the prevalence of transfusion transmitted diseases following instructions of skae (national coordination haemovigilance centre). aims: to investigate the prevalence of the most important blood borne infections in our blood transfusion centre in the state achaia during the last five years (2000) (2001) (2002) (2003) (2004) . materials and methods: the detection of hbsag, anti-hcv, anti-hiv 1/2 was made by automated microparticle enzyme immunoassay (axsym, abbott) and by enzyme-linked immunoassay methods (dade behring, ortho, biomerieux) syphilis tests were made by using rpr kits. confirmation for anti-hcv positive samples was made riba or inno-lia, while the confirmation of anti-hiv1/2 positive samples was made by 'st. andrews' general hospital of patras reference centre. results: all the seropositive donors were first time donors. conclusion: (1) we observe that there is a decrease in all four infections. (2) the absence of anti-hiv seropositive donors is due to the high percentage of volunteer blood donation which approaches 70% in our centre during the last four years. methods: a prospective, one-year study has been set up in order to enrol at least 40 000 out of the estimate of 200 000 first-time donors, involving 21 blood transfusion centres from 9 of the 21 italian regions. each centre was required to enrol all first-time donors born before december 31st, 1978, and thus not included in the hbv mass vaccination campaign. the selected donors were tested for hbsag (mandatory by law) and for anti-hbc by commercial assays. all hbsag and/or anti-hbc positive specimens were stored frozen and sent to a reference laboratory for additional serological testing (anti-hbs, anti-hbe, anti-hbc/igm and anti-hbc avidity index by an experimental procedure) and for the determination of hbv-dna (both qualitative and quantitative) by real-time pcr. results: in the first 9 months of the study the 21 sites saw almost 29 000 first-time donors, of whom 70.5% belonged to the required age groups. among eligible donors, 0.4% were both hbsag and anti-hbc positive, and 10.3% were hbsag negative/anti-hbc positive. hbv positivity rates were higher in southern than in northern regions, although a high variability in hbv prevalence was observed between neighbouring areas in the north. hbsag positives were mostly males, 93% were positive for anti-hbe, 6% had raised alt and 8% were concurrently positive for anti-hbs. among hbsag negative/anti-hbc positive donors, 18% were negative and 82% were positive for anti-hbs. among anti-hbs positives, 35% showed values <100 miu/ml and 65% > 100 miu/ml. the avidity index results suggested that approximately 30% of anti-hbc positive individuals were recently infected. conclusions: our preliminary data indicate that approximately 70% of the italian first-time donors are older than 28 years of age and thus not belonging to the age groups who underwent to the mandatory vaccination against hbv, and that 10.7% of them have serological markers of ongoing or past hbv infection. anti-hbc alone was detected in nearly 2% of the study population. hbv-dna testing is underway at the time of this writing. in our country mandatory tests for each blood donations are: hbsag, anti-hcv, anti-hiv1/2 and tp ab. to c + ns3 + ns4, 9 (18.4%) to c + ns3, 4 (8.2%) to c + ns3 + ns5, 2 (4%) to ns3 + ns4 + ns5, 2 (4%) to c + ns4 + ns5, 2 (4%) to ns3 + ns5. the use of the hcv core ag elisa test system may provide substantially earlier identification of hcv infection than it is possible with current serological assays. although all of six anti-hcv assays are very sensitive and specific screening assays, they didn't detect hcv infection in one patient. majority of anti-hcv positive patients (77.5%) had anti-hcv ab for 3 or more different epitopes of hcv. international comparison of performance of abbott prism assays used for blood donor screening background: the national serology reference laboratory, australia (nrl), coordinates a quality control (qc) programme for laboratories that screen for anti-hiv 1&2, anti-hcv, hbsag and anti-htlv i/ii using the abbott prism assays. nineteen laboratories from australia, belgium, canada, ireland, israel, the netherlands, new zealand, norway, singapore, south africa and thailand have submitted data for this programme. aims: to determine the accuracy and precision of results from laboratories, individual prism instruments and different reagent lots by analysing data accumulated between october 2001 and january 2005. the multi-marker qc sample 'pelispy s2058 type 7' (s2058), produced by viral quality control (now acrometrix-viral quality control), was provided to participants. laboratories tested s2058 in each calibration run, in addition to the manufacturer's controls, on each sub-channel of the instrument. pelispy was used as a 'go/nogo control' and results were required to be reactive (s/co > 1) for a test run to be deemed valid. data were collected and analysed using the nrl's internet-based application edcnet (https://www.nrlqa.net). after submission, laboratories were able to compare their results with those submitted by other laboratories and investigate differences in results from reagent lots and instruments. data for five different s2058 lots were exported from edcnet and analysed. results: nearly 95 000 results were submitted: all results were reactive (s/co > 1). fifty of these results (0.0005%) were excluded from analyses because they were reported from invalid test runs [due to pipetting, aspiration or sampling error (n = 48) or due to unacceptable results (n = 2)]. a further 16 results were excluded because data provided by laboratories were inconsistent or incorrect. a total of 94 189 results, reported using 332 different prism reagent lots (145 for anti-hiv, 79 for anti-hcv, 55 for anti-htlv and 53 for hbsag), were analysed. results from prism hbsag and anti-hiv showed the least variation with coefficient of variations (cv) of <10% for all s2058 lots. results from prism anti-hcv and anti-htlv produced cvs between 9.17% and 15.38% for all s2058 lots. data reported for s2058 lot ps030618 (n = 38 662, range 8080 for anti-htlv to 10 225 for hbsag) were analysed further to review performance of prism reagent lots. hbsag showed the least variability between prism reagent lots with <8% bias for the 14 prism hbsag reagent lots used (bias: the difference between the mean ratio for the reagent lot and the weighted mean ratio for all reagent lots, expressed as a percentage of the weighted mean ratio for all reagent lots). prism anti-htlv showed greater variability between reagent lots with a single reagent lot generating a +63% bias. prism anti-hcv showed the greatest variability within reagent lot with results from 12 of 21 reagent lots showing a cv between 10% and 13%. conclusion: in 94 255 results in a qc sample distributed to 19 laboratories the abbott prism performance was found to be consistent over four assays. edcnet was robust in supporting laboratories' abilities to follow precision and accuracy of the assays in real time. introduction: since hcv rna testing of all blood donors started in finland in 2000, the nat screening process has continuously been improved. investments in process automation have made the work more efficient and blood safety has further increased since hiv-1 rna screening of all blood donors started late 2004. aim of the study: to implement hiv-1 rna testing in the nat screening program cost effectively, without increasing the throughput time of the samples and delay in the result reporting. to study if the sensitivity for both hiv-1 rna and hcv-rna will be sufficient when a single extraction is used and when the pool size of 96 donations and the sample volume are kept unchanged. methods: the nucleic acids were isolated from plasma samples of 1 ml with the magna pure lc instrument using the magna pure lc total nucleic acid isolation large volume kit. the internal controls from the cobas ampliscreen multiprep specimen preparation and control kit and the cobas amplicor hcv specimen preparation kit were added to the lysis/binding buffer (5 ml/ml of each). from the final volume of the nucleic acid eluate (100 ml) 50 ml was used for the detection of hiv-1 rna (roche cobas ampliscreen) and 30 ml for the detection of hcv rna (roche cobas amplicor). a run control containing both hiv-1 rna (200 iu/ml) and hcv rna (50 iu/ml) was included in all extraction runs. sensitivity of the both assays was assessed by testing dilution series of the who standards for hiv-1 rna and hcv rna. specificity was evaluated by testing fractionation plasmapool samples (n = 35) and minipool samples (n = 68). results: detection limits of the hiv-1 and hcv assays (95% hit rate) were calculated to be 100.1 iu/ml and 18.7 iu/ml respectively. specificity for both assays was 100% and during the validation phase also the robustness was good. the sensitivity of both assays with a pool size of 96 was below the recommendations by the council of europe for blood donor screening (for hiv-1 rna 10 000 iu/ml and for hcv 5000 iu/ml per individual donation). specificity of the assays was excellent, false reactive results were not observed. implementation of the hiv-1 nat assay in the screening program did not increase the throughput time of the donor samples when the pool size of 96 donations, 1 ml sample volume and a single extraction for two assays were used. the the very substantial increase in the number of industry-sponsored clinical trials has created challenges for medical schools, academic hospitals, faculty members of these institutions, and the journals that publish the results of these trials. in many cases, authors of reports of industry-sponsored clinical trials are paid consultants to the sponsor, have been paid by the sponsor to lecture on behalf of its products, or have equity in the sponsoring company. these ties to industry create a tension that actually is or can be perceived as • work internationally; • send young volunteers to international youth forums; • employ young people in your organisation; why use volunteers in blood donor recruitment? • they have networks to scout-groups, sports-organizations, tradeunions, rotary, staff of large companies etc. • they bring in fellow volunteers -with different prospects of society; • often paid recruiters are underpaid (!) and tend not to remain • you can not recruit by telephone! • 2 out of 3 donors are recruited by personal contact! • so you need direct personal contact = need many people (e.g. young ambassadors); • a large number of volunteer recruiters is a gift from heaven! paid donation gives the act of blood donation low status. the act of blood donation should be respected, and praised by role models, queens and presidents. efficient work and close cooperation of blood bank staff and volunteer organisations is the key to success in blood donor recruitment and retention! with such a prospect, it was important to evaluate the practices of blood donor selection in the eu. material and methods: a questionnaire was designed and sent in 2003 to the relevant institutions of the 15 eu countries plus switzerland. the questionnaire included questions on the interviewing practices before homologous blood donations, regarding non-specific risks to donors and recipients, identified risks to donors, infectious, bacterial, viral, parasitic and prionic risks to recipients and non-infectious risks to recipients. the questionnaire also inquired about each country's exclusion period for each contraindication (ci) to donation. results: predonation interviews were prepared, in all countries, by circulating informative documents to blood donors. they were supported by written questionnaires in nearly all countries. in half of the countries, those interviews had to be led by physicians (nurses or technologists in the others). the 18-65 age limits for blood donation (18-60 for a first donation) were the rule in 11 countries. in other countries the age limit could be brought forward to 17 and extended to 70 years old. the time interval between donations was identical for men and women in 11 countries, and varied from 8 to 16 weeks according to country. the questions of the questionnaires were very similar as regards the identification of risks to donors and recipients, and very close to the requirements that appeared later in the 2004/33/ec directive. this particularly concerned how to meet the expectations of european donors, so that they come back to your blood center! know your donors: make regular donor surveys. age, gender, number of donations, number of first time donors, media consumption, education, job situation, income brackets. use local donor organisations let volunteers help! they work for free, but donor recruitment and -retention costs money. each blood center should have a local donor organisation, run by volunteers. the donor organisation should receive a payment for each bag collected. the reputation of the blood system tell the donors, what the blood is used for. that all blood is tested. and that blood provides safe medical treatment safety of the donor. insurance is a must good quality and efficiency in blood services decentralized blood collection no waste, and minimal outdating efficient service: • a friendly environment, • donor friendly opening hours, • pleasant rooms, beds and well equipped waiting rooms, • parking-spaces, transport, • beverages and food, • letters with correct data etc. donors expect to be serviced by trained, medical professionals and that the medical check-up is taken seriously. donors should be recognized continuously. use directed press-coverage to higher the self-esteem of the donors. donors should be well informed: • leaflets, posters and questionnaires should be 100% correct; • use e-mail and web-sites for quick up-date of donor information. be visible! • have an offensive and comprehensive media approach; • have a yearly national campaign 14 june up to world blood donor day; • have an attractive home-page, constantly updated; • streamline your lay-out; • mail a donor magazine to all regular donors: • send newsletters regularly to volunteers and the press. • use recruitment cards. • easy phone-and fax numbers, e-mail addresses. directed campaign towards young people: • young ambassadors group; • special training sessions for young volunteers; • advertisements in media catering to young people; • poster competitions; • book and leaflets on blood addressed directly to young people; minimum bodyweight, blood pressure, pulse limits, questions involving viral risks, either sexually transmitted or linked to drug abuse, questions investigating risks of malaria transmission, questions aimed at identifying risk of prionic disease transmission. analyzing ineligibility times, on the other hand, revealed wide differences. for example, ineligibility for current multiple sexual partners, sexual relations with risk individuals, tattooing or body piercing, endoscopy, general anesthesia or invasive surgery, could vary from 3 to 12 months. previous transfusion history could not be a ci or could be one varying from 6 months to indefinite ci (this point recently changed in several countries). the results of that survey have revealed some differences between countries in the questions asked and especially in the ineligibility times. however, the conditions under which donor selection interviews are conducted were similar in all countries. the enquiry tool used in this study proved to be well adapted to evaluate the donor selection practices throughout eu. a next step will be to use it to appreciate their evolutions and especially the impact of the 2004/ec/33 directive on these practices in the 25 eu countries and furthermore to evaluate the results of this selection (rates and motives of deferral) which is a major factor of patients' transfusional safety. background: in europe on average 43 whole-blood donations are performed per 1000 inhabitants and year. whole-blood donation comprises a puncture of a venous vessel and letting of blood (usually 450 ml), which may be repeated several times a year. like other invasive procedures, blood donation has a range of effects on the individual who is subjected to it. aim: the aim of this paper is to review some aspects of the present state of knowledge on effects and complications of whole-blood donations. results: most studies of the effects of whole-blood donations on the donor have focused on negative or unpleasant events and on time in rather close association to the donation. complications related to percutaneous needle insertion (bruise assessed by inspection 0.66% -bruise assessed using post-donation interview 22.7%, sore arm 0-10.0%, nerve irritation 0.02%, arterial puncture/pseudoaneurysm/arteriovenous fistula 0.003-0.009% etc) are most commonly reported, while negative systemic reactions (vasovagal reactions 2.5-7.8%, syncope 0.08-0.34%) occurring in connection to blood donation are less frequent (newman bh: blood donor complications after whole-blood donation. curr opin hematol 2004; 11: 339-345.) serious complications requiring hospitalization (myocardial infarction, stroke) are extremely rare (0.0005%). fatigue (7.8%-10.0%) and diminished physical working capacity (7.0%) are reported to occur during days after the donation. a recent study of a consecutive sample of 528 swedish blood donors (nilsson sojka b, sojka p: the blood-donation experience: perceived physical, psychological and social impact of blood donation on the donor. vox sang 2003; 84: 120-128.) (with a selfadministered questionnaire with an open-ended question: how does blood donation affect you? physically-bodily/psychologicallyspiritually/ethically-morally/socially during or after blood donation?) revealed that perceived negative effects (fatigue, diminished physical working capacity, vertigo/dizziness, susceptibility to infections etc) were less common (25% of the donors) than positive effects (feeling of satisfaction, being more alert, feeling generally better, less migraine, higher physical working capacity, respect from environment, feeling of relaxation etc; 35% of the donors). the duration of positive effects was regularly reported to be weeks, while negative effects lasted only days. investigations on the long-term effects of blood donation are scarce. yet, they may indicate that the donation of blood is associated with e.g. lower blood pressure and a reduced risk of myocardial infarction (nilsson sojka b, sojka p: the blood-donation experience: perceived physical, psychological and social impact of blood donation on the donor. vox sang 2003; 84: 120-128). conclusion: both the panorama and the frequency of occurrence of the different effects and complications of whole-blood donations vary as a function of how the information was gathered (openended questions, observations, interviews etc). serious reactions to whole-blood donations are extremely rare. more studies are needed in particular with respect to the long-term effects of regular wholeblood donations. t-pa-056 establishing a national adverse event reporting system for blood donors -a prospective study of 1. there was no national system and significant regional variation showed that the data was scientifically unsound. a coincidental initiative to remove the mandatory post donation rest period for regular donors further emphasised the lack of reliable, retrospective data to monitor and compare the impact of this new policy. aims: 1. to develop and implement a high quality, reliable national donor adverse events reporting (daer) system; 2. to define and categorise adverse events; 3. to record data systematically and prospectively using the existing computerised donor database. methods: from summer 2002, a small project team of senior clinical and operational staff took 9 months to agree a detailed policy for capturing and recording all donor adverse events, including precise definitions for grades of vasovagal reactions and bruising. detailed training material was written in may 2003 and the new protocol was validated in one region. from july to december a formal one day training programme was delivered to over 3000 staff working on 98 mobile collection teams, 19 static sites and 11 blood centres. daer was fully implemented by january 2004. adverse events are assessed by health care professionals on session and the relevant code entered onto the donor's computer record by clerical staff. information received after the session is entered by centre based doctors using the same system. the database is interrogated monthly for statistics. results: in the first 9 months 1.86 million donors attended sessions throughout england and north wales. results were very consistent month on month. 25 175 donors (1 : 75) had vasovagal symptoms but only 14% of these suffered syncope. 71% of all vasovagal reactions occurred in women. 21% occurred in donors aged 17-20 and a further 30% in donors aged 21-30. (donors aged 17-30 represent only 25% of the total donor base.) 831 donors (1 : 2250) reported a delayed reaction, 52% of whom did lose consciousness. nerve injury, unrelated to haematoma, occurred in 109 donors (1 : 17 000) and, more rarely, arterial puncture was diagnosed in 46 donors (1 : 40 000). bruising was reported after the session by 1 : 1300 donors. summary: a robust system has been developed and successfully implemented across a large, national blood service. based on the data already accumulated our next phase is to develop strategies to minimise adverse events, confident that any intervention will be effectively monitored. the community role in enhancement of voluntary, non-remunerated blood donation in the new millennium introduction: in the developing countries, about 80% of the blood supply comes from paid or replacement donors, where a high number of infected persons are in the donors population. only 16% of the global blood supply is donated as voluntary nonremunerated blood donors in countries with low and medium human development indices. and around 80% of the global blood population has access to only 20% of a safe blood supply. conclusion: blood is a national resource, it is the responsibility of governments through it's communities to ensure that the blood supply is safe and adequate to meet the needs of patients population and available to all who needs it. background: in response to a documented increase in the average age of donors, a survey was conducted to explore if young people had more unfavourable attitudes towards becoming blood donors. aim: to identify if the increasing difficulty in recruitment and retention of young people as donors, is linked to a low level of motivation for donating blood in this age group. methods: a national telephone-survey was conducted among a cross-sectional sample of the adult norwegian population (1000 participants). the survey was performed in november 2004. results: five percent reported being active donors (had donated during the last 12 months), 17% were passive donors (had not donated during the last 12 months), 22% were non-donors with a positive attitude towards becoming donors, and 55% non-donors with no intentions ever to donate blood. in the youngest age group (age 18-29), 1% reported being active donors and 8% were passive donors. however, 36% of the young non-donors reported having intentions of becoming a blood donor. fifty-five percent of young non-donors had a negative attitude towards ever donating blood. all non-donors were asked why they did not donate. thirty-six percent of all non-donors reported health related reasons for not donating blood. thirty-one percent of all non-donors claimed that they did not donate because no one had requested them to do so personally, and 4% reported they did not care about blood donation. in comparison only 28% of young non-donors reported medical reasons for not donating. thirty-eight percent claimed lack of personal request, and 7% reported of lack of interest as the main reason for not donating. summary/conclusion: although the youngest age group was under-represented among active donors, we found that a great proportion (36%) of young non-donors had a positive attitude towards becoming blood donors. the most important reason why young people do not donate was the lack of a personal request. indifference regarding donation was not very widespread. a relatively high proportion of young people considered themselves as not medically disqualified to donate. in light of these findings, efforts to recruit young people as blood donors are strongly recommended. background: the ever-increasing demand for blood, coupled with emerging new threats to blood safety, motivate the strengthening of the blood banking infrastructure. aim: employing new technology as an instrument for building relationships of trust between blood bank and blood donors. material: 1. a new software module supporting the management of magnetic cards was added to e-aima blood bank management application. the magnetic card supports the following information: • front-side: donor's photograph, surname, name and blood group (including rhesus phenotype & kell) and sign of blood collection centre. • magnetic stripe where data concerning donor's serial number, medical history (risk factors), test results for infections and the number of donations is stored. 2. specific hardware allowing reading from and writing to magnetic cards is integrated to the software module: • magnetic card scanners were added to pcs serving to donation collection, including laptop for mobile team collection. • web cameras to capture the photograph of the donor to be printed on the card. • card printer was deemed necessary to produce magnetic cards for donors on a need basis. 3. consumables: blank plastic magnetic cards, ink cartridges for printer. methods: in order to evaluate the performance of magnetic cards compared to the paper-based system, a questionnaire was distributed to 300 first-time and 200 repeat blood donors, in order to be used as an indicant of donor's satisfaction. 1. first-time donors increased 7.8% in the 6 months of application. among them, 4.3% were donors 'for relatives or friends' turned into volunteer donors and 3.5% were first-time volunteer donors. the questionnaire analysis further revealed: • 96% were motivated by the use of magnetic card. • 92% appreciated the presence of their photo on the card and they confessed that they had used it as a spill for recruiting their friends as donors. • 100% were persuaded that employing new technology would result in safer and more trustworthy procedures combined with reduced waiting time. • 100% considered magnetic cards more practical compared to paper cards because of their compact size and improved durability. 2. the turnover of repeat donors also increased 1.3% after replacing their plain old paper cards with new ones. further analysis revealed that: • 100% appreciated the quick cross-checking of donor's identity. • 100% were satisfied with the effectiveness and efficiency of magnetic cards in managing donor's data. conclusions: in 2004, greek health policy provided the legal basis for establishing the electronic national health card. the introduction of the national donor's magnetic card is another step towards this direction, being aligned with the modern national health strategy. apart from the positive impact on the number of both firsttime and repeat blood donors, it should be also pointed out that the use of a unique donor serial number on country level results in less error-prone procedures due to the reduction of administrative process overhead and facilitates interoperability between national blood banks using compatible technological infrastructure. t-pa-060 emerging technologies in transfusion. dna based assays until the late 1990s, mandatory blood screening for transmissible infectious agents depended entirely on antigen/antibody-based detection assays. recent emergence of nucleic acid technologies (nat) has revolutionized viral diagnosis by not only increasing the sensitivity level but also facilitating the detection of several viruses in parallel, by multiplexing specific primers. however, in more complex biological situations when a broad spectrum of pathogens must be screened, the limitations of these first generation technologies became apparent. high throughput systems such as dna arrays permit a conceptually new approach. these miniaturized microsystems allow the detection of hundreds of different targets simultaneously, inducing a dramatic decrease in reagent consumption, in additional confirmation tests and simplify data interpretation. however, the microsystems actually available require additional instrumentation and reagents for sample preparation and target-amplification prior to detection on the dna array. future technologies such as 'lab-on-a-chip' include channels, fluidics and thermal zones allowing extraction, amplification and detection. another major challenge in the area of dna detection is the development of methods that do not rely on target-amplification systems. almost all blood group antigens are bi-allelic and encoded by single nucleotide polymorphisms (snps). to facilitate the direct availability of typed red cells and platelets, we develop a high-throughput technique to genotype by dna microarray the whole donor cohort for all clinically relevant red cell and platelet antigens. methods: a multiplex pcr was developed to both amplify and fluorescently label 28 gene fragments of red cell and platelet antigens in one reaction. each array contains spots of short (17-27 nt) allelespecific oligonucleotides to discriminate between the two alleles of an antigen system. results: two blinded panels encompassing 94 donors were genotyped for hpa-1 through -5 and 15; no discrepancies were found. currently, arrays are prepared for the red cell systems. the fya/fyb, fy-gata1 mutation, jka/jkb, k/k, kpa/kpb, m/n, rhc/c, rhe/e, rhdpseudogen, rhdvi negative, rs, doa/dob, genotypes can be determined. the set up of genotyping assays for rare genotypes is difficult because of lack or insufficient amount of dna. the latter can be overcome by phi29 dna polymerase-mediated isothermal genomic dna amplification, from minute amounts of dna present in stored red cell fractions or antiserum. the results show that the blood group typing dna microarray will provide a reliable and fast genotyping procedure. the method can be further improved to obtain the necessary automated throughput for typing of large donor cohorts. and 15, all other weak d types should be regarded as potential anti-d immunizers. for correct determination of weak d both serological typing (polyclonal and monoclonal), as rhd dna typing are mandatory. when serology indicates weak d, more anti-d antibodies are tested (9 epitope model) to distinguish partial d from weak d. in addition, an rhd mpx pcr is performed to detect the presence of rhd exons 3, 4, 5, 6, 7 and 9. in all known weak d types, all six rhd specific exons are amplified (except for weak d type 4 which lacks rhd exons 4 and 5), whereas partial d phenotypes usually show aberrant patterns. aim: the aim of this study was to evaluate the diagnostic scheme for weak d typing. methods: between 2002 and 2004, 24 samples were investigated for weak d characteristics. four pcr-ssp assays were developed for identification of weak d types 1 (809t > g), 2 (1154g > c), 3 (8c > g) and 5 (446c > a). weak d type 4 was identified by the combination of serology and absence of exons 4 and 5 by rhd mpx pcr. rhd-specific exon sequencing was performed when serology and molecular typing were inconsistent. results: all 24 samples were subjected to the rhd mpx pcr and 1 sample showed absence of rhd exons 4 and 5, indicative of a weak d type 4 when combined with serology. the remaining 23 samples were analyzed by the weak d pcr-ssps, resulting in 12 weak d type 1 samples, 4 weak d type 2 samples, 4 weak d type 3 samples and 1 weak d type 5 sample. two samples remained undetermined and were sequenced for all 10 rhd exons and the rhd promotor region. one sample showed the mutations corresponding to the dau 2 partial d phenotype (209g>a, 998g>a and 1136c>t). the other sample had only one, not previously known mutation (1193a>t), which is located intracellularly at the coohtail. extensive serology using the 37 epitope model showed a pattern matching weak d. this new weak d variant was registered as weak d type 41. conclusions: based on these results it may be concluded that weak d phenotypes should be confirmed on molecular level to avoid misinterpretation of partial d that cannot be detected by rhd mpx pcr analyses. patients with weak d phenotypes, except for types 1, 2 and 3 should be regarded as being at risk for anti-d immunization after transfusion of rhd-positive blood products and should therefore be treated with rhd-negative bloodproducts. in this evaluation, 4 out of 24 patients carried such alleles. introduction: although kell antigens are expressed very early during erythropoiesis and a 0.1% incidence of anti-kel1 is found in obstetric patients, this is a relatively rare cause of hdn. anemia is produced by immune destruction of fetal rbcs and suppression of erythropoiesis. maternal antibody titers or amniotic/cord blood bilirubin levels are not relevant indicators of the severity of the disease, and the measurement of the fetal haemoglobin by cordocentesis is a procedure with risks of miscarriage and sensitization. pcr techniques for the determination of blood groups using fetal dna isolated from maternal plasma, allows the application of noninvasive methods. clinical cases: we describe two cases of pregnancies in women with anti-kel1 acquired by transfusion/previous pregnancies: 1st case: in july 2000, a 30-year-old woman (gravida 2, para 0), rhdnegative, kel1-negative was referred at 20 weeks gestation. the father's phenotype was rhd-positive, kel1-positive. a maternal antibody screen revealed d and kel1 alloantibodies. dna was extracted from amniotic liquid. the kel genotype was determined by pcr-rflp using the bsm i. pcr-ssp was used to studied intron 4 and exon 7 of the rhd gene. the results showed that the fetus was positive for rhd sequences and showed kel2 homozygosity; 2nd case: in august 2004, a 36-year-old woman (gravida 2, para 1) was referred at 28 weeks gestation. she had a history of transfusion with 2 rbcs units in 1996 -one of the donors was kel1positive. the woman typed rhd-negative and her husband typed rhd-positive. rbcs from both were kel1-negative. the maternal antibody screen revealed anti-kel1. doubts existed about the putative father of this child. dna was extracted from maternal plasma using the magna pure lc (roche). real-time pcr was applied to analyse: sequences of intron 4, exons 4, 6, 7 and pseudogene of the rhd gene and the sry gene by sybr green; and the alleles kel1/kel2 by hybridization probes. all rhd sequences were detected (with the exception of the pseudogene) and the kel genotype gave a kel2/kel2 result. in both cases the doctors choose not to use any invasive method to monitoring the fetuses regarding a hdn due to anti-kell antibodies, and the results of the molecular analysis were confirmed by testing the cord rbcs after birth. discussion: these cases illustrate the reliability of the molecular biology results, based on the collection of simple peripheral blood samples. a determination that the fetus lacks the relevant antigen obviates the need for expensive and invasive monitoring throughout the pregnancy. evidence-based medicine (ebm) is defined as: 'the conscientious, explicit and judicious use of current best evidence in making decisions about the care of individual patients. the practice of ebm requires the integration of individual clinical expertise with the best available external clinical evidence from systematic research and our patient's unique values and circumstances. ' otherwise healthy individuals without cardiopulmonary dysfunction (cdf) tolerate acute reduction of haemoglobin concentration to about 5 g/dl, provided that blood volume is kept normal by a volume expander. however, individuals experience physical fatigue, and there is faint reduction of perception as measured by neurophysiological tests. symptoms are reversed upon retransfusion of fresh, autologous erythrocytes. acute, normovolemic anemia seems to be progressively less tolerated with increasing age and cdf. controversy has existed on whether or not to correct hypoalbuminemia in asb or icp by infusion of albumin. recently a large trial showed no outcome differences between icp patients treated with albumin or saline. thus in general there is no indication for albumin in asb or icp. however, albumin may yet be advantageous in e.g. patients with head injuries. furthermore, fractionated albumin is not equivalent to native albumin, since fractionation stabilizers remain bound to the albumin molecule. thus more refined albumin preparations may carry advantages still to be investigated. erythrocytes are given to increase the total oxygen transportation capacity of the organism. the effect of blood bank stored erythrocytes may differ from that of fresh, autologous erythrocytes, since changes of presumably important erythrocyte properties occur during storage. in the only large trial available, a transfusion trigger of 7.0 g/dl was found to be favourable to one of 10 g/dl in icp, except possibly in icp with unstable angina pectoris or heart infarction. however, the erythrocyte concentrates given were not leukocyte filtered, and side effects of infused leukocytes may have hampered the conclusion. on the other hand, a metanalysis showed transfusion as an independent indicator of unfavourable outcome in coronary bypass patients, but again, leukocyte filtered erythrocyte preparations were not applied. the effect on morbidity and mortality of 'top up' transfusions given e.g. to mobilize patients postoperatively has not been studied by trials, although this effect seems evident to many clinicians. grave anemia may reduce the haemostatic effect of thrombocytes because changes of blood rheology reduces the pressure forcing thrombocytes against the walls of small vessels. the transfusion trigger for thrombocytes in asb or icp remains to be established by clinical trials, however. the same applies for fresh frozen plasma, which is infused as a source of coagulation factors. on the other hand, the haemostatic effect of various fibrinolysis inhibitors is well established in asb and icp, but many clinicians appear hesitant to use them. another interesting haemostatic agent is recombinant fviia, the use of which to control asb in blunt trauma is supported by one well controlled clinical trial. evidence by systematic research is insufficient to decide what is optimal transfusion practice. the procurement of such evidence is one of the greatest current challenges to transfusion medicine research. . concerns about the transfusion-related complications, such as infections, tumour behaviour and immuno-modulatory effects, and the costs, necessitated a re-evaluation of the transfusion practice. aims: the goal of this study is to evaluate if a restrictive transfusion policy (hb transfusion trigger <4.5 mmol/l) reduces the amount of red cell transfusion compared to a liberal transfusion trigger (hb < 6.0 mmol/l) without a decrease in hrqol. because of concerns about the feasibility of this study early results were analysed and are presented in this abstract. material and methods: after a run in period of 3 months (hb transfusion trigger of hb < 6.0 mmol/l) patients are randomised for the restrictive or the liberal transfusion policy. patients are followed then 12 months. hrqol is measured after inclusion, after randomisation, 6 weeks, 3, 6, 9, and 12 months after randomisation. also anaemia related complications and red cell antibodies are scored. hb values were blinded for the patients during the study period. results: from july 2002 till june 2004 15 patients were included (4 ra, 5 rars, 4 rcmd, 1 raeb, 1 cmmol) in 2 general hospitals and 1 university hospital. two patients died in the run in period. eight patients were randomised for the restrictive transfusion policy and 5 patients for the liberal transfusion policy. the mean follow up period in the liberal group was 6.2 months (inclusive run in period) and 7.4 months for the restrictive group. two patients in the liberal group died after randomisation. one patient received growth factors. in the restrictive group 2 patients finished the study, 1 received growth factors and 1 patient withdrew informed consent. the mean hb level was lower in the restrictive group and after randomisation about 55% reduction in amount of transfused red cells was found (1.8 units per pt per month in the liberal group vs 0.8 in the restrictive group). no anaemia related complications were found, e.g. cardiac failure and cerebro vascular ischemia nor a decrease in activity performance. conclusion: there were some concerns after introduction of the restrictive transfusion policy. this preliminary results show, however, that a restrictive transfusion policy leads to a diminished use of red cell transfusion without an increase of cardiac complications or a decrease in activity performance. this study will be continued to compare hrqol scores in both groups. introduction: strong evidence supports the efficacy of blood conservation strategies such as autologous blood donation (abd) and erythropoietin (epo) for reducing exposure to allogeneic blood. however, use of these interventions is highly variable among institutions and frequently sub-optimal. the program to reduce orthopedic blood exposure (probe) evaluated a blood conservation program in patients undergoing total hip joint arthroplasty (thja) at ontario hospitals. aim of the study: the objective of probe was to determine whether a comprehensive blood conservation algorithm (bca) was more effective than usual care (uc) for reducing exposure to allogeneic blood in patients undergoing thja. methods: we randomized 30 hospitals that perform high volume elective primary thja to implement either a bca or to continue with uc. the bca consisted of three components: physician and patient education, blood conservation interventions (use of abd or epo), and transfusion guidelines. t-pa-071 table) . mortality for non-transfused patients was significantly lower than for patients receiving either lr-or s-prbc at all time points (p < 0.0001). t-pa-074 thalassaemia: the impact on blood transfusion services thalassaemia major is a genetically determined disease that causes severe chronic anaemia and further complications. it can be managed successfully in the vast majority of cases, so long as public health and other scientific and organizational infrastructures are adequate. although the progress achieved in the field of bone marrow transplantation and other disciplines promises cure of the genetic defect, regular blood transfusion from early childhood remains the cornerstone of treatment of patients with thalassaemia major. this presents national health authorities with the formidable task of assuring an adequate blood supply of high quality and safety for these patients and ensuring that it is transfused in the appropriate way. the basic principle in the modern management of thalassaemia patients is that of a global approach to care. within this approach, a standardized protocol for regular blood transfusions is a prerequisite for the patient's long survival and quality of life. if thalassaemia patients are not transfused effectively, the severe anaemia and over-expansion of bone marrow due to ineffective erythropoesis can lead to poor growth, bone deformities, organomegaly and impairment of normal physical activities. in countries or regions with large numbers of thalassaemic patients, the organizational and technical aspects of meeting their blood requirements represents a heavy additional workload for the blood transfusion services responsible for providing blood for this group of multi-transfused patients. the acquisition and preparation of blood, genotyping the patients' blood group (including at least rh, kell, kidd and duffy systems) preventing the transmission of infectious diseases and other transfusion associated complications, and assessing the patients' blood transfusion indices all have a tremendous impact on blood transfusion and treatment units. blood transfusion services are thus confronted with major challenges that can only be met if appropriate national transfusion policies are in place, both in the laboratory and the clinical setting of blood transfusion. the availability of safe blood is related to the effectiveness of donation programmes aimed at recruiting and retaining voluntary unpaid blood donors who are at low risk for the transmission of infectious diseases. sufficiency is further related to resources, organization and management of the blood transfusion service and continuous education of its staff. high technical standards for the transfused product and quality management systems are required to ensure that the product meets these requirements, as well as pre-transfusion, transfusion and haemovigilance systems and other more stringent quality measures in the whole chain of blood donation and transfusion. additional measures and continuous care are specifically required for the optimal transfusion therapy of the thalassaemic patient. the patients should be transfused with red cell concentrates, (rccs) preferably not more than one week's old and leucodepleted. other processes i.e washing of rccs, use of nutrient additive solutions, irradiation etc may be used to improve the quality and the safety of the transfused product, while other advances in red cell transfusion are expected to improve blood safety by preventing adverse reactions and reducing exposure of the patient to donor blood. should patients with thalassemia intermedia be regularly transfused? thalassemia major (tm) and thalassemia intermedia (ti) share mostly a common basic molecular mechanism, that is the reduced synthesis of the * globin chains. 1 the consequences of the resulting chronic hemolytic anemia are also common and include growth retardation, bone marrow expansion, extramedular hematopoiesis, splenomegaly, increased intestinal iron absorption, susceptibility to infections and hypercoagulability. what differentiates the two forms of the disease is the severity of the clinical phenotype, which in turn depends on a particularly heterogeneous molecular background and imposes diverse therapeutic strategies. the consequences of the genetic defect as well as the effect of the applied therapy seem to be mainly responsible for the clinical course of the disease. in untreated tm cases, the aforementioned consequences occur fast and patients die early in life mainly due to high output heart failure. over the past decades, the gradual adoption of the current transfusion and iron chelation strategies and the patients' compliance with this therapy have resulted in a significant improvement of survival, that according to recent statistics reaches 68% at age 35. this rate is even better in well-treated patients, almost 80% of whom survive at age 40. regular therapy extends survival mainly by preventing early development of cardiac complications. in addition, a multi-organ improvement is accomplished while patients' physical appearance is almost indistinguishable from that of the general population, hence permitting a normal social behavior with a high overall quality of life. bone marrow expansion and extramedular hematopoiesis are prevented; hepatosplenomegaly is substantially restricted and usually there is no need for splenectomy, while thromboembolic complications are rare and pulmonary hypertension is practically absent. patients with ti remain as a rule without regular therapy until a number of severe complications arise. the consequences of chronic anemia develop slowly compared to untreated tm cases and dominate patients' clinical picture usually by the third decade of life. at this time, all patients have developed hepatosplenomegaly and most of them have been splenectomized. bone marrow expansion results to bone deformities and fractures often occur. extramedular hemaotpoietic masses and bone deformities may lead to various complications depending on their bulk and location, such as neurological symptoms from masses arising in the paraspinal area or dyspnea from lung restriction. hypercoagulability, resulting from defects of native erythrocyte membrane phospholipids, together with the coexistent thrombocytosis in splenectomized patients lead to a wide spectrum of thromboembolic events. pulmonary involvement with respiratory dysfunction and hypoxemia as well as pulmonary hypertension leading to congestive heart failure are well documented in ti patients. nowadays, the beneficial effects of regular transfusion and chelation therapy in tm are beyond any doubt. the occasional application of transfusions in ti has a transient effect and does not seem to inhibit the consequences of chronic hypoxia. intensive and regular transfusion and chelation therapy in ti has proved effective in ameliorating the established complications such as spinal cord compression, hypercoagulability and pulmonary hypertension, without however reversing them. given the 30-year experience on intensive therapy in tm and the first encouraging data in ti, the earlier application of such treatment seems to be crucial in ti. the timing however of therapeutic intervention in ti in order to prevent anemia-related complications still remains an open issue that needs to be properly addressed. the impact of prestorage leucodepletion on the immediate transfusion adverse events of patients with thalassaemia major backround: regular blood transfusion therapy in patients with bthalassaemia major decreases the complications of anemia but it is associated to many immediate and delayed side effects. febrile non haemolytic transfusion reactions (fnhtr) are common complications due to alloimmunization of recipients against hla and/or specific antigens on donor's wbcs or to the accumulation during storage of biologic response modifiers (bmrs) that are directly pyrogenic or indirectly by stimulating recipients' white cells to produce pyrogenic mediators. post storage leucoreduction (lr) has reduced the fnhtr in these patients from 13% to 0.5% per unit. it is unknown whether introduction of prestorage leucodepletion (ld) has reduced the incidence of nhftr further. we analyzed the immediate transfusion reactions of 175 adult patients with b-thalassaemia major transfused with a total of 32.990 rbc units from january 1998 to december 2003. all units were fresh, stored less than 7 days. 11.950 units were lr-rbc, 8.370 units were ld-rbc and 12.670 were washed lr-rbc. results: the incidence of fnhtr and allergic reactions in patients receiving lr-rbc was 0.16% and 0.35%/per unit respectively, in those receiving ld-rbc was 0.10% and 0.36%/per unit respectively, while in those receiving washed lr-rbcs was 0.10% and 0.25%/per unit respectively. the relative risk (rr) of fnhrt and allergic reactions following transfusion of ld-rbc and washed lr-rbc compared to lr-rbc is shown in table. conclusion: prestorage leucodepletion and washing of rbcs reduced the risk of fnhrt in regularly transfused b-thalassaemia patients 1.6 times compared to poststorage filtration. these findings show that fnhtr after rbc transfusions are due not only to alloimmunization but also to accumulation of bmrs even in patients transfused with fresh rbcs. washing is as effective as prestorage leucodepletion in reducing fnhtr. prestorage leucodepletion has no effect on allergic reactions or other immediate adverse events in these patients. t-pa-078 viral inactivation/elimination of plasma derived medicinal products the safety of medicinal plasma products (mpps) relies on a whole range of measures from the quality of the source material to the release of the products after manufacturing under cgmp conditions. viral safety relies on careful donor selection, viral testing of the source material and viral inactivation and/or elimination during the manufacturing process. mpp manufacturing processes must include viral safety steps capable of inactivating, and/or eliminating, a large range of viruses covering the known blood borne viruses as well as anticipating possible future pathogens. it is recognized that one single step is often not sufficient to satisfy this requirement and manufacturing processes very often include two or even three complementary viral safety steps. very efficient methods have been implemented by manufacturers, for two decades, for the inactivation of major blood borne enveloped viruses (hiv, hcv and hbv). additional safety steps have also been introduced to provide a second step for enveloped viruses and to extend the efficacy to nonenveloped viruses (hav and parvovirus b19). pasteurisation (liquid heat treatment at 60°c) which has been historically used for viral inactivation of albumin solutions has been applied to some other plasma products. solvent-detergent (sd) treatment which is specific to enveloped viruses is used primarily for coagulation factors. since the introduction of sd-treated products, no hiv, hcv or hbv transmission has been reported. viral inactivation of coagulation factors can also be achieved using various conditions of dry-heating. acidic treatment is also an efficient means of inactivating viruses in igg products. nanofiltration using filters of less than 50 nm pore size was introduced in the early 1990s. this technique for viral elimination is based on the size of the agent and is independent of their resistance to other treatments. this property could be helpful in cases of new emerging pathogenic agents. new inactivation tech-niques are currently under development such as uv treatment or gamma irradiation with efficacy reported on enveloped as well as non-enveloped viruses. these new techniques can complement existing methods after careful validation that they do not have harmful effects on proteins in the product. the efficacy of existing techniques is well documented in controlled clinical studies and pharmacovigilance records. their application to each product is extensively validated at laboratory scale, according to international regulations and then carefully evaluated by health authorities. in this context, a recent european guideline established a viral risk assessment model to quantitatively estimate the theoretical safety margins of mpps, by taking into account the different safety measures, such as viral testing of plasma and the efficacy of viral inactivation/elimination steps. whilst technical limitations and some lack of scientific data lead to very conservative estimates, this model gives an overall assessment of the efficacy of the measures in the manufacture of a given product. developments in viral inactivation/elimination methods, in plasma testing as well as in evaluation procedures have together given mpps an excellent level of safety never previously achieved. to date the important safety measures needed to ensure a high safety margin to pooled plasma products are well understood by the plasma fractionation industry. safety nets rely on carefully done: donor selection to exclude high-risk donors, serological and nat viral testing of single donations and, pooled plasma testing using sensitive validated methods, and most particularly, efficient viral reduction treatments that must be validated and implemented at a large-scale following good manufacturing practices. over the last 25 years, successive key breakthrough in plasma product viral safety have included the use of solvent-detergent treatment to inactivate lipid-enveloped viruses, and nat testing of starting plasma pools and viral nanofiltration of products to reduce the risks associated to small non-enveloped viruses. the excellence of the system currently in place is illustrated by the demonstration that these safety barriers have virtually stopped the transmission of known viruses and avoided that of 'emerging' agents, such as west nile virus (wnv). however, multiple viral reduction treatments have generally decreased product recovery. in addition, although the implementation of viral reduction treatments have forced fractionators to introduce significant changes to product manufacturing methods, this period has been understandably followed by a period of relative conservatism of the plasma fractionation industry against further process changes. as time evolves and market dynamics changes struggle for improved economic balance of the plasma product industry is putting product recovery and diversified product portfolio at the forefront of r&d objectives. these developments in the plasma fractionation scene of the western world have been taking place in a context where many patients in the developing world are still treated with sub-standard, non-virally inactivated crude plasma fractions. in this specific area, one can expect that the developing world will bring innovative thoughts and take actions to find ways to improve the quality and safety of their own local plasma product supply. safety strategies adapted to the infrastructure and economy of less solvable countries may have to be considered. the intercept blood system for plasma uses a synthetic psoralen, amotosalen hcl, and long-wavelength ultraviolet light to photochemically inactivate a broad spectrum of bloodborne pathogens in plasma intended for transfusion (intercept plasma, i-ffp). phase 3 clinical trials have shown that i-ffp retains proteins necessary for hemostasis in the treatment of acquired and inherited coagulopathies, and in support of therapeutic plasma exchange for ttp. a prototype plasma processing set was used for the clinical trials. for commercialization, a new processing set has been developed to improve productivity. the prototype set accommodated approximately 250 ml of plasma, whereas the improved set accommodates up to 635 ml of plasma, resulting in up to three i-ffp doses per treatment. aims: this study was designed to characterize pro-and anti-thrombotic proteins in i-ffp prepared using the improved processing set. proteins of interest included components of the intrinsic and extrinsic coagulation cascade, the fibrinolytic pathway, the contact factor pathway, and the complement system, the vonwillebrand complex, endogenous inhibitors, and markers of thrombin generation. methods: six fresh jumbo (600 ml) apheresis plasma units, collected using the haemonetics pcs device (gambro), were photochemically treated. sodium citrate was used as the anticoagulant. plasma samples for analysis were collected before and after photochemical treatment, and were frozen below -60°c until batch analysis. standardized clinical assays were used for all analyses. results: (results in the table below are expressed as the percent activity in i-ffp in proportion to the activity in plasma before treatment [mean ± sd]). retention of procoagulant factors in i-ffp plasma ranged from 77% to 92%. factor viii and vonwillebrand factor activity, antigen, cleaving protease activity (vwf : cp, adamts-13), and multimeric composition remained within normal ranges after treatment. endogenous inhibitors of coagulation were retained 93% to 100%. plasminogen and alpha 2-antiplasmin were retained 94% and 78%, respectively. retention of contact factors was variable; some factors were below the reference range prior to pct. with the exception of tat, all markers of coagulation activation were well within normal ranges. the tat level in one i-ffp unit was slightly above the normal range; all other units had tat levels that were well within the normal range. the significance of this is unclear. cept plasma is similar to conventional plasma. the improved processing set, intended for commercialization, allows up to 3 doses of i-ffp to be produced from a single photochemical treatment. background: guidelines of the european directive 2004/33/ec require that fresh plasma prior to freezing contains <6109 residual rbcs per litre. this rbcs content is below the sensitivity limit of the automated cell counters used in routine laboratories. aim of the study: it was therefore essential to make available an alternative method to detect and quantify rbcs in plasma. we implemented a method by flow cytometry using a pe conjugated anti-glycophorin a (gpa) monoclonal antibody that recognises rbcs and erythroid precursors. to quantify residual rbcs in fresh plasma, the method uses the same trucount test tubes (becton dickinson) as those used to quantify wbcs and that contain a known number of fluorescent beads. after addition of plasma and pe-gpa antibody, cell counting is performed on flow cytometer (bd facscalibur). validation of the method: assessment of accuracy, linearity, and reproducibility with 2 different pe-gpa antibodies (immunotech and pharmingen) application of the method: quantification of rbcs in 105 fresh plasmas divided into 3 groups: group 1: 45 plasmas from leucoreduced whole blood, group 2: 30 plasmas from packed cells after removal of buffy coat and specific filtration, group 3: 30: apheresis plasmas. results: validation of the method: for both anti gpa antibodies, detection threshold is 0.05 ¥ 10 9 residual rbcs/l; linearity study with concentrations of 0.1, 0.25, 0.50, 1.0, 1.5, 3.0, and 6.0 ¥ 10 9 rbcs/l showed excellent correlation between observed and expected values (r > 0.99); reproducibility study showed c.v of respectively 5.7% and 6%. for values >2 ¥ 10 9 rbcs/l, it appeared necessary to introduce a correction factor of 1.3 for the anti gpa pharmingen. quantification of rbcs in plasma: group 1 (plasma from leucoreduced whole blood): 0.39 10 9 ± 0.23 rbcs/l; group 2 (plasma from packed cells after removal of buffy coat and filtration): <0.05°¥ 10 9 rbcs/l in all the cases; group 3 (apheresis plasma): <0.05°¥ 10 9 rbcs/l in all the cases. conclusion: quantification of rbcs in plasma by flow cytometry is a precise, quick and reproducible test. in addition this study shows that even if there are differences in residual rbcs counts according to the origin of plasma, the obtained values are much lower than regulatory requirements. 1. improving basic transfusion knowledge amongst health workers; 2. improving pre-operative preparation for surgery 3. strategies such as cell salvage autotransfusion combined with a conservative transfusion strategy for the use of allogeneic blood. my presentation will outline the approach adopted to ensure that the use of cell salvage autotransfusion both improves the use of allogeneic blood and preserves allogeneic stores. well-organised training can both minimise the risk of using such advanced techniques and decrease the overall risk involved in undergoing surgery where blood loss may be a significant factor in increasing morbidity and mortality. the various training methods employed to improve knowledge in this area will be described. the increasing current perception that the safety of allogeneic blood transfusion has dramatically been improved during the last decade is challenging autologous haemotherapy methods. in addition, growing concern about the unfavourable cost-effectiveness of most autologous haemotherapy methods requires a refinement of the application of these measures to well defined circumstances. in contrast, newly emerging transfusion-transmissible infections or periods of blood shortage might revive interest in these blood sparing techniques. the first two cases of transfusion transmitted vcjd provide a paradigm for this scenario; not so much with respect to a public fear of infection but rather a waning donor population due to more rigorous recruitment criteria. preoperative autologous blood donation (pabd) still plays a significant role in settings with high individual benefit for the patient, high transfusion probabilities and when all opportunities of cost minimization can be applied. adjustment to the individual situation of the patient is the main aim of a medically reasonable and economic use of autologous haemotherapy. this implies consideration of the patient's haematocrit, blood volume, tolerable blood loss, expected blood loss, etc. in order to choose the optimal method in the individual case. in this respect, double red cell apheresis may play a significant role. with this approach, donation schedules assumed to enhance erythropoiesis can be adopted. moreover, inconveniencies caused by long distances between patient home and donation service can be facilitated by withdrawing two red cell units during one session in selected patients. in conclusion, red cell apheresis can be used to promote the proposed approach towards individualized autologous haemotherapy preoperative plasmapheresis is considered to be a sensible adjunct if intraoperative retransfusion of salvaged and washed red cells is planned. acute normovolaemic haemodilution is valuable when the patient's tolerability of the haemodilution and the expected blood loss are carefully examined beforehand. intraor postoperative salvage of wound blood can also be regarded as useful measures to prevent allogeneic transfusions as long as the specific advantages and disadvantages of the different methods are taken into account. finally, alternative and supplemental measures such as iron or erythropoietin administration should always be considered in order to optimize the efficacy and effectiveness of autologous haemotherapy methods. the goal of a 'bloodless medicine' might not be reached but is supposed to be approached closely with an integrated concept exploiting all measures available. however, in times of restricted health care resources, regular sound costeffectiveness analyses, taking the availability and the cur-rent safety profile of allogeneic blood products into account, are always warranted and needed. compensatory fluid replacement of surgical blood losses: the transfusion of allogeneic blood is expensive and -although safer than ever before -still associated with potential complications. to reduce both, costs and immanent risks, allogeneic transfusion should either be completely avoided or at least minimized during surgical procedures. as a consequence an intraoperative blood loss is initially not replaced by red blood cells, but by erythrocyte-free, i.e. cristalloidal or colloidal solutions. when normovolemia is maintained the resulting dilutional anemia is compensated by an increase of cardiac output and enhanced arterial o2 extraction. however, once the hb has dropped to values recommended as the lower intraoperative limit, or once compensatory mechanisms of acute anemia become exhausted, as a rule transfusion of red blood cells (rbc) is initiated to increase arterial oxygen content (cao2) and to preserve a margin of safety for tissue oxygenation and organ function. as an alternative to immediate rbc transfusion, ventilation with pure o2 (hyperoxic ventilation) can be employed to rapidly raise cao2 by increasing the amount of physically dissolved o2 in plasma (hyperoxia). however, molecular o2 causes vasoconstriction, mediated by products of the arachidonic acid metabolic pathway. as a consequence hyperoxia has been shown to increase systemic vascular resistance and to decrease cardiac output and o2 consumption in subjects with normal hemoglobin concentration ( properly scheduled, three women who did not reach the necessary hct level after the first donation and consequently they got out of the protocol, and one woman who had unexpected intraoperative bleeding and received 5 homologous units in addition. the 3 patients undergoing tkr and the 11 patients undergoing removal of implants predeposited 9 and 21 units respectively, but finally 3 and 12 of them have been used. according to our patients data, the 55.5% of unused autologous blood units belongs to patients with tkr and implant removal. therefore a better schedule is needed for these type of surgery. all the autologous donors were supported by oral iron supplementation throughout the predonation and 1 month past surgery. fourteen of our cases were supported by erythropoietin s.c. in a dose of 300 iu/kg every other day. the majority of these patients was female, only one was male with multiple alloantibodies and was scheduled to predonate 5 autologous units. all of them underwent thr except one woman who also had a tkr 6 months later. the autologous blood donation was well tolerated by all patients and only one woman had a reaction during predonation. furthermore a group of 98 patients matched for age, sex and type of surgery, who did not predeposit blood, received a mean of 3.2 homologous units per patient, that is more than the patients on pabd program. our results show that autologous transfusion can be used in scheduled orthopedic surgical procedures and can reduce the need for homologous blood. however, every effort should be made to render the practice of pabd more efficient and to minimize its costs. colloid solutions and their establishment in clinical practice background: various situations like trauma, critical ill patients, sepsis, major surgical procedures and anaphylactic reactions are associated with disturbances in fluid homeostasis. this disturbance is related with reduced oxygen delivery, subsequent lactic acidosis and imbalance in oxidative status. the final result will most likely be an increased mortality and morbidity. aim: the important issue from clinical aspect is to define the optimal volume and type of fluid therapy. the debate for the ideal resuscitation solution lasts a couple of decades due to inconclusive and conflicting results. method: we searched the literature for clinical trials and major met analyses concerning patients undergoing scheduled surgical procedures, trauma patients and critical ill who received resuscitation fluids. results: dextrans reduce blood viscosity and von willebrand factor levels more, for the same degree of hemodilution, compared to other plasma expanders. in clinical setting they are effective in reducing the incidence of deep vein thrombosis and pulmonary embolism. after the initiation of dextran 1 for prophylaxis against anaphylactic reactions, they are considered the safest plasma substitutes, except maybe during pregnancy. dextran 40 10% is the most like to cause the 'hyperoncotic acute renal failure' syndrome. gelatins also cause a decrease in circulating levels of vwf : ag, vwf r : co, thrombin-antithrombin complexes and f1+2. in clinical aspect however, there are contradicted results about the effect of gelatins in bleeding diathesis, although they appear to exert a greater effect on rbcs protection from mechanical stress. in respect to anaphylactoid reactions, they have the greatest relative risk. hes has the same effectiveness in volume expansion with albumin and has the advantage of remaining intravascular even if there is an increased capillary permeability. in addition, it may improve splanchnic blood flow and tissue oxygenation. hes130/0.4 subsides the inflammatory response in patients undergoing major surgery, compared to a crystalloid-based volume therapy, but has conflicting results about it's effects on neutrophil respiration burst. clinical trials have so far failed to have a unanimous conclusion about the bleeding diathesis after hes administration, especially. caution must be held when administering hes during renal transplantation. albumin became the scapegoat of transfusion strategy during the past 20 years. resent met analysis have contradicted results about the safety of albumin infusion in variouw settings. a positive effect seems to have the early administration of hypertonic solutions in trauma patients, especially in combination with dextrans. conclusions: although some minor conclusions can be extracted, there is still a great lack of large scale multicentre randomized prospective clinical trials for extracting evidence based criteria. instead, we try to extract conclusions through met analysis. results show no evidence that resuscitation with colloids reduces the risk of death compared with crystalloids in patients with trauma, burns, major surgery or sepsis. also, there is lack of evidence that one colloid solution is safer -in clinical aspect -than any other. recombinant human erythropoietin therapy in critically ill patients -a dose response study* objective: the aim of our study was to assess the efficacy of two dosing schedules of recombinant human erythropoietin (rhuepo) in increasing hemoglobin (hb) level and reducing the exposure to red blood cells (rbc) transfusion in critically ill patients. design: a prospective, randomized, multicenter trial. patients: a total of 148 patients who met eligibility criteria were enrolled. intervention: patients were randomly assigned to receive intravenous (i.v.) iron saccharate alone (control group), i.v. iron saccharate and subcutaneous rhuepo 40 000 units once per week (group a) and i.v. iron saccharate and subcutaneous rhuepo 40 000 units three times per week (group b). rhuepo was given for a minimum of 2 weeks or until icu discharge or death. the maximum duration of therapy was 3 weeks. the requirement for rbc transfusions was significantly higher in control group than that in group a and b. no significant difference was observed between group a and b. the mean increase in hematocrit (dhct) and hb (dhb) from baseline to final measurement were significantly higher in group b than these in control group. dhct was significantly higher in group b than that in group a. dhct in group a was significantly higher than that in controls, whereas dhb did not differ significantly between control and group a. conclusion: administration of rhuepo in critically ill patients significantly reduced the need for rbc transfusions. the magnitude of the reduction did not differ between the low and high dose of rhuepo, whereas there was a dose response of hct and hb to rhuepo in these patients. in transfusion medicine, antibodies to antigens in the platelet membrane have traditionally been regarded as less significant compared with antibodies towards red cell antigens. there is an increasing awareness of antibodies towards platelet antigens. detection of autoantibodies to platelets can be a diagnostic challenge, but is seldom a problem in transfusion medicine because patients with such antibodies rarely are candidates for platelet transfusions. also, severe foetal thrombocytopenia is seldom present in pregnancies with autoantibodies to platelet antigens. the real challenge in transfusion medicine is related to patients with severe thrombocytopenia who are refractory to platelet transfusion due to alloantibodies towards platelet antigens. in our department, flow cytometry is used for compatibility testing and the choice of compatible blood donor is done without knowledge of antibody specificity. if crossmatch negative random donors cannot be identified, antibody specificity testing is performed and donors are chosen based on the specificity of the antibodies determined by a modified maipa procedure and with hla class i beads (flowpra from one lambda, usa) in flow cytometry. in some cases both hla class i and human platelet antigen (hpa) specific antibodies are detected and hla class i, hpa matched donors are chosen for crossmatch. if the crossmatch is negative, there is >90% chance of successful transfusions. in some cases drug induced anti-platelet antibodies are suspected and flow cytometry based antibody tests are performed in the presence and absence of the drug. in the case of suspected heparin induced antibodies, a beads assay is performed (diamed, switzerland). two percent of caucasian women have the platelet type hpa 1bb. ten percent of these women make anti-hpa 1a antibodies in their first hpa 1a incompatible pregnancy. 1 in 1000-2000 new-born has thrombocytopenia due to maternal alloantibodies which have crossed the placenta (neonatal alloimmune thrombocytopenia, naitp). results from a screening study covering the outcome of 100 000 pregnancies show that only 2 babies were born with intracranial haemorrhage (ich) and there was no still-born babies in the study. pregnant women with a-hpa 1a antibodies were diagnosed, received careful clinical follow-up and the delivery was performed by caesarean section in week 38 of the pregnancy with immediate transfusion of hpa compatible platelets if the new-born had platelet count <35 ¥ 10 e 9 /l. in previous studies, it is reported the ich appears in 10-30% of the pregnancies where antibodies are present and that 50% of the babies with ich, die. our results are different from what is reported from other studies and this may reflect the prospective approach and the clinical interventions. naitp represent a challenge in transfusion medicine both diagnostically, but also related to compatible blood products for the thrombocytopenic new-born and the mother who may have high level of antibodies towards platelet antigens. methods: apheresis platelets (4 ¥ 10e 11 mean) from donors with same blood group were pooled and divided equally into two bags, po-80 and control (pl2410, baxter), which have 2660 and 2024 ml/m 2 *day*atm of oxygen permeability, respectively. on days 0, 1, 3, 5, 7, and 9 of storage, swirling, mean platelet volume, po2, pco2, ph, glucose, lactate, aggregation, and p-selectin expression were evaluated. six experiments were performed. results: the swirling pattern was preserved better for up to 9 days in po-80 (6/6) than in control (3/6) bags. dropped ph less than 6.2 on day 9 was observed 1/6 in po-80 whereas 3/6 in the control. aggressive drop of glucose (0 mmol/l) with prominent lactate accumulation (327 mg/l) was also observed on day 9 in 1 of control bags. the po2 level in the control dropped more significantly by 45% (46.6 mmhg) on day 3 than in po-80 (65.8 mmhg) compared with the initial level (84.9 mmhg) (p < 0.001). these results suggest that aerobic metabolism of higher concentration platelets was maintained better in a container with higher oxygen permeability. and less lactate generation with slower glucose consumption is also suggested in po-80 bags than in control bags. the %hsr and aggregation decreased gradually in a similar manner in both bags until day 7, and became a detrimental defect in 2 of 6 control bags on day 9. p-selectin expression was higher in control bags than in po-80 on days 7 and 9 with no statistical difference. in two control bags p-selectin expression reached >84% and was accompanied by a loss of swirling. these functional and biochemical characteristics of platelets at a higher concentration were kept better for 7-9 days when stored in a container with higher oxygen permeability than in the best of marketed containers. t-pa-096 background: maintenance of a neutral ph in the range of 6.8-7.4 is essential for preservation of platelet function and viability during storage. furthermore, studies have also indicated that the presence of glucose in the platelet suspending medium is important for maintenance of platelet quality. however, a platelet additive solution (pas) containing glucose having a ph of 6.8-7.2 cannot be manufactured by steam sterilization due to caramelization of glucose. in order to have optimal ph, the currently available pas such as t-sol does not contain any glucose. this study describes a novel twostep approach to provide a glucose containing additive solution (pas-g) by using an acid, glucose containing electrolyte solution (ph 5.5) for resuspension and processing of the pooled buffy coats (bc), followed by transfer of the processed platelet concentrate (pc) into a storage bag containing bicarbonate for ph neutralization and maintenance during extended storage. aim: compare the platelet quality of pooled bc pc stored in pas-g with pooled bc pc stored in t-sol. methods: a paired study design was used, where a pool of 8 bcs obtained from standard day 1-old cpd-wb units, was divided into two equal parts: one part was resuspended and processed with a pall leukoreduction system (atsbc) using t-sol, the other part was processed in a similar manner with the acid part of pas-g. percentage plasma carryover ranged from 20-35%. both processed pc products were transferred and stored in elx tm bags with the pas-g pc elx bag containing a bicarbonate tablet. ten replicate studies were performed. results: the yields (3.2 ± 0.3 vs. 3.2 ± 0.4°¥ 10e 11 ) were similar (pags vs t-sol). statistically significant (p < 0.05 with paired ttest) improved platelet quality at days 5, 7, and 9 of storage was observed with platelets stored in pas-g as compared to t-sol. the table below shows results at 7 and 9 days of storage for ph, extent of shape change (esc) and hypotonic shock response (hsr). the results for t-sol stored platelets correlated highly with initial glucose (% plasma carry over) level (r = 0.91 for esc, and r = 0.68 for hsr at 7 days storage), while no significant correlations were found for pas-g stored platelets. conclusion: this study demonstrated the practicality of using a two step procedure to store bc pc in a glucose containing additive solution with neutral ph during storage, and confirmed the importance of glucose in the storage medium as nutrient for optimal platelet storage quality. the effect of irradiation on white cell reduced platelet concentrates, stored for 7 days background: the storage of white cell (wbc)-reduced platelet concentrates (pcs) can be extended from 5 to 7 days provided the quality has been validated and bacterial screening is performed. irradiation up to 50 gray (gy) does not affect platelet quality, but the effect of pre storage irradiation with subsequent storage up to 7 days is not known. method: two wbc reduced pcs, each made from 5 buffy coats and a unit of plasma, were pooled and divided into control group 'a' and study group 'b' . pcs in group 'b' were irradiated immediately after preparation with 25 gy. pcs in both groups 'a' and 'b' were then stored on a continuous flat bed shaker at 20-24°c. swirl, ph and cd62p expression were determined on day 1, 6 and 8. twelve experiments were performed and compared with a paired t-test, p < 0.05 was considered significant. results: see table (day 8 values; mean ± sd; n = 12). pooling and dividing of the pcs was successful with respect to volume and platelet number. on day 8, the ph in group 'b' was slightly lower than in group 'a', but the difference is not significant. in group 'a', ph on day 8 was <6.8 in 3/12 pcs, versus 5/12 in group 'b' (not significant). the cd62p expression in irradiated pcs is not significantly higher than in non-irradiated pcs. conclusion: irradiation had no significant effect on platelet quality when stored for up to 7 days after blood collection. -1b, -3a, -5b, pra 63%, 12 donors. in patient 3 -three transfu-sions were effective (two crossmatches neg by lct and pift, one pos lct, neg pift) but later on, when the patient was in severe clinical status (shortly before his death) two transfusions were ineffective in spite of neg crossmatches. it is very likely that for the same reason patient 4 and 5 were refractory to two and one hpa compatible platelet units respectively. in patient 6 and 7 compatible platelets were not transfused because they died before the whole procedure (diagnosis and finding a proper donor) was completed. conclusions: 1. the frequency of occurrence of anti-hpa antibodies in transfused patients was: -5b, -1b, -2b, -3a, -1a; in three patients they were monospecific, in four polyspecific. 2. in 2 patients who developed anti-hpa alone, transfusions of platelets without relevant hpa antigens were successful. 3.the effectiveness of compatible platelets in patients with both anti-hpa and -hla was more difficult to assess because of their severe clinical status, which might have been responsible for transfusion failure. in one of these patients, however, the transfusions of compatible platelets were successful when he was in relatively good clinical status, but shortly before death transfusions were ineffective. t-pa-099 the successful implementation of nucleic acid testing (nat) for hiv, hbv, hcv and further viruses as well as improved donor selection led to a dramatic risk reduction for viral transmission via blood transfusion over the last years. today, other risks get into the focus of haemovigilance. bacterial contamination of blood products can occur via the donor, suffering from a (clinically unapparent) bacterial infection, or via the donation process itself, storage and handling of the blood product. particularly platelet concentrates (pc) are vulnerable to bacterial growth due to their storage conditions. patients receiving such products have a potential risk of severe complications or even death. modern hygiene regimes, e.g. improved disinfection of the donors´ skin or preparation of blood products in fully closed systems as well as diversion, led to a significant reduction of bacterial contamination risk in the past. however, a small risk remains. therefore, two possible ways of further reducing the risk of bacterial contamination of blood products are feasible: (a) testing and/or (b) inactivation. testing for bacterial contamination is possible by different methods: direct detection methods for bacteria (microscopy, flow cytometry) have disadvantages regarding sample size and detection limit. bacteria might rapidly grow in a contaminated pc, so testing should be performed as close as possible to transfusion to the recipient. biochemical methods like oxygen consumption might not detect anaerobic germs. automated culture methods are still the most sensitive technique, but they have their downsides as well (e.g. time and size of aliquot drawn). novel molecular genetic test methods for detection of bacterial nucleic acid are in different states of development, but still have to proof their suitability for routine use. three different principles of pathogen inactivation can be distinguished: photodynamic reactions produce oxygen radicals which in turn inactivate bacterial structures by oxidation processes. examples for these chemicals are phenothiazines like methylene blue and thionin as well as vitamins like riboflavin (vitamin b2). photochemical reactants penetrate cell boundaries and irreversibly inhibit nucleic acid, thus blocking replication and proliferation of pathogens. chemicals of this group are psoralens like amotosalen as well as pen-110 or s-303. the third method, the solvent detergent (sd) method, is used for pooled plasma only and consists of the combination of both solvents and detergents, which interact with membranes and destroy bacteria. methods for inactivation of bacterial contaminants have to proof, that they effectively inhibit bacterial growth while maintaining full functionality of the blood product at the same time. these two qualities have to be fulfilled up to the end of the storage period. toxic or mutagenic compounds must not remain in the final product. the technology must be easily integrated into the existing work cycle of a blood bank. finally, costs per product must be acceptable. in summary, both testing and inactivation have their advantages and disadvantages, which have to be weighed up against costs and benefits of both procedures. pros and cons of introduction of inactivation methods in a blood donor service producing 1 600 000 blood components per year will be discussed. bacterial contamination of blood components, particularly platelets, is now recognized as a serious adverse reaction that is preventable. there are many studies that have documented that these reactions occur from platelets stored at room temperature, most commonly arising from a skin contaminant, but originating from donors with asymptomatic bacteremia in about 1/3 of cases. the problem is intensified for patients receiving pools of platelets compared to single donor platelets collected by apheresis. reactions are more commonly noted and more severe with platelets stored for greater lengths of time. previous studies at johns hopkins described a series of 23 reactions in 12 years with reactions more common in platelet pools (1 : 1600 transfusions) than with single donor platelets (1 : 15 000 transfusions). these reactions caused fatalities in 4 of 23 cases. although our data suggests that these reactions are more common than other studies using hemovigilance systems, our case definition requiring culture of all transfusion reactions to platelets led to a more reliable estimate of the incidence of sepsis from platelets, many potential solutions have been proposed to prevent these reactions. improved skin antisepsis should always be sought but will never eliminate the 1/3 of reactions due to asymptomatic bacteremia. the same limitation applies to methods that divert potential skin plugs from the collection bag. antibiotics in the bag would lead to manufacturing concerns or problems for patients with drug allergies. although cold storage of platelets is currently being revisited, it is not yet a practical solution. pathogen eradication systems have been developed but they remain unapproved in most of the world and have led to concerns about toxicity of additives, damage to treated cells, and cost. as a result of increasing recognition of the problem, there has been increased interest in bacterial screening to prevent sepsis from platelets. in march 2004, the aabb standards required testing of platelets for bacterial contamination. testing programs have been implemented in the us widely as a result of the aabb action. licensed systems based upon bacterial culture or growth characteristics are available for single donor platelets and have been commonly employed. although these systems have some difficulty with false positive reactions, the evolving national data suggest an incidence of 1 : 5000 true positive reactions. these data suggest that a number of serious reactions have been averted, although some cases have persisted due to incomplete adoption, problems with slow growing bacteria, or the use of inferior testing methods with inadequate sensitivity. whole blood derived platelets have become a more difficult issue, since the approved testing methods are limited. the use of ph monitoring, gram stain, glucose measurements, and inspection for swirling have all been attempted. these methods are not sufficiently sensitive or specific to interdict many contaminated units, so that screening for bacteria in pooled platelets is less effective. it is anticipated that new methods may become available to make screening of whole blood derived platelets easier to perform in a reliable manner. it is also hoped that bacterial screening of platelets may form the basis to permit seven day storage and prestorage pooling in the us. results: twenty four hcv rna (+)/anti-hcv(-) repeat donors were previously tested in routine hcv rna in mini-pools and were negative. twenty available look back samples were individually tested for hcv rna and in one the virus was detected. to make sure that the failure of hcv rna detection in routine nat was not due to the pooling procedure, the hcv rna was tested in undiluted look back sample and dilutions of this sample by hcv negative plasma: 2/2 repeats of 48x dilution and 2/2 repeats of 24x dilution were hcv rna negative, whereas 1/1 repeat of 8x dilution and 2/2 repeats of undiluted sample were positive. the results of cobas amplicor monitor (sensitivity 600 iu/ml) were negative, which means that viremia in hcv rna mini-pool negative donation was below 600 iu/ml. in the recipient of red blood cell concentrate from this donation hepatitis c was diagnosed. however, the possibility of pretransfusion hcv infection cannot be excluded as no hcv marker tests were performed before transfusion. the patient and the donor were infected with genotype 3a. the low hcv viremia (below 600 iu/ml) in the preseroconversion window period was responsible for no hcv rna detection in routine mini-pool hcv rna testing. introduction and aim of the study: bacterial contamination is a life threatening risk of blood transfusion, especially with platelet transfusions. bacterial culturing (bc) of platelets as well as pathogen reduction (pr) reduce the likelihood of such contamination. where the costs of bacterial contamination are far less than the costs of pathogen reduction, the latter will reduce not only the risk of bacterial contamination but also risks of other pathogens. therefore, the question arises whether this additional expenditure can be justified in the light of the additional effect achieved. this question we will answer by cost-effectiveness and sensitivity analyses. methods: the balance between costs and effects of preventing adverse events due to platelet transfusion is assessed using a mathematical model and assuming optimal effectiveness of pr. model parameters and valuations of health states were obtained from literature and information from dutch sanquin blood banks. . while the estimates in comparison to the situation without bc or pr are surrounded with large uncertainties, the conclusion that pr is not cost-effective in comparison to bc is very robust. the cost-effectiveness of bc and pr are very sensitive to the estimates concerning sepsis probability and associated complication rate, the cost-effectiveness of pr relative to bc is not. this conclusion is also insensitive to a wide range of assumptions regarding residual risks and costs associated with hiv, hcv and hbv. the estimates indicate that culturing in the netherlands is cost-effective, even with the deviation bag in place. the estimates however appear to be very sensitivity to the probability of sepsis. a decision to use pr will, after the introduction of bc and the use of a deviation bag, never meet cost-effectiveness criteria. even when assuming perfect protection, the conclusion that it is not cost-effective in comparison to bc is very robust and does not alter when varying underlying parameters within their margins of uncertainty. table) . of cb collection. two collections have been transplanted to date and this represents a 2.5% take-up rate (3.5% where a sibling is alive). this compares favourably with the numbers transplanted from unrelated cb banks. dcb collection is therefore at least as efficient a method as unrelated cb collection for transplantation albeit in the limited number of cases where a dcb collection is possible. dcb collection has the benefit of a possible immediate transplant combined with the availability of a sibling donor for future donation of both stem cells and lymphocytes. it is therefore a useful service to provide and complements the work of unrelated cord blood banks. increased yield of mature platelets in cultures of cd34-enriched cord blood cells maintained at 39°c introduction: the future use in transplantation of ex vivo expanded hematopoietic stem (hsc) and progenitors cells will facilitate the transplantation of adult patients and speed up hematologic recovery. also ex vivo cultures of hscs may eventually permit to produce donor-free blood components such as platelets for transfusion. culture of animal cells is routinely done at 37°c. however there is previous clinical evidence suggesting that hematopoiesis may be more active in hyperthermic patients. we have therefore compared the effect of hyperthermia on the ex vivo expansion and differentiation of cord bloodderived hsc in megakaryocytes (mk) and mature platelets. the cord blood-derived cd34 cells were cultured continuously at 37°c or 39°c for 14 days in cytokine conditions optimized for mk development and maturation. the cultures were regularly monitored for various parameters. results: compared to 37°c, the cultures maintained at 39°c produced significantly more total cells (4.9 fold) and total mks (7 fold), and showed accelerated and enhanced mk maturation with increased yield of proplatelets and mature platelets (16.6 fold). accordingly, the cells cultured at 39°c contained an increased frequency of cfc-mk (8 fold) at day 14. cultures done at 38°c and 40°c were also more efficient than at 37°c but less than at 39°c. platelets produced in 39°c cultures could be normally activated by thrombin. as expected, the cells cultured at 39°c contained an increased amount of the heat shock protein hsp70. control experiments showed that the culture of several cell lines was inhibited or unaffected by the 39°c temperature. the unexpected resistance of hematopoietic cells to the deleterious effects of heat and the stimulatory effect of >37°c temperatures on hsc proliferation and differentiation indicate that the routine culture of normal human cells at 37°c is a paradigm that needs to be revised. the responsible molecular mechanisms remain to be identified but the observation will facilitate the ex vivo expansion of the progenitors of the mk and possibly other lineages. the synchronous generation of a significant number of mature platelets in vitro will facilitate the study of the mechanisms of platelet formation and ageing and could eventually have important applications in transfusion medicine. it remains to be seen if the stimulatory effects of higher than 37°c temperatures represent a protective response against sustained body fever that is specific to the hematopoietic system. several countries have, in the past few years, included human tissue banking within a regulatory framework similar to that of blood. indeed, tissue safety has come to the forefront of the preoccupations of regulatory agencies after several well publicised morbidity and mortality cases have been reported in the press. tissue safety has many features similar if not identical to blood safety and a review of those common elements will be reported. as well, arguments in favor of integrating tissue banking within a blood system will be discussed, one of the more important aspect of which being the expertise of the blood centre staff with cgmps. the experience of a blood establishment (héma-québec) with the integration of tissue banking such as bone, skin, heart valves within its operations will be reported, emphasizing the medical as well as the management aspects of such an integration. finally, tissue banking is an activity which brings more expertise to a blood centre, expands its knowledge of its customers and gives more opportunities to its personnel. umbilical cord blood (cb) is an important source of stem cells for clinical transplantation and may cause less gvh disease than non-t-depleted bone marrow (bm). the relatively low numerical cell dose available from cb has usually restricted its use for transplantation in adults. only 25-30% of patients have an hla matched sibling and for others an unrelated bm or a stored unrelated cb donation may also not be available. for some children the collection of cb following the birth of a sibling may be the only opportunity for a transplant. directed cb (dcb) donations from matched siblings have been shown to give better long-term overall results than matched unrelated cb or bm. dcb collection is however not as easy to control as cb for banking where dedicated hospitals and trained staff are used. here we review dcb banking in oxford over a 2.5-year period. requests were received for 88 deliveries from 84 mothers and of these, 81 collections were successful including 4 pairs of twins. failed collection was most often due to a damaged cord at delivery. 61 collections were made for 57 siblings possibly requiring transplant (median age 4) with 24 for leukaemia, 16 for erythroid disorders, 13 for immune deficiency, 5 for enzyme deficiency and 3 others. the remaining 20 collections were mostly requested where there was a family history of an inherited disorder (majority scid). three collections tested positive for anti-hcv antibody but negative for hcv by pcr. collections were not excluded on the basis of volume or cell number. mean volume was 80 ml (range 22-174, 86% exceeded 40 mls) and mean tnc count was 1.0 ¥ 10 9 (range 0.2-2.7, 58% exceeded 0.8 ¥ 10^9). the mean cd34+ve count was 3.1 ¥ 10 6 (range 0.2-19.0). all collections were cryopreserved within 24 hours using dmso/dextran/saline without volume reduction. the mean tnc viability prior to freezing was 98% (range 86-100%) and the mean cd34+ve viability post freezing was 82% (range 71-96%). the reliance on the goodwill of midwives and the logistical difficulties that arise when organising collections from many different hospitals do not appear to reduce the success . rhd and rhce typing was performed by multiplex-pcr with 24 fluorescent primer pairs. positive results were obtained for rhd-exons 2-7, 9, 10 and polymorphisms associated with antigens c, c, cw, e and e. sequencing of rhce-sequences, including exons 1 to 10 and intron/exon borders, were done by direct taq cyclesequencing using bigdye-terminators v.1.1 in an abi310 (applied biosystems). background: a number of adverse immune reactions associated with blood transfusion result from contamination of blood products by donor white blood cells. among these reactions, transfusionassociated graft-versus-host disease (ta-gvhd) has a mortality of greater than 90%. mirasol ® pathogen reduction technology (prt) has been developed for the reduction of viruses, bacteria, parasites and white blood cells loads in blood products. the technology is based on light and riboflavin photochemistry. this study was performed in order to evaluate the effectiveness of the mirasol ® prt process for inactivation of human pbmncs. methods: human pbmncs were collected from trima platelet apheresis disposable sets, purified by ficoll-hypaque discontinuous gradient centrifugation and divided into test and control samples. the test cells were treated with mirasol ® prt in autologous plasma on day 0. both test and control samples (n = 3) were tested on day 1 for cellular immunophenotype, t-cell activation using flow cytometry, proliferation in response to mitogen or allogeneic stimulator cells, ability to stimulate the proliferation of allogeneic responder cells and cytokine synthesis in response to lps stimulation was measured using a cba assay kit. results: although mirasol ® prt treatment did not significantly change the distribution of cd3+, cd3+cd4+, cd3+cd8+, cd19+ and cd16+cd56+ human lymphocyte subpopulations there were significant functional change. the expression of the activation marker, cd69, was observed in 65.4% (sd = 15.6%) of control t cells upon activation with pma, while only a 1.7% (sd = 1.3%) of the test t cells increased cd69 expression. proliferation assays showed that 3h-thymidine incorporation did not increase in the test cells in response to either pha or allogeneic stimulator pbmnc compared to the significant increase in thymidine incorporation levels observed with control cells. the test cells, when compared to the controls cells, demonstrated an inability to stimulate allogeneic responder pbmnc proliferation. the release of il-10, il-6, il-1b and il-8 cytokines after 24-h incubation in culture media increased significantly to 128 pg/ml (sd = 73), >5000 pg/ml, 404 pg/ml (sd = 301) and >5000 pg/ml for control cells, respectively. under the same conditions, these cytokines in test samples remained at background levels of 0.4 pg/ml (sd = 0.7) for il-10, 2.4 pg/ml (sd = 1.2) for il-6, 11 pg/ml (sd = 3) for il-1b and 1022 pg/ml (sd = 286) for il-8. addition of lps further stimulated the release of tnf-a, il-10, il-6, il-1b and il-8 in the control samples, but not in the test cell samples. in vitro studies demonstrate that mirasol ® prt treatment does not change lymphocyte immunophenotype, inhibits tcell activation by pma, abolishes pbmnc proliferative activity, eliminates pbmnc stimulatory activity for responder cell proliferation and suppresses the production of cytokines by pbmnc in both the absence or presence of lps. introduction: it has been discovered that vaccination of dendritic cells (dcs) with tumor antigens is a potential strategy to induce tumor-specific immunity in tumor-bearing patients. aim of the study: the purpose of the study was to investigate whether human monocyte-derived dendritic cells (dcs) were able to present p210bcr-abl protein and induce antigen-specific ctl responses in vitro after transfected with total rna of k562 cells (k562-rna). methods: dcs were derived from human pbmncs, which were incubated for 5 days in the presence of gm-csf and il-4, and then were transfected with k562-rna using electroporation or dotap lipofection. to verify the successful transfection of dcs with k562-rna, bcr-abl fusion genes expression of dcs was detected by rt-pcr and western blot. the immune phenotypes of the dcs were analyzed by flow cytometry. the cytotoxicity of ctl was assayed by propidium iodide (pi) staining and flow cytometry. results: it was shown that the bcr-abl fusion gene was detected in the dcs immediately after the transfection, but disappeared 24 hours later, while the cells were expressing p210bcr-abl protein and expressing increased cd80, cd83, cd86, hla-dr. moreover, the transfected dcs could significantly promote the t lymphocytes to kill the target k562 cells. conclusion: human dendritic cells transfected with total rna of k562 cells in vitro could induce effective p210bcr-abl proteinspecific immune responses and be used to induce tumor-specific immunity, which implies potential application of immunotherapy to tumors. appear to be relevant to the clinical response. ivig has a remarkably good safety record for long term administration, however the following side effects have been observed: mild, infusion-rate related reactions such as headaches, myalgia or fever; moderate but inconsequential events, such as aseptic meningitis and skin rash; and severe, but rare, complications such as thromboembolic events and renal tubular necrosis. judicial use of ivig based on results from controlled studies is recommended. t-pl3-09 donor-lymphocyte infusion: transfusion immunotherapy following allogeneic hematopoietic transplantation the notion that bone marrow containing immunocompetent cells is capable of mediating an antitumor effect was determined experimentally almost 50 years ago. subsequently, pooled leukocytes from patients with cml were found to effect responses in patients with advanced leukemia. response correlated with cell dose and with severity of gvhd. in the 1970's, the graft-versus-leukemia (gvl) effect was defined in the transplant setting using a lethallyirradiated mouse model and splenocyte infusions. such studies suggested that gvl could be enhanced without causing severe gvhd. the era of adoptive immunotherapy in the transplant setting began in the 1990's with reports of donor lymphocyte infusions (dli) for relapsed acute and chronic leukemias after bone marrow transplant. it is now clear that chronic myelocytic leukemia (cml) in chronic phase is highly susceptible to gvl effects mediated by dli which induce durable remission in 70-80% of relapsed patients. the success rate is 30% or less in patients with accelerated phase or blast crisis. since dli cell dose appears to be important in this setting, strategies of escalating dose infusions have been investigated to enhance gvl without exacerbating gvhd. unfortunately, the response to dli in relapsed acute leukemia and myeloma is less favorable (<30%) and less durable. dli have been used successfully to treat viral infections and virus-associated malignancies following transplant. both unfractionated dli and ex vivo-generated tcell clones have suppressed reactivated cytomegalovirus and eradicated epstein-barr virus-induced lymphoproliferative disease, a polyclonal proliferation of donor-origin b cells that occurs after transplant. where tumor-specific antigens have been defined, efforts to target dli have been undertaken and donor and patient immunization has been investigated. acute or chronic gvhd develops in approximately 60% of patients receiving dli for relapsed hematologic malignancies and for related, but not unrelated transplants, correlates with the donor t-cell dose. dli-induced pancytopenia occurs in approximately 20% to 50% of patients, is generally mild, and transient, but in <5% of patients, aplasia is severe and prolonged. complications of aplasia include infection, bleeding, increased transfusion requirements. efforts to limit the adverse effects of dli while retaining the therapeutic effects include insertion of 'suicide genes, ' selection of lymphocyte subpopulations, and targetting lineage-specific minor histocompatibility antigens. available clinical and experimental evidence suggests, that in addition to primary and secondary immune deficiencies, a wide spectrum of immune-mediated conditions could benefit from intravenous immunoglobulin (ivig), including acute and chronic/relapsing diseases, autoimmune diseases mediated by pathogenic autoantibodies or by autoaggressive t cells and inflammatory disorders e.g. an imbalance in cytokine networks. trimar-collected apheresis platelet concentrates (pcs) were exposed to 17.2 j/ml uv light in the presence of 50 um riboflavin, followed by storage under blood bank conditions with various concentrations of 2-deoxyglucose from 0 to 20 mm for 5 days. the control platelets were not stressed by uv light exposure and were stored under the same conditions without 2-dog presence. all test and control platelets were measured for in vitro cell quality including rates of glycolysis, morphology score and activation levels at days 0, 1, 3 and 5. results: lactate production and glucose consumption increased from 0.0378 mmol/1012 cells/h (sd = 0.0097) and 0.0268 mmol/1012 cells/h (sd = 0.0114) for control samples to 0.1371 (sd = 0.0281) and 0.0724 (sd = 0.0151) for uv-treated platelets, respectively. uv treatment also caused a decrease in ph from 7.50 (sd = 0.07) for controls to 6.55 (sd = 0.26) for treated platelets at day 5, hsr from 75% (sd = 12.4) to 33% (sd = 25.1), esc from 24.3% (sd = 3.9) to 3.8% (sd = 3.6), swirl from 2.8 (sd = 0.7) to 1.0 (sd = 1.0), and increased p-selectin expression from 21.2% (sd = 7.3) to 85.2% (sd = 9.4). addition of 2-dog up to 20 mm significantly reduced lactate production rate to 0.0515 mmol/1012 cells/h (sd = 0.0045) and glucose consumption rate to 0.0293 mmol/1012 cells/h (sd = 0.0060), and maintained ph above 7.35 (sd = 0.09) for 5 days of storage. the effect of 2-dog exhibited a dose-dependent response. however, the addition of 2-dog had no effects on hsr (28.1 + 11.4% at day 5), esc (2.86 + 2.75% at day 5), swirl (0.8 + 0.5 at day 5) and p-selectin expression (69.9 + 6.4% at day 5) during platelet storage. atp contents in both treated and control groups were maintained at a relatively constant level above 87% of the value seen in fresh platelets. furthermore, an exaggeration of uv-stressed platelet aggregation by addition of 2-dog was also observed. conclusions: increased glycolytic flux is not a direct cause for platelet morphology changes and spontaneous activation incurred during the development of the storage lesion. the results also suggest that a reduction in glucose utilization may foster an increase in platelet loss during storage. aim of the study: was to evaluate analytical sensitivity, sensitivity and inclusivity for subtypes and genotypes of hiv, hcv and hbv, the assay's effectiveness in closing the pre-seroconversion window period, clinical specificity as well as the effect of endogenous substances and microorganisms on the sensitivity and specificity of the assay. methods: secondary standard traceable to who international standard for hiv-1 (97/ 656), international standards for hcv (96/798) and hbv (97/746) were used to determine the analytical sensitivity. sensitivity and inclusivity for hiv-1 subtypes other than hiv-1b, for hiv-2 and for hepatitis b and c genotypes as well as specificity was evaluated with >1000 specimens. results: results from this study indicate that high analytical sensitivities (39 iu/ml hiv-1m, 114 cp/ml hiv-1o and 1.1 cp/ml hiv-2, 7 iu/ml hcv and 3 iu/ml hbv) and a specificity of >99.4% are accomplishable for the mpx test. the 95% detection rate for hiv-1m subtype isolates (a through h) was between 10 to 50 iu/ml, for hcv genotype isolates (1a through 6) between 2 to 10 iu/ml and for hbv genotype isolates (a through g and precore mutant) between 2 to 5 iu/ml. investigating seroconversion panels, hiv-1 rna was detected an average of 6 and 5 days earlier than hiv-1 antigen with abbott hivag-1 monoclonal and coulter p24 antigen tests, respectively, hcv rna an average of 31 or 21 days earlier than hcv antibody with the abbott hcv eia 2.0 or ortho eia 3.0 tests, hbv dna an average of 18 days earlier than hbsag with the abbott hbsag eia imx test. for all targets, no interference was detected with 17 microorganisms tested as well as elevated levels of triglycerides, albumin, hemoglobin, human dna or bilirubin. conclusion: automated pooling, sample preparation, and real time pcr using the blood screening system 200 taqscreen mpx test is an efficient and sensitive method to simultaneously screen for five important viruses in human plasma. the mpx test is another evolution step in the development of pcr automation by roche molecular diagnostics, and further represents roche's commitment to increasing the safety of the global blood supply. aim of the study: a prospective hemovigilance plan was set up in order to establish a registry for future reference, and to detect any unexpected side effect of ip that may occur with significant frequency in populations and indications that were not studied before and outside of a formal trial environment. methods: this plan is proposed to blood establishments and transfusion prescribers who have already decided to implement ip. this is an observational, non randomized, non controlled plan. no patient selection, inclusion or exclusion criteria are required. all ip transfusions are documented using an internet form, whether or not a reaction is observed. patient population data are collected anonymously, for epidemiological purposes. results: between october 2003 and september 2004, 2512 apheresis ip units have been transfused in 2 sites and registered in the database. ip platelets were considered leucocyte inactivated and were not irradiated, but were antigen matched as indicated (3.9%). the population of patients receiving at least one transfusion (n = 271) included 58.3% of males, 40.6% of females, the median age was 64 (range 0-89). the most frequent broad diagnostic categories were hematology-oncology (64.9%) and cardiovascular surgery (19.9%). the patients received their transfusions either in regular hospital wards (63.5%), intensive care units (27.3%) or as outpatients (9.2%). the number of transfusions by patient ranged from 1 to 106 (mean 9.3 ± 16.0, median 2). half of the patients (49.8%) had previous transfusion experience and 5.9% had previous history of transfusion reaction. transfusion reactions, defined as any deterioration of the patient's state of health observed following transfusion, were observed in 1.2% (n = 31) of the transfusions (95% ci 0.84-1.75), and 8.5% of patients. only 2 (0.1%) were considered serious. after further causality analysis including biological and clinical investigations by the transfusion physician, 0.9% (95% ci 0.58-1.37) of the transfusions were confirmed as having caused reactions in 6.3% of patients, none of them serious. the most often reported symptoms were chills (0.9%) and fever (0.6%). itching, skin rash or urticaria was observed in 0.5% of transfusions. of the 2 serious reactions, one was hypotensive shock in a patient with liver cirrhosis and haemorrhage, and one was septic shock, in which the platelet unit bacterial culture was negative. none of the reactions occurred in cardiovascular surgery patients. patients were more likely to experience reactions if they had previous transfusion history (odd ratio 2.17, p = 0.15). the active hemovigilance plan is a valid and feasible method to collect epidemiological data on transfusion safety. the risk profile of ip transfusions appears favorable. quality of theraflex mb-plasma during storage and treatment s reichenberg* and n müller † *maco pharma international gmbh, langen, † inst. for transfusion medicine, essen, germany background: although in the last decades thanks to the implementation of several methods like donor selection and testing procedures the risk of virus transmission from plasma has decreased, infection of patients still exists. additionally new viruses like west nile virus enter the transfusion chain. therefore, the treatment of therapeutic plasma with methylene blue (mb) is a technique used in several european countries for pathogen inactivation. macopharma has developed the proprietary theraflex mb-plasma bag system including a mb pill and a final mb filtration step. aims: aim of the study is to show the quality of the mb plasma during the preparation procedure and during storage using the theraflex system. methods: for the preparation process every single step was evaluated using 18 single donor plasma units. for the evaluation of the plasma factors 5 ml were drawn at different stages (before treatment, after plasma filtration with plas4, after dissolution of the mb pill, after illumination, after treatment). because the sample volume for single sample measurements would be too low the samples were pooled after drawing and measured for the specified factors. six samples of each stage were pooled at three days. a whole panel of plasma factors was measured for the resulting three pools. global tests: quick, inr, aptt, thrombin time coagulation factors: fibrinogen, factor ii, factor v, factor viii:c, factor ix, factor x, factor xi inhibitors: at iii, protein c, protein s fibrinolysis: plasmin inhibitor, alpha1-antitrypsin complement: ch50 activation: tat, factor xiia, d-dimer stability data were generated using three plasma pools. six plasmas were pooled and afterwards divided into six aliquots. each was treated as single unit and then each was divided into six storage samples. the same plasma factors as for the manufacturing process were evaluated. results: a moderate reduction for some coagulation factors during the preparation was found in the illumination step but not in the other preparation stages. this was mainly fibrinogen (17.5%), factor viii (22.2%), and factor x (13.4%). despite this reduction the values were within the ranges found in non-treated plasma. all investigated plasma factors remained stable during the investigated storage time. summary/conclusions: the investigation showed that plasma treated with the theraflex procedure showed slight reduction during treatment and no reduction during storage. all plasma factors remained within the threshold values. the treatment of therapeutic plasma with mb is a valid technique of pathogen inactivation. validation of intercept treatment of pooled platelets g santos, c silva, f pereira and g sousa lisbon regional blood centre, lisbon, portugal background: intercept blood system for platelets uses amotosalen hcl and uva light to inactivate viruses, bacteria, protozoa and leucocytes that may contaminate platelet products. aims: the purpose of the study was to assess the feasibility of introducing this technology in the routine of lisbon regional blood center (crsl) and validate the procedure in our center. material and methods: whole blood units of 450 ml were collected from volunteer blood donors in quadruple top and bottom blood bags (optipure rc soft t& b baxter), kept in n-butanodiol plates; buffy coats with a volume of 55 ml were obtained in the opipress ii and kept overnight at room temperature before pooling. five buffy coats were pooled with 280 ml intersol using the octopus system intercept buffy coat pooling set with an integrated filter. the pools were treated using the intercept. samples were taken before treatment, after cad remotion, on days 2, 5 and 7. the following tests were performed: platelet count, mean platelet volume, ph, swirling. results: all pools met the intercept guardbands. platelet yield pre inactivation was 3.93 ¥ 10 11 (2.95-4.72 ¥ 10 11 ; sd-0.41). platelet pool volume was 362.3 ml (sd-10.7). plasma % was within 32.9% and 36.5%. all pools had leucocytes within council of europe specifications. after photoinactivation the platelet concentrates had 3.58 ¥ 10 11 (±0.37). the average platelet loss was 0.35 ¥ 10 11 . the ph was within specifications during all the storage period. conclusions: intercept treatment of pooled buffy coat platelets is feasible in the routine of crsl and in vitro parameters do not show significant changes, allowing us to proceed to clinical use. shown ip and conventional platelets (cp), stored for up to 5 days, exhibit comparable hemostatic efficacy and safety. extension of platelet storage duration to 7 days has the potential to improve platelet availability and reduce outdating and inventory shortages. clinical efficacy and safety of ip stored for 7 days were investigated. methods: a randomized, controlled, single-center, crossover, noninferiority design (pilot) study evaluated efficacy and safety of buffy coat ip vs buffy coat cp, each stored for 7 days. patients were randomized to receive one 7-day ip transfusion and one 7-day cp transfusion in random order. after each study transfusion, the 1hour platelet count, ci, and cci; time to next transfusion; bleeding response; transfusion reactions; and serious adverse events (saes) were assessed. the primary endpoint, 1-hour cci, was analyzed by a one-sided non-inferiority test for the per protocol population (patients with both transfusions and no major protocol deviations interfering with efficacy evaluation). the per protocol population included 20 patients, 9 randomized to the ip-cp sequence and 11 to the cp-ip sequence. more patients received allogeneic stem cell transplant in the cp-ip sequence than the ip-cp sequence (64% vs 33%; p = 0.37). mean platelet dose (¥10e11) was 2.8 for ip and 3.0 for cp (p = 0.23). there was a significant period by treatment interaction (p = 0.07) at the 0.10 significance level; therefore, the first period only was also analyzed for the primary endpoint. including both treatment periods, mean (±sd) 1-hour cci (¥10e3) was 6.6 ± 4.5 for ip vs 8.9 ± 5.5 for cp. the mean paired difference for both sequences was 2.4 ¥ 10e3 (p = 0.55 by non-inferiority test; upper bound of the 95% confidence interval = 4.0). for the first period only, mean 1-hour cci (¥10e3) was 8.7 ± 3.8 for ip vs 7.4 ± 5.4 for cp) the mean paired difference for the first period sequences was 2.4 ¥ 10e3 (p = 0.06 by non-inferiority test; upper bound of the 95% confidence interval = 2.4). the non-inferiority margin for the study was 2.2 ¥ 10e3 for mean treatment difference in cci (cp-ip). median time to next transfusion was 25 h for ip vs 24 h for cp following the first transfusion (p = 0.87 log-rank test; data censored at 7 days after transfusion) and 16 h ip vs 34 h cp after the second transfusion (p = 0.02). bleeding pre-or post-transfusion was uncommon, usually mucocutaneous, and grade 2 or lower, and responded similarly to ip and cp. no significant transfusion reactions or saes were reported. in this double-blinded, two-treatment crossover study the primary endpoint regarding 1-hour cci was not met. however, transfusion with 7-day-old platelets treated by the intercept blood system showed only a marginally and probably clinically insignificantly lower 1-hour cci compared to 7-day-old conventional platelets. methods: new zealand white rabbits were transfused with syngeneic blood (10 ml/kg), across a major antigen (hgd) mismatch. high anti-s-303 ab titers (≥1 : 1000) were induced after repeated immunization (days 1, 8, 15, 36, 64) with klh-(s-303) hapten (klhhapten) in complete freund's adjuvant. ab titers against srbc were determined by gel card agglutination, or by facscan with fitc-goat_anti-rabbit_igg. survival of infused rbc (4 ml/kg) treated with different methods was assessed by rbc biotinylation. blood samples were taken 1, 3, 7, 15, 21 and 28 days after transfusion, analyzed using streptavidin_pe and facscan to determine the proportion of circulating biotinylated rbc. results: groups (g) of rabbits were transfused with control rabbit rbc (crbc; n = 4, g1), or o-srbc (n = 6, g2). no ab against o-srbc developed after biweekly transfusions over 24 weeks in g2 rabbits. high ab titers to o-srbc could however be induced by klh-hapten immunization in a different group of animals (n = 2; g3). transfused rabbits (g1 & g2) exhibited no change in hematocrit or body weight and maintained good vital signs. high titer anti-s-303 abs were then induced by klh-hapten immunization in rabbits from g1 (n = 2) and g2 (n = 4), and in a group (n = 6, g4) of naïve rabbits. non-immunized rabbits (g1, n = 2), and (g2, n = 2) were maintained on the biweekly transfusion schedule of crbc and o-srbc, respectively. all rabbits treated with klh-hapten developed comparably high ab titers. klh-hapten immunization did not affect the viability of crbc in g1 rabbits. transfusion of o-srbc demonstrated reduced viability in hyper-immune g4 rabbits, but not any of the g2 rabbits. g2 rabbits exposed to 12 o-srbc transfusions prior to hyper-immunization with klh-hapten, had viability of o-srbc comparable to crbc, suggesting induction of immune tolerance by repeated exposure to o-srbc. after depletion of labeled o-srbc from circulation, g2 and g4 were transfused with m-srbc. viability of m-srbc in all g2 rabbits (hyper-immune or not) and the hyperimmune g4 rabbits was equivalent to crbc circulation in g1 rabbits. in pre-immunized rabbits with high titer anti-s-303 ab, o-srbc are cleared faster than control. in contrast, m-srbc survive normally in rabbits with high anti-s-303 ab titers. repeated transfusion of o-srbc does not result in alloimmunization of naive rabbits. the modified s-303 rbc process offers the potential for pathogen inactivation with elimination of immunoreactivity and retention of rbc viability. introduction: the bombay phenotype is extremely rare and characterized by complete absence of abh activity both on erythrocytes and in secretions. those individuals can produce anti-h, which is active over a wide thermal range. method: using liss indirect antiglobulin technique, the patient's serum showed +4 reaction by panel of eleven cells at room temperature phase as well as indirect phase while the auto reaction is negative. a cold adsorption using rabbit erythrocyte stroma was done to remove the cold antibodies from the serum; +2 reaction of an antibody was detected in the patient serum after five folds of rabbit erythrocyte stroma adsorption. introduction: immunohematology reference laboratory in kuwait central blood bank receives samples from all hospitals in kuwait both governmental and private sector. the laboratory performs the tests according to international standards and it is monitored by internal and external quality assessments on periodic basis. material and method: a tube and gel cards are two methods in the reference laboratory for antibody identification. the laboratory can identify the most commonly encountered clinically significant antibodies and investigates causes of positive direct antiglobulin test that occur mostly in autoimmune hemolytic anemia. there are facilities to phenotype most of rare red blood cell antigens. results: records of all patients investigated in the laboratory since the year 1985 are kept in computerized system that has 7528 patient's records. central blood bank has the potential to identify rare phenotypes such as kpb-, jsb-, lan-, bombay, rzr2, rzr1, r¢r¢, r¢r≤, r≤r≤, ge-2,3,4 and rare red blood cell antibodies such as high frequency antibodies anti-k, anti-ge2, anti-h, anti-lan, anti-kpb, anti-jsb, anti-wrb, anti-ena, anti-csa and low frequency antibodies anti-kpa-, anti-jsa-, anti-dia, anti-lua, anti-cob as well as hightiter-low-avidity antibodies such as anti-chido. samples of rare red blood cells and rare serums are kept frozen either by glycerol or liquid nitrogen technique to be used for pre-transfusion compatibility testing and continuing educational program. purpose of the work: to present the substitution of the blood groups o, a, b, ab in abo blood group system and rh (d) blood group in rh (d) blood group system in the population in the gevgelija-valandovo region. material and methods: a retrospective analysis was done on the data of following the blood groups o, a, b, ab and d in the blood group system abo and rh in the gevgelija-valandovo region. the asked population are voluntary blood donors, candidates for drivers, patients, pregnant women and newborn children. the period (1989) (1990) (1991) (1992) (1993) (1994) (1995) (1996) (1997) (1998) (1999) (2000) (2001) (2002) (2003) (2004) was analysed. the examinations were done with two standard methods (on the plate and in tube), to define blood groups from the most represented blood group systems abo and rh in the population. tests were done with different series of commercial anti serum tests from domestic and foreign origin. results: totally 43.395 examinees were typified. with o blood group were 15.761 (36.31%); with blood group a were 17.675 (40.73%); b blood group 7.119 (16.4%) and ab blood group were 3.019 (7.52%) examinees. totally 37.365 (86.11%) were d positive and 6.029 (13.89%) were d negative. discussion: the given results from our examinations for the frequency of o, a, b, ab and rh (d) blood groups from abo and rh (d) blood group systems in the region gevgelija-valandovo show that the most present is the blood group a from abo blood group system 17.675 (40.73%) and d blood group in rh system with 37.365 (86.11) form examined population. the results are in correlation with data from the literature for other european nations. introduction: the policy of our center is to transfuse all heamatologic multitransfused patients with their own rhesus and kell phenotype. in addition, thalassemic and young leukemic patients are being transfused with compatible phenotype of the most clinical important duffy and kidd systems while all the other patients receive blood compatible only with abo and rhesus system. nevertheless, it is observed a significant positivity of the indirect antiglobulin test, due to alloimmunization. aim of the study: in this study we tried to evaluate the prevalence of alloimmunization in patients of our region. the most frequent detectable alloantibody remains the anti-d, with high prevalence 88.9% of anti-d in females versus 11.1% in males, due to alloimmunization during the pregnancy. as a consequence, the high incidence of anti-d is not transfusion related and anti-e is evidenced to be the most frequent transfusion related alloantibody, followed by anti-kell. care must be taken in order to transfuse as more patients as possible with their own phenotype regarding, at least the most immunogenic antigens, like anti-e and anti-kell. severe hemolytic reaction due to anti-j k3 background: red blood cell alloantibodies directed against antigens of the kidd system are notorious for causing delayed hemolytic transfusion reactions. the antibodies are formed because of pregnancy or transfusion. blood donors with the red blood cell (rbc) phenotype jk(a-b-) are extremely rare in the white population and exhibit a frequency of less than 0.1%. however, the rare phenotype jk(a-b-) is more common in polynesians (1.4%). individuals with jk(a-b-) phenotypes typically form anti-jk3 with inseparable anti-jka and anti-jkb activity. some jk(a-b-) patients' sera may show an additional distinct anti-jka or anti-jkb component when examined with adsorption studies. case report: a 63 years-old caucasian female with a negative antibody screen, no prior history of transfusion, presented with gastrorrhagia. it is reported four pregnancies with no history of haemolytic disease of the newborn (hdn). on admission, her haemoglobin was 5.5 g/dl. she was given 4 units of crossmatchcompatible rbc. on day 6 her haemoglobin was 12.1 g/dl, with a total bilirubin of 0.72 mg/dl and lactate dehydrogenase of 141 u/l. on 10th day an unexpected fall in hb (5.6 g/dl) occurred with an increase of bilirubin to 1.9 mg/dl and of lactate dehydrogenase to 1042 u/l. a new blood sample obtained for antibody screening and additional crossmatches showed a pan-agglutination and incompatible crossmatch. anti-jk3 antibody high titer was detected in the plasma by gel-test using liss/coombs cards (id-diamed). the dat was negative and the antibody reacted equally with jk(a-b+), and jk(a-b+) panel cells (jka:1/16 384 and jkb:1/16 384). other alloantibodies could not excluded, because jk(a-b-) cells are not available. she was started with erythropoietin-a (epo), folic acid, fe iv and high dose intravenous immunoglobulin (ivig). the epo was discontinued after four week of therapy when the haemoglobin was 12 g/dl. two months later her haemoglobin was 13.5 g/dl and anti-jk3 was present in the same titer. a year later her blood cell count was normal and the anti-jk3 was detected in a lessened titer (1/16). no additional distinct anti-jka or anti-jkb component was shown after two adsorptions at 37°c using carefully selected phenotyped red cell compatible with patient's rh, fy, mnss, lu, le system and jka(+) and jkb(-), but two additional alloantibodies anti-c and anti-e of low titer (1/4) were revealed. the rare anti-jk3 alloantibody found in this case displayed the erratic nature of many kidd system antibodies. although anti-jk3 may cause mild hemolytic disease of newborn, she did not have a history of hdn. our patient was sensitized to a kidd antigen during pregnancy, but showed no serologically detectable antibody until challenged with a massive transfusion following a gastrorrhagia. the use of epo and high dose intravenous immunoglobulin succeeded to avoid transfusion with incompatible rbc unit. background: differential warm adsorption is used in the investigation of patients with red cell autoantibodies for searching of underlying alloantibodies, but it is also useful in the detection of clinically significant alloantibodies in patients with alloantibodies to high frequency antigens such as k, kpb, lub and inb. this technique is especially useful in cases when patients when patient's phenotype cannot be identified due to recent transfusion. purpose: differential warm adsorption is performing on cases presented with an antibody reacting with all red cells of the panel and having a negative auto control test. in these cases, even rare cells panels, which allow the identification of a pan antibody, are available, other more common clinical significant antibodies cannot be excluded. methods and result: case 1. a 63 years-old caucasian female with preexisting myeloproliferative disorder (polycythemia) presented with pancytopenia. anti-k was detected in the plasma. it is reported two pregnancies and no history of transfusion. the dat was negative and the plasma did not react with one k-cell present on the red cell panel in use. the anti-k specificity was confirmed using additional k-cells. the patient's red cell were group a, d+, k+, k-, c-, e-, fy(a-), s-, le(a-), kp(a-), cw-. she was transfused with two units rbc k-. ten days after the first transfusion the dat became positive and the one k-cell present on the red cell panel reacted with her plasma. two adsorptions were carried out at 37°c using carefully selected phenotyped red cell (compatible with patient's rh, fy, jk, mnss, lu, le system and k positive). an anti-fya was identified in the presence of ant-k. . an antik (titer 1/16) was suspected. because k-red cell was not present in the panel in use, adsorptions were carried out at 37°c using carefully selected phenotyped red cell (compatible with patient's rh, fy, jk, mnss, lu, le system and k positive). after the anti-k antibody was totally removed, no additional alloantibodies were revealed. conclusion: differential adsorption in cases with alloantibodies to high frequency antigens represents a useful application of the technique and helps in the identification of clinical significant antibodies present, allowing a more accurate decision for transfusion. evaluation of validity of the expired enzymetreated 0.8% red cells in antibody identification gel tests using nacl cards p chalkia, s intzepeli, v avgoloupi, a tsoukala, e ntinopoulou and p didoudi ahepa hospital, thssaloniki, greece background: expired red blood cells of required phenotypic profile is often used to identify antibody specificities in patients with multiple anti-erythrocytes antibodies. accurate results depend on the integrity of the antigens. purpose: to validate the expired enzyme-treated 0.8% red cells for use in antibody identification gel tests using nacl cards. the serum of nineteen patients with common specificities antibodies in rhesus, kell, duffy, kidd, and mnss systems tested with commercially prepared 0.8% enzyme treated cells rbc panel (id-diamed). gel tests were performed according to the manufacturer's instructions on in-date rbc and simultaneously on rbc 1 month to 5 months past expiration. reactivity of the expired antigen positive and antigen cells was compared to in-date cells. results: twenty antibodies detected with enzyme treated red cells in neutral gel cards [d(4), c(3), e(2), k(7), cw(4)] and were tested with enzyme treated red cells in use and rbc 1 to 5 months post expiration. seventeen antibodies tested with enzyme treated cells gave acceptable results with antigen positive cells 1 to 5 months post-expiration, except 3 anti-k antibodies (negative with k positive cells 2-4 months post expiration). conclusion: most rbc antigens studied were detectable 5 months after rbcs expiration date. tests with 0.8% cells were valid in gel test (nacl/enzyme) for at least 5 months after manufacturer assigned expiration date and may be helpful for complex identification studies. studies for more antigens specificities are needed to testify the validity of the expired enzyme-treated 0.8% red cells. background: wr(a) is a low-incidence blood group antigen (1 : 1000) in the caucasian population. despite that anti-wr(a) is a common antibody type, it may cause severe transfusion reactions, haemolytic disease of the new-born. anti-wr(a) may occur as an autoantibody or arise without immune stimulus. we report a case of a naturally occurring anti-wr(a) antibody. case report, methods and results: 56-year-old non-transfused male patient with acute pancreatitis and severe anaemia had been transferred to surgery from a county hospital. the serological status identified by their blood bank was: b rhd positive, with anti-wr(a) antibody in the serum (cellbind card method). our results: the patient's cells were group b rhd positive (microplate method), dat negative (tube and gel test method). the antibody identification showed positive antibody reaction with all enzyme treated test cells but negative reactions in liss iat (tube test) and gel iat (scangel and diamed). discussion: our routine tests for antibody detection didn't detect any specific antibody in patient's serum. he was transfused several units of blood, that was wr(a) negative and showed negative crossmatch reactions. the patient had no transfusion reactions. 10 days after the transfusion anti-wr(a) specificity was confirmed in the serum with cellbind test. only few test cell panels contain wr (a) positive cells, which are usually not present in commercial screening cells. in our case the cross-match was only performed for this patient because of the detected nonspecific antibody reaction in enzyme. the risk of transfusion reactions caused by rare antigens are particularly high in the type and screen cases. background: according to requirements of the french committee for accreditation (comité français pour l'accréditation cofrac, iso 17 025 standards), it is essential to use validated and standardised methods in immunohematology. this imposes, among various requirements, the knowledge of metrological tolerances for all the techniques. aim: a multicentre study was carried out to define the maximal acceptable deviations concerning incubation temperature and time, volumes of patient plasma and of tests cells for antibody screening using indirect antiglobulin test (iat) in filtration technique. the antibody screenings were performed manually in 3 blood centres using 3 different filtration systems: id diamed, biovue ortho and scangel biorad, the same tests cells, a standard 20 ng/ml anti rh1 (provided by cnrgs), a positive control anti kel1 and a negative control. all equipment used (oven, chronometer, pipettes) were calibrated according to cofrac standards. each antibody sample was tested under the following combined conditions (243 tests/sample): results: all the tests of antibody screenings from the multiples combinations of the above parameters gave the same results with a 2+ intensity agglutination for positive samples and the absence of agglutination for the negative control. conclusion: this study allowed us to define a range of tolerance for 4 critical physical parameters involved in the antibody screening in iat using commercial filtration systems maryvonne. the authors present a retrospective study involving 12 091 blood donors from the university hospital of coimbra during the year 2004. the incidence of weak d and rh (d) phenotype was determined in 1729 individuals who were rh (d) negative. the ab0 and rh(d) blood grouping was performed using a column gel agglutination card (diamed). the rh (d) typing was done using a anti-d polyclonal and a anti-d monoclonal antibodies. all donors that gave negative or poor agglutination results were tested for weak d with an indirect antiglobulin test, with anti-igg (gel matrix card) plus anti-d serum (diamed). within our target group of blood donors the rh (d) negative represented 14.3% of the total sample. we also describe ab0, rh(d) phenotype (c, c, d, e, e) group frequencies and establish reliable estimates frequency for weak d and rhesus haplotypes. the background: in 80s and 90s several authors tried to assess the relationship between the number od igg molecules per rbc and in vivo haemolysis, but determinations usually concerned small groups of tested patients. some of the investigators suggested that the number of igg autoantibody molecules per rbc was a major determinant of the severity of the haemolysis, whereas others found aiha patients with severe haemolysis and undetectable autoantibodies. aim: presentation of our experience with the quantitative elat performed on a large group of aiha patients during long-term observation. material and methods: six hundred fifty eight blood samples from 268 warm-type aiha patients were randomly tested for the number of igg molecules per rbc. eighty six of the patients were tested periodically from 3 to 15 times at one-month intervals. autoantibodies on rbcs were detected by the direct antiglobulin test (microcolumn technology) and measured by the enzyme-linked antiglobulin test (elat). results: in about 1/3 of tested samples the number of igg molecules per rbc was small (<190) and the laboratory signs of haemolysis were present in 65.7% of them as well as in 70.4% samples with moderately coated red cells (200-980 igg/rbc). the large number of igg molecules per rbc (>1000) was significantly associated with high frequency (87.9%) of severe haemolysis and it was also associated with presence of multiple igg subclasses on rbcs and c3d. in 79% of patients tested periodically, the number of igg molecules per rbc decreased and it significantly correlated with improvement of haemolysis parameters. in 21% of aiha patients the number of igg fluctuated and it was a poor prognostic factor. conclusion: in aiha patients the dynamics of the changing number of igg autoantibody molecules per rbc is a more helpful diagnostic and prognostic parameter than the number of igg molecules per rbc evaluated in one test. -b+) , -h-negative (using the anti-h lectin). antibody work-up showed a positive antibody screen (liss and peg-tube methods) reacting 4+ with o rbcs at all phases and 2+ with a1 rbcs. the direct antiglobulin test (dat) was positive with polyspecific ahg as well as anti-c3b,-c3d (table 1) . prewarming of test system did not change the reactivity (4+ at antiglobulin phase). a treatment with dithiothreitol (dtt) was performed and abolished all reactivity of the serum ( table 2 ). autoabsorption of the patient's plasma was performed. the absorbed plasma showed a decrease in reactivity from 4+ to 2+ when tested with o red cells, as well as a significant reduction in antibody titer from 1 : 256 to 1 : 2 tested at immediate spin (table 3 ). the patient remained crossmatch incompatible with o and a rbcs, but was compatible with oh rbcs. summary: we report an unusually strong igm anti-h antibody in this patient, who may require oh phenotype units. the patient is not a para-bombay since her red cells type strongly as group a. the cause for the auto-anti-h remains unknown at this time. if a thermal amplitude test shows that the antibody appears to be clinically significant the patient should receive h-units if transfusion is required. introduction: fetomaternal haemorrhage may determine an alloimmunization, in fact the transplacental passage of antibodies may cause the haemolytic disease of newborn. for this reason, in pregnant women, a screening for irregular antibodies research is routinely performed. however the indirect antiglobulin test (iat) may result falsely positive or negative for various causes, as operative mistakes or low specificity/sensitivity of the used techniques. aim of the study. in this study we have retrospectively evaluated the real incidence of alloimmunizations occurred in women screened by private laboratories. methods: we have studied 1.560 women, 19-43 years old, resulted iat positive at the first screening and successively assisted by our two hospitals. all women were re-tested, using gel-agglutination technique, for both direct antiglobulin test and iat. results: a positive iat was confirmed only in 64 cases; moreover a rbc autoimmunization was found in 7 women. anti-d (16 cases), e (10), c (8), k (8), c (6), s (5), d + jka (1), d + s + e (2), d + c + k (2), m (3), c + e (1), d + c + g (2) were the identified alloantibody specificities. anti-s, -e, -k, -c and -jka were the specificities in autoimmunized women. conclusion: in conclusion, a real alloimmunization is occurred only in 4.1% of screened women, while in the remaining cases iat resulted falsely positive: this observation forces us to affirm that, in order to minimize errors and alarmisms, the screening for antibody research in pregnant women must be performed only by immunohematology qualified center. background: one of the problems of the rbc transfusion is the alloimmunisation and the delayed haemolytic reactions (dhtr). besides rhesus and kell systems the antibodies against kidd antigens cause both dhtr and difficulties in their detection. aim and methods: the exact recording of all blood units according to abo rhesus kell and kidd systems. the abo-rh-kell systems are identified through automated microcolumn method (autovue, ortho), while kidd antigens are identified manually using microcolumn gel (diamed). results: the percentage of jka+ and jkb+ found in our department (75.8%) and (70%) respectively is similar to that of the caucasian population. conclusions: given the fact that there is lack of available freezing rbc system in greece, detailed recording of all units to the above antigenic systems can be proved extremely useful under circumstances of incompatibility. in the latter case suitable donors can be called and cover the shortage. the identification of all antigentic systems of the donated rbc units is underway. background: in many countries transfusion recipients are currently typed and transfused d-positive, if their red cells are agglutinated by 2 igm monoclonal anti-d that do not react with dvi. the transfusion strategy in weak d patients is not clear defined and it depends on the chosen monoclonal reagents and methods. patients who are carrying dw types 1, 2 and 15 were prone to develop anti-d. aim: the aim of this pilot study was to estimate capability of commercially available monoclonal anti-d reagents to recognize this weak d types as rhd positive. material and methods: edta anticoagulant blood samples were collected from 50 blood donors, previously typed as weak d positive by indirect antiglobulin test. molecular genotyping of rhd gene and weak d alleles by cde-ssp and d weak-ssp kits (inno-train, germany) were performed. direct agglutination was tested in a tubes and microplates using the following antibodies: rum-1, th-28, ms-201 (bioscot/serologicals) and d415 1e401/175-2 (immucor). results: out of 50 samples molecular typing results were as follows: in 5 samples dw were not determined, in 45 samples, 20 weak d type 1; 12 weak d type 2; 8 weak d type 3, 1 weak d type 11; 3 weak d type 14 and 1 weak d type 15 were determined. by all monoclonal reagents 95% weak d type 1, 100% weak d type 2, 10% weak d type 3 and 100% weak d type 15 negative results were given. by all monoclonal reagents weak d type 11 and weak d type 14 positive results were given. conclusion: according to weak d types, which were known to be at risk for anti-d immunization further advances may be brought by improved patient's monoclonal typing reagents with a low and donor's monoclonal typing reagents with high affinity for weak d type 1, type 2 and type 15. such improved typing strategies with novel reagents would enhance the transfusion safety. background: vel is a high-incidence antigen found in >99% of the population. anti-vel can be igm or igg and reacts optimally at iat, although it can also react at immediate spin and 37c. anti-vel may or may not cause severe hemolytic transfusion reactions and mild to severe hdn. autoanti-vel has also been reported. the aabb technical manual 14th edition states that the vel antigen is unaffected by protease and sulfhydryl treatment. we have reason to believe that sulfhydryl treatment may have an effect on the vel antigen as evidenced by a recently referred case. case report: a 67 year-old caucasian female presented with symptoms of anemia and renal vascular hypotension. transfusion history indicated multiple red cell transfusions in 1959. according to the patient, previous attempts to locate compatible units were unsuccessful. the case was referred to our laboratory. the patient's red cells (rbcs) typed as group o, d+ with a negative dat. the serological picture revealed an antibody reacting 2 + -3 + s at 37 c/liss, as well as at the antiglobulin phase. the antibody reacted with all rbcs tested and the autocontrol was negative. further characterization of the antibody showed similar reactivity using enzyme-treated rbcs (0.1% ficin) and no reactivity using 0.2 m dithiothreitol-(dtt)treated rbcs. a high incidence negative red cell panel (untreated) was selected that lacked antigens reported to be destroyed by dtt. the antibody reacted with all rbcs tested. additional rbcs were tested that lacked high-incidence antigens, including vel. the antibody did not react with four vel-rbcs tested using liss and peg methods. the patient's rbcs typed as vel-negative. all other clinically significant antibodies were ruled out using vel-or dtt-treated rbcs. based on the unusual reactivity demonstrated by the anti-vel, we tested 12 different examples of anti-vel (frozen in our rare sera inventory) against two sets of known vel+ and vel-rbcs. one rbc set was dtt-treated; the other set was tested neat. liss enhancement was used to test both sets. one of the 12 antisera failed to react with the positive control and one reacted with the negative control. both were disqualified from the study. four of ten remaining antisera demonstrated a decrease in reactivity >1 grade, between the neat and the dtt-treated rbcs. the remaining six antisera showed no change in reactivity. conclusion: contrary to the statement in the aabb technical manual, we discovered that sulfhydryl treatment (0.2 m dtttreatment) can have an effect on the vel antigen. our experience has demonstrated that in some cases anti-vel may not react or may show reduced reactivity when tested with dtt-treated rbcs. therefore, the presence of anti-vel should not be ruled out if negative reactivity with dtt-treated rbcs is encountered. additionally, dtt treatment may be a useful tool obtaining rule-outs of other clinically significant antibodies in the presence of anti-vel. additional data is needed to confirm these findings. but with no identified specific antibodies were investigated by repeated screening/crossmatch, papainized panel identification, hla antibody screening by lymphocytotoxicity test (lct) and elisa in some cases. patients' age, sex, department, diagnosis, previous transfusions/pregnancies, techniques, reactions' strength, number of positive cells, urgency, subsequent antibody tests, identification and lct were noted. antibody tests were performed: at pretransfusion testing (pt) by liss-coombs (diamed) and at blood grouping (bg) by biovue polyspecific (ortho) microcolumns -manually in urgency and routinely by sampler iif (diamed) and mitis (ortho) systems. results: investigated reactivity was recorded in 241 samples from 186 patients; 29 (15.6%) patients had > 1 episode. these findings comprised 15.1% of 1598 unexpected results found at pt and bg. incidences were 0.18% at routine and 0.21% at urgent bg (37 339 and 16 003 bgs, respectively), and 0.35% both at routine and urgent pt (15 803 and 23 705 pts, respectively). 56.5% patients were female, 44.6% over 60, but 19.9% < 30 years, coming mostly from surgery (19.4%), internal medicine (15.1%), hematology (10.8%), ginecology (10.2%), cardiac diseases (9.1%) and cardiac surgery (8.6% patients). frequent diagnosis were solid tumors (16.1%), cardiac diseases (13.4%), hematologic malignancies (8.6%), uraemia (4.8%), orthopedic surgery (4.3%) and hepatic diseases (3.2% patients). 43.0% patients were previously transfused, with only 9.1% patients proved as not transfused or pregnant. subsequently positive antibody test during the study had 27.6% tested patients. at pt positive crossmatch was found in 38.8%, antibody screening in 43.9% and both tests in 17.3% cases. majority of reactions were '1+' (50% at pt and 38.6% at bg); reactions '3+' or '4+' were found in only 5.1% cases at pt, compared to 17% at bg. one crossmatch only was positive in 75.2% positive crossmatches, with 38/43 patients having > 1 crossmatched unit. ahg identification was positive in 27.6% tested patients; in 26% of them papainized panel was also positive. lymphocytotoxic antibodies were found in 45.5% tested patients; 72.6% (8%-100%) of lymphocytes were reactive. finally, the cause of reactivity in antibody tests was determined as 'laboratory mistake' in 42.5%, hla lymphocytotoxic antibodies in 9.7%, 'igg antibodies of unknown specificity' in 7.5% (hla noncytotoxic antibodies in 2/2 elisa tested samples!), contaminated sample in 5.9%, anti-bga in 4.8%, non-specific cold antibodies in 3.8%, subsequently recognized specific antibodies in 3.2% (2 lua, 2 m, 1 kpa, 1 yka), non-specific autoantibodies in 3.2%, carry-over of dat-positive cells and antibody to reagent in 2.2% cases each, while in 4.3% cases antibody screening and in 4.8% cases crossmatch was repeatedly positive without confirmation in panels. discussion: after introducing of sensitive microcolumns, positive antibody tests without detectable specific antibodies require significant laboratory activities, particularly in older patients with malignancies or surgery. such reactivity was frequently caused by laboratory mistake, but often hla and sometimes specific antibodies were later recognized, or reactivity continued without confirmation in panels. relationships that may be helpful are further discussed in abstract part ii. results: significant differences (p < 0.05) and relationships of interest were noted. sex. in female vs male patients frequent features were: crossmatch as only reactivity at pt (45.8% vs 28.2%), positive lct (59.4% vs 26.1%), lymphocytotoxic hla antibodies (lytxab) (13.3% vs 4.9%) and reactivity 'positive screening, negative panels' (5.7% vs 2.5%); in males non-specific cold antibodies (7.4% vs 1.0%) and antibodies to reagents (4.9% vs 0) were noted. age. in patients > 60 vs < 30 reactivity was often found at pt (65.1% vs 35.8%), caused by lytxab (21.7% vs 5.4%), anti-bga (8.4% vs 2.7%) or 'positive crossmatch, negative panels' (9.6% vs 0), but rarely by laboratory mistake (37.3% vs 59.5%) or later recognized antibody (1 of 6 patients). subsequent antibody tests: tests were subsequently positive more often if reactivity was found at pt (79.3% vs 20.7% at bg), as positive antibody screening (41.7% vs 29.2% if positive crossmatch), with positive panels (48.3% vs 12.2% if negative). subsequent tests were positive in only 43.8% patients with lytxab, 50% with anti-bga, 1 of 3 with antibodies to reagent and in no case with 'positive crossmatch, negative panels' . techniques. lct was positive in 60% and 45.5% tested samples found at urgent and routine pt (diamed), vs 0 at bg. all 18 cases due to lytxab, 8 of 9 anti-bga and 6 of 8 'positive antibody screening, negative panels' were found by diamed. at bg (ortho) 71.4% cold antibodies and 64.6% laboratory mistakes were found. positive antibody test: identification was negative in 78.6% screening-only cases; 69% of them were caused by laboratory mistake. lytxab were found in 67.9% crossmatch-only cases; 77.8% reactivities caused by lytxab were crossmatch-only. identification. ahg panel was positive in 60% cases with lytxab (in 2 with papainized panel) and often due to 'igg antibodies of unknown specificity' (29.7%), anti-bga (24.3%), contaminated sample (16.2%), but also to later recognized specific antibody (10.8% cases). strength of reaction: laboratory mistake was noted in 61.6% of 'w', 41.3% of '1+', 46.4% of '2+' and 58.8% of '3+ and 4+' antibody screenings (ns). diagnosis. in patients with solid and hematologic malignancy reactivity was often found at pt (80% and 81.3% of patients, respectively), due to positive crossmatch (40% and 50%; 0 and 4.3% patients with liver and cardiac diseases) and caused by lytxab (16.7% and 31.3%) or 'crossmatch/screening positive, panels negative' (16.7% patients with solid tumors). in patients with liver and cardiac diseases reactivity was often found at bg (83.3% and 68.0% of patients), due to laboratory mistake (50% and 60%) or cold antibodies (16.7% and 12%), respectively. discussion: features of non-specific reactivity depended on sex, age, positive antibody test, diagnosis and, moreover, used techniques, sometimes in very distinctive manner. this analysis might be of considerable help in planning of laboratory tests, but also in quick analysis of unexpected results and choice of further testing, particularly in urgent situations. background: worldwide screen and type is a very usual method for pre-transfusional testing. the ultimate objective is to prevent not only the clinically expressed delayed hemolytic transfusion reactions but also the serologically revealed ones. aim: the aim of this study was to determine the frequency of red blood cell (rbc) alloantibodies in patients undergoing cardiac surgery or cardiac procedure, during the pre-transfusion screening. materials and methods: blood samples of 16 291 patients (12 628 male and 3663 female) were evaluated. the mean age of the patients was 57 years. pre-transfusion samples were examined for clinically significant alloantibodies, using antibody screening with gel test (liss -enzyme). in case of a positive result, identification was performed (panel with autologous control). in addition, the serological testing included cold agglutinins´ detection (tube test), as well as titration (tube test) and identification (gel test) in case of a positive result. when the result was marginal (1/32) a new test was carried out after a seven days period. in the presence of a positive autologous control or an autoantibody, samples were examined with direct antiglobulin test (dat). results: alloantibodies were detected in 580 patients with the incidence of 3.56%. antibodies were registered more frequently in females (190/3663, 5.18%) than in males (390/12 628, 3.09%). 183 patients (31.49%) developed single antibody with anti-kell being the most frequent. the incidence and the specificity of the detected antibodies are summarized in the following table (table 1 ). in 23 patients (3.96%) multiple antibodies were detected, with most frequent the anti-d and anti-c combination. 35 patients (6.03%) were dat positive. autoantibodies were found in 10 patients (1.72%), all of which had specificity to rhesus system. cold agglutinins were positive in 53 patients (0.32%). no specificity could be assigned in 245 patients (42.24%), while in 110 patients (18.96%) non specific reactions in enzyme treated rbcs, were observed. one patient developed delayed haemolytic reaction 15 days post-transfusion, due to anti-jka. the antibody, however, was not detected in the pretransfusion sample re-testing. the frequency of the pre-transfusion detection of red blood cell alloantibodies in our center, was 3.56%. the most frequently identified were the anti-kell and anti-rh. the high rates of unidentifiable antibodies and non specific reactions in enzyme treated rbcs are probably attributed to the kind of medication that most of these patients receive, as well as to the degree of inflammatory process which usually accompanies such diseases. the high frequency of unidentifiable antibodies indicates that a larger and more complex erythrocyte panel would be useful for routine testing. the routine pre-transfusion screening for alloantibodies probably assures the prevention of dhtrs and provides sufficient time for blood selection for transfusion. introduction: in the united kingdom, about 1% of women form red cell allo-antibodies in pregnancy and 0.07% of all pregnant women produce anti-c. before the introduction of prophylactic anti-d, it was reported that 3% of the total haemolytic disease of the newborn (hdn) cases were due to anti-c. currently, cases of hdn due to anti-c are half as frequent as anti-d and 14% of the uk population are rhc negative. we present two unusual cases of pregnant women who are d and c negative and have anti-c and anti-d detected in their serum. case studies and results: case 1: a 38-year-old asian woman had three previous uneventful pregnancies. in her 4th pregnancy she presented with miscarriage at 19 weeks gestation. she was group b, d and c negative. her serum contained anti-c and anti-d. anti-d was detected by liss tube iat and anti-c was only detected by manual polybrene technique (0.9 iu/ml by quantification using r2r2 cells). in a 5th pregnancy, no antibodies were detected until 34 weeks gestation when this patient presented in early labour. anti-c was then detected by two-stage papain technique only as well as anti-d. case 2: a 33-year-old asian woman had a positive antibody screen post caesarean section in jan 2005. no antibody had been detected during the pregnancy. standard prophylactic anti-d was given at 28 and 34 weeks gestation as this patient was d neg. anti-c and anti-d were confirmed in her serum. the anti-c level was 0.7 iu/ml using rr cells and the anti-d level was <0.1 iu/ml using r1r1 cells (prophylactic anti-d ig). she was phenotyped as o r¢r¢. at delivery the baby was found to have a negative dat and was phenotyped as r¢r. discussion: cde/cde (r¢r¢) is an uncommon phenotype in the uk population with a frequency of 1/10 000. in routine antenatal testing, abo/d grouping is only performed for pregnant women at booking and 28 weeks gestation according to bcsh guidelines. full rh phenotyping is not carried out unless the pregnant women has a positive antibody screen. in routine testing of the above cases, this extremely rare phenotype is missed. prophylactic anti-d was given to both patients and immunisation due to anti-d was prevented. there is currently no prophylactic regime developed to prevent anti-c allo-immunisation by the fetus in pregnancy. antenatal management of patients with anti-c and anti-d during pregnancy can be problematic: (i), problem in antibody identification; (ii) monitoring of antibody level (i.e. quantitation by auto analyser for anti-c and anti-d) with two different cells and (3) provision of blood during pregnancy, at labour and post delivery for both mother and newborn. study on the frequency of red cell phenotypes (e.g. duffy, kidd and mns blood group system) in our local population l leou, yf wong, mbc koh and d teo health sciences authority, singapore, singapore background: the frequency of various red cell antigens in the caucasian population has been well studied. to date, the frequency of these antigens in the local population composed of a multi-racial mixture of chinese, malay, indian and others is still unclear, especially in the malays with paucity of data in the literature. aims: to investigate the frequency of clinically significant red cell antigens duffy, kidd and mns across the local ethnic groups. to investigate the occurrence of rare phenotypes. to be aware of these rare phenotypes so as to facilitate planning of blood inventories and supplies. typing for the duffy, kidd and ss antigen on blood donors was performed using monoclonal as well as polyclonal anti-sera by manual tube method. a total of 1427 blood donor samples were tested using specific anti-sera that will agglutinate red blood cells that have the corresponding antigen. agglutination is demonstrated by the indirect antiglobulin technique. result and discussion: table 1 -a higher frequency of the fy(a+b-) phenotype is seen in chinese, malay and others in contrast to the indian and caucasian population. the clinically significant allo-antibody anti-fya is rarer in our local population. it occurs predominantly in malays and indians and usually in combination with other antibodies. it means that provision of antigen negative blood may be difficult. table 2 -all groups show similarity of distribution of the kidd phenotype with the caucasians and distinct from the american blacks. the jk(a+b-) and jk(a+b+) phenotypes are relatively equal in frequency and there should be no problem looking for such a phenotype in the local population. table 3 -the s-s+ phenotype is most common amongst all races. the indians are more similar to the caucasians with a relatively high frequency of s+s+ phenotype. the chinese and malay distribution are unique with > 99% being s+. conclusion: there is a unique distribution of red cell antigen groups in the 4 races and the data on malays is especially useful. this data will allow the national blood service in its inventory planning and the potential difficulties of providing antigen negative products due to clinically significant allo-antibodies. miltenberger phenotypes among taiwanese table 1 . conjointly, in order to obtain the frequency of mi.v phenotype, we screened 543 samples among 1230 with anti-hil and four additional cases of mi.v were found. conclusion: in this study significant miltenberger polymorphism was seen among the taiwanese population. besides previously described mi.iii phenotype (1.9%), there were also mi.i/ii phenotype (0.4%), mi.v phenotype (0.7%), mi.vi phenotype (0.7%), mi.x phenotype (0.1%), and most interestingly two miltenberger related not yet classified variants (1%). related variant a (0.5%) was phenotypes as mia+, anek+ and hil+. related variant b (0.5%) was phenotyped as mia+, anek+. interestingly, both variants were mur-(negative). the total estimated frequency of miltenberger variants in taiwanese population (including related variants a and b) is therefore 4.8% (table1). the discovery of 2 unclassified variants (most likely not yet described in the literature) is of great interest in the field of immunohaematology and warrant further molecular genetic study. introduction: the use of column technologies for the detection of rbc antibodies improved significantly the screen test sensitivity. each column-based method has its advantages and disadvantages. aim: to compare antibody detection by two column agglutination tests; the fully automated ortho auto vue tm method and the manual diamedᮀ id-micro typing system. material and methods: during the study period 57 375 patient samples were screened, 414 of the positive results were evaluated. 377 blood samples with positive screen tests by the ortho auto vue tm method (av) performed with 3% cell suspension, and 37 patient samples with antibodies identified by the diamedᮀ system (dm), were reciprocally re-screened, respectively. positive samples were tested by diamed panels for antibody identification. sera samples were divided into 6 categories according to antibody specificity; 1. rh system antibodies (n = 150), 2. clinically significant non rh system antibodies (n = 85), 3. clinically non significant antibodies (n = 28), 4. auto antibodies (n = 13), 5. not identified (ni) antibodies (n = 48), 6. negative screening results by the diamed technique (n = 90). the 6 categories were divided, according to the intensity of agglutination in the screen test, into those exhibiting stronger reactions by the auto vue (av > dm), those exhibiting equal strength reactions (av = dm) and those with weaker reactions by the auto vue (av < dm). statistical analysis was carried out using the wilcoxon signed ranks matched-pairs test. results: a total of 414 samples were compared, 31.2% of them gave stronger reactions by av, 45.4% gave equal reaction strength and 23.4% of them gave weaker reactions by av. positive screen tests by av only were detected in 90 samples, no specific antibodies were identified. in contrast to that, positive screen tests by dm only were detected in 9 samples, 5 were rh system related, 3 kell system related and 1 not identified, results are summarized in the table. discussion and summary: the antibodies detected by dm only, are anti-d and antibodies directed to low frequency antigens. the failure of av to detect rh system antibodies is further sustained by the fact that statistically significant weaker reactions for this antibody system were obtained by av technique. recently ortho-clinical diagnostics modified the screening reagent red blood cells to 0.8% suspension, in order to improve the sensitivity of the method (unpublished data). the possible explanation for the failure to detect low frequency antibodies is that ortho screen cells do not consistently carry the low frequency antigens cw and kpa. positive screen tests detected by av only, can be explained either by the fact that antibody identification was carried out on dm panels, or these are false positive reactions. in order to clarify this question we recently repeated these av only positive samples by the manual ortho bio vue technique. preliminary results indicate that no specific antibodies were detected. it can be assumed that these results are false positive. further study of this issue is required. aim of the study: we have detected blood donor with rohar variant which was mistaken as d-and his donations used for d-recipients. we tested these patients in order to evaluate possible anti-d or anti-lfa immunization. methods: rohar variant was tested serologically (commercial and workshop moabs) and on dna level (pcr-ssp). involved recipients were tested by diamed column agglutination (gliat with normal and enzyme treated rbcs) with commercial rbcs and with rh33 and rh50 rbcs. results: rohar variant was confirmed on phenotype and genotype levels. in four d-and two d+ recipients of rohar positive units no anti-d not anti-rh33 or -rh50 antibodies were detected. in one case anti-le(a) antibody was found. conclusion: in our cases massive exposition of recipients (whole transfusion unit) by rohar red cells did not lead to production of detectable anti-d or anti-lfa. the immunogenic potential of this variant seems to be low. unusual ab0 grouping discrepancy -inhibition of anti-b reagent by isolated increase of plasmatic b substance in a patient with group ab and pancreatic cancer m pisacka*, k petrtylova † , m kralova* and h flidrova* *uhkt, prague 2, † blood bank, faculty hospital m, prague 5, czech republic introduction: in rare pathological conditions excess amount of blood-group specific substances can be observed and can cause neutralization of grouping reagents. changes in abh and related histo-blood group antigens in malignant tissues were described but there are few information about similar changes in secreted bloodgroup specific substances. aim of the study: we describe a case of isolated increase of b group substance in plasma of a group ab patient with pancreatic cancer. methods: ab0 grouping was performed with registered immucor reagents (immuclone: anti-a birma 1, anti-b lb2) by slide and tube tests. neutralizing effect was quantified by (i) inhibition od anti-b reaction by titrated patient's serum; and (ii) inhibition of titrated anti-a and anti-b reagents by patient's serum, compared to ab serum of a healthy donor. results: slide test: unwashed rbcs: group a, washed rbcs: ab. tube test (washed rbcs): ab. titrated patient's serum when added in aliquot to anti-b reagent inhibited agglutination up to titre 64. titration of reagents /+ aliquot of serum added/: anti-a: titre 4096 (both patient's serum and control); anti-b + patient's serum: titre 4, anti-b + control: titre 4096. conclusion: pancreas is known as rich source of blood group specific substances. malignant transformation is known to be associated with either loss or re-expression of cell-bound abh antigens. reported excessive increase of b substance in group ab patient could be either due to loss of a-transferase activity in malignant pancreas cells or isolated increase of b-transferase activity. further studies on larger groups of pancreatic cancer patients will help to understand this observation. weakened abh reactions of unwashed rbcs could be of diagnostic importance, because in other case this observation preceded several years the pancreatic cancer clinical manifestation. implementation of the autovue innova, an upgraded column agglutination technology (cat) for pre-transfusion testing in a large blood establishment c politis, k armyros, a antypas, v malamou and p katsea g. gennimatas general hospital, athens, greece objective: automated cat testing in a blood transfusion laboratory aims at standardization and savings in labour as well as reagents. a new technology is evaluated in comparison to standard methods. materials and methods: we used column agglutination technology with the innova autovue system, ortho, ratrian n.j. according to the manufacturers this system provides priority to the management of the stat samples while the random access feature of the system enhances the system throughput. it provides an extended test menu as well as automated antibody identification with the red cells panels. the autovue innova system supports the bi-directional communication with the lis interface for all the tests including the crossmatches and it provides a continuous traceability and notifying of the instrument's status concerning either the required and available resources or the proper function of the system's submodules. in this study we performed 1423 tests including forward and reverse abo group, rh type and phenotype and kell in randomly selected blood donors, as well 895 tests in haematological patients for antibody screening using an untreated three-cell panel and autocontrol in the indirect antiglobulin test (iat). the results and the time performance (specimen handing, operation of testing and recording of results) were compared with those obtained by the semiautomatic id-diamed gel agglutination microtyping system and by standard manual methods. results: test results showed 100% agreement between all three methods. 68 samples were tested per 60 min with the autovue innova, compared to 90 min required for the same number of tests performed with manual testing and 70 min with the semi-automated method. the technical execution was easy with the autovue innova procedure and it appears that the consumption of testing reagents is smaller with the automated system comparing with the other methods. computerization of the test results with the autovue innova provides an important advantage in record keeping in the blood establishment. conclusion: standardization of sample collection and tests performance in pre-transfusion testing, as well as computerized records and time saving are advantages offered by the autovue innova, a new automated column agglutination technology, comparing with a semi-automated and the classical manual methods. a heavy workload is expected to significantly decrease time performance. clearance of senescent erythrocytes in young and old individuals al racca, a ensinck, c cotorruelo, s garcía borrás, l racca and cs biondi universidad nacional de rosario, rosario, argentina introduction: after a lifespan of 120 days, human red blood cells (rbc) are captured and phagocytized by monocytes/macrophages. the accumulation of autologous igg on rbc membrane provides a direct mechanism for the removal of senescent (se) rbc. an alternative pathway, immunoglobulin-independent, with participation of sialic acid, has been proposed. the physiological elimination of serbc might be modified by individual's age. aim of the study: to investigate in young and old individuals, the interaction between monocytes and different erythrocytes suspensions: serbc, rbc stored with or without serum and desialinized rbc. methods: healthy individuals blood samples (20-40 years old, n = 50 and > 70 years old, n = 49) were studied. different suspensions from each sample were obtained: (i) se and young (y) rbc by differential centrifugation; (ii) rbc stored with its own serum (rbcs) and without serum (rbcws); and (iii) rbc desialinized with neuraminidase (ne) and tripsine (t). the suspensions were subjected to the erythrophagocytosis assay: peripheral blood monocytes were incubated with the different erythrocyte suspensions for 3 h at 37°c. two hundred cells were analyzed to determine the percentage of active monocytes (am) with phagocytosed and adherent red cells. non sensitized rbc (nrbc) and ex vivo sensitized rbc (srbc) were used as negative and positive controls respectively. results: the% of am obtained with old individuals were: serbc: 21.9 + 1.0, yrbc: 2.9 + 1.2, rbcs: 16.2 + 1.4, rbcws: 3.1 + 0.8, nerbc: 11.1 + 1.3, trbc: 3.2 + 1.1, nrbc: 3.0 + 1.2; srbc: 31.2 + 1.8. the values of am obtained with young individuals were: serbc: 17.1 + 1.5, yrbc: 3.1 + 0.9, rbcs: 11.1 + 1.1, rbcws: 2.8 + 1.2, nerbc: 10.8 + 1.4, trbc: 3.5 + 1.0. nrbc: 2.9 + 1.3; srbc: 30.5 + 1.7. conclusions: no differences in the% of am were found when compared to positive and negative controls, indicating that this assay would not detect variations in the phagocytic activity of monocytes from young and old donors. the values of am with serbc were higher (p < 0.001) than those obtained with yrbc in both populations analysed. the rate of erythrophagocytosis with serbc in old individuals was significantly higher (p < 0.001) than that obtained in young donors. the increase observed may be due to agedependent changes of rbc that occur with human ageing. the% of am with rbcs were higher (p < 0.001) in old individuals. no modifications were observed with rbcws. the significant increase in the rate of erythrophagocytosis with serbc and rbcs show the involvement of autologous igg in the selective removal of erythrocytes. these values were higher in old individuals indicating that this process would increase in aged donors. the tripsine activity was not enough to modify the% am. the values of am obtained with neuraminidase treated rbc were higher than those observed with yrbc (p < 0.001). however these values were similar between young and old individuals, suggesting that the desialylation would not participate in the increased removal of erythrocytes observed in old donors. cell-cell adhesion is a crucial phenomenon for the relationship between the cell and its environment. therefore, the development of experimental methods to obtain quantitative parameters of cellular adhesion is important. erythrocytes are widely available and their membrane properties are well known, so these cells are used as an ideal model for studying the cellular interaction mechanisms. the study of the formation and break-up of receptor-ligand bonds in sheared erythrocyte suspensions is a subject of considerable importance in the blood circulation, where formation and break-up of blood cell aggregates occur in a variety of physiological and pathological conditions. the main two objectives of this work were to study the cellular adhesion phenomenon using erythrocyte adhesion mediated by monoclonal anti-a antibody as a model and to achieve quantitative values of the parameters involved in the intercellular binding and its possible relationship with cellular deformability parameters. agglutinates of two a erythrocytes (doublets) induced by specific monoclonal antibodies anti-a were used. adhesion energy was indirectly quantified by the study of doublet dissociation under the effect of a given shear stress using a controlled flow chamber system. the chamber was installed on the stage of an optical inverted microscope (union optical, magnification 100¥) in such a way that the cover glass was the floor of the microchannel. a ccd (charge coupled device) camera was placed in the ocular tube of the microscope and connected to a digital image processor (ipplus system) to digitize, record and analyze microscopic images. the doublets were initially immobilized inside the chamber, and then put under different shear stress values by a controlled laminar flow during a definite time. in this way, a shear stress parallel to the contact surface between both cells was applied, observing that the upper cell detached progressively with time and also with a gradual rise of the shear stress. the sequential microscopical images were registered and digitally processed, measuring the geometrical dimensions that the cells acquire during the deformation process, and the dissociation of the antigen-antibody bond. digital image processing allows the analysis and the quantification of the red blood cells doublets dissociation phenomenon. these results show that, while the shear stress applied is raised, the contact area between both cells diminishes. the obtained parameters give important information that lead to the estimation of the value of adhesion energy between two red blood cells agglutinated by monoclonal antibodies. as consequence, they will allow the characterization of the antibodies used, since it would evaluate their association capacity with cellular antigens. glycophorins (gp) a, b and c are abundant transmembrane integral proteins in red blood cell (rbc). their highly glycosylated nature and high sialic acid content account for the net negative charge of mature rbc membrane, which is physiologically important because it impedes any tendency to stick together in the circulation. in this study, we have tested anti-gp specific mouse monoclonal antibodies (moab) as primary antibody to directly agglutinate rbc. fret technique has been developed to characterize the hemoagglutination based on the interaction of fluorophores (alexa488tm, dio and dii) located in the rbc membrane or combined to secondary antibody directed against the primary agglutining moab. combined intensity (spectral) and lifetime (flim) imaging was used to discriminate the fret signal of molecules on their different lifetimes whereas their emission spectra overlap (alexa488tm or dio/dii) as independent phenomena of the fluorophore concentration and photobleaching. furthermore, gp-cytoskeleton interactions were analyzed by 3d-fluorescence microscopy after lipid extraction with triton x-100 in red cell. all the antibody used was found to directly agglutinate the human rbc. in view of the fluorescence properties depicted in rbc for the pair of fluorophore alexa-488tm (donor) and dii (acceptor), it may be expected that upon agglutination of rbc, effective fret should be observed. flim in dynamic-state provides a discrimination of molecules in their fluorescence lifetime, which allows to evaluate the underlying mechanism of energy transfer process in the agglutinated erythrocytes. the results demonstrate that's upon excitation at 780 nm the flim-fret from alexa488tm to dii for the anti-gpb moabs only with a lifetime distribution in the picosecond range. similarly, effective fret was not observed for the anti-gpa moabs. the contrast in measured lifetime image is a reliable indicator for spatial variations in donor-acceptor association. 3d-fluorescence microscopy images showed interactions between gpc or gpb and cytoskeleton and did not show interactions between gpa and cytoskeleton. all together, these results only revealed by fret-flim and 3d-fluorescence microscopy strongly support the importance of the specific reactivity with glycophorin a or b of agglutining moab. introduction: the frequency of alloimmunization to red blood cell antigens in transfused sickle cell patients can range from 10 to 40%, but the development of autoantibodies is much less recognized and descried. aim of the study: in order to evaluate the autoantibody formation and observe the blood transfusion association, we analyzed the direct antiglobulin test (dat) as well as the antibody screening results in 612 transfused sickle cell patients. methods: all the patients included in the study had received at least 1 unit of red blood cell concentrate, on the majority of the cases matched for the c, c, e, e, k antigens. the transfusion range was 1-94 units. the dat performed was a polyspecific gel test. in cases of positive dat, was performed the monospecific gel test (igg, iga, igm, c3c, c3d) . in almost all of cases an acid glicin elution was performed and the eluate was tested against a red cell panel (liss/coombs and papain). an antibody screening by gel test (liss/coombs and papain) was performed in all the 612 patients. the dat was positive in 98 (16%) of the 612 patients. in 96 cases (98%), the dat was igg type, in 1 case (1%) igg + c3d and in 1 case (1%) igg + c3c + c3d. the acid elution was performed in 97 cases. the eluate was positive in 38 cases (39%). in 32 (84%), of the 38 cases, autoantibodies were pointed out whose specificity were 25 specificity public, 5 anti-e (rh5), 1 anti-c (rh2), 1 anti-ce (rh7). in 6 cases (16%) we found alloantibodies whose specificity were 2 anti-e(rh3), 2 anti-k(k1), 1 anti-c (rh2) and 1 anti-jka (jk1). in the 59 cases (61%) no antibody was identified on the eluate and the cause of positive dat is unclear. we could correlate the positive dat with a presence of autoantibodies in 32 (5.2%) of the 612 patients. of these, 13 (40%) had a blood transfusion association. the global frequency of red cell alloimmunization in this set of 612 patients was 15%. seventeen patients (53%) of the 32 patient who had autoantibodies had also alloantibodies associated. conclusion: the mechanism by which erythrocyte antibodies form in association with blood transfusion are not well understood, but even though the medical literature indicates strong association with blood transfusion as well erythrocyte alloantibody formation, we could not find support for this association. the loss of splenic to sickle cell patients could be important because experimental studies suggest that the spleen is involved in the regulation of autoantibody formation. forward and reverse blood grouping with lateral flow based assays p schwind*, i aebischer*, k loester † and p monod* *medion diagnostics gmbh, duedingen, switzerland, † prisma diagnostika gmbh, berlin, germany background: recently, a lateral flow assay for simultaneous typing of abod, rhesus subgroups and kell with stable end-point and without a centrifugation step was presented (loester k, fleischhauer s, schwind p: lateral flow assay for simultaneous typing of of abo, rhesus subgroups and kell. vox sang 2004, 87 (suppl. 3), 40). in many countries, the determination of isoagglutinins in addition to the red cell antigens is mandatory for abo grouping. aims: to develop a lateral flow test for reverse grouping, supplementing the lateral flow typing assay. a lateral flow device was constructed with a separation membrane equipped in a cassette housing having 4 distinct incubation wells, application zones and detection areas. 25 microliters of 5% suspensions of reagent red cells for reverse grouping (reverse-cyte a1, a2, b, 0, medion diagnostics, switzerland) are mixed in each incubation well with 100 microliters of plasma. the resulting suspensions are incubated for 2 min, followed by the transfer of 50 microliters each to the application zones, where migration starts immediately. results can be read after 3 min in the detection areas. a positive result is recognized as a distinct red dot, a negative result is monitored by the absence of a dot. results: the plasmas of 80 donors, previously determined for the respective blood groups and isoagglutinins by the tube technique, have been tested with this method. the results for both methods were in full agreement. conclusions: a simple, rapid and flexible lateral flow method for reverse grouping is presented, allowing now for the determination of forward and reverse typing in similar formats. both methods give results after 5 min with stable end-points without the need of a centrifugation step and are easily applicable to non-laboratory environments. the performance of the autovuetm system for red cell antibody screening of blood donors during background: in israel every donation is tested for the presence of red cell antibodies (rbc abs). screening is performed on the autovuetm system, using ortho 2 screening rbc since the year 2000. aim: (i) to summarize the performance of the autovuetm system for rbc abs, during 4 years (2000) (2001) (2002) (2003) (2004) . (ii) to evaluate if an initial low positive result, with two negative repeats, could be considered a negative result. methods and results: rbc abs screening was performed using igg cassettes. positive results were confirmed by diamed gel cards, with 3 screening rbc, followed by diamed panels for abs identification. results: summary of the results is presented in the table. (i) in 0.57% of 1358,769 blood donations that were screened, during 2000-2004, using the autovuetm system, a positive initial result for rbc abs was detected, which was confirmed in 40.2% (0.22% of the donations). an increased percentage of confirmed tests is noted, since 2003, probably due to an improvement in the manufacturing of the cassettes by ortho. (ii) during the validation, 83 samples with low positive results (agglutination degree of 0.5) were retested twice on the autovuetm. 57/83 (69%) gave negative results in both repeats. only 1/57 was confirmed positive and anti-m was identified by diamed gel test. the remaining 26 samples had at least one positive repeat. 5/26 (19.2%) of those samples were confirmed positive. summary: autovuetm can be used as a competent method for rbc abs screening, in blood services which require high thoughput automated systems. samples with a low positive agglutination, which were negative in two repeats, can be considered negative. retesting these samples on the autovuetm, can reduce the number of tests sent for confirmation and allow early release of blood components. rk tagi-zadeh*, aa karimov*, ar hasanov † , ly novruzova* and si donskov ‡ *hematology and transfusiology, baku, † blood transfusion centre, ganja, azerbaijan, † centre for hematology, moscow, russian federation background: the main method of treatment of severe homozygous thalassemia forms at the moment is still an anemia control with regular rbc transfusions. the use of adequate transfusion regime for these patients not only prolongs their lives but also promotes normal physical development of the children and improves the quality of their lives. however, necessity to do multiple transfusions increases a risk of post-transfusion reactions and complications due to alloimmunization to rbc antigens. in order to prevent posttransfusion reactions it is essential to know the frequency of blood group distribution in the region and then implement organizational procedures to enhance the transfusion services for these patients. objective: examine the distribution of rbc antigens among thalassemic patients and blood donors. methods: 108 blood samples of homozygous betta-thalassemia patients (who stayed at the daytime inpatient wards of scientific research institute of hematology and transfusiology baku, azerbaijan) and 1690 blood donors of azeri nationality were examined. 108 patients' blood samples were typed on rbc rh (c, c, d, e, e) and kell (k, k) antigens. gel test bio-rad (france) was used to detect rbc antigens. results: phenotyping of the patients' rbc rh antigens (d, c, c, e, e, cw) revealed that frequency of occurrence of the antigens in general was higher than in the donors. studying of rh phenotype distribution showed that the patients' ccdee (44.1%) •• ccdee (30.4%) phenotypes were twice as much higher than the occurrence of those in the donors (20% and 16.5% respectively). phenotype ccdee occurs more frequently in the donors (32%), whereas it is significantly rare in the patients (8.8%). the similar picture was observed in relation to ccdee and ccdee phenotypes, which occurred more frequently among donors (11.8% and 10.6%) than in thalassemic patients (6.86% and 2.94%). additionally, the results demonstrated that k antigen of the kell system was not detected in the thalassemic patients whereas k antigen was detected in all the patients. the same picture was observed with two other antigens of this system. thus among the patients typed on rbc antigens kpa antigen was detected in just one case whereas kpb was detected in the rest the patients. the results of the analysis showed that for thalassemic patients with phenotypes ccdee and ccdee it is easier to find compatible blood than for patients with phenotypes ccdee, ccdee and ccdee. furthermore, it is known, that some congenital diseases are genetically connected with the specific group systems, for example, the mcleod syndrome is genetically associated with the sharp suppression of the kell alloantigen expression (absence of gene kx). the fact of the discovered absence of antigen k in homozygous bthalassemic patients makes it possible to assume, that this group of patients suffer from scarcity along the kell system, like the scarcity in mcleod syndrome. all the mentioned above make it possible for us to conclude, that prior to blood transfusion to thalassemic patients phenotyping of rbc rh and kell antigens must be carried out. hyperhaemolysis in sickle cell disease due to complement activation. tessa thorp rci national blood service manchester uk tm thorp national blood service, manchester, uk introduction: sickle haemoglobin is caused by a genetic mutation in codon 6 of the beta globin gene, resulting in the conversion of glutamic acid to valine. when critical amounts of polymer accumulate within the sickled erythrocyte cellular injury results. clinically sickle cell disease is characterised by chronic haemolysis and intermittent vaso-occlusion. mold et al. demonstrated that deoxygenation and sickling of erythrocytes is related to membrane phospholipid changes and these changes result in the activation of the alternative complement pathway. aim: the objective of this study was to measure the amount of c3c and c3d bound in vivo to red cells of homozygous and heterozygous sickle cell patients. these levels were then compared to 'normal' sickle negative blood donors. haemoglobin levels and red cell morphology were also examined for signs of active haemolysis. the hypothesis was that an increase in red cell bound complement in vivo could provide an indication of hyperhaemolysis syndrome is sickle patients. method: flow cytometric analysis using rabbit anti-c3c or anti-c3d and fitc labelled goat anti-rabbit was developed using a beckman epics xlmcl flow cytometer. control samples were prepared using a fruitstone buffer technique of complement coating. results: a total of 16 heterozygous sickle patients and 11 homozygous patients were analysed. of the 11 homozygous patients analysed only one was undergoing a sickle crisis. a positive result was indicated if the mean trait or homozygous sample was coated with complement to a greater degree that the mean normal donor plus two standard deviations. the results obtained in this research project indicate an increase in c3c levels bound to red cells of homozygous sickle patients in vivo. statistical analysis of results obtained suggest that there was no increase in c3d levels in either sickle trait or homozygous sickle patients. conclusion: these findings support research carried out by mold et al. (1995) and are present in more than 90% of caucasian population. it has been reported that anti-chido and anti-rogers don't cause hemolitic reaction but may be responsible for life-threatening anaphylactic reaction during transfusion of plasma proteins. case report: positive iat and dat were detected in a 34 years old swedish woman, a rh negative, at 34th week of pregnancy. previous iat and dat determinations were negative. at time of detection dat wsa weakly positive and igg subclasses identified. antibody identification revealed the presence of high titre (1 : 64) anti-chido and anti-rogers antibodies. a slight decrease in platelet count (from 210.000/ul to 80.000/ul) was observed. this pattern showed no variation until the end of the pregnancy. at 39th week a healthy baby was delivered, dat negative. the mother dat and iat remained positive for four months after delivery. the platelets count raised again to 200.000/ul. the same laboratory findings were detected in a previuos pregnancy in sweden. the patient reported the presence of antibodies at 35th week that disappeared few months after delivery. a healthy baby, dat negative was delivered in this case too. conclusions: this is the first report of the presence of chido and rogers as autoantibodies during the last months of pregnancy. the association with decrease in platelets count and the lack of evident clinical symptoms need further investigation. p-289 rbc alloantibody frequency and their prevalence within chinese, malay and indian community in singapore e widjaja, mbc koh and d teo health science authority, singapore, singapore background and aim: there is a recognised existence of different alloantibodies in different ethnic groups. while this has been studied in the caucasian population, their frequencies remain less well documented in the asian population. the frequency of different alloantibodies and their distribution in terms of age, sex in singapore population is studied, as were their prevalence within the chinese, malay and indian races in singapore. design and method: we conducted a retrospective study for the frequency of 28 alloantibodies on 1856 blood samples over 2004. these blood samples were largely sent by hospitals where preliminary antibody screening had been done and positive result obtained. they were sent to the centre for transfusion medicine for antibody identification. small number of these samples came from hospitals where preliminary antibody testing was not done. the antibody distribution across different ethnic groups (chinese, malays, indians) age and sex were studied. results: the most frequent alloantibodies in the population is mia (40.7%), e (20%), le a (17.5%), le b (14.8%), p1 (6.1%), m (5.4%), d (4.5%), c (2.7%), jka (1.9%) and c (1.4%). within the chinese community, the most frequent alloantibodies were similar: mia (51.9%), e (24.1%), le a (11%), le b (8.8%) and p1 (6.1%). in the malays, the most frequent alloantibodies were le a (35%), le b (26.4%), mia (24.2%), e(13.4%) and p1 (8.2%); while in the indians, these were mia (28.9%), le b (28.9%), le a (27.8%), d (17.5%), and e (12.4%), with the anti-d reflecting the higher incidence of rh d negativity in the indians race. for the lower incidence antibodies, anti-c was more common in the malays and indians (4%) compared to chinese (1.9%). anti jka tended to occur mainly in the malay race and anti-c was rare in all (<2%) reflecting the high prevalence of c in the singapore population (r1r1 phenotype). the ratio of alloimmunised male to female (m : f) is 1 : 3. most alloantibodies demonstrated significant skewing towards to the female, although relatively less so for mia where m : f ratio is almost equal at 1 : 1.9. alloimmunisation increased with age for mia, e, k, p1, jka and fyb while the frequency of alloimmunisation to lea, leb, d, m and c decresed with age. the prevalence of patients with multiple alloantibodies (2 or more) within the alloimmunised subjects is 21.7%. conclusion: anti mia is very common within the asian population especially in the chinese. anti d is common in the indians. most antibodies show increased frequency with age except for anti lea + b, d and m. the majority of alloimmunised patients are females. study aim: to identify the present antibodies in newborns, after a positive direct antiglobulin test (dat). material and method: during a 7 years period, from 1/1/1998-12/31/2004, we studied 2652 newborns and their mothers. each newborn was examined for abo group, rhesus with phenotype, kell and dat. each mother was also tested for abo group, rhesus with phenotype, kell and indirect antiglobulin test (iat). after each positive dat, the study was continued with the elution test, in order to identify the present antibody. dat test was performed with dc screening i-diamed sa, switzerland. for the laboratorial analysis of newborns the sample used was cord blood and in certain cases venous blood. results: the dat test was positive in 132 (5%) of newborns and the antibodies found were igg type immunoglobulin. anti-a antibody was detected in 89 (67.4%) (newborns of group a with mothers of group o), anti-b antibody in 25 (19%) (newborns of group b with mothers of group o), anti-d antibody in 2 (1.5%) (newborns d+ from d-mothers), anti-e in one case and anti-jka in another one. the eluate test was found negative in 3 newborns and in the rest 11, no special antibody could be identified. the results are presented in the following table. conclusion: the majority of antibodies in newborns with a positive dat test, is due to abo incompatibility (the mother belongs to group o and the newborn to group a or b). anti-d (2), anti-e (6), anti-k (7), anti-fya (5), anti-s (2), anti-jkb (1), anti-n (1), anti-kpb (1). in two patients 2 antibodies were identified, while in 24/97 (24.7%) no antibody was identified (unspecific). it is remarkable that only in 10 out of 30 patients with both dat and iat positive, an irregular antibody was identified, while the rest 20 patients had unspecific antibodies. in 19 patients with only iat positive, 15 had an irregular antibody and 4 had unspecific antibodies. in 3 out of 31 patients with both dat and iat negative the cause of incompatibility was the positive dat in the corresponding sample of the blood donor, while in the rest 28 patients the reasons were technical problems that include the inappropriate blood sample of the patient, patients under medication and errors during the crossmatching procedure. the results show that the incidence of red cell incompatibility in our hospital is 2.96% and the most common antibodies are anti-k, anti-e, anti-fya, while anti-d is important for d negative patients. 24.7% of the detected antibodies were unspecific and this is still a problem that possibly was due to the lack of additional panels of reagent red cells or antibodies to low-incidence antigens. finally, in some cases the reasons for incompatibility are due to factors affecting the blood donor or to technical problems in the crossmatching procedure. multiple isoforms excluding normal rhd mrna detected in rh blood group del phenotype with rhd 1227a allele introduction: del phenotype is very common in rh-negative chinese. the rates in hans (more than 91% in china) were reported from 26% to 30%. moreover all del individuals in this population were found mainly carrying a same allele, rhd 1227a, through genomic dna analysis. those individuals always possess one or two of this allele with ccee or ccee phenotypes. aim and the study: we focused on the mrna investigations of del individuals carrying rhd 1227a alleles, in chinese, to expect it could be explained that why a silent mutation is associated with del phenotype. the full-length rhd mrna was analyzed in 2 rh-positive donors with cde/cde and cde/cde genotypes, respectively, and 4 del phenotype individuals carrying rhd 1227a allele with cde/cde, cde/cde, cde/cde and cde/cde genotypes, respectively, through reversed-trancriptase pcrs and cdna direct or cloning sequencing. results: five transcripts and 6 isoforms were detected in rh-positive and del, respectively. among them, 4 isoforms have identical sequences, which are transcripts with exon 9, exons 8 and 9, exons 7 and 9, and exons 7 to 9 spliced out. the normal rhd mrna was only observed in rh-positive, but not in del individuals. in stead, two additional transcripts were found in del individuals. its exon 9 or exons 8-9 were spliced out, but both possess a 170 bp segment of sequence from intron 7 of rhd. through 2 additional reversedtrancriptase pcrs, which amplified exon 8 to 3¢-region and exon 9 to 3¢-region, the results showed that exon 9 did not exist in del anyway. conclusion: (i) a normal rhd protein does not exist in a del individual with rhd 1227a allele since the exon 9 was always spliced out in all isoforms. all 6 transcripts in del maintain a normal open reading frame and encode 6 proteins with different numbers of amino acid residues and different c-terminals (genbank ay751491, ay751492, ay751493, ay751494, ay751495, ay751496). among them, the sequence of del9 (isoform with exon 9 spliced) transcript was the most similar as normal rhd mrna. this isoform was first described by chang et al. in taiwan in 1998 . it encodes 463 amino acid residues and has 46 amino acids more than normal rhd. it is different from rhd after codon 384. in normal rhd protein, the amino acids after 384 (33 residues) are mainly the trans-membrane and intracellular regions. therefore a further study on if a del red cell possesses all epitopes of normal d antigen may be significative. (ii) a normal rh-positive individual has also the transcript of del9 that was found in del. (iii) there is only one polymorphism in the region of 170 bp segment between rhd intron 7 and 2 of the del transcripts, which indicated that other polymorphisms may exist in intron 7 of rhd 1227a allele compared rhd to explain that this situation was not happened in normal rh-positive individuals. total wbc were enumerated by flow cytometry and cell counting. wbc subsets were analyzed by flow cytometry with three-color fluorescence. in this study, the third generation bags and filters are used. results: before filtration, the total number of wbc, was significantly higher in fresh units compared with stored units, whereas in postfiltration samples the number of white cells was significantly lower in the fresh compared with the stored units. although absolute numbers were significantly reduced, filtration also induced significant changes in the proportions of subsets in hoth fresh and storod units, the percentage of t cells was decreased, whereas the percentage of b cells and monocytes was increased after filtration. in conclusion, both pre and post storage wbc filtration affect the proportions of wbc in the final product but pre storage wbc filtration of platelet concentrates is superior than post storage wbc filtration. the effect of pre-and post-storage filtration on platelet rich plasma: derived platelet concentrations background and objective: the white blood cells (wbc) within transfusion products are a major stimulus for a number of detrirmental biological reactions, including febrile nonhemolytic transfusion reactions, alloimmunization against hla antigens and cytomegalovirus transmission. in this work, our objective was to study the effect of storage time on the filtration of platelet concentrates (pcs). the total number of white blood cells as well as the distribution of wbc subsets, in units filtered before and after storage were compared. materials and methods: platelet rich plasma -derived pcs were filtered either fresh (5 pooled we reported earlier that metabolic arrest followed by incubation at 4°c reduces the platelet storage lesion (badlou et al. transfusion 2005) . here we report that this treatment also reduces binding and phagocytosis by macrophages. metabolic suppressed platelets (msp) were prepared by incubation in glucose-free, antimycin a containing medium (40 min, 37°c) followed by storage (48 h, 4°c) and recovery with glucose (1 h, 37°c). controls were (i) platelets in glucose-rich medium stored for 48 h at 22°c and recovery with glucose (c22) and (ii) platelets stored for 48 h at 0°c (c0) with rewarming. platelets were labelled with mepacrine and incubated with pma-matured thp-1 cells (37°c). binding was measured by facs analysis of cd42b/cd14 positive particles, and phagocytosis by counting mepacrine/cd14 positive particles. binding of msp, c22, c0 was 30 ± 4, 39 ± 10, 50 ± 12% of total platelets. phagocytosis of msp, c22 and c0 was 32 ± 9, 40 ± 8, 50 ± 9% of total macrophages (means ± sem, n = 4). before recovery of msp, binding/phagocytosis was 30% higher than thereafter, revealing energy-dependent control of the mechanisms that trigger plateletmacrophage interaction. these data show that metabolic suppression prior to cold storage attenuates binding and phagocytosis by phagocytes and may help to develop means to improve platelet survival post-transfusion. platelet compatibility testing and alloimunization in multiply transfused hematologic patients purpose: multiply transfused patients with heamotological malignancy often become refractory to platelets due to alloimmunization. refractoriness is usually defined as an insufficient platelet increment after consecutive platelet transfusions. two major causes of a decreased platelet increment can be distinguished, immune and nonimmune factors. alloimmunization occurs most frequently against the hla, and rarely against the hpa system. nonimmune factors been identified are splenomegaly, fever, sepsis, and disseminated intravascular coagulation as well as the quality of the transfused platelet concentrates. we performed this study in order to investigate platelet crossmatching, compatibility, and antibody determination among thrombocytopenic patients multiply transfused. we performed 164 crossmatchingcompatibility tests of single donor leucodepleted, abo compatible, platelet concentrates been transfused in 23 patients with leukemia and lymphoma, 18 males and 5 females with mean age 50 ± 25 years old. we also obtained samples from the patients for platelet antibody detection. we evaluated the cci (corrected count increment) 18 h after the transfusion. the solid phase rbc adherence assay (modified capture p system/immucor) was used for platelet compatibility and antibody detection. a total of 164 compatibility tests were performed, of which 84 were compatible. twenty five compatible platelet concentrates out of 84 were clinically evaluated. twenty from 25 compatible crossmatches (80%) were resulted in successful transfusion while only 5 from 25 (20%) in unsuccessful. the 80 incompatible platelets been transfused was resulted in unsuccessful transfusion. we found statistically significant difference among patients successfully transfused with compatible and incompatible platelets (p~0.01). additionally 14 patients out of 23 (60.8%) had been alloimmunized against multiple hla antibodies. three patients transfused with compatible platelets, during the study, developed alloantibodies. we found a large number of incompatible platelet concentrates that result in unsuccessful transfusion and clinical response. the platelet compatibility testing as well as alloantibody determination of multiply transfused patients is necessary for the identification and selection of compatible, with the patients, donors in order to result in succesful transfusion and clinical outcome. furthermore compatible platelet concentrates provide optimal support for refractory patients and it is known that they are acceptable as an alternative component. naitp is a rare clinical syndrome characterized by marked thrombocytopenia shortly after birth. it is caused by maternal immunization against paternally inherited antigens present on foetal platelets. screening and identification of antibodies in the maternal blood sample is the main support in the diagnosis and management of naitp. we have evaluated the frequency of maternal alloimmunization, the role of the antibodies involved (hpa and/or hla systems) and the pertinent risk of naitp in neonates using a fully automated system with a solid phase red cell adherence methodology (sprc-capture p) and paternal or random donors (indirect test). screening started in june 2003 and is still in course: in january 2005, 2300 blood samples were examined. identification of antibodies in maternal serum was carried out using elisa methodologies: maipa and commercial kits (pak-plus and quick screen-gti). of the 2300 blood samples analysed, 43 were reactive and the specificity of the antibodies were: anti hpa1a: 8, anti hpa1b: 2, anti hpa1a + hla: 2, anti hpa5b. 6, anti hla: 24, auto hpa-5b: 1. specificity of hpa antibodies was confirmed by determination of parents' hpa genotype (hpa-1,2,3,4,5,6) using pcr-ssp or pcr-rflp. the infants with hpa 1 immunization suffered from severe (plt count 5-103/ml) and symptomatic naitp (bleeding and petechiae were present), therefore they were treated with platelet transfusion and administration of high doses of intravenous immunoglobulin. we confirm that naitp due to hpa-5 and hla immunization is clinically less severe: all neonates had mild and self limiting thrombocytopenia at birth; no therapy was administered. it would be advisable to carry out pre-natal screening, at reasonable cost, using maternal serum versus paternal platelets and to proceed to the identification of antibodies only in presence of positive results. background: fetal or neonatal alloimmune thrombocytopenia (fmait) results from a maternal alloimmunization against fetal platelet antigens. it is the commonest cause of severe thrombocytopenia in the neonatal period. the diagnosis of fmait is made initially on clinical grounds, depending on exclusion of other causes of neonatal thrombocytopenia. in caucasians, hpa-1a is the most frequently implicated antigen. other antigens such as hpa-3a, or hpa-4a are less often implicated. during the past few years fmait has been reported associated with rare or private antigens. the diagnosis is straightforward when a maternal alloantibody with a corresponding parental antigen incompatibility is present. however it could be equivocal in the absence of such an antibody or difficult when a private antigen is implicated. if the father is heterozygous for the considered antigen, the infant's platelet typing should be performed to confirm the diagnosis. due to the risk of hemorrhage, particularly intracranial hemorrhage (ich), during the course of severe thrombocytopenia, specific therapy is mandatory. because subsequent siblings may be more severely affected, accurate diagnosis will allow better management of subsequent pregnancies. study design and methods: since the first documented case of feto-maternal alloimmune thrombocytopenia (fmait) due to anti hpa-9bw (maxa+), no additional cases have been reported. we present here a retrospective analysis of the cases referred to our laboratories in recent years. since 1999 we have screened for rare or private antigens in suspected cases of fmait when there is no incompatibility for the most frequently implicated antigens. the diagnosis was performed by genotyping and identification of the maternal alloantibody by the maipa technique. results: parental genotyping showed hpa-9bw (maxa+) mismatch as the sole antigenic incompatibility in 7 out of 8 families. in the last one, incompatibility was found for hpa-3 without anti hpa-3b maternal alloantibody. as the father was found to be hpa-9bw (maxa+) heterozygous in all the cases, the infant or fetus was genotyped to ascertain the diagnosis. the maternal alloantibody was identified in the maipa technique. however, our data strongly suggest that recognition of the hpa-9bw (maxa+) epitope is not uniform. the neonatal thrombocytopenia was severe in most cases with bleeding. the outcome was good in all the cases but one. conclusion: this analysis confirms that anti hpa-9bw (maxa+) fmait is not uncommon and was found to be around 2% of our confirmed fmait cases with parental incompatibilities and presence of maternal alloantibodies. it is a clinically severe syndrome which requires prompt diagnosis, albeit difficult, and maternal platelet transfusion therapy. laboratory investigation of a suspected fmait case should be carried out in a specialist laboratory wellexperienced in optimal testing. therapy requires strict collaboration between clinicians and blood bank services. appropriate management and antenatal therapy should be considered for successive pregnancies to prevent fetal bleeding. introduction: the human platelet (plt) antigen (hpa) system is independent of the hla system. therefore, host-or donor derived alloimmune thrombocytopenia can develop after allogeneic haematopoietic stem cell transplantation (hsct) even in hlamatched donor-recipient pairs. we report the first case on a stem cell recipient developing thrombocytopenia due to host-derived hpa-1a antibodies after non-myeloablative allogeneic hsct. a 52year-old male patient was diagnosed with multiple myeloma in 6/2003. treatment consisted of 3 cycles vincristin, adriamycin and dexamethason followed by tandem autologous stem cell transplantation. because of progressive disease he received 4 cycles of bortezomib, and after complete remission a stem cell allograft (7.99 ¥ 106/kgbw cd34 + cells) from his histocompatible (hla a,b,c,dr identical) brother after reduced intensity conditioning regimen with fludarabine (3 ¥ 30 mg/m 2 ) and alkeran (100 mg/m 2 ). he had received only twice packed red blood cell concentrates and one plt concentrate before allogeneic hsct. stable bilinear engraftment occurred around d12 but was accompanied with a continuous decrease of plt counts. between d10 and d19 the patient received seven plt transfusions, containing a median of 2.96 ¥ 1011 plt/unit (range 2,7-3,21 ¥ 1011 plt/unit) from random donors. the corrected plt count increments at 12 to 24 h after these transfusions were <2500/ml. therefore, and because of even a further decline of platelet counts to 1000/ml on d19 we investigated the presence of plt antibodies. methods: the patient's serum was tested by antigen capture elisa assays (pakplus® and pak1®, gti) and a solid phase assay (capture-p®, immucor). the maipa assay was used to confirm the results obtained by the above mentioned assays. in addition, we tested the patient's serum by the maspat kit (clb) against plt from the donor and against homozygous hpa-1a plt obtained from our donor pool. stored recipient's dna from the time before hsct was used for genotyping. genotyping for hpa-1, -2, -3 and -5 of the donor and the recipient was performed by pcr-ssp (hpa, protrans). the patient's serum obtained on d19 after hsct reacted strongly with the donor's plt due to anti-hpa-1a antibodies and antibodies against hla class i antigens. the patient's genotype before transplantation was hpa-1bb, -2aa, -3ab, and -5aa; the donor was hpa-1ab, -2aa, -3aa, and -5aa. thus, the antibodies were host derived and directed against the donor's plt. serum samples obtained on d38, d45 and d65 after hsct contained antibodies against hla class i antigens but hpa-1a antibodies were not anymore detectable. no hla antibodies were detectable on d149 after hsct. the severe thrombocytopenia was caused by hostderived hpa-1a antibodies. fortunately, plt counts started to increase on d20 spontaneously and the patient could be discharged at d27 (plt 51.000/ml) with a complete donor chimerism. the decrease of the serum antibodies parallel to the increase of the plt count strongly suggests a progressive elimination of residual host cells. we conclude that the hpa mismatch between recipient and host affected thrombopoietic engraftment and the success of plt transfusions. severe neonatal alloimmune thrombocytopenia with anti-hpa-5b antibodies: case report p moncharmont, m vignal, y mérieux and d rigal efs rhone alpes site de lyon, lyon cedex 07, france usually, in case of feto-maternal incompatibility, the platelet (plt) specific anti-hpa-5b antibodies (ab) induce only sometimes a mild neonatal alloimmune thrombocytopenia (nait). contrary to this observation here is reported the case of a severe nait. a 34-year old mother, gestation 2/partum 2, gave birth to a male neonate by caesarean section at 34 weeks of gestation because of intra-uterine growth retardation (iugr) and anamniotic fetus. five years before, she had had a first pregnancy with iugr of the fetus but no nait. the second neonate weighted 1.350 g, was 41.0 cm tall and had a head circumference of 27.5 cm. the apgar score was 9,10,10,10 at 1, 3, 5 and 10 min respectively after birth. no bleeding, hepatomegaly, splenomegaly or infectious signs were noted. five hours after birth, a respiratory distress syndrome appeared and an oxygenotherapy was performed during 44 h. the plt count which was 82.0, 79.0 and 73.0 giga/l at day (d) 0, d1 and d2 respectively dropped dramatically at 9.0 giga/l at d5. simultaneously, an intracranial hemorrhage grade ii was diagnosed on ultrasound scan. because of the clinical signs and of the decreasing plt count the mother's serum was tested for plt-specific ab by immunocapture and the ab identified by the monoclonal ab-specific immobilization of plt antigen (maipa) assay. a plt genotyping was performed in the neonate and his parents by sequence-specific primers polymerase chain reaction. the mother was hpa-5a/a and anti-hpa-5b ab were detected in her serum. the baby was heterozygous, hpa-5a/b. plt were transfused to the baby and the plt count rose to 80.0, 115.0 and 315.0 giga/l at d6, 10 and 18 respectively. no further transfusion was needed and the development of the baby was satisfactory with a normal electroencephalogram. in conclusion, when a mild thrombocytopenia with iugr and hypoxia but without bleeding signs is present in a neonate immediately after birth, a maternal plt specific ab screening must be performed in case the thrombocytopenia became severe during the newborn monitoring. anti-hpa-5b ab can be detected. partial results of the incidence of heparin induced thrombocytopenia type ii osc oliveira*, ra rached*, c cavalheiro-filho*, jc nicolau*, shgl pasqualucci*, daf chamone † and sp bydlowski † *heart institute, university of são paulo, † university of são paulo, são paulo, brazil heparin induced thrombocytopenia type ii (hit) is a severe side effect of heparin, associated with heparin-platelet factor 4 antibodies. hit type ii occurs in up to 5% of patients who are exposed to unfractionated heparin (ufh). in our institution patients that are under heparin treatment are mostly cardiac patients. the purpose of this study is to determine the incidence of hit type ii in these patients. material and methods: 111 patients from the intensive care unit and cardiac care unit treated with ufh or low molecular weight heparin (lmwh) for 5 or more days were studied. known causes of thrombocytopenia were excluded. platelet count was monitored pre and post heparin therapy. all selected patients were tested for detection of anti heparin/pf4 antibody test (diamed id-card). results: from the studied patients, 63 (56.8%) developed thrombocytopenia (determined by a decrease in the platelet count below 30%, after the introduction of heparin therapy); 48 (43.2%) did not show decrease in the platelet count. six (5.4%) out of 63 thrombocytopenic patients were positive for anti-heparin/pf4 antibody. three (2.7%) out of 48 non thrombocytopenic patients were positive for anti-heparin/pf4 antibody. the results demonstrate that 9 (8.1%) patients were positive for anti-heparin/pf4 antibody and they were no different from those described in the literature regarding the frequency of heparin induced thrombocytopenia. moreover, a higher frequency of patients with heparin/pf4 antibody was noted without the presence of thrombocytopenia, indicating that other factors should be considered. introduction: neonatal alloimmune thrombocytopenia (natp) due to maternal immunization against fetal platelet antigens affects 0.8-1 in 1000 live births. although it is usually a self-limiting condition, a major complication in cases of severe thrombocytopenia is the occurrence of intracranial haemorrhage leading to death (in up to 10% of reported cases). the commonest antibodies are anti-hpa-1a. treatment consists of ivig, compatible donor platelet concentrates or washed maternal platelets. the administration of random donor platelet transfusions is controversial but has been used successfully in some urgent cases when compatible platelets were not available. case report: a baby born in week 40 of gestation to a healthy mother after first uneventful pregnancy; birth weight 3000 g, apgar score 8. immediately after birth, severe thrombocytopenia (7 ¥ 109/l) and signs of haemorrhagic diathesis (generalized petechiae and ich gr. ii-iii) were observed. coagulation tests were abnormal and k-vitamin, fresh frozen plasma and random donor platelet concentrate (retrospectively genotyped as hpa-1a/a) were given. twenty-four hours later platelet count rose to 26 ¥ 109/l and no new petechiae were observed. on third day of life the blood platelet count was 13 ¥ 109/l and the newborn received ivig 2 g/kg and corticosteroides. twenty-four hours later the platelet count rose to 107 ¥ 109/l and further clinical course was uneventful. natp due to hpa-1a was serologically confirmed. conclusion: optimal therapy for an infant with severe thrombocytopenia during the first 24 h of life is the transfusion of platelets that will not be destroyed by the maternal alloantibody in the infant circulation. random donor platelet concentrates are controversial in a setting where optimal treatment is not available, however, in this case they led to a significant platelet count improvement in spite of hpa-1a incompatibility. accordingly, random donor platelets may be considered appropriate in emergency situations. background: rhd is the most immunogenic blood group antigen, and its correct identification is essential in the blood bank and in the prevention of the haemolytic disease of the newborn. the weak d phenotype is the most common d variant, with a frequency of 0.2% to 1% in caucasian individuals. there are several weak d types, with different frequencies in european countries, which may pose serologic problems and have the potential for alloimmunization. the objective of the study was to determine the frequency of the principal weak d types in portugal. study design and methods: lisbon regional centre of the portuguese blood institute and oporto são joão university hospital selected samples from blood donors and patients. rhd was tested by two (oporto) or three (lisbon) distinct anti-sera, in direct agglutination tests, at room temperature. when discrepant results were observed, the samples were tested with panels of monoclonal anti-d by liss-iat. samples that reacted weakly with igm anti-d but positive with igg anti-d were sent to the molecular biology centre. pcr with sequence-specific primers was performed using two commercially available kits (inno-train and bagene). real-time pcr, carried out on a light cycler, was applied when the interpretation was dubious. results: 101 samples were referred after being characterized as weak d. in 99 cases we obtained a positive result, with a preponderance of weak d type 2 (63.6%) over type 1 (16.2%), 3 (14.1%) and 4 (6.1%). two samples were not categorized. the high incidence of weak d type 2 in our population is in marked contrast to studies performed in other european populations where weak d type 1 was the most frequent. this might be due to our sample selection criteria or ethnic variation in the causes of weak d. there are advantages in genotyping serologically depressed d samples: to avoid the waste of d-negative rbc units and the use of immunoglobulin in pregnant women, who have no risk of alloimmunization. analysis of rhd zygosity in different rh phenotypes cal except for a 4-bp t insertion. the deletion of the rhd gene, found in most rhd-negative caucasians, was theoretically due to recombination of the upstream and downstream rhesus boxes resulting in the formation of a hybrid rhesus box. thus, the detection of a hybrid rhesus box in an rhd-positive individual denotes an rhd heterozygous status. aim of the study: to determine the rhd zygosity in different rh phenotypes. methods: blood samples from 106 white trios (father, mother and child) were studied. the rh phenotype was performed by hemmaglutination and the rhd zygosity was inferred in each member of the family groups. the rhd deleted allele was determined by a pcr strategy using a forward primer complementary to 5¢ end of the identity region of the upstream and hybrid rhesus boxes and a reverse primer complementary to the 3¢ end of identity region of the downstream and hybrid rhesus boxes. these primers selectively amplify a 1980-bp segment of the hybrid rhesus box in rhdnegative and rhd-positive heterozygous samples. serological and pcr inconsistencies were studied by a pcr-rflp method to detect another polymorphic site of the hybrid rhesus box. frequencies were obtained analysing only unrelated individuals (fathers and mothers, n = 212). results: 179 (84.4%) rhd-positive and 33 (15.6%) rhd-negative samples were phenotyped. of the rhd-positive donors, 75 (41.9%) were rhd homozygous and 104 (58.1%) were rhd heterozygous according to pcr. pcr-rflp analysis confirmed the results of pcr in serological and molecular discrepancies. these results were coherent within each family group and did not differ from those published in the literature for caucasians based on the most probable genotype method. however, the homozygosity indexes were significantly higher in the dccee (20.0% vs 6.6%) and dccee (16.6% vs 3.3%) phenotypes due to an increase of the dce haplotype. in all samples with the dce haplotype the rhces allele, frequent in individuals of african descent, was investigated by pcr-ssp. this allele was found in 33.0% of the dce haplotypes. . rhd and rhce typing was performed by multiplex-pcr with 24 fluorescent primer pairs. positive results were obtained for rhd-exons 2-7, 9, 10 and polymorphisms associated with antigens c, c, cw, e and e. sequencing of rhce-sequences, including exons 1 to 10 and intron/exon borders, were done by direct taq cycle-sequencing using bigdye-terminators v.1.1 in an abi 310 (applied biosystems). results: see table 1 . the substitutions of rhce-specific nucleotides in exons 3, 5 and 6 with their rhd-specific counterparts lead to 3 different new rhc-antigens with weakened expression. since one amino acid change in allele 3 lie in extracellular loop of the antigen suggested that this antigen may be involved in allo-immunization. inheritance of a rhd cat vi type ii in a twin pregnancy: a case report introduction: determination of hdn-relevant fetal blood groups in amniocentic fluid with pcr is a routinely used method. misinterpretation of test results -e.g. overlooking rhd category -decreases depending on the number of examinated rhd-specific exons. case report: a 28-year old mother (w.o.g. 23) pregnant with twins was regularly tested for irregular antibodies and was shown to have an anti-d. after amniocentesis of both foetuses tests for the occurrence of rhd intron 4, exons 7 and 10 were performed in the hospital's lab. the sample of fetus i showed pcr-products for intron 4, exon 7 and exon 10 while sample of fetus ii lacked rhd-specific intron 4. therefore we investigated blood samples from the parents as well as amniocentic fluid of both foetuses. methods: total dna was amplified in a multiplex pcr with fluorogenic primers for rhd exons 2-7, 9 & 10; polymorphisms for dweak type 1-5, d-vii, d-hmi and rhce polymorphisms for c, c, cw, e, e. capillary electrophoresis for size fractionation and fluorigeneic analysis was done in an abi 310. rhd zygosity was determined by quantitative real-time pcr with co-amplification of rhdand rhce-specific exon 3 and subsequent calculation of d ct value (ct rhce minus ct rhd). results: while maternal dna sample has been genotyped as rhdnegative, amplification of paternal dna for rhd-specific exons 2, 3, 7, 9 and 10 were possible and failed only in exon 4-6. determination of rhd-zygosity revealed a homozygous constellation of the rhd-gen. investigation of amniocentic fluid from both foetuses resulted in a rhd-wildtyp for fetus i and a rhd cat. vi type ii for fetus ii which was the reason for missing amplification in rhd intron 4 pcr. results: weak d type 3 was not identified in our research population. weak d type 1 was identified in 2 cn, weak d type 2 was identified in 1 cn and weak d type 4 was found in 1 cn, 3 bsa, 4 be, 1 bc and 1 saa. conclusions: although weak d types 1 to 3 represent the majority of all weak d phenotypes in caucasian populations, none of these weak d alleles were found in black populations. since none of the frequent weak d types were identified in non-caucasians one might not expected to find type 4 in all populations. however, regarding rhd phylogeny, the weak d type 4 mutations (602c > g and 667t > g) form the basis of a cluster of aberrant alleles that are predominantly observed in blacks. therefore, it is not surprising that weak d type 4 was identified in non-caucasians. based on these results it may be concluded that weak d phenotypes have evolved relatively recently since they are present in caucasian and asian methods: dna was extracted from 9 a, 14 b, 11 ab subgroups, and 21 provisional cis-ab after serological abo typing. allelespecific pcr, rflp, direct sequencing of exon 6 and 7, and allele separation were performed on these samples. results: abo*a205 allele was observed in an aint subgroup. two new a alleles that showed 784g > a base change and 990c > t of intron 6 and a polymorphism of 532c > t in a(pro) intron 5 were discovered. o04 and o20 alleles were also observed. in b subgroups, a silent substitution 255c > t (leu85leu) was observed as a new b allele. another new b allele which showed 547g > a was also found in 4 b subgroups. conclusion: we discovered new abo alleles and polymorphisms in korean populations. many other polymorphisms and alleles already reported in japanese were also observed in koreans. evaluation of a multiplex snapshot-pcr method for red blood cell marker genotyping c jungbauer*, c hobel*, em dauber † , pk rabitsch*, dwm schwartz* and wr mayr* *austrian red cross, † medical university vienna, vienna, austria once conventional reagents for identification of rare red blood cell phenotypes are scarce, methods using current nucleic acid testing techniques to identify the patient's genotype and possibly to screen for donors would be desirable. the approach of multiplexgenotyping using pcr-ssp has apparently technical constraints so we are currently analyzing whether a modification using snapshot pcr-technique could provide a routine application for such purposes. compared to ssp-technique, the snapshot pcr only requires one single primer (labeling reaction) for the detection of two (or more) alleles instead of one pair of primers for every single allele. briefly, we perform snapshot pcr as follows: in a first step, dna segments covering the essential polymorphic regions for the abo, rhd, kel, jk and fy genes are amplified using a standard pcr step. after purification treatment of the pcr products (roche high pure pcr product purification kit or shrimp alkaline phosphatase (sap)/exo1 treatment according to the abi prism snapshot multiplex kit protocol) the labeling reaction is performed using the abi prism snapshot multiplex kit. after a second purification step the products are analyzed on a abi prism 310 genetic analyser in fragment analysis mode. our preliminary results show the feasibility of this approach as reliable typing results can be obtained for all tested single nucleotide polymorphisms corresponding to alleles. generally a better signal quality from controls and samples is obtained compared to the ssp-technique with consecutive gel electrophoresis. we also consider the possibility of the automated interpretation of the results as an important improvement, especially when aiming for an application for a large number of samples (donors) or markers (patients). in contrast, the method involves more manual steps and higher costs. we may conclude that the implementation of snapshot-pcr techniques for red cell marker genotyping is a promising alternative to pcr-ssp. the obvious quality improvements compared to pcr-ssp might be critical for a routine application in blood banks (donor screening) or complex questions in clinical laboratories. quantification and quality control of monoclonal antibody using an optical sensor based on the laser reflectometry technique the monoclonal antibodies (moab) are biological reagents of homogeneous activity. they are generally used in the recognition and quantification of very small amounts of biological substances (hormones, enzymes) present in a solution, ag identification, blood group determination, oncology, organs transplant, etc. moab can be characterized by its specificity and affinity. affinity may be expressed as the equilibrium association constant (k). several techniques are available to determine the equilibrium constant of the ag-ab interaction. in this work, is shown an optical sensor for monoclonal antibody quantification by reflectometry technique. the laser reflectometry technique (null ellipsometry technique) can give information about the kinetic of the interactions, stoichiometry of molecular binding and the concentration of molecules in a solution, and also offers detailed and accurate determinations of real-time adsorption kinetics of protein without labeling. a silicon wafer was chosen as reflectant surface. once fixed the principal angle of incidence, an amount of anti-ab is added to the sample. the reflected laser intensity is registered in real time as the protein is being adsorbed onto the wafer. the mathematical analysis of the results verifies that the antibodies adsorption follows langmuir's kinetics. from the curve analysis, the parameters related to the anti-ab concentration are extracted. from them, the calibration curve is constructed. this curve allows the desired commercial monoclonal antibody quantification. the developed technique shows to be sensible and precise. the obtained graphics are very well approximated (r > 0.94) verifying that the monoclonal anti-ab associa study aim: to prevent the sensitization to rh0(d), to a d-patient who was transfused with d+ blood. material and method: on september 2003, (9/28/2003), we admitted to our hospital through air-carriage, a female of 22 years old, badly injured after a car-accident. the patient was on an olighemic shock (ht: 16%) due to retroperitoneal, paracolic and procystic hematomas, had multiple fractures on the left feet, the main important of which was an acetabulum one. she had a blood group: o, d-and her phenotype was ccdee with du-. after her arrival she was urgently admitted to the surgery room and our blood bank was asked for condensed red cells. initially she was transfused with blood of the same group (o, d-) but when we were short out of dblood, we were asked for 7 more units. the necessity of blood was imperative, the patient was on a critical condition and the mechanism of blood transport, from another blood bank would take some time to be put in motion, and so it was decided that the patient would be provided with d+ blood. the indirect antiglobulin test (iat) and papaine were negative, so there would be no problem with the transfusion with d+ blood. the patient received finally 3 units (850 ml) of d+ condensed red cells, out of 7 that were initially asked. before the 48h from the surgery had passed, it was decided that human anti-d immunoglobulin (rhesogamma p) should be provided, in order to prevent the sensitization of the patient to rh0(d). the indicted dose was 500-1250 iu/10 ml of the transfused blood, provided piecemeal during a several days period. analyzed by real-time pcr amplification. based on a published report, we selected 12 primer pairs targeting insertion/deletion polymorphisms which are located on different chromosomes, unrelated to each other and not associated with immunocompatibility. we optimized the amplification conditions for all 12 primer pairs using our sybr green real-time quantitative pcr protocols, and investigated analytical sensitivity for each primer pair by performing spiking studies, in which a single copy of positive dna was added into 100 000 copies of negative dna followed by allele-specific pcr amplification. we also created a theoretical panel of donor-recipient pairs (n = 73) to evaluate the clinical sensitivity for detection of ta-mc using both hla-dr and indel panels. results: for the short-term samples, 10 additional mc cases was identified in non-lr group using indel panel; and one additional mc case was detected in lr group (table 1) . for the long-term follow-up samples, 4 additional mc cases were found. when evaluating analytical sensitivity, we were able to detect a single copy of positive dna mixed with 100 000 copies of negative dna in a single amplification tube for all 12 primer pairs. we were also able to calculated the clinical sensitivity that using 73 donor-recipient pairs. 99.5% of donor-recipient had at least one informative allele for detection of ta-mc if we consider both hla-dr and indel panels. conclusion: using our new indel panel, we were able to detect more instances of mc in this cohort of patients. we conclude that the dr assay underestimates the presence of mc. moreover, the tandem use of both panels provides a powerful tool for the detection of mc with 99.5% of recipients having at least one informative allele. background: we reported severe immunesuppression and longterm transfusion-associated microchimerism (ta-mc) in transfused trauma patients. we have also reported, in a murine transfusion model, that sensitivity to 1-chloro-2-4 dinitrobenzene could be transferred, albeit transiently, by transfusion of fresh blood from a sensitized donor to a naïve immunecompetent recipient. in order to mimic the immunecompromised status of trauma patients and further investigate the mechanism underlying ta-mc, we established an animal model using immunedeficient knock-out mice. aim: our objective was to test virus-specific immune functionality of the chimeric donor leukocytes in a murine ta-mc model. material and methods: female rag-2/common gamma double knock-out mice were transfused with fresh blood collected from male balb/c mice, which were either not infected (non-primed, np) or infected twice (primed, p) with 100 million viral particles of murine cmv (mcmv). at different post-transfusion time-points (1 h, 2 weeks, 4 weeks, 6 weeks, 8 weeks), different female recipients plus non-transfused female knock-out controls were challenged with 100 million viral particles of mcmv intra-peritoneally, and then monitored weekly for the concentrations of male donor cells as well as mcmv viral load in recipient's circulation. each female knock-out received only one challenge of mcmv. if the subject died, we quantitated mcmv viral load in the brain, spleen, lung and liver. we used real-time quantitative pcr targeting murine y-chromosome, h2k and mcmv to quantitate male donor cells, transfused recipient cell dna input, and mcmv vrial load, respectively. the number of recipient cell dna input served as a denominator to calculate the concentration of male donor cells and the mcmv viral load. results: results of overall mortality are summarized in the table. all female knock-out recipients transfused with primed donor blood, except for the post-transfusion 6 weeks, are able to survive mcmv infection. all non-transfused control and recipients transfused with non-primed donor blood died after mcmv infection; these two groups also had higher mcmv viral load in blood than the recipients transfused with primed donor blood. when the subject died, we were able to detect mcmv in all four organs we analyzed, with liver having the highest mcmv viral load. there was no significant difference for the concentration of donor cells in recipients' blood between recipients transfused with non-primed donor blood and recipients with primed donor blood. the preliminary data of our study showed that chimeric primed donor cells, but not non-primed donor cells, are able to protect immune compromised knock-out recipients from murine cmv infection. the time-point of 'post-transfusion 6 weeks' might represent a weak window for the functionality of chimeric donor cells, which requires further investigation and confirmation. aims: to compare the effectiveness and the cost of epoetin-a and darbepoetin in patients undergoing pabd. methods: seven adult patients scheduled for operations were administered aranesp (300 mg sc once or q3 weeks if needed) for pabd (aranesp group) and they were compared with a historical epoetin-a group of seven age-matched adults (eprex 300 iu/kg biweekly). the two groups were matched according to the ht, ferritin levels, number of the predonated units and type of the operation performed. cbc count and reticulocytes were measured weekly during the donation period, the day before, day 1 and day 6 after surgery while ferritin and biochemical indices were measured during the first visit. erythropoiesis-stimulating factor was administered when ht £ 36% during blood donations and blood donation was not performed if ht < 34%. results: there was no statistical significant difference in hematological parameters during the donation period, the pre-operation day and after surgery between the two groups. five of seven patients from both groups received one or two autologous blood units. both factors were well tolerated without any side effects. the cost per patient was 609.09€ in the aranesp group and 414.2€ in the epoetin group. conclusion: despite the small number of patients and the limitations of this preliminary retrospective trial we believe that subcutaneous darbepoetin-a is equally effective with epoetin-a in patients undergoing pabd. darbepoetin has the advantage of less frequent administration and it is possibly superior that epoetin-a in terms of patient compliance. however smaller doses should be examined in order to reduce the cost. larger prospective randomized trials are needed to estimate the cost-effectiveness of the use of darbepoetin-a in pabd. background: incompatibility with many blood units is a major problem in transfusion therapy. in selective operations, preoperative autologous blood donation could solve many problems, when of course the patient's condition and his haemoglobin levels are appropriate. we present here the experience of our blood transfusion centre from 4 operations in 3 patients with anti-erythroid antibodies. materials: three patients (1 male and 2 females), aged between 67-80 years old, had to undergo selective operations, 3 total hip replacement surgery and 1 aortic aneurysm. introduction: because of improvements in surgical techniques, preoperative autologous blood donation (pabd) in patients undergoing radical retropubic prostatectomy (rp) is contested. aim of the study: we wanted to develop and validate an algorithm to determine the patients who probably do not benefit from pabd. methods: we calculated the perioperative hb-loss of 153 consecutive patients (group 1) who donated two red cell units (rbc) of autologous blood 5 and 4 weeks before undergoing rp: hb-loss (g) = preop-hb ¥ bv + (n rbc ¥ 65) -(postop-hb ¥ bv) (bv = blood volume (l) = body weight (kg) ¥ 0.08; postop-hb = hb (12-18 h after rp); n rbc = number of transfused autologous and allogeneic rbc). hb of rbc was taken as 65 g. rbc requirement is probably if initial-hb -(hb-loss: bv) -trigger-hb (taken as 80 g/l) < 0 (initial-hb = hb at the first contact with the pabd-unit). this assumption was validated by the next 125 patients who were also assigned for pabd (group 2). pabd was refused if the probability of rbc requirement (prr) was <25%. between 25-50% one rbc was taken after considering the patient's individual risk of pabd. if prr exceeded 50% two donations were planned. results: both groups did not significantly differ in age or initial hb. preop-and postop-hb were significantly lower in group 1 (141 vs 151 and 98 vs 111 g/l). 79% of autologous blood of group 1 were discarded, 3/153 patients needed additional allogeneic rbc. hb-loss caused by rp was 269 ± 111 g. mean prr in group 2 was 8.5%. 5/125 patients donated one rbc, which was later discarded, and no patients donated two rbc. 7/125 of group 2 needed allogeneic rbc. mean prr of these patients was 12% (range 0.65-33.3). conclusion: postop-hb were lower in rp-patients with pabd because of the lower preop-hb and the restrictive indication for transfusion of autologous blood. the individual calculation of prr, for which only body-weight and initial-hb of the patient are necessary, shows that pabd in patients undergoing rp is indicated only in rare cases. the algorithm also may be used in other major operations, if hb-loss is known. use of darbepoetin-alpha in preoperative autologous blood donation: preliminary results background: preoperative autologous blood donation (pabd) is an alternative practice to eliminate complications of allogeneic blood transfusion although its cost-effectiveness has been questioned. darbepoetin-a (aranesp, genesis pharma sa) is a novel erythropoiesis-stimulating factor that it has been shown to be equivalent to epoetin-a (eprex, janssen-cilag) in patients with chronic renal failure and cancer. darbepoetin-a has a longer serum half-life and higher relative potency than epoetin-a. this property leads to less frequent administration and may reduce drug cost. so far, no clinical trials with darbepoetin have been published in patients with surgical anemia. other 44 (15.3%) patients who required autologous and homologous blood, had average predonation hb level of 13 509 (sd ± 9.01) g/l. we found a significant relationship between the need for postoperative transfusion and the predonation hb level (p = 0.003), predonation htc values (p = 0.006), weight (p = 0.003) and gender (p = 0.002): female patients and patients with lower predonation hb and htc, as well as patients with lower body weight more often needed additional homologous blood transfusion. no relationship was found between age of patients and the need for transfusion (p = 0.121). 104 (80.6%) patients with ptka were transfused with autologous blood only, and had average predonation hb level of 13 867 (sd ± 9.06) g/l. other 25 (19.4%) patients transfused with autologous and homologous blood had average predonation hb level of 13 588 (sd ± 8.93) g/l. the significant relationship was found between the need for postoperative transfusion and weight (p = 0.012): patients with lower body weight more often needed additional homologous blood transfusion. no relationships were found between predonation hb level (p = 0.099) predonation htc values (p = 0.243), gender (p = 0.241) and age (p = 0.276) of patients and the need for postoperative transfusion. conclusions: our results show that over 80% of patients needed only autologous blood. in our patients with ptha predonation hb was significant predictive factor for additional transfusion therapy, while in ptka it was not observed. in both groups of patients body weight was significant predictive factor, thus this feature seems important for planning of transfusion therapy in patients with ptha and ptka. aim: prevention results of loosen anastomoses on colon, with fibrin sealent (fs) application and influence on colagen production. materials and methods: investigations were done on rats, weight 350-500 g. in control group, after partial resection of left half of colons termino-terminal anastomosis was derivated. fs was applied in examined group. concentration of colagen was done indirectly, with quantitative l-hydroxyproline determination. place of anastomosis, 1 cm proximal and 1 cm distal of anastomosis, was analyzed iii, v, vii and xiii day postoperatively. results: analysis of hydroxyproline on the place of anastomosis showed higher hydroxyproline value in group with fs application. the highest approximate value of hydroxyproline was registered v day postoperatively. distal, 1 cm of anastomosis, the quantity of hydroxyproline is higher iii day postoperatively in control group but v postoperative day value is intensively growing in group with fs application. electronic microscopical was done v postoperative day in control group at the place of anastomosis detected a defect with detritus and absence of larger colagen fibres. in group with fs application on the place on anastomosis, in the shape of bundle, colagen fibres were grouped and completely fills the place of anastomosis. conclusion: fs application accomplish higher concentration of colagen in all segments of isolated colon, that enables better healing of anastomosis. the study of the use of the safest blood (autologous blood transfusion) through preoperative blood donation (pbd) in 100 surgery patients introduction: the use of the autologous blood is already under consideration in developed countries. thus, it is probable that autologous blood donation would be effective in one way or the other in reducing blood transfusion complications. in this study, pbd as the easiest method to use and the most cost-effective one was selected. aim of the study: it aims at improving blood safety and raising blood inventory. methods: in this study, 100 patients, including 75 males and 25 females, intended to undergo elective surgery were selected as subjects to donate their autologous blood. the subjects with hematocrite level of about 30-33 percent as ordered by their physician donated their blood by this method. blood collection procedure was followed at 3-7 day intervals. the blood volume taken from patients in every collection differed from 100-450 ml according to their weight. results: this study showed that in all patients undergoing plastic, gynaecological, jaw, and ent surgeries autologous blood transfusion was used with no need for allogenic transfusion. in other surgeries, including orthopaedics, the need for allogenic transfusion was estimated to be at about 50 percent of cases. to avoid the complications of allogenic blood transfusion, the safest way is the use of autologous blood which involves low cost and is easy to perform. the introduction: the purpose of this study is to describe a technique to perform labelling of autologous platelet-gel with 111in-oxine and to evaluate its usefulness, after in vivo graft implant, as a marker of bone osteoinduction by means of scintigraphy, in patients with jaws bone defects following the enucleation of cystic lesions and cystic lesion derived from extraction of deeply impacted lower third molar. methods: agp made. consent was obtained by patient to conduct hiv and hbv testing. briefly, 40 cc to 60 cc of blood is withdrawn from the patient. the blood was separated, by means of successive step of centrifugation, in to platelet-rich plasma (prp) , platelet-poor plasma (ppp), and red blood cells (rbc). the red blood cells were discarded. the prp [comprises of approximately 10% of the total blood volume withdrawn] had platelet counts of 500 000-3 000 000/mm 3 . the procedures of agp labelling were performed in laminar flow chamber. to 3 seconds the solution will assume a gel-like consistency forming platelet gel. imaging: the scintigraphy was performed 2 h after application of labelled agp (early scan) and at 24, 48, 72, 96 h (delayed scan) by means of a gamma camera equipped with medium energy collimator. a later scan was performed at 10 days after graft. the platelet uptake index (pui) was then calculated by dividing the cpm/pixel in the graft roi (recognized in and planar and trans-axial slice) for cpm/pixel in a mirror background roi. in vitro sampling: the radioactivity of the plasma samples collected at 2, 24 h and at lapse time = 24 h for 7 days, were used for the plasma clearance determinations and for in vitro studies of the platelet loss from the gel. results: all patients presented early high concentration of 111in oxine agp, at site of the graft, that was easy recognized at scintigraphy performed as in anteroposterior and lateral planar projection of the jaw as in spet slices reconstructions. all labelled agp was well confined within area of original implant and no activity was seen in the surrounding tissues or in the distant organ. conclusion: all patients studied well tolerate the implant of agp; no adverse reactions were observed and follow up -performed 3 months later -showed bone remodelling activity in the site of the graft. serial blood donations in a ko pregnant woman with the use of recombinant erythropoietin for intrauterine transfusions of severe hemolytic disease of the newborn due to anti-ku biweekly) were administered to the mother to ensure an adequate supply of compatible rbcs for intrauterine transfusions and possible perinatal haemorrhage as well. results: intrauterine transfusions were repeated every 1-3 weeks. by the 35th week of gestation the patient had donated four units of blood, her hematocrit was 40%, anti-ku titre was 1/2048 and four intrauterine transfusions had been performed. cesarian section was decided and the apgar of the newborn were 8 and 10 at 1 and 5 min. the newborn was treated with phototherapy but without exchange transfusions and two weeks later he was discharged. by the 15th day of life rh-epo was administrated to him due to anemia. the maternal red cells completely disappeared from the child's blood by the day 100. the experience of the use of erythropoietin in pregnancy is minimal. as illustrated by this case treatment with rh-epo and iv fe has effectively increased mother's capacity to donate rbcs' for autologous use and intrauterine transfusions as well, with no adverse effects to the mother or the child. however, further research is necessary to evaluate if rh-epo crosses the placenta. introduction: blood components should be transported by a system which has been validated. the containers used for transport should be well insulated, some form of temperature indicator should be used to monitor the in-transit temperature. whole blood should be stored at different temperatures above 2°c. aim of the study: bags with whole blood collected in a collection site are transported in containers without active cooling. we tested temperature of blood in containers put into the extreme weather conditions (+50°c, -20°c) during loading test for transport. methods: the container without active cooling was filled with the exact number of bags with blood and the exact number of passive cooling elements (frozen water cubes in plastic) placed in the exact positions without close contact with the blood bags. the bags with blood of the temperature +4°c, +20°c and +37°c were used. temperature indicators were situated in the bottom, centre and top of the container. filled container was placed into thermostat (+50°c) or freezer (-20°c). the temperature was observed in 10 min intervals for three hours, first measurement was 30 min after putting into freezer or termostat. results: (see table 1 ) nt, non tested; tmp, temperature. in the table there are shown minimum and maximum temperature parametres observed during tested time including increasing or decreasing trend. conclusion: loading test for transport of the bags with collected whole blood helps us to optimize transporting system, especially number of cooling elements in relation to the season and its place in the container. in the light of presented data we corrected transporting system to maintain the recommended temperature during transport. background: since 1999, we have produced pooled and filtered platelet concentrates out of four buffy coats in tsol platelet additive solution and have stored them in pall autostop clx bags made out of pvc/totm. the residual plasma content is between 30-40%, the mean volume about 260 ml and the mean platelet-content is 3.1 ¥ 10 11 per unit. for pathogen inactivation or bacterial screening it is necessary to extent the storage time from 5 to 7 days. new foliage like polyolefin is supposed to maintain a good quality environment for prolonged storage of platelets. aims: storage bags made out of polyolefin (pall autostop elx) were tested to prove their suitability for prolonged storage of platelets. methods: 18 twins made out of pools from 8 buffy coats were produced with the standard method, one twin was stored in the conventional bag (cb) the other in the new foliage bag (nfb). the platelet pools were stored on flatbed shakers at 22°c, and sampled at day 1, day 5 and day 7. ph, glucose, lactate, hypotonic shock response (hsr) and p-selectin expression were measured by standard in-house methods. results: mean ph on day 7 with cb was 7.21, with nfb 7.35; glucose with cb 4.4 mmol/l, with nfb 6.0 mmol/l; lactate with cb 17.4 mmol/l, and with nfb 14.8 mmol/l, hsr with cb 79%, with nfb 85%; p-selectin with cb 29% and with nfb 18%. the new platelet storage bag showed better results of in vitro quality markers, especially after day 5 of storage. prolonging storage time will make it easier to introduce bacterial screening or pathogen inactivation techniques into platelet transfusion. the possibility to filter rbc either at 4°c or rt simplifies the preparation process. filtration at +4°c enables to achieve a better leukoreduction performance. the nbs has successfully implemented this project which has the potential for improvement in patient safety and is predicated upon practical application and risk reduction rather than elimination. the impact of this work on the incidence of trali will require detailed, long term analysis of hemovigilance data using existing mechanisms. active communication, a team approach, perceived value of the initiative and the hard work of all staff involved were key success factors. quality assessment of buffy coat-derived platelets prepared from leucoreduced whole blood background: whole blood can be separated into; plasma, buffy coat and red-cell conc (rcc) by differential centrifugation and separation on a separation device. because of the high hematocrit of the rcc, 60% of the process time is needed for expression of the rcc. by increasing the internal diameter of the tubing at the bottom of a t&b system by 0.7 mm. a decrease of the process time is expected. methods: 220 units of whole blood were collected with the new t 3988 'wide boring' blood pack and separated on a routine base. quality control parameters were checked and the whole process time was monitored. free hemoglobine was measured up to 42 days. results: process time of a 'wide boring' bag is significant shorter compared to a standard blood bag. average decrease: 86 s. slightly increase in free hemoglobine is measured probably due to the increased express rate of the red cells. bloodproducts produced with the new t 3988 meet european guidelines. no significant increase of free hemoglobine due to the faster expression is measured. an significant decrease in process time is measured with the wide boring bloodpack. the new fresenius hemocare rcc in-line system: t 3988 can be used for routine production which will speed up the production process considerably. introduction: leukoreduction of blood components is required to prevent several transfusion-associated complications. aim: the aim of this study was the full process validation of the pall leukotrap wb system for the preparation of leukoreduced blood components. we collected 60 whole blood units from donors suitable for donation using a quadruple blood-bag, which includes an wbf3 in-line filter (pall) for the removal of leukocytes and platelets. mixer balances (baxter) were used and donation occurred within 12 min in all cases. after donation whole blood units were stored at room temperature for 2 h. subsequently, whole blood filtration was performed by gravity at a standard height of 150 cm using a blood leukoreduction cart (baby leuko cart, itl-corporation). filtered units were centrifuged at 5000 g ¥ 10 min by an heraeus cryofuge 6000i. an automatic extractor (bag press plus-bioelettronica) was used to prepare red cell concentrates in sag-m solution and fresh plasma units. air in the system was automatically expelled by the extractor. complete cell counts and hemoglobin concentration were evaluated in pre-filtration samples and at the end of the blood components preparation using an automated cell counter (pentra dx120-abx). we enumerated residual leukocytes in red cell units by flow cytometry (becton dickinson-leucocount kit). results: pre-filtration data of whole blood and end-of-process data of red cell and plasma filtered units, are summarized in table 1 . results are given as mean and standard deviation. whole blood filtration was completed within 10 min in all cases. red cell units were transfused after a mean of 6 days to 57 patients affected from transfusion dependent (45%), post-surgery (30%), and post chemotherapy anemias (25%). no cases of transfusion reaction were observed. the pall leukotrap wb system was easily introduced in our setting. all blood components prepared by the system fulfilled the council of europe requirements with regards to hemoglobin content in red cell units and post-filtration residual leukocytes. future studies are needed to evaluate its cost-effectiveness in the setting of routine blood component preparation. background: during an evaluation of the compodock (fresenius hemocare) sterile connection device (scd), we observed irregularities on the inside of the tubing at the site of the weld. it was our aim to investigate the effect of these observations on the quality of blood products. methods: three leukoreduced red cell concentrates (rccs) were pooled and divided over 3 bag systems: one without weld in the connecting tubing, one with a compodock-weld, and one with a weld made with the terumo scd. the rcc was transferred 10 times over this tubing to have maximum result if the weld had deleterious effects. the rccs were stored in pvc containers, and sampled on day 1, 28, 35 and 42, and free hemoglobin (hb) was measured. the same procedure was also performed using platelet concentrates (pcs), but these were stored in polyolefin containers, sampled on day 1, 6 and 8, and cd62 expression was measured. ten experiments were performed per blood component. according to who standards processing of blood into labile components are considered an expression of quality of transfusion service. in our practice, modern transfusion principles are successfully applied. they cover blood collection, serological processing of blood units, technological preparation of blood products (gmp, sop) and rational utilization of blood components and blood derivatives. in the past four years (2001, 2004.) aberrations from these principles have taken place (self-sufficiency). nbti collected x = 62 926 (249 778) blood units into blood bags. in serbia x = 230 537 (691 611) blood units. retrospective analysis: ldpc-bc was administered x = 97% with the satisfactory haemostatic effect. increase of the cyta and plasmapheresis-manual procedures was also noted (100%). increase of the use of leukocyte poor red blood cells was also registered (95% introduction: according to the relevant recommendations of the council of europe, whole blood is a source material used for the preparation of blood components and blood products. basic concept of the therapeutic use of blood components is the compensation of the lacking or deficient blood component. in that way, a possibility of the infusion of unrequired or deleterious components of whole blood is eliminated. objective of this presentation is to analyze the reasons of non-utilization of certain blood units, the actual quantity and ratio. aim of the study: the purpose of our in vitro study is to compare storage of platelet concentrates at 4°c with platelets stored at 22°c, and to determine the in vitro-effects of pre-incubation at 37°c for 1h prior to analysis on the basis of the maintenance of platelet metabolic and cellular integrity. methods: platelets concentrates (pcs) were prepared from pooled buffy coats (bc) for paired studies to be stored into different conditions. (i) at 20-24 degree on a flat bed agitator; (ii) at 20-24 degree on a flat bed agitator and pre-incubated for 1 h prior to analysis; (iii) at 4°c; and (iv) at 4°c and pre-incubated for 1 h prior to analysis. this paired in vitro study (n = 8) over 21 days include volume, platelet counts, mpv, volume, ph, po 2 , pco 2 , bicarbonate, glucose, lactate, swirling, leucocytecount, hsr, esc, atp, ldh and release of a-granule content (rantes, ß-thromboglobulin and pf4). results: platelet count (day 5 and 21; p < 0.05), mpv (day 5; p < 0.01), ph (day 14 and 21; p < 0.01), pco 2 (day 14 and 21; p < 0.01), bicarbonate (day 21; p < 0.01), glucose (day 5, 7, 10, 14 and 21; p < 0.01), atp (day 14 and 21; p < 0.01) was significantly higher in platelets stored at 4°c and platelets stored at 4°c with preincubation. ldh (day 21; p < 0.01), bicarbonate (day 5 and 7; p < 0.01), lactate (day 5, 7, 10, 14 and 21; p < 0.01), ph (day 5 and 7; p < 0.01), esc (day 5, 7, 10, and 14; p < 0.05), hsr (day 5, 7 and 14; p < 0.05) was significantly lower in platelets stored at 4°c and platelets stored at 4°c with pre-incubation. the concentration of rantes, ß-thromboglobulin and pf4 was significantly higher in platelets stored at 22°c than in platelets stored at 4°c (day 5, 7, 10, 14 and 21; p < 0.01). hsr (day 5 and 10; p < 0.05) and esc (day 5, 7, 10 and 14; p < 0.01) was significantly higher in preincubated platelets stored at 22°c compared with platelets stored at 22°c. conclusion: platelets stored at 4°c maintain metabolic and cellular characteristics to a great extent during 21 days of storage. we confirm the loss of platelet discoid shape and have shown that loss of discoid shape in platelets stored at 4°c is associated with decreased metabolic rate and decreased release of a-granule content. aim: as reference centre of the swiss blood transfusion service for new materials and blood products we evaluated that system for routine use and official registration in switzerland. method: 20 whole blood donations were collected in a whole blood filtration set with cpda-1 and stored at room temperature for 1 h before filtration at room temperature. the leucodepleted whole blood was stored for 42 days. following parameters were analysed on day 0, 35, 42: free haemoglobin in%, k. in addition leucocyte count was performed on day 0 and a blood culture on day 49 (see table) . blood cultures on day 49 remained negative and all counts of residual leucocytes were below 1 ¥ 10 (exponential) 6/unit. summary and conclusion: as expected there was a clear increase in k and free haemoglobin after day 35. however the results were within the required specifications from the european and swiss guidelines up to day 42. we conclude that autologous leucodepleted whole blood can be stored in cpda-1-for 42 days without loss of stability of the red cells. we will introduce the system to the offi-cial material list of the swiss blood transfusion service and then implement the procedure to our daily routine. results of ffp production from whole blood, and of ffp and pc produced by use of cell separator over a 5-month period before and after the introduction of measures for trali prevention are presented. the following measures were undertaken: (4) blood of female donors was not used for ffp production, and plasma was only used for fractionation (5) plasma of female donors was not used for kt-bc pools (6) platelets and plasma were produced on a cell separator only from the female donors without a history of pregnancy. female donors of whole blood: 16%*; 16%** ffp produced by plasmapheresis: 2%*; 2.5%** female donor units on cell separator: 9.4%*; 4.5%** ffp from total plasma units: 63%*; 58%** plasma units used for bc-pc pools: 4%*; 6%** *period before and **after the introduction of measures for trali prevention the exclusion of female donors had no major impact on the production of ffp and bc-pc pools from whole blood because of the very low rate of female subjects in the croatian blood donor population. the amount of plasma and pc collected from female donors by use of cell separator was significantly lower (~50%), however, without any major impact on total ffp store because of the small rate of plasma and platelets obtained by apheresis. background: platelet concentrates (pcs) are currently stored for a maximum of 5 days. extended storage to 7 days would increase the supply and reduce the waste of pcs. transfusion-associated graftversus-host-disease (ta-gvhd) is a severe transfusion reaction caused by t-lymphocytes in the transfusion product. the risk of developing ta-gvhd can be prevented by gamma irradiation of the pcs. various in vitro tests can be used to study the quality of pcs such as inspection of the swirling phenomenon, hypotonic shock response (hsr), detection of platelet surface markers (e.g. cd62p and cd42b), metabolic parameters and blood gases. free oscillation rheometry (for) using the instrument reorox® 4 can be used to monitor the coagulation over time in whole blood, pcs and plasma samples, and to obtain information about clotting time and coagulum elasticity. aim of the study: the purpose of this study was to evaluate the quality of pcs obtained by apheresis technique during storage for 7 days and to study the effect of gamma irradiation by using several in vitro methods including for. methods: platelets were collected from healthy donors (n = 12) using apheresis technique. the pc from each donor was divided in 2 units, one served as control and the other was gamma irradiated with 25 gy. the pcs were stored on a flatbed agitator at 22°c for 7 days. samples were taken on day 0 (= day of collection) for analysis of blood gases, metabolic parameters (glucose and lactate), platelet count and swirling. samples taken on day 1, 5 and 7 were also analysed for hrs, cd62p (p-selectin) and cd42b (gpib) expression utilising flow cytometry. evaluation of coagulation by for was performed on day 1, 5 and 7. the maximum elasticity (g'max) and the time to g' were evaluated from the for elasticity curves. results: there was no difference between irradiated and nonirradiated pcs regarding any of the tested parameters during the storage period. swirling, hsr, platelet count and percentage of cd42b expressing cells were well maintained for 7 days of storage. glucose decreased and lactate increased significantly during the storage period, from 2.5 mmol/l to 16.2 mmol/l for lactate and from 16.5 mmol/l to 8.9 mmol/l for glucose. the percent cd62p expressing cells increased significantly during storage from 15% on day 1 to 32% on day 7. po 2 was well maintained but ph increased and pco 2 decreased significantly between day 0 and 1 whereafter ph decreased and pco 2 continued to decrease. the for parameters g'max and time to g'max increased significantly between day 1 and 5 and the time to g'max continued to increase significantly between day 5 and 7. the results indicate a well preserved platelet quality after storage for 7 days. gamma irradiation did not affect the platelet quality. cytokine release during storage of buffy coat platelet concentrates produced manually and automatically background: transfusion reactions following platelet transfusion are still a problem even when leukoreduction is included in the production process. platelet derived cytokines released during storage upon activation or lysis, accumulate in the platelet products and have been suggested to be involved in transfusion reactions. rantes (regulated upon activation, normal t cell expressed and presumably secreted) is a chemokine playing an important role in the inflammatory immune response and causes degranulation of eosinophiles and release of histamines from basophiles, which again can cause allergic reactions. tgf-b1 (transforming growth factor b1) has been shown to be immunosuppressive, inhibits the proliferation of t-and b-lymphocytes and decreases the secretion of igg and igm from b-lymphocytes. aims: as part of our quality control program, we aimed to quantify the amounts of rantes and tgf-b1 released during storage in platelet concentrates produced from pooled buffy coats by our manual routine method (m-pcs) and by an automated method using the orbisac system (gambro) (a-pcs). methods: pcs were produced from 5 buffy coats. following overnight storage at 20-22°c, buffy coats were pooled with 300 ml t-sol (baxter). forty-two pcs were produced either manually (n = 21) using the imugard iii s-pl set (terumo) with integrated soft leuko-reduction filter or by the automated procedure (n = 21) using the orbisac validation bc set (gambro) equipped with the lrp6 leuko-reduction filter (pall). swirling was scored visually, platelet count and mpv were measured on a cell counter (cobas argos, roche), and blood gas analyses, glucose as well as lactate were measured on an abl700 series analyser (radiometer). samples for testing of cytokines were centrifuged for 15 min at 4800 g, 20°c; supernatants were harvested and frozen at -86°c until analysis. cytokines were quantified using quantikine human rantes immunoassay (r&d systems) and human tgf-b1 elisa immunosorbent assay (bender medsystems gmbh). all analyses were performed on days 1, 4 and 7. results: platelet concentrate volume (mean): m-pcs: 316 ml, a-pcs: 328 ml. platelet yield was found to be 3.15 ¥ 10 11 for m-pcs and 3.43 ¥ 10 11 for a-pcs (p < 0.0001). in all pcs ph levels were between 6.9-7.2. glucose consumption and lactate production from days 1-4 and days 4-7 did not differ significantly. rantes levels (pg pr 10 9 plts) were significantly higher in a-pcs than in m-pcs (p = 0.04, repeated measures analysis of variance), but no significant difference was found in tgf-b1 levels (pg pr 10 9 plts). summary and conclusions: preparation of buffy coat platelet concentrates by the automated orbisac system improves platelet yield compared to our manual processing procedure, but the levels of the chemokine rantes were significantly highest in the automatically produced products. the clinical importance of these findings is still unclear, but may be related to the shear stress the platelets are subjected to during the automated production process. the quality of cryopreserved vs liquid stored platelets: a comparative study table 1 . the mismatches can be divided into the two categories. the first of them is characterized by differences in allelic groups, i.e. at low-resolution level. 22 allelic group differences were detected in the group with one mismatch, most of them in hla-c locus (this locus was not concluded in primary donor search). in the other category there are differences in alleles within the same group, i.e. at high-resolution level only. 12 differences within the same group in all tested loci were detected in the group with one mismatch. the mismatches described above were heterogeneous and a correlation of specific mismatch with transplantation outcome was not possible in this group. conclusion: the use of high-resolution dna methods makes the identification of hla match/mismatch more accurate and can affect the outcome of unrelated hsct. this work was supported by the grant iga mz, no. nr/7984-3. pre-freezing and post-thawing quality controls in umbilical cord blood assigned for transplantation p bergamaschi, c perotti, g viarengo, c del fante, c parisi, a marchesi, l bellotti and l salvaneschi irccs policlinico 'san matteo', pavia, italy background and aims: nowadays umbilical cord blood (ucb) represents a well established source of haematopoietic stem cells for unrelated transplantation in children affected with haematological and inherited diseases. thanks to the large-scale banking of unrelated units and the preliminary encouraging results, ucb employ in adults is quickly growing up. in this context, total nucleated cells (tnc) count of the graft is considered the main predictor for clinical outcome; however, other indicators of the haematopoietic potential, such as cd34+ cell content and short-term culture clonogenic assay, are recommended in accordance to netcord-fact stan-dards. in order to guarantee the safety and the prompt availability of a ucb unit assigned to a matched recipient, a pattern of rigorous quality controls should be carried out not only at the time of cryopreservation but also before the release for transplant. we report the results of the quality controls performed on thawed cryovials referring to the 26 units delivered by our ucb bank compared to the pre-freezing values. methods: every ucb unit stored in our bank is accompanied by 3 satellite cryovials available for subsequent controls. for each unit issued for transplantation, one cryotube was thawed in 37°c water bath with gentle agitation without washing out dmso. tnc and mononucleated cells (mnc) were estimated by an automated cell counter; viability and cd34+ cell count were evaluated by flow cytometry with a no-wash, single-platform technique and 7aminoactinomycin d. cfu assay was performed using commercial reagents (methocult gf h4434, stemcell technologies) and colonies were counted after 14 days. the same tests were performed before cryopreservation, taking a sample from each fresh unit. moreover, before the delivery for transplant, a second cryotube was thawed to investigate the bacterial contamination by direct microbial culture, whereas the sterility test before freezing was performed by inoculum into 30 ml media (bact/alert®fa/fn, biomérieux inc). results: the ucb characteristics before freezing and after thawing are detailed in the tables 1, 2 and 3. post-thawing tnc and mnc, as well as cd34+ cells, showed no significant difference in comparison to the pre-freezing values. despite of the expected decrease of the overall viability after thawing, we observed a highly satisfactory viability referred to the cd34+ cells. the colony forming units (cfu) growth after thawing was documented and was always lower as respect to the pre-freezing assay. finally, the results of microbial cultures were negative for all the 26 units on both fresh and thawed specimens. conclusions: in our experience, well standardized evaluation of ucb content could be obtained with regard to tnc, mnc and cd34+ cell. concerning the results of short-term cultures, the presence of dmso as inhibiting factor may be advocated to explain the discrepancies between fresh and thawed samples. finally, rigorous quality controls documented that the procedures of manipulation and cryopreservation did not affect the quality of ucb to be infused for transplant and provided to the physician all the parameters necessary for a safe transplant in a close and appropriate time. bone marrow transplantation or bmt transplantation of progenitor blood cells to regenerate blood normal cells in patients with blood disorders. bone marrow has an organized and structured architecture in which close relationships exist between a regulatory microenvironment and primitive hematopoietic cells. in fact, normal hematopoietic cells depends on critical interactions that occur between stem cells and their microenvironment. this microenvironment is a complex meshwork composed of growth factors, stromal cells, and extracellular matrix. marrow injury can occur as a consequence of a variety of diseases. some diseases could be due to a microenvironment that fails to support hematopoiesis. a possibility is that aplasia and leukemia share a common etiology such as drug, chemical, radiation, virus or other environmental hazards. we can say that microenvironmental abnormalities in interactions between stromal cells and hematopoietic progenitors may be important in the pathogenesis and clinical expression of hematopoietic malignancies in humans. background: intrauterine growth retardation, with associated low birth weight, represents one of the most important cause of baby mortality and morbidity. understanding the genetic bases of this adverse event is still an open goal. there is evidence that motherchild hla compatibility and hla-drb1 foetal genotype are associated with a reduced placental growth and a low birth weight. the recent institution of cord blood banks, with their huge amount of hla types, offers an unique opportunity to look inside the molecular bases of normal birth weight. aims: we investigated whether the baby-linked immunogenetic profile, i.e. hla gene frequencies and homozygosity rate, affects the physiological variance of the size at birth. methods: 1868 cord blood units (961 from males and 907 from females) were hla typed with pcr-ssp and/or reverse pcr-sso techniques and recorded in the cord blood bank database of pavia-italy. all were defined at low resolution level for hla-a and b genes and at high resolution for hla-drb1. 686 blood units were also randomly typed for hla-dqa1 and dqb1 at high resolution. results: mean birth weight was 3430 g and mean relative birth weight (i.e. corrected for gestational age according to the gender) was 0.19 g. 70 babies were <5th centile (2782 g) and 70 were >95th centile (4068 g). comparing the hla allele distribution in these extreme bands we found that hla drb1*08 was significantly associated with high relative birth weight: 5.1% in 95th centile vs 0.7% in 5th centile, p = 0.032. on the contrary, hla-drb1*14 and dqb1*05 were associated with low relative birth weight: 7.9% and 30.4% respectively in 5th centile vs 2.2% and 9.6% in 95th centile p = 0.035 and p = 0.019. all infants were analysed as to the effect of the above mentioned alleles. we confirmed the positive association of hla-drb1*08 and higher relative birth weight (mean 0.17 vs 0.08; p = 0.033) as well as the association of hla-drb1*14 with lower relative birth weight (mean 0.03 vs 0.27, p = 0.04). no significant association was found as far as hla homozygosity was concerned. conclusions: the present findings confirm the role of foetal hla-drb1 gene in the intrauterine growth. about the specific involved alleles, one possible explanation comes from the studies of crystallography and amino acid sequencing of hla-dr binding groove. it has been demonstrated that hla-drb1*08 and hla-drb1*14 genes encode for different amino acid sequences in the pocket 4 of the molecule (aa 70, 71, 74). this implies distinct functional restriction patterns. the sequence motif of hla-dr14 is characteristic of some autoimmune conditions, such as hashimoto's thyroiditis, and preeclampsia which is associated with intrauterine growth retardawhich provides a high yield and excellent purity without lymphocyte and erythrocyte contamination. in a 3 month period, we studied blood samples from bone marrow transplant patients and from normal subjects. the extraction of leukocyte polymorphonuclear was obtained with a 6%-7% dextran solution in 0.8% saline. after incubation at room temperature with lymphopre solution, the mixture was centrifuged. two clear and separate rings of mononuclear and pmn leukocytes were obtained. to eliminate any red blood cells, pmnl ring was separated and washed three times with cold ammonium chloride. after a short period of incubation 4°c, mixture was centrifuged and the pmnls were isolated. the purity and viability of total leukocyte population was counted and the percentage of pmnl obtained was established. the total blood samples studied were divided in two groups, i.e., bone marrow transplant patients and normal subjects. in both cases the pmns isolated were of high purity and viability. the overall percentage of pmnls obtained from both groups under study was 98% to 99% when stained with gimsa or wright staining method. the viability of isolated pmnls was also 98% too, which is excellent for numerous immunological or molecular studies. the pmnls isolated by this method were highly pure and viable in comparison with standard methods used to isolate human pmnls. generation a high amount of pmnls is another advantage of the suggested method. this method to separate pmnls is recommended for in vitro studies of different subjects. et al. 1950 .) the object of exchange transfusion (et) is to remove bilirubin already present in the plasma or remove alloantibody which can cause hemolytic disease (in order anti-d, anti-c and anti-k are easily the most important) or remove anti-d positive red cells. we have studied exchange transfusion in our hospital in neonatal intensive care unit, during 1997 to 2004. at delivery cord samples were taken for determination blood group, rhesus, hb and ht have been counted. also direct antiglobulin test (dat) has been performed. in cases of positive dat, hb and bilirubin levels were monitored. newborn body-weight were weighted (ranged 710 g to 3100 g). the blood for et was of group o, d negative and kell negative and was compatible with the serum of mothers; it was less than 7 days old. the blood was screened for hbs and for anti-cmv as well as being submitted to all usual tests. the method has been determined by using the umbilical vein; the multi-way tap makes it possible to draw blood from the infant into the syringe, to discard the blood into a sterile empty vessel, then to draw blood from a donor unit into the syringe and inject this into the infant. results: 17 exchange transfusions were carried out. four out of 17 et due to abo incompatibility mother-newborn. five out of 17 due to rhesus incompatibility mother-newborn and eight out of 17 due to jaundice undetermined origin and immaturity. the last two years only three et were carried out due to immaturity. conclusions: phototherapy, when applied early enough and with sufficient intensity, can avoid the need for exchange transfusion in many infants. phototherapy alone or phototherapy plus high dose igg therapy has been used to minimize exchange transfusions in this population. detection abo blood group system antibodies of neonatal using fully automated column agglutination technology (auto-vue) background: the abo blood group system remains the most important in transfusion practice. this is because of the regular occurrence of the antibodies anti-a, anti-b and anti-a, b reactive at 37°c, in persons whose red cells lack the corresponding antigens. the regular presence of anti-a and anti-b is used in the routine determination of abo blood groups; in addition to testing red cells for a and b antigens, the group is checked, in serum or reverse grouping, by testing the serum against red cells of known abo groups. methods: 100 samples were taken from 100 newborns at first 24 h of life for abo blood group typing during 2001-2004. simultaneously the presence of anti-a and anti-b antibodies has been studied using fully automated column agglutination technology (auto vue ortho diagnostic systems) with bio-vue cassettes aborh/reverse. the column agglutination technology is based upon the ability of glass beads to form a physical barrier between agglutinated and unagglutinated red cells. to determine the abo serum group, test serum and abo reagent red cells were added to the top of column containing diluents. the abo grouping columns were centrifuged and examined for agglutination. the presence or absence of agglutination has been recorded. results: in 68 out of 100 newborns were found detected antibodies anti-a, anti-b. in 24 out of 100 newborns no antibodies were found. in 8 out of 100 were found antibodies maternal origin. the automated reader detected all positive reactions. positive results were recorded on a scale from +0.5 to +4. conclusions: the technical performance of device allows objectivity and precision to detect abo blood group antibodies of newborn. the origin and the type of antibodies and also factors that influence their presence are to be studied. introduction: diagnosis, management and prevention of red blood cell immunization have improved, so hemolytic disease of the newborn (hdn) has changed from a common to a rare pathology. aim of the study. in this study we have retrospectively evaluated the benefits of the immunohematological screening for the management of pregnancies with alloimmunization. methods: in the last 3 years, we have performed an immunohematological screening on all pregnant women assisted by our hospitals. ab0 and rh typing, antibody screening and, eventually, identification and titration were performed on maternal specimens by microcolumn technique. results: not considering ab0 incompatibilities, we have discovered 64 alloimmunized women with the following specificities: 10 anti-d, 9 anti-c, 7 anti-c, 9 anti-e, 3 anti-jka, 1 anti-d + anti-jka, 1 anti-d + anti-s, 3 anti-d + anti-c, 1 anti-d + anti-c + anti-k, 5 anti-s, 10 anti-k, 2 anti-m, 1 anti-c + anti-e and 2 anti-d + anti-c + anti-g. the most severe hdn were the 2 d + c + g, the c + e and 2 out of 7 c newborns, with mean hemoglobin between 5 and 6 g/l, bilirubin = 20.5 g/l, reticulocyte count = 10%. in these exchange transfusion needed at the delivery. other newborns were only treated with phototherapy. conclusion. thanks to the immunohematological monitoring, the diagnosis of alloimmunization, the correct management of pregnancy and the adequate neonatal therapy were possible. in fact all newborns survived and showed no neurological lesions in the following controls. conclusion: in order to provide a highly specialized perinatal care, immunohematologist, obstetric and pediatric should provide a good antenatal and perinatal screening. this is an interesting case of rhd immunisation in rhd negative woman despite the application of rhd immunoprophylaxis. case report: a blood sample of pregnant woman, 30 years of age, in 12th gestation week, was sent to our laboratory for serological analysis. her blood group was o, ccddee and she had an anti-d antibody reactive only with enzyme treated panel of test erythrocytes. her husband was a, ccdee, and two children were both a, ccddee. on the next visit, she gave the data of one arteficial and one missed abortion before the 12th gestation week covered with 50 mg of rhd immunoprophylaxis, but without the measurement of fetomaternal haemorrhage (fmh). both of abortions were after the deliveries. until the end of the pregnancy, detailed serological analysis showed anti-d specificity of antibody in her sera which remained reactive only with enzyme treated red blood cells. the fetus was under permanent ultrasound control. she delivered a mature, rhd positive, ccdee male child, without any sign of haemolytic disease. the proper personal history, measurement of the size of fmh, distinguishing the anti-d and anti-g specificity of the antibody, administration of rhd immunoprophylaxis and cooperation between transfusion medicine specialists, gyneacologists and neonatologists still remain major principles of prenatal and perinatal care concerning haemolytic deisease of the fetus and newborn caused by anti-d antibody. anti d antibody, reactive only with enzyme treated red blood cells is usually harmless for fetus and the newborn. introduction: red blood cell (rbc) transfusion is widely used in neonatal intensive care units for acute or chronic pathological conditions. clinical indications for rbc transfusion are shock, sepsis and/or anemia with the following laboratory criteria: a) hematocrit (hct) < 20% or hemoglobin (hb) < 7 g/dl and reticulocytes < 4%; b) hct < 30% or hb < 10 g/dl in these conditions: o2 required < 35%, recurrent apnoea and bradicardia, cardiac rate > 180 bpm and respiratory rate > 80 bpm for more 24 h; c) hct < 35% or hb < 12 g/dl with severe respiratory distress. aim of the study. aim of this study has been to evaluate the effectiveness of rbc transfusion therapy in premature and at term newborns independently of initial pathological conditions. methods: our therapeutic objective has been to achieve an hct of 50% after the whole cycle of transfusion therapy. for each little patient, the volume of transfused rbc unit has been calculated, according to international guide lines, using the following criteria: weight (kg) ¥ blood volume (100 ml per kg if premature or 80 ml per kg if at term newborn) ¥ (hct desired -hct observed)/hct of unit transfused. particularly we have considered that premature infants (with a gestation age of 26-36 weeks) show a range of weight from 600 to 1.800 g, while at term newborns from 1.800 to 4.000 g. in order to avoid a circulatory overload, the indicated hemocomponent has been always packed rbc with higher possible hematocrit. for the same reason, the rate of infusion has been always 3-10 ml/kg/hour. methods: this is a descriptive study. the name of the neonates who received transfusion was obtained from the blood bank of beheshti hospital. information concerning the type of blood product, frequency and indication of transfusion, sex, gestational age and weight of infants was recorded in questionnaire and analyzed. results: out of 541 neonates admitted during one year, 118 (68 male, 50 female) received blood components. fifty four percent received one, 35% two and 11% received three types of blood components. the frequency of transfusions were 311 times. the most common used blood products were fresh frozen plasma (49%), red blood cell (33%), whole blood (14%) and platelets (4%). all the blood products except whole blood were used more common in premature and low birth weight infants. appropriateness of transfusion of red cells, fresh frozen plasma, platelets and whole blood were 92%, 88%, 100% and 91% respectively. (hdn) . in accordance to current regulations, this study is carried out in all pregnant women attending in our service. according to our protocol, when an alloantibody of any specificity is detected through the liss-coombs gel technique, the same determination is made using papain-treated screen cells to detect any association with other antibodies which could be of clinical relevance for a hdn. there are scientific evidences that the use of enzymatic techniques increase the test's sensitivity, though clinical relevance of 'enzyme only antibodies' may be questioned. aims: demonstrate the importance of a routine identification of irregular antibodies in pregnant by two methods (liss-coombs -enzymatic) and the prevalence of anti-d associated antibodies, only detected by using enzymatic technique. analyze the need to carry out a follow up of sensitized patients to determine if the associated antibodies only detected with in an enzymatic medium can be detected with a liss-coombs medium during pregnancy, thus acquiring clinical significance for hdn. materials and methods: between january 2000 and december 2004 we studied 1970 d-negative pregnant women. the studies performed were: abo grouping, d and weak d, rh phenotype, direct antiglobulin test and irregular antibody detection (iad) against commercial screen cells with liss-coombs (diamed) ® gel-medium technique, according to the manufacturer's specifications. when iad were positive, an antibody identification using two commercial cell panels with gel techniques (liss-coombs-enzymatic) was performed. along the pregnancy, periodic controls were carried out to determine the exact moment when antibodies, previously only identified in an enzymatic medium, could be detected in a liss-coombs medium (clinically significant antibodies). results: out of 1970 d-negative studied samples, 278 (14.1%) had a positive dai and it was only showed anti-d specificity in a liss-coombs medium. after analyzing this specificity against enzymetreated erythrocytes, it was possible to determine that 79 patients (24.8%) had in their serum other anti-d associated alloantibodies: 5 anti-k (1.8%), 63 anti-c (22.7%), 10 anti-e (3.6%) and 1 anti-c + e (0.3%). during the immunohematologic follow up, it was determined that in 4/79 patients some of the antibodies which were previously only detected in an enzymatic medium, could be identified in a liss-coombs medium and later they were identified in the red cell elution of the newborn. conclusions: these results confirm the relevance of a screening for irregular antibodies of clinical importance by means of a conven-tional technique and one of increased sensitivity in all pregnant women. the detection of an association of antibodies provides information for the undertaking of diagnostic and therapeutic measures, both by the obstetrician, as for any eventual transfusional requirements for hdn. it was also concluded that, although antibodies detected in an enzymatic medium are considered of low clinical significance, its investigation and follow up is suggested in pregnant women to determine the moment in which they can be detected in an antiglobulinic medium, thus revealing their clinical significance. background: fibrin glue is one of the most complex human plasma derivatives both in terms of composition and clinical applications. this product mimics the last step of coagulation cascade through activation of fibrinogen by thrombin, leading to the formation of a fibrin clot in the presence of factor xiii. in contrast to synthetic adhesives, the significant advantage of this plasma-derived sealant is its biocompatibility and biodegradability as well as the fact that it does not induce inflammation, foreign body reaction or extensive fibrosis. readsorption of the fibrin clot is achieved during wound healing within days/weeks following application, depending upon the type of surgery, the amount and type of product used or the proteolytic activity of the treated site. the risk of virus transmission by commercial fibrin glue products is still debated and investigators are looking for alternative fibrinogen sources. many of these studies rely on autologous on single donor cryoprecipitate as source of fibrinogen. aims: the aim of this study was to compare single and double methods of cryoprecipitation of fibrin glue. the influence of different plasma preparation methods and plasma storage temperatures (-30°c and -80°c) on the quality of fibrinogen concentrate was examined. methods: whole blood was collected by standard phlebotomy technique and centrifuged at 4000 ¥ g for 5 min within 2 h of collection. plasma was removed. four units of plasma were pooled into a 2000 ml bag, mixed, divided into 4 parts (aprox. 219.7 ml) and immediately frozen. two of these units were stored at -30°c and 2 units of ffp at -80°c. after one month the plasma was thawed at 4°c during 16-18 h. the fibrinogen concentrates (21-25 ml) were received by single and double cryoprecypitation. to compare single and double methods of cryoprecipitation, the levels of fibrinogen, fibronectin, plasminogen and factor xiii were determined. results and conclusion: the levels of fibrynogen were significantly higher in fibrynogen concentration obtained by double cryoprecypitation from plasma stored at -30°c. there were no significant differences in the level of plasminogen in both tested groups. double cryoprecipitation of a single unit of plasma (stored -30°c) is an efficient, simple and safe method of obtaining fibrin glue. background: talking about professional risk we generally consider the risk of acquiring some kind of infective disease through accidental injury. in this article i would like to point out another side of the problem-the risk of making preventable medical error. with all its consequences. aim: how blood transfusion errors are among the most serious types of medical errors, the final goal is to initiate nationwide, regular, mandatory error reporting. information obtained, distributed and openly discussed at professional meetings will contribute to improving the patient safety. at the same time it would contribute to avoidance of blaming and shaming of many health care providers. prevent what preventable could be! method: retrospective analyze was conducted at the middle size blood transfusion center (10 000 donations per year and utilization of approximately 25 000 of components). results: after clearly distinguishing adverse events due to underlying patient condition from preventable medical error we fined out that: -great majority of adverse events resulted from medical error -every part of blood transfusion center, from blood donation ward, through laboratory testing to component issuing has it' weak points' or vulnerable places -any educational level is equally liable to error there is no significant difference about occurrence time: -working day/holiday -emergency/routine request -routine h/out of routine h -main error cause were as follows: -donor sample misidentification -rhd typing error -abo typing error -incorrectly performed cross match -recipient misidentification -wrong component prepared -sample confusion during freezing preparation conclusion: the truth incidence of transfusion medical errors is underestimated. mandatory report of fatal or 'only' harmful errors to the referent institution and its periodical announcement is the step ahead in preventing errors. those reports should be discussed at professional meetings (not at the 'yellow pages') and served as educational tool. but, as the most of the errors are system related, the key to reduce them is to focus on improvement of the system and nil for plasma. wastage rate was highest for plasma components. the influence of local practices on such discarding and whether avoidable shall be discussed. audit for blood discarding and corrective actions to minimize discarding is essential for all transfusion services and blood centers. designed technical and economic support. options include importing finished products and/or procuring products made from locally collected plasma. one approach is to consider local fractionation of plasma by building and operating a plasma fractionation facility, which may produce, finished products, or may produce intermediate products that are further manufactured in another facility. an alternative approach is the implementation of a plasma fractionation program where local plasma is sent to an established fractionator, and the plasma is fractionated following preagreed terms. the end products are returned to the country of the plasma supplier. in 1954 the national center for the production of blood products was established, under the direction of elias politis and 9 years later in 1963 begun the production of dried plasma from greek donors. by the year 1965 the center started the production of fibrinogen and by the year 1967 the production of antihaemophilc factor. in 1992 all the activities of the center settled down due to administrative aspects. at the beginning of 1990s a contract fractionation program was instituted (under the direction of k. sofroniadou) concerning the fractionation of liquid plasma and production of albumin, which by the end of year 2000 stopped and was replaced with a new contract for the fractionation of source plasma and the production of albumin. the challenge of adapting to the new and more stringent regulations governing the manufacture of blood products was great and brought a lot of changes in the structure of our center. a new bar-coding system ensuring the traceability of blood donations was instituted together with complex software for packaging and preparation of plasma shipments to the fractionation center together with all necessary paper work. a close collaboration with the medicines regulatory authority in order to be able to fulfill all the requirements that regulate issues associated to the quality and safety of human derived medicinal products. collaboration with blood collection establishments was promoted in order to increase the amount of plasma produced. there is a continuous effort from all the implicated parts in order to follow defined quality assurance procedures as highlighted by international guidelines for the blood donor selection, collection procedures, testing methods, donation handling, storage and transportation of plasma. the plasma contract fractionation program may serve, as an initial step prior to switching production to a locally built facility. this lapse of time may be used to expand the plasma collection potential, and to permit appropriate design, qualification and validation of the facility as well as training of local personnel. background: fibrin glue became a reality in the early 1970s, when techniques for the isolation and concentration of clotting factors were improved. in 1972, matras et al. described successful application of fibrin glue for peripheral nerve repair. this encouraging report prompted the use of fibrin glue in wound closure, skin grafting and bone union of osteotomies. the fibrinogen component of fibrin glue is produced from single unit donations of fresh frozen plasma. such procedure helps to reduce the risk of transfusion transmitted infections encountered by exposure to pools from large numbers of donors or by use of fibrinogen prepared from autologous blood prior to surgery. the second component, a mixture of thrombin and cacl2, is commercially available. thrombin is applied to the operation site simultaneously and in equal volume to the fibrinogen but from a separate syringe. there are many methods of fibrinogen concentrate preparation but none of them has been described in detail. aims: the aim of this study was to choose/select the most effective, simple and safe method of obtaining fibrinogen concentrate (basic component of fibrinogen glue) which would also be easy to prepare in blood transfusion centers. methods of precipitation of fibrinogen by polyethylene glycol (peg), ammonium sulphate, ethanol or cryoprecipitation were compared. methods: plasma was obtained after centrifugation (4000 ¥ g for 5 min) of whole blood. four units of plasma were pooled into a 2000 ml bag, mixed, divided into 4 parts and immediately frozen. one of them was stored at -30°c and after one month the plasma was thawed at 4°c during 16-18 h. fibrinogen was obtained by cryprecipitation and each of the three remaining units was precipitated with ethanol, peg and ammonium sulphate. the levels of fibrinogen, fibronectin, plasminogen and factor xiii were determined in each fibrinogen concentrate. results and conclusion: the level of fibrynogen ratio in fibrinogen concentration, obtained by peg and ammonium sulphate was significantly higher. cryoprecipitation is a simple, economic and reproducible procedure with the advantage of being performed in a closed system. plasma fractionation program in greece: an unknown history the provision of safe and sufficient plasma derivatives to meet the needs of local population requires special consideration and a well(light cycler, roche diagnostic systems, nj) was used for the identification of the c282y, h63d and the s65c point mutations of the hemochromatosis gene, and were based on protocols developed, for c282y by the unidad de medicina molecular (ingo, santiago de compostela, espanha), and for the other two mutations by bolhalder m et al. the primers and probes were designed by tib molbiol (berlin, germany). results: the analysis of the percentages of genotypes and allele frequencies of the hemochromatosis gene mutations are described in the table. no differences were found between the patients and the controls. when we compared subgroups of patients based on their hepatitis c genotypes, a higher value for the c282y allele was obtained (9.0%) in individuals with genotype 1, however without statistical significance. discussion: hereditary hemochromatosis is a common disorder and is associated, in some studies, with a worst prognosis in patients with viral hepatitis. follow-up studies are necessary in order to evaluate if the presence of these mutations can cause a more severe course of the illness (greater risk to develop fibrosis or cirrhosis) and a different outcome when treated with antiviral drugs. also, it will be important to evaluate if aggressive phlebotomies will modify their clinical evolution. introduction: portugal has a higher prevalence of viral hepatitis, with probably more than 250.000 patients chronic infected with hepatitis b and/or c. hereditary hemochromatosis (hfe) is one of the most common causes of known hereditary illnesses with hepatic repercussion. hfe mutations are also found in linkage desiquilibrium with particular hla haplotypes, conferring, eventually, a different response to viral agents and antiviral drugs. in this study we evaluated the prevalence of the main mutations c282y, h63d and s65c for the hfe in a population with chronic hepatitis b and/or c and in a cohort control. background: haemolytic disease of the newborn (hdn) is the destruction of the red blood cells of the fetus and neonate by antibodies produced by the mother. although postpartum rhig prophylaxis reduced the incidence of alloimmunization from pregnancy from 16% to 1-2%, the doubt subsists if it is appropriate to use it as routine antenatal prophylaxis. material and methods: a total of 1836 samples (918 mothers and 918 newborns), from 01/05/2003 and 30/04/2004, were studied. all abo, rh typing, antibody tests and dat were carried out in column agglutination tests. results: from the 918 cases studied it was found 778 cases without incompatibility (85%). from the 140 incompatibilities, 88.6% were abo, 8.6% were rhd, 1.4% were other rh incompatibilities, and 1.4% were due to auto-antibodies. 80% of the mothers were rhd+ and 20% rhd-. conclusion: of the 918 pregnant women studied, only 184 were rhd-. from this group 83 (45%) delivered rhd-newborns, what revealed that the antenatal prophylaxis they were submitted was unnecessary. from the 184 pregnant women rhd-, 13% had incompatibility abo, which decreases to near 2% the risk of development of rhd immunization. being anti-d immunoglobulin a product that has the potential risk of infection transmission, is it appropriate to use indiscriminately as a routine antenatal prophylaxis? the introduction of molecular methods to determine the fetal rhd genotype could rationalize the use of antenatal anti-d immunoglobulin prophylaxis. introduction: the frequency of hla a201 haplotype expression has been found about 44-46% in caucasian and in greek population particularly, 44.3%. because of the high frequency, it is used widely in anticancer immunization. the immune system plays an important role in the defense against neoplastic disease and immune response show temporal chances related to circadian variation of antibodies and total lymphocytes in the peripheral blood. aim: the probable difference in the frequency of hla a201 expression and their lymphocyte phenotype into a group of cancer patients and a group of healthy donors, during screening of immunization with hla-combined peptides. materials and methods: 323 healthy donors who proceeded in the department of transfusion medicine, university hospital of heraklion crete were tested for the hla a201 expression. in 11 of these donors the expression of cd3, cd4, cd8, cd2, cd19, cd20, hla dr, cd3+cd4+, cd3+cd8+, cd16-cd56+, cd3+cd16-cd56+, cd3-cd16-cd56+, cd3-cd16-cd56+ was examined. meanwhile, 132 patients with metastatic cancer who were hospitalized in the department of medical oncology, university hospital of heraklion crete, were tested for their hla a201 expression, while in 50 of them for their lymphocyte phenotype. the antigens expression was examined in flow cytometry. the hla a201 expression in healthy donors was 44.8% and in cancer patients 45% (p > 0.05). in table 1 the mean, standard error, t-test and p of the two groups are included. (see table 1 ). the two groups (healthy donors and cancer patients) revealed no statistical significant difference on lymphocyte phenotype, except of the cd20 expression, which was higher in cancer patients. summary and conclusion: the expression of hla a201 in cancer patients and in healthy donors was comparable. also, the lymphocyte phenotype among the two groups has not statistical significant difference, except of the cd20 (total b-cells). the significance of this result has to be investigated. in the course of original documents research i found out that dr. kalic, head of the first organized blood transfusion institution in the balkan region (at beograd, serbia, in 1936), set himself a professional goal: blood should be awaiting all patients and transfusion should not be a privilege of large city inhabitants only. dr. kalic's idea was that blood transfusion should be administered according to clearly given instructions and using simple blood sets. encouraged by the conclusions of the congress held in paris in 1937, dr. kalic started preparations for the transport of blood to the inland. he concluded bravely that citrated blood could be sent by regular mail, as an ordinary parcel, without particular protection from the outside temperature. he advised his colleagues to use blood as an intravenous injection. blood was taken from voluntary female donor in belgrade (capital), march 1938. after keeping it for 10 days at storage, blood was forwarded on a two-day journey to a small town, 150 kilometres away from belgrade. there it was kept on a room temperature before its final use for a treatment of a patient suffering from secondary anaemia. the patient underwent the procedure without side effects and responded to the transfusion with blood sent in this manner much better in comparison to earlier methods of direct blood transfused. reminding ourselves of the courage of our ancestors to implement their professional knowledge and personal original ideas in a new way with the desire to help the patient as successfully as possible, we pay them the deserved respect and gratitude for inspiring and encouraging us in this way to try the same. conclusions: automation leads to increased standardization, faster specimen processing and reporting, elimination of manual specimen identification, uniform interpretation of serological reaction patterns and objective reading of haemagglutination endpoints. using auto-vue allowed the staff uninterrupted time to perform quality assurance duties, extended antibody identifications, preventative maintenance, inventory control. the instrument allowed us to leverage current staff to a more productive, less stressful level. introduction: exosomes are 60-100 nm secreted vesicles produced by antigen-presenting cells (apcs). the finding that exosomes from dc pulsed with tumor-derived peptides elicited potent antitumor tcell responses and tumor regression in mice has led to the proposal that human exosomes could be effective vectors for antigen delivery in the context of cancer immunotherapy. aim of the study: to establish the method of producing a new kind of tumor vaccine -exosomes secreted by dc, pulsed with tumor peptides. methods: exosomes used in this study were generated from monocyte-derived dc pulsed with peptides from k562 tumor cell lines. exosomes were purified by the methods of ultrafiltration and ultracentrifugation. the methods of dynal magnetic beads, flow cytometry and western-blotting were used to determine the surface molecules of the exosomes. the function of the exosomes was deterobjective: to develop an immunoheatological technique for the study of erythrocyte hyaluronic acid sodium salt (cd44) receptor expression in red blood cells (rbcs) from adults and newborns. materials and methods: samples of anticoagulated blood from adults (n = 30) and umbilical cordon (n = 30) were used. several dilutions oh hailuronic acid sodium salt solution 2% (sigma l-77h0544) were confronted with 2% erythrocyte suspension in phosphate saline buffer (pbs) ph 7.4. the rbcs were previously treated with an enzymatic solution of 5% bromeline in pbs ph 7.4 (sigma l60h0168). agglutination readings' were been by slow sharking after of 12 h incubation at 4°c. the results were expressed through the sensibility parameter which involves titer and score. this is defined by a mathematical expression a = à6 si. di-1. 10-3 (i = 1, 2, 3 . . .) where si represent the score and di-1 is dilution inverse. the adult' rbcs showed a = 26 ± 8, while en the newborn the parameter was a = 3 ± 1. our results showed significant differences between both groups. conclusions: in this work, we present a simple immunohematological technique for the hyaluronic acid sodium salt (cd44) receptor expression in red blood cells, which could be a useful tool to evaluate the alterations of the receptor's expression in rbc. a new technology for crossmatching tests adapted to a fully automated system l gaillard, v desvigne, a boulet, l fauconnier and jm pelosin diagast, loos, france we have developed a new automated technology for crossmatching (compatibility) test suitable for automation and high throughput. the method does not require centrifugation steps thanks to the use of magnetised red blood cells (rbc). all the steps described are performed on the fully automated qwalys system. this methodology requires washing steps under magnetic field and is based on the fixation of sensitised rbc on the surface of a well coated with monoclonal anti-human globulins. in a first step, the red blood cells from target blood bags were magnetised during 10 min. then the patient plasma is distributed on a microplate and incubated with the previously magnetised rbc during 20 min at 37°c. excess of unbound immunoglobulins is removed by washing steps. in a third step, sensitised magnetised rbc were transferred in the antiglobulins coated plate and placed 5 min on a magnet plate. wells in which antigen-antibody interactions have occurred display a confluent layer of rbc (positive reaction). the negative reaction appeared as a pellet in the middle of the well. the test can be read by an automatic reader or by naked eye. the patterns in the well are stable for at least 24 h at room temperature. the plasma samples are provided by the laboratory of haematology of the chru of lille. the red blood cells are collected from segment of tubing of blood bags coming from the laboratory of blood donors of the efs (french blood services) nord de france-lille. the results are obtained in 40 min. comparative studies showed that our new technology, without any centrifugation steps, is reliable and sufficiently sensitive and specific enough to perform cross matching tests using a high throughput automated system. the mechanisms of p53-dependent apoptosis involve a set of genes that possess the ability to modulate oxidative stress. one of them pig3, is induced by p53 through a microsatellite in its promoter region. this microsatellite has been proposed to represent an evolutionary adaptation of tumor suppressor mechanisms. microsatellite instability and genetic constitution, comprising the presence of the low repetition allele (10 tgycc repeats), at this locus have been hypothesized to provide an increased risk for cancer development. aim: in the present analysis we examined this polymorphism in blood samples from voluntary health donors and compared it with human lung cancer samples, employing two different ethnic groups, greek and british. results: analysis of this locus in both types of samples showed: (i) the homozygous presence of the 10 repeats allele only in the samples from healthy blood donors; (ii) a very low frequency of microsatellite instability (<1%) and no loss of heterozygosity in matched normal-tumor tissues; and (iii) a non-significant increase of the most frequent allele (15 repeats) in the cancer groups as compared to samples from healthy blood donors. the last two observations were found in both greek and british populations. conclusion: taken together, these data do not support the notion that this pig3 polymorphism is associated with an increased risk for cancer susceptibility. background: blood group determinations are routinely performed by the sensitive technique 'gel test' for the last few years. many weak d and partial d phenotypes which react as d negative or weak d by slide test, are assigned the rh d + status by gel test. this is most desirable in the case of blood donors but creates concern in case of patients and antenatal women with a partial d phenotype. case report: we report a female patient (blood group o, c+, c+, e-, e+) whose red blood cells gave a positive reaction of different strength and speed with different anti-d antibodies in slide tests. we were asked to type the patient and provide the appropriate blood units. the patient's cells gave a 4+/3+ reaction in the standard screening procedure for the rh d in gel test micro-typing system that contains a polyclonal reagent of human origin (which allows a direct detection of most weak ds), a 4+ reaction in a test with monoclonal anti-d and a 4+/3+ reaction in the gel test micro-typing system destined to detect du and which contains polyclonal anti-d of human origin. however, since the slide test gave a rather slow onset of agglutination with one commercial reagent (made up of a blend of polyclonal and monoclonal anti-d) we tested the patient's red cells against 6 anti-d reagents in the id-partial d typing system. one of these (number 4) gave negative reactions and the remaining five gave positive reactions (ranging from 3+/2+ to 4+), indicative of a partial d category vii phenotype. the patient's red cells were also tested in the id-card 'diaclon abo/d' . this card provides the complete profile for abo/rh d in one single procedure step, including the confirmation of rh d. it contains two different anti-d reagents within the gel matrix in two consecutive microtubes. the first anti-d (polyclonal human) is expected to give a positive result with d+ red cells and partial d category vi, while the second (monoclonal rabbit) is expected to give a negative result with dvi+ red cells. our patient's cells gave a negative reaction with the first and a 3+/2+ reaction with the second anti-d in this system, indicating a d variant other than dvi. finally the patient was assigned the partial d category vii phenotype (according to the pattern of the reactions obtained with the id-partial d typing set) and rhesus d negative blood units were issued. this case illustrates the diversity of reagents used for rhesus typing in different laboratories. failure to disclose some d variants is a disadvantage when typing patients. a combination of techniques is often needed to reveal the real rh d phenotype. the only single system that could have revealed a d variant in our patient from the beginning, is the id-card 'diaclon abo/d' with two different anti-d reagents in two consecutive microtubes as described above. a cost-benefit analysis should be undertaken to show whether it should replace other screening tests for abo and rh d when typing patients. who cares about the quality of life of the chronic patients treated with blood products? d ilcenco*, e hanganu-turtureanu † , c burcoveanu † , c vartolomei ‡ and d azoicai § *blood transfusion center, † hospital 'sfantul spiridon', ‡ institute of hygiene, § university of medicine, iasi, romania quality of life is one of the methods used to appreciate the quality of the health system. romania is going to join soon the european union, so there must be a concern regarding the improvement of the national health system. blood receiver's life quality never been researched before in romania. we have been chosen a batch of chronic ill patients who have been received blood transfusion with blood or blood components, and asked them to complete two types of questionnaires regarding their life. we used nottingham health profile and beck's depresion index. results shows that this kind of patients need special care, because they all (with one single exception) feel frustrated and feel like a burden to the other normal persons. evolution of the pain index, mobility index, energy index, emotions index, sleep index and social isolation index was in concordance with the depression index. in conclusion, this type of patients needs special attention and medical authorities should make more efforts to assure their life quality support. transfusion medicine practice in surgically treated urology patients: our experience il ilincic*, bm bozovic* and ts tadic † *clinical center dr dragisa misovic, † natio. blood transfusion inst., belgrade, serbia objective: multiple studies demonstrate that the use of blood/blood products in patients undergoing elective urology surgeries, as well as the actual needs assessment, present the issue of numerous debates. method: using the retrospective method, utilization of blood/blood products was analyzed, as well as the ratio of prepared/used blood units in urology patients in the surgical ward, in the intensive care unit (icu) and at the urology center within the cc dr dragisa conclusion: due to a rather liberal use of primarily ffp in certain cases (cystectomiae in the first place), and a discrepancy between the prepared and actually used blood units, hospital transfusion committees should be an imperative in order to solve current dilemmas regarding justified use and proper administration of blood and blood products. background: the safe collection, production, distribution and application of blood and blood products in a high quality needs logistic on a high level. since the seventies computers, special software and barcode are used in transfusion medicine and improved the safety of processing data. in the last years a new technology was developed for industrial use, the radio frequency identification (rfid). aim: the aim of our studies was to check whether rfid can use reasonable in transfusion medicine. methods: at first we developed a flow chart, where we can use the technology and where are the problems by introduction. so we tested in the red cross donation centre in saxony about 1000 passive rfid smart label under real conditions. in cooperation between the akh vienna and novatech research a new handheld pc software 'labelview' for all steps around the transfusion was developed, including the identification of the patient and the processing of the haemovigilance data, and tested in first time. results: passive and semiactive (with temperature control) rfid labels survives all hard steps during the working up of the whole blood (e.g. centrifugation by 5000 g, separation, etc.). as a result of the contactless identification they are help to make easier the documentation of all processing steps according good manufacturing practice. in clinical practice they are a good supplement to bed side transfusion software. conclusion: for all lot of problems by the logistic and the safe identification around the transfusion existing various single point solutions such as patient-wristband, bed-side test, double check of blood group typing and donor -donation registry in software, etc. the lecture will deal with new developments in logistics and data management, which can help to reduce the problems associated with documentation, safe identification and reporting of haemovigilance data. our experiences with the immunohaematological analyser olympus pk 80 applied conventional and no conventional (hemolytic medium time) techniques in 23 sera of patients with ascariasis. results: the ai and hk tests showed: b epithopes in 4 ae from b patients and in 3 ae from ab patients; a epithopes in 1 ae from ab patient and in 3 ae from a patients; p and p1 epithopes in 18 ae and only p1 epithopes in 3 ae. these 21 patients had both epithopes in their erythrocytes. the hemolytic techniques showed: anti b immune antibodies in 8 sera and anti a immune antibodies in 8 sera. the presence of abo and p epithopes in ae and immune antibodies in patients with ascariasis show a relation about blood groups and ascariasis. the fact of to find the same abo and p antigens in a. umbricoides and in its hosts suggests that the parasite might absorb them during its life cycle. these epithopes would be involved in the molecular mimicry. the use of filters for leucocyte depletion in anemic patients on maintenance hemodialysis g poposki*, s kovaceski*, b krstanoski*, s mena* and n solaz † *institute of nephrology, struga, macedonia, † ankara university, faculty of medicine, ankara, turkey introduction: renal anemia is one of the major chronic complications in end stage renal disease. it is caused by reduced production of erythropoietin (epo) due to uremic toxin effects, reduced halflife of rbc, iron deficiency, aluminum intoxication, blood loss during hemodialysis, gastrointestinal hemorrhage, epistaxis, infections etc. allogenic blood transfusion is transplantation of certain or all cell types. however, allogenic blood transfusion can contribute to many immune system disturbances with clinical side effects. besides erythrocytes, mononuclear, t and b-lymphocytes, are also transfused, which cause immunomodulatory disturbances in immune system of recipient. leukocytes are responsible for frequent febrile non-hemolytic transfusion reactions, alloimmunization toward leukocytes and hla antigen and transmission of cmv. anti-le antibodies, forming of immune-complexes, complement activation with pirogenic c3a and c5a immunoinflamatoric citokines cause febrile reactions. commercial use of filters for leukocyte depletion with removal of leukocytes and degraded products of microagregates and cytokines, cause minimum harmful immunomodulatory effects and prevent transmission of cmv. aim: the aim of the study was to present the effects of transfusion of erythrocytes with residual number of leukocytes in anemic patients on chronic hemodialysis at institute of nephrology in struga. matherial and methods: during 1997-2004 period all anemic patients on hemodialysis were divided in 4 groups. the first group 34 pts with febrile non-hemolitic transfusion reaction. the second group-25 pts immunized toward leukocyte and hla antigen. the third group 57 young candidates for kidney transplantation for prevention of hla immunization. the fourth group 16 pts with sle (for immune-complexes and autoantibodies). total 132 patients (65 males and 67 females) received 319 units of rbc with residual number of leukocytes. commercial filters of baxterô (lekostop 4 lds) and terumoô (imugard iii rc) of second and third generation with microagregate filter and synthetic polyurethane fibers, with 20-40 microns pores that remove leukocytes, platelets, microagregates and fibrin were used. erythrocyte concentrates are filtered until 5 days of collection. result: aabb permits maximum <5 ¥ 106 wbcs/unit for prevention of febrile non-hemolytic reaction. the filters we used reach residual leukocyte number of 2 ¥ 10 5 the le reduction of 99-99.99%. the number of rbc after filtration is minimum 90% -40 g hb per unit. in none of the patients who have received the leuco-filtered blood, no adverse post transfusion reactions were noticed. conclusion: the used filters for leucocyte depletion are characterized with superior biocompatibility, excellent elimination of all types of leucocytes and high 'recovery' of erythrocytes. the use of filters for le depletion reduces and minimizes the side effects of allogenic blood transfusion in patients on chronic hemodialysis who are alloimmunized, in patients with sle, and particularly in young patients candidates for kidney transplantation. background: fv leiden, prothrombin g20210a, mthfr c677t are three most common and important prothrombotic inherited mutations. aims: the aim of the case-control study was to assess the prevalence of mutations and their single or combined effects as risk factors for thrombosis. methods: the study included 103 thrombotic patients (venous thromboembolism, chronical venous diseases, different etiology) and 200 asymptomatic healthy individuals as control group. extraction of genomic dna was followed with genotyping of fvl by pcr-ssp, prothrombin and mthfr mutation by pcr-rflp. results: a statistically significantly higher prevalence of fvl mutation was found in thrombotic patients (32.0% heterozygous, 1.9% homozygous) compared to controls (3.0% heterozygous), p < 0.0001. the or for heterozygous carriers was 15.24 (95% ci 6.13-37.94), confirming the association of fvl mutation with the risk of thrombosis. there was no statistically significant difference in the prevalence of the prothrombin mutation in patients (5.8%) and controls (3.5%), or 1.71 (95% ci 0.56-5.21), p = 0.3441. although the group of thrombotic patients showed a higher prevalence of homozygous carriers of c677t mthfr than the control group (17.5% vs 10.5%), or was not significant (1.81, 95% ci 0.91-3.57), p = 0.0859. analysis of combined effects of mutations showed an additional thrombotic risk for carriers of fvl mutation and both mutated alleles of c677t mthfr gene (tt and ct) (or 9.40, 95% ci 3.41-25.80), p < 0.0001. conclusions: fv leiden mutation was detected as significant single risk factor for thrombosis in studied patients group. additional prothrombotic risk have carriers of fvl mutation and c677t mthfr gene mutation. a female patient in a high fever due to urinary tract infection does not respond being given antibiotics. on the contrary, leukocytes rose (to 30 ¥ 10 9 /l), anaemia became even deeper, as well as thrombocytopenia. hemocultures were negative. hematologist decided to search for hematological disease. the first citology results of bone marrow aspirate suggested lymphoproliferative disease (15% atipical plasma cells). to treat heavy anaemia (hgb 53 g/l) hematologist asked for red blood cell concentrate. pretransfusion testing revealed warm autoantibodies in the patient serum and on red blood cells. antibodies had no apparent specificity. biochemical parameters (bilirubin, ldh, haptoglobin) suggested mild hemolitic process. electroforesis revealed polyclonal hypergamaglobulinaemia. the 9th day of hospital treatment, the therapy with corticosteroids was introduced (solu-medrol 125 mg per day). coagulation parameters were tested: pt 1.75 inr, aptt 41s, fibrinogen 0.5 g/l, trb 66 ¥ 109/l, d-dimer 251 mg/l, atiii 52%. dic was suspected. liver enzymes showed mild liver dysfunction (normal ast, alt, elevated ggt, low che). substitution therapy started with 1 dose of cryoprecipitate, 1 dose of fresh frozen plasma, 1000 iu atiii, 2 doses of red blood cells and vitamin k 10 mg. two days after the substitution therapy we saw pt 1.28 inr, aptt 31s, fibrinogen <0.5 g/l, trb 75 ¥ 10 9 /l, atiii 71%. during the next few days erythrocytes and thrombocytes rose, but due only to corticosteroid therapy and not to substitution therapy. the patient had neither signs of con-sumptive coagulopathy, nor hypoproduction of coagulation factors, except for fibrinogen. till 17th day of therapy, fibrinogen was below 0.5 g/l. there was no hemorrhagic diathesis. after that, fibrinogen rose, and on the 21st day the patient was recovered, in both clinical and laboratory terms. the results of immunological tests, collected later, confirm the diagnosis of systemic lupus erythematosus. we did not have any specific test to confirm antibody mediated hypofibrinogenaemia, but in the setting of sle, without any specific treatment except corticosteroids, fibrinogen recovered. we assume it is quite enough for highly suspected immunological hypofibrinogenaemia. results: twenty-four-year-old male patient with severe hemophilia type a suffering from low incoercible digestive bleeding secondary to ischemic colitis caused by autoimmunity (vasculitis) without response to current management. treatment was initiated with 90 mg/kg/dose of rfviia (*) for 7 days, after which there was clinical and endoscopic recovery, and an inh decrease to 1.0 ub/ml (fviii dosage 19%) . he began to take meprednisone (1 mg/kg/day) for 45 days, after which the inh titre was 12.0 ub/ml. (table 1a ,b) the patient underwent surgery the following year (correction of equinus foot). he entered the operating room with an inh of 26.7 ub/ml and was treated with 120 mg/kg/dose of rfviia (*) for 10 days, obtaining an excellent hemostatic response. he had two autologous blood units, but it was not necessary to be administered. the inh titre decreased again (down to 5.7 ub/ml) during the intratreatment stage. thirty-five days after rfviia, the inh titre was 7.5 ub/ml. (table 2a ,b) the presence of high titre inh against fviii is a critical problem in cases of bleeding or surgery need due to the inefficacy of the available therapeutic options and the severity of the events. in this case, we have observed that, apart from inducing hemostasia through the activation of the coagulation extrinsic path, rfviia could reduce the inh titre in sequential dosages. we have introduced the case of a 24-year-old patient with hemophilia complicated by a high titre inh against fviii. in this case, we have observed that, apart from inducing an effective hemostasia through the activation of the coagulation extrinsic path, rfviia could reduce the inh titre in sequential dosages. there was also a decrease in the inh titre concomitantly with an increase of the plasma fviii level during its use. this phenomenon suggests that rfviia could produce a modulation in the immune response. evaluation of an automated blood collection system with standard ratio of anti-coagulation and integrated filter for whole blood leucodepletion l dadiotis, a kolokytha, m dimou, a perdiou, c alepi, p spyropoulou, e igoumenides, c velidou, v panagopoulou and s matsagos tzaneion general hospital, pireas, greece automated blood collection system (abc) is a device manufactured by macopharma which collects by gravity a preset volume of blood and mixes it with anticoagulant (ac) in standard ratio (7 : 1). this is managed by passing the ac, which is stored in a special bag, anti-erythrocyte antibodies are immunoglobulins that belong to the igg, igm and iga classes. their common characteristic is a specific reaction with antigens that are located on the erythrocyte surface. they can emerge as auto antibodies and alloantibodies. the blood transfusion in patients may induce a post-transfusion hemolytic reaction (pthr). in order to avoid or reduce the danger of the pthr it is necessary to examine whether there are irregular anti-erythrocyte antibodies in the patient's serum as well as in the serum of the voluntary blood donors. all the irregular anti-erythrocyte antibodies are not clinically relevant. the experience shows that the clinically significant antibodies most often belong to abo, rh, kell, kidd, duffy and ssu blood groups. in the period from april, 2003 to november, 2004, we monitored and examined, at the institute for blood transfusion, clinically significant antibodies in the serum of the patients who are treated with blood transfusions as well as in the serum of the voluntary blood donors. we used the following tests for detecting irregular anti-erythrocyte antibodies: enzyme test, indirect coombs test, screening test by the commercial test erythrocytes and gel filtration method. the detected irregular antierythrocyte antibodies are identified by means of the commercial test erythrocytes for identification. our results are the following: voluntary blood donors: anti d, anti c + d and anti-leb antibodies. patients: anti-d, anti-k, anti-fya, anti-c and anti-e antibodies. in nine patients, anti-erythrocyte antibodies were discovered, namely, those that react at the temperature higher than 30°b ut whose specificity we could not discover with the existing techniques. improved predictive factors of response for myelodysplastic syndrome patients treated by the combination of erythropoietin and g-csf s park*, c kelaidi † , s grabar ‡ , v bardet ‡ , d vassilieff ‡ , f picard ‡ , m guesnu ‡ , mc quarre ‡ , p fenaux § and f dreyfus ‡ *service hématologie, hopital cochin, † hématologie, hopital avicenne, ‡ service hématologie, hopital cochin, § hématologie, hopital avicenne, paris, france it has previously been shown that serum epo level and number of previous red blood cell transfusions are predictive factors of response to epo + g-csf treatment of myelodysplastic syndromes (mds). in a subgroup of patients with mds having sepo <500 ui/l, known to be good responders to epo + g-cscf, the gfm group wanted to refine the model predicting the response to epo + g-csf, especially with cytology (who classification with dysplasia and percentage of erythroblasts and blasts). in a population of 64 patients (ra, rars and raeb <10% blasts) receiving epo ± gcsf between 2000 and 2004 and having serum epo <500 ui/l, the response rate at week 12 (iwg criteria) was 60%. six variables were associated with response to epo ± g-csf for mds: age >70 years (p = 0.02), number of prior red blood cell transfusions <2 packs/months (p = 0.005), serum epo level <200 ui/l (p = 0.02), percentage of blasts <5% (p = 0.05), percentage of erythroblasts >30% (p = 0.02) and low ipss score (p = 0.001). we did not found any influence of dysplasia, type of rhepo (darbopoietin alfa or epoietin alfa) and karyotype on response rate. in multivariate analysis, age through a rotating pump. the abc can be used with all types of p-417 pan-european blood safety alliance the pan-european blood safety alliance is a unique alliance of patient organizations, formed to promote the highest level of blood safety for all in europe. it was formerly established on 11 february 2005, during the course of the first general meeting of the pbsa, which comprised of 8 founding patient organizations. the objectives of the pbsa are: 1. to promote the fundamental right and duty to safety of all patients in need of blood transfusions and blood products. 2. to ensure the availability of sufficient amounts of safe blood, to meet all treatment need through: -the education of all staff handling blood components, to reduce human error. -the implementation of and access to, proactive blood safety technologies, for each patient across europe. -haemovigilance -the adequate access to blood transfusion services, which should be provided free of charge to the patient. other objectives are to raise awareness on a local and european level regarding blood safety, to promote eu legislation that improves safety standards of blood transfusion services, including stem cell preparation and storage across europe and to lobby for increased patient influence on eu health policy makers. very importantly, the alliance aims at providing a forum for patients, healthcare professionals, health policy makers and relevant industry, as well as acting as a point of reference to the national health authorities, the european commission and other european institutions, when seeking the opinions of patients on blood safety. cerns of insertional mutagenesis and the safety of some viral vectors that randomly insert genes through the genome have been recently resurfaced following the development of a haematological malignancy in a child treated with a retroviral vector. particularly questions also remain as to, whether gene therapy and the production of ectopic factor viii and ix will be a risk for inhibitor development or indeed whether it might promote tolerance in those patients with inhibitors. w-pl4-11 gene therapy for thalassemia: will it become reality? university of washington, seattle, wa, usa experiments aimed to develop gene therapy approaches for the beta chain hemoglobinopathies, sickle cell disease and beta thalassemia started about 25 years ago. in the beginning results were dismal because of the extremely low and variable expression of globin genes contained in the therapeutic vectors. a major development occurred in 1987 with the discovery of powerful regulatory elements that could guarantee high level of globin gene expression. these elements when incorporated into viral vectors allow expression of therapeutic levels of the transferred globin genes. a second major progress was achieved with the development of safe lentiviral vectors that can efficiently infect the human pluripotent repopulating hemopoietic stem cells. as a result of this progress, today beta thalassemia and sickle cell disease can be cured in murine models of these disorders. considerable effort is already being devoted into further improvement of lenti viral vectors with emphasis on incorporating elements which will decrease the probability of insertional mutagenesis and leukemogenesis. the major challenge for the clinical application of stem cell gene therapy of thalassemia is the need for genetic correction of large numbers of mutant stem cells. in vivo selection of corrected stem cells is being investigated but there are questions about its safety because of the possibilities of clonal expansion of stem cell lines carrying undesirable integrants. other major challenges have to do with logistics: production of therapeutic vectors, infrastructure required for stem cell gene therapy delivery, and sponsoring and funding of the clinical trials. gene therapy trials on limited number of patients are expected to be initiated relatively soon. if these trials are successful and cures of beta thalassemia ensue, the major challenge will be the delivery of this molecular therapy in the context of medical practice. w-pl4-10 gene therapy for haemophilia haemophilia is an ideal target for gene therapy because only a small rise in factor levels to 1-2 u/dl would achieve the goals of prophylaxis without regular infusions of concentrate and deliver a substantial improvement in lifestyle for patients with severe haemophilia. gene therapy for haemophilia today relies upon addition of normal factor viii or ix genes. with present technology gene therapy can offer the prospect of a true 'cure' for haemophilia in animal models, although this may not be currently realizable in man. more than 30 patients with haemophilia have now been treated in phase 1 gene therapy protocols. all studies have failed to conclusively show that therapeutic levels of factor viii and ix can be reliably obtained. the first trial reported used im injection of a factor ix containing recombinant adeno associated virus (raav) in adult patients with severe haemophilia b. only very modest increases in factor ix level, <2 u/dl rise, in 4/8 patients enrolled were observed, although less factor ix concentrate was needed in 3/6 subjects. a similar study using the same raav vector via intrahepatic artery infusion has been conducted. this has been complicated by the observation of aav vector in the semen of subjects. in six patients enrolled, no durable levels of ix above 1 u/dl were seen. further development of this raav vector is suspended. for haemophilia a, three systems are have been tried. the first study was an ex vivo addition of factor viii gene to autologous fibroblasts and then laparoscopic reimplantation. preclinical assessments demonstrated durable expression of factor viii (>5% of normal) for >1 year in mice following a single treatment. in 4/6 patients treated repeated factor viii rises (0.5-3.5 u/dl) were seen, but no improvements lasted beyond 10 months. the second protocol used a murine leukemia retrovirus containing factor viii, injected intravenouslya development of preclinical data in rabbits and haemophilic dogs. 0/13 patients enrolled sustained levels of factor viii >1 u/dl. the third study, using a modified, 'gutless', adenovirus containing factor viii gene has recruited one patient. this patient demonstrated transient liver toxicity and thrombocytopenia at doses lower than those that cause toxicity in primates. sustained levels of factor viii of ~1 u/dl have been observed over a number of months. accrual to the study has been poor. haemophilia remains a prime target for gene therapy. however, haemophilia is no longer a life threatening disease with current therapy that is both safe and efficacious. a balance between the benefits and theoretical risks must be borne in mind when considering gene-based approaches to therapy. conreference: petz ld, garratty g. immune hemolytic anemias. 2nd ed. philadelphia: churchill livingstone, 2004, pp 375-400. w-pa-114 autoimmune neutropenia introduction: autoimmune neutropenia (ain), a granulocytic disorder due to the presence of anti-neutrophil antibodies, may present as neutropenia of varying degree with or without recurrent infections un previously healthy individuals (primary or idiopathic ain) or in patients with a known underlying disease such as lupus erythematosus, lymphoid malignancies, etc (secondary ain). the condition affects more frequently infants of small ages while it is rare in adults [1] [2] [3] [4] [5] [6] [7] . in some patients, diagnosis is established in occasion of a respiratory, urinary or cutaneous infection, but in many cases is simply a finding of cell blood counting performed for unrelated reasons [6] . clinical and laboratory findings: in general, physical examination is negative. laboratory investigation reveals the existence of isolated neutropenia. association of the disorder with autoimmune hemolytic anemia or autoimmune thrombocytopenia is rarely seen [8] . blood biochemistry is normal while serologic tests for bacterial, viral or other pathogens may be positive depending on the underlying infection. bone marrow is hypercellular without maturation arrest of granulocytic series. hemopoietic stem cell reserves and function are normal or increased, and stromal cell function is within the range of the normality [9]. methods for the detection of granulocyte-specific antibodies: serology for the detection of granulocyte-specific antibodies has been marred, compared to erythrocyte serology, because the target cell here, the granulocyte, is short-lived, fragile and becomes easily activated. the former two of these difficulties require absolutely freshly (<6 h old) isolated neutrophils from a panel of donors to be used every day to run the tests with sera from patients, while the third difficulty is more important since spontaneous cell clumping in vitro is very common and nay mimic the specific aggregation caused by cross-linkage of surface bound antibodies in the granulocyte agglutination test (gat). in order to overcome these problems, the second international granulocyte serology workshop [10] recommended a combination of two tests as the best screening procedure for the detection of granulocyte autoantibodies in patient sera, gat and granulocyte immunofluorescence test (gift). gat is mainly mediated by igm antibodies and is positive in about 2% of cases. gift detects igg antibodies and is positive in about 98% of cases. it is to be noted that flow-cytometry fluorescence may arise not only from the surface but also from the cytoplasm of neutrophils, necessitating assessment of membrane fuorescence by microscopy. a good direct anti-granulocyte test is not available today. this is due to the fact that too few neutrophils can be obtained from the blood of neutropenic patients, and also to the observation that neutrophils are often activated in vivo because if an underlying infection or other inflammatory process, thus expressing fcgrii and fcgriiib to which nonspecific binding of w-pa-113 practical approach to transfusion in autoimmune hemolytic anemia (aiha) g garratty american red cross blood services, pomona, ca, usa a major problem when transfusing patients with aiha is that often all units are incompatible. this may be due to autoantibodies (autoab) and/or alloantibodies (allo-ab). if the incompatibility is due to only auto-ab, then transfusion of incompatible blood will not usually result in a clinically significant reaction, but if due to alloab, the result may be similar to that seen in any other patient (i.e. a hemolytic transfusion reaction ranging from mild to severe). thus, it is essential (as in any other patient) to exclude the presence of allo-ab. it is wise to phenotype all patients, for as many antigens as possible, before the patient receives transfusion. there are two popular approaches to determine if allo-abs are present but being masked by 'warm' auto-ab activity. the preferred method is to remove the auto-ab by adsorbing the patient's serum with autologous rbcs treated with enzymes, or preferably, with zzap reagent. the latter reagent contains an enzyme leading to optimal adsorption of auto-ab, and dtt. these two chemicals will destroy significant antigens other than rh and kidd (e.g. mns, duffy, kell, lutheran, dombrock, cromer, lw, some yta and ge, inb, jmh, ch, rg, pr antigens), thus will not adsorb alloantibodies to these antigens. if autoadsorption is not possible (e.g. patient has been transfused recently or there are too few rbcs), one has to perform adsorption with enzymes or zzap-treated allogeneic rbcs. one does not have to be concerned with covering any antigens destroyed by zzap (e.g. kell and duffy systems). we use a rough guide relating the strength of the indirect antiglobulin test to the number of adsorptions needed to remove auto-ab (1+ = 1 adsorption; 2+ = 2 adsorptions; 3+ = 3 adsorptions; 4+ = 3 or more adsorptions). if there is no activity left after the adsorptions, then one can suspect that the incompatibility was due to auto-ab, but on rare occasions one can be wrong and an allo-ab to a high-frequency antigen has been removed. this is a major disadvantage of using allogeneic adsorptions, and is why adsorptions with autologous rbcs are preferred. if time (2-8 h) does not allow for adsorptions, one can dilute the patient's serum (e.g. 1 in 4, or 1 in 5) and test the dilution against a panel to see if any alloantibody specificity becomes obvious. another approach is to select units matching the patient's phenotype as closely as possible. when dealing with cold agglutinin syndrome one can usually exclude allo-ab activity by testing strictly at 37c. this can be helped by performing adsorptions with enzyme-treated autologous rbcs at 4c, but it is difficult to adsorb all of the powerful cold autoagglutinin activity. it is reported that 30-40% of aiha have allo-abs; the incidence is even higher in patients who have received multiple transfusions. thus, we feel that procedures such as those discussed above must be performed before transfusing incompatible blood if time allows. one should always negotiate with the attending physician regarding the time it will take to perform adsorptions. a decision may be made not to perform adsorptions if the patient has life-threatening hemolysis, and especially if the patient has never been transfused, or pregnant. serum igg may occur (naig). the presence of immune-comlexes in the serum, such as in patients with felty's syndrome, lupus erythematosus and other diseases, as well as the presence of immune aggregates formed in sera stored frozen for long time, may give false-positive tests given that they may bind to fcgriiib molecules expressed on the surface of neutrophils. elimination of immunecomplexes and immune aggregates can be easily obtained by ultracentrifugation [11] . another cause of false-positive results may be the presence of anti-hla antibodies because of allo-immunization. these allo-antibodies react with hla molecules found not only on neutrophils but also on the surface of many other cells including lymphocytes. these allo-antibodies can be eliminated by platelet absorption. it seems that the best method in the search of true antigranulocyte antibodies is the monoclonal antibody-specific immobilization of granulocyte antigens (maiga) [12] . with this method one can specify anti-granulocyte antibodies using a panel of known granulocyte antigenic specificity. finally, it is notable that the levels of serum antibodies to neutrophils may vary considerably over the time. one negative test does not exclude ain. usually, two to three tests have to be run over a period of months [13] . antigenic specificity: human neutrophil antigens (hna) are classified according to an international granulocyte antigen working party [14] . three glycoproteins have been found to be involved in the determination of antigenic specificity, fcgriiib, gpnb1 (cd177) and gp70-95. the respective antigens, frequencies and alleles are illustrated in table 1 . antigenic specificity can also be studied by using methods applied in molecular biology. a promising approach is transfection of mammalian cells by cdna derived from granulocyte antigen specific mrna. cell lines have been established with cells expressing the respective human granulocyte antigen, making the detection of anti-granulocyte antibodies more easier. genotyping of hna antigens can also be stydied with the pcr technique [15] . references are available from the author upon request. w-pa-115 a rare case of 'coombs negative' autoimmune haemolytic anaemia due to red cell autoantibodies of iga class warm autoimmune haemolytic anaemia (waiha) is usually associated with red cell auto-antibodies of the igg class, which can be detected by polyspecific direct antiglobulin test (dat). routine polyspecific direct antiglobulin tests contain anti-igg and anti-c3d components, and are not standardized to react with iga-or igmsensitized red blood cells. haemolytic anaemia caused by warmreacting auto-antibodies solely of the iga class is exceedingly rare. those cases of autoimmune haemolytic anaemia can be difficult to diagnose because of the negative polyspecific coombs' test, which is a standard in investigation of possible causes of haemolysis. we present a case of severe warm autoimmune haemolytic anaemia caused by iga class autoantibodies. a 40-yr old male patient was admitted with anaemia, haemoglobinuria, and other signs of severe haemolytic disease. he received multiple transfusions but haemoglobin level did not rise above 7 g/dl. the initial polyspecific direct antiglobulin test, containing an anti-igg and anti-c3d antiserum, was negative. tests for cold agglutinins and other possible causes of haemolysis were negative. only by using a monospecific, anti-iga antiserum could we show that the warm iga auto-antibodies against red blood cells were present on patient's erythrocytes. we have not detected signs of complement activation by iga autoantibodies in this patient. the patient received corticosteroids with good initial effect. his haemoglobin level stabilized and he did not require more transfusions. anti-iga direct antiglobulin test became negative about to weeks after the therapy was initiated. however, in spite for the initial effect of steroid therapy haemolysis continued, and splenectomy was performed 6 months after diagnosis was made. it has been shown that human lymphocytes, granulocytes and monocytes contain specific fc receptors for iga, and both monocyte-mediated phagocytosis and antibody-dependent cellular cytotoxicity due to iga auto-antibodies has been demonstrated. there is also increasing evidence that iga auto-antibodies can activate complement, both via the classical and the alternative pathway. a phenomenon of 'reactive haemolysis', which involves c3-independent binding of c5b6 complexes to 'bystander' red blood cells, has also been described. we emphasize the importance of performing additional testing in cases of apparent 'coombs' negative' haemolytic anaemia due to iga, igm or 'low affinity' igg autoantibodies, and serological aids that are available for that purpose. described. both siblings were born on term, in good general clinical status, free from any signs of infection, and with isolated severe neutropenia (170 and 123 neutrophils/ml). the diagnosis of annanti hna-2a was made upon exclusion of other possible causes of neonatal neutropenia, and confirmed by serological testing of granulocyte antigens and antigranulocyte antibodies. in both cases, the course of the disease was mild, with bacterial omphalitis on day 15 and 5, respectively. omphalitis was successfully treated with 7-day antibiotic therapy according to antibiotic sensitivity report. the first neonate received standard dosage of intravenous gammaglobulins for 5 days without success. this was followed by an attempt at neutrophil count increase with 2-week corticosteroid therapy, also without response. the second neonate received no specific therapy for neutrophil count increase. the children were discharged for home care with clinical and laboratory control examinations at 2-week intervals. in spite of prolonged neutropenia (2 and 6 months, respectively), no other infections were recorded. discussion and conclusion: in our patients, the therapeutic approach to ann was individualized, based on standard antibiotic therapy, intravenous gammaglobulins, corticosteroids, available literature data, and our own clinical experience. although in the last few years rh-gcsf is successfully used in patients with neutropenia, we decided to postpone its use in case the neonatal sepsis developed. the reasons for such decision were: (1) the fact that both neonates were in good general clinical status, with a mild course of the disease with only short-term umbilical infection successfully managed with antibiotic therapy; (2) literature reports suggesting the unexpected failure to respond to rh-gcsf therapy in patients with neutropenia induced by anti hna-2a immunization, and (3) the unknown effect of rh-gcsf on developing tissues of the neonate. the choice and efficacy of specific therapy for neutrophil count increase in the management of alloimmune neonatal neutropenia have not yet been fully defined and require additional evaluation in the majority of cases. male donors for the production of fresh frozen plasma: a special issue for trali patients trali is a significant cause of transfusion associated morbidity and mortality, and has been reported as the third most common cause of fatal transfusion reactions. there is no good evidence on which to base transfusion support policy for patients who have experienced trali. the hypothesis that there may be patient associated factors that contribute to the risk of trali is generally accepted. for this reason it seems reasonable to try to avoid further transfusion during the period of illness. if this is unavoidable the next best solution to reduce the risk of recurrence seems to be the avoidance of using plasma containing blood components (especially ffp) as there is a high chance of positivity for leucocyte antibodies especially for those coming from female donors. as fresh frozen plasma transfusion accounts for up to half of all trali cases and as our center is the only in greece responsible for the testing and processing of blood from blood donors representing military recruits, the last two years we tried to set up a project in order to provide components from male donors on request. our donor base consists predominantly from males donors (98.7%), aged between 18 and 25 years old, with a small chance of having a positive history for transfusion the difficulty of the project consisted on the fact that these donors are assigned to military camps throughout greece, which makes difficult the on time arrival of the units to our establishment in order to be processed for the production of ffp conform the european council quality requirements. this was the main reason why, till now, all plasma produced from these donations was regarded as plasma for fractionation. the first step for implementing the new project was to evaluate the number of donations that, by minor changes on the time of arrival, could be processed for ffp production. the next step was to re-schedule the shifts of the personnel for the on time production of ffp. during 2003, 25% of donations were fulfilling the specifications for the production of ffp and with the flexibility of the schedule the 97% of them were successfully processed to ffp. during 2004, 30% of the donations were fulfilling the specifications and 97.8% of them were processed to ffp. so it is feasible to increase the proportion of male ffp by organizing better the transportation of blood from the donation sites to the blood establishment and by retaining available specialized personnel to cover the extra shifts. maximising the blood supply chain in times of shortage shortages in the blood supply chain may occur for a variety of reasons. they may be temporary e.g. due to a flu epidemic or prolonged e.g. due to the exclusion of a high proportion of donors due to new pre-donation tests or because of a lack of volunteer donors. increasing awareness of the possibility of blood shortages mainly related to increased precautions associated with the possible transmission of vcjd by transfusion has been the driver for the development of blood shortage contingency plans in the uk. in england and north wales, hospitals and the national blood service (nbs) have worked together to develop an integrated blood shortage plan (ibsp) designed to ensure that hospitals and the nbs work together within a consistent, integrated framework giving patients equal access to available blood on the basis of need. an essential element of the plan is the principle that shortages can, in most cases be avoided by reducing the current usage of blood through appropriate use programmes. the impetus for hospitals to implement these programmes was a government circular (hsc 2002/009). hospitals have embraced the circular and have recruited specialist hospital transfusion practitioners, introduced lower hb triggers, cell salvage and hospital transfusion teams and are participating in the blood stocks management scheme (bsms). audits of compliance with the circular have taken place, and a web based tool kit is available. the demand for blood has declined for the last three years, with a decrease of about 5% during 2004-05, suggesting that the drive for improvement has been successful. the shortage plan introduced in england and north wales has two key aims: that the national pool of blood is available for all essential transfusions for all patients and that overall usage is reduced to ensure the most urgent cases receive blood. the plan is structured to provide actions for the nbs and hospitals in three phases, 'normal' circumstances, reduced availability and severe prolonged shortage. hospitals should have documented emergency blood management arrangements for each of the phases. the national plan is activated when the nbs red cell stock level falls to pre-defined levels, hospitals are informed by fax that they should reduce their normal stock holding levels according to guidance in the ibsp and comply with the daily hospital usage budget. the bsms has used its knowledge of hospital inventory levels and demand to provide guidance on appropriate inventory levels for normal and reduced status, it also provides the daily hospital budget. to monitor progress against the recommendations in hsc 2002/009 hospitals will be benchmarked against a number of performance indicators. these include the presence of emergency blood management arrangements, median red cell usage for a number of surgical procedures and percentage wastage of blood. there have been no shortages within the nbs for more than six years, it is hoped that the implementation of the ibsp will help to ensure that in the unlikely event of reduced availability blood will be available to the maximum number of patients requiring a blood transfusion. w-pa-120 transfusion during disaster g klein nih, bethesda, md, usa publicity given to blood donation during wartime has created a powerful association between the need for blood and occurrence of a disaster. blood is rarely needed in excessive quantities at the moment a disaster occurs. the outpouring of blood donors, especially at the site of a disaster, often proves counterproductive. the terrorist attacks on the world trade center on september 11, 2001, with almost 3000 deaths and more than 4000 injuries, provides an instructive model. more than a million potential donors contacted blood-collection centers. hundreds of thousands of prospective blood donors crowded collection facilities and many waited for hours, often to be turned away. qualified staff were in short supply and screening errors occurred as minimally qualified staff were recruited and as collection personnel fatigued. supplies and storage capabilities were pushed to their limit. some blood was inadequately processed and stored. resources were diverted from needed apheresis collections and component preparation to whole blood collection. in the aftermath of the disaster, blood outdated and volunteer donors became disillusioned as their 'gift of life' was refused or unused. similar responses have occurred numerous times over the 60-year period since blood-donor programs were introduced. in virtually every civilian disaster in the u.s. during the past century, all the blood that was needed was immediately available from blood inventory. in only four cases were more than 100 units of blood used in the first 24 to 30 h. in 2001 in new york, the five hospitals closest to the disaster site admitted only 139 disaster victims. the new york blood center, which supplies 80 percent of blood for the city's hospitals, added 600 units to routine inventory at hospitals. the center received 12 000 telephone calls and collected more than 5000 units of blood in the first 12 h. in the area of the pentagon, the chesapeake and potomac red cross blood center supplemented hospital blood inventories within h of the disaster. meanwhile, spurred by well-meaning media and federal officials, lines of blood donors were being processed at local hospitals, makeshift collection centers, the small research hospital at nih, and at a building next to the white house. in the week after september 11, america's blood centers collected 167 000 more units of blood, and the american national red cross collected 287 000 more units than in the same period the previous year. more than 475 000 units were collected for the disaster victims, but only 258 units were used. u.s. blood collectors and federal agencies have created a disaster plan that acknowledges the need for altruistic people to volunteer for blood donation in the time of disaster and speaks with a single voice to avoid needless collection activity while harnessing the good will of well-intentioned people to supplement the ongoing need for volunteer blood donation. rehabilitation of blood transfusion service in azerbaijan cd asadov, ga huseynov and ab hagiyev institute of hematology and transfusiolo, baku, azerbaijan at the end of 80th years of the last century in azerbaijan as well as in other republics of the former ussr began process of progressive deterioration of blood service parameters. in result there was an essential reduction of prepared blood and blood components quantity, manufacture of preparations from blood's plasma has completely stopped. it is connected by that our republic experiences a heavy transition period from scheduled to market economy. after reception by azerbaijan of the sovereignty on development of a national policy the big work has been lead to areas blood transfusion and development of national rules and the standards regulating functioning of establishments of blood service. in 1996 the law about ' the donorship of blood and its components in the azerbaijan republic' has been accepted, instructions on physical examination of donors and preparations of blood and its components are authorized, and also the new speciality transfusiology has been entered into the nomenclature of medical specialities. now the national program of blood service development is developed. at drawing up of the program social and economic conditions of the country, ethnic both cultural traditions and a mental potential of the nation are considered. within the framework of this program is planned to refuse gradually a paid blood donation during the certain period of time to reach 100%s' voluntary unpaid blood donorship. however in connection with limitation of resources, the state is not capable to allocate enough of means for its realization. the big work on attraction of the international organizations has been carried out. now the project of the united nations development program (undp) 'rehabilitation of blood transfusion service in azerbaijan' is carried out at sponsor's support of the norwegian government. realization of this project will lead to reorganization of blood transfusion service in our country according to practice of the european countries. within the framework of project realization it is planned to make changes and additions to a existing law about a blood and its components donorship to bring it into accord with recommendations of the europe council. updating of the russian law 'concerning the donation of blood and blood components' on june 9, 1993, a law, 'concerning the donation of blood and blood components, ' was signed by the first russian president, boris eltsin. now, after more than ten years of market economy and democratic evolution in russia, this law was significantly changed on august 22, 2004, as shown in the following sections: 1. the development of a voluntary blood donor system. 2. removal of the upper age limit for blood donors. 3. funding for blood donations. from january 1, 2005, each level of the state power budgets for a blood donor service to supply blood products for federal, regional, or municipal hospitals. costs of these drugs and the need of prolonged growth factor treatment in these disorders. w-pa-127 can iron administration reduce peripartum blood transfusion c breymann university of zurich, zurich, switzerland the prevalence of iron-deficiency anemia in different regions of the world ranges from 12 to 43%. the increased iron requirement in pregnancy and the puerperium carry with it an increased susceptibility to iron deficiency and iron-deficiency anemia and perioperative or peripartal blood transfusion. however, if ever possible administration of blood transfusion should be avoided for several reasons which will be pointed out in the talk. infections: it is well known that various pathogens such as bacteria and virus can be transmitted by administration of blood. around 0.03% of are contaminated by bacteria such as yersinia or pseudomonas species but are not screened routinely for bacteria. in addition there is no donor screening for hepatitis a, herpes species (cmv, ebv, hhv 7, hhv 8), parvovirus b19, hepatitis g (3.4%) and tt (transfusion transmitted) virus (1.9%). numbers for positive testings for 'classic' virus such as hiv, hep. b and hep. c vary from country to country and lie around 1 : 30 000 to 1 : 5000 000 depending on quality of donor screening programs, pcr sensitivity etc. recently there is increasing evidence that even prions which cause the jakob creutzfeld disease variation ('mad cow disease') might be transmitted by transfusions. therefore the fda has determined that blood donors from countries with high prevalence of prion positive persons are not permitted to give blood in the us (e.g. donors from uk). beside infections, other well known effects of transfusion are problems due to incorrect blood or components transfused, post transfusion purpura, acute and delayed lung injury, graft versus host disease and other acute and delayed allergic reactions. beside these negative effects it was also shown that patients who receive blood transfusion liberally after operations or in icu show higher morbidity and mortality compared to patients with restrictive transfusion policy. this might be due to negative effects on immune functions and inflammatory reactions and lack of stored blood to efficiently improve organ oxygenation. for example it is known that stored blood has worse capillary perfusion and worse viscosity properties compared to fresh blood. taken together there is increasing scientific evidence that blood transfusion is not the gold standard for anaemia management and alternatives such as endogenous blood pooling and efficient treatment of any anaemia must be enforced in the clinical settings. prevention and correction presuppose reliable laboratory parameters and a thorough understanding of the mechanisms of iron therapy. in order to correctly diagnose the type and degree of anaemia, a prerequisite for selection of the proper therapy, one must first of all correctly differentiate between the relative, i.e. the physiological anaemia of pregnancy due to the normal plasma volume increase during pregnancy, and 'real anaemias' with various different pathophysiological causes. when defining the hb cutoff value for anaemia in pregnancy, the extent of the plasma volume changes with respect to the gestational age must be taken into consideration. it has been found that haemoglobin values <11.0 g/dl in the first and third trimesters, and <10.5 g/dl in the second trimester may point to an anaemic situation which should be further clarified. the first important steps for diagnosing anaemia in a pregnant patient include a thorough check of her medical w-pa-126 impact of epo treatment on transfusion requirements in myelodysplasia c gardin and p fenaux hopital avicenne, aphp, university of paris 13, bobigny, france myelodysplastic syndromes (mds) are clonal disorders of hematopoeisis, associated with bone marrow failure and an increased risk of evolution to acute myeloid leukemia (aml). despite an normal or increased bone marrow cellularity in most cases, cytopenias worsen with time due to increased apoptosis and defective differentiation of blood lineage precursors. incidence of mds increases with age and reach 15/100 000 above 70 years of age. bone marrow cytogenetics number of cytopenia and percentage of bone marrow blasts are strong predictors of survival and evolution to aml. a composite international prognosis scoring system (ipss) is used in everyday practice to guide the management of these diseases. these disorders are heterogeneous and include 'low risk' patients (less than 10% bone marrow blasts) with a prolonged evolution marked by chronic anemia, and 'high-risk' patients (excess of bone marrow blasts >10%) evolving in a short timespan with severe cytopenias, and to aml in approximately 50% of cases. at diagnosis, 80% of mds patients are anemic, with an hemoglobin level less than 100 g/l, and 80% of them will require chronic blood components transfusion, during the evolution of their disease. chronic anemia and multiple blood products transfusions are associated with an altered quality of life, clinical iron overload, and important health care costs. although transfusion practices and patient's transfusion need are variable, elderly mds patients require a mean of 10-20 units/year of follow-up, in recent surveys. therapies able to diminish or abolish the need for rbc transfusion have therefore a major role in the management of mds, as allogeneic bone marrow transplantation, the only curative therapy of these diseases, is limited to a small subset of mds patients. high-doses of recombinant erythropoetin (epo) (150-300 u/kg tiw, or a 40 000-60 000 u as single weekly dose) are typically used in low-risk mds. the response rate to epo is 25-40%, including major responses (suppression of rbc dependency or rise of hemoglobin level of more than 20 g/l). absent or low rbc transfusion needs and a serum epo level less than 500 u/l are strongly predictive of response to epo, in patients with low-risk mds. the duration of response is variable (12-20 months) in most studies, with some long-term responders. the use of higher doses of epo or its prolonged administration may be associated with higher response rates, although no randomized studies are available combination of epo and low-dose granulocyte-colony stimulating factor (g-csf) increases the response rate to 40-60%, including in patients not responding to several weeks of treatment with epo alone. two randomized trials published in 2004, compared g-csf-epo to rbc transfusions and confirmed the efficacy of this combination, and a longer survival of epo-g-csf responding patients. studies are ongoing in mds, including with darbepoetin, a modified erythropoetin with longer half-life, administered once a week. two such studies have been recently reported, (darbepoetin 150 or 300 ug/week) with response rates varying from 35% to 60% in low-risk mds. in both studies, a response to darbepoetin was observed in some patients, who failed to respond to previous treatments with alpha or beta epoetin. further assessment of the optimal dosage, administration schedule of these drugs, and validation of their likely impact on qol are required, in order to epo and its derivatives to gain acceptance in mds, due the high history and a medical examination. this procedure often lays the basis for a correct diagnosis. the current gold standard to detect iron deficiency remains the serum ferritin value. to be reliable, this requires the ruling out of an infection (chronic or acute) as a cause of the anaemia. we recommend a complete laboratory test for the exact haematological status as well as the assessment of specific chemical laboratory parameters. these should the hb level alone is insufficient to guide management. a complete work-up (ferritin, transferrin saturation) is essential, preferably with haematological indices such as hypochromic and microcytic red cells and reticulocytes, classified by degree of maturity, in particular, before parenteral therapy is given. since ferritin acts as both an iron-storage and acute-phase protein, it cannot be used to evaluate iron status in the presence of inflammation. a high ferritin level thus requires the presence of an inflammatory process to be eliminated before it can be taken at face value. if the c-reactive protein level is also raised, the soluble tfr concentration can be used, since it is unaffected by inflammation. inadequate understanding of the complex chemistry of parenteral iron administration was previously responsible for serious side effects, such as toxic and allergic reactions, and even anaphylactic shock, in particular with dextran preparations. however, the current type ii iron complexes that release iron to the endogenous iron-binding proteins with a half-life of about 6 h are not only effective but carry a minimal risk of allergic accident and overload, especially after a comprehensive pretreatment work-up. after correct diagnosis, major emphasis should be put on safe and effective treatment of anemia which again depends on severity of anemia, time for restoration and patients characteristics. today effective alternatives to oral iron only or blood transfusion such as parenteral iron sucrose complex and in selected cases also recombinant erythropoietin have been investigated and show promising results concerning effective treatment of anemia during pregnancy and postpartum. our departmental data collected over 10 years and backed by postmarketing experience in 25 countries indicate that iron sucrose complex therapy is a valid first-line option for the safe and rapid reversal of iron-deficiency anemia. w-pa-128 iron therapy in orthopaedic surgery surgery of the vertebral column, hip or knee is considered a bloody procedure (blood loss >1 l) and as a consequence represents the main indication for red blood cell transfusion in orthopaedics. because of the non-negligible residual risk of transmission of infectious agents by transfusion, but mainly because of immunologic complications induced by the administration of foreign proteins and cells, an alternative solution has been actively sought. studies have clearly shown that in patients undergoing such surgery, transfusion risk correlates inversely with pre-operative hemoglobin level. correction of even slight preoperative anemia is thus mandatory. in the elderly, iron and vitamin deficiency (b12 and/or folic acid) should be looked for as a matter of routine. we recommend the use of iron + epo whenever a rapid correction (<4 weeks) of the anemia is desirable in cases with transferrin saturation <30% and ferritin levels <200 mg/l. with this regime it is possible to collect up to 6 autologous blood units in cases of increased perioperative blood loss (e.g. double hip replacement). in the post-operative period, anemia worsens because of the existing inflammatory state. this inhibits iron absorption from the intestine and iron release from the macrophages while it affects epo function and production. there is increasing evidence that i.v. iron combined with epo induces a rapid correction of post-operative anemia. it is thus recommended to stimulate erythropoiesis by i.v. iron and epo starting on the first post-operative day and to avoid transfusions in asymptomatic patients even in cases with hb as low as 70 g/l. background: hereditary hemochromatosis is one of the most common inherited disorders in which an excessive amount of iron is absorbed from the diet and then deposited in organs. the effective treatment is the regular whole blood removal which causes erythropoesis activation and leads to decrease of iron stores. red cell apheresis is an optional method for removing of higher amount of erytrocytes in one session. we performed 262 red cell apheresis in 15 patients with diagnosis of hereditary hemochromatosis (14 ¥ c282y homozygotes, 1 ¥ c282y + h63d heterozygote) using haemonetics mcs 3p cell separator (protocol tae) in which red cells are removed from patients in 2-5 cycles; plasma and buffy-coat are reinfused. collection time, donor convenience, side effects and red cell yield were recorded and analysed. samples for hematology and iron studies in patients were drawn, analyzed and compared to baseline levels. background: the collection of 2 units of red blood cells by apheresis (drbc) has been reported to be safe and effective in increasing the yield of rbc units from a donor population. however several reports demonstrated the risk of inducing iron depletion when the interval between a drbc donation and a subsequent rbc donation is shorter than 180 days. aims: to evaluate the recovery from anaemisation and iron stores depletion after drbc donation. methods: donors who underwent drbc donation between december 01, 2002 and february 28, 2003 have been enrolled in a follow up program to monitor haemoglobin (hb), htc, serum iron and ferritin values. these parameters have been assessed on the day of donation and, thereafter 7 and 180 days after drbc procedure. donors suitable to drbc apheresis had to have: age between 18 and 60 years, weight > 70 kg, hb > 15.5 g/dl and serum ferritin between 50 and 400 ng/ml. a written informed consent about the collection procedure and the follow-up program has been obtained from all the enrolled donors. drbc collection procedures have been performed by using a mcs + (haemonetics) cell separators. results: out of 130 donors who donated drbc during the study period, only 14 males completed the follow up program and have been analysed. baseline haematological values and iron metabolism parameters were: mean hb 16.3 ± 0.5 g/dl, ferritin 85 ± 31 ng/ml, serum iron 121 ± 42 microg/dl. on day 7 mean hb was 14.3 ± 0.5 g/dl (p < 0.001). on day 180 mean hb was 15.3 ± 0.8 g/dl (p < 0.001), ferritin 53 ± 22 ng/ml (p < 0.05), serum iron 111 ± 27 (p ns). only 6 out of 14 donors (43%) had a ferritin value > 50 ng/ml. in the studied donors the collection of 2 units of rbcs induced an expected reduction of about 2 grams of hb, however only 50% of this reduction was recovered after 180 days (p < 0.001). similarly, also iron stores have not been restored after 6 months from donation, as shown by a 38% reduction in mean serum ferritin value. according to these data it appear that the amount of iron 'lost' with the donation of 2 units of rbcs (approximately 320-420 mg of elemental iron) could not be completely compensated by iron absorption from the diet intake. further data are necessary to define the risk of iron depletion after the donation of a drbc, however, at least in areas where iron intake by diet is not very high, the opportunity to prolong the interval between a drbc and a subsequent rbcs donation beyond six months or to provide adequate iron supplementation therapy should be carefully considered. background: increased transferrin saturation and/or serum ferritin have been observed in italy in approximatively 5% of subjects at first blood donation and, in these subjects, hfe mutations prevalence was 0.11 for c282y and 0.26 for h63d (velati et al., 2003) . aims: the role of the c282y mutation is well known in the patho-genesis of iron overload, whereas the role of the h63d mutation remains uncertain. the aims of the present study were first to study the main hfe mutations prevalence in a random group of repeat blood donors and second to evaluate iron parameters and iron depletion in repeat blood donors heterozygous for the h63d mutation in comparison to a population of blood donors wt/wt for the h63d mutation. methods: a total of 1165 repeat blood donors were examined in 10 italian transfusion centers (6 in northern italy and 4 in southern) for c282y and h63d mutations. out of those, 50 blood donors heterozygous for the h63d mutation and 31 wt/wt for the same hfe mutation, both groups wt/wt for the c282y, were enrolled to evaluate iron parameters and iron depletion. these two groups were similar for number of blood donations (expressed as iron loss) and for sex distribution. serum ferritin (sf) was the iron index recorded at first and second observation. results: table 1 summarizes the allelic frequencies in the 900 blood donors. table 2 reports the haematological evaluation in the 50 subjects heterozigous for h63d mutation and the 31 wt/wt for the same mutation. conclusions: these data suggest that subjects with h63d mutation of the hfe gene have, at first observation, a higher ferritin levels than subjects wt/wt. this seems to be more evident in blood donors of southern italy than in northern. blood donation induces significant reduction of the iron stores both in h63d heterozygous and in wt/wt subjects. although our observation is preliminary and restricted to a limited number of subjects, it seems worthwhile to extend the follow-up of blood donors h63d heteroxygotes or even homozygotes when available, in order to get further insights on the h63d role in iron metabolism. background: cd 43 is a sialylated glycoprotein expressed on the surface of most hematopoietic cells and has been implicated in cell adhesion and signaling. consequently the levels of soluble cd43 as well as the expression on the cell surface is a marker of cell activation. furthermore, downregulation of this molecule has been correlated with increased susceptibility to infections. the myelodysplastic syndromes (mds) are a group of stem cell disorders characterized by ineffective hematopoiesis, refractory cytopenias and an increased risk of leukemic transformation. the mds patients are often introduced to transfusions for anemia improvement and present increased susceptibility to infections. aims: we studied cd43 expression in transfusion-dependent and non-transfused mds patient in an effort to investigate mechanisms of regulation of this molecule. we also studied other activationassociated antigens in the absence of manifest infection. material and methods: forty-two patients were included in the study suffering from refractory anaemia (ra). thirty-one were males and 11 females aged 33 to 85 (median 75). twenty of them had never been transfused (group a) and 22 were regularly transfused (group b). nineteen age matched healthy individuals were used as controls (group c). cell surface antigens were detected by direct immuno-fluorescence evaluated by flow cytometer. the following mouse monoclonal antibodies were tested: anti-cd11b, anti-cd18, anti-cd43, and anti-cd45. leukocytes were gated according to cd45. we used a sensitive sandwich enzyme linked immunoassay to measure the level of soluble vascular adhesion molecule as an indicator of endothelial cell activation. the r&d elisa kit was used according to the manufacturer's instructions. results: the cd43 was found down-regulated in the transfusiondependent mds patients compared with the non-transfused ones (p < 0.001) and controls (p = 0.012). this downregulation concerned the proportion of cd43 + cells, that was lower in the transfused patients than the non-transfused (p < 0.01) and controls (p = 0.006), and the rfi (relative fluorescence intensity) value that was also lower in the group a compared to the group b (p < 0.01) and group c (p = 0.001). negative correlation was observed between the cd43 expression and cd11b (p = 0.001) and cd18 (p = 0.001). cd11b was found up-regulated in the transfused patients. the rfi value was significantly elevated in the transfused patient compared with the non-transfused and controls (p = 0.001 and 0.001 respectively) while the percentage of cd11b cells did not differ significantly between the various groups. increased expression of cd 18 was also found in the group a compared to group b (p < 0.01) and c (p = 0.008). the proportion of cd18 + cells did not differ between the various groups. the levels of immuno-reactive svcam-1 as determined by elisa were found 512.02 + 35.56 in group a, 3.42 + 65 in group b and 194.11 + 45.48 in the control group. conclusions: activated hemopoietic and endothelial cells are found in mds that may be associated to the vascular disorders found in these patients. cd43 downregulation may also be associated to increased susceptibility to infections in these patients. despite improved safety of the blood supply, allogeneic blood transfusion continues to be associated with risks that can be eliminated or reduced by autologous transfusion. preoperative autologous blood donation (pad) prevents transfusion-transmitted viral infection, red cell alloimmunization, and some adverse transfusions reactions. it may decrease the risk of postoperative wound infection because immunosuppression as a result of allogeneic blood transfusion is avoided. pad also supplements the blood supply, provides compatible blood for patients with alloantibodies and rare red cell phenotypes, accelerates erythropoiesis, and provides peace of mind to patients. as any medical intervention, pad has both advantages and disadvantages. with proper patient selection and dedicated attention to process control and quality assurance, the advantages outweigh. background: prestorage pooling of whole blood derived (wbd-pc's) buffy coat platelet concentrates (pc) is common practice in europe event-free survival was significantly better in patients who responded to epo + g-csf. we have reviewed data in 2 centers and the gfm has the intention to extend the study to a larger population in at least 10 centers in france blood components and preparations. the new law prohibits the mixing of different blood products, i.e. blood components and blood fractions. different methods are necessary for the quality control of blood components and blood preparations privileges for blood donors include: -a paid day off work on the day of blood donation and medical examination for blood donation additional paid day off work after blood donation an extra paid day off work if blood is given during vacation or on a holiday this award will be given to non-remunerated donors after 40 blood donations or 60 plasma donations. before 2005, each 'honoured donor of russia' or 'honoured donor of the ussr' had three privileges: free use of public transportation, receipt of certain pharmaceuticals free of charge, and a discount on apartment utilities previously, municipalities also could have their own blood establishments. this resulted in more than 1500 blood establishments in the russian federation. from both administrative and financial points of view, many of these are too small to be costeffective, and should be discontinued. services, and wider implementation of modern technology for blood collection, testing, processing, storage, and distribution acknowledgements: we thank ksw microtec ag, dresden/ germany for sponsoring the rfid-labels and novatech research gmbh, vienna/austria for developing the clinic-software. background: the national preparation human immunoglobulin g 5% for intravenous use (ivig) that is produced at the serbian institute for blood transfusion is used in therapy of neurological, heartand haemolytic diseases and on patients that have undergone surgery. aims: it is our aim to prove the impact of this national medical preparation human immunoglobulin g 5% for intravenous use on patients that have been infected with sepsis as a consequence of surgery. material and methods: human immunoglobulin g 5% for intravenous use (ivig) has been used in the study. the preparation is liquid, 5% stabilised with glucose of a ph value of 4.25 ± 0.25. it is used in those cases where sepsis developed after surgery. both an ivig group (n = 40) and a control group (n = 40) were viewed; the control group not being treated with ivig. the number of specimens with the ivig therapy cholecystitis is (n = 3), and the control group (n = 2); pancreatitis (n = 8) control group (n = 7); intestinal obstruction (n = 7) control group (n = 8); abdominal organ perforation (n = 5) control group (n = 8); abdominal perforate injuries (n = 12) control group (n = 13); serious abdominal interventions (n = 5) control group (n = 6). the period of hospitalisation of the patients in the ivig group was 25 ± 9 days while the period of hospitalisation in the control group was 28 ± 12 days. the mortality rate in the ivig group was 40% counter 63.7% in the control group. summary: toxic gram -negative bacteria caused synergistic damage of human tissues and generalized inflammatory responsesepsis. by using human immunoglobulin g 5% for intravenous use, in cases of severe disease, the mortality rate is significantly lowered, depending of course on the anamnesis of the patient prior to surgery and the presence of other diseases such as diabetes mellitus, neoplasma, cardiac diseases etc. background: manual production pc from buffy coats (bc) is a procedure with some consecutive manipulations. the orbisac system (gambro bct) automates the steps and we assessed its performance. material and methods: 160 pc were produced by this device and some parameters were studied. for the preparation of pc, 5 bc were pooled using the orbisac set, with an integrated filter (pall lrp6). bc pool was resuspended in the additive solution t-sol in order to obtain a final ratio plasma/t-sol 35/65. the pc was stored in a gambro elp bag. results: the average platelet count per unit was 3.41 ¥ 10e11. the platelet recovery from pooled bc was 85.12% (range 79.75%-93.21%). all products of the tested pc containing <1 ¥ 10e6 wbc (by flow cytometry). the values of ph on day 1 and 5 of storage were 7.45 and 7.43. the swirling phenomenon was good until day 5°. the average loss of haemoglobin per bc was 6.8 g.conclusions:the orbisac system is very suitable for routine pc preparation and it allows increased productivity and better standardization method for pc preparation. platelet concentrates met the requirements for leucodepleted product. increased production of plasma components from male donors background: we routinely separate whole blood (wb) after hard centrifugation into a red cell concentrate (rcc), a buffy coat (bc) and plasma (pl) by an automated expresser (compomat, fresenius). the bcs are subsequently processed into platelet concentrates (pcs) by soft centrifugation and an additional (manual) expression step. the atreus 3c system (gambro bct) eliminates several of those hand-on steps by combining them into one integrated process. a processing 'circular' bag is placed in the device and filled with the wb. while the bag is centrifuged, the system expresses pl, pc and rcc into separate containers. the rccs are subsequently leukoreduced (manually) with a filter (lr-rccs). this study was designed to evaluate the storage characteristics of the rccs obtained with a prototype of the atreus system in comparison to rccs obtained by routine procedure. methods: whole blood (500ml) was collected in top-and-bottom bags, and randomly selected to be processed by either (1) current routine or (2) atreus 3c. rccs were leukoreduced with the integrated inline filter: fresenius (routine group) or pall rc2d (atreus). lr-rccs were stored at 4°c and sampled until day 42. various in vitro measurements were performed (n = 20 per group).results: see table (mean ± sd). the lr-rccs contained significantly more leukocytes in the atreus group. despite the rbc loss in the bc, hemoglobin (hb) content was 6% lower in the atreus group, but met the requirements. in vitro storage characteristics for the rccs were similar in both groups. the atreus pcs contained 73 ± 28 ¥ 10 9 platelets in 78 ± 6 ml. although plasma volume was higher in the routine group, subsequent preparation of pcs would have resulted in an additional loss of 50 ml per unit in the control group. atreus plasma had extremely low levels of residual wbc and rbc. 0.02 ± 0.02 4.0 ± 4.2 <30 <0.001 aims: the aim of this study was to examine platelet quality of prestorage pooled prp-derived pc's for up to 7 days storage. methods: pc's were manufactured from wbd-pc's using in-line filtration of prp on day 0. on day 1, either 4, 5, 6, or 8 pc's were pooled into an elx® container using a sterile connecting device. studies were performed on days 1, 5 and 7 for the following measure of platelet quality. ph, morphology score (ms), extent of shape change (esc), hypotonic shock response (hsr), percent in surface expression of p-selectin (p-sel), phosphatidyl serine (ps), glycoprotein 1b (gp1b) and by thromboelastography of the prp (maximum aplitude, ma). results: a total of 20 pools were studied, 5 each of 4, 5, 6 and 8 pc's. the mean platelet yield was 4.3 ¥ 10e11 with a range of 2.4-7.0 ¥ 10e11. the five 8 pc's had a mean yield of 6.0 ¥ 10e11 and all maintained a ph > 7.0 on day 7. all products had less than 1 ¥ 10e6 residual wbc. platelet quality data is presented in the table. data are the mean ± 1 sd, n = 20. conclusion: platelet pools manufactured from pc's produced by inline filtered prp and stored in elx® containers show good quality preservation to day 7 over a range of platelet yields. introduction: the big progress in treatment of critically ill children significantly increases the need for blood and blood products. loss of blood (lowering of the total erythrocyte mass), as well as decreasing of oxygen capacity of blood that can influence cardiovascular function, is main indication for the erythrocyte transfusion. aim of the study: to present the number of erythrocyte concentrates (ek) that were issued to the pediatric clinic in skopje, as well as to point out how they were distributed. material and method: this is a retrospective study performed in nitm-skopje from january till may 2004. the following criteria were followed: hemoglobin (hb), hematocrit (htc), as well the clinical evaluation, and then final decision for transfusion was made.results: there were 254 blood units (ek) issued for the mentioned period to pediatric clinic for 73 pediatric patients (~3, 5% units/per child). the biggest consumers are children at intensive care unit and at the hematology-oncology unit. one unit of leukodepleted erythrocytes (er) was split equally to 3-5 bags. for small and prematurely born children and for some other selected patients er unit was filtered and irradiated. the dosage was 25-100 ml er (depends on age and body weigh). 173 ek was issued as washed concentrates, 15 ek were filtered and 25 ek were resuspended in ab plasma. distribution among abo system was the following: conclusion: gynecologic patients consumed rbc more than 4 times than obstetric ones (159 vs 44) and the number of given transfusions is high. the a blood group is the most needed one. we should insist on using the who guidelines for the proper clinical use of blood and try to minimize the percentage of given transfusion. and z. cermakova university hospital, ostrava, czech republic background: fully automated system olympus pk 80 is an immunohaematological analyser for detection of red blood cells antigens of ab0, rh (d, c, c, e, e) and kell systems without centrifugation by mam (microplate agglutination method) on unique terraced microplate olympus. in the czech republic analyser pk 80 is used only in blood center ostrava. aim: to evaluate the validity of results, sensitivity of microplate agglutinaton method, cause of abortive tests, requirements for analyst, capacity and reliability. methods: 95 880 blood samples of donors were tested between july 2002 and january 2005. all samples were analysed for ab0 blood group. 69 052 samples were tested for rhd antigen and 15 220 ones for rh (c, c, e, e) and k antigen. the validity of the results was evaluated for ab0 with parallel testing antigens and antibodies, while for rh (d, c, c, e, e) and k using two diagnostic serums. sensitivity of mam i.e. occurrence false negative or positive results were found out when results were confronted with previous ones in our data bank acquired testing classical manual tube or microplate methods. requirements for analyst were evaluated in according to demands for needful knowledge for new analyst, necessity of control pk during testing and maintenance. capacity were evaluated as a number of samples tested per day. reliability determine by occurrence disorders. results: ab0, rh (d, c, c, e, e) and k were investigated truly by first testing at 99.5% samples. two diagnostic serums anti-d olymp igm and totem differentiate directly rhd negative and rhd positive donors. false negative or positive results were not founded out due to mam or quality of diagnostic serums. about 0.5% samples with abortive tests were analysed next time the same testing or manual technique. causes of abortive tests were microagglutination several samples except for anticoagulative edta, weak solution of red blood cells prepared by analyser, damage of microplate, hemolysis due to impurity of microplate. in one case analyser evaluated false ab blood group due to hemolysis. analyser has friendly software, simple maintenance and sound control during testing, capacity about 400 samples per day and minimal occurrence of weighty disorders. conclusion: analyser olympus pk 80 is an effective alternative full automation for medium serological laboratory and together with mam easy and truly proves blood groups of majority samples with minimal necessity repetition due to abortive tests. introduction and aim of the study: the purpose of this study is to establish nested-pcr for the detection of hepatitis b virus (hbv) in blood and blood products. methods: the primer pair set was designed to amplify 513 bp in sregion of hbv genome in the first pcr and 233 bp of first pcr amplicon with rubisco (internal control) in the second pcr. to assess the specificity of pcr results, all the samples were tested cross-reactivity or interference in the assay. results: in case of hbv spiked blood products such as immunoglobulin and coagulation factors, this method could detect hbv dna up to 62.5 iu/ml. nested-pcr was compared with pcr-elisa and hybrid capture ii (hc-ii), the pcr-elisa showed a sensitivity of 96% (hc-ii; 77%) and a specificity of 98% (hc-ii; 100%) (p < 0.005). the results of the study show that nested-pcr and pcr-elisa could be used equally in the management for hbv detection in blood and blood products. p-398 blood component therapy: slow improvement a mrdja health center subotica, subotica, serbia background: transfusion department at general hospital was founded in 1953. since that time till now it has answered to all demands in blood and blood components. aim: the aim is to present development of the transfusiology department in the last 20 years, so that we could see how much of scientific knowledge we have adopted and in which direction our department goes at the moment. method: retrospective analysis of blood/component utilization in period from 1. january 1984 to 31. december 2004. results: in 1984 whole blood participated in the consumption with 84.18%, packed red blood cells with (rbc) only 5.94%, washed rbc were used in 9.87% of the cases. in 2004 whole blood participated in the consumption with 31.5%, packed rbc with 61.65%, washed rbc with 0.14% and rbc in additive solution with 6.71%. as far as plasma preparations are concerned, there has been, since 1984, a great consumption of plasma -witch was separated from whole blood in period up to five day in 97.1% cases, and small consumption of fresh frozen plasma (ffp) only 2.9%. since 2003, there has completely been cancelled the production of five day old plasma, only ffp is being used. from 1984 to 2002 for the patients who needed platelets, platelet rich plasma (prp) was prepared and applied right after preparation. the consumption rate was from 35 units to 103 units per year. in 2002, after the purchase of platelet shaker, began the production of platelet concentrates (pc) and consumption suddenly rose from 58 units in 2002 to 462 units in 2004. conclusions: it is obvious from the analysis that irrational consumption of whole blood was reduced to more acceptable values and therefore the use of component therapy got increased. variation in blood consumption and its slight increase is obvious though application red blood cells was conducted according to strict indications. in 2003 the production of plasma old up to five days was cancelled and instead we produced only ffp. pc we prepared for patients only in agreement of treating physician. although very slow progress in development of transfusion therapy in our department can be seen in accepting scientific knowledge. transfusion specialist are active participants in patient treatment and by accepting scientific achievement are able to set standards and help our colleagues, clinics, in successful hemotherapy. introduction: blood groups may act as receptors of parasites, bacteria and viruses. there is evidence that they perform a function and play a biological role. objective: the aim was to detect abo and p epithopes in ascaris lumbricoides extracts (ae) and to study the presence of immune antibodies in patients with ascariasis. materials and methods: 50 ae were prepared by refrigerated mechanical rupture of adult specimens. agglutination inhibition (ai) and haemogglutination kinetics (hk) tests were made with the ae. the patients´ abo and p blood groups were determined. we 1998 1999 2000 2001 2002 2003 2004 total febrile non haemolitic 2 3 2 3 2 4 2 5 2 4 2 4 2 9 4 34 16 male transfusion reaction 2 1 1 2 3 2 2 5 1 8 f e m a l e alloimunisation on le/hla 3 1 2 2 1 3 1 3 3 4 1 5 2 25 6 male antigens 2 2 1 2 3 3 3 3 1 9 f e m a l e kandidates of renal 4 3 4 4 5 4 6 4 6 5 10 8 12 8 10 7 57 43 male transplantation 1 1 2 1 2 4 3 14 female lupus nephritis 3 3 2 3 2 1 1 1 1 6 male 3 3 2 3 2 1 1 1 1 6 f e m a l e total 12 12 12 16 16 18 21 25 132 65 female 67 male introduction: irradiation of blood product has been in routine use to prevent graft-versus-host disease (gvhd) in certain recipients for many yeas. gamma irradiation can abrogate the ability of lymphocytes to proliferate in vitro, 1500 cgy of gamma radiation reduce lymphocyte response to mitogens by 90%.the aim of the study: 1. to estimate potassium level increment in stored irradiation blood units. 2. to compare the increment in potassium level between leucodepleted and non leucodepleted, irradiated stored blood units. 3. to evaluate the expiratory date of blood units post irradiation. the study included 40 units of blood collected in cpd-adsol (as-1). in twenty units the blood collection bag was with inline leucodepletion, while the other 20 units were non leucodepleted. all the 40 units were irradiated using caesium 137 as a source of irradiation, with a dose of 1500-2500 cgy. baseline samples from the bags were obtained for measuring of extra cellular potassium (k+). control samples included. results: there is statistically significant increment in potassium level in the irradiated samples compared to the non irradiated samples starting from 1st day post irradiation and continues to day 35 post irradiation. comparing the group of irradiated leucodepleted, with irradiated non leuconondepleted, for potassium level estimation during the days of storage post irradiation. there is no statistically significant difference between the two groups during all the days of storage, starting from base line samples and other samples post irradiation until day 35, p value of more than 0.05. 1. gamma irradiation of bloods units can cause cell damage that the use of such components needs to be modified. 2. there is a significant increment in the extra cellular potassium level in irradiated blood units that shows doubling value within 48 h post irradiation. 3. there is no significant difference in extra cellular potassium level increment post irradiation when prestorage leucodepleted units are compared with non leucodepleted units. 4. an out date of 28 days post collection (unless they expired before) for irradiated red blood units seems reasonable to ensure transfusion of irradiated units without serious complications, except in neonates and massive transfusion cases where irradiated blood units should be fresh and used within 48-72 h post irradiation. 5. the percentage of irradiated blood units requested by our physicians (0.09%) is very less that reflects the needs of physicians awareness of the indications for requesting irradiated components that can prevent serious post transfusion complications. the use of whole blood and blood components in treatment of surgical patients in ten years period was analyzed in iran. in accordance with world trend of using blood component therapy, in medical centers throughout the country in ten years period, there are decreasing trend of using whole blood from 26% (1994) to 9.37% (2003) and increasing trend of using packed red cells component therapy from 70.3% (1994) to 88. 54% (2003) . there is also increasing trend of using fresh frozen plasma (ffp) from 26.4% (1994) to 55. 2% (2003) . comparing 1994 and 2003 year, in use of blood therapy related to hospitalized patients at surgical department who received blood and patients who did not received blood; it appears that there is statistically significant difference between these two years. results: during 1 year period, a total of 5310 units of blood and 867 units of f.f.p were used. more specifically, the results can be shown in the following table 1 . a high rate of f.f.p usage is observed both in surgery and pathology clinics. the main causes of its usage are: haemodynamic disorders -volume depletion, and coagulation disorders and low blood protein, for the two clinics respectively. conclusion: the only way for rational usage of f.f.p is the regular reminding of plasma transfusion indications to the clinical doctors, so that undesirable side-effects caused by plasma transfusion will be reduced and the percentage of plasma used for fractionation will increase. acquired factor v inhibitor is extremely rare and is associated with diverse clinical symptomatology that varies from asymptomatic forms of the disease to very severe hemorrhagic episodes with a potentially lethal outcome. it may occur spontaneously or as a result of various clinical conditions. a 50-year-old man was admitted to our hospital with a diagnosis of left-sided periscrotal abscess and scheduled for an incision procedure. during the routine preoperative procedure screening coagulation tests showed pathologic values: aptt 41s, pt 35%, fibrinogen 2.8 g/l, fv 15% (other factors were in normal range), platelet count 189 ¥ 10 9 /l. factor v inhibitor was detected by a modified bethesda assay. the assay showed a low level of inhibitor of about 1.30 bethesda units (bu). the patient's medical history showed no major morbidity except appendectomy performed 22 years ago. the patient was prepared for operative procedure, with preventive preoperative administration of fresh frozen plasma (ffp) in a dose of 15 mg/kg (~1500 ml). upon ffp transfusion, repeated determination of the factor v plasma was unchanged from the initial finding (15%), indicating a failure of therapeutic response. as the measured level of factor v activity was at the borderline hemostatic level, and the operative procedure was not associated with a high risk of hemorrhage, the patient underwent abscess incision. the procedure and postoperative course were uneventful and without major hemorrhage. laboratory testing for the possible systemic autoimmune disorder produced normal findings. control examination performed two years later revealed no major clinical or laboratory variation, while a low factor v level persisted (17%) along with the presence of factor v inhibitor at a level of 1.15 bu. we have evaluated two groups of rcc's, one we routinely use (quadruple leucoflex lcr5 t/t cpd/sagm) (macopharma) and one using an automated collection device which gives the ability to collect whole blood in cpd with a rate of 7 : 1 respectively during the whole donation. the mentioned system has been evaluated using the suitable, quadruple leu-coflex lcr5 t/t cpd/sagm (macopharma). whole blood 450 ml in cpd was collected from random donors. in both groups the whole system was stored at scaled r.t. 20 ± 2°c for 2 to 5 h. after component separation (beckman coulter j6mi-optipress i-baxter), the red cell concentrates were filtered immediately at r.t. 20 ± 2°c. sampling was done after filtration and wbc measurements were determinated using nageotte champer (bright line-detection limit 0.05 wbc/ml) with leucoplate solution (sobioda). the other parameters were measured with (celldyne 1700 abbott). conclusion: all products met the accordance of national and european norms for blood components quality. leucodepletion with leucoflex lcr5 and abc leucoflex lcr5 (5.040 and 5.015) is highly efficient. the use of abc leucoflex lcr5 showed better scaled donation in terms of collection (statistical analysis mann-whitney, minitab p-value = 0.0392 < 0.05). additionally less hb-loss occurred, due to the filtration process, most probably due to the total absence of clots (analysis man-whitney, minitab, p-value = 0.0382 < 0.05). this new generation of collection gives the ability in blood services to collect well calibrated donations, indoors or outdoors. smaller quantities of donations theoretically can be valid because of the stable 7 : 1, rate of donation. the abc system gives the ability of fully traceability during donation. macopharma's blood collection bags, with or without integrated filters, provided they have this modified system of storing the ac. the device can keep records for many parameters and can transfer them to the data base server of the blood center. to evaluate the performance of the abc, we conducted a comparative study between abc leucoflex lst1 system and leucoflex lst1 system we currently use. the later is a well known macopharma's system for collection of whole blood with integrated whole blood filter and the final production of one unit of leucodepleted crcs and one unit of leucodepleted plasma. the abc was handled with the same system modified with the storage bag for the ac. issues of comparison were the accuracy in donation volume, the duration of filtration, the loss of blood in the filter and the residual wb cells after filtration. we performed 24 donations with the lst1 system and 28 donations with the abc lst1 system. all the donors were random male volunteers and they were meant to donate 450 mls of whole blood. our results analysed by mann-whitney, minitab statistical analysis have as follows:1. no significant difference was found between the two systems concerning the whole blood volume, but there was broader distribution of the values in the lst1 system compared to the abc lst1 system. 2. the duration of filtration has been found without statistically significant differences between the two systems. 3. the loss of volume in the filter of lst1 is higher than in the abc lst1 (p = 0.007 < 0.05, which is statistically significant). 4. there was very good leucodepletion with the lst1 systems (median reduction of the wb cells 4.7 log) but there was a superiority of the abc lst1 over the lst1 concerning the leucodepletion per litter and per unit (p: 0.0368 and p: 0.0398 respectively). 5. the personnel after a very short period of training accepted fully the abc procedure.in conclusion abc is an easy to handle device which provides with high quality blood products in combination with leucoflex lst1. evaluation of a post-storage filter for wbcs with an incorporated waste bag for washing rccs leucolab lcg4 is a system manufactured by macopharma for poststorage filtration of a rcc unit (with or without an incorporated waste bag for further washing of the filtered product). to evaluate the efficiency and reliability of the above system we conducted a study with twenty three random units of rccs stored in cpd/sag-m, aged 2-6 days, filtered and consecutively washed with 200 ml of normal saline, span down in the regular way and the supernatant extracted in the waste bag. issues for evaluation were:1. the duration of priming and filtering the rccs. 2. the loss of volume in the filter.3. the efficiency of the filtration. 4. the acceptance of the personnel of a new (to them) filtration system.our results have as follows:1. we counted the duration of priming and filtration. median time of priming was 90 s (range 40-300) and of filtration was 12 min (range 8-20). 2. the median volume lost in the filter (as calculated) was 23.4 ml (ranging from 22.4 to 28.1). this narrow range is apparently due to the non-flexible cell of the filter. 3. the efficiency of the leucodepletion was counted by flow cytometry. the median wbc counted per lt was 0.9 ¥ 106 (range 0.3-2.1 ¥ 106) and per unit was 0.17 ¥ 106 (range 0.05-0.5 ¥ 106). the median reduction of the wbc count was 4.2 log (range 3.9-4.7). 4. the personnel involved in the procedure found the system easy to handle, even without specific training. in conclusion the lcg4 is a reliable and easy to handle system, for leucodepleting (and washing) rccs, very efficient in removing wbcs with negligible loss of volume. objective: standardization of blood banks and establishing quality assurance are important landmarks in the new era of transfusion medicine. as the number of blood banks grows and the capacities of them changes, centralization need arises and trends to nationally coordinated blood services eventually appear. aim: to investigate the types and capacities of blood bank in the country and evaluate the statistics of them. material and method: blood banks and transfusion society of turkey conducted a nation-wide survey of a comprehensive questionnaire. this is a preliminary report of this investigation and illustrates the capacities of the most blood banks in turkey. it also guides the nationally planned renewing structure of blood banks. results: there are nearly 340 blood banks and blood stations in whole country. the overall blood collected at those centers is about 1.800.000 units. nearly one in third is being collected at government hospitals (31.5%), nearly the same is collected at university hospital blood banks (29.6%). the third major group is the red crescent society blood centers-rcsbc (23.5%), followed by the social security hospitals (12.9%). blood collecting capacities are not appearing in the same order. the major blood banks belong to the rcsbcs, whereas the small ones are mostly government hospitals. the only donor recruitment organization is run by the rcsbcs. yearly blood collecting capacity blood banks (%) >50.000 0.4 20.000-50.000 4.8 10.000-20.000 8.7 5.000-10.000 13.8 <5.000 72.3conclusion: there are many steps for improving blood safety in a country, and the prior ones are structuring the blood transfusion system and donor organization on a national basis, and then establishing good manufacturing practices. these are only possible after centralization of all existing blood banks. in our country, we should first arrange all small capacity blood banks and standardize them. controversial clinical questions considered in a medical opinion forum by physicians for the advancement of transfusion medicine (patm) patm is a newly formed group of close to 300 physicians drawn from pathology and hematology, transfusion services, hospital blood banks and blood centers. its mission is to address the concern that patient oriented medical opinion and influence has been diminished in transfusion medicine (tm). they believe that a patient oriented voice should be distinct from institutional, commercial or regulatory weight and have a common focus on patients and the therapeutics of transfusion and related therapies. therefore, their first objective is to create a new medical, patient oriented voice that weighs in on national policy pertaining to treatments related to tm. as such, patm held its first medical opinion forum where members of the group debated important questions pertaining to tm clinical practice. the forum was held just prior to the aabb annual meeting and attracted 58 physicians. the questions debated were preselected by patm membership via an email survey. respondents were asked to select their top four preferences from among 17 topics in five broad categories. the top two topics were selected for the forum from the 80 completed surveys. the subjects selected and debated were (i) what are the medical considerations for reducing the rate of mistransfusion? and (ii) what are the medical considerations for managing a limited blood inventory? the participants were divided into four groups, with each topic assigned to two groups. all the groups were given two h to debate and arrive at consensus on their topic. each group then presented a summary of their discussions along with specific recommendations for addressing these clinical practice issues. there was remarkable consensus between the groups debating the same issue. the conclusions and recommendations on these two topics will be presented in detail. patm is a new organization that will add an important medical voice and opinion on current topics in tm. at its first meeting, two topics were successfully discussed and debated with broad consensus achieved on current issues confronting the field. key: cord-023354-f2ciho6o authors: nan title: tuesday plenary session 3 tuesday: posters date: 2005-06-08 journal: vox sang doi: 10.1111/j.1423-0410.2005.00654.x sha: doc_id: 23354 cord_uid: f2ciho6o nan from the perspective of causal inference, there is a hierarchy of evidence, ranging from case-series to large randomized controlled trials (rcts). in addressing a particular clinical or policy problem, clinicians or policy-makers can base their decisions on the types of clinical reports that have been published, along with an assessment of the strengths and weaknesses of each study. rcts are controlled clinical experiments in which patients are randomly allocated by the investigators to receive a treatment under study. if randomization is used, all participants are equally likely to be allocated to either the treatment or the control arm of the study. rcts should be distinguished from observational studies in which investigators passively observe patients who happen to receive a treatment under study. because the allocation of subjects to the treatment and control arms of an rct is random, the play of chance should distribute all confounding factors equally between these two arms. thus, the treatment and control arms of an rct should be equivalent with respect to all confounding factors except for the treatment under study. randomization removes selection bias, because neither the investigator nor the participant knows what the treatment allocation will be before a patient enters a study. moreover, if an rct is double-blind, observation bias is also removed, because preconceived notions about benefit from the treatment cannot color the reporting of symptoms by a patient or the assessment of disease activity by an investigator. when the undertaking of rcts is deemed unlikely, meta-analyses of individual patient data, that is, of the data recorded previously on subjects enrolled in published, small rcts may allow investigators to address issues of possible biases 2ed-01-01 standardizing blood components would allow better monitoring of the effectiveness of transfusion d davenport university of michigan, ann arbor, mi, usa in routine transfusion of red blood cells (rbc) or platelet concentrates (pc), we have only a very rough idea of the dose we administer. the minimum expected hemoglobin content of rbc should be 50 g (fda criteria). however, actual measurements have found a mean hemoglobin content of 46.6 ± 11.7 g per unit, with variability between manufacturers. 25 percent of units may contain less than 34.9 g of hemoglobin while 25 percent may contain more than 56.3 g. the effect of storage senescence can magnify these differences. the resulting hematocrit increase, depending on size of recipient and age of a unit, may be 1.5 to 9 percent. apheresis collection of rbc can help standardized unit contents, but this technology is relatively expensive and time consuming. knowledge of actual hemoglobin content can be used to optimize red cell transfusion. one trial, using a simple formula incorporating the desired hemoglobin and estimated blood volume, showed that nearly 60 percent of transfusion orders could be met with one unit while achieving a mean target hemoglobin of 9.3 g/dl. for pc transfusion, about 66 percent of transfusions achieve the targeted dose of 3-6 ¥ 10 11 platelets in actual practice, with considerable variability between institutions. without knowledge of the actual content of pc, determination of refractoriness and response to matched pc is problematic. standardization and labeling of rbc and pc units for content will permit accurate dosage and quantification of transfusion practice. are transfusion guidelines evidence-based? jp aubuchon dartmouth-hitchcock medical center, lebanon, nh, usa application of the results of randomized, controlled clinical trials to similar clinical situations should increase the predictability of the outcomes of transfusions. unfortunately, too few such trials have been conducted to investigate all the potential situations where transfusion might be applied. however, the ones that have been reported indicate that, in general, transfusion is unlikely to provide substantial benefit in many of the situations where it is routinely used. in the intensive care setting, transfusing red cells at a trigger point of 7 g/dl rather than 10 g/dl not only did not lead to increased anemic morbidity but was actually associated with a reduction in overall mortality. subgroup analyses indicated transfusion at the higher trigger point did not decrease morbidity in patients with cardiac disease nor decrease the time until weaning from a ventilator. following cardiac surgery, a transfusion trigger of a hematocrit of 25% yielded the same outcomes as one of 32%. an observational study suggested that patients with peripheral vascular disease and a post-operative hematocrit below 28% were more likely to suffer a morbid cardiac event, but this group of patients had more evidence of anemia and cardiac ischemia preoperatively, illustrating the pitfalls of observational studies. most would agree that transfusion is necessary below a hb of 7 g/dl and unlikely to be necessary at a hb above 10 g/dl, but most patients fall between these limits, and there are insufficient data in the and residual confounding factors, and may permit them to make a judgment of a causal relationship. sackett proposed that the evidence generated from meta-analyses of individual patient data be regarded as being equivalent in strength to that generated from rcts. meta-analysis is the structured and systematic integration of information from different studies of a given problem. when the results of individual studies are discrepant, the purpose of an overview is to investigate reasons for disagreements among studies. when the results are concordant, the goal of meta-analysis is to derive, through application of a number of quantitative techniques, a measure of the effect of the intervention across combined investigations. this measure is referred to as the 'summary' effect of the treatment under study. meta-analysis differs from the traditional, narrative reviews of the literature, in that: (1) all completed investigations of the efficacy of an intervention that meet specific, eligibility criteria are retrieved and included in the overview; (2) the quality of retrieved studies is assessed systematically; (3) the degree of agreement among studies is evaluated, both conceptually and based on statistical criteria, and the synthesis of the findings proceeds only if the variation in reported results is sufficiently modest to be attributed to chance; and (4) quantitative methods are used to calculate the summary effect of the intervention and to test that effect for statistical significance. 2ed-02-04 biology of stem cell mobilization th papayannopoulou university of washington, seattle, wa, usa at baseline hematopoiesis, the majority of developing hematopoietic cells (precursors, progenitors and stem cells) is actively retained in bone marrow (bm), whereas fully mature cells emigrate to peripheral circulation. a small number of stem/progenitor cells are also present in blood, serving presumed physiologic roles for the repopulation of remote damaged areas. however, in several hematopoietic perturbations, i.e. post irradiation, post chemotherapy or by using empiric treatments, a heightened emigration (mobilization) of progenitor/stem cells was noted over 30 years ago. the introduction of g-csf as an efficient mobilizing agent not only had a major clinical impact, but has triggered a flurry of studies exploring the mechanisms of mobilization. from these studies, significant insight has been gained about the molecular pathways leading to mobilization, especially by studying the altered environment within bm post mobilization, and less so by studying cells mobilized in blood. several attractive scenarios have been proposed and their importance was further bolstered by studying genetic mouse models. a prominent role of the sdf-1/cxcr4 pathway has been emphasized, either by down regulation of cxcr4, changes in sdf-1 gradients, or by disruption of sdf-1/cxcr4 signaling. whether this pathway is disrupted by a proteolytic mechanism prevailing within bm post g-csf or by other protease-independent mechanisms has not yet been settled. in addition to sdf-1/cxcr4, disruption of other pathways responsible for the retention of primitive cells in bm can lead to mobilization. for example, disengagement of the vla4/vcam-1 pathway by anti-functional antibodies or its genetic deficiency in mice results in egress of stem/progenitor cells. mobilization is also seen following the use of other cytokines (i.e. kit-l, flt-3 l, il-3) or chemokines (i.e. il-8, gro?), complement activation, etc., but the detailed mechanisms or the interdependence of these other pathways with the ones already proposed have not been worked out. finally, efforts to improve mobilization efficiency in man has led to the use of combination treatments with g-csf, either by adding another cytokine (i.e. growth hormone), agonistic sdf-1 molecules (i.e. ctce0021), or cxcr4 antagonists (i.e. amd3100) which have yielded synergistic increments, and these will be discussed. a full understanding of the principles of mobilization, as well as the bm homing characteristics of mobilized cells after their i.v. infusion in transplantation, should lead to targeted, more efficient experimental protocols in the future. the possibility of deriving all kinds of mature cell types from embryonic stem (es) cells for the purpose of cell replacement therapies will probably face a major obstacle; a limited supply of available hla types for an ever growing population in demand. one possible solution is to use adult sources of stem cells such as the haematopoietic stem cells (hsc) that can repopulate the entire haematopoietic system after transplantation in a myeloablated host. however cells with the repopulating ability of hscs are still elusive for tissues such as muscle, heart, cns and pancreas. it was therefore exciting news when it was shown that hscs could repopulate other non-haematopoietic tissues arguing for a general role of bmderived cells in tissue regeneration. the term plasticity was thus coined to describe the phenomenon whereby cells of one lineage could trans-differentiate into cells of another tissue. this in turn implied that a certain degree of developmental plasticity was still available in the adult hsc, or their derived progeny, that allowed them to present with novel phenotypes. how exactly this was accomplished was a matter of speculation. in order to address the mechanism we used an animal model of liver failure where bmt with normal (wild type, wt) hsc was shown to rescue the liver failure; the repopulating hepatocytes apparently differentiated from the sole source of wt cells, the bm compartment. by studying the genetic composition of the regenerating liver nodules we observed that both wt and mutant dna was present in the nodules, a finding that was consistent with bm-derived cells fusing with host hepatocytes. the mechanism of plasticity was therefore directly related to the exposure of the donor bm-derived wt nucleus to the transcriptional environment of the hepatocyte and the expression of hepatocyte-specific genes. this fusion mechanism was also shown to underlie perceived cases of haematopoietic cell transdifferentiation to purkinje cells in the cerebellum and to cardiomyocytes. however, there are carefully executed studies that strongly support the alternative transdifferentiation mechanism in tissues such as epithelia or the epidermis. what these observations may imply is that in tissues with high rates of cell turnover, there may be a potent stem cell niche that can reprogram incoming cells to follow relevant cell lineages. if this hypothesis is correct, then the major research effort should focus on what makes the niche and whether it can be recreated faithfully in vitro so as to educate hscs to diverse lineages opening the way for realistic cell replacement therapies. collection and distribution of blood and its products to meet the medical needs of industrialized countries are typically managed through large-scale national or nationally-supported blood programs. the development, maintenance, and ultimate success of such programs are all components of a process that involves the delicate balance of continuously competing pressures, e.g. social, economic, ethical, political, regulatory/legislative. as with all endeavors that directly affect human life, safety is a central, unifying theme of this process. because transactions of blood inherently involve risks at both donation and reception/transfusion ends, efforts to enhance and maintain an acceptable level of safety generally rely on a focused program of risk management (rm). rm involves three equally important elements: identification/evaluation of risk factors in a process, control of exposure to them, and continuous monitoring to assess the effectiveness of countermeasures and the emergence of new risk factors. specific approaches to rm and quality assurance in transfusion medicine will be presented. examples from blood programs currently in effect in selected countries will be discussed. rm efforts within the corresponding blood program in greece will then be examined. the lack of data to adequately assess the status of this program suggests that at least one of the elements of rm -monitoring -can still be improved. a preliminary research effort aims to survey blood donors, transfusion recipients, and physicians in order to build an understanding of the specific factors that drive the perception of risk in the greek population. the findings form important stepping points upon which specific recommendations can be made for the development and maintenance of a comprehensive, effective rm program in greece. 2ed-03-08 the use of haemovigilance data to increase safety in transfusion medicine haemovigilance is a surveillance of all the procedures in the transfusion chain. the intension is to collect and assess information on unexpected or undesirable effects in bleeding of donors and transfusion of blood components. in europe haemovigilance was introduced as a concept in the middle of the nineties. the first national reports on haemovigilance appeared in the late nineties, and were only dealing with complications related to transfusion. later on other kind of events like 'near miss' events were added. nowadays national reports are dealing with all steps in the transfusion line, and to some extend also with complications in blood donors. the state of the art is haemovigilance with retrospective registration of unwanted events that has happened, but also a more active prospective part with an early warning in a rapid alert system about new threats in the transfusion world. the aim of the different national haemovigilance systems has been to improve safety in the transfusion line. after the first 10 years with haemovigilance in the european countries, what has been the outcome of this big effort? data from national reports do not signify major improvements, but safety is suggested to have improved due to many minor changes of procedures and awareness of dangerous situations. however, in most countries nothing really effective has been done to avoid failures, the most important cause of serious complications in transfusion of patients. systems which can prevent most of the failures are on the market. the cost of these equals the cost of nat screening for virus introduced in the same period of time. the common perception of transfusion risks is still much more focused on the hypothetical risk of virus transmission than to the demonstrated magnitude of severe complications related to bleeding of donors and transfusion of patients. therefore, to increase safety in transfusion medicine the nature and occurrence of the risks should be revealed by haemovigilance and not less important the results should be published with the aim to get a realistic common perception of the transfusion risks. the role of diagnostic testing to identify congenital vs acquired disorders of hemostasis and to optimize the management of perioperative bleeding g despotis washington university school of medicine, st louis, mo, usa excessive bleeding with trauma or after surgery can result in hypoperfusion and anemia related end-organ dysfunction and mortality as well as transfusion-related complications. several large studies have demonstrated that if bleeding is excessive after cardiac surgery to the point that reexploration is required, that overall mortality increases by 3-4 fold. transfusion related complications include the following potentially lethal complications: disease transmission of pathogens, acute hemolytic reactions, allergic reactions, transfusion associated acute lung injury, allo-immunization related disease (e.g. post-transfusion purpura, platelet refractoriness, transfusion-associated graft-vs-host disease) and other potential complications (e.g. increased perioperative infection or multi-organ system failure) related to transfusion associated immune modulation. in addition, blood shortages related to increasing consumption (i.e. expanding geriatric population) and/or a shrinking supply (i.e. related to exclusion of donors related to donor exclusion criteria designed to prevent disease transmission) may limit our ability to adequately manage our anemic and bleeding patients. this highlights the relative importance of accurate diagnosis and optimal management of excessive bleeding to minimize bleeding related complications and conserve our blood supply. patients at risk for excessive bleeding include those with an established hereditary disorder (e.g. vwd, hemophilia, connective tissue disorders), end-stage hepatic or renal disease as well as patients with acquired defects of the hemostatic system. in specific, patients with platelet (i.e. platelet refractoriness) or coagulation factor inhibitors at increased risk, extracorporeal circulation related defects or as related to certain pharmacologic agents). patients who require longer periods of extracorporeal circulation for cardiac surgical procedures (e.g. repeat, combined procedures or use of deep hypothermic circulatory arrest) are at a greater risk of developing excessive bleeding (e.g. cpb-related abnormalities). in addition, patients receiving one or more longacting anti-thrombotic medications in the immediate preoperative period (e.g. plavix, reopro, low molecular weight heparin etc) are at increased risk for bleeding. clinicians in the past have resorted to empiric and 'shot gun' approaches when managing excessive bleeding due lack of immediate availability of results from laboratory-based coagulation tests. on this basis, the use of point-of-care (poc) tests of hemostatic function to facilitate the optimal management of excessive bleeding, help differentiate between microvascular vs surgical bleeding and reduce transfusion have been investigated. five of six recently published studies have demonstrated that implementation of a standardized approach to manage bleeding (e.g. algorithm) which when coupled with either point-ofcare or laboratory tests of hemostatic function can optimize the management of bleeding and reduce total donor exposures by 50%. ddavp has bee shown to be beneficial with uremia-induced platelet dysfunction and with type i von willbrand's disease. although previous studies have not been able to conclusively show that ddavp can reduce bleeding and transfusion when administered prophylactically, more recent evidence indicates that this agent may be useful in preventing excessive bleeding when a test (point-of-care) reveals platelet dysfunction. recombinant activated factor vii (rfviia) is licensed for use in bleeding episodes in hemophiliac patients with inhibitors. although, only anecdotal reports and results from small clinical studies have shown that this agent can reverse life-threatening bleeding after major surgery, other reports indicate that there is variability in the effectiveness of rfviia as well as highlight lifethreatening thrombotic complications in a subset of high risk patients (i.e. patients with congenital or acquired thrombotic disorders or systemic activation of the hemostatic system such as with dic or after cardiac surgery). therefore, large clinical trials evaluating the efficacy and safety of rfviia are needed before any widespread use can be recommended. in recent years, molecular methods of blood grouping have become routine diagnostic procedures in many laboratories throughout the world. they have proved especially valuable for the determination of fetal blood groups. the ability to detect rhd in pregnancies where the fetus is at risk from haemolytic disease of the newborn represents a significant advance in obstetric care. furthermore, the occurrence of fetal dna in the mothers' peripheral blood has allowed this diagnostic procedure to be carried out without the risks that accompany amniocentesis (lo ymd. (1999) ann med 31:308-312). determination of the coding sequence of all the genes giving rise to antigens within the 29 blood group systems currently recognised is virtually complete. by correlating the coding sequence of these genes with the phenotype of red cells from individuals with different blood groups it has been possible to infer the molecular bases of most of the antigens comprising each blood group system (daniels gl (2002) human blood groups, 2nd ed. blackwell science). the polymorphic antigens of most systems other than abo and rh, are defined by single nucleotide substitutions (snps) which effect a single amino acid sequence change in the gene product. numerous methods, both manual and automated, are available to determine snps and these have been applied to the determination of blood groups. useful applications of snps detection methods include, determination of the blood group phenotype of patients who have been transfused recently and still have donor blood in their circulation (rozman p, dove t, gassner c et al. (2000) transfusion 40:936-942), and donor screening to find compatible units for patients with multiple blood group antibodies or for the management of patients with diseases like the haemoglobinopathies who are going to be transfusion-dependent over many years (castilho l, rios m, bianco c et al. (2002) transfusion 42:232-238). other applications of molecular methods include zygosity determination for rhd, determination of the blood group phenotype of patients with a positive direct antiglobulin test, selection of donors with rare blood group phenotypes, and as aids to the solution of complex blood grouping problems in the reference laboratory. the molecular bases of abo and rh antigens are complex and cannot be determined reliably by a single snp. nevertheless, methodologies are available that allow the comprehensive sequence analysis of individual genes and could be applied to abo and rh typing. whether or not this will ever be a cost-effective procedure is a moot point. it is clear that molecular methods are a useful addition to the range of diagnostic procedures available to transfusion medicine practitioners but it is important to remember that methods based on dna analysis determine phenotype by inference and not by direct measurement. the results of molecular tests should be interpreted with this caveat in mind. 2ed-06-18 the hla system g stavropoulos general hospital 'g. gennimatas', athens, greece since its discovery in the mouse in 1936 the major histocompatibility complex (mhc) has become one of the most important region in the vertebrate genome with respect to infection, autoimmunity and transplantation. mhc primary function is to provide protection against pathogens. this is achieved through sophisticated pathways in which mhc class i molecules present endogenous antiges to cd8+ t cells and class ii molecules present exogenous antigens to cd4+ t cells. an increasing number of other proteins are being found that support these two pathways; many of these proteins, together with the class iii complement proteins, also map to the mhc. the mhc molecules were originally studied for their ability to cxonfer tolerance (histocompatibility) following tissue grafts or later, organ transplants. nowadays, the success of unrelated hematopoetic cell transplantation is influenced by the degree of mhc compatibility between the donor and patient. thus, for patients who lack matched donors, the rules that govern permissibility of mhc mismatching still need to be identified. for patients with high risk disease who lack matched donors, use of donors with a single mhc mismatch may permit early treatment before disease progression. scientific evidence to fully answer the questions 'when are platelet components clinically effective' and 'what do we really know?' is limited. despite this limitation and the level of uncertainty it generates with regard to treatment options and policies, it is reassuring to note that current prophylactic platelet transfusion protocols -mostly derived from empirical observations collected during the 1960's and 1970's -protect oncology patients from clinically relevant bleeding in more than 95% of thrombocytopenic days spent in hospital or at home, even in aggressive conditions such as leukemia, lymphoma and other severe blood diseases. this evidence seems to justify the prevalent policy of transfusing platelets when the patient's platelet count falls below 10 000 per microliter (or 20 000 in 'unstable' patients). this policy is based on positive outcomes of prospective and retrospective studies performed during the 1990's including some hundred non-surgical patients and on the desire of balancing the will of reducing patient exposure to limited, expensive, potentially infectious and immunogenic blood products with the objective of preventing clinically relevant hemorrhage. several national and international organizations endorse the above policy. although the role of platelet support in surgery or in specific clinical situations requiring invasive procedures or in patients affected by multi-organ and system co-morbidity suffers from even more limited evidence, the latter has been carefully collected and summarized in the guidelines for platelet transfusion published in j clin oncol 2001;19:1519-1538. additional data on lumbar puncture are reported in ann hematol 2003;82:570-573. another important topic where there is room for improvement and need for further investigation is platelet refractoriness, a condition developed by a proportion of chronic platelet recipients, which causes high hemorrhage risk if compatible platelets are not provided and in which consensus on the diagnosis and treatment is lacking. with regard to the type of platelet product, it will be necessary to determine the clinical impact of buffy-coat derived platelets, consistently used in europe and current object of increased interest in canada, as compared to the platelet-rich plasma method traditionally used in the us, of viral inactivation procedures and of laboratory methods for detection of bacterial contamination of platelet concentrates. in addition, ongoing studies on the clinical impact of high versus low platelet doses will provide novel elements to determine the relative merits of apheresis versus whole-blood derived platelets. as far as the established procedure of white cell reduction by filtration, which shows high technical efficiency and has been implemented as a standard by a number of institutions, debate is still ongoing on its equivalence with serologic screening for the prevention of cmv transmission, whereas general consensus supports its pre-storage use to prevent febrile, non hemolytic transfusion reactions. finally, although the high cost of filters do not allow a generalized use of this technology in many settings, white cell reduced platelets have been unequivocally shown to reduce alloimmune refractoriness from about 15% to about 5% of patients. 2ed-07-20 ffp: appraisal of the evidence for the clinical use of ffp although the indications for transfusion of plasma (fresh frozen plasma; ffp) are limited, the use of ffp continues to rise in the united kingdom and has risen by over 20% in the past few years. local uk audits continue to document that a significant proportion of ffp transfusions are not consistent with indications reported in guidelines. a systematic review of the evidence base for the effectiveness of ffp was therefore undertaken to identify, select and appraise all relevant randomised controlled trials, as the most robust form of study to assess effectiveness of ffp. in the systematic review of ffp, the criteria for inclusion of full-published studies were: there must have been at least two groups in the study; • allocation to the groups must have been either by formal randomisation or by a quasi random method e.g. alternation; • one of the arms of trial must include ffp or plasma as an intervention; results on the relevant clinical or laboratory outcome must be presented. the main analysis was qualitative, and differentiated between: 1. studies of interventions comparing ffp with no ffp; 2. studies of interventions comparing ffp with a non-blood product e.g. solutions of colloids and/or crystalloids; 3. studies of interventions comparing ffp with a different blood product; 4. studies comparing different formulations of ffp, e.g. solvent detergent and methodine blue treated. an evaluation of studies comparing ffp with no ffp would be expected to provide the clearest direct evidence for a positive effect of ffp. studies comparing ffp with colloids or crystalloids were separately appraised because these latter products may have variable effects on coagulation tests. studies comparing different formulations of ffp do not directly evaluate effectiveness of ffp but were included because there is now a uk national policy to use these products in younger patients and therefore these trials might identify negative outcomes. the search strategy identified a total 57 publications. although it may appear that this number of randomised trials might provide a reasonable evidence base to help inform clinical policy and decision making, the review identified a number of important concerns about the published trials. few of the identified studies included details of the study methodology (method of randomisation, blinding of patients and study personnel). the sample size of many included studies was very small (range 8-78 patients per arm). few studies took adequate account of the extent to which adverse events might negate the clinical benefits of treatment with ffp. many of the identified trials in groups such as cardiac, neonatal, and other clinical conditions, evaluated a prophylactic transfusion strategy. however, when these trials evaluating prophylactic usage were assessed together as a single grouping in the review, it appeared there was no consistent support for a beneficial effect of prophylactic ffp, irrespective of clinical setting. there is a pressing need to develop new trials to determine the effectiveness of ffp, including for those clinical situations in which it has become an accepted part of current transfusion practice. a law decided upon by the riksdag (the swedish parliament) often sets the general standards as a framework and authorises the central government authority concerned to elaborate and decide upon the more detailed regulations. laws and regulations define the level of quality and safety that has to be reached and state what must be done, while establishments and their managers are kept responsible for fulfilling the requirements and how this is done. guidelines (non-binding) present detailed instructions on ways how to fulfil the requirements. making a law is complicated and time-consuming. the ministry draws up a legislative proposal, refers the proposal to relevant bodies for consideration and comments, and then drafts a bill which is referred to the council on legislation for consideration before it is submitted to the riksdag. a parliamentary committee deals with the bill before it is put to the chamber of the riksdag for approval. when adopted, the bill becomes law. regulations elaborated by central government authorities are handled in a principally similar way. however, regulations are more readily adjusted to scientific and technical progress, and when legislation need requirements for technical details, these are preferably presented in a regulation. the ministry of health and social affairs is responsible for elaborating the bill to be submitted to the riksdag on the blood safety law. two central government authorities, designated as competent authorities, are responsible for preparing the appropriate complimentary regulations as well as for inspecting and licensing the blood establishments: • the national board of health and welfare (nbhw), responsible for requirements related to blood components intended for transfusion, and • the medical product agency (mpa), responsible for requirements related to blood components intended for the manufacture of medicinal products. sweden became a member of the eu ten years ago. since then, experts in transfusion medicine have been appointed by the ministry, nbhw and mpa for advice on scientific and technical issues and for representing sweden at expert meetings arranged by the commission. some of these experts are also members of the handbook committee of the national society for transfusion medicine, preparing and revising the national guidelines. consequently, the requirements of the directives are readily implemented in the daily work of the blood establishments before (due to legal and technical reasons) the new blood safety law and the revised nbhw and mpa regulations can be promulgated. to a great extent, requirements of the blood directives are covered by existing swedish legislation. no major problems or obstacles seem to arise when implementing new requirements into law and regulations and into the blood establishments' daily service. a few detailed requirements have been found difficult to follow precisely in the routine work. these minor difficulties, however, will not compromise the high quality and safety of blood components prepared according to the standards set by the blood directives. the landscape for blood collection and distribution includes availability and safety. true international availability of blood has not been reached. the landscape of safety has been mountainous, if viewed by the degree of frustration among the transfusion specialists. the reasons for frustration have varied from serological problems to those of transmissible infections, to which no end is in sight. mistrust in the safety of blood taken by others is one reason for lack of international landscape in blood collection and distribution. increasing demands on safety, availability and economy force the health care providers to reorganise blood services. national systems are winning ground. this may make it easier to achieve international collaboration. the council of europe has pioneered in creation of international recommendations for blood collection and distribution. in 1958 a european agreement on the exchange of therapeutic substances of human origin, including human blood, was published. its purpose was to make blood available to other parties of the agreement in case of urgent need. many countries ratified the agreement rapidly, some did it first in the nineties. in practice little exchange of blood components has taken place. the council of europe recommendations lack legal power. the european union is different, and it has taken on its agenda activities aiming at improving confidence in the safety of the blood transfusion chain in the community (commission communication 1994) . the agenda is based on an agreement reached at a high level eu meeting in adare, ireland in 1994. first in the commission agenda was a recommendation on donor selection criteria, given in 1998. then came the european blood directive 2002/98/ec, the aim of which is to improve the safety of blood and blood components within the union so that enough mutual trust between member countries can be achieved to make the exchange of blood components possible. being a legally binding document the directive is undoubtedly helpful in reaching a harmonised standard in europe. hopefully the european commission has the resources to follow its implementation. there are concerns, however. the epidemiological differences among the eu member countries and frequent appearance of new infectious agents potentially transmitted by blood make it difficult to foresee that even effective viral inactivation of blood components could totally erase the national differences in donor approval. blood collection and preparation of components are already the same in eu member countries and the directive helps in their further standardisation. there has not been much distribution of blood components internationally, with the exception of export of red cell concentrates to the us from switzerland and the netherlands. the european legislation opens new horizons and widens the european landscape as its purpose is to simplify the crossing of borders between the member states, but before true movement of blood components is achieved more work is needed on the eu commission blood agenda. the eu directive implemented into the legal act for polish blood transfusion service blood transfusion service (bts) in poland is an integral part of the public polish health service. in the country of nearly 39 million people, approximately one million units of blood and plasma are collected every year from voluntary, nonremunarated donors, which is statistically over 25 donations per 1000 inhabitants. blood and plasma are collected in strictly appointed centers and no private collection sites are permitted. the legal basis for the activity of polish bts is polish blood transfusion act of 22nd august 1997 which came into force as of january 1st 1999. this act was then updated in november 2003 according to eu directive 2002/98/ec and came into force as of january 13th 2004. this act introduced the principles for organization, collection, processing, storage, transport and quality assurance in bts. it specified -among others -the system of accreditation, haemovigilence, as well as requirements for bts employees. according to this act the polish bts is obliged to monitor and supervise immunohematology testing and transfusion procedures in all hospitals where blood and blood products are transfused. polish bts consists of 21 regional blood transfusion centers (rbtc), one military center and one ministry of internal affairs and administration center as well as the institute of hematology and blood transfusion (ihbt) which acts as supervisor. the 21 rbtcs have a uniform organization structure, uniform quality assurance system and act according to uniform guidelines issued by ihbt. they have two financing sources -the central budget and hospital reimbursement for distributed blood and blood products. the polish blood transfusion act of 22nd august 1997, in force since january 1st 1999, has been supplemented by 8 decrees: 1. procedures for external bts audits; 2. requirements for donor selection; 3. requirements and procedures for organization and safe management of blood transfusion in hospitals; 4. requirements for implementing of national and regional donor registers; 5. employment criteria for bts personnel; 6. training requirements for hospital personnel involved in blood and blood product administration; 7. national, uniform price list for blood and blood products; 8. organization requirements for setting up of a national committee for blood and blood transfusion. introduction: several technical aspects must be considered in pediatric apheresis due to the size of the patient. factors that must be evaluated are extracorporeal circuit volume, blood flow rates, type of anticoagulant and vascular access. adverse events are mainly related either to vascular access or to metabolic or hemodynamic changes. aim of the study: in this study we show our experience using fresenius hemocare com.tec for pbsc collection in children. methods: twelve pediatric patients (median age 8 years, range 2-16; median weight 38 kg, range 12.2-60.5 kg) with solid tumors at onset or on relapse underwent collections with the p1y kit of the fresenius hemocare com.tec blood cell separator. our cd34+ cells target was 10 ¥ 10 e 6 /kg. collections were started if a peak of at least 0.02 10e9/l cd34+ cells (20 per microlitre) were reached in the peripheral blood. in all the patients, leukapheresis were performed through a central venous catheter and temporary peripheral venous access. acd ratio 1 : 14-1 : 16 was combined with heparin 40 u/kg. in 3 children with <15 kg the separator was initialized with a compatible filtered and irradiated red blood cell unit, suspended in 4% albumin, up to the patient's hematocrit, to avoid transient hypovolemia, due to the volume sequestered in the separator. results: twenty-five procedures were performed. a median blood volume of 5400 ml (range 2.100-12.400 ml) was processed in a separation time of 270 min (range 180-330 min). the median product weight was 108 g (range 37-258 g) and yield of cd34+ cells was 3.2 ¥ 10e 6 /kg body weight (range 0.7-12.5 ¥ 10e 6 /kg body weight). three poor mobilizing patients (peripheral blood cd34+ peak of 20-26 cells per microlitre) underwent more than two apheresis to collect the desired transplantation dose (3.5 and 4). all collection procedures were well tolerated. children never required sedation to perform the leukapheresis. only mild hypocalcemia-related symptoms, promptly responding to small i.v. boluses of calcium gluconate, were reported. no circulatory side effects were observed. blood flow alarms occurred in every procedures but no collection had to be terminated due to insufficient flow. conclusion: in summary, leukapheresis in children can be safely and effectively performed with the fresenius hemocare com.tec separator with minimal technical difficulties even in patients under 15 kg. m-pa-007 alternative methods for prevention of infection transmission (pathogen inactivation etc.), cost-benefit considerations of the procedure itself. however, one must also take the loss of plasma into consideration -and more difficult: calculate the effects of changes in product quality, as reduced content of factor viii, protein s and other labile proteins. for methods involving pooling, there are also concerns about the risks due to the pool sizes. the major benefit of the methods will be the potential reduction of infectious transmission, but also possible advantages as reduction of allergic transfusion reactions and trali must be evaluated. the study types involved in cost-benefit considerations are costeffectiveness analysis where the cost and effects of an intervention and an alternative are presented in a ratio of incremental cost to incremental effect and cost-utility analysis, where quality-adjusted life years (qaly) are used as the effectiveness endpoint. qualityadjusted life years is a method that assigns a preference weight to each health state and estimates life-expectancy as the sum of these products of each preference weight and time spent for each state. a complete glossary of terms is found at http://www.hsph.harvard.edu/cearegistry. in literature, there are several publications on cost-benefit considerations within the field of transfusion medicine. concerning plasma products, the costeffectiveness of solvent-detergent plasma has been the major focus. however, the conclusions of different authors have been conflicting. in 1994, aubuchon and birkmeyer published a paper (jama 1994; 272(15) :1210-1214) where they concluded that the cost was usd 340 000 per qaly, which is far above the 'acceptable limit' of usd 50 000. this estimate was adjusted to usd 9.7 mill. per qaly in a 1999 letter to jama (jackson, jama 282). in 2003 , riedler et al. published (vox sang 2003 85:88-95 ) that the discounted cost/life year saved for sd-ffp use in the uk was gbp 22 278 for neonates and gbp 98 465 for patients aged 70. the main reason for the differences between the two papers (and others) was considered to be different calculation of non-infectious complications. the papers cited above underline the difficulties of the cost-benefit considerations. the age of the patient, the outcome of the treatment, the quality of life in an infected patient and the cost of side effects will differ. in addition, how much are we willing to pay for protection against emerging viruses? this paper will not provide the answers, but introduction: the photodynamic treatment of therapeutic plasma using methylene blue (mb) in combination with visible light is a well established procedure for the inactivation of blood borne viruses. aim of the study: evaluation of the quality and stability of mb/light-treated plasma (mb plasma) prepared under worst case conditions for routine processing. methods: single donor units (n = 12) were treated using the macopharma theraflex mb-plasma system which includes plasma membrane filtration (plas4) and addition of mb prior to illumination followed by mb and photoproduct filtration (blueflex). samples were taken before treatment and from the final product. additionally mb-treated plasma was prepared from four different plasma pools and stored for up to 9 months. treatment was done under worst case conditions for the preservation of coagulation factors: maximum mb concentration during illumination (1.15 mmol/l), maximum storage time of whole blood before separation (4°c, 17 h), maximum storage time of mb plasma before freezing (1 h). several plasma parameters and the concentration of mb and its photoproducts (azure a, azure b, azure c and thionine) were determined. results: mb/light treatment had a significant influence on thrombin time (+20.5%), fibrinogen (clauss) (-20 .4%), factor v (-15.6%), factor viii (-22.3%), factor xi (-13.3%) and protein c (-10.3%). no significant changes were detected for at iii, vwf : rco, vwf cleaving protease, plasmin inhibitor and alpha-1-antitrypsin. the entire virus inactivation procedure including the filtration steps for leukocyte depletion and mb and photoproduct depletion had no significant effect on activation markers (prothrombin fragment 1 + 2, thrombin-antithrombin-complex, d-dimers). no further essential loss of coagulation factor activity was observed during storage of the plasma at 30°c for 9 months. mb and its photoproducts (azure a, azure b, azure c) were depleted to a final concentration of <0.1 mmol/l. thionine was undetectable in all samples. conclusion: photodynamic treatment of fresh frozen plasma (ffp) using the theraflex mb-plasma system leads to only moderate decreases in the activities of different coagulation factors even under worst case conditions for routine production. mb and its photoproducts were effectively removed from the plasma by the blue-flex-filter integrated in the theraflex-system. the quality of mb plasma is well preserved during storage. pulmonary complications, particularly transfusion-related acute lung injury and circulatory overload, are the most common causes of transfusion-associated morbidity and mortality in the developed world. trali is a syndrome characterized by acute respiratory distress, hypoxemia, hypotension and pulmonary edema, occurring within 6 hours (usually 1-2 hours) of transfusion of a plasmacontaining blood product. other signs, including hypertension, leucopenia and hypocomplementemia, are less frequent. all blood products, except for albumin and solvent/detergent plasma, have been associated with trali, but red blood cells, platelets and ffp are the most common. the incidence is unknown, but 1 : 5000 plasma-containing transfusions is the most commonly cited figure. in 80% of patients, recovery is well underway within 96 hours, and leads to complete resolution. death occurs in 6-23%. the profile of the at-risk recipient has not been identified. recurrent cases have been infrequently described. there are two prevailing theories of pathogenesis: (1) antibody-mediated and (2) 2-hit hypothesis. evidence supports both concepts and neither is mutually exclusive. more than 60% of reported cases are associated with blood components containing either hla-specific (class i or ii) or hnaspecific antibodies. in 50% these antibodies correspond to at least one epitope in the recipient. most implicated components are donated by multiparous women. five percent of patients have hla or hna antibodies in their pre-transfusion serum. treatment requires prompt, assertive respiratory intervention, frequently necessitating mechanical ventilation. because trali has a much better prognosis than ards, it is an important diagnosis. some blood collectors are diverting plasma from multiparous donors away from ffp production or routinely screening for hla antibodies. the most effective method for identifying 'high risk' components has not been identified. introduction: trali is a life threatening adverse reaction of blood transfusion. it is characterized by noncardiogenic pulmonary edema developed soon after blood transfusion. in japan, we built hemovigilance system in 1993 and have been collecting the voluntary reports of severe adverse reactions of blood transfusion including trali cases since 1997. since the diagnostic criteria of trali have not been established until recently and no specific diagnostic markers for trali have been discovered so far, it is very difficult to make proper diagnosis of trali in each reported case. we have been collecting the cases with respiratory distress and pulmonary edema developed after blood transfusion as suspected case of trali. we reevaluated each report whether it meet the recommended diagnostic criteria for trali published in transfusion journal in dec. 2004 . aim of the study: purpose of this study is to select trali cases which met internationally recognized criteria so that it will reveal the possible causes of trali with laboratory testing for anti-leukocyte antibodies in donors and recipients. methods: the cases with respiratory failure and pulmonary edema are selected for evaluation from the adverse reaction case reports voluntarily reported to japanese red cross. in order to make a proper diagnosis of trali, we have been utilizing the respiratory distress questionnaire which has recently revised. this helps us eliminate other adverse reactions such as circulatory overload, cardiac failure, anaphylaxis and bacterial contamination, which results in selecting out the internationally recognized trali cases properly. for laboratory testing, flowpra and labscreen for hla antibodies and gift-fcm for hna antibodies are performed. the cross-matching test is also performed if possible. results: during past 8 years, 95 cases of trali and 29 cases of possible trali have been confirmed by critical review of each questionnaire. of 95 cases of definite trali, donor specimens were obtained in 84 cases. of 84 cases, anti-leukocyte antibodies were detected in 43 cases (51%) of donors' blood, which was significantly higher than the positive rate of anti-leukocyte antibodies in donors' blood of other adverse transfusion reactions (<10%). of 43 cases of antibody positive donors, anti hla antibodies were detected in 33 cases, anti hna antibodies were detected in 18 cases, and both were detected in 8 cases. of 33 cases of positive anti hla antibodies, class i antibodies were detected in 22 cases, class ii antibodies were detected in 28 cases, and both were detected in 17 cases. on the other hand, the anti-leukocyte antibodies were detected in 38% of trali recipients, and this rate is almost the same with that of positive rate of other adverse reactions of blood transfusion (39%). these results indicate anti-leukocyte antibodies in the blood donors are one of the prerequisites for developing trali from the antibody-hypothesis-oriented point of view. other cases with no detectable antibodies should be investigated in more detail in the future. thus, reevaluating trali cases based on recommended trali criteria will allow us to reveal new information about trali. of 2087 adverse events analysed by the serious hazards of transfusion (shot) scheme (1996) (1997) (1998) (1999) (2000) (2001) (2002) (2003) , 556 (27%) were haemolytic transfusion reactions (htrs). 303 were due to incorrect blood component transfused (ibct): 226/303 were abo incompatible and 77/303 caused by other red cell antibodies. a further 40 cases were reported as acute htrs (ahtrs; i.e. occurring within 24 hours of transfusion) whilst 213 were recognised more than 24 hours after transfusion and reported as delayed htrs (dhtr). 21/556 (4%) patients died and 58 suffered major morbidity. htrs associated with ibct result from clinical or laboratory errors and are all preventable. it has been assumed that other htrs are unavoidable. closer scrutiny reveals that this may not always be the case, though review is hampered by incomplete investigations. 9/40 ahtrs occurred in group a (8/9) or group b (1/9) patients given group o platelets. of the 31 ahtrs related to red cell transfusion, 4 were due to errors, 9 were in patients with auto-antibodies, in only 4 of whom alloantibodies had been adequately excluded/identified. at least 24/213 dhtrs were potentially avoidable; in 18 cases the antibody was detectable retrospectively in the pre-transfusion sample; in 6 cases the presence of a previous antibody was not communicated to the laboratory. two patient deaths related to dhtr might have been avoided by earlier diagnosis and clinical involvement. 14% htrs reported to shot would have been avoided by compliance with pretransfusion testing guidelines and provision of group a platelets for all group a recipients. htrs can be clinically overlooked and inadequately investigated. national guidelines are needed for the investigation and management of htr, with focus on the identification of underlying causes to guide the choice of future component therapy. reference laboratories can provide valuable support in elucidating complex serological problems. patients treated with high dose chemotherapy and autologous blood progenitor cell (bpc) support may get malignant cells reinfused together with stem cells. we analyzed bpc products collected from patients suffering from germ cell cancer measuring malignant contamination and followed these patients for up to 98 months after transplantation also with the question if transplanted malignant cells influence survival. aliquots of stem cell apheresis products containing one million mononuclear cells were sedimented on glass slides and by immunocytochemistry quantitation of cytokeratin expressing cells was performed manually with light microscopy and by automated image analysis. in 11 of 35 patients (31%) cytokeratin expressing cells were detected in bpc apheresis products of patients treated for germ cell cancer. we followed the activity of the malignant disease of patients for more than eight years in median 60 months after transplantation. no significant difference in survival was demonstrated for our two patient groups. background: the use of adequate number of peripheral blood stem cells (pbsc), collected by mnc apheresis is essential for effective treatment of hematological malignancies, solid tumors, and other disorders by transplantation. aims: the goals of this study were: (a) to obtain an effective apheretic protocol for mnc harvesting, and (b) to compare the hematopoietic reconstitution after hspc transplantations in different clinical settings. methods: in this study, pbsc transplantations -60 allogeneic from matched sibling healthy donors and 60 autologous -were performed in the management of patients with severe aplastic anemia, leukemias (all, anll, cml), multiple myeloma, hodgkin's and non-hodgkin's lymphoma, breast and ovarial cancer, and extragonadal non-seminal germ cell tumor. pbsc mobilization was achieved with rhg-csf (5-16 g/kgbm/day). mnc-apheresis procedures (generally one and occasionally two) were performed using blood cell separator cobe-spectra. the first mnc-apheresis was accomplished when the leukocyte cont was 5-10 ¥ 10e9/l (autologous setting) or on the 5th day 4-5 hour after the last rhg-csf administration (allogeneic setting). the processed blood volume during one mnc-apheresis was 12. 7-22.2 l, and 24 .3 l for one pbsc transplantation in average. results: using a minimal target dose of cd34+ cell count (3 ¥ 10e6/kgbm), performing one mnc-apheresis procedure for 86% recipients sufficient number of pbscs were obtained. the mnc yield was 10.1 ¥ 10e8/kgbm in allogeneic and 8.1 ¥ 10e8/kgbm in autologous setting in average. the mean cd34+ yields for allogeneic and autologous transplantations were 12.1 ¥ 10e6/kgbm and 6.5 ¥ 10e6/kgbm, respectively. hematopoietic reconstitution was achieved on the 9.4th day for leukocytes and the 13.05th day for platelets when pbsc transplantation was applied. summary/conclusion: improved mnc and cd34+ cell yield, as well as rapid hematopoietic reconstitution were observed when: (a) the intention of auditing any clinical practice is, ostensibly, to improve patient care. the term 'audit' implies comparison against a standard. although absolute indications for transfusion have not been defined by clinical trials applicable to most clinical situations, many institutions have established their own transfusion triggers based on their reading of the literature and common practice at that hospital. assuming these represent prudent guidelines, comparison of actual practice to them may allow physicians to re-assess their pattern of hemotherapy and bring it into conformance with the guideline. there are several common ways of performing transfusion audits. retrospective analysis of transfusions allows appreciation of the clinical situation (and its ultimate outcome) but reviews the event through a 'retrospectoscope' that assesses clinical information in a different manner than that available to the clinician making the decision to transfuse. furthermore, the time lapse between the decision to transfusion and feedback about that decision may render the feedback from the reviewing body (e.g., a transfusion committee or blood utilization review committee) of little practical import. to speed the provision of feedback and emphasize educational rather than any punitive outcomes of the audit, some facilities have abolished attempts at determining whether the decision to transfuse was supportable and have used electronic means to feed back to clinicians a non-judgmental informational message summarizing the literature regarding the indications for transfusion. this appears to be at least as effective as the traditional retrospective audit system. prospective review of requests for blood components have the potential to redirect practice in a manner that immediately helps patients. however, such interactions with clinicians may come at inopportune times, may require considerable (unscheduled) time, and are most likely to be fruitful if a knowledgeable transfusion medicine expert can serve as the intermediary. both approaches (or a combination) have been shown to be beneficial in altering practice, but efforts must be diligent and sustained. providing data comparing a physician's practice to colleagues in the same specialty may prompt additional introspection and practice change, particularly if physician leadership of the institution supports the effort as a quality improvement tool. extending the comparison to a group of physicians and contrasting their transfusion habits with benchmark data from other institutions may also be helpful, but one needs to be ready to counter arguments that differences in patient groups are the reason for the differences in practice (studies have shown that, in general, practice patterns are primarily related to training and habit rather than large differences in patient acuity). increased focus on the performance of hospitals as expressed in outcome data may soon extend to transfusion practices as well. the public or governmental institutions may ask to see data illustrating the transfusion practice of an institution and its improvement over time. carefully conducted, diligent, and ongoing transfusion audits are an integral part of an institution's quality improvement program. informed consent: the term informed consent, appeared for the first time in the late 40s but it was only in the 70s that it attracted attention with regard to health care. numerous discussions and publications have attempted to define the meaning and the justification of informed consent in recent years. initially it consisted in the obligation of the physician to disclose information to the patient, regarding the procedure he was to undergo, but more recently, ethicists have emphasized the need to ensure the patient's understanding and his autonomous decision to consent. current institutional rules of ethics demand that the physician must obtain the informed consent of a patient 'prior to any substantial intervention' . what is however the meaning of informed consent? is it a mutual decision making between physician and patient? in 'principles of biomedical ethics' beauchamp and childress claim that' it is critically important to distinguish informational exchanges through which patients elect medical interventions, from acts of approving and authorizing those interventions. the elements of consent include: disclosure, voluntariness, decision and authorization. when applying the concept of informed consent in transfusion medicine one can distinguish it into donors' and patients' consent. donor informed consent: information to blood donors constitutes a sensitive issue. it refers to their protection from side-effects of the donation, as well as to protection of the recipient. with regard to whole blood donors a detailed history and information as to side effects are necessary for first-time donors. for repeat donors one needs mainly an updated history. because of time-pressure, whole blood donors are usually given written information and are asked to answer written questions. donors however differ in literacy and even literate ones do not always understand medical terminilogy; so, during the interview one should probe the degree of understanding of each donor. with first time apheresis donors more time is needed in order to explain the procedure and potential side effects. since granulocyte and stem-cell donors require premedication with growth factors and or corticosteroids, the responsibility for detailed information is even greater. the fact that all donors sign the informed consent does not mean that they are all adequately informed! interviewers must be familiar with side effects, their frequency and sequelae and must pass on this information. misses and near-misses, (serious) adverse events and failures in medical practice seem to be not preventable. in medical interventions, preparing and prescribing of medication, assistance by doctors and nurses, medical treatment and follow-up, unwanted and unexpected events occur (http://www.mederrors.com.). these events happen also in transfusion medicine and focus on safety is not unique. haemovigilance which is defined in the eu blood directive as 'a set of organised surveillance procedures relating to serious adverse or unexpected events or reactions in donors or recipients, and the epidemiological follow-up of donors' (eu directive 2002/98/ec off. j. european union.8.2.2003:l33/30-l33/40) is established to help in trying to identify and minimise the misses and (serious) adverse events in the chain from donor to recipient of blood components. the causes or reasons should be studied in order to prevent re-occurrence. adverse event reporting in blood transfusion and transfusion medicine is complex. it depends on the cooperation between blood establishments with clinicians and hospitals. it implies knowledge of blood banking, transfusion medicine and routine clinical care of all gender and ages, of potential hazards of transfusion, of immune-haematology, of microbiology, and of epidemiology. an adverse event may have its cause in every single part of the blood chain and reference may take place to a proven problem, a potential problem, or to a justified doubt. in almost all blood transfusion centres, a single donation will be processed into a number of different products, and these units might be divided or processed into more products. blood components are produced from whole blood or apheresis donations, and depending of the blood drawing and processing techniques, a high number of products with different specifications is prepared. the products' shelf life is not equal and therefore the moment in time of actual use of each unit prepared from the same donation may differ. in case the unit of platelets harms the recipient, a rapid alert can warn in order not to issue or to transfuse the unit of red cells or the unit of ffp prepared from the same donation because of the potential adverse reaction, which was detected during or after the transfusion of the first unit used. haemovigilance is not only important to blood establishments and to patients and prescribers, but it is also to clinical scientists, and to the public at large. it should provide a basis for minimising adverse reactions on blood components, and it should enable the therapeutic potential of new or established treatments with components to be maximised, since demonstrations of safety during widespread use may lead to extended usage, and wider availability. it is quite worrisome that underreporting is a general problem in medical care. it might be expected that in transfusion medicine the same rates of underreporting can be found, but also that the same mechanisms for improvement are applicable. although medical misses occur and do not seem to be preventable, the handling of these misses is often quite poor. many patients like to hear a detailed explanation, and the majority expects even apologies from the treating physician. it seems desirable to look for new routes in the prevention of unwanted events of transfusion medicine. for problem solving, analysis and improvement of working methods, where needed and possible, are often the most effective methods. attention should be focused on improvement and not on identification of the person who caused the problem ('bad apple'). lack of communication and insufficient insight in each other work are often the causes of problems and unwanted events. it should be recognised that advices given by the haemovigilance officer or the blood transfusion committee about prevention without the input and commitment of the direct responsible persons will lead in most cases to advices which are not effective or which will not be accepted. setting up a haemovigilance system or appointing of haemovigilance officers or installation of blood transfusion committees will not be sufficient. it will be necessary to develop ways of registration, data collection and analysis, but more importantly to support by giving advice and training to prevent reoccurrence of the adverse events or not optimal use of blood components. the confidentiality of the information should be guarded sufficiently. for a physician-patient relation, even after a medical failure, a 'blame-free culture' with a central role for openness and transparency is necessary. for blood establishments and hospitals, there is an important role in the right assistance and help of the physicians concerned both on a practical and on an emotional way. m-pa-032 8 years of shot data 1996-2004: a view of transfusion safety in the uk and reactions. this will require investment in infrastructure, for which there must be a trade-off in improved transfusion safety. transfusion-transmitted infections and serious immunological reactions are rare; shot has highlighted the need for blood services to implement strategies to minimise bacterial contamination and transfusion related acute lung injury and will monitor their effectiveness. from the inception of shot it has been clear that the most frequent transfusion hazard is 'incorrect blood component transfused' i.e. a patient receiving a blood component intended for another person or not meeting appropriate requirements. only a minority of these events results in patient harm and is reportable under the terms of the directive. haemovigilance schemes such as shot, that analyse no-harm errors and near-misses, can reveal clues as to the root causes of 'wrong blood', which contributed to 16 deaths and 85 cases of major morbidity in the uk between 1996 and 2003 . analysis of such events shows that most errors occur in clinical areas, the most frequent being failure of the 'bedside check' . clinical audit data indicates that 10% of patients are transfused without a wristband or other form of identification, whilst anecdotal reports suggest that urgent clinical situations, massive transfusions and nocturnal transfusions are particularly error-prone. strategies aimed at reducing errors include structured education and competency testing, and methodologies, both high and low-tech, to ensure accurate patient identification in all circumstances. onethird of errors occurs in hospital laboratories; denominator data on laboratory workload shows that work done outside of 'core hours' accounts for 20% of all pre-transfusion testing but 40% of errors, suggesting that biomedical scientists 'on-call' or on shift work are working under pressure and beyond their competency. 85% of hospitals reported that they participated in shot in 2003, but only 47% of eligible hospitals reported adverse events suggesting that transfusion hazards remain under-recognised and under-reported. however, benchmarking of 'wrong blood' incidents against transfusion activity shows that the number of observed incidents is roughly proportional to blood use. if haemovigilance data is to contribute to improved transfusion safety, clinicians must be encour-aged to report all events, thus contributing to an evidence base that can be used to effect change and facilitate learning. barriers to reporting include cumbersome systems, lack of time and resource, lack of feedback and fear of blame. transfusion practitioners have a vital role in the recognition and reporting of adverse events, education and clinical audit, but must be adequately resourced and supported by senior clinicians and managers through an active hospital transfusion committee. *provisional. m-pa-033 passing the borders: when, how and where d pirc-tiljak croatian institute of transfusion med., zagreb, croatia there may come that moment in professional life when you have to follow a strong professional and ethical need and confront your evidence-based statements with the leadership, passing the border of your own small society. in order to protect patient's health and respect human right to be informed about all possible consequences of irregular medical therapy, insisting on professional dignity and truth, you feel responsible and follow-up processing of your serious error report. once you pass the border, trying to warn authorities and find ethical resonance and critical confirmation of your professional fears, you are 'persona non grata' . methods/results: reality checking, personal experiences and observations studying the path of serious error report. although the qc system functions, yet omissions happen . . . the possible reasons could be: lack of knowledge, lack of experience, lack of independency, personal confront of interest, immature leadership, political influence, even corruption . . . how strict do the authorities manage a fault, bearing in mind the responsibility toward the patients under the risk. there is a need to create an available, effective international expert's board which will react and give professional counselling support-asylum for endangered professionals who found enough power to blow the whistle. who will hear it? transfusion transmitted infections (tti) are a major source of concern given the repercussions of hiv, hepatitis c, bacteria and vcjd transmission by blood components. large amounts of resource have been expended in making products safer and in maintaining public confidence in the blood supply. identifying emerging infections of concern is a major activity for many transfusion services. of the long list of emerging infections identified by disease control agencies around the world, identifying those responsible for tti requires, amongst other things, that: • the agent is identifiable. • it is present in blood • it causes a disease of concern. • it is transmitted by transfusion. • it is present at relatively low frequency. • if a test is available (nat, serology or immunoassay) what the infection window period is. once an agent has been identified various approaches are possible, including: • donor selection by testing, geography or lifestyle e.g. wnv; • product selection e.g. erythrovirus (b19) antibody or bacterial testing; • product treatment e.g. pathogen inactivation; • patient selection e.g. cmv matching, immune status. against this background where should our attentions focus? some agents of initial concern are now known to be ubiquitous and have minimal disease association (ttv, gbv) although transmitted by transfusion. for others (coronavirus -sars or the possible kawasaki disease agent, dengue, flavivirus encephalopathies, avian flu, etc.) this is less certain, with agents arising from species crossover being of particular concern (avian flu, vcjd, hiv). • enhancement of automation/computerisation; • process control to provide an 'error-free pathway'; • (national) surveillance and trend analysis of results, preferably based on national working standards; • significantly increased sensitivity, especially from development of antigen/antibody 'combi' assays (e.g. for hiv, and recently, for hcv); • awareness of hbsag vaccine-escape mutants and design of assays to cope with this; • extension of range of agents and markers tested for (varies in different countries); • increasing range of assays available for testing donors with a relevant history of exposure to malaria or chagas' disease infection (for retrieval of otherwise wasted blood); • european union's in vitro diagnostics directive: this has caused some problems and reduced flexibility. nucleic acid testing (nat): nat continues to increase in blood service usage world wide, although not (as yet) to replace serological methods. trends include: • reduction in sample pool size; • increased automation (and process control); • increased multiplexing to detect 2 or more agents in the same assay; • increased number of agents being tested by nat (varies in different countries); • introduction of rapid and flexible nat to detect west nile virus, in north america. bacterial screening of platelet preparations: several countries have introduced (or will introduce) routine screening of platelet concentrates either with biomerieux, bactalert or pall ebds (02 depletion assessment). other bacterial testing methods are under active assessment, some rapid enough for possible 'point of use' testing. m-pa-036 evaluation of in vivo red blood cell recovery after processing with a new filter designed to reduce prions e nelson*, h taylor † , p whitley † and t lieu* *pall medical, covina, ca, † american red cross and evms, norfolk, va, usa background: a filter, called the leukotrap affinity prion reduction filter (prf1b filter, pall medical), has been developed to reduce the level of infectious prions, associated with several fatal neurodegenerative diseases including variant creuztfeldt-jakob disease (vcjd), from leukocyte-reduced red cell products. aim: the objective of this study was to evaluate the quality of leukocyte-reduced red cells (lr-rbc) processed through this filter and stored for 42 days. red cell quality was determined by measuring the in vivo red blood cell recovery 24 hours after re-infusion of the 42-day stored red cells. storage hemolysis and atp were also determined. methods: units of blood (450 ml) were collected from normal volunteers into the leukotrap wb system containing cp2d/as-3 anticoagulant/preservative solutions (pall medical). units were either processed to lr-rbc within 8 hours at room temperature (rt units), or after 24 hours at 1-6°c (cold units). the prion filter set was sterilely connected to the units on day 1, and the units were filtered and stored for 42 days. samples were taken pre-and post-prion filtration and post-storage for plasma hemoglobin and atp determinations. post-storage samples were taken for labeling with 51-cr radioisotope, re-infusion, and determination of the 24-hour in vivo rbc recovery. a donor sample was also labeled with 99m-tc to allow for red cell mass determination. thus, both single-and double-label 24-hour recovery values were calculated. results: twelve units were collected. in vitro testing was completed on all units. in vivo testing was completed on 11 units. the mean single-label 24-hour recoveries were 84.4% and 85.7% for the rt and cold units, respectively. the mean double-label recoveries were 81.9% and 84.1% for the rt and cold units, respectively. the overall combined mean in vivo and in vitro results are shown in the table. conclusion: the 24-hour in vivo red cell recovery means are well above the fda and council of europe's requirement of achieving a mean post-transfusion survival of no less than 75% of the transfused red cells, and they are comparable to this center's previous results of red cells filtered using the licensed leukotrap rc system with cp2d/as-3 (pall medical introduction: to reduce the risk of platelet transfusion-associated sepsis (tas), methods to routinely screen for bacterial contamination have been implemented. pathogen inactivation treatment of labile blood components provides an alternative means to prevent tas. the intercept blood system for platelets (baxter healthcare) has received the ce mark and has been introduced into clinical practice. aims: this study compared the efficacy of bacterial screening using a culture method (bact/alert system, biomerieux) with pathogen inactivation (intercept blood system) for prevention of transfusion of platelet components contaminated with bacteria. methods: seven strains of bacteria associated with tas, including gram-positive staphylococcus epidermidis, streptococcus agalactiae, and staphylococcus aureus, gram-negative escherichia coli, and klebsiella pneumoniae, and the anaerobes propionibacterium acnes and clostridium perfringens were studied. for each strain, three double-dose platelet concentrates (~6 ¥ 10e 11 platelets in 600 ml of 35% plasma and 65% intersol) were collected using the amicus® cell separator. on day of collection, calibrated stocks of bacteria (20, 200, 2000 cfu) were added to the double units. each double unit was divided into two identical products containing 10, 100, or 1000 cfu of bacteria and stored overnight under conventional blood bank conditions. the control platelet concentrate was not treated. the test platelet concentrate was treated with the intercept process (150 mm amotosalen + 3 j/sq cm uva). both units were cultured using the bact/alert system at the time of bacterial inoculation and on days 1, 2 and 5 of storage. samples (10 ml) were taken for both the aerobic and anaerobic cultures. a platelet sample was considered contaminated with bacteria if a positive signal was registered within 120 hours of culture. results: for control platelet concentrates, cultures failed to detect low-dose inocula. the time to positive culture varied with the bacterial strain, contamination level, and time of sampling. at 10 and 100 cfu per product, 4 strains (s. epidermidis, e. coli, c. perfringens, s. agalactiae) and 2 strains (e. coli, c. perfringens) tested negative after 5 days of platelet storage, respectively. k. pneumoniae tested positive after 7-8 hours of culture when sampled on day 2 of platelet storage for both 10 and 100 cfu per product. at 1000 cfu per product, p. acnes tested negative in aerobic culture and c perfringens tested negative in anaerobic culture after 5 days of platelet storage. the anaerobic cultures of p. acnes became positive after 50 hours of culture when sampled on day 5 of platelet storage. of the 7 strains studied, only s. aureus consistently tested positive after 7-16 hours of culture. in contrast, all test platelet concentrates treated with intercept remained negative by bact/alert cultures throughout the entire 5-day observation period regardless of the strain and the contamination level. conclusions: bacterial detection using cultures may fail to detect low levels of bacteria typically associated with platelet contamination at time of collection and processing. failure to detect bacteria will result in the release of contaminated platelet products with 'test negative-to-date' status. in contrast, inactivation of bacteria is capable of preventing release of contaminated platelet components. background: since nat implementation for hiv-1 and hcv rna in france, the residual risk (rr) of transfusion-transmitted infections (tti) has dramatically decreased. the rr estimates, for a threeyear period from 2001 to 2003 showed a significant decrease from 1/1 700 000 and 1/1 560 000 before nat implementation to 1/3 150 000 and 1/10 000 000 after nat implementation for hiv and hcv respectively. as for hbv, the serological screening is only based on both hbsag and anti-hbc assays. for the same period, the rr estimate for hbv is 1/640 000, five times higher than hiv one and 16 times higher than hcv one. aims: as the overall rr of tti is mainly related to hbv, and given the availability of hbv nat assays, a study was conducted to determine whether hbv nat has the ability to further reduce the hbv rr and then should be implemented in blood donor screening in france. we have estimated the wp reduction by nat in comparison with one of the most sensitive hbsag screening assays, on 10 commercial seroconversion panels (bioclinical partners, franklin, ma, usa). the nat test was the procleix ultrio assay (genprobe/chiron, san diego, usa). the hbs ag test was the prism hbsag (abbott, france). the comparison was performed on both neat samples and diluted samples 1/8, 1/16, and 1/24, in order to simulate minipools of different sizes. then, we have calculated the yield of hbv-infected donations detected by nat relative to prism hbsag assay. results: on the basis of a window period (wp) of 56 days, ultrio assay is projected to close the wp by an average of 15 days on undiluted samples, 5 days in minipools of 8 samples, 4 days in minipools of 16 samples and only 2 days in minipools of 24 samples. the projected yield calculated on the basis of 2.4 million donations collected per year in france, would be 0.65 unit per year for minipool-nat and 2 to 3 units per year for individual donation nat. conclusion: introduction of minipool-nat will offer only a little added benefit to transfusion safety relative to current serological screening strategies based on both hbsag and anti-hbc assays. hbv minipool-nat is then unsuitable for hbv screening in french blood donors. single-sample nat or minipool-nat with smaller pool sizes and/or modified procedures (genome enrichment or test improvement) would be more relevant. automation when technologically and practically feasible is a prerequisite for single-donation nat. therefore, decision has been made not to implement hbv nat in the french transfusion network until fully automated systems will be available. however, as the prevalence of hbv infections is higher in the overseas territories than in continental france, and as nat is performed on individual donations in these sites, hbv-nat has been implemented since december 2004 in these territories. combined detection of hepatitis c virus core antigen and antibody as an alternative to nucleic acid testing in blood screening grating the capillary cytometer with a robotic workstation and a small footprint centrifuge. significantly, there was no decrement in system performance following automation: 474 of 501 clinical samples (94.6%) typed identically with this system and cat, and 25 of the 27 discrepant results were eventually resolved in favor of the automated cytometry method. testing showed high-throughput capabilities (currently 280 samples/day) and was inexpensive. to demonstrate the flexibility of this testing platform, we also developed a method to perform completely automated counting of residual wbcs (rwbc) following leukoreduction of blood components. there were no significant differences in accuracy and precision when rwbc in analytical controls and authentic clinical samples were quantitated by the automated capillary cytometry method or the leucocount method performed manually. given the flexibility of this system, it is very likely that additional blood bank assays could be modified for high performance automated testing on this platform. noninvasive prenatal genotyping on cell free fetal dna in maternal plasma ce van der schoot sanquin research, amsterdam, netherlands in 1997 lo et al. demonstrated that in the maternal circulation small amounts of cell free fetal dna are present, concentrations ranging from on average 25 genome equivalents(geq)/ml early in pregnancy to about 100 geq/ml at the end of pregnancy. most likely this dna is derived fom apoptotic syncitiorophoblasts. the human placenta is hemichorial, which means that the syncitiotrophoblast is in direct contact with the maternal blood flow, and apoptotic nuclei are directly released into the maternal circulation. the cell free dna is very rapidly cleared from the circulation, the t1/2 being only 15 minutes. in 1998 we have shown that this cell free fetal dna could be used for rhd genotyping. in the last 5 years many groups have shown the successful application of different prenatal genotyping assays such as fetal sexing, thalassemia, achondroplasia, duchenne's disease, adrenogenital syndrome etc. on this source of dna. importantly, no false positive result have been described due to the presence of fetal dna from previous pregnancies, the main draw back of prenatal diagnostics on circulating fetal cells. at present prenatal rhd genotyping has been introduced in routine diagnostics in the united kingdom, france and the netherlands. in large scale high throughput studies it has been shown that the diagnostic accuracy of prenatal rhd genotyping is over 99%, and it is to be expected that in the netherlands this screening will soon be introduced to restrict the antenatal anti-d immunoprophylaxis to women carrying rhd-positive fetuses. in a large european project (safe, co-ordinator maj hulten, warwick uk) many researchers collaborate to further explore the possibilities of cell free fetal dna for future diagnostics. standard operating procedures for the isolation of plasma dna have been established. control pcrs for the presence of fetal dna have been developed. recent findings on differences in methylation status of placental genes in fetal dna opens new possibilities. the main technical problem that hampers wide application of prenatal genotyping is the impurity of the fetal dna, only 1-5% of the cell free dna in plasma is from fetal origin. this makes diagnostic assays on numerical chromosomal abnormalities impossible. and also for many single nucleotide polymorphisms (snps) such as almost all blood group antigens, assays are hampered by aspecific amplification from maternal dna. our own preliminary results indicate that this latter problem can be solved by pna clamping. the addition of a pna probe specific for the k-allele partial d feature d antigen alteration, often identified as distinct 'partial' d epitope loss. the clinical impact of partial ds is due to the ability of their carriers to form anti-d antibodies upon confrontation with regular d after transfusion, or pregnancy. this leads to the -naively spoken -contradictory finding of an allo anti-d antibody in a d positive individual in connection with a negative autocontrol. the antibodies themselves include the same fatal clinical potential as anti-d antibodies of d negative individuals, but may be even more hazardous since unexpected in d positive individuals a priori. d categories (ii to vii) represent a nomenclatorily defined subgroup of partial ds. the molecular cause of partial d lies within single (caused by point mutation in the respective rhd gene sequence), or multiple amino acid exchanges (caused by gene conversion events leading to rhd-rhce-rhd hybrid genes) which determines a qualitative d antigen alteration, rendering them distinguishable from regular d by a partial d carriers immune system. nowadays, transfusion specialists and gynaecologists are more or less aware of these facts and are taking them into consideration in the clinical setting. most partial d exhibit decreased d antigen density, enabling principal recognition of them. however, routine serological methods may not properly recognise all partial ds and will identify their carriers after immunisation only, which represents a reactive diagnostic/therapeutic attitude second best to an actively prognostic one. this actively prognostic proceeding with respect to early detection of partial ds became widely feasible by rhd dna typing techniques. currently, routine rhd dna typing techniques offer an affordable, accurate and fast approach to an unambiguous identification of partial ds and their reliable discrimination from weak d types, not at risk for allo anti-d immunisation. a reasonable proactive proceeding could e.g. demand for (once in a lifetime) routine rhd dna typing of all weakly expressed ds as defined by serology, since most partial ds also meet this phenotype. rhd allele frequencies and their geographical and regional prevalence will certainly have an important impact on dna typing strategies and their (mandatory) specificities. fluorescence cytometry for completely automated immunohematology testing d roback*, b barclay † and d hillyer † *emory university school of medicine, atlanta, † transfusion & transplantation technologi, decatur, ga, usa we previously described a methodology for accurate immunohematology testing by fluorescence cytometry [roback, j.d. et al. (2004) transfusion 44 (2), 187]. this system utilized low-speed centrifugation of 96-well filter plates for red cell staining, and a smallfootprint capillary cytometer for data acquisition. when authentic clinical samples from hospitalized patients were tested for abo group, the presence of d antigen, and red cell alloantibodies, the results were well-correlated with those obtained by commerciallyavailable column agglutination technology (cat). this system determined the correct abo group and d type for 98.7% of 520 samples, compared to 98.8% for cat (p > 0.05). when samples were tested for unexpected alloantibodies, fc determined the correct result for 98.6% of 215 samples, as compared to 96.3% for cat (p > 0.05). this novel method was better than cat at detecting weak anti-a (p < 0.0001) and alloantibodies. based on these promising results, we sought to completely automate this method by inte-prevents the aspecific amplification of the k-allele, and makes it possible to detect the fetal k-allele in the presence of excess of maternal k-alleles. furthermore, it has been shown that fetal dna is in the plasma present in shorter fragments (<300 bp) than maternal dna. size separation of cell free fetal dna can therefore be used to increase the relative concentration of fetal dna, which will help the development of new genotyping assays. in conclusion, cell free fetal dna in maternal plasma is nowadays routinely used for prenatal rhd typing and fetal sexing. new technical developments will make it possible to extend these indications to other blood group antigens in the near future. more insight in the characteristics of fetal dna might finally lead to wider applications, including numerical chromosomal aberrations. furthermore, it might become possible to apply genomic dna microarrays for the screening on many different inherited diseases, including hemoglobinopathies. determination of the affinity of anti-d present in the serum of immunized subjects and in anti-d ig preparations by a method using unlabeled antibodies p lambin*, m debbia* and y brossard † *institut national de la transfusion, † chp hopital saint antoine, paris, france introduction: few data are available concerning the affinity of maternal anti-d responsible for the hemolytic disease of the fetus and the newborn (hdn), and the affinity of anti-d immunoglobulin used for the prophylaxis of that disease. we recently described a method to measure the affinity (ka) of untagged anti-d monoclonal antibodies. aims of the study: in this work, a similar method was applied to determine the affinity constant (ka) of polyclonal anti-d present in the serum from d-immunized mothers and donors and from anti-d ig preparations. methods: a constant amount of o r1r rbcs was sensitized with increasing concentrations of anti-d present in the sera from immunized subjects, and in anti-d ig preparations. at equilibrium, the amount of anti-d bound to rbcs was measured by elisa. the scatchard equation (linear regression) and the langmuir equation (hyperbolic regression) were used to determine the ka of anti-d. the experimental data fitted well with the scatchard equation (mean r † = 0.95) but a better correlation was observed with the langmuir equation (mean r † = 0.99). in 11 maternal sera, the mean ka of anti-d was 5.6 ¥ 10 to the 8 m-1 (from 2.8 to 12 ¥ 10 to the 8 m-1). in the sera from 6 immunized donors, the mean ka was 3.9 ¥ 10 to the 8 m-1 (from 1.5 to 6.8 ¥ 10 to the 8 m-1) and in 5 lots of anti-d ig, the mean ka was 3.4 ¥ 10 to the 8 m-1 (from 3.1 to 4.2 ¥ 10 to the 8 m-1). the comparison of anti-d affinity measured in 5 cases of hdn in which infants presented a fetal anemia and in 6 cases of hdn in which infants presented only a postnatal anemia showed no significant difference. the mean value of ka in the cases of fetal anemia was 5.4 ¥ 10 to the 8 m-1 whereas in the cases of postnatal anemia the mean value of ka was 5.8 ¥ 10 to the 8 m-1. conclusion: the method previously described for monoclonal anti-d was applied to polyclonal anti-d present in the serum of d-immunized subjects and in ig preparations. the experimental data fitted well with the langmuir equation, and the affinity of polyclonal of anti-d was measured with accuracy. in addition, no significant difference was observed (at least in the 11 cases of this study) between the affinities of anti-d measured in the most severe cases of hdn (fetal anemia) and in the less severe cases of hdn (post-natal anemia). introduction: cryopreservation of platelets is widely used in platelet immunology to ensure the availability of well characterised panel cells for the detection of hpa antibodies. but recovered platelets do not express the hpa-15 alloantigens. aim of the study: here we describe a method for the successful preservation of platelets by lyophilization. we report the value of this new reagent for the detection of hpa alloantibodies and especially anti hpa-15 alloantibodies. methods: rehydrated lyophilised platelets (lyo p) were tested for their reactivity with monoclonal antibodies against gpiibiiia, gpibix, gpiaiia and cd109 by flow cytometry. the levels of reactivity were comparable with the ones obtained with fresh platelets. the rehydrated platelets were used in the maipa with a panel of hpa antibodies (anti-hpa-1a, 30; anti-hpa-1b, 3; anti-hpa-3a, 3; anti-hpa-5a, 3; anti-hpa-5b, 50; anti-hpa-15a, 2 and anti-hpa-15b, 4). results: all hpa antibodies showed the expected pattern of reactivity and in several cases absorbance reading were well above those obtained with fresh platelets. absorbance values produced by inert sera were comparable with those obtained with fresh platelets (ranges 0.001-0.008). interestingly, we used lyophilized platelet with a high expression of cd 109 bearing the hpa-15 system and we have detected 24 anti hpa 15 antibodies among 1000 sera previously negative with fresh platelets. nineteen sera concerned patients suffering from hematological diseases and 5 from pregnancy women. conclusion: lyophilized platelets are possibly an ideal reagent for the platelet immunologist to be used for the detection of hpa antibodies. moreover, this work bring new insights on the hpa-15 system in platelet transfusion. we are now pursuing more extensive validation studies with a larger number of samples representing all known hpa specificities and several diseases. the diagnosis and treatment of sick infants and children requires a broad knowledge of physiology, biochemistry, genetics and the application of sophisticated testing and treatment options. one of these options is transfusion of blood and blood products. transfusion of the infant, especially the premature infant, and sick child, especially those with major organ dysfunction, requires careful consideration of their unique metabolic, hepatic and renal clearance mechanisms. guidelines that direct the indications for transfusion differ from those in adults. non-invasive measures of oxygen delivery and oxygen offloading may assist in guidelines for red blood transfusion. metabolic complications from massive transfusion and/or the manipulation of blood products must also be considered. evidence from a high quality randomised controlled trial suggests that anaemia is also well tolerated by critically ill patients. a restrictive approach to rbc transfusion that maintained the hb concentration between 70 and 90 g/l was found to be as effective as and possibly superior to a more liberal strategy of maintaining the hb concentration between 100 and 120 g/l. there are concerns that some groups of critically ill patients, such as those with cardiovascular disease and patients who are difficult to wean from mechanical ventilation, may benefit from higher hb levels. rbcs also have a role in primary haemostasis and higher triggers may be appropriate in coagulopathic patients. it is important to realise that blood is not a uniform product and the clinical efficacy of rbc transfusion may vary. one factor that may have a considerable effect on the quality of the rbc product is the storage time. rbcs undergo marked changes during refrigerated storage. the implications of these changes on tissue oxygenation are not known but these concerns have led some clinicians to request 'fresh' blood for critically ill patients. there is insufficient evidence to support such practice. it is of great practical importance to determine if, or when, fresh rbcs could be superior to stored rbcs. background: with a decreasing blood donor base, fully tested, fresh unrefrigerated whole blood (fuwb) has been found to be a more efficient and effective use of a limited resource in place of, or as an adjunct to, traditional blood component therapy in surgical situations associated with massive blood loss. aims: to outline the use of fuwb in situations where there is potential for intractable bleeding associated with major surgery, and evaluate platelet function in fuwb versus platelet components. methods: outcomes of fuwb and traditional blood component use were examined for 20 cases of cardiac bypass surgery. in addition, exclusive use of fuwb for 23 burns debridement cases was analysed. an evaluation of platelet function in whole blood compared to platelet components was also performed by measuring platelet aggregation and activation parameters. results: there was a decreased requirement for blood components following administration of whole blood post cardiac surgery. whole blood usage for burns debridement surgery eliminated the requirement for additional blood components. platelet activation was markedly reduced in whole blood compared to component platelets, and this may be one reason for the increased efficacy of whole blood in these clinical settings. conclusion: fuwb appears to have a role in minimising blood product requirements and consequent donor exposure in situations associated with massive blood loss. m-pa-051 transfusion practice for coronary artery bypass surgery in greece s lakoumenta, m vassili, g hatzidimitriou, t asteri, p stratigi, s kanellas and g palatianos hellenic society of blood transfusion, athens, greece cardiac surgery is associated with a demand for allogenic blood and blood product availability as well as a considerable consumption. the impact of consensus guidelines for allogenic blood transfusion during coronary artery bypass graft surgery (cabg) in us attracted great attention 1. the present study is conducted in order to reveal the transfusion practice in greece on a similar population i.e. patients undergoing cabg operations. methods: five participating centers collected data concerning transfusion of allogenic blood and blood products in patients undergoing elective first time cabg procedures, as well as parameters that may influence blood loss, such as duration of the operation, cardiopulmonary bypass time (cpb) etc. the total estimated blood loss was calculated as the sum of red blood cell volume reduction [(body weight in kg ¥ 60 ml/kg) ¥ (admission haematocrit-discharge haematocrit)]+(red blood cell volume transfused). results: results are shown in table 1 : means, standard deviations, and p-values of the wilcoxon test comparisons between the hospitals. a preliminary analysis of data from 5 centres (268 patients) showed no difference in patient characteristics (age, body weight, male to female ratio). there is a statistically significant difference (p < 0.0001) between the five centers in duration of the operation, cpb, estimated blood loss and volume of transfused plasma. red cell use showed also a variation which however did not reach statistical significance (p < 0.02). the center with the highest figure for blood loss has the lowest volume of allogeneic red cell transfusion because of the use of cell salvage. conclusions: although variations, as those observed in greek cardiac surgery centers, have been documented in other countries, the variation in the use of plasma is striking and we are in the process of trying to identify the reasons. our study is in progress and additional data are being collected and will be presented. introduction: premature infants and at term newborns have an higher circulating blood volume per kilogram than the adults (100 ml/kg in premature; 80 ml/kg in at term), for this reason, in case of neonatal thrombocytopenia, a specific hemocomponent, with a very high platelet concentration, needs for transfusion therapy. the laboratory criteria for platelet transfusion are the following: (a) a plt count < 150 ¥ 10 9 cells/l if bleeding is observed; (b) a plt count < 20 ¥ 10 9 cells/l without bleeding; (c) a plt count < 50 ¥ 10 9 cells/l in newborns showing critical clinical conditions. aim of this study: in this study, we have monitored the plt transfusion therapy in our neonatal intensive care unit (nicu) in the last four years. methods: effects of plt transfusions have been followed in 37 children (31 premature infants and 6 at term newborns). the weight of premature infants ranged from 600-1800 g and at term newborn from 1800-4000 g. gestation age of premature infants ranged from 26-36 weeks and of at term ones, of course, from 37-42 weeks. for every platelet transfusion in these newborns, the volume of platelet concentrate has been of 10-20 ml/kg, with a plt count < 700 ¥ 10 9 cells/l. results : in the study period, 77 plt transfusions have been performed: 23 children have been only transfused one time, while multiple plt transfusions (ranged 2-10) needed for 14 children according to clinical conditions. the observed clinical indications for transfusions have been the sepsis with haemorrhagic syndrome (26 cases) , haemorrhagic syndrome without sepsis (7 cases) and neonatal alloimmune thrombocytopenia without haemorrhagic syndrome (4 cases). after 24 hours from transfusion therapy, the absolute plt count and the correct count increment have increased in all 37 little patients. the highest increase in plt count was 45 ¥ 10 9 cells/l, while the lowest 5 ¥ 10 9 cells/l. no difference in the efficacy of therapy has been detected between premature group and at term group. 94% of children have been discharged from hospital in good general conditions without complications in following controls. in conclusion, we can affirm that plt transfusion in premature infants and in at term newborns is an efficient and safe treatment of severe haemorrhagic conditions. however a collaboration between nicu and transfusion center is necessary to choice the adequate platelet concentrate's volume for transfusion and the best plt donor for the newborn. developing transfusion strategies 27 fusion society of turkey (bbtst) in 2004 with contribution of 253 blood transfusion centers. according to these figures 51% of centers attended operates apheresis procedures. two centers informed us that apheresis in the hospital was carried out at blood bank. there was not enough information from one center, so it was excluded. of the 51 blood banks performing apheresis, 30 were university hospital blood banks. another 38 blood banks were producing both productive and therapeutic, 10 produce only productive and 2 produce only therapeutic procedures. one center did not respond. all centers reported to prepare and separate erythrocyte and plasma. however only 48 centers reported to prepare random platelets as well. each center had apheresis machines between 8-15. a total of 19 centers was carrying out around <500 procedures, 12 around 501-1000, at 9 centers about 1001-2000, at a further 10 centers around 2001-5000 procedures a year (one center was excluded). of the responders to the survey 36, all procedure were done at blood banks, whereas at 6 of them all were carried out by the hematology clinics. at 8 other centers, productive procedures were conducted by the blood bank, and therapeutics were performed by the hematology division. a total of 48 blood banks stated that they have not kept the platelet suspensions produced and used them straightaway. productive apheresis center capacities were as shown: 13 centers < 5000, 16 centers 5000-10000, 13 centers 10001-20000 and 6 centers > 20000 units have donations a year. around 68% of all apheresis procedures were carried out by large well run blood banks. conclusion: as the use and production of random platelets increase, and settle of apheresis devices in big centers will eventually decrease the demand of apheresis procedures and keep the welltrained staff at big centers, decrease the cost thereafter. • planning of resources for the financing of the bts, adoption of a methodology for creating and adjusting the price list of products, adoption of the yearly plan of needs for blood/products and services of health institutions which use blood/products. • achieving recognition of real costs of products and services from the health insurance fund and ministry of health. • harmonization of low level of acclaimed costs and real costs of basic transfusion activities. results: acclaimed costs for activities in transfusion practice (collection, testing, processing, storage, distribution and transport) as a reflection on the price of the products are 60% lower than real costs. the prices of health services in the official price list are much lower than the proportion of costs of material resources needed for the realization of these services. this especially affects the management of independent blood establishments (bti's in serbia) with core blood transfusion activities as their basic field of work, in comparison with the 44 hospital based transfusion services, which are financed within the budget of the whole hospital. the 44 hospitals with hospital based transfusion services involved partly in core transfusion activities are completely financed by the health insurance fund, while independent blood establishments are financed through the price of products and services they provide. conclusion: in order to provide adequate quantities of safe blood/products for the end user -the patient, it is elementary to create stabile and equal financial management conditions for the whole blood transfusion service in serbia. this can be achieved only by continuous cooperation of the health insurance fund, ministry of health and independent blood establishments. sion centers (rbtc). the activities on promotion and organization of voluntary, nonremunerated blood donation, blood collection and patients' services are carried out in the rbtc and in 24 departments of blood transfusion (dbt), part of the district hospitals. the collected units in dbt are transported by special cars to the ncht and the rbtc for processing, testing and control. the same transport is used for the requested by dbt blood components for storage and distribution to hospital departments. thus the issued components are with an equal quality and safety for all patients throughout the country. lbbdbt introduces hemovigilance as a mandatory system, covering the whole chain of the blood transfusion process. it includes as well the creation of registries at a national, regional and district level of blood donors, recipients of blood products and all activities of the blood transfusion service. . seventy hospitals are exclusively users of blood, blood products and services. the current organization of blood transfusion services faces the following problems: fragmented transfusion service, lack of a national blood policy, the blood program is not nationally coordinated, limited knowledge on quality management, inadequately distributed human resources, limited material resources, lack of it system, lack of planned, continuous skill upgrading. as a direct consequence we have: suboptimal blood collection activities, inadequate blood supplies significantly vary between seasons, high percentage of replacement donors, outdated methodologies, old, even obsolete equipment, the quality of blood products is not standard, there is a lack of traceability. aim of study: to reorganize blood collection activities in serbia to increase collection of safe blood up to 4% (304 000 blood units). methods: division of responsibilities between blood establishments and hospital based transfusion services by: • optimizing organizational structure • implementing blood collection standards to enhance blood safety and donor care • gradually replacing family donors with a network of voluntary non-remunerated blood donors from low risk population groups • creating and implementing a training strategy. results: through the eu funded project support to a national blood transfusion service in serbia, we are in the effort of integrating the services and standardizing their work. the blood collection working group began by dividing serbia into 3 blood collection regions: north, central, and south. each region is divided into sub regions covering approximately half a million population (4 in the north, 8 in the central and 4 in the south region). each sub region will have one standard mobile blood collection team to collect 80 blood units daily, i.e. 19 000 annually. the 19 000 blood units per 16 teams provide the 304 000 blood units (4%) to cover hospital needs in serbia. to this effect, the following has been achieved: • blood collection activities in serbia analyzed • performance analysis of bte and hbts mobile teams in place • two model standard mobile teams tested in the field • national blood collection sop's written • national donor questionnaire form prepared • national set of blood collection standards prepared • list of donor deferral criteria prepared • blood collection equipment renewed • regional reorganization plans in progress. the objectives and results can be achieved by the participation and mutual cooperation of all institutions involved in blood transfusion within an integrated, standardized system with clearly delegated responsibilities. p-005 60 years of the national blood transfusion institute in serbia n nedeljkovic national blood transfusion institute, belgrade, yugoslavia nbti was founded in 1944 . since 1953 and unpaid blood donation is mandatory, organized in cooperation with red cross. blood donation is regulated by the law in 1974, 1993/94. codex of voluntary blood donation and health care staff has also been established; 300 000 blood donors donates blood annually. in the past 60 years, there was over 3 million blood donations, performed in accordance with who regulations. over 250 transfusion medicine specialists and 700 technicians specially trained for the work in blood transfusion service (n = 53), perform transfusion medicine doctrine of rational labile blood component and stable blood derivatives therapy, based on the selfsufficiency concept in fr yugoslavia with 10.3 million inhabitants in serbia, montenegro and kosovo. plastic blood containers and tests are imported or given as humanitarian aid gift and from 2003. now, they have been regulated by tender. in 1951, test to lues was introduced, to hbsag in 1971, to a-hiv in 1985 and to a-hcv in 1993. information system was introduced in 1987. nbti includes: national haemovigilance coordinatoin center, center for medical care of haemophiliacs, tissue typing center, center for prenatal and perinatal protection of pregnant women and newborns. activities of nbti are organized through: center for planning, organization and development of blood transfusion service, center for blood collection, preparation and distribution, center for immunology and immunochemistry, plasma fractionation center for plasma in 8 west balkan countries, center for diagnostics means, center for quality control of drugs and medical and diagnostic means, center for education and training and scientific research work. nbti is the third year of gmp, sop, yus iso 9002 implementation. in the current reform of transfusiology system we are aiming for 4 percent of voluntary blood donation. nbti is the publisher of the national bulletin of transfusion medicine and it is included in the education system of the belgrade university medical faculty and the estm in belgarde 2003. the problems of blood service in russia ea selivanov and t danilova russian inst. of hematol. & transfusiol., st. petersburg, russian federation background: the blood transfusion service (bts) development as a platform for providing the hospitals with blood and blood derivatives is an important national problem. aims: russian blood service assessment with international comparison. methods: a study was conducted on the base of the reports from all regions of russia followed by a computer statistical analysis. results: blood and blood components were collected in the russian federation in 2003 in 190 stations of blood transfusion and in 1065 blood transfusion departments at big hospitals. amount of donors in 2003 was equal to 2 202 853, voluntary donors being 87.2% of them. the average number of whole blood donations in relation to the general population is 25 per 1000 inhabitants, and on average 28 percent of the donor base consists of first time donors. the average number of blood collected in relation to the general population and health care system is 11.4 ml per inhabitant and 1240 ml per one bed. an average volume of one blood donation is 396 ml. blood was collected into plastic bags containing domestic or foreign anticoagulants. about 93.2% of collected blood is used for procurement of blood components and preparations, 0.8% of banked blood is used for transfusions. amount of donors and the volume of whole blood have been significantly decreased for the last 15 years. at present in russia all donations are tested for abo blood group, rh(d) type, anti-hiv-1/2, hbsag, anti-hcv and syphilis. the total percentage of blood discarded after testing for transfusion-transmissible infections is 4.5%. 32% of plasma is obtained by plasmapheresis. blood components collected are as ffp, rbc, frozen rbc, 1eukocyte-and platelet-depleted rbc, rbc suspension, and preparations: 10% albumin, immunoglobulins, and cryoprecipitate. as to blood safety measures -implementation of blood components leucodepletion and ffp and rbc quarantine in process. the new national strategy of bts reorganization has been developed. it includes the following: increasing the visibility and resource commitment to blood issues at the national, regional and municipal levels; the national voluntary donor programme promoting; blood safety increasing; blood collection, testing and pro-cessing concentration in federal and regional bts establishments, appropriate blood and blood components usage. calculating the cost of blood in turkey n solaz, s kemahli and s cin ankara university, ankara, turkey background: like other fields of the medicine cost efficacy is gaining importance in blood banking and transfusion medicine since last few years. since last 5 years even the most developed countries started to discuss about the cost of blood. in turkey ministry of health determines the cost of blood annually. aim: to establish a safe, cost effective and reliable prices for blood components. methods: turkish ministry of health (moh) started to determine the cost of blood components as 'all inclusive' principle. this means that cost of a unit of blood component will cover all conventional expenses such as; blood typing, infectious screening, labour, consumables, etc. this system has provided uniformity to blood component costs but if the system is not controlled and followed properly it will cause serious risks. there might be some blood banks which will not respect the safety regulations and may modify the test standards for decreasing the cost of tests. conclusion: current blood product pricing system looks generally reasonable and reliable but moh should establish close follow up systems for avoiding any abuse on the safety of blood. background: a positive direct antiglobulin test occasionally occurs in normal blood donors, and is often discovered when the donor's red cells are found incompatible in a compatibility test. the incidence of a positive dat was expected to increase since more sensitive techniques (gel test) were installed. the aim of our study was to examine whether dat positive otherwise healthy donors presented any clinical or laboratory abnormalities. methods: in the first 19.000 cross-matches last year (in 10 months) 38 were found incompatible due to dat positivity of blood donors' red cells (0.02%). dat positive [(1+)-(3+)] samples were only igg positive in 32 cases, only c3d positive in 5 and igm positive and c3d positive in 1 case. all blood donors were notified and thirty two of them responded to a request for a further sample. a complete blood count, a reticulocyte count, bilirubin (total, direct, indirect), transaminases, serologic immunological tests (ana, anti-dna, anti-ena, rf, anticardiolipin antibodies), quantitative assessment of immunoglobulins, aptt and lupus anticoagulant were performed, as well as serologic tests for markers of viral infections. dat and iat were performed by gel test (id-diamed) according to the manufacturer's instructions. dat were performed with polyvalent and monovalent reagents (anti-igg, -igm, -iga, -c3c, -c3d). the blood donors were also examined clinically. the donors who had positive immunological tests were referred to a rheumatologist for further investigation. results: among the thirty one blood donors eight had received medication the last 24 hours before blood donation, two had been vaccinated for hepatitis b recently, four presented signs of a viral infection soon after blood donation, three had evidence of an allergic condition, five had positive tests for anticardiolipin antibodies and ana, two were positive for anticardiolipin antibodies only and two had a positive ana test only. in six blood donors we did not find any abnormality that might be interrelated to dat positivity. conclusions: all blood donors with positive dat should be requested to undergo further investigation. some of them are possibly candidates to long medical follow-up, especially those with other immunologic abnormalities such as positive ana and/ or anticardiolipin antibodies. the eligibility of such donors for future donation of whole blood, platelets or plasma needs to be elucidated. tions: usefulness, frequency and sincerity in answering questions. donors could choose one of the offered answers and elaborate in writing the answer they have chosen. results: of the 1000 donors that participated in the survey 947 (97.4%) answered the questionnaire, 820 (86.6%) men and 127 (13.4%) women. that the survey was useful thought 91% and 9% that it was not. opinions were elaborated by 61.1%. that the questionnaire should be completed before each blood donation was the opinion of 81.2%, 17% thought it should be filled out only the first time blood is donated and 1.8% that the questionnaire should not be completed at all. the answers given were sincere in 95.4% of blood donors, 2% were not and 2.6% were given automaticallywithout comprehension. conclusion: most donors believe that completing the questionnaire before each blood donation is an effective way to increase safety by preventing potentially infected individuals from donating blood. they are also aware of the importance of answering questions truthfully because the end result is protecting the wellbeing of both blood donors and receivers. analysis of blood donor's deferral in national institute for transfusion medicine -skopje for the last five years (2000) (2001) (2002) (2003) (2004) p blagoevska*, i nikolovska † and r grubovik* *national institute for transfusion medic, skopje, † medical center, prilep, macedonia introduction: safety of blood and blood products depends on many different factors, starting with selection of blood donors. the aim of this study is to analyze the number of deferred blood donors and the reasons for their deferral, as well as the total number of blood donors in nitm and their correlation (voluntary/family donors). materials and methods: this is a retrospective, epidemiological study and data were taken from the blood donor's registry in nitm from 01.01.2000 till 31.12.2004. statistical mass includes blood donors who came to nitm to donate blood in the mentioned period. results: there were total 14 229 donors in nitm and 541 (3.8%) deferrals. 71.5% of deferred ones are male, as well as in the group with donated blood (males are predominant). the most common reason for deferral is low hb level in 232 (42.9%) blood donors, use of drugs -78 (14.4%), low blood pressure -47 (8.7%), high blood pressure -43 (7.9%), infections -30 (5.5%), cardiovascular diseases -21 (3.9%) and others. relation voluntary/family donors is almost equal (49. 6 : 50.4 ). in the last two years the number of voluntary blood donors is increasing (60 : 40), which is good sign. conclusion: percentage of deferred blood donors in first three years is ~2%, which is result of insufficient data and it is increasing in the last two years (>6%). reasons for deferral are predominantly from temporary character (98.9%). permanent deferrals are only 6 (1.1%), which is probably due to good education of the population and self-deferral. we should establish the national registry for deferred donors, as well as for the donors with positive markers for tti. we should design a strategy for returning of temporary deferred donors. regruting blood donors in multiethnical environment p blagoevska*, r grubovik* and k elezi † *national institute for transfusion medic, skopje, † medical center, gostivar, macedonia introduction: population in r.macedonia consists of 75% macedonians, 22% albanians and 3% others (serbs, gypsies, turks). over 80% of blood donors are voluntary non-remunerated and ~20% are family donors. transfusion service and red cross should recognize the values and cultural differences of minors groups and recruiters should developed methods for reaching and motivating them to donate blood. the aim of the study is to present the ethnical structure of our donors and to develop strategy for their regrutation and retention. the study reviews the results from the blood donation actions among the high schools and university students in west part of the country (multiethnical environment) from 2002 till 2004. results: there were 3704 blood donations for the mentioned period. predominant blood donors are employed and high school students in 75%. family blood donors are ~20%; between them 70% are from albanian population. the ratio between blood donors macedonians vs. albanians is 80 : 20. woman blood donors are presented with 14%. first time blood donors are 53%, and regular donors arẽ 13%. conclusion: first step in planning the blood donation in multiethnic society is creation of special teams of important and devoted volunteers, such as religious leaders, teachers, doctors and businessman. for a successful campaign it is necessary to design special promotional material and address personally to the target population on their mother language. background: pursuit of pharmaceutical purity of the blood in the bag has led to a shrinking donor base and a significantly more expensive product. decisions regarding new infectious marker testing and donor deferrals have typically been made emphasizing decreasing one specific risk without considering the effect the intervention will have on the overall safety of blood transfusion. regulations have been formulated by governmental agencies with limited input from the medical community. the decision making process has lacked risk benefit analyses and has not had the robustness associated with spirited discussions. policies made in this manner may result in certain risks being decreased but can also have adverse unintended consequences. discussion: in the u.s., the fda's implementation of donor exclusions to prevent possible transfusion transmitted vcjd has reduced the donor base by more than 2%. given the demographics of the deferred donors, the impact on plateletpheresis donations has been even greater. to compensate for the loss of donors, blood services will have to persuade present donors to donate more frequently, to recruit new donors, or both. one study has indicated that two-thirds of donors have no intention of donating more frequently. new donors have higher rates of infectious disease markers with positivity for hiv and hcv twice as high as repeat donors. despite sensitive testing techniques, window periods still exist and not all potentially infectious donors will be excluded. another area of concern is the aggressive use of inducements to attract new donors. some blood services are offering lavish incentives such as enrolling donors into drawings to win automobiles. most donors entering the lottery will be low risk; however, it is reasonable to worry that such extreme tactics might also attract persons who should not be donatconclusion: (a) blood donors who were patients' relatives were many more than volunteers as well as more were men than women. also people of young ages were more than those from older ages. (b) the frequency of the diseases for which the blood units were tested was found to be in low levels in the population of the area. specifically as concerns hcv, it seems that transmission frequency has been reduced after the obligatory testing of hcv in blood transfusion centres and stations. genotype 1b of hepatitis c virus is the most frequent in blood donors 2d, from 3a to 3f, from 4a to 4k, 5a and 6a. these are differently distributed in the world: types 1 and 3 are the most common in europe and in usa. aims: considering that, in our region, anti-hcv antibody positivity is variable from 0.87 to 4% of general population, aim of this study has been to evaluate the prevalence of hcv genotypes in blood donors. methods: in period from may 2003 to december 2004, 83 362 blood units were analyzed by nat for viral rna research. nat has been performed on single sample by tma technique. on rna-positive samples, the hcv genotype has been identified by reverse hybridisation with line probe assay. results: 143 blood donors have resulted hcv-rna positive with identification of the following genotypes: 1a = 11 cases (7.7%); 1b = 89 (62.3%); 1a + 1b = 3 (2.1%); 2a/2c = 35 (24.4%); 3 = 1 (0.7%); 4 = 4 (2.8%); none was 5a or 6a. we have also analyzed the differences between the two sexes in hcv-genotypes distribution. hcv-1a has showed a double prevalence in men (8 cases, 5.6%) respect in women (3 cases, 2.1%), while genotype 1b is more frequent in women (48 cases, 33.6%) than in men (41 cases, 28.7%), moreover genotypes 3 and 4 do not compare in women. although an accurate pre-donation selection, discharging all subjects with alt >40 iu, our results show that 1.7 : 1000 donors, apparently healthy and without risk factors, have resulted hcv-positive. analyzing our data, the genotype 1b has resulted the most frequent in blood donors' population, followed by type 2, while the others have showed a very low prevalence. the high frequency of genotype 1 in blood donors is explained by the observation that hcv 1 is usually associated with low alt levels, for this reason affected subjects may escape to donor's screening only based on dosage of alt. on the contrary subjects affected by other hcv types, associated with high alt levels, may be deferred increasing the hcv 1b relative prevalence. at the end, the different distribution of hcv genotypes between men and women and between age's classes probably reflects differences in the pathogenic characteristics of the virus, in the transmission way and in the risk factors. in fact, it has been demonstrated that genotype 1 is principally linked to a not transfusion transmission way; genotype 2 is linked to old age, to female sex and to post-transfusion transmission; genotypes 3 and 4 are associated to young age and to an history of drugs abuse, respectively with high and low viral load; genotypes 5 and 6 are still little known because extremely rare in europe. p-023 kell blood group system and rare blood donors v fakitsa*, p karyda*, s giannoulea † , c antoniou*, j flesiopoulou*, e haliou*, m papakonstantinou*, h dessilla † , e katsadorou*, g lyrakos* and k sofroniadou* *general hospital of nikea, pireas, † blood transfusion center, athens, greece background: the kell blood group system is a compound antigen system exclusively of red blood cells. some of the kell antigens are highly immunogenic. the commoner kell antibody is anti-kel1. the kel2 (cellano) antigen is a high frequency antigen and the blood donors lacking this antigen are quite rare. the blood donors who have not factor cellano are classified in the rare blood donors. rare blood by its very nature is required rarely, but when needed that blood has to be ensured to specified patients. there are other blood donors in their family -74 (35.57%) students, but the number of persons that donate blood from their neighborhood and close environment is much bigger -175 (84.13%). motives for their donation are the following: their wish to help the ones that need blood -142 (68.26%), concern that some day everyone can be a potential recipient of blood -38 (18.26%), because of offered benefits -27 (12.98%), for a friend or relative -23 (11.05%), care for their health -16 (7.69%), because of citizen duty -8 (3.84%), because the others donate -6 (2.88%), curiosity -1 (0.48%). they want to be invited every 6 months -68 (%) students, every 12 months -38 (18.26%), every 3 months -16 (7.69%) and 16 ( the mean age of case group was 30/55 ± 9/33 and the mean weight of them was 71/23 ± 9/3, 72/6% was male and the mean number of blood donation was 2/34 ± 2/19. the mean age of control group was 35/27 ± 10/72 and the mean weight of them was 78/61 ± 12/77. 90/3% of them was male and the mean number of blood donation was 6/58 ± 7/08. the blood donors who were female, first time blood donor low wt the rate of vasovegal rx was higher in female, first time, low weight, younger blood donors (p < 0.005). the rate of vasovegal rx was higher in blood donor (p < 0.005) who were fatigue or first time blood donor, low wt blood donation, fatigue of them and starvation of them had higher absolute donation reaction than other donors. when each variable was adjusted for other variable by regression analysis. young age, first time blood donation, anxiety, fatigue, starvation were significant (p < 0.005) and the others were not. conclusion: donation -related vasovegal syncopal reactions are a multi factorial process. these reaction are more prevalent in blood donors who are young, first time donor, anxiety, fatigue, starvation. these reactions might be predicted vasovegal reaction and these some facth donors need more care. with better donation care, syncopal reaction may be decrease this would be improved donor safety, better donor retention, higher donor satisfaction, and reduce cost and increase regular blood donors. to avoid iron deficiency in blood donors, iron compensation is necessary in most females and males who donate more than 1-2 and 3-4 whole blood units per year, respectively. we present 3 studies dealing with different dose and duration of iron compensation. in the first randomized placebo controlled study iron decreased continuously in males and females at donation intervals of two (males) and three months (females) without iron compensation. 40 mg and 20 mg daily combined with 400 mg ascorbic acid over 2 months (males) or 3 months (females) compensated for iron loss or even overcompensated in females. in the second open study we reduced iron dose to 20 mg daily over one month for both genders. this iron dose was sufficient for compensation of iron loss. a further reduction of iron dose to 20 mg daily over half a month led to negative iron balance in the majority of donors. in all three studies donors with exhausted iron stores profit more from iron compensation, whereas donors with high ferritin values (>50 mg/ml) tend to loose storage iron. aim of the study: one of our campaign strategy how to increase blood donation among adolescents are periodical seminars and excursions for students of secondary schools (more than 40 per year). the aim of this study is to analyze impact of our campaign educational system on adolescents in period 2000-2004. methods: the donation of whole blood and aphaeresis platelets from donors of age from 18 to 20 (max. 3 years for each class) were count for the period of five years (2000) (2001) (2002) (2003) (2004) . the percentage of the man´s donation was calculated for each target class (1982) (1983) (1984) (1985) (1986) . results please see tables 1 and 2. in the tables there is shown observed data in relation to the total number of births in the czech republic in reviewed years. the study showed that number of donation from donors of age from 18 to 20 decreased during objected years. unfavourable state of total number of births in the czech republic (153 801 birth in 1980 republic (153 801 birth in , 133 942 birth in 1986 ) and its decreasing tendency (92 786 birth in 2002!) is with high probability a major demographic factor affected number of young donors. despite energy invested in our campaign educational system our recruitment efforts should be intensified to decrease influence of demographic factors. we should find new ways and methods to attract new blood donors and keep the regular ones, too. the aim of the research was to investigate women's attitudes towards blood donation in cyprus. a statistical sample was selected using stratified sampling and consisted of 369 women from the district of limassol (the second largest urban center of cyprus) between the ages of 19 and 60. using linear logistic regression, the analysis of the data collected revealed that there is a greater probability for a woman to be a blood donor if she is of a higher educational level, a member of an organized group or association, or if she is acquainted with other blood donors. the percentage of female blood donors is higher in rural areas than in urban centers. 40% of women do not donate blood and attribute their reluctance to do so to health-related problems, while about 25% of those who have never donated blood claim to fear the blood donation procedure. in addition, more than half of the women who have stated they would never donate blood again have attributed their denial to healthrelated problems. the research revealed that there could be an increase of up to 30% of the percentage of female blood donors if they were given time off work for a few hours or one or two days afterwards. even though very few female blood donors expressed a preference for the blood donation to take place on a particular weekday, half of them prefer the donation to take place on the discussion: it is about small group of students. the impression is that the altruistic behaviour is present at most of the questioned students. the fact about free school days is not underestimated because it is one of the most important motives of blood donoring of the young population. families where the blood donoring is a tradition have a great influence for young children because the children in these families are better informed for blood donoring. conclusion: including the children in the process of education for young children is of particular importance because the altruistic behaviour as a higher feeling is from an early age of the child and it is under the influence of the environment (family and friends). active participation of the department for transfusion medicine in the educational process, especially in the education of young children, is a guarantee to achieve longlasted positive results. adverse reactions in 2 blood donors taking betablocking antihypertensive medications l paesano*, m d'onofrio*, s misso † , g fratellanza* and e d'agostino* *university federico ii, naples, † hospital san sebastiano, caserta, italy one aim of blood donor's selection is to avoid an adverse reaction to phlebotomy (as vasovagal reaction, syncope and/or hypovolemic cardiac insufficiency). blood donation is surely contraindicated in various pharmacologic therapies, but not in all. in fact a certain degree of discretionarily exists about the assumption, or the period of suspension, relative to a numerous pharmaceutical products, as the antihypertensive agents. according to literature, the deferral of donors taking antihypertensive medication is not indicated when blood pressure is normal, symptoms are absent, and diuretics or similar agents are the only drugs used. on the contrary, it is a common opinion that an antihypertensive therapy by betablockers is not compatible with blood donation for its cardiac effects. nevertheless, in our daily activity, the observation of a blood donor taking beta-blocking drugs may occur for various causes. a possible error is a superficial pharmacological anamnesis, as it can occur in donations on autohemotheca, for a too fast medical visit (due to a large number of donors), or for the inexperience of the selector (often a not specialist of transfusion medicine young doctor). another possibility of observation is constitute by patients, undergoing to elective surgery, included in a program of autologous blood donation, suffering hypertension treated with betablockers. in fact, in this last case, the risk/benefit balance justify the blood letting procedure. in the last year we have just observed two severe post-donation reactions in donors suffering hypertension treated with atenolol. the reactions have been similar, in fact both donors showed lypotimia followed by convulsions about past half hour by the end of phlebotomy. no prodromic symptoms have been observer or referred. cardiac frequencies (cf) before donation were respectively 60 and 64 beat per minutes and blood pressures (bp) were both in the normal range (130/80 and 120/80 mmhg). after donation, during adverse reaction, cf showed no substantial variations, while bp have been decreased respectively to 80/45 and 60/30 mmhg. immediate treatment has consisted in putting the donors in the trendeleburg's position and in applying a dolorous stimulation. in the first case this treatment has been sufficient to report the bp to 100/65 mmhg (with disappearing of all symptoms) in only half hour time. in the second one, the marked hypotension showed a very slow remission, for this reason the subministration of a plasma expander needed, with the complete resolution of the symptoms after two hours. these two donors were not deferred from donation because they were periodic donors that had modified their antihypertensive therapy, without referring it neither in the questionnaire nor during anamnesis. our experience confirms that the blood donation don't must be permitted to subjects taking betablocking antihypertensive drugs. in fact these medications act on cardiac pump decreasing the cardiac rhythm and limiting the postdonation cardiac recover. this effect is very dangerous because it appears relatively in retard respect to the end of donation, when donor may have just leaved the transfusion center. introduction and aim of the study: in society under transition privatisation and marketisation probe all areas of life. transition to market economy is extremely important and sensitive issue in health and welfare services in general, and specifically in the case of blood transfusion service. the aim of the study was to analyze effects of confusing publicity which introduced possible ways of transforming blood transfusion service in serbia (ideas about privatization of some parts of national blood transfusion institute, buying blood from blood donors, selling blood from voluntary blood donors to private clinics, exporting blood from vbd, stories about tradition of paid blood donations in some european countries). publicity was restricted to a small number of sporadic outbreaks concerted in a limited period of time. table. conclusion: surveillance of adverse reactions and injuries or accidents during or after blood donation is essential for maintaining the well being of active blood donors, as well as for the safety and quality of the donated blood components. information on other activities and parameters affecting the quality of blood including materials, reagents and equipment should be collected and any serious deviations from standard operating procedures should be notified to the competent authority using haemovigilance infrastructures. skae has built up such procedures working along the lines of the european haemovigilance network. improvement of existing national haemovigilance systems is expected to follow from the implementation of the eu directive. although inevitable, blood donor deferrals lead to losses in donated blood supply and may affect donor-return rates and subsequent blood donations. to estimate the scope of blood donor deferrals and their causes, we analyzed the 2003-2004 data from regional blood centers using standardized criteria for temporary and permanent blood donor deferrals. within this period (2003) (2004) , 14.6 percent of persons who presented for donation were deferred; 85.9% were temporary deferrals (50% due to laboratory test results, among others low hemoglobin, 6.4% due to risk of acquiring a transfusiontransmissible infection) and 14.1% were permanent (20% due to the infectious diseases markers, 8.7% due to cardiovascular diseases). for regional blood centers the temporary deferral rates varied widely (see the table below ). in the case of individual regional centers, the differences as well as the most common causes were often difficult to explain. according to our analysis, some blood centers have a more restrictive approach to donor acceptance than others and this results in increased donated-blood loss. to some extent such losses could be avoided. further studies are recommended to elucidate the problem and eliminate unnecessary deferrals. caption 1: percentage of deferrals aims: from our experience in selecting blood donors, a certain number of issues have been noticed that remain obscure and need to clarification since those seem to 'haunt' the whole process of blood donation. methods: many first time blood donors and especially volunteers think that rejection reasons are permanent and they are completely incapable of donating blood their entire life. this is a 'tragic' misunderstanding since the doctor did not explain that the reason of the rejection is only temporary and in the future this man is capable of donating blood. those potential donors will never even approach again blood donation centre and when in the future they are asked why they do not donate blood, they repeat the cause of the past rejection. results: one of these rejection reasons is for example low blood pressure (12.54% of total causes of rejection). as we all know blood pressure must be determined according to age, sex, weight and from other factors as sleep, emotional status, food and liquid intake. therefore blood pressure is very important but should be evaluated with all the above factors and must not be alone the only reason for rejection. even when one blood donor is rejected it should be made clear to him that this is only temporary and if in the future he is in better physical condition, he could donate blood. in fact 85-90% of those donors rejected for hypotension are readmitted in blood donation after meeting the above mentioned criteria. another matter of equal importance is anemia (25.04% of total causes of rejection), especially concerning young women. since most of those women tend to develop anemia due to depletion of iron stores, they should be advised to donate blood at longer periods than regular, to receive proper medication and diet according to their needs. the doctor must explain the donor the reasons for iron depletion, so blood donation should not be considered as the only cause for this situation from the donor. there are many factors contributing to anemia, menses, specific diets, overwhelming stress and exercise, not to mention other medical reasons. it is the duty of the doctor to correct those factors that resulted in iron depletion or anemia and readmits those donors in blood donation in the future (75-80% of those rejected are readmitted in our centre). summary/conclusions: at our blood centre we have created a program of regular tests (blood tests-physical examination) for all our blood donors. our experienced and well taught personnel offers advice and provides useful information in every aspect of blood donation and more. we have created a friendly environment for all our volunteers with love, understanding and appreciation and believe that this is the only way to keep a constant 'flow' of blood in our region. introduction: an innovative perception for blood donation in a new and evolving environment must focus on specific matters and ideas and adopt in a certain level lifestyles and concerns of society. aims: the purpose of this study is to find methods and ideas that can help blood donation centers throughout our country to create new blood donors, give a motive and inspiration for blood donation by adopting new trends of society and finally accomplish national need. methods: by having a personal interview with many volunteers about their feelings for healthier life, their nutritional habits, daily physical activity, sports, vitamins, smoking, weight, cholesterol levels. we investigated whether they believe that blood donation has, if any role towards a more hygienic life. results: we divided blood donor volunteers according to their age, educational level, and number of blood donations per year. our results indicated that there is a tendency among young educated people to adopt a personal lifestyle that includes consuming healthier food, keeping their weight low close to the ideal, having some kind of personal activity, not smoking, watching cholesterol levels, following doctors advice and concerning seriously about their health. this dynamic group of blood donor volunteers considers blood donation as a contributing factor to well being and donates blood at specific intervals. besides the yearly run lab tests that are done by our blood centre they also seek advice and discuss any matter concerning their health with the blood centre doctor. it appears that they are extremely sensitive in those matters and they seem to appear well informed about issues concerning their health, they also believe that blood donation is part of the plan they have to keep fit and being well. in our blood centre we encourage this belief and we also provide information concerning this new trend towards healthier habits. summary/conclusions: this approach has already shown some positive results in our blood centre as many people especially young educated women have joined our blood donorship program and donate blood at scheduled intervals. in order to achieve our goal which is to raise the percentage of blood donors in the region we have to be flexible, innovative according to new habits and lifestyles. we have to move with society and modernize the way we attract various groups of people. blood donation against prejudice as saltamavros*, s dimitrakopoulos † , v zacharaki*, p giannaros*, s markou* and p tseliou* *st. andrews hospital patras greece, patras, † pyrgos general hospital, pyrgos, greece introduction: in order to achieve a greater population to be admitted in blood donation we have to provide information concerning any obscure issues that presents in selecting donors. to examine the accuracy of hb measurements obtained by the noninvasive clinical device, as compared to values detected by standard methods, (cell-dyn 1600, abbott laboratories, usa), in a blood donor setting. methods: the nbm-100 device utilizes a finger base sensor using occlusion red/near-infrared spectroscopy (o-rnirs) to detect and analyze the hb/hct levels. the clinical trials were conducted at two blood donor centers (israel and usa). studies were carried out on a group of 159 subjects (86 females, 73 males) aged 19-64. subjects were healthy volunteers who had come to donate whole blood or aphaeresis components. after obtaining informed consent, hb/hct levels of all the study volunteer participants was tested non-invasively, using the nbm-100 device, followed by a venous blood sample. additionally, the usa center tested a capillary blood sample using the hematastat hct measurement device (71 donors). hb levels were considered normal when readings were equal to or >12.5 g/dl. results: venous hb measurements ranged from 9.9-16.2 g/dl. the mean nbm-100 hb level was 13.20 ± 1.35 g/dl, only 0.28 g/dl lower than the mean hb result obtained by venous sampling, which reached 13.48 ± 1.22 g/dl. the standard deviation of the difference between the invasive and noninvasive hb readings was found to be ±1.04 g/dl. the mean absolute error (mae) of their difference was 0.81 g/dl. when checked against the cell-dyn in the usa center, where 21 subjects had hb of 12.5 g/dl or lower, the nbm-100 and hematastat devices showed comparable sensitivity results. the nbm-100 using o-rnirs is a promising noninvasive technique for hb screening in blood donors. the device is easy to use and agreeable for both blood donors and personnel. the technique reduces the need for the invasive finger prick or venous blood sampling, thereby enhancing safety, reducing costs, and improving the experience of blood donation. the effect of short-term, temporary deferral on blood donor return rates and subsequent blood donations background: blood donors are deferred for numerous reasons. some deferrals like intravenous drug use, male homosexual contact or certain positive test results are permanent. the majority of donor deferrals, however, are short-term temporary deferrals (sttds) that are resolved in a matter of days, weeks or months, after which time the person is again an eligible blood donor. the effect of sttds on blood donor return rates and subsequent blood donations is studied. materials and methods: donors given sttds during the 15 december 1999 to 15 march 2000 were computer-matched with non deferred donors on the basis of age, sex, and donation date (case group: 804 donors -control group: 295 donors). computer records were evaluated during the next 3 years (21 march 2000 to 21 march 2003 to determine donor return rates. significance for comparison between the two groups was based on chi-square analysis. results: the most common reasons sttds were elevated blood pressure (52%), deferred for medication (29%) and colds and/or sore throats (4.2). non deferred donors were a little more likely than donors with sttds to return over the next 3 years (36.6% vs. 34.8% pv = 0.0001) and non deferred donors donated more whole blood units. . according to ethnic structure, women -ethnic macedonians donate blood in largest numbers -1096 (14%), while all other ethnic groups are present with only 3%. the most prevalent is the group of adults aged 41-50 (358 -27.5%), with high school education -800 (61.4%) and mostly those who donated blood 1-5 times (719 -55%). conclusion: having in mind that 50% of the population in macedonia is female, the obtained results reveal a significant, yet insufficient participation of women in blood donation with 17% in relation to the total number of blood donors surveyed in the period 1999-2003. this is due to insufficient motivation and education of women from all ethnic groups especially those from the younger population and with elementary education. incorporating them in education and organization would contribute to their more extensive participating in blood donation. comparison of serum beta 2-microglobulin (b2-mg) between hbsag positive donor and healthy control f tarabadi*, m shaeigan*, g babaee † , a talabiean* and m khadir* *iranian blood transfusion center, † tarbiat modarrs university, tehran, iran background: beta 2-microglobulin (b2-mg) is a low molecular weight protein (11 800 daltons) and found in all biological fluids it is light chain of histocompatibility class1-human present on the most membranes of cells. in the hepatitis infection the viral antigen presentation on the hepatocyte in the presence of class 1-hla antigen plays a role in the elimination of the virus. method & samples: beta 2-micro globulin was measured in serum drawn from 45 hbs ag positive blood donors include 5 (11.1%) female and 40 (89.9%) male in age between 17-56 years, and 50 healthy 16 (32%) female and 34 (68%) male in the same age we detected serumic b2-mg by enzyme immunoassay (ela). results: our studies showed b2 mg level increased in 7 (15.6%) hbs ag positive donor that was significant differences with healthy control (p = 0.0001). conclusions: it seems that serum b2mg is a good marker for hbs ag replication. the role of b2mg in monitoring of response therapy needs to be more evaluated. and 1635 (0.8%) were contributed by vd, rd and dd respectively. over the last 5 1 / 2 years, voluntary donations have shown a rising trend from 54.3% to 60.9%, where as rd (44.9% to 38.4%) and dd (0.9% to 0.8%) have shown a declining trend. the percentage of female donors was maximum in voluntary group as compared to rd and dd (11.01% vs. 2.02% vs. 6.17%) respectively. the rates of all tti markers reactivity were significantly higher in rd as compared to others donors. the hbsag and anti hcv reactivity in vd and dd is comparable (0.84% vs. 0.85% and 0.45% vs. 0.49%). hiv antibodies was found more frequently in vd as compared to dd [0.15% vs. 0.06% (p < 0.05)] whereas, vdrl reactivity was lower in formal as compared to latter [0.26% vs. 0.37% (p < 0.05)]. conclusion: voluntary blood donation has shown a rising trend over a last few years, thus highlighting efficient donor motivational strategies. these strategies need to be strengthened to increase the female donor base. the safety of dd is equivalent to vd when the rates of tti are compared. thus, dd should be advised to donate blood regularly as voluntary blood donors. blood safety depends on a number of factors. the chain of safe blood starts with the donor. one of the procedures for obtaining safe blood for transfusion is the medical selection based on the completed questionnaire and the possibility of self-exclusion from the process of blood donation, the medical history of the potential donor and the medical examination. donor selection consists of two sets of information necessary for protection of the blood recipient as well as the donor himself. aim: to present the most frequent reasons for declining volunteer blood donors. material: the materials used for analysis were the questionnaires completed by all the potential blood donors at the transfusion department of the medical center in strumica as well as the record books of the blood donors which contain the results of the analysis we make for the potential donors. these donors donated blood in the period between 2001 and 2003. results: during this period 4129 people volunteered to donate blood, out of which 3940 were allowed to donate blood, while 189 were declined. out of the total number of blood donors 3313 were male and 627 female donors. the reasons for declining potential donors were the following: 42.3% had low levels of hb, 22.36% were taking antibiotics, 9.37% were ill, 7.83% had low blood pressure, 6.75% had high blood pressure, 11.39% for other reasons. conclusion: donor selection and their care on one side and obtaining safe blood for transfusion on the other side entails obligatory organized medical control. the obligatory completion of questionnaires, the medical examination of the potential donor and their self-exclusion as a result of the feeling of personal responsibility as well as the obtained information are very important for the selection of quality blood donors and obtaining safe blood for transfusion. questionnaire on 2700 subjects-students, their knowledge and motivation on blood donation f vladareanu, a bugner and s sirian national institute of heamatology transf, bucharest, romania the research theme of this questionnaire is as follows: 'what is the level of knowledge and of motivation in the non-remunerated and voluntary blood donation at students?' we also tried to see the practical implications that this study will have and how it will influence the knowledge in this area. the purpose of this questionnaire was not dissimulated. the general theme of the knowledge and motivation on blood donation had been studied before through two big questionnaires applied in 1987 and 1992, but the general population was their target. students had never been an investigated lot up to now. the hypothesis referring to this problem is as follows: students are not informed either on the act of donation, or on the crisis of blood. 1. the lack of information is a first cause of the indifference of the studied lot towards the idea of donation. 2. the lack of motivation of the studied lot is another cause. the questionnaire was applied on a 2700 lot of students from seven different cities: bucharest, iasi, constanta, cluj, sibiu, brasov, timisoara. the number of the questions was limited to 22, which we consider best for a questionnaire applied on the street or at college. as a conclusion, we can say that a passive-defensive attitude towards the blood donation was revealed after this questionnaire. not knowing the issue caused by their lack of information sometimes determines indifference at the statement of the subject. on a general dissolution environment of the responsibility of the youth, the donation problem is not in their aria of preoccupations, the general attitude being of non-involvement for the moment, at this idea which is not yet in every individual conscience and which is normally administrated at an institutional level. the donor data and the details of blood application of the north west transdanubian region of hungary k vörös*, c bercsényi † , o petró † , r jáger † and e miskovits ‡ *hungarian national blood transfusion s., györ, † blood bank, tatabánya, ‡ headquarters hungarian n.b.s., budapest, hungary the ongoing fundamental reorganization of the blood service began on the 01.07.1998 in hungary. as the consequence of reorganization till 01.07.2000, 28 blood banks had been established instead of 63 existing before, under direction of the hhnbts. the working profile of the 6 regional blood centers and 22 local blood banks will be changed step by step. virus screening, blood group serology and processing will be made in the 6 regional centers. one of the regions is the 'north west transdanubian region' (nwtr, city györ as the center, with about 930 000 inhabitants and 8000 hospital beds). 3 local blood banks (tatabánya, sopron, and szombathely) are belonging to nwtr. the regional center and the local blood banks provide the labile blood products and high level clinical-transfusion service (cross-matching, antibody screening, outpatient immunhematology investigations, etc.) for the hospitals. annually 56 000 donors donate blood in this region. this donation activity covers about the 6% of all inhabitants. the acceptance ratio of the donors is good (6-8% of the donors were deferred). there are 14 hospitals in our region. the regional demand on rbcc is 30-45.000 u/year, on ffp is 5.500-7.000 u/year and on pc is 8-10.000 u/year. the poster shows the donor data and the details of blood application of this region since 1999. p-054 implementation of 2rbc collection using haemonetics mcs ® +: medical staff training, donor recruitment and acceptance g woimant, c fretz, d puydupin, e pélissier and jl beaumont efs ile de france, paris, france background: single donor 2rbc collection is an approved apheresis technique in france. aims: our goal was to evaluate the implementation of 2rbc collection in our center in terms of donor recruitment and acceptance, as well as medical staff training and adaptation. methods: donors were selected according to the french requirements for 2rbc collection (weight ≥ 65 kg, height ≥ 165 cm, hb ≥ 13.5 g/dl, ferritin ≥ 20 ng/ml for repeat 2rbc donors). all personnel were trained on adequate communication with donors. eligible donors were contacted by mail, by phone or during pre-donation interview. among the 102 recruited donors, all donors were male, 63% were regular whole blood donors, 36% were regular whole blood or apheresis donors and 3% were new donors. the medical staff was trained on 2rbc collection with the sdr protocol and disposable set ln 948pf on the mcs ® +. most of the medical staff was already used to autologous rbc donation with similar apheresis devices. blood samples were taken from donors pre-and post-donation, as well as 2 to 4 months later for those returning for a subsequent donation. donors were asked to fill out a post-donation survey for assessing donor comfort and information. results: donor profile and clinical follow-up are summarized in table 1 . six percent of the donors had a ferritin level below 20 ng/ml; these donors were regular whole blood donors. the collections were well tolerated and no changes in vital signs were noted. four reactions were reported: 2 hematomas and 2 citrate reactions. no reaction was observed post-donation and hemoglobin levels measured before next donation were back to normal. the technique was easily implemented by the medical staff and fitted well in the existing blood center processes. the medical staff as well as the donors found collection duration short (average of 35 min). the results of the survey were very favorable as more than 95% of the donors considered their donation and the information they received as satisfying. most of them agreed to donate again and several actually donated twice during the evaluated period. conclusion: the implementation of 2rbc collection in our center, using haemonetics mcs ® +, was successful in terms of ease of use of the technique, as well as user and donor acceptance. we now plan to evaluate donor loyalty in the longer term. risk from first-time blood donors e zhiburt, s golosova and p reizman federal blood center, moscow, russian federation introduction: each third dose of whole blood in russia is donated by first-time blood donor. there are two reasons for attention to this kind of donors: (1) possible risk of infectious disease in seronegative study; (2) possible risk of donation for person with contraindication. aim of the study: we investigated role of regional deferred donors registry (rddr) in by first-time donor selection. methods: moscow rddr includes parts: hiv, viral hepatitis, syphilis, tuberculoses, malaria, drug users, psychiatry, 60 days after blood donation. rddr was complete and our center began actively work with it since last year. each donor has to be registered in rddr and automatically checked for deferral reason. effectiveness of rddr was investigated. results: first-time donors donate less than 10% blood in our center. about a quarter of them are deferred before possible donation. part of donors deferred by rddr has been significantly increase in 2003 (c2 = 19.6; p < 0.001) at the expense of seropositive people. conclusion: rddr is effective for blood donor selection and decreases necessity in laboratory screening. first-time blood donors have to be examined before blood donation. if they have not contraindications, donation can be performed up to 10 days before examination and screening. the double unite platelet production is important especially if the relatives of patient find the donors. we evaluated the effectiveness two apheresis machine for platelet collection. in our blood bank, one fenvall amicus and one cs3000+ apparatus were used for platelet apheresis. 1531 apheresis were performed between 2/12/2002 and 13/2/2005. including criteria of donors are that estimated process time is smaller than 90 minute and estimated postapheresis platelet count is higher than 100 ¥ 10 9 /l. donors firstly was enrolled to amicus. if amicus was busy, then it was enrolled to cs. the properties of our donor populations were given in blood and plasma cell components are obtained either by traditional manual method from whole blood or by apheresis. modern medical treatment is based on transfusion of deficient components such as erythrocytes, leukocytes or plasma proteins. this involves new solutions to achieve higher yields and better quality of such components. the aim of our study was to estimate the efficacy of blood cell separator cobe trima in obtaining platelet concentrates (pcs) as compared to older-generation cobe spectra blood separator. apheresis procedures were performed on both these blood cell separators. the quality of platelet concentrates was tested during 5 day storage period (see table below ). we have tested the effect of apheresis procedure on donors and estimated the operating comfort of both separators. the tolerance of both separators was satisfactory except for more frequent hypocalcemia when trima separator was used. most donors were more satisfied with trima procedure because of single venipuncture although it involved special donor selection (good vein access). in general we may say that trima is undoubtedly a more modern and more friendly separator. however, cobe spectra may continue to be used with success especially when a more versatile cell separator is necessary (leukocyte concentrates, peripheral blood stem cells or therapeutic apheresis). methods and results: tls (587 procedures on 67 patients) were used successfully in patients with acute or chronic leukemia with hyperleukocytosis (white cell count >100 ¥ 10e9/l or blast count >50 ¥ 10e9/l) when high cell count would promote leukostasis with vascular occlusion in the microcirculation. performed tl procedures were rapidly reduced both the white cell count and the whole blood viscosity. average fall in white cell count after treatment was 73.3%. tp-treatments (186 procedures on 32 patients with symptomatic thrombocythemia and/or platelet count higher than 1000 ¥ 10e9/l) were applied in order to prevent the development of 'thrombotic-hemorrhagic syndrome' . the tps performed resulted with rapid platelet counts reduction (84.3% in average) and with clearly noted clinical improvements, subsequently. tes (429 procedures on 196 patients) were performed using manually technique in patients with 'cellular hyperviscosity syndrome' induced by high red blood cell count. it was shown that te procedures resulted to red blood cell number lowering and decreasing of blood hyperviscosity. average fall in hemoglobin and red blood cell concentrations after te treatments was from 20.5% till 35.7%. rbcx treatment (8 procedures on five patients with malaria and two with severe aiha crysis) was performed on an urgent basis, particularly when clinical symptoms indicate life-threatening situations and resulted with rapid and significant reduction of concentration of unwanted pathogen affected rbcs and summary/conclusion: the effects of tcs depended on the nature and stage of the basic hds, of adequate selection of patients and of timely applied apheresis. rapid cytoreduction is obtained justly in patients with excessively high cell count, and this effect did not associated with bone marrow remission. thus, tc should be looked upon as adjunct to the standard treatment of different cithemias, but not as replacement therapy. the present study indicates that the best therapeutic effects were obtained by rbcx. were carried out with continuous flow blood cell separator cobe spectra and all patients underwent large volume leukapheresis (lvl). in all procedures, a blood warmer was connected to the return line and a continuous calcium infusion was administered preventively. six patients, who were under 25 kg body weight, had the extracorporeal circuit primed with irradiated, filtered packed red cells diluted with 5% albumin solution. seven children had vital signs and ecg continuously monitored during the procedure. results: each patient underwent a median of 2 collections (range 1-6). the inlet blood flow ranged between 16.0 and 95.4 ml/min (median 54.5 ml/min). the median blood volume processed was 11 754 ml (range 2700-22 500). leukapheresis lasted a median of 240 min (range 120-270). the median total nucleated cell yield was 11.86 ¥ 10e8/kg (range 1.94-21.21), mononuclear cell (mnc) yield was 6.01 ¥ 10e8/kg (range 0.97-12.73) and cd34+ cell yield was 3.5 ¥ 10e6/kg (range 0.19-28.01). the median of mnc collection efficiencies was 56.11% (range 12.34-158.09). in 9 (39.1%) patients, in only one apheresis procedure more than 2 ¥ 10e6 cd34+ cell/kg were collected. during 6 (9.09%) procedures patients had experienced apheresis-related side effects. the citrate-induced reactions were most commonly observed. the reactions were mild and cessation of collection was required only in one case, because of catheter related complication. mild sedation was required only in few very small children. post-donation platelet count was less than 30 ¥ 10e9/l in 3 cases and these patients required platelet transfusion before subsequent procedure. our results show that lvl in pediatric patients is relatively safe procedure, well tolerated and with a very low risk of serious adverse events. close monitoring of blood counts, especially platelets, between pbsc collections is necessary. the cessation of procedure was required in only one case and no life threatening side effects occurred. neonatal alloimmune thrombocytopenia (natp) caused by fetomaternal mismatch for human platelet (plt) alloantigens (hpas) worsens approximately 1/2000 pregnancies and can lead to a serious bleeding diathesis, intracranial hemorrhage (ich) and sometimes death of the fetus or newborns. we describe the successful management of a 37-year-old pregnant woman, alloimmunized to the hpa-1a (p1a1, zwa) antigen, with a history of two previously children with severe thrombocytopenia and ich. the pregnant woman was at her terminal pregnancy and was suddenly admitted. to evaluate the risk of ich in the fetus, cordocente was performed to demonstrate fetal thrombocytopenia (plt 4.000/mmc). to ensure a rapid provision of compatible negative-antigen platelets, we decide to collect platelets from the mother using apheresis. plateletapheresis was performed using com.tec separator, fresenius. blood processed was 1.100 ml in a short time procedure (35 minutes). no significant adverse effects were observed in the mother and fetus, during and after the procedure. platelets collected (1.5 ¥ 10e11) were transferred to the preparation set and plasma was removed after centrifugation to resuspend the platelets in octaplas ab. then we separated the platelets into two units containing 0.7 ¥ 10e11 each. the day after the donation, the mother gave birth to a girl by caesarean section. after the transfusion, the plt account increased from 4.000/mmc to 35.000/mmc and after a week the child had plt 75.000/mmc without hemorrhagic complication. according to the literature data and our observations of the patients, there are changes of the hemostasis system indexes in the most patients with the endogenous intoxication syndrome and immune disturbances. in the number of cases medicamentous therapy appears to be not enough to normalize the changes, but it is especially important for pregnant women and women in childbirth, because on the background of these disturbances different complications of pregnancy and postnatal period take place. the aim of our study was the substantiation of plasmapheresis using in complex therapy of purulent inflammatory complications in obstetrics and immunoincompatible pregnancy with hemostasiologic disturbances. 10 patients with hemostasis system disturbances: one woman in childbirth with exacerbation of chronic pyelonephritis, who had in the first 24 hours some signs of hypocoagulation on the background of permissible blood loss (prolonged coagulation time up to 20-30 minutes with episodes of its absence on the background of the normal indexes of general coagulogramm, quantity and function of thrombocytes and the dilute fibrin monomer complex level in 7 times higher than the norm) and nine pregnant women with the perinatal losses in anamnesis severed by the pregnancy (threat of abortion, places of fetal egg detachment). these women were examined, the following was revealed: the high antibody titer to chorionic gonadotropin, parameters of partially activated thrombin time were higher than the norm (47-52 seconds), thrombin time (18-20 seconds), the dilute fibrin monomer complex (8-12 mg%), coagulation time (15-20 min). in all these cases the conservative methods of treatment (antibacterial, hemostatic, hormonal therapy) were effective for a short period of time and they didn't succeed to correct the given parameters of hemostasiogramm. the discrete centrifugation plasmapheresis was included in the complex of medical treatment. the woman in birth 5 operations were done, in the programme of plasma replacement during the first two plasmapheresis procedures donor fresh-frozen plasma was included. six pregnant women on the given stage one course consisting of 3 plasmapheresis procedures for plasma replacement with crystalloids was done, the volume of the removed plasma was 25-30% of the circulating plasma volume. three pregnant women before delivery were required two courses of plasmapheresis more consisting of 3-4 procedures each. the system heparin was not used. in all patients already after the first procedure of plasmapheresis the normalization of hemostasis indexes was marked, that allowed to prolong pregnancy, to prevent the coagulopathy bleeding and the development of disseminated intravascular syndrome. four women are discharged from the hospital, the other patients are observed with progressive pregnancy. thus, the using of discrete centrifugation plasmapheresis is effective at the signs of hypocoagulation in patients with isoimmunisation with fetal antigens and infectious pathology, and is the reserve in prevention and treatment of obstetric complications. extracorporeal photochemotherapy: an alternative therapeutic approach to control graft versus host disease after allotransplant with reduced intensity conditioning regimen c del fante*, c perotti*, gl viarengo*, p bergamaschi*, p pedrazzoli † and l salvaneschi* *irccs policlinico s. matteo, pavia, † ospedale niguarda cà granda, milano, italy background: extracorporeal photochemotherapy (ecp) can be defined as an immunomodulatory therapy that demonstrated to be efficacious in treating patients affected with graft versus host disease (gvhd) after allotransplants for oncohematological diseases. reduced intensity conditioning regimen (ricr) for allotransplant is a relatively new practice in patients (pts) ineligible for a conventional myeloablative conditioning regimen. the use of immunosuppressive therapy (ist) to control gvhd is limited for the high risk of developing infections and disease relapse due to the strong reduction of graft versus tumor (gvt) effect. aims: to evaluate the effectiveness and safety of ecp in treating pts affected with gvhd post rcr and the possibility to taper, at the same time, the ist. methods: 6 pts (5 females, 1 male), median age 47.5 years (25-63), affected with agvhd grade ii (1) and extensive cgvhd (5) gtx with median total granulocyte doses of 55 (21-150) ¥ 10 9 per gtx corresponding to 1.46 ¥ 10 9 granulocytes/kg in children and 0.58 ¥ 10 9 granulocytes/kg in adults. the wbc counts increased from baseline values of 0.2 (0-0.5 ¥ 10 12 ) g/l for both pediatric and adult patients to peak values of 3.8 (0.4-18.2) ¥ 10 12 g/l (children) and 0.9 (0.3-9.4) ¥ 10 12 g/l (adults) at one hour after gtx and to 1.4 (1.1-13.6) ¥ 10 12 g/l (children) and 0.4 (0.3-8.6) ¥ 10 12 g/l (adults) at 24 hours after gtx. in 42 out of 54 patients (80%), the crp levels significantly declined (60 (11-91)%; p£0.001) during the granulocyte transfusion period; in almost all cases (39/42; 90%) after the initial or 2nd transfusion. thirty-eight patients (71%) were alive at day +30 after termination of neutropenia and gtx. patients without crp response to gtx (6/9, 66%) and patients with severe viral infections 6/7 (86%) were not among the day +30 survivors. background: in recent years, the use of platelet concentrates obtained from single donors by automated apheresis has grown steadily. plateletpheresis donation is considered to be a safe procedure with modern instruments. so far, no studies have identified donor or procedure specific factors that may be associated with serious adverse events. aim: to evaluate the incidence of adverse events during plateletpheresis procedure, over a five-year period in our hospital. materials and methods: eight hundred single-needle plateletpheresis collections were performed by using two automated intermittent-flow cell separators: 700 of them with mcs3p and 100 with mcsplus (haemonetics), according to automatic standard protocols a3p and ldplp, respectively (with the collection of an additional plasma unit). acd-a was used as the anticoagulant in all apheresis procedures (acd-a: blood ratio was 1 : 11). most of the donors (84% men and 16% women) were patient´s relatives. half-hour before the initiation of the procedure, 250 mg of calcium (1 tablet cal-c-vita) were administered to each donor. the mean platelet yield was 3.2e11 /unit. the overall rate of the donor related adverse events was 3.4%. feeling faint was the most frequent event, which was occurred in 1.62% of donations. hypotension and citrate related rates were 0.77% and 0.73%, respectively. all citrate related symptoms were only transient perioral paresthesias, which were relieved by slowing the i.v. rate, without additional administration of oral calcium. donor unconsciousness was the only observed severe event, the rate of which was 0.25%. other adverse events were venipuncture related (2.37%), machine related (2.7%) and miscellaneous complications (1.25%). (1) plateletpheresis using the mcs3p and the mcsplus automated cell separators is a safe procedure, with a low risk of serious adverse effects. (2) with the used acd-a-to-blood ratio (1 : 11) satisfactory platelet concentrates were obtained with very low incidence of citrate-related events. (3) the peros administration of calcium before the initiation of the procedure, probably lowers the rate and the severity of hypocalcemia symptoms. quality assessment of ffp collected as a byproduct of plateletpheresis 1. from the donors immediately with the initiation of the procedure (citrated whole blood) and 2. from the final platelet concentrates after one hour rest at room temperature without agitation. in vitro platelet response to the aggregation-inducing agonists adp, collagen, ristocetin and arachidonic acid was investigated by means of an aggregometer (pap-4c, bio/data). results: there were no significant differences between the groups of donors with respect to age, sex, smoking habits, preapheresis wbc and plt counts and hemoglobin concentration, as well as in the harvesting time between the two cell separators. our findings are shown in the following table. mildly decreased response to all agonists was observed (mainly to adp and arachidonic acid) in the samples taken right after the initiation of the procedure, in both groups. platelets from the final component showed a further slight decrease in response to adp, which was more prominent in the mcs3p device (p = 0.025). on the contrary, an increase in platelet response to the other three agonists was observed in both devices, which, however, was statistically significant upon collagen and ristocetin stimulation. conclusions: reduced response to aggregation stimuli is possibly caused immediately with the initiation of the apheresis process. literature reports regarding further platelet traumatisation due to the procedure, are rather conflicting. in our study, such traumatisation was observed only in the case of adp in the mcs3p obtained collections and this could be correlated with the technological differences between the two devices. recovery of platelet aggregability, as it was expressed by the upregulation in platelet response in the other stimuli, could be attributed to the resting period and seems not to be affected by the timing of the leucodepletion procedure. background: lupus erythematosus often is accomplished with severe symptoms, such as polyarthritis, nephritis, pericarditis or dermal alterations. in pregnancy cytostatic therapy affects gestation. on the other hand the course of disease can be refractory to corticosteroid therapy. elimination of autoantibody and immuncomplexes by plasmapheresis could be an efficient way to amend the severity of symptoms. a 24 year old pregnant woman in the 24th gestation week with systemic lupus erythematosus showed severe symptoms like polyarthritis, nephritis and pericarditis. treatment was initially 1 mg/kg bw prednisolone for 3 weeks and subsequently 8 mg/kg bw for 2 weeks. plasmapheresis was applied daily in the beginning and continued depending on the condition of the patient ( table 1) . the eliminated plasma was substituted by fresh frozen plasma. the medium volume was 2.951 ml per apheresis. after day 11 plasmapheresis treatment was suspended to avoid problems with coagulation and was followed by a cycle of 3 immunoabsorptions to eliminate circulating immuncomplexes. results: prednisolone therapy alone brought no effect even after changing to high-dose treatment. a significant amelioration of all symptoms could be observed after the first plasmapheresis. good condition of the patient remained stable over the period of daily plasmapheresis for 4 days. intermitting apheresis treatment for one day lead to a significant aggravation of symptoms. apheresis no. 6 again lead to a recovery of the patient which held on until day 11. conclusions: treatment of systemic lupus erythematosus in pregnancy especially in combination with resistance to corticosteroid therapy, is an effective therapy to ease severe symptoms such as polyarthritis, pericarditis and nephritis. exposure to cytostatic drugs can be avoided and therefore the impairment of the fetus can be reduced. background: the collection of mnc represents the first step of photopheresis procedures and could be of critical importance in achieving a therapeutic goal. in this work we compare the cell yield of two collection programs on cobe spectra device: the mnc versus the autopbsc program using the ecp procedure modified by andreu. methods: 39 procedures were carried out with mnc program and 19 procedures with autopbsc on 4 patients with cgvhd. both hemoglobin increased from 35.9 ± 5.7 to 204 ± 100 mg/tu, k+ from 1.7 ± 0.7 to 44.8 ± 13.9 mmol/l, level of glucose decreased from 26.65 ± 2.06 to 9.75 ± 2.06 to 9.75 ± 0.91 mmol/l, ldh from 1.81 ± 0.48 to 6.24 ± 1.89 ukat/, lactate from 2.77 ± 0.47 to 22.72 ± 14.02 mmol/l, ph from 7.50 ± 0.14 to 6.74 0.08. the volume of apheresis units was lower than wb-rbc, the leucocyte count was normal in all units. the rbc loss by filtration was 24.9 ± 3.1 ml/tu and was lower than at wb-rbc. in apheresis rbc there were the differences in hb and ht value between the day of storage 0 and 40, in wb-rbc there were no differences. during the storage period we found no differences in k+ increasing value and no change in ph value between apheresis rbc and wb-rbc, the increasing of lactate was higher in wb -rbc, increasing of ldh correlated to hemolysis. the plasma hb value increase was higher at apheresis rbc in contradistinction to literature. hb and ht correlation in apheresis units according to predonate value in donors was lower than at wb -rbc. the method is a useful alternative to conventional whole blood donation, we get rbc units with high standard of quality and low correlation according to predonate hb and ht value in donors. acknowledgement: the study is supported by grant iga ministry of healthy cr n. nr/8015-3. background: in life-threatening exacerbations of sle a satisfying efficient therapy is lacking. despite intensive immunosuppressive therapy some patients are resistant or contraindicated to conventional treatment. in particular circulating antibodies and immune complexes play an important role in the pathogenesis of sle and mctd. an extracorporeal removal of these pathological substances may be effective in the treatment of active disease. methods: five patients with severe therapy-resistant sle/mctd underwent immunoadsorption onto protein a. blood was drawn from patients by using a jugular catheter or a peripheral intravenous catheter. anticoagulation was performed with acd-a and heparine or acd-a and r-hirudine. plasma was separated by centrifugation. the 1.5 to 2-fold total plasma volume was treated in every immunoadsorption. the columns were floated with a maximal plasma flow of 20 ml/min. the procedure was carried out every second day. additionally supplementary intravenous immunglobulin therapy was given only once. results: remission of the disease was achieved in four patients. see table below . conclusion: pa-ia is highly effective regarding the elimination of autoantibodies and circulating immune complexes, might induce a remission in patients with sle/mctd. it is an acceptable alternative treatment option in patients when other therapies are ineffective or contraindicated. background: purification of bone marrow from erythrocytes is used to prevent early hemolysis in major abo incompatible allogeneic hemopoietic cell transplantations. erythrocyte depletion is strongly recommended to reduce product volume and stem cell purification before storing autologous and even allogeneic bone marrow in order to prevent early hemolysis and dmso toxicity that might develop after thawing. centrifugation, sedimentation with hes, and cell separating devices are methods for erythrocytes depletion. aim: in our center, we prefer to use cell separation device, since it is a reliable method and has a high-yield and risk of contamination with erythrocytes is low. success of the process is retrospectively analyzed for high and low volumes. method: erythrocytes depletion of bone marrow harvest was done in hemapharesis unit with cobe spectra device in the last five years in 20 cases with bone marrow volume over 750 ml, and 17 cases with bone marrow volume under 750 ml. fifteen of these cases were allogeneic, and 22 were autologous procedures; a software uploaded with cobe pbsc coll vers 5.1 and (catalog no: 777-006-000) set was used in the procedure, and at the same time, double bag system with intermediate connectors were used to prevent re-circulation (catalog no: 777-006-300). results: the mean volume reduction was 90.48% (83.81-93.54) for volumes over 750 ml, and 89.79% (84.03-94.02) for volumes less than 750 ml. regarding the success of the procedure no statistically significant difference was found between procedures with high and low volumes. no complication developed related to the device or product, and waste bag never had to be re-used. in none of the patients early massive intravascular hemolysis was observed. conclusion: erythrocyte depletion and volume reducing with cell separation device is a reliable method. this process is successfully applied with high volumes (over 750 ml); and in low volumes as well for reducing erythrocytes, and gain of mononuclear cells and cd34+ cells. platelet concentrates obtained by apheresis procedure-correlation between the initial count and the final concentration v srejic*, g bogdanovic*, z garic*, n vavic* and b balint † *national blood transfusion institute, † military medical academy, belgrade, serbia apheresis team of the national blood transfusion institute processed and classified data of 100 donors who donated platelets by apheresis procedure from january till april 2004. procedures were performed in accordance with the ldplp protocol, using haemonetics mcs+. initial donors' platelet count and the absolute platelet concentration in the final preparation were followed, as well as red blood cell and leukocyte contamination and the volume of the processed blood. donors' initial platelet count was not less than 191 ¥ 10 9 /l and the volume of the processed blood was not less than 2300 ml. according to histogram, the most frequent donors' initial platelet value ranged from 210 ¥ 10 9 /l to 308 ¥ 10 9 /l (70%). final concentration of the samples of 100 tested donors ranged from 3.9 ¥ 10 11 to 6.1 ¥ 10 11 in the average volume of 400 ml. regression analysis demonstrated that there was a correlation between the initial donors' platelet count and the obtained final concentrate. student's t test showed p < 0.0011. leukocyte contamination of the final concentrate prepared without the filter ranged from 0.0 ¥ 10 9 /l to 0.4 ¥ 10 9 /l. presence of red blood cells in the final concentrate ranged from 0.01 ¥ 10 12 /l to 0.02 ¥ 10 12 /l. p-077 therapeutic apheresis (ta) in croatian hospitalsadherence to respectable guidelines z zivkovic*, b jeren strujic*, s boras † , i bojanic † , b golubic cepulic † and z ivankovic † *clinical hospital dubrava, † clinical hospital center zagreb, zagreb, croatia introduction: besides considerable resources, ta requires high costs and risk for patients. therefore, indication for ta often considers interests of patient, hospital and requesting physician. the most respectable guidelines for the implementation of ta were defined by aabb and asfa, classifying total of 58 diseases into 4 categories (ctg), ranging from 'standard therapy' to 'lack of efficacy' . the objective of this study was to determine indications for ta performed in 6 croatian hospitals in the period 2001-2004, respecting aabb/asfa guidelines. results: during the observed period in croatia, ta was performed in 777 patients suffering from 26 various diseases. in 514 (66%) patients ta was performed by membrane filtration, while in 263 (34%) separation by centrifugation was used (table 1) . according to the 4 ctg, s of the aabb/asfa guidelines, ta was performed in 573 (74%) diseases from ctg i, 126 (17%) ctg ii, 54 (6%) ctg iii, and 25 (3%) ctg iv of patients. the most frequent indications included in ctg i were: myasthenia gravis (32%), collection of pbpcs (31%), sy. guillain-barré (17%), and plasmacytoma (6%). in ctg ii frequent indications were: poisonings (35%), systemic lupus erythematodes (30%), and rapidly progressing glomerulonephritis (14%), and in ctgs iii and iv: cytoreduction-polycythaemia (52%), thyroid storm (15%), gvhd (10%), and reumatoid arthritis (8%). (1) the time spent for resolving h 52/40, (2) mtp 0/0, (3) discarded blood units 10/4. iii group: wrong data input 5/4, donor replacement 4/1, marking errors 3/10, error in determining blood group at the first blood taking 28/13, errors in input medical consulting 9/12 and disregard of prohibitions 2/13. the consequences are: (1) the time spent for resolving h 35/40, (2) mtp 2.5/3.5, (3) discarded blood units 9/18. conclusion: the analysis of errors has showed that the number of errors can be decreased by implementation of corrective/preventative action based on continual education of the staff, appropriate sop, effective organization, qmp for equipment. the conclusion of our study is that reducing the rate of work errors will decrease the waste of material and time, also that will decrease the number of discarded blood units. an iso standard for blood transfusion? background: in our search for an independent, objective assessment at western province blood transfusion service, we were unable to find one single model that met the specific requirements of blood transfusion. we therefore resorted to developing our own model but ask the question: why not have one international standard for blood transfusion? aims: our aim was to have a standardised system for the independent, objective assessment of our blood transfusion service with audits carried out by an internationally recognised body. we wanted a formalised, professional system of accreditation with inspection checklists, reports, certificates etc. methods: having moved away from accreditation by the american association of blood banks in mid 1990's, we evaluated various other options such as inspection by our government department of health or the world health organisation but neither organisation had trained inspectors or systems in place. we also investigated iso 9000 certification but, although this was acceptable on the quality management side, it did not cover the technical parameters relating to blood transfusion. results: we therefore developed our own model for accreditation that consists of three parts: • a quality management section incorporating iso 9000 principles; • a technical section incorporating specifications from the south african standards of practice (we also consulted the european, american, canadian and australian guides); • a laboratory section incorporating iso 17025 parameters (soon to be updated with iso 15189). we then chose to be accredited by the south african national accreditation system, sanas, an internationally recognised institution. once we had written a national accreditation checklist, sanas submitted this to two international accreditation bodies (iaf and ilac) for approval. the system has been in place for three years now during which time we have had four successful assessments. summary/conclusions: in developing our system, we reviewed what was being done elsewhere in the world and it became evident that, although there are great similarities between countries, there p-078 software for the management of the scansystem bacterial detection method the scansystem tm was developed for bacterial detection in blood products. to be implemented in blood banks, a specific software is now available in compliance with blood bank regulation in order to manage sample traceability and data file transfer. the software is divided in 3 main levels: an administrator level to create an application configuration in compliance with customer needs (product bar code characteristics, frequency of the positive controls, manual or automatic data file transfer . . .), a technical level to manage the operators (password, id) and to validate some specific results, an operator level for routine testing. the software assess sample traceability when testing pools of samples from 1 to more than 20. indeed, bacterial detection is performed for pools of 1 to 3 platelet samples and 1 to 20 red cell samples. each sample in the pool is traced through its barcode until the final result. the system is compatible with most of the barcode standard including isbt 128. in addition, the system checks each barcode protecting the sample against duplicate testing. the software assists and monitors the bacterial detection process from the sampling to the end of the test (final result), each step of the procedure is identified through its barcode and at each time, it is possible to know the test status for each sample. for a pool of samples, 2 results can be obtained: 'negative' or 'on hold' for a positive result. for a 'negative' pool, each sample constituting the pool are determined as 'negative' . for an 'on hold' pool each sample constituting the pool must be tested as a single sample and the final result is 'negative' or 'positive' . data transfer may be manual or automatic. a final technical validation is necessary before the transfer through an active selection of results to download. final results are provided in a compliant format for an easy import into the blood bank database. all necessary information are displayed: 'machine id' 'product/sample barcode id' 'date' 'time of transfer' 'operator id' 'result' ('negative' or 'positive'). the main advantage of this software is a continuous check of each step reducing the risk of error in testing. it makes the scansystem tm test compatible with a routine use in blood banks according to the current regulations and quality assurance programs. is no overall consistency. we feel it would be of benefit to establish an international working group to investigate the feasibility of writing an iso standard for blood transfusion. the standard would harmonise quality management parameters based on iso principles and technical/laboratory parameters specific to blood transfusion. minimum technical specifications would need to be agreed upon based on the various standards and guidelines available around the world. this iso standard could then be used for the purposes of certification/accreditation or government inspections. this would ensure global standardisation of world-class best practices. first world countries would be able to achieve compliance and a subsequent step could be the establishment of an international forum to assist developing countries to work towards compliance in the longer term. residual leukocytes in leukoreduced cellular blood products -evaluation by flow cytometry web-based outcome review: do you know how productive your trima® can be? background: web-based outcome review is a new software tool developed by gambro bct for the management and the interpretation of data from the trima and trima accel tm automated blood collection systems. aim: does the interpretation of reports obtained through outcome review lead to an increase in the number of products per run and the overall productivity of the apheresis center? method: run data files (rdf) from trimaᮀ were collected and transferred onto the outcome review server. these rdf do not contain any donor related data that can lead to possible donor identification. the reports were generated on the outcome review website (gambro bct intranet), interpreted by a gambro bct employee and presented and discussed with the management of blood centers. results: a total of 21 different reports can be generated on the website as a pdf file. for this study, 9 reports were investigated. they are: doses per collection, doses per collection trend, platelet collection trend, platelet procedure performance, platelet procedure performance trend, product distribution, average procedure time, procedure time, machine productivity trend. the results obtained for a center can be compared to word-wide, national, regional or to other individual trima devices in the centre (benchmarks). this benchmarking allows the management of an apheresis center to compare the results, to draw the right conclusions and to develop and implement corrective actions. implementing successfully these corrective action plans will lead over time to productivity results that are more in line with the figures that are generally accepted to be the optimal production capabilities of trima. the reports also help to monitor the effects of the corrective action plan over time and to adjust this plan if the results are not in line with the expectations. reaching and maintaining the optimal production capabilities of trima will also increase the net revenue by procedure or decrease the cost by procedure for the blood centre. conclusion: web-based outcome review allows getting more products from the existing donor base by interpretation of the multiple reports and implementing the required corrective actions until optimal production capabilities of trima are reached and maintained. introduction: to examine the cell vitality of packed rbc's during storage several parameters like atp, free hb or 2,3-dpg are used. less kits for the determination of atp are available and they need either a large sample volume and/or are time consuming. here we present the modification of a commercial testkit for a time-and cost saving detection of atp. methods: samples were analysed with (a) 3-phosphoglyceratekinase reaction according to bergmeyer, h. methods of enymatic analysis 2nd. edition. academic press, new york 1965; (b) detection via hplc and c) using a commercial testkit (r. greiner bio-chemica, germany). the atp-kit were minimized from 3500 ml to a total volume of 200 ml and tests were performed in microtiterplates. results: 245 samples were analysed in hplc and modificated commercial testkits, another 20 samples have been examined in all tests. comparison of the results showed no discrepancies in the above mentioned methods. standard curves have been performed (range 5-1000 mm atp) and statistical analysis demonstrated a given linearity (r = 9.97). variability has been calculated as 1.2% (intraassay; n = 25) and 4.6% (inter-assay; n = 70). the hand-on-time calculated for 96 samples has been decreased from 4.5 hours to 30 minutes. at least the costs of atp-determination have been reduced from €3.45 to €0.22 per sample. conclusion: performance of test kits in microtiter format is a fast and rapid method, reliable for high-throughput determination of atp in packed rbc's. background: in order to preserve both blood safety and availability it is mandatory that a minimal amount of blood units would be discarded due to defects in the materials and supplies used for blood collection, or to deviations in blood processing or storage. aims: (1) to monitor the derangements of different materials and disposables used during blood collection and processing, and to study the suppliers' responses and corrective actions taken. (2) to asses the relative contribution of different defective materials (dm) to the need to discard valuable blood components. materials and methods: about 831 000 whole blood units were collected and processed by mda national blood services in [2002] [2003] [2004] . as part of the routine quality control activities, derangements of the dm used were recorded and analyzed. some 40 different types of dm were defined. out of 68 reports sent to the corresponding manufacturers for investigation, 45 responses (66%) were received and analyzed. about 70% of dm were detected during blood collection. manufacturer defects of different materials were the reason for components discard in nearly 25% of cases. conclusion: defective materials are one of the major causes of the infringements of blood collection and blood component preparation processes. analysis and monitoring of the different defects and of the suppliers' responses and corrective actions are essential to improve products' safety and availability. establishment of a network for the exchange of information among international blood centers would enable the blood banking community to compare between different suppliers and to use the documented cases for training of personnel of both the blood services and the manufacturers. such a system may contribute to the improvement in quality of materials used and might lower the discard rate of valuable blood units. results: table 1 analysis and characteristics of t (1) comprehension of the blood bank's processes and the interaction between them and between the processes of the whole hospital. (2) monitor, measure and analyze these processes, in order to improve their effectiveness continuously. (3) implementation of internal and external quality controls for blood and blood products and implementation of appropriate statistical techniques for monitoring their results. (4) identification of interested parties (doctors, donors, patients) satisfaction and taking up the necessary preventive or corrective actions to improve their satisfaction. the qms of our blood bank was certified by tuv rheinland in 16/11/2004 and the scope of the certification is: 'blood collection, testing of infections markers, production of blood components, compatibility screening for blood transfusion and other immunological tests and implementation of therapeutic schemes in thalassaemia patients' . the implementation of the qms based on iso 9001:2000 standards ensures the improvement of services provided by the blood bank and the increase in customer satisfaction, whether donors or patients are concerned. the former enjoy the respect and recognition of their social contribution, while the latter are assured of very high levels of service and health protection. finally, we shall not underestimate the positive impact of qms in the motivation of blood bank personnel. quality becomes integrated both in their professional and personal attitude and allows for achieving increased satisfaction from their work. equipment management in the national blood transfusion service in serbia introduction: new equipment was urgently needed in three blood transfusion establishments (bte) in serbia. equipment was mostly inadequate for core blood transfusion activities, placed in inappropriate facilities, very old without routine maintenance or calibration. also, technical documentation for most of the equipment did not exist, and procedures for equipment management and responsibilities were not defined. further more, coordination on equipment issues with the qa department was not recognized. service funded by the european agency for reconstruction, provided various equipment for the three blood transfusion establishments. the new equipment includes blood collection equipment, centrifuges, refrigerators, incubators, automated testing equipment, genetic analyzer and it equipment of 1.6 million euros value. before the new equipment is installed the bte's agreed to have the national procedures on installation, validation, calibration and preventative maintenance in place. this will ensure that the equipment can be properly installed and validated before use. the project has provided training on validation to the working group (wg) on quality. the wg has created national procedures related to the equipment, including quite new term validation. the same problems in implementation of procedures were present in all three bte's. significant efforts are made to explain to the staff how the equipment has an impact on quality, how to ensure that the equipment does what it is supposed to do, how to be confident that the results obtained are accurate and how important it is to generate records. qa managers played an important role in the preparation of facilities for equipment installation, making plans for equipment layouts, creating documents (master cards, instruction for use) and designing the validation protocols. the same procedures and records enable an exchange of results, comparability, sharing information on what works and what does not work between bte's. the qa managers also prepared an introduction of equipment requirements to the heads of departments, with special attention to the validation process so that they are able to fully understand what is required and why to validate their equipment. results: national standards in equipment management in serbia are set and are being implemented. qa managers carefully managed that the new, numerous equipment, delivered in the short period of 2 months was correctly installed, validated, obtained with necessary documentation, followed by previously trained staff, so that equipment can be considered as controlled. the additional, positive effect is that validation is performed in one establishment for all bte in the country, allowing a more prompt response to problems and presenting of joint request to suppliers, as well as an easier way of monitoring equipment performance of the three bte's. organized equipment management has affect on every aspect of blood activities and finally to the quality of blood and blood components. background: the vista information system (vista tm ) is used in centers in europe as an apheresis management system. with vista tm it is possible to increase productivity, donor comfort and loyalty and therefore simultaneously improving the overall process in the center. aim: a calculation tool (microsoft excel) was developed to evaluate the added value of vista tm . three blood centers in different european countries completed a questionnaire using their local data. summarizing these figures gives us an idea about the impact of vista tm on the daily work and budget of a blood centre. method: the excel calculation tool that was used investigated 3 major areas where vista tm could show added value: improvement of regulatory compliance, increase the efficiency in operations and improve productivity. results: regulatory compliance: the number of infringements decreased, causing a considerable direct financial gain because these events are very expensive to deal with. the time spent on regulatory reviews decreased with a mean value of 35%. operational efficiency: the number of reports is very site dependent: sometimes a report is made for every procedure together with the printout of a blood loss history form. because vista tm tracks all procedure related data, some sites decided to stop printing these types of reports and to go completely paperless. the percent time reduction in reporting is therefore very variable. however the % of errors related to these reports decreased considerably with a mean value of 30%. efficiency in operations was also obtained because of the number of reports that are available in vista tm . productivity, management and process reports allow verifying and correcting the daily operations of the blood center. increased productivity: depending on the center, also an increase in the number of platelet and plasma products collected was detected. the number of product discards caused by infiltration reduced with a mean value of 50%, mainly due to the possibility to have customized and more donor adapted trima settings. the percentage of whole blood donors targeted for conversion was very site dependent (min 0.5%-max 4%). but because with vista any procedure brings between 2.0 and 3.0 products in general, the financial gain was considerable when donors could be converted from whole blood to apheresis. the use of vista allows the apheresis center to work with a reduced error rate and to increase the operational efficiency and the productivity. the financial impact of this has been estimated by the centers between 4€ and 26€ (mean value 10€) per procedure. establishment of national quality system in blood transfusion service in serbia introduction: the production of blood products is a semiautomated process in which the manual steps may be difficult to control and standardize. aim of the study: we introduced a specialised team for the blood production to test if this improved the control of the quality of the blood products. methods: the blood products tested for statistical process control were red cells in additive solution, buffy coat removed, and leukodepleted (ld) platelet pools prepared from 4 buffy coats. the products were collected in t&b triple opti-pac from baxter and the platelet pools were ld using plx-5 filters from asahi and stored in platelet bags from baxter. using control charts, namely x-mrchart, exponentially weighted moving average ewma chart and for autocorrelated stationary data the ewmast chart, we examined if time series of quality control values were in statistical control. if not we examined if autocorrelation and/or differences between the technologists producing the blood products could explain the lack of control. data included approximately biweekly measurements of volume, haemoglobin (hb) concentration, hb/unit, haematocrit and log leukocyte count (wbc)/unit of 352 units of red cells, measurements of volume, platelet concentration and platelet count/pool of 79 ld platelet pools produced by a team of 13 technologists and of 79 ld platelet pools produced by a specially trained team of four technologists. results: log wbc/unit was out of statistical control due to systematic differences between technologists. apparent lack of control of volume, hb-concentration, hb/unit caused by autocorrelation disappeared when the ewmast chart was used. platelet concentration and volume of the platelet pools produced by the 13 technologists were out of control. in that some technologists systematically produced low values. this could be explained by inappropriate handling of the platelet product between centrifugation and separation. systematic differences between the four specially trained technologists could not be demonstrated and they produced platelet pools with a significantly higher platelet count/pool. however, standard deviations of the four technologists differed significantly causing occasional outlying values. conclusion: training and routine in blood production or process automation, and also importantly, feed back to the technologists based on control chart quality control data, is recommended. background: one important principle of the use of blood and blood products is the ability to trace the units from donor to the recipient. this study set out to establish whether or not there was sufficient reporting on transfusions from the hospitals supplied by fort portal regional blood bank in western uganda as a means of establishing sound haemovigilance and look back systems. were reported with no unit number and could not be traced to the patients. there was sufficient reporting on the data requested by the blood bank. these results suggest that it is possible to establish effective 'look back' and haemovigilance systems. capture of data on outcomes and adverse effects will be necessary to fully establish the system. further efforts are required to educate those involved in transfusing blood about the need for adequate and accurate documentation. external quality assessment of blood grouping were misinterpreted as rhd-positive samples without the use of control reagent. rbc phenotyping was made correctly by 79.7% of participants. the remaining 20.3% of participants carried out the phenotyping incorrectly, while false-positive and false-negative results were derived in 8.91% and 11.39% of cases correspondingly. polyspecific human sera and monoclonal antibodies were used for abo, rh and antigens typing. the reason of errors in antigen detection was low quality of reagents. antibodies identification was carried out in six distributed exercises. 75% of participants detected anti-d-k-c alloantibody correctly. the rest of participants did not found alloantibodies or detected their specificity incorrectly. results of testing depended on quality of screening cells. thus, the participants using homemade pooled screening cells had a significant lower detection rate of antibodies comparing with those using diamed ag cell panel. consequently, the results of the first federal external quality assessment scheme show the necessity of improving the quality of red cell reagents produced in russia. in addition, the more appropriate training of staff is required. the importance of iso 15189-quality system in increasing safety of blood transfusion introduction: hadassah hospital transfusion medicine department received on 3/2004 iso 15189-quality system accreditation. an essential element of this standard is the development of a reliable system to identify, document, analyze and correct actual and near miss events and assess the effectiveness of corrective and preventive actions. aim of the study: assessment of events and corrective actions following implementation of iso 15189 quality system. methods: events in the blood bank were identified by the staff, by internal or external audits and by complaints from the wards. all events were recorded and classified into two categories; quality system and technical. the latter were further classified into preanalytical (sample receipt), analytical (abo rh typing, antibody screen and identification, cross matching and phenotyping) and post-analytical (issue of components to wards). all events were graded into 4 levels; 1-most severe, potentially harmful to patient. 2-severe, damage to process and result. 3-moderatly severe, damage to process only. 4-benign, no harm. events were corrected and effectiveness of corrective actions was assessed by monitoring recurrence of the event. results: during the years 2003-2004, 248 events were detected and recorded in the blood bank, they comprised 0.072% of all tests performed (343 595). most of the events were technical. all events were detected before causing harm to the patients. results are summarized in the table. *the percent analytical value is a summary of rates of events per test types included in this category. events detected in the quality system were mainly of severity level 2&4, whereas technical events were mainly of severity levels 2&1. analysis of event recurrence in the quality system revealed that 97% of events were resolved, whereas only 14%-31% of technical problems were completely solved. the main source of event identification and documentation, in the quality system were audits whereas in the technical system, staff members revealed most events. the implementation of iso 15189 quality system provided a powerful means for recognition, analysis and study of patterns of near misses and actual events. understanding the root causes of events enables to choose the most effective corrective and preventive action to control event recurrence. evaluation of the frequency of events confined to the blood bank revealed a very low rate of 0.072%. these results are in agreement with data in the literature. creating a non-punitive, non-stressing open environment, motivates personnel to identify and document events, which are regarded as opportunities for improvement and serve as important tools for upgrading transfusion safety. the explosive use of information technology and the speed with which it has spread into all life activities has created vulnerabilities for all organizations. those vulnerabilities are compounded by the complexity of information technology, limited time to market, development constraints, and constantly changing relationships between organizations and suppliers. growth in the sophistication of security threats makes it imperative that organizations remain equally competent in identifying vulnerabilities and mitigating security risks. aim: the goal of this document is to explain how isbt intends to provide guidance to the blood banking community on implementation of effective information security policy. when using information for critical activities, blood banks should consider information security as an important aspect of their management policies. evaluation of existing standards, such as iso 17799 and hipaa, allows us to establish a framework for information security without regard to the type of organization. it remains very difficult however, due to the complexities involved, to establish an information security policy without guidance. method: the isbt information task force was created to provide guidelines on information security for blood banking organizations of all sizes. the intent is to help them understand existing standards as well as provide tools for implementing information security policy. these guidelines are based on existing standards that are followed by most worldwide countries: iso 17999 and hipaa. results: information security can be defined as the 'protection of systems, information and services from accidental and deliberate threats to confidentiality, integrity and availability' . understanding existing information security standards was the first step for establishing a structure for the guidelines. the core is organized within an implementation framework and presented under the three following layers: 1. administrative for defining the it security organization, the information security policy, and information security awareness and training. 2. physical for providing solutions that relate to physical environment protection and access, equipment and it infrastructure security, and control for accessing computerized equipment. 3. technical for maintaining confidentiality of electronic information and ensuring that authorized access to information systems is maintained (technology relating to identification and authentication, logical access, operating system, network management, application access, etc.). strategy guidance is also included for senior managers in charge of establishing organization policy, including responsibilities and methods to successfully implement policy. further, the task force is addressing both risk analysis and management including identification of potential dangers to information systems (threat-source) and existing controls (risk description), as well as a plan to address identified vulnerabilities and mitigation of specific risks. conclusions: information security standards are prerequisite to understanding the issues involved when considering information vulnerability. international guidelines for information security, specifically directed to the blood banking community, are equally necessary if we are to identify, plan for, and mitigate risks associated with vulnerabilities to critical blood banking information. the isbt task force is committed to providing such guidelines. introduction: the important part of quality planning and quality assurance within production of blood components is measurement system analysis (msa). measurement system analysis was performed on microscopic counting of blood cells in our study. aim of the study: aim of this study is determination if microscopy counting of residual elements in whole blood plasma is suitable for quality control of blood components. methods: we practiced measurement system analysis in counting of residual elements (leukocytes and erythrocytes) in whole blood plasma. the counting performed two lab technicians in ten samples of plasma from whole blood. leukocytes and erythrocytes were measured in naggeotte counting chamber. we used the method of mean and range for the determination of reproducibility and repeatability (r&r) and analysis of variance for complex measurement system analysis. regulation diagrams were applied for the graphic statement. we determined the value of repeatability, reproducibility, coefficient r&r, variability among samples of plasma and total variability of measurement system. the important conclusion was to determinate if the microscopic counting of samples of blood components is sufficient with regard to quality parameters specified in guide to the preparation of blood components (erythrocytes: <6.0 ¥ 10 9 /l, leukocytes: <0.1 ¥ 10 9 /l). our results show that microscopy counting of residual elements in whole blood plasma is suitable for quality control of blood components. aim of the study: to provide transfusion services with a tool for proper qms implementation, an international collaborative study on qms applications, employing the 'process approach', has been undertaken by a group of transfusion services of varying sizes and structures with experience of qms, in collaboration with a university institute offering a master in qms implementation in health services, and an expert from a quality association. the 'process approach' serves as a tool to manage transfusion activities as a system based upon a network of processes and their interactions. guide-lines have been produced based upon this principle and will be published (volume and cd-rom) and distributed at no cost to all transfusion services of nations participating in the study. the main contents of the guide-lines' chapters are: through definition/analysis of single processes and the correlation network amongst these, the 'process approach' methodology renders the transfusion centre's functioning units completely interdependent, eliminates process interface barriers, provides personnel with a unified focus on the main transfusion objectives, and lays the basis for improvement of transfusion service quality, organization and performance through efficient control of processes' interactions. the blood screening system 400 automated high-throughput nat system for simultaneous screening of hcv, hbv and hiv nucleic acids: full process surveillance e pfeifer*, b alessandri † , hr bachmann † , t barker † , y ohhashi*, c parkhouse*, j pinsl-ober ‡ , p wenzig ‡ and g ziegler ‡ *roche molecular systems, inc., pleasanton, usa, † roche instrument center ag, rotkreuz, switzerland, ‡ roche diagnostics gmbh, penzberg, germany introduction: the blood screening system 400 combines on one deck both automation of dna/rna extraction from blood/plasma samples and multiplex pcr amplification and detection of nucleic acid targets. the system is designed for high-throughput single unit testing and pooled specimen processing. a data management system supervises and controls the complete process from initial sample pipetting through to result compilation and reporting. the objective of this project was to present the system 400 assay and device built-in quality control measures that guarantee process safety and reliability. the study shows how internal control, external batch controls, pipetting sensors, validation & maintenance procedures, and a controlled development & manufacturing process yield an optimal test method and system stability. method: failure modes and effects criticality analysis (fmeca) was used to evaluate a mathematically derived safety metric for optimizing risk reduction. this method makes use of a system risk objective-function (srof), which provides a multivariate description of sample processing, amplification and detection steps. the method for analyzing system behaviour first employs product classification into risk domains, followed by ranking of process steps that are determined to be linked to known system hazards. this provides objective means for directing system design leading to risk minimization. the srof responses associated with redundant liquid sensing channels were shown to substantially reduce risk during sample or reagent transfer steps. the system 400 liquid flow, airpressure-based (plld) and capacity-coupled liquid (clld) sensors detect aspiration and dispensing inaccuracies. sensor signal tolerance band widths studied for fluid classes representative of system 400 reagents and for plasma samples from lipemic, icteric, and hemolized sample sources were shown to correlate with srof multivariate modelling. the impact of surveillance design elements on the srof response demonstrates that risk is functionally dependent on design elements. additional surveillance examples are presented that describes temperature sensing, robotic positioning and motion control. treatment of residual risk is addressed by introducing external controls that are configurable to specific workflow scenarios. the negative external control (nc) is used to check for contamination of reagents. five low concentration armored external controls, i.e. hiv-1 group m arna, hiv-1 group o arna, hiv-2 arna, hcv arna, and protected hbv dna are run at the beginning of a batch as a run-control measure. in addition to these roche controls the system 400 supports running of user-defined external controls for co-validating the batch. the internal control (ic) is based on armored hiv-1 group m rna that is co-extracted and co-amplified with the external controls and the target nucleic acids potentially present in the sample. lastly, the system 400 resource management monitors the status of samples, ready-to-use reagents and disposables via rack sensors and bar-coded bottles and tubes. the fmeca methodology provides a risk-minimized, comprehensive system design. redundant sensors with internal and external controls are evaluated through comparison of modelled versus actual run results. the system 400 brings a new level of surveillance and throughput for automated pcr testing, and emphasizes roche's commitment to increasing the safety of the global blood supply. technical standards for safe storage and transport of blood components and blood samples objective: to implement standardization of technical specifications for safe storage, transport and distribution of blood samples and blood components intended for transfusion, as part of a quality system in blood transfusion medicine. methods: in the course of implementing a quality system in our blood establishment (distributing 10% of the national blood supply), we have developed standard operating procedures (sops) for temperature and hygienic conditions to maintain and control storage of blood components during their shelf life and to ensure their safe distribution to other blood services in the country and abroad, in compliance with eu directives 2002/98/ec and 2004/33/ec and the recommendations of the council of europe and who. procedures are validated and relevant records are kept. a statistical process control is also in place to monitor deviations from specified temperature and time range throughout the period of transportation. blood components collected and prepared for specific purposes (e.g. directed donations, irradiated units, hla-typed units, anti-cmv negative blood components and blood for neonates) are stored separately, and alarms and warning systems are in place. packing and transport conditions of red cells, platelets and ffp are submitted to the tests and criteria of adr (european agreement concerning the international carriage of dangerous goods by road). in greece, the adr legal framework has applied since 1999. blood samples are classified according to adr in division 6.2 under un 3373 as diagnostic specimens, and the criteria for safe carriage include packaging, specific labelling and vehicle requirements as well as carrier obligations and personnel training. our blood establishment has a contract with biotrans, a private company accredited for packaging, storing and transporting blood, organs, tissues and cells as well as potentially infectious biologic substances and blood samples for diagnostic purposes. assurance system). the approach is specified in the form of requirements of art. 4.1 of the standard: the organization shall: (a) identify the process needed for the quality management system and their application throughout the organization, (b) determine the sequence and interactions of these processes, (c) determine criteria and methods needed to ensure that both the operation and control of these processes are effective. regional centre for transfusion medicine in biaĺystok was the first transfusion service in poland which was certified according to iso 9001:2000 standard. our expected profits from the implementation qms were: possibility to overview organization's pathways of operation and to inspire corrective and improvement actions, emphasis on the role of staff in the system, focus on self-control and responsibility for one's own work as a factor of staff's mentality creation/modification. our one year of experience with iso 9001:2000 standard proved the system to be handy tool of management for the organization collecting, processing, testing blood and releasing of blood products for the hospitals and to be well accepted by the staff. the implementation and certification of the internationally nor-malized quality management system simplify and shorten all accreditation and registration procedures required for legal activities of transfusion service as well as for any supplemental medical activities which may be performed by the centre. the implementation of qms facilitates the implementation of other quality systems and simplifies procedures required for the ce certification for the products. red blood cells stored in blood banks, normally undergo a series of chemical alterations, or storage lesions. the ultimate consequence of these lesions is a decrease in the viability of the red cells following transfusion. the chemical alterations are mainly changes in levels of na+, k+, cl-concentrations, ph and 2,3 dpg levels and they affect the electrical impedance of blood. the electrical impedance, cole-cole parameters, is determined mainly by the resistance of the red cell extracellular fluid (re), the resistance of the intracellular fluid (ri) and the capacitance of the cell membranes (cm). in this study we aimed to investigate the relation between blood parameters and electrical impedance changes, and their further clinical implication. all parameters were measured on 51 erythrocyte suspension (es) samples during 42 days of storage at 4oc on days 0, 10, 21, 35 and 42. for 31 whole blood (wb) samples during 21 days of storage, same parameters were measured on days 0, 10, and 21. the measurement of the complex impedance of blood samples were performed in the frequency range from 100 khz to 1 mhz. by using the 4284a hp lcr meter, the impedance z, and the phase angle a for each sample were read. these values were corrected according to the gain and phase characteristics of the amplifier; and the resistance r and the reactance x were calculated. each data was first fit to the cole-cole model; hence the cole-cole parameters, intracellular resistance ri, extracellular resistance re, characteristic frequency fc and phase angle a were obtained by using lms software. afterwards, cm was calculated by using ri, re, a and fc. whereas ri and cm decreased progressively with time on both wb and es, re changes showed some differences. the electrical impedance alterations were explained by measurements of na+, k+, clconcentrations, ph and 2,3 dpg by indicating days. storage of red cells resulted in a rise in extracellular k+ and a fall in extracellular na+, cl-, ph and 2,3 dpg. anova was used to evaluate differences in blood measures in relation to storage time. the results were presented as the mean ± sd. according to the regression analysis in spss, the intracellular resistance (ri) on both es and wb was affected more efficiently from all blood parameters among all other electrical parameters. although ri and re were correlated with na+, k+, cl-, ph and 2,3 dpg more significantly, cm measurements were failed to show correlations with blood parameters because of the intervening parameters, a and fc. the best relationship between the parameters mentioned above on both es and wb was ri and k+. ph changes were the same for ri and re both for es and wb. the correlation between parameters on es was better than those on wb, because whole blood consists of several particles that may affect our measurements. our study showed that 2,3 dpg has an effect on ri and re as efficiently as other blood parameters and therefore electrical impedance measurements may serve future implications. background: issue of quality blood products and donor safety are the main aims of blood transfusion services. a comprehensive quality system should be in place to fulfill these aims, which can be attained through strict adherence to the established standard operating procedures (sops). the drugs and cosmetics act of india, which controls the licensing of blood transfusion services, does not provide clear guidelines regarding plateletpheresis procedure. aim: we therefore established our own sop and operational flow chart for plateletpheresis that can be easily followed by other centers in india. methods: a total of 100 plateletpheresis procedures performed using two cell separators (cs3000 baxter, usa, mcs3p, hemonetics, usa) were evaluated following our established sop. the mean platelet yield in cs3000 was 2.9 ± 0.84 ¥ 10 11 and in mcs3p, it was 2.88 ± 0.75 ¥ 10 11 per unit, however, only 4-7% of sdps showed wbc levels <5 ¥ 10 6 . six of 100 donors complained of hypocalcemic symptoms. the operational flow chart designed in this study was found to be simple and easy to adapt by blood transfusion services in this country. the first advanced quality management training course for blood transfusion services in the western pacific region mk tan*, jp yu † and d teo* *health sciences authority, singapore, † who regional office for wpr, manila, singapore background: blood transfusion is a key part of modern medicine. a well-organised blood transfusion service (bts) is a prerequisite for the safe and effective use of blood and blood products. in 2000, the world health organization (who) introduced a new initiative of quality management project (qmp) to achieve the goal of safe and adequate global supply of blood. through qmp, regional training centers were identified and quality management training (qmt) courses were established. in 2001, centre for transfusion medicine (ctm) of health sciences authority singapore was appointed as a collaborating center for qmt in the western pacific region (wpr background: spectrophotometric method according to harboe was traditionally used at our institute for determination of free haemoglobin in supernatants of rbc blood components at the end of storage time. in the year 2002 hemocue plasma low hb system (hemocue, sweden) was introduced as a replacement method. aim: the aim of the study was to examine reliability and suitability of the hemocue method for measurement of free haemoglobin in supernatants of rccs. methods: hemocue plasma low hb method was validated and compared with harboe method. supernatants of 36 rbc products were tested by two methods and results compared using regression analysis. additional testing was performed to investigate precision of hemocue method (within-run and between-day variation), trueness using reference material and to define optimal sample handling. results: regression analysis showed high correlation between two methods (r = 0.98), with higher values obtained with hemocue method. immediately after centrifugation one supernatant was measured 6 times in order to determine within-run imprecision. coefficient of variation (cv) calculated from 6 consecutive measurements was 6.1%. to investigate between-day imprecision of the hemocue method one sample was divided in 5 aliquots and frozen. one sample was thawed each day and measured. cv of five measurements was 1.95%. during the period of validation 37 measurements of reference material (low, medium and high) were performed. cv calculated for these measurements were 7.86% (low), 1.88% (medium) and 1.04% (high). in order to investigate possibility of batch testing, supernatants of 5 different rbc products were divided in aliquots and measured periodically during 1-month period. cvs calculated from 7 measurements of each sample were in range 3.1-9.9%. conclusion: hemocue plasma low hb method appears to be an excellent replacement for the harboe method. it is consistent, easy to use, measurements are performed in short time, and errors are minimized by eliminating dilutions and manual calculations. background: determination of haemolysis in red cell concentrates (rccs) at the end of storage time is routine method in quality control of blood components at our institute. for this purpose, haemoglobin concentration in supernatant of rccs is measured using hemocue plasma/low hb system (hemocue, sweden). according to manufacturer recommendation, visually turbid samples should be filtered before analysis with a 0.2 mm filter. because visual estimation of turbidity is highly subjective and unreliable, filtration of all supernatants before analysis using appropriate filters is possible solution for standardisation of the method. aim: the aim of the study was to investigate the effect of filtration of visually non-turbid samples on results of free haemoglobin measurement. methods: haemoglobin concentrations were measured in 42 visually non-turbid supernatants before and after filtration using hemocue plasma/low hb photometer (7 wb negative controls were used for each blood component. in all blood components tested bacterial contamination was detected using both types of bottles. in comparison with sa/sn bottles, nearly all enrolled microorganisms were detected faster in bpa/bpn bottles (see table 1 ). all negative controls were negative (no false positive). the results of the study performed support the use of bact/alert bpa and bpn plastic bottles in quality control testing of different blood components. objective: management of returned blood products is important part of quality assurance activities in transfusion medicine. blood products are returned most frequently because of routine rotation of stock and because of nonconformities discovered after the product has been received. methods: qa data about returned blood products were retrospectively analysed for the 5-year period (2000-2004) . only blood products returned because of nonconformities were taken into consideration. data about the reasons for returns are presented and discussed. the top 4 reasons for returns were positive dat, labelling errors, blood bag defects and visual appearance of blood units (see table 1 ). in all cases positive dat was confirmed in our institute, and blood donors managed accordingly. nonconformities related to visual appearance of blood component and other nonconformities related to the quality of blood products were investigated and corrective actions conducted. in case of blood components returned because of damaged bag, significant problem is to investigate the cause of nonconformity (usually inappropriate transport conditions). conclusion: management of returned blood products is important tool in improving the quality of blood products. introduction: hemovigilance is the systematic monitoring of the blood transfusion chain for side effects and adverse incidents from the moment of blood collection until after administration of the unit to the recipient, and comprises all activities that can lead to a safer and more effective use of blood components. attempts to achieve a more safe and effective use of blood components constitute a huge task given the number of interventions and array of medical and not-medical personnel involved in the transfusion process. registration of the impact of all participants is a tremendous job as such. information management may enhance the chances for a successful hemovigilance. aim: the aim is, to establish a basis for all the information related to the chain, such as standard operating procedures, (inter)national guidelines, local transfusion protocols and forms, and procedures to direct the process. additional factors that contribute to the final results are the profile and role of employees, incidents, points of care, product information, prices, budget, contracts, etc. by clarifying and explaining the basis to all participants by means of an existing infra structure, everyone will gain access to identical, consistent and up-to-date information at all times. the primary activity is to create the basis by an outline of every individual link in the transfusion chain from donor to recipient. secondly, the resources, responsibilities, actions, and internal controls (what task, who is performing, why, which help, where, when) are documented. by creating distinct databases for these 6 'w's' in combination with a separate database for the hemovigilance process itself, it is possible to establish relations, hyperlinks, between the different databases. all the information collected in the foundation, complete with all the resources, responsibilities, actions, and internal controls is made available to target groups by means of the intranet in our hospital as well as in a printed handbook format. collecting and displaying the information on hemovigilance this way, creates a transparent and more easy-to-manage process. it establishes a basis, which incorporates all resources, responsibilities, actions, and internal controls to all participants and enables the organisation to operate both efficiently and effectively. it allows us to show auditors (both internal and external) which processes are related to which guidelines as well as the results in daily. in addition, it reveals which factors determine the genuine application of the guidelines in clinical practice. conclusion: by outlining the process, followed by defining, analysing, securing, on the 'w6' method, the hemovigilance process offers a stable basis and framework for all participants and may stimulate a more safe and effective use of blood components. background: transfusion medicine is a basic post-graduate specialty for medical doctors with a specific national curriculum in bulgaria. in order to improve the post-graduate training of medical doctors in transfusion medicine, a new curriculum was elaborated in 2004. purpose of the new program: independent work of transfusion medicine specialists on all levels of the blood transfusion service (bts) -organization of bts, promotion of voluntary, nonremunerated blood donation, collection, processing, testing, storage and transport of blood and blood components, immunohematologic testing of patients, laboratory hematology, clinical experience, assistance and advice on diagnostic and therapeutic problems of patients, requiring treatment with blood products, teaching transfusion medicine to bts and clinical personnel. admittance and duration to training -medical doctors are admitted to post graduate specialization after a successful state examination. the overall duration of training is 4 years, of which at least 2 are obligatory for training in the national or regional blood transfusion centers. main sections of the curriculum: 1. organization of blood donation and blood transfusion, including promotion of voluntary, nonremunerated blood donation; organization, planning and information systems; organization of blood transfusion; quality management. 2. collection, processing, storage and transport of blood and blood components 3. laboratory hematology and specialized methods (general laboratory methods, immunology, immunohematology, transmissible infections, hemostaseology, nat techniques, flowcytometry) 4. clinical use of blood components and plasma products (treatment with blood products, adverse events and reactions, alternatives to blood transfusion). the new curriculum of transfusion medicine guarantees a better training of medical doctors, thus leading to safe and effective blood products their proper clinical use. introduction: transfusion medicine is integrated medical discipline based on clinical and laboratory practice. it is closely connected with molecular biology, genetics, as well as with apheresis, automatically techniques and transplantation. the aim of studies in transfusion medicine is proper education, skills, attitudes and knowledge of students at the medical faculty and of doctors on residency in transfusion medicine and other specialties about blood and its usage. material and methods: there will be presented how transfusion medicine is implemented in educational and health sector in r. results: there is 3-year specialization in transfusion medicine, established 30 years ago (over 90 specialists finished this specialization till now). we have postgraduate studies in tm started 15 years ago. transfusiology, as subject at the medical faculty, is now included in new curricula of medical student, consists of 15 theoretical and 15 practical lectures. also, medical doctors on specialization in surgery, anesthesiology, obstetrics and gynecology, pediatrics, internal medicine, maxillo-facial surgery and others have obligatory 1-month education in tm. transfusion medicine is integral part in education of nurses and laboratory technicians; all personnel that work in transfusion field in our country finished 6-mounths course in tm and get certificate. conclusion: although we have already done so much in educational field, we will continue with our efforts to improve our collaboration with clinicians and to involve ourselves more in clinical disciplines. introduction: in hospitals of saint petersburg donor blood, its components and preparations, autoblood and its components, hemocorrectors are applied with the medical purpose at 20-30% of hospital patients. the service of blood and the existing organization transfusion therapy in hospitals should provide maximal immunological and infectious safety and also its high medical efficiency. it demands special preparation for all doctors: general physicians on clinical transfusiology and doctors of blood service on all sections of the general, industrial and clinical transfusiology. clinical gemostasiology, pharmacology of hemotransfusion means and hemocorrectors, the organization of the blood service, the donor service; the organization, technique and technics of preparation of blood, its components and preparations, quality assurance, transfusion therapy programs, technique and technics of transfusion medicine, a problem of maintenance of immunological and infectious safety, preventive maintenance, diagnostics and treatment of posttransfusion complications, feature transfusion therapy in pediatrics and medicine of accidents). clinical physicians master all basic questions of clinical transfusiology, thus the special attention is given questions of clinical immunohematology and clinical gemostasiology, programs and a technique of transfusion therapy, the prevention of posttransfusion complications. employment are carried out in departments of city blood bank, hospitals blood departments. final examination under the test program and at interview shows sufficient mastering a theoretical and practical material (right answers of 80-98%). the described system of postgraduate studies provides an opportunity of successful independent work of doctors on the workplaces both regular increase and qualifications not less often than 1 time in 3-5 years. conclusion: the existing system of postgraduate education for doctors on transfusiology provides the blood services and hospital by the qualified staff. introduction: nowadays in hospitals of saint-petersburg more and more it is frequently applied the extracorporal hemacorrection (haemapheresis, haemasorbtion, plasmasorbtion, krhyoplasmasorbtion, ultrafiltration etc.) and photohemotherapy (optical radiation influence on blood) at various diseases and traumas. they are used at treatment almost 6% from the general number of patients of hospitals with positive results at 85% from them. for this purpose in hospitals there are specialized branches or cabinets with staff of the doctors -transfusiologists. therefore an actual problem is organization of postgraduate special education for them. because there is no specific blood banking and transfusion medicine training in either under or post graduate medical education those general practitioners have limited knowledge about this field. aim: to give basic knowledge and practice to those general practitioners moh has established a special training programme with the collaboration of medical schools for 5 years. methods: this is a 6 months programme which has 2 basic components; theoretical education, laboratory and blood banking practice. the trainees are evaluated by a final examination. each group has limited cadets and the training is provided in selected blood banks with the collaboration of universities and moh training hospitals. conclusion: almost 100 general practitioners are trained by this programme in the last 5 years and those doctors helped to increase the quality of blood banking and transfusion service at their hospitals. background: even the history of public blood banking in turkey has dates back to 1957, whole blood was comprising the majority of transfused units (more then 96%) in turkey until the last few years. aim: the main target was to decrease the whole blood use in a short time and establish countrywide effective blood transfusion practice. methods: there were no specific blood banking and transfusion education either at undergraduate and postgraduate medical education in turkey until the last few years. blood banks and transfusion society of turkey (bbtst) was established at 1997 with the main aim of education of the related people about blood banking and transfusion medicine. bbts has organized 35 symposiums, 13 national courses, 1 national and 1 international congresses since 1997. due to increased knowledge about the blood components and improved infrastructure of blood banks whole blood consumption has decreased to national average to 60%. in most of the university hospitals and training hospitals this is around 19%. conclusion: like most of the public based behaviours dedicated efforts of civil initiative has changed the traditional habit of blood consumption in turkey by the direct affect of well organized educational activities. training of general practitioners in blood banking and transfusion medicine n solaz*, b keskinkilic † and c oruc † *ankara university, ankara, † ministry of health, ankara, turkey background: turkish ministry of health runs majority of the hospital blood banks in turkey. most of the moh hospital blood banks give service under the medical supervision of general practitioners. because there is no specific blood banking and transfusion medicine training in either under or post graduate medical education those general practitioners have limited knowledge about this field. aim: to give basic knowledge and practice to those general practitioners moh has established a special training programme with the collaboration of medical schools for 5 years. methods: this is a 6 months programme which has 2 basic components; theoretical education, laboratory and blood banking practice. the trainees are evaluated by a final examination. each group has limited cadets and the training is provided in selected blood banks with the collaboration of universities and moh training hospitals. conclusion: almost 100 general practitioners are trained by this programme in the last 5 years and those doctors helped to increase the quality of blood banking and transfusion service at their hospitals. background: it has been accepted that during cardiac surgery the complement-system is activated which enhances ischemia-reperfusion injury, leading to postoperative complications as infections and organ failure. the lectin pathway is one of the mechanisms that can activate the complement-system, this pathway can be mediated by mannose-binding-lectin (mbl). the level of mbl in serum shows a wide variation. a low level of mbl can lead to more infections in some conditions and a high level to tissue damage after ischemiareperfusion injury. the effect of transfusions of erythrocyteconcentrates and plasma on the mbl levels and thereby on complications after cardiac surgery are not known. aim of the study: the role of pre-and postoperative mbl levels and the effect of transfusions on postoperative complications after cardiac surgery. in a randomized controlled trial 474 cardiac surgery patients were included and blood samples were taken pre-and postoperatively. mbl measurements were performed by elisa assays. the data were linked with postoperative complications as mortality, infections and multiple-organ-dysfunction-syndrome-mods and with peri-operative erythrocyte and plasma transfusions. results: the mean pre-operative mbl-level was 837 ± 796 and postoperative 456 ± 347 mg/ml. there were no differences in preand post-operative mbl levels between patients with infections or mods compared with non-infected and non-mods patients. the difference in pre-operative mbl between non-survived (672 ± 587) and survived patients (853 ± 812) was not significant (p = 0.20). postoperative mbl levels between survived and non-survived patients was smaller. conclusions: pre-and postoperative mbl levels in cardiac surgery are not related to postoperative infections, mods and hospitalmortality. whether large amount of blood transfusions are affecting the mbl-levels and thereby the patients outcome is not known. introduction: patients undergoing cardiac surgery are receiving high amount of blood transfusions and are at risk for the development of infections and multiple-organ-dysfunction-syndrome (mods), these complications are influencing the survival of the patients. during cardiac surgery pro-and anti-inflammatory cytokines are released by different mechanisms. we found in a randomized trial in cardiac surgery a significant reduction in infections and mortality due to mods by transfusion of leukocytedepleted erythrocyte concentrates (ld) compared to leukocyte-containing, buffy-coat depleted erythrocytes (pc). aim of the study: the effect of ld on the concentration of proinflammatory cytokine il-12 and anti-inflammatory cytokine il-10 in relation to clinical outcome and complications after cardiac surgery. methods: in 474 participating patients blood samples were taken before and after surgery. using elisa il-10 and il-12 were measured in these samples. the results were linked with the endpoints of the randomized trial: postoperative infections, mods and in-hospital mortality. results: all pre-operative concentrations of il-10 and il-12 were low. mean postoperative level for il-10 was 70 ± 121 and for il-12 57 ± 74. compared with patients without mods and without infections and survived patients only the il-10 was significant higher in patients who died and in patients with mods. there were no differences in mean levels related to ld and pc. the levels of il-10 were higher in patients receiving more then 10 units blood transfusions compared to 0 transfusions. conclusion: there is an association between mods and mortality and il-10, not with il-12. ld has no influence on mean levels of il-10 and il-12. there is a correlation between transfusion of more then 10 units and il-10, not with il-12. these cytokine-profiles are not associated with the beneficial effect of ld on the postoperative outcome in cardiac surgery patients. anemia and blood transfusion practice in critically ill patients e grouzi*, e tsigou*, p evagelopoulou † , g baltopoulos † and i spiliotopoulou* *kat general hospital, transfusion service, athens, † athens university school of nursing icu, athens, greece background: anemia is a common problem in critically ill patients admitted to intensive care unit (icu), but the consequences of anemia on mortality and morbidity in the critically ill is poorly defined. aim: to define the incidence of anemia and red blood cell (rbc) transfusion practice in critically ill patients in the icu) of our hospital, and to examine the relationship of anemia and rbc transfusion to clinical outcome. patients and methods: the period study was from july 2003 to december 2004. patients were enrolled within 48 h of icu admission. follow-up time was 30 days, hospital discharge or death, whichever occurred first. results: a total of 169 patients (116 male, 53 female, mean age 57.6 ± 21.1 years, range 15-96) were included in the study. the mean hemoglobin (hb) level at baseline was 11.2 ± 2.1, which level was descending during the study. overall 63.9% (108/169) of the patients received one or more rbc units while in the icu stay (mean 3.1 ± 3.8 units per patients). the mean pretransfusion hb was 8.7 ± 1.8 g/dl and the mean time to first icu transfusion was 2.6 ± 4.1 days. more rbc transfusions were given in the first week of the icu stay (331 units vs 110, 61, 36 units in the second, third and forth week respectively). the number of rbc units which a patient received during the study was positive associated with longer icu length of stay and an increase in mortality (r = 0.19, p < 0.05 and r = 0.26, p < 0.05 respectively). baseline hb level was significantly related to the number of rbc transfusion (r = 0.55, p < 0.001), but was not an independent predictor risk factor of length of stay or mortality (r = 0.16, p > 0.05 and r = 0.13, p > 0.05 respectively). the mean baseline apache ii and saps scores were 19.6 ± 7.4 and 49.8 ± 17.4 respectively. furthermore both baseline apache ii and saps scores were significantly higher for patients with a baseline hb level of <10 g/dl (23.2 ± 7.9 vs 18.3 ± 6.7 and 54.6 ± 18.8 vs 48.1 ± 16.5 respectively), while the apache ii values were positive associated with a significantly increased likelihood of rbc transfusion (pearson correlation p < 0.005). conclusions: anemia is common in the critically ill patients, it appears early in the icu course and persists throughout the duration of the icu stay. rbc transfusion seems to be associated with worse clinical outcome. despite the intensive research for the transfusion practice, which taken place worldwide during recent years, data from our country is limited. the results of our study suggest that approaches to reduce rbc transfusion would be desirable. however, further well designed prospective studies with large number of patients are required to efficiently explore the risk of anemia, optimal transfusion hb threshold and the risk and benefit of rbc transfusion in the critically ill. consumption of blood products in a large, general hospital he heier*, j pillgram-larsen † , m hestnes ‡ , b gran*, a krog* and no skaga ‡ *ullevaal university hospital, oslo, † ullevaal university hospital, dept. of thoracic surgery, oslo, ‡ ullevaal university hospital, dept. of anestesiology, oslo, norway uuh is the largest general hospital in norway, and trauma referral centre for half of the norwegian population (2.5 mill) the trauma team performed initial assessment and resuscitation of 323 patients, 75% males, during the first six months of 2002. the aim of our study was to analyze transfusion practice at uuh with specific focus on trauma. materials and methods: blood consumption during this period was recorded. clinical data of trauma patients were collected from our trauma registry and anonymized before analysis. results: 7012 units of erythrocytes (er), 914 of thrombocytes (thr) (buffy coat preparations from 4 donors) and 903 of s/d-treated whole plasma (op) (octaplasò) were transfused in uuh during this period. 56.6% of er were given to surgical, 21.3% to medical and 12.2% to gynaecological and obstetric patients. 62% of er were given to patients above 45 years. er units were given to 1475 patients (mean 4.75 units/patient; range 1-63). mean age of trauma patients was 33 ± 18, median 30, range 1-89 years. for transfusion of this group local guidelines state that haemodynamically unstable patients should receive er if hgb <9 g/dl, irrespective of age and sex. eighty-eight patients (27.8%) received er, 15 of them as massive transfusion ( 3 10 er units in <24 hours) (17.0%), 6 of these died (40%). altogether trauma patients received 851 units of er (12.1% of total uuh consumption), 51.5 thr (6.1% of total) and 150 units of op (16.5% of total). massive transfusion consumed 340 er (40%), 29.5 thr (57.3%) and 110 op (73.3%) units. fourteen adult trauma patients (15.9% of those transfused) received 1 or 2 er units only. lowest pre-transfusion hgb was 3.3-11.3 g/dl, median 7.6, mean 7.5 ± 1; highest post-transfusion hgb 7.8-14.3 g/dl, median 9.5, mean 9.8 ± 1. five patients received er transfusion at higher pretransfusion hgb level than 9 g/dl, but in 4 er were given because of a hyperacute clinical situation. fifty-five% of issued er units had been stored for >15 days, while only 10% were issued before 10 days of storage. discussion: life-saving effect of transfusion would seem evident in the massively transfused survivors, while in the other transfused patients documentation of clinical effect is inadequate. transfusion practice in trauma at uuh seems fairly well in accordance with internationally accepted guidelines. the trauma unit consumed a surprisingly small part of total uuh transfusion resources. trauma patients deviate from the general uuh patient population by sex and age; transfusion is otherwise mainly given to more elderly patients. further analysis is needed on transfusion indications and results to optimize the total use of blood products in uuh. special focus should be on transfusion to non-bleeding patients without haematological disease. it seems that only a small part of transfusion efforts results in the saving of life. in croatia national guidelines for the use of rhd gamma globulin were laid down in the year 2000. in accordance with the guidelines, rhd gamma globulin is administered intramuscularly in doses of 250-300mg and should be given to rhd negative women after delivery of rh positive children, after abortions, in week 28 of their first pregnancy, as well as in cases pregnancies with an increased risk of fetomaternal hemorrhage. as to the latter, detection and measurement of fetomaternal hemorrhage are recommended. three years after the issuance of the guidelines a survey was conducted regarding the use of rhd gamma globulin that involved 33 health institutions across the country. as many as 38 601 deliveries and 10 974 abortions were reported in 2003. the institutions reported the usage of 5103 doses of 250 mg rhd gamma globulin or 110 doses/1000 deliveries + abortions. we compared our data with those obtained from similar surveys conducted in 1999, i.e. before the issuance of the guidelines. in that year 4407 deliveries and 1400 abortions were reported, and 6125 doses of rhd gamma globulin or 100 doses/1000 deliveries + abortions were administered. institutions were then divided into four categories: clinical hospitals, general hospitals with the capacity of 400-600 beds, general hospitals with the capacity of 100-400 beds, and institutions of rather limited capacity with gynaecology department. the range of the consumption of rhd gamma globulin/1000 deliveries + abortions in the first category was 73-167 with the median being 119; in the second category there were 73-115 doses with the median being 92, in the third category 55-128 doses with the median being 88, while in the fourth category the range was 10-191 doses with the median of 79 doses. the number of pregnancies which should have been protected with rhd immunization in 2003 year was obtained by the following formula: (frequency of rhd negative subjects) ¥ (frequency of the r gene) = 0.17 ¥ 0.59 = 0.10. if a complete antenatal and postnatal preventive measures involving rhd immunization had been taken in 2003, 12 287 doses of rhd gamma globulin or 250 mg/1000 deliveries + abortions should have been given. our research has shown that, despite of the national guidelines adopted in 2000, the prevention of the rhd immunization in pregnant women is still not adequate in croatia. in fact, it has not improved since a year prior to the adoption of the guidelines. it has also been established that the category of the institution is a factor influencing the implementation of the protective measures. we consider that much more should be done on informing both women and gynaecologists about the importance of prophylaxis of the rhd immunization. consumption of plasma derivates in croatia g jaklin*, b golubic-cepulic † and m dondur* *general hospital, varazdin, † clinical hospital center zagreb, zagreb, croatia a increasing consumption of plasma derivates, their limited supply and insufficient national reserves are problems that croatia is faced with nowadays, as well as many other countries. in 2003 a study was carried out regarding the usage of plasma derivates. as many as 34 health institutions took part having the capacity of 15 487 acute beds out of a total of 16 279. the data regarding the use of plasma derivates were collected as follows: albumin, i.v. gamma globulin, and concentrate of coagulation factor viii in two periods of time. we divided institutions into three categories: clinical hospitals, general hospitals with the capacity of 400-600 beds and general hospitals with the capacity of 100-400 beds. institutional practice patterns regarding the use plasma derivates were compared among them. the parameters considered were the total consumption of plasma derivates, consumption per bed and consumption per inhabitant. data on the use of plasma derivates was obtained from hospital transfusion departments and pharmacies across the country. we compared our data with similar study carried out in 1997. the consumption of albumin was 441.44 kg or 98 kg/million inhabitants in 2003 and consumption per bed for the first category institutions was in range 14.27-72.27 kg with the median being 39.42, for the second category the range was 1.24-44.64 kg with the median being 4.36, while for the third category the range was 0.32-23.13 kg with the median being 6.63. in 1997 in croatia the consumption of albumin was 436.50 kg or 97 kg/million inhabitants. the consumption of i.v. gamma globulin was 59.92 kg or 13.32 kg/million inhabitants in 2003 and the consumption per bed for the first category institutions was in range 0.03 g-8.89 g. with the median being 2.78, for the second category was in range 0.03-3.95 g. with the median of 1.56 and for the third category was in range 0.00-12.75 with the median 1.56 g. in 1997 in croatia the consumption of i.v. gamma globulin was 22.20 kg or 4.93/million inhabitants. the consumption of the concentrate of factor viii was 8807.000 iu or 1.96 iu per inhabitant in 2003. 1539.500 iu, i.e. 17.48%, was a recombinant factor viii. in 1997 the consumption of factor viii was 4876.350 iu or 1.08 iu per inhabitant. discussion: the use of albumin showed stagnation. however, the use of i.v. gamma globulin increased 2.6 times and the use of the concentrate of factor viii increased 1.8 times if compared with the results in 1997. self-sufficiency has not been reached in plasma derivates in croatia we needed 20 000 l plasma for gamma globulin and 58 000 l plasma for concentrate of factor viii for the level of usage of these plasma derivates in 2003. significant differences in the level of usage among hospitals have been observed. these reflected a considerable difference in hospital policy regarding the use of these products in defined clinical settings. therefore, we would recommend that the national society sets out guidelines for the use of the products. background: blood bank good practice requires avoidance of rh alloimmunization. several studies report a probability of immunization of more than 80% following transfusion with rhd+ rbc's and as many as 19% in the case of platelets. aims: the purpose of this study was to investigate the development of anti-d and the causes of alloimmunization in a university hospital (1300 beds), reviewing our practice on the use of rhd+ blood components in rhd-recipients. method: a retrospective study was performed whereby 15.872 rhd-patients, from 1990 to 2004, were evaluated for the development of anti-d analysing the data on the blood bank information system. results: from the 15.872 rhd-patients we found out 274 identified anti-d, 77% (211) detected before any transfusion in our hospital. of the 63 left, nine were excluded because no information was available during some years. 5 had history of pregnancy related to the immunization. 36 patients were exposed to rhd+ blood components: 12 were transfused only with rhd+ platelets (including two women of childbearing age); 24 with rhd + rbc's. the remaining 13 had been exposed exclusively to rhd -rbc's suggesting that some units could be mistyped. this idea was corroborated in three patients where there was a common donor (he was called for investigation). conclusions: transfusion services avoid as far as possible administration of rhd+ blood components to rhd-patients, although there are situations in which such transfusions are necessary. the retrospective review of records revealed the need for specific recommendations regarding the use of rh immunoglobulin to prevent anti-d immunization. the possibility of mistyping blood components also appeared in this review, emphasizing the role of these studies in the evaluation of methodologies used at blood banks. the purpose of the work: to present the usage of whole blood (wb) and blood products (bp) in the treatment of patients who are hospitalized in the internal disease department in gevgelija. material and methods: retrospective analysis is done according the data which were analysed in five years period (2000) (2001) (2002) (2003) (2004) . statistical methods which were taken from the history of the hospitalized patients in the internal department were used for analysis and the data of transfused wb units and units of bp were taken from the department of transfusion medicine. results: from the total 4285 hospitalized patients who have been analyzed, 759 (17.71%) received wb and bp, and there have been transfused 1411 units wb and bp. every patient, on an average, has been transfused with 1.86 units wb and bp. at the same time 326 (23.1%) units have been transfused as a wb, 928 (65.77%) have been transfused as red blood cells (rbcs) and 157 (11.13%) units have been transfused as a fresh frozen plasma (ffp). the data for every year particularly will be presented in our paper to the congress. the collaboration between doctors of transfusion medicine and health workers from the other medical branches is the most important thing in the medical practice. our aim is to raise the awareness among the health workers for the importance of blood safety and their role though adequate clinical usage of wb and rbcs, minimizing the non-useful transfusions, evaluation of reflexive information from undesirable reactions in the usage of blood and blood products, use the alternatives etc. the necessity of direct involvement of the doctors in transfusion medicine in the process of healthy workers' education from the other branches for regularly transfusion therapy via meetings and lectures is an imperative for regular function of the department of transfusion medicine. femoral neck fracture repair is one of the commonest orthopedic procedures. it mainly concerns intracapsular or intratrochanteric fractures and it may be considerably hemorrhagic, requiring blood transfusion perioperatively. the aim of our study was to investigate blood transfusion requirement in patients with femoral neck fractures repair at katerini general hospital during 2002-2003 and to compare them with other studies. one hundred sixty five (165) unselected patients of whom 116 (70%) were women and 49 (30%) were men with a mean age of 76 years (range 26-94 years) were studied retrospectively. seventy six (46%) patients had intracapsular fracture and 89 (54%) intratrochanteric fracture. the mean hb concentration on admission was 12.25 g/dl (range 6.8-17 g/dl) and the mean hb concentration at discharge was 10.78 g/dl (range 7.2-14.3 g/dl). a total of 297 units of rbc were transfused (a mean of 1.8 units per patient). blood transfusion occurred in 125 patients (75.8%), (45-69% in other studies) with a mean of 2.38 units per patient (range 1-10 units), (1.47-2.6 in other studies). patients with preoperative hb values <10 g/dl were transfused more often than those with hb values >10 g/dl (100% versus 73%) p: 0.017. women were transfused more often men (80.2% versus 65.3%) p: 0.042. patients aged >70 years were transfused more often than those aged <70 years (81% versus 60%) p: 0.008. finally patients with intratrochanteric fractures were transfused more often than those with intracapsular fractures (88.65% versus 60%) p: 0.001. conclusions: in our study blood transfusion requirement for femoral neck fracture repair are similar with these reported in other studies. blood transfusion frequency is greater in patients with hb <10 g/dl, in patients older than 70 years, in women and in intratrochanteric fractures repair. fresh frozen plasma guidelines and practices as saltamavros*, g talampouka*, c koumoundourou*, s dimitrakopoulos † and p tseliou* *st. andrews hospital patras greece, patras, † pyrgos general hospital, pyrgos, greece introduction: fresh frozen plasma transfusions should be done according to specific standards and guidelines. aims: we have reviewed the ffp transfusion practices followed in our hospital in an attempt to provide a set of guidelines and principles that will assist physicians and health care workers to make the right decisions for appropriate use of ffp transfusions. methods: retrospective review of ffp transfusions practices during the entire year of 2004. we classified transfusion practices according to the diagnosis of the patient and also of the clinical depart-ment that demanded transfusion. then we analyzed our results by comparing the requests with the internationally approved guidelines for ffp transfusion and counted the percentage of ffp to rbc units. finally we discussed with our fellow physicians the proper use of ffp and informed them about the accepted guidelines. as we can see from the underneath table, diagnoses and departments with high consumption were: internal medicine 22%, plasmapheresis to patients suffering from guillain barre and ttp (thrombotic thrombopenic purpura) 21.04% and trauma/surgery 20.68%, cancer 13.60%. summary/conclusions: the use of ffp corresponds to 27.88% of the rbc units transfused in our hospital. plasma units should be used properly and according to internationally accepted standards. plasma use should be justified by the physician, in order to avoid unnecessary risks on behalf of the patient for effective treatment and care. to show gradual discontinuation of using whole blood and increased use of red blood cells (rbc) in the management of patients in the transfusion department of the clinical hospital 'zvezdara' . methods: data of wb and rbc use from 1982 to 2004 in our hospital. results: our data are presented in table 1 . conclusion: after 1982, wb usage rates systematically decreased from 51% to 1.3%. however, in the period of 1991-2004, the wb usage rates increased slightly, and we arrived at a generally very low rate of wb use, namely only in about 3% of the cases. education about transfusion medicine and rational use of blood in the medical curriculum was generally insufficient and very poor. therefore, we started an education program and we established a hospital transfusion committee (htc) with the task of medical control and monitoring the quality of clinical transfusion practice in our hospital. members of the htc are a specialist in transfusion medicine, an internal medicine specialist, an anesthesiologist, surgeon and gynecologist. we have been organizing courses for the medical staff, doctors and medical technicians since 1995. background: platelet transfusions are given mainly to thrombocytopenic patients with malignant haematological diseases. currently the decision to give a prophylactic platelet transfusion is based almost exclusively on the number of circulating platelets in the patient although it is known that various clinical factors might influence the bleeding tendency. by free oscillating rheometry (for), using a reorox® 4 instrument, it is possible to monitor the coagulation over time in whole blood and obtain information about clotting time and coagulum elasticity. aim of the study: the aim of this study was to find out if for using the reorox® 4 instrument could be used to evaluate the hemostatic status of thrombocytopenic patients. methods: the change in elasticity over time in non-anticoagulated whole blood from leukaemia/lymphoma patients with thrombocytopenia was measured in the reorox® 4 pre and post a platelet transfusion (n = 10) and was compared with healthy controls (n = 40). the effect of platelet concentration on coagulation was studied by diluting platelet rich plasma from healthy subjects with autologous plasma to various platelet counts whereafter the coagulation was monitored with for (n = 8). the change in elasticity per minute (g'max slope) and maximum elasticity (g'max) were evaluated from the elasticity curves. results: the g'max, g'max slope and platelet count increased after transfusion for all patients. the thrombocytopenic patients had significantly lower g'max and g'max slope values both pre and post transfusion compared with healthy control subjects (p < 0.01). the measurement in platelet rich plasma showed that g'max and g'max slope increased with increasing platelet concentration. however patients with similar platelet count developed different clot elasticity. the reorox ® 4 instrument responds to changes in haemostatic function in form of the clot elasticity. the fact that patients with similar platelet concentration developed different elasticity shows that clot elasticity is a function of not only platelet concentration but also of functional properties. the for method seems promising to evaluate platelet function. quality control of pre-storage leukodepleted red cell concentrates vu urlep salinovic, k perbil lazic and l lokar teaching hospital maribor, maribor, slovenia background: the system of quality assurance including quality control (qc) in transfusion medicine is most important for safe blood supply. the safety and quality of blood components are increased by pre-storage leukodepletion of whole blood (wb). aim: the qc of leukodepleted red cell concentrates (rcc), prepared from pre-storage filtration of wb (quadruple blood bag systems imuflex wb-rp terumo) is performed with the aim to be sure that the quality of leukodepleted rcc is in accordance with the guidelines of council of europe. in three years (2002) (2003) (2004) , 35 559 units of wb were collected, among them 7765 (21.6%) units were collected for pre-storage filtration and in 376 (4.8%) of these qc was done. within 4 hours after collection, wb was filtered at room temperature. filtered wb was centrifuged at 4000 g for 20 minutes and separated in rcc and plasma. the samples for qc were collected before and after filtration. for each unit, volume, hemoglobin, hematocrit, % of hemolysis and residual white blood cells (wbc) count were measured. the number of residual wbc after filtration was determined in the nageotte chambre. for all parameters the mean value and standard deviation were calculated. the results of qc for the following parameters were: volume 320 ± 25.1 ml (89% corresponds with the guidelines of council of europe), hematocrit 0.61 ± 0.06 (96%), hemoglobin 60.9 ± 8.0 g/unit (99%), number of residual leukocytes 0.028 ± 0.16 ¥ 10 6 (100%), hemolysis 0.30 ± 0.19% (100%), the test of sterility was 100% negative. during filtration of wb, wbc were removed in approximately 99.99%, the duration of filtration was 16 ± 6 minutes and the loss of hemoglobin was 8.2 ± 4.3%. the pre-storage filtration of wb is highly efficient, 99.99% of wbc were removed and the loss of hemoglobin was small. background: the patients of cardiosurgery use big amount of blood components. there is no uniform approach by treatment with blood components in these patients. the safest and the most rational use of blood is based on the individual treatment of a patient and the evaluation of the most clinical and laboratory factors. aim: the aim of our study is to analyse the use of blood components from 1998, when the cardiosurgery department was founded in our hospital, to 2004. the data about use of red cell concentrates (rcc), platelet concentrates (pc) and fresh frozen plasma (ffp) in the period from 1998 to 2004 were collected from information system datec. the average use of all three blood components per patient is presented. we were interested, if the average use per patient was diminished in the analysed period. results: the use of three blood components and average use of all blood components are presented in the table. the data from the table shows, that the number of patients increased more than three times, but the average use of blood components per patient was not essentially diminished. conclusion: during seven years period the use of blood components per patient in cardiosurgery was not diminished. for more rational use it is necessary to apply all methods of autologous blood transfusion because of the increasing number of cardiac operations and decreased number of voluntary blood donors. background: the presence of leucocytes in blood components is cause for appearance of various adverse posttransfusion reactions such as nhfptrs, urticary, anaphylactic shock, alloimunization and platelet refractorines, infection with bacteria and leucothropyc viruses (cmv, htlv). the removal of leucocytes from blood components through filtration is especially important in the treatment of patients with malignant diseases who need frequent transfusions of blood and blood components. this is because of their lowered immunologic status which is a result of the disease and received immunosuppressive chemotherapy and radiotherapy. aim: to show the prevention of posttransfusion reactions and the positive effect from transfusion of leucoreduced er. concentrates, produced with filtration in therapy of anaemia in patients with malignant diseases, treated in our daily transfusion hospital at medical center -stip. methods: 123 patients with malignant diseases have been treated in our daily transfusion hospital in the last four years. most of these patients suffer from ca pulmonum, ca collonis, ca uteri, ca mammae. also, because of the secondary anaemia, the same patients were transfunded with at least two doses of leucoreduced er. concentrates. the blood donored by voluntary repeated blood donors was filtrated the same day after the donation, separation and the control of the same one. the blood was collected in baxter and terumo bags, and it was filtrated with baxter -sepacell rs -2000 and pall -purecell rn filters. the analyses of leucoreduction were made for every filtrated and transfunded unit before and after filtration. the analyses samples were taken from the tubing system before and after the filter. haemathologic parameters were automatically made in the central clinic laboratory. results: er. concentrates poor with the leucocytes for about 98-99.9% were produced with the use od these filters, and platelets from 88-100% with which side effects from frequent transfusions at these patients were prevented. with the routine monitoring of all patients none posttransfusion reactions were registered. the therapeutic effect is also important because of the fact that the number of er and the level of hg and hct remain almost unchanged. the aim of the blood banks is to help and to increase the safety of the blood components. the leucoreduction through of er. concentrates poor with leucocytes is a regular procedure in the therapy of malignant patients with remarkable clinical picture of anaemic syndrome and prevention the cancer recurrence end infection. the time of filtration is also very important which has to be shorter after collection of blood, because the leucocytes should be removed before they become disintegrated and relapse potentially dangerous substances end metabolites in the blood components. we have been applying so called ' prestorage filtration' because it has been proved that it is more efficient that bed -side filtration. background: the effect of leukocyte reduction of rbcs by filtration has been the topic of several rcts. these rcts investigated the effect of leukocyte reduction on mortality; post-operative infections and hospital stay in different patient populations. some rcts came up with answers that conflicted with the results of other rcts. aim of the study: with including the individual patient datasets of several rcts in one database, combined analyses are possible that may explain the differences in the reported results. using this technique, we may come up with answers (or questions) that cannot be obtained by performing standard meta-analyses of these rcts. methods: we coordinated several rcts comparing the perioperative use of buffy-coat depleted rbcs with the use of filtered rbcs. cardiac surgery patients were included in three rcts (1¥ cabg and/or valve; 1¥ re-cabg and/or valve; 1¥ valve with or without cabg). oncologic surgery patients were also included in three rcts (1¥ colorectal cancer; 1¥ gi-oncology or vascular surgery; 1¥ gi-oncology, vascular surgery or orthopedic surgery). the electronic data files of these rcts were uniformly recoded and entered in a single database. as different primary endpoints were investigated in the rcts, we focused on common endpoints, recorded in 4 or more of the rcts. multivariate analyses were performed on: in-hospital mortality, 30-day mortality, hospital stay, stay on icu, postoperative infections, and mods. results: in the rcts, individual datasets from 3875 surgery patients were collected (1630 onco; 1272 cardiac; 569 vascular; 352 orthopedic, 52 other). in the multivariate analyses, patients in the buffycoat depleted trial arm showed a higher mortality rate both in-hospital (p = 0.01) and at 30 days post-surgery (p = 0.03). no association between randomization and stay in hospital (p = 0.10) or stay on icu (p = 0.56) was seen in the combined study population. also, no association of randomization with the incidence or duration of mods was seen. the analyses of post-operative infections in the total population showed the trial arm to be associated (p = 0.016), and 'hospital' to be far stronger associated (p < 0.001). however, when the oldest rct, that had not yet used our standard definition list for scoring post-operative infections, was excluded, the association with the hospital was lost (p = 0.34) and the trial arm became more strongly associated with infections (p = 0.002). in the analyses of 3875 surgery patients, the use of filtered rbcs, compared to the use of buffy-coat depleted rbcs, resulted in reduced mortality (both in-hospital and at 30 days postsurgery) and a reduction in post-operative infections. the association of the variable 'hospital' with post-operative infections, as is frequently reported in literature, was initially confirmed in our analyses. however, when a standard definition for post-operative infections was used (excluding the datasets from one of six studies), this association was lost. background/aims: uk neqas for blood transfusion laboratory practice (btlp) operates an eqa service for 472 uk laboratories (including eire) and participants throughout europe, including major groups in denmark (n = 52) and portugal (n = 32). in november 2004 a questionnaire was distributed to determine the criteria used for selecting phenotyped blood for different patient groups, including pre-menopausal women (pmfs). results were analysed by country to establish any variation in practice relating to the selection of k negative (k-) and rhc negative (c-) blood, since antibodies to these antigens are now the major cause of hdn. results: overall return rate was 84% (uk) and 64% (non-uk), although only centres treating pmfs were included in this analysis. k-blood was selected for pmfs by 81% in wales (n = 16) and 71% in england (n = 291), but only 6% in scotland (n = 32), 8% in northern ireland (n = 12) and 10% in eire (n = 31). in mainland europe, variation was also observed: 12% in denmark (n = 17), 75% in portugal (n = 20) and 85% in other countries (n = 27 from 10 countries). fewer laboratories selected c-blood for pmfs: 8% in england and northern ireland, 0% in scotland and eire, rising to 81% in wales. mainland europe showed similar variation: 12% in denmark, 75% in portugal (all ccee matched) and 51% in other countries. there are no guidelines requiring selection of k-and c-blood for pmfs, but in the uk d negative blood is required for d negative females aged <60 years. trend analysis of questionnaire data in the uk, using theoretical clinical scenarios, shows a decrease in the number of laboratories that would select k + blood for a 30 year old female (with pre-existing antibodies other than anti-k), from 58% (1996), 42% (1999) to 26% in 2004. in this survey, k + blood was selected for a female aged 30 (no antibodies) by 31%, whilst 81% selected a k + unit for a female aged 54 (no antibodies), despite this patient being treated as a pmf for provision of d negative blood. a similar distinction was made between the 30 and 54 year old females in portugal and mainland europe. conclusions: variation in practice may at least in part be due to the availability of blood routinely labelled for rh and k. in the uk all donations are labelled, except for those from new donors, as are most units in portugal. uk questionnaire data (1999) suggested that >50% laboratories would change to selecting k-blood for pmfs if all units were labelled. however, labelling alone does not account for the differences seen within the uk, and perhaps the trend towards providing k-(and to a lesser extent c-) blood for pmfs is influenced by advice from transfusion services. it would be of great interest to monitor and compare the incidence of hdn due to anti-k and anti-c in different countries to measure the outcome of differing practices for provision of blood to pmfs and to thereby inform future policy. there is no ultimate in ex-vivo assay described, which can predict the outcome of plt transfusion in vivo. current in ex-vivo assays and animal studies are rather very complicated to carry out, cost effective and time consuming. objective: we hypothesized that the quantitative measurement of gpib expression by facs can be used to predict the outcome of platelet survival post transfusion. in our previous studies we demonstrated that our phagocytosis assay can predict the plt survival sensitively (blood dec-2004) . we isolated human washed plts by centrifugation and labelled with mepacrine and then incubated with pma-matured thp-1 cells (37°c). binding was measured by facs analysis of cd42b/cd14 positive particles, and phagocytosis by counting mepacrine/cd14 positive particles. we measured gpib expression before and after plt-macrophages interaction by facs flowcytometry. results: gpib expression at surface of c22 fresh showed 90 ± 5 and after 48 hours storage 50 ± 15% expression. the gpib expression at surface of plt decreased and showed three populations with different densities high (gpib-1), low (gpib-2) and in-between (gpib-3). the binding and phagocytosis of plt showed an increase of 40 ± 10% which implicates an indirect and negative relation to gpib-1, and direct relation to gpib-2 expression. anti-human pselectin (cd62p) delayed 45 ± 10% the binding and annexing v 60 ± 12% the phagocytosis of stored plts, after 48-hour storage. these results show that gpib expression is rather easy, reproducible test which can be standardised and be used as a very sensitive in vitroassay to predict platelet survival posttransfusion. transfusion and lung injury jd dodig*, d sovic † , m tomicic † and h sager* *university hospital 'sestre milosrdnice', zagreb, † institute for transfusion med, zagreb, croatia background: the respiratory tree has been viewed as an infrequent site of injury arising as serious complication of transfusion. in recent years, this view has changed as investigators have shown that two complications-circulatory overload and transfusion related acute lung injury-are relatively frequent events. case report: a 59-year-old men was admitted due to chronic macrohematuria. he had no history or current evidence of cardiac failure. the hb level was measured 36 g/l. he received 500 ml 0.9% nacl and 500 ml ringer solutions. after that, he was transfused with 2 units of rbcs. during transfusion second unit he developed following symptoms: tachycardia, dyspnea, hypertension. massive pulmonary edema was noted. he was treated with mechanical ventilation, oxygen, diuretics, aminophillin, antihistaminic and corticosteroides. the patient recovered after being on ventilation for 6 hours. two days after the patient was operated. after surgery he was transfused with 4 units of rbcs. all transfusions were regularly performed. results: chest x-ray confirmed bilaterally pulmonary edema. the samples (patient and donors of first two units rbcs) tested were negative for the presence of hla specific and granulocyte antibodies. granulocyte agglutination and lymphocytotoxicity test were negative. tnf-a in recipient serum was slightly increased. conclusion: our patient is the first reported case suspected of trali, but all of the investigations didn't give us the answer. against neutrophils using flowcytometric detection of cell surface cd11b expression and l-selectin shedding. methods: a hundred microl of volunteers' heparinized whole blood were incubated for 15 minutes at 37°c with fmlp, lps or pma. monoclonal antibodies against hna1a and hla-i, or patient serum were incubated with whole blood for 30 min. surface cd11b and l-selectin were detected using facscalibur. results: neutrophil activation was detected after fmlp, lps or pma stimulation. p38 map kinase inhibitor reduced activation induced by fmlp and lps. anti-hna1a monoclonal antibodies induced neutrophil activation, which were also inhibited by p38 map kinase inhibitor. ten serum samples obtained from patients or donors who caused transfusion reactions were evaluated. five sera out of 8 samples having anti-neutriphil and/or hla antibodies exhibited neutrophil activation, while two samples without leukocyte antibodies had no effect on any activation. conclusion: these findings indicate that neutrophils activation is regulated through mak kinase and detection of neutrophil activation may be useful to predict transfusion-related reactions. results: 38 out of 50 transfusion reactions were febrile, 19 were anaphylactic, 1 were due to circulatory overload, 1 out of 50 transfusion reactions concerned the transfusion of incorrect blood component. 1 out of 50 transfusion reactions concerned acute haemolytic reaction. post transfusion purpura or suspected trali was not seen. fatal complication was not seen. the reactions to plasma were predominately anaphylactic. we detected no case of bacterial contamination among the cultures of transfusion bags. conclusions: the incidence of reactions among patients malignancy was high, while among surgical patients was lower. the incidence of transfusion reactions during the months had no statistically significant difference. we suggest that the improvement in prevention of transfusion reactions require a continual vigilance system for rapid recognition and information regarding these complications. measurements of ige immunoglobulin in thalassemic patients, before and after blood transfusion background: many studies have reported the implications of proinflammatory cytokines including interleukin (il)-1 beta, il-8 and tumor necrosis factor alpha (tnf-alpha) in febrile nonhemolytic transfusion reactions. il-8 has been shown to accumulate in packed rbcs even after the procedure of filtration, which is explained by the release of il-8 from rbc receptors into the packed rbc super-natant. stress induced elevation in tnf-alpha levels was demonstrated in healthy individuals. aim: to assess the level of proinflammatory cytokines in peripheral blood of blood donors. material and methods: immediately upon blood collection, plasma was separated from postdonation blood samples obtained from 75 blood donors and frozen at -80°c. upon thawing, the level of the il-1 beta, il-8 and tnf-alpha cytokines was determined in plasma samples by elisa method using commercial kits for cytokine determination (roche molecular biochemicals). the level of il-1 beta was at the test detection limit in all donor plasma samples. in 1 (1.3%) bd, the level of il-8 was 36.8 pg/ml, exceeding the test sensitivity limit of 6.2 pg/ml. in 12 (16.6%) bd, the level of tnf-alpha was within the range of 14-60 pg/ml, with a test sensitivity limit of 12 pg/ml. tnf-alpha levels >800 pg/ml were measured in plasma samples of 3 (4.1%) blood donors. the increased activity of blood donor's immune cells, indicated by elevated levels of the proinflammatory cytokines il-8 and tnf-alpha in peripheral circulation some blood donors, may lead to the occurrence of febrile nonhemolytic transfusion reactions in the recipients of the blood products manufactured from the blood of these blood donors. this hypothesis will be thoroughly investigated in our future studies. background: transfusion-related acute lung injury (trali) is a life-threatening complication of transfusion, under-recognized and underreported possibly lacking a consensus definition. aim: we report here a 'probable' case of trali syndrome in an elderly female patient. case presentation: an eighty-two year old female patient was transferred to the intensive care unit on the third postoperative day (pod) following the abrupt onset of acute pulmonary insufficiency (pao 2 = 49 mmhg, o 2 saturation = 79%), hypotension (bp = 90/50 mmhg) and fever (38°c). this occurred ten minutes after initiation of an infusion of a unit of packed red blood cells (prc). the patient had no history of any cardiac or pulmonary disease. she was intubated and placed on mechanical respiration and supported hemodynamically. the cvp (12 cm h 2 o) ruled out fluid overload. the chest x-ray revealed bilateral pulmonary oedema, the echo cardiogram and blood cultures ruled out cardiac and infectious aetiology of the episode. after treatment for 48 hours the patient improved significantly, was extubated and returned to the surgical unit on the 6th pod. reviewing the transfusion history we discovered that the patient did not receive any blood or blood component prior or during the correction of her ileum, but she was transfused a unit of ffp (fresh frozen plasma) five hours prior to the incident. the donor review revealed that the unit of ffp was from a 29-year old female with a history of multiple abortions whereas the prc unit was from a 52-year old male, a volunteer of several years who had no history of transfusions. there are some prerequisites for the implementation of a haemovigilance network and some of them have been met, so we can present the first results. methods: traceability of blood components is possible because there is unique information system in place in the whole country, identifying the donor, donation, each single blood component and identification of the recipient, but feed back information of the performed transfusion is still missing. cooperation between blood transfusion service and hospitals was established by introduction of hospital transfusion committees; the intensity and quality of their work is very different. homogeneity of reporting is achieved by the introduction of unique reporting form. a reporting route was defined: patients physician sends notification of atr to the local blood transfusion service. atr reporting form is prepared there and sent to the national blood transfusion service, where the data is collected for the centre for haemovigilance, which is going to be established very soon. data analysis will be responsibility of the centre for haemovigilance at the governmental level. type of adverse transfusion reactions and events is defined. education and information was passed to the health personnel by inclusion of haemovigilance in under and postgraduate programmes as well as seminars, scientific meetings and some publications. results: in the years 2002-2004 there were 410.000 blood components issued in slovenia and 333 atrs were reported (1 in 1232 blood components issued), in 25 of them the severity grade was 2 (1 in 16.412 blood components issued). in the year 2004 there were considerable differences between the number of atr reports compared to the number of blood components issued among slovenian hospitals, ranging from 1 in 3375 to 1 atr in 490 blood components issued. 9.6% of all atr were not classified. conclusions: although the number of reported atrs was increased by 24% and 31% a year in 2003 and 2004 respectively, it can be assumed that the collected data is not complete. feed back reports, much more information and cooperation is needed, especially in some hospitals, which should contribute to a better registration of atrs and events in the future. the slovenian haemovigilance system still needs upgrading, but despite this, some important work has been done in building a national haemovigilance system. . considering the multiplicity groups in cattle and since there was no research on repeated blood transfusions reactions in iran's native cattle, we decided to consider crossmatching in different processes of repeated blood transfusion from a head of blood donor to five recipients and observe the clinical and hematological alterations. six healthy iran's native cow, 2.5 years old, with average weight of 210 kg were used. the animals were dewormed by albendazole (15 my/kg bw) and were kept for two weeks under uniform managemental condition. three days prior to blood transfusion, vital signs registration (temperature, heart rate and respiratory rate) blood collection via jugular vein was done to indicate the baseline of research parameters. after that, blood transfusions were performed from one donor cow to five recipients three times at one-week intervals. cross matching was done at each transfusion. after each transfusion the research parameters do determined. results indicated that one of the recipients cows experienced anaphylactic shock, in the first step of blood transfusion and another cows in the second step and finally in the third steps two other cows showed the serious shock. this is in the contrary of this opinion that the first transfusion can be given safely without crossmatching in cattle practice ( van der valt, et al. (1969) background and objectives: blood transfusion may lead to the manifestation of anti-hla and platelet-specific antibodies that may in turn bring about different problems like platelet refractoriness. it appears that the study of antibodies against hla-class i and platelet-specific antigens are useful for the selection and success of the appropriate treatment protocol. the aim of this study was to detect anti-hla and anti-platelet-specific antibodies by flowcytometry in patients with hematologic disorders (including acute leukemia, aplastic anemia) and patients with itp. in this descriptive study, anti-hla and platelet-specific antibodies were detected by flowcytometric technique, using 62 sera drawn from patients with different haematological disorders who showed a poor response to platelet transfusion and 20 from patients with itp. the results of anti-hla antibodies were then compared by panel reactive antibodies (pra). results: our results showed 44 (53.7%) out of 82 (53.7%) patients had anti-hla class-i antibodies in their sera. the frequency of each antibody isotype was found to be as follows: igm (51.2%), igg (32.9%) and iga (1.2%). 36 (43.9%) out of 82 patients had platelet specific antibodies and the frequency of each antibody isotype was found to be as follows: igm (40.2%), igg (30.5%) and iga (12.2%). 27 (31.7%) out of 82 patients had both antibodies. no difference was found between the two groups in platelet specific antibodies. despite significant correlation between flowcytometry and pra methods, pra can only detect antibodies which react with complement. conclusions: with increase in the number of platelet transfusion, immunization to hla antigens occurs; moreover, immunization against platelet specific antigens may also occur during autoimmunity. the presence of these antibodies may be one of the reasons of poor response to platelet transfusion and platelet refractoriness in patients under study. conducting similar studies with higher number of samples, platelet cross-match, and the use of hlamatched platelets for these patients are recommended. post transfusion purpura (ptp) is a rare, severe thrombocytopenia that results from alloimunization to platelet specific alloantigens, following blood transfusion. in this condition, the patient's own platelets are destroyed by the alloantibody even though they are not supposed to carry the 'guilty ' antigen. the disease is rare occurring mainly in multiparous women. the majority of reported cases involved antibodies against the platelet specific alloantigen hpa-1a in a homozygous hpa-1b patient. in a minority of cases the offending antibodies were directed against hpa-1b, -3a, -3b, 4a, -5a, -5b. although self-limiting, the syndrome is characterized by severe bleeding with high morbidity and mortality; therefore prompt diagnosis and appropriate therapy is crucially important. ptp is a challenging diagnosis, because the patients are often critically ill or post-surgery, and have alternative explanations for thrombocytopenia such as infections or drugs. we present three patients with severe thrombocytopenia initially misdiagnosed. the first patient, a 54-year old women, had a past history of systemic lupus erythematosus and coombs positive autoimmune hemolytic anemia. at the present hospitalization, after antibiotic therapy for endocarditis, a severe hemolytic episode occurred and she need blood transfusion. when severe thrombocytopenia appeared, she was wrongly diagnosed as evans syndrome. the second patient, a 73-year old man, suffered from sepsis after vascular surgery and revealed clinical and laboratory picture of dic. the third patient, a 58-year old women, had end-stage renal failure and received heparin during hemodialysis, thus heparin-induced thrombocytopenia was first suspected. history of recent blood transfusion rose the suspicion of ptp in all this cases, and appropriate therapy with high dose iv immunoglobulin was started. adequate laboratory work-up confirmed the diagnosis. three different anti hpa-antibodies were identified: anti hpa-3a, anti hpa-1b and anti hpa-3b, respectively. the platelets genotype of the first patient was hpa-3b/3b, of the second hpa-1a/1a and of the third patient, hpa-3a/3a. the reported cases emphasized the importance of keeping in mind the possibility of ptp. incorrect diagnosis may lead to wrong treatment and fatal outcome. health sciences authority, singapore, singapore introduction: the haemovigilance programme in singapore was started by the centre for transfusion medicine (ctm) in 2002. the system covers registration of collected, produced and transfused blood components, and monitors adverse transfusion reactions (atr). the programme runs on a voluntary, non-punitive and confidential basis. aims of the study: (1) to gather and analyse reports of all adverse and untoward events occurring during transfusion of blood and components. (2) to use the information acquired to determine the morbidity of transfusion. (3) to provide guidance on corrective measures to prevent the recurrence of some accidents, and to improve transfusion safety. (4) to improve public confidence by demonstrating to public, patients and professionals the safety of the existing transfusion system. methods: (1) a common report form is used and made available to all participating hospitals. within the reporting system, the identification of the patient and staff involved are not required, to ensure confidentiality and protection of information belonging to the hospital. (2) reportable events include immediate reactions during transfusion (haemolysis, non-haemolytic febrile transfusion reaction, urticaria, anaphylactic shock, bacterial contamination, trali), delayed untoward effects after transfusion (haemolysis, post-transfusion purpura, acute gvhd), transfusion-transmitted infections, incorrect components transfused, and near misses. (3) within the hospitals, a responsible person ensures that all adverse events and untoward effects of transfusion are reported on the haemovigilance forms and provided to ctm for collation. within the ctm, the haemovigilance coordinator is designated to assist hospitals in investigating serious adverse events and advise on the reporting formats. results: (1) the total number of reported cases has steadily increased since the introduction of the programme. (2) the number of participating healthcare institutions has also increased to 100% (n = 12). please refer to table entitled 'summary of the haemovigilance report 2002-2004' . (1) the implementation of the haemovigilance programme in singapore is feasible with respect to the asian setting, and can significantly contribute to blood safety. (2) there has been very good participation from the 12 participating healthcare institutions, signifying greater awareness and willingness to partake in the programme. (3) the results obtained from the programme have given rise to initiatives and recommendations aimed at reducing (71%) were rated for seriousness. of these, 50 (6.5%) were rated as grade 2 (moderate to serious morbidity) or worse. 793 (72.6%) were rated for imputability to the blood transfusion. of these, a relationship to the transfusion was graded as 'certain' or 'probable' in 470 (59.3%) and as 'possible' in 245 (30.9%). overall relatively few errors were reported in comparison to other systems. a small number of reports concern (possibly) infected blood components, and imputability was deemed probable or certain only in a minority of these reports. autologous blood components gave rise to five reports (errors as well as mild transfusion reactions) which shows a relatively high risk associated with their use (9.98 per 1000 the rate of post transfusion hepatitis (pth) in israel in unknown. this information is important in order to learn about the residual infection risk in blood recipients. aim: to summarize the data on reported cases of pth. methods: suspected cases of pth are reported to mda blood services. the investigation procedure includes follow up testing of implicated donors and retesting of an archive sample of the transfused unit, if available. donors involved in suspected pth-b are tested for hbsag and anti-hbc. anti-hbc+ donors are tested for anti-hbs. hbv-dna testing is done if anti-hbs is less than 10 miu/ml. donors involved in suspected pth-c, are tested for anti-hcv and alt. since 2000 hcv-ag or hcv-rna are performed, when appropriate. investigation is considered complete if all the involved donors are retested >6 months following the implicated unit. results: between 1993 between -2004 suspected pth cases were reported: 74 (57%) were pth-b, 54 were pth-c (42%) and in 1 patient both hbv and hcv infections were reported. investigation was completed in 53/129 (41%) cases, with 70% of the involved donors (1222/1749) being retested >6 months after the implicated donation. hbsag was not detected in any of the 773 retested donors. anti-hbc was detected in 26 donors involved in 7 pth-b cases of which 21 were also positive for anti-hbs. pcr for the detection of hbv-dna was performed on the 5 'anti-hbc+ only' donors, and none was found positive. only in 1/449 donors suspected to be involved in pt-hcv, anti-hcv antibodies were subsequently detected. this donation was collected and transfused before the introduction of anti-hcv testing in israel, which was implemented in 1991. in another pth-c case, the implicated donor is still negative for anti-hcv, in follow up samples of up to 28 months, but was found positive for hcv-ag and hcv-rna. pth investigation of all the donors involved was completed in 49% (52/105) of the cases where the patient received up to 10 blood components. conclusion: in the past 12 years an average of 11 cases/year of suspected pth were reported to the israeli national blood services. investigation was completed in 41% of the reported cases. so far, there was no clear evidence of hbv transmission. in 2 cases (out of 2.99 million blood units collected nationwide during 1993-2004) hcv seemed to be associated with blood transfusion: one caseprior to the implementation of anti-hcv testing and the otherprior to the implementation of hcv-ag testing in a donor that did not develop anti-hcv antibodies. these findings suggest that other modes of hbv and hcv transmission should be sought in blood recipients in israel. 3-2.9, p < 0.05). the seroprevalence for a-hiv in the bd population was 0.096% (se: 0.028; ci: 0.034-0.153; p < 0.01) whereas in the a-hbc positive bd was 1.08% (p < 0.01) and in the a-hbc negative bd was 0.07%. from 113 a-hiv positive donations, 33 were also positive for a-hbc, that means that the sensitivity of the a-hbc in the detection of a donation a-hiv positive was 29.2%. the relative prevalence (percentaje of positive donations for a-hiv in the a-hbc positive population divided the percentage of this marker in the donations a-hbc negative: rp) was 15.43, which indicates that the number of bd a-hiv positive is 15.43 times higher in the a-hbc positive that in the a-hbc negative population of bd. as regards a-htlv, the seroprevalence in the bd population was 0.042% (se: 0.019; ci: 0.01-0.084, p < 0.01), in the a-hbc positive bd was 0.42% (p < 0.01) and in the a-hbc negative bd was 0.031%. from 49 a-htlv positive donations, 13 were also positive for a-hbc, that means that the sensitivity of the a-hbc in the detection of a donation a-htlv positive was 26.5%. the rp for a-hbc as regards a-htlv was 13.54, which indicates that the number of bd a-htlv positive is 13.54 times higher in the a-hbc positive that in the a-hbc negative population of bd. our results suggest that, in our bd population the screening with a-hbc would be useful to prevent other infections transmissible by blood transfusion, like retroviruses, because of the high sensitivity and the relative prevalence. 1. despite the high specificity of currently available elisas, the positive predictive value is lower in blood donors. therefore, immunoblot tests and polymerase chain reaction (pcr) have been adopted for routine testing in elisa +ve blood donors, by our service. 2. our data show that the real anti-hcv prevalence of our donor population is very low (0.04%). 3. the selection and evaluation of appropriate assays of all donated blood for hcv infection ensure good laboratory practice and accurate post-notification counselling of infected donors. 4. given that donors who are elisa positive but persistently negative or indeterminate probably do not represent a risk for transmission, their deferral from donation increases the problem of availability of blood supply. 5. donor re-entry in the pool of donors is an issue for further discussion. 6. the introduction of nat technology may elicit more accurate responses and improve the screening process. background: the introduction of hcv antibody screening of all donor blood in 1992 represented a major step in the prevention of transfusion-associated hcv hepatitis and in identification of infected donors. the study of infected individuals provides a unique opportunity to define behavioral factors associated with infection. evaluating risk factors in hcv infected blood donors is essential for monitoring blood supply safety, donor screening effectiveness and developing appropriate prevention programs objectives: 1. to recognize the epidemiology of hepatitis c and how it differs geographically; 2. to investigate the risk factors for presence of anti-hcv antibody in blood donors; 3. to evaluate the effectiveness of our donor selection program in local level. methods: serological testing for hcv was performed according to standard procedures. initial screening was performed using secondgeneration eia and, after january 1996, using third-generation eia. our study included a confirmation test (riba) a questionnaire was used to collect data concerning demographic, social and sexual behaviors, and number and type of donations of blood donors. the study also included testing of sexual partners and family members. changes in rates of hcv infections were evaluated by comparing yearly prevalence estimates. the overall prevalence of anti-hcv (eia) was 0.11% out of 168 374 blood donations. the average prevalence of hcv infection by riba was 0.04%, which reaffirms the very low risk of transfusion-transmitted disease. the cumulative number of hcv infected donors was 68, with 54 cases in males and 14 cases in females. most infections were found among older persons (32% were aged 22-39, and 68% aged 40-59). the seropositivity was higher in family/replacement donors (63%) than in volunteers (37%). the annual prevalence decreased throughout years. the relative importance of risk factors for hepatitis c was: transfusion 10%, hospitalization 8%, immigrants 8%, occupational 5%, sexual transmission 6%, injection drug use 3%, household contacts 5%, other 3%, tattooing 2%, unknown 50%. according to the criteria for blood donation, certain donors should have been excluded in the predonation interview but these donors had denied risky behaviour when questioned. the importance of sexual activity in the transmission of hcv has not been well-established as we tested 30 sexual partners and 14 family members and none of them was found positive. conclusions: our results suggest that major improvement in the safety and quality of our blood supply has been made in our area. introduction: apart from immuno-haematological complications, blood transfusion recipients are exposed to the risk of viral and bacterial contamination of donor blood. the latter infectious risks are generally associated with the number of donations that are needed in the production of blood products: the pooling effect. one measure recently being discussed is the generalisation of the use of trombocytapheresis for the production of trombocyte concentrates. normally, the trombocyte product is a concentration of trombocyte extractions from a pool of 5 buffy coats: pooled platelet concentrates (ppc). in case of trombocytapheresis sufficient trombocytes for one transfusion can be collected from one single donor. aim of the study: in this presentation the effect of using 100% trombocytapheresis for the production of single donor platelets (sdp) instead of pooled platelet concentrates (ppc) on contamination risks will be assessed. these risks can be divided in 1) the risk of bacterial contamination, 2) the risk of viral contamination (e.g. hcv, hbv, hiv) resulting from window period donations, and 3) the risk of contamination with tse or emerging infections for which no screening test exists. the contamination probability of sdp versus ppc is assessed on the basis of the production characteristics of both products, e.g. the presumption that the contamination risk per trombocyte product will be reduced by a factor equal to the number of pooled donations (five in our case). reduction of the bacterial contamination risk was estimated using the results of the bacterial testing of pooled and apheresis platelet products. as patients are likely to obtain multiple blood products during treatment, the contamination risk reduction through sdp is not only dependent on the reduction of risk in trombocyte products, but also will also dependent on the total number of blood products transfused and their associated contamination risks. the platelet recipient risk reduction was calculated on basis of the distribution of blood products received by the patient population of the university medical center utrecht (umcu) in the year 2001. results: in the attached table the estimated risk reduction through 100% trombocytapheresis is shown for the general blood recipient patient and for the trombocyte recipient patients only. our analysis indicate that in platelet recipients, general application of sdp instead of ppc will reduce the risk for transfusion acquired tse infections by 49%, the risk of known viral infections by 56%, and transfusion acquired bacterial infections by 59%. the confidence intervals surrounding the results were obtained by bootstrapping. the large confidence intervals surrounding the reduction of bacterial infection risk is caused by the fact that only a limited set of 4300 apheresis trombocyte products were tested. conclusion: our analysis indicates that general application of sdp instead of ppc will not reduce the risk of transmitting infections to platelet recipients as linearly (1 : 5) as expected. whether it is a costeffective precautionary measure will have to be evaluated by a costbenefit analyses consideration clinical benefits and additional costs and risks of apheresis donations. introduction: hepatitis b is serious health problem world wide. its prevention, particularly in the population of blood donors is essential for providing good health care and protection. aim: the aim of this study is to present the distribution of hbsag(+) and hbsag(-) blood donors according to their profession. methods: specially designed questionnaires are used for interviewing the blood donors who has previously given signed consent for participation in this study. results: table 1 shows the distribution of the blood donors in different professions. administrative clerks with 43 (34.12%) and the workers with 40 (31.73%) registered in the group of hbsag(+) blood donors, as well as workers with 50 (50%) and administrative clerks with 30 (30%) from the hbsag(-) group of blood donors are dominantly more frequent than the other categories of professions. health care professionals, housewives and farmers in both groups of blood donors are least frequent. taking in consideration the distribution of blood donors by the given professions in both groups, for u = 21 and p > 0.05 there is no significant difference found. the analysis of the differences among the different frequencies, in distribution of blood donors according the profession, for d = 0.436 and p < 0.05 shows a significant difference, where the workers with 90 (39.82%) are the most dominantly represented. in relation to the issue of whether the type of profession of the blood donors is production or non production the results are presented on table 2 . in the group of hbsag(+) blood donors the number of those who work in production profession-92 (73.01%), is dominant over the number of those that has non-production profession-34 (26.98%). in the group of hbsag(-) blood donors there is no significant difference between the types of professions. conclusion: having in consideration the professions of blood donors in both groups, for c 2 = 14.8 and p < 0.01 there is a significant difference in the presented distribution. according to our study, which shows the horizontal transmission of hbv infection in the family in which there is an index case, the biggest number of participants in the study is workers, and the least number of participants are the farmers. introduction: blood donors, as part of the healthy population are tested for hbsag with each blood unit they give. therefore, they can be an epidemiological model for exploring the appearance of hbv infection in general population. aim: this study aims to show the distribution of hbv infection in blood donors in relation with their living conditions, space and facilities. the material needed for the study consists of the data obtained from 126 confirmed hbsag(+) blood donors and 100 confirmed hbsag(-) blood donors as control group. results: the table 1 shows almost equal number of hbsag(+) blood donors that live in houses or flats. in the hbsag(-) group 64 (64%) donors that live in a flat dominate compared to 36 (36%) of those that live in a house. having in consideration the presented distribution (table 10) for c 2 = 3.96 and p < 0.05 there is a significant difference, that comes from the bigger number of blood donors (128) that live in flat. as for the distribution of blood donors according the living space they use by member of the family (table 2) , we can show that 24 (19.04%) of the hbsag(+) donors have less than 10 m 2 living space, in compared to 5 (5%) from the hbsag(-) group. the bigger number of hbsag(+) blood donors with small living space gives bigger possibility for transmission of hbv infection in the family. the differences in the two groups for donors that have between 10 and 30 m 2 , and between 31 and 40 m 2 of living space per person, obviously are not very big. for u = 19 and p > 0.05 there is no significant difference in the number of the donors in the two groups, when the available living space in m 2 is discussed. introduction: when one of the sexual partners has hbv infection the other is also infected in 1 from 3 cases. aim: the aim of this study is to outline that the risk from transmission of hbv infection between sexual partners is smaller if they use condom as protection. methods: two groups of blood donors-hbsag(+) and hbsag(-) have been interviewed whether they are using condoms as protection, or not. results: table1 shows the distribution of blood donors concerning the use of condoms. table1: in the group of hbsag(+) blood donors those who have not used condoms dominate with number of 81 (64.28%), in compared to those 45 (35.71%) who used condoms. these data are in favor of eventually possible sexual transmission of hbv infection in hbsag(+) blood donors. in the group of hbsag(-) blood donors dominate those who have used condom-57 (57%), compared to 43 (43%) who have not. the given distribution of blood donors concerning the use of condoms for c 2 = 10.2 and p < 0.01, shows significant difference, which is due to the prevailing of the number of blood donors (124), who have not used condom. of er -concentrates are leukodepleted. the choice of bacteriological control of empty bags for blood, bags with er -concentrates in additive solution, universal and iso group plasma, as well as the systems for taking of blood are taken on free choice. the control of the erytrocyte concentrates is performed on the first day after the preservation and dekanting, and again between the 15th-21st day and 30th-35th day after the preservation. the pulled plasma is controlled on the day of pouring (spreading), and the control of the iso group plasma on the day of deplasming. three months later the iso group and the universal plasma kept on the temperature of -30°c is bacteriologically controlled again. bacteriological control is performed with standard procedures in the institute for health protection in stip. the transfusion transmitted infections are potentially dangerous complications of transfusion therapy in immunocompromised patients. the aim of this study was to determine the prevalence of transmissible infections in blood donor population in kashan, iran. a total of 600 consecutive sera were tested for cmv-igm antibody, hbsag, hepatitis b core (hbc) antibody, hepatitis c (hcv) antibody, and hiv antibody with standard methods. of the sera tested, 14 specimens (2.3%) were cmv-igm positive. the frequency of seropositive revealed no significant differences between male and female donors. the frequency rates of cmv-igm seropositive tests tend to decline with increasing the age. there was no relation between the frequency rates of cmv-igm seropositive with the educational level, socioeconomic status, marital status, urban dweller and rural resident patients. the prevalence of hbv, hcv, and hiv antibody were 0.5%, 0.5%, and 0%, respectively. these findings implied important clinical applications because detection of cmv positive sera may reduce the risk for transmission of cmv in blood transfusion and thereby decrease the risk on cmv-induced complications. introduction: the worlds problem, aids, steel can't be said that is a problem in these three centers in r. macedonia, in which blood is collected, controlled, and distributed. found negative and 5 of them were found positive for one of the three viruses (hiv-1, hcv, hbv). with the elisa/axsym assay 29 of the 992 blood units which were negative by the procleix ultrio assay were positive for anti-hbcag and negative for anti-hbsag and hbsag. from those 29 blood units 23 units were given for transfusion following our blood centre protocol and the remaining 6 units were discarded. the protocol consists of a good medical history, liver enzymes (ast, alt, ggt). we must take into consideration that from those 5 that were found positive by the procleix ultrio assay 1 was positive for anti-hcv and 4 were positive for anti-hbsag. summary/conclusions: despite the fact of the short period of time we perform this method, the ability of the nat technique for rapid use, reliability and sensitivity in detecting three viruses simultaneously, indicates the need for immediate use in blood donation as a screening method. in spite of the high cost of the method, it is clear that this assay is a valuable tool in our blood centre to provide fast and safer blood. bacterial contamination of blood products is a persistent, but often overlooked, problem in transfusion medicine. in greece it is recommended that platelets (plt) must be used or discarded within five days post-collection. recent reports from europe have advocated the use of bacterial culturing of platelets on day 2 or 3 and, in case of negative result, prolongation of their storage time to 7 days. aim of the study: to assess the prevalence of bacterial contamination of standard platelet units from whole blood, and to provide evidence that with the use of bacterial culturing it is feasible to extend the self life of platelets to 7 days. materials and methods: eligible blood donors were bled according to standard operating procedures used in greece. plt were prepared from whole blood, solely for the purpose of the present study, by the platelet-rich plasma method. plts were stored for up to 7 days at 20 to 24°c with end-over-end agitation. other 111 plts prepared from blood collected in triple-pack container system also provided with a predonation sampling device were also tested. plts were sampled in the bacteriology laboratory. plts were sampled on day 2, 5 and 7. both aerobic and anaerobic culture bottles were inoculated with a 7-ml platelet sample. culture bottles were incubated at 35°c in an automated microbe -detection system (bact/alert system) until a positive reaction was detected or for 10 days. all samples that were reactive were confirmed by routine culture. each reactive sample with bacteria growth on the routine culture was sub cultured for identification of the bacteria. results: a total of 1889 plt concentrates were cultured and bacterial contamination was assessed in each unit at day 2, 5 and 7 after collection. on of storage day 2 two out of 1889 (0.1%) plt units were found to be positive for bacterial growth. 7 cases of unconfirmed positive results were noted at the beginning of the study. out of the other 1887 units which were negative on day 2 and continued to be cultured for the next days, the assessment at day 5 found no other positive. after further storage, at day 7, defined as the end of the prolonged incubation period, 5 out of the 1887 plt concentrates (0.26%) grew bacteria although testing of the same units on day 2 and 5 gave no signal. from the 111 platelets units that were prepared from blood collected with a predonation sampling device, none of the plt concentrates gave a positive signal although 3 pouches were found to be positive, and subculture showed bacterial growth of coagulase -negative staphylococcus. despite the relative small number of tested platelet concentrate units, our findings discourage specialists in attempting platelet storage time prolongation to 7 days. bacterial contamination testing on day 2 and a storage time of maximum 5 days seems to be still the safest practice. bacterial screening of platelet concentrates using bact background: bacterial screening of blood components is a routine measure in the evaluation of blood product quality. at our institute bacterial screening is performed using bact/alert system. sampling and culturing of blood products is performed according to paul-erlich institute recommendations. methods: quality control data on the bacterial screening of platelet concentrates performed from 1999-2004 were retrospectively analysed. an initially positive (ip) and true positive (cp) rate, organisms isolated and time of detection are presented. results: a total of 5359 platelet products were tested during the 6year period. thirty (0.56%) were found initially positive by bact/alert. the cultures screening positive were subjected to bac-terial identification to distinguish false positive from real positive signals. bacterial contamination was confirmed in 14 (0.26%) plt concentrates. positive cultures were confirmed and identified in an independent laboratory ( hbsag, anti-core, anti-hbs, anti-hcv, anti-hiv i/ii, anti-htlv i/ii, rpr. these patients were transfused with 5-131 units of concentrated red cells, depending on their problem. a total 530 units were delivered from the beginning of their problem until december 2003. the control were done using last generation enzyme-linked immunoassay (dade behring, ortho, biomeurieux), as also using automated enzyme-linked immunoassay (axgym). the same patients were checked by their physicians before the initiation of transfusions for the same diseases. results: we found: 12 patients anti-hbc (+) and anti-hbs (+). 5 patients anti-hbc (-) and ánti-hbs (-) 3 patients anti-hbc (-) and anti-hbs (+) 1 patient anti-hbc (+) and anti-hbs (-) 1 patient hbsag (+), anti-hbc (+) and anti-hbs (-). all patients were negative for hcv, hiv, htlv, and rpr. the same results were found also from patients' physicians. conclusions: we conclude that the blood supply for blood transfusion-transmitted diseases is 100% safe in our centre. these results are in accordance with current international literature. this is due to careful selection of blood donors, to high quality of corporation between departments as also to internal and external quality control. all these factors contribute to safety of transfusions, the quality of life of the patients and the protection of patient's environment. neither in the pipetor nor in the extractor runs was found contamination. no false positive were detected and all the positive samples confirmed. (see tables 1 and 2) . for hiv and hcv the specificity was 99.99%. the validation criterion were met, so the system was implemented routinely in our laboratory. the purpose of our study was to analyse the applicability of the pall enhanced bacterial detection system (ebds) in the routine of our transfusion unit which is totally focused on apheresis platelet collection. methods: apheresis pcs, obtained by trima (cobe) and amicus (baxter) separators and re-suspended in 30% plasma and 70% ssp solution (macopharma), were submitted to microbiologic control using pall ebds system which uses oxygen percentage decrease as a surrogate marker of bacterial growth. the working steps were the following: 16-24 hour after donation, about 3 ml of pc were sampled into the ebds collection pouch and then incubated at 35°c under continuous agitation (incubator helmer 900) for 24 hours. after this period, oxygen percentage was measured using an oxygen analyser (pall bdso2). the test is based on the 'pass/fail' principle. in case of 'fail' result the microbiology department has drawn up the procedure to follow in order to confirm the data and to allow the micro-organism to be identified. the incidence of post transfusion hepatitis has been reduced by blood donor screening for hbsag, but the hbv infection is still responsible for a certain cases of post-transfusion hepatitis in world-wide. in this study the hbsag negative blood units were evaluated for anti-hbc and hbv dna by pcr method. an extra sample was collected from 2000 hbsag, anti-hcv, anti-hiv and rpr-negative blood donors. all of samples were examined by approved anti-hbc assay. all of anti-hbc positive samples were tested by hbsab assay and evaluated for hbv dna (pcr). the sensitivity of the hbv dna (pcr) assay was estimated 50 geq/ml according to vqc proficiency and run control panels. 212 (10.6%) out of 2000 samples were positive for anti-hbc.166 (78.3%) out of 212 anti-hbc positive samples were hbsab positive, and 46 (21.7%) were hbsab negative. all of 212 samples were assayed for hbv dna (pcr) single and all of them were negative for hbv dna (pcr). further study for evaluation of anti-hbc test as a screening assay for blood unites in high hbv infection prevalence area strongly recommended. early detection of hepatitis b surface antigen: a comparison of ten assays hbsag detection is the corner stone of detection of hepatitis b virus infection in blood donors and patients with hbv infection. one of the most challenges is sensitivity of the technique and kit. in this study ten different assays evaluated by seroconversion and performance panels. some of them can not be used as screening assay due to low sensitivity. the sensitivity of ten hbsag assays from biorad, dade behring, biomeriux, diasorn, radim, diesse, thermo. biokit, gb and shanghai companies were evaluated by two or three seroconversion and two performance panels from boston biomedica ink. seroconversion panel is a series of samples that collected over a period of time from individual developing antibodies due to a primary infection. for evaluation of the assay sensitivity who and other notified body in the world-wide recommended the seroconversion and performance panels. the hbsag assays are two groups. group one with high sensitivity included six assays. they can detect ad and ay subtypes from 0.1 ng/ml bbi to 0.4-0.5 ng/ml bbi respectively. low sensitivity group included four assays and they can detect ad and ay subtypes more than 0.7 and 0.5 ng/ml bbi respectively. for blood safety, the high sensitivity hbsag assays recommended for blood screening and all assays should be evaluated by seroconversion and performance panels. diagnosis of chronic hdv infection is usually by antibody testing and hbv dna detected by pcr method. it is rare to find patients with two replicating hepatotropic viruses and if the accompanying hbv is replicating, prognosis will be very poor. to clarify the correlation between hepatitis delta virus infection and hepatitis b virus dna positivity, sensitive hbv dna (pcr) assay was used. the presence of hbv dna was investigated in 274 patients referred during the 21 aug. to 28 dec. 2003. all of them were hbsag positive. all samples were evaluated for hbv dna (pcr). the sensitivity of the hbv dna (pcr) assay was estimated 50 geq/ml according to vqc proficiency panel and run control. anti-hdv was tested by commercial available enzyme immunosorbent assay. 179 (65.3%) were hbv dna positive and 95 (34.6%) were negative. 8 (4.4%) out of 171 patients had evidence of delta infection and 13 (13.6%) samples of hbv dna (pcr) negative patients were positive for delta agent. the serum alanine aminotransferase (alt) levels in 5 out of 8 hbv dna (pcr) and anti-hdv positive patients were higher than reference interval, but only in 2 out of 13 hbv dna (pcr) negative and anti-hdv positive samples were higher than reference interval. the present data indicate that 7.7% of patients with chronic hepatitis b have hepatitisdelta infection. patients with hbv dna (pcr) negativity had a significantly higher prevalence of delta marker (13.6%) than those with hbv dna (pcr) positivity (4.4%). delta rna testing in positive hbv dna and anti-hdv patients is recommended. introduction: reduction of the window period of hepatitis c virus (hcv) infection represents an important goal in the transfusional and diagnostic setting and nucleic acid technology-based tests have been introduced in some developed countries to reduce the potential risk of transfusion-associated infection. a prototype assay designed to simultaneously detect circulating hcv antigen and anti-hcv has been developed by biorad (biorad laboratories limited, marnes la coquette, france). aim of the present study: to define the cut-off (co) value of the assay and to evaluate the specificity and sensitivity of this new assay in the detection both of antibody and antigen comparing its efficacy with commercial assays. methods: in order to establish the co value and to evaluate the specificity of the assay, we tested 400 sera samples from the general population and 100 'difficult' sera from haemodialysis patients (n conclusion: the new assay shows high sensitivity and specificity and could be a useful tool not only in the diagnostic setting, where procedures to reduce the window period, such as antigen or hcv-rna detection, are not currently recommended, but also in the screening of blood donations, when nucleic acid technologies is not feasible due to costs, organization, emergency and/or logistic difficulties. introduction: in recent years the concern with the blood safety regarding the transmission of blood-borne viruses has been improved. this safety has been achieved with the combining of different strategies, such as a careful selection of donors, the screening for relevant virological markers and the viral inactivation/ removal methods. more recently, the implementation of the nucleic acid amplification technologies for the detection of hiv-1, hcv and hbv, has increase this aim by reducing the 'window period' of the infections. other viruses, such as parvovirus b19 (pb19) and hepatitis a virus (hav), can raise problems to the blood safety. these infections could provoke serious complications in some risk groups, like pregnant women, patients with haematological problems, children and patients with immunodeficiency. material and methods: an observational study was performed to determine the prevalence of pb19 and hav in portuguese blood donors. we gather, during four months, 5025 plasma donations and joined them into 505 pools, with no more than 10 donations each. [1999] [2000] [2001] [2002] [2003] in voluntary donors the anti-hcv prevalence ranged from 0.3% to 0.7%, in family replacement donors from 0.4% to 1.2%, autologous donors from 1% to 2.02%. we observed that the anti-hcv prevalence has a decline tendency during years in blood donors. according to sex the anti-hcv prevalence in men is 0.7% and women 0.6% (p = 0.004). over the 5 year periods the prevalence in men has a decline tendency (1.2% to 0.4%; p = 0.033) and increasing tendency in women (0.4% to 0.9%; p = 0.007). according to age group the anti-hcv is 0.6% in 17-29 age group, 0.65% in 30-39 age group, 0.8% in 40-49 age group, 0.86% in 50-60 age group (p = 0.001). the prevalence of anti-hcv is higher in fds than vds, but not statistical significant. [1999] [2000] [2001] [2002] [2003] . a total 14627 ftd and 684 ad have been tested for hbsag. a sample was considered as hbsag positive when found repeatedly reactive by 3rd generation. immunoassay method (elisa). the chi-square test was used for statistical analysis. results: the 5-year overall hbsag prevalence among first time blood donors was 8.9%. and ad 6.7%. among autologous blood donors was observed a decreasing hbv prevalence from 6.3% to 3.7% in 2003. according to age the prevalence was higher in 17-29 year group 7.7%, while according to sex was higher in man (8.8%) than female 6.2% (p < 00.5). among ad, a decreased hbsag prevalence according to age was observed in men and women. the same trends by sex and age were observed in ftd. the prevalence of hbsag in ad was lower than in ftd. however, from 1999-2003 hbsag prevalence has decreased in the same proportion in both population. this decreased can explain by to main factor: the improvement hbsag screening method in blood donors and decreased the hbsag prevalence in general population. (1 : 30 250) . the hbv dna-emia in hbsag negative samples was 1.14 ¥ 10 2 -9.55 ¥ 10 4 copies/ml. in two donors anti-hbc total was positive and in one anti-hbe was also detected. in one donor the glycin 145 alanin mutation in the s region was identified. the frequency of hbv dna pos/hbsag neg donors in poland is high (0.002%) therefore the decision to introduce routine hbv nat screening is justified. (1/1001) with stored apheresis and whole blood derived platelet concentrates. of these 118 failed results there were 23 confirmed positives (presence of bacteria in both the ebds pouch and the platelet mother bag by culture) representing 1/5133. the bacteria detected were staphylococcus or streptococcus sp. 76 of the 118 fail results were false positives (no presence of bacteria in the ebds pouch and the platelet mother bag by culture) representing 0.064% or 1/1554, and 18 were not confirmed initial positives (no bacteria in the mother bag by culture, ebds pouch not tested) representing 1/6559. there was one reported case of a missed detection with confirmed presence of bacteria (staphylococcus epidermidis) in the mother bag by culture. subsequently, the bacteria strain was isolated and inoculated into platelet units in our laboratory at levels as low as 5 cfu/ml. in all cases the pall ebds was able to detect. this supports the hypothesis that this missed detection was the result of a statistical sampling error rather than a system failure. the results from blood centers routinely using pall ebds demonstrated effective detection of bacteria in platelet products stored under routine conditions with a true positive rate of 1/5133, and with a low false positive rate (<0.1%). this is comparable to a recent survey result with other culture based systems. summary/conclusions: the minority group of pregnant women who come to labor without prenatal testing of hepatitis b and c revealed essentially similar prevalence of anti-hcv with healthy bd even if definitive confirmation is probably increased in this minority group. there is however markedly higher prevalence of hbv infection in the pw so that screening for hbv is essential for the prevention of vertical transmission. the systematic screening of bd with anti-hbc serves as further assurance for the prevention posttransfusion hepatitis eliminating only 1.21% of the possibly infectious, a percentage which can be restored to the blood pool after proving their immunity. methods: blood samples were screened for the presence of hbsag, hcv and hiv antibodies using enzyme immune assay and for syphilis using the tpha test. the results were analysed retrospectively. all samples with results at or above the minimum positive value were considered reactive. the tests for hbsag, anti-hiv and anti-hcv were repeated in duplicate in all reactive donations. blood units that were reactive in the primary or secondary assays were discarded. hiv positivity was confirmed by western blot analysis using hiv blot 2.2 (genelab diagnostics) results: results from a total of 77 903 screened donors were analysed. hepatitis b surface antigen rates was 1.99%; anti-hcv seropositivity was 0.54%; anti-hiv seropositivity was 0.01% and tpha seropositivity was 0.06%. one study calculated this risk to be one in 63 000 for hbv, one in 493 000 for hiv and one in 103 000 for hcv. it is therefore important to take a careful history from blood donors to eliminate those at high risk of infection. in view of the high infectivity of hiv positive blood, it is important not only to screen donated blood but also to exclude donations from high-risk individuals, such as males who have engaged in homosexual activity and intravenous drug users. a careful history should identify those who should not give blood. in turkey, among blood donors the average hbsag prevalence in 1985-1998 was 5.1%. but it had decreased to approximately 2.1% in 2003. anti-hcv positivity has been reported to be 0.6% between 1992 and 1999. but it was approximately 0.5% in 2003. rpr positivity in blood donors in turkey was reported to be <0.1% in 1973 and 0.0026% in 1990. in 2003, the rpr rates was 0.09%. in our study these rates are 1.99%, 0.54%, 0.09% and 0.06% respectively. anti-hiv seropositivity was found around 0. introduction: the serological detection of specific antibodies to treponema pallidum (tp) is an effective means of diagnosing syphilis, and an automated chemiluminescent assay is ideally suited to testing large numbers of specimens for the laboratory diagnosis of the disease. aims of the study: to develop a qualitative syphilis assay for the detection of tp immunoglobulin m (igm) and g (igg) antibodies. the assay will be used for the serological diagnosis of syphilis using the architect platform, which has the capacity to test 200 specimens/hour. the two-step assay is based on paramagnetic microparticle chemiluminescent technology, utilising microparticles coated with three recombinant tp antigens (tpn15, tpn17 and tpn47) and acridinium labelled anti-human igg and igm monoclonal antibodies as conjugates. in the first step, specimens, microparticles and diluent are incubated together, prior to a wash step; in the second step, acridinium labelled antibodies are added and after washing, pre-trigger and trigger are added to produce chemiluminescence, which is measured as relative light units (rlu). specimens yielding rlus less than the cut-off are considered negative, while those yielding rlus greater than the cut-off are considered positive. the sensitivity of architect syphilis tp was determined to be 100%, after testing 426 specimens that were previously screened as syphilis positive in fujirebio tppa; no prozoning was observed with high positive specimens (over 2 16 titer by tppa). the specificity generated from testing 564 hospitalised patients previously screened as tppa negative, was 99.6%. testing a mixture of sera and plasma from 5000 random donor specimens, generated donor specificity figures of 99.8%. the precision (cv%) with a positive control was 5.8% (95% confidence interval: 4.9-7.0%) by the standard 20-day nccls analysis (ep5a2). in a study conducted at asahikawa medical college hospital, in which, 81 positive and 82 negative specimens were tested, concordance with fujirebio tppa was determined to be 100%. no significant interference to the assay was observed from bilirubin (conjugated type and free type), haemoglobin or lipid. the architect syphilis tp assay is an automated, specific and sensitive test for the detection of antibodies to t. pallidum. background: hcv exposure of blood donors is serologically determined by the detection of anti-hcv antibodies in serum or plasma. however a 'window' period of 30-70 days after exposure exists during which specific antibodies to hcv antigens cannot be detected. hcv rna detection and/or hcv core protein testing result in dramatic reductions in the preseroconversion window period. the new bio-rad test, based on the simultaneous detection of hcv core antigen and anti-hcv (core, ns3, ns4) antibodies, improves the detection of hcv infection in the early phase. aims: the aim of this study is to assess the performance characteristics of this new screening microplate immunoassay, monolisa hcv ag-ab ultra, by using the bio-rad evolis automated microplate processor system. methods: this two-step elisa assay is based on the combination of an indirect test for the detection of antibodies (core, ns3, ns4) and a sandwich test for core antigen detection. results are available within 2.5 hours, with sample addition monitoring and color coded reagents. no specimen pretreatment is required. evolis is a self-contained microplate processor designed for full automation of microplate-based eia techniques. the walkaway system can process four microplates at a time with continuous loading of samples and reagents. positive identification of samples, reagents and microplates, usage of disposable tips with clot detection, integrated quality control and complete traceability provide a high level of safety management. the monolisa hcv ag-ab ultra/evolis system performance is evaluated for clinical sensitivity on 30 commercially available and well-documented seroconversion panels. the results are compared to viral rna detection and conventional hcv ab screening assays. specificity is evaluated by using random blood donor samples. results: among the seroconversion panels that begining with samples negative for hcv rna and anti-hcv antibodies, the monolisa hcv ag-ab ultra assay detects exposure to hcv an average of 25 days earlier than the monolisa hcv plus v2 test. the mean delay of the monolisa hcv ag-ab assay in detecting hcv infection compared to hcv rna testing is around 2.5 days. the monolisa hcv ag-ab ultra/evolis system allows simultaneous detection of hcv core antigen and anti-hcv (core, ns3, ns4) antibodies, thus significantly reducing the time gap between the initial detection of hcv rna and the first appearance of detectable anti-hcv antibodies. the fully automated system combines high degree of assay performance with optimization of laboratory workflow and safety management. operational evaluation of pall ebds bacterial detection system l larrea gonzalez, ma soler and rj roig centro de transfusion, valencia, spain introduction: regulatory bodies are increasingly mandating the use of bacterial detection systems for platelet products (22 ed standards for blood banks and transfusion services). one system currently available is the pall ebds bacterial detection system which utilises percentage oxygen as a surrogate marker for bacterial growth. aims: to evaluate the pall ebds in routine use in our blood centre. in particular, to assess feasibility and adaptability to daily labour routines. the orbisac (gambro bct) system was used to produce leucocyte depleted buffy coat (bc) platelet pools (4 bc/pool) stored in platelet additive solution (ssp macopharma). mean platelet count was 3.2 ¥ 10e 11 /pool with mean leucocyte count 0.09 ¥ 10e 6 /pool. ebds installation and training occurred over a 2 day period. 500 platelet pools were tested for bacterial contamination over the subsequent 5 weeks. ebds pouches were sterile connected onto platelet pools 22 hours after blood donation. platelet samples were taken into the pouches and then the pouches were incubated for 24 hours on a shaking agitator at 35°c. after this time, percentage oxygen was measured. no positive results were found in this study. this was as expected due to the relatively low number of platelet pools tested and it also highlights the absence of false positive results. minimal training was required to use the ebds. the system was easy to use and did not require the use of a laminar flow cabinet to take samples. it was quick and simple to take samples and perform oxygen measurement. after pouch incubation, 1 technician was able to make 20 oxygen measurements in less than 13 minutes. the data management system allowed full traceability of product and work flow. results were very easy to interpret. conclusion: the pall ebds was found to adapt perfectly to a routine blood centre environment. (2004) was 8470. the percentage collected from volunteer blood donors was 56% (n = 4783) and the rest 44% (n = 3687) was given from patient-related donors. the age of donors ranged from 18 to 65 years old. the assay used for the detection of hbsag, hbeag, anti-hbc igg/total, anti-hbc igm, anti-hbe and anti-hbs was the automated microparticle enzyme immunoassay (axsym) of the abbot company. all the units were tested for hbsag, and anti-hbc igg. if the anti-hbc igg was detected, the specimens were automatically tested for anti-hbs. the units were wasted if the anti-hbs was negative, and the specimens were manually programmed for the testing of the anti-hbc igm, hbeag, and anti-hbe. from the total of 8470 tested units, 1195 of them were found to be positive to at least one marker of hbv infection, that means the 14.11% of the health adult population was infected in the past by the hbv. the 13.69% (n = 1160) was previously infected and now immunized with hbsag(-) and anti-core igg(+) and 0.41% (n = 35) were chronic carriers of the hbv with hbsag(+). the 68.57% (n = 24) of the positive donors were patient related donors and 31.42% (n = 11) were volunteer donors. in other words, 24 of the 3687 not volunteers (0.65%) and 11 of the 4783 volunteers (0.23%) were detected to be infectious. the combinations of the serologic markers for hbv are illustrated in the table attached. these results indicate that the incidence of hbv infection in the northeastern department of greece is equivalent to the incidence of hbv in other greek regions (0.42%) as it is referred to the national haemovigilance data and moreover, the percentage of infectious donors is bigger among replacement donors, 0.65% compared with the 0.23% of voluntary donors. as a consequence, the best source for safe blood collection is the population of volunteers. earlier detection of human immunodeficiency type 1, hepatitis c and hepatitis b viruses using the procleix® ultrio tm assay on the procleix® system and the study objective was to assess the ability of the ultrio assay and associated discriminatory assays to reduce the detection windows for hiv-1, hcv, and hbv. commercially available seroconversion panels were used for testing. methods: hiv-1 (n = 13), hcv (n = 12), and hbv (n = 16) seroconversion panels were tested neat and diluted (1 : 8 and 1 : 16) in the ultrio assay. panels were tested neat in the appropriate discriminatory assay. times to detection of hiv-1, hcv, and hbv nucleic acids in seroconversion panels were compared to the vendor's historical data on time to detection of antibody and/or antigen using licensed or validated serologic tests. p-210 effectiveness and limitations of methods for platelet bacteria screening -how to apply which screening method? the successful concept of virus safety in transfusion medicine is not suitable in bacterial contamination. bacteria can grow up in blood components to enormous amounts, whereas the initial number of contaminating bacteria is typically very low. therefore, sample drawing for bacteria screening must not be done immediately after blood donation. the established concept of relevance of clinical microbiology (pathogenic, non-pathogenic, facultative pathogenic species) is not valid for bacteria contaminating blood. here, the currently discussed criterion of clinical relevance is the ability of bacteria strains (not species!) to grow up in blood components. the paul ehrlich institute (pei) developed pei bacteria standards, which are characterized concerning their behavior in blood components. they contain a defined number of living bacteria, they are deep frozen, ready to use and shippable. there are two strategies to improve bacteria safety of blood: screening and pathogen reduction. neither of them is perfect, but screening methods are successfully established since several years in routine (belgium, the netherlands), and represent the current state of the art. further development and collecting of experience will produce the basis for assessments in the future. it is of high importance to apply the screening methods in dependence on their properties. methods implying an incubation/cultivation step ('early methods') have to be distinguished carefully from 'rapid methods' . for example, it is unreasonable to compare (or to advertise with) different sensitivities of methods not considering their detection principle or their informative value. both principles, cultivation methods as well as rapid methods, show advantages and disadvantages. selection of the method has to consider the respective conditions of the given blood service (including logistics up to time frame between issue and transfusion). results from the procleix hiv-1/hcv and hiv-1/hcv/hbv (procleix ultrio) assays for the detection of hiv-1 rna, hcv rna and hbv dna in blood donors of two blood transfusion centers of sw greece in discriminatory assay testing, 3 out of 5 (60% of the positive, 0.020% of total) were reactive for hcv rna only and 2 out of 5 (40% of the positive, 0.013% of total) were reactive for hiv-1 rna only. none were positive for both hiv-1 and hcv. the standard serological assays gave the same results for the above positive samples. two samples that tested positive by the standard serological assays tested negative in the procleix hiv-1/hcv assay. of the 4151 samples tested by the ultrio assay, 21 (0.5%) tested reactive for hiv-1/hcv/hbv. in discriminatory assay testing, 1 out of 21 (4.76% of the positive, 0.024% of total) was reactive for hiv-1 rna, 4 out of 21 (19% of the positive, 0.096% of total) were reactive for hcv rna, and 16 out of 21 (76.2% of the positive, 0.38% of the total) were reactive for hbv dna. all were single positive i.e. none tested positive for more than 1 virus. three out of 16 positive samples for hbv dna tested negative by the standard serological tests. the opposite was not observed. the procleix ultrio assay is a definite improvement over the procleix assay in a region with a high incidence of hbv carriers. up until its use, it is obvious that hbv positive blood with very low antibody titers was transfused into patients. more results will show whether procleix ultrio can eventually replace the standard serological tests. the introduction: patients with hemophilia represent a high-risk group for post-transfusion hepatitis whose frequency is closely linked with the number and quantity of blood products used. in albania, the frequency of hepatitis is also linked with hbsag testing with elisa (introduced in1993), and hcv testing (introduced in 1997). aim of the study: evaluation of the prevalence of the markers of hepatitis b, c, and d in patients with hemophilia. methods: our study included 145 patients with hemophilia treated with cryoprecipitate and commercial clotting factors. blood testing for anti-hcv, anti-hdv, and hbsag was performed with elisa -gen. iii. results: of 145 patients tested, 26 cases (17%) were hbsag positive, 87 cases (60%) were anti-hcv positive, and 7 cases (5%) were anti-hdv positive. co-infection of hbsag and hcv was found in 16 cases (11%), whereas co-infection of hcv, hdv, and hbv was found in 4 persons (3%). the highest rates of infections and coinfections were found in patients above 30 years of age. conclusion: mandatory blood testing has decreased the levels of post-transfusion hepatitis. in albania, hemophilia is also still treated with cryo-precipitation, thus patients are at a particularly high risk during the 'window period' . results: 2/16 760 (0.012%) samples from rbd were anti hiv 1 + 2 nonreactive and rr for p24 ag both being nonreactive in the neutralization test, they were interpreted as false positives. 1/134 617 (0.0007%) sample from fbd was rr for p24ag/anti hiv 1 + 2 nonreactive and it was confirmed positive by neutralization. this bd had been autoexcluded himself after blood donation. he showed seroconvertion 21 days later: p24ag nonreactive, anti hiv 1 + 2 reactive and western blot positive. the only bd p24ag positive/anti hiv 1 + 2 nonreactive during the analized period, was an first time donor and the post donation autoexclusion was effective en this case. although a larger populations of bd is necessary to be studied and in spite of the low prevalence we have found, we consider p24ag screening is an alternative up to implementation of nucleic acid testing and simultaneously we should increase the quantity of altruist repeat blood donors, undoubtedly, the best population to give blood. owing to the rather short interval between successive donations (~70 days), this suggests that some 260-310 infectious units escape the screening annually. to these, one has to add the (now unknown) proportion of potentially hbsag negative + hbv dna positive ftbds. hcv: since the introduction of the screening in 1995, the general incidence in rbd has dropped from 13.1‰ to 0.001‰, suggestive of a 1 : 10 000 escape rate. the prevalence in ftbd has stabilized at 12 ± 2‰. based on reasons similar to these employed for hbv, the residual incidence in rbd suggests that 1 potentially infectious donation in 10 000 rbd escapes the screening (= to a total of aprox. 40, annually). a limited investigation using hcv-antigen eia evidenced a 1‰ escape rate in ftbds (= to a total of aprox. 50, annually table 1 and 2 are concerned to the fist two months of the implementation, where we had to adjust the volume of the eluate. conclusion: these system adjusts to the laboratory daily routine in the blood bank, with the pools released after first analysis in less than 18 hours. background: the hbsag, anti-hcv, anti-hiv1/2, p24 antigen, alt and syphilis tests are performed for blood donations in czech republic. no nat tests are mandatory in czech republic. the aim of this pilot study was: 1. hcv rna pcr testing in anti-hcv negative blood donations; 2. correlation between hcv nat and anti-hcv testing results. methods: blood samples (anti-hcv serologically negative, alt not elevated) were pooled using the guardian plus spii into pools of 24 samples. pools of 1 ml were tested using the cobas ampliscreen hcv test v. 2.0 (roche). results: 891 pools of 24 samples from 21 384 a-hcv serologically negative donations were tested from october 2002 to july 2004. no one pool was initially reactive. invalid tests: 14 (1.6%) run failures were observed, due to: invalid internal controls 10 (1.1%) and invalid positive controls 4 (0.5%). invalid tests were repeated. in none of 891 pools a positive hcv nat result was observed. conclusions: no discrepancy between hcv nat and a-hcv results was observed in our study. all of the nat tested donors in our study were regular voluntary whole blood or plasma donors who were repeatedly a-hcv serologically negative. the hcv incidence in the czech republic blood donor population is low but it is slightly growing up in general population. hcv nat testing could improve the safety of blood supply by reducing the window period for hcv. introduction: parvovirus b19 is the only parvovirus known to be a human pathogen. most commonly, it causes a mild childhood rash, erythema infectiosum, but in some cases more serious symptoms can be linked to b19, such as acute or persistent arthropathies, critical failures of red cell production, hydrops fetalis, fetal loss, myocarditis or hepatitis. inactivation of the non-enveloped virus has proven difficult. as a consequence, manufacturers of blood products have implemented screening measures to reduce the load of parvovirus b19 in manufacturing plasma pools by the use of nucleic acid amplification techniques (nat). in our institute all blood donations were screened for human parvovirus b19 by nat since april 2000. methods: over the last 5 years 5.5 million donations were screened for b19 by nat. samples with a virus load over 105 iu/ml were defined as positive, whereas samples with a virus load between the detection limit (103 iu/ml) and 105 iu/ml were defined as weak positive. weak positive products were released, whereas positive products were discarded. in addition infection markers of 50 b19 positive donors (case group) were determined over a time period of one year. virus load and b19 antibody status was compared with 100 b19 negative donors (randomised control group). b19 antibodies (igg vp2, igm vp2, ns1) were analysed by two commercial antibody tests. results: overall 496 b19 nat-positive donors were identified with a virus load over 105 iu/ml out of 5.5 million tested. there was a seasonal accumulation during spring and summer, whereas a large epidemic occurred throughout the last year. vp2 igg was detected in 90.9% and 74% of the case and control group, respectively (p = 0.01). these data demonstrated statistically significance (p = 0.01). all donor samples which were b19 nat positive for more than three months developed neutralizing vp2 antibodies. in contrast, ns1 antibodies were observed in 83% of the case group and in 15% of the control group (p < 0.01). ns1 antibodies were detected more frequently in samples, which were b19 nat positive for more than six months. conclusion: b19 nat could be implemented in blood donor screening as release criterion without causing a shortage in blood supply. all b19 positive donors of the case group developed neutralizing antibodies within three months and virus load was dropped rapidly below 105 iu/ml. these data support our testing algorithm all components of high positive donations (virus load over 105 iu/ml) were discarded. donors with ns1 antibodies showed more often signs of a chronic disease with detectable levels of parvovirus b19 longer than six months. background: on recent years, the syphilis screening of blood donors has become increasingly important not only because of the transmission risk of this infection but also due to the risk behavior that this implies. on account of the importance of this screening the tests used are becoming more and more sensitive. aims: to evaluate an elisa screening test (the tmpa test recombinant is based on the sandwich principle, an immunoenzymatic technology in solid phase, for the measure of anti-treponema pallidum in serum or plasma). methods: in this study 1696 samples from blood donors were tested by the rotine 'cardiolipidic reagent for syphilis screening on microplates' -diagast laboratories as well as with 'hdtmpa recombinant' -hoslab diagnostics. positive samples were then confirmed with fta abs/tpha. results: using the mentioned tests we obtained the following results: 1. 1666 (98.23%)cases turned out negative with both technologies; 2. 3 (0.17%) cases were positive in both methods; 3. 27 cases were positive only using tmpa recombinant [of which 23 (1.36%) were confirmed positive by tpha/fta abs. as seen we found 23 samples (1.36%) that were only positives by tmpa recombinant test and that were confirmed by tpha/fta abs. we concluded that tmpa recombinant seems to be a suitable test for a quick and automated syphilis screening of blood donors and provides maximum safety for the recipients. background: in recent years, there has been substantial evidence indicating that typing and subtyping for hcv is clinically important in understanding hcv disease and its therapeutycal options. 'naive' viral load also seems to influence disease severity and responsiveness to therapy. therefore, viremia and genotype identification have been done routinely in molecular biology laboratory units. aims: the university hospital of coimbra studies and tests his own patients and patients from 11 other hospitals in the central portugal. we also collect and test blood donor candidates from this region. we proposed to analyse the distribution of hcv genotypes in this region, among 1578 patients with cronic hcv infection. we have simultaneously analysed the viremia and correlated it with age and severity of liver disease. methods: nucleic acid extraction was done using the semiautomatic 'xstractor' from biomerieux laboratories (boom method). the genotyping used reverse hybridization and was performed using probes from the 5¢ non-coding region (innulipa introduction: bacterial contamination of blood products remains a persistent problem. various techniques for the detection of bacteria in blood products exist but none of them has been widely accepted. bacterial detection systems could be divided into culture systems and rapid technologies. hemosystem has developed a rapid and sensitive technology for bacteria detection named scansystem tm . bacterial contamination of platelet concentrates is a rare event with an incidence between 1 : 2000 to 1 : 3000 per donation. therefore hemosystem developed a positive control in order to validate the scansystem tm platelet kit before use. aim of the study: the current study was designed to evaluate the performance of the scansystem tm positive control. the scansystem tm positive control is a capsule containing lyophilised lactobacillus casei subsp rhamnosus. the bacteria concentration per capsule is at least 8 ¥ 10 8 cfu. the positive control has to be stored at room temperature and is stable for 2 years. after dilution in pbs, the preparation has to be used within 1 hour. two capsules were tested for ten consecutive days with scansystem tm platelet kit as well as with optimised scansystem tm platelet kit. in an independent experiment three capsules were diluted in platelets stored in additive solution and were tested each with scansystem tm platelet kit and optimised scansystem tm platelet kit. results: 25 microscopic fields were analysed for bacteria specific fluorescence for each sample. the ratio between bacteria specific fluorescence signals and analysed signals was 1.0 in all samples for both scansystem tm platelet kit and optimized scansystem tm platelet kit. therefore by definition all tested capsules were positive. the lyophilized positive control capsules enable the user to validate the scansystem tm platelet kit before use. because bacterial contamination of platelet products occurs rarely, the routine use of positive controls improves safety of the screening method. scansystem tm is currently the only method that provides this safety measure. introduction: whereas implementation of nat for blood donor screening reduced the risk for transfusion transmitted hiv and hcv infections currently below one per 23 million transfusions, the risk for bacterial infections is estimated to be 1 : 200 to 1 : 3000. especially platelet products, which are stored at room temperature, are prone to bacterial contamination. aim of the study: several methods are currently developed to prevent the transfusion of bacterial contaminated platelet concentrates. the study investigates a new rapid bacterial detection method. material/methods: pool platelet concentrates were spiked with seven transfusion relevant bacteria strains under sterile conditions at concentrations of 100 cfu/ml to 106 cfu/ml. bacterial concentration was verified on blood agar plates immediately after spiking. five millilitres of spiked platelet concentrates were centrifuged, stained with thiazole orange dye and analysed directly by facs within five minutes after staining. aliquots of pool platelets spiked with concentration of 102 cfu/ml and 101 cfu/ml of each bacteria strain were incubated for two to eight hours in special bouillon at 37°c and were analysed by facs immediately after incubation. results: sensitivity of facs analysis differed between 103 cfu/ml for e. coli and 105 cfu/ml for klebsiella pneumoniae without preincubation and was enhanced to 101 cfu/ml when a pre-incubation step of two to four hours was included. conclusion: bacteria detection by facs analysis combined with a short pre-incubation (2-4 h) at 37°c is a quick and simple method with sensitivity comparable to other commercially available detection systems. the advantage of this new method is the rapid analysis, easy handling, high sensitivity and less expensive price. introduction: detection of bacterial contamination of platelet concentrates represents a major challenge in transfusion medicine. for blood transfusion services the method must have a high sensitivity, an easy performance and a low price. aim of the study: in this spiking study we evaluated the new optimised scansystem tm platelet kit detection method for use in apheresis platelets. methods: apheresis platelet concentrates (apcs) were spiked with strains of ten different bacteria species. after different incubation periods, apcs spiked with 10 cfu/ml were analysed by the optimised scansystem tm platelet kit. the number of bacteria was monitored by plating on blood agar. results: all bacteria strains were detected with the optimised scansystem tm platelet kit when the sample was collected 24 h after spiking. identity of the spiked bacteria was confirmed by gram staining and dna fingerprints. conclusion: in summary, the optimised scansystem tm platelet kit was able to reliably detect ten transfusion relevant bacteria species in apheresis platelet concentrates within 70 minutes when the sample was taken 24 hours after spiking. background: since year 2000 our laboratory started routine screening of hcv-rna in plasma minipools for all plasma intended for fractionation. although nat testing is not yet mandatory all blood products are released depending upon nat results. aim: to test and compare two different methods of rna extraction in order to make all the necessary adjustments to the test procedures while preserving the availability of blood products. methods: plasma minipools of 24 donations are prepared either on a tecan genesis robot or on a hamilton at plus. hcv-rna is isolated from 2 ml plasma by using either the qiagen biorobot 9604 and qiamp virus biorobot 960 kit or the manual extraction with cobas ampliscreen hcv pcr kit v 2.0. results: between march 2000 and december 2004 a total of 238 000 seronegative donations (9916 pools) were tested for the presence of hcv-rna. four pools were found to be positive for hcv-rna. of the four nat-positive pools with no eia-positive donor, four were confirmed as true positive by donor follow-up testing and/or testing of an independent sample from the index donation. all the positive donations were detected independently of the extraction method used (manual or automated). our experience shows that although the automated extraction method is 'off label' and it has to be validated, the use of biorobot 9604 does not pose a detectable contamination risk and it is possible to achieve a detection level for hcv less than 50 iu/ml. the advantage of the manual method is that it has better recovery of nucleic acids than the qiagen extraction. concerning the time needed for the extraction process the automated method runs 96 samples in 2 hours where the manual method needs 3 hours for 24 samples, needing, prior to extraction, an extra centrifugation step for one hour. the automated extraction method results in an assay with a high sample throughput, fast time, sufficiently sensitive, that can be successfully introduced into routine use in laboratories which have more than 800 samples/day while preserving the availability of blood products. anti-hcv similarly was high till 2002 (0.5-0.8%), but in trend to decrease afterwards (0.6%). anti-hiv reflected the low endemicity of the disease in public setting and was 0% through the mentioned years. rpr test for syphilis was around 0.1%. directed donors were 95% of all and volunteer donors consisted nearly 5%. donors in our blood center are being informed about donation prior to giving their blood and donor questionnaire forms (dqf) are filled out by the donor candidates. using dqfs have been mandatory at all blood banks in turkey by law since 1996. from that time infectious disease marker rates were dramatically reduced at all centers. donor information about the risks of transfusion and the importance of safe blood supply were detailed by the donation staff and physicians, consequently self-exclusion by the donor candidates who have risky behaviors was encouraged at our center. the interviewing staff was trained specifically for this topic. this steps were particularly emphasized in the last three years and the infectious screening results were displayed the outcome of this efforts. conclusion: education of the prospective donors, and recruit the voluntary, non-remunerated and regular donors will be the utmost goal of all blood banks. rigorous donor selection will contribute this ultimate success. we should spend more efforts to maximize enrolling voluntary donors to lower the serological marker results, consequently achieve safe blood. background: human t cell lymphotropic virus type i is endemic in japan, the caribbean, southeastern united states and parts of south america and africa. in non-endemic areas such as europe, htlv-i is less common and most infections are identified in immigrants. the epidemiology of htlv-ii is different, being predominantly found among indigenous american-indian populations and among ivdus, but the routes of transmission are the same. aim: our study's aim was to ascertain the prevalence of htlv i/ii in blood donors in order to understand the epidemiology of htlv in greece and initiate discussions of an acceptable level of risk and appropriate level of screening for rare transfusion-transmitted diseases. overall, anti-htlv seroprevalence levels among blood donors, are low. although the number of annual donations in this study is relatively small, the data for htlv indicate that rates of this infection are low and that infected donors will be seen infrequently. as all blood donations are screened for htlv i/ii during the last six years, a national survey is necessary in order to define the epidemiology of htlv in greece. introduction: toxoplasma gondii is the causative organism of toxoplasmosis. the disease transmitted by ingestion of either oocysts (in the feces of cats) or bradyzoites (in raw or undercooked meat). the parasite can also be acquired transplacentally by organ transplantation or from blood transfusion. the purpose of this study was survey of toxoplasma antibodies in some iranian blood donors at tehran blood center. blood samples were randomly collected for detecting of igg and igm antibodies (by elisa technique).the total numbers of donors was 217 of 52 (%24) were female and 165 (%76) male in age ranged from 18 to 55 years. results: 217 sera tested, 128 (59%) were found to be positive for toxoplasma igg antibodies and 6 (2.8%) were igm antibodies positive and 2 of them (0.9%) were borderline for igm antibodies. among males the frequency of positivity was higher than woman but this different was not significant. the most frequency of seropositivity was found in age group 41 to 50 years. conclusions: diagnosis of toxoplasmosis can be aided by serologic or histocytologic examination. the acute infection in healthy individuals is generally asymptomatic and not associated with any morbidity but in an immunocompromised host, toxoplasmosis be a very serious disease, and this can occur if a person is infection with toxoplasmosis before or after his/her immunosystem is compromised. in spite of the progress in the development of diagnostic, therapeutic and prophylactic methods, virus hepatitis still presents a serious global health problem. the possibility of transmission of these infections through transfusion of blood and blood derivates implies obligatory control of the donated blood. post-transfusion hepatitis is an important health problem in everyday practice, especially in patients who have to receive transfusion of erythrocyte concentrates as the only possible treatment for many years. objective: to show the prevalence of hepatitis b (hbsag) and hepatitis c (anti hcv antibodies) in multitransfused thalassemic patients. in our region there are 5 patients suffering from thalassemia major who are aged between 9 and 17, and who have been receiving erythrocytic transfusion 1-2 times a month since the age of one or two. they receive washed red blood cells, and in certain periods filtered red blood cells, controlled for viral markers and they mostly receive blood from voluntary, periodic and regular donors. the patients are tested periodically for the presence of viral markers (hbsag, anti hcv antibodies), using tests for hbsag (abbott auxyme monoclonal eia) and for anti hcv (abbott hcv eia 3.0). the presence of markers for hepatitis b and hepatitis c has not been detected in any of these multitransfused thalassemic patients who receive at least 20 transfusions a year. the tests in all 5 patients were negative. the blood used for transfusion must be tested for viral markers, and for the patients who have to receive blood for their whole life, the blood should be from voluntary, regular and periodic donors who donate blood at least three times a year, because then the risk of transfusion transmissible infections is very small. introduction: we observe yearly the prevalence of transfusion transmitted diseases following instructions of skae (national coordination haemovigilance centre). aims: to investigate the prevalence of the most important blood borne infections in our blood transfusion centre in the state achaia during the last five years (2000) (2001) (2002) (2003) (2004) . materials and methods: the detection of hbsag, anti-hcv, anti-hiv 1/2 was made by automated microparticle enzyme immunoassay (axsym, abbott) and by enzyme-linked immunoassay methods (dade behring, ortho, biomerieux) syphilis tests were made by using rpr kits. confirmation for anti-hcv positive samples was made riba or inno-lia, while the confirmation of anti-hiv1/2 positive samples was made by 'st. andrews' general hospital of patras reference centre. results: all the seropositive donors were first time donors. conclusion: (1) we observe that there is a decrease in all four infections. (2) the absence of anti-hiv seropositive donors is due to the high percentage of volunteer blood donation which approaches 70% in our centre during the last four years. methods: a prospective, one-year study has been set up in order to enrol at least 40 000 out of the estimate of 200 000 first-time donors, involving 21 blood transfusion centres from 9 of the 21 italian regions. each centre was required to enrol all first-time donors born before december 31st, 1978, and thus not included in the hbv mass vaccination campaign. the selected donors were tested for hbsag (mandatory by law) and for anti-hbc by commercial assays. all hbsag and/or anti-hbc positive specimens were stored frozen and sent to a reference laboratory for additional serological testing (anti-hbs, anti-hbe, anti-hbc/igm and anti-hbc avidity index by an experimental procedure) and for the determination of hbv-dna (both qualitative and quantitative) by real-time pcr. results: in the first 9 months of the study the 21 sites saw almost 29 000 first-time donors, of whom 70.5% belonged to the required age groups. among eligible donors, 0.4% were both hbsag and anti-hbc positive, and 10.3% were hbsag negative/anti-hbc positive. hbv positivity rates were higher in southern than in northern regions, although a high variability in hbv prevalence was observed between neighbouring areas in the north. hbsag positives were mostly males, 93% were positive for anti-hbe, 6% had raised alt and 8% were concurrently positive for anti-hbs. among hbsag negative/anti-hbc positive donors, 18% were negative and 82% were positive for anti-hbs. among anti-hbs positives, 35% showed values <100 miu/ml and 65% > 100 miu/ml. the avidity index results suggested that approximately 30% of anti-hbc positive individuals were recently infected. conclusions: our preliminary data indicate that approximately 70% of the italian first-time donors are older than 28 years of age and thus not belonging to the age groups who underwent to the mandatory vaccination against hbv, and that 10.7% of them have serological markers of ongoing or past hbv infection. anti-hbc alone was detected in nearly 2% of the study population. hbv-dna testing is underway at the time of this writing. in our country mandatory tests for each blood donations are: hbsag, anti-hcv, anti-hiv1/2 and tp ab. to c + ns3 + ns4, 9 (18.4%) to c + ns3, 4 (8.2%) to c + ns3 + ns5, 2 (4%) to ns3 + ns4 + ns5, 2 (4%) to c + ns4 + ns5, 2 (4%) to ns3 + ns5. the use of the hcv core ag elisa test system may provide substantially earlier identification of hcv infection than it is possible with current serological assays. although all of six anti-hcv assays are very sensitive and specific screening assays, they didn't detect hcv infection in one patient. majority of anti-hcv positive patients (77.5%) had anti-hcv ab for 3 or more different epitopes of hcv. international comparison of performance of abbott prism assays used for blood donor screening background: the national serology reference laboratory, australia (nrl), coordinates a quality control (qc) programme for laboratories that screen for anti-hiv 1&2, anti-hcv, hbsag and anti-htlv i/ii using the abbott prism assays. nineteen laboratories from australia, belgium, canada, ireland, israel, the netherlands, new zealand, norway, singapore, south africa and thailand have submitted data for this programme. aims: to determine the accuracy and precision of results from laboratories, individual prism instruments and different reagent lots by analysing data accumulated between october 2001 and january 2005. the multi-marker qc sample 'pelispy s2058 type 7' (s2058), produced by viral quality control (now acrometrix-viral quality control), was provided to participants. laboratories tested s2058 in each calibration run, in addition to the manufacturer's controls, on each sub-channel of the instrument. pelispy was used as a 'go/nogo control' and results were required to be reactive (s/co > 1) for a test run to be deemed valid. data were collected and analysed using the nrl's internet-based application edcnet (https://www.nrlqa.net). after submission, laboratories were able to compare their results with those submitted by other laboratories and investigate differences in results from reagent lots and instruments. data for five different s2058 lots were exported from edcnet and analysed. results: nearly 95 000 results were submitted: all results were reactive (s/co > 1). fifty of these results (0.0005%) were excluded from analyses because they were reported from invalid test runs [due to pipetting, aspiration or sampling error (n = 48) or due to unacceptable results (n = 2)]. a further 16 results were excluded because data provided by laboratories were inconsistent or incorrect. a total of 94 189 results, reported using 332 different prism reagent lots (145 for anti-hiv, 79 for anti-hcv, 55 for anti-htlv and 53 for hbsag), were analysed. results from prism hbsag and anti-hiv showed the least variation with coefficient of variations (cv) of <10% for all s2058 lots. results from prism anti-hcv and anti-htlv produced cvs between 9.17% and 15.38% for all s2058 lots. data reported for s2058 lot ps030618 (n = 38 662, range 8080 for anti-htlv to 10 225 for hbsag) were analysed further to review performance of prism reagent lots. hbsag showed the least variability between prism reagent lots with <8% bias for the 14 prism hbsag reagent lots used (bias: the difference between the mean ratio for the reagent lot and the weighted mean ratio for all reagent lots, expressed as a percentage of the weighted mean ratio for all reagent lots). prism anti-htlv showed greater variability between reagent lots with a single reagent lot generating a +63% bias. prism anti-hcv showed the greatest variability within reagent lot with results from 12 of 21 reagent lots showing a cv between 10% and 13%. conclusion: in 94 255 results in a qc sample distributed to 19 laboratories the abbott prism performance was found to be consistent over four assays. edcnet was robust in supporting laboratories' abilities to follow precision and accuracy of the assays in real time. introduction: since hcv rna testing of all blood donors started in finland in 2000, the nat screening process has continuously been improved. investments in process automation have made the work more efficient and blood safety has further increased since hiv-1 rna screening of all blood donors started late 2004. aim of the study: to implement hiv-1 rna testing in the nat screening program cost effectively, without increasing the throughput time of the samples and delay in the result reporting. to study if the sensitivity for both hiv-1 rna and hcv-rna will be sufficient when a single extraction is used and when the pool size of 96 donations and the sample volume are kept unchanged. methods: the nucleic acids were isolated from plasma samples of 1 ml with the magna pure lc instrument using the magna pure lc total nucleic acid isolation large volume kit. the internal controls from the cobas ampliscreen multiprep specimen preparation and control kit and the cobas amplicor hcv specimen preparation kit were added to the lysis/binding buffer (5 ml/ml of each). from the final volume of the nucleic acid eluate (100 ml) 50 ml was used for the detection of hiv-1 rna (roche cobas ampliscreen) and 30 ml for the detection of hcv rna (roche cobas amplicor). a run control containing both hiv-1 rna (200 iu/ml) and hcv rna (50 iu/ml) was included in all extraction runs. sensitivity of the both assays was assessed by testing dilution series of the who standards for hiv-1 rna and hcv rna. specificity was evaluated by testing fractionation plasmapool samples (n = 35) and minipool samples (n = 68). results: detection limits of the hiv-1 and hcv assays (95% hit rate) were calculated to be 100.1 iu/ml and 18.7 iu/ml respectively. specificity for both assays was 100% and during the validation phase also the robustness was good. the sensitivity of both assays with a pool size of 96 was below the recommendations by the council of europe for blood donor screening (for hiv-1 rna 10 000 iu/ml and for hcv 5000 iu/ml per individual donation). specificity of the assays was excellent, false reactive results were not observed. implementation of the hiv-1 nat assay in the screening program did not increase the throughput time of the donor samples when the pool size of 96 donations, 1 ml sample volume and a single extraction for two assays were used. the the very substantial increase in the number of industry-sponsored clinical trials has created challenges for medical schools, academic hospitals, faculty members of these institutions, and the journals that publish the results of these trials. in many cases, authors of reports of industry-sponsored clinical trials are paid consultants to the sponsor, have been paid by the sponsor to lecture on behalf of its products, or have equity in the sponsoring company. these ties to industry create a tension that actually is or can be perceived as • work internationally; • send young volunteers to international youth forums; • employ young people in your organisation; why use volunteers in blood donor recruitment? • they have networks to scout-groups, sports-organizations, tradeunions, rotary, staff of large companies etc. • they bring in fellow volunteers -with different prospects of society; • often paid recruiters are underpaid (!) and tend not to remain • you can not recruit by telephone! • 2 out of 3 donors are recruited by personal contact! • so you need direct personal contact = need many people (e.g. young ambassadors); • a large number of volunteer recruiters is a gift from heaven! paid donation gives the act of blood donation low status. the act of blood donation should be respected, and praised by role models, queens and presidents. efficient work and close cooperation of blood bank staff and volunteer organisations is the key to success in blood donor recruitment and retention! with such a prospect, it was important to evaluate the practices of blood donor selection in the eu. material and methods: a questionnaire was designed and sent in 2003 to the relevant institutions of the 15 eu countries plus switzerland. the questionnaire included questions on the interviewing practices before homologous blood donations, regarding non-specific risks to donors and recipients, identified risks to donors, infectious, bacterial, viral, parasitic and prionic risks to recipients and non-infectious risks to recipients. the questionnaire also inquired about each country's exclusion period for each contraindication (ci) to donation. results: predonation interviews were prepared, in all countries, by circulating informative documents to blood donors. they were supported by written questionnaires in nearly all countries. in half of the countries, those interviews had to be led by physicians (nurses or technologists in the others). the 18-65 age limits for blood donation (18-60 for a first donation) were the rule in 11 countries. in other countries the age limit could be brought forward to 17 and extended to 70 years old. the time interval between donations was identical for men and women in 11 countries, and varied from 8 to 16 weeks according to country. the questions of the questionnaires were very similar as regards the identification of risks to donors and recipients, and very close to the requirements that appeared later in the 2004/33/ec directive. this particularly concerned how to meet the expectations of european donors, so that they come back to your blood center! know your donors: make regular donor surveys. age, gender, number of donations, number of first time donors, media consumption, education, job situation, income brackets. use local donor organisations let volunteers help! they work for free, but donor recruitment and -retention costs money. each blood center should have a local donor organisation, run by volunteers. the donor organisation should receive a payment for each bag collected. the reputation of the blood system tell the donors, what the blood is used for. that all blood is tested. and that blood provides safe medical treatment safety of the donor. insurance is a must good quality and efficiency in blood services decentralized blood collection no waste, and minimal outdating efficient service: • a friendly environment, • donor friendly opening hours, • pleasant rooms, beds and well equipped waiting rooms, • parking-spaces, transport, • beverages and food, • letters with correct data etc. donors expect to be serviced by trained, medical professionals and that the medical check-up is taken seriously. donors should be recognized continuously. use directed press-coverage to higher the self-esteem of the donors. donors should be well informed: • leaflets, posters and questionnaires should be 100% correct; • use e-mail and web-sites for quick up-date of donor information. be visible! • have an offensive and comprehensive media approach; • have a yearly national campaign 14 june up to world blood donor day; • have an attractive home-page, constantly updated; • streamline your lay-out; • mail a donor magazine to all regular donors: • send newsletters regularly to volunteers and the press. • use recruitment cards. • easy phone-and fax numbers, e-mail addresses. directed campaign towards young people: • young ambassadors group; • special training sessions for young volunteers; • advertisements in media catering to young people; • poster competitions; • book and leaflets on blood addressed directly to young people; minimum bodyweight, blood pressure, pulse limits, questions involving viral risks, either sexually transmitted or linked to drug abuse, questions investigating risks of malaria transmission, questions aimed at identifying risk of prionic disease transmission. analyzing ineligibility times, on the other hand, revealed wide differences. for example, ineligibility for current multiple sexual partners, sexual relations with risk individuals, tattooing or body piercing, endoscopy, general anesthesia or invasive surgery, could vary from 3 to 12 months. previous transfusion history could not be a ci or could be one varying from 6 months to indefinite ci (this point recently changed in several countries). the results of that survey have revealed some differences between countries in the questions asked and especially in the ineligibility times. however, the conditions under which donor selection interviews are conducted were similar in all countries. the enquiry tool used in this study proved to be well adapted to evaluate the donor selection practices throughout eu. a next step will be to use it to appreciate their evolutions and especially the impact of the 2004/ec/33 directive on these practices in the 25 eu countries and furthermore to evaluate the results of this selection (rates and motives of deferral) which is a major factor of patients' transfusional safety. background: in europe on average 43 whole-blood donations are performed per 1000 inhabitants and year. whole-blood donation comprises a puncture of a venous vessel and letting of blood (usually 450 ml), which may be repeated several times a year. like other invasive procedures, blood donation has a range of effects on the individual who is subjected to it. aim: the aim of this paper is to review some aspects of the present state of knowledge on effects and complications of whole-blood donations. results: most studies of the effects of whole-blood donations on the donor have focused on negative or unpleasant events and on time in rather close association to the donation. complications related to percutaneous needle insertion (bruise assessed by inspection 0.66% -bruise assessed using post-donation interview 22.7%, sore arm 0-10.0%, nerve irritation 0.02%, arterial puncture/pseudoaneurysm/arteriovenous fistula 0.003-0.009% etc) are most commonly reported, while negative systemic reactions (vasovagal reactions 2.5-7.8%, syncope 0.08-0.34%) occurring in connection to blood donation are less frequent (newman bh: blood donor complications after whole-blood donation. curr opin hematol 2004; 11: 339-345.) serious complications requiring hospitalization (myocardial infarction, stroke) are extremely rare (0.0005%). fatigue (7.8%-10.0%) and diminished physical working capacity (7.0%) are reported to occur during days after the donation. a recent study of a consecutive sample of 528 swedish blood donors (nilsson sojka b, sojka p: the blood-donation experience: perceived physical, psychological and social impact of blood donation on the donor. vox sang 2003; 84: 120-128.) (with a selfadministered questionnaire with an open-ended question: how does blood donation affect you? physically-bodily/psychologicallyspiritually/ethically-morally/socially during or after blood donation?) revealed that perceived negative effects (fatigue, diminished physical working capacity, vertigo/dizziness, susceptibility to infections etc) were less common (25% of the donors) than positive effects (feeling of satisfaction, being more alert, feeling generally better, less migraine, higher physical working capacity, respect from environment, feeling of relaxation etc; 35% of the donors). the duration of positive effects was regularly reported to be weeks, while negative effects lasted only days. investigations on the long-term effects of blood donation are scarce. yet, they may indicate that the donation of blood is associated with e.g. lower blood pressure and a reduced risk of myocardial infarction (nilsson sojka b, sojka p: the blood-donation experience: perceived physical, psychological and social impact of blood donation on the donor. vox sang 2003; 84: 120-128). conclusion: both the panorama and the frequency of occurrence of the different effects and complications of whole-blood donations vary as a function of how the information was gathered (openended questions, observations, interviews etc). serious reactions to whole-blood donations are extremely rare. more studies are needed in particular with respect to the long-term effects of regular wholeblood donations. t-pa-056 establishing a national adverse event reporting system for blood donors -a prospective study of 1. there was no national system and significant regional variation showed that the data was scientifically unsound. a coincidental initiative to remove the mandatory post donation rest period for regular donors further emphasised the lack of reliable, retrospective data to monitor and compare the impact of this new policy. aims: 1. to develop and implement a high quality, reliable national donor adverse events reporting (daer) system; 2. to define and categorise adverse events; 3. to record data systematically and prospectively using the existing computerised donor database. methods: from summer 2002, a small project team of senior clinical and operational staff took 9 months to agree a detailed policy for capturing and recording all donor adverse events, including precise definitions for grades of vasovagal reactions and bruising. detailed training material was written in may 2003 and the new protocol was validated in one region. from july to december a formal one day training programme was delivered to over 3000 staff working on 98 mobile collection teams, 19 static sites and 11 blood centres. daer was fully implemented by january 2004. adverse events are assessed by health care professionals on session and the relevant code entered onto the donor's computer record by clerical staff. information received after the session is entered by centre based doctors using the same system. the database is interrogated monthly for statistics. results: in the first 9 months 1.86 million donors attended sessions throughout england and north wales. results were very consistent month on month. 25 175 donors (1 : 75) had vasovagal symptoms but only 14% of these suffered syncope. 71% of all vasovagal reactions occurred in women. 21% occurred in donors aged 17-20 and a further 30% in donors aged 21-30. (donors aged 17-30 represent only 25% of the total donor base.) 831 donors (1 : 2250) reported a delayed reaction, 52% of whom did lose consciousness. nerve injury, unrelated to haematoma, occurred in 109 donors (1 : 17 000) and, more rarely, arterial puncture was diagnosed in 46 donors (1 : 40 000). bruising was reported after the session by 1 : 1300 donors. summary: a robust system has been developed and successfully implemented across a large, national blood service. based on the data already accumulated our next phase is to develop strategies to minimise adverse events, confident that any intervention will be effectively monitored. the community role in enhancement of voluntary, non-remunerated blood donation in the new millennium introduction: in the developing countries, about 80% of the blood supply comes from paid or replacement donors, where a high number of infected persons are in the donors population. only 16% of the global blood supply is donated as voluntary nonremunerated blood donors in countries with low and medium human development indices. and around 80% of the global blood population has access to only 20% of a safe blood supply. conclusion: blood is a national resource, it is the responsibility of governments through it's communities to ensure that the blood supply is safe and adequate to meet the needs of patients population and available to all who needs it. background: in response to a documented increase in the average age of donors, a survey was conducted to explore if young people had more unfavourable attitudes towards becoming blood donors. aim: to identify if the increasing difficulty in recruitment and retention of young people as donors, is linked to a low level of motivation for donating blood in this age group. methods: a national telephone-survey was conducted among a cross-sectional sample of the adult norwegian population (1000 participants). the survey was performed in november 2004. results: five percent reported being active donors (had donated during the last 12 months), 17% were passive donors (had not donated during the last 12 months), 22% were non-donors with a positive attitude towards becoming donors, and 55% non-donors with no intentions ever to donate blood. in the youngest age group (age 18-29), 1% reported being active donors and 8% were passive donors. however, 36% of the young non-donors reported having intentions of becoming a blood donor. fifty-five percent of young non-donors had a negative attitude towards ever donating blood. all non-donors were asked why they did not donate. thirty-six percent of all non-donors reported health related reasons for not donating blood. thirty-one percent of all non-donors claimed that they did not donate because no one had requested them to do so personally, and 4% reported they did not care about blood donation. in comparison only 28% of young non-donors reported medical reasons for not donating. thirty-eight percent claimed lack of personal request, and 7% reported of lack of interest as the main reason for not donating. summary/conclusion: although the youngest age group was under-represented among active donors, we found that a great proportion (36%) of young non-donors had a positive attitude towards becoming blood donors. the most important reason why young people do not donate was the lack of a personal request. indifference regarding donation was not very widespread. a relatively high proportion of young people considered themselves as not medically disqualified to donate. in light of these findings, efforts to recruit young people as blood donors are strongly recommended. background: the ever-increasing demand for blood, coupled with emerging new threats to blood safety, motivate the strengthening of the blood banking infrastructure. aim: employing new technology as an instrument for building relationships of trust between blood bank and blood donors. material: 1. a new software module supporting the management of magnetic cards was added to e-aima blood bank management application. the magnetic card supports the following information: • front-side: donor's photograph, surname, name and blood group (including rhesus phenotype & kell) and sign of blood collection centre. • magnetic stripe where data concerning donor's serial number, medical history (risk factors), test results for infections and the number of donations is stored. 2. specific hardware allowing reading from and writing to magnetic cards is integrated to the software module: • magnetic card scanners were added to pcs serving to donation collection, including laptop for mobile team collection. • web cameras to capture the photograph of the donor to be printed on the card. • card printer was deemed necessary to produce magnetic cards for donors on a need basis. 3. consumables: blank plastic magnetic cards, ink cartridges for printer. methods: in order to evaluate the performance of magnetic cards compared to the paper-based system, a questionnaire was distributed to 300 first-time and 200 repeat blood donors, in order to be used as an indicant of donor's satisfaction. 1. first-time donors increased 7.8% in the 6 months of application. among them, 4.3% were donors 'for relatives or friends' turned into volunteer donors and 3.5% were first-time volunteer donors. the questionnaire analysis further revealed: • 96% were motivated by the use of magnetic card. • 92% appreciated the presence of their photo on the card and they confessed that they had used it as a spill for recruiting their friends as donors. • 100% were persuaded that employing new technology would result in safer and more trustworthy procedures combined with reduced waiting time. • 100% considered magnetic cards more practical compared to paper cards because of their compact size and improved durability. 2. the turnover of repeat donors also increased 1.3% after replacing their plain old paper cards with new ones. further analysis revealed that: • 100% appreciated the quick cross-checking of donor's identity. • 100% were satisfied with the effectiveness and efficiency of magnetic cards in managing donor's data. conclusions: in 2004, greek health policy provided the legal basis for establishing the electronic national health card. the introduction of the national donor's magnetic card is another step towards this direction, being aligned with the modern national health strategy. apart from the positive impact on the number of both firsttime and repeat blood donors, it should be also pointed out that the use of a unique donor serial number on country level results in less error-prone procedures due to the reduction of administrative process overhead and facilitates interoperability between national blood banks using compatible technological infrastructure. t-pa-060 emerging technologies in transfusion. dna based assays until the late 1990s, mandatory blood screening for transmissible infectious agents depended entirely on antigen/antibody-based detection assays. recent emergence of nucleic acid technologies (nat) has revolutionized viral diagnosis by not only increasing the sensitivity level but also facilitating the detection of several viruses in parallel, by multiplexing specific primers. however, in more complex biological situations when a broad spectrum of pathogens must be screened, the limitations of these first generation technologies became apparent. high throughput systems such as dna arrays permit a conceptually new approach. these miniaturized microsystems allow the detection of hundreds of different targets simultaneously, inducing a dramatic decrease in reagent consumption, in additional confirmation tests and simplify data interpretation. however, the microsystems actually available require additional instrumentation and reagents for sample preparation and target-amplification prior to detection on the dna array. future technologies such as 'lab-on-a-chip' include channels, fluidics and thermal zones allowing extraction, amplification and detection. another major challenge in the area of dna detection is the development of methods that do not rely on target-amplification systems. almost all blood group antigens are bi-allelic and encoded by single nucleotide polymorphisms (snps). to facilitate the direct availability of typed red cells and platelets, we develop a high-throughput technique to genotype by dna microarray the whole donor cohort for all clinically relevant red cell and platelet antigens. methods: a multiplex pcr was developed to both amplify and fluorescently label 28 gene fragments of red cell and platelet antigens in one reaction. each array contains spots of short (17-27 nt) allelespecific oligonucleotides to discriminate between the two alleles of an antigen system. results: two blinded panels encompassing 94 donors were genotyped for hpa-1 through -5 and 15; no discrepancies were found. currently, arrays are prepared for the red cell systems. the fya/fyb, fy-gata1 mutation, jka/jkb, k/k, kpa/kpb, m/n, rhc/c, rhe/e, rhdpseudogen, rhdvi negative, rs, doa/dob, genotypes can be determined. the set up of genotyping assays for rare genotypes is difficult because of lack or insufficient amount of dna. the latter can be overcome by phi29 dna polymerase-mediated isothermal genomic dna amplification, from minute amounts of dna present in stored red cell fractions or antiserum. the results show that the blood group typing dna microarray will provide a reliable and fast genotyping procedure. the method can be further improved to obtain the necessary automated throughput for typing of large donor cohorts. and 15, all other weak d types should be regarded as potential anti-d immunizers. for correct determination of weak d both serological typing (polyclonal and monoclonal), as rhd dna typing are mandatory. when serology indicates weak d, more anti-d antibodies are tested (9 epitope model) to distinguish partial d from weak d. in addition, an rhd mpx pcr is performed to detect the presence of rhd exons 3, 4, 5, 6, 7 and 9. in all known weak d types, all six rhd specific exons are amplified (except for weak d type 4 which lacks rhd exons 4 and 5), whereas partial d phenotypes usually show aberrant patterns. aim: the aim of this study was to evaluate the diagnostic scheme for weak d typing. methods: between 2002 and 2004, 24 samples were investigated for weak d characteristics. four pcr-ssp assays were developed for identification of weak d types 1 (809t > g), 2 (1154g > c), 3 (8c > g) and 5 (446c > a). weak d type 4 was identified by the combination of serology and absence of exons 4 and 5 by rhd mpx pcr. rhd-specific exon sequencing was performed when serology and molecular typing were inconsistent. results: all 24 samples were subjected to the rhd mpx pcr and 1 sample showed absence of rhd exons 4 and 5, indicative of a weak d type 4 when combined with serology. the remaining 23 samples were analyzed by the weak d pcr-ssps, resulting in 12 weak d type 1 samples, 4 weak d type 2 samples, 4 weak d type 3 samples and 1 weak d type 5 sample. two samples remained undetermined and were sequenced for all 10 rhd exons and the rhd promotor region. one sample showed the mutations corresponding to the dau 2 partial d phenotype (209g>a, 998g>a and 1136c>t). the other sample had only one, not previously known mutation (1193a>t), which is located intracellularly at the coohtail. extensive serology using the 37 epitope model showed a pattern matching weak d. this new weak d variant was registered as weak d type 41. conclusions: based on these results it may be concluded that weak d phenotypes should be confirmed on molecular level to avoid misinterpretation of partial d that cannot be detected by rhd mpx pcr analyses. patients with weak d phenotypes, except for types 1, 2 and 3 should be regarded as being at risk for anti-d immunization after transfusion of rhd-positive blood products and should therefore be treated with rhd-negative bloodproducts. in this evaluation, 4 out of 24 patients carried such alleles. introduction: although kell antigens are expressed very early during erythropoiesis and a 0.1% incidence of anti-kel1 is found in obstetric patients, this is a relatively rare cause of hdn. anemia is produced by immune destruction of fetal rbcs and suppression of erythropoiesis. maternal antibody titers or amniotic/cord blood bilirubin levels are not relevant indicators of the severity of the disease, and the measurement of the fetal haemoglobin by cordocentesis is a procedure with risks of miscarriage and sensitization. pcr techniques for the determination of blood groups using fetal dna isolated from maternal plasma, allows the application of noninvasive methods. clinical cases: we describe two cases of pregnancies in women with anti-kel1 acquired by transfusion/previous pregnancies: 1st case: in july 2000, a 30-year-old woman (gravida 2, para 0), rhdnegative, kel1-negative was referred at 20 weeks gestation. the father's phenotype was rhd-positive, kel1-positive. a maternal antibody screen revealed d and kel1 alloantibodies. dna was extracted from amniotic liquid. the kel genotype was determined by pcr-rflp using the bsm i. pcr-ssp was used to studied intron 4 and exon 7 of the rhd gene. the results showed that the fetus was positive for rhd sequences and showed kel2 homozygosity; 2nd case: in august 2004, a 36-year-old woman (gravida 2, para 1) was referred at 28 weeks gestation. she had a history of transfusion with 2 rbcs units in 1996 -one of the donors was kel1positive. the woman typed rhd-negative and her husband typed rhd-positive. rbcs from both were kel1-negative. the maternal antibody screen revealed anti-kel1. doubts existed about the putative father of this child. dna was extracted from maternal plasma using the magna pure lc (roche). real-time pcr was applied to analyse: sequences of intron 4, exons 4, 6, 7 and pseudogene of the rhd gene and the sry gene by sybr green; and the alleles kel1/kel2 by hybridization probes. all rhd sequences were detected (with the exception of the pseudogene) and the kel genotype gave a kel2/kel2 result. in both cases the doctors choose not to use any invasive method to monitoring the fetuses regarding a hdn due to anti-kell antibodies, and the results of the molecular analysis were confirmed by testing the cord rbcs after birth. discussion: these cases illustrate the reliability of the molecular biology results, based on the collection of simple peripheral blood samples. a determination that the fetus lacks the relevant antigen obviates the need for expensive and invasive monitoring throughout the pregnancy. evidence-based medicine (ebm) is defined as: 'the conscientious, explicit and judicious use of current best evidence in making decisions about the care of individual patients. the practice of ebm requires the integration of individual clinical expertise with the best available external clinical evidence from systematic research and our patient's unique values and circumstances. ' otherwise healthy individuals without cardiopulmonary dysfunction (cdf) tolerate acute reduction of haemoglobin concentration to about 5 g/dl, provided that blood volume is kept normal by a volume expander. however, individuals experience physical fatigue, and there is faint reduction of perception as measured by neurophysiological tests. symptoms are reversed upon retransfusion of fresh, autologous erythrocytes. acute, normovolemic anemia seems to be progressively less tolerated with increasing age and cdf. controversy has existed on whether or not to correct hypoalbuminemia in asb or icp by infusion of albumin. recently a large trial showed no outcome differences between icp patients treated with albumin or saline. thus in general there is no indication for albumin in asb or icp. however, albumin may yet be advantageous in e.g. patients with head injuries. furthermore, fractionated albumin is not equivalent to native albumin, since fractionation stabilizers remain bound to the albumin molecule. thus more refined albumin preparations may carry advantages still to be investigated. erythrocytes are given to increase the total oxygen transportation capacity of the organism. the effect of blood bank stored erythrocytes may differ from that of fresh, autologous erythrocytes, since changes of presumably important erythrocyte properties occur during storage. in the only large trial available, a transfusion trigger of 7.0 g/dl was found to be favourable to one of 10 g/dl in icp, except possibly in icp with unstable angina pectoris or heart infarction. however, the erythrocyte concentrates given were not leukocyte filtered, and side effects of infused leukocytes may have hampered the conclusion. on the other hand, a metanalysis showed transfusion as an independent indicator of unfavourable outcome in coronary bypass patients, but again, leukocyte filtered erythrocyte preparations were not applied. the effect on morbidity and mortality of 'top up' transfusions given e.g. to mobilize patients postoperatively has not been studied by trials, although this effect seems evident to many clinicians. grave anemia may reduce the haemostatic effect of thrombocytes because changes of blood rheology reduces the pressure forcing thrombocytes against the walls of small vessels. the transfusion trigger for thrombocytes in asb or icp remains to be established by clinical trials, however. the same applies for fresh frozen plasma, which is infused as a source of coagulation factors. on the other hand, the haemostatic effect of various fibrinolysis inhibitors is well established in asb and icp, but many clinicians appear hesitant to use them. another interesting haemostatic agent is recombinant fviia, the use of which to control asb in blunt trauma is supported by one well controlled clinical trial. evidence by systematic research is insufficient to decide what is optimal transfusion practice. the procurement of such evidence is one of the greatest current challenges to transfusion medicine research. . concerns about the transfusion-related complications, such as infections, tumour behaviour and immuno-modulatory effects, and the costs, necessitated a re-evaluation of the transfusion practice. aims: the goal of this study is to evaluate if a restrictive transfusion policy (hb transfusion trigger <4.5 mmol/l) reduces the amount of red cell transfusion compared to a liberal transfusion trigger (hb < 6.0 mmol/l) without a decrease in hrqol. because of concerns about the feasibility of this study early results were analysed and are presented in this abstract. material and methods: after a run in period of 3 months (hb transfusion trigger of hb < 6.0 mmol/l) patients are randomised for the restrictive or the liberal transfusion policy. patients are followed then 12 months. hrqol is measured after inclusion, after randomisation, 6 weeks, 3, 6, 9, and 12 months after randomisation. also anaemia related complications and red cell antibodies are scored. hb values were blinded for the patients during the study period. results: from july 2002 till june 2004 15 patients were included (4 ra, 5 rars, 4 rcmd, 1 raeb, 1 cmmol) in 2 general hospitals and 1 university hospital. two patients died in the run in period. eight patients were randomised for the restrictive transfusion policy and 5 patients for the liberal transfusion policy. the mean follow up period in the liberal group was 6.2 months (inclusive run in period) and 7.4 months for the restrictive group. two patients in the liberal group died after randomisation. one patient received growth factors. in the restrictive group 2 patients finished the study, 1 received growth factors and 1 patient withdrew informed consent. the mean hb level was lower in the restrictive group and after randomisation about 55% reduction in amount of transfused red cells was found (1.8 units per pt per month in the liberal group vs 0.8 in the restrictive group). no anaemia related complications were found, e.g. cardiac failure and cerebro vascular ischemia nor a decrease in activity performance. conclusion: there were some concerns after introduction of the restrictive transfusion policy. this preliminary results show, however, that a restrictive transfusion policy leads to a diminished use of red cell transfusion without an increase of cardiac complications or a decrease in activity performance. this study will be continued to compare hrqol scores in both groups. introduction: strong evidence supports the efficacy of blood conservation strategies such as autologous blood donation (abd) and erythropoietin (epo) for reducing exposure to allogeneic blood. however, use of these interventions is highly variable among institutions and frequently sub-optimal. the program to reduce orthopedic blood exposure (probe) evaluated a blood conservation program in patients undergoing total hip joint arthroplasty (thja) at ontario hospitals. aim of the study: the objective of probe was to determine whether a comprehensive blood conservation algorithm (bca) was more effective than usual care (uc) for reducing exposure to allogeneic blood in patients undergoing thja. methods: we randomized 30 hospitals that perform high volume elective primary thja to implement either a bca or to continue with uc. the bca consisted of three components: physician and patient education, blood conservation interventions (use of abd or epo), and transfusion guidelines. t-pa-071 table) . mortality for non-transfused patients was significantly lower than for patients receiving either lr-or s-prbc at all time points (p < 0.0001). t-pa-074 thalassaemia: the impact on blood transfusion services thalassaemia major is a genetically determined disease that causes severe chronic anaemia and further complications. it can be managed successfully in the vast majority of cases, so long as public health and other scientific and organizational infrastructures are adequate. although the progress achieved in the field of bone marrow transplantation and other disciplines promises cure of the genetic defect, regular blood transfusion from early childhood remains the cornerstone of treatment of patients with thalassaemia major. this presents national health authorities with the formidable task of assuring an adequate blood supply of high quality and safety for these patients and ensuring that it is transfused in the appropriate way. the basic principle in the modern management of thalassaemia patients is that of a global approach to care. within this approach, a standardized protocol for regular blood transfusions is a prerequisite for the patient's long survival and quality of life. if thalassaemia patients are not transfused effectively, the severe anaemia and over-expansion of bone marrow due to ineffective erythropoesis can lead to poor growth, bone deformities, organomegaly and impairment of normal physical activities. in countries or regions with large numbers of thalassaemic patients, the organizational and technical aspects of meeting their blood requirements represents a heavy additional workload for the blood transfusion services responsible for providing blood for this group of multi-transfused patients. the acquisition and preparation of blood, genotyping the patients' blood group (including at least rh, kell, kidd and duffy systems) preventing the transmission of infectious diseases and other transfusion associated complications, and assessing the patients' blood transfusion indices all have a tremendous impact on blood transfusion and treatment units. blood transfusion services are thus confronted with major challenges that can only be met if appropriate national transfusion policies are in place, both in the laboratory and the clinical setting of blood transfusion. the availability of safe blood is related to the effectiveness of donation programmes aimed at recruiting and retaining voluntary unpaid blood donors who are at low risk for the transmission of infectious diseases. sufficiency is further related to resources, organization and management of the blood transfusion service and continuous education of its staff. high technical standards for the transfused product and quality management systems are required to ensure that the product meets these requirements, as well as pre-transfusion, transfusion and haemovigilance systems and other more stringent quality measures in the whole chain of blood donation and transfusion. additional measures and continuous care are specifically required for the optimal transfusion therapy of the thalassaemic patient. the patients should be transfused with red cell concentrates, (rccs) preferably not more than one week's old and leucodepleted. other processes i.e washing of rccs, use of nutrient additive solutions, irradiation etc may be used to improve the quality and the safety of the transfused product, while other advances in red cell transfusion are expected to improve blood safety by preventing adverse reactions and reducing exposure of the patient to donor blood. should patients with thalassemia intermedia be regularly transfused? thalassemia major (tm) and thalassemia intermedia (ti) share mostly a common basic molecular mechanism, that is the reduced synthesis of the * globin chains. 1 the consequences of the resulting chronic hemolytic anemia are also common and include growth retardation, bone marrow expansion, extramedular hematopoiesis, splenomegaly, increased intestinal iron absorption, susceptibility to infections and hypercoagulability. what differentiates the two forms of the disease is the severity of the clinical phenotype, which in turn depends on a particularly heterogeneous molecular background and imposes diverse therapeutic strategies. the consequences of the genetic defect as well as the effect of the applied therapy seem to be mainly responsible for the clinical course of the disease. in untreated tm cases, the aforementioned consequences occur fast and patients die early in life mainly due to high output heart failure. over the past decades, the gradual adoption of the current transfusion and iron chelation strategies and the patients' compliance with this therapy have resulted in a significant improvement of survival, that according to recent statistics reaches 68% at age 35. this rate is even better in well-treated patients, almost 80% of whom survive at age 40. regular therapy extends survival mainly by preventing early development of cardiac complications. in addition, a multi-organ improvement is accomplished while patients' physical appearance is almost indistinguishable from that of the general population, hence permitting a normal social behavior with a high overall quality of life. bone marrow expansion and extramedular hematopoiesis are prevented; hepatosplenomegaly is substantially restricted and usually there is no need for splenectomy, while thromboembolic complications are rare and pulmonary hypertension is practically absent. patients with ti remain as a rule without regular therapy until a number of severe complications arise. the consequences of chronic anemia develop slowly compared to untreated tm cases and dominate patients' clinical picture usually by the third decade of life. at this time, all patients have developed hepatosplenomegaly and most of them have been splenectomized. bone marrow expansion results to bone deformities and fractures often occur. extramedular hemaotpoietic masses and bone deformities may lead to various complications depending on their bulk and location, such as neurological symptoms from masses arising in the paraspinal area or dyspnea from lung restriction. hypercoagulability, resulting from defects of native erythrocyte membrane phospholipids, together with the coexistent thrombocytosis in splenectomized patients lead to a wide spectrum of thromboembolic events. pulmonary involvement with respiratory dysfunction and hypoxemia as well as pulmonary hypertension leading to congestive heart failure are well documented in ti patients. nowadays, the beneficial effects of regular transfusion and chelation therapy in tm are beyond any doubt. the occasional application of transfusions in ti has a transient effect and does not seem to inhibit the consequences of chronic hypoxia. intensive and regular transfusion and chelation therapy in ti has proved effective in ameliorating the established complications such as spinal cord compression, hypercoagulability and pulmonary hypertension, without however reversing them. given the 30-year experience on intensive therapy in tm and the first encouraging data in ti, the earlier application of such treatment seems to be crucial in ti. the timing however of therapeutic intervention in ti in order to prevent anemia-related complications still remains an open issue that needs to be properly addressed. the impact of prestorage leucodepletion on the immediate transfusion adverse events of patients with thalassaemia major backround: regular blood transfusion therapy in patients with bthalassaemia major decreases the complications of anemia but it is associated to many immediate and delayed side effects. febrile non haemolytic transfusion reactions (fnhtr) are common complications due to alloimmunization of recipients against hla and/or specific antigens on donor's wbcs or to the accumulation during storage of biologic response modifiers (bmrs) that are directly pyrogenic or indirectly by stimulating recipients' white cells to produce pyrogenic mediators. post storage leucoreduction (lr) has reduced the fnhtr in these patients from 13% to 0.5% per unit. it is unknown whether introduction of prestorage leucodepletion (ld) has reduced the incidence of nhftr further. we analyzed the immediate transfusion reactions of 175 adult patients with b-thalassaemia major transfused with a total of 32.990 rbc units from january 1998 to december 2003. all units were fresh, stored less than 7 days. 11.950 units were lr-rbc, 8.370 units were ld-rbc and 12.670 were washed lr-rbc. results: the incidence of fnhtr and allergic reactions in patients receiving lr-rbc was 0.16% and 0.35%/per unit respectively, in those receiving ld-rbc was 0.10% and 0.36%/per unit respectively, while in those receiving washed lr-rbcs was 0.10% and 0.25%/per unit respectively. the relative risk (rr) of fnhrt and allergic reactions following transfusion of ld-rbc and washed lr-rbc compared to lr-rbc is shown in table. conclusion: prestorage leucodepletion and washing of rbcs reduced the risk of fnhrt in regularly transfused b-thalassaemia patients 1.6 times compared to poststorage filtration. these findings show that fnhtr after rbc transfusions are due not only to alloimmunization but also to accumulation of bmrs even in patients transfused with fresh rbcs. washing is as effective as prestorage leucodepletion in reducing fnhtr. prestorage leucodepletion has no effect on allergic reactions or other immediate adverse events in these patients. t-pa-078 viral inactivation/elimination of plasma derived medicinal products the safety of medicinal plasma products (mpps) relies on a whole range of measures from the quality of the source material to the release of the products after manufacturing under cgmp conditions. viral safety relies on careful donor selection, viral testing of the source material and viral inactivation and/or elimination during the manufacturing process. mpp manufacturing processes must include viral safety steps capable of inactivating, and/or eliminating, a large range of viruses covering the known blood borne viruses as well as anticipating possible future pathogens. it is recognized that one single step is often not sufficient to satisfy this requirement and manufacturing processes very often include two or even three complementary viral safety steps. very efficient methods have been implemented by manufacturers, for two decades, for the inactivation of major blood borne enveloped viruses (hiv, hcv and hbv). additional safety steps have also been introduced to provide a second step for enveloped viruses and to extend the efficacy to nonenveloped viruses (hav and parvovirus b19). pasteurisation (liquid heat treatment at 60°c) which has been historically used for viral inactivation of albumin solutions has been applied to some other plasma products. solvent-detergent (sd) treatment which is specific to enveloped viruses is used primarily for coagulation factors. since the introduction of sd-treated products, no hiv, hcv or hbv transmission has been reported. viral inactivation of coagulation factors can also be achieved using various conditions of dry-heating. acidic treatment is also an efficient means of inactivating viruses in igg products. nanofiltration using filters of less than 50 nm pore size was introduced in the early 1990s. this technique for viral elimination is based on the size of the agent and is independent of their resistance to other treatments. this property could be helpful in cases of new emerging pathogenic agents. new inactivation tech-niques are currently under development such as uv treatment or gamma irradiation with efficacy reported on enveloped as well as non-enveloped viruses. these new techniques can complement existing methods after careful validation that they do not have harmful effects on proteins in the product. the efficacy of existing techniques is well documented in controlled clinical studies and pharmacovigilance records. their application to each product is extensively validated at laboratory scale, according to international regulations and then carefully evaluated by health authorities. in this context, a recent european guideline established a viral risk assessment model to quantitatively estimate the theoretical safety margins of mpps, by taking into account the different safety measures, such as viral testing of plasma and the efficacy of viral inactivation/elimination steps. whilst technical limitations and some lack of scientific data lead to very conservative estimates, this model gives an overall assessment of the efficacy of the measures in the manufacture of a given product. developments in viral inactivation/elimination methods, in plasma testing as well as in evaluation procedures have together given mpps an excellent level of safety never previously achieved. to date the important safety measures needed to ensure a high safety margin to pooled plasma products are well understood by the plasma fractionation industry. safety nets rely on carefully done: donor selection to exclude high-risk donors, serological and nat viral testing of single donations and, pooled plasma testing using sensitive validated methods, and most particularly, efficient viral reduction treatments that must be validated and implemented at a large-scale following good manufacturing practices. over the last 25 years, successive key breakthrough in plasma product viral safety have included the use of solvent-detergent treatment to inactivate lipid-enveloped viruses, and nat testing of starting plasma pools and viral nanofiltration of products to reduce the risks associated to small non-enveloped viruses. the excellence of the system currently in place is illustrated by the demonstration that these safety barriers have virtually stopped the transmission of known viruses and avoided that of 'emerging' agents, such as west nile virus (wnv). however, multiple viral reduction treatments have generally decreased product recovery. in addition, although the implementation of viral reduction treatments have forced fractionators to introduce significant changes to product manufacturing methods, this period has been understandably followed by a period of relative conservatism of the plasma fractionation industry against further process changes. as time evolves and market dynamics changes struggle for improved economic balance of the plasma product industry is putting product recovery and diversified product portfolio at the forefront of r&d objectives. these developments in the plasma fractionation scene of the western world have been taking place in a context where many patients in the developing world are still treated with sub-standard, non-virally inactivated crude plasma fractions. in this specific area, one can expect that the developing world will bring innovative thoughts and take actions to find ways to improve the quality and safety of their own local plasma product supply. safety strategies adapted to the infrastructure and economy of less solvable countries may have to be considered. the intercept blood system for plasma uses a synthetic psoralen, amotosalen hcl, and long-wavelength ultraviolet light to photochemically inactivate a broad spectrum of bloodborne pathogens in plasma intended for transfusion (intercept plasma, i-ffp). phase 3 clinical trials have shown that i-ffp retains proteins necessary for hemostasis in the treatment of acquired and inherited coagulopathies, and in support of therapeutic plasma exchange for ttp. a prototype plasma processing set was used for the clinical trials. for commercialization, a new processing set has been developed to improve productivity. the prototype set accommodated approximately 250 ml of plasma, whereas the improved set accommodates up to 635 ml of plasma, resulting in up to three i-ffp doses per treatment. aims: this study was designed to characterize pro-and anti-thrombotic proteins in i-ffp prepared using the improved processing set. proteins of interest included components of the intrinsic and extrinsic coagulation cascade, the fibrinolytic pathway, the contact factor pathway, and the complement system, the vonwillebrand complex, endogenous inhibitors, and markers of thrombin generation. methods: six fresh jumbo (600 ml) apheresis plasma units, collected using the haemonetics pcs device (gambro), were photochemically treated. sodium citrate was used as the anticoagulant. plasma samples for analysis were collected before and after photochemical treatment, and were frozen below -60°c until batch analysis. standardized clinical assays were used for all analyses. results: (results in the table below are expressed as the percent activity in i-ffp in proportion to the activity in plasma before treatment [mean ± sd]). retention of procoagulant factors in i-ffp plasma ranged from 77% to 92%. factor viii and vonwillebrand factor activity, antigen, cleaving protease activity (vwf : cp, adamts-13), and multimeric composition remained within normal ranges after treatment. endogenous inhibitors of coagulation were retained 93% to 100%. plasminogen and alpha 2-antiplasmin were retained 94% and 78%, respectively. retention of contact factors was variable; some factors were below the reference range prior to pct. with the exception of tat, all markers of coagulation activation were well within normal ranges. the tat level in one i-ffp unit was slightly above the normal range; all other units had tat levels that were well within the normal range. the significance of this is unclear. cept plasma is similar to conventional plasma. the improved processing set, intended for commercialization, allows up to 3 doses of i-ffp to be produced from a single photochemical treatment. background: guidelines of the european directive 2004/33/ec require that fresh plasma prior to freezing contains <6109 residual rbcs per litre. this rbcs content is below the sensitivity limit of the automated cell counters used in routine laboratories. aim of the study: it was therefore essential to make available an alternative method to detect and quantify rbcs in plasma. we implemented a method by flow cytometry using a pe conjugated anti-glycophorin a (gpa) monoclonal antibody that recognises rbcs and erythroid precursors. to quantify residual rbcs in fresh plasma, the method uses the same trucount test tubes (becton dickinson) as those used to quantify wbcs and that contain a known number of fluorescent beads. after addition of plasma and pe-gpa antibody, cell counting is performed on flow cytometer (bd facscalibur). validation of the method: assessment of accuracy, linearity, and reproducibility with 2 different pe-gpa antibodies (immunotech and pharmingen) application of the method: quantification of rbcs in 105 fresh plasmas divided into 3 groups: group 1: 45 plasmas from leucoreduced whole blood, group 2: 30 plasmas from packed cells after removal of buffy coat and specific filtration, group 3: 30: apheresis plasmas. results: validation of the method: for both anti gpa antibodies, detection threshold is 0.05 ¥ 10 9 residual rbcs/l; linearity study with concentrations of 0.1, 0.25, 0.50, 1.0, 1.5, 3.0, and 6.0 ¥ 10 9 rbcs/l showed excellent correlation between observed and expected values (r > 0.99); reproducibility study showed c.v of respectively 5.7% and 6%. for values >2 ¥ 10 9 rbcs/l, it appeared necessary to introduce a correction factor of 1.3 for the anti gpa pharmingen. quantification of rbcs in plasma: group 1 (plasma from leucoreduced whole blood): 0.39 10 9 ± 0.23 rbcs/l; group 2 (plasma from packed cells after removal of buffy coat and filtration): <0.05°¥ 10 9 rbcs/l in all the cases; group 3 (apheresis plasma): <0.05°¥ 10 9 rbcs/l in all the cases. conclusion: quantification of rbcs in plasma by flow cytometry is a precise, quick and reproducible test. in addition this study shows that even if there are differences in residual rbcs counts according to the origin of plasma, the obtained values are much lower than regulatory requirements. 1. improving basic transfusion knowledge amongst health workers; 2. improving pre-operative preparation for surgery 3. strategies such as cell salvage autotransfusion combined with a conservative transfusion strategy for the use of allogeneic blood. my presentation will outline the approach adopted to ensure that the use of cell salvage autotransfusion both improves the use of allogeneic blood and preserves allogeneic stores. well-organised training can both minimise the risk of using such advanced techniques and decrease the overall risk involved in undergoing surgery where blood loss may be a significant factor in increasing morbidity and mortality. the various training methods employed to improve knowledge in this area will be described. the increasing current perception that the safety of allogeneic blood transfusion has dramatically been improved during the last decade is challenging autologous haemotherapy methods. in addition, growing concern about the unfavourable cost-effectiveness of most autologous haemotherapy methods requires a refinement of the application of these measures to well defined circumstances. in contrast, newly emerging transfusion-transmissible infections or periods of blood shortage might revive interest in these blood sparing techniques. the first two cases of transfusion transmitted vcjd provide a paradigm for this scenario; not so much with respect to a public fear of infection but rather a waning donor population due to more rigorous recruitment criteria. preoperative autologous blood donation (pabd) still plays a significant role in settings with high individual benefit for the patient, high transfusion probabilities and when all opportunities of cost minimization can be applied. adjustment to the individual situation of the patient is the main aim of a medically reasonable and economic use of autologous haemotherapy. this implies consideration of the patient's haematocrit, blood volume, tolerable blood loss, expected blood loss, etc. in order to choose the optimal method in the individual case. in this respect, double red cell apheresis may play a significant role. with this approach, donation schedules assumed to enhance erythropoiesis can be adopted. moreover, inconveniencies caused by long distances between patient home and donation service can be facilitated by withdrawing two red cell units during one session in selected patients. in conclusion, red cell apheresis can be used to promote the proposed approach towards individualized autologous haemotherapy preoperative plasmapheresis is considered to be a sensible adjunct if intraoperative retransfusion of salvaged and washed red cells is planned. acute normovolaemic haemodilution is valuable when the patient's tolerability of the haemodilution and the expected blood loss are carefully examined beforehand. intraor postoperative salvage of wound blood can also be regarded as useful measures to prevent allogeneic transfusions as long as the specific advantages and disadvantages of the different methods are taken into account. finally, alternative and supplemental measures such as iron or erythropoietin administration should always be considered in order to optimize the efficacy and effectiveness of autologous haemotherapy methods. the goal of a 'bloodless medicine' might not be reached but is supposed to be approached closely with an integrated concept exploiting all measures available. however, in times of restricted health care resources, regular sound costeffectiveness analyses, taking the availability and the cur-rent safety profile of allogeneic blood products into account, are always warranted and needed. compensatory fluid replacement of surgical blood losses: the transfusion of allogeneic blood is expensive and -although safer than ever before -still associated with potential complications. to reduce both, costs and immanent risks, allogeneic transfusion should either be completely avoided or at least minimized during surgical procedures. as a consequence an intraoperative blood loss is initially not replaced by red blood cells, but by erythrocyte-free, i.e. cristalloidal or colloidal solutions. when normovolemia is maintained the resulting dilutional anemia is compensated by an increase of cardiac output and enhanced arterial o2 extraction. however, once the hb has dropped to values recommended as the lower intraoperative limit, or once compensatory mechanisms of acute anemia become exhausted, as a rule transfusion of red blood cells (rbc) is initiated to increase arterial oxygen content (cao2) and to preserve a margin of safety for tissue oxygenation and organ function. as an alternative to immediate rbc transfusion, ventilation with pure o2 (hyperoxic ventilation) can be employed to rapidly raise cao2 by increasing the amount of physically dissolved o2 in plasma (hyperoxia). however, molecular o2 causes vasoconstriction, mediated by products of the arachidonic acid metabolic pathway. as a consequence hyperoxia has been shown to increase systemic vascular resistance and to decrease cardiac output and o2 consumption in subjects with normal hemoglobin concentration ( properly scheduled, three women who did not reach the necessary hct level after the first donation and consequently they got out of the protocol, and one woman who had unexpected intraoperative bleeding and received 5 homologous units in addition. the 3 patients undergoing tkr and the 11 patients undergoing removal of implants predeposited 9 and 21 units respectively, but finally 3 and 12 of them have been used. according to our patients data, the 55.5% of unused autologous blood units belongs to patients with tkr and implant removal. therefore a better schedule is needed for these type of surgery. all the autologous donors were supported by oral iron supplementation throughout the predonation and 1 month past surgery. fourteen of our cases were supported by erythropoietin s.c. in a dose of 300 iu/kg every other day. the majority of these patients was female, only one was male with multiple alloantibodies and was scheduled to predonate 5 autologous units. all of them underwent thr except one woman who also had a tkr 6 months later. the autologous blood donation was well tolerated by all patients and only one woman had a reaction during predonation. furthermore a group of 98 patients matched for age, sex and type of surgery, who did not predeposit blood, received a mean of 3.2 homologous units per patient, that is more than the patients on pabd program. our results show that autologous transfusion can be used in scheduled orthopedic surgical procedures and can reduce the need for homologous blood. however, every effort should be made to render the practice of pabd more efficient and to minimize its costs. colloid solutions and their establishment in clinical practice background: various situations like trauma, critical ill patients, sepsis, major surgical procedures and anaphylactic reactions are associated with disturbances in fluid homeostasis. this disturbance is related with reduced oxygen delivery, subsequent lactic acidosis and imbalance in oxidative status. the final result will most likely be an increased mortality and morbidity. aim: the important issue from clinical aspect is to define the optimal volume and type of fluid therapy. the debate for the ideal resuscitation solution lasts a couple of decades due to inconclusive and conflicting results. method: we searched the literature for clinical trials and major met analyses concerning patients undergoing scheduled surgical procedures, trauma patients and critical ill who received resuscitation fluids. results: dextrans reduce blood viscosity and von willebrand factor levels more, for the same degree of hemodilution, compared to other plasma expanders. in clinical setting they are effective in reducing the incidence of deep vein thrombosis and pulmonary embolism. after the initiation of dextran 1 for prophylaxis against anaphylactic reactions, they are considered the safest plasma substitutes, except maybe during pregnancy. dextran 40 10% is the most like to cause the 'hyperoncotic acute renal failure' syndrome. gelatins also cause a decrease in circulating levels of vwf : ag, vwf r : co, thrombin-antithrombin complexes and f1+2. in clinical aspect however, there are contradicted results about the effect of gelatins in bleeding diathesis, although they appear to exert a greater effect on rbcs protection from mechanical stress. in respect to anaphylactoid reactions, they have the greatest relative risk. hes has the same effectiveness in volume expansion with albumin and has the advantage of remaining intravascular even if there is an increased capillary permeability. in addition, it may improve splanchnic blood flow and tissue oxygenation. hes130/0.4 subsides the inflammatory response in patients undergoing major surgery, compared to a crystalloid-based volume therapy, but has conflicting results about it's effects on neutrophil respiration burst. clinical trials have so far failed to have a unanimous conclusion about the bleeding diathesis after hes administration, especially. caution must be held when administering hes during renal transplantation. albumin became the scapegoat of transfusion strategy during the past 20 years. resent met analysis have contradicted results about the safety of albumin infusion in variouw settings. a positive effect seems to have the early administration of hypertonic solutions in trauma patients, especially in combination with dextrans. conclusions: although some minor conclusions can be extracted, there is still a great lack of large scale multicentre randomized prospective clinical trials for extracting evidence based criteria. instead, we try to extract conclusions through met analysis. results show no evidence that resuscitation with colloids reduces the risk of death compared with crystalloids in patients with trauma, burns, major surgery or sepsis. also, there is lack of evidence that one colloid solution is safer -in clinical aspect -than any other. recombinant human erythropoietin therapy in critically ill patients -a dose response study* objective: the aim of our study was to assess the efficacy of two dosing schedules of recombinant human erythropoietin (rhuepo) in increasing hemoglobin (hb) level and reducing the exposure to red blood cells (rbc) transfusion in critically ill patients. design: a prospective, randomized, multicenter trial. patients: a total of 148 patients who met eligibility criteria were enrolled. intervention: patients were randomly assigned to receive intravenous (i.v.) iron saccharate alone (control group), i.v. iron saccharate and subcutaneous rhuepo 40 000 units once per week (group a) and i.v. iron saccharate and subcutaneous rhuepo 40 000 units three times per week (group b). rhuepo was given for a minimum of 2 weeks or until icu discharge or death. the maximum duration of therapy was 3 weeks. the requirement for rbc transfusions was significantly higher in control group than that in group a and b. no significant difference was observed between group a and b. the mean increase in hematocrit (dhct) and hb (dhb) from baseline to final measurement were significantly higher in group b than these in control group. dhct was significantly higher in group b than that in group a. dhct in group a was significantly higher than that in controls, whereas dhb did not differ significantly between control and group a. conclusion: administration of rhuepo in critically ill patients significantly reduced the need for rbc transfusions. the magnitude of the reduction did not differ between the low and high dose of rhuepo, whereas there was a dose response of hct and hb to rhuepo in these patients. in transfusion medicine, antibodies to antigens in the platelet membrane have traditionally been regarded as less significant compared with antibodies towards red cell antigens. there is an increasing awareness of antibodies towards platelet antigens. detection of autoantibodies to platelets can be a diagnostic challenge, but is seldom a problem in transfusion medicine because patients with such antibodies rarely are candidates for platelet transfusions. also, severe foetal thrombocytopenia is seldom present in pregnancies with autoantibodies to platelet antigens. the real challenge in transfusion medicine is related to patients with severe thrombocytopenia who are refractory to platelet transfusion due to alloantibodies towards platelet antigens. in our department, flow cytometry is used for compatibility testing and the choice of compatible blood donor is done without knowledge of antibody specificity. if crossmatch negative random donors cannot be identified, antibody specificity testing is performed and donors are chosen based on the specificity of the antibodies determined by a modified maipa procedure and with hla class i beads (flowpra from one lambda, usa) in flow cytometry. in some cases both hla class i and human platelet antigen (hpa) specific antibodies are detected and hla class i, hpa matched donors are chosen for crossmatch. if the crossmatch is negative, there is >90% chance of successful transfusions. in some cases drug induced anti-platelet antibodies are suspected and flow cytometry based antibody tests are performed in the presence and absence of the drug. in the case of suspected heparin induced antibodies, a beads assay is performed (diamed, switzerland). two percent of caucasian women have the platelet type hpa 1bb. ten percent of these women make anti-hpa 1a antibodies in their first hpa 1a incompatible pregnancy. 1 in 1000-2000 new-born has thrombocytopenia due to maternal alloantibodies which have crossed the placenta (neonatal alloimmune thrombocytopenia, naitp). results from a screening study covering the outcome of 100 000 pregnancies show that only 2 babies were born with intracranial haemorrhage (ich) and there was no still-born babies in the study. pregnant women with a-hpa 1a antibodies were diagnosed, received careful clinical follow-up and the delivery was performed by caesarean section in week 38 of the pregnancy with immediate transfusion of hpa compatible platelets if the new-born had platelet count <35 ¥ 10 e 9 /l. in previous studies, it is reported the ich appears in 10-30% of the pregnancies where antibodies are present and that 50% of the babies with ich, die. our results are different from what is reported from other studies and this may reflect the prospective approach and the clinical interventions. naitp represent a challenge in transfusion medicine both diagnostically, but also related to compatible blood products for the thrombocytopenic new-born and the mother who may have high level of antibodies towards platelet antigens. methods: apheresis platelets (4 ¥ 10e 11 mean) from donors with same blood group were pooled and divided equally into two bags, po-80 and control (pl2410, baxter), which have 2660 and 2024 ml/m 2 *day*atm of oxygen permeability, respectively. on days 0, 1, 3, 5, 7, and 9 of storage, swirling, mean platelet volume, po2, pco2, ph, glucose, lactate, aggregation, and p-selectin expression were evaluated. six experiments were performed. results: the swirling pattern was preserved better for up to 9 days in po-80 (6/6) than in control (3/6) bags. dropped ph less than 6.2 on day 9 was observed 1/6 in po-80 whereas 3/6 in the control. aggressive drop of glucose (0 mmol/l) with prominent lactate accumulation (327 mg/l) was also observed on day 9 in 1 of control bags. the po2 level in the control dropped more significantly by 45% (46.6 mmhg) on day 3 than in po-80 (65.8 mmhg) compared with the initial level (84.9 mmhg) (p < 0.001). these results suggest that aerobic metabolism of higher concentration platelets was maintained better in a container with higher oxygen permeability. and less lactate generation with slower glucose consumption is also suggested in po-80 bags than in control bags. the %hsr and aggregation decreased gradually in a similar manner in both bags until day 7, and became a detrimental defect in 2 of 6 control bags on day 9. p-selectin expression was higher in control bags than in po-80 on days 7 and 9 with no statistical difference. in two control bags p-selectin expression reached >84% and was accompanied by a loss of swirling. these functional and biochemical characteristics of platelets at a higher concentration were kept better for 7-9 days when stored in a container with higher oxygen permeability than in the best of marketed containers. t-pa-096 background: maintenance of a neutral ph in the range of 6.8-7.4 is essential for preservation of platelet function and viability during storage. furthermore, studies have also indicated that the presence of glucose in the platelet suspending medium is important for maintenance of platelet quality. however, a platelet additive solution (pas) containing glucose having a ph of 6.8-7.2 cannot be manufactured by steam sterilization due to caramelization of glucose. in order to have optimal ph, the currently available pas such as t-sol does not contain any glucose. this study describes a novel twostep approach to provide a glucose containing additive solution (pas-g) by using an acid, glucose containing electrolyte solution (ph 5.5) for resuspension and processing of the pooled buffy coats (bc), followed by transfer of the processed platelet concentrate (pc) into a storage bag containing bicarbonate for ph neutralization and maintenance during extended storage. aim: compare the platelet quality of pooled bc pc stored in pas-g with pooled bc pc stored in t-sol. methods: a paired study design was used, where a pool of 8 bcs obtained from standard day 1-old cpd-wb units, was divided into two equal parts: one part was resuspended and processed with a pall leukoreduction system (atsbc) using t-sol, the other part was processed in a similar manner with the acid part of pas-g. percentage plasma carryover ranged from 20-35%. both processed pc products were transferred and stored in elx tm bags with the pas-g pc elx bag containing a bicarbonate tablet. ten replicate studies were performed. results: the yields (3.2 ± 0.3 vs. 3.2 ± 0.4°¥ 10e 11 ) were similar (pags vs t-sol). statistically significant (p < 0.05 with paired ttest) improved platelet quality at days 5, 7, and 9 of storage was observed with platelets stored in pas-g as compared to t-sol. the table below shows results at 7 and 9 days of storage for ph, extent of shape change (esc) and hypotonic shock response (hsr). the results for t-sol stored platelets correlated highly with initial glucose (% plasma carry over) level (r = 0.91 for esc, and r = 0.68 for hsr at 7 days storage), while no significant correlations were found for pas-g stored platelets. conclusion: this study demonstrated the practicality of using a two step procedure to store bc pc in a glucose containing additive solution with neutral ph during storage, and confirmed the importance of glucose in the storage medium as nutrient for optimal platelet storage quality. the effect of irradiation on white cell reduced platelet concentrates, stored for 7 days background: the storage of white cell (wbc)-reduced platelet concentrates (pcs) can be extended from 5 to 7 days provided the quality has been validated and bacterial screening is performed. irradiation up to 50 gray (gy) does not affect platelet quality, but the effect of pre storage irradiation with subsequent storage up to 7 days is not known. method: two wbc reduced pcs, each made from 5 buffy coats and a unit of plasma, were pooled and divided into control group 'a' and study group 'b' . pcs in group 'b' were irradiated immediately after preparation with 25 gy. pcs in both groups 'a' and 'b' were then stored on a continuous flat bed shaker at 20-24°c. swirl, ph and cd62p expression were determined on day 1, 6 and 8. twelve experiments were performed and compared with a paired t-test, p < 0.05 was considered significant. results: see table (day 8 values; mean ± sd; n = 12). pooling and dividing of the pcs was successful with respect to volume and platelet number. on day 8, the ph in group 'b' was slightly lower than in group 'a', but the difference is not significant. in group 'a', ph on day 8 was <6.8 in 3/12 pcs, versus 5/12 in group 'b' (not significant). the cd62p expression in irradiated pcs is not significantly higher than in non-irradiated pcs. conclusion: irradiation had no significant effect on platelet quality when stored for up to 7 days after blood collection. -1b, -3a, -5b, pra 63%, 12 donors. in patient 3 -three transfu-sions were effective (two crossmatches neg by lct and pift, one pos lct, neg pift) but later on, when the patient was in severe clinical status (shortly before his death) two transfusions were ineffective in spite of neg crossmatches. it is very likely that for the same reason patient 4 and 5 were refractory to two and one hpa compatible platelet units respectively. in patient 6 and 7 compatible platelets were not transfused because they died before the whole procedure (diagnosis and finding a proper donor) was completed. conclusions: 1. the frequency of occurrence of anti-hpa antibodies in transfused patients was: -5b, -1b, -2b, -3a, -1a; in three patients they were monospecific, in four polyspecific. 2. in 2 patients who developed anti-hpa alone, transfusions of platelets without relevant hpa antigens were successful. 3.the effectiveness of compatible platelets in patients with both anti-hpa and -hla was more difficult to assess because of their severe clinical status, which might have been responsible for transfusion failure. in one of these patients, however, the transfusions of compatible platelets were successful when he was in relatively good clinical status, but shortly before death transfusions were ineffective. t-pa-099 the successful implementation of nucleic acid testing (nat) for hiv, hbv, hcv and further viruses as well as improved donor selection led to a dramatic risk reduction for viral transmission via blood transfusion over the last years. today, other risks get into the focus of haemovigilance. bacterial contamination of blood products can occur via the donor, suffering from a (clinically unapparent) bacterial infection, or via the donation process itself, storage and handling of the blood product. particularly platelet concentrates (pc) are vulnerable to bacterial growth due to their storage conditions. patients receiving such products have a potential risk of severe complications or even death. modern hygiene regimes, e.g. improved disinfection of the donors´ skin or preparation of blood products in fully closed systems as well as diversion, led to a significant reduction of bacterial contamination risk in the past. however, a small risk remains. therefore, two possible ways of further reducing the risk of bacterial contamination of blood products are feasible: (a) testing and/or (b) inactivation. testing for bacterial contamination is possible by different methods: direct detection methods for bacteria (microscopy, flow cytometry) have disadvantages regarding sample size and detection limit. bacteria might rapidly grow in a contaminated pc, so testing should be performed as close as possible to transfusion to the recipient. biochemical methods like oxygen consumption might not detect anaerobic germs. automated culture methods are still the most sensitive technique, but they have their downsides as well (e.g. time and size of aliquot drawn). novel molecular genetic test methods for detection of bacterial nucleic acid are in different states of development, but still have to proof their suitability for routine use. three different principles of pathogen inactivation can be distinguished: photodynamic reactions produce oxygen radicals which in turn inactivate bacterial structures by oxidation processes. examples for these chemicals are phenothiazines like methylene blue and thionin as well as vitamins like riboflavin (vitamin b2). photochemical reactants penetrate cell boundaries and irreversibly inhibit nucleic acid, thus blocking replication and proliferation of pathogens. chemicals of this group are psoralens like amotosalen as well as pen-110 or s-303. the third method, the solvent detergent (sd) method, is used for pooled plasma only and consists of the combination of both solvents and detergents, which interact with membranes and destroy bacteria. methods for inactivation of bacterial contaminants have to proof, that they effectively inhibit bacterial growth while maintaining full functionality of the blood product at the same time. these two qualities have to be fulfilled up to the end of the storage period. toxic or mutagenic compounds must not remain in the final product. the technology must be easily integrated into the existing work cycle of a blood bank. finally, costs per product must be acceptable. in summary, both testing and inactivation have their advantages and disadvantages, which have to be weighed up against costs and benefits of both procedures. pros and cons of introduction of inactivation methods in a blood donor service producing 1 600 000 blood components per year will be discussed. bacterial contamination of blood components, particularly platelets, is now recognized as a serious adverse reaction that is preventable. there are many studies that have documented that these reactions occur from platelets stored at room temperature, most commonly arising from a skin contaminant, but originating from donors with asymptomatic bacteremia in about 1/3 of cases. the problem is intensified for patients receiving pools of platelets compared to single donor platelets collected by apheresis. reactions are more commonly noted and more severe with platelets stored for greater lengths of time. previous studies at johns hopkins described a series of 23 reactions in 12 years with reactions more common in platelet pools (1 : 1600 transfusions) than with single donor platelets (1 : 15 000 transfusions). these reactions caused fatalities in 4 of 23 cases. although our data suggests that these reactions are more common than other studies using hemovigilance systems, our case definition requiring culture of all transfusion reactions to platelets led to a more reliable estimate of the incidence of sepsis from platelets, many potential solutions have been proposed to prevent these reactions. improved skin antisepsis should always be sought but will never eliminate the 1/3 of reactions due to asymptomatic bacteremia. the same limitation applies to methods that divert potential skin plugs from the collection bag. antibiotics in the bag would lead to manufacturing concerns or problems for patients with drug allergies. although cold storage of platelets is currently being revisited, it is not yet a practical solution. pathogen eradication systems have been developed but they remain unapproved in most of the world and have led to concerns about toxicity of additives, damage to treated cells, and cost. as a result of increasing recognition of the problem, there has been increased interest in bacterial screening to prevent sepsis from platelets. in march 2004, the aabb standards required testing of platelets for bacterial contamination. testing programs have been implemented in the us widely as a result of the aabb action. licensed systems based upon bacterial culture or growth characteristics are available for single donor platelets and have been commonly employed. although these systems have some difficulty with false positive reactions, the evolving national data suggest an incidence of 1 : 5000 true positive reactions. these data suggest that a number of serious reactions have been averted, although some cases have persisted due to incomplete adoption, problems with slow growing bacteria, or the use of inferior testing methods with inadequate sensitivity. whole blood derived platelets have become a more difficult issue, since the approved testing methods are limited. the use of ph monitoring, gram stain, glucose measurements, and inspection for swirling have all been attempted. these methods are not sufficiently sensitive or specific to interdict many contaminated units, so that screening for bacteria in pooled platelets is less effective. it is anticipated that new methods may become available to make screening of whole blood derived platelets easier to perform in a reliable manner. it is also hoped that bacterial screening of platelets may form the basis to permit seven day storage and prestorage pooling in the us. results: twenty four hcv rna (+)/anti-hcv(-) repeat donors were previously tested in routine hcv rna in mini-pools and were negative. twenty available look back samples were individually tested for hcv rna and in one the virus was detected. to make sure that the failure of hcv rna detection in routine nat was not due to the pooling procedure, the hcv rna was tested in undiluted look back sample and dilutions of this sample by hcv negative plasma: 2/2 repeats of 48x dilution and 2/2 repeats of 24x dilution were hcv rna negative, whereas 1/1 repeat of 8x dilution and 2/2 repeats of undiluted sample were positive. the results of cobas amplicor monitor (sensitivity 600 iu/ml) were negative, which means that viremia in hcv rna mini-pool negative donation was below 600 iu/ml. in the recipient of red blood cell concentrate from this donation hepatitis c was diagnosed. however, the possibility of pretransfusion hcv infection cannot be excluded as no hcv marker tests were performed before transfusion. the patient and the donor were infected with genotype 3a. the low hcv viremia (below 600 iu/ml) in the preseroconversion window period was responsible for no hcv rna detection in routine mini-pool hcv rna testing. introduction and aim of the study: bacterial contamination is a life threatening risk of blood transfusion, especially with platelet transfusions. bacterial culturing (bc) of platelets as well as pathogen reduction (pr) reduce the likelihood of such contamination. where the costs of bacterial contamination are far less than the costs of pathogen reduction, the latter will reduce not only the risk of bacterial contamination but also risks of other pathogens. therefore, the question arises whether this additional expenditure can be justified in the light of the additional effect achieved. this question we will answer by cost-effectiveness and sensitivity analyses. methods: the balance between costs and effects of preventing adverse events due to platelet transfusion is assessed using a mathematical model and assuming optimal effectiveness of pr. model parameters and valuations of health states were obtained from literature and information from dutch sanquin blood banks. . while the estimates in comparison to the situation without bc or pr are surrounded with large uncertainties, the conclusion that pr is not cost-effective in comparison to bc is very robust. the cost-effectiveness of bc and pr are very sensitive to the estimates concerning sepsis probability and associated complication rate, the cost-effectiveness of pr relative to bc is not. this conclusion is also insensitive to a wide range of assumptions regarding residual risks and costs associated with hiv, hcv and hbv. the estimates indicate that culturing in the netherlands is cost-effective, even with the deviation bag in place. the estimates however appear to be very sensitivity to the probability of sepsis. a decision to use pr will, after the introduction of bc and the use of a deviation bag, never meet cost-effectiveness criteria. even when assuming perfect protection, the conclusion that it is not cost-effective in comparison to bc is very robust and does not alter when varying underlying parameters within their margins of uncertainty. table) . of cb collection. two collections have been transplanted to date and this represents a 2.5% take-up rate (3.5% where a sibling is alive). this compares favourably with the numbers transplanted from unrelated cb banks. dcb collection is therefore at least as efficient a method as unrelated cb collection for transplantation albeit in the limited number of cases where a dcb collection is possible. dcb collection has the benefit of a possible immediate transplant combined with the availability of a sibling donor for future donation of both stem cells and lymphocytes. it is therefore a useful service to provide and complements the work of unrelated cord blood banks. increased yield of mature platelets in cultures of cd34-enriched cord blood cells maintained at 39°c introduction: the future use in transplantation of ex vivo expanded hematopoietic stem (hsc) and progenitors cells will facilitate the transplantation of adult patients and speed up hematologic recovery. also ex vivo cultures of hscs may eventually permit to produce donor-free blood components such as platelets for transfusion. culture of animal cells is routinely done at 37°c. however there is previous clinical evidence suggesting that hematopoiesis may be more active in hyperthermic patients. we have therefore compared the effect of hyperthermia on the ex vivo expansion and differentiation of cord bloodderived hsc in megakaryocytes (mk) and mature platelets. the cord blood-derived cd34 cells were cultured continuously at 37°c or 39°c for 14 days in cytokine conditions optimized for mk development and maturation. the cultures were regularly monitored for various parameters. results: compared to 37°c, the cultures maintained at 39°c produced significantly more total cells (4.9 fold) and total mks (7 fold), and showed accelerated and enhanced mk maturation with increased yield of proplatelets and mature platelets (16.6 fold). accordingly, the cells cultured at 39°c contained an increased frequency of cfc-mk (8 fold) at day 14. cultures done at 38°c and 40°c were also more efficient than at 37°c but less than at 39°c. platelets produced in 39°c cultures could be normally activated by thrombin. as expected, the cells cultured at 39°c contained an increased amount of the heat shock protein hsp70. control experiments showed that the culture of several cell lines was inhibited or unaffected by the 39°c temperature. the unexpected resistance of hematopoietic cells to the deleterious effects of heat and the stimulatory effect of >37°c temperatures on hsc proliferation and differentiation indicate that the routine culture of normal human cells at 37°c is a paradigm that needs to be revised. the responsible molecular mechanisms remain to be identified but the observation will facilitate the ex vivo expansion of the progenitors of the mk and possibly other lineages. the synchronous generation of a significant number of mature platelets in vitro will facilitate the study of the mechanisms of platelet formation and ageing and could eventually have important applications in transfusion medicine. it remains to be seen if the stimulatory effects of higher than 37°c temperatures represent a protective response against sustained body fever that is specific to the hematopoietic system. several countries have, in the past few years, included human tissue banking within a regulatory framework similar to that of blood. indeed, tissue safety has come to the forefront of the preoccupations of regulatory agencies after several well publicised morbidity and mortality cases have been reported in the press. tissue safety has many features similar if not identical to blood safety and a review of those common elements will be reported. as well, arguments in favor of integrating tissue banking within a blood system will be discussed, one of the more important aspect of which being the expertise of the blood centre staff with cgmps. the experience of a blood establishment (héma-québec) with the integration of tissue banking such as bone, skin, heart valves within its operations will be reported, emphasizing the medical as well as the management aspects of such an integration. finally, tissue banking is an activity which brings more expertise to a blood centre, expands its knowledge of its customers and gives more opportunities to its personnel. umbilical cord blood (cb) is an important source of stem cells for clinical transplantation and may cause less gvh disease than non-t-depleted bone marrow (bm). the relatively low numerical cell dose available from cb has usually restricted its use for transplantation in adults. only 25-30% of patients have an hla matched sibling and for others an unrelated bm or a stored unrelated cb donation may also not be available. for some children the collection of cb following the birth of a sibling may be the only opportunity for a transplant. directed cb (dcb) donations from matched siblings have been shown to give better long-term overall results than matched unrelated cb or bm. dcb collection is however not as easy to control as cb for banking where dedicated hospitals and trained staff are used. here we review dcb banking in oxford over a 2.5-year period. requests were received for 88 deliveries from 84 mothers and of these, 81 collections were successful including 4 pairs of twins. failed collection was most often due to a damaged cord at delivery. 61 collections were made for 57 siblings possibly requiring transplant (median age 4) with 24 for leukaemia, 16 for erythroid disorders, 13 for immune deficiency, 5 for enzyme deficiency and 3 others. the remaining 20 collections were mostly requested where there was a family history of an inherited disorder (majority scid). three collections tested positive for anti-hcv antibody but negative for hcv by pcr. collections were not excluded on the basis of volume or cell number. mean volume was 80 ml (range 22-174, 86% exceeded 40 mls) and mean tnc count was 1.0 ¥ 10 9 (range 0.2-2.7, 58% exceeded 0.8 ¥ 10^9). the mean cd34+ve count was 3.1 ¥ 10 6 (range 0.2-19.0). all collections were cryopreserved within 24 hours using dmso/dextran/saline without volume reduction. the mean tnc viability prior to freezing was 98% (range 86-100%) and the mean cd34+ve viability post freezing was 82% (range 71-96%). the reliance on the goodwill of midwives and the logistical difficulties that arise when organising collections from many different hospitals do not appear to reduce the success . rhd and rhce typing was performed by multiplex-pcr with 24 fluorescent primer pairs. positive results were obtained for rhd-exons 2-7, 9, 10 and polymorphisms associated with antigens c, c, cw, e and e. sequencing of rhce-sequences, including exons 1 to 10 and intron/exon borders, were done by direct taq cyclesequencing using bigdye-terminators v.1.1 in an abi310 (applied biosystems). background: a number of adverse immune reactions associated with blood transfusion result from contamination of blood products by donor white blood cells. among these reactions, transfusionassociated graft-versus-host disease (ta-gvhd) has a mortality of greater than 90%. mirasol ® pathogen reduction technology (prt) has been developed for the reduction of viruses, bacteria, parasites and white blood cells loads in blood products. the technology is based on light and riboflavin photochemistry. this study was performed in order to evaluate the effectiveness of the mirasol ® prt process for inactivation of human pbmncs. methods: human pbmncs were collected from trima platelet apheresis disposable sets, purified by ficoll-hypaque discontinuous gradient centrifugation and divided into test and control samples. the test cells were treated with mirasol ® prt in autologous plasma on day 0. both test and control samples (n = 3) were tested on day 1 for cellular immunophenotype, t-cell activation using flow cytometry, proliferation in response to mitogen or allogeneic stimulator cells, ability to stimulate the proliferation of allogeneic responder cells and cytokine synthesis in response to lps stimulation was measured using a cba assay kit. results: although mirasol ® prt treatment did not significantly change the distribution of cd3+, cd3+cd4+, cd3+cd8+, cd19+ and cd16+cd56+ human lymphocyte subpopulations there were significant functional change. the expression of the activation marker, cd69, was observed in 65.4% (sd = 15.6%) of control t cells upon activation with pma, while only a 1.7% (sd = 1.3%) of the test t cells increased cd69 expression. proliferation assays showed that 3h-thymidine incorporation did not increase in the test cells in response to either pha or allogeneic stimulator pbmnc compared to the significant increase in thymidine incorporation levels observed with control cells. the test cells, when compared to the controls cells, demonstrated an inability to stimulate allogeneic responder pbmnc proliferation. the release of il-10, il-6, il-1b and il-8 cytokines after 24-h incubation in culture media increased significantly to 128 pg/ml (sd = 73), >5000 pg/ml, 404 pg/ml (sd = 301) and >5000 pg/ml for control cells, respectively. under the same conditions, these cytokines in test samples remained at background levels of 0.4 pg/ml (sd = 0.7) for il-10, 2.4 pg/ml (sd = 1.2) for il-6, 11 pg/ml (sd = 3) for il-1b and 1022 pg/ml (sd = 286) for il-8. addition of lps further stimulated the release of tnf-a, il-10, il-6, il-1b and il-8 in the control samples, but not in the test cell samples. in vitro studies demonstrate that mirasol ® prt treatment does not change lymphocyte immunophenotype, inhibits tcell activation by pma, abolishes pbmnc proliferative activity, eliminates pbmnc stimulatory activity for responder cell proliferation and suppresses the production of cytokines by pbmnc in both the absence or presence of lps. introduction: it has been discovered that vaccination of dendritic cells (dcs) with tumor antigens is a potential strategy to induce tumor-specific immunity in tumor-bearing patients. aim of the study: the purpose of the study was to investigate whether human monocyte-derived dendritic cells (dcs) were able to present p210bcr-abl protein and induce antigen-specific ctl responses in vitro after transfected with total rna of k562 cells (k562-rna). methods: dcs were derived from human pbmncs, which were incubated for 5 days in the presence of gm-csf and il-4, and then were transfected with k562-rna using electroporation or dotap lipofection. to verify the successful transfection of dcs with k562-rna, bcr-abl fusion genes expression of dcs was detected by rt-pcr and western blot. the immune phenotypes of the dcs were analyzed by flow cytometry. the cytotoxicity of ctl was assayed by propidium iodide (pi) staining and flow cytometry. results: it was shown that the bcr-abl fusion gene was detected in the dcs immediately after the transfection, but disappeared 24 hours later, while the cells were expressing p210bcr-abl protein and expressing increased cd80, cd83, cd86, hla-dr. moreover, the transfected dcs could significantly promote the t lymphocytes to kill the target k562 cells. conclusion: human dendritic cells transfected with total rna of k562 cells in vitro could induce effective p210bcr-abl proteinspecific immune responses and be used to induce tumor-specific immunity, which implies potential application of immunotherapy to tumors. appear to be relevant to the clinical response. ivig has a remarkably good safety record for long term administration, however the following side effects have been observed: mild, infusion-rate related reactions such as headaches, myalgia or fever; moderate but inconsequential events, such as aseptic meningitis and skin rash; and severe, but rare, complications such as thromboembolic events and renal tubular necrosis. judicial use of ivig based on results from controlled studies is recommended. t-pl3-09 donor-lymphocyte infusion: transfusion immunotherapy following allogeneic hematopoietic transplantation the notion that bone marrow containing immunocompetent cells is capable of mediating an antitumor effect was determined experimentally almost 50 years ago. subsequently, pooled leukocytes from patients with cml were found to effect responses in patients with advanced leukemia. response correlated with cell dose and with severity of gvhd. in the 1970's, the graft-versus-leukemia (gvl) effect was defined in the transplant setting using a lethallyirradiated mouse model and splenocyte infusions. such studies suggested that gvl could be enhanced without causing severe gvhd. the era of adoptive immunotherapy in the transplant setting began in the 1990's with reports of donor lymphocyte infusions (dli) for relapsed acute and chronic leukemias after bone marrow transplant. it is now clear that chronic myelocytic leukemia (cml) in chronic phase is highly susceptible to gvl effects mediated by dli which induce durable remission in 70-80% of relapsed patients. the success rate is 30% or less in patients with accelerated phase or blast crisis. since dli cell dose appears to be important in this setting, strategies of escalating dose infusions have been investigated to enhance gvl without exacerbating gvhd. unfortunately, the response to dli in relapsed acute leukemia and myeloma is less favorable (<30%) and less durable. dli have been used successfully to treat viral infections and virus-associated malignancies following transplant. both unfractionated dli and ex vivo-generated tcell clones have suppressed reactivated cytomegalovirus and eradicated epstein-barr virus-induced lymphoproliferative disease, a polyclonal proliferation of donor-origin b cells that occurs after transplant. where tumor-specific antigens have been defined, efforts to target dli have been undertaken and donor and patient immunization has been investigated. acute or chronic gvhd develops in approximately 60% of patients receiving dli for relapsed hematologic malignancies and for related, but not unrelated transplants, correlates with the donor t-cell dose. dli-induced pancytopenia occurs in approximately 20% to 50% of patients, is generally mild, and transient, but in <5% of patients, aplasia is severe and prolonged. complications of aplasia include infection, bleeding, increased transfusion requirements. efforts to limit the adverse effects of dli while retaining the therapeutic effects include insertion of 'suicide genes, ' selection of lymphocyte subpopulations, and targetting lineage-specific minor histocompatibility antigens. available clinical and experimental evidence suggests, that in addition to primary and secondary immune deficiencies, a wide spectrum of immune-mediated conditions could benefit from intravenous immunoglobulin (ivig), including acute and chronic/relapsing diseases, autoimmune diseases mediated by pathogenic autoantibodies or by autoaggressive t cells and inflammatory disorders e.g. an imbalance in cytokine networks. trimar-collected apheresis platelet concentrates (pcs) were exposed to 17.2 j/ml uv light in the presence of 50 um riboflavin, followed by storage under blood bank conditions with various concentrations of 2-deoxyglucose from 0 to 20 mm for 5 days. the control platelets were not stressed by uv light exposure and were stored under the same conditions without 2-dog presence. all test and control platelets were measured for in vitro cell quality including rates of glycolysis, morphology score and activation levels at days 0, 1, 3 and 5. results: lactate production and glucose consumption increased from 0.0378 mmol/1012 cells/h (sd = 0.0097) and 0.0268 mmol/1012 cells/h (sd = 0.0114) for control samples to 0.1371 (sd = 0.0281) and 0.0724 (sd = 0.0151) for uv-treated platelets, respectively. uv treatment also caused a decrease in ph from 7.50 (sd = 0.07) for controls to 6.55 (sd = 0.26) for treated platelets at day 5, hsr from 75% (sd = 12.4) to 33% (sd = 25.1), esc from 24.3% (sd = 3.9) to 3.8% (sd = 3.6), swirl from 2.8 (sd = 0.7) to 1.0 (sd = 1.0), and increased p-selectin expression from 21.2% (sd = 7.3) to 85.2% (sd = 9.4). addition of 2-dog up to 20 mm significantly reduced lactate production rate to 0.0515 mmol/1012 cells/h (sd = 0.0045) and glucose consumption rate to 0.0293 mmol/1012 cells/h (sd = 0.0060), and maintained ph above 7.35 (sd = 0.09) for 5 days of storage. the effect of 2-dog exhibited a dose-dependent response. however, the addition of 2-dog had no effects on hsr (28.1 + 11.4% at day 5), esc (2.86 + 2.75% at day 5), swirl (0.8 + 0.5 at day 5) and p-selectin expression (69.9 + 6.4% at day 5) during platelet storage. atp contents in both treated and control groups were maintained at a relatively constant level above 87% of the value seen in fresh platelets. furthermore, an exaggeration of uv-stressed platelet aggregation by addition of 2-dog was also observed. conclusions: increased glycolytic flux is not a direct cause for platelet morphology changes and spontaneous activation incurred during the development of the storage lesion. the results also suggest that a reduction in glucose utilization may foster an increase in platelet loss during storage. aim of the study: was to evaluate analytical sensitivity, sensitivity and inclusivity for subtypes and genotypes of hiv, hcv and hbv, the assay's effectiveness in closing the pre-seroconversion window period, clinical specificity as well as the effect of endogenous substances and microorganisms on the sensitivity and specificity of the assay. methods: secondary standard traceable to who international standard for hiv-1 (97/ 656), international standards for hcv (96/798) and hbv (97/746) were used to determine the analytical sensitivity. sensitivity and inclusivity for hiv-1 subtypes other than hiv-1b, for hiv-2 and for hepatitis b and c genotypes as well as specificity was evaluated with >1000 specimens. results: results from this study indicate that high analytical sensitivities (39 iu/ml hiv-1m, 114 cp/ml hiv-1o and 1.1 cp/ml hiv-2, 7 iu/ml hcv and 3 iu/ml hbv) and a specificity of >99.4% are accomplishable for the mpx test. the 95% detection rate for hiv-1m subtype isolates (a through h) was between 10 to 50 iu/ml, for hcv genotype isolates (1a through 6) between 2 to 10 iu/ml and for hbv genotype isolates (a through g and precore mutant) between 2 to 5 iu/ml. investigating seroconversion panels, hiv-1 rna was detected an average of 6 and 5 days earlier than hiv-1 antigen with abbott hivag-1 monoclonal and coulter p24 antigen tests, respectively, hcv rna an average of 31 or 21 days earlier than hcv antibody with the abbott hcv eia 2.0 or ortho eia 3.0 tests, hbv dna an average of 18 days earlier than hbsag with the abbott hbsag eia imx test. for all targets, no interference was detected with 17 microorganisms tested as well as elevated levels of triglycerides, albumin, hemoglobin, human dna or bilirubin. conclusion: automated pooling, sample preparation, and real time pcr using the blood screening system 200 taqscreen mpx test is an efficient and sensitive method to simultaneously screen for five important viruses in human plasma. the mpx test is another evolution step in the development of pcr automation by roche molecular diagnostics, and further represents roche's commitment to increasing the safety of the global blood supply. aim of the study: a prospective hemovigilance plan was set up in order to establish a registry for future reference, and to detect any unexpected side effect of ip that may occur with significant frequency in populations and indications that were not studied before and outside of a formal trial environment. methods: this plan is proposed to blood establishments and transfusion prescribers who have already decided to implement ip. this is an observational, non randomized, non controlled plan. no patient selection, inclusion or exclusion criteria are required. all ip transfusions are documented using an internet form, whether or not a reaction is observed. patient population data are collected anonymously, for epidemiological purposes. results: between october 2003 and september 2004, 2512 apheresis ip units have been transfused in 2 sites and registered in the database. ip platelets were considered leucocyte inactivated and were not irradiated, but were antigen matched as indicated (3.9%). the population of patients receiving at least one transfusion (n = 271) included 58.3% of males, 40.6% of females, the median age was 64 (range 0-89). the most frequent broad diagnostic categories were hematology-oncology (64.9%) and cardiovascular surgery (19.9%). the patients received their transfusions either in regular hospital wards (63.5%), intensive care units (27.3%) or as outpatients (9.2%). the number of transfusions by patient ranged from 1 to 106 (mean 9.3 ± 16.0, median 2). half of the patients (49.8%) had previous transfusion experience and 5.9% had previous history of transfusion reaction. transfusion reactions, defined as any deterioration of the patient's state of health observed following transfusion, were observed in 1.2% (n = 31) of the transfusions (95% ci 0.84-1.75), and 8.5% of patients. only 2 (0.1%) were considered serious. after further causality analysis including biological and clinical investigations by the transfusion physician, 0.9% (95% ci 0.58-1.37) of the transfusions were confirmed as having caused reactions in 6.3% of patients, none of them serious. the most often reported symptoms were chills (0.9%) and fever (0.6%). itching, skin rash or urticaria was observed in 0.5% of transfusions. of the 2 serious reactions, one was hypotensive shock in a patient with liver cirrhosis and haemorrhage, and one was septic shock, in which the platelet unit bacterial culture was negative. none of the reactions occurred in cardiovascular surgery patients. patients were more likely to experience reactions if they had previous transfusion history (odd ratio 2.17, p = 0.15). the active hemovigilance plan is a valid and feasible method to collect epidemiological data on transfusion safety. the risk profile of ip transfusions appears favorable. quality of theraflex mb-plasma during storage and treatment s reichenberg* and n müller † *maco pharma international gmbh, langen, † inst. for transfusion medicine, essen, germany background: although in the last decades thanks to the implementation of several methods like donor selection and testing procedures the risk of virus transmission from plasma has decreased, infection of patients still exists. additionally new viruses like west nile virus enter the transfusion chain. therefore, the treatment of therapeutic plasma with methylene blue (mb) is a technique used in several european countries for pathogen inactivation. macopharma has developed the proprietary theraflex mb-plasma bag system including a mb pill and a final mb filtration step. aims: aim of the study is to show the quality of the mb plasma during the preparation procedure and during storage using the theraflex system. methods: for the preparation process every single step was evaluated using 18 single donor plasma units. for the evaluation of the plasma factors 5 ml were drawn at different stages (before treatment, after plasma filtration with plas4, after dissolution of the mb pill, after illumination, after treatment). because the sample volume for single sample measurements would be too low the samples were pooled after drawing and measured for the specified factors. six samples of each stage were pooled at three days. a whole panel of plasma factors was measured for the resulting three pools. global tests: quick, inr, aptt, thrombin time coagulation factors: fibrinogen, factor ii, factor v, factor viii:c, factor ix, factor x, factor xi inhibitors: at iii, protein c, protein s fibrinolysis: plasmin inhibitor, alpha1-antitrypsin complement: ch50 activation: tat, factor xiia, d-dimer stability data were generated using three plasma pools. six plasmas were pooled and afterwards divided into six aliquots. each was treated as single unit and then each was divided into six storage samples. the same plasma factors as for the manufacturing process were evaluated. results: a moderate reduction for some coagulation factors during the preparation was found in the illumination step but not in the other preparation stages. this was mainly fibrinogen (17.5%), factor viii (22.2%), and factor x (13.4%). despite this reduction the values were within the ranges found in non-treated plasma. all investigated plasma factors remained stable during the investigated storage time. summary/conclusions: the investigation showed that plasma treated with the theraflex procedure showed slight reduction during treatment and no reduction during storage. all plasma factors remained within the threshold values. the treatment of therapeutic plasma with mb is a valid technique of pathogen inactivation. validation of intercept treatment of pooled platelets g santos, c silva, f pereira and g sousa lisbon regional blood centre, lisbon, portugal background: intercept blood system for platelets uses amotosalen hcl and uva light to inactivate viruses, bacteria, protozoa and leucocytes that may contaminate platelet products. aims: the purpose of the study was to assess the feasibility of introducing this technology in the routine of lisbon regional blood center (crsl) and validate the procedure in our center. material and methods: whole blood units of 450 ml were collected from volunteer blood donors in quadruple top and bottom blood bags (optipure rc soft t& b baxter), kept in n-butanodiol plates; buffy coats with a volume of 55 ml were obtained in the opipress ii and kept overnight at room temperature before pooling. five buffy coats were pooled with 280 ml intersol using the octopus system intercept buffy coat pooling set with an integrated filter. the pools were treated using the intercept. samples were taken before treatment, after cad remotion, on days 2, 5 and 7. the following tests were performed: platelet count, mean platelet volume, ph, swirling. results: all pools met the intercept guardbands. platelet yield pre inactivation was 3.93 ¥ 10 11 (2.95-4.72 ¥ 10 11 ; sd-0.41). platelet pool volume was 362.3 ml (sd-10.7). plasma % was within 32.9% and 36.5%. all pools had leucocytes within council of europe specifications. after photoinactivation the platelet concentrates had 3.58 ¥ 10 11 (±0.37). the average platelet loss was 0.35 ¥ 10 11 . the ph was within specifications during all the storage period. conclusions: intercept treatment of pooled buffy coat platelets is feasible in the routine of crsl and in vitro parameters do not show significant changes, allowing us to proceed to clinical use. shown ip and conventional platelets (cp), stored for up to 5 days, exhibit comparable hemostatic efficacy and safety. extension of platelet storage duration to 7 days has the potential to improve platelet availability and reduce outdating and inventory shortages. clinical efficacy and safety of ip stored for 7 days were investigated. methods: a randomized, controlled, single-center, crossover, noninferiority design (pilot) study evaluated efficacy and safety of buffy coat ip vs buffy coat cp, each stored for 7 days. patients were randomized to receive one 7-day ip transfusion and one 7-day cp transfusion in random order. after each study transfusion, the 1hour platelet count, ci, and cci; time to next transfusion; bleeding response; transfusion reactions; and serious adverse events (saes) were assessed. the primary endpoint, 1-hour cci, was analyzed by a one-sided non-inferiority test for the per protocol population (patients with both transfusions and no major protocol deviations interfering with efficacy evaluation). the per protocol population included 20 patients, 9 randomized to the ip-cp sequence and 11 to the cp-ip sequence. more patients received allogeneic stem cell transplant in the cp-ip sequence than the ip-cp sequence (64% vs 33%; p = 0.37). mean platelet dose (¥10e11) was 2.8 for ip and 3.0 for cp (p = 0.23). there was a significant period by treatment interaction (p = 0.07) at the 0.10 significance level; therefore, the first period only was also analyzed for the primary endpoint. including both treatment periods, mean (±sd) 1-hour cci (¥10e3) was 6.6 ± 4.5 for ip vs 8.9 ± 5.5 for cp. the mean paired difference for both sequences was 2.4 ¥ 10e3 (p = 0.55 by non-inferiority test; upper bound of the 95% confidence interval = 4.0). for the first period only, mean 1-hour cci (¥10e3) was 8.7 ± 3.8 for ip vs 7.4 ± 5.4 for cp) the mean paired difference for the first period sequences was 2.4 ¥ 10e3 (p = 0.06 by non-inferiority test; upper bound of the 95% confidence interval = 2.4). the non-inferiority margin for the study was 2.2 ¥ 10e3 for mean treatment difference in cci (cp-ip). median time to next transfusion was 25 h for ip vs 24 h for cp following the first transfusion (p = 0.87 log-rank test; data censored at 7 days after transfusion) and 16 h ip vs 34 h cp after the second transfusion (p = 0.02). bleeding pre-or post-transfusion was uncommon, usually mucocutaneous, and grade 2 or lower, and responded similarly to ip and cp. no significant transfusion reactions or saes were reported. in this double-blinded, two-treatment crossover study the primary endpoint regarding 1-hour cci was not met. however, transfusion with 7-day-old platelets treated by the intercept blood system showed only a marginally and probably clinically insignificantly lower 1-hour cci compared to 7-day-old conventional platelets. methods: new zealand white rabbits were transfused with syngeneic blood (10 ml/kg), across a major antigen (hgd) mismatch. high anti-s-303 ab titers (≥1 : 1000) were induced after repeated immunization (days 1, 8, 15, 36, 64) with klh-(s-303) hapten (klhhapten) in complete freund's adjuvant. ab titers against srbc were determined by gel card agglutination, or by facscan with fitc-goat_anti-rabbit_igg. survival of infused rbc (4 ml/kg) treated with different methods was assessed by rbc biotinylation. blood samples were taken 1, 3, 7, 15, 21 and 28 days after transfusion, analyzed using streptavidin_pe and facscan to determine the proportion of circulating biotinylated rbc. results: groups (g) of rabbits were transfused with control rabbit rbc (crbc; n = 4, g1), or o-srbc (n = 6, g2). no ab against o-srbc developed after biweekly transfusions over 24 weeks in g2 rabbits. high ab titers to o-srbc could however be induced by klh-hapten immunization in a different group of animals (n = 2; g3). transfused rabbits (g1 & g2) exhibited no change in hematocrit or body weight and maintained good vital signs. high titer anti-s-303 abs were then induced by klh-hapten immunization in rabbits from g1 (n = 2) and g2 (n = 4), and in a group (n = 6, g4) of naïve rabbits. non-immunized rabbits (g1, n = 2), and (g2, n = 2) were maintained on the biweekly transfusion schedule of crbc and o-srbc, respectively. all rabbits treated with klh-hapten developed comparably high ab titers. klh-hapten immunization did not affect the viability of crbc in g1 rabbits. transfusion of o-srbc demonstrated reduced viability in hyper-immune g4 rabbits, but not any of the g2 rabbits. g2 rabbits exposed to 12 o-srbc transfusions prior to hyper-immunization with klh-hapten, had viability of o-srbc comparable to crbc, suggesting induction of immune tolerance by repeated exposure to o-srbc. after depletion of labeled o-srbc from circulation, g2 and g4 were transfused with m-srbc. viability of m-srbc in all g2 rabbits (hyper-immune or not) and the hyperimmune g4 rabbits was equivalent to crbc circulation in g1 rabbits. in pre-immunized rabbits with high titer anti-s-303 ab, o-srbc are cleared faster than control. in contrast, m-srbc survive normally in rabbits with high anti-s-303 ab titers. repeated transfusion of o-srbc does not result in alloimmunization of naive rabbits. the modified s-303 rbc process offers the potential for pathogen inactivation with elimination of immunoreactivity and retention of rbc viability. introduction: the bombay phenotype is extremely rare and characterized by complete absence of abh activity both on erythrocytes and in secretions. those individuals can produce anti-h, which is active over a wide thermal range. method: using liss indirect antiglobulin technique, the patient's serum showed +4 reaction by panel of eleven cells at room temperature phase as well as indirect phase while the auto reaction is negative. a cold adsorption using rabbit erythrocyte stroma was done to remove the cold antibodies from the serum; +2 reaction of an antibody was detected in the patient serum after five folds of rabbit erythrocyte stroma adsorption. introduction: immunohematology reference laboratory in kuwait central blood bank receives samples from all hospitals in kuwait both governmental and private sector. the laboratory performs the tests according to international standards and it is monitored by internal and external quality assessments on periodic basis. material and method: a tube and gel cards are two methods in the reference laboratory for antibody identification. the laboratory can identify the most commonly encountered clinically significant antibodies and investigates causes of positive direct antiglobulin test that occur mostly in autoimmune hemolytic anemia. there are facilities to phenotype most of rare red blood cell antigens. results: records of all patients investigated in the laboratory since the year 1985 are kept in computerized system that has 7528 patient's records. central blood bank has the potential to identify rare phenotypes such as kpb-, jsb-, lan-, bombay, rzr2, rzr1, r¢r¢, r¢r≤, r≤r≤, ge-2,3,4 and rare red blood cell antibodies such as high frequency antibodies anti-k, anti-ge2, anti-h, anti-lan, anti-kpb, anti-jsb, anti-wrb, anti-ena, anti-csa and low frequency antibodies anti-kpa-, anti-jsa-, anti-dia, anti-lua, anti-cob as well as hightiter-low-avidity antibodies such as anti-chido. samples of rare red blood cells and rare serums are kept frozen either by glycerol or liquid nitrogen technique to be used for pre-transfusion compatibility testing and continuing educational program. purpose of the work: to present the substitution of the blood groups o, a, b, ab in abo blood group system and rh (d) blood group in rh (d) blood group system in the population in the gevgelija-valandovo region. material and methods: a retrospective analysis was done on the data of following the blood groups o, a, b, ab and d in the blood group system abo and rh in the gevgelija-valandovo region. the asked population are voluntary blood donors, candidates for drivers, patients, pregnant women and newborn children. the period (1989) (1990) (1991) (1992) (1993) (1994) (1995) (1996) (1997) (1998) (1999) (2000) (2001) (2002) (2003) (2004) was analysed. the examinations were done with two standard methods (on the plate and in tube), to define blood groups from the most represented blood group systems abo and rh in the population. tests were done with different series of commercial anti serum tests from domestic and foreign origin. results: totally 43.395 examinees were typified. with o blood group were 15.761 (36.31%); with blood group a were 17.675 (40.73%); b blood group 7.119 (16.4%) and ab blood group were 3.019 (7.52%) examinees. totally 37.365 (86.11%) were d positive and 6.029 (13.89%) were d negative. discussion: the given results from our examinations for the frequency of o, a, b, ab and rh (d) blood groups from abo and rh (d) blood group systems in the region gevgelija-valandovo show that the most present is the blood group a from abo blood group system 17.675 (40.73%) and d blood group in rh system with 37.365 (86.11) form examined population. the results are in correlation with data from the literature for other european nations. introduction: the policy of our center is to transfuse all heamatologic multitransfused patients with their own rhesus and kell phenotype. in addition, thalassemic and young leukemic patients are being transfused with compatible phenotype of the most clinical important duffy and kidd systems while all the other patients receive blood compatible only with abo and rhesus system. nevertheless, it is observed a significant positivity of the indirect antiglobulin test, due to alloimmunization. aim of the study: in this study we tried to evaluate the prevalence of alloimmunization in patients of our region. the most frequent detectable alloantibody remains the anti-d, with high prevalence 88.9% of anti-d in females versus 11.1% in males, due to alloimmunization during the pregnancy. as a consequence, the high incidence of anti-d is not transfusion related and anti-e is evidenced to be the most frequent transfusion related alloantibody, followed by anti-kell. care must be taken in order to transfuse as more patients as possible with their own phenotype regarding, at least the most immunogenic antigens, like anti-e and anti-kell. severe hemolytic reaction due to anti-j k3 background: red blood cell alloantibodies directed against antigens of the kidd system are notorious for causing delayed hemolytic transfusion reactions. the antibodies are formed because of pregnancy or transfusion. blood donors with the red blood cell (rbc) phenotype jk(a-b-) are extremely rare in the white population and exhibit a frequency of less than 0.1%. however, the rare phenotype jk(a-b-) is more common in polynesians (1.4%). individuals with jk(a-b-) phenotypes typically form anti-jk3 with inseparable anti-jka and anti-jkb activity. some jk(a-b-) patients' sera may show an additional distinct anti-jka or anti-jkb component when examined with adsorption studies. case report: a 63 years-old caucasian female with a negative antibody screen, no prior history of transfusion, presented with gastrorrhagia. it is reported four pregnancies with no history of haemolytic disease of the newborn (hdn). on admission, her haemoglobin was 5.5 g/dl. she was given 4 units of crossmatchcompatible rbc. on day 6 her haemoglobin was 12.1 g/dl, with a total bilirubin of 0.72 mg/dl and lactate dehydrogenase of 141 u/l. on 10th day an unexpected fall in hb (5.6 g/dl) occurred with an increase of bilirubin to 1.9 mg/dl and of lactate dehydrogenase to 1042 u/l. a new blood sample obtained for antibody screening and additional crossmatches showed a pan-agglutination and incompatible crossmatch. anti-jk3 antibody high titer was detected in the plasma by gel-test using liss/coombs cards (id-diamed). the dat was negative and the antibody reacted equally with jk(a-b+), and jk(a-b+) panel cells (jka:1/16 384 and jkb:1/16 384). other alloantibodies could not excluded, because jk(a-b-) cells are not available. she was started with erythropoietin-a (epo), folic acid, fe iv and high dose intravenous immunoglobulin (ivig). the epo was discontinued after four week of therapy when the haemoglobin was 12 g/dl. two months later her haemoglobin was 13.5 g/dl and anti-jk3 was present in the same titer. a year later her blood cell count was normal and the anti-jk3 was detected in a lessened titer (1/16). no additional distinct anti-jka or anti-jkb component was shown after two adsorptions at 37°c using carefully selected phenotyped red cell compatible with patient's rh, fy, mnss, lu, le system and jka(+) and jkb(-), but two additional alloantibodies anti-c and anti-e of low titer (1/4) were revealed. the rare anti-jk3 alloantibody found in this case displayed the erratic nature of many kidd system antibodies. although anti-jk3 may cause mild hemolytic disease of newborn, she did not have a history of hdn. our patient was sensitized to a kidd antigen during pregnancy, but showed no serologically detectable antibody until challenged with a massive transfusion following a gastrorrhagia. the use of epo and high dose intravenous immunoglobulin succeeded to avoid transfusion with incompatible rbc unit. background: differential warm adsorption is used in the investigation of patients with red cell autoantibodies for searching of underlying alloantibodies, but it is also useful in the detection of clinically significant alloantibodies in patients with alloantibodies to high frequency antigens such as k, kpb, lub and inb. this technique is especially useful in cases when patients when patient's phenotype cannot be identified due to recent transfusion. purpose: differential warm adsorption is performing on cases presented with an antibody reacting with all red cells of the panel and having a negative auto control test. in these cases, even rare cells panels, which allow the identification of a pan antibody, are available, other more common clinical significant antibodies cannot be excluded. methods and result: case 1. a 63 years-old caucasian female with preexisting myeloproliferative disorder (polycythemia) presented with pancytopenia. anti-k was detected in the plasma. it is reported two pregnancies and no history of transfusion. the dat was negative and the plasma did not react with one k-cell present on the red cell panel in use. the anti-k specificity was confirmed using additional k-cells. the patient's red cell were group a, d+, k+, k-, c-, e-, fy(a-), s-, le(a-), kp(a-), cw-. she was transfused with two units rbc k-. ten days after the first transfusion the dat became positive and the one k-cell present on the red cell panel reacted with her plasma. two adsorptions were carried out at 37°c using carefully selected phenotyped red cell (compatible with patient's rh, fy, jk, mnss, lu, le system and k positive). an anti-fya was identified in the presence of ant-k. . an antik (titer 1/16) was suspected. because k-red cell was not present in the panel in use, adsorptions were carried out at 37°c using carefully selected phenotyped red cell (compatible with patient's rh, fy, jk, mnss, lu, le system and k positive). after the anti-k antibody was totally removed, no additional alloantibodies were revealed. conclusion: differential adsorption in cases with alloantibodies to high frequency antigens represents a useful application of the technique and helps in the identification of clinical significant antibodies present, allowing a more accurate decision for transfusion. evaluation of validity of the expired enzymetreated 0.8% red cells in antibody identification gel tests using nacl cards p chalkia, s intzepeli, v avgoloupi, a tsoukala, e ntinopoulou and p didoudi ahepa hospital, thssaloniki, greece background: expired red blood cells of required phenotypic profile is often used to identify antibody specificities in patients with multiple anti-erythrocytes antibodies. accurate results depend on the integrity of the antigens. purpose: to validate the expired enzyme-treated 0.8% red cells for use in antibody identification gel tests using nacl cards. the serum of nineteen patients with common specificities antibodies in rhesus, kell, duffy, kidd, and mnss systems tested with commercially prepared 0.8% enzyme treated cells rbc panel (id-diamed). gel tests were performed according to the manufacturer's instructions on in-date rbc and simultaneously on rbc 1 month to 5 months past expiration. reactivity of the expired antigen positive and antigen cells was compared to in-date cells. results: twenty antibodies detected with enzyme treated red cells in neutral gel cards [d(4), c(3), e(2), k(7), cw(4)] and were tested with enzyme treated red cells in use and rbc 1 to 5 months post expiration. seventeen antibodies tested with enzyme treated cells gave acceptable results with antigen positive cells 1 to 5 months post-expiration, except 3 anti-k antibodies (negative with k positive cells 2-4 months post expiration). conclusion: most rbc antigens studied were detectable 5 months after rbcs expiration date. tests with 0.8% cells were valid in gel test (nacl/enzyme) for at least 5 months after manufacturer assigned expiration date and may be helpful for complex identification studies. studies for more antigens specificities are needed to testify the validity of the expired enzyme-treated 0.8% red cells. background: wr(a) is a low-incidence blood group antigen (1 : 1000) in the caucasian population. despite that anti-wr(a) is a common antibody type, it may cause severe transfusion reactions, haemolytic disease of the new-born. anti-wr(a) may occur as an autoantibody or arise without immune stimulus. we report a case of a naturally occurring anti-wr(a) antibody. case report, methods and results: 56-year-old non-transfused male patient with acute pancreatitis and severe anaemia had been transferred to surgery from a county hospital. the serological status identified by their blood bank was: b rhd positive, with anti-wr(a) antibody in the serum (cellbind card method). our results: the patient's cells were group b rhd positive (microplate method), dat negative (tube and gel test method). the antibody identification showed positive antibody reaction with all enzyme treated test cells but negative reactions in liss iat (tube test) and gel iat (scangel and diamed). discussion: our routine tests for antibody detection didn't detect any specific antibody in patient's serum. he was transfused several units of blood, that was wr(a) negative and showed negative crossmatch reactions. the patient had no transfusion reactions. 10 days after the transfusion anti-wr(a) specificity was confirmed in the serum with cellbind test. only few test cell panels contain wr (a) positive cells, which are usually not present in commercial screening cells. in our case the cross-match was only performed for this patient because of the detected nonspecific antibody reaction in enzyme. the risk of transfusion reactions caused by rare antigens are particularly high in the type and screen cases. background: according to requirements of the french committee for accreditation (comité français pour l'accréditation cofrac, iso 17 025 standards), it is essential to use validated and standardised methods in immunohematology. this imposes, among various requirements, the knowledge of metrological tolerances for all the techniques. aim: a multicentre study was carried out to define the maximal acceptable deviations concerning incubation temperature and time, volumes of patient plasma and of tests cells for antibody screening using indirect antiglobulin test (iat) in filtration technique. the antibody screenings were performed manually in 3 blood centres using 3 different filtration systems: id diamed, biovue ortho and scangel biorad, the same tests cells, a standard 20 ng/ml anti rh1 (provided by cnrgs), a positive control anti kel1 and a negative control. all equipment used (oven, chronometer, pipettes) were calibrated according to cofrac standards. each antibody sample was tested under the following combined conditions (243 tests/sample): results: all the tests of antibody screenings from the multiples combinations of the above parameters gave the same results with a 2+ intensity agglutination for positive samples and the absence of agglutination for the negative control. conclusion: this study allowed us to define a range of tolerance for 4 critical physical parameters involved in the antibody screening in iat using commercial filtration systems maryvonne. the authors present a retrospective study involving 12 091 blood donors from the university hospital of coimbra during the year 2004. the incidence of weak d and rh (d) phenotype was determined in 1729 individuals who were rh (d) negative. the ab0 and rh(d) blood grouping was performed using a column gel agglutination card (diamed). the rh (d) typing was done using a anti-d polyclonal and a anti-d monoclonal antibodies. all donors that gave negative or poor agglutination results were tested for weak d with an indirect antiglobulin test, with anti-igg (gel matrix card) plus anti-d serum (diamed). within our target group of blood donors the rh (d) negative represented 14.3% of the total sample. we also describe ab0, rh(d) phenotype (c, c, d, e, e) group frequencies and establish reliable estimates frequency for weak d and rhesus haplotypes. the background: in 80s and 90s several authors tried to assess the relationship between the number od igg molecules per rbc and in vivo haemolysis, but determinations usually concerned small groups of tested patients. some of the investigators suggested that the number of igg autoantibody molecules per rbc was a major determinant of the severity of the haemolysis, whereas others found aiha patients with severe haemolysis and undetectable autoantibodies. aim: presentation of our experience with the quantitative elat performed on a large group of aiha patients during long-term observation. material and methods: six hundred fifty eight blood samples from 268 warm-type aiha patients were randomly tested for the number of igg molecules per rbc. eighty six of the patients were tested periodically from 3 to 15 times at one-month intervals. autoantibodies on rbcs were detected by the direct antiglobulin test (microcolumn technology) and measured by the enzyme-linked antiglobulin test (elat). results: in about 1/3 of tested samples the number of igg molecules per rbc was small (<190) and the laboratory signs of haemolysis were present in 65.7% of them as well as in 70.4% samples with moderately coated red cells (200-980 igg/rbc). the large number of igg molecules per rbc (>1000) was significantly associated with high frequency (87.9%) of severe haemolysis and it was also associated with presence of multiple igg subclasses on rbcs and c3d. in 79% of patients tested periodically, the number of igg molecules per rbc decreased and it significantly correlated with improvement of haemolysis parameters. in 21% of aiha patients the number of igg fluctuated and it was a poor prognostic factor. conclusion: in aiha patients the dynamics of the changing number of igg autoantibody molecules per rbc is a more helpful diagnostic and prognostic parameter than the number of igg molecules per rbc evaluated in one test. -b+) , -h-negative (using the anti-h lectin). antibody work-up showed a positive antibody screen (liss and peg-tube methods) reacting 4+ with o rbcs at all phases and 2+ with a1 rbcs. the direct antiglobulin test (dat) was positive with polyspecific ahg as well as anti-c3b,-c3d (table 1) . prewarming of test system did not change the reactivity (4+ at antiglobulin phase). a treatment with dithiothreitol (dtt) was performed and abolished all reactivity of the serum ( table 2 ). autoabsorption of the patient's plasma was performed. the absorbed plasma showed a decrease in reactivity from 4+ to 2+ when tested with o red cells, as well as a significant reduction in antibody titer from 1 : 256 to 1 : 2 tested at immediate spin (table 3 ). the patient remained crossmatch incompatible with o and a rbcs, but was compatible with oh rbcs. summary: we report an unusually strong igm anti-h antibody in this patient, who may require oh phenotype units. the patient is not a para-bombay since her red cells type strongly as group a. the cause for the auto-anti-h remains unknown at this time. if a thermal amplitude test shows that the antibody appears to be clinically significant the patient should receive h-units if transfusion is required. introduction: fetomaternal haemorrhage may determine an alloimmunization, in fact the transplacental passage of antibodies may cause the haemolytic disease of newborn. for this reason, in pregnant women, a screening for irregular antibodies research is routinely performed. however the indirect antiglobulin test (iat) may result falsely positive or negative for various causes, as operative mistakes or low specificity/sensitivity of the used techniques. aim of the study. in this study we have retrospectively evaluated the real incidence of alloimmunizations occurred in women screened by private laboratories. methods: we have studied 1.560 women, 19-43 years old, resulted iat positive at the first screening and successively assisted by our two hospitals. all women were re-tested, using gel-agglutination technique, for both direct antiglobulin test and iat. results: a positive iat was confirmed only in 64 cases; moreover a rbc autoimmunization was found in 7 women. anti-d (16 cases), e (10), c (8), k (8), c (6), s (5), d + jka (1), d + s + e (2), d + c + k (2), m (3), c + e (1), d + c + g (2) were the identified alloantibody specificities. anti-s, -e, -k, -c and -jka were the specificities in autoimmunized women. conclusion: in conclusion, a real alloimmunization is occurred only in 4.1% of screened women, while in the remaining cases iat resulted falsely positive: this observation forces us to affirm that, in order to minimize errors and alarmisms, the screening for antibody research in pregnant women must be performed only by immunohematology qualified center. background: one of the problems of the rbc transfusion is the alloimmunisation and the delayed haemolytic reactions (dhtr). besides rhesus and kell systems the antibodies against kidd antigens cause both dhtr and difficulties in their detection. aim and methods: the exact recording of all blood units according to abo rhesus kell and kidd systems. the abo-rh-kell systems are identified through automated microcolumn method (autovue, ortho), while kidd antigens are identified manually using microcolumn gel (diamed). results: the percentage of jka+ and jkb+ found in our department (75.8%) and (70%) respectively is similar to that of the caucasian population. conclusions: given the fact that there is lack of available freezing rbc system in greece, detailed recording of all units to the above antigenic systems can be proved extremely useful under circumstances of incompatibility. in the latter case suitable donors can be called and cover the shortage. the identification of all antigentic systems of the donated rbc units is underway. background: in many countries transfusion recipients are currently typed and transfused d-positive, if their red cells are agglutinated by 2 igm monoclonal anti-d that do not react with dvi. the transfusion strategy in weak d patients is not clear defined and it depends on the chosen monoclonal reagents and methods. patients who are carrying dw types 1, 2 and 15 were prone to develop anti-d. aim: the aim of this pilot study was to estimate capability of commercially available monoclonal anti-d reagents to recognize this weak d types as rhd positive. material and methods: edta anticoagulant blood samples were collected from 50 blood donors, previously typed as weak d positive by indirect antiglobulin test. molecular genotyping of rhd gene and weak d alleles by cde-ssp and d weak-ssp kits (inno-train, germany) were performed. direct agglutination was tested in a tubes and microplates using the following antibodies: rum-1, th-28, ms-201 (bioscot/serologicals) and d415 1e401/175-2 (immucor). results: out of 50 samples molecular typing results were as follows: in 5 samples dw were not determined, in 45 samples, 20 weak d type 1; 12 weak d type 2; 8 weak d type 3, 1 weak d type 11; 3 weak d type 14 and 1 weak d type 15 were determined. by all monoclonal reagents 95% weak d type 1, 100% weak d type 2, 10% weak d type 3 and 100% weak d type 15 negative results were given. by all monoclonal reagents weak d type 11 and weak d type 14 positive results were given. conclusion: according to weak d types, which were known to be at risk for anti-d immunization further advances may be brought by improved patient's monoclonal typing reagents with a low and donor's monoclonal typing reagents with high affinity for weak d type 1, type 2 and type 15. such improved typing strategies with novel reagents would enhance the transfusion safety. background: vel is a high-incidence antigen found in >99% of the population. anti-vel can be igm or igg and reacts optimally at iat, although it can also react at immediate spin and 37c. anti-vel may or may not cause severe hemolytic transfusion reactions and mild to severe hdn. autoanti-vel has also been reported. the aabb technical manual 14th edition states that the vel antigen is unaffected by protease and sulfhydryl treatment. we have reason to believe that sulfhydryl treatment may have an effect on the vel antigen as evidenced by a recently referred case. case report: a 67 year-old caucasian female presented with symptoms of anemia and renal vascular hypotension. transfusion history indicated multiple red cell transfusions in 1959. according to the patient, previous attempts to locate compatible units were unsuccessful. the case was referred to our laboratory. the patient's red cells (rbcs) typed as group o, d+ with a negative dat. the serological picture revealed an antibody reacting 2 + -3 + s at 37 c/liss, as well as at the antiglobulin phase. the antibody reacted with all rbcs tested and the autocontrol was negative. further characterization of the antibody showed similar reactivity using enzyme-treated rbcs (0.1% ficin) and no reactivity using 0.2 m dithiothreitol-(dtt)treated rbcs. a high incidence negative red cell panel (untreated) was selected that lacked antigens reported to be destroyed by dtt. the antibody reacted with all rbcs tested. additional rbcs were tested that lacked high-incidence antigens, including vel. the antibody did not react with four vel-rbcs tested using liss and peg methods. the patient's rbcs typed as vel-negative. all other clinically significant antibodies were ruled out using vel-or dtt-treated rbcs. based on the unusual reactivity demonstrated by the anti-vel, we tested 12 different examples of anti-vel (frozen in our rare sera inventory) against two sets of known vel+ and vel-rbcs. one rbc set was dtt-treated; the other set was tested neat. liss enhancement was used to test both sets. one of the 12 antisera failed to react with the positive control and one reacted with the negative control. both were disqualified from the study. four of ten remaining antisera demonstrated a decrease in reactivity >1 grade, between the neat and the dtt-treated rbcs. the remaining six antisera showed no change in reactivity. conclusion: contrary to the statement in the aabb technical manual, we discovered that sulfhydryl treatment (0.2 m dtttreatment) can have an effect on the vel antigen. our experience has demonstrated that in some cases anti-vel may not react or may show reduced reactivity when tested with dtt-treated rbcs. therefore, the presence of anti-vel should not be ruled out if negative reactivity with dtt-treated rbcs is encountered. additionally, dtt treatment may be a useful tool obtaining rule-outs of other clinically significant antibodies in the presence of anti-vel. additional data is needed to confirm these findings. but with no identified specific antibodies were investigated by repeated screening/crossmatch, papainized panel identification, hla antibody screening by lymphocytotoxicity test (lct) and elisa in some cases. patients' age, sex, department, diagnosis, previous transfusions/pregnancies, techniques, reactions' strength, number of positive cells, urgency, subsequent antibody tests, identification and lct were noted. antibody tests were performed: at pretransfusion testing (pt) by liss-coombs (diamed) and at blood grouping (bg) by biovue polyspecific (ortho) microcolumns -manually in urgency and routinely by sampler iif (diamed) and mitis (ortho) systems. results: investigated reactivity was recorded in 241 samples from 186 patients; 29 (15.6%) patients had > 1 episode. these findings comprised 15.1% of 1598 unexpected results found at pt and bg. incidences were 0.18% at routine and 0.21% at urgent bg (37 339 and 16 003 bgs, respectively), and 0.35% both at routine and urgent pt (15 803 and 23 705 pts, respectively). 56.5% patients were female, 44.6% over 60, but 19.9% < 30 years, coming mostly from surgery (19.4%), internal medicine (15.1%), hematology (10.8%), ginecology (10.2%), cardiac diseases (9.1%) and cardiac surgery (8.6% patients). frequent diagnosis were solid tumors (16.1%), cardiac diseases (13.4%), hematologic malignancies (8.6%), uraemia (4.8%), orthopedic surgery (4.3%) and hepatic diseases (3.2% patients). 43.0% patients were previously transfused, with only 9.1% patients proved as not transfused or pregnant. subsequently positive antibody test during the study had 27.6% tested patients. at pt positive crossmatch was found in 38.8%, antibody screening in 43.9% and both tests in 17.3% cases. majority of reactions were '1+' (50% at pt and 38.6% at bg); reactions '3+' or '4+' were found in only 5.1% cases at pt, compared to 17% at bg. one crossmatch only was positive in 75.2% positive crossmatches, with 38/43 patients having > 1 crossmatched unit. ahg identification was positive in 27.6% tested patients; in 26% of them papainized panel was also positive. lymphocytotoxic antibodies were found in 45.5% tested patients; 72.6% (8%-100%) of lymphocytes were reactive. finally, the cause of reactivity in antibody tests was determined as 'laboratory mistake' in 42.5%, hla lymphocytotoxic antibodies in 9.7%, 'igg antibodies of unknown specificity' in 7.5% (hla noncytotoxic antibodies in 2/2 elisa tested samples!), contaminated sample in 5.9%, anti-bga in 4.8%, non-specific cold antibodies in 3.8%, subsequently recognized specific antibodies in 3.2% (2 lua, 2 m, 1 kpa, 1 yka), non-specific autoantibodies in 3.2%, carry-over of dat-positive cells and antibody to reagent in 2.2% cases each, while in 4.3% cases antibody screening and in 4.8% cases crossmatch was repeatedly positive without confirmation in panels. discussion: after introducing of sensitive microcolumns, positive antibody tests without detectable specific antibodies require significant laboratory activities, particularly in older patients with malignancies or surgery. such reactivity was frequently caused by laboratory mistake, but often hla and sometimes specific antibodies were later recognized, or reactivity continued without confirmation in panels. relationships that may be helpful are further discussed in abstract part ii. results: significant differences (p < 0.05) and relationships of interest were noted. sex. in female vs male patients frequent features were: crossmatch as only reactivity at pt (45.8% vs 28.2%), positive lct (59.4% vs 26.1%), lymphocytotoxic hla antibodies (lytxab) (13.3% vs 4.9%) and reactivity 'positive screening, negative panels' (5.7% vs 2.5%); in males non-specific cold antibodies (7.4% vs 1.0%) and antibodies to reagents (4.9% vs 0) were noted. age. in patients > 60 vs < 30 reactivity was often found at pt (65.1% vs 35.8%), caused by lytxab (21.7% vs 5.4%), anti-bga (8.4% vs 2.7%) or 'positive crossmatch, negative panels' (9.6% vs 0), but rarely by laboratory mistake (37.3% vs 59.5%) or later recognized antibody (1 of 6 patients). subsequent antibody tests: tests were subsequently positive more often if reactivity was found at pt (79.3% vs 20.7% at bg), as positive antibody screening (41.7% vs 29.2% if positive crossmatch), with positive panels (48.3% vs 12.2% if negative). subsequent tests were positive in only 43.8% patients with lytxab, 50% with anti-bga, 1 of 3 with antibodies to reagent and in no case with 'positive crossmatch, negative panels' . techniques. lct was positive in 60% and 45.5% tested samples found at urgent and routine pt (diamed), vs 0 at bg. all 18 cases due to lytxab, 8 of 9 anti-bga and 6 of 8 'positive antibody screening, negative panels' were found by diamed. at bg (ortho) 71.4% cold antibodies and 64.6% laboratory mistakes were found. positive antibody test: identification was negative in 78.6% screening-only cases; 69% of them were caused by laboratory mistake. lytxab were found in 67.9% crossmatch-only cases; 77.8% reactivities caused by lytxab were crossmatch-only. identification. ahg panel was positive in 60% cases with lytxab (in 2 with papainized panel) and often due to 'igg antibodies of unknown specificity' (29.7%), anti-bga (24.3%), contaminated sample (16.2%), but also to later recognized specific antibody (10.8% cases). strength of reaction: laboratory mistake was noted in 61.6% of 'w', 41.3% of '1+', 46.4% of '2+' and 58.8% of '3+ and 4+' antibody screenings (ns). diagnosis. in patients with solid and hematologic malignancy reactivity was often found at pt (80% and 81.3% of patients, respectively), due to positive crossmatch (40% and 50%; 0 and 4.3% patients with liver and cardiac diseases) and caused by lytxab (16.7% and 31.3%) or 'crossmatch/screening positive, panels negative' (16.7% patients with solid tumors). in patients with liver and cardiac diseases reactivity was often found at bg (83.3% and 68.0% of patients), due to laboratory mistake (50% and 60%) or cold antibodies (16.7% and 12%), respectively. discussion: features of non-specific reactivity depended on sex, age, positive antibody test, diagnosis and, moreover, used techniques, sometimes in very distinctive manner. this analysis might be of considerable help in planning of laboratory tests, but also in quick analysis of unexpected results and choice of further testing, particularly in urgent situations. background: worldwide screen and type is a very usual method for pre-transfusional testing. the ultimate objective is to prevent not only the clinically expressed delayed hemolytic transfusion reactions but also the serologically revealed ones. aim: the aim of this study was to determine the frequency of red blood cell (rbc) alloantibodies in patients undergoing cardiac surgery or cardiac procedure, during the pre-transfusion screening. materials and methods: blood samples of 16 291 patients (12 628 male and 3663 female) were evaluated. the mean age of the patients was 57 years. pre-transfusion samples were examined for clinically significant alloantibodies, using antibody screening with gel test (liss -enzyme). in case of a positive result, identification was performed (panel with autologous control). in addition, the serological testing included cold agglutinins´ detection (tube test), as well as titration (tube test) and identification (gel test) in case of a positive result. when the result was marginal (1/32) a new test was carried out after a seven days period. in the presence of a positive autologous control or an autoantibody, samples were examined with direct antiglobulin test (dat). results: alloantibodies were detected in 580 patients with the incidence of 3.56%. antibodies were registered more frequently in females (190/3663, 5.18%) than in males (390/12 628, 3.09%). 183 patients (31.49%) developed single antibody with anti-kell being the most frequent. the incidence and the specificity of the detected antibodies are summarized in the following table (table 1 ). in 23 patients (3.96%) multiple antibodies were detected, with most frequent the anti-d and anti-c combination. 35 patients (6.03%) were dat positive. autoantibodies were found in 10 patients (1.72%), all of which had specificity to rhesus system. cold agglutinins were positive in 53 patients (0.32%). no specificity could be assigned in 245 patients (42.24%), while in 110 patients (18.96%) non specific reactions in enzyme treated rbcs, were observed. one patient developed delayed haemolytic reaction 15 days post-transfusion, due to anti-jka. the antibody, however, was not detected in the pretransfusion sample re-testing. the frequency of the pre-transfusion detection of red blood cell alloantibodies in our center, was 3.56%. the most frequently identified were the anti-kell and anti-rh. the high rates of unidentifiable antibodies and non specific reactions in enzyme treated rbcs are probably attributed to the kind of medication that most of these patients receive, as well as to the degree of inflammatory process which usually accompanies such diseases. the high frequency of unidentifiable antibodies indicates that a larger and more complex erythrocyte panel would be useful for routine testing. the routine pre-transfusion screening for alloantibodies probably assures the prevention of dhtrs and provides sufficient time for blood selection for transfusion. introduction: in the united kingdom, about 1% of women form red cell allo-antibodies in pregnancy and 0.07% of all pregnant women produce anti-c. before the introduction of prophylactic anti-d, it was reported that 3% of the total haemolytic disease of the newborn (hdn) cases were due to anti-c. currently, cases of hdn due to anti-c are half as frequent as anti-d and 14% of the uk population are rhc negative. we present two unusual cases of pregnant women who are d and c negative and have anti-c and anti-d detected in their serum. case studies and results: case 1: a 38-year-old asian woman had three previous uneventful pregnancies. in her 4th pregnancy she presented with miscarriage at 19 weeks gestation. she was group b, d and c negative. her serum contained anti-c and anti-d. anti-d was detected by liss tube iat and anti-c was only detected by manual polybrene technique (0.9 iu/ml by quantification using r2r2 cells). in a 5th pregnancy, no antibodies were detected until 34 weeks gestation when this patient presented in early labour. anti-c was then detected by two-stage papain technique only as well as anti-d. case 2: a 33-year-old asian woman had a positive antibody screen post caesarean section in jan 2005. no antibody had been detected during the pregnancy. standard prophylactic anti-d was given at 28 and 34 weeks gestation as this patient was d neg. anti-c and anti-d were confirmed in her serum. the anti-c level was 0.7 iu/ml using rr cells and the anti-d level was <0.1 iu/ml using r1r1 cells (prophylactic anti-d ig). she was phenotyped as o r¢r¢. at delivery the baby was found to have a negative dat and was phenotyped as r¢r. discussion: cde/cde (r¢r¢) is an uncommon phenotype in the uk population with a frequency of 1/10 000. in routine antenatal testing, abo/d grouping is only performed for pregnant women at booking and 28 weeks gestation according to bcsh guidelines. full rh phenotyping is not carried out unless the pregnant women has a positive antibody screen. in routine testing of the above cases, this extremely rare phenotype is missed. prophylactic anti-d was given to both patients and immunisation due to anti-d was prevented. there is currently no prophylactic regime developed to prevent anti-c allo-immunisation by the fetus in pregnancy. antenatal management of patients with anti-c and anti-d during pregnancy can be problematic: (i), problem in antibody identification; (ii) monitoring of antibody level (i.e. quantitation by auto analyser for anti-c and anti-d) with two different cells and (3) provision of blood during pregnancy, at labour and post delivery for both mother and newborn. study on the frequency of red cell phenotypes (e.g. duffy, kidd and mns blood group system) in our local population l leou, yf wong, mbc koh and d teo health sciences authority, singapore, singapore background: the frequency of various red cell antigens in the caucasian population has been well studied. to date, the frequency of these antigens in the local population composed of a multi-racial mixture of chinese, malay, indian and others is still unclear, especially in the malays with paucity of data in the literature. aims: to investigate the frequency of clinically significant red cell antigens duffy, kidd and mns across the local ethnic groups. to investigate the occurrence of rare phenotypes. to be aware of these rare phenotypes so as to facilitate planning of blood inventories and supplies. typing for the duffy, kidd and ss antigen on blood donors was performed using monoclonal as well as polyclonal anti-sera by manual tube method. a total of 1427 blood donor samples were tested using specific anti-sera that will agglutinate red blood cells that have the corresponding antigen. agglutination is demonstrated by the indirect antiglobulin technique. result and discussion: table 1 -a higher frequency of the fy(a+b-) phenotype is seen in chinese, malay and others in contrast to the indian and caucasian population. the clinically significant allo-antibody anti-fya is rarer in our local population. it occurs predominantly in malays and indians and usually in combination with other antibodies. it means that provision of antigen negative blood may be difficult. table 2 -all groups show similarity of distribution of the kidd phenotype with the caucasians and distinct from the american blacks. the jk(a+b-) and jk(a+b+) phenotypes are relatively equal in frequency and there should be no problem looking for such a phenotype in the local population. table 3 -the s-s+ phenotype is most common amongst all races. the indians are more similar to the caucasians with a relatively high frequency of s+s+ phenotype. the chinese and malay distribution are unique with > 99% being s+. conclusion: there is a unique distribution of red cell antigen groups in the 4 races and the data on malays is especially useful. this data will allow the national blood service in its inventory planning and the potential difficulties of providing antigen negative products due to clinically significant allo-antibodies. miltenberger phenotypes among taiwanese table 1 . conjointly, in order to obtain the frequency of mi.v phenotype, we screened 543 samples among 1230 with anti-hil and four additional cases of mi.v were found. conclusion: in this study significant miltenberger polymorphism was seen among the taiwanese population. besides previously described mi.iii phenotype (1.9%), there were also mi.i/ii phenotype (0.4%), mi.v phenotype (0.7%), mi.vi phenotype (0.7%), mi.x phenotype (0.1%), and most interestingly two miltenberger related not yet classified variants (1%). related variant a (0.5%) was phenotypes as mia+, anek+ and hil+. related variant b (0.5%) was phenotyped as mia+, anek+. interestingly, both variants were mur-(negative). the total estimated frequency of miltenberger variants in taiwanese population (including related variants a and b) is therefore 4.8% (table1). the discovery of 2 unclassified variants (most likely not yet described in the literature) is of great interest in the field of immunohaematology and warrant further molecular genetic study. introduction: the use of column technologies for the detection of rbc antibodies improved significantly the screen test sensitivity. each column-based method has its advantages and disadvantages. aim: to compare antibody detection by two column agglutination tests; the fully automated ortho auto vue tm method and the manual diamedᮀ id-micro typing system. material and methods: during the study period 57 375 patient samples were screened, 414 of the positive results were evaluated. 377 blood samples with positive screen tests by the ortho auto vue tm method (av) performed with 3% cell suspension, and 37 patient samples with antibodies identified by the diamedᮀ system (dm), were reciprocally re-screened, respectively. positive samples were tested by diamed panels for antibody identification. sera samples were divided into 6 categories according to antibody specificity; 1. rh system antibodies (n = 150), 2. clinically significant non rh system antibodies (n = 85), 3. clinically non significant antibodies (n = 28), 4. auto antibodies (n = 13), 5. not identified (ni) antibodies (n = 48), 6. negative screening results by the diamed technique (n = 90). the 6 categories were divided, according to the intensity of agglutination in the screen test, into those exhibiting stronger reactions by the auto vue (av > dm), those exhibiting equal strength reactions (av = dm) and those with weaker reactions by the auto vue (av < dm). statistical analysis was carried out using the wilcoxon signed ranks matched-pairs test. results: a total of 414 samples were compared, 31.2% of them gave stronger reactions by av, 45.4% gave equal reaction strength and 23.4% of them gave weaker reactions by av. positive screen tests by av only were detected in 90 samples, no specific antibodies were identified. in contrast to that, positive screen tests by dm only were detected in 9 samples, 5 were rh system related, 3 kell system related and 1 not identified, results are summarized in the table. discussion and summary: the antibodies detected by dm only, are anti-d and antibodies directed to low frequency antigens. the failure of av to detect rh system antibodies is further sustained by the fact that statistically significant weaker reactions for this antibody system were obtained by av technique. recently ortho-clinical diagnostics modified the screening reagent red blood cells to 0.8% suspension, in order to improve the sensitivity of the method (unpublished data). the possible explanation for the failure to detect low frequency antibodies is that ortho screen cells do not consistently carry the low frequency antigens cw and kpa. positive screen tests detected by av only, can be explained either by the fact that antibody identification was carried out on dm panels, or these are false positive reactions. in order to clarify this question we recently repeated these av only positive samples by the manual ortho bio vue technique. preliminary results indicate that no specific antibodies were detected. it can be assumed that these results are false positive. further study of this issue is required. aim of the study: we have detected blood donor with rohar variant which was mistaken as d-and his donations used for d-recipients. we tested these patients in order to evaluate possible anti-d or anti-lfa immunization. methods: rohar variant was tested serologically (commercial and workshop moabs) and on dna level (pcr-ssp). involved recipients were tested by diamed column agglutination (gliat with normal and enzyme treated rbcs) with commercial rbcs and with rh33 and rh50 rbcs. results: rohar variant was confirmed on phenotype and genotype levels. in four d-and two d+ recipients of rohar positive units no anti-d not anti-rh33 or -rh50 antibodies were detected. in one case anti-le(a) antibody was found. conclusion: in our cases massive exposition of recipients (whole transfusion unit) by rohar red cells did not lead to production of detectable anti-d or anti-lfa. the immunogenic potential of this variant seems to be low. unusual ab0 grouping discrepancy -inhibition of anti-b reagent by isolated increase of plasmatic b substance in a patient with group ab and pancreatic cancer m pisacka*, k petrtylova † , m kralova* and h flidrova* *uhkt, prague 2, † blood bank, faculty hospital m, prague 5, czech republic introduction: in rare pathological conditions excess amount of blood-group specific substances can be observed and can cause neutralization of grouping reagents. changes in abh and related histo-blood group antigens in malignant tissues were described but there are few information about similar changes in secreted bloodgroup specific substances. aim of the study: we describe a case of isolated increase of b group substance in plasma of a group ab patient with pancreatic cancer. methods: ab0 grouping was performed with registered immucor reagents (immuclone: anti-a birma 1, anti-b lb2) by slide and tube tests. neutralizing effect was quantified by (i) inhibition od anti-b reaction by titrated patient's serum; and (ii) inhibition of titrated anti-a and anti-b reagents by patient's serum, compared to ab serum of a healthy donor. results: slide test: unwashed rbcs: group a, washed rbcs: ab. tube test (washed rbcs): ab. titrated patient's serum when added in aliquot to anti-b reagent inhibited agglutination up to titre 64. titration of reagents /+ aliquot of serum added/: anti-a: titre 4096 (both patient's serum and control); anti-b + patient's serum: titre 4, anti-b + control: titre 4096. conclusion: pancreas is known as rich source of blood group specific substances. malignant transformation is known to be associated with either loss or re-expression of cell-bound abh antigens. reported excessive increase of b substance in group ab patient could be either due to loss of a-transferase activity in malignant pancreas cells or isolated increase of b-transferase activity. further studies on larger groups of pancreatic cancer patients will help to understand this observation. weakened abh reactions of unwashed rbcs could be of diagnostic importance, because in other case this observation preceded several years the pancreatic cancer clinical manifestation. implementation of the autovue innova, an upgraded column agglutination technology (cat) for pre-transfusion testing in a large blood establishment c politis, k armyros, a antypas, v malamou and p katsea g. gennimatas general hospital, athens, greece objective: automated cat testing in a blood transfusion laboratory aims at standardization and savings in labour as well as reagents. a new technology is evaluated in comparison to standard methods. materials and methods: we used column agglutination technology with the innova autovue system, ortho, ratrian n.j. according to the manufacturers this system provides priority to the management of the stat samples while the random access feature of the system enhances the system throughput. it provides an extended test menu as well as automated antibody identification with the red cells panels. the autovue innova system supports the bi-directional communication with the lis interface for all the tests including the crossmatches and it provides a continuous traceability and notifying of the instrument's status concerning either the required and available resources or the proper function of the system's submodules. in this study we performed 1423 tests including forward and reverse abo group, rh type and phenotype and kell in randomly selected blood donors, as well 895 tests in haematological patients for antibody screening using an untreated three-cell panel and autocontrol in the indirect antiglobulin test (iat). the results and the time performance (specimen handing, operation of testing and recording of results) were compared with those obtained by the semiautomatic id-diamed gel agglutination microtyping system and by standard manual methods. results: test results showed 100% agreement between all three methods. 68 samples were tested per 60 min with the autovue innova, compared to 90 min required for the same number of tests performed with manual testing and 70 min with the semi-automated method. the technical execution was easy with the autovue innova procedure and it appears that the consumption of testing reagents is smaller with the automated system comparing with the other methods. computerization of the test results with the autovue innova provides an important advantage in record keeping in the blood establishment. conclusion: standardization of sample collection and tests performance in pre-transfusion testing, as well as computerized records and time saving are advantages offered by the autovue innova, a new automated column agglutination technology, comparing with a semi-automated and the classical manual methods. a heavy workload is expected to significantly decrease time performance. clearance of senescent erythrocytes in young and old individuals al racca, a ensinck, c cotorruelo, s garcía borrás, l racca and cs biondi universidad nacional de rosario, rosario, argentina introduction: after a lifespan of 120 days, human red blood cells (rbc) are captured and phagocytized by monocytes/macrophages. the accumulation of autologous igg on rbc membrane provides a direct mechanism for the removal of senescent (se) rbc. an alternative pathway, immunoglobulin-independent, with participation of sialic acid, has been proposed. the physiological elimination of serbc might be modified by individual's age. aim of the study: to investigate in young and old individuals, the interaction between monocytes and different erythrocytes suspensions: serbc, rbc stored with or without serum and desialinized rbc. methods: healthy individuals blood samples (20-40 years old, n = 50 and > 70 years old, n = 49) were studied. different suspensions from each sample were obtained: (i) se and young (y) rbc by differential centrifugation; (ii) rbc stored with its own serum (rbcs) and without serum (rbcws); and (iii) rbc desialinized with neuraminidase (ne) and tripsine (t). the suspensions were subjected to the erythrophagocytosis assay: peripheral blood monocytes were incubated with the different erythrocyte suspensions for 3 h at 37°c. two hundred cells were analyzed to determine the percentage of active monocytes (am) with phagocytosed and adherent red cells. non sensitized rbc (nrbc) and ex vivo sensitized rbc (srbc) were used as negative and positive controls respectively. results: the% of am obtained with old individuals were: serbc: 21.9 + 1.0, yrbc: 2.9 + 1.2, rbcs: 16.2 + 1.4, rbcws: 3.1 + 0.8, nerbc: 11.1 + 1.3, trbc: 3.2 + 1.1, nrbc: 3.0 + 1.2; srbc: 31.2 + 1.8. the values of am obtained with young individuals were: serbc: 17.1 + 1.5, yrbc: 3.1 + 0.9, rbcs: 11.1 + 1.1, rbcws: 2.8 + 1.2, nerbc: 10.8 + 1.4, trbc: 3.5 + 1.0. nrbc: 2.9 + 1.3; srbc: 30.5 + 1.7. conclusions: no differences in the% of am were found when compared to positive and negative controls, indicating that this assay would not detect variations in the phagocytic activity of monocytes from young and old donors. the values of am with serbc were higher (p < 0.001) than those obtained with yrbc in both populations analysed. the rate of erythrophagocytosis with serbc in old individuals was significantly higher (p < 0.001) than that obtained in young donors. the increase observed may be due to agedependent changes of rbc that occur with human ageing. the% of am with rbcs were higher (p < 0.001) in old individuals. no modifications were observed with rbcws. the significant increase in the rate of erythrophagocytosis with serbc and rbcs show the involvement of autologous igg in the selective removal of erythrocytes. these values were higher in old individuals indicating that this process would increase in aged donors. the tripsine activity was not enough to modify the% am. the values of am obtained with neuraminidase treated rbc were higher than those observed with yrbc (p < 0.001). however these values were similar between young and old individuals, suggesting that the desialylation would not participate in the increased removal of erythrocytes observed in old donors. cell-cell adhesion is a crucial phenomenon for the relationship between the cell and its environment. therefore, the development of experimental methods to obtain quantitative parameters of cellular adhesion is important. erythrocytes are widely available and their membrane properties are well known, so these cells are used as an ideal model for studying the cellular interaction mechanisms. the study of the formation and break-up of receptor-ligand bonds in sheared erythrocyte suspensions is a subject of considerable importance in the blood circulation, where formation and break-up of blood cell aggregates occur in a variety of physiological and pathological conditions. the main two objectives of this work were to study the cellular adhesion phenomenon using erythrocyte adhesion mediated by monoclonal anti-a antibody as a model and to achieve quantitative values of the parameters involved in the intercellular binding and its possible relationship with cellular deformability parameters. agglutinates of two a erythrocytes (doublets) induced by specific monoclonal antibodies anti-a were used. adhesion energy was indirectly quantified by the study of doublet dissociation under the effect of a given shear stress using a controlled flow chamber system. the chamber was installed on the stage of an optical inverted microscope (union optical, magnification 100¥) in such a way that the cover glass was the floor of the microchannel. a ccd (charge coupled device) camera was placed in the ocular tube of the microscope and connected to a digital image processor (ipplus system) to digitize, record and analyze microscopic images. the doublets were initially immobilized inside the chamber, and then put under different shear stress values by a controlled laminar flow during a definite time. in this way, a shear stress parallel to the contact surface between both cells was applied, observing that the upper cell detached progressively with time and also with a gradual rise of the shear stress. the sequential microscopical images were registered and digitally processed, measuring the geometrical dimensions that the cells acquire during the deformation process, and the dissociation of the antigen-antibody bond. digital image processing allows the analysis and the quantification of the red blood cells doublets dissociation phenomenon. these results show that, while the shear stress applied is raised, the contact area between both cells diminishes. the obtained parameters give important information that lead to the estimation of the value of adhesion energy between two red blood cells agglutinated by monoclonal antibodies. as consequence, they will allow the characterization of the antibodies used, since it would evaluate their association capacity with cellular antigens. glycophorins (gp) a, b and c are abundant transmembrane integral proteins in red blood cell (rbc). their highly glycosylated nature and high sialic acid content account for the net negative charge of mature rbc membrane, which is physiologically important because it impedes any tendency to stick together in the circulation. in this study, we have tested anti-gp specific mouse monoclonal antibodies (moab) as primary antibody to directly agglutinate rbc. fret technique has been developed to characterize the hemoagglutination based on the interaction of fluorophores (alexa488tm, dio and dii) located in the rbc membrane or combined to secondary antibody directed against the primary agglutining moab. combined intensity (spectral) and lifetime (flim) imaging was used to discriminate the fret signal of molecules on their different lifetimes whereas their emission spectra overlap (alexa488tm or dio/dii) as independent phenomena of the fluorophore concentration and photobleaching. furthermore, gp-cytoskeleton interactions were analyzed by 3d-fluorescence microscopy after lipid extraction with triton x-100 in red cell. all the antibody used was found to directly agglutinate the human rbc. in view of the fluorescence properties depicted in rbc for the pair of fluorophore alexa-488tm (donor) and dii (acceptor), it may be expected that upon agglutination of rbc, effective fret should be observed. flim in dynamic-state provides a discrimination of molecules in their fluorescence lifetime, which allows to evaluate the underlying mechanism of energy transfer process in the agglutinated erythrocytes. the results demonstrate that's upon excitation at 780 nm the flim-fret from alexa488tm to dii for the anti-gpb moabs only with a lifetime distribution in the picosecond range. similarly, effective fret was not observed for the anti-gpa moabs. the contrast in measured lifetime image is a reliable indicator for spatial variations in donor-acceptor association. 3d-fluorescence microscopy images showed interactions between gpc or gpb and cytoskeleton and did not show interactions between gpa and cytoskeleton. all together, these results only revealed by fret-flim and 3d-fluorescence microscopy strongly support the importance of the specific reactivity with glycophorin a or b of agglutining moab. introduction: the frequency of alloimmunization to red blood cell antigens in transfused sickle cell patients can range from 10 to 40%, but the development of autoantibodies is much less recognized and descried. aim of the study: in order to evaluate the autoantibody formation and observe the blood transfusion association, we analyzed the direct antiglobulin test (dat) as well as the antibody screening results in 612 transfused sickle cell patients. methods: all the patients included in the study had received at least 1 unit of red blood cell concentrate, on the majority of the cases matched for the c, c, e, e, k antigens. the transfusion range was 1-94 units. the dat performed was a polyspecific gel test. in cases of positive dat, was performed the monospecific gel test (igg, iga, igm, c3c, c3d) . in almost all of cases an acid glicin elution was performed and the eluate was tested against a red cell panel (liss/coombs and papain). an antibody screening by gel test (liss/coombs and papain) was performed in all the 612 patients. the dat was positive in 98 (16%) of the 612 patients. in 96 cases (98%), the dat was igg type, in 1 case (1%) igg + c3d and in 1 case (1%) igg + c3c + c3d. the acid elution was performed in 97 cases. the eluate was positive in 38 cases (39%). in 32 (84%), of the 38 cases, autoantibodies were pointed out whose specificity were 25 specificity public, 5 anti-e (rh5), 1 anti-c (rh2), 1 anti-ce (rh7). in 6 cases (16%) we found alloantibodies whose specificity were 2 anti-e(rh3), 2 anti-k(k1), 1 anti-c (rh2) and 1 anti-jka (jk1). in the 59 cases (61%) no antibody was identified on the eluate and the cause of positive dat is unclear. we could correlate the positive dat with a presence of autoantibodies in 32 (5.2%) of the 612 patients. of these, 13 (40%) had a blood transfusion association. the global frequency of red cell alloimmunization in this set of 612 patients was 15%. seventeen patients (53%) of the 32 patient who had autoantibodies had also alloantibodies associated. conclusion: the mechanism by which erythrocyte antibodies form in association with blood transfusion are not well understood, but even though the medical literature indicates strong association with blood transfusion as well erythrocyte alloantibody formation, we could not find support for this association. the loss of splenic to sickle cell patients could be important because experimental studies suggest that the spleen is involved in the regulation of autoantibody formation. forward and reverse blood grouping with lateral flow based assays p schwind*, i aebischer*, k loester † and p monod* *medion diagnostics gmbh, duedingen, switzerland, † prisma diagnostika gmbh, berlin, germany background: recently, a lateral flow assay for simultaneous typing of abod, rhesus subgroups and kell with stable end-point and without a centrifugation step was presented (loester k, fleischhauer s, schwind p: lateral flow assay for simultaneous typing of of abo, rhesus subgroups and kell. vox sang 2004, 87 (suppl. 3), 40). in many countries, the determination of isoagglutinins in addition to the red cell antigens is mandatory for abo grouping. aims: to develop a lateral flow test for reverse grouping, supplementing the lateral flow typing assay. a lateral flow device was constructed with a separation membrane equipped in a cassette housing having 4 distinct incubation wells, application zones and detection areas. 25 microliters of 5% suspensions of reagent red cells for reverse grouping (reverse-cyte a1, a2, b, 0, medion diagnostics, switzerland) are mixed in each incubation well with 100 microliters of plasma. the resulting suspensions are incubated for 2 min, followed by the transfer of 50 microliters each to the application zones, where migration starts immediately. results can be read after 3 min in the detection areas. a positive result is recognized as a distinct red dot, a negative result is monitored by the absence of a dot. results: the plasmas of 80 donors, previously determined for the respective blood groups and isoagglutinins by the tube technique, have been tested with this method. the results for both methods were in full agreement. conclusions: a simple, rapid and flexible lateral flow method for reverse grouping is presented, allowing now for the determination of forward and reverse typing in similar formats. both methods give results after 5 min with stable end-points without the need of a centrifugation step and are easily applicable to non-laboratory environments. the performance of the autovuetm system for red cell antibody screening of blood donors during background: in israel every donation is tested for the presence of red cell antibodies (rbc abs). screening is performed on the autovuetm system, using ortho 2 screening rbc since the year 2000. aim: (i) to summarize the performance of the autovuetm system for rbc abs, during 4 years (2000) (2001) (2002) (2003) (2004) . (ii) to evaluate if an initial low positive result, with two negative repeats, could be considered a negative result. methods and results: rbc abs screening was performed using igg cassettes. positive results were confirmed by diamed gel cards, with 3 screening rbc, followed by diamed panels for abs identification. results: summary of the results is presented in the table. (i) in 0.57% of 1358,769 blood donations that were screened, during 2000-2004, using the autovuetm system, a positive initial result for rbc abs was detected, which was confirmed in 40.2% (0.22% of the donations). an increased percentage of confirmed tests is noted, since 2003, probably due to an improvement in the manufacturing of the cassettes by ortho. (ii) during the validation, 83 samples with low positive results (agglutination degree of 0.5) were retested twice on the autovuetm. 57/83 (69%) gave negative results in both repeats. only 1/57 was confirmed positive and anti-m was identified by diamed gel test. the remaining 26 samples had at least one positive repeat. 5/26 (19.2%) of those samples were confirmed positive. summary: autovuetm can be used as a competent method for rbc abs screening, in blood services which require high thoughput automated systems. samples with a low positive agglutination, which were negative in two repeats, can be considered negative. retesting these samples on the autovuetm, can reduce the number of tests sent for confirmation and allow early release of blood components. rk tagi-zadeh*, aa karimov*, ar hasanov † , ly novruzova* and si donskov ‡ *hematology and transfusiology, baku, † blood transfusion centre, ganja, azerbaijan, † centre for hematology, moscow, russian federation background: the main method of treatment of severe homozygous thalassemia forms at the moment is still an anemia control with regular rbc transfusions. the use of adequate transfusion regime for these patients not only prolongs their lives but also promotes normal physical development of the children and improves the quality of their lives. however, necessity to do multiple transfusions increases a risk of post-transfusion reactions and complications due to alloimmunization to rbc antigens. in order to prevent posttransfusion reactions it is essential to know the frequency of blood group distribution in the region and then implement organizational procedures to enhance the transfusion services for these patients. objective: examine the distribution of rbc antigens among thalassemic patients and blood donors. methods: 108 blood samples of homozygous betta-thalassemia patients (who stayed at the daytime inpatient wards of scientific research institute of hematology and transfusiology baku, azerbaijan) and 1690 blood donors of azeri nationality were examined. 108 patients' blood samples were typed on rbc rh (c, c, d, e, e) and kell (k, k) antigens. gel test bio-rad (france) was used to detect rbc antigens. results: phenotyping of the patients' rbc rh antigens (d, c, c, e, e, cw) revealed that frequency of occurrence of the antigens in general was higher than in the donors. studying of rh phenotype distribution showed that the patients' ccdee (44.1%) •• ccdee (30.4%) phenotypes were twice as much higher than the occurrence of those in the donors (20% and 16.5% respectively). phenotype ccdee occurs more frequently in the donors (32%), whereas it is significantly rare in the patients (8.8%). the similar picture was observed in relation to ccdee and ccdee phenotypes, which occurred more frequently among donors (11.8% and 10.6%) than in thalassemic patients (6.86% and 2.94%). additionally, the results demonstrated that k antigen of the kell system was not detected in the thalassemic patients whereas k antigen was detected in all the patients. the same picture was observed with two other antigens of this system. thus among the patients typed on rbc antigens kpa antigen was detected in just one case whereas kpb was detected in the rest the patients. the results of the analysis showed that for thalassemic patients with phenotypes ccdee and ccdee it is easier to find compatible blood than for patients with phenotypes ccdee, ccdee and ccdee. furthermore, it is known, that some congenital diseases are genetically connected with the specific group systems, for example, the mcleod syndrome is genetically associated with the sharp suppression of the kell alloantigen expression (absence of gene kx). the fact of the discovered absence of antigen k in homozygous bthalassemic patients makes it possible to assume, that this group of patients suffer from scarcity along the kell system, like the scarcity in mcleod syndrome. all the mentioned above make it possible for us to conclude, that prior to blood transfusion to thalassemic patients phenotyping of rbc rh and kell antigens must be carried out. hyperhaemolysis in sickle cell disease due to complement activation. tessa thorp rci national blood service manchester uk tm thorp national blood service, manchester, uk introduction: sickle haemoglobin is caused by a genetic mutation in codon 6 of the beta globin gene, resulting in the conversion of glutamic acid to valine. when critical amounts of polymer accumulate within the sickled erythrocyte cellular injury results. clinically sickle cell disease is characterised by chronic haemolysis and intermittent vaso-occlusion. mold et al. demonstrated that deoxygenation and sickling of erythrocytes is related to membrane phospholipid changes and these changes result in the activation of the alternative complement pathway. aim: the objective of this study was to measure the amount of c3c and c3d bound in vivo to red cells of homozygous and heterozygous sickle cell patients. these levels were then compared to 'normal' sickle negative blood donors. haemoglobin levels and red cell morphology were also examined for signs of active haemolysis. the hypothesis was that an increase in red cell bound complement in vivo could provide an indication of hyperhaemolysis syndrome is sickle patients. method: flow cytometric analysis using rabbit anti-c3c or anti-c3d and fitc labelled goat anti-rabbit was developed using a beckman epics xlmcl flow cytometer. control samples were prepared using a fruitstone buffer technique of complement coating. results: a total of 16 heterozygous sickle patients and 11 homozygous patients were analysed. of the 11 homozygous patients analysed only one was undergoing a sickle crisis. a positive result was indicated if the mean trait or homozygous sample was coated with complement to a greater degree that the mean normal donor plus two standard deviations. the results obtained in this research project indicate an increase in c3c levels bound to red cells of homozygous sickle patients in vivo. statistical analysis of results obtained suggest that there was no increase in c3d levels in either sickle trait or homozygous sickle patients. conclusion: these findings support research carried out by mold et al. (1995) and are present in more than 90% of caucasian population. it has been reported that anti-chido and anti-rogers don't cause hemolitic reaction but may be responsible for life-threatening anaphylactic reaction during transfusion of plasma proteins. case report: positive iat and dat were detected in a 34 years old swedish woman, a rh negative, at 34th week of pregnancy. previous iat and dat determinations were negative. at time of detection dat wsa weakly positive and igg subclasses identified. antibody identification revealed the presence of high titre (1 : 64) anti-chido and anti-rogers antibodies. a slight decrease in platelet count (from 210.000/ul to 80.000/ul) was observed. this pattern showed no variation until the end of the pregnancy. at 39th week a healthy baby was delivered, dat negative. the mother dat and iat remained positive for four months after delivery. the platelets count raised again to 200.000/ul. the same laboratory findings were detected in a previuos pregnancy in sweden. the patient reported the presence of antibodies at 35th week that disappeared few months after delivery. a healthy baby, dat negative was delivered in this case too. conclusions: this is the first report of the presence of chido and rogers as autoantibodies during the last months of pregnancy. the association with decrease in platelets count and the lack of evident clinical symptoms need further investigation. p-289 rbc alloantibody frequency and their prevalence within chinese, malay and indian community in singapore e widjaja, mbc koh and d teo health science authority, singapore, singapore background and aim: there is a recognised existence of different alloantibodies in different ethnic groups. while this has been studied in the caucasian population, their frequencies remain less well documented in the asian population. the frequency of different alloantibodies and their distribution in terms of age, sex in singapore population is studied, as were their prevalence within the chinese, malay and indian races in singapore. design and method: we conducted a retrospective study for the frequency of 28 alloantibodies on 1856 blood samples over 2004. these blood samples were largely sent by hospitals where preliminary antibody screening had been done and positive result obtained. they were sent to the centre for transfusion medicine for antibody identification. small number of these samples came from hospitals where preliminary antibody testing was not done. the antibody distribution across different ethnic groups (chinese, malays, indians) age and sex were studied. results: the most frequent alloantibodies in the population is mia (40.7%), e (20%), le a (17.5%), le b (14.8%), p1 (6.1%), m (5.4%), d (4.5%), c (2.7%), jka (1.9%) and c (1.4%). within the chinese community, the most frequent alloantibodies were similar: mia (51.9%), e (24.1%), le a (11%), le b (8.8%) and p1 (6.1%). in the malays, the most frequent alloantibodies were le a (35%), le b (26.4%), mia (24.2%), e(13.4%) and p1 (8.2%); while in the indians, these were mia (28.9%), le b (28.9%), le a (27.8%), d (17.5%), and e (12.4%), with the anti-d reflecting the higher incidence of rh d negativity in the indians race. for the lower incidence antibodies, anti-c was more common in the malays and indians (4%) compared to chinese (1.9%). anti jka tended to occur mainly in the malay race and anti-c was rare in all (<2%) reflecting the high prevalence of c in the singapore population (r1r1 phenotype). the ratio of alloimmunised male to female (m : f) is 1 : 3. most alloantibodies demonstrated significant skewing towards to the female, although relatively less so for mia where m : f ratio is almost equal at 1 : 1.9. alloimmunisation increased with age for mia, e, k, p1, jka and fyb while the frequency of alloimmunisation to lea, leb, d, m and c decresed with age. the prevalence of patients with multiple alloantibodies (2 or more) within the alloimmunised subjects is 21.7%. conclusion: anti mia is very common within the asian population especially in the chinese. anti d is common in the indians. most antibodies show increased frequency with age except for anti lea + b, d and m. the majority of alloimmunised patients are females. study aim: to identify the present antibodies in newborns, after a positive direct antiglobulin test (dat). material and method: during a 7 years period, from 1/1/1998-12/31/2004, we studied 2652 newborns and their mothers. each newborn was examined for abo group, rhesus with phenotype, kell and dat. each mother was also tested for abo group, rhesus with phenotype, kell and indirect antiglobulin test (iat). after each positive dat, the study was continued with the elution test, in order to identify the present antibody. dat test was performed with dc screening i-diamed sa, switzerland. for the laboratorial analysis of newborns the sample used was cord blood and in certain cases venous blood. results: the dat test was positive in 132 (5%) of newborns and the antibodies found were igg type immunoglobulin. anti-a antibody was detected in 89 (67.4%) (newborns of group a with mothers of group o), anti-b antibody in 25 (19%) (newborns of group b with mothers of group o), anti-d antibody in 2 (1.5%) (newborns d+ from d-mothers), anti-e in one case and anti-jka in another one. the eluate test was found negative in 3 newborns and in the rest 11, no special antibody could be identified. the results are presented in the following table. conclusion: the majority of antibodies in newborns with a positive dat test, is due to abo incompatibility (the mother belongs to group o and the newborn to group a or b). anti-d (2), anti-e (6), anti-k (7), anti-fya (5), anti-s (2), anti-jkb (1), anti-n (1), anti-kpb (1). in two patients 2 antibodies were identified, while in 24/97 (24.7%) no antibody was identified (unspecific). it is remarkable that only in 10 out of 30 patients with both dat and iat positive, an irregular antibody was identified, while the rest 20 patients had unspecific antibodies. in 19 patients with only iat positive, 15 had an irregular antibody and 4 had unspecific antibodies. in 3 out of 31 patients with both dat and iat negative the cause of incompatibility was the positive dat in the corresponding sample of the blood donor, while in the rest 28 patients the reasons were technical problems that include the inappropriate blood sample of the patient, patients under medication and errors during the crossmatching procedure. the results show that the incidence of red cell incompatibility in our hospital is 2.96% and the most common antibodies are anti-k, anti-e, anti-fya, while anti-d is important for d negative patients. 24.7% of the detected antibodies were unspecific and this is still a problem that possibly was due to the lack of additional panels of reagent red cells or antibodies to low-incidence antigens. finally, in some cases the reasons for incompatibility are due to factors affecting the blood donor or to technical problems in the crossmatching procedure. multiple isoforms excluding normal rhd mrna detected in rh blood group del phenotype with rhd 1227a allele introduction: del phenotype is very common in rh-negative chinese. the rates in hans (more than 91% in china) were reported from 26% to 30%. moreover all del individuals in this population were found mainly carrying a same allele, rhd 1227a, through genomic dna analysis. those individuals always possess one or two of this allele with ccee or ccee phenotypes. aim and the study: we focused on the mrna investigations of del individuals carrying rhd 1227a alleles, in chinese, to expect it could be explained that why a silent mutation is associated with del phenotype. the full-length rhd mrna was analyzed in 2 rh-positive donors with cde/cde and cde/cde genotypes, respectively, and 4 del phenotype individuals carrying rhd 1227a allele with cde/cde, cde/cde, cde/cde and cde/cde genotypes, respectively, through reversed-trancriptase pcrs and cdna direct or cloning sequencing. results: five transcripts and 6 isoforms were detected in rh-positive and del, respectively. among them, 4 isoforms have identical sequences, which are transcripts with exon 9, exons 8 and 9, exons 7 and 9, and exons 7 to 9 spliced out. the normal rhd mrna was only observed in rh-positive, but not in del individuals. in stead, two additional transcripts were found in del individuals. its exon 9 or exons 8-9 were spliced out, but both possess a 170 bp segment of sequence from intron 7 of rhd. through 2 additional reversedtrancriptase pcrs, which amplified exon 8 to 3¢-region and exon 9 to 3¢-region, the results showed that exon 9 did not exist in del anyway. conclusion: (i) a normal rhd protein does not exist in a del individual with rhd 1227a allele since the exon 9 was always spliced out in all isoforms. all 6 transcripts in del maintain a normal open reading frame and encode 6 proteins with different numbers of amino acid residues and different c-terminals (genbank ay751491, ay751492, ay751493, ay751494, ay751495, ay751496). among them, the sequence of del9 (isoform with exon 9 spliced) transcript was the most similar as normal rhd mrna. this isoform was first described by chang et al. in taiwan in 1998 . it encodes 463 amino acid residues and has 46 amino acids more than normal rhd. it is different from rhd after codon 384. in normal rhd protein, the amino acids after 384 (33 residues) are mainly the trans-membrane and intracellular regions. therefore a further study on if a del red cell possesses all epitopes of normal d antigen may be significative. (ii) a normal rh-positive individual has also the transcript of del9 that was found in del. (iii) there is only one polymorphism in the region of 170 bp segment between rhd intron 7 and 2 of the del transcripts, which indicated that other polymorphisms may exist in intron 7 of rhd 1227a allele compared rhd to explain that this situation was not happened in normal rh-positive individuals. total wbc were enumerated by flow cytometry and cell counting. wbc subsets were analyzed by flow cytometry with three-color fluorescence. in this study, the third generation bags and filters are used. results: before filtration, the total number of wbc, was significantly higher in fresh units compared with stored units, whereas in postfiltration samples the number of white cells was significantly lower in the fresh compared with the stored units. although absolute numbers were significantly reduced, filtration also induced significant changes in the proportions of subsets in hoth fresh and storod units, the percentage of t cells was decreased, whereas the percentage of b cells and monocytes was increased after filtration. in conclusion, both pre and post storage wbc filtration affect the proportions of wbc in the final product but pre storage wbc filtration of platelet concentrates is superior than post storage wbc filtration. the effect of pre-and post-storage filtration on platelet rich plasma: derived platelet concentrations background and objective: the white blood cells (wbc) within transfusion products are a major stimulus for a number of detrirmental biological reactions, including febrile nonhemolytic transfusion reactions, alloimmunization against hla antigens and cytomegalovirus transmission. in this work, our objective was to study the effect of storage time on the filtration of platelet concentrates (pcs). the total number of white blood cells as well as the distribution of wbc subsets, in units filtered before and after storage were compared. materials and methods: platelet rich plasma -derived pcs were filtered either fresh (5 pooled we reported earlier that metabolic arrest followed by incubation at 4°c reduces the platelet storage lesion (badlou et al. transfusion 2005) . here we report that this treatment also reduces binding and phagocytosis by macrophages. metabolic suppressed platelets (msp) were prepared by incubation in glucose-free, antimycin a containing medium (40 min, 37°c) followed by storage (48 h, 4°c) and recovery with glucose (1 h, 37°c). controls were (i) platelets in glucose-rich medium stored for 48 h at 22°c and recovery with glucose (c22) and (ii) platelets stored for 48 h at 0°c (c0) with rewarming. platelets were labelled with mepacrine and incubated with pma-matured thp-1 cells (37°c). binding was measured by facs analysis of cd42b/cd14 positive particles, and phagocytosis by counting mepacrine/cd14 positive particles. binding of msp, c22, c0 was 30 ± 4, 39 ± 10, 50 ± 12% of total platelets. phagocytosis of msp, c22 and c0 was 32 ± 9, 40 ± 8, 50 ± 9% of total macrophages (means ± sem, n = 4). before recovery of msp, binding/phagocytosis was 30% higher than thereafter, revealing energy-dependent control of the mechanisms that trigger plateletmacrophage interaction. these data show that metabolic suppression prior to cold storage attenuates binding and phagocytosis by phagocytes and may help to develop means to improve platelet survival post-transfusion. platelet compatibility testing and alloimunization in multiply transfused hematologic patients purpose: multiply transfused patients with heamotological malignancy often become refractory to platelets due to alloimmunization. refractoriness is usually defined as an insufficient platelet increment after consecutive platelet transfusions. two major causes of a decreased platelet increment can be distinguished, immune and nonimmune factors. alloimmunization occurs most frequently against the hla, and rarely against the hpa system. nonimmune factors been identified are splenomegaly, fever, sepsis, and disseminated intravascular coagulation as well as the quality of the transfused platelet concentrates. we performed this study in order to investigate platelet crossmatching, compatibility, and antibody determination among thrombocytopenic patients multiply transfused. we performed 164 crossmatchingcompatibility tests of single donor leucodepleted, abo compatible, platelet concentrates been transfused in 23 patients with leukemia and lymphoma, 18 males and 5 females with mean age 50 ± 25 years old. we also obtained samples from the patients for platelet antibody detection. we evaluated the cci (corrected count increment) 18 h after the transfusion. the solid phase rbc adherence assay (modified capture p system/immucor) was used for platelet compatibility and antibody detection. a total of 164 compatibility tests were performed, of which 84 were compatible. twenty five compatible platelet concentrates out of 84 were clinically evaluated. twenty from 25 compatible crossmatches (80%) were resulted in successful transfusion while only 5 from 25 (20%) in unsuccessful. the 80 incompatible platelets been transfused was resulted in unsuccessful transfusion. we found statistically significant difference among patients successfully transfused with compatible and incompatible platelets (p~0.01). additionally 14 patients out of 23 (60.8%) had been alloimmunized against multiple hla antibodies. three patients transfused with compatible platelets, during the study, developed alloantibodies. we found a large number of incompatible platelet concentrates that result in unsuccessful transfusion and clinical response. the platelet compatibility testing as well as alloantibody determination of multiply transfused patients is necessary for the identification and selection of compatible, with the patients, donors in order to result in succesful transfusion and clinical outcome. furthermore compatible platelet concentrates provide optimal support for refractory patients and it is known that they are acceptable as an alternative component. naitp is a rare clinical syndrome characterized by marked thrombocytopenia shortly after birth. it is caused by maternal immunization against paternally inherited antigens present on foetal platelets. screening and identification of antibodies in the maternal blood sample is the main support in the diagnosis and management of naitp. we have evaluated the frequency of maternal alloimmunization, the role of the antibodies involved (hpa and/or hla systems) and the pertinent risk of naitp in neonates using a fully automated system with a solid phase red cell adherence methodology (sprc-capture p) and paternal or random donors (indirect test). screening started in june 2003 and is still in course: in january 2005, 2300 blood samples were examined. identification of antibodies in maternal serum was carried out using elisa methodologies: maipa and commercial kits (pak-plus and quick screen-gti). of the 2300 blood samples analysed, 43 were reactive and the specificity of the antibodies were: anti hpa1a: 8, anti hpa1b: 2, anti hpa1a + hla: 2, anti hpa5b. 6, anti hla: 24, auto hpa-5b: 1. specificity of hpa antibodies was confirmed by determination of parents' hpa genotype (hpa-1,2,3,4,5,6) using pcr-ssp or pcr-rflp. the infants with hpa 1 immunization suffered from severe (plt count 5-103/ml) and symptomatic naitp (bleeding and petechiae were present), therefore they were treated with platelet transfusion and administration of high doses of intravenous immunoglobulin. we confirm that naitp due to hpa-5 and hla immunization is clinically less severe: all neonates had mild and self limiting thrombocytopenia at birth; no therapy was administered. it would be advisable to carry out pre-natal screening, at reasonable cost, using maternal serum versus paternal platelets and to proceed to the identification of antibodies only in presence of positive results. background: fetal or neonatal alloimmune thrombocytopenia (fmait) results from a maternal alloimmunization against fetal platelet antigens. it is the commonest cause of severe thrombocytopenia in the neonatal period. the diagnosis of fmait is made initially on clinical grounds, depending on exclusion of other causes of neonatal thrombocytopenia. in caucasians, hpa-1a is the most frequently implicated antigen. other antigens such as hpa-3a, or hpa-4a are less often implicated. during the past few years fmait has been reported associated with rare or private antigens. the diagnosis is straightforward when a maternal alloantibody with a corresponding parental antigen incompatibility is present. however it could be equivocal in the absence of such an antibody or difficult when a private antigen is implicated. if the father is heterozygous for the considered antigen, the infant's platelet typing should be performed to confirm the diagnosis. due to the risk of hemorrhage, particularly intracranial hemorrhage (ich), during the course of severe thrombocytopenia, specific therapy is mandatory. because subsequent siblings may be more severely affected, accurate diagnosis will allow better management of subsequent pregnancies. study design and methods: since the first documented case of feto-maternal alloimmune thrombocytopenia (fmait) due to anti hpa-9bw (maxa+), no additional cases have been reported. we present here a retrospective analysis of the cases referred to our laboratories in recent years. since 1999 we have screened for rare or private antigens in suspected cases of fmait when there is no incompatibility for the most frequently implicated antigens. the diagnosis was performed by genotyping and identification of the maternal alloantibody by the maipa technique. results: parental genotyping showed hpa-9bw (maxa+) mismatch as the sole antigenic incompatibility in 7 out of 8 families. in the last one, incompatibility was found for hpa-3 without anti hpa-3b maternal alloantibody. as the father was found to be hpa-9bw (maxa+) heterozygous in all the cases, the infant or fetus was genotyped to ascertain the diagnosis. the maternal alloantibody was identified in the maipa technique. however, our data strongly suggest that recognition of the hpa-9bw (maxa+) epitope is not uniform. the neonatal thrombocytopenia was severe in most cases with bleeding. the outcome was good in all the cases but one. conclusion: this analysis confirms that anti hpa-9bw (maxa+) fmait is not uncommon and was found to be around 2% of our confirmed fmait cases with parental incompatibilities and presence of maternal alloantibodies. it is a clinically severe syndrome which requires prompt diagnosis, albeit difficult, and maternal platelet transfusion therapy. laboratory investigation of a suspected fmait case should be carried out in a specialist laboratory wellexperienced in optimal testing. therapy requires strict collaboration between clinicians and blood bank services. appropriate management and antenatal therapy should be considered for successive pregnancies to prevent fetal bleeding. introduction: the human platelet (plt) antigen (hpa) system is independent of the hla system. therefore, host-or donor derived alloimmune thrombocytopenia can develop after allogeneic haematopoietic stem cell transplantation (hsct) even in hlamatched donor-recipient pairs. we report the first case on a stem cell recipient developing thrombocytopenia due to host-derived hpa-1a antibodies after non-myeloablative allogeneic hsct. a 52year-old male patient was diagnosed with multiple myeloma in 6/2003. treatment consisted of 3 cycles vincristin, adriamycin and dexamethason followed by tandem autologous stem cell transplantation. because of progressive disease he received 4 cycles of bortezomib, and after complete remission a stem cell allograft (7.99 ¥ 106/kgbw cd34 + cells) from his histocompatible (hla a,b,c,dr identical) brother after reduced intensity conditioning regimen with fludarabine (3 ¥ 30 mg/m 2 ) and alkeran (100 mg/m 2 ). he had received only twice packed red blood cell concentrates and one plt concentrate before allogeneic hsct. stable bilinear engraftment occurred around d12 but was accompanied with a continuous decrease of plt counts. between d10 and d19 the patient received seven plt transfusions, containing a median of 2.96 ¥ 1011 plt/unit (range 2,7-3,21 ¥ 1011 plt/unit) from random donors. the corrected plt count increments at 12 to 24 h after these transfusions were <2500/ml. therefore, and because of even a further decline of platelet counts to 1000/ml on d19 we investigated the presence of plt antibodies. methods: the patient's serum was tested by antigen capture elisa assays (pakplus® and pak1®, gti) and a solid phase assay (capture-p®, immucor). the maipa assay was used to confirm the results obtained by the above mentioned assays. in addition, we tested the patient's serum by the maspat kit (clb) against plt from the donor and against homozygous hpa-1a plt obtained from our donor pool. stored recipient's dna from the time before hsct was used for genotyping. genotyping for hpa-1, -2, -3 and -5 of the donor and the recipient was performed by pcr-ssp (hpa, protrans). the patient's serum obtained on d19 after hsct reacted strongly with the donor's plt due to anti-hpa-1a antibodies and antibodies against hla class i antigens. the patient's genotype before transplantation was hpa-1bb, -2aa, -3ab, and -5aa; the donor was hpa-1ab, -2aa, -3aa, and -5aa. thus, the antibodies were host derived and directed against the donor's plt. serum samples obtained on d38, d45 and d65 after hsct contained antibodies against hla class i antigens but hpa-1a antibodies were not anymore detectable. no hla antibodies were detectable on d149 after hsct. the severe thrombocytopenia was caused by hostderived hpa-1a antibodies. fortunately, plt counts started to increase on d20 spontaneously and the patient could be discharged at d27 (plt 51.000/ml) with a complete donor chimerism. the decrease of the serum antibodies parallel to the increase of the plt count strongly suggests a progressive elimination of residual host cells. we conclude that the hpa mismatch between recipient and host affected thrombopoietic engraftment and the success of plt transfusions. severe neonatal alloimmune thrombocytopenia with anti-hpa-5b antibodies: case report p moncharmont, m vignal, y mérieux and d rigal efs rhone alpes site de lyon, lyon cedex 07, france usually, in case of feto-maternal incompatibility, the platelet (plt) specific anti-hpa-5b antibodies (ab) induce only sometimes a mild neonatal alloimmune thrombocytopenia (nait). contrary to this observation here is reported the case of a severe nait. a 34-year old mother, gestation 2/partum 2, gave birth to a male neonate by caesarean section at 34 weeks of gestation because of intra-uterine growth retardation (iugr) and anamniotic fetus. five years before, she had had a first pregnancy with iugr of the fetus but no nait. the second neonate weighted 1.350 g, was 41.0 cm tall and had a head circumference of 27.5 cm. the apgar score was 9,10,10,10 at 1, 3, 5 and 10 min respectively after birth. no bleeding, hepatomegaly, splenomegaly or infectious signs were noted. five hours after birth, a respiratory distress syndrome appeared and an oxygenotherapy was performed during 44 h. the plt count which was 82.0, 79.0 and 73.0 giga/l at day (d) 0, d1 and d2 respectively dropped dramatically at 9.0 giga/l at d5. simultaneously, an intracranial hemorrhage grade ii was diagnosed on ultrasound scan. because of the clinical signs and of the decreasing plt count the mother's serum was tested for plt-specific ab by immunocapture and the ab identified by the monoclonal ab-specific immobilization of plt antigen (maipa) assay. a plt genotyping was performed in the neonate and his parents by sequence-specific primers polymerase chain reaction. the mother was hpa-5a/a and anti-hpa-5b ab were detected in her serum. the baby was heterozygous, hpa-5a/b. plt were transfused to the baby and the plt count rose to 80.0, 115.0 and 315.0 giga/l at d6, 10 and 18 respectively. no further transfusion was needed and the development of the baby was satisfactory with a normal electroencephalogram. in conclusion, when a mild thrombocytopenia with iugr and hypoxia but without bleeding signs is present in a neonate immediately after birth, a maternal plt specific ab screening must be performed in case the thrombocytopenia became severe during the newborn monitoring. anti-hpa-5b ab can be detected. partial results of the incidence of heparin induced thrombocytopenia type ii osc oliveira*, ra rached*, c cavalheiro-filho*, jc nicolau*, shgl pasqualucci*, daf chamone † and sp bydlowski † *heart institute, university of são paulo, † university of são paulo, são paulo, brazil heparin induced thrombocytopenia type ii (hit) is a severe side effect of heparin, associated with heparin-platelet factor 4 antibodies. hit type ii occurs in up to 5% of patients who are exposed to unfractionated heparin (ufh). in our institution patients that are under heparin treatment are mostly cardiac patients. the purpose of this study is to determine the incidence of hit type ii in these patients. material and methods: 111 patients from the intensive care unit and cardiac care unit treated with ufh or low molecular weight heparin (lmwh) for 5 or more days were studied. known causes of thrombocytopenia were excluded. platelet count was monitored pre and post heparin therapy. all selected patients were tested for detection of anti heparin/pf4 antibody test (diamed id-card). results: from the studied patients, 63 (56.8%) developed thrombocytopenia (determined by a decrease in the platelet count below 30%, after the introduction of heparin therapy); 48 (43.2%) did not show decrease in the platelet count. six (5.4%) out of 63 thrombocytopenic patients were positive for anti-heparin/pf4 antibody. three (2.7%) out of 48 non thrombocytopenic patients were positive for anti-heparin/pf4 antibody. the results demonstrate that 9 (8.1%) patients were positive for anti-heparin/pf4 antibody and they were no different from those described in the literature regarding the frequency of heparin induced thrombocytopenia. moreover, a higher frequency of patients with heparin/pf4 antibody was noted without the presence of thrombocytopenia, indicating that other factors should be considered. introduction: neonatal alloimmune thrombocytopenia (natp) due to maternal immunization against fetal platelet antigens affects 0.8-1 in 1000 live births. although it is usually a self-limiting condition, a major complication in cases of severe thrombocytopenia is the occurrence of intracranial haemorrhage leading to death (in up to 10% of reported cases). the commonest antibodies are anti-hpa-1a. treatment consists of ivig, compatible donor platelet concentrates or washed maternal platelets. the administration of random donor platelet transfusions is controversial but has been used successfully in some urgent cases when compatible platelets were not available. case report: a baby born in week 40 of gestation to a healthy mother after first uneventful pregnancy; birth weight 3000 g, apgar score 8. immediately after birth, severe thrombocytopenia (7 ¥ 109/l) and signs of haemorrhagic diathesis (generalized petechiae and ich gr. ii-iii) were observed. coagulation tests were abnormal and k-vitamin, fresh frozen plasma and random donor platelet concentrate (retrospectively genotyped as hpa-1a/a) were given. twenty-four hours later platelet count rose to 26 ¥ 109/l and no new petechiae were observed. on third day of life the blood platelet count was 13 ¥ 109/l and the newborn received ivig 2 g/kg and corticosteroides. twenty-four hours later the platelet count rose to 107 ¥ 109/l and further clinical course was uneventful. natp due to hpa-1a was serologically confirmed. conclusion: optimal therapy for an infant with severe thrombocytopenia during the first 24 h of life is the transfusion of platelets that will not be destroyed by the maternal alloantibody in the infant circulation. random donor platelet concentrates are controversial in a setting where optimal treatment is not available, however, in this case they led to a significant platelet count improvement in spite of hpa-1a incompatibility. accordingly, random donor platelets may be considered appropriate in emergency situations. background: rhd is the most immunogenic blood group antigen, and its correct identification is essential in the blood bank and in the prevention of the haemolytic disease of the newborn. the weak d phenotype is the most common d variant, with a frequency of 0.2% to 1% in caucasian individuals. there are several weak d types, with different frequencies in european countries, which may pose serologic problems and have the potential for alloimmunization. the objective of the study was to determine the frequency of the principal weak d types in portugal. study design and methods: lisbon regional centre of the portuguese blood institute and oporto são joão university hospital selected samples from blood donors and patients. rhd was tested by two (oporto) or three (lisbon) distinct anti-sera, in direct agglutination tests, at room temperature. when discrepant results were observed, the samples were tested with panels of monoclonal anti-d by liss-iat. samples that reacted weakly with igm anti-d but positive with igg anti-d were sent to the molecular biology centre. pcr with sequence-specific primers was performed using two commercially available kits (inno-train and bagene). real-time pcr, carried out on a light cycler, was applied when the interpretation was dubious. results: 101 samples were referred after being characterized as weak d. in 99 cases we obtained a positive result, with a preponderance of weak d type 2 (63.6%) over type 1 (16.2%), 3 (14.1%) and 4 (6.1%). two samples were not categorized. the high incidence of weak d type 2 in our population is in marked contrast to studies performed in other european populations where weak d type 1 was the most frequent. this might be due to our sample selection criteria or ethnic variation in the causes of weak d. there are advantages in genotyping serologically depressed d samples: to avoid the waste of d-negative rbc units and the use of immunoglobulin in pregnant women, who have no risk of alloimmunization. analysis of rhd zygosity in different rh phenotypes cal except for a 4-bp t insertion. the deletion of the rhd gene, found in most rhd-negative caucasians, was theoretically due to recombination of the upstream and downstream rhesus boxes resulting in the formation of a hybrid rhesus box. thus, the detection of a hybrid rhesus box in an rhd-positive individual denotes an rhd heterozygous status. aim of the study: to determine the rhd zygosity in different rh phenotypes. methods: blood samples from 106 white trios (father, mother and child) were studied. the rh phenotype was performed by hemmaglutination and the rhd zygosity was inferred in each member of the family groups. the rhd deleted allele was determined by a pcr strategy using a forward primer complementary to 5¢ end of the identity region of the upstream and hybrid rhesus boxes and a reverse primer complementary to the 3¢ end of identity region of the downstream and hybrid rhesus boxes. these primers selectively amplify a 1980-bp segment of the hybrid rhesus box in rhdnegative and rhd-positive heterozygous samples. serological and pcr inconsistencies were studied by a pcr-rflp method to detect another polymorphic site of the hybrid rhesus box. frequencies were obtained analysing only unrelated individuals (fathers and mothers, n = 212). results: 179 (84.4%) rhd-positive and 33 (15.6%) rhd-negative samples were phenotyped. of the rhd-positive donors, 75 (41.9%) were rhd homozygous and 104 (58.1%) were rhd heterozygous according to pcr. pcr-rflp analysis confirmed the results of pcr in serological and molecular discrepancies. these results were coherent within each family group and did not differ from those published in the literature for caucasians based on the most probable genotype method. however, the homozygosity indexes were significantly higher in the dccee (20.0% vs 6.6%) and dccee (16.6% vs 3.3%) phenotypes due to an increase of the dce haplotype. in all samples with the dce haplotype the rhces allele, frequent in individuals of african descent, was investigated by pcr-ssp. this allele was found in 33.0% of the dce haplotypes. . rhd and rhce typing was performed by multiplex-pcr with 24 fluorescent primer pairs. positive results were obtained for rhd-exons 2-7, 9, 10 and polymorphisms associated with antigens c, c, cw, e and e. sequencing of rhce-sequences, including exons 1 to 10 and intron/exon borders, were done by direct taq cycle-sequencing using bigdye-terminators v.1.1 in an abi 310 (applied biosystems). results: see table 1 . the substitutions of rhce-specific nucleotides in exons 3, 5 and 6 with their rhd-specific counterparts lead to 3 different new rhc-antigens with weakened expression. since one amino acid change in allele 3 lie in extracellular loop of the antigen suggested that this antigen may be involved in allo-immunization. inheritance of a rhd cat vi type ii in a twin pregnancy: a case report introduction: determination of hdn-relevant fetal blood groups in amniocentic fluid with pcr is a routinely used method. misinterpretation of test results -e.g. overlooking rhd category -decreases depending on the number of examinated rhd-specific exons. case report: a 28-year old mother (w.o.g. 23) pregnant with twins was regularly tested for irregular antibodies and was shown to have an anti-d. after amniocentesis of both foetuses tests for the occurrence of rhd intron 4, exons 7 and 10 were performed in the hospital's lab. the sample of fetus i showed pcr-products for intron 4, exon 7 and exon 10 while sample of fetus ii lacked rhd-specific intron 4. therefore we investigated blood samples from the parents as well as amniocentic fluid of both foetuses. methods: total dna was amplified in a multiplex pcr with fluorogenic primers for rhd exons 2-7, 9 & 10; polymorphisms for dweak type 1-5, d-vii, d-hmi and rhce polymorphisms for c, c, cw, e, e. capillary electrophoresis for size fractionation and fluorigeneic analysis was done in an abi 310. rhd zygosity was determined by quantitative real-time pcr with co-amplification of rhdand rhce-specific exon 3 and subsequent calculation of d ct value (ct rhce minus ct rhd). results: while maternal dna sample has been genotyped as rhdnegative, amplification of paternal dna for rhd-specific exons 2, 3, 7, 9 and 10 were possible and failed only in exon 4-6. determination of rhd-zygosity revealed a homozygous constellation of the rhd-gen. investigation of amniocentic fluid from both foetuses resulted in a rhd-wildtyp for fetus i and a rhd cat. vi type ii for fetus ii which was the reason for missing amplification in rhd intron 4 pcr. results: weak d type 3 was not identified in our research population. weak d type 1 was identified in 2 cn, weak d type 2 was identified in 1 cn and weak d type 4 was found in 1 cn, 3 bsa, 4 be, 1 bc and 1 saa. conclusions: although weak d types 1 to 3 represent the majority of all weak d phenotypes in caucasian populations, none of these weak d alleles were found in black populations. since none of the frequent weak d types were identified in non-caucasians one might not expected to find type 4 in all populations. however, regarding rhd phylogeny, the weak d type 4 mutations (602c > g and 667t > g) form the basis of a cluster of aberrant alleles that are predominantly observed in blacks. therefore, it is not surprising that weak d type 4 was identified in non-caucasians. based on these results it may be concluded that weak d phenotypes have evolved relatively recently since they are present in caucasian and asian methods: dna was extracted from 9 a, 14 b, 11 ab subgroups, and 21 provisional cis-ab after serological abo typing. allelespecific pcr, rflp, direct sequencing of exon 6 and 7, and allele separation were performed on these samples. results: abo*a205 allele was observed in an aint subgroup. two new a alleles that showed 784g > a base change and 990c > t of intron 6 and a polymorphism of 532c > t in a(pro) intron 5 were discovered. o04 and o20 alleles were also observed. in b subgroups, a silent substitution 255c > t (leu85leu) was observed as a new b allele. another new b allele which showed 547g > a was also found in 4 b subgroups. conclusion: we discovered new abo alleles and polymorphisms in korean populations. many other polymorphisms and alleles already reported in japanese were also observed in koreans. evaluation of a multiplex snapshot-pcr method for red blood cell marker genotyping c jungbauer*, c hobel*, em dauber † , pk rabitsch*, dwm schwartz* and wr mayr* *austrian red cross, † medical university vienna, vienna, austria once conventional reagents for identification of rare red blood cell phenotypes are scarce, methods using current nucleic acid testing techniques to identify the patient's genotype and possibly to screen for donors would be desirable. the approach of multiplexgenotyping using pcr-ssp has apparently technical constraints so we are currently analyzing whether a modification using snapshot pcr-technique could provide a routine application for such purposes. compared to ssp-technique, the snapshot pcr only requires one single primer (labeling reaction) for the detection of two (or more) alleles instead of one pair of primers for every single allele. briefly, we perform snapshot pcr as follows: in a first step, dna segments covering the essential polymorphic regions for the abo, rhd, kel, jk and fy genes are amplified using a standard pcr step. after purification treatment of the pcr products (roche high pure pcr product purification kit or shrimp alkaline phosphatase (sap)/exo1 treatment according to the abi prism snapshot multiplex kit protocol) the labeling reaction is performed using the abi prism snapshot multiplex kit. after a second purification step the products are analyzed on a abi prism 310 genetic analyser in fragment analysis mode. our preliminary results show the feasibility of this approach as reliable typing results can be obtained for all tested single nucleotide polymorphisms corresponding to alleles. generally a better signal quality from controls and samples is obtained compared to the ssp-technique with consecutive gel electrophoresis. we also consider the possibility of the automated interpretation of the results as an important improvement, especially when aiming for an application for a large number of samples (donors) or markers (patients). in contrast, the method involves more manual steps and higher costs. we may conclude that the implementation of snapshot-pcr techniques for red cell marker genotyping is a promising alternative to pcr-ssp. the obvious quality improvements compared to pcr-ssp might be critical for a routine application in blood banks (donor screening) or complex questions in clinical laboratories. quantification and quality control of monoclonal antibody using an optical sensor based on the laser reflectometry technique the monoclonal antibodies (moab) are biological reagents of homogeneous activity. they are generally used in the recognition and quantification of very small amounts of biological substances (hormones, enzymes) present in a solution, ag identification, blood group determination, oncology, organs transplant, etc. moab can be characterized by its specificity and affinity. affinity may be expressed as the equilibrium association constant (k). several techniques are available to determine the equilibrium constant of the ag-ab interaction. in this work, is shown an optical sensor for monoclonal antibody quantification by reflectometry technique. the laser reflectometry technique (null ellipsometry technique) can give information about the kinetic of the interactions, stoichiometry of molecular binding and the concentration of molecules in a solution, and also offers detailed and accurate determinations of real-time adsorption kinetics of protein without labeling. a silicon wafer was chosen as reflectant surface. once fixed the principal angle of incidence, an amount of anti-ab is added to the sample. the reflected laser intensity is registered in real time as the protein is being adsorbed onto the wafer. the mathematical analysis of the results verifies that the antibodies adsorption follows langmuir's kinetics. from the curve analysis, the parameters related to the anti-ab concentration are extracted. from them, the calibration curve is constructed. this curve allows the desired commercial monoclonal antibody quantification. the developed technique shows to be sensible and precise. the obtained graphics are very well approximated (r > 0.94) verifying that the monoclonal anti-ab associa study aim: to prevent the sensitization to rh0(d), to a d-patient who was transfused with d+ blood. material and method: on september 2003, (9/28/2003), we admitted to our hospital through air-carriage, a female of 22 years old, badly injured after a car-accident. the patient was on an olighemic shock (ht: 16%) due to retroperitoneal, paracolic and procystic hematomas, had multiple fractures on the left feet, the main important of which was an acetabulum one. she had a blood group: o, d-and her phenotype was ccdee with du-. after her arrival she was urgently admitted to the surgery room and our blood bank was asked for condensed red cells. initially she was transfused with blood of the same group (o, d-) but when we were short out of dblood, we were asked for 7 more units. the necessity of blood was imperative, the patient was on a critical condition and the mechanism of blood transport, from another blood bank would take some time to be put in motion, and so it was decided that the patient would be provided with d+ blood. the indirect antiglobulin test (iat) and papaine were negative, so there would be no problem with the transfusion with d+ blood. the patient received finally 3 units (850 ml) of d+ condensed red cells, out of 7 that were initially asked. before the 48h from the surgery had passed, it was decided that human anti-d immunoglobulin (rhesogamma p) should be provided, in order to prevent the sensitization of the patient to rh0(d). the indicted dose was 500-1250 iu/10 ml of the transfused blood, provided piecemeal during a several days period. analyzed by real-time pcr amplification. based on a published report, we selected 12 primer pairs targeting insertion/deletion polymorphisms which are located on different chromosomes, unrelated to each other and not associated with immunocompatibility. we optimized the amplification conditions for all 12 primer pairs using our sybr green real-time quantitative pcr protocols, and investigated analytical sensitivity for each primer pair by performing spiking studies, in which a single copy of positive dna was added into 100 000 copies of negative dna followed by allele-specific pcr amplification. we also created a theoretical panel of donor-recipient pairs (n = 73) to evaluate the clinical sensitivity for detection of ta-mc using both hla-dr and indel panels. results: for the short-term samples, 10 additional mc cases was identified in non-lr group using indel panel; and one additional mc case was detected in lr group (table 1) . for the long-term follow-up samples, 4 additional mc cases were found. when evaluating analytical sensitivity, we were able to detect a single copy of positive dna mixed with 100 000 copies of negative dna in a single amplification tube for all 12 primer pairs. we were also able to calculated the clinical sensitivity that using 73 donor-recipient pairs. 99.5% of donor-recipient had at least one informative allele for detection of ta-mc if we consider both hla-dr and indel panels. conclusion: using our new indel panel, we were able to detect more instances of mc in this cohort of patients. we conclude that the dr assay underestimates the presence of mc. moreover, the tandem use of both panels provides a powerful tool for the detection of mc with 99.5% of recipients having at least one informative allele. background: we reported severe immunesuppression and longterm transfusion-associated microchimerism (ta-mc) in transfused trauma patients. we have also reported, in a murine transfusion model, that sensitivity to 1-chloro-2-4 dinitrobenzene could be transferred, albeit transiently, by transfusion of fresh blood from a sensitized donor to a naïve immunecompetent recipient. in order to mimic the immunecompromised status of trauma patients and further investigate the mechanism underlying ta-mc, we established an animal model using immunedeficient knock-out mice. aim: our objective was to test virus-specific immune functionality of the chimeric donor leukocytes in a murine ta-mc model. material and methods: female rag-2/common gamma double knock-out mice were transfused with fresh blood collected from male balb/c mice, which were either not infected (non-primed, np) or infected twice (primed, p) with 100 million viral particles of murine cmv (mcmv). at different post-transfusion time-points (1 h, 2 weeks, 4 weeks, 6 weeks, 8 weeks), different female recipients plus non-transfused female knock-out controls were challenged with 100 million viral particles of mcmv intra-peritoneally, and then monitored weekly for the concentrations of male donor cells as well as mcmv viral load in recipient's circulation. each female knock-out received only one challenge of mcmv. if the subject died, we quantitated mcmv viral load in the brain, spleen, lung and liver. we used real-time quantitative pcr targeting murine y-chromosome, h2k and mcmv to quantitate male donor cells, transfused recipient cell dna input, and mcmv vrial load, respectively. the number of recipient cell dna input served as a denominator to calculate the concentration of male donor cells and the mcmv viral load. results: results of overall mortality are summarized in the table. all female knock-out recipients transfused with primed donor blood, except for the post-transfusion 6 weeks, are able to survive mcmv infection. all non-transfused control and recipients transfused with non-primed donor blood died after mcmv infection; these two groups also had higher mcmv viral load in blood than the recipients transfused with primed donor blood. when the subject died, we were able to detect mcmv in all four organs we analyzed, with liver having the highest mcmv viral load. there was no significant difference for the concentration of donor cells in recipients' blood between recipients transfused with non-primed donor blood and recipients with primed donor blood. the preliminary data of our study showed that chimeric primed donor cells, but not non-primed donor cells, are able to protect immune compromised knock-out recipients from murine cmv infection. the time-point of 'post-transfusion 6 weeks' might represent a weak window for the functionality of chimeric donor cells, which requires further investigation and confirmation. aims: to compare the effectiveness and the cost of epoetin-a and darbepoetin in patients undergoing pabd. methods: seven adult patients scheduled for operations were administered aranesp (300 mg sc once or q3 weeks if needed) for pabd (aranesp group) and they were compared with a historical epoetin-a group of seven age-matched adults (eprex 300 iu/kg biweekly). the two groups were matched according to the ht, ferritin levels, number of the predonated units and type of the operation performed. cbc count and reticulocytes were measured weekly during the donation period, the day before, day 1 and day 6 after surgery while ferritin and biochemical indices were measured during the first visit. erythropoiesis-stimulating factor was administered when ht £ 36% during blood donations and blood donation was not performed if ht < 34%. results: there was no statistical significant difference in hematological parameters during the donation period, the pre-operation day and after surgery between the two groups. five of seven patients from both groups received one or two autologous blood units. both factors were well tolerated without any side effects. the cost per patient was 609.09€ in the aranesp group and 414.2€ in the epoetin group. conclusion: despite the small number of patients and the limitations of this preliminary retrospective trial we believe that subcutaneous darbepoetin-a is equally effective with epoetin-a in patients undergoing pabd. darbepoetin has the advantage of less frequent administration and it is possibly superior that epoetin-a in terms of patient compliance. however smaller doses should be examined in order to reduce the cost. larger prospective randomized trials are needed to estimate the cost-effectiveness of the use of darbepoetin-a in pabd. background: incompatibility with many blood units is a major problem in transfusion therapy. in selective operations, preoperative autologous blood donation could solve many problems, when of course the patient's condition and his haemoglobin levels are appropriate. we present here the experience of our blood transfusion centre from 4 operations in 3 patients with anti-erythroid antibodies. materials: three patients (1 male and 2 females), aged between 67-80 years old, had to undergo selective operations, 3 total hip replacement surgery and 1 aortic aneurysm. introduction: because of improvements in surgical techniques, preoperative autologous blood donation (pabd) in patients undergoing radical retropubic prostatectomy (rp) is contested. aim of the study: we wanted to develop and validate an algorithm to determine the patients who probably do not benefit from pabd. methods: we calculated the perioperative hb-loss of 153 consecutive patients (group 1) who donated two red cell units (rbc) of autologous blood 5 and 4 weeks before undergoing rp: hb-loss (g) = preop-hb ¥ bv + (n rbc ¥ 65) -(postop-hb ¥ bv) (bv = blood volume (l) = body weight (kg) ¥ 0.08; postop-hb = hb (12-18 h after rp); n rbc = number of transfused autologous and allogeneic rbc). hb of rbc was taken as 65 g. rbc requirement is probably if initial-hb -(hb-loss: bv) -trigger-hb (taken as 80 g/l) < 0 (initial-hb = hb at the first contact with the pabd-unit). this assumption was validated by the next 125 patients who were also assigned for pabd (group 2). pabd was refused if the probability of rbc requirement (prr) was <25%. between 25-50% one rbc was taken after considering the patient's individual risk of pabd. if prr exceeded 50% two donations were planned. results: both groups did not significantly differ in age or initial hb. preop-and postop-hb were significantly lower in group 1 (141 vs 151 and 98 vs 111 g/l). 79% of autologous blood of group 1 were discarded, 3/153 patients needed additional allogeneic rbc. hb-loss caused by rp was 269 ± 111 g. mean prr in group 2 was 8.5%. 5/125 patients donated one rbc, which was later discarded, and no patients donated two rbc. 7/125 of group 2 needed allogeneic rbc. mean prr of these patients was 12% (range 0.65-33.3). conclusion: postop-hb were lower in rp-patients with pabd because of the lower preop-hb and the restrictive indication for transfusion of autologous blood. the individual calculation of prr, for which only body-weight and initial-hb of the patient are necessary, shows that pabd in patients undergoing rp is indicated only in rare cases. the algorithm also may be used in other major operations, if hb-loss is known. use of darbepoetin-alpha in preoperative autologous blood donation: preliminary results background: preoperative autologous blood donation (pabd) is an alternative practice to eliminate complications of allogeneic blood transfusion although its cost-effectiveness has been questioned. darbepoetin-a (aranesp, genesis pharma sa) is a novel erythropoiesis-stimulating factor that it has been shown to be equivalent to epoetin-a (eprex, janssen-cilag) in patients with chronic renal failure and cancer. darbepoetin-a has a longer serum half-life and higher relative potency than epoetin-a. this property leads to less frequent administration and may reduce drug cost. so far, no clinical trials with darbepoetin have been published in patients with surgical anemia. other 44 (15.3%) patients who required autologous and homologous blood, had average predonation hb level of 13 509 (sd ± 9.01) g/l. we found a significant relationship between the need for postoperative transfusion and the predonation hb level (p = 0.003), predonation htc values (p = 0.006), weight (p = 0.003) and gender (p = 0.002): female patients and patients with lower predonation hb and htc, as well as patients with lower body weight more often needed additional homologous blood transfusion. no relationship was found between age of patients and the need for transfusion (p = 0.121). 104 (80.6%) patients with ptka were transfused with autologous blood only, and had average predonation hb level of 13 867 (sd ± 9.06) g/l. other 25 (19.4%) patients transfused with autologous and homologous blood had average predonation hb level of 13 588 (sd ± 8.93) g/l. the significant relationship was found between the need for postoperative transfusion and weight (p = 0.012): patients with lower body weight more often needed additional homologous blood transfusion. no relationships were found between predonation hb level (p = 0.099) predonation htc values (p = 0.243), gender (p = 0.241) and age (p = 0.276) of patients and the need for postoperative transfusion. conclusions: our results show that over 80% of patients needed only autologous blood. in our patients with ptha predonation hb was significant predictive factor for additional transfusion therapy, while in ptka it was not observed. in both groups of patients body weight was significant predictive factor, thus this feature seems important for planning of transfusion therapy in patients with ptha and ptka. aim: prevention results of loosen anastomoses on colon, with fibrin sealent (fs) application and influence on colagen production. materials and methods: investigations were done on rats, weight 350-500 g. in control group, after partial resection of left half of colons termino-terminal anastomosis was derivated. fs was applied in examined group. concentration of colagen was done indirectly, with quantitative l-hydroxyproline determination. place of anastomosis, 1 cm proximal and 1 cm distal of anastomosis, was analyzed iii, v, vii and xiii day postoperatively. results: analysis of hydroxyproline on the place of anastomosis showed higher hydroxyproline value in group with fs application. the highest approximate value of hydroxyproline was registered v day postoperatively. distal, 1 cm of anastomosis, the quantity of hydroxyproline is higher iii day postoperatively in control group but v postoperative day value is intensively growing in group with fs application. electronic microscopical was done v postoperative day in control group at the place of anastomosis detected a defect with detritus and absence of larger colagen fibres. in group with fs application on the place on anastomosis, in the shape of bundle, colagen fibres were grouped and completely fills the place of anastomosis. conclusion: fs application accomplish higher concentration of colagen in all segments of isolated colon, that enables better healing of anastomosis. the study of the use of the safest blood (autologous blood transfusion) through preoperative blood donation (pbd) in 100 surgery patients introduction: the use of the autologous blood is already under consideration in developed countries. thus, it is probable that autologous blood donation would be effective in one way or the other in reducing blood transfusion complications. in this study, pbd as the easiest method to use and the most cost-effective one was selected. aim of the study: it aims at improving blood safety and raising blood inventory. methods: in this study, 100 patients, including 75 males and 25 females, intended to undergo elective surgery were selected as subjects to donate their autologous blood. the subjects with hematocrite level of about 30-33 percent as ordered by their physician donated their blood by this method. blood collection procedure was followed at 3-7 day intervals. the blood volume taken from patients in every collection differed from 100-450 ml according to their weight. results: this study showed that in all patients undergoing plastic, gynaecological, jaw, and ent surgeries autologous blood transfusion was used with no need for allogenic transfusion. in other surgeries, including orthopaedics, the need for allogenic transfusion was estimated to be at about 50 percent of cases. to avoid the complications of allogenic blood transfusion, the safest way is the use of autologous blood which involves low cost and is easy to perform. the introduction: the purpose of this study is to describe a technique to perform labelling of autologous platelet-gel with 111in-oxine and to evaluate its usefulness, after in vivo graft implant, as a marker of bone osteoinduction by means of scintigraphy, in patients with jaws bone defects following the enucleation of cystic lesions and cystic lesion derived from extraction of deeply impacted lower third molar. methods: agp made. consent was obtained by patient to conduct hiv and hbv testing. briefly, 40 cc to 60 cc of blood is withdrawn from the patient. the blood was separated, by means of successive step of centrifugation, in to platelet-rich plasma (prp) , platelet-poor plasma (ppp), and red blood cells (rbc). the red blood cells were discarded. the prp [comprises of approximately 10% of the total blood volume withdrawn] had platelet counts of 500 000-3 000 000/mm 3 . the procedures of agp labelling were performed in laminar flow chamber. to 3 seconds the solution will assume a gel-like consistency forming platelet gel. imaging: the scintigraphy was performed 2 h after application of labelled agp (early scan) and at 24, 48, 72, 96 h (delayed scan) by means of a gamma camera equipped with medium energy collimator. a later scan was performed at 10 days after graft. the platelet uptake index (pui) was then calculated by dividing the cpm/pixel in the graft roi (recognized in and planar and trans-axial slice) for cpm/pixel in a mirror background roi. in vitro sampling: the radioactivity of the plasma samples collected at 2, 24 h and at lapse time = 24 h for 7 days, were used for the plasma clearance determinations and for in vitro studies of the platelet loss from the gel. results: all patients presented early high concentration of 111in oxine agp, at site of the graft, that was easy recognized at scintigraphy performed as in anteroposterior and lateral planar projection of the jaw as in spet slices reconstructions. all labelled agp was well confined within area of original implant and no activity was seen in the surrounding tissues or in the distant organ. conclusion: all patients studied well tolerate the implant of agp; no adverse reactions were observed and follow up -performed 3 months later -showed bone remodelling activity in the site of the graft. serial blood donations in a ko pregnant woman with the use of recombinant erythropoietin for intrauterine transfusions of severe hemolytic disease of the newborn due to anti-ku biweekly) were administered to the mother to ensure an adequate supply of compatible rbcs for intrauterine transfusions and possible perinatal haemorrhage as well. results: intrauterine transfusions were repeated every 1-3 weeks. by the 35th week of gestation the patient had donated four units of blood, her hematocrit was 40%, anti-ku titre was 1/2048 and four intrauterine transfusions had been performed. cesarian section was decided and the apgar of the newborn were 8 and 10 at 1 and 5 min. the newborn was treated with phototherapy but without exchange transfusions and two weeks later he was discharged. by the 15th day of life rh-epo was administrated to him due to anemia. the maternal red cells completely disappeared from the child's blood by the day 100. the experience of the use of erythropoietin in pregnancy is minimal. as illustrated by this case treatment with rh-epo and iv fe has effectively increased mother's capacity to donate rbcs' for autologous use and intrauterine transfusions as well, with no adverse effects to the mother or the child. however, further research is necessary to evaluate if rh-epo crosses the placenta. introduction: blood components should be transported by a system which has been validated. the containers used for transport should be well insulated, some form of temperature indicator should be used to monitor the in-transit temperature. whole blood should be stored at different temperatures above 2°c. aim of the study: bags with whole blood collected in a collection site are transported in containers without active cooling. we tested temperature of blood in containers put into the extreme weather conditions (+50°c, -20°c) during loading test for transport. methods: the container without active cooling was filled with the exact number of bags with blood and the exact number of passive cooling elements (frozen water cubes in plastic) placed in the exact positions without close contact with the blood bags. the bags with blood of the temperature +4°c, +20°c and +37°c were used. temperature indicators were situated in the bottom, centre and top of the container. filled container was placed into thermostat (+50°c) or freezer (-20°c). the temperature was observed in 10 min intervals for three hours, first measurement was 30 min after putting into freezer or termostat. results: (see table 1 ) nt, non tested; tmp, temperature. in the table there are shown minimum and maximum temperature parametres observed during tested time including increasing or decreasing trend. conclusion: loading test for transport of the bags with collected whole blood helps us to optimize transporting system, especially number of cooling elements in relation to the season and its place in the container. in the light of presented data we corrected transporting system to maintain the recommended temperature during transport. background: since 1999, we have produced pooled and filtered platelet concentrates out of four buffy coats in tsol platelet additive solution and have stored them in pall autostop clx bags made out of pvc/totm. the residual plasma content is between 30-40%, the mean volume about 260 ml and the mean platelet-content is 3.1 ¥ 10 11 per unit. for pathogen inactivation or bacterial screening it is necessary to extent the storage time from 5 to 7 days. new foliage like polyolefin is supposed to maintain a good quality environment for prolonged storage of platelets. aims: storage bags made out of polyolefin (pall autostop elx) were tested to prove their suitability for prolonged storage of platelets. methods: 18 twins made out of pools from 8 buffy coats were produced with the standard method, one twin was stored in the conventional bag (cb) the other in the new foliage bag (nfb). the platelet pools were stored on flatbed shakers at 22°c, and sampled at day 1, day 5 and day 7. ph, glucose, lactate, hypotonic shock response (hsr) and p-selectin expression were measured by standard in-house methods. results: mean ph on day 7 with cb was 7.21, with nfb 7.35; glucose with cb 4.4 mmol/l, with nfb 6.0 mmol/l; lactate with cb 17.4 mmol/l, and with nfb 14.8 mmol/l, hsr with cb 79%, with nfb 85%; p-selectin with cb 29% and with nfb 18%. the new platelet storage bag showed better results of in vitro quality markers, especially after day 5 of storage. prolonging storage time will make it easier to introduce bacterial screening or pathogen inactivation techniques into platelet transfusion. the possibility to filter rbc either at 4°c or rt simplifies the preparation process. filtration at +4°c enables to achieve a better leukoreduction performance. the nbs has successfully implemented this project which has the potential for improvement in patient safety and is predicated upon practical application and risk reduction rather than elimination. the impact of this work on the incidence of trali will require detailed, long term analysis of hemovigilance data using existing mechanisms. active communication, a team approach, perceived value of the initiative and the hard work of all staff involved were key success factors. quality assessment of buffy coat-derived platelets prepared from leucoreduced whole blood background: whole blood can be separated into; plasma, buffy coat and red-cell conc (rcc) by differential centrifugation and separation on a separation device. because of the high hematocrit of the rcc, 60% of the process time is needed for expression of the rcc. by increasing the internal diameter of the tubing at the bottom of a t&b system by 0.7 mm. a decrease of the process time is expected. methods: 220 units of whole blood were collected with the new t 3988 'wide boring' blood pack and separated on a routine base. quality control parameters were checked and the whole process time was monitored. free hemoglobine was measured up to 42 days. results: process time of a 'wide boring' bag is significant shorter compared to a standard blood bag. average decrease: 86 s. slightly increase in free hemoglobine is measured probably due to the increased express rate of the red cells. bloodproducts produced with the new t 3988 meet european guidelines. no significant increase of free hemoglobine due to the faster expression is measured. an significant decrease in process time is measured with the wide boring bloodpack. the new fresenius hemocare rcc in-line system: t 3988 can be used for routine production which will speed up the production process considerably. introduction: leukoreduction of blood components is required to prevent several transfusion-associated complications. aim: the aim of this study was the full process validation of the pall leukotrap wb system for the preparation of leukoreduced blood components. we collected 60 whole blood units from donors suitable for donation using a quadruple blood-bag, which includes an wbf3 in-line filter (pall) for the removal of leukocytes and platelets. mixer balances (baxter) were used and donation occurred within 12 min in all cases. after donation whole blood units were stored at room temperature for 2 h. subsequently, whole blood filtration was performed by gravity at a standard height of 150 cm using a blood leukoreduction cart (baby leuko cart, itl-corporation). filtered units were centrifuged at 5000 g ¥ 10 min by an heraeus cryofuge 6000i. an automatic extractor (bag press plus-bioelettronica) was used to prepare red cell concentrates in sag-m solution and fresh plasma units. air in the system was automatically expelled by the extractor. complete cell counts and hemoglobin concentration were evaluated in pre-filtration samples and at the end of the blood components preparation using an automated cell counter (pentra dx120-abx). we enumerated residual leukocytes in red cell units by flow cytometry (becton dickinson-leucocount kit). results: pre-filtration data of whole blood and end-of-process data of red cell and plasma filtered units, are summarized in table 1 . results are given as mean and standard deviation. whole blood filtration was completed within 10 min in all cases. red cell units were transfused after a mean of 6 days to 57 patients affected from transfusion dependent (45%), post-surgery (30%), and post chemotherapy anemias (25%). no cases of transfusion reaction were observed. the pall leukotrap wb system was easily introduced in our setting. all blood components prepared by the system fulfilled the council of europe requirements with regards to hemoglobin content in red cell units and post-filtration residual leukocytes. future studies are needed to evaluate its cost-effectiveness in the setting of routine blood component preparation. background: during an evaluation of the compodock (fresenius hemocare) sterile connection device (scd), we observed irregularities on the inside of the tubing at the site of the weld. it was our aim to investigate the effect of these observations on the quality of blood products. methods: three leukoreduced red cell concentrates (rccs) were pooled and divided over 3 bag systems: one without weld in the connecting tubing, one with a compodock-weld, and one with a weld made with the terumo scd. the rcc was transferred 10 times over this tubing to have maximum result if the weld had deleterious effects. the rccs were stored in pvc containers, and sampled on day 1, 28, 35 and 42, and free hemoglobin (hb) was measured. the same procedure was also performed using platelet concentrates (pcs), but these were stored in polyolefin containers, sampled on day 1, 6 and 8, and cd62 expression was measured. ten experiments were performed per blood component. according to who standards processing of blood into labile components are considered an expression of quality of transfusion service. in our practice, modern transfusion principles are successfully applied. they cover blood collection, serological processing of blood units, technological preparation of blood products (gmp, sop) and rational utilization of blood components and blood derivatives. in the past four years (2001, 2004.) aberrations from these principles have taken place (self-sufficiency). nbti collected x = 62 926 (249 778) blood units into blood bags. in serbia x = 230 537 (691 611) blood units. retrospective analysis: ldpc-bc was administered x = 97% with the satisfactory haemostatic effect. increase of the cyta and plasmapheresis-manual procedures was also noted (100%). increase of the use of leukocyte poor red blood cells was also registered (95% introduction: according to the relevant recommendations of the council of europe, whole blood is a source material used for the preparation of blood components and blood products. basic concept of the therapeutic use of blood components is the compensation of the lacking or deficient blood component. in that way, a possibility of the infusion of unrequired or deleterious components of whole blood is eliminated. objective of this presentation is to analyze the reasons of non-utilization of certain blood units, the actual quantity and ratio. aim of the study: the purpose of our in vitro study is to compare storage of platelet concentrates at 4°c with platelets stored at 22°c, and to determine the in vitro-effects of pre-incubation at 37°c for 1h prior to analysis on the basis of the maintenance of platelet metabolic and cellular integrity. methods: platelets concentrates (pcs) were prepared from pooled buffy coats (bc) for paired studies to be stored into different conditions. (i) at 20-24 degree on a flat bed agitator; (ii) at 20-24 degree on a flat bed agitator and pre-incubated for 1 h prior to analysis; (iii) at 4°c; and (iv) at 4°c and pre-incubated for 1 h prior to analysis. this paired in vitro study (n = 8) over 21 days include volume, platelet counts, mpv, volume, ph, po 2 , pco 2 , bicarbonate, glucose, lactate, swirling, leucocytecount, hsr, esc, atp, ldh and release of a-granule content (rantes, ß-thromboglobulin and pf4). results: platelet count (day 5 and 21; p < 0.05), mpv (day 5; p < 0.01), ph (day 14 and 21; p < 0.01), pco 2 (day 14 and 21; p < 0.01), bicarbonate (day 21; p < 0.01), glucose (day 5, 7, 10, 14 and 21; p < 0.01), atp (day 14 and 21; p < 0.01) was significantly higher in platelets stored at 4°c and platelets stored at 4°c with preincubation. ldh (day 21; p < 0.01), bicarbonate (day 5 and 7; p < 0.01), lactate (day 5, 7, 10, 14 and 21; p < 0.01), ph (day 5 and 7; p < 0.01), esc (day 5, 7, 10, and 14; p < 0.05), hsr (day 5, 7 and 14; p < 0.05) was significantly lower in platelets stored at 4°c and platelets stored at 4°c with pre-incubation. the concentration of rantes, ß-thromboglobulin and pf4 was significantly higher in platelets stored at 22°c than in platelets stored at 4°c (day 5, 7, 10, 14 and 21; p < 0.01). hsr (day 5 and 10; p < 0.05) and esc (day 5, 7, 10 and 14; p < 0.01) was significantly higher in preincubated platelets stored at 22°c compared with platelets stored at 22°c. conclusion: platelets stored at 4°c maintain metabolic and cellular characteristics to a great extent during 21 days of storage. we confirm the loss of platelet discoid shape and have shown that loss of discoid shape in platelets stored at 4°c is associated with decreased metabolic rate and decreased release of a-granule content. aim: as reference centre of the swiss blood transfusion service for new materials and blood products we evaluated that system for routine use and official registration in switzerland. method: 20 whole blood donations were collected in a whole blood filtration set with cpda-1 and stored at room temperature for 1 h before filtration at room temperature. the leucodepleted whole blood was stored for 42 days. following parameters were analysed on day 0, 35, 42: free haemoglobin in%, k. in addition leucocyte count was performed on day 0 and a blood culture on day 49 (see table) . blood cultures on day 49 remained negative and all counts of residual leucocytes were below 1 ¥ 10 (exponential) 6/unit. summary and conclusion: as expected there was a clear increase in k and free haemoglobin after day 35. however the results were within the required specifications from the european and swiss guidelines up to day 42. we conclude that autologous leucodepleted whole blood can be stored in cpda-1-for 42 days without loss of stability of the red cells. we will introduce the system to the offi-cial material list of the swiss blood transfusion service and then implement the procedure to our daily routine. results of ffp production from whole blood, and of ffp and pc produced by use of cell separator over a 5-month period before and after the introduction of measures for trali prevention are presented. the following measures were undertaken: (4) blood of female donors was not used for ffp production, and plasma was only used for fractionation (5) plasma of female donors was not used for kt-bc pools (6) platelets and plasma were produced on a cell separator only from the female donors without a history of pregnancy. female donors of whole blood: 16%*; 16%** ffp produced by plasmapheresis: 2%*; 2.5%** female donor units on cell separator: 9.4%*; 4.5%** ffp from total plasma units: 63%*; 58%** plasma units used for bc-pc pools: 4%*; 6%** *period before and **after the introduction of measures for trali prevention the exclusion of female donors had no major impact on the production of ffp and bc-pc pools from whole blood because of the very low rate of female subjects in the croatian blood donor population. the amount of plasma and pc collected from female donors by use of cell separator was significantly lower (~50%), however, without any major impact on total ffp store because of the small rate of plasma and platelets obtained by apheresis. background: platelet concentrates (pcs) are currently stored for a maximum of 5 days. extended storage to 7 days would increase the supply and reduce the waste of pcs. transfusion-associated graftversus-host-disease (ta-gvhd) is a severe transfusion reaction caused by t-lymphocytes in the transfusion product. the risk of developing ta-gvhd can be prevented by gamma irradiation of the pcs. various in vitro tests can be used to study the quality of pcs such as inspection of the swirling phenomenon, hypotonic shock response (hsr), detection of platelet surface markers (e.g. cd62p and cd42b), metabolic parameters and blood gases. free oscillation rheometry (for) using the instrument reorox® 4 can be used to monitor the coagulation over time in whole blood, pcs and plasma samples, and to obtain information about clotting time and coagulum elasticity. aim of the study: the purpose of this study was to evaluate the quality of pcs obtained by apheresis technique during storage for 7 days and to study the effect of gamma irradiation by using several in vitro methods including for. methods: platelets were collected from healthy donors (n = 12) using apheresis technique. the pc from each donor was divided in 2 units, one served as control and the other was gamma irradiated with 25 gy. the pcs were stored on a flatbed agitator at 22°c for 7 days. samples were taken on day 0 (= day of collection) for analysis of blood gases, metabolic parameters (glucose and lactate), platelet count and swirling. samples taken on day 1, 5 and 7 were also analysed for hrs, cd62p (p-selectin) and cd42b (gpib) expression utilising flow cytometry. evaluation of coagulation by for was performed on day 1, 5 and 7. the maximum elasticity (g'max) and the time to g' were evaluated from the for elasticity curves. results: there was no difference between irradiated and nonirradiated pcs regarding any of the tested parameters during the storage period. swirling, hsr, platelet count and percentage of cd42b expressing cells were well maintained for 7 days of storage. glucose decreased and lactate increased significantly during the storage period, from 2.5 mmol/l to 16.2 mmol/l for lactate and from 16.5 mmol/l to 8.9 mmol/l for glucose. the percent cd62p expressing cells increased significantly during storage from 15% on day 1 to 32% on day 7. po 2 was well maintained but ph increased and pco 2 decreased significantly between day 0 and 1 whereafter ph decreased and pco 2 continued to decrease. the for parameters g'max and time to g'max increased significantly between day 1 and 5 and the time to g'max continued to increase significantly between day 5 and 7. the results indicate a well preserved platelet quality after storage for 7 days. gamma irradiation did not affect the platelet quality. cytokine release during storage of buffy coat platelet concentrates produced manually and automatically background: transfusion reactions following platelet transfusion are still a problem even when leukoreduction is included in the production process. platelet derived cytokines released during storage upon activation or lysis, accumulate in the platelet products and have been suggested to be involved in transfusion reactions. rantes (regulated upon activation, normal t cell expressed and presumably secreted) is a chemokine playing an important role in the inflammatory immune response and causes degranulation of eosinophiles and release of histamines from basophiles, which again can cause allergic reactions. tgf-b1 (transforming growth factor b1) has been shown to be immunosuppressive, inhibits the proliferation of t-and b-lymphocytes and decreases the secretion of igg and igm from b-lymphocytes. aims: as part of our quality control program, we aimed to quantify the amounts of rantes and tgf-b1 released during storage in platelet concentrates produced from pooled buffy coats by our manual routine method (m-pcs) and by an automated method using the orbisac system (gambro) (a-pcs). methods: pcs were produced from 5 buffy coats. following overnight storage at 20-22°c, buffy coats were pooled with 300 ml t-sol (baxter). forty-two pcs were produced either manually (n = 21) using the imugard iii s-pl set (terumo) with integrated soft leuko-reduction filter or by the automated procedure (n = 21) using the orbisac validation bc set (gambro) equipped with the lrp6 leuko-reduction filter (pall). swirling was scored visually, platelet count and mpv were measured on a cell counter (cobas argos, roche), and blood gas analyses, glucose as well as lactate were measured on an abl700 series analyser (radiometer). samples for testing of cytokines were centrifuged for 15 min at 4800 g, 20°c; supernatants were harvested and frozen at -86°c until analysis. cytokines were quantified using quantikine human rantes immunoassay (r&d systems) and human tgf-b1 elisa immunosorbent assay (bender medsystems gmbh). all analyses were performed on days 1, 4 and 7. results: platelet concentrate volume (mean): m-pcs: 316 ml, a-pcs: 328 ml. platelet yield was found to be 3.15 ¥ 10 11 for m-pcs and 3.43 ¥ 10 11 for a-pcs (p < 0.0001). in all pcs ph levels were between 6.9-7.2. glucose consumption and lactate production from days 1-4 and days 4-7 did not differ significantly. rantes levels (pg pr 10 9 plts) were significantly higher in a-pcs than in m-pcs (p = 0.04, repeated measures analysis of variance), but no significant difference was found in tgf-b1 levels (pg pr 10 9 plts). summary and conclusions: preparation of buffy coat platelet concentrates by the automated orbisac system improves platelet yield compared to our manual processing procedure, but the levels of the chemokine rantes were significantly highest in the automatically produced products. the clinical importance of these findings is still unclear, but may be related to the shear stress the platelets are subjected to during the automated production process. the quality of cryopreserved vs liquid stored platelets: a comparative study table 1 . the mismatches can be divided into the two categories. the first of them is characterized by differences in allelic groups, i.e. at low-resolution level. 22 allelic group differences were detected in the group with one mismatch, most of them in hla-c locus (this locus was not concluded in primary donor search). in the other category there are differences in alleles within the same group, i.e. at high-resolution level only. 12 differences within the same group in all tested loci were detected in the group with one mismatch. the mismatches described above were heterogeneous and a correlation of specific mismatch with transplantation outcome was not possible in this group. conclusion: the use of high-resolution dna methods makes the identification of hla match/mismatch more accurate and can affect the outcome of unrelated hsct. this work was supported by the grant iga mz, no. nr/7984-3. pre-freezing and post-thawing quality controls in umbilical cord blood assigned for transplantation p bergamaschi, c perotti, g viarengo, c del fante, c parisi, a marchesi, l bellotti and l salvaneschi irccs policlinico 'san matteo', pavia, italy background and aims: nowadays umbilical cord blood (ucb) represents a well established source of haematopoietic stem cells for unrelated transplantation in children affected with haematological and inherited diseases. thanks to the large-scale banking of unrelated units and the preliminary encouraging results, ucb employ in adults is quickly growing up. in this context, total nucleated cells (tnc) count of the graft is considered the main predictor for clinical outcome; however, other indicators of the haematopoietic potential, such as cd34+ cell content and short-term culture clonogenic assay, are recommended in accordance to netcord-fact stan-dards. in order to guarantee the safety and the prompt availability of a ucb unit assigned to a matched recipient, a pattern of rigorous quality controls should be carried out not only at the time of cryopreservation but also before the release for transplant. we report the results of the quality controls performed on thawed cryovials referring to the 26 units delivered by our ucb bank compared to the pre-freezing values. methods: every ucb unit stored in our bank is accompanied by 3 satellite cryovials available for subsequent controls. for each unit issued for transplantation, one cryotube was thawed in 37°c water bath with gentle agitation without washing out dmso. tnc and mononucleated cells (mnc) were estimated by an automated cell counter; viability and cd34+ cell count were evaluated by flow cytometry with a no-wash, single-platform technique and 7aminoactinomycin d. cfu assay was performed using commercial reagents (methocult gf h4434, stemcell technologies) and colonies were counted after 14 days. the same tests were performed before cryopreservation, taking a sample from each fresh unit. moreover, before the delivery for transplant, a second cryotube was thawed to investigate the bacterial contamination by direct microbial culture, whereas the sterility test before freezing was performed by inoculum into 30 ml media (bact/alert®fa/fn, biomérieux inc). results: the ucb characteristics before freezing and after thawing are detailed in the tables 1, 2 and 3. post-thawing tnc and mnc, as well as cd34+ cells, showed no significant difference in comparison to the pre-freezing values. despite of the expected decrease of the overall viability after thawing, we observed a highly satisfactory viability referred to the cd34+ cells. the colony forming units (cfu) growth after thawing was documented and was always lower as respect to the pre-freezing assay. finally, the results of microbial cultures were negative for all the 26 units on both fresh and thawed specimens. conclusions: in our experience, well standardized evaluation of ucb content could be obtained with regard to tnc, mnc and cd34+ cell. concerning the results of short-term cultures, the presence of dmso as inhibiting factor may be advocated to explain the discrepancies between fresh and thawed samples. finally, rigorous quality controls documented that the procedures of manipulation and cryopreservation did not affect the quality of ucb to be infused for transplant and provided to the physician all the parameters necessary for a safe transplant in a close and appropriate time. bone marrow transplantation or bmt transplantation of progenitor blood cells to regenerate blood normal cells in patients with blood disorders. bone marrow has an organized and structured architecture in which close relationships exist between a regulatory microenvironment and primitive hematopoietic cells. in fact, normal hematopoietic cells depends on critical interactions that occur between stem cells and their microenvironment. this microenvironment is a complex meshwork composed of growth factors, stromal cells, and extracellular matrix. marrow injury can occur as a consequence of a variety of diseases. some diseases could be due to a microenvironment that fails to support hematopoiesis. a possibility is that aplasia and leukemia share a common etiology such as drug, chemical, radiation, virus or other environmental hazards. we can say that microenvironmental abnormalities in interactions between stromal cells and hematopoietic progenitors may be important in the pathogenesis and clinical expression of hematopoietic malignancies in humans. background: intrauterine growth retardation, with associated low birth weight, represents one of the most important cause of baby mortality and morbidity. understanding the genetic bases of this adverse event is still an open goal. there is evidence that motherchild hla compatibility and hla-drb1 foetal genotype are associated with a reduced placental growth and a low birth weight. the recent institution of cord blood banks, with their huge amount of hla types, offers an unique opportunity to look inside the molecular bases of normal birth weight. aims: we investigated whether the baby-linked immunogenetic profile, i.e. hla gene frequencies and homozygosity rate, affects the physiological variance of the size at birth. methods: 1868 cord blood units (961 from males and 907 from females) were hla typed with pcr-ssp and/or reverse pcr-sso techniques and recorded in the cord blood bank database of pavia-italy. all were defined at low resolution level for hla-a and b genes and at high resolution for hla-drb1. 686 blood units were also randomly typed for hla-dqa1 and dqb1 at high resolution. results: mean birth weight was 3430 g and mean relative birth weight (i.e. corrected for gestational age according to the gender) was 0.19 g. 70 babies were <5th centile (2782 g) and 70 were >95th centile (4068 g). comparing the hla allele distribution in these extreme bands we found that hla drb1*08 was significantly associated with high relative birth weight: 5.1% in 95th centile vs 0.7% in 5th centile, p = 0.032. on the contrary, hla-drb1*14 and dqb1*05 were associated with low relative birth weight: 7.9% and 30.4% respectively in 5th centile vs 2.2% and 9.6% in 95th centile p = 0.035 and p = 0.019. all infants were analysed as to the effect of the above mentioned alleles. we confirmed the positive association of hla-drb1*08 and higher relative birth weight (mean 0.17 vs 0.08; p = 0.033) as well as the association of hla-drb1*14 with lower relative birth weight (mean 0.03 vs 0.27, p = 0.04). no significant association was found as far as hla homozygosity was concerned. conclusions: the present findings confirm the role of foetal hla-drb1 gene in the intrauterine growth. about the specific involved alleles, one possible explanation comes from the studies of crystallography and amino acid sequencing of hla-dr binding groove. it has been demonstrated that hla-drb1*08 and hla-drb1*14 genes encode for different amino acid sequences in the pocket 4 of the molecule (aa 70, 71, 74). this implies distinct functional restriction patterns. the sequence motif of hla-dr14 is characteristic of some autoimmune conditions, such as hashimoto's thyroiditis, and preeclampsia which is associated with intrauterine growth retardawhich provides a high yield and excellent purity without lymphocyte and erythrocyte contamination. in a 3 month period, we studied blood samples from bone marrow transplant patients and from normal subjects. the extraction of leukocyte polymorphonuclear was obtained with a 6%-7% dextran solution in 0.8% saline. after incubation at room temperature with lymphopre solution, the mixture was centrifuged. two clear and separate rings of mononuclear and pmn leukocytes were obtained. to eliminate any red blood cells, pmnl ring was separated and washed three times with cold ammonium chloride. after a short period of incubation 4°c, mixture was centrifuged and the pmnls were isolated. the purity and viability of total leukocyte population was counted and the percentage of pmnl obtained was established. the total blood samples studied were divided in two groups, i.e., bone marrow transplant patients and normal subjects. in both cases the pmns isolated were of high purity and viability. the overall percentage of pmnls obtained from both groups under study was 98% to 99% when stained with gimsa or wright staining method. the viability of isolated pmnls was also 98% too, which is excellent for numerous immunological or molecular studies. the pmnls isolated by this method were highly pure and viable in comparison with standard methods used to isolate human pmnls. generation a high amount of pmnls is another advantage of the suggested method. this method to separate pmnls is recommended for in vitro studies of different subjects. et al. 1950 .) the object of exchange transfusion (et) is to remove bilirubin already present in the plasma or remove alloantibody which can cause hemolytic disease (in order anti-d, anti-c and anti-k are easily the most important) or remove anti-d positive red cells. we have studied exchange transfusion in our hospital in neonatal intensive care unit, during 1997 to 2004. at delivery cord samples were taken for determination blood group, rhesus, hb and ht have been counted. also direct antiglobulin test (dat) has been performed. in cases of positive dat, hb and bilirubin levels were monitored. newborn body-weight were weighted (ranged 710 g to 3100 g). the blood for et was of group o, d negative and kell negative and was compatible with the serum of mothers; it was less than 7 days old. the blood was screened for hbs and for anti-cmv as well as being submitted to all usual tests. the method has been determined by using the umbilical vein; the multi-way tap makes it possible to draw blood from the infant into the syringe, to discard the blood into a sterile empty vessel, then to draw blood from a donor unit into the syringe and inject this into the infant. results: 17 exchange transfusions were carried out. four out of 17 et due to abo incompatibility mother-newborn. five out of 17 due to rhesus incompatibility mother-newborn and eight out of 17 due to jaundice undetermined origin and immaturity. the last two years only three et were carried out due to immaturity. conclusions: phototherapy, when applied early enough and with sufficient intensity, can avoid the need for exchange transfusion in many infants. phototherapy alone or phototherapy plus high dose igg therapy has been used to minimize exchange transfusions in this population. detection abo blood group system antibodies of neonatal using fully automated column agglutination technology (auto-vue) background: the abo blood group system remains the most important in transfusion practice. this is because of the regular occurrence of the antibodies anti-a, anti-b and anti-a, b reactive at 37°c, in persons whose red cells lack the corresponding antigens. the regular presence of anti-a and anti-b is used in the routine determination of abo blood groups; in addition to testing red cells for a and b antigens, the group is checked, in serum or reverse grouping, by testing the serum against red cells of known abo groups. methods: 100 samples were taken from 100 newborns at first 24 h of life for abo blood group typing during 2001-2004. simultaneously the presence of anti-a and anti-b antibodies has been studied using fully automated column agglutination technology (auto vue ortho diagnostic systems) with bio-vue cassettes aborh/reverse. the column agglutination technology is based upon the ability of glass beads to form a physical barrier between agglutinated and unagglutinated red cells. to determine the abo serum group, test serum and abo reagent red cells were added to the top of column containing diluents. the abo grouping columns were centrifuged and examined for agglutination. the presence or absence of agglutination has been recorded. results: in 68 out of 100 newborns were found detected antibodies anti-a, anti-b. in 24 out of 100 newborns no antibodies were found. in 8 out of 100 were found antibodies maternal origin. the automated reader detected all positive reactions. positive results were recorded on a scale from +0.5 to +4. conclusions: the technical performance of device allows objectivity and precision to detect abo blood group antibodies of newborn. the origin and the type of antibodies and also factors that influence their presence are to be studied. introduction: diagnosis, management and prevention of red blood cell immunization have improved, so hemolytic disease of the newborn (hdn) has changed from a common to a rare pathology. aim of the study. in this study we have retrospectively evaluated the benefits of the immunohematological screening for the management of pregnancies with alloimmunization. methods: in the last 3 years, we have performed an immunohematological screening on all pregnant women assisted by our hospitals. ab0 and rh typing, antibody screening and, eventually, identification and titration were performed on maternal specimens by microcolumn technique. results: not considering ab0 incompatibilities, we have discovered 64 alloimmunized women with the following specificities: 10 anti-d, 9 anti-c, 7 anti-c, 9 anti-e, 3 anti-jka, 1 anti-d + anti-jka, 1 anti-d + anti-s, 3 anti-d + anti-c, 1 anti-d + anti-c + anti-k, 5 anti-s, 10 anti-k, 2 anti-m, 1 anti-c + anti-e and 2 anti-d + anti-c + anti-g. the most severe hdn were the 2 d + c + g, the c + e and 2 out of 7 c newborns, with mean hemoglobin between 5 and 6 g/l, bilirubin = 20.5 g/l, reticulocyte count = 10%. in these exchange transfusion needed at the delivery. other newborns were only treated with phototherapy. conclusion. thanks to the immunohematological monitoring, the diagnosis of alloimmunization, the correct management of pregnancy and the adequate neonatal therapy were possible. in fact all newborns survived and showed no neurological lesions in the following controls. conclusion: in order to provide a highly specialized perinatal care, immunohematologist, obstetric and pediatric should provide a good antenatal and perinatal screening. this is an interesting case of rhd immunisation in rhd negative woman despite the application of rhd immunoprophylaxis. case report: a blood sample of pregnant woman, 30 years of age, in 12th gestation week, was sent to our laboratory for serological analysis. her blood group was o, ccddee and she had an anti-d antibody reactive only with enzyme treated panel of test erythrocytes. her husband was a, ccdee, and two children were both a, ccddee. on the next visit, she gave the data of one arteficial and one missed abortion before the 12th gestation week covered with 50 mg of rhd immunoprophylaxis, but without the measurement of fetomaternal haemorrhage (fmh). both of abortions were after the deliveries. until the end of the pregnancy, detailed serological analysis showed anti-d specificity of antibody in her sera which remained reactive only with enzyme treated red blood cells. the fetus was under permanent ultrasound control. she delivered a mature, rhd positive, ccdee male child, without any sign of haemolytic disease. the proper personal history, measurement of the size of fmh, distinguishing the anti-d and anti-g specificity of the antibody, administration of rhd immunoprophylaxis and cooperation between transfusion medicine specialists, gyneacologists and neonatologists still remain major principles of prenatal and perinatal care concerning haemolytic deisease of the fetus and newborn caused by anti-d antibody. anti d antibody, reactive only with enzyme treated red blood cells is usually harmless for fetus and the newborn. introduction: red blood cell (rbc) transfusion is widely used in neonatal intensive care units for acute or chronic pathological conditions. clinical indications for rbc transfusion are shock, sepsis and/or anemia with the following laboratory criteria: a) hematocrit (hct) < 20% or hemoglobin (hb) < 7 g/dl and reticulocytes < 4%; b) hct < 30% or hb < 10 g/dl in these conditions: o2 required < 35%, recurrent apnoea and bradicardia, cardiac rate > 180 bpm and respiratory rate > 80 bpm for more 24 h; c) hct < 35% or hb < 12 g/dl with severe respiratory distress. aim of the study. aim of this study has been to evaluate the effectiveness of rbc transfusion therapy in premature and at term newborns independently of initial pathological conditions. methods: our therapeutic objective has been to achieve an hct of 50% after the whole cycle of transfusion therapy. for each little patient, the volume of transfused rbc unit has been calculated, according to international guide lines, using the following criteria: weight (kg) ¥ blood volume (100 ml per kg if premature or 80 ml per kg if at term newborn) ¥ (hct desired -hct observed)/hct of unit transfused. particularly we have considered that premature infants (with a gestation age of 26-36 weeks) show a range of weight from 600 to 1.800 g, while at term newborns from 1.800 to 4.000 g. in order to avoid a circulatory overload, the indicated hemocomponent has been always packed rbc with higher possible hematocrit. for the same reason, the rate of infusion has been always 3-10 ml/kg/hour. methods: this is a descriptive study. the name of the neonates who received transfusion was obtained from the blood bank of beheshti hospital. information concerning the type of blood product, frequency and indication of transfusion, sex, gestational age and weight of infants was recorded in questionnaire and analyzed. results: out of 541 neonates admitted during one year, 118 (68 male, 50 female) received blood components. fifty four percent received one, 35% two and 11% received three types of blood components. the frequency of transfusions were 311 times. the most common used blood products were fresh frozen plasma (49%), red blood cell (33%), whole blood (14%) and platelets (4%). all the blood products except whole blood were used more common in premature and low birth weight infants. appropriateness of transfusion of red cells, fresh frozen plasma, platelets and whole blood were 92%, 88%, 100% and 91% respectively. (hdn) . in accordance to current regulations, this study is carried out in all pregnant women attending in our service. according to our protocol, when an alloantibody of any specificity is detected through the liss-coombs gel technique, the same determination is made using papain-treated screen cells to detect any association with other antibodies which could be of clinical relevance for a hdn. there are scientific evidences that the use of enzymatic techniques increase the test's sensitivity, though clinical relevance of 'enzyme only antibodies' may be questioned. aims: demonstrate the importance of a routine identification of irregular antibodies in pregnant by two methods (liss-coombs -enzymatic) and the prevalence of anti-d associated antibodies, only detected by using enzymatic technique. analyze the need to carry out a follow up of sensitized patients to determine if the associated antibodies only detected with in an enzymatic medium can be detected with a liss-coombs medium during pregnancy, thus acquiring clinical significance for hdn. materials and methods: between january 2000 and december 2004 we studied 1970 d-negative pregnant women. the studies performed were: abo grouping, d and weak d, rh phenotype, direct antiglobulin test and irregular antibody detection (iad) against commercial screen cells with liss-coombs (diamed) ® gel-medium technique, according to the manufacturer's specifications. when iad were positive, an antibody identification using two commercial cell panels with gel techniques (liss-coombs-enzymatic) was performed. along the pregnancy, periodic controls were carried out to determine the exact moment when antibodies, previously only identified in an enzymatic medium, could be detected in a liss-coombs medium (clinically significant antibodies). results: out of 1970 d-negative studied samples, 278 (14.1%) had a positive dai and it was only showed anti-d specificity in a liss-coombs medium. after analyzing this specificity against enzymetreated erythrocytes, it was possible to determine that 79 patients (24.8%) had in their serum other anti-d associated alloantibodies: 5 anti-k (1.8%), 63 anti-c (22.7%), 10 anti-e (3.6%) and 1 anti-c + e (0.3%). during the immunohematologic follow up, it was determined that in 4/79 patients some of the antibodies which were previously only detected in an enzymatic medium, could be identified in a liss-coombs medium and later they were identified in the red cell elution of the newborn. conclusions: these results confirm the relevance of a screening for irregular antibodies of clinical importance by means of a conven-tional technique and one of increased sensitivity in all pregnant women. the detection of an association of antibodies provides information for the undertaking of diagnostic and therapeutic measures, both by the obstetrician, as for any eventual transfusional requirements for hdn. it was also concluded that, although antibodies detected in an enzymatic medium are considered of low clinical significance, its investigation and follow up is suggested in pregnant women to determine the moment in which they can be detected in an antiglobulinic medium, thus revealing their clinical significance. background: fibrin glue is one of the most complex human plasma derivatives both in terms of composition and clinical applications. this product mimics the last step of coagulation cascade through activation of fibrinogen by thrombin, leading to the formation of a fibrin clot in the presence of factor xiii. in contrast to synthetic adhesives, the significant advantage of this plasma-derived sealant is its biocompatibility and biodegradability as well as the fact that it does not induce inflammation, foreign body reaction or extensive fibrosis. readsorption of the fibrin clot is achieved during wound healing within days/weeks following application, depending upon the type of surgery, the amount and type of product used or the proteolytic activity of the treated site. the risk of virus transmission by commercial fibrin glue products is still debated and investigators are looking for alternative fibrinogen sources. many of these studies rely on autologous on single donor cryoprecipitate as source of fibrinogen. aims: the aim of this study was to compare single and double methods of cryoprecipitation of fibrin glue. the influence of different plasma preparation methods and plasma storage temperatures (-30°c and -80°c) on the quality of fibrinogen concentrate was examined. methods: whole blood was collected by standard phlebotomy technique and centrifuged at 4000 ¥ g for 5 min within 2 h of collection. plasma was removed. four units of plasma were pooled into a 2000 ml bag, mixed, divided into 4 parts (aprox. 219.7 ml) and immediately frozen. two of these units were stored at -30°c and 2 units of ffp at -80°c. after one month the plasma was thawed at 4°c during 16-18 h. the fibrinogen concentrates (21-25 ml) were received by single and double cryoprecypitation. to compare single and double methods of cryoprecipitation, the levels of fibrinogen, fibronectin, plasminogen and factor xiii were determined. results and conclusion: the levels of fibrynogen were significantly higher in fibrynogen concentration obtained by double cryoprecypitation from plasma stored at -30°c. there were no significant differences in the level of plasminogen in both tested groups. double cryoprecipitation of a single unit of plasma (stored -30°c) is an efficient, simple and safe method of obtaining fibrin glue. background: talking about professional risk we generally consider the risk of acquiring some kind of infective disease through accidental injury. in this article i would like to point out another side of the problem-the risk of making preventable medical error. with all its consequences. aim: how blood transfusion errors are among the most serious types of medical errors, the final goal is to initiate nationwide, regular, mandatory error reporting. information obtained, distributed and openly discussed at professional meetings will contribute to improving the patient safety. at the same time it would contribute to avoidance of blaming and shaming of many health care providers. prevent what preventable could be! method: retrospective analyze was conducted at the middle size blood transfusion center (10 000 donations per year and utilization of approximately 25 000 of components). results: after clearly distinguishing adverse events due to underlying patient condition from preventable medical error we fined out that: -great majority of adverse events resulted from medical error -every part of blood transfusion center, from blood donation ward, through laboratory testing to component issuing has it' weak points' or vulnerable places -any educational level is equally liable to error there is no significant difference about occurrence time: -working day/holiday -emergency/routine request -routine h/out of routine h -main error cause were as follows: -donor sample misidentification -rhd typing error -abo typing error -incorrectly performed cross match -recipient misidentification -wrong component prepared -sample confusion during freezing preparation conclusion: the truth incidence of transfusion medical errors is underestimated. mandatory report of fatal or 'only' harmful errors to the referent institution and its periodical announcement is the step ahead in preventing errors. those reports should be discussed at professional meetings (not at the 'yellow pages') and served as educational tool. but, as the most of the errors are system related, the key to reduce them is to focus on improvement of the system and nil for plasma. wastage rate was highest for plasma components. the influence of local practices on such discarding and whether avoidable shall be discussed. audit for blood discarding and corrective actions to minimize discarding is essential for all transfusion services and blood centers. designed technical and economic support. options include importing finished products and/or procuring products made from locally collected plasma. one approach is to consider local fractionation of plasma by building and operating a plasma fractionation facility, which may produce, finished products, or may produce intermediate products that are further manufactured in another facility. an alternative approach is the implementation of a plasma fractionation program where local plasma is sent to an established fractionator, and the plasma is fractionated following preagreed terms. the end products are returned to the country of the plasma supplier. in 1954 the national center for the production of blood products was established, under the direction of elias politis and 9 years later in 1963 begun the production of dried plasma from greek donors. by the year 1965 the center started the production of fibrinogen and by the year 1967 the production of antihaemophilc factor. in 1992 all the activities of the center settled down due to administrative aspects. at the beginning of 1990s a contract fractionation program was instituted (under the direction of k. sofroniadou) concerning the fractionation of liquid plasma and production of albumin, which by the end of year 2000 stopped and was replaced with a new contract for the fractionation of source plasma and the production of albumin. the challenge of adapting to the new and more stringent regulations governing the manufacture of blood products was great and brought a lot of changes in the structure of our center. a new bar-coding system ensuring the traceability of blood donations was instituted together with complex software for packaging and preparation of plasma shipments to the fractionation center together with all necessary paper work. a close collaboration with the medicines regulatory authority in order to be able to fulfill all the requirements that regulate issues associated to the quality and safety of human derived medicinal products. collaboration with blood collection establishments was promoted in order to increase the amount of plasma produced. there is a continuous effort from all the implicated parts in order to follow defined quality assurance procedures as highlighted by international guidelines for the blood donor selection, collection procedures, testing methods, donation handling, storage and transportation of plasma. the plasma contract fractionation program may serve, as an initial step prior to switching production to a locally built facility. this lapse of time may be used to expand the plasma collection potential, and to permit appropriate design, qualification and validation of the facility as well as training of local personnel. background: fibrin glue became a reality in the early 1970s, when techniques for the isolation and concentration of clotting factors were improved. in 1972, matras et al. described successful application of fibrin glue for peripheral nerve repair. this encouraging report prompted the use of fibrin glue in wound closure, skin grafting and bone union of osteotomies. the fibrinogen component of fibrin glue is produced from single unit donations of fresh frozen plasma. such procedure helps to reduce the risk of transfusion transmitted infections encountered by exposure to pools from large numbers of donors or by use of fibrinogen prepared from autologous blood prior to surgery. the second component, a mixture of thrombin and cacl2, is commercially available. thrombin is applied to the operation site simultaneously and in equal volume to the fibrinogen but from a separate syringe. there are many methods of fibrinogen concentrate preparation but none of them has been described in detail. aims: the aim of this study was to choose/select the most effective, simple and safe method of obtaining fibrinogen concentrate (basic component of fibrinogen glue) which would also be easy to prepare in blood transfusion centers. methods of precipitation of fibrinogen by polyethylene glycol (peg), ammonium sulphate, ethanol or cryoprecipitation were compared. methods: plasma was obtained after centrifugation (4000 ¥ g for 5 min) of whole blood. four units of plasma were pooled into a 2000 ml bag, mixed, divided into 4 parts and immediately frozen. one of them was stored at -30°c and after one month the plasma was thawed at 4°c during 16-18 h. fibrinogen was obtained by cryprecipitation and each of the three remaining units was precipitated with ethanol, peg and ammonium sulphate. the levels of fibrinogen, fibronectin, plasminogen and factor xiii were determined in each fibrinogen concentrate. results and conclusion: the level of fibrynogen ratio in fibrinogen concentration, obtained by peg and ammonium sulphate was significantly higher. cryoprecipitation is a simple, economic and reproducible procedure with the advantage of being performed in a closed system. plasma fractionation program in greece: an unknown history the provision of safe and sufficient plasma derivatives to meet the needs of local population requires special consideration and a well(light cycler, roche diagnostic systems, nj) was used for the identification of the c282y, h63d and the s65c point mutations of the hemochromatosis gene, and were based on protocols developed, for c282y by the unidad de medicina molecular (ingo, santiago de compostela, espanha), and for the other two mutations by bolhalder m et al. the primers and probes were designed by tib molbiol (berlin, germany). results: the analysis of the percentages of genotypes and allele frequencies of the hemochromatosis gene mutations are described in the table. no differences were found between the patients and the controls. when we compared subgroups of patients based on their hepatitis c genotypes, a higher value for the c282y allele was obtained (9.0%) in individuals with genotype 1, however without statistical significance. discussion: hereditary hemochromatosis is a common disorder and is associated, in some studies, with a worst prognosis in patients with viral hepatitis. follow-up studies are necessary in order to evaluate if the presence of these mutations can cause a more severe course of the illness (greater risk to develop fibrosis or cirrhosis) and a different outcome when treated with antiviral drugs. also, it will be important to evaluate if aggressive phlebotomies will modify their clinical evolution. introduction: portugal has a higher prevalence of viral hepatitis, with probably more than 250.000 patients chronic infected with hepatitis b and/or c. hereditary hemochromatosis (hfe) is one of the most common causes of known hereditary illnesses with hepatic repercussion. hfe mutations are also found in linkage desiquilibrium with particular hla haplotypes, conferring, eventually, a different response to viral agents and antiviral drugs. in this study we evaluated the prevalence of the main mutations c282y, h63d and s65c for the hfe in a population with chronic hepatitis b and/or c and in a cohort control. background: haemolytic disease of the newborn (hdn) is the destruction of the red blood cells of the fetus and neonate by antibodies produced by the mother. although postpartum rhig prophylaxis reduced the incidence of alloimmunization from pregnancy from 16% to 1-2%, the doubt subsists if it is appropriate to use it as routine antenatal prophylaxis. material and methods: a total of 1836 samples (918 mothers and 918 newborns), from 01/05/2003 and 30/04/2004, were studied. all abo, rh typing, antibody tests and dat were carried out in column agglutination tests. results: from the 918 cases studied it was found 778 cases without incompatibility (85%). from the 140 incompatibilities, 88.6% were abo, 8.6% were rhd, 1.4% were other rh incompatibilities, and 1.4% were due to auto-antibodies. 80% of the mothers were rhd+ and 20% rhd-. conclusion: of the 918 pregnant women studied, only 184 were rhd-. from this group 83 (45%) delivered rhd-newborns, what revealed that the antenatal prophylaxis they were submitted was unnecessary. from the 184 pregnant women rhd-, 13% had incompatibility abo, which decreases to near 2% the risk of development of rhd immunization. being anti-d immunoglobulin a product that has the potential risk of infection transmission, is it appropriate to use indiscriminately as a routine antenatal prophylaxis? the introduction of molecular methods to determine the fetal rhd genotype could rationalize the use of antenatal anti-d immunoglobulin prophylaxis. introduction: the frequency of hla a201 haplotype expression has been found about 44-46% in caucasian and in greek population particularly, 44.3%. because of the high frequency, it is used widely in anticancer immunization. the immune system plays an important role in the defense against neoplastic disease and immune response show temporal chances related to circadian variation of antibodies and total lymphocytes in the peripheral blood. aim: the probable difference in the frequency of hla a201 expression and their lymphocyte phenotype into a group of cancer patients and a group of healthy donors, during screening of immunization with hla-combined peptides. materials and methods: 323 healthy donors who proceeded in the department of transfusion medicine, university hospital of heraklion crete were tested for the hla a201 expression. in 11 of these donors the expression of cd3, cd4, cd8, cd2, cd19, cd20, hla dr, cd3+cd4+, cd3+cd8+, cd16-cd56+, cd3+cd16-cd56+, cd3-cd16-cd56+, cd3-cd16-cd56+ was examined. meanwhile, 132 patients with metastatic cancer who were hospitalized in the department of medical oncology, university hospital of heraklion crete, were tested for their hla a201 expression, while in 50 of them for their lymphocyte phenotype. the antigens expression was examined in flow cytometry. the hla a201 expression in healthy donors was 44.8% and in cancer patients 45% (p > 0.05). in table 1 the mean, standard error, t-test and p of the two groups are included. (see table 1 ). the two groups (healthy donors and cancer patients) revealed no statistical significant difference on lymphocyte phenotype, except of the cd20 expression, which was higher in cancer patients. summary and conclusion: the expression of hla a201 in cancer patients and in healthy donors was comparable. also, the lymphocyte phenotype among the two groups has not statistical significant difference, except of the cd20 (total b-cells). the significance of this result has to be investigated. in the course of original documents research i found out that dr. kalic, head of the first organized blood transfusion institution in the balkan region (at beograd, serbia, in 1936), set himself a professional goal: blood should be awaiting all patients and transfusion should not be a privilege of large city inhabitants only. dr. kalic's idea was that blood transfusion should be administered according to clearly given instructions and using simple blood sets. encouraged by the conclusions of the congress held in paris in 1937, dr. kalic started preparations for the transport of blood to the inland. he concluded bravely that citrated blood could be sent by regular mail, as an ordinary parcel, without particular protection from the outside temperature. he advised his colleagues to use blood as an intravenous injection. blood was taken from voluntary female donor in belgrade (capital), march 1938. after keeping it for 10 days at storage, blood was forwarded on a two-day journey to a small town, 150 kilometres away from belgrade. there it was kept on a room temperature before its final use for a treatment of a patient suffering from secondary anaemia. the patient underwent the procedure without side effects and responded to the transfusion with blood sent in this manner much better in comparison to earlier methods of direct blood transfused. reminding ourselves of the courage of our ancestors to implement their professional knowledge and personal original ideas in a new way with the desire to help the patient as successfully as possible, we pay them the deserved respect and gratitude for inspiring and encouraging us in this way to try the same. conclusions: automation leads to increased standardization, faster specimen processing and reporting, elimination of manual specimen identification, uniform interpretation of serological reaction patterns and objective reading of haemagglutination endpoints. using auto-vue allowed the staff uninterrupted time to perform quality assurance duties, extended antibody identifications, preventative maintenance, inventory control. the instrument allowed us to leverage current staff to a more productive, less stressful level. introduction: exosomes are 60-100 nm secreted vesicles produced by antigen-presenting cells (apcs). the finding that exosomes from dc pulsed with tumor-derived peptides elicited potent antitumor tcell responses and tumor regression in mice has led to the proposal that human exosomes could be effective vectors for antigen delivery in the context of cancer immunotherapy. aim of the study: to establish the method of producing a new kind of tumor vaccine -exosomes secreted by dc, pulsed with tumor peptides. methods: exosomes used in this study were generated from monocyte-derived dc pulsed with peptides from k562 tumor cell lines. exosomes were purified by the methods of ultrafiltration and ultracentrifugation. the methods of dynal magnetic beads, flow cytometry and western-blotting were used to determine the surface molecules of the exosomes. the function of the exosomes was deterobjective: to develop an immunoheatological technique for the study of erythrocyte hyaluronic acid sodium salt (cd44) receptor expression in red blood cells (rbcs) from adults and newborns. materials and methods: samples of anticoagulated blood from adults (n = 30) and umbilical cordon (n = 30) were used. several dilutions oh hailuronic acid sodium salt solution 2% (sigma l-77h0544) were confronted with 2% erythrocyte suspension in phosphate saline buffer (pbs) ph 7.4. the rbcs were previously treated with an enzymatic solution of 5% bromeline in pbs ph 7.4 (sigma l60h0168). agglutination readings' were been by slow sharking after of 12 h incubation at 4°c. the results were expressed through the sensibility parameter which involves titer and score. this is defined by a mathematical expression a = à6 si. di-1. 10-3 (i = 1, 2, 3 . . .) where si represent the score and di-1 is dilution inverse. the adult' rbcs showed a = 26 ± 8, while en the newborn the parameter was a = 3 ± 1. our results showed significant differences between both groups. conclusions: in this work, we present a simple immunohematological technique for the hyaluronic acid sodium salt (cd44) receptor expression in red blood cells, which could be a useful tool to evaluate the alterations of the receptor's expression in rbc. a new technology for crossmatching tests adapted to a fully automated system l gaillard, v desvigne, a boulet, l fauconnier and jm pelosin diagast, loos, france we have developed a new automated technology for crossmatching (compatibility) test suitable for automation and high throughput. the method does not require centrifugation steps thanks to the use of magnetised red blood cells (rbc). all the steps described are performed on the fully automated qwalys system. this methodology requires washing steps under magnetic field and is based on the fixation of sensitised rbc on the surface of a well coated with monoclonal anti-human globulins. in a first step, the red blood cells from target blood bags were magnetised during 10 min. then the patient plasma is distributed on a microplate and incubated with the previously magnetised rbc during 20 min at 37°c. excess of unbound immunoglobulins is removed by washing steps. in a third step, sensitised magnetised rbc were transferred in the antiglobulins coated plate and placed 5 min on a magnet plate. wells in which antigen-antibody interactions have occurred display a confluent layer of rbc (positive reaction). the negative reaction appeared as a pellet in the middle of the well. the test can be read by an automatic reader or by naked eye. the patterns in the well are stable for at least 24 h at room temperature. the plasma samples are provided by the laboratory of haematology of the chru of lille. the red blood cells are collected from segment of tubing of blood bags coming from the laboratory of blood donors of the efs (french blood services) nord de france-lille. the results are obtained in 40 min. comparative studies showed that our new technology, without any centrifugation steps, is reliable and sufficiently sensitive and specific enough to perform cross matching tests using a high throughput automated system. the mechanisms of p53-dependent apoptosis involve a set of genes that possess the ability to modulate oxidative stress. one of them pig3, is induced by p53 through a microsatellite in its promoter region. this microsatellite has been proposed to represent an evolutionary adaptation of tumor suppressor mechanisms. microsatellite instability and genetic constitution, comprising the presence of the low repetition allele (10 tgycc repeats), at this locus have been hypothesized to provide an increased risk for cancer development. aim: in the present analysis we examined this polymorphism in blood samples from voluntary health donors and compared it with human lung cancer samples, employing two different ethnic groups, greek and british. results: analysis of this locus in both types of samples showed: (i) the homozygous presence of the 10 repeats allele only in the samples from healthy blood donors; (ii) a very low frequency of microsatellite instability (<1%) and no loss of heterozygosity in matched normal-tumor tissues; and (iii) a non-significant increase of the most frequent allele (15 repeats) in the cancer groups as compared to samples from healthy blood donors. the last two observations were found in both greek and british populations. conclusion: taken together, these data do not support the notion that this pig3 polymorphism is associated with an increased risk for cancer susceptibility. background: blood group determinations are routinely performed by the sensitive technique 'gel test' for the last few years. many weak d and partial d phenotypes which react as d negative or weak d by slide test, are assigned the rh d + status by gel test. this is most desirable in the case of blood donors but creates concern in case of patients and antenatal women with a partial d phenotype. case report: we report a female patient (blood group o, c+, c+, e-, e+) whose red blood cells gave a positive reaction of different strength and speed with different anti-d antibodies in slide tests. we were asked to type the patient and provide the appropriate blood units. the patient's cells gave a 4+/3+ reaction in the standard screening procedure for the rh d in gel test micro-typing system that contains a polyclonal reagent of human origin (which allows a direct detection of most weak ds), a 4+ reaction in a test with monoclonal anti-d and a 4+/3+ reaction in the gel test micro-typing system destined to detect du and which contains polyclonal anti-d of human origin. however, since the slide test gave a rather slow onset of agglutination with one commercial reagent (made up of a blend of polyclonal and monoclonal anti-d) we tested the patient's red cells against 6 anti-d reagents in the id-partial d typing system. one of these (number 4) gave negative reactions and the remaining five gave positive reactions (ranging from 3+/2+ to 4+), indicative of a partial d category vii phenotype. the patient's red cells were also tested in the id-card 'diaclon abo/d' . this card provides the complete profile for abo/rh d in one single procedure step, including the confirmation of rh d. it contains two different anti-d reagents within the gel matrix in two consecutive microtubes. the first anti-d (polyclonal human) is expected to give a positive result with d+ red cells and partial d category vi, while the second (monoclonal rabbit) is expected to give a negative result with dvi+ red cells. our patient's cells gave a negative reaction with the first and a 3+/2+ reaction with the second anti-d in this system, indicating a d variant other than dvi. finally the patient was assigned the partial d category vii phenotype (according to the pattern of the reactions obtained with the id-partial d typing set) and rhesus d negative blood units were issued. this case illustrates the diversity of reagents used for rhesus typing in different laboratories. failure to disclose some d variants is a disadvantage when typing patients. a combination of techniques is often needed to reveal the real rh d phenotype. the only single system that could have revealed a d variant in our patient from the beginning, is the id-card 'diaclon abo/d' with two different anti-d reagents in two consecutive microtubes as described above. a cost-benefit analysis should be undertaken to show whether it should replace other screening tests for abo and rh d when typing patients. who cares about the quality of life of the chronic patients treated with blood products? d ilcenco*, e hanganu-turtureanu † , c burcoveanu † , c vartolomei ‡ and d azoicai § *blood transfusion center, † hospital 'sfantul spiridon', ‡ institute of hygiene, § university of medicine, iasi, romania quality of life is one of the methods used to appreciate the quality of the health system. romania is going to join soon the european union, so there must be a concern regarding the improvement of the national health system. blood receiver's life quality never been researched before in romania. we have been chosen a batch of chronic ill patients who have been received blood transfusion with blood or blood components, and asked them to complete two types of questionnaires regarding their life. we used nottingham health profile and beck's depresion index. results shows that this kind of patients need special care, because they all (with one single exception) feel frustrated and feel like a burden to the other normal persons. evolution of the pain index, mobility index, energy index, emotions index, sleep index and social isolation index was in concordance with the depression index. in conclusion, this type of patients needs special attention and medical authorities should make more efforts to assure their life quality support. transfusion medicine practice in surgically treated urology patients: our experience il ilincic*, bm bozovic* and ts tadic † *clinical center dr dragisa misovic, † natio. blood transfusion inst., belgrade, serbia objective: multiple studies demonstrate that the use of blood/blood products in patients undergoing elective urology surgeries, as well as the actual needs assessment, present the issue of numerous debates. method: using the retrospective method, utilization of blood/blood products was analyzed, as well as the ratio of prepared/used blood units in urology patients in the surgical ward, in the intensive care unit (icu) and at the urology center within the cc dr dragisa conclusion: due to a rather liberal use of primarily ffp in certain cases (cystectomiae in the first place), and a discrepancy between the prepared and actually used blood units, hospital transfusion committees should be an imperative in order to solve current dilemmas regarding justified use and proper administration of blood and blood products. background: the safe collection, production, distribution and application of blood and blood products in a high quality needs logistic on a high level. since the seventies computers, special software and barcode are used in transfusion medicine and improved the safety of processing data. in the last years a new technology was developed for industrial use, the radio frequency identification (rfid). aim: the aim of our studies was to check whether rfid can use reasonable in transfusion medicine. methods: at first we developed a flow chart, where we can use the technology and where are the problems by introduction. so we tested in the red cross donation centre in saxony about 1000 passive rfid smart label under real conditions. in cooperation between the akh vienna and novatech research a new handheld pc software 'labelview' for all steps around the transfusion was developed, including the identification of the patient and the processing of the haemovigilance data, and tested in first time. results: passive and semiactive (with temperature control) rfid labels survives all hard steps during the working up of the whole blood (e.g. centrifugation by 5000 g, separation, etc.). as a result of the contactless identification they are help to make easier the documentation of all processing steps according good manufacturing practice. in clinical practice they are a good supplement to bed side transfusion software. conclusion: for all lot of problems by the logistic and the safe identification around the transfusion existing various single point solutions such as patient-wristband, bed-side test, double check of blood group typing and donor -donation registry in software, etc. the lecture will deal with new developments in logistics and data management, which can help to reduce the problems associated with documentation, safe identification and reporting of haemovigilance data. our experiences with the immunohaematological analyser olympus pk 80 applied conventional and no conventional (hemolytic medium time) techniques in 23 sera of patients with ascariasis. results: the ai and hk tests showed: b epithopes in 4 ae from b patients and in 3 ae from ab patients; a epithopes in 1 ae from ab patient and in 3 ae from a patients; p and p1 epithopes in 18 ae and only p1 epithopes in 3 ae. these 21 patients had both epithopes in their erythrocytes. the hemolytic techniques showed: anti b immune antibodies in 8 sera and anti a immune antibodies in 8 sera. the presence of abo and p epithopes in ae and immune antibodies in patients with ascariasis show a relation about blood groups and ascariasis. the fact of to find the same abo and p antigens in a. umbricoides and in its hosts suggests that the parasite might absorb them during its life cycle. these epithopes would be involved in the molecular mimicry. the use of filters for leucocyte depletion in anemic patients on maintenance hemodialysis g poposki*, s kovaceski*, b krstanoski*, s mena* and n solaz † *institute of nephrology, struga, macedonia, † ankara university, faculty of medicine, ankara, turkey introduction: renal anemia is one of the major chronic complications in end stage renal disease. it is caused by reduced production of erythropoietin (epo) due to uremic toxin effects, reduced halflife of rbc, iron deficiency, aluminum intoxication, blood loss during hemodialysis, gastrointestinal hemorrhage, epistaxis, infections etc. allogenic blood transfusion is transplantation of certain or all cell types. however, allogenic blood transfusion can contribute to many immune system disturbances with clinical side effects. besides erythrocytes, mononuclear, t and b-lymphocytes, are also transfused, which cause immunomodulatory disturbances in immune system of recipient. leukocytes are responsible for frequent febrile non-hemolytic transfusion reactions, alloimmunization toward leukocytes and hla antigen and transmission of cmv. anti-le antibodies, forming of immune-complexes, complement activation with pirogenic c3a and c5a immunoinflamatoric citokines cause febrile reactions. commercial use of filters for leukocyte depletion with removal of leukocytes and degraded products of microagregates and cytokines, cause minimum harmful immunomodulatory effects and prevent transmission of cmv. aim: the aim of the study was to present the effects of transfusion of erythrocytes with residual number of leukocytes in anemic patients on chronic hemodialysis at institute of nephrology in struga. matherial and methods: during 1997-2004 period all anemic patients on hemodialysis were divided in 4 groups. the first group 34 pts with febrile non-hemolitic transfusion reaction. the second group-25 pts immunized toward leukocyte and hla antigen. the third group 57 young candidates for kidney transplantation for prevention of hla immunization. the fourth group 16 pts with sle (for immune-complexes and autoantibodies). total 132 patients (65 males and 67 females) received 319 units of rbc with residual number of leukocytes. commercial filters of baxterô (lekostop 4 lds) and terumoô (imugard iii rc) of second and third generation with microagregate filter and synthetic polyurethane fibers, with 20-40 microns pores that remove leukocytes, platelets, microagregates and fibrin were used. erythrocyte concentrates are filtered until 5 days of collection. result: aabb permits maximum <5 ¥ 106 wbcs/unit for prevention of febrile non-hemolytic reaction. the filters we used reach residual leukocyte number of 2 ¥ 10 5 the le reduction of 99-99.99%. the number of rbc after filtration is minimum 90% -40 g hb per unit. in none of the patients who have received the leuco-filtered blood, no adverse post transfusion reactions were noticed. conclusion: the used filters for leucocyte depletion are characterized with superior biocompatibility, excellent elimination of all types of leucocytes and high 'recovery' of erythrocytes. the use of filters for le depletion reduces and minimizes the side effects of allogenic blood transfusion in patients on chronic hemodialysis who are alloimmunized, in patients with sle, and particularly in young patients candidates for kidney transplantation. background: fv leiden, prothrombin g20210a, mthfr c677t are three most common and important prothrombotic inherited mutations. aims: the aim of the case-control study was to assess the prevalence of mutations and their single or combined effects as risk factors for thrombosis. methods: the study included 103 thrombotic patients (venous thromboembolism, chronical venous diseases, different etiology) and 200 asymptomatic healthy individuals as control group. extraction of genomic dna was followed with genotyping of fvl by pcr-ssp, prothrombin and mthfr mutation by pcr-rflp. results: a statistically significantly higher prevalence of fvl mutation was found in thrombotic patients (32.0% heterozygous, 1.9% homozygous) compared to controls (3.0% heterozygous), p < 0.0001. the or for heterozygous carriers was 15.24 (95% ci 6.13-37.94), confirming the association of fvl mutation with the risk of thrombosis. there was no statistically significant difference in the prevalence of the prothrombin mutation in patients (5.8%) and controls (3.5%), or 1.71 (95% ci 0.56-5.21), p = 0.3441. although the group of thrombotic patients showed a higher prevalence of homozygous carriers of c677t mthfr than the control group (17.5% vs 10.5%), or was not significant (1.81, 95% ci 0.91-3.57), p = 0.0859. analysis of combined effects of mutations showed an additional thrombotic risk for carriers of fvl mutation and both mutated alleles of c677t mthfr gene (tt and ct) (or 9.40, 95% ci 3.41-25.80), p < 0.0001. conclusions: fv leiden mutation was detected as significant single risk factor for thrombosis in studied patients group. additional prothrombotic risk have carriers of fvl mutation and c677t mthfr gene mutation. a female patient in a high fever due to urinary tract infection does not respond being given antibiotics. on the contrary, leukocytes rose (to 30 ¥ 10 9 /l), anaemia became even deeper, as well as thrombocytopenia. hemocultures were negative. hematologist decided to search for hematological disease. the first citology results of bone marrow aspirate suggested lymphoproliferative disease (15% atipical plasma cells). to treat heavy anaemia (hgb 53 g/l) hematologist asked for red blood cell concentrate. pretransfusion testing revealed warm autoantibodies in the patient serum and on red blood cells. antibodies had no apparent specificity. biochemical parameters (bilirubin, ldh, haptoglobin) suggested mild hemolitic process. electroforesis revealed polyclonal hypergamaglobulinaemia. the 9th day of hospital treatment, the therapy with corticosteroids was introduced (solu-medrol 125 mg per day). coagulation parameters were tested: pt 1.75 inr, aptt 41s, fibrinogen 0.5 g/l, trb 66 ¥ 109/l, d-dimer 251 mg/l, atiii 52%. dic was suspected. liver enzymes showed mild liver dysfunction (normal ast, alt, elevated ggt, low che). substitution therapy started with 1 dose of cryoprecipitate, 1 dose of fresh frozen plasma, 1000 iu atiii, 2 doses of red blood cells and vitamin k 10 mg. two days after the substitution therapy we saw pt 1.28 inr, aptt 31s, fibrinogen <0.5 g/l, trb 75 ¥ 10 9 /l, atiii 71%. during the next few days erythrocytes and thrombocytes rose, but due only to corticosteroid therapy and not to substitution therapy. the patient had neither signs of con-sumptive coagulopathy, nor hypoproduction of coagulation factors, except for fibrinogen. till 17th day of therapy, fibrinogen was below 0.5 g/l. there was no hemorrhagic diathesis. after that, fibrinogen rose, and on the 21st day the patient was recovered, in both clinical and laboratory terms. the results of immunological tests, collected later, confirm the diagnosis of systemic lupus erythematosus. we did not have any specific test to confirm antibody mediated hypofibrinogenaemia, but in the setting of sle, without any specific treatment except corticosteroids, fibrinogen recovered. we assume it is quite enough for highly suspected immunological hypofibrinogenaemia. results: twenty-four-year-old male patient with severe hemophilia type a suffering from low incoercible digestive bleeding secondary to ischemic colitis caused by autoimmunity (vasculitis) without response to current management. treatment was initiated with 90 mg/kg/dose of rfviia (*) for 7 days, after which there was clinical and endoscopic recovery, and an inh decrease to 1.0 ub/ml (fviii dosage 19%) . he began to take meprednisone (1 mg/kg/day) for 45 days, after which the inh titre was 12.0 ub/ml. (table 1a ,b) the patient underwent surgery the following year (correction of equinus foot). he entered the operating room with an inh of 26.7 ub/ml and was treated with 120 mg/kg/dose of rfviia (*) for 10 days, obtaining an excellent hemostatic response. he had two autologous blood units, but it was not necessary to be administered. the inh titre decreased again (down to 5.7 ub/ml) during the intratreatment stage. thirty-five days after rfviia, the inh titre was 7.5 ub/ml. (table 2a ,b) the presence of high titre inh against fviii is a critical problem in cases of bleeding or surgery need due to the inefficacy of the available therapeutic options and the severity of the events. in this case, we have observed that, apart from inducing hemostasia through the activation of the coagulation extrinsic path, rfviia could reduce the inh titre in sequential dosages. we have introduced the case of a 24-year-old patient with hemophilia complicated by a high titre inh against fviii. in this case, we have observed that, apart from inducing an effective hemostasia through the activation of the coagulation extrinsic path, rfviia could reduce the inh titre in sequential dosages. there was also a decrease in the inh titre concomitantly with an increase of the plasma fviii level during its use. this phenomenon suggests that rfviia could produce a modulation in the immune response. evaluation of an automated blood collection system with standard ratio of anti-coagulation and integrated filter for whole blood leucodepletion l dadiotis, a kolokytha, m dimou, a perdiou, c alepi, p spyropoulou, e igoumenides, c velidou, v panagopoulou and s matsagos tzaneion general hospital, pireas, greece automated blood collection system (abc) is a device manufactured by macopharma which collects by gravity a preset volume of blood and mixes it with anticoagulant (ac) in standard ratio (7 : 1). this is managed by passing the ac, which is stored in a special bag, anti-erythrocyte antibodies are immunoglobulins that belong to the igg, igm and iga classes. their common characteristic is a specific reaction with antigens that are located on the erythrocyte surface. they can emerge as auto antibodies and alloantibodies. the blood transfusion in patients may induce a post-transfusion hemolytic reaction (pthr). in order to avoid or reduce the danger of the pthr it is necessary to examine whether there are irregular anti-erythrocyte antibodies in the patient's serum as well as in the serum of the voluntary blood donors. all the irregular anti-erythrocyte antibodies are not clinically relevant. the experience shows that the clinically significant antibodies most often belong to abo, rh, kell, kidd, duffy and ssu blood groups. in the period from april, 2003 to november, 2004, we monitored and examined, at the institute for blood transfusion, clinically significant antibodies in the serum of the patients who are treated with blood transfusions as well as in the serum of the voluntary blood donors. we used the following tests for detecting irregular anti-erythrocyte antibodies: enzyme test, indirect coombs test, screening test by the commercial test erythrocytes and gel filtration method. the detected irregular antierythrocyte antibodies are identified by means of the commercial test erythrocytes for identification. our results are the following: voluntary blood donors: anti d, anti c + d and anti-leb antibodies. patients: anti-d, anti-k, anti-fya, anti-c and anti-e antibodies. in nine patients, anti-erythrocyte antibodies were discovered, namely, those that react at the temperature higher than 30°b ut whose specificity we could not discover with the existing techniques. improved predictive factors of response for myelodysplastic syndrome patients treated by the combination of erythropoietin and g-csf s park*, c kelaidi † , s grabar ‡ , v bardet ‡ , d vassilieff ‡ , f picard ‡ , m guesnu ‡ , mc quarre ‡ , p fenaux § and f dreyfus ‡ *service hématologie, hopital cochin, † hématologie, hopital avicenne, ‡ service hématologie, hopital cochin, § hématologie, hopital avicenne, paris, france it has previously been shown that serum epo level and number of previous red blood cell transfusions are predictive factors of response to epo + g-csf treatment of myelodysplastic syndromes (mds). in a subgroup of patients with mds having sepo <500 ui/l, known to be good responders to epo + g-cscf, the gfm group wanted to refine the model predicting the response to epo + g-csf, especially with cytology (who classification with dysplasia and percentage of erythroblasts and blasts). in a population of 64 patients (ra, rars and raeb <10% blasts) receiving epo ± gcsf between 2000 and 2004 and having serum epo <500 ui/l, the response rate at week 12 (iwg criteria) was 60%. six variables were associated with response to epo ± g-csf for mds: age >70 years (p = 0.02), number of prior red blood cell transfusions <2 packs/months (p = 0.005), serum epo level <200 ui/l (p = 0.02), percentage of blasts <5% (p = 0.05), percentage of erythroblasts >30% (p = 0.02) and low ipss score (p = 0.001). we did not found any influence of dysplasia, type of rhepo (darbopoietin alfa or epoietin alfa) and karyotype on response rate. in multivariate analysis, age through a rotating pump. the abc can be used with all types of p-417 pan-european blood safety alliance the pan-european blood safety alliance is a unique alliance of patient organizations, formed to promote the highest level of blood safety for all in europe. it was formerly established on 11 february 2005, during the course of the first general meeting of the pbsa, which comprised of 8 founding patient organizations. the objectives of the pbsa are: 1. to promote the fundamental right and duty to safety of all patients in need of blood transfusions and blood products. 2. to ensure the availability of sufficient amounts of safe blood, to meet all treatment need through: -the education of all staff handling blood components, to reduce human error. -the implementation of and access to, proactive blood safety technologies, for each patient across europe. -haemovigilance -the adequate access to blood transfusion services, which should be provided free of charge to the patient. other objectives are to raise awareness on a local and european level regarding blood safety, to promote eu legislation that improves safety standards of blood transfusion services, including stem cell preparation and storage across europe and to lobby for increased patient influence on eu health policy makers. very importantly, the alliance aims at providing a forum for patients, healthcare professionals, health policy makers and relevant industry, as well as acting as a point of reference to the national health authorities, the european commission and other european institutions, when seeking the opinions of patients on blood safety. cerns of insertional mutagenesis and the safety of some viral vectors that randomly insert genes through the genome have been recently resurfaced following the development of a haematological malignancy in a child treated with a retroviral vector. particularly questions also remain as to, whether gene therapy and the production of ectopic factor viii and ix will be a risk for inhibitor development or indeed whether it might promote tolerance in those patients with inhibitors. w-pl4-11 gene therapy for thalassemia: will it become reality? university of washington, seattle, wa, usa experiments aimed to develop gene therapy approaches for the beta chain hemoglobinopathies, sickle cell disease and beta thalassemia started about 25 years ago. in the beginning results were dismal because of the extremely low and variable expression of globin genes contained in the therapeutic vectors. a major development occurred in 1987 with the discovery of powerful regulatory elements that could guarantee high level of globin gene expression. these elements when incorporated into viral vectors allow expression of therapeutic levels of the transferred globin genes. a second major progress was achieved with the development of safe lentiviral vectors that can efficiently infect the human pluripotent repopulating hemopoietic stem cells. as a result of this progress, today beta thalassemia and sickle cell disease can be cured in murine models of these disorders. considerable effort is already being devoted into further improvement of lenti viral vectors with emphasis on incorporating elements which will decrease the probability of insertional mutagenesis and leukemogenesis. the major challenge for the clinical application of stem cell gene therapy of thalassemia is the need for genetic correction of large numbers of mutant stem cells. in vivo selection of corrected stem cells is being investigated but there are questions about its safety because of the possibilities of clonal expansion of stem cell lines carrying undesirable integrants. other major challenges have to do with logistics: production of therapeutic vectors, infrastructure required for stem cell gene therapy delivery, and sponsoring and funding of the clinical trials. gene therapy trials on limited number of patients are expected to be initiated relatively soon. if these trials are successful and cures of beta thalassemia ensue, the major challenge will be the delivery of this molecular therapy in the context of medical practice. w-pl4-10 gene therapy for haemophilia haemophilia is an ideal target for gene therapy because only a small rise in factor levels to 1-2 u/dl would achieve the goals of prophylaxis without regular infusions of concentrate and deliver a substantial improvement in lifestyle for patients with severe haemophilia. gene therapy for haemophilia today relies upon addition of normal factor viii or ix genes. with present technology gene therapy can offer the prospect of a true 'cure' for haemophilia in animal models, although this may not be currently realizable in man. more than 30 patients with haemophilia have now been treated in phase 1 gene therapy protocols. all studies have failed to conclusively show that therapeutic levels of factor viii and ix can be reliably obtained. the first trial reported used im injection of a factor ix containing recombinant adeno associated virus (raav) in adult patients with severe haemophilia b. only very modest increases in factor ix level, <2 u/dl rise, in 4/8 patients enrolled were observed, although less factor ix concentrate was needed in 3/6 subjects. a similar study using the same raav vector via intrahepatic artery infusion has been conducted. this has been complicated by the observation of aav vector in the semen of subjects. in six patients enrolled, no durable levels of ix above 1 u/dl were seen. further development of this raav vector is suspended. for haemophilia a, three systems are have been tried. the first study was an ex vivo addition of factor viii gene to autologous fibroblasts and then laparoscopic reimplantation. preclinical assessments demonstrated durable expression of factor viii (>5% of normal) for >1 year in mice following a single treatment. in 4/6 patients treated repeated factor viii rises (0.5-3.5 u/dl) were seen, but no improvements lasted beyond 10 months. the second protocol used a murine leukemia retrovirus containing factor viii, injected intravenouslya development of preclinical data in rabbits and haemophilic dogs. 0/13 patients enrolled sustained levels of factor viii >1 u/dl. the third study, using a modified, 'gutless', adenovirus containing factor viii gene has recruited one patient. this patient demonstrated transient liver toxicity and thrombocytopenia at doses lower than those that cause toxicity in primates. sustained levels of factor viii of ~1 u/dl have been observed over a number of months. accrual to the study has been poor. haemophilia remains a prime target for gene therapy. however, haemophilia is no longer a life threatening disease with current therapy that is both safe and efficacious. a balance between the benefits and theoretical risks must be borne in mind when considering gene-based approaches to therapy. conreference: petz ld, garratty g. immune hemolytic anemias. 2nd ed. philadelphia: churchill livingstone, 2004, pp 375-400. w-pa-114 autoimmune neutropenia introduction: autoimmune neutropenia (ain), a granulocytic disorder due to the presence of anti-neutrophil antibodies, may present as neutropenia of varying degree with or without recurrent infections un previously healthy individuals (primary or idiopathic ain) or in patients with a known underlying disease such as lupus erythematosus, lymphoid malignancies, etc (secondary ain). the condition affects more frequently infants of small ages while it is rare in adults [1] [2] [3] [4] [5] [6] [7] . in some patients, diagnosis is established in occasion of a respiratory, urinary or cutaneous infection, but in many cases is simply a finding of cell blood counting performed for unrelated reasons [6] . clinical and laboratory findings: in general, physical examination is negative. laboratory investigation reveals the existence of isolated neutropenia. association of the disorder with autoimmune hemolytic anemia or autoimmune thrombocytopenia is rarely seen [8] . blood biochemistry is normal while serologic tests for bacterial, viral or other pathogens may be positive depending on the underlying infection. bone marrow is hypercellular without maturation arrest of granulocytic series. hemopoietic stem cell reserves and function are normal or increased, and stromal cell function is within the range of the normality [9]. methods for the detection of granulocyte-specific antibodies: serology for the detection of granulocyte-specific antibodies has been marred, compared to erythrocyte serology, because the target cell here, the granulocyte, is short-lived, fragile and becomes easily activated. the former two of these difficulties require absolutely freshly (<6 h old) isolated neutrophils from a panel of donors to be used every day to run the tests with sera from patients, while the third difficulty is more important since spontaneous cell clumping in vitro is very common and nay mimic the specific aggregation caused by cross-linkage of surface bound antibodies in the granulocyte agglutination test (gat). in order to overcome these problems, the second international granulocyte serology workshop [10] recommended a combination of two tests as the best screening procedure for the detection of granulocyte autoantibodies in patient sera, gat and granulocyte immunofluorescence test (gift). gat is mainly mediated by igm antibodies and is positive in about 2% of cases. gift detects igg antibodies and is positive in about 98% of cases. it is to be noted that flow-cytometry fluorescence may arise not only from the surface but also from the cytoplasm of neutrophils, necessitating assessment of membrane fuorescence by microscopy. a good direct anti-granulocyte test is not available today. this is due to the fact that too few neutrophils can be obtained from the blood of neutropenic patients, and also to the observation that neutrophils are often activated in vivo because if an underlying infection or other inflammatory process, thus expressing fcgrii and fcgriiib to which nonspecific binding of w-pa-113 practical approach to transfusion in autoimmune hemolytic anemia (aiha) g garratty american red cross blood services, pomona, ca, usa a major problem when transfusing patients with aiha is that often all units are incompatible. this may be due to autoantibodies (autoab) and/or alloantibodies (allo-ab). if the incompatibility is due to only auto-ab, then transfusion of incompatible blood will not usually result in a clinically significant reaction, but if due to alloab, the result may be similar to that seen in any other patient (i.e. a hemolytic transfusion reaction ranging from mild to severe). thus, it is essential (as in any other patient) to exclude the presence of allo-ab. it is wise to phenotype all patients, for as many antigens as possible, before the patient receives transfusion. there are two popular approaches to determine if allo-abs are present but being masked by 'warm' auto-ab activity. the preferred method is to remove the auto-ab by adsorbing the patient's serum with autologous rbcs treated with enzymes, or preferably, with zzap reagent. the latter reagent contains an enzyme leading to optimal adsorption of auto-ab, and dtt. these two chemicals will destroy significant antigens other than rh and kidd (e.g. mns, duffy, kell, lutheran, dombrock, cromer, lw, some yta and ge, inb, jmh, ch, rg, pr antigens), thus will not adsorb alloantibodies to these antigens. if autoadsorption is not possible (e.g. patient has been transfused recently or there are too few rbcs), one has to perform adsorption with enzymes or zzap-treated allogeneic rbcs. one does not have to be concerned with covering any antigens destroyed by zzap (e.g. kell and duffy systems). we use a rough guide relating the strength of the indirect antiglobulin test to the number of adsorptions needed to remove auto-ab (1+ = 1 adsorption; 2+ = 2 adsorptions; 3+ = 3 adsorptions; 4+ = 3 or more adsorptions). if there is no activity left after the adsorptions, then one can suspect that the incompatibility was due to auto-ab, but on rare occasions one can be wrong and an allo-ab to a high-frequency antigen has been removed. this is a major disadvantage of using allogeneic adsorptions, and is why adsorptions with autologous rbcs are preferred. if time (2-8 h) does not allow for adsorptions, one can dilute the patient's serum (e.g. 1 in 4, or 1 in 5) and test the dilution against a panel to see if any alloantibody specificity becomes obvious. another approach is to select units matching the patient's phenotype as closely as possible. when dealing with cold agglutinin syndrome one can usually exclude allo-ab activity by testing strictly at 37c. this can be helped by performing adsorptions with enzyme-treated autologous rbcs at 4c, but it is difficult to adsorb all of the powerful cold autoagglutinin activity. it is reported that 30-40% of aiha have allo-abs; the incidence is even higher in patients who have received multiple transfusions. thus, we feel that procedures such as those discussed above must be performed before transfusing incompatible blood if time allows. one should always negotiate with the attending physician regarding the time it will take to perform adsorptions. a decision may be made not to perform adsorptions if the patient has life-threatening hemolysis, and especially if the patient has never been transfused, or pregnant. serum igg may occur (naig). the presence of immune-comlexes in the serum, such as in patients with felty's syndrome, lupus erythematosus and other diseases, as well as the presence of immune aggregates formed in sera stored frozen for long time, may give false-positive tests given that they may bind to fcgriiib molecules expressed on the surface of neutrophils. elimination of immunecomplexes and immune aggregates can be easily obtained by ultracentrifugation [11] . another cause of false-positive results may be the presence of anti-hla antibodies because of allo-immunization. these allo-antibodies react with hla molecules found not only on neutrophils but also on the surface of many other cells including lymphocytes. these allo-antibodies can be eliminated by platelet absorption. it seems that the best method in the search of true antigranulocyte antibodies is the monoclonal antibody-specific immobilization of granulocyte antigens (maiga) [12] . with this method one can specify anti-granulocyte antibodies using a panel of known granulocyte antigenic specificity. finally, it is notable that the levels of serum antibodies to neutrophils may vary considerably over the time. one negative test does not exclude ain. usually, two to three tests have to be run over a period of months [13] . antigenic specificity: human neutrophil antigens (hna) are classified according to an international granulocyte antigen working party [14] . three glycoproteins have been found to be involved in the determination of antigenic specificity, fcgriiib, gpnb1 (cd177) and gp70-95. the respective antigens, frequencies and alleles are illustrated in table 1 . antigenic specificity can also be studied by using methods applied in molecular biology. a promising approach is transfection of mammalian cells by cdna derived from granulocyte antigen specific mrna. cell lines have been established with cells expressing the respective human granulocyte antigen, making the detection of anti-granulocyte antibodies more easier. genotyping of hna antigens can also be stydied with the pcr technique [15] . references are available from the author upon request. w-pa-115 a rare case of 'coombs negative' autoimmune haemolytic anaemia due to red cell autoantibodies of iga class warm autoimmune haemolytic anaemia (waiha) is usually associated with red cell auto-antibodies of the igg class, which can be detected by polyspecific direct antiglobulin test (dat). routine polyspecific direct antiglobulin tests contain anti-igg and anti-c3d components, and are not standardized to react with iga-or igmsensitized red blood cells. haemolytic anaemia caused by warmreacting auto-antibodies solely of the iga class is exceedingly rare. those cases of autoimmune haemolytic anaemia can be difficult to diagnose because of the negative polyspecific coombs' test, which is a standard in investigation of possible causes of haemolysis. we present a case of severe warm autoimmune haemolytic anaemia caused by iga class autoantibodies. a 40-yr old male patient was admitted with anaemia, haemoglobinuria, and other signs of severe haemolytic disease. he received multiple transfusions but haemoglobin level did not rise above 7 g/dl. the initial polyspecific direct antiglobulin test, containing an anti-igg and anti-c3d antiserum, was negative. tests for cold agglutinins and other possible causes of haemolysis were negative. only by using a monospecific, anti-iga antiserum could we show that the warm iga auto-antibodies against red blood cells were present on patient's erythrocytes. we have not detected signs of complement activation by iga autoantibodies in this patient. the patient received corticosteroids with good initial effect. his haemoglobin level stabilized and he did not require more transfusions. anti-iga direct antiglobulin test became negative about to weeks after the therapy was initiated. however, in spite for the initial effect of steroid therapy haemolysis continued, and splenectomy was performed 6 months after diagnosis was made. it has been shown that human lymphocytes, granulocytes and monocytes contain specific fc receptors for iga, and both monocyte-mediated phagocytosis and antibody-dependent cellular cytotoxicity due to iga auto-antibodies has been demonstrated. there is also increasing evidence that iga auto-antibodies can activate complement, both via the classical and the alternative pathway. a phenomenon of 'reactive haemolysis', which involves c3-independent binding of c5b6 complexes to 'bystander' red blood cells, has also been described. we emphasize the importance of performing additional testing in cases of apparent 'coombs' negative' haemolytic anaemia due to iga, igm or 'low affinity' igg autoantibodies, and serological aids that are available for that purpose. described. both siblings were born on term, in good general clinical status, free from any signs of infection, and with isolated severe neutropenia (170 and 123 neutrophils/ml). the diagnosis of annanti hna-2a was made upon exclusion of other possible causes of neonatal neutropenia, and confirmed by serological testing of granulocyte antigens and antigranulocyte antibodies. in both cases, the course of the disease was mild, with bacterial omphalitis on day 15 and 5, respectively. omphalitis was successfully treated with 7-day antibiotic therapy according to antibiotic sensitivity report. the first neonate received standard dosage of intravenous gammaglobulins for 5 days without success. this was followed by an attempt at neutrophil count increase with 2-week corticosteroid therapy, also without response. the second neonate received no specific therapy for neutrophil count increase. the children were discharged for home care with clinical and laboratory control examinations at 2-week intervals. in spite of prolonged neutropenia (2 and 6 months, respectively), no other infections were recorded. discussion and conclusion: in our patients, the therapeutic approach to ann was individualized, based on standard antibiotic therapy, intravenous gammaglobulins, corticosteroids, available literature data, and our own clinical experience. although in the last few years rh-gcsf is successfully used in patients with neutropenia, we decided to postpone its use in case the neonatal sepsis developed. the reasons for such decision were: (1) the fact that both neonates were in good general clinical status, with a mild course of the disease with only short-term umbilical infection successfully managed with antibiotic therapy; (2) literature reports suggesting the unexpected failure to respond to rh-gcsf therapy in patients with neutropenia induced by anti hna-2a immunization, and (3) the unknown effect of rh-gcsf on developing tissues of the neonate. the choice and efficacy of specific therapy for neutrophil count increase in the management of alloimmune neonatal neutropenia have not yet been fully defined and require additional evaluation in the majority of cases. male donors for the production of fresh frozen plasma: a special issue for trali patients trali is a significant cause of transfusion associated morbidity and mortality, and has been reported as the third most common cause of fatal transfusion reactions. there is no good evidence on which to base transfusion support policy for patients who have experienced trali. the hypothesis that there may be patient associated factors that contribute to the risk of trali is generally accepted. for this reason it seems reasonable to try to avoid further transfusion during the period of illness. if this is unavoidable the next best solution to reduce the risk of recurrence seems to be the avoidance of using plasma containing blood components (especially ffp) as there is a high chance of positivity for leucocyte antibodies especially for those coming from female donors. as fresh frozen plasma transfusion accounts for up to half of all trali cases and as our center is the only in greece responsible for the testing and processing of blood from blood donors representing military recruits, the last two years we tried to set up a project in order to provide components from male donors on request. our donor base consists predominantly from males donors (98.7%), aged between 18 and 25 years old, with a small chance of having a positive history for transfusion the difficulty of the project consisted on the fact that these donors are assigned to military camps throughout greece, which makes difficult the on time arrival of the units to our establishment in order to be processed for the production of ffp conform the european council quality requirements. this was the main reason why, till now, all plasma produced from these donations was regarded as plasma for fractionation. the first step for implementing the new project was to evaluate the number of donations that, by minor changes on the time of arrival, could be processed for ffp production. the next step was to re-schedule the shifts of the personnel for the on time production of ffp. during 2003, 25% of donations were fulfilling the specifications for the production of ffp and with the flexibility of the schedule the 97% of them were successfully processed to ffp. during 2004, 30% of the donations were fulfilling the specifications and 97.8% of them were processed to ffp. so it is feasible to increase the proportion of male ffp by organizing better the transportation of blood from the donation sites to the blood establishment and by retaining available specialized personnel to cover the extra shifts. maximising the blood supply chain in times of shortage shortages in the blood supply chain may occur for a variety of reasons. they may be temporary e.g. due to a flu epidemic or prolonged e.g. due to the exclusion of a high proportion of donors due to new pre-donation tests or because of a lack of volunteer donors. increasing awareness of the possibility of blood shortages mainly related to increased precautions associated with the possible transmission of vcjd by transfusion has been the driver for the development of blood shortage contingency plans in the uk. in england and north wales, hospitals and the national blood service (nbs) have worked together to develop an integrated blood shortage plan (ibsp) designed to ensure that hospitals and the nbs work together within a consistent, integrated framework giving patients equal access to available blood on the basis of need. an essential element of the plan is the principle that shortages can, in most cases be avoided by reducing the current usage of blood through appropriate use programmes. the impetus for hospitals to implement these programmes was a government circular (hsc 2002/009). hospitals have embraced the circular and have recruited specialist hospital transfusion practitioners, introduced lower hb triggers, cell salvage and hospital transfusion teams and are participating in the blood stocks management scheme (bsms). audits of compliance with the circular have taken place, and a web based tool kit is available. the demand for blood has declined for the last three years, with a decrease of about 5% during 2004-05, suggesting that the drive for improvement has been successful. the shortage plan introduced in england and north wales has two key aims: that the national pool of blood is available for all essential transfusions for all patients and that overall usage is reduced to ensure the most urgent cases receive blood. the plan is structured to provide actions for the nbs and hospitals in three phases, 'normal' circumstances, reduced availability and severe prolonged shortage. hospitals should have documented emergency blood management arrangements for each of the phases. the national plan is activated when the nbs red cell stock level falls to pre-defined levels, hospitals are informed by fax that they should reduce their normal stock holding levels according to guidance in the ibsp and comply with the daily hospital usage budget. the bsms has used its knowledge of hospital inventory levels and demand to provide guidance on appropriate inventory levels for normal and reduced status, it also provides the daily hospital budget. to monitor progress against the recommendations in hsc 2002/009 hospitals will be benchmarked against a number of performance indicators. these include the presence of emergency blood management arrangements, median red cell usage for a number of surgical procedures and percentage wastage of blood. there have been no shortages within the nbs for more than six years, it is hoped that the implementation of the ibsp will help to ensure that in the unlikely event of reduced availability blood will be available to the maximum number of patients requiring a blood transfusion. w-pa-120 transfusion during disaster g klein nih, bethesda, md, usa publicity given to blood donation during wartime has created a powerful association between the need for blood and occurrence of a disaster. blood is rarely needed in excessive quantities at the moment a disaster occurs. the outpouring of blood donors, especially at the site of a disaster, often proves counterproductive. the terrorist attacks on the world trade center on september 11, 2001, with almost 3000 deaths and more than 4000 injuries, provides an instructive model. more than a million potential donors contacted blood-collection centers. hundreds of thousands of prospective blood donors crowded collection facilities and many waited for hours, often to be turned away. qualified staff were in short supply and screening errors occurred as minimally qualified staff were recruited and as collection personnel fatigued. supplies and storage capabilities were pushed to their limit. some blood was inadequately processed and stored. resources were diverted from needed apheresis collections and component preparation to whole blood collection. in the aftermath of the disaster, blood outdated and volunteer donors became disillusioned as their 'gift of life' was refused or unused. similar responses have occurred numerous times over the 60-year period since blood-donor programs were introduced. in virtually every civilian disaster in the u.s. during the past century, all the blood that was needed was immediately available from blood inventory. in only four cases were more than 100 units of blood used in the first 24 to 30 h. in 2001 in new york, the five hospitals closest to the disaster site admitted only 139 disaster victims. the new york blood center, which supplies 80 percent of blood for the city's hospitals, added 600 units to routine inventory at hospitals. the center received 12 000 telephone calls and collected more than 5000 units of blood in the first 12 h. in the area of the pentagon, the chesapeake and potomac red cross blood center supplemented hospital blood inventories within h of the disaster. meanwhile, spurred by well-meaning media and federal officials, lines of blood donors were being processed at local hospitals, makeshift collection centers, the small research hospital at nih, and at a building next to the white house. in the week after september 11, america's blood centers collected 167 000 more units of blood, and the american national red cross collected 287 000 more units than in the same period the previous year. more than 475 000 units were collected for the disaster victims, but only 258 units were used. u.s. blood collectors and federal agencies have created a disaster plan that acknowledges the need for altruistic people to volunteer for blood donation in the time of disaster and speaks with a single voice to avoid needless collection activity while harnessing the good will of well-intentioned people to supplement the ongoing need for volunteer blood donation. rehabilitation of blood transfusion service in azerbaijan cd asadov, ga huseynov and ab hagiyev institute of hematology and transfusiolo, baku, azerbaijan at the end of 80th years of the last century in azerbaijan as well as in other republics of the former ussr began process of progressive deterioration of blood service parameters. in result there was an essential reduction of prepared blood and blood components quantity, manufacture of preparations from blood's plasma has completely stopped. it is connected by that our republic experiences a heavy transition period from scheduled to market economy. after reception by azerbaijan of the sovereignty on development of a national policy the big work has been lead to areas blood transfusion and development of national rules and the standards regulating functioning of establishments of blood service. in 1996 the law about ' the donorship of blood and its components in the azerbaijan republic' has been accepted, instructions on physical examination of donors and preparations of blood and its components are authorized, and also the new speciality transfusiology has been entered into the nomenclature of medical specialities. now the national program of blood service development is developed. at drawing up of the program social and economic conditions of the country, ethnic both cultural traditions and a mental potential of the nation are considered. within the framework of this program is planned to refuse gradually a paid blood donation during the certain period of time to reach 100%s' voluntary unpaid blood donorship. however in connection with limitation of resources, the state is not capable to allocate enough of means for its realization. the big work on attraction of the international organizations has been carried out. now the project of the united nations development program (undp) 'rehabilitation of blood transfusion service in azerbaijan' is carried out at sponsor's support of the norwegian government. realization of this project will lead to reorganization of blood transfusion service in our country according to practice of the european countries. within the framework of project realization it is planned to make changes and additions to a existing law about a blood and its components donorship to bring it into accord with recommendations of the europe council. updating of the russian law 'concerning the donation of blood and blood components' on june 9, 1993, a law, 'concerning the donation of blood and blood components, ' was signed by the first russian president, boris eltsin. now, after more than ten years of market economy and democratic evolution in russia, this law was significantly changed on august 22, 2004, as shown in the following sections: 1. the development of a voluntary blood donor system. 2. removal of the upper age limit for blood donors. 3. funding for blood donations. from january 1, 2005, each level of the state power budgets for a blood donor service to supply blood products for federal, regional, or municipal hospitals. costs of these drugs and the need of prolonged growth factor treatment in these disorders. w-pa-127 can iron administration reduce peripartum blood transfusion c breymann university of zurich, zurich, switzerland the prevalence of iron-deficiency anemia in different regions of the world ranges from 12 to 43%. the increased iron requirement in pregnancy and the puerperium carry with it an increased susceptibility to iron deficiency and iron-deficiency anemia and perioperative or peripartal blood transfusion. however, if ever possible administration of blood transfusion should be avoided for several reasons which will be pointed out in the talk. infections: it is well known that various pathogens such as bacteria and virus can be transmitted by administration of blood. around 0.03% of are contaminated by bacteria such as yersinia or pseudomonas species but are not screened routinely for bacteria. in addition there is no donor screening for hepatitis a, herpes species (cmv, ebv, hhv 7, hhv 8), parvovirus b19, hepatitis g (3.4%) and tt (transfusion transmitted) virus (1.9%). numbers for positive testings for 'classic' virus such as hiv, hep. b and hep. c vary from country to country and lie around 1 : 30 000 to 1 : 5000 000 depending on quality of donor screening programs, pcr sensitivity etc. recently there is increasing evidence that even prions which cause the jakob creutzfeld disease variation ('mad cow disease') might be transmitted by transfusions. therefore the fda has determined that blood donors from countries with high prevalence of prion positive persons are not permitted to give blood in the us (e.g. donors from uk). beside infections, other well known effects of transfusion are problems due to incorrect blood or components transfused, post transfusion purpura, acute and delayed lung injury, graft versus host disease and other acute and delayed allergic reactions. beside these negative effects it was also shown that patients who receive blood transfusion liberally after operations or in icu show higher morbidity and mortality compared to patients with restrictive transfusion policy. this might be due to negative effects on immune functions and inflammatory reactions and lack of stored blood to efficiently improve organ oxygenation. for example it is known that stored blood has worse capillary perfusion and worse viscosity properties compared to fresh blood. taken together there is increasing scientific evidence that blood transfusion is not the gold standard for anaemia management and alternatives such as endogenous blood pooling and efficient treatment of any anaemia must be enforced in the clinical settings. prevention and correction presuppose reliable laboratory parameters and a thorough understanding of the mechanisms of iron therapy. in order to correctly diagnose the type and degree of anaemia, a prerequisite for selection of the proper therapy, one must first of all correctly differentiate between the relative, i.e. the physiological anaemia of pregnancy due to the normal plasma volume increase during pregnancy, and 'real anaemias' with various different pathophysiological causes. when defining the hb cutoff value for anaemia in pregnancy, the extent of the plasma volume changes with respect to the gestational age must be taken into consideration. it has been found that haemoglobin values <11.0 g/dl in the first and third trimesters, and <10.5 g/dl in the second trimester may point to an anaemic situation which should be further clarified. the first important steps for diagnosing anaemia in a pregnant patient include a thorough check of her medical w-pa-126 impact of epo treatment on transfusion requirements in myelodysplasia c gardin and p fenaux hopital avicenne, aphp, university of paris 13, bobigny, france myelodysplastic syndromes (mds) are clonal disorders of hematopoeisis, associated with bone marrow failure and an increased risk of evolution to acute myeloid leukemia (aml). despite an normal or increased bone marrow cellularity in most cases, cytopenias worsen with time due to increased apoptosis and defective differentiation of blood lineage precursors. incidence of mds increases with age and reach 15/100 000 above 70 years of age. bone marrow cytogenetics number of cytopenia and percentage of bone marrow blasts are strong predictors of survival and evolution to aml. a composite international prognosis scoring system (ipss) is used in everyday practice to guide the management of these diseases. these disorders are heterogeneous and include 'low risk' patients (less than 10% bone marrow blasts) with a prolonged evolution marked by chronic anemia, and 'high-risk' patients (excess of bone marrow blasts >10%) evolving in a short timespan with severe cytopenias, and to aml in approximately 50% of cases. at diagnosis, 80% of mds patients are anemic, with an hemoglobin level less than 100 g/l, and 80% of them will require chronic blood components transfusion, during the evolution of their disease. chronic anemia and multiple blood products transfusions are associated with an altered quality of life, clinical iron overload, and important health care costs. although transfusion practices and patient's transfusion need are variable, elderly mds patients require a mean of 10-20 units/year of follow-up, in recent surveys. therapies able to diminish or abolish the need for rbc transfusion have therefore a major role in the management of mds, as allogeneic bone marrow transplantation, the only curative therapy of these diseases, is limited to a small subset of mds patients. high-doses of recombinant erythropoetin (epo) (150-300 u/kg tiw, or a 40 000-60 000 u as single weekly dose) are typically used in low-risk mds. the response rate to epo is 25-40%, including major responses (suppression of rbc dependency or rise of hemoglobin level of more than 20 g/l). absent or low rbc transfusion needs and a serum epo level less than 500 u/l are strongly predictive of response to epo, in patients with low-risk mds. the duration of response is variable (12-20 months) in most studies, with some long-term responders. the use of higher doses of epo or its prolonged administration may be associated with higher response rates, although no randomized studies are available combination of epo and low-dose granulocyte-colony stimulating factor (g-csf) increases the response rate to 40-60%, including in patients not responding to several weeks of treatment with epo alone. two randomized trials published in 2004, compared g-csf-epo to rbc transfusions and confirmed the efficacy of this combination, and a longer survival of epo-g-csf responding patients. studies are ongoing in mds, including with darbepoetin, a modified erythropoetin with longer half-life, administered once a week. two such studies have been recently reported, (darbepoetin 150 or 300 ug/week) with response rates varying from 35% to 60% in low-risk mds. in both studies, a response to darbepoetin was observed in some patients, who failed to respond to previous treatments with alpha or beta epoetin. further assessment of the optimal dosage, administration schedule of these drugs, and validation of their likely impact on qol are required, in order to epo and its derivatives to gain acceptance in mds, due the high history and a medical examination. this procedure often lays the basis for a correct diagnosis. the current gold standard to detect iron deficiency remains the serum ferritin value. to be reliable, this requires the ruling out of an infection (chronic or acute) as a cause of the anaemia. we recommend a complete laboratory test for the exact haematological status as well as the assessment of specific chemical laboratory parameters. these should the hb level alone is insufficient to guide management. a complete work-up (ferritin, transferrin saturation) is essential, preferably with haematological indices such as hypochromic and microcytic red cells and reticulocytes, classified by degree of maturity, in particular, before parenteral therapy is given. since ferritin acts as both an iron-storage and acute-phase protein, it cannot be used to evaluate iron status in the presence of inflammation. a high ferritin level thus requires the presence of an inflammatory process to be eliminated before it can be taken at face value. if the c-reactive protein level is also raised, the soluble tfr concentration can be used, since it is unaffected by inflammation. inadequate understanding of the complex chemistry of parenteral iron administration was previously responsible for serious side effects, such as toxic and allergic reactions, and even anaphylactic shock, in particular with dextran preparations. however, the current type ii iron complexes that release iron to the endogenous iron-binding proteins with a half-life of about 6 h are not only effective but carry a minimal risk of allergic accident and overload, especially after a comprehensive pretreatment work-up. after correct diagnosis, major emphasis should be put on safe and effective treatment of anemia which again depends on severity of anemia, time for restoration and patients characteristics. today effective alternatives to oral iron only or blood transfusion such as parenteral iron sucrose complex and in selected cases also recombinant erythropoietin have been investigated and show promising results concerning effective treatment of anemia during pregnancy and postpartum. our departmental data collected over 10 years and backed by postmarketing experience in 25 countries indicate that iron sucrose complex therapy is a valid first-line option for the safe and rapid reversal of iron-deficiency anemia. w-pa-128 iron therapy in orthopaedic surgery surgery of the vertebral column, hip or knee is considered a bloody procedure (blood loss >1 l) and as a consequence represents the main indication for red blood cell transfusion in orthopaedics. because of the non-negligible residual risk of transmission of infectious agents by transfusion, but mainly because of immunologic complications induced by the administration of foreign proteins and cells, an alternative solution has been actively sought. studies have clearly shown that in patients undergoing such surgery, transfusion risk correlates inversely with pre-operative hemoglobin level. correction of even slight preoperative anemia is thus mandatory. in the elderly, iron and vitamin deficiency (b12 and/or folic acid) should be looked for as a matter of routine. we recommend the use of iron + epo whenever a rapid correction (<4 weeks) of the anemia is desirable in cases with transferrin saturation <30% and ferritin levels <200 mg/l. with this regime it is possible to collect up to 6 autologous blood units in cases of increased perioperative blood loss (e.g. double hip replacement). in the post-operative period, anemia worsens because of the existing inflammatory state. this inhibits iron absorption from the intestine and iron release from the macrophages while it affects epo function and production. there is increasing evidence that i.v. iron combined with epo induces a rapid correction of post-operative anemia. it is thus recommended to stimulate erythropoiesis by i.v. iron and epo starting on the first post-operative day and to avoid transfusions in asymptomatic patients even in cases with hb as low as 70 g/l. background: hereditary hemochromatosis is one of the most common inherited disorders in which an excessive amount of iron is absorbed from the diet and then deposited in organs. the effective treatment is the regular whole blood removal which causes erythropoesis activation and leads to decrease of iron stores. red cell apheresis is an optional method for removing of higher amount of erytrocytes in one session. we performed 262 red cell apheresis in 15 patients with diagnosis of hereditary hemochromatosis (14 ¥ c282y homozygotes, 1 ¥ c282y + h63d heterozygote) using haemonetics mcs 3p cell separator (protocol tae) in which red cells are removed from patients in 2-5 cycles; plasma and buffy-coat are reinfused. collection time, donor convenience, side effects and red cell yield were recorded and analysed. samples for hematology and iron studies in patients were drawn, analyzed and compared to baseline levels. background: the collection of 2 units of red blood cells by apheresis (drbc) has been reported to be safe and effective in increasing the yield of rbc units from a donor population. however several reports demonstrated the risk of inducing iron depletion when the interval between a drbc donation and a subsequent rbc donation is shorter than 180 days. aims: to evaluate the recovery from anaemisation and iron stores depletion after drbc donation. methods: donors who underwent drbc donation between december 01, 2002 and february 28, 2003 have been enrolled in a follow up program to monitor haemoglobin (hb), htc, serum iron and ferritin values. these parameters have been assessed on the day of donation and, thereafter 7 and 180 days after drbc procedure. donors suitable to drbc apheresis had to have: age between 18 and 60 years, weight > 70 kg, hb > 15.5 g/dl and serum ferritin between 50 and 400 ng/ml. a written informed consent about the collection procedure and the follow-up program has been obtained from all the enrolled donors. drbc collection procedures have been performed by using a mcs + (haemonetics) cell separators. results: out of 130 donors who donated drbc during the study period, only 14 males completed the follow up program and have been analysed. baseline haematological values and iron metabolism parameters were: mean hb 16.3 ± 0.5 g/dl, ferritin 85 ± 31 ng/ml, serum iron 121 ± 42 microg/dl. on day 7 mean hb was 14.3 ± 0.5 g/dl (p < 0.001). on day 180 mean hb was 15.3 ± 0.8 g/dl (p < 0.001), ferritin 53 ± 22 ng/ml (p < 0.05), serum iron 111 ± 27 (p ns). only 6 out of 14 donors (43%) had a ferritin value > 50 ng/ml. in the studied donors the collection of 2 units of rbcs induced an expected reduction of about 2 grams of hb, however only 50% of this reduction was recovered after 180 days (p < 0.001). similarly, also iron stores have not been restored after 6 months from donation, as shown by a 38% reduction in mean serum ferritin value. according to these data it appear that the amount of iron 'lost' with the donation of 2 units of rbcs (approximately 320-420 mg of elemental iron) could not be completely compensated by iron absorption from the diet intake. further data are necessary to define the risk of iron depletion after the donation of a drbc, however, at least in areas where iron intake by diet is not very high, the opportunity to prolong the interval between a drbc and a subsequent rbcs donation beyond six months or to provide adequate iron supplementation therapy should be carefully considered. background: increased transferrin saturation and/or serum ferritin have been observed in italy in approximatively 5% of subjects at first blood donation and, in these subjects, hfe mutations prevalence was 0.11 for c282y and 0.26 for h63d (velati et al., 2003) . aims: the role of the c282y mutation is well known in the patho-genesis of iron overload, whereas the role of the h63d mutation remains uncertain. the aims of the present study were first to study the main hfe mutations prevalence in a random group of repeat blood donors and second to evaluate iron parameters and iron depletion in repeat blood donors heterozygous for the h63d mutation in comparison to a population of blood donors wt/wt for the h63d mutation. methods: a total of 1165 repeat blood donors were examined in 10 italian transfusion centers (6 in northern italy and 4 in southern) for c282y and h63d mutations. out of those, 50 blood donors heterozygous for the h63d mutation and 31 wt/wt for the same hfe mutation, both groups wt/wt for the c282y, were enrolled to evaluate iron parameters and iron depletion. these two groups were similar for number of blood donations (expressed as iron loss) and for sex distribution. serum ferritin (sf) was the iron index recorded at first and second observation. results: table 1 summarizes the allelic frequencies in the 900 blood donors. table 2 reports the haematological evaluation in the 50 subjects heterozigous for h63d mutation and the 31 wt/wt for the same mutation. conclusions: these data suggest that subjects with h63d mutation of the hfe gene have, at first observation, a higher ferritin levels than subjects wt/wt. this seems to be more evident in blood donors of southern italy than in northern. blood donation induces significant reduction of the iron stores both in h63d heterozygous and in wt/wt subjects. although our observation is preliminary and restricted to a limited number of subjects, it seems worthwhile to extend the follow-up of blood donors h63d heteroxygotes or even homozygotes when available, in order to get further insights on the h63d role in iron metabolism. background: cd 43 is a sialylated glycoprotein expressed on the surface of most hematopoietic cells and has been implicated in cell adhesion and signaling. consequently the levels of soluble cd43 as well as the expression on the cell surface is a marker of cell activation. furthermore, downregulation of this molecule has been correlated with increased susceptibility to infections. the myelodysplastic syndromes (mds) are a group of stem cell disorders characterized by ineffective hematopoiesis, refractory cytopenias and an increased risk of leukemic transformation. the mds patients are often introduced to transfusions for anemia improvement and present increased susceptibility to infections. aims: we studied cd43 expression in transfusion-dependent and non-transfused mds patient in an effort to investigate mechanisms of regulation of this molecule. we also studied other activationassociated antigens in the absence of manifest infection. material and methods: forty-two patients were included in the study suffering from refractory anaemia (ra). thirty-one were males and 11 females aged 33 to 85 (median 75). twenty of them had never been transfused (group a) and 22 were regularly transfused (group b). nineteen age matched healthy individuals were used as controls (group c). cell surface antigens were detected by direct immuno-fluorescence evaluated by flow cytometer. the following mouse monoclonal antibodies were tested: anti-cd11b, anti-cd18, anti-cd43, and anti-cd45. leukocytes were gated according to cd45. we used a sensitive sandwich enzyme linked immunoassay to measure the level of soluble vascular adhesion molecule as an indicator of endothelial cell activation. the r&d elisa kit was used according to the manufacturer's instructions. results: the cd43 was found down-regulated in the transfusiondependent mds patients compared with the non-transfused ones (p < 0.001) and controls (p = 0.012). this downregulation concerned the proportion of cd43 + cells, that was lower in the transfused patients than the non-transfused (p < 0.01) and controls (p = 0.006), and the rfi (relative fluorescence intensity) value that was also lower in the group a compared to the group b (p < 0.01) and group c (p = 0.001). negative correlation was observed between the cd43 expression and cd11b (p = 0.001) and cd18 (p = 0.001). cd11b was found up-regulated in the transfused patients. the rfi value was significantly elevated in the transfused patient compared with the non-transfused and controls (p = 0.001 and 0.001 respectively) while the percentage of cd11b cells did not differ significantly between the various groups. increased expression of cd 18 was also found in the group a compared to group b (p < 0.01) and c (p = 0.008). the proportion of cd18 + cells did not differ between the various groups. the levels of immuno-reactive svcam-1 as determined by elisa were found 512.02 + 35.56 in group a, 3.42 + 65 in group b and 194.11 + 45.48 in the control group. conclusions: activated hemopoietic and endothelial cells are found in mds that may be associated to the vascular disorders found in these patients. cd43 downregulation may also be associated to increased susceptibility to infections in these patients. despite improved safety of the blood supply, allogeneic blood transfusion continues to be associated with risks that can be eliminated or reduced by autologous transfusion. preoperative autologous blood donation (pad) prevents transfusion-transmitted viral infection, red cell alloimmunization, and some adverse transfusions reactions. it may decrease the risk of postoperative wound infection because immunosuppression as a result of allogeneic blood transfusion is avoided. pad also supplements the blood supply, provides compatible blood for patients with alloantibodies and rare red cell phenotypes, accelerates erythropoiesis, and provides peace of mind to patients. as any medical intervention, pad has both advantages and disadvantages. with proper patient selection and dedicated attention to process control and quality assurance, the advantages outweigh. background: prestorage pooling of whole blood derived (wbd-pc's) buffy coat platelet concentrates (pc) is common practice in europe event-free survival was significantly better in patients who responded to epo + g-csf. we have reviewed data in 2 centers and the gfm has the intention to extend the study to a larger population in at least 10 centers in france blood components and preparations. the new law prohibits the mixing of different blood products, i.e. blood components and blood fractions. different methods are necessary for the quality control of blood components and blood preparations privileges for blood donors include: -a paid day off work on the day of blood donation and medical examination for blood donation additional paid day off work after blood donation an extra paid day off work if blood is given during vacation or on a holiday this award will be given to non-remunerated donors after 40 blood donations or 60 plasma donations. before 2005, each 'honoured donor of russia' or 'honoured donor of the ussr' had three privileges: free use of public transportation, receipt of certain pharmaceuticals free of charge, and a discount on apartment utilities previously, municipalities also could have their own blood establishments. this resulted in more than 1500 blood establishments in the russian federation. from both administrative and financial points of view, many of these are too small to be costeffective, and should be discontinued. services, and wider implementation of modern technology for blood collection, testing, processing, storage, and distribution acknowledgements: we thank ksw microtec ag, dresden/ germany for sponsoring the rfid-labels and novatech research gmbh, vienna/austria for developing the clinic-software. background: the national preparation human immunoglobulin g 5% for intravenous use (ivig) that is produced at the serbian institute for blood transfusion is used in therapy of neurological, heartand haemolytic diseases and on patients that have undergone surgery. aims: it is our aim to prove the impact of this national medical preparation human immunoglobulin g 5% for intravenous use on patients that have been infected with sepsis as a consequence of surgery. material and methods: human immunoglobulin g 5% for intravenous use (ivig) has been used in the study. the preparation is liquid, 5% stabilised with glucose of a ph value of 4.25 ± 0.25. it is used in those cases where sepsis developed after surgery. both an ivig group (n = 40) and a control group (n = 40) were viewed; the control group not being treated with ivig. the number of specimens with the ivig therapy cholecystitis is (n = 3), and the control group (n = 2); pancreatitis (n = 8) control group (n = 7); intestinal obstruction (n = 7) control group (n = 8); abdominal organ perforation (n = 5) control group (n = 8); abdominal perforate injuries (n = 12) control group (n = 13); serious abdominal interventions (n = 5) control group (n = 6). the period of hospitalisation of the patients in the ivig group was 25 ± 9 days while the period of hospitalisation in the control group was 28 ± 12 days. the mortality rate in the ivig group was 40% counter 63.7% in the control group. summary: toxic gram -negative bacteria caused synergistic damage of human tissues and generalized inflammatory responsesepsis. by using human immunoglobulin g 5% for intravenous use, in cases of severe disease, the mortality rate is significantly lowered, depending of course on the anamnesis of the patient prior to surgery and the presence of other diseases such as diabetes mellitus, neoplasma, cardiac diseases etc. background: manual production pc from buffy coats (bc) is a procedure with some consecutive manipulations. the orbisac system (gambro bct) automates the steps and we assessed its performance. material and methods: 160 pc were produced by this device and some parameters were studied. for the preparation of pc, 5 bc were pooled using the orbisac set, with an integrated filter (pall lrp6). bc pool was resuspended in the additive solution t-sol in order to obtain a final ratio plasma/t-sol 35/65. the pc was stored in a gambro elp bag. results: the average platelet count per unit was 3.41 ¥ 10e11. the platelet recovery from pooled bc was 85.12% (range 79.75%-93.21%). all products of the tested pc containing <1 ¥ 10e6 wbc (by flow cytometry). the values of ph on day 1 and 5 of storage were 7.45 and 7.43. the swirling phenomenon was good until day 5°. the average loss of haemoglobin per bc was 6.8 g.conclusions:the orbisac system is very suitable for routine pc preparation and it allows increased productivity and better standardization method for pc preparation. platelet concentrates met the requirements for leucodepleted product. increased production of plasma components from male donors background: we routinely separate whole blood (wb) after hard centrifugation into a red cell concentrate (rcc), a buffy coat (bc) and plasma (pl) by an automated expresser (compomat, fresenius). the bcs are subsequently processed into platelet concentrates (pcs) by soft centrifugation and an additional (manual) expression step. the atreus 3c system (gambro bct) eliminates several of those hand-on steps by combining them into one integrated process. a processing 'circular' bag is placed in the device and filled with the wb. while the bag is centrifuged, the system expresses pl, pc and rcc into separate containers. the rccs are subsequently leukoreduced (manually) with a filter (lr-rccs). this study was designed to evaluate the storage characteristics of the rccs obtained with a prototype of the atreus system in comparison to rccs obtained by routine procedure. methods: whole blood (500ml) was collected in top-and-bottom bags, and randomly selected to be processed by either (1) current routine or (2) atreus 3c. rccs were leukoreduced with the integrated inline filter: fresenius (routine group) or pall rc2d (atreus). lr-rccs were stored at 4°c and sampled until day 42. various in vitro measurements were performed (n = 20 per group).results: see table (mean ± sd). the lr-rccs contained significantly more leukocytes in the atreus group. despite the rbc loss in the bc, hemoglobin (hb) content was 6% lower in the atreus group, but met the requirements. in vitro storage characteristics for the rccs were similar in both groups. the atreus pcs contained 73 ± 28 ¥ 10 9 platelets in 78 ± 6 ml. although plasma volume was higher in the routine group, subsequent preparation of pcs would have resulted in an additional loss of 50 ml per unit in the control group. atreus plasma had extremely low levels of residual wbc and rbc. 0.02 ± 0.02 4.0 ± 4.2 <30 <0.001 aims: the aim of this study was to examine platelet quality of prestorage pooled prp-derived pc's for up to 7 days storage. methods: pc's were manufactured from wbd-pc's using in-line filtration of prp on day 0. on day 1, either 4, 5, 6, or 8 pc's were pooled into an elx® container using a sterile connecting device. studies were performed on days 1, 5 and 7 for the following measure of platelet quality. ph, morphology score (ms), extent of shape change (esc), hypotonic shock response (hsr), percent in surface expression of p-selectin (p-sel), phosphatidyl serine (ps), glycoprotein 1b (gp1b) and by thromboelastography of the prp (maximum aplitude, ma). results: a total of 20 pools were studied, 5 each of 4, 5, 6 and 8 pc's. the mean platelet yield was 4.3 ¥ 10e11 with a range of 2.4-7.0 ¥ 10e11. the five 8 pc's had a mean yield of 6.0 ¥ 10e11 and all maintained a ph > 7.0 on day 7. all products had less than 1 ¥ 10e6 residual wbc. platelet quality data is presented in the table. data are the mean ± 1 sd, n = 20. conclusion: platelet pools manufactured from pc's produced by inline filtered prp and stored in elx® containers show good quality preservation to day 7 over a range of platelet yields. introduction: the big progress in treatment of critically ill children significantly increases the need for blood and blood products. loss of blood (lowering of the total erythrocyte mass), as well as decreasing of oxygen capacity of blood that can influence cardiovascular function, is main indication for the erythrocyte transfusion. aim of the study: to present the number of erythrocyte concentrates (ek) that were issued to the pediatric clinic in skopje, as well as to point out how they were distributed. material and method: this is a retrospective study performed in nitm-skopje from january till may 2004. the following criteria were followed: hemoglobin (hb), hematocrit (htc), as well the clinical evaluation, and then final decision for transfusion was made.results: there were 254 blood units (ek) issued for the mentioned period to pediatric clinic for 73 pediatric patients (~3, 5% units/per child). the biggest consumers are children at intensive care unit and at the hematology-oncology unit. one unit of leukodepleted erythrocytes (er) was split equally to 3-5 bags. for small and prematurely born children and for some other selected patients er unit was filtered and irradiated. the dosage was 25-100 ml er (depends on age and body weigh). 173 ek was issued as washed concentrates, 15 ek were filtered and 25 ek were resuspended in ab plasma. distribution among abo system was the following: conclusion: gynecologic patients consumed rbc more than 4 times than obstetric ones (159 vs 44) and the number of given transfusions is high. the a blood group is the most needed one. we should insist on using the who guidelines for the proper clinical use of blood and try to minimize the percentage of given transfusion. and z. cermakova university hospital, ostrava, czech republic background: fully automated system olympus pk 80 is an immunohaematological analyser for detection of red blood cells antigens of ab0, rh (d, c, c, e, e) and kell systems without centrifugation by mam (microplate agglutination method) on unique terraced microplate olympus. in the czech republic analyser pk 80 is used only in blood center ostrava. aim: to evaluate the validity of results, sensitivity of microplate agglutinaton method, cause of abortive tests, requirements for analyst, capacity and reliability. methods: 95 880 blood samples of donors were tested between july 2002 and january 2005. all samples were analysed for ab0 blood group. 69 052 samples were tested for rhd antigen and 15 220 ones for rh (c, c, e, e) and k antigen. the validity of the results was evaluated for ab0 with parallel testing antigens and antibodies, while for rh (d, c, c, e, e) and k using two diagnostic serums. sensitivity of mam i.e. occurrence false negative or positive results were found out when results were confronted with previous ones in our data bank acquired testing classical manual tube or microplate methods. requirements for analyst were evaluated in according to demands for needful knowledge for new analyst, necessity of control pk during testing and maintenance. capacity were evaluated as a number of samples tested per day. reliability determine by occurrence disorders. results: ab0, rh (d, c, c, e, e) and k were investigated truly by first testing at 99.5% samples. two diagnostic serums anti-d olymp igm and totem differentiate directly rhd negative and rhd positive donors. false negative or positive results were not founded out due to mam or quality of diagnostic serums. about 0.5% samples with abortive tests were analysed next time the same testing or manual technique. causes of abortive tests were microagglutination several samples except for anticoagulative edta, weak solution of red blood cells prepared by analyser, damage of microplate, hemolysis due to impurity of microplate. in one case analyser evaluated false ab blood group due to hemolysis. analyser has friendly software, simple maintenance and sound control during testing, capacity about 400 samples per day and minimal occurrence of weighty disorders. conclusion: analyser olympus pk 80 is an effective alternative full automation for medium serological laboratory and together with mam easy and truly proves blood groups of majority samples with minimal necessity repetition due to abortive tests. introduction and aim of the study: the purpose of this study is to establish nested-pcr for the detection of hepatitis b virus (hbv) in blood and blood products. methods: the primer pair set was designed to amplify 513 bp in sregion of hbv genome in the first pcr and 233 bp of first pcr amplicon with rubisco (internal control) in the second pcr. to assess the specificity of pcr results, all the samples were tested cross-reactivity or interference in the assay. results: in case of hbv spiked blood products such as immunoglobulin and coagulation factors, this method could detect hbv dna up to 62.5 iu/ml. nested-pcr was compared with pcr-elisa and hybrid capture ii (hc-ii), the pcr-elisa showed a sensitivity of 96% (hc-ii; 77%) and a specificity of 98% (hc-ii; 100%) (p < 0.005). the results of the study show that nested-pcr and pcr-elisa could be used equally in the management for hbv detection in blood and blood products. p-398 blood component therapy: slow improvement a mrdja health center subotica, subotica, serbia background: transfusion department at general hospital was founded in 1953. since that time till now it has answered to all demands in blood and blood components. aim: the aim is to present development of the transfusiology department in the last 20 years, so that we could see how much of scientific knowledge we have adopted and in which direction our department goes at the moment. method: retrospective analysis of blood/component utilization in period from 1. january 1984 to 31. december 2004. results: in 1984 whole blood participated in the consumption with 84.18%, packed red blood cells with (rbc) only 5.94%, washed rbc were used in 9.87% of the cases. in 2004 whole blood participated in the consumption with 31.5%, packed rbc with 61.65%, washed rbc with 0.14% and rbc in additive solution with 6.71%. as far as plasma preparations are concerned, there has been, since 1984, a great consumption of plasma -witch was separated from whole blood in period up to five day in 97.1% cases, and small consumption of fresh frozen plasma (ffp) only 2.9%. since 2003, there has completely been cancelled the production of five day old plasma, only ffp is being used. from 1984 to 2002 for the patients who needed platelets, platelet rich plasma (prp) was prepared and applied right after preparation. the consumption rate was from 35 units to 103 units per year. in 2002, after the purchase of platelet shaker, began the production of platelet concentrates (pc) and consumption suddenly rose from 58 units in 2002 to 462 units in 2004. conclusions: it is obvious from the analysis that irrational consumption of whole blood was reduced to more acceptable values and therefore the use of component therapy got increased. variation in blood consumption and its slight increase is obvious though application red blood cells was conducted according to strict indications. in 2003 the production of plasma old up to five days was cancelled and instead we produced only ffp. pc we prepared for patients only in agreement of treating physician. although very slow progress in development of transfusion therapy in our department can be seen in accepting scientific knowledge. transfusion specialist are active participants in patient treatment and by accepting scientific achievement are able to set standards and help our colleagues, clinics, in successful hemotherapy. introduction: blood groups may act as receptors of parasites, bacteria and viruses. there is evidence that they perform a function and play a biological role. objective: the aim was to detect abo and p epithopes in ascaris lumbricoides extracts (ae) and to study the presence of immune antibodies in patients with ascariasis. materials and methods: 50 ae were prepared by refrigerated mechanical rupture of adult specimens. agglutination inhibition (ai) and haemogglutination kinetics (hk) tests were made with the ae. the patients´ abo and p blood groups were determined. we 1998 1999 2000 2001 2002 2003 2004 total febrile non haemolitic 2 3 2 3 2 4 2 5 2 4 2 4 2 9 4 34 16 male transfusion reaction 2 1 1 2 3 2 2 5 1 8 f e m a l e alloimunisation on le/hla 3 1 2 2 1 3 1 3 3 4 1 5 2 25 6 male antigens 2 2 1 2 3 3 3 3 1 9 f e m a l e kandidates of renal 4 3 4 4 5 4 6 4 6 5 10 8 12 8 10 7 57 43 male transplantation 1 1 2 1 2 4 3 14 female lupus nephritis 3 3 2 3 2 1 1 1 1 6 male 3 3 2 3 2 1 1 1 1 6 f e m a l e total 12 12 12 16 16 18 21 25 132 65 female 67 male introduction: irradiation of blood product has been in routine use to prevent graft-versus-host disease (gvhd) in certain recipients for many yeas. gamma irradiation can abrogate the ability of lymphocytes to proliferate in vitro, 1500 cgy of gamma radiation reduce lymphocyte response to mitogens by 90%.the aim of the study: 1. to estimate potassium level increment in stored irradiation blood units. 2. to compare the increment in potassium level between leucodepleted and non leucodepleted, irradiated stored blood units. 3. to evaluate the expiratory date of blood units post irradiation. the study included 40 units of blood collected in cpd-adsol (as-1). in twenty units the blood collection bag was with inline leucodepletion, while the other 20 units were non leucodepleted. all the 40 units were irradiated using caesium 137 as a source of irradiation, with a dose of 1500-2500 cgy. baseline samples from the bags were obtained for measuring of extra cellular potassium (k+). control samples included. results: there is statistically significant increment in potassium level in the irradiated samples compared to the non irradiated samples starting from 1st day post irradiation and continues to day 35 post irradiation. comparing the group of irradiated leucodepleted, with irradiated non leuconondepleted, for potassium level estimation during the days of storage post irradiation. there is no statistically significant difference between the two groups during all the days of storage, starting from base line samples and other samples post irradiation until day 35, p value of more than 0.05. 1. gamma irradiation of bloods units can cause cell damage that the use of such components needs to be modified. 2. there is a significant increment in the extra cellular potassium level in irradiated blood units that shows doubling value within 48 h post irradiation. 3. there is no significant difference in extra cellular potassium level increment post irradiation when prestorage leucodepleted units are compared with non leucodepleted units. 4. an out date of 28 days post collection (unless they expired before) for irradiated red blood units seems reasonable to ensure transfusion of irradiated units without serious complications, except in neonates and massive transfusion cases where irradiated blood units should be fresh and used within 48-72 h post irradiation. 5. the percentage of irradiated blood units requested by our physicians (0.09%) is very less that reflects the needs of physicians awareness of the indications for requesting irradiated components that can prevent serious post transfusion complications. the use of whole blood and blood components in treatment of surgical patients in ten years period was analyzed in iran. in accordance with world trend of using blood component therapy, in medical centers throughout the country in ten years period, there are decreasing trend of using whole blood from 26% (1994) to 9.37% (2003) and increasing trend of using packed red cells component therapy from 70.3% (1994) to 88. 54% (2003) . there is also increasing trend of using fresh frozen plasma (ffp) from 26.4% (1994) to 55. 2% (2003) . comparing 1994 and 2003 year, in use of blood therapy related to hospitalized patients at surgical department who received blood and patients who did not received blood; it appears that there is statistically significant difference between these two years. results: during 1 year period, a total of 5310 units of blood and 867 units of f.f.p were used. more specifically, the results can be shown in the following table 1 . a high rate of f.f.p usage is observed both in surgery and pathology clinics. the main causes of its usage are: haemodynamic disorders -volume depletion, and coagulation disorders and low blood protein, for the two clinics respectively. conclusion: the only way for rational usage of f.f.p is the regular reminding of plasma transfusion indications to the clinical doctors, so that undesirable side-effects caused by plasma transfusion will be reduced and the percentage of plasma used for fractionation will increase. acquired factor v inhibitor is extremely rare and is associated with diverse clinical symptomatology that varies from asymptomatic forms of the disease to very severe hemorrhagic episodes with a potentially lethal outcome. it may occur spontaneously or as a result of various clinical conditions. a 50-year-old man was admitted to our hospital with a diagnosis of left-sided periscrotal abscess and scheduled for an incision procedure. during the routine preoperative procedure screening coagulation tests showed pathologic values: aptt 41s, pt 35%, fibrinogen 2.8 g/l, fv 15% (other factors were in normal range), platelet count 189 ¥ 10 9 /l. factor v inhibitor was detected by a modified bethesda assay. the assay showed a low level of inhibitor of about 1.30 bethesda units (bu). the patient's medical history showed no major morbidity except appendectomy performed 22 years ago. the patient was prepared for operative procedure, with preventive preoperative administration of fresh frozen plasma (ffp) in a dose of 15 mg/kg (~1500 ml). upon ffp transfusion, repeated determination of the factor v plasma was unchanged from the initial finding (15%), indicating a failure of therapeutic response. as the measured level of factor v activity was at the borderline hemostatic level, and the operative procedure was not associated with a high risk of hemorrhage, the patient underwent abscess incision. the procedure and postoperative course were uneventful and without major hemorrhage. laboratory testing for the possible systemic autoimmune disorder produced normal findings. control examination performed two years later revealed no major clinical or laboratory variation, while a low factor v level persisted (17%) along with the presence of factor v inhibitor at a level of 1.15 bu. we have evaluated two groups of rcc's, one we routinely use (quadruple leucoflex lcr5 t/t cpd/sagm) (macopharma) and one using an automated collection device which gives the ability to collect whole blood in cpd with a rate of 7 : 1 respectively during the whole donation. the mentioned system has been evaluated using the suitable, quadruple leu-coflex lcr5 t/t cpd/sagm (macopharma). whole blood 450 ml in cpd was collected from random donors. in both groups the whole system was stored at scaled r.t. 20 ± 2°c for 2 to 5 h. after component separation (beckman coulter j6mi-optipress i-baxter), the red cell concentrates were filtered immediately at r.t. 20 ± 2°c. sampling was done after filtration and wbc measurements were determinated using nageotte champer (bright line-detection limit 0.05 wbc/ml) with leucoplate solution (sobioda). the other parameters were measured with (celldyne 1700 abbott). conclusion: all products met the accordance of national and european norms for blood components quality. leucodepletion with leucoflex lcr5 and abc leucoflex lcr5 (5.040 and 5.015) is highly efficient. the use of abc leucoflex lcr5 showed better scaled donation in terms of collection (statistical analysis mann-whitney, minitab p-value = 0.0392 < 0.05). additionally less hb-loss occurred, due to the filtration process, most probably due to the total absence of clots (analysis man-whitney, minitab, p-value = 0.0382 < 0.05). this new generation of collection gives the ability in blood services to collect well calibrated donations, indoors or outdoors. smaller quantities of donations theoretically can be valid because of the stable 7 : 1, rate of donation. the abc system gives the ability of fully traceability during donation. macopharma's blood collection bags, with or without integrated filters, provided they have this modified system of storing the ac. the device can keep records for many parameters and can transfer them to the data base server of the blood center. to evaluate the performance of the abc, we conducted a comparative study between abc leucoflex lst1 system and leucoflex lst1 system we currently use. the later is a well known macopharma's system for collection of whole blood with integrated whole blood filter and the final production of one unit of leucodepleted crcs and one unit of leucodepleted plasma. the abc was handled with the same system modified with the storage bag for the ac. issues of comparison were the accuracy in donation volume, the duration of filtration, the loss of blood in the filter and the residual wb cells after filtration. we performed 24 donations with the lst1 system and 28 donations with the abc lst1 system. all the donors were random male volunteers and they were meant to donate 450 mls of whole blood. our results analysed by mann-whitney, minitab statistical analysis have as follows:1. no significant difference was found between the two systems concerning the whole blood volume, but there was broader distribution of the values in the lst1 system compared to the abc lst1 system. 2. the duration of filtration has been found without statistically significant differences between the two systems. 3. the loss of volume in the filter of lst1 is higher than in the abc lst1 (p = 0.007 < 0.05, which is statistically significant). 4. there was very good leucodepletion with the lst1 systems (median reduction of the wb cells 4.7 log) but there was a superiority of the abc lst1 over the lst1 concerning the leucodepletion per litter and per unit (p: 0.0368 and p: 0.0398 respectively). 5. the personnel after a very short period of training accepted fully the abc procedure.in conclusion abc is an easy to handle device which provides with high quality blood products in combination with leucoflex lst1. evaluation of a post-storage filter for wbcs with an incorporated waste bag for washing rccs leucolab lcg4 is a system manufactured by macopharma for poststorage filtration of a rcc unit (with or without an incorporated waste bag for further washing of the filtered product). to evaluate the efficiency and reliability of the above system we conducted a study with twenty three random units of rccs stored in cpd/sag-m, aged 2-6 days, filtered and consecutively washed with 200 ml of normal saline, span down in the regular way and the supernatant extracted in the waste bag. issues for evaluation were:1. the duration of priming and filtering the rccs. 2. the loss of volume in the filter.3. the efficiency of the filtration. 4. the acceptance of the personnel of a new (to them) filtration system.our results have as follows:1. we counted the duration of priming and filtration. median time of priming was 90 s (range 40-300) and of filtration was 12 min (range 8-20). 2. the median volume lost in the filter (as calculated) was 23.4 ml (ranging from 22.4 to 28.1). this narrow range is apparently due to the non-flexible cell of the filter. 3. the efficiency of the leucodepletion was counted by flow cytometry. the median wbc counted per lt was 0.9 ¥ 106 (range 0.3-2.1 ¥ 106) and per unit was 0.17 ¥ 106 (range 0.05-0.5 ¥ 106). the median reduction of the wbc count was 4.2 log (range 3.9-4.7). 4. the personnel involved in the procedure found the system easy to handle, even without specific training. in conclusion the lcg4 is a reliable and easy to handle system, for leucodepleting (and washing) rccs, very efficient in removing wbcs with negligible loss of volume. objective: standardization of blood banks and establishing quality assurance are important landmarks in the new era of transfusion medicine. as the number of blood banks grows and the capacities of them changes, centralization need arises and trends to nationally coordinated blood services eventually appear. aim: to investigate the types and capacities of blood bank in the country and evaluate the statistics of them. material and method: blood banks and transfusion society of turkey conducted a nation-wide survey of a comprehensive questionnaire. this is a preliminary report of this investigation and illustrates the capacities of the most blood banks in turkey. it also guides the nationally planned renewing structure of blood banks. results: there are nearly 340 blood banks and blood stations in whole country. the overall blood collected at those centers is about 1.800.000 units. nearly one in third is being collected at government hospitals (31.5%), nearly the same is collected at university hospital blood banks (29.6%). the third major group is the red crescent society blood centers-rcsbc (23.5%), followed by the social security hospitals (12.9%). blood collecting capacities are not appearing in the same order. the major blood banks belong to the rcsbcs, whereas the small ones are mostly government hospitals. the only donor recruitment organization is run by the rcsbcs. yearly blood collecting capacity blood banks (%) >50.000 0.4 20.000-50.000 4.8 10.000-20.000 8.7 5.000-10.000 13.8 <5.000 72.3conclusion: there are many steps for improving blood safety in a country, and the prior ones are structuring the blood transfusion system and donor organization on a national basis, and then establishing good manufacturing practices. these are only possible after centralization of all existing blood banks. in our country, we should first arrange all small capacity blood banks and standardize them. controversial clinical questions considered in a medical opinion forum by physicians for the advancement of transfusion medicine (patm) patm is a newly formed group of close to 300 physicians drawn from pathology and hematology, transfusion services, hospital blood banks and blood centers. its mission is to address the concern that patient oriented medical opinion and influence has been diminished in transfusion medicine (tm). they believe that a patient oriented voice should be distinct from institutional, commercial or regulatory weight and have a common focus on patients and the therapeutics of transfusion and related therapies. therefore, their first objective is to create a new medical, patient oriented voice that weighs in on national policy pertaining to treatments related to tm. as such, patm held its first medical opinion forum where members of the group debated important questions pertaining to tm clinical practice. the forum was held just prior to the aabb annual meeting and attracted 58 physicians. the questions debated were preselected by patm membership via an email survey. respondents were asked to select their top four preferences from among 17 topics in five broad categories. the top two topics were selected for the forum from the 80 completed surveys. the subjects selected and debated were (i) what are the medical considerations for reducing the rate of mistransfusion? and (ii) what are the medical considerations for managing a limited blood inventory? the participants were divided into four groups, with each topic assigned to two groups. all the groups were given two h to debate and arrive at consensus on their topic. each group then presented a summary of their discussions along with specific recommendations for addressing these clinical practice issues. there was remarkable consensus between the groups debating the same issue. the conclusions and recommendations on these two topics will be presented in detail. patm is a new organization that will add an important medical voice and opinion on current topics in tm. at its first meeting, two topics were successfully discussed and debated with broad consensus achieved on current issues confronting the field. key: cord-010119-t1x9gknd authors: nan title: abstract presentations from the aabb annual meeting san diego, ca ctober 7‐10, 2017 date: 2017-09-04 journal: transfusion doi: 10.1111/trf.14286 sha: doc_id: 10119 cord_uid: t1x9gknd nan background/case studies: zika virus (zikv) is associated with severe neurological consequences in fetuses and adults and potential for transfusion transmission (tt). rna persistence has been reported in whole blood (wb) long after clearance of viremia in plasma, raising concerns over the risk of tt with plasma based nucleic-acid amplification testing (nat). the dynamics of zikv persistence in asymptomatic infection are not well understood and are needed for understanding of the natural history of zikv infection. we sought to characterize the dynamics of infection through prospective enrollment of zikv rna1 blood donors. study design/method: donors identified through investigational zikv nat screening were enrolled into longitudinal follow up and assessed for viral and serological persistence and clinical outcomes. plasma and rbc were obtained from index donations and blood, urine, saliva and semen samples were collected prospectively at weeks 1, 3, 6, 12 and 24 following index donations from 50 donors and detailed symptom questionnaires were administered at each study visit. blood compartments and body fluids were tested for zika rna by real time rt-pcr. plasma samples were tested for zika specific igm and igg antibodies results/finding: the percent of zikv rna1 samples, followed by the number of samples tested in parenthesis, for each sample type during each sampling interval is summarized in the table. plasma viremia declined rapidly after index donations whereas rbc-and wb-associated viral rna persisted for up to 3 months and peripheral blood mononuclear cell (pbmc) associated virus was detected intermittently at low levels and waning by 3 months. urine and saliva detection decreased significantly after 2 weeks and was undetectable by 3 months. of donors who were enrolled in the acute pre-seroconversion stage of infection 65% (15/23) developed multiple zikv related symptoms 1 week post index donation, compared to only 30% (7/27) for donors detected post-seroconversion. conclusion: zikv rna persists in cellular blood compartments for several months following clearance from plasma and body fluids, with higher rates of symptoms than previously reported. the persistence of zika rna in rbcs has unknown implications for blood screening, which currently relies on plasma testing; infectivity studies are in progress. wb testing may be of value to extend detection of acute infection and for diagnostics and monitoring of pregnant women. iron status and novel risk factors for iron depletion in a diverse donor population bryan r. spencer* 1 , yuelong guo 2 , ritchard g. cable 3 , joseph e. kiss 4 , michael paul busch 5 , grier page 2 , stacy endres-dighe 2 , steve kleinman 6 , simone glynn 7 , alan mast 8 and for the nhlbi recipient epidemiology and donor evaluation study-iii (reds-iii) 9 . 1 american red cross, 2 rti international, 3 american red cross blood services, 4 blood systems inc., 5 blood systems research institute, 6 university of british columbia, 7 nih/ nhlbi, 8 blood research institute, 9 nhlbi background/case studies: blood centers and regulators in the united states (us) are evaluating strategies for minimizing iron depletion in blood donors. the logistics of donor management might differ across blood centers, but the optimal approach may also vary according to biological or behavioral differences across sub-populations of donors. studies donors have been conducted in predominantly caucasian populations, which may differ from racial/ethnic minority donors in iron metabolism and capacity to undergo repeat phlebotomy. study design/method: over 12,600 donors were enrolled from 4 us blood centers for ferritin testing. the study population was enriched for racial minorities [1600 african-american (aa), 1600 asian (as), 1000 hispanic (hisp)] and for "super donors" (1600, who had completed 101 donations in two years without low hemoglobin deferral). the minority donors and the remaining 6800 non-hispanic white (nhw) donors were an unselected population with no specific eligibility criteria. subjects completed questionnaires on risk factors for iron depletion. logistic regression was used to identify demographic and behavioral predictors of absent iron stores (ais, ferritin <12 ng/ml) and low ferritin (lf, ferritin <26 ng/ml). results/findings: across all subjects, 19% had ais and 42% had lf, with a high degree of variability based on demographic factors and donation behavior. in models stratified by race, expected patterns common to all 4 groups included a sharp increase in risk with increasing donation intensity, and a large decrement in risk for females > 50 years old. in models including all subjects, race was an independent predictor of both ais and lf controlling for age, sex, body weight, donation frequency, and other factors (table) . aa and as donors showed %20% decreased risk for ais compared to nhw, while hisp donors had 25% higher risk. daily use of exogenous iron reduced risk for lf and ais by 30 to 40%, respectively, while the estimated benefit from less-than-daily use was lower (5 to 19% protection). regular use of antacids was associated with a 20% or greater increment to risk. reported use of hormone supplements showed opposing effects in males and females. use of oral contraceptives or estrogen in females reduced risk by %15-20%, while males who reported current use of supplemental testosterone had twice the estimated risk for ais. conclusion: this large study confirms the high prevalence of lf and ais in us donors and the principal risk factors of age, sex, and donation frequency. the diverse population studied and the questionnaire data from donors identify additional demographic and behavioral risk factors of secondary importance. in developing iron mitigation strategies, practices based on age and gender could be further refined depending on a given blood center's operational context and donor population. data are reported as mean (6sd) *p < 0.05 compared to batf31/1, 200ul hod rbcs mfi, median fluorescence intensity background/case studies: during storage, red blood cells (rbcs) undergo multiple morphological, biochemical and molecular modifications, collectively called the storage lesion. the proportion of cleared rbcs is correlated with storage duration, which may be responsible for the rapid clearance of up to 25% of transfused rbcs, reducing transfusion yield. it has been shown, using imaging flow cytometry that a subpopulation of morphologically altered rbcs accumulates during storage. the reduced surface area of these small rbcs (srbcs) suggests their rapid elimination by the spleen in the hours following transfusion. this hypothesis remains to be clarified, since the physiological mechanisms of rbc clearance remain to be precisely identified. study design/method: murine "young" and "old" rbcs (respectively on d1 and d14 of storage) were transfused into different models including splenectomized or macrophage-depleted mice. flow cytometry was used to determine the kinetics of clearance, the transfusion yield and to quantify rbcs retention in organs. the accumulation, during storage, and the posttransfusion disappearance of srbcs were analyzed by imaging flow cytometry. results/finding: using a murine model of transfusion, we confirmed that the post-transfusion yield decreases with storage duration (62% on d14 vs 100% on d1 of storage). a clearance of the storage-damaged rbcs mediated by spleen and macrophages is shown by significant improvements in post-transfusion yield observed in the splenectomized (74%) and macrophage-depleted (79%) groups. as in humans, we observed the accumulation of a subpopulation of small rbcs (mouse small rbc: msrbc) of reduced projected surface area with altered morphology. these msrbcs disappear rapidly from the circulation in control or splenectomized mice with a decrease of more than 50% at 2h post-transfusion. in contrast, in macrophage-depleted mice, msrbcs are kept in circulation at 2h posttransfusion. at 24h, these msrbcs completely disappear in all models, suggesting the importance of their elimination and the presence of compensation clearance mechanisms. in control mice, storage-damaged rbcs are mostly retained in the spleen but also in the bone marrow (bm) . no retention is observed in the liver, kidney or lung. in macrophage-depleted mice, retention is decreased in the spleen and bm. conversely, elevated retention is observed in the bm of splenectomized mice, associated with a transient retention in the kidney and liver. conclusion: during storage of murine rbcs, damaged rbcs accumulate, and are eliminated following transfusion via spleen/macrophage-mediated mechanisms. they include, as observed in humans, a subpopulation of small rbcs which undergoes a rapid macrophage-mediated clearance. the increase in transfusion yield in the absence of spleen or macrophages suggests that the recipient's functional state is one of its determining factors. age dependent relapsing and remitting autoimmune hemolytic anemia in a murine model andrea sut ling wong* 1 , amanda l richards 2 and krystalyn e hudson 2 . background/case studies: breakdown of tolerance to rbc antigens may result in development of pathogenic autoantibodies (autoab) and lead to autoimmune hemolytic anemia (aiha), a severe and sometimes fatal disease. aiha in humans has a number of known features, including increased frequency with age, and tendency to relapse and remit. however, the mechanisms behind such observations are not understood. to gain insight into tolerance (or loss thereof) to an rbc autoantigen, we utilized the hod mouse, which expresses an rbc-specific triple fusion protein consisting of hen egg lysosyme (hel), ovalbumin (ova), and, duffy (hod). hod mice were bred to a transgenic mouse that expresses a t cell receptor specific for an ova peptide in hod presented by mhcii (otii mice). thus, hod 1 otii 1 mice are predisposed to have autoreactive cd4 1 t cells. study design/method: four cohorts of hodxotii f1 mice (16-48 mice/ cohort) were bled monthly for 15 months to assess for autoab production. peripheral rbcs were stained with anti-complement (c3) and mouse immunoglobulin ab. spleens were weighed and splenocytes were stained with anti-cd71 and ter119 to assess for the presence of rbc progenitors. statistical analysis between hod 1 otii 1 autoab 1 vs. hod 1 otii 1 autoabvs. hod -otii 1 was performed using kruskal-wallis test and corrected for multiple testing with dunn's test. results/finding: otii cd4 1 t cells were not deleted in the thymus of hod 1 otii 1 mice; rather, they matured to the periphery. despite these peripheral autoreactive t cells, no detectable autoab were observed in hod 1 otii 1 . however, as they aged, 15-50% of hod 1 otii 1 were positive for rbc autoab by 6 months. thereafter, $50% of the autoab 1 mice stopped producing autoab within two months after onset and remained autoab free throughout the study. in 3 of the 4 cohorts, 60-100% of autoab 1 mice were female. hod 1 otii 1 autoab 1 mice also had enlarged spleens compared to hod 1 otii 1 autoaband hod -otii 1 mice (0.42g vs. 0.21g and 0.14g, resp., p<0.04). this may due to rbc consumption, extramedullary erythropoiesis, or both. consistent with increased erythropoiesis, elevated numbers of rbc progenitors (cd71 hi ter119 inter ) were observed in the spleens of hod 1 otii 1 autoab 1 mice but not in hod 1 otii 1 autoaband hod -otii 1 (2.86% vs. 0.06% and 0.05% resp., p<0.03). moreover, autoab and c3 deposition were found (0.1-2% and 3-10%, resp.) on ter119 1 rbcs in all of the hod 1 otii 1 autoab 1 mice analyzed. conclusion: several features known to exist in human aiha were observed, including age-dependant autoab production, relapsing of autoimmunity after onset, and an increased frequency in females. this model may serve as an experimental system to investigate the mechanisms of aiha. b5-a01a reduction in neutrophil numbers is a risk factor for rbc alloimmunization amanda l richards 1 , christopher a tormey 2 and krystalyn e hudson* 1 . background/case studies: red blood cell (rbc) alloimmunization occurs in up to 10% of transfusion recipients (excluding abo and rhd). the underlying factors that influence alloimmunization are poorly understood; thus, there is currently no reliable way to predict who will make an alloantibody and who will not. patients who receive multiple rbc units or several separate transfusions are at higher risk of alloimmunization; likewise, certain disease states have higher rates of alloimmunization, such as myelodysplasctic syndrome (mds) and sickle cell disease patients. however, despite chronic transfusions, some patients never develop rbc alloantibodies. it has been recently reported that poly (i:c)-elicited inflammation leads to enhanced alloimmunization rates and is correlated with increased splenic neutrophil (pmn) numbers. additionally, rbc transfusion into an inflamed recipient leads to enhanced erythrophagocytosis by pmns. here, we test the hypothesis that pmns regulate rbc alloantibody generation. study design/method: mice: c57bl/6 (b6) mice were treated with pbs, or anti-ly6g to deplete pmns, followed by poly (i:c) to elicit inflammation, and finally a transfusion of allogeneic dio-labeled rbcs expressing a synthetic antigen, hod (hel-ova-duffy). multiple splenic cellular subsets were evaluated for dio fluorescence, an indirect measure of rbc consumption, at 18-24 hours post-transfusion. anti-hod alloantibody generation was assessed 14 days post-transfusion by flow cytometry. humans:retrospectively, mean white blood cell (wbc) and pmn counts were collected on chronically transfused mds patients at va connecticut healthcare. for alloimmunized patients (n55), wbc and pmn counts were assessed on the day of exposure to the alloimmunizing rbc unit, whereas counts were averaged for the entirety of rbc therapy for non-alloimmunized patients (n55). patients were matched for numbers of rbc transfusions. results/finding: mice: the mfi of anti-hod antibodies was significantly increased in pmn-depleted mice, compared to controls (3/3 experiments, p<0.01). while many control mice made no alloantibody (non-responders), all pmn-depleted mice made detectable anti-hod. pmn depletion also led to a significant reduction in dio1 leukocytes, suggesting a lack of compensatory mechanism(s) for rbc consumption. absence of pmns also shifted rbc consumption from macrophages to immune-stimulating dendritic cell subsets. flow cytometric analysis revealed that pmns with internalized rbcs upregulated expression of co-inhibitory molecules (e.g. pd-l1), compared to pmns (without internalized rbcs) from the same mouse; thus, pmns may regulate alloimmunization through antigen presentation and/or inhibitory signals. humans:. alloimmunized mds patients had a significant decrease in pmns, compared to non-alloimmunized (p<0.05); no significant differences were detected in mean wbc counts between the two arms. conclusion: these data demonstrate that in both murine and human settings, pmns may play a significant role in regulating rbc alloimmunization and may provide key insights into predicting which patients will become alloimmunized. b9-a03b cxcr5 1 pd1 1 and ccr7 1 expressions characterize responders to rbc immunization benoît vingert* 1,2,3 , marie tamagne 1,2,3 , sadaf pakdaman 1,2,3 , anoosha habibi 2,3,4 , philippe bierling 1,2,3,4,5 , rachid djoudi 1 and france pirenne 1,2,3,5 . 1 efs ile de france, 2 laboratory of excellence gr-ex, 3 imrb u955 -eq2, 4 ap-hp, 5 universit e paris est background/case studies: post-transfusion alloimmunization can induce life-threatening hemolytic transfusion reaction. in human, mechanisms responsible of rbc alloimmunization are not fully defined. cd4 1 t cells are major for antibodies production. we have already shown in responder patients that the majority of anti-rbc cd4 1 t cells have a th17 profile. in contrast, in whole blood of non-responder patients, there is an unexpected expression of circulating cd4 1 t cells with a cxcr5 1 pd1 1 phenotype. this phenotype is usually associated with the presence of tfh cells, specialized in the production of antibodies. it has been suggested that some of the activated circulating tfh could have a cxcr5 1 pd1 hi profile, with a differentiated expression of ccr7. ccr7 is essential for t cells domiciliation in lymph nodes where the encounter t and b cells is major for b cell differentiation and antibody production. others chemokines receptor like ccr6 and cxcr3 can also differentiate circulating tfh subpopulations. in this study, we were interested in the phenotype and function of these cxcr5 1 pd1 1 lymphocytes which were paradoxically highly represented in non-responder patients. study design/method: the membrane and functional phenotype of the circulating cxcr5 1 pd1 1 cells were compared in 2 groups of transfused sickle cell patients : alloimmunized (n514) and non-alloimmunized patients (n510). the analysis was also performed in non-transfused healthy controls (n512). all assays were performed on whole blood without separation procedures that are known to alter the expression of chemokine receptors results/finding: the cxcr5 1 pd1 hi subpopulation expression was identical between transfused groups and controls. ccr6 and cxcr3 expressions show no difference between the transfused groups or the controls. however, in non-responder patients, ccr7 expression was very strong independently of the expression of pd1. in the aim to determine the help of the circulating cxcr5 1 pd1 1 cells in the production of antibodies, these cells were purified by flow cytometry and co-cultured for 5 days with b cells, and in the presence of seb protein. the levels of antibodies after seb stimulation were identical with the cxcr5 1 pd1 1 subpopulations from transfused groups or controls. conclusion: the paradoxical presence of circulating cxcr5 1 pd1 1 cells in non-responder transfused patients do not appear to have any particular functions that can promote the absence of a humoral response. however, in responder patients, the high expression of ccr7 on circulating cxcr5 1 pd1 1 cells suggests remarkable migratory properties towards secondary lymphoid organs, and could facilitate allo-immune responses. in conclusion, the study of the cxcr5 1 pd1 1 profile and the ccr7 1 expression in these cells could help to differentiate responder and non-responder patients to rbc immunization. primed cd4 t cells to one rbc alloantigen can enhance subsequent alloimmunization seema r patel* 1 , ashley bennett 1 , kathryn girard-pierce 1 , connie arthur 1 , amanda mener 1 , patty zerra 1 , christopher a tormey 2 , jeanne hendrickson 3 and sean stowell 4 . 1 emory university, 2 yale-new haven hospital, 3 yale university, 4 emory university school of medicine background/case studies: while red blood cell (rbc) alloantibodies can increase the probability of transfusion-related complications, not all patients become alloimmunized following transfusion. however, individuals that do generate alloantibodies appear to experience an increased rate of additional alloantibody formation following subsequent transfusion. however, how immunity to one rbc alloantigen primes immunization to a completely distinct alloantigen remains unknown. though cd4 t cell help classically occurs through direct recognition of a peptide that resides within a target b cell antigen, individuals who develop antibodies toward one rbc alloantigen experience increased rates of antibody formation against completely distinct rbc alloantigens. these observations suggest that cd4 t cells that respond to one alloantigen may directly facilitate immunity to a completely distinct rbc alloantigen. study design/method: b6 recipients were transfused with kel rbcs in the presence or absence of poly i:c (pic), followed by transfusion of hod rbcs, kel rbcs, rbcs expressing hod and kel (hod x kel), or a mixture of hod and kel rbcs (hod 1 kel). to examine the role of cd4 t cells, pic/kel primed b6 recipients were cd4 t cell depleted prior to transfusion. in addition, b6 recipients were adoptively transferred with cd4 t cells from na€ ıve or pic/kel primed donors, followed by transfusion of hod rbcs or (hod x kel) rbcs. anti-hod and anti-kel alloantibody formation was evaluated using indirect immunofluorescence staining. results/findings: kel rbc transfusion in the presence of pic (pic/kel) not only enhanced anti-kel antibody production through a cd4 t celldependent process, but this same priming event directly facilitated anti-hod antibody formation following subsequent (hod x kel) rbc transfusion (p < 0.001); pic/kel primed recipients transfused with (hod 1 kel) rbcs or hod rbcs alone failed to impact anti-hod antibody formation. the ability of immunity to kel to boost a humoral response to the hod antigen following (hod x kel) rbc transfusion required kel priming in the presence of pic. cd4 t cell depletion prevented pic/kel primed recipients from boosting an anti-hod antibody response (p < 0.0001) and transfer of cd4 t cells from pic/kel primed recipients likewise directly facilitated anti-hod antibody formation following a (hod x kel) rbc transfusion (p < 0.001). conclusion: these results demonstrate that cd4 t cells primed to one rbc alloantigen can directly enhance the immune response to a completely distinct rbc alloantigen, suggesting a mechanism whereby alloantibody responders may exhibit an increased rate of additional alloantibody background/case studies: platelet refractoriness remains a significant clinical problem, yet the mechanisms by which it occurs are incompletely understood. immune-mediated platelet clearance by anti-platelet alloantibodies plays a significant role, and patients with detectable alloantibodies can be managed with transfusion of hla-matched platelets. still, many patients are refractory even after receiving hla-matched platelets. it was shown previously that cd8 t cells can play a direct role in platelet clearance, as allogeneic platelets are cleared within 24 hours post transfusion in b celldeficient mmt recipient mice (ie in the absence of anti-platelet alloantibodies) and depletion of cd8 t cells prevents such clearance. since minor antigenic differences still exist between donor hla-matched platelets and a recipient, we hypothesized that minor antigens alone may mediate clearance of otherwise hla-matched platelets. study design/method: to test whether minor antigens can stimulate cd8 t cell-dependent platelet clearance we examined platelet refractoriness using mova and oti transgenic mice. leukoreduced donor platelets from mova mice, which express a membrane-bound form of chicken ovalbumin and thus present ovalbumin peptides complexed with murine mhc class i h2kb, were labelled in vivowith the fluorescent dye cfse and transfused into wildtype (wt, c57bl/6) mice or oti mice, whose cd8 t cell receptors recognize a specific ovalbumin peptide in the context of mhc class i h2kb. in some experiments oti mice were primed with mova or wt splenocytes one week prior to mova platelet transfusion, and in others wt mice were adoptively transferred with oti splenocytes 48 hours before mova platelet transfusion. platelet recovery was measured immediately after transfusion as well as after 2, 4, 8, 16, and 24 hours and on days 2-5. results/finding: transfusion of mova platelets into oti mice results in significant platelet clearance as compared to transfusion with wt platelets. clearance kinetics demonstrate platelet loss starting after 8 hours and peaking at 24 hours, and are similar whether oti mice are na€ ıve or previously primed with mova splenocytes. specifically, mova platelet recovery in oti recipients is 28-35% versus >60% in wt recipients at 24 hours (p<0.05), whereas transfusion of wt platelets into either oti or wt recipients is approximately 60% at 24 hours after transfusion. adoptive transfer of oti cd8 t cells into wt mice recapitulates the effect, with significant mova platelet clearance at 24 hours compared to wt platelet clearance (p<0.05). conclusion: this work extends the ability of cd8 t cells to mediate platelet clearance to a minor antigen, providing insight into the potential etiology of platelet refractoriness in patients receiving hla-matched products. this study also holds implications for the clinical management of any nonantibody-mediated platelet refractory patient, as therapies directed toward immunomodulation of t cell responses may prove beneficial. background/case studies: alloimmunization against major histocompatibility (mhc) antigens is a common complication of transfusion, and can negatively impact subsequent transfusions and transplants. we have previously demonstrated that pathogen reduction with riboflavin and uv light (uv1r) is effective both at rapidly killing donor white blood cells (wbcs) and at blocking their ability to stimulate an allogeneic response in vitro. furthermore, uv1r treatment of allogeneic platelet rich plasma (prp) prevents alloimmunization in mice, and provides partial antigen-specific tolerance to subsequent transfusions. as cells that die through different pathways can be either tolerizing or inflammatory, we sought to determine which cell death pathways are triggered by uv1r, as well as evaluate the immunogenicity of prp containing wbcs killed by other methods. study design/method: wbc-rich prp was prepared from c57bl/6 mouse blood and treated with uv1r, and wbcs prepared in parallel from the same blood were treated with known inducers of either apoptosis or necrosis. membrane integrity, phosphatidylserine exposure, caspase activity, and chromatin condensation were evaluated by flow cytometry. balb/c recipients were transfused with either uv1r treated wbc-rich prp, or uv1r treated wbc-poor prp either alone or with added untreated, apoptotic, or necrotic wbcs, all generated from allogeneic c57bl/6 donor blood. a second transfusion of untreated wbc-rich c57bl/6 prp was given 2 weeks later, and alloresponses were compared against mice given no transfusion or only the second untreated transfusion. results/finding: uv1r treated wbcs have a pattern of phosphatidylserine exposure and loss of membrane integrity consistent with early apoptosis, but fail to demonstrate significant caspase activity or clear chromatin condensation. alloantibody responses to transfusion were significantly higher in mice previously exposed to untreated (p<0.01) or necrotic (p<0.05) wbcs, but not those given uv1r treated or apoptotic wbcs. ex vivo cytokine responses to stimulation with c57bl/6 wbcs were reduced in recipients of either uv1r or apoptotic wbcs, and enhanced in recipients of untreated or necrotic wbcs. conclusion: the mechanism of wbc death following uv1r treatment shares some membrane characteristics of early apoptosis, but is distinct from classic apoptosis. however, both uv1r treated and apoptotic wbcs fail to trigger an alloresponse, and offer some protection against subsequent alloexposures. background/case studies: in mitochondria-less red blood cells (rbcs), oxygen is the main substrate for oxidative reactions and resulting oxidative damage is considered as one of the major causative factors in the development of rbc storage lesion. oxygen saturation (so 2 ) of venous blood is generally assumed to be around 70-80% as measured from a central venous line. however, a recent investigation of so 2 levels in freshly prepared leukocyte-reduced red cell concentrates (lr-rccs) revealed unexpectedly wide so 2 distribution (mean 45.9%617.5% [yoshida et al. 2017; blood transfusion 15, 172] . the present study was undertaken to determine the distribution of so 2 in lr-rcc produced at a medium-size blood center using a novel non-invasive so 2 probe. additionally, quantitative metabolomics were carried out to examine the redox status of the stored rbc under various so 2 levels. study design/method: the so 2 from 977 units of lr-rcc were examined on five consecutive days representing 78% of the collected units during the period at a regional blood center where all the units were processed at room temperature within 8 hours of blood collection. so 2 was measured noninvasively through the pvc bag immediately prior to refrigeration by employing a resonance raman spectrometry (pendar microvascular oximeter a3u11; pendar technologies, cambridge ma). in addition to so 2 , process methods, rcc volumes, blood types, gender and process times were recorded for analyses. in a separate study, lr-rcc (n54) from human volunteers were stored in as-3 under normoxic, hyperoxic, or hypoxic conditions for up to 42 days (so 2 ranging from <3 to >95%) prior to uhplc-ms metabolomics analyses in presence of 13 c, 15 n or deuterated internal stable-isotope labeled standards for absolute quantitation. results/finding: measurements of so 2 carried out non-invasively at a blood center yielded a similar wide distribution as previous study from 497 units of lr-rcc procured and sampled invasively within 24 hours after blood collection [yoshida ibid]. the shape of the so 2 distribution appeared near normal with the mean of 47.0%621.0%, median 45.2%, range < 5% to > 95% and inter-quartile range (iqr) of 31.4%-61.9%. male donors showed higher so 2 compared to female donors (p<0.04). no correlations were observed between so 2 levels and processing time, donor age or blood types. metabolomics workflow indicated that lower so 2 levels ameliorate the energy and oxidative metabolic lesion. lower so 2 levels yielded higher rate of gsh synthesis, higher nadph concentration, higher gsh / gssg and nadph/nadp 1 ratios, lower supernatant urate consumption and lower purine oxidation. the surprisingly wide distribution of starting %so 2 levels was observed from lr-rcc manufactured at a blood center using 8-hour room background/case studies: cellular prion protein (prp c ) is a gpi-anchored cell surface glycoprotein that is expressed mainly in the brain but also in peripheral organs including blood, bone marrow (bm), and lymphoid tissue. prp c can be converted post-translationally into scrapie-prp (prp sc ), which is involved in the pathogenesis of neurodegenerative diseases including creutzfeldt-jakob disease, kuru in humans, and scrapie and bovine spongiform encephalopathy in animals. however, biological functions of prp c have yet to be conclusively elucidated. study design/method: in this study, prp c knockout mice (ko) are utilized to investigate the role of prp c in the hematopoietic system with controls of age and sex-matched prp c transgenic mice harboring a slightly augmented prp c expression. peripheral blood was examined by hematology analyzer to establish counts. bone marrow, thymus, spleen, lymph nodes, and peripheral blood were harvested and analyzed by flow cytometry using a comprehensive panel of fluorochrome-conjugated antibodies specific for all hematologic cell precursors/ lineages. histology of bone marrow, spleen, thymus and lymph nodes were evaluated by light microscopy. results/finding: complete blood count (cbc) showed a significant increase of wbc in ko mice. closer analysis of wbc differential revealed that the elevated number of wbc in ko mice was due to lymphocytosis. specifically, ko mice had a 3-fold increase in the absolute lymphocyte count (ko 7.59 6 4.63 x10 9 /l vs. wt 2.90 6 1.32 x10 9 /l, p 5 0.0303), as well as a higher lymphocyte percentage compared to controls. ko mice also had a trend toward higher hemoglobin, rbc, and hematocrit compared to wt mice. additionally, platelet count in ko mice was higher than control mice. of interest, the mean platelet volume indicating platelet size was significantly increased in ko mice compared to controls (ko 6.00 6 0.29 fl vs. wt 5.24 6 0.56 fl, p50.0140). a comprehensive flow cytometric analysis of all cell lineages revealed no significant differences in the numbers of rbc and megakaryocyte in bm, and of lymphocytes in the thymus, spleen and lymph nodes. histological analysis of bm, thymus, spleen and lymph node tissue from ko and wt animals failed to show morphological differences between the two groups. conclusion: absence of prp c resulted in significant leukocytosis and specifically higher absolute count and percentage of lymphocytes, as well as larger platelets in peripheral blood, but does not appear to affect hematopoiesis and lymphopoiesis. our findings indicate that prp c might be critical in the survival and trafficking of lymphocytes in peripheral blood. the molecular mechanisms underlying the observed changes in lymphocytes and platelets, and whether these involve functional changes in these cells will be subject of future studies. potential role of cd81foxp31 regulatory t cells derived exosomes in their immune modulation yiming yang*, rufeng xie and jie yang. blood engineering laboratory, shanghai blood center background/case studies: exosomes are defined as one type of membrane vesicles secreted into extracellular space by most types of cells and are reported to involve in intercellular communications, mediate biological process. human periphery blood cd8 1 foxp3 1 tregs cells are reported as more stable regulatory cells with greater inhibition effects. however, cd8 1 foxp3 1 tregs derived exosomes and their functions involved in cd8 1 tregs mediated immune-modulation were seldom reported. study design/method: cd8 1 t cells were freshly purified from pbmcs, cultured with anti-cd3/cd28 antibody packaged beads and il-2, and then polarized with tgf-b and rapamycin into cd8 1 foxp3 1 treg cells. the harvest cells were co-cultured with cd3/cd28 beads stimulating cd41cd25effector cells in the transwell plate. the supernatant derived from cd81 tregs was collected and ultrafiltrated by centrifugation and the remaining solution was precipitated with peg. the harvest precipitation was resuspended in pbs and exosomes were analyzed by sem and nta. exosome surface marker cd63, cd81, tsg101 and other proteins expression were evaluated by flow cytometry and western blot. microrna was isolated with mircute mirna kit and mir-155, let-7b, let-7d were measured by qpcr. the precipitated exosomes were further purified by cd63 immunoaffinity capture and co-cultured with effector cells to investigate their function in immune modulation. results/finding: as compared with direct contact co-culture, separated cd8 1 treg cells could suppress the proliferation of effector cells with a small decline (p>0.05), which means some non-contact factors involved in the cd8 1 treg mediated immune modulation. a total number of 4.57 6 0.52 3 10 8 /10 6 cells exosomes were harvest. electron microscope analysis demonstrated a kind of round-shaped membrane vesicle 50-150nm in diameter (145.16 6.7nm by nta). cd63 and cd81 were expressed on these background/case studies: regulatory t cells (tregs), containing cd4 1 and cd8 1 subtypes, play an essential role in immune regulation and autoimmune disease prevention which makes it a potential candidate for cell therapy on autoimmune disease (aids). unfortunately, due to the instability of natural cd4 1 foxp3 1 regulatory t cells (ntregs) in inflammation conditions (including instability of foxp3, conversion to pro-inflammatory effector cells and was unable to modify established disease), thus, it is needed to investigate cd81 regulatory t cells stability both in vitro and in vivo. in our previous works, we found that cd8 1 treg has an effective therapeutic function on cia mice. in this study we aim to investigate the stability of induced polyclonal human cd8 1 regulatory t cells in inflammation and transfusion. study design/method: human cd8 1 tregs were induced with tgf-b1 and rapamycin from cd8 1 t lymphocytes in vitro. collagen-induced arthritis (cia) mice were induced with type-two collagen as an autoimmune disease model. in vitro the stability of cd8 1 tregs when encountering with inflammation were test by foxp3 expression, th1 and th17 cells conversion in inflammations conditions (il21tgf-b11il211il23 and il21tgf-b11il1b1il6) on day3, day6 and day9. in vivo, cd8 1 tregs were transfused into cia mice and then their survival in mice and foxp3 express were evaluated to reveal the stability of cd8 1 tregs in an inflammation condition model. additionally, we also investigate the stability maintenance of cd8 1 tregs when induced factor tgf-b1 and rapamycin were removed by testing the foxp3 expression on day3, day6 and day9. results/finding: ex vivo induced human cd8 1 treg were foxp3 1 (90.40 6 1.40%) and did not secret il17a (both in supernatant and % of cells). foxp3 express in cd8 1 tregs were maintained after induced factor tgf-b1 and rapamycin were removed on day3, day6 and day9. in vitro, foxp3, il2 and ifn-c expression has no significant difference when compared with controlled tregs on day3, day6 and day9 and did not secret il17a when encounter with inflammation conditions (il21tgf-b11il211il23 and il21tgf-b11il1b1il6). in vivo, cd81 treg cells were transfused into cia mice on the peak of disease onset (35 days after the first collagen immunization, has inflammation condition in vivo) to test cd8 1 tregs survival. cd8 1 tregs were found in cia mice foot (27.4 6 2.03%), blood (4.55 6 1.03%) and spleen cells (1.90 6 0.05%) 72 hours after transfusion and their % of foxp3 1 were remained. conclusion: the results revealed that ex-vivo induced and expanded human cd8 1 tregs are stable in inflammation and transfusion and can maintain foxp3 expression when induced factor were removed, these make cd8 1 treg a novel and stable cell for potential cell therapy on aids. this research can provide some instructive reference and improve the utilization of blood components. tolerogenic dendritic cells induced by mtor suppression and control inflammation in chs model through s6k related proteins translation inhibition. li gao*. shanghai blood center background/case studies: tolerogenic dendritic cells (tdcs) adoptive cellular immunotherapy is a cutting edge strategy for treating hypersensitivity response disease, in which immune responses are directed against selfantigens, such as atopic dermatitis, systemic lupus erythematosus (sle), rheumatoid arthritis (ra),et al. however, the traditional strategy base on the tdcs was usually unstable and inconspicuous through cytokines inducing processing so that might be the limitations on tdc adaptive cell therapy in future clinical use. study design/method: human tdcs were derived from fresh purified monocytes from pbmncs isolated from buffy coat and induced by mtor inhibitors (rapamycin and temsirolimus) in safe concentrations confirmed by apoptosis assay when the cells were completely differentiated. the mature markers and endocytisis were detected by flow cytometry. the production of cytokines and chemokines was measured using elisa. mechanism investigation was analysis by real-time pcr and western blotting. contact hypersensitivity (chs) model, an atopic dermatitis animal model, was treated with tdcs induced via mtor suppression and analyzed by ear thickness and tissue leukocytes number calculating. results/finding: human tdcs treated with mtor inhibitors had a lower mature marker cd83/cd80/cd86 expression after tlr signaling activation, accompanied with a set of cytokines and chemokines remarkably downregulated in a concentration dependent manner but not the lps absent group. moreover, mtor suppression extremely reduced the capacity of lps treated human dcs to stimulate autogenic na€ ıve t cell proliferation, which is one of the most important characteristics of tdc. beyond expectation, the common signal transduction pathway, mapk and nf-jb pathway, were not the signal target so that it could hardly be the explanation for the tolerogenic performance of tdc when exposure to lps stimulation. however, the p70s6k and its downstreanm proteins, especially the protein s6, which controls the protein translation, were shown in charge of the tolerogenic induction mechanism. the data were also supporting the suggestion that rare difference on mrna transcription of the related functional proteins in tdcs induced by mtor inhibitors when exposure to lps stimulation from the non-induced cells, although there was more transcription of ido induced by mtor inhibitors. more important, edema responses of ears were clearly weakened in the chs model and recruited less leukocytes to the tissue when co-sensitized with mtor inhibitors or with tdcs induced by mtor inhibitors suggested that the tdc induced by mtor suppression were able to control hypersensitivity inflammation response in vivo. conclusion: accordingly, tdc induced by mtor suppression is a potent adoptive cellular immunotherapy strategy for treating hypersensitivity response disease and the induction mechanism of it might be through suppressing systematically effective function proteins by mtor-s6 related protein translation inhibition. xiaoyun fu* 1,2 , mikayla anderson 1 and james c zimring 1,2 . 1 bloodworksnw research institute, 2 university of washington school of medicine background/case studies: red blood cells (rbcs) undergo many changes when stored under blood banking conditions, collectively known as the storage lesion. bioactive lipids generated during rbc storage have been implicated in certain adverse outcomes. recently, we reported that bioactive lipids, especially polyunsaturated fatty acids (pufas) and their oxidized products (oxylipins) accumulate during rbc storage despite leukoreduction. to evaluate the extent of membrane lipid degradation and oxidation in stored rbc units among the donor population with different blood groups, we quantified pufas and lysophospholipids (lpls) in 405 leukoreduced rbc units. study design/method: rbc units from different donors were acquired and processed on day 43 (one day past their expiration). 35 bioactive lipids including 10 common fatty acids, 10 oxylipins, and 15 lpls were analyzed by liquid chromatography-tandem mass spectrometry with multiple reaction monitoring (lc-ms/ms-mrm). total fatty acid concentrations of selected units were also analyzed. a one-way anova test was used to determine significant difference of analytes amongst the different blood groups. results/finding: we observed a wide distribution in concentration of major pufas in 405 stored rbc units. for example, arachidonic acid (aa) ranges from 0.5 -10.7 mm, linoleic acid (la) (1.4-28 mm), dihomo-c-linolenic acid (dgla) (0.1-0.8 mm), eicosapentaenoic acid (epa) (0.03-3.1 mm), docosahexaenoic acid (dha) (0.2-3.0 mm), and alpha-linolenic acid (ala) (0.06-2.3 mm). ten oxylipins including hetes, hodes, and dihomes, and 15 lpls including lpcs, lpss, and lpes all showed a large variation in concentration among donors. of 35 analytes quantified, 25 showed a significant difference in concentration among different blood types by one-way anova testing (fdr<0.05). the ab rh1 blood group consistently exhibited the lowest concentration of major pufas, while the o rh-blood group showed the highest, averaging a two-fold difference in concentration (o rh-/ab rh1). the fold increase of o rh-/o rh1 among pufas ranges from 1.3 to 2.1, suggesting the rh blood group, independent of the abo blood group, correlates with donor to donor variation in lipid metabolism. conclusion: the wide distribution in the concentration of bioactive lipids among 405 stored rbc units suggests that lipid degradation is highly donor-background/case studies: to ensure availability of biological products to hospitals, blood banks have developed and validated multiple storage conditions for each of their products to maximize shelf life and quality. in the case of labile products, their metabolism is known to remain active during storage, leading to storage lesions. micrornas (mirnas) levels are modulated by these storage-related damages, which makes mirnas ideal candidates as potential biomarkers of quality monitoring. lately, nanoparticles have been widely studied and used for biosensing applications. the objective of this work is to develop biocompatible gold nanosensors for sensitive, selective and direct detection of biomarkers to characterize and assess the quality of blood products delivered to hospitals. study design/method: gold nanoparticles (gnps) surrounded by a fluorescent silica shell were prepared using a wet chemistry method. mirna-223 was chosen as a potential target, since it is strongly expressed in platelet concentrates and its concentration fluctuates according to storage lesions. custom rna and dna molecular beacons were designed and used as a probe for the specific detection of mirna-223 targets in pbs and human plasma. these fluorescent transducer probes were conjugated at the surface of fluorescent silica shell-gnps using an edc/nhs cross-linking reaction. the hybridization reaction between the target and the probe initiates an energy transfer mechanism which can be recorded by fluorescence. results/finding: gnps (49 6 6 nm) surrounded by a thick fluorescent silica shell (22 6 2 nm) were prepared and used as nanosensors because of their optimal luminescence properties and long-term stability. conjugation of the probe onto the nanoparticles was confirmed by fluorescence spectroscopy and microscopy, as well as nanoparticle tracking analysis. the fluorescent response of the molecular beacons was studied and showed a reproducible and linear relationship (r 2 rnaprobe 5 0.96 and r 2 dnaprobe 5 0.98) with mirna-223 concentration, down to a 10-nm limit of detection. hybridization assays in 1% human plasma appear to demonstrate denaturation of rna probes and targets. conclusion: biocompatible fluorescent gnps were prepared and used as tools for blood product characterization. the conjugation of a molecular beacon at the surface of nanoparticles was achieved and characterized using spectroscopic and microscopic techniques. the functionalization of the probe is still being optimized. the fluorescence response of the molecular beacon was characterized for the detection of a model mirna target in pbs and in 1% human plasma. energy metabolism profile of erythrocytes during storage suping ren*, qun yu, yanbing wang, changlan li and yu wang. background/case studies: the moment the mature red blood cells (rbcs) leave the bone marrow, it is optimally adapted to perform the binding and transport of oxygen and its delivery to all tissues. red blood cells modulate oxygen transport, protect hemoglobin from oxidant-induced damage, and maintain the osmotic environment of the cell. glycolysis is the only energetic metabolic pathway for mature rbcs to obtain atp which is the energy for rbcs to maintain a number of vital cell functions. generally, the current methods used to measure rbcs glycolysis are not in living state in realtime, or are destructive to cells or require radioactivity.xf technology can be applied to different types of cells, in which the red blood cells are suspended and the cell shape and size are different from other cells, and more importantly, rbcs have no nucleus, mitochondria and other organelles, so application of the xf technology in erythrocytes and exploration of the assay conditions are necessary. 6.7 6 0.0 a 6.9 6 0.0 6.7 6 0.0 a 6.6 6 0.0 6.7 6 0.0 a 6.9 6 0.0 total atp,lm/ghb 7.6 6 0.3 a 5.2 6 0.3 7.3 6 0.2 a 5.5 6 0.3 7.3 6 0.4 a 4.9 6 0.5 extracellular lactate,mm 5 6 1 a 6 6 1 7 6 0 8 6 0 6 6 0 6 6 1 extracellular glucose,mm 32 6 1 a 50 6 3 2 9 6 1 a 38 6 1 2 5 6 0 a 28 6 1 extracellular na 1 ,mm 142 6 2 143 6 2 138 6 1 a 137 6 1 144 6 1 141 6 1 extracellular k 1 ,mm 1 6 0 a 4 6 0 1 6 0 a 5 6 0 1 6 0 a 4 6 0 a p<0.05, paired t-test b intercept blood system for red blood cells is not approved for commercial use. c this project has been funded in whole or in part with federal funds from the dhhs; aspr; barda; contract no. hhso100201600009c. background/case studies: pathogen inactivation methods for platelet concentrates are increasingly being used in blood banks worldwide to make transfusion safer. in vitro studies have demonstrated the effects of pathogen inactivation on storage lesion, but little routine quality control data on blood banking outcomes have been reported. study design/method: swirling of distributed products was monitored one year before and one year after implementation of intercept pathogen inactivation. metabolic parameters like ph, glucose and lactic acid were determined in a random sample of expired pathogen inactivated products. furthermore, indicators of platelet storage lesion were measured in apheresis concentrates with premature low swirling and compared to controls with normal swirling. results/finding: in an experimental phase on a limited number of products (n56) to validate the intercept pathogen inactivation method, ph and glucose levels decreased faster in apheresis platelet concentrates with high platelet content than with low platelet content or than in pooled buffy coat derived products. once pathogen inactivation was implemented, routine products showed glucose exhaustion more often when prepared by apheresis compared to buffy coat derived platelet concentrates despite more plasma carryover in the former. furthermore, the number of apheresis products with premature low swirling increased by 50% (63/12,492) compared to the previous year without pathogen inactivation (42/12, 931, p50.030, chisquare) . in contrast, the incidence of premature low swirling in platelet concentrates prepared by the buffy coat method decreased (2/15,286 vs 10/ 13,488). of note, apheresis concentrates with premature low swirling had a significantly higher median platelet count (5.0x10 11 ) than unaffected controls (3.5x10 11 ) and showed signs of increased storage lesion compared to controls expiring on day five without swirling defects. these signs included lower ph, higher lactic acid concentration, increased mean platelet volume, phosphatidylserine exposure and alpha-degranulation. conclusion: the risk of increased storage lesion rates following intercept pathogen inactivation is higher for apheresis than for buffy coat derived platelet concentrates, especially when platelet content is above 5.0x10 11 . in vitro quality of single dose amotosalen/uva treated platelets in 35% plasma/65% pas-3 after 5 days of storage crystal stanley 1 , marguerite kelher 2 , nero evero 1 , melissa vongoetz 3 , betsy donnelly 3 and anna erickson* 3 . 1 belle bonfils memorial blood center, 2 university of colorado, 3 cerus corporation background/case studies: the interceptv r blood system for platelets is fda approved for the ex vivo preparation of pathogen-reduced amicus o apheresis platelet components (pc) in pas-3 to reduce the risk of tti, including sepsis, and to potentially reduce the risk of transfusion-associated gvhd. registration studies (clinicaltrials.gov nct02298842) are in progress to support approval of the trima o apheresis platform for collection of platelets components (pc) suspended in pas-3 and plasma. the objective of this study was to evaluate in vitro function of platelets suspended in 35% plasma/65% pas-3, collected using the trima platform, after treatment with the intercept blood system for platelets. study design/method: double dose apheresis pc, 7.5 6 0.6 310 11 platelets in 602 6 52 ml, were collected on the trima apheresis platform in 35% plasma/65% pas-3. a sample was taken from each donation prior to dividing the donation to produce intercept treated apheresis pc (t), using the small volume (sv) set, and an untreated control pc (c). input volumes for replicates, n56, were 302 6 26ml (t) and 300 6 27ml (c) with doses of 3.8 6 0.3 3 10 11 (t) and 3.7 6 0.3 3 10 11 (c). all pc were stored under the same conditions and evaluated on day 5 and day 7 for physical/metabolic characteristics. results/finding: on days 5 and 7 all t and c pc had ph228c !6.2. the dose recovery for t was 87%63%. on day 5, t had lower count, volume, dose, bicarbonate and glucose compared to c pc; however, parameters predictive of in vivo function (atp, morphology score, hsr, and esc) were equivalent between t and c (table 1) . conclusion: trima pc in 35% plasma/65% pas-3 treated with the inter-cept blood system for platelets using the sv set and stored for 5 days retained in vitro metabolic and functional properties consistent with in vivo functionality. induction of pluripotent stem cell-derived cardiomyocyte toxicity by supernatant of long term-stored red blood cells in vitro feng-yan fan 1,2 , yang yu 1 , li-ping sun 1 , shu-fang wang 1 , rui wang 1 , lei-ying zhang 1 and deqing wang* 1 . 1 the department of blood transfusion, the pla general hospital, 2 the department of blood transfusion, air force general hospital, pla background/case studies: recently, multi researches have reported that longer term-stored red blood cells(rbcs) units were associated with increased risks of clinically adverse events, especially in critically ill patients. however, other studies have concluded the negative results. whether rbcs storage duration was associated with increased risks of clinically adverse events is uncertain and had become a popular topic. to study the adverse effects of longer term-stored rbcs directly, we aim to look at the pluripotent stem cell-derived cardiomyocyte toxicity induced by supernatant of suspended red blood cells(ssrbcs), and study the possible mechanism. study design/methods: 1 five doses of leuko-reduced rbcs were prepared, and supernatant was isolated by centrifugation on d0, d14 and d35. we looked at the cardiotoxicity of ssrbcs on human-induced pluripotent stem cell-derived cardiomyocytes (hips-cms). hips-cms were treated with ssrbcs in 17% final volume simulating the large volume blood transfusion. using real-time cellular analysis (rtca) technology the beating of hips-cms was recorded in real time in detail. 2 levels of k and lactic acid (la) were tested using automatic biochemical analyzer.3 k and la solution with concentrations being consistent with ssrbcs were prepared and cocultured with hips-cms. we analyzed the cardiotoxicity of k and la solution on hips-cms. 4 treated hips-cms with d35 ssrbcs, d35 k and cell culture media for 48h. the nuclear shape and integrity of filament and sarcomere was examined by immunofluorescence. total rna of hips-cms was isolated and mrna analysis microarray was implemented. screened for toxic effects related signaling pathways through bioinformatics analysis. results/findings: 1 d0 ssrbcs had no obvious influence on beating state of hips-cms-hips-cms treated with d14 ssrbcs stop beating, but beating patterns restored at 48h. hips-cms treated with d35 ssrbcs stop beating, and beating patterns did not restored at 48h. 2 levels of k and la in ssrbcs changed most obviously. 3 only d35 k solution made hips-cms stop beating and can restore in 48h; d0 k, d14 k and la solution did not influence the beating pattern in at the end of the treatment for 24h, hips-cms treated with d35 ssrbcs show obvious shrinkage. at the end of the treatments for 48h, cells treated with d35 k and d35 ssrbcs both show obvious shrinkage, the shrinkage in d35 ssrbcs group was more serious. the immunofluorescence results show the integrity of filament and sarcomere was complete and no nuclear pyknosis was detected. 5 gene expression array results show a total of 140 genes were differentially expressed in d35 ssrbcs group compared with naive group. there was no consistent separation within the d35 k and naive group. fifteen differently expressed genes were selected with bioinformatics method which were likely to play an important role in the cytotoxic effect. under the condition of simulating the large volume blood transfusion, ssrbcs of long term-stored rbcs have toxic effect on myocardial cells. 2 in addition to high potassium that induced cardiotoxicity, there must be other elements are involved in the toxic effects. 3 further study should be applied to signal pathways on ssrbcs induced cytotoxicity. 4 large volume transfusion of long term-stored rbcs may be a risk factor for adverse clinical outcomes, and clinical should pay attention to it. background/case studies: processing thawed, deglycerolized red cell concentrates (rcc) in a functionally closed system allows for a prolonged storage after thawing. thawed cells are better maintained in as-3 as compared to sagm. the presence of citrate in as-3 seems to be necessary to prevent hemolysis of thawed cells. during storage in as-3, atp and 2,3-dpg levels rapidly decline. recently developed additive solutions like pag3m and as-7 have shown to better maintain 2,3-dpg and atp levels during storage of normal, unfrozen, rcc. however, most probably due to the absence of citrate, these solutions are not suitable for storage of thawed cells. we therefore designed pag3c in which the mannitol of pag3m was replaced by citrate. the aim of this study was to investigate the in vitroquality of thawed, deglycerolized rbc during storage at 2-68c in pag3c. study design/method: leukoreduced rcc (n56) in pag3c (phosphate, adenine, glucose, guanosine, gluconate, citrate) were stored at 2-68c. on day 8, rccs were glycerolized using acp215 (haemonetics v r , braintree, ma) to a final concentration of 40% (w/v), frozen and stored for at least two weeks at -808c. after thawing and deglycerolization using acp215, rcc were resuspended in pag3c. during storage at 2-68c, stability (hemolysis), atp and 2,3-dpg levels were determined. results were compared with thawed rcc (prefreeze storage in sagm, n58) resuspended in or sagm (n54). results/finding: pre-freeze storage in pag3c resulted in increased 2,3-dpg levels at day 8 as compared to storage in sagm, resp. 9.1 6 7.6 mmol/g hb and 1.9 6 0.7 mmol/g hb. hemolysis during post-thaw storage in pag3c remained below 0.8% for 35 days and was comparable with storage in as-3. in sagm, hemolysis remained below 0.8% for 7 days. during the first 2 weeks of post-thaw storage in pag3c, both atp and 2,3-dpg levels increased, followed by a gradual decline during prolonged storage. during the whole postthaw storage period, rccs in pag3c showed significantly higher atp and 2,3-dpg levels compared to as-3 or sagm. while in sagm and as-3, 2,3-dpg levels were undetectable after 7 days post-thaw storage, in pag3c, 2,3-dpg levels only decreased to 6.1 lmol/g hb after 35 days of storage. conclusion: pre-freeze storage in pag3c resulted in increased 2,3-dpg levels. as compared to as-3, post-thaw storage in pag3c showed comparable hemolysis while atp and 2,3-dpg levels were much better maintained. based on a maximum allowed hemolysis of 0.8% and an atp content of >2.7mmol/g hb, thawed rcc can be stored at 2-68c for 35 days in pag3c. background/case studies: platelets (plts) are vital for effective treatment of hemorrhage. cold (48c, 4c) storage of plts in platelet additive solution (pas) is a promising alternative to conventional storage at room temperature (rt) due to a lower risk of bacterial concerns, preservation of plt function, and mitigation of plt activation. currently only 2 apheresis (ap) and pas systems are fda-approved for use in the us: trima and isoplate-pas (iso; terumo) and amicus and intersol (int; fenwal) . the goal of this study was to assess the adhesive function of long-term cold-stored plts collected by fda-approved ap/pas methods. study design/method: plts were collected (n54-5) in 65% iso using a trima or in 65% int using an amicus and stored for 15 days at rt and 4c. samples were tested on day 1 (baseline, bl), 5, 10, and 15 of storage to assess plt adhesion under shear flow (bioflux). acd vacutainer tubes were collected from donors and centrifuged to obtain red blood cells (rbcs) for all bioflux runs. simulated whole blood was created by combining plts labeled with calcein-am with rbcs at 40% hct. labeled blood was perfused through microfluidic channels (fluxion) coated with 100 ug/ml type-1 collagen at 720s -1 shear rate. images were acquired every 30 sec for 10 min using a fluorescent microscope and % surface coverage was reported. data were analyzed using two-way anova and posthoc tukey test with significance at p<0.05. results/finding: both rt-int and rt-iso plts showed significantly decreased adhesion by day 5 of storage compared to bl (bl: 11.6 6 1.7%, rt: 4.9 6 1.2%; p<0.005). 4c-int samples showed no difference in adhesion at any timepoint compared to bl-int but significantly enhanced adhesion compared to both rt-int and rt-iso. in contrast, 4c-iso plts showed significant enhancement of surface coverage compared to bl-iso by day 5 (p50.03) and compared to 4c-int by day 10 (p<0.01). conclusion: our work suggests that 4c storage of plts collected with a trima ap system in iso for up to 15 days offers a significant enhancement in adhesive function compared to plts collected with an amicus system in int and stored at 4c. these results are surprising since both 4c-int and 4c-iso have been shown to express similar levels of cd62p, pac-1, and phosphatidylserine and may suggest differences in pas plt intracellular signaling. as expected, storage at 4c of plts collected on either platform demonstrated superior function to rt storage. a plt product with superior hemostatic function and a shelf-life 3x longer than the current standard-ofcare provides the potential for shipment of products to underserved areas and may bolster plt availability for trauma care in the us. table 1. comparisons of white blood cell counts and percentages of apoptotic cells in whole blood components after 2-week storage between unirradiated and irradiated groups (n 5 29) tang, is an anti-inflammatory agents and has a good safety records in clinic. it could reduce the severity of experimental autoimmune encephalomyelitis (eae), asthma, colitis, systemic lupus erythematosus(sle) and other immune diseases.however,its potential in inducing transfusion tolerance remains to be explored.the aims of our study are to find if baicalin could inhibit red blood cell (rbc) immunization and to elucidate the possible mechanism of yin-chen-tang in preventing hdn. study design/method: we used human red blood cells with adjuvant lipopolysaccharide (lps) and transfused mice to induce antibodies, as an experimental system to study the effect of baicalin on rbc immunization. mice were divided into a normal control group, a human rbc transfused positive control group receiving human rbc and lps intravenously weekly for five weeks, a control group receiveing dexamethasone (10mg/kg/day) intraperitonealy daily for five weeks,a treatment group receiving baicalin (250mg/kg/day) intraperitonealy daily for five weeks. assessment of human rbc immunization was performed by measuring serum immunoglobulin g (igg) and immunoglobulin m (igm) against human rbc weekly. and the lymphocyte changes in spleen are also monitored by flow cytometry. results/finding: we found that baicalin treatment decreased serum igg but not igm production significantly since the second week, with a concomitant reduction in th17 cells and increase in cd4 regulatory t cells in both spleen and mesenteric lymph nodes. and there are no significant differences in the percentage of th1,th2,tfh and tfr cd4 subpopulation among all groups.in addition, baicalin treatment didn't decrease the size of spleen and the percentage of cd4 positive cells in spleen in baicalin treatment mouse but in dexamethasone treated mouse. our results indicate that baicalin could inhibit rbc immunization especially igg production without the damage to the function of spleen,while dexamethasone as a wildly used immune-suppressive drug in blood transfusion could damage the function of spleen.considering its good safety records in clinic, it may be exploited for suppressing transfusion immunization events. in addition, our results elucidate the inhibitory effect in antibody production of baicalin may be a possible mechanism for yin-chen-tang as a widely used chinese herbal medicines in preventing hdn. comparison of immucor's pak plus and pak lx assays for the detection of human platelet alloantibodies randy m schuller* 1 , sarah kloss 1 , sara crew 1 and sandra j nance 2 . 1 american red cross, 2 american red cross and american rare donor program background/case studies: alloantibodies directed against human platelet membrane glycoproteins (gp) ia, iia, iib, iiia, ib, ix, iv, and cd109 have been implicated in several clinically significant disorders such as fetal and neonatal alloimmune thrombocytopenia (fnait), post-transfusion purpura (ptp), refractoriness to platelet transfusions, and passive transfer of antibodies in donor plasma. polymorphic epitopes on these gps give rise to 28 unique human platelet antigens (hpa). identification of the specific platelet alloantibody is crucial in diagnosing and treating these bleeding disorders. currently the only 510k fda approved test permits the identification of these hpa antibodies to the glycoprotein level. immucor has recently released pak lx, a research use only (ruo) assay in the united states that has the ability to identify hpa antibodies to a single nucleotide polymophism (snp). we compared the performance of pak lx to the fda approved immucor pakplus. study design/method: we compared pakplus and pak lx results from 40 plasma and serum clinical specimens. group 1 contained a single hpa alloantibody specificity with or without hla antibodies (n526). group 2 included 5 specimens with hla antibodies alone and group 3 consisted of 9 patient samples that were negative for both hpa and hla antibodies. pak lx utilizes a luminex bead based assay which allows the user to report antibodies to the platelet specific antigen (hpa-1, hpa-2, hpa-3, hpa-4, hpa-5, gpiv) and hla class i. pakplus uses an elisa method and results can only be reported to the glycoprotein location (gpiib/iiia, gpia/ iia, gpib/ix, gpiv) along with hla class i. however, based upon the pattern of reactivity observed in the pakplus and pak lx assays it is possible to determine the most probable hpa antibody specificity to the hpa snp. results/finding: conclusion: when analyzing hpa antibody specificity, there is 100% concordance observed for hpa-1a, hpa-1b and hpa-5b antibodies. the pak-plus assay had difficulty discriminating hpa-5b from hpa-5a antibody when hpa-5a antibody was present (3 false positive samples) although the pak-plus signal od to cutoff od ratio was significantly higher for hpa-5a when compared to hpa-5b in these samples. the discordant hla class i antibody results between the assays was isolated to very weakly positive antibody (within 10% of the cut-off for pakplus and <2.0 adjusted ratio for pak lx). we conclude that pak lx is an easy to use platelet alloantibody screening method that has the ability to differentiate hpa antibodies to the allele level. histo-blood group antigen lewis y promotes cell migration via regulation of microtubule acetylation huijun zhu* and ping lu. shanghai blood center background/case studies: blood group antigens are critical for transfusion practices as antibodies raised against them can cause severe transfusion reaction. beside this, blood group antigens themselves are composed of sugar chains, proteins, lipids, etc, which may be involved in various biological processes. lewis y is a histo-blood group antigen belonging to abh family. ley consists of carbohydrate chains which may play important roles in cell recognition, adhesion as well as migration, which are all critical steps in tumor progression and thus attracts wide researches focusing on its relevance in tumor biology. ley is demonstrated to affect cell mirgration via various mechanisms. however. although changes in cytoskeleton organization is the basis for cell motility, little is known about the association between cytoskeleton and ley. as microtubule and its construction unit tubulin participate in various steps of cell migration, we aim to explore the role of ley in microtubule and cell migration using breast cancer cells, which may provide reference to clinical study of other histo-blood group antigens and change the way of thinking in transfusion practice. study design/methods: we first manipulate ley expression in breast cancer cells by overexpression or sirna knockdown of fucosyltransferases, and block ley activity in mda-mb231 cells using anti-ley antibody, to verify the effect of ley on cell migration. then, we detect acetyl-a-tubulin level change as microtubule acetylation is a sign for stability. to establish the role of ley in cell migration via microtubule modification, we use hdac6 specific (tubacin) and nonspecific (tsa) inhibitors to minimize deacetylation of acetyl-atubulin and test again the effect of fut1 overexpression on cell motility. results/findings: fut1 overexpression increases both ley expression and cell migration, while fut1 knockdown leads to the opposite. ley activity blockade by anti-ley antibody also significantly inhibits cell migration. western blot and immunostaining results show a-tubulin acetylation level is negatively related with ley expression. tubacin or tsa treatment increases the acetyl-a-tubulin level while inhibits cell migration; in the meantime, the significance of fut1 overexpression in promoting cell migration is eliminated. conclusion: it can be concluded from the results above that ley can promote cell migration via regulation of a-tubulin acetylation, wherein ley may have interaction with deacetylase hdac6. as tumor promoter, hdac6 becomes the target of many anti-cancer drugs. we demonstrated the potential association of ley and hdac6 function in this study. many blood group antigens are also carbohydrate chains, which are not only critical in blood group determination, compatible transfusion and immunological reaction, but may also have an effect in the initiation and development of diseases as tumor, similar to ley; they can even be components in a network with other important molecules and contribute to the destiny of diseases. transfusion of blood products is frequently needed by tumor patients. most attention is focused on the search of compatible blood for reducing transfusion reaction. however, it may lower the chance for the disease to advance to take account background/case studies: reducing the risk of bacterial contamination in platelet (plt) products is of great concern since plt storage occurs at room temperature (rt). pathogen reduction technologies (prt) were developed to inactivate pathogens prior to transfusion; however, studies have shown that prt may damage plts over the course of extended storage at rt resulting in a greater loss of function than what is normally concomitant with platelet storage lesion. storage of plts in platelet additive solution (pas) at 48c helps to preserve plt function and reduces the risk of contamination. in this study, we established the impact of prt performed after long-term coldstorage of plts in pas, instead of before storage, on plt function, mitochondrial respiration, and cell death parameters. study design/method: plt units were collected in pas (n53) and stored at 48c for up to 10 days. after this time period, the bag was treated using mirasol prt (riboflavin and uv). samples were obtained and tested on the day of collection (baseline, bl), pre-mirasol (pre), post-mirasol (post), and 30 minutes post-mirasol . aggregometry (adp, collagen, trap), rotem, flow cytometry (cd62p [p-selectin] , lactadherin [ps] , , and gpib), high-resolution respirometry (oroboros), and imaging flow cytometry (amnis) were used for analysis. data are reported as means6sem, and paired student's t-tests were used to determine statistical significance (p<0.05). results/finding: on day 10, p-selectin levels were significantly higher in pre than bl (p50.03). mirasol treatment caused a significant increase in pac-1 expression compared to pre (pre: 10.5 6 3.1%, post: 28.1 6 4.7%; p50.04), which remained after incubation. a significant drop in both collagen and trap aggregation was observed in post samples compared to pre, but adp aggregation response was preserved. no differences in p-selectin, gpib expression, and mitochondrial respiration were observed between pre and post samples. post-30 samples displayed significantly less function, higher activation levels, and lower mitochondrial respiration compared to pre and post. conclusion: prt treatment of plt units in pas after 10 day storage at 48c presents a unique alternative to prt treatment of plts prior to rt storage. in addition to providing a lower risk of bacterial contamination, 48c-stored pas plts may provide better preservation of hemostatic function than standard-of-care rt plts, even after mirasol prt treatment. however, we show here that mirasol prt of day 10 4c-stored pas plts followed by incubation (30 minutes or more) results in widespread cell damage and should be avoided. safety evaluation of lyophilized canine platelets in a model of coronary artery bypass graft (cabg) todd m. getz* 1 , arthur p. bode 1 , anne s hale 2 , michael stanton 3 , mark johnson 4 and g. michael fitzpatrick 3 . 1 cellphire, 2 bodevet, inc, 3 cellphire, 4 background/case studies: cellphire has completed a micro dose clinical safety trial using lyophilized human platelets. cellphire also evaluated the safety of lyophilized canine platelets (lcp) in comparison to liquid stored canine platelets, following intravenous administration in a model of on-pump coronary artery bypass graft (cabg) in the canine. this safety study was in support of a future phase ii human clinical trial in cardiac patients. study design/method: three groups of eight mixed breed hounds underwent cabg to create an anastomosis and were administered lcps equal to 33, 10, and 3.3% of the total circulating platelet count (tcpc). one group of four animals served as the vehicle group which received lyophilization platelet-formulation buffer, and another group of four animals received control (2-day old liquid-stored platelets). safety was assessed through the collection of blood loss data, evaluation of blood flow through the bypass graft, evaluation of the development of acute thrombosis, and maintenance of patency through the graft over the 4 hr evaluation period. full necropsies with complete tissue analysis were also performed. efficacy signals were evaluated through the collection of blood loss data and coagulation endpoints (pt, aptt, fibrinogen, and teg). the results demonstrated that administration of the test article at doses up to 33% of the tcpc was not associated with any unexpected mortality, adverse changes in hematology or coagulation parameters, development of thrombosis at the anastomosis sites, or evidence of adverse thrombosis formation either clinically or microscopically regardless of group. the mortality noted on study was considered to be related to the surgical model and not a result of test article administration. the results also demonstrated that administration of doses of 10% and 33% of the tcpc produced a significant decrease in blood loss. the lcps at 10% and 33% tcpc were as effective in mitigating blood loss as 2-day old liquid-stored platelets and trended towards being more effective. no appreciable differences in coagulation parameters were observed between groups. conclusion: the results of the study demonstrate that administration of lcp up to 33% of the tcpc was safe in a canine cabg model. the data also demonstrate that administration of lcp at doses of 10% and 33% of the tcpc reduced blood loss. these results suggest a starting dose above 3.3% tcpc may be required to achieve an effective dose in future human phase ii trials in cardiac patients. although the study was not powered for efficacy, these data indicate a level of safety, as 10% tcpc had similar efficacy signals as 33% tcpc with no observable severe adverse events. the starting effective dose may vary depending on the clinical indication. future studies will be required. this study was funded under barda contract hhso100201300021c. the study on pcr-ssp technique for the genotyping of cd36 329-330del.ac mutation and the genetic polymorphism of cd36 329-330del.ac in chinese population lilan li* and guoguang wu. nan-ning institute of transfusion medicine background/case studies: cd36 (platelet glycoprotein iv, scarb3) is an important and characteristic platelet antigen implicated in immune-mediated thrombocytopenia in chinese population. except anti-hla, anti-cd36 is the most common antibody of clinically relevant platelet antibodies in chinese population, which is associated with the high frequency of cd36 deficiency in china. cd36 gene mutation is the main reason that leads to cd36 deficiency. cd36 329-330del.ac (frameshift at aa110) mutation is one of the cd36 mutations that causes cd36 deficiency. have had natural mumps, measles and rubella infections, resulting in lower antibody levels in their blood. the recommendations may thus be unfounded and outdated, and prevent valuable vaccination opportunities for children with frequent blood transfusions. this places an already highly vulnerable pediatric population at risk for acquiring preventable infections. the primary aim of this project was to determine mmr vaccination immunogenicity in patients chronically transfused with rbc. study design/method: medical charts were reviewed for vaccination and transfusion histories. mmr-specific antibodies were quantified in 28 pediatric patients who received both doses of the mmr vaccine at 12 and 18 months of age while they were on a chronic rbc transfusion program for sickle cell disease, b-thalassemia major, diamond-blackfan anemia or pyruvate kinase deficiency. there was no formal control group; long-term immunity rates in the literature are !90% for all mmr components. results/finding: table 1 shows immunogenicity to vaccine components. delays between vaccination and serology testing averaged 6.8 years (0.5 to 16.5 years). thirteen patients (46%) were chronically transfused at the time of serology. twenty-three patients (82%) seroconverted to at least one of the vaccine components. conclusion: to the best of our knowledge, this is the first study designed to measure the effect of rbc transfusions on mmr vaccine immunogenicity. although lower than the rates reported in the literature, the results suggest a high rate of immunogenicity to each component of the mmr vaccine in chronically transfused patients immunized prior to 6 months posttransfusion. weighing the risks and benefits of disease prevention in a highly vulnerable population, and taking into account the aforementioned results, a reevaluation of immunization delays post rbc transfusions is called for in chronically transfused infants. post-vaccination serology should be considered. cold stored uncrossmatched whole blood can be safely administered to pediatric trauma patients christine m leeper 1,2 , franklyn cladis 2 , richard saladino 2 , darrell triulzi 3 , barbara a gaines 2 and mark yazer* 1 . 1 university of pittsburgh, 2 children's hospital of pittsburgh of upmc, 3 institute for transfusion medicine background/case studies: the use of uncrossmatched cold stored whole blood (wb) is becoming increasingly popular in the initial resuscitation of trauma patients without a current abo group. wb has advantages over conventional component therapy including greater platelet and factor concentrations, as well as less saline and additive solution compared to an equivalent volume of reconstituted whole blood. this report details the initial use of wb in pediatric trauma patients. study design/method: pediatric trauma patients !3 years old and !15 kg with evidence of hemorrhagic shock were eligible to receive up to 20 cc/kg of cold stored, leukoreduced group o negative wb during their initial resuscitation. all wb units had a low titer of anti-a and -b (<50) to reduce the likelihood of hemolysis in non-group o recipients. biochemical markers of hemolysis were measured on the day of wb transfusion and the following two days. admission thromboelastograms were obtained and repeated as necessary during the resuscitation. after receipt of the maximum quantity of wb, conventional components were utilized. results/finding: in approximately 11 months, 15 trauma patients received wb: 7 group o and 8 group a recipients, 53% male, median (iqr) age was 11 (4.5-14) and 73% blunt trauma mechanism. patients were severelyinjured with a median (iqr) injury severity score of 36 (22-51) and 47% mortality rate. the median (iqr) quantity of wb transfused to group o recipients was 21.9 (14.8-24.3) ml/kg versus 13.4 (9-18) ml/kg to non-group o recipients. no transfusion reactions were reported. the mean 6 standard deviation haptoglobin concentrations for non-group o recipients was 51.3 6 14.4 mg/dl on day 0, 86.3 6 36.8 mg/dl on day 1, and 126.9 6 45.8 mg/dl on day 2; the corresponding haptoglobin concentrations for group o recipients were 51.4 6 38.0 mg/dl, 84.7 6 61.5 mg/dl, and 134.8 6 68.3 mg/dl, respectively (p>0.42 for all comparisons). similarly there were no significant differences in total bilirubin, ldh, creatinine, and potassium at any time point. regarding evaluation of cold platelet function, we compared the subset of patients who received wb but no warm platelets (n57) to a historical group of pediatric trauma patients who received conventional components including warm platelets (n514). the mean 6 standard deviation platelet volume administered was 112 6 24 cc for whole blood recipients versus 147 6 68 cc for warm platelet recipients. when pre-and posttransfusion teg and platelet counts were analyzed, there was no difference in median platelet count or teg maximum amplitude (ma) between cold and warm platelet groups. conclusion: use of cold-stored uncrossmatched whole blood for the resuscitation of pediatric trauma patients is feasible, acceptable, and appears to be safe. receipt of low titer group o wb did not lead to detectable hemolysis amongst the non-group o recipients. given this finding, the maximum quantity of wb per patients will be increased to 30 ml/kg. identification of red blood cell antibodies in human breast milk by novel adaptation of serological method philippe p pary*, alexis leonard, lauren hittson, naomi lc luban, deepika s darbari, yunchuan delores mo, cyril jacquot, valli criss and jennifer webb. children's national medical center background/case studies: human breast milk contains immunoglobulins that are present in maternal serum and secretions. data in mice has demonstrated the potential for kell antibodies to be absorbed enterally from breast milk and impact the survival of transfused kell positive cells; however, methods to test and titer human breast milk for red cell antibodies are lacking. a two week old infant with a history of rh-d hemolytic disease of the fetus and newborn (hdfn), previously treated with intravenous immunoglobulin and phototherapy, was referred for anemia and reticulocytosis. patient was o positive, positive direct antiglobulin test (dat) 41 with anti-human igg only, and a 31 positive antibody screen by gel method. antibody identification showed anti-d in both the plasma and eluate. patient was transfused o negative red cells and discharged. over several weeks, the patient returned twice for persistent anemia requiring additional transfusions. at eight weeks of age, evaluation showed a persistent dat igg reactivity concerning for continued antibody exposure. maternal breast milk was evaluated as a potential source. study design/method: based on similar properties of human breast milk and plasma, testing to identify igg antibodies using a stantard tube saline method was performed with a 60 minute 378c incubation, followed by 3 automated washes prior to the addition of anti-human igg reagent. as a control, breast milk from an o positive, antibody screen negative mother was used to assess for interference by milk proteins. antibody screens were performed on the plasma of the patient, the patient's mother and the control concurrently using the same method. antibody identification and titers were also performed when indicated. only freshly collected breast milk stored at room temperature for less than 3 days was found suitable for this technique. results/finding: the patient's mother showed plasma anti-d with a titer 4096 and the breast milk showed anti-d with a titer between 16 and 64. the patient had a consistent plasma anti-d titer of 8. the patient's mother chose to stop breast feeding after 8 weeks, and the patient's hemoglobin was improved at 12 and 16 weeks of age. using this method, we identified two additional cases of breast milk induced hemolysis: another anti-d and an anti-jka. conclusion: testing showed that it is possible to identify red cell igg antibodies in human breast milk using a standard tube saline method. we identified implicated antibodies in the breast milk received by infants with persistent anemia due to hdfn. breast milk titers were generally lower than maternal serum titers, but titers varied depending upon the timing and frequency of breast feeding. cessation of breast feeding correlated with improved hemoglobin in affected infants. background/case studies: red blood cell (rbc) transfusion is lifesaving for patients with sickle cell disease (scd), but is commonly complicated by rbc alloimmunization. despite transfusion protocols serologically matching for c,e, and kell antigens, alloimmunization to rh antigens continues. scd patients often exhibit a hybrid rhd-ce-d gene which is often characterized by the production of a partial c antigen. it has been previously documented that 30% of c1 scd patients from the west indies and west and central africa are partial c and at (30%) risk for alloimmunization to the c antigen through transfusion of c1 rbcs. this study sought to determine the prevalence within a cohort of children with scd at a u.s comprehensive scd center. study design/method: rbc genotyping results performed on all scd patients using precisetype hea array (immucor, norcross, ga) at children's healthcare of atlanta were reviewed and compared to the serologic type for rh (c/c, e/e) antigens. the prevalence of c-antigen positive patients (serologically) was determined overall, and compared to the prevalence partial c antigen based on the detection of the rhce*ce(733g,1006t) allele in the absence of an rhce gene encoding a conventional c antigen in trans, since this allele is commonly linked to the hybrid rhd*diiia-ce(4-7)-d gene which encodes the partial c antigen. review of the blood bank information system was performed to identify the number of c-antigen positive transfusion exposures and frequency of alloimmunization to the c antigen. results/finding: out of a total of 255 patients with genotype/rh phenotype data available, 78 (30.6%) were c antigen positive serologically. the allele frequency of rhce*ce(733g,1006t) was 0.071. in total, 15 (5.9%) patients possessed rhce*ce(733g, 1006t) in the absence of conventional c gene in trans. of the 78 c antigen positive patients, 15 individuals (19.2%) were predicted to be partial c based on four molecular profiles [rhce*ce(733g, 1006t)/rhce*ce:12; rhce*ce(733g, 1006t)/rh*ce:1; rhce*ce(733g, 1006t)/rh*ce(733g):1; rhce*ce(733g, 1006t)/rh*ce(733g, 1006t):1]. in these 15 partial c patients, no anti-c alloantibodies (or other rh antibodies) were detected after 60 transfusion exposures (57 c-antigen negative units; mean: 4, range: 0-36), likely from placement of a c-negative rbc restriction upon detection of the rhce*ce(733g, 1006t) allele. conclusion: this report confirms previous data of a high prevalence of the partial c antigen in scd patients historically typed as c-positive serologically, and demonstrates the benefits of rbc genotyping to prevent alloimmunization to a highly immunogenic rh antigen by identifying individuals who should receive c-negative blood. all patients with scd should have rbc genotyping performed for determination of their rbc phenotype, preferably prior to receiving transfusions. investigational detection of zika virus rna in us blood donors paula p sa a* 1 , megan l nguyen 1 , melanie c proctor 1 , david e krysztof 1 , gregory a foster 1 , erin k sash 1 , sandy s dickson 1 , joua yang 1 , jeffrey m linnen 2 , kui gao 3 , jaye p brodsky 4 and susan l stramer 1 . 1 american red cross, 2 grifols diagnostic solutions inc., 3 grifols diagnostic solutions, inc, 4 quality analytics, inc background/case studies: zika virus (zikv), an emerging flavivirus, is primarily transmitted by infected aedes aegypti mosquitoes, but recent outbreaks have revealed non-vector transmission routes including the unprecedented sexual transmission of an arbovirus. acute zikv infection is mainly asymptomatic or presents as a self-limited disease but also includes severe congenital defects and neurologic disorders. the large proportion of asymptomatic cases, high numbers of returning travelers from zikv-active areas, severe clinical consequences to developing fetuses, the detection of rna in asymptomatic donors during the french polynesia epidemic, and 4 suspected cases of transfusion transmission in brazil led fda to release guidance documents to minimize the risk of zikv transmission via blood/ blood components. study design/method: investigational testing by mini-pool (mp)-nat using the procleix zika virus assay (tma) was implemented on collections from five presumed high-risk us states on 6/20/16 (fl, ga, sc, ms, al). following revised guidance on 8/26/16, testing was extended to all blood donations; conversion from mp-nat to individual donation (id)-nat was implemented in phases and completed on 12/12/16. travel history questions were discontinued on 1/23/17. confirmatory testing included repeat tma; in addition, rt-pcr, serology and red cell (rbc) tma were performed. estimates of viral loads were performed by end-point tma on plasma and rbcs. results/finding: as of 4/8/17, 2,288,855 donations were tested including 393,713 (17%) in 24,611 mps. no reactive donations were identified by mp-nat. of the 1,895,142 id-nat donations, 72 were initial reactive (ir) of which 8 (11%) confirmed positive (cp) by subsequent testing (cp rate of 1:286,107; positive predictive value of 11%; specificity of 99.997%). five (62%) cp donations were id-nat repeat reactive (rr); 3 (38%) donations were id-nat ir only, igm positive and rna positive in rbcs. cp donors resided in ma, tx, ca, ny, wv and 3 in fl, 2 of which were local transmissions. six donors had traveled to a zikv-active area returning to the us from 2 to 73 days prior to donation. two donors with a travel risk reported clinical symptoms; 6 cp donors (75%) remained asymptomatic. zikv rna was detected in rbcs from all cp index donations with estimated levels varying from less than 40 copies (c)/ml to about 8ê 5 c/ml. at the time of writing, the longest period of detection in rbcs was 91 days vs. 17 days in plasma from the same tma-rr donor. zikv rna levels in plasma were obtained from 1 ir and all rr donors, ranging from 12 to 2000 c/ml. study design/method: plasma from blood donors were screened by individual donation (id-nat) for the presence of zikv rna with the cobasv r zika test. id-nat1 samples were repeated in duplicate and further tested by a second nat to confirm infection and estimate vl, and for anti-zikv igm. simulated mps of 6 were prepared by diluting nat1 plasma 1:6 and tested to discriminate id-nat only detectable donations. nat yield samples for which simulated mp and conclusive igm results were available (n5308) were sorted into 4 categories corresponding to sequential stages of acute zikv infection: igm-/low vl; igm-/high vl; igm1/high vl; igm1/low vl. results/finding: of 52,942 donations collected april 3-december 31, 352 were reactive for zikv rna. igm-index donations had higher vls (mean 1.1 x 10 6 vs 8.3 x 10 4 iu/ml) and higher proportions of simulated mp-detectable results (93% vs 23%) than igm1 donations. the distribution by stage of infection was evaluated as the epidemic evolved. over the course of the epidemic, the rates of id-nat only detectible and igm1 donations increased (table 1) . conclusion: this study demonstrates how the viral and immunological profiles of zikv infection in the index donations shifted through the course of the 2016 pr epidemic. categorization of index samples into stages of infection is important for blood safety considerations, since infectivity and utility of mp vs id-nat screening likely correlate with vl and serological stages of infection. staging of infections also has implications for diagnostic testing and understanding the durations of zikv viral and immunological markers in blood and persistence of zikv in body fluids and tissues. cobasv r zika is not commercially available for blood screening. data generated under the cobasv r zika ind is preliminary and has not been reviewed by fda. this project has been funded in whole or in part with federal funds detection of zika virus rna in united states blood donations using cobas v r zika on the cobas v r 6800/8800 systems lisa lee pate* 1 , phillip c williamson 2 , michael paul busch 3 , susan rossmann 4 , scott jones 5 , ann butcher 1 , john duncan 1 , jean stanley 1 and susan a galel 1 . 1 roche molecular systems, inc., 2 creative testing solutions, 3 blood systems research institute, 4 gulf coast regional blood center -sugar land, 5 qualtex laboratories background/case studies: in february 2016, the us fda recommended that all blood donations in areas with active zika virus (zikv) transmission be tested with an fda approved nucleic acid test (nat) for zikv rna or treated with an fda approved pathogen reduction technology. the cobasv r zika test was approved under an investigational new drug application on march 30, 2016 and testing of puerto rico donations began on april 3, 2016. as a precautionary measure some blood centers in the us states also began nat testing for zikv. in august 2016, the fda recommended universal screening of all blood donations. the aim of this study is to describe the detection of zikv rna in blood donations collected in us states between april 3, 2016 -february 28, 2017 using the investigational cobasv r zika for use on the cobas v r 6800/8800 systems. study design/methods: donations were screened with cobasv r zika by individual donation testing. all initial reactive (ir) results were repeated in duplicate. supplemental testing included an alternative nat (altnat) assay which is less sensitive than cobasv r zika and serology testing for anti-zika igm and igg. reactive donors were invited to enroll in follow-up, which included cobasv r zika and serology testing. a donor was considered to be zika confirmed positive if at least one replicate of the repeat testing by cobasv r zika was reactive on index donation or follow-up, reactive by altnat on the index donation, or positive for anti-zika igm on index or follow-up. all ir donations were also retested at a 1:6 dilution to simulate mini-pool testing. results/findings: a total of 1,776,190 blood donations were screened using cobasv r zika. of 56 ir donations, 12 were repeat reactive (rr), 39 non-rr and 5 had no repeat testing. of the 12 rr donations, 7 were positive by altnat; 3 of these were igm positive. all 4 altnat negative donors were igm positive. one donor was alt-nat equivocal and igm negative. of the 5 rr donors that were not igm positive on index, 3 enrolled in follow-up and all seroconverted. of 39 non-rr donations, 38 were altnat negative and 1 is pending supplemental testing. 8/38 donors were igm positive on index. 30 donors were igm negative on index; 15/30 enrolled in follow-up; 14 remained igm negative and 1 was 1gm inconclusive. of 5 donations without repeat testing results, 2 met criteria for positive (1 was altnat positive, igm negative and 1 altnat negative, igm positive). 1 donation is pending additional testing. altogether, 22/56 ir donations met the criteria for true positive on the index donation. 9/22 (41%) true positive donations were reactive when retested in a simulated minipool. 16/22 were igm positive. conclusion: 0.001% of the 1,776,190 donations in us states screened for zikv rna were confirmed as true positives. cobas v r zika is not commercially available for blood screening use. using monte carlo simulation luiz amorim* 1 , marc germain 2 , gilles delage 3 , maria esther lopes 1 and yves gr egoire 3 . 1 hemorio, 2 hemaquebec, 3 h ema-qu ebec background/case studies: zika virus was implicated in very large and recent outbreaks, in french polynesia (2013) , and in brazil (2015/2016), which was followed by outbreaks in south america, central america and caribbean. four probable transfusion transmitted cases were reported in brazil; since 80% of zika cases are asymptomatic, the actual transfusion rates can be much higher than reported. in this study, we used a monte carlo simulation for risk estimation during the brazilian outbreak. study design/method: the data feeding the monte carlo simulation were collected from january 1 st , 2016 through november, 26 th , 2016, from brazil (the whole country) and for rio de janeiro state, one of the outbreak epicenters. the data came from brazilian epidemiologic bulletins and from brazilian blood donation figures. the risk assessment was performed separately for whole blood (wb) donation and for apheresis platelets (ap). the model took into account the following parameters: zika incidence in brazil and in rio; lognormal distribution symptomatic viremia (period: 5 days, with 99% of the values lower than 18 days); 20% of infected donors with symptoms lasting 2 days; 1.2 donation/donor/year for wb and 1.75 for ap. the formula for transfusion risk calculation was: incidence x infectious period x average donation number per donor per year (wb, x/y; aph, z/y) x (1 -proportion of refused donors) x (1proportion of discarded donations due to post donation -pd -information). results/finding: the table below shows the results. the estimated risk of transfusion transmitted zika is very important in brazil and in rio de janeiro, where it can attain 1:13,598, for apheresis platelets. the severe consequences of zika in vulnerable populations -pregnant women and newborn -indicate that interventions to reduce this unfavorable outcome, such as donor testing and pathogen inactivation, should be considered in brazil dengue (denv) arboviruses in the population are not available in brazil. the objective of this study was to assess the contemporaneous incidence of these agents in donors at 4 large geographically dispersed blood centers located in the southeast and northeast of brazil. study design/method: in the brazil public blood bank system, nat screening for hiv, hcv and hbv is performed on minipools (mp from 6 donations). the residual volume of mp plasma, 0.35 -0.45 ml, is routinely discarded. beginning in april 2016 each blood center saved $67 mps/week for retrospective testing using the triplex zikv, chikv, denv transcription mediated amplification (tma) assay developed by grifols/hologic. mps were shipped to the usa and batch tested at grifols. in the first two weeks (april 3-15) 3 mp6 were combined into pools of 18 donations; thereafter mp6 were tested without additional pooling. to estimate the percent positive donors, the denominator was adjusted to account for the number of donations included in each pool each month and 95% confidence intervals (ci) calculated using the method developed by biggerstaff. results/finding: the triplex assay performance was shown to have very high sensitivity (95% limit of detection <20 copies/ml for zikv/chikv/ denvs) and to accurately discriminate each of the arboviruses. testing of the first 6 months of samples is complete for 6,292 mp, comprised of 37,752 donations collected from april 3 to october 9, 2016. a total of 77 pools were positive, with 76 detected between april-june 2016. the table summarizes the highest monthly estimated percent positive donors for each virus in each city. months with highest percent postive donors were april or may. at the peak over 0.6% of donors in belo horizonte and rio were viremic for zikv, whereas zika was not evident in donors in recife, but over 0.4% of donors in that city were viremic for chivk during the peak. conclusion: during the latter part of the arbovirus outbreak season in brazil in 2016, zikv, chikv, and denv were being transmitted by mosquitoes to donors with asymptomatic donors donating, indicating that blood recipients in brazil were extensively exposed to viremic blood components. the use of donor mps for surveillance may be one of the most efficient approaches for public health monitoring of the onset and magnitude of arbovirus infections. universal zika screening for blood donors in singapore sally lam* 1 , sze sze chua 1 , mars stone 2 , michael paul busch 2 and ai leen ang 1 . 1 health sciences authority, blood services group, 2 blood systems research institute background/case studies: singapore reported its first locally transmitted zika case on 26 august 2016. the numbers rose rapidly to 386 cases by the end september, with eight clusters (hotspots) of cases island-wide. zika virus (zikv) shares the same mosquito vector, aedes aegypti, as the dengue viruses and can caused microcephaly in unborn fetuses of infected pregnant women and guillan-barr e syndrome, which hastened singapore's blood services group (bsg) to look into securing the safety of blood supply from the zika threat. we aimed to assess the assay performance of usa-fda investigational (ind) procleix zikv nucleic acid technology (nat) assay for universal blood donation screening in singapore to prevent transfusion-transmitted zika infection. study design/method: all blood donations were screened for zika with the procleix zikv nat assay since 1 october 2016. zika nat reactive samples were tested at blood system research institute (bsri) for zika rna in plasma and red cells by pcr and for zika and dengue igm and igg antibodies. a zika confirmed case was defined by the presence of zika rna by pcr and/or zika antibodies. the analytical sensitivity was evaluated using 300 blinded frozen samples consisting of 25 replicates of 11 half log dilutions of the who international standard for zikv and 25 replicates of negative controls prepared by bsri. probit analysis was performed to determine the 50% and 95% limits of detection (lod) . clinical performance of the procleix zikv assay was also assessed with local patient samples obtained from institute of infectious disease and epidemiology, singapore and a 14 member blinded zikv reference panel from the usa-fda. results/finding: a total of 63,144 donations were screened from 1 october 2016 to 31 march 2017, with 1 false positive case and 1 zika confirmed donation detected. alternative zikv pcr tested positive in both the plasma and red cells with an estimated plasma viral load of 9.54x10 5 copies/ml. zika igm was negative in the index donation sample but present in the 10-day post-donation follow up sample.. the donor reported no clinical symptoms. the analytical sensitivity for the procleix zikv assay was determined to be 2.1 copies/ml at 50% lod and 10.0 copies/ml at 95% lod. the procleix zikv assay detected rna in 6 out of 9 patient samples and provided 85.7% agreement to the results of the usa-fda zikv reference material. conclusion: the investigational procleix zikv assay showed good analytical sensitivity and clinical performance, suitable for blood screening of zika infection especially in asymptomatic donor populations. bsg commenced universal zika nat screening by individual donation testing following the zika outbreak with 1 confirmed zika donation (high-titer and seronegative) interdicted, which translates to a risk incidence of 1 in 25,888 donations in singapore. background/case studies: a cap/aabb work group suggested that steps be taken to phase in rhdgenotyping for patients with a serologic weak d phenotype. weak d types 1, 2 and 3 express all the major rhd epitopes and these patients can be managed as rhd-positive, which may lead to a reduction in unnecessary rh immunoglobulin (rhig) administration and conservation of rhd-negative rbcs. study design/method: rhd genotyping was performed on all patient samples with weaker than expected or discrepant rhd typing results, utilizing a commercially available genotyping kit manufactured by immucor (rhd beadchip). initially, testing was performed at a reference lab while the rhd beadchip was validated and implemented at this institution. a serologic weak d phenotype is defined as weak to 21 reactivity on initial gel testing. if genotyping demonstrated weak d types 1, 2 or 3, the intent was to manage the patient as rhd-positive. if weak d types 1, 2 or 3 were not detected, the patient is considered at risk for alloimmunization and treated as rhdnegative. while rhd genotyping results were pending, rhd-negative rbcs were used and if pregnant, the patient was eligible for rhig. results were generally available in 2 to 4 weeks. results/finding: rhdgenotyping was performed on 22 patient samples over 15 months. of these 22 patient samples, 13 (59%) were weak d types 1 or 2. the remaining samples demonstrated a variety of alleles including known partial d variants (see table) . one patient identified as weak d type 1 required multiple transfusions over the study period, and refused rhd-positive rbcs. the remaining weak d types 1 and 2 patients have not received transfusions at this institution since they were genotyped. four of 4 obstetric weak d types 1 and 2 patients received rhig while genotyping was pending. conclusion: testing and management of patients with serologic weak d phenotypes is not standardized. rhd genotyping may lead to more consistent, personalized patient care and appropriate management of resources. in this 15 month study period 13 serologic weak d patients were identified who could be managed as rhd-positive, however this did not result in withholding any doses of rhig nor conservation of rhd-negative rbcs. genotyping results pertaining to the management of an obstetric patient were discussed with each obstetrician and it is possible this information may impact management of future pregnancies. these outcomes highlight the limitations of current genotyping processes, including long turn-around-time background/case studies: the rh blood group is highly immunogenic and the most clinically significant blood group secondary only to abo. currently, in the united states, blood donors who type rhd-negative by serology undergo weak-d testing to identify some weak and partial states of rhd expression. however, not all rhd expression can be detected serologically. it has been suggested that investigation of serologic rhd-negative blood donors using genotyping methods can more accurately identify units that may lead to alloimmunization in rhd-negative recipients. study design/method: rhd genotyping of all serologic rhd-negative blood donors presenting to our blood donor center was implemented to identify units with altered rhd alleles that should be characterized as rhdpositive. repeat donations were not tested. initial serologic testing of blood donors was performed using 3 fda approved anti-d reagents. when reactivity with all 3 reagents was negative, rhd genotyping was performed using a commercially available genotyping kit manufactured by immucor (rhd beadchip). this assay detects over 80 rhd variant alleles and additional dna sequencing was performed in selected cases. to maximize efficiency samples were batched for testing; testing was generally performed once a month. if an rhd variant known or suspected to be associated with an increased risk of alloimmunization was detected, recipients of previous donations were investigated for evidence of alloimmunization, and all future donations were restricted to rhd-positive recipients. results/finding: over a period of 8 months we tested 509 rhd-negative blood donors. there were 3 (0.6%) partial-d, 1 weak d (0.2%), and 3 (0.6%) del donors. in one donor sample a novel rhd allele was identified through dna sequencing (rhd*ivs5-46_42deltctc). the phenotype associated with this allele variant is unknown. investigation of previous donations from these 8 donors showed that 6 rhd-negative recipients received rbcs from 4 of these donors. five of these recipients underwent antibody screening after an average follow-up period of 5 months; anti-d was not detected in any sample (see table) . conclusion: serologic testing occasionally fails to identify some rhdpositive donor units, which could place rhd-negative recipients at risk for alloimmunization. dna-based testing can be used to identify donors who have the potential to sensitize rhd-negative individuals. in this limited study period a small number of serologic rhd-negative donors, whose genotype indicated potential to sensitize recipients, were found. however, review of recipient transfusion records indicated that prior exposure to these donors' rbcs did not lead to detectable immunization to date. future potential sensitizing events will be avoided by restricting these units to rhd-positive recipients. grifols diagnostic solutions labs, 5 grifols immunohematology center background/case studies: pregnant women with rhd variants may be candidates for rhig prophylaxis if molecular analysis reveals a genotype associated with possible anti-d formation. proposed testing algorithms advocate molecular characterization of weak d types but if a patient types as rhd-positive, no further action is proposed. women with partial d variants who may also be at risk of anti-d formation have not been included in algorithms proposed to date yet molecular testing may unmask this hidden subpopulation of women who type as d-positive but who may be candidates for rhig prophylaxis. our hospital is in an urban setting in which 63% of deliveries are to african-american patients. we initiated routine, full-gene rhdsequencing for obstetric patients whose serology demonstrated not only weak d, but also those who were categorized as "d1" with 31 reactivity to determine the prevalence of partial d patients in an ethnically-mixed population who may be at risk of anti-d formation. study design/methods: from october 2016 to march 2017, we performed routine d typing (neo, immucor) on 1875 obstetric specimens followed by rhd sequencing on samples with either a serologic weak d phenotype or anti-d testing strength of 31 using at least 1 antibody. solid phase and manual testing used the series 4 and series 5 reagents. four additional anti-d reagents manufactured by grifols (dg gel anti-d), quotient (anti-d blend), biorad (anti-d (rh1) blend), and ortho (bioclone anti-d) were also used for supplemental testing. rhd sequencing was performed by sanger methodology using routine clinical protocols. results/findings: rhd polymorphisms or variations were identified in all 13 samples. two of 13 (15.4%) were d1 with an rhd gene with only common, known intronic variants that is predicted to produce the "reference" rhd protein (ivs1-29c, rs2301153; ivs3 1 117c, rs28521909; and ivs3 1 124a, rs28562109). two (15.4%) were d1 and heterozygous for two apparently new rhd coding variations which we are confirming by further testing. four (30.8%) patients had rhd alleles with known potential to make anti-d (rhd*dol2, rhd*dar1.2, and 2 with weak d type 4.0). one had weak d type 96, which has uncertain susceptibility to alloimmunization and one was weak d type 1, which has not yet been associated with anti-d. interestingly, two (15.4%) had variable d expression associated with apparently new alleles, pending ongoing confirmatory testing and cloning. one patient background/case studies: a weak d type 2 is a variant of the rhd protein that comprises an amino acid substitution located in the 12th transmembrane segment and expresses a reduced amount of the d antigen. this variant is known to be associated with the missense mutation c.1154 g>c which is the first nucleotide of the exon 9 of the rhd gene and thus could be implicated in exon 9 skipping when it is mutated. when performing ngs (next generation sequencing) analysis to fully genotype known patients, we identified an additional variant. study design/method: dna samples were studied by beadchip technology (immucor/bioarray solutions) and ngs using the sureselect human all exon v6 (agilent) and the nextseq500 platform. in silico analysis with different bioinformatic tools was used to predict splicing events. furthermore, a functional splicing assay was performed to determine the impact of the nucleotide variations on exon 9 skipping of rhd gene. this study was completed by the comparative modeling between the wild type and the weak type 2 rhd proteins. results/finding: by a targeted analysis of full exome sequencing, we have confirmed the blood group genotype of 10 patients previously characterized by beadchip technology. interestingly, 4 out of 10 carry the c.1154-31c>t intronic variation on the rhd gene, already described and associated with a del allele. among these last 4 patients, one has been previously characterized as rhd weak type 2 carrying the c.1154g>c (p.gly385ala). independently, sanger sequencing on 50 unrelated rhd weak type 2 samples pinpoint to a linkage disequilibrium between c.1154g>c (exac, maf 5 0.001145) and the c.1154-31c>t (exac, maf 5 0.2496). in silico analysis of both mutation located close to the splice acceptor site of the exon 9 does not predict a significant reduction of its strength score. with minigene vectors harboring rhd wildtype exon 9, mutant rhd c.1154g>c, mutant rhd c.1154-31c>t and double rhd mutants c.1154g>c plus c.1154-31c>t, we showed no influence on skipping of exon 9 due to these mutations. comparative modeling of rhd proteins pointed out an additional hydrophobic interaction on the rhd weak type 2 between ala385 (transmembrane helix 12) and val183 (transmembrane helix 6) hampering membrane insertion. conclusion: the c.1154-31c>t variation is always associated in cis with the missense mutation c.1154g>c on the allele rhd weak type 2. the c.1154-31c>t can be found alone on the rhd gene as a neutral polymorphism. we assess that these two mutations isolated or combined do not lead to abnormal rhd transcripts. our results clearly demonstrate that the weak d antigen reactivity observed with rhd type 2 red blood cells is due to the substitution of alanine at amino acid position 385 to glycine. topology of jk-weak or jk-negative single-nucleotide missense variants in the kidd protein glenn ramsey*. northwestern university background/case studies: the human urea transporter-b (hut-b) protein carrying the kidd blood group has 10 transmembrane (tm) and 2 tilted ureapore a-helices, a long extracellular connector segment, and 2 cytoplasmic segments at each end. numerous single-nucleotide missense variants (snmvs) weaken or abolish expression of jk a/b antigens determined at p.280. we mapped all reported jk-weak or jk-negative (jk-neg) snmvs onto the hut-b structure to explore topological correlates of jk antigen expression. study design/methods: jk*a and jk*b snmvs affecting jk expression were compiled from dbrbc and isbt registries, literature searches and 2010-2016 aabb, isbt and british blood transfusion society meeting abstracts. snmv locations were correlated with the human homolog of the x-ray-crystallographic structure of mammalian ut-b derived for analysis of ut function (levin ej, 2012) . results/finding: seven snmvs located within 1 amino acid (aa) from the exofacial or internal end of a tm helix are mostly weak variants (table) . all 3 at the exofacial ends (p.a93t, p.w240r, p.v333d) are jk-weak; the two jkneg exceptions p.g298e and p.g299e are at the internal end of the tm helix bearing jk a/b . four snmvs in the cytoplasmic n-terminal segment are mostly weak variants. in contrast, 13 snmvs within membrane helices are mostly jk-neg variants. three jk-weak snmvs (p.v10m, p.e44k, p.v76i) have been associated with allo-anti-jk a/b to the antigen on their alleles ("weak partial"). six of the 13 jk-neg variants are within 19 aa (p.270-p.299) of jk a/b at p.280. none of these snmvs are in the long extracellular connector region or the cytoplasmic c-terminal segment. jk-neg variants p.n289s and p.s291p are adjacent to p.288f and p.292l which line part of the urea transporter pore. conclusion: in the transporter-structured rhd and rhce proteins, snmvs with weak d, c, c, e or e expression are mostly within the rbc membrane, and non-canonical antigen-negative snmvs are unusual. in the structurally similar kidd hut-b, most jk-weak snmvs are at the ends of the tm helices or in the n-terminal cytoplasmic segment. among 13 jk-neg snmvs, most are in membrane helices. however, whether a variant appears jk-weak or jk-neg may depend on the extent of testing. next-generation sequencing may provide more complete structure-antigen correlations. background/case studies: the kidd-null blood group is most often inherited as a recessive genetic trait due to biallelic mutations in the slc14a1 gene, which encodes the urea transporter ut-b1. the kidd-null phenotype is associated with transfusion risk and also is associated with abnormalities in the ability to concentrate urine. the cause of the identical kidd-null phenotype with dominant inheritance [in(jk)] has not yet been defined, though it was first described in 1965. in contrast to recessively inherited kidd-null phenotype, this is not associated with mutations in the slc14a1 gene. the aims of the studies was to identify and characterize the causative gene for dominant kidd-null red blood cell phenotype (injk). jk-weak (bold)/ jk-neg expression n within 1 aa from tm a-helix end v76i, a93t, w171r, w240r, g298e, g299e, v333d* cytoplasmic n-terminal v10m, g40s, e44k, l45p in membrane tm and urea-pore a-helices r64w, r64q, g65d, i117t, a183v, l246r, a248t ‡, a270a §, l272f, n289s, s291p, t319m 2/10 * second nucleotide variant in this allele is synonymous (p.p196p). ‡reported as jk-neg but considered jk-weak by isbt. §near splice point. study design/method: we identified several families with dominant inheritance of the kidd-null phenotype in multiple kindreds in spain. we performed whole-genome linkage analysis, exome sequencing, expression (rt-pcr and western) analyses, and urea lysis using patients' cells. in addition, two probands underwent urine concentration tests. results/finding: using molecular approaches, we mapped the affected locus to a 5 mbp region in 19q13.11-13.2 with an lod score of 9.6. using deep sequencing, we identified a potential deleterious mutation in the znf850 gene, which deletes 84 bp resulting in loss of an entire zing finger domain. the identical del84-znf850 mutation is present in all affected individuals, and is absent from all controls tested (n>2000). in addition, two adult individuals who are homozygous for the entire haplotype including the deletion within the znf850 locus, thus completely lacking the common allele, were identified. we also obtained dna from an unrelated injk individual reported from japan. in this individual, there was a similar, though not identical, znf850del84. none of the other potential genetic variants identified in the spanish kindreds was present in the dna from the injk individual from japan. consistent with the fact that the kidd antigen, encoded by the slc14a1gene, is a urea transporter that has been associated with renal function, we found that people with the znf850del84 in spain had an inability to concentrate their urine. conclusion: a predicted zinc finger deletion at znf850, prevalent in southern spain due to a founder mutation, leads to ut-b1 dysfunction and underlies the dominantly inherited kidd-null blood phenotype. the phenotype associates subnormal urine concentrating ability. in background/case studies: di-(2-ethylhexyl) phthalate (dehp) makes pvc film flexible and useful for blood products. during storage, dehp can leach from the bag film into solution and be metabolized. studies in rodents have suggested that exposure to dehp may be associated with adverse health effects, albeit at high dosages. attempts to find dehp alternatives for blood bags have been difficult due to the rbc membrane-stabilizing effect of dehp. bis(2-ethylhexyl) terephthalate (deht) a non-ortho-phthalate is structurally and functionally similar to dehp, but distinct from a metabolic and toxicological standpoint. deht can undergo complete hydrolysis and has an excellent safety profile; it is not classified as a carcinogen, mutagen, reproductive toxicant or endocrine disruptor. the study objective was to evaluate the quality of fresh frozen plasma (ffp) stored in deht containers versus ffp stored in dehp containers at 30 days and 1 year. study design/methods: thirty-six wb units were collected into cpd solution, leukoreduced, centrifuged, and separated into rbc and plasma. abo identical plasma units were pooled together in groups of three. the 12 pools included 5 group a, 6 group o and 1 group ab. each plasma pool was weighed, mixed, sampled, divided into dehp and deht pairs, and frozen at less than -208c within 8 hours of collection. in vitro plasma testing (pt, aptt, factor v, factor viii, fibrinogen, protein c, and protein s) was done on day 0 (pool), day 30, and 1 year of storage. dehp and deht paired plasmas were thawed and tested at the same time. plasticizer concentrations were determined on day 0, day 30, and 1 year of ffp storage. dehp and deht and their monoesters were analyzed by liquid chromatography-mass spectrometry. internal standards were deuterated-dehp, mehp, deht and meht. the lower limits of quantification (lloq) were: dehp 5 2.9 ppm; mehp 5 0.3 ppm; deht 5 0.9 ppm; and meht 5 0.2 ppm. results/findings: mean and standard deviation (sd) for key clotting factors and plasticizer results are summarized in the table. there was no statistical difference in any plasma parameter between dehp and deht bags at the same time period. factor viii retained greater than 80% of its initial value. plasma stored in deht bags had an average plasticizer content 90% lower than that of the dehp bags. background/case studies: plasma prevents dilutional coagulopathy in trauma victims by replacing coagulation factors and substrates during resuscitation with red blood cells (rbcs) and/or crystalloid solutions. spray-dried plasma (spdp) is lightweight and can be reconstituted in minutes making it ideal for use in combat and pre-hospital settings to rapidly provide plasma in situations where it is impractical to administer fresh frozen plasma (ffp). the spray-drying process preserves coagulation proteins, but high molecular weight multimers (hmwm) of von willebrand factor (vwf) are decreased. the objective of this study was to compare spdp and ffp in reconstituted whole blood (rwb) to test the hypothesis that spdp is not inferior to ffp in facilitating platelet adhesion and thrombus formation. study design/method: under an irb-approved protocol, whole blood from healthy volunteers was collected into sodium citrate and centrifuged at 100 g to separate rbcs from platelet-rich plasma (prp). prp was diluted 3-fold in pipes-saline with 1.4mm pge1 and centrifuged at 1900 g. the platelet pellet was resuspended in either spdp or ffp and recombined with the packed rbcs to create rwb with hematocrit of 34-40% and 150,000-250,000 platelets/ml. in addition, two rwb pairs were reconstituted with spdp diluted 1:1 (spdp50%) with plasma from a patient with type 3 vw disease (t3vwd). samples were fluorescently labeled with a gpiibiiia-specific antibody and the sample was flowed through a type i collagen-coated microchannel at a shear rate of 1600 s -1 for 180 seconds. still images of adherent platelets and thrombi were captured in order to calculate surface area coverage (sa) along the length of the channel. ratio paired t-test was used to compare sa in samples reconstituted with spdp vs. ffp. the margin of noninferiority was 20% (spdp/ffp > 0.8). results/finding: six batches of spdp/ffp were evaluated using 17 subjects. there was no statistical difference between the spdp/ffp pairs (p50.7558). the mean ratio of spdp/ffp was 1.21 with a 95% ci of 0.84 -1.57. comparing spdp vs. spdp50%, there was no difference (median ratio 5 1.045, range: 0.95-1.14) in sa. two-way anova demonstrated that batch did not significantly affect ratio of sa in spdp vs. ffp. conclusion: spdp, despite a decrease of vwf hmwm, was not inferior to ffp in ability to support platelet adhesion and thrombus formation. on average, sa in samples reconstituted with spdp was 20% greater than in samples reconstituted with ffp. the lower limit of the 95 th % ci is a difference of 16%, which is less than the a priori determined margin of noninferiority of 20%. even with 50% dilution with t3vwd plasma, there was no reduction in platelet adhesion and thrombus formation in the spdp rwb samples. these data support the development of in-human studies to evaluate the efficacy and safety of spdp in preventing and reversing trauma-related coagulopathy. spray-dried plasma deficient in high molecular weight multimers of von willebrand factor retains hemostatic properties michael a. meledeo* 1 , qiyong peter liu 2 , grantham c. peltier 1 , ryan c. carney 2 , ashley s. taylor 1 , colby s. mcintosh 1 , james a. bynum 1 and andrew p cap 1 . 1 u.s. army institute of surgical research, 2 velico medical inc background/case studies: restoring coagulation factors is key in acute resuscitation after traumatic hemorrhage, but blood products are frequently unavailable in emergency response due to shelf-life restrictions and storage needs. a single unit spray dried plasma (spdp) process has been developed that produces a long-lived and readily stored product that has a reduction in high molecular weight multimers of von willebrand factor (vwf) and an increase in low molecular weight multimers. vwf is critical in platelet adhesion and thrombus formation. following work demonstrating enhanced function with use of glycine-based reconstitution solutions for spdp, this study examines two different spdp pretreatment conditions. study design/method: the samples were: (1) ffp; (2) ffp with 70mm glycine; (3) regular spdp without pretreatment (rspdp), rehydrated with glycine-hcl:glycine; (4) spdp pretreated with glycine-hcl (20mm); and (5) spdp pretreated with glycine-hcl:glycine (20mm:50mm; both pretreated were rehydrated in water). six donor-matched plasmas of each type were tested. vwf activity was measured by ristocetin cofactor assay. fibrin polymerization kinetics were analyzed by turbidimetry. thrombin generation (tg) was observed by thrombogram. chemistry was evaluated by i-stat. residual cell material was quantified by flow cytometry. coagulation properties were measured by thromboelastography (teg) in plasma and reconstructed whole blood (40% hct with 200 platelets/nl from typematched donors). platelet adhesion to collagen under shear was measured by bioflux. results/finding: pretreated spdp showed enhanced vwf activity over rspdp (p < .05). fibrin polymerization density was slightly diminished in rspdp vs. ffp (0.879 vs. 0.742 o.d., p < .01), but tg was unchanged. bicarbonate/base excess were lower in spdp samples vs. ffp (p < 0.001). residual cellular material (especially platelet-derived) was reduced threefold in rspdp vs. ffp (p < .01) and an additional twofold in pretreated spdps vs. rspdp (p < .05). teg results were unchanged in plasma-only samples; in reconstructed wb there was a reduction in amplitude (clot strength) in all spdp samples vs. ; p < 0.01). platelet adhesion was equivalent in pretreated spdps and ffp, while rspdp was improved vs. all other samples (71.53% surface coverage vs. 30.26-43.87%, p < .05). conclusion: spdp has a longer shelf life and easier storage requirements than ffp and was equivalent or superior to ffp in most of these in vitro assays. spdp pretreated with glycine solutions was similar to ffp in most assays and showed superior vwf activity and fewer residual cellular materials but inferior support for platelet adhesion to collagen while under flow compared with untreated spdp. clinical significance of these findings is unclear, but overall in vitro outcomes suggest clinical studies are warranted. the interaction between red blood cell transfusion and lung injury: the influence of blood component manufacturing methods mathijs wirtz* 1 , anita tuip-de boer 1 , ruqayyah almizraq 2 , jason p. acker 3 , philip j. norris 4 , jennifer a muszynski 5 and nicole juffermans 1 . 1 academic medical center, 2 university of alberta, 3 canadian blood services, 4 blood systems research institute, 5 nationwide children's hospital background/case studies: red blood cell (rbc) transfusion is associated with acute lung injury, in particular in patients on mechanical ventilation. the causative factor is not known but may include residual cells or extracellular vesicles (evs) . in this study we investigated the functional effect of different manufacturing methods of rbc products on the response of pulmonary cells in an in vitro model of mechanical ventilation. study design/methods: groups of rbc products (whole blood filtered [wbf] , red cell filtered [rcf] , apheresis derived [ad] and whole blood derived [wbd]) were manufactured from 8 donors (blood type a or b). supernatants were prepared after 4-5 (fresh) and 41-42 days of storage (stored) for measurement of thrombin generation and ev analysis. a549 type ii alveolar cells were seeded onto flexible membranes and incubated with rbc supernatant. cells were subjected to 25% stretch using a cellstretcher. control cells were not stretched. after 24 hours, il-8 and il-6 production were measured. results/findings: both fresh and stored supernatants from ad products significantly increased pulmonary cell il-6 and il-8 production compared to incubation with other rbc products and non-incubated controls, which was further exacerbated by cell stretching. ad products also had significantly increased thrombin generating ability compared to other rbc products, as well as a significantly increased number of rbc-derived evs compared to rcf and wbd products (p<0.05) . incubation of stretched cells with stored wbf products resulted in higher il-8 production compared to other blood products and stretched controls. rcf products did not activate pulmonary cells, had an absence of tg and had low levels of evs compared to other products. conclusion: manufacturing methods markedly influence the interaction of rbc products with lung cells. ad products activate lung cells, which is further aggravated by cell stretching. this may in part be mediated by rbc-background/case studies: investigators previously demonstrated immunosuppressive effects of rbc supernatant on monocytes in vitro, with greater effects seen in response to older units. recent clinical data suggest that rbc manufacturing method may influence immunomodulatory potential, but this has not been directly measured. we used in vitro models to test the hypothesis that rbc supernatants obtained by different manufacturing methods will have differential effects on monocyte function. study design/method: rbc products were manufactured by 4 different methods from 5 individual donors, each: (whole blood filtration [wbf] , red cell filtration [rcf] , apheresis, and whole blood derived [wbd] ). rbc products were stored in sagm (wbf and rcf) or adsol-containing preservative solution (apheresis and wbd). supernatants were obtained after 4-5 days (fresh) and 41-42 days (expiry). monocytes were co-cultured in media plus 20% rbc supernatant or media only (control) followed by lps stimulation. experiments were performed in 5 replicates, each with a distinct monocyte donor. comparisons between groups by anova with dunnett's post-test for multiple comparisons. data are mean 6 sd of % of control values. results/finding: exposure to apheresis or wbd rbc supernatants suppressed monocyte lps-induced tnfa production capacity compared to controls (table 1) . this was true for fresh units and those at expiry. for monocytes exposed to rbc supernatant alone without lps, interleukin-8 production was higher after exposure to fresh wbf (248 6 115 % control, p 5 0.02) or wbd at expiry (292 6 111 % control, p 5 0.0005). conclusion: manufacturing method and/or storage solution significantly alters immunomodulatory effects of rbc supernatant on monocytes in vitro and may confound analyses of clinical effects of rbc storage duration, particularly within international multi-center studies. a magnetic levitation system to study the impact of donor gender, age and blood storage conditions on red blood cell density profile gozde durmus* 1 , alessandro tocchio 1 , anita howell 2 , kaushik sridhar 1 , jason p. acker 3 and utkan demirci 1 . 1 stanford university, 2 canadian blood services, centre for innovation, 3 background/case studies: the amount of hemolysis in red blood cell units increases as the product ages and has been shown to be lower in female blood donors than in males. it is hypothesized that female donors possess, on average, a younger population of red cells, which results in the lower hemolysis that is observed in the pre-menopausal population. it is also hypothesized that the differences between donor populations are mitigated by lysis of older cells when whole blood units undergo processing steps to produce red cell concentrate (rcc) units. as red blood cells (rbcs) age in circulation, they undergo characteristic changes in density and membrane composition that allows for them to be separated from younger cells. study design/method: our aim is to study the effect of donor factors and method of manufacturing and storing conditions on the average rbc age and density of red cell units. we have recently developed a powerful yet simple and inexpensive magnetic levitation-based platform, which allows realtime, high-resolution imaging and monitoring of various cell populations. this label-free system allows density profiling for individual red blood cells, with an unprecedented resolution of 10 -4 g/ml. first, to determine the effect of rcc storage on the density profile of rbcs, levitation and single-cell density profiles were measured at 7, 14, 21, 28, 35 and 42 days. in addition, to determine the effect of donor age and sex on the rbc density profile, blood samples from 24 volunteers with four different age and sex categories (male, 18-40 years; male, >60 years; female, 18-40 years; female, >60 years) were profiled. results/finding: first, we observed that the levitation and density profiles as well as morphology of rbcs within rcc units change significantly during storage. in addition, rbc density was significantly different between young (1.098 g/ml) and older female donors (1.109 g/ml) (p < 0.01). moreover, rbcs from young males (1.096 g/ml) were significantly less dense compared to rbcs profiled from older female donors (1.109 g/ml) (p < 0.05). conclusion: we have developed a magnetic levitation system for the point-of-care, real-time evaluation of rbc and red cell concentrate (rcc) quality. we envision our results might inform decision makers about impact that donor deferral criteria may be having on the quality of red cell concentrates available in the blood banks, for the optimal clinical outcomes. cytokine production of pulmonary cells il-6 (pg/ml) il-8 (pg/ml) background/case studies: oxidation reduction potential (orp) or redox is the ratio of activity between oxidizers and reducers. redox imbalance caused by a higher production of reactive oxygen species (ros) and reactive nitrogen species or a decrease in endogenous protective antioxidants results in oxidative stress (os). while os can cause cellular injury and death, it is also important in the regulation of a healthy immune response to injury or disease. in the present study we investigated changes in hemoglobin, free heme, and orp as red blood cells (rbc) age and the effects of red blood cell age on icu patient morbidity and mortality. study design/method: 120 icu patients were enrolled in this prospective observational trial investigating the effect of transfused rbc age on icu patient morbidity and mortality. all rbcs were pre-storage leukoreduced and abo identical. citrated blood samples were collected from each rbc unit prior to issue. the rbc supernatants were tested for free hemoglobin/ heme and orp. the patients were followed prospectively. results/finding: a total of 426 rbc units were transfused. patients and rbc characteristics are shown in the table. significant reductions were detected in orp values over storage duration (p<0.001). substantial correlations were also found between orp and free hemoglobin (p<0.05) and orp and free heme (p<0.05). interestingly, there was a statistically significant difference between the average orp values of the transfused rbc in patients who developed infection with higher orp values measured in rbc units given to patients who developed post-transfusion infections 132 6 10 vs 127 6 13 (p<0.05). no significant differences were observed between orp and patient mortality, hospital/icu days, or thrombosis. also, no correlations were detected between free heme/hemoglobin or rbc age and infection development. conclusion: these data demonstrate that older blood has lower orp values as well as increased free heme/hemoglobin. there were no differences in orp values between the different blood groups once rbc age was controlled for and there were no statistically significant differences in patient mortality associated with orp, free heme/hemoglobin, or rbc age. the decreased orp values observed in the older blood are likely attributable to the "storage lesion". higher transfused rbc orp values were associated with subsequent development of infection, and younger rbcs were found to have higher orp values. thus, this data supports that young/fresher blood may predispose to subsequent development of infection in critically ill patients. further studies are needed. background/case studies: no randomized trials in humans have addressed whether only exposure to red blood cells (rbcs) that have been stored for a long time is associated with harm. we explore the effect on inhospital mortality of transfusing rbcs stored for more than 35 days compared to rbcs stored for 7 days or less. study design/method: data from a multi-national randomized controlled trial were used for this exploratory analysis. the patients were hospitalized adults who required transfusions and were randomly allocated to receive the freshest rbcs in inventory or the oldest (standard issue) rbcs providing a large cohort of patients receiving rbcs with storage durations along the entire rbc storage continuum of 1 to 42 days. using a time dependent variable patient exposure was defined by the maximum storage duration of rbcs received. this was then used to classify individuals on each day of hospitalization into one of three mutually exclusive exposure categories: freshest (exclusively exposed to rbcs less than or equal to 7 days storage duration -reference group), medium age (at least 1 rbc of 8-35 days storage), and oldest (at least 1 rbc greater than 35 days storage). the primary outcome was all-cause in-hospital mortality. cause-specific cox regression models of in-hospital death assessed the effect of exposure of rbcs in each category to exclusive exposure to rbcs stored for 7 days or less. the effects of fixed and time-dependent confounders were dealt with through stratification and regression. sensitivity analyses were conducted with a) weekly partition with cut-points every 7 days, and b) a finer partition using cut-points every 3 days. results/finding: 24,726 patients receiving 90,530 rbcs were included in the analysis. exposure to rbcs stored for more than 35 days was not associated with increased risk of in-hospital death compared with exposure exclusively to the freshest rbc units (stored for 7 days or less) after adjusting for several fixed and time-dependent potential confounders (hr 5 0.91; 95% ci: 0.72, 1.14; p 5 0.400). exposure to blood stored for at most 8-35 days yielded a similar hazard ratio (hr 5 0.90; 95% ci: 0.73, 1.10; p 5 0.295). in the sensitivity analyses using weekly partitions, exposure to rbcs stored for greater than 35 days compared to exclusive exposure to rbcs stored 7 days or less was not significant (hr 0.90; 95% ci 0.72, 1.14; p 5 0.381). the confidence intervals around the hazard ratios for the other 7-day intervals all include 1. similar findings were obtained with partitioning exposure data into 3 day intervals where exposure to rbcs stored for 40-42 days was not associated with increased risk of death compared with exclusive exposure to rbcs stored for 1-3 days (hr 0.82; 95% ci 0.37, 1.83; p 5 0.635). the confidence intervals around the hazard ratios for the other 3-day intervals all include 1. conclusion: individuals exposed to rbcs stored for more than 35 days were not at increased risk of in-hospital death compared to individuals exposed exclusively to rbcs stored for 7 days or less. transfusion of anaerobically stored red blood cells improves recovery in experimental rat hemorrhagic shock model alexander williams* 1 , cynthia walser 1 , tatsuro yoshida 2 , andrew dunham 2 and pedro cabrales 1 . 1 university of california san diego, 2 new health sciences inc. background/case studies: hemorrhagic shock (hs) severely decreases oxygen (o 2 ) delivery and induces cardiovascular collapse. in parallel to controlling the hemorrhage, clinicians respond by infusing large volumes of red blood cells (rbcs) to restore blood volume, o 2 carrying capacity, and hemodynamic stability. the quality of the transfused rbcs determines the recovery from hs, and extent of clinical sequelae prompted by the hs. this study compares the ability to recover from hs with conventionally stored rbcs, anaerobically (o 2 saturation <10%) stored rbcs, or anaerobic/hypercapnic (o 2 saturation <10% and pco 2 (@378c) $70mmhg) stored rbcs. study design/method: packed red blood cells (prbcs) stored in as-3 after leukorfiltration were created from donor sprague-dawley rats. prbc units were randomly stored under either 1) conventional; 2) anaerobic; or 3) anaerobic/hypercapnic conditions. rats (150-200g) were hemorrhaged to 50% of blood volume, held in hypovolemia for 30 minutes, and resuscitated to recover blood pressure to 90% pre-hemorrhage with prbc stored for either 1 or 3 weeks. systemic hemodynamics, cardiac function, and blood gas parameters were monitored during shock and resuscitation; and vital organ inflammation, oxygenation, and function were evaluated post resuscitation. data were analyzed using two-way anova, followed by the appropriate post hoc analyses. 1(11%) neg patient showed short term response and 6(67%) patients showed progressive disease. at the neg group standard eval 1(11%) patient showed response and 3(33%) had progressive disease. 1(11%) neg patient had long term response compared to 11(21%) pos patients. at the pos short term eval 22(42%) patients showed response and 20(38%) patients had progressive disease. at the pos group standard eval, 20(38%) patients showed response and 6(11%) patients had progressive disease. overall, 28(53%) pos patients responded compared to 2(22%) neg. conclusion: there is a trend in lower response rate in patients with negative antibody screens compared to positive controls. these findings suggest that an anti-cd38 neutralizing substance could play a role in treatment response. alternatively, reduced cd38 expression may also contribute. the low response rates seen in both groups may result from biased selection. the need for repeat t&s and presumed repeat transfusions may be preselecting patients with more aggressive disease. also, only a small number of patients were suitable for review. a larger prospective study that controls for such variables is needed. a review of blood utilized during provider-activated and critical administration threshold-triggered massive transfusion events patrick ramos* and john hess. division of transfusion medicine, harborview medical center background/case studies: traditional definitions of massive transfusions -e.g., the transfusion of ten or more units of red blood cells (rbcs) in a 24-hour period -are limited in prospectively identifying patients requiring massive transfusions, excluded patients who may not survive long enough to meet criteria, or ignored the acuity of the event. to address these issues, a level i trauma center adopted the critical administration threshold (cat) as an additional indication for activating its massive transfusion protocol (mtp). this study reviewed blood utilized during massive transfusion events based upon whether the mtp was provider-activated versus cat-triggered. study design/method: all massive transfusion events between january and april 2017 were reviewed to identify the start time, termination time, number of components transfused, and the start time of each component transfused. the transfusion of three or more blood components in an hour defined cat. a massive transfusion was any event in which the concern for hemorrhagic shock either necessitated a provider to activate the mtp or blood components were transfused at a rate that met cat criteria. the massive transfusion start time is based on either the time the provider activated the mtp or the time the first blood component was transfused, whichever came first. unless the patient expired first, the termination of the massive transfusion event was determined by identifying the point in time in which the patient went three or more hours without the transfusion of any additional blood components. this information was tabulated to determine the monthly number of provider-activated mtps, cat-triggered mtps, and average blood component transfused per massive transfusion. conclusion: blood utilization is lower within the cat-triggered mtps even though it outnumbered provider-activated mtps. however, the mode for both groups suggests that most massive transfusion require less blood components than the average rate. using the mode provides an approximate 24% replacement of blood volume. this should be enough to counter the early signs and symptoms of hemorrhagic shock. though this study did not review the appropriateness of provider-activated mtps, using cat as an indicator ensures clinicians are prepared for a potential massive transfusion. further investigation is needed to determine the factors contributing to the downward trend of the average blood components transfused. the mode would suggest optimistically that patients are being stabilized faster and resuscitated more efficiently. if this is the case, defining massive transfusion should include the rate of components transfused in addition to the total volume transfused. the long term storage effect of 0.2m dithiothreitol on red cell antigen integrity in reagent red blood cells heike carrel* 1 , laurie sutor 1,2 , germ an leparc 3 , marjorie doty 3 and william crews 1 . 1 carter bloodcare, 2 ut southwestern medical center, 3 oneblood background/case studies: anti-cd38 drugs, such as daratumumab, pose a problem for the transfusion service. they may cause a number of false positives, including positive direct antiglobulin tests (dat), indirect antiglobulin tests (iat), and panreactivity in eluates. such results can prolong compatibility testing and delay delivery of blood products for patients. treating reagent red cells (rrbcs) with 0.2m dithiothreitol (dtt) removes drug interference due to daratumumab and allows for the detection of underlying alloantibodies. this study aimed to investigate the effect of dtt-treatment on rrbc antigen integrity over a 28 day period. study design/method: twelve aliquots of human plasma, each containing an antibody of a single, known specificity (anti-d, -c, -e, -c, -e, -m, -s, -s, -fy a , -fy b , -jk a , and -jk b ), were tested against untreated and 0.2m dtttreated rrbcs (immucor panoscreen i, ii, iii; dtt from acros organics). dtt treatment of rrbcs was performed using the methodology described in the aabb technical manual (18 th edition). each of the 12 plasma aliquots was further separated into 28 aliquots and stored at -208c until day of use. fresh aliquots were thawed each day to avoid unintended antibody integrity degradation. a polyethylene glycol (immucor) enhancement technique was used and reactions were read at the iat phase. hemolysis, if present, was observed in the diluent each day prior to mixing the cell suspension and given a grade based on the haemonetics color comparator chart. serological antibody reaction strengths were observed and documented each day. (25) a monthly breakdown for both groups also displayed a downward trend in the average use of blood components. results/finding: there was noticeably more hemolysis with the dtttreated cells over time compared to the untreated cells. red cell antigens remained serologically detectable on the dtt-treated cells throughout the study, despite a greater degree of observed hemolysis. there was minimal difference in reactivity strength between untreated and dtt-treated cells for antigens not affected by dtt. in most instances, the dtt-treated cells reacted slightly more strongly. none of the antibodies produced reactivity strengths of less than 11 with the untreated or dtt-treated cells during the study. conclusion: long term storage of 0.2m dtt-treated rrbcs does not compromise antigen integrity. advance dtt-treatment and storage of a large aliquot of rrbcs may serve to increase efficiency in the transfusion service. background/case studies: monocyte monolayer assay (mma) is a cellular bioassay used to evaluate the hemolytic significance of blood group antibodies and aid in the selection of rbcs for alloimmunized patients. the requirement for fresh auto/allogenic monocytes for mma is highly restrictive due to tedious processing of fresh peripheral blood (pb). our previous study described processing and cryopreservation of buffy-coat (bc) derived and fresh pb-monocytes for mma assay. the aim was to evaluate the functional properties of cryopreserved bc-monocytes as substitute for fresh pbmonocytes in mma in evaluation of previously reported clinically significant rbc alloantibodies. study design/methods: peripheral blood mononuclear cells (pbmcs) were isolated from buffy-coats (histopaque-10771), pooled, suspended in cryopreservation media (20% dmso; 1:1) and stored in liquid nitrogen. pbmc membrane integrity post-thaw was determined by trypan blue exclusion. pbmcs were cultured on poly-l-lysine-treated coverslips (378c, 5% co 2 , 1 h) and monocyte monolayers incubated with fresh or cryopreserved antigen positive (o1) rbcs sensitized with either anti-d (positive control), anti-scianna-2 (sc2) or anti-anwj or lipopolysaccharide stimulated for 2 h. aliquots of the sensitized rbcs were tested for opsonization by indirect antiglobulin test (iat). phagocytosis index (pi) was determined microscopically as the number of fully phagocytosed rbcs/100 monocytes. supernatants were analyzed for cytokines using luminex technique. results/findings: cryopreserved pbmcs showed 96.2 6 1% viability postthaw. we report no significant difference in phagocytosis of anti-d sensitized rbcs by cryopreserved monocytes vs fresh monocytes. we show a significant increase in tnf-a, il-1b, il-6, il-8, mip-a (p < 0.01), mip-b and gro (p < 0.05) secretion from cryopreserved bc monocytes vs both fresh bc and pb-monocytes. sc2-and anwj-sensitised rbcs resulted in a pi of 9.2 6 2% and 60.2 6 6.4% respectively vs anti-d sensitized rbcs (pi: 72 6 8.7%). a weak (11) reactivity by iat was observed for anti-anwj sensitized rbcs while anti-d sensitized rbcs resulted in 41 iat reactivity. these results correlated with previously reported results for clinical significance and mma when using freshly obtained autologous or healthy donor monocytes. conclusion: this study shows that cryopreservation preserved monocyte viability and phagocytosis function for mma. as previously reported with fresh monocytes mma assay, the two alloantibodies tested with cryopreserved bc monocytes were shown to have a phagocytic index of clinical significance (pi>5%). the use of cryopreserved bc-monocytes has the ability we describe antigen typing discrepancies in 5 patients, involving 3 antigens (c, jk a , s), revealed when serologic results differed from the phenotype predicted by dna testing. all 5 patients had 3-41 positive dat with anti-igg and warm autoantibodies identified in the plasma. investigation of the antigen typing discrepancies showed both false negative and false positive results using monoclonal reagents. study design/method: standard tube hemagglutination methods were used for antigen typing. rbcs were treated with edta glycine-acid (ega) using gamma ega kit. genomic dna was isolated from wbcs and hea precisetype performed. results/finding: the rbcs of patients 1 and 2 typed c-on initial testing with immucor gamma-clone anti-c, but were predicted c1 by hea precise-type. ega-treated rbcs gave 31 reactions with the same anti-c reagent. patient 1 rbcs gave variable reactivity (vw-11) with bio-rad seraclone and ortho bioclone anti-c. patient 2 rbcs gave 11 reactivity with all 3 anti-c reagents when incubated for the maximum incubation time allowed. patient 3 rbcs were jk(a1) with immucor gammaclone anti-jk a , which the manufacturer states is suitable for testing dat1rbcs, but predicted jk(a-) by hea. ega-treated rbcs tested jk(a-) with the same reagent. rbcs from patients 4 and 5 tested s1 with bio-rad seraclone anti-s (3-41), but predicted s-by hea. further testing with immucor gammaclone anti-s showed rbcs from both patients were s-. ega-treated rbcs from both were non-reactive with both anti-s reagents. conclusion: commercial monoclonal reagents are valuable resources, especially when phenotyping dat1 rbcs but not all manufacturers include reagent limitations regarding testing of dat1 rbcs. we describe 2 cases of false negative tests with monoclonal anti-c due to antigen blocking by igg, and 3 cases with false positive tests with anti-s (n52) and anti-jk a (n51) typing. false positive tests would potentially be anticipated, but false negative results due to antigen blocking are unexpected. extended incubation as indicated in the reagent insert may reveal weak reactivity when antigen blocking is involved. results concordant with dna testing were obtained with ega-treated rbcs, but it is generally accepted that this is not necessary when using a direct-agglutinating monoclonal reagent. these cases caution the potential for both false negative and false positive results for samples with 3-41 positive dat and supports testing to dissociate igg from rbcs strongly dat1 before antigen typing. in addition, this report highlights the benefits of dna testing as part of the routine reference laboratory workup. background/case studies: sensitization to antigens expressed on transfused cells, by triggering premature antibody-mediated clearance, diminishes the therapeutic effectiveness of transfusion and may also lead to serious delayed hemolytic transfusion reactions. accepted us clinical practice, while providing that sensitized patients receive only cells lacking "offending" antigens, nevertheless ensures continued alloexposure, and thus possible sensitization, to additional antigens, thereby complicating patient management. to mitigate sensitization risk, especially in an era of increasingly cost-conscious procurement, a quantitative assessment of the immunogenicity of specific antigens will be desirable. giblett, long ago, introduced a relative scale relating the rbc antigen immunogenicities to (an assumed) immunogenicity of "k" (http://bit.ly/2opqfew ). here, we show that an absolute estimate of immunogenicities may be extracted directly from observed antibody counts provided these are properly normalized to the fraction of recipients at risk (namely those lacking a specific antigen) and the expected fraction of donors expressing that antigen. study design/method: we define immunogenicity, or sensitization risk, r, for any antigen ("ag") of interest, as the conditional probability of alloantibody ("ab") formation, given allo-exposure to ag, i.e. r :5 prob(ab|al-loexp), so that prob(ab) 5 prob(ab|alloexp)*prob(alloexp) and 0 r 1; rewriting prob(alloexp) 5 prob(recipient, "r", lacks ag)*prob(donor , "d" has ag); and estimating prob(ab) 5 nab/nr, nab denoting the number of ab in nr recipients, we obtain: nab/(nr*prob(r lacks ag)) 5 r * prob(d has ag), the left-hand side representing the observed sensitized fraction, u, i.e. the number of observed ab in relation to the number of recipients at risk. conclusion: several antigens, though corresponding antibodies may be rare (e.g. "jsa", "e", "u"), nevertheless are highly immunogenic, requiring only a single exposure (on average) for sensitization; in contrast, others (on average) will require many exposures and thus pose a relatively low risk. in conjunction with patient genotypes, our r -scale will facilitate the selection of patient-specific cells so as to minimize the risk of (proliferating) alloimmunization even when perfectly matched cells are not available. our approach may be readily extended to additional rbc antigens and other antigen systems. background/case studies: aabb and fda require a 12 month deferral of donors with a tattoo applied using non-sterile needles or reusable ink. we review state regulations to ascertain if tattoo establishments are licensed and required to use sterile or single-use needles and single-use ink. we recently added two large states in which we collect blood to the acceptable states list (asl). we compared the rates of donors deferred before and after the addition of these states to determine potential donor gain with changes in state tattoo licensing regulations. study design/method: we analyzed allogeneic interview responses to the screening question, "in the past 12 months have you had a tattoo?" and if 'yes', whether the tattoo was applied by a state regulated entity. blood centers in 2 states were selected for the analysis before and after state tattoo regulation. in state a, a comparison period of similar 3 months before (2/ 2015 -4/2015) and 3 months after (2/2016 -4/2016) was selected; for state b, a similar 4 months before (12/2015 -3/2016) and 4 months after (12/2016 -3/2017) was selected. frequency and rate of responses were compared in before and after periods. among those who responded to having a tattoo in a regulated state, donations were reviewed for presence of infectious disease markers including hiv, hbv and hcv. results/finding: a higher proportion of donors presenting to give blood admitted to having a recent (<12 months) tattoo in the post period in both states. this increase occurred immediately following the addition of states a and b to the asl (data not shown). among those who responded yes to having a tattoo, in states a and b respectively, there was a 13-and 3-fold increase in accepted donors (table) . the absolute number of accepted donors with tattoos increased from 13 to 567 (state a) and 151 to 1,496 (state b), which annualized, represents a potential gain of 2,216 (state a) and 4,035 (state b) additional donations. all donors who had a tattoo in regulated states (asl) tested negative for hiv, hbv and hcv. conclusion: to counter rising numbers of ineligible donors resulting from recently added deferrals, we considered recovery of donors deferred for tattoos as a way to enhance our donor base. the immediate rise in the number of donors reporting a tattoo following the addition of the 2 states may reflect a decline in self-deferrals based on having had a recent tattoo. we demonstrated an increase in the potential number of donations without compromising safety. background/case studies: transgender donors represent a small fraction of blood donors. determining their eligibility to donate has been challenging for blood centers. to assess behavioral risk, the donor is required to answer gender specific questions. the same is true when assessing trali risk where the donor is asked about a history of prior pregnancies. prior to the implementation of the fda's final rule, blood centers asked donors for their birth gender and determined eligibility based on that gender. if the donor changed their gender they were asked to answer both the male and female questions. the final rule now allows blood centers to accept the donor's stated gender and to determine eligibility based on that gender. in order to assess the risk of failing to ask a transgender male donor (birth gender female) the pregnancy question, a review was done to determine the number of transgender males who were actively donating with a large blood center. and tracked. donors were contacted to resolve any descrepancies. donors who had changed their gender from female to male and who had answered yes to prior pregnancies were identified. hla antibody test results were reviewed for these donors to see if they had been tested and whether they had tested positive or negative. results/finding: from 2013 -2015 , there were 181 donors identified who had changed their gender from their birth gender; 121 female donors changed their gender to male and 60 male donors changed their gender to female. there were 7 (6%) transgender male donors, birth gender female, who had answered yes to the pregnancy question at one of their donations. three of these donors were apheresis donors who had been tested for hla antibodies. one tested positive and the other two tested negative for hla antibodies. the four other donors were whole blood donors and had not been tested. an hla test was added to these donors' records so that the test could be performed the next time they presented to donate. conclusion: transgender male donors may have had prior pregnancies and are also choosing to become pregnant after having transitioned from female to male. six percent of transgender males that we identified reported a prior history of pregnancy. at our center, when a donor requests a gender change from female to male, an hla test is requested for the next donation. first time donors are qualified based on their stated gender so transgender donors with a history of pregnancy will not be identified unless they volunteer this information. consideration should be given to using educational materials to prompt the donor to reveal a history of pregnancy at the time of donation so that hla antibody testing can be performed. effect of variable volume scale introduction in a large multi-site blood center ralph r vassallo*, marjorie d bravo and hany kamel. blood systems, inc. background/case studies: regulations allow whole blood donation [wbd] of up to 10.5 ml/kg or 15% of estimated blood volume [ebv] . traditional measuring/mixing devices are set to halt blood flow at fixed volumes which, with testing samples, are consistently below the 15% limit. variable volume scales [vvs] can be programmed to vary unit volume (up to 550 ml) by donor ebv. this maximizes transfusable rbcs and plasma and recovered plasma [rp] volume. rp from wbds is a small but important source of derivatives and blood center cost recovery. we report the effect of introducing the hemoflow vvs on donor reaction rates and rp volume in a large blood center. compared to previous fixed settings, variable collection volumes were expected to decrease by 10 ml at ebvs <3.5l in donors !23 yo, but increase by 5-40 ml for all others. study design/method: donor vasovagal reaction [vvr] rates (prefaints, prolonged/offsite reactions, and loss of consciousness [loc]) for successful wbds were obtained from the center's hemovigilance database for the 18 mos. before a 6 mo. phased implementation of the vvs, and the subsequent 24 mos. multivariable analysis [mva] by 6-mo. periods was performed in a model incorporating donor sex, age, first-time [ftd] vs. repeat status, ebv and donation site. both the volume and number of units of plasma sent for fractionation were available for the same time periods from the blood center's data warehouse. results/finding: compared to the baseline period, a significant increase in prefaint reaction rates were noted in pre-implementation (impl) periods 1 & 2, continued during impl and post-impl periods 1 & 2, returning to the baseline rate in post-impl periods 3 & 4 (table) . more severe reactions showed an increasing trend that only became significant in post-impl periods 3 & 4. the mva showed the vvs as independent factor contributing to the increased prefaint and more severe reactions. however, its contribution, as measured by odds ratios, was consistently lower than those exerted by known donor determinants of reaction rates: young age, low ebv, ftd status and collection site (not shown). plasma unit volume increased an average of 3.8 ml during post-impl periods 1 & 2 from the temporally matched baseline & pre-impl period 1. conclusion: following an initial increase in mild vvrs during and immediately after implementation of the vvs, vvr rates fell back to baseline, suggestive of transient staff distraction from usual donor care, or a minor effect of increased blood loss with a superimposed improvement trend. the subsequent increase in prolonged/offsite reactions and loc after prefaint reactions had already returned to baseline suggests that staff training, work load, donor compliance with mitigation strategies and other determinants of donor reactions have a far greater effect than the small additional blood loss due to the vvs. small but significant increments in rp volume improve derivative availability and offset the cost of the vvs. comparison of vasovagal and citrate reaction rates in donors according to type of apheresis procedure pierre robillard* 1 and yves gr egoire 2 . 1 hema-quebec, 2 h ema-qu ebec background/case studies: apheresis procedures expose donors to various volumes of citrate depending upon type and length of procedure and type of machine used. citrate reaction (cr) results from various degrees of hypocalcemia in donors. blood volumes taken from donors vary according to type of procedure and use of volume replacement. loss of blood volume is in part responsible for the occurrence of vasovagal reactions (vvr). this analysis was conducted to estimate the incidence of cr and vvr according to various types of apheresis procedures performed at our blood center. (yfv) were reported in some brazilian states -rio de janeiro, sao paulo, minas gerais and espírito santo, mainly. the vectors of those cases were mosquitoes from the haemagogus and sabethes genders, whose habitat is the tropical forests. since many brazilian urban areas are very close to rain forests, there is an outbreak risk in those areas, where the infection is transmitted by the aedes mosquitoes. in order to minimize this risk, rio de janeiro health authorities decided to promote a mass vaccination in late march, 2017. the vaccine is produced with live and attenuated yfv, which can circulate for at least 4 weeks after vaccination. in some individuals, the vaccine can elicit viscerotropic effects and sometimes severe diseases. due to that, brazilian blood regulation authority established a 4 week deferral period after yfv vaccination. this action could dramatically affect the availability of blood donors. this study shows the measures taken by rio de janeiro blood center to circumvent this risk and attract more donors. study design/method: the strategy consisted in offering the population, at a single place -the blood center -the possibility to donate blood and, immediately after donation, to get vaccinated against yfv. there were no financial advantages to the donors, since yfv vaccine is completely free of charge for any brazilian citizen. the vaccine was administrated by trained nurses, in an office close to the donors session. if, for any reason, the prospective donors were not able to donate, the vaccine was also offered to them, provide there were no contraindications. the blood center annnounced just before the mass vacination campaign launching that it would vaccinate 600 people who came to the blood center to donate blood. if, for any reason, the prospective donors were not able to donate, the vaccine was also offered to them, provide there were no contraindications. results/finding: during the five days of campaign, we received 3,351 blood donors candidates; from those, 2,449 were accepted as a blood donor, after medical interview. the deferral rate was 26.9%. at the same period of the year 2016, there were 1,215 prospective donors, and 883 blood donations. the deferral rate was 27.3%. the "get vaccinated against yfv . . .but give blood before" campaign was able to attract, in a five day period, 1,566 additional donors, compared to 2016 same dates. that represents a 177.34% increase in the number of blood donations, without deferral rate increment. there was a slight increase in the proportion of first-time donors, from 42.7% in 2016 to 45.8% in 2017. conclusion: the strategy was more than successful, and it allowed the blood center to build a blood inventory large enough to avoid risks of shortage due to mass vaccination against yfv. dose loss which must be accommodated when collecting plt donations to ensure the us plt dose of !3.0x10 11 is met. currently, triple set kits for pr are only approved in europe. plt loss, and adjusted apheresis targeting parameters may impact split rate (sr) or products per apheresis procedure. inventory suitable for pr without impacting us blood center srs warrants evaluation and optimization. study design/method: 1,000 apheresis collections from 4 centers with different srs were analyzed. a baseline sr for conventional pc was calculated assuming i) a minimum dose (allowing for production loss) of 3.1 x10 11 for single (s), 6.3x1011 11 for double (d), and 9.5x10 11 for triple (t) conventional pcs, ii) concentration and volume requirements from apheresis device manufacturer were used. for each collection, dose, volume, and concentration were assessed for pr kit compatibility, based on storage medium (pas or 100% plasma) assuming i) a minimum dose (allowing for production loss) of 3.5 x10 11 for s and 6.7x10 11 for d for pr units, ii) removing small quantities from units with excess volume or dose to meet pr specs., iii) if all or part of an out of parameter d or t collection could be divided into one or more kits for pr, eligible parts undergo pr, and the remainder treated conventionally, iv) collections unsuitable for pr specs. or would decrease sr if treated would be counted as conventional pcs. results/finding: conclusion: blood centers today can adopt pr for a significant percent of their current supply (as high as 99%) without affecting their sr. compatibly increases further by dividing t and large d donations. percent achievable depends on their current s, d, t proportion of collections and practices. changes to d and t collection parameters, optimized donation and counting accuracy, and volume reduction will improve pr compatibility further. individual analysis is warranted for each blood center. rbc 2rbc plt/p plt plt/rbc/p plt/rbc 2plt 2plt/rbc 2plt/p #donations 47990 63 1775 10832 968 476 67 1150 142 10252 citrate exposure (mls) 41-85 71 138 263 266 300 322 349 478 study design/method: a randomized (2:1), placebo-controlled, single blind, 15 subject, single-site study of ascending microdoses of autologous (apheresis-derived) thrombosomes was conducted. subjects were divided into 5 cohorts, receiving increasing doses, ranging from 1/1,000 -1/10 of the lowest effective dose found in the above rabbit model. cohorts 4 and 5 received the 1/10th dose, but cohort 5 received two 1/20th doses one hour apart. the primary end points were safety and tolerability. subjects were monitored in-hospital for 24hrs post infusion and followed for up to 60 days for adverse events, global neurological assessments, abbreviated physical exams, and laboratory tests. results/findings: there were no serious adverse events (saes) or subject discontinuation post-infusion due to a significant decrease in platelet count from baseline. there were a total of 68 aes: 40 were treatment emergent (teae), of which 8 were treatment-related (6 thrombosomes and 2 control). all teaes were mild or moderate in severity. in cohorts 4 and 5, 3/4 thrombosomes subjects had treatment related adverse events. one cohort 4 subject developed an upper respiratory infection and elevated wbcs within 8 hours post infusion, which resolved by 24 hours, and an elevated d-dimer at 24 hours post infusion, which resolved by day 7. this subject also had an elevation of prothrombin fragment 1 1 2 at baseline, which increased post transfusion and peaked at 24 hours with resolution by day 14. one cohort 5 subject developed non-specific t-wave changes at 1 and 2 hours following her 2nd infusion that resolved by day 21 without clinical symptoms. troponin levels and echo stress tests were normal. ekgs were considered possibly a normal variant or related to placement of the ekg leads. another cohort 5 subject developed an igg platelet autoantibody on days 7-21, which was undetectable on days 42-60; there was no change in platelet counts. the thrombosomes autoantibody assay was positive at baseline, days 7-14, and negative on days 21-60. background/case studies: cryopreservation of platelets (plts) could extend the shelf life from 5-7 days to over two years. cryopreserved plts (cryoplts) appear to have a greater in vivo hemostatic effect than liquidstored plts. plts have been shown to require protein synthesis capabilities for certain functions such as clot signaling and immune responses. this study was designed to assess whether reconstituted cryo-plts carry out protein synthesis upon thawing and short term storage. study design/methods: apheresis plts were cryopreserved with 5% dmso and stored at 2808c. after thawing, the unit was reconstituted in thawed ffp spiked with either 500 lm puromycin (pm) or 250 nm biotinlabeled pm. plts were stored at room-temperature with agitation. samples were drawn immediately after reconstitution as well as after 2, 4 and 24 hours to assess pm incorporation as a measure of protein synthesis, and for in vitro assays to determine platelet activation by cd62p binding, phosphatidylserine exposure by annexin-v binding and microvesicle count in the supernatant. plt microvesicles (pmv) were prepared from the supernatant by ultracentrifugation. plts and pmv were lysed in a triton x-100containing buffer and qualitative proteomics was performed on samples following affinity-purification with streptavidin beads. results/findings: in vitro parameters of reconstituted and subsequently stored platelets were in line with previously published results, with high surface levels of cd62p and phosphatidylserine. pmvs were generated during cryopreservation and the count increased by 11-fold during 24 hour storage. immunoblot analyses of the plts showed a 2-and 4-fold increase in pm incorporation after 4 and 24 hours of storage, respectively. massspectrometry revealed 23 unique proteins that were synthesized after 4 hours of storage, which was confirmed for gtpase and gtpase-regulatory proteins rac1, rap1 and rhogdi by immunoblot analyses. analyses of the pmv translatome also revealed the presence of synthesized proteins; however, these did not change throughout storage. this finding suggests that a defined panel of proteins is packaged into pmvs upon freezing and thawing. additionally, the pmv translatome profile comprised a smaller subset of synthesized proteins compared to the cryo-plt translatome, including the proteins rac1, rap1 and rhogdi. conclusion: this study has demonstrated that cryo-plts can synthesize proteins upon reconstitution in ffp and subsequent storage. discovery of a subset of these proteins in the pmv suggests their encapsulation, possibly in a selective manner. this observation provides novel insights into the capacity for protein synthesis in cryo-plts and the potential regulation of protein packaging into pmv. background/case studies: in 2015, the authors' hospital-based blood bank received variances from the fda and aabb for the use of cold stored platelets (csps) with a shelf life of 3 days. these group a csps, stored in a refrigerator in the emergency department, were used to support the trauma program for use in massively bleeding patients. the placement of the csps on the air ambulances, stored in coolers, was the next logical step in providing platelet therapy sooner to these patients. study design/methods: eight double unit csps were collected using the trima accelv r . two double csps were pathogen reduced using the inter-ceptv r pathogen reduction system. half of the csp pairs were subjected to flat storage in a refrigerator; the other half were loaded into a credov r 4-496 cooler with 2 units of ffp, 2 units of rbcs, and 1 unit of whole blood. three to 5ml of platelets were collected via syringe from each unit at 0 min (before storage in cooler or refrigerator) and after 0.5, 1, 3, 5, and 6 hours of storage for functional validation of platelets. the platelet count, agonists (thrombin receptor agonist peptide (trap), adenosine diphosphate (adp) and collagen stimulated platelets aggregation), non-activated and agonists activated platelet surface expression of phosphatidylserine (ps, annexin-v binding), p-selectin, fibrinogen receptor (pac-1 binding) were measured by coulter counter, 2 channel aggregometer, and digital flow cytometer. paired wilcoxon rank sum tests were used to analyze differences in degradation rates with p<0.05 deemed significant. conclusion: platelets, including pathogen reduced, stored in an oxygendeprived environment, (cooler), do not lose functional capabilities when compared to those platelets stored in a refrigerator with adequate oxygen for 6 hours. therefore, cold stored platelets transported in a cooler are a viable option for providing timelier platelet intervention for severely injured patients prior to hospital arrival. c49-a03h molecular sieving: beyond genotyping ghazala hashmi 1 , reinhard klemm 2 and michael seul* 1,2 . 1 biomolecular analytics, 2 immunoinformatica background/case studies: more than a decade after its commercial introduction (hashmi2005 http://bit.ly/2ohlehe), blood group genotyping, though available in several formats, has remained a tool for special tasks, e.g. the profiling of difficult patient samples or the identification of rare antigen combinations, while serology has remained the tool of choice for routine antigen typing. here, we introduce molecular sieving as an alternative to the current approach of managing special donor unit inventories. this novel process for dna analysis combines the "multiplexing" of markers offered by existing genotyping methods with the pooling of multiple samples in manner permitting the step -wise refinement of candidate sets by molecular attribute patterns. study design/method: molecular sieving is a special format of leansequencing, a proprietary process that permits the simultaneous analysis of up to four samples for alleles encoding 401 rbc antigens in mns,rhce, lu,kel,fy,jk, di,yt,do and co, including the identification of 30 rhce alleles. molecular sieving extends these capabilities to the analysis of pools to attain large scale. thus, in one format of the process, 4*4*96 samples are accommodated in a single run. following the completion of the sieving step, candidates may be directly assigned to requests, or may be selected to enrich a subsequent profiling step for samples with rare or otherwise desirable attributes. here, molecularsieving was used to identify suitable donor units for 135 sensitized sickle cell anemia ("sca") patients (tb1 in cas-tro2002, http://bit.ly/2oplxhr, excluding le and e(variant) and assuming 1 request per patient), presenting with up to 9 allo-antibodies ("ab") in multiple combinations. proprietary "greedy" algorithms were invoked to optimally pair candidate units with requests. results/finding: sieving of only 1 =2 plate holding 4*48 candidate units from actual black donors, followed by profiling of 44 samples selected to enrich for "e neg" and "c neg" and "c-e-k-fya neg", produced assignments for 127 of 135 requests (594.1%), as indicated by colors, and shown in the row "assigned" below: thus, the number of assignments substantially exceeded the number of wells processed. moreover, the remaining 66 pooled samples produced 44 additional assignments to a second set of 135 requests, for a total of 171 assignments from only 92 wells. in another scenario, sieving of a full plate of 4*96 samples, produced $250 assignments for two successive batches of 271 requests from sca patients, a yield exceeding 2.5x. sieving alone typically fills 65-75% of requests of moderate complexity ( 5 ab). conclusion: molecularsieving, by widening the "funnel" while focusing the search for candidate donor units, attains a new level of efficiency in procuring suitable units for patients with hemoglobinopathies. molecular sieving for identifying red blood cells with special phenotype attributes kristopher fernandez 1 , monica kalvelage 2 , ghazala hashmi* 1 and michael seul 1 . 1 biomolecular analytics, 2 lifeshare blood centers background/case studies: providing transfusion support to patients with sickle cell anemia and other hemoglobinopathies remains a challenging logistical task that must accommodate pre-existing allo-antibodies in multiple combinations preferably while minimizing the risk of (continued) transfusion-related sensitization. the allelic diversity of the predominantly black patient population, especially at the rh locus which encodes a variety of "partial" phenotypes further complicates the problem (chou2013 http://bit. ly/2ppvfeq ). study design/method: molecular sieving is a proprietary new process that, in order to rapidly probe candidate donor units in large numbers for multiple phenotype patterns, permits the analysis of pools and pools of pools of samples for a multiplicity of alleles (including 30 at the rhce locus) that encode 401 mns, rh, lu, kel, fy, jk, di, yt, do and co antigens. based on sieving, samples may be grouped by molecular attribute patterns ranging from single "ag2" (e.g. e2,c2,e2,c2) to specific combinations of "ag2" (e.g. c2e2k2fya2 and c2e2jsa2) or combinations of alleles such as those encoding partial rh phenotypes. sieving, optionally, may be followed by profiling of samples selected for desirable attribute patterns. genomic dna from 384 (predominantly) black donors, independently genotyped by one of two commercial methods were provided by lbc. at bmx, 96 pools were prepared prior to amplification, and analyzed by a novel leansequencing method. results/finding: all pool genotypes were consistent with available individual sample genotypes. antigen patterns of particular interest included two groups, namely: several for which pools were homozygous and certain others t for which pools were heterozygous. illustrative of the former pattern type are these: appropriate pool queries revealed that sieving alone identified, among the 124 c2 samples, 24 that were also v2 and vs2 and, among the e2 samples, 12 that were also negative for any partial_e phenotype. illustrative of the latter pattern type are pools identified as heterozygous ("het") for alleles encoding antigens of high or low prevalence. by segregating het pools into subpopulations, we were able to select 6 specific "ee" pools of which 4 were demonstrated (in subsequent profiling) to contain an e-sample. we also identified pools "het" for alleles indicating the possible presence of a rare donor, for example yta|b (8 pools), co a|b (8) and others. conclusion: molecularsieving of a single 96-well plate identified many desirable "antigen-negative" phenotypes and permitted selection of pools for combinations of "ag-neg" patterns including "partial:" rh phenotypes and combinations of c2, e2 and jsa2. these samples are thus confirmed "ag neg" and available for assignment. sieving also facilitated the enrichment of subsequent refinement of molecular attribute profiles in accordance with pending or anticipated demand. "antigen-neg" pattern partial_c partial _c, _e samples available after sieving 124 132 100 100 360 208 192 40 28 92 52 background/case studies: sequence information generated from next generation sequencing (ngs) is often computationally phased using haplotype-phasing algorithms. utilizing experimentally derived haplotype information improves this prediction, as routinely used in hla typing. among the 36 blood group systems, however, experimentally derived haplotypes are known for short genes only, such as icam4 (landsteiner-wiener) and ackr1 (duffy). for longer genes, such as abo of >20 kb, most haplotypes are only statistically derived. we recently established a large dataset of long ermap haplotypes, which code for the scianna blood group system. study design/methods: the nucleotide sequence of >21 kb each was used for all physically confirmed 48 ermap alleles that we previously published. full-length sequences were aligned and variant sites were extracted manually. the bayesian coalescent algorithm implemented in beast v1.8.3 was used to estimate a coalescent phylogeny for these variants and the allelic ancestral states at the internal nodes of the phylogeny. results/findings: we found at least 4 clades representing clusters of 5 to 11 alleles. for each clade, one observed allele was identified as the ancestral allele for its cluster of alleles. using the 4 alleles, we were able to predict alleles with high posterior probability, which were ancestral to the observed alleles and, while not yet observed, may be extant. conclusion: we explored the phylogenetic structure and evolutionary events underlying the origin of different ermap alleles and predict ancestral alleles. in the present study, we show means to predict alleles and to calculate the distinct probabilities of correctness for such predicted alleles. the probabilities can be instrumental in defining a cut-off value to determine which computationally predicted alleles are worth confirming by physical evidence. the alleles identified by studies like ours may be utilized in designing of microarray technologies, imputing of genotypes and mapping of ngs data. the new alleles with nucleotide insertions would be predicted to cause complete loss of expression of the corresponding antigen from a bioinformatics perspective and to encode group o. rather very weak expression of the respective antigen and lack of the corresponding antibody in the plasma was found, confirming these represent subgroups of a and b and suggesting that transcriptional slippage, which has been observed before, is responsible for low level antigen expression. abo genotyping is powerful when both serology and molecular results are evaluated together, and these studies are needed to inform development of bioinformatics tools to accurately associate abo genotypes with phenotypes. background/case studies: evolutionarily related abo and gbgt1 genes encode a and b glycosyltransferases (at and bt) and forssman glycolipid synthase (fs), which catalyze the biosynthesis of a and b, and forssman (fors1) oligosaccharide antigens responsible for the abo and fors blood group systems, respectively. human at and bt possess leuglygly and metglyala, respectively, at codons 266-268, and these tripeptides are important in determining the sugar specificity of enzymes, n-acetyl-d-galactosamine (galnac) for at and galactose for bt. functional fss possess gly-glyala at the corresponding codons, and exhibit galnac specificity. it has been recently shown that human at gained weak fs activity when the leu-glygly was substituted by glyglyala, suggesting that the tripeptide is involved in the recognition/binding of acceptor substrates, in addition to donor nucleotide-sugar substrates. study design/methods: we have searched for additional mechanisms that might enable human at to express fors1. a variety of amino acid substitution constructs of human at were prepared. additionally, exon deletion constructs of at mrna transcripts were also prepared. dna from those expression constructs was transfected into cos1(b3galnt1) cells, and cell-surface expression of fors1 antigen was immunologically monitored with a monoclonal anti-fors1 antibody. results/findings: we found that met69thr/ser substitutions also conferred human at with weak fs activity. we also found that the deletion of exon 3 or 4 of human at transcripts bestowed weak fs activity. because altered rna splicing is frequent in cancer, this mechanism may explain, at least partially, the appearance of fors1 antigen on certain cancer cells and tumors in forssman antigen-negative human species. furthermore, the co-introduction of one of those changes together with the glyglyala substitution synergistically conferred strong fs activity, in addition to strong at and bt activities. conclusion: the substitution of the glyglyala tripeptide codon in the catalytic domain may modify the acceptor specificity of the enzyme. met69thr/ ser or exon 3/4 deletion may alter the intra-glogi localization of the enzyme. and those mechanisms function in synergy. the overlapping usage of acceptors by glycosyltransferases encoded by abo and gbgt1 genes is reminiscent of common ancestral origin of alpha 1,3-gal(nac) transferase genes. the finding that at can synthesize fors1 implicates that the boundary between abo and fors systems may not be as strict as was previously delineated due to the crosstalk in-between. rh typing is required by the fda and fact/aabb for identity testing. since most antibodies in cb plasma are maternal in origin, the abo/rh phenotype relies only on the red cell typing. a and b antigens are not fully developed at birth, presenting about one third of a or b antigen expression levels compared to adult cells. this can result in indeterminate abo results for some cb products. we evaluated the use of dna-based methods for abo typing to aid the resolution of inconclusive ("indeterminate") or discrepant serologic typing results. study design/methods: a total of 29,308 cb units (cbu) were typed for abo/rh (beckman coulter pk system blood grouping and phenotyping) during the period 7/1/2008 -4/1/2017. abo genotyping targeting specific snps for groups a, a2, b, o1, and o2 and, if needed, gene sequencing was conducted in cases with indeterminate results, and in 4 cbu that were provided for transplantation with abo discrepancy found at the transplant center. results/findings: sixty-two (0.21%) cb samples had no reportable abo/ rh phenotype on initial testing, and therefore the cbu could not be used clinically. molecular abo/rh typing resolved all but one. all cases were heterozygous (a/o, b/o, or a/b); in 53% the predicted abo phenotype was a rh neg (table 1a ). the predominant donor race was caucasian (65%). four cbu with abo discrepancy were also evaluated by genotyping (table 1b) . in 3 of those, abo typing performed at the hospital on the day of transplant differed from that reported by the cb bank; the fourth was identified by posttransplant abo typing of the recipient. molecular genotyping resolved the discrepancies. cbu identity was always verified by confirmatory hla typing. conclusion: there is currently no fda approved dna-based abo assay. however, abo genotyping is a useful method for samples where antibody tests alone cannot be conclusive, and can "rescue" cbu that could not be used otherwise. further, genotyping can help resolve abo discrepancies. abstract cobas v r hev for use on the cobasv r 6800/8800 systems is a qualitative pcr test for the detection of hev rna in human plasma. the purpose of this study was to evaluate the prevalence of hev rna among us blood donations collected in the midwest, a region reported to have a higher prevalence of hev infection, and the eastern us. study design/methods: 30,695 fresh and 20,029 frozen edta plasma samples from american red cross donors, collected from february 2015-2016, were de-identified and screened by individual donation testing (id-nat) using cobasv r hev for use on the cobasv r 8800 system under a research protocol. samples were primarily from midwestern and eastern regions of the us. samples reactive on cobasv r hev were further tested by an alternate hev nat, hev rna quantitation, hev genotyping, and for hev antibodies. results/findings: of 50,724 valid results, a total of 3 donations were reactive on cobasv r hev and all were confirmed positive. the confirmed donations were from a 65-year old male in indiana, a 21-year old male in california, and a 55-year old female in kentucky. all 3 donations were positive by hemi-nested pcr and alternative hev nat; however, only the kentucky donation had a high level of hev rna (1440 iu/ml), and was strongly positive for both igm and igg hev antibodies. the indiana donation was genotyped as 3a, the california donation genotype 3b, and no genotype determined for the kentucky donation (see table) . the clinical specificity for the cobasv r hev test in id-nat was 100% (95% exact ci: 99.993% to 100%). conclusion: based on the 3 confirmed-positive donations of 50,724 tested, the hev prevalence was 0.006% (95% exact ci: 0.001% to 0.017%) with a detection rate of 1:16,667 (95% ci, 1:588-1:100,000). to date, no cases of tt-hev have been documented in the us. however, based on the prevalence observed, immunosuppressed transfusion recipients may be at increased risk for transfusion-transmitted hev. background/case studies: monitoring the epidemiology of ttis within the donor population is critical to provide an ongoing assessment of infection risks associated with fda policy changes such as the msm deferral criteria. ttims is a multi-center, federally-funded program intended to derive hbv, hcv and hiv prevalence, incidence, viral genotypes, and donor risk factors for greater than 50% of blood collected in the us. ttims is supported by two distinct coordinating centers (laboratory and risk factor, lrcc, and donation database, ddcc). here we report 13 months of prevalence along with demographic trends from the ddcc. study design/methods: four blood providers and their respective testing laboratories participated. standardized consensus-positive (cp) monitoring definitions were established for donor test results for hbv, hcv and hiv. these results, along with demographics for each donor and donation status (first-time vs repeat) were assembled into a single data set. rates of nucleic acid test (nat) yield (seronegative) and concordant positives (serologic plus nat positives) were combined to comprise cps, were computed overall for donors and donations and by demographic, geographic and temporal characteristics. where appropriate, rates were compared for differences using 95% confidence intervals. this analysis contains data from 9/1/15-9/30/16. results/findings: among 7,578,462 donations reported (16.2% from firsttime and 83.8% from repeat donors), there were respectively 483, 1489 and 194 cp results for hbv, hcv and hiv with corresponding rates of 6.37,19.63 and 2.56 per 100,000 (pht) donations. prevalence among firsttime donors was, as expected, higher than among donations from repeat donors with ratios of 23:1, 24:1 and 5.4:1 for hbv, hcv and hiv. rates (pht) among males were higher than among females for all markers (hbv 8.3 vs 4.2; hcv 23.5 vs 15.2; hiv 3.9 vs 1.0). in general, higher rates for all markers were seen among minority donors, those in the 25-39-year age group (also 18-24 year for hiv), and those from the southeast (and south central for hiv and hcv, and southwest for hbv). no trends were noted over time when 3-month periods were compared. conclusion: data from 4 major us blood systems were successfully combined and are a baseline for monitoring purposes. demographic trends are similar to those observed in other donor studies and generally agree with community trends. changes in rates will require analyses in the context of potential changes in the demographic structure of the donor population. screening donated blood from babesia endemic regions of the united states using a transcription-mediated amplification assay on a fully automated system vanessa bres* 1 , melanie c proctor 2 , deanna self 1 , monique portugal 1 , adrian gurrola 1 , laura tonnetti 2 , sonia bakkour 3 , cheryl lobo 4 , michael paul busch 3 , susan l stramer 2 and jeffrey m linnen 1 . 1 grifols diagnostic solutions inc., 2 american red cross, 3 blood systems research institute, 4 new york blood center background/case studies: the procleix v r babesia assay on the procleix panther v r system is a qualitative in vitro nucleic acid test currently under development. the assay, which is based on transcription-mediated amplification (tma), detects four clinically relevant babesia species (b. microti, b. divergens, b. duncani, and b. venatorum) in human whole blood specimens. this test is intended to screen blood donations individually and in pools of up to 16 donations. whole blood samples are lysed and then pooled on the automated procleix xpress v r system prior to testing on the procleix panther system. these studies evaluated the preliminary analytical and clinical performance of the procleix babesia assay on the panther system. study design/method: analytical sensitivity was determined by diluting in vitro synthesized rna transcripts for the four babesia species. fresh b. microti-infected hamster whole blood, cryopreserved b. duncani-infected hamster whole blood and fresh b. divergens-infected human erythrocytes were tested to determine the limit of detection (lod) of parasites/ml (p/ml) by probit analysis. clinical sensitivity and specificity were determined by screening 32,274 unlinked whole blood donations collected from august 25 th 2016 to april 7 th 2017 in the northeastern united states. initial reactive donations were confirmed by repeat testing, pcr, and/or igg immunofluorescence assay (ifa). reactive individual donor lysates were tested in pools of 16. results/finding: the procleix babesia assay detected all four babesia species with a 95% lod ranging from 7.10-13.51 copies/ml. the preliminary 95% lod in parasites/ml ranged from 0.64-3.61 p/ml for b. microti (n59), from 0.92-1.52 p/ml for b. duncani (n52), and from 0.62-4.95 p/ml for b. divergens (n52). of the 32,274 donations screened, 17 initial reactive and 14 confirmed positive donations were identified for specificity of 99.991% (95%ci: 99.972-99.997%). of the confirmed positive specimens, 8 were reactive by both ifa and pcr, 5 by ifa only and 1 by pcr only. all confirmed positive samples were reactive in lysate pools of 16. donors of reactive donations resided in ct (11), nj (1), nh (1) and me (1) for an overall incidence of 1:2,305, and 1:1,433 in ct. conclusion: the procleix babesia assay on the procleix panther system demonstrated high clinical specificity and sensitivity and detected all four babesia species with similar sensitivity. all confirmed positive donations were also detected in pools of 16 thus demonstrating the effectiveness of pooled lysate screening. conclusion: use of the lag avidity assay shows that in both first-time and repeat hiv-positive us blood donors, newly-acquired (i.e., incident) hiv infections are more frequent in younger donors. the use of this approach provides an additional monitoring tool to assess changes in characteristics of donors whose risk exposure was proximate to the date of donation and will also complement traditional incidence methods by allowing derivation of incidence by donor type. epidemiology of hepatitis b virus, hepatitis c virus and human immunodeficiency virus in united states blood donors lauren a crowder* 1 , whitney r steele 1 , ed p notari 1 , james haynes 1 , roger y dodd 2 and susan l stramer 1 . 1 american red cross, 2 american red cross (retired) background/case studies: from 2004 -2012 , the prevalence of hbv and hcv in us blood donors decreased, while hiv rates remained constant. however, incidence has not been recently calculated. here we report the prevalence, incidence and residual risk (rr) of hbv, hcv, and hiv in a large us blood system from 2008-2015. study design/methods: prevalence was calculated in 2-year intervals. incidence was measured as the number of positives among repeat donors divided by the total time at risk, in person-years (py). rr was calculated using the window periods of 18.5, 7.4 and 9.1 days for hbv, hcv and hiv, respectively. linear regressions were calculated with p<0.05 (*) as significant. results/findings: from 1/1/08-12/31/15, there were more than 48 million donations from 13,204,447 donors (51.4% female, 33% first-time (ft), 81.4% caucasian). there were significant decreases in donation prevalence for hbv and hcv (p50.014 and 0.044), but no significant decrease in hiv during the 8 years (see table for f and r 2 values). a significant decrease was seen in ft donor prevalence for hbv and hcv (p50.026 and 0.042). prevalent ft donors were significantly more likely to be male (68.3% -hbv, 59.8% -hcv, 79.7% -hiv; p<0.001). incidence for all agents declined (significant only for hbv; p50.035). the decrease in hcv incidence was not significant, but there were fewer incident donors in the last 2-year period (74 in 2012-2013 vs. 19 in 2014-2015) . hcv incident donors in 2014-2015 were more likely to be male (79.0% vs 46.0% in 2012-2013, p<0.001) and were younger (84.2% vs. 67.6% in 2012-2013 <40 years, p50.011). overall, incident donors were more likely to be caucasian males (p<0.01). rrs for all 3 agents decreased over time with rrs in 2014-2015 of 1 in 1,565,000; 1 in 2,680,000; and 1 in 2,435,000 for hbv, hcv and hiv, respectively. conclusion: prevalence, incidence and rr of hbv, hcv and hiv have generally decreased within this blood system over the 8-year time frame. as donor screening and deferral regulations evolve, it is important to monitor these risks. it is critical to note that even in a large population, small changes to the number of positives can have a significant impact on prevalence and incidence rates. furthermore, in 2015, mayv was isolated from a patient in haiti, suggesting the virus is already circulating in the caribbean. the extent of mayv transmission could be underestimated due to limited surveillance and diagnostic capabilities; therefore, it is necessary to be prepared for mayv emergence and the potential risk for the blood supply in case it can be transmitted through blood transfusion. study design/method: platelet components (pc) prepared in pas were spiked with mayv and treated with amotosalen and uva illumination. samples were collected pre-uva and post-uva illumination for infectious titer determination. as-5 rbcs were spiked with mayv, mixed with glutathione (gsh)/processing solution, dosed with 200lm amustaline, and incubated for 18hrs at room temperature. samples were collected prior to the addition of amustaline (pre-treatment) and following the 3hr incubation (post-treatment) to determine infectious titers. infectious titers for all samples were determined by plaque assay on vero76 cells. the extent of inactivation was determined by comparing the infectious titers (plaque forming units (pfu)/ml) in pre-vs. post-treatment samples. results/finding: mayv was inactivated to the limit of detection in both pc and rbcs. in platelets, >6.9 log 10 , or >6.2 log 10 pfu/ml, inactivation of mayv was achieved. in rbcs, inactivation of mayv was >6.2 log 10 , or >5.5 log 10 pfu/ml. conclusion: this study demonstrates robust inactivation of mayv by both amotosalen/uva treatment in pc and amustaline/gsh treatment in rbcs. these systems are efficient at inactivating alphaviruses that have demonstrated or have the potential for transfusion-transmission, including mayv, chikv and rrv. prt offers potential as a mitigation strategy for maintaining blood component availability in areas where multiple alphaviruses are epidemic or endemic, and testing is not feasible. (data have not been submitted for fda review and intercept for red blood cell is not approved for commercial use). thrombotic thrombocytopenic purpura with high adamts-13 inhibitor may represent a distinct disease subset in response to therapy based on immature platelet count (a-ipc) dynamics hamza n gokozan* 1,2 , hollie m reeves 1,2 and robert w maitta 1,2 . 1 case western reserve university school of medicine, 2 university hospitals cleveland medical center background/case studies: thrombotic thrombocytopenic purpura is a lifethreating consumptive thrombocytopenia and microangiopathic hemolytic anemia causing diffuse ischemic damage to tissues. early therapeutic plasma exchange (tpe) initiation has improved survival. absolute immature platelet count (a-ipc) has been found to aid in diagnosis and follow-up of ttp patients. a-ipc changes in response to therapy in patients with low adamts13 activity and high inhibitor have not been analyzed in a patient cohort. we analyzed a-ipc response to therapy in five patients with adamts13 deficiency and high inhibitor at a large tertiary academic medical center. study design/method: patients had adamts13 activity of <5% and high inhibitor (1.4-8). mean age of cohort 22.8 years (range 17-64). four patients were female and one was male. patients presented with microangiopathic hemolytic anemia, thrombocytopenia (mean 15.2 x 10 9 /l, range 9 -27 x 10 9 /l) and low a-ipc (mean 1.5 x 10 9 /l, range 0.5 -3.6 x 10 9 /l). patients were initiated on daily tpe and prednisone; additional immunosuppression during hospital stay for cohort consisted of rituximab 375 mg/m 2 (4 patients) and cyclophosphamide 400 mg/m 2 (one patient). tpe continued until platelet count reached 150 x 10 9 /l for at least two consecutive days. immature platelet fraction (%-ipf) and a-ipc (%-ipf x platelet count) were obtained with daily pre-tpe cbc. a-ipc ratio was calculated from baseline. results/finding: patients responded rapidly to daily tpe (mean of 2.4 days [range 1-4 days]) when they achieved a three-fold increase in a-ipc from baseline (mean 11.1 x 10 9 /l, range 2.2 -25.3 x 10 9 /l) and a rapid improvement in platelet count. however, this improvement in platelet count was not accompanied by expected decreases in a-ipc, suggestive of recovery from disease. all patients experienced platelet (mean 217.6 x 10 9 /l, range 200 -294 x 10 9 /l) and a-ipc (mean 19.4 x 10 9 /l, range 13 -28.5 x 10 9 /l) decreases that occurred concurrently while receiving daily tpe so that after a mean of 11.6 days (range 8-14 days) mean platelet count was 65.4 x 10 9 /l (range 14 176 x 10 9 /l) and mean a-ipc 3.2 x 10 9 /l (range 0.7 -6.6 x 10 9 /l). patients were initiated in either rituximab or cyclophosphamide therapy in conjunction with tpe after a mean of 20.8 days of a-ipc and platelet count instability. a-ipc trended to levels indicative of restoration of a negative feedback after this time. conclusion: rapid decreases in platelet counts after a good response in ttp patients may raise suspicion for presence of high adamts13 inhibitor. patients with a high inhibitor have similar a-ipc dynamics during which initial high a-ipc production is followed by unexpected decreases in a-ipc concurrent with platelet counts. recovery occurs once negative feedback between platelet and a-ipc production is re-established. patients with a high inhibitor may represent a distinct subset of ttp as suggested by a-ipc responses. benchmarking the centralized urgent plasma exchange service for patients admitted with a diagnosis of thrombotic thrombocytopenic purpura at a multi-hospital healthcare system jansen n seheult* 1 , michelle n stram 1 , joan sevcik 2 , alesia kaplan 2,3 and joseph e. kiss 2,3 . 1 department of pathology, university of pittsburgh medical center, 2 blood systems inc., 3 university of pittsburgh background/case studies: consensus guidelines recommend that therapeutic plasma exchange (tpe) must be started as early as possible and within 4-8 hours after the diagnosis of thrombotic thrombocytopenic purpura (ttp) has been made; however, there are limited data documenting actual practice. there are several operational facets of delivering a centralized urgent tpe program in a multi-hospital healthcare system, including: central venous (cv) access, ordering, release and delivery of thawed plasma, and transportation of personnel and equipment to perform the procedure. this study analyzes the time elapsed between major steps from diagnosis to initiation of tpe in patients admitted with ttp. study design/method: a retrospective review of the electronic medical record and laboratory information systems from january 1, 2013 to november 1, 2016 was conducted to identify all ttp patients undergoing urgent tpe. demographics, comorbidities, and other pertinent laboratory tests (such as adamts-13 activity levels, complete blood count, biochemical markers of hemolysis and coagulation studies) were reviewed on all identified patients. temporal data for tpe request, cv access placement, plasma product release (which usually happens after cv access), arrival of tpe team and initiation of the procedure were extracted from procedure notes and the blood bank information system. descriptive and summary statistics were generated using stata version 14 (statacorp, tx). group comparisons were made based on hospital location, level of care and history of ttp using a wilcoxon rank-sum test. results/finding: of the 96 ttp patients identified, 22 were excluded due to missing temporal data for important variables. the majority (85%) of patients were treated at central academic centers, with the remainder being treated at peripheral sites. fifteen patients (20%) had a prior history of ttp and 26% had severe adamts13 deficiency on admission. the median time from tpe request to initiation was 5.6 hours (interquartile range: 4.7-7.2 hours). there were non-significant trends to shorter time intervals from request to cv access and request to tpe initiation in patients admitted to the intensive care unit (icu) versus non-icu patients (table 1) . treatment was not started within an 8-hour window in 13 patients; the median time to cv access was significantly longer in these patients (5.8 vs 2.47 hours, p<0.001). two of these patients had a prior history of ttp and only four patients had severe adamts-13 deficiency. the majority (more than 70%) of the time interval between tpe request and tpe initiation was spent obtaining cv access and plasma products. there were no significant differences in time intervals comparing patients with a new diagnosis of ttp versus patients with recurrent/ relapsed disease (table 1) or between patients treated at a central academic center versus a peripheral hospital. conclusion: the consensus 4-8 hour target window from tpe request to initiation appears feasible for a centralized tpe program servicing a multi-48a transfusion 2017 vol. 57 supplement s3 hospital healthcare system. addressing limitations in availability of cv access would likely yield the greatest improvement in timeliness of urgent tpe. cytoreductive therapy for cellular hyperviscosity: utility of cytapheresis treatment for chronic myelogenous leukemia and essential thrombocythemia. jan c hofmann* and dobri d kiprov. california pacific medical center background/case studies: several retrospective, case series have suggested that cytoreductive therapy to treat cellular hyperviscosity and prevent thrombotic events in patients (pts) with chronic myelogenous leukemia with accelerated transformation (cml-at) or essential thrombocythemia (et) may improve short-term outcomes. however, no randomized controlled trial (rct) assessing the efficacy of cytapheresis treatment in this group of pts has been performed. study design/method: from january, 2006 through january, 2017, we performed cytapheresis (cy) treatments (txs) for 123 pts with either cml-at or et, and clinical and/or laboratory evidence of cellular hyperviscosity. 84 pts (68%) had cml-at and received 319 leukapheresis (lp) txs; 39 pts (32%) had et and received 124 thrombocytapheresis (tc) txs. cml-at pts presented with median wbc 398 x 10 9 /l (range 193-689 x 10 9 /l), of which 63% had blast percent >75% or blast count >100 x 10 9 /l. median age was 42 years (8-79 years); 62% were male. cns symptoms (sxs) of leukostasis (lks) were defined as: headache, cognitive decline, confusion, somnolence, visual abnormalities, or seizure; pulmonary (pulm) sxs of lks were defined as: dyspnea, hypoxia, or bilateral chest infiltrates. 22% of cml-at pts had no sxs of lks; 40% pts had sxs of either cns or pulm lks (1 sxs), and 38% pts had sxs of both cns and pulm lks (2 sxs). et pts presented with median platelet (plt) count of: 1738 x 10 9 /l (642-3510 x 10 9 /l)and 71% pts had sxs of thrombosis (evidence of cva or tia, mi, or dvt). median age was 66 years (31-89 years); 58% pts were male. results/finding: all pts received a course of cy tx with following objectives: 1) decreasing the risk of thrombotic/ hemorrhagic complications related to hyperviscosity, and 2) stabilizing cml-at pts for induction chemotherapy (ind chemo). wbc (or plt ct) tx goals were: wbc count (ct) <100 x 10 9 /l for cml-at pts, and plt ct <450 x 10 9 /l for symptomatic et pts and <750 x 10 9 /l for asymptomatic et pts. cml-at pts received median of 3 lp txs (mean 3.9 txs/pt; range 2-8 txs). et pts underwent median of 2 tc txs (mean 3.4 txs/pt; 1-7 txs). outcomes were evaluated by percentage of pts who: 1) reached wbc (or plt ct) tx goal, and 2) received ind chemo. "improved" outcome was defined as pts who reached their wbc (or plt ct) tx goal during cy tx; "stabilized" were pts who achieved >50% reduction in wbc (or plt ct) without reaching goal; and "unchanged" were pts who achieved neither. in cml-at cohort, 76% pts improved, 21% pts stabilized; and 3% pts worsened. in et cohort, 85% improved, 14% stabilized, and 1% were unchanged. for cml-at pts, median final wbc ct 5 96 x 10 9 /l (range 66-307 x 10 9 /l); 94% pts received ind chemo. for et pts, median final plt ct 5 705 x 10 9 /l (263-1087 x 10 9 /l); 95% pts had resolution of thrombotic 49a transfusion 2017 vol. 57 supplement s3 symptoms. 4% of cml-at pts and 0% of et pts expired within 1-4 days after course of cy tx. of 3 expired pts, 2 pts had both blast crisis and sxs of cns/ pulm lks; 1 pt had intracranial hemorrhage or cva; and 2 pts were hypotensive, intubated, or unable to tolerate ind chemo. conclusion: pts with cml-at or et and evidence of impending thrombosis may benefit from cytoreductive therapy. a limited number of cytapheresis treatments (median 2-3 txs) can enable a high percentage of pts to receive definitive treatment and may improve short-term clinical outcomes. a rct to assess efficacy of cytapheresis treatment versus induction chemotherapy (or platelet inhibitor tx) alone in this subset of pts would be very useful. background/case studies: partial normal saline replacement during plasma exchange procedures is common practice. benefits of using normal saline as a replacement fluid include reduced procedure costs and possible reduction of the hypothetical hyper-oncotic effects of standard albumin formulations. however, the use of normal saline may increase the risk of undesired, and potentially costly, adverse events, such as hypotension and citrate reactions. the goal of this study was to compare the frequency of reported adverse outcomes for patients that received all albumin versus albumin/ saline as replacement fluid for plasma exchange at our institution. study design/method: a four year retrospective chart review was done of all therapeutic apheresis procedures performed by our apheresis service that used 100% albumin or 80% albumin-20% normal saline (80/20) as replacement. patients who received plasma entirely or partially as replacement were excluded. the procedure type ordered (100% albumin vs 80/20), the percent of normal saline actually used during the procedure, age, gender, and any noted adverse events during the procedure were recorded in all cases. repeated procedures were modeled using a generalized linear mixed model to examine the risk of having hypotension and/or citrate toxicity where 100% albumin was used versus those that used 80/20. covariates included were fluid types, age and gender. odds ratios (or) and 95% confidence intervals (ci) were used as a measure of risk. we used the term significant for a two-sided p-value < 0.05. results/finding: during the study period, 3650 procedures were documented for 414 subjects (46% female), age range 0-93 years, of which 2,470 (67.7%) received 80/20. the type of fluid used as replacement had a significant effect on the risk of having either hypotension or citrate toxicity. replacement with 100% albumin had a significantly lower risk of having either event than by using 80/20, [p50.002, or (ci): 0.40(0.22, 0.72)] , and also had a significantly lower risk of causing hypotension [p50.023, or (ci):0.45 (0.22, 0.89)] in addition to a lower risk of causing citrate toxicity [p50.042, or (ci): 0. 24 (0.06, 0.95)]. age had a significant effect on having a hypotensive event [p50.04, or (ci):1.1 (1.0, 1.1)] but no effect on citrate toxicity or the combined outcome. gender had no effect on frequency of any event. conclusion: partial saline use as a replacement fluid with albumin during plasma exchange significantly increases the risk of hypotension and citrate toxicity during the procedure. age also increases the risk of hypotension. use of saline as replacement fluid during plasma exchanges should be minimized to maximize patient safety especially in older patients. background/case studies: therapeutic apheresis (ta) is a complex procedure that is mostly well-tolerated and rarely associated with adverse events (aes). there are few studies published on aes associated with ta but they lack uniformity of data. moreover, there is no common database in the united states (us) to report ta-associated aes. we evaluated the annual incidence rates of aes associated with ta at a large tertiary academic medical center over a 10 year period and compared it to published literature. study design/method: we conducted a 10-year retrospective study of ta procedures performed and aes were classified according to criteria described in table 1 . during the study period, ta were performed using cobe spectra (software versions 4.7 and 6.1) and since 2013 the spectra optia apheresis system (version 8.0). literature search was conducted for data published on aes associated with ta. four studies from us and 13 non-us studies (canada, europe and japan) were analyzed. trend for ae rates from 2007-16 was also analyzed. statistical analysis was performed using chi square and spearman rho tests. results/finding: the overall ae incidence was 6.9% (396 of 5,684 procedures) during 10 year period. frequency of aes associated with therapeutic plasma exchange (tpe) was significantly higher (8.5%, p<0.00001) compared to other ta procedures. we found significant correlation between number of tpe and aes (spearman rho 0.7, p50.002) over the 10 years and significant down trend of moderate and severe aes with a spearman rho of -0.64 (p50.04) and -0.83 (p50.003) respectively. there were no fatalities during the study period. majority of aes were grade i (60%) and grade ii (28%): 32/5684 (0.6%) procedures were not completed due to aes. comparison of aes [6.9% (396/5,684)] to both european [11.2% (n513, 12, 256/ 109, 842) ] and other us studies [13.6% (n54, 860/6,324)] showed a statistically significant difference (p<0.0001). conclusion: overall incidence of aes was significantly lower than current published literature. incidence of aes published in other countries is significantly lower than rates published in us. differences in incidence of aes in literature emphasizes need for uniform reporting and stratification of aes and development of a common database to report ta-associated aes. we propose a grading rationale in order to standardize reporting of ae (table 1) . variations in biochemical markers of bone metabolism during plateletpheresis: impact of socio-demographic and lifestyle factors? markus dettke*. akh vienna university hospital background/case studies: plateletpheresis is associated with short-term variations in biochemical markers of bone turnover. socio-demographic factors and lifestyle behaviors are recognized factors which influence mineral metabolism and bone health. in the present study we analyzed the influence of demographic and lifestyle factors on the observed changes in bone markers in a large cohort of routine platelet donors. study design/method: altogether 200 platelet donors with a donation activity of up to 150 platelet donations participated in the study. after a detailed anamnesis all participants underwent a standardized questioner asking for several lifestyle factors known to affect bone metabolism. blood was sampled before and after plateletpheresis and was analyzed for the bone formation marker osteocalcin (oc) and the bone resorption marker cross-linked telopeptides of type i collagen (ctx), among other parameters. the effect of calcium supplementation on bone metabolism was tested in a placebocontrolled crossover study involving ten donors. results/finding: plateletpheresis resulted in an increase in the serum levels of the bone resorption marker ctx and the bone formation marker oc. both parameters returned to base levels within 2 hours after the end of the collection. multiple regression analysis including the parameters sex, age , positive family history of bone disease, but also individual factors like hormonal contraception, smoking, regular alcohol consumption or sportive activity revealed no influence of socio-demographic or lifestyle factors on the observed variation in ctx or oc. there was no association between individual donor career or the number of previous donation and the observed increase in bone turnover. the only predictive parameter we could identify was the amount of citrate exposure during plateletpheresis. increase in serum ctx, showed an inverse correlation to changes of serum ionized calcium. continuous iv supplementation of calcium-gluconate throughout plateletpheresis reduced the variations in bone markers, although this effect was more pronounced for ctx compared to oc. conclusion: the amount of citrate infused during routine plateletpheresis is a predictive parameter for the transient increase in serum markers of bone metabolism. known risk factors for bone diseases, including sex, age, smoking or alcohol consumption, seems to have a low impact on the observed citrate-related variations in serological biomarkers of bone turnover. transfusion with optimized blood products versus transfusion with standard products in a trauma-transfusion rat model mathijs wirtz* 1 , jordy jurgens 1 , jacoline buchner-doeven 1 , joris roelofs 1 , philip spinella 2 , jennifer a muszynski 3 , carel goslings 1 and nicole juffermans 1 . 1 academic medical center, 2 washington university school of medicine, 3 nationwide children's hospital background/case studies: transfusion is associated with nosocomial infection and organ dysfunction in trauma patients, which may be mediated by soluble bioactive substances in blood products. we hypothesized that removing these bioactive substances improves host immune response and reduces organ dysfunction. study design/methods: blood products were prepared from syngeneic rat blood according to blood bank standards. soluble mediators were removed from red blood cells (14 days old) and platelets (5 days old) by washing. plasma was filtered through a 0.22um filter. rats ($350 grams) were poly-traumatized by crush injury to the small intestines, the liver lobes, and by fracture of the right femur and hemorrhaged $30% of their estimated blood volume, which was calculated to be 57ml/kg. hemorrhage continued until a mean arterial pressure of 40mmhg was reached. rats were randomized to resuscitation with standard blood products, washed/filtered blood products or sham. blood samples were taken up to 4h after trauma to assess biochemistry and coagulation status. ex vivo whole blood stimulation tests with lps were performed after sacrifice, and organ damage was assessed by histopathology. blood products were sampled to assess for biochemical changes. comparisons between groups was done by anova and dunnett's post-test for multiple comparisons. results/findings: filtering or washing of blood products significantly stabilized ph, sodium and potassium concentrations and decreased lactate levels in the products compared to standard products. both resuscitation groups received an average of 17ml/kg of blood products in a 1:1:1 ratio. however, use of washed/filtered products did not improve organ failure, as assessed by histopathologic score and levels of creatinine, asat and alat. the coagulation status as assessed by thromboelastometry was deranged in all groups and normalized during transfusion, showing no significant differences between washed/filtered products and standard care. immune response to lps was decreased following trauma compared to healthy controls but did not differ between groups. conclusion: filtering or washing of blood products reduces some aspects of storage lesion of blood products, without affecting the hemostatic capacity of the products, but does not improve organ injury in a rat trauma and transfusion model, nor does it improve the immunosuppressive host response. these results suggest that washing or filtering of blood products may have no relevant clinical effects in a rat polytrauma model. safety and efficacy of tranexamic acid during cardiovascular surgery: a single center before-and-after study takuma maeda* and shigeki miyata. national cerebral and cardiovascular center background/case studies: tranexamic acid (txa), an antifibrinolytic agent, has been widely used in cardiovascular surgery, since several studies have shown that prophylactic use of txa is effective in reducing blood loss after cardiovascular surgery. however, there is concern about the risk of thromboembolic events and adverse neurological effects such as seizures, which might worsen patient outcomes. consequently, we stopped using txa in april 2013, which enabled us to conduct a before-and-after study. the present study aimed to examine the association between txa and adverse effects (seizures, thromboembolism, and renal dysfunction) in patients undergoing cardiovascular surgery using a propensity score matching model. we also assessed the association between txa and other clinical outcomes (reoperation for bleeding, transfusion volume, blood loss, ventilation time, intensive care unit stay, and 30-day mortality). study design/method: this single center retrospective cohort study involved patients who underwent cardiovascular surgery with cardiopulmonary bypass or offpump coronary artery bypass grafting between january 2008 and july 2015 (n53535). because of missing data on patient characteristics, 257 patients were excluded. the incidence of adverse effects associated with txa and other clinical outcomes were evaluated before (january 2008 to march 2013, n51987) and after (april 2013 to july 2015, n51291) using a propensity score model. we estimated propensity scores using a logistic regression model for txa use as a function of 18 baseline variable, generating 969 pairs of patients who received or did not receive txa. we also evaluated the adverse effects of txa using segmental regression analysis. results/finding: propensity-matched analysis showed that seizures were more common (8.7% vs 3.7%, p<0.001) and ventilation time was longer (15 h vs 13 h, p50.04) significantly in the txa group than in the non-txa group. in contrast, transfusion volume and blood loss were significantly lower in the txa group than in the non-txa group (2000 ml vs 2200 ml, p50.009; and 1265 ml vs 1460 ml, p<0.001, respectively). however, 30-day mortality was not statistically different between the groups (1.6% vs 1.4%, p50.82). none of the other outcomes were significantly different. segmental regression analysis yielded similar results. conclusion: even though txa may be associated with an increased rate of seizures and longer ventilator time, it does not increase mortality. the use of txa is significantly associated with decreased blood loss and transfusion volume, providing social benefit by reducing the need for blood transfusion because the supply of blood components will be limited with the aging of japanese society. it seems to be advantageous to use txa because decreased blood loss and transfusion volume and the associated social benefit outweigh the disadvantages of an increased rate of seizures and longer ventilator time. sustained impact of blood management strategies in orthopedics: continuous quality improvement linda levinus* and michele deeney. new england baptist hospital background/case studies: transfusions are one of the most over-utilized treatments performed in any hospital setting (choosing wisely campaign, april 2014, www.choosingwisely.org/societies/american-association-of-bloodbanks). costs and risks associated with transfusions are high and may have a significant impact on patient safety. in our institution we perform over 12,000 joint replacements and spine surgeries per year, making transfusion-associated costs very high. since our last formal evaluation of the metrics used post implementation of patient blood management (pbm) strategies, questions regarding the feasibility of continued transfusion reduction and sustainability of the program were raised by administration and key stakeholder physicians. the objective of this study is to determine what, if any, sustainable improvement to our blood utilization dashboard table 1 ). the data collected show that there has continued to be a reduction in transfusion rate, and blood expenditures through fy16. length of stay has also shown a continued reduction, which is an indicator that the pbm strategies implemented have not compromised quality outcomes. further, continued review and monitoring of the chosen metrics, evaluating changes to policy and practice related to transfusion medicine, and communication of findings to providers/administration upon immediate restrospective analysis, are integral to the continued success and sustainability of our pbm program. going forward, these practices, along with investigating use of additional pbm strategies, will provide the basis for an effective continuous quality improvement program in transfusion medicine for orthopedics. safety and efficacy of 4-factor prothrombin complex concentrate: a retrospective review of outcomes at an academic hospital stephanie jalaba*, hollie benson, nan zhang, jill adamski and theresa kinard. mayo clinic arizona background/case studies: 4-factor prothrombin complex concentrate (pcc) contains factors ii, vii, ix, x, proteins c and s and is used for reversal of vitamin k antagonists in acute major bleeding or urgent, invasive procedures. occasionally, it is used off-label when plasma is not optimal for achieving hemostasis. this study compares the efficacy of on-label and off-label use of pcc in correcting coagulation parameters and reducing allogeneic blood transfusion. study design/methods: a retrospective chart review was performed for pcc use at our institution in 2015. marginal modeling (gee method) was used to account for within patient correlation and assess changes in lab values and products transfused. logistic regression (gee method) was used to evaluate potential risk factors for unsuccessful hemostasis (uh5 rate of transfusion after pcc ! rate before pcc) or thrombotic complications. results/findings: the reduction in pt (p5.005) and ptt (p5.05) was significantly greater in on-label than off-label use. interestingly, transfusion reduction in rbc (p5.03) and plasma (p5.04) after off-label use was significantly greater than on-label use. 20 cases, both on-label and off-label, with uh were associated with cell saver, acute normovolemic hemodilution (anh), or cardiopulmonary bypass (cpb). the odds of having uh were 5.5 times (p5.0072) more with cell saver or anh, and 5.3 (p5.0130) times more with cpb. post-pcc thromboses were identified in 6 cases, but no association was found with potential risk factors: use of antifibrinolytics, vitamin k, factor viia, or extracorporeal support. background/case studies: when a pregnant woman with high risk pregnancy (diagnoses such as abnormal placentation, multiple gestation) is admitted to inpatient bedrest the obstetrical team would like to assure ability to crossmatch red blood cells (rbc) at all times by always having an in-date type and screen specimen. per current aabb standards, this necessitates a new sample every 3 days. this can lead to excessive iatrogenic blood loss and increasing difficulty with obtaining intravenous access in the patient, to the point that an invasive catheter such as a picc line may be placed. in order to mitigate these issues, we chose to extend the type and screen specimen to expire after 7 days in patients without rbc alloantibodies other than passively acquired anti-d due to rh immune globulin administration. study design/method: patients expected to have an antenatal hospitalization of at least 4 days with high risk for transfusion need are identified by the obstetrical service, which submits a request to the transfusion service for extension of pre-transfusion specimens to 7 days. the transfusion service medical director reviews the case and gives final approval. we observed only 1 patient did not have an in-date specimen when the extended out-dating was requested. thirty-eight (38) patients were in-patients continuously until delivery. five patients were discharged prior to delivery-1 moved to another state, 1 was admitted later at another local hospital, and three were readmitted for later deliveries. the mean interval from approval to delivery was 17 days (range 0-63). six (6) patients delivered within 3 days of approval. after approval, the mean number of additional specimens per patient was 2.1 (range, 0-9). no patient required transfusion prior to delivery. five patients received transfusion of at least 1 rbc at the time of delivery, and none had evidence of transfusion reaction. conclusion: since no new antibodies were identified prior to discharge or delivery and no transfusion reactions were observed, the process appears safe. with only 6 patients delivering within 3 days of approval for extended specimens, 37 patients avoided collection of at least 1 specimen each, and 16 patients avoided at least 4 collections each. since new antibodies are not detectable for at least 10 days after immunization, even longer extension of pre-transfusion specimen out-date may be considered. although this requires further study, we believe our practice of extending the pre-transfusion testing sample expiration date to 7 days is safe and is justified, when weighed against the risk of excess iatrogenic blood loss and placing an invasive line for blood sampling in a pregnant patient. iron metabolism in critically ill patients developing anemia of inflammation margit boshuizen* 1,2 , jan m. binnekade 1 , benjamin nota 2 , pieter r tuinman 3 , kirsten van de groep 4 , olaf l cremer 4 , janneke horn 1 , marcus j schultz 1 , robin van bruggen 2 and nicole p juffermans 1 . 1 academic medical center, 2 sanquin research and landsteiner laboratory, 3 vu university medical center, 4 university medical center utrecht background/case studies: anemia due to inflammatory processes (anemia of inflammation, ai) frequently occurs in critically ill patients. in ai, inflammation-induced hepcidin decreases iron availability, a process that is thought to be regulated by erythroferrone, which impact erythropoiesis. knowledge on changes in iron metabolism during the course of ai is limited, hampering the development of strategies to counteract ai. this study aimed to investigate the dynamics of parameters of iron metabolism during the development of ai in critically ill patients. study design/methods: a case control study was performed in 2 tertiary icus in the netherlands comparing 30 patients who developed ai during icu stay with 3 control groups: 30 non-anemic patients with sepsis, 30 non-anemic patients without sepsis, and 10 patients with anemia due to acute blood loss. patients were matched on age and sex. a linear mixed model was used to assess differences in parameters of iron metabolism between groups and over time. results/findings: in patients with ai, levels of iron, transferrin and transferrin saturation decreased already prior to the development of anemia, with lower levels compared to controls (table) . ferritin and hepcidin were increased in ai compared to controls. in the course of ai development, erythroferrone decreased. differences in iron metabolism between groups were not influenced by disease severity. patients with ai differed from patients with anemia due to acute blood loss, the latter was characterized by high iron (15.4 vs. 2.9 mmol/l, p<0.001) and transferrin saturation (53 vs. 9 %, p<0.001), and low ferritin (104 vs. 645 mg/l, p<0.001). conclusion: in critically ill patients with ai, iron metabolism is already altered prior to the development of anemia, suggesting a potential window of opportunity for therapy. iron metabolism in ai is more disturbed than in non-anemic septic controls, irrespective of disease severity, indicating that ai is not solely determined by severity of inflammation. iron metabolism in ai patients differs from patients with acute blood loss, suggesting that efforts to modulate iron metabolism in anemic icu patients should take the cause of anemia into account. clinical oral abstract session: novel approaches to processing and assessing cell therapy products a paradigm shift in stem cell isolation and storage jeffrey drew*. cells4life group llp background/case studies: widespread use of umbilical cord blood is limited by processing yield and post-thaw recovery of viable nucleated cells. the recommended therapeutic cell dose is approximately 2.5 x 10 7 cells per kg body weight indicating that a single cord unit may be insufficent to treat larger individuals. cell isolation methods were developed to remove erythrocytes whilst recovering the white cell fraction (wcf). however, all current methods result in significant loss of the wcf, some up to 65%, whilst leaving 25% of the starting volume of erythrocytes. additionally, there is an almost total loss of potentially important, low abundance cellular subsets. the use of cord blood for hematopoietic reconsititution and in regenerative medicine would be widened if processing methods improved postprocessing and post-thaw viable cell recovery. study design/methods: we have developed a solution consisting of a defined concentration of reagents routinely used in blood therapy. on combination with blood, this solution results in the selective sedimentation of erythrocytes by gravity within 30 minutes. the wcf remains in solution and can be easily separated from the erythrocyte sediment. the wcf can then be concentrated by gentle centrifugation into a small volume containing less than 1% of the original erythrocyte content. the addition of dmso for cryogenic storage and controlled freezing using standard procedures then completes this simple process. results/findings: we have clearly demonstrated that this method allows almost the entire wcf to be isolated and/or concentrated with only modest loss of any of the cellular sub-sets thus far examined. in addition to improving pre-freeze yields, post-thaw recoveries of viable cells are markedly increased, with a yield of approximately 65% of the cd341 fraction post separation and freeze thaw (table 1) . possibly more important, the cfu assay results reproducibly yield higher counts of cfu-gm, cfu-gemm and bfu colonies (table 1) which is a strong indicator that this method will improve patient outcomes. in addition, our separation method isolates and preserves the megakaryocyte-like cells (cd451cd611) and early projenitor cells expressing oct4 and nanog (markers for vsels) which are two examples of cellular subsets usually lost using current separation techniques. conclusion: these results demonstrate that our method achieves: 1. routine recovery of the wcf at levels higher than current methods, independent of volume. 2. higher percentage recoveries of all cell types tested than can be achieved with existing methods. 3. markedly higher post-thaw recovery of viable nucleated cells than any current methodology. 4. almost complete removal of hematocrit. as a result units of cord blood separated using this new method will contain cell yields that could only otherwise be achieved through pooling multiple separate units. therefore, this new method has the potential to increase the demand for cord blood in therapy, expanding to larger individuals and adults, where up until now, it has been suppressed due to limited cell yields delivered by existing methods. effects of implementation of an absolute lymphocyte count target, in addition to cd341 target, for hematopoietic progenitor cell collection edwin a burgstaler*, luis f porrata, dennis a gastineau, eapen k jacob and jeffrey l winters. mayo clinic background/case studies: lymphoma patients receiving >0.5x10 9 lymphocytes(lymph)/kg during peripheral blood stem cell transplant have superior survival. in addition to a cd341 cell target of 4.0x10 6 /kg, a lymph target was also implemented. fifty patients before (no alc) and after (alc) implementation were retrospectively evaluated. study design/method: lymph and cd341 yields, number of collections, lymph target reached, and days to engraftment were examined. mobilization was g-csf (g) or g-csf 1 plerixafor (g1pl). consecutive no alc and alc procedures were examined. the mann-whitney and chi square tests were used for statistical comparison, p< 0.05 considered significant. results/finding: 110 no alc and 159 alc collections occurred among the 50 patients. fenwal amicus was used for 91% of the no alc and 99% of the alc collections (terumobct spectra optia cmnc used for remaining). diagnosis was 5 hodgkin's and 45 non-hodgkin's lymphoma (no alc); 7 hodgkin's and 43 non-hodgkin's lymphoma (alc). pre procedure wbc and lymph counts were significantly higher for no alc (wbc 49.3, lymph 2.0x10 9 /l) than alc (wbc 39.1, lymph 1.2x10 9 /l). equivalent whole blood (corrected for ac) was processed for no alc (16.4l) and alc (17.1l). for alc group, extra collections beyond cd341 target were: 0 days: 24%, 1 day: 36%, 2 days: 22%, 3 days: 16%, and 5 days: 2%. significantly more patients were mobilized with g1pl in no alc group (n581) than alc group (n560) and 42 collections in alc group had mobilization discontinued after cd341 cell target reached. there was no significant difference in g (13.2x10 9 lymph) compared to g1pl mobilized collections (13.0x10 9 lymph); both were significantly higher than the collections where mobilization had been discontinued (5.9 x10 9 lymph). days to wbc engraftment (13.5 no alc vs 13.0 alc) and platelet engraftment (13.0 no alc vs 12.0 alc) were not significantly different. median number of collections for no alc (2) and alc (3) were not significantly different. data (medians) in the table. conclusion: not all patients achieved the 0.5x10 9 lymph/kg or even the 0.3x10 9 lymph/kg targets. implementation of a lymph target increased patients obtaining 0.5x10 9 lymph/kg from 40% to 54%. only 12% had <0.3x10 9 lymph/ kg. discontinuation of mobilization once cd341 cell target was reached significantly reduced lymph yield. the median increase of one collection per patient following implementation was less than had been expected. extended preprocessing storage impairs cord blood hematopoietic stem cell activity suria jahan* 1,2 and nicolas pineault 2,3 . 1 canadian blood services, 2 university ottawa, 3 canadian blood services, centre for innovation background/case studies: large distances between collection and processing sites combined with staff availability can result in long processing delays of umbilical cord blood (ucb) unit. current net-cord-fact standards specify that units can be stored for almost 48 hours at room temperature (rt) as long as units are cryopreserved by 48-hours post-collection. the impact of such delay on hematopoietic stem cell (hsc) function is unclear since most studies have not used transplantation assays that measure hsc key properties and activities. we hypothesized that such processing delay reduces the engraftment activities of ucb units. we set out to measure the loss in engraftment activities associated with preprocessing storage. study design/method: ucb units (n53) were split with one half processed immediately (baseline 8-12 hours) and the second after 43 hours storage at rt. ucb were then processed with hetastarch and buffy coat maintained cryopreserved in liquid nitrogen until use. viability was assessed post-thaw, and thawed ucb buffy coat cells were transplanted into nsg mice. serial transplantation was used to test the self-renewal and differentiation activities of hsc, while limiting dilution (ld) assay and poisson statistic were used to estimate the frequency of scid repopulating cells (src) in thawed units. results/finding: storage before processing had no significant impact on the recovery of viable post-thaw cd451 cells and cd341 cell (n53). primary nsg mice were transplanted with a ucb cell dose that contained a total of 7,500 annexinv neg viable cd341 cells. the latter was done to avoid any bias towards one group or another. short term platelets (190 vs. 140 hplt/ml, p50.06) and leucocytes (1.2% vs. 0.2% hcd451, p<0.02) engraftment at 4-weeks were significantly reduced in stored mice vs. baseline (n53), and similar results were observed long-term at 16-weeks. long-term human bone marrow (bm) engraftment was also reduced in primary transplants from stored samples ( myeloid engraftment was however confirmed in both groups. bm cells from primary mice were transplanted into secondary recipients and human engraftment investigated 3 months post-transplant. strikingly, the frequency of human cd451 bm cells was 10-fold greater in baseline vs. stored mice (p<0.01, n52). hence, storage at rt of ucb units is associated with a deficit in engraftment activity likely due to a loss in hsc activity and/or numbers. to distinct between both possibilities, the net number of src in baseline and stored samples for two units were calculated by ld transplantation assay. the net number of src measured 22-weeks post-transplants were reduced by 30% in unit 1, and by 80% in unit 2. conclusion: prolonged preprocessing rt storage significantly impairs the engraftment activities of ucb units. the reduced engraftment in secondary transplants coupled with the results from the ld assays suggest that this engraftment deficit origins from loss of hsc numbers. our results stress the importance of rapid ucb processing to avoid loss of engraftment activity. acoustic microfluidic separation of blood components charles lissandrello, ryan dubay, kenneth kotz and jason fiering*. draper background/case studies: new cell therapies require efficient and automated methods for purification of target cells prior to subsequent processing. while apheresis, density gradient centrifugation, and magnetic separation achieve some of the requirements, no method is currently available that fully meets clinical needs for a closed, automated, and scalable process. continuous acoustic separation in microchannels is emerging as a versatile method for sorting, separating, and concentrating cells from blood. it has advantages over centrifugation because it is scalable to small or large quantities and can discriminate cells by size as well as density. meanwhile, unlike magnetic methods, acoustophoresis is "label free" and adds no reagents to the therapeutic cells. it has been shown previously that acoustic separation can separate blood components including purification of lymphocytes. however, these studies used devices that were constructed from silicon or glass and have limited potential for scale-up or production as disposable cartridges. in contrast, we report the first ever demonstration of acoustic lymphocyte enrichment along with rbc and platelet depletion in a disposable plastic chip, and we present a cartridge concept that enables clinical scale throughput by linking microchannels in parallel. study design/method: acoustophoresis uses ultrasonic waves to oscillate a rectangular microchannel having a cross section on the scale of the ultrasonic wavelength ($1mm). this results in an acoustic force across the channel that drives cells toward the axial center stream. because the force increases with a cell's size and density, lymphocytes experience a weaker force than rbcs and other classes of wbcs. thus, as blood product flows through the device, the lymphocyte population is enriched at the sides of the channel and can be captured in a branching outlet. likewise, platelets can by separated from lymphocytes. initial and output cell counts are measured by a standard hematology analyzer. results/finding: in our acoustic system, lymphocyte purity (% of total wbcs) was enriched up to 97%, using leukapheresis product as the starting material. this enrichment was achieved in a single pass through the device (residence time of 1sec). total lymphocyte recovery was 43% and monocyte concentration was reduced 76%. furthermore, in a two-pass process platelets were reduced by 75%. in a 12-fold parallel system we tested rbc separation from plasma and achieved 90% separation at 72ml/hr. conclusion: acoustic lymphocyte enrichment along with platelet depletion from standard blood product was demonstrated for the first time in plastic microchannels. such disposable devices are suitable for scale up to clinical bioprocessing systems. lymphocyte purity is comparable to existing methods with the advantage of monocyte and platelet depletion and potential for an automated instrument. background/case studies: the use of natural killer (nk) cells as a cellular immunotherapy has increased over the past several years, specifically their use in patients with hematologic malignancies. nk cells have been used at our institution for the past 15 years. most patients have a reaction with nk cell infusion with some reactions being quite severe. we retrospectively analyzed the reactions associated with nk cell infusions to help address why some patients have more severe reactions than others. study design/method: retrospective chart review of nk cell infusions performed at our institution from 9 clinical protocols from 2008-2016. an infusion reaction was defined as any symptom from the time of nk cell infusion up to 4 hours afterwards. a severe reaction was defined as any symptom with grade 3 or higher severity (graded on common terminology criteria for adverse events-ctcae). preliminary data was analyzed using r 3.3.1. two major endpoints of interest were: 1) infusion reaction with any symptom and 2) severe infusion reaction. to numerically summarize the association of continuous variables with our endpoints, the median, (range) and interquartile range (iqr) were used. a wilcoxon test was performed to test the association between the continuous variables and our end points. a chi-square test was used to test the association between categorical variables and our endpoints of interest. results/finding: there were a total of 127 nk cell infusions. there were 119 (94%) patients with an infusion reaction of any symptom and there were 37 (29%) patients with a severe reaction. infusion rate (ml/min) was similar among those with any reaction (median52.55, p50.42) and those with severe reaction (median52.52, p5 0.42). infusion rate (ml/min/kg) was also similar among those with any reaction (median50.03, p50.43) and those with severe reaction (median50.03, p50.15 respectively). incubation of nk cell product overnight in il-2 vs il-15 had similar reaction rates for those with any symptom (88% had reaction with il-2, 86% had reaction with il-15, p50.94) and those with severe reaction (28% had severe reaction with il-2, 24% had severe reaction with il-15, p50.80). patients with severe reaction had a higher calculated monocyte dose (monocytes/kg) in the nk cell product (median52.44 x 107) versus those without (median51.92 x 107, p50.02). conclusion: our preliminary data analysis reveals that a higher number of monocytes in the nk cell product may contribute to severe infusion reactions, causing patients to have a grade 3 or higher symptom. limitations to this study include this was a retrospective review at a single institution. a streamlined mixed lymphocyte reaction (mlr) assay for evaluation of human mesenchymal stem cell immunomodulation activity christopher p delavan 1 , maryanne c herzig* 1 , barbara a christy 1 , james a. bynum 2 and andrew p cap 2 . 1 us army institute of surgical research, 2 u.s. army institute of surgical research background/case studies: mesenchymal stem cells (msc) have been investigated for treatment of acute respiratory distress syndrome (ards), graft versus host disease (gvhd), wound healing and trauma. a consensus is building that the immunomodulation by mscs is key to their therapeutic potential. mscs suppress peripheral blood mononuclear cells (pbmc) proliferation in vitro, suggesting a correlation for suppressing pbmc inflammatory responses in vivo. current mixed lymphocyte reaction (mlr) assays generally rely on either direct co-culture or indirect culture using transwell systems for monitoring the proliferation of isolated pbmcs in the presence of mitotically inactive mscs. in the study detailed here, mscs are analyzed in a direct co-culture with pbmcs using a luminescent atp assay. study design/method: blood was obtained from an in house blood bank and pbmcs were separated by centrifugation over ficoll-paque in leuco-sep tubes as specified by the manufacturer. the pooled donor pbmcs were stored at -80. mscs derived from bone marrow, adipose tissue or umbilical cord (bm-msc, ad-msc, uc-msc, respectively) or human umbilical cord endothelial cells (huvec) were serially diluted starting at 50-60,000 cells/ well and cultured in 96 well plates for 4-48 h in their respective medias. on day 0, mscs were washed, resuspended in pbmc media and incubated with or without 150,000 freshly thawed pbmcs/well, in the presence or absence of phytohemagglutinin a (pha, 0-5 lg/ml). proliferation of both mscs and pbmcs was assessed in triplicate wells by quantitation of atp levels using the bioluminescent reagent cell titer-glo (promega). results/finding: pbmc proliferation in response to pha gave a robust atp signal by 72 h, with >6 fold increase over control pbmcs. no increase in atp response or proliferation was seen in the absence of pha. co-culture with mscs inhibited pbmc proliferation dependent upon msc passage, source, msc media additive. intra-assay variance of triplicate samples was 10.0%. inter-assay variation of msc preps run under identical conditions was 7.5%. inhibition of pbmc proliferation was graded from 0-100% over the range msc concentrations therefore an ec50 of msc cell number resulting in 50% suppression of pbmc could be determined for each msc prep. this ec50 however was dependent upon pbmc donor pool. conclusion: direct co-culture of live mscs with freshly thawed pbmcs give a robust determination of immunosuppression by mscs. graded responses can be determined, allowing comparison of potency between msc preparations. this streamlined assay can be performed within 72 h, without irradiating cells and with minimal equipment outlay. background/case studies: a high prevalence of iron depletion (id) in blood donors has been documented by recent studies, but none targeted high school aged donors, who consistently contribute 10% or more of the us blood supply. differences between donors 16-18 years old (yo) and adults in baseline and donation-altered iron status are important to understand because teenagers need increased iron for physiological growth and development and may be more susceptible to harm from iron depletion. study design/method: donors aged 16-49 were eligible for ferritin testing if they donated at a high school (hs) blood drive at the start of the 2015/16 academic year at two blood centers. samples from return donations over the remainder of the school year were also tested. the prevalence of absent iron stores (ais, ferritin <12 ng/ml) and low ferritin (lf, ferritin <26 ng/ml) were estimated for 16, 17, 18 and 19-49yo groups separately for both genders. linkage to operational databases established first-time (ft) vs repeat (rpt) donor status. linear regression analysis tested for differences in natural log of enrollment ferritin values by age. multiple logistic regression assessed whether young age independently predicts iron depletion controlling for donation frequency and other factors. results/finding: a total of 4265 donors contributed 6219 donations. donors were evenly split by gender, 66% were ft donors, and 87% were 16-18yo. ft and rpt 16-18yo donors had on average lower ferritin values at enrollment (p<.0001), and a greater percentage were iron-depleted than donors 19-49yo (table) . in repeated measures logistic regression analysis using data from all visits, female sex, greater number of previous donations, shorter interval since last donation, and lower body weight were risk factors for both ais and lf. controlling for these covariates, donors aged 16-18 have sharply higher risk for iron depletion than donors 19-49yo. odds for lf were 4 to 6 times greater in the younger donors, and for ais were 3-to 4fold higher. preliminary statistical models indicate 16yo donors may have greater risk for lf than 17 or 18yo by 4 to 5 percentage points, controlling for other factors (p5.06). conclusion: the prevalence of iron depletion varies markedly by age, sex, and donation frequency, but was considerably higher in 16-18yo donors than in adult controls. logistic regression analysis confirms lower age as an independent risk factor for iron depletion. blood centers should implement measures to mitigate higher risk for iron depletion and the potential adverse consequences for this population of vulnerable donors. mitigation of iron deficiency in young donors -a preliminary report ralph r vassallo*, marjorie d bravo, mary townsend and hany kamel. blood systems, inc. background/case studies: iron deficiency is observed in blood donors who meet regulatory hemoglobin (hb) requirements for blood donation. frequent donations result in negative iron balance and eventually lead to anemia. young donors may be at risk for adverse health consequences (cognitive dysfunction, pregnancy-related complications, fatigue, decreased exercise endurance and pica) even before anemia occurs. study design/method: serum ferritin testing was implemented on 12/19/ 2016 by a large blood collector. testing was performed on successful 16-18 y/o whole blood and apheresis donations. low ferritin (lf) was defined as a value < 20 ng/ml in females (f) and < 30 ng/ml in males (m). donors with low ferritin were notified of deferral from red blood cell (rbc) donations (12 months for f and 6 months for m) and counseled to take 18-28 mg of elemental iron daily for 60 days. for m and f, a ferritin < 12 ng/ml indicated absent iron stores (ais) and < 26 ng/ml indicated iron deficient erythropoiesis (ide). ferritin levels ! 20 ng/ml in f and ! 30 ng/ml in m were considered as indicating an iron-replete state. conclusion: ferritin testing of young donors identified individuals with lf who would benefit from risk mitigation, e.g., delaying subsequent rbc donations and/or taking iron supplements. lf is more common in f than in m donors. lf is more prevalent in m and f donors with any rbc donations in the prior 24 months. an appreciable number of donors with no rbc donations in the prior 24 months presented with lf. these data may be useful in conducting a riskbased decision making exercise to establish recommendations for risk mitigation which could be different for m than for f, e.g., universal iron replacement in teen male donors may not be warranted above a certain hb value. ferritin blood screening in minor or young adult donors jennifer l ritter* 1 , joan williams 1 , michelle humphries 1 , nancy haubert 1 , ben reynolds 1 , michael phillips 1 , randall spizman 1 , ralph r vassallo 2 , hany kamel 2 , sally caglioti 1 , german leparc 1,3 and phillip c williamson 1 . abstract completely investigated. the adolescent growth spurt, poor nutrition and onset of menses increase the risks of iron depletion in young donors. 1 new studies show that teenage donors who give blood frequently may be more susceptible to becoming iron deficient than older repeat donors. 2 study design/methods: over 28,000 serum samples from donors aged 16, 17 and 18 years were analyzed for ferritin levels using the beckman coulter au680 instrument and reagent kit. the anti-ferritin reagent is a suspension of polystyrene latex particles, of uniform size, coated with polyclonal rabbit anti-ferritin antibody. immune complexes formed in solution scatter light in proportion to their size, shape and concentration. the decrease in light intensity is measured spectrophotometrically. 3 results/findings: background/case studies: the risk of cardiovascular (cv) disease in adults can often be identified during adolescent years. the presence of even borderline levels of multiple risk factors increases the likelihood of a cv event. our blood program routinely provides a total non-fasting cholesterol (tc) and blood pressure (bp) measurement for all blood donors. we added glycated hemoglobin (hba1c) determination and performed analyses of the prevalence of abnormal (borderline or elevated) levels of multiple risk factors among 21,007 adolescents (ages 16-19; 61.5% female) who donated blood from 2015 to 2016. study design/method: abnormal risk factor levels were defined as hba1c ! 5.7%, sbp/dbp ! 120/80 mm hg and tc !170mg/dl, as suggested by the american heart association for adolescents. the presence of isolated risk factors was defined as one single abnormal risk factor per individual. clustering of risk factors was defined as the presence of 2 or more abnormal risk factors in the same individual. donor sex was recorded at the time of donation. results/finding: table 1 shows the prevalence of isolated abnormal risk factors and the prevalence of abnormal risk factor clustering in the study cohort. overall, 11,283 (53.7%) adolescents had at least one abnormal risk factor (61.8% of males, 48.6% of females). of these, 8,709 adolescents had isolated abnormal risk factors, and 2,574 adolescents had clustering risk factors. higher proportions of males were in the abnormal bp alone, background/case studies: pre-donation determination of hemoglobin (hb) level in candidate blood donors is a pre-requisite in the majority of blood services and is used to ensure donor safety and blood product quality. however, a variety of hb testing strategies are used across blood services to satisfy this selection criterion. this study aimed to identify how hb screening practices vary across blood donation services and to what extent they influence deferral rates for low hb. study design/method: an online survey was performed among members of the biomedical excellence for safer transfusion (best) collaborative. additionally, data from literature were used to extend the dataset. the survey involved a detailed assessment of hb screening practices, numbers of donations and low hb deferrals for male and female donors separately. multivariable negative-binomial regression models were built to estimate the adjusted effects of minimum donation intervals, hb cutoffs (high/low with high defined as !13.5 g/dl for men and !12.5 g/dl for women), iron monitoring (y/n), iron supplements (y/n providing or prescribing), and geographical location on deferral rates due to low hb. results/finding: data were included from 52 blood services worldwide and complete data were available for 25 blood services. deferral percentages for low hb varied from 0.01% to 8.81% among male donors and 0.03% to 46.73% among female donors. hb deferral rates were notably higher in asian blood services. overall, iron monitoring was associated with 53% lower hb deferral rates in men (95% confidence interval [ci] 11% to 75%) and 61% lower rates in women (95%ci 15% to 82%). iron supplementation was associated to 57% lower hb deferral rates among women (95%ci 22% to 76%) but there was no evidence of such an effect among men (p50.680). each one-week increase in minimum donation intervals resulted in 8% lower hb deferral rates among women (95%ci 1% to 14%) but not among men (p50.454). at the 5% level of significance, higher hb cutoffs do not appear to have an effect among men or women. conclusion: the variation in hb deferral rates across blood donation services can be, particularly in female donors, explained by differences in hb screening and deferral practices. mitigation strategies should consider the variable response among men and women. these insights can help improve both blood service efficiency and donor care. were: characteristics of donors (age, sex, size, weight, region); hb levels, date and volume of donation for index application and previous donation; and number of previous donations (in the 5 previous years and the lifetime). data were analyzed using logistic regression stratified by sex. results/finding: 9.15% of all candidates for wb donation were deferred in continental france in 2015. deferral was significantly more frequent in women (11.16%) than in men (7.29%), due to anemia in 24.41% of deferred women and 9.79% of deferred men. plotting mean hb recovery against time showed mean recovery times ranging from 20 to 30 weeks. analysis (table) identified 3 main factors associated with a higher likelihood of hb recovery: higher logarithm of time since previous donation, lower levels of hb at previous donation, higher number of blood donations in the 5 previous years. conclusion: the 3 main factors associated with higher likelihood of hb recovery after wb donation are probably linked with hematopoiesis stimulation and selection bias among high-frequency donors. mean times required for hb recovery were long enough to require further studies to assess interdonation intervals in france. background/case studies: red blood cell (rbc) transfusion has been related to thrombo-embolic events. microvesicles in the rbc product may support coagulation, which in part may depend on storage time because microvesicles have procoagulant effects in vitro and the amount of microvesicles increase with storage duration. study design/method: we investigated whether transfusion of rbcs containing microvesicles promotes coagulation in human recipients. as transfusion is mostly administered to ill patients, we used a model of mild endotoxemia. eighteen healthy volunteers were randomized to receive either saline, 2 days stored or 35 days stored autologous rbc transfusion two hours after infusion of lipopolysaccharide (lps, from e.coli, 2 ng/kg). blood was sampled every 2 hours up to 8 hours after lps infusion. results/finding: lps resulted in a mild increase in thrombin generation. during storage, the total number of microvesicles increased from 1.4e108 (iqr 8.3e107-1.9e108) /ml in the fresh product to 1.7e110 (iqr 7.9e109-2.3e110/ml; p<0.01) in the stored product (p <0.001), which were mostly rbc derived vesicles. after transfusion, microvesicles from stored rbc products, but not from fresh products, could be detected in the circulation of healthy volunteers and were cleared within 6 hours. however, infusion of stored rbc microvesicles did not augment thrombin generation. levels of d-dimer and thrombin-antithrombin complex were also unaffected. conclusion: transfusion of autologous rbcs containing high levels of microvesicles does not enhance coagulation in human volunteers with mild endotoxemia. background/case studies: transfusion-associated circulatory overload (taco) is characterized by hydrostatic pulmonary edema related to blood transfusion. we sought to examine contemporary risk factors and outcomes for taco during a period where patient blood manaement has led to declines in blood utilization. study design/methods: at four academic hospitals, cases of taco were detected by active surveillance of all adult hospitalized patients who received a blood transfusion, and transfused controls were matched to cases by transfusion intensity. taco incidence was calculated, and clinical characteristics were compared with control patients. odds ratios (or) were calculated using multivariable logistic regression. hospital mortality and length of stay were modeled using cumulative incidence functions in proportional hazards regression. results/findings: 200 cases of taco and 405 matched controls were enrolled from 20,845 transfused patients who received 128,263 blood components from may 2015 until july 2016. taco incidence was 1 case per 100 patients transfused. in addition to well described cardiac and renal comorbidities, multivariable analysis identified the following independent predictors of taco: number of plasma units, emergency surgery, pre-transfusion diuretic use, and higher post-transfusion hemoglobin levels (see table) . compared to controls, taco cases were more likely to require mechanical ventilation (71% vs. 49%; p < 0.001), experienced longer intensive care (4 vs. 3 days; p50.04) and hospital length of stay following transfusion (10 vs. 7 days; p< 0.001), and had higher mortality (21% vs. 11%; p50.02). conclusion: the incidence of taco was lower than what has been reported by prior active surveillance studies. despite declines in its incidence and the number of blood components transfused per case, taco remains a complication of transfusion with significant associated morbidity and mortality. in addition to risk factors for cardiovascular and kidney disease, plasma transfusion and higher post-transfusion hemoglobin levels were associated with taco after controlling for other covariates in the model. additional research is needed to examine the utility of these risk factors in the development of real-time predictive algorithms and the benefit of reduced erythrocyte or plasma exposure in patients at high risk for taco. background/case studies: the residual risk of bacterial contamination of single-donor apheresis platelets (ap) was recently addressed by the march 2016 fda draft guidance to enhance the safety of platelet transfusion. this document also describes an existing pathway for ap outdate extension from 5 to 7 days using an fda cleared rapid test (rt). our hospital based transfusion service has used this rt to enhance the safety of ap transfusion since july 2008 and to routinely extend ap outdate to day 7 since february 2016. this study reports a 103 month experience of secondary screening of ap using a rt. study design/methods: all ap were obtained from our hospital-based donor center or one of four external suppliers. ap were screened by culture based methods post-collection and prior to entry into our inventory. from july 2008-january 2016, ap underwent rt on day 4. day 6 and 7 units were transfused with physician approval when deemed medically necessary. any units remaining in inventory on day 8 had a second rt performed. from february 2016-january 2017, ap underwent rt on day 5 with routine outdate extension to 7 days by performing a second rt on day 6 and a third rt on day 7, as per manufacturer instructions. any positive rts were repeated in triplicate. repeat rt positive units were quarantined and cultured to identify true positives. false positives (fp) were defined as repeat rt negative (type 1) or repeat rt positive with negative confirmatory culture (type 2). all rt results were reviewed during both study periods. ap transfusion and outdate rates were also summarized. results/findings: since july 2008, 20,010 ap were entered into inventory. of these, 11,840 (59%) were transfused prior to rt testing. the remaining 8170 (41%) underwent rt on day 4 or day 5. of these 43 (0.5%) were rt positive (29 type 1 fp, returned to inventory; 14 type 2 fp, discarded), leaving a total available inventory of 8156 units tested by rt. of these, 5631 (28% of original inventory) were transfused before the end of day 5 and the remaining 2525 (13% of original inventory) reached a day 5 outdate. a total of 1561 (8% of original inventory) were transfused on day 6 or day 7. of these, 768 underwent a second rt on day 6 (2 rt positives; 1 fp type one and 1 fp type 2) and 233 underwent a third rt on day 7 (no positive results). a total of 964 (5% of original inventory) outdated on day 7. of these, 754 underwent a second rt on day 8 (no positive results). conclusion: to date we have performed 9925 rts on ap at our hospital. no true positives have been identified. use of rt over the study period decreased our outdate rate from a predicted 13% to only 5%. a total of 1522 ap have been tested twice by rt (768 on day 5 and 6; 754 on day 4 and 8) with 2 (0.1%) positive results, both of which were deemed fp by repeat testing or culture. a total of 233 units have been tested 3 times (day 5, day 6 and day 7) with no additional positives identified. we have not yet identified any units with an initial negative rt result that subsequently converted to a true positive. there is a low fp rate which should also be expected when performing repeat testing on the same unit. these data suggest that the yield for repeating the rt every 24 hours, as currently specified by the manufacturer instructions, is quite low. additional studies are needed to clarify how rt can optimally be used to enhance detection of ap bacterial contamination. survival of trypanosoma cruzi in human blood components laura tonnetti*, aaron thorp and susan l stramer. american red cross background/case studies: trypanosoma cruzi, the agent of chagas disease, is associated with 8 to 10 million infections worldwide, mostly in latin america. despite the extensive immigration from endemic areas, only 5 cases of transfusion-transmission (tt) t. cruzi have been reported in the us, before blood donor screening was implemented in 2007. contributing factors to the low number of tt cases are a possible association between parasite lineage and tt, and high numbers of unreported cases. platelets are almost exclusively involved in t. cruzi tt cases; however, during preparation of components a large fraction of the parasites can be found in red blood cells (rbcs). we investigated if blood component preparation and storage time affect the survival of the parasite and thus play a role in tt of t. cruzi. study design/method: whole blood (wb) units were spiked with t. cruzi trypomastigotes to a final concentration between 10-10,000 parasites/ml. each parasite concentration in wb was tested x2. an aliquot of contaminated wb was used to prepare hemocultures to detect live parasites before preparation of components. rbcs were separated and half of the components leukoreduced (lr) by filtration. platelets and plasma were separated, along with one aliquot of plasma collected before lr. rbcs were stored at 48c for up to 42 days; platelets were stored at 228c (rt) under agitation for 5 days and plasma was frozen at -208c. aliquots for culture were removed weekly from rbcs, daily from platelets and after 30 days from frozen plasma. all samples were cultured in liver infusion tryptose (lit) media at 278c for detection of live parasites for up to 16 weeks. results/finding: hemocultures from spiked-wb were positive at all concentration of parasites. lr'd and non-lr'd rbcs cultured before storage were positive at all concentrations. after storage at 48c, rbcs from all units spiked with 10,000 parasites/ml were positive for up to 21 days; all further times yielded negative results. at lower concentrations, only non-lr'd rbcs spiked with 1000 parasites/ml were positive for up to 7 days. plasma samples cultured before freezing were positive at the highest concentration in one non-lr'd sample, while all others were negative. platelets obtained from wb spiked with 10,000 and 1000 parasites/ml were positive up to 5 days at rt. no parasites were observed in plasma or platelets prior to storage at lower concentrations. molecular analysis to determine the presence of parasite dna in each component is on-going. conclusion: platelet storage conditions offer a suitable environment for t. cruzi survival; however, high concentrations of parasites also survived in rbcs at 48c for up to 3 weeks. leukoreduction offers partial protection, while freezing conditions appears unsuitable for t. cruzi survival. hemovigilance monitoring of platelet septic transfusion reactions (str) after treatment with intercept tm pathogen reduction or large volume, delayed bact/alert tm bacterial culture screening richard benjamin* 1 , marion lanteri 2 and larry corash 1 . 1 cerus corporation, 2 scientific affairs department, cerus corporation background/case studies: amotosalen/ultraviolet a (uva) light (inter-cept tm blood system, cerus corporation) pathogen reduction (pr) and delayed, large volume, bacterial culture with the bact/alert tm system (dlvbc) (biomerieux, inc) represent respective best-in-class systems to reduce the risk of str associated with platelet concentrates (pc). where implemented, hemoviligance (hv) programs continue to receive reports of suspected str, most of which have low imputability as other causes are more likely or insufficient information is available to impute system failure. study design/methods: united kingdom (2006 -2015 ), french (2006 -2015 , swiss (2011 -2015 ), and belgium(2009 -2015 hv reports, and cerus corporation's adverse event records were reviewed to assess the residual risk and imputability of str with amotosalen/uva-treated or dlvbc-screened pc. results/findings: approximately 1.35 million dlvbc-screened were issued with a 7 day outdate after release into inventory 3 days after collection, and $2.3 million amotosalen/uva-treated pc were released into inventory on day 1 or 2, with a 5 to 7 day shelf-life. no septic fatalities were reported with either technology. the french, belgium and swiss hv programs monitored >2.83 million conventional, non-dlvbc-screened pc and recorded 58 str and 9 fatalities. concurrently, zero definite and 2 possible str were reported with 607,871 amotosalen/uva-treated pc, significantly fewer than with conventional pc (table 1 ) (20.5 str per million vs. 0.0 per million, p<0.001). one definite, 1 possible, 7 undetermined/indeterminate non-fatal str and 5 contaminated "near miss" pc were reported with 1.35 million dlvbc-screened pc between 2010 and 2015, for a reduced falsenegative rate compared with the prior five years (3.7 str per million vs. 16.3 per million, p <0.05). hv programs highlight a major weakness when reporting str. stringent criteria are used to determine definite imputability, including evidence of patient infection, pc contamination and irrefutable evidence of a donor source, with confirmation of strain identity. reports with incomplete investigations are considered undetermined or indeterminate, or possible sepsis. some of these cases are almost certainly due to bacterial contamination of pc, suggesting that the actual rates of sepsis are considerably higher than that reported by hv programs. conclusion: best-in-class pathogen reduction and bacterial culture systems reduce str risk, although underreporting and inadequate clinical data may result in underestimation of the true rates. pathogen reduction of background/case studies: despite extant mitigation measures (e.g. diversion pouches and primary platelet culture at the collection facility), bacterial contamination of platelets and associated septic transfusion reactions remains a leading cause of transfusion-associated fatalities in the united states (us). consequently, the us food and drug administration has recommended adoption of additional measures such as point of release testing (port) and/or pathogen reduction to safeguard against transfusionassociated sepsis. however, port poses logistical challenges, particularly in institutions with high-volume platelet utilization, while pathogen reduction is a high cost intervention. we evaluated a second bacterial culture to contend with residual risk. study design/method: phased implementation of secondary bacterial culture testing (bact/alert tm ,biomerieux, inc., durham, nc) was initiated in october 2016 for all platelets received at our institution. at time of receipt at the blood bank (day 3 post collection), products were sampled using a sterile connection device (tscd tm , terumo, elkton, md) and a sampling kit (sam-plok tm sampling kit, 10 ml, itl biomedical, malaysia). five mls of product was transferred aseptically to bact/alert bpa (aerobic) culture bottles using the same sampling device. inoculated culture bottles were loaded into the bact/alert incubator modules and incubated at 35c for three days. results/finding: a total of 9473/11,066 (85.6%) platelet products were successfully cultured (934/1373 [68.03%] and 1842/1912 [96.3%] in october 2016 and march 2017 respectively). over the 6-month period, two true positive cultures were obtained (incidence of 1 in 4736 platelet products). the cultures grew acinetobacter species (case a) and coagulase negative staphylococcus species (case b); both positive results were obtained four days following collection. repeat testing of cases a and b grew the same organisms identified in the initial cultures. there was a co-component in our inventory (case a) with negative initial and repeat cultures. none of the products were released for transfusion. the initial post-collection product cultures remained negative at the collection facility. over the same time period, no false positives were detected. implementation required hiring one additional dedicated fte; the total cost (technologist time, equipment and related supplies) was calculated to be $us16.83 per product tested. the cost per averted case was $us79,707. conclusion: we demonstrate the feasibility of implementation of a secondary bacterial culture test of apheresis platelets to interdict bacterially contaminated units and prevent septic transfusion reactions. this presents a low-cost strategy (as compared to pathogen reduction) to mitigate risk of septic transfusion reactions. importantly, it offers a viable alternative to port in high volume institutions where logistic (e.g. time and personnel) constraints impede practical adoption of port. an increase in cases of blood culture positive transfusion reactions (bcptr) was noted at our hospital; bcptr was defined as bacterial culture positivity in the transfusion recipient and/or associated transfused blood product during investigation of a transfusion reaction. we sought to characterize the risk and clinical presentation of bcptr at our institution. study design/method: an analysis was conducted of all reported transfusion reactions at johns hopkins hospital (jhh) between january 2009 and december 2016. the data, extracted from hemovigilance records, were evaluated to determine the incidence of bcptr; the severity and symptoms were evaluated in concordance with recipient data, including patient diagnosis, medications and clinical manifestations of the reaction. bacterial culture results were evaluated for both patients and associated blood products (i.e. partially transfused or residual product in blood bag). results/finding: in the 7-year study period, a total of 3280 transfusions reactions were reported, 18 of which were bcptr (0.55% of transfusion reactions). of the 18 bcptr, 15 (83%) were associated with apheresis platelets, 2 (11%) with red blood cells, and 1 (6%) with plasma. recipient diagnoses spanned hematologic/oncology (n512), renal (n53), cardiac (n51), autoimmune (n51), and obstetrics (n51). an organism was identified in both the blood product and recipient in 10 (56%) cases; in 6 (33%) cases an organism was grown in the blood product but not the recipient; and in 2 (11%) cases an organism was isolated from the recipient only, due to inability to culture the product. the transfusion recipients in 5 of the 6 cases that did not isolate organisms in the recipients were on broad-spectrum antibiotics at the time of transfusion. symptoms of bcptrs included fever (83%), chills (67%), nausea and vomiting (50%), pain (27%) and dyspnea (22%). blood pressure (bp) decreased in 22%, increased in 17%; 61% of reported bcptrs had no change in bp. conclusion: the signs and symptoms of bcptrs are not specific and overlap both with underlying disease as well as other types of adverse transfusion associated events, thus contributing to delayed diagnosis and under-reporting. furthermore, high rates of antibiotic use in transfusion recipients can mask symptoms of true septic transfusion reactions. hospitals should consider expanding the clinical indications for culturing blood components that are implicated in transfusion reactions. furthermore, excessively stringent criteria (cdc/nhsn blood safety surveillance) for transfusion-transmitted infection, may contribute to misclassification of septic events in some recipients, particularly if on antibiotics. clinical oral abstract session: immonohematology and genetics --sickle cell disease and beyond blindspots and cross-reactivities of anti-human globulin specific for igg subtypes heather howie 1 , jenna lebedev 1 , linda kapp 1 , xiaohong wang 1 , meghan delaney 2 , lay see er 1 and james c zimring* 3 . 1 bloodworksnw research institute, 2 bloodworks nw, 3 university of washington school of medicine background/case studies: there are four different subclasses of human igg (igg1-igg4), each with different effector function. essentially all existing data on the effect of igg subclass on hemolytic transfusion reactions and hdfn, were generated using ahg specific for igg subclasses. in recent decades, it has become appreciated that there are at least 29 natural human variants of igg. in this study, the reactivity of igg specific ahg was tested against all 29 known variants. study design/methods: the heavy and light chain variable regions of an anti-k1 monoclonal antibody were sequenced and cloned into expression plasmids that fused variable regions (in frame) with each of the known 29 igg variants. plasmids were expressed by co-transfection into cho cells. the resulting panel of antibodies were pre-incubated with k11 rbcs and were then subjected to testing with currently available igg subtype specific ahg (monoclonal ahgs from southern biotech and sanquin, polyclonal ahgs from sanquin and the bindingsite). all testing was carried out by flow cytometry. results/findings: polyclonal reagents against igg2, igg3, and igg4 had cross-reactivity with variants found in other igg subclasses, and specific amino acids responsible were identified by site directed mutagenesis (table 1). titrations of the ahgs did not identify a dilution at which crossreactivities were lost, but authentic targets were still detected. however, cross-reactivity could be neutralized by pre-incubating ahg with the crossrecognized igg forms (against a third party antigen); the remaining reactivity recognized the intended igg subtype without detectable cross-reactivity. no cross-reactivity was detected for polyclonal anti-igg1 or for any of the monoclonal ahgs tested. monoclonal anti-igg3 had a blindspot for igg3-04, due to the shorter hinge region on igg3-04. no blindspots were detected in other monoclonal or polyclonal ahg. conclusion: the relative quantitation of different igg subtypes has been studied in multiple immune settings, and plays important roles in diagnosis and research of human disease, including immunohematology. herein, we demonstrate that the reagents used to generate this body of knowledge suffer problems of cross-reactivities and blindspots. as such, the existing data regarding igg subtype biology may have some inaccuracies as a result of these defects in igg specific ahg. genotype matching for pediatric sickle cell disease patients nancy robitaille* 1 , yves dominique pastore 1 and maryse st-louis 2 . 1 chu sainte-justine, 2 hema-quebec background/case studies: among the different treatment modalities available for sickle cell disease (scd), blood transfusion is frequently used. however, alloimmunisation remains a significant problem, even if prophylactic antigen matching is performed for c, e and kell antigens. this is partly explained by different antigen frequency among caucasian blood donors and african-american recipients, and by variants in the rh blood group of people of african-descent. blood group genotyping has been proposed as a potential way to alleviate this problem. the scd cohort of a pediatric academic hospital was genotyped for rhd, rhce and fy genes. the primary objective of our study was to evaluate whether compatible genotyped blood donors presenting similar rh variants could be identified. study design/methods: since 2008, our local blood provider intensified recruitment of african-descent blood donors. these donors were phenotyped and genotyped for clinically relevant antigens by different means: genomelab snp stream, laboratory-developped assays and idcorext. as of 2014, 205 scd children were genotyped by sequencing rhd, rhce and fy cdnas after obtaining informed consent. extended red blood cell phenotypes were done at diagnosis at the hospital. patients' genotypes were compared to h ema-qu ebec's donor database to attribute blood donors to specific patients. results/findings: from diagnosis until september 2016, 117 (57%) patients had been transfused and 14 had antibodies with known blood group antigen specificity: anti-c, anti-e (2), anti-hrb, anti-fya, anti-jka, anti-jkb (2), anti-s, anti-m, anti-sc2, anti-leb (2). seventeen patients (8.3%) were either d2 or partial d. rhce results showed that 163 patients expressed a normal c antigen and 32 expressed partial c. as for e antigen, 163 had a normal antigen, 38 bore a partial antigen and 3 were weakly expressed. fy(a2b2) phenotype was found in 182 (89%) patients. a total of 2606 genotyped blood donors of african-descent were available. the table below indicates the compatibility with these donors. conclusion: this study shows that several patients have rhce variants difficult to match, even with available genotyped blood donors from their community. although this measure is probably beneficial to decrease alloimmunisation, a larger donor pool is still needed to fulfill the patients' needs. the continued effort put towards recruitment and pheno/genotyping should improve the situation. using genetic markers to select responders and non-responders sickle cell disease (scd) patients for transfusion with rh haplotype matching red blood cell (rbc) units tamires delfino dos santos 1 , emilia sippert 1 , mayra dorigan de macedo 1 , sheila fatima perecin menegati 1 and lilian castilho* 1,2 . 1 hemocentro unicamp, 2 university of campinas background/case studies: rbc alloimmunization has been associated with several factors and with individual characteristics of each patient. we recently found that tnfa-308a, il1b-511t cytokine polymorphisms, rhag 808g>a and hla-drb1*15 alleles may predict a good responder phenotype (sippert et al, transfusion 2017) and that rhag 808a and hla-drb*15 alleles are closely linked to rh alloimmunization. based on this and considering the challenge to fulfill the transfusion needs of the patients with rh variants, we used these genetic markers to select responders and nonresponders scd patients for transfusion with rh haplotype matching rbc units and evaluated the risk of alloimmunization. study design/method: our study included 96 non-alloimmunized patients with scd, homozygous for hbs, receiving a range of 5-289 rbc units. rbc antigen phenotypes of each patient and history of rbc antibodies were obtained from the medical records and transfusion service computerized database. rbc genotyping was performed using whea, wrhd and wrhce beadchip arrays (bioarray solutions, immucor) in accordance with the manufacturer's instructions. cytokine gene polymorphisms (tnfa-308g>a, il1b-511c>t) and the rhag 808g>a gene polymorphism were analysed by pcr-rflp and taqman assays. hla class ii genotyping was performed using pcr-sso. results/finding: among 96 non-alloimmunized patients, 21 were homozygous or compound heterozygous for rh variant alleles. from those, 6 had rhag 808a and/or hla-drb*15 alleles and at least one cytokine polymorphism (tnfa-308a or ilb1-511t) associated with risk of alloimmunization and were transfused with extended and rh haplotype matching rbc units. the other 15 patients with no risk factors associated with rbc alloimmunization were considered non-responders and were not transfused with extended and rh matching units. all patients were followed for one year and did not develop rbc antibodies. conclusion: these findings contributed to the development of a transfusion strategy for non-alloimmunized scd patients as typing for these polymorphisms could potentially help in the classification of responder and nonresponder scd patients, allowing blood with high level of compatibility to be five discrepant samples required sequencing. id core xt identified three rhce*cear samples encoding a partial c, and a partial e (predicted phenotype: vweak, vs-) and 2 were confirmed by sequencing. the third sample was found to be rhce*cevs.01,rhce*cebi on sequencing (predicted phenotype v1,vs1). the 3 samples were typed as v1 (or ce s ) and vs1 (or e s ) by hea. in addition, id core xt accurately identified rhce*ce[712g]in 2 samples. this snp has been linked to various allelic variants affecting c and e antigenic expression. both samples were predicted to be c1 by hea. conclusion: blood group genotyping platforms vary depending on the specific snps that are included in each assay. such variations may be clinically significant when genotyping is used as a tool for providing matched blood. discrepancies leading to differences in the predicted phenotype could affect unit selection. despite the discrepancies between the 2 methods, the high concordance rate and the limitations of serology warrant further reconsideration for the need for serologic confirmation of extended phenotypes. background/case studies: over three decades ago, two independent groups published work suggesting a novel categorization of warm autoimmune hemolytic anemia (waiha) on the basis of dat scores of agefractionated rbcs: type i waiha, comprising 80% of patients, showed increased binding of autoantibodies to aged rbc, whereas type ii waiha autoantibodies (20% of patients) bound young and old rbcs with no apparent prejudice. band-3 is a ubiquitously expressed rbc transmembrane protein which plays a vital role in maintenance of rbc structural integrity, cellular hemostasis, and regulation of senescence; and, has been suggested to be targeted by autoantibodies from patients with waiha. band-3 is regulated through phosphorylation of key residues; its hyperphosphorylation is a hallmark of normal rbc senescence, which causes band-3 to disengage from the cytoskeleton, increasing its lateral diffusion, thereby permitting the formation of band-3 aggregates forming new epitopes which are recognized by natural igg autoantibodies causing phagocytosis and destruction of senescent rbcs. type i waiha has been postulated to be caused by an exacerbation of normal rbc senescence. study design/methods: in an effort to confirm and characterize the two waiha subtypes we age-fractionated whole blood samples from 22 patients with waiha on discontinuous percollv r gradients and looked for differences in dat results between less (young rbcs) and more dense (aged rbcs) fractions, fractionation patterns and band-3 tyrosine phosphorylation. results/findings: we confirm that two distinct types of waiha can be identified based on autoantibody reactivity with the youngest and oldest autologous rbcs. further, comparing 5 type i and 5 type ii patients, we found that type i is characterized by 5 percollv r fractions (similar to healthy storage-matched controls) but increased band-3 tyrosine phosphorylation compared to healthy storage-matched controls, with phosphorylation occurring during younger stages of rbc development. type ii patients were characterized by 3-4 percollv r fractions, lacking the fraction containing the oldest rbcs, and showed a complete lack of, or dramatic decrease in, band-3 tyrosine phosphorylation compared to healthy storage-matched controls. conclusion: these results confirm the two distinct types of waiha. in type i waiha, the increased binding of autoantibodies to older rbcs coupled with increased tyrosine phosphorylation of band-3 suggests that rbcs from type i patients are aging faster than rbcs from normal healthy controls; this may represent an accelerated and pathogenic form of normal rbc senescence. in contrast, type ii waiha where autoantibodies bind strongly to either young or old rbcs coupled with a lack of fractionated bands that represent the oldest rbcs and a dramatic diminution in tyrosine phosphorylation of band 3 suggests faster destruction of rbcs, consistent with the early published data, and metabolic changes that could affect rbc function. microbial pathogen primary sequence correlates with blood group antigen immunogenicity ian baine* 1 , burak bahar 1 , jeanne hendrickson 2 , krystalyn e hudson 3 and christopher a tormey 1 . 1 yale-new haven hospital, 2 yale university, 3 background/case studies: it is known that specific groups of patients immunologically respond more readily than others to rbc antigens. while rbc antigenic differences between donors and recipients are required for humoral immune responsiveness, other variables are also involved. studies have shown that there is significant primary sequence identity between common rbc antigens and microbes, and that cross-reactivity is possible between antigens in experimental models. we hypothesize that responder populations may be immunologically primed to form rbc alloantibodies via environmental exposure to cross-reactive microbial antigens, and that such a correlation may be linked to observed blood group antigen immunogenicity. study design/method: we performed peptide homology searches of the most immunogenic rbc antigens, based on previously published antigenicity findings. thirteen amino acid peptides containing the polymorphic residues of k, jk a , lu a , e, c, m, c, fy a , and s antigens were queried for identity with microbial peptides using the blast database (blastp, pam30 abstract algorithm, e value51x10 -6 , word size5 6, gap costs: existence59 exten-sion51). search results were restricted to bacteria and fungi, with a selective threshold of >80% identity set for inclusion criteria. to corroborate with observed patient data, we also examined preceding cultures from 162 alloimmunized patients to explore agreement between specific pathogens and rbc alloantibodies. results/finding: significant peptide identity was found between rbc antigens and pathogenic organisms including b. fragilis, p. aeruginosa, candida spp. among others. linear regression analysis of the number of genuses in microbial kingdoms meeting inclusion criteria showed a statistically significant inverse trend in predicting the degree of immunogenicity when fy a (an outlier) was removed (b5-0.0017, r 2 50.624 & p50.0197); that is, lower immunogenicity antigens were associated with larger number of kingdoms. k-medoids cluster analysis comparing immunogenicity and kingdoms showed that antigens clustered to low (c), moderate (e, c, s, m) and high (k, jk a , lu a , fy a ) immunogenicity groups, suggesting that an antibody response is inversely associated with environmental antigenic prevalence. of 162 alloimmunized patients reviewed, 105 were culture-positive. of these, 76% of the anti-c/c group (13 of 17 patients) and 16% of the anti-k group (7 of 43 patients) had microbe-antibody agreement. remaining microbe-rbc antibody agreements ranged from 0 -11.1%. overall, 21.9% (23 of 105 patients) demonstrated agreement. interestingly, we observed a particularly strong agreement between infection with klebsiella species and anti-k, despite the lack of >80% sequence identity. while 27.6% (29 of 105) patients reviewed had positive cultures for klebsiella species, 62.1% of these (18 of 29 patients) demonstrated an anti-k. conclusion: our study highlights the potential connection between microbial infection and rbc alloimmunization, based on shared epitopes. we speculate that low-level antigenic exposure to highly prevalent microbial antigens such as commensals may promote immunotolerance, providing a model for the inverse relationship between rbc antigen immunogenicity and prevalence of microorganisms. longitudinal studies of microbial carriage (or acute microbial infection) and rbc alloimmune responses in larger patient cohorts may be informative. background/case studies: thromboelastogram (teg) has been incorporated into many hospital armories to manage transfusions during cardiovascular (cv) surgeries. some institutions use well-defined protocols for teg utilization at different stages of surgery (baseline, rewarming, postprotamine, and post-operative). on the other hand, at some institutions teg utilization is driven mainly by clinical judgment. when teg is ordered based on clinical judgment (clinical bleeding in most cases), some patients receive blood transfusions before teg is performed. there is no published literature on how pre-teg transfusions impact teg results and guide further transfusion requirements during cv surgeries. in this study, we have tried to address this issue. study design/method: we retrospectively reviewed 109 tegs performed on 76 patients undergoing cv surgeries at our institution from jan 1 to dec 31, 2016. no specific teg protocol was used to direct transfusions (plasma, platelets, and cryoprecipitate) during that period. only the first teg performed during surgery was included in the analysis. we excluded the patients that received only red blood cell (rbc) transfusions during the surgery because rbc transfusions are usually not based on teg results. for the 56 tegs analyzed, teg results were divided into three categories: "normal" (reaction time (r), kinetics (k), angle (a), maximum amplitude (ma), and lysis at 30 minutes (min) all within reference range), "hypocoagulable" (r>10 min, k>3 min, a<53 degrees, ma<50 mm) and "hypercoagulable" (r<5 min, k<1 min, a>72 degrees, ma>70 mm). fisher's exact tests and z-scores for two population proportions were used to identify statistically significant differences in teg results and blood product utilization. results/finding: out of 56 tegs analyzed, 37 patients (66%) received pre-teg transfusions. we found significantly fewer hypocoagulable teg results in pre-teg transfused patients than nontransfused patients (8% vs. 32%, p50.02). the data also reflected a trend suggesting that there may be more normal teg results in pre-teg transfused patients compared with nontransfused (32% vs. 11%, p50.07). there was no statistically significant difference in transfusions after obtaining teg results in both groups. however, there was a trend suggesting that hypocoagulable state was more likely to be corrected by transfusion in patients who were already transfused pre-teg compared to nontransfused (100% versus 33%, p50.06). conclusion: pre-teg transfusions impact teg results (transfusions correct/normalize coagulopathy) but do not significantly impact further blood product utilization during cv surgeries. the decreased threshold (more transfusions) for correcting hypocoagulable state in patients who already received pre-teg transfusions may be due to more clinical significant bleeding in these patients to begin with. background/case studies: orthotopic liver transplantation (olt) is associated with significant blood loss, due to the complexity of the procedure and extensive liver vascularity, demanding blood transfusion. in this setting, cell salvage autotransfusion (cs) is been used as an alternative to decrease allogeneic red blood cell transfusion. however, as long as some studies have shown that cs in olt decreases allogeneic blood transfusion, others reported that cs presented little benefit or might have been associated with increased blood loss through fibrinolysis. in this study, we evaluate cs efficacy in reducing allogeneic blood transfusion in the intraoperative period. study design/method: we retrospectively evaluated data from 670 liver transplants, performed from 2011 to 2015 in a single-center. patients were divided in two groups: one with cell salvage (cs) and another without cs (ncs). study endpoint included the requirement of allogeneic blood components transfusion during intraoperative period in both groups. cs was used in all liver transplant recipients but patients with malignancy and sepsis. blood transfusions were indicated based on clinical and hemodynamic criteria. clinical data included age, gender, diagnosis, body weight, height, warm and cold ischemic time and model for end-stage liver disease (meld) score. statistical analyses were performed using t-test, chi-square test, mann whitney test. results/finding: in this study period, 670 olts were performed. a total of 345 patients was submitted to cs. the median age was 51 years (range 10-78 yo). cirrhosis caused by chronic hepatitis c virus infection was the main etiology of liver disease. hepatocellular carcinoma (hcc) was found in 31,6% of the patients. the average meld score was 29,6 6 9,4 and it was slightly higher in the cs group (31,3 vs 27,9, p<0,001) . there was no statistically significant difference in other variables such as body weight, height and cold ischemic time. the mean salvaged blood volume was 8856 6 4503 ml and mean reinfused blood volume was 914 6909 ml. allogeneic blood transfusion was required in 71,8% patients in the cs group, compared to 46,7% patients in the ncs group. however, average red blood cells (rbc) and fresh frozen plasma (ffp) units transfused were lower in the cs group. the threshold for rbc transfusion was significantly lower in the cs group (2,4 units vs 3,39 units, p<0,001 background/case studies: hemorrhage is a leading cause of mortality in trauma patients and morbidity in non-trauma patientsaddin en.cite.data. massive transfusion protocols (mtp) reduce mortality in trauma and nontrauma settings; however, this may be at the cost of blood product wasta-geaddin en.cite.data. blood product wastage benchmarks are loosely established, and data on wastage associated with mtps especially sparse. with a redesign of mtp and obstetric massive transfusion protocols (obp) which have different blood product preparation schedules, we assessed wastage, delivery method, and product utilization to identify differences in wastage during these protocols. study design/method: following institutional review board approval, a retrospective study on blood product wastage associated with the mtp and obp between july 2015-december 2016 was performed. data on numbers of products dispensed and wasted were manually collected from transfusion service paper and electronic records and an automated data report from the electronic medical record. results/finding: the mtp resulted in higher total number of wasted products than the obp (27 and 15 products, respectively) however, obp wastage occurred more frequently in the 18 month period. this reflects automatic thawing of cryoprecipitate in the first round of deployed products in the opb. mtp-trauma activations contributed higher wastage than non-trauma activations (23 versus 4 products). this is skewed by one month when 20 products were wasted due to expiration of product on the floor. cooler-related issues (6) and products dwelling too long out of a controlled environment (5) were common reasons reported for wastage. the overall product wastage rates for mtp: trauma, mtp: nontrauma, and obp were 1.7%, 0.3%, and 2.3%, respectively, with a total exsanguination protocol waste rate of 1.33%. the difference between the overall proportion of waste between the mtp and obp protocols was insignificant (p50.176). conclusion: wastage associated with both protocols was low and there is no statistical difference between mtp versus obp wastage. coolerrelated issues accounted for most product wastage, allowing for targeted waste reduction strategies including educational outreach and improved product delivery methods. better documentation of waste events identifies wastage trends for further product utilization optimization during these protocols. a 17 year old female with multiple gun shots was admitted to a level one trauma center and received uncrossmatched group o, rh negative (d-) red blood cells (rbcs) through a rapid infuser during resuscitation. transfusion of uncrossmatched products before sample collection can lead to errors and confusion in blood typing, as can the venipuncture site used for collecting the patient's blood sample. the current fda guidance and aabb standard of two samples for determination of blood type to prevent cases wrong blood in tube (wbit) or electronic identification systems do not always catch or clarify these errors. study design/methods: patient was tested by manual tube method. two different technologists using two different reagent racks performed initial testing with matching results. results/findings: two samples were collected during resuscitation from the patient and typed as o d-. patient was transfused with 5 units of o d-rbcs before stabilizing. two days later another sample was collected and typed as o rh positive (d1) with mixed field being seen on the anti-d. a weak d testing was performed to see if the negative result with anti-d could be strengthened through incubation. both original samples still resulted as d(table a) . after consulting the patient care team it was discovered the samples were collected above the iv site after one unit had been completed and while the second unit was being transfused. it was also discovered all other clinical laboratory samples were rejected due to possible line contamination when results for the sodium, potassium, and glucose appeared inaccurate. the transfusion service laboratory is in a different area of the hospital and was unaware those samples had been rejected. conclusion: the initial samples were collected above the iv site and were contaminated with the d-blood product being rapidly transfused during resuscitation. the samples collected during the initial trauma response should have been rejected and a request made for samples drawn below the iv site. because both samples were collected while the unit was being transfused, contamination was in both. use of a handheld barcode system would not have caught this error because the patient had been correctly identified. future prevention of the above anomaly would be the education of transfusion testing staff to recognize an abnormal high hematocrit: secondly reminding the staff collecting samples to be aware of the proper collection procedures for laboratory testing, which would include type and screen. facilities also should strive to perform collection of the confirmatory sample from a completely different venipuncture site. impact of cell saver usage during solid organ transplants at a major institution holly ross* 1 , edward smith 2 , thomas brown 2 , foeks jeremy 2 , metcalf suzanne 2 , james johnson 2 , peter davis 2 , karafa sw badjie 1 and abba zubair 1 . 1 department of laboratory medicine and pathology, transfusion medicine, mayo clinic, 2 department of anesthesia, mayo clinic background/case studies: our institution performs an average of 398 solid organ transplants (sots) yearly. transfusion support for transplants can be tremendous, accounting for a large percentage of red blood cell (rbc) transfusions annually. even the best practices for allogeneic transfusion are not without risk. transmission of pathogens is possible with even the strictest screening methods, and each transfusion increases the risk of alloimmunization. the advent of intraoperative blood recovery has reduced the need for allogeneic donor rbcs during surgeries expected to bleed heavily. with the cell saverv r (haemoneticsv r , braintree, ma), patients' own blood shed during surgery is collected, washed, concentrated, and reinfused, lessening the need for transfusion support. this study sought to examine the amount of allogeneic donor rbc units saved during sots through the use of the cell saver for intraoperative blood recovery. study design/methods: data was collected for sots which utilized the cell saver. these included liver, liver/kidney combination, lung, and heart transplants. data a 29 y.o. female was admitted to the trauma department after a motor vehicle collision (mvc) and transfused 2 o(1) rbc units from the kiosk. her blood type was determined as o(-) with a negative rbc antibody screen (as). she was transfused 10 more units of o(-) rbc. two months later, a repeat as identified two new rbc alloantibodies, anti-d and anti-e. the anti-d formation resulted from the 2 o(1) rbc transfused from the kiosk, but the source of the anti-e was undetermined since e antigen is expressed in 1% of rh(-) individuals. the trauma department staff was notified of delayed serologic transfusion reaction and asked to investigate further since a 29 y.o. female patient should not have received o(1) rbcs. study design/method: an investigative plan was developed by the trauma staff involving a patient census, review of the chart and kiosk inventory, obtaining feedback from clinical providers, and review of information provided by emergency services (ems). results/finding: the trauma unit was busy with 10 admissions during the 5 hours preceding the patient's arrival. the chart review found the following physical attributes; patient was overweight (107 kg) with obvious facial deformities from the mvc, that compromised age assessment. it was determined that the kiosk was fully stocked with both o(-) and o(1) rbc units. one clinical provider recalls that the patient identification (id) might have been unknown. review of the ems communication states "patient is a 50 y.o. female." conclusion: use of visual examination to determine age was significant in the selection of o(1) rbc for this patient. the trauma staff proposed and implemented a change in policy to prevent future incidents. any female patient that arrives without id or written confirmation of age will be transfused o(-) uncrossmatched rbc until a blood type can be determined. after being notified of the incident, the trauma staff took the lead in investigating and providing a process improvement resolution. this is credited to the excellent collaborative relationship between the transfusion service and trauma department on ensuring patient safety during emergent, uncrossmatched rbc transfusions. rate of abo/rh confirmation in outpatient pelvic organ prolapse surgery alexis r peedin*, taylor brueseke, yara park and jay s raval. university of north carolina background/case studies: approximately 375,000 surgeries for urinary incontinence or pelvic organ prolapse (pop) are performed annually. for abdominal pelvic floor disorder (pfd) surgeries, transfusion rates historically range from 6-16%, whereas transfusion rates for vaginal and robotic pfd surgeries range from 0.2-1.6% and 0.3-1.4%, respectively. since the implementation of college of american pathologists (cap) requirements for abo/ rh confirmation, approximately 15% of patients who receive a transfusion in our hospital required a second abo/rh specimen to be drawn; however, limited data are available regarding the impact of this new requirement on patients preparing to undergo outpatient surgery that currently require preoperative type & screen (t&s). the primary objective of our study was to assess the rate of abo/rh confirmation in women who underwent outpatient pop surgery. study design/method: this was a planned secondary analysis of a retrospective cohort study of consecutive patients undergoing pop surgical repair from may 2015 -may 2016 in our academic tertiary care institution. among this sample, patients were excluded if their first t&s was drawn before our institution implemented the abo/rh confirmation requirement. fisher's exact test was used, and statistical significance was defined as p<0.05. results/finding: we identified 66 patients for analysis, of whom 65 (98.5%) had a preoperative t&s ordered. two (3.1%) of these 65 patients had positive antibody screens; one patient had an anti-k and one had a warm-reacting autoantibody. fifty-nine (90.8%) of the 65 patients required a second abo/rh specimen per hospital protocol; 51 (86.4%) of these actually had a second specimen drawn. in patients for whom abo/rh confirmation was indicated, there were no differences between those who did and did not have abo/rh confirmed when comparing age, body mass index (bmi), pre-operative hemoglobin (hgb), or surgical approach (table 1) . no abo/rh discrepancies were identified. one patient received 1 unit of red cells after abdominal pop surgery. conclusion: the rate of requiring abo/rh confirmation before pop surgery was markedly higher than that seen in all patients receiving transfusions at our institution (90.8% vs. 15%, respectively). because the vast majority of women undergoing vaginal or robotic pop surgery are not transfused perioperatively, hospital transfusion services should consider eliminating routine pre-operative t&s for this low-risk population in the maximum surgical blood ordering schedule, avoiding this unneeded test and subsequent abo/rh confirmation. volume reduction of red cells to reduce transfusion-associated adverse events related to hyperkalemia maressa t pollen*, laura knicks, linda van tol and c. michael knudson. background/case studies: one attribute of older blood is an increase in supernatant potassium level which can contribute to transient hyperkalemia. this problem is exacerbated in conditions of massive transfusion and in patients with renal failure. washing rbcs can effectively remove free potassium but is time consuming and can often only be performed on one unit at a time. here, we estimate the amount of potassium that is removed by volume reduction of red cell units. we also examined whether this technique would be feasible in the setting of massive transfusion in a patient with hyperkalemia. study design/method: expired or over temperature units (n527) that had been removed from inventory were utilized for these studies. each unit was weighed and a volume reduction procedure was performed. the supernatant was weighed and the potassium of the supernatant was measured using routine laboratory assays. for all formulas, weight was converted to volume using a specific gravity of 1.05 g/ml. the hematocrit (hct) of the volume reduced rbc was measured using a sysmex xs-1000i instrument. the percentage of supernatant removed was calculated by dividing the residual supernatant in the volume reduced unit (rbc hct x rbc volume) by the total supernatant prior to the procedure (residual supernatant 1 removed supernatant). the remaining free potassium (meq) was calculated as the (concentration of potassium in the supernatant (mmol/l) x the estimated red blood cell residual supernatant volume. to simulate the process that would occur in the setting of a massive transfusion protocol (mtp), 5 units were subjected to the volume reduction while recording the time needed to process all 5 units. this was performed twice for a total of 10 units processed in this manner. results/finding: the volume reduction procedure reduced the supernatant volume by an average of 72% (range 49%-87%). in units between 21 and 42 days (n510), the estimated mean residual k1 was 1.89 meq (range 0.61 to 2.21). in the two mock mtp trials, the time to complete the procedure was approximately 50 minutes and we estimate an additional 5-10 minutes would be required to modify and issue the units in our lis/emr. conclusion: a manual volume reduction protocol in red cell units significantly reduces the amount of potassium administered in a unit of red cells. this procedure may be useful when only older red cell units are available for a patient at risk for hyperkalemia. the procedure can be performed in less than one hour and may be useful under the conditions of massive transfusion. processing, cryopreservation, and non-specialized hospital collection. preliminary studies of three shipping conditions after collection were tested using sterile containers with sterile normal saline (ns) alone, ns plus antibiotic/antimycotic (ab/am) and a dry container. prolonged exposure to ab/am solution retarded outgrowth of mscs, but control of microbial growth in cultured tissue samples was needed. these findings were used to construct a validation study. study design/methods: a validation study designed to test procedures to collect, transport, process, and store umbilical cord tissue was measured by post-thaw outgrowth. collected uc tissue from consenting mothers was transported to the distant lab in validated shipping containers in a dry, sterile cup from vaginal (3) and caesarian (2) births. uc collections were divided into 3 segments to test 3 conditions. segment explants were placed on 0.1% gelatin-coated gridded tissue culture plates (32 explants per plate) in enriched medium specified for msc outgrowth containing antibiotic only with an endpoint of 21 days. growth was scored as the number of squares with explants exhibiting outgrowth compared to the total planted explants. one segment (fresh control) was dissected and planted without further processing. the 2 remaining tissue segments were soaked in (ab/am) saline solution for 1 hr and 24 hrs at 48c, respectively. tissue segments were frozen in cryo bags with a proprietary 10% dmso/large molecular weight sugar solution. background/case studies: it has been the practice in our institution to process 3 or 4 times the total blood volume (bv) of the patient, up to a maximum of 25 liters (l) per procedure, to obtain peripheral blood cd341 stem cells. as a consequence, a patient often would need to spend 6 hours or more on the machine. it would be desirable to be able to specify the exact volume of blood to process to achieve the desired cd341 cell yield, thus minimizing the patient's time on the machine, the nurse's time performing the procedure, and the number of bags that have to be submitted for cryopreservation and storage. study design/methods: our institution recently implemented the new spectra optia cmnc collection protocol, a continuous flow and continuous collection procedure that uses the automated interface management (aim) system to precisely manage the separation interface. an analysis of our 2016 collection data suggested a highly reliable collection process, so a prediction algorithm (pa) based on the linear regression between the patient's cd341 pre-count and cd341 yield, normalized per liter of blood processed, was derived utilizing the patient's cd341 pre-count, the patient's weight in kilograms (kg), and the target cd341 dose/kg. this pa calculated the exact volume of whole blood to be processed to achieve the requested dose of peripheral cd341 stem cells. the initial equation was modified to add an additional 15% to the predicted volume, to account for the natural variability of the process. this pa was then tested prospectively in the clinical setting. results/findings: in 8 patients, representing both allogeneic and autologous donors, the average blood volume processed was 14.8 l. the range was 4.9 l -21.6 l. the target dose was achieved in all patients. our previous practice for these 8 patients would have required, assuming a standard 4 bv procedure, processing an average of up to 28 l per patient, with a range of 20-62 l. to quantify how well the new pa works, it was decided to evaluate the ratio between actual and predicted volume vs. the ratio between the actual and expected cd341 yield. the result was a high correlation between these two ratios (r 2 5 0.92), indicating that the algorithm produces very consistent results. conclusion: the predictability of our collection process during the time period analyzed was a robust r 2 5 0.92, confirming the findings in the first data analysis. the blood volumes processed and patient time on the machine decreased substantially, with some patients only needing 2 hours or less to achieve their target dose. nurses and lab medical technologists have seen a dramatic change in their workflow. the number of bags to process has dropped for the lab, with the consequent freezer space savings and the shorter collection times allowing the lab medical technologists to finish with their work earlier in the day. all in all, implementation of this pa has produced huge increases in patient and provider satisfaction. important factors that likely contributed to the success of the protocol included the precision and consistency of the aim system of the apheresis device, as well as the small number of nurses (1-2) who performed the procedures, resulting in less variability. the economic impact of this pa has not been quantified, but might be an interesting area for future studies. background/case studies: zarziov r , a biosimilar granulocyte colonystimulating factor (g-csf) has recently been introduced into clinical practice. its use has stimulated a certain debate regarding their possible less efficacy and security on cd341 mobilization. the aim of this study is to evaluate if there are differences between good and bad mobilizers and assess the need for plerixafor when a biosimilar as g-csf is used. study design/method: we retrospectively evaluated autologous mobilization processes performed between june 2015 and march 2016. patients (n525) evaluated were diagnosed with malignant lymphoma (n515), multiple myeloma (n59) and primary amyloidosis (n51) and were mobilized according to standard protocols. collection cd341 cellularity target was established ! 2x10e6/kg. two groups, good and bad mobilizers, have been determined. predictors of unsuccessful mobilization were defined by >65 years old, previous fludarabine, lenalidomide, or bendamustine treatments or !2 previous regimens, present peripheral cytopenias, active disease and previous mobilization failure. mann-whitney u test was used to compare means and comparisons of medians were performed by the median test. cd341 count was performed according ishage protocol. adverse events (ae) were analysed according to ctcae v4.0. results/finding: the media (range) general collection parameters were: cd341 (day 5) 27.50/ml (4.5-157.5/ml), blood volume processed 23204ml (9718-39618ml) and 4.96 (2-7.30) exchanged volemias. seventeen patients were considered bad mobilizers, 7 needed plerixafor and 5 had to undergone a collection procedure twice. there were statistically significant differences between both groups on mobilization characteristics and product cellularity [mean (sd) ); p50.071]. there were no significant differences on mobilization characteristics and product cellularity between both groups. five mobilization ae were observed [muscle pain (n52), fever (n52) and flu syndrome; all grade 1]. two patients could not undergo hematopoietic stem cell transplantation due low cd341 cellularity. conclusion: there are differences between products collected from the good mobilizer (rich in gm and cd34) versus poor mobilizer (with plerixafor) rich in cn and cmn. the mobilization with zarziov r could be smaller than expected since there are no significant differences if we compare the good mobilizers versus the bad mobilizers although the number of cases studied can be limiting. background/case studies: mesenchymal stem cells (mscs) have been widely studied and have shown beneficial effects on tissue regeneration, immunomodulation, and improvement of multiple organ failure caused by infection, sepsis, and trauma. however, mscs express tissue factor, which may be a risk factor for thrombosis especially if administrated systemically following trauma when coagulopathies are common. before applying mscs in a preclinical animal model, we sought to determine the procoagulant properties of rat mscs in vitro. study design/methods: bone marrow and adipose derived mscs (bmsc and amsc) were isolated from bones (femur and tibia) and visceral fat tissue in normal young sprague dawley rats respectively. both bmscs and amscs were cultured and passaged using dmem medium with 20% fetal bovine serum. bmsc and amsc at passage 2-5 were used in this study. the tissue factor expression of mscs was determined by immunohistochemistry. citrated whole blood collected from normal rats was treated with rat bmscs and amscs at low, medium and high doses (1.5 3 10 4 /ml, 7 3 10 4 /ml and 1.5 3 10 5 /ml respectively). the prothrombin time (pt), coagulation properties and platelet aggregation (response to adp, collagen and par4) were measured by hemostasis analyzer, rotational thromboelastometry (rotem) and impedance aggregometry (multiplate) respectively within 30min and 2hr after incubation. results/finding: tissue factor was significantly expressed among both bmsc and amsc at all passages in vitro. bmsc and amsc at any dose and time of treatment neither shortened nor elongated pt in whole blood. however, both bmsc and amsc significantly shortened the clotting time (ct) (none: 334 6 35 seconds, versus low, medium and high doses of amsc (145 6 2, 111 6 6, and 75 6 12 seconds), and bmsc (155 6 2, 90 6 10, 80 6 7.0 seconds), p<0.05), clot formation time (cft, p<0.05) and increased alpha angle (p<0.05) by natem measurement, but did not significantly affect the ct, cft and alpha angle by extem. maximum clot firmness (mcf) and fibrinolytic index were not affected by mscs. there was no significant impact of both bmsc and amsc on platelet aggregation simulated by adp, collagen and par4. no significant differences of hemostatic and platelet function were found between the treatments of bmsc and amsc. conclusion: consistent with reports from human derived msc, both rat bmsc and amsc significantly expressed tissue factor in both early and late passages, which led to a significant decrease in clotting time at various dose and time of treatment. however, mscs had no direct impact on platelet aggregation in vitro. as considering the procoagulant capability of mscs, future study will be necessary to determine the optimal dose and safety of using mscs for systemic application in vivo. comparison of the terumo bct mnc and cmnc protocols for peripheral blood stem cell collections lindsey westbrook* 1 , neil bagamasbad 2 , reynold dilag 2 , melissa nasser 2 , nicole bauer 2 , jennifer wheeler 3 and mary berg 1 . 1 department of pathology, university of colorado -anschutz medical campus, 2 department of medicine, division of hematology, university of colorado hospital, 3 scientific support, terumo bct background/case studies: terumo bct recently offered a new method of peripheral blood stem cell (pbsc) collection using the spectra optia, an apheresis instrument. the new protocol, continuous mononuclear cell collection (cmnc) collects cells continuously as opposed to the older protocol, the mononuclear cell collection (mnc) protocol, which is batch collection or dual stage collection, involving an additional step where platelets are separated from mnc within a cell separation chamber. our institution has used both protocols and the purpose of this study was to compare pbsc product characteristics and run times between the cmnc and the mnc protocols. study design/method: a retrospective review and comparison of parameters from 120 collection procedures using the mnc protocol and 173 collection procedures using the cmnc protocol was done using the t-test. data from patients/donors (including 36 allogeneic donors) as well as procedure details including run time, flow cytometry marker for stem cells (cd34)-positive (cd341) throughput, cd341 collection efficiency (ce%), platelet loss 71a transfusion per total blood volume processed (plt loss/tbv), and collection product characteristics were included in the analysis. results/finding: numerical results are summarized in the table. the mnc and cmnc donor groups included 14 and 22 allogeneic donors, respectively. donor weight was not significantly different between the two groups. pre-procedure wbc values were also similar between the two groups. run time was found to be significantly shorter using the cmnc protocol compared to the mnc protocol. product volume was also significantly lower in the cmnc group compared to the mnc group. although the volume was lower, the cmnc product had significantly higher percentages of mononuclear cells (mono%) and lymphocytes (lymph%) collected when compared to the mnc product. the cd341 throughput was significantly higher in the cmnc group than the mnc group. the cd341 ce% was found to be slightly increased in the cmnc group, though not significantly. the platelet loss was not significantly different between the protocols when normalized for total blood volume. product hematocrit (hct%) was significantly higher using the cmnc protocol; however, the red blood cell volume never exceeded 20 ml due to the lower product volume with the cmnc protocol. the cmnc protocol collects a smaller volume of a purer product when compared to the mnc protocol with comparable platelet and red blood cell loss. staff members who perform apheresis procedures are pleased by the shorter run time. background/case studies: hematopoietic stem cell (hsc) donors and their recipients need not have a matching blood type. eventually, the hsc recipient will become the blood type of the hsc donor. this scenario can become quite a conundrum if the hsc recipient becomes a patient in need of an organ transplant. in order for a patient to receive a donor organ, the patient and donor's blood type and hla typing must be compatible. study design/methods: blood type was determined using gel test cards. hla typing was determined by using sequence-specific oligonucleotide (sso), sequence-specific primer (ssp), and sequence based typing (sbt) technologies. hsc sources were bone marrow and umbilical cord blood. results/findings: patient #1, originally typed as an a2, had 1 bone marrow donor and 2 cord blood transplants. one of the cord blood transplants successfully engrafted. the engrafted unit was from a type o donor. patient #1 is now typing as type o. patient #2 was originally typed as a2 and received a bone marrow transplant from a type b donor. patient #2 is now front-typing as a b and backtyping as an ab. since the patient's abo front and back-type do not match, a note must be made, that when confirming abo during crossmatch, the abo will not match. the patient now has an hla and abo identical kidney match (his father who is a type b). previously, the patient and his father were abo incompatible. the abo and hla results on both patient #1 and patient #2 indicate that the hsc transplants have engrafted. results also indicate that the abo and hla now match that of the donor and differ from the recipient's original abo and hla type. due to various reasons, for example, a side effect of the immunosuppression, both patients now need a kidney transplant. both patients will be entered into the unet system according to their "new" abo and hla types, as unos regulations require patients to be listed as per the results of two separate abo typing tests. the patients' antibodies will be monitored as per lab policy and communication with the transplant centers and blood banks is crucial. background/case studies: mesenchymal stem cells (msc) are beneficial for tissue regeneration, immunomodulation and improvement of multiple organ failure caused by infection, sepsis, and trauma. mscs express tissue factor (tf) that activate the clotting cascade and interfere hemostasis. hypoxia is a condition that occurs after trauma globally during shock or at the site of injury, and is known to change or influence the phenotypes of cells, including mscs. in this study, we want to determine if hypoxia changes the expression of tissue factor and the pro-coagulant properties of rat msc in vitro. study design/method: bone marrow and adipose derived mscs (bmsc and amsc) were isolated from bones (femur and tibia) and visceral fat tissue in normal young sprague dawley rats respectively. both bmscs and amscs were cultured using dmem medium with 20% fetal bovine serum under either normoxia (20% o 2 ) or hypoxia (3.5% o 2 ). msc growth curves were measured by cell counter. the tf expression was determined by immunohistochemistry. cd90/cd29 and cd45 were measured as positive and negative markers of msc respectively by flow cytometry. the citrated rat whole blood was treated with msc (1.5 3 10 5 /ml) either from normoxia or hypoxia. the coagulation properties were measured by hemostasis analyzer and rotational thromboelastometry (rotem). results/finding: hypoxia potentiated the growth of bmsc by 15%, but depressed the growth of amsc by 30% at day 5 in comparison to normoxia. both bmsc and amsc equally expressed cd90 and cd29 but not cd45 under any culture condition. tissue factor was significantly expressed among bmscs and amscs from both normoxia and hypoxia. whole blood treated with bmscs and amscs from normoxia significantly shortened the clotting time (ct: 468 6 64 (control), versus 170 6 13 (bmsc), and 195 6 60 (amsc) seconds) by natem. hypoxia also significantly shortened ct (165 6 20 (bmsc), 169 6 50 (amsc) seconds, p<0.05 as compared to control), but the changes in ct were not significantly different between bmscs and amscs. maximum clot firmness (mcf) and fibrinolytic index did not change after treatment with bmsc and amsc regardless of the normoxia or hypoxia conditions. conclusion: tissue factor is constitutively expressed in rat bmscs and amscs. adjustment of the msc culture condition to hypoxia did not affect tissue factor expression or the procoagulant properties of msc (bmsc and amsc). this study also suggests that the procoagulant properties will not be affected if mscs are recruited into injured tissues with hypoxic environments. future study will be necessary to determine the optimal dose msc and whether it is safe to use mscs for systemic application in trauma. effect of double-end cryopreservation on gene-transduced human hematopoietic stem and progenitor cells sandeep k srivastava*, jiaqiang ren, steven highfill, narda theobald, suksee deravin, andre larochelle, david f stroncek and sandhya r panch. national institutes of health background/case studies: current early-phase clinical gene therapy trials use freshly collected or cryopreserved cd341 cells as the starting fraction prior to gene manipulation. following gene-transduction and culture, the end product is infused fresh into recipients. for wider applicability and scale-up, gene therapy manufacturing protocols would benefit from double-end cryopreservation (dec) of cd341 cells during manufacture (i.e. immediately post-collection and again, post-gene modification). dec helps delink patients' preparative conditioning phase from cell manufacture, eases logistics of inter-facility cell transportation, and ensures fulfillment of regulatory product release criteria before infusion. our objective was to study the effects of dec on gene transduced mobilized peripheral blood (mpb) cd341 cells. study design/method: cryopreserved cd341 cells from 2 healthy adult donors were thawed and transduced (tr) in retronectin coated tissue culture bags with an ef1-alpha-yfp lentivirus (2.5% concentration) and media (x-vivo-10, human serum albumin(hsa), 100 ng/ml each of cytokines (scf, tpo and flt3-l) over 2 days. untransduced (utr) cells were cultured as controls. tr and utr fractions were re-cryopreserved. a standard freeze-mix of 5% dmso, 6% pentastarch, hsa, plasma-lyte a was used for cryopreservation. viability, hematopoietic stem cell (hsc) (cd34 1 cd38 -cd45ra -cd90 1 cd49f 1 cells) phenotyping and cfu assays were done following first thaw (pt1), post-transduction (ptxn) and second cryopreservation-thaw (pt2). results/finding: tnc recovery decreased gradually in the donor samples at each step. transduction efficiency, cd34%, cfus were similar before and after pt2. hscs ranged from 824 to 1655 cells/10 6 cd341 cells in the pt2-tr arm compared to a range of 286 to 1416 /10 6 cd341 cells after pt1. viability, % cd341 and cfus were lower in the tr compared to the utr arm. this difference was not altered after pt2 (table) . conclusion: dec of mpb human cd341 cells decreases tnc recovery, but has minimal effects on cd341 cell phenotype, transduction efficiency and cell function. hsc numbers were within acceptable range after recryopreservation. lower viability and cd34% in the tr arm compared to the utr arm is likely due to vector toxicity. this was unaffected by recryopreservation. additional studies to assess dec mediated changes on cd341 cell early apoptotic markers, telomere lengths, gene expression and engraftment potential in nod/scid mice will inform clinical trials. background/case studies: autologous peripheral blood stem cell (pbsc) transplantation has been used as a powerful resource during the treatment of some hematological malignancies. cryopreservation of these cells is routinely performed to allow for patient adequate conditioning and chemotherapy. in some cases, pbsc are harvested as a backup option and remain stored for several years, although effect of storage lesion in this product is still controversial. our work presents retrospective data on pbsc infusion after long-term storage. study design/method: all products were harvested after patient mobilization with g-csf by apheresis with cobe spectra v r . flow cytometry analysis of cd341 cells was performed prior to cryopreservation. the cryoprotective solution was freshly prepared by addition of 20% hydroxyethyl starch, 18% human serum albumin and 10% dmso at final concentration. pbsc were cryopreserved by direct immersion on -808c mechanical freezer (dump freeze) and stored until transplantation. post-thaw viability was determined from stored cryotube samples by trypan blue exclusion minutes prior to infusion. cells were thawed and infused on bedside. engraftment was defined as the first day of 03 consecutive days of neutrophil count >0.5 x10 9 /l and platelet count >20 x10 9 /l after 07 days. with g-csf for four days and patients with g-csf for five days with use of mozobil when cd341 was below 10 x 10 6 cells/l on the fourth day. hpc collection was performed on the fourth day of mobilization for healthy donors and on the fifth day for patients. all procedures were realized based on a prediction algorithm using pre-cd341 on the day of the collection and estimating wbc liters to be processed to obtain sufficient stem cells for the transplant. this algorithm was designed using linear regression of peripheral blood cd341 on the day of the collection versus collected cd341 per liter of blood processed. there was no distinction between patients and donors, once the efficiency coefficient was used for both. collected material was sent to analysis and total cd341 was calculated. final laboratory count of cd341 per kilogram was compared with the number predicted by the algorithm with spearman's correlation to evaluate whether the formula is effective. calculations were made using ibm spss 23 software. results/findings: among patients collecting hpc for autologous transplantation, 69,23% needed only one day of hpc harvesting, while 25,64% needed two days and 5,13% needed three or more days. our collection efficiency (ce) and standard error of the mean (sem) was 49 1-2,91%. after comparing predicted values with cd341 collected in the final product, we found a very strong correlation of 0.873 (p<0.01) for patients and a strong correlation 0.653 for healthy donors (p<0.01). conclusion: the use of a mathematical model with a prediction algorithm is safe, has low cost and provides a good tool to estimate wb liters to process and avoid unnecessary procedures in both patients and healthy donors. this study evaluated the phenotypic characteristics of uc-mscs derived from fresh and cryopreserved cord tissues (ct), as described in isct's position paper on minimal characteristics of mesenchymal stem cells (plastic adherent; !95% cd90, cd105, cd73 and 2% cd14, cd19, cd34, cd45, hla-dr) study design/method: umbilical cord tissue (n510) was washed, blood vessels removed, cut into 0.5-3mm pieces, and washed twice in saline. fresh tissue was immersed in 0.9% saline for same day culture, while frozen tissue was cryopreserved for at least 24 hours prior to culture. for colony forming unit (cfu) testing tissue was plated directly in a 25cm 2 tissue culture flask following a wash in pbs with antibiotic/antimycotic. the tissue was allowed to adhere for 10 minutes prior to the addition of cell culture media. media was changed several times a week. cells were passed when robust colony growth was observed and in subsequent cultures >80% confluence. all cells were tested on an msc flow panel at passage 2 just prior to confluence. results/finding: both fresh and cryopreserved tissue showed excellent colony forming capabilities. average time for cellular emergence of 8 days (fresh 5 7.8, frozen 5 8.1), and 13 days (total) for the msc's to reach passage 1 (fresh 5 12.6, frozen 13.4). all cells were ready for flow analysis in approximately 3 weeks time. there was no statistical difference between fresh and frozen tissue in their colony emergence (p 5 0.81), or their growth rates (p > 0.05 for all). flow cytometry showed average !95% for positive markers and 2% negative markers. there was no statistical difference between fresh and frozen flow result (p > 0.05). conclusion: uc-msc's show excellent adherence to plastic in both fresh and frozen explant cultures, with a consistent fibroblast-like morphology. flow cytometry analysis showed strong msc phenotype in both fresh and frozen samples. the data show that the cryogenic process does not appear to have any detrimental effects on the ability to obtain msc colonies. studies have shown that hsct improves survival and disease-free survival rates when compared to conventional chemotherapy treatments. the increase in the number of hscts over the last years has demanded quality and safety improvements of cell processing and cryopreservation services. cell recovery and viability are crucial parameters to assess ucb quality as a viable hsct graft source. study design/method: twenty-five ucb units cryopreserved for periods of 2 up to 11 years (2004 to 2017) were analyzed. units underwent red cell and plasma depletion and then subjected to controlled rate freezing and subsequent cryopreservation using dmso (dimethylsulfoxide) cryoprotectant with 10% concentration. informed consent and the unit discard terms for all units were obtained. units were thawed in a 378 c water bath and 0.5ml aliquots were diluted at a 1:1 proportion with 5% human albumin solution and plasmin were prepared, enabling dmso stabilization and concentration reduction. the following analysis were performed: nucleated cell count (tnc) in an automated hematologic counter and cell viability using flow citometry. post-processing (pre-cryopreservation) cell viability was tested using trypan blue as exclusion dye, while post-thaw cell viability was assessed using 7-aad marker through flow cytometric analysis. results/finding: ucb storage period was 7.24 years (mean) and cell recovery was 86.31% (mean). there was no statistically significant correlation between storage period and post-processing cell recovery (p 5 0.11). post-thaw cell viability of 63.13% (mean) showed no statistically significant correlation with unit storage period (p 5 0.07). post thaw cell viability results are within parameters defined in other studies. background/case studies: umbilical cord (uc) tissue is a rich source of mesenchymal stem cells (mscs) that can be collected noninvasively at birth and stored for potential future use. as such, a growing number of stem cell banks have established uc storage programs based on mounting preclinical evidence of its therapeutic potential. however, little has been reported on the ability to isolate msc-like cells from uc tissue after extended periods of cryopreservation. this work describes and characterizes the isolation of mscs from uc tissue cryopreserved as a composite material at a family stem cell bank for 5 years. study design/method: donated uc units from consenting mothers were evaluated. units had been cryopreserved as composite tissue pieces in ln 2 vapor in a dmso-based cryoprotectant for 5yrs. (5.49 6 0.431; n54). units were rapidly thawed and rinsed in dpbs, then 25 pieces were excised from each using a biopsy punch. pieces from each unit were explanted in a 5x5 grid pattern in msc-supportive medium and incubated for 7 days, after which the tissue was discarded and media exchanged. cells were isolated on the 14 th day, counted, and subcultured for two passages. at the end of each passage, cells were collected, counted and population doubling time was calculated. isolated cells from each unit were also evaluated for msc immunomarkers. results/finding: small, proliferative cells with fibroblastic morphology were obtained from all explants, yielding a 100% success rate. cells were positive for the msc markers cd73, cd90, and cd105 (98.8 6 0.7%, 98.7 6 0.6%, and 97.8 6 0.6%, respectively) and negative for the hematopoietic markers cd34/45 (1.1 6 0.7%). passage 1 and passage 2 doubling times were 1.92 6 0.47 days and 2.07 6 0.43 days, respectively, which are in line with values reported for mscs isolated from fresh uc tissue. conclusion: due to their immature status, ease of collection, and potential therapeutic value, uc mscs are an appealing candidate for future clinical 75a transfusion 2017 vol. 57 supplement s3 research and treatment. the present work demonstrates that the long-term cryopreservation of uc tissue does not disrupt the ability to isolate functional mscs from the tissue at a later date. importantly, growth characteristics of isolated mscs appear to be comparable to those reported for mscs from fresh uc tissue. based on the consistent isolation and lack of apparent impact on proliferation kinetics, it is reasonable to expect cell yields in the range anticipated for therapeutic requirements and more than sufficient for moving to clinical grade bioreactors for expansion. these results support the feasibility of storage of uc as a composite material for future potential cell isolation and expansion to clinically relevant doses. large volume leukapheresis with spectra optia cmnc protocol in adult and pediatric patients: performance and determination of cd34 yield prediction algorithm ines bojanic* 1 , nelly besson 2 , ivana vidovic 1 and branka golubic cepulic 1 . 1 department of transfusion medicine and transplantation biology, university hospital centre zagreb, 2 terumo bct background/case studies: large volume leukapheresis (lvl) have shown to enhance cd341cell yield collected. this study evaluated performance and safety of the spectra optia cmnc protocol (version 11) in adult and pediatric lvl. a prediction algorithm for cd341cell yield was also tested. study design/method: we evaluated retrospectively 67 lvl performed in 46 adult patients, and 14 lvl in 11 pediatric patients treated in uhc zagreb from march 2016 till september 2016. mobilization regimen combined chemotherapy and filgrastim; 2 poor mobilizes received plerixafor additionally. a combination of acd-a and heparin was used as anticoagulant (acd-a:whole blood ratio 1:24). in patients weighting 25kg (n59), a rbc prime was performed. cd34, lymphocyte(ly) and monocyte(mo) collection efficiencies (ces) were calculated. a customized prediction algorithm was determined on linear regression between pre-cd341cell count and cd341cells collected / blood volume processed. prediction accuracy was evaluated by comparing predicted cd34 values to real cd34 yield. results are presented as median (iqr). results/finding: in both groups, cd34, ly and mo ces were high. target cd34 dose was successfully reached in 1 procedure in 30 (65,2%)adults and in 9 (81.8%) children. all procedures were well tolerated: adverse reactions were restricted to mild citrate toxicity symptoms in 5 (7.5%) adults, while all pediatric apheresis went uneventful. no bleeding episodes occurred, and no transfusion was needed. product and procedure characteristics* a high correlation between precd341cells and cd341cells collected/ blood volume was observed in both groups (r 2 50.97 and 0.83 in adults and children respectively, p<0.0001) suggesting cd34 yield could be predicted based on precd341cells and blood volume to process. linear regression equations served as prediction algorithm. the high correlation between predicted cd34 yield and observed cd34 yield (r 2 50.95 and 0.82 in adults and children respectively, p<0.001) showed accuracy of the algorithm. implementation of the algorithm could have allowed sparing a median of 10.1(8.9-12.9)l of blood in 20 adult procedures, and 5.9(3.5-7.8)l in 7 pediatric procedures. conclusion: lvl performed using spectra optia cmnc protocol is safe and efficient in adults and in low body weight children. high cd34, ly and mo ce1 were observed in both groups. implementation of a predictive algorithm can reliably minimize blood volume processed, shorten procedure duration, reduce anticoagulant volumes infused, and improve patient comfort. mesenchymal stem cell therapy in steroid refractory graft-versus-host disease (gvhd) emese molnar* 1 , aniko barta 2 , arpad batai 2 , zoltan csukly 2 , zita farkas 2 , laszlo gopcsa 2 , gabor tatai background/case studies: steroid refractory acute graft-versus-host disease (gvhd) is a serious complication of allogeneic hematopoietic stem cell transplantation (hsct). more experience accumulates in the immunomodulatory effect of mesenchymal stem cell (msc) infusion in numerous immunopathological disorders -such as gvhd -and signals. mscs have a hlarestrictive and non-immunogenic nature. study design/method: we have evaluated the efficacy of msc transfusions in cases of acute gvhd refractory to conventional immunosuppressive treatment. the patients with steroid-resistant gvhd had received third-party mscs (derived from wharton's jelly and bone marrow) 4 times per case weekly at a dose of 1 million cells/kg. clinical response was assessed 28 days after administering the first dose. complete remission was defined as the complete disappearance of symptoms. partial remission was assessed by the significant relief of symptomsand by the general improvement of the patient's condition. results/finding: in all 12 patients had received 13 cycles of msctreatment (4 dose per cycle). the median age was 47 years old (19-56) with a male/female ratio of 1:2. distribution of the original malignancies (n): acute myeloid leukemia: 6; acute lymphoblastic leukemia: 2; myelofibrosis: 1; myelodysplastic syndrome: 1; multiple myeloma: 1; t-cell lymphoma: 1. nine patients had undergone allogeneic hsct with matched unrelated donors, the other three had stem cells derived from hla-identic relatives. the first episode of gvhd after hsct was started on the median 63rd day (7-455). the involved organs were skin (2), gut (4), skin and gut combined (7) and even lung in 3 cases. the median time of msc's first infusion was 274 days after the stem cell transplantation (hsct) and 165 (19-1974) days after the first episode of gvhd. 4 of the 13 cycles of msc-treatment led to complete remission (30.8%) and 7 resulted inpartial remission (53.8%). conclusion: we have evaluated msc-therapy as an effective treatment of gvhd in the majority of the observed cases with 83% overall cumulative response rate. the application of third-party mscs offers a promising alternative in the therapy of gvhd and other gvhd-associated complications after hsct. further research is needed to determine the optimal start of the treatment, along with the issue of long-term safety. background/case studies: stem cell collection by leukapheresis for transplantation is a significant endeavor for the patient and the clinical team. whether the collection is allogenic or autologous, the patient undergoing the collection and the physicians caring for the patient are always concerned whether they will be able to harvest enough cells for transplantation and engraftment. a typical goal for most adult procedures is 2 million cd341 cells/kg. if a patient does not reach this goal on the day of the procedure, they will likely have to return the following day to undergo a second procedure to reach the desired goal. given the logistical challenges in planning transplantation, it is reasonable to attempt to optimize the number of cells collected while minimizing the number of collections. measuring a patient's cd341 cells/ml in their peripheral blood before the leukapheresis procedure has been used to predict if the collection will successfully reach the 2 million cells/kg goal. the ideal minimum cd341 cells/ml that will lead to successful harvest has not been conclusively identified. study design/methods: we analyzed the collection data from 55 patients to evaluate the predictive value of the cd341 cells/ml level. data was collected over 6 months from every patient who underwent a stem cell collection. four patients were allogenic donors and 51 were autologous donors. the patients' weight, diagnosis, and pre-procedure cd341 cells/ml level were all collected. the run time, amount of volume processed, and the absolute viable cd341 cells collected were recorded. the collection efficiency and the cd341 cells/kg were calculated for each patient. results/findings: our data showed a strong linear correlation between pre-procedure cd341 cells/ml and post-procedure cd341 cells/kg (r50.95). any patient who had a pre-procedure cd341 cells/ml count of 29 or greater had a collection of at least 2 million cells/kg. any patient who had a pre-procedure cd341 cells/ml count of 16 or less collected less than 2 conclusion: the pre-procedure cd341 cells/ml level in the peripheral blood has a very strong predictive value for the post-procedure cd341 cells/kg level. to confidently know that a patient will be able to produce the desired 2 million cells/kg, a pre-procedure cd341 cells/ml count of at least 29 should be obtained. for any patient with a count below 16, they should be counseled that their collection is likely to take at least a second day and a second procedure. further studies, including potentially lengthening the run time and the volume processed, to evaluate how to handle the patients who fall between 16 and 29 cd341 cells/ml should be conducted. heidi elmoazzen 1 , antonio giulivi 1 , michael halpenny* 1 , lisa martin 1 , donna perron 1 , chris bredeson 2 , lin yang 1 , locksley mcgann 1 , paul birch 1 and jason p. acker 1 . 1 canadian blood services, 2 ottawa hospital background/case studies: a critical aspect of hematopoietic progenitor cell processing is the cryopreservation method. our program uses a "dump" freeze method consisting of product placement directly into liquid nitrogen vapour after addition of a cryopreservation solution containing dmso (5% final concentration) and hes (hydroxyethyl starch). pentastarch (hes source) a critical component of the cryoprotectant formulation was discontinued by the commercial vendor. this required that an alternative cryoprotectant formulation be validated to minimize the risk to patient safety without compromising engraftment quality. study design/method: the validation study consisted of 3 phases; firstevaluation of the efficacy of four different cryoprotectant formulations, second -evaluation of full scale production and crypreservation and third -a concurrent validation for clinical transplant. phase i -samples from four different cryoprotectant formulations were tested for tnc, cd34, viability and cfu at three points during manufacturing (fresh, post processing and post thaw). phase ii -mock hpc, apheresis units were used for a side-by-side comparison of freezing curves for the control and replacement formulations. phase iii -five clinical transplants were performed with hpc, apheresis products cryopreserved using the recommended replacement (hetastarch). results/finding: phase i -results indicate that aliquots cryopreserved in 5% dmso and 1.7% hes (hetastarch) did not behave significantly different than cells cryopreserved in the control in terms of cell recovery, viability or cell proliferation assay (cfu). phase ii -the majority of freezing profiles displayed typical or expected bulk freezing profiles for both formulations. phase iii -transplants performed resulted in a mean engraftment time of 12.6 days for anc500 with no adverse patient reactions observed. engraftment times using the new hetastarch formula were compared to the previous engraftment times with no significant difference. conclusion: a change in the formulation of a cryoprotectant solution represents a major change that could have a significant impact on quality. in addition, maintaining the current 5% dmso final concentration was critical as post thaw washing is not performed at the clinical site, history demonstrating a very low toxicity rate with the existing formulation. this study demonstrated the acceptability of the hetastarch formulation using 5% dmso and 1.7% hetastarch to replace pentastarch in the cryoprotectant formulation used for cryopreservation of hpc, apheresis products. background/case studies: autologous stem cell transplantation is usually performed with mobilized peripheral blood stem cells (pbscs). traditional mobilization regimens include granulocyte colony stimulating factor (g-csf) with or without chemotherapy, but have failure rates ranging from 5% to 40%. plerixafor is an adjunct agent used to improve mobilization in many clinical settings. however, its high cost is a significant concern. the manufacturer-recommended dose is 0.24 mg/kg, therefore patients weighing >100 kg would require a second vial, thus doubling the drug cost. in 2013 we implemented a policy of capping plerixafor at 24 mg for patients weighing >100 kg. this retrospective study compares the mobilization of patients >100kg who received capped doses (2013) (2014) (2015) (2016) , with historical control patients (2010-2013) who received full or uncapped doses. study design/method: patients weighing >100 kg with crcl >50ml/min who received capped and full doses of plerixafor were identified in the pharmacy database. electronic medical records were used to collect baseline characteristics and cell collection data. results/findings: a total of 47 and 40 consecutive patients were included in the capped and full dosing groups, respectively. they showed comparable baseline distributions of age, weight, gender and diagnoses. plerixafor was given upfront, or as a rescue agent due to suboptimal mobilization in both groups. in the capped dosing group, fewer patients received chemomobilization or plerixafor upfront. when compared to historical controls, they used half of the number of vials of plerixafor, but collected similar numbers of cd341/cells kg and achieved a comparable collection success rate. the strategy dose capping plerixafor at 24 mg for patients >100 kg is cost-effective and achieves comparable mobilization outcomes while decreasing the drug cost by half. mean and range of %cd34 in peripheral blood were calculated. the data show that in the non-hispanic group, the youngest donors (<30yrs) have a higher pre-apheresis %cd34 level than any of the other groups, reaching statistical significance when comparing the %cd34 pre-apheresis between the youngest group (<30 yrs) and the oldest group (>540 yrs). hispanic donors show statistically similar %cd34 pre-apheresis levels over all age groups. moreover, the hispanic older age group (>540yrs) had a statistically higher %cd34 pre-apheresis level than the non-hispanic older age group. conclusion: in this analysis of 121 sequential unrelated pbsc donors, hispanic donors maintain a similar pre-apheresis %cd34 level even as the donor ages, while non-hispanic donors show a decreasing pre-apheresis %cd34 level as they age. if proven, this data would suggest there are genetic factors that modulate a person's ability to mobilize stem cells as they age and that these genetic factors differ between ethnic groups. this small data set would suggest that people of hispanic ethnicity maintain a more robust and quickly responsive stem cell pool, even as they age. further studies of larger cohorts are needed to validate this observation. if proven, this has far reaching implications within the stem cell research and therapy arena. background/case studies: an update in hpc apheresis collection software led to higher collection volume in the organization's human progenitor cell (hpc) products without a corresponding increase in total cellular counts. incorporation of a volume reduction step was therefore warranted as larger product volumes require additional time to transfuse and lead to a larger dmso load to the recipient, often resulting in the need to transfuse over several days. the objectives of this study were to develop suitable mock hpc (mhpc) products and evaluate the effectiveness of the biosafe pericell volume reduction technology on white blood cell (wbc) recovery and viability. study design/method: hpc products are not readily available for development. mhpc were created from whole blood buffy coats (bcs). fresh abo compatible bcs were pooled and concentrated using centrifugation and manual extraction of supernatant and red cells. the mhpc products were then diluted in plasma to produce an appropriate concentration and volume. hpc collection data from last 3 years was analyzed to determine the 95 th percentile, median and 5 th percentile values for both hpc volume and wbc concentration. six mhpc products were tested; three high wbc (234 x 10 6 cell / ml) and three low wbc (114 x 10 6 cells / ml) concentrations, each at high (505 ml), low (265 ml) and median (355 ml) volumes. each unit was processed sequentially from high, median and low volumes. hence, the highest mhpc volume was processed for volume reduction first with a sepax 2 (pericell protocol, cs.430.1 kits), analyzed and then reconstituted and volume adjusted to the next volume target before being volume reduced again, and so forth. one additional mock product was prepared for a reproducibility study and was volume reduced three times. wbc concentration and 7-aad viability was determined before and after each volume reduction. a control sample was removed from the product prior to processing and sat on the bench top until the end of the protocol to assess the change in cell concentration and viability over time. results/finding: mock hpc products had a mean starting 7-aad viability of 76 6 8% [range 64-85]% and a hematocrit of 14 6 5% [9-19] which is well below the maximum allowable limit of the pericell. no significant differences in wbc recovery or change in viability were seen between the 6 mhpc products. aggregate data showed that the mean wbc recovery of the volume reduction process was 97 6 8% [64-105] with a 3 6 3% [-2-11] change in viability. the recovery protocol used to salvage product after each volume reduction gave a recovery of 99 6 4 [92, 104] % and a change in 7-aad viability of 2 6 2 [0, 11] % from the input product. the method was found to have a cv of 2.0%. the change in wbc concentration and wbc viability of the test products was not significantly different from the unprocessed control samples. conclusion: mock bc products are a suitable alternative where hpc products are not available for development and are a good use of product otherwise directed for rejection and disposal. the volume reduction protocol evaluated had minimal impact on the wbc concentration and wbc viability in the mock products and was found to be highly reproducible, giving confidence that it will be a valuable processing step with hpcs and will facilitate transfusion of hpc products into the recipient. the protocol is now in use with patient hpc products and engraftment kinetics will be tracked in a postimplementation study. validating 78a transfusion clinical assessment. in the first phase, 2 cryopreserved pbsc products were tested. two aliquots were thawed simultaneously for each product: one was passed through a pre-set infusion pump and a second control aliquot was drained by gravity. each aliquot was tested for baseline total nucleated cell (tnc) count and viability, and for final tnc recovery, trypan blue (tb) viability, cd34 7-aad viability, and potency (cfu). the effect of longterm exposure to dmso was assessed by visually inspecting the product for aggregates and measuring viability up to 3 hours post thaw. the second in vivo phase included use of an infusion pump for 10 consecutive autologous patients, with comparison of infusion and transplant outcomes to 18 previous infusions by gravity drip. comparison variables included infusion rate, adverse events (ae), and engraftment time. results/finding: no significant differences were observed between infusion pump and drip for the 2 products tested in vitro, including tnc recovery, cell viabilities, and potency. for both methods tnc tb viability decreased by more than 20% within 1 hour, while cd341 cell viability remained stable up to 3 hours post thaw. small aggregates appeared after 1 hour for both methods and increased by a similar rate over time. comparison of infusion and transplant outcomes between drip and infusion pump patients showed no significant differences for all measured variables. engraftment time was similar for both groups. anc days to engraftment for pump and drip were 10.8 6 1.3 and 11.6 6 1.0, respectively (p-value50.075). platelet days to engraftment for pump and drip were 17.9 6 2.2 and 20.2 6 5.0, respectively (p-value50.207). infusion rates were slightly higher for the pump group. for control patients, 2 required transfer of products to syringes due to slow infusion rate and 2 others experienced allergic and hypotension infusion adverse events. conclusion: no significant in vitro or clinical differences were observed between thawed pbscs infused by gravity or an infusion pump. these results demonstrate that the use of a pump for pbsc infusion is safe, provides consistent infusion rates, eliminates the need to transfer products to syringes, and results in comparable engraftment times. donor racial distribution among the 278 zikv ineligible cbus was: caucasian 52%, asian 9%, black/aa 20%, and multi-race 21%. racial distribution of all clinical cbu donors was caucasian 49%, asian 15%, black/aa 20%, and multi-race 17%, suggesting there is no race correlation for this risk factor driven by cultural habits such as family travel. there were no cases in which onlythe sexual partner's potential exposure determined donor's ineligibility. conclusion: our study indicates that currently the leading risk factor for ineligible cb donors is potential exposure to zikv: 78% of all ineligible cbus and 21% of all banked cbus in the study period. we anticipate the number of cases to decrease following maternal education and travel warnings. recognizing the importance of zikv in public health, and its potential transmission via hct/p products, an fda approved screening test for hct/ p donors becomes a timely necessity. acknowledgments: funded by zimmer biomet, a zimmer biomet company, ibgrl red cell reference and nhsbt reagents background/case studies: during storage, red blood cells (rbcs) become less deformable, deplete 2,3-diphosphoglycerate (2,3-dpg) and adenosine triphosphate (atp), release pro-coagulation phospholipids, accumulate pro-inflammatory molecules, free iron and haemoglobin and increase their potential for adhesion to a recipient's vascular endothelium. longer rbc storage may impair transfusion outcome due to impaired oxygen delivery, promotion of oxidative stress, increased pro-inflammatory state and coagulation. a sterile, non-pyrogenic rejuvenation solution, containing pyruvate, inosine, phosphate, and adenine (citra labs, llc, braintree, ma), is approved by the u.s. food and drug administration for the rejuvenation of stored rbcs. the solution acts by restoring 2,3-dpg and atp in stored rbcs to levels equivalent to those in the circulation. the aim of the study was to investigate the effect treatment with this rejuvenation solution had on the crossmatch reaction profile and phenotypic state of stored rbcs. study design/method: a 10 ml aliquot was removed from abo/rh grouped, leucocyte depleted rbc units (n 5 20), which were stored in sagm for 22 days, to act as untreated controls. the remainder of each unit ($270 ml) underwent treatment with the rejuvenation solution (50ml, 60minutes at 37 o c), followed by cell washing twice in sagm ('manual' centrifuge-based process). to represent current transfusion laboratory practice, units were crossmatched against plasma from 39 random donors, using both diamed gel column and glass tube technique. phenotype investigation with commercial antisera was performed to identify the effect the rejuvenation solution treatment exerted on rbc surface antigens (a, b, d, c, c, e, e, k, m, n, s, s, p 1 , lu a , k, kp a , kp b , le a , le b , fy a , fy b , jk a , and jk b ), including whether it exposed crypt antigens (t, tn, tk*, th, tx*, and cad). crossmatch and phenotype agglutination scores observed for the untreated and treated rbcs were then compared. results/finding: crossmatch findings were defined as compatible, suitable, and incompatible. the study identified no difference between the crossmatch reaction profiles of untreated and treated rbcs. furthermore, no difference was observed in the phenotypic state between untreated and treated rbcs. conclusion: treatment of 22 day old stored rbcs with the rejuvenation solution had no effect on crossmatch reaction profiles or phenotypic state when compared to matched untreated samples. background/case studies: cryopreserved platelet production is burgeoning worldwide. currently, there are no automated platelet cryopreservation methods. by contrast, red blood cell cryopreservation using the acp 215 (haemonetics corp., baintree, ma) has automated the processing within a closed system, increased labour productivity and provided high quality blood components. purpose: to automate platelet cryopreservation procedure. study design/method: apheresis platelet concentrates (pc) were collected on the trima accel system. platelet counts were performed using an abx micros 60. pc were centrifuged at 1250g in a sorvall rc3c1 centrifuge (sorvall, usa) for 10 min. the combination cryoprotectant dmso1dextran (cryosure dex40, germany) was used for pc cryopreservation. cryopreserved pc (cpc) were frozen and stored in a kelvinator chest freezer. cpc were thawed at 37 degrees c (barkey plasmatherm) for 10 min. cpc osmolality was measured with an osmomat 030 osmometer. results/finding: staged platelet cryopreservation technology has been developed. platelets were cryopreserved in a closed system (patent no.: ru 169287 u1). during the first stage, cpc were spun to separate a plateletrich plasma (prp) fraction from platelet-poor plasma (ppp). the second step was to resuspend the prp by adding a combination of dmso1dextran (cryosure dex40) , as a cryoprotectant, to obtain a final concentration of 5% dmso in the platelet suspension. the injectomat mc agilia and npbi compomixer m3 were instrumental in automating that phase. pc to be frozen had an osmolality of no less than 1500 mosm/l. prp and ppp were frozen at a cooling rate of 1-38c/min and stored at -85 0 in the chest freezer for up to 24 months. pre-transfusion defrosted platelets were also processed in a closed system (patent no.: ru 167874 u1). our transfer set made it possible to automate platelet resuspension in plasma through the agency of the exadrop v r . post-thaw prp was resuspended in plasma, which lowered the osmolality to 380 mosm/l. freeze-thaw recovery of platelets was 80% or more of the original population. defrosted pc were stored at 20-24 0 with continuous gentle stirring from a helmer platelet agitator for no longer than 4 hours before transfusion. it took no more than 30 min to cryopreserve pc and process pre-transfusion thawed platelets. the automated processing accounted for the bulk of the time (over 20 min). conclusion: the automated technique developed reduced the workload while offering reproducibility of the procedure and high cpc quality. the use of closed systems ruled out bacterial contamination. employing the infusion pump, platelet stirrer and precision flow regulator enabled adequate osmolality monitoring. bacterial detection in leukoreduced apheresis platelets on day 4 and day 5 evelyn c. oyler*. suncoast blood bank background/case studies: the recently published fda draft guidance describing bacterial testing to enhance the safety and availability of platelets outlined the steps for blood collection establishments and transfusion services to extend apheresis platelets dating for up to 7 days. this evaluation will compare culture based and rapid based test methods for detecting bacterial contamination in apheresis platelets. study design/method: a large community blood center and transfusion service collects leukoreduced apheresis platelets (lrap) using amicus separator system (fenwal, lake zurich, il) and trima accel system (terumo bct, lakewood, co). previously-cultured lrap units were sampled on day 4 for secondary culture using bact/alert (biomerieux, durham, nc) and rapid bacterial tests using bactx (immunetics, boston, ma) and pgd (verax, marlborough, ma). if lrap unit is still available, it is also sampled and tested for rapid testing on day 5. a total of 60 lrap units were tested over a 3-month period: 50 were cultured and rapid tested on day 4; 10 were rapid tested on day 5. the rapid test methods were also evaluated based on cost, ease of use, incubation time and indication for use. results/finding: of the lrap units evaluated for this study, there were 59 true negatives (tn) and 1 false positive (fp) on day 1 when tested by bact/alert, with 60 tns on day 4. bactx testing results showed 50 tns on day 4 and 10 tns on day 5. testing using the pgd kit showed 50 tns on day 4; and 8 tns and 2 fps on day 5. fp results were confirmed by performing a secondary culture, which were found to be negative. bactx requires a specific analyzer and 30 minutes are required for result interpretation. there is no instrument requirement for pgd and reactions can be read within 20 minutes. conclusion: the results of this evaluation makes pgd the best fit for this blood center based transfusion service. pgd offers a shorter time for reading of results, does not need an initial investment for an analyzer and is indicated for lrap in 100% plasma and lrap in pas/plasma. its ease of use allows for testing of lrap on day 4 and day 5 during the night shift to be accomplished without additional staffing and allows to extend outdate to 7day storage of lrap. change in growth factor content of human serum for use as eye drops during frozen storage for 1 year jos lorinser 1 , pieter f van der meer 1 , hans van der heiden 2 and dirk de korte* 1 . 1 department of product and process development, sanquin blood bank, 2 mu-drop background/case studies: growth factors are thought to be among the active components in serum used for treatment of dry-eye syndrome. stability of growth factors during frozen storage in mini containers (140 ml) is unknown. if these products can be stored at -188c it will be feasible to store this product in 3-star household freezers, making the product available for patients in need of serum eye drops. the purpose of this study is to demonstrate stability of growth factor content in human serum during longtime storage at -188c or <-25 to -358c packed in a new micro dose device for single use as eye drops. study design/method: serum produced from 500 ml whole blood donations from non-remunerated healthy donors was quickly frozen. after frozen storage at <-258c for 3-12 months and controlled thawing, six different sera were used to fill a large number of mini (140 ll) containers, which were refrozen and stored at either -188c or <-258c. during storage at 3 months intervals, samples were tested for several growth factors, using magpixv r luminex multiplex assays and compared to control samples stored at <-808c. growth factors tested were pdgf-aa&ab/bb, tgf-ß1/2/3, vegf, 80a transfusion 2017 vol. 57 supplement s3 egf, fgf2. the study was a fact-finding study, without preset acceptance criteria. results/finding: pdgf-ab/bb and tgf-ß1 were the most abundant growth factors, on average 35, resp. 40 ng/ml. also pdgf-aa was detected at relatively high concentration in human serum, on average 11 ng/ml. tgf-ß2, egf and vegf were detected at relatively low values, resp. 3 ng/ml, 0.5 ng/ml and 0.3 ng/ml. average levels of fgf2 and tgf-ß3 were close to detection limit (< 0.2 ng/ml). the controls stored at <-808c showed for all growth factors close to 100% of the initial values in samples at t50 (moment of filling mini containers). for serum stored at <-258c for up to 12 months, most factors showed less than 2 % decrease, except for pdgf-aa and tgf-ß2, showing 6% resp. 3% lower values. for serum stored at -188c the values for tgf-ß1, egf and vegf were stable, whereas pdgf-ab/bb, pdgf-aa and tgf-ß2 showed a decrease of resp. 9, 17 and 3%. conclusion: human serum eye drops can be stored in the new micro dose device at -188c (3-star household freezers) or <-258c (professional freezers) for at least one year after preparation without large decreases in growth factor content. the maximum decrease was found for pdgf-aa in serum stored at -188c. it is yet unknown if the tested components add to the in vivo effectiveness of serum eye drops and what the minimal concentration is to ensure in vivo effectiveness. further stability testing in combination with in vitro and in vivo application is required to extend the shelf-life beyond 1 year. ruqayyah almizraq* 1 , heather inglis 2 , phillip norris 2,3 , jennifer a muszynski 4 , nicole juffermans 5 , jelena holovati 1 and jason p. acker 1,6 . 1 university of alberta, 2 blood systems research institute, 3 university of california, san francisco, 4 nationwide children's hospital, 5 academic medical center, 6 canadian blood services background/case studies: different blood manufacturing methods can influence residual cell numbers and membrane vesiculation, which may affect quality and safety of blood components. the aim was to identify, quantify and characterize residual cells and extracellular vesicles (evs) in stored rbc products produced by different blood manufacturing methods. study design/methods: thirty-two rbc units produced using whole blood filtration (wbf), red cell filtration (rcf), apheresis, and whole blood derived (wbd) methods were examined (n58 per method). residual platelets and white blood cells (wbcs) were measured on day 5 using flow cytometer (fc). on storage day 5 and 42, number and cell of origin/surface markers of evs were assessed with fc, and concentration and size-profile of evs were examined using tunable resistive plus sensing (trps). results/findings: on day 5, apheresis and wbd units had significantly greater residual platelets in comparisons to rcf (vs: apheresis p<0.01, wbd p<0.05) and wbf (vs: apheresis p<0.0001, wbd p<0.01) methods. while rcf units yielded the lowest count of platelet-evs (cd41a1) on day 5 and 42, the highest number of platelet-evs were in apheresis (day 5) and in wbd (day 42). similarly, there was significant difference among methods in the number of wbc-evs (cd31, cd141, cd161, cd191, cd66b1) and rcf contained the smallest concentration. moreover, both trps and fc showed an increase in the total number of evs on day 42 vs day 5 in all of the processing methods. noteworthy, trps showed that the number of small evs/exosomes (< 200 nm) was greater than large evs (! 200 nm) in all of the products on day 5 and 42, and the highest level of evs < 200 nm were in apheresis units. trps results also showed a significant difference in the evs size-profile amongst all rbc products (p<0.05). conclusion: this study shows that the method of manufacturing significantly affects rbc and non-rbcs evs characteristics throughout storage, which has the potential to impact quality and safety of rbc products. the differences in the evs cell-of-origin, concentration, and size-profile observed between manufacturing methods, warrants further examination of their potential immunomodulatory effects and clinical consequences. coagulation and complement assays in whole blood stored at 48 centigrade maryanne c herzig* 1 , crystal lafleur 2 , chriselda g fedyk 1 , sherrill j. slichter 3 and andrew p cap 2 . 1 us army institute of surgical research, 2 u.s. army institute of surgical research, 3 university of washington background/case studies: whole blood has been demonstrated to retain hemostatic activity, including platelet aggregation function, over at least 2 weeks of storage at 48c without agitation. it may be possible to extend the preservation of platelet function by agitating wb. in order to more fully characterize the quality of wb stored at 48c with or without agitation, we evaluated complement activation as a marker of inflammatory potential. study design/method: subjects donated one unit of wb collected in cpd-a2 (citrate phosphate dextrose anticoagulant with adenine). the wb was not leukoreduced nor was it separated into components. units were stored under refrigerated conditions for 10, 12, 15, or 22 days after collection. units were stored for 12 days without agitation. units stored for 10, 15 or 22 days were agitated during storage with a model 400 hybridization incubator at 48c set for end over end rotation at 2-3 rpms. at the appropriate time point, platelet free plasma was obtained from the wb sample and stored at -808c. the frozen plasma was analyzed by elisa assays to determine: thrombinantithrombin complex (tat) as a marker of coagulation; soluble cd40l as a measure of platelet activation and granule release; plasmin anti-plasmin complex (pap) as a marker of fibrinolysis; plasminogen activator inhibitor (pai-1) as another fibrinolytic measure; and complement activation markers c3a, c4d, c5a and c5b-9. data was analyzed by one way repeated measure anova. results/finding: only 49 6 12% of the platelets were recovered in units stored for 12 days without agitation. these levels did not meet fda requirements of 5.5 x 10 10 platelets per wb unit. subsequently, wb was agitated and platelet recovery was 71-76%. no difference was seen in elisa analysis for agitated or non-agitated samples. no change was seen in tat or pap levels between t0 (day of collection) and t10, 12, 15, or 22 measurements. significant elevations of pai-1 and scd40l indicate activation of platelets and inhibition of fibrinolysis (p<0.001). activated complement peptides c3a, c5a, and c4d were all elevated over time (p<0.001) while sc5d-9 was not. however, only c3a and c4d levels at t22 were above normal reference ranges at 1.30 and 1.41 times maximum reference, respectively. conclusion: whole blood agitation appeared necessary to recover platelets at or above fda requirements. whole blood stored at 48c for 10-22 days did show some activation of complement proteins. in contrast to studies in stored red blood cells with elevations of sc5d-9 reported, wb showed elevation of c3a, 5a and c4d and not sc5d-9. complement was gradually and modestly activated with most levels remaining within reference ranges over whole blood shelf life. meredith lummer* 1 and christian todd 2 . 1 cerus corporation, 2 community blood serivces background/case studies: the interceptv r blood system for platelets (cerus, concord ca) is used for the pathogen reduction (pr) of platelet collections, and replaces irradiation, cmv testing, bacterial culture and point of issue bacterial testing. to better understand pr compatibility and impact to split rate, data were analyzed from a mid-size blood center with roughly 1.1x10 10 6 5.6x10 9 9.8x10 10 6 5.6x10 10 310 6 330 430 6 440 900 6 260 28000 6 33000 3 6 3 186 31 30 6 22 110 6 97 rcf 1.9x10 10 6 7.4x10 9 4.2x10 10 6 1.1x10 10 13 6 4 316 14 530 6 160 5100 6 2000 3 6 3 96 7 176 7 346 11 apheresis 2.4x10 10 6 2.0x10 10 1.0x10 11 6 6.1x10 10 520 6 320 700 6 310 2200 6 1900 9800 6 4100 14 6 17 7 6 5 466 15 120 6 24 wbd 6.4x10 9 6 3.1x10 9 4.6x10 10 6 1.5x10 10 350 6 140 760 6 360 1000 6 180 4400 6 2400 3 6 2 426 23 57 6 24 120 6 56 platelet collections must meet specific volume, concentration, and dose ranges to qualify for intercept pr. changes made to apheresis devices included adding the following 4 collection targets: 4.4x10 11 in 350ml, 6.6x10 11 in 400ml, 6.8x10 11 in 400ml, and 7.0x10 11 in 400ml. study design/methods: four months of collections were retrospectively analyzed. platelet collections were evaluated to determine eligibility for pr treatment, and all products meeting pr processing specifications (unless intended for an hla matched recipient at a hospital not able to accept pr products) underwent pr treatment regardless of potential impact to split rate. a minimum post-treatment dose of 3.0x10 11 or 6.0x10 11 was required to classify collections as singles or doubles respectively. volume/dose mitigation (removal of volume to increase the number of products eligible for pr) was not utilized during this study. thus units were treated conventionally if volume, dose, and/or concentration did not meet pr specifications without further manipulation. results/findings: 64% of all single and double collections were eligible for and underwent pr treatment. split rate for single and double collections was 1.34. conclusion: it is possible to treat 64% of single and double platelet donations with intercept pr at the blood center's current state with only a slight impact to split rate if centers are willing to make alterations to their targeting practices. platelet collections that fall outside of the specifications for pr are processed and distributed as conventional products. strategies to increase eligibility toward 100% while minimizing impact to split rate are being investigated, including incorporating new collection settings, splitting triples, and volume/dose mitigation. further evaluation is needed to determine the additional quantity of pr eligible products resulting from such changes. monique p gelderman* 1 , andrey skripchenko 1 , fei xu 1 , ying li 1 , stephen j wagner 2 , pamela h whitley 3 and jaroslav g vostal 1 . 1 fda/cber/ obrr/dbcd/lch, 2 american red cross holland laboratory, 3 american red cross mid-atlantic research facility background/case studies: platelets (plts) stored at room temperature (rt) can support bacterial proliferation in contaminated units and therefore septic transfusion reactions may occur. storing plts at cold temperature (4-6 o c [ct]) limits bacterial growth but results in rapid clearance upon transfusion. the development of alternate storage conditions usually involves costly radiolabeling human studies but success in these studies is difficult to predict based on in vitro studies. thus, an animal model of plt circulation that could predict performance of human plts in human volunteers would positively impact the development of alternate storage conditions. study design/method: we designed an immunodeficient (scid) mouse model to evaluate recovery of human plts and compared this side by side to a radiolabeling study in human volunteers that was conducted for evaluating a new plt storage condition: thermocycling plts (11 hrs ct: 1 hr 37 o c [tc]). autologous apheresis plts stored for 7-days at rt, tc and ct were radiolabeled and infused into healthy human volunteers (n59) and the same non-labeled plts were also infused into mice (n590). blood samples from humans and mice were collected over time to generate survival and clearance curves of the plts in circulation. flow cytometry was used to detect and analyze the human plts in the mouse samples to generate such curves; counts <5% were considered background. results/finding: the mean recoveries of infused plts were 51.2 6 16.7% for rt, 37.7 6 12.3% for tc and 23.1 6 8.8% for ct in humans. in mice, mean recoveries of the same plts were 24.9 6 10.3% for rt, 19.1 6 9.8% for ct and 16.2 6 6.9 for ct (mean6sd). to compare performance of the plts in humans and mice we expressed all recoveries as a percentage of the rt recoveries. in humans tc was $74% and ct was $45% of rt. in mice tc was $76% and ct was $64% of rt. the area under the survival curve (auc) was calculated for the individual mouse study and human trial data sets. the results of both auc were normalized to 100% for rt plts. human tc plts had 26% auc while ct plts had 11% auc compared to rt plts in humans. in comparison, the same tc plts had 39% auc and ct plts had 26% auc of the rt auc in the mice. the calculated ratios of the auc between the tc plts and ct plts of the human data set and mouse model data set are 2.4 and 1.5, respectively. conclusion: the scid mouse model differentiates between rt plts and ct plts similar to humans based on auc and plt recovery data. however, the mouse model cannot differentiate between ct plts and tc plts as occurs in humans. even though the mouse model cannot differentiate between ct plts and tc plts, it may still be a useful tool to screen other novel storage conditions for human plts. converting the component manufacturing from a manual process to automation nicole peters* 1 and geeta paranjape 1,2 . 1 coastal bend blood center, 2 carter blood care background/case studies: initiatives focused on improvements to donor collection processes drove us to investigate opportunities in our component manufacturing processes. our goal was to maintain blood quality while streamlining manufacturing and automating the in-process documentation. the compomat g5 was evaluated using a multi-team approach including component manufacturing staff, equipment management, qa, regulatory affairs and it. study design/method: after a comprehensive evaluation, the team decided to purchase the compomat g5 with the compomaster net software for data management. implementation was planned for a november 2014 go-live. . to centralize processing, new work counters were installed. fresenius kabi installed the compomat g5s and compomaster in june 2014. training and validations were successfully completed and a full launch occurred mid-march 2015. device and sop training was performed. training qualification checklists were completed for each technician with a required number of successful units processed and completed december 2014. validation was completed and signed off in march of 2015. manufacturing data was collected using the compomaster net data management system and our quality control software for platelet (plt) parameters, including plt count, plt weight, and plt yield from before implementation (bi) and after implementing (ai) of the compomat g5 system. data points were collected from 210 units bi and 302 units ai. results/finding: upon initial implementation, staff training and use, the compomat g5 was found to be easy. plt weight spread was reduced from an average of 22gm to an average of 15 gm. actual plt weights were reduced from an average of 63gm to 59gm, resulting in an average increase in recovered plasma of 3.78ml per unit. plt count on average increased from a count of 1435 to 1506 (10 3 /mm 3 ) with a negligible change in plt yield. conclusion: plt weight spread was reduced by 31.8% after implementation of the compomat g5 and our plt concentrations increased on average by 5%. we were able to consistently produce a smaller volume plt (average 59 gm), which gave us 3.78ml more plasma per unit for recovered plasma. the team intends to review a dryer cryo as a next step for potential additional plasma yields for recovered plasma. deglycerolization of manually glycerolized, frozen rccs using a closed system cell processor anita howell 1 , angela hill 1 , brandie dennis 2 and jason p. acker* 2,3 . 1 canadian blood services, centre for innovation, 2 canadian blood services, 3 university of alberta background/case studies: upon implementation of a closed system cell processor for glycerolization and deglycerolization of red cell concentrates (rccs), many rare rccs frozen using the current manual, open system glycerolization method will remain in the organization's frozen inventory. a study was undertaken to assess the feasibility of deglycerolizing this existing inventory on the closed cell processor and to evaluate how the change may impact post-thaw red blood cell (rbc) in vitro quality. as the closed cell processor uses a fixed centrifuge bowl for deglycerolization and rbc resuspension, both large and small units were assessed to determine the impact of cellular loss and variability in hematocrit on the post-thaw product. study design/methods: 13 abo/rh matched lr sagm rccs were pooled and split to produce 6 large (354 ml) and 6 small (244 ml) rccs. the rccs were stored to 14 d and glycerolized manually by mixing 400 ml of glycerol with the rcc in a 2000 ml freezing bag. units were frozen at -658c for ! 72 h before being removed from frozen storage and thawed in a 378c water bath. 3 large rccs and 3 small rccs were deglycerolized using the organization's current procedure on the cobe 2991 cell processor prior to re-suspension in 0.9% saline, 0.2% dextrose. the remaining rccs were transferred into a 1l bag, spun to allow removal of excess glycerol by manual extraction to achieve a hematocrit of 75 6 5%, and deglycerolized in a 275 ml centrifuge bowl on the acp-215 with re-suspension in as-3. rbc quality was tested at 24 6 2 h post-deglycerolization. results/findings: large rccs had significantly higher hemoglobin per unit (cobe: p50.006, acp215: p50.007) and lower cell recovery (cobe: p50.002, acp215: p<0.001) post-deglycerolization than smaller rccs on both cell processors. large rccs deglycerolized on the cobe 2991 had higher hemolysis (p<0.001) and supernatant potassium (p50.001) than did small volume rccs. large cobe 2991 rccs had higher hematocrits (p50.033), hemoglobin (p50.006), and recovery (p50.001) than did large acp-215 rccs. however, all cobe 2991 rccs had higher (p<0.001) hemolysis (0.99 6 0.24 %) levels than did acp-215 rccs (0.31 6 0.02 %). cobe 2991 rccs failed to meet regulatory hemolysis standards of 0.8%. conclusion: addition of a 400 ml bolus dose of glycerol to rccs of different volumes results in different concentrations of glycerol in the frozen rcc product and may lead to differences in frozen rcc quality. additionally, the size of the rcc impacts quality for rccs processed on the closed cell processor due to centrifuge bowl volume limitations which result in lower recovery, hemoglobin, and hematocrits. use of the closed cell processor with resuspension in as-3 and storage for 24 6 2 h, met in vitro quality standards for recovery, hemoglobin, and hematocrit, and drastically reduced hemolysis levels in rccs glycerolized manually. the acp-215 cell processor can therefore be used to deglycerolize rccs glycerolized using a manual, open system glycerolization method. background/case studies: washed platelets may be indicated for thrombocytopenic patients who experience severe allergic/anaphylactic or febrile reactions to conventional platelet transfusions. platelet washing process is time-consuming which may delay transfusion. this study was conducted to evaluate the manual platelet washing method (mm) using 0.9% saline and centrifugation and the semi-automated washing method (sam) using the cobe 2991blood cell processor. study design/method: in this study, 20 units of single donor platelets were evaluated (10 washed using the mm and 10 washed using the sam. the collected data included product weights (pre-and post-wash), platelet counts (pre-and post-wash), total plasma protein (pre-and post-wash), presence/absence of platelet clumps, calculated % protein removal, and calculated % platelet recovery rate. the platelet counts were measured on the sysmex exn and the total plasma protein samples were measured on the roche cobas 6000. results/finding: table 1 shows that the average platelet recovery for the sam (92%) was significantly higher compared to the mm (82%). the mm had a slightly higher average protein removal compared to the sam. no platelet clumps were observed in either the mm or the sam. it was observed that the hands-on time for the mm took 10-15 minutes longer than the sam. background/case studies: the interceptv r blood system for platelets is currently licensed for pathogen reduction (pr) of amicus platelets in inter-sol (pas-3) for input platelet doses of 2.9 to 8.0 3 10 11 platelets in 255 to 420 ml of 47 to 68% plasma and 32-53% pas. a new platelet processing set was designed with three storage containers (ts) to process apheresis platelet components in pas-3 containing doses of 6.0 to 12.0 3 10 11 platelets in a volume of 420 to 650 ml. study design/methods: apheresis pcs (amicus v r ) were collected in 35% plasma and 65% pas-3. one study was performed at the nominal dose (9.2 -10.0 x10 11 platelets), volume (558 -629 ml) in 65% pas/35% plasma using single donor apheresis collections. two studies were performed to evaluate the high dose and high volume condition (9.7 -11.8 x 10 11 platelets in 593 -659 ml) using either single or pooled donations. input pcs (n520) were treated with the intercept ts set by the end of day 1 post collection; the incubation time in the compound adsorption device (cad) container ranged from 4 to 16 hours and the intercept treated pcs were stored in 3 containers (n560). day 5 and 7 post-donation pcs were evaluated using a panel of in vitro platelet function assays results/findings: in vitro function data for apheresis pcs in pas-3 treated in the intercept ts set demonstrated acceptable in vitro function (table 1 ). all intercept treated pcs had ph(228c) !6.2. platelet dose and volume recovery post-treatment ranged from 82% to 99% and 88% to 92%, respectively. conclusion: pathogen reduced platelet components processed using the intercept ts set from either single or pooled apheresis donations maintained acceptable in vitro quality through 7 days of storage. intercept blood system for platelets ts set is currently not approved for use in the us. background/case studies: the possibility of transmitting infectious organisms via blood products, plasma and their derivatives is a major public health concern. while current screening measures have considerably improved transfusion safety by reducing the risks associated with known pathogens, they cannot protect from emerging infectious threats. the pathogen reduction technology (prt) represents a proactive strategy to further reduce transfusion-transmitted infectious risk. however, the scientific community broadly agrees over the fact that prt has negative impacts on the product's quality markers. this study aims at evaluating the impacts of the mirasol prt on platelet (plt) quality and plt processing. study design/method: two abo-compatible platelet concentrates (pcs) containing 100% plasma obtained from either apheresis or sagm whole blood (wb)-derived processing were paired, pooled and then split into two equal units. one unit was used as a non-treated control (ctrl) (n56). riboflavin was added to the other pc unit and then exposed to uv light according to the manufacturer's instructions for the mirasol prt (teru-mobct) (test) (n56). numerous in-vitro quality markers (plt concentration, atp, po2, pco2, ph, glucose, lactate, sodium, and potassium) were measured for both mirasol-treated and non-treated pcs on days 1, 3, 5 and 7 for apheresis pcs, and on days 2, 3, 5 and 7 for wb-derived pcs. two flow cytometry assays were used to evaluate cd62p expression with and without thrombin activation, and to measure the percent annexin vpositive plt. transfusion 2017 vol. 57 supplement s3 results/finding: platelet recovery was 92 6 5% and 81 6 10% for apheresis and wb-derived pcs, respectively. mirasol-treated pcs showed higher levels of annexin v-positive cells (3% 6 1 (test), vs. 1.7% 6 0.5 (ctl) on day 5) and a higher rate of cd62p expression than control pc units (58% 6 7 (test), vs. 23% 6 6 (ctl)) on day 5). the mirasol treatment generates changes in ph, glucose and lactate for pcs during storage. conclusion: the mirasol treatment induces a loss in the net number of plts/unit and elevated platelet activation. changes in ph, glucose and lactate suggest that prt affects plt metabolism. finally, prt has numerous impacts on logistic, storage and processing time constraints of blood bank operations. nevertheless, the mirasol prt is routinely used in europe with acceptable clinical outcomes. evaluation of a test method to detect bacterial contamination in platelets; bactx tm assay ji hye park sexton* 1 , lorraine blagg 2 , christi e marshall 1 , herman woodson 1 , sean erony 1 , krishna patel 1 and eric gehrie 3 . 1 the johns hopkins hospital, 2 johns hopkins hospital transfusion medicine dept, 3 johns hopkins university school of medicine background/case studies: bacterial contamination of platelets (plts) is the leading infectious risk of platelet transfusion therapy and it is the most significant infectious cause of transmission-associated morbidity and mortality. therefore, detecting various potential bacterial contaminants in platelets in a timely manner is critical. the bactx assay is a rapid colorimetric assay that detects peptidoglycan, a cell wall component of both gram-positive and gram-negative bacteria. here, we report an analysis of the bactx assay at our hospital. study design/method: we aimed to determine the sensitivity and specificity of the bactx assay. 340 intact leukoreduced apheresis plt (lrap) units were tested by bactx at storage day 4. as a control, each intact lrap was also cultured by an automated bacterial detection system (bact culture) on storage day 3. the results of the bactx test were compared to the results of the bact culture system. results/finding: a total of 340 lrap were tested. 335 lraps initially tested negative by bactx, while 5 lraps initially tested positive by bactx. all 5 initial positive bactx tests were negative when subjected to repeat testing. in contrast, all lraps tested negative with the bact culture system. the specificity of the bactx test was 98.5%. we did not have any true positive test results; therefore, the sensitivity of the bactx could not be determined. conclusion: this is a small study of only 340 platelet units. the expected rate of bacterial contamination of platelets is less than 1 per 2000 units. the 1.5% initial positive rate was therefore higher than expected, but given the small sample size, it is clear that further study is needed to more rigorously assess the true sensitivity and specificity of the bactx assay. in vitro quality of rejuvenated and washed cpd/as-1 and cp2d/as-3 rbc alan d. gray* 1 , matt landrigan 2 , pamela whitley 3 , michael wellington 3 , sherrie sawyer 3 , shalene hanley 3 , emily rondeau 4 , louise herschel 4 , neeta rugg 5 , patricia a.r. brunker 3 , shawnagay nestheide 5 , jose cancelas-perez 5 , larry dumont 6 and zbigniew m. szczepiorkowski 7 . and 2,3-dpg to fresh levels. the objective was to demonstrate that in vitro quality measures are maintained for rbc when stored for >24 hours after treatment with an fda approved rejuvenation solution. study design/method: whole blood (530-550 ml) was collected and processed at 3 sites into leukocyte-reduced rbc (a total of n563 cpd/ as-1 and n564 cp2d/as-3). 50 ml of rejuvenation solution (citra labs) was added to each rbc on day 35 (d-35), incubated for 60 minutes with agitation at 378c water bath (helmer dh4), washed (haemonetics acp215), and stored in as-3 at 1-6 oc for 7 days (d-36 through d-42). in vitro recovery (%) was calculated and hemolysis, atp, and 2,3-dpg were determined on day 0, d-35, d-35 after rejuvenation and washing (postrjv), d-36, d-38, d-40, and d-42. all units were cultured on d-35 postrjv and on d-42, and then concentrated by centrifugation on d-42. results/finding: in vitro rbc recoveries were 95.7% and 95.5% (as-1 and as-3, respectively) and no bacterial growth was observed. hemolysis on d-42 was maintained <1% in 58/63 (92%) as-1 units and 63/64 (98.4%) as-3 units. all as-1 and as-3 units (100%) had hemolysis <1% following concentration by centrifugation. morphology score was reduced to 77% (as-1) and 74% (as-3) by d-35, restored after rejuvenation (91%, 92%, respectively) and maintained through d-42 (>90%). atp was restored and maintained above fresh levels after rejuvenation. 2,3-dpg was restored above fresh levels and was maintained !80% of fresh levels through d-38. all values were significantly different compared to d-35 except as noted (p<0.001, paired ttest) ( table 1) . conclusion: rejuvenation of stored rbc restores atp and 2,3-dpg above fresh values and morphology to near fresh levels while maintaining improved in vitro rbc quality measures through d-42 when compared to nonrejuvenated rbc on d-35. this study is funded by zimmer biomet. storage >24 hours is not fda approved for use at the time of this publication. liposomes and rejuvenation: new approach for improving quality of stored red blood cells luciana da silveira cavalcante 1 , jason p. acker* 1,2 and jelena holovati 1 . background/case studies: liposomes have been shown to minimize rbc membrane damage occurring during 42-day hypothermic storage (hs), while rejuvenation solutions have been shown to restore rbc metabolism by maintaining atp and 2,3-dpg levels. this study aimed to evaluate the effect of combining liposomes and rejuvenation on the quality of stored rbcs. study design/methods: five leukoreduced packed rbc units obtained were pooled and split. the units produced were segregated into four experimental groups: sham control (s), liposome-treated (l), rejuvesol-treated (r) and liposome 1 rejuvesol-treated (l1r). the prbcs were incubated for 1 h at 378c with hepes-nacl (sham), liposomes (dopc:chol, 7:3 mol%, 2 mm lipid), rejuvesol or liposomes plus rejuvesol. the in vitro quality was accessed by hemolysis, deformability, aggregation, atp and 2,3-dpg at day 42 hs. results/findings: hemolysis was significantly decreased in all treatments compared to sham control (0.60 6 0.06%): l (0.53 6 0.01%, p50.042), r (0.43 6 0.02%, p50.004), l1r (0.48 6 0.06%, p50.020). ektacytometry analysis showed an increase in maximum elongation (ei max ) in r (0.55 6 0.01, p50.010) and l1r (0.55 6 0.01, p50.010) treatments compared to s (0.53 6 0.01) but not l (0.53 6 0.01, p50.936). rbc rigidity (kei) increased in all treatments compared to sham (1.19 6 0.07): l (1.28 6 0.06, p50.025), r (1.44 6 0.17, p50.010) and r1l (1.44 6 0.06, p50.004). aggregation amplitude was significantly increased by r treatment only (24.07 6 1.67 au vs. 19.12 6 1.38 au, p50.004). atp levels were significantly higher in all treatments compared to sham (1.64 6 0.14 mmol/g hb): l (2.00 6 0.21 mmol/g hb, p50.010), r (4.70 6 1.20 mmol/g hb, p50.004), l1r (5.00 6 1.56 mmol/g hb, p50.004). the levels of 2,3-dpg were no longer detectable in s and l treatments at day 42. the combined treatment was comparable to r (2.38 6 3.26 mmol/g hb vs. 2.62 6 2.20 mmol/g hb, p50.868). conclusion: both rejuvenation and liposome treatments improved the quality of stored rbcs compared to sham control. the combined treatment (l1r) did not have a greater impact in improving in vitro quality of stored rbcs compared to rejuvenation alone. step toward a unique and adaptable thermoregulation system lucie boyer 1 , eric ducas 1 , patricia landry 1 , nathalie dussault 1 , jacques bernier 1 , danny brouard* 1 and anne maltais 2 . 1 h ema-qu ebec, 2 institut de technologie des emballages et du g enie alimentaire background/case studies: h ema-quebec (hq) is facing major logistic challenges in the transportation and distribution of blood components over a large geographic area. in collaboration with the institut de technologie des emballages et du genie alimentaire, our applied research group is working on the development and optimization of a transport packaging for the 500-ml whole blood leukotrap rc system (haemonetics corp.). the objective is to design a packaging system for the rapid cooling (t < 108c) of one to six 500-ml whole blood units (wbu) within 8h from collection. moreover, the insulating and thermoregulation system must maintain the internal temperature of wbu between 18c and 108c for 24h under extreme external conditions (-308c to 408c), including the initial blood cooling period. study design/method: the proposed packaging design is based on an external coroplast box containing six vacuum insulated panels (vip) for increased insulating efficiency. preservation of the initial cooling period and extended thermoregulation properties were ensured by an assembly of preconditioned 58c phase change material (pcm). the number of pcm, their position and conditioning were optimized and tested in order to meet the expected performance criteria. preconditioned pcm were stored into vip boxes for 24h at 20-248c before each test to mimic a worst-case scenario for remote blood drives. for the experimental testing, 500-ml wb bags were filled with 555 ml saline 0.9% at t 5 308c to mimic freshly collected wb. probes were positioned inside the saline-filled bags to monitor temperature profiles of wbu under extreme winter (-308c) and summer (408c) conditions. shipping boxes were filled with either one or six bags (n5 2). results/finding: the results showed that the thermoregulation box prototype is able to cool wbu bags under 108c in 4.55 6 0.62h and maintain their internal temperature between 18c and 108c for 24h with final values ranging between 6.38c and 9.38c for the extreme summer scenario. similar results were obtained for the extreme winter scenario; units reached the 108c threshold value in 2.4 6 0.2h and the bags' internal temperatures were within the acceptable range for 24h. conclusion: the insulating and thermoregulation system met hq performance criteria. preliminary results showed that pcm could be conditioned at temperatures higher than -138c without any significant impact on the system performances. hq is currently validating the shipping box prototype performances. additionally, we are working on reducing the pcm conditioning time to optimize logistic operations. as this packaging has many advantages in terms of durability, price and convenience, hq intends to evaluate this system for the packaging and transport of other lines of blood products. stuart weisberg* 1 , christopher c. c silliman 2 , beth shaz 1 , marguerite kelher 2 and claudia s. cohn 3 . 1 new york blood center, 2 bonfils blood center, 3 department of laboratory medicine and pathology, university of minnesota background/case studies: platelets collected and stored in platelet additive solution (pas) reduce recipient exposure to donor plasma components. to better define the effects of pas on platelet supernatant composition, we compared total protein, isohemagglutinin titers, hla antibodies and in vitro neutrophil (pmn) priming activity in supernatants of pas-c platelets to plasma platelets. study design/methods: apheresis platelets from group o blood donors were collected into either 100% donor plasma (n550) or 65% pas-3 / 35% donor plasma (n550). within 12 hours of collection, samples of the product supernatant were frozen, assayed for total protein concentration, anti-a and anti-b titer, and pmn priming activity within the total and lipid extractable fractions. all samples were screened for hla antibodies. screen-positive samples were tested using luminex single bead assays for antibody strength and specificity. soluble cd40 ligand (scd40l) was measured using solid-phase elisa. results/findings: supernatants of pas-c platelets had significantly lower total protein concentration, anti-a and anti-b titers compared to plasma platelets. there was no significant difference in the number of hla-antibody screen positive pas-c (3/50 products) compared to plasma platelets (2/50 products); however, the hla-antibody screen-positive supernatants of pas-86a transfusion 2017 vol. 57 supplement s3 abstract c platelets had fewer hla specificities (2 specificities) compared to those of the plasma platelets (18 specificities). pmn priming activity was significantly increased in the supernatant of pas-c platelets. the lipid extractable fraction was not affected; however scd40l levels were increased in the supernatant of pas-c compared to plasma platelets (table 1) . conclusion: decreased plasma proteins likely underlie lower rates of allergic and febrile non-hemolytic transfusion reactions seen with use of pas-c platelets. decreased anti-a and anti-b titers may prevent hemolysis from minor abo mismatch. lower hla-antibody specificities may mitigate transfusion related acute lung injury (trali). increased pmn priming by pas-c platelets is likely due to platelet membrane release of scd40l and not bioactive lipids. although scd40l has been associated with trali, only pmn priming with lipid -not cytokine -agents has been causally linked with trali. the mechanism and clinical impact of increased scd40l in pas-c platelets remain to be elucidated. background/case studies: current guidelines require a reduction of residual white blood cells (rwbc) below 5x10 6 wbc in us and 1x10 6 wbc in europe, per unit. the established reference method for testing rwbc in platelet (plt) and red blood cell (rbc) products is flow cytometry. alternative technologies have been developed including hemocytometry and microfluorometry. study design/methods: this study compared performance and workflow efficiency of the facsvia, a flow cytometer with a simplified workflow and automated loader to the adam automatic microscopic cell counter based on imaging technology. nonfiltered whole blood (wb) samples, apheresis platelet units (n52) and leukoreduced (lr) rbc units (n52) were used to generate spiked samples. apheresis platelets and lr rbc were filtered to deplete wbcs and were used as a diluent. nonfiltered wb samples were the source of wbcs to prepare a sample of 1000 wbc/ul. the spiked samples of 5, 12.5, 5, 25, 50 and 100 wbc/ul were prepared from the source sample of 1000 wbc/ul and filtered platelet and rbc units. to evaluate linearity, wbc concentrations (0, 12.5, 5, 25, 50, 100 wbc/ul) were measured using adam and facsvia. samples were stained and run in triplicate on each analyzer. data was analyzed using linear regression. the results were proportional to the wbc concentration in the spiked samples. reproducibility of the two systems was measured by running spiked samples (0, 5, 25, 50 wbc/ul). 10 tubes of each sample were stained and run per system. the %cv and %diff were calculated. a batch of 20 samples (plt and rbc) were run on both analyzers, repeated for 5 days. workflow efficiency was assessed observationally by measuring the time of tasks performed. tasks recorded were instrument qc, assay controls and sample testing and analysis. results/findings: the wbc concentration results for plt and rbc samples on facsvia correlated well with adam (r-plt50.996, slope50.972), (r-rbc50.999, slope50.992). the %diff-plt at 5, 25, 50 wbc/ul were 7.8, 4.7 and 10, respectively. the %diff-rbc at 5, 25, 50 wbc/ul were 10.8, 3.2 and 14.7, respectively. the average total testing time was similar on both instruments; 89 min for the facsvia and 92 min for the adam. of the total testing time, adam required continuous hands-on time, while facsvia demonstrated 62% (56 of 89 min) hands-off time. conclusion: both instruments showed comparable precision, linearity and accuracy. while the average total testing time was similar on both instruments, facsvia offered a significant workflow efficiency advantage. users saved an average hands-on time of 56 minutes that could be used on other tasks. platelet rich plasma and quality control: is there a role for the blood bank? claudia s. cohn* 1 and mickey koh 2 . 1 department of laboratory medicine and pathology, university of minnesota, 2 st george's hospital and medical school background/case studies: autologous platelet-rich plasma (aprp) is a poorly regulated blood component often produced at the patient's bedside and used for indications such as chronic and acute orthopedic injuries, wound and incision-healing and rheumatologic diseases. prp isolation can be done by apheresis, which yields a consistent, platelet-rich fraction; however, most aprp is made using small bench-top centrifuges with cartridges that deliver uneven platelet enrichment. thus, the consistency and quality of aprp is questionable and the lower yielding prp may have decreased efficacy. study design/methods: a survey was designed to assess aprp manufacture, usage and quality control (qc) measures taken prior to its use. a survey was developed with input from content experts. the survey was sent to members of best and isbt. survey respondents were encouraged to forward the survey to colleagues, thus a true denominator is unknown. a total of 62 completed and partially completed surveys were received. results/findings: responses came from 13 countries, but the majority of responses came from the united states (us). of the respondents, 35% reported aprp use in their hospital. aprp was used predominantly for outpatients, though >40% of hospitals also used aprp in the in-patient setting. in most hospitals, aprp was used by 1-5 mds; however, 3 hospitals had >10 mds using aprp. the aprp was used for orthopedics, wound/incision repair, rheumatology and other indications. in the us the aprp was manufactured outside of the blood bank, while outside the us aprp was isolated by blood bank personnel. nearly all the aprp manufacturing was done with no quality control (qc) measures (97%); however, 3 respondents assessed the final product prior to release. these qc measures included a platelet count to measure the enrichment of the platelet fraction, culturing the product and infectious serology testing. in some cases, if the aprp failed qc it could still be used, pending an md's approval. in the 3 hospitals conducting qc on the final aprp, the testing was done by the blood bank. a subset of respondents from african nations also used allogeneic prp (allprp). in contrast to the patterns of use with aprp, allprp was used primarily for inpatients for indications including orthopedics, wound/incision repair and 'other'. the allprp was manufactured in the blood bank or the donor center with no qc other than a regular check of the centrifuge used to isolate the prp fraction. conclusion: prp is used in hospitals throughout the world for a wide variety of indications. the blood bank is involved in its manufacture in some countries, but in the us aprp is made outside of the blood bank. quality control of aprp production and the final product is not done in most hospitals. to improve the consistency and efficacy of prp, more stringent qc measures need to be in place. background/case studies: the morphology of donated red blood cells (rbc) change with storage, along with a loss of deformability, increased surface exposure of phosphatidylserine (ps), and decreased intracellular atp. these changes have been associated with increased rbc clearance within hours of transfusion. analysis of morphological alterations of stored rbc with imaging flow cytometry (ifc) has identified a subpopulation of small rbc that accumulates upon storage. this rbc subpopulation has a reduced projected surface area and undergoes a spherocytic shift which is expected to induce their retention in the spleen (roussel, dussiot et al, 2017) . some of the storage alterations are reversible when the rbc metabolism is reestablished. as such, treatment with a rejuvenation solution (citra labs) before transfusion is expected to restore some of the rbc properties and thus potentially increase their capacity to stay in circulation and operate effective tissue oxygenation following transfusion. study design/methods: a multi-parametric analysis of rbc alterations was performed to evaluate the effect of rejuvenation on rbcs stored in sagm (n56) under blood bank conditions at day 3 (d3), at day 42 (d42), after rejuvenation (r), and after rejuvenation and washing (rw). morphological alterations of stored rbcs were evaluated with ifc (imagestream x mark ii, amnis v r ). results/finding: rejuvenation increased the level of intracellular atp, confirming the metabolic effect of this process. population distribution as per rbc projected surface area measured by ifc depicted a well-demarcated subpopulation of small rbc that increased with storage from 2.1-8.8% at d3 to 8.3-68.1% at d42. rejuvenation markedly reduced this storage-induced spherocytic shift (1.7-29.3%) and partially restored rbc morphology, an effect confirmed by differential interference contrast microscopy. the restoration effect of the rejuvenation process did not correct the storage-related loss of rbc elongation but was associated with a decrease in ps exposure (table) . conclusion: our multi-parametric analysis shows that some but not all storage-related alterations are therefore corrected by metabolic rejuvenation. the impact of these effects while generally positive at the cellular scale requires further analysis by specific clinical studies assessing transfusion yield and tissue oxygenation. red cell concentrate volume and manufacturing method impact post-thaw quality in cryopreserved products processed using a closed cell processor anita howell 1 , angela hill 1 , tracey turner 2 , april xu 2 , brandie dennis 2 and jason p. acker* 2,3 . 1 canadian blood services, centre for innovation, 2 canadian blood services, 3 university of alberta background/case studies: the blood service uses both top/top with whole blood filtration (wbf) and top/bottom with red cell filtration (rcf) methods to prepare cpd/sagm lr red cell concentrates (rccs). mean volume (ml) is higher in wbf units (314 6 15) than in rcf units (275 6 16), with similar hematocrits. a closed system cell processor is currently being implemented for cryopreservation of rccs. post-deglycerolization re-suspension in as-3 additive solution is performed on-instrument to a defined total end volume, as dictated by the centrifuge bowl size. the impact of the resulting variation in hematocrit on post-thaw in vitrorbc quality was evaluated to ensure that regulatory standards can still be met for rccs at the extreme edge of the input volume range. study design/methods: 12 small rcf (252-263 ml) and 12 large wbf (322-353 ml) rccs were stored for 21 d before being glycerolized and frozen at -658c for !72 h. large rccs whose red cell mass exceeded the capacity of the 275 ml deglycerolization centrifuge bowl were volume reduced prior to glycerolization. rccs were thawed in a 378c water bath, deglycerolized and re-suspended in as-3. rccs were stored 14 d and then tested for in vitrorbc quality. results/finding: small rcf rccs had lower (p<0.05) hematocrit, specific gravity, hemoglobin per unit, supernatant k 1 and na 1 concentration, deformability (ei max ), and higher (p<0.001) recovery than did large wbf units. no significant differences in hemolysis, atp, 2,3-dpg, p50, rbc indices, rbc morphology, or residual glycerol were seen between groups. the majority of units met acceptance criteria (table 1) , however 8 of 12 large wbf units had rbc recoveries < 80% due to pre-glycerolization volume reduction, and 2 of the small rcf units had hemoglobin values < 35 g per unit. when the recovery and hemoglobin failure rates are analyzed against the organization's rcc production volume distribution, the mean recovery is projected to be well above 80% and the hemoglobin failure rate would be below 10% of units tested; compliant with regulatory standards. conclusion: the differences between groups in the cryopreserved rcc physical characteristics were expected due to the re-suspension method and differences in the input product red cell mass. the lack of significant metabolic differences between groups indicates that the differences in postdeglycerolization hematocrits are not adversely affecting product quality. 0.03 6 .02 0.33 6 .28 0.83 6 .3 0.32 6 .14 elongation index (30pa) 0.602 6 .008 0.585 6 .017 0.580 6 .017 0.578 6 .017 this study is funded by zimmer biomet. (hasan 1994) . the objective was to determine the effect of rbc rejuvenation on rbc oxygen release capacity (orc) and estimated oxygen consumption (vo 2 ) after simulating a single unit transfusion of either standard or rejuvenated rbc stored for 42 days. study design/method: oxygen dissociation curves (odc) (hemox analyzer, tcs scientific) were generated from fifty-two (52) rbc units (leukocyte-reduced), cpd/as-1 or cp2d/as-3, on day 0, day 42, and after rejuvenation and washing (pw). the odc for each sample was used to determine orc (ml o 2 /g hb) and total releasable oxygen (tro) of the unit (ml o 2 ). orc was determined by assessing the change in % o 2 saturation from 100 mm hg po 2 (e.g., lung) to 40 mm hg po 2 (e.g., venous blood) multiplied by 1.34 ml o 2 /g hb (li 2016). a simulated baseline pretransfusion vo 2 of 115 ml o 2 /min was estimated using the day 0 orc and assuming a 7 g/dl transfusion trigger with a cardiac output of 5 l/min and 5 l blood volume. paired student's t tests were used for comparative statistical analyses. results/finding: rbc rejuvenation on day 42 restored orc and tro to levels greater than day 0 ( table 1) . orc of the rejuvenated unit was 1.5 6 0.2 times and 3.4 6 0.5 times greater than rbc on day 0 and day 42, respectively (p<0.001). vo 2 increased after a simulated single unit transfusion of rbc (day 0, day 42, and pw) by 19.3%, 8.9%, and 28.8% over the pre transfusion vo 2 , respectively (p<0.001). conclusion: these results suggest a transfusion with rejuvenated rbcs has the potential to release 3.3 times the volume of o 2 compared to standard, untreated rbcs stored for 42 days. inferior oxygen delivery to tissues (vo 2 max) has been observed during exercise in healthy human volunteers after transfusion of two autologous rbc units stored for 42 days vs 7 days which seem dependent on genetic variability and storage time (bennett-guerrero 2017). therefore, transfusion practices to correct anemia may be less effective than intended due to the variable orc of standard stored rbc units. transfusion strategies should consider whether the use of rbc with increased orc may be physiologically advantageous. disclosure: this study was funded by zimmer biomet. rejuvenation solution as an adjunct storage solution maintains physiological hemoglobin oxygen affinity during rbc unit storage andrea ansari* 1 , jay srinivasan 1 , gustaaf de ridder 2 , alan d. gray 3 , matt landrigan 4 , keaton charles stoner 5 , angela crabtree 6 , jessica poisson 7 and ian welsby 8 . 1 duke university school of medicine, 2 duke health pathology, 3 citra labs, a zimmer biomet company, 4 zimmer biomet, 5 duke university, 6 department of pathology, durham veterans affairs medical center, 7 duke university hospital, 8 duke university medical center background/case studies: deleterious changes develop during the storage of packed red blood cells (rbcs) collectively called the "storage lesion". these include altered membrane composition and decreased deformability, increased in-bag and post-transfusion hemolysis, loss of atp, snitrosohemoglobin, vasodilatory capacity, and cell surface ps expression, and depleted 2,3-diphosphoglycerate (2,3-dpg). the loss of 2,3-dpg increases the oxygen affinity of hemoglobin, resulting in lower p50 (partial pressure of oxygen at 50% hemoglobin saturation). decreased p50 may negatively impact the ability for transfused rbcs to release oxygen to peripheral tissues. an fda-approved rejuvenation solution (citra labs) can restore normal levels of atp and 2,3-dpg, normalizing membrane function and oxygen affinity, respectively. this process requires incubation at 378c for an hour, an impractical step in time-sensitive situations, followed by washing of the rbcs. we tested the hypothesis that rejuvenation without the incubation step ("cold rejuvenation") could prevent or reverse changes in oxygen affinity, deformability, and susceptibility to hemolysis of rbcs. study design/method: eight units of group a1, leukoreduced prbc stored in as-1 were obtained from our local blood center. after 3 days of storage, units were divided into 4 separate aliquots: control (ctl), wash (w), standard rejuvenation (sr), and cold rejuvenation (cr). the rejuvenation solution (50ml) was added to the cr group, and all groups were then stored for another 12 days at 1-68c. on day 15 of storage, the sr group was incubated for 1 hour at 378c with rejuvenation solution, after which the w, sr, and cr groups were separately washed on a c.a.t.s v r (fresenius kabi) using the high quality wash setting. hemoglobin p50 was measured by tonometry using a hemox analyzer (tcs scientific). deformability (elongation index or ei) was measured by ektacytometry (lorrca mechatronics). supernatant plasma free hemoglobin (pfhb) was measured using visiblelight spectrophotometry. cell surface ps expression (ps1) was measured by annexin v flow cytometry. all group results were compared using nonparametric wilcoxon signed-rank tests with a 5 0.05. results/finding: significant differences in p50 were noticed between all groups (table 1) . ei, ps1, and pfhb did not differ between groups. conclusion: cold rejuvenation prevents the increased oxygen affinity (lower p50) seen over 15 days of rbc storage without adverse effects on deformability or hemolysis. this offers an alternative to incubated rejuvenation to provide clinicians with ready access to rbcs with a high/normal p50 that may better release oxygen to the tissues. cause transient and potentially fatal cardiac arrhythmias upon transfusion, particularly in infants, and massively-transfused patients, and those with compromised renal function. reactive antibodies and other inflammatory agents in rbcs can also elicit life-threatening reactions, potentially causing high fever, transfusion-related acute lung injury (trali), anaphylaxis, and even death. in this study, a multifunctional bead-based filter was evaluated for removal of k 1 , along with free hemoglobin (hb) and other prbc contaminants that can contribute to transfusion related adverse events. study design/method: ten leukocyte-reduced prbc (300ml) units stored in as-1, obtained from a regional blood donor center at expiration (42 6 2 days), were passed by gravity through sorbent-devices containing 50 ml of multifunctional polymer bead, at a flow rate of 20 ml/min. supernatants were analyzed for k 1 removal as well as free hb, antibodies and cytokines (27-plex, biorad). rbcs were analyzed for viability and integrity via flow cytometry and osmotic fragility assay, respectively. results/finding: filtration of the aged prbc units through the sorbent device reduced [k 1 ] from 54.2 6 5.0 to 1.98 6 1.3 meq/l; equivalent to an 84.6% reduction. free hb was reduced by 96.3% from 2.5 6 1.0 to 0.39 6 0.2 mg/ml. antibodies, specifically igg, iga, and igm decreased from 9.91 6 3.1 to 2.40 6 1.1 mg/ml (77.7%), 0.48 6 0.1 to 0.25 6 0.01 mg/ml (48.9%), and 0.73 6 0.2 to 0.49 6 0.1 mg/ml (31.5%), respectively. inflammatory cytokines were significantly reduced, specifically: ip-10 from 144.27 6 16.2 to 18.43 6 2.7 pg/ml (87.2%), mip-1b from 37.37 6 5.7 to 7.23 6 2.5 pg/ml (80.7%), and pdgf from 1348.3 6 291.9 to 77.91 6 22 pg/ml (94.2%). filtration had no significant impact on cell surface markers of rbc viability (<0.1% decrease) or sensitivity to osmotic changes. values listed represent mean 6 sem (p < 0.01 for all analytes tested). a paired ttest was used to assess significance. conclusion: the sorbent filter was highly effective in reducing the levels of extracellular k 1 as well as free hb, antibodies, and cytokines from prbcs without impact on rbc viability or integrity. this study demonstrates the viability of a multifunctional sorbent filter for removal of k 1 along with other detrimental components from stored prbcs that can readily be incorporated into transfusion practices to minimize adverse effects. background/case studies: platelets carry no rh antigens, but residual red blood cell (rbc) in platelet products can immunize d negative recipients if the donor is d positive. current recommendation is to give rh immunoglobulin (rhig) to rh negative patient if they receive rh positive platelet unit to avoid potential alloimmunization to d antigen. a recent study has shown a very low frequency (1.5%) of d alloimmunization when a rh mismatch platelet is transfused. restricting d negative patients to receive only d negative platelets could create shortage and cause inventory challenges. higher yields of platelets with minimum to none residual rbcs are obtained with new generations of apheresis machines. as a consequence, the need for prophylactic rh immunoglobulin (rhig) may be unnecessary with the use of apheresis derived platelets. the accurate determination of residual rbc in a platelet unit is important for patient safety to prevent rh alloimmunization. hemocytometer is considered the gold standard for cell counting. however, the rapidity and convenience offered by automated methods resulted in widespread use of automated hematology analyzers. currently there are no standardization and/or guidelines to advise what system to use for rbc quantification in platelet products. study design/method: we designed this study to quantify the residual rbc in apheresis platelets and whole blood derived platelets comparing hemocytometer and automated methods. we measured the amount of red blood cells per microliter in 50 apheresis and 50 whole blood derived platelet units using hemocytometer and two different automated hematology analyzers, namely, sysmex (sysmex america, lincolnshire, il) and advia 2120 (siemens healthcare diagnostics, tarrytown ny). the whole blood derived platelet units were produced using acrodose tm system technology. we conducted non-parametric permutation test based on 10000 permutations to compare sysmex and advia between apheresis and whole blood derived groups. abstract collection) rbcs to rbcs stored for 42 days and after treatment with an fda approved rejuvenation solution. study design/method: the addition of a rejuvenation solution to stored red blood cells (rbcs) has been shown to increase atp and 2,3-dpg profiles to fresh levels. the objective was to compare 50% hemoglobin-oxygen saturation (p50) and morphology profiles of fresh(day of collection) rbcs to rbcs stored for 42 days and after treatment with an fda approved rejuvenation solution. results/finding: in vitro rbc recovery (overall) was 97.2 6 2.2%. hemolysis (%) was similar on day 42 before and after dry-air incubation with the rejuvenation solution (0.34 6 0.14% vs 0.35 6 0.14%). percent hemolysis (%) decreased after washing (0.24 6 0.07%) and was maintained below <1% for all units during storage for 24hr (0.51 6 0.19%). average atp and 2,3-dpg were restored above the average fresh values. the morphology score decreased $25% by day 42, which was restored to near fresh values following rejuvenation and washing and storage 24hr (93.7% and 95.1%, respectively). rbc oxygen affinity, as assessed by p50, was restored above fresh values. all values were significantly different compared to day 42 (p<0.001, paired t-test) ( table 1) . conclusion: rbc morphology was restored to near fresh and average atp, 2,3-dpg, and p50 were restored above fresh values when incubated with a rejuvenation solution using the dry-air incubation process. rbc morphology, atp and 2,3-dpg were maintained during storage 24hr. rejuvenation of refrigerated rbcs may offer avenues to improve rbc quality prior to transfusion. vandi ly*, dimath alyemni, warren r korn, matthew j brune and julie katz karp. thomas jefferson university hospital background/case studies: blood donors are screened with a donor history questionnaire that includes questions regarding behavioral risk factors, but none that specifically screen for the use of marijuana. therefore, there is the theoretical possibility of transfer of active cannabis metabolites through transfusion. donor plasma collected at an urban, hospital-based blood donor center was examined for the presence of active cannabis metabolites, d9tetrahydrocannabinol (thc) and 11-oh-d9-tetrahydrocannabinol (11-oh-thc). study design/method: de-identified donor plasma segments were sequestered and stored frozen until time of testing. testing for thc and 11-oh-thc was performed by liquid chromatography-tandem mass spectrometry (lc-ms/ms) based on a method modified from lacroix and saussereau. in summary, this method used dabsyl chloride derivatization of thc and 11-oh-thc to produce samples for lc-ms/ms analysis. lc used a c18 column. post-column detection by ms/ms used positive ion electrospray with q1:q3 ion pairs of m/z 5 605.3:225.3 (internal standard (is), d3-thc), m/ z 5 602.2:225.2 (thc), and m/z 5 618.3:256.1 (11-oh-thc). quantitative results for thc and 11-oh-thc were obtained from a standard curve (ratio of analyte integrals to integrals of internal standard) ranging from 0-50 ng/ ml for both thc and 11-oh-thc. limits of quantitation, defined as 5 standard deviations above background, were 0.7 ng/ml for thc and 7 ng/ml for 11-oh-thc. results/finding: a total of 424 donor plasma samples were tested for thc and 11-oh-thc. no samples tested positive for either thc or 11-oh-thc. theoretical calculations according to statistics of a poisson distribution indicated that there would be a 50% probability of one or more positives at a prevalence of 0.16% positive samples, and a 95% probability of one or more positives at a prevalence of 0.71% positive samples. results thus indicated a boundary of prevalence of the presence of active thc-metabolites in plasma samples to be less than 1% among this donor population. standard pharmacokinetics of cannabis metabolism in previous studies indicate a likely time window of less than 12 hours for post-exposure detection of thc and/or 11-oh-thc in plasma. conclusion: testing of 424 donor plasma samples for active metabolites of cannabis at one urban, hospital-based blood donor center produced no testpositives. statistically, results indicated that prevalence of positivity, if greater than zero, is at most less than 1%. probability of occurrence of cannabis metabolites in blood donor samples is likely to be highly variable across donor centers and is largely dependent on blood donor demographics. elisabeth maurer-spurej* 1 , ruqayyah almizraq 2 , daniel millar 3 and jason p. acker 4 . 1 university of british columbia, 2 university of alberta, 3 lightintegra technology inc., 4 canadian blood services background/case studies: the controversy around the quality and clinical impact of aged red blood cell concentrates (rcc) is ongoing. current studies are limited by the lack of quality measures suitable for routine screening of rcc. based on evidence that fragments called microparticles (mp) or extracellular vesicles are markers of cellular activation or degradation, this study investigated the utility of mp screening to characterize the effect of rcc production methods and storage. study design/method: red blood cell concentrates were prepared by whole blood filtration (wbf; top/top) or red cell filtration (rcf; top/bottom) methods, centrifuged to prepare a supernatant and tested for mp content (as measured with dynamic light scattering or a tunable resistive pulse sensing technique), hemolysis, atp and red cell deformability on days 7, 21, and 42 of storage. one rcf rcc was tested on days 1, 5, 14, 21, and 43 and six 10 ml aliquots were stored in parallel and tested on days 14, 21, and 43. all samples were tested for mp content and compared to the other quality indicators. results/finding: mp content showed a linear increase with storage time with statistically significant differences between days 14, 21 and 43 (p<0.001) and correlated with supernatant hemoglobin, and inversely with atp or rbc elasticity. both mp testing methods agreed with respect to total mp content. starting levels of the quality indicators varied between donations, preparation methods (wbf rcc contained much higher levels of mp), and storage time. mp content in the 6 aliquots were consistent at each time point but statistically higher than in the original rcc on and after day 21 of storage. conclusion: mp content correlates with measures of hemolysis and other rbc quality indicators and could be implemented as a routine screening tool. differences in mp content between donors, processes and age could be monitored and used to inform component production decisions. measuring mp content would allow 100% screening of rcc products in studies and pragmatic qc initiatives which are needed to settle the controversy about the clinical effect of rcc age. single donor spray-dried plasma: the future of plasma therapy? qiyong peter liu*, jihae sohn, ryan c. carney, sruthi sundaram and mark a popovsky. velico medical inc background/case studies: frozen plasma is integral to hemostasis management in many situations but logistically cumbersome because of frozen storage and long thawing time. spray-dried plasma (odp, on demand plasma) is potentially superior because it may be stored under refrigeration near the patient and reconstituted in minutes at the point-of-care. the objective of this study is to determine if odp can be consistently manufactured at a blood center with key proteins and coagulation function comparable to ffp. study design/methods: units of never frozen plasma collected at a blood center were processed on-site at a fixed volume into odp using velico's spray dryer. odp (n 5 60) and paired ffp aliquots were stored for 31-33 days at 2-68c and -188c, respectively, reconstituted with a fixed volume of rehydration fluid (sterile water for injection), and extensively characterized with respect to the levels of hemostatic proteins, coagulation and complement activation markers, and clotting performance. the volumes of processed plasma and rehydration fluid were pre-determined ensuring similar total protein concentration in reconstituted odp and ffp for direct comparison. results/findings: compared to ffp, odp had ! 80% levels of functional clotting factors (fibrinogen, factors ii, v, vii, viii, ix, x, xi and xii), plasminogen, and protease inhibitors (antithrombin iii, protein c, protein s; plasmin, c1 esterase and alpha 1-proteniase inhibitors). the level of factor xiii in odp was slightly lower, about 70% of ffp by both activity and antigen assays. odp was identical to ffp in the levels of albumin, immunoglobulins (iga, igg and igm), lipoproteins, calcium, citrate, and coagulation proteins evaluated by antigen assays except for factor xiii. the levels of the markers for coagulation (thrombin-antithrombin, prothrombin fragments i1ii and ddimer) and complement (c3a and c5a) activation in odp remained similar to ffp. odp was equivalent to ffp when assessed by aptt, pt and thrombelastography. transfusion 2017 vol. 57 supplement s3 abstract spray-drying fragmented a substantial number of high molecular weight von willebrand factor (vwf) multimers into smaller ones, leading to a net increase of vwf multimers in odp. the size re-distribution reduced the vwf ristocetin cofactor activity (vwf:rco) to 62% in odp relative to ffp, but had no impact on vwf antigen and factor viii function (stabilized by vwf). vwf-specific studies have shown that odp retains hemostatic function in supporting platelet adhesion and aggregation (see abstracts by meledeo et al/us army institute of surgical research and bercovitz et al/blood center of wisconsin). conclusion: odp can be manufactured at a blood center with a quality comparable to that of ffp. future studies will determine if the product is bioequivalent to ffp and comparable in safety and efficacy. background/case studies: the collection time of whole blood is, according to european guidelines, limited to 15 minutes. in addition, donations with collection times between 12 and 15 minutes should not be used for preparation of platelet (plt) concentrates (pc) because of the chance of too much activation of plt. it seems justified to re-evaluate the quality of plt from these donations because new generations collection systems and mixers were introduced, including a more efficient needle. the aim of this study was to investigate the in vitro quality of pc prepared from 12-15 minutes buffy coats (bc) with the aim to prevent unnecessary discarding of bc and to simplify the total blood bank process. study design/method: single-donor pc (spc, n56) were prepared from one 12-15 minutes bc and 60 ml of autologous plasma in a 600 ml pvc-dehp container. as a reference, spc from donations with collection times of <12 minutes were prepared (n55). in addition, pc were prepared from 5 bc, of which at least 4 bc were from 12-15 minutes donations (n55). after pooling of the bc, 300 ml of pas-e was added and a standard pooling set with a pvc-bthc storage container was used for storage of pc. all pc were stored for 8 days at 22 6 28c and sampled at regular intervals for determination of the in vitro quality. aggregation tests were performed with chronolog (adp or collagen) and multiplate (arachidonic acid) aggregometers. thromboelastography (teg), using kaolin as an activator, was applied for assessment of the overall clotting capacity. values are expressed as mean6 sd. a non-paired t-test or a mann-whitney u test was applied for statistical analyses of normal or non-normal distributed data respectively. results/finding: volume (67 6 5 vs. 66 6 16 ml) and platelet content (74 6 11 vs. 71 6 15 x10 9 ) were similar in both groups. at the end of storage, both groups showed comparable in vitro quality (day 8, ph(378c): 6.84 6 0.16 vs 6.83 6 0.17, other data not shown). no differences in aggregation response after stimulation with arachidonic acid, adp or collagen were measured. teg parameters in both groups were also comparable. the five-donor pc fulfilled all requirements of european guidelines, aside from occurrence of small aggregates at day 6 and/or 8 in 2/5 pc (possibly because sometimes ab0 incompatibility was accepted). on day 8, plt showed low cd62p expression (17.1 6 1.8%) and phosphatidylserine exposure (annexin v binding, 8.9 6 1.9%). hypotonic shock response of platelets was comparable with historical data. conclusion: single-pc in plasma as well as five-donor pc in pas-e, prepared from 12-15 minutes whole blood donations had a normal composition and showed good in vitro quality during 8 day storage. to substantiate that the exclusion of 12-15 minutes donations for pc preparation could be stopped, further studies will be performed. the effects of a pneumatic tube system on red blood cell units amy mata* 1 , jessie miller 1 , ranee marie wannarka-farlinger 1 , sandra bryant 1 , scott a hammel 1 , sherry stern 1 and camille van buskirk 2 . 1 mayo clinic, 2 mayo clinic rochester background/case studies: the use of pneumatic tube systems (pts) has become commonplace in many healthcare facilities throughout the world. the purpose of these systems is to transport products and specimens, resulting in reduced turnaround time for laboratory testing and to aid in the timely delivery of patient care. a downfall of ptss is that they have the potential to play a role in increased hemolysis. while several studies have been published on the effects of ptss on blood specimens, there are very few that address the effects on blood products, specifically red blood cells (rbc). the objective of this study was twofold: to determine if the pts that is in use at our facility contributes to an increase in hemolysis of rbc units and to evaluate how the pts system affects red cell microparticle (rmp) levels. study design/method: forty-one units of as-3 rbcs, 20 irradiated and 21 non-irradiated, were selected for the study. the units varied in age, ranging from 2 to 42 days old. specimens were obtained from each unit both prior to and after being transported through the pts, which runs underground and spans the length of a mile and a half. specimens were spun down and the plasma supernatant was removed. all specimens were evaluated for plasma hemoglobin (hgb), potassium (k), hemolysis index (hi), and rmps. the wilcoxon signed-rank test and p value were used to compare the pre and post values. additional statistical analysis was performed to compare the values after adjusting for age and irradiation. results/finding: after sending the rbc units through the pts, hgb, hi, and rmps were statistically (p< 0.05) higher than before. when adjusted for irradiation, the same analytes remained statistically higher, however when adjusted for age, the p-value was only significant for hgb and hi. the k values did not significantly change. rmps significantly increased, but only if the units were irradiated (p50.02). (table) conclusion: the use of a pts provides an effective means to transport blood products; however, it can contribute to biological changes within rbc units. it is uncertain at this time how those changes can affect the outcome of patients who receive these products. each pts system is different in its specifications and should be validated prior to being used to transport blood products. validation of factor viii levels of thawed fresh frozen plasma after 5 days of storage pei lun karen lim* 1 , erma sofia sumardi 1 , isamar eduardo ancheta 1 , susan lim 2 , christina yip 1 , lip kun tan 2 and shir ying lee 3 . 1 national university hospital singapore, 2 national university hospital, 3 national university hospital, singapore background/case studies: plasma transfusion is indicated in patients with coagulation factor deficiencies and active bleeding, or who are about to undergo an invasive procedure. fresh frozen plasma (ffp) has to be placed in the freezer within 8 hours of processing and stored at -188c or colder in order to preserve its coagulation factors. thawed ffp has an expiration period of 24 hours hence to reduce wastage, this study aims to investigate factor viii (fviii) activity in thawed plasma stored for 5 days and kept at 1 to 68c. fviii was chosen as it is an important coagulation factor in correcting coagulopathies. arbitrary fviii level acceptance limit was set as not less than 50 iu/dl. study design/method: randomly selected units of ffp (n510) were measured for fviii concentration based on clotting assay (stav r -deficient 92a transfusion 2017 vol. 57 supplement s3 viii diagnostica stago). fviii levels were measured at five time points: prefreezing, 0, 24, 72 and 120 hours post-thawing. ffp were thawed using helmer plasma thawer (helmer scientific) at 30 to 378c for 35 minutes. an aliquot of thawed ffp from each unit was removed and measured for fviii before refrigeration (0 hours post-thaw). thawed plasma (tp) units were kept in a refrigerator at 1 to 68c for 5 days for subsequent testing. results/finding: results obtained were listed in table 1 . units 7 to 9 were not tested for fviii at post thaw-24 hour due to operational issues. the overall fviii concentration decreased at an average of 13% from pre-freezing to post thaw 0 hour. after further storage of tp post thaw-24 hour and -72 hour, residual fviii level remain to be above 50 iu/dl except unit 10 which had a lower initial fviii concentration. at post thaw-120 hour, 7 out of 10 units tested had residual fviii activity within the pre-set standard of 50 iu/ dl. the average decline from 0-hour post-thaw to 24-hour, 72-hour and 120hour post-thaw was 36.5%, 42.7% and 47.9% respectively. there was no observed trend of any blood group having higher or lower pre-freezing fviii and this is likely due to small sample size. conclusion: decrease of coagulation factor such as fviii in ffp is expected due to its diminishing stability. nevertheless, our data showed that majority of the tp retained at least 50 iu/dl of fviii. typically patients with factor levels below 30 iu/dl may start to show abnormal coagulation profile. while tp is not used for specific factor replacement therapy, it may be indicated for patients with general coagulopathies and active bleeding. further study extending to measurement of other labile factor such as fv may add value to the validation study. validation of the pathogen reduction method using amotosalen/ uva: comparing pathogen-reduced pooled prp-platelets and conventional single prp platelets for quality and bacterial inactivation efficacy lubna ahmed almenawi 1 , ayman mohamad sabri 1 , ali abdullah alajeafi 1 , ashwaq hasan alhekri 1 , saleem bin mahfouz 1 , ali hasan alkhodari 1 , rawya saeed shealy 1 , marcus picard-maureau* 2 and hussain bana almalki 1 . 1 king abdulaziz hospital and oncology center, 2 cerus europe bv background/case studies: the growing number of transfusiontransmitted infectious (tti) risks, including emerging and endemic pathogens, is a constant challenge for blood centers in saudi arabia. while for a limited number of these pathogens tti risk can be reduced using blood screening assays, alternative solutions are anticipated. pathogen reduction (pr) technology was identified as a potential solution. validation of amotosalen/uva photochemical treatment in our blood center was performed by comparing the platelet component (pc) quality of the standard "control" single-donor prp-concentrate in 100% plasma over a 5 day storage period and the new "test" pathogen-reduced, pooled (pools of 5) prp pc in 100% plasma over a 7 day storage period. the efficacy of the bacterial inactivation was also assessed in our setting. study design/method: the quality parameters of 4 leucoreduced test pcs were assessed at day 7 of storage and compared to leucoreduced control pc at day 5 of storage. the test pcs were pathogen-reduced with the intercept blood system (cerus corporation, concord, u.s.a.) at day 0; the process was completed by day 1 post-collection. samples were taken daily for quality analysis from test and control pc until day 5 and day 7, respectively. for bacterial spiking, additional pc were spiked with each receiving 4 ml of 1 mcfarland ($ 1.2x10 9 cfu) s. aureus, s. epidermidis, e. coli, p. aeruginosa or s. viridans, respectively, to challenge pr efficacy. results/finding: the average platelet loss in the test pc post pr treatment was 4.7% 6 2.0, the total average platelet loss at day 7 was 11.2% 62.8. the average platelet loss in the control units at day 5 was 9.5% 61.4. the average ph of the test units at day 7 was 6.64 60.04 and in the same range as the control pc, ph 5 6.89 60.09. glucose concentration in test pc at day 7 (13.8 63.0 mmol/l) was lower than in the day 5 control units (18.32 61.06 mmol/l). lactate levels increased during the course of storage; lactate levels at days 5 and 7 were outside the range of the assay (>15 mmol/l). cultures inoculated with pathogen reduced, bacterially spiked units were negative after 7 days of incubation, in contrast to those inoculated with nonpathogen reduced samples from the control units, which were positive for bacterial growth. conclusion: the quality parameters of the pathogen reduced test pc were within specifications and comparable to the conventional control pc. the high efficacy of bacterial inactivation together with comparable quality parameter values suggests the use of amotosalen/uva pathogen reduction is safe and efficient to enhance pc transfusion safety. keaton charles stoner* 1 , jay srinivasan 2 , jessica poisson 3 and ian welsby 4 . 1 duke university, 2 duke university school of medicine, 3 duke university hospital, 4 duke university medical center background/case studies: the coagulation cascade relies on a complex interaction between proteins known as clotting factors. cryoprecipitate (cryo) is a plasma-derived blood product that contains several of the proteins central to the clotting cascade and is typically used as a fibrinogen replacement in bleeding patients. however, cryo contents tend to be variable, and little quantitative evidence exists regarding the exact therapeutic effect of cryo on coagulation. my study aimed to better characterize cryo for consistency across and within sources in terms of its functional effect on in vitroclot formation. study design/method: the duke proteomics core conducted a semiquantitative liquid chromatography-mass spectrometry/mass spectrometry transfusion developed an in vitromodel for a coagulopathic patient using serial dilutions of pooled normal plasma with saline and then added the equivalent of one, two, and three cryoprecipitate doses. a tissue factor-activated test on the rotemv r delta hemostasis analyzer (extem) was performed on each condition. for each source, dose-response curves for clotting time (ct), alpha angle, and maximum clot formation (mcf) were generated using linear regression models. inter-source unit variability was determined by anova and tukey's hsd post-hoc analysis (rstudio inc.). results/finding: lc-ms/ms identified 256 proteins in cryo; of the 10 most abundant, only fibrinogen was relevant to coagulation. notably, the american red cross (arc) single donor source had the steepest slope for mcf (4.44 mm/dose), indicating a greater per dose potency than the other sources. the arc single donor source had the highest mean mcf across all dosing levels, but also the highest standard deviations and response variability. the arc single donor source was significantly more potent than the australian source. conclusion: paired with our estimates regarding the variability of clot formation responses to cryo, the quantitative dose-response curves provided in this study for ct, mcf, and alpha angle can provide physicians with more information regarding cryo dosing. future studies that evaluate the therapeutic effect of cryoprecipitate versus fresh frozen plasma or fibrinogen concentrate would be of clinical importance and give us further insight into the relative utility of and dose requirements for cryo to correct dilutional coagulopathy. viral inactivation and enrichment of factor viii, factor xiii, fibrinogen and von willebrand factor (vwf) multimers from fresh frozen plasma (ffp)using, "vips plasma, virus inactivation treatment system". background/case studies: the solvent/detergent (sd) process used for plasma can safely inactivate all lipid-enveloped viruses. the method proved effective in the processing of coagulation factor concentrates by disrupting the membranes of lipid-enveloped viruses, cells and most protozoa, while leaving the labile coagulation factors intact. this study is done to assess viral inactivation and, factor viii, factor xiii, fibrinogen and von willebrand factor (vwf) multimers enrichment capacity of, "vips plasma, virus inactivation treatment system". study design/method: "vips plasma, virus inactivation treatment system" comprise of interconnected bag system where the s/d reagents are removed by filtration and the final products subjected to bacterial (0á2 lm) filtration. cryoprecipitate mini-pools (400 6 20 ml) were subjected to doublestage s/d viral inactivation, followed by one oil extraction and a filtration on a s/d and phthalate [di(2-ethylhexyl) phthalate (dehp)] adsorption device and a 0á2 lm filter. the initial and the final products were compared for visual appearance, blood cell count, factor viii, factor xiii, fibrinogen and von willebrand factor (vwf) multimers. initial and final products were also checked for hiv, hbv, hcv, dengue, malaria and bacterial contaminations. results/finding: our analysis showed that the treated cryoprecipitate were very clear, with negative blood count and the protein content of factor viii, factor xiii, fibrinogen and von willebrand factor (vwf) multimers were well conserved (table 1) . kit ensured bacterial sterility (table 3 ) and most importantly, final product was free of hbv, hcv and hiv (table 2) . conclusion: it's the first time, "vips plasma, virus inactivation treatment system", is used in south asia for product enrichment and viral inactivation. results showed effective product enrichment and viral inactivation in our conditions. but further investigation is needed to characterize functional activity of the enrich component. irrespective of that the process may offer one additional option to blood establishments for the production of virally inactivated plasma components especially in low income countries. background/case studies: buffy coats (bc) from donors who used pain medication like aspirin and ibuprofen up to 4 days prior to the donation are discarded, because a known side effect of these non-steroidal anti-inflammatory drugs (nsaids) is inhibition of platelet (plt) aggregation. these nsaids inhibit the enzyme cyclooxygenase-1, thereby blocking synthesis of thromboxane a 2 from arachidonic acid. however, the quality of platelet concentrates (pc), prepared from this bc is not known. the aim of the study was to investigate the in vitro quality of pc prepared from nsaid-bc and autologous plasma during storage. study design/method: single-donor pc (spc, n518) were prepared from a nsaid-bc and 60 ml of autologous plasma. information about the type of pain medication was extracted from the anamneses form. the spc were stored for 8 days at 22 6 28c and sampled at regular intervals. aggregation tests were performed with chronolog (adp or collagen) and multiplate (arachidonic acid) aggregometers. thromboelastography (teg, kaolin) was applied for assessment of the overall clotting capacity. spc in plasma from normal controls (n55) were investigated as a reference. values are expressed as mean6sd or as median & iqr. a non-paired t-test or a mann-whitney u test was applied for statistical analyses of normal or nonnormal distributed data respectively. results/finding: volume (69 6 4 vs. 66 6 16 ml) and plt content (67 6 14 vs. 71 6 15x10 9 ) were similar in both groups. on day 8, both groups showed comparable ph and changes in plt content (data not shown). phosphatidylserine exposure on day 8 was significant higher in a subset of donors who had used ibuprofen (n55). aggregation tests with arachidonic acid revealed in general a low or absent response for spc with aspirin (0,0-30, p<0.05), diclofenac (31,1-76) and naproxen (0,0-24, p<0.05), compared to normal controls (76, . no differences were detected in aggregation with adp or collagen. with teg, slightly longer r-times (initiation phase) were measured on day 1 in spc with aspirin, diclofenac and naproxen, compared to the normal controls (only significant for naproxen). these differences disappeared during storage. conclusion: storage properties of spc prepared from nsaid-bc were comparable with spc from normal controls. main differences were observed in aggregation and coagulation properties for donors who used aspirin, diclofenac or naproxen. plt from donors who used ibuprofen showed little or no deviations. this is most likely caused by the fast (<24 hour) disappearance of ibuprofen from the blood circulation and the reversible binding to plt. the use of bc from donors who used ibuprofen will be further investigated in a 'worst case' (pc in plasma) and 'best case' (pc in additive solution) scenario. the effects of ibuprofen on aggregation and coagulation properties will be further investigated in a dose-response study design adding different levels of ibuprofen to plt. background/case studies: previously it was shown that donors could be classified as having platelets (plt) with good, average or poor storage properties [bontekoe, transfusion, 2014] . a main difference between 'good' and 'poor' storage properties involved metabolic activity, resulting in a faster decline of ph during storage of 'poor' plt concentrates (pc). this might be caused by a different functionality of the plt mitochondria and there are indications that donors with a history of 'poor' pcs are more likely to have health issues, pointing towards metabolic syndrome and type 2 diabetes (t2d). because of the strong rise of people with t2d in the dutch population, the aim of this study was to characterize plt from whole blood donors diagnosed for t2d, but accepted as donor. study design/method: twelve whole blood donors with t2d, not using insulin, were selected and buffy coat (bc) and plasma were, after overnight hold, used for preparation of a single-donor pc (spc). an equivalent number of spc was prepared from age and sex matched control donors, derived from the same collection sessions. spc were stored for 8 days at 22 6 28c and sampled on day 1, 4 or 5 and 8. the diabetic marker hba1c was determined in red cells and cholesterol and triglyceride levels in plasma. from both groups 3 'good' (ph day8 >6.6) and 3 'poor' (ph day8 <6.3) storing spc were selected and analysed in more detail. results/finding: donors were of age 57 6 10 year and primarily men (75%). donors with t2d had a higher mean bmi (30.3 6 4.6 vs.25.4 6 3.4 kg/m 2 ) and higher hba1c than controls. the spc of both groups had the same volume (70 6 5 vs 726 2 ml) and plt content (71 6 9 vs 73 6 11x10 9 ) but on day 1 glucose concentration was higher in the diabetic group (20.5 6 1.7 vs 18.9 6 1.4 mm, p<0.05). on day 8, the average in vitro quality was comparable in both groups (data not shown). when combining 94a transfusion 2017 vol. 57 supplement s3 the selected 'good' and 'poor' storing plt from both groups, a large difference in lactate production was observed (0.14 6 0.04 vs 0.36 6 0.03 mmol/ day/10 11 plt). the 'poor' plt showed a faster decline of the mitochondrial membrane potential (as measured with jc-1) during storage than 'good' plt. remarkably, a difference in triglyceride levels was detected on day 1 ('poor':2.2 6 0.7 vs 'good':1.1 6 0.2, p<0.01). conclusion: bc from donors with t2d who did not use insulin and fulfilled all donor criteria, were comparable with bc from age and sex matched controls, and seem suitable for preparation of pc. when selecting the 'good' and 'poor' storing plt from the combined groups, the results of our previous study were confirmed, with significant differences in glycolysis rate and functionality of mitochondria. metabolic syndrome and t2d are still suspected as health issues involved in 'poor' storage of plt because donors were of high mean age and because of the observed differences in triglyceride levels between 'good' and 'poor' stored pcs. whole blood leukoreduction failures --following manufacturer's instructions may not be enough karen klinker*, nancy m. dunbar and zbigniew m. szczepiorkowski. background/case studies: our hospital based blood donor program uses a blood collection system which leukoreduces the unit at room temperature prior to centrifugation. the manufacturer recommends minimum wait time of 30 minutes prior to filtration. anecdotally, the vendor states waiting an hour improves the leukoreduction. we experienced leukoreduction failures in january and february of 2017 detected by our routine qc. we initiated an investigation as to the cause of these unexpected failures. study design/method: for each of the leukoreduction failures, the following factors were analyzed: collection time, length of filtration, length of wait time prior to filtration, platelet count, staff performing the process, the lot number of the collection system bag, and whether or not units collected from the same donor failed leukoreduction in the past. hemoglobin s determinations were not sought out as no repeat donor failures were noted and our donor population would suggest a minimal number of donors would be found to be hemoglobin s positive. results/finding: a relationship was established between the length of time the product rested or waited prior to filtration and leukoreduction failure. we found that shorter wait times increased the percentage of leukoreduction failures (see table 1). all units that failed had wait times less than one hour. a similar trend was noticed for the previous year. the investigation showed no relationship between length of collection time, or the length of filtration time and leukoreduction failure. staff performing the filtration was ruled out as possible cause as the failures were spread out among numerous personnel and observation of their technique displayed no sample collection issues. platelet counts on the donors involved were available and none were outside of the normal range. various lot numbers of the collection sets were involved, and no donors were repeat failures. conclusion: in our small study, we found that following manufacturer's recommendations for the resting or wait time prior to filtration was insufficient to avoid excessive leukoreduction failures. we extended our minimum wait time to 60 minutes based on our data. we have not experienced any leukoreduction failures after this change. absolute immature platelet count in diagnostic algorithm and management of pediatric thrombotic microangiopathy hamza n gokozan* 1,2 , katharine a downes 1,2 , hollie m reeves 1,2 and robert w maitta 1,2 . 1 case western reserve university school of medicine, 2 university hospitals cleveland medical center background/case studies: prior studies highlighted the utility of absolute immature platelet count (a-ipc) and a-ipc ratio once therapeutic plasma exchange (tpe) is initiated to differentiate thrombotic thrombocytopenic purpura (ttp) from other thrombotic microangiopathies. this can be helpful to determine those who may benefit from prompt initiation of tpe when tests such as adamts13 are not readily available. we report a young pediatric patient presenting with diarrhea in the setting of laboratory results suggestive of a microangiopathic thrombocytopenia suspicious for ttp in which a-ipc measurement was clinically useful. study design/methods: previously healthy 12 month old unvaccinated girl presented with history of diarrhea for 5 days which was bloody at onset, accompanied by fever and dehydration. laboratory results showed: white blood cell count: 32 x 10 9 /l, platelets: 62 x 10 9 /l, bun: 77mg/dl, creatinine: 2.4mg/dl, lactate dehydrogenase 1940 u/l. hospital course was complicated by tonicclonic seizure episodes that stopped with anti-convulsants and acute kidney injury requiring hemodialysis. peripheral blood smear revealed schistocytes. on third day of hospitalization, platelet count decreased to 44 x 10 9 /l, adamts13 sample was sent out and tpe was initiated for clinical suspicion of ttp versus hemolytic uremic syndrome, atypical versus shiga-toxin mediated. immature platelet fraction (%-ipf) and calculated a-ipc (%-ipf x platelet count) were obtained with daily pre-tpe cbc. a-ipc ratio was calculated from baseline. results/findings: platelet count began to increase prior to tpe initiation (74 x 10 9 /l and a-ipc of 4.7 x 10 9 /l). two consecutive tpe were completed which resulted in a platelet count decrease to 54 x 10 9 /l and a-ipc of 5.1 x 10 9 /l. a-ipc ratio was 1.1 below the ratio of 3 which has been reported for ttp patients. similarly a-ipc count was not below 5 x 10 9 /l threshold reported in setting of ttp with severe adamts13 deficiency. at this time stool culture obtained prior to start of tpe came back positive for e. coli o157:h7 toxin. testing of c3, c4, factor h, factor h autoantibody, factor i and factor b were normal. adamts13 activity was 93%. patient was treated for the infection and platelet count improved within 10 days to 315 x 10 9 /l, with resolution of her renal failure: bun: 42 mg/dl, creatinine: 0.65 mg/dl. no additional seizures were observed during follow-up. conclusion: measurement of a-ipc can be used to aid clinical decisions in pediatric patients suspected of ttp especially when adamts13 testing and those for other etiologies are still pending. tpe did not seem to have a significant effect in a-ipc but decreased platelet counts in this patient. a-ipc is rapid to obtain and can provide helpful information in the setting of potentially overlapping etiologies in the setting of other testing with longer turnaround time. background/case studies: thrombotic thrombocytopenic purpura (ttp) is a thrombotic microangiopathy characterized by low adamts13 activity. many patients with severe autoantibody-mediated adamts13 deficiency at initial disease presentation may suffer from one or more recurrent episodes over the following months or years. it is unclear if disease course and characteristics of recurrent/relapsed ttp may be different from that seen at initial presentation. since absolute immature platelet counts (a-ipc) have been shown to be useful in the diagnosis and to follow response to therapy of ttp patients, we proceeded to evaluate if a-ipc pattern was different in relapsed verse initial presentation. study design/methods: our study cohort consisted of three patients (two female and one male) with acquired ttp (adamts13 activity <5%) who underwent daily therapeutic plasma exchange (tpe). clinical course and laboratory values were reviewed. platelet count (plt), immature platelet fraction (%-ipf) and a-ipc (%-ipf x platelet count) were analyzed during treatment course. a-ipc values at presentation and peak, a-ipc peak time (days), and plt count recovery time (days) were compared between initial onset and relapse episode for each patient. a-ipc percent change in relapse episodes compared to initial presentation was calculated. results/findings: all patients had an increased %-ipf, and decreased a-ipc and plt count at presentation in both initial and recurrent episodes. once tpe treatment was initiated, a-ipc rapidly increased and reached a peak value 2-3 days prior to plt count recovery, consistent with that previously described in ttp patients. however, compared to first onset, recurrent episodes featured lower a-ipc at presentation (results shown as percent decrease, column 1), increased peak a-ipc value (results shown as percent increase, column 2), delayed a-ipc peak, and delayed plt recovery (table 1) . moreover, recurrent episodes required more procedures compared to initial presentation (table 1) . conclusion: recurrent/relapsed ttp demonstrate lower a-ipc at presentation and a delayed and increased a-ipc peak value in response to tpe compared to initial presentation. a longer treatment course was observed in recurrent patients. future studies of more relapsed ttp patients are needed. donors undergoing frequent plateletpheresis and its effect on the hematological parameters sweta nayak*, poonam coshic and r.m pandey. all india institute of medical sciences background/case studies: frequent plateletpheresis donors are assets for the blood banks. the well-being of these donors has been a matter of concern. in our study we intend to analyze the effect of plateletpheresis on the hematological parameters of these donors assessed prior to each subsequent procedure. we also try to compare the effect 3 cell separators used for plateletpheresis on the post donation hematological parameters. study design/method: the study was conducted during february 2016 to march 2017 on all the repeat plateletpheresis donors coming to the department of transfusion medicine for the 2 nd time within a month of the first plateletpheresis. the values of the hematological parameters including red cell and platelet indices tested prior to each plateletpheresis were entered into the excel sheet and gap between each donations were calculated. the plateletpheresis were done either on hemonetics mcs1 separator (hemonetics corporation, braintree, massachusetts, usa), fresinius separator (com.tec), dn (fresinius hemocare gmbh, bad homburg v.d.h, germany) and gambro trima accel, software version 5.0 after taking consent from the donors. the target collection of each procedure was a dose of 3 x 10 11 platelets in 200-250 ml of plasma. to compare the effect of the cell separators on the hematological parameters due to the plateletpheresis, parameters at 2 consecutive donations within 7 days were considered. data was analyzed by stata 14. within change in the continuous variables were assessed by paired t-test and between two groups comparison was done by independent t-test or wilcoxon rank sum test. the comparison among the cell separators was done by kruskal-wallis test or one way anova. results/finding: of the 98 donors, 35 repeated the plateletpheresis within a week (group i) and 63 underwent 2 nd plateletpheresis within 8-30 days (group ii). no significant alteration was found in the red cell or the platelet indices within either group but a significant difference in the variation of platelet counts of the 2 groups (p50.025). though above the eligibility cutoff of 1.5 lakhs/ml, platelet counts were lower than baseline in group i donors whereas it was higher at 2 nd plateletpheresis in group ii donors. there were 49 donors who presented to us for the 3 rd time for plateletpheresis with a mean gap between 1 st and 3 rd plateletpheresis being 46 days. no significant difference in the parameters assessed prior to any of the plateletpheresis was found except the platelet distribution width (p50.000). plateletpheresis through all the 3 cell separators had similar effects on the hematological parameters. conclusion: there was no significant change in the hematological parameters in the plateletpheresis donors who underwent frequent plateletpheresis. post donation follow-up hematological parameters were not affected by the cell separators used for plateletpheresis. efficacy of therapeutic plasma exchange on angiotensin ii type 1 receptor antibodies in two kidney transplant recipients chisa yamada*, silas p. norman, milagros samaniego and laura cooling. background/case studies: some kidney transplant recipients develop antibody mediated rejection (amr) without detected hla donor specific antibodies (dsas) in sera. in recent years, angiotensin ii type-1 receptor antibody (at1rab) has been reported to cause amr, especially refractory amr, possibly by contraction of renal arteries. at our institution, therapeutic plasma exchange (tpe) followed by ivig every other day has been applied to reduce at1rabs in kidney transplant recipients, and we here report efficacy of tpe treatments in two cases. study design/methods: two kidney transplant recipients who received tpe treatment followed by ivig to decreased at1r ab are reviewed. results/findings: case 1: the patient is a currently 43-year-old female with focal segmental glomerulosclerosis who received her first kidney transplant from a living related donor at age 22, and a second deceased donor transplant due to a rejection of the transplanted kidney at age 38. three years post-transplant, her creatinine (cr) started to rise from 0.7 to 1.35 mg/dl and a biopsy showed banff criteria grade 2 amr, grade 2a t-cell mediated rejection (tcmr) and grade 3 interstitial fibrosis and tubular atrophy. hla dsa had been negative in serum, but high level at1rab was identified at >40 u/ ml (high: >17 u/ml, intermediate: 12-17 u/ml, negative: <12 u/ml). she received 6 tpe treatments every other day and started losartan. after a course of tpe, at1rab decreased to 32 u/ml and histology showed improvement of amr and tcmr, however, cr kept increasing slowly to 1.9 ml/dl. in one month, her at1rab increased again to >40 u/ml, therefore, she received 3 more tpe treatments with a decrease in her at1rab to 16 u/ml. although at1rab level increased slightly to 20 u/ml after 3 months, her cr has been stable at 1.3-1.6 ml/dl. case 2: the patient is a 25-year-old mean 1/-se -54.71/-12.9% * 183.1%1/-12.8%* * p<0.05 96a female with malignant hypertension who received a deceased donor kidney transplant at age 24. her cr started to rise 2 weeks post-transplant from 1.4 to 2.68 mg/dl without detectable hla dsa. although biopsy showed no amr or tcmr, there was focally severe arteriopathy. she was found to have high at1rab level at 18 u/ml. she received 6 tpe procedures every other day and at1rab decreased to 8 u/ml with a decrease of cr to 1.98 mg/dl and improved arteriopathy in histology. because her at1rab level slightly increased to 12 u/ml over the next 2 weeks, she started weekly tpe treatment. after 5 weekly tpe, tpe treatment was stopped because her at1rab level remained relatively unchanged. her cr has been stable at around 1.5 ml/dl to date. conclusion: we present 2 kidney transplant recipients who received tpe treatments for high at1rab levels. a course of tpe procedures followed by ivig every other day was effective to decrease at1rab levels; however, weekly tpe had no effect on reducing at1rabs. tpe treatment may be also beneficial to improve histological amr and clinical kidney function. experience in management of thyroid storm by plasmapheresis tatiana belousova*, vanya jaitly, brian castillo, hlaing tint, kimberly klein and yu bai. university of texas health sciences center at houston background/case studies: thyroid storm (ts) is an extreme manifestation of thyrotoxicosis that is a serious complication occurring primarily in patients with graves' disease. clinically they may present with a wide range of hypermetabolic symptoms which may be fatal if not managed appropriately. we report two cases where ts with severe cardiac complications was managed by plasmapheresis (plex) with excellent effect. study design/method: a 36 year old man (patient a) with a medical history of hyperthyroidism present with ts complicated with cardiogenic shock [ejection fraction (ef) < 10%], renal and hepatic dysfunction as well as coagulopathy. patient was persistent tachycardic while being intubated, sedated and requiring tandem heart support. a 33 year old man (patient b) with a medical history of hypothyroidism (on synthroid for 2 years), end stage renal disease and non-ischemic cardiomyopathy (ef of 20-25%) presented for evaluation of dual kidney-heart transplant. he subsequently developed ts with multiorgan failure. standard steroid medication treatment showed little response. results/finding: both patients underwent urgent plex along with standard medication administration as soon as the clinical suspicion of thyroid storm was raised. a 1-1.5 plasma volume, iso-volumic procedure using fresh frozen plasma as replacement was performed in the intensive care unit where the procedure associated hemodynamic impact could be easily managed. both patients showed significant clinical improvement within 12 hours of the procedure completion. their total t4, t3 and free t4 levels trended to normal or near normal range within 24 hours (table) . in addition, the plex effect on hormone and the associated antibody removal seemed remained and no "rebound" phenomenon was observed in both cases, making repeated plex unnecessary. both patients had total thyroidectomy 3-4 weeks after the event with great clinical outcome. conclusion: our cases demonstrate that plex is a safe, effective treatment option in managing ts patient with severe cardiac dysfunction. the procedure can not only lead rapid decrease in thyroid hormone and its associated antibody levels, but also lessen the severity of tissue injury by moderating the inflammatory process and correcting complications. extracorporeal photopheresis in s ezary syndrome treatment: hospital-based blood bank experience sandra ortega s anchez* 1 , laura martínez molina 2 , cristina muniesa montserrat 2 , octavio servitje bedate 2 , silvia cosano navarro 1 and maria isabel gonz alez medina 1 . 1 banc de sang i teixits, 2 dermatology service. background/case studies: extracorporeal photopheresis (ecp) is an immunomodulatory therapy widely used since 30 years in cutaneous t cell lymphoma, several autoimmune diseases and organ transplant rejection, and in the last 20 years, also used in graft versus host disease treatment. the use of ecp in cutaneous t cell lymphoma (ctcl), mycosis fungoides (mf) and s ezary syndrome (ss) in their erytrodermic form are recently categorized by the american society for a pheresis (asfa) 2016, as first line treatment alone or in combination with other therapies, with a strong recommendation: grade ib, category 1. since mf and ss are incurable diseases current therapies are focus in controlling skin symptoms and minimizing immunosuppression. the objective of this observational study is to assess outcomes of 10 patients diagnosed with ss and compare them in their first evaluation once the 20 th procedure is been performed. study design/method: ecp is a leukapheresis-based therapy, ex vivo exposition to a photosensitizer drug ( 8-methoxypsoralen, 8-mop) and uva light, and subsequent reinfusion of the treated cells which are now induced to apoptosis. volume treated varies from 1.5 to 2 total body volume (tbv) and the schedule for ss disease is one cycle (two daily ecp procedures) twice per month. the venous access was peripheral in all cases except in 2 where central catheter was needed. the procedures were performed with optia or amicus devices for the aphaeresis and external uva irradiation for off-line system (in 7/10 patients) and with online system (therakos) just in 3. main parameters for evaluation were cutaneous response rate, number of s ezary cells, previous treatments, duration of the response and possible complications during ecp treatment. results/finding: global response rate is 77'7% (partial remission 66.6% and complete remission 11.1% with maintained response). no severe side effects related with the procedure were found. the patient outcomes analyzed are similar to results in published literature. conclusion: cases treated in our hospital confirm the efficacy of ecp in ss treatment, with a good safety profile. another great advantage of ecp is the relative lack of immune suppression. many questions remain still unanswered about ecp: which schedule is the most suitable one, how we must continue or stop when partial or complete remission is achieved; and the number of leukocytes to be treated, as techniques as mini-photopheresis are also getting good results. all these questions and more make prospective studies necessary to be performed. : 3835 u/l) requiring transfusions, mild thrombocytopenia (144 x 10 9 /l), acute kidney injury (bun 175 mg/dl, creatinine 2.51 mg/dl). by the third hospitalization day hgb improved to 10 g/dl, however with worsening thrombocytopenia (16 x 10 9 /l) that led to clinical concern for ttp. peripheral smear showed many red cell fragments. patient was transfused with platelets day prior to first tpe. immature platelet fraction (%-ipf) and a-ipc (%-ipf x platelet count) were obtained with daily pre-tpe cbc. a-ipc ratio was calculated from baseline. results/finding: four tpe in five days were performed (hospital days 6-10). platelet count and a-ipc improved to 52 x 10 9 /l and 6.6 x 10 9 /l respectively just prior to first tpe. response to four tpe led to a decrease in both platelet count (30 x 10 9 /l) and a-ipc 1.98 x 10 9 /l. these dynamics did not resemble those which had been described for ttp patients with adamts13 deficiency. adamts13 obtained prior to tpe initiation was resulted at this time and was 67%. no causative organism or toxin was identified after urine, blood, and stool examination and culture. based on these results, tpe was discontinued which led to an immediate increase in a-ipc (2.64 x 10 9 /l) that preceded platelet count increase to 80 x 10 9 /l three days later when patient was discharged. other laboratory values at this time were ldh of 635 u/l, hgb: 11.2 g/dl in the setting of recovery of renal function. conclusion: timely diagnosis of ttp is essential to start of tpe. a-ipc dynamics differ in ttp compared to other thrombotic microangiopathies. in our patient a-ipc failed to improve despite tpe and improved once procedures were discontinued and were followed by increases in platelet counts three days later. when ttp is not the causative etiology, a-ipc can help adjust therapy and lead to clinical improvement. further research is needed to characterize immature platelet dynamics in non-ttp microangiopathies. infection and its role in the clinical course of idiopathic thrombotic thrombocytopenic purpura associated with severe adamts13 deficiency eiman hussein* 1 and jun teruya 2 . 1 department of clinical pathology, cairo university, 2 texas children's hospital background/case studies: ttp is a life threatening disease, defined by microangiopathic hemolytic anemia, thrombocytopenia and severely deficient adamts13. since the introduction of therapeutic plasma exchange (tpe) as a treatment modality for ttp, its prognosis has improved dramatically. nonetheless, some patients may develop relapse or refractoriness, with potentially fatal outcomes. despite the notable progress that has been made with studies that emphasized the pivotal role of adamts13, the epidemiology of ttp remains uncertain. previous studies have suggested that many factors appear to influence its pathogenesis. some studies point toward infection as a possible trigger which may contribute to the development and can ultimately influence its clinical course. one of the theories to explain this association is the possible cross reactivity between antibodies targeting infectious pathogens and those directed against adamts13. the aim of this study was to prospectively examine the potential association between infection and the clinical outcome in a cohort of patients with idiopathic ttp. study design/method: patients with idiopathic ttp who underwent tpe from january 2008 through march 2017 were studied. sessions were performed daily until platelets and reticulocytes had been normal, then sessions were gradually tapered. we only included patients with adamts13 activity of less than 10%. data on infections that occurred at or within a week prior to the development of ttp were analyzed. results/finding: thirty-two patients were categorized as idiopathic ttp with severe adamts13 deficiency. eight patients (25%) were associated with suspected bacterial infection. four of the 8 patients (50%) showed acute relapse coincident with bacterial infections. central line associated staphylococcus aureus infections occurred in three patients and acinetobacter urinary tract infection was reported in one patient. one patient had symptoms of respiratory infection before the development of ttp, on his initial as well as his relapsing episode. refractoriness to treatment was demonstrated in 3 patients. it was associated with dental abscess in one patient. the other two were associated with mycoplasma pneumonia. tpe sessions were continued in all refractory patients until their death. conclusion: in patients with idiopathic ttp refractory to conventional treatment, a serious consideration should be given to non-idiopathic causes, particularly the presence of a remote source of infection, which can be an additional triggering factor for their initial and / or recurrent episodes. sandra satoe kayano*, marcos paulo colella, rafaela guerra maciel, ingrid priscila ribeiro paes ferraz and rafael colella. a c camargo cancer center background/case studies: therapeutic leukapheresis (tl) has become an ordinary procedure in low body weight children with cancer, and its use over the time has been replacing exchange transfusion. leukodepletion preceding chemotherapy helps preventing leukostasis and hiperviscosity, and aims to reduce metabolic and renal complications associated with cell lysis. the objective of this study is to evaluate the efficacy and safety of leukapheresis procedure in pediatric patients with less than 10 kilograms using a single apheresis procedure. study design/method: in october 2015 and june 2016, two children with possible leukemia were submitted to tl procedure. they were 6 and 9 months old, and weighted 7,0 and 9,1 kilograms. central venous catheters were placed, and apheresis were performed using a continuous flow apheresis system. the device was primed with 285 ml of abo, rh and kell compatible, leukocyte-reduced, irradiated, 64% hematocrit packed rbcs, and the anticoagulant used was acd-a plus heparin (750 ml of acd-a and 7,500 units of heparin), at a blood to anticoagulant ratio of 25:1. a complete blood count was determined before and after apheresis. the room was heated to avoid hypothermia, and ionized calcium was measured every 30 minutes to prevent hypocalcemia. during the collection, changes in blood pressure, oxygen saturation and heart rate were observed. net fluid balance was calculated as the sum of the volume of anticoagulant, cation and nondiverted apheresis prime solutions minus the product volume. when the procedure was completed, the blood that filled the apheresis tubing was discarded. the patients were in the intensive care unit (icu) under the supervision of a pediatric physician and icu nurse who were aware of potential adverse events, and the procedure were performed by two hematology physicians and the nurse practitioner. results/finding: the white blood cell (wbc) in blood was counted immediately before apheresis in both subjects, and were 120.000 and 150.000/ mm 3 . the formula "collection pump flow 5 0,0003 x inlet flow x preapheresis wbc count" was used with the goal of removing up to 3 x 10 9 leukocytes/ml. a single leukapheresis procedure was performed with 2 total blood volume processed per patient. immediately after the 2-hour procedures, wbc count were 74.000 and 92.000 wbc/mm3, and 12-hour post tl, wbc count were respectively 45.000 and 70.000/mm3. net fluid balance was zero in both procedures, and the patients required no transfusion. conclusion: tl was safe and efficient. experience with leukodepletion in infants is limited, and a procedure in children weighing 10 kg or less needs forethought and a multidisciplinary effort, hence operators need to customize procedures for safe collection. however, despite the potential complications that may occur (placement of adequate vascular access, management of low extracorporeal blood volume, anticoagulant-related toxicity with metabolic and hematologic issues), remains an excellent source for leukoreduction in hematologic malignant diseases. background/case studies: nationwide apheresis registry can give us information on the current status and trend regarding apheresis procedures. data can be compared with other regions to find and understand differences in perspectives, indications, technology, and clinical practice. the korean society for apheresis (ksfa) has launched an online web based registry system for apheresis procedures since 2006. we report the data from the year 2016. study design/method: the registry is consisted of two sub-registries. one addresses the overall aspects of apheresis procedures performed at each institute, and the other is focused on therapeutic plasmapheresis procedures. data is registered by voluntarily participating hospitals in korea. results/finding: a total of 13,302 apheresis procedures were performed at 37 hospitals. therapeutic plasmapheresis was the most frequent procedure (50.4%) followed by autologous peripheral blood stem cell (pbsc) collection (23.9%), allogeneic pbsc collection (11.0%), donor leukapheresis (4.0%), and therapeutic leukapheresis (3.9%). cobe spectra (37.4%) and amicus (16.8%) were the most widely distributed instruments. centrifugation was the dominant technique (92.2%) for therapeutic plasmapheresis. detailed information was given for 4,199 therapeutic plasmapheresis procedures performed on 786 patients (some items were not completely filled out). spectra optia (42.7%) and cobe spectra (26.6%) were the most frequently used instruments for therapeutic plasmapheresis. fresh frozen plasma (ffp) was used most frequently (47.2%) as the replacement fluid followed by 5% albumin (26.3%), 4% albumin (13.3%), and 5% albumin 1 ffp (11.1%). most of the procedures were performed for 1 plasma volume (72.4%). acd (88.4%) and heparin (11.5%) were used for anticoagulation. central venous catheter (91.9%) was the dominant type of vascular access. major clinical indications were desensitization for abo incompatible renal transplantation (24.1%), antibody mediated rejection in renal transplantation (19.9%), thrombotic microangiopathy (11.5%), desensitization for abo compatible renal transplantation (4.7%), neuromyelitis optica spectrum disorders (4.6%), and hyperviscosity in monoclonal gammopathies (4.6%). adverse reactions were observed in 8.5% of the procedures. allergic reaction (55.2%), hypocalcemic symptom (20.4%), and hypotension (6.9%) were frequently reported. therapeutic effect was achieved in 86.5% of the patients. our apheresis registry has been well run for 10 years. recent data reflects the increase of abo incompatible transplantation in korea. revision and update of the registry planned this year will help us achieve better understanding on the apheresis status of our region. plasma exchange may not always be necessary in patients with severe hypertriglyceridemia and acute pancreatitis. jan c hofmann* and dobri d kiprov. california pacific medical center background/case studies: hypertriglyceridemic pancreatitis (hp) is characterized by severe hypertriglyceridemia (shtg: triglyceride >1000-2000 mg/dl), acute pancreatitis (ap), and absence of other causes. hp is a potentially fatal complication of acute pancreatitis with an incidence of $18 deaths/100,000 cases/year. complications of shtg include: abdominal pain (nausea/vomiting), acute pancreatitis, hepatosplenomegaly, eruptive xanthomas, lipemia retinalis, memory loss, dementia, and peripheral neuropathy. we report on the effective use of plasma exchange (pe) to treat patients (pts) with hp refractory to conventional medical therapy (lipid-free diet plus pharmaceutical interventions). study design/method: we reviewed the medical records of 41 pts who were diagnosed with hp from january, 2009 through january, 2017, and referred for immunotherapy evaluation. 27/41 (66%) pts received conventional therapy (ct) and pe (pe group), and 14/41 (34%) pts received ct alone (ct group). mean age was 36 years (range 16-79), and 56% were female. baseline mean triglyceride level (normal <150 mg/dl) for pe group was 6,728 mg/dl (4,652-12,486) versus 3,142 mg/dl (1,697-5,120) for ct group. baseline mean lipase level (normal <393 u/l) for pe group was 1,798 u/l (797-2,745) versus 923 u/l (472-1,796) for ct group. results/finding: all pts were treated with dietary restriction (lipid-free diet, or nothing by mouth) and aggressive lipid lowering protocols involving 2-3 medications. 24/27 (89%) of pe group and 11/14 (79%) of ct group received insulin therapy to manage symptoms (sxs) of hyperglycemia and/or diabetic ketoacidosis. 20/27 (75%) of pe group and 6/14 (43%) of ct group received heparin therapy to stimulate lipoprotein lipase release. the pe group underwent an average of 2.85 pe treatments (txs) (median of 2, range 1-4 daily txs) using 5% albumin; 7/27 (26%) required ffp to treat dilutional coagulopathy. in most cases, we did not perform pe txs when baseline triglyceride levels were <3000-4000 mg/dl and lipase <950-1375 u/l (2.5-3.5 x upper limit of normal). mean triglyceride levels after 2 pe txs were 1,976 mg/dl (627-3,968) for pe group (mean decrease 72%); mean triglyceride levels after 48 additional hours of ongoing ct were 1,576 mg/dl (487-2,873) for ct group (mean decrease 50%). while the pe group achieved a greater mean decrease in triglyceride levels after 2 pe txs (compared to the ct group after 48 hours of ct), both groups experienced marked improvement in clinical sxs of pancreatitis and hyperglycemia (p>0.05). limitations of the retrospective cohort study include lack of long-term follow-up. conclusion: this small study adds to the literature which demonstrates that plasma exchange is very effective in rapidly lowering triglyceride levels in pts with acute pancreatitis and hypertriglyceridemia. it suggests that there may be a threshold (or range) of triglyceride and lipase levels below which conventional therapy may be nearly as effective in achieving clinical resolution of symptoms. randomized controlled trials would further elucidate the appropriate use of adjunctive plasma exchange in the setting of hypertriglyceridemic pancreatitis. role of plasma replacement in therapeutic plasma exchange for hypertriglyceridemia: a single patient study geoffrey wool* and angela treml. university of chicago background/case studies: our apheresis service performs chronic therapeutic plasma exchanges (tpe) for a 47-year-old man with a chronic history of hypertriglyceridemia >1000 mg/dl, diabetes mellitus type ii, and chronic abdominal pain. his abdominal pain is severe and persistent, but there is not overt evidence of chronic pancreatitis on imaging or fecal elastase testing. targeted sequencing has not revealed a pathogenic mutation to explain the patient's hypertriglyceridemia. hypertriglyceridemic pancreatitis is a category iii indication for tpe by asfa 2016 guidelines, in a patient unresponsive to optimal medical management. asfa 2016 guidelines for this disorder state that "some have used plasma as it contains lipoprotein lipase and could enhance triglyceride (tg) removal. no direct comparisons of replacement fluids have been reported". there are three apheresis physicians on our service and use of partial plasma replacement has been variable. we undertook a retrospective study of the efficacy of partial plasma replacement in this patient. study design/method: we have performed 39 tpe on this patient. we performed a chart review to capture replacement fluid use and pre-and post-tg levels, if drawn. tpe was performed using spectra optia (terumo, lakewood, co) exchanging approximately one plasma volume, using entirely 5% albumin for exchange fluid (100% albumin procedures) or partial plasma replacement (2-3 units of thawed plasma). twenty-six tpe had pre-and post-procedure tg values available. we determined the percent tg reduction achieved by the tpe. we also determined the daily rate of tg increase until the next tpe appointment (to assess any long-term effects of plasma preventing tg rebound). significance was assessed by student's t-test (one-tailed, heteroscedastic). results/finding: twelve tpe were performed with partial plasma replacement, while 27 were performed with 100% albumin replacement. table shows that partial plasma replacement was associated with significantly greater % tg reduction. the rate of subsequent daily tg increase was also lower with partial plasma replacement, but this did not meet significance. one mild allergic reaction has occurred during partial plasma replacement which responded quickly to additional iv diphenhydramine. conclusion: we have performed an ad hoc cross-over study on the efficacy of partial plasma replacement in tpe for hypertriglyceridemia. in this patient without lipoprotein lipase mutations, plasma was significantly associated with improved % tg reduction, but not with prevention of post-tpe tg rebound. safety and efficacy of local albumin replacement for therapeutic plasma exchange phandee watanaboonyongcharoen* 1,2 , metha apiwattanakul 3 , sompis santipong 2 , jutaluk jaipian 2 , jettawan siriaksorn 2 and ponlapat rojnuckarin 1 . 1 chulalongkorn university, 2 king chulalongkorn memorial hospital, 3 prasat neurological institute background/case studies: therapeutic plasma exchange (tpe) with albumin replacement has been used to treat a variety of diseases. however, there had been rising cost and supply shortage of imported albumin in our country. to solve the problem, our national blood centre had established a plasma fractionation plant to manufacture plasma derivatives including albumin. the objective of the study was to evaluate the safety and efficacy of local albumin as a replacement for tpe. study design/method: all tpes using local albumin as a replacement from two tertiary care hospitals performed from june 2016 through february 2017 were included. complete blood count and serum calcium were tested before tpe. serum albumin was tested before and after tpe. local albumin is available as a 20% solution. before using, it was diluted to a 4% albumin concentration with normal saline. all the patients were hospitalized and received oral calcium before tpe to prevent hypocalcemia. the adverse effects were recorded. results/finding: the total of 156 tpes in 38 patients were included as shown in the table. neurologic disorders were the most common indication for tpe, followed by autoimmune diseases. the median total plasma volume was 3,000 (range 1,750-4,200) ml. although the corrected calcium level was low (<8 mg/dl) in 3.2% (5/156) before the procedure, no clinical manifestation of hypocalcemia was detected. adverse effects were observed during the tpe procedure in 2 patients. the first patient had 2 events of mild symptomatic hypotension. he previously took angiotensin converting enzyme inhibitor. the second patient complained nausea after finishing tpe. all reactions were mild. the incidence of adverse effects was 1.9% (3/ 156). in 2014, the incidence of tpe adverse effects was 1.6% (2/125) when commercial albumin was used. the difference was not statistically different (p 5 1.000). median serum albumin levels pre-tpe and post-tpe were 3.6 (1.9-4.4) and 3.9 (2.4-5.0) g/dl. the increase in serum albumin after tpe was statistically significant (p<0.001). eighty-two percent of pre-tpe serum albumin levels were lower than 4.0 g/dl explaining the rises of albumin after the procedures. we demonstrated that local albumin was safe and effective in maintaining albumin levels in patients undergoing tpes. safety, efficancy and cost-effectiveness of mononuclear cell collections for autologous immunotherapies: experience from a private outpatient collection facility within the eu markus dettke*. akh vienna university hospital, cyto-care.eu background/case studies: within the eu the collection of mononuclear cells (mnc) as starting source for the manufacturing of autologous cell therapies are mainly performed in hospitals or hospital-associated apheresis centers. we report about the challenges to perform the leukapheresis procedure (la) at a private held medical practice, with specific emphases on safety, cell collection efficiency, and cost-effectiveness. study design/method: we reviewed the records of altogether 60 outpatients who underwent a total of 100 la procedure at cyto-care, a private held medical practice/ certified cell collection facility located in vienna, austria. all patients participated in various industry-sponsored clinical p i-iii trials; the study sponsors were responsible for the manufacturing of the active cell product. disease entities were mainly prostatic cancer (75%) and ovarian cancer (20%). based on differences in the study protocols la was performed either one-time (41%), two-times (27%) or three-times (32%), with an interval of at least 2 weeks between repeated collections. results/finding: all patients successfully completed the apheresis course. because of poor venous access, 3 out of 60 patients (5%) required a shortterm femoral catheter insertion. there were no serious side effects in patients who required a femoral catheter, or in patients with repeated la procedures. side effects of the la procedure mainly consisted on mild hypocalcaemia-related symptoms in 16% of patients. a follow-up survey one week after completion of the la revealed no infectious complications, and no patient required hospitalization. median cell yield collected per single apheresis was 1.4x10 10 wbc consisting of 1.1x10 10 mnc. mnc cell yields remained stable even in repeated la collections. all cell products were successful transformed into an active cellular product. analysis of the cost structure showed that the total cost of care was 32% lower in the setting of a private collection center compared to hospital-based apheresis centers. conclusion: leukapheresis performed in a private medical practice/ certified cell collection facility is safe and effective, with low rates of complications and high levels of patient satisfaction. this service model is costeffective and can help to reduce the cost of manufactured goods in the production of innovative cellular products. although typically associated with monoclonal gammopathies (e.g. waldenstrom's macroglobulinemia and multiple myeloma), hvs has rarely been reported in patients with disorders of immune system such as rheumatoid disease, sjogren's syndrome, hiv and igg4-related diseases. therapeutic plasma exchange (tpe) is indicated in hvs due to monoclonal gammopathy (asfa category 1 indication). however, there are limited data for the utility of tpe in hvs due to polyclonal gammopathy. study design/methods: a 70 year old female patient with a medical history significant for seropositive erosive rheumatoid arthritis, hypertension, diabetes mellitus, cutaneous lupus and diffuse parenchymal lung disease, presented to our institution with complaints of progressive fatigue, muscle weakness, poor appetite, headache and epistaxis for a few months. fundoscopic examination showed dilated and tortuous vasculature as well as bilateral retinal hemorrhages (mixed flame-shaped and dot-blot patterns). pertinent laboratory findings included a positive anti-nuclear antibody screen with anti-histone antibodies and anti-ro antibodies. serum rheumatoid factor was markedly elevated to 57,000 iu/mls (ref. range < 35) and anti-cyclic citrulline peptide antibody was elevated to 34,339 units (ref. range < 20) . serum protein electrophoresis and immunofixation demonstrated a polyclonal hypergammaglobulinemia; protein precipitates were noted at the point of application, suggestive of circulating immune complexes. serum igg, igm and iga were 4610, 2890 and 1320 mg/dl respectively. a cryoglobulin screen was negative. serum free kappa to lambda ratio was 1.74. peripheral blood flow cytometry did not identify any monoclonal bcell population. plasma viscosity was noted to be 8.5 centipoise (cp) at admission (ref. range 1.6 -1.9). pet-ct imaging was negative. the patient was treated with high dose steroids; a single tpe procedure was performed using the following parameters: volume treated -1 total plasma volume; replacement fluid -5% albumin and normal saline in a 50:50 ratio; replacement fluid volume: 110% of the total volume processed. the procedure was tolerated without complication. results/findings: immediately post-tpe her plasma viscosity level dropped to 2.4 cp. serum igg, igm and iga levels decreased to 2040, 1510 and 672 mg/dl respectively. her rf had decreased to 19,900 iu/ml. the patient reported subjective improvement in strength. she subsequently received two infusions of rituximab separated by two weeks. her plasma viscosity has remained less than 3 cp since tpe. conclusion: polycolonal gammomathy (e.g. secondary to ra) is a rare cause of hvs. tpe can provide transient relief of symptoms in unusual cases of hvs and may facilitate therapy to prevent recurrent hvs episodes. therapeutic plasma exchange in neuromyelitis optica spectrum disorders -experience from tertiary care centre in north india ratti ram sharma*, rekha hans, satya prakash, naveen sankhyan and neelam marwaha. postgraduate institute of medical education and research background/case studies: neuromyelitis optica spectrum disorder (nmosd) is an idiopathic inflammatory demyelinating disorder of central nervous system preferentially involving optic nerve and upper segments of the spinal cord leading to optic neuritis and myelitis. tpe is indicated in acute phase or as a maintenance therapy to treat or prevent relapses in chronic phase. study design/method: to assess the efficacy of plasma exchange in patients of nmosd not responding to high dose intravenous steroids. we did a retrospective review of tpe records for patients with nmosd over a period of three years (jan 2013 -dec 2016). tpe was done using, cobe spectra (terumo bct, lakewood co. usa), replacing one to one and half patient plasma volume with 5% human serum albumin or fresh frozen plasma on alternate days. the improvement in clinical signs and symptoms was recorded after each tpe procedure and at the end of the therapy. adverse reactions if any were also recorded results/finding: eleven patients of nmosd between 4 to 35 years age (m: f; 1:2) underwent 62 tpe procedures with an average of 5.6 per patient. all the patients were on high dose immunosuppressant therapy without much clinical improvement. three (27%) patients had only visual symptoms, 5 (46%) had both visual as well as muscular symptoms whereas 3 (27%) patients had muscular symptoms only. three (27%) out of the seven tested, were positive for aqp4-igg. all the patients showed significant improvement in their visual symptoms post exchange, from no vision/light perception to finger counting in two patients, recovery of colour vision and diplopia in six patients. post exchange recovery in the muscle power was observed in 8 patients with grade-1, in 1 patient, and by grade-2, in seven. adverse events were observed in 8% (5/62) of the procedures with allergic reactions to replacement fluid as most common event (n-3) followed by hypotension (n-2). follow up was available in 55% (6/11) of patients and are doing well on immunosuppressive therapy. one patient died due to respiratory failure after 3 months and another had relapse for which he underwent second tpe cycle and continue to do well. conclusion: tpe is a safe and effective adjunct therapy to high dose immunosuppression in nmosd. trima accel software upgrade from 6.0 to 6.4 for platelet collections rachel m beck*, kimberly j duffy, sandra bryant, audrey e traun, mary m benike, james r stubbs and justin d kreuter. mayo clinic background/case studies: terumobct released trima accel software version 6.4 as an enhancement to allow for the collection of platelets (plt) with platelet additive solution (pas) and provide additional improvements to increase overall reliability. additionally, the manufacturer identified a slower centrifuge speed at low draw flow rates. this software was expected to function similarly to version 6.0. the objective of this retrospective study is to identify any variances with the software upgrade influenced the plt products collection process or products collected. study design/methods: prior to 1/16/2016, plt collections were performed on nine trima accel machines operating with version 6.0. upgrading and validating all nine machines to version 6.4 occurred from 1/16/2016 to 4/30/ 2016. the trimas were programmed with the same plt configurations both before and after software update. platelet collection data from version 6.0 (5/1/2015 to 9/30/2015) was compared to version 6.4 (5/1/2016 to 9/30/ 2016). incomplete collections, runs identified as having possible leukocyte contamination, duration of collection, and plt split rate were evaluated for each time period. generalized estimating equations (gee) were used to assess differences between plt collections with version 6.0 and 6.4, adjusting for multiple visits per donor, with significance defined as p-value < 0.05. results/findings: following the upgrade to version 6.4, staff observed a number of changes including an increased centrifuge recovery time on a donor with a low flow and a notable increase in possible leukocyte contamination products. version 6.4 of the trima accel showed a statistically significant increase in possible leukocyte contamination from 3% to 5% of collections as compared with version 6.0. both the duration of collections and the plt split rate remained constant even with centrifuge speed adjustments in version 6.4. conclusion: due to fda limitations not allowing for the implementation of trima accel pas plts with the currently available pathogen reduction system, the institution decided to implement only the pathogen reduction system at this time. subsequently, the version 6.4 software is no longer required. with the noted slight increase in possible leukocyte contamination as well as the lack of enhancements for plt collection, the upgrade to version 6.4 currently does not provide added value over version 6.0 for plt collection. pulmonary and neurologic symptoms due to leukostasis. therapeutic leukocytaphersis (tl) is used as an adjuvant therapeutic modality in these patients with symptoms suggestive of leukostasis. tl procedures are performed using cell separators where anticoagulated blood is subjected to centrifugal force resulting in separate layers of cells and plasma depending on their density. there are two programs in the cell separator, a mononuclear (mnc)program which has greater centrifuge speed and efficiency for the collection of mncs and a polymorphonuclear (pmn)cell program with lower centrifuge speed for the collection of pmns. hydroxyethyl starch(hes) is preferred for the collections of granulocytes for transfusion from healthy donors. use of hes facilitates the sedimentation of the granulocyte layer and increases the efficiency of collection. though use of hes in tl was not associated with adverse events with its use as a volume expander (pagano) its use in tl varies and no reports are available on the efficiency of leukodepletion using hes for tl. study design/method: we received a request for leukoreduction in 32 yearold lady with chronic myelogenous leukemia (cml) who had a good response to imatinib. she is 30 weeks pregnant with an increased wbc count due to the discontinuation of imatinib. we performed tl with the cobe spectra using a replacement fluid of 500 ml 5% albumin. wbc counts were monitored pre and post tl in the patient and in the collected product. we modified the collection based on these results using the mnc program with acd-a or the pmn program with acd-a . as leukodepetion was not adequate with these programs we elected to use hes after discussion with the patient and her physician. tl was performed using 500 ml of hes with citrate and the pmn program. wbc pre procedure, immediate post procedure and the product was obtained and the efficiency of leukodepletion with the different programs was calculated. results/finding: the efficiency of % wbc depletion was calculated by product wbc to patient wbc based on blood volume and also pre to post wbc the patient tolerated the procedures well and there were no adverse reactions in the patient and in fetal monitoring during the procedures conclusion: therapeutic leukocytapheresis in cml patients is safe and more effective in reducing the wbc count with the use of 500 ml of hydroxyethyl starch with anticoagulant. post procedure patient wbc counts sometimes may not provide the data on the efficiency of leucodepletion. background/case studies: early recognition of hypertriglyceridemia (htg) in the setting of acute pancreatitis (ap) is critical to initiate effective therapy. the role of plasmapheresis as an early/adjuvant approach in acute htg-induced pancreatitis is controversial. currently, there are no consensus guidelines in optimal therapy and is asfa category iii. reported here is a case where the tg level as well as clinical symptoms improved after one therapeutic plasma exchange (tpe). study design/method: a 45 years old male with history of hypertension, htg, and diabetes mellitus (dm) presented to our emergency department with excruciating abdominal pain. the patient was diagnosed with htg at 20 years old. he was treated initially with diet and lifestyle modification. however, his clinical course has been compromised after developing pancreatitis with 3 acute episodes requiring prolong hospital admission of approximately 2 months each which were successfully treated medically. however, the recurrent episodes resulted in chronic pancreatitis which was complicated with pancreatic pseudocyst and pancreatic insufficiency. since the first episode of pancreatitis, he was then medically managed with fenofibrate, lovaza, lisinopril, levemir and novolog. during evaluation on current admission, he was found to have a tg level of 4980 mg/dl, lipase 92 u/l, glucose 250 mg/dl, bicarbonate 24 mmol/l, anion gap 12. ct findings were consistent with ap without evidence of necrosis and stable pancreatic pseudocyst. medical therapy was started with omega 3 fatty acid, fibrate, statin, hydration as well as pain control. statin therapy was suspended on day 3 of hospitalization, because he was noted to have elevated liver function tests (lft) and tpe was requested and started on day 3 after admission. results/finding: the patient tg decreased by 52% (2365 mg/dl) with medical therapy, followed by additional 67% (767 mg/dl) after one volume of tpe. his symptoms significantly improved and was discharged with medical treatment on day 6 after admission. compared to previous episodes, his hospital stay was significantly decreased. tg levels remained below 1000 mg/dl at 20 days follow up after discharge. conclusion: early tpe may be of value in treating patients with elevated tg associated with recurrent pancreatitis. plasmapheresis might be an effective early adjuvant therapy to mitigate length of hospital stay, improve cost-effectiveness and patient safety. background/case studies: from 2009 to 2013, a national blood donor center in southeast asia conducted a program to monitor the ferritin levels of platelet blood donors. the aim of this study was to explore the trend of changes in ferritin. study design/method: in this study, we collected 5,129 cases whose ferritin levels have been monitored more than twice with an interval of detection in 150-160 days. the collected plasma samples were tested for ferritin by chemiluminescence using a commercial assay. inclusion criteria included apheresis platelet blood donors with over two results of ferritin, and first time ferritin test result was over 50 lg/l. and the upper limit was set to be 244 lg/ l in male and 158 lg/l in female as described in manufactures insert. the impact on ferritin from gender, age, and the blood donation frequency were examined with anova test. the blood donations frequency was categorized into five groups: 0 times, 1 to 3 times, 4 to 6 times, 7 to 9 times and more than 10 times. the high frequency (more than 10 times group) blood donors were analyzed ferritin changes in longitudinal data. results/finding: there were 5,129 donors included in the study, of which 4,944 were male (96.4%) and 185 were female (3.6%). the mean ferritin was 82.0 lg/l in male (95% ci: 80.7-83.2 lg/l) and 66.5 lg/l in female (95% ci: 60.9-72.0 lg/l). the result of anova indicates that the group with the highest frequency (more than 10 times) has the significant lowest ferritin level (p<0.05). the average change of ferritin if donation over 10 times would up to 13.4 and 14.1 lg/l in younger and elder 50 y/o male and 18 and 23 lg/l in female. and then for high frequency (half a year more than 10 times the group of blood donors) for longitudinal analysis and found that the long-term sustained high frequency of blood donation caused a significant decline in ferritin. the average change about ferritin in high frequencies donors (over 10 times in 150$160 days) was reduced from 21.5 lg/l in the first period to 4.1 lg/l in the third period (1 period5150$160 days). along with the more and more period, the decline of ferritin decreased. conclusion: this analysis revealed that frequent apheresis platelet donation would decrease ferritin of donors. but the high frequency of platelet blood donors who continue to donate after a year, the decline of ferritin slowed down. a rare case of blood donation precipitating acute delirium joseph griggs* 1 , mary townsend 2 and lizabeth rosenbaum 3 . 1 university of new mexico hospital, 2 blood systems, inc., 3 blood systems inc. background/case studies: we report a case of whole blood (wb) donation that precipitated a transient agitated delirium. a 22 year-old first time male donor presented to the local blood center, completed the donor health questionnaire, mini-physical exam, and hemoglobin check, and was deemed eligible for blood donation. approximately 10 minutes after an uncomplicated wb donation, the donor had an observed, brief loss of consciousness in the post-donation area. no fall or injury was seen. shortly after regaining consciousness, the donor became agitated, confused, and was not oriented to month or year; was unable to remember the names of friends and family members; was unable to read an analog clock; and had difficulty with word finding. the donor was transported to the local university hospital where he was noted to be combatively delirious and had altered mental status; he had to be forcibly restrained. he ultimately was sedated and intubated, and transferred to the intensive care unit. study design/method: an extensive laboratory investigation was performed including standard hematologic and chemistry panels; serologic and pcr-based studies for multiple organisms including west nile, herpes, hiv, varicella zoster, and syphilis; aerobic and anaerobic blood cultures; and a urine drug screen for multiple drugs of abuse. radiographic imaging was performed including a chest x-ray, and a ct and mri of head and spine. in addition, an eeg was performed. the inpatient neurology and psychiatry services were consulted for this patient. results/finding: after the sedation was discontinued, the patient was successfully extubated and rapidly improved. he completely returned to baseline within 24 hours of onset of the event. laboratory investigation revealed no signs of infectious organisms or evidence of drugs of abuse. radiographic imaging and eeg studies showed no abnormalities. in addition, infectious disease marker testing performed by the blood center laboratory was negative. investigation revealed that the donor was experiencing high levels of stress at school, had an aversion to the sight of blood, and was coerced into donating by his girlfriend and peers. a week following hospital discharge, the blood center medical director contacted the donor by phone; the donor had resumed his normal routine and was attending his graduate level classes. conclusion: to our knowledge, this is the first report of blood donation precipitating a transient acute delirium. at the time of donation, the health status of all potential blood donors is assessed to help ensure the safety of the donor and the recipient. the health questionnaire, physical exam, vital signs, hemoglobin level, and infectious disease testing help to identify overt signs of medical illness that may disqualify a donor. however, routine donor screening does not explicitly evaluate mental health issues, both diagnosed and undiagnosed. although exceedingly rare, this case highlights the limitations of donor screening to identify donors who may be at risk for mental health adverse reactions when donating blood. a targeted approach to increasing the african american blood donor pool arnethea sutton* 1 , william korzun 1 , teresa nadder 1 , susan roseff 2 and elizabeth ripley 1 . 1 virginia commonwealth university, 2 virginia commonwealth university medical center background/case studies: a continuous need for blood products for those who require frequent transfusions, such as individuals with sickle cell disease who could benefit from products collected from african american donors, warrants the need for targeted interventions to increase blood donations from underrepresented populations. one population in particular, african americans, only account for 1% of blood donors in the united states. literature indicates numerous reasons why this population is underrepresented amongst donors, including fear, lack of knowledge about the blood donation, and specific to this population, lack of trust in the medical community. study design/method: african americans in richmond and norfolk, virginia were recruited through churches and local universities. the study's aims were to develop, implement, and assess a targeted educational approach incorporating the theory of planned behavior and various teaching methods, to develop and implement a survey to evaluate participants' feelings, attitudes, and intent to donate, and to motivate african americans non-donors to attempt to donate blood. participants attended a 1-hour educational session where they were educated on the importance of red blood cell donations from african americans. participants completed three surveys -one before the session, one directly after the session and one, two months after the session. a two-proportion z-test was used to compare the known proportion of african americans who present to donate in the study areas to those who presented to donate in this study, while regression analysis was used to estimate the relationships among survey variables. results/finding: a total of 142 subjects were included in the data analysis. sixteen percent of the study participants presented to donate as a result of attending the educational session. this resulted in a statistically significantly higher proportion of african americans presenting to donate than the current proportion in the areas of the state where this study was conducted. results from the first two surveys indicated that subjective norm and attitude were significant predictors of one's intent to donate blood, while perceived behavioral control was not a factor. the educational session increased survey scores related to intent to donate in comparison to scores obtained prior to the session. conclusion: this study shows that a targeted educational program can change attitudes toward blood donations in african americans resulting in an increase in new blood donors. additional studies are needed to see if this behavior will continue and whether african americans can influence their community to increase awareness and motivation for life-long blood donation. were from female basic trainees conclusion: the significant increase in hemoglobin deferrals at basic training site a from 2015 to 2016 could be a result of a change in the blood drive timing of the training schedule of that location. in 2015, basic trainees at site a were scheduled at day 57 of 70. in january 2016, the blood drive date changed to day 60 of 70. the extra three days in the basic training atmosphere, and its associated diet changes and increased physical activity may have had an effect on the hemoglobin levels in that population. at basic training site b, the significant increase from 2015 to 2016 of hemoglobin deferrals can be attributed to a larger male population presenting at this site for basic training. additionally, the percentage of female recruits donating at the blood drives decreased in 2016. these observations support the hypothesis that the increase in hemoglobin deferrals in 2016 resulted from the implementation of the male hemoglobin standard change from 12.5 to 13.0 g/dl at basic training site b. when planning for blood drives at basic training site b, screening of an additional 24% of recruits must be considered when performing these blood drives, in order to meet the same collection goals set prior the implementation of the change in the male hemoglobin standard. blood donation in the donor with spinal cord injury joan-ramon grífols* 1 , eva alonso 1 , oscar bascuñana 1 , monica romero 1 , teresa vich 1 , elena castaño 1 , laura carbonell 1 , eva palomas 1 , saray almerge 1 , francesc carpio 1 and xavier curia 2 . 1 banc de sang i teixits, 2 institut guttmann background/case studies: donation of blood components (bc) in donors with spinal cord injuries (sci) is poorly studied. paralysis is a state, not a disease, after a reasonable time since its acquisition these people should not be differentiated from the rest of the non-paralytic population in terms of bc donation. the literature reviews of blood donation suitability criteria among these people are scarce and the vegetative lability that they may present depending on the type of their sci it's obvious. in daily practice these potential donors are often rejected for donation with no specific criteria related to their sci. the objectives of this study are to establish the selection criteria for bc donation in people with sci based on medical criteria. to evaluate the rate of adverse donation blood reactions of these donors against a donor control group without sci. study design/method: our organization regularly organizes a donation campaign at a rehabilitation center for patients with sci. in this campaign some donors with sci as donors without (professionals of the center, relatives, etc.) donate blood. from january 2015 to december 2016 we analyzed the number of donors who came to give blood, the number and reasons for exclusion of those who could not make the donation, whether or not they had sci and number and typology of adverse reactions to the donation detected in both groups. donors with sci higher than t5 due to the high risk of autonomic dysreflexia were excluded for donation. donors with sci below t5 and less than one year of evolution were set as temporary exclusion criteria. the presence of neurogenic bladder was not considered a reason for exclusion. results/finding: in the analyzed period, 219 donors came to give blood, of these, 15 (7%) were excluded for donation for various reasons. two of the donors excluded suffered sci higher than t5 excluding them due their high risk of dysreflexia. another one donor excluded suffered sci lower than t5 but his hemoglobin levels were lower than our selection criteria. of the 204 donors selected for donation 16 (7.8%) had sci lower than t5 and t6. adverse reactions to donation (1.4%) were recorded in our haemovigilance program, none of them in donors with sci. conclusion: according to our experience donors with sci lower than t5 have not had any type of adverse reaction to the blood donation. there should be selection / exclusion criteria based on the donor's paralytic conditions. the vagal syndrome that could appear as a complication to the donation in these sci donors should be approached differently to the usual protocols that we use. blood donor center's experience with changing from manual to automated blood pressures kimberly j duffy*, sandra bryant, audrey e traun, kristine i borth, mary m benike, james r stubbs and justin d kreuter. mayo clinic background/case studies: blood pressure (bp) is important for determining the health and suitability of blood donors. the manual method of reading bp can result in variability due to minor variances in the way staff perform the manual procedure. automated bp devices are able to reduce the variability in bp determination. in december of 2013, automated bp devices were validated and replaced the manual bp method in our blood donor center. the objective of this retrospective study is to determine if the change from a manual to an automated bp process has impacted the average systolic and diastolic pressures and, additionally, if a differences in the deferral and reaction rate can be observed. study design/methods: data for the manual bp process was accumulated for an 11 month period from january 2013 to november 2013. the same information was assembled for the automated bp process for the 11 month period of january 2014 to november 2014. the automated bp process implemented in mid-december 2013; so the december data for both 2013 and 2014 has been excluded from the study. bp, bp deferrals, reactions, donor weights and demographics were evaluated for each time period. a donor may be included multiple times in each year and could be in both sets of data. generalized estimating equations were used to assess differences between automated and manual bp with significance defined as p < 0.05. results/findings: significantly more people were deferred using automated bp compared to manual bp readings (p50.006). both systolic and diastolic bp measured significantly higher by automated bp method than by manual method. although donors in the automated bp group experienced fewer reactions than those in the manual bp group, the reduction was not large enough to reach statistical significance. even after adjusting for gender, weight and age at donation, bp deferrals, systolic and diastolic bps all remained significantly higher (all p < 0.03) with the automated bp while and reactions remained non-significantly lower (p 5 0.086). conclusion: automated bp devices have improved convenience for both staff and donors. with a statistically significant increase in deferrals and marginal decrease in reactions, the use of automated bp devices may play a minor role in the safety of blood donors. for the purpose of this study, only the hemoglobin values that were below 12.5 g/dl will be compared as a surrogate for deferral. to adjust for multiple visits per donor, generalized estimating equations were used to assess significance between lancet a and lancet b, using the appropriate distribution for the data type, defining statistical significance as p-value < 0.05. results/findings: the average hgb was slightly lower with lancet b but there was a larger change with the number of donors under 12.5. statistically more visits with hgb less than 12.5 g/dl used lancet b than lancet a. additionally, fewer first time donors were seen during the lancet b time than during the lancet a time. after adjusting for the effects of both gender and first-time donation by using logistic regression, the risk of hgb under 12.5 was 16.5% higher with lancet b than with lancet a. conclusion: donor's hgb was slightly lower with lancet b than lancet a, but not clinically different. slightly more lancet bs were used per visit than lancet as. in addition, more hgb deferrals were obtained using lancet b than lancet a. even after adjusting for the effects of gender and repeat donors, we saw more potential deferrals with lancet b than lancet a. the slight difference in the gauge of the lancet may have some association to free-flowing amount of blood and may affect hgb levels. prior to implementing materials at a lower cost, an evaluation of downstream consequences would be recommended. blood donors' acceptance and response towards implementation of automatic appointment booking yi lin ang*, ching lian toh and william choon hong sim. health science authority background/case studies: with surges in demand for blood due to an aging population and more hospitals being built, it is becoming increasingly important to be able to ensure that donors return on a regular basis to improve blood supply and blood stock management. disliking the obligation imposed by appointments, singaporean donors generally prefer "walk-ins" as opposed to appointment bookings. blood services group (bsg) singapore, has made a move to change donors' mindset by introducing automatic appointment scheduling. this paper aims to study donors' level of acceptance towards this initiative. study design/method: to determine the donors' acceptance rate, data was collected from 1 january to 31 march 2017. after completing their donation, donors were automatically given the next earliest eligible date for their next donation. those who do not wish to accept the recommended appointment can either decline this arrangement or log into the blood bank's donor appointment booking system (donor-care) to make changes to the appointment offered. a reminder will be sent to their phone via sms and/or email to their account three days before the appointment date. data was collected from donor-care and was used to measure the number of appointments made and declined over the three months period. donors who declined appointment scheduling were verbally interviewed for their reasons. results/finding: a total of 6680 donors who has donated blood in the blood bank's main branch were used as the baseline for this study. 85% of donors (n55678) accepted automatic appointment booking, whereas some donors (n51002) were not comfortable with it. 77% of those who declined still preferred walk-ins (n5771) based on their own time schedule, the rest decided that variable situations (n5112), donation frequency (n569) and choice of preferred donation locations (n550) were reasons for declining automatic appointment booking. prior implementation of appointment booking at other blood bank branches showed that donors who booked appointment through donor-care was 19%. a comparison was made and found that this study shown a significant increase of acceptance rate by 66%. conclusion: generally, the results were positive and the automatic appointment booking system enabled bsg to predict donor attendance, ensure better manpower management to reduce donor turnaround time and thus hopefully improve donor retention. bsg is still monitoring this automatic appointment system and future study are still required to determine the effectiveness of automatic appointment booking, donor return and retention rate. currently bsg has 4 collection centers, each managing its own appointment system. the eventual aim is to be able to have a centralized appointment booking system whereby donors can book appointments and still be able to donate at any collection site. 4) , 3.28 0.4940 4 1 poisson distribution, 2 normal distribution, 3 logistic distribution, 4 lognormal distribution 105a transfusion (p>0.05) in donor and reference populations except in younger (20-44 yrs) male donors (p<0.0021; donor 4.9%, reference 10.0%). mean donor sbp, dbp, and pulse were 125 6 14.7 mmhg, 75.1 6 9.6 mmhg, and 75.9 6 11.2 bpm, respectively. screening blood pressure levels consistent with hypertension (29.4% male; 16.6% female) in the 20-44 year donor group, significantly (p<0.0001) higher than the reference population (11.2% male; 8.7% female). no differences were observed in the 45-64 year groups. conclusion: normal source donor demographic and physiologic characteristics often paralleled those of the reference usa populations. however there were differences including lower cholesterol levels and a higher rate of high blood pressure in younger donors and higher weights in 20-49 year old females. developing blood donor educational materials gay wehrli* 1 , susan rossmann 2 , louis m. katz 3 and dan a waxman 4 . 1 university of virginia health system, 2 gulf coast regional blood center -sugar land, 3 americas blood centers, 4 indiana blood center background/case studies: donors must have sufficient information to make a decision, time to consider options before making a decision and an opportunity to make a choice of whether to proceed with or decline donating. donor education (de) materials must address mandates set forth by regulatory agencies. these materials must be accessible and understandable by the general population. the goal of this non-experimental, qualitative design study was to evaluate knowledge acquired through standardized de materials. this study was irb approved as an exempt protocol. study design/method: we developed a de document written at an 8 th grade comprehension level. a convenience sample of volunteers was identified for this two-part study. a focus group (fg) incorporated a pre-and post-quiz for knowledge acquisition from reading the four-page de document. the quiz was followed by a group discussion for feedback. the preand post-quiz contained the same 10 multiple choice questions with single best answers including the option to answer, "i don't know." the de document was revised based upon the fg feedback and quiz results. the revised, 3.5 page, de document was then tested using the same pre-and post-quiz during individual interviews (ii). results/finding: demographics and quiz results are summarized in table 1. results from the fg and ii revealed a lack of knowledge in four areas: a donor might be asked not to donate at any time during the donation process, the need for photo identification to donate, iron helps increase a low red blood cell level, and not to donate for the sole purpose to obtain hiv testing. post-quizzes from the ii group revealed an improvement in knowledge acquisition for all four areas. feedback from both groups reiterated that the document was too long. conclusion: developing de materials requires a complicated balance of providing critical information, concisely and at an appropriate comprehension level (8 th grade). testing de materials is an essential step in the development process to ensure the intended knowledge is acquired by the end user population. the next steps for this group will be to pilot the further revised, two-page de document at donation sites. effect analysis of the 'rh(-) blood supply program' establishment hyesung han*, deokja oh, buja hur and chulyong kim. korean red cross blood services background/case studies: the rh(-) blood supply program was developed in 2011 for the purpose of prompt and stable blood supply. based on the computerized system, the program operates the emergency contact/ communication. this program has 4 major functions such as the request of the emergency blood, the recruitment and management of the rh(-) blood donors for the emergency blood donation, real-time blood supply status monitoring program and statistics program. the aim of the research is to validate the effect of rh(-) blood supply program operations and the responsiveness of the emergency blood supply under the rh(-) blood supply program. study design/method: researchers investigated the database from 2011 to 2016 after the rh(-) blood supply program was developed. investigators analyzed and compared the recruitment and blood donation of the rh(-) blood donors for the emergency blood donation and securing the blood supply upon request. results/finding: the data shows that the number of voluntary blood donors who pledge to give blood for the emergency blood donation has increased from 5.6% to 21.4% in 2011 and 2016, respectively. also, the actual participation rate of rh(-) blood donations among the group who pledge to give blood for the emergency blood donation has increased from 26.8% in 2011 to 57% in 2016. moreover, the data has indicated that the blood supply has fully met the demand for the emergency blood request. conclusion: the result showed that the rh(-) blood supply program was effective for the recruitment/management of the rh(-) blood donors for the emergency blood donation. this system contributes to recruiting and managing rh(-) blood donors who pledge to donate blood and securing rh(-) blood in emergency situation . the institution that needs to meet the demand of rare blood type could possibly use the rh(-) blood supply program which leads to securing special type blood. hanwei chen*. wuhan blood center background/case studies: in china, volunteer blood donors can donate platelets by apheresis (ap) up to 24 times per year. however, the awareness and knowledge of ap donation is much lower than whole blood donation among the chinese population. there are approximately 1.3 million doses of ap transfused within 1.375 billion people each year in china; it is one challenge to recruit new ap donors and retention them as frequency ap donors in china. study design/method: one stratified recruitment and retention strategy established and applicate at wuhan blood center since 2006. firstly, "one-to-one" telephoning model for whole blood donors instead to donate platelet; secondly, group message for permanent ap donors and had not donated with an interval of more than 180 days in low inventory. thirdly, specific recruiter telephone for those ap donors who had donated aps for more than 4 times and had not donated for more than 90 days or less than 4 times with an interval of more than 60 days from the last donation; the last one is preparing one letter of thanks for those ap donors who gave more than 8 times annually which advise them to voluntarily come to the blood center for ap donation when they were available. results/finding: over the past decade, the overall donation time of ap donors increased by 7.46 times from 5550 to 41420 and the doses of ap increased by 7.41 times from 7363 to 54553 within 10 years. the aps collected fulfilled the clinical needs. according to the donation frequency, ap donors were divided into 5 groups: those who donated ap once, those who donated 2-4 times, 5-9 times, 10-29 times, and those who donated more than 30 times, respectively. it was found that the number of permanent ap donors who donated ap more than 30 times was only 965 (2.1%), but they denoted a total of 76432 doses of ap (29.2%) from 2006 to 2016. conclusion: aps increased at a rapid and steady pace in wuhan blood center from 2006 to 2016, which not only met the clinical needs but also were supplied to other region outside wuhan. and in addition, the permanent ap donors who gained more attention donated the greatest percentage of platelets. in conclusion, stratified recruitment is one effective approaches to meet clinical needs for platelets and worth to popularize to other region. years were evaluated at 4 sites on 2 consecutive donations for finger stick (fs) hemoglobin (hb) per site policy. venous (ven) and capillary (cap) zpp and ven ferritin (fer) were performed per manufacturers' direction. donors were assessed for subclinical iron deficiency using ranges (fer <26 ng/ml and zpp levels >100 umol/mol heme) at 3 hb levels. participants completed an online survey between donations to collect data on symptoms of anemia. univariate linear regression analysis was used to determine relationship between tests. results/finding: subclinical iron deficiency was present among first-time and repeat blood donors at all 3 hb levels with both genders and all age groups. (table) there was a highly significant correlation between fs zpp and ven zpp 87.8% (r50.937) at first and 86.5% (r50.93) at second donations. at first donation when compared to fs hb, only 10.4% (r50.323) of variation could be explained by variation in fs zpp, 12.3 % (r50.35) by ven zpp and 9.4% (r50.307) by ven fer. at second donation, when compared to fs hb, only 9% (r50.30) of variation could be explained by variation in fs zpp, 14.4% (r50.38) by ven zpp and 20.1% (r50.448) by ven fer. for each donation, variation among tests (fs hb, ven fer, ven zpp and fs zpp) was significant (p<0.001) suggesting strong evidence against correlation. 55% (181) responded to the survey of which 4% (13) reported not feeling well after donation. it should be noted that noted that 1% (3) female study participants reported feeling unwell after the first donation and had ferritin levels below 26ng/ml but the zpp levels were less than 100 umol/mol heme. of the 3% (10) male participants that reported not feeling well none had ferritin levels below 26 ng/ml nor ven or fs zpp levels above 100 umol/mol heme. conclusion: subclinical iron deficiency was present at all hemoglobin levels. there was insufficient correlation with fs hb and ven fer to support use of fs or ven zpp analysis as measurement of iron stores for blood donors. symptoms reported by study participants were not consistent with laboratory results. the minimum male hb was raised from 12.5 to 13.0 gm/dl. fda imposed specific vs ranges for acceptable pulse (p) and blood pressure (bp), removing center-by-center discretion. a survey of members of america's blood centers (abc) was performed to assess the impact on donor deferrals resulting from these changes. study design/method: online survey software (surveygizmo, boulder, co) was used to solicit collections and deferral information from 59 blood centers over two intervals, july-dec. 2015 and july-dec. 2016 (i.e., before and after the implementation deadline for the final rule respectively). information on deferral at presentations for whole blood (wb) donations and apheresis platelet (ap) donations was requested for hb thresholds and vs. the information was stratified by gender (male5m, female5f), and abo type. statistical analysis included t-tests for numerical and chi-square for categorical data (minitab 17.0, chicago il). p <.05 was considered significant. results/findings: data were provided by 40 of 59 centers invited, representing 2,420,886 and 2,945,802 wb donations and 272,094 and 319,161 ap donations in aggregate during the two intervals respectively. gender and abo distributions appeared representative of the us donor base. among m wb donors the rate of deferral rose from 1.5% to 2.9% in the two intervals among aggregated donation attempts (p<.001), and for m ap from 1.8 to 3.5% (p<.001). the mean "by center" deferral rates (table) were similar to that and significant (p<.001). mean by center hb deferral rates among f donations during the two intervals were 11.6 and 11.9% (p50.241) for wb, 11.8 and 13.0% (p5.041) for ap, respectively, absent any change in their acceptable hb thresholds. data on vs deferrals were much sparser. for p deferrals, only 12 centers could provide specific high vs. low vs. irregular pulse deferrals; 27 provided only a summary (i.e total pulse deferrals), and 1 could provide none. for bp, 8 provided detail (high vs. low), 28 summary and 4 none. p deferrals increased in the successive intervals among f wb donors from a center mean of 0.57 to 1.49% (p5.018) and for m wb donors from 0.78 to 1.16% (p5.006). where details were available, high and irregular pulses were responsible for most of the changes for both genders. bp deferrals were not significantly increased among wb donors, regardless of gender. the data sets and deferral rates re: vs in ap donors were quite small, possibly reflecting culling during their prior donation experience. conclusion: substantial additional donor deferrals attended the increased hb thresholds for m in the final rule, for both wb and ap. changes were more modest among female donors, consistent with the absence of changes in allowable hb levels. modest but significant changes attended more stringent requirements for vs, though data limitations restrict this aspect of the analysis. background/case studies: diabetes mellitus is reaching potentially epidemic proportions in india. given the disease is now highly visible across all sections of society within india, there is now the demand for screening of diabetes and urgent research and intervention -at regional and national levels -to try to mitigate the potentially catastrophic increase in diabetes that is predicted for the upcoming years. due to its ease of use, several studies have found that hba1c testing can identify patients in the community who might otherwise go undiagnosed. we took an initiative to find out the incidence of diabetes by random blood sugar (rbs) measurement among indian blood donors and measure the hba1c levels among those with rbs >180 mg/dl study design/methods: a prospective study was done at department of transfusion medicine and department of biochemistry from 1 st march 2017 to 31 st march 2017. total of 1,861 blood donors were tested for rbs. those with rbs > 180 mg/dl were further tested for hba1c by gold standard hplc method using variant ii biorad. blood donors with >180 mg/dl rbs and hba1c > 6.5% were advised to consult a physician for further evaluation. results/findings: of the 1,861 donors tested, 44 (2.36%) donors showed a rbs of > 180 mg/dl. forty two (95.45%) were males and 2 (4.54%) females with a mean age of 40.55 years (26-56 years). of these, 14 (31.81%) were known case of type-ii diabetes mellitus (dm) on oral medications and were excluded. of the remaining 30, 8 (26.66%) of them had a family history of dm. of these 30 donors, 8 donors did not give a consent for testing for hba1c. among the 22 donors tested for hba1c levels, 16 (72.72%) had hba1c > 6.5%. all the 16 donors were counselled and referred to a physician for further management. the overall incidence of donors having dm in the population is 0.87% (16 of 1839 donors). conclusion: screening for blood glucose level by targeting the blood donors can go a long way in curbing the diabetes burden on the society. incidence of low ferritin levels in regular male blood donors with acceptable hemoglobin levels in singapore ramir alcantara* 1 , hwee huang tan 1 and ai leen ang 2 . 1 health sciences authority blood services group, 2 health sciences authority, blood services group background/case studies: iron deficiency is a known complication of regular blood donation. in order to protect the donor's health and prevent iron deficiency, aabb increased the minimum acceptable hemoglobin level for male whole blood and apheresis donors from 12.5 to 13.0 g/dl last may 2016. the current minimum acceptable hemoglobin for male donors in singapore is 12.5 g/dl. the aim of the study is to determine the incidence of low ferritin levels in regular whole blood and apheresis male blood donors with acceptable borderline hemoglobin levels (12.5-12.9) and in donors with hemoglobin 13 g/dl and above. study design/method: during a 4 month period, serum ferritin testing was performed on 350 regular male whole blood and 250 regular male apheresis donors who made at least 3 donations in the last two years with an acceptable hemoglobin level. the donors were divided into groups according to donation type and hemoglobin range; group a (whole blood with hemoglobin 12.5-12.9) group b (whole blood with hemoglobin !13, group c (apheresis with hemoglobin 12.5-12.9) and group d (apheresis with hemoglobin !13). the serum ferritin levels of the four donor groups were compared and analyzed. a ferritin level below 30 ug/l is considered low and levels below <12 ug/l are considered having absent iron stores. results/findings: 55.1% of donors in the study have ferritin levels below 30 ug/l. there were more donors with low ferritin in group a compared to group b, 80% and 53% respectively (p<0.05). in apheresis donors, low ferritin rates were higher in group c donors compared with group d, 49% and 30% respectively (p50.001876). ferritin results for the 4 groups can be seen in table 1. conclusion: more than half of the donors in the study have low ferritin and of the donors with low ferritin, more than half or 54.3% have absent iron stores. donors with low ferritin were immediately informed of their result, given iron supplements and advised to come back for donation after 4 months or more. since donor health and safety is of paramount importance, measures to limit and prevent iron deficiency in blood donors must be implemented. due to the high incidence of low ferritin levels in whole blood and apheresis donors with hemoglobin 12.5-12.9 g/dl, it is recommended that the minimum hemoglobin level cut off for male blood donors in singapore be increased to 13.0 g/dl. other measures to be implemented includes better donor education on the risk of iron deficiency and the need for iron supplementation using our website and social media. background/case studies: safe blood is a crucial and irreplaceable component in the medical management of many diseases. the voluntary nonremunerated blood donation is the ideal sources of quality blood, which forms less than 15 % of the demand of the blood in pakistan. motivation among the youth, particularly students, is essential to make voluntary blood movement more successful. to assess the knowledge, attitude and practice regarding the voluntary blood donation among the young student population of karachi so that an effective approach can be made regarding motivation enrolment of voluntary non remunerated blood donors in future in pakistan study design/method: a cross sectional prospective study was conducted among 600 students from different universities and colleges of karachi. a well-structured and pre-tested questionnaire, in english, was used to access the knowledge, attitudes and practices about voluntary blood donation. a scoring mechanism was used to understand overall knowledge level. obtained data was analyzed. results/finding: the sample population consisted of 54% male and 46% female students in the age group of 18-28 years. only 65 % of the students have heard about voluntary blood donation and 28 % of the students have given blood once in their lifetime and among them 19 % are blood donors at the moment. 42 % of the participants believed that there is a specific reason why they don't donate blood and 59 % believed that there is a risk involved for the donors, when donating blood. 80 % students wanted to promote voluntary blood donation. fear and lack of awareness on blood donation are the reasons for not donating blood. students gather information about voluntary blood donation from several sources mostly schools, colleges, family and friends. (21); miscellaneous effects were reported in 23 courses. side effects led to interruption of supplementation in 55 instances. ferritin levels (mgt6sd) at entry into the program and at the last visit were 48.9 6 2 and 65.4 6 1.7 mg/l in participants, vs 64.1 6 2.2 and 56.3 6 2.2 mg/l in controls. the positive impact of iron supplementation on ferritin levels was observed only in those who took !75% of the tablets. ferritin levels<26mg/l were found in 4,8% of participants and 14.7% of controls. deferral for low hemoglobin was below 1% in both groups. conclusion: an iron supplementation program in a drbcd program is feasible.however, when taking into account acceptance to participate and compliance with supplementation, only 50% of donors obtain full benefit from such a program. using an iron preparation which is better tolerated may increase compliance. background/case studies: hereditary hemochromatosis (hh) patients are permitted to donate blood for the allogeneic blood supply as long as they are eligible for donation under 21cfr630.10 and the collection is a physician-ordered therapeutic phlebotomy. blood collections establishments do not need an exception or alternative under § 640.120 to make a collection under this provision if the requirements set forth in § 630.15(a)(2) are met. the objective is to describe current hh donors and long-term contributions of to our hospital-based donor center and hospital blood supply. study design/method: in 2001, an irb protocol was approved for the enrollment and therapeutic phlebotomy of hh patients/subjects. this required filing an fda variance to permit hh donor blood for use in our allogeneic supply without disease labeling. the frequency of therapeutic bleeds are guided by routine clinical assessment, mcv/hemoglobin, serum ferritin, and transferrin % saturation monitoring. serum ferritin levels of 50 -75 ng/ ml are targeted for maintenance phlebotomy. operationally, a custom, computerized database application is employed to ease phlebotomy management. results/finding: since inception, the cumulative number of hh subjects enrolled in the hemochromatosis protocol reached 547, of whom 365 (67%) are c282y homozygotes. without active recruitment, accrual rate is about 7 per quarter, with 69% of subjects qualifying as allogeneic donors. the mean current age is 59.7 years, 65% male, 96% caucasian. the majority of hh donors (276 of an active cohort of 318) are in the maintenance phase of therapy with an average of 2.6 donations/year and a 4% deferral rate. over the last 5 years, hh donors contributed approximately 8-11% of the hospital's allogeneic blood supply, averaging 475 whole blood units for transfusion per year. moreover, hh donor's whole blood (wb) donations provided 30-40% of blood for in vitro research at our institution with an average of 180 wb research donations/year. there have been no hh donor-derived transfusion-transmitted infections over 16 years. since 5/23/16, with an increase in male hgb deferral threshold to 13g/dl, there has been only 1 hh male deferral from blood donation. conclusion: a simple, safe system for donor evaluation, phlebotomy management, and transfusion of blood drawn from hh subjects was established. blood donated by hh donors remains an important resource at our hospital. hh donors benefit from careful medical follow-up of their iron status. this mutually beneficial relationship is feasible and sustainable. testing for accuracy of non-invasive blood hemoglobin methodology in a blood donor setting michele walker*, sharon garcia and mythili ram. gulf coast regional blood center background/case studies: the objective of the study was to assess the accuracy of hemoglobin (hb) levels measured on the orsense nbm-200 non-invasive occlusion spectroscopy device by comparing them to hb levels measured on venous samples with a laboratory hematology analyzer. in addition, the study examined operator ease of use and donor satisfaction with a finger stick-free method. study design/method: study procedures and protocol, including acceptance criteria, were defined in conjunction with the device manufacturer to determine the standard deviation (sd) of the difference between the nbm-200 non-invasive sample results and the sysmex hematology analyzer venous sample results. staff were provided training on the use of the nbm-200 non-invasive occlusion spectroscopy device. over a span of 7 days, 200 eligible blood donors, both male and female, were first screened by the nbm-200 non-invasive occlusion spectroscopy device followed by performance testing utilizing a capillary blood screening method. a venous sample was collected from each of the 200 blood donors for the performance of hb measurement on the sysmex hematology analyzer within 1-3 hours of collecting the venous samples. results/finding: the sd of the difference between the nbm-200 non-invasive sample results and the sysmex hematology analyzer venous sample results was not to exceed 1.1g/dl. the hb measurements obtained from the nbm-200 and the sysmex hematology analyzer were analyzed using the statistical software minitab and the sd of the difference was reported to be 0.978 g/dl. the precision of the nbm-200 yielded a co-efficient of variation of .02 g/dl and a standard deviation of .33 g/dl. conclusion: the operators found the nbm-200 easy to install, maintain, and operate with minimal training. the nbm-200 non-invasive occlusion spectroscopy technology showed accurate performance compared with the venous sample results. it was comparable to the capillary finger stick method and deemed suitable for screening donors. donors were satisfied with the process and appreciated the safe, painless methodology. ronel swanevelder 1 , ravi reddy 1 , dhuly chowdhury 2 , don brambilla 2 and edward l. murphy* 3 . 1 sanbs, 2 rti international, 3 ucsf/bsri background/case studies: to maintain an adequate blood supply, south african blood centers need to collect more blood from their majority black african population. success in recruiting first-time black blood donors has been tempered by lower suboptimal return rates. study design/method: we performed a prospective cohort study of firsttime, black blood donors donating during a four-month period in 2014 and followed them for one year. within 56 days post donation, a questionnaire including questions on blood donation motivators and deterrents was administered by telephone. questions used 4-point likert scales to assess agreement with statements relating to domains of altruism, collectivism, selfesteem and marketing derived from local focus groups (muthivhi et al. 2015) . linking questionnaires to a blood donation database allowed logistic regression analysis to predict return for a second donation within one year. results/finding: we included 2,902 first-time black donors with median age 23 and female predominance (59%). within one year, 1,786 donors (62%) attempted at least one additional donation. when likert scales were analyzed as an ordinal variable (45 strongly agree to 15 strongly disagree), donor return was associated with the following motivators "blood donation is an easy way to make a difference" (odds ratio for each likert increment (or) 5 1.16, 95% ci 1.06-1.28), "i donated in response to adverts/campaigns on the radio, tv or newspapers" (or51.11, 95% ci 1.00-1.23). responses to altruism-associated statements were not associated with return. among deterrents, donors were less likely to donate if they agreed with the statement "i am afraid of the sight of blood" (or50.83, 95% ci 0.72-0.96) and "i wasn't treated well by the <blood center> staff" (or50.85, 95% ci 0.74-0.97). surprisingly, donors were more likely to return if they agreed with the statement "i was afraid of finding out about my hiv status" (or51.19, 95% ci 1.03-1.37). a secondary analysis treating the likert scales as 4-level categorical variables revealed generally similar results, with the additional finding that donors who disagreed with the statements "if i give blood then blood will be available when i need it" and "i don't know where the nearest blood collection point is" were more likely to return. conclusion: this novel design allowed us to study the link between donation motivators and deterrents and actual rather than intended return for donation. it is interesting that self-esteem and marketing predicted return better than altruism. fear and poor customer experience are recognized deterrents which could be addressed. we plan to use these data to construct black donor recruitment interventions which may be tested using randomized trial designs. willingness to donate blood during the summer christopher d bernard 1 , ramya ghantasala 1 , obhijit d hazarika 1 , nicole leonard 1 , cori a polonski 1 , zachary b wunrow 1 , michelle heleba 2 , jan k carney 1 and mark k fung* 1 . 1 university of vermont larner college of medicine, 2 american red cross blood services background/case studies: each year donation rates fall in the summer months straining blood banks' capacities to meet local demands. in hopes of identifying factors to increase summer donations, our study investigated donor reported barriers which influence summer donations habits. study design/method: an anonymous 16 question survey investigating various donation factors was administered across multiple blood donor centers in a state-wide region. questions addressed donor demographics, frequency of blood donation, preference in appointment making modalities including smartphone app use, summer travel habits, willingness to donate during vacation, and factors that deter donors from donating on vacation. results/finding: a total of 292 surveys were received. survey respondents across multiple demographic groups cited similar barriers to summer donation, namely "too busy" (27.5 %) and "traveling is a time for me to relax." (30.6 %). of the respondents who travel in the summer, very few reported donating while traveling (3.4 %). summer donation rates between summertime travelers (36.5 %) and non-travelers (36.4 %) were essentially equivalent. the most preferred methods of scheduling appointments were via the regional blood donor center website (45.6 %) and phone (28.4%). willingness to use a regional blood donation smartphone app was highest among respondents ages of 18 to 34 (45-55%) and lowest among ages 55 and older (13-15%). of respondents with no prior knowledge of summer seasonal shortages (22 %), 2/3rds indicated newfound motivation to donate. background/case studies: viral infections (adenovirus, ebv, cmv, bk, hhv6, and rsv etc.) have been implicated as major contributors to posttransplant morbidity and mortality in hematopoietic stem cell transplantation (hsct) from unrelated donors. investigators have shown that in-vitro expanded virus specific cytotoxic t lymphocytes (ctls) generated from donors with specificity for one or more viruses are safe and effectively treat viral infections in the hsct setting in recent clinical trials. present clinical trials have shown that ctls can be rapidly produced by a single stimulation of donor peripheral blood mononuclear cells (pbmcs) with a peptide-mixture spanning the target antigens in the presence of potent prosurvival cytokines interleukin-4(il-4) and il7. others have used banked third party epstein barr virus (ebv)-specific ctls generated from third party ebv-seropositive blood donors with encouraging results. study design/methods: eligible and consented blood donors were tested for cmv antibodies by serology. cmv-seropositive whole blood (wb) units underwent buffy coats processing from non-leucocyte reduced wb units collected in fenwal triple blood-packs tm that underwent 2 hard spins at 3800 rpm for 7 minutes with separation after each spin on a compomatev r g5. plasma and buffy coat was separated from red cells after the first spin. the second spin lead to the separation of the buffy coat from plasma. the buffy coats were submitted to the gmp stem cell lab for processing of cytomegalovirus-specific ctls. hla typing at high resolution for hla-a/-b/-drb1 loci was obtained for all donors. results/findings: forty five eligible healthy blood volunteers (13m [29%]: 32 [71%] f); median age 42 years (range 21-70) donated a unit (500 ml) blood from which buffy coats (average volume 56 ml) were processed. the buffy coat process was previously validated on 20 wb units. the mononuclear cells (lymphocytes and monocytes) recovered from the buffy coats are listed in figures 1 and 2. all of the buffy coats received by the gmp stem cell lab were adequate in cell numbers to be processed. the processing of buffy coats from whole blood is a viable option for the concentration of pbmcs specifically for production of viral specific ctls as third party off the shelf products as well as use in other research projects that require pbmcs from healthy adults. background/case studies: the goal of this presentation is to describe the journey and challenges towards tjc, patient blood management (pbm) certification. transfusion-related health risks and increasing economic pressures have driven hospitals to recognize evidence-based blood management as an important cost-saving strategy. providence holy cross medical center (phcmc), as the providence california region alpha site, has embarked on this journey. our goals are pbm certification and reduction of the number of unnecessary transfusions by 20% within 12 months of the program launch while improving patient outcomes. this paper will discuss our journey toward certification and the various hurdles being overcome. study design/method: tjc, aabb, and the society for the advancement of blood management have served as our primary resources for identifying current evidence-based transfusion practices and management methods. we needed to identify our organizational gaps in data gathering and analysis. then we could determine baseline performance and set improvement targets. from our internal assessment, we learned we had to start from scratch as we had no easily accessible data metrics and gaps in education to our staff. we took the following steps to develop our pbm program: formed an interdisciplinary pbm team consisting of physicians, nurses, blood bank staff, and data analysts constructed a report on rbc transfusions to help identify outliers and opportunities background/case studies: the maximum surgical blood ordering schedule (msbos) is a list of surgical procedures performed at a hospital along with a recommendation for pre-transfusion testing and rbc allocation before each surgery. the extent to which hospitals have an msbos and its design was explored in this survey. study design/methods: the survey was designed, piloted and refined by members of the best collaborative and invited colleagues. it was then encoded in online survey software and the link distributed to best members and colleagues who were encouraged to respond and to further distribute it. the survey was open for 34 days. results/findings: there were 158 completed responses, of which 73 (46%) indicated that their hospital had an msbos and 85 (54%) did not. the majority of hospitals without an msbos were academic centers (36/85, 42%) from oceania (26/85, 31%) or europe (23/85, 27%), had between 500-999 beds (30/85, 35%); the majority of these hospitals transfused between 1,001-4,999 rbcs (21/85, 25%) per year. 15/85 (18%) are going to implement an msbos in 2017. of those with an msbos, the majority 23/73 (32%) were from north america. the majority were academic hospitals (39/ 73, 53%) with 500-999 beds (43/73, 59%) that transfused !20,000 rbc units per year (21/73, 29%) offering a wide range of surgical services. on average there were 207 6 577 procedures listed in the msbos'. the msbos recommended no pre-transfusion testing for a mean of 30% of the procedures listed, a pre-operative type and screen for 38%, crossmatching rbc units for 28%, and for 4% of procedures a different recommendation was made. most (32/73, 44%) of the msbos' were created by a combination of obtaining consensus between the surgical services and blood bank and use of procedure-specific transfusion data; only 5/73 (7%) of msbos' were created solely by using procedure-specific data, and most (35/73, 48%) do not use patient-specific data in making a testing recommendation. most msbos' are updated less frequently than annually (30/73, 41%), and the hospital transfusion committee is often (39/73, 53%) involved in updating it. the msbos' are generally available electronically in both the operating rooms and in the blood banks. it was the opinion of the majority of respondents (30%) that the msbos was used regularly by only a limited number of surgeons and anesthesiologists, 23% of respondents felt that it was regularly used by all surgeons and anesthesiologists; 10% felt that it was not used at all at their hospital, 36% did not respond. conclusion: an msbos was available in only about half of the respondent's hospitals and in only the minority of cases was it felt to be regularly used. however, 18% of the hospitals currently without one indicated that it would be implemented in 2017 suggesting that these hospitals perceive the value of having one in place. implementing and following an msbos can be an important step in peri-operative patient blood management and in streamlining the operations of the blood bank vis-a-vis pre-operative testing. blood management -one hospital system experience leana serrano rahman*, mallika gupta, susan solometo, ronald walsh and joan uehlinger. montefiore medical center background/case studies: our system, a pioneer aco, is a 1490-bed tertiary-care referral center dedicated to serving patients from across the new york city area and beyond. the comprising four hospitals see 93,000 hospital admissions and nearly 300,000 emergency department visits annually. we have active programs in high risk ob, stem cell transplant, solid organ transplant (heart, liver, and kidney), ct surgery, ecmo, oncology and critical care. transfusion medicine plays a key role in the support of these services. blood product spending in 2015 was approximately $15.8m. in nov. 2015, an interdisciplinary committee was created in an effort to improve patient care (by reducing blood product exposure) and reduce blood product expenditures. the vice president-sponsored multidisciplinary committee was composed of representatives of: surgery, anesthesia, blood bank, pediatrics, perfusion, cardiothoracic surgery, critical care, medicine, and emergency department. study design/method: first important step: "know your numbers"-although the committee had multiple sources of data, there was no "one report" that could display all of the pertinent information. baseline numbers were imperative to the committee's ability to effect change. a home grown one time only report revealed which services and clinicians were the highest volume users. the initial plan was to target their use with education. an initial goal was set to reduce expenditure by $1.2m. the journey continued with regular bimonthly meetingsto brainstorm strategies and monitor utilization. utilization was analyzed using a home grown crystal report "transfused patients by location". this report was further compared to utilization patterns (2014 and 2015), by "dollars spent" and "total units per patient" by the project manager using excel. key initiatives developed by the committee 1. development of evidence based transfusion triggers. 2. education on evidence based transfusion triggers across multiple campuses, specialties and resident programs 3. clinical information system (cis) "soft stops" when ordering blood products outside guidelines. rbc order set defaulting to "1" unit instead of "2" units. 4. updated guidelines posted to easy to find internal intranet spots results/finding: despite higher patient volumes and a more complicated patient mix in 2016, we were still able to reduced blood product expenditures by $933,874 when compared to 2015. conclusion: in spite of limited resources, the committee was able to effect change by capitalizing on current stakeholders fully supported by leadership and project management. cord blood pathway to reduce iatrogenic blood loss in neonatal intensive care patients tracy shachner* 1 , anna w rains 2 and christopher t clark 3 . 1 university of tennessee graduate school of medicine, 2 univeristy of tennessee medical center, 3 univeristy of tennessee graduate school of medicine background/case studies: anemia due to iatrogenic blood loss in preterm and low birth weight infants is a major contributory factor leading to red blood cell transfusion in this patient population. methods to reduce phlebotomy for laboratory testing can reduce iatrogenic anemia. at a universitybased teaching hospital, a pathway to collect cord blood samples on all newborn deliveries was established. the cord blood sample is used for initial blood bank laboratory testing on newborn patients transferred to the neonatal intensive care unit (nicu), preventing need for additional blood draw. the blood tubes are saved for 1 week post-delivery, with cost of $1.10 per delivery tray for sterile tubes. with an initial negative antibody screen on cord blood sample, no additional phlebotomy is required for blood product selection or compatibility testing in this population until four months of age. study design/method: labor and delivery data from our facility in 2016 was analyzed, and the gestational age and birth weight of all infants transferred to the nicu was collected. from this data, we were able to calculate the total blood volume of these infants using medcalc 3000 system. by using the blood volume values, and assigning a value of 1.5 ml as the minimum amount of blood that would be drawn to perform an antibody screen, we calculated the percent of an infant's blood that would have to be drawn if the cord blood pathway was not established. transfusion results/finding: in 2016, there was a total of 3,331 infants delivered at our facility. out of all the deliveries, 487 (14%) infants were transferred to the nicu. of those infants, 27% received at least one red blood cell transfusion and 7% received at least one platelet transfusion. of the 487 infants transferred to the nicu, 98 (20%) had a percentage of blood volume that would have had to be drawn for blood bank testing greater than or equal to 1% (which we considered to be significant), had the cord blood pathway not been in effect. the percentage of blood volume preserved in these infants ranged from 1.0% all the way up to 3.9%. in those 98 infants, the birth weight ranged from 400-1650 grams, and the gestational age ranged from 22 weeks to 36 weeks and 4 days. conclusion: the established cord blood pathway has proven to be a relatively cost-effective method to prevent iatrogenic blood loss secondary to blood bank testing in a population of nicu infants who are most susceptible to iatrogenic anemia. the infants that were most likely to benefit from this policy are premature infants who are low birth weight (less than 2500 grams). development of a standardized response team for massive hemorrhage events outside of an operating room setting james burner* 1 , shannon davis 2 , suzan new 2 , vaishali patel 2 and oren guttman 2 . 1 university of texas southwestern medical center, 2 ut southwestern medical center background/case studies: managing a massive transfusion protocol (mtp) in an operating room (or) is a relatively frequent occurrence with team members well trained in their specific roles. however, in the event of mtp activation outside of an or, sufficient and/or appropriately trained individuals may not be present. this can lead to a scene of confusion and chaos with potential for patient harm. study design/method: a failure mode effects analysis was performed to develop a standardized process for managing mtp outside of an or setting. with participation from anesthesia, surgery, transfusion medicine, patient safety and quality and nursing, every step of the hospital's mtp was analyzed for potential errors. the results were used to create a "code hemorrhage" team trained to respond to any massively hemorrhaging non-or patient. results/finding: code hemorrhage represents a multi-system team critical event requiring coordination of different sub-teams (primary resuscitation, surgical/interventional, transfusion services, blood preparation, equipment management, medication management, and lab requisition/monitoring). our code hemorrhage protocol utilizes critical care trained nurses from the hospital's rapid response team who play two key new coordination roles: hemorrhage coordinator and electronic medical record (emr) coordinator. their combined roles serve to reduce the cognitive load of the various teams, prevent duplication of resources/efforts during mtp and enable enhanced closed loop task performance. the hemorrhage coordinator establishes reliable 1:1 communication between the primary resuscitation team and transfusion services, and aids in multi-team on-site coordination. the emr coordinator enters all orders into the emr, sends/communicates laboratory results and ensures blood products are available to the resuscitation team. the primary resuscitation team includes a team leader (medical decision making and cardiac life-support management); a proceduralist (establishing venous and/or arterial access), event documenter (real-time documentation of actions, medications, events, etc.), medication manager (registered nurse who prepares and administers medications) and equipment technologist (managing rapid blood product infusion devices). additional secondary roles will also be assigned, such as blood product checker(s) (verifies blood product prior to transfusion) and blood bank runner (courier sent to retrieves blood product shipments). conclusion: the code hemorrhage protocol is designed to ensure timely, efficient delivery of blood products to massively bleeding patients outside of an or setting. future work will assess its overall effectiveness by comparing blood product utilization/wastage and patient outcomes before and after implementation. background/case studies: preoperative anemia affects up to 50% of surgical patients and increases the risk of red blood cell (rbc) transfusion. both preoperative anemia and perioperative rbc transfusion are associated with increased risk of adverse outcomes following surgery. preoperative treatment of anemia includes oral and intravenous (i.v.) iron and erythroid stimulating agents (esa) such as erythropoietin (epo); however, the optimal treatment strategy for preoperative anemia remains to be established. our objectives were to evaluate the efficacy and safety of esa and iron therapy based on their effects on the prevalence of rbc transfusions and adverse thrombotic events. study design/method: we searched the cochrane central register of controlled trials, medline and embase from inception to july 2016; reference lists of published guidelines, reviews and associated papers, as well as conference proceedings. no language restrictions were applied. we included randomized controlled trials in which adult patients undergoing surgery received either an esa and/or iron before surgery, versus iron or no intervention. three authors independently reviewed the studies and extracted data from included trials. risk of bias was assessed for all included studies. where applicable, we pooled risk ratios of dichotomous outcomes and mean differences of continuous outcomes across trials using randomeffects models. our primary outcome was the number of patients transfused with red blood cells. secondary outcomes included risk of mortality and other thrombovascular events (stroke, myocardial infarction, deep vein thrombosis, and pulmonary embolism). results/finding: a total of 79 randomized controlled trials (8, conclusion: amongst patients undergoing surgery, the administration of an esa in addition to oral or i.v. iron was associated with a reduction in patients requiring rbc transfusion. intravenous iron was less effective at reducing rbc transfusion. neither treatment was associated with any clear increase in risk of adverse thrombotic events. additional large prospective randomized controlled trials are required to determine the optimal management strategy for patients undergoing surgery with iron restricted anemia. evidence based blood therapeutics scott neeley* 1 and stephanie rogers 2 . 1 dignity health st joseph's medical center, 2 dignity health background/case studies: over 12 million units of packed red blood cells (prbc) are transfused annually in the united states and there is no clinical basis for as many as half of these transfusions. no randomized prospective trial has ever demonstrated a clinical benefit for transfusion in mild to moderately anemic patients and yet there is a large body of evidence which has shown that due to a variety of reasons including an immunomodulatory effect and the storage lesion, blood transfusions can cause considerable harm, including higher risk of hospital acquired bacterial infections, transfusion related acute lung injury/acute pulmonary edema, acute myocardial infarction, higher recurrence of rebleeding and higher cancer recurrence. study design/methods: a system wide goal was launched across 39 hospitals to decrease the number of prbc transfusions given to clinically stable patients with hemoglobin (hgb) levels >5 7.0 g/dl. the numerator consisted of all prbc units transfused to patients with a hgb of 7.0 g/dl or greater prior to transfusion and the denominator consisted of all prbc units transfused. exclusions included cardiac surgery, nursery, nicu, pregnancy, post-partum hemorrhage, massive transfusion protocol and transfusions in which 4 or more prbc units were transfused in one episode. data was extracted directly from the electronic medical record and hospitals received patient level detail every month for all prbc units transfused to patients with a hgb of 7.0 g/dl or higher prior to transfusion. an extensive educational campaign re: evidence-based transfusion practice was launched for physicians and nurses, including the development of a blood therapeutics toolkit, development of standardized dignity health blood therapeutics guidelines, a one day blood therapeutics advanced training symposium, on-site visits to 21 hospitals including 16 cme presentations, online physician and nursing educational videos, communication tools including infographics and "7 is the new 10" buttons, development of a patient education resource and bi-monthly webinars with various educational topics and speakers. additionally, the ehr powerplans were revised to ensure available selections for "transfusion indication" (required field) were aligned with evidence based guidelines. facilties were encouraged to develop multi-disciplinary blood therapeutics committees to review all transfusions given to patients with pre-transfusion hgb >5 7.0 g/dl on a routine basis, providing feedback to providers whose transfusions were deemed not in accordance with current evidence-based guidelines. results/findings: from fy2015 to fytd2017, there was a 26% reduction in prbc units transfused to patients with hgb >5 7.0 g/dl, starting at a baseline of 67% down to 41%. this represents an fy17 annualized savings of $9.732m, from a baseline of 82 units per 1,000 patients days down to an average 71 units and approximately 2,000 fewer units transfused per month. conclusion: blood transfusions, while life saving, should be regarded as an organ transplant and as such they carry considerable risk. transfusions to stable, non-bleeding patients with hgb levels >5 7.0 g/dl are not in accordance with evidence-based guidelines and should be avoided due to the associated potential harm. furthermore, this potential harm is dose dependent, so if the decision to transfuse is made, one unit of prbc should be transfused rather than two. three af studies (sdm5-0.258) reduced rbc units and two studies decreased the percentage of patients transfused (or5 0.700). forty-three studies showed that intravenous tranexamic acid reduced the percentage of patients (or 50.264) and rbc units transfused (sdm5-0.553). qualitative/meta-analyses were translated into recommendations by an expert panel and approved by the lmbp workgroup for reducing rbc transfusion. recommendations are: early assessment and effective am; rt, hb alerts in cpoe/cds; reduction of blood loss and af assessing the percentage of patients and rbc units transfused across cases, physicians and service areas over discrete periods of time with feedback to physicians for continuous quality improvement. conclusion: conclusion: the lmbp a-6 method led to evidence-based recommendations for reducing transfusion. critical laboratory support is needed to achieve continuous quality and patient safety. background/case studies: reducing the inappropriate use of blood products via the implementation of evidence based guidelines is a main tenet of patient blood management. the use of electronic decision support tools such as best practice alerts (bpas) to enforce red blood cell (rbc) transfusion thresholds have been shown to reduce use by informing ordering providers when or when not to transfuse. the tools in use to date have not provided a dose of rbcs to transfuse, so in fact providers can continue to over-transfusion based on the number of units of rbc given. a therapeutic hemoglobin/hematocrit (hgb/hct) targeted approach to rbc indications/ orders allows for the calculation of a dose of rbcs to achieve the desired target and could further reduce the use of rbc units. our group has developed a computer algorithm to calculate rbc dose based on patient specific data drawn from the electronic medical record (emr) that has been used in select patient populations but has not been prospectively applied to hospital wide clinical practice. this study describes our initial experience with the use of this algorithm in non-surgical rbc transfusion. study design/method: the blood utilization calculator (buc) is a mathematical formula that draws patient specific information including index hgb/ hct and calculates a dose in number of units of rbcs to transfuse in order to achieve a selected target hgb/hct. hgb/hct target based indications for rbc transfusion were designed and used as the basis for rbc order set with in the ethe buc was embedded within the emrs rbc order set to provide a recommended transfusion dose in number of units when any nonsurgical rbc indication was selected. the target hgb/hct for these indications was 7g/dl/21% or 8g/dl/24%. the number of rbc units ordered and transfused were tracked prospectively for each of the orderable indications. comparison of units transfused per month before and after the buc implementation was performed using student's t-test. results/finding: historically, the three non-surgical rbc indications represented approximately 42% of the total rbc transfused. prior to the buc the mean number of non-surgical rbc units transfused was 590 1 24 units/ month. after the first 5 months of buc activation the mean number of units was 439 1 50 units/month a reduction of 151 units/month or 26% of nonsurgical blood use (p50.003 by t-test). non-surgical rbc use now represents approximately 29% of the total rbc use hospital wide a 13% reduction. this change represents a significant cost savings in rbcs over time. conclusion: the use of target based transfusion indications and an electronic decision support algorithm to calculate a recommended transfusion dose can significantly reduce the non-surgical rbc transfusion rate providing enhanced patient blood management and potential cost savings. implementation of patient blood management at a community hospital -30 month report card richard gammon*. oneblood, inc. background/case studies: a collaboration between blood center between (bc) as consultant and three hospital (4001 beds) healthcare system (hcs) to implement a patient blood management (pbm) program was undertaken. this is a review of the first 30 months. study design/method: during year one pbm working group was established. achievements included physician engagement programs, creation of transfusion committee and providing nursing education. auditing processes were implemented with nonconformance letters sent to physicians and nurses when compliance with informed consent, transfusion tags and thresholds and discharge instructions was not achieved. in year two, it created best practice alerts (bpa) when an order did not meet transfusion threshold criteria. bpa showed first line of associated procedure, link to the full procedure, three most recent lab results (e.g., hemoglobin & hematocrit for red blood cells (rbc)) and allowed ordering physician to cancel order after review. a blood administration video was created. it was mandatory that all physicians granted privileges complete within six months. low vital sign compliance required action that included reducing requirement from five to three during transfusion and formation of working group (wg) to address knowledge and practice gaps. in year three, as historically at this hcs very few jehovah's witness patients (jwp) presented, pbm wg was involved with implementation of a bloodless medicine program. all steps of care were addressed including identifying jwp at registration, creating 115a transfusion special arm bands, forming a bloodless medicine physician group, implementing nursing bpa in the electronic medical record, creating advanced directives and marketing to the public. results/finding: the following were monitored for compliance (2q14 vs. 1q17): present and completed consents (66 vs. 94%), present and completed nursing flow sheets (19 vs. 96%), transfusion thresholds supported (73 vs. 100%), discharge instructions provided (17 vs. 86%); (3q15 vs.1q17) vital sign compliance (39% vs.71%). jwp increased from 27 to 225 (04/16-03/17). cost savings were realized by decreased utilization and implementation of bpa. (table 2016 -1q17) conclusion: pbm implementation at a hcs is a continuous and multiyear process. even with a robust program challenges such as vital sign compliance remain. improving patient outcomes in the golden-hour beatrice lebeuf*. medical city plano background/case studies: in emergency medicine, "the golden hour" refers to the critical one-hour time period following traumatic injury in which the patient has a higher likelihood of survival. nearly half of all trauma related deaths occur in the first hour after injury -half of those deaths are the result of major hemorrhaging. rapid administration of blood products is vital to the survival of these patients. we implemented bloodtrack emerge (haemonetics, braintree, ma) in our trauma emergency department (ed) as part of a quality improvement initiative to more efficiently provide group o rbcs and thawed/liquid plasma for incoming trauma patients to support ratio-based transfusions and ensure the proper handling and traceability of this regulated resource. study design/methods: we treat approximately 30-40 trauma patients monthly. an assessment of our current blood supply chain revealed a multistep, manual process that took about 8 minutes to prepare and physically transport a cooler from the blood bank to the ed. coolers of blood were provided for incoming trauma patients, whether they ended up needing transfusions or not. this practice worked to ensure available blood supplies during critical moments, but resulted in inefficiencies and unnecessary inventory tie-ups, with only 10 percent of coolers fully used. it also consumed valuable staff time as technologists typically made 20-45 trips per month from the blood bank to the ed. plus, there was no effective way to maintain traceability, control access to coolers or monitor usage. results/findings: since our november 2016 implementation, bloodtrack emerge has freed up technologists to perform important tasks, tightened traceability and inventory control procedures and contributed to the medical city plano's verification as a level 1 trauma center. rather than preparing coolers of blood in case they may be needed in emergency situations, bloodtrack emerge provides ed staff ready access to emergency units whenever they're actually needed -and frees up an estimated 6-10 hours of tech time per month during which they can perform other tasks. audio and visual alerts notify the blood bank when emergency units are removed, allowing a quick response. plus, by stocking emergency blood supplies in the ed, the blood bank isn't unnecessarily tying up group o rbc units. today, the blood bank stocks and maintains 2-4 units of group o rhd negative, 4 units of o rhd positive, and 4 units of group a thawed plasma/ liquid plasma in bloodtrack emerge. conclusion: implementing bloodtrack emerge has enabled us to more effectively provide blood products for incoming trauma patients to support ratio-based transfusions, improve staff efficiencies and proactively respond to emergency situations. background/case studies: platelets are a limited resource for which the benefits of transfusion must be weighed against the risks. in 2015, the aabb published platelet transfusion guidelines to assist providers. at our academic medical center, a computer provider order entry (cpoe) system combines institutional transfusion guidelines with a patient's most recent lab results to guide transfusion decisions. discordant information activates an "override" system, in which providers are prompted to select a prefixed indication for transfusion (e.g. count < 10 k/ml [prophylaxis]) with the option to add a free-text comment. the order is placed and data is stored for later review. study design/method: override platelet orders placed from june 2015-october 2016 were reviewed using the following data: prefixed indication, most recent platelet count, free-text comment, and ordering service/department. one of five "codes" was assigned to each order: i-indicated or ni-not indicated (based on institutional/aabb guidelines); nmi-need more information; p-protocol (e.g. liver transplant), and nic-non-indication comments (e.g. reserve for or). free-text comments were categorized and assigned one or more keywords in order to determine the common reasons for overrides. results/finding: over a 17-month period, 1,270 cpoe override platelet orders occurred. the percentages of code assignments by month are provided in table 01 below. overall, 532 (42%) were assigned as not indicated (ni). the top keywords assigned to free-text comments were "platelet count less than. . ." (325), "active bleeding" (303), "platelet count of . . ." (173), and "downtrend" (92), many with specified platelet count goals. certain platelet count goals and reasons for transfusion (e.g. "downtrend," "anticipate drop," or "per service,") are not included in institutional or aabb guidelines. of note, 618 (49%) of overrides were placed by hematology-oncology providers. conclusion: a majority of override platelet orders were determined to not be indicated based on institutional and aabb guidelines. of concern were keywords such as "downtrend" and "anticipate drop," as these are not indications for transfusion and expose patients to unnecessary transfusions. it is unclear whether trainee progression throughout the year had any effects on ordering practices and associated override patterns. this review suggests the potential benefits of provider education initiatives at all levels of experience (with particular emphasis on hematology-oncology) in order to improve blood product utilization practices. background/case studies: early diagnosis of iron deficiency anemia (ida) by clinical laboratories (cl), with effective prevention and treatment in primary care may have an impact on packed red blood cell (prbc) transfusion, as well as intravenous iron therapy and, most importantly, applying lower transfusion triggers. they all help to avoid not essential transfusions, but also promote health and wellbeing by improving iron status in the population. results are described after implementing a process to prevent ida, its early detection and treatment for years 2014-2016. study design/methods: performance measure after educational and organizational intervention. setting: public integrated healthcare system located in north africa bordering morocco, isolated by 207 km sea distance to nearest continental spain airport, with a general hospital blood transfusion service and a establishment for blood donation and component production. cl involved in anemia detection and diagnosis receives four primary care centers and hospital based samples, and shares common leadership with both blood establishments. process: guidelines for first step cl diagnosis of ida and call for attention, primary oral iron prevention and treatment in first level care, and early intravenous iron complex for inpatients (sucrose) and outpatients (carboxymaltose). transfusion was avoided for stable ida patients without active bleeding or coronary heart disease, with a safety hemoglobin (hb) threshold of 5,5g/dl. severely anemic patients were closely followed to asses hb increase and referred for etiology studies when hb> 9 g/dl. background/case studies: bedside nurses are critical in safeguarding the delivery of appropriate patient care. more recently, nurses have also begun to play an important role in patient blood management (pbm) programs at the administrative level, although to our knowledge little has been published on the influence nurses may have on transfusion practice at the bedside. the goal of this study was to evaluate the impact nurses have on patient expectations and physician ordering practice. study design/method: a short electronic survey (12 questions) was prepared to assess how often bedside nurses discussed transfusion necessity and the persons (patient or physician) with whom they discussed it with, as well as what was discussed, and what they felt were appropriate lab thresholds for transfusion. the survey was distributed to all registered nurses via email from floor leaders. responses were also solicited by hospital volunteers and lab staff with electronic tablets and included coverage of the night shift. results/finding: there were a total of 32 complete responses (16%). the nurses had a range of experience from less than one year to forty years. ninety percent stated they discussed transfusion necessity with patients, 81% with physicians, and of these, 59% reported doing so proactively before an order was placed. ninety-six percent said they would discuss transfusion to suggest their patient required a blood product; only 3% responded that they would suggest product was not needed. nursing perception of acceptable transfusion thresholds had a wider distribution, with the most commonly reported values being hemoglobin of 7-8 g/dl (56%), platelet count of 20-50,000 (38%), and inr of greater than 2.0 (69%). conclusion: this study demonstrates that nurses are willing to discuss transfusions with both patients and providers, although they appear to be most comfortable doing so in the setting of perceived transfusion necessity. the limited number of survey responses suggests a discomfort with their level of education in transfusion practice. this, along with the distribution of perceived thresholds and the reluctance to recommend against transfusions, presents an opportunity for education to further empower nurses in providing appropriate patient care within the guidelines of pbm programs. background/case studies: the use of red blood cell per 1,000 inhabitants may vary 3 folds between european countries, revealing that there may be substantial room for blood optimization strategies. patient blood management (pbm) is an evidence-based, multidisciplinary approach aiming to preserve and optimise patients' own blood in order to improve clinical outcomes. the objective of our study was to assess the effect of a nationwide pbm program on public health in portugal. study design/method: the first phase of this research project involved a group of 18 key opinion leaders (kol) in a stated preference inquiry to assess the relative value of specific pbm strategies, grouped in pbm pillars, to highlight the need for strategy prioritization in the implementation of a nationwide pbm policy. adaptive conjoint analysis techniques were used to elicit kol preferences. in the second phase a decision analysis model was used to estimate the impact of pbm implementation in the following therapeutic areas: surgery (orthopaedic, cardiac and urologic), cardiology, oncology, gastrointestinal bleeding, abnormal uterine bleeding, haemodialysis, inflammatory bowel disease and pregnancy. model inputs included effectiveness data regarding transfusion utilization, health resource consumption and mortality obtained from portuguese national health databases and literature review. the public health value of pbm implementation in portugal derives from the comparison of two scenarios: "current clinical practice" and "with pbm implementation". results/finding: kol elicited iron administration followed by restrictive transfusion of red blood cell as the most preferred pbm strategies (14.4% and 14.0%), for the remaining strategies weights varied between 7.0% and 10.6%. we estimate that 384,704 patients would be eligible for pbm strategies in one year time horizon, resulting in 594 premature death avoided (3.8% reduction) corresponding to a gain of approximately 1,500 life years and a reduction of 3,660 (6.0%) disability adjusted life years (daly) relative to the current clinical practice. a decrease of 233,141 in-hospital days is expected mainly due to a 8.4% reduction in hospital length of stay and a 37.3% reduction in 30-day readmission rate. in this population the overall transfusion rate could decrease to 4.3% from the current 8.7% (51.2% reduction) implying 17,202 blood transfusion avoided and 65,214 red blood cells units spared. conclusion: we anticipate that the implementation of a nationwide patient blood management program will represent a paramount improvement in clinical outcomes in terms of morbidity and mortality and may have a substantial public health impact while contributing a more efficient use health resources. results/finding: 237 adult liver transplants were performed during the evaluation period. preoperative hemoglobin, creatinine, meld score, spontaneous bacterial peritonitis (sbp), preoperative hemodialysis, gender, and portal vein thrombosis (pvt) gave the strongest model predicting rbc usage. if the model predicted <1250ml of rbcs, all cases with 0ml transfused were captured and only 7.8% of the time >1250ml were used. if 1250-2000ml rbcs were predicted to be transfused, >2000ml were used 25% of the time. if predicted usage was >2000ml, 53% of the time it exceeded 2000ml. conclusion: a model using specific preoperative factors can be used to predict intraoperative rbc usage. patients at risk for >1250ml of rbc transfusion can be identified with reasonable accuracy using this model at our institution. use of this model might help improve preparation and utilization of the blood bank. review of blood ordering practice for elective surgeries in a maternity hospital qi raymond fu*. kk women's and children's hospital background/case studies: pre-operative over-ordering of blood is common, resulting in waste of blood bank resources. blood units are withdrawn from the pool, leading to constraints in allocating the limited blood resources to meet the needs of other patients. the cross-match to transfusion (ct) ratio is often used in benchmarking efficient blood utilization within the hospital blood transfusion service. according to the american association of blood banks (aabb), a ct ratio of less than 2.0 is favorable, and anything above indicates over-ordering and cross-matching of blood. to achieve this, it is necessary to review pre-surgical blood ordering practice in a maternity hospital. study design/methods: data on elective surgeries requiring blood for standby was collected retrospectively over a 3 month period (jan to mar 2017). details of total blood cross-matched, issued, transfused and returned were analyzed along with the ct ratio. results/findings: during the 3 month period, there were 274 patients undergoing obstetrics and gynecology procedures requiring blood on standby. a total of 494 units of blood were requested. 154 units were crossmatched, of which 138 units were sent to the operating theatre (ot). only 33.3% of blood issued to ot were transfused (n546) while the rest were unutilized. the observed ct ratio was 3.35. conclusion: although only 31% of total blood requested was crossmatched, the ct ratio remains above the recommended guideline of !2.0, with almost 70% of cross-matched blood unutilized. there is a need to improve and standardize the blood ordering practice to achieve costeffectiveness and reduce unnecessary workload. establishing and adhering to a maximum surgical blood order schedule (msbos) could help in conserving blood and prevent over-ordering of blood. background/case studies: total knee arthroplasty (tka) is a major orthopaedic procedure with increased perioperative blood loss. this perioperative blood loss could be more significant in patients undergoing bilateral tka in a single stage. the increased blood loss in bilateral tka often requires blood transfusion which results in high post-operative morbidities. study design/methods: in this retrospective study 35 patients who received tranexamic acid (txa) (study group) and 31 patients who did not receive txa during surgery (control) were evaluated for blood loss and transfusion requirement. the study group received a single bolus dose of txa 1gm iv before tourniquet deflation on first side knee. statistical background/case studies: blood product utilization is an increasing concern for hospital systems attempting to reduce transfusion-associated risks. one strategy to optimize utilization is to employ clinical decision support in the form of alerts to clinicians ordering blood products. we investigated whether an alert targeted to a patient's transfusion indication could alter provider ordering behavior. study design/method: this retrospective, observational study over the course of seven months included the inpatient adult medicine floors and intensive care units at a large academic hospital. each time a crossmatch for packed red blood cells (prbcs) was ordered via the hospital's electronic ordering system, an indication (e.g. "hemodynamically stable with hemoglobin < 7.0 g/dl") must be selected. if the indication selected contains a threshold hemoglobin concentration, and the patient's most recent hemoglobin on record was greater than this threshold, an interruptive alert displaying the patient's hemoglobin was activated. ordering providers were then given three options: cancel the order, select a more appropriate indication from a list, or provide an explanation via free text as to why transfusion was being requested outside of approved indications. an alert encounter was defined as all activations on a patient within a six hour period without an intervening transfusion results/finding: over seven months, there were 1732 unique alert encounters. of these, 1531 (88.4%) led to a crossmatch being ordered while 201 (11.6%) led to the order being canceled. providers were more likely to cancel transfusions in response to alerts for hemodynamically stable patients with lower hemoglobin thresholds (7.0 g/dl) than for more complicated patients (bleeding, cardiovascular disease, or preoperative) with higher hemoglobin thresholds (8.0 or 9.0 g/dl background/case studies: the maximum surgical blood ordering schedule (msbos) is a list of surgical procedures performed along with a recommendation for the extent of pre-transfusion testing to be completed before the surgery begins. with improved patient data management systems it is now possible to create an msbos based on actual red blood cell (rbc) utilization data on a per-patient basis. this study investigated the transfusion patterns at 4 academic hospitals with data-derived msbos. study design/method: the 4 hospitals were in 2 groups, with one shared msbos for each group. three of these hospitals were large academic centers while one was a children's hospital. at each center the msbos recommended no pre-transfusion testing if 5% of patients had been transfused for a specific procedure in the previous year, a pre-operative type and screen (t&s) if 5-24% of the patients had been transfused, and a crossmatch of the median number of rbcs transfused if !25% of the patients had been transfused. data were collected at each center over a 1 month period between january to march 2017 and included a maximum of 400 cases per hospital during that one month to ensure equal representation between centers results/finding: between these 4 centers there were a total of 1599 cases analyzed. some of the more frequently performed surgeries included orthopedics (23% of cases), general surgery (16%) and cardiac surgery (11%). there were 1362 t&s ordered for these cases, of which 5 were positive for antibodies on the day of surgery. of all the t&s ordered, 52% were ordered in accord with the msbos recommendation, 26% were ordered when the msbos did not recommend one, and in 0.2% a t&s was not ordered when the msbos recommended one. background/case studies: peripartum blood transfusion is more common in south africa than in the usa and recent studies have demonstrated that antenatal anemia is a strong risk factor for such transfusion (odds ratio 5 6.12 for prenatal hemoglobin (hgb) 8-8.9). we therefore analyzed the etiology and characteristics of antenatal anemia according to hiv status at a large hospital with a hiv prevalence of 29% among obstetric patients. study design/method: we studied a sample of anemic (hgb<10.0 g/dl) pregnant women who were referred to an antenatal anemia clinic at a large hospital in south africa. clinical information was abstracted and blood was sent for laboratory studies. t-tests were used to compare continuous variables between groups. results/findings: a total of 301 women were enrolled, with median age 27 (interquartile range 23-32) years, median gravida 2 / para 1 and median gestational age 28 weeks. mean hgb before referral was 7.5 g/dl and most were already taking oral iron therapy. a total of 169 women were hiv positive with mean cd41 lymphocytes counts of 394 cells/ul; 29 (12%) of hiv positive subjects were on anti-retroviral therapy (art) prior to the pregnancy and 156 (92%) were on art during the current pregnancy. iron deficiency anemia was the overwhelmingly prevalent diagnosis, present in 292 (97%) of women. there was concurrent chronic disease (n52), infection (n52), vitamin b12 deficiency (n52) and antenatal hemorrhage (n56); 10 had other/unknown/missing causes of anemia. there were few pregnancy related complications. hiv positive women had higher levels of c-reactive protein but slightly lower levels of transferrin, soluble transferrin receptor and rbc folate than hiv negative women (table) . conclusion: iron deficiency is the overwhelming cause of antenatal anemia among south african pregnant women. compared to hiv-negative women, hiv-positive women had evidence of increased inflammation, relatively little differences in iron studies after early treatment with iron and lower red cell folate. a high proportion of hiv positive women were receiving art, consistent with national guidelines. future studies will examine longer-term responses to iron therapy to assess its potential in decreasing the incidence of peripartum blood transfusion. background/case studies: a 2 month old boy presented to our institution after a 1 month hospitalization in japan. he was admitted there, several weeks after his unremarkable term birth to an ab rh positive woman, with lethargy, failure to thrive, bloody mucoid stools with eosinophilia, and an elevated serum white count. he was found to be anemic and thrombocytopenic and required multiple transfusions. also, he had a diffuse, scaling, erythematous rash over his inner thighs. study design/method: initial workup was suspicous for an allergic/necrotizing enterocolitis. the patient had an elevated ldh and potassium, and concern was raised for leukemia with possible tumor lysis syndrome. a sample sent to our blood bank showed an anti-e, with a positive dat (igg and complement), and was positive for e, e, and c antigens. concern for a maternally-induced antibody was raised, as was the possibility of a red cell antigen passively transfused from blood products administered at the japanese hospital; both possibilities were excluded. further workup revealed no infection or hematologic proliferation. biopsy of his rash showed spongiotic dermatitis. his clinical course deteriorated, and he developed hepatomegaly and jaundice. a concern for wiskott-aldrich syndrome was raised, and workup showed normal immunoglobulin levels, but with elevated ige (15270 ku/l; rr: 0-2.9). anti-platelet antibodies were identified. three days after admission, testing was sent for genetic alterations of foxp3, while a japanese-speaking physician at our institution read a prior flow cytometry study showing a deficiency of foxp31 cd41 lymphocytes. the majority of these indications are seen in adults and for which a reported plasma wastage is $1.8%. fortunately in pediatrics the incidence of these indications is low despite the heterogeneity of the patient population. during the utilization review process at our primary pediatric institution, we noted a mean wastage of 8.2% over the last 5 years. with recent changes in clinical practice (liver transplants and increased trauma) and recent evidence that faster plasma improves massive transfusion protocol (mtp) outcomes, our facility decided to implement the use of thawed plasma and benchmark mtp plasma wastage. study design/method: blood utilization review revealed an increase in the overall percentage of plasma wastage from 2014 to 2016, with a peak of 11.6% (range 3.2%-11.6%). a single cause could not be readily identified prompting us to query children's hospital association (cha), as our initial external pediatric benchmarking, to determine if our wastage was comparable to other children's hospitals in addition to reviewing our "time of plasma availability" for 2016 mtps. results/finding: in 2016, mtp was activated 28 times. in 6 cases the patient did not receive any blood product and in 11 cases plasma was already available at the time of rbc allocation/issue. this left 11 cases to evaluate. the median time to plasma availability was 29 minutes (range 4 minutes -61 minutes). the mean plasma wastage for mtp activations was 32% (range 0-100%). of the 9 cha replies, 3 were using thawed plasma and their wastage was </5 5%. conclusion: increasing clinical evidence supports the prompt transfusion of plasma in the setting of mtp to decrease morbidity and mortality. our brief review suggests that implementing thawed plasma will allow a potential decrease in plasma availability of 29 minutes. our implementation began with thawing a single unit of plasma for "level 1 neuro trauma" alerts, and we are now keeping a single unit of ab plasma thawed at all times. plasma is now available at the same time as rbcs thus providing more effective and balanced blood product support while reducing wastage (preliminary estimates are 9.5 %) to a level consistent with external benchmarking. prospective evaluation of this practice is ongoing at our institution and includes collaborative clinical assessment with the trauma team. ultimately prospective, multi-institutional studies in pediatric institutions are necessary to define best clinical practice and effectively benchmark blood bank practice. background/case studies: bacterial and fungal infections remain a significant cause of morbidity and mortality in severely neutropenic patients with hematological disease receiving high-dose chemotherapy and hematopoietic stem cell transplantation (hsct). granulocyte transfusions (gtx) have been used for over 40 years, although effectiveness, indications, and both patient and donor safety remain debated, particularly in children. to contribute to this discussion we demonstrated 5 cases of gtx in pediatric patients with age from 8 months to 16 years old, neutropenic, with severe infection resistant to antibiotics and/or antifungal therapy, undergoing hsct and chemotherapy. study design/method: donors: data concerning a total of 56 granulocytes donations from 2015 to 2016 were collected from 43 health donors (21 male/ 22 female). donors had matched blood type and serology status for cytomegalovirus when the patient was negative. granulocytes were mobilized with a single dose of rhu-g-csf (filgrastim) subcutaneously, 5mcg/kg/dose (donors up to 70kg, maximum of 600mcg) and oral dexamethasone (8mg), 12h prior to apheresis. apheresis was performed using cobe spectra v r or spectra optia v r with citrate as anticoagulant. all products were irradiated with 40gy. results/finding s: granulocytes number increased 5 times from basal levels and the median of cells collected were 4.89 x10 10 (range 1.09-9.85). in general donors reported no significant adverse reactions. from 56 donations, only in 4 was reported light paresthesia during the procedure. patients: all patients were medicated prior to transfusions with diphenhydramine and acetaminophen. the product was transfused within 24 hours after collection, with efforts to transfuse as soon as possible. details about the transfusions are described on table 1 . adverse reactions for patients were minor and not frequent (4 in 66 gtx) including vomiting, hypotension and headache, which resolved without serious or lasting complications, assuming that gtx is well tolerated. conclusion: our study provides further evidence that gtx transfusions can be safely performed on pediatric patients. irina kumukova* 1 , elena kurnikova 1 and pavel trakhtman 2 . 1 russian institute for paediatric haematology, oncology and immunology, 2 instituteffor pediatric hematology, oncology and immunology background/case studies: infections cause major mortality among persons with prolonged neutropenia related to hsct and chemotherapy and among patients with granulocyte disorder. the granulocyte transfusion therapy, a logical approach in treating patients with infections, who are refractory to antibiotics. we want to demonstrate our granulocyte transfusions experience in children who have neutropenia or defects of the immune system, with severe infections study design/method: we analyzed the retrospective data for the period 04/2012-02/2016. donors underwent granulocyte mobilization with g-csf in a dose 3-5 mg/kg s.c. 12-16 hrs prior to donation; granulocytes were collected with cobe spectra, caridianbct. the analysis was performed through microsoft excel, nonparametric mann-whitney's and spearman tests results/finding: was performed 169 granulocytes collections in 153 donors (82 f, 71 m), ages 18 to 58. the mean increment of wbc after stimulation was 22,6x10 9/l (9,2-41,9 x10 9/l; f-23,2 x10 9/l; m-21.9 x10 9/l); in the age groups 18-39 years (n5121) and over 40 years (n532), the mean increment of wbc was respectively 22,2 x10 9/l (9,2-41,9x10 9/l) and 24,02x10 9/l (12,6-35 x10 9/l). the mean volume of treated blood was 566, a50,05) . the mean number of total wbc in the product was 38,43x10 9/l (13.38-77x10 9/ l; f-34,8x10 9/l, m-42,7x10 9/l). only three donors (1,96%) had apheresisrelated adverse effects the analysis included 244 granulocyte transfusions in 35 cases of infectious complications in 32 patients. the mean number of transfusions was 6.9 (1-60). each patient received transfusions from mean 5 donors (1-32); the granulocyte transfusions started at the mean 8 days (2-30) after infection; the mean dose of wbc on the transfusion was 11.6x10 8/kg (2-30.7x10 8/kg). wbc increment was able to estimate after 144 transfusions. wbc increment was recorded after 96 transfusions (66.7%); the mean increment was 1,12x10 9/l (0,01-11,38 x10 9/l). efficacy was determined by the number of patients who survived an infection episode and amounted to 80.6%. in the cases of severe sepsis, the efficacy of transfusions was 76.2%. the frequency of post-transfusion reactions was 22%, (n 5 8): febrile nonhemolytic reactions 8,3% (n 5 3); pulmonary complications 11,1% (n 5 4); the anti-hla antibodies formation 2,7% (n 5 1) conclusion: no significant differences in the mobilization of granulocytes and products' volume, between men and women, or between age groups and we did not find significant relationship between baseline wbc and their increment after stimulation. the mean total wbc in the collected product and the mean volume of treated blood from men and women were significantly different. perhaps the reason for lower cell products from women is to treat smaller blood volume. the lack of increment wbc after transfusion is not equivalent to lack of its efficacy. we found a significant association between transfused dose and increment of wbc. we deliberately did not estimate our data on the treatment of patients with sepsis and other severe infections between patients receiving and not receiving granulocyte transfusions because the need for transfusions of granulocytes indicates more severe patients, when they have infections in a neutropenia or dysfunction of the immune system, and refractory to antibiotics cp150 hemolytic disease of the newborn due to anti-rh17 keegan barry-holson* 1 and jennifer andrews 2 . 1 stanford university, dept of pathology, 2 stanford university, dept of pathology & pediatrics background/case studies: anti-rh17 is a rare antibody against a high incidence red blood cell (rbc) antigen (ag) present in people with common rh phenotypes. this ag is missing in individuals who are rh null or have rh deletion phenotypes, including d--/d--. d--individuals strongly express d and lack c, c, e, and e ags. the clinical relevance of anti-rh17 has primarily been reported in pregnant women, causing mild to fatal hemolytic disease of the newborn (hdn). we present a rare case of hdn due to anti-rh17 alloimmunization during pregnancy in a d--mother. study design/methods: an o-positive full term infant was born to an opositive g3p1 > 2 mother with a negative 1 st trimester antibody screen and no prior transfusions. she had two prior pregnancies, the first resulted in a normal term singleton, and the second resulted in a spontaneous miscarriage during the 1 st trimester. father's blood type is unknown but presumably he has rh antigens. the infant was transferred to our institution at 6 hours of life because he was found to have anemia (hemoglobin 12.0 g/dl), severe hyperbilirubinemia (total bilirubin (t bili) 9.0 mg/dl), reticulocytosis (8%) and a positive direct antiglobulin test (igg 21). he was admitted to our neonatal intensive care unit for potential need for exchange transfusion given concern for hdn. he was treated with intravenous immunoglobulin and triple phototherapy on the day of admission, temporarily blunting his hemolysis. t bili rose to a maximum of 16.7mg/dl on day 8 of life and phototherapy was restarted. his t bili subsequently stabilized and he was discharged home and followed in clinic. meanwhile, his mother donated blood given there were no compatible red blood cells available in the united states via rare donor query. nine days after discharge, he was readmitted for worsening anemia (hemoglobin 6.3 g/dl) and was given steroids and washed maternal red blood cells. he was discharged and followed in clinic for several months with ultimate resolution of his anemia and hyperbilirubinemia. results/findings: at delivery, the mother's antibody screen was positive and anti-rh17 was identified; no other alloantibodies were detected. antibody identification was performed using polyethylene glycol, low ionic strength solution and ficin enhancement. maternal serum was pan reactive against panel cells and non-reactive against d--cells. anti-rh17 sera did not react against maternal rbcs. phenotyping of the mother revealed that she was d1 c-e-c-e-. molecular testing confirmed her d--genotype; molecular beadchip test yielded no type due to low signal for e, e, v and vs ags. genotyping for rh variant and targeted genomic rhce testing failed to detect several rhce exons. father was unavailable for further testing. conclusion: we report a rare case of hdn due to anti-rh17 antibody in a d --mother. we hope to obtain further laboratory studies in maternal relatives given the rarity of this phenotype in the general population. these studies have important implications for genetic counseling for mother's sisters. management of severe autoimmune hemolytic anemia: a case report of an infant treated with manual whole blood exchange with rapid clinical improvement yunchuan delores mo* 1 , cyril jacquot 1 , valli criss 1 , philippe p pary 1 , jay greenberg 1 , naomi lc luban 1 and edward cc wong 2 . 1 children's national medical center, 2 quest diagnostics background/case studies: management of severe autoimmune hemolytic anemia (aiha) presenting with life-threatening anemia is challenging, particularly in the pediatric population. mortality rates in aiha are typically low; however, in children, the rate may be as high as 4-11%. although corticosteroids and immunomodulatory therapies are first line modalities, several case reports describe the use of manual whole blood exchange (wbex) to successfully treat aiha in older children and adults refractory to first line treatment. to our knowledge, this is the first case report in which an infant with severe aiha has been successfully treated with manual wbex in an acute care setting. study design/methods: case report format. results/findings: a 2 month-old previously healthy female patient presented to the emergency department with hemodynamic instability and a nadir hemoglobin (hb)/hematocrit (hct) of 1.6 g/dl/4.9%. wbc counts (19 x 10 9 /l) were mildly elevated and platelet counts (410 x 10 9 /l) were within normal limits. her history was notable for upper respiratory tract infection 6 days prior to the onset of anemia. laboratory studies on admission showed hyperbilirubinemia (total 7.1 mg/dl, direct 1.4 mg/dl), normal ldh (318 u/l), and undetectable haptoglobin (<7 mg/dl) indicative of ongoing hemolysis. clinical symptoms included diffuse jaundice, hemoglobinuria, lethargy, and emesis. she was admitted to the pediatric intensive care unit for further management, including right internal jugular central venous catheter placement due to poor peripheral vascular access. the patient's blood group was o, rh (d)-negative with a positive antibody screen and panel demonstrating a strong panagglutinin (3-41 reactivity) with positive autocontrol. dat was 41 positive for anti-igg and negative for c3 despite a positive cold antibody screen. the patient weighed 6.9 kg with an estimated total blood volume of 620 ml. she initially received simple transfusions totaling 20 ml/kg of least incompatible group o rh(d)-negative rbcs with no incremental response. manual wbex was then performed with 463 ml of reconstituted whole blood consisting of o, rh(d)-negative rbcs and ab fresh frozen plasma (ffp) to an hct of 40%, utilizing the central venous catheter. no adverse events took place over the course of the 2 hour exchange. her one hour post-exchange hb was 9.5 g/ dl and a subsequent antibody screen demonstrated reduced intensity of the panagglutinin (21). after initiation of steroid therapy (methylprednisolone, 2 mg/kg/day), she continued to improve clinically. one week later, the patient was discharged home with a hb of 11 g/dl. one month later, she experienced recurrent hemolysis requiring re-hospitalization, at which time she had normal igm and iga levels with markedly elevated igg levels (3168 mg/dl). at a subsequent follow-up visit 3 months after her initial presentation, her anemia had resolved and she had been completely weaned off steroids. conclusion: we demonstrate a case of severe neonatal aiha successfully treated with manual wbex. the main advantages of wbex include removal of both autologous rbcs and plasma as well as infusion of allogeneic rbcs. in this case, manual exchange transfusion avoided the need for an automated apheresis procedure requiring citrate anticoagulation. in summary, manual wbex is a potentially safe procedure that may be performed in young children with severe aiha. abstract operating room, each experienced blood-colored urine, laboratory evidence of hemolysis, and acute kidney injury. clerical and serologic investigations revealed no cause for hemolysis. mechanical hemolysis from transfusion rate, catheter gauge, or a recently introduced one-way valve was considered. study design/methods: in vitro simulated transfusions were performed via syringe. measurements included hematocrit (hct), free hemoglobin, and visual hemolysis index. washed and unwashed red blood cells (rbcs) were tested with or without a one-way valve, using a 24 or 16 gauge (g) intravenous (iv) catheter. each one-way valve was used to test three identical samples. constant pressure was applied manually (rapidly, 1.431/-0.49 ml/ second) or with a mechanical syringe pump (slowly, 2 ml/min). a subset of the manual transfusions was timed. control samples for baseline measurements were collected by gravity drip, without passing through the one-way valve or catheter. results/findings: the one-way valve increased hemolysis markedly during rapid transfusion using both catheters as well as both washed and unwashed rbcs (see table) . with the 24g catheter, the mean change in hct was -3.531/-0.69% with the one-way valve and 0.221/-0.13% without (p<0.00001). comparing the one-way valves tested, differences in hemolysis were observed (change in hct; p<0.0001). during rapid manual transfusion with a 24g catheter and unwashed rbcs, hemolysis was greater for samples that took longer to transfuse 4.5ml when using a one-way valve (change in hct versus time: r5-0.75, p<0.0001) compared to a significantly different (p50.0085) slight increase in hemolysis for samples that took less time to transfuse 4.5ml when not using a one-way valve (change in hct versus time: r50.58, p50.23). correlations between time and hemolysis were similar, but insignificant using 24g with washed rbcs and the 16g iv catheter. conclusion: mechanical hemolysis should be considered when investigating possible hemolytic transfusion reactions, especially with high rates of transfusion and use of a one-way valve. during rapid manual transfusion with the one-way valve, greater resistance was associated with increased hemolysis. background/case studies: gerbich (ge) antigens expressed on glycophorin c are present in 99.9% of the population. ge antibodies cause delayed hemolytic transfusion reactions and hemolytic disease of the fetus and newborn (hdfn). ge antibodies also suppress erythropoiesis resulting in late-onset anemia. we report a case of hdfn due to anti-ge3. study design/methods: a woman of paraguayan origin with prior terminated pregnancies presented at 24 weeks gestation with passive anti-d and an anti-ge3 titer of 256. she was d-and ge:-2,-3, 4 by antigen typing. her obstetrician scheduled maternal blood collection near her due date for possible neonatal transfusion, but the woman went into labor at 37 weeks. cord blood was dat positive for igg; the eluate confirmed anti-d and anti-ge3. the birth hemoglobin (hgb) was 12.6 g/dl, reticulocyte (retic) was 8.6%, bilirubin (bili) was 2.8 mg/dl; the infant was discharged. on day 7 of life, the infant was referred to pediatric hematology for lethargy and poor feeding, with hgb 7.6 g/dl, retic 2.6%, and bili 6.6 mg/dl. ge3-blood was not available from the blood center or rare donor registry. the mother was b rh-and baby was b rh1. obstetrics had to authorize maternal blood donation due to her hgb of 10.9 g/dl. maternal blood collection and rbc washing was expedited and the infant received 40ml of maternal rbcs within 24 hours, at which time his hgb was 6.1 g/dl. post-transfusion hgb was 10.8 g/dl. one week later, the infant was symptomatic with hgb 7.1 g/dl, retic 1.0%, bili 2.1 mg/dl. a 2 nd aliquot of 60ml washed maternal cells was transfused. two weeks thereafter, the infant had hgb 7.8 g/dl, retic 0.7%, anti-ge3 titer 8, and needed another transfusion. the maternal blood stored for just 3 weeks had hemolyzed necessitating a 2nd maternal donation for baby's 3 rd transfusion. at 6 weeks, the infant's anti-ge3 titer was 2, hgb 9.2 g/dl, retic 1.7%; no transfusion was necessary. at 8 weeks of life, hgb was 10.2 g/dl, retic was 3.3%, and the baby was thriving. results/findings: serologic studies at the hospital and reference blood center confirmed the antibodies and risk of anti-ge3 hdfn. molecular analysis revealed that the mother was homozygous ge3-negative ge*01.-03, the father had homozygous wild type ge*01, and the infant was heterozygous ge*01/ge*01.-03. conclusion: the infant had hdfn due to antibodies to the high prevalence ge3 antigen. the continued need for transfusion was consistent with hemolysis and suppression of erythrocyte production caused by anti-ge3. hemolysis of stored maternal blood was consistent with the absence of glycophorin c. this case demonstrates that cooperative multidisciplinary care among the blood bank, donor center, obstetrics, and hematology in a rare case of hdfn resulted in a successful neonatal outcome. background/case studies: patient blood management is a collaborative approach to optimize transfusion therapies to improve patient outcomes. in pediatrics, blood management is not 'one size fits all' given the paucity of clinical trials to guide evidence-based practice. in addition, pediatric care encompasses a very heterogeneous patient population such that applying one set of guidelines is difficult. because there are no standard, evidence-based clinical best practices regarding blood product usage in all children, unnecessary variation is occurring at our institution. we designed a robust analytics process to study baseline clinical practice and examine blood product usage, and plan to target the three pediatric sub-specialties with highest usage to establish standards in order to decrease variation/unnecessary transfusions. study design/methods: a data base encompassing all admissions and outpatient visits to a large, tertiary care academic children's and women's hospital was established, and included all relevant patient demographics, diagnostic and procedural codes, attending physician and specialty for each visit/admission, relevant hematology/coagulation laboratory results and blood product orders. we focused on rbc orders given the tripicu randomized clinical trial results (1) supporting a hemoglobin trigger of 7 g/dl in stable critically ill children and ffp since anecdotally we noted many children receiving this product for only minimally elevated international normalized ratio (inr) values without bleeding. results/findings: in 2016, 14, 247 rbc orders occurred and the top three patient groups were: 34% in congenital heart disease patients, 25% in hematology/oncology patients and 14% in neonates in the neonatal intensive care unit (nicu). average hemoglobin of every patient was 9.85 g/dl as measured in the 72 hours prior to rbc order placement. in 2016, 3105 ffp orders occurred and the top three patient groups were: 46% in neonates in the nicu, 28% in congenital heart disease patients and 13% in pediatric intensive care patients. average inr of every patient was 2.09 as measured in the 72 hours prior to ffp order placement. conclusion: we have designed a robust data base that is continually updated for children in a large, tertiary care academic children's hospital. this serves as an important benchmark in pediatric blood utilization, and we plan to leverage usage patterns to make relevant practice changes in the care of children with a heterogeneous set of illnesses. background/case studies: bacterial contamination of plts remains an ongoing threat to transfusion recipients. recently, a psoralen-based pr technology that reduces the replication potential of pathogens in stored plts was fda approved. we describe our approach to phasing pr-plts into our inventory, including preliminary results of an ongoing qa study of neonatal and pediatric (peds) recipients of pr-plts. study design/methods: before the arrival of pr-plt, we undertook an educational campaign for hospital administrators, it staff, laboratory staff, clerical/clinical aides, nurses, and physicians. we also contacted risk management and the hospital ethics committee. phototherapy devices used at our hospital were confirmed to be compatible with the psoralen-based pr-plt product. shortly following the arrival of pr-plt, we introduced day 5 bacterial "safety measure" testing of our conventional (c-plt) supply. a peds qa study monitored plt utilization and adverse transfusion event reporting relating to both pr-and c-plt transfusions. this study evaluated neonates (0-4 months of age), infants (>4-12 months of age) and children (>12 months-18 years of age) who received at least one transfusion of pr-plts. results/findings: risk management and the ethics committee agreed that both pr-plts and bacteria tested c-plts would be the hospital standard of care. pr-plts were phased in and transfused to patients based on abo compatibility and expiration date, per routine, without regard for patient age or medical condition. after 4 months, pr-plt represented 30% of our platelet inventory (average daily plt inventory: 45 units). we encountered no complications with the pr platelet phase-in, either from a clinical, informatics or logistical perspective. due to the dual inventory, many peds patients in all age groups were transfused with both pr-and c-plts (table) . two potential transfusion reactions (trs) were reported over the study period in teenage recipients, one associated with a c-plt and the other with a pr-plt. in both cases, the symptoms were ultimately attributed to an underlying medical condition. no rashes were observed among 16 transfused neonates (0-4 m) who received any pr-plts and phototherapy. background/case studies: packed red blood cell (prbc) transfusions are believed to improve oxygen delivery particularly in vulnerable patients such as neonates and children. however, evidence shows that hemoglobin (hgb) in prbcs has increased oxygen affinity and thus reduced oxygen delivery to tissues due to decreased 2,3 dpg levels. standardization of prbc transfusion practices in this population and the scientific evidence on which current practice is based is limited. additionally, due to small transfusion volumes, infants may be exposed to multiple blood donors, increasing their potential for adverse events. study design/method: medical records of 60 pediatric patients receiving prbc transfusion over a 12 month period were retrospectively reviewed. a total of 44 patients were identified as receiving allogeneic prbc transfusion. 16 patients who received autologous blood (cell salvage) were excluded. patient characteristics, length of stay, prbc transfusion volume, pre-and post-transfusion hgb, and adverse events were collected. results/finding: the average pre-transfusion hgb was 10.6 g/dl with post-transfusion hgb rising to 14.5 g/dl. the mean prbc volume transfused was 46.3 ml using a dose of 15ml/kg for all patients. complications noted were; volume overload, thrombosis, fever/infection, hemolysis, necrotizing enterocolitis (nec), and death (table) . conclusion: evidence based transfusion guidelines are lacking in neonates and infants. a typical dose of 10-15 ml/kg in a 2 kg patient, for instance, would translate into 3 full prbc units (about 1000 ml) in an average size adult. the current standard dose of 10-15 ml/kg yields very high increases in hgb and may put these patients at risk of adverse outcomes, especially thrombosis due to increased blood viscosity. additionally, many of these patients received volume reduced products which delivers a higher hgb concentration per transfusion. dosing should be based on goal hgb and patient condition rather than weight based, though the hematocrit level at which the benefits outweigh the risks remains unclear. pneumoniae has rarely been associated with warm autoimmune hemolytic anemia, with only 3 case reports suggesting this association. however, each of these cases is confounded by other findings in addition to a mycoplasma infection. we describe a unique case in which a pediatric patient has clear evidence of severe hemolysis, a very strongly reactive warm autoantibody, and clinical and laboratory evidence of a mycoplasma infection without a detectable cold agglutinin. study design/methods: the patient is a 7month-old, previously healthy female infant who presented to the hospital with a 1-week history of fever, fatigue, decreased appetite, and pallor. she was only treated with acetaminophen. she also developed clear rhinorrhea the day before hospital admission. at the time of her admission, laboratory testing (outside hospital) revealed a hemoglobin and hematocrit of 2.7 g/dl and 9.1%, respectively, platelets of 635,000, and a reticulocyte count of 10.3%. all other elements of the complete blood count were within the normal reference range for age. a complete metabolic panel revealed no abnormalities except for a total bilirubin of 4.9 mg/dl with a direct fraction of 0.43 mg/dl. a filmarray respiratory panel (biofire diagnostics; salt lake city, ut) detected mycoplasma pneumoniae, while all other pathogens (19 total) were non-detectable. the patient was started on a 5-day course of azithromycin (zithromax). results/findings: prior to rbc transfusion, blood bank evaluation revealed that the patient was o-positive and had a stronglyreactive antibody screen. further testing demonstrated an antibody reactive with all reagent red blood cells. the dat was strongly reactive for igg but very weakly reactive for c3. an eluate was reactive with all reagent red cells tested. finally, a cold agglutinin study was negative with undiluted serum. in addition to starting azithromycin, the patient was given iv methylprednisolone. during her 8-day hospital course, the patient received 2 rbc transfusions on the day of admission and several rbc transfusions thereafter (see table 1 ). despite transfusion, her hemolytic process persisted, so she was infused with a dose of iv immunoglobulin on hospital day 6. her hemoglobin rose to 8.4 g/dl on hospital day 7 and increased to 9.5 g/dl on hospital day 8. at that time, the patient was discharged from the hospital with instructions to wean her oral steroid dose over the next 2 weeks. she was followed closely by the hematology clinic and was found to have a stable hemoglobin (up to 11.2 g/dl on day 57 after her hospital admission) with no recurrence of her hemolytic process. conclusion: m. pneumoniae infection is a typical cause of cad and has only rarely been associated with warm autoimmune hemolytic anemia. our case demonstrates clear evidence of severe warm autoimmune hemolysis in a previously healthy infant. with the increasing use of multiplex respiratory viral and bacterial pathogen detection systems, the once rare phenomenon of a m. pneumoniae infection associated with warm autoimmune hemolytic anemia may become a more recognized entity. ) and may serve to more reliably reflect when the neonates at risk for hyperbilirubinemia. the difficulty in eliminating the cord blood testing is the neonatologists' reliance of using abo incompatibility as part of the neonates risk assessment rather than using the point of care bilirubin testing. currently the ts requires all positive dat tests to be communicated to the nursing staff immediately. given that the dat strength positively correlates with the percentage of neonates diagnosed with hyperbilirubinemia, the ts staff may also consider notifying nursing staff only for those patients whose dat is 3 or 41. platelet and leukocyte immunohematology, testing and genetics table 1 . of 53 pairs, 7 pairs were complete match (2/2), 26 pairs were partial match (1/2), 20 pairs were complete mismatch (0/2). the matching rate of hla-dpb1 in our study is 13%. conclusion: the matching rate of hla-dpb1 in 10/10 hla matched unrelated hematopoietic stem cell transplantation is low and the gene frequency of hla-dpb1 in unrelated hematopoietic stem cell transplantation was obtained ,which will help to study on the relationship between hla-dpb1 and unrelated hematopoietic stem cell transplantation. this work was sponsored by national science foundation of china (81401732) background/case studies: thrombotic thrombocytopenic purpura (ttp) is caused by severely reduced activity of the von willebrand factor-cleaving protease adamts13. therapeutic plasma exchange (tpe) as well as immunosuppression minimize the morbidity and potential mortality of this presentation. absolute immature platelet counts (a-ipc) have been shown to help diagnose and follow ttp patients' responses to therapy. we report the case of a man with relapsing ttp, low adamts13 with high inhibitor, treated with mycophenolate mofetil in which a-ipc-indicated an unexpected response to therapy. study design/method: a 56 year old male with a 7-year history of ttp, presented with status epilepticus complicated by acute respiratory failure admitted with suspicion for relapsing ttp. patient had been treated in prior admissions with tpe, prednisolone, rituximab, and cyclophosphamide with clinical improvement. he was on mycophenolate mofetil maintenance therapy which he last received just prior to day of admission due to consistently low platelet counts, adamts13 <5% and inhibitor of 3.6. on day of admission platelet count was 95 x 10 9 /l which decreased within five days to 14 x 10 9 /l leading to initiation of daily tpe along with mycophenolate mofetil discontinuation just prior to tpe start. immature platelet fraction (%-ipf) and calculated a-ipc (%-ipf x platelet count) were obtained with daily pre-tpe cbc. a-ipc ratio was calculated from baseline. abstract results/finding: a-ipc and platelet count were 1 x 10 9 /l and 14 x 10 9 /l respectively. counts improved rapidly post-tpe initiation and after one tpe his a-ipc tripled to 3.2 x 10 9 /l achieving the ratio of 3 previously shown to be diagnostic of ttp. on day 5 his a-ipc and platelet counts had improved to 7.5 x 10 9 /l and 218 x 10 9 /l respectively. absence of anti-pf4 antibodies ruled out heparin-induced thrombocytopenia at this time. on day 6 he had an unexpected decrease in both a-ipc and platelet count to 4.8 x 10 9 /l and 132 x 10 9 /l respectively, worsening by day 8 to 1.7 x 10 9 /l and 40 x 10 9 /l respectively despite daily tpe. patient received 25 additional tpes that failed to improve a-ipc or platelets which on day 32 were 0.4 x 10 9 /l and 13 x 10 9 /l respectively. a-ipc had remained at this level for 16 days suggesting that the observed decrease was irreversible. adamts13 activity remained <5% low with a high inhibitor. patient's clinical condition continued to deteriorate and family placed patient on comfort care. conclusion: ttp patients have low a-ipc and plt counts at presentation, with the former improving first post-tpe initiation. despite appropriate therapy leading to early improvement of platelet count, patient's counts declined rapidly leading to suspicion for platelet production suppression as indicated by the sustained very low a-ipc. in the setting of ttp, or relapsing ttp use of immunosuppression should be closely followed and a-ipc may aid in establishing early if therapy is affecting platelet production. application of luminex bead technology to detect hpa-1a, hpa-3a, and hpa-5a antibodies su-dan tao*, ying liu, yan-min he, ji he and fa-ming zhu. blood center of zhejiang province, key laboratory of blood safety research, ministry of health background/case studies: detection of antibodies against human platelet antigens (hpas) is crucial for patients' refractory to platelet transfusion therapy. in the text, luminex bead coupled with anti-gpiib/iiia and anti-gpia/iia monoclonal antibody was implied to detect hpa-1a, hpa-3a, and hpa-5a antibodies, and the sensitivity of luminex bead technology was compared with monoclonal antibody immobilization of platelet antigens (maipa) assay. study design/method: monoclonal antibodies p2 and gi9, specific for platelet glycoproteins gpiib/iiia and gpia/iia, were separately coupled to luminex xmap beads. four standard sera, containing anti-hpa-1a, anti-hpa-3a, anti-hpa-5a and anti-hpa-5b respectively, were bought from nibsc; three negative sera without hpa antibodies were prepared from ab type blood donors. platelets (containing hpa-1aa, hpa-3ab and hpa-5aa) were collected and reacted with anti-hpa-1a, anti-hpa-3a, anti-hpa-5a and anti-hpa-5b standard sera respectively, then the antigen-antibody reaction complexes were lysed and the lysates were incubated with luminex beads to specifically capture antigen-antibody complexes via the epitopes on platelet glycoproteins. the beads-antigen-antibody complexes were then subjected to flow cytometric analysis on a luminex100. the hpa-1a serum was diluted to 10 serial dilutions (from neat to 1/502) to test the sensitivities of maipa and luminex beads assay. the two methods were then used to test five blinded samples which were collected from fmait patients. results/finding: luminex bead technology showed that the mfi values of hpa-1a, hpa-3a, hpa-5a standard sera samples reacted with the coupled beads were significantly higher than the negative controls ( .08 vs 37.05), which implied that the luminex bead technology could specifically identify negative and positive sera of anti-hpa-1a, anti-hpa-3a, anti-hpa-5a. furthermore, because the platelet was hpa-5aa, the hpa-5b serum did not react with the coupled beads with mfi was comparable to negative control (286.59 vs 127.25). the sera were re-tested by maipa and the results of which were comparable to luminex bead technology, illustrating that detecting hpa antibodies by luminex beads technology was successful. the sensitivity of luminex bead assay and maipa to detect anti-hpa-1a was 1/128 (0.78iu/ml) and 1/64 (1.56iu/ml), respectively. no cross-reactivity was observed with the samples containing hla, abo or other platelet antibodies. all results of five blinded samples tested by luminex assay showed that four sera were positive for gpiib/iiia antibodies which were consistent with maipa results. conclusion: the luminex beads coupled with gpiib/iiia and gpia/iia monoclonal antibodies could be successfully used to detect hpa-1a, hpa-3a and hpa-5a antibodies via the epitopes on platelet glycoproteins. the sensitivity of luminex technology was higher than maipa technology. (ahus) is a thrombotic microangiopathy (tma) characterized by the triad of microangiopathic hemolytic anemia, thrombocytopenia and renal failure in the absence of infectious toxin. the literature suggests the presence of pathogenic mutations in complement proteins in 50% of cases of ahus. there is a lack of well-defined recommendations regarding testing for genetic ahus. complement pathway mutation analysis is an expensive test so appropriate utilization is crucial to prevent undue health care costs. we reviewed the indications for genetic testing to understand physician ordering practice and determine the frequency of pathogenic mutations in the population. study design/method: we performed a retrospective review of all cases referred for complement pathway mutation analysis to a national reference laboratory from 1 january 2014 to 31 december 2016. clinical history was solicited by genetic counselors. cases were classified by the authors as primary ahus (tma and renal failure without identifiable cause), secondary tma (tma and renal failure with identifiable cause previously associated with tma) or non-tma. the test panel identified variants in complement proteins (cfh, cfi, mcp, factor b, c3, c4bp, thbd, dgke, cfhr3, cfhr1, cfhr4 and cfhr5) that were classified as vus (variances of uncertain significance), pathogenic or benign by the american college of medical genetics. chi square analysis/fishers' exact test was used to determine differences in proportion of patients with pathogenic mutations and primary ahus versus secondary tma. independent sample t-test was used to compare differences in continuous variables between primary ahus and secondary tma. results/finding: of 134 patients tested, pathogenic mutations were detected in 13% (18/134) and vus in 35% (47/134). 20% (27/134) of patients did not fulfill criteria for tma; no pathogenic mutations were found in this group and 9 (33%) had vus. 31% (42/134) of patients had primary ahus; of these, 28% (12/42) had pathogenic mutations and 40% (17/42) had vus. 48% (65/134) of patients had secondary tma; of these, 9% (6/65) had pathogenic mutations and 32% (21/65) had vus. in patients with pathogenic mutations, 39% (7/18) were children, 22.5% (4/18) had a positive family history of ahus and 28% (5/18) had recurrent disease. patients with primary ahus had a significantly lower age at presentation (22 6 18 vs. 33 6 20 yrs; p-value: 0.005) and a higher proportion of pathogenic mutations (28% vs. 9% p-value: 0.009) compared to patients with secondary tma. gender distribution, hemoglobin nadir and serum creatinine levels were similar between the two groups. conclusion: we found a lower frequency of patients with pathogenic mutations compared to reported literature. our data suggests that patients with secondary tma should be carefully evaluated prior to ordering genetic testing and those without tma should not undergo this test. counting of platelets in platelet concentrates on hematology analyzers pentraxl80 and sysmex xn9000 compared with a flow cytometric method farshid ezligini 1 , kjersti roen eriksen 1 , annette vetlesen 1 , thomas larsen titze 1 and geir hetland* 1,2 . 1 oslo university hospital, 2 university of oslo background/case studies: hematology analyzers are made for counting of whole blood samples but are often used for quality control of blood components such as platelet (plt) concentrates (pcs). a flow cytometric method for counting of plt in pcs has been developed as validation tool (van der meer et al, transfusion 2012). therefore, it is pertinent to evaluate plt counting in bcs on hematology analyzers with this validation method in a flow cytometer. study design/methods: samples from ten apheresis pcs and 33 buffy coat-derived pcs were subjected to plt counting on hematology analyzers pentraxl80 (horiba abx, montpelier, france) and xn9000 sysmex toa (kobe, japan) (both impedance score), and additionally, diluted and stained with anti-cd41a fitc in truecount tubes (bd biosciences)(internal bead standard) for measuring in a gallios flow cytometer (beckman coulter, indianapolis in, usa). results were analyzed by paired samples test and shown in bland-altmann plots. results/findings: mean plt values x10 9 /l 6 sd were 819 6118, (<) 1106 6137, (<) and 1195 6176 for counting by sysmex toa, pentraxl80, and the gallios flow cytometer, respectively. sysmex count was the very lowest 129a transfusion 2017 vol. 57 supplement s3 abstract (31.4% less than for flow cytometry), but all plt counts were significantly different (p<0.001), although least so (7.4%) between pentra and flow cytometry. conclusion: as validated by the flow cytometric method, pentraxl80 seems suitable for routine quality control of pcs both because of the small difference and lower counts compared with flow cytometric method, which is too cumbersome in a routine setting. the much lower plt count on sysmex may reflect its optimization for plt counting in whole blood rather than in pcs. fast, precise & easy hpa typing with real-time pcr jonathan downing 1 , arishma lata 1 , roland russnak 2 , zachary antovich 2 , heather dunckley 1 and thierry viard* 2 . 1 new zealand blood service, 2 linkage biosciences background/case studies: the interaction of membrane-bound plateletspecific glycoproteins with the extracellular matrix plays a significant role in hemostasis. human platelet antigens (hpa) found within these glycoproteins can stimulate production of antibodies in recipients of transfused platelets or in fetus of mothers with incompatible hpa. thus, platelet incompatibility is associated with various forms of thrombocytopenia, posttransfusion purpura and other blood disorders. the new zealand blood service performs hpa typing on a pool of platelet donors to provide compatible transfusions where the need arises. the molecular basis of most hpas has been characterized as generally caused by a single-nucleotide polymorphism (snp). hpa typing has typically been performed using pcr-ssp, a method that utilizes time-consuming post pcr analysis steps. the aim of this study was to evaluate the use of real-time pcr-based techniques in a transfusion laboratory setting. study design/method: we evaluated a commercially available solution which consists of 24 reactions that identify both variants of 12 relevant snps located within hpa genes (hpa-1 through hpa-11, and hpa 15). genomic dna purified from 48 blood samples, previously genotyped for hpa-1,-2,-3,-4,-5 and -15 by our in house pcr-ssp method were used in this study as validation samples. results/finding: results of the validation samples were 100% concordant with typing obtained by pcr-ssp. the real-time pcr approach overcomes the major challenges of hpa molecular typing by providing an automated solution resulting in increased laboratory productivity and decreased turn-around time. the analysis is facilitated by a software which generates the results. with less than 10 minutes of hands-on set-up and no further operator intervention with the reagents, complete molecular genotyping results are provided in approximately 90 minutes. further, since amplified products are never handled, the risk of laboratory contamination is significantly reduced. the real-time pcr approach with automated analysis was implemented by the new zealand blood service tissue typing laboratory in late 2016 and to date has tested 749 dna samples from 400 blood donors (349 donors were tested in duplicate). concordance between the sample replicates was 100%. there were 24 occasions where the assay had to be repeated, giving a repeat rate of 3.2%. occasionally a reaction peak was insufficient to trigger the software automatic allele call and a manual interpretation was required. this occurred most commonly with the hpa-3 (4.7%) and hpa-5 (1.2%) assays. conclusion: real-time pcr with automated analysis provides an effective, robust an accurate method for molecular hpa genotyping. with its minimal hands-on time workflow, it is also very easy to implement and offers a cost effective alternative to classical methods used in a transfusion laboratory setting. genetic variation of cd36 antigen deficiency expression in jiangsu chinese han population qing chen* 1 , jianyu xiao 1 and chengyin huang 2 . 1 jiangsu province blood center, 2 jiangsu province blood center background/case studies: cd36 has been implicated in the platelet refractoriness, neonatal alloimmune thrombocytopenia, and posttransfusion purpura, especially in the non-caucasian. cd36 deficiency varies widely among different ethnic populations, with the frequency of 3-11% in asians and 2.4% of african americans, respectively. however, there is little information on the molecular basis of individuals with cd36 deficiency in jiangsu chinese han population. study design/method: to investigate platelet cd36 expression levels and to determine the molecular basis of cd36 deficiency on the platelet surface of the han population in jiangsu region. cd36 expression levels on platelets were detected by flow cytometry among 243 blood donors in jiangsu region. donors without cd36 antigen expression on their platelet surface were further to be determined the expression of cd36 antigen on their peripheral blood monocyte cells. the coding exons of cd36 gene and adjacent introns were amplified and sequenced in cd36 deficient individuals. results/finding: among these 243 blood donors, cd36-deficient and cd36-expression individuals were 2.47% (6/243) and 97.53% (237/243), respectively. the frequencies of type i and type ii cd36 deficiency among the study population were 0.41% (1/243) and 2.06 % (5/243), respectively. among 237 individual with platelet cd36 expression, according to mean fluorescence intensity (mfi) value, 45, 141 and 51 individuals showed low, moderate and high expression levels of cd36, respectively, and their mfis were 1725.9 6 343.6, 3876.1 6 788.5 and 8431.6 6 529.9 (p<0.05), respectively. the type i cd36 deficiency individual were heterozygous for 1200-13a>g and 430-14c>g, respectively. among type ii cd36 deficiency individuals, two harbored a t insertion at position 560 in exon 6 which caused frameshift at codon 187; one has a t>c exchange at position 538 in exon 6 which resulted in a tryptophan to arginine substitution at codon 180; one has a a insertion before the 17th bp of the start codon atg in the promoter region; one were heterozygous for 748 1 2t>c and 1006 1 2t>g, respectively. conclusion: platelet cd36 surface expression levels were diversified in the jiangsu chinese han population. the frequency of the type ii cd36 deficiency was higher than that in type i. the study findings indicated that the frequency of cd36 deficiency in the chinese population is slightly lower than that in other asian countries. background/case studies: cd36-deficient phenotype can be immunized by pregnancy or transfusion, and involved in neonatal alloimmune thrombocytopenia, platelet transfusion refractoriness and other disorders. the frequency of platelet cd36-deficient individuals widely varies among ethnic groups, with 3% to 11% in japanese, 8% in sub-saharan africans, 2.4% in african americans, and 0.3% in caucasians. although some studies of cd36 deficiency are focused on the asian populations, relatively little information has been reported in the chinese population. here we investigated the cd36 expression on platelets in large samples of the eastern chinese donors. study design/methods: peripheral blood samples were collected from 1282 unrelated platelet-apheresis donors in the eastern china. the expression of cd36 antigen on platelets was determined by flow cytometry using fluorescein conjugated monoclonal antibodies (fitc-anti-cd36 and peanti-cd41). the isotype control (fitc-mouse igg) was also analyzed to calculate a reference range of cd36-nagtive phenotype. for those donors with cd36-negative platelets, cd36 antigen expression on monocytes was analyzed further to distinguish between cd36 type i and type ii deficiency. flow cytometric parameters were statistically analyzed by mann-whitney test. the work was supported by national natural science foundation of china (81570170) and zhejiang high-level innovative health talents. results/findings: the mfi (mean fluorescence intensity) of platelet cd36 in all 1282 samples showed a continuous distribution profile, and no obvious fluorescence-gap could be utilized to distinguish negative from positive phenotype. on account of this limitation, we classified the cd36 phenotypes using the (mean 1 3sd) of the background mfi observed in isotype controls. forty-three samples were detected as cd36 deficiency on platelet, in which one sample was cd36 negative both on platelet and monocyte. the frequency of cd36 type i and type ii deficiency in the eastern chinese donors was 0.08% and 3.3%, respectively. the average mfi of cd36 deficiency samples was significantly lower than cd36 positive samples (15.2 6 7.9 vs 79.8 6 37.8, p< 0.0001). conclusion: the frequency of platelet cd36 deficiency in the eastern chinese donors was close to japanese and african americans. it means that the possibility of cd36 antibody occurred by pregnancy and transfusion in this population is existed. it is useful to find and register cd36-deficient donors by large-samples screening for potential immune thrombocytopenia patients with cd36 antibody. background/case studies: cd36 (gpiv, chromosome 7q11.2) is an 88 kda glycoprotein expressed on multiple cell types including platelets (plts), monocytes (mono), & erythroblasts. although rare among whites, cd36 deficiency (cd36-n) is observed in 3-10% of africans (t1264g) & is classified as either type i (cd36-n plt, cd36-n mono) or type ii (cd36-n plt, cd361 mono). an acquired type ii cd36-n phenotype can also be observed in the setting of myelodysplastic syndrome (mds). type 1 cd36-n individuals can develop anti-cd36 alloantibodies with plt refractoriness & neonatal alloimmune thrombocytopenia. we report a case of profound plt refractoriness caused by anti-cd36 in a patient with newly diagnosed mds. study design/method: hla antibody testing was performed with a commercial bead-based fluorescent assay. cd36 phenotyping (plt, mono) of patient & family members was performed by flow cytometry (fc). cd36 staining of bone marrow was performed by immunohistochemistry. plt crossmatching (plt-xm) was performed by the american red cross. pltspecific alloantibody testing & cd36 dna sequencing were performed at a commercial reference laboratory. results/finding: the patient was an 80 year-old, group o1 african-american male who presented with blurry vision & lightheadedness. complete blood count findings were significant for hemoglobin 4.4 g/dl & plt count 5k/ml. bone marrow biopsy & cytogenetic analysis revealed multilineage dysplasia, 5-10% blasts & a complex karyotype with del(7)(q22q34) consistent with mds. plt refractory work-up was initiated after repeated plt transfusion failures with corrected count increments (ccis) < 5. hla antibody testing was negative (class i panel reactive antibody (pra)50%). the patient was plt-xm-incompatible with most donors (10/14). a trial of 4 group o, plt-xm-compatible plts was unsuccessful (cci 1). subsequent testing for plt-specific alloantibodies identified anti-cd36. fc-phenotyping showed no cd36 on patient's mono or plt, consistent with type i cd36-n. preliminary dna results show that the patient is heterozygous for t1264g. because cd36-n apheresis plt were unavailable from blood suppliers, the patient's 3 children & grandson were screened as possible donors: all showed normal cd36 expression on plts. trial of eltrombopag & romiplostim was attempted with no improvement in plt count. repeat hla antibody testing (day 16) demonstrated new class i alloantibodies (pra 5 55%) in response to transfusion (21 apheresis plts, 5 rbcs). given his plt refractoriness & poor prognosis, the patient opted for hospice. conclusion: we describe a patient with cd36-n & severe plt refractoriness in the setting of new mds, and 7q-chromosomal abnormalities. the absence of cd36 on plt & mono support congenital type 1 cd36-n although a contribution by the patient's underlying mds cannot be excluded. rapid platelet donor classification: hla & hpa profiles by "leansequencing" without dna purification dipika patel 1 , kristopher fernandez* 1 , eric senaldi 2 , pascal george 2 , michael seul 1 and ghazala hashmi 1 . 1 biomolecular analytics, 2 central jersey blood center background/case studies: prophylactic platelet transfusion is the standard of care for managing thrombocytopenia. in the emerging paradigm of personalized medicine, the selection of cellular products in accordance with patient immunomolecular signatures has the potential to reduce the rate of antibody-mediated platelet clearance and thus to improve treatment efficacy. while the benefits of customizing transfusion therapy have long been recognized (gmur1978 http://bit.ly/2q51heq), the routine, real-time selection of platelets by immunogenetic profile has remained impractical by current methods of dna analysis. to address this issue, we evaluated a process of platelet donor classification using buccal swab samples from apheresis platelet donors for determining the combined hla class i and hpa signature without dna purification using a novel "leansequencing" process. study design/method: under a study protocol and informed consent, we evaluated a process for collecting and classifying buccal swab samples from $100 adult donors who had made ! 6 donations in the previous 12 months. samples (labeled with study barcodes) were shipped weekly to biomolecular analytics ("bmx") for preparation of "crude extracts" for leansequencing: this novel process combines a proprietary sample pooling strategy with a protocol that eliminates many traditional sample "clean-up" steps. briefly, after preparation of crude extracts, samples were amplified, pooled and analyzed (in separate runs) for 2, 3, 4, 5, 6, 7, 8, 9, 11, 15 and for hla class 1 (a,b,c) , the latter using a proprietary design that limits analysis to informative alleles in the hla sequence; this "information-theoretic" design permits direct allele and haplotype reconstruction using bmx-proprietary software. a subset of crude extracts was purified and analyzed side by side with positive and negative controls. results/finding: crude extracts from buccal swabs produced viable profiles for hpa as well as hla class i with significant savings in time-to-result. as an illustration, the table reports allele frequencies for platelet-antigens ("hpa") that are consistent with a predominantly caucasian or hispanic platelet donor population (http://bit.ly/2pdplf8) in hw equilibrium. similarly, hla-class i haplotype frequencies were determined. conclusion: leansequencing lends itself to the rapid determination of hla-class i and hpa signatures of platelets; the process with its streamlined lean protocol achieves additional time (and cost) savings by accommodating crude extracts produced from buccal swab samples collected and handled in accordance with the process validated in this project. the process could be readily implemented to another site using the elements and process developed. the "pool & plex" process and the early donor recruitment enables economies of scale for matched donor procurement. the serological characteristics and heritage background of a novel hla allele, hla-a *26:82 chuan-fu zhu*, yong-hong song, xiang-min nie and wen-ben qiao. blood center of shandong province background/case studies: there are 16,429 hla alleles documented according to the imgt / hla sequence database in janury2017, and more than 80% of them were identified in the last 10 years. besides sequences many of the novel hla alleles have not been analyzed their serological reactivities. hla-a *26:82 allele was fist detected in our laboratory during our hla typing for china bone marrow donor program(cmdp). for further study, the serological characteristics and heritage investigation were performed. study design/methods: the routine hla tying for the potential donors from cmdp were performed by bi-allelic sequence-based typing method,using a commercial kit (rose europe gmbh, frankfurt, germany). in the case of no full matched hla typing results, group specific hlassure-se sbt typing kit (texas biogene inc., taipei, taiwan) was employed to identify the nucleotide sequences of the novel allele. fresh blood samples were collected of the proband and his family members with the consent, in order to nanalysis the serological reactivities and the possible haplotype associations to the novel allele. the hla serological specificity was indicated by one lambda(asn72d)hla kit. results/findings: no full matched result was obtained at hla-a locus in hla typing for a donor,which suggested the possible existence of a novel allele. the latter nanalysis indicated that the proband have a nove1 nucleotide sequences at hla-a locus, the new sequences was most close to those of hla-a *26:01:01:01, but 1 nucleotide substitution in exon 4, by nt 746 c-a (codon225 acc-aac), which resulted in one aminoacid substitution ,thr-asn. the novel hla-a allele was officially named as hla-a background/case studies: anti-d is a frequent cause of hemolytic disease of the fetus and newborn (hdfn). as a rule, immunization occurs in d negative pregnant women, but occasionally anti-d is also observed in carriers of d variants. currently, maternal plasma analysis for determination of the fetal rhd status became an exciting new tool for the management of d-negative pregnant women, but one of the challenges in non invasive fetal rhdgenotyping is the presence of d variants in the pregnant women. we present a case of a 13 year-old pregnant woman typed as ab1, who delivered a baby affected by severe hdfn. the newborn was typed as b1 and presented a positive direct antiglobulin test (dat) with an anti-d identified in the eluate. the baby was treated by exchange transfusion and the mother's sample was investigated. study design/method: serologic testing was done by hemagglutination in gel cards. genomic dna was extracted from whole blood by spin column and all rhd exons were sequenced by sanger sequence method. results/finding: the mother's rbcs reacted 41 with the four monoclonal anti-d used (igm clones p3x61 and rum 1 and the blends clones th281ms26 and d1751d415) and were typed as c-c1e-e1. an anti-d was identified in her serum. molecular analysis showed the 410c>t and 455a>c in exon 3, the snp 509t>c changes in exon 4 and the 667t>g nucleotide change in exon 5. the set of snps found is similar to the molecular background of dol3, except for 455a>c change. conclusion: this novel set of snps found in this mother is related to a novel rhd allele leading to a partial d antigen involved in the production of an anti-d that can cause severe hdfn. this finding shows the need to elucidate the clinical significance of different rhd genotypes in various ethnic backgrounds. the and erytra v r (the routine reference platform) was performed. a total of 1089 immuno hematological tests (465 abo/d grouping (including 33 newborn samples), 12 extended erythrocytic phenotype, 562 antibody screening, 14 antibody identification, 16 dat) and 20 crossmatches were performed on patient's whole blood samples. the erytra eflexis v r performance was evaluated according to a protocol that was designed to simulate the routine workload using the system in its two different configurations. concordance between systems was assessed and discrepancies were analyzed. the following performance metrics were assessed: time to first result (ttfr), turn-around time (tat) for the total workload from first result to last result (throughput, results/h), and manual "handson" time required as well as walk-away time, considering the two different configurations of the system. for the ease of use evaluation, different usability features were ranked and the number of steps and timing of the following activities were tracked: sample sort and loading, routine testing, post-run procedures, consumables used, and space requirements. a threshold for in vitrodetection of anti-d gamma globulin was also determined. v r analyzer and the reference method were obtained in 99.2% of the abo/d tests (n5265), 99,7% of the antibody screening tests (n5377), 88,8% of the antibody identification tests (n59) and 100% of the dat tests (n510). there were 4 discrepancies (2 abo/d for the same patient, 1 for antibody screening and 1 antibody identification: in both cases, the erytra eflexis v r could conclude whereas erytra could not due to a poor reaction. use of the stat mode (incubator is reserved for urgent tests) proved its usefulness when testing several samples (time saving was more than 10 min). detection threshold of the d antibody was assessed at 2.5 ng/ml (0.0125 ui/ml) whereas the french recommendations are 20 ng/ml. the possibility of interchanging the trays (reagents/sample) makes also possible to optimize the analyzer operation. the impressions of the technical staff were positive regarding esthetic and functional design, intuitive and easy use, as well as flexibility. v r results demonstrated velocity, sensitivity, as well as the ability to easily perform the routine workload of a medical analysis laboratory. erytra eflexis v r meets both the requirements for french regulatory in immunohematology and for iso 15189 accreditation. background/case studies: kell system antibodies inhibit erythropoiesis causing severe anemia in hemolytic disease of the fetus and newborn (hdfn). we report a case of hdfn secondary to anti-kpb that resulted in multiple intrauterine transfusions of kp (b-) donor cells and hemolytic anemia upon birth. case: a 31 year old g5p3 presented during her fifth pregnancy with anti-kpb with an initial titer measured of 64. by history, the anti-kpb developed during her third pregnancy which ended in a spontaneous abortion before antibody titers could be initiated. the patient's antibody titers peaked at 16 during the fourth pregnancy which resulted in a healthy male without anemia or jaundice. . in the latest pregnancy, ultrasound was initiated with elevated middle cerebral artery doppler exams (1.7 moms) peaking at 27 weeks. this resulted in three intrauterine transfusions. due to potential labor and the finding of reversed diastolic flow on middle cerebral artery doppler studies, a finding that has been associated with impending intrauterine fetal demise, caesarean delivery was performed at 35 weeks gestation. the baby boy required phototherapy for hyperbilirubinemia. the indirect bilirubin at birth was 3.4 mg/dl with 13.6 g/dl hemoglobin. the baby typed as o positive, kp (b1) with a micro positive dat. the antibody workup revealed an anti-kpb. continued hemolysis required one more transfusion at 6 weeks of age. the positive dat and passively acquired anti-kpb were no longer detected by 8 weeks of age. his hemoglobin recovered to 9.0 g/dl with an indirect bilirubin of 1.4 mg/dl at 9 weeks of age. all clinical signs of hemolytic anemia were resolved. study design/method: serologic testing included peg iat by tube methods. acid elution was performed using immucor gamma elu-kit ii. molecular testing was performed using immucor bio-array hea platform. results/finding: antibody identification on the mother was performed as well as alloadsorption studies to rule out other underlying alloantibodies. a new weakly reacting anti-s was detected on the day of the delivery. the baby typed as s positive however the anti-s was not detected in an eluate prepared from the baby's red cells. all of the intrauterine transfusion units were s negative. conclusion: to our knowledge only five case reports have been described for anti-kpb which resulted in moderate to severe hdfn. pregnant mothers with anti-kpb detected should be monitored closely. background/case studies: in some clinical cases, the c3d-specific dat may be too insensitive to detect low, but significant levels of c3d, or it may be inconclusive due to spontaneous red cell (rbc) aggregation. further, the dat is not well suited to quantify the number of immunoprotein molecules on rbcs, since a "1111" reaction corresponds to about 500 molecules/ cell. a number of flow cytometric methods for the detection of rbc-bound c3d have been published. however, these are mainly designed to quantify the fraction of rbcs with c3d-sensitization. the aim of this study is to present a flow cytometric method for the quantification of the level of rbcbound c3d. study design/method: ten microliters (ul) of 1:80 (after documenting experimentally that this amount ensured maximum binding of anti-c3d) mouse monoclonal anti-human anti-c3d (abcam, clone 7c10) were added to 5 ul of a 2.5% rbc suspension. after incubation for 60 minutes at 4c, samples were washed x3, and 25 ul of 1:10 diluted anti-mouse-f(ab)2-pe (ro480, dako) were added. after incubation at 4c, samples were washed and resuspended before being acquired on a flow cytometer (becton dickinson facscanto ii). to enable calibration of fluorescence signals in antibody binding capacity (abc), a calibration standard (dako qifikit) stained with ro480 was run in parallel with all experiments. background fluorescence (in abc) was subtracted to yield net abc values corresponding to specific staining with anti-c3d. the assay, in parallel with our routine dat (dc-screening i, id-card, gel card, biorad) was applied to a series of a1 rbcs stained with 10 levels (2fold dilution, 1:1 -1:512) of o serum with high titer anti-a. to estimate the normal range of rbc-bound c3d, edta-stabilized samples from 4 healthy donors were tested. finally, the assay was applied to a sample from a patient with clinical aiha with an inconclusive dat due to unspecific dat polyreactivity. results/finding: the correlation of the net level of rbc-bound c3d (values ranging from 0 to 3,393 abc) with level of 0-serum dilution (used to sensitize a1 rbcs) proved to be highly linear (logarithmic vs. logarithmic plot; r2 5 0.97, p < 0.0001). compared with dc-screening 1, the sensitivity of the flow cytometric assay was superior. it detected c3d sensitization at least 4 dilution steps further. the median normal level of rbc-bound c3d was 11 abc (range 7-20 abc, n54). the assay enabled demonstration of specific c3d-sensitization in the patient; the level of rbc-bound c3d in the sample was significantly elevated (1,907 abc). conclusion: the presented flow cytometric assay is capable of quantifying the level of rbc-bound with a high degree of linearity and analytical sensitivity. further, it is capable of quantifying the level of rbc-bound c3d in dat polyreactive samples. background/case studies: abo blood group system of red blood cells (rbcs) consists of a and b oligosaccharide antigens and anti-a and anti-b antibodies against these antigens, which are present in the sera of individuals who do not express the antigen(s)(landsteiner's law). because of the expression of those antigens on some epithelial and endothelial cells in the body, the abo matching is critical not only in blood transfusion, but also in cell/tissue/organ transplantation. in spite of the fact that both antigens and antibodies are involved, these genetic traits are specified by a single genetic locus of abo. forssman (fors) system is another rbc blood group system which consists in a glycosylation polymorphism specified by the gbgt1 gene. in humans, the abo and gbgt1 genetic loci are located on chromosome 9q34, and the functional alleles encode a and b glycosyltransferases (at and bt) and forssman glycolipid synthase (fs), which catalyze the last biosynthetic steps of a and b, and forssman (fors1) oligosaccharide antigens. the molecular genetic bases for allelism of those two systems in humans have been well-elucidated. the abo and gbgt1 genes are also present in some other species in addition to humans. however, the presence/absence and functionality/non-functionality are species-dependent. molecular mechanisms/forces that created this species divergence, including human polymorphism, were unknown. study design/methods: utilizing genomic information available from gen-bank and ensembl databases, the gene maps of the chromosomal region surrounding the abo and gbgt1 genes have been constructed of 88 vertebrate species. results/findings: extensive similarities were observed in the kinds, numbers, and orders of genes, as well as their chromosomal locations. however, numerous differences were also identified. these include chromosomal rearrangements, as well as the insertions and amplifications of specific genes. interestingly, the abo and gbgt1 genes were found located at the boundaries of chromosomal fragments that seem to have undergone frequent inversions/translocations during species evolution. conclusion: genetic alterations, such as deletions and duplications, are known to be prevalent at the ends of rearranged chromosomal fragments. therefore, the species-dependent divergence and polymorphism within species of those clinically important glycosyltransferase genes may have been resulted, at least partially, from unstable chromosomal structures neighboring those genes. alloimmunization despite phenotype matching in a patient with sickle cell disease and a complex rhce genotype jessica kneib* and emily coberly. university of missouri health care background/case studies: red blood cell transfusion plays an important role in the treatment of patients with sickle cell disease. sickle cell patients have a significantly increased risk of alloimmunization compared to the general population, and the standard of care is to provide phenotypically matched units for at least c, e, and k1 antigens to reduce this risk. unfortunately, the genotype and true alloimmunization risk may not always be accurately represented by the red blood cell phenotype, particularly in patients with complex partial rhce variants. study design/method: a 14 year old female with a history of sickle cell disease, stroke, and iron overload presented for routine exchange transfusion. transfusion 2017 vol. 57 supplement s3 the patient's blood type was o positive and her red cells had been previously phenotyped as c-, c1, e-, e1 and k1-. an antibody screen was positive, and antibodies against c and e antigens were identified in the plasma. the patient had only received phenotypically matched units negative for c and e antigens for all previous transfusions at our institution, based on her known red blood cell phenotype. blood samples were sent to a reference laboratory for molecular testing to look for partial rhce variants that might explain the antibody development. results/finding: molecular testing was performed to reveal the presence of two different partial rhce alleles, resulting in a predicted phenotype of d1, c-, e-, partial c1, partial e1. the probable rhce genotype, rhce*ce-jal/rhce*ce733g, results in partial expression of both c and e antigens. in addition to the known risk of alloimmunization against the absent c and e antigens, this result indicates that the patient is also capable of forming alloantibodies against the absent portions of both c and e antigens. based on these results, the patient's anti-e was determined to be an alloantibody and not an autoantibody. conclusion: although phenotypically matched units are standard of care for patients with sickle cell disease, the red blood cell phenotype may not accurately represent the alloimmunization risk in patients with complex partial rhce genotypes. in this case, molecular testing confirmed that the patient is at risk of developing alloantibodies against c, c, e, and e antigens. as the patient had already made alloantibodies against c and e antigens, it was determined that she would require units that were molecularly matched to her rhce variants for all future transfusions. this case demonstrates that phenotype matching for sickle cell disease patients may not be adequate to prevent alloimmunization in individuals with partial rhce variants. altered splicing in the rhd*weak d type 2 allele associated with the skipping of exon 9 in a pregnant woman and her newborn carolina bonet bub* 1 , maria giselda aravechia 1 , thiago costa 1 , marilia sirianni 1 , eduardo bastos 1 , leandro santos 1 , lilian castilho 2 and jos e kutner 1 . 1 hospital israelita albert einstein, 2 hemocentro unicamp background/case studies: rhd*weak d type 2 is a variant commonly found in caucasians associated with a weak d phenotype. as previously reported (vege et al, transfusion 2007) the c.1154g>c change (p.g385a), which characterizes the rhd*weak d type 2 allele is a splicing variant that induces skipping of the whole rhd exon 9. we report an altered splicing in the rhd*weak d type 2 allele associated with the skipping of exon 9 in a pregnant women and her newborn with weak d expression. study design/method: the d antigen expression was evaluated with 4 commercially available monoclonal anti-d reagents: 1 blended igm/igg (clones th-28/ms-26), 2 igm (clones ms201 and p3x61) and 1 igg (ms26) in tube and on gel cards. c, c, e and e phenotyping were performed in gel. rhd genotyping was performed with the rhd beadchip platform from immucor. direct automated sequencing of the 10 rhd exons and flanking intron regions was performed by the sanger dideoxy method. results/finding: both pregnant women and newborn samples were phenotyped as d1 w c-c1e1e1. the samples showed weak hemagglutination reactions (11/21) with all anti-d clones used. rhd beadchip results showed the ls* signal indicating a possible deletion of exon 9 in both dna samples. sequencing showed the c.1154g>c change and the intronic c.1154-8t>a and c.1154-31t>c substitutions, which are associated to the rhd*weak d type 2 allele. conclusion: our results showed that c.1154g>c associated with c.1154-8t>a and c.1154-31t>c variations had probably a functional impact on splicing inducing exclusion of exon 9 in both dna from mother and newborn. this finding is important to develop assays and interpret genotyping results, as current guidelines do not recommend anti-d igg prophylaxis for women with weak d type 2. background/case studies: sickle cell disease (scd) patients require red blood cell (rbc) transfusions to minimize disease-specific symptomatology. previous studies have shown that more than 50% of children with scd receive at least one rbc transfusion in their lifetime. both simple transfusions and erythrocytapheresis are associated with increased risk of rbc alloimmunization. published literature is lacking on the frequency of alloimmunization and geographical associations in pediatric populations, which is made difficult to compare due to lack of standardized categorization of what represents a pediatric patient population across studies. therefore, we looked at the alloimmunization rates of pediatric patients with scd in the unites states (us) and other countries. study design/method: a literature search was performed for studies published on alloimmunization rates of scd pediatric patients including hbss, sickle beta-thalassemia and hbsc. we evaluated the overall alloimmunization rates as number of alloantibodies per transfused patient and alloantibodies per 100 transfused units across world literature and compared them using chi-square analysis. results/finding: fourteen studies reporting data to derive alloimmunization rates of pediatric scd patients were found. these included eleven us studies with 1,057 patients and 3 studies from other regions (brazil, egypt and france) with 641 patients. majority of patients included in the studies had hbss disease. patients received either episodic, chronic simple transfusions or erythrocytapheresis. age range for the us studies was 0 to 26 years and for the other countries 0 to 20 years. available data from 5 us studies included a total of 91 alloantibodies, the most frequent of which were antibodies to c, e, kell, m, s and kidd antigens (18.7%, 16.5%, 15.4%, 7.7%, 7.7% and 6.5% respectively). alloimmunization rates were calculated as antibodies per patient in some studies and antibodies per 100 transfused units in other studies. we evaluated rates using both approaches as per available data. us had an alloimmunization rate of 16.5 % (14.1 to 19.2, 95% ci) vs. 9.4% for non-us studies (7.3-11.8, 95% ci) (p50.0008) and 134a transfusion 2017 vol. 57 supplement s3 more alloantibodies per transfused patient (0.25 vs. 0.096, p50.0001). similarly, the number of alloantibodies per 100 transfused units in the us, evaluated from five studies, was higher compared to a large french patient cohort (0á68 vs. 0.33, p50á0005). average number of rbc units transfused per patient in the us was also higher compared to data from france (77 vs. 45, p50.0001). conclusion: despite limited studies available to compare alloimmunization rates in pediatric scd patients in the us and other countries, the overall rates are higher in the us. though no definitive reasons could be concluded from the available data, limiting the number of rbc exposures, i.e. units transfused in non-critical conditions could lead to lower alloimmunization rates. results/findings: a post-transfusion sample was referred to the irl for a trxn investigation. there were no clerical errors; however, hemolysis was present in the post-transfusion plasma/serum. abo/rh and crossmatches using lo-ion tm were repeated on the pre-and post-transfusion samples with no discrepancies. the post-transfusion dat was positive with a negative eluate. the hospital requested another unit before the investigation was complete. antibody identification on the post transfusion sample with lo-ion tm was negative. suspecting a weak antibody, additional investigation using peg tm on both samples revealed an anti-c. no additional clinically significant alloantibodies detected in the pre-or post-transfusion samples using peg tm . conclusion: the patient experienced an acute hemolytic transfusion reaction due to anamnestic interaction of anti-c in the patient's plasma/serum against c antigen on the transfused cells. anti-c was not detected by our routine antibody identification techniques. the mma confirmed anti-e, -m and -c were clinically significant. laura bailey* 1 , melissa grohotolsky 2 , lisa deblass 1 , bala carver 1 and kip kuttner 2 . 1 health network laboratories, 2 miller keystone blood center background/case studies: the en a antigen is a high prevalence antigen in the mns blood group system. the antigens of the mns system are carried on glycophorin a (gpa) and glycophorin b (gpb). anti-en a is a rare immune igm/igg antibody made by individuals who lack all or part of the gpa protein. anti-en a has been implicated in fatal htr and hdfn. the en(a-)phenotype can result from either a rare deletion of the gpa protein or the even rarer m k phenotype. because individuals with the m k phenotype lack both the gpa and gpb protein their red blood cells type as m-, n-, s-, s-, u-, en(a-), wr(b-) and have reduced sialic acid. study design/method: 23 year old white mennonite female g1,p0 presented to her midwife for prenatal care with the intent of home delivery. she had a positive antibody screen by solid phase at the hospital transfusion service. an antibody identification panel was done in gel. testing for antibodies against selected cells (u-and u var ) in tube with peg enhancement and phenotyping was done. based on mns phenotype, anti-en a was suspected. the specimen was referred to an immunohematology reference laboratory (irl). the testing included phenotyping with unlicensed antisera, ficin treated panels by tube technique, allogeneic adsorptions for antibody exclusion and identification and antibody titration. following identification of anti-en a by the irl the midwife was advised to refer the patient to a maternal fetal medicine specialist at an academic center close to the patients' home. the midwife was also advised to consider autologous blood donation and /or testing of siblings. results/finding: testing by the hospital blood bank demonstrated positive reactivity in the antibody screen. the gel antibody panel ahg phase resulted in 21 panagglutination and a negative autocontrol, suggesting a high prevalence antibody. the phenotype was performed and determined to be m-, n-, s-, s-, u -. outdated u variant reagent cells reacted in peg igg phase ruling out anti-u. anti-en a was suspected and the sample was referred to the irl. allogeneic adsorptions were performed to rule out antibodies to common red cell antigens. lack of reactivity on a ficin panel eliminated the presence of anti-u,-wr b . phenotyping with unlicensed anti-u was negative and unlicensed glycine soja demonstrated 11 reactivity, suggesting that the patient is en(a-). the patient's phenotype is consistent with the m k phenotype. based on the lack of reactivity on the ficin panel, the antibody was identified as anti-en a fs. since anti-en a is extremely rare, this specificity could not be confirmed due to the lack of en(a-) cells and appropriate antisera. the baseline antibody titer was 2 at igg phase without enhancement. conclusion: this case study describes the workup of a rare antibody in a prenatal patient at a tertiary care hospital. studies performed after the patient was transferred closer to home confirmed the anti-en a (fs) and genotyping was performed. three titers were performed for the remainder of the pregnancy and held at 2. although anti-en a has been implicated in hdfn, a healthy infant was delivered without complications. this patient should be monitored closely through future pregnancies. autologous donation and/or sibling testing should be considered in order to provide compatible blood for intrauterine transfusion or transfusions at or after delivery. background/case studies: a 15 year old caucasian male diagnosed with hemolytic anemia and no previous transfusions was referred to the immunohematology reference laboratory (irl) for antibody identification and rbc genotyping. initial serologic testing by the referring facility and the irl demonstrated anti-d, anti-c and/or anti-g specificity with a positive auto control and igg dat. anti-g has an anti-d, -c specificity and is most frequently found in rr individuals exposed to r'r cells. the g antigen is present on rbcs expressing either rhd and/or c and very rarely on d-c-g1 (r g r) cells. both rhce*c and rhd genes encode ser103 which determines g expression. rare rhd variant antigens lacking ser103 are g-. study design/methods: serologic evaluation included tube testing using gamma lo-ion tm (immucor, inc., norcross, ga) enhancement, elution studies (gamma elu-kitv r ii (immucor, inc.)), edta glycine acid treatment (gamma ega tm kit (immucor, inc.)), allogeneic adsorptions with papain treated intact rbcs, reagent and patient-derived rbcs and antisera. molecular testing was performed with bioarray precise type ivd hea assay (immucor, inc.). results/findings: molecular testing revealed an rhce*ce genotype (with a c-e1c1e-predicted phenotype) and an otherwise unremarkable rbc typing report. serologically, the antibody(ies) demonstrated an anti-d, -c, -g specificity in the serum and eluate using r o r, r 2 r 2 , r'r, r g r and rr cells. this patient is predicted to be r 2 r 2 (dce/dce) therefore, anti-c is possible but an allogeneic anti-d or -g is exceptionally unlikely. allogenic adsorptions using papain treated r o r and r'r cells excluded anti-c and anti-d, leaving anti-g as the only explanation of the initial findings. reactivity with the patient's ega treated (dat negative) cells against the "neat" serum, eluate and anti-g antisera confirmed auto anti-g. conclusion: warm autoantibodies are common findings and often have an rh specificity; however, these antibodies usually demonstrate a broad but weaker specificity in the eluate or in the serum when enhancements are used. this anti-g had no reactivity with g-cells. the differentiation of anti-g from anti-d and anti-c is generally academic as transfusion recommendations are the same: provide rhd-, c-units. it is relevant and clinically important to determine the presence or absence of anti-d in rhd negative women of childbearing age who present with an anti-g specificity. if anti-d is 135a transfusion 2017 vol. 57 supplement s3 excluded these women should receive rhig as part of their prenatal care. in this case differentiating anti-d, -c from an auto anti-g was necessary to provide transfusion recommendations. providing rhd-and c-units to give serologically compatible rbcs could result in formation of an allogeneic anti-e. automated eluates: comparison of solid-phase red cell adherence and gel automated eluate testing jayanna slayten* 1 , christa voliva 1 , kathy fletcher 1 , heather vaught 2 and tracie ingle 1 . 1 indiana university health, 2 indiana university health (iu health) background/case studies: acid eluates (elu kit ii. immucor. norcross, ga) are to be tested via tube iat method in parallel with the recovered last wash per the manufacturer's package insert. finck et al (immunohematology 2011; 27:1-5) demonstrated acid eluates may be tested in other platforms such as manual gel microcolumn assay (id-mts.igg card. ortho clinical diagnostics. raritan, nj) and automated solid-phase red cell adherence systems (echo. immucor. norcross, ga). our study looked to compare the use of the automated gel microcolumn analyzer (vision, ortho clinical diagnostics. raritan, nj) to the solid-phase red cell adherence analyzer (echo, immucor. norcross, ga) for the testing of acid eluates in a regional midwestern transfusion service. study design/methods: twenty patient samples, less than 7 days from collection and drawn in edta, were used to prepare acid eluates (elu-kit ii. immucor. norcross, ga) while retaining the last wash to be tested in parallel. two samples were >21 dat positive, 2 were weakly dat positive and 16 were dat negative. the prepared eluates were observed for color (bluegreen/bg, blue-brown/bb, blue-purple/bp), and the ph was documented for the prepared eluate (whatman 6.0-8.1ph. whatman international. maidstone, england). the prepared eluates and last washes were tested on the vision and echo against an antibody screen. if the antibody screen was positive, the sample was tested against an antibody panel to determine specificity/pan-reactivity. prior to the eluates being tested on the automated platforms, they were spun for 5 minutes twice to remove any rbc debris which could cause false positive reactions. results/findings: the eluates prepared ranged in color: 5 bb, 14 bg and 1 bp. the ph of all eluates ranged from 6.9-8.1 with the highest percentage of eluates at a ph of 8.1 (35%). sixteen of the 20 eluates tested yielded the same results in both automation platforms (concordance of 80%). four eluates with different results are summarized in table 1 . conclusion: the study demonstrated that both analyzers may be used for eluate investigations. both methods yielded apparent false positive results on samples which were initially dat negative. the echo was more sensitive, yielding false positive results (3) when the vision was negative, while the vision was false positive with one eluate with echo negative. there was no apparent association in the non-correlating eluate results in relation to color of eluate, age of sample when eluate was prepared, or ph of the eluate. a larger study may be able to better elucidate the apparent false positive results noted in this study between echo and vision eluate study. background/case studies: blood agglutination observed by landsteiner in 1900 led to the discovery of human blood groups. in the abo system >200 alleles have been described. the glycosyltransferase encoded by most results in weakened expression of a or b or the null (group o) phenotype. as testing methods and reagents improve, donors may appear to change their abo type. here we describe a frequent group o blood donor (38 units over 17 years) who is actually a w . study design/methods: donations were tested with the pk7300 instrument (beckman coulter inc.). routine forward and reverse abo testing was used to investigate the discrepancy. molecular studies were performed by dna sequencing of abo introns 1,2 and 4 and exons 6 and 7. specific primers located in the flanking intron regions of the blood group gene were used to amplify relevant exons by pcr. the template used is genomic dna extracted from whole blood collected in edta. pcr-amplified exons are subjected to bidirectional dna sequence analysis using standard sanger dideoxy chemistry. seqscape software (abi) was used to analyze sequence data by comparing the obtained sequence to a reference sequence from ncbi. results/findings: serologic results are shown in table 1 . tests with anti-a, -a1, -b anti-a,b were negative as were the a2 cells in reverse testing. the results of dna sequencing of abo introns/exons are shown in table 2 . the significant changes were found in exons 6 and 7. in exon 6 there was a nucleotide (nt) deletion of 261g which resulted in a shortened transcript due to a stop codon, and another nt substitution lead to the amino acid change gly117ala. mutations in exon 7 included a nt substitution causing a pro156-leu change and a nt deletion 1061c resulting in shortened transcript. conclusion: serologic testing of the donor plasma with a2 cells was nonreactive revealing the abo discrepancy. molecular testing confirmed the donor genotype is heterozygote a/o [abo*o.01.01/abo*aw.02] which predicts a w phenotype. normally, donor rbcs are tested with anti-a and -b and the reverse type confirmed by testing the with a1 and b cells. this abo discrepancy was caused by the presence of anti-a1 in the plasma causing the forward and reverse type to be interpreted as group o. according to fda guidelines, the donor is technically group a, and as such all donations need to be labeled as group a. the donor was contacted and instructed to cease donating blood for transfusion. if donations continue, the unit labeled group a would likely test as group o at the transfusion facility resulting in an fda reportable error. there are numerous reports in the literature of the relative insusceptibility of a2 cells to destruction by anti-a, however, there is one hemolytic transfusion reaction to a x blood transfused to a patient with a potent anti-a titer >1:1000. (schmidt, nacarrow et al. 1959) . a review of transfusion recipients of the donor reported here did not reveal any untoward reaction after transfusion. 136a transfusion 2017 vol. 57 supplement s3 extraction of gdna from edta-anticoagulated whole blood from pilot tubes derived from the unit. dna extraction from whole blood is performed on up to 96 blood tubes using the biorobot universal system (qiagen). there is no information on the maximum acceptable age of the blood for this purpose, either from the vendor or in peer-reviewed literature. we set out to assess if blood up to 15 days post collection yielded suitable gdna for downstream rbc genotyping. study design/method: 92 edta blood tubes collected from random blood donors were used to extract dna from 200 microliters of whole blood on day 5, 12 and 15 days post collection. blood samples were stored at 2-8c before and after extraction. tubes were brought to room temperature and rocked before loading on the biorobot. extraction was performed using the mdx blood minikit (qiagen). resulting dna samples were assessed for gdna yield and absorbance a260/a280 using a nanodrop 2000 (thermo scientific). the extracted gdna was tested using precisetype hea molecular beadchip ("hea", immucor) and failure rates on both the biorobot and the hea were assessed. results/finding: all three extractions were successful with no invalids (result50) on the biorobot universal report. no evidence of visible clots or splatter during extraction was noted by the technologist. out of the 92 samples, 20 samples were chosen at random and concentrations were measured using nanodrop for each of the extracted plates. dna concentrations ranged from 10.8 to 62.6 ng/ul. all readings with the exception of 1 (10.8ng/ ul) had concentrations >5 15ng/ul. interestingly, the one that was <15ng/ ul on day 5, yielded >515ng/ul on day 12 and 15 post collection. over the next 3 months, 67 sets of 92 samples were extracted and tested by hea. eighty-three (1.3%) failed extraction and 82 (1.3%) failed hea. none of the samples that failed extraction were 12 or 15 days post collection; none of those that failed hea were 15 days post collection; 3.7% were >10 <15 days post collection. conclusion: based on these results it can be concluded that edta blood tubes up to 15 days post collection can be used as a source of gdna for rbc genotyping without negatively effecting the concentration of the resulting dna samples and the validity of the resulting genotyping. case study: investigation of persistent negative antibody screens on patients receiving daratumumab raeann thomas 1 , carlos villa 1 , rachel davis-rauser* 1 , helen carpenter 1 and vrunda patel 2 . 1 university of pennsylvania, 2 hospital of the university of pennsylvania background/case studies: daratumumab is an anti-cd38 monoclonal antibody therapy that received fda approval for treatment of multiple myeloma in 2015. communications suggest all patients receiving therapy would have a positive antibody screen because cd38 is a common antigen expressed on red blood cells. currently, 154 patients have been treated with daratumumab at a large academic medical center. a wide variation of reactivity was observed, including patients who were found to have consistently negative antibody screens. while there are several potential causes, neutralization of anti-cd38 antibodies could easily be tested by applying established techniques used for neutralizing antibody reactivity. study design/method: samples received were drawn as a standard of care. indirect antiglobulin testing was performed using solid phase red cell adherence and gel. neutralization was performed by adding equal volumes of negative daratumumab treated patients' plasma with positive daratumumab treated patients' plasma. a dilution control was made by adding saline to each positive patient's plasma. samples were incubated for 1 hour at room temperature and antibody screens were repeated. serial two-fold dilutions were also tested to determine if the neutralization could be titered. testing was repeated using various positive patient samples to determine if negative/positive combinations resulted in different reactivity. results/finding: all control samples remained positive. positive/negative samples were negative in solid phase testing across all patient combinations at 1:1 dilutions. variable reactivity was observed in gel. serial dilutions showed that neutralization for 2 negative patients was observed up to a 1:4 dilution. conclusion: results suggest that patients' plasma may have a substance that neutralizes the antibodies. there is a possible correlation with patients who have persistent negative antibody screens and patient response to daratumumab. additional studies are necessary to uncover how this correlates to patient outcomes. further studies using a standardized daratumumabspiked sample will be conducted. background/case studies: the mns blood group is a red cell antigen system located on glycophorin a (gypa) and glycophorin b (gypb). individuals lacking gypa or both gypa and gypb on their red blood cells may develop a rare antibody against the en (a) antigen. the en (a) antigen is a highprevalence antigen, located on gypa. we present a case with a rare red cell phenotype and alloimmunization to the en (a) antigen. a 28 y/o g1p0 at approximately 23 weeks gestation was discovered to have an anti-en (a) antibody in her plasma on a prenatal type and screen. this was worrisome for both mother and fetus, as the en (a) antibody is of igg isotype and has been implicated in both acute and delayed hemolytic transfusion reactions and hemolytic disease of the fetus and newborn (hdfn) [1, 2] . further testing with red cell antisera revealed that the patient lacked m, n, s, s, and u antigens. a multiplex, allele-specific, pcr platform we commonly use to detect the presence or absence of red cell gene sequences failed to amplify genes specific for the m, n, s, s, and u antigens. these findings were consistent with a null phenotype for both gypa and gypb antigens, i.e. m (k) m (k) phenotype. the patient's husband and father of her unborn baby demonstrated a m1n-s1s1 phenotype by the same serological and molecular means. given the exceedingly rare incidence of en (a-) individuals (positive frequency >99.9), clinical encounters with alloantibodies to this antigen are limited in our experience and in the literature [1, 2] . however, the existing data gives credence to its association with transfusion reactions and hemolytic disease of the fetus and newborn (hdfn). the consensus in this case was to work her up as a high-risk pregnancy with frequent intensive monitoring which involved frequent monitoring of antibody titers. if transfusions were required for the mother or fetus, our options were to either search for rare units lacking the en(a) antigen via rare blood donor registries or directed donations from family members who match the patient's phenotype. at term, the patient underwent induction of labor and successfully delivered a health baby boy by vaginal route. the delivery was without event. no transfusions were necessary antepartum or postpartum. study design/methods: n/a results/findings: n/a conclusion: anti-en(a) is a rare antibody and there is limited data about its potential clinical sequelae, which is concerning in a pregnant woman. providing this patient with rare en(a) negative red cells via national or international blood donor registries would have been an arduous task if needed. this patient had many compatible family members available and willing to donate blood. the m(k) null allele (s) within this family is likely due to a genetic recombination among the gypa and gype genes rather than a mutation in both the gypa and gypb genes [3] . this results in the absence of glycophorins a and b and the constitutive antigens of the mns blood group system. our patient was exposed to the en(a) present on glycophorin a on her unborn baby's red cells (inherited from father) in utero with subsequent alloimmunization. in conclusion, this case report demonstrates a clinical approach in identifying a rare anti-en(a) antibody in a prenatal sample. the clinical finding of a rare antibody in which there is limited data requires leveraging every resource available in order to predict its behavior and provide safe blood products to patients who may require it. background/case studies: transfusions are essential for patients with scd and thalassemia to maintain growth and development during childhood and to sustain good quality of life during adulthood; however, the development of red blood cell (rbc) alloantibodies and autoantibodies complicates transfusion therapy in such patients. routine phenotyping of blood recipients and the use of phenotype-matched blood units for transfusion has been useful to lower the occurrence of red cell alloantibodies in chronically transfused patients with thalassemia and scd. nevertheless, extensive phenotyping is expensive, laborious and cannot be performed in certain situations. the molecular understanding of blood groups has enabled the design of assays 137a transfusion 2017 vol. 57 supplement s3 that are being used to better guide matched red blood cell transfusions and to maintain an inventory of units dna typed. based on this, our aim was to evaluate the clinical outcomes of molecular matching performed at different levels during 3 years for patients with scd and thalassemia. study design/method: blood group genotypes were determined in 67 dna samples from chronically transfused patients with scd, in 65 patients with thalassemia and in 3000 dna samples from blood donors. laboratory developed tests (ldts), hea beadchip tm , rhd beadchip tm , rhce bead-chip tm , and sequencing were used to determine the genotypes among patients and donors. molecular matching was performed in 3 levels: (1) rh and k matching; (2) extended matching and (3) extended matching including rh variants. we considered the total of red blood cell units requested for each patient and a number of 2 donations per year for the compatible donors. results/finding: according to the patients needs we performed molecular matching for 100% of our thalassemic and scd patients at level 1, 90% for scd patients and 70% for patients with thalassemia at level 2 and 30% for patients with scd and 90% for patients with thalassemia at level 3. the patients were transfused with a median of 36.4 rbc units. after three years of molecular matching, we observed that this transfusion strategy avoided new alloantibodies development and hemolytic transfusion reactions in all studied patients. conclusion: molecular matching has shown clinical benefits to the patients with scd and thalassemia, contributing significantly to reduce the rates of alloimmunization to 5-10% with c e k matching and <1% with extended matching. improvements in the clinical outcomes of the patients have also been observed as shown by an increase in their hb levels and reduction in the % of hbs in scd patients, better in vivo rbc survival and diminished frequency of transfusions. allahna lilly elahie* and sandra fazari. hamilton regional laboratory medicine program background/case studies: the ideal manual backup method for an automated antibody detection system is an important choice. currently, our backup method is saline tube (6 drops plasma, 30 minutes incubation). the change to either a low ionic strength solution (liss) or polyethylene glycol (peg) method would reduce incubation time to 10 minutes and specimen volume to 2 drops, both important laboratory considerations. objectives of this study were to compare the relative sensitivity, specificity, positive predictive value (ppv) and negative predictive value (npv) of peg and liss, and to determine the most appropriate manual backup method for the existing automated solid phase system. study design/method: a total of 202 specimens were compared utilizing: automated solid phase red cell adherence assay (sprca) with manual tube peg and liss, some samples were not sufficient quantity to test in liss. identification panels were used to determine: clinically significant antibodies, warm autoantibodies, and nonspecific reactions. calculations were based upon comparison to sprca. results/findings: a total of 164 clinically significant antibodies were detected using sprca technique, as well as 9 warm autoantibodies and 97 nonspecific reactions. peg demonstrated the highest sensitivity and lowest specificity while liss was least sensitive and most specific for clinically significant antibodies. for warm autoantibodies, liss was more sensitive than peg with both being 100% specific. both reduced the detection of nonspecific reactions. while peg had more nonspecific reactions (30 versus 13), it identified more clinically significant antibodies (129) than liss (93). (table) conclusion: ultimately, the decision to choose a manual backup method must be based upon the highest sensitivity for clinical significant antibodies so as to minimize failure to detect one. peg was selected as the backup manual method even though peg has a higher sensitivity to nonspecific reactions. this study clearly demonstrates the interplay and tradeoffs between methods, which are important to understand and consider when making method choice decisions. comparison of thiol reagents in denaturing cd38 on rbcs patricia a arndt* 1 , anthony salazar 2 and regina m. leger 1 . 1 american red cross blood services, 2 long beach memorial medical center background/case studies: monoclonal anti-cd38, e.g., daratumumab (dara), which is used to treat patients with multiple myeloma, causes positive indirect antiglobulin tests (iats) due to expression of cd38 on red blood cells (rbcs). this serologic reactivity cannot be removed by adsorption so other methods have been developed to detect/identify underlying alloantibodies. one popular method is to denature the cd38 antigen by treatment of rbcs with thiol reagents, e.g., dithiothreitol (dtt) or 2aminoethylisothiouronium bromide (aet). chapuy et al described (2015) and validated (2016) 2), and 6% aet (ph 8.0) as per the aabb technical manual, 17th ed. these treated and untreated rbcs were stored in alsevers at 4c and tested on days 1, 2, 4, 5 and 8 by two methods: 1) polyethylene glycol (peg) iat using plasma from two myeloma patients who had received dara (plasmas from 8 total dara patients were tested with reactivity 5 1-31), and 2) flow cytometry using phycoerythrin (pe)labeled anti-cd38. rbcs were also tested on days 1 and 5 or 8 with a serum containing anti-k by peg iat. results/findings: the 0.2m dtt in ph 8.0 pbs had a final ph of 7.3 and the ph of the commercial 0.2m dtt was 6.5. results are in table 1 ; flow cytometry results from days 2, 4 and 5 (data not shown) were similar to those from days 1 and 8. rbcs treated with 0.2m dtt (both sources) or aet were nonreactive with anti-k and plasma from all dara patients and gave very low results (% positive events) with pe anti-cd38 by flow cytometry for up to 8 days after treatment. rbcs treated with 0.01m dtt reacted similarly to untreated rbcs with anti-k and dara plasmas, and showed only some weakening (10-30%) of reactivity with pe anti-cd38. background/case studies: clinically significant hemolytic disease of the fetus and the newborn (hdn) is often caused by feto-maternal rhd incompatibility. with the discovery 1997 of cell-free fetal dna (ccfdna) in maternal plasma, it became possible to determine the rhd genotype of the fetus using non-invasive techniques. however, the reliability of the non-invasive prenatal rhd test (nip rhd) is dependent on sufficient amounts of cffdna in the maternal plasma sample. recent studies show that the fraction of ccfdna in maternal plasma varies significantly between pregnant women and is inversely related to maternal body mass index (bmi). thus, high maternal bmi, may impair the validity of nip rhd. the aim of this study was to examine the effect of maternal bmi on the correctness of nip rhd and the correlation of maternal bmi with fraction of ccfdna to total free dna in the sample. study design/method: measurements of body height and weight of pregnant rhd negative women in gestational week 12 were obtained from patient records and used for the calculation of maternal bmi. data on bmi were combined with the results from nip rhd (real-time pcr targeting rhd exon 5 and 10) and sample fraction of ccfdna (measured as threshold cycle [ct] value of rhd) to total free dna (measured as ct of ccr5) in gestational week 25. the correctness of nip rhd was determined by correlation with postnatal serological rhd determination. results/finding: a total of 1618 pregnant women were included. nip rhd was positive in 987/1618 (61%), negative in 582/1618 (36%) and inconclusive in 49/1618 (3.0%). compared to the postnatal rhd type, 9/987 (0.1%) of nip rhd results were false positive (fp) and 4/582 (0.7%) were false negative. in 5/49 (10%) of inconclusive nip rhd, the postnatal rhd type was positive. mean bmi (n51618) at gestational week 12 was 25.3 (10-and 90-percentiles: 20.0 -32.4). there was no difference in mean bmi between individuals who tested inconclusive or false negative by nip rhd compared to the remainder (p50,71). the fraction of ccfdna was calculated for 150 randomly selected nip rhd true positive cases. median ccfdna ratio was 5.47 (the distribution had a highly positive skew, 10-and 90-percentiles: 0.64 -27.2). there was no statistical correlation between bmi and fraction of ccfdna to total free dna (r2 50,012; p50.49). conclusion: neither the correctness of nip rhd test result nor the fraction of cffdna to total free dna appear to be correlated to maternal bmi with regard to maternal plasma samples drawn in the 25th gestational week. delayed hemolytic transfusion reaction due to anti-lan antibody: a case report. adla dh angelina*, suneeti sapatnekar and suzanne bakdash. cleveland clinic background/case studies: lan is a high-prevalence antigen and the sole member of the lan blood group system. anti-lan is a very rare igg antibody, with conflicting information regarding its clinical significance and potential for hemolysis. we report a case of delayed hemolysis in a patient with anti-lan antibody. study design/method: the patient's medical record and available literature were reviewed. results/finding: an 83 year old man, o-positive, with a history of heart disease and bladder cancer was admitted for radical cystectomy. the antibody screen and panel were panreactive by multiple test methods (gel, liss, peg) with negative autocontrols and dat and a saline antibody titer of 1, suggestive of an antibody to a high-frequency antigen. anti-lan antibody was identified by a reference laboratory. only 1 in 20,000 donors are lan-, but two frozen rbc units were locally available and transfused postoperatively. the patient's siblings were tested; one o-positive, lan-sibling was identified. nine months later, the patient was admitted for surgical management of metastases. at this time, the antibody screen was weakly reactive with 1 cell and new antibodies were ruled out. blood conservation measures were instituted, including limited blood draws and cell salvage for surgery. due to bleeding during and after surgery, 4 lan-rbc units were transfused over 4 days, including rare donor units and units from the sibling donor. another surgical procedure was then performed; by post-operative day 2, the patient had symptomatic anemia with hemoglobin (hb) 5.3 g/dl and serially increasing troponin. no lan-rbc units were available. four rbc units untested for lan were transfused without adverse event; the units were presumed lan1 but crossmatch compatible and phenotypically matched for the patient's other antigens. a post-transfusion hb of 10.2 g/dl was maintained for 4 days. the antibody screen was negative on day 3 post-transfusion, but strongly panreactive on day 6, with a positive dat (igg 21, c3 11) and anti-lan antibody identified in the plasma and eluate. there was also evidence of extra-vascular hemolysis, including a progressive decrease in hb from 10.9 g/dl on day 5 to 7.4 g/dl by day 8 with no bleeding identified, and increase in total bilirubin and ldh (peak 2.4 mg/dl and 304 u/l on day 7) with normal haptoglobin. the patient was febrile with leukocytosis, but had negative cultures and no other evidence of infection. a lan-rbc unit was transfused on day 8 with good response (hb 8.1 g/dl). the patient remained stable and was discharged to a skilled nursing facility 6 days later. conclusion: transfusion of lan1 rbcs caused a resurgence of anti-lan antibody and a delayed hemolytic transfusion reaction 6 days after transfusion. the rarity of lan-units may require a patient with anti-lan to be transfused with lan1 units, but close monitoring for delayed hemolysis is necessary even if the antibody is not demonstrable at the time of transfusion. delayed serologic transfusion reaction caused by auto-anti-f. karen yunker* 1 , andrea gerner 1 , lynne stewart 1 , carol sostok 1 , mollie bell 2 and gregory r halverson 2 . 1 st. elizabeth healthcare, 2 hoxworth blood center background/case studies: anti-f was first described in 1953 by rosenfield and coworkers in the serum of a hemophiliac who had been multiply transfused. the f antigen is comprised of the c and e antigens alligned in cis on the same chromosome, and is the 6th antigen assigned to the rh blood group system (isbt rh6). it is capable of causing significant transfusion reactions and mild hdfn. we report in this case a 56 year old caucasian male, admitted for evaluation of suspected t-cell lymphoma, who appears to have had a delayed serologic transfusion reaction (dstr) due to auto anti-f. abstract study design/method: antibody screen and compatibility testing was performed by automated solid phase (echo and neo, lmmucor, inc). red cell phenotyping was done by standard tube testing with commercial reagents following the manufacturers instructions. molecular genotyping was performed using the bloodchip assay (grifols, san marcos tx). elution studies were performed using the elu-kit ii (lmmucor, inc.) results/finding: the initial antibody screen (as) was negative and the patient was transfused 1 unit o-rbcs. two weeks later the patient received an additional o-rbc. within 4 days the hgb had decreased from 8.3 to 7.1 g/dl, the as and direct antiglobulin test (dat) were now positive, and ounits were incompatible. anti-f was identified in the patient's plasma and eluate. three additional units were requested for transfusion. due to the rarity of o-f-rbcs, the patient was transfused 3 r 1 r 1 (dce/dce) rbcs with no reported complications. the patient was discharged to follow up in clinic. molecular genotyping showed the patient was rhd deleted (rho* del) and had normal rhce (rhce*ce/rhce*ce) genes which predict a d-c-e-c1e1f1 phenotype. the rh phenotype and as was repeated on a sample collected 18 days later. the c typing was micro positive, mixed field only after 5 minute incubation. the other rh antigens were not mixed filed, and the as was non reactive. however, the dat was weakly positive with anti-lgg. no elution study was performed. conclusion: the expected post 24 hour hgb increment from the receipt of a standard unit of blood should be near 1 g/dl (or 3% hct.) throughout this patients hospitalization, the post-transfusion increments did not fully achieve this expectation. the first transfuion resulted in a 0.9 g/dl increase, and the second unit was only 0.5 g/dl. the last transfusion of 3 units increased by only 1.2 g/dl. less than three weeks later, the rhc antigen typing was microscopic/mixed field only after extended incubation, indicating the removal of 3 r 1 r 1 units was nearly complete. in a case from 1989, ohto and kariyone (transf. 1989; vol29, no. 3) reported a 51 cr ásurvival study of f1 rbcs in a patient with anti-f. they showed that the initital survival of f1 cells was fairly normal, however, after 18 days, there was a sudden increase of red cell destruction, and by day 27 all f1 cells were cleared from the circulation. it is not unusual to find auto-anti-f as many have been reported, however, it is unusual to find the auto-antibody has caused the clearance of three units of f-negative blood. this patient will be monitored to see if the autoantibody recurs and determine if it still has anti-f specificity. background/case studies: use of dithiothreitol (dtt) treated reagent red cells (rrbc) is increasing in blood banks as an effective way to negate the interfering panreactivity caused by daratumumab, an anti-cd38 drug for treatment of multiple myeloma. daily preparation of dtt-treated rrbc for testing of individual patients is burdensome for the laboratory and may delay patient care. we evaluated the effectiveness of batch-prepared dtt-treated rrbc, stored up to 9 days after treatment, in antibody detection tests. study design/methods: in-date rrbc (ortho clinical diagnostics, raritan nj) were selected based on phenotype to match the antisera to be tested. rrbc were treated with 0.2m dtt (sigma-aldrich, st. louis mo) and stored in reagent red cell diluent. rrbc were tested with commercial antisera (ortho clinical diagnostics, raritan nj and immucor, norcross ga) per the manufacturer's instructions for specificities from the rh, duffy, kidd and mns blood groups (see table 1 ). patient source antibodies (anti-d, anti-c) were also tested. testing was performed before dtt treatment, on the day of dtt treatment and up to 9 days following the dtt treatment of rrbc. reactions were graded using standard serological grading of 0 (negative) to 41 (positive) reaction strength. stored dtt-treated rrbc were also observed for hemolysis during the storage period. results/findings: see table 1 for a summary of results. commercial monoclonal and human source antisera, and patient source antibody, were reactive with the dtt-treated rrbc throughout the storage period. reactivity decreased by less than one reaction grade for all antisera and patient source antibodies tested. mild to moderate hemolysis was noted in the dtttreated rrbc's during the storage period. conclusion: dtt-treated rrbc showed adequate reactivity with various red cell antisera after storage for up to 9 days. this suggests that dtttreated reagent red cells can be stored for at least 9 days and used for the detection of alloantibodies with minimal effect on detection ability. batch preparation and storage of dtt-treated rrbc can increase testing efficiency and decrease turn-around-time when performing pre-transfusion testing for patients receiving anti-cd38 therapy. interference: more than just kell? marilyn stewart*, angela treml and geoffrey wool. university of chicago background/case studies: daratumumab (dara) is an anti-myeloma and anti-lymphoma agent that is known to interfere with routine blood bank antibody screening tests. dara is an igg monoclonal antibody that binds cd38 that is present on the red cell surface. at the university of chicago blood bank, we have seen many patients treated with dara and were showing this interfering reactivity. it has been well described that cd38 is a disulfidelinked molecule and its immune epitopes are disrupted by reducing agents such as dtt. we performed a validation of dtt-treatment of reagent rbc to abrogate dara interference. study design/methods: the validation was done to prove that dtt treated red cells could be used to screen patients receiving dara and still detect clinically significant allo-antibodies. screening cells and panel cells selected for dtt treatment were those rbc homozygous for clinically significant antigens, therefore allowing rule-outs of clinically significant antibodies in patient plasma. several patients that had received the dara drug protocol were selected for testing as well as many patients that had allo-and auto-antibodies (but not dara treatment). reagent screening cells and panel cells were treated with 0.2m dtt prepared using the sop from judd's methods in immunohematology and the aabb technical manual. the treated cells were preserved between testing episodes using alsever's solution, stored at abstract 2-5c, and observed for hemolysis (none was seen) for up to 21 days. all immunohematology testing using dtt-treated cells was performed using gel methodology. untreated and dtt treated cells were tested with anti k before any patient testing was done. the untreated cells reacted 2-41 with the anti k, and the treated cells were negative. these controls were run and tested each time dtt treatment was done. thirty eight patient samples, including six dara patient samples were tested. results/finding: of the six patients who had dara interference in their untreated antibody screens, all samples had negative reactions with the dtt treated cells except one patient, which had weak reactions in one cell. this specimen was repeated three times and all repeats had weak positive reactions in the same cell. this sample was sent to the arc reference lab for dtt treatment and all clinically significant antibodies were ruled out. patients with allo-antibodies present in their plasma did react with the dtt treated cells as would be expected based on the underlying alloantibody, with the exception of newly formed anti-e antibodies in 4 patients. plasma from these four patients with a nascent anti -e all showed no reactivity with dtt treated cells. plasma from fourteen patients with a long history of anti-e (greater than 6 months) did react with the dtt treated cells. conclusion: dtt treatment eliminates dara interference as previously described, but also unexpectedly lessens the ability of treated cells to react with nascent anti-e. because of the negative testing with some of the alloanti e antibodies, dara-treated patients at ucm will be given both kell and e negative blood if they have immunohematology testing performed using dtt reagent cells. mahboubeh rahmani* 1 , monique scott 2 , garcia curtis 2 , ellice wong 2 , alexa j siddon 1 and christopher a tormey 1 . 1 yale-new haven hospital, 2 va connecticut healthcare background/case studies: benign ethnic neutropenia (ben) seen in approximately 25% to 50% of persons of african descent is characterized by neutrophil count of <1.5x10 9 /l with no obvious cause and no increased susceptibility to infection or any other adverse effect. at present, there is no laboratory assay used to identify this condition and it is generally diagnosed on a clinical basis. in this study, we investigated whether duffy (fy) blood group phenotyping would be a potentially useful modality to help identify patients with ben; such testing could potentially be used as a surrogate test to prevent unnecessary further work up including bone marrow biopsy in the correct ethnic and clinical setting. study design/method: cases included patients clinically diagnosed with ben; and controls were chosen randomly from the pools of patients from whom a cbc and type and screen were checked for any other reason. cases and controls were tested for the rbc antigens fy a and fy b phenotype using serologic methods. the fy phenotype, absolute neutrophil count (anc), white blood cell (wbc), hemoglobin level, platelet count, and medical diagnoses were extracted from the medical record. where appropriate, data were compared statistically using the mann-whitney u test with significance set at p<0.05. results/finding: subjects who were clinically identified as having probable ben included 7 patients (mean age 48.7; all self-identified as african-american; 6/7 were male) and controls included 50 patients (mean age 68.5; 10 self-identified as african american; (50/50 male). all of the cases (100%) diagnosed with ben had fy(a-b-) phenotype. mean anc (1.95x10 3 /ul) and wbc counts (4.04x10 3 /ul) were significantly lower in the cases with ben and fy(a-b-) phenotype (p50.0008 and 0.001, respectively) compared with controls (mean anc 5 5.46x10 3 /ul ; mean wbc count 5 8.14x10 3 /ul). there was no significant difference in mean platelet counts (161x10 3 /ul vs 213x10 3 /ul; p50.2301) or mean hemoglobin levels (12.4 g/dl vs 11.7 g/dl; p50.6031) between the two groups. none of the patients with ben had an accompanying marrow-suppressive hematologic disorder based on record review; however, 18 subjects in the control group had accompanying conditions that were potentially marrow-suppressive including hepatocellular carcinoma, acute myeloid leukemia, and myelodysplastic syndrome. conclusion: testing for fy phenotype could potentially be used as a surrogate test in patients with chronic neutropenia in a correct ethnic and clinical setting for the diagnosis of ben. further studies regarding fy phenotyping comparing controls with neutropenia for any reason to our ben population are in progress to better determine the positive predictive value. these tests were compared to the provue for concordance. additional samples tested with anti-igg,-c3d were correlated against tube testing for the dat and antigen typing for: c, c, e, e, and k. results/findings: the ih-1000 had 100% concordance for all blood grouping assays. for ahg assays, the ih-1000 detected an anti-jka1e, anti-fya 1 warm antibody, antibody to a high incidence antigen and a warm antibody that were missed by the provue. the ih-1000 identified one additional anti-e not identified on the provue. discrepancies were also noted with the non-cord dat results. five samples were positive on the ih-1000 with anti-igg,-c3d vs. tube testing; reflecting the increased sensitivity of gel methodology over tube. the table below summarizes the results. conclusion: this study demonstrated that the ih-1000 analyzer and associated ih-system tm gel cards are equivalent to the ortho provue. with random access capability, minimal operator touchpoints, broad test menu and excellent assay performance, the ih-1000 is an ideal immunohematology system for the hospital transfusion service environment. chris elliott*, susan barnes, fiona lisle, debra smith and whitehouse natalie. background/case studies: the erytra eflexisv r (grifols) is a new fully automated, mid-size analyzer that performs pre-transfusion compatibility testing using dg gelv r technology. erytra eflexisv r analyzer performance, usability and adaptability to different workflows was evaluated in the routine environment of a large uk acute hospital transfusion laboratory. study design/methods: a comparison study was performed between the erytra eflexisv r and erytra (our routine system providing the reference platform). a total of 2944 tests were performed on 1,214 adult patient samples and 208 donor red cell units. erytrav r eflexis performance was evaluated according to a series of scenarios designed to simulate routine workload using the system in different configurations. concordance between systems was assessed and discrepancies analyzed. time to first result (ttfr), overall turn-around time (tat) total workload from first result to last result (throughput, results/h), manual "hands-on" time and walk-away time were all recorded. for ease of use evaluation, we ranked usability features with number of steps and timing of activities including sample sort and loading, routine testing, post-run procedures, consumables used, and space requirements. fault recognition and messaging was assessed by simulating failures e.g. reagent absence. results/findings: blood grouping, antibody screening, antibody identification (using panels), direct antiglobulin test, red cell phenotyping and serological crossmatching were successfully tested. concordant results between the erytra eflexisv r analyzer and reference method were obtained in 99.9% of samples tested. there were 4 discrepancies, all antibody screening (2 false positives, 1 failure to detect a very weak prophylactic anti d and 1 positive reaction not detected on the erytra but panels on both systems suggested a genuine anti cw). ttfr and tat depended significantly on a number of factors including; number and variety of tests requested and whether the stat functions were activated. the analyser seemed to prioritise antibody screening prioritization of the group, especially for stat samples, was considered preferable the laboratory team found the software easy to use with some improvements over existing ertyra software. physical design of the analyser was considered good with easy access to almost all areas. probe changing was quick and simple. while the analyser successfully flagged all error scenarios some messages were considered misleading and could be better phrased. conclusion: results showed the erytra eflexisv r offered a robust automated solution for routine transfusion testing. the device could comfortably deal with a medium laboratory (processing 80-100 group and screens per day). it is very flexible being able to deliver grouping, antibody screening and identification, dat, phenotyping and serological crossmatching ,compensating for its' single probe and wash station by clever use of incubators, centrifuges and design features. this allows a compact design with maximum flexibility without compromising on turnaround times cp201 evaluation of two monoclonal anti-e as reagents for the detection of the rh e antigen and its variants gregory a. denomme* 1 , kathleen bensing 1 , michael schanen 1 , cindy piefer 1 , randall w. velliquette 2 , christine lomas-francis 2 and connie m. westhoff 2 . 1 immunohematology reference laboratory, versiti/bloodcenter of wisconsin, 2 immunohematology and genomics laboratory, new york blood center background/case studies: monoclonal antibodies are used as reagents for automated and manual phenotyping. false negative phenotypings have implications for variant antigens; e.g. altered c antigen mistyped as a cblood unit stimulating anti-c in a c-recipient. the development of new 7/0 7/0 7/0 cecf 1/1 2/0 2/0 rhce*ce or rhce*ce compound heterozygotes ce254g 1 ce733g or ce48c,733g or ces or ceti 6/0 6/0 6/0 ce733g 1 ce48c,712g or 48c,733g 4/0 4/0 4/0 ce733g 1 ces or cemo or ceek or ceek(var) or cern 8/0 8/0 8/0 ce48c,733g 1 ce48c,712g or cemo or ceti 2/0 2/0 2/0 ce48c,712g/ce254g/733g; ces/ceti; cear/ceek; ceek/cejal; cemo/cebi 7/0 7/0 7/0 total 106/8 110/4 113/2 142a transfusion 2017 vol. 57 supplement s3 reagents should include an evaluation of antigen variants to confirm fidelity. we evaluated two monoclonal anti-e reagents with comparator reagent using a large panel of molecular confirmed rh e variants. study design/method: two monoclonal anti-e clones, rd9/4 and rd12/2, and a licensed comparator anti-e (p3gd5121ms63), all from diagast (loos, france), were evaluated. rbc samples were either recovered from frozen storage (n 5 42) or edta blood from donors (n 5 72) and were tested using a manual tube method or on a pk7300 automated platform. a score 6 (11) or greater was deemed acceptable for manual tube and a positive call for automated testing. results were tabulated by complexity of rhce*ce alleles (table 1) . results/finding: the specificity of the monoclonal anti-e were confirmed using common rhce haplotypes: r 1 r 1 , r 2 r 2 , r 1 r, and rr. twenty-one different rhce*ce alleles were included in the extensive panel: 40 were rhce*ce that were in trans to rhce*ce; 16 were various rhce*ce plus rhce*ce48c compound heterozygotes; 31 were rhce*ce or rhce*ce homozygotes; 27 were various rhce*ce and rhce*ce compound heterozygotes. the comparator reagent was negative or unacceptably weak for 6 rhce*ce alleles in trans to rhce*ce (rhce*cear, rhce*cemo, rhce*-cejal, rhce*cehar), with 1 rhce*cear/rhce*ce48c compound heterozygote, and with 1 rhce*cecf homozygote. rhce*cear, rhce*cemo, rhce*cejal, and 1 of 2 rhce*cecf homozygotes were detected using the comparator reagent. rd9/4 and rd12/2 failed with 4 and 1 e variants, respectively (table 1) . failure to detect the e variants was observed using both manual tube and automated methods for the comparator and the rd9/4 clone. none of the reagents detected e antigen variant expressed on 1 example of rhce*cehar/rhce*ce. conclusion: rd9/4 and rd12/2 anti-e reacted with more e variants than the comparator reagent. the e antigen encoded by rhce*jal and rhce*ar is not always detected when in trans to rhce*ce. however, double-dose expression was detected suggesting that the 3 monoclonal reagents bind weakly to the respective altered e antigen epitopes. the e antigen encoded by rhce*cehar continues to be a challenge to detect. meihong liu*, teresita mercado, orieji illoh, maria rios and zhugong liu. obrr, cber, fda background/case studies: extended molecular typing of a large number of blood donors can increase the likelihood of identifying donor red blood cells (rbcs) that match those of the recipient. this is especially important in the management of chronically-transfused patients and patients with rbc alloantibodies. several high-throughput multiplex blood group molecular typing platforms have been developed to determine blood group antigen phenotypes. targeted next-generation sequencing (ngs) provides comprehensive sequence information focusing on specified genomic regions, and allows the simultaneous detection of genetic variants from multiple genes in a large number of samples. we developed and evaluated targeted ngs assays using two different target enrichment platforms for extended blood group genotyping. study design/method: two custom design platforms sureselect and halo-plex were used independently for preparation of probes that target the entire genes of 19 blood group genes associated with the expression of 56 blood group antigens from 17 blood group systems. we used the illumina's hiseq 2000/2500 system to perform next generation sequencing first on sureselect-enriched genes from 16 dna reference samples with average target design coverage of 97.5%, and then on haloplex-enriched genes from 32 dna reference samples with average target design coverage above 97.0%. twelve samples were enriched and sequenced in both methods to allow a direct comparison. all reference samples were previously characterized for 38 blood group genetic variants in these 19 genes using taqman snp assay and sanger sequencing assay. serological data were also available for these samples. the ngs data were analyzed by clc genomic workbench. sequencing variants were detected and annotated using dbsnp database. blood group genotype calls by the two targeted ngs methods were compared with the reference results. results/finding: for the two targeted ngs methods, we evaluated and compared the target enrichment efficiency, off-target enrichment, quality of ngs, sequencing coverage, and genotype concordance. a higher percentage of the haloplex reads (80.54%) were mapped to the target regions relative to the sureselect reads (29.23%). the mean sequence coverage depth of the targeted bases was around 200x for sureselect method and 300x for haloplex method. some exons, such as rhd exons 4 and 8, 10, rhce exon 10, ermap exons 5 and 12, cd55 exons 10 and 11, cr1 gene (most exons) and gypb exon 5, are consistently covered with less than 10x coverage by both sureselect and haloplex targeted ngs methods. both methods detected rhd gene deletion in a few representative samples. the genotype call concordance on 38 blood group genetic variants was assessed by comparing ngs results to taqman genotyping and sanger sequencing results, and more than 90% concordance was obtained for both targeted ngs methods. incorrect calls were restricted to four complex blood group genes: mns, rhd, rhce and abo, and involved mainly heterozygous variants and indels. conclusion: using two targeted ngs methods, we have correctly detected more than 90% blood group genetic variants in 19 selected genes. evidence rhce*cehar does not encode for rh34 (hr b ) antigen debra j bailey* 1 , trina horn 2 , paul mansfield 2 , najmi qazi 1 , pamela nickle 2 , jessica keller 2 , margaret a keller 2 and jan r hamilton 1 . background/case studies: the rhce gene has many variant forms, yet for many, the phenotypes encoded by these variant alleles is unknown or incomplete. new information can be elucidated when two altered alleles or haplotypes are expressed in an individual with subsequent alloantibody formation. the rhce*cehar allele was first described in 1996 and has a phenotype of c2e2c1e1 w f1 w , g2, hr 0 1 w , hr2, hr s 2, rh:33, rh:50 with a partial d antigen expression. we describe new information regarding an rh haplotype that includes an rhce*cehar allele and its apparent rh:234 (hr b 2) expression. study design/method: rbc typing was performed by standard tube methods with polyclonal and monoclonal antisera. antibody identification studies were performed by standard tube hemagglutination methods by published techniques. molecular immunohematology testing was performed on genomic dna extracted from whole blood and included hea, rhd and rhce beadchips (immucor) and pcr-rflp analysis for rhce c.254c>g and rhd c.1136c>t. results/finding: a sample from an african american female with a history of an anti-e and anti-k was evaluated for unexpected antibodies. her red cell serologic rh phenotype on an untransfused sample was d1c1e2c1e1. her plasma contained an alloanti-s and an antibody that reacted strongly with all random e2k2s2 reagent red cells except her own. the unidentified reactivity persisted following ficin and dtt pretreatment of reagent red cells. only d22 and dc2 red cells were non-reactive in initial tests. differential adsorption studies excluded antibodies to all other common antigens and hr b except e, s and k. when subsequent examples of e2s2k2 red cells homozygous for the rhd*diiia-ce(4-7)-d, rhce*-ce48c,733g,1006t haplotype (i.e., r' s /r' s ) and rhd*diiia, rhce*-ce48c,733g,1006t/ rhd*diiia-ce(4-7)-d, rhce*ce48c,733g,1006t (i.e., bastiaan genotype) were found to be non-reactive with the patient's plasma, the antibody specificity was determined to be anti-hr b . the patient's red cell antigen genotype identified the following probable rh haplotypes: rhd*01, rhce*cehar and rhd*diiia-ce(4-7)-d, rhce*ce48c,733g,1006t. additional antigen typing of the patient's red cells with unlicensed antisera indicated an hr1 (2 of 2 sources) and hr b 2 (2 of 3 sources) phenotype. conclusion: the rhd*diiia-ce(4-7)-d, rhce*ce48c,733g,1006t haplotype is one of the rh haplotypes expressed by the original hr b 2 individual bastiaan. the hr b antigen status of red cells of individuals with the rhce*-cehar allele has not been described. we report an individual with the probable rhd*01, rhce*cehar and rhd*diiia-ce(4-7)-d, rhce*ce48c,733g,1006t rh haplotypes and production of alloanti-hr b . the specificity of the alloantibody produced and the red cell hr b serologic antigen type supports the conclusion the variant allele rhce*cehar does not encode for the hr b antigen. excluding clinically significant alloantibodies in the presence of interfering antibodies with high-titer, low-avidity characteristics. background/case studies: high-titer, low-avidity antibodies (htla) are a group of clinically insignificant antibodies (ab) directed against highprevalence red cell antigens. they interfere with the exclusion of clinically significant red cell ab and crossmatch testing, leading to long work-ups and potential transfusion delays. we often use automated solid phase red cell adherence assay antibody panels (sp) when htla interference is seen by other methods, and undertook this study to determine its efficacy. study design/methods: a search of the laboratory information system database was conducted for patients with htla between 1/1/2006 and 3/ 31/2017. all patient samples with available records of the full serological investigation were reviewed for testing method and results, with specific attention to the value of a given test method in permitting exclusion of clinically significant ab (rule out). results/findings: over approximately 11 years, 81 patients had htla established at least once by titration studies. serological investigations on a total of 118 samples using a combination of gel, sp, and peg and liss tube methods, and occasional dtt and ficin panels, found that htla interference noted most frequently in gel (primary method) was, indeed, less often seen with sp. however, the proportion of cases achieving rule out on sp was no greater than that with peg testing (table) . for samples where rule out could not be performed with a combination of methods, patients were assigned to phenotype-matched transfusions, or testing was referred to a reference laboratory. reference testing on 20 samples was successful in rule out in 60% of cases. in an additional 12 patient samples, with negative antibody screens, htla were identified upon work-up for incompatible crossmatches. conclusion: sp is useful in avoiding interference from htla, but this conclusion is limited because sp was performed in only 40% of samples, and the inability to use select cell panels with sp made it difficult to complete rule out on samples containing multiple ab. peg testing was available for 71% of samples, and was at least as effective. further, manual testing allowed flexibility in selecting test cells when other ab were present. both sp and peg testing may be used alone or in combination to avoid interference due to htla, and can potentially decrease the number of patients requiring phenotype-matched units due to incomplete serological evaluations. background/case studies: the dau family of rhd alleles is characterized by c.1136c>t (p.thr379met). the dau0 allele harbors only this change, is not associated with depressed or altered d antigen expression, and is the ancestral allele from which other dau alleles are purported to have evolved. srivastava et al (transfusion 2016, 56:2520) recently summarized serologic characteristics and associated anti-d alloimmunization for 18 dau family alleles. we investigated two samples with the c.1136c>t change referred with weak d antigen expression. study design/method: serologic testing was performed by standard tube methods using licensed anti-d reagents and the albaclone partial rhd typing kit. genomic dna was isolated from wbcs and used in manual and array assays and for amplification and sequencing rhd. results/finding: sample 1 was from a 17 yo multiracial female. her rbcs reacted 11 s at immediate spin (is) and 31 in iat with immucor gammaclone and series 4 and 5, and mi1 at is and 41 in iat with ortho bioclone anti-d. rbcs did not react with 2 of 12 (lhm 174/102 & 57/17) anti-d in the partial d typing kit. this pattern did not match any of the defined partial d epitope patterns. rhd beadchip found no changes but rflp detected c.1136c>t characteristic of dau. rhd sequencing confirmed c.1136c>t and identified two adjacent changes, c.787g>t and c.788g>t (c.787_788delinstt), in exon 5 encoding p.gly263leu. sample 2 rbcs reacted 1w at iat with both ortho bioclone and quotient albaclone delta, but were non-reactive with immucor gamma-clone, series 4 and 5, and quotient albaclone blend and alpha anti-d. papain treated rbcs were 11s in iat with ortho bioclone. these results suggested a d el like phenotype. rhd beadchip found no changes but rflp detected c.1136c>t. sequencing confirmed c.1136c>t and found a new c.761c>t change (p.ser254-leu) in exon 5. the c.761t has not been reported, but c.761g encodes a stop codon (p.ser254stop) in japanese (vox sang 2015, 109:359). conclusion: we report two new alleles: rhd with c.787_788delinstt (p.gly263leu) and rhd with c.761c>t (p.ser254leu), both also carrying the c.1136c>t (p.thr379met) characteristic of the african dau cluster. d antigen associated with p.263leu is a partial d antigen with a novel epitope pattern. the p.254leu change is associated with a del-like phenotype, the first observed to our knowledge for a dau allele, and d antigen on the rbcs is not detected in iat by 5/7 commercial anti-d. the rhd nucleotide changes reported here are not in dbsnp database. this study brings the dau family of alleles to 21. the number and diversity of alleles in the dau cluster supports that the c.1136c>t change is a major ancestral african background allele (wagner et al, blood 2002,100:306). tae eun kim*. krc btri background/case studies: there have been the cases of anti-d alloimmunization caused by the transfusion of serologically d negative blood component. by analysis of genotype of the blood component, all of them were confirmed as asian type del. for that reason, the application of genetic analysis for the blood donor has been required in addition to serological assay. we established the algorithm for the genetic analysis of rhdin blood donors. in this study, we would introduce the experience of the application of the algorithm and the results in the preliminary test. study design/method: from september 2016 to present day we got 130 samples of repeated blood donors who are known to be d negative, c positive and/or e negative from 15 blood centers. we obtained the consent for the test from all of the donors who provided samples. as a genetic analysis, we accomplished polymerase chain reaction with sequence-specific primers (pcr-ssp) for the region of promoter, exon 4, exon 7 and exon 10 in rhd gene. based on the results of pcr-ssp, we discriminated the results into total rhd deletion, rhd-ce-d hybrid and rhd variant. when the results were discriminated to be rhd variant, we additionally analyzed the sequence of exon 9 to confirm the existence of c.1227g>a and c.1222t>a variations. for the sample with indeterminate results, we performed sequencing for the full region of exon. when the result was confirmed to be rhd deletion or rhd-ce-d hybrid, the blood components were regarded as rhd negative. when the result was confirmed to be rhdvariant, the blood components were regarded as rhd positive. blood components were not supplied until the final results were obtained. results/finding: for the 130 sample, we identified 71 cases (54.6%) of total rhd deletion, 18 cases (13.8%) of rhd-ce-d hybrid, and 41 cases (31.5%) of rhd variant. 39 of rhd variant were determined to be asian type del with c.1227g>a variation. 2 cases of rhdvariant were regarded to be unknown variation. conclusion: the frequency of rhd variant in this study was 10 % higher than that of the general d negative donors not considering rhce phenotype in a previous study. for that reason, we considered that the genetic analysis of rhd targeting the donors of d negative, c positive and/or e negative is more efficient approach to identify rhd variant and better way to improve blood safety in the transfusion medicine related with rhd negative blood donors. lei fang tsai*, ping chun wu, shu hui feng, yi wen tsai, ming hung chen and shun chung pai. taipei blood center, taiwan blood services foundation background/case studies: certain abo subgroups or physiologic conditions may lead to mixed-field agglutination on abo typing among blood donors. the b 3 phenotype was found to be the most common subgroup in taiwanese. however, it is hard to distinguish the b 3 phenotype from other b subtypes also with mixed-field agglutination using routine serology without the genotype. this study aimed to evaluate if flow cytometric method could alternatively differentiate different b subtypes with mixed-field agglutination rather than using molecular genotyping. study design/method: blood samples from 30 taiwanese blood donors exhibiting known common abo phenotypes were included to establish normal flow cytometric patterns and genotyped. blood samples (n552) from b subtype donors with mixed-field agglutination by routine serology (tube method and gel card) were further analyzed by flow cytometry and genotyping. flow cytometric method was performed by facscalibur flow cytometry using the gamma-clone anti-a and -b. for genotyping, exon 6 and exon 7 of the abo gene were amplified and sequenced. the abo*b3.03 allele was confirmed by pcr-rflp analysis. results/finding: among 52 subjects with b 3 or ab 3 phenotypes, 47 were genotyped as abo*b3.03. the abo*b3.03 group performed similar characteristic flow cytometric pattern and the profile was reproducible over time. the pattern showed the main population of cells expressed no b antigen, while a percentage (37.81 6 6.62) of the rbcs exhibited b antigen levels diminishing with increasing of fluorescence. other 5 subjects with b 3 or ab 3 subjects, genotyped as abo*b3.06(n51), abo*bw.03(n51), abo*bw.11(n51), abo*bw.12(n51) and abo*bw.29(n51), displayed flow patterns differed from the abo*b3.03 group. the abo*bw.03, abo*bw.11 and abo*b3.06 subjects also showed a main population of cells expressed no b antigen and, however, less percentage of rbcs exhibited b antigen levels (<10% in abo*bw.03 and abo*bw.11 subjects and <20% in abo*b3.06 subject). both abo*bw.12 and abo*bw.29 displayed a wedge-shaped pattern. conclusion: the flow cytometric method for the detection of b antigens on rbc might be useful in discriminating between b subtypes with mixed-field agglutination, especially abo*b3.03 genotype. this approach could assist the serological abo subgrouping in clinical reference laboratory. frequencies and specificities of "solid-phase only" detected erythrocyte antibodies: is solid phase testing worth the headache? karen finegan*, karen gray, jill adamski, theresa kinard and qun lu. background/case studies: an effort to re-evaluate automated testing platforms (automated solid-phase red blood cell adherence vs automated gel column agglutination) was recently initiated due to the perception of excessive equivocal reactions from the solid-phase resulting in "unnecessary" workup at one site of a hospital system. the data available from parallel testing on solid-phase, gel, and peg performed at another cite of the same hospital system was collected and evaluated to determine the frequencies and specificities of "solid-phase only" detected erythrocyte antibodies and to see if solid-phase only antibody workup is necessary for patient care. study design/methods: throughout 2016, the transfusion service used automated solid-phase red blood cell (rbc) adherence as the primary method for antibody screening and identification. all solid-phase antibody screen positive samples were re-tested using both gel column agglutination and peg method manually in order to determine which method should be used for antiglobulin phase crossmatch of rbc products. all antibody screen results on three methods and final antibody identification results were transcribed into a spread sheet and analyzed. results/findings: a total of 398 patients were positive on solid-phase antibody screen and re-tested on gel and peg antibody screen. in 12% (n549) patients antibody reactivity observed in solid phase only and the concurrent gel and peg testing were completely negative. of them clinically significant rbc alloantibodies, warm autoantibodies, clinically insignificant antibodies were identified in 22% (n511), 4% (n52), and 74% (n536) of the cases, respectively. rbc alloantibodies identified in solid-phase only included anti-e (n54), anti-jka (n53), anti-k (n52), anti-jkb (n51), both anti-e and anti-c (n51) (see table 1 ). conclusion: solid-phase only rbc antibodies are clinically important in a significant portion of cases (roughly 1 in 4 cases). workup for solid-phase only antibodies is not "unnecessary" workload. transfusion of corresponding antigen negative rbcs to these patients prevented possible hemolytic transfusion reactions. full-length nucleotide sequence of ackr1 alleles encoding duffy (fy) antigens in africans of ethiopia qinan yin*, kshitij srivastava, addisalem taye-makuria and willy a flegel. background/case studies: the human ackr1 gene (previously known as darc), comprising two exons and a single intron, encodes a multi-pass trans-membrane glycoprotein expressing the duffy blood group antigens (fy). the duffy protein acts as a chemokine receptor for various proinflammatory cytokines and for the malaria parasites plasmodium vivax and p. knowlesi. the study of fy variants in the low altitude and tropical gambela region is important, as malaria is endemic and the endogenous population is living in this region for a long time. in the present study, we determined the full length nucleotide sequence of the ackr1 gene encoding fy antigens in donors from ethiopia's southwestern gambela region. study design/method: edta-anticoagulated whole blood was collected from study 60 volunteers in the gambela region (nct01282021). the whole ackr1 gene was amplified in one reaction covering 12,125 base pairs (bp). this primary amplicon was re-amplified using nested primers covering 5782 nucleotides. nucleotide sequence was obtained by 14 sequencing reactions and manually annotated using ncbi refseq ng_011626.2. the sequencing covered 1008 bp of both exons, 480 bp of intron, 2101 bp of 5'-flanking region, 947 bp of 5'-utr, 53 bp of 3'-utr and 1092 bp of 3'-flanking region and encompassed all the 470 variations present in dbsnp and nhlbi esp databases. results/finding: among the 60 samples, a total of 15 snps, including one novel snp in 5'-utr were observed. 4 snps occurred in the exons, 5 in 5'and 3'flanking region, 4 in 5'-utr and 2 in the intron. all 60 individuals carried the snp indicative of the common fy:2 phenotype; while 58 individuals were homozygous and 1 was heterozygous for the gata box mutation. no splice site mutation was detected. as 46 individuals were observed as being homozygous or heterozygous for 1 snp, we could unambiguously assign 8 distinct alleles. in the remaining 14 individuals with 2 or more heterozygous snps, allele specific pcr is required to identify the alleles. conclusion: we sequenced more than 5.5 kb of the ackr1 gene and identified at least 8 different alleles. the present study found that the vast majority of alleles (117/120) in the gambela population as defined by 15 snps, were similar to the clinically relevant fy*02n.01 allele, which in turn is defined by only 2 snps at positions c.1-67t>c and c.125g>a. out of the remaining 3 alleles, 2 were similar to fy*02 with the fy(b1) phenotype and 1 was similar to fy*02w.01 with the fy x phenotype. the high frequency of fy*02n.01 (95%) in this study is similar to other studies conducted in western, central and south-eastern regions from gambia to mozambique (95%-100%). a more detailed analysis, including other regions of ethiopia, will be useful to support transfusion care in the us for ethiopian-americans, the majority of whom may be of mixed ethiopian ethnical background. judith aeschlimann*, sunitha vege, christine lomas-francis and connie m. westhoff. immunohematology and genomics laboratory, new york blood center background/case studies: the homology, proximity, and inverted orientation of rhd and rhce on the chromosome favor gene conversion events. regions of rhd are transferred into rhce and conversely, resulting in hybrid alleles that encode novel or the absence of high prevalence antigens. rhd*diiia-ce(4-7)-d is the most common hybrid and is found in african blacks. it arose by conversion of exons 4-7 of rhce*ces into rhd*diiia and no longer encodes d antigen, rather (somewhat confusingly) encodes partial c antigen from the rhd locus. this hybrid allele is in cis to rhce*-ces, together known as the r's haplotype. we investigated atypical rh genotyping results in three samples; two associated with weak d typing and one patient with sickle cell disease (scd). study design/method: serologic testing was by standard methods. genomic dna was isolated from wbcs. all samples were investigated by hea precisetype, rhd and rhce beadchip, rflp, and rh-cdna sequencing. snp-specific sequencing was used to establish linkage/phasing. results/finding: sample 1 (male) and sample 2 (multiracial female), both c1c2e2e1 (presumed r1r1), presented with weaker than expected d typing; 11 is and 31/41 at iat. rhd beadchip identified the common african rhd*diiia-ce(4-7)-d hybrid encoding partial c antigen with apparent conventional rhd in trans. these results did not provide an explanation for weak d antigen. hea indicated rhce*ce /ce, concordant with the rh phenotype, but c.733c>g and c.1006g>t (heterozygous) was also detected. as rhce*ce with 733g and 1006t has not been reported, rh-cdna analysis was done. transcripts from the rhce locus included one conventional rhce*ce in trans to rhce*ces with exons 2 and 3 replaced with rhd*diiia, and from the rhd locus, one conventional rhd and the hybrid rhd* diiia-ce(4-7)-d were found in both samples. sample 3 (scd male), d1c2e2c1e1, by rh beadchip had one conventional rhd and rhd*diii type 8, and rhce*ce733g/ces. as rhd*diiia type 8 has never been found with either of these rhce alleles, rh-cdna analysis was performed. transcripts representing a unique conversion event at the rhd locus, specifically rhce*ce(48c) exons 1 and 2 had replaced those exons in the common hybrid rhd*diiia-ce(4-7)-d and expression of partial c antigen was lost these unique hybrid alleles have been deposited as genbank#: ky926711and ky926710. we report two different and novel complex rh rearrangements: two samples thought to be r1r1 had a unique rhce locus representing a gene conversion into rhce*ces, designated rhce*ces-diiia(2-3)-ce. in kind, a sample genotyped as diii type 8 rather had a novel rhd locus representing a gene conversion into the common hybrid, designated as rhd*ce48c(1-2)-diiia(3)-ces(4-7)-d . these represent novel events on the r's haplotype that can confound rh genotyping interpretations. interestingly, samples 2 and 3 have weaker than expected d antigen typing, despite the presence of a conventional rhd with rhce*ce [r1 haplotype (dce)]. it is important to further investigate samples with unconventional results when interpreting rh genotypes. high-frequency antibodies anti-lu(b-) and anti-yt(a-) in a multi-transfused patient: a case study nadia baillargeon*, carole ethier, cynthia parent, jessica constanzo-yanez, maryse st-louis, marie-claire chevrier and andre lebrun. hema-quebec background/case studies: a 81-year-old caucasian female was referred to our immunohematology reference laboratory (irl) for serological investigation. she was diagnosed with anemia, renal failure and cardiac history. her hemoglobin level was recorded at 81g/l. her pregnancy history was not provided. she had received 5 units of packed red blood cells (rbcs) in the past including 1 unit within the last 3 months. none of the transfused unit was phenotypically-matched. the referring hospital obtained panreactivity in gel with liss-suspended rbcs and ficin-treated rbcs and negative direct antiglobulin test (dat) and autocontrol (at). study design/methods: abo/rh, dat and antibody identification were performed by h ema-qu ebec's irl according to approved techniques. in addition to liss-suspended rbcs and papain-treated rbcs, trypsin-treated and chemical-treated reagent rbcs such as dithiothreitol (dtt) were tested. alloadsorption were done using papain-treated allogeneic rbcs (r 1 r 1 , r 2 r 2 , rr). id core xt platform (progenika biopharm / grifols, vizcaya, spain) was used to analyse 29 polymorphisms which determine 37 antigens including carthright and lutheran blood groups. pcr-ssp (sequence specific primer) and pcr-rflp (restriction fragment length polymorphism) were also performed to verify the absence of the high frequency antigens yt a and lu b . sibling samples were also requested to conduct a family study. results/findings: initial serologic testing showed strongly reactive panels in gel with liss suspended rbcs, papain-treated rbcs as well as trypsintreated rbcs and dtt-treated rbcs but negative at in each media leading to a probable antibody directed against high-frequency antigen. alloadsorption procedure allowed the identification of an anti-jk a . a select panel of high frequency antigens absent in caucasian population was tested. the patient's sera react weakly with one jk(a-), lu(a-b-) reagent cell. in the meantime, genotyping results confirm the probable phenotype of the patient as jk(a-) lu(b-) yt(a-). additional testing in gel using trypsin and dtt differential effects on antigens lu(b) and yt(a) were performed to confirm antibody specificities. no rbcs unit jk(a-) lu(b-) yt(a-) were available for transfusion. selected units were jk(a-) and lu(b-) as alloanti-yt a are known to cause none to moderate transfusion reactions. her daughter' sample were types as yt(a1) and lu(b1). conclusion: serological study showed the presence of an anti-jk a in addition to two antibodies directed against high prevalence antigen namely anti-lu b and anti-yt a . the association of various selected serologic procedures combined with ethnic clues and genotyping results serves to solve this uncommon antibody combination. background/case studies: the kel blood group system, consisting of 36 antigens encoded by the kel gene, is organized into 19 exons. there are approximately 30 kel alleles associated with a kell null phenotype (k 0 ) in which no kell antigens are expressed, and 12 alleles associated with a kell mod phenotype (k mod ). individuals with the k mod phenotype express very weak amounts of antigen on the surface of the rbc, and expression levels vary based on the allele present. here we describe the molecular and serologic testing that was performed in the case of a 53 year-old hispanic male blood donor whose rbcs phenotyped k-k-js(b-) kp(b-). study design/method: the blood donor was phenotyped for k, k, kp b and js b antigens using standard tube agglutination methods. adsorption and elution studies of the donor red cells were performed using commercial anti-k antisera (american national red cross). genomic dna (gdna) was isolated from an edta blood tube using standard techniques. dna was genotyped for human erythrocyte antigens using the precisetype tm hea molecular beadchip (immucor). exons 10, 11, 12,13 and 14 and flanking intron sequences were amplified and sequenced. total rna was extracted using rneasy lipid tissue mini kit (qiagen) and kel cdna was amplified and the resulting pcr product was subjected to sanger sequence analysis and aligned using sequencher (genecodes). results/finding: precisetype tm hea molecular beadchip testing predicted the sample to be k-k1 kp(a-b1) js(a-b1). kel-cdna sequence analysis was performed and detected a single transcript species with c.578c, c.841c, 1790t, and missing the sequences corresponding to exons 11, 12 and 13. amplification of the exons from gdna did not identify any nucleotide changes when compared to the reference sequence and the splice sites were intact. cdna analysis was repeated and the same aberrant transcript was detected. adsorption and elution studies of the k antigen demonstrated weak anti-k reactive after 37c incubation at the peg-igg-agt phase. conclusion: here we describe a donor homozygous for a novel kel*02 allele. this donor was presumed to have a k 0 phenotype based on serology, but after molecular testing, has been reclassified as a k mod phenotype with extremely weak expression of k. the discovery of the aberrant transcript led to adsorption and elution studies to confirm the presence of weakly expressed k antigen on the red cells. the 11 variant alleles reported to date (http://www.isbtweb.org/working-parties/red-cell-immunogenetics-and-bloodgroup-terminology/) are associated with missense mutations. in contrast, the allele reported here is associated aberrant mrna transcript. we propose that this allele be named kel*02m.12. here we report a case of a possible novel b subgroup observed in a pregnant black female. the patient specimen was referred to our reference laboratory to investigate a possible abo discrepancy. the referring facility reported the patient's red blood cells were nonreactive with reagent anti-a and anti-b and the patient's plasma was reactive with a cells, but nonreactive with b cells using automated gel methodology. study design/methods: serological testing of the patient's red blood cells was performed using routine and enhancement methods. molecular testing by pcr-rflp was performed to determine the patient's genetic abo typing and predicted abo phenotype. results/findings: serological testing of the patient's red blood cells is summarized in table 1 ; similar results were obtained with multiple sources of antisera. enzyme treatment failed to enhance reactivity. patient sera strongly agglutinated a1 and a2 cells, but failed to agglutinate multiple sources of b cells at all phases of testing. molecular testing by pcr-rflp resulted in an uncommon banding pattern and indicated the presence of c.261 deleted g, characteristic of o alleles, c.467t, characteristic of a2 and some uncommon o alleles, and c.703a and c.1096a, characteristic of b alleles. genomic sequencing of exons 6 and 7 confirmed the presence of an o allele, abo*o09 261del g, 318t, 467t), and the presence of a b allele (297g, 526g, 657t, 703a, 796a, 803c, and 930a), but did not reveal any changes associated with previously reported weak subgroups of b. conclusion: while serologic abnormalities in pregnancy have been reported due to decreased antigen expression, the unusually weak reactions observed when testing this patient are unlikely due to pregnancy alone. additional abo gene sequencing is required to determine the specific allele mutation responsible for this weakened antigen expression. carine arnoni* 1 , tatiane vendrame 2 , janaína muniz 3 , diana gazito 2 , afonso cortez 2 , lilian castilho 4 and flavia latini 2 . 1 associa, 2 associac¸ão beneficente de coleta de sangue, 3 hemocentro de são jos e do rio preto, 4 hemocentro unicamp background/case studies: after the elucidation of the molecular basis of vel, molecular tools have been used to explain the reduced expression of vel antigen in different populations. negative or weak reactions are generally related to the 17-bp deletion in smim1 in homozygous or heterozygous status. however, other nucleotide changes have been described to reduce the vel expression, as for example, the major a allele of the snp rs1175550 located in the second intron of the gene, a regulatory region in erythroblasts. this study aimed to characterize the genetic changes related to atypical vel expression in a brazilian population. study design/method: a total of 400 blood donor samples from the southeast region of brazil were typed for vel with an anti-vel serum from our inventory in gel-iat. samples typed as vel-negative were further analyzed by adsorption-elution. molecular study was performed in samples with negative results, in samples reacting 21 and in samples with reactivity of 31. dna was isolated from peripheral blood and smim1 was sequenced. results/finding: from 400 donor samples studied, 4 were serologically vel negative by gel-iat but positive by adsorption-elution, 158 presented a 21 reaction and the remained samples showed a reactivity of 31. genotyping results showed that the 4 samples with negative results and 5 of 26 samples that presented 21 reaction were heterozygous for the 17 bp deletion and presented the a allele rs1175550 in homozygous status. from the 21 of 26 remaining samples with reactivity of 21, 19 (90%) had the a allele of rs1175550 and 14 (66.7%) had the a allele of rs6673829. in contrast, in the 16 samples with stronger reactions we found the a allele of rs1175550 in 5 (31.25%) samples and the a allele of rs6673829 in 3 (18.75%) samples. conclusion: the molecular changes rs6673829 and rs1175550 are located in intron 2 distancing 38 nucleotides. this study reinforces the association of the a allele of rs1175550 with reduction of vel expression and suggests the involvement of a new rs6673829 change in vel expression. in conclusion, the several patterns of vel expression found in different populations can be influenced by different molecular changes. background/case studies: the d antigen is the most immunogenic antigen after abo. consequences of misclassification of the d-antigen in patients or donors can be severe. some persons inherit mutations resulting in quantitative reductions of d antigen on the cell surface (weak d), some inherit rh haplotypes that result in biochemical effects that reduce the availability of the d antigens to reagent anti-sera (ceppellini effect), and others inherit d genes which are qualitatively different than wild type d. these latter individuals are often not identified until after they have formed anti-d. we hypothesize that some of these persons at risk of forming anti-d might be uncovered if they have weak and/or disparate d typing results with reagents that recognize different epitopes of the d antigen. study design/methods: all testing was performed using microtiter-well direct agglutination on the galileoneo or galileoecho (immucor, norcross,ga). any specimen that did not react as 0 (rh negative), or !31 on the neo or !21 on the echo (rh positive) for both series 4 and series 5 anti-d antisera were included. patients with discrepant historical types also were evaluated. any specimens meeting the inclusion criteria were tested on the neo, echo, and by saline tube method using series 4 and series 5 anti-d antisera. genotyping was performed from whole blood samples sent to immucor genotyping laboratory in warren, nj using an algorithm of: rhd beadchip, rhdxp (prototype assay), rhd zygosity, and rhce beadchip. results/findings: 80 patients met inclusion criteria for molecular testing for the d antigen. weak or rhd variants were identified in 51 of 80 (63.7%) of the samples. ceppellini effect (i.e. c in trans to rhd) resulting in weak d reactivity was seen in 16 of 80 (20%) of samples. 67 of 80 (83.8%) of the samples that resulted in weak or discrepant reactivity had some type of genetic cause that was resolved by using our algorithm. 40 of 80 (50%) of tested samples had results indicating weak/variant d proteins with the potential to cause alloimmunization to the d antigen. the remaining 13 of 80 (17.3%) samples did not have identified genetic cause for the weak and/or discrepant d test results and were presumptively classified as wild type d. conclusion: transfusion services that use the galileoneo or galileoecho to perform rh typing should consider molecular testing of patients whose rh typing results are discrepant, or positive but <31 on the galileoneo or positive but <21 on the galileoecho, as about half of these patients can develop anti-d. this is particularly relevant for females of child-bearing potential where avoidance of d-positive transfusions and administration of rhig during pregnancy is prudent until their d typing can be confirmed by molecular testing. carine arnoni* 1 , tatiane vendrame 2 , janaína muniz 3 , rosangela person 2 , lilian castilho 4 , afonso cortez 2 and flavia latini 2 . 1 associa, 2 associac¸ão beneficente de coleta de sangue, 3 hemocentro de são jos e do rio preto, 4 hemocentro unicamp background/case studies: rhd and rhce, are major protein constituents of red blood cell membrane, composing a complex together with rhag. many variant rh proteins have been described and most of them affect the integration of rh proteins in the membrane. d antigen expression can be affected by several molecular changes and also by the rhcehaplotypes. the present study investigated the score of reactivity of samples presenting a strong reduction in d expression. study design/method: a total of 108 samples were included in the study, being 69 previously genotyped as rhd*dar1.2, 37 rhd*dar3.1 and 2 rhd*dau6. the samples were phenotyped in neov r (immucor) to d, c/c and e/e antigens by direct agglutinationin microplate. results obtained in neov r were expressed in a score from 0 -99 corresponding to the reaction intensity. zygosity assay was performed by a multiplex real-time quantitative pcr using a set of rhd-specific primers in rhd exon 10. rhce genotyping was performed by pcr-rflp and ssp-pcr. the presence of a d-cehybridexon 3 was identified by amultiplex pcr. sequencing and identification of rhce variants were also performed when necessary. results/finding: zygosity results showed that 10 of 108 samples (5 dar1.2, 4 dar3.1 and 1 dau6) had 2 rhd genes, were phenotyped as c1e-c1e1 and genotyped as rhce*ce/rhcece. rhd and rhce genotyping in these 10 samples showed the presence of the d-ce-d s hybrid gene. rhce variants investigated in 2 dar1.2 samples showed the rhce*-cear/ce s genotype, in 2 dar3.1 samples the rhce*cevs.02/ce s genotype and in the dau6 sample the rhce*ce s /ce genotype. table 1 describes the differences found in the reactivity of d among the samples carrying the (c)ce s allele and in the samples homozygous for rhce*ce. the results showed that the presence of rhce*(c)ce s significantly reduces the expression of d antigen, probably due to the expression of the partial c partial antigen in trans to rhd. additionally, the samples with reduction on d expression carrying rhce variant alelles phenotype can be useful to provide compatible blood to some patients with rarerh variant alleles. background/case studies: drugs are known to interfere with routine blood bank testing. a novel monoclonal humanized 5f9 antibody (hu5f9-g4) that binds human cd47 has been entered into clinical trials for patients with acute myeloid leukemia, non-hodgkin lymphoma and solid tumors. we describe two cases of patients treated with hu5f9-g4 (anti-cd47) who had abo discrepancy with extra-reactivity in the reverse typing and a panaggutinin in the plasma. study design/method: this is a retrospective review of two cases with immunohematology work-up showing abo discrepancy and plasma panagglutinin. the first case is of a 69 year old female with progressive follicular lymphoma who was enrolled in phase 1b/2 trial of hu5f9 g4 in combination with rituximab designed for patients with relapsed/refractory b cell nhl. she had no prior transfusion history and her historical blood type was not known. two rbc units were requested in anticipation for a surgical procedure. the second case is of a 48 year old male with refractory diffuse large b cell lymphoma enrolled in hu5f9-g4 clinical trial. his historical blood type was a rh d positive with a negative antibody screen. he received three rbc units within the past month prior to testing and receiving the anti-cd47 therapy. results/finding: the abo typing in the first case showed a discrepancy between the forward typing (41 with anti-a, non-reactive with anti-b) and the reverse typing (31 with both a 1 cells and b cells). rhd typing was positive. the extended reagent rbc panel tested with the patient's serum reacted with all cells tested at the immediate spin (is) phase (11 to 41), at liss-37c (11 to 31), at liss-polyspecific ahg (m1), and at peg-anti-igg (m1 to 11). plasma reactivity at is persisted with dtt or ficin treated red cells and was not removed by cold autoadsorption, cold alloadsorption, or rest adsorption. repeat testing, which avoided the is and 37c readings, was non-reactive in the antihuman globulin (ahg) phase using both liss and peg enhancements, ruling out clinically significant alloantibodies directed toward common red blood cell antigens. the direct antiglobulin test (dat) and autocontrol were negative. the rbc units issued to the patient were crossmatch compatible at 37 o c ahg phase. the abo typing of the second case performed after anti-cd47 administration showed a discrepancy between the forward (41 with anti-a) and the reverse (41 with both a 1 and b cells). rhd typing was positive. the antibody screen performed in solid phase technology was positive with all reagent red cells. his plasma reacted with all reagent red cells at is (21), at 37c in liss (21), and liss-polyspecific ahg (m1). the dat and autocontrol were negative. his genotype was determined to be a1/o and full rbc phenotype by dna analysis was obtained. repeat testing which avoided the is phase did not show reactivity at peg-ahg excluding all alloantibodies directed toward common red blood cell antigens. conclusion: anti-cd47 therapy interferes with blood bank testing by causing abo discrepancies and panagglutinin reactivity in the plasma at is, 37c liss, but not at ahg phase using gamma-clone anti-igg, unlike the anti-cd38 interference. knowledge of patient's blood type and phenotype before starting this therapy is critical for providing safe blood. background/case studies: a middle-aged male with discrepant abo typing results was investigated. initial forward typing was group o but no anti-b was seen in the reverse typing. an unexpected 31 reaction was noted with an anti-a,b reagent. genotyping surprisingly showed abo*o.01.01/ o.01.01, consistent with group o. after initial testing at the referring center, samples were sent for extended analysis. study design/method: standard serological methods and flow cytometry were used. a panel of abo reagents (n523) and lectins were tested with both native and papain-treated red blood cells (rbcs). lewis phenotyping was performed, as was genetic testing for abo, gbgt1 and a4galt. papain-treated patient rbcs were used to screen donor plasmas (n578) and two reactive plasmas were dtt-treated. results/finding: positive reactions were obtained with 3 polyclonal anti-a,b and a monoclonal anti-b (clone g1/2) when tested with the patient's papain-treated rbcs. a panel of lectins gave negative results. genetic testing confirmed the predicted group o and ruled out the presence of fors1 or nor antigens. the patient was le(a-b1) and thus a secretor. a positive crossmatch was seen with 47% of group o plasmas, while no reactivity was obtained with a or b plasmas. dtt treatment of crossmatchpositive plasmas indicated the antibody to be mainly of igg type. this was confirmed by positive flow cytometry cross match using anti-human igg secondary antibodies. reactivity remained after b-zyme treatment, thus excluding the normal (type 1 or 2) b antigen to be the underlying reason. inhibition with lewis substance significantly decreased reactivity. enzyme activity assay showed the patient's plasma to contain a fully functional b glycosyltransferase. on the suspicion that the patient had non-erythroid cells producing breactive type 1 chains, a sample from a hematopoietic stem cell transplant (hsct) patient (group b secretor receiving group o donor cells) was included as a control and gave the same type of reactions. conclusion: the medical history of the patient was queried and he had indeed undergone an hsct $15 years earlier. the reactions are likely due to uptake of recipient-derived ble b (type 1) antigen (isbt no. 007006), which is the dominant lewis antigen in the recipient's original blood group, b le(a-b1). interestingly, b-zyme did not affect ble b . anti-ble b is not simply anti-b plus anti-le b but an inseparable and rarely reported specificity, which appears to be common among group o donors. the phenomenon reported here has unknown clinical implications but highlights the complexities of carbohydrate blood groups. background/case studies: the provuev r and visionv r (ortho scientific, raritan new jersey) automated analyzer use mts-gel tm card technology to perform immunohematology testing. benefits of automated testing include improved efficiency and enhanced reliability. after eight years of using the provuev r our transfusion medicine service switched to the ortho visionv r analyzer in january of 2017. shortly after implementation, technologists reported increased time spent performing manual resolution of indeterminate (designated as "?") results. additionally, some test columns were noted to be visually negative but called positive (11) by the analyzer. the objective of this study was to investigate the cause of "?" and apparent false positive results on visionv r three-cell antibody screens. study design/methods: with assistance from ortho diagnostics, analyzer archives were queried to identify the number of gel card columns used for screens, the number of columns with "?" results, and the gel card lot numbers used for testing from 1/29/2017 to 3/31/2017. reactivity was determined to be false positive based on supervisory review of digital images and antibody panel results. investigation also included review of daily qc records, instrument maintenance, instrument diagnostics, and camera calibration. results/findings: of 19,647 columns run as part of antibody screens, 1,633 (8.3%) columns generated "?" results. assuming 30 seconds of technologist time per "?", we estimate that 13.6 hours were needed to resolve and update these results. among all potential causes investigated, only the gel card lot number was associated with the number of "?" generated (table 1) . in 26 cases, all three columns were visually negative but the analyzer reported 11 reactivity with 1 of 3 cells. all cases had mts-gel tm antibody identification panels performed, 25 of 26 also had a mts-gel tm ficin panel. the yield for the 51 panels performed was two routine panels with weak reactivity against hla1 cells, and four ficin panels with weak reactivity with no apparent specificity. fourteen patients coincidentally had a subsequent type and screen; 13 were negative. one patient newly demonstrated anti-jka. fifty percent (13/26) of visually negative but analyzer positive samples were tested with gel card lot number 3, 38% (10/26) with lot 1, and 12% (3/ 26) with lot 2. conclusion: the incidence of "?" and visually negative analyzer positive results is dependent on the specific lot of mts-gel tm cards used. the difference between the lots is being investigated by ortho diagnostics, and remains to be explained. to avoid unnecessary waste of technologist time and other resources, with assistance from ortho diagnostics, we have results/finding: of the methods evaluated, the dtt method proved the most useful for mitigating dara interference. cord cells were effective but in limited supply and alloadsorption was ineffective. of the three different dtt methods evaluated, the tube method initially failed which led to re-evaluation with the addition of liss (passed). the gc method was the most sensitive method. following release of dara, samples from 28 patients (137 cross-match samples, 301 units issued) were tested using both liss tube and gc iat methods. despite dtt treatment, the gc method remained positive by iat in 16/28 patients. further testing was performed in 9/16. eight were tested for the presence of antibodies at 188c and confirmed in 8/8. rouleaux formation was observed in 5/9 patients, 4/5 had reactivity detectable at 188c. no transfusion reactions have been reported to date nor has alloantibody formation been observed to date. conclusion: as previously reported, the dtt method was the most useful for mitigating dara interference. the observed interference seems to be due to rouleaux and/or cold reactive antibodies -seen least in the liss tube iat. this may be due to the washing phase in this technique which dissipates rouleaux formation. reactivity due to cold reactive antibodies can be eliminated by performance at strict 378c. our practice is now to use both dtt iat methods on initial patient referral, if residual reactivity in gc is observed use liss tube in preference thereafter in these patients. a further observation is that investigation of pan-agglutination could include the use of cord cells to confirm/exclude dara use if suspected. wendy beres* 1 , sandra nance 2 , david moolten 3 and p. dayand borge 3 . (2):47-53). our laboratory tested random allogeneic blood donors, and autologous donors (which were intended to represent a hospitalized patient population), to determine a mean and range of "normal" levels. this 2.5 year retrospective study was performed to assess levels of rbc-bound igg, iga and igm in normal donors. study design/method: residual edta-anticoagulated aliquots from 150 random allogeneic and 20 autologous blood donors were sequestered and tested per institutional review board approved protocol. the samples were tested by fc with fluorescein isothiocyanate (fitc)-labeled anti-human igg and iga (jackson immunoresearch lab, west grove, pa) or fitc-labeled anti-human igm (life technologies, carlsbad, ca) at optimized dilutions in dulbecco's pbs containing 0.6% bsa. the becton dickinson facscalibur tm or facscan tm (san jose, ca) fc analyzed 50k rbcs from each sample. edta-anticoagulated samples and ig coated control rbcs were tested to determine fc settings and control for validity and cross-reactivity. controls reacted as expected. there were less autologous donors tested and with a mean age of 62 these donors could have been older than the allogeneic donors, but the mean age of the allogeneic donors was not captured. despite the relatively small number of samples tested there was a higher than expected instance of allogeneic donors having elevated rbc-bound iga, igg, and igm levels. this emphasizes the need to include testing of normal donor populations in establishing expected reactivity, thus normal and abnormal ranges for flow cytometric testing. long range pcr reveals the genetic basis of an antibody in pregnancy to a high prevalence mns antigen judith aeschlimann* 1 , anna burgos 1 , virginia lew 2 , sunitha vege 1 , susan veneman 3 , christopher j gresens 3 , jonathan hughes 3 and connie m. westhoff 1 . 1 immunohematology and genomics laboratory, new york blood center, 2 blood centers of the pacific, 3 bloodsource background/case studies: recombination events have generated many gypa and gypb hybrids giving rise to glycophorin (gp) variants that express low-prevalence antigens (e.g. mia, miny, mur). in rare individuals who are homozygous these alleles are associated with lack of highprevalence antigens (e.g. enkt, eneh, enav). complex hybrid recombination events can make it challenging to elucidate specific alleles present in samples, particularly heterozygotes. we investigated samples from a pregnant asian (hmong) woman with an antibody to an unidentified highprevalence mns antigen, and samples from her sister. study design/method: standard methods were used for rbc typing with licensed and unlicensed reagents and for antibody identification. dna was isolated from wbcs, and hea precisetype, exon-specific amplification and sequencing gypb exons 1-6, and long range sequencing of exon 2-6 (5.4kb amplicon) were performed. snp-specific primers were used to associate changes (phasing) to specific alleles. results/finding: rbcs of the pregnant proband typed s-s-(gammaclone anti-s), and the plasma reactivity was consistent with an antibody to a high background/case studies: the rhd antigen is clinically significant and immunogenic and therefore individuals who develop anti-d are at risk of haemolytic transfusion reaction. rhd polymorphism shows substantial ethnic variability and at least 100 rhd variants associated with weak d alleles have been reported. in this study, we report two new rhd alleles in brazilian blood donors associated with weak d antigen expression. study design/method: the d status was evaluated with 4 commercially available monoclonal anti-d reagents: 1 blended igm/igg (clones th-28/ ms-26), 2 igm (clones ms201 and p3x61) and 1 igg (ms26) in tube and on gel cards. c, c, e and e phenotyping were performed in gel. most common weak d and partial d alleles were investigated by allele specific (as) pcr and with the rhd beadchip platform from immucor. direct automated sequencing of the 10 rhd exons and flanking intron regions was performed by the sanger dideoxy method. in order to determine rhd allelic combinations, we also performed rh-cdna cloning and sequencing. background/case studies: donors negative for multiple common antigens or lacking a high prevalence antigen are efficiently identified using a red blood cell (rbc) genotyping panel. when serology is used to confirm antigen negative status, discrepancies are identified, albeit rarely. investigation of the discrepancy often leads to identification of variant antigens. it is known that the set of gyp variants associated with expression of the st a antigen can also be associated with n typing discrepancies in m1n-individuals (meyer et al. br j haematol. 2016; 174:624-36) . the st a allele, also described as gyp*401, is a hybrid gypb-gypatranscript with the crossover in intron 3. we sought to investigate five n typing discrepancies for which alternative genotyping methodology was performed and found to be concordant with the initial panel. background/case studies: a sample from a 24 years old pregnant, african american female g1p0 was sent to the blood bank for abo/rh and antibody screen. the sample was analyzed using the provue analyzer (ortho diagnostics). the patient was typed as o pos with no reverse type discrepancy. a retype of the same sample was performed using tube method with biorad reagents per hospital policy due to no previous abo/rh history on file. the retype showed that the patient was a subgroup with anti-a 1 antibody present in the plasma. the sample was referred for genotyping, with the suspicion of a 3 like phenotype. genetic testing did not support the serological findings of a 3 subgroup and a new abo allele, abo*784c that has never been reported in correlation with an a 3 like subgroup was detected study design/method: the patient rbcs were typed with anti-a 1 (immucor) and anti-a,b (biorad and grifols dg gel). an anti-a 1 antibody work up was performed using three different lots of a 1 cells and three lots of a 2 cells, as well as a type o screening cell and auto control . the tubes were read at is and also incubated at rt and 48c for 30min. the patient 's initial antibody screen using ortho gel was negative. conclusion: the patient delivered a healthy baby boy at 35 weeks of gestation. the baby cord was sent to the laboratory. the baby serological type showed an a 3 b phenotype and it was referred for genetic testing. the baby rbcs showed the same abo*784c found in the mother. the previously reported abo*784a allele encoded an aspartic acid to asparagine change at position p.256 consistent with an a weak phenotype. also, at least five other alleles encoding an a 3 phenoytpe consisted of polymorphisms at positions c.745 through c.871, giving special characteristics to this new and unreported abo allele. from the data collected, it can be concluded that this a3/ aweak phenotype is encoded by the variant allele abo*784c. this highlights the clinical relevance of confirming the serology of abo subgroups by molecular methods. philip berardi*, jacqueline cote, gwen clarke, vito scalia, robert liwski and mindy goldman. canadian blood services background/case studies: elucidation of the molecular basis of blood group expression has led to the development of high throughput molecular methods for predicting blood group antigens. the commonly used single nucleotide polymorphism (snp) arrays require nucleic acid isolation which is typically achieved by extracting genomic dna from whole blood. this method requires venipuncture and may not be an ideal approach for severely anemic patients or potential donors that are unable to provide a sample of whole blood due to their remote location. dna extracted from buccal swab samples offers a noninvasive alternative to venipuncture and may provide a safe and efficient means of transporting samples from remote locations to reference laboratories for extended blood type prediction. canadian blood services (cbs) has performed large scale dna extraction and hla genotyping for the onematch stem cell and marrow registry using buccal swabs since 2007; buccal swabs are also used by other unrelated donor stem cell registries, such as the us nmpd. we sought to assess the accuracy and reliability of using dna extracted from buccal swabs in predicting blood group antigen expression. study design/method: we performed parallel red cell genotyping on an automated typing platform, the progenika/grifols idcorext assay (progenika biopharma-grifols, bizkaia, spain) using dna extracted from blood and buccal tissue from volunteers. for antigen systems with available serologic reagents, we also compared results with serologic typing. we evaluated three different methods of dna extraction and performed testing regardless of dna yield or purity. two buccal swabs (puritan medical products, guilford, maine) were used for each test. swabs were stored at room temperature, and dna extraction was performed within six days of collection. in the initial phase of the study, buccal swab samples (n 5 15) were processed with the automated biorobot m48 robot using the magattract dna mini m48 extraction method (qiagen, venlo, the netherlands). extracted dna had a mean concentration and purity of 38.1ng/ml and 1.83 respectively. in the second phase of the study (n 5 39), dna extractions from buccal swabs were performed using methods available in our national red cell immunohematology reference laboratory: the qiaamp dna mini kit, using either manual or an automated qiacube robotic workstation (qiagen, venlo, the netherlands). results/finding: the manufacturer's recommended analytical range for dna concentration was 20-80ng/ll and the recommended purity was an absorbance ratio of 1.63-2.1 (a260/280) for use of the id corext platform. dna extraction from buccal swab samples did not meet these specifications in several cases. however, in most cases, a lower concentration of dna was adequate for prediction of phenotype. the dombrock system was the most susceptible to failure of interpretation in the samples with a low dna concentration, with "no call" results reported. there was 100% concordance in genotyping results when source dna was extracted from whole blood or buccal tissue; there was also 100% concordance between predicted phenotype and serologic testing results. conclusion: this study supports the use of genomic dna extracted from buccal tissue on the id corext for predicting rbc phenotype with high accuracy. extraction methods may require optimization to achieve dna yields within the recommended analytical range of the assay. performance evaluation of id rhd xt, a genotyping assay for the detection of high-prevalence rhd negative and weak d types araitz molano 1 , izaskun apraiz 1 , maría azcarate 2 , miguel angel vesga 2 , montserrat rubia 2 , mercedes piedrabuena 2 , fernando puente 3 , barbera veldhuisen 4 , ellen van der schoot 4 and m onica l opez* 1 . 1 progenika biopharma, a grifols company, 2 centro vasco de transfusi on y tejidos humanos, 3 banco de sangre y tejidos de arag on, 4 sanquin blood supply research background/case studies: it is well established that weak d 1, 2 and 3 phenotypes are not at risk for forming allo-anti-d, whereas a few weak d and all partial d and negative phenotypes are. routine serologic d typing does not distinguish among them, consequently rhd genotyping is recommended, especially in patients. id rhd xt (progenika, grifols) is a qualitative, pcr/luminex v r xmap hybridization-based genotyping test for the identification of the following rhd gene allelic variants: rhd*weak d type 1, rhd*weak d type 2, rhd*weak d type 3, rhd deletion, rhd*pseudogene and rhd*diiia-ce(3-7)-d and itgb3 gene: hpa1a and hpa1b, in genomic dna extracted from whole blood specimens. in this study the performance of id rhd xt genotyping assay was evaluated in terms of whole system failure rate, call rate and accuracy for rh and hpa-1 blood group typing. study design/method: a cohort of 1000 previously serotyped samples for d antigen obtained from three european blood centers were analyzed with id rhd xt at progenika. samples were distributed as recommended by the annex of the common technical specifications 2009/108/ce for a ivd product of list a (!10% clinical samples, >2% neonatal specimens and !2% weak d donors). for the intended use of the product, weak d serotyped donors were enriched (n5160, 16%). commercial serology tests for d antigen predicted phenotype and bi-directional-sequencing (bds) for weak d type confirmation and hpa-1 predicted phenotype were used for comparison. transfusion results/finding: no system failure, 100% call rate and no inconclusive results were obtained. discrepancies were found for d antigen between serology and id rhd xt predicted phenotype results, although a 100% concordance was obtained when analyzed by bds, considering id rhd xt result correct. concordance between id rhd xt and bds results for the weak d type was 100%. the following id rhd xt predicted phenotype results were obtained: d negative (n5361), no amplification variant (n515), weak d type 1 (n522), weak d type 1 heterozygous (n51), weak d type 2 (n532), weak d type 2 heterozygous (n51), weak d type 3 (n534), weak d type 3 heterozygous (n51), weak d types 1, 2 or 3 not detected (n5533). regarding hpa-1 blood group, the predicted phenotype results obtained by id rhd xt were 100% concordant with bds results: hpa-1a positive (n5157) and hpa-1a negative (n56), hpa-1b positive (n546) and hpa-1b negative (n5117). conclusion: id rhd xt genotyping assay performed as a reliable and accurate method for predicting the genotype and phenotype of high prevalence rhd negative and weak d types (100% specificity and 100% sensitivity for d antigen, hpa-1a and hpa-1b antigens). that makes it a useful tool for the implementation of the rhdgenotyping recommendation on patient blood transfusion and anti-d prophylaxis. background/case studies: scd patients form red blood cell (rbc) antibodies at higher rates than other transfused populations. multiple predictors of alloimmunization have been reported but not well replicated in large scd cohorts. we investigated the clinical, laboratory and genetic predictors of alloimmunization. study design/method: a large scd cohort was established in brazil to investigate disease outcomes. at participating sites, patients are currently transfused with abo/d/cc/ee/kell matched rbcs prophylactically and extended phenotypically matched rbc after first antibody forms. policies for matching are center-specific and evolved to increased levels of matching over the exposure period included in this study. transfused subjects with 11 rbc alloantibody of defined specificity within the cohort were compared to transfused antibody negative subjects using chi squared to compare categorical variables and t-test or wilcoxon rank-sum tests as appropriate to compare continuous variables. backward elimination multivariable logistic modeling was used to generate odds ratios (or) and identify independent predictors of alloimmunization using results of univariate analyses. all subjects had peripheral blood whole genome snp typing performed using an affymetrix array, which included enhanced content for blood related snps. genome wide association (gwa) analyses were conducted using a logistic model to identify additive genetic effects associated with alloimmunization. a p value <50.05 (clinical analysis) or <5x10 -8 (gwa) was considered statistically significant. results/finding: of the 2795 cohort patients, 2272 (81.3%) transfused subjects were included with 129 alloimmunized children <18 years (11.0% of 1172) and 224 alloimmunized adults (20.4% of 1100). in multivariable logistic regression models, age (or 4.2, p50.009, for age 501 compared to 0-4), gender (or 1.3, p50.04, for female compared to male), transfusion history (or 3.5, p<0.0001, for 811 transfusions compared to 1-5), site, hemolysis (or 1.3, p50.05, for log transformed lactate dehydrogenase) and presence of autoimmune disorders (or 4.5, p<0.0001) were independent predictors of alloimmunization. gwa identified a single snp of unclear biologic significance associated with alloimmunization (eefsec gene responsible for incorporation of selenocysteine into proteins). conclusion: rbc alloimmunization is primarily driven by transfusion burden in this scd cohort. hemolysis remained significantly associated with alloimmunization after controlling for transfusions. presence of an autoimmune disease was also associated with rbc alloimmunization, indicating more systemic immune dysregulation may be present in scd patients who develop rbc alloantibodies. however, the gwa did not identify snps in immunoregulatory genes significantly associated with rbc antibody formation in the study population. background/case studies: physiologic anemia is more severe in preterm infants and worsened by the blood loss required for laboratory tests. to reduce iatrogenic anemia, placental blood, which otherwise would be discarded, can be used for laboratory testing. mother and infant blood are mixed in the placenta during delivery and pre-transfusion test results potentially can be altered due to fetal-maternal hemorrhage. there has been no published study to show if pre-transfusion test results of placental blood give the same result as the heel stick samples, which is the standard of practice. study design/methods: transfusion service tested sample pairs from 32 newborns less than 2,000 gr birth weight. one of the samples was collected from the newborn as a heel stick sample, the other from the placenta. the following tests were performed on the sample pairs: abo, rh, antibody screen and direct antiglobulin test with igg (dat). results/findings: abo, rh and dat tests were performed on 32 sample pairs. dat test was negative on 30 sample pairs and two were positive. there was 100% concordance with the abo, rh and dat tests performed on these sample pairs. antibody screen was performed on 32 placental blood samples and 29 heel stick samples. twenty eight sample pairs were negative with the antibody screening test. there was one positive heel stick sample, which was also positive using the placental sample. one heel stick sample was negative for the presence of an antibody but found to be positive with the placental blood sample. this antibody which was detected only in the placental sample was a passive anti-d mother received during pregnancy. this discrepant result indicates that the placental blood sample was more sensitive to detect a weak antibody. conclusion: this study shows that placental blood sample is not inferior to heel stick sample regarding abo, rh and dat testing. based on this comparison study placental blood can be used for pre-transfusion testing for < 2,000 g birth weight newborns. o-(7.4%), ab1(6.8%), b-(2.7%), and a-(0.6%). among the tested donors, 89.2% were d positive with r1r being the most common rh phenotype. in the kell blood group system, 4.5% of the donors were k positive, while the k antigen was found to be 99.4%. the most common phenotype in the duffy blood group system was fy(a-b-), while the fy(a1b1) was found at a higher frequency compared to what has been reported in the black population. (table) the commonest phenotypes for the kidd and mns blood group systems were jk(a1b1) and m1n-s1s1 at 47% and 22.6% respectively. the le a 1 and le b 1 alleles were seen in 21.7% and 67.3% of donors respectively, while lu b -phenotype was found in 3.3% of the donors. the frequencies of the rare phenotypes jk(a-b-), le(a1b1) and lu(a-b-) were 0.3% , 0.3% and 2.7% respectively, while the m1n-s-s-and m-n1s-s-phenotypes were not found. the frequency of the p1 antigen was found to be at 78.9% similar to what has been reported in caucasians. conclusion: this is the first study to examine the frequencies of rbc blood group phenotypes among the omani blood donors. the results show higher frequencies of the rare null phenotypes fy(a-b-), jk(a-b-) and lu(a-b-) compared to what has been reported in caucasians. the frequencies of the duffy blood group system resemble what has been reported in the black population. this data is helpful in understanding the influence of the arab ethnic background on the rbc blood group systems and warrants large genotype-phenotype studies in the region. quantitation of anti-d in serum using flow cytometry amanda whitelonis*, izekial butler, karen leighton, scott jones and anand srinivasan. qualtex laboratories background/case studies: rh(d) antibodies (anti-d) are developed in rhnegative individuals when exposed to d antigens. this scenario is commonly observed in alloimmunized antenatal and volunteer immunized patients. quantitation of anti-d in serum is important in the clinical setting to predict the risk of hemolytic disease of the newborn. quantitation of anti-d is also performed in quality control operations of organ procurement organizations and plasma fractionators. it is a common practice to report the strength of anti-d in serum as antibody titer values but quality control operations require a quantitative value. we have developed a screening assay using flow cytometry to quantitate anti-d in serum. study design/method: we have developed a method to quantitate anti-d in serum using flow cytometry, by modifying the protocols of christensson et al., and hilden et al.. red blood cells from rh-positive blood samples were washed three times in phosphate-buffered saline (pbs) at ph 7.2 and the supernatant was discharged. a dilution buffer containing 2% human serum albumin (v/v) in phosphate buffered saline was prepared. serum samples or who anti-d standards, suspended in dilution buffer were mixed with 10ll of washed red cells. the cell suspensions were incubated for 30 min at 378c. following incubation, fitc-labeled anti-igg diluted in buffer was added and the mixture was incubated for an additional 15 min at 228c. the samples were then analyzed by flow cytometry using gates for a typical red cell based on the forward and side-scatter signals. green fluorescence was collected using a band-pass filter set for 515-548nm. events were recorded at a frequency of 1000 cells. results/finding: multiple dilutions of who anti-d reference standard were tested against rh-positive red blood cells from five different donors. the reproducibility of the assay was determined by measuring the change in coefficient of variance due to dilution procedure, machine variation and sample storage condition. after optimizing these factors, a linear regression was calculated to establish the standard curve. the fluorescent intensity emitted by probes demonstrated a linear correlation with the concentration of rh(d) antigens in reference standard. serum from thirty rh(d)-immunized volunteers were analyzed for concentration of anti-d and the results were benchmarked with antibody titer values. conclusion: based on our study, we conclude that the quantitation of rh antigens by flow cytometry can be used as a reliable assay to measure the concentration of anti-d antibodies in serum. the method is reproducible and advantageous over reporting antibody titer values. the operations of this platform can be translated to a well-plate based high-throughput flow cytometry. sarah k harm* 1 , mark yazer 2 , nancy m. dunbar 3 and biomedical excellence for safer transfusion (best) collaborative 1 . 1 university of vermont medical center, 2 university of pittsburgh, 3 dartmouth-hitchcock medical center background/case studies: the use of emergency issued group a plasma and uncrossmatched group o whole blood (wb) in patients without a valid abo group is becoming increasingly common in the usa. it is unclear if low titer products should be provided in this situation and indeed a universally agreed upon threshold that would qualify as "low titer" has not been established. this study was designed to determine the rate of high titer donors using a titer threshold of 50. study design/methods: three academic hospitals that routinely issue group a plasma units for emergency issue participated in this study. before issuing this plasma to patients, a 1:50 dilution of the donor's plasma in saline was produced and added to group b reagent red blood cells (rbc). if any degree of macroscopic agglutination after immediate spin was observed, the unit was considered high titer and it would only have been issued to group a or o recipients. at these three centers no temperature, plasma volume or time enhancements were performed in the titer procedure, and anti-human globulin was not added. at one center samples were taken from the plasma of group o wb units and the same procedure was followed using a1 and b reagent rbcs; if at least one antibody demonstrated macroscopic agglutination after immediate spin, the wb unit was considered high titer and it was then centrifuged into an rbc unit for transfusion while the plasma and platelet components were discarded. two centers provided plasma testing data for a 4-year and 5-year period, respectively. one center provided plasma and wb testing data for a 1-year period. results/findings: in total there were 7106 group a plasma units tested and 654 (9.2%) had a high titer anti-b. the range of high titer group a plasma units between these three centers was 7.4%-12.5%. of the 1778 wb units tested, 388 (21.8%) units had a high titer; 221/1778 (12.4%) of the units had a high titer anti-a, 61/1778 (3.4%) had a high titer anti-b, and 106/1778 (6%) had high titers of both anti-a and anti-b. background/case studies: dithiothreiol (dtt) is a sulfhydryl reagent that denatures selective blood group antigens. reagent red blood cells (rbcs) treated with 0.2m dtt is used as a tool in identifying antibodies to high frequency antigens. recently, dtt has become widely used in destroying cd38 on reagent rbcs and render them free from plasma anti-cd38 drug interference. procedures for the preparation of 0.2m dtt has been published advocating the use of buffered saline at different ph levels. in this study, an effect of ph on 0.2m dtt treatment time is investigated. study design/methods: non-buffered saline (nbs, thermo fischer scientific inc, middletown, va), used in the preparation of 0.2m dtt, was adjusted to ph7.16, ph 7.56, ph 7.96 using sodium phosphate dibasis (sigma aldrich, saint louis, mo). reagent rbcs (immucor, norcross, ga)(n53) were treated with the 0.2m dtt solutions in parallel by mixing 1:4 ratio of packed rbcs to 0.2m dtt solution followed by incubation at 378c. for up to 45 minutes during treatment, the expression of k antigens was measured every 5 minutes by tube method using 2 different sources of anti-k. to assure uniformity, all reactions were graded by the same investigator. each reaction grade (in each rbc and each antiserum) is converted into a semiquantitive score and an average score was calculated every 5 minutes for each ph level. the reduction in average scores between different phs were also calculated at every 5 minutes to measure the impact of 0.2m dtt reagent ph on the rate of k antigen destruction. results/findings: the expression of k antigen, measured by agglutination grades with two different k antisera, is significantly weakened (by !11) after 15 minutes of dtt treatment at ph 7.96; 15 minutes at ph7.56 and 20 minutes at ph7.16. complete loss of k expression was seen after 25 minutes of dtt treatment at ph7.96; 35 minutes at ph7.56 and 35 minutes at ph 7.16. the reactivity patterns of k antigen tested with 2 sources of anti-k correlate with each other. the reductions in average scores were seen between 15 to 30 minutes range of dtt treatment time when ph 7.16 was raised to ph7.56; 15 to 30minutes range when ph7.56 was raised to ph7.96; and 15 to 30 minutes range when ph7.16 was raised to ph7.96. conclusion: the use of higher ph buffered saline may shorten the treatment time it takes to weaken or destroy k antigen. based on the comparison of reaction scores between different ph levels, the ph levels did not have an impact on dtt treatment up to 15 minutes and/or beyond 35 minutes of incubation. the ph of the 0.2m dtt reagent relative to the treatment time is a factor to consider during the validation of dtt-treatment process and qualification of 0.2m dtt reagent in a laboratory. background/case studies: data on the characteristics and frequencies of clinically significant red cell antibodies within the prenatal population have not been well established in the united states. the aim of this study was to determine if frequencies of red cell antibodies differed between geographically distinct regions within the continental united states. study design/ method: the aim of this retrospective study was to evaluate a cohort of prenatal patients (n 5 756,221) drawn between july 1, 2013 and june 30, 2014. these patients were divided into united states census bureau regional and divisional categories according to their place of residence. prenatal blood work was collected which included an abo, rh(d) and a screen for unexpected alloantibodies. samples found to be positive for red cell antibodies were sent to one of nine regional laboratories for identification. results/ finding: in total, 11,647 patients were found to possess clinically significant red cell antibodies for an overall incidence of 1.5 percent. the three most commonly encountered antibodies were anti-d (n 5 7639) 63.1%, anti-m (n 5 1288) 0.6%, and anti-e (n 5 1227) with a frequency of 10.1%. a total of 455 (3.9%) prenatal women were found to possess two or more antibodies. in general, the combination of anti-d and anti-c proved to be the most common, with 182 instances (40.0%) followed by anti-e and anti-c with 79 (17.4%), and anti-c, anti-e with 26 (5.7%). of the multiple antibodies identified, 435 (95.6%) included at least one antibody from the rh blood group. the south region had the largest number of antibodies identified with 7474 or 61.8% of the total. the west had 2111 (17.4%), the midwest 1538 (12.7%) and the northeast with 979 (8.1%). a contingency table, using the two-sided fisher's exact test, was performed comparing the northeast, south, midwest and west regions. the p value of anti-d was calculated to determine nonrandom associations and values of 0.05 and below was deemed significant from a region-to-region perspective. with regard to anti-d, the pacific division comprised of california, oregon, washington, and alaska, had p values below the 0.05 thresholds when compared against seven of the eight other divisions. the west south central division (texas, oklahoma, arkansas, and louisiana) did not show statistically significant results when compared against the pacific division (p 5 0.17). conclusion: depending upon the antibody, statistically significant variations between geographical regions and divisions within the united states were observed. this relationship between antibody and locality requires further investigation but may be attributed to the presence or absence of red cell antigens among different racial and ethical populations. reduction in repeat testing using gel technology amy mata* 1 , lindsy rich 1 , sherry stern 1 , sharon wangen 1 and camille van buskirk 2 . 1 mayo clinic, 2 mayo clinic rochester background/case studies: our institution currently uses the immucor neo (immucor, inc., norcross, georgia) to perform abo/rh and antibody screen (absc) testing utilizing solid phase technology. when results are unable to be obtained from the immucor neo, testing is repeated on the manual testing bench using tube agglutination. this repeat testing can lead to significant expenses including reagents, supplies, and technologist time. it was decided by leadership in our laboratory that it would be beneficial to observe how other methodologies perform in this regard. a side-by-side evaluation was performed between the immucor neo and the ortho vision (ortho clinical diagnostics, rochester ny) to determine if there was a significant difference in the amount of repeat manual tube testing that needed to be performed. the evaluation looked at abo/rh and absc testing as those are the only tests that are currently automated in our laboratory. study design/method: thirty specimens that were processed on the immucor neo and resulted in no type determined (ntd) for abo/rh testing were selected to be tested on the ortho vision. twenty-three specimens that were processed on the immucor neo and produced positive results for absc testing were selected to be tested on the ortho vision. all specimens were edta tubes and were collected within the previous 4 days. the timeframe between when the specimen was tested on the immucor neo and the ortho vision was 1 to 2 days. results/finding: of the 30 ntd specimens from the immucor neo, 8 resulted in valid abo/rh typings on the ortho vision. three results were flagged indicating possible extra reactivity. upon performing a visual review of all 3 results, it was determined that there was no reactivity and a valid result was present. the other 19 samples required manual tube testing to interpret the abo/rh and were due to mixed field, weak isoagglutinins, unexplained extra reactivity, and hemolysis. of the 23 absc specimens that were resulted out as positive on the immucor neo, 11 specimens produced a negative result on the ortho vision and were confirmed to be negative with manual tube testing using peg as the enhancement media. one specimen was flagged for fibrin, but upon performing a visual review, was determined to be negative. nine specimens that were positive on the immucor neo were also positive on the ortho vision. one specimen proved to be an anti-m that was seen in gel but not in tube and one specimen displayed unexplained reactivity in gel as it was negative in tube and all clinically significant antibodies were ruled out. all showed discrepant results with monoclonal anti-c reagents, with a similar pattern of reactivity: 3-41 with ms24 (n515), 1-31 s with ms23 (n59), no reaction with ms273, dgc02, p3x255 (n514). 14 samples tested with a polyclonal anti-c showed a 1-31 reactivity. 3 d1c1e1c1e1 cases tested with a polyclonal and monoclonal anti-e (ms16, ms21, ms62, ms63) showed no weakened reactivity. rhce sequencing (genomic dna or cdna) showed a c.286g>a mutation in exon 2, predicted to encode the p.gly96ser substitution. for 2 apparent r 1 r 2 donors, a f-negative type allowed the prediction of a rhce*ce286a/rhce*ce genotype. altogether, our results are consistent with the presence of a very likely rhce*ce286a allele (c and e in cis) in all samples. 3 d1c1e1c1e1 individuals were reactive 11 s with the original source of anti-rh55, slightly weaker when compared to rhce*ce286a/rhce*ce rbc samples available from our cryobank (21). conclusion: our results confirm that the c.286g>a mutation alters the conformational properties of the rhce protein, either on a ce or ce background, and encodes the low-prevalence locr antigen (rh55). the locr reactivity appears to be rather similar when coded by rhce*ce286a or rhce*ce286a alleles. this was quite an unexpected finding, since the p.gly96ser substitution is close to the critical amino-acid for c/c expression (p.pro103ser). none of our 15 cases made anti-c and/or anti-e but few were subject to a potential alloimmunization background. however, as rhce*-ce286a was reported to code for a partial c (rh:-26), we consider that rhce*ce286a likely encodes partial c and e, this being also supported by the predicted localization of the p.gly96ser change on the second extracellular loop of the rhce protein. background/case studies: weak d genotyping is recommended for transfusion recipients, pregnant women, and newborns who had a rhd typing discrepancy, or a serological weak d phenotype, to determine if they carried the weak d genotypes 1, 2 or 3. the purpose of this study was to analyze the underlying rhd genotypes of the patient samples received for weak d genotyping since published recommendations, in particular those found to not carry the weak d 1, 2, or 3 genotypes. study design/methods: between 9/2015 and 2/2017 50 samples were received for weak d genotyping. testing was performed using pcr-rflp targeting the sequence variants in the rhd gene that have been previously defined. samples that did not have weak d types 1, 2, or 3 genotypes, but 156a transfusion 2017 vol. 57 supplement s3 had evidence of rhd genetic sequences in exon 7 and/or intron 4 in preliminary testing were evaluated by sanger sequencing for rhd and rhce exons 1-10 to determine the underlying rh genotype. when provided, the patient's ethnicity and presence of anti-d was recorded. results/findings: the majority of the samples were from obstetrical patients (62%) followed by transfusion patients (28%); 10% had no clinical indication provided. 34 samples (68%) were found to be weak d type 1, 2, or 3 (24, 6, and 4 samples, respectively). 5 samples (10%) appear to be genetically rhd negative. genetic sequencing was performed on 11 samples; 9 had rhd genetic variants that were not weak d types 1, 2, or 3 (table) . all of these variant rhd samples also showed some variation in the rhce gene. two samples (4%) had wild type rhd alleles; further evaluation is ongoing. conclusion: most samples tested by weak d genotyping were found to be weak d types 1-3. of the 11 samples that had evidence of an rhd gene and did not carry the known weak d types 1-3 polymorphisms, 9 (82%) of were found to have other rhd variants, and 2 (18%) did not have underlying genetic variation detected in the rhd gene. the majority of the non weak d types 1-3 variants were dar alleles, which are often associated with anti-d production. background/case studies: rhd genotyping has been recommended to guide transfusion of d-negative rbcs and administration of rh immunoglobulin to patients with discordant or weaker than expected d typing, particularly for females and ob patients . the recommendation is based on observational evidence, primarily from europe (flegel 2006, curr opin hematol13:476) , that individuals with weak d types 1, 2, and 3 are not at risk for clinically significant anti-d. the implications and utility of this approach for the diverse u.s. population are not yet clear. here we report 15 months experience with rhd genotyping on 352 samples referred with discrepant or weak d typing investigated from january 2016 to april 2017. study design/method: serologic testing was performed by standard tube agglutination with licensed reagents. dna isolated from wbcs was used in manual rflp and rhd beadchip assays and rhd sequencing for some. ethnicity was known for 153 samples (53.3% caucasian, 32.2% african american/african, 6.6% multiracial, 4.6% hispanic, 2% asian, and 1.3% other). results/finding: rhd genotyping identified weak d types 1, 2, and 3 in 155/352 (44%) and alleles known to encode partial d phenotypes in 168/ 352 (47.7%) (table) . uncommon or rare weak d alleles including types 6, 15, 40, 42, 45, 51, 57 (n52), 59, 61, 78, 91 , and 119 were found in 13 (3.7%). the partial d alleles found were diverse, but the largest number included partial rhd*d 4.0 (n562) and *dar ( conclusion: in a multiracial cohort of 352 individuals with weaker than expected d typing 44% were due to weak d types 1, 2, or 3 and would not be considered at risk of clinical significant anti-d, but for 56% there is potential or unknown risk. these studies are important to gain insight into the prevalence of specific alleles in the u.s. multiethnic population and to continue to evaluate and refine rhd genotyping for clinical practice. cp242 rhd*07.02 allele causes discrepant genotyping results for rhce small c sabine scholz* 1 , sandra schneider 1 , sabrina k€ onig 1 , susanne helmig 1 and vicky van sandt 2 . 1 inno-train diagnostik gmbh, 2 rode kruis vlaanderen background/case studies: in the human rh blood group system the c, c, e, e and d antigens are expressed by the two highly homologous genes rhce and rhd. after d, c is the most immunogenic rh antigen. the difference between c (307c) and c (307t) is caused by the snp on position 307 on the rhce gene. the rhd*07.02 allele (also known as rhd cat vii type 2) carries the snp 307t>c on the rhd gene and additionally the snp 329t>c. this rhd*07.02 allele has been described to partially express rhc on the d polypeptide (faas, transfusion, 2001) . aims: genotyping was performed to clarify the cause of the weak c expression. serology of a patient sample (male, 81938) indicated a partial c phenotype with a cde. study design/method: rhd and rhce phenotyping was done by accredited routine protocols (monoclonal ab id card: diaclon rh subgroups, seraclone anti-c). genotyping was performed with a taqman probe assay (rbc-fluogene veryfy, inno-train diagnostik gmbh), sso (rbc-lifecodes, gen-probe inc.), in-house ssp-pcr (hila, rode kruis-vlaanderen) and commercial ssp-pcr (rbc-ready gene cde, inno-train diagnostik gmbh). sanger sequencing of the rhd gene was performed using an inhouse method (inno-train diagnostik gmbh). results/finding: discrepant genotyping results were generated by different test systems: the taqman probe based assay showed in repetition a ccee genotype, while the sso system rbc-lifecodes predicted in repetition a ccee phenotype. in ssp-pcr the sample showed a weak c band with the in-house method, while there was no band visible with the commercial test kit. the parallel analysis of the rhd gene with rbc-ready gene cde test system revealed a variant d cat vii rhd allele. sequencing of the dna sample identified two snps on one of the rhd alleles (307t>c, 329t>c) confirming a rhd*07.02 and one rhd*01 allele. hispanic female in preparation for surgery resulted in variable reactivity and weakly positive d reactions when using microtiter-well agglutination versus tube testing. determination of whether the d antigen expression represented a weak d or a variant d could not be resolved by serologic testing alone. here we report the characterization of a novel rhd gene mutation identified by rhd gene sequencing. study design/method: serologic typing was initially performed by microtiter-well agglutination by automated analyzer platforms galileo neo and galileo echo (immucor, norcross,ga) and by standard tube testing using the immucor series 4 and 5 anti-d reagents. further immunohematologic evaluation was performed by standard tube testing (immediate spin -is, and indirect antiglobulin -iat) using orthobioclone, immucor gammaclone, immucor series 4 and series 5, and albaclone anti-d reagents. dna isolated from wbcs was used in manual rflp and rhd beadchip assay (immucor, bioarray) and rhd sequencing. results/finding: rbc reactivity is summarized in the table. dna testing detected a hybrid rhesus box associated with the rhd gene deletion, indicating the patient was hemizygous for rhd. rflp assay and rhd beadchip did not identify any changes. rhd gene sequencing identified a new c.463a>g change in exon 3 encoding an amino acid change p.met155val. the predicted location of this change is within the fourth transmembrane segment of the rhd protein. conclusion: we identified a novel rhd allele with c.463a>g (p.met155val) change in exon 3. several snps, deletions, and insertions have been reported with changes in exon 3. phenotypes of these genetic variations result in rh negative, weak d types, and variant d. since this change has not been previously identified, we are unable to determine if this confers a risk of anti-d alloimmunization, but the rhd c.463a>g snp results in serologically weak phenotypic expression of d antigen when tested by microtiterwell agglutination on the neo/echo platforms. in this case the combination of microtiter-well agglutination and dna sequencing helped identify a new allele which would be missed by standard tube serologic testing and the current commercially available array assays. serologic and molecular detection of an antibody to a high incidence antigen in patient with history of chronic transfusions georgia spanos* 1 , juan merayo-rodriguez 2 , christopher lough 1 and nancy eckert 3 . 1 lifesouth community blood centers, 2 life south community blood centers, 3 lifesouth community blood center-headquarters background/case studies: the jo a antigen is one of three high incidence antigens in the dombrock system. the prevalence of this antigen is 100% in most populations and greater than 99% in the black population. the jo a antigen can be resistant or enhanced with enzyme treatment (ficin/papain) and typically variable with dithiothreitol (dtt), w.a.r.m. tm (immucor) and zzap treatment. anti-jo a is an igg antibody that demonstrates at ahg phase. hemolytic transfusion reactions to the jo a antigen vary from none to moderate/severe. hemolytic disease of the fetus and newborn (hdfn) has not been observed with any antibody associated in the dombrock system. there are two common phenotypes present in the black population:hy negative/ jo a negative and hy weakly expressed/jo a negative. study design/methods: an antibody identification and red blood cell (rbc) units were requested for an o positive, 57 year old, african-american female with a history of sickle cell disease and no history of pregnancy. the patient was not recently transfused, however, had a history of chronic transfusions. last reported transfusion was three years prior to the current specimen. there were no known rbc antibodies at the time of the request. facility reports that the patient's hemoglobin(g/dl)/hematocrit(%) (hgb/hct) is 6.4/ 18.8 and does not appear to be in sickle cell crisis. a request for phenotypically matched units, as per hospital policy, for c, e, k and s was received by our immunohematology reference laboratory (irl). results/findings: anti-jo a was detected in patient plasma reacting with liss and peg (tube method) and manual gel-iat. the antibody was resistant when tested with dtt treated red cells. in-house frozen reagent rbcs negative for the jo a antigen (positive for hy) were used to serologically prove the presence of the antibody to this high incidence antigen. an allogenic peg adsorption was performed to rule out other common clinically significant antibodies. anti-kp a was identified using this adsorbed plasma. further testing with molecular genotyping (grifols idcore xt ) confirmed the patient's genotyping as antigen negative for the jo a , kp a and positive for hy. conclusion: molecular testing is frequently performed on patients and retained donor samples from our local community donor pool throughout florida, georgia and alabama. staff is able to search our database for any combination of antigen negative phenotypes using the internal 510(k) blood establishment computer software (becs) integrated blood bank information system (ibbis). this enabled us to locate one refrigerated and three cryogenically preserved jo a negative rbc units. we found 251 eligible blood donors that could be recruited via an automatically generated call list. the request for rbcs was cancelled. patient's clinical symptoms improved without transfusion and repeat hgb/hct increased to 7.1/21. the patient's sibling is historically negative for the jo a antigen and should future transfusions be required, it was recommended that a directed donation be made on the patient's behalf. in order to continue having blood components available to meet all our patient's needs, irl staff is consistently screening and searching our inventory for blood components that are negative for rare antigens to retain for patients needing antigen negative units in a timely fashion. rbcs of two females whose samples were referred for rhd genotyping with previously reported alleles for which serologic reactivity had never been investigated. study design/method: serologic testing was performed by automated analyzer, galileo echo and neo (immucor, norcross, ga), and by standard tube testing with licensed anti-d reagents and the albaclone advanced partial rhd typing kit. genomic dna isolated from wbcs was used for immucor rhd beadchip assay, pcr-rflp, and rhd sequencing. results/finding: patient 1 was a 29 yo female, c2e2c1e1, whose rbcs reacted 11 by echo and 31 by neo with anti-d4, and '?' with anti-d5. testing with d4 and d5 by tube gave 21 and 11 w on initial spin (is) respectively and 41 by indirect antiglobulin test (iat). rbcs were non-reactive at is with ortho bioclone and biorad seraclone, and 1 w with immucor gammaclone anti-d, and all were 21 at iat. rbcs did not react with two (lhm 174/102 & 57/17) of 12 anti-d in the alba partial d kit. this pattern did not match any partial d identified by these clones. rhd beadchip detected an inactive rhd pseudogene in trans to rhd. gene sequencing confirmed the presence of the pseudogene, but rhd had a c.780c>a change encoding p.his260gln. patient 2 was a 20 yo pregnant female, c2e2c1e1, whose rbc were 1 w at is and 31 at iat with immucor series 4 and 5 and gammaclone, and moderately reactive, 21 is and 41 iat, with alba alpha, alba blend and delta anti-d. rbcs did not react with two (lhm 174/102 & 170/45) anti-d in the partial d kit with no known partial d pattern. dna testing predicted she was rhd hemizygous and rhd beadchip detected markers for rhd*dar but exon 2 gave low signal (ls). sequencing found a hybrid dar with ce-specific nucleotides in exon 2 from c.150 to c.203 encoding amino acid changes p.ile60leu and ser68asn. conclusion: we found two previously reported rare alleles: rhd with a c.780c>a (p.his260gln), previously found in france (lefloch et al. genbank ku363612), and rhd*dar with part of exon 2 replaced by rhce, reported in sub-sahara africa (granier et al. transfusion 53:3009) designated rhd*dar(ce2:v505v-s68n) with an allele frequency of 0.002 to 0.016. blood samples were not available to test for alterations in d expression for either allele. we provide serologic evidence that these alleles, found in two females evaluated by rhd genotyping, inform transfusion and rh immune globulin prophylaxis, as they encode partial d phenotypes with novel epitope expression patterns, meaning these patients are at risk of forming allo anti-d. background/case studies: hu5f9-g4 is a human monoclonal igg4 antibody recognizing cd47 that is in clinical trials to treat hematologic or solid malignancies. cd47 is a transmembrane glycoprotein that binds to signalregulatory protein a (sirpa) on macrophages and functions to regulate phagocytosis. blocking cd47 is thought to enhance phagocytosis and promote anti-tumor responses. cd47 is also highly expressed on rbcs, and the purpose of this study was to evaluate anti-cd47 drug interference in blood bank testing. study design/method: serologic testing was performed by standard methods. serial samples (n57) from 2 patients were tested over the course of 1 month treatment. plasma was tested at immediate spin (is) and by iat with r2r2, rr, d--, rh mod and rh null rbcs, as cd47 expression levels vary depending on rh phenotype. dtt and enzyme treated rbcs were also tested. both immucor gamma-clone anti-igg (does not detect igg4) and ortho bioclone anti-igg (total igg) were used. for titration plasma was diluted in pbs. allo-adsorptions were performed with papain treated rr rbcs and eluates were made using gamma elu-kit ii. results/finding: anti-cd47 was observed in plasma as soon as 1 hour post infusion. plasma reacted 31 to 41 at is and 41 with all panel cells in peg iat using ortho anti-igg. d--, rh mod and rh null rbcs were nonreactive at is and weaker (31 and 21) in peg iat with ortho reagent. reactivity with all panel cells by ortho igg gel card was 31. in contrast, iat reactivity using gamma-clone anti-igg was only 1 w to 11, and this reactivity was confirmed to be carry-over agglutination. d--, rh mod and rh null were non-reactive in peg iat using gamma-clone anti-igg . the anti-cd47 titer was 1 at is and peg iat with gamma-clone anti-igg, but was ! 256 with ortho anti-igg. plasma reacted with dtt, trypsin, papain, a-chymotrypsin or w.a.r.m. treated rbcs. somewhat unexpected, autocontrols were negative and dats were non-reactive or microscopic only. acid eluates (n54) were 31 reactive with ortho, and non-reactive with gamma-clone anti-igg. plasma reactivity was removed after 4x allo-adsorption with papain treated rr cells, but in some samples low level (micro-11) reactivity remained. peg adsorption was invalid due to precipitation/complexing of antibody. robust plasma reactivity interferring in abo reverse typing was observed, and weak spontaneous agglutination of the rbcs in the abo forward and rh typing. conclusion: hu5f9-g4 anti-cd47 therapy interferes with routine pretransfusion testing, not only antibody screening and crossmatch, but abo and rh typing. high levels of cd47 expression on rbcs results in plasma agglutination at is, mimicking reactivity observed with igm antibodies although hu5f9-g4 is igg4. reactivity was observed in all phases and with all test methods. cd47 is not cleaved from rbcs by dtt, trypsin, papain/ ficin, dtt with ficin (w.a.r.m.) or a-chymotrypsin, and treatment of rbcs with these does not mitigate interference. numerous adsorptions with papain treated rr rbcs were required to remove anti-cd47 reactivity from plasma. use of immucor gamma-clone anti-igg, which does not detect igg4, can mitigate interference in iat although carryover reactivity may be observed. due to blocking by anti-cd47 on the patient rbcs, dat and autocontrols were weak or non-reactive; however eluates prepared from the dat1 rbcs were strong and pan-reactive using ortho anti-igg. background/case studies: a caucasian woman with history of a caesarean section and a rbc tx in 1985. in august 2016, she was admitted to hospital for trauma surgery, ab screening was negative and two units were transfused without transfusion reactions. five days later she was referred to a tertiary care trauma center due to a severe postop infection and need for a reoperation. ab screening was now positive, with an antibody reacting with all panel cells detected. because of the urgent need for rbc tx, two weakly cross-match positive rh1k matched units were transfused with a warning of possible alloantibodies. the patient got acute hemolysis. study design/method: a gel technique was used in the hospital transfusion laboratory. in addition, various antibody identification panels and special serological and genotyping methods were used in the reference laboratory. kel sequencing was done by the international immunohematology center. results/finding: the hospital transfusion laboratory results were o rhd neg, dat neg, and the ab identification was 21 with untreated and 31 with enzyme-treated cells, with weakly positive autocontrols. a sample was submitted to the reference laboratory for additional investigation. dat was weakly positive, while ab identification results were similar to the hospital results. different pheno-and genotyping methods were used in addition to several identification panels to exclude rare blood groups. after pk, vel neg, jk:-3 etc. had been excluded, k-phenotyping revealed a k 0 -phenotype. a total of 38 silencing mutations are known for the kel gene and the genotyping kits used did not recognize these. the anti-ku antibody reacts with all cells apart from the k 0 -phenotype. the presence of dtt-sensitive anti-ku was confirmed with dtt-treated panel cells. anti-ku may cause immediate and delayed hemolytic transfusion reactions. samples were taken from the patient's two siblings and daughter. kel sequencing revealed kel*02n.19 with c.2023t encoding p.675ter (reported in an individual from austria in 2007). there are two known k 0 -patients in our country, both homozygous for c.2023t. the daughter was a c.2023t heterozygote, while the siblings did not have this variant. a new operation is necessary but no k 0 -donors are available in our country. with the help of the isbt rare donor working party, a k 0 o rhd neg donor was found in japan and one unit was delivered to us for use in the next operation. conclusion: an alloantibody should always be suspected when autocontrol is weaker than panel cell reactions, even if the direct coombs is positive. a combined serological and genotyping approach offers the best solution for problematic antibody cases. compatible blood is not always available in rare blood group cases, but international co-operation may be of help in finding a suitable donor. transfusion strategy for the serologic weak d phenotype in tunisia based on rhd alleles and rh haplotypes mouna ouchari* 1 , kshitij srivastava 2 , houda romdhane 3 , saloua jemni yacoub 4 and willy albert flegel 1 . 1 nih, 2 dtm/cc/nih, 3 regional blood transfusion center sousse, 4 regional blood transfusion center sousse, tunisia background/case studies: d antigen variants have been studied molecularly in many arab populations, including gaza, tunisia, egypt and libya, 159a transfusion 2017 vol. 57 supplement s3 since 2009. the tunisian population has the largest known prevalence of weak d type 4.0 alleles, occurring in 1 of 105 rh haplotypes, compared to 1 in 6,060 or less in europe. a systematic study was missing for samples with the serologic weak d phenotype routinely found in blood donor and patient testing in tunisia. the study was designed to obtain data on weak d type 4.0 in a population known to harbor the greatest prevalence of such allele worldwide. study design/methods: a total of 13,431 random blood donors were serologically screened for the d antigen using 3 routine techniques. samples with weak reactivity were tested with a panel of 6 monoclonal anti-d (partial rhd-typing set) to identify partial d phenotypes. the rhd gene was sequenced in all samples with serologic weak d phenotype. the rhce gene was also tested molecularly by either direct sequencing or using the rhce beadchip kit to ascertain the rhce allele linked to the rhd allele. results/findings: a total of 67 discrepant samples (0.5%) were observed and expressed the serologic weak d phenotype. among them, 60 carried an allele of the weak d type 4 cluster (89.6%), of which 53 samples (88.3%) showed the weak d type 4.0 allele. only 1 sample each was found for the weak d types 1, 3 and 100 and the dvii, while 3 samples showed the consensus rhd sequence. no mutation in any of the 10 rhd exons was detected in another 3 samples. the molecular analysis of the rhce gene showed that 59 out of 67 samples with serologic weak d phenotype (88.06%) had a variant rhce allele and the most common associations were: weak d type 4.0 linked to rhce*cevs.04.01; weak d type 4.2.2 with cear; and weak d type 4.1 to rhce*cevs.02, while the other rhd alleles were linked to one of the common rhce alleles. conclusion: almost 90% of the weak d phenotypes in tunisia were caused by alleles of the weak d type 4 cluster, of which 88% represented the weak d type 4.0 allele. based on established rh haplotypes for variant rhd and rhce alleles and the lack of adverse clinical reports in tunisia, we recommend d positive transfusions for patients and no rhig administration for pregnant women with weak d type 4.0 in tunisia. we propose this strategy as a pragmatic clinical decision, even if eventually a rare allo-anti-d immunization would occur in tunisia associated with weak d type 4.0 phenotype. there is a possibility that the rhce*cevs.04.01 allele, typically associated in tunisian individuals, may protect from allo-anti-d immunizaton and other rhce alleles, such as rhce*ce more often associated in individuals of other ethnic groups, may not. however, we conclude that this conjecture has not much evidence in support at this time and would need corroboration by experimental and clinical data, before used to guide clinical recommendations. martha rae combs* 1 , heather simmons 1 , christine lomas-francis 2 , gayane shakarian 2 , sunitha vege 2 , lauren hutelmyer 3 , sandra nance 4 , jessica poisson 1 , nicholas bandarenko 1 and connie m. westhoff 2 . 1 duke university hospital, 2 immunohematology and genomics laboratory, new york blood center, 3 arc pennjersey, 4 american red cross, immunohematology reference laboratory, biomedical services background/case studies: plasma from a transfused, a1, 2 year old white female, post liver transplant with rbc aplasia, reacted at rt and in peg iat with all rbc samples tested except her own. study design/method: standard hemagglutination methods were used for antibody id and antigen typing. acid eluates were prepared using gamma elu-kit ii (immucor). genomic dna was isolated from wbcs and used for hea precisetype array and kel and sc gene sequencing. samples from the proband and her mother were tested, as applicable. results/finding: the patient's dat was negative. her plasma reacted with 0.2m dtt-treated and papain-treated rbcs, all available rbc samples lacking high-prevalence antigens, and with phenotypically similar rbc samples [c2, k2, fy(a2),s2]. reactivity was detected to a titer of 64; it was not removed by prewarm technique or by 4x peg alloadsorption. the adsorbed plasma reacted with 0.2m dtt-treated rbcs. extensive rbc phenotype results were unremarkable except for the following: k2, k2, js(b2), kp(a2b2) and sc:21,23. her plasma reacted with k o , mcleod, sc:21,22 rbc samples and dtt-treated sc:21 rbcs at rt and peg iat but her diluted plasma and pretransfusion eluate showed relative kp b specificity. the patient was transfused 4 aliquots of crossmatch incompatible kp(b2), s2 rbcs. her post-transfusion dat was 21 with anti-igg, 11 with anti-c3d. the eluate reacted with all rbc samples except 1 kp(b2) sample. she tolerated additional aliquots from 4 phenotypically similar rbcs untested for high-prevalence kell or scianna antigens. the hea precise-type predicted k2, k1, kp(a2b1), js(a2b1) and sc:1,22, discordant with her rbc phenotype. kel gene sequencing identified a homozygous change, c.1481a>t (p.glu494val) (kel*02.10) encoding the low prevalence antigen, ul a , but no changes associated with lack of kell system antigens; however, her rbcs typed ul(a2). sc sequencing found heterozygosity for a 5'-2g>a change (rs12124733, 24 to 30% prevalence) and conventional sc*01, predicting sc:1,22,3. kel and sc results on the mother were kel*02/kel*02.10, heterozygous for the sc change 5'-2g>a, and her rbcs typed k2k1 kp(a2b1), sc31, ula1, consistent with dna predictions. plasma collected 7 months later was nonreactive at rt and in peg iat. her rbcs were dat2 and now typed k1, kp(a2b1), ul(a1) sc11 and sc31,concordant with predicted kell and sc phenotypes. conclusion: we report an example of kell and scianna antigen suppression or blocking in the presence of autoantibody or an alloantibody in the kel system. to our knowledge, this is the first report of a ul a kel*02.10 homozygote. the rbcs may lack a high-prevalence antigen antithetical to ul a . without dna testing and gene sequencing, the patient would be presumed to have kell null and sc null phenotypes, a search for k o , and/or sc:21,23 rbc units would be performed and we would not have been prompted to re-type her rbcs when the dat was negative. background/case studies: anti-jka is a common antibody identified in the blood bank and providing phenotypically characterized red cells lacking this antigen is important in avoiding an acute or delayed hemolytic transfusion reaction. in nearly all cases, this antibody is identified in the context of a phenotypically homozygous jkb patient, jk(a-b1). other scenarios are quite rare. we present two cases of anti-jka in which this phenotype was not observed. study design/method: patient a is a 55-year-old multiparous female with no known transfusion history. her blood typed as o positive with a positive antibody screen, negative dat, and a clearly identified anti-jka in plasma. the patient phenotyped as jk(a-b-). genotyping revealed the presence of the jk*b allele, but not the jk*a allele. complete sequencing of the jk gene showed an intron 5 polymorphism in homozygosity. specifically, the patient showed a jk*b(ivs5-1a)genotype, associated with a jkb null phenotype. anti jk3 was not identified. the conclusion was an allo-anti-jka in a jk null patient. the patient did not receive any transfusions. patient b is a multiply transfused 64 old female. her blood typed as a positive with a positive dat and antibody screen. both the plasma and eluate revealed an anti-jka. despite the recent transfusion, the patient phenotyped as jk(a1) 41 and jk(b1) 21. genotyping showed the presence of both jk*a and jk*balleles. whole gene sequencing was not performed. there was no hematologic or biochemical evidence of hemolysis. the patient was considered to have an auto-anti-jka and jka negative cells used for transfusion. results/findings: patients a and b both developed anti-jka while having uncommon phenotypes/genotypes. conclusion: it is common for jk null patients to develop anti-jk3. however, we speculate that expression of the kidd glycoprotein with the jkb epitope was below the threshold of serological detection, but enough to prevent the formation of anti-jk3 or anti-jkb. auto-anti-jka is usually reported in the context of an active hemolytic process, but patient b illustrates an auto-anti-jka without hemolysis which is more commonly observed with autoantibodies exhibiting specificity for rh epitopes. these rare cases of anti-jka require phenotypic and genetic analysis for the jkb epitope and jk*b allele respectively, and in more complex cases whole gene sequencing. background/case studies: donor genotyping for red blood cell antigens has become common practice in many blood bank laboratories. package inserts for commercial assays indicate false negative results may be generated when unexpected rare mutations affect primer or probe binding and cause allele dropout or failed amplification. these outcomes may go unrecognized unless serological results are available for comparison. study design/method: a routine blood donor, self-identified as african american, was selected for red blood cell genotyping. dna was extracted and genotyping was performed using two commercial platforms 160a transfusion 2017 vol. 57 supplement s3 (precisetype, bioarray, warren nj; idcore xt , grifols, emeryville, ca). genotype results were compared to historical serological results. discrepancies were resolved by sanger sequencing (grifols ih, san marcos, tx). results/finding: genotyping results showed variants in both the duffy (fy) and kell (kel) blood group systems. the donor's genotype was concordant on both platforms, fy*a/fy*b_gata, kp*a/kp*a, for a predicted phenotype: fy(a1b-); kp(a1b-). when genotype results were compared to historical serology, it was noted that the donor previously typed fy(a-) on 3 separate donations. no previous kpa or kpb serotyping was available. sequencing of fy exon 2 revealed a 287g>a mutation, fy01*n.04, known to silence fya. sequencing of kel exons 1-19 exposed a silent polymorphism in exon 8, 846g>c. this polymorphism causes a dropout artifact yielding a false negative kpb interpretation. conclusion: the discrepant fy*a result, as well as the unlikely kp(b-) type prompted the request for sequencing. the rare fy01*n.04 mutation has been reported in people of caucasian descent. this is the first example of this fy mutation identified in this regional population. the kpb antigen is present in nearly 100% of all populations. however, kp(b-) is most frequently seen in people of caucasian descent. to date, 64 self-identified african american donors have been genotyped as kp*a/kp*b at this blood center. given the diversity of regional heterogeneity, it is feasible to identify a kp(b-) donor, self-reporting as african american. red blood cell genotyping offers an abundance of information, but cannot replace serology as the sole means of red cell antigen characterization. donor ethnicity continues to play a key role in selection for genotyping and the search for rare and unusual red cell types. in this case, a donor selected for genotyping based on ethnicity was initially thought to have 2 genetic variants not previously reported in those of african descent. only 1 was proven to be present. this case acts as a reminder that genotype limitations must be considered even when using licensed methodologies. this case report presents two group o pediatric patients who had been on enteral feeds and had absent/weak anti-b that became strong over time in patient 1. study design/methods: patient 1 was a 7 year-old male born prematurely with short gut syndrome who underwent a small bowel and liver transplant at 3 years of age. anti-b changed from undetectable/weak to strong at the age of 7 years. patient 2 was a 17 month-old female with a metabolic urea cycle disorder who underwent a liver transplant. anti-b was 0/11. both patients were on total parenteral nutrition (tpn) since birth and had strong anti-a and normal immunoglobulin testing. abo typing with enhancing techniques is presented in table 1 . results/findings: both patients typed as group o on forward typing. anti-a was strong in both patients. anti-b varied in strength in patient 1 with 0-11 reactions up to 7 years of age. thereafter, abo typing showed mainly strong anti-b. patient 2 had 0/11 anti-b. conclusion: intestinal bacteria stimulates production of anti-a and -b. unexpected changes in anti-b that caused abo discrepancies are reported here for 2 children on long-term tpn. patient 1 had absent/weak anti-b since birth up to 7 years of age, then developed strong anti-b with no change in feeding regiment and medications. patient 2 had consistently strong anti-a and absent/weak anti-b. these findings support the notion that normal colonization of the gut is important in the development of anti-a and -b and suggests that microflora of the gut in patients on prolonged tpn is different leading to the delayed formation of these antibodies compared to individuals on normal enteral diet. difference in strength of anti-a and-b could be due to stronger a than b antigen expression on gut bacteria. results/finding: a daratumumab protocol was established that incorporated use of the cord panel. multiple myeloma patients selected as candidates for daratumumab treatment were baseline tested for blood type and antibody screen, dat and genotype. after daratumumab infusion, a two unit crossmatch was order as a precaution in the event the patient developed a reaction to the medication. repeat of the antibody screen demonstrated panagglutination which served as a positive control for the medication. the cord panel ruled out underlying alloantibodies. selected red cell units were crossmatched at immediate spin phase to avoid expected indirect antiglobulin reactivity. conclusion: the cord panel was used 28 times over a five month period to rule out underlying alloantibodies. tests for the daratumumab protocol consisted of a routing antibody screen followed by a cord panel for resolution. the daratumumab protocol significantly reduced testing time and allowed for the provision of compatible blood products in an efficient and cost effective manner. teresa gorey* 1 and elizabeth hart 2 . 1 brigham and women's faulkner hospital, 2 university of massachusetts-dartmouth background/case studies: the purpose of performing a pre-transfusion antibody screen is to detect clinically significant unexpected antibodies and to decrease the probability of detecting clinically insignificant antibodies. several antibody detection methods (polyethylene glycol (peg), liss, and albumin) are routinely used in small transfusion services. the utility of peg is to enhance the sensitivity of detecting clinically significant antibodies by the indirect antiglobulin procedure. the code of federal regulations, title 42, cfr part 493.1271(a), states the manufacturer's instructions are followed when testing for unexpected antibodies. the package insert for gamma peg tm (immucor inc., norcross, ga), states that negative reactions may be examined with an optical aid. based on these directions, our institutional policy is to confirm all negative reactions using the microscope. study design/method: a one-year retrospective document review was performed on all patient samples in which a positive antibody screen (absc) triggered the antibody identification (abid) to be performed in 2016. a total of 232 samples were evaluated. each abid was subcategorized; (1) as being a new antibody for our facility or in the patient's shared electronic health record within the partnersv r healthcare system and (2) whether a microscopic absc result triggered the abid. also, patients with known antibodies were grouped according to a microscopic absc result. a comparison of the new patients and the previously known antibody patients with microscopic results were reviewed to determine if the antibodies were clinically significant. results/finding: a total of 83 abids were performed on new patient samples. of the new abid samples, 29 (35%) had microscopic absc results. for the previously known antibody patients, there were 35 which accounted for 15% of the total abids performed. when reviewing the total abid workups, a total of 64 (28%) of the abscs had microscopic results which resulted in an abid being performed. the antibodies identified in the 29 new antibody samples were: conclusion: a total of 86% of the new antibodies identified based on a microscopic absc were clinically insignificant. the manufacturer's directions were followed but they do not state that an optical aid is required to confirm all negative results. due to the results of this study, a decision will be made to: (1) discontinue the use of the microscope, (2) switch to a peg manufacturer whose directions indicate to observe macroscopically for agglutination, or (3) define the use of the agglutination viewer as the optical aid. decreasing the number of abids will save time and money while providing potential rbcs for transfusion in a timely and efficient manner. anton") has a prevalence greater than 99% in all populations. hereditary absence of anwj has only been described once (in a single family). however, red cell expression of anwj may be markedly decreased to near undetectable levels in blood donors of the in(lu) (or "dominant lutheran inhibitor") phenotype. similarly, anti-anwj antibody formation is rare, with only 10 cases reported in the literature. the antibody developed in the context of hereditary absence of anwj (i.e., a true alloantibody) in only one of the cases. in the other nine cases, the antibody occurred in the context of autoimmune or lymphoproliferative disease, where, in this context, it is believed to have developed secondary to transient anwj antigen suppression. most of the reported cases lacked clinical or laboratory evidence of hemolysis. however, in the most recently reported case, involving a 56-yearold woman with aplastic anemia, the antibody was associated with acute hemolytic reactions after rbc transfusions, necessitating transfusion support with anwj-negative and in(lu) rbcs. the case was also unique in that the anti-anwj resulted in a direct antiglobulin test (dat) that was positive for complement only, rather than igg like all previous cases in which the dat was performed and was positive. study design/method: a 59-year-old woman with severe aplastic anemia experienced acute hemolytic transfusion reactions (ahtr) with development of a panagglutinin on indirect antiglobulin test (iat) screens. prior to identifying the specificity of the panreactive antibody, the patient received 10 rbc transfusions and showed signs of hemolysis with six of them. the first three transfusions were prior to her positive iat and were electronically crossmatched. the next seven transfusions were incompatible by antihuman globulin (ahg) phase crossmatch, but were extended phenomatched for clinically significant antigens. the patient's ahtr signs and symptoms included fever, rigors, nausea, vomiting, dark urine, flank pain and "impending doom" anxiety; while her laboratory findings included hemoglobin decreasing below pre-transfusion levels, and increased total bilirubin and ldh. the dat, while initially negative during the immediate posttransfusion workup of the transfusion reactions, eventually became positive for igg only (1-21), and negative with anti-c3b, c3d reagent. the antibody showed a peak gel-igg iat titer of 32. results/finding: the antibody was identified as having anwj specificity. the patient's pre-transfusion sample showed weak anwj expression (w1), altogether suggesting an auto-anti-anwj. monocyte monolayer assay testing using the patient's plasma and rbcs from the ahtr-implicated units yielded monocyte indices ranging from 33 to 83%, consistent with the clinical hemolysis observed. given the patient's group o, rh d negative blood type and continuing transfusion dependence, in order to avoid further ahtrs, international collaboration was necessary in order to procure and provision group o, rh d negative rbcs that were also serologically negative for anwj. the patient was successfully transfused three such units without further incident. conclusion: this is the second documented case of anti-anwj in a patient with aplastic anemia and, overall, the third anti-anwj case associated with ahtr. this case also underscores the importance of international collaboration. cold auto-antibody 12 anti-p 1 4 anti-m 2 anti-sd a 6 anti-le b 1 anti-jk a 1 anti-k 1 anti-e 1 anti-c 1 results/finding: three hundred and ninety weak d genotypes have been determined to this day with frequencies of 21% (type 1), 5% (type 2), 9% (type 3), 25% (type 42) and 40% other than 1, 2, 3 or 42. further investigation was conducted to determine the molecular identity of the «others». out of 157 samples, 119 (75%) were confirmed to be legitimate serological weak or partial d, mainly deletions of exon 5 or both exons 4 and 5. a surprising amount of 38 samples were discovered to be normal rhd. conclusion: along with sandler et al. (2015) data, our findings highlight the difficulties hospitals face in interpreting serological weak d. trend analysis was conducted regarding the reagents and technologies used by each hospital, the origin of the request and the ethnicity of the concerned patient, but no significant correlation could be identified at this point. altogether, our findings allow to share the frequency of weak d types 1, 2, 3 and 42 obtained in serological weak d, 45 years old quebec's women, and also highlight the need for further investigation of standard practices amongst hospitals regarding the management and interpretation of atypical d typing. were classified as fnhtr. taco incidence was 0,6%. no trali happened in the period. prophylaxis were used in 98% of patients. conclusion: fnhtr is described as the most common adverse event related to transfusion, but our data showed a higher incidence of allergic reactions. fnhtr occurred 3 times less than allergic reactions. this might be explained by universal leukoreduction and universal prophylaxis adopted at our institution. further studies are necessary to evaluated the benefit of this approach. that cannot be associated with a specific rbc unit or were deemed unrelated to transfusion, 358 rbc transfusion aes were analyzed. chi-square test and logistic regression were used to compare the ae incidences among transfusion groups. results/finding: univariate and multivariate logistic analyses showed that irradiated rbcs were associated with a significantly increased incidence of transfusion-related aes (p <0.05). there was a significant difference in febrile non-hemolytic transfusion reaction (fnhtr) (0.27% vs 0.11%, p <0.001) or aes with a non-allergic type inflammation etiology (0.30% vs 0.14%, p <0.001) including transfusion-related acute lung injury, transfusion-associated dyspnea, but not transfusion-associated circulatory overload, infections or hemolytic transfusion reactions, between irradiated rbcs and non-irradiated rbcs. in contrast, the incidences of allergic aes (0.028% vs 0.024%, p 5 0.614) were similar between these two groups. the incidences of inflammation aes after transfusion of irradiated rbcs that were stored for 1, 2, 3, and 4 weeks were 0.25%, 0.32%, 0.39% and 0.41%, respectively (p 5 0.084, logistic regression) but there was a significant difference in the incidence of inflammation aes caused by irradiated rbcs stored for a week (0.25%) and longer than a week (0.35%) (p < 0.05). conclusion: irradiated rbcs associated with a higher incidence of transfusion inflammation aes compared to non-irradiated rbcs and this risk increased when rbcs were stored longer than 1 week after irradiation. while it is likely the patient population is a factor in ae caused by irradiated rbcs, it is also possible that rbc radiation damage, as shown in previous studies, contributed to this increased ae incidence. a list of patients with one of these icd10 codes was generated. the emr was searched to find the clinical scenario in which trali was mentioned. these patients' records were then searched within our laboratory information system (copath), to determine if they had a transfusion reaction reported to our transfusion medicine service. results/finding: the search of our electronic medical record found 11 patients from 2011-2016, who had trali mentioned in their chart as a diagnosis or possible/likely diagnosis. one patient was excluded from our study because trali was mentioned as a past medical history from an outside hospital. only the patients who had trali listed as a diagnosis or possible diagnosis were included in this study. these 10 patients had clinical scenarios in which a transfusion of a blood product occurred which was followed by various forms of respiratory distress. the clinical teams caring for these patients were either giving a diagnosis of trali or considering trali as a possible diagnosis. of these 10 cases, only 2 of them were reported to our transfusion medicine service as transfusion reactions. of the reported cases, one was determined to be trali and the other one was consistent with taco. eight out of those 10 cases were never reported. background/case studies: despite diligent efforts to transfuse the safest product available to patients, undetected alloantibodies may cause delayed hemolytic transfusion reactions (dhtr). this transfusion reaction is seen in as many as 1 out of 100 transfused products. therapeutic plasma exchange (tpe) may be employed to mitigate ongoing immune mediated hemolysis, but few reports in the literature describe tpe for clinical management after profound hemolysis. study design/method: case review of a patient was performed after diagnosis and treatment of severe dhtr. results/finding: a man with a history of gastrointestinal bleeding presented to the emergency room with shortness of breath and "hematuria". he had a known history of anti-d and anti-c, and was transfused two units of crossmatch compatible rbcs seven days prior during a previous admission. readmission hemoglobin (hb) was 8.7 g/dl but declined to 7.3 g/dl the next day. an antibody screen was consistent with anti-d, anti-c, and direct antiglobulin test (dat) was negative. he received three units of crossmatch compatible rbcs over days 2 and 3 with poor responses. on day 4, routine labs could not be reported due to marked hemolysis, he had "worsening hematuria", creatinine rose from 1.0 mg/dl to 1.8 mg/dl (reference 0.8-1.3 mg/dl), and lactate dehydrogenase was above reportable linearity, >2500 u/l (reference 122-222 u/l). testing revealed additional anti-e, anti-jkb, dat c31, plasma free hb 64.4 mg/dl (reference 1-15.2 mg/dl), and hemoglobinuria. four of five transfused rbc units were jk(b1), one of which was also e1. one volume tpe was performed to remove free hb on days 5, 6, and 7 using fresh frozen plasma as replacement fluid for haptoglobin supplementation. creatinine peaked at 3.7 mg/dl on day 13, decreased to 2.3 mg/dl before discharge on day results/findings: twenty three cases were identified, of which 20 had medical records available for analysis. ten (50%) patients were male, the mean age was 50.4 years (range 24-76 years), 15 (75%) had an underlying hematologic malignancy or bone marrow disorder, and 3 (15%) had a history of coronary artery disease (cad). the implicated units included 14 (70%) red blood cells and 6 (30%) platelets; 17 (85%) patients received a single unit, and 3 (15%) received two or more within the previous 6 hours; the mean volume transfused was 153.3 ml (range 20-280 ml). the mean time to onset of chest pain was 92.15 minutes (sd 85 minutes), with 90% of patients presenting within 2.5 hours and 100% within 6 hours of starting the transfusion. chest pain was present as the only symptom in 35% of the cases, and for the other cases the accompanying symptoms included dyspnea (30%), fever (25%), back pain (20%), and hypo-and hypertension (10%). a post-transfusion chest x-ray was performed in 65% of cases, and all showed no evidence of pulmonary edema to suggest possible volume overload/transfusion associated circulatory overload (taco). electrocardiogram was performed in 70% of cases and showed no findings to suggest acute ischemia. three (15%) patients had a minimal increase in their troponin levels, although 1 had a history of chronically elevated troponin due to stress cardiomyopathy. fourteen (70%) patients received some form of treatment, including increased oxygen supplementation, metoprolol, acetaminophen, morphine, and oral calcium carbonate; the pain resolved after more than 10 minutes in the majority of patients (90%). no cases resulted in new admission to the icu or procedure cancelation. conclusion: chest pain associated with transfusion was infrequent, but several such cases were identified during the review period. this symptom is not a diagnostic criterion for any of the other hemovigilance categories and merits further characterization to determine whether blood product transfusion could be the cause of the chest pain. larger observational studies to power clinical characterization could help to further inform hypotheses regarding a transfusion-related mechanism, which could be interrogated by translational research studies. background/case studies: thrombotic microangiopathy (tma) in children is most commonly seen in the form of hemolytic uremic syndrome (hus). however, tma may be seen in the presence of streptococcus pneumoniae (spn). the action of bacterial neuraminidase of spn results in exposure of the normally "hidden" thomsen-freidenreich antigen (t-antigen) found on erythrocytes and other tissues. ultimately, this may lead to spn induced hemolytic uremic syndrome (phus) with subsequent hemolysis and end organ damage by naturally occurring anti-t antibodies against the exposed t antigen. specific lectins or anti-sera can confirm exposure of the t antigens in phus. alternatively, phus can be identified by minor crossmatch incompatibility resulting from agglutination of exposed t antigens on recipient's erythrocytes to anti-t antibodies in the plasma portion of blood products. we present a case of suspected phus that resulted in a compatible minor crossmatch leading to concern and eventually diagnosis of atypical hus (ahus). study design/method: a 6 months old boy presented with respiratory failure. he was found to have blood cultures positive for spn as well as hemolytic anemia, thrombocytopenia, and acute renal failure. he was shiga toxin negative and had normal levels of adamts 13. based on the findings, the clinical team was concerned for phus. therefore, he received washed erythrocytes. for his thrombocytopenia, our institution does not routinely provide washed platelets due to decrease quality of the platelet product. as a result, a minor crossmatching was suggested and performed to determine if t activation was present. results/finding: minor crossmatch was performed with patient's erythrocytes and plasma of abo-identical platelets to be transfused. no agglutination was seen at immediate spin, 37 degree, or anti-human globulin phase. check cells were found to be 21. these findings were conveyed to the clinical team and platelets were issued without washing. due to the lack of identification of t activation by minor crossmatching and poor clinical response despite appropriate antibiotic treatment, additional studies were performed by the primary team for complement mutations and found to be consistent with ahus. the patient was then treated with eculizumab with clinical and laboratory improvement. we present a case clinically consistent with phus. confirmation of this diagnosis is done with lectins or anti-sera that are not readily available. an alternative means of identifying phus is by minor crossmatch incompatibility. by demonstrating minor crossmatch compatibility, we further elucidated a definitive diagnosis of ahus with appropriate management. background/case studies: orthotopic liver transplantation (olt) is a complex and technically challenging procedure that can be complicated by severe intraoperative bleeding. we report a case of massive transfusion in an olt patient necessitating an abo blood group switch (from o1 to a1) to sustain transfusion support and minimum group o rbc inventories. study design/methods: type & screen (ts, gel) and anti-a titers (tube) were performed using routine methods. a chart review was performed for pertinent medical and laboratory findings. results/findings: the patient was a 63-year-old o1 man with cirrhosis secondary to nonalcoholic steatohepatitis and alpha-1 antitrypsin deficiency who presented for olt (donor o1). during olt, the patient endured substantial bleeding from retroperitoneal collateral vessels complicated by post-transplant coagulopathy. he required rapid high volume rbc and plasma support, which strained hospital inventories. after receiving 46 units of o1 rbcs and 26 units of o1 plasma with ongoing severe hemorrhage, he was switched to group a products. ten units of a1 plasma were transfused to wash out anti-a antibody prior to transfusion of a1 rbcs. due to difficulty controlling the bleeding, biliary reconstruction and fascial closure were delayed for 7 hours post-transplant. the patient's total estimated blood loss was >20l. he received a total of 71 units of rbcs (including 23 a1), 63 units of plasma (including 37 a1), 6 units of cryoprecipitate, and 9 units of platelets. towards the end of the second procedure, the patient's hemorrhage was stabilized and the final two rbc units he received were o1. on postoperative day (pod) 1, a ts showed predominantly a1 rbcs with trace o1 rbcs, as well as very low anti-a igm and igg titers (table 1) . he received two additional o1 rbc units (1 each on pod 4 and pod 12) with increasing o1 rbcs on ts and rising anti-a titers. his blood type was unequivocally o1 by pod 13. the patient showed recovery of liver synthetic function on pod 1 (factor 5 activity 5 58%) complicated by cholestasis. conclusion: this study shows successful switching of a group o patient to group a in the setting of rapid hemorrhage and massive transfusion. by pod 13, the patient had reverted to o1 with recovery of anti-a titers. at 3 months post-olt, the patient is alive with signs of improving biliary graft function. a new rfid transfusion safety system anna millan* 1 , alfred mingo 1 , maria isabel gonzalez 1 , antoni mena 2 and juan pedro benitez 2 . 1 bst, 2 at-biotech background/case studies: a new transfusion safety system (tss), based on processes and technologies, especially, identification by radio frequency (rfid), is currently implemented in two hospitals, a general one (h1) and an oncology center (h2). the tss is fully effective in protecting against incidents, and specifically offers mechanisms to detect near misses (nm) by using procedural and physical barriers, assuring that the pretransfusional sample extraction (pse) and the blood components administration (bca) only take place at bed side, using a location control and interacting with the clinical and transfusion information systems (tis). the tss allows to analyze the transfusional activity information in real time to project organizational changes in both transfusion services and hospital units, and to create a new classification of nm. study design/method: retrospective analysis of 2016 transfusion activity in both h1 and h2 shows 7970 pse and 12572 bca, out of 13163 and 20676 respectively, since the tss deployment in 2015. retrospective analysis and classification of 6700 security events has been done. results/finding: activity results for both hospitals are shown in the table below. the safety events have been classified in pretransfusion sample extraction (pse), blood component assignment (bcas) in the transfusion service and blood component administration (bca) near misses (nm). for h1, nm related to pse accounted for 42.39% of all, being the mistake in concordance between patient identification and prescription order the most frequent (52.03%). the nm detected in bcas were 12.1% of all and mostly (74.52%) occur when the patient information in the tis does not match the one registered in the tss. the nm detected in bca are 45.51% of all and mostly (36%) the systems detects a not assigned bracelet. for h2, nm related to pse accounted for the 47.49% of all, being the error in concordance between the transfusion security number in the bracelet and in pretransfusion sample the most frequent (65.84%). the nm detected in bcas accounts for 24.48% of all and in 69.08% occurs when the patient information in the tis does not match with the one registered in the tss. the nm detected in bca are 28.02% of all and in 65.61% of them the blood components were assigned to another patient. (1, 3, 4, 5, 8, 9, 12, 14, 19, 23, 26, 51, 56, 68) were analyzed via a commercially available elisa. comparison of adequate response to ppv23, defined as ! 2 mcg/ml for >7 serotypes, was perform based on alloimmunization status. statistical significance was determined by comparing means of subgroups using paired and non-paired t-tests. results/findings: pre-vaccine sp titers were available in 72 patients (alloimmunized, 15); pre-and post-vaccine titers were available for 19 patients (alloimmunized, 6). of the 72 patients, 25 were on chronic transfusions, 24 were on hydroxyurea, 11 were surgical splenectomized, 58 patients had no history of surgical splenectomy or status was unknown. forty-four patients had a previous history of ppv23 in the previous 10 years; 9/44 also reported previous history 13-valent sp conjugate vaccine within the last 5 years. baseline pre-vaccination titers (n572) showed no difference between alloimmunized and non-alloimmunized patients (all p-values >0.13). in the group with pre-and post-vaccination (n519) titers available, 11 out of 13 (85%) non alloimmunized patients had an adequate response versus 4 out of 6 in the alloimmunized group (67%, p 5ns background/case studies: blood transfusion is the most common procedure performed in the hospital setting and the transfusion process is monitored to ensure regulatory compliance. to safeguard safety, efficacy and regulatory compliance, transfusion services actively benchmark transfusionrelated errors (tres) as they occur from "vein-to-vein", i.e. from collection of pre-transfusion sample to final infusion of product -with the goal of ensuring that the right product/dose goes to the right patient at the right time. multiple over-lapping error documentation processes are needed to capture and report tres from within and outside of blood bank (bb). we present a comprehensive error management program along with data on five years of benchmarking tres at a large academic medical center. study design/method: tres were detected by capturing and reporting of sample suitability, testing variances and biologic product deviations. in addition, tres as observed and reported by providers and clinical staff (i.e. blood delays/undertransfusions, transfusions without consent, infusions with wrong fluids) were reported to the bb and hospital quality through the veritas system, a hospital based reporting system that enables reporting any occurrence with potential for causing patient harm. all serious errors were reviewed daily and summation of tres was discussed on a monthly basis. mapping tres within the "vein-to-vein" was performed by reviewing the fiveyear of transfusion medicine quality records (from 2012 to 2016). patient harm events recorded within the veritas system from january to july 2016 were investigated in depth. transfusion reactions were excluded in this analysis. results/finding: an average of 114 tres per month and 1300 per year were found over five years. 81% of tres are associated with pre-bb activities, 10% occur within bb, and 9% are post-bb events. sample collection and handling represent 80% of total tres. most tres (96%) were reported by bb staff, 4% were reported by non-bb staff. patient harm analysis revealed an average of four level 0 (near miss), three level 1 (no known harm), and 0.3 level 2 (patient harm) per month. no deaths related to tre were detected over the seven month january to july 2016 period. patient harm was associated with tres occurring in the bb (17%) and post-bb (83%). these events were reported externally (78%) and by bb staff (22%). conclusion: although most tres were detected in the pre-bb phase, no patient harm was associated with these events indicating an efficient capture prior to causing patient harm. the tres causing patient harm, including near miss events, were mostly reported externally and they occurred entirely in the post-bb and bb phases. these results suggest that significant opportunities for quality improvement may be achieved in two areas: the pre-bb phase aimed at reduction of waste associated with sample collection and handling, and the post-bb and bb phases aimed at improving tre detection and decreasing patient harm. background/case studies: uncrossmatched red blood cells (rbc) and emergency issued platelets (plt), plasma and cryoprecipitate (cryo) are lifesaving in a bleeding patient without a valid type and screen. collectively termed "emergently issued products" they are issued as a bridge until pretransfusion testing is completed. this study evaluated the utilization and wastage rates of blood products during pregnancy-related hemorrhage where the first products issued were emergently issued. study design/methods: a list of patients on whom blood products had been emergently issued between january 1, 2013 and march 28, 2017 was obtained from the blood bank at a regional maternity care hospital. patients who were not experiencing a pregnancy-related bleed (e.g., postpartum hemorrhage or bleeding relating to a complication of pregnancy such as a ruptured ectopic pregnancy or bleeding post spontaneous or therapeutic abortion) were excluded. the total number of products (emergently issued plus crossmatched or non-emergently issued products) that were transfused, returned back into the blood bank's inventory, and wasted within 6 hours of the first emergently issued products were enumerated. apheresis plt units were multiplied by 5 and added to the number of individual whole blood plts; apheresis plasma units were multiplied by 2 and added to the number of whole blood plasma units. results/findings: seventy women who received emergently issued blood products during a pregnancy-related hemorrhage were identified. average age was 31. the majority of these patients with pregnancy-related hemorrhage who received at least one unit of emergently issued blood products received at least one unit of the product that was issued to them and few units were wasted. that plt wastage was higher than the other products was likely due to the 4-hour post-pooling room temperature shelf life. keeping wastage rates low while meeting the clinical needs of these patients is the ideal situation for the blood bank. . patient blood platelets were higher before prophylactic than therapeutic transfusions (18[10 9 /l vs. 14[10 9 /l, p50.029). there were no significant differences in the frequency of effective therapeutic (55% vs. 72%, p50.1) and prophylactic (63% vs. 54%, p50.09) transfusions between the prcs and 25gypcs. we did not find significant differences between prcs and 25gypcs in cci1 after prophylactic (16.0 6 7.1 vs. 19.2 6 8.7) and therapeutic (11.3 6 9.0 vs. 11.8 6 5.8) transfusions, in cc24 after prophylactic (20.0 6 9.2 vs. 22.5 6 12.8) and therapeutic (13.3 6 8.9 vs. 13.9 6 8.) transfusions. there were no significant differences between prcs and 25gypcs also in ma1 after prophylactic (62.2 6 8.5 vs. 60 6 8.5, p50.7) and therapeutic (61.3 6 9.9 vs. 60.9 6 12.7, p50.08) pc transfusions. reduction of the severity of bleeds was obtained in 78 (86%) of the 91 cases after prpc transfusions and in 51 (84%) of 61 cases after 25gypc transfusions. there were no significant differences in the frequency of adverse post-transfusion reactions between the groups (respectively, 3 and 2 cases). background/case studies: an 'end-to-end' electronic transfusion management process including a bedside administration system was developed and implemented in this large multi-site academic center in 2006. it enables the safe administration of blood components at the patient bedside and provides an audit trail for all blood components. an error was identified in the electronic bedside transfusion process which was reported to our national hemovigilance scheme in 2014 under the category 'errors relating to information technology'. this error was the incorrect use of the emergency transfusion process for non-emergency transfusions. the standard (non-emergency) process requires a scan of the barcode on the patient's wristband containing their identification details which is verified against the same details from the barcode on the compatibility label attached to the blood bag. the emergency transfusion option is only intended for use with 'emergency group o rhd negative blood units' which, unlike non-emergency units allocated to specific patients, do not have a compatibility label. the emergency transfusion option skips the compatibility label barcode scan as the emergency units can be transfused to any patient needing urgent transfusion. it was found that the emergency blood option was being misused for non-emergency transfusions, leading to blood units not being checked to ensure they were for the correct patient. study design/method: this center worked with the software supplier to develop a solution which corrects the weakness in the process. the revised process involves providing a universal compatibility label for emergency units so that all units (emergency and non-emergency) require a scan of the compatibility label on the blood bag and the patient's wristband at the bedside before transfusion. the use of the emergency process was audited pre and post implementation of the new process to determine whether it was being used correctly or not. results/finding: there were 593 units administered using the emergency transfusion process in the 3 months before the change was implemented. it was found that 51/593 (9%) units were non-emergency units administered incorrectly without a bedside compatibility check. following the implementation of the change there were no instances of incorrect administration of non-emergency units in the next month (2530 components administered), 109/2530 (4%) were emergency units which were administered correctly. users of the system reported the revised process was quicker, safer and unified with other functions on the device. conclusion: the improved process for the administration of blood in an emergency now prevents users from following the incorrect procedure for non-emergency transfusions and missing the essential final bedside electronic check. this report indicates the need for continued vigilance of the functionality of electronic transfusion processes, and the correction of any weaknesses compromising patient safety. background/case studies: recent recommendations indicate one red blood cell (rbc) unit should be transfused at a time with reassessment after each transfusion to determine the need for more. however, the practices of canadian transfusion medicine (tm) experts and what constitutes a reassessment are unknown. therefore, we conducted a survey of tm experts across canada to gather information on their practices and criteria for reassessment. study design/method: tm experts were identified and contact information obtained from the canadian national advisory committee (nac) and from contacting least one tm expert per province. each respondent was assigned a unique study id after consenting to the survey, allowing for anonymity on analysis. the survey contained demographics, general practice questions, and questions regarding transfusion in: 1) a stable anemic inpatient, 2) a stable anemic inpatient to be discharged, and 3) an asymptomatic post-operative inpatient. results/finding: we identified 67 canadian tm experts: 48 (71.6%) provided a response and most had a primary place of practice in a laboratory setting (38/48; 79.2%). for a stable, non-bleeding, anemic inpatient, 87.5% of respondents recommended transfusing one rbc unit, then reassessing. recommendations were more variable in outpatient settings, with 31.2% generally recommending transfusing two rbc units then reassessing. recommendations for reassessment were mainly functional status/symptoms and vitals within a short time period (1-2 hours), a repeat hemoglobin >18 hours later dependent on the clinical scenario, and a search for an underlying cause of anemia in outpatient settings. lab practitioners emphasized volume status, cardiac examination, and transfusion at lower hemoglobin thresholds. with an asymptomatic patient to be discharged, fewer respondents chose to transfuse (38.1%) compared to an inpatient potentially symptomatic due to anemia (72.1%). none of the respondents suggested transfusion in an asymptomatic post-operative patient who had a hemoglobin trending down. conclusion: tm experts generally recommend transfusing one unit at a time in stable inpatients. assessment for transfusion should focus on patient symptoms, pertinent physical exam, hemoglobin levels, and an underlying cause. "top-up" transfusions were not recommended. these recommendations may help guide clinicians, but further research is needed to generate higher quality evidence around the clinical benefits and cost effectiveness of these practices. background/case studies: current evaluation of red blood cell (rbc) post transfusion recovery is based on ex vivo labeling of stored rbcs with radioactive chromium-51 ( 51 cr). this method has several limitations including the risks associated with radioactivity, and the inability to evaluate multiple rbc populations in the recipient. rbc labeling with s-nhs-biotin (bio-rbcs) overcomes many of these limitations and offers safe and longitudinal tracking of multiple transfused rbcs in vivo. the purpose of this study was to scale up and optimize the biotinylation procedures to the current good manufacturing practice (gmp) environment. study design/method: packed rbc units (n514) were divided into two 150ml aliquots, which were labeled with selected concentrations of s-nhsbiotin (3 and 30 lg/ml) in a cgmp closed system (average bio-rbcs hematocrit of 38.4 6 1.6%). optimization of labeling efficacy was determined by flow cytometric analysis of bio-rbcs using fluorochrome-conjugated streptavidin (sa). approximately 2 million rbcs were measured in triplicate. quantum simply cellular beads were used to quantify fluorochrome (molecules of equivalent soluble fluorescence, mesf) and infer number of biotin molecules per rbc. the lower limit of detection was determined for rbc labeled with varying amounts of biotin. product quality and safety were evaluated by endotoxin and sterility testing, and by determining the levels of spontaneous hemolysis before and after rbc biotin labeling. results/finding: investigation of different fluorochromes, laser excitation wavelengths and laser power to maximize the signal to noise ratio of labeled and unlabeled rbcs revealed that 561nm excitation of phycoerythrin (pe)-sa and high laser power (150mw) provided the best separation between the two bio-rbc populations, and between labeled and unlabeled rbcs. labeling with 3lg/ml of biotin resulted in $50,000 mesf/rbc, and were detectable among unlabeled rbc at a lower limit of detection (lld, 95% ci) of 1 in 380,000 (0.0003%). the lld95 for rbc labeled with biotin at 30lg/ ml was $ 1 in 1 million (0.0001%). biotinylation was not associated with increased levels of hemolysis (0.40 6 0.22% before labeling versus 0.34 6 0.12% after labeling; p50.09) or bacterial contamination. conclusion: the resulting manufacturing process produces large volumes (150ml/transfusion) of bio-rbcs with low risk of contamination or hemolysis. the flow cytometry assay can detect bio-rbc in unlabeled blood at very low frequency. we plan to use this technology to study the impact of donor characteristic on rbc storage stability and post-transfusion survival. background/case studies: blood products offer resuscitation benefits in trauma over crystalloid/colloid volume expanders (which provide no hemostatic benefit or oxygen delivery), but usage is often hampered by supply or storage needs. hemoglobin-based oxygen carriers (hbocs) are not red cell replacements but may supplement oxygen delivery and expand volume during transport until blood is available. since hemostasis is critical in resuscitation, this study evaluated bovine hemoglobin glutamer-250 (hboc-201) effects on coagulation parameters alongside freeze-dried plasma (fdp) in an in vitro model of hemorrhage/resuscitation. study design/method: whole blood (wb) was collected from healthy donors under an approved institutional standard operating procedure. in the first study (limited resuscitation), samples were: (1) wb, (2) wb110% hboc volume (model of two units in an adult), (3) wb110% fdp, and (4) wb110% hboc110% fdp. samples (5)-(8) simulated autoresuscitation by adding 25% plasmalyte to 1-4. susceptibility to lysis was tested with 75ng/ml tissue plasminogen activator (tpa). follow-up studies were performed with severe resuscitation simulations of 50%, 60%, 75%, and 100% volume replacement with hboc and/or fdp, with or without prior 25% plasmalyte dilution. coagulation parameters were obtained with a coagulation analyzer and thromboelastography (teg). rbcs/hemoglobin were measured on a hematology analyzer. thrombin generation was quantified by thrombogram. platelet aggregation was measured in multiplate and adhesion to collagen under shear in bioflux. viscosity was evaluated by rheology. results/finding: a limited resuscitation model with hboc and/or fdp had no effects on fibrinogen, pt, aptt, ph, hct, or hemoglobin. in teg, wb, wb1hboc, and wb1hboc1fdp had reduced clot strength with dilution and tpa. there was increased susceptibility to tpa-induced lysis between wb and wb1hboc in autodilution simulation (mean lysis 4.79% vs. 16.36%; p<.05). hboc and fdp had no statistically significant impact on thrombin generation. no effects on platelet aggregation were observed; no significant differences within diluted v. undiluted groups were seen in platelet adhesion under flow. hboc (10%) did not significantly change viscosity. severe resuscitation simulations had increased pt/ptt and reduced clot strength, particularly in hboc-only resuscitation; however, even 75% hboc volume replacement produced clots with acceptable teg parameters. conclusion: in a limited resuscitation model with hboc-201, there were no significant in vitro effects on hemostatic parameters (except increased susceptibility to lysis); more severe resuscitations impacted coagulation parameters but did not prevent clotting. considering the large impact healthy platelets have on coagulation function, further in vitro studies with impaired platelets are warranted alongside in vivo studies of hboc1plasma as initial resuscitation of hemorrhagic shock. therapy in patients with acute major bleeding. while published literature has largely focused on the efficacy and safety of pcc, actual usage practices are less characterized. our aim was to describe the pcc usage practices within a tertiary care center. study design/method: we conducted a retrospective review of the electronic medical records of patients who received pcc between its addition to our institution's formulary in 8/2013 and 2/2015. we compiled information about the usage of pcc in these patients. descriptive statistics were generated with microsoft excel. results/finding: of 81 patients, 24 were on warfarin. pcc was most frequently prescribed for hemorrhage due to surgery (43%). pcc was given for warfarin reversal in 31% of cases. a subset of patients received plasma within 2 hours prior to pcc (40%) or 24 hours after (47%). pcc was most frequently ordered in the or/perioperative service (25%). conclusion: the majority of pcc usage was "off-label" in terms of being prescribed for indications other than warfarin reversal. the most frequent indication was hemorrhage due to surgery, and pcc was most often ordered in the or/perioperative service. although guidelines recommend the use of pcc as a plasma alternative, plasma was administered within hours of pcc in a notable subset of patients. background/case studies: in emergent situations, when a patient's life may be jeopardized by delaying transfusion, a physician may decide to transfuse blood emergently. however, in some cases, poor communication and lack of clear expectations between the blood bank and patient care areas can lead to frustration and delays in the timely provision of blood products. an incident prompted an appraisal of our emergency release protocol (erp), which revealed gaps in communications and expectations by both the blood bank and nursing personnel. thus, it is imperative that there is a standardized er protocol with clear communications for both the blood bank and nursing personnel. reported here is the outcome of a process improvement that resulted in improved communication, expectations, and turnaround (tat) for our er protocol. study design/method: in 2016, several meetings were conducted with stakeholders (critical care units (icu), emergency department (ed), internal medicine, interventional radiology (ir) etc.) in an effort to identify process gaps, improve communications, and expectations for er episodes. the goals was to design a process for emergent blood product request and release in life threatening situations that will; 1) simplify and expedite the process; 2) improve communication and expectations to decrease tat; 3) improve patient safety and meet compliance. in order to achieve these goals, a series of activities were conducted. these included meetings with all stakeholders to ensure process improvement meet the needs intended. a series of training sessions with nurse educators in icu, ed, ir and surgery managers were conducted. during the meetings, communication goals, and expectations were defined and agreed upon. training sessions included powerpoint presentations to educate staff members and performance of dry runs, to identify weaknesses and strengths with the process flow. the impact on the current process was analyzed and, as a result, led to the revision of the current sop, addition of pre-labeled emergency pack blood (4 units of o neg rbc's) and implementation of an electronic emergency blood order set. results/finding: in the ten months post implementation of our improved, standardized er pack protocol, a total of 61 er episodes were received. the average tat from order to delivery at the bedside was reduced by 50% (7.0 minutes compared to 14 minutes previously), while the compliance rate for er orders and physician documentation was 100% (61/61), with no current wastage of blood products. conclusion: the implementation of the improved standardized er protocol significantly improved communications and expectations, decreased tat and delays in transfusions while ensuring patient safety and compliance to regulatory requirements. background/case studies: massive transfusion (mt) in the trauma setting has been extensively studied. yet, the literature in non-trauma areas, especially oncology is rather sparse. the following study was conducted to understand the background and outcomes of mt in cancer patients. study design/methods: this was a single center retrospective study performed at a large cancer center between february 2016 -february 2017. mt was defined as the transfusion of ! 10 rbc units in a 24-hour period. the following data were collected included: age, gender, primary diagnosis, surgery or acute care type, amount and type of blood components transfused, whether or not a massive transfusion protocol (mtp) was activated, and survival at 30 days. results/findings: thirty mts occurred during a one year period. a total of 192,441 blood products were transfused during that time period. gender distribution was 21/30 (70%) males, and the average age of all patients was 68 with a range of 21 to 70 years of age. surgical patients accounted for 26/30 (86.7%) mts, and 4/30 (13.3%) were critical care patients. tumor categories included carcinomas (14/30), sarcomas (13/30), leukemias (2/30) and lymphoma (1/30). resection of tumor followed by complex reconstruction was the cause of the majority of mts. metastatic renal cancer (6/30) was the most common disease seen followed by sacral chordoma (4/30). mtps were activated in only 8/30 (26.7%) cases. thirty-day survival was seen in 25/30 (83.3%) patients. only 1 of 5 mortalities was a surgical case (peritoneal mesothelioma), and the remainder were caused by gi hemorrhage (3/ 5) or perisplenic hematoma (1/5). the overall ratio of rbc:ffp in the entire patients ( background/case studies: plasma is a straw-colored supernatant of blood that is used for type and screen (t&s) and crossmatch. in the analytic phase of testing, plasma is examined prior to processing. plasma occasionally becomes discolored, interfering with crossmatch procedures. timely identification of the etiology allows for corrective actions and minimizes delay in transfusion. study design/method: during the analytic phase of blood bank testing, samples were evaluated for t&s and crossmatch; this identified three samples with discolored plasma. we present a series of cases that illustrate the testing process. results/finding: a 54-year-old woman diagnosed with breast cancer presented for mastectomy with sentinel lymph node biopsy. a preoperative t&s specimen contained bright green plasma. review of her preoperative case revealed exposure to intravenous methylene blue. this dye is known to alter the color of urine, tears, and blood with no known pharmacologic effects. alternative causes of green plasma include other dyes used to locate sentinel nodes and oral contraceptive use. although not ideal, this sample could be used for crossmatch by tube method, but not automated gel technique. a specimen drawn one week later contained clear plasma. a 26-year-old woman diagnosed with a warm autoimmune hemolytic anemia was refractory to blood transfusions secondary to alloantibodies. administration of a synthetic blood product resulted in dark maroon colored plasma. the most common cause of a dark red color is hemolysis of the sample, which is usually discarded. in this instance, the hemoglobin color was due to the infused product, an experimental bovine pegylated carboxyhemoglobin that affects colorimetric evaluation of blood samples. with this in mind, the sample was not discarded and testing was completed by tube method. a 70-year-old woman admitted with acute stroke was treated with a thrombolytic. her t&s revealed cloudy white plasma that could not be used for the crossmatch procedure. common causes of white plasma include purulence, hypertriglyceridemia, and sampling of blood drawn proximal to administration of radiopaque agents such as propofol. although an etiology could not be identified a repeat specimen drawn several hours later was clear. conclusion: these cases highlight the importance of an appropriate evaluation of discolored plasma. once a discolored sample is identified, a repeat sample is required to confirm the change in color. in the first two cases, the discoloration persisted, prompting further clinical investigation. once the etiology was identified, need for further testing and eligibility for further transfusion was determined. testing by tube method could be performed in two cases. in the third case, repeated sampling revealed a clear sample and the transfusion process continued without delay. decisions regarding the analytic phase of testing must include reevaluation of the sample, identification of the etiology, and comprehension regarding how to proceed when discoloration persists. caleb wei-shin cheng* 1,2 , rebecca ross 2 , christopher a tormey 1,2,3 and amit gokhale 1,2 . 1 yale university school of medicine, 2 yale-new haven hospital, 3 va connecticut healthcare background/case studies: daratumumab (dara) is a igg1 monoclonal antibody therapy that specifically targets cd38, a glycoprotein highly expressed on plasma cells, where it has been successfully used in patients with refractory or relapsed multiple myeloma. dara interferes with blood bank testing as it binds to cd38 expressed on red blood cells, causing pan reactivity. the dara interference can be overcome with the use of dithiothreitol (dtt) treated reagent red blood cells. to minimize alloimmunization and to provide crossmatch compatible blood to treated patients, we instituted a dara protocol in our blood bank. the purpose of this retrospective study was to identify the outcomes of our protocol, with a particular focus on the development of de novo alloantibodies during dara treatment at our institution. study design/method: all dara patients' antibody workups were completed using dtt pre-treated reagent red blood cells. if the antibody screen was negative, k antigen negative rbc products are provided. if an antibody is identified, k negative along with that particular antigen negative blood is provided. our electronic medical record (emr) was searched for patients who received dara over the past eight months. study subjects were examined to see if they had pre-existing alloantibodies before dara treatment and whether they formed new alloantibodies during dara treatment. the age, gender, type and screen pre-dara treatment, type and screen post-dara, intervening blood transfusions, and the date of first dara treatment was recorded. results/finding: overall, 54 subjects were identified for analysis. their mean age was 67.8 years, with 29 male and 25 female subjects; all were diagnosed with multiple myeloma. we found an alloimmunization rate of 0% (0/54) prior to administration of dara. of these patients, 22 were transfused with red blood cells (rbcs) after initiation of dara therapy. following our testing/matching protocol, none of these (0%; 0/22) patients formed a confirmed, new alloantibody during dara treatment; each of these patients underwent at least one follow-up screen after their first rbc unit. we also found no complications in providing crossmatch compatible units to any of the 22 patients. conclusion: to our knowledge, this is the largest case series reporting on results of overcoming dara interference with blood bank type and screen testing. the protocol implemented in our laboratory appears to be successful in providing compatible units and preventing alloimmunization in patients receiving dara therapy. it is possible that the drug, targeting antibody forming cells, may have an immunosuppressive effect on the humoral response; further studies of this effect may be warranted. background/case studies:a multi-facility transfusion service began stocking liquid plasma in september of 2015 for use in massive transfusion and trauma situations. due to the infrequent occurrence of these incidents, the liquid plasma would outdate before use. a policy to use liquid plasma in nonemergent situations when the units were nearing their expiration dates was implemented. this study evaluated the effects of that policy on inr values of plasma recipients. study design/methods:a retrospective analysis was developed to compare the effectiveness of fresh frozen plasma (ffp) and liquid plasma (lqp) in changing inr values of recipients. all plasma units transfused within the facility from september 1, 2015 through april 7, 2017 were identified. the following data was obtained from the hospital and laboratory information systems for each unit: the recipient, primary reason for transfusion of plasma, number of plasma units transfused, type of plasma transfused, preand post-transfusion inr values, and whether or not vitamin k was administered. patients were divided into groups based on the type of plasma units transfused and were evaluated based on primary reason for transfusion, number of units transfused, and administration of vitamin k. the change in inr for each recipient was calculated, along with the average change in inr for each group. background/case studies: in gynaecological settings, most but not all relatively young anaemic women are iron deficient due to blood loss associated with menstruation. transfusion could generally be avoided in those without haemodynamic instability. the oral antifibrinolytic drug tranexamic acid is an effective and well tolerated treatment for menorrhagia. besides, iron replacement is often necessary for a prolonged period of time after normalization of haemoglobin (hb). the present study attempted to look into transfusion appropriateness and the use of iron and tranexamic acid in transfused women in hong kong. study design/methods: anonymous data of gynaeological patients age 60 was retrieved from a central database of public hospitals which included age, number of units of red cell transfused, pre-and posttransfusion hb, the use of iron and/or tranexamic acid during hospitalization and upon discharge. all transfusion episodes associated with surgical operations during same admission are excluded. results/findings: in 2016, 2,523 unique women receiving a total of 5,889 units of red cells (rc) in 2,906 transfusion episodes were identified. their median age was 45 (range 11 -60). the distribution of pre-and post-transfusion hb and units of rc transfused were summarized below: in this cohort, pre-and post-transfusion hb were absent in 46 (1.6%) and 283 (9.7%). 635 (21.9%) transfusion episodes were associated with the use of 3 units or more rc. as a result, 1385 (47.7%) episodes resulted in a post transfusion hb ! 9g/dl. parenteral iron or tranexamic acid was uncommon during hospitalization and was given 2 (<0.1%) and 116 (4.6%) women respectively. upon discharge, 442 (15.0%), 84 (3.2%) and 1,994 (65.8%) women were prescribed with oral iron alone, oral tranexamic acid alone or both respectively. however, neither were given to 386 (16.0%) women. conclusion: in the present study, it is observed that 49.7% transfusion episodes were given at hb ! 7g/dl. a substantial number of episodes (71.7%) were transfused with multiple units and resulted in almost half having a post transfusion hb level (! 9g/dl). for iron replenishment and bleeding control, up to 16.0% transfused women were not given iron or tranexamic acid at discharge. the results indicate that awareness of both transfusion appropriateness and iron deficiency anaemia management have to be improved. it is recommended that in-depth education and training should be provided for a better gynaecological patient blood management. background/case studies: granulocyte transfusions may be utilized to boost the immune response in patients with life-threatening neutropenia or neutrophil dysfunction and evidence of treatment-refractory bacterial or fungal infection. however, granulocytes are rarely administered due to uncertainty regarding efficacy, difficulty in collection, and increased propensity for adverse reactions. we report a case of granulocyte transfusion therapy following chimeric antigen receptor t-cell (car-t) therapy in a patient with severe neutropenia and multiple infections in the context of relapsed b-cell acute lymphoblastic leukemia (b-all). study design/method: granulocytes (0.8-1.3x10 10 per unit) were collected from abo-identical unstimulated donors at a regional blood center. each unit was irradiated with 25 gy and transfused over 3-4 hours within 24 hours after the time of collection. the patient's response and laboratory data were reviewed in the medical record. conclusion: this data suggests that a diagnosis of aml is associated with anti-hla antibodies. an increased frequency of blood group a in patients with aml has been reported, but here no statistically significant difference between abo blood group frequencies was found in any category except the patient's with hla antibodies. blood group b has a significant association with hla alloimmunization in the studied patients. it has been reported in a large study of female blood donors that no difference in hla antibody frequency was observed based on abo blood group at centers using the flow-based assay. although the reasons for the higher rate of group b blood type among patients with anti-hla antibodies and hematologic malignancies is unknown, this could be due to variation in immunizing events (pregnancy vs transfusion) or immune dysregulation related to the hematologic malignancy, especially aml. females with aml who are blood group b appear to be most likely to have hla alloimmunization among patients with hematologic malignancies. implementation of electronic solution to reduce risk of mistransfusion in a regional transfusion service debra lane* 1 , lee grabner 1 , brenda herdman 2 , robert fallis 1 , amin kabani 3 and charles musuka 3 . 1 canadian blood services, 2 kenora rainy-river regional laboratory program, 3 diagnostic services manitoba background/case studies: patient misidentification and improper sample labeling has been an ongoing risk for the safety of blood transfusion. the rate of mistransfusion has remained unchanged in over 50 years. attempts have been made to reduce mistransfusion including barrier devices, barcoding and rfid. within a regional background/case studies: the role of donor age and sex on hemoglobin content and susceptibility to hemolysis during storage of red blood cell (rbc) units is receiving increased attention. however, the impact of donor characteristics on efficacy of rbc transfusion has not been studied in largescale donor-recipient outcomes databases. study design/methods: we conducted an analysis using blood donor data routinely collected by a blood center and transfusion recipient data from a large community hospital network between 2008 and 2011 before patient blood management initiatives. linkage was performed between blood donor characteristics and hospitalized rbc transfusion recipients who received a single rbc unit. studied exposures for this analysis were blood donor sex and age in addition to rbc storage age. the wilcoxon test was used to examine changes in hemoglobin level following rbc transfusion, and , and 38% were male. recipients of rbc's from male and female donors had similar pre-transfusion hemoglobin levels (8.7 g/dl; p50.94); however, transfusion recipients of male donor rbc units had higher post-transfusion hemoglobin levels and larger increments in hemoglobin compared to those of female rbc units (9.9 vs 9.8 g/dl; 1.2 vs. 1.1 g/dl; both p50.02). female recipients had a larger rise in hemoglobin per rbc unit compared to male recipients (1.3 g/dl vs. 1.0 g/dl; p<0.001). female sex of the recipient remained a significant predictor of change in hemoglobin after accounting for recipient age and estimated circulating blood volume in multivariable analysis (p50.01). rbc storage age and the age of the donor were not significant factors in changes in hemoglobin levels in multivariable analysis, p50.53 and p50.32, respectively. conclusion: rbc units from male donors resulted in a larger rise in hemoglobin levels compared to those from female donors, and these changes were more apparent in female recipients even after accounting for effective circulating blood volumes. this suggests that the dose of hemoglobin is lower in female than male rbc units. this analysis demonstrates the feasibility of using this approach to study the association between donor characteristics and rbc efficacy, hemolysis and other donor-component-recipient interactions. background/case studies: people who identify as jehovah's witnesses (jw) comprise less than 1% of the population of the united states. however, as a group they can present a special challenge in medicine due to a religious aversion to blood products, based on biblical readings. the degree of this religious refusal can vary from individual to individual, but as institutional policy, a conservative approach is warranted. however, in large institutions where multiple teams manage a single patient, blood refusal information can be lost or poorly communicated from provider to provider. as such, a system to alert providers of patient blood refusal was recently implemented through the electronic medical record in a large west-coast institution. study design/method: the electronic medical record (emr) utilized in this study in an institutionally modified version of epic ea best practices alert (bpa) was designed to trigger each time an end user attempted to place orders related to blood transfusion, transfusion-related lab testing, or human-derived pharmacy items on patients with blood refusal codes in their history, problem list, or religion (jehovah's witness) discrete data fields. the alert constitutes a "soft-stop" in which the ordering provided is prompted to either cancel the triggering orders or acknowledge the blood refusal/religious history and override the warning with an option to select a reason for the override. data on the triggers are automatically collected through the emr systems and generated into a report by informatics personnel. results/finding: the available data covers triggers in the two month postimplementation of the bpa. the bpa triggered 33 times in total, affecting 15 patients and 21 users. stratified by location, the majority of triggers occurred in the perioperative areas (18 times) and the liver icu (6 times) with a minority occurring on the regular hospital floors and emergency department. nurses, attendings, residents, pharmacists, and nurse anesthesiologists were the users affected. orders that triggered the bpa included type & screens, human albumin 25% iv solution, human albumin 5% iv solution, immune globulin (human) solution. conclusion: despite the limited and very preliminary data, the user action findings seem to indicate that the bpa is effective in halting up to half of the contraindicated orders for blood-derived products and type & screens orders. given the limited types of orders that the bpa is triggering on, the pattern suggests that the bpa is potentially alerting some previously unaware providers of the patient's religious status and/or the fact that certain pharmacy items are blood-derived, and therefore unacceptable to many jw patients. despite these positive initial findings, this is an ongoing study to track the efficacy of the bpa and more data needs to be collected for better metrics of the institutional sensitivity to patient blood refusal. intervention to address inappropriate cryoprecipitate-ahf orders at a tertiary medical center sirisha kundrapu* 1,2 , mahmut akgul 1,2 , hollie m reeves 1,2 , robert w maitta 1,2 , marcie pokorny 2 , anne capetillo 2 and katharine a downes 1,2 . background/case studies: although introduced for the management of hemophilia a, now cryoprecipitate is primarily indicated for low fibrinogen levels. at our institution the transfusion medicine service (tms) reviews and makes recommendations to clinicians for all inappropriate cryoprecipitate orders. we aimed at analyzing the effectiveness of this intervention in reaching target fibrinogen levels in under-estimated and over-estimated orders. study design/method: we conducted a 7-month retrospective study (january-july 2016) of adult cryoprecipitate order quality assurance forms. the reference range for fibrinogen was 200-400 mg/dl with critical value of 50mg/dl. cryoprecipitate orders for massive transfusion protocol, from operating rooms and for extracorporeal membrane oxygenation were not reviewed by the tms. during the study period, tms evaluated orders for appropriateness of dosing and agreement with estimated required doses. post-transfusion fibrinogen levels due to intervention were compared with hypothesized no intervention levels. statistical analysis was performed using chi-square and t-tests. results/finding: there were 301 adult (>18 years) orders reviewed by tms out of which 299 were approved. of the 299 approved orders, 136 (45.5%) were in agreement with tms's estimated dose. of 163 (54.5%) orders that were not in agreement with the tms's estimate, 142 (47%) were underestimated and 21 (7%) were overestimated. seventeen of 299 orders had no post-transfusion fibrinogen levels. without intervention, there would have been a median deficit of 23.6 mg/dl (range 0.3 to 124 mg/dl) and a median excess of 13.3 mg/dl (range 0.6 to 155 mg/dl) of fibrinogen from the target. median difference between target and actual post-transfusion fibrinogen level was 12 mg/dl above target, which is significantly higher with intervention than without (which could have been 12 mg/dl below the target; p<0.0001). median differences between target and post-transfusion fibrinogen levels for the group with agreement between approved and requested units was not significantly different from possible differences without intervention (11 vs. 2.7 mg/dl, p50.07). median differences between target and post-transfusion fibrinogen levels for the group with non-agreement between approved and requested units was significantly different from possible difference without intervention (15 vs. -23.5 mg/dl, p<0.0001). seven of 299 (17) 40 (24) orders were for critically low fibrinogen (<50 mg/dl) and 4 of these were under-estimated requests and reached target fibrinogen with tms's estimate and approval of required units to be transfused. overall most frequent orders were 10 and 5 units (59.5% and 25%) i.e. 2 and 1 pools and the most frequent orders in the disagreement group were 10, 1, 5 and 2 units (33%, 20%, 17% and 16%). there is a significant difference between agreement and disagreement groups based on clinical service ordering the units (table) . conclusion: intervention by tms to review and approve cryoprecipitate orders was associated with increased accuracy of orders and achievement of desired target fibrinogen levels. further studies are needed to develop multidisciplinary strategies for accurate cryoprecipitate dosage. patient characteristics, medical records, vitamin k administration, and adverse events, were collected (table) . results/finding: the average pre-transfusion inr was 2.36 and posttransfusion was 1.91. only 22% of patients had their inr corrected to 1.5, while 28% had no change, or had increased inr. (table) . the majority (67%) of patients received 2 units of plasma. the mean plasma dose was 6 ml/kg. there were 4 transfusion reactions reported, 1 non-hemolytic and 3 transfusion associated circulatory overload reactions in which 1 required admission to the icu. two patients experienced bleeding during ir procedures (tips) and 1 developed a hematoma (tunneled central line). the median of inr correction in this study was 1.9 with no relationship to the number of units of plasma transfused and/or if vitamin k was administered. this study suggests it may not be beneficial and may be harmful to transfuse plasma for correction when inr is 1.9. randomized trials are needed to assess whether the inr is a rational tool to measure bleeding risk, and whether prophylactic treatment with plasma yields any benefit. 3 of the 111 patients experienced bleeding complications indicating that inr of 1.9 may be considered safe in some lower risk procedures. current practices may provide little or no benefit, with substantial risk of life threatening complications. background/case studies: group ab plasma, which lacks anti-a and anti-b antibodies, is considered to be the universal plasma donor and is used in the emergency setting before the patient's blood group is available. approximately 4% of the population is group ab, which limits the available inventory of group ab plasma. of group ab population, only plasma from male donors are considered suitable for transfusion since females, especially multiparous female donors, have a greater propensity to develop antibodies that can cause transfusion related acute lung injury (trali). this makes type ab plasma a limited resource. our hospital is a level one trauma center, where a significant amount of plasma transfusion is required for severely bleeding patients before their blood type is known. group o individuals make up 45 % of the population and have no a or b antigens on their cells. group a is the second most prevalent blood group in the us population (40%) and has no b antigen on their cells. so, group a plasma is compatible with both group o and a patients, approximately 85% of the patient population. before patient's blood type is known, type o red cell units are transfused with a plasma, which decreases the chance of hemolysis. to conserve ab plasma, we instituted a policy effective july 1, 2014 as follows: 2 units of group a plasma and 3 units of group ab plasma is provided for the massive transfusion protocol (mtp) along with 5 units of o negative rbc until patient blood type is known. study design/method: this prospective study is designed to monitor the use of group a plasma in mtps at our institution and to evaluate the risk and severity of hemolysis in patients transfused with incompatible plasma. direct antiglobulin test (dat) is performed if patient received incompatible plasma. if dat is positive, lactate dehydrogenase (ldh), haptoglobin and bilirubin levels are obtained to detect possible hemolytic transfusion reaction. results/finding: we reviewed 235 mtps at our institution between july 2014 and march 2017. twenty patients (8.5 %) were transfused with incompatible group a plasma (5 group ab and 15 group b patients). five patients died due to severe injury, and follow-up testing of these patients could not be performed. the remaining 15 patients had negative dat, indicating the lack of significant amount of antibody coating their red cells, which could lead to hemolysis. none of these patients developed acute hemolytic reaction, or any other adverse effects of incompatible plasma transfusion. conclusion: our study adds more evidence of the safety of group a plasma transfusion in trauma patients requiring emergent massive transfusion before the patient blood type is known. based on this and other recently published studies, starting in april 2017, our institute will provide only group a plasma for emergency release and mtp cases before the patient blood type is known. average ( background/case studies: in 2013, bonfils immunohematology reference lab (irl) sent out approximately 245 special platelets for patients with hla antibodies. by 2016, hla platelet orders increased dramatically and the irl sent out over 650 special platelet products. the purpose of this abstract is to illuminate the methods used to fulfill increased client need that occurred in a short period of time. study design/methods: bonfils blood center has over 10,000 donors in the database with historical hla typing. however, only approximately 3500 of those donors actively donate. in the denver area, one of the most common hla types is a1 a2 b7 b8. only 81 of the 10,000 donors have this type (0.81%). therefore, to fill an hla platelet order request for a common hla type, only 28 donors in the system would be a perfect hla match. with that low number of donors, it is not likely that there would be a platelet on the shelf ready to fill the order. after a donor is recruited and donates, it takes at least two days to fill an order. for a less uncommon hla type like a9 a11 b17 b35, there is only 1 out of 10,000 donors (0.01%) that match perfectly. in those cases, there are no donors to recruit to fill such an order. in some complicated cases, the irl was provided with an hla antibody list or panel reactive antibody test (pra). in order to find product for these patients, lists of platelets in inventory with corresponding hla types were printed. if a patient had an antibody to a1 for example, all of the a1 positive platelets were crossed off the list. this cross-out process would continue manually until the only platelets on the list were the ones positive for hla antigens to which the patient did not have antibodies. these platelets are pra matched to the patient. in order to automate this process a report linked to the donor database was created to find both pra platelets in inventory and donors for recruitment. the blood center medical director began suggesting that hospital clients order a pra for each patient with platelet refractoriness. the pra test is fast and it is a definitive method to discern hla antibody mediated refractoriness from platelet refractoriness due to other causes. results/findings: in all but the most complicated cases with rare hla patient phenotypes, it was much easier to find a pra patient matched platelet on the shelf than an hla match donor. in 2012, approximately 27% of these special order platelets were pra matched and the remaining 73% were hla matched by donor recruitment. by 2017, approximately 59% of special platelets sent are pra matched. this change resulted in a 2.2 fold increase of finding product in inventory to fill orders quickly. conclusion: developing a system to provide pra matched platelets is a faster alternative to finding hla matched platelets thus contributing to better patient care. background/case studies: in urgent cases where large amounts of blood products are needed quickly, maintaining a standard massive transfusion protocol (mtp) is critical to the timely delivery of these products. each mtp pack at ucm contains 6 packed red blood cells (prbcs), 4 fresh frozen plasma (ffp) units, and 1 plateletpheresis pack; a unit of prepooled cryoprecipitate is also given if the patient is in labor and delivery (l&d) or if one is requested. at ucm, blood products are generally transported through the pneumatic tube system (pts). we undertook a review of our mtp issuing practices and efficiency patterns over the last three and half years. study design/method: the electronic archives of the blood bank laboratory information system and electronic medical record at our institution were queried for patients who had mtp activations. the archives were correlated to paper copies of these activations to collect data pertaining to the relevant information such as where the order originated from, how quickly the first product was sent out, how many products were transfused, and so on. results/finding: between august 2013 to march 2017 mtps were activated at ucm, of which 251 orders could be traced to the origin: 118 on inpatient floor (including icus), 58 in the operating rooms, 46 in the emergency department, 25 in labor and delivery, and 4 in other procedure rooms. of the 2207 prbcs that were issued, 1406 were transfused (64% utilized); of the 1446 units of ffp that were issued, 901 were transfused (62% utilized); of the 359 platelet packs that were issued, 246 were transfused (69% utilized); of the 64 units of cryoprecipitate that were issued, 49 were transfused (77% utilized). since march 2016, the time of first product issue after the initiation of an mtp has also been tracked. of the 84 events that fall within this time period, 39 (46%), had the first product issued in 5 minutes or less. another 31 (37%) were issued between 5-10 minutes, resulting in over 80% of patients being issued their first blood product within the first 10 minutes. only 15 of 84 (17%) events had an initial time greater than 10 minutes and none were greater than 21 minutes. conclusion: the majority of our activations currently come from inpatient floors (primarily icus). as our institution anticipates the introduction of an adult level 1 trauma center, we anticipate this balance will shift. in addition, the data shows that (with the exception of cryoprecipitate) the utilization rate is nearly identical among the blood products sent during mtp activations ($60-70%). again, we anticipate utilization rate of issued mtp products to increase with the introduction of a new adult trauma center. we have recently begun tracking time to last product issued during an mtp, but cannot report on that variable at this time. overall, our data show that our transfusion service is generally performing adequately to issue the first product within 10 minutes of mtp protocol activation. this data only reflects time to issue in the pts; patient care areas can experience additional minutes delay in pts delivery and arrival of product at bedside. we must continue to collaborate with our clinical colleagues to collect accurate data to provide the best and most efficient mtp care. mehreen yasin* 1 , shailesh macwan 1 , arline stein 1 , jane fischman 1 , nancy nikolis 1 , matthew bank 1 , lennart logdberg 1 , alexander indrikovs 2 , sherry shariatmadar 1 and vishesh chhibber 1 . 1 north shore university hospital, 2 northwell health background/case studies: massive bleeding is generally defined as any patient who requires 1 blood volume replacement within 24 hours and/or receives transfusion of greater than or equal to 4 units in one hour with 177a transfusion 2017 vol. 57 supplement s3 ongoing bleeding. our mtp was officially implemented in 2013 in preparation for an initial verification as a level 1 trauma center by acs. our mtp has the following packages: 1st pack has a ratio of 4:4:1 (rbcs, plasma & platelets) and subsequent packs a ratio of 6:6:1. our mtp also includes prothrombin time (pt), activated partial thromboplastin time (aptt) and fibrinogen testing after each pack is transfused. this data is used to assess the patient and allows the transfusion service and clinical team to identify coagulopathies. however, attempts to supplement mtp packs with cryoprecipitate (cryo) and prothrombin complex concentrate (pcc) were challenging to accomplish in a timely manner. study design/methods: due to challenges in timely supplementation of mtp packages with cryo and pcc, the protocol was modified in march 2016 to add cryo and pcc at a defined point in the mtp (cryo is included in the 3rd pack and pcc in the 4th pack). in order to validate this modification of adding these products at defined intervals regardless of laboratory data, we decided to review all patients that received >20 rbc at our institution as these massively hemorrhaging patients would receive pcc based on our current protocol. we reviewed the blood products received by these patients and their available laboratory data. results/findings: we had 8 patients who received >20 rbc in 2015 and 2016. mtp had been activated for all patients and all patients received between 0.5 to 1 unit of plasma for each rbc unit transfused. despite receiving these ratios of blood products, all patients had elevations of their pt >16 seconds and many had elevations of the aptt and fibrinogen levels less than our institution's target of 200 mg/dl (table 1) . as anticipated, improvement in the coagulation parameters was noted with cryo and pcc supplementation. conclusion: our data on massively hemorrhaging patients supports a role for supplementation of our mtp with cryo and pcc in patients who require transfusion of >20 rbc. our current protocol with the addition of cryo and pcc at defined intervals has streamlined the process and improved timely provision of these products in bleeding coagulopathic patients. background/case studies: red blood cell hemolysis is a key finding for a diagnosis of transplant-associated passenger lymphocyte syndrome (ta-pls). however, whether a hematopoietic stem cell or organ transplant recipient experiences hemolysis when a transplant contains unintended antibody-forming passenger lymphocytes depends, by chance, on the recipient's blood group phenotype. a living donor liver segment transplant resulted in a case of ta-pls with donor-derived anti-d that had the potential for causing a clinically significant hemolytic event. the donor's plasma contained anti-d. anti-d was absent in the recipient's pre-transplant plasma, but present in the recipient's 5-day and 114-day post-transplant plasma. although these findings established a diagnosis of ta-pls, hemolysis did not occur because the recipient's blood group phenotype was d-. the conventional focus on hemolysis, rather than on the transfer of antibody-forming lymphocytes, is a diversion from the primary pathophysiology of pls and limits capturing the true scope of the syndrome. study design/method: to determine the standard of practice for detecting and diagnosing ta-pls, a retrospective 10-year pubmed search for peerreviewed english-language journal articles was conducted using key words "passenger lymphocyte syndrome." cases were categorized according to the presence or absence of hemolysis and whether there was a routine antibody screen to detect donor-derived, passenger lymphocyte-formed blood group antibodies. results/finding: of 63 published cases (31 reports) of ta-pls, 8 (4 reports) were stem cell and 55 (27 reports) were organ transplants. all 8(100%) stem cell transplants and 52 (95%) organ transplants were associated with hemolysis, reflecting an overwhelming bias for identifying ta-pls associated with hemolysis. of the 4 reports of stem cell ta-pls, 3 actively screened for antibodies in the immediate post-transplant period, and of the 27 reports of organ ta-pls, 1 actively screened for antibodies. these screens detected 5 cases of stem cell ta-pls before hemolysis became apparent and 2 cases of organ ta-pls with antibodies without hemolysis. it can be inferred that ta-pls is currently under-diagnosed, because hemolysis is not consistently present and/or antibody screens are not performed routinely. conclusion: a new category of "non-hemolytic ta-pls" is recommended to capture otherwise undiagnosed cases where ta-passenger lymphocytes form blood group antibodies in the recipient, but hemolysis does not occur, as in our aforementioned case. to ensure including the full scope of ta-pls, an antibody screen should be performed routinely one week after transplant and repeated as clinically indicated. occult hemolytic anemia due to anti-mur in a patient receiving blood from a region with a prominent asian donor population jean oak* 1 , rosario mallari 2 , marc de asis 2 , elaine shu 3 , jonathan hughes 4 and tho pham 1,3 . 1 stanford university, 2 stanford health care, 3 stanford blood center, 4 bloodsource background/case studies: mur antigen is present in 7-10% of individuals in southeast asia, taiwan, and parts of southeastern china, but is rare elsewhere. antibodies against mur antigens are clinically significant, hence many countries in asia routinely screen for it while other countries, including the us, does not include mur in the standard screen. we describe a case of an occult anti-mur antibody causing anemia and donor ethnicity distribution in a regional blood center with a large asian donor population. 41 year old hispanic male with chronic myelomonocytic leukemia and plasma cell dyscrasia developed anemia. initial antibody screen and dat were negative, and the patient received 1-2 rbc units every 1-2 weeks to maintain a hemoglobin (hb) level of 8 g/dl. the patient remained stable for 5 months when his hb level acutely dropped to 6.6 g/dl. the antibody screen remained negative for an additional 2 months when it became positive for anti-jka and anti-mur. donor ethnicity data was available for 30 of the 33 rbc units he received. 3 units were from an asian donor, and a unit transfused 13 days prior to the hb drop was from a caucasian/chinese donor. study design/method: we reviewed the ethnicity data of 64,495 donors at a hospital-associated blood center located in a region where asians comprise approximately 30% of the population. results/finding: 6.6% of donors identified as chinese, vietnamese, filipino, or other southeastern asian. these donors account for 5245 of 37933 (13.8%) rbc collections. conclusion: identification of anti-mur in this patient was triggered by the presence of a concurrent anti-jka alloantibody. since over 10% of the rbc supply in the local blood center was collected from chinese or southeast asian donors, chronically transfused patients are at risk of developing anti-mur-mediated hemolysis that could be missed on a standard screen. this finding raises a possible need for blood banks located in regions with a prominent asian population to implement screening for anti-mur. brian adkins* 1 , princess maynie 1 , carol chandler 1 , shelia garret 1 and pampee young 2 . 1 vanderbilt, 2 vanderbilt university medical center, department of pathology, microbiology and immunology background/case studies: antibody titration is a testing modality vital to both obstetric and transplant services. manual direct tube testing is associated with variability in results (poor reproducibility/precision) and is also time and resource intensive. in fact, studies have shown a three-to eightfold inter-institutional difference between the antibody titers from the same samples using manual tube method. the orthovision automated analyzers offers automated titering of patient plasma using gel technology. although there is intense interest in adopting automated testing technology for titering, it is well-appreciated that titers obtained in manual gel testing are much higher than those obtained by manual/direct tube testing. the higher titer results lack clinical fetal anemia and outcome correlations, which is a barrier to their implementation. moreover, despite the increased sensitivity of gel testing, prior studies have found variable results with regard to reproducibility and precision. [3] [4] [5] there is minimal information on the comparisons of tube titers to orthovision automated titers or assessment of the reproducibility of this automated method. study design/method: rh and non rh minor rbc antibody titrations were performed by manual direct tube method on clinical samples and the same samples were analyzed on three different ortho vision analyzers to assess precision and inter-instrument reproducibility. results/finding: a total of 26 samples have been analyzed (table) , 17 rh and 9 non-rh antigens. titers via automated testing on orthovision resulted in a mean titration being 2.77 (range 1-7) times higher. the average fold change for rhd/c/e antibody titers were 3.2, whereas the average fold change for non rh titers was 1.03 (range 1-2). the range for anti d titers was particularly variable, 2-7, whereas for c/e, it was 1-3. the overall reproducibility/precision of the automated analyzer was $90%. to correlate the 178a transfusion 2017 vol. 57 supplement s3 increased titers observed with some classes of antibodies, particularly anti d, we will be performing parallel testing of obstetric samples and correlating with pregnancy outcome/fetal testing the obtained values. conclusion: automated titration of antibodies using the orthovision analyzers resulted in highly reproducible results between different instruments using the same sample. however, the automated analyzers consistently yielded higher values, particularly with rh d, with results $3 times higher than in manual tube testing. interestingly, the difference in titers of non rh antibodies between manual tube and automated testing was not statistically significant, although our n thus far is small. in order to leverage the efficiency and reproducibility benefits of automated titering we will need to establish "critical titer ranges" which require active monitoring of the fetus. platelet additive solution reduces the isoagglutinin titer in apheresis platelet units maxim tynuv*, elizabeth j furlong and willy a flegel. dtm/cc/nih background/case studies: isoagglutinins in the plasma of apheresis platelets are a concern during transfusion, as high titer anti-a and/or anti-b may cause a hemolytic transfusion reaction (htr) in a recipient with cognate antigen. apheresis platelet collections are usually reconstituted with donor plasma, however most facilities do not test for high titer of isoagglutinins, exposing recipients to the risk of htr due to plasma incompatibility if given based on short outdate and not abo type. at our facility testing is performed on all apheresis platelets with a cutoff titer of 250. units above the cutoff are marked as "high titer" and only given to abo plasma-compatible recipients or washed with saline to reduce plasma. however, washing platelets is a time consuming process that results in a loss of up to 33% of the platelets. platelet additive solution (pas) is used as an alternative collection and storage solution, replacing approximately 65% of donor plasma in the final product. the goal of this study was to determine what affect pas has on isoagglutinin titers and whether using pas could lead to a revision of one facility's procedure for management out of group platelet transfusions. study design/method: isoagglutinin titers of whole blood edta samples were compared to the final apheresis platelet unit collected in pas (intersol, fresenius kabi, lake zurich, il). using two-fold dilution steps, plasma was tested with pooled red cells (equal mix 0.8% suspension of a1 and b cells, ortho, raritan, nj) in a gel matrix test (mts buffered card, ortho, raritan, nj) with 15 min incubation (room temperature) prior to centrifugation (mts ortho workstation). fifty two donors were group o, 32 group a, and 16 group b. results/finding: of the 100 whole blood edta samples tested, 26 (25 group o and 1group b) exceeded a high titer threshold of 250. when the pas samples of these 26 donors were tested, only one (group o) exceeded the same threshold. pas specimens showed a consistent two-fold decrease in titer compared with whole blood specimens. nearly half of the group o donors exceeded a titer of 250 when whole blood specimens were tested. conclusion: only one sample from apheresis platelets collected in pas exceeded our clinically applied titer threshold of 250, a 96% decrease from the number of whole blood specimens exceeding the threshold. testing the platelet bag collected in pas instead of plasma from whole blood specimens would lower the number of units exceeding the high titer threshold, and reduce products needing to be washed. furthermore, facilities not collecting platelets on site or without access to whole blood specimens from donors could implement the process described here and screen platelet apheresis collections for potentially clinically adverse isoagglutinin titers, whether collected using pas or not. other components. the majority of blood components in israel are collected and distributed by magen david adom (mda), from 2 main locations. several hospitals in israel also collect platelets in-house. as part of an effort to understand plt utilization, a nationwide survey of plt transfusion and expiration was conducted. study design/methods: data on the disposition of all plt units, acquired from mda and collected in-house, during the calendar year 2016 was requested from all hospitals in israel. the number of plt distributed to hospitals by mda was also collected. plt wastage was defined as the sum of plt that were returned and not reissued from the hospital blood banks and plt that expired on blood bank shelves. results/findings: sixteen of the 27(59%) hospitals in israel, along with mda, participated in the survey, listed as a to p. the results are presented in the table along with each hospital's distance from the 2 mda facilities. for some hospitals, the sum of transfused and wasted plt was slightly less than the number of plt supplied by mda; this is likely due to the small number of plt that had not either been transfused or expired by the time the data collection period ended. three of the largest hospitals (c, b and a) collected plt in-house in addition to acquiring units from mda. these 3 hospitals had a lower overall rate of wastage including their own donations than the other 13 hospitals that did not collect in-house plt. the other 13 hospitals had wastage rates ranging between 9-54%. no correlation was apparent between the hospital's distance from the mda facility or its number of beds and the plt wastage rate. conclusion: there is considerable platelet wastage in israel. large hospitals in israel with in-house donations had the lowest overall wastage rates in comparison to the other hospitals. factors known to affect plt utilization and wastage such as patient diagnosis mix, policies about how plt are issued and accepted back into hospital inventory, plt inventory size and the time of pooling of whole blood platelets relative to the time they are issued and returned to the blood bank need to be investigated and optimized in order to reduce wastage rates. possible immune-mediated hemolysis due to platelet transfusion masked by underlying hemolysis in a patient with blast crisis sirisha kundrapu* 1,2 , christopher j gresens 3 , anne capetillo 2 , hollie m reeves 1,2 and katharine a downes 1,2 . 1 case western reserve university school of medicine, 2 university hospitals cleveland medical center, 3 bloodsource background/case studies: transfusion-related hemolysis with abomismatched platelets is rare with a reported incidence of <0.1%. most commonly in such cases group o platelets having high titer anti-a result in clinically significant hemolysis when transfused to a group a or ab recipient. we present a patient with a possible hemolytic reaction following transfusion of abo mismatched platelets presenting in the setting of underlying disease associated hemolysis. study design/method: a 58-year-old male with chronic myelogenous leukemia in blast crisis was evaluated for possible transfusion reaction to a single donor platelet (sdp). two hours post transfusion he developed chills, rigors, and increased blood pressure (117/65 mm hg to 205/89 mm hg) followed by hematuria (500 ml). chills and rigors resolved; blood pressure stabilized after 15 min with diphenhydramine, solumedrol, and acetaminophen. negative. patient abo group, rh (d) type and antibody screen on pre-and post-transfusion specimens showed no discrepancies. laboratory indicators of hemolysis are summarized in table. notably, while total/ indirect bilirubin increased and hemoglobin decreased after transfusion other tests were indeterminate for hemolytic transfusion reaction with abnormal pretransfusion levels. despite underlying disease associated hemolysis, the blood supplier of the unit was contacted to investigate into the possibility of high titer donor anti-a. this revealed donor anti-a titer results of 256 (igm) and 1,024 (igg); donor was deferred from future platelet donations. conclusion: while the post-transfusion sample had no visible hemolysis and a negative dat, increased total/ indirect bilirubin after transfusion and high titer donor anti-a are supportive of immune mediated hemolytic transfusion reaction. the key unique aspect in this case is baseline underlying hemolysis, which may mask needs for further investigation of donor for high titer anti-a. ana paula hitomi yokoyama* 1 , leila patricia de sousa fontenele 1 , isabel nagle reis 1 , carolina bonet bub 1 , araci sakashita 1 , raffael zamper 1 , cristiane nakazawa 1 , tatiane almeida omura paula 1 , patricia silva batista 1 , marcio dias almeida 1 , fernanda loureiro de andrade orsi 2 and jose mauro kutner 1 . 1 hospital israelita albert einstein, 2 hemocentro unicamp-universidade estadual de campinas background/case studies: orthotopic liver transplantation (olt) is a high complex procedure, fundamental to therapeutic approach for end-stage liver disease. despite improvements in hemostatic , surgical, and anaesthetic techniques, liver transplantation is still associated with massive blood loss and high rates of transfusion requirements. peri and intraoperative transfusion of red blood cells (rbc) have been previously reported as major predictors of post -operative mortality . identifying predictive factors for transfusion requirements may help optimise patient blood management strategies in olt. we conducted a single center retrospective analysis of 671 cases of olt performed between 2011 and 2015 in brazil in order to identify predictive factors for red blood cell transfusion study design/method: a retrospective analysis in a single institution was performed, and charts of 671 consecutive patients submitted to liver transplantation between 2011 and 2016 were reviewed. the following variables were collected for each patient: gender, race, primary diagnosis, presence of hepatocellular carcinoma, age, body mass index, corrected model for end-stage liver disease (meld), duration of warm and cold ischemia. categorical variables were analysed using pearson chi-square test. continuous variables were analysed using t-student test. a forward logistic regression model was used to analyse data in a multivariate fashion, to identify independent contribution of variables previously found to be significant. results/finding: in univariate analysis, female patients, absence of hepatocellular carcinoma (hcc), primary diagnosis, corrected meld and warm ischemia time were significantly associated with consumption of rbc use in the intraoperative period. multivariate logistic regression of these factors showed that female patients (or 1,726 -95% ci: 1,147-2,597, p:0,009), absence of hcc (or 0,295 -95% ci:0,199-0,437, p:0,0), cirrhosis of any cause (or 4,161 -95% ci 1,816-9,534 -p:0,001), miscellaneous diagnosis (auto-immune, metabolic diseases, familial amyloid polyneuropathy, vascular complications) (or 5,236 95%ic 2,212-12,394) and retransplantation due to primary non function of the graft (or 5,791 95%ci 1,33-4,25,206, p: 0,019) were independently associated with rbc transfusion requirements. conclusion: in this study, female patients, absence of hcc, specific primary diagnosis and retransplantation due to primary non function of the graft were significantly associated with rbc consumption in intraoperative period. determination of rbc transfusion predictors before surgery might provide important information regarding management of blood components and help optimise utilisation of resources for blood conservation strategies. prevalence of high-titer anti-a1/b in group o platelet products. charles k. childers* 1 , mark destree 2 , ashley rose 2 and theresa nester 3,4 . 1 madigan army medical center, 2 bloodworks northwest, 3 bloodworks nw, 4 dept of laboratory medicine, university of washington background/case studies: with platelet substitution policies, minor aboincompatible platelets (where donor's plasma may contain antibodies to recipient's red blood cells) are often issued in an effort to best utilize the community supply. however, rare reports of acute intravascular hemolysis have been reported from such transfusions, and can be attributed to high anti-a1 or anti-b titers, typically in a group o donor. one method to reduce the risk of hemolysis is to identify high titer platelet units prior to transfusion with a subsequent intervention. the percentage of high titer anti-a1/b in group o platelet products is presented from a large regional blood center collected over 10-12 months. data from both pre-storage pooled platelet units (pspp) and apheresis derived platelet units (aplt) is shown. study design/method: platelet component samples were collected in 2 ml edta sample tubes. a single 1:150 dilution of plasma was prepared using a hamilton microlab 600 series dilutor using 2235.0 ml saline diluent and 15.0 ml platelet component sample. using a standard transfer pipette, two drops of diluted sample were transferred to each reaction tube along with one drop of a1 or b red blood cell reagent. reaction tubes were centrifuged immediately in a serological centrifuge at 3175 rpm for 20 seconds. reactions were read using a lighted agglutination reader. the presence of macroscopic agglutination (weak or greater) with either the a1 cells or b cells was recorded as a positive reaction, indicative of a high titer anti-a1 or anti-b. retesting of samples was performed to confirm high titers. results/finding: the above results indicate that, when a titer cut-off of 150 is used, approximately 3% of group o apheresis platelets will have a high titer, most commonly with anti-a1. less than half of a percent of pspp units will have a high titer. testing units for the titer can help to change abo out-of-group platelet substitution policies. in our example, the bloodworks transfusion service was able to change from a policy of volume reducing any group o apheresis platelets being issued to a group a or ab patient, to giving high titer products to only group o patients. the subsequent decrease in episodes of volume reduction helped to improve overall availability of apheresis platelets, by maintaining their 5 day outdate. after 10 months of testing pspp units and verifying that the products rarely had a high titer (0.28%), the blood center stopped performing this testing for pspp units. rh1) ] started complaining of worsening back pain two and half hours after receiving one unit of rbc for a drop in hematocrit to 19% (from 24% on the previous day). his hematocrit did not increase (18%), and over the ensuing 12 hours, he became anuric and jaundiced. clerical checks confirmed that his forward type was a positive, which was also the type of the rbc unit transfused, but revealed anti-a at a titer of 8 in his plasma. furthermore, the direct antiglobulin tests (dat) were positive for c3 in the pre-and post-transfusion blood samples. anti-a was not detected in his plasma collected three days earlier, however. although his plasma color was amber, he had signs of intravascular hemolysis: undetectable haptoglobin, increased lactate dehydrogenase (ldh) and total and indirect bilirubin results/finding: the positive dat in the pre-transfusion sample pointed to ongoing hemolysis prior to the transfusion of the a rbc unit. in the setting of recent abo-mismatched transplant, his picture was consistent with hemolysis from newly formed anti-a by proliferation of donor lymphocytes, or pls. we performed an emergent rbc exchange using o rbcs with a goal hematocrit of 24% while reducing the number of a rbcs in his circulation by approximately 70%. his pain improved rapidly thereafter, and he had complete recovery of renal function. conclusion: pls should be in the differential diagnosis when suspecting/ investigating clinically significant hemolysis in abo-mismatched hpc transplant recipients, especially when the hpc source is from peripheral blood. as in our patient, it usually takes 7-14 days for antibodies to develop and they are short-lived (3-5 weeks). due to the severity of his manifestations, we performed an emergent rbc exchange successfully. furthermore, this patient's event exposed a vulnerability in our system of issuing the proper blood type for abo-mismatched transplant recipients, which has since been remediated electronically background/case studies: group o rhd negative (oneg) red blood cells (rbcs) are a precious resource. to conserve the oneg inventory while minimizing the risk of rhd alloimmunization in oneg females of childbearing age, transfusion services may automatically provide group o rhd positive (opos) rbcs to rhd negative males and/or rhd negative postmenopausal females during bleeding emergencies. despite these conservation strategies, shortages of oneg rbcs occur. the goal of this study was to determine how the utilization of oneg rbcs can be optimized using agebased opos switching for routine transfusions in oneg patients. study design/methods: recipient age and abo/rhd group were obtained for all allogeneic rbc transfusions during the 2016 calendar year from 9 hospitals. an additional hospital* provided data for august-december 2016. rbc transfusions in patients <1 year of age, and in patients whose age and/ or abo group were unknown, were excluded from analysis. the abo/rhd group of each rbc unit was compared to that of the recipient to determine the number of oneg rbcs transfused to all patients, the number of rbcs transfused to oneg patients and the number of oneg rbcs transfused to oneg patients. the number of oneg rbcs transfused specifically to oneg patients >/5 70 years was also determined. results/findings: see table 1. the fraction of all transfused rbcs that were oneg ranged from 5-14% (row f). the percentage of oneg rbcs transfused to oneg patients ranged from 37-89% (row g); thus, non-oneg patients received 11-63% of the oneg units transfused (row h). hospitals differed widely in the practice of issuing oneg rbcs to oneg patients (68%-100%; row i). overall use of oneg rbcs could have been reduced by 10%-39% if opos units had been given to all oneg patients >/ 5 70 years old (row j). conclusion: during times of oneg shortage, age based opos switching rules may be applied for routine transfusions. this would help to ensure the availability of oneg rbc units for oneg females of childbearing age. rasha eldeeb mohammed* 1 , nehad mohammed 2 , marwa aly 2 and nashwa fahmy 2 . 1 national blood transfusion services, 2 nbts background/case studies: sensitization to the transfused red cell may complicate further transfusion& make it increasingly difficult to find compatible blood components for those patients. splenectomy has been shown to increase human leucocyte antigen immunization. the aim of the study is to evaluated the effect of splenectomy on the occurrence of red cell alloimmunization in humans. study design/method: this study was conducted on 206 multitransfused patients who received blood transfusion chronically at our central blood center. they were 129 thalassemia patients (128 bthalassemia patients, one patients with a thalassemia), 10 sickle cell anemia patients and 6 immune hemolytic anemia patients (4 auto immune hemolytic anemia patients, one paroxysmal nocturnal hemoglobinemia patient, one immune thrombocytopenic purpra patients). 29 oncology patients, 32 chronic diseases patients. history and demographic data were documented. all the patients who received blood are examined for the presence of the spleen.our patients were subjected to direct & reverse blood grouping (abo& rh) tests, alloantibody screening and detection. results/finding: statistical study is done to determine what is the effect of splenectomy in increasing the rate of red cell sensitization in chronically transfused hemolytic patients. the study revealed that: 32 out of 48 (67%) alloimmunized patients and 16 out of48(33%) non alloimunized patients(p<0.001) . statistical analysis show that there is high statistical significant difference between patients who performed splectomy& who did not perform splenectomy as regard form conclusion: patients who had splenectomy had a higher alloimmunization rate removal of the spleen is not recommended in those patients who are periodically in need of blood and blood components. restrictive transfusion triggers rather than specific evidence. therefore, two systematic reviews of a) rbc transfusion guidelines and review articles to determine if single or multiple unit transfusion strategies are recommended and b) to identify studies comparing strategies were performed. study design/method: methods medline, embase, cinahl, web of science, national guideline clearinghouse, and the trip database were searched from inception to june 2016. screening and data abstraction were done independently by two assessors. for review a, the proportion of articles with recommendations and articles recommending single unit strategies were assessed; stratified by guidelines, systematic reviews, and other review articles. for review b, the primary outcome was rbc utilization. secondary outcomes included proportion of units transfused using a single unit strategy, length of stay, and mortality. meta-analysis was done using the mantel haenszel random effects model. results/finding: review a identified 136 articles for data abstraction, where 48 articles were transfusion guidelines. there were 12 guidelines (25%) that made a recommendation, 11 for a single unit and 1 for multiple unit transfusion strategy (table 1) . review b identified 3 retrospective cohort studies that were eligible and data abstraction was performed. all utilized a policy encouraging single unit transfusion strategies and compared a pre-implementation period to a post-implementation period. meta-analysis could only be performed on the secondary outcome of the proportion of units transfused using a single unit strategy, which was higher after the policy intervention (or 9.4, 95% ci 5.02-17.60), although heterogeneity was high (i 2 597%). conclusion: our systematic reviews demonstrated a lack of recommendations amongst guidelines pertaining to transfusing single units of rbcs and only a few retrospective cohort studies to support benefits of the use of single unit transfusion strategies. additional high quality studies are needed to identify the benefits of a single unit transfusion strategy and when it should be used. guidelines groups should review research in this area to determine if a recommendation can be made. background/case studies: platelets made with platelet additive solution c (pas c) and treated for pathogen reduction (pr) have been shown to have decreased post transfusion platelet counts from platelets stored in all plasma. with the advent of multiple types of platelets, we are evaluating whether a mixed platelet inventory has had an effect on component use. the literature from europe has shown that platelet and red cell use does not increase when pr and pas products are used. evaluation of rbc use at our institution has shown no change in the number of products transfused per patient per month. we are evaluating whether the mixed inventory has led to more platelet transfusions. study design/method: we looked at occasions when patients received all of their platelet transfusions on a single day. by doing this we were able to exclude refractory patients from the analysis. the information obtained from routine quality management audits of transfusions between december 2016 and february 2017 was used for this analysis. the information included the ordering service, product release time, product code, pre and post counts. statistical analysis was performed using minitab. results/finding: during the 3 months, 1723 units of platelets were transfused to 238 recipients. over the 3 months, a median of 4 units was given to each patient with a range of 1 to 69. the overall distribution of products used was 58% plasma, 24% pr, 7% pas f and 11% pas c. thirty percent of patients (n572) received all of their products on a single day. single units were given to 54 patients while 14, 3 and 1 received 2, 3, and 4 units respectively. the distribution by product type was 56% plasma, 25% pr, 13% pas c and 4% pas f. this same percentage was present for single and multiple products and was not statistically significantly different from the overall distribution of the products given during the 3 month period (p5 1.00). the distribution by service was different for the groups receiving multiple units. for single units the distribution was 44% hematologic malignancy, 22% infusion clinic (nos), 13% solid tumor medicine, 11% surgery, and 9% pediatrics. for those receiving multiple units the distribution was 50% surgery and 16% each for solid tumor, hematology and infusion (nos). the chi-square test for associations showed the increase in multiple units to surgical patients to be significant with a p value of 0.022. conclusion: the distribution of the type of platelets given during a single event of transfusion was not significantly different from the overall distribution of platelets given during the 3 month period. the patient's clinical service was a better predictor of the use of multiple products than the type of product given. this suggests that surgical losses or the need to have a higher platelet count during a procedure was the leading factor in the use of multiple products in this transfusion scenario. the effect of red blood cell transfusion on iron metabolism in critically ill patients margit boshuizen* 1,2 , yvemarie b.o. somsen 2 , maike e. van hezel 2 , marleen straat 2 , robin van bruggen 1 and nicole p juffermans 2 . 1 sanquin research and landsteiner laboratory, 2 academic medical center background/case studies: anemia of inflammation (ai) has a high prevalence in critically ill patients. in ai, iron metabolism is altered, as high levels of inflammation-induced hepcidin reduces the amount of iron that is available for erythropoiesis. ai is treated by red blood cell (rbc) transfusions. it is known that rbc transfusions increase iron level in neonates and thalassemia patients, but the effect of rbc transfusion on iron metabolism during inflammatory processes is unknown. since one unit of rbcs contains 220 mg of iron and 25% of the rbcs are cleared by macrophages within 1 hour following transfusion, rbc transfusion could increase iron levels and iron availability for erythropoiesis. we investigated the effect of rbc transfusion on iron metabolism in icu patients, and additionally compared the effect in septic patients to non-septic patients. study design/method: in a prospective cohort study in 52 icu patients who received one rbc transfusion, different iron parameters were measured before and 24 hours after transfusion, to determine the effect of a rbc transfusion over a period of time. next, the impact of a rbc transfusion on plasma iron parameters in septic patients compared to that in non-septic patients was analyzed. plasma iron concentration, transferrin (saturation), ferritin, haptoglobin, hepcidin and il-6 levels were determined. results/finding: in this cohort, serum iron levels were low and did not change following transfusion (4.1 vs. 4.3 mmol/l, p50.69). also, the transfusion had no effect on transferrin saturation (12 vs. 13 %, p50.13), ferritin (531.0 vs. 599.0 mg/l, p50.74) and il-6 levels (35.0 vs. 25.5 pg/ml, p50.09). hepcidin levels increased in these icu patients after rbc transfusion (223 vs 332 ng/ml, p50.01). in septic patients, rbc transfusion induced a decrease in haptoglobin levels compared to baseline, which did not occur in non-septic patients (-2.7 vs. 3.7 % change, p50.05). other iron parameters did not differ between septic and non-septic patients. conclusion: transfusion of one unit of rbcs does not increase iron levels in icu patients. the increase of hepcidin suggests rbc transfusion induced upregulation of hepcidin, despite the absence of a significant increase in il-6 or plasma iron levels. this increase in hepcidin levels after transfusion can potentially further hamper iron availability for erythropoiesis. in sepsis, rbc transfusion decreases haptoglobin levels, suggestive of hemolysis. in conclusion, rbc transfusion might have a negative effect on erythropoiesis, due to the increase in hepcidin levels that are observed after transfusion. the effects of pas and pr on platelet use barbara mendez, judith delmonte, elizabeth mccabe and joanne becker*. roswell park cancer institute background/case studies: with the anticipated release of the fda guidance: bacterial risk control strategies for blood collection establishments and transfusion services to enhance the safety and availability of platelets for transfusion, the use of pathogen reduced platelets (pr) which are often produced from products made with platelet additive solution (pas) may become more common. our institution has been transfusing platelets made with additive solutions since 2011 and pathogen reduced platelets have been available since 2016. in our data validating pas and pr, the post counts from transfusion of pas-c and pr products have been statistically lower than platelets in all plasma (pp) or pas f products. our study looks at whether this difference has led to a corresponding increase in the number of units of platelets transfused. study design/method: the data was obtained from the routine quality reports produced for the blood utilization committee at our facility between 2012 and 2016. during this time pas c, pas f and pr went from 13% to 40% of all platelet products given. all recipients had an oncology diagnosis. the data collected included the service, unit number and product code. the number of unique recipients was determined monthly. the data was converted to plt/month/recipient for analysis. statistical analysis was performed using the two sample t-test results/finding: the data was normalized to plt/recipient/month. in 2011 patients received an average of 5.41 units/recipient/month and in 2016 the average was 5.39 units/recipient/month. the intervening data points for 2013, 2014, and 2015 were 5.92, 5.66, and 5.92 respectively. the 5 year average was 5.66. the slope of the graph for all 5 points was y5 -0.004 15.672. the two sample t-test showed that the plt/recipient/month from 2012 to 2016 was not statistically different with a p value of 0.81. conclusion: the implementation of pas and pr platelets in the oncology environment has not increased in the number of platelet transfusions given. in additional analysis, the red cell use has decreased (data not shown). this can be interpreted as indicating that patients have not had increased episodes of bleeding. although the post platelet count from pas/pr platelets may be lower, we do not have evidence from our platelet transfusion data that this is leading to clinical outcomes necessitating additional products to be given. background/case studies: it is reported that the incidence of alloimmunization in aml patients is unrelated to the number of transfusions the patient receives and most patients who have hla antibodies do not exhibit platelet refractoriness. many cases are also found not to have any anti-platelet antibodies detectable by standard laboratory tests. recent data in leukemia and hematopoietic stem cell (hsct) recipients transfused exclusively with leukoreduced products show that 4% to 8% develop alloimmune platelet refractoriness. objective: to determine an improvement in platelet count with the match grade and/or the abo blood group of the hla matched platelets in highly alloimmunized patients with concomitant non-immune causes for platelet destruction. study design/method: clinically documented platelet refractory patients, who received hla matched irradiated sda platelets with their hla typings for hla-a/-b and hla antibody identification were reviewed. there were two strategies utilized, the hla strategy (matching recipient and donor hla-a/ -b types) and the antibody specificity prediction (patient provided with platelets from donors lacking only those hlas to which the patient had antibodies) strategy. statistical analysis: a one sample t-test using minitab 17 statistical software was performed comparing the mean against a platelet increment of a hypothetical difference of at least 5 k/ul. the analysis revealed that the mean of 9.35 k/ul (n584) had a 95 percent lower bound confidence interval platelet increment of 7 k/ul (p<50.001) results/findings: 123 (median 4 range [1-43]) hla matched leucoreduced irradiated sda platelets were transfused to 17 (6m/11f) patients, median age 60 years (range 27-83). 15/17 (88%) patients showing broad alloimmunization to hla class i/class ii antigens. 2/17(12%) patients had anti-hpa antibodies (gp iib/iiia and gp iib/iiia and gp ia/iia). the majority 16/17 (94%) had a diagnosis of hematologic malignancy (aml/mds/mpn/ cmml/mm); 9/11 (81%) female patients had prior exposure via pregnancy and 4/11 (24%) had a history of hsct. 63 (51%) platelets were abo identical-platelet increment median 7 k/ul (range -14 to 61), 53 (43%) were abo compatible -platelet increment median of 2k/ul (range -10 to 46) and 7(6%) were abo incompatible with platelet increments median 8k/ul ( range -10 to 32). platelet counts were performed within 24 hours in 73 (57%) transfusions. the hla match grade of the transfused platelets were as follows: the use of massive transfusion protocol (mtp) in a community hospital rohini patel* 1 , renee leblanc 2 , dongfu xie 2 , alice cabe 1 and yanyun wu 2 . 1 overlake hospital, 2 bloodworks northwest the use of massive transfusion protocol (mtp) in a community hospital background/case studies: the establishment and use of massive transfusion protocol (mtp) have become common practice, especially in trauma centers and tertiary hospitals due to significant number of patients with massive bleeding. however, it is not well established if the use of mtp also has value in small hospitals and community hospitals, and how mtp is used in fig. 1 these settings, such as indication for mtp, blood products used, and the outcomes of these patients. study design/method: retrospective review of transfusion data from a community hospital with a bed size of about 350 for 3 years (from 2014 to 2016) was performed. patients with mtp requested are included in this study. results/finding: please see the table below for the summary of data. notably, patients with gi bleed and ob bleed are the two most common indications for mtp, and 68 % of patients survived with the support of mtp. in one case, no blood product was used. the establishment and readiness of mtp can be very important in supporting patients who experience massive bleed in small hospitals and community hospitals. in these settings, mtp is most commonly used for patients with massive gi bleed and ob bleed. if the patient develops antibodies to a high incidence antigen, finding compatible units may become impossible. included in the mns system, and residing on glycophorin b (gpb), the u antigen is absent in less than 0.25% of the black population. those with altered forms of gpb, known as u variants, can produce a diverse group of antibodies capable of causing mild to severe hemolytic transfusion reactions and hemolytic disease of the fetus and newborn (hdfn). this case illustrates the balance between the need to transfuse and avoiding complications thereof. a 32-year-old ghanaian woman with scd and history of chronic transfusion presented with diffuse pain and a hemoglobin value of 6.4 g/dl (baseline 9-10 g/dl). she is known to be e, c, k, fya, jkb, s, s negative, u variant, and has anti-e, c and u antibodies. there were no eligible family donors and a nationwide search for compatible blood yielded four crossmatch compatible u variant units. the decision to transfuse was made. the patient had no change in symptoms or vital signs during transfusion but post-transfusion hemoglobin was 5.9 g/dl. a transfusion reaction work-up was ordered. post-transfusion serum sample was negative for hemolysis and no new antibodies were identified. the post-transfusion dat was weakly positive only with complement and laboratory data revealed a decrease in total bilirubin (8.8 to 6.2 mg/dl). two additional u variant, crossmatch compatible units were transfused over the next two days restoring her hemoglobin to 6.2 g/dl. the patient was discharged to home in stable condition and follow-up hemoglobin levels continued to rise back to baseline. study design/methods: molecular genotyping was used in donor unit selection prior to compatibility testing by transfusion services. conventional methods were used to monitor the patient's condition pre and posttransfusion. results/findings: each donor unit came from a different donor but all were the same gpb genotype as the patient. the patient did not experience an acute or delayed hemolytic transfusion reaction and genotype matching successfully facilitated donor unit selection in this case. conclusion: transfusion of u variant red cells to a u variant patient should be undertaken with great caution due to epitope and antibody heterogeneity. this case highlights the importance of genotype compatibility in selecting donor units for a chronically transfused, scd patient with anti-u. sound transfusion management of such patients requires planning and good communication on the part of clinicians and the laboratory staff. background/case studies: patients with decompensated waiha may require transfusion with red blood cell (rbc) products that are cross-match incompatible due free autoantibodies. the feasibility of blood transfusions in waiha patients is controversial because of difficulty in cross-matching and increased risk of transfusion reactions, since transfused rbcs may be destroyed more rapidly in patients with active hemolysis. to study the actual vs. theoretical risk of increased hemolysis in waiha patients, we investigated the post-transfusion (post-tfn) hematocrit (hct) change in waiha patients who were transfused compatible rbcs compared to those who received li blood. we further hypothesized that a post-tfn hct would be inversely related to the degree of ahg-phase incompatibility. study design/method: we reviewed all transfusions to patients in our quaternary-care hospital with a history of waiha from october 2015 to march 2017. patient hcts were ordered by prescribing physicians for clinical purposes. a transfusion episode was defined as all units released in the interval before a post-tfn cbc. ahg-phase crossmatch was tube tested in saline per clinical procedure. transfusion medicine physicians determined the release of least-incompatible units. statistical tests were performed with statcalc (epiinfo, cdc) and www.socscistatistics.com. results/finding: there were 139 rbc products transfused to 40 waiha patients. twenty-three (57.5%) patients received at least 1 incompatible unit. the mean age was 51.4 years (range 4-93 yrs) with 50% women. ethnic composition was 55% african-american, 40% caucasian, and 5% patients of mixed/other ethnicity. one hundred fourteen (82%) of these products were released as li products and 25 (18%) were compatible. ninetythree (81.6%) of the li product transfusions had a post-tfn hct change of <3% whereas only 14 (56%) of the compatible product transfusions resulted in a post-tfn hct change of <3% (p50.0092, v 2 (1), exact methods). the mean hct increase in the compatible group was 1.83% per unit vs. a slightly lesser per-unit increase of 1.71% in the li group (p50.82, t-test, 2-tailed) within the li group, there was no difference in the per-unit hct change according to strength of incompatibility (table) . strength of ahg incompatibility was not available for 12 units. units that were 31 incompatible had a lower mean post-tfn hct rise compared to all other li units (1.49% vs. 2.15%); however, this difference was not statistically significant (p50.38). conclusion: the post-tfn hct change for transfusions of li units to patients with waiha was less than the expected 3% per unit more frequently than it was for waiha patients who received compatible products (81.6% vs. 56%). however, likely due to our small sample size, the mean differences were not statistically significant. interestingly, there was no difference in the per-unit post-tfn hct according to differing strengths of incompatibility in our sample, although the mean increase for the 31 li products was less than all other li products combined. the increase was unexpectedly low for weaklyincompatible units, which we are further studying. future work includes consideration of inpatient vs. outpatient clinical status, effect of co-incident alloantibodies, comorbidities, and medications. transfusion management was summarised by individual hospital, type and total cases. in-hospital mortality (adjusted for age, sex, comorbidity, bleeding context and number of rbcs in the first 4-hours from mt onset) was calculated with 95% and 99.8% control limits to indicate potential outliers. data were analyzed using statistical software (stata). results/finding: there were 5482 mt cases from 25 hospitals (17 tertiarylevel, 6 smaller/medium sized acute-care and 2 specialist women's). number of mt cases per hospital ranged from 5 to 721. patient median age was 65 years (iqr 49, 76), 62% were male and 73% required admission to intensive care. the most common clinical groups were cardiac surgery (21% cases), trauma (20%) and gastrointestinal hemorrhage (13%); however there was marked variation between hospitals. ratios of transfused products, analyzed according to bleeding context, varied between hospital types. the pooled average adjusted in-hospital mortality for the 17 tertiary-level hospitals was 21% (range 13% to 33%) and 16/17 (94%) were within the 95% control limit. cb that required !10 rbcs within 24-hours of mt onset occurred in 40% of cases. comparison of transfusion management for this subset of mt cases showed that patients treated in smaller/medium sized acute-care were less likely to receive cryoprecipitate than patients treated in tertiary-level hospitals (67% versus 78%; p50.03). conclusion: patient characteristics and transfusion practice varied between hospitals and hospital types, however in-hospital mortality outcomes were comparable. results are made available to participating hospitals in the anz-mtr to initiate discussion, practice review, and examination of compliance with national standards, patient blood management guidelines and to highlight areas for further investigation. data are also available for review by governance and policy bodies at state and national level to support practice improvement activities and highlight priority areas for future research. background/case studies: in hospitals and medical centers, in case of big traumas often an intraosseous entrance via a bone needle is combined with a fast flow fluid warmer. with this, infusion fluids, including blood products, are administered under pressure. this is done because veins of trauma patients are often not suitable for infusion of fluids. suppliers of pump and needles describe the possible transfusion of blood products, but this is mainly limited to plasma and erythrocytes. there is no information available concerning transfusion of platelets under pressure via a bone needle. the aim of the study was to investigate the effects of warming and administration of a platelet concentrate (pc) under pressure via a bone needle on the in vitro quality of platelets. study design/method: pools of 5 bcs and 280 ml of platelet additive solution iii (pasiii) were used to produce pcs (n55). pcs were stored on a flatbed agitator (60 cycles/min) in a temperature-controlled cabinet at 22 6 28c for 4-7 days. to mimic hospital conditions, pcs were warmed using a blood warmer and transfused via a bone needle to a transfer bag. on the pcs a pressure of 300 mm hg was applied. using clamps, a flow velocity of 90-120 ml/minute was realized. platelet quality before and after pressurized simulated transfusion was determined by means of various in vitro parameters. results/finding: due to priming of the transfusion disposable with saline, the pcs were diluted 10-30%, resulting in a significantly increased pc volume and decreased platelet concentration after simulated transfusion. because of loss of platelets in the disposable set, also the total number of platelets was decreased after simulated transfusion. after simulated transfusion, the pcs still fulfilled the requirements for platelet concentration (0.8-1.6x10 11 /l) and number (>250x10 9 /unit). simulated transfusion had no effect on the percentages of cd62p and annexin v positive cells, indicating no activation or induction of apoptosis. ph was not influenced by simulated transfusion. due to the dilution effect, glucose and lactate concentrations were slightly lower after simulated transfusion. conclusion: warming and simulated transfusion of pcs under high pressure via a bone needle has no negative effect on the in vitro quality parameters of platelets. transfusion of warmed pcs via an intraosseous entrance via a bone needle is not expected to have a negative effect on the in vivo functionality of platelets. it is recommended to study the in vivo effects in a limited clinical study. alesia kaplan* 1,2 , joan sevcik 2 and joseph e. kiss 1,2 . 1 university of pittsburgh, 2 blood systems inc. background/case studies: low titer a plasma has been safely used as a substitute for ab plasma in trauma patients. low inventories of ab plasma can cause a delay in life saving therapeutic plasma exchange (tpe) procedures in ab patients needing plasma replacement. here, 2 ab non-bleeding patients are presented who safely received ab and low titer a plasma for tpe. one ab patient who received ab plasma only was used as control to compare hemolysis laboratory data over tpe course. study design/method: a retrospective review of tpe procedures for 3 patients was conducted from medical records. number of procedures, volume replaced, total number of plasma units, number of a plasma units, quantity of a plasma and hemolysis laboratory data were recorded. average quantity (ml) for a plasma and % of a plasma out of total volume of plasma used were calculated. all a plasma units were low anti-b titer units. in the laboratory, plasma dilution 1:50 is prepared and tested with reagent b cells. if agglutination is not observed, the unit is labeled as "low titer anti-b". hemolysis laboratory data was traced with linear graphs and trends were compared between patient 1 and 2 and 3 (control). results/finding: all 3 patients were ab blood type. patient 1, a 57 year old female with recurrent adamts13 deficient ttp, received 2 courses of tpe (total 12 tpe procedures) for relapse and exacerbation. ten out of 12 procedures were performed with ab and a plasma (average 916 ml of a plasma or 24% of total plasma volume for 10 tpe procedures). patient 2, a 27 year old female with thrombocytopenia, schistocytes and presumed ttp, received a total of 12 tpe procedures. four out of 12 procedures were performed with ab and a plasma (average 1210.5 ml of a plasma or 48% of total plasma volume for 4 tpe procedures). patient 3, a 33 year old female with adamts13 deficient ttp who served as a control, received a total of 10 procedures with ab plasma only. haptoglobin, ldh, hemoglobin and total bilirubin were graphed and compared between 3 patients. the trends of hemolysis laboratory data for patient 1 and 2 were comparable with patient 3. all 3 patients had negative dat. only patient 3 received 2 rbc transfusions. all 3 patients had a favorable clinical outcome with tpe treatments and adequate platelet recovery. conclusion: in this study, tpe was effectively performed without evidence of increased hemolysis using up to 48% of low titer a plasma. this approach can reduce strains on limited supplies of ab plasma while providing a vital treatment alternative for ab patients undergoing tpe who require plasma replacement. when cd36 negative platelet unit is not available for a patient with anti-cd36 antibodies sameer khatri* 1 , charles harmon 1 , brian r curtis 2 and chisa yamada 1 . background/case studies: refractoriness to platelet (plt) transfusion can be caused by antibodies (abs) against human leukocyte antigen (hla) class i antigens (ags) or less frequently against plt specific ags (psas). glycoprotein iv (cd36) is one of the identified plt surface ags and deficiency is rare, but found in asians (3-11%), sub-saharan africans (7-8%) and also in some people from mediterranean descent. two types of cd36 deficiency have been described. type 1 deficiency is the complete lack of cd36 on both plts and monocyte-macrophages whereas type 2 deficiency lacks cd36 on plts with variable expression (12-99%) on monocytemacrophages. transfusing plts in a patient with cd36 deficiency is challenging given the rarity of cd36 negative phenotype and risk of further immunization when giving ag non-matched platelets. study design/method: a patient with cd36 negative phenotype who received multiple plt units was reviewed in the electronic medical record. results/finding: a 21 year old man developed aplastic anemia following liver injury possibly due to a supplement for body building and required multiple plt and rbc transfusions. he received more than 20 units of apheresis plt units over a 2 week period without any significant increase in plt count. cross-match compatible plt unit found in 1 of 32 units and hla matched units were tried without success. at that point, a cd36 ab was identified in the serum and the patient's type 1 cd36 deficiency was confirmed by flow cytometry. his hla class i panel reactive ab (pra) was 95% due to multiple plt transfusions, although all abs were low levels. the patient initially received high-dose prednisone and thymocyte immune globulin infusions without significant improvement in plt increase. following three doses of ivig, he received a cd-36 negative (but blood type different and hla 187a transfusion 2017 vol. 57 supplement s3 unmatched) plt unit from his relative with only a slight increase in plt count. however, he started to respond to cd36 non-tested apheresis plts after receiving a fourth ivig and two rituximab infusions. since then, he has received ivig every 2 weeks. other medications include filgrastim, eltrombopag, and cyclosporine for treatment of aplastic anemia. the mean corrected count increments (cci) when post-transfusion plt count was available are shown in table. with desensitization therapy, his cd36 antibody positive reactivity in serial dilutions has reduced from 1:32 to 1:2 dilutions and his hla class i pra has decreased to 37%. he is currently receiving 2 apheresis plt units twice a week and rbc units periodically. his bone marrow (bm) has been slowly recovering evidenced by increased wbc count from zero to up to 1.0 k/ml and slow increase of reticulocyte counts. current plan is rbc/plt transfusion support until bm recovers or a haplo-identical transplant if bm recovery fails. conclusion: we report a case with anti-cd36 abs that received multiple plt transfusions. this case demonstrates that decreasing ab level with immunomodulation can be an alternative option for successful plt transfusion when compatible plts are not available for patients with rare or multiple abs to plts. table: mean available cci for plt transfusions a blood center's experience screening donations for babesia microti using enzyme-linked immunoassay methodology nancy van buren*, jed gorlin, vanessa reynolds and deborah anderson. background/case studies: our blood center, located in an area considered to be moderately endemic for babesia microti, implemented universal screening of red cell collections from minnesota and wisconsin under an investigation new drug (ind) study in oct 2015 utilizing the immunetics investigational enzyme-linked immunoassay (elisa) performed by creative testing solutions (cts). this test was selected as the most cost-effective approach for universal screening of blood donors, as opposed to the investigational ifa/pcr test combination. study design/methods: we performed a retrospective analysis of our screening test results and deferral rates for 2016 to evaluate for seasonality, donor abo bias, deferral rates, and outcomes of lookback investigations. since an opt-out of this research test was originally offered, we report donor opt-out rates. results/findings: from jan through dec 2016, 101,854 blood donations were screened for b microti by immunetics elisa. of those, 267 (0.26%) were positive. the percent of positive donations was evaluated monthly revealing a variable reaction rate between 0.08% and 0.42%. no patient babesia transmission has been reported since implementing this test, but we only had 4 documented babesia ttd cases from 2007-2017. donors who previously tested negative demonstrated an increased seroconversion rate during the summer months, consistent with historical seasonal variation corresponding with tick season in minnesota and wisconsin. test performance characteristics were analyzed by abo group with no demonstrable differences in positive rates. the opt-out rate of donors who chose not to be tested significantly decreased over time, reflecting an increased acceptance of this test. of 267 positive test results, 160 lookback investigations were initiated representing 59% of positive donations. lookbacks were only performed when there was a donation within 12 months of the new positive screening test, according to ind protocol. no confirmatory testing was performed per ind protocol or for donor counseling, so the true positive rate is unknown. in the prior ind trial, up to 80% were unlikely to transmit infection in our region, i.e. were pcr and blood smear negative. although a small number of antibody positive, pcr negative donors may be actively infected, no transfusion-transmitted babesia infections were identified by lookback investigations. notification of blood donors with positive screening results was also performed and information provided for healthcare provider followup. overall, donor deferral represented 0.25% loss of eligible donors during this follow-up period. deferred donors were invited to participate in other research collections not requiring volunteer donor eligibility. conclusion: testing for b microti may help improve blood safety, particularly in endemic regions. although only 0.25% of donors have a positive reaction, this represents a significant loss of eligible donors over time, most of whom are unlikely to transmit infection. a direct test capable of detecting babesia in individuals with very low levels of organisms without the need for concurrent antibody testing would be ideal. a reinstatement protocol for donors who test positive should also be considered. nonetheless, the current method of screening is inexpensive compared to pcr-based methods. background/case studies: human anelloviruses are the smallest in particle size, smallest in genome size, and least complex in genetic organization of all human pathogens. they establish a chronic persistent infection in infancy or early childhood and produce a constantly detectable load in plasma thereafter. some studies suggest they are ubiquitous, present in >90% of the human population, and that immune surveillance is required to control the level of the virus load. study design/methods: we have developed a quantitative dna pcr assay for the most conserved region of the anellovirus genome that detects all known genotypes of the virus. we used this assay to examine viral loads in the plasma of us blood donors and transplant recipients pre-transplant and three months post-transplant. results/findings: for 53 blood donors, 51 were positive with an average load of 1.49x10 2 copies/ml of plasma, a median value of 80.5 copies/ml of plasma, ranging from 0 to 1.87x10 3 copies/ml. pre-transplant viral loads were similar. for 41 transplant candidates, 40 were positive with an average of 3.70x10 2 copies/ml of plasma, a median value of 88 copies/ml of plasma, ranging from 0 to 1.18x10 5 copies/ml. post-transplant viral loads were remarkably different. for 94 transplant recipients, all were positive with an average of 3.14x10 5 copies/ml of plasma, a median value of 1.25x10 5 copies/ml of plasma, ranging from 0 to 4.6x10 7 copies/ml. conclusion: these results validate the pcr assay that was developed and confirm that detectable viral loads of around 100-200 copies were present in >90% of the blood donors surveyed. in addition, the effect of post-transplant immunosuppressive therapy has caused an increase in the viral load of at least 2 orders of magnitude above that of non-immunosuppressed individuals. background/case studies: the screening of blood donors and travelers returning from endemic/epidemic areas has highlighted the importance of multiplex diagnostic approaches for the simultaneous analysis of various pathogens. furthermore, in the context of similar clinical signs, the differential diagnosis of arboviruses during acute infection is essential to discriminate the causative agent for patient management and epidemiological surveillance. the development of a flexible diagnostic approach is a key challenge to face the continuing emergence of arboviruses, belonging to flavivirus and alphavirus, such as dengue virus (denv), west nile virus (wnv), zika virus (zikv), yellow fever virus (yfv), usutu virus (usuv) and chikungunya virus (chikv). study design/method: an innovative diagnostic approach combining generic rt-pcr amplification and identification on low cost microarrays has been developed. we have patented original polythiolated probes grafted on maleimide-activated microplates for the robust, sensitive and specific mean cci pre-ivig: all plt (17) 0.2 post-ivig: all plt (41) 5.5 post-ivig: cd36-negative plt from relative (1) 0.8 post-ivig: single donor apheresis (23) 4.3 post-ivig: cross-match compatible (15) 6.1 post-ivig: flow cross-match compatible plt (2) 12.6 188a transfusion 2017 vol. 57 supplement s3 detection of the viral genomes. analytical performances of the test were evaluated on viral standards and on clinical samples: denv (1/2/3/4), wnv, zikv and chikv. forty human plasmas from blood donors with no history of contact with arboviruses were used as negative controls. we have designed two sets of degenerated primers for the generic rt-pcr amplification of all flaviviruses and for chikv. biotinylated amplicons were captured on complementary grafted polythiolated probes on microplate. after addition of streptavidin-europium label, the molecular hybridization events were detected by time-resolved fluorescence using a microplate reader. results/finding: one original generic probe for denv and specific probes designed for each of the four denv serotype, wnv, the two zikv lineages and for chikv, were validated. the use of our methodology combining the amplification of the viral genomes and their identification using polythiolated probes shows 100% of specificity, with no false positive results on the 40 control samples, and no cross reactions. using viral reference standards, we have observed sensitivities of 1 tcid 50 /ml for denv-1, denv-3 and chikv and of 10 tcid 50 /ml for denv-2, denv-4 and zikv. finally, the first results obtained on 110 denv(1), 69 zikv(1) and 50 chikv(1) clinical samples show 85%, 87% and 96% correlation respectively between our approach and commercial or in house real time rt-pcr methods. conclusion: this innovative strategy allows the development of flexible, highly sensitive and easy to handle platforms dedicated to the multiplex screening and identification of emerging viruses. this methodology is adapted for the easy inclusion of additional molecular targets to improve the surveillance and the prevention of arboviral infections. babesia microti serological testing with pooled samples: a feasibility study laura tonnetti*, aaron thorp, letitia dixon and susan l stramer. background/case studies: blood donation screening for babesia microti, a tick-borne intraerythrocytic parasite endemic in the northeast and upper midwest us, is performed under an investigational study using nucleic acid and immunofluorescence assays (ifa). however, ifa is a time consuming and labor intensive procedure. with the possibility of an fda licensed screening assay(s) in the near future, we investigated if b. microti testing by ifa in pools of plasma or serum could be a feasible screening approach. study design/method: to test if the increased amount of plasma or serum interferes with background fluorescence, pools of 4, 8, 16 and 32 were prepared from 192 plasma or 192 serum samples determined to b. microti-negative by individual ifa screening. the pools were tested by ifa with or without a blocking step using bovine serum albumin (bsa) and goat serum to minimize background fluorescence. potential interference from multiple pooled plasma or serum samples on the endpoint titer of positive samples was investigated by including positive samples with endpoint titers from 1:128 to 1:1024 (2-fold dilutions) in the pools. results/finding: non-specific fluorescence was visible in pools of 16 or higher and was not eliminated by the addition of a blocking step. pools of 4 or 8 samples did not show significant increased background. there was no difference between testing of pooled serum or plasma samples. when one single positive sample was included in the pools of 4 or 8 samples, the pool tested positive and the final titer was the same as the positive sample tested individually. when two or more positive samples were included in the pools, the final titer of the pools was equal to the sample with the highest titer. conclusion: this study represents a proof of concept that serological testing for b. microti by ifa in pools of up to 8 plasma or serum samples does not increase false positivity while maintaining the sensitivity of the test. background/case studies: the rapid detection of bacterial contamination in platelets is key to reducing the risk of infection in transfusion of blood components. the bact/alert virtuo* is an advanced, next generation system with improved automation, connectivity and with data management systems. the virtuo's new algorithm significantly reduces the time to detection (ttd) of microorganisms during quality control testing of platelet preparations using bact/alert (bta) bpa (aerobic) and bpn (anaerobic) bottles. as plasma is known to be bactericidal, a study was completed to evaluate plasma susceptibility/resistance for organisms considered for virtuo studies. study design/method: human plasma (thawed and pooled) and saline controls were seeded with $100 cfu/ml of 12 organisms associated with platelet contamination and incubated at room temperature for 18-24 hours. colony counts were performed initially and after incubation. plasma resistance was determined if the colony count of the seeded plasma was equivalent or higher (1 1 log) than the colony count of the seeded saline after incubation. results/finding: the serially diluted strains and all bioball tm strains except p. aeruginosa, nctc 12924, were determined to be plasma resistant. the bioball tm p. aeruginosa was susceptible to the antimicrobial effects of human plasma, but when spiked into 4 ml of leukocyte reduced apheresis platelets (lrap) and inoculated into bta bpa bottles and loaded into the bta 3d and virtuo the organism was recovered 100% . conclusion: results confirm that previously tested organisms and additional strains are plasma resistant with the exception of p. aeruginosa, nctc 12924. however, the bpa bottles still recover p. aeruginosa in the presence of lrap. bpa/bpn bottles inoculated with select organisms from this panel in the presence of 4ml lrap demonstrated 100% recovery when loaded onto the virtuo and 3d ( table 1) . further studies may be required to determine if higher test volumes of lrap could affect the recovery of plasma sensitive strains. * virtuo is not fda cleared for platelet testing a. notoscriptus, identified as a major urban vector of rrv, is also capable of transmitting dengue virus 1-4, and has recently been found in los angeles, illustrating an expansion in range. with the growing geographical distribution of aedes species mosquitoes, the potential for rrv to enter local transmission cycles outside of australia is significant. in 2014, a probable transfusiontransmission (tt) was confirmed as the cause for an rrv infection in australia, validating the reality that rrv tt can occur. rrv morbidity leads to clinical manifestations that are similar to chikv infection, with varying degrees of arthralgia, which can become debilitating. various asymptomatic to symptomatic infection ratios have been reported, but this further increases the risk of additional tt in endemic areas and could mask the spread of the disease globally. study design/method: platelet concentrates (pc) prepared in pas were inoculated with rrv, amotosalen was added to final concentration of 150 mm and the units were treated with uva light. pre-and post-treatment illumination samples were collected for titration. as-5 rbc units were contaminated with rrv, mixed with processing solution/glutathione (gsh) and treated with amustaline at a final 200 mm concentration. pre-and post-treatment samples were removed prior to amustaline treatment and 3hrs after amustaline addition, respectively, for titration by plaque assay on vero76 cells. log reduction was calculated as the difference between the mean infectious titer in pre-vs. post-treatment samples. results/finding: inactivation of rrv was achieved to the limit of detection in pc and rbc. in pc, >5.1 log 10 or log 10 /ml of rrv was achieved, with >5.5 log 10 or >5.2 log 10 /ml of rrv inactivated in rbc. conclusion: these studies illustrate that amotosalen/uva and amustaline/ gsh treatments are effective at inactivating rrv in pc and rbc, respectively. these data corroborate previous results achieved with other alphaviruses, including chikv and mayaro virus which are inactivated at high titers in pc and rbc, demonstrating the ability for these systems to mitigate tt potential and maintain safe blood component availability in endemic areas. (data have not been submitted for fda review and intercept for red blood cell is not approved for commercial use). background/case studies: the interceptv r blood system for platelets is designed to inactivate pathogens and contaminating leukocytes. this photochemical treatment process utilizes amotosalen and low energy ultraviolet a (uva) light. the current available sets include small volume (sv; 255-325 ml), large volume (lv; 300-390 ml) and dual storage containers (ds; 300-420 ml) designed to treat platelet doses between 2.9 and 8.0x10 11 . the new triple storage (ts) set was designed to expand the dose range to 12.0x10 11 and the maximum volume to 650 ml, generating either 2 or 3 doses of pathogen reduced platelet components (pc). the objective of this study was to evaluate the effectiveness of the system by performing log reduction assays using representative gram positive and negative bacteria and enveloped and non-enveloped viruses in platelets suspended in pas, or 100% plasma using ts set. study design/methods: for each experiment, a platelet pool was prepared either in 47% plasma/53% pas or 100% plasma with a final volume of $650 ml and a dose of 9-12 3 10 11 platelets. these conditions represent inactivation using the lowest amotosalen concentration (135 mm) and highest concentration of platelets. platelet units were inoculated with high titers of viruses, or bacteria and treated. control (pre-uva) and test (post-uva) samples were serially diluted and cultured. plates with suitable media were used for bacteria, whereas viral titers were determined using plaque assays. log reduction was calculated as the difference between the log 10 titers in control (pre-uva) and test (post-uva) samples. conclusion dromedary camels were identified to be the reservoir of mers cov, transmission to humans occurs through direct and indirect contact. mers cov has been detected with high genomic titers of 6-10 logs in respiratory secretions of mers patients, and with lower genomic titers of 4-5 logs in blood. the presence viral particles in the blood of acute patients gives rise to concerns, especially in endemic areas. the high mortality rate, especially for critically ill patients, which often require blood transfusion, raises the need for a method to safely exclude mers cov contamination of blood products. pathogen reduction with amotosalen/uva technology is a widely established technology with a broad range of data supporting clinical efficacy and safety of amotosalen/uva treated blood products. the aim of the study is the assessment of the mers cov inactivation efficacy in human plasma with amotosalen/uva pathogen inactivation technology to safely exclude the presence of infectious virus in human plasma units. pre-uva titer post-uva titer log 10 reduction/ml (log 10 /ml) 47%plasma/ 53% pas e .coli 6.0 <-1.0 >6.0 e. cloacae 6.4 <-1.0 >6.4 k. pneumoniae 6.6 <20.1 >6.5 s. aureus 6.7 <-1.0 >6.7 blue tongue virus 4.9 <-1.0 >4.9 bovine viral diarrhea virus 4.6 <-1.0 >4.6 adenovirus-5 1 3.9 <-0.6 >3.9 100%plasma k. pneumoniae 1 6.5 -0.5 >6.5 s. aureus 1 6.2 <-0.7 >6.2 adenovirus-5 1 4.5 <-0.6 >4.5 1 n53 190a transfusion 2017 vol. 57 supplement s3 abstract study design/method: four therapeutic human plasma units were spiked with a fully characterized mers cov clinical isolate followed by pathogen inactivation with amotosalen/uva (intercept blood system, cerus corporation) at four different days. pathogen reduced samples were taken preand post-pathogen reduction after various processing steps to assess the infectious titer by plaque assay titration and the genomic titer by real-time-pcr. samples post pathogen reduction have been passaged 3 times up to 9 days, assessing the infectious titer and genomic titer every 3 rd day to exclude the presence of low-titer infectious particles. results/finding: all viral particles in the plasma units were completely inactivated with an average efficacy of !5.8 log infectious titer. no viral replication was observed after 9 days of passaging post inactivation. the genomic titer was only slightly affected by pathogen inactivation, which is designed to target the infectious titer, but not the physical titer. conclusion: amotosalen alone had a slight effect on the infectious titer while amotosalen/uva effectively inactivated all infectious mers cov viral particles in the plasma units with an inactivation efficacy above 5 logs infectious titer, giving evidence for improved blood safety of amotosalen/uva treated plasma in mers cov endemic regions. estimating the prevalence and incidence in a national blood service in taiwan for hcv eradication program yun-yuan chen* 1 , jen-wei chen 1 , chi-ling chen 2 , sheng-nan lu 3 and pei-jer chen 2 . 1 department of research, head office, taiwan blood services foundation, 2 graduate institute of clinical medicine, college of medicine, national taiwan university, 3 division of hepatogastroenterology, department of internal medicine, kaohsiung chang gung memorial hospital background/case studies: world health organization (who) has set a goal to eliminate hcv by 2030, and the epidemiological indicators generated from a national blood service is useful to monitor the effectiveness. this study aimed to evaluate the prevalence and incidence of hcv infection in taiwan. study design/method: in taiwan, anti-hcv (since 1992) and 8-sample mini-pools triplex nucleic acid test of hcv, hbv and hiv (since 2013) have been used in the routine blood screening. prevalence of anti-hcv and hcv rna were estimated in the first-time donors during 1999-2016 and 2013-2016, respectively. age-standardized prevalence and its 95% confidence interval (95% ci) were calculated with adjustment of who world standard population 2000-2025. for the incidence study, donors who have donated blood two or more times during 2013-2016 and who were without a history of anti-hcv positive before the follow-up period were included. the incidence and its 95% confidence interval was estimated from the number of new hcv rna positive cases divided by the person-years of follow-up. results/finding: the crude prevalence of anti-hcv in the first-time donors was dramatically decreased from 15.2 per 1,000 donors (95% ci: 14.8-15.7) to 4.0 per 1,000 (95% ci: 3.7-4.3) during 1999-2016, and the agestandardized prevalence was also decreased from 27.0 per 1,000 donors (95% ci: 25.6-28.4) to 7.7 per 1,000 (95% ci: 6.9-8.5). the agestandardized prevalence of anti-hcv was generally higher in female donors before 2015, but it was significantly higher in male donors at 2016 (p-value50.03). a total of 1,036 hcv rna positive cases, 1.9% of them were anti-hcv negative, identified from 579,286 first-time donors during 2013-2016, and the crude and age-standardized prevalence of hcv rna was 1.8 per 1,000 (95% ci: 1.7-1.9) and 5.0 per 1,000 (95% ci: 4.3-5.7), respectively. crude prevalence of hcv rna was significantly higher in female donors (p value <0.0001), but no significant difference was found after age standardization (p value50.93). both the prevalence of anti-hcv and hcv rna were increased with age (p for trend<0.0001). in the incidence study, a total of 68 new hcv rna positive cases, 23.5% of them were anti-hcv negative, found from 1,202,165 donors followed for 2,415,668 person-years. the incidence of hcv rna was 2.8 per 100,000 person-years (95% ci: 2.2-3.5), and no significant difference was observed between both genders (p-value50.41) and between age groups (p for trend 0.37). conclusion: the prevalence of hcv infection has been dramatically decreased by 71.5% during 1999-2013. it becomes significantly higher in male donors and that needs to monitor in the future. incidence of hcv rna is low in repeat blood donors and it needs to identify more incident cases to observe the epidemiological characteristics. dalia ashour* 1 , sahar muhmmad 2 and dalia el dewi 2 . 1 national blood transfusion services, 2 azhar university background/case studies: blood safety is a challenge in egypt because of the high prevalence of hcv and hbv. nucleic acid amplification test (nat) technologies have the potential to detect viremia earlier than current screening methods, which are based on seroconversion. the primary benefit of nat is the ability to reduce residual risk of infectious wp donations. the estimated reduction of the wp utilizing nat for hcv is 70-12 days, hiv from 22 to 11 days, and hbv from 25-30 days. study design/method: this cross sectional study was conducted in national blood transfusion center (giza, egypt) from 2012 to 2015, the total number of donor samples to be screened is 178685, the age of the donors ranged from 18 to 50 years, and they were of both sexes (m: f 5 3:1).screening by nat ulterio assay (grifols diagnostics; formerly novartis diagnostics) was done in parallel with eia testing for hbsag, hcv-ab and hiv ag/ ab. using individual donation nat (id-nat). multiplex nat yield samples are further tested using the discriminatory assay in order to ascertain which viral nucleic acid is present in the donor sample. statistical analysis chi-square (v2) test was used to measure the association between two qualitative variables. results/finding: nat screening detected a total of 75 nat yield donations among 178685 (0.04%) seronegative donors. among these 75 nat yields cases, 53 (0.03%) were reactive for hbv, 20 (0.011%) were reactive for hcv and 2 (0.001%) were reactive for hiv-1. we stratified the age of the donors into 3 groups; group a (18 -28 years), group b (29 -39 years) and group c (40 -50 years). the prevalence of nat yield to the three viruses was significantly higher in either group b or c, compared to group a (p 5 0.0089; with 95% confidence interval (ci) 5 0.0085 -0.0520 & p 5 0.0247; with 95% ci 5 0.0025 -0.0534 respectively). prevalence of nat-hbv; was significantly higher in age group b, as compared with group a (p 5 0.0224; with 95% ci 5 0.0032 -0.0413). on the other hand, there was no statistically significant difference between groups c and a and between groups b and c. comparing groups b and c combined with group a found a significantly higher prevalence of hbv in the former (p 5 0.0335; with 95% ci 5 0.0015 -0.0352). nat-hcv; did not differ significantly between the three groups (p 5 0.3222; with 95% ci 5-0.0089 to 0.0161 between groups a and b & p 5 0.1340; with 95% ci 5 -0.0055 to 0.0270 between groups a and c & p 5 0.4277; with 95% ci 5 -0.0080 to 0.0215 between groups b and c). nat-hiv; did not also differ significantly between the three groups (p 5 0.3801; with 95% ci 5-0.0077 to 0.0077 between groups a and b & p 5 0.3172; with 95% ci 5 -0.0056 to 0.0077 between groups b and c). in either group a and c, no nat-hiv detected. nat yield to the three viruses was significantly higher in males than in females (p 5 0.0013; with 95% ci 5 0.0136 to 0.0507). nat hbv was significantly higher in males (p 5 0.002; with 95% ci5 0.0103 -0.0413), but the prevalence of either hcv or hiv did not differ significantly between males and females (p 5 0.3835; with 95% ci 5 -0.0077 -0.0145 & p 5 0.2751; with 95% ci 5 -0.0044 -0.0066; respectively). conclusion: in this study the nat yield of 75 in 178685 assumes more significance when one considers the fact that single donation is used for generating 3 components that can be used by 3 recipients. hence, in effect the nat yield becomes 3 times that is, 225 in 178685. saving 225 recipients from tti out of 178685 (0.13%) is indeed very significant. results/finding: of the 303,569 donors who were tested by our donor center, 1,386 (0.460%) were repeat reactive. a seasonal pattern in the prevalence was observed with the highest number of donors being positive in summer, and then progressively declining during the fall and winter months and increasing again in spring. there was a single case of transfusion transmitted babesiosis reported from our center during this period. a patient who was transfused with two units of packed red blood cells (rbcs) from two donors in the beginning of july presented in august for further transfusion and was found to have parasitemia in the peripheral blood smear and was subsequently diagnosed with babesiosis. the donors were called back, however one of them could not be tracked. samples were sent to the state for further testing: an immunofluoresence assay was performed (combination of igg, igm and iga). the test was positive at 1:128 titer. the screening elia s/co of this donor was 0.2782. both donors were indefinitely deferred as blood donors. conclusion: our data confirm a decreased risk in transfusion transmission with the use of a screening assay. prior to implementation of the screening there were 6-10 transfusion transmitted babesia cases per year from 2008-2015 (table 2 ). in the 11 months after implementation of pre-transfusion babesia screening, one break through case of transfusion transmitted babesia was observed (1 in 303,569 donors tested). thus the babesia eia screening test effectively prevents ttb. however, there was a substantial loss of donors due to being screen positive. four years of experience with id-nat at a tertiary care centre in north india: implications for transfusion transmission and donor screening. jasmeet singh*, amarjit kaur, gurpreet kaur, rajesh kumar and sonia gupta. dayanand medical college and hospital background/case studies: transfusion transmitted diseases are a challenge for transfusion medicine specialists and patient care providers around the globe. blood safety is a formidable task especially in a high population country like india. newer technologies like id-nat equip us to screen and prevent transfusion transmitted viral infections and prevent their transmission by improving over the sensitivity and specificity of conventional methods. this study aims at examining the effect of id-nat as an additional test on the safety of blood supply. study design/method: a retrospective observational study was conducted to analyze the data of 4 years of additional nat testing at blood bank, dmch, ludhiana from september 2012 to december 2016. results/finding: results 1.73% (2041 of 118021) units were initially nat reactive. these units were further tested, of which 90.98% were discriminated (70 hiv, 1051 hcv, 726 hbv and 10 co-infections). the remaining 6.71% (137) were repeat non-reactive and 1.91% (39) could not be discriminated. overall, nat yield rate was one in 837, whereas virus-specific nat yield rates were one in 59,010 for hiv, one in 1873 for hcv, one in 1639 for hbv and one in 29,505 for hbv/hcv coinfections, respectively. conclusion: id-nat screening of all blood donations at our institution over past 4 years has increased the screening sensitivities to check viral load and prevented transmission of 141 probable transfusion transmitted viral infections. assuming 100 % component preparation it saved 423 transfusion recipients from harm. implementation of nat along with routine serological tests for screening of the blood donations definitely improves the transfusion safety and should be mandated across all transfusion centers. min xu 1,2 , wei mao 3 , tao he 3 , yashan yang 1,2 , zhan gao 1,2 , chunhong zhang 3 , hongmei liao 3 , jingxing wang 1,2 and miao he* 1,2 . 1 institute of blood transfusion, chinese academy of medical sciences & peking union medical college, 2 sichuan blood safety and blood substitute international science and technology cooperation base, 3 chongqing blood center background/case studies: many emerging infectious pathogens are known to be existed in heathy blood donations, and could be transmitted via transfusion with potential hazardous consequences against recipients. with more convenient application of high through put sequencing, it becomes much easier to investigate uncultured microbiome in qualified blood donations. therefore, metagenomics analyses were used to reveal emerging and re-emerging infectious diseases in healthy donations which might potentially threat the blood safety. study design/method: pooled plasma sample were collected from 5,000 voluntary blood donors from chongqing, china. total dna and rna were extracted and amplified with random primers pcr respectively in order to construct a 250pe library to peform deep sequencing by illumina miseq. all reads were trimmed to remove low quality bases and adapter sequences. the fully overlapping paired-end reads passing the quality filter were concatenated using pear. we classified the final reads using kraken and a kraken database made from complete refseq bacterial, archaeal and viral genomes, along with the grch37 human genome. the unclassified reads by kraken were aligned to ncbi nt database using blastn with cut-off evalue as 1e -5 . the best alignment hits were used to classify the reads. krona was used to generate all taxonomic distribution plots. finally, the potential emerging and re-emerging infectious pathogens were identified out of the classified microbiome by experience. abstract results/finding: 1.23 gb raw data with 2,450,046 reads were generated in the dna library. meanwhile, 1.98gb raw data with 3,967,242 reads were generated in the rna library. after cleaning the human background, 211 reads from bacteria, 98 reads from viruses, and 341 reads from parasites were identified (table 1) . no hazardous viruses were identified as potential threats to blood safety. except for viruses and bacterias which would do limited hazards to blood safety, plenty of parasites were identified in which some were already considered as threats to blood safety in some developed countries were also discovered such as plasmodium sp. and leishmania infantum (table 1) . conclusion: the investigation has revealed the metagenomics of the qualified blood donations in chongqing, china. the results showed a thoughprovoking discovery of genomic fragments of some microbes which might threat the blood safety. the displayed serious results let us have to think about regulating some reasonable screening methods as well as donor recruitment strategy in certain epidemic areas or seasons to ensure the blood safety. however, on the contrary, the results should be considered more cautiously because the existing of genomic fragments could not represent the existing of infectious pathogens. the validity of the metagenomics hints were suggested to go through epidemiological investigations and specifically tested under laboratory ways such as bacteria or virus culturing to ensure the vitality of those pathogens. background/case studies: the caribbean has become an endemic region for several emerging viruses in the last decade. after a chikungunya outbreak in 2015 most recently zika was shown to be endemic on the caribbean island of curacao. to effectively provide safe blood products in an endemic region the conventional international recommendations of donor exclusion and testing do not seem a viable option and could severely affect the local blood supply. pathogen reduction (pr) is considered an important new approach with potential benefits. the introduction and experience of use of pr platelets in the dutch caribbean over a period of one year is presented. study design/method: pathogen reduction of thrombocyte concentrates by use of riboflavin and ultraviolet treatment (mirasol prt, terumo, belgium) was introduced. all thrombocyte concentrates provided to the general hospitals on the dutch caribbean islands of curaçao, bonaire and sint maarten were pr and data collected over the period of 1 february 2016 to 1 february 2017. thrombocyte concentrates are prepared out of 4 single donation units by the buffycoat method. results/finding: over the period 260 platelet concentrates were provided to adult and pediatric patients. these included patients on the intensive care and neonatal intensive care departments. no adverse events were reported and the cci for each transfusion was within the expected outcome. introduction of pr had minimal impact on the logistics of thrombocyte concentrate preparation and availability. furthermore no transfusion related bacterial contaminations were reported. conclusion: pr of platelet concentrates seems viable and safe for use in a small scale caribbean setting with endemicity for emerging viruses like chikungunya and zika. it offers a realistic alternative for conventional recommendations of donor exclusion and testing, thereby helping to maintain sufficient labile blood product availability. michael phillips* 1 , germ an leparc 2 , phillip c williamson 1 , lani palmer 1 , ben reynolds 1 , maria noedel 1 and lindsey houghton 1 . 1 creative testing solutions, 2 oneblood background/case studies: due to the risk of travel and sexually transmitted zika infections, the food and drug administration issued a guidance document on february 16, 2016 recommending that blood centers in puerto rico cease distribution of locally collected blood products unless donors are tested or products are pathogen reduced by march 1, 2016. with the high incidence of zika virus (zikv) in puerto rico and uncertainty of the impact to the continental u.s. blood supply, there was intense pressure to implement a donor screening test for zikv. the project was initiated on february 16, 2016 and included clinical trial requirements, client onboarding and laboratory operations. stakeholders consisted of clients, the manufacturer, institutional review boards (irb), informational technology (client and lab based), the food and drug administration (fda), the centers for disease control cdc, and the florida department of health. clinical trial requirements included development of instrument and assay validations, sop creation, result reporting, assay and clinical trial training, deviation management, donor notification, and follow up sample handling. client onboarding began with confidentiality agreements between the client and the sponsor. a zika based webinar was created to provide an overview of the sponsor protocol, lab test system and client responsibilities. the complexity of the project increased when mosquito borne zika transmission was identified in two counties in florida. this required zikv testing to be performed on collections in both florida and puerto rico. the zikv-nat is performed in singlet, unlike the mpx and wnv assays which are run in minipools. this had a significant impact on instrument capacity. despite these obstacles and the changing regulatory requirements, the zikv screening test was implemented within six weeks. study design/method: one metric used to measure client service levels is our ability to meet established upload time goals for individual clients. the percentage of samples released on time is evaluated daily with a running monthly total. our upload time goals were negatively impacted from july through september due to the unexpected increase in zikv testing, the requirement to perform testing in singlets and the resulting instrument capacity issues. additional instruments were sourced in october and operations stabilized. conclusion: on february 16, 2016, the project to implement a zikv ind test was initiated. six weeks later, testing was performed on the first batch of samples. despite the changing regulatory requirements over time, the implementation was extremely successful. initiating a new ind testing within 6 weeks is unprecedented and required exceptional collaboration between all participants and stakeholders. background/case studies: plasmodium falciparum (pf), an intraerythrocytic protozoan parasite, is accountable for nearly all malaria mortality in africa. in 2015, who reported $212 million new cases worldwide, resulting in >400,000 deaths. malaria prevalence is highest in sub-saharan africa, home to 90% of all infections accounting for 92% of mortalities. both the incidence and prevalence of malaria in africa significantly increase the potential for transfusion-transmission (tt), with little to no screening of products in developing countries. the objective of this study was to evaluate the inactivation of pf in whole blood (wb) using a system specifically developed for the realities of the developing world and in support of the swiss red cross humanitarian foundation for whole blood pathogen inactivation for africa. the inability to consistently supply blood components leads to routine wb transfusion, and as transfusion-transmitted diseases are prevalent in the developing world, the establishment of a robust wb pathogen inactivation system is desirable. the approach uses the small molecule amustaline to form covalent adducts and crosslinks within nucleic acids of leukocytes and contaminating pathogens to prevent replication. the process includes addition of 0.2 mm amustaline and 2 mm glutathione (gsh) and a 24h at room temperature (rt) incubation after which the treated wb unit is suitable for storage up to 7 days at rt. study design/method: for each experiment, a wb unit was spiked with ring-stage pf-infected red blood cells (irbc). a pre-treatment sample was removed prior to addition of amustaline and a post-treatment sample was removed 24h after amustaline addition to determine the pre-and posttreatment titers to calculate the level of inactivation. these samples were serially diluted in flasks containing medium with 5% fresh rbcs. the diluted samples were used to inoculate flasks in quadruplicate and monitored for parasitemia by counting irbc in blood smears and by flow cytometry. pretreatment cultures were terminated after reaching >1% parasitemia, while no residual pf was detected in post-treatment cultures. log reduction was calculated as the difference between the mean titer in pre-and posttreatment samples. results/finding: robust inactivation of pf in wb was achieved to the limit of detection, at >7.5 log 10 or >6.0 log 10 /ml. conclusion: pf was inactivated to the limit of detection in wb after treatment with amustaline/gsh, illustrating that the system has potential to mitigate the risk for pf transfusion transmission in endemic regions that lack testing capacity and operate under the constraint of a very limited blood component supply and rely on wb transfusion. (this system for wb is not approved for commercial use). increased patient safety and improved inventory management with 7 day apheresis platelets nancy m. dunbar* and zbigniew m. szczepiorkowski. background/case studies: a pathway currently exists for apheresis platelet (ap) outdate extension from 5 to 7 days using an fda cleared rapid test (rt). in february 2016, our hospital based transfusion service implemented the use of rt on day 5, 6 and 7 to routinely extend ap shelf life to 7 days. prior to this, we tested aps by rt on day 4 and transfused day 6 or day 7 units with physician approval when deemed medically necessary. this report describes changes observed in transfusion practice and platelet inventory management one year following routine use of 7 day platelets. study design/methods: data were obtained for two 12-month study periods: october 2014-september 2015 (pre-implementation) and february 2016-january 2017 (post-implementation). the interval transition period was intentionally excluded. for each study period, we determined the total number of aps transfused, rt status on the day of transfusion, total number of rts performed, expired ap units, and aps obtained from suppliers using ad-hoc ordering. we also obtained hospital data including inpatient admissions, surgical volumes, average length of stay and case mix index. results/findings: data are shown in table 1 . the number of ap transfusions increased by 7% post-implementation, comparable to a 4% increase in inpatient admissions and an 11% increase in surgical volumes. the hospital length of stay and case mix index were similar for both periods. the average number of platelet transfusions per patient was not statistically different (3.16 pre; 3.12 post, p50.91). the number of rts performed increased by 130%. the percentage of transfused units tested at least once by rt prior to transfusion increased by 21% (p<0.0001). the outdate rate decreased from 5% to 2% (p<0.0001). ad-hoc ordering decreased from 21% to 9% (p<0.0001). conclusion: use of an approved rt for routine ap outdate extension to day 7 was associated with increased patient safety as more transfused units underwent secondary testing prior to transfusion. increased cost of rt was offset by reduced ap waste and less frequent need for ad-hoc ordering. sheila o'brien* 1 , vito scalia 1 , carla osiowy 2 , michael carpenter 2 , anton andonov 2 and margaret fearon 1 . 1 canadian blood services, 2 public health agency of canada background/case studies: the rates of hepatitis b (hbv) and hepatitis c virus (hcv) positive donations are low (6.6 and 4.9 per 100,000 donations, respectively) and most are among first time donors. we aimed to determine the frequency of various genotypes of hbv and hcv in canadian blood donors confirmed positive for hbv and hcv. study design/methods: in 2011 the roche multiplex assay (hcv/hiv/ hbv) was implemented in minipools of 6 units. hcv nat was in place since 1999 (using minipools of 24) but this is the first time donors have been screened by hbv nat. hbsag, anti-hbc and anti-hcv were tested using the abbott prism assay. confirmatory testing for hbsag was by the prism neutralization assay. anti-hcv repeat reactivity was confirmed by the inno-lia hcv score line immunoassay. since march 2016 all samples testing hbv nat positive, or confirmed positive for hbsag and all hcv nat positive or anti-hcv confirmed positive samples were considered positive and samples were sent to phac for sequencing. a sample from each positive donation was aliquoted and frozen at -20 o c. genotyping was carried out by sequence and phylogenetic analysis of the hbv surface antigen coding region. hcv viral rna was extracted and subjected to reverse transcription and pcr amplification in the 5' ntr-e1 and ns5b regions. sanger sequencing of these regions represents approximately 15% of the genome. results/findings: all confirmed positive donations were whole blood donations. there were 42 hbv positive donations. of these, 37 had tested hbv nat positive. genotypes were 8 type a, 6 b, 4 c, 17 d and 2 e. there were 5 samples hbv nat negative but hbsag positive (2 were anti-hbc reactive). of these, 4 could not be sequenced and one was genotype a (also anti-hbc reactive). there were 30 samples considered hcv positive. of these, 17 samples were hcv nat positive. genotypes were 5 type 1a, 3 1b, 3 2c, 2 2b and 4 3a. there were also 13 samples hcv nat negative but anti-hcv positive. none of these could be sequenced. conclusion: the first 8 months of molecular surveillance show a range of genotypes for hbv and hcv for samples identified as nat positive. to date no samples that were nat negative anti-hcv reactive could be sequenced, however one nat negative sample that was positive for hbsag and anti-hbc reactive was hbv genotype a. surveillance over a longer period is background/case studies: the bact/alert 3d microbial detection system (bta 3d) is currently fda cleared for the quality control testing of leukocyte reduced apheresis platelets (lraps). the bact/alert virtuo microbial detection system (virtuo) (biom erieux, st. louis, mo) is a new generation of bact/alert instrumentation. the underlying colorimetric technology used in previous generations of bact/alert is used in the vir-tuo and incorporates new instrument architecture to improve temperature stability, workflow improvement via automation of processes that are currently performed manually, an improved user interface and an enhanced algorithm to shorten time to detection. the objective of this study was to compare the performance of the virtuo and bact/alert 3d (bta 3d) instruments, using bact/alert bpa (aerobic) and bact/alert bpn (anaerobic) bottles, for the detection of a range of typical bacterial contaminants seeded into leukocyte reduced apheresis platelets (lraps).* study design/method: the study was performed at two institutions, one in the us and the other in the uk. aliquots of lraps were seeded with low levels (1-20 cfu/ml) of 11 bacterial species commonly associated with platelet contamination, and 20 replicates (10 per instrument) of 4 ml aliquots per bottle were inoculated into bpa and bpn bottles. one set of bottles was loaded into bta 3d and the other into virtuo and incubated until signaled positive by the instruments or for up to 7 days. overall detection rates and time to detection of bacterial contaminants between instruments were compared. additionally 98 bottles were tested in each instrument (lraps only, no organism) to evaluate differences in the overall negative agreement rates (detection of false positives) between instruments and to serve as sterility controls for the platelet preparations. background/case studies: the implementation of nucleic acid testing (nat) blood screening is still a challenge in resource-limited countries. at the same time, in these countries, higher to similar proportions of replacement to voluntary blood donors are recruited. a higher prevalence of infections is observed in relation to developed countries. as a consequence, more incident cases of infections can be expected. in our country, some hospital blood banks could not afford nat due to high costs, but belong to a net that centralizes nat in a reference blood center. the process to consolidate small blood banks in regional blood centers, which will be able to implement nat, is not yet complete. although efforts to reduce replacement /familiar blood donations are in progress, these goals have not been completely achieved. the aims were to compare the prevalence of hiv, hcv and hbv by nat screening in a blood center recruiting only voluntary blood donors with the prevalence in centers recruiting replacement and voluntary blood donors, and describe the nat yield rates for hiv, hcv and hbv in a period of three and a half year experience. study design/method: a regional blood donor center (rbdc) has centralized nat screening from centers in different regions of the country due to since august 2013. this process required to achieve adequate laboratory conditions and staff qualification and a development of software to assure sample traceability and interface for transmission of results. when a window period was suspected, the nat screening was repeated from the plasma unit and a second sample of the blood donor was required to confirm nat results. this rbdc have also been developed a 100% voluntary donor program since 2011 and is the only center in the country that has achieved this goal. results/findings: a total of 264,343blood donations were studied from august 2013 to december 2016. in the rbdc, where only voluntary blood donations are recruited, the prevalence was 18 per 100,000 donations for hiv (ic95% 8-34:100,000); 14 per 100,000 for hbv (ic95% 7-29:100,000) and 18 per 100,000 for hcv (ic95% 8-34:100,000). in all other centers together, where voluntary and replacement blood donations are recruited, the prevalence was 89 per 100,000 donations for hiv (ic95% 77-103:100,000); 70 per 100,000 for hbv (ic95% 59-83:100,000) and 78 per 100,000 for hcv (ic95% 66-91:100,000). window period infections were detected only in centers recruiting voluntary and replacement blood donations, giving nat yield rates of 1: 66,086 for hbv; 1: 132,172 for hiv and 1: 264,343 for hcv. conclusion: the hiv, hbv and hcv prevalence was lower in a center where the tasks to sustain a voluntary blood donor program were developed. nat yield rates could be reduced in the region if this program could completely be applied in all centers. mechanisms leading to obi include various factors such as imperfect host's immune response and viral variation factors. this study was to determine the viral loads of obi under currently recruitment and screening among blood donors in five blood services of zhejiang province, china. study design/method: before donation, the donors were screened and precluded with hbsag preliminary test positive and alt level abnormal. following, the samples were detected for hbsag twice using different elisa reagents and hbv dna using tma or qt-pcr techniques. then, the samples with hbv dna positive and elisa negative were tested for the viral loads using taqman technique in cobas s201 system. hbv s region was also sequenced. results/finding: 234 obi were found in the 230,000 donations. in the viral loads assay, 43 samples were negative and 104 samples' viral loads were lower 20iu/ml. the mean viral loads was 1.85 6 0.41 (log10) iu/ml in other 87 samples,while the mean viral loads with hbsag1/hbv dna1 samples was 2.38 6 0.83 (log10) iu/ml. 60 samples of obis have analyzed the hbv genotype, which b was the most prevalent subtype (69.0%) and the other was hbv c genotype(31.0%). compared the samples with hbsag1/hbv dna1 ,we found two obi samples carrying with 318t>c mutation, which could cause an amino acid s55f. conclusion: in this study, the viral loads of obi infection in donors was much low than hbsag1/hbv dna1, and some unique variation was identified in the obi individuals. 195a transfusion in general population. screening of blood donors for hbv in india is primarily based upon detection of hepatitis b surface antigen (hbsag) in donor's sera. the current study was undertaken to determine the prevalence of occult hbv infection (obi) in voluntary blood donors and to analyze the burden of hbv window period donations. study design/method: this is a prospective, observational, mono-centric study performed in a national accreditation board for hospitals (nabh) accredited apex blood bank, located in maharashtra state, india. monolisa hbsag ultra (bio-rad, france)sandwich type elisa using monoclonal and polyclonal antibodies was used for hbsag detection in donor's sera. all the elisa non-reactive samples were also tested by an additional real time multiplex polymerase chain reaction (mpx-pcr) by cobasv r taqscreen mpx test. the donors which were found to be positive for hbv dna were followed upat 15 th days, 1month, 3 months& 6 months by monolisa hbsag ultra (bio-rad, france) to analyze interval of window period and to delineate the window period donations (wpd) & true obi. background/case studies: occult hepatitis b infection (obi) is characterized by hepatitis b virus (hbv) dna-positive, but hbv surface antigen (hbsag) -negative. since may 2015, we have been testing apheresis donors for hbv nucleic acids and improvements in laboratory testing have reduced the risk of transfusion-transmitted infection. the number of apheresis collections increased significantly year by year, however, data on hepatitis b virus marker rates among these donors continue to be lacking. the aim of this study is to evaluate the epidemic characteristics, incidence and estimate the risk factors of obi among apheresis donors in a region of central china. study design/method: apheresis donors' data from may 2015 to dec 2016 was retrospectively analyzed. all samples were tested for hbsag, hbv dna, and other markers. nucleic acids testing (nat) was performed on the roche cobas s201 platform using pools of 6 serologically negative samples and any pools positive would undergo nat again individually. hbsag negative, but hbv dna positive were further tested for hbv dna quantitative pcr, antibody to hepatitis b surface antigen (hbsab), antibody to hepatitis b core antigen (hbcab), hepatitis b e antigen (hbeag) and antibody to hepatitis b e (hbeab). results/finding: in the evaluation, 68547 seronegative donations were screened by nat and a total of 20 hbv dna-reactive/hbsag-negative donors were detected. no hiv rna -reactive or hcv rna -reactive sample was detected. complete serologic screening of the index donations indicated that the majority of these donors had an occult hbv infection and the majority of which were married men and the fixed donors with many whole blood or apheresis donations. age distribution of the age group 31-55 years old showed a large proportion, who accounted for 80% of reported infections. most of the hbv dna cases (about 80.0%) reached senior high school education. the average hbsag dna positive rate was 0.029% (20/ 68547). incidence among apheresis donors in this period for hbsag dna were 2.91/10000. these estimates were comparable to those among repeat whole blood donors. we developed pathogen reduced (pr) cryo derived from ffp and pf24 with 5 day stability at 228c. study design/method: six replicates of type-matched pools of whole blood derived (wbd) and apheresis (aph) plasma were split to produce conventional control (225 610 ml) and test components (625 ml 625 ml). test components were pr with amotosalen and uva light. aph and wbd ffp were produced by freezing plasma within 8 hr and wbd pf24 within 24 hr. cryo was manufactured according to site sops and frozen at -308c (test 62 6 2 ml, control 22 6 2 ml ). test and control cryos were thawed at 378c, and characterized immediately post thaw (t50), and after 5 d storage at 228c and tested for fb and fviii function, thromboelastography (rotem) and thrombin generation (cat). results/finding: pr cryo retained sufficient fb and fviii activity post thaw and over 5d at 228c (table) for hemostatic capacity. rotem (extem) showed retention of fibrin formation (a angle) and clot quality (mcf) (table) . thrombin generation was robust as demonstrated by multiple parameters (lag time, peak thrombin, endogenous total thrombin potential (etp), and time to peak (tt) despite lower fviii levels. these parameters were maintained through 5d storage at 228c. conclusion: pr cryo can be processed from 3 plasma sources, including pf24, and stored at rt for 5 days. pr plasma provides adequate levels of fb with hemostatic capacity equivalent to control as demonstrated by rotem and cat. use of pf24 with stability over 5 days can increase the availability of cryo with a reduced risk of transfusion-transmitted infection. cryo produced with psoralen-treated (pr) plasma is not approved for use in the us. performance of a new automated alinity s assay for antibodies to t. cruzi darwin smith* 1 , ed bakker 2 , anton van weert 2 , jane bryant 1 , mark paradowski 1 , lynne fleischmann 1 , mirjana sarac 3 , george chen 1 , george schlauder 1 and gregg williams 1 . 1 abbott diagnostics, 2 sanquin diagnostics, 3 abbott gmbh & co. kg background/case studies: the parasite, trypanosoma cruzi (t. cruzi), is the cause of chagas disease which is endemic to the americas and infects 6-8 million people. in order to prevent transfusion mediated transmission of this parasite in endemic countries, blood collection centers require high throughput anti-t. cruzi assays with good specificity and sensitivity. in nonendemic countries, selective testing of at risk donors is a strategy to avoid temporary donor deferrals. in addition, continued pressures on laboratory operations demand that assays perform on platforms capable of increased walk away time and enhanced automation in areas of reagent management, retest options, and commodity/waste management. study design/method: the performance of the new automated chemiluminescence immunoassay for the detection of antibodies to t. cruzi was evaluated on the alinity s automated platform and compared to another onmarket chemiluminescent immunoassay. precision was assessed over 20 days using a panel of positive and negative samples. sensitivity was evaluated on 407 presumed antibody positive specimens and specificity was evaluated on 7621 random blood donor samples. results/finding: precision was 7% cv or less for positive samples over 20 days. the overall specificity in a blood donor population was 99.99% (7620/ 7621). sensitivity was 100.00% for 407 presumed antibody positive samples. conclusion: these results indicated that the new automated alinity s chagas assay provided very good performance in sensitivity and specificity, comparable to the current on-market anti-t. cruzi assay, and is equally suitable for use of universal screening in endemic and selective donor screening in non-endemic countries. performance of a new automated alinity s assay for hepatitis b surface antigen and hepatitis b surface antigen confirmatory randal makela* 1 , anton vanweert 2 , ed bakker 2 , jane bryant 1 , mark paradowski 1 , lynn martin 1 , daniela kaleve 3 , george chen 1 , gregg williams 1 and george schlauder 4 . 1 abbott laboratories, 2 sanguin diagnostics, 3 abbott gmbh & co. kg, 4 abbott diagnostics background/case studies: despite the development of sensitive nat methods, blood transfusion in many parts of the world relies on serologic screening for hepatitis b surface antigen (hbsag) to prevent transfusion transmitted hbv infection. sensitive hbsag assays must be capable of coping with a wide range of mutants while exhibiting an uncompromised specificity. in addition, continued pressures on laboratory operations demand that assays perform on platforms capable of increased walk away time and enhanced automation in areas of reagent management, retest options, and commodity/waste management. study design/method: the performance of a new automated chemiluminescence immunoassay for the detection and confirmation of hbsag was evaluated on a next generation automated platform, abbott alinity s. precision was assessed over 20 days. sensitivity was evaluated using 511 known positive samples, 30 commercially available seroconversion panels, the who standard, 23 hbsag mutants, and 94 hbsag genotyped specimens (a through h). specificity was evaluated on random blood and plasmapheresis donors. results/finding: precision was less than 8% cv for positive samples over 20 days. the blood donor specificity was 99.98% (7998/8000). sensitivity was 100% for 511 presumed positive samples. sensitivity was 100% for all genotypes. 100% of the mutants were detected vs 83% for the comparator assay. seroconversion detection was equivalent to the comparator assay with 157 reactive samples detected with the alinity s assay and 154 reactive samples detected by the comparator assay. analytical sensitivity ranged from 0.015 to 0.016 iu/ml. the alinity s hbsag confirmatory assay confirmed all known positive hbsag specimens, including 3 hbsag mutant samples that were not confirmed by the comparator hbsag confirmatory assay. conclusion: the new automated alinity s hbsag assay provided precision, specificity, and seroconversion sensitivity comparable to the current onmarket comparator assay. however, the alinity s hbsag assay demonstrated a gain in sensitivity over the comparator assay through the detection and confirmation of a wider range of mutants. performance of a new automated alinity s immunoassay assay for hiv darwin smith* 1 , ed bakker 2 , anton van weert 2 , jane bryant 1 , mark paradowski 1 , kevin callear 1 , susan sullivan 1 , george chen 1 , george schlauder 1 and gregg williams 1 . 1 abbott diagnostics, 2 sanquin diagnostics background/case studies: blood donations are commonly screened to detect the presence of antibodies (or antibody and antigen) to human immunodeficiency virus types 1 and 2 (anti-hiv-1/2). blood centers require very high throughput anti-hiv-1/2 assays with high specificity and sensitivity to prevent unnecessary donor deferrals while maintaining a safe blood supply. in addition, continued pressures on laboratory operations demand that assays perform on platforms capable of increased walk away time and enhanced automation in areas of reagent management, retest options, and commodity/waste management. in the response for the need for such screening assays, we have evaluated an improved automated assay for the detection of anti-hiv-1/2 antibodies and hiv-1 p24 antigen. study design/method: the performance of the new chemiluminescence combination immunoassay for the detection of anti-hiv-1/2 antibodies and hiv-1 p24 antigen was evaluated on the abbott alinity s system. precision was assessed over 20 days evaluating positive samples. specificity was evaluated on samples obtained from random blood donors and plasmapheresis donors. sensitivity was evaluated using presumed positive samples for hiv-1, hiv-2 and hiv group o antibodies and hiv-1 p24 antigen. seroconversion sensitivity was evaluated with 41 commercial seroconversion panels. results/finding: precision was less than 8% cv for positive samples over 20 days. the blood donor specificity was 99.96% (8082/8085). sensitivity was 100% for 813 presumed antibody positive samples comprised of hiv-1, hiv-2 and hiv-1 groups o, n, p, crf and urf samples. also, sensitivity was 100% for 102 antigen positive viral isolate samples comprised of hiv-1, hiv-2 and hiv-1 groups o, n, p, crf and urf samples. seroconversion detection was equivalent to the comparator assay with 136 reactive samples detected with the alinity s assay and 135 reactive samples detected by the comparator assay. conclusion: these results indicate that the new automated alinity s hiv ag/ab combo assay provided acceptable performance in specificity, sensitivity and precision, while providing similar seroconversion sensitivity as the comparator assay. performance of a new automated alinity s immunoassay for the detection of anti-hbc antibodies randal makela* 1 , anton vanweert 2 , ed bakker 2 , jane bryant 1 , mark paradowski 1 , joyce siregar 1 , angela vockel 3 , george chen 1 , gregg williams 1 and george schlauder 4 . 1 abbott laboratories, 2 sanguin diagnostics, 3 abbott gmbh & co. kg, 4 abbott diagnostics background/case studies: in countries with a low prevalence of hepatitis b, blood donations are commonly screened to detect the presence of antibodies to hepatitis b core antigen (anti-hbc) alongside hbsag and hbv nat to detect donors with occult hepatitis b infections (obi). blood centers require anti-hbc assays with high specificity and sensitivity to prevent unnecessary donor deferrals while maintaining a safe blood supply. in addition, continued pressures on laboratory operations demand that assays perform on platforms capable of increased walk away time and enhanced automation in areas of reagent management, retest options, and commodity/waste management. in the response for the need for such screening assays, we have developed an improved automated assay for the detection of anti-hbc on the alinity s system. study design/method: the performance of a new chemiluminescence anti-hbc assay for the detection of anti-hbc antibodies was evaluated on the next generation automated abbott alinity s system. precision was assessed over 20 days evaluating positive samples. specificity was evaluated on samples obtained from random blood donors. sensitivity was evaluated using specimens characterized as anti-hbc positive by means of serologic methods. analytical sensitivity was assessed using the who 1st international standard. seroconversion sensitivity was evaluated using 10 commercial seroconversion panels. results/finding: precision was less than 6% cv for positive samples over 20 days. the blood donor specificity was 99.93% (6946/6951). sensitivity was 100% for 500 samples presumed to be anti-hbc positive. analytical sensitivity results on the alinity s anti-hbc assay ranged from 0.57 to 0.62 iu/ml. seroconversion detection was equivalent to the comparator assay with 136 reactive samples detected with the alinity s assay and 134 reactive samples detected by the comparator assay. conclusion: these results indicate that the new automated alinity s anti-hbc assay provided good performance in specificity, sensitivity and precision versus the comparator assay. performance of a new automated alinity s immunoassay for the detection of htlv i and htlv ii antibodies melanie anderson* 1 , anton vanweert 2 , ed bakker 2 , mark paradowski 1 , jane bryant 1 , tuan bui 1 , joyce siregar 1 , george chen 1 , george schlauder 3 and gregg williams 1 . 1 abbott laboratories, 2 sanguin diagnostics, 3 abbott diagnostics background/case studies: in endemic countries, universal blood screening is necessary to prevent transfusion transmitted htlv infections (anti-htlv i/htlv ii). in non-endemic countries, selective testing may avoid unnecessary temporal deferrals for donors at high risk, such as returning travelers from or donors born in countries with a high htlv prevalence. blood centers require high throughput anti-htlv i/htlv ii assays with high specificity and sensitivity to prevent unnecessary donor deferrals while maintaining a safe blood supply. in addition, continued pressures on laboratory operations demand that assays perform on platforms capable of increased walk away time and enhanced automation in areas of reagent management, retest options, and commodity/waste management. in response for the need of an assay with high specificity on a high throughput instrument we have developed a new assay for the detection of antibodies against htlv-i/ii antibodies for the alinity s system. study design/method: precision was assessed over 20 days using htlv i and htlv ii positive samples. specificity was evaluated using 8,001 blood donor specimens from europe and 200 diagnostic samples obtained from the united states. sensitivity was evaluated using 500 preselected htlv i and htlv ii positive samples. sensitivity and specificity samples were split across 3 reagent lots during testing. confirmation of repeatedly reactive samples was done using the mp diagnostic htlv blot 2.4. results/finding: imprecision was less than 7.0% for positive samples over 20 days. clinical sensitivity was 100.00% (500/500) on preselected htlv i and htlv ii positive samples. the specificity was 99.98% (7,999/8,001) on a blood donor population and 100.00% (200/200) on diagnostic samples. conclusion: these results indicate that the new alinity s automated htlv i/ ii assay provided very good performance in specificity, sensitivity, and precision. sensitivity and specificity were comparable to the comparator assay. claudia ramirez 1 , michel garcia* 2 , fernando palomino 3 and guillermo orjuela-falla 1 . 1 national blood bank colombian red cross, 2 universidad del rosario, 3 fuats background/case studies: current hepatitis c virus (hcv) supplemental testing algorithm for blood donations in colombia, requires that an immunoblot assay be performed on every hcv enzyme immunoassay (eia) repeatreactive sample. a higher proportion of indeterminate (ind) results by immunoblot assays has been documented for non-us donor samples, affecting donor counseling and eventually increasing costs and opportunity for the notification of infected donors. this work aimed to establish the distribution of immunoblot results in colombian repeat-reactive samples, as well as the frequency of band detection in both positive and indeterminate blots. study design/method: in total, 387 anti-hcv-reactive donor samples (signal-to-cutoff (s/co) ratio greater than 1.0; abbott architect i2000sr) underwent supplemental testing by immunoblot (either chiron riba hcv 3.0 sia or hcv blot 3.0 test, mp diagnostics). negative (neg), indeterminate (ind) and positive (pos) blot results were grouped by s/co ranges as follows: 1-4.99, 5-9.99, >10. band detection and intensity were independently analyzed for indeterminate and positive results. results/finding: immunoblot results were negative in 57.9% (224/387) of samples, indeterminate in 30.7% (119/387) and were positive in 11.4% (44/ 387). a direct relationship was observed between positive immunoblot and increased s/co. the proportion of ind results were higher in the s/co group 5-9.99 (63.2%) compared with the 1-4.99 (29.9%). in samples with indeterminate results, ns3_2 was the most frequent band detected (52,9%). in contrast, the most frequent band in the group of positive results was core (93,2%). only one sample from the indeterminate group (0.8%) had a strong band intensity (31), compared with 10 samples from the positive group (22.7%). conclusion: the proportion of indeterminate immunoblot results in this sample of colombian donors is one of the highest ever reported, being twice as much as the proportion found in larger samples of us donors. the high proportion of ind results found in the s/co group (5-9.99) suggests that the optimal s/co ratio for predicting a confirmed anti-hcv result in this population should be higher than the one recommended by the cdc for us population (>5). overall, these results suggest that the supplemental testing algorithm for blood donations in colombia could be improved not only by using high s/co ratios as an alternative to immunoblot, but also by introducing hcv genomic assays instead of immunoblots, at least for samples with intermediate s/co ratios. ns3_1 and ns3_2 cross-reactivity in colombian population warrants further investigation. performance of the alinity s immunoassay for the detection of syphilis antibodies melanie anderson* 1 , ivanka mihaljevic 2 , manuela miletic 2 , miljana stojic vidovic 2 , irena jukic 2 , jane bryant 1 , mark paradowski 1 , angela vockel 3 , george chen 1 , gregg williams 1 and george schlauder 4 . 1 abbott laboratories, 2 croatian institute of transfusion medicine, 3 abbott gmbh & co. kg, 4 abbott diagnostics background/case studies: blood donations are commonly screened for syphilis in order to detect the presence of antibodies to the bacterium treponema pallidum. in addition, continued pressures on laboratory operations demand that the full panel of ttid assays perform on a single platform capable of increased walk away time and enhanced automation in areas of reagent management, retest options, and commodity/waste management. in response to those needs, we have evaluated a new automated immunoassay for the detection of antibodies to t. pallidum. study design/method: performance of the new automated chemiluminescence immunoassay for the detection of antibodies to treponema pallidum was evaluated on the alinity s system. precision was assessed over 20 days using positive samples. specificity was evaluated on samples obtained from 9,101 blood and plasmapheresis donors from the united states and europe and 200 diagnostic samples obtained from the united states. sensitivity was evaluated using 514 preselected positive samples. sensitivity and specificity samples were split across 3 reagent lots during testing. confirmation of repeatedly reactive samples was done using a testing algorithm with 3 confirmatory assays, inno-lia tm syphilis score, and mikrogen recomline treponema igg and igm blots. results/finding: imprecision was less than 6.0% cv for positive samples over 20 days. clinical sensitivity was 100.00% (514/514) on preselected syphilis positive samples. the specificity was 99.97% (9,063/9,066) for blood donor specimens and 100.00% (200/200) on diagnostic samples. conclusion: these results indicate that the new automated alinity s syphilis assay provided good performance in precision, specificity and sensitivity in line with data found for the comparator assay. performance of the new automated alinity s assay for anti-hcv melanie anderson* 1 , ed bakker 2 , anton vanweert 2 , jane bryant 1 , mark paradowski 1 , tuan bui 1 , lynn martin 1 , george chen 1 , gregg williams 1 and george schlauder 3 . 1 abbott laboratories, 2 sanguin diagnostics, 3 background/case studies: serological screening for antibodies to hepatitis c virus (hcv) often in conjunction with nucleic acid testing (nat) is used worldwide to prevent transfusion transmitted hcv infections. while nat provides improved sensitivity and detection of hcv in the pre-seroconversion window, serological testing provides continued detection of hcv in infected individuals and individuals with resolved infections with no detectable hcv rna. blood and plasma centers require very high throughput anti-hcv assays with high specificity and sensitivity to prevent unnecessary donor deferrals while maintaining the safety of the blood and plasma supply. in addition, continued pressures on laboratory operations demand that assays perform on platforms capable of increased walk away time and enhanced automation in areas of reagent management, retest options, and commodity/waste management. study design/method: the performance of a new automated chemiluminescence immunoassay for the detection of antibodies to hcv was evaluated on the alinity s system. precision was assessed over 20 days evaluating positive samples. sensitivity was evaluated using 501 preselected positive samples and 30 seroconversion panels. specificity was evaluated on samples obtained from 8,113 blood and plasmapheresis donors from the united states and europe and 200 diagnostic samples obtained from the united states. sensitivity and specificity samples were split across 3 reagent lots during testing. confirmation of repeatedly reactive samples was done using a testing algorithm consisting of the inno-lia tm hcv score and nat/hcv discriminatory nat assays. results/finding: imprecision was less than 7.0% cv for positive samples over 20 days. overall clinical sensitivity was 100% on 501 preselected anti-hcv positive samples. seroconversion sensitivity was better than the comparator as evidenced by the new anti-hcv assay identifying 5 more bleeds than the comparator assay. the specificity was 99.99% (8,111/8,112) for blood donor specimens and 98. 98% (194/196) background/case studies: zika virus (zikv), which has been outbroken in south america and the united states since middle of 2015, was declared as the public health emergency of international concern by who in feb 2016. in addition to mosquito, zikv can be transmitted via maternalneonatal relationship, sexual intercourse or blood transfusion. the potential for transfusion-transmitted zika virus was shown in french polynesia where 2.8% of asymptomatic blood donors tested were positive for zika virus rna using nucleic acid test (nat). several case reports have confirmed that zikv can be transmitted by transfusion. it has been shown that among blood donors, 73.8% of the zikv infections were asymptomatic and the ratio of symptomatic to asymptomatic patients observed in micronesia was approximately 1:5 to 1:6. thus zikv has raised a great challenge to transfusion safety. measures should be taken to prevent transfusion-transmitted zikv, including temporary deferral of blood donors in epidemic locations, donor self-reporting of zikv symptoms after donation with or without quarantine of blood components, supply by blood collected from non-endemic areas to epidemic regions, nat of blood donations, and pathogen inactivation of blood products. in this study, we evaluated zikv inactivation in plasma by using methylene blue photochemical treatment (mbpt). study design/methods: plasma units from randomly selected healthy donors were collected and spiked with zikv. samples were added by mb at a final concentration of 1lm and assayed after illumination with visible light from both sides for 5, 15, and 30min. viral infectivity and zikv rna loads (reverse transcription pcr) were measured in spiked plasma before and after mbpt and confirmed using repetitive passages in cell culture. control was zikv spiked plasma without photochemical treatment. results/findings: zikv titer of control sample was 4.5 log 50% tissue culture infectious dose (tcid 50 )/ml. no viral infectivity was detected after mb photochemical inactivation treatment for 5min, 15min or 30min and the losses of the infectivity were further demonstrated by 3 repetitive passages of cell culture. meanwhile, zikv rna loads decreased significantly during the initial 5min of treatment whereby ct-value jumped from 18.25 (control) to 25.50 (mbpt for 5min) (table 1) . conclusion: it showed that mb photochemical treatment could effectively inactivate zikv in plasma. rna lesions were induced during mbpt process so that nucleic acid reverse transcription and amplification were inhibited. mbpt is proved to be an efficient method to prevent plasma transfusiontransmitted zikv infections. gilles delage* 1 , margaret fearon 2 , susan l stramer 3 , megan l nguyen 3 , france bernier 1 , sheila o'brien 2 , vito scalia 2 , sakina smith 3 , yves gr egoire 1 and boris hogema 4 . 1 h ema-qu ebec, 2 canadian blood services, 3 american red cross, 4 sanquin background/case studies: hepatitis e virus (hev) is known to be transfusion-transmissible. as part of the risk assessment for this infection, a study was carried out in 14,000 canadian blood donors in 2013. in a subset of 4,000 donor samples the seroprevalence was 5.9%. however, no donor samples were positive for hev by an in-house nucleic acid test (hev-nat). since that study suggested exposure to hev in canada but used an hev-nat with a limit of detection of 250 iu/ml, a larger study was performed using a more sensitive hev-nat assay. study design/method: donors were informed about the study in the predonation reading materials. linked samples from approximately 50,000 canadian whole blood donors including 30,000 from canadian blood services (cbs) and 20,000 from h ema-qu ebec (hq) were collected. clinics were selected to ensure representative sampling of the donor population. all 199a transfusion 2017 vol. 57 supplement s3 donations with available plasma samples were tested by individual donation nat at the american red cross laboratory in gaithersburg, md, using the cobas v r hev test (95% lod 18.6 iu/ml, 95% ci 15.9-25.6) for use on the cobas v r 6800/8800 system. this test is not currently approved in canada or the usa, but is available as a ce marked test. all nat-reactive donors are questioned concerning risk factors for recent hev infection (travel, animal contact, food and water exposure), undergo confirmatory testing (alternate nat, viral load, genotyping and igm/igg serology), are notified by letter, and deferred from donating for 6 months; in-date products collected from the donor, and any frozen red blood cells or plasma from the previous 6 months are destroyed. recipients will be traced in the event of any products transfused in the previous 6 months. results/finding: as of april 10, 2017, 9 of 39,834 (19,395 cbs, 20,439 hq) tested samples with valid results have been found hev-nat reactive: 8 donors have been confirmed by further testing to date. confirmation is pending in 1 donor. of the 9 donors, 7 were from quebec, and one each from nova scotia and alberta (7 male, 2 female). ages ranged from 21 to 70 years. only two donors reported non-specific symptoms (fatigue). in terms of risk factors: 6 ate pork (including 3 who ate pork liver), 4 ate shellfish, 2 ate venison, and 3 drank well water. one donor had no identifiable risk factor. viral loads ranged from 3 to 151 iu/ml, of which 2 were <10, 3 were 10-50, and 3 were >50 iu/ml; 2 were anti-hev igm positive and 4 anti-hev igg positive at index (wantai assay). conclusion: the prevalence rate of acute hev infection in this donor population appears to be around 1/4400. the data from this study will contribute to the ongoing risk assessment of transfusion-transmitted hev infection in canada. prevalence of malaria parasite in donated blood at nakasero blood bank, uganda gerald nsubuga* and musiisi ezra. uganda blood transfusion service background/case studies: introduction infectivity of donated blood with malaria is a significant health problem facing humanity. in uganda, screening for malaria parasite is neither routinely done in blood banks, nor stipulated in the current uganda national blood transfusion service (ubts) guidelines by the ministry of health. as a result, the proportion of donated blood that is infected with malaria is largely unknown. malaria infection places more than half of the world's population at risk and in majority of the tropical and sub-tropical regions of the world and about 300 to 500 million cases and 2 to 3 million death occur per year. however the study aimed at determining the prevalence of malaria parasites in donated blood at nakasero blood bank, kampala, uganda study design/method: a cross sectional study was carried out in nakasero blood bank, kampala, uganda in four hundred and seventy randomly selected donor samples at the blood bank between june and august 2014. both thin and thick glass stained blood smears of 417 blood samples with giemsa was examined using microscope. results/finding: of the 417 donated blood samples, 17 (4.1%) tested positive for malaria parasite (p. falciparum), although there was no significant difference in occurrence of plasmodium in relation to sex, age and blood group (p>0.05), majority of the blood donors that tested positive belonged to blood group o (64.71%). the prevalence of malaria parasite in the study was 4.1%. regardless of the prevalence, the presence of malaria parasite (plasmodium falciparum) in donated blood from donors that were presumed to be healthy raises a serious concern on the safety of donated blood in uganda. the ministry of health should review the existing guidelines for screening malaria and mandatory universal blood donor screening policy for malaria, for exclusion of blood donors with plasmodia parasitaemia. using methods like pathogen inactivation compared to tedious microscopic procedure to screen donated blood to be introduced to further enhance blood safety in our communities. components. the bact/alert virtuo* (virtuo) is an advanced, next generation system with improved automation, connectivity, and with data management systems. most importantly, the virtuo's new algorithm significantly reduces the time to detection (ttd) of microorganisms during quality control testing of platelet preparations using bact/alert bpa (aerobic) and bpn (anaerobic) bottles. bpa and bpn bottles were tested on virtuo and bact/ alert 3d (bta 3d) to evaluate repeatability to detect growth in seeded leukocyte reduced apheresis platelets (lrap) without platelet additive solution (pas), throughout platelet shelf life (3, 4 and 5 days after collection). study design/method: pooled lrap were seeded with low levels of 6 organisms commonly associated with platelet contamination at 3, 4 and 5 days post collection. the seeded lrap were inoculated into bpa and bpn bottles on 10 different days (not consecutive) alternating between 2 teams of 2 people each. seeded bottles were loaded into a virtuo and a bta 3d and incubated until declared positive or negative (up to 7 days). additionally, bpa and bpn bottles inoculated with 4 ml of unseeded lrap were tested on the virtuo and the bta 3d (120 and 40 bottles respectively), to serve as negative controls, sterility controls, and to evaluate the risk of false positives caused by lrap results/finding: the repeatability of the virtuo to detect organisms in lrap was demonstrated by a recovery rate of seeded bottles of 99.9% for the virtuo and 99.5% for the bta 3d. the virtuo demonstrated an average improved ttd of 3.2 hours, when compared to the bta 3d in the presence of 4 ml lrap platelets. the lrap did not cause false positives. additionally, the age of the lrap units (within 5 day expiry),did not impact the ttd when seeded with organism background/case studies: zika virus (zikv) is an emerging flavivirus that is transmitted by the aedes aegypti mosquito and sometimes a. albopictus mosquito. most infections are asymptomatic. zikv nucleic acid testing (nat) became a required test for blood donors per the fda guidance entitled, "revised recommendations for reducing the risk of zika virus transmission by blood and blood components". based on our geographical location, implementation of this testing began 12 weeks after this guidance was issued. we performed zikv nat for donors of whole blood and blood components under an investigational new drug (ind) study (sponsored by hologic, inc.). we performed a retrospective analysis on all nat results as there is a potential to defer donation due to false positive screening results. study design/method: donors that consented to donate blood and be tested for the zikv were obtained from three blood banks in colorado and nebraska. nat was performed using the procleix virus assay which is a qualitative in vitro nucleic acid assay system that detects zikv rna in plasma specimens. the assay was performed on the automated procleix panther system. all testing was performed according to the manufacturer package insert. results/findings: in the event of a reactive result, donors would be retested by nat in addition to other testing (igm antibody testing, neutralization test). donors are deferred for 120 days barring continued zikv testing and nonreactive results. a total of 2,485 donors were screened for zikv. all donors screened for zikv were nonreactive by nat. no invalid test results were obtained. in addition the number of failed test runs due to instrument or assay issues were experienced were quite low (1.0%). this data indicates that both the assay and instrument are robust. there was a low frequency for additional testing which allows the laboratory to publish timely infectious disease results for our blood bank customers. conclusion: the reactive rate data presented here demonstrate that there is a low/zero incident rate in our region for whole blood and blood component discard due to reactive results. this screening is important to continue to ensure blood safety in the united states. robust inactivation of the yellow fever virus 17d strain can be achieved using amotosalen and uva light for pathogen reduction treatment (prt) of platelet components andrew laughhunn 1 , felicia santa maria 1 , yvette girard 1 , peter bringmann 1 , marion lanteri* 2 and adonis stassinopoulos 2 . 1 microbiology department, cerus corporation, 2 scientific affairs department, cerus corporation background/case studies: yellow fever virus (yfv) is known to cause explosive outbreaks, such as the one in angola in 2015. the rapidly increasing number of infections in brazil, with hundreds of fatalities since december 2016, is of concern. yfv is a flavivirus transmitted by aedes mosquitoes and could spread, like zika virus, to other parts of the americas where the vector is endemic. with no effective antivirals and only supportive therapy available, the best mitigation strategy is through vaccination with live attenuated vaccine strains, like the 17d-yfv strain. yfv vaccine is considered an effective and safe vaccine; however major adverse events have been reported including neurologic and visceral adverse effects. in addition, transfusion transmission (tt) of live attenuated yfv has been reported with severe clinical outcomes, especially in immunosuppressed patients. in order to prevent tt by yfv vaccine strain, the aabb recommends a 2 weekperiod deferral after yfv vaccination. yfv outbreaks and vaccination campaigns may therefore reduce blood availability. this pilot study evaluated the ability to inactivate 17d-yfv using amotosalen (s-59) and uva light prt of platelet components (pc). study design/method: pc in 65%pas (n53) or 100% plasma (n51) were spiked with high titers of 17d-yfv and treated with s-59/uva prt. samples were taken pre-and post-uva illumination and infectious titers were determined, by plaque assay using vero76 cells. the extent of inactivation was quantified by comparing titers before and after inactivation. results/finding: pre-prt infectious titers were 4.71 6 0.7 log 10 pfu/ml for pc in 65% plasma and 5.19 log 10 pfu/ml for pc in 100% plasma while titers in post-prt samples were <-0.7 6 0.0 log 10 pfu/ml for pc in 65% plasma and <-0.7 log 10 pfu/ml for pc in 100% plasma. inactivation to the limit of detection of >5.41 6 0.7 log 10 or inactivation of >4.71 6 0.7 log 10 pfu/ml was achieved for pc in 65% plasma. inactivation to the limit of background/case studies: the use of biotin as a supplement has increased in recent years and many health care professionals may not be aware of the high dosage intake by their patients. this high dosage has resulted in an increased prevalence of individuals being exposed to biotin levels much greater than the recommended daily dose and as a consequence, has led to inaccurate lab results for assays that utilize the free capture biotin-streptavidin methodology. although abbott's alinity s assays do not utilize this free capture biotin-streptavidin methodology, eight assays developed for blood screening on the alinity s system were evaluated for biotin interference to ensure there are no unknown consequences of high biotin levels. study design/methods: the purpose of this study was to determine if the eight developed abbott alinity s assays would be susceptible to biotin interference by evaluating their performance in the presence of a high concentration of biotin. for each of the alinity s assays evaluated (hiv ag/ab combo, htlv i/ii, anti-hcv, chagas, hbsag, anti-hbc, syphilis, and cmv igg), samples spiked with a concentration of biotin at approximately 1000 ng/ml were tested against a control (unspiked) sample preparation to determine if there was a difference between the control and biotin containing samples. two samples, one negative and one positive, were tested with all assays, except the hiv and htlv assays, which each tested two positive samples (1 hiv-1 antibody and 1 hiv-1 p24 antigen, and 1 htlv-i antibody and 1 htlv-ii antibody, respectively). results/findings: for the negative samples, the sample to cutoff (s/co) differences between the biotin spiked and control were 0.00 for hcv, hbc, syphilis, cmv igg, and chagas, 0.01 for hiv ag/ab and htlv i/ii, and 0.03 for hbsag. for the positive samples, the mean s/co % differences between the biotin spiked and control were 0.00 % (antibody sample) and 0.36% (antigen sample) for hiv ag/ab combo; 0.90% (htlv i antibody sample) and 0.32% (htlv ii antibody sample); -1.43% for anti-hcv, -2.52% for chagas, -0.71% for hbsag, -0.37% for anti-hbc, -1.62% for syphilis, and -0.59% for cmv igg. conclusion: eight abbott alinity s assays were evaluated to determine if they were susceptible to biotin interference. these results indicate that the eight alinity s assays do not show susceptibility to biotin interference at an approximate concentration of 1000 ng/ml. robustness of the abbott prism methods to biotin interference c fischer 1 , r schneider 2 , w leonard 2 , m cobb 3 , g schlauder 3 , g williams 3 , m zuske 2 m janulis* 2 . 1 transfusion medicine, abbott diagnostics, wiesbaden, germany, 2 add diagnostics, 3 transfusion medicine, abbott laboratories, chicago, united states background/case studies: the use of biotin as a dietary supplement has increased significantly in recent years and many health care professionals do not realize their patients are taking high doses. the increase has resulted in an increased prevalence of people being exposed to biotin levels much higher than the recommended daily dose and as a consequence, potentially inaccurate lab results for assays that utilize the free capture biotin-streptavidin methodology. the purpose of this study was to identify any abbott prism assays that may be susceptible to biotin interference based on assay design and then evaluate the performance of those assays with high concentrations of biotin. after a comprehensive review of abbott's current on market prism assays, no assays were identified that utilize biotin-streptavidin capture; however, 3 assays were identified for subsequent testing as they contain biotin in their assay design. background/case studies: bacterial contamination of platelets is the highest residual infectious risk in transfusion despite the current preventive strategies. while bacterial contamination may affect any blood component, the ambient storage temperature conditions for platelets make them most likely to facilitate bacterial growth. based on all the precautionary measures, the final platelet concentrates include in the worst cases a very limited viable bacteria number estimated from 10 to 100 colony forming units (cfus)/bag (i.e. 0.03 to 0.3 cfu/ml). one major difference between viruses and bacteria is that bacteria have the ability to grow up to a concentration of 10 8 -10 9 cfu/ml over the 5 days product shelf-life. moreover, a large diversity of strains is found in contaminated platelets representing a key challenge for the development of a generic bacterial test. the aim of this study was to develop an economic and easy diagnostic approach for the early, rapid, sensitive and generic detection of bacteria in platelet concentrates. the adaptability of the process with the blood transfusion services requirements was of major concern. hence, attention was focused on an easy to automate technique able to deliver results on day 2 after collection. study design/method: a large panel of bacteria involved in transfusion reactions including clinical isolates and reference strains was established and used for mouse immunizations, antibody screening and platelet spiking steps. an original approach was used to produce and select monoclonal antibodies directed against bacteria to develop our generic immunoassay. as recommended, 24 hours (day 1) after collection a sampling volume of spiked platelets (0.1-1 cfu/ml) was tested after a short generic culture, lysis and capture of bacteria on magnetic microparticles in a microplate format. an immunoassay was performed for the detection of the captured bacteria. results/finding: this approach was tested on a panel of 25 bacterial strains involved in transfusion reactions. the pre-analytical steps and the capture of bacteria on microparticles were improved to avoid false negative results and to enhance the sensitivity of detection. the full test developed in this study combining a pre-analytical culture step followed by an there are many stakeholders are involved in hcv eradication program, including government authority such as centers for disease control and prevention, national health insurance and health promotion administration, and private property like hospitals, medical societies, pharmaceutical and vaccine industries, npos and academia. results/finding: tbsf is a private nationwide single blood services program in taiwan, and performs anti-hcv screening test and nat confirmatory test on every collected blood, which is a large-scale population screening of hcv in taiwan because of its high blood donation rate (7.5%). tbsf confirmed positive test result of repeated blood donors, and can identify hcv rna seroconversion cases as recently-infected hepatitis patients. those infected patients would be referred to physician for further medical care and deferred permanently by tbsf to secure blood safety. by interviewing the newly-infected cases, the risk factors of hcv patients can be studied and then help identifying and eliminating sources of hcv infection. tbsf also contribute to health education by teaching our donors being aware of potential risks of hcv infection and keep monitoring every parameters of hcv epidemiology to evaluate the efficacy of hcv eradication program. conclusion: in hcv eradication program, tbsf can not only secure blood safety but also participate in health education, disease screening, etiology finding and prevention, surveillance and evaluation. thus, among all stakeholders, tbsf is particularly important and can play a pivotal role in eradicating hcv by 2030 in taiwan. the theraflex uv-platelets technology efficiently inactivates transfusion-relevant bacteria species in contaminated platelet concentrates ute gravemann 1 , frank tolksdorf 2 , wiebke handke 1 , thomas h. m€ uller 1 and axel seltsam* 1 . 1 german red cross blood service nstob, 2 maco pharma international, gmbh background/case studies: the theraflex uv-platelets system (macopharma) is a uvc-based pathogen inactivation system for platelet concentrates (pcs). inactivation efficiency has been shown for a broad range of viruses, bacteria, and protozoans. previous studies with the first set of bacteria species of the who international repository of platelet transfusion relevant bacterial reference strains revealed a high inactivation capacity for clinically relevant bacteria. aim of the current study was to investigate the bacteria inactivation efficacy of the theraflex uv-platelets system for enterobacter cloacae, pseudomonas fluorescens, staphylococcus aureus and streptococcus bovis which have recently been added to the who international repository. study design/method: pcs were produced from 5 buffy coats using the additive solution ssp1 (macopharma) with a residual plasma content of 35%. for inactivation kinetics, pcs (n53) were spiked with bacteria to a final concentration of approx.10 6 colony forming units (cfu)/ml and irradiated with increasing doses until the full uvc dose was achieved. samples were taken for the bacterial titer determination after each irradiation step. for sterilization studies, two pcs were pooled and inoculated with bacteria to a final concentration of approximately 0.3 cfu/ml. bacteria were allowed to grow for 6 h in the pcs at 22 6 28c under agitation. after splitting, one pc remained untreated (growth control) while the other one was uvc-treated. after storage for seven days, samples were taken from both bags for sterility testing by bactalert (biomerieux) and for determination of the bacterial titer in the untreated control units. results/finding: bacteria in pcs were inactivated in a dose-dependent manner by treatment using the theraflex uv-platelets system. mean log 10 reduction factors ranged from 6 to 7 for enterobacter cloacae (6.3 6 0.6, pei-b-p-43), pseudomonas fluorescens (7.1 6 0.4, pei-b-p-77), staphylococcus aureus (6.6 6 0.4, pei-b-p-63), and streptococcus bovis (7.0 6 0.3, pei-b-p-61). pcs (n512 for each species) spiked with these different bacteria species were efficiently sterilized (12 out of 12). treated pcs remained sterile during storage for 7 days, while bacteria in non-treated pcs grew to high titers of 10 6 -10 8 cfu/ml. the theraflex uv-platelets system efficiently inactivates a broad range of different bacteria species, including the who reference strains. sterility is maintained over a storage period of 7 days. these results suggest that the uvc-based pathogen inactivation technology will significantly improve the bacterial safety of platelet transfusions. transfusion transmissible infections among blood donors and strategy on direct laboratory testing cost of blood screening at national blood bank center, addis ababa, ethiopia abraham zewoldie*. national blood bank service background/case studies: blood and its components are life saving; however, they are also associated with life threatening hazards such as transfusion transmitted infections (ttis). hepatitis b virus (hbv), hepatitis c virus (hcv), human immunodeficiency virus (hiv) and syphilis are the most serious infections transmitted during blood transfusion. serious of blood shortages especially in developing countries and reliance on unsafe family replacement or paid donors also contribute to an increased risk of ttis. knowing the current prevalence of ttis among blood donors will be crucial in donor program strategy development and cost effective alternative strategies of blood screening are highly required especially in resource limited setup. study design/method: a retrospective analysis of blood donors' record covering the period from july 1, 2008 to july 30, 2013 was conducted. the data was collected from the nation al blood bank (nbb) center donor data base. in addition, direct laboratory costs of parallel versus sequential strategy of blood screening were compared using the current price of the laboratory costs. data was first exported to spss version 16 software for analysis. data analysis was performed using scores and odds ratio using same software to look for an association between dependent and independent variables. p values less than 0.05 were considered significant. results/finding: a total of 173, 207 consecutive blood donors were screened between 2008 and 2013. the overall seroprevalence rate of hbv, hiv, hcv and syphilis of blood donors was 5.0%, 1.6%, 1.4% and 0.1% respectively. the hiv-hbv co-infection was higher among blood donors 135(41.79%) followed by hbv-hcv co-infection whish accounts about 103(31.89%). significantly increased sero-prevalence of ttis was observed in among family replacement donors, factory workers, daily labors and the age group of 26-35. in this study the difference in cost between the current in use strategy (parallel) versus the newly proposed designed sequential testing algorism was 746,773.9 ethiopian birr. conclusion: a significant percentage of the blood donors harbor ttis. the nbb center should work on voluntary blood donor mobilization and develop culture of voluntarism. the direct laboratory cost analysis using current in use strategy (parallel) was higher than the newly designed sequential testing algorithm. thus, the new strategy can be implemented to make screening of ttis cost effective in nbb center. transfusion transmitted malaria in a 14 month old infant patricia davenport* 1 , geeta paranjape 1 and laurie sutor 1,2 . 1 carter bloodcare, 2 ut southwestern medical center background/case studies: in 2017 at a large pediatric hospital, a 14 month old infant was supported for 31 days by extracorporeal membrane oxygenation (ecmo). over this time 113 blood products were transfused. about 10 days after end of ecmo support, a routine blood smear examination revealed inclusions in some of the patient's red cells. the patient had also been having intermittent fever. malaria was confirmed by pcr as plasmodium ovale (p. ovale). because the patient had no other risks, the infection was suspected to be transfusion related and was reported to our blood center which had supplied all transfused products. study design/method: the investigation began by focusing on donors of red cell products, since the chance of an apheresis platelet product transmitting malaria is relatively small, and that of a frozen product is remote. we identified 27 donors of red cell products. each donor was contacted and was asked four questions. additional questions were asked for clarification if needed. based on donor response, risk for active malaria infection was assessed. we also considered areas where p. ovale is, or is not found. donors identified as having possible risk were tested for antibodies and parasitic dna. results/finding: the five donors who had been ill all had common cold or bronchitis like symptoms. donors who traveled went only to non-risk areas. three donors were former residents of another country and may have risk because they lived in malaria endemic countries since birth and came to the u.s. as adults. it was discovered that one of these three did not meet all donor criteria. the donor had failed to disclose that he had not completed 3 years stay in the u.s. after emigrating from cameroon, an area endemic for p. ovale. he had not travelled anywhere after coming to the united states in october 2014 and answered "no" to travel. antibody tests on this donor were positive for p. ovale and p. falciparum, but pcr tests were negative. another possible at-risk donor, a former resident of iran was tested and was pcr and antibody negative. the third donor has not yet been tested but the country of residence does not have p. ovale malaria. conclusion: while it could not be definitively proven that the donor with antibodies to p. ovale had active malaria at the time of donation, the donor was indefinitely deferred and referred to an outside physician for treatment. transfusion-transmitted babesiosis outside an endemic area: a case report german felix leparc*. oneblood background/case studies: an 81 y.o. male patient was admitted to the emergency room for severe acute gastrointestinal bleeding, caused by an arterio-venous malformation later located in the proximal jejunum that was clipped endoscopically. during this admission, he received a total of 13 units of red blood cells. approximately 4 weeks later, he was re-admitted due to another episode of gi bleed manifested by melena. as part of his routine evaluation, a cbc was performed in which a blood smear revealed the presence of intraerythrocytic parasites consistent with babesia sp. study design/method: upon notification of a suspected case of transfusion-transmitted babesiosis, lookback of all donors involved in prior transfusion event was initiated. results/finding: to confirm the presumptive diagnosis of babesiosis, pcr was performed and babesia microti dna was detected. an evaluation of the patient's risk factors revealed that prior to the gi bleed episode for which he received transfusions, eight months earlier he was also transfused during open heart surgery. no travel history to the us midwest, and while he travelled to new england two years ago he did not spend time outdoors. he was splenectomized in his mid 20's. donor lookback identified a donor who lived in new london county, connecticut but spent the winter season in central florida, where the blood donation (double rbc collected by apheresis) took place. he had never been diagnosed with babesiosis, but participated regularly in outdoor activities in connecticut that put him at risk for tick bites (although he never noticed being bitten or showing signs of it). upon testing, he was found to be negative for b. microti on pcr as well as igm antibodies, but had igg antibody titers of 1:256. the recipient of the other rbc unit collected in the same donation was deceased within hours of transfusion, so no follow up could be performed. during phone interviews, none of the remaining donors had risk factors for babesiosis, and all but four were tested and found serologically negative. conclusion: while transmission of babesiosis through the zoonotic route is confined to regions were the appropriate hosts and vector coexist, people from areas where it is endemic may establish temporary residency and donate blood in non-endemic locations facilitating transmission through transfusion as illustrated in this case. once licensed assays for babesia microti become available, testing schemes will have to be formulated through policies that take this issue into consideration. transfusion-transmitted stenotrophomonas maltophilia from a red cell unit: a case report ashley c gamayo* 1 , andrea j linscott 2 and donny dumani 3 . background/case studies: transfusion-transmitted bacterial infections (ttbi) are rare, but serious complications of blood product transfusions. from 2011-2015, 8% of 173 transfusion-associated fatalities reported to the fda were attributed to bacterial contamination. red cell units are rarely implicated in severe and fatal ttbi. when present, contaminants are often gram-negative rod (gnr) bacteria with psychrophilic properties. we present a case of a sickle cell patient who developed definitive sepsis after receiving a red cell unit contaminated with stenotrophomonas maltophilia (s. maltophilia). study design/methods: a 27-year-old female with sickle cell disease was admitted to the hospital for possible pain crises. pre-transfusion blood and urine cultures collected on day 1 of hospitalization showed no growth after five days. on day 3, the patient required a blood transfusion for which she was issued a cmv-safe, irradiated, hbs-negative, crossmatched, o-negative red cell unit. the 318 ml unit had been aliquoted via sterile connecting device 12 days prior for a pediatric patient. all 26 ml of the pediatric aliquot were transfused without adverse effects. the patient's pretransfusion temperature was 37.18c. within 45 minutes of starting the transfusion, the patient's temperature increased to 39.38c and subsequently reached a maximum of 39.58c. the transfusion was stopped and the blood bank notified immediately. gram stain of the remainder of the transfused component revealed gnr bacteria. blood was collected from the patient for culture and antibiotic treatment initiated. results/findings: initial transfusion reaction work-up revealed no evidence of clerical errors with negative post-transfusion antibody screen and direct antiglobulin test. blood cultures from both the patient post-transfusion and the implicated red cell unit grew gnr bacteria identified as s. maltophilia. further microbial testing revealed the cultured pathogen was able to proliferate at 4-68c; a finding not characteristically observed in s. maltophilia. conclusion: this is the first definitive case of ttbi with s. maltophilia. this bacterium is a globally emerging gnr that is widely spread in the environment, causing both community-acquired and nosocomial infections in immunocompromised and debilitated patients. contamination was unlikely due to an asymptomatic donor. there was laboratory evidence of the pathogen in both the transfusion recipient and the transfused component. the patient was not infected with the pathogen prior to transfusion, and no other potential exposures could be identified. the patient recovered following appropriate antibiotic treatment, but endured prolonged hospitalization. the transfusion reaction was classified as definitive, severe tti of definite imputability. validation of commercial immunoassays for detecting hbsag and hiv antibodies in production pools karen leighton, izekial butler and scott jones*. qualtex laboratories background/case studies: plasma fractionators test plasma production pools for hbsag and hiv antibodies as a qualitative limit test for the control of impurities, to safeguard against errors in donation testing or pooling. the european medicines agency (ema) has published guidelines for the validation of immunoassays for the detection of hbsag and hiv antibodies in production pools. the aim was to validate commercial immunoassays for the testing of production pools for hbsag and hiv antibodies utilizing the ema guidelines. study design/method: a lower calculated cutoff value for the abbott prism hbsag and hiv o plus assays was determined by calculating the mean signal-to-cutoff ratio (s/co) plus 3 standard deviations of four different types of plasma production pool samples. the calculated cutoff values were utilized for the rest of the validation. the detection limit was determined by testing in triplicate, serial dilutions of who hbsag and hiv antibody standards diluted in plasma. a normalized detection limit was calculated for the hbsag assay using production pools containing low, typical and high anti-hbsag titers. intra-assay variability was determined by testing a minimum of 6 determinations of a low positive control in 1 run. inter-assay variability was determined by testing at least 3 representative negative production pool samples, at least 1 low positive sample (about 3 s/co) and a titration series of who standard spiked into plasma production samples. runs were performed on six separate days using two different instruments and two different lots of assay reagents. results/finding: the lower calculated cutoff values for the hbsag and anti-hiv assays were both below the manufacturer cutoffs of 1.00 and were 0.72 and 0.48 respectively. the hbsag assay detection limit was 0.065 iu/ ml for source plasma and 0.120 iu/ml for recovered plasma samples. the normalized detection limit study demonstrated that one and a half hours was the maximum amount of time the pool samples could sit at 15-25 0 c where all samples were still reactive for hbsag. the anti-hiv lowest positive dilution for all replicates varied between 1:10,000 to 1: 1,250,000 depending on subtype and group. the % cv of the s/co values of the replicates of the intra-assay variability validation were less than 5% for both assays. the %cv of the s/co values of the panel of samples of the inter-assay variability validation were less than 14%. conclusion: a lower calculated cutoff value could be determined for commercially available immunoassays for hbsag and anti-hiv. these immunoassays could meet all of the recommendations in ema validation guidelines. the abbott prism hbsag and hiv o plus assays can be utilized to test production pool samples. was performed on 6 donors (3-17 days after the index donation) -3 donors in the follow up study and 3 tested by the doh. no donors tested by the doh participated in the follow up study. follow up testing was negative for all 6 donors. denv antibodies were negative in 9 donations and equivocal in 1. our initial reactive rate is higher than that reported to date for the procleix zikv tma of 1 per 23, 342 [p. williamson, et al transfusion, in press] . conclusion: universal testing under ind was successfully implemented and incorporated into blood center operations. we have noted an initial reactive other demographics that should be analyzed for their potential to be used to predict cmv seroconversion rate include gender, age, race, ethnicity or a combination of these. background/case studies : growing the geographic footprint has been a priority for the organization since 2014. over a four year period, the organization doubled the number of blood centers, with continued growth expected. with the current challenges in the blood industry, the audit program needed to be flexible, maximizing efficiency and capacity utilization, and without increasing compliance risk. the internal audit function was centralized in late 2011, for which the program consisted of 2 types of audits, an operational compliance audit and a support systems compliance audit. each type was performed twice per year at each main center. this model was no longer serving the changing organization. study design/methods: lean six sigma concepts were applied to this project. survey results and brainstorming aided in capturing the strengths of the current program, opportunities for improvement, and ideas for a redesigned program. this information was the primary input to the swot analysis (strengths, weaknesses, opportunities, and threats) for the purpose of understanding performance of the current program, as well as elements that could impact the future design. potential solutions were placed into a pugh matrix, which was used to facilitate a disciplined, team-based process for concept generation and selection. each potential solution was compared to criteria for evaluation and selection of the best solution. results/findings: the program was re-designed to perform internal audits annually as a single, team-based comprehensive audit. remote auditing was incorporated to require less on-site time, less disruption, improved auditor work/life balance, and cost savings. a formula was created to determine on-site audit time that included adjustable risk factors. the audit reporting process was also automated for simplification, efficiency, and to meet stakeholder needs. the team-based approach leverages auditor strengths, fosters a learning environment, and increases detectability of organization-wide concerns. conclusion: the comprehensive team-based approach, and other program improvements, has been effective in responding to organizational growth without sacrificing quality or increasing compliance risk. external inspection performance has achieved record performance levels the past year. diversity of auditor skills led to a stronger skill presence, which was consistently applied across system. auditing is more efficient and effective. stronger collaboration among audit team members provided stronger objectivity, fairness, and consistency across the system. auditors and auditees have increased in knowledge, and the internal quality audit program has improved. background/case studies: in many places, blood banking is using semiautomated systems to perform fractioning in different blood components (red blood cells, platelets and plasma). banc de sang i teixits (bst), adopted the fully automated reveos system (terumo bct inc, lakewood, co) few years ago to manufacture blood components. in june 2016, bst started a validation of new blood bags manufactured by terumo bct with different variables on platelet volume after processing and a kit to perform platelets pools with a new filter. study design/method: to perform this validation, 300 blood donations were used under different conditions (see table below ). the current filter evaluated for the platelet pool (lrf-xl, haemonetics corporation) was compared to a new filter (terumo bct inc.). the new blood bags were manufactured using a new vinyl supplier. a portion of these processed blood components (red blood cells, platelets and plasma) was used for different quality control (qc) tests (routine qc performed at bst following european directorate for quality of medicines & healthcare; cytokine analysis, such as p-selectin and platelets recovery through the filter). results/finding: the results are very similar between both bags, current and new one, as well as filters. all the analysis done to evaluate the quality of the blood components were similar in all conditions. also, it was shown a better performance on platelets pools, when they came from bags centrifuged with 60 ml of plasma, vs. 30 ml of plasma and additive solution. conclusion: these new bags and filter have shown a similar behavior when using them for manufacturing blood donations with reveos system in our blood bank. regarding the new platelets pooling kits, a better manipulation by the operator was observed; although the tubing is shorter and it meant being more difficult when manipulating the pools. no issues should be found if they are implemented in routine use. it's planned to start this implementation during this year, 2017; so then there will be larger results in order to have a proper procedure qualification. conclusion: patients requiring rare blood products are rare, and those lacking high prevalence antigens are the most challenging for whom to obtain antigen negative blood. it is clear that some requests for exquisitely rare types are not able to be filled with current donors. molecular testing of large numbers of donors has likely helped to identify more rare donors in recent years. it is recognized that commercial platforms do not include many of these making these rare types even more challenging to find. consideration should be given to testing more donors of all ethnicities to identify more rare donors. recommendation #1: updating donor educational material to provide more comprehensive information on risks of iron deficiency and recommendations on iron supplementation. updating our educational materials will likely have a minor impact. recommendation #2: implementing strategies such as iron supplementation, ferritin testing or increasing interdonation intervals for all donors or those groups most at risk for iron deficiency. initial implementation would likely be either iron supplementation or ferritin testing for at risk groups only and implementation of either one of these strategies would potentially affect over 120, 000 donors. the recommendation to limit the number of donations would have a substantial impact. for this analysis, the focus was on 16-18 year olds and premenopausal women (ages 19-55) donors. on average, 16-18 year olds donate 1.3 times a year and premenopausal women donate 1.49 times a year. if both of these groups were limited to donating once a year, a total of 4,845 donations from 16-18 year olds and 9,272 donations from premenopausal donors would not be collected. conclusion: after analyzing the impact of the aabb association bulletin #17-02, the bulletin will have a significant impact on both donors and our local blood supply. more than half of donors would receive either ferritin testing or iron supplementation. if the only measure employed is limiting the number of times a donor could donate for 16-18 year olds and premenopausal women, this recommendation would have a substantial impact on our ability to provide blood products to local hospitals. background/case studies: transfusion medicine knowledge deficits are apparent among medical students, residents and practicing physicians. these deficiencies may be due to the frequency and type of education. the majority of medical students in the united states receive four or fewer hours of transfusion medicine education. the transfusion medicine academic award group published educational content guidelines for medical school, residency and fellowships. however, the frequency and educational methods remain poorly evaluated and with little guidance. we investigated the effects of different educational techniques on transfusion medicine knowledge acquisition in novice learners. study design/method: three educational pathways were developed to teach principles of transfusion medicine while allowing learners to recognize problems and develop solutions for transfusion medicine complications. the simulation group received all educational activities within a 2.5 hour inperson, high-fidelity live session. the hybrid group received some educational component online and also attended an in-person high-fidelity simulation session. the online only group received all educational materials online, including a pre-recorded-video simulation session. the learners were second year medical students enrolled at one institution. the same faculty members taught all live sessions and developed all online materials ensuring the content was the same. a pre-and post-test was created to address blood groups, blood donation, blood testing, blood component indications and transfusion complications. the educational session was evaluated by the likert scale survey which ranges from zero (poor/unsatisfactory) to five (outstanding). results/finding: 97% (101/104) of the simulation group students improved their post-test scores and had an average likert scale rating of 4.1 (very good). 89% (63/71) of hybrid group students improved their post-test scores and had an average likert scale rating of 4.2 (very good). 89% (90/101) of online only students improved their post-test scores and had an average likert scale rating of 3.0 (good). the average changes in scores were statistically significant within all training groups (p value < 0.0001). additionally, the simulation group had a larger increase in average post-test scores when compared to the online only group (p<0.0001) and the hybrid group (p<0.0001). conclusion: our study demonstrated that a faculty taught high-fidelity transfusion medicine simulation curriculum consisting of an in-person didactic session and simulation session for second year medical students produces greater knowledge acquisition compared to an online only or hybrid curriculum. the high-fidelity simulation curriculum is also preferred over the online only education as indicated by the likert survey results. aaron j wyble*, yeon mi kim and barbara j bryant. university of texas medical branch background/case studies: diagnostic management teams (dmts) are an innovative way to bridge the communication gap between the laboratory and clinical services thereby facilitating the delivery of improved patient care. dmts employ a multidisciplinary approach which integrates clinical and laboratory data into succinct interpretations and recommendations. the interpretations must be of moderate to high complexity in order to be clinically valuable. recommendations are made regarding future testing, timing of testing prior to blood component needs, and other pertinent concerns to allow for improved coordination of patient care. the timeliness of the dmt reporting is vital to patient management. the inherent design of a dmt also provides an educational opportunity for trainees at academic centers. study design/method: the transfusion medicine service at a large university-based academic medical center implemented a dmt in 2016. all cases involving complex antibody identification workups, transfusion reactions, deviations from standard operating procedures, consultations for blood component utilization, and massive transfusion protocols from july 2016 through january 2017 were evaluated by transfusion medicine residents. the electronic medical record (emr) of each patient was also reviewed to determine relevant clinical history. all significant findings were presented at the transfusion medicine dmt conferences. the dmt was comprised of physicians from transfusion medicine, hematology/oncology, anesthesiology, transfusion service technical staff as well as visiting clinical staff from surgery, obstetrics and gynecology, transplant services, and pediatrics. the dmt integrated the clinical and laboratory data to formulate relevant interpretations and recommendations. the final dmt reports were placed into the emr for access by health care providers. financial benefits of a transfusion medicine dmt were also evaluated. results/finding: in a 7-month period, 504 cases of complex antibody identification workups (65%), transfusion reactions (2%), consultations for blood component utilization (6%), and deviations from standard operating procedures and massive transfusion protocols (27%) were presented at the transfusion medicine dmt conferences. the placement of dmt narratives in the emr as progress notes and laboratory reports provided informative and timely communications. residents participating in dmts demonstrated improved clinical and laboratory correlation skills. as a result, resident competency in transfusion medicine was enhanced. over $68,000 of revenue was generated utilizing the standard professional component cpt codes. conclusion: dmts encompassing multiple aspects of transfusion medicine improved patient care through enhanced communication between laboratory and clinical services. additional benefits of a dmt program include resident, clinician, and technical staff education and the generation of revenue for the institution. streamlining a blood center and hospital transfusion service supply-chain with an informatics vendor-managed inventory solution hamilton c. tsang* 1 , david lancaster 2 , dianne geary 2 , robert scott 1 , anh thu nguyen 1 , adam garcia 2 , raina shankar 1 , leslie buchanan 1 and tho pham 2 . 1 stanford health care, 2 stanford blood center background/case studies: inventory management is both a major challenge and an integral part of hospital transfusion service (hts) and blood centers (bc) operations. the current process at our institution involves twice-per-day shipments from the bc to the hts, with each shipment predicated upon current stock levels at hts. manually obtaining inventory levels for each product is time-consuming. the manual determination is also errorprone. we aim to enhance inventory management operations by developing an informatics solution to (1) streamline the ordering process to accurately reflect inventory status and transfusion practices and (2) re-allocate valuable hts tech time. study design/method: at our hts, the general inventory accounts for over 50 product categories broken down by component, blood type, irradiated status, and cmv-serology status. we therefore sought to establish an electronic method to reliably infer the general inventory level. since the raw electronic inventory report comprised both the general inventory and physically sequestered units (e.g. special antigen units, cross-matched units), over a 5-month calibration period we performed linear regression between electronic and the gold-standard manual count to impute from the electronic census the number of units of each product category in the general inventory. once we had a reliable electronic method to determine inventory levels, we implemented a 3-month pilot period. we analyzed various metrics pre and post pilot implementation to ensure non-inferiority of our electronic system: (1) the ratio of units transfused per week to the number stocked (t:s), (2) the number of products ordered as stat, and (3) the number of expired products. we created in-house programs on visual basic for applications (microsoft, redmond, wa) for both the calibration and pilot periods. 2486 lines of code were written for both programs, including 2 class modules and 34 distinct subroutines. results/finding: during the pilot period, we investigated our system's noninferiority. the average weekly t:s ratio for cryoprecipitate, plasma, and rbc, respectively, were 1.03, 1.21, and 1.48 before the pilot period compared with 0.88, 1.17, and 1.40 during the pilot period. these differences did not reach statistical significance (p50.28). we also monitored the number of stat ordered products before and during the pilot period, which were 27 and 31 stat units per week, respectively (p50.86). lastly, we also monitored the number of monthly wasted products due to expiration as an indicator of inventory mismanagement before and during the pilot period, which were 226 and 196 units, respectively (p 5 0.28). an estimated 7 hours per week of technologist time was reallocated to other tasks once the electronic census was adopted. this translates to 0.175 fte and $18,200 per year saved from labor costs per year if permanently adopted. conclusion: we created an in-house electronic ordering system to enhance information fidelity, re-allocate technologist time, and further standardize ordering. our system showed non-inferiority to the labor-intensive manual system, by not changing the number of stat orders, having the same t:s ratio, and not increasing the number of expired products. this is achieved while freeing up over 360 hours of staff time per year. future directions include full automation with involvement from hts informatics department. transfusion practice improvement: gaining traction through the use of a provincial transfusion quality improvement plan denise evanovitch* 1 , yulia lin 2 , troy thompson 1 , allison collins 1 and sheena scheuermann 1 . 1 ontario regional blood coordinating network, 2 sunnybrook health sciences centre background/case studies: a provincial regional blood coordinating network (prbcn) held a "quality focus day" (qfd) in 2014 to explore transfusion quality indicators to be included in a province wide quality improvement plan (qip). the plan's main goal is to reduce patient harm by improving transfusion practice in hospitals through the reduction of inappropriate use. the following recommendations were made: select a blood component that most hospitals could monitor display progress in a public forum so that hospitals could compare themselves to peers strike a province-wide transfusion qip committee to guide the development of the plan, supporting resources and ongoing improvement initiatives study design/method: a provincial transfusion quality improvement plan (ptqip) committee was formed and included broad representation: the provincial patient blood management coordinators, physicians, technologists, nurses, administrators, clinicians, quality/risk managers from all regions of the province and the provincial blood advisory committee, the blood supplier and a patient. there was further collaboration with other organizations such as the provincial health quality division, choosing wisely after the launch, an informal survey indicated that 74 of the province's 158 hospitals were interested or had already adopted portions of the ptqip. to further assist hospitals in advancing their qips, a technologist prospective screening educational module was developed in addition to an electronic tracking tool with which hospitals can enter their baseline data and subsequent audit data and track their success. both hospital and provincial reports can be generated from the tracking tool. a more formal survey conducted in 2017 indicated that 93% plan to implement or already have implemented the ptqip and 43% of the respondents already have put prospective order screening by technologists in place. conclusion: helping hospitals through the development of standardized templates, instructions, education and other tools for transfusion quality improvement increases the ability of hospitals to uptake quality improvement initiatives. taking a standardized approach across the province allows for both aggregate and hospital data comparison analyses. background/case studies: military and civilian trauma-based studies have demonstrated the advantages of transfusing blood products prior to a patient's hospital arrival, a process known as pre-hospital transfusion (pht). helicopter emergency medical services (hems) worldwide have implemented this protocol with great success, despite a current lack of guidance or advisory publications. there is a need for literature that addresses the regulatory requirements and logistical challenges associated with developing a pht program. herein, we report our experience as a large hospital system embarking on the development of a multi-state pht service. study design/method: in october 2016 a work group was formed to establish pht services for the hems providing care to over thirty regional hospitals. composed of flight care staff, emergency physicians and transfusion medicine specialists, the group identified the major tasks to be addressed: federal/state regulations; inventory structure/management; product storage/testing; tracking/traceability; emergency release protocol; and staff training. while there are no specific regulations governing pht, the regulations pertaining to blood product storage, validation, and monitoring apply. the fda, aabb, and state agencies were each consulted to ensure compliance with all directives. results/finding: the largest hospital within this system, already acting as a reference site, was designated to perform all confirmatory testing on products supplied to the multi-state hems. similarly, this hospital was tasked with remote monitoring of all blood refrigerators at the helipad sites. the system's fda licensed blood supplier was deemed responsible for product consignment and transport between the four hems sites. the blood inventory at each site was designed to contain: group o positive rbcs, group a low anti-b titer liquid plasma, and four-factor prothrombin complex concentrate. a military-tested in-flight medical record system will be used to transfer transfusion information to non-affiliated hospitals as needed. validated inflight coolers, protocol for product emergency release, inventory tracking system, and re-stocking schedule were also requisite to this plan. staff competencies regarding emergency release guidelines, transfusion reactions, and the handling/storage of products are maintained by the hems medical director with additional oversight provided by transfusion medicine physicians. conclusion: our work group successfully identified the challenges associated with a multi-state pht helicopter based service, which spans blood product management, adaptation of existing transfusion procedures and operating policies, licensing requirements, and personnel training. our pht service will go live in 2017. publishing this experience may benefit future sites as they launch similar pht initiatives. blood transfusion during humanitarian emergencies yetmgeta e. abdella* 1 , rana hajjeh 1 and cees th. smit sibinga 2 . 1 world health organization regional office for the eastern mediterranean, 2 international quality management (iqm) consulting background/case studies: more than 76 million people are affected by humanitarian emergencies in the eastern mediterranean region of the world health organization (who), where some of the most affected countries in the world are located. in these countries, the health systems have been weakened or destroyed and health workers provide health services under difficult circumstances. humanitarian emergencies increase the demand for blood transfusion and make its delivery challenging and complex. despite these obvious needs, across the region, there is a lack of information on the emergency preparedness and response capacity of blood transfusion and on the challenges countries and health responder's face in meeting the needs of the patients during emergencies. study design/method: we searched pubmed and index medicus for the who eastern mediterranean region for data on availability and safety of blood transfusion in humanitarian emergencies. we conducted a structured survey of blood transfusion services (bts) in all countries in the region to identify the following: type of humanitarian emergencies between 2006 and 2016; current strategies to ensure availability and safety of blood transfusion during emergencies; coordination and collaboration between countries; and gaps and challenges. additional information was collected during a regional consultation (eastern mediterranean region) held in may 2016 in tunisia. results/finding: we found 24 publications on disaster from five countries in the region and 16 publications on disaster preparedness and blood transfusion in casualties and severe trauma outside the region. however, none dealt with the questions of availability and safety of blood transfusion during emergencies. twelve countries (54.5%) responded to the survey. armed conflicts and terrorism are the commonest types of emergencies with estimated 10-85% of the injured requiring blood transfusion. nine countries have emergency preparedness and response plans for bts. potential blood donors are mobilized through public calls, besides a direct appeal on regular and replacement donors. seven of the responding countries keep an emergency blood stock. collaboration between the different stakeholders exists in seven countries. lack of adequate and competent human resource, transport and cold chain deficits, shortages in supply of consumables and maintenance of equipment, lack of reliable power supply, and shortage in finances are the gaps identified. conclusion: there is a need to integrate bts in the overall national emergency preparedness and response, collect and disseminate updated information on factors affecting provision of blood transfusion in humanitarian emergencies, provide technical and financial assistance to affected countries, strengthen mechanisms for coordination and collaboration among different parties, and develop a regional emergency blood services system and management expertise. (63, 85, and 108 for 2014-2016) . the number of collections per registered trt donor varied significantly, ranging from 0 to 12 therapeutic draws/donor per year. excluding those that didn't present for a therapeutic blood collection, the average number of trt collections/donor per year decreased from 3.8 to 2.8 between 2014 and 2016. conclusion: our blood center has experienced an increasing number of therapeutic phlebotomies, as well as individuals on trt referred for therapeutic phlebotomy due to elevated hemoglobin values from 2014 through 2016. it is not clear from information provided by the ordering physician whether this is intended as a temporary measure to decrease the hemoglobin while the patient is on trt, or whether the dose was being adjusted or discontinued due to the known risk factor of cardiovascular disease in patients with polycythemia; however, the average number of donations per trt donor decreased during this timeframe. the percentage of men on testosterone who present as regular blood donors at our blood center is not known, since this hormone is not reason for deferral. our findings raise the concern, however, that regular phlebotomy is necessary to reduce the risk of testosterone-associated polycythemia in this population. as it is our duty to provide a safe and adequate blood supply, our blood center also has concerns about perpetuating the misperception that repeat phlebotomy, particularly if required more frequently than 56 days, is sufficient to mitigate the risks of testosterone therapy. hence, we have made the decision to discontinue offering phlebotomy services to this population of donors other than for those on testosterone that meet all donor eligibility requirements. approaches involving the use of a vein illumination device in a blood donor center sara matheson*, kimberly j duffy, audrey e traun, mary m benike, james r stubbs and justin d kreuter. mayo clinic background/case studies: venipuncture is a critical step in blood collection and locating a suitable vein for this procedure can be a challenge. unacceptable vein selection or incorrect needle placement can lead to incomplete collection or infiltration. in a blood donor center, the primary selection of a vein is done by palpation within the antecubital area. prior to needle insertion, the skin at the site must be prepared and contact avoided until after needle is placed. vein illuminator (vi) devices are available to aid in visual display of potentially suitable veins. such a device was made available to staff in march of 2010. after an initial testing and instructional period, the vi has since not been used by staff. the objective of this study is to discover reasons why staff does not use the vi to identify potentially suitable veins. study design/method: a staff survey was developed and distributed to staff in march 2017 to inquire about usage of the vi and obtain feedback about the device. at the time that the survey was sent, the device had been available for several years. the survey included questions involving frequency of use, adequacy of training, comfort with using the device, knowledge of the device's storage location, willingness try the device, and general feedback. results/finding: the survey had a 77% response rate (n533). of these, 78.8% have never or very rarely utilized the vi. self-reported reasons for low utilization focused on two dominant themes. first, that the device is not needed and second that it doesn't accurately show veins. 87.9% of respondents are aware of where the vi is stored and a more accessible location to share the device was not identified. although 93.9% of respondents have been provided training on using the vi, the group was mixed regarding their comfort level in using the device independently. only 48% of the group was willing to try vi. conclusion: infiltration and incomplete collection account for approximate 3% (770 units/year) of qualified blood donors, yet vi does not appear to be a viable solution for our blood donor program. there seems to be both an opportunity and challenge with vi implementation. the opportunity is to create critical awareness of problems with vein cannulation. the challenge is to identify a device that is more effective at visualizing deeper veins necessary for blood donation. benefits of converting from mcs1 to alyx penny schroeder* and elizabeth parker. indiana blood center background/case studies: in 2015, apheresis red cells (arc) represented 4.7 % of total red cell collections at our center. hae mcs1 ln8150 was utilized to collect arc. due to the age of the instruments, challenges with collections on mobiles as well as the need to increase collection of right type products, the decision was made to change technologies. study design/method: fresenius kabi demonstrated the fenwal alyx technology as well as the business case to the primary stakeholders. all implicated departments were involved in the initial impact assessment. a multidepartment kick off meeting was held and project team formed. due to product demands, the decision was made to validate arc and plasma apheresis. the primary departments affected were blood collection and production. fresenius kabi provided sample validation plans, sops, training and training materials for use. four mobile-carts were purchased for easy transportation of alyx and quick-connect feet for installation on mobile buses. the lead trainer and the bc technical administrator traveled to an affiliate blood center to observe their alyx program and identify best practices. a team of blood collection trainers and preceptors were the initial group trained and validation performed. this team also served as the subject matter experts and field preceptors. fresenius kabi returned for advanced alyx operator training. the training plan targeted previous mcs1 operators first and then operators new to apheresis with a training goal of 30% of mobile staff. validation of the 12 alyx began 06/01/16 and took approximately 45 days to complete. during this time, fresenius kabi conducted alyx education and apheresis recruitment training to all collection and recruitment staff. the mcs1 machines were removed from service 07/08/16. alyx go-live occurred 07/13/16. additional operator training continued through september 2016. results/finding: due to ease of mobility and use of alyx, reduced procedure time compared to mcs1 and donor conversion training we increase components collected. alyx disposable kit includes pre-attached solution containers reducing ancillary items required to pack and carry to mobiles. this decreased kit cost by $19.21 each providing an estimated annual savings of $239,000. conclusion: with the multiple alyx donation types we were able to increase our collection of right type procedures by approximately 2.5% and decrease our kit costs by 22%. with alyx the collection plasma on mobile blood drives is now possible. due to ease of use, operators have embraced this technology and we have consistently met our monthly collection goals from october 2016-march 2017. background/case studies: high frequency of donation is a risk factor for iron deficiency. because females' iron stores are generally lower than males' before they start their donation career, females who donate frequently are particularly high risk. minimum hemoglobin (hb) has long been the same for males and females at 125g/l, but for males this falls below the normal limit. as a first step to mitigate iron deficiency, criteria for whole blood donors were modified for males (minimum hb increased to ! 130 g/l) and for females (minimum interdonation interval increased from 56 to 84 days). the longer interdonation interval in females was gradually implemented, starting with donor messaging in october 2016, changes in rebooking of donation appointments in december 2016 and culminating with eprogesa criteria changes on march 5, 2017. both these changes are expected to initially result in donation loss, but may be partly counteracted by a decrease in hb deferral rates in female donors. we aimed to assess the impact of these changes on hb deferral rates. study design/method: percentages of hb deferrals were calculated as the number of donation attempts that resulted in hb deferral divided by the number of successful donations plus hb deferrals multiplied by 100. percentages were calculated for male and female donors before and after changes were made. results/finding: the percentage of hb deferrals increased in male donors from 0.89% in the 3 weeks pre-implementation to 2.16% in the 3 weeks post-implementation of the change in the hb criterion. hb deferral rates for female donors were 12.6% in september, 12.0 % in october/november, and 9.9 % from december to march, 2017. conclusion: hb deferral was more frequent in male donors after the minimum hb was increased to 130 g/l. the gradual implementation of increased interdonation interval for females resulted in a reduction in deferrals, thus the initial donation loss associated with this change may be partly offset over time by decreased hb deferrals. a longer observation period is necessary to confirm these findings and assess impact on phenotyped blood and donor retention. in the past 8 years, 59,223 blood products, derived from 10,509 procedures, were distributed to 185 different investigators in over 200 laboratories. whole blood was the most common product (45.2%), followed by unmanipulated mononuclear cell collections (28.6%), and elutriated monocytes or lymphocytes (19.8%). less common requests included platelets (2.5%), plasma (2.5%) and granulocytes (0.8%). adverse donor reactions were infrequent (0.33% of procedures). conclusion: we report the feasibility of a program for collecting and distributing blood for investigators to obtain blood components for in vitro research use, utilizing the staff and resources of a hospital-based blood bank. research blood donation is essential to support laboratory research and to maintain positive relationships with donors who have been deferred from allogeneic transfusion. hospital-based blood donor center's experience with implementing platelet pathogen reduction system kimberly j duffy*, mary m benike, james r stubbs and justin d kreuter. background/case studies: the safety of platelet products has been continually improving due to testing despite the continued emergence of microbial threats. the recent fda approval of platelet pathogen reduction technology will protect transfusion recipients regardless of the new microbial dangers. in order for platelet products to use the pathogen reduction technology, the volume, platelet yield (dose), and concentration must be collected within tight specifications. the objective of this study was to determine the optimal collection settings to enable 100% collection of pathogen reduced platelets while limiting the loss of products. study design/methods: the collection instrument evaluated for this study has fda approval for platelets suspended in 100% plasma. the corresponding pathogen reduction system used for the study has 3 kits with 3 different collection specifications. all apheresis collections occurred at a fixed site and pre-platelet counts were performed on a hematology analyzer. the yield scale factor has been established for correlation between the hematology analyzer and apheresis collection device. in order to determine the optimal collection targets, the apheresis collection instrument had a variety of multiple yields and volumes established for collections. staff was instructed to collect the highest available yield per donor. after collection, volume, platelet yield, and concentration data was obtained. this data was used to determine if the product met the specifications for one of the available kits, and if the actual platelet yield was higher than 6.8 x10 11 , thus meeting the criteria for a double product. results/findings: a higher platelet concentration product is ideal to produce a double product, but targeting products with a platelet concentration greater than 1800 x 10 3 /ml was more likely to be outside the specification of the pathogen reduction kit. the platelet concentration target of 1867 x 10 3 / ml results in discarding products and was quickly removed from instrument settings. collections with a platelet yield as low as of 3.5 x10 11 and platelet concentration of 1167 x10 3 /ml were more likely to produce a product that was not within the specification of the pathogen reduction kit. abstract conclusion: the loss of both triple platelet products and lowered postprocessing platelet recovery requires the collection of platelets to be far more precise. the goal of platelet collection has shifted from simply maximizing each platelet collection to an approach that considers optimal collection within the limits of kit specifications. final collection instrument configurations are platelet yield of 7.0 x10 11 and 6.8 x10 11 at the volume of 400 mls and platelet yield of 4.2 x10 11 , 4.0 x10 11 , and 3.5 x10 11 at the volume of 300 mls. moving from subjective to objective donor eligibility screening platforms: a blood center's journey angela dirr* 1 and steve cihura 2 . 1 bonfils blood center, 2 bbc / bsi background/case studies: in 2012, the device used by bonfils blood center to determine donor hemoglobin and donor eligibility was reaching its end of life, and bbc needed to define a path forward for a reliable replacement device. study design/method: bbc evaluated 3 devices with the following criteria in mind: 1) device disposable costs, 2) reagents/controls/quality control, 3) objective hgb/hct measurement, 4) portability and durability for a mobile environment, 5) ease of use, 6) donor experience, 7) battery life, 8) validation requirements plans, 9) blood center suitability, and 10) ability to link to becs. multiple departments including donor care, equipment management and validation, quality, and regulatory affairs were involved in the evaluation and product selection. bbc tested 50 donors per each device at both a fixed and a mobile site. bbc also considered donor feedback for the choice of replacement technology. the project started february 2013 with a targeted implementation date of july 2013. after creating necessary sops and adopting existing sops, bbc successfully completed the validation of the devices, and chose the compolab technology from fresenius kabi as the new device for bbc blood bank. results/finding: the compolab was selected as it met project scope and selection criteria. it was important for bbc to reduce paperwork and daily tasks. the compolab eliminates daily qc reducing paperwork, time and improves error management. after converting to the new technology, bbc donor deferral rates increased by approximately 15%. as a consequence to this increase, bbc conducted reminder training with bbc staff to ensure proper sampling technique and higher sample quality. over time, bbc deferral rates stabilized to 4.59% in 2014 and 4.29% in 2015. during this time period, bbc also successfully recruited new blood donors to bbc program, which may have contributed to an increase in deferral rates. in 2016, the deferral rate increased again, probably due in part to the fda final rule "requirements for blood and blood components intended for transfusion or for further manufacturing use", which went into effect in may 2016. conclusion: during the evaluation for new equipment, bbc learned that it is critical to understand the equipment's life cycle and the effect the equipment has on all aspects of the business. after comprehensive evaluation of multiple donor eligibility screening platforms, the compolab device was selected at bbc facility. it met the majority of all aspects of the project scope and qualifying criteria. bbc also learned that continuous refresher training of the staff ensured optimal device performance, and how external factors such as changes to the regulatory environment may impact deferral rates. flowmetry on platelet apheresis. tetsu yamamoto* 1 , ayumi araki 1 , hiromi sanyoshi 1 , hiromi kanai 1 , hiroya kikuchi 2 , katsushi tsukada 2 and kazuhide mure 2 . 1 hokkaido red cross blood center, 2 japanese red cross hokkaido blood center background/case studies: vasovagal reaction (vvr) is known to be the most common adverse reaction to blood collection, but effective measures for preventing vvr have not yet been developed. effective timing of interventions during apheresis donations in particular should hold the key to predicting vvr, but no research has been done on the topic. study design/methods: this study investigated the potential to predict vvr from fluctuations in peripheral blood flow measured by laser doppler flowmetry in platelet apheresis donors, a population highly likely to experience vvr. data were collected from 354 individuals who donated platelets during the 6-month period between february and august 2015, and data from the 30 donors who experienced vvr were analyzed. to calculate the level for issuing vvr alert, the percent decrease in blood flow (dbf) and the percent decrease in heart rate (dhr) were calculated, the time from alert to vvr was estimated for three dbf levels, and the detection performance of each alert level was calculated. results/findings: eight of the 156 men (5.1%) and 22 of the 198 women (11.1%) experienced vvr. one donor did not experience vvr during blood collection, but had a delayed reaction while resting afterward. mean maximum dbf in the 30 donors in the vvr group was 64.7 6 13.7%, which was significantly higher than the 25.6 6 11.7% in the non-vvr group. at a maximum dbf threshold of 45%, sensitivity for discriminating between vvr and non-vvr donors was 93.3% and accuracy was 94.4%. when 45% dbf was used as the alert level, alerts were issued for 44 donors, including 25 in the vvr group. therefore sensitivity for predicting vvr was 83.3% and specificity was 94.1%. mean time from alert to diagnosis in the vvr group was 4.03 6 4.35 minutes, and accuracy of the alert was 56.8%. some of the vvr could not be predicted even the value of maximum dbf exceeded 45%. the reason was supposed to be the difference of donor susceptibility on dbf. conclusion: we investigated whether vvr in platelet apheresis donors can be prevented by prediction and found that it is possible to predict vvr early enough before onset to intervene by monitoring dbf in real time during blood collection using laser doppler flowmetry. future research must also investigate whether the incidence of vvr can actually be reduced by interventions such as adjusting extracorporeal circulation. the risks of alloimmunization in sickle cell patients using c, e, k negative blood: experience of a hospital apheresis and transfusion service grace banez sese* 1,2 , salam abdus 3 and shabrina shah 3 . 1 inova blood donor services, 2 inova fairfax medical campus, 3 inova fairfax medical campus transfusion services background/case studies: red blood cell (rbc) transfusion is often a lifesaving measure for patients with sickle cell disease (scd). it is critical in the management of scd complications such as splenic sequestration, stroke, priapism, iron overload and acute chest syndrome. a wellrecognized complication of chronic transfusion in scd patients is alloimmunization to rbc antigens. to prevent alloimmunization, transfusion with rbcs negative for c, e, and k antigens has been advocated. this has led to reports of reduction in the rate of alloimmunization and a decrease in hemolytic transfusion reactions. we report a summary of our three year experience with the prophylactic transfusion of rbc units negative for c, e, k antigens for scd patients during red blood cell exchange transfusions (rbcx). study design/method: retrospective review of 10 scd patients with a history of stroke, refractory sickle pain crisis and priapism was done. rbcx was performed every 4 to 8 weeks from december 2013 to march 2017. blood bank work-up used the mts gel method for antibody screen and identification. our hospital-based donor center proactively works with the hospital blood bank in preparing these units in a timely manner. results/finding: a total of 10 patients, 3 females and 7 males, who underwent a total of 178 rbcx from october 2013 to march 2017, using an average number of 7 rbc units per rbcx. rbc units negative for c, e, and k antigens were used during rbcx for 8 patients. two patients positive for c antigen underwent rbcx, using e and k antigen negative rbc units. review of the antibody screen test results performed prior to each of the 178 rce showed that no new clinically significant alloantibodies were formed after exposure to multiple rbc units. conclusion: although there is no consistent standard of care in transfusion practice related to the extent of antigen matching for scd patients, studies suggest that the standard of care for transfusion of all patients with scd is to provide rbc negative for c, e, and k antigens. this ability to find these rare units is also affected by the characteristics of one's institution and blood supplier. it is an advantage to have a hospital based donor center to work with, as we proactively collaborate with them to provide these rare units. the approach by our institution to transfuse rbc units negative for c, e, k or study design/method: venous blood specimens of 168 healthy volunteers were collected before blood donation and after blood donation immediately, 1 day, 1 week, 4 weeks, and 12 weeks among men and 16 weeks among women. immunoglobulin g (igg), immunoglobulin m ( igm) , immunoglobulin a ( iga)and complement component 3 ( c3) , red blood cell (rbc), white blood cell count ( wbc) , hemoglobin (hb), hematocrit (hct), and serum iron (fe) , were measured to monitor he dynamic changes of these biomarkers and blood quality. results/finding: the level of igg slightly decreased after blood donated immediately, iga and c3 decreased significantly but still within their normal ranges, igm did not change after blood donation. the level of iga significantly decreased at 12 weeks among men and 16 weeks among women, while c3 significantly increased at the same time period. igg, rbc, hb, hct and fe started to recover 1 week after blood donated and reached their levels before blood donated within 12 weeks among men and 16 weeks among women. conclusion: the biomarkers mutually changed over the course of 12 weeks among men and 16 weeks among women. donating 400 ml blood will not significantly affect overall blood quality. utilizing amicus dxt relay data managment solution to increase platelet split rate and improve amicus productivity janelle wilhelm* and jennifer kaluza. memorial blood centers background/case studies: with the increase in platelet demand and the opportunity to export products we set an initiative to increase the platelet products collected form our existing donor base. we also faced the challenge of managing multiple collection sites in multiple states. the decision was made to implement amicus dxt relay data management solution to provide us insight into procedure details to make data driven decisions. day to day variability previously dipped as low 40% split forcing reactive planning. study design/method: incorporate dxt to strategically plan our day to day operations. dxt reports were monitored by management and with the fresenius kabi team for productivity by site, phlebotomist and device. reports measured target vs actual yield, donor parameters, and procedure events to perform a donation opportunity analysis. this allowed us to adjust configuration settings when appropriate to improve the accuracy of the yield prediction. reports by phlebotomist were utilized for training on how to optimize the donor's gift to donate an additional platelet or plasma product(s) and increase procedure success rate. results/finding: the monthly dxt report analysis resulted in device configuration improvements, phlebotomist and center manager accountability, effective training, and donation optimization we increased our overall platelet split rate 15 percent and increased concurrent plasma collections by 24 percent. with utilization of the dxt reports we are able to take a proactive approach allowing us to predict product availability, with day to day variability dropping no lower than 62 percent split. phlebotomist qns rates were easily monitored regularly (daily, weekly monthly) resulting in a decrease in our overall qns rate to consistently below 3 percent. conclusion: dxt was easy to implement, is very user friendly and will continue to help improve our platelet collection and process improvements between donor centers. dxt provides invaluable tools for the operational supervisors to monitor their staff and improve productivity at their multiple sites. next step is to develop the plan for implementation of paperless documentation with dxt and healthcare-id. the ability to immediately review data directly from amicus was key in the productivity improvements realized. evaluating the impact of a background/case studies: as blood and blood products are limited and expensive resources, they are prescribed, handled, stored and transfused according to hospital guidelines established to ensure that the best practice standards are maintained for patient safety. it is a prerequisite for all registered nurses (rns) involved in blood and blood product administration to possess fundamental knowledge of transfusion practice. aim: the aim of this study is to evaluate the impact of a hospital-based transfusion practice training program among registered nurses, through administration of a knowledge-based questionnaire before and after implementation of the program. the results gathered would identify gaps in assimilation of knowledge and suggest improvements to the design and implementation of specific content in the nurse-led transfusion training programme. study design/method: all rns from various units and departments were invited to participate in the blood transfusion knowledge questionnaire in october 2015. after which, a formal transfusion practice training programme was introduced, consisting of an online learning platform and in-service training sessions. the same questionnaire was administered to the rns one year later in september 2016 for post-training programme evaluation. individual item scores and proportion of nurses with perfect scores was compared pre-and post-implementation. results/finding: in 2015 and 2016, a total number 1,097 rns and 965 rns completed the questionnaires, giving a response rate of 78.5% and 67.4% respectively. the overall mean score in 2015 was 6.24 points (range 0 to 8). the mean score in 2016 was 6.57 points (range 2 to 8). the percentage of rns having perfect scores of 8 increased from 8.8% in 2015 to 20.5% in 2016. table i below shows the results for each question item. the implementation of a hospital-based, nurse-led transfusion practice training programme has led to encouraging improvement in blood transfusion knowledge amongst rns. further training may be needed in the preparation of blood sets and management of fever. background/case studies: clinical use of blood has shown to be the least developed part in the vein-to-vein transfusion chain. this global survey was therefore carried out in order to investigate the level of awareness, accessibility and utilization of e-continuous learning and quality of blood use among blood prescribing clinicians and nurses. study design/methods: a descriptive 'ex-post facto' survey design was used; 264 purposively selected blood prescribing clinicians and nurses from 60 hospitals in 13 countries of the 4 human development index (hdi) groups (low, medium, high, and very high) participated. three research questions were answered, while seven null hypotheses were tested at .05 level of significance. descriptive statistical tools (frequency counts and percentage) were used to analyze the demographic backgrounds, while inferential statistics -pearson product-moment correlation coefficient (ppmc), analysis of variance (anova), were used to analyse the hypotheses. results/findings: quality of clinical use of blood was positively and significantly correlated with levels of awareness (r5 .137; p5.03; df5262) and accessibility (r5.184; p5.01; df5262) to e-continuous learning among blood prescribing clinicians/nurses. there was significant difference in levels of awareness [f(3,260) conclusion: today e-continuous learning has become a conditio sine qua non to effective and quality clinical use of blood. the higher the hdi level the better the awareness, accessibility and utilization of continuous education, both through e-learning and conventional programs. there is a better awareness among clinicians routinely prescribing blood as compared to others involved only incidentally in blood transfusion. accessibility of e-learning depends highly on the presence of a sustained societal infrastructure which is less guaranteed in the low and medium hdi countries; reliable power supply, maintenance of hardware tools and updated software programs, together with the necessary knowledge and skills of e-technology are prominent factors. the results are used for policy and strategy recommendations to improve knowledge and clinical practice through continuous e-learning programs eg, starting at undergraduate medical and nursing schools and continuing at postgraduate vocational medical specialization institutes, principles of clinical transfusion practice should be comprehensively included through appropriate and timely curricula; creation of a technical climate to guarantee access to e-learning courses and materials; stimulation of national and international exchange of e-learning programs focused on continuing education; creation of an e-learning mentoring network through professional societies, associations and education institutes. background/case studies: transfusion medicine (tm) didactic teaching materials for pathology residents are not widely available to share among residency training programs. the advancing blood knowledge (abo) leaders project is a novel approach wherein education materials are created collaboratively through a community of practice (cop). educational theorist etienne wenger defined cops as groups of people who share a concern or passion for something they do and learn how to do it better as they interact regularly. study design/method: as a pilot project, 7 junior faculty co-investigators from 5 west coast institutions each had 2 months to create a 30 minute powerpoint presentation on a fundamental tm topic, after which 2 other members had 2 months to review and edit. therefore, each member created 1 and reviewed 2 presentations (three total steps). during each step, members wrote 2 multiple-choice questions for those particular topics. in the end, each topic would have 6 quiz questions to assess learning. at completion, 7 evidence-based, peer reviewed presentations would be available for all members to use for teaching pathology residents. three methods were planned to measure effectiveness of these materials: 1) pre and postlecture abo leaders exam using the questions made for each topic to assess learning; 2) pre and post-lecture 20 question validated examination (best collaborative) to assess learning; 3) resident in-service examination trends specific to tm. results/finding: six presentations were developed as 6 of the 7 abo leaders members continue to participate in this cop for tm education. abo leaders and best pre-test results are shown in tables 1 and 2. abo leaders pre-test data could not be obtained for institution b, and 3 trainees declined to participate in the examinations at institution a. challenges experienced by the cop have included heterogeneity between institutionsõ resident schedules, balancing time dedicated to the group given busy schedules, and difficulty in giving all 6 presentations during the defined institution-specific teaching period. post-test results will be included when assessments are complete. conclusion: despite logistical and organizational challenges, it is feasible to create a multicenter cop for tm education. the impact of such a group on resident learning will be assessed and plans for growth will be evaluated. background/case studies: the traditional educational curriculum for the pathology residency program is primarily based on didactic lectures, casebased presentations, and discussion of on-call cases. the use of dramatic vignettes has proven to be an effective educational tool to illustrate complex and multidisciplinary topics in medicine. our goal is to use and evaluate the relevance of this approach in resident education. study design/method: a clinical vignette based on a placenta accreta case was written by a pathology resident during the transfusion medicine rotation. during a two-week laboratory management course, residents prepared for the dramatic vignette performance with a focus on transfusion medicine and laboratory management topics. each resident completed a 10 question preand post-test on topics related to the vignette. several meetings for review and adaptation of the script, topic discussion, and rehearsals were held. there were several commonly encountered problems and deviations from the standard operating procedures that the residents in the audience were asked to identify prior to the performance. during the skit, each resident presented at least one major transfusion management teaching point. results/finding: the educational activity, including the 40 minute vignette performance and the 20 minute discussion, was completed with a focus on: communication between the operating room and the blood bank during surgery, maximum surgical blood order schedule, pre-transfusion testing, transfusion safety, informed consent, massive transfusion protocol, emergency release blood products, thromboelastometry interpretation, patient safety, adverse events, and root cause analysis. all performers significantly improved their scores in the post-test (mean 95 1 4%) when compared to the pre-test scores (mean 67 1 26%) ttest p<0.017. during the vignette discussion, residents together identified all the intended non-conformances and answered related questions. residents in the audience actively participated in the post skit discussion and 90% reported a satisfactory learning experience. conclusion: dramatic clinical vignettes can illustrate multidisciplinary complex interactions that are of pivotal importance in the daily activities and professional development of pathology residents. with specific structured goals, clinical dramatic vignettes can be used as a complementary educational tool to illustrate challenging topics in an integrative way that is enjoyable and easy to understand and remember. the skit performers benefit from the activity further by preparing and extensively studying the topics to deliver a multifaceted and coherent presentation with emphasis on the integral role of the laboratory and transfusion medicine in patient care. hannele sareneva*, susanna sainio, inna sareneva, tiia kivipuro and taru jaske. finnish red cross blood service background/case studies: the finnish red cross blood service (frcbs) is the nationwide blood service provider in finland, responsible for collection, testing, processing and distribution of blood products to all hospitals and health care providers. the frcbs serves as the national blood group reference laboratory and provides a wide range of other laboratory services e.g. tests for hemostasis and tissue typing for possible donors as well as patients waiting for organ or stem cell transplantation. frcbs also performs antenatal blood group and rbc antibody tests covering whole country. as a sole national operator we are providing educational services to ensure the safe use of blood products as well as accurate use of our laboratory services. study design/methods: we have performed customer surveys to healthcare professionals to assemble the needs for education. based on these results and continuous feedback frcbs provides hospital customers in blood banks and clinics the following additional services: * regular education * e-learning application of transfusion medicine * handbook for blood products on the web site * reports to hospitals for their use of blood products * annual national blood safety reports regular elements of our educational program are the practical, problem solving course for blood bank personal and safe transfusion training day for clinicians. for every education we collect numerical feedback as following: "how did the education responded your expectations" and "can you utilize the knowledge in practice". we also inquire "how likely you would recommend the training for your colleges" indicating net promoter score (nps). results/findings: more than 350 healthcare professionals participate training days at frcbs annually. in addition our experts give tens of lectures at hospitals across finland. feedback from educations has been very good, varying between 8.3 to 9.4 (in the range of 4-10). nps varies between 83 and 98. according to customer surveys frcbs provides appropriate education to healthcare professionals. this score has increased 2011-2016 from 8.4 to 9.0. conclusion: feedback, nps scores and surveys ensure that education and training program of frcbs responses to customer needs. hospitals can utilize annual courses of frcbs in their own initiation programs. together with clinical contact persons in hospitals our aim is to ensure patient blood management (pbm) and to optimize use of blood products. we also have plans to increase e-learning applications and the courses of transfusion medicine for nurses and medical students. educational outreach and effect on reporting septic transfusion reactions kathleen m grima* 1 , anne eder 2 , beth a. dy 1 and mary o'neill 1 . 1 american red cross, 2 georgetown university background/case studies: hemovigilance programs to monitor adverse events after transfusion depend on clinicians' ability to recognize and report reactions to the blood center. about 1 in 100,000 apheresis platelet donations are implicated in septic transfusion reactions (strs), but this could underestimate the risk because of the difficulty in recognizing delayed or mild reactions. a large blood center designed an educational outreach program to increase awareness of strs and assessed its effect on the rate of str reporting to its national hemovigilance program. study design/method: in dec. 2015, a large blood center developed a web based course on strs for cme/ceu credit. letters were sent to 2,300 hospital customers about recognizing and reporting strs, and alerting them to the availability of the course. blood center physicians and staff in sales and marketing also engaged hospital customers directly in discussions about recognizing and reporting strs, using the online educational content. the physicians tracked their interactions. the blood center's national hemovigilance program compared the number of strs reported in the 12 months before and after launching the educational outreach. results/finding: the web based course was completed by more than 700 participants; 117 were physicians. based on a review of the evaluations, the course was highly valued with 93% of participants rating it excellent or very good. the blood center physicians gave over 200 presentations to hospital customers. reporting of suspected strs in 2016 increased by 23% compared to the prior year. the increased reporting came from 2 specific regions. the total number of strs that met the hemovigilance definitions for definite (culture-confirmed) and probable strs in the nationwide system increased but did not change significantly compared to the previous years. the educational initiative was designed to deliver a consistent message on the risks, recognition and reporting of strs. while the number of reports of suspected strs in two regions increased, there was no meaningful change in the overall reporting of suspected or confirmed strs across the national blood system. this finding could reflect that hospitals already recognize and report medically significant reactions or that the target audience was laboratory personnel and physicians in transfusion medicine, but not the clinicians closest to patient care at the bedside. more targeted educational efforts provided by personnel who interface with hospitals could be used to address identified professional practice gaps in transfusion medicine. implementation of subscription-based cgmp e-learning laurie mcgraw*, courtney saphier, helene belton, sallie bittner and ward scott. gulf coast regional blood center background/case studies: previous cgmp e-learning courses we developed required 30-45 minutes for learners to complete. while feedback was positive, manufacturing areas struggled to schedule time for staff to complete courses within their assigned schedules. at the same time, a shift in design trends suggest that subscription-based learning is more effective (thalheimer, 2014.) subscription-based e-learning utilizes 5-7 minute modules, delivered at regular intervals. this changes the learning process from a singular event to a regular interaction that reinforces learning and keeps the content at the top of the learner's mind. study design/method: we began developing cgmp subscription-based elearning in 2016 by selecting our first five series topics: equipment, personnel, labeling, sops, and records. the first topic, equipment, was divided into modules on selection, validation, calibration, quality control, and maintenance. these modules, and pre-and post-quizzes for the equipment series, were developed and assigned to employees in manufacturing-related jobs using our learning management system. the pre-quiz was assigned to employees in june 2016, with a new equipment module assigned each month for the following five months. the series concluded in december 2016 with the post-quiz. results/finding: using surveys, assessments and incident reports, we evaluated the training effectiveness using three of the four kirkpatrick levels. while our previous cgmp courses received good ratings from learners, the equipment series received the highest rating of 3.5 on a 4-point scale. of employees who completed all versions of our cgmp courses, the majority preferred the equipment series over all previous courses combined. comments clearly demonstrated that learners preferred the short, subscription format over the previous courses with 21 positive and 1 negative comment. level 2: learning the average score of users increased 13% from the pre-test to the posttest, with the greatest improvements noted in the scores from laboratory employees. a two-sample t-test determined the result to be statistically significant with a t-critical value of 1.647 and a t-stat value of 5.641. level 4: results while equipment-related errors decreased by 20% after training, there is not enough data to demonstrate a statistical significance. conclusion: our level 1 and 2 evaluation data validated that the subscription approach was effective. knowledge increased from the pre-to postquiz, learners reported that they appreciated the shorter training, and they completed the modules without special scheduling requirements. as a result, we are continuing development of the remaining series. background/case studies: the interdisciplinary nature of transfusion medicine requires the collaboration of multiple work units for efficient patient care, but departmental "silos" impede collaboration between transfusionrelated care teams. we hypothesized that regular educational meetings would improve knowledge and awareness of each department's role, so in october 2013, a multidisciplinary educational meeting called friday blood conference (fbc) began as a collaborative, interprofessional forum involving frontline staff of our transfusion practice. during these monthly meetings, which are also broadcast online for those unable to attend in person, presenters from different work units share background information and patient cases before opening the floor to constructive discussion. study design/method: a survey was sent to fbc participants (n5151) to retrospectively capture the effect of fbc on interdepartmental collaboration. the survey was structured to obtain formative feedback using the published interprofessional collaborative practice competencies (icpc) as a guide. these core competencies target maintaining a climate of mutual respect, communicating within and between departments, fostering teamwork, and understanding everyone's role in patient care. results/finding: our survey response rate was 35%. of those, 96% endorse that fbc creates a climate of respect within our transfusion practice, 94% believe it has improved communication between work units, and 98% feel that fbc leads to increased understanding of interdepartmental processes. notably, laboratory scientists and transfusion nurses have the highest attendance rate. furthermore, those attending via the online broadcast report the lowest satisfaction, with only 56% responding positively. the main reasons individuals attend fbc are to increase knowledge about transfusion medicine, interact with and learn from other departments, hear about patient case studies, and understand the "big picture" of one's role in patient care. suggestions for improvement include preparing questions to help initiate discussion, increasing representation of other areas for broader perspectives during interdepartmental dialogue, and posting recordings of fbc for later viewing. conclusion: the application of icpc in transfusion medicine was an effective lens to assess the value of interprofessional collaboration. although there is room for improvement, the results support that fbc has contributed to better communication between transfusion-related care teams and has increased understanding of interdepartmental processes within our transfusion practice. novel approach to curriculum development: demystifying transfusion medicine ritcha saxena* and ananya saxena. all saints university school of medicine background/case studies: transfusion medicine is an essential element of education required for the future physicians in various disciplines like surgery, internal medicine and anesthesiologists to work effectively with the blood bank personnel. transfusion carries considerable advantages as well as risks. consequently, educational initiatives are required to identify the particular knowledge deficits in transfusion medicine and subsequently, bridge the gaps. and the challenge is to update the undergraduate medical curriculum to reflect the latest enhancements in transfusion medicine. study design/methods: 41 students of undergraduate semester 3 and 59 students of semester 4 participated in the study. self-directed learning resources combined with modules of interactive instruction were implemented in a tbl course design. five education modules focusing on quality management, blood collection, transfusion reactions, precise utilization of blood products and innovations in component safety were designed for the students. the students' reaction to tbl in transfusion medicine was evaluated using qualitative and quantitative assessment tools to analyze knowledge attainment and critical thinking development along with team continuity. the participants were first assessed with readiness assurance testing (rat) to guarantee that they understood the concepts and their application followed by case study based test questions. results/findings: students' reaction to tbl was primarily positive, with 86% of students giving a positive feedback. evaluation through readiness assurance testing (rat) illustrated improved team knowledge acquisition in implementation of effective quality management systems over knowledge acquired through individual study. students grasped a conceptual knowledge of principles of transfusion medicine and achieved confidence in dealing with transfusion-related complications. anecdotally, students significantly attained perception in blood component preparation, storage and their optimal utilization along with developments in safety techniques in blood donation. conclusion: our study suggests that reforming the medical curricula for undergraduate medical students, with specific educational modules designed to focus on blood banking and blood transfusion principles and latest advances in transfusion medicine, is much required in the interest of patient care and safety, by the future physicians. tbl is an interesting and efficient way to deliver the key aspects of transfusion medicine to the students. results/finding: open house attendees were given tours of the bb, led by a bb attending, bb residents, bb supervisor, or bb quality coordinator. the patient blood management nurse was also in attendance to answer attendee questions and educate about patient blood management. light refreshments were offered to the attendees in the bb break room. the first bb open house was held on wednesday, 12/7/16 from 9-11am. there were 17 attendees, including a second-year medical student, four regular blood donors at the hospital blood donor center (who were also employees in facilities management and the university office of admissions, respectively), a hospital senior vice-president, six apheresis nurses, two clinical laboratory staff, two medical laboratory science students, and one additional staff member from the university office of admissions. the second bb open house was held on thursday, 4/20/17 from 7-11am. there were 14 attendees, including 2 regular blood donors (who were also employees in the office of international affairs and supply chain, respectively), a hematology/oncology fellow, and 11 surgical residents. background/case studies: simple, partial, and exchange transfusions are routinely performed in patients with sickle-cell disease (scd) with the goal to increase the oxygen carrying capacity of the blood and reduce the relative percentage of sickled cells. it is essential for clinicians to be able to rapidly estimate the effects of the available therapeutic modalities using clinical information to minimize the risk of red blood cell exposure. given that the formulas for these calculations are complicated, we developed and validated an online calculator to assist physicians with such tasks. study design/method: a web application was generated (www.phamcalcs.com). the performance of the simple transfusion and partial manual exchange calculators were validated by comparing the predictions to clinical data. the performance of the automated and depletion rbcx calculators was validated using the terumo bct (lakewood, co) calculator up to a fraction cells remaining (fcr) 50% as patients with fcr 50% may benefit from delaying the procedure for performance in the future. validation process included (1) a deming regression to globally assess the predicted vs. actual results and (2) an individual comparison wherein validation was contingent on the (predicted-expected results)/(expected results) demonstrating |d| 15%. validation was performed for hematocrit (hct) and hemoglobin s (hgbs) level post-transfusion for simple and partial manual exchange and volume of replacement fluid for automated and depletion rbc exchange. results/finding: see table 1 background/case studies: with the focus on new technologies the modern medicine requires more expenses. despite the increase in the target impact on patients there is still a risk of adverse reactions to medical treatment. the issues that are currently under discussion: the use of standardized or personalized approach, for doctors -being multidisciplinary or having a narrow specialization, integration of new technologies, the need for more trainings resulted from knowledge deficiency. in russia, the development of insurance medicine creates the demand for more intensive and cost-effective treatment programs. as a multidisciplinary approach, pbm optimizes the transfusion practice reducing the risk of adverse effects and improving the financial performance of a health care institution. however, the prosperous implementation of pbm also requires supplemental medical competencies that provide harmonization of dialogue logistics: administrator -clinician -transfusiologist. study design/method: at the medical simulation centre of hospital there has been a unique opportunity to launch an educational program for the medical specialists practicing blood components transfusion. the main innovative features of the training course are an interdisciplinary approach, intensive learning performance, comprehensiveness of learning methods. during 2 days (18 academic hours) the trainees can attend 6 lectures, discuss the methodical materials, participate in 3 seminars, 2 interactive clinical discussions, a master class and a game that presents the modelling of working processes. since initiating the project in june, 2016 with the group capacity 220a transfusion 2017 vol. 57 supplement s3 of up to 35 people the number of medical specialists who have attended the training is nearly 450. results/finding: the medical competencies gained: knowledge of modern recommendations on the use of blood components the ability to interpret all parameters of the haemogram, coagulogram and tromboelastogram the ability to unveil the indications and contraindications for urgent and scheduled blood component transfusion personalization of the blood transfusion risks using a personalized approach on selecting the type and the dosing of transfusion habitat predicting the efficacy of transfusion the correction of anemia and hemostasis system malfunctions using the medicinal treatment performing the macroscopic assessment of blood component before the transfusion procedure performing the differentiated diagnostics and ability to prescribe the adverse effect treatment ability to carry out the auditorial check of health cards conclusion: the launch of the program "guidance for safe and effective blood use in adult patients of multi-field hospitals" is aimed to meet the educational and professional needs of medical specialists, develop the algorithmic thinking and a range of useful motivations in case of patient blood management and reach the compliance in practice. the effect of emergent situation drills on technologist teamwork and comfort levels abigail neils*, raeanne stensgard, rebecca wren, elisabeth greer, amy mata and camille van buskirk. mayo clinic rochester background/case studies: teamwork and composure are essential for technologists when dealing with emergent situations in a large hospitalbased blood bank where multiple situations can occur simultaneously. in an effort to reduce errors and improve emergency response, a group was formed to evaluate the effectiveness of emergency situation drills (esd). the esd were based on common emergent situations encountered in the lab and were run once per month per shift. the main goal of esd was to improve teamwork and comfort level during real emergent situations; therefore reducing the amount of unplanned standard operating procedure (sop) deviations. study design/method: prior to esd implementation, a survey was sent to all technologists to determine baseline comfort levels associated with various emergent situations. one year post esd implementation the same survey was sent to all technologists to reassess the comfort levels for the same situations. the surveys asked employees to rate satisfaction and comfort level on a grading scale of 1-100; 1 being least satisfied/comfortable and 100 being most satisfied/comfortable. the pre and post survey results were evaluated by calculating lab average comfort levels per situation and survey. in addition, unplanned sop deviations related to emergent situations were counted for one year before and one year after esd implementation. results/findings: out of 35 total technologists, 31 technologists took the pre esd survey and 25 technologists took the one year post esd implementation survey. table 1 shows the lab averages from the pre and post surveys as well as the percent difference. out of the employees who responded to the post survey, 19 (76.0%) answered "true" to the statement "esd have improved my comfort level with emergent situations." in the year prior to esd implementation there were 14 unplanned sop deviations; in the year after esd implementation there were only 5 deviations. conclusion: all but one area increased in comfort level post esd implementation. also most technologists agreed that the esd helped improve their overall comfort level with emergent situations. the goal of implementing esd has been met based on the unplanned sop deviation decrease and technologist satisfaction increase; therefore esd were deemed effective. monthly esd will continue to be run with the hope of continual improvement in teamwork, comfort levels and deviation levels. therapeutic background/case studies: category i indications for red blood cell exchange (rbc exchange) in children with sickle cell disease include following acute stroke and for stroke prophylaxis, as well as for iron overload prevention. as described in the first installment of this series about therapeutic plasma exchange (tpe), the challenges of access, volume management, and instrumentation persist, as along with the need to address the psychological and emotional well being of this population. rbc exchange is a complicated procedure to explain to adults and becomes an even more intimidating task when translating into the language of childhood. nevertheless, pre-treatment education is shown to decrease the anxiety associated with medical care. providing age appropriate specific treatment information to pediatric patients decreases negative behaviors, reduces stress and promotes faster recovery. a previous project explaining tpe to the pediatric population revealed the lack of age specific literature for apheresis procedures in general, including tpe and rbc exchange. study design/method: in collaboration with a child life specialist, an ageappropriate story-driven explanation of the rbc exchange procedure was adapted from a previously implemented project related to tpe. artwork was produced with the aid of a medical illustrator to complement the story-line. results/finding: the story board addresses why rbc exchange is performed, the steps involved in preparing for and performing the procedure, and strategies for coping before, during and after the procedure. the idea of long-term therapy is also briefly addressed, to prepare these children for the concept of ongoing therapy. the booklet is in production in concert with our hospital's medical illustrator and will be available on our hospital website for patient use. conclusion: using the previously illustrated story as a guide, an explanation of red cell exchange was created to provide education and reduce anxiety. this second installment continues the pediatric series helping to explain apheresis procedures to pediatric populations in the hopes of reducing patient stress and promoting age appropriate coping strategies. transfusion safety officer resource manual leonor de biasio*. it is also intended to be utilized by hospitals that do not have a formal tso position but which have delegated the responsibilities to other staff. the resource provides helpful information to assist with education in transfusion safety, adverse event investigation and reporting, product administration guidelines or monographs, and links to information about the equipment used for infusion of blood. the resource manual will serve as a useful reference tool to assist with a healthcare professional's transition into the tso role. turning on pathogen reduction: a case of flipping the switch kassandra poffenberger*, darla wendt, jennifer vrieze and james r stubbs. mayo clinic background/case studies: a critical aspect of implementing a new method in manufacturing blood products is to develop a training plan that adequately prepares staff but doesn't interfere with production or cause delays in patient care. with the implementation of pathogen reduction technology (prt) using interceptv r blood system for platelets it was understood that we would need more collections to make up for the loss of products, specifically our triple collections. our institution collects the majority of its blood products and supplements inventory from a major blood collection center. it was crucial for the component laboratory to maintain daily processing levels while learning the new method in order to sustain optimal platelet inventory levels without relying on purchasing additional platelets from external vendors. our approach in introducing prt for apheresis platelets was to "flip the switch" and process all products with the new method rather than a step wise roll out with a dual inventory. study design/method: it was essential to prioritize who would be trained first. collections occur monday through friday from 0600 to 1600. the first group to be trained was those who would be performing training (a two person team) and product validation; they were trained by cerus deployment team. the second group was those who would process platelets on weekends and evening hours without direct management support. the last group was the technologists who would be working during normal hours with direct management support. prt processing for platelets in 100% plasma is broken up in to two days. on day 0 platelets are treated with amotosalen and placed in a compound adsorption device to remove residual amotosalen for 12-24 hours. on day 1 products are removed from the cad and modified into final product codes and labeled. each technologist was trained one on one, over a one week period. the trainers alternated training processing days for day 0 and day 1. in the weeks following training it was important that each technologist rotated back thru prt processing to maintain proficiency. results/finding: 13 of 18 employees were trained in a two month time period. prior to "flipping the switch" the daily average of products collected was 21. for the two month training period the daily average rose to 23. conclusion: our "flip the switch" training plan for implementing prt platelets in 100% plasma has been highly successful for our laboratory; training while implementing the new technology did not create a bottle-neck in the process. it was imperative to prioritize who would be trained first to insure complete coverage during off hour shifts. technologists were able to become proficient with the new process while maintaining daily processing expectations and sustaining an optimal platelet inventory. accepted depending on each individual's conscience. due to these unique medical challenges, it is important for caretakers to have an understanding of their beliefs in order to provide optimal care. we describe the process of identifying jw in our hospital and communicating treatment needs to staff. study design/methods: proper treatment of jw requires the ability to identify the patient and his/her needs. when a jw is admitted to our hospital, our electronic medical record (emr) triggers several processes based on the patient's listed religion. one process creates an order that reminds caretakers to complete the declining blood consent (dbc) with the patient. the dbc contains language declining mabf and reviews the mibf with the patient to identify any that would be accepted. the emr order regarding the dbc provides educational links that include a bloodless policy, step-by-step instructions on obtaining the dbc, and information on alternatives to transfusion. a second emr process triggers a stop-gate to prevent the completion of any mabf order or mibf order for a product that the patient has declined. a third enrolls patients in the minimal blood volume labs protocol which uses microtainers, partial-fill vacutainers, and blood reservoir sets to reduce blood loss during draws. additionally, at registration, a bloodless packet is added to the patient's paper chart. this packet contains the dbc, a glossary of dbc terms, a bloodless sign to be placed over the patient's bed, a bloodless wristband to be worn by the patient, and two bloodless chart stickers that are added to the outside of the chart. these steps remind the caretakers of the patient's special requests. finally, the patient blood management (pbm) department receives emr developed reports which identify jw presenting to the hospital. these patients are followed by the pbm nurses and medical director during the duration of care. treatment plans to optimize hemoglobin, oxygen carrying capacity, and hemostasis are discussed with the bedside caretakers and implemented as needed. results/findings: nearly 100% of jw that enter our hospital have a dbc completed. this has resulted in increased education of the medical staff. in addition, patients have reported better communication with caretakers leading to a more inviting environment for the patients. conclusion: our hospital has found success by using an education-based team-oriented approach involving emr, pbm, and caretakers when caring for the jw patient. this approach has set up a foundation for treating other bloodless medicine patients. background/case studies: transfusion services should provide safe blood components from vein to vein with donors acting as suppliers and patients as final customers. this process involves labor-intensive activities, critical materials, human resources, facilities and highly coordinated processes. cost management has a great impact on technical processes guiding decisions upon supplies and technical staff. activity-based costing (abc) is a method to determine cost drivers within activities and determine process or product final cost allowing managers to take precise decisions. we demonstrate how an effective abc approach can result in financial savings without compromising process quality in a mid-size transfusion service. study design/method: materials costs can represent as far as 90% of an activity. in 2015 we had a central storage supplying satellite storages at each department and replacement was done independent of residual stock. purchases were performed on demand. at the end of 2015 we performed a supply inventory on all departments to plan future purchases and control residual stocks. in 2016, we implemented annual purchases and satellite storages were supplied only to replenish programmed stock. cost drivers were defined upon activities on standard operational procedures (sops) resulting in a 2016 cost estimate. technical staff was involved in cost driver calculations to indicate possible changes to sops, supplier and deliveries. to minimize seasonal fluctuations we compared last quarter 2015 (q4/15) with last quarter 2016 (q4/16). in this work we present activity data from blood collections to illustrate abc method. results/finding: in q4/15 1756 blood bags were used compared to 1998 in q4/16, demonstrating an activity "13.78%. price negotiation resulted in 12.58% readjustment. both indicated an estimated cost "28.10% with a possible impact of over us$ 35,000. we have identified a real cost #2.31% in q4/16, representing an overall #14.89% and us$ 3,716.72 (r$12,235.16) savings. conclusion: economy had deteriorated in our country in 2016 with higher inflation and exchange rate variations, directly impacting imported materials, most of them critical. even with adverse economy, abc showed to be an effective tool that allowed cost decrease without significant changes in critical materials and processes. cost drivers calculations demanded review of sops and suppliers by technical staff resulting in optimization of activities. also, staff involvement reduced discharged materials since costs were wellknown to area supervisors and satellite stocks were reviewed briefly. automated verification of immunohematology results and the impact to donor testing barbara j bachman* 1 , candace williams 1 , carmen meyer 2 , paul lamonby 1 , anne cleverley 1 and silke milbradt-pohan 1 . 1 bio-rad laboratories, 2 diamed gmbh title: automated verification of immunohematology results and the impact to donor testing background/case studies: staffing challenges in today's blood banks require instrumentation with minimal operator intervention. technology advances have developed where every immunohematology result does not necessarily require operator visual review. this study evaluates the impact of automated result verification on the bio-rad ih-1000 tm immunohematology system through the ih-com tm data management system (dms) for donor processing laboratories. study design/method: a multi-center study was performed on donor samples as shown in table a evaluating two of the most commonly used ih-system gel cards available in the us. workflow data was analyzed using process modellar app (ipad). this study focused on post-analytical steps of result verification, evaluating with and without automated result verification to determine the impact on quality (# operator touchpoints, visual result review occurrence), result accuracy, and speed (time from result interpretation to lis data transfer). operator touchpoints during the post-analytical phase are only required when doing visual result verification and are software defined. speed metrics were analyzed using minitab v17, statistical the transfusion team collaborated with multiple user groups to educate them regarding the new processes. a gap analysis was performed to determine the optimal delivery process for blood products, with key stakeholders invited to review the options. the use of the pneumatic tube system to deliver blood throughout the entire campus was investigated to determine whether it would be a viable option given the expanded size of the new campus. results/finding: user groups requested additional training sessions as questions arose regarding use of the ehr for blood ordering. because the pneumatic tube system would be heavily used, and due to concerns that blood products could become "lost", it was decided this would not be the best route for delivery of blood. department educators requested support to create job aids specific to workflow changes impacting their departments, such as how to order rh immune globulin, a cord blood workup, etc. conclusion: leadership was challenged to provide a stable and positive environment during a complex set of changes. the simultaneous hospital move/merger and implementation of a new ehr constituted an arduous task that would not have been possible had substantial preparations not been initiated a year in advance. training is essential to the success for a scope of change this big and should not be minimized. while training was thorough prior to the move, gaps were nonetheless discovered following the move. abstract conclusion: strategic development partners funding and support based on newly developed government strategy on blood service with commitment of the government has brought a positive impact in establishing sustainable and safe national blood service program in ethiopia. even though the identified positive impacts mentioned are achieved, the bts remains with multiple challenges and needs continuity of funding and more partner support and government commitment. pilot implementation of a comprehensive hybrid performance management system at national blood service zimbabwe blessing mukwada*, judith j parirewa and tonderai mapako. national blood service zimbabwe background/case studies: the national blood service zimbabwe (nbsz) introduced its first performance management system (pms) in 2006. in the 2015-2018 nbsz strategic plan it was noted that the current pms lacked objectivitety and there was no relationship between perfomance and remuneration. in order to revise the pms, the nbsz set up a three membered committee at the executive management level to spearhead the revamp of the nbsz pms. the aim of the new pms was to achieve a shared vision of the purpose and objectives of the organization, helping each staff member to understand and recognize the contribution to the strategic plan. in this paper, we share how nbsz revamped and implemented its new hybrid pms that derived its inputs from established pmss and nbsz monitoring and evaluation (m&e) process that have been linked together. one-selected departmental results for one quarter are shared to demonstrate how the system works. study design/method: pms committee developed and shared with executive management a pms conceptual and implementation framework. consultations including field visits were done on three established pms to assess suitability for nbsz adoption. a hybrid pms was adopted for nbsz and a pilot application for one quarter on selected department was done. review of policy, procedures to including hybrid pms templates and forms were done. pms committee trained all staff on how to implement an integrated scorecard, how to conduct appraisal, how to develop scorecards, how to measure performance using the new pms, how weighted performance reward systems based on all layers of performance for bonus payments works using standardised tools. throughout the process risk assessment were done. results/finding: the nbsz hybrid pms is based on five levels of planning namely strategic, departmental, branch, sectional and individual. the fourcoloured traffic light reporting system is central in uniformly assessing performance at all levels. the levels of accountability were properly defined for each level of planning. a weighted overall integrated individual scorecard (iis) is determined based on 60% individual and 40% for the other four levels (10% for each). the bonus (%) is calculated based on the iis as follows; category a: 100% (iis >575%), category b: 75% (iis: 50 -<75%), category c: 50% (iis: 25-<50%) and category d: 0% (iis < 25%). on the pilot implementation, the individual scores for 12 staff ranged from 71% to 100%. the iis were 76% to 81%. the number of staff in each bonus categories were 11, 92% (category a) and 1, 8% (category b). conclusion: the new hybrid pms was generally accepted by all staff and it was easily implemented at various staff levels. this provides a basis for the full implementation of the new pms and this simplified pms can be easily be adapted in similar settings to ensure all staff contribute sufficiently and objectively to the realisation of the organisation strategic vision. rare donor engagement with american rare donor program (ardp) margaret c manigly* 1 , deborah r fludd 1 and sandra j nance 2 . background/case studies: rare donors are defined as a blood type occurring in less than 1 in 1000 people in a given population. these donors are discovered by testing new donors in a random or targeted way and require testing many donors to find one rare donor. once found, if the facility is a member of the american rare donor program (by being an aabb accredited or american red cross accredited irl), the donor is registered in the ardp database as a rare donor. in 2016, there were 65,801 active rare donors in the ardp. with the mobility of the population in the usa, it is important that as donors relocate, that they are recognized as a rare donor when they donate and their unit can be identified and used for a patient with a rare blood need. in addition, when recruitment is needed for a patient need, correct contact information on the donor is required. study design/method: the ardp procedure for ardp members requires that donors be contacted every six months to ensure that ardp (or the facility) has their latest contact information. the timing is determined by the postal service time limit of six months to forward mail to a new address. this contact ensures that if recruitment is required to obtain blood for a patient with a rare blood need, the donor can be contacted by the collecting center to donate. this contact is achieved by ardp sending a contact card by postal mail twice yearly to all donors for whom the ardp has address demographics. results/finding: the ardp reports on the information obtained from the contact cards returned in the ardp annual activity report to the ardp members at the aabb annual meeting. of the 6398 (9.7% of total active donors) returned contact cards alerting ardp of changes in calendar year 2016, 355 (5.5%) were donors moving from one ardp facility to another, 1369 (21.4%) were donors no longer eligible to donate, and an additional 4324 (68.4%) were address changes. other changes were 115 (1.8%) reactivated donors and 235 (3.5%) donors who we were notified were deceased, or did not want to be listed in the ardp. in 2016, 5390 new rare donors were submitted to ardp for registration. the number of donors that could potentially be lost to follow-up in 2016 was 4709 (355 1 4324), which would be 87.4% of the new donors submitted. conclusion: with nearly a 10% response rate for donors receiving the mailed contact cards, it is clear that rare donors (and their families) are responsive to the ardp contact card, and inform ardp of address changes and changes in their health status that affects their ability to donate. this is evidence of the importance of the card in ensuring correct donor contact information. in 2016, 4709 donors changed their addresses which often are not known to the collecting facility until the donors donates again, after their move. the ardp contact card is effective in retaining the relationship with the ardp registered donors and keeps the address information of rare donors current. workflow comparison of two gel analyzers in a large transfusion service j peter pelletier* 1 , barbara j bachman 2 , mike leamy 2 , susan olson 2 and candace williams 2 . 1 university of florida college of medicine, 2 bio-rad laboratories background/case studies: vendor-assisted workflow studies are becoming more popular as analyzer choices and capabilities vary in the market. the purpose of this study was to evaluate the provue (ortho clinical diagnostics) against the ih-1000 (bio-rad laboratories, inc.) in a large volume transfusion service using lean process flow. study design/method: twenty-two (22) runs of one to six (6) samples per run were observed for two ortho provues alternating testing at a large transfusion service performing 153,000 types, screens, type & screens (t&s) annually. the workflow patterns observed were then repeated on the ih-1000 and compared. each process was mapped in detail by direct observation using process modellar app (ipad). the evaluation started at sample centrifugation completion and ended with results sent from analyzer to lis (lab information system). each was evaluated for quality (testing process steps, biohazardous exposure episodes, and maintenance tasks), speed (operator/analyzer time) and cost (testing/maintenance personnel hours recaptured). time studies were analyzed using minitab v17, and statistical significance was assessed using the paired t-test, with p values of <0.05 considered significant. regardless of quality or speed metrics evaluated, the ih-1000 demonstrated a significant reduction (improvement) in process steps and associated times when compared against the ortho provue (p < 0.001). ih-1000 process steps and time studies addressed in the table below did not account for the ih-1000 reagent storage capacity. in reality, the improvements would be greater than what was displayed here in a real-life operation. evaluating the total number of maintenance tasks required annually, as well as the times associated with maintenance performance, there was a significant reduction on the ih-1000 (77% reduction, a difference of 43 hours/year). conclusion: this study verified the ih-1000 provided significant efficiencies and cost avoidance over the ortho provue for a large volume transfusion service. workflow comparison of two high volume, high throughput analyzers aaron samson* 1 , kimberly monnin 1 , barbara j bachman 2 , kyla warren 2 , susan olson 2 and candace williams 2 . 1 clinical pathology labs, 2 bio-rad laboratories background/case studies: few workflow studies have been performed on high volume, high throughput blood bank analyzers in large volume testing facilities. the purpose of this study was to evaluate the galileo v r neo (immucor) against the ih-1000 tm (bio-rad laboratories, inc.) using lean process flow. study design/method: a total of 12 separate test runs of 72 or 144 samples per run were observed over a three day period on the galileo neo at a reference laboratory annually performing approximately 211,500 type & screens (t&s). the workflow patterns observed were then repeated on the ih-1000 and compared. each process was mapped in detail by direct observation using process modellar app (ipad). the evaluation started at sample centrifugation completion and drop-off in testing area and ended with results sent from analyzer to lis (lab information system). each was evaluated for quality (process steps, biohazardous exposure), speed (operator/analyzer time) and cost (testing/maintenance personnel hours recaptured). time studies were analyzed using minitab v17, and statistical significance was assessed using the paired t-test, with p values of <0.05 considered significant. results/finding: detailed process steps, biohazardous exposures, and published analyzer maintenance tasks were evaluated/compared (table, part a) . time studies focused on operator time, analyzer time, and maintenance time (table, part b). regardless of quality or speed metrics evaluated, the ih-1000 demonstrated significant reduction (improvement) in process steps and associated times when compared against the galileo neo (p < 0.001). evaluating the total number of maintenance tasks required annually, as well as the times associated with maintenance performance and downtime, was a significantly reduced on the ih-1000 (difference of 120 hours/year). conclusion: this study verified the ih-1000 provided significant efficiencies and cost avoidance over the galileo neo for high volume/high throughput testing facilities. workflow impact of automated result verification for patient and donor blood typing barbara j bachman* 1 , candace williams 1 , carmen meyer 2 , paul lamonby 1 , anne cleverley 1 and silke milbradt-pohan 1 . 1 bio-rad laboratories, 2 diamed gmbh background/case studies: immunohematology facilities face many challenges including standardization, process control, productivity, staffing and patient safety. to alleviate these challenges, the ih-1000 tm instrument and complementary ih-com tm data management system (dms) were designed to provide lean automation to enhance blood testing facility workflow. the purpose of this study was to focus on the lean functionality of automated result verification on the ih-1000 and ih-com dms and determine its impact on workflow. study design/methods: internal and external studies using the ih-1000 with the ih-com dms were performed with patient and donor samples. assays included abo/rh blood grouping and antibody screening (abs) as shown in table a . workflow data was analyzed using process modellar app (ipad). the evaluation focused on post-analytical steps of result verification, evaluating with and without automated result verification to determine the impact on quality (operator touchpoints, visual result review occurrence, result accuracy), and speed (time from result interpretation to lis data transfer). operator touchpoints during the post-analytical phase are only required when doing visual result verification and are software defined. speed metrics were analyzed using minitab v17. statistical significance was assessed using the paired t-test, with p values of <0.05 considered significant. results/findings: using automated result verification, only 0.93% out of 6,339 samples evaluated for abo/rh testing would require visual verification, resulting in a 98% reduction in operator touchpoints (p < 0.001) and a labor saving of 444 minutes (7:01 hh:mm) for abo/rh testing. for 8,750 antibody screens, automatic validation of results would result in 99.5% reduction in operator touchpoints (p < 0.001) and a labor savings of 378 minutes (6:18 hh:mm). no false positive or false negative typing results or false negative screenings occurred with results auto-verification. (rbc) has remained, and in fact is proportionally increasing while blood usage has notably declined in the era of patient blood management. over the past 2 years a steady increase in demand for o neg rbc compared to other blood types has been observed at our blood center. utilization metrics for hospital customers are monitored monthly for overall trending and forecasting and the data shared with them during regular visits. despite heightening awareness, percent o neg rbc sales continued to rise by 1% annually and peaked at 16% in mid 2016. to better understand this increased demand a survey was conducted to gather insight for improved utilization. we speculated that during the survey an observer effect, or change in the staff behavior, would result in reduction of o neg rbc sales. study design/methods: a tie tag was designed as a survey tool and attached to each o neg rbc distributed to hospital customers for an 8week period in late 2016. hospital transfusion service staff were asked to record the final disposition of the o neg rbc (transfused, wasted, returned) on the tie tag. information on the survey objective and instructions for tie tag completion were communicated via customer meetings, emails and reminders sent by blood center drivers. completed tags were returned to the blood center. customers are allowed to return rbc units with greater than 10 day shelf life remaining. units with tie tags attached were in hospital inventories for up to 3 months due to the shelf life of rbc. return rates and percent of net sales (gross sales minus returns) by abo/rh type were tracked monthly before, during and after the survey. results/findings: participation was 100% of the 56 hospitals surveyed. mean percent o neg rbc gross sales for a 3 month period before, during, and after the survey was 16.6%, 16.0% and 16.5%, respectively. mean percent o neg net sales during the 3-month survey fell to 13.5% compared to an average of 15.4% in the 3 months prior. during the 3-month survey period o neg rbc monthly return rate increased to an average of 28.4% compared to an average of 23.0% in the 3 months prior. for the 3 months after the survey the average o neg rbc return rate further increased to 28.9% while mean percent o neg rbc net sales trended slightly upward to 14.1%. when customer hospitals were queried whether any process changes occurred, no major changes to policy or inventory levels were reported. conclusion: during and after the survey percent o neg rbc gross sales was fairly constant indicating that target inventory levels and transfusion service staff ordering practices remained unchanged. however, during the same period the increase in o neg rbc return rate and corresponding decline in percent net sales suggests improved o neg rbc utilization. increased awareness from participating in the survey and staff knowing they were being observed likely played a role in the lowering of percent o neg rbc net sales. tracking of monthly metrics will provide ongoing review to determine if the effect is transient or sustained and identify other opportunities for improving o neg rbc utilization. acoustophoretic separation of platelets from whole blood: a relevant and practical alternative to centrifugation pierre bohec* 1 , jeremie gachelin 1 , veronique ollivier 2 , thibaut mutin 1 , xavier telot 1 , benoit ho tin noe 2 and sandra sanfilippo 1 . 1 aenitis technologies, 2 hôpital bichat, inserm u1148 background/case studies: shear-induced platelet activation is an unwanted side effect of the centrifugation-based procedure currently used in blood banks to prepare platelet concentrates. transfusion of partly activated platelets could indeed increase the risk of adverse transfusion reactions. aims: here we evaluated the effectiveness of an innovative acoustic-based fractionation device by carrying out a qualitative and functional in vivo analysis of isolated human platelets. study design/method: whole blood was obtained from 14 donors and fractionated using an acoustic-based device. platelet recovery and purity were determined by quantifying blood cell subpopulations in the microchannel outlet samples. quality of isolated platelets was evaluated using the surface expression of two activation markers (p-selectin, pac1) using flow cytometric methods while their procoagulant ability was investigated using in vivo experimentation. platelets isolated using a soft-spin protocol, were used as inactivated control. results/finding: fractionation using the acoustic-based device led to a red blood cell clearance ratio from whole blood greater than 80 % (p< 0.001) and a purity of platelets close to 91.0 %. we did not find any difference in terms of quality and functionality of platelets from the same donors isolated using the acoustic device versus the soft-spin protocol. conclusion: this acoustic-based blood processing method led to excellent preservation of platelet quality and functionality providing a novel promising technique for whole blood fractionation in clinical settings. automation in blood bank processing: where we go? robert fernandez, lluis puig, pilar ortiz, joan ovejo, nuria martinez, elena valdivia and susana g gomez*. banc de sang i teixits background/case studies: nowadays, blood banking is requiring new strategies to manufacture blood components, due to the increase on their production. at banc de sang i teixits (bst), we have implemented during the last years automation manufacturing, including lean management methods, to be able to process our needs of over 250.000 blood donations for an area with more than 7 million people. study design/method: the automation of blood donations process, bst has done different changes on the equipment. in 2005, orbisac (terumo bct) was the equipment to obtain buffy coats and from this product, we got platelets concentrates. it was in 2007, when we moved from this equipment to atreus 2c (terumo bct), to get red blood cells, buffy coat and fresh frozen plasma. then we did some updated on atreus; in 2011 we changed to atreus 3c (terumo bct) and finally in 2013, we moved to reveos system (terumo bct). since the changes in 2007, our blood components were red cell concentrate, plasma, platelets and a leukocyte residue. while all these changes in processing equipment, we added also some automation in our registration (donation id, weight and temperature) and labeling steps, implementing two homemade robots. and finally, to get better results and more efficacy in our production, in 2009, bst incorporated an engineer to introduce lean manufacturing methods. these methods are based on the identification and analysis of problems, and then chose all these activates that add some value to the procedure. results/finding: once all these changes have been updated, we have evaluated the quality of blood components, such as red cells and platelets, also the number of donations that we were missing and working hours that were necessary to process our blood donations. this evaluation was done for processes during 2008 and 2016. conclusion: with these results, it's obvious that automation in blood banking makes more efficient the manufacturing of blood components, getting better quality of them and also in a cheaper way. we encourage maintaining lean philosophy in order to keep improving our methods and identifying those activities that add value to our processes and get rid of those ones that are not necessary. in a globalized and industrialized world, where everything changes very fast, these improvements are necessary to be on top of the field and be a state of the art blood bank. background/case studies: the laboratory envisioned an automated blood product delivery system that extended blood access to the bedside through the use of remote blood allocation devices, or "smart blood refrigerators" to improve patient safety, provide timely access to blood products, and potentially reduce laboratory workload. as part of this initiative, bloodtrack haemobanks (hb) (haemonetics, braintree, ma) were installed and interfaced to the existing safetrace tx (haemonetics, braintree, ma) laboratory information system. one hb was installed in the methodist hospital (rmh) campus which includes a busy outpatient infusion therapy center (itc). study design/method: an assessment of the current blood supply chain revealed improvement opportunities for both nursing and blood bank staff. frequent daily trips to and from the blood bank take nurses away from the patient beside and can create congestion at the blood bank window during peak times. for the itc, with a daily outpatient volume of 140-160 patients and an average, round-trip travel time of approximately 15-minutes, even small delays waiting in line at the blood bank window would produce profound ripple effects. itc nurses faced the additional challenge of maintaining nurse-to-patient ratios and providing timely patient care. about 30% of patients in the itc have same-day transfusion orders, adding to the blood bank workload and creating unpredictability in workflow. often for patients in the itc, nurses had to repeat pre-transfusion vital signs because too much time had elapsed between gathering vitals and obtaining the blood. these inefficiencies resulted in longer patient wait times and, ultimately, a longer stay in the itc. results/finding: hb devices allow nursing staff to access red blood cells (rbc) for the majority of their patients at the point of care. since implementing in november 2015, the hb has significantly improved the turnaround time of rbc issue -from 15-minutes to less than 60-seconds-and helped maintain nurse to patient ratios and reduced traffic at the blood bank issue window. prior to hb implementation, blood bank staff at rmh were issuing approximately 750 rbc per month out of the window for non-surgical patients. this has been reduced to approximately 300 rbc per month, a 60% average monthly reduction. conclusion: having the hb located in the itc has helped to expedite the care of patients and more easily manage blood products for patients with same-day orders. the use of hb devices has not resulted in a reduction in blood bank fte, but rather a shift in workload; from issuing products to monitoring inventory and restocking. consists of registered paramedics that are pararescue specialists and helicopter personnel. when in combat, the squadron conducts personnel recovery operations and rescues downed airmen. when stationed in the us, they mitigate in state emergencies and perform aeromedical evacuations. in 2015, they supported a civilian medical emergency and the patient needed a transfusion in the field. they procured blood products from a distant air force base with adjacent medical facility. at the debriefing, members of the 131 st rescue squadron (131 rqs) decided to find a local civilian blood supplier. the master sergeant contacted our blood center and set up a contract for blood supply. study design/method: blood center representatives met with the 131 rqs master sergeant in january 2016. we asked what 131 rqs's order and delivery expectations were. he said sporadic use and the blood order would be 2 rbcs. we wrote a procedure for consignment and packaging, using standard blood transport boxes. we developed a communication template for staff to anticipate the 131 rqs needs. staff was trained based on data from january 2016 meeting. we contacted the 131 rqs in september 2016 to perform a trial run. at that time, we learned the master sergeant was shipped out to military theater. we invited his replacement to the blood center. this pararescue senior airman had just returned from syria and was assigned to civilian duty. he had no prior knowledge of the 131 rqs association with a civilian blood center. based on his field experiences, he changed the blood order from 2 to 6 rbcs. he introduced blood transport containers, used in military operations, saying they were easier to carry during water and land pararescue missions. we rewrote the procedures, incorporated his transport containers, and made a pictorial job aid to assist staff on packaging blood using these containers. the blood center and 131 rqs performed a mock run on october 31, and we felt prepared for any future events. results/finding: on november 11, 2016, the 131 rqs was deployed to a civilian aeromedical evacuation. we anticipated a 6 rbc order. the actual order was 7 rbcs and 4 ffp. staff was preparing frozen ffp to ship, as was their norm for filling hospital orders. realizing that they could not thaw plasma in flight, we contacted the 131 rqs and offered liquid plasma instead, which they accepted. product was consigned and picked up at 4:30am by the 131 rqs. the patient was transfused in the field and then taken to a nearby hospital. at our joint debriefing on november 28 th , we established a maximum blood order of 10 rbc and 4 liquid plasma, noting future orders may request fewer products, yet meet the preferred 2 rbc; 1 plasma transfusion ratio. conclusion: military personnel are adapted to instantly adjust to an ever changing environment. regulated blood centers are not as adaptable. with clear and comprehensive communication and anticipation on the blood center's part, we now supply civilian blood products to the air national guard. (table 1 ). the highest mean fib concentration was 535 mg/donor unit; lowest mean fib concentration was 264 mg/donor unit. all sites had a mean fib concentration at least 100 mg/donor unit above the fda minimum requirement of 150 mg/donor unit. fifteen of 17 blood centers completed the manufacturing process survey. one used a leukocyte reduction filter with ahf destined plasma. all blood centers manufactured single donor cryoprecipitate; 12 manufactured pooled donor cryoprecipitate. most froze plasma in a -188c or colder blood bank freezer. one froze plasma using dry ice, and one used a blast freezer. two blood centers method of thawing frozen plasma took longer than 10 hours. conclusion: blood centers consistently met the overall fib minimum requirement with a mean of 345 mg/donor unit, over double the fda requirement. however there is variability in fib levels amongst blood centers. in general, manufacturing processes were similar with a few exceptions. blood centers should inform their hospital customers of their average fib level in cryoprecipitate in order to most appropriately care for patients receiving this product. compliance & productivity improvement via engineered-staffing/ scheduling calculator application (app) mary deck, mark angelelli and kevin lee*. american red cross background/case studies: the healthcare industry, particularly the blood banking industry continues to experience tremendous pressures not only with ensuring patient safety and quality daily, but managing and maintaining an efficient operation with a cost competitive structure. applications of basic industrial engineering tools, coupled with lean-six sigma techniques such as time study analysis, bottleneck elimination & process standardization to transfusion reduce variation has been transformed into an application (for short "app"), which can be utilized to determine process and staffing optimization and provide flexibility to the dynamic nature of changing needs in blood banking. study design/methods: a time study analysis offers valuable data about the process requirements. once this baseline has been established, translating the data into a user-friendly app would enable ease and practical use to facilitate business decision-making as well as effectively manage daily operations. important concepts such as lean-pull production system, bottleneck elimination, work-load balancing together with basic development of the app using ms excel software will be demonstrated. results/findings: successful rollouts and implementations of the staffing/ scheduling calculator app across pilot facilities, then onto facilities nationwide, has yielded improved productivity together with a sustainable compliance scorecard. the app interactive-based approach, programmed via a commonly used software, ms excel, was used to analyze how to optimize staffing requirements together with staff-scheduling (i.e. match incoming volume/work content to staffing availability). the staffing/scheduling calculator app has been utilized by executives to evaluate "what-if" scenarios (sensitivity analysis) as well as a planning toolkit to proactively manage the changing demands of blood banking. conclusion: besides providing a key mechanism for increased productivity and sustained compliance -a top priority for blood banks -the staffing/ scheduling calculator app will highlight continuous improvement opportunities and spring-board to system-wide acceptance and standardization. all coolers were prepared in a walk-in refrigerator. two scoops of wet ice or two ice packs were placed at the bottom of large/medium or small coolers, respectively, with rbc units on top of the ice. a quality-controlled thermometer was placed on top of the rbc units. a control thermometer was place at the interface between the ice and the rbc units in one large and one medium cooler. the start temperature was recorded and then the temperature was recorded every 15 minutes for a 12 hour period or until the temperature exceeded 68c. results/finding: the temperature recorded from the thermometer on top of the units in all five coolers reached >68c in 75 minutes as shown in table 1 . the control thermometer recorded temperatures maintained at 1-68c for the entire 12 hour observation period in both the large and medium cooler. conclusion: when units are placed on top of the ice in a cooler, the temperature is not reliably maintained at 1-68c for more than 45 minutes. these data support a policy of wasting units that are returned to the blood bank with rbc units on top of the ice. background/case studies: an fda draft guidance has highlighted the need to reduce the risk of bacterial contamination of platelet components (pc) via pathogen reduction (pr) or secondary rapid testing (rt). hospitals must understand the cost implications that may result. our objective was to create an interactive model to analyze the budget impact for different pc types across the range of existing us hospitals. study design/methods: an excel model was built and populated with base case costs and probabilities identified through literature search as well as through a survey administered to 27 us hospital transfusion service directors. the model was reviewed and refined by a panel of seven transfusion medicine physicians. the model allows base-case assumptions to be overwritten with values specific to the institution. three scenarios were generated to compare annual costs of plt acquisition, testing, wastage, dispensing /transfusion, adverse events (ae), shelflife, and reimbursement for a hospital that purchases all of its pcs: 100% conventional (c-pc), 100% pr-pc, and mix of 75% c-pc/25% pr-pc. the model predicts a modest ($4%) cost increase for pr-pc compared to c-pc depending on the degree of pr conversion; this takes into account cost offsets such as elimination of bd and irradiation, decreased waste due to increased shelf-life, and outpatient reimbursement. the effective pc shelf-life is potentially increased with pr due to elimination of bd, and is dependent on nat turnaround time. benefits not captured by the model include transfusion-transmitted infectious risk mitigation from emerging pathogens, which may impact cost/benefit analyses. future iterations of this model will also enable hospitals to consider scenarios in which rt is used. this model can serve as an important tool for hospitals considering pr adoption. in january 2011. a report was created to identify donors previously classified as rare according to the american rare donor program (ardp) criteria. donors are classified as rare by meeting one of the following: highprevalence antigen negative, multiple common antigen negative, or iga deficient. the new process utilized the report and involved sending a letter to the donors notifying them of their rare donor status and encouraging them to continue to donate. a database was created to track the letters sent to rare donors. in august 2013, inventory reduction efforts were implemented to gradually decrease the number of allogeneic red blood cells (rbc) collected to minimize unit age at transfusion. the inventory reduction occurred in phases and was completed by january 2015. a study was performed to determine the impact of the inventory reduction on the number of rare donor donations. study design/methods: the total number of allogeneic rbc donations, rare donor donations, and number of rare donor letters sent was analyzed from 2010 to 2016 (see table) . the percentage of rare donor donations per year was calculated. background/case studies: blood centers (clients) often carry low inventory of blood and blood components. laboratories performing donor screening therefore, have limited time to determine the presence or absence of infectious disease within these products. in order to measure and ensure expedited donor screening we implemented a daily performance metric consisting of upload time goals for release of results to clients. in 2016, zkv-nat testing was implement for travel deferral donors (july), followed by universal individual donor screening in september and november in response to the fda recommendations for "reducing the risk of zkv transmission by blood and blood components". per the fda guidance we implemented mandatory zkv testing for clients with proximity to areas with locally acquired mosquito-borne cases of zkv within 4 weeks (sept. phase 1) and nationwide within 12 weeks (nov. phase 2). zkv testing is performed on individual samples, unlike all other nat tests that are performed in minipools (16-donations). therefore zkv testing has a disproportionate impact on the turnaround times for testing, which we analyzed in this study. study design/method: within two regional testing labs, participating in the same clinical trial, lab 1 had 86% and lab 2 had 77% of clients requiring universal zkv testing. we evaluated a 12-month test result upload performance period to determine the impact of zkv test implementation. results/finding: during 2016, lab 1 upload time performance ranged from 92% to 94.2% from january to july; upload time performance fell between august through november, returning to 94.2% performance in december. lab 2 upload time performance ranged from 91.4% to 95.3% january to august. performance fell september through december 83.3% -88.5%. lab 1 experienced a low of 75% upload time performance during phase 1 when there was a rapid implementation; 69% clients required zkv nat. improved performance was observed during phase 2, with a 16% increase in zkv clients. for lab 2: phase 1 experienced a modest decline of upload performance ranging from 83.3% to 88.5% with 33.3% of clients implementing zkv nat. performance was 87.1% in phase 2, when an additional 43.3% of clients implemented zkv testing. conclusion: with an unprecedented rapid implementation of zkv testing our laboratories experienced a short period of reduced ability to maintain our upload time performance metric. enhanced platelet bacterial screening in an eight-hospital system robin larson* 1 and colleen a. aronson 2 . 1 advocate lutheran general hospital, 2 acl laboratories/ advocate hospitals background/case studies: in response to two platelet-related septic transfusion reactions and the draft fda guidance released in march 2016 regarding bacterial risk control strategies for transfusion services, an eight-hospital system implemented the verax pgd enhanced platelet bacterial screening test in 6 of the 8 hospital transfusion services. the 2 sites that did not implement the test arranged for fresh platelets to be rotated in from the blood supplier. the 6 sites which implemented the verax pgd test perform testing on all day 4 and day 5 platelets to be issued for transfusion. this abstract summarizes the data collected for the first 5 weeks of testing. study design/methods: platelet bacterial testing logs were reviewed over the entire time period studied for platelets tested on day 4, day 5, and those that were tested twice. inventory reports were reviewed for platelets issued on day 2 or day 3 that did not require testing, and for the total number of platelets issued over the time period studied. results/findings: in the month of february (1 week of performing the test), 48.1% of all platelets issued by the 6 participating transfusion services were day 2 or day 3 platelets. in march that number dropped to 29.9%. it is expected that this number will level off at some percentage at or below 29.0% with further data collection. in february 25.9% of platelets were tested twice prior to final issue from the transfusion services. in march conclusion: the percent of platelets issued fresh (day 2 or day 3) will likely level off at some number at or below 29.9% due to inventory management from both the blood supplier and the individual transfusion services. testing platelets twice is undesirable. ideally, no platelets would be tested twice as this represents a high cost for both the test reagents as well as the staff time to complete the testing. in addition, 5 of the 6 sites performing testing are level 1 trauma centers and need to have tested platelets available at all times. this will require some amount of double testing, but the goal is to have this number be as low as possible, so that the percent of tested vs issued platelets does not exceed 100%. as the transfusion service staff becomes more comfortable with judging inventory levels and performing testing, it is expected that the amount of double testing will decrease. background/case studies: in order to make up for the deficiency of the apheresis platelets in clinical application, and also to improve the comprehensive utilization of blood, we investigate the feasibility of preparation of pooled platelet concentrates(pcs) for providing a reliable source for clinical application. to speed up the storage research of pooled pcs in china, we evaluate the changes in platelet function after filtering leukocytes with leukocytes filter for pcs and the quality changes during storage in pvc-bthc blood bags. study design/method: pcs were prepared from 400 ml virus free whole blood by platelet-rich plasma (prp) method. five or six bags of abomatched pcs were pooled and filtered with leukocytes filter for pcs(n57). the swirling phenomenon, ph, automatic blood count, platelet aggregation, hypotonic shock response (hsr), the extent of shape change(esc), cd62p expression, atp level in platelet, glucose and lactate concentration were detected before and after filtering, and on days1, 3, 5 and 7 of storage, respectively. results/finding: the platelet recovery ratio of a therapeutic dose of pooled platelet concentrates after filtering leukocytes was (86.7 6 1.6)%, relative change rate of hsr was (3.87 6 12.75)%, the residual leukocytes were (0.15 6 0.15)310 6 . the ph, hsr, and the cd62p expression of pooled platelet concentrates before and after filtering were (7.00 6 0.17) vs (7.06 6 0.16), (66.96 6 12.35)% vs (63.22 6 8.26)% and (28.94 6 14.25) % vs (31.60 6 16.77)%. there is significant change for wbc after filtering (p<0.01). during storage in pvc-bthc blood bags, the biochemical parameters of pooled platelet concentrates changed with increasing storage time, as shown in table 1. conclusion: storage in pvc-bthc blood bags for five days, the quality of pooled pcs met the requirements of chinese standards (gb 18469-2012) . it can be a complementary source for apheresis platelets supplement in china. evaluation of samplokv r segment sampler to obtain and measure samples from blood component tubing segments abbejane blair*. ajblair laboratory consulting background/case studies: current methods used to obtain samples from blood component tubing segments are cumbersome and present a significant risk for exposure to biohazards, sharps injury and cross contamination. itl biomedical has developed samplokv r segment sampler (ss), a device for obtaining measured samples from sealed tubing segments that is less cumbersome and offers improved safety, eliminating the need to manually cut and squeeze tubing segments. ss was evaluated with the goals of reducing the number of steps required to obtain a measured sample, and, reduce biohazards and sharps exposure. study design/method: ss obtains fluid samples from sealed tubing segments into a needleless syringe. it consists of two chambers with recessed internal needles located at the top of the device and a female port located at the bottom of the device. a needleless syringe is attached to the female port, the sealed tubing ends are then aligned with the ss chambers and, gently pushed onto the needles to pierce each end of the segment. the sample from the segment is then withdrawn into the syringe. the study was performed at rhode island blood center (providence, ri) using tubing segments from three bag manufacturers to demonstrate ease of use on the following processes: segment alignment over needles and piercing, ability to draw sample into syringe, ability to expel air bubbles from syringe, fluid leaks, ease of transfer of sample from syringe to tube and to collect user feedback. two lengths of tubing segments were filled to contain sample volumes of 500ml and 1000ml. two users then evaluated the ss tubing segment types with 500ml or 1000ml samples for a total of 10 data points. samples were collected into the attached 1ml or 3ml syringe then a measured sample was transferred from the syringe into a test tube or microcentrifuge tube. results were tabulated as pass or fail. results/finding: a total of 10 ss were evaluated by two users. all samples were successfully collected and transferred into tubes. insertion of the segment edge requires observation to ensure placement onto the needles. any air bubbles collected into the syringe could easily be moved to the top by background/case studies: the management of platelet inventory is crucial due to a number of factors including the 5 day product outdate, the allocation of staff due to the lengthy donation process, the increasingly small donor pool, and the high cost of production (e.g. platelet collection kits, testing, product processing). the use of a platelet inventory management tool has the potential to enhance the understanding of units transfused, optimize inventory, increase efficiency, and reduce waste. the objectives of this assessment were to decipher if the platelet inventory management tool has reduced the amount of outdated platelet products, total cost of platelet production, and full time equivalent (fte) allocation. study design/method: in january 2015, a platelet inventory management process was implemented which uses a spreadsheet based tool to predict the amount of platelet collection procedures needed to be scheduled each day. the tool uses daily historical transfusion data from the last five weeks. additional calculations are included to account for deferrals, no shows, incomplete collections, and product split rate. the number generated from the calculations correlates to how many platelet collection procedures to schedule for the specific day of the week considering testing release and historical daily transfusion trends. the effectiveness of the tool was verified by comparing platelet collections, platelet products outdated, and fte information for a one year period prior to the implementation of platelet inventory management to one year period following implementation. results/finding: by implementing a platelet inventory management tool, collections have been lowered or shifted to accommodate the transfusion needs. the staffing adjustments and targeted collections have lowered fte and outdate cost by 22%. the platelet outdate rates dropped after implementing the platelet inventory tool from 14% (1324 units) to 11% (874 units); a 21% decrease. fte was able to be monitored closely with the donor schedule and lowered from a yearly average of 7 fte to 6.2 fte, lowering fte by 11%. conclusion: considering historical transfusion data for potential platelet demand has had a positive impact on scheduling platelet collections. staffing requirements and outdating products have decreased since implementation of the platelet management spreadsheet tool, leading to less waste both in terms of staffing and platelets. given these positive results, we are beginning to develop a similar tool for our whole blood collections. identifying opportunities to right-size hospital inventory using compotrace radio frequency id inventory management system nanci fredrich* 1 , jaclyn mckay 1 , jennifer curnes 1 and rowena punzalan 1,2 . 1 bloodcenter of wisconsin, 2 children's hospital of wisconsin background/case studies: the ability to track inventory of blood components in real time is challenging for both hospital transfusion services (ts) and blood centers (bc) using current blood bank information systems (bbis). in addition, determining if established par levels of individual components meet or exceed daily transfusion needs is difficult to ascertain. a pilot was designed to track and monitor all blood components from distribution at the bc to issue in a hospital ts using fresenius kabi compotrace radio frequency id (rfid) enabled inventory management system. the objectives were to determine feasibility of the compotrace system and analyze compotrace data for real-time usage and optimal inventory levels. study design/method: a 3 month pilot was conducted at a pediatric hospital and its bc using both bbis and compotrace systems to track all adult-size blood components. staff were trained on use of compotrace system. upon receipt of order from pilot hospital, bc staff applied rfid tags to all component bags and scanned components into the compotrace system. components were transported and delivered to ts following established procedures. upon receipt at the hospital, components were scanned into inventory using both the ts bbis and compotrace systems. dual scanning of components occurred upon issue to or return from floor, component modification or return to bc. products for emergency use or at time of high demand were not rfid-scanned. a priori, the pilot would stop if the compo-trace system hampered current workflow, component issue was delayed or if ts errors increased. no inventory changes were made during the pilot. results/findings: real-time data from compotrace system provided actual usage for all blood components including component disposal and shipment to and from bc. average daily rbc inventory levels and usage for selected blood types is shown in table. lessons learned related to equipment and workflow: (1) use of smaller irradiation canister may damage rfid tag, which was resolved by relocating tag, (2) ts workflow and stat orders challenged consistent use of dual processes to track component status. however no increase in ts errors or delay in issue of components occurred. conclusion: use of rfid to track blood components from bc to final disposition is feasible. real-time data from compotrace system identified optimal inventory levels for rbc at the pilot ts. use of real-time rfid to track inventory and adjust target levels based on actual daily usage over time may reveal seasonal influences that affect target inventory. background/case studies: physicians expect blood to be available at all times. following a national appeal in july 2016 for donors based on a predicted summer shortage with high likelihood of extending into the fall, our transfusion service (ts) recognized a potentially dire situation given the institution's patient acuity. our hospital-based ts supports a full range of services: a level i trauma service; stem cell and solid organ transplant services; a brisk cardiothoracic surgical program; a high risk obstetrical service; and high acuity medical/surgical services. a regional donor center supplies our blood products. to insure appropriate response to patient needs, the ts created a management plan, with input from multiple stakeholders, to assist with product management in times of extreme shortages. the approach is described herein. study design/method: at the direction of the transfusion committee (tc), ts directors presented the concern for impending shortages to the hospital quality directors (qd) committee. the qd committee consists of clinicians and non-clinicians trained in health care quality/regulatory affairs who are responsible for institutional health care quality (hcq) activities. the qd recommended creation of a multidisciplinary team: "the blood shortage task force (bstf)", analogous to an existing task force started for management of drug shortages. results/finding: with hcq and tc support, the ts created the bstf and blood shortage management algorithm (bsma). standing members of the bstf include ts medical director (chair), senior vice president (svp) of hcq, svps of clinical services director of regulatory affairs, legal counsel, and representatives from ethics, social work, pharmacy, patient referrals, and communications. ad hoc members include those whose patients would be most impacted by the specific shortage. the bsma designed by the bstf provides a framework for ts's to conduct operational and therapeutic assessments of potential impact and defines criteria for convening the bstf. trigger criteria include: marked ts concern; essential product; high likelihood of inventory depletion; broad patient impact. once convened, the bstf is responsible for situational assessment and formulation of a management plan, with a goal of maintaining quality patient care. conclusion: faced with the potential for limited blood supply, the ts reached beyond the laboratory and engaged the tc and members of hcq to assemble a robust, multidisciplinary task force. this resulted in an inclusive plan which can be activated at any time to address shortages, and assist in management of impacted patients. abstract background/case studies: cryoprecipitate (for short "cryo") plays a critical role in clotting and controlling hemorrhaging, and is often used in the treatment of massive trauma and major diseases, including metastasized cancers, cardiac diseases, hepatic failures, and organ transplants. the collection process of cryo is particularly challenging; due to fact to be processed into cryo units, the collected whole blood has to be shipped to the production facility and be processed within 8-hours after collection. this tight 8hour time constraint between collection and production can only be satisfied with precision collection planning and extra courier services; which makes the collection for cryo units more costly than other products. study design/methods: the american red cross (arc), in partnership with researchers from the georgia institute of technology (gt), has developed a blood collection model to increase the amount of whole blood that can be processed into cryoprecipitate. after reviewing blood collecting and processing schedules, collection locations, and other factors, arc-cryo subject matter experts together with gt researchers were able to analyze the problem structurally with several analytic/dynamic programming properties, and developed a near optimal solution algorithm or mathematical model. results/findings: to facilitate implementation, a decision support tool (dst) was developed to systematize the selection of the collection sites; determining when and from which mobile collection sites to collect blood for cryo production and how to schedule the courier services such that the collection targets are met and the total collection costs are minimized. the implementation of the dst led to an increase in the number of whole blood units satisfying the tight 8-hour completion time constraint for cryo production (capacity expansion). in particular, during the 4 th -quarter of 2016, a blood processing region was able to process about 1000 more cryo units/month (an increase of 20%) at a slightly lower collection cost (cost avoidance), resulting in an approximately 40% reduction in the per unit collection cost for cryo. conclusion: by utilizing operations research toolkits, a mathematical model or near-optimal algorithm could be developed to optimize the cryoprecipitate collection process, ensuring the time constraints and product consistency levels are achieved. this interdisciplinary improving cryoprecipitate collections collaborative project has been selected as a finalist on the 2017-the franz edelman award, recognizing outstanding achievements and practices in operations research. inventory management and transfusion practice before and after 7-day apheresis platelets sarah k harm*. university of vermont medical center background/case studies: the shelf life of apheresis platelet (ap) units stored in plasma may be extended from 5 to 7 days in the usa using an fda cleared rapid test (rt). in august 2016, our hospital based transfusion service began using a rt on day 6 and 7 to routinely extend ap shelf life to 7 days. this report describes changes in platelet inventory management and transfusion practice six months following routine use of 7-day ap. study design/methods: data were obtained for two study periods: september 2015-february 2016 (pre-implementation) and september 2016-february 2017 (post-implementation). the study periods were intentionally made to span the same months of the year due to seasonal variability in platelet transfusion rates in our region. the transition period from 5-day to 7-day ap inventory was excluded. the following data was collected for each study period: the total number of ap transfusion recipients, ap units transfused, expired ap units, ap units ordered ad-hoc from suppliers, inpatient admissions, surgical volumes, and average length of stay. results/findings: data are shown in the table. the number of ap transfusions decreased by 3% post-implementation while inpatient admissions and surgical volume increased by 1% and 3%, respectively. the hospital length of stay was similar for both periods. ap inventory decreased by 36% post-implementation and the outdate rate decreased from 29% to 15% (p<0.0001). ad-hoc ordering was not statistically different between study periods (p50.10). the average number of ap transfusions per patient between pre-and post-implementation periods was not statistically different (2.1 and 1.9, respectively, p50.65). furthermore, a new "rejection threshold" for lipaemic products will be implemented. this threshold represents the tg concentration above which viral marker testing for donor screening will be affected. in kcbb abbott's prism assays are used for: hbsag, anti-hcv ab, anti-hiv ab, anti-htlvi/ii . results/finding: using data management system and file records in kcbb as regard discarding blood components due to lipaemia during the last five years (2012) (2013) (2014) (2015) (2016) , it was demonstrated that number of discarded rbcs due to lipaemia during the whole period was 4892 units. number of discarded different plasma, platelets, and cryoprecipitate components during the last two years due to lipaemia was 8546, 24, and 2 units respectively. the mean number of discarded rbc units of the five years of the study exceeds 50% of the tested ones. literature about guidelines on the management of lipaemic donations were reviewed in order to minimize donation loss, and establish an accurate rejection threshold for lipaemic donations. by reviewing sample requirements for viral marker testing in kcbb, the accepted level for tg in blood samples is below 1000 mg/dl, and so the rejection threshold for lipaemia is level equal to or more than 1000 mg/dl. conclusion: many blood product units are discarded needlessly in kcbb due to lipaemia in the last five years (including 4892 rbcs, 8546 plasma products and 24 apheresis platelet units). in an effort to reduce the waste of potentially lifesaving products, the rejection threshold for lipaemic products is recommended to be changed from 200 mg/dl to 1000 mg/dl which does not affect blood safety. a follow up study is recommended after applying the new threshold to evaluate the new policy. logistical management of the incorporation of pathogen reduced single donor platelets (pr-sdp) into inventory at a u.s. tertiary care medical center eric gehrie* 1,2 , rebecca ross 3 , debra mraz 3 , anne baker 3 , zenna neal 3 , melanie champion 3 and edward l. snyder 2,3 . 1 johns hopkins university school of medicine, 2 yale university, 3 yale-new haven hospital background/case studies: the approval of pr-sdp by the fda provided an opportunity to improve the safety of our platelet inventory across all patient demographics. we outline our approach and address issues we faced during the first 4 months of pr-sdp availability. study design/methods: our nursing education team provided presentations to the nursing and clinical unit support staff. a company-sponsored trainer staffed sessions for the evening/night shifts on the clinical wards. presentations to physicians were made by the blood bank medical staff. information technology personnel created a new product type in the blood bank computer system, tested the abo/rh truth tables, and ensured that billing codes were in place. the necessity for transiently supporting a dual inventory of pr-sdp and conventional platelets led to consultation with the ethics committee and risk management, to confirm that pr-sdp and conventional platelets (c-plts) tested for bacteria ("safety measure" testing) could both be considered the hospital standard of care. we chose to not gamma irradiate any unit of pr-sdp, consistent with the package insert. results/findings: the ethics committee and risk management confirmed that informed consent was not needed for transfusion of pr-sdp. pr-sdp available from our blood supplier incremented monthly. over the first four months of pr-sdp availability, 777 pr-sdp were transfused at our hospital (out of a total of 3286 platelets transfused). after 4 months of scale-up, pr-sdp were approximately 30% of inventory. questions received during the nursing and medical conferences related to: the risk of bacterial contamination with c-plts vs. pr-sdp; toxicology of the pr process; scanning pr-sdp labels into the electronic medical record; and the need to irradiate pr-sdp. our use of a "safety measure" addressed concern over bacterial contamination of c-plts. published pr-sdp toxicology data comparing the content of psoralens in food products such as grapefruit ($12 mg per 100g) to the content in pr-sdp (<1 ng per ml) addressed toxicology concerns. nursing/it allayed concern over scanning issues with a simple demonstration. finally, we ensured that all parties were aware that fda did not require irradiation of pr-sdp. presentations at the medical conferences were also used as an opportunity to provide transfusion-transmitted disease training and information on platelet utilization. company personnel did not present at medical or nursing conferences per institutional policy. no background/case studies: ensuring platelet supply capability represents a challenge in terms of donor recruitment and inventory management operations. in september 2017, the apheresis collection process (acp) was completely revised to increase the number of products per donation by maximizing the rate of double-platelet donations (dpd). the process review has led to several changes, including the substitution of the pre-donation platelet (plt) count measurement before donation type allocation, in favor of the use of the donor's past donation records. multiple processing steps were eliminated, and the evaluation of plt concentration as a function of time, deduced from complete blood count (cbc) measurements, allowed the centralization of the analysis at the qc department. finally, introducing the concept of non-optimal donations has led to an increase in the proportion of dpd. study design/method: at the donation centers, whole blood (wb) from 10 donors was collected in k 3 edta tubes. plt concentrations were determined at the qc department using the coulter act 5 diff hematology analyzer (beckman coulter). sample tubes were stored at 20-248c and measured at 24, 48 and 72 hours post-collection. single platelet donations (spd) or dpd were collected using the trima accel. units were pooled and split in elp (extended life platelet, terumo bct) storage bags to mimic spd (250 ml; n58) or dpd units (500 ml; n54). plt pools were stored at 20-248c under mild agitation for seven days except for dpd, which were split in two 250-ml bags after 18 6 1 h. samples were taken on days 1 and 7. ph, po 2 and pco 2 , hypotonic shock response (hsr), extent of shape change (esc), cd62p expression, atp content, lactate and glucose concentrations were used as in vitroquality markers. results/finding: plt concentration as a function of time, determined from wb cbc measurements, showed no significant difference at 24h (247 6 32 pltx10 9 /l), 48h (247 6 27 pltx10 9 /l) and 72h (247 6 32 pltx10 9 /l) postdonation. dpd can be stored in the same collection bag for 24h after donation without any significant impact on plt quality markers. plt concentrations were within the manufacturer's acceptable limits (1141-1526 pltx10 9 / l) before splitting. on day 1, lactate and pco 2 concentrations increased, and po 2 decreased in dpd. however, these values normalize to those of control units at the expiration day. conclusion: this project was approved by health canada and implemented in our organization in march 2017. there are numerous operational and cost benefits from this process optimization initiative, without significant impact on safety and quality. post-implantation efficiency data will be compared to the targeted 12% increase in the targeted number of plt units per donation ratio. phased implementation of pathogen-reduced platelets in a health system elizabeth s. allen* 1 , colleen vincent 2 and patricia kopko 1 . 1 university of california -san diego, 2 american red cross background/case studies: pathogen reduced platelets (prp) provide improved safety compared to conventional apheresis platelets, but collection and manufacturing are complex. early evidence shows only 40-45% of double platelet collections meet requirements for pathogen reduction treatment. 1 blood centers need hospitals to implement prp to start manufacturing, but hospitals may not wish to use prp until they can provide the product to all patients. scaling up manufacturing at the blood center and phasing in prp across patient populations meets both parties' needs. we evaluated this strategy at our university health system (transfusion volume: 9,500 apheresis platelets annually), which includes two hospitals (750 inpatient beds) and an outpatient cancer center. study design/method: before initiation, approval and funding were obtained from the hospital quality council and administration, and stakeholder groups such as hematology/oncology were educated and consensus gained. live training was provided for nurses in the outpatient cancer center (week 0) and the bone marrow transplant (bmt) ward (week 6). an e-mail communication explained the change to all physicians and nurses. in phase 1, we implemented prp in the outpatient cancer center. these patients are immunocompromised and do not have access to the immediate advanced critical care of the inpatient environment should a septic reaction occur. in phase 2, we expanded usage to include the inpatient bmt ward. in phase 3, we lifted all restrictions so prp could be used throughout the health system, with the goal to reach 100% prp within 6 months. results/finding: in phase 1 (weeks 1-6), we requested 31 prp products weekly, based on typical usage in the outpatient cancer center. our blood supplier provided an average of 23 prp weekly (range 9-33), and prp constituted 44% of platelet transfusions in the cancer center. in week 2, excess prp inventory required use of prp in the inpatient bmt ward ahead of schedule, a practice which continued throughout phase 1. in phase 2 (weeks 7-8), we formally expanded issuing of prp to include the inpatient bmt ward and requested 91 prp products weekly. our blood supplier provided an average of 57 prp weekly (range 44-69), and prp constituted 53% of platelet transfusions in the phased-in areas. in phase 3 (weeks 9-10), we began issuing prp throughout the health system. our supplier provided an average of 70 prp weekly (range 61-78), and prp constituted 43% of all platelet transfusions. scaling-up is ongoing. conclusion: phased implementation of prp by patient group prioritizes patients who stand to benefit most from the product, and allows time for the blood center to scale up manufacturing. background/case studies: maintaining adequate inventory of platelets without significant outdating and waste of product is a constant challenge for many institutions, especially for smaller community hospitals. our health system comprises 21 hospitals including smaller community hospitals (sch) and larger tertiary care medical centers (tcmc). for several years, we have been using a limited internal process of platelet sharing between some of our institutions to successfully reduce platelet wastage. this encouraged us to analyze platelet usage throughout our health system and devise an expanded novel concept of platelet distribution, in partnership with our blood supplier that would allow us to maintain an inventory of 2 apheresis platelet (ap) units at our smaller community hospitals without significantly increasing platelet waste and the associated cost. study design/methods: a "round robin" (rr) transportation system for platelet delivery and pick up was strategically developed with the regional blood center to align with routine delivery of red blood cell (rbc) standing orders. an efficient delivery system was implemented so that the regional blood center would realize reduced supplemental and emergency deliveries of blood components to our hospitals. platelets are transferred at the time of rbc standing order delivery based on a predetermined route schedule. each day, 2 ap are delivered to the sch and the previous day's platelets retrieved (if not transfused), packed in blood center transport boxes, and then picked up by the blood center driver. these platelets typically have a 48 hour shelf life remaining. the same process occurs at the next sch on the route. all retrieved platelets from the sch are delivered to the tcmc which is the last stop on the route. thus, the sch has adequate number of units available for regular transfusion and massive transfusion protocol. results/findings: review of our rr process revealed a significant benefit to our smaller community hospitals as we were able to routinely maintain an ap inventory for patients requiring urgent platelet transfusion. an additional benefit was further decrease in ap waste (table 1 ) resulting in a cost savings of $50k. an additional cost savings of approximately $25k was noted due to decreased cost of emergent platelet transportation. conclusion: our novel rr process of platelet distribution has resulted in improved platelet availability at our smaller community hospitals while maintaining the reduced level of ap waste at our health system from our previous platelet sharing process. we anticipate additional decreases in ap waste as 237a transfusion 2017 vol. 57 supplement s3 we further streamline our process. with the trending merge of health delivery systems, we predict that other health systems will adopt similar processes to improve platelet availability and reduce waste. post implementation adjustments of our pathogen reduction process jacqueline carlson* 1 , james r stubbs 1 , scott a hammel 1 and manish gandhi 2 . 1 mayo clinic, 2 mayo clinic-rochester background/case studies: the implementation of pathogen reduction for apheresis platelets using cerusv r intercept system for apheresis platelets was a substantial endeavor encompassing many different areas. as with any process change, adjustments and modifications can occur along the way. after implementing 100% pathogen reduction technology (prt) for apheresis platelets, we made two additional adjustments to our sampling processes to ensure accurate labeling/categorization/branding of our final products. study design/method: our prt validation consisted of 100 apheresis platelet products. each product was tested pre-processing for white blood cell (wbc) content and platelet yield, along with post processing platelet yield. this data was used to calculate our yield and volume retention during processing. we anticipated products with preprocessing yields of 3.0, 3.1, and 3.3 x10 11 may end up below a 3.0 in the final storage bag and would need a post-processing sample to ensure the product met criteria at !3.0x10 11 platelets. results/finding: during the validation, we discovered one collection was not leukoreduced and two collections started at a 3.4 yield but ended with a yield below 3.0. these two discoveries led to adjustments in our prt platelet process. with the wbc failure, we reviewed the wbc count on the sysmex xe-2100d preprocessing report to see if it would alert us to a potential wbc failure. the review discovered that 99 of 100 results were 0.00 or 0.01x10 3 / mcl with the exception being the wbc failure with a count of 0.29. further monitoring of the wbc counts discovered a result of 0.04 which was tested on the adam r-wbc for wbc count and determined to not be leukoreduced. we decided all sysmex wbc results from the pre-processing sysmex report would be reviewed prior to processing and a wbc result of 0.03 will be tested on the adam to confirm a leukoreduced product. we also discovered 2 of 4 (50%) of the 3.4 preprocessing yields products ended with a post processing yield <3.0. we decided to increase the yields requiring post processing samples to include the 3.4. conclusion: we are continuing to sample all collections for a post processing yield so we can be confident that we are releasing products into inventory with a yield of !3.0x10 6 platelets and to have enough data to accurately determine our volume and yield loss during processing background/case studies: the university of kentucky medical center (ukmc), a large academic hospital with level i trauma center, is supplied with blood products by the kentucky blood center (kbc) on a consignment agreement-based contract. ukmc is kbc's largest consumer of blood products. as platelet usage can vary widely day to day platelet usage projections are provided to kbc by the ukmc blood bank, thereby allowing kbc to act accordingly with a given day's stock (i.e. import vs export). daily platelet projections are based on phone calls asking clinicians working in high-demand locations to estimate their needs. this process can be easily confounded by multiple factors and has undergone multiple adjustments to improve its accuracy. study design/method: daily platelet projection forms from 9/16-12/16 were retrospectively reviewed and compared to actual usage data over that same time. the prediction system used in the ukmc bb up to that time (estimated clinical need 1 6) was evaluated for effectiveness based on: total number of days under-predicted, number of days with large underprediction, average number of units under-predicted, and average difference between prediction and usage. the prediction system was subsequently changed based on this data in 2017; the revised prediction method (estimated clinical need 1 11) was then evaluated retrospectively using the same data sources covering 1/17-4/17 and then compared to the prior method. results/finding: the average number of platelets transfused from 9/16-12/ 16 was 18.2 u/d with a standard deviation of 5.3 u/d; the predicted amount was 13.7 u/d. the difference between the predicted amount and the number of units used was -4.5 u/d. 79% days (23d/month) were under-predicted (average: 6 u/d). 17% of days (10) were under-predicted by !10 u (average: 12 u; max: 15 u (4x)). the average number of platelets used from 1/17-4/17 was 17.5 u/d with a standard deviation of 4.4 u/d; the predicted amount was 18.3 u/d. the difference between the predicted and units used was a 10.8 u/d. 38% days (11d/ month) were under-predicted (average: 3.5 u/d). one day (1%) over this period was under-predicted !10 u (11 u). conclusion: review of clinical platelet usage over this time identified a relatively stable average daily clinical demand. adjustment of our prediction system to ensure that no, or as few as possible, days were projected for less than that average has markedly reduced catastrophic shortages (17% a 1%), reduced the number of days under-predicted (79% a 38%), and decreased the discrepancy on those under-predicted days (5.9 u a 3.5 u). these improvements in estimating usage allow for an increased ability to handle unpredictable events without suddenly straining kbc's supply flexibility or severely limiting ukmc clinical settings. rapid implementation of zika virus (zkv) nat blood donor screening joan dunn williams* 1 , maria noedel 1 , nancy haubert 1 , kenneth hudson 1 , larry morgan 1 , robert shaw 1 , tracy fickett 1 , jamie jue 1 , valerie winkelman 1 , sally caglioti 1 , german leparc 2,3 and phillip c williamson 1 . background/case studies: on 08/26/16, fda issued a guidance document for "reducing the risk of zkv transmission by blood and blood components". in response, a plan was implemented for mandatory zkv testing for all clients with locally acquired mosquito-borne cases of zkv within 4 weeks; nationwide in 12 weeks. this organization performs testing for clients (blood centers, hospitals) across the country. we report on 1 of 2 manufacturers' (sponsor) provided investigational new drug (ind) protocols. a single project management (pmo) system was used to control all required processes. study design/method: project focus included: clinical trial requirements, client onboarding, lab operations (labs). our objectives were to implement zkv testing for 44 clients within 4 weeks, and an additional 21 clients within 12 weeks. to minimize the impact to labs a staggered implementation was used with tracked/streamlined communications from stakeholders: vendors, institutional review board (irb), it (client and lab based), client services and labs. results/finding: clinical trial requirements increased the complexity of implementing an unlicensed test. documents included donor notification, informed consent, protocol training, staff certification, deviation management, and result reporting. multiple irb documents were required. to ensure accuracy in ind commitments a principle investigator was assigned to labs with client sub-investigators. deliverables were multiple including client requirements, vendor responsibilities and labs. client onboarding included confidentiality agreements between client and sponsor. an immediate zkv based webinar provided materials and understanding of sponsor protocol, lab test system, and client/donor based responsibilities. to facilitate and ensure effective communication, twice weekly conference calls were held. clients sent questions which were facilitated by labs and directed to sponsor. specific to clients were irb documents, it updates/validation for zkv test ordering and result receipt. labs were multifaceted: vendor instruments, assay materials, package inserts, staff training. assessments included: zkv sample volume, throughput, instrument capability/capacity. work requirements included vendor installation, equipment, assay and reagent qualifications, staff training, competency assessments, result reporting. all clients were provided with zkv testing within required timeframes. conclusion: the success in meeting a rapid implementation of zkv testing was largely due to a centralized pmo system which provided a controlled process for sponsor, client, vendor and labs. within lessons learned strength was found in a multi-client onboarding process. a weakness was in understanding instrument test volume capacity throughput which was exceeded during the 4-week period but overcome during the 12-week cycle. red blood cells baby units traceability and discard in kuwait central blood bank and five hospitals marwa moemen al deeb* 1 , hala samuel boules 1 , fatemah saleh al matroud 1 , rabab hussien ali dashti 1 , hanan alawadhi 2 and reem al radwan 3 . 1 kuwait central blood bank, 2 kuwait central blood bank, 3 kuwait central blood bank background/case studies: ill children are more likely to receive red blood cells (rbcs) transfusion than any other patient age group. rbcs are the component most often transfused during neonatal period. small volume aliquots are used to limit donor exposure, prevent circulation overload and decrease donor related risk. traceability is the ability to trace each individual unit from donor to recipient or disposal. blood component should be fully traceable from collection to final disposition. the kuwait central blood bank (kcbb), is preparing baby units and distributing it to all hospitals all over the country. kcbb, being accredited by the american association of blood banks (aabb), is following the aabb's regulations in tracing every component. study design/method: this is a retrospective study to assess final deposition and the percentage of discard of prepared packed rbcs baby units in the kcbb and five hospital blood banks (hbb). also, to assess the levels of traceability as a reflection of the improvement in the efficient use of these blood products. methods: a total of 3000 rbcs baby units were randomly chosen to be traced to their final deposition from the year 2012 till 2016. half of them (1500 units) were traced in kcbb. tables showing the numbers of the chosen units were distributed to the five governmental hbb (60 units for each year of the study period). results/finding: preliminary results show that the tracing of rbcs baby units in the kcbb is 100% efficient. results from other hospitals are under process. statistical analysis of the traceability will be done as soon as the data is collected. the study will analyze the usage of the baby units in different departments and the percentage of discarded units. the traceability of rbcs baby units in the kcbb is excellent, this is due to good management and training of the working staff and the use of an electronic system in registration and issuing. most of the kuwait governmental hospitals are using electronic systems, so the traceability should be up to the recommended levels. the percentage of discard of the baby units in the hospitals is very high. this may be due to the practice of using fresh blood (<5 days of donation) and the reservation of the baby units of the same donor to the same baby to reduce the hazards of multiple donor exposure. the creation of a national policy for using rbcs baby units is highly recommended to reduce the discard of such units. we also calculated the number of false positive results. the study traced all products through mid-march 2017. results/findings: a total of 339 products were tested. fifteen units (4%) had a false positive result and could not have their life span extended. of the fifteen reactive units, two repeat donors were identified and their charts were marked to not test subsequent donations. cross-reactive antibodies were identified in all 15 by the vendor and none were true positives by re-culture. of the 324 units that were successfully tested, 200 were tested again on day 6 for use on day 7(62%). there were 166 platelets transfused (51%) and 158 expired after day 7 (49%). the cost to test the products including controls was $12,970 and our calculated cost to produce 324 products would be $77,436. if we had needed to import products to meet needs, the cost would be roughly $91,300 without shipping costs which are estimated at $14,815.50. we averaged 40 expired platelet products per month (range 6-67) before verax testing and 26 (range 9-40) after implementation. conclusion: using verax point-of-care testing saved 166 platelet products from discard. the cost savings were $93,145.50 from importing and $64,466 from producing a replacement for those 324 products. the average discard rate per month went from 40 to 26 after verax implementation. extending platelet shelf life to 7 days more than paid for the cost of testing and ensured products were available for patients who needed them. secure text messaging in transfusion medicine: can texting decrease wastage? melanie estrella* and elsie lee. george washington university hospital background/case studies: secure text messaging in hospital settings allows for quick, easy, and hipaa compliant communication between members of patient care teams. it works on a mobile phone or computer, and provides read-receipt confirmation and a temporary record of team communications. secure texting has potential to be a useful management tool in transfusion medicine in reducing blood product wastage. for example, it provides a relatively low-burden means for busy clinicians to provide feedback to the transfusion service about scenarios of potential wastage. this information can be used to identify areas in which management strategies could be developed. it also allows for personalized educational opportunities between clinicians and the blood bank about usage guidelines and how to reduce future wastage. the goal of this study is to use secure texting to investigate wastage, evaluate the responses from clinicians, and evaluate the potential effects on reducing wastage. it is hoped that the results will identify secure texting as a useful management tool in transfusion medicine. study design/method: wastage records that were investigated without the assistance of secure texting from july to december 2016 were reviewed to identify the most common scenarios of preventable blood product wastage. wastage records from january to april 2017 were reviewed, and wasted products that were considered preventable were investigated using secure texting to communicate with the ordering physician. results/finding: for 2016 data, 129 units were investigated without the use of secure texting. of these, 118 units were identified as preventable wastage, and 11 wasted units were considered beyond the control of the clinician. the categories for preventable wastage were defined as follows: 1) product not released after procedure/ or when patient stabilized (42) 2) product returned outside of appropriate temperature range (40) 3) clinician unaware product was assigned (36). thus far in 2017, wastage records have identified 31 units of preventable wastage. secure texting was used by a transfusion service physician to investigate. twelve responses provided useful feedback for future management strategies, 11 responses thanked the transfusion service for the information, and in 8 instances, the message was read with no reply. conclusion: secure text messaging has the potential to improve communication in transfusion medicine. it is easy to use, hipaa compliant, and helps identify strategies for reducing wastage by improving communication and allowing personalized educational opportunities between ordering physicians and the transfusion service. sequence of reagent adding for cryopreservation freezing solution guoling chen*, xu zhao, andrew tiss, sasha turner, devin emerson, manijeh shemirani, sharon novak, david garvin, john eng and wanxing cui. medstar georgetown university hospital background/case studies: dimethyl sulfoxide (dmso), plasmalyte-a (plas-a), human serum albumin (hsa) are widely used to prepare cryopreservation freezing solution. some use autologous plasma instead of plas-a and hsa. this study is to identify the choice of reagents and the optimal sequence of adding these reagents when making freezing solution. study design/methods: materials: 99.9% dmso, plas-a, 25% hsa, autologous plasma extracted. containers: transfer pack (bag) and polystyrene tubes. the freezing solution recipe used in this study is (volume ratio) 99.9%dmso: plas-a : 25%hsa51:2:2. plas-a and hsa are kept at room temperature (20-258c, rt) and refrigerated at 48c, plasma at rt (to simulate the end-of-centrifuge temperature), dmso at rt (due to high freezing point 18.58c). different combinations of the reagents choice, storage temperature, adding sequence, are tested with photo taken. total 14 tests. at least 10 minutes cooling after dmso, before adding the next reagent. see table: (1) after directly adding 99.9% dmso alone to bag, the bag turned from transparent to white, so dmso should not add first. (2) in tube, autologous plasma first, dmso next, powder-like precipitates. (3) in tube, dmso first, hsa next, precipitated instantly, a layered appearance. (4) & (5) in tube, plas-a first, then hsa, dmso at last, precipitates formed; rt plas-a and hsa combination formed a thicker precipitate than those kept at 48c. (6)&(7) in tube, hsa first, dmso next: precipitation formed heavily, sculpture shape. precipitation in the 48c group is slightly milder/slower than rt group. so hsa should not be added first. (8)&(9) trace of hsa(<1ml) was mixed into the plas-a bag (500ml). in tube, such "hsa-contaminated" plas-a was added first, then dmso, small fragments of precipitates formed, so dmso should not add last. background/case studies: maintaining a robust blood product supply is an essential requirement to guarantee optimal patient care for all major hospitals. however, daily blood product use is difficult to anticipate. platelet products are the most variable in daily usage, have short shelf lives, and are also one of the more expensive products to produce, test, and store. due to the combination of absolute need, uncertain daily demand, and short shelflife, platelet products are also frequently wasted due to expiration. sophisticated data analysis has the potential to accurately predict hospital wide platelet needs and therefor reduce wastage. study design/method: we have investigated platelet usage patterns at our institution, and specifically interrogated the relationship between platelet usage and aggregated hospital-wide patient data over a recent consecutive 29-month period. using a convex statistical formulation, we have found that platelet usage is highly dependent on several factors. these include day of week, number of abnormal cbc, location-specific hospital census data, and other less important factors. we exploited this relationship to develop a mathematical model to guide collection and ordering strategy. results/finding: this model minimizes waste due to expiration while never allowing for a shortage; the number of remaining platelet units at the end of any day never drops below 10 in our model. compared with historical expiration rates during the same period, our model reduces the expiration rate from 10.5% to 3.2%. with an annual platelet usage of approximately 13,000 units, this reduction equates to approximately 950 units saved from expiration annually. depending on platelet pricing in different regions, this accounts for annual savings between $450,000 to $650,000, per institution. conclusion: to our knowledge our research is the first such use of hospital wide data to inform real-time donor recruitment strategies based on anticipated patient demand. thawed plasma implementation: signficant cost savings and decreased plasma wastage morvarid moayeri* 1 , russell thorsen 1 , rosaline ma 1 , antonio g insigne 1 , amy decourten 1 , florence panganiban 1 , patricia mckean 1 , cyril jacquot 2 , sara bakhtary 1 and ashok nambiar 1 . 1 ucsf health, 2 children's national medical center background/case studies: plasma (ffp, pf24, pf24rt24) stored at 1-6c outdates 24 hours after thawing. if collected in a functionally closed system, it may be relabeled as thawed plasma (tp), extending expiration to 5 days from the thaw date. although coagulation factor levels decrease over this period, they remain above hemostatic levels. as tp can be safely used for the vast majority of patients requiring coagulation support, we implemented use of tp in our multi-site tertiary care system, with the aim of decreasing costs and minimizing wastage. study design/methods: the massive transfusion protocol at our instiution already allowed the use of group ab tp. following a review of literature and practice at other large centers, the transfusion committee extended the approval of tp to all patients. neonates (<4 months), patients undergoing plasmapheresis and those with factor deficiency or other disorders for which we also noted a significant decrease (not quantified) in technologist time and effort, as less time was expended on the following: thawing units, printing inventory reports and reporting/record-keeping for discarded units. conclusion: in many large facilities, providers frequently order more plasma units than are ultimately transfused, leading to high plasma wastage rates due to limited (24 hr) shelf-life. tp has an extended shelf-life, and can be used interchangeably with ffp and pf24 for most patients. implementing tp in a multi-site tertiary health care system resulted in sustained decrease in plasma wastage, saving thousands of dollars and helping conserve a precious resource. the merging of immunohematology reference lab's (irl) inventories-using technology to create advanced search functions alexander delk 1 and richard gammon* 2 . 1 oneblood, 2 oneblood, inc. background/case studies: immunohematology reference labs (irls) must maintain diverse inventory of antisera to aid in antibody identification, antigen type rbc units, and meet regulatory requirements. when our current organization was established, two irl sites had independent inventory management systems. although the purpose of maintaining the antisera inventory was the same, organization, storage, & access to instructions for use (ifus) were not. our irl developed a synergistic method to organize and store antisera coupled with in-house designed custom excel spreadsheet to organize and search antisera and view ifus. study design/method: a list of similarities and differences was constructed. best practices of both methods were identified. we determined that our antisera could be broadly classified/organized into two main categories: rare and bulk for screening. sequential lab assigned numbers were given to antiserum for each category: s (rare sera) and b (bulk sera). a dynamic/static freezer box storage system that was inter-box static and intra-box dynamic was determined to be best option to combine two inventories while conserving elements of each allowing for library growth. antisera assigned to a box remained in that box, but may be moved within the box. the box itself may be moved among freezers. to track boxes, location and movement within the box, a custom excel spreadsheet was created. its location tracking feature allowed for two different storage methods to function in one spreadsheet. the spreadsheet had a tab for s and b antisera categories. abo group, desired and unwanted antibodies filters allowed quick search for appropriate antisera. the spreadsheet also had hyperlinks to scanned ifus. results/finding: sequential lab s and b numbers were assigned to new additions using a dynamic/static storage system. an excel spreadsheet with scanned ifus (hyperlinks) was used. pre-merger systems, it took on average 5-8 minutes to choose an antiserum and obtain the appropriate ifu. post-merger system was reduced to on average 2-5 minutes. (table) conclusion: the merging of two irl's antisera inventories resulted in a need for innovation to create an inventory management system with an advanced search function and hyperlinked ifus. this process saved valuable technologist time and organized the antisera more efficiently. abstract continue to flash until they are removed from shelf and their status updated in our database. 'units allocated' tab includes truncated patient name (to protect privacy), unit number, component type, allocation and expiration date/time, and time since allocation, with a flashing alert for units expiring in < 4 hours. the xm/hla platelets tab provides patient names and status of units allocated. a 'trxn/xmplat' tab lists pending transfusion reactions and platelet cross match reports. dashboard eliminated the printing (several times/shift) of lengthy computer-generated reports, simplified thawed plasma inventory management and helped decrease plasma wastage (from 34% to 14%). conclusion: using in-house talent and minimal capital expenditure, we designed and implemented a dynamic web-based dashboard for managing blood product inventory across a multi-site transfusion service. the dashboard is stable, customizable and requires little maintenance. initially built to optimize inventory display for thawed plasma implementation, the dashboard was expanded to include all allogeneic blood products. over the past year, this tool has replaced manual processes for monitoring and rotating inventory and directly helped decrease plasma wastage. use of deglycerolized red blood cells for hospital transfusion service inventory management ronnie l. hill*, jason corley and lizabeth ostiguin. us army background/case studies: regional blood shortages have been documented across the united states during the winter holiday timeframe. deglycerolized red blood cells (drbcs) have been shown to be an effective alternative though more expensive to manufacture. this study looks into the fiscal and inventory efficacy of using drbcs to meet the needs of transfusion services during times of blood shortage. study design/method: on three separate occasions, a medium sized dod donor center used its frozen blood inventory to produce type o drbcs to meet the needs of two regional transfusion services. all frozen red cells were manufactured by an offsite facility with the haemonetics acp-215 using the low glycerol (40%) freezing method and frozen at -658c within six days of collection. thawing occurred in a 328c water bath in the following order: 7 o positive and 1 o negative on 3 january 2017; 7 o positive and 1 o negative on 7 february 2017; and 8 o negative on 22 february 2017. deglycerolization occurred on site using the acp-215 with all units passing internal qc requirements. drbcs were shipped the same day to a hospital transfusion service, allowing for 13 days of shelf life prior to expiration. results/finding: during the three events, all supported transfusion services and the blood center were below minimum inventory requirements for standard type o red blood cells (rbcs). o positive rbcs were only available through nbe at $240-280 and had the limitation of arrival on the next business day. collection and processing time of liquid rbcs takes approximately two days including: donor screening, phlebotomy, component processing, testing, and labeling. drbcs cost the dod on average $400 per unit to produce and distribute. drbcs have a shorter shelf life, 14 days versus the 211 days for other rbcs, but are washed during deglycerolization and thus produce fewer transfusion reactions. one tech can operate up to four acp-215's and deglycerolize four units at a time. in january and february 2017, it took one tech four hours per iteration of eight units to include thawing, labeling, and packing for shipment. conclusion: while not as readily available as traditional rbcs, drbcs can be an effective product to bridge the inventory gap when small numbers of units are needed due to reduced inventory. collection and processing of whole blood into components takes approximately two days, but can produce greater numbers of units in that timeframe. based on this, drbcs can be ready faster than freshly collected units of blood. there is an increased cost associated with manufacturing frbcs which is compensated for by the longer available shelf life of 10 years. having a small contingency supply of frozen red cells and deglycerolization equipment has been effective on three occasions in ensuring availability of type o red blood cells for hospital transfusion services. validation of a human anti-tetanus toxoid immunoglobulin assay performed on the abbott c8000 izekial butler* 1 , karen leighton 1 , scott jones 1 and rachel beddard 2 . 1 qualtex laboratories, 2 biobridge global background/case studies: plasma fractionators require anti-tetanus quantitative testing to be performed on plasma samples collected from individual donors or plasma production pools. this testing serves as a quality control test and helps estimate the antibody potency of the product. the binding site, human anti-tetanus toxoid immunoglobulin liquid reagent kit is for use on a turbidimetric analyzer. the aim was to optimize and validate the human anti-tetanus toxoid immunoglobulin liquid reagent kit for use on a photometric analyzer. study design/method: experiments were performed in order to determine the optimal amount required of reagent buffer and latex reagent from the anti-tetanus toxoid immunoglobulin kit utilizing the abbott c8000 instrument. precision of the new assay parameters was determined by testing 10 replicates of a panel of samples at three concentrations of tetanus antibody in a single testing run. the panel samples were created by spiking appropriate amount of a who tetanus antibody standard into sodium citrate plasma. accuracy was determined by testing a series of samples ranging from 1 iu/ml to 60 iu/ml of tetanus antibody. the samples for the accuracy study were created by diluting an appropriate amount of a who tetanus antibody standard with sample diluent from the reagent kit. linearity regression was determined by using the accuracy study values within the range of 2.0 to 45.0 iu/ml. stability of samples was determined by testing samples stored at 2-8 0 c and -20 0 c in triplicate at various time intervals. results/finding: the %cv for the optimized anti-tetanus assay for all antibody levels determined in the precision study varied from 1.2855 to 1.3142. so, precision was acceptable since the %cv for all samples tested was 5%. the mean values for the samples tested in the accuracy study were all 610% of the expected value which was much lower than the acceptance criteria which was 615% of the expected value. the linearity of the assay was acceptable with a r2 ! 99.0%. the linearity study established that the known tetanus concentration was a statistically significant predictor of the observed concentration. the sample stability studies demonstrated the ability to quantitate tetanus antibody concentrations in samples stored up to 14 days at 2-8 0 c and up to 1 month at -20 0 c. conclusion: the data presented shows the successful optimization of the human anti-tetanus immunoglobulin reagent kit for use on a photometric analyzer. validation studies of this optimized assay demonstrate excellent accuracy, precision and linearity using samples stored for 14 days at 2-8 0 c and stored up to one month at -20 0 c. a deep dive audit of intravenous immunoglobulin use for immune thrombocytopenia: is its use inappropriate? jiajia liu*. university of toronto background/case studies: intravenous immunoglobulin (ivig) is a generally safe and effective therapy for immune thrombocytopenia (itp) but is only suggested for scenarios when a rapid increase in platelet count is desired or as first line therapy if steroids are contraindicated. due to concerns regarding adverse effects, cost and resource availability, an ivig request form was implemented in our jurisdiction in 2010 to track utilization and appropriateness. a recent audit of these request forms from four academic institutions found a lack of compliance with form requirements and inadequate documentation of efficacy which led the authors to conclude that the use of ivig was broadly inappropriate (shih et al, 2017) . as such, we aimed to conduct an extensive chart review of patients who received ivig for itp at our institution to assess appropriateness of use. study design/method: we conducted a retrospective chart review of all patients with itp who received ivig in our institution from april 1, 2014 to march 31, 2015. local research ethics board approval was obtained. results/finding: 40 patients received ivig for itp at smh over the study period for a total of 76 unique ivig infusions. the most common indications for ivig within currently accepted guidelines were: active bleeding (13, 17%), pre-operative or antepartum care (22, 29%), a platelet count of less than 10 and contraindication to corticosteroids (8, 11%). additional indications that still fell within accepted guideline recommendations included: patients with arterial/venous thromboembolism or risk thereof requiring initiation of antithrombotic therapy; and patients requiring myelosuppressive chemotherapy. indications that fell outside of guidelines included: use of ivig as a diagnostic challenge where the etiology of thrombocytopenia was unclear and use prior to international travel for patients with difficult-to-treat chronic itp despite a platelet count between 30-50 x 109/l. 6 patients received ivig for a likely diagnosis itp while 245a transfusion being investigated for alternative explanations for thrombocytopenia. three patients were refractory to all other therapy for itp and were dependent on regular ivig infusions. 18/76 (24%) of infusions consisted of 2g/kg over 2 days; the remainder of infusions consisted of 1g/kg. of those who received 2g/kg,3 of patients (17%) had evidence of partial remission after a first 1g/kg dose. ivig was generally well tolerated and infusion reactions were mitigated with use of corticosteroids, antipyretics and/or antihistamines. conclusion: we found, at our institution, that use of ivig for itp was generally appropriate and carefully considered even in cases that did not meet current guideline recommendations. we believe that ivig remains an important treatment for itp particularly in the aging population where prevalence of conditions complicating bleeding risk is increasing. detailed utilization/ knowledge data inquiries are required to develop tools and policies to enhance appropriate ivig use in multiple settings. we believe that there is an opportunity to promote administration of a single 1g/kg dose to minimize unnecessary utilization of ivig amongst hematologists who manage itp. a process for improving crossmatch bench ergonomics janet dornfeld*, sheng-chung cheng, ann eggebrecht, beth greer, savannahsue rondeau, brian rognholt and beth taylor. mayo clinic background/case studies: a mission of our institution is to reduce the risk of work-related injuries. accordingly, each year an ergonomic survey is undertaken as a component of a general department of laboratory medicine and pathology safety audit. our 2016 survey identified potential musculoskeletal risks that suggested a redesign of our crossmatch benches. study design/methods: a seven item ergonomics survey of the working environment was sent to 32 staff members in early february of 2017. twenty-two technologists responded for a 69% response rate. the below table below reports the survey items and responses. results/findings: the most problematic area was the available workspace. of the respondents, 81% indicated that workspace size was insufficient and 71% that the chairs at the fixed height benches were problematic. problems noted were difficulty with climbing up into a chair and backing down and with the chairs holding the chosen height. our laboratory lean team operational support group was tasked to aid with the bench redesign and to choose products for improving the workspace. our goals were to design a layout to streamline testing workflow and better utilize lab space, including our plasma thawing and sink space, eliminating dead space. the configuration of the new workspace was guided by the survey findings. adjustable height workstations were recommended to replace our fixed height bench. we worked with our facilities design contactor to purchase adjustable benches and plan add-on cabinet shop work. the benches were assembled off site, which allowed a bench top layout to be determined and installation of cabinet shop add-ons of a drawer for supplies and a pull out breadboard as a writing surface. the opportunity to assemble off site streamlined the process of installation, resulting in minimal disruption of testing. conclusion: the survey was effective in identifying working areas for improvement. employee comments have been positive for the new workstations. an effectiveness assessment will follow, using the original survey, to assess the success of the project. a retrospective study of emergency department initiated type and screen testing: were patients transfused after testing? sandra lamm* 1 , neil bangs 1 and kimberly sanford 2 . 1 vcu health system, 2 virginia commonwealth university background/case studies: type and screen (t&s) testing is often ordered on patients presenting in the emergency department (ed). if the patient does not have a historical type, a second sample is drawn with an additional phlebotomy for type confirmation. if the patient does not need a transfusion of red blood cells (rbcs), the testing and second phlebotomy is an inefficient use of resources and time. study design/method: as part of a performance improvement initiative in transfusion medicine, we performed a retrospective study of all t&s orders that were initiated in the ed from 1/1/2015 to 6/30/2015 to determine if testing was subsequently followed by transfusion of blood products. patients were stratified by ed department, time from t&s draw (tsd) to transfusion (<4 hours, > 4 hours < 24 hours), and if a second sample was required. results/finding: a total of 3144 t&s orders were initiated from the ed in this time period. 2787 (88.7%) patients were not subsequently transfused any type of blood product within 4 hours of tsd and 2584 (82.2%) patients were not subsequently transfused any type of blood product within 24 hours of tsd. a total of 2119 (67.3%) patients required a second sample. of these patients requiring a second sample, 1960 (92.5%) were not subsequently transfused any type of blood product within 4 hours of tsd and 1886 (89%) were not subsequently transfused any type of blood product within 24 hours of tsd. conclusion: routine ordering of t&s testing is not an efficient use of resources and time as many patients are not subsequently transfused. ultimately unnecessary t&s and second sample collection and testing for those patients not subsequently transfused within 24 hours of tsd amounted to an estimated $699,706 in unnecessary patient charges and approximately 628.7 nursing hours for phlebotomies in a six month period. anti-d from alloimmunization versus rh immune globulin: detective work in the blood bank and transfusion medicine services (bbtms) margaret diguardo* 1 , debra berry 1 , yunchuan delores mo 2 and gay wehrli 1 . 1 university of virginia health system, 2 children's national medical center background/case studies: the institute for healthcare improvement triple aim incorporates enhancing patient satisfaction by providing high quality, safe care. towards these goals the bbtms is charged with communicating to obstetric physicians (obs) a patient's antibody specificity with associated hemolytic disease of the fetus/newborn risk. thus, when anti-d is detected in a female of childbearing age, it is critical to determine whether this represents rh immune globulin (rhig) or alloimmunization (alloanti-d). review of a patient's electronic health record (ehr) helps quickly identify rhig administration, but if this documentation is missing, then it is easy to assume presence of alloanti-d. rhd alloimmunization impacts mom, fetus, newborn and future pregnancies. therefore, without a national, comprehensive health information exchange (hie) system, it is imperative to investigate beyond the on-site ehr whether a patient received rhig at an outside hospital (oh). we report an irb approved (exempt) case series where detective work revealed rhig administration at ohs. study design/method: over a two month time period, anti-d was identified in four pregnant women. review of their ehrs did not reveal a history of abstract rhig administration; nor did subsequent direct communication with their obstetricians (ob) reveal a history of rhig. based on each patient's home address, the bbtms of any nearby ohs were contacted as was a primary care physician if listed in the ehr. results/finding: investigations beyond the ehr and obs revealed each of the four patients received discontinuous prenatal care with presentations at multiple sites. through phone calls to the bbtms of ohs, a history of one or more rhig administrations within the preceding three months was found for each patient. our bbtms records and ehr were amended to reflect the presence of a passive anti-d due to rhig, rather than alloanti-d. the changes were also directly communicated with the ob caring for each patient. conclusion: when a new anti-d is identified in a pregnant female, investigation is required to determine whether it is passive rhig versus alloanti-d. when neither the ehr patient history or ob reveal a rhig history, it remains in the patient's best interest to investigate further. through phone calls to oh we revealed a history of rhig administration in four patients. finding and communicating this critical information helps enhance the quality and safety of patient by ensuring subsequent rhig administrations when indicated, at our institution. future strategies for avoiding similar situations include expanding our national hie for critical information such as bbtm history and allergy history and expanding use of wallet-size patient identification cards with rhig and alloantibody histories. auditing massive transfusion protocol colleen a. aronson* 1 , elizabeth halperin 2 , sharon breining 2 and mona papari 3 . 1 acl laboratories/ advocate hospitals, 2 advocate health care, 3 itxm background/case studies: a large midwest hospital system with 5 level i trauma sites evaluated how to audit the massive transfusion protocol (mtp). the possibility of real time audits is impractical due to the unpredictability of these events. a search of the internet found an example from new zealand for post process evaluation. this was shared with a team as a starting point and then adjusted for system specific priorities. to start the audit, the initiation of the mtp needed to be determined as events are often started as a verbal request but then followed up with either downtime or computer orders. study design/method: the transfusion service (ts) was determined to be the source of truth for all of the mtp events. a tracking sheet was created to capture the patient demographics, start and stop time, number and type of products issued and wastage. this was then passed onto nursing quality staff that used the tracking form and the patient chart to enter an event into the error management data base as a focused event. the focused event was built to include patient demographics and other information from the tracking form as well as where the event was called (surgery (or), emergency (ed), labor and delivery (l&d), etc.), type of event, use of tranexamic acid (txa), calcium chloride (cacl), temperature monitoring and pre/ post lab results. a trial was started and 3 months of data were evaluated that contained 29 events. results/finding: there was an equal number of events that were initiated in the ed and the or (12). male patients were involved 69% of the time and 31% of time the patients expired. trauma of some type was the majority of the cause but 13.8% of the cases involved gi bleed and only 6.9% were obstetric cases (see chart). the lowest hemoglobin (hgb) was found to average 7.1 with the post hgb average of 9.7. ratios of 1:1 for red blood cells (rbc) to plasma as well as rbc to platelets (plt) and cryoprecipitate (cryo) were also determined with a target of 4:1. it was found that the rbc: plasma was 1.9:1, rbc: plt was 5.9:1 and rbc to cryo was 7.4:1. use of txa was only 24.1% and cacl was utilized in 58.6% of cases. conclusion: although this data is for a short period of time it has pointed out several opportunities for improvement. the use of mtp in gi cases was not previously understood but opens up a new group of people for which education and understanding of the mtp process is needed. the low use of txa needs to be evaluated and already has started conversations about how this drug should be stored and accessed for the mtp process. the product ratio numbers were suspected of being off but now that data is available, it is much easier to speak to this issue and look for improvement. the process will now be expanded to the level ii trauma sites in the system and routine evaluation will be shared with all sites. automated report significantly reduces turnaround time for rbc antibody alert jessica l dillon* 1 , jody a barna 1 , donald e ulinski 1 and nancy m. dunbar 2 . 1 dartmouth hitchcock medical center, 2 dartmouth-hitchcock medical center background/case studies: clinically significant antibodies should be promptly and clearly communicated to the patients' healthcare team to avoid potential transfusion delays in blood availability or complications of incompatible transfusion. at our institution, all newly identified clinically significant antibodies are immediately resulted in the electronic medical record (emr). an interpretative comment is also entered by the transfusion medicine service (tms) physician after the antibody work-up has been reviewed (this may be up to 2 weeks after the antibody is identified). this comment describes the antibody(ies) identified, indicates the need for crossmatch compatible blood and alerts clinicians of possible delays in providing crossmatched units. since clinicians may not always review these results, the tms physician also simultaneously adds an "allergy to red blood cells" alert in the patient emr at the time the interpretive comment is entered. study design/methods: in july 2016, we implemented an automated report to reduce the turnaround time (tat) for entry of the allergy alert. the report contains all detected red cell antibodies in the prior 24 hours and is provided to the tms physician during daily morning rounds (monday through friday) for manual entry of allergy alerts. this study describes a three month comparison both before and after the automated report intervention, to evaluate the tat for allergy alert entry into the emr. age ( abstract results/findings: between august 2015 and november 2015 (pre-implementation) , newly identified clinically significant antibodies were resulted for 56 patients compared to 51 patients between the months of august 2016 and november 2016 (post-implementation). the tat for allergy alert entry for both periods is shown in table 1 . we observed that 57% of allergy comments were performed within 24 hours in the post-implementation period versus only 30% pre-intervention (p50.0067). using the new process, nearly all of the alerts were entered into the emr within 72 hours of antibody resulting and none of the entries were missed. conclusion: there was a significant improvement in the tat for allergy comment entry following implementation of an automated report. this project illustrates how information technology can be leveraged to facilitate timely communication of antibody identification. blood bank verbal tool implementation for cardiovascular surgery rita louie* 1 , shailesh macwan 1 , nancy nikolis 1 , arline stein 1 , janelle richardson 1 , manju bagu 1 , lennart logdberg 1 , alexander indrikovs 2 , vishesh chhibber 1 and sherry shariatmadar 1 . 1 north shore university hospital, 2 northwell health background/case studies: our institution is a tertiary care facility performing over 1500 cardiovascular surgeries (cvs) in 2016, an increase of 117% after the healthcare system cvs integration in 2015. transfusion support of these patients includes preoperative preparation of prbcs according to a maximum surgical blood order schedule. additional blood components are issued as orders are placed. until december 2016, the additional written orders were submitted to the blood bank via the pneumatic tube system without further communication. after 2 reported events in q3 2016 that resulted in delays in blood transfusion, we examined our process very closely and identified opportunities for improvements. in collaboration with cvs, the blood bank implemented a new workflow process to enhance communication with the cvs team, reduce turnaround time and improve patient safety. study design/method: 1. open discussions and collaboration between blood bank and cvs nursing teams 2. mapping the process using flowcharts for additional blood orders from cvs. 3. identify bottlenecks and brainstorm solutions. 4. a verbal cvs order process and form was implemented to improve communication between cvs and blood bank, which solidified communication by including the time of the order, patient identifiers, caller identification, ordering prescriber, staff receiving order, the quantity and kinds of products ordered, the mode of order delivery, and anticipated future orders. a read back was also documented for verification of the order. 5. the blood bank staff immediately processes this order while waiting for the written order to arrive. upon receipt of the written order the blood is issued to the or. 6. follow plan-do-check-act. the transfusion safety officer reviews each order for the following parameters: number/type of products, turn around times (tat), wastage/returned products and overall efficacy since implementation of this process. results/finding: a significant improvement was noted in communication and tat after implementation of the process described above. for the period 12/23/16-4/7/17 the blood bank has received 327 verbal orders with varying product combinations. the table below represents average turn-around times to issue blood products: conclusion: the introduction of the verbal order tool for cvs has streamlined the blood ordering process leading to increased efficiency and lower tat. effective communication between the or team and transfusion service is the key to timely provision of blood products for these critical patients. challenge of blood type testing for multiply transfused sickle cell disease patients jayanna slayten* 1 , tracie ingle 1 and heather vaught 2 . 1 indiana university health, 2 indiana university health (iu health) background/case studies: we report our midwestern, university transfusion service challenge of obtaining the correct blood types in rbc exchanged sickle cell disease (scd) patients tested by our primary testing method, solid-phase red cell adherence analyzer echo (immucor. norcross, ga). the echo operation manual in chapter 12-6 and appendix d it states: "warning: the galileo echo cannot reliably detect hemagglutination reactions that are graded as 11 or less in tube methodology. the galileo echo does not generate as interpretation of mixed-field. such a mixed-field reaction will be interpreted as positive, negative, or equivocal." we report of a challenge with this analyzer limitation which impacts the assignment of the correct blood type for multiply transfused scd patients. study design/method: two scd when initially tested by the echo as o, d negative; however, each patient was historically o, d positive. both patients had received a rbc exchange transfusion with 8-11 o, d negative red blood cells over 30 days previously. repeat testing of the samples was completed by the vision (ortho clinical diagnostics. raritan, nj), neo (immucor. norcross, ga), and by standard abo/rh manual testing (anti-a, anti-b, anti-d series 4, anti-d series 5, a1 cell and b cells. immucor. norcross, ga). the repeat testing was compared to verify the patient's abo/rh typing and the results were entered into the computer system to allow for assigning the patient's abo/rh typing and electronic crossmatch. results/finding: table 1 summarizes the initial and repeat testing with the two patient samples. although the echo failed to interpret or flag the blood type as mixed-field, the other methods identified the transfusion of o, d negative blood with the detection of mixed-field in the d typing or by failing to interpret the abo/rh as not type determined (ntd). the vision and manual abo/rh typing yielded the easiest mixed-field to interpret macroscopically. conclusion: our results agree with the findings of summers et al (trans-fusion 2009; 49:1672 -1677 who reported the challenge detection of mixed-field with the use of the echo compared to improved detected with automated gel column agglutination. when the samples were tested by multiple automated and manual abo/rh methods, the expected mixed-field was detected. the failure of the echo to detect the mixed-field is acknowledged by manufacturer, but there is a risk that a facility may mistype the abo/rh when there is not a historical abo/rh to compare. to avoid this risk, it may be appropriate to re-type first time scd patients by other methods rather than the echo to avoid this challenge. consistent with summers do not account for regional distribution. many large hospitals acting as regional hubs for redistribution may appear to have optimized inventory based on odr and bsr, but we hypothesized that these are crude key performance indicators (kpis) requiring redevelopment. study design/method: kpi redevelopment occurred in a large tertiary care hospital blood bank in canada, responsible for 75% and 20% of transfusions in the region and province respectively. rbc supply, inventory, and disposition data were retrospectively assessed from february 2014-june 2015 as the baseline period. a "demand-driven inventory planning policy" (ddip) was instituted to assess and implement the optimal rbc reorder quantity based on the difference between the historical maximum and minimum rbc inventories during weekdays; that would not lead to blood shortages. shelflife inventory (sli) was chosen as the main surrogate marker for the assessment of efficiency of the supply chain process, calculated by the differences between age of blood transfused (abt) and received (abr). iterative simulation modeling (r statistical software) was then performed to optimize sli in a post-implementation period from june 2015-october 2016. results/finding: modeling predicted observed rbc disposition. through simulation, optimization of sli was found to occur by optimizing a set of kpis for each abo blood group (table 1) . this led to a reduction in observed overall sli (7.2 6 1.8 days vs 6.0 6 1.5 days, p<0.01) and odr (0.9% vs 0.5%). the bsr was not significantly increased during the postimplementation period. conclusion: optimization using simulation modeling of multiple factors other than bsr and odr led to further efficiency gains in a large tertiary care hospital blood bank. hospital blood banks should use an integrative approach with a set of kpis to optimize the supply chain. this approach requires validation in other blood banks and jurisdictions. (6)) requires that the hospital make reasonable attempts to notify the patient (or the patient's physician), counsel the patient, and offer testing. the hospital must maintain records of this lookback notification as part of the patient's medical record. paper records of lookback notifications are less accessible than electronic records and are at greater risk of being damaged or lost. to facilitate the lookback process and reduce paper documentation we sought to use the electronic medical record (emr) to perform and document notifications. study design/method: representatives from transfusion medicine (tm) and information technology (it) worked together to define minimum and optimal emr solutions. minimally, a completed paper packet could be scanned into the emr. this solution had no advantages in terms of ease of use, process control, or transparency. desired optimal functionality includes the ability to send letters in the emr, document control so that original communications may not be altered, opportunity for patient's physician to electronically sign and return responses, letter and form templates that can be individualized, and the ability to track when and by whom notifications were sent and received. the emr system at our institution, epic (epic systems corp., verona, wi), has a function called "letters" with the capacity to do all these tasks. a series of five templates were developed: hiv and hcv letters to physicians, response forms for physicians to return to the transfusion service, and a blank letter template to be used for specially tailored letters. templates are opened within the patient's emr and demographic information is automatically populated by epic (eliminating many possibilities for clerical errors), the blood product transfused (e.g. rbcs or plasma) is selected from a drop-down menu, and the date of transfusion is manually entered by the sender. the completed letter is then routed to the patient's physician; it shows up automatically (and instantly) in their electronic in basket as well as in the patient's emr. physicians may electronically complete and return the response form within epic, or print it and return the form by fax. results/finding: between january 2014 and december 2016 thirty-five (35) notifications were sent to physicians using epic letters and of those, fourteen (14) responded to the epic notification and five (5) used the provided electronic response form. for these cases the time to mail or handdeliver paper notifications was avoided. the remaining 21 cases required follow-up paper notification, but the electronic letter remains as permanent, easily accessible documentation of when the transfusion service first notified the physician. conclusion: lookback notifications within the emr makes compliance with government requirements more transparent and records more accessible to caregivers, patients, and assessors. secondarily, efficiency may be improved by reducing the need to print and mail/deliver letters. evaluation of ordering practice in the operating rooms and its impact on product wastage alexandra budhai* 1 , denden benabdessadek 2 , annu george 2 and alexandra jimenez 2 . 1 westchester medical center, 2 new york blood center background/case studies: blood product wastage is an issue that many hospitals aim to address. the or was identified to have the highest rate of wastage within our hospital. in this study, we assessed the appropriateness of the product order and utilization by the or to understand its impact on wastage. study design/methods: data on product orders, issue, and return for two months were analyzed. the hospital cpoe and product requisition forms were used to collect this data. the surgical procedures and number of ordered units were compared to the hospital's maximum surgical blood order schedule (msbos). trends for inappropriate orders for products by physicians were evaluated. results/findings: a total of 941 orders were reviewed. approximately, 30% of these products were issued to the or. we found that the physician orders were within the guidelines of the msbos for most cases (89%), but of the issued products, all were returned to the blood bank in 40% of cases. we observed that the percentage of products ordered and used compared with the products ordered and returned in cardiac surgeries are nearly equal. in addition, all of the products ordered for c-sections were not used; albeit ordering frequency being significantly lower than for cardiac cases. conclusion: the data analyzed demonstrates that the majority of surgeons are adhering to our institutional msbos guidelines. it was noted that surgeons are requesting products be issued for invasive procedures where rapid exsanguination is possible. our analysis revealed that the hospital's msbos does allow for an excess in blood ordering for some surgical procedures. the msbos should be updated to reduce the suggested maximum product order. in general, the data does not imply that the blood product wastage in the or is due to the ordering practices of the surgeons. a larger period of surgical blood ordering practices should be analyzed to detect blood product ordering, utilization and wastage trends in other subspecialties. background/case studies: the visionv r and visionv r max (ortho diagnostics, raritan new jersey) are id-mts tm gel card-based automated immunohematology analyzers marketed for small to medium, and high-volume [> 50 type and screens (t&s) per day] blood banks 1 , respectively. our laboratory which serves a large 1278-bed multispecialty academic hospital and receives 275-300 t&s specimens per day needed to replace three provue analyzers prior to the availability of the visionv r max. we implemented three visionv r analyzers to work with our existing neov r and echov r (immucor inc, norcross georgia). a recent multicenter field application trial of the visionv r reported a mean turnaround time (tat) for t&s and abo, rh typing (abo/rh) of 32.2 6 4.5 and 27.5 6 5.6 minutes 2 , respectively. the objective of this study was to determine visionv r tats under routine daily high-volume practice. study design/methods: one visionv r was in operation during a five-week period (phase i), and then two additional analyzers were brought into service (phase ii). tats are defined as the time when the order is received by the instrument to when the test is completed and available for review. three-cell screen and abo/rh tats, and number of visionv r antibody panels were collected for a nine-week period. the tat for the screen was used as the tat for the t&s because the screen is the rate determining step. all testing was performed using in-service analyzers on routine patient samples by trained technologists. samples were not deliberately batched but were placed on the analyzer based on the volume and flow of work at the time. results/findings: under the high volume conditions of our laboratory with three visionv r analyzers, the mean t&s tat was 30% longer and had a larger standard deviation (s.d.) than the published trial result of 32.2 6 4.5. transfusion 2017 vol. 57 supplement s3 abstract during phase i visionv r 1 performed 263 panels. during phase ii visionv r 1 performed 351 of the 361 visionv r panels. conclusion: our visionv r analyzers are used under high volume conditions more suitable for the visionv r max. when balanced with the testing menu, including ability to perform select cell panels, our tats using three analyzers were satisfactory. the large standard deviation indicates that opportunities remain for improving tats through workflow improvement. from west nile virus to the emergence of zika virus: a nationwide survey of how regulators are keeping the blood supply safe and available falisha atwell* 1 , john roback 2 , ronald arkin 1 , michael bartlett 1 , robert geiger 1 and jaxk reeves 1 . 1 university of georgia, 2 emory hospital background/case studies: with the emergence of zikv in the united states, it is important to assess the fda's response time in providing guidance to ensure the safety and availability of blood products in the face of newly emerging infectious diseases. this research compares the responsiveness of the fda during west nile virus (wnv) and zika virus (zikv) outbreaks to evaluate our current preparedness. study design/methods: the literature review was conducted to analyze fda's response time during the wnv crisis and determine if it was effective and efficient. the research survey was performed to determine if the donor history questionnaire (dhq) adequately screens donors for zikv as the sole preventive method (as per the february 2016 guidance for industry: recommendations for donor screening, deferral and product management to reduce the risk of transfusion-transmission of zika virus) and to determine if the current regulatory practices (including the august 2016 guidance for industry: revised recommendations for reducing the risk of zika virus transmission by blood and blood components) are perceived to be effective and efficient in the face of the current zikv outbreak. survey monkey was used and participation was anonymous. over 4,000 emails and web-links were sent to members of aabb, scabb, seabb, and personal network with a 10% target response rate. participants self-selected or deselected based on the inclusion and exclusion criteria listed in the consent letter. results/findings: the literature review revealed that the fda's response was slow during the wnv outbreak, while the zikv response is efficient thus far. a total of 317 participants responded to the survey (7.94% response rate). statistically, participant agreement with fda's decisions was performed by "t" test (with n-15317-15316 df) of the null hypothesis that the mean50 vs. the alternative that the true mean is> 0. overall participants had favorable opinions of the fda's decisions. statistically, whether participants in different levels of the demographic variables (region, profession, and years of experience) answer significantly differently, one way anova models were used with likert-scale question responses as if they were continuous. the f-statistic and p-value are for the null hypothesis that all levels of the explanatory variable have the same mean for the response variable. there were no significant differences in the years of experience and profession variables for participants. region was determined to be unreliable due to undefined states for each region listed. conclusion: the research revealed that industry experts conclude that the current system of dhq and fda guidance documents, if issued timely, are adequate. background/case studies: when evaluating a new instrument solution for pre-transfusion testing, it is important to consider the operational impact of the system on the lab. there are a variety of operational, performance and system metrics that can be evaluated to determine this impact including: test workflow, hands on time, and automation time. study design/methods: the study involved a current state to a future state comparison of testing processes with an instrument ortho provuev r (pv) and manual testing vs. an instrument ortho visionv r (ov). data collection methods included direct observation, time studies, and interviews. the pv bench performs type & screens (ts) on the pv and manual abid/selected cell panels in the gel test. all other testing; cord blood(cb), dat, unit confirm(uc), patient type confirm(pc) and crossmatch(xm), etc. are done manually in tube. the future state incorporated the ov. ts, abid and uc were evaluated in both states. cycle time(ct) was averaged based on 3 run cycles. ct was comprised of 3 metrics; instrument time(it), standby time(st) and labor time(lt). st may be comprised of 2 components, time that could be utilized as "walkaway" time or vigilant time (vt) which requires operator presence but not operator action. for automated instruments, vt for each cycle was measured as instrument access unavailable. instrument daily maintenance (dm) ct was evaluated as well. similarly, timing of manual tube test processes used these metrics. for repetitive activities within a process, such as uc or xm, a time per individual process was captured and then multiplied per unit. results/findings: table 1 provides details about the metrics of current state and future state processes. tube based test timing is as follows: pc (2:50), xm (32:23), cb (13:18) and dat (10:00). by implementing the future state, an average $1.3 min. lt and vt is saved on each sample loaded for ts equating to a 73% labor reduction over the current state. a 19% improvement in tat on the ts was achieved in the future state. moving from manual abid to automated processing resulted in a 59% lt reduction. on average, a 38 min. continuous walk-away time is achieved for each automated abid. uc had less impact on labor time with minimal difference however allowed for focus on consistency and quality metrics. conclusion: based on the metrics evaluated and compared between current state and future state, the ov has demonstrated improvement in lab operations to both the labor required and result tat delivery. opportunity exists to automate workflows on other tests that are still manually performed. background/case studies: high throughput and efficient automation of serologic tests is crucial in the workflow of a blood bank that tests $100 type and screen samples per day. the erytrav r (grifols) is a fully-automated walkaway analyzer utilizing 8-column gel cards for pretransfusion testing. the blood bank validated and implemented the use of erytrav r for abo/d typing, antibody screening and identification of patient samples as a replacement for a solid phase testing platform. the blood bank also validated automation of donor unit retypes. the instrument has bidirectional interface to the blood bank lab information system (lis), hcll tm (hemocare life line, mediware). instrument validation and implementation were done in conjunction with the software version upgrade of hcll tm and an interface system change to maestro tm . study design/method: correlation testing of the erytrav r results with the manual tube testing (peg iat; reference method) was performed on 100 patient samples for abo/d typing and antibody screening; of which at least 10 had a positive antibody screen. out of the 10, 5 had known antibody specificities. forty-two rbc units were also tested for abo/d confirmation; of which 17 were d(-) and 25 were d(1). calculations of concordance, sensitivity, and specificity were performed. precision studies were also done. interface testing of erytrav r , hcll and the hospital's information system using the maestro tm interface system was performed and validated. results/finding: concordant results between both methods were obtained in all of the 100 patient and 42 donor samples tested (100% concordance). all 10 samples with positive antibody screens were obtained by both methods. all clinically significant antibodies were detected by both systems. erytrav r gave 100% sensitivity and specificity. the precision studies showed that both methods gave the same type and screen results for 5 samples at 3 different testing events. after validation of the lis upgrade and interface system change, a bidirectional interface with hcll tm was established. the instrument has been operational in our lab for over 3 months. conclusion: erytrav r was found to be reliable and accurate and can handle the high workload of our lab. users found the instrument easy to use; hence training, proficiency, and competency of the users are achievable and manageable. the validation of the the instrument is straightforward. the major challenge and delay in the implementation experienced by this blood bank were attributed to the concurrently occurring lis upgrade and migration of the data integration system. a post-implementation workflow assessment would be ideal to perform to ensure that the instrument is being used at its full potential. implementation of a system-wide platelet inventory report optimizes platelet utilization and reduces unit wastage elly landolfi* 1 , craig fletcher 2 and peter millward 1 . 1 beaumont hospital, 2 beaumont health system background/case studies: a sufficient number of blood components should be available to meet routine and emergent hospital needs. this must be assured while minimizing outdating of scarce and expensive blood components -an inherent challenge with platelet units which have a short 5-day shelf-life. we report the results of a quality improvement project implementing a custom computerized platelet inventory report designed to mitigate the most common cause for platelet wastage at our institution: high platelet outdate rates. the report includes blood type, product code, unit number, respective product attributes, supplier and availability status of all platelet units for each hospital location. all system blood banks receive a morning fax of the report which facilitates transfer of units prior to expiration and adjustments are more readily made for product orders to the supplier. study design/method: the study was conducted in the hospital-based blood bank and based on available platelet inventory and wastage quality data. the report went live october 2014 and quality data was reviewed from august 2013 to december 2016. the collected data was then analyzed using descriptive statistical methods. results/finding: data from 2016 indicates platelet wastage comprised 1% of total received platelets and 79% of these wasted platelet units were due to expiration. other reasons included failed visual inspection, blood dispensed but not used and wasted on the floors, potential tube station problems or short-dated units transferred into our blood bank from another facility. the mean of monthly wasted platelet units 12 months preimplementation of the report was 13 units, compared to 11 units 12 months post-implementation and 5 units 24 months post-implementation. wastage rates improved from 6% (wasted yearly platelets/total received yearly platelet units) in 2014, the year of report implementation, to post-implementation rates of 3% in 2015 and 1% in 2016 (see table) . importantly, this occurred despite a greater than 30% increase in platelet inventory between 2014 and 2016 and resulted in cost savings of over $60,000 in this period. conclusion: study limitations included restricting data collection to one campus. the option to transfer expiring platelet units to another blood bank was available to all 4 participating sister hospitals. it would have been interesting to see the effect of the report on those hospitals which have lower transfusion rates and different ordering practices. aside from lowering platelet wastage within 2 years of implementation, additional benefits to the report included facilitating ordering from the blood supplier. cornerstones of a successful inventory management plan include daily inventory monitoring and, ideally, coordinated system-wide efforts to share platelet units. we have shown achievement of this end is facilitated by a customized daily platelet inventory report -an efficacious and easily adaptable tool with demonstrable gains. valerie halling* 1 , lisa marie button 2 , lori scanlan-hanson 2 , karen koch 2 , janet finley 2 , deepi goyal 2 and camille van buskirk 3 . 1 mayo clinic-rochester, 2 mayo clinic, 3 mayo clinic rochester background/case studies: transfusions in the emergency department of a level i trauma center were ordered using a handwritten order form. the transfusion lab's (tl) management team and medical director met with emergency department (ed) leadership and it resources in 2014 to define the needs of a successful electronic blood transfusion system. the handwritten order forms had several potential error sources which could lead to a delay in filling the order pending correction (in the best of circumstances) or could lead to transfusing the wrong patient or the wrong product if the error was not detected. the potential error sources included clerical errors involving the patient's name or medical record number (mrn), writing two different names on the order form (because there were two locations to record patient name), two product types ordered on one form when the requirement is for one product type per order, no priority indicated (stat or routine), or not including the prescriber call-back information. the number of ed reported transfusion related events in 2013 and 2014 were 63/1187 (events/ed transfusions 2013-2014). study design/method: electronic ordering for the ed was implemented march 31 st 2015. any transfusion orders generated from the ed are now electronic, unless in the case of electronic downtime. the system electronically fills in the patient's name and mrn, controls for the type of blood product being ordered, requires an order priority and provides service contact information. it was designed to accommodate transfusion ordering needs for adults, pediatric patients <35kg and pediatric patients >35kg. 252a transfusion orders had three critical fields identified that are required for the order to be processed including patient weight, product volume, and infusion rate. the electronic system was designed so that an order cannot be submitted unless all critical fields are completed. results/finding: the electronic ordering system has been in place for 2 years (april 2015 -march 2017), and during that time there was 1 instance of blood being ordered for an unintended patient 0.09% (1/1081). this was because a previous patient's medical record was accessed rather than the intended patient's medical record. there have been no instances of clerical errors (name misspelled or mrn transposition etc.), missing service contact information, missing order priority information, more than one product type ordered on a single order, or two patient names on one order. electronic ordering also provided a place for the transfusionist to chart against, leading to increased transfusion documentation compliance. prior to electronic order implementation, in 2013, 17/651 (2.61%) units were transfused in the ed but not charted in the patient's medical record. in 2014, 18/536 (3.36%) transfusions were not charted. however, in 2016, the first full year of electronic transfusion order capability, only 4/462 (0.87%) transfusions were not charted in the patient's medical record. conclusion: electronic ordering in the ed has essentially eliminated ordering errors in this area resulting in less rework for both technologists and physicians. it allowed the order to be processed more quickly by tl, resulting in a faster turnaround time. improvement in the overall quality of transfusion ordering through electronic ordering reduced the influence of human factors in order placement and provided an added benefit of having a specific order to chart against. implementation of blood bank automated attendant lok tse*, gerald motta and maria aguad. brigham and women's hospital background/case studies: the blood bank receives numerous nonemergent phone calls on a daily basis. these calls not only occupied valuable time but also made the lines unavailable when a real emergency occurred. the hospital is categorized as a level 1 trauma center, with over 700 inpatient beds and over 50 operating rooms. a proposal to implement a blood bank automated attendant was recommended to decrease phone calls, minimize errors due to distraction from phone calls, free team members to perform other duties and have a direct line designated for requesting trauma coolers, massive transfusion protocol (mtp) and emergency release of blood products. study design/method: the first step was to categorize the types of phone calls received by the blood bank by creating a phone log. data were collected and analyzed for four weeks. the blood bank collaborated with nursing, hospital administrative staff and telecommunication team to evaluate the possibility of implementing an automated attendant to minimize phone calls. it was very important to maintain patient safety and quality of service at the same time. the automated attendant consist of: option 1 (urgent) for trauma, emergency release, mtp and obstetric hemorrhage emergency release; option 2 (verbal) for verbal orders and coolers set up; and option 3 (staff) to speak with staff member. instructions were also given for specimen inquiry and product availability in the hospital information system. results/finding: the data in table 1 showed that most of the incoming calls fall into three categories (specimen inquiries, product order inquiries, and other inquiries). most of the calls were from nursing staff inquiring about the length of wait time for blood products and specimen availability. there was an overall decrease in phone calls by 68% with the implementation of an automated attendant. conclusion: with the implementation of an automated attendant, the blood bank team was able to identify and respond accordingly and efficiently to urgent requests and verbal phone orders. the decrease in phone calls freed up team members to perform other critical tasks in the department. improved detection of wrong blood in tube errors: implementation of a two-sample blood type verification process ariana king* 1 , steven zibrat 1 , geoffrey wool 2 and angela treml 2 . 1 university of chicago medicine, 2 university of chicago background/case studies: our organization used a blood bank identification (bbid) band system for pre-transfusion testing and detection of wrong blood in tube errors (wbit). additionally, type & screen results were compared to patient's historical records; the specimen was retyped by a second technologist if historical results were not available. the bbid bands were prone to clerical errors and excessive specimen rejections, and believed to miss some wbit errors. in 2015, blood bank accounted for 48% of all rejected clinical laboratory samples, yet comprised only 5% of total laboratory volume; 88% of rejected blood bank samples were due to bbid band issues. the wbit error rate detected by bbid-based system was 0.006%. study design/method: a multidisciplinary workgroup was formed to review data and best practices. the decision was made to discontinue bbid bands and implement a two-sample verification process, in keeping with standards. a new laboratory test order was created in the emr system and embedded into the existing t&s order. providers are prompted to order the abo verification test only when no previous abo/rh typing results are found. education was provided to all clinical staff in the form of in-services, emails, and annual competencies completed electronically. the new process went live in september 2016. results/finding: in the five months following implementation, four wbit errors were detected with the second sample. these may have been missed using the bbid band system. improved detection revealed a wbit error rate of 0.128%, three times the national average of 0.043%. under the new system, rejected blood bank samples decreased from an average of 50% to 28% of all rejected laboratory samples, a 43% decrease. implementation of the new process produced a net savings of $55.8k. conclusion: replacing the bbid band system with two-sample verification successfully improved our ability to detect wbit errors among patients who lacked historical blood bank results. additionally, discontinuation of the bbid system decreased the incidence of clerical errors and unnecessary specimen rejections, and also saved money for the organization. next steps are for blood bank and laboratory quality leaders to partner with nursing leadership to drive down wbit error incidence. 253a addendum with the final culture results. we used a student's t test to determine whether there was a statistically significant difference in the mean tat for result addendum entry in the post-implementation period compared to the pre-implementation period. results/findings: in the pre-implementation period, we cultured 19 residual products for suspected str. the tat for final culture result entry into the patient's emr was 5-78 days (mean 19 days, sd 20). in the postimplementation period, we cultured 22 residual products for suspected str. the tat for final culture result entry into the patient's emr was 5-12 days (mean 7 days, sd 2; p50.0082). there were no positive cultures during either study period. conclusion: our study demonstrates that tat for documentation can improve with the use of information technology to notify the transfusion medicine physician when results are available for documentation in a patient's emr. improved turnaround time of type and screen samples michaelene hultman* 1 , marcus holme 1 , johnathan bakst 1 , gunta musa 1 and angela treml 2 . 1 university of chicago medicine, 2 university of chicago background/case studies: the primary test performed in the blood bank with regard to pre-transfusion testing is the type and screen (tys). the current target for this institution's blood bank is an 80 minute turnaround time (tat). in april of 2016, the blood bank was forced to move to a temporary location due to building construction, which necessitated a switch from automated solid phase methodology to manual gel method. the average number of outliers increased 104%. tat analysis of a representative one week sampling per month showed an increase in outliers from 28 per month to 57 per month. average monthly tys samples performed is 2758. these numbers did not improve even upon returning to the original facilities. study design/method: two ortho clinical diagnostics visionv r analyzers (raritan, n.j.) were purchased for the blood bank. the instruments were set up with a bi-directional interface allowing for samples to be continuously loaded without manually ordering the tests. batch testing was eliminated allowing samples to be run as received. the results were auto interpreted, and transmitted to the laboratory information system (lis) based on predetermined rules. only results in need of manual review or interpretation were held back. final verification of results was performed by the technologist within the lis. reagents and other needed consumables could be preloaded on the instruments eliminating the need to repeatedly load consumables with each sample run. key quality indicators including tat continued to be monitored throughout implementation. data was monitored for significant changes and improvements in patient care. the go-live date was 12/20/ 2016. results/finding: the average number of outliers decreased 61% from 57 per month to 22. further benefits include a reduction in the number of technologists needed to perform tys testing. additionally, reduced waste due to better utilization of supplies by the instruments along with less repeat testing has resulted in projected cost savings of $134,000 for fiscal year 2017. conclusion: the use of gel technology, in combination with a two way interface and a continuous load instrument can result in a significant decrease in tat over manual gel method. improvements in the timely reporting of final product culture results in the patient's emr. barbara a. hewitt*. dartmouth hitchcock medical center background/case studies: in certain transfusion reactions it is required that a culture of the returned blood product be performed. these cultures are reported in our cerner operating system but those results do not cross over to the patient's emr . the finalized product culture results are entered into the patient's emr as an addendum to the transfusion reaction clinical note. a review of the transfusion reaction database revealed that there were occasions when the final product culture results were not entered into the patient's emr in a timely manner. it is important to the patient's care for the transfusion medicine service and the patient's primary provider to know if a transfusion reaction is related to a contaminated product or the patient's general overall health. this information is also crucial to the supplier of the product to determine if others have received components of the affected unit and to possibly determine if there are any quality control issues at the donor facility. study design/methods: a review of a specific 7 month period revealed that the timeframe in which the finalized product culture results were entered into the patient's emr ranged from 0-65 days with a mean of 13.64 days. it was determined that this was not in the interest of improving patient care. in collaboration with laboratory information services a report was created in which once product culture results were finalized an email would be generated notifying the medical director and the transfusion safety officer that results were available. results/findings: data was collected for 7 months following the implementation of this report and it was noted that timeliness of finalized product culture results being entered into the patient's emr improved to a range of 0-7 days with a mean of 2 days. conclusion: improvements in patient care require diligence and timely reporting of finalized culture reports to determine potential causes of transfusion reactions. this process can be made easier when the correct tools are used. omer ilyas* 1 and randy levine 2 . 1 northwell health, 2 lenox hill hospital background/case studies: transfusion of non-irradiated blood in patients with hematologic malignancies and those receiving cytotoxic chemotherapy can result in life-threatening graft versus host disease (gvhd). after noting several instances where non-irradiated blood was transfused in patients requiring irradiated blood, we designed a quality improvement project with educational sessions involving the oncology unit and blood bank. study design/method: the project was separated into three parts. in the first part, data on transfusion practices was retrospectively collected over a four month period on the oncology unit. the variables collected included date and time of transfusion, pre-and post-transfusion hemoglobin, patient diagnoses, and whether or not blood was ordered to be irradiated and if so, whether or not irradiated blood was issued by the blood bank. all patients with hematological malignancies and all patients receiving cytotoxic chemotherapy were candidates for irradiated blood. the second part of this project was an educational intervention. residents, oncology floor nurses, and blood bank staff were given lectures on the importance of transfusing irradiated blood on the oncology floor. residents were also instructed to order irradiated blood for all patients on the oncology unit. in the third part of this project, repeat data was collected over a two month period to assess whether the intervention was successful. results/finding: pre-intervention, 67 units were transfused on the oncology floor with 38 units (57%) requiring irradiation and only 22 of those 38 units (57%) ordered as irradiated. since the blood bank occasionally issues irradiated blood without a specific order, 9 additional irradiated units were issued (31/67; 46%). post-intervention, 29 units were transfused on the oncology floor with 15 units (52%) requiring irradiation and all 15 of those units (100%) ordered irradiated specifically to prevent gvhd. eight additional irradiated units were ordered with no requirement for irradiation; thus 23 of the 29 (79%) total units were ordered as irradiated. again, 4 additional irradiated units were issued (27/29;93%) without a specific order by the blood bank. the results are summarized in the accompanying table. conclusion: this quality improvement project demonstrates that educational intervention can succeed in changing clinical practices. continued monitoring of ordering practices will ensure that compliance continues. we plan to expand the quality improvement project to other settings, including the emergency department and surgical floors. we expect that adherence to transfusion guidelines in this patient population will reduce the incidence of adverse events. samantha ngamsuntikul* 1 , charlotte van dyke 2 , dina garza van hoose 2 and rachel beddard 1 . 1 biobridge global, 2 south texas blood and tissue center background/case studies: at our blood center, apheresis platelets and red cells are collected on trima accels and double red cells on haemonetics 8150s. in addition to routine quality control (qc), qc is performed for instrument flags on collection instruments. quality control for apheresis platelets includes: volume variance and rwbc; quality control for apheresis red cells includes: product volume, volume variance, hemoglobin and red blood cell mass. study design/methods: during the period of january 1, 2016 to april 19, 2017, 2,097 total collections were flagged for additional qc by our trima accels and haemonetics 8150 instruments. quality control at our center is tracked by our quality control software management system, hematerra's hemacomply which allows the ability to track and retrieve this information. the majority of products flagged for instrument qc pass and are released for distribution. a small percentage, however do fail qc leading to loss of product. quality control data can be retrieved and monitored for trends using a quality control software management system. background/case studies: in 2015, the centers for medicare & medicaid services (cms) rolled out a plan for implementing iqcp (individualized quality control plan) as a new quality control option based on a risk management plan for clia laboratories performing non-waived testing. this plan was meant for clia approved tests, but serves as a good tool for labs performing non-traditional and traditional tests on non-traditional samples. study design/method: clia clinical laboratories can either follow traditional clinical clia qc requirements according to the regulations or implement iqcp. while we perfrom traditional qc assessments on all the tests we perfrom on our cellular products we did decide to implement the iqcp program within in our quality control laboratory. we followed the iqcp process for assessing some of our qc tests used to assess the safety, purity and potency of our cell based products. one test in particular where we applied this tool was in the review of our qc sterility testing method and found it to be a very useful in improving the overall process. the tool walks you through three process requirements: 1) risk assessment, 2) quality control plan and 3) quality assessment for the preanalytical, analytical and post analytical phases of testing. abstract conclusion: the integration of the iqcp into the quality control laboratory was determined to be a success. the iqcp tool was successful in identifying gaps within the sterility testing process. this tool will be used on additional quality control tests and manufacturing processes. the implementation of the iqcp program ensure regulatory qc requirements appropriate for testing performed. we were able to revise our procedures, reeducate those involved in the process and hopefully minimize potential sources of error. objective performance of massive transfusion protocols at a single institution gustaaf de ridder* 1 , rachel jug 1 , kimberly ingersoll 1 , nicholas bandarenko 2 , nicole guinn 3 and jessica poisson 2 . 1 duke health pathology, 2 duke university hospital, 3 duke health anesthesiology background/case studies: hemorrhage is both a leading cause of mortality in trauma patients and morbidity in non-trauma patients. using a balanced 6:6:1 transfusion ratio (tr) for massive resuscitation is recommended based on trauma data. objective performance during massive transfusion protocol (mtp) activations is poorly studied and there may be differences based on site or medical service of mtp initiation. with the impending release of a unified, redesigned exsanguination protocol (exp) at our institution, we established baseline performance characteristics for our existing mtp and obstetric massive transfusion protocol (obp). study design/method: following institutional review board approval, we performed a retrospective study on blood product utilization and outcomes of mtp and obp activations from july 2015-december 2016. data were manually collected from transfusion service paper records, electronic (safe-trace) records, and an automated data report from the electronic medical record (epic). conclusion: we observed considerable variability in transfusion practices during acute hemorrhage depending on the service and location of activation. trauma activations demonstrated the sharpest deficit in platelet transfusion, whereas all groups lagged somewhat in transfused plasma relative to packed red blood cells. los and mortality varied among groups, likely reflecting underlying medical conditions and indications for massive transfusion. we have identified an opportunity for improvement in mtp transfusion ratios observed in trauma cases, the specific environment from which the 6:6:1 ratio was derived, and in which the impact of protocol-driven blood resuscitation is most efficacious. patient identification improvement strategy to help reduce unacceptable specimens arline stein* 1 , nancy nikolis 1 , linda benison 1 , ruthmire thelusca 1 , renee liberty 1 , sherry shariatmadar 1 , alexander indrikovs 2 and vishesh chhibber 1 . 1 north shore university hospital, 2 northwell health background/case studies: our blood bank (bb) processes approximately 60,000 specimens per year. bb specimens are unacceptable when they are unlabeled, unsigned or missing necessary documentation. in such cases, a new specimen is requested to be drawn as per protocol. our investigation of unacceptable specimens previously included generation of a report by the blood bank staff that was subsequently submitted to the bb supervisor for completion. following completion, the report was sent to the nurse manager of the patient care unit (pcu) for follow-up and investigation with the staff members involved. this process was cumbersome, taking a few days before the staff member of the pcu was alerted to the deviation in protocol. at times, residents or float staff involved were difficult to identify and it was often challenging to track down the staff and do the necessary investigations and in-services. study design/method: in june 2016, a patient identification improvement strategy was implemented jointly by the department of nursing and the bb to address mislabeled, unlabeled and unsigned specimens as part of a patient safety initiative. currently, following this strategy, when an unacceptable specimen is received, the nurse manager (nm) of the pcu is immediately notified by bb staff. the nm promptly initiates a debrief process with the staff involved in drawing the specimen. a debrief form (tool) was created to guide the discussion. this process is followed 24/7. the nm will also engage other available staff in a huddle to review the incident and reinforce the policy. the debrief form is then submitted to hospital qa and the bb with preventative actions included. we believe in using the just culture model to help us understand the reasons why the staff did not label the specimen according to policy. just culture helps promote shared accountability to ensure we have the proper systems and processes in place to deliver high quality care. results/finding: the table below represents the percentage of unacceptable specimens identified by the bb since the second quarter of 2016. the implementation of this new process has led to a decrease in the number of unacceptable specimens up to 30% quarterly following its implementation. the opportunity for direct intervention by the nm with the staff involved has risen from 75% to 96%, due to the immediate debrief process. abstract conclusion: the patient identification improvement strategy allows for real time engagement of the bb and pcu staff to promptly investigate and institute corrective/preventive actions when there is a deviation from policies related to specimen collection. the heightened awareness of correcting patient specimen labeling errors can only improve patient safety and the patient experience. platelet transfusion practices among pediatric oncology patients: a single institutional experience nicole m crews* 1,2 , morgan rockwell 2 , joseph hagan 1 , jun teruya 1,2 and shiu-ki hui 2 . 1 texas children's hospital, 2 baylor college of medicine background/case studies: despite advances in adult platelet transfusion (ptx) literature, questions persist regarding pediatric transfusion thresholds, dosage and responses. therefore, ptx are commonly guided by local institutional recommendations (ir). the aim of this study was to determine the degree of adherence of ptx practice to ir at a pediatric tertiary institution. study design/method: retrospective review of ptx practices including transfusion thresholds, responses and dosages were collected. platelet counts within 24 hours pre and post transfusion were evaluated. patients (0-18 years) receiving prophylactic ptx from july to december 2016 admitted to the oncology acute care unit with diagnosis of leukemia or lymphoma were included. for prevention of volume overload, the ir for ptx were < 10ml/kg for patients < 35kg and one apheresis unit (au) for patients >35kg; therefore, patients were separated into 2 groups: < 35 kg and > 35 kg. a significant proportion of orders for both < 35 kg and > 35 kg did not meet patient platelet threshold criteria (p<0.001). conclusion: ptx threshold above ir for both groups were 31 ( 35 kg) and 48% (> 35 kg). most common reason for above ir threshold was an invasive procedure or low molecular weight heparin therapy. greater than 10% of ptx dosage in both groups were above ir, however the platelet response did not increase significantly (p>0.05) with a higher dose vs. ir dose. this study demonstrated that there were still considerable deviations from ir in ptx practice among pediatric oncological patients. in addition, the false assumption that a higher dose will yield a better response can put patients at increased risk for transfusion related adverse events. each institution should conduct a quality assurance review to determine ptx practice. pre-surgical sample process improvement to enhance patient safety and compliance lisa marie button*, stephanie saathoff, jered luedke, benjamin colvin, umalkair amare and james r stubbs. mayo clinic background/case studies: our institution provides the option for presurgical samples (pss) to be drawn up to 56 days prior to surgery as long as the patient reports not being transfused with a blood or blood component containing allogeneic red cells and they have not been pregnant in the preceding 3 months from the date of pss collection. when pss patients returned for surgery, the patient's service was required to ask the patient again about their transfusion and pregnancy history to determine if there had been any new opportunities for allogeneic red cell exposure, however, there was no process to capture the information the patient reported for the time between the pss draw to the day of surgery/possible transfusion. study design/method: an electronic fix was designed that was applied to the surgical intake process. a new set of questions was added to the a.m. admit questionnaire that must be completed prior to the patient's surgical procedure. the questions ask the patient if they have been pregnant or transfused in the preceding three months and if the answer is affirmative, the computer system runs a blaze rule causing an alert in the blood bank. the blood bank techs review the alert and inactivate the patient's pss based on the new transfusion/pregnany information. one year post-implementation of the electronic fix, transfusion lab performed a retrospective review of all pss alerts generated during a three-month period. results/finding: the results of the review were analyzed and are displayed in the table. it was determined that only 2.1% of patients with a pss alert had an active sample requiring inactivation. conclusion: implementing an electronic solution that requires documentation about pss eligibility upon return for surgery has resulted in an estimated 3220 (805x4) pss alerts in the blood bank each year. of these alerts, it is estimated that approximately 68 patients (17x4) per year are identified as no longer eligible for pss status. once this retrospective review was performed, it was shared with the project stakeholders to determine if the electronic questionnaire could be further tailored to patient's based on age, gender, and pss status. while the benefit of having fewer false positive pss alerts (97.9%) was recognized as an ideal future state, it was not compatible with the institution's current it project of implementing a new electronic medical record (emr) system. the safety enhancement provided by the current electronic fix will remain as is and the improvement suggestions were shared with the team creating the parameters for the new emr with the intention of targeting only patients with an active pss in the blood bank, rather than all surgical patients. weill cornell medicine, 7 columbia university school of medicine background/case studies: blood ordering is a complex, high-risk process with multiple steps that have the potential for errors and delays. risks associated with this process, from ordering through pick-up, require evaluation and strategies for mitigation. given the complexity and high-risk nature of blood ordering a proactive risk assessment (pra) for blood product ordering using the fmea methodology was conducted. the goal of the project was to proactively assess the effect of a redesigned electronic order set on the quality and safety of blood ordering study design/method: to evaluate the electronic blood ordering redesign process, a pra was completed using the fmea methodology. the team identified each step and sub-step of the electronic blood ordering process, all failure modes and causes, and then scored each by severity occurrence and detectability to determine the risk priority number (rpn). all rpns with a score above the threshold were reviewed and rescored based on mitigation strategies designed to address the failure mode. results/finding: the group scored the identified failure modes by categories used in root cause analyses. the electronic blood order process has internal logic and alerts that improve communication and reduce the risk score. several mitigation strategies that will reduce the risk of the identified failure modes include type and screen status within the rbc order, streamlined alerts when the order does not meet the laboratory threshold, a nursing task list for transfusion, and a change to the pickup process that is linked to the product ready status in the laboratory information system. a transfusion history will be available to providers when ordering blood products to further reduce communication risks. categories for failure modes included clinical,communication,equipment people,process and system. the average overall failure mode rpn was reduced by 29% with the communication category average rpn having the greatest reduction of 72%. conclusion: an fmea of an electronic blood ordering process can proactively improve quality and patient safety by preventing transfusion delays and errors in blood product administration. accurate and timely information in the blood ordering process has the potential to reduce risks associated with ordering,preparing and dispensing blood. reducing turn around time for type and screens in the blood bank kimberly ouellette* 1 , karen king 1 and joseph sweeney 2 . 1 rhode island hospital, 2 lifespan academic medical center background/case studies: expeditious turn-around times (tat) in the blood bank are critical to provide fully tested and crossmatch compatible blood in a timely manner. the blood bank at rhode island hospital, a level 1 trauma center and teaching hospital associated with brown university, was originally designed to accommodate tube testing by all technologists. the original setup of the lab was split into three sections allowing for preparation and issuing of units in the first section, bench testing in the second, and the receipt of components in the third. as technology changed, the blood bank adopted first the manual gel station and then the automated gel system (ortho provuev r ) but did not adapt the space. the second section of the blood bank contained the manual and subsequently automated gel stations with no other changes. the process of sample receipt through completion of testing and issuing of units remained segmented and inefficient. the average tat for type and screens was 80 6 32 minutes. study design/method: the blood bank design was remodeled to make for a more open concept to allow for collaboration amongst technologists as well as the best use of space and technology. the first section of tables were removed and replaced with a center console to allow for movement about the entire front of the laboratory. a wall was constructed to separate the main work flow, automated gel testing and issuing units, from the area for complicated workups and inventory receipt. the third section remained, but was repurposed for teaching medical technology students and residents. in addition to the remodel, the blood bank retired the ortho provuev r for the ortho visionv r , which is considered a true continuous feed machine. although the inter-device tat is not significantly different (30 minutes for the provuev r and 28 for the visionv r ) the visionv r is built with a scheduler that effectively handles the system and processes samples efficiently. the visionv r is also equipped with two centrifuges to process samples, which further reduces tat when multiple samples are onboard. a bi-directional interface was designed to allow for test orders (type and screens) to go to the visionv r and test results to go directly from the visionv r to the lis without the need to manually order the tests or transmit the results. data on tat were collected and analyzed using independent t tests and chi square. results/finding: the mean tat pre-and post-reconfiguration and implementation of the ortho visionv r and a fraction of samples with tat over 90 minutes are shown in the table. the results show a reduction in tat by 14 minutes with a 20% reduction of tat greater than 90 minutes. conclusion: a combination of new technology and space remodeling can lead to a significant reduction of tat of testing in the blood bank. caleb wei-shin cheng* 1,2 , lorna orengo 3 , monique scott 3 and christopher a tormey 1,2,3 . 1 yale university school of medicine, 2 yale-new haven hospital, 3 va connecticut healthcare background/case studies: the type and screen (t&s) is a fundamental laboratory test that allows the blood bank to provide compatible blood for patients. despite this, erroneous blood product administration may occur as much as 1 in 19,000 blood transfusions. to prevent errors, adequate specimens such as those lacking hemolysis and those with proper specimen labeling are necessary; otherwise the specimen is rejected, leading to a second blood draw and a delay in medical/surgical management. hemolysis rates for t&s specimens are reported to be as high as 25% prior to interventions, but may potentially be reduced to as little as 1.5%. however, there is little published data on non-hemolysis-related type and screen rejections. an initiative was undertaken to reduce the rejection rate in the blood bank to a sustained rate of <5%, with a particular emphasis on non-hemolysisassociated forms of rejection. study design/method: a root cause analysis (rca) was performed over the preceding 6 months to obtain a baseline understanding of the errors involved. t&s submission at our facility involves standard completion of the specimen label plus completion of a unique witness form to confirm the identity of the patient from whom the specimen was collected; specimen and witness form must be submitted simultaneously. when a specimen was rejected, we recorded the patient name, medical record number, and the reason for rejection. following rca, an intervention was created to resolve the most common issues documented that resulted in rejection. approval for the intervention was granted by the department chair, transfusion committee, forms committee, and the medical executive committee. after implementation, prospective data will be collected for several months in the same manner as before to determine the effectiveness of the intervention. results/finding: over the study period, the t&s rejection rate averaged 8.2%. reasons for specimen rejection were divided into 5 groups: 1) hemolysis, 2) blood bank witness collection form errors, 3) quantity not sufficient, abstract 4) duplicate sample, and 5) specimen tube labelling errors. the highest percentage of rejections was due to improperly-filled witness forms (table 1) . after multiple form redesigns and approval by appropriate committees the new form was implemented. preliminary data collected thus far demonstrates a 6.8% rejection rate with only 1 rejection relating to witness form errors. conclusion: rejected t&s specimens are an impediment to safe clinical care as it may delay medical/surgical management. rejection rates could be reduced through simplification of blood bank specimen collection forms. care providers have multiple tasks that need to be performed in a short amount of time, therefore, simplification is often times necessary to reduce human error. future quality initiatives will aim to simplify complex healthcare processes without compromising patient care. reduction of failed whole blood donor testing runs on the roche cobas s 201 system christopher shahan* 1 , christina dejesus 1 , mosi mccall 1 , fallon hampton 1 , tangi herring 1 , judy davis 1 , anjali patel 1 , sonya gomillion 1 and bonnie maltby 2 . 1 qualtex laboratories, 2 qualtex laboratories background/case studies: as part of our quality control program, we track the number of technician related failed runs observed on the roche cobas s 201 system. this system is used to test whole blood donor samples for human immunodeficiency virus (hiv) rna, hepatitis c virus (hcv) rna, hepatitis b virus (hbv) dna and west nile virus (wnv) rna. technician related failed testing runs can cause the laboratory to report results outside of the contractual 10-12 hour turnaround time. failed runs also cause retesting which increases reagent utilization for the system. currently 42% of whole blood donor testing turnaround time delays are due to issues and failed runs on the s 201 system and we have 3 technician related failures per week. a lean six sigma approach for process improvement was utilized to identify root causes and develop countermeasures in order to decrease the number of technician related failures on the s 201 system. study design/method: the number of technician related failed runs on the s 201 system were tracked from 10/11/2015 thru 12/11/2016. a pareto chart was used to determine that technician related errors was the largest controllable factor causing run failures. the 5 whys were performed to determine root causes of technician related failed runs. a gemba walk was performed on all of the lab testing processes to help identify areas for improvement. the process improvement team talked, met, observed, and worked directly with staff that operate the s 201 system. roche was also contacted to provide guidance on how to help decrease technician related failures. results/finding: the main root cause determined was that there was no current process flow map for whole blood donor testing using the s 201 system. counter measures implemented included creating a two phase process map. one phase was related to the processes related to start-up of the system and the second phase was related to the processes involved in processing of samples. roche provided a job aid for the technicians which provides clear steps technicians should take when handling and cleaning up crashes and failed runs on the s 201 system. after counter measures were implemented, the number of technician related failed runs decreased from 3 to 1.2 failures per week, which was a 58% decrease. conclusion: a lean six sigma approach for process improvement was utilized to identify root causes and develop countermeasures in order to decrease the number of technician related failed runs on the cobas s 201 system. this lean six sigma approach and counter measures significantly decreased the number of technician related failed runs by 58%. patients who were transfused for pre-transfusion hgb >7g/dl with resulting post-transfusion hgb >9g/dl were reviewed. demographics, medical history, provider identity, indication and transfusion complications were abstracted & compiled individually by 11 volunteer internal medicine residents. group discussion for each case ensued before determination of transfusion appropriateness occurred. principal investigator/attending physician then made final determination of appropriateness of rbc transfusion. results/finding: 265 patient charts were reviewed. 91 were excluded for bleeding and cardiovascular instability. 106/174 (60.9%) were determined to be transfused inappropriately. there was no difference in appropriateness of transfusion with respect to age or sex. patients with solid tumors (76.19% vs 55.73%, p50.0181) and anemia of chronic disease (76.47% vs 54.1%, p50.006) were more likely to be inappropriately transfused. patients who had higher pre-transfusion hemoglobin were more likely to be inappropriately transfused (median hgb 7.7 g/dl vs 7.3 g/dl, p<0.0001). inappropriately transfused patients also had higher median post-transfusion hemoglobin (9.9 g/dl vs 9.4 g/dl, p<0.0001). moreover, lab evalutions revealed association with lower folate levels (median 8.1 nmol/l vs 15.7 nmol/l, p50.029). 29/106 (27.3%) patients were inappropriately transfused at least in part because they received more than one unit without an interval hemoglobin check in between. 7/64 providers were responsible for 32.3% of all inappropriate transfusions. 1/68 appropriately-transfused patients experienced an fnhtr. 3 deaths unrelated to transfusion occurred (1 in appropriate, 2 in inappropriate group). conclusion: physicians in training are interested in promulgating optimal rbc transfusion practice. this study identified patient factors (such as solid tumors and anemia of chronic disease) that correlate with a higher likelihood of receiving an inappropriate transfusion. beyond cpoe, educational intervention at individual level should be designed for specific providers responsible for more inappropriate transfusions. successful implementation of a blood bank information system in a small-scale caribbean blood bank: a structured step-wise approach. luigi sille* 1 , willem martin smid 2 and ashley john duits 1 . 1 red cross blood bank foundation, 2 sanquin consulting services background/case studies: an important tool for complying with gmp quality standards is the effective use of a blood bank information system (bis). validation and implementation of a bis is described for centralized large blood bank and literature and guidelines are lacking for the nonautomated small scale blood bank environment. . small-scale blood banks face specific challenges for computerization in relation to economies of scale and existing processes requiring special attention. for the introduction of a bis at the blood bank of the dutch caribbean island of curaçao a specific procedure was designed based on existing guidelines and adapted to the local setting. study design/method: the red cross blood bank foundation curaçao is the sole provider of labile blood components for the dutch caribbean islands of curaçao, bonaire and sint maarten. after selection of the bis provider for implementation isbt and bcsh guidelines for validation of information systems in blood establishments were carefully analyzed to prepare the design of local procedures. these procedures were meant to evaluate and validate the features of a bis (e-delphyn, hemasoft america, miami, usa) before introduction. the outcome of the approach was entered in worksheets that were evaluated by the implementation team and management. from this the implementation plan was designed and implemented. an external auditor (sanquin consulting services, amsterdam, netherlands) was invited to evaluate the implementation and validation plan and its practical implementation. the evaluation was performed according to risk assessment of critical process steps. results/finding: based on the isbt and bcsh guidelines a process flow chart describing the relevant phases and critical steps for introduction and validation of a bis was designed. comparison of the current processes and procedures were compared to the bis characteristics making use of 8 worksheets. with these worksheets the existing gaps with the bis procedures were carefully described. these gaps and the appropriate procedural changes for bis or blood bank were effectuated. the worksheets also provided the basis for staff training in a separate training environment before bis introduction. during the early validation phase all procedures and processes were audited by an external auditor. with the feedback of the expert several improvements were added for the validation and subsequent implementation processes. conclusion: with the use of existing international guidelines a validation and implementation plan was designed to prepare for successful introduction of a bis in a small scale caribbean blood bank. the program as designed seems well suited for small scale blood banks contemplating introduction of a bis. time and cost savings through implementation of a remote blood fridge jessica peters* 1 , dee dee cassidy 1 , jed b gorlin 1,2 and nancy l van buren 1,2 . 1 hennepin county medical center, 2 innovative blood resources background/case studies: rapid delivery of emergency release group o red blood cells (rbc) are vital to patient care. commercial remote blood fridge packages are available but have large upfront and maintenance costs. we implemented a remote blood fridge directly in our emergency department (ed) using an under counter fridge requiring id access, and a selfdeveloped ios application that scans, tracks and real-time alerts transfusion service (ts) to products used and to whom they were dispensed. prior to ed fridge implementation, rbc units were verbally requested and an ed blood runner would pick up and return the cooler. given that our ed is located in a separate building from the ts, this meant 10 or more minutes may be required for transit of units often released in less than 5 minutes. the net effect was that providers would routinely order products to ensure they were at the bedside for patient arrival as a precaution, only to return them when not required. implementing a blood fridge at bedside resulted in the predicted outcome of delivering emergency release rbcs more quickly, with the observed benefit of decreasing wasted staff time. study design/method: the remote blood fridge was implemented in july 2016. data for rbc requests in coolers, rbc returns and rbc transfusions from the ed was collected and compared. baseline data included january 2015-june 2016, and post change included august 2016-december 2016. july 2016 data was excluded as it included both the pre and post processes. results/finding: baseline data shows that the ed requested an average of 99 rbc/month in coolers. post change this dropped to 62 rbc/month, thus less blood was requested from the transfusion service in coolers as units were being used from the fridge. baseline data also shows that an average of 54 rbc/month were returned (55%). post change, the average rbc/ month returned was 27 (44%), this represents an absolute 50% reduction in number of returned products. each rbc dispensed and returned takes approximately 20 minutes to complete paperwork and transport, therefore this change saved an average of 540 minutes per month. it was also noted that the average rbc/month transfused was 44 for baseline and 57 post change. this confirms that the decreased requests and returns were not due to decreased patient volume or severity. the fridge was also successful at decreasing delivery time of blood to patient bedside, as baseline delivery time of 15-20 minutes (estimated) was reduced to 3-5 minutes. conclusion: implementing a remote blood fridge and moving blood access closer to patient bedside ensures a faster delivery of blood to the patient. this change has an additional benefit of decreasing wasted time, and hence cost, by decreasing unnecessary product requests and returns. implementing a blood fridge can also be done at a reduced cost through homegrown processes. transfusions are everyday procedures and over 250 patent-applications have been filed related to "transfusion medicine" and over 400 related to "transfusion alarm", during the last 30 years, employing numerous technical settings, aiming to support automated supervision of the mentioned actions. the aim of this contribution is to present a developed low-cost real-time individual intravenous blood-transfusion monitoring system, based on the internet of things. study design/method: the designed system is based on a commercially available pan-tilt-zoom (ptz) camera, employing an 1/4 inch color cmos sensor, providing effectively 1.0 mp, a 3.6 mm lens, ir-cut, day/night minimum illumination 0.1 lux/f 1 and 808 viewing angle. the camera is focused on the droplets and acts as vis/ir detector with a 24 hz sampling-rate. custom-developed software supports droplets' ratemonitoring, causing acoustic alarm-signals if necessary (e.g. clotting, blood 260a transfusion 2017 vol. 57 supplement s3 abstract or other suspensions depletion etc.) and enables, if necessary, wide-angle image-capturing. the video image-audio settings provide for compression h.264, video frame rate (fps) 1-30/s, refresh rate 50 hz and audio input, through bidirectional built-in microphone. the acquires an ip-address, the connection mode is wireless, the network interface is wi-fi/802.11/b/g, the supported protocols include dhcp, tcp/ip, upnp, http, smtp and p2p is provided. typical 5v power-supply, sized 165x125x101mm and weighing 370 g. client software is required. the ir range is 10-15 m; ir-cut filters, remote access, dual stream, motion detection, day/night and ir night vision distance of 10 m are offered. two-way radio-link is provided, as well as, trans-flash (tf) recording and storage on a 32 gb sd-card. pan/tilt-horizontal 355 o and pan/tilt-vertical 90 o movements can be performed. the system facilitates, if needed, also patient's position monitoring and readings of other monitoring displays, such as nibp, ecg, and spo 2 , if present. results/finding: the system and is being presently tested in a laboratory (non-clinical) environment, by simulating the virtual patient, with a custommade "phantom", combining flow-rate, negative pressure and viscosity resistance regulation. conclusion: the system can measure infusion-speed with a deviation lower than 6 3 %. the developed iot-system takes advantage of the existing hospital wi-fi networked environment and offers a low-cost solution, under 20 $ for each monitoring-set. it allows for even multi-platform (ios, android, windows) smart-phone, short-range connectivity, for up to 4 participants, for example nurse, physician etc. two potential approaches for the quality control of bact/alertv r culture media using various bioball tm organism preparations patricia rule*, michelle keener and christine crawford. biomerieux inc. background/case studies: the bact/alertv r bpa and bpn culture bottles are used with the bact/alert microbial detection system for rapid screening and detection of microbial contamination in leukocyte reduced apheresis platelets (lrap). recent changes in the clia quality control guidelines and aabb accreditation program will require additional quality control of manufactures media that is both lot specific and shipment specific to ensure recovery of bacterial growth. a study was conducted using commercially prepared organisms evaluating both a comprehensive organism panel as well as a streamlined method utilizing only two organisms from the panel. study design/method: the general protocol consisted of three replicates each of each organism inoculated into two lots each of bpa and bpn by two different analyst. the study was two part in that aspergillus brasiliensis, candida albicans, bacillus subtilis subsp. spizizenii, pseudomonas aeruginosa, escherichia coli, clostridium sporogenes, staphylococcus aureus and streptococcus pyogenes were prepared from bioball singleshot (30 cfu), multishot 550 cfu or highdose 10k organism preparations at a low level (< 50 cfu) and evaluated on the same day of preparation as method validation. the second part of the study utilized escherichia coli and staphylococcus aureus prepared and frozen at a higher level and then evaluted over a 14 day study as a stream line approach to routine quality control testing of the bact/alert culture bottles. inoculation preparations were enumerated in duplicate to confirm the level at each inoculation time point. inoculated bpa and bpn bottles were loaded into the bact/alert microbial detection system at 36 c for automatic monitoring of growth. negative bpa and bpn bottles were included in duplicate at each day of testing. results/finding: escherichia coli, staphylococcus aureus, streptoococcus pyogenes and bacillus subtilis subsp. spizizenii were positive in both the bpa and bpn culture bottles. the aerobic aspergillus brasiliensis, candida albicans, and pseudomonas aeruginosa grew and were reported positive in only the bpa aerobic culture bottle as expected. while the obligate anaerobe, clostridium sporogenes was positive only in the anaerobic bpn culture bottles. bacterial cultures were positive in the bact/alert bpa and bpn bottles < 1 days and the fungal organisms in < 2 days. the overall agreement was 99.8 % in 698 bottles tested here. no significant differences were observed in the time to detection between the different lots or between the different analyst. conclusion: the bioball prepared organisms demonstrated a reproducible method as both a comprehensive and streamline approach for the quality control of bact/alert bpa and bpn culture media. the method was simple and did not require additional microbial preparations or storage of live organisms by the laboratory. use of an electronic patient identification system for blood banking specimen labeling found to be superior over historical armband approaches annie newton* 1 , diane schafer 2 , debra brown 1 , jesse cox 3 , scott koepsell 3 and sara shunkwiler 3 . 1 nebraska medicine, 2 the nebraska medical center, 3 university of nebraska medical center background/case studies: anticipating the implementation of the new (30 th addition) aabb standard concerning the confirmation of patient abo blood typing of type and screen (crossmatch) specimens performed prior to the issue of crossmatched blood products, laboratory and organizational leadership evaluated the practical application of an electronic patient identification system to label blood bank specimen collections versus the traditional use of blood bank armbands. continued use of the armbands would require a second sample for abo confirmation of patients that did not have a historical blood type on file. concern was raised regarding the amount of increased workload of staff and delayed results availability based on the number of increased specimens that would be generated, as well the potential for increased iatrogenic blood loss and patient dissatisfaction. moreover, 2 nd sample collection alone would not improve the rate of mislabeled specimens observed, which is of supplementary concern. study design/method: current organization employment of an electronic patient identification system for the labeling of other laboratory specimen collections made it feasible for applying this technology to the blood bank as well. an in-depth evaluation, including a failure modes and effects analysis (fmea) spanning several days, was completed to ensure that the use of the electronic system would produce comparable or superior safety results to its armband counterpart. an alternate process for specimen labeling and abo confirmation (which would satisfy the new standard) was established to support care areas that did not have the capability of using the electronic system. extensive education was provided to all staff (physicians, advanced practice providers, phlebotomist and nurses) to ensure comprehension as well rational for the new process. alerts were congruently built into the electronic health record (ehr) to supplement any information regarding crossmatch testing expiration that may not be readily available by the elimination of the armband use. results/finding: within days of implementing the new process (september 11, 2016), there was a noticeable reduction in the amount of mislabeled blood bank specimens received, totaling 4 in 6 months post implementation compared to 144 in the 6 months prior. in addition, the vast majority of specimens received into the blood bank are henceforth collected and labeled using the electronic system and thus have reduced the amount of potential 2 nd specimen collections needed for abo confirmation. conclusion: use of an electronic patient identification system for labeling blood bank specimen collections in lieu of traditional blood bank armbands has proven to improve patient safety and department efficiency by substantially reducing the occurrence of mislabeled specimens and negate the need for 2 nd specimen collections, reducing potential iatrogenic blood loss and improving patient satisfaction. background/case studies: based on a few small randomised controlled trials (rcts) performed in the late '90s and in early 2000, intravenous immunoglobulin (ivig) use has been suggested as a potential treatment to avoid exchange transfusion (et) for rh hemolytic disease of the newborn (hdn). this treatment modality is now routinely used for rh-hdn and has been extended to hdn caused by abo incompatibility or by other red blood cell antibodies. however, larger rcts performed since 2010 have shown that prophylactic ivig did not reduce the need for et, the duration of hyperbilirubinemia, the maximum bilirubin levels nor the need for top-up red blood cell transfusions. the primary objective of this study was to describe the usage of ivig for hdn at a tertiary academic referral hospital. study design/methods: a retrospective chart review was performed of all neonates who received ivig for hdn in the neonatal intensive care unit (nicu) from january 1, 2005 to june 30, 2016. data collected included patient demographics features and diagnosis, indications for ivig, neonatal laboratory results, treatment details, adverse events and patient outcomes. results/findings: ninety-seven neonates received ivig during the study period: 57% were female and 7% were less than 34 weeks of gestational age. none had co-existing g6pd deficiency, pyruvate kinase deficiency or spherocytosis. all neonates received phototherapy prior to ivig treatment. indications for ivig were abo-hdn (41%) and rhesus-hdn (59%). antibodies most often implicated in rh-hdn were anti-d (22/57), anti-d and anti-c (22/57) and anti-c (5/57). sixteen infants with rh-hdn had received intrauterine transfusions. the mean cumulative dose of ivig was 1 g/kg (range from 0,3 g/kg to 3,8 g/kg). neonates received one to four ivig administrations. table 1 shows the number of patients receiving ivig during two time periods. three adverse reactions were noted during ivig administration: cutaneous rash, hypotension and fever. of all neonates, 14 required an et for rh-hdn and 3 for abo-hdn. forty-five (46%) patients needed top-up transfusions during hospitalisation and until three months of age: 8 with abo-hdn and 37 with rh-hdn. the mean number of transfusions was three (range:1 to 7). conclusion: although initially described for rh-hdn, abo-hdn is now one of the most frequent indications for ivig in neonates. the optimal use of ivig in abo-hdn needs to be better characterized. our study shows a wide variation of ivig dosing and a significant proportion of neonates requiring top-up transfusions. further research is required to evaluate whether anemia in abo-hdn might be exacerbated by hemolysis from ivig isohemagglutinins and if it is dose-dependent. background/case studies: background: one of the most serious adverse reactions to transfusion is the development of graft versus host disease. symptoms include the development of a characteristic cutaneous rash, enteritis often resulting in watery diarrhea, elevated liver function tests and ultimately pancytopenia. the clinical course is rapid with an over 90% case fatality rate. the patient population at risk is reasonably well-defined including patients who are immunocompromised due to disease process or therapy, the fetus and low birth-weight neonates, recipients of hla-matched cellular blood products and the recipients of cellular blood products donated by blood relatives. the basic etiology of ta-gvhd is the inability of the transfusion recipient to mount an effective immune response against donor t-lymphocytes. treatment options for ta-gvhd are ineffective, making it imperative that cellular blood components be irradiated prior to transfusion which virtually eliminates the risk of the complication. study design/methods: most transfusion service information systems have mechanisms to alert transfusion service staff to patients who have been previously identified as needing irradiated blood components. however, if these patients are not identified to the transfusion service at the time of the initial hospital visit or the time at which the qualifying diagnosis, these patients can erroneously receive non-irradiated blood components. following a "near-miss" situation, our hospital information department developed a 4-part program to minimize the risk that the transfusion service is not notified of patients newly requiring irradiated blood components. results/findings: our blood products ordering system has been redesigned to include 3 specific queries to identify those patients who required irradiated cellular blood products. first, physicians have been notified to include the need for blood product irradiation in the patient problems list. once this is included in the list, the transfusion service will be notified of the need for irradiation on all subsequent transfusion orders until the problem list is modified by the clinical staff. second, if irradiated blood components have ever been requested on a patient, an alert will be generated for the ordering physician even if the requirement for irradiated products has not been included in the problem list. finally our system will automatically default to request irradiation on all cellular products ordered for children less than 4 months of age to comply with local irradiation policies. conclusion: we believe that our approach can be further enhanced by including a list of specific diagnoses typically requiring blood product irradiation within our computer algorithm. we believe that this list will provide an additional level of safety in insuring that patients receive irradiated blood components when appropriate. using lab information system and a dynamic dashboard for labeling and tracking coolers russell thorsen, rosaline ma, peter suslow, gina giannarelli, sara bakhtary, ashok nambiar and morvarid moayeri*. ucsf health background/case studies: our tertiary-care transfusion service routinely issues blood products in validated coolers to high acuity areas such as ors, icus, cath-lab, etc. coolers are also used for emergency release and massive transfusion protocols, and for shipping products between our different hospital sites. a robot that can hold one cooler delivers products to locations not served by the pneumatic tube. on average, 30 coolers are issued every day. cooler set-up is a multi-step, labor-intensive process. transfusion service staff track cooler location and elapsed time-in-use and notify clinical teams to return/recharge coolers to avoid product wastage. we developed a lab information system (lis)-based solution to manage cooler labelling and tracking more efficiently. study design/method: nine cooler test batteries were built; the batteries for rbc, plasma, platelet and cryoprecipitate (2 each) are identical, whereas the final battery designated for the cooler delivered by robot (containing plasma and rbcs with variable expiration times) is slightly different. the second battery in each pair was built to avoid duplicate test cancellation by lis when a second cooler (for same component type) is being set up for the same patient. each battery consists of tests capturing the following information: cooler location, cooler id, number of units issued, and expiration. custom barcodes representing each test battery and 45 different locations can be scanned from a 'quick-pick list', avoiding need for manual entry. when coolers are returned, a final entry is made in the test battery, updating lis. a dynamic cooler tracking dashboard with live-feed from lis displays data captured in the test battery. elapsed time, starting from cooler set-up (which is identical to time cooler battery is ordered in lis) is captured automatically. color codes alert users to coolers that have less than 1 hour before expiration. a flashing alert pops up for coolers that have expired. results/finding: we replaced our manual process (hand-write patient information and expiration time on 2 separate tags; affix one tag to cooler and retain second one to track cooler location and expiration) with a novel lis-driven labeling and tracking system. each time a cooler is set up, a test battery is ordered and resulted in lis by scanning the related custom barcodes. a single lis-generated label is printed and attached to each cooler. cooler expiration is defaulted to 10 hours (per our current cooler validation) from the time the test battery is ordered during cooler set up. techs pay attention to expiration of each product they place in a cooler. if an individual product outdates before the cooler expiration time, this information is entered in the test battery and gets displayed on the dashboard as a cooler expiration time, distinct from the system-driven countdown. color-coded visual display and alerts greatly simplify cooler monitoring, and the elimination of some manual steps has improved staff satisfaction. conclusion: using lis for cooler set-up and deploying a linked dynamic dashboard to display cooler locations and expiration time makes cooler management more efficient. these tools reduce manual steps and decrease likelihood of wastage by aiding cooler tracking. improving cryoprecipitate collection operations using operations research and analytics-based methods american red cross, 2 georgia institute of technology ap72 reduction in unnecessary use of type o-negative rbcs in a level i bellevue hospital-nyulmc our hospital is a level i trauma center serving a diverse predominately non-caucasian population. historically we stocked our trauma blood bank monitored refrigerator with 2 o-negative rbcs. trauma requested that we stock additional rbcs to be able to initiate a mtp for multiple patients at the same time. believing that most of our trauma patients are male, elderly, or rh-positive, we agreed add 2 type o-positive rbcs to the stock. rules for determining which units to use were established. o-positive rbcs are to be given to 1a) all adult males (am), 1b) women of non-childbearing age (wncba), and 1c) if both o-negative rbcs were used but not yet restocked, and o-negative rbcs are to be given to 2a) women of childbearing age (wcba) and 2b) children until the patient's aborh type are determined. we sought to assess the impact of this change on our usage and purchases of o-negative rbcs. study design/method: all patients issued emergency release trauma rbcs following the addition of o-positive rbcs were assessed 3%) would have needed to be o-negative. the addition of o-positive rbcs to our trauma refrigerator will enable us to markedly reduce our purchases of o-negative rbcs. ap73 saving apheresis platelets through use of verax point of care testing jennifer rhamy* 1 and rebecca wride 2 . 1 st. mary's regional blood donor center, 2 st. mary's regional medical center background/case studies: our rural hospital-based blood center serves 17 hospitals and a diverse patient population including acute trauma. because of the varying need for platelet products (varies between 0 and 8 per day in 2017), we investigated the use of the verax point-of-care test to better manage our valuable inventory barrett lawson 2 and jun teruya 1,2 . 1 texas children's hospital, 2 baylor college of medicine ap127 vision titers --easier or problematic? (table 1) . results/findings: post intercept, t had volumes of 261-320 ml, with 98 6 4% hemoglobin (hb) recovery. t had 10-fold less extracellular protein than c. after 35 days of storage t had higher atp and na 1 than c while lactate and hemolysis were lower. hct, ph, k 1 and glucose were equivalent between t and c on d35. d35 hemolysis for t was 0.08-0.31%, while for c it was 0.10-0.57%. t and c atp was >2mmol/g hb, the level of atp associated with effective rbc viability, throughout storage (table 1) . hematocrit (hct, %) 58.5 6 2.5* 56.3 6 1.7 60.8 6 2.7 61.8 6 2.9 hemoglobin (g/unit) 59 6 7 6 0 6 4 not measured hemolysis (%) 0.02 6 0.01* 0.06 6 0.04 0.16 6 0.07* 0.29 6 0.14 ph (378c) 6.9 6 0.1* 7.3 6 0.1 6.7 6 0.1 6.6 6 0.1 total atp (mmol/g hb) 7.7 6 0.5* 5.0 6 0.4 4.6 6 0.6* 3.9 6 0.5 k1 (mm) 1.5 6 0.3* 5.7 6 2.5 53. total tested total plts issued feb 87 121 64 48 24 185 181 mar 226 516 342 276 133 858 757 totals 352 730 471 375 174 1201 table: 2. resident reports to the intranet "drop box" increased from 53.7% to 69.3% to 100%, each over 2 month time spans. conclusion: safe transfusion ordering requires a team approach to ensure the right information is available to the ordering provider at the right time. safe ordering prevented recurrent allergic reactions in our patient population. the tso plays a pivotal role in ensuring the full circle of communication occurs. processes that integrated the pathology resident improved with pdsa cycles and impacted the quality and timeliness of hand off. finally, the data provided from the residents enabled efficient participation in hemovigilance. decreasing results/finding: the main root cause determined was that there was no standard work process. sops were being followed but there was no standard work process that included the details so testing was not following the most efficient work flow. counter measures implemented included implementing a standard work process, visual cues were added to the work process, and a samples awaiting testing report was created for the batch release department. specific locations were identified within the work cells in the lab to place samples based on their phase/stage of testing. after counter measures were implemented, the number of exceptions decreased from 17.4 per day or 10,370 dpmo to 6.3 per day or 3,539 dpmo. this is a statistically significant difference since the p-value calculated was 0.002. conclusion: a lean six sigma approach for process improvement was utilized to identify root causes and develop countermeasures in order to decrease the number of exceptions related to the testing of whole blood samples in the laboratory. this approach and counter measures statistically significantly decreased the number of exceptions seen in the whole blood testing process. background/case studies: our blood bank processes approximately 4,400 specimens per month. since 1998, the requirement of having a second blood type on record was met by:1. utilizing the historical blood type and the current specimen, or 2. having second type performed on same specimen by different technologist, and 3. each type and screen specimen signed by 2 staff, one being a licensed practitioner attesting the identification of the patient was done accurately at bedside.to comply with the aabb standards 29 th edition, #5.16.2.2 a decision was made to change our practices. we considered challenges encountered at other hospitals and collaborated with nursing and it to create a streamlined and safe process. in april 2016, the second specimen procedure was implemented addressing the following: ii. extensive education was provided to all involved in the process prior to implementation including a learning module prepared by blood bank and nursing collaboratively. results/finding:1. there was a minor adjustment period with more phone calls made to blood bank to explain the process. 2. there was minimal impact on turn around times for release of components. 3. aborh retype workload decreased from 1500 to 950 (35% to 20% of t&s volume) per month. 4. unnecessary blood draws minimized, improving patient experience. 5. no emergency release requests due to absence of a second specimen. the second specimen process with the conditional order has been beneficial to our blood bank as well as patient care services. overall feedback from staff on the process has been positive. our workload has decreased which results in cost savings and increased efficiency allowing us to devote more resources to the growing services at our institution. background/case studies: the hazards of transfusion are well recognized and in certain cases restrictive transfusion strategies compared to liberal transfusion strategies may be associated with better clinical outcome. with this in mind, aabb and others published guidelines for transfusion, but even with guidelines in place, rate of inappropriate blood transfusions is reported to be as high as 15 to 48%. computerized provider order entry (cpoe), is a process of electronic medical order entry for medical practitioners with instructions and guidelines for treatment. the objective of this study was determination of transfusion practice quality by thorough chart and electronic medical record review, with measures in place to avoid inappropriate transfusion. additionally, factors associated with inappropriate transfusion were examined. study design/method: in our 926 bed hospital, a retrospective chart review was performed (07/01/15-12/31/15) on hospitalized internal medicine patients. cpoe with hospital guidelines for rbc transfusions were in place. transfusion thresholds in different clinical settings were determined by a thorough literature review of studies analyzing restrictive transfusion strategy, and transfusion guidelines by various medical societies. charts for background/case studies: our midwestern university-based transfusion service (ts) evaluated the appropriateness the automated platform vision (ortho clinical diagnostics. raritan, nj) for prenatal titration studies. it has been established from previous publications that the micro-column assay, of which the vision is based, may lead to higher titer results compared to standard tube titrations. this study sought to evaluate the transition from manual to automated titer studies from a sensitivity as well as cost perspective. study design/method: twenty-three prenatal retention plasma samples were tested as part of the evaluation of titration studies of the vision. the samples were manually tested with a standard two-fold serial dilution. the titer was reported as the last tube to demonstrate a 11 reaction by macroscopic observation. the titer studies were then repeated using the vision. the results of the manual and automated processes were compared and categorized as "< 2 grade" or "> 2 grade" difference between endpoints. this analysis is similar to the acceptable ranges used for evaluating college of american pathologists (cap) proficiency survey challenges. a cost analysis was completed based on the direct and indirect cost for each method, excluding the cost of an analyzer. results/finding: table 1 demonstrates a summary of the samples tested by manual titer study and vision titration method. the vision titer results (mean, median, and mode) were higher than the manual tube titer results. less than half of the samples (48%) were > 2 titer results higher, while the majority was 2 titer results different (52%). the cost analysis is summarized in table 2 . the indirect cost (labor) was significantly lower with the use of the vision. the reduction in pre-analytical technical time for manual preparation of the titration is eliminated with the vision completing the titration as part of the profile of the titer study of the analyzer. conclusion: with an estimated 41% decrease in the cost of a vision titer compared to manual tube method, the change in practice would clearly be a cost and efficiency measure in the blood bank. however, the vision demonstrated the expected increase in titer results compared to manual tube titer results. this would impact the critical values currently utilized. an impact assessment for clinical staff would be necessary to adequately implement the change in method. consideration must be given to changes in the computer logic for critical values on titer studies and training of physician and nurse obstetric practitioners for changes in the critical values. in addition, as part of changing to the vision an implementation period will be necessary to ensure that manual titers are compared to previous manual titers and not to vision titer results which would be higher and may be interpreted as a significant change for clinical care of the patient. what is the best practice for testing residual white blood cells in blood components for monthly routine quality control? janja pajk*. general hospital celje background/case studies: we wanted to discover what is the best routine quality control practice for testing residual white blood cells in blood components. our aim was to validate the adam device for counting thne number of residual white blood cells (wbc) in leucocyte depleted and in non-leucocyte depleted blood components (bc) and to compare with standard counting method by microscopy in fuchs rosenthal chamber (frc) used in ghc and with flow cytometry (fc). study design/method: after 23 samples of red blood cells (rbc), platelets (plt) and fresh frozen plasma (ffp) (leucocyte depleted in top and top (t/ t) bags and non-leucocyte depleted in top and bottom (t/b) bags) were stained with propidium iodide (pi) on r-slides; adam -rwbc device was messured fluorescent images of stained wbc nucleus. data were analised by image analysis software and later compared with results of testing samples in frc by microscopy and with fc.15 samples of bc were microscopic tested in frc at department of laboratory medicine in ghc; another 8 samples were measured with fc in ucc maribor. results/finding: 15 samples (6 rbc, 3 plt, 3 ffp-all leucocyte depleted and 3 non-leucocyte depleted ffp) were tested in triplicates on adam and with frc once.coefficient of variation of (kv%) of samples measured on adam for leucocyte depleted bc varied for: rbc from 44,10 -173,21; plt from 86,60 -100,00; ffp from 86,60 -173,21; and for non-leucocyte depleted ffp from 0,69 -8,85 (table 1) . 8 samples (2 rbc, 2 plt, 2 ffp -all leucocyte depleted and 2 nonleucocyte depleted ffp) were tested in triplicates on adam and with fc once.kv% of samples measured on adam for leucocyte depleted bc varied for: rbc from 91, 56; plt from 78, 21, ffp from 66, 21 ; and for non-leucocyte depleted ffp from 11,17 -15,91 (table 2) .high percentage of kv was noticed in samples with low numbers of wbc (in leucocyte depleted bc; low percentage of kv in non-leucocyte depleted ffp, with higher amount of wbc was observed. conclusion: all samples tested with adam met expected criteria for wbc in bc in european union (less than 1x10 9 /unit for leucocyte depleted or 1x10 6 / unit for non-leucocyte depleted) and were comparable with those tested with fc; the correlation with microscopy in frc was worse.with use of disposable r-slides, the risk of exposure to the potential hazardous blood samples is grately reduced, the method is more precise and not time consuming.from january 2017 we changed our protocol for testing residual wbc in bc with adam device and we advise it as the best practice for monthly routine quality control. key: cord-005350-19za0msu authors: o’regan, suzanne m.; drake, john m. title: theory of early warning signals of disease emergenceand leading indicators of elimination date: 2013-05-31 journal: theor ecol doi: 10.1007/s12080-013-0185-5 sha: doc_id: 5350 cord_uid: 19za0msu anticipating infectious disease emergence and documenting progress in disease elimination are important applications for the theory of critical transitions. a key problem is the development of theory relating the dynamical processes of transmission to observable phenomena. in this paper, we consider compartmental susceptible–infectious–susceptible (sis) and susceptible–infectious–recovered (sir) models that are slowly forced through a critical transition. we derive expressions for the behavior of several candidate indicators, including the autocorrelation coefficient, variance, coefficient of variation, and power spectra of sis and sir epidemics during the approach to emergence or elimination. we validated these expressions using individual-based simulations. we further showed that moving-window estimates of these quantities may be used for anticipating critical transitions in infectious disease systems. although leading indicators of elimination were highly predictive, we found the approach to emergence to be much more difficult to detect. it is hoped that these results, which show the anticipation of critical transitions in infectious disease systems to be theoretically possible, may be used to guide the construction of online algorithms for processing surveillance data. infectious diseases are among the most visible and costly threats to individual and public health. antibiotics, vaccines, and the molecular revolution in biology have not erased their mark. millions of persons die every year from treatable ancient diseases, such as malaria (gething et al. 2011; who 2012) , tuberculosis (dye et al. 2008) , and measles (orenstein and hinman 2012; simons et al. 2012) . sometimes, elimination of these diseases through vaccination, prophylaxis, and/or vector control is possible, but sustaining elimination campaigns is difficult as pathogens approach the point of elimination (cohen et al. 2012) . conversely, emerging pathogens such as sars and swineorigin influenza a (h1n1) cause excess mortality, disrupt business, terrorize vulnerable populations, and lead to new, sometimes ineradicable endemic diseases (e.g., hiv/aids). the benefits of accurately forecasting disease emergence would be tremendous: in the case of a low-incidence sarslike pathogen, the savings could be tens of billions of $us (rossi and walker 2005; smith et al. 2009 ); an illness resembling the 1918 influenza virus might take millions of lives and impose costs of the same order as a year's gross domestic product (osterholm 2005) . elimination and emergence of infectious disease both involve a transmission system that is pushed over a critical point. in most cases, criticality occurs at the point where the basic reproduction number, r 0 , the number of secondary infected cases arising from a single infected case in an entirely susceptible population, is equal to one (heffernan et al. 2005) . similar critical points occur in other complex systems (strogatz 1994; sole 2011) . particularly, in noisy (stochastic) systems, these critical points manifest as transitions between alternative modes of fluctuation (scheffer 2009; scheffer et al. 2009; lenton 2011; scheffer et al. 2012) . we call such a stochastic transition a critical transition if there exists a bifurcation in a suitably constructed limit case of the mean field model. a central problem in the study of critical transitions is the identification of phenomena indicating the proximity to a critical transition in the absence of a detailed understanding on the system's dynamical equations and/or the forcing variables causing the change scheffer et al. 2012; boettiger and hastings 2012) . recent studies have established that some noise-induced phenomena may signal the approach to a critical transition in a slowly forced dynamical system (ives and dakos 2012; dakos et al. 2012a, b; dakos et al. 2008; carpenter and brock 2006; donangelo et al. 2012; seekell et al. 2011; carpenter and brock 2011; brock and carpenter 2010; dakos et al. 2010; carpenter et al. 2008; jayaprakash 2008, 2009) . if this property would apply to infectious diseases, it would suggest a model-independent route to forecasting infectious disease emergence in subcritical systems with r 0 < 1 (i.e., crossing the critical point "from below") and documenting the approach to elimination in endemic (supercritical) disease systems where r 0 > 1 (crossing the critical point "from above"). many of these characteristic noise-induced phenomena involve critical slowing down, a decline in the resilience of the system to perturbations, which generally gives rise to an increase in the variance and autocorrelation of fluctuations as the system approaches the transition (van nes and scheffer 2007; dakos et al. 2012a) . but, these properties require that the transition be suitably regular (hastings and wysham 2010) . specifically, to observe critical slowing down requires that the potential function of the system, if it exists, be smooth. also, for systems with multiple attracting sets (e.g., bistable systems), a sufficient level of noise gives rise to "flickering" which exhibits the characteristic increase in variance (but for a different reason) and not the increase in autocorrelation dakos et al. 2012b) . other obstacles to anticipating critical transitions in infectious diseases include that epidemiological systems are complicated by amplification of transients and oscillatory dynamics bauch and earn 2003) , that infectious diseases are often seasonally forced (altizer et al. 2006; fraser and grassly 2006) and propagate in demographically open systems subject to imported cases , and that diseases that are close to elimination or close to emerging are, by definition, characterized by low prevalence and, therefore, both subject to demographic stochasticity and difficult to observe (lloyd-smith et al. 2009 ). therefore, to determine whether diagnostic noise-induced phenomena accompany the critical transitions that occur in infectious disease dynamics, namely the transition to endemicity of emerging infectious diseases and the transition to extinction in disease elimination, it will be useful to have a quantitative theory, both to guide the selection of statistical quantities to be investigated and to predict the form that observable quantities should take as the transition is approached. this paper is a contribution to this theory. we consider the compartmental susceptible-infectious-susceptible (sis) and susceptible-infectious-recovered (sir) models, which are widely used to represent the dynamics of a range of nonimmunizing and immunizing pathogens and may be viewed as approximations to an even broader class of infectious diseases. in their deterministic formulations, these models are characterized by a transcritical bifurcation at r 0 = 1, where the endemic and disease-free steady states meet and exchange stability. the epidemic models we investigate are generalizations of the closed population sis and sir models that allow for immigration from external sources, of which the more familiar models without immigration that are characterized by a transcritical bifurcation are special cases. to appropriately model the slowly forced dynamical system, we first develop mean field theory for the nonstationary systems gradually approaching a bifurcation. we develop the mean field theory here because slowly forced dynamical systems approaching a bifurcation have rarely been investigated (but see kuehn (2011) ). we then develop the full stochastic description in terms of a master equation. our strategy is to use the van kampen system size expansion to separate the mean field dynamics from the fluctuations induced by demographic stochasticity. these fluctuations are described by a fokker-planck equation, from which the power spectrum, variance, autocorrelation, and coefficient of variation may be analytically obtained. our main result is contained in table 4 , which provides formulas expressing these theoretical predictions in terms of the dominant eigenvalue of the mean field solutions, which goes to zero as the system approaches the critical transition. hence, these formulas express how epidemiologically measurable quantities are expected to change as the system approaches the critical transition. a number of approximations are introduced in the derivation of these formulas, including separation of time scales, second-order description of the fluctuations, and continuum representation of the state space. we, therefore, performed simulations to study the possible detrimental effects of these approximations. specifically, we examined the agreement between the theoretical predictions and "measured" statistics obtained in a moving window, as one would analyze real-world data, in both discrete state-space and continuous state-space simulations. we show that the analytical expressions are robust, provided the conditions of fast-slow systems are met. these results demonstrate that the critical transitions associated with disease emergence and elimination may be anticipated even in the absence of a detailed understanding of their underlying causes. we first consider a general sis model that allows for immigration. denoting the proportions of susceptible and infectious populations by x(t) and y(t), respectively, the sis model in a population of size n is given bẏ where β is the the transmission rate, γ is the rate of transfer from the infectious class to the susceptible class, and a dot denotes the time derivative. the model assumes that infection may also occur through contact with a trickle of infectious imports at a rate η, either by susceptibles briefly leaving the population and making contact with infectious individuals located elsewhere or through infectious visitors briefly entering the population and making contact with susceptibles, so that the total force of infection is = βy + η. since the population size n is constant, i.e., x + y = 1, the sis model may be described by a single equation, we note that eq. (2) can encompass a variety of infectious disease systems, including closed population si models. thus, the system can be used to model diseases that confer no long-lasting immunity, e.g., sexually transmitted diseases or acute infections such as influenza or diseases that are fatal. if η = 0, eq. (2) has two equilibria: the disease-free equilibrium y 0 = 0 and the endemic equilibrium y * = (1 − 1/r 0 ), where r 0 = β/γ denotes the basic reproduction number. when r 0 > 1, the infection can persist, but if r 0 < 1, then the infection dies out. the equilibria y 0 and y * meet and exchange stability when the basic reproduction number r 0 = 1, i.e., a transcritical bifurcation occurs. we will refer to the sis model with no immigration as the limiting case. however, it is usually more realistic to assume that a low level of immigration occurs, i.e., η is positive. if η > 0, then eq. (2) has a single positive equilibrium that is always locally stable, provided β, γ > 0, since the slope at the equilibrium λ = β − 2βy * − γ − η is negative. moreover, whether η = 0 or η > 0, the return time −1/λ decreases in the limit β → γ . at the critical point in the limiting case sis model, λ = 0. therefore, the transcritical bifurcation occurs in a suitably constructed limiting case (η = 0), which we suggest gives an indication for the behavior of the fluctuations for the stochastic version of the model with immigration, provided η is small. slow changes in the transmission rate β, through demographic or evolutionary means, may induce a transcritical bifurcation of eq. (2). for example, transmission may decrease as a result of slow increases in vaccination uptake among the population, leading to extinction of the pathogen, or it may increase due to slow decreases in vaccination uptake, potentially causing a transition to endemicity. to formally incorporate slow changes in transmission into the model, we rewrite eq. (2) as a fast-slow system: where 0 < 1 and the function f describes the change in transmission rate. in this paper, we assume that transmission is a slowly changing linear function of time, i.e., β = β(t) = β(1 − p(t)). the proportion of the population that are vaccinated is modeled by the function p(t) = p s + p 0 t, with p 0 an incremental change in β. hence,β = p 0 . if p 0 > 0, then the rate of transmission is slowly declining over time, and if p 0 < 0, it is slowly increasing. the limit case → 0 of eq. (3) is eq. (2), and thus, the endemic equilibrium of the limit case assumes β = β(1−p s ), which arises from the level of vaccination uptake p(t) (the bifurcation parameter). figure 1 shows the transcritical bifurcation diagram for the sis model. for small η, the plot of the stable endemic equilibrium y * as a function of vaccination uptake is similar to the bifurcation diagram for η = 0, except in the vicinity of the critical point p * (inset figure of fig. 1 ). for p > p * , the disease-free equilibrium is stable in the limiting case, but for the model with immigration, the infectious population is sustained at a low level due to importation of the disease from external sources (fig. 1) . the inset plot shows a close-up of the diagram near the transcritical bifurcation point in the limiting case where η = 0. the dashed line indicates the location of the critical point, p * = 1 − 1/r 0 = 0.9. the bifurcation diagram was plotted using the parameters given in table 7 stochastic description of sis model to investigate the effect of noise on this transition, we assume that fluctuations in the infectious population are caused by demographic stochasticity (intrinsic noise). to understand these fluctuations, we require an individual-level description rather than a population-level description. we assume that all individuals have identical attributes, and individuals may move from the susceptible state s(t) to the infectious state i (t), or from the infectious state to the susceptible state. we assume that the population size is constant, i.e., s(t) + i (t) = n. since the population size is constant, we need only model the transitions into and out of the infectious state. furthermore, since the sis system moving towards a bifurcation is a fast-slow system, the transmission rate β is not constant in time but is a slowly changing function of time. because the probability of transmission between an infectious and susceptible individual is slowly changing over time, we treat the transmission rate as constant for each small time increment dt, β. table 1 shows the variables and parameters of the sis model. we assume that in a sufficiently small time increment dt, the number of infectious individuals i can either (a) increase by 1, (b) decrease by 1, or (c) not change in number. infection and removal are the events in an sis process that lead to these changes in state. we denote the number of individuals i at time t or the state of the system by α = i at time t and the alternative states byα, where eitherα = i + 1 orα = i − 1 in the sis model. the system of individuals goes from a state with α = i individuals at time t to a state withα individuals at time t + dt with transition probability per unit time t i (α|α). the system can also go from a state withα individuals at time t to i individuals at time t + dt critical point p * = 1 − 1/r 0 with a probability fluxt i (α|α). table 2 outlines the events, changes in state, and the transition probability fluxes into and out of state i that can occur in a stochastic sis process with a slowly changing transmission rate. since the transitions in table 2 describe a markov process, we can write down a master equation describing how the probability of there being i individuals at time t evolves with time. the master equation represents the continuous time version of the dynamics of the markov process. derivations of master equations can be found in van kampen (1981) and renshaw (1991) . letting p (i, t) = prob (i (t) = i ) be the probability that the infectious state variable i (t) is equal to some nonnegative integer i , the master equation for the evolution of the probability of the infectious population being in state α = i at time t is whereα describes all other states (table 3) , and the probability fluxes into and out of state i are as described in table 2 . this is a system of n +1 ordinary differential equations, α = 0, 1, . . . n, which can be solved with an initial condition of i 0 individuals at time t = 0, i.e., p (α, 0) = 1 for α = i 0 and p (α, 0) = 0 for all α = i 0 . thus, the probability distribution at time t = 0 is a delta function. table 3 summarizes the quantities in eq. (4). we will apply the van kampen system size expansion to the master equation. the system size expansion takes advantage of the fact that, for large population size n, the demographic fluctuations in the population are small, and so n is the expansion parameter. thus, the key assumption for the expansion to be valid is that the system size n is sufficiently large (see appendix a for details). here, we pause to note that that the sis model is equivalent to a simple logistic process, and hence, there are analytical expressions note that β is treated as constant in a small time increment dt, but the transmission depends on a slowly changing linear function of time, β(t) = β(1 − p(t)) = β(1 − (p s + p 0 t)). table 1 summarizes the parameters in the rates table 3 master equation terms. the transition probability of infection per unit time is given as an example of a transition into and out of state α for the sis and sir stochastic processes. the other transition probability fluxes can be found in tables 2 and 6 system state α alternative statesα sis i i + 1, i − 1 sir (s, i ) (s + 1, i − 1), (s − 1, i ), (s + 1, i ), (s, i − 1), (s, i + 1) system probability flux into α probability flux out of α for the mean and variance (allen 2003) . the i = 0 state is absorbing, and the expected time to extinction is finite (allen 2003) . nevertheless, if the population size is large, the probability distribution is approximately stationary for a very long time. it is the statistics of this quasi-stationary distribution that we are interested in. following expansion of the master equation, the leading order and next-to-leading order terms are collected, giving rise to a deterministic system that describes the evolution of the trend and its stochastic correction, respectively, see appendix a for details. the deterministic system turns out to be the fast-slow system (3). the distribution of the fluctuations about the solution of eq. (3) is given by a linear fokker-planck equation. thus the fluctuations are gaussian distributed about the mean, and thus, i (t) ∼ normal(nϕ(t), nσ 2 ), see appendix a for details and definitions. the fokker-planck equation is equivalent to a stochastic differential equation (gardiner 2004 ). this is the key advantage of applying the system size expansion. using the stochastic differential equation, we can obtain analytical expressions for statistical signatures of leading indicators and early warning signals, including the power spectrum and autocorrelation function (see appendix a for details). table 4 summarizes the indicator statistics calculated using the methods described in appendix a for the stable limiting case → 0 for the sis model, in terms of the eigenvalue λ. we will examine how the statistics change over a range of vaccination uptake values in section "results". since the system size expansion involves approximating the discrete random variable i with a normal random variable, its assumptions may break down when i is small. for small numbers of infectious individuals, e.g., if the system is subcritical and, thus, incidence of a disease is low, the assumption that the fluctuations are normally distributed about the mean is likely to be inappropriate. the van kampen expansion is valid when the system is far from the absorbing boundary at i = 0. however, the approximation upon which the approach is built (eq. (10) in appendix a) cannot describe chance extinctions, which can occur if i is small. if η = 0 and p > 1 − 1/r 0 , the disease-free equilibrium is stable, and so the approximation given by eq. (10) is not valid. due to the absorbing boundary, the probability distribution about the disease-free state for a given time t will be one-sided. on the the other hand, if η > 0, then, close to the system boundary, the normal distribution approximation about the deterministic mean may be poor because the distribution is bimodal due to extinction events. however, the models with immigration do not have a disease-free state; just a small infectious population when p is approximately greater than 1/r 0 . therefore, predictions for the quasi-stationary statistics about this state can be obtained. denoting the proportions of susceptible, infectious, and recovered populations by x(t), y(t), and z(t) respectively, the sir model with immigration in a closed population of size n iṡ where β is the transmission rate, γ is the recovery rate, η is the immigration rate, and μ is the per capita birth rate. to maintain a constant population size, the per capita death rate is set equal to the birth rate. a proportion p of the population are chosen at random for vaccination at birth and are recruited into the recovered class. the remaining unvaccinated proportion of the population enter the susceptible class at birth. since the population size n is constant, x + y + z = 1, system (5) is equivalent to the following system of ordinary differential equationṡ the sir model is particularly appropriate for acute immunizing infections such as measles and pertussis. in the absence of vaccination and immigration, the basic reproduction number is given by r 0 = β/(γ + μ). if table 4 analytical expressions for quasi-stationary statistics about the endemic infectious quasi-steady state expressed in terms of the eigenvalues. expressions for the endemic equilibrium ϕ * of the sis and sir models are found by solving equations (11) and (22) in appendices a and b power spectrum s i (ω) autocorrelation the eigenvalues of the jacobian of the sir model evaluated about ϕ * are complex conjugates when the equilibrium is a stable spiral, i.e., reλ 1 = reλ 2 = reλ and |imλ 1 | = |imλ 2 | = |imλ|. when ϕ * is a stable node, the jacobian matrix has two real negative eigenvalues, λ 1 and λ 2 . when there is no immigration, η = 0 and the power spectrum collapses to zero. variables for each model are described in tables 8 and 9 . the expressions for the power spectrum are multiplied by 2 because they are evaluated over the frequency domain [0, ∞). no closed-form expression for the lag-τ autocorrelation is known, and so it must be evaluated numerically η = 0, the sir model (6) has two equilibria: the disease free state (x 0 , y 0 ) = (1 − p, 0) and the endemic equilibrium the sir model undergoes a transcritical bifurcation at r 0 = 1. the endemic equilibrium is locally stable if r 0 > 1 and is not biologically feasible if r 0 < 1, when the disease-free equilibrium is locally stable. as the vaccination uptake p increases, the basic reproduction number reduces by a factor (1 − p), i.e., the effective reproduction number is r 0 (1 − p). the vaccination uptake p for the effective reproduction number is 1 at the critical vaccination threshold, p * = 1 − 1/r 0 (anderson and may 1991), and it is at this critical threshold that the transcritical bifurcation occurs. again, the sir model with no immigration is the relevant limiting case. temporary importation of pathogen often occurs in infectious disease systems (keeling and rohani 2008 ). assuming that a low level of immigration occurs, i.e., η > 0, only a single positive equilibrium is biologically feasible. this equilibrium is a stable spiral when the square of the trace of the jacobian matrix evaluated about the equilibrium is less than four times its determinant and is a stable node if it is greater than or equal to this quantity. complex eigenvalues of the jacobian matrix of eq. (6) characterize a stable spiral, and the eigenvalues are real and negative if the equilibrium is a stable node. the stable node equilibrium can be thought of as a "disease-free" equilibrium if η is small, in the sense that the infectious population is sustained at a low level due to importation of the disease from external sources. therefore, while the transcritical bifurcation occurs in a suitably constructed limiting case (η = 0), the limit case may give an indication for the behavior of the fluctuations for the stochastic version of the model with immigration, provided that η is small. gradual changes in the vaccination uptake rate p may induce a transition from endemicity or to extinction. recruitment of susceptibles may slowly vary over long time scales as a result of demographic or evolutionary changes. vaccination uptake rates are often not constant over time and may exhibit trends, e.g., percentage uptake of pertussis vaccine in the usa (rohani and drake 2011). mathematically, we may express the sir model approaching a transcritical bifurcation as a fast-slow system: where 0 < 1 and the function f describes the change in vaccination uptake p. again, we model vaccination uptake as a linear function of time p(t) = p s + p 0 t, with p 0 an incremental change in p. consequently,ṗ = p 0 . if p 0 > 0, recruitment into the susceptible class is slowly declining over time and if p 0 < 0, then recruitment is slowly increasing over time. in the limit → 0, the vaccination uptake is fixed at a constant rate p s , and the system is stable. figure 2 shows the transcritical bifurcation diagram for the sir model. the infectious equilibrium y * is plotted as a function of vaccination uptake p. the bifurcation diagram indicates that the endemic infectious equilibrium declines linearly with p in the models with and without immigration. however, the inset plot indicates that in the vicinity of the η = 0 critical point, the infectious equilibrium of the immigration model is elevated relative to the disease-free equilibrium, since the infectious population is sustained at a low level due to immigration. model (7) is the deterministic description of the sir system approaching a transition, but there will be stochastic fluctuations in the state of the system as the transition is approached. as before, we assume that these fluctuations result from demographic stochasticity. to quantify these fluctuations, we assume individuals are identical. individuals may be recruited into the susceptible state and can transition out of this state through infection or death. infectious individuals may recover or die. assuming that the population size s(t)+i (t)+r(t) = n is constant, we need only consider the transitions into and out of the susceptible and infectious states. furthermore, since the sir system moving towards a bifurcation is a fast-slow system, the vaccination uptake p is not constant in time but is a slowly changing function of time. therefore, the recruitment rate into the susceptible class is slowly changing, but, for each small time increment dt, we can treat the vaccination uptake as constant p. table 5 presents the variables and parameters of the sir model. events that occur in an sir process in a small time increment dt moving slowly towards a transition include infection, recovery, recruitment to the susceptible class, and death due to natural causes. table 6 outlines the events, changes in state, and the transition probabilities per unit time for each change in state. we can construct a master equation in the same manner as for the sis model. letting p (s, i, t) = prob((s(t), i (t)) = (s, i )) be the probability is equal to some nonnegative integer (s, i ), the master equation for the evolution of the probability of the population being in state α = (s, i ) whereα describes all other states (table 3) . the master eq. (8) is nonlinear and, therefore, cannot be solved analytically. to make analytical progress with eq. (8), again, we can use the van kampen system size expansion (see appendix b for details). the approach gives rise to the deterministic sir fast-slow system (7) and a linear fokker-planck equation that describes the evolution of the fluctuations, which may be written as a system of stochastic differential equations. these equations can be analyzed using fourier transformation. the solution of the fokker-planck equation is a bivariate normal distribution. table 4 summarizes the indicator statistics in the stable system limiting case → 0 for the sir model, in terms of its eigenvalues. the preceding sections present an analytical theory of early warning signals for emergence and leading indicators of elimination. to investigate the results of this theory for a particular parameter set (table 7) , we calculated leading indicators of elimination and emergence, assuming alternatively that (a) the mean proportion of infectious individuals is given by the deterministic endemic equilibrium ( → 0 theory) or (b) assuming it is given by the current state of the fast-slow system approaching a transition. we selected parameters consistent with sexually transmitted table 6 transition probability fluxes for sir model. transitions can occur into and out of state α = (s, i ) change in state transition probability per unit time dt note that p is treated as constant in a small time increment dt, but the vaccination uptake is a slowly changing linear function of time, p = p(t) = p s + p 0 t at time t. table 5 outlines the parameters used in each transition rate sis diseases with long infectious period and large r 0 and parameters typical for childhood infectious diseases with sir dynamics. to examine how different changes in vaccination uptake p 0 affect the statistics, we varied p 0 by 1/500 sufficiently large population sizes n were chosen to ensure validity of the van kampen system size expansion. the per capita immigration rate η on the approach to elimination was calculated assuming η = δr 0 /n, where δ is the number of imports per year (keeling and rohani 2008) . on the approach to emergence, r 0 is approximately one, and the number of infectious individuals is near zero. therefore, η = δ/n (sis model) and η = δ/(n(1 − p s )) (sir model). all emergence simulations begin with the forcing variable p s = 0.96, beyond the threshold p * for emergence, p * = 1 − 1/r 0 . the immigration rate remains fixed during simulations. the initial rate of transmission is calculated from r 0 and 1/30 year −1 in the model with immigration. we also compared the elimination indicators with those calculated assuming that the mean proportion of infectious individuals was given by the deterministic endemic equilibrium from the limiting case models with no immigration. the early warning signals of emergence were not calculated for the limiting case because the disease-free equilibrium is stable. to test the robustness of this theory to the range of approximations that were introduced (fast-slow approximation, continuum description, van kampen expansion), we simulated the approach to elimination and emergence in a variety of cases. to simulate the approach to elimination, we followed the "bottom-up" approach of allen (2003) to derive stochastic differential equations that incorporated demographic stochasticity. this approach uses the rates in tables 2 and 6 to build a system of stochastic differential equations. stochastic differential equations formulated in this way are appropriate, provided that the population size is sufficiently large, because then changes in the state variables are assumed to be normally distributed. simulations were compared with output from gillespie's direct method (gillespie 1977) . the simulations were qualitatively similar for population sizes greater than 50,000. to simulate the approach to emergence, we used gillespie's direct method as this is most appropriate for small population sizes. we simulated the sis and sir stochastic models with immigration approaching elimination and emergence 500 times. to compare the statistics to the theoretical predictions in table 4 , the infectious time series approaching elimination were sampled at yearly intervals. the transcritical bifurcation in these scenarios was approached over a long time frame (e.g., 470 years when p 0 = 1/500 in the sir model). however, the transcritical bifurcation to emergence was approached over a relatively short time frame for the sir model (10 years). to obtain a better sampled time series, the data from the gillespie simulations were aggregated over monthly intervals for the sir model. the infectious time series were aggregated over yearly intervals for the sis model approaching emergence because events did not always occur at a monthly frequency. thus, time series obtained from the sis system approaching emergence allowed us to examine the issues that arise from poorly sampled time series. to investigate the robustness of the early warning predictions over a moving window, i.e., as they would be used in online analysis of surveillance data, the influence of the slowly varying trend must be removed. the van kampen approach (appendices a and b) leads to a natural expression for the fluctuations from the quasi-stationary state nϕ(t), where ϕ(t) is determined by the mean field equations (11) (sis) and (22) (sir) in appendices a and b respectively. to obtain the fluctuations, we subtracted the current mean, which we assumed to be determined by the current state of the fast-slow system nϕ(t) , from the state of the system at the start of each year and divided this quantity by the square root of the population size. we refer to this as van kampen detrending. gaussian filtering is another, more common, method used to remove the influence of a slowly varying mean of a data series. to compare the performance of gaussian smoothing to van kampen detrending, for each time series, we fit a gaussian kernel smoothing function across the entire infectious case record up to the time that the transcritical bifurcation was predicted using a fixed bandwidth. lenton et al. (2012) have shown that the results obtained from applying the gaussian filter across the entire time series do not differ significantly from detrending within windows. to obtain the residuals, we subtracted the fit from each time series and divided by the square root of the population size to be consistent with van kampen detrending. the choice of bandwidth was informed by the resemblance of the gaussian residuals to the fluctuations obtained from the van kampen approach. to study the changes in the statistics up to the critical transition, we calculated the lag-1 autocorrelation and the variance of the fluctuations obtained using the two detrending methods over a moving window half the length of the time series. we calculated the lag-1 autocorrelation coefficient of each replicate using the acf function in r. the coefficient of variation (cv) was calculated by calculating, over a moving window, the mean and standard deviation of each infectious replicate. the median and 95 % prediction intervals for each of the statistics were calculated over the 500 replicates of each model. the prediction intervals were calculated using the quantile function in r. to quantify trends in each statistic for each replicate, we used kendall's correlation coefficient τ . to determine the distribution of kendall's τ , we calculated the coefficient for the trend in the test statistic for each realization. to assess the performance of the leading indicators, we followed the approach of boettiger and hastings (2012) and calculated receiver operating characteristic (roc) curves from the distribution of kendall's τ calculated from realizations of the models with and without transitions. the model without a transition is quasi-stationary, and we refer to it as the baseline or null model. the null models for elimination assume vaccination uptake p = 0 and are simulated beginning from the deterministic equilibrium at p = 0. the null models for emergence assume vaccination uptake p = 0.96 and are simulated beginning from the deterministic equilibrium at p = 0.96. the baseline models were simulated for the same length of time as it takes for the transition to be approached in the test models. the model with a transition is the test model. an roc curve enables investigation of the sensitivity of leading indicators to detect differences between quasi-stationary systems and those approaching a critical transition. we simulated the baseline models 500 times and obtained fluctuations using the van kampen approach and from gaussian filtering. we then quantified the trend in each indicator using kendall's τ for each baseline simulation. in our results, indicator statistics typically exhibited increasing trends or no trend, but for those that decreased, we multiplied kendall's τ for each realization by -1 to calculate the roc curve. the area under the roc curve (auc) was also calculated. an auc close to one indicates near-perfect detection. the mean field fast-slow dynamics drive the transitions to elimination and emergence in stochastic sis models with gradual changes in transmission. figure 3a shows a solution y(t) of the fast-slow system (3) with η = 0. the solution is the current "pool" of infectious individuals (the proportion infectious y(t)), not the number of new cases per time interval that comprise the traditional epidemic curve or recorded case reports of infectious individuals. at time t = 450, the limiting case model predicts elimination of the table 7 pathogen. the limit case plotted in the figure is that arising from the → 0 mean field theory, not the η = 0 critical case. here, we note that the limit case → 0 and the solution of the fast-slow system diverge from one another close to the proximity of the transcritical bifurcation of the η = 0 limit case. figure 3b shows a typical realization of the stochastic counterpart of the fast-slow sis system with η = 0, assuming a change in vaccination uptake of p 0 = 1/500 per year. the description of the stochastic process is outlined in table 2 . the infectious population slowly declines and closely follows the limiting case → 0 up until time t = 400 before eventually diverging. the inset figure indicates that the infectious population remains elevated 50 years after the transcritical bifurcation has occurred in the model with η = 0. therefore, the transition to elimination in this system is characterized by a slow change in the mean, followed by rapid fall off in incidence that precedes a very slow fadeout to extinction. on the other hand, if a disease is emerging due to small increases in transmission, or the level of vaccination uptake in the population is declining, solutions of the fast-slow system may exhibit a "delay" in emergence relative to the occurrence of the transcritical bifurcation. figure 4a shows the delay in emergence in a solution of eq. (3) with p 0 = 1/500. at time t = 30, the limiting case model predicts emergence. again, the limit case indicated in the figure is the limit → 0 of eq. (3), not the η = 0 critical case. figure 4b shows a typical realization of the stochastic counterpart of the solution shown in fig. 4a . the delay in the take-off of the epidemic is apparent. the rapid increase in the number of cases, is preceded by a low level of incidence long after the transition to endemicity has occurred. of course, the question is, does the low level of infection in the time series give any clue, through early warning signals, of the eventual rapid increase of infectious cohorts? the transitions to disease elimination and endemicity in the stochastic sir models with gradually changing vaccination uptake are also driven by the mean field fast-slow dynamics. to eradicate a disease, the effective reproduction number must be reduced by increasing the vaccination uptake p. figure 5a shows a solution of the fast-slow system (7) with η = 0. the mean field → 0 theory and the solution of eq. (7) agree closely. the timing of the transcritical bifurcation in the η = 0 model occurs at t = 470, and the number of infectious individuals has reached a low level by this time. figure 5b shows a stochastic realization of the sir model with immigration approaching elimination (the details of the stochastic process are described in table 6 ). the infectious population exhibits amplified oscillations, unlike the deterministic trajectory in fig. 5a . demographic stochasticity is expected to excite the transient oscillations of the sir model (bauch and earn 2003) , and we will see this in detail later when we examine the power spectrum of the fluctuations. on the other hand, if a disease is emerging through an increase in recruitment into the susceptible class, the effective reproduction number is increasing. figure 6a shows a solution of the fast-slow system (7) table 7 of emergence. after the transcritical bifurcation, the solution of the system does not agree with the stable endemic equilibrium at time t until approximately t = 55 years. the solution grows more slowly than the stable equilibrium. the delay in the dynamics is also exhibited by stochastic realizations, e.g., fig. 6b . outbreaks remain small and sporadic for at least 10 years after the bifurcation has occurred. the delay before emergence arises because the mean predicted by system (7) remains small following the bifurcation. however, the bifurcation delay is not as marked as that in the sis model (compare fig. 4 with fig. 6 ). power spectrum predictions figure 7 compares the power spectra of the fluctuations in the stable models with and without immigration. the patterns in the power spectra with immigration (fig. 7c, d) and without (fig. 7a, b) are similar. for sis systems with low transmission rates, and sir models with low recruitment rate into the susceptible class, the power in the peak of the spectrum is low. the power spectrum for sis and table 7 (emergence parameters) sir systems is expected to shift to lower frequencies as the pathogen approaches extinction. in the sis systems, the power at smallest frequencies increases as the critical point is approached, but this effect is subtle. in contrast, in the sir case, there is a dramatic reduction in the frequency at which the highest power is observed. the approach to endemicity is expected to be indicated by a shift to higher frequencies in the power spectrum. in the sis model, the spectrum encompasses a broader range of frequencies as emergence is approached. on the other hand, in subcritical sir limiting case → 0 prediction for the power spectrum for the models with and without immigration. the predictions for η > 0 agree closely with those generated from the η = 0 models. in the sis case, the power at smallest frequencies increases as the critical point is approached, but this effect is subtle, whereas in the sir case, there is a dramatic reduction in the frequency at which the highest power is observed . each power spectrum is evaluated about the mean field infectious equilibrium ϕ * , given in tables 8 and 9 . the parameters used to calculate the power spectrum are given in table 7 . importation, vaccination uptake, and population sizes were chosen according to the elimination scenario. the power spectrum has been log-transformed for clarity systems approaching endemicity, fluctuations should become less noisy as the threshold is approached. the power spectrum transforms from a flat "white-noise" spectrum in the subcritical scenario to a spectrum exhibiting resonant peaks for sufficiently decreased vaccination uptake. oscillations are expected to be amplified at these resonant frequencies. it has been shown that complex eigenvalues of the jacobian matrix of system (6) guarantee the existence of the power spectrum peak (alonso et al. 2007 ). systems approaching a critical transition are expected to exhibit rising variance and rising autocorrelation (scheffer 2009 ). in general, the predictions for the leading indicators of sis disease elimination follow these expectations for one-dimensional stochastic systems. figure 8a -c shows the leading indicators of elimination for the sis model. as the threshold to pathogen extinction is approached by gradually increasing the vaccination uptake, all of the statistics evaluated about the endemic equilibrium of the stable system increase as the bifurcation parameter is increased, as expected from the analytical formulas for the limiting case (table 4 ). the lag-1 autocorrelation rises, indicating an increase in system memory, and the fluctuation variance increases, as suggested by the plot of the power spectrum for increasing vaccination uptake values. the coefficient of variation also increases, indicating that infectious time series should become noisier as the transition is approached. the signal predictions for the sis systems with and without immigration in the limit case → 0 (in green) are in close agreement, which makes sense from the analytical formulae, provided η is small. to examine how different changes in vaccination uptake p 0 in the fast-slow model with immigration affect the statistics, we varied p 0 by 1/500 and 1/30 year −1 . predictions are in close agreement far from the η = 0 critical point but they differ close to it. the statistics evaluated about the endemic equilibrium the current state of the mean field fast-slow system are shown in blue (dotted) and black lines. the gray dashed line indicates the location of the critical point when η = 0. the theoretical predictions in the η > 0 sis case are not as dramatic as one expects from the limit case but are qualitatively consistent with standard expectations, unlike the sir limit case predictions, which do not conform with standard expectations. the variance in the sir case is predicted to decline, which is not unexpected here, but does not conform to standard expectations. the coefficient of variation increases as the critical point is approached, but the increase is not as dramatic as one may expect from the limiting case. in contrast to expectations, the lag-1 autocorrelation is predicted to decline close to the critical point if η > 0. the predictions for the statistics evaluated about the current state of the fast-slow system differ from those evaluated about the endemic equilibrium. even if p 0 = 1/500 year −1 , there are discrepancies in the predictions near the transcritical bifurcation point. the autocorrelation of the fluctuations with a lag of 1 year is shown, and the variance shown is the fluctuation variance. all calculations used the parameter values in table 7 sir leading indicator predictions leading indicators for sir systems approaching elimination ( fig. 8d-f ) are not always consistent with the standard expectations of increasing variance, increasing autocorrelation, and increasing coefficient of variation. in addition, the predictions for the leading indicators of sir disease elimination evaluated about the endemic equilibrium of the stable system behave differently to sis leading indicators, due to the presence of resonant frequencies. as the threshold to elimination is approached, the lag-1 autocorrelation increases with vaccination uptake, indicating an increase in system memory, but in contrast to the sis model, the fluctuation variance declines. we observe in fig. 7b , d that the area under the power spectrum declines as vaccination uptake increases, particularly in the model with η > 0, where the height of the peak of the power spectrum, which reflects amplification of fluctuations, declines close to the transition. the coefficient of variation also increases, indicating that infectious time series should become noisier as the transition is approached but does so more dramatically in the model without immigration, because the power spectrum peak height increases as the transcritical bifurcation is approached. to examine how different changes in vaccination uptake p 0 affect the statistics, we varied p 0 by 1/500 and 1/30 year −1 in the fast-slow model with immigration. predictions generated from the p 0 = 1/500 model are in close agreement with predictions arising from the η > 0 stable model. however, when vaccination uptake is increasing an order of magnitude faster (i.e., p 0 = 1/30), there can be temporary fluctuations, including increases, in the variance (fig. 8e) . such a pattern is not predicted when we evaluate the statistics about the endemic equilibrium. generally, the analytical predictions for leading indicators for sis disease systems approaching elimination are robust over a moving window, and they perform well in comparison to statistics generated from the null stable models, as indicated by the roc curves in fig. 10 . the roc curves lie above the black line, showing that the indicators behave better than chance in distinguishing between realizations that have been generated by the null and test models and the reported auc values are close to 1. furthermore, the trends in the statistics agree with the theoretically predicted trends (compare fig. 10a , c, e with fig. 8a-c) . however, the gaussian smoother may not be appropriate when the trend declines rapidly, as occurs for the sis system approaching elimination. various bandwidths were used, but while smaller bandwidths removed the slowly varying trend successfully, they did not capture the fluctuations relevant for critical slowing down. larger bandwidths captured these fluctuations but did not successfully remove the slowly varying trend from the last 10 years of the time series, as can be seen in the plots. this problem with gaussian filtering has been noted before by dakos et al. (2008) . the theoretical predictions for leading indicators for sir disease systems approaching elimination are robust over a moving window. the trends in the statistics shown in figs. 11a, c, e, and 12a , c, e agree with the theoretical predictions ( fig. 8d-f) . notably, the median fluctuation variance for the system with p 0 = 1/30 also exhibits more variability, as predicted theoretically. the roc analysis for the sir models indicates that the statistics from the test models perform well in comparison to statistics generated from the null stable models, but the fluctuation variance for the system with p 0 = 1/30 is an exception. the performance is poor because the trend in the variance exhibits wider variation (fig. 12c) . finally, the gaussian filtering performs well in comparison to the van kampen detrending, because the mean of the sir model declines linearly and does not exhibit any rapid changes. as noted, the standard expectations for early warning signals of systems approaching a critical transition include increasing autocorrelation, increasing variance, and increasing coefficient of variation. prior to emergence, the theoretical predictions for the sis model generally agree with these standard expectations . for emerging sis diseases with immigration, the lag-1 autocorrelation is predicted to increase, indicating an increase in memory in the system. the variance is also predicted to increase, as suggested by the increasing area under the power spectrum in fig. 7c . however, the coefficient of variation is predicted to decline as p(t) decreases, indicating that the mean of the stable sis system rises more rapidly than the variance. the rapid rise in the mean can be seen in fig. 4a . however, the coefficient of variation is predicted to increase as the transition is approached if the statistic is evaluated about the solution of the fast-slow system. this is because the mean remains small long after the predicted critical transition (fig. 4a) . however, the limiting case theoretical predictions for the trends in the statistics for systems on the threshold of emergence do not always agree with the predictions for the corresponding fast-slow systems beyond the critical threshold for emergence. this finding results from the predicted mean given by fast-slow system being lower than that of the stable system in the → 0 theory (c.f., fig. 4a ). due to the in agreement with standard theory, the variance and autocorrelation increase in the sir and sis systems as the bifurcation is approached (the peaks in the variance and cv predictions have been cut off for clarity). to aid comparison with fig. 14c , the inset plot of (e) shows a close-up of increasing variance prior to the transition in the sir case. however, the → 0 theory predicts the cv declines on the approach to emergence. the effect of the bifurcation delay is also seen in the statistics predicted by the fast-slow theory. an order of magnitude difference in the change in vaccination uptake p 0 affects the predictions; e.g., the cv in the sis model is predicted to rise before the transcritical bifurcation by the fast-slow theory. increases in autocorrelation (a) and in variance (b) can occur in this model after the transcritical bifurcation. the autocorrelation of the fluctuations with a lag of 1 year (sis) and 1 month (sir) is shown, and the variance shown is the fluctuation variance. all calculations used the parameter values in table 7 ; the sir model calculations used the parameters in table 7 scaled to rates per month rapid rise in the mean following the bifurcation delay, each statistic exhibits another increase following the transcritical bifurcation. in subcritical sir systems approaching endemicity, the theoretical predictions for the sir model agree with standard expectations, except for the coefficient of variation, which is predicted to decline (figs. 9d-f ). the limiting case theoretical predictions for the trends in the statistics for sir systems on the threshold of emergence agree more closely with the predictions for the fluctuations about the solution of the corresponding sir fast-slow system, in contrast to the sis model, because the bifurcation delay, although present, is not as marked as that in the sis model. figure 9d , e shows that the monthly lag-1 autocorrelation and the variance increase as the bifurcation is approached. it is not surprising that the variance increases because the power spectrum increases in power as the critical threshold for emergence is approached (fig. 7d) . however, the coefficient of variation is predicted to decline, i.e., the signal should become less noisy with decreasing vaccination uptake. in the context of the power spectrum result, this again makes sense because when the system transitions from subcritical to endemic, the power spectrum changes from a relatively flat shape to one with a peak about a resonant frequency. to examine how differences in vaccination uptake p 0 affect the statistics, we varied p 0 by 1/500 and 1/30 year −1 in the fast-slow model. the statistics predicted by the fast-slow theory do not change as rapidly as predicted in the → 0 theory. finally, we did not calculate the early warning signals for the disease systems without immigration because the disease-free state is stable. in contrast to disease systems approaching elimination, the early warning signals for diseases on the verge of emergence do not perform well. no marked trends are present in the median statistics calculated from either of the van kampen or gaussian detrending approaches, with the exception that the variance of the sis and sir systems approaching emergence (figs. 13c, d and 14c , d) increases slightly. the 95 % prediction intervals for each statistic further show that all the statistics are highly variable. moreover, the roc curves indicate that it may, in general, be difficult to distinguish between subcritical systems with r 0 < 1 and endemic disease systems with r 0 > 1 (figs. 13b, d, f and 14b, d, f) . the roc curves show that the distributions of kendall's correlation coefficient generated from stable systems and systems approaching a critical transition overlap significantly. however, we are conservative in our approach because we take only the years up to the critical transition in the absence of immigration, and not the years after, when a bifurcation delay may occur, during which time prevalence remains low and so the mean prevalence may not increase for a long time after the transition. therefore, it is not surprising that it is difficult to distinguish between the subcritical and supercritical systems. the transmission of infectious diseases is a paradigm example of a low-dimensional, noisy, nonlinear dynamical process. critical transitions in infectious disease systems, which correspond to such socially important events as the emergence of novel pathogens and the elimination of disease, are of special interest. the development of early warning signals of emerging infectious diseases and leading indicators for disease elimination, particularly, would be of tremendous value to the advance of public health. the goal of our study was to develop the theory of such early warning signals and leading indicators for infectious disease transmission systems that meet the assumptions of the familiar sis and sir models and which are forced through a critical transition by changes in transmission. our main result-analytical expressions for the change in observable statistics as the transmission system approaches the critical transition-are reported in table 4 . b a d c f e fig. 10 performance of the statistics over a moving window for the sis system approaching elimination, assuming that immigration occurs (η > 0). panels a, c, and e show the median statistics (thick lines) and 95 % prediction intervals (shaded regions). the lag-1 autocorrelation and variance have been calculated from the fluctuations obtained from van kampen and gaussian detrending. the coefficient of variation (cv) is marked in dashed green line to indicate that it was calculated from the raw time series, not the deviations from the mean. the dashed vertical line marks the time of the transcritical bifurcation in the → 0 limiting case with η = 0. panels b, d, and f show the corresponding roc curves. the auc value indicates the area under the corresponding roc curve. all curves are above the black line, showing that the indicators behave better than chance in distinguishing between realizations that have been generated by the null and test models. however, the gaussian filtering may not be an appropriate method to obtain the fluctuations, because it does not remove the slowly varying trend close to the transition, as exhibited by the rapid rise in the indicators. all calculations used the parameter values in table 7 . a bandwidth of 20 years was chosen for the gaussian filtering because this theory depends on a sequence of approximations, we investigated the robustness of our results in a sequence of simulations. for the sis model, we found that the approach to elimination (fig. 10) was indicated by an increase in the autocorrelation, variance, and the coefficient of variation, as predicted by the theory. for the sir model, the approach to elimination (figs. 11 and 12) was indicated by an increase in the autocorrelation, a decrease in the variance, and an increase in the coefficient of variation, as predicted by the theory. for the sis and sir models, the approach to emergence was indicated by an increase in variance, but the effects on the autocorrelation and the coefficient of variation were imperceptible (figs. 13 and 14) . further, since the theoretical patterns may be difficult to distinguish from random noise or fail to provide a sufficiently reliable signal under realistic conditions for data collection, we also examined the suitability of these statistics as an online algorithm for early warning. following boettiger and hastings (2012) , we summarized the results of online analysis using the receiver operator characteristic. these results showed that the approach to elimination in both the sis model (fig. 10 ) and the sir model (figs. 11 and 12) could be detected with a high level of reliability (large auc), although variance was a much poorer indicator in the sir model than autocorrelation and the coefficient of variation. however, the approach to emergence was very difficult to detect in both the sis model and the sir model, with estimated auc values hovering just above the null value of 0.5 (figs. 13 and 14) . in summary, our simulation studies show the approximations required to obtain the theoretical predictions to be acceptable but indicate that reliable prediction is likely only in the case of leading indicators for elimination, not early warning signals of emergence. besides the practical goal of indicating patterns that function as early warning signals and leading indicators, our study also provides some basic insights into the dynamics of infectious diseases. particularly, our simulations illustrated the ubiquity of bifurcation delays-changes in system state that lag behind the bifurcation of the limiting case-during disease emergence and elimination. delays are of fundamental interest because they provide a mechanism for the frequent realization of far-from-equilibrium situations and because they are a dramatic example of tipping point phenomena in infectious diseases. indeed, delays can cause a system to appear to undergo a catastrophic shift (e.g., the b a d c f e fig. 11 performance of the statistics over a moving window for the sir system approaching elimination, assuming that immigration occurs (η > 0). panels a, c, and e show the median statistics (thick lines) and 95 % prediction intervals (shaded regions). the lag-1 autocorrelation and variance have been calculated from the fluctuations obtained from van kampen and gaussian detrending. the coefficient of variation is marked in green line to indicate that it was calculated from the raw time series, not deviations from the mean. the dashed vertical line marks the time of the transcritical bifurcation in the → 0 limiting case with η = 0. panels b, d, and f show the roc curves. the auc value indicates the area under the corresponding roc curve. all curves are above the black line, showing that the indicators behave better than chance in distinguishing between realizations that have been generated by the null and test models. a bandwidth of 5 years was chosen for the gaussian filtering. all calculations used the parameter values in table 7 b a d c f e fig. 12 performance of the statistics over a moving window for the sir system rapidly approaching elimination, assuming that immigration occurs (η > 0). panels a, c, and e show the median statistics (thick lines) and 95 % prediction intervals (shaded regions). the lag-1 autocorrelation and variance have been calculated from the fluctuations obtained from the van kampen and gaussian detrending. the coefficient of variation (cv) is marked in dashed green line to indicate that it was calculated from the raw time series, not deviations from the mean. the dashed vertical line marks the time of the transcritical bifurcation in the → 0 limiting case with η = 0. panels b, d, and f show the roc curves. the auc value indicates the area under the corresponding roc curve. all curves are above the black line, showing that the indicators behave better than chance in distinguishing between realizations that have been generated by the null and test models. however, the variance performs poorly compared with the lag-1 autocorrelation and cv. a bandwidth of 5 years was chosen for the gaussian filtering. all calculations used the parameter values in table 7 except for p 0 = 1/30 year −1 dramatic upturn of infectious cases associated with emergence) when, in fact, only a noncatastrophic transcritical bifurcation has occurred. our results show that two kinds of delays occur in disease emergence and elimination. the first delay is a deterministic phenomenon referred to as a canard (diener 1984) . canards occur in fast-slow systems where the solution follows an attracting slow manifold of the fast-slow system, passing close to a bifurcation point, and then follows a repelling slow manifold for a considerable period of time. thus, for instance, in fig. 3a , which depicts the emergence of an sis infection, the → 0 limit case branch rapidly increases close to the η = 0 critical point, but the solution of the fast-slow system ( > 0) tracks the disease-free equilibrium for a noticeable amount of time even after it has become unstable. the influence that the canard has on the stochastic dynamics can be observed in fig. 3b , where the number of infectious individuals remains in the vicinity of the precritical level for a noticeable period of time. a related phenomenon, but not so dramatic, is observed in the elimination case (fig. 2a) . following the bifurcation, the infectious population remains elevated, but unlike the emergence case, no dramatic shift to extinction occurs. analogous phenomena are observed in the sir model (figs. 5 and 6) . the second class of delays is a stochastic phenomenon, caused by the nonzero time interval that occurs between the time at which the critical point is reached and the time of the event that initiates the transition (i.e., the index case of a major outbreak). this phenomenon is most obvious in the emergence of an immunizing pathogen (the emergence scenario for the sir model). in this case, the number of infectious individuals in the population prior to the critical transition is most commonly zero. assuming that there are no infectious individuals in the population at the time of the bifurcation event, then the number of infections in the population will remain zero until the time that an imported case arises. in view of the assumption of demographic stochasticity, importation is a poisson process with nonzero rate parameter, which similarly ensures that a nonzero period of time must elapse before the first case appears. indeed, even after the first case appears, there is no guarantee that the system will undergo transition, since even b a d c f e fig. 13 performance of the statistics over a moving window for the sis system approaching endemicity, assuming that immigration occurs (η > 0). panels a, c, and e show the median statistics (thick lines) and 95 % prediction intervals (shaded regions). the lag-1 autocorrelation and variance have been calculated from the fluctuations obtained from the van kampen detrending and gaussian filtering. the coefficient of variation is marked in green (dashed) line to indicate that it was calculated from the raw time series, not deviations from the mean. the dashed vertical line marks the time of the transcritical bifurcation. panels b, d, and f shows the roc curves. the auc value indicates the area under the corresponding roc curve. there are no marked trends in the median statistics, although the variance exhibits a slight increasing trend. the roc curves show that the distributions of kendall's τ overlap greatly, suggesting that it is difficult to distinguish between a quasi-stationary system and one approaching an emergence threshold. due to the bifurcation delay, it is not surprising that it is difficult to distinguish between null and test replicates. a bandwidth of 10 years was selected for the gaussian filtering. all calculations used the parameter values in table 7 in a supercritical population, there is some chance of rapid extinction (allen 2003 ). when the system is only slightly supercritical, this chance may not be small. for measurement purposes, we might define this stochastic bifurcation delay as the time elapsed between the occurrence of the bifurcation and the final occurrence of zero infectious individuals in the population. clearly, this quantity is a random variable. the distributional properties of this stochastic bifurcation delay are an important problem for further research. delays are also important for the immediate practical reason that their existence implies that the state of the system, i.e., the presence or absence of disease, cannot be taken as reliable evidence concerning whether the system is subcritical or supercritical. in the case of disease emergence, a system may have developed to the supercritical state-a significant concern for public health-although no cases have occurred. in such a scenario, any introduction of infectious individuals may spark a major outbreak. in the case of disease elimination, a system may already exist in the subcritical state, with only lingering infections and short transmission chains enabling the pathogen to transiently persist. in such cases, the battle has already been won, but it is crucial that vaccination campaigns and other elimination activities be continued until the remaining lines of transmission are snuffed out. otherwise, gains that may have come at great cost will fail to be maintained, and the pathogen may resurge in the population, sparked by its own embers still smoldering in the face of relaxed intervention. the development of statistical methods to identify such situations is an urgent problem for further research. limiting case predictions can sometimes be misleading predictions for the statistics derived from the limiting case (η = 0) version of the sir model can sometimes be misleading. as the extinction threshold is approached, the predicted sir (η > 0) lag-1 autocorrelation increases as expected, but this phenomenon gives way to a decrease close to the η = 0 critical point (fig. 8d) . this finding is not so surprising when one recalls that the single biologically feasible equilibrium in the η > 0 model changes from a stable spiral to a stable node near the critical point as in addition, the roc curves indicate that the distributions of kendall's τ overlap greatly, suggesting that it is difficult to reliably distinguish between a stable system and one undergoing a critical transition using the lag-1 autocorrelation coefficient. the coefficient of variation also performs poorly, as predicted theoretically. due to the bifurcation delay, it is not surprising that it is difficult to distinguish between null and test replicates. a bandwidth of 20 months was selected for the gaussian filtering. all calculations used the parameter values in table 7 vaccination is increased. the magnitude of the real part of the eigenvalue, which approximates the rate of decay of the amplitude of the oscillations to the equilibrium, decreases as elimination is approached, but, near the threshold, the magnitude of the real part begins to increase once more, indicating a shorter return time to equilibrium and increased resistance of the system to perturbations, as the spiral begins to resemble a node. this subsequent decline in system memory is reflected in the decrease in the autocorrelation function near the critical point. the more rapid decay of the oscillations is also reflected in the power spectrum, in that the the spectrum peak declines as vaccination uptake increases, meaning that the resonant frequencies become less amplified. on the other hand, the recovery rate of the stable spiral in the limiting case decreases much more dramatically on the transition to elimination, and oscillations at the resonant frequencies become more amplified. thus, while the limiting case is an important guide in understanding more general disease models, it is also necessary to thoroughly investigate these models to understand the behavior of systems approaching a critical transition. this study has followed kuehn (2011) and boettiger and hastings (2012) in the use of stochastic differential equations to understand noise-induced phenomena that occur during a critical transition. it is understood, of course, that for discrete systems (such as infectious disease transmission), this continuum representation is only an approximation, and, moreover, that the approximation breaks down as the system size-or even if the size of just one important compartment-becomes small. the case of disease emergence, where the number of infectious individuals in a population prior to the critical transition is typically zero and only occasionally one or more, is precisely this situation. it would not be surprising, therefore, to discover the approach taken in this paper to perform poorly in this situation. but, this is not the case. as figs. 13 and 14 show, in both the sis and sir systems, the "measured" statistics obtained in simulation are qualitatively similar to the analytic expressions reported in table 4 , with the exception of the coefficient of variation in the sir model. we note, further, that although emergence was difficult to predict (auc values were not much greater than 0.5), the van kampen detrending, which we think of as the theorydependent approach, typically performed better than generic gaussian detrending, which may be computed without any theoretical assumptions. it is, nevertheless, a concern that the approach taken here will be misleading under some scenarios: for instance, a very slow approach to the critical transition, when the time between immigration events may be large and the van kampen approximation is likely to break down. moreover, the stochastic features of bifurcation delay, particularly important in the emergence scenarios, depend heavily on (a) the sequence of immigration events by infectious individuals, and (b) the probability of nonextinction for a transmission chain initiated by an infectious individual after the critical point has been passed. these are both intrinsically discrete aspects of the emergence process. for this reason, we suggest that the study of (nonstationary) discrete transmission models may be a fruitful direction for further study. there is, of course, a large body of work on (stationary) discrete contagion models on which such a theory could build (bailey 1964; renshaw 1991; daley and gani 1999; allen 2003) . in conclusion, early warning systems for emerging infectious diseases and leading indicators of elimination, which would be of tremendous benefit to society, now appear to be potentially achievable. their realization depends on two key developments: (1) better understanding of noiseinduced phenomena exhibited by nonlinear contagion processes in the vicinity of their critical points, and (2) surveillance methods that acquire data of sufficiently high accuracy, resolution, and timeliness. toward the first of these goals, our studies have indicated that anticipation of critical transitions is theoretically possible. it is hoped that these results, which include expressions for the key observable phenomena (table 4) , may help to guide achievement of the second, which is now the most important outstanding problem, i.e., empirical demonstration in experimental or surveillance data. the deterministic system describes the evolution of the trend or the location of the peak of the probability distribution of the infectious state i at a given time t. the variable ϕ(t) is defined as the proportion i (t)/n in the limit n → ∞. the fokker-planck equation describes the evolution of the fluctuations, i.e., the standard deviation of the probability distribution about nϕ(t). since the fokker-planck equation is linear, the solution for the probability distribution of the fluctuations (ζ, t) in the deterministic state is a normal distribution. we note that this is a result of our second-order truncation, not a derivative property of the original model, per se. our aim is to quantify the fluctuations in the quasistationary state given by the solution of the sis fast-slow system at time t. we can do this by using the fact that eq. (12) is equivalent to the following stochastic differential equation: (13) where dw is a wiener process with mean zero and variance dt (gardiner 2004) . analytical analysis of eq. (13) enables us to establish the quasi-stationary statistics that potentially could be used as leading indicators of a critical transition in sis infectious disease systems. as a special case, we consider the limit → 0, whereby the quasi-stationary state is given by the deterministic endemic equilibrium of eq. (11), ϕ * (defined in table 8 ). since eq. (11) dictates the evolution of the deterministic state and eq. (12) determines fluctuations in it, we may let ϕ(t) = ϕ * in eq. (12), and therefore, eq. (13) becomes analysis of eq. (13) leads to expressions for the fluctuation statistics, whereby the influence of the mean has table 8 variable substitutions for the sis model. to calculate the statistics of the fluctuations about the deterministic endemic equilibrium ϕ * , replace ϕ(t) with ϕ * in each variable variable expression β β ( 1 − (p s + p 0 t)) ϕ * (β − γ − η + (β − (γ + η) 2 + 4βη))/2β λ β − 2βϕ(t) − γ − η q βϕ(t)(1 − ϕ(t)) + η(1 − ϕ(t)) + γ ϕ(t) note that β is constant in a small time increment and is evaluated at the current time t already been removed. to analyze eq. (13), it is easier to rewrite it as follows: where λ and q are defined in table 8 in the main text and (t) denotes gaussian white noise. fourier transformation of eq. (15) yields iζ (ω) = λζ (ω) + qγ (ω), whereζ (ω) is the fourier transform of ζ . the definition of the fourier transform of a continuous function is provided in nisbet and gurney (1982) , and those authors extensively discuss the meaning of the fourier transform of white noise. rearranging eq. (15), we obtaiñ the quasi-stationary power spectrum of the fluctuations is given by the long-term average of the square of the magnitude ofζ (ω). the power spectrum is (nisbet and gurney 1982) s i (ω) = |ζ (ω) 2 | = q ω 2 + λ 2 , where . is the expectation. we can integrate this expression over the frequency domain to obtain the quasi-stationary variance, the quasi-stationary autocorrelation is given by the integral 1 π ∞ 0 s i (ω) cos(ωτ )dω = exp(λτ ). finally, we can use eq. (10) to obtain an expression for the coefficient of variation of i (t). since ζ ∼ normal(0, σ 2 ), where the variance σ 2 is given by eq. (19), then i (t) = nϕ(t) + ζ n 1 2 ∼ normal(nϕ(t), nσ 2 ). therefore, the theoretical coefficient of variation is (n − 1 2 σ )/ϕ(t). to make analytical progress with eq. (8) in the main text, we use the van kampen system size expansion. we approximate the number of susceptibles and the number of infectives by respectively. thus, we have transformed the discrete variables in terms of continuous stochastic variables σ and ζ , and, as in appendix a, we anticipate that the probability distribution will depend on the system size n. the van kampen system size expansion has been applied to sir models previously (alonso et al. 2007) , and so, we do not include the full details of the expansion here. using the approximations (21) to leading order, the system size expansion gives rise to the systeṁ which is simply the sir system approaching a transition (7). at next-to-leading order, the fokker-planck equation for the fluctuations in the solution of the system (22) at time t is where x = (σ (t), ζ (t)) denotes the vector of fluctuations from the susceptible and infectious states, respectively. the mean vector is given by μ i (x) = a i1 σ (t)+a i2 ζ(t), i = 1, 2. the matrices a and d are obtained from the system size expansion. the coefficients a ij of the mean vector are the entries of the jacobian matrix obtained from linear stability analysis. the d ij coefficients are the entries of the noisecovariance matrix and can only be obtained from the system size expansion (van kampen 1981) . equation (22) describes the evolution of the trend, and the fokker-planck equation models the evolution of the fluctuations. the solution of eq. (23) is a bivariate normal distribution since the fokker-planck equation is linear (van kampen 1981). we note that when incidence of a disease is low, as occurs when the system is subcritical, the assumption that the fluctuations are normally distributed about the mean is likely to not be appropriate because the approximation (21) cannot describe chance extinctions, which can occur if i is small. finally, it is possible to numerically obtain the variance-covariance matrix for the fluctuations in s and i around the values of the solution of eq. (22), but, in this paper, we are interested only in fluctuations in i since s is unobservable. to quantify the fluctuations, we use the fact that the following system of stochastic differential equations is equivalent to eq. (23): dσ dt = a 11 σ (t) + a 12 ζ(t) + 1 (t) dζ dt = a 21 σ (t) + a 22 ζ(t) + 2 (t), where 1 (t), 2 (t) are white-noise processes with covariance matrix d. the entries of the matrices a and d are included in table 9 in the main text. if the mean field → 0 theory applies, the coefficients a ij are approximately the entries in the jacobian matrix of eq. (6) evaluated at the since we could not obtain an analytical expression for the solution of eq. (29), we had to obtain this integral numerically. finally, an expression for the coefficient of variation of i may also be obtained, using eq. (21). since ζ ∼ normal(0, σ 2 ), where the variance σ 2 is given by eq. (28), then i (t) ∼ normal(nϕ(t), nσ 2 ). therefore, the theoretical coefficient of variation is (n − 1 2 σ )/ϕ(t). an introduction to stochastic processes with applications to biology stochastic amplification in epidemics seasonality and the dynamics of infectious diseases infectious diseases of humans: dynamics and control the elements of stochastic processes with applications to the natural sciences quantifying limits to detection of early warning for critical transitions interacting regime shifts in ecosystems: implication for early warnings rising variance: a leading indicator of ecological transition early warnings of unknown nonlinear shifts: a nonparametric approach leading indicators of trophic cascades malaria resurgence: a systematic review and assessment of its causes slowing down as an early warning signal for abrupt climate change spatial correlation as leading indicator of catastrophic shifts robustness of variance and autocorrelation as indicators of critical slowing down methods for detecting early warnings of critical transitions in time series illustrated using simulated ecological data epidemic modelling: an introduction the canard unchained or how fast/slow systems bifurcate early warnings of catastrophic shifts in ecosystems: comparison between spatial and temporal indicators measuring tuberculosis burden, trends, and the impact of control programmes seasonal infectious disease epidemiology handbook of stochastic methods for physics, chemistry and the natural sciences exact stochastic simulation of coupled chemical reactions changing skewness: an early warning signal of regime shifts in ecological systems spatial variance and spatial skewness: leading indicators of regime shifts in spatial ecological systems regime shifts in ecological systems can occur with no warning perspectives on the basic reproductive ratio detecting dynamical changes in nonlinear time series using locally linear state-space models estimating spatial coupling in epidemiological systems: a mechanistic approach modeling infectious diseases in humans and animals a mathematical framework for critical transitions: bifurcations, fastslow systems and stochastic dynamics early warning of climate tipping points early warning of climate tipping points from critical slowing down: comparing methods to improve robustness epidemic dynamics at the human-animal interface stochastic models in population biology and their deterministic analogs measles: the burden of preventable deaths preparing for the next pandemic modelling biological populations in space and time the decline and resurgence of pertussis in the us the interplay between determinism and stochasticity in childhood infectious diseases assessing the economic impact and costs of flu pandemic originating in asia critical transitions in nature and society early warning signals for critical transitions anticipating critical transitions heteroscedasticity as a leading indicator of ecological regime shifts assessment of the 2010 global measles mortality reduction goal: results from a model of surveillance data the economywide impact of pandemic influenza on the uk: a computable general equilibrium modelling experiment nonlinear dynamics and chaos with applications to physics, biology, chemistry and engineering who (world health organization) (2012) who malaria report 2012 acknowledgments this research was funded by a grant from the james s. mcdonnell foundation.open access this article is distributed under the terms of the creative commons attribution license which permits any use, distribution, and reproduction in any medium, provided the original author(s) and the source are credited. to make the description of the probability distribution given by eq. (4) in the main text more amenable to analytical analysis, we approximate the discrete infectious state random variable by a continuous random variable ζ ,we are anticipating that the probability distribution will depend on the population size n. we do not assume what equation ϕ(t) satisfies, but we will see later that it is equal to the proportion of infectious individuals at time t, in the limit of infinitely large population size n; the system size expansion will give rise to the mean field theory discussed in the "mean field theory of sir model" section. equation (10) says that fluctuations (denoted by ζ ) about the deterministic state i/n are expected to be of the order of n −1/2 , which is expected from the central limit theorem, and so the fluctuations in i are of the order n 1/2 (van kampen 1981). if the population size n is large, we intuitively expect fluctuations to be small. consequently, the probability distribution for the system being in a certain state at time t is expected to have a peak located at the deterministic value nϕ(t) and a standard deviation of the order n 1/2 . the approximation (10) turns out to be a normal random variable.the system size expansion is described in detail by the van kampen (van kampen 1981) and has been applied to one-dimensional population biology models (mckane and newman 2004) . therefore, we do not include the full details of the expansion of eq. (4) in this paper. in summary, there are five essential steps: (1) write down transition rates (e.g., table 2 ); (2) derive the master equation for the markov process; (3) rewrite the master equation in terms of jump functions; (4) substitute the ansatz (10) into the master equation and perform the expansion; and (5) collect deterministic (leading order) and stochastic (next-to-leading order) terms to obtain the deterministic system and its stochastic correction.rewriting the master equation as described in detail in (van kampen 1981) and performing the system size expansion using eq. (10), at leading order, give rise to the differential equation that ϕ(t) satisfiesthis is simply the fast-slow system (3). at the next-toleading order, the method leads to a linear fokker-planck equation for the distribution of the fluctuations about the solution ϕ(t), note that p is assumed to be constant in a small time increment and is evaluated at the current time t. other symbols in the table:endemic equilibrium. to analyze the system (24), we take its fourier transform, leading towhereσ (ω),ζ (ω),˜ 1 (ω) and˜ 2 (ω) are the fourier transforms of σ , ζ , 1 , and 2 , respectively. since we are interested in the fluctuations about the infectious state, we solve eq. (25) for the fourier transformζ (ω), obtainingwhere t and d are the trace and determinant of the jacobian matrix, respectively, given in tables 9. using eq. (26), we can establish the power spectrum of the fluctuations |ζ (ω) 2 | ,where α i is given in table 9 . the variance and autocorrelation of the fluctuations in i (t) around the solution of system (22) may be obtained using eq. (27) through integration. the variance of the fluctuations isthe lag-τ autocorrelation is given by the integral 1 π ∞ 0 s i (ω) cos(ωτ )dω.